Chiara Cremolini

KRAS codon 61, 146 and BRAF mutations predict resistance to cetuximab plus irinotecan in KRAS codon 12 and 13 wild-type metastatic colorectal cancer

Background:KRAS codons 12 and 13 mutations predict resistance to anti-EGFR monoclonal antibodies (moAbs) in metastatic colorectal cancer. Also, BRAF V600E mutation has been associated with resistance. Additional KRAS mutations are described in CRC.Methods:We investigated the role of KRAS codons 61 and 146 and BRAF V600E mutations in predicting resistance to cetuximab plus irinotecan in a cohort of KRAS codons 12 and 13 wild-type patients.Results:Among 87 KRAS codons 12 and 13 wild-type patients, KRAS codons 61 and 146 were mutated in 7 and 1 case, respectively. None of mutated patients responded vs 22 of 68 wild type (P=0.096). Eleven patients were not evaluable. KRAS mutations were associated with shorter progression-free survival (PFS, HR: 0.46, P=0.028). None of 13 BRAF-mutated patients responded vs 24 of 74 BRAF wild type (P=0.016). BRAF mutation was associated with a trend towards shorter PFS (HR: 0.59, P=0.073). In the subgroup of BRAF wild-type patients, KRAS codons 61/146 mutations determined a lower response rate (0 vs 37%, P=0.047) and worse PFS (HR: 0.45, P=0.023). Patients bearing KRAS or BRAF mutations had poorer response rate (0 vs 37%, P=0.0005) and PFS (HR: 0.51, P=0.006) compared with KRAS and BRAF wild-type patients.Conclusion:Assessing KRAS codons 61/146 and BRAF V600E mutations might help optimising the selection of the candidate patients to receive anti-EGFR moAbs.

PTEN Expression and KRAS Mutations on Primary Tumors and Metastases in the Prediction of Benefit From Cetuximab Plus Irinotecan for Patients With Metastatic Colorectal Cancer

PURPOSE PTEN, AKT, and KRAS are epidermal growth factor receptor (EGFR) downstream regulators. KRAS mutations confer resistance to cetuximab. This retrospective study investigated the role of PTEN loss, AKT phosphorylation, and KRAS mutations on the activity of cetuximab plus irinotecan in patients with metastatic colorectal cancer (mCRC). PATIENTS AND METHODS A cohort of patients with irinotecan-refractory mCRC who were treated with cetuximab plus irinotecan was tested for PTEN immunoreactivity (ie, immunohistochemistry; IHC), pAKT IHC, and KRAS mutations. Analyses were performed both on primary tumors and on related metastases, and the association among IHC, mutational results, and treatment outcomes was investigated. RESULTS One-hundred two patients were eligible. Ninety-six primary tumors, 59 metastases, and 53 paired samples were available. Forty-nine primary tumors (58% of assessable samples) had a preserved PTEN expression (PTEN-positive), whereas 35 (40% of assessable samples) were pAKT-positive. Levels of concordance between primary tumors and metastases were 60%, 68%, and 95% for PTEN, pAKT, and KRAS, respectively. PTEN status on primary tumors and pAKT status both on primary tumors and on metastases did not predict response or progression-free survival (PFS). On metastases, 12 (36%) of 33 patients with PTEN-positive tumors were responders compared with one (5%) of 22 who had PTEN-negative tumors (P = .007). The median PFS of patients with PTEN-positive metastases was 4.7 months compared with 3.3 months for those with PTEN-negative metastases (hazard ratio [HR], 0.49; P = .005). Patients with PTEN-positive metastases and KRAS wild type had longer PFS compared with other patients (5.5 months v 3.8 months; HR, 0.42; P = .001). CONCLUSION PTEN loss in metastases may be predictive of resistance to cetuximab plus irinotecan. The combination of PTEN IHC and KRAS mutational analyses could help to identify a subgroup of patients with mCRC who have higher chances of benefiting from EGFR inhibition.

Initial Therapy with FOLFOXIRI and Bevacizumab for Metastatic Colorectal Cancer

BACKGROUND A fluoropyrimidine plus irinotecan or oxaliplatin, combined with bevacizumab (a monoclonal antibody against vascular endothelial growth factor), is standard first-line treatment for metastatic colorectal cancer. Before the introduction of bevacizumab, chemotherapy with fluorouracil, leucovorin, oxaliplatin, and irinotecan (FOLFOXIRI) showed superior efficacy as compared with fluorouracil, leucovorin, and irinotecan (FOLFIRI). In a phase 2 study, FOLFOXIRI plus bevacizumab showed promising activity and an acceptable rate of adverse effects. METHODS We randomly assigned 508 patients with untreated metastatic colorectal cancer to receive either FOLFIRI plus bevacizumab (control group) or FOLFOXIRI plus bevacizumab (experimental group). Up to 12 cycles of treatment were administered, followed by fluorouracil plus bevacizumab until disease progression. The primary end point was progression-free survival. RESULTS The median progression-free survival was 12.1 months in the experimental group, as compared with 9.7 months in the control group (hazard ratio for progression, 0.75; 95% confidence interval [CI], 0.62 to 0.90; P=0.003). The objective response rate was 65% in the experimental group and 53% in the control group (P=0.006). Overall survival was longer, but not significantly so, in the experimental group (31.0 vs. 25.8 months; hazard ratio for death, 0.79; 95% CI, 0.63 to 1.00; P=0.054). The incidences of grade 3 or 4 neurotoxicity, stomatitis, diarrhea, and neutropenia were significantly higher in the experimental group. CONCLUSIONS FOLFOXIRI plus bevacizumab, as compared with FOLFIRI plus bevacizumab, improved the outcome in patients with metastatic colorectal cancer and increased the incidence of some adverse events. (Funded by the Gruppo Oncologico Nord Ovest and others; ClinicalTrials.gov number, NCT00719797.).

Clonal evolution and resistance to EGFR blockade in the blood of colorectal cancer patients

Colorectal cancers (CRCs) evolve by a reiterative process of genetic diversification and clonal evolution. The molecular profile of CRC is routinely assessed in surgical or bioptic samples. Genotyping of CRC tissue has inherent limitations; a tissue sample represents a single snapshot in time, and it is subjected to spatial selection bias owing to tumor heterogeneity. Repeated tissue samples are difficult to obtain and cannot be used for dynamic monitoring of disease progression and response to therapy. We exploited circulating tumor DNA (ctDNA) to genotype colorectal tumors and track clonal evolution during treatment with the epidermal growth factor receptor (EGFR)-specific antibodies cetuximab or panitumumab. We identified alterations in ctDNA of patients with primary or acquired resistance to EGFR blockade in the following genes: KRAS, NRAS, MET, ERBB2, FLT3, EGFR and MAP2K1. Mutated KRAS clones, which emerge in blood during EGFR blockade, decline upon withdrawal of EGFR-specific antibodies, indicating that clonal evolution continues beyond clinical progression. Pharmacogenomic analysis of CRC cells that had acquired resistance to cetuximab reveals that upon antibody withdrawal KRAS clones decay, whereas the population regains drug sensitivity. ctDNA profiles of individuals who benefit from multiple challenges with anti-EGFR antibodies exhibit pulsatile levels of mutant KRAS. These results indicate that the CRC genome adapts dynamically to intermittent drug schedules and provide a molecular explanation for the efficacy of rechallenge therapies based on EGFR blockade.

FOLFOXIRI plus bevacizumab versus FOLFIRI plus bevacizumab as first-line treatment of patients with metastatic colorectal cancer: updated overall survival and molecular subgroup analyses of the open-label, phase 3 TRIBE study

BACKGROUND In the TRIBE study, FOLFOXIRI (fluorouracil, leucovorin, oxaliplatin, and irinotecan) plus bevacizumab significantly improved progression-free survival of patients with metastatic colorectal cancer compared with FOLFIRI (fluorouracil, leucovorin, and irinotecan) plus bevacizumab. In this updated analysis, we aimed to provide mature results for overall survival-a secondary endpoint-and report treatment efficacy in RAS and BRAF molecular subgroups. METHODS TRIBE was an open-label, multicentre, phase 3 randomised study of patients (aged 18-70 years with Eastern Cooperative Oncology Group [ECOG] performance status of 2 or less and aged 71-75 years with an ECOG performance status of 0) with unresectable metastatic colorectal cancer who were recruited from 34 Italian oncology units. Patients were randomly assigned (1:1) via a web-based procedure to receive FOLFIRI plus bevacizumab or FOLFOXIRI plus bevacizumab. Bevacizumab was given as a 5 mg/kg intravenous dose. FOLFIRI consisted of a 180 mg/m(2) intravenous infusion of irinotecan for 60 min followed by a 200 mg/m(2) intravenous infusion of leucovorin for 120 min, a 400 mg/m(2) intravenous bolus of fluorouracil, and a 2400 mg/m(2) continuous infusion of fluorouracil for 46 h. FOLFOXIRI consisted of a 165 mg/m(2) intravenous infusion of irinotecan for 60 min, followed by an 85 mg/m(2) intravenous infusion of oxaliplatin given concurrently with 200 mg/m(2) leucovorin for 120 min, followed by a 3200 mg/m(2) continuous infusion of fluorouracil for 48 h. Tissue samples for RAS and BRAF mutational status analyses were centrally collected. In this updated analysis, we assessed the secondary endpoint of overall survival in the main cohort and treatment efficacy in RAS and BRAF molecular subgroups. All analyses were by intention to treat. TRIBE was concluded on Nov 30, 2014. The trial is registered with ClinicalTrials.gov, number NCT00719797. FINDINGS Between July 17, 2008, and May 31, 2011, 508 patients were randomly assigned. At a median follow-up of 48·1 months (IQR 41·7-55·6), median overall survival was 29·8 months (95% CI 26·0-34·3) in the FOLFOXIRI plus bevacizumab group compared with 25·8 months (22·5-29·1) in the FOLFIRI plus bevacizumab group (hazard ratio [HR] 0·80, 95% CI 0·65-0·98; p=0·03). Median overall survival was 37·1 months (95% CI 29·7-42·7) in the RAS and BRAF wild-type subgroup compared with 25·6 months (22·4-28·6) in the RAS-mutation-positive subgroup (HR 1·49, 95% CI 1·11-1·99) and 13·4 months (8·2-24·1) in the BRAF-mutation-positive subgroup (HR 2·79, 95% CI 1·75-4·46; likelihood-ratio test p<0·0001). Treatment effect was not significantly different across molecular subgroups (pinteraction=0·52). INTERPRETATION FOLFOXIRI plus bevacizumab is a feasible treatment option for those patients who meet the inclusion criteria of the present study, irrespective of baseline clinical characteristics and RAS or BRAF mutational status.

Bevacizumab with FOLFOXIRI (irinotecan, oxaliplatin, fluorouracil, and folinate) as first-line treatment for metastatic colorectal cancer: a phase 2 trial

BACKGROUND The FOLFOXIRI (irinotecan, oxaliplatin, fluorouracil, and folinate) regimen has been shown to be better than FOLFIRI (fluorouracil, folinate, and irinotecan) in a phase 3 trial in patients with metastatic colorectal cancer. Results of various studies have shown that the addition of bevacizumab to chemotherapy increases treatment efficacy. We therefore assessed the safety and activity of the combination of FOLFOXIRI plus bevacizumab in patients with colorectal cancer. METHODS In a phase 2 study, patients (aged 18-75 years) with colorectal cancer, which was judged to be unresectable for metastatic disease, were given the combination of intravenous bevacizumab (5 mg/kg on day 1) and intravenous FOLFOXIRI (irinotecan 165 mg/m(2) on day 1, oxaliplatin 85 mg/m(2) on day 1, folinate 200 mg/m(2) on day 1, and fluorouracil 3200 mg/m(2) for 48 h continuous infusion starting on day 1 and repeated every 2 weeks) as first-line treatment in seven centres in Italy. Induction treatment (FOLFOXIRI and bevacizumab) was administered for a maximum of 6 months, followed by maintenance treatment with bevacizumab (5 mg/kg intravenously on day 1, repeated every 2 weeks). The primary endpoint was progression-free survival (PFS) at 10 months from study entry in the intention-to-treat population. This study has been completed and is registered with ClinicalTrials.gov, number NCT01163396. FINDINGS From July 2, 2007, to April 1, 2008, 57 patients were enrolled; all patients were assessed for safety and efficacy. Median follow-up time was 28.8 months (95% CI 24.9-32.5). PFS at 10 months was 74% (95% CI 62-85). Main grade 3 or 4 adverse events during induction treatment were neutropenia (n=28 [49%], including one case of febrile neutropenia), diarrhoea (n=8 [14%]), stomatitis (n=2 [4%]), neurotoxicity (n=1 [2%]), deep-vein thrombosis (n=4 [7%]), and hypertension (n=6 [11%]). No treatment-related deaths occurred. Six serious adverse events occurred during the induction treatment: febrile neutropenia (n=1 [2%]), grade 3 diarrhoea with dehydration (n=2 [4%]), grade 4 stomatitis (n=1 [2%]), grade 4 hypertension (n=1 [2%]), and fluorouracil-related cardiac ischaemia (n=1 [2%]). The most common grade 3 or 4 adverse events noted in the 37 patients who received maintenance treatment were hypertension (n=5 [14%]) and neurotoxicity (n=3 [8%]). One case of acute myocardial infarction due to coronary thrombosis was noted during the maintenance treatment. INTERPRETATION Bevacizumab can be safely used with FOLFOXIRI without causing unforeseen adverse events. Treatment achieved promising results in terms of PFS. A phase 3 study for the comparison of FOLFOXIRI plus bevacizumab with FOLFIRI plus bevacizumab is in progress. FUNDING Gruppo Oncologico Nord Ovest, ARCO Foundation, and Roche.

Primary tumor location as a prognostic factor in metastatic colorectal cancer.

BACKGROUND We sought to clarify the prognostic impact of primary tumor location in metastatic colorectal cancer (mCRC). METHODS We evaluated the association between tumor location and survival parameters in patients with previously untreated mCRC receiving first-line chemotherapy ± bevacizumab in three independent cohorts: a prospective pharmacogenetic study (PROVETTA) and two randomized phase III trials, AVF2107g and NO16966. Cancers proximal or distal of the splenic flexure were classified as right-sided or left-sided, respectively. The primary end point was overall survival (OS). Data were analyzed with Cox proportional hazards and logistic regression models. All statistical tests were two-sided. RESULTS Among evaluable patients in the PROVETTA (n = 200), AVF2107g (n = 559), and NO16966 (n = 1268) studies, 72.0%, 63.1%, and 73.7% had left-sided tumors, respectively. In PROVETTA, patients with left-sided tumors had superior OS (left-sided vs right-sided: hazard ratio [HR] = .44, 95% confidence interval [CI] = .28 to .70, P < .001) and progression-free survival (HR = .52, 95% CI = .36 to .75, P < .001) outcomes. Multivariable analyses confirmed right-sided location as a negative prognostic variable, independent of mucinous histology and BRAF mutational status. Data from the AVF2107g (HR for OS = .55, 95% CI = .43 to .70) and NO16966 trials (HR for OS = .71, 95% CI = .62 to .82 both P < .001) also showed favorable outcomes in patients with left-sided tumors. In both randomized studies, the efficacy of bevacizumab was independent of tumor location. CONCLUSIONS These data demonstrate that primary tumor location is an important prognostic factor in previously untreated mCRC. Given the consistency across an exploratory set and two confirmatory phase III studies, side of tumor origin should be considered for stratification in randomized trials.

Genetic modulation of the Let-7 microRNA binding to KRAS 3′-untranslated region and survival of metastatic colorectal cancer patients treated with salvage cetuximab–irinotecan

There is increasing evidence that the Let-7 microRNA (miRNA) exerts an effect as a tumor suppressor by targeting the KRAS mRNA. The Let-7 complementary site (LCS6) T>G variant in the KRAS 3′-untranslated region weakens Let-7 binding. We analyzed whether the LCS6 variant may be clinically relevant to patients with metastatic colorectal cancer (MCRC) treated with anti-epidermal growth factor receptor (EGFR) therapy. LCS6 genotypes and KRAS/BRAF mutations were determined in the tumor DNA of 134 patients with MCRC who underwent salvage cetuximab–irinotecan therapy. There were 34 G-allele (T/G+G/G) carriers (25%) and 100 T/T genotype carriers (75%). G-allele carriers were significantly more frequent in the KRAS mutation group than in patients with KRAS wild type (P=0.004). In the 121 patients without BRAF V600E mutation, overall survival (OS) and progression-free survival (PFS) times were compared between carriers of the LCS6 G-allele genotypes and carriers of the wild-type T/T genotype. LCS6 G-allele carriers showed worse OS (P=0.001) and PFS (P=0.004) than T/T genotype carriers (confirmed in the multivariate model including the KRAS status). In the exploratory analysis of the 55 unresponsive patients with KRAS mutation, LCS6 G-allele carriers showed adverse OS and PFS times. These findings deserve additional investigations as they may open novel perspectives for the treatment of patients with MCRC.

Randomized Trial of Two Induction Chemotherapy Regimens in Metastatic Colorectal Cancer: An Updated Analysis

BACKGROUND In a randomized trial with a median follow-up of 18.4 months, 6 months of induction chemotherapy with a three-drug regimen comprising 5-fluorouracil (by continuous infusion)-leucovorin, irinotecan, and oxaliplatin (FOLFOXIRI) demonstrated statistically significant improvements in response rate, radical surgical resection of metastases, progression-free survival, and overall survival compared with 6 months of induction chemotherapy with fluorouracil-leucovorin and irinotecan (FOLFIRI). METHODS From November 14, 2001, to April 22, 2005, we enrolled 244 patients with metastatic colorectal cancer. To evaluate if the superiority of FOLFOXIRI is maintained in the long term, we updated the overall and progression-free survival data to include events that occurred up to February 12, 2009, with a median follow-up of 60.6 months. We performed a subgroup and a risk-stratified analysis to examine whether outcomes differed in specific patient subgroups, and we analyzed the results of treatment after progression. Survival curves were estimated by the Kaplan-Meier method. Multivariable Cox regression models were fit to estimate hazard ratios (HRs) and 95% confidence intervals (CIs). All statistical tests were two-sided. RESULTS FOLFOXIRI demonstrated statistically significant improvements in median progression-free survival (9.8 vs 6.8 months, HR for progression = 0.59, 95% CI = 0.45 to 0.76, P < .001) and median overall survival (23.4 vs 16.7 months, HR for death = 0.74, 95% CI = 0.56 to 0.96, P = .026) with a 5-year survival rate of 15% (95% CI = 9% to 23%) vs 8% (95% CI = 4% to 14%). The improvements in progression-free survival and, to a lesser extent, in overall survival were evident even when the analysis excluded patients who received radical resection of metastases. With regard to the risk-stratified analysis, FOLFOXIRI results in longer progression-free survival and overall survival than FOLFIRI in all risk subgroups. CONCLUSIONS Six months of induction chemotherapy with FOLFOXIRI is associated with a clinically significant improvement in the long-term outcome compared with FOLFIRI with an absolute benefit in survival at 5 years of 7%.

FOLFOXIRI plus bevacizumab as first-line treatment in BRAF mutant metastatic colorectal cancer

BACKGROUND BRAF V600E mutation plays a negative prognostic role in metastatic colorectal cancer (mCRC), leading to a median Progression Free Survival (PFS) of 4-6months with first-line conventional treatments. Our group recently reported in a retrospective exploratory analysis of a phase II trial that FOLFOXIRI (5-FU/LV+Oxaliplatin+Irinotecan) plus bevacizumab might allow to achieve remarkable results in terms of PFS and Overall Survival (OS) also in this poor-prognosis subgroup. The aim of this work was to prospectively validate our retrospective finding. PATIENTS AND METHODS This phase II trial was designed to detect an increase in 6month-Progression Free Rate (6m-PFR) from 45% to 80% in a population of BRAF mutant mCRC patients treated with first-line FOLFOXIRI plus bevacizumab. Secondary end-points were PFS, OS, response rate (RR) and the analysis of outcome parameters in the pooled population consisting of both retrospectively and prospectively included patients. This trial is registered with ClinicalTrials.gov, number NCT01437618. RESULTS Two-hundred-fourteen potentially eligible mCRC patients were screened for BRAF mutational status. Fifteen BRAF mutant patients (7%) were included in the validation cohort. At a median follow up of 25.7months, 6m-PFR was 73%. Median PFS and OS were 9.2 and 24.1months, respectively. In the pooled population, at a median follow up of 40.4months, 6m-PFR was 84%. Median PFS and OS were 11.8 and 24.1months, respectively. Overall RR and disease control rate were 72% and 88%, respectively. CONCLUSION Lacking randomised trials in this specific molecular subgroup, FOLFOXIRI plus bevacizumab might be a reasonable option for the first-line treatment of BRAF mutant mCRC patients.