Comparative sequencing analysis reveals high genomic concordance between matched primary and metastatic colorectal cancer lesions

Abstract

Background: Colorectal cancer is the second leading cause of cancer death in the United States, with over 50,000 deaths estimated in 2014. Molecular profiling for somatic mutations that predict absence of response to anti-EGFR therapy has become standard practice in the treatment of metastatic colorectal cancer; however, the quantity and type of tissue available for testing is frequently limited. Further, the degree to which the primary tumor is a faithful representation of metastatic disease has been questioned. As next-generation sequencing technology becomes more widely available for clinical use and additional molecularly targeted agents are considered as treatment options in colorectal cancer, it is important to characterize the extent of tumor heterogeneity between primary and metastatic tumors.

Results: We performed deep coverage, targeted next-generation sequencing of 230 key cancer-associated genes for 69 matched primary and metastatic tumors and normal tissue. Mutation profiles were 100% concordant for KRAS, NRAS, and BRAF, and were highly concordant for recurrent alterations in colorectal cancer. Additionally, whole genome sequencing of four patient trios did not reveal any additional site-specific targetable alterations.

Conclusions: Colorectal cancer primary tumors and metastases exhibit high genomic concordance. As current clinical practices in colorectal cancer revolve around KRAS, NRAS, and BRAF mutation status, diagnostic sequencing of either primary or metastatic tissue as available is acceptable for most patients. Additionally, consistency between targeted sequencing and whole genome sequencing results suggests that targeted sequencing may be a suitable strategy for clinical diagnostic applications.

Figure 1

Mutation patterns are concordant between primary and metastatic tumors and consistent with TCGA. (A) Most commonly mutated gene frequencies are similar to those of the TCGA non-hypermutated cohort, with minor differences likely due to increased sequencing depth and more advanced disease. (B, C) Mutations are highly concordant between primary and metastatic tumors. Shared mutations are in dark purple, private to primary is light red, private to metastasis in light blue. Mutations that are loss-of-function (nonsense, frameshift, or splice site) or that occur in at least five samples in Cosmic are marked with an orange dot.

Figure 2

Phenotypic concordance of mutations. In patient 10, the primary tumor harbors a nonsense mutation not found in the metastatic tumor (A). (B) However, the tumor/normal ratio per exon of coverage on chromosome 5 shows that the exons of APC (red dots) are deleted in the metastatic tumor, yielding identical phenotypic results. Similar results were found for PIK3CA and TP53 (Additional file 2: Figure S2).

Figure 3

Metastatic-specific RTK-RAS activating events in RAS/RAF wildtype tumors. In several tumors lacking KRAS, NRAS, or BRAF mutations, additional events in the RTK-RAS pathway were identified. (A) In patient 19, a metastatic-specific MAP2K1 p.Q56P mutation was identified. Transfection of GFP-tagged MAP2K1 plasmids demonstrate that the p.Q56P mutation hyperactivates downstream signaling to the same level as the known p.K57N mutation. (B) In patient 3, chromosome 7p is specifically amplified in the metastatic tumor. (C) FISH analysis confirms regions of high level amplification of EGFR in the metastatic tumor (right) while the primary tumor only shows 7p polysomy (left).

Figure 4

Whole genome analysis of mutational concordance. (A) Concordant and discordant non-synonymous mutations and indels for four CRC patients. (B) Percent of protein coding alterations per sample for IMPACT and WGS results. Patients 3 and 19 were discordant by IMPACT and remain so by WGS, while patients 14 and 54 remain largely concordant.