CANCER RESEARCH AND TREATMENT continued
The following are real-world examples of ddPCR’s impact across the full spectrum of cancer research from bench to bedside.
A starting point for personalized medicine The ability to measure genomic amplifications of oncogenes is critical
to developing targeted cancer therapies and demands high precision. However, generating accurate copy number (CN) measurements from small amounts of poor-quality DNA found in formalin fixed paraffin embedded (FFPE) tumor samples—already a mix of normal and tumor cells—makes the task even more challenging.
Traditionally, scientists have used qPCR and microarray-based assays to quantify these FFPE cancer tissue samples, but these methods are limited because they inherently lack the necessary precision as well as the tolerance to degraded FFPE DNA and its associated inhibitors. In contrast, the inherent measurement precision of ddPCR is more robust to the suboptimal PCR conditions afforded by FFPE samples.
In a recent study,1 researchers in the Ji Research Group at the Stanford
University School of Medicine demonstrated that ddPCR is superior to qPCR for DNA copy number analysis of archival material. Using ddPCR, they measured a sevenfold amplification of the FGFR2 locus in an FFPE- processed gastric tumor, which was very similar to the value determined using a microarray analysis on a “matched” flash-frozen sample. Analysis of the same FFPE tumor sample with qPCR found a drastically different copy number. These results show that ddPCR is more accurate than qPCR for determining copy number variants in FFPE-derived samples.
The next frontier: microRNAs as cancer biomarkers MicroRNAs (miRNAs) regulate the expression of thousands of genes in
both normal and pathophysiological processes, including cancer, mak- ing them an intensive area of biological research. Numerous studies have shown that these small, noncoding RNAs have great potential as biomarkers, as researchers can monitor them in samples from tissues, cells, and body fluids—such as urine, cerebrospinal fluid, blood, plasma, serum, and sputum.
qPCR has traditionally been used to measure miRNAs; however, the high interlaboratory and interday variability of measurements in serum or plasma undermines their use as reliable blood-based biomarkers.
In a study2 published in Nature Methods, Dr. Muneesh Tewari, a research-
er at the University of Michigan, evaluated the diagnostic potential of miRNA as an early biomarker for prostate cancer. The study showed that ddPCR increases reproducibility of the miRNA quantification process, making it feasible for results to be compared at different times. When they tested ddPCR versus qPCR on cDNA from six different synthetic miRNAs, in both water and serum, on three separate days, they found that ddPCR demonstrated greater precision—48 to 72% lower coeffi- cients of variation—with respect to qPCR-specific variation.
They also found that ddPCR improved day-to-day reproducibility sevenfold relative to qPCR and, importantly, ddPCR enabled better dis- crimination of cancer samples from controls based on the level of a single miRNA.
The team’s ultimate goal is to develop biofluid-based approaches for disease detection and monitoring with simplicity and reproducibility in mind.
T-lymphocyte levels predicting patient outcomes in
ovarian cancer Tumor-infiltrating T-lymphocytes (TILs) are a type of tumor-attacking immune cell that is strongly associated with improved cancer survival. Clinicians would like to use TIL counts in tumors to predict survival outcomes and determine which patients may benefit from targeted im- munotherapies. However, current immunohistochemical methods for scoring their abundance are too subjective and variable between labs to allow for their rigorous evaluation as a prognostic factor.
Dr. Jason Bielas, Associate Member of the Public Health Sciences and Human Biology Divisions at the Fred Hutchinson Cancer Research Center, found that ddPCR has the ability to comprehensively quantify the number of TILs in a tumor based on the shared genomic signature on the surface of each TIL.3
Researchers first demonstrated that their QuanTILfy assay can be used to accurately and reproducibly characterize T-cell clonality in patients with T-cell acute lymphoblastic leukemia—results that they validated using deep sequencing.
The team then demonstrated that the assay could robustly quantify the abundance of TILs in ovarian cancer from different patients, as well as spatial differences in TIL numbers within a single tumor. Finally, they per- formed the assay on primary tumor samples from 30 ovarian carcinoma patients with known survival outcomes. They found that TIL frequency was approximately threefold higher in those who survived more than five years as compared with those who survived less than two years, showing that TIL levels correlate positively with patient survival.
The robustness of the ddPCR-based QuanTILfy assay may surmount the limitations of existing immunohistochemical methods for TIL enu- meration and enable this biomarker to be developed for prognosis and treatment of ovarian and other cancers.
Quantification of plasma cfDNA by liquid biopsy in
a melanoma patient An increasing number of translational studies are finding that ddPCR is well-suited for investigating the use of liquid biopsy in patient treatment and monitoring (see sidebar). In a recent illustrative study in Cancer Discovery,4
researchers described a patient with metastatic BRAF-mutant
melanoma. Treatment with vemurafenib led to a dramatic shrinkage of his melanoma, but it induced proliferation of a chronic myelomonocytic leukemia (CMML) due to a previously unrecognized NRAS mutation. The researchers hypothesized that treating the patient with combined therapy of RAF and MEK inhibitors would treat the melanoma and re- duce proliferation of the patient’s CMML, respectively.
Based on white blood cell counts and radiographic imaging, they were able to arrive at an effective treatment regimen that beat back both cancers with less toxicity. During 20 months of evolving and improved
AMERICAN LABORATORY • 28 • SEPTEMBER 2014
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