Prognostic significance of K-Ras mutation rate in metastatic colorectal cancer patients. Bruno Vincenzi Università Campus Bio-Medico di Roma

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1 Prognostic significance of K-Ras mutation rate in metastatic colorectal cancer patients Bruno Vincenzi Università Campus Bio-Medico di Roma

2 Colorectal cancer 3 rd most common cancer worldwide Approximately 25% of patients present with metastatic colorectal cancer (mcrc) Metastatic disease develops in 30-45% of newly diagnosed patients Treatment is typically by surgery and chemotherapy Now includes EGFR targeted monoclonal antibodies e.g. cetuximab & panitumumab Karapetis, NEJM, 2009

3 Key Drivers in Colorectal Cancer RAS PI3K RAF Normanno et al. Nat Rev Clin Oncol, 200

4 RAS, RAF and PIK3CA Mutations in Colorectal Cancer Mutation frequency KRAS: 40% NRAS: 3% BRAF: 5% PIK3CA: 15% BRAF and KRAS/NRAS mutations are mutually exclusive KRAS and NRAS mutations are mutually exclusive De Roock, Lancet Oncol 2010

5 K-RAS Status and Prognosis in CRC RASCAL I. KRAS mutation associated with increased risk of recurrence and death (1) RASCAL II. RASCAL II (2001) confirmed but only in Dukes C (Stage 3) tumors (2) posthoc PETACC-3, EORTC 40993, SAKK KRAS mutation was not a prognostic marker (3) posthoc PETACC-8. MSS, KRAS mut and BRAF mut associated with worse outcomes 1. Andreyev HJ, J Natl Cancer Inst Andreyev HJ et al. Br J Cancer. 2001

6 Is KRAS Gene Mutation a Prognostic Factor for Colorectal Cancer: A Systematic Review and Meta-analysis. Ren, JiaoJiao; Li, GuangXiao; Ge, Jie; Li, Xia; Zhao, YaShuang STUDY SELECTION: The prognostic value of KRAS gene mutations was examined in patients with colorectal cancer who did not receive preoperative chemotherapy or radiation. After exclusion of duplicate and irrelevant studies, 23 eligible published studies that included 25 sets of data were finally retrieved for the metaanalysis. RESULTS: The pooled HR for the association between KRAS gene mutations and overall survival in patients with colorectal cancer was 1.04 (95% CI: , p = 0.11). Subgroup analysis showed significant reductions in the overall survival associated with mutations at KRAS codon 12, the articles that reported HR directly, and the studies published before and after 2005, although publication bias was present. All the associations disappeared after adjustment with the trimand-fill method. The pooled HR of 3 studies examining mutations at KRAS codon 13 was 1.47 (95% CI: , p = 0.02), and no publication bias was observed. No significant association was observed in different study regions. CONCLUSIONS: Overall KRAS gene mutations seem not to correlate with the prognosis of patients with colorectal cancer. The association remains to be confirmed with a more precise analysis of a large sample. JJ Ren, Diseases of the Colon & Rectum.

7 K-RAS Status and Prognosis in mcrc Modest DP, Ann Oncol

8 K-RAS Status and Prognosis in mcrc Modest DP, Ann Oncol

9 K-Ras mutation status has been always considered a dichotomic information, so far. BUT In the cancers genetic heterogeneity scenario is important to take into account the percentage of K-Ras mutated cells into the tumour Mutation rate defined as a measure of the rate of mutant alleles

10

11 Study Design Type: Multicentric Retrospective Study Aim: Evaluation of the prognostic significance of K-RAS mutation rate in mcrc patients treated with bevacizumab-containing chemotherapy Network: Policlinico Universitario Campus Bio-Medico di Roma Azienda Ospedaliero-Universitaria Pisana Ospedale Niguarda Ca Granda, Milano Oncologia Medica, Università di Palermo Università Cattolica del Sacro Cuore, Roma Azienda Ospedaliero-Universitaria, Udine Università G. D Annunzio, Chieti Methods : Pirosequencing on FFPE tumor tissue (>60% tumor cells) Statistical analyses: Univariate Analyses Kaplan-Meier (KRAS Mutation Rate dichotomized >40% vs <40%) Multivariate Analyse Cox-regression analyses (KRAS Mutation Rate as continue variable) Endpoints : PFS and OS

12 Methods: Patients population Absolute number Number of evaluable patients Available data about Bevacizumab-chemotherapy Gender (male/female) 144/ /43.4 K-Ras determination (primary vs met) 194/ /26.2 Second Line % Third Line Irinotecan based/oxaliplatin based 201/ /23.6 Number of involved organs or more Cumulative response rate (cpr + ccr) 49% - PFS 8.25 months OS 26 months B.Vincenzi, Oncotarget 2016

13 Methods: Patients population Diagram of the patients enrolled 397 patients (K-Ras mutated/beva in 1line) 92 out (<60% cancer cells) 305 patients 27 out (incomplete FUP data) 278 patients 15 out (miscellaneous) 263 evaluable patients B.Vincenzi, Oncotarget 2016

14 Laboratory methods FFPE tumor samples selection and dissection by pathologist (presence of tumor cells at least 60%) RNA extraction and purification Pyrosequencing analysis of K-Ras status (codons 12, 13, 61, 117 and 146)

15 Results: univariate survival analysis POPULATION (K-Ras mutated codons) Codon 12, all patients (pts) Codon 12, Pts with >40% vs 40% mutant alleles PFS(95% CI) [mts] OS(95% CI) [mts] 8.25 (95% CI: ) 28 (95% CI: ) 7.45 (95% CI: ) vs 9.45 (95% CI: ) 23 (95% CI: ) vs 31 (95% CI: ) Codon 13, all patients (pts) 8.90 (95% CI: ) 21 (95% CI: ) Codon 13, Pts with >40% vs 40% mutant alleles Rare codons, all patients (pts) Rare codons, Pts with >40% vs 40% mutant alleles All K-Ras mutated codons, Pts with >40% vs 40% mutant alleles 7.25 (95% CI: ) vs (95% CI: ) 5.50 (95% CI: ) 5.5 (95% CI: ) vs 7.25 (95% CI: ) 7.25 (95% CI: ) vs 9 (95% CI: ) 17 (95% CI: ) vs 24 (95% CI: ) 21 (95% CI: ) vs 31 (95% CI: ) Log Rank P P=0.000 P=0.002 A mutation rate higher than 40% in all codons analyzed was significantly associated with a worse survival B.Vincenzi, Oncotarget 2016

16 Results: Kaplan-Meyer analysis B.Vincenzi, Oncotarget 2016

17 Results: multivariate survival analysis Survival Analysis (unadjusted) PFS HR: 0.63 ( ) P<0.001 OS HR: 0.66 ( ) P=0.004 Survival Analysis (adjusted) HR: 0.67 ( ) P=0.003 HR: 0.66 ( ) P=0.012 Codon 12 Codon 13 Rare codons HR: 0.71 ( ) P=0.003 HR: 0.62 ( ) P=0.066 HR: 0.19 ( ) P=0.397 HR: 0.69 ( ) P=0.011 HR: ( ) P=0.179 HR: 0.43 ( ) P=0.397 Only codon 12 mutation rate remained significantly associated with PFS and OS in multivariate analyses B.Vincenzi, Oncotarget 2016

18 Conclusions Despite of the well known importance of K-Ras mutations as predictive factor to anti-egfr drugs administration, there is no consensus about the prognostic value of mutational status of K-Ras in CRC patients One of the reasons of these inconclusive results can be also attributed to cancer heterogeneity The use of pyrosequencing technology allow us to identify a novel parameter the mutation rate defined as the measure of the rate of mutant alleles We demonstrated for the first time a correlation between the K-Ras mutation rate (presence of more or less than 40% mutant alleles) in advanced CRC population treated with bevacizumab containing first-line regimens The limit of the study derives from its retrospective nature, thus our data need to be confirmed in a validation prospective set The cut-off of 40% (median value) was arbitrarily chosen and should be

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