High Concordance and Negative Prognostic Impact of RAS/BRAF/PIK3CA Mutations in Multiple Resected Colorectal Liver Metastases

Abstract

We investigated the intra-patient heterogeneity of driver gene mutations among colorectal liver metastases by sequencing 479 tumor samples from 106 patients. A near-perfect intra-patient concordance was found in the mutation status of the primary tumor and multiple metastatic lesions of KRAS/NRAS/BRAF and PIK3CA when high-sensitivity methods were applied. Mutations in KRAS alone and KRAS/NRAS/BRAF combined had a negative prognostic impact after liver resection. Background: The prevalence and clinical implications of genetic heterogeneity in patients with multiple colorectal liver metastases remain largely unknown. In a prospective series of patients undergoing resection of colorectal liver metastases, the aim was to investigate the inter-metastatic and primary-to-metastatic heterogeneity of mutations in KRAS, NRAS, BRAF, and PIK3CA and their prognostic impact. Patients and Methods: We analyzed the mutation status among 372 liver metastases and 78 primary tumors from 106 patients by methods used in clinical routine testing, by Sanger sequencing, by next-generation sequencing (NGS), and/or by droplet digital polymerase chain reaction. The 3-year cancer-specific survival (CSS) was analyzed using the Kaplan-Meier method. Results: Although Sanger sequencing indicated inter-metastatic mutation heterogeneity in 14 of 97 patients (14%), almost all cases were refuted by high-sensitive NGS. Also, heterogeneity among metastatic deposits was concluded only for PIK3CA in 2 patients. Similarly, primary-to-metastatic heterogeneity was indicated in 8 of 78 patients (10%) using Sanger sequencing but for only 2 patients after NGS, showing the emergence of 1 KRAS and 1 PIK3CA mutation in the metastatic lesions. KRAS mutations were present in 53 of 106 patients (50%) and were associated with poorer 3-year CSS after liver resection (37% vs. 61% for KRAS wild-type; P ¼ .004). Poor prognostic associations were found also for the combination of KRAS/NRAS/BRAF mutations compared with triple wild-type (P ¼ .002). Conclusion: Intrapatient mutation heterogeneity was virtually undetected, both between the primary tumor and the liver metastases and among the metastatic deposits. KRAS mutations separately, and KRAS/NRAS/BRAF mutations combined, were associated with poor patient survival after partial liver resection.

Genomic and Prognostic Heterogeneity Among RAS/BRAFV600E/TP53 Co-Mutated Resectable Colorectal Liver Metastases

“…Intra-patient molecular heterogeneity is anticipated to have clinical implications (33), and current evidence in metastatic CRC suggests that heterogeneity on the DNA copy number level is more widespread than heterogeneity of single nucleotide variants (SNVs) and small insertions/deletions (indels), at least in cancer critical genes (21,23,24). We have shown that mutations in KRAS, NRAS, BRAF V600E (17), and TP53 are predominantly homogeneously present among multiple resected CRLM from each patient. The DNA copy number states of the four genes were more heterogeneous among metastases and correlated with the genome-wide inter-metastatic CNA heterogeneity, consistent with a lower selection pressure for these genes on the DNA copy number level than on the point mutation level.…”

“…A total of 355 metastatic tumor samples from 103 patients have previously been analyzed for hotspot mutations in BRAF exon 15 and KRAS and NRAS exons 2-4 by Sanger sequencing (17). The remaining 86 tumor samples and 68 patients were analyzed in the present study.…”

“…Five cases with intra-patient mutation heterogeneity of TP53 among metastatic lesions were re-analyzed. Heterogeneous cases were rst veri ed by a second Sanger sequencing, and two of the patients were subsequently re-analyzed also with ultra-deep targeted sequencing with the Illumina TruSight Tumor 15 gene panel as described in (17). High-sensitivity analyses discon rmed TP53 mutation heterogeneity in one patient.…”

“…CRLM commonly presents with multiple distinct liver lesions. Cancer-critical genes with a high mutation prevalence in CRC generally have a homogenous mutation pattern across metastatic lesions from the same patient (16,17), although treatment pressure may cause subclonal expansion, as illustrated by the emergence of resistant subclones with pre-existing or acquired KRAS mutations during anti-EGFR therapy (18,19). More extensive mutation heterogeneity has been demonstrated in other protein-coding genes, both in intra-tumor and inter-tumor comparisons (20)(21)(22).…”

Genomic and prognostic heterogeneity among RAS/BRAFV600E/TP53 co‐mutated resectable colorectal liver metastases

“…CRLM commonly presents with multiple distinct liver lesions. Cancer-critical genes with a high mutation prevalence in CRC generally have a homogenous mutation pattern across metastatic lesions from the same patient [18,19], although treatment pressure may cause subclonal expansion, as illustrated by the emergence of resistant subclones with pre-existing or acquired KRAS mutations during anti-EGFR therapy [20,21]. More extensive mutation heterogeneity has been demonstrated in other protein-coding genes, both in intra-tumor and inter-tumor comparisons [22][23][24].…”