Impact of circulating tumor DNA mutant allele fraction on prognosis in RAS-mutant metastatic colorectal cancer.

Despite major advances in the treatment of metastatic colorectal cancer (mCRC), the survival rate remains very poor. This study aims at exploring the prognostic value of RAS-mutant allele fraction (MAF) in plasma in mCRC. Forty-seven plasma samples from 37 RAS-mutated patients with nonresectable metastases were tested for RAS in circulating tumor DNA using BEAMing before first- and/or second-line treatment. RAS MAF was correlated with several clinical parameters (number of metastatic sites, hepatic volume, carcinoembryonic antigen, CA19-9 levels, primary site location, and treatment line) and clinical outcome [progression-free survival (PFS) and overall survival (OS)]. An independent cohort of 32 patients from the CAPRI-GOIM trial was assessed for clinical outcome based on plasma baseline MAF. RAS MAF analysis at baseline revealed a significant correlation with longer OS [Hazard ratios (HR) = 3.514; P = 0.00066]. Patients with lower MAF also showed a tendency to longer PFS, although not statistically significant. Multivariate analysis showed RAS MAFs as an independent prognostic factor in both OS (HR = 2.73; P = 0.006) and first-line PFS (HR = 3.74; P = 0.049). Tumor response to treatment in patients with higher MAF was progression disease (P = 0.007). Patients with low MAFs at baseline in the CAPRI-GOIM group also showed better OS [HR = 3.84; 95% confidence intervals (CI) 1.5-9.6; P = 0.004] and better PFS (HR = 2.5; 95% CI: 1.07-5.62; P = 0.033). This minimally invasive test may help in adding an independent factor to better estimate outcomes before initiating treatment. Further prospective studies using MAF as a stratification factor could further validate its utility in clinical practice.

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.

Current and advancing treatments for metastatic colorectal cancer.

INTRODUCTION: Colorectal cancer (CRC) is one of the leading causes of cancer deaths worldwide. Despite the introduction of several new drugs targeting the vascular endothelial growth factor or epidermal growth factor receptor (EGFR) signaling pathways, survival and disease control in metastatic CRC remains poor. AREAS COVERED: Chemotherapy based on fluoropyrimidines and irinotecan or oxaliplatin has been the cornerstone of CRC standard of care for several decades. Optimal regimens are selected according to toxicity profiles and patient characteristics. The addition of targeted drugs inhibiting angiogenesis, notably bevacizumab, aflibercept and ramucirumab, has improved chemotherapy outcomes in metastatic CRC. Anti-EGFR agents, cetuximab and panitumumab, in combination with chemotherapy have also improved survival in patients with wild-type RAS tumors. In the refractory setting, there are emerging drugs such as regorafenib or TAS-102 that also have demonstrated impact on outcomes. EXPERT OPINION: Drugs targeting signaling pathways involved in tumorigenesis improve patient outcomes over chemotherapy alone. Determining the most suitable combination and sequence should be carefully selected, with studies yet to provide a definitive solution to this unknown. Molecular mechanisms of colorectal cancer are at the forefront of research. Knowledge in this domain will help overcome resistance to therapies and introduce new drugs in the personalized CRC therapeutic scenario.

Intrinsic cancer subtypes--next steps into personalized medicine.

Recent technological advances have significantly improved our understanding of tumor biology by means of high-throughput mutation and transcriptome analyses. The application of genomics has revealed the mutational landscape and the specific deregulated pathways in different tumor types. At a transcriptional level, multiple gene expression signatures have been developed to identify biologically distinct subgroups of tumors. By supervised analysis, several prognostic signatures have been generated, some of them being commercially available. However, an unsupervised approach is required to discover a priori unknown molecular subtypes, the so-called intrinsic subtypes. Moreover, an integrative analysis of the molecular events associated with tumor biology has been translated into a better tumor classification. This molecular characterization confers new opportunities for therapeutic strategies in the management of cancer patients. However, the applicability of these new molecular classifications is limited because of several issues such as technological validation and cost. Further comparison with well-established clinical and pathological features is expected to accelerate clinical translation. In this review, we will focus on the data reported on molecular classification in the most common tumor types such as breast, colorectal and lung carcinoma, with special emphasis on recent data regarding tumor intrinsic subtypes. Likewise, we will review the potential applicability of these new classifications in the clinical routine.

TGF-beta specifically enhances the metastatic attributes of murine lung adenocarcinoma: implications for human non-small cell lung cancer.

Lung cancer is the most frequent and one of the most deadly cancer types and is classified into small cell lung cancer and non-small cell lung cancer (NSCLC). Transforming growth factor beta (TGFß) regulates a wide array of cell functions and plays a major role in lung diseases, including NSCLC. TGFß signals through the complex of TGFß type I and type II receptors, triggering Smad and non-Smad signaling pathways such as PI3K/Akt and MEK1/ERK. We investigated the role of TGFß1 on the progression of the murine lung adenocarcinoma cell line LP07. Furthermore, we undertook a retrospective study with tissue samples from stage I and II NSCLC patients to assess the clinical pathologic role and prognostic significance of TßRI expression. We demonstrated that although lung cancer cell monolayers responded to TGFß1 anti-mitogenic effects and TGFß1 pulse (24 h treatment) delayed tumor growth at primary site; a switch towards malignant progression upon TGFß1 treatment was observed at the metastatic site. In our model, TGFß1 modulated in vitro clonogenicity, protected against stress-induced apoptosis and increased adhesion, spreading, lung retention and metastatic outgrowth. PI3K and MEK1 signaling pathways were involved in TGFß1-mediated metastasis stimulation. Several of these TGFß responses were also observed in human NSCLC cell lines. In addition, we found that a higher expression of TßRI in human lung tumors is associated with poor patient's overall survival by univariate analysis, while multivariate analysis did not reach statistical significance. Although additional detailed analysis of the endogenous signaling in vivo and in vitro is needed, these studies may provide novel molecular targets for the treatment of lung cancer.