By Anthony M Magliocco MD
Digital Droplet PCR, or ddPCR is a phenomenally sensitive, specific, and most importantly, precise method to measure target DNA.
One important application for this technology that is now reaching the clinical laboratory is the development of tests for monitoring circulating cell free DNA that is released from cancer cells.
The challenge of detecting cfDNA is monumental. A tube of blood is loaded with a rich variety of complex biomolecules and cells including lymphocytes, neutrophils, platelets, and of course red corpuscles. These cells, including RBCs, are loaded with nucleic acids and whats worse, they can easily rupture during blood draws or pre analytical handling.
Advanced lung adenocarcinoma, (NSCLC) is probably the single tumor type that has caused the largest advances in personalized oncology of solid tumors. Investigations into the molecular biology of this disease has uncovered the fact that NSCLC is a heterogeneous disease defined by distinct molecular subtypes and clear molecular driving pathways. Further the discovery of targetable driver mutations including presence of EGFR mutations among others has led to pivotal clinical trials proving the efficacy of the Tyrosine Kinase inhibitor Drugs (TKIs). Further it seems that lung tumors with EGFR mutations are more likely to arise in women and non-smokers.
Despite the remarkable efficacy of TKI therapy in many patients with advanced NSCLC, the disease is relentless and frequently overcomes treatment by developing resistance mechanisms. The commonest mechanism of resistance is acquisition of EGFR T790M mutation which further enhances the activity of the EGFR pathway, driving growth of the lung cancer cells.
Fortunately, third generation non-competitive inhibitors of EGFR were developed that can overcome the development of T790M resistance -osimertinib or Tagresso from AZD.
When Osimertinib first became available it was necessary to develop an ultra sensitive assay to detect the mutation in tissues and blood.
The Moffitt Cancer Center Morsani Laboratories, when faced with this challenge, decided to use a digital PCR approach because of its superior sensitivity, specificity and reasonable cost of operation.
Dr John Puskas worked diligently to develop and validate a cfDNA assay for EGFR T790M mutation using ddPCR for CLIA at the Moffitt Cancer Center. Different PCR platforms were evaluated but the Bio-Rad QX200 was eventually selected for use.
The assay was superbly sensitive and precise as well as convenient to run. The assay can detect mutant EGFR T790M down to 0.1% and can easily monitor blood concentration over time to give assessment of disease progression
The development of accurate and specific cfDNA assays to precisely monitor cancer progression in the metastatic setting is an important tool to potentially enable oncologists the opportunity to determine more rapidly f tumors are responding to treatment or not and make adjustments.
This novel opportunity to trace tumor evolution in real time, at a molecular level, could play a role in trials and treatments of the future where treatments are carefully adjusted to avoid the inadvertent development of treatment resistance due to over treatment.