Clinical applications using digital PCR.

Molecular diagnostics and disease-specific tailored treatments are now being introduced to patients at many hospitals and clinics throughout the world (Strain and Richman, Curr Opin HIV AIDS 8:106-110, 2013) and becoming prevalent in the nonscientific literature. Instead of generically using a "one treatment fits all" approach that may have varying levels of effectiveness to different patients, patient-specific molecular profiling based on the genetic makeup of the disease and/or a more accurate pathogen titer could provide more effective treatments with fewer unwanted side effects. One commonly known example of this scenario is epidermal growth factor receptor (EGFR). EGFR is upregulated in many cancers, including many lung and colorectal cancers. Commonly used treatments for these include the receptor blockers cetuximab or panitumumab and tyrosine kinase inhibitors erlotinib or gefitinib. These agents are effective at reducing out-of-control cell cycling and tumor proliferation, but only if downstream signaling kinases and phosphatases are not mutated. Known oncogenes such as BRAF V600E and KRAS G12/13 that are constitutively activated render these treatments ineffective. The use of known ineffective drugs and treatments can thus be avoided reducing time to more effective treatments, reducing cost, and increasing patient well-being. Although digital PCR is for all practical purposes a "new" technology, there is already tremendous interest in its potential for the clinical diagnostics arena. Specificity of the information acquired, accuracy of results, time to results, and cost per sample analyzed are making dPCR an attractive tool for this field. Three areas where dPCR will have a noticeable impact are pathogen/viral detection and quantitation, copy number variations, and rare mutation detection and abundance, but it will inevitably expand from these as the technology becomes more and more prevalent. This chapter discusses digital PCR assay optimization and validation, pathogen/viral detection and quantitation, copy number variation, and rare mutation abundance assays. The sample methods described below utilize the QX100/QX200 methodologies, but with the exception of reaction sub-partitioning (dependent on the instrumentation used) most other parameters remain the same.