(Philadelphia, PA) - Researchers at the University
of Pennsylvania School of Medicine have developed a paradigm-shifting
method for detecting small amounts of proteins in the blood. Applications
of this method will make discerning low-abundance molecules associated
with cancers (such as breast cancer), Alzheimer’s disease,
prion diseases, and possibly psychiatric diseases relatively easy
and more accurate compared with the current methodology, including
the widely used ELISA (enzyme-linked immunoadsorbent assay).
ELISA is a common immune-system-based assay that uses enzymes linked
to an antibody or antigen as a marker for picking out specific proteins.
For example, it is used as a diagnostic test to determine exposure
to infectious agents, such as HIV, by identifying antibodies present
in a blood sample.
The sensitivity of detecting molecules by the new method, called
FACTT, short for Fluorescent Amplification Catalyzed by T7-polymerase
Technique, is five orders of magnitude (100,000 times) greater than
that of ELISA, the Penn researchers found.
Senior author Mark I. Greene MD, PhD, the John
Eckman Professor of Medical Science, Hongtao Zhang, PhD
research specialist; Xin Cheng, PhD, research investigator,
and Mark Richter, a research technician in Greene’s
lab, report their findings in the advanced online publication of
“The current ELISA tests can only detect proteins when they
are in high abundance,” says Zhang. “But the problem
is that many of the functional proteins - those that have a role
in determining your health - exist in very low amounts until diseases
are apparent and cannot be detected or measured at early stages
of medical pathology. It was important to develop a technique that
can detect these rare molecules to detect abnormalities at an early
The FACTT technology uses a different enzyme amplification system
so quantitative signals can be obtained from even a few protein
molecules compared to ELISA. “The technology is remarkably
adaptable to any protein and can be performed in an automated format,”
notes Greene. He states that the technology will soon be robotized
so as to be able to screen for many rare disease-causing proteins
using tiny amounts of blood. “It is even possible that one
could screen for multiple diseases at the same time and produce
a precise accounting of whether disease-causing molecules are present
at an early time when disease can be readily treated,” adds
Greene also noted that the FACTT technology represents the further
evolution of an earlier approach that was developed in collaboration
with Professor of Pharmacology James Eberwine, PhD,
also from Penn. The earlier technique employed radioisotopes.
Development of a test for the cancer marker Her2/neu
The researchers compared detection of Her2/neu in the blood between
ELISA and FACTT. Her2/neu proteins were in fact first identified
by the Greene laboratory in the early 1980s, and the Her2/neu gene
was found by other scientists to be overexpressed in breast cancer.
Her2/neu is normally a low-abundance molecule that becomes overexpressed
in more than 30 percent of primary breast, ovarian, and pancreatic
tumors. Part of the Her2/neu molecule is shed from the surface of
tumor cells and has been detected in the blood of breast-cancer
patients. Higher blood concentrations of Her2/neu correlate with
a lower response rate to chemotherapy and shorter survival time
The Greene lab developed mouse models that carry cancer cells overexpressing
Her2/neu. When these cells are implanted into animals they form
tumors exactly like breast tumors in humans. Using ELISA, the researchers
could not detect Her2/neu from mouse blood until the tumors reached
an inoperable size, but with the new FACTT technology they could
detect Her2/Neu in some mice when tumors were barely visible and
within two days of implantation. These results indicate that it
is possible to detect tumors at very early stages so that tumor
emergence or reoccurrence can be rapidly treated or even prevented.
Greene’s laboratory established many of the principles of
targeted therapy for Her2/neu tumors and the prototype antibodies
that led to the development of Herceptin, a similar antibody molecule
that was created by Genentech. The Greene laboratory also previously
showed that early treatment of Her2/neu tumors with targeted monoclonal
antibodies in animal models led to far more significant prevention
of tumor growth as well as tumor emergence and reoccurrence.
Greene stresses that early treatment is far more effective than
treating advanced tumors with the same antibodies. Recent clinical
trials support the notion that early treatment prevents tumor reoccurrence
in women with breast tumors. FACTT technology represents a way to
couple early diagnosis with early treatment to prevent tumor emergence.
Detecting Her2/neu in humans for breast cancer
The most widely used clinical Her2/neu tests are IHC (immunohistochemistry)
and FISH (fluorescence in situ hybridization). However, both FISH
and IHC are complex, time-consuming tests.
Patients who test positive for Her2/neu using FISH or IHC have responsive
rates of about 35 percent to the cancer drug Herceptin. Monitoring
Her2/neu status from the blood with a powerful technology such as
FACTT represents an alternative approach compared to IHC or FISH,
say the researchers.
Pre-treatment Her2/neu levels correlate with tumor size and the
extent of disease. Post-treatment Her2/neu levels predict disease-specific
survival. A more sensitive assay could more accurately allow treatment
of humans with breast cancer and allow treatment more quickly if
the tumor reoccurs.
The researchers collected blood samples from healthy women and breast
cancer patients who did or did not overexpress Her2/neu, as detected
by IHC and FISH. When using the new FACTT method her2/neu positive
cancer patients showed dramatically elevated Her2/neu levels (average:
384 ng/ml), while the level in Her2/neu-negative breast cancer patients
(19.5 ng/ml) were close to the levels of the healthy control participants
Using FACTT, nine out of 10 of the Her2/neu positive patients had
elevated Her2/neu levels and one out of four in the Her2/neu negative
group had elevated Her2/neu levels. Using ELISA only two out of
10 in the Her2/neu positive group showed elevated Her2/neu levels.
“Clearly the sensitivity of the ELISA assay does not satisfy
the current need for the clinical detection of marker proteins that
determine whether a patient has breast cancer or not,” says
The researchers have also tried the FACTT method on other rare,
but medically important molecules, such as the prion protein (for
mad cow disease with Mansun Sy at Case Western University) and TNF-alpha
(for autoimmune diseases), and will be developing tests for other
cancer markers including lung cancer and colon cancer. All proteins
tested so far with FACTT have been detected with an over 1000-fold
higher sensitivity compared to current technologies.
The researchers say this points to FACTT’s broad applicability
and compatibility with current high-throughput testing technology.
This, in turn, will facilitate the detection of rare markers and
not-so-rare targets from much smaller sample volumes, as well as
aid in monitoring marker levels at much earlier stages of disease.
“The importance of FACTT is that we can still get an accurate
description of the number of molecules that cause disease even when
other assays cannot,” says Greene. The researchers surmise
that FACTT could be used to monitor levels of Her2/neu in already-diagnosed
breast cancer patients to monitor recurrence or treatment effectiveness.
“The critical issue arises when women are diagnosed with early
breast cancer,” adds Greene. “They often have a lumpectomy
and are sometimes treated with radiation or chemotherapy, but despite
this conventional therapy the cancer still can occasionally reoccur,”
says Greene. Detection of very early recurrence is important and
Greene feels the power of this technology will facilitate recognizing
early phases of tumor emergence.
Rational targeted therapy has shown in animal models - over 10 years
ago - and more recently in clinical trials that treatment of small
or incipient tumors is a way to prevent tumor emergence or reoccurrence.
“Prevention of the consequences of recurrence is critical
since treating advanced tumors is very complex and difficult,”
This research was funded in part by The Abramson Family Cancer Research
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