Clinical Sequencing Defines the Genomic Landscape of Metastatic Colorectal Cancer

Abstract

Metastatic colorectal cancers (mCRCs) are clinically heterogeneous, but the genomic basis of this variability remains poorly understood. We performed prospective targeted sequencing of 1,134 CRCs. We identified splice alterations in intronic regions of APC and large in-frame deletions in CTNNB1, increasing oncogenic WNT pathway alterations to 96% of CRCs. Right-sided primary site in microsatellite stable mCRC was associated with shorter survival, older age at diagnosis, increased mutations, and enrichment of oncogenic alterations in KRAS, BRAF, PIK3CA, AKT1, RNF43, and SMAD4 compared with left-sided primaries. Left-sided tumors frequently had no identifiable genetic alteration in mitogenic signaling, but exhibited higher mitogenic ligand expression. Our results suggest different pathways to tumorigenesis in right- and left-sided microsatellite stable CRC that may underlie clinical differences.

Keywords: APC; CTNNB1; colorectal cancer; integrative genomics analysis; intronic alterations; location; prognosis.

Figure 1

Cohort characteristics and significantly recurrently altered genes identified by MSK-IMPACT testing. (A) Mutation burden versus fraction of genome altered in the 1134 CRC samples sequenced. (B) Classification schema for molecular subtypes: POLE-mutated, MSI-H/hypermutated, and MSS. (C) Comparison of clinical characteristics between the MSK and TCGA CRC cohorts. (D) Significantly recurrently mutated genes identified in MSS CRC and the breakdown of genetic changes within these genes. As the MSK-IMPACT gene panel was successively expanded during the study period, all frequencies and p values were adjusted by the number of samples in which the gene was sequenced. TCF7L2, INHBA, and HIST1H3C were not included in the earliest gene panel. See also Figures S2 and S3 and Tables S2 and S3.

Figure 2

WNT pathway alterations. (A) Alteration frequencies, types of alterations and alteration patterns of genes in the WNT pathway in MSI-H and MSS CRC cases. (B) A mutation in the intron upstream of exon 9 of APC introduces a splice acceptor site, which leads to a change in protein frame (left). Representative images of β-catenin immunohistochemistry in tumors with this APC splice variant (right). (C) Genomic alterations identified in CTNNB1 in MSI-H and MSS CRC cases and representative pictures of β-catenin immunohistochemistry in tumors with the indicated genotypes. Genomic alterations in exons 2–4 in the MSS CRC lollipop plot are shown in detail below, with in-frame deletions identified in CTNNB1 aligned to their start and stop sites. Bar graph shows relative frequency and spectrum of CTNNB1 alterations across tumor types, including hepatocellular carcinoma (HCC), endometrial carcinoma (UCEC), and skin cutaneous melanoma (SKCM). See also Figure S4.

Figure 3

Potentially actionable oncogenic drivers identified by MSK-IMPACT testing. (A) Highest level alterations (alt) for potential clinical actionability in MSI-H and MSS right-sided and left-sided metastatic CRC. (B) Frequency, associated level of actionability, and co-mutation pattern of genomic alterations in mitogenic signaling pathways in metastatic CRC. Star (*) indicates statistically significant difference at p<0.05 level. (C) Frequency and location of oncogenic mutations identified in EGFR. (D) Oncoprint (Top) showing concurrent mutations in the RAS pathway, and lollipop plot (Bottom) showing relative frequency of BRAF mutations in CRC. (E) Bar graphs indicating relative frequency of BRAF mutations and mutation classes in CRC, lung adenocarcinoma (LUAD), and skin cutaneous melanoma (SKCM).

Figure 4

Genomic alterations by primary tumor site. (A) Kaplan-Meier analysis of overall survival (OS) from time of diagnosis of metastatic disease by primary tumor site. (B) Sankey diagram illustrating relative flow of first site of metastasis from left- and right-sided metastatic CRC. (Gyn refers to ovaries, fallopian tubes, uterus, cervix, and vagina; PAO refers to peritoneum, abdominal wall or omentum) (C) Genomic alteration enrichment analysis by primary tumor site. (D) Multivariate model for OS for patients with metastatic MSS CRC. Included in cases that underwent complete metastasectomy are 6 cases with ablation of isolated metastasis and 2 cases with radiation to a single site of metastasis. Square boxes correspond to the hazard ratio (HR) and the ranges indicate the confidence intervals for the HRs. See also Figure S5.

Figure 5

Genomic pathway subgroups and associated outcomes. (A) Kaplan-Meier analysis of overall survival by genomic pathway (pw) subgroups from time of diagnosis of metastatic disease. (B) Bar graphs illustrating relative proportion of genomic subtypes by clinical variables. Star (*) indicates statistically significant difference at p<0.05 level. See also Figure S6 and Table S4.