Oncomine tumor mutation load assay

Plasma Cell-free circulating DNA samples were analysed using Oncomine™ Tumor Mutation Load Assay, according to the manufacturer recommendation, covering 1.65 Mb across 409 genes relevant cancer (Thermo Fisher Scientific, Les Ulis, France). Briefly, circulating the DNA were extracted by Maxwell® RSC ccfDNA Plasma Kit (Promega, Charbonnières-les-Bains, France). Libraries were performed using Ion Chef™ Instrument and Ion 550 Chip, then sequenced with Ion S5™ System (Thermo Fisher Scientific). The median coverage depth 751 X (from 636X to 817X) was obtained. Variant callings and Tumor mutational burden (TMB; count of mutations / megabase) were calculated using Ion reporter software package (version 5.14.1.0) with the pipeline: Oncomine Tumor Mutation Load - w3.1 - DNA - Single Sample (Thermo Fisher Scientific).

20 ng DNA quantified with the Qubit dsDNA HS Assay (Thermo Fisher Scientific, Waltham, MA, USA) on the Qubit 2.0 Fluorometer (Thermo Fisher Scientific) was used for library preparation with the Oncomine Tumor Mutation Load Assay (Thermo Fisher Scientific) according to manufacturer’s instructions. Library concentrations were quantified with the Ion Library TaqMan Quantification Kit (Thermo Fisher Scientific). Libraries were loaded on the Ion Chef for template preparation and chip loading using the Ion 540 Kit (Thermo Fisher Scientific), followed by sequencing on the Ion S5 XL System (Thermo Fisher Scientic).
Quality of the Ion S5 XL run was assessed with the Ion Torrent Suite 5.10 (Thermo Fisher Scientific). Data were analyzed with the Ion Reporter 5.10 (Thermo Fisher Scientific).

Co‐existing somatic mutation profiling and tumor mutation burden (TMB) estimation were performed using the Oncomine Tumor Mutation Load Assay (Thermo Fisher Scientific, Wilmington, DE, USA) in accordance with the manufacturer's recommended protocol. The assay covers 1.65 Mb across 409 oncogenes relevant across major cancer types (full list of the genes is available at https://www.thermofisher.com/order/catalog/product/A37909). Sequencing was performed using IonS5™ XL platform (Thermo Fisher Scientific). Sequencing data were analyzed with Ion Torrent Suite and Ion Reporter (Thermo Fisher Scientific). In order to evaluate the quality of the sequencing, on‐target alignment rate > 90%, mean depth > 500, and deamination ≤ 10 were set as the reference values. Samples that deviated from the reference values for any of the items were considered unassessable and subsequently excluded from further investigations in this study.

DNA was extracted from FFPE samples using the MagMAX™ FFPE DNA/RNA Ultra kit (Thermo Fisher Scientific, Waltham, MA, USA). Manufacturer protocol was followed except for the magnetic bead drying step that was performed at RT for 4–6 min, instead of shaking for 2 min.
The Oncomine™ tumor mutation load assay (Thermo Fisher Scientific, Waltham, MA, USA) was used to determine tumor mutation burden, as instructed by the manufacturer. Library quantification was performed by quantitative PCR using the Ion Library TaqMan quantitation kit (Thermo Fisher Scientific, Waltham, MA, USA).). The Ion Chef system was used for template preparation and chip loading before sequencing using the Ion S5™ XL sequencer (Thermo Fisher Scientific, Waltham, MA, USA). The data were analyzed with Ion Reporter™ software version 5.18.0.1 (Thermo Fisher Scientific, Waltham, MA, USA), and tumor mutation burden was determined as number of mut/Mbp using variants with a minimum of 10% allelic frequency.

A prospective case-control study was conducted based on a large academic hospital's cancer center between January 2017 and August 2019 (Hadassah Medical Center). CRC patients with mucinous histology were recruited, along with conventional adenocarcinoma histology controls. Clinical and pathological data were extracted from digital records. Genetic data was analyzed and validated by the pathology department in Hadassah Medical Center or Foundation Medicine tests. Mismatch repair (MMR) status was evaluated by immunostaining for the mismatch repair proteins hMLH1, hPMS2, hMSH6, and hMSH2. Next-generation sequencing tests were conducted to identify alternations in hotspot regions in a few key factor functioning genes by Ion Torrent system. For library construction of KRAS, BRAF, and PI3K genes, Oncomine^TM Solid Tumour DNA Kit was used; for BRCA1/2 genes, Ion AmpliSeq™ Oncomine BRCA primers were used.
Tumor mutation burden (TMB) results were based on either (1) commercial kits (such as 324-gene panel assay FoundationOne® CDx test, validated comparing to whole-exome sequencing (WES) [20 (link)]) or (2) local analysis by Pathology Department with Ion Torrent system sequencing and assessed by the Oncomine Tumor Mutation Load Assay (Thermo Fisher Cat. No. A37910), also validated comparing to WES [21 (link)].