Spatio-temporal tumor heterogeneity in metastatic CRC tumors: A mutational-based approach

It is well known that activating mutations in the KRAS and NRAS genes are associated with poor response to anti-EGFR therapies in patients with metastatic colorectal cancer (mCRC). Approximately half of the patients with wild-type (WT) KRAS colorectal carcinoma do not respond to these therapies. This could be because the treatment decision is determined by the mutational profile of the primary tumor, regardless of the presence of small tumor subclones harboring RAS mutations in lymph nodes or liver metastases. We analyzed the mutational profile of the KRAS, NRAS, BRAF and PI3KCA genes using low-density microarray technology in samples of 26 paired primary tumors, 16 lymph nodes and 34 liver metastases from 26 untreated mCRC patients (n=76 samples). The most frequent mutations found in primary tumors were KRAS (15%) and PI3KCA (15%), followed by NRAS (8%) and BRAF (4%). The distribution of the mutations in the 16 lymph node metastases analyzed was as follows: 4 (25%) in KRAS gene, 3 (19%) in NRAS gene and 1 mutation each in PI3KCA and BRAF genes (6%). As expected, the most prevalent mutation in liver metastasis was in the KRAS gene (35%), followed by PI3KCA (9%) and BRAF (6%). Of the 26 cases studied, 15 (58%) displayed an overall concordance in the mutation status detected in the lymph node metastases and liver metastases compared with primary tumor, suggesting no clonal evolution. In contrast, the mutation profiles differed in the primary tumor and lymph node/metastases samples of the remaining 11 patients (48%), suggesting a spatial and temporal clonal evolution. We confirm the presence of different mutational profiles among primary tumors, lymph node metastases and liver metastases. Our results suggest the need to perform mutational analysis in all available tumor samples of patients before deciding to commence anti-EGFR treatment.This work has been partially supported by grants from the Instituto de Salud Carlos III (ISCIII; Ministerio de Sanidad y Consumo, Madrid, Spain) (PI18/00282), the Gerencia Regional de Salud de Castilla y León, Valladolid, Spain (GRS1302/A/16), the RTICC of the ISCIII (RD12/0020/0035-FEDER, RD12/0036/0048-FEDER) and CIBERONC (CB16/12/00400 and CB16/12/00233), the Fundación Memoria de Don Samuel Solórzano Barruso (Salamanca, Spain) and the Fundación Eugenio Rodríguez Pascual, (Madrid, Spain). JM Sayagués and ME Sarasquete are supported by grants (CES11/004 and CP13/00080) from the ISCIII, Ministerio de Ciencia e Innovación, Madrid, Spain

Spatio-temporal tumor heterogeneity in metastatic CRC tumors: a mutational-based approach

[EN] It is well known that activating mutations in the KRAS and NRAS genes are associated with poor response to anti-EGFR therapies in patients with metastatic colorectal cancer (mCRC). Approximately half of the patients with wild-type (WT) KRAS colorectal carcinoma do not respond to these therapies. This could be because the treatment decision is determined by the mutational profile of the primary tumor, regardless of the presence of small tumor subclones harboring RAS mutations in lymph nodes or liver metastases. We analyzed the mutational profile of the KRAS, NRAS, BRAF and PI3KCA genes using low-density microarray technology in samples of 26 paired primary tumors, 16 lymph nodes and 34 liver metastases from 26 untreated mCRC patients (n=76 samples). The most frequent mutations found in primary tumors were KRAS (15%) and PI3KCA (15%), followed by NRAS (8%) and BRAF (4%). The distribution of the mutations in the 16 lymph node metastases analyzed was as follows: 4 (25%) in KRAS gene, 3 (19%) in NRAS gene and 1 mutation each in PI3KCA and BRAF genes (6%). As expected, the most prevalent mutation in liver metastasis was in the KRAS gene (35%), followed by PI3KCA (9%) and BRAF (6%). Of the 26 cases studied, 15 (58%) displayed an overall concordance in the mutation status detected in the lymph node metastases and liver metastases compared with primary tumor, suggesting no clonal evolution. In contrast, the mutation profiles differed in the primary tumor and lymph node/metastases samples of the remaining 11 patients (48%), suggesting a spatial and temporal clonal evolution. We confirm the presence of different mutational profiles among primary tumors, lymph node metastases and liver metastases. Our results suggest the need to perform mutational analysis in all available tumor samples of patients before deciding to commence anti-EGFR treatment

Prognostic implications of EGFR protein expression in sporadic colorectal tumors: Correlation with copy number status, mRNA levels and miRNA regulation

[EN]Sporadic colorectal cancer (sCRC) is the third most frequent cancer worldwide and the second most common cause of cancer-related deaths (mainly due metastatic dissemination). We investigated the immunohistochemical expression of frequently altered proteins in primary tumors from 51 patients (25 liver metastatic and 26 non-metastatic cases) with a median 103 months follow-up (103 months). We evaluated EGFR copy number (using SNP arrays and FISH) and its expression and regulation (by mRNA and miRNA arrays). We found differences between metastatic and non-metastatic sCRCs for MLH1 (p = 0.05), PMS2 (p = 0.02), CEA (p < 0.001) and EGFR (p < 0.001) expression. EGFR expression was associated with lymph node metastases (p = 0.001), liver metastases at diagnosis (p < 0.001), and advanced stage (p < 0.001). There were associations between EGFR expression-, EGFR gene copy number- and EGFR mRNA levels. We found potential interactions of two miRNAs targeting EGFR expression, (miR-134 and miR-4328, in non-metastatic and metastatic tumors, respectively). EGFR expression was associated with a worse outcome (p = 0.005). Multivariate analysis of prognostic factors for overall survival identified that, the expression of EGFR expression (p = 0.047) and pTNM stage (p < 0.001) predicted an adverse outcome. EGFR expression could be regulated by amplification or polysomies (in metastatic tumors), or miRNAs (miRNA-134, in non-metastatic tumors). EGFR expression in sCRC appears to be related to metastases and poor outcome.This work has been partially supported by grants from the Gerencia Regional de Salud de Castilla y León, Valladolid, Spain (GRS2051/A/19), CIBERONC from the ISCIII (CB16/12/00400) and the Fundación Memoria de Don Samuel Solórzano Barruso, Salamanca, Spai