Sample collection and ctDNA analysis
Mutect, Tumor
TUMOR
At baseline (C1D1), on treatment (C5D1 and every subsequent 3 cycles), and at the end of treatment (EOT), blood samples were collected in K2-ethylenediaminetetraacetic acid (EDTA)–coated collection tubes and processed within 30 min of collection. ctDNA was extracted from approximately 4 mL of plasma with the QIAamp Circulating Nucleic Acid Kit (Qiagen, Hilden, Germany) per the manufacturer’s instructions and constructed into sequencing libraries with end repair, A-tailing, polymerase chain reaction (PCR) amplification using the TruSeq Nano Library Preparation Kit (Illumina, San Diego, CA). Samples with low ctDNA concentrations were flagged and eliminated from further analysis at 3 steps: ability to make a library compatible with capture (> 200 ng), captured library compatible with sequencing (2 nM), and unique target coverage > 300x. The constructed libraries were sequenced using a targeted panel of 572 cancer-relevant genes (Additional file Single-nucleotide variants were identified using Mutect (Broad Institute, Cambridge, MA). Indels were identified using Pindel (EMBL Outstation European Bioinformatics Institute, Cambridge, UK) [Mutations and indels with an allelic fraction of > 40% and no presence in the COSMIC database were also removed because matched normal DNA was not sequenced. Synonymous mutations and indels were removed. Tumor mutational burden calculated from plasma samples (bTMB) was estimated using callMutationBurden in PureCN after removing common and private germline mutations. All comparisons between mutated vs nonmutated (e.g., Lead time to progression was calculated as the difference between a patient’s progression-free survival time (days from baseline) and the first time point between C5D1 and EOT at which the ctDNA fraction was greater than 0. Furthermore, we required that the ctDNA fraction was greater than 0 in at least two consecutive visits. We reason that including only patients that clinically progressed and satisfied the above requirements regarding consecutively confirmed ctDNA fractions greater than 0 would reduce errors due to mutation artifacts that confound ctDNA fraction estimation algorithms.