Julieta Grasselli

Molecular landscape of acquired resistance to targeted therapy combinations in BRAF mutant colorectal cancer

Although recent clinical trials of BRAF inhibitor combinations have demonstrated improved efficacy in BRAF-mutant colorectal cancer, emergence of acquired resistance limits clinical benefit. Here, we undertook a comprehensive effort to define mechanisms underlying drug resistance with the goal of guiding development of therapeutic strategies to overcome this limitation. We generated a broad panel of BRAF-mutant resistant cell line models across seven different clinically relevant drug combinations. Combinatorial drug treatments were able to abrogate ERK1/2 phosphorylation in parental-sensitive cells, but not in their resistant counterparts, indicating that resistant cells escaped drug treatments through one or more mechanisms leading to biochemical reactivation of the MAPK signaling pathway. Genotyping of resistant cells identified gene amplification of EGFR, KRAS, and mutant BRAF, as well as acquired mutations in KRAS, EGFR, and MAP2K1 These mechanisms were clinically relevant, as we identified emergence of a KRAS G12C mutation and increase of mutant BRAF V600E allele frequency in the circulating tumor DNA of a patient at relapse from combined treatment with BRAF and MEK inhibitors. To identify therapeutic combinations capable of overcoming drug resistance, we performed a systematic assessment of candidate therapies across the panel of resistant cell lines. Independent of the molecular alteration acquired upon drug pressure, most resistant cells retained sensitivity to vertical MAPK pathway suppression when combinations of ERK, BRAF, and EGFR inhibitors were applied. These therapeutic combinations represent promising strategies for future clinical trials in BRAF-mutant colorectal cancer. Cancer Res; 76(15); 4504-15. ©2016 AACR.

Nanofluidic Digital PCR and Extended Genotyping of RAS and BRAF for Improved Selection of Metastatic Colorectal Cancer Patients for Anti-EGFR Therapies.

The clinical significance of low-frequent RAS pathway–mutated alleles and the optimal sensitivity cutoff value in the prediction of response to anti-EGFR therapy in metastatic colorectal cancer (mCRC) patients remains controversial. We aimed to evaluate the added value of genotyping an extended RAS panel using a robust nanofluidic digital PCR (dPCR) approach. A panel of 34 hotspots, including RAS (KRAS and NRAS exons 2/3/4) and BRAF (V600E), was analyzed in tumor FFPE samples from 102 mCRC patients treated with anti-EGFR therapy. dPCR was compared with conventional quantitative PCR (qPCR). Response rates, progression-free survival (PFS), and overall survival (OS) were correlated to the mutational status and the mutated allele fraction. Tumor response evaluations were not available in 9 patients and were excluded for response rate analysis. Twenty-two percent of patients were positive for one mutation with qPCR (mutated alleles ranged from 2.1% to 66.6%). Analysis by dPCR increased the number of positive patients to 47%. Mutated alleles for patients only detected by dPCR ranged from 0.04% to 10.8%. An inverse correlation between the fraction of mutated alleles and radiologic response was observed. ROC analysis showed that a fraction of 1% or higher of any mutated alleles offered the best predictive value for all combinations of RAS and BRAF analysis. In addition, this threshold also optimized prediction both PFS and OS. We conclude that mutation testing using an extended gene panel, including RAS and BRAF with a threshold of 1% improved prediction of response to anti-EGFR therapy. Mol Cancer Ther; 15(5); 1106–12. ©2016 AACR.

Are Gene Signatures Ready for Use in the Selection of Patients for Adjuvant Treatment

Fluoropyrimidine-based chemotherapy improves survival in stage III colon cancer patients in the adjuvant setting, whereas its clinical benefit in stage II is limited. Adjuvant therapy could be considered in patients with high-risk stage II disease, who are more likely to benefit from chemotherapy. Clinicopathological factors have been routinely used for risk stratification in stage II, as well as microsatellite instability (MSI) analysis, which has been recently incorporated in clinical guidelines as a prognostic marker. Other molecular markers, such as KRAS and BRAF mutations, suggested improving accuracy in prognostic classification in non-metastatic disease. Recent data derived from randomized clinical trial demonstrated that KRAS/BRAF gene mutations are associated with worse outcome depending on MSI status and tumor localization. Similarly, supervised gene expression signatures have refined recurrence risk stratification in several prospective studies although the clinical utility is still debatable. In this review, we focus on the new data on molecular and gene expression profiling in non metastatic colon cancer and the impact on prognostication and treatment decision.

Molecular prescreening (MP) to treat patients (pts) with advanced pancreatic cancer (PC) in early clinical trials.

272 Molecular prescreening (MP) to treat advanced pancreatic cancer (PC) patients (pts) in early clinical trials Background: Advanced PC has a dismal prognosis, with conventional therapies having poor impact on disease course. The aim of this study was to investigate the impact of a MP program to identify potentially targetable alterations (PTa) in PC pts eligible for clinical trials with matched targeted therapies. Methods: From Jan 2011 to June 2015, a total of 86 chemorefractory PC pts were referred to MP at the VHIO Phase I Unit. Archived tumor samples were analyzed for selected gene mutations (mut) in using mass spectrometry (MassARRAY, Sequenom) until June 2014 or next-generation sequencing (Amplicon, MiSeq) thereafter. PTEN and PDL1 expression levels were measured with immunohistochemistry. All demographic, treatment and survival data were extracted retrospectively from electronic medical records. Results: Median age was 61 years (range 29-78) and 64% were male. Median treatment lines before MP was...

Tenth anniversary of bevacizumab in colorectal cancer: has it fulfilled its promise?

149 ISSN 1479-6694 10.2217/FON.14.6