Droplet Digital PCR for Absolute Quantification of EML4-ALK Gene Rearrangement in Lung Adenocarcinoma

Abstract

Crizotinib treatment significantly prolongs progression-free survival, increases response rates, and improves the quality of life in patients with ALK-positive non-small-cell lung cancer. Droplet Digital PCR (ddPCR), a recently developed technique with high sensitivity and specificity, was used in this study to evaluate the association between the abundance of ALK rearrangements and crizotinib effectiveness. FFPE tissues were obtained from 103 consecutive patients with lung adenocarcinoma. Fluorescent in situ hybridization (FISH) and ddPCR were performed. The results revealed that 14 (13.6%) of the 103 patients were positive by dual-color, break-apart FISH. Three variants (1, 2, and 3) of the EML4-ALK gene rearrangements were detected. Thirteen of 14 ALK-positive cases identified by FISH were confirmed by ddPCR (four with variant 1, two with variant 2, and seven with variant 3). The case missed by ddPCR was identified as KIF5B-ALK gene rearrangement by PCR-based direct sequencing. Sixteen patients were detected with low copy numbers of EML4-ALK gene rearrangement, which failed to meet the positive cutoff point of FISH. Two of them responded well to crizotinib after unsuccessful chemotherapy. Our study indicates that ddPCR can be used as a molecular analytical tool to accurately measure the EML4-ALK rearrangement copy numbers in FFPE samples of lung adenocarcinoma patients.

Detection of human papillomavirus type 16 in oropharyngeal squamous cell carcinoma using droplet digital polymerase chain reaction

“…ddPCR can be used to detect rare mutations, can quantify gene expression with very low copy numbers, and can be multiplexed for enhanced efficiency . When applied to biomarker analysis of individuals with cancer, ddPCR has demonstrated superior diagnostic performance compared with other available molecular techniques . To our knowledge, the use of ddPCR technology has not yet been reported for the detection of oncogenic HPV in head and neck squamous cell carcinoma (SCC).…”

Comprehensive profiling and quantitation of oncogenic mutations in non small-cell lung carcinoma using single molecule amplification and re-sequencing technology

“…In addition to the ability to detect point mutations and small deletions and insertions, the SMART assay also showed proof of concept for detection, quantitation and precise breakpoint identification of different EML4-ALK fusion variants and, may provide an alternative to recently developed PCR methods [32, 45]. Moreover, through the construction of the paired end allelic reads, the resulting DNA sequence allowed precise mapping of the fusion site and identified the nature of each EML4-ALK fusion variant.…”

ROS-1 Rearrangements in Circulating Tumor Cells

“…Whether tumor tissue is truly the criterion standard for molecular analysis is currently disputed, as blood tests can reveal alterations not discovered in tumor tissue. 3 Our study underscores the need to define an analytical criterion standard for identifying the largest possible amount of druggable alterations. In conclusion, we report that CTC analysis can identify ROS1 rearrangements.…”