The importance of next generation sequencing (NGS) rises in cancer research as accessing this key technology becomes easier for researchers. The sequence data created by NGS technologies must be processed by various bioinformatics algorithms within a pipeline in order to convert raw data to meaningful information. Mapping and variant calling are the two main steps of these analysis pipelines, and many algorithms are available for these steps. Therefore, detailed benchmarking of these algorithms in different scenarios is crucial for the efficient utilization of sequencing technologies. In this study, we compared the performance of twelve pipelines (three mapping and four variant discovery algorithms) with recommended settings to capture single nucleotide variants. We observed significant discrepancy in variant calls among tested pipelines for different heterogeneity levels in real and simulated samples with overall high specificity and low sensitivity. Additional to the individual evaluation of pipelines, we also constructed and tested the performance of pipeline combinations. In these analyses, we observed that certain pipelines complement each other much better than others and display superior performance than individual pipelines. This suggests that adhering to a single pipeline is not optimal for cancer sequencing analysis and sample heterogeneity should be considered in algorithm optimization.

Advanced-stage liver metastatic Colorectal Cancer (CRC) disease remission rates are markedly low, often with less time for life-saving but challenging medical procedures and physically demanding regimens of neoadjuvant chemotherapies. Medical decisions supported by patient-specific genomic information may improve cancer prognosis, long-term remission rates, and overall patient health. Understanding the genomic landscape of liver metastasis with respect to primary tumors can help clinical decision-making that may result in added life years for patients with advanced disease. For unresectable metastatic disease, prediction of the possible targetable mutations of both primary colon tumors and liver metastasis could have an impact on treatment decisions and survival rates. In this study 16 samples were collected from five different CRC patients with cooccurring liver metastases. Whole exome sequencing (WES) was performed on an Illumina Hi-Seq 2000. Samples included primary tumor, tumor micro-environment and metastasis (with one patient undergoing a polypectomy). At the biopsy date, treatment naïve samples allowed observation of the natural genomic makeup. Analysis confirmed 70x average coverage. Data were processed with multiple mapping and variant discovery algorithms. In these samples, a putative driver variant, FAM25C was identified, in addition to observing canonic driver mutations for colorectal cancer such as TP53, APC, KRAS. After variant discovery, microsatellite instability and tumor mutation burden (TMB) analyses were conducted. One patient showed high TMB with almost 10-fold more mutations observed, when compared to other samples. All samples of the High-TMB patient share almost half of the same variants, which can be indicative of early expansion of the major clone. Average probabilistic Jaccard similarity coefficient percentages of somatic variants of only primary CRC tumor and liver metastasis tissues were 41.08% with a standard deviation of 4.05% across all patients. Whole exome sequencing of samples with subsequent analysis of clonal variation between CRC tumors and liver metastasis show different mutational composition trends in dissimilarity to the primary tumor. To understand tumor evolution, phylogenetic analysis was conducted for each patient sample. Although different studies were conducted on understanding the genomic properties of metastasis through pairwise comparison, detailed studies which investigate the genomic patterns of tumor expansion through detailed sampling are crucial for effective therapy development. We believe our results in combination with other multi-sample sequencing studies will help the CRC community to better understand this deadly disease, leading to better treatment strategies. These authors hypothesize that investigating clonal diversity via synchronous biopsy in primary and metastatic tumors may be one facet in a larger conversation of treatment decisions and responses of patients who will undergo neoadjuvant chemotherapies for nonresectable liver metastatic CRC disease. Citation Format: Duygu Altinok Dindar, Batuhan Kısakol, Sahin Sarihan, Mehmet Arif Ergun, John Cheney, Emel Ergul, Nihal Uren, Ender Dulundu, Emine Bozkurtlar, Nihat Zafer Utkan, Mehmet Baysan. Investigating primary colorectal cancer tumor and liver metastasis clonality through whole exome sequencing of synchronous biopsies [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr A029.