- Tailoring cancer therapy with the help of PCR analysis
In the latest campaign in the struggle against cancer, researchers
are working to devise ways to individualize cancer treatment. One attempt involves
therapies that block the epidermal growth factor receptor. Signaling of this receptor
leads to increases in proliferation, angiogenesis and metastasis, and to decreases
in apoptosis. Clinical trials have shown that compounds such as gefitinib and erlotinib
effectively inhibit epidermal growth factor receptor activity, but the factors that
determine and predict their efficacy — knowledge that could aid in individualization
of treatment — remain largely unknown.
Investigators with Johns Hopkins School of Medicine
in Baltimore are exploring these factors. In ongoing work, they are using a quantitative
polymerase chain reaction (PCR) amplification technique to probe genes of interest
in the RNA of patients treated with targeted compounds. They hope to develop a methodology
that will allow clinicians to predict the efficacy of drugs in individual patients
by measuring specific biomarkers in ex vivo samples and, subsequently, to tailor
cancer treatment for them.
Researchers are using a quantitative PCR method
to predict how effective specific drugs will be in individual cancer patients. Ultimately,
this may help to tailor therapies to those patients.
They have looked at the c-fos
oncogene, which often is used to identify and characterize factors affecting cancer
cell growth, including in assessments of epidermal growth factor receptor activation
and anti-epidermal growth factor receptor therapy. Specifically, they examined
whether variations in this gene corresponded to the receptor’s inhibition
both in vitro and in vivo as well as the extent to which it can be used as a biomarker
for predicting a patient’s sensitivity to epidermal growth factor receptor
therapy based on an ex vivo approach.
The researchers are using an MX3000P
quantitative PCR system from Stratagene Inc. of La Jolla, Calif., for real-time
reverse transcription analysis. The fluorescence-based instrument features four
optical channels, and a specific set of four filters can be chosen at the time of
purchase for flexibility. A quartz tungsten-halogen lamp provides excitation in
the 350- to 700-nm range, while a single photomultiplier tube provides detection.
The company introduced this line of
instruments in 2003. Rena McClory, director of product marketing of instrumentation,
noted that the system used in the current study was one of the first to offer its
particular set of features for less than $40,000. At the time, she said, most other
instruments cost in the area of $45,000 to $55,000; this system was launched at
Since the original launch, the company
has released regular software upgrades — at least one per year — to
incorporate new features and meet new demands, often as requested by customers.
The upgrades are generally provided to customers at no cost.
Peter Kulesza, one of the Johns Hopkins
researchers, said that he and his colleagues chose the system over comparably priced
instruments because of the quality of its software and its convenience — it’s
very fast and has a small footprint, he explained. Also, the instrument delivers
the reproducibility that is needed for their assays.
The scientists have probed the inhibitory
and c-fos-modulating effects of gefitinib and erlotinib in human cancer cell
lines. To do this, they xenografted the cell lines in mice, which were treated with
the drugs for 14 days. They performed fine-needle-aspiration biopsy of tumors at
the end of the 14 days, as well as at baseline, to determine the efficacy of the
therapy. They also looked at the biomarker in five paired tumor samples from a clinical
trial of gefitinib to assess its potential for clinical translation.
As detailed in the Feb. 15 issue of
Cancer Research, the experiments showed that the compounds inhibited c-fos
expression in cell lines that are inherently sensitive to epidermal growth factor
receptor inhibitors (though not in cell lines that are intrinsically resistant).
They also found that they could reliably measure c-fos levels in clinical
materials. This suggested that ex vivo assays could help to determine a patient’s
potential level of responsiveness prior to cancer therapy, which would represent
a considerable step forward in developing individualized treatments.
They emphasized, however, that the
latter findings should be viewed with caution: Validation in a much larger tumor
population will be needed before the ex vivo paradigm can be established for clinical
purposes. And the technique itself requires further development. The researchers
are working on another soon-to-be-submitted paper that is methodology-based. In
it, they use data from more than 200 samples of human cancer cell lines xenografted
in mice, acquired with the same instrument, to demonstrate an approach to normalization.
This protocol is based on Kulesza’s experience with housekeeping genes. “The
idea is to not have any genes affected by therapy and be able to compare between
specimens,” he said. “That is why we use the equation.”
As for the quantitative PCR system,
the company continues to develop it, preparing the next software upgrade and plotting
the next generation of the instrument. In addition, it recently reached an agreement
with Bayer Health Care to develop it as a platform for medical diagnostics. Specifically,
Bayer will generate customer software for such applications and market the system
for clinical use. It plans to launch the system in 2008.
Contact: Rena McClory, Stratagene
Inc., La Jolla, Calif.; e-mail: email@example.com; or Peter Kulesza, Johns
Hopkins School of Medicine, Baltimore, and The University of Alabama at Birmingham;
tel.: +1 (205) 996-5410.
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