Abstract
Circulating cell-free DNA from blood plasma of cancer patients can be used to non-invasively interrogate somatic tumor alterations. Here we develop MSK-ACCESS (Memorial Sloan Kettering - Analysis of Circulating cfDNA to Examine Somatic Status), an NGS assay for detection of very low frequency somatic alterations in 129 genes. Analytical validation demonstrated 92% sensitivity in de-novo mutation calling down to 0.5% allele frequency and 99% for a priori mutation profiling. To evaluate the performance of MSK-ACCESS, we report results from 681 prospective blood samples that underwent clinical analysis to guide patient management. Somatic alterations are detected in 73% of the samples, 56% of which have clinically actionable alterations. The utilization of matched normal sequencing allows retention of somatic alterations while removing over 10,000 germline and clonal hematopoiesis variants. Our experience illustrates the importance of analyzing matched normal samples when interpreting cfDNA results and highlights the importance of cfDNA as a genomic profiling source for cancer patients.
Highlights
Circulating cell-free DNA from blood plasma of cancer patients can be used to non-invasively interrogate somatic tumor alterations
The use of circulating-tumor DNA (ctDNA) as an analyte, has its inherent limitations. It is usually found in low concentrations in the plasma[19], which may be the result of low disease burden in earlystage tumors, disease control in response to treatment, or low tumor DNA shedding in blood
Sensitive assays that are limited to single mutation ctDNA profiling assays such as droplet digital PCR20 are not practical for broad clinical use given the increasing number of genomic alterations that are predictive of response to FDA-approved targeted therapies or required as inclusion criteria for clinical trial enrollment
Summary
We clinically reported a total of 1697 SNVs and indels in 486 samples from 435 patients, with a median VAF of 1.9% (range 0.02–99%) (Fig. 2c) Of these mutation calls, 95% (n = 1606) were called de novo without the aid of prior molecular profiling results for the tested patient. EML4-ALK and KIF5B-RET fusions were detected, de novo and by genotyping, in this cohort, along with rearrangements of ROS1 with multiple partners Both clinically actionable and oncogenic alterations were found in the five most represented tumor types prospectively sequenced by MSK-ACCESS (Fig. 2e). The alteration rates in select genes and cancer types between MSK-ACCESS and MSK-IMPACT were comparable, with some notable exceptions such as a decrease in KRAS mutations in pancreatic cancer, an increase of EGFR mutations in the lung, AR mutations in prostate cancer, and NF1 mutations in Breast cancer in the ctDNA (Fig. 2f).
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