A sub set of these genes had high MAF values, indicating a higher likelihood of being present in all clonal populations in the primary tumor or metastasis. As we describe later, these genes were prioritized for further experimental test ing in gastric organoids. Mutations affecting gene function Among the mutations that were externally validated, we focused on the subset of mutations leading to amino acid substitutions, premature stop codons and indels that altered the open reading frame. Subsequently, we deter mined if these coding mutations were potentially deleteri ous to gene function using a number of prediction algorithms such as Polyphen and SIFT among others. Based on the MAF information for each mutation, we determined whether these mutations with a possible deleterious impact on the gene products were common or exclusive to the primary tumor and metastasis.

On the subset of deleterious mutations, we conducted additional biological pathway analysis, literature review and comparison against the Cancer Genome Atlas data available for diffuse gastric cancer. This identified a set of known cancer genes and likely cancer related candi dates with mutations that likely had an impact on pro tein function. We focused on a number of candidate driver genes that had previously been demon strated to have oncogenic potential or were known tumor suppressors with biallelic changes present in the cancer genomes. Nadauld et al. Genome Biology 2014, 15 428 Page 6 of 17 Copy number variations and allelic imbalances distinguishing the primary tumor from the metastasis We noted larger scale genomic aberrations that differenti ated the primary from the metastasis.

This included copy number changes and LOH events. Unique to the primary tumor were two genomic amplifi cations on chromosomes 5 and 10, and two inversions on chromosomes 15 and 16. The chromosome 10 amplification covered a 1. 66 Mb interval. When considering deletions or allelic imbalances, the only major event that was noted involved a loss of the p arm of chromosome 17. In contrast to the primary tumor, the metastatic tumor had numerous chromosomal scale LOH events and gen omic deletions affecting 12 different chromosomes, the majority of which were unique to the metastatic tumor. This included multiple deletions and copy neutral LOH events that are detailed in Additional file 1 Table S3.

There was a five fold genomic amplification in chromosome 2 but no specific known genes existed in the affected interval. There were no detectable inter chromosomal Brefeldin_A translocations in either the primary tumor or metastasis genomes. Other cancer rearrangements were identified but did not point towards aberrations in any candidate driver genes. There were indications of large scale genomic instability based upon allelic imbalance analysis.