Alexander D. Le, Bing Zhang, Kim C. Worley, David Wheeler, Benjamin L. Musher, Heidi Dowst, Amy Wood, Jeff Schaeffer, Nick Olsen, Lorenzo Grego, Matt Sonnleitner, and William E. Fisher
Background: The Cancer Genome Atlas (TCGA) characterizes somatic mutations of pancreatic cancer (PC) patients (pts) using whole-exome sequencing (WES, mean coverage 405x) of fresh frozen tumor tissues and matched blood samples. In addition, it includes a confirmation of the key genomic findings by a targeted sequencing panel (~644x) and microfluidic PCR-based sequencing (~30,000x). For the vast majority of patients who do not undergo resection, however genomic characterization of formalin-fixed, paraffin-embedded (FFPE) biopsy samples is the only available assessment. In order to assess this NGS testing process at Baylor College of Medicine, we sought to confirm that routinely collected diagnostic FFPE specimens could be used for sequencing without compromising the detection rates of key gene alterations in PC pts.
Methods: Matched tumor and normal specimens (peripheral blood or saliva) from 59 Baylor College of Medicine PC pts were sequenced using Tempus|xT (Tempus Labs, Chicago, IL), a 648-gene DNA sequencing panel (avg. depth of 500x) integrated with whole-transcriptome RNA sequencing. Retrospective analysis of detection rates of all clinically reported pathogenic mutations was performed using LENS, a proprietary application that allows interrogation of large de-identified molecular and clinical datasets.
Results: The tumor cellularity of the 59 FFPE samples as assessed by board-certified pathologists ranged from 10% to 80%. Approximately 88% (52/59) of these samples had detectable KRAS alterations, relative to 93% identified in the TCGA database. KRAS G12D, G12V, and G12R were the most frequent findings (29/59, 11/59, and 8/59, respectively). The average KRAS variant allele fraction (VAF) was 16.87% (range 1.7%- 42.2%). Alterations in CDKN2A were detected with a noticeably higher incidence as compared to the TCGA database, 37.2% (22/59) vs 17.4%, respectively. Conversely, TP53 and SMAD4 had similar detection incidence between the two platforms. In addition, 13.5% (8/59) of PC pts had a somatic or germline mutation in the homologous recombination (HR) pathway, with 4 pts having a BRCA2 alteration (6.7%). Finally, one patient was tumor mutational burden (TMB)-High and microsatellite instability (MSI)-High, both of which are tumor-agnostic indications for checkpoint inhibition.
Conclusions: An analysis of PC FFPE tissues using the Tempus|xT panel at a clinical depth of 500x revealed a detection rate of KRAS, TP53, and SMAD4 alterations comparable to the TCGA database.
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