As science and medical innovation churns at an unprecedented pace, cancer rates are falling. According to the National Cancer Institute, death rates for all cancers combined decreased by 1.8% per year among men and by 1.4% per year among women between 2001 and 2010.
In its Annual Report to the Nation on the Status of Cancer, the institute attributes this encouraging decline to many factors, including more effective therapies and earlier detection capabilities. Both of these have been made possible by the rapid advancement of medical research and availability of new technologies.
One of the more exciting new technologies contributing to current advancements in cancer is droplet digital PCR (ddPCR), a method that is already being shown to track both favorable and unfavorable responses to therapy more rapidly than current imaging methods and to enable improved treatments. Here’s a look at researchers around the country who are fueling improved cancer survival through discoveries made with ddPCR.
Mining New Knowledge from Archival Cancer Samples
At the Stanford University School of Medicine, the Ji Research Group has adopted ddPCR to quantify cancer genome amplifications in archival cancer tissue samples. Identifying and measuring these amplifications is important to treating cancer.
Unfortunately, detecting amplifications in cancer tissue can be technically challenging for several reasons. First, there is the biology: genomic amplifications are diluted both by the genetic heterogeneity of the cancer cells and by the presence of normal tissue within the tumor sample. This requires greater sensitivity to small differences in gene copy number (i.e. greater precision). Second, the conventional method of tumor sample preparation and preservation raises additional challenges: clinical samples are traditionally processed as formalin fixed paraffin embedded (FFPE) tissues, which leads to irreversible damage to the genomic DNA and to the presence of PCR inhibitors. Traditionally, scientists have used qPCR to quantify genomic amplifications in FFPE cancer tissue samples. However, this method has neither the greater tolerance of ddPCR to inhibitors and other factors influencing PCR amplification—and thus accurate quantification—nor the precision to detect amplifications or deletions necessitated by dilution of affected cancer cells in the tumor DNA.
Researchers in the Ji group demonstrated the superiority of ddPCR over qPCR for copy number analysis of archival material in DNA, thanks to its capacity for accuracy, reproducibility and sensitivity. This will allow them to conduct a variety of genomic studies using ddPCR that could not have been done otherwise. And in turn, these amplified oncogenes could move us closer to long-sought-after personalized therapies for cancer treatment.
Quantifying miRNA as Cancer Biomarkers
Dr. Muneesh Tewari, a University of Michigan researcher and former member of the Fred Hutchinson Cancer Research Center, is using ddPCR to investigate microRNA (miRNA) as an early biomarker for prostate cancer.
miRNAs play an important role in the body, regulating the expression of thousands of genes in both normal and pathophysiological processes, making them an area of intense interest for cancer research. These small, non-coding RNAs have tremendous potential as biomarkers, because researchers are able to non-invasively monitor miRNA in samples from tissues, cells and body fluids, such as urine, cerebrospinal fluid, blood, plasma, sputum and serum.
While quantitative real-time PCR has been the standard for detection of miRNA in blood samples, researchers have found that qPCR measurements of miRNAs in serum or plasma can’t be reliably compared from one day to the next. This can undermine the use of miRNAs as blood-based biomarkers.
Tewari and colleagues found that, compared to qPCR, ddPCR demonstrated greater precision—up to 72% lower coefficients of variation—with respect to qPCR-specific variation. Furthermore, ddPCR reduced variation of miR-141 biomarker quantification in serum samples by seven-fold and enabled superior discrimination of prostate cancer cases from controls. Tewari said the precision and reproducibility of ddPCR will pave the way for further development of miRNA and other nucleic acids as circulating biomarkers.
In addition to studying miRNAs as biomarkers of cancer, Tewari and his lab are working to learn more about other types of RNA and DNA biomarkers. Their goal is to develop simple biofluid-based approaches for disease detection and monitoring.
Noninvasive Monitoring of Cancer Treatment
Liquid biopsies, which use blood—or other body fluids such as urine—instead of tissue, have the potential to noninvasively detect cancer, track its progress and guide treatment decision-making. One of the challenges preventing the test from becoming a clinical reality is finding the cancerous DNA in the vast sea of healthy DNA that is also continuously shed from the body’s cells.
At the Dana-Farber Cancer Institute, researchers Geoffrey Oxnard, Cloud Paweletz and colleagues showed that genotyping cell-free DNA (cfDNA) using ddPCR can detect resistance to cancer treatment up to 16 weeks earlier than the standard clinical measure, radiographic imaging.
DFCI researchers measured cfDNA in blood plasma from advanced non-small cell lung cancer (NSCLC) patients receiving targeted therapy (erlotinib).The investigators relied on ddPCR to simultaneously measure several mutations in the plasma of these cancer patients. Based on prior tumor genotyping of patients, they developed ddPCR assays for each mutation of interest using plasma collected from patients with advanced lung cancer or melanoma. These assays allowed them to serially measure levels of the mutations at different stages in treatment, over more than a year for some patients.
In each patient, they observed notable differences in the count of mutant DNA copies per milliliter of blood across time. They saw decreases in cases of favorable response to therapy, and increases where a known resistance mutation triggered disease progression. These results demonstrate that non-invasive genotyping of cell-free plasma DNA has potential as a clinical biomarker, and can help medical professionals personalize the treatment of genotype-defined solid tumors.
The ddPCR technology not only helped in terms of speed, cost and ease of use compared to other PCR-based assays, but also opened up broader clinical applications due to sensitivity and the quantitative nature of the assays. The quantitative nature of plasma genotyping with ddPCR offers a mechanism for serially monitoring the prevalence of tumor clones harboring a specific genotype, giving new insight into the pharmacodynamics of a targeted therapy. Researchers will continue to use ddPCR in their efforts to bring non-invasive genotyping to the clinic, allowing oncologists to personalize cancer therapies throughout the course of the disease.
Propelling the Future of Cancer Research
Even as cancer rates fall, it remains the second-leading cause of death in the United States. However, armed with the power of ddPCR, researchers will continue to drive important new breakthroughs in the detection, monitoring and effective treatment of cancer.
Filed Under: Drug Discovery