Defining tissue requirements for a reliable detection of tumor mutational burden (TMB) in clinical FFPE samples.

Abstract

e14265 Background: In the context of immuno-oncology related cancer treatment, tumor mutational burden (TMB) is currently explored as a predictive biomarker for several human malignancies. Several sequencing assays are increasingly commercially available. Established methodologies require rather large amounts of DNA input (in the range of 100 ng) which, however, are frequently not available from small biopsies. We aim to investigate how tissue size and DNA input influence TMB scores. Methods: DNA from 20 specimens (12 biopsies of non-small cell lung cancer (NSCLC); 8 surgical resection specimens from NSCLC, colorectal cancer and endometrial carcinomas) was manually extracted by using the Qiagen GeneRead DNA FFPE kit. Cases were selected to provide a wide range of relative tumor cell content (from < 10% to > 50%) and to include microsatellite-stable and –instable (MSI) cases. Samples were analyzed in triplicates from predefined numbers of unstained sections of a standardized tissue size. DNA quantification was done fluorometrically and by qPCR. Up to 40 ng of DNA were analyzed with the QIASeq TMB Panel (incl. MSI primer boosters; Qiagen). Sequencing was done on an Illumina NextSeq platform, TMB scores and MSI status were determined by using the CLC workbench 5.0.1 (Qiagen). Results: Biopsy samples generated less numbers of DNA molecules (as detected by unique molecular identifiers, UMIs) and less overall reads compared to resection samples. UMI coverage was > 500x in all samples with > 15 ng DNA input. TMB scores were highly reproducible among all samples with > 15 ng DNA but differed significantly among samples with limited DNA input. Interestingly, TMB scores were inversely correlated with DNA input among those samples with < 15ng. Thus, valid TMB scores could also be obtained from only one slice of 1 cm2 tissue from tumor resections or 3 slices of an endoscopic biopsy (each of 5µm thickness). All pre-characterized MSI carcinomas could be detected correctly. Conclusions: We provide first evidence that TMB can be reliably determined also in small biopsies yielding limited DNA content. However, false high TMB scores can occur in samples with < 15ng DNA input. Our results contribute to the definition of minimum requirements (tissue size and DNA input) for valid TMB measurement on clinical FFPE samples.