Patent application title: METHODS AND COMPOSITIONS FOR THE DETECTION OF CERVICAL DISEASE
Timothy J. Fischer (Raleigh, NC, US)
Douglas P. Malinowski (Hillsborough, NC, US)
Douglas P. Malinowski (Hillsborough, NC, US)
Adriann J. Taylor (Durham, NC, US)
Margaret R. Parker (Raleigh, NC, US)
TriPath Imaging, Inc
IPC8 Class: AG01N3353FI
Class name: Chemistry: molecular biology and microbiology measuring or testing process involving enzymes or micro-organisms; composition or test strip therefore; processes of forming such composition or test strip involving antigen-antibody binding, specific binding protein assay or specific ligand-receptor binding assay
Publication date: 2009-06-11
Patent application number: 20090148864
Methods and compositions for identifying high-grade cervical disease in a
patient sample are provided. The methods of the invention comprise
detecting overexpression of at least one biomarker in a body sample,
wherein the biomarker is selectively overexpressed in high-grade cervical
disease. In particular claims, the body sample is a cervical smear or
monolayer of cervical cells. The biomarkers of the invention include
genes and proteins that are involved in cell cycle regulation, signal
transduction, and DNA replication and transcription. In particular
claims, the biomarker is an S-phase gene. In some aspects of the
invention, overexpression of a biomarker of interest is detected at the
protein level using biomarker-specific antibodies or at the nucleic acid
level using nucleic acid hybridization techniques. Kits for practicing
the methods of the invention are further provided.
1. A kit comprising at least three antibodies, wherein each antibody
specifically binds to a nuclear biomarker protein that is selectively
overexpressed in high-grade cervical disease, and wherein a first and a
second antibody in the kit specifically bind to the biomarker protein
MCM2, and wherein a third antibody specifically binds to the biomarker
2. The kit of claim 1, wherein said kit further comprises a peroxidase blocking reagent, a protein blocking reagent, chemicals for the detection of antibody binding to said biomarker proteins, a counterstain, a bluing agent, and instructions for use.
3. The kit of claim 2, wherein said chemicals for the detection of antibody binding comprise a chromogen and a secondary antibody conjugated to a labeled polymer, wherein the chromogen comprises 3',3'-diaminobenzidine, and wherein the labeled polymer comprises horseradish peroxidase conjugated to a dextran polymer.
4. The kit of claim 2, wherein said counterstain comprises hematoxylin.
5. The kit of claim 2, wherein said bluing agent comprises a solution comprising Tris buffered saline, pH 7.4, Tween-20, and sodium azide.
6. The kit of claim 2 further comprising a positive control sample.
7. The kit of claim 6, wherein said positive control sample comprises SiHa cells.
8. The kit of claim 1 further comprising reagents for Papanicolaou (Pap) staining.
9. The kit of claim 8, wherein the reagents for Pap staining comprise EA50 and Orange G.
10. The kit of claim 1, wherein the at least three antibodies are provided as separate reagents.
11. The kit of claim 1, wherein the at least three antibodies are provided as a cocktail.
12. A method for diagnosing high-grade cervical disease in a patient independent of the patient's HPV infection status, the method comprising:a) obtaining a body sample from the patient;b) contacting the sample with at least three antibodies, wherein each of the antibodies specifically binds to a nuclear biomarker protein that is selectively overexpressed in high-grade cervical disease, wherein a first and a second antibody specifically bind to the biomarker protein MCM2, and wherein a third antibody specifically binds to the biomarker protein MCM7; and,c) detecting binding of the antibodies to the biomarker proteins MCM2 and MCM7 to determine if the biomarker proteins are overexpressed in the sample, and thereby diagnosing high-grade cervical disease in the patient.
13. The method of claim 12, wherein the method comprises performing immunocytochemistry.
14. The method of claim 12, wherein the method is performed manually.
15. The method of claim 12, wherein the method is performed in an automated manner.
16. The method of claim 12, wherein the sample comprises cervical cells.
17. The method of claim 16, wherein the sample comprises a monolayer of cervical cells.
18. The method of claim 12, wherein the sensitivity of the method for diagnosing high-grade cervical disease is at least 90% and the specificity of the method for diagnosing high-grade cervical disease is at least 75%.
19. The method of claim 12, wherein the method is performed in response to the patient having an abnormal Pap smear result.
20. The method of claim 12, wherein the method is performed as a primary screen for high-grade cervical disease in a general patient population.
21. The method of claim 12 further comprising Papanicolaou (Pap) staining of the sample.
22. The method of claim 12, wherein the antibodies are contacted with the sample sequentially as individual antibody reagents.
23. The method of claim 12, wherein the antibodies are contacted with the sample simultaneously as an antibody cocktail.
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a divisional of U.S. patent application Ser. No. 11/521,144, filed Sep. 14, 2006, which is a continuation of U.S. patent application Ser. No. 11/087,227, filed Mar. 23, 2005, now U.S. Pat. No. 7,157,233, which claims the benefit of U.S. Provisional Application Ser. No. 60/556,495, filed Mar. 24, 2004, all of which is incorporated herein by reference in their entirety.
REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS-WEB
The official copy of the sequence listing is submitted concurrently with the specification as a text file via EFS-Web, in compliance with the American Standard Code for Information Interchange (ASCII), with a file name of 364711SequenceListing.txt, a creation date of Oct. 30, 2008, and a size of 116 KB. The sequence listing filed via EFS-Web is part of the specification and is hereby incorporated in its entirety by reference herein.
FIELD OF THE INVENTION
The present invention relates to methods and compositions for the detection of high-grade cervical disease.
BACKGROUND OF THE INVENTION
Carcinoma of the cervix is the second most common neoplasm in women, accounting for approximately 12% of all female cancers and causing approximately 250,000 deaths per year. Baldwin et al. (2003) Nature Reviews Cancer 3: 1-10. In many developing countries where mass screening programs are not available, the clinical problem is more serious. Cervical cancer in these countries is the number one cause of cancer deaths in women.
The majority of cases of cervical cancer represent squamous cell carcinoma, although adenocarcinoma is also seen. Cervical cancer can be prevented by population screening as it evolves through well-defined noninvasive intraepithelial stages, which can be distinguished morphologically. Williams et al. (1998) Proc. Natl. Acad. Sci. USA 95:14932-14937. While it is not understood how normal cells become transformed, the concept of a continuous spectrum of histopathological change from normal, stratified epithelium through cervical intraepithelial neoplasia (CIN) to invasive cancer has been widely accepted for years. The precursor to cervical cancer is dysplasia, also known in the art as CIN or squamous intraepithelial lesions (SIL). Squamous intraepithelial abnormalities may be classified by using the three-tiered (CIN) or two-tiered (Bethesda) system. Under the Bethesda system, low-grade squamous intraepithelial lesions (LSIL), corresponding to CINI and HPV infection, generally represent productive HPV infections with a relatively low risk of progression to invasive disease. High-grade squamous intraepithelial lesions (HSIL), corresponding to CINII and CINIII in the three-tiered system, show a higher risk of progression to cervical cancer than do LSIL, although both LSIL and HSIL are viewed as potential precursors of malignancy. Patient samples may also be classified as ASCUS (atypical squamous cells of unknown significance) or AGUS (atypical glandular cells of unknown significance) under this system.
A strong association of cervical cancer and infection by high-risk types of human papilloma virus (HPV), such as types 16, 18, and 31, has been established. In fact, a large body of epidemiological and molecular biological evidence has established HPV infection as a causative factor in cervical cancer. Moreover, HPV is found in 85% or more of the cases of high-grade cervical disease. However, HPV infection is very common, possibly occurring in 5-15% of women over the age of 30, but few HPV-positive women will ever develop high-grade cervical disease or cancer. The presence of HPV alone is indicative only of infection, not of high-grade cervical disease, and, therefore, testing for HPV infection alone results in many false positives. See, for example, Wright et al. (2004) Obstet. Gynecol. 103:304-309.
Current literature suggests that HPV infects the basal stem cells within the underlying tissue of the uterine-cervix. Differentiation of the stem cells into mature keratinocytes, with resulting migration of the cells to the stratified cervical epithelium, is associated with HPV viral replication and re-infection of cells. During this viral replication process, a number of cellular changes occur that include cell-cycle de-regulation, active proliferation, DNA replication, transcriptional activation and genomic instability (Crum (2000) Modern Pathology 13:243-251; Middleton et al. (2003) J. Virol. 77:10186-10201; Pett et al. (2004) Cancer Res. 64:1359-1368).
Most HPV infections are transient in nature, with the viral infection resolving itself within a 12-month period. For those individuals who develop persistent infections with one or more oncogenic subtypes of HPV, there is a risk for the development of neoplasia in comparison to patients without an HPV infection. Given the importance of HPV in the development of cervical neoplasia, the clinical detection of HPV has become an important diagnostic tool in the identification of patients at risk for cervical neoplasia development. The clinical utility of HPV-based screening for cervical disease is in its negative predictive value. An HPV negative result in combination with a history of normal Pap smears is an excellent indicator of a disease-free condition and a low risk of cervical neoplasia development during the subsequent 1-3 years. However, a positive HPV result is not diagnostic of cervical disease; rather it is an indication of infection. Although the majority of HPV infections is transient and will spontaneously clear within a 12-month period, a persistent infection with a high-risk HPV viral subtype indicates a higher risk for the development of cervical neoplasia. To supplement HPV testing, the identification of molecular markers associated with cervical neoplasia is expected to improve the clinical specificity for cervical disease diagnosis.
Cytological examination of Papanicolaou-stained cervical smears (Pap smears) currently is the method of choice for detecting cervical cancer. The Pap test is a subjective method that has remained substantially unchanged for 60 years. There are several concerns, however, regarding its performance. The reported sensitivity of a single Pap test (the proportion of disease positives that are test-positive) is low and shows wide variation (30-87%). The specificity of a single Pap test (the proportion of disease negatives that are test-negative) might be as low as 86% in a screening population and considerably lower in the ASCUS PLUS population for the determination of underlying high-grade disease. See, Baldwin et al., supra. A significant percentage of Pap smears characterized as LSIL or CINI are actually positive for high-grade lesions. Furthermore, up to 10% of Pap smears are classified as ASCUS (atypical squamous cells of undetermined significance), i.e., it is not possible to make a clear categorization as normal, moderate or severe lesion, or tumor. However, experience shows that up to 10% of this ASCUS population has high-grade lesions, which are consequently overlooked. See, for example, Manos et al. (1999) JAMA 281:1605-1610.
Thus, a method for diagnosing high-grade cervical disease that is independent of or works in conjunction with conventional Pap smears and molecular testing for high-risk HPV infection is needed. Such a method should be able to specifically identify high-grade cervical disease that is present in all patient populations, including those cases classified as LSIL or CINI by Pap staining that are actually positive for high-grade lesions (i.e., "false negatives"). Therefore, there is a need in the art for specific, reliable diagnostic methods that are capable of detecting high-grade cervical disease and of differentiating high-grade disease from conditions that are not considered clinical disease, such as early-stage HPV infection and mild dysplasia.
SUMMARY OF THE INVENTION
Compositions and methods for diagnosing high-grade cervical disease are provided. The methods of the invention comprise detecting overexpression of at least one biomarker, particularly a nuclear biomarker, in a body sample, wherein the detection of overexpression of said biomarker specifically identifies samples that are indicative of high-grade cervical disease. The present method distinguishes samples that are indicative of high-grade cervical disease from samples that are indicative of benign proliferation, early-stage HPV infection, or mild dysplasia. Thus, the method relies on the detection of a biomarker that is selectively overexpressed in high-grade cervical disease states but that is not overexpressed in normal cells or cells that are not indicative of clinical disease.
The biomarkers of the invention are proteins and/or genes that are selectively overexpressed in high-grade cervical disease, including those that result from HPV-induced cell cycle dysfunction and activation of certain genes responsible for S-phase induction. Biomarkers of particular interest include S-phase genes, whose overexpression results from HPV-induced cell-cycle dysfunction and the subsequent activation of the transcriptional factors SP-1 and E2F. The detection of overexpression of the biomarker genes or proteins of the invention permits the differentiation of samples that are indicative of high-grade disease, such as moderate to severe dysplasia and cervical carcinomas, from normal cells or cells that are not indicative of clinical disease (e.g., early-stage HPV infection absent dysplasia and mild dysplasia).
Biomarker overexpression can be assessed at the protein or nucleic acid level. In some embodiments, immunocytochemistry techniques are provided that utilize antibodies to detect the overexpression of biomarker proteins in cervical cytology samples. In this aspect of the invention, at least one antibody directed to a specific biomarker of interest is used. Overexpression can also be detected by nucleic acid-based techniques, including, for example, hybridization and RT-PCR. Kits comprising reagents for practicing the methods of the invention are further provided.
The methods of the invention can also be used in combination with traditional gynecological diagnostic techniques that analyze morphological characteristics or HPV infection status. Thus, for example, the immunocytochemistry methods presented here can be combined with the Pap test so that all the morphological information from the conventional method is conserved. In this manner, the detection of biomarkers that are selectively overexpressed in high-grade cervical disease can reduce the high false-negative rate of the Pap test and may facilitate mass automated screening.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 provides a schematic summary of proliferation and cell cycle de-regulation in cervical dysplasia. Cell cycle alterations and proliferation control defects in cervical neoplasia. HPV infection and over-expression of the E6 and E7 oncoproteins produces a series of alterations in the cell cycle and proliferation control. The HPV E6 oncoprotein abrogates cell cycle checkpoints at the G1/S and G2/M boundaries with subsequent replication of DNA with somatic mutations. E7 promotes the acceleration into the S-phase with prolonged expression of the S-phase genes required for DNA replication (aberrant S-phase induction). Likewise, E6 promotes expression of telomerase ensuring continued chromosomal telomere integrity during proliferation and cellular immortalization. Finally, E7 abrogates the TGF-beta signaling pathway and abrogates this control mechanism for G1 arrest and control of proliferation.
FIG. 2 provides a schematic representation of aberrant S-phase induction in cervical neoplasia. The effects of HPV proteins on cell cycle control and proliferation include inactivation of the p53 and Rb tumor suppressor pathways, activation of E2F-1 transcription, induction of the S-phase genes MCM-2, MCM-6, MCM-7, TOP2A and Cyclin E1 along others. In addition, E2 interacts with the Sp-1 transcription factor to activate gene expression of p21-waf-1.
FIG. 3 provides a schematic representation of the feedback loop on cell proliferation in aberrant S-phase of the cell cycle. Overexpression of Cyclin E and CDK2 in the S-phase results in an independent mechanism to permit induction of the S-phase genes.
FIG. 4 provides a schematic representation of the role of c-myc in aberrant S-phase induction. C-myc is an important transcriptional activator in cellular proliferation. The gene encoding c-myc is located on the chromosome. This is the same site that HPV 18 integration has been documented with a corresponding amplification of this gene region. Amplification of the c-myc gene would result in over-expression of the encoded protein and increased levels of c-myc would independently contribute to S-phase gene transcription further accelerating cellular proliferation.
FIG. 5 provides a schematic representation of TaqMan® primers directed to MCM7 transcript variants.
FIG. 6 illustrates the differential staining pattern of an antibody directed to Claudin 1 in an IHC assay for a patient with mild dysplasia and a patient with squamous cell carcinoma.
FIG. 7 illustrates the differential staining pattern of an antibody directed to Claudin 1 in an IHC and ICC format. Normal cells and cells indicative of CINIII and HSIL are shown.
FIG. 8 illustrates nuclear staining patterns obtained with a nuclear biomarker (i.e., MCM2) and cytoplasmic staining patterns obtained with a cytoplasmic biomarker (p16). Results are from an immunocytochemistry (ICC) assay of a high-grade cervical disease patient sample.
FIG. 9 illustrates desirable and undesirable antibody staining in an immunohistochemistry (IHC) assay using two different antibodies directed to MCM6 on cervical tissue from a patient with high-grade cervical disease.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides compositions and methods for identifying or diagnosing high-grade cervical disease. The methods comprise the detection of the overexpression of specific biomarkers that are selectively overexpressed in high-grade cervical disease (e.g., moderate to severe dysplasia and cervical cancer). That is, the biomarkers of the invention are capable of distinguishing between HPV-infected cells and HPV-infected cells that are pre-malignant, malignant, or overtly cancerous. Methods for diagnosing high-grade cervical disease involve detecting the overexpression of at least one biomarker that is indicative of high-grade cervical disease in a tissue or body fluid sample from a patient. In particular embodiments, antibodies and immunocytochemistry techniques are used to detect expression of the biomarker of interest. Kits for practicing the methods of the invention are further provided.
"Diagnosing high-grade cervical disease" is intended to include, for example, diagnosing or detecting the presence of cervical disease, monitoring the progression of the disease, and identifying or detecting cells or samples that are indicative of high-grade cervical disease. The terms diagnosing, detecting, and identifying high-grade cervical disease are used interchangeably herein. By "high-grade cervical disease" is intended those conditions classified by colposcopy as premalignant pathology, malignant pathology, moderate to severe dysplasia, and cervical cancer. Underlying high-grade cervical disease includes histological identification of CINII, CINIII, HSIL, carcinoma in situ, adenocarcinoma, and cancer (FIGO stages I-IV).
As discussed above, a significant percentage of patients presenting with Pap smears classified as normal, CINI, or ASCUS actually have lesions characteristic of high-grade cervical disease. Thus, the methods of the present invention permit the identification of high-grade cervical disease in all patient populations, including these "false negative" patients, and facilitate the detection of rare abnormal cells in a patient sample. The diagnosis can be made independent of cell morphology and HPV infection status, although the methods of the invention can also be used in conjunction with conventional diagnostic techniques, e.g., Pap test, molecular testing for high-risk types of HPV, etc.
HPV types have been divided into high and low-risk categories based on their association with cervical cancer and precancerous lesions. Low-risk HPV types include types 6, 11, 42, 43, 44 and are not associated with an increased risk of cervical cancer. In contrast, high-risk HPV types, including types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, have been strongly associated with cervical cancer and squamous intraepithelial lesions. See, for example, Wright et al. (2004) Obstet. Gynecol. 103:304-309. In fact, over 99% of cervical cancers are associated with high-risk HPV infection. Persistent high-risk HPV infection leads to the disruption of the cell cycle and mitotic checkpoints in cervical cells through the action of HPV genes E2, E6, and E7. In particular, HPV E7 causes an increase in cyclin E and the subsequent release of the transcription factor E2f from the retinoblastoma (Rb) protein. The released E2f transcription factor then triggers the transcription of a variety of S-phase genes, including topoisomerase II alpha (Topo2A), MCM proteins, cyclins E1 and E2, and p14arf, resulting in loss of cell cycle control. HPV E2 further stimulates overexpression of S-phase genes such as p21.sup.waf-1 by activating the Sp-1 transcription factor. The cell cycle disruption caused by persistent HPV infection can lead to mild cervical dysplasia that may then progress to moderate or severe dysplasia and eventually to cervical cancer in some cases. By "cervical cancer" is intended any cancer or cancerous lesion associated with cervical tissue or cervical cells.
HPV infection within cervical keratinocytes results in a number of alterations that disrupt the activities within the cell cycle. The E6 and E7 oncoproteins of the high-risk HPV subtypes have been implicated in a number of cellular processes related to increased proliferation and neoplastic transformation of the infected keratinocytes. The E6 protein has been implicated in two critical processes. The first is the degradation of the p53 tumor suppressor protein through ubiquitin-mediated proteolysis. Removal of functional p53 eliminates a major cell cycle checkpoint responsible for DNA repair prior to entry into DNA replication and mitosis (Duensing and Munger (2003) Prog Cell Cycle Res. 5:383-391). In addition, E6 has been shown to interact with the c-myc protein and is responsible for direct transcriptional activation of the hTERT gene with subsequent expression of telomerase (McMurray and McCance (2003) J Virol. 77:9852-9861; Veldman et al. (2003) Proc Natl Acad Sci U.S.A. 100: 8211-8216). Activation of telomerase is a key step in cancer biology responsible for the maintenance of telomere length on replicating chromosomes and this enzyme ensures functionally intact chromosomes during cellular immortalization.
The HPV oncoprotein E7 is known to contribute to cellular proliferation through two independent mechanisms. The first is the inactivation of the TGF-beta tumor suppressor pathway responsible for cell cycle arrest at the G1 phase through direct interaction of E7 with the Smad proteins (Smad 2, 3 and 4), thereby inhibiting their ability to bind to DNA (Lee et al. (2002) J Biol. Chem. 277:38557-38564). Likewise, E7 is known to specifically interact with the Rb tumor suppressor protein. Within the G1 phase of the cell cycle, Rb complexes the E2F transcription factor and prevents E2F from activating gene transcription. At the G1/S boundary, the Rb protein is phosphorylated with release of the E2F transcription factor--thereby initiating E2F gene transcription and entry into the S phase of the cell cycle. The HPV E7 oncoprotein abrogates this control mechanism by directly binding with Rb and displacing E2F from the complex. This results in E2F driven gene transcription independent of normal cell cycle control (Duensing and Munger (2003) Prog Cell Cycle Res. 5:383-391; Duensing and Munger (2004) Int J Cancer 109:157-162; Clarke and Chetty (2001) Gynecol Oncol. 82:238-246). This release of E2F uncouples gene transcription from cell cycle control and results in prolonged and aberrant transcription of S-phase genes responsible for DNA synthesis and cellular proliferation. In addition, the combined actions of both E6 and E7 have been shown to contribute to centrosome abnormalities and the subsequent genomic instability in cervical neoplasia (Duensing and Munger (2004) Int J Cancer 109:157-162).
While not intending to be limited to a particular mechanism, in some embodiments, the molecular behavior of high-grade cervical disease can be characterized as the overexpression of discrete genes, normally expressed only during the S-phase of the cell cycle, as a result of infection by oncogenic strains of HPV. The subsequent uncontrolled activation of gene transcription and aberrant S-phase induction is mediated through the E2F-1 transcription factor pathway. This behavior appears to be indicative of high-grade cervical disease and provides a link between oncogenic HPV infections and the molecular behavior of cervical neoplasia. The use of these molecular biomarkers of cervical neoplasia in molecular diagnostic assay formats can improve the detection of cervical disease with an improved sensitivity and specificity over current methods. See generally FIGS. 1-4 and Malinowski (2005) BioTechniques 38:1-8 (in press), which is herein incorporated by reference in its entirety. Thus, in particular embodiments, a method for diagnosing high-grade cervical disease comprises detecting overexpression of a biomarker, wherein overexpression of the biomarker is indicative of aberrant S-phase induction, as described herein. In still other embodiments, the methods comprise detecting overexpression of a biomarker, wherein overexpression of the biomarker is indicative of active transcription or overexpression of the HPV E6 and HPV E7 genes.
Dysplasia is conventionally defined in morphological terms by a loss of normal orientation of epithelial cells, accompanied by alterations in cellular and nuclear size, shape, and staining characteristics. Dysplasia is graded according to the degree of the cellular abnormalities (i.e., mild, moderate, severe) and is widely accepted to be an intermediate stage in the progression from normal tissue to neoplasia, as evidenced by the identification of pre-malignant dysplastic conditions such as CIN. The methods of the present invention permit the identification of high-grade cervical disease, which includes moderate to severe dysplasia and cervical cancer (i.e., CINII conditions and above), based on the overexpression of biomarkers that are specific to high-grade cervical disease.
The methods disclosed herein provide superior detection of high-grade cervical disease in comparison to PAP smears and/or HPV infection testing. In particular aspects of the invention, the sensitivity and specificity of the present methods are equal to or greater than that of conventional Pap smears. As used herein, "specificity" refers to the level at which a method of the invention can accurately identify samples that have been confirmed as NIL by colposcopy (i.e., true negatives). That is, specificity is the proportion of disease negatives that are test-negative. In a clinical study, specificity is calculated by dividing the number of true negatives by the sum of true negatives and false positives. By "sensitivity" is intended the level at which a method of the invention can accurately identify samples that have been colposcopy-confirmed as positive for high-grade cervical disease (i.e., true positives). Thus, sensitivity is the proportion of disease positives that are test-positive. Sensitivity is calculated in a clinical study by dividing the number of true positives by the sum of true positives and false negatives. See Examples 1-3 below. In some embodiments, the sensitivity of the disclosed methods for the detection of high-grade cervical disease is preferably at least about 70%, more preferably at least about 80%, most preferably at least about 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more. Furthermore, the specificity of the present methods is preferably at least about 70%, more preferably at least about 80%, most preferably at least about 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more.
The term "positive predictive value" or "PPV" refers to the probability that a patient has high-grade cervical disease when restricted to those patients who are classified as positive using a method of the invention. PPV is calculated in a clinical study by dividing the number of true positives by the sum of true positives and false positives. In some embodiments, the PPV of a method of the invention for diagnosing high-grade cervical disease is at least about 40%, while maintaining a sensitivity of at least about 90%, more particularly at least about 95%. The "negative predictive value" or "NPV" of a test is the probability that the patient will not have the disease when restricted to all patients who test negative. NPV is calculated in a clinical study by dividing the number of true negatives by the sum of true negatives and false negatives.
The biomarkers of the invention include genes and proteins, and variants and fragments thereof. Such biomarkers include DNA comprising the entire or partial sequence of the nucleic acid sequence encoding the biomarker, or the complement of such a sequence. The biomarker nucleic acids also include RNA comprising the entire or partial sequence of any of the nucleic acid sequences of interest. A biomarker protein is a protein encoded by or corresponding to a DNA biomarker of the invention. A biomarker protein comprises the entire or partial amino acid sequence of any of the biomarker proteins or polypeptides.
A "biomarker" is any gene or protein whose level of expression in a tissue or cell is altered compared to that of a normal or healthy cell or tissue. Biomarkers of the invention are selective for underlying high-grade cervical disease. By "selectively overexpressed in high-grade cervical disease" is intended that the biomarker of interest is overexpressed in high-grade cervical disease but is not overexpressed in conditions classified as LSIL, CINI, HPV-infected samples without any dysplasia present, immature metaplastic cells, and other conditions that are not considered to be clinical disease. Thus, detection of the biomarkers of the invention permits the differentiation of samples indicative of underlying high-grade cervical disease from samples that are indicative of benign proliferation, early-stage HPV infection, or mild dysplasia. By "early-stage HPV infection" is intended HPV infection that has not progressed to cervical dysplasia. As used herein, "mild dysplasia" refers to LSIL and CINI where no high-grade lesion is present. The methods of the invention also distinguish cells indicative of high-grade disease from normal cells, immature metaplastic cells, and other cells that are not indicative of clinical disease. In this manner, the methods of the invention permit the accurate identification of high-grade cervical disease, even in cases mistakenly classified as normal, CINI, LSIL, or ASCUS by traditional Pap testing (i.e., "false negatives"). In some embodiments, the methods for diagnosing high-grade cervical disease are performed as a reflex to an abnormal or atypical Pap smear. That is, the methods of the invention may be performed in response to a patient having an abnormal or atypical Pap smear result. In other aspects of the invention, the methods are performed as a primary screening test for high-grade cervical disease in the general population of women, just as the conventional Pap test is performed currently.
The biomarkers of the invention include any gene or protein that is selectively overexpressed in high-grade cervical disease, as defined herein above. Such biomarkers are capable of identifying cells within a cytology cell suspension that are pre-malignant, malignant, or overtly cancerous. The biomarkers of the invention detect cells of CINII conditions and above, but do not detect CINI and HPV-infected cells where there is no underlying high-grade disease. Biomarkers of particular interest include genes and proteins involved in cell cycle regulation, HPV disruption of the cell cycle, DNA replication and transcription, and signal transduction. In some embodiments, the biomarkers are S-phase genes, including those genes whose expression is stimulated by the E2f transcription factor or the Sp-1 transcription factor. Nuclear biomarkers may be used to practice certain aspects of the invention. By "nuclear biomarker" is intended a biomarker that is predominantly expressed in the nucleus of the cell. A nuclear biomarker may be expressed to a lesser degree in other parts of the cell. Although any biomarker indicative of high-grade cervical disease may be used in the present invention, in certain embodiments the biomarkers, particularly nuclear biomarkers, are selected from the group consisting of MCM2, MCM6, MCM7, p21.sup.waf1, topoisomerase II alpha (Topo2A), p14arf, and cyclin E. More particularly, the biomarker may comprise an MCM protein.
Minichromosome maintenance (MCM) proteins play an essential part in eukaryotic DNA replication. The minichromosome maintenance (MCM) proteins function in the early stages of DNA replication through loading of the prereplication complex onto DNA and functioning as a helicase to help unwind the duplex DNA during de novo synthesis of the duplicate DNA strand. Each of the MCM proteins has DNA-dependent ATPase motifs in their highly conserved central domain. Levels of MCM proteins generally increase in a variable manner as normal cells progress from G0 into the G1/S phase of the cell cycle. In the G0 phase, MCM2 and MCM5 proteins are much less abundant than are the MCM7 and MCM3 proteins. MCM6 forms a complex with MCM2, MCM4, and MCM7, which binds histone H3. In addition, the subcomplex of MCM4, MCM6, and MCM7 has helicase activity, which is mediated by the ATP-binding activity of MCM6 and the DNA-binding activity of MCM4. See, for example, Freeman et al. (1999) Clin. Cancer Res. 5:2121-2132; Lei et al. (2001) J. Cell Sci. 114:1447-1454; Ishimi et al. (2003) Eur. J. Biochem. 270:1089-1101, all of which are herein incorporated by reference in their entirety.
Early publications have shown that the MCM proteins, and in particular, MCM-5, are useful for the detection of cervical disease (Williams et al. (1998) Proc Natl Acad Sci U.S.A. 95:14932-14937), as well as other cancers (Freeman et al. (1999) Clin Cancer Res. 5:2121-2132). The published literature indicates that antibodies to MCM-5 are capable of detecting cervical neoplastic cells. The specificity for detection of high-grade cervical disease has not been demonstrated for MCM-5 (Williams et al. (1998) Proc Natl Acad Sci U.S.A. 95:14932-14937). The detection of MCM-5 expression is not restricted to high-grade cervical disease but is also detected in identified low-grade dysplasia and proliferative cells that have re-entered the cell cycle following infection with high-risk HPV. The detection of cervical neoplasia with antibodies to MCM-5 is shown in FIG. 4. In addition to MCM-5, other members from the MCM family, including MCM-2 and MCM-7 have been shown to be potentially useful markers for the detection of cervical neoplasia in tissue samples (Freeman et al. (1999) Clin Cancer Res. 5:2121-2132; Brake et al. (2003) Cancer Res. 63:8173-8180). Recent results have shown that MCM-7 appears to be a specific marker for the detection of high-grade cervical disease using immunochemistry formats (Brake et al. (2003) Cancer Res. 63:8173-8180; Malinowski et al. (2004) Acta Cytol. 43:696).
Topoisomerase II alpha (Topo2a) is an essential nuclear enzyme involved in DNA replication and is a target for many anti-cancer drugs used for cancer therapy. Decreased expression of Topo2a is a predominant mechanism of resistance to several chemotherapeutic agents. A significant variation in the range of expression of this protein has been noted in many different tumors. Topo2a is predominant in proliferating cells and is modified in M phase by phosphorylation at specific sites, which is critical for mitotic chromosome condensation and segregation.
p21 is a protein encoded by the WAF1/Cip1 gene on chromosome 6p. This gene was shown to inhibit the activity of several cyclin/cyclin-dependent kinase complexes and to block cell cycle progression. The expression of p21.sup.waf1 mediates the cell cycle arrest function of p53. Because p21 appears to mediate several of the growth-regulatory functions of p53, its expression may reflect the functional status of p53 more precisely than p53 accumulation. Furthermore, p21.sup.waf1 can inhibit DNA replication by blocking the action of proliferating cell nuclear antigen (PCNA).
Cyclin E is a regulatory subunit of cdk-2 and controls G1/S transition during the mammalian cell cycle. Multiple isoforms of Cyclin E are expressed only in tumors but not in the normal tissues, suggesting a post-transcriptional regulation of Cyclin E. In vitro analyses indicated that these truncated variant isoforms of Cyclin E are able to phosphorylate histone H1. Alterations in Cyclin E proteins have been implicated as indicators of poor prognosis in various cancers.
Although the above biomarkers have been discussed in detail, any biomarker that is overexpressed in high-grade cervical disease states (e.g., CINII, CINIII, and cervical carcinomas) may be used in the practice of the invention. Other biomarkers of interest include cell cycle regulated genes that are specific to the G1/S phase boundary or to S-phase. Such genes include but are not limited to helicase (DDX11), uracil DNA glycolase (UNG), E2F5, cyclin E1 (CCNE1), cyclin E2 (CCNE2), CDC25A, CDC45L, CDC6, p21 WAF-1(CDKN1A), CDKN3, E2F1, MCM2, MCM6, NPAT, PCNA, stem loop BP (SLBP), BRCA1, BRCA2, CCNG2, CDKN2C, dihydrofolate reductase (DHFR), histone H1, histone H2A, histone H2B, histone H3, histone H4, MSH2, NASP, ribonucleotide reductase M1 (RRM1), ribonucleotide reductase M2 (RRM2), thymidine synthetase (TYMS), replication factor C4 (RFC4), RAD51, chromatin Factor 1A (CHAF1A), chromatin Factor 1B (CHAF1B), topisomerase III (TOP3A), ORC1, primase 2A (PRIM2A), CDC27, primase 1 (PRIM1), flap structure endonuclease (FEN1), fanconi anemia comp. grp A (FNACA), PKMYT1, and replication protein A2 (RPA2). See, for example, Whitfield et al. (2002) Mol. Biol. Cell 13:1977-2000, herein incorporated by reference in its entirety. Other S phase genes of interest include cyclin-dependent kinase 2 (CDK2), MCM3, MCM4, MCM5, DNA polymerase I alpha (DNA POL1), DNA ligase 1, B-Myb, DNA methyl transferase (DNA MET), pericentrin (PER), KIF4, DP-1, ID-3, RAN binding protein (RANBP1), gap junction alpha 6 (GJA6), amino levulinate dehydratase (ALDH), histone 2A Z (H2A.Z), spermine synthase (SpmS), proliferin 2, T-lymphocyte activation protein, phospholipase A2 (PLA2), and L6 antigen (L6). See, for example, Nevins et al. (2001) Mol. Cell. Biol. 21:4689-4699, herein incorporated by reference.
In some aspects of the invention, the biomarkers comprise genes that are induced by the E2f transcription factor. Such genes include but are not limited to thymidylate synthase, thymidine kinase 1, ribonucleotide reductase M1, ribonucleotide reductase M2, CDK2, cyclin E, MCM3, MCM7, PCNA, DNA primase small subunit, topoisomerase II A (Topo2A), DNA ligase 1, flap endonuclease 1, RAD51, CDC2, cyclin A2, cyclin B1, cyclin B2, KI-67, KIFC1, FIN16, BUB1, importin alpha-2, HMG2, enhancer of zeste, STK-1, histone stem-loop BP, Rb, P18-INK4C, annexin VIII, c-Myb, CDC25A, cyclin D3, cyclin E1, deoxycytosine kinase, DP-1, endothelin converting enzyme, enolase 2, P18 INK4C, ribonucleotide reductase, and uracil DNA glycolase 2. See, for example Nevins et al., supra; Muller et al. (2000) Genes and Dev. 15:267-285. In particular embodiments the biomarker of interest is a gene induced by E2f transcription factor that is involved in cell cycle regulation and DNA replication, such as, for example, cyclin E2, Ki-67, p57KIP2, RANBPM, and replication protein A1. Some E2f-induced genes of interest are involved in apoptosis, including APAF1, Bcl-2, caspase 3, MAP3 Kinase 5, and TNF receptor associated factor. Other E2f-induced genes are involved in the regulation of transcription and include, for example, ash2 like, polyhomeotic 2, embryonic ectoderm protein, enhancer of zeste, hairy/enhancer of split, homeobox A10, homeobox A7, homeobox A9, homeodomain TF1, pre-B-cell leukemia FT3, YY1 TF, POU domain TF, TAFII130, TBP-factor 172, basic TF3, bromodomain/zinc finger, SWI/SNF, ID4, TEA-4, NFATC1, NFATC3, BT, CNC-1, MAF, MAFF, MAFG, core binding protein, E74-like factor 4, c-FOS, JUNB, zinc finger DNA BP, and Cbp/p300 transactivator. E2f-induced genes involved in signal transduction are also potential biomarkers of interest and include TGF beta, follistatin, bone morphogenetic protein 2, BMP receptor type 1A, frizzled homolog 1, WNT10B, sphingosine kinase 1, dual specificity phosphatase 7, dual specificity (Y) phosphatase, FGF Receptor 3, protein tyrosine phosphatase, dual specificity (Y) phosphatase D6655, insulin receptor, mature T-cell proliferation 1, FGF receptor 2, TGF alpha, CDC42 effector protein 3, Met, CD58, CD83, TACC1, and TEAD4.
Although the methods of the invention require the detection of at least one biomarker in a patient sample for the detection of high-grade cervical disease, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more biomarkers may be used to practice the present invention. It is recognized that detection of more than one biomarker in a body sample may be used to identify instances of high-grade cervical disease. Therefore, in some embodiments, two or more biomarkers are used, more preferably, two or more complementary biomarkers. By "complementary" is intended that detection of the combination of biomarkers in a body sample results in the successful identification of high-grade cervical disease in a greater percentage of cases than would be identified if only one of the biomarkers was used. Thus, in some cases, a more accurate determination of high-grade cervical disease can be made by using at least two biomarkers. Accordingly, where at least two biomarkers are used, at least two antibodies directed to distinct biomarker proteins will be used to practice the immunocytochemistry methods disclosed herein. The antibodies may be contacted with the body sample simultaneously or concurrently. In certain aspects of the invention, the overexpression of MCM2 and Topo2A is detected using three antibodies, wherein two of the antibodies are specific for MCM2 and the third antibody is specific for Topo2A.
In particular embodiments, the diagnostic methods of the invention comprise collecting a cervical sample from a patient, contacting the sample with at least one antibody specific for a biomarker of interest, and detecting antibody binding. Samples that exhibit overexpression of a biomarker of the invention, as determined by detection of antibody binding, are deemed positive for high-grade cervical disease. In particular embodiments, the body sample is a monolayer of cervical cells. In some aspects of the invention, the monolayer of cervical cells is provided on a glass slide.
By "body sample" is intended any sampling of cells, tissues, or bodily fluids in which expression of a biomarker can be detected. Examples of such body samples include but are not limited to blood, lymph, urine, gynecological fluids, biopsies, and smears. Body samples may be obtained from a patient by a variety of techniques including, for example, by scraping or swabbing an area or by using a needle to aspirate bodily fluids. Methods for collecting various body samples are well known in the art. In particular embodiments, the body sample comprises cervical cells, as cervical tissue samples or as cervical cells in suspension, particularly in a liquid-based preparation. In one embodiment, cervical samples are collected according to liquid-based cytology specimen preparation guidelines such as, for example, the SurePath® (TriPath Imaging, Inc.) or the ThinPrep® preparation (CYTYC, Inc.). Body samples may be transferred to a glass slide for viewing under magnification. Fixative and staining solutions may be applied to the cells on the glass slide for preserving the specimen and for facilitating examination. In one embodiment the cervical sample will be collected and processed to provide a monolayer sample, as set forth in U.S. Pat. No. 5,346,831, herein incorporated by reference.
The monolayer method relates to a method for producing a monolayer of cytological material on a cationically-charged substrate. The method comprises the steps of separating the cytological material by centrifugation over a density gradient, producing a packed pellet of the cytological material, mixing the pellet of the cytological material, withdrawing an aliquot of a predetermined volume from the mixed pellet, depositing the aliquot and a predetermined volume of water into a sedimentation vessel, which is removably secured to the cationically-charged substrate, allowing the cytological material to settle onto the substrate under the force of gravity, and after settlement of the cytological material, removing the water from the sedimentation vessel. For automated analysis, the sedimentation vessel may be detached from the substrate. Disaggregation may be by any methods known in the art, such as syringing, trypsinizing, ultrasonication, shaking, vortexing, or by use of the device described in copending U.S. Pat. No. 5,316,814, the contents of which are herein incorporated by reference. In some embodiments, slides comprising a monolayer of cervical cells are prepared from SurePath® (TriPath Imaging, Inc.) samples using the PrepStain® slide processor (TriPath Imaging, Inc.).
Any methods available in the art for identification or detection of the biomarkers are encompassed herein. The overexpression of a biomarker of the invention can be detected on a nucleic acid level or a protein level. In order to determine overexpression, the body sample to be examined may be compared with a corresponding body sample that originates from a healthy person. That is, the "normal" level of expression is the level of expression of the biomarker in cervical cells of a human subject or patient not afflicted with high-grade cervical disease. Such a sample can be present in standardized form. In some embodiments, particularly when the body sample comprises a monolayer of cervical cells, determination of biomarker overexpression requires no comparison between the body sample and a corresponding body sample that originates from a healthy person. In this situation, the monolayer of cervical cells from a single patient may contain as few as 1-2 abnormal cells per 50,000 normal cells present. Detection of these abnormal cells, identified by their overexpression of a biomarker of the invention, precludes the need for comparison to a corresponding body sample that originates from a healthy person.
Methods for detecting biomarkers of the invention comprise any methods that determine the quantity or the presence of the biomarkers either at the nucleic acid or protein level. Such methods are well known in the art and include but are not limited to western blots, northern blots, southern blots, ELISA, immunoprecipitation, immunofluorescence, flow cytometry, immunocytochemistry, nucleic acid hybridization techniques, nucleic acid reverse transcription methods, and nucleic acid amplification methods. In particular embodiments, overexpression of a biomarker is detected on a protein level using, for example, antibodies that are directed against specific biomarker proteins. These antibodies can be used in various methods such as Western blot, ELISA, immunoprecipitation, or immunocytochemistry techniques. Likewise, immunostaining of cervical smears can be combined with conventional Pap stain methods so that morphological information and immunocytochemical information can be obtained. In this manner, the detection of the biomarkers can reduce the high false-negative rate of the Pap smear test and may facilitate mass automated screening.
In one embodiment, antibodies specific for biomarker proteins are utilized to detect the overexpression of a biomarker protein in a body sample. The method comprises obtaining a body sample from a patient, contacting the body sample with at least one antibody directed to a biomarker that is selectively overexpressed in high-grade cervical disease, and detecting antibody binding to determine if the biomarker is overexpressed in the patient sample. A preferred aspect of the present invention provides an immunocytochemistry technique for diagnosing high-grade cervical disease. Specifically, this method comprises antibody staining of biomarkers within a patient sample that are specific for high-grade cervical disease. One of skill in the art will recognize that the immunocytochemistry method described herein below may be performed manually or in an automated fashion using, for example, the Autostainer Universal Staining System (Dako) or the Biocare Nemesis Autostainer (Biocare). One protocol for antibody staining (i.e., immunocytochemistry) of cervical samples is provided in Example 1.
In a preferred immunocytochemistry method, a patient cervical sample is collected into a liquid medium, such as, for example, in a SurePath® collection vial (TriPath Imaging, Inc.). An automated processor such as the PrepStain® system (TriPath Imaging, Inc.) is used to collect cells from the liquid medium and to deposit them in a thin layer on a glass slide for further analysis. Slide specimens may be fixed or unfixed and may be analyzed immediately following preparation or may be stored for later analysis. In some embodiments, prepared slides are stored in 95% ethanol for a minimum of 24 hours. Alternatively, in other embodiments, slides are stored in a pretreatment buffer, as described below.
Samples may need to be modified in order to make the biomarker antigens accessible to antibody binding. In a particular aspect of the immunocytochemistry methods, slides are transferred to a pretreatment buffer, for example the SureSlide® Preparation Buffer (TriPath Imaging, Inc.) and Optionally Heated to Increase Antigen accessibility. Heating of the sample in the pretreatment buffer rapidly disrupts the lipid bi-layer of the cells and makes the antigens (i.e., biomarker proteins) more accessible for antibody binding. The pretreatment buffer may comprise a polymer, a detergent, or a nonionic or anionic surfactant such as, for example, an ethyloxylated anionic or nonionic surfactant, an alkanoate or an alkoxylate or even blends of these surfactants or even the use of a bile salt. In particular embodiments, the pretreatment buffer comprises a nonionic or anionic detergent, such as sodium alkanoate with an approximate molecular weight of 183 kD blended with an alkoxylate with an approximate molecular weight of 370 kD (hereafter referred to as RAM). In a particular embodiment, the pretreatment buffer comprises 1% RAM. In some embodiments, the pretreatment buffer may also be used as a slide storage buffer, as indicated above. In another embodiment a solution of 0.1% to 1% of deoxycholic acid, sodium salt, monohydrate was used as both a storage buffer as well as a pretreatment buffer. In yet another embodiment of the invention a solution of sodium laureth-13-carboxylate (e.g., Sandopan LS) or and ethoxylated anionic complex or even an alkylaryl ethoxlate carboxylic acid can be used for the storage and pretreatment buffers. In a particular aspect of the invention, the slide pretreatment buffer comprises 0.05% to 5% sodium laureth-13-carboxylate, particularly 0.1% to 1% sodium laureth-13-carboxylate, more particularly 0.5% sodium laureth-13-carboxylate. In one embodiment the slides can be stored in the buffer for up to 72 hours prior to the pretreatment and staining process. The pretreatment buffers of the invention may be used in methods for making antigens more accessible for antibody binding in an immunoassay, such as, for example, an immunocytochemistry method or an immunohistochemistry method. See Example 14. The terms "pretreatment buffer" and "preparation buffer" are used interchangeably herein to refer to a buffer that is used to prepare cytology or histology samples for immunostaining, particularly by increasing antigen accessibility for antibody binding.
Any method for making antigens more accessible for antibody binding may be used in the practice of the invention, including the antigen retrieval methods known in the art. See, for example, Bibbo et al. (2002) Acta. Cytol. 46:25-29; Saqi et al. (2003) Diagn. Cytopathol. 27:365-370; Bibbo et al. (2003) Anal. Quant. Cytol. Histol. 25:8-11, herein incorporated by reference in their entirety. In some embodiments, antigen retrieval comprises storing the slides in 95% ethanol for at least 24 hours, immersing the slides in 1× Target Retrieval Solution pH 6.0 (DAKO S1699)/dH2O bath preheated to 95° C., and placing the slides in a steamer for 25 minutes. See Example 2 below.
Following pretreatment or antigen retrieval to increase antigen accessibility, samples are blocked using an appropriate blocking agent, e.g., a peroxidase blocking reagent such as hydrogen peroxide. In some embodiments, the samples are blocked using a protein blocking reagent to prevent non-specific binding of the antibody. The protein blocking reagent may comprise, for example, purified casein. An antibody, particularly a monoclonal antibody, directed to a biomarker of interest is then incubated with the sample. As noted above, one of skill in the art will appreciate that a more accurate diagnosis of high-grade cervical disease may be obtained in some cases by detecting more than one biomarker in a patient sample. Therefore, in particular embodiments, at least two antibodies directed to two distinct biomarkers are used to detect high-grade cervical disease. Where more than one antibody is used, these antibodies may be added to a single sample sequentially as individual antibody reagents or simultaneously as an antibody cocktail. See Example 3 below. Alternatively, each individual antibody may be added to a separate sample from the same patient, and the resulting data pooled. In particular embodiments, an antibody cocktail comprises at least three antibodies, wherein two antibodies specifically bind to MCM2 and a third antibody specifically binds to Topo2A.
Techniques for detecting antibody binding are well known in the art. Antibody binding to a biomarker of interest may be detected through the use of chemical reagents that generate a detectable signal that corresponds to the level of antibody binding and, accordingly, to the level of biomarker protein expression. In one of the immunocytochemistry methods of the invention, antibody binding is detected through the use of a secondary antibody that is conjugated to a labeled polymer. Examples of labeled polymers include but are not limited to polymer-enzyme conjugates. The enzymes in these complexes are typically used to catalyze the deposition of a chromogen at the antigen-antibody binding site, thereby resulting in cell staining that corresponds to expression level of the biomarker of interest. Enzymes of particular interest include horseradish peroxidase (HRP) and alkaline phosphatase (AP). Commercial antibody detection systems, such as, for example the Dako Envision+ system and Biocare Medical's Mach 3 system, may be used to practice the present invention.
In one particular immunocytochemistry method of the invention, antibody binding to a biomarker is detected through the use of an HRP-labeled polymer that is conjugated to a secondary antibody. Antibody binding can also be detected through the use of a mouse probe reagent, which binds to mouse monoclonal antibodies, and a polymer conjugated to HRP, which binds to the mouse probe reagent. Slides are stained for antibody binding using the chromogen 3,3-diaminobenzidine (DAB) and then counterstained with hematoxylin and, optionally, a bluing agent such as ammonium hydroxide or TBS/Tween-20. In some aspects of the invention, slides are reviewed microscopically by a cytotechnologist and/or a pathologist to assess cell staining (i.e., biomarker overexpression) and to determine if high-grade cervical disease is present. Alternatively, samples may be reviewed via automated microscopy or by personnel with the assistance of computer software that facilitates the identification of positive staining cells.
The terms "antibody" and "antibodies" broadly encompass naturally occurring forms of antibodies and recombinant antibodies such as single-chain antibodies, chimeric and humanized antibodies and multi-specific antibodies as well as fragments and derivatives of all of the foregoing, which fragments and derivatives have at least an antigenic binding site. Antibody derivatives may comprise a protein or chemical moiety conjugated to the antibody.
"Antibodies" and "immunoglobulins" (Igs) are glycoproteins having the same structural characteristics. While antibodies exhibit binding specificity to an antigen, immunoglobulins include both antibodies and other antibody-like molecules that lack antigen specificity. Polypeptides of the latter kind are, for example, produced at low levels by the lymph system and at increased levels by myelomas.
The term "antibody" is used in the broadest sense and covers fully assembled antibodies, antibody fragments that can bind antigen (e.g., Fab', F'(ab)2, Fv, single chain antibodies, diabodies), and recombinant peptides comprising the foregoing.
The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts.
"Antibody fragments" comprise a portion of an intact antibody, preferably the antigen-binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (Zapata et al. (1995) Protein Eng. 8(10):1057-1062); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, whose name reflects its ability to crystallize 35 readily. Pepsin treatment yields an F(ab')2 fragment that has two antigen-combining sites and is still capable of cross-linking antigen.
"Fv" is the minimum antibody fragment that contains a complete antigen recognition and binding site. In a two-chain Fv species, this region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. In a single-chain Fv species, one heavy- and one light-chain variable domain can be covalently linked by flexible peptide linker such that the light and heavy chains can associate in a "dimeric" structure analogous to that in a two-chain Fv species. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab fragments differ from Fab' fragments by the addition of a few residues at the carboxy terminus of the heavy-chain CH1 domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments that have hinge cysteines between them.
Polyclonal antibodies can be prepared by immunizing a suitable subject (e.g., rabbit, goat, mouse, or other mammal) with a biomarker protein immunogen. The antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized biomarker protein. At an appropriate time after immunization, e.g., when the antibody titers are highest, antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler and Milstein (1975) Nature 256:495-497, the human B cell hybridoma technique (Kozbor et al. (1983) Immunol. Today 4:72), the EBV-hybridoma technique (Cole et al. (1985) in Monoclonal Antibodies and Cancer Therapy, ed. Reisfeld and Sell (Alan R. Liss, Inc., New York, N.Y.), pp. 77-96) or trioma techniques. The technology for producing hybridomas is well known (see generally Coligan et al., eds. (1994) Current Protocols in Immunology (John Wiley & Sons, Inc., New York, N.Y.); Galfre et al. (1977) Nature 266:550-52; Kenneth (1980) in Monoclonal Antibodies: A New Dimension In Biological Analyses (Plenum Publishing Corp., NY); and Lerner (1981) Yale J. Biol. Med., 54:387-402).
Alternative to preparing monoclonal antibody-secreting hybridomas, a monoclonal antibody can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with a biomarker protein to thereby isolate immunoglobulin library members that bind the biomarker protein. Kits for generating and screening phage display libraries are commercially available (e.g., the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; and the Stratagene SurfZAP θ Phage Display Kit, Catalog No. 240612). Additionally, examples of methods and reagents particularly amenable for use in generating and screening antibody display library can be found in, for example, U.S. Pat. No. 5,223,409; PCT Publication Nos. WO 92/18619; WO 91/17271; WO 92/20791; WO 92/15679; 93/01288; WO 92/01047; 92/09690; and 90/02809; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum. Antibod. Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffiths et al. (1993) EMBO J. 12:725-734.
Detection of antibody binding can be facilitated by coupling the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive material include 125I, 131I, 35S, or 3H.
In regard to detection of antibody staining in the immunocytochemistry methods of the invention, there also exist in the art, video-microscopy and software methods for the quantitative determination of an amount of multiple molecular species (e.g., biomarker proteins) in a biological sample wherein each molecular species present is indicated by a representative dye marker having a specific color. Such methods are also known in the art as a colorimetric analysis methods. In these methods, video-microscopy is used to provide an image of the biological sample after it has been stained to visually indicate the presence of a particular biomarker of interest. Some of these methods, such as those disclosed in U.S. patent application Ser. No. 09/957,446 to Marcelpoil et al. and U.S. patent application Ser. No. 10/057,729 to Marcelpoil et al., incorporated herein by reference, disclose the use of an imaging system and associated software to determine the relative amounts of each molecular species present based on the presence of representative color dye markers as indicated by those color dye markers' optical density or transmittance value, respectively, as determined by an imaging system and associated software. These techniques provide quantitative determinations of the relative amounts of each molecular species in a stained biological sample using a single video image that is "deconstructed" into its component color parts.
The antibodies used to practice the invention are selected to have high specificity for the biomarker proteins of interest. Methods for making antibodies and for selecting appropriate antibodies are known in the art. See, for example, Celis, ed. (in press) Cell Biology & Laboratory Handbook, 3rd edition (Academic Press, New York), which is herein incorporated in its entirety by reference. In some embodiments, commercial antibodies directed to specific biomarker proteins may be used to practice the invention. The antibodies of the invention may be selected on the basis of desirable staining of cytological, rather than histological, samples. That is, in particular embodiments the antibodies are selected with the end sample type (i.e., cytology preparations) in mind and for binding specificity.
In some aspects of the invention, antibodies directed to specific biomarkers of interest are selected and purified via a multi-step screening process. In particular embodiments, polydomas are screened to identify biomarker-specific antibodies that possess the desired traits of specificity and sensitivity. As used herein, "polydoma" refers to multiple hybridomas. The polydomas of the invention are typically provided in multi-well tissue culture plates. In the initial antibody screening step, a tumor tissue microarray comprising multiple normal (i.e., no CIN), CINIII, squamous cell carcinoma, and adenocarcinoma samples is generated. Methods and equipment, such as the Chemicon® Advanced Tissue Arrayer, for generating arrays of multiple tissues on a single slide are known in the art. See, for example, U.S. Pat. No. 4,820,504. Undiluted supernatants from each well containing a polydoma are assayed for positive staining using standard immunohistochemistry techniques. At this initial screening step, background, non-specific binding is essentially ignored. Polydomas producing positive results are selected and used in the second phase of antibody screening.
In the second screening step, the positive polydomas are subjected to a limiting dilution process. The resulting unscreened antibodies are assayed for positive staining of CINIII or cervical carcinoma samples using standard immunohistochemistry techniques. At this stage, background staining is relevant, and the candidate polydomas that only stain positive for abnormal cells (i.e., CINIII and cancer cells) only are selected for further analysis.
To identify antibodies that can distinguish normal and CINI samples from those indicative of high-grade cervical disease (i.e., CINII and above), a disease panel tissue microarray is generated. This tissue microarray typically comprises multiple no CIN, CINI, CINII, CINIII, squamous cell carcinoma, and adenocarcinoma samples. Standard immunohistochemistry techniques are employed to assay the candidate polydomas for specific positive staining of samples indicative of high-grade cervical disease only (i.e., CINII samples and above). Polydomas producing positive results and minimal background staining are selected for further analysis.
Positive-staining cultures are prepared as individual clones in order to select individual candidate monoclonal antibodies. Methods for isolating individual clones are well known in the art. The supernatant from each clone comprising unpurified antibodies is assayed for specific staining of CINII, CINIII, squamous cell carcinoma, and adenocarcinoma samples using the tumor and disease panel tissue microarrays described herein above. Candidate antibodies showing positive staining of high-grade cervical disease samples (i.e., CINII and above), minimal staining of other cell types (i.e., normal and CINI samples), and little background are selected for purification and further analysis. Methods for purifying antibodies through affinity adsorption chromatography are well known in the art.
In order to identify antibodies that display maximal specific staining of high-grade cervical disease samples and minimal background, non-specific staining in cervical cytology samples, the candidate antibodies isolated and purified in the immunohistochemistry-based screening process above are assayed using the immunocytochemistry techniques of the present invention. Exemplary protocols for performing immunocytochemistry are provided in Examples 1 and 2.
Specifically, purified antibodies of interest are used to assay a statistically significant number of NIL (i.e., no invasive lesion), ASCUS, LSIL, HSIL or cancerous cervical cytology patient samples. The samples are analyzed by immunocytochemistry as described herein and classified as positive, negative, or indeterminate for high-grade cervical disease on the basis of positive antibody staining for a particular biomarker. Sensitivity, specificity, positive predictive values, and negative predictive values for each antibody are calculated. Antibodies exhibiting maximal specific staining of high-grade cervical disease in cervical cytology samples with minimal background (i.e., maximal signal to noise ratio) are selected for the present invention.
Identification of appropriate antibodies results in an increase in signal to noise ratio and an increase in the clinical utility of the assay. Assay format and sample type to be used are critical factors in selection of appropriate antibodies. Many antibodies directed to biomarkers do not produce a desirable signal to noise ratio in an immunocytochemistry format with cytology preparations or in an immunohistochemistry format with formalin-fixed paraffin-embedded samples. Moreover, biomarker antibodies that produce a maximal signal to noise ratio in an immunohistochemistry format may not work as well in immunocytochemistry assays. For example, an antibody that produces the desired level of staining in an immunocytochemistry format may not produce the appropriate level of staining in an immunohistochemistry assay (data not shown). Likewise, an antibody that produces an acceptable signal to noise ratio when used in the immunohistochemistry assay may result in overstaining of immunocytochemistry samples (data not shown). Thus, antibody selection requires early consideration of the assay format and the end sample type to be used.
Cytology-based assays (i.e., immunocytochemistry) differ from tissue-based assays (i.e., immunohistochemistry) insofar as the tissue architecture is not available to assist with staining interpretation in the immunocytochemistry format. For example, in an immunohistochemistry assay performed on samples from patients with mild dysplasia or squamous cell carcinoma with an antibody directed to Claudin 1, the results indicated that Claudin 1 was expressed in the lesion of the mild dysplasia sample (i.e., light brown staining) but was significantly overexpressed (i.e., dark brown staining) in the cancer lesion (FIG. 12). The results obtained with the same Claudin 1 antibody in an immunocytochemistry assay format were indeterminate (FIG. 13). While abnormal cells are easily detectable using a Claudin 1 antibody in an immunohistochemistry assay, the results obtained by the staining of Claudin 1 in the immunocytochemistry assay of the invention were more difficult to interpret. Therefore, biomarkers that are appropriate in an immunohistochemistry format may not be suitable in an immunocytochemistry assay and, thus, are not included in the preferred embodiment of the invention.
Furthermore, the location of biomarkers within the cell is also an important consideration in immunocytochemistry assays. Biomarkers that display nuclear, cytoplasmic, or membrane staining patterns can be confirmed morphologically and are appropriate for immunohistochemistry methods. Cytoplasmic and membrane staining, however, make it difficult to identify critical morphological characteristics of cervical disease (e.g., nuclear to cytoplasmic ratio) in immunocytochemistry assays. See FIG. 15. In contrast, biomarkers that are expressed in the nucleus and show a nuclear staining pattern facilitate detection of antibody staining and also permit morphological analysis. See FIG. 15. Thus, in some preferred embodiments, only biomarkers that are selectively expressed in the nucleus are used in an immunocytochemistry assay of the invention.
One of skill in the art will recognize that optimization of antibody titer and detection chemistry is needed to maximize the signal to noise ratio for a particular antibody. Antibody concentrations that maximize specific binding to the biomarkers of the invention and minimize non-specific binding (or "background") will be determined. In particular embodiments, appropriate antibody titers for use in cervical cytology preparations are determined by initially testing various antibody dilutions on formalin-fixed paraffin-embedded normal and high-grade cervical disease tissue samples. Optimal antibody concentrations and detection chemistry conditions are first determined for formalin-fixed paraffin-embedded cervical tissue samples. The design of assays to optimize antibody titer and detection conditions is standard and well within the routine capabilities of those of ordinary skill in the art. After the optimal conditions for fixed tissue samples are determined, each antibody is then used in cervical cytology preparations under the same conditions. Some antibodies require additional optimization to reduce background staining and/or to increase specificity and sensitivity of staining in the cytology samples.
Furthermore, one of skill in the art will recognize that the concentration of a particular antibody used to practice the methods of the invention will vary depending on such factors as time for binding, level of specificity of the antibody for the biomarker protein, and method of body sample preparation. Moreover, when multiple antibodies are used, the required concentration may be affected by the order in which the antibodies are applied to the sample, i.e., simultaneously as a cocktail or sequentially as individual antibody reagents. Furthermore, the detection chemistry used to visualize antibody binding to a biomarker of interest must also be optimized to produce the desired signal to noise ratio.
In other embodiments, the expression of a biomarker of interest is detected at the nucleic acid level. Nucleic acid-based techniques for assessing expression are well known in the art and include, for example, determining the level of biomarker mRNA in a body sample. Many expression detection methods use isolated RNA. Any RNA isolation technique that does not select against the isolation of mRNA can be utilized for the purification of RNA from cervical cells (see, e.g., Ausubel et al., ed., (1987-1999) Current Protocols in Molecular Biology (John Wiley & Sons, New York). Additionally, large numbers of tissue samples can readily be processed using techniques well known to those of skill in the art, such as, for example, the single-step RNA isolation process of Chomczynski (1989, U.S. Pat. No. 4,843,155).
The term "probe" refers to any molecule that is capable of selectively binding to a specifically intended target biomolecule, for example, a nucleotide transcript or a protein encoded by or corresponding to a biomarker. Probes can be synthesized by one of skill in the art, or derived from appropriate biological preparations. Probes may be specifically designed to be labeled. Examples of molecules that can be utilized as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules.
Isolated mRNA can be used in hybridization or amplification assays that include, but are not limited to, Southern or Northern analyses, polymerase chain reaction analyses and probe arrays. One method for the detection of mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to the mRNA encoded by the gene being detected. The nucleic acid probe can be, for example, a full-length cDNA, or a portion thereof, such as an oligonucleotide of at least 7, 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to an mRNA or genomic DNA encoding a biomarker of the present invention. Hybridization of an mRNA with the probe indicates that the biomarker in question is being expressed.
In one embodiment, the mRNA is immobilized on a solid surface and contacted with a probe, for example by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane, such as nitrocellulose. In an alternative embodiment, the probe(s) are immobilized on a solid surface and the mRNA is contacted with the probe(s), for example, in an Affymetrix gene chip array. A skilled artisan can readily adapt known mRNA detection methods for use in detecting the level of mRNA encoded by the biomarkers of the present invention.
An alternative method for determining the level of biomarker mRNA in a sample involves the process of nucleic acid amplification, e.g., by RT-PCR (the experimental embodiment set forth in Mullis, 1987, U.S. Pat. No. 4,683,202), ligase chain reaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189-193), self sustained sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi et al. (1988) Bio/Technology 6:1197), rolling circle replication (Lizardi et al., U.S. Pat. No. 5,854,033) or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers. In particular aspects of the invention, biomarker expression is assessed by quantitative fluorogenic RT-PCR (i.e., the TaqMan® System). Such methods typically utilize pairs of oligonucleotide primers that are specific for the biomarker of interest. Methods for designing oligonucleotide primers specific for a known sequence are well known in the art.
Biomarker expression levels of RNA may be monitored using a membrane blot (such as used in hybridization analysis such as Northern, Southern, dot, and the like), or microwells, sample tubes, gels, beads or fibers (or any solid support comprising bound nucleic acids). See U.S. Pat. Nos. 5,770,722, 5,874,219, 5,744,305, 5,677,195 and 5,445,934, which are incorporated herein by reference. The detection of biomarker expression may also comprise using nucleic acid probes in solution.
In one embodiment of the invention, microarrays are used to detect biomarker expression. Microarrays are particularly well suited for this purpose because of the reproducibility between different experiments. DNA microarrays provide one method for the simultaneous measurement of the expression levels of large numbers of genes. Each array consists of a reproducible pattern of capture probes attached to a solid support. Labeled RNA or DNA is hybridized to complementary probes on the array and then detected by laser scanning. Hybridization intensities for each probe on the array are determined and converted to a quantitative value representing relative gene expression levels. See, U.S. Pat. Nos. 6,040,138, 5,800,992 and 6,020,135, 6,033,860, and 6,344,316, which are incorporated herein by reference. High-density oligonucleotide arrays are particularly useful for determining the gene expression profile for a large number of RNA's in a sample.
Techniques for the synthesis of these arrays using mechanical synthesis methods are described in, e.g., U.S. Pat. No. 5,384,261, incorporated herein by reference in its entirety for all purposes. Although a planar array surface is preferred, the array may be fabricated on a surface of virtually any shape or even a multiplicity of surfaces. Arrays may be peptides or nucleic acids on beads, gels, polymeric surfaces, fibers such as fiber optics, glass or any other appropriate substrate, see U.S. Pat. Nos. 5,770,358, 5,789,162, 5,708,153, 6,040,193 and 5,800,992, each of which is hereby incorporated in its entirety for all purposes. Arrays may be packaged in such a manner as to allow for diagnostics or other manipulation of an all-inclusive device. See, for example, U.S. Pat. Nos. 5,856,174 and 5,922,591 herein incorporated by reference.
In one approach, total mRNA isolated from the sample is converted to labeled cRNA and then hybridized to an oligonucleotide array. Each sample is hybridized to a separate array. Relative transcript levels may be calculated by reference to appropriate controls present on the array and in the sample.
Kits for practicing the methods of the invention are further provided. By "kit" is intended any manufacture (e.g., a package or a container) comprising at least one reagent, e.g., an antibody, a nucleic acid probe, etc. for specifically detecting the expression of a biomarker of the invention. The kit may be promoted, distributed, or sold as a unit for performing the methods of the present invention. Additionally, the kits may contain a package insert describing the kit and methods for its use.
In a particular embodiment, kits for practicing the immunocytochemistry methods of the invention are provided. Such kits are compatible with both manual and automated immunocytochemistry techniques (e.g., cell staining) as described below in Example 1. These kits comprise at least one antibody directed to a biomarker of interest, chemicals for the detection of antibody binding to the biomarker, a counterstain, and, optionally, a bluing agent to facilitate identification of positive staining cells. Any chemicals that detect antigen-antibody binding may be used in the practice of the invention. In some embodiments, the detection chemicals comprise a labeled polymer conjugated to a secondary antibody. For example, a secondary antibody that is conjugated to an enzyme that catalyzes the deposition of a chromogen at the antigen-antibody binding site may be provided. Such enzymes and techniques for using them in the detection of antibody binding are well known in the art. In one embodiment, the kit comprises a secondary antibody that is conjugated to an HRP-labeled polymer. Chromogens compatible with the conjugated enzyme (e.g., DAB in the case of an HRP-labeled secondary antibody) and solutions, such as hydrogen peroxide, for blocking non-specific staining may be further provided. In other embodiments, antibody binding to a biomarker protein is detected through the use of a mouse probe reagent that binds to mouse monoclonal antibodies, followed by addition of a dextran polymer conjugated with HRP that binds to the mouse probe reagent. Such detection reagents are commercially available from, for example, Biocare Medical.
The kits of the present invention may further comprise a peroxidase blocking reagent (e.g., hydrogen peroxide), a protein blocking reagent (e.g., purified casein), and a counterstain (e.g., hematoxylin). A bluing agent (e.g., ammonium hydroxide or TBS, pH 7.4, with Tween-20 and sodium azide) may be further provided in the kit to facilitate detection of positive staining cells.
In another embodiment, the immunocytochemistry kits of the invention additionally comprise at least two reagents, e.g., antibodies, for specifically detecting the expression of at least two distinct biomarkers. Each antibody may be provided in the kit as an individual reagent or, alternatively, as an antibody cocktail comprising all of the antibodies directed to the different biomarkers of interest. Furthermore, any or all of the kit reagents may be provided within containers that protect them from the external environment, such as in sealed containers. An exemplary kit for practicing the methods of the invention is described below in Example 8.
Positive and/or negative controls may be included in the kits to validate the activity and correct usage of reagents employed in accordance with the invention. Controls may include samples, such as tissue sections, cells fixed on glass slides, etc., known to be either positive or negative for the presence of the biomarker of interest. In a particular embodiment, the positive control comprises SiHa cells. This is a human cervical squamous cancer cell line that is hypertriploid and positive for HPV-16 infection and, therefore, serves as a positive control for the overexpression of biomarkers in high-grade cervical disease states. SiHa control cells may be provided in the kits of the invention as prepared slides or as a cell suspension that is compatible with slide preparation. The design and use of controls is standard and well within the routine capabilities of those of ordinary skill in the art.
In other embodiments, kits for identifying high-grade cervical comprising detecting biomarker overexpression at the nucleic acid level are further provided. Such kits comprise, for example, at least one nucleic acid probe that specifically binds to a biomarker nucleic acid or fragment thereof. In particular embodiments, the kits comprise at least two nucleic acid probes that hybridize with distinct biomarker nucleic acids.
In some embodiments, the methods of the invention can be used in combination with traditional cytology techniques that analyze morphological characteristics. For example, the immunocytochemical techniques of the present invention can be combined with the conventional Pap stain so that all the morphological information from the conventional method is conserved. In this manner the detection of biomarkers can reduce the high false-negative rate of the Pap smear test and may facilitate mass automated screening. In a particular embodiment, the immunocytochemistry methods disclosed herein above are combined with the conventional Pap stain in a single method, as described below in Example 6-7. A combined immunocytochemistry and Pap staining method permits visualization of both biomarkers that are selectively overexpressed in high-grade cervical disease and cell morphology in a single sample (e.g., a microscope slide comprising a monolayer of cervical cells). The combined immunocytochemistry and Pap staining method may permit the more accurate identification and diagnosis of high-grade cervical disease, particularly in cases mistakenly classified as normal, LSIL, or ASCUS by conventional Pap testing. Analysis of both biomarker overexpression and cell morphology in a single method could replace the Pap smear as the primary screening method for cervical cancer.
One of skill in the art will recognize that the staining parameters (e.g., incubation times, wash conditions, chromogen/stain concentrations, etc.) for this combined methodology will need to be optimized such that a sufficient contrast between the immunocytochemistry output (e.g., chromogen staining) and the Pap stain is obtained. The design of assays to optimize staining parameters is standard and well within the routine capabilities of those of ordinary skill in the art. Kits for performing the combined immunocytochemistry and Pap staining method are also encompassed by the present invention. Such kits comprise the reagents needed for immunocytochemistry, as described herein above, and the reagents for conventional Pap staining, particularly EA50 and Orange G.
One of skill in the art will further appreciate that any or all steps in the methods of the invention could be implemented by personnel or, alternatively, performed in an automated fashion. Thus, the steps of body sample preparation, sample staining, and detection of biomarker expression may be automated.
The following examples are offered by way of illustration and not by way of limitation:
Detection of Biomarker Overexpression Using Immunocytochemistry
Slide Preparation and Pretreatment
Patient cervical samples are collected and placed into a SurePath® collection vial (TriPath Imaging, Inc.). Cervical cells are collected from the liquid medium and deposited in a thin layer on a glass slide using the PrepStain® slide processor system (TriPath Imaging, Inc.). Prepared slides are immediately transferred to a pretreatment buffer (1% RAM) and heated for 45 minutes at 95° C. The slides are cooled to room temperature and rinsed three times (2 minutes per rinse) in TBS (tris buffered saline).
To prevent non-specific background staining, slides are not permitted to dry out during the staining procedure. Furthermore, in order to block non-specific staining, hydrogen peroxide is applied to the slides for 5 minutes, followed by a TBS rinse. An antibody directed to MCM6 is applied to the slide for 1 hour at room temperature. Following incubation with the MCM6 antibody, the slide is washed three times with TBS for 2 minutes per wash. The Dako Envision+HRP-labeled polymer secondary antibody is applied to the slide for 30 minutes at room temperature, followed by a TBS rinse. The HRP substrate chromogen DAB is applied for 10 minutes, and then the slides are rinsed for 5 minutes with water. Each slide is counterstained with hematoxylin and then rinsed with water until clear. Following counterstaining, the slides are soaked in ammonium hydroxide for 10 seconds and then rinsed with water for 1 minute.
Samples are dehydrated by immersing the slides in 95% ethanol for 1 minute and then in absolute ethanol for an additional minute. Slides are cleared by rinsing 3 times in xylene for 1 minute per rinse. Slides are then coverslipped with permanent mounting media and incubated at 35° C. to dry. Positive staining cells are visualized using a bright-field microscope.
The Dako Autostainer Universal Staining system is programmed according to the manufacturer's instructions, and the necessary staining and counterstaining reagents described above for manual immunocytochemistry are loaded onto the machine. The prepared and pretreated slides are loaded onto the Autostainer, and the program is run. At the end of the run, the slides are removed and rinsed in water for 5 minutes. The slides are dehydrated, cleared, coverslipped, and analyzed as described above.
Detection of Biomarkers in Clinical Samples
Approximately 180 cervical cytology patient samples representing various diagnoses were collected. The presence or absence of cancerous cells or lesions indicative of high-grade disease in these patients was previously confirmed by colposcopy. The following table indicates the number of samples within each diagnosis group analyzed in this study, as well as a description of the colposcopy findings (e.g., presence or absence of high-grade lesions).
TABLE-US-00001 TABLE 1 Specimens analyzed Diagnosis Count Description NIL 72 HPV Negative ASC-US 26 26 without lesion 0 with lesion or high risk HPV LSIL 48 42 negative for high grade lesion 6 positive for high grade lesion HSIL 25 Cancer 10 Squamous Cell Carcinoma and Adenocarcinoma
The samples were analyzed by immunocytochemistry methods to identify high-grade cervical disease. Antibodies were used to detect the overexpression of six biomarkers of interest: MCM2, MCM6, MCM 7, p21.sup.waf1, Cyclin E, and Topo2A. Assay controls included MCM2, MCM6, MCM7, p21.sup.waf1, Cyclin E, Topo2A and a mouse IgG negative run on the SiHa cell line. Samples were also analyzed by traditional Pap staining techniques.
Preparation of Slides
Each sample was removed from storage and allowed to come to room temperature. 6 ml of TriPath CytoRich® preservative was added to each vial, and the vials were vortexed. Samples were processed on the TriPath PrepMate® automated processor, and any remaining fluid in the vial was transferred to a centrifuge tube. The samples were centrifuged for 2 minutes at 200×g, and the supernatant was aspirated. The samples were then centrifuged for 10 minutes at 800×g, and the supernatant was decanted. Sample tubes were loaded onto the TriPath PrepStain® system and the system software (version 1.1; Transfer Only) was run. Eight slides for each patient sample were prepared and stored in 95% ethanol for at least 24 hours but not longer than 2 weeks prior to use in Pap staining and immunocytochemistry methods.
Pap Staining Method
Prepared slides were incubated in 95% ethanol for 30 seconds and then rinsed with water for an additional 30 seconds. Hematoxylin was applied to the slides for 6 minutes. Slides were rinsed in water for 1 minute, acid water for 2 seconds, and water for 30 seconds. A bluing agent (ammonium hydroxide) was applied for 30 seconds, and the slides were rinsed first in water and then in 95% ethanol for 30 seconds each. EA 50 and Orange G (Autocyte®) were applied for 6 minutes. The slides were rinsed 2 times in 95% ethanol, 3 times in 100% ethanol, and 3 times in xylene for 30 seconds per rinse.
The slides were then coverslipped using Acrytol mounting media and incubated at 35° C. to dry. Samples were reviewed by a pathologist using a bright-field microscope.
Prepared slides were removed from the 95% ethanol and rinsed with deionized water for approximately 1 minute. Slides were placed in a 1× Target Retrieval Solution pH 6.0 (DAKO S1699)/dH2O bath preheated to 95° C. and placed in a steamer for 25 minutes. Samples were allowed to cool for 20 minutes at room temperature, rinsed well in deionized water, and placed in TBS. Pretreated slides were stained for biomarker expression essentially as described above in Example 1, "Automated Immunocytochemistry." Commercial antibodies directed to MCM2 (1:200), MCM7 (1:25), p21waf1 (1:100), and cylcin E (1:100) were diluted as indicated and used to detect biomarker expression. Purified MCM6 antibody, identified by polydoma screening as described in Example 4, was used at a 1:6000 dilution.
Interpretation of Slides
Each slide was screened and reviewed by a cytotechnologist and a pathologist. Samples were classified as positive, negative, or indeterminate for high-grade cervical disease according to the following parameters: Non-cellular artifacts and inflammatory cells staining brown (DAB) were disregarded. Mature, normal-appearing squamous cells and normal-appearing glandular cells were not counted as positive when staining with DAB. Squamous metaplastic cells along with abnormal cells were considered positive. A staining intensity of less than 1.5 was considered negative. Discrepant results were resolved through joint review of slides.
The immunocytochemistry results were compared with the results previously obtained by colposcopy. Each slide was then given a final result of true positive (TP), true negative (TN), false positive (FP), false negative (FN), or indeterminate. Sensitivity, specificity, positive predictive values, and negative predictive values for each biomarker were calculated.
The results for each biomarker are summarized below.
TABLE-US-00002 TABLE 2 MCM2 TP FP FN TN Indeter. Totals NIL 0 0 0 71 1 72 ASC-US (No Lesion) 0 0 0 25 1 26 ASC-US (Lesion) 0 0 0 0 0 0 LSIL (No HSIL) 0 7 0 31 4 42 LSIL (HSIL) 3 0 3 0 0 6 HSIL 24 0 1 0 0 25 Cancer 7 0 1 0 2 10 34 7 5 127 8 181 Sensitivity 0.8718 Specificity 0.9478 PPV 0.8293 NPV 0.9621
TABLE-US-00003 TABLE 3 MCM6 TP FP FN TN Indeter. Totals NIL 0 0 0 68 4 72 ASC-US (No Lesion) 0 3 0 22 1 26 ASC-US (Lesion) 0 0 0 0 0 0 LSIL (No HSIL) 0 14 0 24 4 42 LSIL (HSIL) 3 0 2 0 1 6 HSIL 22 0 0 0 3 25 Cancer 10 0 0 0 0 10 35 17 2 114 13 181 Sensitivity 0.9459 Specificity 0.8702 PPV 0.6731 NPV 0.9828
TABLE-US-00004 TABLE 4 MCM7 TP FP FN TN Indeter. Totals NIL 0 0 0 67 5 72 ASC-US (No Lesion) 0 2 0 21 3 26 ASC-US (Lesion) 0 0 0 0 0 0 LSIL (No HSIL) 0 12 0 28 2 42 LSIL (HSIL) 4 0 2 0 0 6 HSIL 24 0 1 0 0 25 Cancer 9 0 0 0 1 10 37 14 3 116 11 181 Sensitivity 0.9250 Specificity 0.8923 PPV 0.7255 NPV 0.9748
TABLE-US-00005 TABLE 5 Cyclin E TP FP FN TN Indeter. Totals NIL 0 0 0 72 0 72 ASC-US (No Lesion) 0 0 0 26 0 26 ASC-US (Lesion) 0 0 0 0 0 0 LSIL (No HSIL) 0 3 0 35 4 42 LSIL (HSIL) 2 0 4 0 0 6 HSIL 15 0 4 0 6 25 Cancer 7 0 2 0 1 10 24 3 10 133 11 181 Sensitivity 0.7059 Specificity 0.9779 PPV 0.8889 NPV 0.9301
TABLE-US-00006 TABLE 6 p21.sup.waf1 TP FP FN TN Indeter. Totals NIL 0 2 0 61 9 72 ASC-US (No Lesion) 0 1 0 22 3 26 ASC-US (Lesion) 0 0 0 0 0 0 LSIL (No HSIL) 0 12 0 23 7 42 LSIL (HSIL) 3 0 3 0 0 6 HSIL 21 0 1 0 3 25 Cancer 7 0 2 0 1 10 31 15 6 106 23 181 Sensitivity 0.8378 Specificity 0.8760 PPV 0.6739 NPV 0.9464
TABLE-US-00007 TABLE 7 TOPO2A TP FP FN TN Indeter. Totals NIL 0 0 0 68 4 72 ASC-US (No Lesion) 0 1 0 24 1 26 ASC-US (Lesion) 0 0 0 0 0 0 LSIL (No HSIL) 0 4 0 27 11 42 LSIL (HSIL) 3 0 3 0 0 6 HSIL 21 0 1 0 3 25 Cancer 9 0 0 0 1 10 33 5 4 119 20 181 Sensitivity 0.8919 Specificity 0.9597 PPV 0.8684 NPV 0.9675
Approximately 180 cases were analyzed for the presence of high-grade cervical disease using the immunocytochemistry methods of the invention. Of that number, the MCM biomarkers produced an indeterminate rate ranging from 4% to 7%. Additionally, MCM2 showed a specificity of 95% with a sensitivity of 87%. The MCM6 and MCM7 biomarkers produced comparable sensitivity results of 95% and 93%, respectively. The specificity for these two biomarkers ranged from 87% to 89%.
Cyclin E produced the highest specificity value of 98%. Although the indeterminate rate was 6%, the sensitivity was only 71%. The indeterminate rate for p21.sup.waf1 was the highest of all markers tested at 13%. p21.sup.waf1 produced a sensitivity of 84% and a specificity of 88%. 96% specificity was observed with the biomarker Topo2A. The indeterminate rate for Topo2A was 11%, with a sensitivity of 89%.
Detection of Biomarkers in Clinical Samples Using Antibody Cocktails
Approximately 180 colposcopy-confirmed cervical cytology samples were analyzed by immunocytochemistry methods to identify high-grade cervical disease. Each sample was analyzed for the expression of multiple biomarkers of interest. Specifically, various combinations of antibodies directed to MCM2, MCM6, MCM 7, p21waf1, Cyclin E, and Topo2A were analyzed for their ability to detect high-grade cervical disease. These samples were evaluated for the expression of multiple biomarkers of interest using the immunocytochemistry methods and slide interpretation guidelines described in Example 2.
The immunocytochemistry results were compared with the results previously obtained by colposcopy. Each slide was then given a final result of true positive (TP), true negative (TN), false positive (FP), false negative (FN), or indeterminate. Sensitivity, specificity, positive predictive values, and negative predictive values for each biomarker were calculated.
The results for each biomarker are summarized below.
TABLE-US-00008 TABLE 8 MCM2 and MCM7 TP FP FN TN Indeter. Totals NIL 0 0 0 66 6 72 ASC-US (No Lesion) 0 2 0 20 4 26 ASC-US (Lesion) 0 0 0 0 0 0 LSIL (No HSIL) 0 13 0 25 4 42 LSIL (HSIL) 4 0 2 0 0 6 HSIL 24 0 1 0 0 25 Cancer 10 0 0 0 0 10 38 15 3 111 14 181 Sensitivity 0.9268 Specificity 0.8810 PPV 0.7170 NPV 0.9737
TABLE-US-00009 TABLE 9 MCM6 and MCM7 TP FP FN TN Indeter. Totals NIL 0 0 0 65 7 72 ASC-US (No Lesion) 0 3 0 21 2 26 ASC-US (Lesion) 0 0 0 0 0 0 LSIL (No HSIL) 0 16 0 23 3 42 LSIL (HSIL) 4 0 2 0 0 6 HSIL 24 0 0 0 1 25 Cancer 10 0 0 0 0 10 38 19 2 109 13 181 Sensitivity 0.9500 Specificity 0.8516 PPV 0.6667 NPV 0.9820
TABLE-US-00010 TABLE 10 MCM7 and TOPO2A TP FP FN TN Indeter. Totals NIL 0 0 0 64 8 72 ASC-US (No Lesion) 0 2 0 21 3 26 ASC-US (Lesion) 0 0 0 0 0 0 LSIL (No HSIL) 0 12 0 29 1 42 LSIL (HSIL) 4 0 2 0 0 6 HSIL 20 0 2 0 3 25 Cancer 8 0 0 0 2 10 32 14 4 114 17 181 Sensitivity 0.8889 Specificity 0.8906 PPV 0.6957 NPV 0.9661
TABLE-US-00011 TABLE 11 MCM7 and Cyclin E TP FP FN TN Indeter. Totals NIL 0 0 0 67 5 72 ASC-US (No Lesion) 0 2 0 21 3 26 ASC-US (Lesion) 0 0 0 0 0 0 LSIL (No HSIL) 0 12 0 28 2 42 LSIL (HSIL) 4 0 2 0 0 6 HSIL 24 0 1 0 0 25 Cancer 10 0 0 0 0 10 38 14 3 116 10 181 Sensitivity 0.9268 Specificity 0.8923 PPV 0.7308 NPV 0.9748
TABLE-US-00012 TABLE 12 MCM7 and p21waf1 TP FP FN TN Indeter. Totals NIL 0 2 0 57 13 72 ASC-US (No Lesion) 0 3 0 20 3 26 ASC-US (Lesion) 0 0 0 0 0 0 LSIL (No HSIL) 0 14 0 21 7 42 LSIL (HSIL) 4 0 2 0 0 6 HSIL 24 0 1 0 0 25 Cancer 9 0 0 0 1 10 37 19 3 98 24 181 Sensitivity 0.9250 Specificity 0.8376 PPV 0.6607 NPV 0.9703
TABLE-US-00013 TABLE 13 MCM2 and MCM6 TP FP FN TN Indeter. Totals NIL 0 0 0 67 5 72 ASC-US (No Lesion) 0 3 0 21 2 26 ASC-US (Lesion) 0 0 0 0 0 0 LSIL (No HSIL) 0 17 0 21 4 42 LSIL (HSIL) 3 0 2 0 1 6 HSIL 24 0 0 0 1 25 Cancer 10 0 0 0 0 10 37 20 2 109 13 181 Sensitivity 0.9487 Specificity 0.8450 PPV 0.6491 NPV 0.9820
TABLE-US-00014 TABLE 14 MCM2 and TOPOIIA TP FP FN TN Indeter. Totals NIL 0 0 0 67 5 72 ASC-US (No Lesion) 0 1 0 23 2 26 ASC-US (Lesion) 0 0 0 0 0 0 LSIL (No HSIL) 0 8 4 18 12 42 LSIL (HSIL) 3 0 3 0 0 6 HSIL 25 0 0 0 0 25 Cancer 9 0 0 0 1 10 37 9 7 108 20 181 Sensitivity 0.8409 Specificity 0.9231 PPV 0.8043 NPV 0.9391
TABLE-US-00015 TABLE 15 MCM2 and Cyclin E TP FP FN TN Indeter. Totals NIL 0 0 0 71 1 72 ASC-US (No Lesion) 0 0 0 25 1 26 ASC-US (Lesion) 0 0 0 0 0 0 LSIL (No HSIL) 0 9 0 27 6 42 LSIL (HSIL) 3 0 3 0 0 6 HSIL 24 0 1 0 0 25 Cancer 8 0 2 0 0 10 35 9 6 123 8 181 Sensitivity 0.8537 Specificity 0.9318 PPV 0.7955 NPV 0.9535
TABLE-US-00016 TABLE 16 MCM2 and p21waf1 TP FP FN TN Indeter. Totals NIL 0 2 0 60 10 72 ASC-US (No Lesion) 0 1 0 21 4 26 ASC-US (Lesion) 0 0 0 0 0 0 LSIL (No HSIL) 0 13 0 21 8 42 LSIL (HSIL) 3 0 3 0 0 6 HSIL 24 0 1 0 0 25 Cancer 9 0 1 0 0 10 36 16 5 102 22 181 Sensitivity 0.8780 Specificity 0.8644 PPV 0.6923 NPV 0.9533
TABLE-US-00017 TABLE 17 TOPO2A and Cyclin E TP FP FN TN Indeter. Totals NIL 0 0 0 68 4 72 ASC-US (No Lesion) 0 1 0 24 1 26 ASC-US (Lesion) 0 0 0 0 0 0 LSIL (No HSIL) 0 5 0 27 10 42 LSIL (HSIL) 3 0 3 0 0 6 HSIL 22 0 1 0 2 25 Cancer 9 0 0 0 1 10 34 6 4 119 18 181 Sensitivity 0.8947 Specificity 0.9520 PPV 0.8500 NPV 0.9675
TABLE-US-00018 TABLE 18 TOPO2A and p21waf1 TP FP FN TN Indeter. Totals NIL 0 2 0 58 12 72 ASC-US (No Lesion) 0 2 0 21 3 26 ASC-US (Lesion) 0 0 0 0 0 0 LSIL (No HSIL) 0 13 0 19 10 42 LSIL (HSIL) 3 0 3 0 0 6 HSIL 25 0 0 0 0 25 Cancer 10 0 0 0 0 10 38 17 3 98 25 181 Sensitivity 0.9268 Specificity 0.8522 PPV 0.6909 NPV 0.9703
TABLE-US-00019 TABLE 19 p21waf1 and Cyclin E TP FP FN TN Indeter. Totals NIL 0 2 0 61 9 72 ASC-US (No Lesion) 0 1 0 22 3 26 ASC-US (Lesion) 0 0 0 0 0 0 LSIL (No HSIL) 0 12 0 23 7 42 LSIL (HSIL) 3 0 3 0 0 6 HSIL 22 0 1 0 2 25 Cancer 8 0 1 0 1 10 33 15 5 106 22 181 Sensitivity 0.8684 Specificity 0.8760 PPV 0.6875 NPV 0.9550
TABLE-US-00020 TABLE 20 MCM2, MCM6, and MCM7 TP FP FN TN Indeter. Totals NIL 0 0 0 64 8 72 ASC-US (No Lesion) 0 3 0 20 3 26 ASC-US (Lesion) 0 0 0 0 0 0 LSIL (No HSIL) 0 17 0 21 4 42 LSIL (HSIL) 4 0 2 0 0 6 HSIL 24 0 0 0 1 25 Cancer 10 0 0 0 0 10 38 20 2 105 16 181 Sensitivity 0.9500 Specificity 0.8400 PPV 0.6552 NPV 0.9813
TABLE-US-00021 TABLE 21 MCM2, MCM7, and TOPO2A TP FP FN TN Indeter. Totals NIL 0 0 0 63 9 72 ASC-US (No Lesion) 0 2 0 20 4 26 ASC-US (Lesion) 0 0 0 0 0 0 LSIL (No HSIL) 0 13 0 21 8 42 LSIL (HSIL) 4 0 2 0 0 6 HSIL 25 0 0 0 0 25 Cancer 10 0 0 0 0 10 39 15 2 104 21 181 Sensitivity 0.9512 Specificity 0.8739 PPV 0.7222 NPV 0.9811
TABLE-US-00022 TABLE 22 MCM6, MCM7, and TOPO2A TP FP FN TN Indeter. Totals NIL 0 0 0 63 9 72 ASC-US (No Lesion) 0 3 0 21 2 26 ASC-US (Lesion) 0 0 0 0 0 0 LSIL (No HSIL) 0 16 0 20 6 42 LSIL (HSIL) 4 0 2 0 0 6 HSIL 25 0 0 0 0 25 Cancer 10 0 0 0 0 10 39 19 2 104 17 181 Sensitivity 0.9512 Specificity 0.8455 PPV 0.6724 NPV 0.9811
TABLE-US-00023 TABLE 23 MCM6, MCM7, and Cyclin E TP FP FN TN Indeter. Totals NIL 0 0 0 65 7 72 ASC-US (No Lesion) 0 3 0 21 2 26 ASC-US (Lesion) 0 0 0 0 0 0 LSIL (No HSIL) 0 16 0 23 3 42 LSIL (HSIL) 4 0 2 0 0 6 HSIL 24 0 0 0 1 25 Cancer 10 0 0 0 0 10 38 19 2 109 13 181 Sensitivity 0.9500 Specificity 0.8516 PPV 0.6667 NPV 0.9820
TABLE-US-00024 TABLE 24 MCM2, MCM7, and Cyclin E TP FP FN TN Indeter. Totals NIL 0 0 0 66 6 72 ASC-US (No Lesion) 0 2 0 20 4 26 ASC-US (Lesion) 0 0 0 0 0 0 LSIL (No HSIL) 0 13 0 25 4 42 LSIL (HSIL) 4 0 2 0 0 6 HSIL 24 0 1 0 0 25 Cancer 10 0 0 0 0 10 38 15 3 111 14 181 Sensitivity 0.9268 Specificity 0.8810 PPV 0.7170 NPV 0.9737
TABLE-US-00025 TABLE 25 MCM2, MCM7, and p21waf1 TP FP FN TN Indeter. Totals NIL 0 2 0 56 14 72 ASC-US (No Lesion) 0 3 0 18 5 26 ASC-US (Lesion) 0 0 0 0 0 0 LSIL (No HSIL) 0 14 0 20 8 42 LSIL (HSIL) 4 0 2 0 0 6 HSIL 24 0 1 0 0 25 Cancer 10 0 0 0 0 10 38 19 3 94 27 181 Sensitivity 0.9268 Specificity 0.8319 PPV 0.6667 NPV 0.9691
TABLE-US-00026 TABLE 26 MCM2, TOPOIIA and Cyclin E TP FP FN TN Indeter. Totals NIL 0 0 0 67 5 72 ASC-US (No Lesion) 0 1 0 23 2 26 ASC-US (Lesion) 0 0 0 0 0 0 LSIL (No HSIL) 0 9 0 22 11 42 LSIL (HSIL) 3 0 3 0 0 6 HSIL 25 0 0 0 0 25 Cancer 9 0 0 0 1 10 37 10 3 112 19 181 Sensitivity 0.9250 Specificity 0.9180 PPV 0.7872 NPV 0.9739
TABLE-US-00027 TABLE 27 MCM2, Cyclin E and p21waf1 TP FP FN TN Indeter. Totals NIL 0 2 0 60 10 72 ASC-US (No Lesion) 0 1 0 21 4 26 ASC-US (Lesion) 0 0 0 0 0 0 LSIL (No HSIL) 0 13 0 21 8 42 LSIL (HSIL) 3 0 3 0 0 6 HSIL 24 0 1 0 0 25 Cancer 9 0 1 0 0 10 36 16 5 102 22 181 Sensitivity 0.8780 Specificity 0.8644 PPV 0.6923 NPV 0.9533
TABLE-US-00028 TABLE 28 MCM2, TOPOIIA and p21waf1 TP FP FN TN Indeter. Totals NIL 0 2 0 57 13 72 ASC-US (No Lesion) 0 2 0 20 4 26 ASC-US (Lesion) 0 0 0 0 0 0 LSIL (No HSIL) 0 13 0 18 11 42 LSIL (HSIL) 3 0 3 0 0 6 HSIL 25 0 0 0 0 25 Cancer 10 0 0 0 0 10 38 17 3 95 28 181 Sensitivity 0.9268 Specificity 0.8482 PPV 0.6909 NPV 0.9694
TABLE-US-00029 TABLE 29 MCM7, TOPO2A, and Cyclin E TP FP FN TN Indeter. Totals NIL 0 0 0 64 8 72 ASC-US (No Lesion) 0 2 0 21 3 26 ASC-US (Lesion) 0 0 0 0 0 0 LSIL (No HSIL) 0 12 0 23 7 42 LSIL (HSIL) 4 0 2 0 0 6 HSIL 25 0 0 0 0 25 Cancer 10 0 0 0 0 10 39 14 2 108 18 181 Sensitivity 0.9512 Specificity 0.8852 PPV 0.7358 NPV 0.9818
TABLE-US-00030 TABLE 30 MCM7, p21waf1, and Cyclin E TP FP FN TN Indeter. Totals NIL 0 2 0 57 13 72 ASC-US (No Lesion) 0 3 0 19 4 26 ASC-US (Lesion) 0 0 0 0 0 0 LSIL (No HSIL) 0 14 0 21 7 42 LSIL (HSIL) 4 0 2 0 0 6 HSIL 24 0 1 0 0 25 Cancer 10 0 0 0 0 10 38 19 3 97 24 181 Sensitivity 0.9268 Specificity 0.8362 PPV 0.6667 NPV 0.9700
TABLE-US-00031 TABLE 31 MCM7, p21waf1, and TOPO2A TP FP FN TN Indeter. Totals NIL 0 2 0 54 16 72 ASC-US (No Lesion) 0 3 0 19 4 26 ASC-US (Lesion) 0 0 0 0 0 0 LSIL (No HSIL) 0 14 0 18 10 42 LSIL (HSIL) 4 0 2 0 0 6 HSIL 25 0 0 0 0 25 Cancer 10 0 0 0 0 10 39 19 2 91 30 181 Sensitivity 0.9512 Specificity 0.8273 PPV 0.6724 NPV 0.9785
TABLE-US-00032 TABLE 32 MCM2, MCM7, Cyclin E, and p21waf1 TP FP FN TN Indeter. Totals NIL 0 2 0 56 14 72 ASC-US (No Lesion) 0 3 0 18 5 26 ASC-US (Lesion) 0 0 0 0 0 0 LSIL (No HSIL) 0 14 0 20 8 42 LSIL (HSIL) 4 0 2 0 0 6 HSIL 24 0 1 0 0 25 Cancer 10 0 0 0 0 10 38 19 3 94 27 181 Sensitivity 0.9268 Specificity 0.8319 PPV 0.6667 NPV 0.9691
TABLE-US-00033 TABLE 33 MCM2, MCM7, Cyclin E and TOPOIIA TP FP FN TN Indeter. Totals NIL 0 0 0 63 9 72 ASC-US (No Lesion) 0 2 0 20 4 26 ASC-US (Lesion) 0 0 0 0 0 0 LSIL (No HSIL) 0 13 0 21 8 42 LSIL (HSIL) 4 0 2 0 0 6 HSIL 25 0 0 0 0 25 Cancer 10 0 0 0 0 10 39 15 2 104 21 181 Sensitivity 0.9512 Specificity 0.8739 PPV 0.7222 NPV 0.9811
TABLE-US-00034 TABLE 34 MCM2, MCM7, Cyclin E, p21waf1, and TOPO2A TP FP FN TN Indeter. Totals NIL 0 2 0 53 17 72 ASC-US (No Lesion) 0 3 0 18 5 26 ASC-US (Lesion) 0 0 0 0 0 0 LSIL (No HSIL) 0 14 0 18 10 42 LSIL (HSIL) 4 0 2 0 0 6 HSIL 25 0 0 0 0 25 Cancer 10 0 0 0 0 10 39 19 2 89 32 181 Sensitivity 0.9512 Specificity 0.8241 PPV 0.6724 NPV 0.9780
TABLE-US-00035 TABLE 35 MCM2, MCM6, MCM7, TOPO2A, Cyclin E, and p21waf1 TP FP FN TN Indeter. Totals NIL 0 2 0 52 18 72 ASC-US (No Lesion) 0 4 0 18 4 26 ASC-US (Lesion) 0 0 0 0 0 0 LSIL (No HSIL) 0 18 0 16 8 42 LSIL (HSIL) 4 0 2 0 0 6 HSIL 25 0 0 0 0 25 Cancer 10 0 0 0 0 10 39 24 2 86 30 181 Sensitivity 0.9512 Specificity 0.7818 PPV 0.6190 NPV 0.9773
Data was compiled on 28 antibody cocktails, as described above. Biomarker expression was analyzed using cocktails comprising antibodies directed to 2, 3, 4, 5 or even all 6 of the biomarkers of interest. Twenty-one of the 28 antibody cocktails showed sensitivities greater than 92%. Four of the 28 cocktails produced specificities above 90% with the lowest value at 78%. The highest values were achieved with a combination of MCM2, TOPOIIA and Cyclin E. This cocktail yielded a sensitivity of 93% along with a specificity of 92%. It appears that a combination of at least 3 biomarkers should yield a sensitivity greater than 90%. It is recognized that adjustments to the assay would further increase the sensitivity and specificity of the assay.
Detection of Biomarker Expression Using Antibody Cocktails
Antibody cocktails were prepared using various combinations of antibodies directed to Cyclin E, MCM2, MCM6, MCM7, p21waf1, and TOPO2a. The composition of each cocktail is listed in the table below.
TABLE-US-00036 TABLE 36 Composition of Antibody Cocktails Cocktail ID Biomarkers Cocktail 1 Cyclin E, MCM2, MCM7 Cocktail 2 Cyclin E, MCM6, MCM7 Cocktail 3 Cyclin E, MCM7, p21waf1 Cocktail 4 Cyclin E, MCM7, TOPO2a Cocktail 5 MCM2, MCM7, p21waf1 Cocktail 6 MCM6, MCM7, p21waf1 Cocktail 7 MCM7, p21waf1, TOPO2a Cocktail 8 MCM2, MCM7, TOPO2a Cocktail 9 MCM6, MCM7, TOPO2a Cocktail 10 MCM2, MCM6, MCM7 Cocktail 11 Cyclin E, MCM2, MCM6, MCM7, p21waf1, TOPO2a Cocktail 12 Cyclin E, MCM2, MCM7, p21waf1 Cocktail 13 MCM2 and MCM 7 Cocktail 14 MCM7 and p21waf1 Cocktail 15 MCM7 and Cyclin E Cocktail 16 MCM2 and p21waf1 Cocktail 17 Cyclin E and p21waf1 Cocktail 18 MCM2 and Cyclin E Cocktail 19 MCM7 and TOPO2a Cocktail 20 MCM2 and TOPO2a Cocktail 21 Cyclin E and TOPO2a Cocktail 22 p21waf1 and TOPO2a
Two sets of cervical cytology specimens were prepared by pooling HSIL cases (HSIL pool) and NIL cases (NIL pool). Each antibody cocktail was then tested on the HSIL pool and the NIL pool. Biomarker antibodies were also tested individually as a control. Slide preparation and automated immunocytochemistry were performed as described in Example 2.
Slides were screened and reviewed by a cytotechnologist and a pathologist. Specific staining of cells indicative of high-grade cervical disease, staining of glandular cells, bacteria cross-reactivity, and the location of cell staining were all variables that were recorded during the screening process.
The immunocytochemistry results indicated an increase in staining of cells indicative of high-grade cervical disease in the HSIL pool with the biomarker antibody cocktails when compared to the results obtained with detection of a single biomarker. Additionally, there was no significant increase in background when the number of antibodies in the cocktails increased from 2 to 3, 4 or 6. Furthermore, the various antibody cocktails did not show an increase in background staining when tested on the NIL pool.
Detection of Biomarker Overexpression in Cervical Samples Using Immunocytochemistry
Slide Preparation and Pretreatment
Patient cervical samples were collected as described above in Example 1. Slides comprising a monolayer of cervical cells were prepared by the AutoPrep® System using "prep only" mode. Prepared slides were immediately placed in 1× SureSlide® Pretreatment Buffer (0.5% sodium laureth-13-carboxylate (Sandopan LS) in deionized H20) for a minimum of 1 hour and a maximum of 72 hours. The pretreated slides were placed in a steamer at 95° C. for 45 minutes without preheating. Slides were removed from the steamer, allowed to cool at room temperature for 20 minutes, and then rinsed well in deionized water. Slides were rinsed in TBST (TBS/Tween-20) twice at 2 minutes per rinse. The slides were tapped to remove excess buffer and placed into a humid chamber. Slides were subjected to manual or automated immunocytochemistry, as described below.
200 μl of peroxidase block reagent (0.03% hydrogen peroxide) was applied to each slide to cover the cell deposition area for a period of 5 minutes. The slides were then rinsed with TBST three times at 2 minutes per rinse. Excess buffer was removed, and the slides were placed into a humid chamber. 200 μl of protein block reagent (purified casein and surfactant) was applied to each slide and incubated for 5 minutes. After the excess protein block reagent was removed, the slides were placed in a humid chamber.
200 μl of primary monoclonal antibody cocktail comprising two mouse anti-human antibodies directed to MCM2 (clone 27C5.6 at 0.39 mg/ml, 1:800 dilution; clone 26H6.19 at 0.918 mg/ml, 1:10,000 dilution) and a third mouse anti-human antibody specific for Topo2A (clone SWT3D1 at 100 μg/ml, 1:10,000 dilution) was applied to each slide to completely cover the cell deposition area. Slides were incubated for 30 minutes and then rinsed with TBST three times at 2 minutes per rinse. Excess buffer was removed, and the slides were returned to the humid chamber. 200 μl of mouse probe reagent that binds to mouse monoclonal antibodies was applied as above for 20 minutes. The slides were rinsed with TBST three times at 2 minutes per rinse. Excess buffer was removed, and the slides were again placed into the humid chamber.
200 μl of polymer reagent comprising a dextran polymer conjugated with HRP and a secondary goat anti-mouse antibody that binds to the mouse probe reagent was applied as above for 20 minutes and then the slides rinsed with TBST 3 times at 2 minutes per rinse. After the excess buffer was removed, the slides were returned to the humid chamber. 200 μl of DAB substrate-chromgen solution was applied as above for 5 minutes. The slides were rinsed with deionized water for 5 minutes and then rinsed with TBST for 2 minutes with one change of buffer. Excess buffer was removed and the slides were placed in a humid chamber as before. 200 μl of hematoxylin was added for 1 minute, followed by 3 rinses with deionized water at 2 minutes per rinse. Excess deionized water was removed, and the slides were placed into the humid chamber. 200 μl of bluing agent (i.e., TBS, pH 7.4 with tween-20 and sodium azide) was applied to each slide for 1 minute. The slides were then rinsed in one change of TBST and 1 change of deionized water for 2 minutes each. The slides were then dehydrated, cleared, coverslipped, and analyzed as described in Examples 1 and 2.
The autostainer was programmed according to manufacturer's instructions to include the following sequence of steps:
a. 2 buffer rinses (TBST)
b. 5 min peroxidase block
c. 2 buffer rinses (TBST)
d. 5 min protein block, blow
e. 30 min primary antibody cocktail incubation
f. 3 buffer rinses (TBST)
g. 20 min mouse probe reagent
h. 3 buffer rinses (TBST)
i. 20 min polymer-HRP
j. 3 buffer rinses (TBST)
k. 5 min DAB (1 drop of chromagen to 1 ml of buffer)
l. 3 H2O rinses
m. 2 buffer rinses (TBST)
n. 1 min Mayer's hematoxylin
o. 3H2O rinses
p. 1 min bluing agent
q. 1 buffer rinse (TBST)
r. 1 H2O rinse
The necessary staining and counterstaining reagents were loaded onto the machine. The prepared and pretreated slides were loaded onto the autostainer, and the above program was run. At the end of the run, the slides were removed and rinsed briefly in tap water. The slides were dehydrated, cleared, coverslipped, and analyzed as described in Examples 1 and 2.
TABLE-US-00037 TABLE 37 NIL Cases (n = 45) Pap Result NIL Other Unsatisfactory 44 1 (ASC-US) 0 ICC Result Positive Negative Unsatisfactory 0 44 1
TABLE-US-00038 TABLE 38 HSIL Cases (n = 45) Pap Result HSIL Other Unsatisfactory 45 0 0 ICC Result Positive Negative Unsatisfactory 45 0 0
Of the 45 NIL cases tested, a review of the Pap stained slides revealed one ASC-US case. The immunocytochemistry (ICC) results for the NIL samples were negative, with one case deemed unsatisfactory for evaluation. Regarding the HSIL cases, each of the 45 cases was confirmed to be high-grade cervical disease based upon the review of the Pap stained slides. Additionally, each of the 45 HSIL cases was also positive in the ICC assay. The negative control, i.e., a universal mouse IgG control applied to the SiHa cell line control, produced negative results in the ICC assay. The positive control, i.e., the primary antibody cocktail applied to the SiHa cell line control, produced positive results in the ICC assay.
Combined Immunocytochemistry and Pap Staining Procedure
Patient cervical samples were collected as described above in Example 1. Slides comprising a monolayer of cervical cells were prepared and pretreated as indicated in Example 5. Each pretreated slide was subjected to automated immunocytochemistry and Pap staining, thereby permitting visualization of both biomarker overexpression and cell morphology on a single slide.
Automated immunocytochemistry was performed on each slide as described above in Example 5. At the end of the staining program, the slides were removed from the autostainer and rinsed in tap water for 3-5 minutes. Each slide was then stained according to conventional Pap staining methods, as described below.
Pap Staining Method
Following automated immunocytochemistry, each slide was further stained with Pap stain. The slides were first rinsed in 95% ethanol for 30 seconds. EA50 and Orange G were applied to half of the slides for 3 minutes and to the remaining slides for 6 minutes. All of the slides were then rinsed 2 times in 95% ethanol, 3 times in 100% ethanol, and 3 times in xylene for 30 seconds per rinse. The slides were then coverslipped with permanent mounting media and analyzed as described above in Examples 1 and 2.
A panel of 5 NIL and 5 HSIL cases were each subjected to 3 minutes or 6 minutes of staining with EA50 and Orange G in the Pap staining method. Results indicated minimal difference between the 3 minute and the 6 minute staining protocols. The slides subjected to 3 minutes of Pap staining displayed slightly less intense staining. Furthermore, the ICC positive staining HSIL cells were readily observed with the Pap counterstain.
Combined Immunocytochemistry and Pap Staining Procedure (Optimization of Pap Staining)
The combined immunocytochemistry and Pap staining procedure outlined in Example 6 was modified to optimize the Pap staining parameters in order to maximize the contrast between the chromogen (i.e., DAB) staining of the immunocytochemistry method and the level of Pap staining.
Slides were prepared, pretreated, and subjected to automated immunocytochemistry as described above in Example 6. The slides were then stained with a conventional Pap stain essentially as described in Example 6, with the following modifications. Hematoxylin was tested using the Harris formulation along with Myers formulation. EA/Orange G was applied for 3 minutes or 6 minutes. Additionally, there were 3 changes of 95% ethanol after the EA/Orange G addition.
Slides received a determination of positive, negative, or unsatisfactory based upon the immunocytochemistry staining. Additionally, slides were evaluated morphologically for comparison with the incoming Pap diagnosis.
TABLE-US-00039 TABLE 39 Results of Combined Immunocytochemistry and Pap Staining Method Incoming Pap ICC Diagnosis Results Comments NIL Negative All cases confirmed as NIL. n = 7 LSIL Negative 5 of the 6 LSIL cases did not have LSIL n = 6 cells on the slides. These cases were either NIL or ASC-US. HSIL Positive All cases confirmed as HSIL. n = 6 Cancer Positive All cases were squamous cell carcinoma. n = 4
The combined ICC and Pap staining procedure permitted both morphological analysis and assessment of biomarker overexpression. Additional experimentation will be required to further optimize the method.
Immunocytochemistry Kit for the Detection of Biomarker Overexpression in Cervical Samples
I. Principles of the Procedure
An immunocytochemical test kit contains reagents required to complete a three-step immunocytochemical staining procedure for routinely prepared monolayer cervical specimens. Following incubation with the monoclonal antibody cocktail, this kit employs a ready-to-use visualization reagent based on dextran technology. This reagent consists of a secondary goat anti-mouse antibody molecule and horseradish peroxidase molecules linked to a dextran polymer backbone. The enzymatic conversion of the subsequently added chromogen results in the formation of a visible reaction product at the antigen(s) site. The specimen is then counterstained with hematoxylin, a bluing agent is applied, and the slide is coverslipped. Results are interpreted using a light microscope. A positive result indicative of cervical high-grade is achieved when cells of interest are stained brown.
A gallery of potentially positive cells may be created using automated imaging equipment. The gallery then can be reviewed to determine a positive result or negative result.
The immunocytochemical test kit is applicable for both manual and automated staining.
II. Reagents Provided
The following materials, sufficient for 75 monolayer preparations using 200 μL of the ready-to-use mouse monoclonal cocktail per preparation, were included in the immunocytochemical test kit:
TABLE-US-00040 TABLE 40 Immunocytochemistry Kit Components Vial No. Quantity Description 1a 1 × 15 mL Peroxidase-Blocking Reagent: Buffered hydrogen peroxide plus stabilizer and proprietary components 1b 1 × 15 mL Protein Blocking Reagent: Purified casein plus proprietary combination of proteins in modified PBS with preservative and surfactant 2 1 × 15 mL Mouse Anti-Human Antibody Cocktail: Ready-to-use monoclonal antibody cocktail supplied in TRIS buffered solution with Tween 20, pH 7.4. Contains 0.39 mg/mL MCM2 mAb clone 27C5.6 (1:800 dilution), 0.918 mg/mL MCM2 mAb clone 26H6.19 (1:10,000 dilution), 100 μg/mL Topo2a mAb clone SWT3D1 (1:10,000 dilution), stabilizing proteins and anti-microbial agent. 3a 1 × 15 mL Mouse Probe Reagent: Binds to mouse monoclonal antibodies 3b 1 × 15 mL Polymer Reagent: Polymer conjugated with horseradish peroxidase that binds to Mouse Probe Reagent 4a 1 × 18 mL DAB Substrate Buffer: Substrate buffer used in the preparation of the DAB Chromogen 4b 1 × 1 mL DAB Chromogen: 3,3'-diaminobenzidine chromogen solution 5 1 × 18 mL Hematoxylin Counterstain: aqueous based Mayers Hematoxylin 6 1 × 18 mL Bluing Agent: Tris buffered saline, pH 7.4 with Tween 20 and 0.09% NaN3
The following materials and reagents were required to perform the immunocytochemistry methods but were not supplied in the kit: Absorbent Wipes SiHa Cell Line (TriPath Imaging, Inc.) Deionized or Distilled Water Ethanol (95% and 100%) Glass Coverslips Gloves Humid Chamber Light Microscope (10×, 20×, 40× objectives) Mounting Media Pipettes and Pipette Tips (capable of delivering 20 μl, 200 μl and 1000 μl volumes) SureSlide Preparation Buffer (TriPath Imaging, Inc.)-Pretreatment Buffer (0.5% sodium laureth-13-carboxylate (Sandopan LS) in deionized H20) Staining Jars or Baths Timer (capable of 1-60 minute intervals) Tris Buffered Saline (TBS) Tween 20 Universal Mouse IgG Negative Control Vortexer Xylene or Xylene Substitutes Steamer/waterbath
III. Instructions for Use
The following steps were followed for the preparation of cervical samples: Consult the Operator's Manual for the SurePath PrepStain System® for the preparation of slides from residual specimens. Add 8 mL of SurePath® preservative fluid to the residual sample in the SurePath® vial (approx. 2 mLs). The diluted sample is processed on the PrepMate® using the standard technique and on the PrepStain® using the GYN version 1.1, Slide Preparation.
Prepared slides are immediately placed into the pretreatment buffer for a minimum of 1 hour with a maximum of 72 hours prior to immunostaining.
Epitope retrieval must be used for optimal kit performance. This procedure involves soaking prepared slides in the pretreatment buffer for a minimum of 1 hour at room temperature followed by heating slides in the pretreatment buffer to 95° C. Slides are held at 95° C. for 15 minutes and allowed to cool down at room temperature for 20 minutes. The use of a calibrated waterbath or vegetable steamer capable of maintaining the required temperature is recommended. Laboratories located at higher elevations should determine the best method of maintaining the required temperature. The staining procedure is initiated immediately following epitope retrieval and cool down. Deviations from the described procedure may affect results.
The following reagents were prepared prior to staining:
Tris Buffered Saline with 0.05% Tween 20 (TBST) Prepare TBS according to manufacturer's specifications. Add Tween 20 to a final concentration of 0.05%. Store at room temperature if used within one week. Unused solution may be stored at 2-8° C. for 3 months. Solution is clear and colorless. Discard diluted solution if cloudy in appearance.
Substrate-Chromogen Solution (Dab) (Volume Sufficient for 5 Slides)
 Transfer 1 mL of DAB Buffered Substrate to a test tube. Add one drop (20-30 uL) of DAB+Chromogen. Mix thoroughly and apply to slides with a pipette. Prepare Substrate-Chromogen solution fresh daily. Any precipitate developing in the solution does not affect staining quality.
IV. Staining Protocol (Performed at Room Temperature, 20-25° C.)
The following steps were performed for immunostaining of the cervical cytology samples:
Staining Procedural Notes
The user should read these instructions carefully and become familiar with all components prior to use. All reagents are equilibrated to room temperature (20-25° C.) prior to immunostaining. All incubations are performed at room temperature. Do not allow slides to dry out during the staining procedure. Dried cellular preparations may display increased non-specific staining. Cover slides exposed to drafts. Slides should be placed in a humid chamber for prolonged incubations.
 Place the prepared slides in the pretreatment buffer for a minimum of 1 hour to a maximum of 72 hours. Incubate for 15 minutes at 95° C. Remove the entire coplin jar with slides from the waterbath or steamer and allow slides to cool in the buffer for 20 minutes. Rinse the slides with diH2O and transfer to a TBST bath.
 Tap off excess buffer. Load slides into prepared humidity chamber (filled with water moistened paper towels or gauze). Apply 200 μL Peroxidase Block reagent to cover the cell deposition area. Incubate 5 minutes (±1 minute). Rinse slides in TBST, 3 changes, 2 minutes each.
 Tap off excess buffer. Load the slides into the prepared humidity chamber (filled with water moistened paper towels or gauze). Apply 200 μL of Protein Block to completely cover cell deposition area. Incubate 5 minutes (±1 minute). Do not rinse slides.
Primary Antibody Cocktail
 Tap off excess Protein Block. Load the slides into the prepared humidity chamber (filled with water moistened paper towels or gauze). Apply 200 μL primary antibody cocktail (to completely cover cell deposition area. Incubate 30 minutes at room temperature. Rinse each slide individually with TBST using a wash bottle (do not focus the flow directly on the cell deposition area). Load slides into a slide rack. Rinse slides in TBST, 3 changes, 2 minutes each.
 Tap off excess buffer. Load slides into prepared humidity chamber (filled with water moistened paper towels or gauze). Apply 200 μL Mouse Probe to completely cover cell deposition area. Incubate 20 minutes (±1 minute). Rinse slides in TBST, 3 changes, 2 minutes each. Tap off excess buffer. Load slides into prepared humidity chamber (filled with water moistened paper towels or gauze). Apply 200 μL of Polymer to cover cell deposition area. Incubate for 20 minutes (±1 minute). Rinse slides in TBST bath, 3 changes, 2 minutes each. Tap off excess buffer. Load the slides into the prepared humidity chamber (filled with water moistened paper towels or gauze). Apply 200 μL of DAB working solution to completely cover cell deposition area. Incubate for 5 minutes (+1 minute). Rinse slides for 5 minutes in diH2O for 5 minutes.
 Rinse slides in TBST, 1 change for 2 minutes. Load slides into prepared humidity chamber (filled with water moistened paper towels or gauze). Apply 200 μL of hematoxylin to completely cover cell deposition area. Incubate for 1 minute (±10 seconds). Rinse slides for 3 minutes in running H2O. Load slides into prepared humidity chamber (filled with water moistened paper towels or gauze). Blue slides by applying 200 μL Bluing Agent for 1 minute (±10 seconds). Repeat running water rinse for 1 minute.
 Immerse slides in 95% ethanol, 1 minute or 25 dips. Immerse slides in absolute alcohol, 4 changes, 1 minute each or 25 dips. Clear with xylene, 3 changes, 1 minute each or 25 dips. Coverslip slides with non-aqueous, permanent mounting media using glass coverslips.
V. Quality Control
The following quality control issues were considered when using the immunocytochemistry kit described in this example:
Variability in results is often derived from differences in specimen handling and changes in test procedures. Consult the proposed quality control guidelines of the NCCLS Quality Assurance for Immunocytochemistry for additional information.
Control Cell Line is available from TriPath Imaging, Inc. Each vial contains a cervical cancer cell line, which is processed in a similar manner as the clinical specimens. Two slides should be stained in each staining procedure. The evaluation of the control slide cell line indicates the validity of the staining run.
VI. Interpretation of Staining
The control slide stained with the immunocytochemical test kit were examined first to ascertain that all reagents functioned properly. The presence of a brown (3,3'-diaminobenzidine tetrahydrochloride, DAB) reaction product in the nuclei of the cells were indicative of positive reactivity.
Slide evaluation was performed by a cytotechnologist or pathologist using a light microscope. Cells were reviewed manually or electronically stored in an image gallery derived from a light microscope.
Approximately 1610 cervical samples representing various diagnoses were collected. The following table indicates the number of samples analyzed using the immunocytochemistry kit within each diagnosis group, as determined by conventional Pap staining or biopsy.
TABLE-US-00041 TABLE 41 Patient Specimens within Each Diagnosis Group (Pap Staining) Cytology Results Number % NIL 671 41.7% LSIL 395 24.53% ASCUS 349 21.68% HSIL 150 9.32% ASC-H 38 2.36% AGUS 6 0.37% SCC 1 0.06% Total 1610
TABLE-US-00042 TABLE 42 Patient Specimens within Each Diagnosis Group (Biopsy) Biopsy Results Number % NIL 968 60.20% CIN1 369 22.95% CIN2 140 8.71% CIN3 131 8.15% Missing 2 Total 1610
Slide Scoring Guide
The following procedure was followed for the scoring of all slides analyzed by the immunocytochemistry methods described in this example:
Step 1: Is it an Adequate Specimen?
The Bethesda System for Reporting Cervical Cytology (second edition) states, "An adequate liquid-based preparation should have an estimated minimum of at least 5000 well-visualized/well-preserved squamous cells." These same criteria were applied when evaluating all of the slides. However, as with a routine Pap preparation, any specimen with abnormal cells, which are exhibiting a positive molecular reaction, was, by definition, satisfactory for evaluation. If the answer to this step was "yes", the cytotechnologist proceeded to the next step; if the answer was "no," the result was Unsatisfactory for Evaluation.
Step 2: Is there Moderate to Intense Brown Nuclear Staining in Epithelial Cells?
The detection chemicals used in the immunocytochemistry kit of this example (e.g., SureDetect Detection Chemistry Kit) stains dysplastic nuclei associated with ≧CIN 2 with a brown chromagen, DAB. To answer "yes" to this step, samples were analyzed for brown staining that was easily visualized. If just a faint amount, or "blush," of brown was seen, this was not enough to warrant a rendering of positive. If no brown nuclear stain was seen, this was deemed a negative test result. If there was adequate brown stain, the analysis proceeded to the next step.
Step 3: Is this a Squamous (or Glandular) Cell with Brown Nuclear Staining and is the Cell ≧ASC (AGC)?
Using the same morphological criteria outlined in The Bethesda System for Reporting Cervical Cytology (2nd Ed.) ("TBS"), it was determined if the squamous cell containing the brown nucleus was ≧ASC (atypical squamous cells). This would include ASC-US, ASC-H, LSIL, HSIL, and cancer. If the cell was glandular in appearance, the TBS criteria for determining if a cell is ≧AGC (atypical glandular cells) applied. This would include endocervical AGC, endometrial AGC, AIS, and adenocarcinoma. If the cell was considered to be ≧ASC (or ≧AGC) than this would result in a positive test result. If the cells in question were consistent with NILM (negative for intraepithelial lesion or malignancy) this would be a negative test result.
27 cases that were originally classified as NIL by conventional Pap staining methods stained positive in the immunocytochemistry test. Of these 27 cases, 7 were classified as HSIL, 10 as ASC-H, 3 as ASC-US, and 3 as indeterminate upon review by aboard certified pathologist. The 7 HSIL cases are considered high-grade cervical disease. These 27 cases were identified by positive immunostaining in the immunocytochemistry assay, thereby indicating the value of the methods disclosed herein for identifying patients misclassified as NIL by Pap staining.
Biopsy results were not obtained for all NIL specimens. Estimates of sensitivity and positive predictive value (PPV) for the immunocytochemistry method described in this example were calculated based on comparison with the "gold standard" biopsy results. Single biopsy has limitations as a gold standard. PPV for the ICC assay will improve by serial monitoring of the patient or utilizing a more aggressive surgical endpoint such as loop electrosurgical excision procedure or cone biopsy. Single biopsy is known to have a false negative result for disease of at least 31%. See Elit et al. (2004) J. Lower Genital Tract Disease 8(3):181-187.
TABLE-US-00043 TABLE 43 Estimated sensitivity and positive predictive value of ICC test based on the biopsy results ASC-H ASCUS LSIL HSIL ≧ASCUS Sensitivity 76.5% 92.6% 97.7% 98.5% 96.2% (52.7%, 90.4%)* (76.6%, 97.9%) (92.1%, 99.4%) (94.6%, 99.6%) (93.1%, 97.9%) PPV 59.1% 26.0% 31.0% 90.1% 46.9% (38.7%, 76.7%) (18.3%, 35.6%) (25.9%, 36.7%) (84.1%, 94.0%) (42.8%, 51.2%) *(95% confidence interval)
The sensitivity and PPV of the immunocytochemistry method was also compared to those obtained with conventional Pap staining. Two clinical endpoints for Pap staining (i.e., ≧LSIL and ≧HSIL) were used. Again, the standard for all calculations was the biopsy result.
TABLE-US-00044 TABLE 44 Comparison of Pap Test and Immunocytochemistry Method ≧LSIL (with ≧HSIL (with Pap test) Pap test) ≧ASCUS (with ICC) Sensitivity 76.5% 92.6% 97.7% (52.7%, 90.4%)* (76.6%, 97.9%) (92.1%, 99.4%) PPV 59.1% 26.0% 31.0% (38.7%, 76.7%) (18.3%, 35.6%) (25.9%, 36.7%) *(95% confidence interval)
The results presented in Table 42 indicate that the immunocytochemistry method detected more high-grade cervical disease samples, while maintaining a high PPV.
There were 14 false negatives in this study using the immunocytochemistry kit. HPV testing was conducted on 13 of the 14 patient samples. No remaining sample was available for one of the false negative patients.
Genomic DNA was isolated from the cervical cytology samples using the NucleoSpin® Tissue DNA Kit (BD Clontech, Cat#635967). For quality control purposes, PCR analysis of beta-globin, a housekeeping gene, was performed.
HPV L1 gene amplification was performed as described in the art by both conventional L1 PCR with MY09/11 primer set and by nested PCR with MY09/11 and GP5+/6+ primer sets to improve detection sensitivity. DNA sequencing of the L1 amplicon was further performed to identify the type(s) of HPV(s) present.
Good quality genomic DNA was isolated from 10 out of the 13 clinical cytology samples. 3 samples had poor quality genomic DNA as indicated by beta-globin PCR analysis. HPV DNA was either undetectable or negative in 10 of the 13 samples using both conventional L1 PCR (with MY09/11 primers) and nested L1 PCR (with MY09/11 and GP5+/6+ primers). This data indicates that a sampling error occurred for a majority of the false negative samples, given that HPV is positive for high-grade cervical disease (sensitivity of >92%).
MCM6 Antibody Selection
Polydomas provided in multi-well tissue culture plates were screened to identify MCM 6 biomarker-specific antibodies that possess the desired traits of sensitivity and specificity. A tissue microarray comprising multiple normal (i.e., no CIN), CINIII, squamous cell carcinoma, and adenocarcinoma samples on a single slide was generated. Undiluted supernatants from each well containing a polydoma were assayed for positive staining of the tissue microarray. Background, i.e. non-specific binding, was essentially ignored at this stage. Eleven of the 35 polydomas tested produced positive staining results and were selected for further analysis.
In order to determine the specificity of the selected polydomas, the staining patterns obtained with the polydoma supernatants were compared with those obtained with a commercially available MCM 6 antibody (BD Transduction Laboratories). The staining patterns obtained with the polydoma supernatants appeared to be more specific than those observed with the commercial MCM 6 antibody (FIG. 17).
The 11 selected polydomas were then subjected to a limiting dilution process. Thirty limiting dilutions, resulting from the supernatants of the selected polydomas, were assayed for positive staining of a tissue microarray comprising multiple normal (i.e., no CIN), CINIII, squamous cell carcinoma, and adenocarcinoma samples. Two limiting dilution clones, 9D4.3 and 9D4.4, were selected as the best supernatants based on positive staining of abnormal and cancerous cervical tissue samples. Varying dilutions of these clones were then tested for their reactivity to NIL, LSIL, HSIL tissue and pooled liquid based cytology samples. Clone 9D4.3 at a 1:100 dilution produced the maximal signal to noise ratio and was selected for further characterization.
Characterization of MCM 6, Clone 9D4.3
In order to further characterize clone 9D4.3, the clone was assayed for positive staining of 40 liquid based cytology samples selected from the following diagnostic categories: NIL (7), LSIL (10), HSIL (18), and cervical carcinoma (5). Slides were prepared using the PrepStain® slide processor (TriPath Imaging, Inc.) for each of the 40 samples. Two slides per sample were each stained with an MCM 2 antibody (Dako) and clone 9D4.3. The remaining slides were used for PAP staining or as a negative control.
To prepare slides, each sample was centrifuged for 2 minutes at 200×g to form a pellet, and the supernatant was decanted. 2 mL of deionized water was added to each sample, and the samples were vortexed and then centrifuged for 5 minutes at 600×g. After decanting the supernatant, an additional 700 μL of tris buffered water was added. Finally the samples were loaded onto the PrepStain® slide processor (Tripath Imaging, Inc.), version 1.1, and the Transfer Only program was run.
All slides were held in 95% ETOH for at least 24 hours and no more than 3 days after preparation. Antigen retrieval for MCM2 was achieved by placing the slides in a 1× Target Retrieval Solution pH 6.0 (DAKO S1699)/dH2O bath, preheated to 95° C., for 25 minutes in a steamer. For MCM6, antigen accessibility was achieved by placing the slides in a 1× Tris pH 9.5 buffer (Biocare)/dH2O bath, preheated to 95° C., for 25 minutes in a steamer. After steaming, all slides were allowed to cool at room temperature for 20 minutes.
Slides were stained by immunocytochemistry using the DAKO Universal Autostainer as described in Example 1, "Automated Immunocytochemistry." The slides were screened and evaluated by an experienced cytotechnologist for a morphological determination of diagnostic category. The samples were assessed for marker staining intensity (0-3), percentage of positive-staining cells, and the location of the marker staining (nuclear, cytoplasmic, membrane, or a combination). Intensity of cell staining was given a score of 0-3. Cells scoring ≧1.5 were counted. Mature normal-appearing squamous cells and normal-appearing glandular cells were not counted as positive when staining brown. However, squamous metaplastic cells were counted as positive along with abnormal cells. The immunocytochemistry slides were then given a designation of TN (true negative), FN (false negative), TP (true positive), or FP (false positive).
TABLE-US-00045 TABLE 45 Clone 9D4.3 (MCM6) TP FP FN TN Indet. Total NIL 0 0 0 1 0 1 Sensitivity 0.9655 LSIL 0 1 0 9 0 10 Specificity 0.9091 HSIL 23 0 1 0 0 24 PPP 0.9655 Cancer 5 0 0 0 0 5 NPP 0.9091 28 1 1 10 0 40
TABLE-US-00046 TABLE 46 MCM2 TP FP FN TN Indet. Total NIL 0 0 0 1 0 1 Sensitivity 0.9310 LSIL 0 1 0 9 0 10 Specificity 0.9091 HSIL 23 0 1 0 0 24 PPP 0.9643 Cancer 4 0 1 0 0 5 NPP 0.8333 27 1 2 10 0 40
Positive Predictive Power(PPP)=TP/(TP+FP)
Negative Predictive Power(NPP)=TN/(FN+TN)
The sensitivity and specificity for clone 9D4.3 was comparable to that obtained with the commercially available MCM2 antibody. One NIL case was negative for both antibodies. 9 of 10 LSIL cases were negative with clone 9D4.3 and the commercial MCM2 antibody. 23 of 24 HSIL cases were positive with clone 9D4.3 and the commercial MCM2 antibody. With the cervical cancer samples, 5 of 5 were positive with clone 9D4.3, and 4 of 5 were positive with the MCM 2 antibody.
Purification of MCM 6, Clone 9D4.3
Because of its sensitivity, specificity, and the presentation of a nuclear staining pattern, clone 9D4.3 was purified for further analysis. Purified antibody was obtained using Streamline rProteinA (Amersham Biosciences) affinity adsorption chromatography, in accordance with standard methods. The resulting antibody solution was then tested for reactivity against HSIL liquid-based cervical cytology pools at various dilutions between 1:500 and 1:6000. Signal was evident out to a titer of 1:6000.
Real-Time PCR Detection of Biomarkers in Clinical Tissue Samples
TaqMan® real-time PCR was performed with the ABI Prism 7700 Sequence Detection System (Applied Biosystems). The primers and probes were designed with the aid of the Primer Express® program, version 1.5 (Applied Biosystems), for specific amplification of the targeted cervical biomarkers (i.e., MCM7, p21.sup.waf1, p14ARF/p16, cyclin E1, and cyclin E2) in this study. The sequence information for primers and probes is shown below:
TABLE-US-00047 MCM7: Primer Name: MCM7_T1T3-F (SEQ ID NO:25) Sequence: CTCTGAGCCCGCCAAGC Primer Name: MCM7_T1T3-R (SEQ ID NO:26) Sequence: TGTAAGAACTTCTTAACCTTTTCCTTCTCTA Probe Name: MCM7_T1T3-Probe (SEQ ID NO:27) Sequence: CCCTCGGCAGCGATGGCACT Primer Name: MCM7_T2T4-F (SEQ ID NO:28) Sequence: GAGGAATCCCGAGCTGTGAA Primer Name: MCM7_T2T4-R (SEQ ID NO:29) Sequence: CCCGCTCCCGCCAT Probe Name: MCM7_T2T4-Probe (SEQ ID NO:30) Sequence: CCCATGTGCTTCTTTGTTTACTAAGAGCGGAA Primer Name: MCM7_T2-F (SEQ ID NO:31) Sequence: GTCCGAAGCCCCCAGAA Primer Name: MCM7_T2-R (SEQ ID NO:32) Sequence: CCCGACAGAGACCACTCACA Probe Name: MCM7_T2-Probe (SEQ ID NO:33) Sequence: CAGTACCCTGCTGAACTCATGCGCA Primer Name: MCM7_T3T4-F (SEQ ID NO:34) Sequence: CGCTACGCGAAGCTCTTTG Primer Name: MCM7_T3T4-R (SEQ ID NO:35) Sequence: CCTTTGTTTGCCATTGTTCTCTAA Probe Name: MCM7_T3T4-Probe (SEQ ID NO:36) Sequence: TGCCGTACAAGAGCTGCTGCCTCA p21.sup.waf1: Primer Name: p21T1T2-F (SEQ ID NO:37) Sequence: CAAACGCCGGCTGATCTT Primer Name: p21T1T2-R (SEQ ID NO:38) Sequence: CCAGGACTGCAGGCTTCCT Probe Name: p21T1T2-Probe (SEQ ID NO:39) Sequence: CAAGAGGAAGCCCTAATCCGCCCA Primer Name: p21T2-F (SEQ ID NO:40) Sequence: GAGCGGCGGCAGACAA Primer Name: p21T2-R (SEQ ID NO:41) Sequence: CCGCGAACACGCATCCT Probe Name: p21T2-Probe (SEQ ID NO:42) Sequence: CCCAGAGCCGAGCCAAGCGTG Primer Name: p21T3-F (SEQ ID NO:43) Sequence: TGGAGACTCTCAGGGTCGAAA Primer Name: p21T3-R (SEQ ID NO:44) Sequence: TCCAGTCTGGCCAACAGAGTT Probe Name: p21T3-Probe (SEQ ID NO:45) Sequence: CGGCGGCAGACCAGCATGAC p14ARF/p16: Primer Name: p16T4-F (SEQ ID NO:46) Sequence: GCC CTC GTG CTG ATG CTA CT Primer Name: p16T4-R (SEQ ID NO:47) Sequence: TCA TCA TGA CCT GGT CTT CTA GGA Probe Name: p16T4-Probe (SEQ ID NO:48) Sequence: AGC GTC TAG GGC AGC AGC CGC Primer Name: p16T1-F (SEQ ID NO:49) Sequence: TGCCCAACGCACCGA Primer Name: p16T1-R (SEQ ID NO:50) Sequence: GGGCGCTGCCCATCA Probe Name: p16T1-Probe (SEQ ID NO:51) Sequence: TCGGAGGCCGATCCAGGTCATG Primer Name: p16T2-F (SEQ ID NO:52) Sequence: AAGCTTCCTTTCCGTCATGC Primer Name: p16T2-R (SEQ ID NO:53) Sequence: CATGACCTGCCAGAGAGAACAG Probe Name: p16T2-Probe (SEQ ID NO:54) Sequence: CCCCCACCCTGGCTCTGACCA Primer Name: p16T3-F (SEQ ID NO:55) Sequence: GGAAACCAAGGAAGAGGAATGAG Primer Name: p16T3-R (SEQ ID NO:56) Sequence: TGTTCCCCCCTTCAGATCTTCT Probe Name: p16T3-Probe (SEQ ID NO:57) Sequence: ACGCGCGTACAGATCTCTCGAATGCT Primer Name: p16Universal-F (SEQ ID NO:58) Sequence: CACGCCCTAAGCGCACAT Primer Name: p16 Universal-R (SEQ ID NO:59) Sequence: CCTAGTTCACAAAATGCTTGTCATG Probe Name: p16 Universal-Probe (SEQ ID NO:60) Sequence: TTTCTTGCGAGCCTCGCAGCCTC Cyclin E1: Primer Name: CCNE1T1T2-F (SEQ ID NO:61) Sequence: AAAGAAGATGATGACCGGGTTTAC Primer Name: CCNE1T1T2-R (SEQ ID NO:62) Sequence: GAGCCTCTGGATGGTGCAA Probe Name: CCNE1T1T2-P (SEQ ID NO:63) Sequence: CAAACTCAACGTGCAAGCCTCGGA Primer Name: CCNE1T1-F (SEQ ID NO:64) Sequence: TCCGCCGCGGACAA Primer Name: CCNE1T1-R (SEQ ID NO:65) Sequence: CATGGTGTCCCGCTCCTT Probe Name: CCNE1T1-Probe (SEQ ID NO:66) Sequence: ACCCTGGCCTCAGGCCGGAG Cyclin E2 Primer Name: CCNE2T1T2-F (SEQ ID NO:67) Sequence: GGAATTGTTGGCCACCTGTATT Primer Name: CCNE2T1T2-R (SEQ ID NO:68) Sequence: CTGGAGAAATCACTTGTTCCTATTTCT TaqMan Probe Name: CCNE2T1T2-P (SEQ ID NO:69) Sequence: CAGTCCTTGCATTATCATTGAAACACCTCACA Primer Name: CCNE2T1T3-F (SEQ ID NO:70) Sequence: TCAACTCATTGGAATTACCTCATTATTC Primer Name: CCNE2T1T3-R (SEQ ID NO:71) Sequence: ACCATCAGTGACGTAAGCAAACTC TaqMan Probe Name: CCNE2T1T3-P (SEQ ID NO:72) Sequence: CCAAACTTGAGGAAATCTATGCTCCTAAACTCCA Primer Name: CCNE2T2-F (SEQ ID NO:73) Sequence: TTTTGAAGTTCTGCATTCTGACTTG Primer Name: CCNE2T2-R (SEQ ID NO:74) Sequence: ACCATCAGTGACGTAAGCAAGATAA TaqMan Probe Name: CCNE2T2-P (SEQ ID NO:75) Sequence: AACCACAGATGAGGTCCATACTTCTAGACTGGCT
The probes were labeled with a fluorescent dye FAM (6-carboxyfluorescein) on the 5' base, and a quenching dye TAMRA (6-carboxytetramethylrhodamine) on the 3' base. The sizes of the amplicons were around 100 bp. 18S Ribosomal RNA was utilized as an endogenous control. An 18S rRNA probe was labeled with a fluorescent dye VIC®. Pre-developed 18S rRNA primer/probe mixture was purchased from Applied Biosystems. 5 μg of total RNA extracted from normal (N) or cancerous (T) cervical tissues was quantitatively converted to the single-stranded cDNA form with random hexamers by using the High-Capacity cDNA Archive Kit (Applied Biosystems). The following reaction reagents were prepared:
TABLE-US-00048 20X Master Mix of Primers/Probe (in 200 μl) 180 μM Forward primer 20 μl 180 μM Reverse primer 20 μl 100 μM TaqMan probe 10 μl H2O 150 μl Final Reaction Mix (25 μl/well) 20X master mix of primers/probe 1.25 μl 2X TaqMan Universal PCR master mix (P/N: 4304437) 12.5 μl cDNA template 5.0 μl H2O 6.25 μl
20× TaqMan Universal PCR Master Mix was purchased from Applied Biosystems. The final primer and probe concentrations, in a total volume of 25 μl, were 0.9 μM and 0.25 μM, respectively. 10 ng of total RNA was applied to each well. The amplification conditions were 2 minutes at 50° C., 10 minutes at 95° C., and a two-step cycle of 95° C. for 15 seconds and 60° C. for 60 seconds for a total of 40 cycles. At least three no-template control reaction mixtures were included in each run. All experiments were performed in triplicate.
At the end of each reaction, the recorded fluorescence intensity is used for the following calculations: Rn.sup.+ is the Rn value of a reaction containing all components. Rn.sup.- is the Rn value of an unreacted sample (baseline value or the value detected in NTC). ΔRn is the difference between Rn.sup.+ and Rn.sup.- and is an indicator of the magnitude of the signal generated by the PCR. The comparative CT method, which uses no known amount of standard but compares the relative amount of the target sequence to any reference value chosen (e.g., 18S rRNA), was used in this study. The TaqMan® Human Endogenous Control Plate protocol was used to convert raw data for real-time PCR data analysis.
The results obtained with each biomarker and with the specific primers are listed below in tabular form. Results obtained with normal cervical tissue samples (i.e., NIL) are designated N; those obtained with cervical cancer tissues are labeled T.
TABLE-US-00049 TABLE 47 MCM7 TaqMan ® Results Sample T2 T5 T1T3 T2T4 T3T4 CV01-T 4 0.04 29.9 4.5 1.4 CV03-T 5.7 0.02 36.8 6.1 2.6 CV05-T 4.13 0.08 17.3 1.35 3.68 CV07-T 2.6 0.06 18.77 0.88 3.27 CV09-T 4.96 0.08 15.01 3.69 3.22 CV11-T 5.9 0.01 7.37 3.08 1.75 CV13-T 6.74 0.04 19.74 4.55 4.11 CV15-T 3.04 0.05 3.65 3.43 1.25 CV17-T 5.21 0.02 20.07 2.74 1.56 CV19-T 3.34 0.09 21.17 2.88 6 CV21-T 6.7 0.08 10.64 4.75 4.59 CV23-T 7.08 0.33 32.17 5.6 4.25 CV25-T 4.87 0.03 18.11 4.58 4.51 CV27-T 4.24 0.03 36.25 4.6 2.82 MEAN 4.89 0.07 20.50 3.77 3.22 MEDIAN 4.89 0.05 19.74 3.77 3.22 STD 1.32 0.07 9.46 1.39 1.32 CV02-N 2.5 0.02 10.6 2.6 1.1 CV04-N 4.6 0.02 7.1 4.8 2.4 CV06-N 1.75 0.01 2.14 1.36 2.63 CV08-N 1.35 0.01 4.8 1.71 1.54 CV10-N 5.6 0.03 5.07 5.12 1.85 CV12-N 5.68 0.02 7.34 3.19 2.29 CV16-N 4.35 0.08 3.72 2.75 1.78 CV18-N 3.98 0.01 4.74 3.63 1.7 CV20-N 2.03 0.03 5.42 1.4 2.78 CV22-N 2.66 0.02 4.33 2.26 2.42 CV24-N 4.88 0.09 9.03 1.53 2.77 CV28-N 2.71 0.01 10.38 1.36 1.7 MEAN 3.51 0.03 6.22 2.64 2.08 MEDIAN 3.51 0.02 5.42 2.60 2.08 STD 1.40 0.03 2.48 1.21 0.50
TABLE-US-00050 TABLE 48 p21.sup.waf1TaqMan ® Results Patients T1T2 T2 T3 Pt01-T 23.33 0.06 0.00 Pt02-T 14.66 0.01 0.00 Pt03-T 11.86 0.00 0.00 Pt04-T 27.04 0.01 0.00 Pt05-T 14.72 0.00 0.00 Pt06-T 22.84 0.01 0.00 Pt07-T 14.04 0.00 0.00 Pt08-T 31.93 0.01 0.01 Pt09-T 35.02 0.00 0.00 Pt10-T 13.2 0.00 0.00 Pt11-T 24.87 0.01 0.00 Pt12-T 10.85 0.00 0.00 Pt13-T 36.51 0.02 0.01 Pt14-T 12.72 0.00 0.00 Pt15-T 10.64 0.00 0.00 Pt16-T 22.58 0.04 0.00 Pt17-T 39.64 0.14 0.04 Pt01-N 4.57 0.03 0.00 Pt02-N 5.57 0.00 0.00 Pt03-N 3.54 0.00 0.00 Pt04-N 8.18 0.00 0.00 Pt05-N 5.4 0.10 0.00 Pt06-N 11.01 0.00 0.00 Pt08-N 10.39 0.00 0.00 Pt09-N 9.11 0.00 0.00 Pt10-N 4.41 0.00 0.00 Pt11-N 8.64 0.00 0.00 Pt12-N 3.03 0.00 0.00 Pt14-N 3.55 0.00 0.00 Pt15-N 2.42 0.01 0.00 Pt17-N 11.46 0.05 0.01 T-mean 21.5559 N-mean 6.52 St. T-test = 7.3E-06
TABLE-US-00051 TABLE 49 p14ARF/p16 TaqMan ® Results Patient T1 T2 T3 T4 UNIVERSAL Pt01-T 0.2 0.1 0.2 0.2 0.2 Pt02-T 16.3 11.2 5.1 21.7 36.5 Pt03-T 16.5 6.2 3.1 15.1 29.6 Pt04-T 10.1 2.8 2.6 13.2 27.7 Pt05-T 12.7 3.6 2.1 11.3 23.1 Pt01-N 0.1 0.1 0.1 0.1 0.1 Pt02-N 2.5 2.6 1.6 2.7 6.8 Pt04-N 2.6 0.6 0.8 2.4 5.8 Pt05-N 2.1 0.8 0.7 4.1 4.6 T-Mean 11.2 4.8 2.6 12.3 23.4 N-Mean 1.8 1.0 0.8 2.3 4.3
TABLE-US-00052 TABLE 50 Cyclin E1 TaqMan ® Results T1 T1T2 Cancer Cancer T1T2 Normal Cancer Cancer T1 Normal Normal Patient M. SD Normal M. SD M. SD M. SD Pt 01 12.19 0.12 4.11 0.13 1.34 0.04 0.5 0.03 Pt 02 16.72 0.21 4.44 0.34 1.35 0.02 0.47 0.05 Pt 03 11.45 0.41 2.81 0.13 1.17 0.01 0.06 0.02 Pt 04 21.33 0.45 5.33 0.09 0.76 0.1 0.23 0.01 Pt 05 11.17 0.25 3.68 0.15 0.95 0.05 0.15 0.03 Pt 06 21.65 0.24 3.11 0.22 0.89 0.03 0.13 0.02 Pt 07 23.26 0.54 0 0 0.75 0.06 0 0.01 Pt 08 8.37 0.24 3.1 0.01 0.12 0.01 0.13 0.02 Pt 09 17.74 0.43 2.17 0.08 0.73 0.02 0.09 0.01 Pt 10 18.51 0.29 4.56 0.17 1.37 0.03 0.41 0.04 Pt 11 10.58 0.52 3.92 0.12 0.57 0.01 0.23 0.03 Pt 12 33.67 0.58 7.87 0.1 0.78 0.01 0.28 0.05 Pt 13 36.9 0.41 0 0 1.05 0.04 0 0 Pt 14 31.01 0.29 6.01 0.26 1.68 0.05 0.24 0.03 Pt 15 7.35 0.23 1.24 0.09 0.34 0.08 0.08 0.02 Pt 16 12.71 0.61 3.72 0 1.1 0.06 0.07 0.01 Pt 17 12.13 0.21 11.46 0.15 0.34 0.07 0.05 0.01 Pt 18 14.22 0.14 5.94 0.06 0.73 0.08 0.26 0.04 Pt 19 12.69 0.81 3.52 0.02 0.41 0.04 0.24 0.02 Pt 20 16.56 0.16 6.1 0.12 0.17 0.02 0.06 0 Pt 21 11.63 0.23 3.01 0.06 0.54 0.04 0.23 0.01 Pt 22 17.39 0.34 2.36 0.02 0.47 0.02 0.24 0.05 Pt 23 16.56 0.16 2.1 0.02 0.18 0.03 0.09 0.01 Pt 24 22.23 0.33 4.06 0.28 1.9 0.17 0.52 0.01 Pt 25 13.98 0.48 3.72 0.05 0.54 0.04 0.23 0.01 Pt 26 22.71 0.76 4.48 0.07 0.47 0.02 0.24 0.05 Pt 27 16.17 0.4 5.64 0.3 0.18 0 0.12 0.01 Pt 28 12.6 0.56 3.8 0.06 0.29 0.03 0.05 0 Pt 29 13.69 0.34 3.1 0.18 0.29 0.03 0.11 0 Pt 30 17.69 0.61 4.3 0.11 0.36 0.01 0.03 0 Pt 31 20.46 0.3 3.91 0.21 0.47 0.03 0.08 0 Pt 32 18.38 0.18 3.16 0.06 0.42 0.02 0.17 0.01 Pt 33 21.1 0.62 4.52 0.33 1.07 0.05 0.24 0.01 Pt 34 21.5 1.37 4.56 0.13 0.24 0.01 0.11 0.01 Average 17.54 4.26 0.68 0.20 T/N 4.1 t-test 7.80E-14 P =
TABLE-US-00053 TABLE 51 Cyclin E2 TaqMan ® Results T1T2 T1T3 Std. Std. T2 Std. Patients T1T2 Dev. T1T3 Dev. T2 Dev. Pt01-T 13.17 1.02 16.11 0.39 0.01 0.00 Pt02-T 13.42 0.3 18.12 2.21 0.15 0.02 Pt03-T 13.64 0.50 17.40 2.16 0.05 0.01 Pt04-T 19.37 1.41 24.26 1.01 0.01 0.00 Pt05-T 10.59 1.1 14.71 1.58 0.17 0.02 Pt06-T 7.96 0.91 9.32 0.51 0.06 0.01 Pt07-T 14.1 1.73 16.92 0.84 0.54 0.06 Pt08-T 8.11 0.67 9.50 0.66 0.34 0.07 Pt09-T 13.04 0.72 18.27 0.99 0.02 0.00 Pt10-T 19.56 2.29 23.42 0.00 0.02 0.01 Pt11-T 16.8 1.57 18.71 2.15 0.08 0.01 Pt12-T 16.05 0.85 18.81 0.74 0.91 0.01 Pt13-T 14.91 0.87 18.51 1.59 0.61 0.16 Pt14-T 14.89 0.32 20.49 0.86 0.42 0.03 Pt15-T 12.44 0.47 15.26 1.00 0.68 0.18 Pt16-T 11.54 1.58 13.13 0.75 1.02 0.14 Pt17-T 6.78 0.47 7.91 0.45 0.85 0.10 Pt01-N 4.89 0.21 5.94 0.53 0.00 0.00 Pt02-N 6.32 0.47 8.91 0.61 0.13 0.00 Pt03-N 4.8 0.31 5.89 0.30 0.04 0.00 Pt04-N 13.28 0.74 15.28 1.37 0.01 0.00 Pt05-N 6.51 1.2 9.04 0.82 0.16 0.02 Pt06-N 4.96 0.83 6.41 0.84 0.05 0.01 Pt08-N 6.48 0.73 6.82 0.60 0.07 0.02 Pt09-N 3.74 0.48 4.63 0.66 0.03 0.01 Pt10-N 10.32 0.93 11.31 0.89 0.02 0.00 Pt11-N 10.34 0.26 13.90 0.53 0.04 0.04 Pt12-N 13.81 1.69 16.60 1.45 0.24 0.07 Pt14-N 6.92 0.63 9.07 0.95 0.14 0.03 Pt15-N 4.8 0.73 8.55 1.40 0.10 0.03 Pt17-N 5.33 0.2 5.78 0.27 0.23 0.07 T-mean 13.32 16.52 0.35 N-mean 7.32 9.15 0.09 St. 4.16E-05 3.31742E-05 0.008813 T-test
Real-Time PCR Detection of Biomarkers in Clinical Tissue Samples
TaqMan® real-time PCR was performed as described in Example 9 using cervical cancer tissue samples (e.g., adenocarcinoma, squamous cell carcinoma) and normal cervical tissue samples. The primers and probes were designed with the aid of the Primer Express® program, version 1.5 (Applied Biosystems), for specific amplification of the targeted cervical biomarkers (i.e., MCM2, MCM6, MCM7, and Topo2A) in this study. The sequence information for primers and probes is shown below:
TABLE-US-00054 TaqMan Primers MCM2: Primer Name: MCM2-F (SEQ ID NO:80) Sequence: 5'-GGAGGTGGTACTGGCCATGTA-3' Primer Name: MCM2-R (SEQ ID NO:81) Sequence: 5'-GGGAGATGCGGACATGGAT-3' TaqMan Probe Name: MCM2-P (SEQ ID NO:82) Sequence: 5'-CCAAGTACGACCGCATCACCAACCA-3' MCM6: Primer Name: MCM6-F (SEQ ID NO:83) Sequence: 5'-CATTCCAAGACCTGCCTACCA-3' Primer Name: MCM6-R (SEQ ID NO:84) Sequence: 5'-ATGCGAGTGAGCAAACCAATT-3' TaqMan Probe Name: MCM6-P (SEQ ID NO:85) Sequence: 5'-ACACAAGATTCGAGAGCTCACCTCATCCA-3' MCM7: Primer Name: MCM7_T1T3-F (SEQ ID NO:25) Sequence: CTCTGAGCCCGCCAAGC Primer Name: MCM7_T1T3-R (SEQ ID NO:26) Sequence: TGTAAGAACTTCTTAACCTTTTCCTTCTCTA Probe Name: MCM7_T1T3-Probe (SEQ ID NO:27) Sequence: CCCTCGGCAGCGATGGCACT Primer Name: MCM7_T2T4-F (SEQ ID NO:28) Sequence: GAGGAATCCCGAGCTGTGAA Primer Name: MCM7_T2T4-R (SEQ ID NO:29) Sequence: CCCGCTCCCGCCAT Probe Name: MCM7_T2T4-Probe (SEQ ID NO:30) Sequence: CCCATGTGCTTCTTTGTTTACTAAGAGCGGAA Primer Name: MCM7_T2-F (SEQ ID NO:31) Sequence: GTCCGAAGCCCCCAGAA Primer Name: MCM7_T2-R (SEQ ID NO:32) Sequence: CCCGACAGAGACCACTCACA Probe Name: MCM7_T2-Probe (SEQ ID NO:33) Sequence: CAGTACCCTGCTGAACTCATGCGCA Primer Name: MCM7_T3T4-F (SEQ ID NO:34) Sequence: CGCTACGCGAAGCTCTTTG Primer Name: MCM7_T3T4-R (SEQ ID NO:35) Sequence: CCTTTGTTTGCCATTGTTCTCTAA Probe Name: MCM7_T3T4-Probe (SEQ ID NO:36) Sequence: TGCCGTACAAGAGCTGCTGCCTCA TOPO2A: Primer Name: TOP2A_F (SEQ ID NO:86) Sequence: 5'-GGCTACATGGTGGCAAGGA-3' Primer Name: TOP2A _R (SEQ ID NO:87) Sequence: 5'-TGGAAATAACAATCGAGCCAAAG-3' TaqMan Probe Name: TOP2A _P (SEQ ID NO:88) Sequence: 5'-TGCTAGTCCACGATACATCTTTACAATGCTCAGC-3'
The results obtained for each biomarker are listed below in tabular form. The data is also summarized below.
TABLE-US-00055 TABLE 52 Snap-frozen Cervical Cancer Tissue Samples TPO HPV MCM2 MCM6 TOP2A Patient ID Path. Diag Type TaqM TaqMan MCM7 TaqM TaqM Pt 01 CV- Sq. Cell CA HPV16 8.93 11.31 29.9 23.76 001 Pt 02 CV- Adeno CA HPV18 10.94 14.29 36.8 25.28 003 Pt 03 CV- Adeno CA HPV18 17.67 13.84 17.3 23.18 005 Pt 04 CV- Sq. Cell CA HPV16 23.61 13.3 18.77 23.26 007 Pt 05 CV- Sq. Cell CA HPV16 9.3 11.26 15.01 20.33 009 Pt 06 CV- Sq. Cell CA HPV16 13.86 11.58 7.37 8.37 011 Pt 07 CV- Adeno CA HPV18 27.03 16.32 19.74 34.29 013 Pt 08 CV- Sq. Cell CA HPV16, 8.28 8.16 3.65 8.57 015 HPV18, + Pt 09 CV- Sq. Cell CA HPV18 12.61 13.56 20.07 11.31 017 Pt 10 CV- Sq Cell CA HPV18 31.88 23.38 21.17 27.48 019 Pt 11 CV- Sq. Cell CA HPV16 11.27 14.76 10.64 12.73 021 Pt 12 CV- Sq. Cell CA HPV16 11.39 11.29 32.17 21.11 023 Pt 13 CV- Sq. Cell CA HPV16 23.88 18.98 18.11 27.96 025 Pt 14 CV- Sq. Cell CA HPV18, 12.26 15.53 36.25 26.63 027 HPV16, + Pt 15 CV- Sq Cell HPV16 6.56 7.92 9.64 7.81 029 Carcinoma Pt 16 CV- Sq Cell HPV73 28.12 12.21 27.3 21.4 031 Carcinoma Pt 17 CV- Sq Cell HPV16 8.76 7.59 14.37 12.42 033 Carcinoma Pt 18 CV- Sq Cell HPV16 21.4 12.65 23.63 27.57 035 Carcinoma Pt 19 CV- Sq Cell HPV18 12.59 13.06 14.37 9.24 037 Carcinoma Pt 20 CV- Adenosqu. HPV16, 7.24 8.17 16.97 15.13 039 Cell CA HPV18, + Pt 21 CV- Sq Cell CA HPV16 9.61 11.84 13.88 11.92 041 Pt 22 CV- Sq Cell CA HPV16 21.57 13.21 18.31 24.19 043 Pt 23 CV- Sq Cell CA HPV16 21.19 13.18 18.76 19.97 045 Pt 24 CV- Sq Cell CA HPV18 24.61 19.09 20.19 28.14 047 Pt 25 CV- Sq Cell CA HPV18 11.43 10.2 13.70 10.55 049 Pt 26 CV- Sq Cell CA HPV16 24.25 20.54 23.26 33.26 051 Pt 27 CV- Sq Cell CA HPV45 26.74 21.34 20.96 20.34 053 Pt 28 CV- Sq Cell CA HPV16, 12.65 12 14.42 12.17 055 HPV18, + Pt 29 CV- Sq Cell CA HPV16 16 14.72 25.46 22.16 057 Pt 30 CV- Sq Cell CA HPV16, 22.55 17.87 15.30 25.54 059 HPV18, + Pt 31 CV- Sq Cell CA HPV16 24.08 21.88 23.11 25.28 061 Pt 32 CV- Sq Cell CA HPV18, 24.16 12.55 21.63 22.39 063 HPV16, + Pt 33 CV- Sq Cell CA HPV16 26.63 16.05 27.56 28.84 065 Pt 34 CV- Sq Cell CA HPV16 19.61 23.28 19.03 25.57 067
TABLE-US-00056 TABLE 53 Adjacent Normal Tissue Samples TPO HPV MCM2 MCM6 MCM7 TOP2A Patient ID Type TaqM TaqMan TaqM TaqM Pt 01 CV- Negative 3.04 4.4 10.6 10.52 002 Pt 02 CV- Negative 6.26 6.28 7.1 9.06 004 Pt 03 CV- HPV18 2.06 2.53 2.14 3.86 006 Pt 04 CV- Negative 3.14 4.15 4.8 8.03 008 Pt 05 CV- Negative 2.2 3.45 5.07 6.91 010 Pt 06 CV- Negative 2.06 2.29 7.34 6.82 012 Pt 07 CV- Negative N/A N/A N/A N/A 014 Pt 08 CV- Negative 2.55 3.13 3.72 2.02 016 Pt 09 CV- Negative 2.09 3.09 4.74 1.24 018 Pt 10 CV- Negative 8.15 6.76 5.42 10.41 020 Pt 11 CV- Negative 4.53 5.34 4.33 6.64 022 Pt 12 CV- Negative 1.94 2.45 9.03 6.13 024 Pt 13 CV- Negative N/A N/A N/A N/A 026 Pt 14 CV- Negative 2.62 2.95 10.38 5.3 028 Pt 15 CV- Negative 1.14 1.28 2.06 1.54 030 Pt 16 CV- Negative N/A N/A N/A N/A 032 Pt 17 CV- Negative 1.24 1.91 1.32 0.42 034 Pt 18 CV- Negative 3.4 1.89 4.01 4.32 036 Pt 19 CV- Negative 3.48 4.98 5.60 7.92 038 Pt 20 CV- Negative 1.84 3.28 3.73 1.38 040 Pt 21 CV- Negative 1.53 3.3 4.77 1.01 042 Pt 22 CV- Negative 2.65 4.03 2.74 2.59 044 Pt 23 CV- Negative 3.09 3.53 5.90 3.42 046 Pt 24 CV- HPV18 2.57 5.19 3.82 5.32 048 Pt 25 CV- Negative 5.84 4.64 7.78 9.14 050 Pt 26 CV- Negative 5.11 5.22 5.37 5.13 052 Pt 27 CV- Negative 2.91 3.29 5.10 0.76 054 Pt 28 CV- Negative 4.14 3.74 5.54 4.15 056 Pt 29 CV- HPV16 2.83 4.98 10.13 7.57 058 Pt 30 CV- Negative 6.41 5 5.39 10.05 060 Pt 31 CV- Negative 5.72 4.93 9.29 9.95 062 Pt 32 CV- Negative 8.06 5.41 7.64 9 064 Pt 33 CV- Negative 9.93 7.94 10.78 9.95 066 Pt 34 CV- Negative 2.36 6.39 5.73 1.81 068
Summary of Results
TABLE-US-00057 TABLE 54 Tumor vs adjacent normal Marker Tumor (M ± SD) Normal (M ± SD) R P MCM2 17.43 ± 7.34 3.71 ± 2.21 4.70 <0.0001 MCM6 14.32 ± 4.32 4.12 ± 1.56 3.48 <0.0001 MCM7 19.38 ± 6.94 5.85 ± 2.59 3.31 <0.0001 TOP2A 20.53 ± 7.54 5.56 ± 3.33 3.69 <0.0001 M: Mean; SD: Standard Deviation; R: Ratio of the means of tumor versus normal; P: P value of t-test.
TABLE-US-00058 TABLE 55 HPV-16 vs HPV-18 Marker Tumor HPV type Cases Tumor (M ± SD) Normal (M ± SD) MCM2 16 18 16.77 ± 6.78 3.29 ± 2.13 18 8 17.23 ± 8.16 3.99 ± 2.40 16 + 18 6 14.52 ± 7.18 4.27 ± 2.47 MCM6 16 18 14.19 ± 4.44 3.97 ± 1.75 18 8 14.24 ± 4.10 4.35 ± 1.54 16 + 18 6 12.38 ± 3.89 3.92 ± 1.04 MCM7 16 18 19.39 ± 6.94 6.07 ± 2.98 18 8 17.23 ± 4.16 5.07 ± 1.91 16 + 18 6 18.04 ± 7.71 6.07 ± 2.56 TOP2A 16 18 20.92 ± 7.38 5.46 ± 3.26 18 8 19.78 ± 9.52 6.19 ± 3.33 16 + 18 6 18.41 ± 7.49 5.32 ± 3.57
TABLE-US-00059 TABLE 56 Squamous Cell Carcinoma vs Adenocarcinoma Marker Histopathology Cases Tumor (M ± SD) Normal (M ± SD) MCM2 SCC 30 17.66 ± 7.28 3.74 ± 2.23 AC 4 15.72 ± 8.69 3.39 ± 2.49 MCM6 SCC 30 14.48 ± 4.44 4.13 ± 1.55 AC 4 13.16 ± 3.49 4.03 ± 1.98 MCM7 SCC 30 19.27 ± 7.25 6.01 ± 2.58 AC 4 20.20 ± 4.57 4.32 ± 2.53 TOP2A SCC 30 20.01 ± 7.47 5.65 ± 3.34 AC 4 21.47 ± 7.87 4.77 ± 3.92 SCC: Squamous Cell Carcinoma; AC: Adenocarcinoma.
Real-Time PCR Detection of Biomarkers in Cervical and Breast Cancer Cell Lines
TaqMan® real-time PCR was performed to detect MCM2, MCM6 and MCM7 expression levels in cervical and breast cancer cell lines.
Experimental Design and Protocols
Three human cervical cancer cell lines of SiHa, Caski and HeLa and three human breast cancer cell lines of MCF-7, SK-BR3 and CAMA were purchased from ATCC and used in this experiment. Total cellular RNA was extracted from freshly cultured cells by RNeasy® Protect Mini kit (Qiagen, Valencia, Calif.) and converted into the single stranded cDNA form with random hexamers using the High-Capacity cDNA Archive Kit (Applied Biosystems, P/N: 4322171). Real-time PCR was performed on the ABI Prism® 7700 Sequence Detection System using TaqMan® Universal PCR Master Mix (Applied Biosystems, Inc., Foster City, Calif.).
The primers and probes for specific amplification of MCM2, MCM6 and MCM7 were designed with ABI Primer Express® program, v1.5. MCM7 contains four transcriptional variants: transcript variant 1 (T1, refseq NM--005916) and transcript variant 2 (T2, refseq NM--182776) were identified in NCBI Entrez nucleotide database. Variant T3 and T4 have alternate exons near the 5'-end as analyzed by EST assembly through NCBI's Model Maker. Primers and probes were designed as T1T3, T2T4, T2 and T3T4 specifically for detecting variants T1 and T3, T2 and T4, T2, and T3 and T4, respectively. The sequences of primers and probes are shown above in Example 10 and 11.
The probes were labeled with a fluorescent dye FAM (6-carboxyfluorescein) on the 5' base, and a quenching dye TAMRA (6-carboxytetramethylrhodamine) on the 3' base. 18S ribosomal RNA was utilized as endogenous control. 18S rRNA probe was labeled with a fluorescent dye VIC. Pre-developed 18S rRNA primer/probe mixture was purchased from Applied Biosystems. 10 ng of cDNA were applied to the reaction mixture containing 0.9 μM and 0.25 μM of the primers and probes, respectively, in a total volume of 25 μl. The amplification conditions were: 2 minutes at 50° C., 10 minutes at 95° C., and a two-steps cycle of 95° C. for 15 seconds and 60° C. for 60 seconds, for a total of 40 cycles. At least three no-template control reaction mixtures were included in each run. All experiments were performed in duplicate. The relative quantification method was employed to calculate the expression levels of target genes relative to the 18S endogenous control, based on their CT values following the ABI's user manual (P/N 4303859).
The results obtained for each biomarker are listed below in tabular form.
TABLE-US-00060 TABLE 57 Biomarker Expression Cervical and Breast Cancer Cell Lines SiHa Caski HeLa MCF7 SK-BR3 CAMA MCM2 21.4 5.01 8.79 18.84 7.65 17.32 MCM6 12.34 5.77 6.46 12.6 5.44 13.14 MCM7 20.53 17.27 8.31 26.91 30.38 25.36
The cervical HeLa cell line was shown to have low-expression levels of MCM2, MCM6 and MCM7 biomarkers. The cervical SiHa, breast MCM7, and CAMA cell lines all showed overexpression of MCM2, MCM6 and MCM7 biomarkers. Cervical Caski and breast SK-BR3 cell lines showed overexpression of MCM7, but low-expression for MCM2 and MCM6.
Induction of Cervical Biomarker Expression in 293 Cells by Transient HPV16 E6/E7 Gene Transfection
TaqMan® real-time PCR assay was used to investigate the linkage of cervical biomarker expression with high-risk HPV oncogene transcription in an HEK 293 cell line system.
Experimental Design and Protocols
A tetracycline regulated expression system (T-Rex system, Invitrogen, Inc) was adapted in this experiment. T-Rex vectors expressing HPV16 E2, E6 or E7 protein were constructed. Vectors containing mutant E2, E6 or E7 genes were utilized as negative controls. T-Rex 293 cells were then transfected with the HPV plasmids, and expression of HPV genes were activated by tetracycline for 4 hours, 24 hours and 72 hours. Total cellular RNA was extracted from the transfected cells by RNeasy® Protect Mini kit (Qiagen, Valencia, Calif.) and converted into the single stranded cDNA form with random hexamers using the High-Capacity cDNA Archive Kit (Applied Biosystems, P/N: 4322171). Real-time PCR was performed on the ABI Prism® 7700 Sequence Detection System using TaqMan® Universal PCR Master Mix (Applied Biosystems, Inc., Foster City, Calif.).
The primers and probes for specific amplification of MCM2, MCM6, MCM7, TOP2A, Cyclin E1, p21, p14, HPV16 E2, E6 and E7 were designed with ABI Primer Express® program, v1.5. MCM7 contains four transcriptional variants: transcript variant 1 (T1, refseq NM--005916) and transcript variant 2 (T2, refseq NM--182776) were identified in NCBI Entrez nucleotide database. Variant T3 and T4 have alternate exons near the 5'-end as analyzed by EST assembly through NCBI's Model Maker. Primers and probes were designed as T1T3, T2T4, T2 and T3T4 specifically for detecting variants T1 and T3, T2 and T4, T2, and T3 and T4, respectively. The sequences of primers and probes are shown as shown in Examples 10 and 11.
The probes were labeled with a fluorescent dye FAM (6-carboxyfluorescein) on the 5' base, and a quenching dye TAMRA (6-carboxytetramethylrhodamine) on the 3' base. 18S ribosomal RNA was utilized as endogenous control. 18S rRNA probe was labeled with a fluorescent dye VIC. Pre-developed 18S rRNA primer/probe mixture was purchased from Applied Biosystems. 10 ng of cDNA were applied to the reaction mixture containing 0.9 μM and 0.25 μM of the primers and probes, respectively, in a total volume of 25 μl. The amplification conditions were: 2 minutes at 50° C., 10 minutes at 95° C., and a two-steps cycle of 95° C. for 15 seconds and 60° C. for 60 seconds, for a total of 40 cycles. At least three no-template control reaction mixtures were included in each run. All experiments were performed in duplicate. The relative quantification method was employed to calculate the expression levels of target genes relative to the 18S endogenous control, based on their CT values following the ABI's user manual (P/N 4303859).
Expression of HPV16 E2, E6 and E7 genes in T-Rex 293 cells was observed to increase through the time-course of transfection. mRNA expression of Topo2A, MCM2, MCM6, MCM7 and cyclin E in T-Rex 293 cells was significantly induced by HPV16 E6 or E7 genes, post-transfection from 4 hours up to 72 hours. However, there were no elevated expression levels detected for p21 and p14 post HPV gene transfection. Expression of E6 or E7 did not appear to be repressed by co-transfection of E2 gene. This is because the expression of E6 or E7 was purely driven by the external CMV promoter instead of the natural HPV promoters. The latter are not present in this model system.
TABLE-US-00061 TABLE 58 Topo2A 0 h 24 h 72 h Transfection 0 h SD 4 h 4 h SD 24 h SD 72 h SD 293-H16E2 6.91 0.07 5.22 0.13 5.68 0.14 6.61 0.36 293-H16E6 6.91 0.07 11.31 0.22 18.13 0.89 17.39 0.85 293-H16E7 6.91 0.07 20.33 0.9 28.94 0.71 35.02 1.03 293- 6.91 0.07 6.43 0.35 8.18 0.64 7.39 0.18 H16dE7 293-LacZ 6.91 0.07 7.4 0.07 7.36 0.22 7.25 0.67
TABLE-US-00062 TABLE 59 MCM2 0 h 24 h 72 h Transfection 0 h SD 4 h 4 h SD 24 h SD 72 h SD 293-H16E2 4.79 0.23 5.25 0.36 5.24 0.31 4.44 0.3 293-H16E6 4.79 0.23 6.04 0.21 9.38 0.37 12.08 0.18 293-H16E7 4.79 0.23 10.81 0.16 12.29 0.36 16.34 0.8 293- 4.79 0.23 5.72 0.36 4.98 0.27 5.03 0.39 H16dE7 293-LacZ 4.79 0.23 5.67 0.61 5.68 0.47 5.98 0.79
TABLE-US-00063 TABLE 60 MCM6 4 h 24 h 72 h Transfection 0 h 0 h SD 4 h SD 24 h SD 72 h SD 293-H16E2 3.62 0.2 3.5 0.22 4.72 0 4.44 0.26 293-H16E6 3.62 0.2 4.74 0.07 9.03 0.04 9.68 0.43 293-H16E7 3.62 0.2 7.7 0.04 13.5 0.33 14.03 0.41 293- 3.62 0.2 5.23 0.28 4.6 0.32 4.73 0.37 H16dE7 293-LacZ 3.62 0.2 4.77 0.12 4.66 0.14 5.34 0.39
TABLE-US-00064 TABLE 61 MCM7 24 h 72 h Transfection 0 h 0 h SD 4 h 4 h SD 24 h SD 72 h SD 293-H16E2 4.2 0.04 6.3 0.28 5.3 0.18 5.8 0.31 293-H16E6 4.2 0.04 4.99 0.05 9.55 0.23 15.24 0.3 293-H16E7 4.2 0.04 10.11 0.84 14.23 0.84 21.18 0.31 293- 4.2 0.04 3.65 0.3 6.06 0.3 4.64 0.07 H16dE7 293-LacZ 4.2 0.04 5.74 0.45 5.31 0.55 5.66 0.17
TABLE-US-00065 TABLE 62 Cyclin E1 0 h 24 h 72 h Transfection 0 h SD 4 h 4 h SD 24 h SD 72 h SD 293-H16E2 6.02 0.00 5.06 0.10 5.03 0.35 5.72 0.31 293-H16E6 6.02 0.00 9.19 0.18 8.95 0.79 9.38 0.18 293-H16E7 6.02 0.00 12.91 0.38 17.63 0.17 17.32 0.25 293- 6.02 0.00 5.45 0.24 6.87 0.20 5.11 0.08 H16dE7 293-LacZ 6.02 0.00 5.72 0.31 6.28 0.37 5.65 0.64
TABLE-US-00066 TABLE 63 p21 0 h 24 h 72 h Transfection 0 h SD 4 h 4 h SD 24 h SD 72 h SD 293-H16E2 4.76 0.19 4.05 0.30 5.19 0.61 4.92 0.60 293-H16E6 4.76 0.19 5.56 0.19 5.60 0.08 7.21 0.07 293-H16E7 4.76 0.19 7.52 0.29 5.22 0.13 6.45 0.13 293- 4.76 0.19 4.38 0.26 5.60 0.66 5.10 0.05 H16dE7 293-LacZ 4.76 0.19 3.86 0.00 4.53 0.27 5.37 0.29
TABLE-US-00067 TABLE 64 p14 0 h 24 h 72 h Transfection 0 h SD 4 h 4 h SD 24 h SD 72 h SD 293-H16E2 4.78 0.30 4.44 0.09 5.04 0.44 5.04 0.07 293-H16E6 4.78 0.30 4.77 0.12 5.48 0.13 4.52 0.11 293-H16E7 4.78 0.30 6.38 0.62 5.60 0.25 6.43 0.35 293- 4.78 0.30 5.08 0.12 5.53 0.35 5.10 0.15 H16dE7 293-LacZ 4.78 0.30 4.54 0.40 4.68 0.16 5.76 0.25
TABLE-US-00068 TABLE 65 HPV16 E2 E2 E6 E7 dE2 dE6 dE7 E2 + E6 E2 + E7 dE2 + E6 dE2 + E7 LacZ Mock 4 h 130.22 0 0 110.7 0 0 95.34 36.6 3.94 12.86 0 0 24 h 162.12 0 0 111.41 0 0 118.17 90.19 19.77 7.7 0 0 72 h 251.55 0 0 141.57 0 0 162.54 128.41 32.94 9.89 0 0
TABLE-US-00069 TABLE 66 HPV16 E6 E2 E6 E7 dE2 dE6 dE7 E2 + E6 E2 + E7 dE2 + E6 dE2 + E7 LacZ Mock 4 h 0 205 0 0 219.87 0 128.41 0 199.65 0 0 0 24 h 0 329.67 0 0 225.96 0 158.31 0 188.03 0 0 0 72 h 0 757.26 0 0 315.22 0 392 0 271.55 0 0 0
TABLE-US-00070 TABLE 67 HPV16 E7 E2 E6 E7 dE2 dE6 dE7 E2 + E6 E2 + E7 dE2 + E6 dE2 + E7 LacZ Mock 4 h 0 0 330.76 0 0 165.48 0 120.65 0 201.19 0 0 24 h 0 0 1514.6 0 0 239.63 0 857.89 0 600.57 0 0 72 h 0 0 2806.8 0 0 355.9 0 1444.25 0 809.11 0 0
Increasing Antigen Accessibility in Immunocytochemistry and Immunohistochemistry Methods Using a Slide Pretreatment Buffer
Specimen Selection and Reagent Description
Paired cytology and histology specimens, from the same patient, were subjected to immunoassays to detect biomarker overexpression. Paraffin block tissue samples and SurePath® cytology specimens from patients categorized as ASCUS (3), LSIL (6), and HSIL (5) were analyzed. The reagents used were the Antibody Cocktail (for cytology), the Modified Antibody Cocktail (for histology), Detection Reagents, Counterstains, and SureSlide® Preparation Buffer 10× (pretreatment buffer).
Cytology Slide Preparation and Automated Immunocytochemistry
For immunocytochemistry, slide preparation and pretreatment was conducted as indicated in Example 5. Automated immunocytochemistry was then performed on each cytology specimen as described in Example 5 with one exception. The primary antibody cocktail (MCM2 Clone 26H6.19 1:10,000, MCM2 Clone 27C5.6 1:800, TOPOIIA Clone SWT3D1 1:1000) incubation was reduced to 30 minutes for this experiment.
Histology Slide Preparation and Automated Immunohistochemistry
For each case, 4 micron sections were cut and dried overnight or for 20 minutes in a 70° C. forced air oven. Sections were deparaffinized in 3 changes of xylene for 5 minutes each. Slides were then cleared in absolute alcohol for 5 minutes each. Slides were brought down to water and rinsed thoroughly. Slides were transferred to a preheated solution of 1× SureSlide Preparation Buffer and incubated in the steamer for 25 minutes. The slides were removed from the steamer and allowed to cool at room temperature for 20 minutes. Slides were slowly rinsed in water until the buffer was completely exchanged. A TBST rinse was applied for 2 changes at 2 minutes each.
Automated immunohistochemistry was conducted as described in Example 5 for immunocytochemistry, with two exceptions. The primary antibody cocktail incubation was reduced to 30 minutes for this experiment. Additionally, the primary antibody cocktail was modified with the following dilutions (MCM2 Clone 26H6.19 1:4,000, MCM2 Clone 27C5.6 1:200, TOPOIIA Clone SWT3D1 1:400).
The anticipated staining patterns were observed on both the histology and cytology specimens with the use of the RUO reagents. Specifically, the ability to immunostain both histology and cytology specimens with the SureSlide® Preparation Buffer, Detection Reagents and the Counterstain Reagents was successfully demonstrated.
TABLE-US-00071 TABLE 68 Biomarker Nucleotide and Amino Acid Sequence Information Nucleotide Sequence Amino Acid Sequence Accession Sequence Accession Sequence Biomarker Name No. Identifier No. Identifier Cyclin E1 (Isoform 1) NM_001238 SEQ ID NO: 1 NP_001229 SEQ ID NO: 2 Cyclin E1 (Isoform 2) NM_057182 SEQ ID NO: 3 NP_476530 SEQ ID NO: 4 Cyclin E2 (Isoform 1) NM_057749) SEQ ID NO: 5 NP_477097 SEQ ID NO: 6 Cyclin E2 (Isoform 2) NM_057735 SEQ ID NO: 7 NP_477083 SEQ ID NO: 8 Cyclin E2 (Isoform 3) NM_004702 SEQ ID NO: 9 NP_004693 SEQ ID NO: 10 MCM2 NM_004526 SEQ ID NO: 11 NP_0045417 SEQ ID NO: 12 MCM6 NM_005915 SEQ ID NO: 89 NP_005906 SEQ ID NO: 90 MCM7 (Isoform 1) NM_005916 SEQ ID NO: 13 NP_005907 SEQ ID NO: 14 MCM7 (Isoform 2) NM_182776 SEQ ID NO: 15 NP_877577 SEQ ID NO: 16 p21/waf1 (Variant 1) NM_000389 SEQ ID NO: 17 NP_000380 SEQ ID NO: 18 p21/waf1 (Variant 2) NM_078467 SEQ ID NO: 19 NP_510867 SEQ ID NO: 20 p14ARF NM_058195 SEQ ID NO: 21 NP_478102 SEQ ID NO: 22 Topo2a NM_001067 SEQ ID NO: 23 NP_0010568 SEQ ID NO: 24
In light of the above description and examples, one skilled in the art will appreciate that the methods of the invention permit superior detection of high-grade cervical disease, independent of age, in comparison to conventional practice. The methods of the invention may find particular use as described below: For women over the age of thirty, the test may be a reflex from either an HPV positive result or as a reflex from an ASCUS+ cytology result. For women under the age of 30, the test may be used in combination with cytology for the detection of high-grade cervical disease. For women over the age of 30, the test may be used in combination with cytology for the detection of high-grade cervical disease. For women under the age of 30, the test may be used as a primary screen to detect high-grade cervical disease. For women over the age of 30, the test may be used as a primary screen to detect high-grade cervical disease. The test may be a replacement for the Pap smear in women under the age of thirty. Ultimately, the test may be a replacement for the Pap smear, independent of age.
Other potential advantages stemming from the practice of the present invention include: Detection of histologic high-grade abnormality in women 30 years old and above with NIL/HPV positive results. Superior specificity for the detection of high-grade cervical disease in women over the age of 30 who are positive to the DNA+Pap test. Superior detection for high-grade cervical disease in women within the ASC-US, ASC-H, and LSIL categories, independent of age. Superior specificity for the detection of high-grade cervical within HSIL category. Detection of high-grade cervical disease in conjunction with cytology-based diagnosis in women under the age of 30. Detection of high-grade cervical disease in conjunction with cytology-based diagnosis, independent of age. Improved specificity for the detection of high-grade cervical disease as a primary screen in women under the age of 30. Improved specificity for the detection of high-grade cervical disease as a primary screen, independent of age. Identification of cervical disease and differentiation of HPV infection and high-grade cervical disease. Acceptable assay performance can be established using manual interpretation or assisted interpretation via automated microscopy.
All publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended embodiments.
9011958DNAHomo sapiens 1agcagccggc gcggccgcca gcgcggtgta gggggcaggc gcggatcccg ccaccgccgc 60gcgctcggcc cgccgactcc cggcgccgcc gccgccactg ccgtcgccgc cgccgcctgc 120cgggactgga gcgcgccgtc cgccgcggac aagaccctgg cctcaggccg gagcagcccc 180atcatgccga gggagcgcag ggagcgggat gcgaaggagc gggacaccat gaaggaggac 240ggcggcgcgg agttctcggc tcgctccagg aagaggaagg caaacgtgac cgtttttttg 300caggatccag atgaagaaat ggccaaaatc gacaggacgg cgagggacca gtgtgggagc 360cagccttggg acaataatgc agtctgtgca gacccctgct ccctgatccc cacacctgac 420aaagaagatg atgaccgggt ttacccaaac tcaacgtgca agcctcggat tattgcacca 480tccagaggct ccccgctgcc tgtactgagc tgggcaaata gagaggaagt ctggaaaatc 540atgttaaaca aggaaaagac atacttaagg gatcagcact ttcttgagca acaccctctt 600ctgcagccaa aaatgcgagc aattcttctg gattggttaa tggaggtgtg tgaagtctat 660aaacttcaca gggagacctt ttacttggca caagatttct ttgaccggta tatggcgaca 720caagaaaatg ttgtaaaaac tcttttacag cttattggga tttcatcttt atttattgca 780gccaaacttg aggaaatcta tcctccaaag ttgcaccagt ttgcgtatgt gacagatgga 840gcttgttcag gagatgaaat tctcaccatg gaattaatga ttatgaaggc ccttaagtgg 900cgtttaagtc ccctgactat tgtgtcctgg ctgaatgtat acatgcaggt tgcatatcta 960aatgacttac atgaagtgct actgccgcag tatccccagc aaatctttat acagattgca 1020gagctgttgg atctctgtgt cctggatgtt gactgccttg aatttcctta tggtatactt 1080gctgcttcgg ccttgtatca tttctcgtca tctgaattga tgcaaaaggt ttcagggtat 1140cagtggtgcg acatagagaa ctgtgtcaag tggatggttc catttgccat ggttataagg 1200gagacgggga gctcaaaact gaagcacttc aggggcgtcg ctgatgaaga tgcacacaac 1260atacagaccc acagagacag cttggatttg ctggacaaag cccgagcaaa gaaagccatg 1320ttgtctgaac aaaatagggc ttctcctctc cccagtgggc tcctcacccc gccacagagc 1380ggtaagaagc agagcagcgg gccggaaatg gcgtgaccac cccatccttc tccaccaaag 1440acagttgcgc gcctgctcca cgttctcttc tgtctgttgc agcggaggcg tgcgtttgct 1500tttacagata tctgaatgga agagtgtttc ttccacaaca gaagtatttc tgtggatggc 1560atcaaacagg gcaaagtgtt ttttattgaa tgcttatagg ttttttttaa ataagtgggt 1620caagtacacc agccacctcc agacaccagt gcgtgctccc gatgctgcta tggaaggtgc 1680tacttgacct aagggactcc cacaacaaca aaagcttgaa gctgtggagg gccacggtgg 1740cgtggctctc ctcgcaggtg ttctgggctc cgttgtacca agtggagcag gtggttgcgg 1800gcaagcgttg tgcagagccc atagccagct gggcaggggg ctgccctctc cacattatca 1860gttgacagtg tacaatgcct ttgatgaact gttttgtaag tgctgctata tctatccatt 1920ttttaataaa gataatactg tttttgagac aaaaaaaa 19582410PRTHomo sapiens 2Met Pro Arg Glu Arg Arg Glu Arg Asp Ala Lys Glu Arg Asp Thr Met1 5 10 15Lys Glu Asp Gly Gly Ala Glu Phe Ser Ala Arg Ser Arg Lys Arg Lys20 25 30Ala Asn Val Thr Val Phe Leu Gln Asp Pro Asp Glu Glu Met Ala Lys35 40 45Ile Asp Arg Thr Ala Arg Asp Gln Cys Gly Ser Gln Pro Trp Asp Asn50 55 60Asn Ala Val Cys Ala Asp Pro Cys Ser Leu Ile Pro Thr Pro Asp Lys65 70 75 80Glu Asp Asp Asp Arg Val Tyr Pro Asn Ser Thr Cys Lys Pro Arg Ile85 90 95Ile Ala Pro Ser Arg Gly Ser Pro Leu Pro Val Leu Ser Trp Ala Asn100 105 110Arg Glu Glu Val Trp Lys Ile Met Leu Asn Lys Glu Lys Thr Tyr Leu115 120 125Arg Asp Gln His Phe Leu Glu Gln His Pro Leu Leu Gln Pro Lys Met130 135 140Arg Ala Ile Leu Leu Asp Trp Leu Met Glu Val Cys Glu Val Tyr Lys145 150 155 160Leu His Arg Glu Thr Phe Tyr Leu Ala Gln Asp Phe Phe Asp Arg Tyr165 170 175Met Ala Thr Gln Glu Asn Val Val Lys Thr Leu Leu Gln Leu Ile Gly180 185 190Ile Ser Ser Leu Phe Ile Ala Ala Lys Leu Glu Glu Ile Tyr Pro Pro195 200 205Lys Leu His Gln Phe Ala Tyr Val Thr Asp Gly Ala Cys Ser Gly Asp210 215 220Glu Ile Leu Thr Met Glu Leu Met Ile Met Lys Ala Leu Lys Trp Arg225 230 235 240Leu Ser Pro Leu Thr Ile Val Ser Trp Leu Asn Val Tyr Met Gln Val245 250 255Ala Tyr Leu Asn Asp Leu His Glu Val Leu Leu Pro Gln Tyr Pro Gln260 265 270Gln Ile Phe Ile Gln Ile Ala Glu Leu Leu Asp Leu Cys Val Leu Asp275 280 285Val Asp Cys Leu Glu Phe Pro Tyr Gly Ile Leu Ala Ala Ser Ala Leu290 295 300Tyr His Phe Ser Ser Ser Glu Leu Met Gln Lys Val Ser Gly Tyr Gln305 310 315 320Trp Cys Asp Ile Glu Asn Cys Val Lys Trp Met Val Pro Phe Ala Met325 330 335Val Ile Arg Glu Thr Gly Ser Ser Lys Leu Lys His Phe Arg Gly Val340 345 350Ala Asp Glu Asp Ala His Asn Ile Gln Thr His Arg Asp Ser Leu Asp355 360 365Leu Leu Asp Lys Ala Arg Ala Lys Lys Ala Met Leu Ser Glu Gln Asn370 375 380Arg Ala Ser Pro Leu Pro Ser Gly Leu Leu Thr Pro Pro Gln Ser Gly385 390 395 400Lys Lys Gln Ser Ser Gly Pro Glu Met Ala405 41031787DNAHomo sapiens 3gtgctcaccc ggcccggtgc cacccgggtc cacagggatg cgaaggagcg ggacaccatg 60aaggaggacg gcggcgcgga gttctcggct cgctccagga agaggaaggc aaacgtgacc 120gtttttttgc aggatccaga tgaagaaatg gccaaaatcg acaggacggc gagggaccag 180tgtgggagcc agccttggga caataatgca gtctgtgcag acccctgctc cctgatcccc 240acacctgaca aagaagatga tgaccgggtt tacccaaact caacgtgcaa gcctcggatt 300attgcaccat ccagaggctc cccgctgcct gtactgagct gggcaaatag agaggaagtc 360tggaaaatca tgttaaacaa ggaaaagaca tacttaaggg atcagcactt tcttgagcaa 420caccctcttc tgcagccaaa aatgcgagca attcttctgg attggttaat ggaggtgtgt 480gaagtctata aacttcacag ggagaccttt tacttggcac aagatttctt tgaccggtat 540atggcgacac aagaaaatgt tgtaaaaact cttttacagc ttattgggat ttcatcttta 600tttattgcag ccaaacttga ggaaatctat cctccaaagt tgcaccagtt tgcgtatgtg 660acagatggag cttgttcagg agatgaaatt ctcaccatgg aattaatgat tatgaaggcc 720cttaagtggc gtttaagtcc cctgactatt gtgtcctggc tgaatgtata catgcaggtt 780gcatatctaa atgacttaca tgaagtgcta ctgccgcagt atccccagca aatctttata 840cagattgcag agctgttgga tctctgtgtc ctggatgttg actgccttga atttccttat 900ggtatacttg ctgcttcggc cttgtatcat ttctcgtcat ctgaattgat gcaaaaggtt 960tcagggtatc agtggtgcga catagagaac tgtgtcaagt ggatggttcc atttgccatg 1020gttataaggg agacggggag ctcaaaactg aagcacttca ggggcgtcgc tgatgaagat 1080gcacacaaca tacagaccca cagagacagc ttggatttgc tggacaaagc ccgagcaaag 1140aaagccatgt tgtctgaaca aaatagggct tctcctctcc ccagtgggct cctcaccccg 1200ccacagagcg gtaagaagca gagcagcggg ccggaaatgg cgtgaccacc ccatccttct 1260ccaccaaaga cagttgcgcg cctgctccac gttctcttct gtctgttgca gcggaggcgt 1320gcgtttgctt ttacagatat ctgaatggaa gagtgtttct tccacaacag aagtatttct 1380gtggatggca tcaaacaggg caaagtgttt tttattgaat gcttataggt tttttttaaa 1440taagtgggtc aagtacacca gccacctcca gacaccagtg cgtgctcccg atgctgctat 1500ggaaggtgct acttgaccta agggactccc acaacaacaa aagcttgaag ctgtggaggg 1560ccacggtggc gtggctctcc tcgcaggtgt tctgggctcc gttgtaccaa gtggagcagg 1620tggttgcggg caagcgttgt gcagagccca tagccagctg ggcagggggc tgccctctcc 1680acattatcag ttgacagtgt acaatgcctt tgatgaactg ttttgtaagt gctgctatat 1740ctatccattt tttaataaag ataatactgt ttttgagaca aaaaaaa 17874395PRTHomo sapiens 4Met Lys Glu Asp Gly Gly Ala Glu Phe Ser Ala Arg Ser Arg Lys Arg1 5 10 15Lys Ala Asn Val Thr Val Phe Leu Gln Asp Pro Asp Glu Glu Met Ala20 25 30Lys Ile Asp Arg Thr Ala Arg Asp Gln Cys Gly Ser Gln Pro Trp Asp35 40 45Asn Asn Ala Val Cys Ala Asp Pro Cys Ser Leu Ile Pro Thr Pro Asp50 55 60Lys Glu Asp Asp Asp Arg Val Tyr Pro Asn Ser Thr Cys Lys Pro Arg65 70 75 80Ile Ile Ala Pro Ser Arg Gly Ser Pro Leu Pro Val Leu Ser Trp Ala85 90 95Asn Arg Glu Glu Val Trp Lys Ile Met Leu Asn Lys Glu Lys Thr Tyr100 105 110Leu Arg Asp Gln His Phe Leu Glu Gln His Pro Leu Leu Gln Pro Lys115 120 125Met Arg Ala Ile Leu Leu Asp Trp Leu Met Glu Val Cys Glu Val Tyr130 135 140Lys Leu His Arg Glu Thr Phe Tyr Leu Ala Gln Asp Phe Phe Asp Arg145 150 155 160Tyr Met Ala Thr Gln Glu Asn Val Val Lys Thr Leu Leu Gln Leu Ile165 170 175Gly Ile Ser Ser Leu Phe Ile Ala Ala Lys Leu Glu Glu Ile Tyr Pro180 185 190Pro Lys Leu His Gln Phe Ala Tyr Val Thr Asp Gly Ala Cys Ser Gly195 200 205Asp Glu Ile Leu Thr Met Glu Leu Met Ile Met Lys Ala Leu Lys Trp210 215 220Arg Leu Ser Pro Leu Thr Ile Val Ser Trp Leu Asn Val Tyr Met Gln225 230 235 240Val Ala Tyr Leu Asn Asp Leu His Glu Val Leu Leu Pro Gln Tyr Pro245 250 255Gln Gln Ile Phe Ile Gln Ile Ala Glu Leu Leu Asp Leu Cys Val Leu260 265 270Asp Val Asp Cys Leu Glu Phe Pro Tyr Gly Ile Leu Ala Ala Ser Ala275 280 285Leu Tyr His Phe Ser Ser Ser Glu Leu Met Gln Lys Val Ser Gly Tyr290 295 300Gln Trp Cys Asp Ile Glu Asn Cys Val Lys Trp Met Val Pro Phe Ala305 310 315 320Met Val Ile Arg Glu Thr Gly Ser Ser Lys Leu Lys His Phe Arg Gly325 330 335Val Ala Asp Glu Asp Ala His Asn Ile Gln Thr His Arg Asp Ser Leu340 345 350Asp Leu Leu Asp Lys Ala Arg Ala Lys Lys Ala Met Leu Ser Glu Gln355 360 365Asn Arg Ala Ser Pro Leu Pro Ser Gly Leu Leu Thr Pro Pro Gln Ser370 375 380Gly Lys Lys Gln Ser Ser Gly Pro Glu Met Ala385 390 39552748DNAHomo sapiens 5agcgggtgcg gggcgggacc ggcccggcct atatattggg ttggcgccgg cgccagctga 60gccgagcggt agctggtctg gcgaggtttt atacacctga aagaagagaa tgtcaagacg 120aagtagccgt ttacaagcta agcagcagcc ccagcccagc cagacggaat ccccccaaga 180agcccagata atccaggcca agaagaggaa aactacccag gatgtcaaaa aaagaagaga 240ggaggtcacc aagaaacatc agtatgaaat taggaattgt tggccacctg tattatctgg 300ggggatcagt ccttgcatta tcattgaaac acctcacaaa gaaataggaa caagtgattt 360ctccagattt acaaattaca gatttaaaaa tctttttatt aatccttcac ctttgcctga 420tttaagctgg ggatgttcaa aagaagtctg gctaaacatg ttaaaaaagg agagcagata 480tgttcatgac aaacattttg aagttctgca ttctgacttg gaaccacaga tgaggtccat 540acttctagac tggcttttag aggtatgtga agtatacaca cttcataggg aaacatttta 600tcttgcacaa gacttttttg atagatttat gttgacacaa aaggatataa ataaaaatat 660gcttcaactc attggaatta cctcattatt cattgcttcc aaacttgagg aaatctatgc 720tcctaaactc caagagtttg cttacgtcac tgatggtgct tgcagtgaag aggatatctt 780aaggatggaa ctcattatat taaaggcttt aaaatgggaa ctttgtcctg taacaatcat 840ctcctggcta aatctctttc tccaagttga tgctcttaaa gatgctccta aagttcttct 900acctcagtat tctcaggaaa cattcattca aatagctcag cttttagatc tgtgtattct 960agccattgat tcattagagt tccagtacag aatactgact gctgctgcct tgtgccattt 1020tacctccatt gaagtggtta agaaagcctc aggtttggag tgggacagta tttcagaatg 1080tgtagattgg atggtacctt ttgtcaatgt agtaaaaagt actagtccag tgaagctgaa 1140gacttttaag aagattccta tggaagacag acataatatc cagacacata caaactattt 1200ggctatgctg gaggaagtaa attacataaa caccttcaga aaagggggac agttgtcacc 1260agtgtgcaat ggaggcatta tgacaccacc gaagagcact gaaaaaccac caggaaaaca 1320ctaaagaaga taactaagca aacaagttgg aattcaccaa gattgggtag aactggtatc 1380actgaactac taaagtttta cagaaagtag tgctgtgatt gattgcccta gccaattcac 1440aagttacact gccattctga ttttaaaact tacaattggc actaaagaat acatttaatt 1500atttcctatg ttagctgtta aagaaacagc aggacttgtt tacaaagatg tcttcattcc 1560caaggttact ggatagaagc caaccacagt ctataccata gcaatgtttt tcctttaatc 1620cagtgttact gtgtttatct tgataaacta ggaattttgt cactggagtt ttggactgga 1680taagtgctac cttaaagggt atactaagtg atacagtact ttgaatctag ttgttagatt 1740ctcaaaattc ctacactctt gactagtgca atttggttct tgaaaattaa atttaaactt 1800gtttacaaag gtttagtttt gtaataaggt gactaattta tctatagctg ctatagcaag 1860ctattataaa acttgaattt ctacaaatgg tgaaatttaa tgttttttaa actagtttat 1920ttgccttgcc ataacacatt ttttaactaa taaggcttag atgaacatgg tgttcaacct 1980gtgctctaaa cagtgggagt accaaagaaa ttataaacaa gataaatgct gtggctcctt 2040cctaactggg gctttcttga catgtaggtt gcttggtaat aacctttttg tatatcacaa 2100tttgggtgaa aaacttaagt accctttcaa actatttata tgaggaagtc actttactac 2160tctaagatat ccctaaggaa tttttttttt taatttagtg tgactaaggc tttatttatg 2220tttgtgaaac tgttaaggtc ctttctaaat tcctccattg tgagataagg acagtgtcaa 2280agtgataaag cttaacactt gacctaaact tctattttct taaggaagaa gagtattaaa 2340tatatactga ctcctagaaa tctatttatt aaaaaaagac atgaaaactt gctgtacata 2400ggctagctat ttctaaatat tttaaattag cttttctaaa aaaaaaatcc agcctcataa 2460agtagattag aaaactagat tgctagttta ttttgttatc agatatgtga atctcttctc 2520cctttgaaga aactatacat ttattgttac ggtatgaagt cttctgtata gtttgttttt 2580aaactaatat ttgtttcagt attttgtctg aaaagaaaac accactaatt gtgtacatat 2640gtattatata aacttaacct tttaatactg tttattttta gcccattgtt taaaaaataa 2700aagttaaaaa aatttaactg cttaaaagta aaaaaaaaaa aaaaaaaa 27486404PRTHomo sapiens 6Met Ser Arg Arg Ser Ser Arg Leu Gln Ala Lys Gln Gln Pro Gln Pro1 5 10 15Ser Gln Thr Glu Ser Pro Gln Glu Ala Gln Ile Ile Gln Ala Lys Lys20 25 30Arg Lys Thr Thr Gln Asp Val Lys Lys Arg Arg Glu Glu Val Thr Lys35 40 45Lys His Gln Tyr Glu Ile Arg Asn Cys Trp Pro Pro Val Leu Ser Gly50 55 60Gly Ile Ser Pro Cys Ile Ile Ile Glu Thr Pro His Lys Glu Ile Gly65 70 75 80Thr Ser Asp Phe Ser Arg Phe Thr Asn Tyr Arg Phe Lys Asn Leu Phe85 90 95Ile Asn Pro Ser Pro Leu Pro Asp Leu Ser Trp Gly Cys Ser Lys Glu100 105 110Val Trp Leu Asn Met Leu Lys Lys Glu Ser Arg Tyr Val His Asp Lys115 120 125His Phe Glu Val Leu His Ser Asp Leu Glu Pro Gln Met Arg Ser Ile130 135 140Leu Leu Asp Trp Leu Leu Glu Val Cys Glu Val Tyr Thr Leu His Arg145 150 155 160Glu Thr Phe Tyr Leu Ala Gln Asp Phe Phe Asp Arg Phe Met Leu Thr165 170 175Gln Lys Asp Ile Asn Lys Asn Met Leu Gln Leu Ile Gly Ile Thr Ser180 185 190Leu Phe Ile Ala Ser Lys Leu Glu Glu Ile Tyr Ala Pro Lys Leu Gln195 200 205Glu Phe Ala Tyr Val Thr Asp Gly Ala Cys Ser Glu Glu Asp Ile Leu210 215 220Arg Met Glu Leu Ile Ile Leu Lys Ala Leu Lys Trp Glu Leu Cys Pro225 230 235 240Val Thr Ile Ile Ser Trp Leu Asn Leu Phe Leu Gln Val Asp Ala Leu245 250 255Lys Asp Ala Pro Lys Val Leu Leu Pro Gln Tyr Ser Gln Glu Thr Phe260 265 270Ile Gln Ile Ala Gln Leu Leu Asp Leu Cys Ile Leu Ala Ile Asp Ser275 280 285Leu Glu Phe Gln Tyr Arg Ile Leu Thr Ala Ala Ala Leu Cys His Phe290 295 300Thr Ser Ile Glu Val Val Lys Lys Ala Ser Gly Leu Glu Trp Asp Ser305 310 315 320Ile Ser Glu Cys Val Asp Trp Met Val Pro Phe Val Asn Val Val Lys325 330 335Ser Thr Ser Pro Val Lys Leu Lys Thr Phe Lys Lys Ile Pro Met Glu340 345 350Asp Arg His Asn Ile Gln Thr His Thr Asn Tyr Leu Ala Met Leu Glu355 360 365Glu Val Asn Tyr Ile Asn Thr Phe Arg Lys Gly Gly Gln Leu Ser Pro370 375 380Val Cys Asn Gly Gly Ile Met Thr Pro Pro Lys Ser Thr Glu Lys Pro385 390 395 400Pro Gly Lys His72613DNAHomo sapiens 7agcgggtgcg gggcgggacc ggcccggcct atatattggg ttggcgccgg cgccagctga 60gccgagcggt agctggtctg gcgaggtttt atacacctga aagaagagaa tgtcaagacg 120aagtagccgt ttacaagcta agcagcagcc ccagcccagc cagacggaat ccccccaaga 180agcccagata atccaggcca agaagaggaa aactacccag gatgtcaaaa aaagaagaga 240ggaggtcacc aagaaacatc agtatgaaat taggaattgt tggccacctg tattatctgg 300ggggatcagt ccttgcatta tcattgaaac acctcacaaa gaaataggaa caagtgattt 360ctccagattt acaaattaca gatttaaaaa tctttttatt aatccttcac ctttgcctga 420tttaagctgg ggatgttcaa aagaagtctg gctaaacatg ttaaaaaagg agagcagata 480tgttcatgac aaacattttg aagttctgca ttctgacttg gaaccacaga tgaggtccat 540acttctagac tggcttttag aggtatgtga agtatacaca cttcataggg aaacatttta 600tcttgcttac gtcactgatg gtgcttgcag tgaagaggat atcttaagga tggaactcat 660tatattaaag gctttaaaat gggaactttg tcctgtaaca atcatctcct ggctaaatct 720ctttctccaa gttgatgctc ttaaagatgc tcctaaagtt cttctacctc agtattctca 780ggaaacattc attcaaatag ctcagctttt agatctgtgt attctagcca ttgattcatt 840agagttccag tacagaatac tgactgctgc tgccttgtgc cattttacct ccattgaagt 900ggttaagaaa gcctcaggtt tggagtggga cagtatttca gaatgtgtag attggatggt 960accttttgtc aatgtagtaa aaagtactag tccagtgaag ctgaagactt ttaagaagat 1020tcctatggaa gacagacata atatccagac acatacaaac tatttggcta tgctggagga 1080agtaaattac ataaacacct tcagaaaagg gggacagttg tcaccagtgt gcaatggagg 1140cattatgaca ccaccgaaga gcactgaaaa accaccagga aaacactaaa gaagataact 1200aagcaaacaa gttggaattc accaagattg ggtagaactg gtatcactga actactaaag 1260ttttacagaa agtagtgctg tgattgattg ccctagccaa ttcacaagtt acactgccat 1320tctgatttta aaacttacaa ttggcactaa agaatacatt taattatttc ctatgttagc 1380tgttaaagaa acagcaggac ttgtttacaa agatgtcttc attcccaagg ttactggata 1440gaagccaacc acagtctata ccatagcaat gtttttcctt taatccagtg ttactgtgtt 1500tatcttgata aactaggaat tttgtcactg gagttttgga
ctggataagt gctaccttaa 1560agggtatact aagtgataca gtactttgaa tctagttgtt agattctcaa aattcctaca 1620ctcttgacta gtgcaatttg gttcttgaaa attaaattta aacttgttta caaaggttta 1680gttttgtaat aaggtgacta atttatctat agctgctata gcaagctatt ataaaacttg 1740aatttctaca aatggtgaaa tttaatgttt tttaaactag tttatttgcc ttgccataac 1800acatttttta actaataagg cttagatgaa catggtgttc aacctgtgct ctaaacagtg 1860ggagtaccaa agaaattata aacaagataa atgctgtggc tccttcctaa ctggggcttt 1920cttgacatgt aggttgcttg gtaataacct ttttgtatat cacaatttgg gtgaaaaact 1980taagtaccct ttcaaactat ttatatgagg aagtcacttt actactctaa gatatcccta 2040aggaattttt ttttttaatt tagtgtgact aaggctttat ttatgtttgt gaaactgtta 2100aggtcctttc taaattcctc cattgtgaga taaggacagt gtcaaagtga taaagcttaa 2160cacttgacct aaacttctat tttcttaagg aagaagagta ttaaatatat actgactcct 2220agaaatctat ttattaaaaa aagacatgaa aacttgctgt acataggcta gctatttcta 2280aatattttaa attagctttt ctaaaaaaaa aatccagcct cataaagtag attagaaaac 2340tagattgcta gtttattttg ttatcagata tgtgaatctc ttctcccttt gaagaaacta 2400tacatttatt gttacggtat gaagtcttct gtatagtttg tttttaaact aatatttgtt 2460tcagtatttt gtctgaaaag aaaacaccac taattgtgta catatgtatt atataaactt 2520aaccttttaa tactgtttat ttttagccca ttgtttaaaa aataaaagtt aaaaaaattt 2580aactgcttaa aagtaaaaaa aaaaaaaaaa aaa 26138359PRTHomo sapiens 8Met Ser Arg Arg Ser Ser Arg Leu Gln Ala Lys Gln Gln Pro Gln Pro1 5 10 15Ser Gln Thr Glu Ser Pro Gln Glu Ala Gln Ile Ile Gln Ala Lys Lys20 25 30Arg Lys Thr Thr Gln Asp Val Lys Lys Arg Arg Glu Glu Val Thr Lys35 40 45Lys His Gln Tyr Glu Ile Arg Asn Cys Trp Pro Pro Val Leu Ser Gly50 55 60Gly Ile Ser Pro Cys Ile Ile Ile Glu Thr Pro His Lys Glu Ile Gly65 70 75 80Thr Ser Asp Phe Ser Arg Phe Thr Asn Tyr Arg Phe Lys Asn Leu Phe85 90 95Ile Asn Pro Ser Pro Leu Pro Asp Leu Ser Trp Gly Cys Ser Lys Glu100 105 110Val Trp Leu Asn Met Leu Lys Lys Glu Ser Arg Tyr Val His Asp Lys115 120 125His Phe Glu Val Leu His Ser Asp Leu Glu Pro Gln Met Arg Ser Ile130 135 140Leu Leu Asp Trp Leu Leu Glu Val Cys Glu Val Tyr Thr Leu His Arg145 150 155 160Glu Thr Phe Tyr Leu Ala Tyr Val Thr Asp Gly Ala Cys Ser Glu Glu165 170 175Asp Ile Leu Arg Met Glu Leu Ile Ile Leu Lys Ala Leu Lys Trp Glu180 185 190Leu Cys Pro Val Thr Ile Ile Ser Trp Leu Asn Leu Phe Leu Gln Val195 200 205Asp Ala Leu Lys Asp Ala Pro Lys Val Leu Leu Pro Gln Tyr Ser Gln210 215 220Glu Thr Phe Ile Gln Ile Ala Gln Leu Leu Asp Leu Cys Ile Leu Ala225 230 235 240Ile Asp Ser Leu Glu Phe Gln Tyr Arg Ile Leu Thr Ala Ala Ala Leu245 250 255Cys His Phe Thr Ser Ile Glu Val Val Lys Lys Ala Ser Gly Leu Glu260 265 270Trp Asp Ser Ile Ser Glu Cys Val Asp Trp Met Val Pro Phe Val Asn275 280 285Val Val Lys Ser Thr Ser Pro Val Lys Leu Lys Thr Phe Lys Lys Ile290 295 300Pro Met Glu Asp Arg His Asn Ile Gln Thr His Thr Asn Tyr Leu Ala305 310 315 320Met Leu Glu Glu Val Asn Tyr Ile Asn Thr Phe Arg Lys Gly Gly Gln325 330 335Leu Ser Pro Val Cys Asn Gly Gly Ile Met Thr Pro Pro Lys Ser Thr340 345 350Glu Lys Pro Pro Gly Lys His35592536DNAHomo sapiens 9agcgggtgcg gggcgggacc ggcccggcct atatattggg ttggcgccgg cgccagctga 60gccgagcggt agctggtctg gcgaggtttt atacacctga aagaagagaa tgtcaagacg 120aagtagccgt ttacaagcta agcagcagcc ccagcccagc cagacggaat ccccccaaga 180agcccagata atccaggcca agaagaggaa aactacccag gatgtcaaaa gaagtctggc 240taaacatgtt aaaaaaggag agcagatatg ttcatgacaa acattttgaa gttctgcatt 300ctgacttgga accacagatg aggtccatac ttctagactg gcttttagag gtatgtgaag 360tatacacact tcatagggaa acattttatc ttgcacaaga cttttttgat agatttatgt 420tgacacaaaa ggatataaat aaaaatatgc ttcaactcat tggaattacc tcattattca 480ttgcttccaa acttgaggaa atctatgctc ctaaactcca agagtttgct tacgtcactg 540atggtgcttg cagtgaagag gatatcttaa ggatggaact cattatatta aaggctttaa 600aatgggaact ttgtcctgta acaatcatct cctggctaaa tctctttctc caagttgatg 660ctcttaaaga tgctcctaaa gttcttctac ctcagtattc tcaggaaaca ttcattcaaa 720tagctcagct tttagatctg tgtattctag ccattgattc attagagttc cagtacagaa 780tactgactgc tgctgccttg tgccatttta cctccattga agtggttaag aaagcctcag 840gtttggagtg ggacagtatt tcagaatgtg tagattggat ggtacctttt gtcaatgtag 900taaaaagtac tagtccagtg aagctgaaga cttttaagaa gattcctatg gaagacagac 960ataatatcca gacacataca aactatttgg ctatgctgga ggaagtaaat tacataaaca 1020ccttcagaaa agggggacag ttgtcaccag tgtgcaatgg aggcattatg acaccaccga 1080agagcactga aaaaccacca ggaaaacact aaagaagata actaagcaaa caagttggaa 1140ttcaccaaga ttgggtagaa ctggtatcac tgaactacta aagttttaca gaaagtagtg 1200ctgtgattga ttgccctagc caattcacaa gttacactgc cattctgatt ttaaaactta 1260caattggcac taaagaatac atttaattat ttcctatgtt agctgttaaa gaaacagcag 1320gacttgttta caaagatgtc ttcattccca aggttactgg atagaagcca accacagtct 1380ataccatagc aatgtttttc ctttaatcca gtgttactgt gtttatcttg ataaactagg 1440aattttgtca ctggagtttt ggactggata agtgctacct taaagggtat actaagtgat 1500acagtacttt gaatctagtt gttagattct caaaattcct acactcttga ctagtgcaat 1560ttggttcttg aaaattaaat ttaaacttgt ttacaaaggt ttagttttgt aataaggtga 1620ctaatttatc tatagctgct atagcaagct attataaaac ttgaatttct acaaatggtg 1680aaatttaatg ttttttaaac tagtttattt gccttgccat aacacatttt ttaactaata 1740aggcttagat gaacatggtg ttcaacctgt gctctaaaca gtgggagtac caaagaaatt 1800ataaacaaga taaatgctgt ggctccttcc taactggggc tttcttgaca tgtaggttgc 1860ttggtaataa cctttttgta tatcacaatt tgggtgaaaa acttaagtac cctttcaaac 1920tatttatatg aggaagtcac tttactactc taagatatcc ctaaggaatt ttttttttta 1980atttagtgtg actaaggctt tatttatgtt tgtgaaactg ttaaggtcct ttctaaattc 2040ctccattgtg agataaggac agtgtcaaag tgataaagct taacacttga cctaaacttc 2100tattttctta aggaagaaga gtattaaata tatactgact cctagaaatc tatttattaa 2160aaaaagacat gaaaacttgc tgtacatagg ctagctattt ctaaatattt taaattagct 2220tttctaaaaa aaaaatccag cctcataaag tagattagaa aactagattg ctagtttatt 2280ttgttatcag atatgtgaat ctcttctccc tttgaagaaa ctatacattt attgttacgg 2340tatgaagtct tctgtatagt ttgtttttaa actaatattt gtttcagtat tttgtctgaa 2400aagaaaacac cactaattgt gtacatatgt attatataaa cttaaccttt taatactgtt 2460tatttttagc ccattgttta aaaaataaaa gttaaaaaaa tttaactgct taaaagtaaa 2520aaaaaaaaaa aaaaaa 253610296PRTHomo sapiens 10Met Ser Lys Glu Val Trp Leu Asn Met Leu Lys Lys Glu Ser Arg Tyr1 5 10 15Val His Asp Lys His Phe Glu Val Leu His Ser Asp Leu Glu Pro Gln20 25 30Met Arg Ser Ile Leu Leu Asp Trp Leu Leu Glu Val Cys Glu Val Tyr35 40 45Thr Leu His Arg Glu Thr Phe Tyr Leu Ala Gln Asp Phe Phe Asp Arg50 55 60Phe Met Leu Thr Gln Lys Asp Ile Asn Lys Asn Met Leu Gln Leu Ile65 70 75 80Gly Ile Thr Ser Leu Phe Ile Ala Ser Lys Leu Glu Glu Ile Tyr Ala85 90 95Pro Lys Leu Gln Glu Phe Ala Tyr Val Thr Asp Gly Ala Cys Ser Glu100 105 110Glu Asp Ile Leu Arg Met Glu Leu Ile Ile Leu Lys Ala Leu Lys Trp115 120 125Glu Leu Cys Pro Val Thr Ile Ile Ser Trp Leu Asn Leu Phe Leu Gln130 135 140Val Asp Ala Leu Lys Asp Ala Pro Lys Val Leu Leu Pro Gln Tyr Ser145 150 155 160Gln Glu Thr Phe Ile Gln Ile Ala Gln Leu Leu Asp Leu Cys Ile Leu165 170 175Ala Ile Asp Ser Leu Glu Phe Gln Tyr Arg Ile Leu Thr Ala Ala Ala180 185 190Leu Cys His Phe Thr Ser Ile Glu Val Val Lys Lys Ala Ser Gly Leu195 200 205Glu Trp Asp Ser Ile Ser Glu Cys Val Asp Trp Met Val Pro Phe Val210 215 220Asn Val Val Lys Ser Thr Ser Pro Val Lys Leu Lys Thr Phe Lys Lys225 230 235 240Ile Pro Met Glu Asp Arg His Asn Ile Gln Thr His Thr Asn Tyr Leu245 250 255Ala Met Leu Glu Glu Val Asn Tyr Ile Asn Thr Phe Arg Lys Gly Gly260 265 270Gln Leu Ser Pro Val Cys Asn Gly Gly Ile Met Thr Pro Pro Lys Ser275 280 285Thr Glu Lys Pro Pro Gly Lys His290 295113453DNAHomo sapiens 11acttttcgcg cgaaacctgg ttgttgctgt agtggcggag aggatcgtgg tactgctatg 60gcggaatcat cggaatcctt caccatggca tccagcccgg cccagcgtcg gcgaggcaat 120gatcctctca cctccagccc tggccgaagc tcccggcgta ctgatgccct cacctccagc 180cctggccgtg accttccacc atttgaggat gagtccgagg ggctcctagg cacagagggg 240cccctggagg aagaagagga tggagaggag ctcattggag atggcatgga aagggactac 300cgcgccatcc cagagctgga cgcctatgag gccgagggac tggctctgga tgatgaggac 360gtagaggagc tgacggccag tcagagggag gcagcagagc gggccatgcg gcagcgtgac 420cgggaggctg gccggggcct gggccgcatg cgccgtgggc tcctgtatga cagcgatgag 480gaggacgagg agcgccctgc ccgcaagcgc cgccaggtgg agcgggccac ggaggacggc 540gaggaggacg aggagatgat cgagagcatc gagaacctgg aggatctcaa aggccactct 600gtgcgcgagt gggtgagcat ggcgggcccc cggctggaga tccaccaccg cttcaagaac 660ttcctgcgca ctcacgtcga cagccacggc cacaacgtct tcaaggagcg catcagcgac 720atgtgcaaag agaaccgtga gagcctggtg gtgaactatg aggacttggc agccagggag 780cacgtgctgg cctacttcct gcctgaggca ccggcggagc tgctgcagat ctttgatgag 840gctgccctgg aggtggtact ggccatgtac cccaagtacg accgcatcac caaccacatc 900catgtccgca tctcccacct gcctctggtg gaggagctgc gctcgctgag gcagctgcat 960ctgaaccagc tgatccgcac cagtggggtg gtgaccagct gcactggcgt cctgccccag 1020ctcagcatgg tcaagtacaa ctgcaacaag tgcaatttcg tcctgggtcc tttctgccag 1080tcccagaacc aggaggtgaa accaggctcc tgtcctgagt gccagtcggc cggccccttt 1140gaggtcaaca tggaggagac catctatcag aactaccagc gtatccgaat ccaggagagt 1200ccaggcaaag tggcggctgg ccggctgccc cgctccaagg acgccattct cctcgcagat 1260ctggtggaca gctgcaagcc aggagacgag atagagctga ctggcatcta tcacaacaac 1320tatgatggct ccctcaacac tgccaatggc ttccctgtct ttgccactgt catcctagcc 1380aaccacgtgg ccaagaagga caacaaggtt gctgtagggg aactgaccga tgaagatgtg 1440aagatgatca ctagcctctc caaggatcag cagatcggag agaagatctt tgccagcatt 1500gctccttcca tctatggtca tgaagacatc aagagaggcc tggctctggc cctgttcgga 1560ggggagccca aaaacccagg tggcaagcac aaggtacgtg gtgatatcaa cgtgctcttg 1620tgcggagacc ctggcacagc gaagtcgcag tttctcaagt atattgagaa agtgtccagc 1680cgagccatct tcaccactgg ccagggggcg tcggctgtgg gcctcacggc gtatgtccag 1740cggcaccctg tcagcaggga gtggaccttg gaggctgggg ccctggttct ggctgaccga 1800ggagtgtgtc tcattgatga atttgacaag atgaatgacc aggacagaac cagcatccat 1860gaggccatgg agcaacagag catctccatc tcgaaggctg gcatcgtcac ctccctgcag 1920gctcgctgca cggtcattgc tgccgccaac cccataggag ggcgctacga cccctcgctg 1980actttctctg agaacgtgga cctcacagag cccatcatct cacgctttga catcctgtgt 2040gtggtgaggg acaccgtgga cccagtccag gacgagatgc tggcccgctt cgtggtgggc 2100agccacgtca gacaccaccc cagcaacaag gaggaggagg ggctggccaa tggcagcgct 2160gctgagcccg ccatgcccaa cacgtatggc gtggagcccc tgccccagga ggtcctgaag 2220aagtacatca tctacgccaa ggagagggtc cacccgaagc tcaaccagat ggaccaggac 2280aaggtggcca agatgtacag tgacctgagg aaagaatcta tggcgacagg cagcatcccc 2340attacggtgc ggcacatcga gtccatgatc cgcatggcgg aggcccacgc gcgcatccat 2400ctgcgggact atgtgatcga agacgacgtc aacatggcca tccgcgtgat gctggagagc 2460ttcatagaca cacagaagtt cagcgtcatg cgcagcatgc gcaagacttt tgcccgctac 2520ctttcattcc ggcgtgacaa caatgagctg ttgctcttca tactgaagca gttagtggca 2580gagcaggtga catatcagcg caaccgcttt ggggcccagc aggacactat tgaggtccct 2640gagaaggact tggtggataa ggctcgtcag atcaacatcc acaacctctc tgcattttat 2700gacagtgagc tcttcaggat gaacaagttc agccacgacc tgaaaaggaa aatgatcctg 2760cagcagttct gaggccctat gccatccata aggattcctt gggattctgg tttggggtgg 2820tcagtgccct ctgtgcttta tggacacaaa accagagcac ttgatgaact cggggtacta 2880gggtcagggc ttatagcagg atgtctggct gcacctggca tgactgtttg tttctccaag 2940cctgctttgt gcttctcacc tttgggtggg atgccttgcc agtgtgtctt acttggttgc 3000tgaacatctt gccacctccg agtgctttgt ctccactcag taccttggat cagagctgct 3060gagttcagga tgcctgcgtg tggtttaggt gttagccttc ttacatggat gtcaggagag 3120ctgctgccct cttggcgtga gttgcgtatt caggctgctt ttgctgcctt tggccagaga 3180gctggttgaa gatgtttgta atcgttttca gtctcctgca ggtttctgtg cccctgtggt 3240ggaagagggc acgacagtgc cagcgcagcg ttctgggctc ctcagtcgca ggggtgggat 3300gtgagtcatg cggattatcc actcgccaca gttatcagct gccattgctc cctgtctgtt 3360tccccactct cttatttgtg cattcggttt ggtttctgta gttttaattt ttaataaagt 3420tgaataaaat ataaaaaaaa aaaaaaaaaa aaa 345312904PRTHomo sapiens 12Met Ala Glu Ser Ser Glu Ser Phe Thr Met Ala Ser Ser Pro Ala Gln1 5 10 15Arg Arg Arg Gly Asn Asp Pro Leu Thr Ser Ser Pro Gly Arg Ser Ser20 25 30Arg Arg Thr Asp Ala Leu Thr Ser Ser Pro Gly Arg Asp Leu Pro Pro35 40 45Phe Glu Asp Glu Ser Glu Gly Leu Leu Gly Thr Glu Gly Pro Leu Glu50 55 60Glu Glu Glu Asp Gly Glu Glu Leu Ile Gly Asp Gly Met Glu Arg Asp65 70 75 80Tyr Arg Ala Ile Pro Glu Leu Asp Ala Tyr Glu Ala Glu Gly Leu Ala85 90 95Leu Asp Asp Glu Asp Val Glu Glu Leu Thr Ala Ser Gln Arg Glu Ala100 105 110Ala Glu Arg Ala Met Arg Gln Arg Asp Arg Glu Ala Gly Arg Gly Leu115 120 125Gly Arg Met Arg Arg Gly Leu Leu Tyr Asp Ser Asp Glu Glu Asp Glu130 135 140Glu Arg Pro Ala Arg Lys Arg Arg Gln Val Glu Arg Ala Thr Glu Asp145 150 155 160Gly Glu Glu Asp Glu Glu Met Ile Glu Ser Ile Glu Asn Leu Glu Asp165 170 175Leu Lys Gly His Ser Val Arg Glu Trp Val Ser Met Ala Gly Pro Arg180 185 190Leu Glu Ile His His Arg Phe Lys Asn Phe Leu Arg Thr His Val Asp195 200 205Ser His Gly His Asn Val Phe Lys Glu Arg Ile Ser Asp Met Cys Lys210 215 220Glu Asn Arg Glu Ser Leu Val Val Asn Tyr Glu Asp Leu Ala Ala Arg225 230 235 240Glu His Val Leu Ala Tyr Phe Leu Pro Glu Ala Pro Ala Glu Leu Leu245 250 255Gln Ile Phe Asp Glu Ala Ala Leu Glu Val Val Leu Ala Met Tyr Pro260 265 270Lys Tyr Asp Arg Ile Thr Asn His Ile His Val Arg Ile Ser His Leu275 280 285Pro Leu Val Glu Glu Leu Arg Ser Leu Arg Gln Leu His Leu Asn Gln290 295 300Leu Ile Arg Thr Ser Gly Val Val Thr Ser Cys Thr Gly Val Leu Pro305 310 315 320Gln Leu Ser Met Val Lys Tyr Asn Cys Asn Lys Cys Asn Phe Val Leu325 330 335Gly Pro Phe Cys Gln Ser Gln Asn Gln Glu Val Lys Pro Gly Ser Cys340 345 350Pro Glu Cys Gln Ser Ala Gly Pro Phe Glu Val Asn Met Glu Glu Thr355 360 365Ile Tyr Gln Asn Tyr Gln Arg Ile Arg Ile Gln Glu Ser Pro Gly Lys370 375 380Val Ala Ala Gly Arg Leu Pro Arg Ser Lys Asp Ala Ile Leu Leu Ala385 390 395 400Asp Leu Val Asp Ser Cys Lys Pro Gly Asp Glu Ile Glu Leu Thr Gly405 410 415Ile Tyr His Asn Asn Tyr Asp Gly Ser Leu Asn Thr Ala Asn Gly Phe420 425 430Pro Val Phe Ala Thr Val Ile Leu Ala Asn His Val Ala Lys Lys Asp435 440 445Asn Lys Val Ala Val Gly Glu Leu Thr Asp Glu Asp Val Lys Met Ile450 455 460Thr Ser Leu Ser Lys Asp Gln Gln Ile Gly Glu Lys Ile Phe Ala Ser465 470 475 480Ile Ala Pro Ser Ile Tyr Gly His Glu Asp Ile Lys Arg Gly Leu Ala485 490 495Leu Ala Leu Phe Gly Gly Glu Pro Lys Asn Pro Gly Gly Lys His Lys500 505 510Val Arg Gly Asp Ile Asn Val Leu Leu Cys Gly Asp Pro Gly Thr Ala515 520 525Lys Ser Gln Phe Leu Lys Tyr Ile Glu Lys Val Ser Ser Arg Ala Ile530 535 540Phe Thr Thr Gly Gln Gly Ala Ser Ala Val Gly Leu Thr Ala Tyr Val545 550 555 560Gln Arg His Pro Val Ser Arg Glu Trp Thr Leu Glu Ala Gly Ala Leu565 570 575Val Leu Ala Asp Arg Gly Val Cys Leu Ile Asp Glu Phe Asp Lys Met580 585 590Asn Asp Gln Asp Arg Thr Ser Ile His Glu Ala Met Glu Gln Gln Ser595 600 605Ile Ser Ile Ser Lys Ala Gly Ile Val Thr Ser Leu Gln Ala Arg Cys610 615 620Thr Val Ile Ala Ala Ala Asn Pro Ile Gly Gly Arg Tyr Asp Pro Ser625 630 635 640Leu Thr Phe Ser Glu Asn Val Asp Leu Thr Glu Pro Ile Ile Ser Arg645 650 655Phe Asp Ile Leu Cys Val Val Arg Asp Thr Val Asp Pro Val Gln Asp660 665 670Glu Met Leu Ala Arg Phe Val Val Gly Ser His Val Arg His His Pro675 680 685Ser Asn Lys Glu Glu Glu Gly Leu Ala Asn Gly Ser Ala Ala Glu Pro690 695 700Ala Met Pro Asn Thr Tyr Gly Val Glu Pro Leu Pro Gln Glu Val Leu705 710 715 720Lys Lys Tyr Ile Ile Tyr Ala Lys Glu Arg Val His
Pro Lys Leu Asn725 730 735Gln Met Asp Gln Asp Lys Val Ala Lys Met Tyr Ser Asp Leu Arg Lys740 745 750Glu Ser Met Ala Thr Gly Ser Ile Pro Ile Thr Val Arg His Ile Glu755 760 765Ser Met Ile Arg Met Ala Glu Ala His Ala Arg Ile His Leu Arg Asp770 775 780Tyr Val Ile Glu Asp Asp Val Asn Met Ala Ile Arg Val Met Leu Glu785 790 795 800Ser Phe Ile Asp Thr Gln Lys Phe Ser Val Met Arg Ser Met Arg Lys805 810 815Thr Phe Ala Arg Tyr Leu Ser Phe Arg Arg Asp Asn Asn Glu Leu Leu820 825 830Leu Phe Ile Leu Lys Gln Leu Val Ala Glu Gln Val Thr Tyr Gln Arg835 840 845Asn Arg Phe Gly Ala Gln Gln Asp Thr Ile Glu Val Pro Glu Lys Asp850 855 860Leu Val Asp Lys Ala Arg Gln Ile Asn Ile His Asn Leu Ser Ala Phe865 870 875 880Tyr Asp Ser Glu Leu Phe Arg Met Asn Lys Phe Ser His Asp Leu Lys885 890 895Arg Lys Met Ile Leu Gln Gln Phe900132821DNAHomo sapiens 13cgccccttcc cagccccaag ggtctaggat acagtctttg tagatgagcg ggtccccctt 60ggaggacaga atgaagaatt gggaaatcat ggccgttctg gagagtagac aagaagacgg 120cgaaagtcgg gcctgccccg ccctgcggcc ccggaacaaa agaacgcgtg tgcgctggcc 180ctttaagagc gattctcctc cgcccgcgcc agctcggacc gcgggaaacc cggcgcctgc 240actaccccgc ccggagattc ccttccgacg cccgcaccgc ctccccgtca ctcattctag 300gcccgcacgg tgattggctt gcggctagcg ggaggtgaag aaggccgcct tgtccgattg 360gcccgcacgc agtggcgccg gtcacgtggg gggcgacgtt tcgcgccaat ttcggttggc 420cggccacagt ccaccgcgcg gagattctca gcttccccag gagcaagacc tctgagcccg 480ccaagcgcgg ccgcacggcc ctcggcagcg atggcactga aggactacgc gctagagaag 540gaaaaggtta agaagttctt acaagagttc taccaggatg atgaactcgg gaagaagcag 600ttcaagtatg ggaaccagtt ggttcggctg gctcatcggg aacaggtggc tctgtatgtg 660gacctggacg acgtagccga ggatgacccc gagttggtgg actcaatttg tgagaatgcc 720aggcgctacg cgaagctctt tgctgatgcc gtacaagagc tgctgcctca gtacaaggag 780agggaagtgg taaataaaga tgtcctggac gtttacattg agcatcggct aatgatggag 840cagcggagtc gggaccctgg gatggtccga agcccccaga accagtaccc tgctgaactc 900atgcgcagat ttgagctgta ttttcaaggc cctagcagca acaagcctcg tgtgatccgg 960gaagtgcggg ctgactctgt ggggaagttg gtaactgtgc gtggaatcgt cactcgtgtc 1020tctgaagtca aacccaagat ggtggtggcc acttacactt gtgaccagtg tggggcagag 1080acctaccagc cgatccagtc tcccactttc atgcctctga tcatgtgccc aagccaggag 1140tgccaaacca accgctcagg agggcggctg tatctgcaga cacggggctc cagattcatc 1200aaattccagg agatgaagat gcaagaacat agtgatcagg tgcctgtggg aaatatccct 1260cgtagtatca cggtgctggt agaaggagag aacacaagga ttgcccagcc tggagaccac 1320gtcagcgtca ctggtatttt cttgccaatc ctgcgcactg ggttccgaca ggtggtacag 1380ggtttactct cagaaaccta cctggaagcc catcggattg tgaagatgaa caagagtgag 1440gatgatgagt ctggggctgg agagctcacc agggaggagc tgaggcaaat tgcagaggag 1500gatttctacg aaaagctggc agcttcaatc gccccagaaa tatacgggca tgaagatgtg 1560aagaaggcac tgctgctcct gctagtcggg ggtgtggacc agtctcctcg aggcatgaaa 1620atccggggca acatcaacat ctgtctgatg ggggatcctg gtgtggccaa gtctcagctc 1680ctgtcataca ttgatcgact ggcgcctcgc agccagtaca caacaggccg gggctcctca 1740ggagtggggc ttacggcagc tgtgctgaga gactccgtga gtggagaact gaccttagag 1800ggtggggccc tggtgctggc tgaccagggt gtgtgctgca ttgatgagtt cgacaagatg 1860gctgaggccg accgcacagc catccacgag gtcatggagc agcagaccat ctccattgcc 1920aaggccggca ttctcaccac actcaatgcc cgctgctcca tcctggctgc cgccaaccct 1980gcctacgggc gctacaaccc tcgccgcagc ctggagcaga acatacagct acctgctgca 2040ctgctctccc ggtttgacct cctctggctg attcaggacc ggcccgaccg agacaatgac 2100ctacggttgg cccagcacat cacctatgtg caccagcaca gccggcagcc cccctcccag 2160tttgaacctc tggacatgaa gctcatgagg cgttacatag ccatgtgccg cgagaagcag 2220cccatggtgc cagagtctct ggctgactac atcacagcag catacgtgga gatgaggcga 2280gaggcttggg ctagtaagga tgccacctat acttctgccc ggaccctgct ggctatcctg 2340cgcctttcca ctgctctggc acgtctgaga atggtggatg tggtggagaa agaagatgtg 2400aatgaagcca tcaggctaat ggagatgtca aaggactctc ttctaggaga caaggggcag 2460acagctagga ctcagagacc agcagatgtg atatttgcca ccgtccgtga actggtctca 2520gggggccgaa gtgtccggtt ctctgaggca gagcagcgct gtgtatctcg tggcttcaca 2580cccgcccagt tccaggcggc tctggatgaa tatgaggagc tcaatgtctg gcaggtcaat 2640gcttcccgga cacggatcac ttttgtctga ttccagcctg cttgcaaccc tggggtcctc 2700ttgttccctg ctggcctgcc ccttgggaag gggcagtgat gcctttgagg ggaaggagga 2760gcccctcttt ctcccatgct gcacttactc cttttgctaa taaaagtgtt tgtagattgt 2820c 282114719PRTHomo sapiens 14Met Ala Leu Lys Asp Tyr Ala Leu Glu Lys Glu Lys Val Lys Lys Phe1 5 10 15Leu Gln Glu Phe Tyr Gln Asp Asp Glu Leu Gly Lys Lys Gln Phe Lys20 25 30Tyr Gly Asn Gln Leu Val Arg Leu Ala His Arg Glu Gln Val Ala Leu35 40 45Tyr Val Asp Leu Asp Asp Val Ala Glu Asp Asp Pro Glu Leu Val Asp50 55 60Ser Ile Cys Glu Asn Ala Arg Arg Tyr Ala Lys Leu Phe Ala Asp Ala65 70 75 80Val Gln Glu Leu Leu Pro Gln Tyr Lys Glu Arg Glu Val Val Asn Lys85 90 95Asp Val Leu Asp Val Tyr Ile Glu His Arg Leu Met Met Glu Gln Arg100 105 110Ser Arg Asp Pro Gly Met Val Arg Ser Pro Gln Asn Gln Tyr Pro Ala115 120 125Glu Leu Met Arg Arg Phe Glu Leu Tyr Phe Gln Gly Pro Ser Ser Asn130 135 140Lys Pro Arg Val Ile Arg Glu Val Arg Ala Asp Ser Val Gly Lys Leu145 150 155 160Val Thr Val Arg Gly Ile Val Thr Arg Val Ser Glu Val Lys Pro Lys165 170 175Met Val Val Ala Thr Tyr Thr Cys Asp Gln Cys Gly Ala Glu Thr Tyr180 185 190Gln Pro Ile Gln Ser Pro Thr Phe Met Pro Leu Ile Met Cys Pro Ser195 200 205Gln Glu Cys Gln Thr Asn Arg Ser Gly Gly Arg Leu Tyr Leu Gln Thr210 215 220Arg Gly Ser Arg Phe Ile Lys Phe Gln Glu Met Lys Met Gln Glu His225 230 235 240Ser Asp Gln Val Pro Val Gly Asn Ile Pro Arg Ser Ile Thr Val Leu245 250 255Val Glu Gly Glu Asn Thr Arg Ile Ala Gln Pro Gly Asp His Val Ser260 265 270Val Thr Gly Ile Phe Leu Pro Ile Leu Arg Thr Gly Phe Arg Gln Val275 280 285Val Gln Gly Leu Leu Ser Glu Thr Tyr Leu Glu Ala His Arg Ile Val290 295 300Lys Met Asn Lys Ser Glu Asp Asp Glu Ser Gly Ala Gly Glu Leu Thr305 310 315 320Arg Glu Glu Leu Arg Gln Ile Ala Glu Glu Asp Phe Tyr Glu Lys Leu325 330 335Ala Ala Ser Ile Ala Pro Glu Ile Tyr Gly His Glu Asp Val Lys Lys340 345 350Ala Leu Leu Leu Leu Leu Val Gly Gly Val Asp Gln Ser Pro Arg Gly355 360 365Met Lys Ile Arg Gly Asn Ile Asn Ile Cys Leu Met Gly Asp Pro Gly370 375 380Val Ala Lys Ser Gln Leu Leu Ser Tyr Ile Asp Arg Leu Ala Pro Arg385 390 395 400Ser Gln Tyr Thr Thr Gly Arg Gly Ser Ser Gly Val Gly Leu Thr Ala405 410 415Ala Val Leu Arg Asp Ser Val Ser Gly Glu Leu Thr Leu Glu Gly Gly420 425 430Ala Leu Val Leu Ala Asp Gln Gly Val Cys Cys Ile Asp Glu Phe Asp435 440 445Lys Met Ala Glu Ala Asp Arg Thr Ala Ile His Glu Val Met Glu Gln450 455 460Gln Thr Ile Ser Ile Ala Lys Ala Gly Ile Leu Thr Thr Leu Asn Ala465 470 475 480Arg Cys Ser Ile Leu Ala Ala Ala Asn Pro Ala Tyr Gly Arg Tyr Asn485 490 495Pro Arg Arg Ser Leu Glu Gln Asn Ile Gln Leu Pro Ala Ala Leu Leu500 505 510Ser Arg Phe Asp Leu Leu Trp Leu Ile Gln Asp Arg Pro Asp Arg Asp515 520 525Asn Asp Leu Arg Leu Ala Gln His Ile Thr Tyr Val His Gln His Ser530 535 540Arg Gln Pro Pro Ser Gln Phe Glu Pro Leu Asp Met Lys Leu Met Arg545 550 555 560Arg Tyr Ile Ala Met Cys Arg Glu Lys Gln Pro Met Val Pro Glu Ser565 570 575Leu Ala Asp Tyr Ile Thr Ala Ala Tyr Val Glu Met Arg Arg Glu Ala580 585 590Trp Ala Ser Lys Asp Ala Thr Tyr Thr Ser Ala Arg Thr Leu Leu Ala595 600 605Ile Leu Arg Leu Ser Thr Ala Leu Ala Arg Leu Arg Met Val Asp Val610 615 620Val Glu Lys Glu Asp Val Asn Glu Ala Ile Arg Leu Met Glu Met Ser625 630 635 640Lys Asp Ser Leu Leu Gly Asp Lys Gly Gln Thr Ala Arg Thr Gln Arg645 650 655Pro Ala Asp Val Ile Phe Ala Thr Val Arg Glu Leu Val Ser Gly Gly660 665 670Arg Ser Val Arg Phe Ser Glu Ala Glu Gln Arg Cys Val Ser Arg Gly675 680 685Phe Thr Pro Ala Gln Phe Gln Ala Ala Leu Asp Glu Tyr Glu Glu Leu690 695 700Asn Val Trp Gln Val Asn Ala Ser Arg Thr Arg Ile Thr Phe Val705 710 715152900DNAHomo sapiens 15agtgtcgtgt aaacagtgtc cttccgcgcg gcggccgcgg agagagctgc ggcccggggg 60ggcgtgcctg ggatccggag cttcgctcgg gcccgggaaa ggcggcagtg ggctgggatc 120gcggtgtctc tgggtgtgat ggccaatggc tggactggct cccgccctgg gcggaggaat 180cccgagctgt gaagcggctg gaatccgggc ccatgtgctt ctttgtttac taagagcgga 240agcgatggcg ggagcggggg tggggtgcgg tggcggggtg cggtggcgga ggtcccggtg 300aaatcagggg ctaaggggac ccaaagaagg cgggggatca taggggtgga aagaaagctg 360agaaccttga gaccggagtg tgaggggcca acggggaagg gcgctagaat tttaaactaa 420agtagggacc ggaattcccc tggggagatg ttggatggcc ctgtgcactg ccacgggctc 480tttattcttc gctggttaga aacagacttg tgaaaaagag ttatgcccac tttggggaga 540cttcgaaaag gttaagaagt tcttacaaga gttctaccag gatgatgaac tcgggaagaa 600gcagttcaag tatgggaacc agttggttcg gctggctcat cgggaacagg tggctctgta 660tgtggacctg gacgacgtag ccgaggatga ccccgagttg gtggactcaa tttgtgagaa 720tgccaggcgc tacgcgaagc tctttgctga tgccgtacaa gagctgctgc ctcagtacaa 780ggagagggaa gtggtaaata aagatgtcct ggacgtttac attgagcatc ggctaatgat 840ggagcagcgg agtcgggacc ctgggatggt ccgaagcccc cagaaccagt accctgctga 900actcatgcgc agattgtgag tggtctctgt cgggaaagat gtagggattg gttctccagg 960atcttgtttg tgactgtttt ctccccttag tgagctgtat tttcaaggcc ctagcagcaa 1020caagcctcgt gtgatccggg aagtgcgggc tgactctgtg gggaagttgg taactgtgcg 1080tggaatcgtc actcgtgtct ctgaagtcaa acccaagatg gtggtggcca cttacacttg 1140tgaccagtgt ggggcagaga cctaccagcc gatccagtct cccactttca tgcctctgat 1200catgtgccca agccaggagt gccaaaccaa ccgctcagga gggcggctgt atctgcagac 1260acggggctcc agattcatca aattccagga gatgaagatg caagaacata gtgatcaggt 1320gcctgtggga aatatccctc gtagtatcac ggtgctggta gaaggagaga acacaaggat 1380tgcccagcct ggagaccacg tcagcgtcac tggtattttc ttgccaatcc tgcgcactgg 1440gttccgacag gtggtacagg gtttactctc agaaacctac ctggaagccc atcggattgt 1500gaagatgaac aagagtgagg atgatgagtc tggggctgga gagctcacca gggaggagct 1560gaggcaaatt gcagaggagg atttctacga aaagctggca gcttcaatcg ccccagaaat 1620atacgggcat gaagatgtga agaaggcact gctgctcctg ctagtcgggg gtgtggacca 1680gtctcctcga ggcatgaaaa tccggggcaa catcaacatc tgtctgatgg gggatcctgg 1740tgtggccaag tctcagctcc tgtcatacat tgatcgactg gcgcctcgca gccagtacac 1800aacaggccgg ggctcctcag gagtggggct tacggcagct gtgctgagag actccgtgag 1860tggagaactg accttagagg gtggggccct ggtgctggct gaccagggtg tgtgctgcat 1920tgatgagttc gacaagatgg ctgaggccga ccgcacagcc atccacgagg tcatggagca 1980gcagaccatc tccattgcca aggccggcat tctcaccaca ctcaatgccc gctgctccat 2040cctggctgcc gccaaccctg cctacgggcg ctacaaccct cgccgcagcc tggagcagaa 2100catacagcta cctgctgcac tgctctcccg gtttgacctc ctctggctga ttcaggaccg 2160gcccgaccga gacaatgacc tacggttggc ccagcacatc acctatgtgc accagcacag 2220ccggcagccc ccctcccagt ttgaacctct ggacatgaag ctcatgaggc gttacatagc 2280catgtgccgc gagaagcagc ccatggtgcc agagtctctg gctgactaca tcacagcagc 2340atacgtggag atgaggcgag aggcttgggc tagtaaggat gccacctata cttctgcccg 2400gaccctgctg gctatcctgc gcctttccac tgctctggca cgtctgagaa tggtggatgt 2460ggtggagaaa gaagatgtga atgaagccat caggctaatg gagatgtcaa aggactctct 2520tctaggagac aaggggcaga cagctaggac tcagagacca gcagatgtga tatttgccac 2580cgtccgtgaa ctggtctcag ggggccgaag tgtccggttc tctgaggcag agcagcgctg 2640tgtatctcgt ggcttcacac ccgcccagtt ccaggcggct ctggatgaat atgaggagct 2700caatgtctgg caggtcaatg cttcccggac acggatcact tttgtctgat tccagcctgc 2760ttgcaaccct ggggtcctct tgttccctgc tggcctgccc cttgggaagg ggcagtgatg 2820cctttgaggg gaaggaggag cccctctttc tcccatgctg cacttactcc ttttgctaat 2880aaaagtgttt gtagattgtc 290016543PRTHomo sapiens 16Met Val Val Ala Thr Tyr Thr Cys Asp Gln Cys Gly Ala Glu Thr Tyr1 5 10 15Gln Pro Ile Gln Ser Pro Thr Phe Met Pro Leu Ile Met Cys Pro Ser20 25 30Gln Glu Cys Gln Thr Asn Arg Ser Gly Gly Arg Leu Tyr Leu Gln Thr35 40 45Arg Gly Ser Arg Phe Ile Lys Phe Gln Glu Met Lys Met Gln Glu His50 55 60Ser Asp Gln Val Pro Val Gly Asn Ile Pro Arg Ser Ile Thr Val Leu65 70 75 80Val Glu Gly Glu Asn Thr Arg Ile Ala Gln Pro Gly Asp His Val Ser85 90 95Val Thr Gly Ile Phe Leu Pro Ile Leu Arg Thr Gly Phe Arg Gln Val100 105 110Val Gln Gly Leu Leu Ser Glu Thr Tyr Leu Glu Ala His Arg Ile Val115 120 125Lys Met Asn Lys Ser Glu Asp Asp Glu Ser Gly Ala Gly Glu Leu Thr130 135 140Arg Glu Glu Leu Arg Gln Ile Ala Glu Glu Asp Phe Tyr Glu Lys Leu145 150 155 160Ala Ala Ser Ile Ala Pro Glu Ile Tyr Gly His Glu Asp Val Lys Lys165 170 175Ala Leu Leu Leu Leu Leu Val Gly Gly Val Asp Gln Ser Pro Arg Gly180 185 190Met Lys Ile Arg Gly Asn Ile Asn Ile Cys Leu Met Gly Asp Pro Gly195 200 205Val Ala Lys Ser Gln Leu Leu Ser Tyr Ile Asp Arg Leu Ala Pro Arg210 215 220Ser Gln Tyr Thr Thr Gly Arg Gly Ser Ser Gly Val Gly Leu Thr Ala225 230 235 240Ala Val Leu Arg Asp Ser Val Ser Gly Glu Leu Thr Leu Glu Gly Gly245 250 255Ala Leu Val Leu Ala Asp Gln Gly Val Cys Cys Ile Asp Glu Phe Asp260 265 270Lys Met Ala Glu Ala Asp Arg Thr Ala Ile His Glu Val Met Glu Gln275 280 285Gln Thr Ile Ser Ile Ala Lys Ala Gly Ile Leu Thr Thr Leu Asn Ala290 295 300Arg Cys Ser Ile Leu Ala Ala Ala Asn Pro Ala Tyr Gly Arg Tyr Asn305 310 315 320Pro Arg Arg Ser Leu Glu Gln Asn Ile Gln Leu Pro Ala Ala Leu Leu325 330 335Ser Arg Phe Asp Leu Leu Trp Leu Ile Gln Asp Arg Pro Asp Arg Asp340 345 350Asn Asp Leu Arg Leu Ala Gln His Ile Thr Tyr Val His Gln His Ser355 360 365Arg Gln Pro Pro Ser Gln Phe Glu Pro Leu Asp Met Lys Leu Met Arg370 375 380Arg Tyr Ile Ala Met Cys Arg Glu Lys Gln Pro Met Val Pro Glu Ser385 390 395 400Leu Ala Asp Tyr Ile Thr Ala Ala Tyr Val Glu Met Arg Arg Glu Ala405 410 415Trp Ala Ser Lys Asp Ala Thr Tyr Thr Ser Ala Arg Thr Leu Leu Ala420 425 430Ile Leu Arg Leu Ser Thr Ala Leu Ala Arg Leu Arg Met Val Asp Val435 440 445Val Glu Lys Glu Asp Val Asn Glu Ala Ile Arg Leu Met Glu Met Ser450 455 460Lys Asp Ser Leu Leu Gly Asp Lys Gly Gln Thr Ala Arg Thr Gln Arg465 470 475 480Pro Ala Asp Val Ile Phe Ala Thr Val Arg Glu Leu Val Ser Gly Gly485 490 495Arg Ser Val Arg Phe Ser Glu Ala Glu Gln Arg Cys Val Ser Arg Gly500 505 510Phe Thr Pro Ala Gln Phe Gln Ala Ala Leu Asp Glu Tyr Glu Glu Leu515 520 525Asn Val Trp Gln Val Asn Ala Ser Arg Thr Arg Ile Thr Phe Val530 535 540172140DNAHomo sapiens 17agctgaggtg tgagcagctg ccgaagtcag ttccttgtgg agccggagct gggcgcggat 60tcgccgaggc accgaggcac tcagaggagg cgccatgtca gaaccggctg gggatgtccg 120tcagaaccca tgcggcagca aggcctgccg ccgcctcttc ggcccagtgg acagcgagca 180gctgagccgc gactgtgatg cgctaatggc gggctgcatc caggaggccc gtgagcgatg 240gaacttcgac tttgtcaccg agacaccact ggagggtgac ttcgcctggg agcgtgtgcg 300gggccttggc ctgcccaagc tctaccttcc cacggggccc cggcgaggcc gggatgagtt 360gggaggaggc aggcggcctg gcacctcacc tgctctgctg caggggacag cagaggaaga 420ccatgtggac ctgtcactgt cttgtaccct tgtgcctcgc tcaggggagc aggctgaagg 480gtccccaggt ggacctggag actctcaggg tcgaaaacgg cggcagacca gcatgacaga 540tttctaccac tccaaacgcc ggctgatctt ctccaagagg aagccctaat ccgcccacag 600gaagcctgca gtcctggaag cgcgagggcc tcaaaggccc gctctacatc ttctgcctta 660gtctcagttt gtgtgtctta attattattt gtgttttaat ttaaacacct cctcatgtac 720ataccctggc cgccccctgc cccccagcct ctggcattag aattatttaa acaaaaacta 780ggcggttgaa tgagaggttc ctaagagtgc tgggcatttt tattttatga aatactattt 840aaagcctcct catcccgtgt tctccttttc ctctctcccg gaggttgggt gggccggctt 900catgccagct acttcctcct ccccacttgt ccgctgggtg gtaccctctg gaggggtgtg 960gctccttccc atcgctgtca caggcggtta tgaaattcac cccctttcct ggacactcag
1020acctgaattc tttttcattt gagaagtaaa cagatggcac tttgaagggg cctcaccgag 1080tgggggcatc atcaaaaact ttggagtccc ctcacctcct ctaaggttgg gcagggtgac 1140cctgaagtga gcacagccta gggctgagct ggggacctgg taccctcctg gctcttgata 1200cccccctctg tcttgtgaag gcagggggaa ggtggggtac tggagcagac caccccgcct 1260gccctcatgg cccctctgac ctgcactggg gagcccgtct cagtgttgag ccttttccct 1320ctttggctcc cctgtacctt ttgaggagcc ccagcttacc cttcttctcc agctgggctc 1380tgcaattccc ctctgctgct gtccctcccc cttgtctttc ccttcagtac cctctcatgc 1440tccaggtggc tctgaggtgc ctgtcccacc cccaccccca gctcaatgga ctggaagggg 1500aagggacaca caagaagaag ggcaccctag ttctacctca ggcagctcaa gcagcgaccg 1560ccccctcctc tagctgtggg ggtgagggtc ccatgtggtg gcacaggccc ccttgagtgg 1620ggttatctct gtgttagggg tatatgatgg gggagtagat ctttctagga gggagacact 1680ggcccctcaa atcgtccagc gaccttcctc atccacccca tccctcccca gttcattgca 1740ctttgattag cagcggaaca aggagtcaga cattttaaga tggtggcagt agaggctatg 1800gacagggcat gccacgtggg ctcatatggg gctgggagta gttgtctttc ctggcactaa 1860cgttgagccc ctggaggcac tgaagtgctt agtgtacttg gagtattggg gtctgacccc 1920aaacaccttc cagctcctgt aacatactgg cctggactgt tttctctcgg ctccccatgt 1980gtcctggttc ccgtttctcc acctagactg taaacctctc gagggcaggg accacaccct 2040gtactgttct gtgtctttca cagctcctcc cacaatgctg aatatacagc aggtgctcaa 2100taaatgattc ttagtgactt taaaaaaaaa aaaaaaaaaa 214018164PRTHomo sapiens 18Met Ser Glu Pro Ala Gly Asp Val Arg Gln Asn Pro Cys Gly Ser Lys1 5 10 15Ala Cys Arg Arg Leu Phe Gly Pro Val Asp Ser Glu Gln Leu Ser Arg20 25 30Asp Cys Asp Ala Leu Met Ala Gly Cys Ile Gln Glu Ala Arg Glu Arg35 40 45Trp Asn Phe Asp Phe Val Thr Glu Thr Pro Leu Glu Gly Asp Phe Ala50 55 60Trp Glu Arg Val Arg Gly Leu Gly Leu Pro Lys Leu Tyr Leu Pro Thr65 70 75 80Gly Pro Arg Arg Gly Arg Asp Glu Leu Gly Gly Gly Arg Arg Pro Gly85 90 95Thr Ser Pro Ala Leu Leu Gln Gly Thr Ala Glu Glu Asp His Val Asp100 105 110Leu Ser Leu Ser Cys Thr Leu Val Pro Arg Ser Gly Glu Gln Ala Glu115 120 125Gly Ser Pro Gly Gly Pro Gly Asp Ser Gln Gly Arg Lys Arg Arg Gln130 135 140Thr Ser Met Thr Asp Phe Tyr His Ser Lys Arg Arg Leu Ile Phe Ser145 150 155 160Lys Arg Lys Pro192281DNAHomo sapiens 19agctgaggtg tgagcagctg ccgaagtcag ttccttgtgg agccggagct gggcgcggat 60tcgccgaggc accgaggcac tcagaggagg tgagagagcg gcggcagaca acaggggacc 120ccgggccggc ggcccagagc cgagccaagc gtgcccgcgt gtgtccctgc gtgtccgcga 180ggatgcgtgt tcgcgggtgt gtgctgcgtt cacaggtgtt tctgcggcag gcgccatgtc 240agaaccggct ggggatgtcc gtcagaaccc atgcggcagc aaggcctgcc gccgcctctt 300cggcccagtg gacagcgagc agctgagccg cgactgtgat gcgctaatgg cgggctgcat 360ccaggaggcc cgtgagcgat ggaacttcga ctttgtcacc gagacaccac tggagggtga 420cttcgcctgg gagcgtgtgc ggggccttgg cctgcccaag ctctaccttc ccacggggcc 480ccggcgaggc cgggatgagt tgggaggagg caggcggcct ggcacctcac ctgctctgct 540gcaggggaca gcagaggaag accatgtgga cctgtcactg tcttgtaccc ttgtgcctcg 600ctcaggggag caggctgaag ggtccccagg tggacctgga gactctcagg gtcgaaaacg 660gcggcagacc agcatgacag atttctacca ctccaaacgc cggctgatct tctccaagag 720gaagccctaa tccgcccaca ggaagcctgc agtcctggaa gcgcgagggc ctcaaaggcc 780cgctctacat cttctgcctt agtctcagtt tgtgtgtctt aattattatt tgtgttttaa 840tttaaacacc tcctcatgta cataccctgg ccgccccctg ccccccagcc tctggcatta 900gaattattta aacaaaaact aggcggttga atgagaggtt cctaagagtg ctgggcattt 960ttattttatg aaatactatt taaagcctcc tcatcccgtg ttctcctttt cctctctccc 1020ggaggttggg tgggccggct tcatgccagc tacttcctcc tccccacttg tccgctgggt 1080ggtaccctct ggaggggtgt ggctccttcc catcgctgtc acaggcggtt atgaaattca 1140ccccctttcc tggacactca gacctgaatt ctttttcatt tgagaagtaa acagatggca 1200ctttgaaggg gcctcaccga gtgggggcat catcaaaaac tttggagtcc cctcacctcc 1260tctaaggttg ggcagggtga ccctgaagtg agcacagcct agggctgagc tggggacctg 1320gtaccctcct ggctcttgat acccccctct gtcttgtgaa ggcaggggga aggtggggtc 1380ctggagcaga ccaccccgcc tgccctcatg gcccctctga cctgcactgg ggagcccgtc 1440tcagtgttga gccttttccc tctttggctc ccctgtacct tttgaggagc cccagctacc 1500cttcttctcc agctgggctc tgcaattccc ctctgctgct gtccctcccc cttgtccttt 1560cccttcagta ccctctcagc tccaggtggc tctgaggtgc ctgtcccacc cccaccccca 1620gctcaatgga ctggaagggg aagggacaca caagaagaag ggcaccctag ttctacctca 1680ggcagctcaa gcagcgaccg ccccctcctc tagctgtggg ggtgagggtc ccatgtggtg 1740gcacaggccc ccttgagtgg ggttatctct gtgttagggg tatatgatgg gggagtagat 1800ctttctagga gggagacact ggcccctcaa atcgtccagc gaccttcctc atccacccca 1860tccctcccca gttcattgca ctttgattag cagcggaaca aggagtcaga cattttaaga 1920tggtggcagt agaggctatg gacagggcat gccacgtggg ctcatatggg gctgggagta 1980gttgtctttc ctggcactaa cgttgagccc ctggaggcac tgaagtgctt agtgtacttg 2040gagtattggg gtctgacccc aaacaccttc cagctcctgt aacatactgg cctggactgt 2100tttctctcgg ctccccatgt gtcctggttc ccgtttctcc acctagactg taaacctctc 2160gagggcaggg accacaccct gtactgttct gtgtctttca cagctcctcc cacaatgctg 2220aatatacagc aggtgctcaa taaatgattc ttagtgactt taaaaaaaaa aaaaaaaaaa 2280a 228120164PRTHomo sapiens 20Met Ser Glu Pro Ala Gly Asp Val Arg Gln Asn Pro Cys Gly Ser Lys1 5 10 15Ala Cys Arg Arg Leu Phe Gly Pro Val Asp Ser Glu Gln Leu Ser Arg20 25 30Asp Cys Asp Ala Leu Met Ala Gly Cys Ile Gln Glu Ala Arg Glu Arg35 40 45Trp Asn Phe Asp Phe Val Thr Glu Thr Pro Leu Glu Gly Asp Phe Ala50 55 60Trp Glu Arg Val Arg Gly Leu Gly Leu Pro Lys Leu Tyr Leu Pro Thr65 70 75 80Gly Pro Arg Arg Gly Arg Asp Glu Leu Gly Gly Gly Arg Arg Pro Gly85 90 95Thr Ser Pro Ala Leu Leu Gln Gly Thr Ala Glu Glu Asp His Val Asp100 105 110Leu Ser Leu Ser Cys Thr Leu Val Pro Arg Ser Gly Glu Gln Ala Glu115 120 125Gly Ser Pro Gly Gly Pro Gly Asp Ser Gln Gly Arg Lys Arg Arg Gln130 135 140Thr Ser Met Thr Asp Phe Tyr His Ser Lys Arg Arg Leu Ile Phe Ser145 150 155 160Lys Arg Lys Pro211275DNAHomo sapiens 21cctccctacg ggcgcctccg gcagcccttc ccgcgtgcgc agggctcaga gccgttccga 60gatcttggag gtccgggtgg gagtgggggt ggggtggggg tgggggtgaa ggtggggggc 120gggcgcgctc agggaaggcg ggtgcgcgcc tgcggggcgg agatgggcag ggggcggtgc 180gtgggtccca gtctgcagtt aagggggcag gagtggcgct gctcacctct ggtgccaaag 240ggcggcgcag cggctgccga gctcggccct ggaggcggcg agaacatggt gcgcaggttc 300ttggtgaccc tccggattcg gcgcgcgtgc ggcccgccgc gagtgagggt tttcgtggtt 360cacatcccgc ggctcacggg ggagtgggca gcgccagggg cgcccgccgc tgtggccctc 420gtgctgatgc tactgaggag ccagcgtcta gggcagcagc cgcttcctag aagaccaggt 480catgatgatg ggcagcgccc gagtggcgga gctgctgctg ctccacggcg cggagcccaa 540ctgcgccgac cccgccactc tcacccgacc cgtgcacgac gctgcccggg agggcttcct 600ggacacgctg gtggtgctgc accgggccgg ggcgcggctg gacgtgcgcg atgcctgggg 660ccgtctgccc gtggacctgg ctgaggagct gggccatcgc gatgtcgcac ggtacctgcg 720cgcggctgcg gggggcacca gaggcagtaa ccatgcccgc atagatgccg cggaaggtcc 780ctcagacatc cccgattgaa agaaccagag aggctctgag aaacctcggg aaacttagat 840catcagtcac cgaaggtcct acagggccac aactgccccc gccacaaccc accccgcttt 900cgtagttttc atttagaaaa tagagctttt aaaaatgtcc tgccttttaa cgtagatata 960tgccttcccc cactaccgta aatgtccatt tatatcattt tttatatatt cttataaaaa 1020tgtaaaaaag aaaaacaccg cttctgcctt ttcactgtgt tggagttttc tggagtgagc 1080actcacgccc taagcgcaca ttcatgtggg catttcttgc gagcctcgca gcctccggaa 1140gctgtcgact tcatgacaag cattttgtga actagggaag ctcagggggg ttactggctt 1200ctcttgagtc acactgctag caaatggcag aaccaaagct caaataaaaa taaaataatt 1260ttcattcatt cactc 127522173PRTHomo sapiens 22Met Gly Arg Gly Arg Cys Val Gly Pro Ser Leu Gln Leu Arg Gly Gln1 5 10 15Glu Trp Arg Cys Ser Pro Leu Val Pro Lys Gly Gly Ala Ala Ala Ala20 25 30Glu Leu Gly Pro Gly Gly Gly Glu Asn Met Val Arg Arg Phe Leu Val35 40 45Thr Leu Arg Ile Arg Arg Ala Cys Gly Pro Pro Arg Val Arg Val Phe50 55 60Val Val His Ile Pro Arg Leu Thr Gly Glu Trp Ala Ala Pro Gly Ala65 70 75 80Pro Ala Ala Val Ala Leu Val Leu Met Leu Leu Arg Ser Gln Arg Leu85 90 95Gly Gln Gln Pro Leu Pro Arg Arg Pro Gly His Asp Asp Gly Gln Arg100 105 110Pro Ser Gly Gly Ala Ala Ala Ala Pro Arg Arg Gly Ala Gln Leu Arg115 120 125Arg Pro Arg His Ser His Pro Thr Arg Ala Arg Arg Cys Pro Gly Gly130 135 140Leu Pro Gly His Ala Gly Gly Ala Ala Pro Gly Arg Gly Ala Ala Gly145 150 155 160Arg Ala Arg Cys Leu Gly Pro Ser Ala Arg Gly Pro Gly165 170235698DNAHomo sapiens 23aggttcaagt ggagctctcc taaccgacgc gcgtctgtgg agaagcggct tggtcggggg 60tggtctcgtg gggtcctgcc tgtttagtcg ctttcagggt tcttgagccc cttcacgacc 120gtcaccatgg aagtgtcacc attgcagcct gtaaatgaaa atatgcaagt caacaaaata 180aagaaaaatg aagatgctaa gaaaagactg tctgttgaaa gaatctatca aaagaaaaca 240caattggaac atattttgct ccgcccagac acctacattg gttctgtgga attagtgacc 300cagcaaatgt gggtttacga tgaagatgtt ggcattaact atagggaagt cacttttgtt 360cctggtttgt acaaaatctt tgatgagatt ctagttaatg ctgcggacaa caaacaaagg 420gacccaaaaa tgtcttgtat tagagtcaca attgatccgg aaaacaattt aattagtata 480tggaataatg gaaaaggtat tcctgttgtt gaacacaaag ttgaaaagat gtatgtccca 540gctctcatat ttggacagct cctaacttct agtaactatg atgatgatga aaagaaagtg 600acaggtggtc gaaatggcta tggagccaaa ttgtgtaaca tattcagtac caaatttact 660gtggaaacag ccagtagaga atacaagaaa atgttcaaac agacatggat ggataatatg 720ggaagagctg gtgagatgga actcaagccc ttcaatggag aagattatac atgtatcacc 780tttcagcctg atttgtctaa gtttaaaatg caaagcctgg acaaagatat tgttgcacta 840atggtcagaa gagcatatga tattgctgga tccaccaaag atgtcaaagt ctttcttaat 900ggaaataaac tgccagtaaa aggatttcgt agttatgtgg acatgtattt gaaggacaag 960ttggatgaaa ctggtaactc cttgaaagta atacatgaac aagtaaacca caggtgggaa 1020gtgtgtttaa ctatgagtga aaaaggcttt cagcaaatta gctttgtcaa cagcattgct 1080acatccaagg gtggcagaca tgttgattat gtagctgatc agattgtgac taaacttgtt 1140gatgttgtga agaagaagaa caagggtggt gttgcagtaa aagcacatca ggtgaaaaat 1200cacatgtgga tttttgtaaa tgccttaatt gaaaacccaa cctttgactc tcagacaaaa 1260gaaaacatga ctttacaacc caagagcttt ggatcaacat gccaattgag tgaaaaattt 1320atcaaagctg ccattggctg tggtattgta gaaagcatac taaactgggt gaagtttaag 1380gcccaagtcc agttaaacaa gaagtgttca gctgtaaaac ataatagaat caagggaatt 1440cccaaactcg atgatgccaa tgatgcaggg ggccgaaact ccactgagtg tacgcttatc 1500ctgactgagg gagattcagc caaaactttg gctgtttcag gccttggtgt ggttgggaga 1560gacaaatatg gggttttccc tcttagagga aaaatactca atgttcgaga agcttctcat 1620aagcagatca tggaaaatgc tgagattaac aatatcatca agattgtggg tcttcagtac 1680aagaaaaact atgaagatga agattcattg aagacgcttc gttatgggaa gataatgatt 1740atgacagatc aggaccaaga tggttcccac atcaaaggct tgctgattaa ttttatccat 1800cacaactggc cctctcttct gcgacatcgt tttctggagg aatttatcac tcccattgta 1860aaggtatcta aaaacaagca agaaatggca ttttacagcc ttcctgaatt tgaagagtgg 1920aagagttcta ctccaaatca taaaaaatgg aaagtcaaat attacaaagg tttgggcacc 1980agcacatcaa aggaagctaa agaatacttt gcagatatga aaagacatcg tatccagttc 2040aaatattctg gtcctgaaga tgatgctgct atcagcctgg cctttagcaa aaaacagata 2100gatgatcgaa aggaatggtt aactaatttc atggaggata gaagacaacg aaagttactt 2160gggcttcctg aggattactt gtatggacaa actaccacat atctgacata taatgacttc 2220atcaacaagg aacttatctt gttctcaaat tctgataacg agagatctat cccttctatg 2280gtggatggtt tgaaaccagg tcagagaaag gttttgttta cttgcttcaa acggaatgac 2340aagcgagaag taaaggttgc ccaattagct ggatcagtgg ctgaaatgtc ttcttatcat 2400catggtgaga tgtcactaat gatgaccatt atcaatttgg ctcagaattt tgtgggtagc 2460aataatctaa acctcttgca gcccattggt cagtttggta ccaggctaca tggtggcaag 2520gattctgcta gtccacgata catctttaca atgctcagct ctttggctcg attgttattt 2580ccaccaaaag atgatcacac gttgaagttt ttatatgatg acaaccagcg tgttgagcct 2640gaatggtaca ttcctattat tcccatggtg ctgataaatg gtgctgaagg aatcggtact 2700gggtggtcct gcaaaatccc caactttgat gtgcgtgaaa ttgtaaataa catcaggcgt 2760ttgatggatg gagaagaacc tttgccaatg cttccaagtt acaagaactt caagggtact 2820attgaagaac tggctccaaa tcaatatgtg attagtggtg aagtagctat tcttaattct 2880acaaccattg aaatctcaga gcttcccgtc agaacatgga cccagacata caaagaacaa 2940gttctagaac ccatgttgaa tggcaccgag aagacacctc ctctcataac agactatagg 3000gaataccata cagataccac tgtgaaattt gttgtgaaga tgactgaaga aaaactggca 3060gaggcagaga gagttggact acacaaagtc ttcaaactcc aaactagtct cacatgcaac 3120tctatggtgc tttttgacca cgtaggctgt ttaaagaaat atgacacggt gttggatatt 3180ctaagagact tttttgaact cagacttaaa tattatggat taagaaaaga atggctccta 3240ggaatgcttg gtgctgaatc tgctaaactg aataatcagg ctcgctttat cttagagaaa 3300atagatggca aaataatcat tgaaaataag cctaagaaag aattaattaa agttctgatt 3360cagaggggat atgattcgga tcctgtgaag gcctggaaag aagcccagca aaaggttcca 3420gatgaagaag aaaatgaaga gagtgacaac gaaaaggaaa ctgaaaagag tgactccgta 3480acagattctg gaccaacctt caactatctt cttgatatgc ccctttggta tttaaccaag 3540gaaaagaaag atgaactctg caggctaaga aatgaaaaag aacaagagct ggacacatta 3600aaaagaaaga gtccatcaga tttgtggaaa gaagacttgg ctacatttat tgaagaattg 3660gaggctgttg aagccaagga aaaacaagat gaacaagtcg gacttcctgg gaaagggggg 3720aaggccaagg ggaaaaaaac acaaatggct gaagttttgc cttctccgcg tggtcaaaga 3780gtcattccac gaataaccat agaaatgaaa gcagaggcag aaaagaaaaa taaaaagaaa 3840attaagaatg aaaatactga aggaagccct caagaagatg gtgtggaact agaaggccta 3900aaacaaagat tagaaaagaa acagaaaaga gaaccaggta caaagacaaa gaaacaaact 3960acattggcat ttaagccaat caaaaaagga aagaagagaa atccctggtc tgattcagaa 4020tcagatagga gcagtgacga aagtaatttt gatgtccctc cacgagaaac agagccacgg 4080agagcagcaa caaaaacaaa attcacaatg gatttggatt cagatgaaga tttctcagat 4140tttgatgaaa aaactgatga tgaagatttt gtcccatcag atgctagtcc acctaagacc 4200aaaacttccc caaaacttag taacaaagaa ctgaaaccac agaaaagtgt cgtgtcagac 4260cttgaagctg atgatgttaa gggcagtgta ccactgtctt caagccctcc tgctacacat 4320ttcccagatg aaactgaaat tacaaaccca gttcctaaaa agaatgtgac agtgaagaag 4380acagcagcaa aaagtcagtc ttccacctcc actaccggtg ccaaaaaaag ggctgcccca 4440aaaggaacta aaagggatcc agctttgaat tctggtgtct ctcaaaagcc tgatcctgcc 4500aaaaccaaga atcgccgcaa aaggaagcca tccacttctg atgattctga ctctaatttt 4560gagaaaattg tttcgaaagc agtcacaagc aagaaatcca agggggagag tgatgacttc 4620catatggact ttgactcagc tgtggctcct cgggcaaaat ctgtacgggc aaagaaacct 4680ataaagtacc tggaagagtc agatgaagat gatctgtttt aaaatgtgag gcgattattt 4740taagtaatta tcttaccaag cccaagactg gttttaaagt tacctgaagc tcttaacttc 4800ctcccctctg aatttagttt ggggaaggtg tttttagtac aagacatcaa agtgaagtaa 4860agcccaagtg ttctttagct ttttataata ctgtctaaat agtgaccatc tcatgggcat 4920tgttttcttc tctgctttgt ctgtgttttg agtctgcttt cttttgtctt taaaacctga 4980tttttaagtt cttctgaact gtagaaatag ctatctgatc acttcagcgt aaagcagtgt 5040gtttattaac catccactaa gctaaaacta gagcagtttg atttaaaagt gtcactcttc 5100ctccttttct actttcagta gatatgagat agagcataat tatctgtttt atcttagttt 5160tatacataat ttaccatcag atagaacttt atggttctag tacagatact ctactacact 5220cagcctctta tgtgccaagt ttttctttaa gcaatgagaa attgctcatg ttcttcatct 5280tctcaaatca tcagaggcca aagaaaaaca ctttggctgt gtctataact tgacacagtc 5340aatagaatga agaaaattag agtagttatg tgattatttc agctcttgac ctgtcccctc 5400tggctgcctc tgagtctgaa tctcccaaag agagaaacca atttctaaga ggactggatt 5460gcagaagact cggggacaac atttgatcca agatcttaaa tgttatattg ataaccatgc 5520tcagcaatga gctattagat tcattttggg aaatctccat aatttcaatt tgtaaacttt 5580gttaagacct gtctacattg ttatatgtgt gtgacttgag taatgttatc aacgtttttg 5640taaatattta ctatgttttt ctattagcta aattccaaca attttgtact ttaataaa 5698241531PRTHomo sapiens 24Met Glu Val Ser Pro Leu Gln Pro Val Asn Glu Asn Met Gln Val Asn1 5 10 15Lys Ile Lys Lys Asn Glu Asp Ala Lys Lys Arg Leu Ser Val Glu Arg20 25 30Ile Tyr Gln Lys Lys Thr Gln Leu Glu His Ile Leu Leu Arg Pro Asp35 40 45Thr Tyr Ile Gly Ser Val Glu Leu Val Thr Gln Gln Met Trp Val Tyr50 55 60Asp Glu Asp Val Gly Ile Asn Tyr Arg Glu Val Thr Phe Val Pro Gly65 70 75 80Leu Tyr Lys Ile Phe Asp Glu Ile Leu Val Asn Ala Ala Asp Asn Lys85 90 95Gln Arg Asp Pro Lys Met Ser Cys Ile Arg Val Thr Ile Asp Pro Glu100 105 110Asn Asn Leu Ile Ser Ile Trp Asn Asn Gly Lys Gly Ile Pro Val Val115 120 125Glu His Lys Val Glu Lys Met Tyr Val Pro Ala Leu Ile Phe Gly Gln130 135 140Leu Leu Thr Ser Ser Asn Tyr Asp Asp Asp Glu Lys Lys Val Thr Gly145 150 155 160Gly Arg Asn Gly Tyr Gly Ala Lys Leu Cys Asn Ile Phe Ser Thr Lys165 170 175Phe Thr Val Glu Thr Ala Ser Arg Glu Tyr Lys Lys Met Phe Lys Gln180 185 190Thr Trp Met Asp Asn Met Gly Arg Ala Gly Glu Met Glu Leu Lys Pro195 200 205Phe Asn Gly Glu Asp Tyr Thr Cys Ile Thr Phe Gln Pro Asp Leu Ser210 215 220Lys Phe Lys Met Gln Ser Leu Asp Lys Asp Ile Val Ala Leu Met Val225 230 235 240Arg Arg Ala Tyr Asp Ile Ala Gly Ser Thr Lys Asp Val Lys Val Phe245 250 255Leu Asn Gly Asn Lys Leu Pro Val Lys Gly Phe Arg Ser Tyr Val Asp260 265 270Met Tyr Leu Lys Asp Lys Leu Asp Glu Thr Gly Asn Ser Leu Lys Val275
280 285Ile His Glu Gln Val Asn His Arg Trp Glu Val Cys Leu Thr Met Ser290 295 300Glu Lys Gly Phe Gln Gln Ile Ser Phe Val Asn Ser Ile Ala Thr Ser305 310 315 320Lys Gly Gly Arg His Val Asp Tyr Val Ala Asp Gln Ile Val Thr Lys325 330 335Leu Val Asp Val Val Lys Lys Lys Asn Lys Gly Gly Val Ala Val Lys340 345 350Ala His Gln Val Lys Asn His Met Trp Ile Phe Val Asn Ala Leu Ile355 360 365Glu Asn Pro Thr Phe Asp Ser Gln Thr Lys Glu Asn Met Thr Leu Gln370 375 380Pro Lys Ser Phe Gly Ser Thr Cys Gln Leu Ser Glu Lys Phe Ile Lys385 390 395 400Ala Ala Ile Gly Cys Gly Ile Val Glu Ser Ile Leu Asn Trp Val Lys405 410 415Phe Lys Ala Gln Val Gln Leu Asn Lys Lys Cys Ser Ala Val Lys His420 425 430Asn Arg Ile Lys Gly Ile Pro Lys Leu Asp Asp Ala Asn Asp Ala Gly435 440 445Gly Arg Asn Ser Thr Glu Cys Thr Leu Ile Leu Thr Glu Gly Asp Ser450 455 460Ala Lys Thr Leu Ala Val Ser Gly Leu Gly Val Val Gly Arg Asp Lys465 470 475 480Tyr Gly Val Phe Pro Leu Arg Gly Lys Ile Leu Asn Val Arg Glu Ala485 490 495Ser His Lys Gln Ile Met Glu Asn Ala Glu Ile Asn Asn Ile Ile Lys500 505 510Ile Val Gly Leu Gln Tyr Lys Lys Asn Tyr Glu Asp Glu Asp Ser Leu515 520 525Lys Thr Leu Arg Tyr Gly Lys Ile Met Ile Met Thr Asp Gln Asp Gln530 535 540Asp Gly Ser His Ile Lys Gly Leu Leu Ile Asn Phe Ile His His Asn545 550 555 560Trp Pro Ser Leu Leu Arg His Arg Phe Leu Glu Glu Phe Ile Thr Pro565 570 575Ile Val Lys Val Ser Lys Asn Lys Gln Glu Met Ala Phe Tyr Ser Leu580 585 590Pro Glu Phe Glu Glu Trp Lys Ser Ser Thr Pro Asn His Lys Lys Trp595 600 605Lys Val Lys Tyr Tyr Lys Gly Leu Gly Thr Ser Thr Ser Lys Glu Ala610 615 620Lys Glu Tyr Phe Ala Asp Met Lys Arg His Arg Ile Gln Phe Lys Tyr625 630 635 640Ser Gly Pro Glu Asp Asp Ala Ala Ile Ser Leu Ala Phe Ser Lys Lys645 650 655Gln Ile Asp Asp Arg Lys Glu Trp Leu Thr Asn Phe Met Glu Asp Arg660 665 670Arg Gln Arg Lys Leu Leu Gly Leu Pro Glu Asp Tyr Leu Tyr Gly Gln675 680 685Thr Thr Thr Tyr Leu Thr Tyr Asn Asp Phe Ile Asn Lys Glu Leu Ile690 695 700Leu Phe Ser Asn Ser Asp Asn Glu Arg Ser Ile Pro Ser Met Val Asp705 710 715 720Gly Leu Lys Pro Gly Gln Arg Lys Val Leu Phe Thr Cys Phe Lys Arg725 730 735Asn Asp Lys Arg Glu Val Lys Val Ala Gln Leu Ala Gly Ser Val Ala740 745 750Glu Met Ser Ser Tyr His His Gly Glu Met Ser Leu Met Met Thr Ile755 760 765Ile Asn Leu Ala Gln Asn Phe Val Gly Ser Asn Asn Leu Asn Leu Leu770 775 780Gln Pro Ile Gly Gln Phe Gly Thr Arg Leu His Gly Gly Lys Asp Ser785 790 795 800Ala Ser Pro Arg Tyr Ile Phe Thr Met Leu Ser Ser Leu Ala Arg Leu805 810 815Leu Phe Pro Pro Lys Asp Asp His Thr Leu Lys Phe Leu Tyr Asp Asp820 825 830Asn Gln Arg Val Glu Pro Glu Trp Tyr Ile Pro Ile Ile Pro Met Val835 840 845Leu Ile Asn Gly Ala Glu Gly Ile Gly Thr Gly Trp Ser Cys Lys Ile850 855 860Pro Asn Phe Asp Val Arg Glu Ile Val Asn Asn Ile Arg Arg Leu Met865 870 875 880Asp Gly Glu Glu Pro Leu Pro Met Leu Pro Ser Tyr Lys Asn Phe Lys885 890 895Gly Thr Ile Glu Glu Leu Ala Pro Asn Gln Tyr Val Ile Ser Gly Glu900 905 910Val Ala Ile Leu Asn Ser Thr Thr Ile Glu Ile Ser Glu Leu Pro Val915 920 925Arg Thr Trp Thr Gln Thr Tyr Lys Glu Gln Val Leu Glu Pro Met Leu930 935 940Asn Gly Thr Glu Lys Thr Pro Pro Leu Ile Thr Asp Tyr Arg Glu Tyr945 950 955 960His Thr Asp Thr Thr Val Lys Phe Val Val Lys Met Thr Glu Glu Lys965 970 975Leu Ala Glu Ala Glu Arg Val Gly Leu His Lys Val Phe Lys Leu Gln980 985 990Thr Ser Leu Thr Cys Asn Ser Met Val Leu Phe Asp His Val Gly Cys995 1000 1005Leu Lys Lys Tyr Asp Thr Val Leu Asp Ile Leu Arg Asp Phe Phe Glu1010 1015 1020Leu Arg Leu Lys Tyr Tyr Gly Leu Arg Lys Glu Trp Leu Leu Gly Met1025 1030 1035 1040Leu Gly Ala Glu Ser Ala Lys Leu Asn Asn Gln Ala Arg Phe Ile Leu1045 1050 1055Glu Lys Ile Asp Gly Lys Ile Ile Ile Glu Asn Lys Pro Lys Lys Glu1060 1065 1070Leu Ile Lys Val Leu Ile Gln Arg Gly Tyr Asp Ser Asp Pro Val Lys1075 1080 1085Ala Trp Lys Glu Ala Gln Gln Lys Val Pro Asp Glu Glu Glu Asn Glu1090 1095 1100Glu Ser Asp Asn Glu Lys Glu Thr Glu Lys Ser Asp Ser Val Thr Asp1105 1110 1115 1120Ser Gly Pro Thr Phe Asn Tyr Leu Leu Asp Met Pro Leu Trp Tyr Leu1125 1130 1135Thr Lys Glu Lys Lys Asp Glu Leu Cys Arg Leu Arg Asn Glu Lys Glu1140 1145 1150Gln Glu Leu Asp Thr Leu Lys Arg Lys Ser Pro Ser Asp Leu Trp Lys1155 1160 1165Glu Asp Leu Ala Thr Phe Ile Glu Glu Leu Glu Ala Val Glu Ala Lys1170 1175 1180Glu Lys Gln Asp Glu Gln Val Gly Leu Pro Gly Lys Gly Gly Lys Ala1185 1190 1195 1200Lys Gly Lys Lys Thr Gln Met Ala Glu Val Leu Pro Ser Pro Arg Gly1205 1210 1215Gln Arg Val Ile Pro Arg Ile Thr Ile Glu Met Lys Ala Glu Ala Glu1220 1225 1230Lys Lys Asn Lys Lys Lys Ile Lys Asn Glu Asn Thr Glu Gly Ser Pro1235 1240 1245Gln Glu Asp Gly Val Glu Leu Glu Gly Leu Lys Gln Arg Leu Glu Lys1250 1255 1260Lys Gln Lys Arg Glu Pro Gly Thr Lys Thr Lys Lys Gln Thr Thr Leu1265 1270 1275 1280Ala Phe Lys Pro Ile Lys Lys Gly Lys Lys Arg Asn Pro Trp Ser Asp1285 1290 1295Ser Glu Ser Asp Arg Ser Ser Asp Glu Ser Asn Phe Asp Val Pro Pro1300 1305 1310Arg Glu Thr Glu Pro Arg Arg Ala Ala Thr Lys Thr Lys Phe Thr Met1315 1320 1325Asp Leu Asp Ser Asp Glu Asp Phe Ser Asp Phe Asp Glu Lys Thr Asp1330 1335 1340Asp Glu Asp Phe Val Pro Ser Asp Ala Ser Pro Pro Lys Thr Lys Thr1345 1350 1355 1360Ser Pro Lys Leu Ser Asn Lys Glu Leu Lys Pro Gln Lys Ser Val Val1365 1370 1375Ser Asp Leu Glu Ala Asp Asp Val Lys Gly Ser Val Pro Leu Ser Ser1380 1385 1390Ser Pro Pro Ala Thr His Phe Pro Asp Glu Thr Glu Ile Thr Asn Pro1395 1400 1405Val Pro Lys Lys Asn Val Thr Val Lys Lys Thr Ala Ala Lys Ser Gln1410 1415 1420Ser Ser Thr Ser Thr Thr Gly Ala Lys Lys Arg Ala Ala Pro Lys Gly1425 1430 1435 1440Thr Lys Arg Asp Pro Ala Leu Asn Ser Gly Val Ser Gln Lys Pro Asp1445 1450 1455Pro Ala Lys Thr Lys Asn Arg Arg Lys Arg Lys Pro Ser Thr Ser Asp1460 1465 1470Asp Ser Asp Ser Asn Phe Glu Lys Ile Val Ser Lys Ala Val Thr Ser1475 1480 1485Lys Lys Ser Lys Gly Glu Ser Asp Asp Phe His Met Asp Phe Asp Ser1490 1495 1500Ala Val Ala Pro Arg Ala Lys Ser Val Arg Ala Lys Lys Pro Ile Lys1505 1510 1515 1520Tyr Leu Glu Glu Ser Asp Glu Asp Asp Leu Phe1525 15302517DNAArtificial SequencePrimer 25ctctgagccc gccaagc 172631DNAArtificial SequencePrimer 26tgtaagaact tcttaacctt ttccttctct a 312720DNAArtificial SequencePrimer 27ccctcggcag cgatggcact 202820DNAArtificial SequencePrimer 28gaggaatccc gagctgtgaa 202914DNAArtificial SequencePrimer 29cccgctcccg ccat 143032DNAArtificial SequencePrimer 30cccatgtgct tctttgttta ctaagagcgg aa 323117DNAArtificial SequencePrimer 31gtccgaagcc cccagaa 173220DNAArtificial SequencePrimer 32cccgacagag accactcaca 203325DNAArtificial SequencePrimer 33cagtaccctg ctgaactcat gcgca 253419DNAArtificial SequencePrimer 34cgctacgcga agctctttg 193524DNAArtificial SequencePrimer 35cctttgtttg ccattgttct ctaa 243624DNAArtificial SequencePrimer 36tgccgtacaa gagctgctgc ctca 243718DNAArtificial SequencePrimer 37caaacgccgg ctgatctt 183819DNAArtificial SequencePrimer 38ccaggactgc aggcttcct 193924DNAArtificial SequencePrimer 39caagaggaag ccctaatccg ccca 244016DNAArtificial SequencePrimer 40gagcggcggc agacaa 164117DNAArtificial SequencePrimer 41ccgcgaacac gcatcct 174221DNAArtificial SequencePrimer 42cccagagccg agccaagcgt g 214321DNAArtificial SequencePrimer 43tggagactct cagggtcgaa a 214421DNAArtificial SequencePrimer 44tccagtctgg ccaacagagt t 214520DNAArtificial SequencePrimer 45cggcggcaga ccagcatgac 204620DNAArtificial SequencePrimer 46gccctcgtgc tgatgctact 204724DNAArtificial SequencePrimer 47tcatcatgac ctggtcttct agga 244821DNAArtificial SequencePrimer 48agcgtctagg gcagcagccg c 214915DNAArtificial SequencePrimer 49tgcccaacgc accga 155015DNAArtificial SequencePrimer 50gggcgctgcc catca 155122DNAArtificial SequencePrimer 51tcggaggccg atccaggtca tg 225220DNAArtificial SequencePrimer 52aagcttcctt tccgtcatgc 205322DNAArtificial SequencePrimer 53catgacctgc cagagagaac ag 225421DNAArtificial SequencePrimer 54cccccaccct ggctctgacc a 215523DNAArtificial SequencePrimer 55ggaaaccaag gaagaggaat gag 235622DNAArtificial SequencePrimer 56tgttcccccc ttcagatctt ct 225726DNAArtificial SequencePrimer 57acgcgcgtac agatctctcg aatgct 265818DNAArtificial SequencePrimer 58cacgccctaa gcgcacat 185925DNAArtificial SequencePrimer 59cctagttcac aaaatgcttg tcatg 256023DNAArtificial SequencePrimer 60tttcttgcga gcctcgcagc ctc 236124DNAArtificial SequencePrimer 61aaagaagatg atgaccgggt ttac 246219DNAArtificial SequencePrimer 62gagcctctgg atggtgcaa 196324DNAArtificial SequencePrimer 63caaactcaac gtgcaagcct cgga 246414DNAArtificial SequencePrimer 64tccgccgcgg acaa 146518DNAArtificial SequencePrimer 65catggtgtcc cgctcctt 186620DNAArtificial SequencePrimer 66accctggcct caggccggag 206722DNAArtificial SequencePrimer 67ggaattgttg gccacctgta tt 226827DNAArtificial SequencePrimer 68ctggagaaat cacttgttcc tatttct 276932DNAArtificial SequencePrimer 69cagtccttgc attatcattg aaacacctca ca 327028DNAArtificial SequencePrimer 70tcaactcatt ggaattacct cattattc 287124DNAArtificial SequencePrimer 71accatcagtg acgtaagcaa actc 247234DNAArtificial SequencePrimer 72ccaaacttga ggaaatctat gctcctaaac tcca 347325DNAArtificial SequencePrimer 73ttttgaagtt ctgcattctg acttg 257425DNAArtificial SequencePrimer 74accatcagtg acgtaagcaa gataa 257534DNAArtificial SequencePrimer 75aaccacagat gaggtccata cttctagact ggct 3476156PRTHomo sapiensVARIANT(1)...(156)Partial amino acid sequence for p16/p14ARF isoform 1 76Met Glu Pro Ala Ala Gly Ser Ser Met Glu Pro Ser Ala Asp Trp Leu1 5 10 15Ala Thr Ala Ala Ala Arg Gly Arg Val Glu Glu Val Arg Ala Leu Leu20 25 30Glu Ala Gly Ala Leu Pro Asn Ala Pro Asn Ser Tyr Gly Arg Arg Pro35 40 45Ile Gln Val Met Met Met Gly Ser Ala Arg Val Ala Glu Leu Leu Leu50 55 60Leu His Gly Ala Glu Pro Asn Cys Ala Asp Pro Ala Thr Leu Thr Arg65 70 75 80Pro Val His Asp Ala Ala Arg Glu Gly Phe Leu Asp Thr Leu Val Val85 90 95Leu His Arg Ala Gly Ala Arg Leu Asp Val Arg Asp Ala Trp Gly Arg100 105 110Leu Pro Val Asp Leu Ala Glu Glu Leu Gly His Arg Asp Val Ala Arg115 120 125Tyr Leu Arg Ala Ala Ala Gly Gly Thr Arg Gly Ser Asn His Ala Arg130 135 140Ile Asp Ala Ala Glu Gly Pro Ser Asp Ile Pro Asp145 150 15577105PRTHomo sapiensVARIANT(1)...(105)Partial amino acid sequence for p16/p14ARF isoform 2 77Met Met Met Gly Ser Ala Arg Val Ala Glu Leu Leu Leu Leu His Gly1 5 10 15Ala Glu Pro Asn Cys Ala Asp Pro Ala Thr Leu Thr Arg Pro Val His20 25 30Asp Ala Ala Arg Glu Gly Phe Leu Asp Thr Leu Val Val Leu His Arg35 40 45Ala Gly Ala Arg Leu Asp Val Arg Asp Ala Trp Gly Arg Leu Pro Val50 55 60Asp Leu Ala Glu Glu Leu Gly His Arg Asp Val Ala Arg Tyr Leu Arg65 70 75 80Ala Ala Ala Gly Gly Thr Arg Gly Ser Asn His Ala Arg Ile Asp Ala85 90 95Ala Glu Gly Pro Ser Asp Ile Pro Asp100 10578116PRTHomo sapiensVARIANT(1)...(116)Partial amino acid sequence for p16/p14ARF isoform 3 78Met Glu Pro Ala Ala Gly Ser Ser Met Glu Pro Ser Ala Asp Trp Leu1 5 10 15Ala Thr Ala Ala Ala Arg Gly Arg Val Glu Glu Val Arg Ala Leu Leu20 25 30Glu Ala Gly Ala Leu Pro Asn Ala Pro Asn Ser Tyr Gly Arg Arg Pro35 40 45Ile Gln Val Gly Arg Arg Ser Ala Ala Gly Ala Gly Asp Gly Gly Arg50 55 60Leu Trp Arg Thr Lys Phe Ala Gly Glu Leu Glu Ser Gly Ser Ala Ser65 70 75 80Ile Leu Arg Lys Lys Gly Arg Leu Pro Gly Glu Phe Ser Glu Gly Val85 90 95Cys Asn His Arg Pro Pro Pro Gly Asp Ala Leu Gly Ala Trp Glu Thr100 105 110Lys Glu Glu Glu11579173PRTHomo sapiens 79Met Gly Arg Gly Arg Cys Val Gly Pro Ser Leu Gln Leu Arg Gly Gln1 5 10 15Glu Trp Arg Cys Ser Pro Leu Val Pro Lys Gly Gly Ala Ala Ala Ala20 25 30Glu Leu Gly Pro Gly Gly Gly Glu Asn Met Val Arg Arg Phe Leu Val35 40 45Thr Leu Arg Ile Arg Arg Ala Cys Gly Pro Pro Arg Val Arg Val Phe50 55 60Val Val His Ile Pro Arg Leu Thr Gly Glu Trp Ala Ala Pro Gly Ala65 70 75 80Pro Ala Ala Val Ala Leu Val Leu Met Leu Leu Arg Ser Gln Arg Leu85 90 95Gly Gln Gln Pro Leu Pro Arg Arg Pro Gly His Asp Asp Gly Gln Arg100 105 110Pro Ser Gly Gly Ala Ala Ala Ala Pro Arg Arg Gly Ala Gln Leu Arg115 120 125Arg Pro Arg His Ser His Pro Thr Arg Ala Arg Arg Cys Pro Gly Gly130 135 140Leu Pro Gly His Ala Gly Gly Ala Ala Pro Gly Arg Gly Ala Ala Gly145 150 155 160Arg Ala Arg Cys Leu Gly Pro Ser Ala Arg Gly Pro Gly165
1708021DNAArtificial SequencePCR primer 80ggaggtggta ctggccatgt a 218119DNAArtificial SequencePCR primer 81gggagatgcg gacatggat 198225DNAArtificial SequencePCR primer 82ccaagtacga ccgcatcacc aacca 258321DNAArtificial SequencePCR primer 83cattccaaga cctgcctacc a 218421DNAArtificial SequencePCR primer 84atgcgagtga gcaaaccaat t 218529DNAArtificial SequencePCR primer 85acacaagatt cgagagctca cctcatcca 298619DNAArtificial SequencePCR primer 86ggctacatgg tggcaagga 198723DNAArtificial SequencePCR primer 87tggaaataac aatcgagcca aag 238834DNAArtificial SequencePCR primer 88tgctagtcca cgatacatct ttacaatgct cagc 34893769DNAHomo sapiens 89aaagctgcag cgtctggaaa aaagcgactt gtggcggtcg agcgtggcgc aggcgaatcc 60tcggcactaa gcaaatatgg acctcgcggc ggcagcggag ccgggcgccg gcagccagca 120cctggaggtc cgcgacgagg tggccgagaa gtgccagaaa ctgttcctgg acttcttgga 180ggagtttcag agcagcgatg gagaaattaa atacttgcaa ttagcagagg aactgattcg 240tcctgagaga aacacattgg ttgtgagttt tgtggacctg gaacaattta accagcaact 300ttccaccacc attcaagagg agttctatag agtttaccct tacctgtgtc gggccttgaa 360aacattcgtc aaagaccgta aagagatccc tcttgccaag gatttttatg ttgcattcca 420agacctgcct accagacaca agattcgaga gctcacctca tccagaattg gtttgctcac 480tcgcatcagt gggcaggtgg tgcggactca cccagttcac ccagagcttg tgagcggaac 540ttttctgtgc ttggactgtc agacagtgat cagggatgta gaacagcagt tcaaatacac 600acagccaaac atctgccgaa atccagtttg tgccaacagg aggagattct tactggatac 660aaataaatca agatttgttg attttcaaaa ggttcgtatt caagagaccc aagctgagct 720tcctcgaggg agtatccccc gcagtttaga agtaatttta agggctgaag ctgtggaatc 780agctcaagct ggtgacaagt gtgactttac agggacactg attgttgtgc ctgacgtctc 840caagcttagc acaccaggag cacgtgcaga aactaattcc cgtgtcagtg gtgttgatgg 900atatgagaca gaaggcattc gaggactccg ggcccttggt gttagggacc tttcttatag 960gctggtcttt cttgcctgct gtgttgcgcc aaccaaccca aggtttgggg ggaaagagct 1020cagagatgag gaacagacag ctgagagcat taagaaccaa atgactgtga aagaatggga 1080gaaagtgttt gagatgagtc aagataaaaa tctataccac aatctttgta ccagcctgtt 1140ccctactata catggcaatg atgaagtaaa acggggtgtc ctgctgatgc tctttggtgg 1200cgttccaaag acaacaggag aagggacctc tcttcgaggg gacataaatg tttgcattgt 1260tggtgaccca agtacagcta agagccaatt tctcaagcac gtggaggagt tcagccccag 1320agctgtctac accagtggta aagcgtccag tgctgctggc ttaacagcag ctgttgtgag 1380agatgaagaa tctcatgagt ttgtcattga ggctggagct ttgatgttgg ctgataatgg 1440tgtgtgttgt attgatgaat ttgataagat ggacgtgcgg gatcaagttg ctattcatga 1500agctatggaa cagcagacca tatccatcac taaagcagga gtgaaggcta ctctgaacgc 1560ccggacgtcc attttggcag cagcaaaccc aatcagtgga cactatgaca gatcaaaatc 1620attgaaacag aatataaatt tgtcagctcc catcatgtcc cgattcgatc tcttctttat 1680ccttgtggat gaatgtaatg aggttacaga ttatgccatt gccaggcgca tagtagattt 1740gcattcaaga attgaggaat caattgatcg tgtctattcc ctcgatgata tcagaagata 1800tcttctcttt gcaagacagt ttaaacccaa gatttccaaa gagtcagagg acttcattgt 1860ggagcaatat aaacatctcc gccagagaga tggttctgga gtgaccaagt cttcatggag 1920gattacagtg cgacagcttg agagcatgat tcgtctctct gaagctatgg ctcggatgca 1980ctgctgtgat gaggtccaac ctaaacatgt gaaggaagct ttccggttac tgaataaatc 2040aatcatccgt gtggaaacac ctgatgtcaa tctagatcaa gaggaagaga tccagatgga 2100ggtagatgag ggtgctggtg gcatcaatgg tcatgctgac agccctgctc ctgtgaacgg 2160gatcaatggc tacaatgaag acataaatca agagtctgct cccaaagcct ccttaaggct 2220gggcttctct gagtactgcc gaatctctaa ccttattgtg cttcacctca gaaaggtgga 2280agaagaagag gacgagtcag cattaaagag gagcgagctt gttaactggt acttgaagga 2340aatcgaatca gagatagact ctgaagaaga acttataaat aaaaaaagaa tcatagagaa 2400agttattcat cgactcacac actatgatca tgttctaatt gagctcaccc aggctggatt 2460gaaaggctcc acagagggaa gtgagagcta tgaagaagat ccctacttgg tagttaaccc 2520taactacttg ctcgaagatt gagatagtga aagtaactga ccagagctga ggaactgtgg 2580cacagcacct cgtggcctgg agcctggctg gagctctgct agggacagaa gtgtttctgg 2640aagtgatgct tccaggattt gttttcagaa acaagaattg agttgatggt cctatgtgtc 2700acattcatca caggtttcat accaacacag gcttcagcac ttcctttggt gtgtttcctg 2760tcccagtgaa gttggaacca aataatgtgt agtctctata accaatacct ttgttttcat 2820gtgtaagaaa aggcccatta cttttaaggt atgtgctgtc ctattgagca aataactttt 2880tttcaattgc cagctactgc ttttattcat caaaataaaa taacttgttc tgaagttgtc 2940tattggattt ctttctactg taccctgatt attacttcca tctacttctg aatgtgagac 3000tttccctttt tgcttaacct ggagtgaaga ggtagaactg tggtattatg gatgaggttt 3060ctatgagaag gagtcattag agaactcata tgaaagctag aggccttaga gatgactttc 3120caaggttaat tccagttgtt tttttttttt tttaagttta taaaagttta ttatactttt 3180ttaaaattac tctttagtaa tttattttac ttctgtgtcc taagggtaat ttctcaggat 3240tgttttcaaa ttgctttttt aggggaaata ggtcatttgc tatattacaa gcaatcccca 3300aattttatgg tcttccagga aaagttatta ccgtttatga tactaacagt tcctgagact 3360tagctatgat cagtatgttc atgaggtgga gcagttcctg tgttgcagct tttaacaaca 3420gatggcattc attaaatcac aaagtatgtt aaaggtcaca aaagcaaaat aactgtctga 3480ggctaaggcc cacgtgggac agtctaatac ccatgagtac tcaacttgcc ttgatgtctg 3540agctttccag tgcaatgtga atttgagcag ccagaaatct attagtagaa agcaagacag 3600attaatatag gttaaaacaa tgatttaaat atgtttctcc caataattat ctctttccct 3660ggaatcaact tgtatgaaac cttgtcaaaa tgtactccac aagtatgtac aattaagtat 3720tttaaaaata aatggcaaac attaaaaaca aaaaaaaaaa aaaaaaaaa 376990821PRTHomo sapiens 90Met Asp Leu Ala Ala Ala Ala Glu Pro Gly Ala Gly Ser Gln His Leu1 5 10 15Glu Val Arg Asp Glu Val Ala Glu Lys Cys Gln Lys Leu Phe Leu Asp20 25 30Phe Leu Glu Glu Phe Gln Ser Ser Asp Gly Glu Ile Lys Tyr Leu Gln35 40 45Leu Ala Glu Glu Leu Ile Arg Pro Glu Arg Asn Thr Leu Val Val Ser50 55 60Phe Val Asp Leu Glu Gln Phe Asn Gln Gln Leu Ser Thr Thr Ile Gln65 70 75 80Glu Glu Phe Tyr Arg Val Tyr Pro Tyr Leu Cys Arg Ala Leu Lys Thr85 90 95Phe Val Lys Asp Arg Lys Glu Ile Pro Leu Ala Lys Asp Phe Tyr Val100 105 110Ala Phe Gln Asp Leu Pro Thr Arg His Lys Ile Arg Glu Leu Thr Ser115 120 125Ser Arg Ile Gly Leu Leu Thr Arg Ile Ser Gly Gln Val Val Arg Thr130 135 140His Pro Val His Pro Glu Leu Val Ser Gly Thr Phe Leu Cys Leu Asp145 150 155 160Cys Gln Thr Val Ile Arg Asp Val Glu Gln Gln Phe Lys Tyr Thr Gln165 170 175Pro Asn Ile Cys Arg Asn Pro Val Cys Ala Asn Arg Arg Arg Phe Leu180 185 190Leu Asp Thr Asn Lys Ser Arg Phe Val Asp Phe Gln Lys Val Arg Ile195 200 205Gln Glu Thr Gln Ala Glu Leu Pro Arg Gly Ser Ile Pro Arg Ser Leu210 215 220Glu Val Ile Leu Arg Ala Glu Ala Val Glu Ser Ala Gln Ala Gly Asp225 230 235 240Lys Cys Asp Phe Thr Gly Thr Leu Ile Val Val Pro Asp Val Ser Lys245 250 255Leu Ser Thr Pro Gly Ala Arg Ala Glu Thr Asn Ser Arg Val Ser Gly260 265 270Val Asp Gly Tyr Glu Thr Glu Gly Ile Arg Gly Leu Arg Ala Leu Gly275 280 285Val Arg Asp Leu Ser Tyr Arg Leu Val Phe Leu Ala Cys Cys Val Ala290 295 300Pro Thr Asn Pro Arg Phe Gly Gly Lys Glu Leu Arg Asp Glu Glu Gln305 310 315 320Thr Ala Glu Ser Ile Lys Asn Gln Met Thr Val Lys Glu Trp Glu Lys325 330 335Val Phe Glu Met Ser Gln Asp Lys Asn Leu Tyr His Asn Leu Cys Thr340 345 350Ser Leu Phe Pro Thr Ile His Gly Asn Asp Glu Val Lys Arg Gly Val355 360 365Leu Leu Met Leu Phe Gly Gly Val Pro Lys Thr Thr Gly Glu Gly Thr370 375 380Ser Leu Arg Gly Asp Ile Asn Val Cys Ile Val Gly Asp Pro Ser Thr385 390 395 400Ala Lys Ser Gln Phe Leu Lys His Val Glu Glu Phe Ser Pro Arg Ala405 410 415Val Tyr Thr Ser Gly Lys Ala Ser Ser Ala Ala Gly Leu Thr Ala Ala420 425 430Val Val Arg Asp Glu Glu Ser His Glu Phe Val Ile Glu Ala Gly Ala435 440 445Leu Met Leu Ala Asp Asn Gly Val Cys Cys Ile Asp Glu Phe Asp Lys450 455 460Met Asp Val Arg Asp Gln Val Ala Ile His Glu Ala Met Glu Gln Gln465 470 475 480Thr Ile Ser Ile Thr Lys Ala Gly Val Lys Ala Thr Leu Asn Ala Arg485 490 495Thr Ser Ile Leu Ala Ala Ala Asn Pro Ile Ser Gly His Tyr Asp Arg500 505 510Ser Lys Ser Leu Lys Gln Asn Ile Asn Leu Ser Ala Pro Ile Met Ser515 520 525Arg Phe Asp Leu Phe Phe Ile Leu Val Asp Glu Cys Asn Glu Val Thr530 535 540Asp Tyr Ala Ile Ala Arg Arg Ile Val Asp Leu His Ser Arg Ile Glu545 550 555 560Glu Ser Ile Asp Arg Val Tyr Ser Leu Asp Asp Ile Arg Arg Tyr Leu565 570 575Leu Phe Ala Arg Gln Phe Lys Pro Lys Ile Ser Lys Glu Ser Glu Asp580 585 590Phe Ile Val Glu Gln Tyr Lys His Leu Arg Gln Arg Asp Gly Ser Gly595 600 605Val Thr Lys Ser Ser Trp Arg Ile Thr Val Arg Gln Leu Glu Ser Met610 615 620Ile Arg Leu Ser Glu Ala Met Ala Arg Met His Cys Cys Asp Glu Val625 630 635 640Gln Pro Lys His Val Lys Glu Ala Phe Arg Leu Leu Asn Lys Ser Ile645 650 655Ile Arg Val Glu Thr Pro Asp Val Asn Leu Asp Gln Glu Glu Glu Ile660 665 670Gln Met Glu Val Asp Glu Gly Ala Gly Gly Ile Asn Gly His Ala Asp675 680 685Ser Pro Ala Pro Val Asn Gly Ile Asn Gly Tyr Asn Glu Asp Ile Asn690 695 700Gln Glu Ser Ala Pro Lys Ala Ser Leu Arg Leu Gly Phe Ser Glu Tyr705 710 715 720Cys Arg Ile Ser Asn Leu Ile Val Leu His Leu Arg Lys Val Glu Glu725 730 735Glu Glu Asp Glu Ser Ala Leu Lys Arg Ser Glu Leu Val Asn Trp Tyr740 745 750Leu Lys Glu Ile Glu Ser Glu Ile Asp Ser Glu Glu Glu Leu Ile Asn755 760 765Lys Lys Arg Ile Ile Glu Lys Val Ile His Arg Leu Thr His Tyr Asp770 775 780His Val Leu Ile Glu Leu Thr Gln Ala Gly Leu Lys Gly Ser Thr Glu785 790 795 800Gly Ser Glu Ser Tyr Glu Glu Asp Pro Tyr Leu Val Val Asn Pro Asn805 810 815Tyr Leu Leu Glu Asp820
Patent applications by Adriann J. Taylor, Durham, NC US
Patent applications by Douglas P. Malinowski, Hillsborough, NC US
Patent applications by Margaret R. Parker, Raleigh, NC US
Patent applications by Timothy J. Fischer, Raleigh, NC US
Patent applications by TriPath Imaging, Inc
Patent applications in class Involving antigen-antibody binding, specific binding protein assay or specific ligand-receptor binding assay
Patent applications in all subclasses Involving antigen-antibody binding, specific binding protein assay or specific ligand-receptor binding assay