Abstract
Researchers at Memorial Sloan Kettering Cancer Center (MSKCC) in New York are investigating the use of Droplet Digital PCR™ (ddPCR™) to personalize melanoma treatment using liquid biopsies in the clinical setting.
Although a common practice for detecting and monitoring cancer is through tissue biopsy, more recently, researchers have looked for a less invasive means of investigating cancerous cells to assess disease progression and treatment response. By harnessing the power of advanced DNA testing to perform biopsies using easily obtained bodily fluids such as urine and blood—liquid biopsy—these minimally invasive blood tests have the potential to detect cancer biomarkers found within cell-free DNA (cfDNA).
The hope is that this new approach will accelerate research into what makes cancers tick, including how tumors spread, as well as which therapies patients will respond to and how they are responding, offering clues into recurrence and treatment resistance.
However, despite the promise of liquid biopsies, conventional digital and real-time PCR assays have been seen as cumbersome and vulnerable to false positive results or insufficiently sensitive, making it nearly impossible to translate this technique for routine clinical use. 1
The Droplet Approach
ddPCR has entered into the spotlight in the past few years as a resource for cancer investigation, given its ability to rapidly interrogate the cancer genome with high levels of sensitivity and precision. In fact, researchers using Bio-Rad Laboratories QX200 ddPCR system for mutation detection report that they can detect a point mutation at 0.0005% to 0.001% of wild type, which is more than 1,000 times more sensitive than conventional quantitative real-time PCR (qPCR). Essentially, what was previously undetectable with other methods, can now be detected and quantified.
At its core, ddPCR counts nucleic acid molecules by partitioning the sample in such a way that either zero or at most a small number of nucleic acid molecules of interest (i.e., targets) are present in each partition. The QX200 system partitions samples into 20,000 droplets containing either target-specific Taqman assays or target-specific primers and the fluorescent DNA-binding dye, EvaGreen. These droplets are then amplified to endpoint using normal PCR conditions in a conventional thermocycler to achieve an absolute measurement of the target concentration through elevated droplet fluorescence.
The level of sensitivity and precision offered by Bio-Rad's ddPCR system allows researchers to quantify cancer biomarkers, which could be the key to bringing liquid biopsies into the clinic.
Using ddPCR to Monitor Treatment Response in Plasma of Patients with Melanoma
Omar Abdel-Wahab, M.D., an oncologist at MSKCC, and his team used ddPCR to track disease progression and identify an effective treatment regimen for a patient initially diagnosed with BRAF-mutant positive melanoma. Treatment with the targeted drug vemurafenib—effective against BRAF mutations but also known to activate ERK signaling in RAS-mutant cells—induced proliferation of chronic myelomonocytic leukemia (CMML). While vemurafenib shrank that patient's metastatic BRAF-mutant melanoma dramatically, it also accelerated the progression of a previously unrecognized NRAS-mutant leukemia.
Dr. Abdel-Wahab and his team's research, which was published in the May issue of Cancer Discovery 2 , set out to prove that combination therapy inhibiting the RAF and MEK pathways would not only treat the melanoma but would reduce proliferation of the patient's CMML. Through Genentech, they were able to arrange a single-patient investigational use of a promising but not-yet approved MEK inhibitor, cobimetinib.
ddPCR was used to serially quantify melanoma-derived (BRAFV600K) and CMML-derived (NRASG12R) DNA in patient plasma over a 20-month treatment period. This technique proved essential for analyzing the regions of interest and determining the number of mutant copies at different times and under different treatment conditions. Using the information gathered from ddPCR alongside traditional assays—white blood cell counts and radiographic imaging—they identified an effective treatment regimen that beat back both cancers with less toxicity.
This research foreshadows how ddPCR can contribute to the monitoring and adjustment of patient therapy according to molecular responses in the blood. While there remains a need for more testing and clinical validation of this biopsy method, Dr. Abdel-Wahab's research and other notable studies, demonstrate how ddPCR is paving the way for future success, given its ability to noninvasively and affordably interrogate the cancer genome with high levels of sensitivity and precision.