Assessing tumor mutational burden and profiling variants from FFPE samples using a PCR-based next-generation sequencing assay

Introduction: High Tumor Mutational Burden (TMB) has shown association with benefit from immune checkpoint blockade therapies. While TMB as computed from whole exome sequencing (WES) is still a gold standard, the high input material (tumor and germline DNA) requirement and complex bioinformatics refrains exploring this biomarker in individual labs. Herein, we develop a PCR-based targeted panel for computing TMB and detecting important variants from FFPE research samples. Methods: A targeted panel was designed that covered 1.7 Mb of genomic region from 409 key cancer genes. First an UDG treatment is applied to repair samples with FFPE fixation error. Utilizing Ion AmpliSeq chemistry, the workflow requires very little (only 20 ng) input DNA. The assay enables a 3-day turn-around time from sample to the final report. The workflow enables T:A mutations below 15% allelic frequency was called deamination. Results: In-silico analyses using 466 lung adenocarcinoma, 375 skin cutaneous melanoma, and 274 colon adenocarcinoma samples from TCGA MC3 dataset displayed high correlation between WES TMB and predicted panel TMB (r2 = 0.90 on lung, r2 = 0.96 on melanoma, and r2 = 0.98 on colon). Deamination reduced by average 85.67% (SD 16.87) on FFPE samples after applying UDG repair. Mean TMB of first six samples was 6.97 (SD 2.00), and TMB of last two samples was 43.58 and 87.14. Among two high TMB samples, first had gain-of-function and loss-of-function mutations in BRAF and NF1 genes and had 59.4% C:G>A:T somatic mutations relating to molecular smoking signature. On the second high TMB sample, the assay detected gain-of-function and loss-of-function mutations in FGFR3, NOTCH1, KRAS, HNF1A, and CREBBP genes and estimated 64.7% C:G>T:A somatic mutations at CpG sites consistent with deamination of 5-methylcytosine. Conclusions: We have developed a workflow on the Ion Torrent sequencing platform with Ion AmpliSeq chemistry to estimate TMB from FFPE research samples. This solution will advance research in immuno-oncology. Citation Format: Ruchi Chaudhary, Charles Scafe, Dinesh Cyanam, Vinay Mittal, Warren Tom, Janice Au-Young, Seth Sadis, Fiona Hyland. Assessing tumor mutational burden and profiling variants from FFPE samples using a PCR-based next-generation sequencing assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5132.