NRAS Status Research Articles

Prospective study of a molecular selection profile for RAS wild type colorectal cancer patients receiving irinotecan-cetuximab.

BackgroundThe aim of our study was to evaluate whether a panel of biomarkers, prospectively analysed might be able to predict patients’ clinical outcome more accurately than RAS status alone.MethodsK-RAS (exons 2, 3, 4) wild type colorectal cancer patients, candidates to second/third-line cetuximab with chemotherapy were prospectively allocated into 2 groups on the basis of their profile: favourable (BRAF and PIK3CA exon 20 wild type, EGFR GCN ≥ 2.6, HER-3 Rajkumar score ≤ 8, IGF-1 immunostaining < 2) or unfavourable (any of the previous markers altered or mutated). After the introduction of N-RAS status (exons 2, 3, 4) only RAS wild type patients were considered eligible.Primary aim was response rate (RR). To detect a difference in terms of RR among patients with an unfavourable profile (estimated around 25%) and patients with a favourable profile (estimated around 60%), with a probability alpha of 0.05 and beta of 0.05, required sample size was 46 patients. Secondary endpoints were progression free survival (PFS) and overall survival (OS).ResultsForty-six patients were enrolled. Seventeen patients (37%) were allocated to the favourable and 29 patients (63%) to the unfavourable profile. RR in the favourable and unfavourable group was 11/17 (65%) and 2/29 (7%) (p = 0.007) respectively. The favourable group also showed an improved PFS (8 months vs. 3 months, p < 0.0001) and OS (15 months vs. 6 months, p < 0.0001).ConclusionsOur results suggest that prospective selection of optimal candidates for cetuximab treatment is feasible and may be able to improve clinical outcome.

Combining a BCL2 Inhibitor with the Retinoid Derivative Fenretinide Targets Melanoma Cells Including Melanoma Initiating Cells

Investigations from multiple laboratories support the existence of melanoma initiating cells (MICs) that potentially contribute to melanoma's drug resistance. ABT-737, a small molecule BCL-2/BCL-XL/BCL-W inhibitor, is promising in cancer treatments, but not very effective against melanoma, with the anti-apoptotic protein MCL-1 as the main contributor to resistance. The synthetic retinoid fenretinide (4-HPR) has shown promise for treating breast cancers. Here, we tested whether the combination of ABT-737 with 4-HPR is effective in killing both the bulk of melanoma cells and MICs. The combination synergistically decreased cell viability and caused cell death in multiple melanoma cells lines (carrying either BRAF or NRAS mutations), but not in normal melanocytes. The combination increased the NOXA expression and caspase-dependent MCL-1 degradation. Knocking-down NOXA protected cells from combination-induced apoptosis, implicating the role of NOXA in the drug synergy. The combination treatment also disrupted primary spheres (a functional assay for MICs) and decreased the percentage of ALDHhigh cells (a marker of MICs) in melanoma cell lines. Moreover, the combination inhibited the self-renewal capacity of MICs, measured by secondary sphere forming assays. In vivo, the combination inhibited tumor growth. Thus, this combination is a promising treatment strategy for melanoma, regardless of mutation status of BRAF or NRAS.

Personalized Treatment for Patients With Colorectal Cancer: Role of Biomarkers

The systemic treatment of patients with colorectal cancer (CRC) has traditionally been based on clinical and tumor histological criteria. Recently however, several prognostic and predictive biomarkers have been proposed for patients with newly diagnosed CRC, including the subgroup with stage II disease. Among the best validated prognostic biomarkers for CRC are CEA levels, MS instability status and certain gene signatures. Although no biomarker currently exists for identifying patients likely to benefit from chemotherapy, the mutational status of KRAS and NRAS is used to predict response to cetuximab and panitumumab. For upfront identification of patients at high risk of suffering from severe therapy-related toxicity, specific variants of dihydropyrimidine dehydrogenase may be measured for predicting toxicity from fluoropyrimidines and uridine diphosphate glucuronosyltransferase*28 (UGT1A1*28) for predicting toxicity from irinotecan.

RAS mutations and their correlation with survival of patients with advanced pancreatic adenocarcinoma.

325 Background: The prognosis of pancreatic adenocarcinoma (PA) remains poor despite FOLFIRINOX and nab-paclitaxel-gemcitabine based chemotherapy. The epidermal growth factor receptor (EGFR) has a major role in pancreatic cancer carcinogenesis.The RAS status showed to be a predictive factor for response to anti-EGFR therapy in colon cancer. We aim to analyze the K-RAS and N-RAS status in PA and its prognostic impact. Methods: Retrospective analysis included 24 patients with metastatic (67%) or locally advanced (29%) PA at diagnosis (AEMOC, Brazil). K-RAS and N-RAS profile were performed by polymerase chain reaction and bidirectional sequencing (codons 12, 13, 61, 117, 146). The results were then analyzed in regards to overall survival (OS) and progression-free survival (PFS) in PA. Results: The sample showed: median age of 63 years (28-86), 62.5% male, 45.8% smoker, head site (67%), ductal (68%), and mild differentiation features (45%). The first-line therapy was FOLFIRINOX (62.5%) and gemcitabine (33.3%). The median PFS and OS were 6.5 and 13 months (mo), respectively. Nineteen patients (79.1%) presented mutation in K-RAS: four c.35G&gt;A (G12D), four c.34G&gt;C (G12R), four c.35G&gt;T (G12V), three c.35G&gt;C (G12A), one c.437C&gt;T (A146V), and one c.34G&gt;T (G12C). Mutation in N-RAS (c.38G&gt;A (G13D) was detected in only one patient (4%). The only independent factor for survival was K-RAS status: the c.35G&gt;A (G12D) polymorphism yielded worse PFS and OS when compared to non-c.35G&gt;A (G12D) mutation: 3 vs 7 mo [HR 0.25, 95% CI, (0.04–1.63)] for PFS (p&lt;0,0043) and 3.5 vs13 mo [HR 0.17, 95% CI, 0.01-1.58)] for OS (p&lt;0,0001), respectively. Conclusions: K-RAS was the only prognostic factor for PFS and OS, with the polymorphism c.35G&gt;A (G12D) being related to a worse prognostic. Further studies are necessary to better evaluate whether K-RAS and N-RAS status is prognostic and/or predictive factor.

Molecular selection for colorectal cancer (CRC) patients receiving cetuximab: Final results of a prospective study.

596 Background: Many markers for a “beyond-RAS” selection of CRC patients receiving cetuximab have been suggested, but none entered clinical practice mainly because prospective validation was lacking. Aim of our study was to evaluate if a molecular profile prospectively analysed was able to predict patients’ clinical outcome more accurately than RAS status alone. Methods: K-RAS (exons 2, 3, 4) wild-type CRC patients, candidates to second/third-line cetuximab with chemotherapy were prospectively allocated, after informed consent, into 2 groups on the basis of their molecular profile: favourable (BRAF and PIK3CA exon 20 wild type, EGFR GCN &gt;2.6, HER-3 Rajkumar score &lt;8, IGF-1 immunostaining &lt;2) and unfavourable (any of the previous markers altered or mutated). After the introduction of N-RAS status (exons 2, 3, 4) only RAS wild type patients were considered eligible for the study. Primary aim was response rate (RR). To detect a difference in terms of RR among patients with an unfavourable profile (estimated around 25%) and patients with a favourable profile (estimated around 60%) required sample size was 46 patients. Secondary endpoints were PFS and OS. Results: 46 patients were enrolled. 17 patients (37%) were allocated to the favourable and 29 patients (63%) to the unfavourable profile. Patients resulted comparable for clinical characteristics (age, sex, ECOG PS, previous treatments, number of metastatic sites). Patients with the unfavourable profile showed 2 BRAF mutations, 3 PIK3CA exon 20 mutations, 18 cases of EGFR GCN &lt;2.6, 20 cases of HER-3 and 16 cases of IGF-1 overexpression respectively. RAS analysis of patients enrolled before the introduction of all RAS status (35 patients) revealed 1 N-RAS mutation (3%) in a patient already allocated to the unfavourable group for HER-3 overexpression. RR in the favourable and unfavourable group was 11/17 (65%) and 2/29 (7%) (p= 0.007) respectively. The favourable group also showed an improved median PFS (8 months vs. 3 months, p &lt;0.0001) and OS (15 months vs. 6 months, p &lt;0.0001). Conclusions: A beyond RAS selection may further improve clinical outcome. This approach may also allow an early switch to alternative treatment for unfavourable profile patients.

Extended KRAS and NRAS Mutation Profiling by Pyrosequencing®.

With the advent of targeted therapies-drugs that specifically target molecules of tumor-driving signalling pathways-and the availability of biomarkers that predict the response of an individual patient on such a targeted therapy, the analysis of the status of the biomarker became an integral part of the therapy. For metastatic colorectal cancer, anti-EGFR-targeted antibodies (Cetuximab/Erbitux(®), Panitumumab/Vectibix(®)) fall into this category of drugs as it was shown in several clinical studies that oncogenic mutations in exons 2-4 of the RAS genes KRAS or NRAS result in therapeutic resistance of the metastatic colorectal cancers against the action of both targeted drugs. Therefore, mutations in the RAS genes exclude patients from this kind of targeted therapy (negative biomarker). Thus the molecular-pathological testing of the mutational status of KRAS and NRAS has become an important cornerstone in planning oncological strategies in the treatment of metastatic colorectal cancer. As the profile of mutations in the RAS genes is characterized by hotspot mutations in only a small number of codons (12, 13 in exon 2-59, 61 in exon 3-117, 146 in exon 4). Pyrosequencing(®) is an ideal and robust tool in the molecular-pathological detection. A detailed protocol for this detection procedure employing Pyrosequencing® is given here.

Synergistic anti-tumor activity and inhibition of angiogenesis by cotargeting of oncogenic and death receptor pathways in human melanoma.

Improving treatment of advanced melanoma may require the development of effective strategies to overcome resistance to different anti-tumor agents and to counteract relevant pro-tumoral mechanisms in the microenvironment. Here we provide preclinical evidence that these goals can be achieved in most melanomas, by co-targeting of oncogenic and death receptor pathways, and independently of their BRAF, NRAS, p53 and PTEN status. In 49 melanoma cell lines, we found independent susceptibility profiles for response to the MEK1/2 inhibitor AZD6244, the PI3K/mTOR inhibitor BEZ235 and the death receptor ligand TRAIL, supporting the rationale for their association. Drug interaction analysis indicated that a strong synergistic anti-tumor activity could be achieved by the three agents and the AZD6244–TRAIL association on 20/21 melanomas, including cell lines resistant to the inhibitors or to TRAIL. Mechanistically, synergy was explained by enhanced induction of caspase-dependent apoptosis, mitochondrial depolarization and modulation of key regulators of extrinsic and intrinsic cell death pathways, including c-FLIP, BIM, BAX, clusterin, Mcl-1 and several IAP family members. Moreover, silencing experiments confirmed the central role of Apollon downmodulation in promoting the apoptotic response of melanoma cells to the combinatorial treatments. In SCID mice, the AZD6244–TRAIL association induced significant growth inhibition of a tumor resistant to TRAIL and poorly responsive to AZD6244, with no detectable adverse events on body weight and tissue histology. Reduction in tumor volume was associated not only with promotion of tumor apoptosis but also with suppression of the pro-angiogenic molecules HIF1α, VEGFα, IL-8 and TGFβ1 and with inhibition of tumor angiogenesis. These results suggest that synergistic co-targeting of oncogenic and death receptor pathways can not only overcome melanoma resistance to different anti-tumor agents in vitro but can also promote pro-apoptotic effects and inhibition of tumor angiogenesis in vivo.

Abstract 4331: Lowering glucose increases nutlin-3 toxicity against melanomas irrespective of BRAF,NRAS or p53 mutations

Abstract BACKGROUND: Melanomas are commonly characterized by mutually exclusive BRAF V600Emutations (∼60%) or NRASQ61 mutations (∼15%). Although targeted therapy with vemurafenib is initially efficient in melanoma with BRAF mutations, tumor recurrence occurs including emergence of resistant melanoma cells with secondary N-RASQ61K mutations. Recently it was demonstrated that nutlin-3, an HDM2 antagonist capable of restoring p53 tumor suppressor function synergized with vemurafenib against BRAF V600E wt p53 melanomas. Although nutlin-3 was initially believed to be non-genotoxic, it can originate p53 mutations which are infrequent in primary melanomas. OBJECTIVES: Since tumor growth increases metabolic demand and glucose consumption, we investigated whether lowering glucose with glycolytic inhibitors increases nutlin-3 toxicity against melanomas irrespective of BRAF, NRAS or p53 status. EXPERIMENTAL: Metabolic activity/proliferation of melanoma cells harbouring BRAF, NRAS or p53 mutations was quantitated by fluorometric studies, crystal violet staining was used to determine differential cell survival, and mechanisms of cell death were studied by protein array immune blotting. RESULTS: Restricting glucose increases nutlin-3 toxicity against wt p53 melanoma cells harbouring BRAF or NRAS, even if the latter have p53 mutations. Responses occur through mechanisms dependent and independent of p53 serine 15-phosphorylation and p21/CIP1/CDNK1A. CONCLUSION: Elimination of melanoma cells irrespective of their BRAF, NRAS or p53 mutational status may be helped by joint use of glucose-lowering agents together with nutlin-3. Citation Format: Manuel Rieber, Valery Chavez-Perez, Mary Strasberg-Rieber. Lowering glucose increases nutlin-3 toxicity against melanomas irrespective of BRAF,NRAS or p53 mutations. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4331. doi:10.1158/1538-7445.AM2014-4331

Dermoscopic criteria associated with BRAF and NRAS mutation status in primary cutaneous melanoma.

The identification of BRAF mutations in melanoma led to the development and implementation of new and effective therapies. Few clinical and histological features have been associated with this mutational status. The main objective of this study was to investigate clinical, histopathological and dermoscopic characteristics of primary melanomas according to BRAF or NRAS mutational status. An observational retrospective study including melanoma dermoscopy images assessed for somatic mutations in BRAF and NRAS. Seventy-two patients were included, 30 women (42%) and 42 men (58%), mean age was 59 ± 15.51 years. BRAF-mutated melanomas were more frequently located on the trunk (n = 18, 64% for BRAF-mutated vs. n = 11, 29% for wild-type melanomas, P = 0.013). Histological ulceration was associated with the presence of BRAF mutations [odds ratio (OR) 3.141; 95% confidence interval (CI) 1.289-7.655; P = 0.002]. The Breslow index tended to be thicker in BRAF-mutated compared with wild-type (P = 0.086). BRAF mutations were present in 28 (39%) patients and only four cases were positive for NRAS mutations (6%), BRAF and NRAS mutations being mutually exclusive. The presence of dermoscopic peppering was associated with MAPK mutations (BRAF and NRAS) (OR 1.68; 95% CI 1.089-2.581; P = 0.015). Dermoscopic ulceration was also associated with BRAF mutations excluding acral and facial melanomas (OR 2.64; 95% CI 1.032-6.754). This study showed a correlation between BRAF and NRAS status and dermoscopic findings of 'peppering' as an expression of regression and melanophages in the dermis, suggesting a morphological consequence of immune behaviour in BRAF-mutated melanomas.

Mutational status of KRAS, NRAS, and BRAF in primary clear cell ovarian carcinoma.

Ovarian clear cell carcinoma (OCCC) is a subtype of epithelial ovarian cancer with characteristic biological features and aggressive clinical behavior. OCCCs show a pattern of gene mutations different from other type I ovarian malignancies, notably a higher frequency of PIK3CA mutations. In low grade serous ovarian cancer, KRAS and BRAF mutations are frequent, but little data are available on the mutational status of these genes in OCCCs. To clarify this issue, we designed a clinicopathological study with the aim to establish the incidence of KRAS, NRAS, and BRAF hot spot mutations in OCCC. Between December 2006 and June 2012, 22 patients with a proven diagnosis of OCCC were admitted to our Institutions. In all cases, final diagnosis was established according to FIGO and WHO criteria. All women received complete surgical staging. The PyroMark Q24 system (Qiagen GmbH, Hilden, Germany) was used for pyrosequencing analysis of KRAS, NRAS, and BRAF hot spot regions on 2.5-μm sections of formalin-fixed paraffin-embedded tissue from primary OCCC. Pyrosequencing analysis of KRAS, NRAS, and BRAF hot spot regions revealed the presence of mutations only at codon 12 in exon 2 of KRAS in 3 of 22 (14 %) cases. We found no mutations in the hot spot regions of NRAF (exons 2, 3, 4) or BRAF (exon 15). The median age of women with a KRAS mutated OCCC was 74 years. These OCCC were unilateral FIGO stage IA lesions in two cases associated with foci of endometriosis. We conclude that in 14 % of OCCCs, a KRAS mutation occurs in codon 2 exon 2. NRAS and BRAF mutations were not found.

Predicting cetuximab efficacy in patients with advanced colorectal cancer

Predicting cetuximab efficacy in patients with advanced colorectal cancer Ibrahim H Sahin, Christopher R Garrett Department of Gastrointestinal Medical Oncology, University of Texas MD Anderson Cancer Center, Houston, TX, USA Abstract: Cetuximab has demonstrated activity, both as monotherapy, and in combination with cytotoxic chemotherapy, albeit modest. Efforts over the last decade have focused on determining which patient populations are most likely to benefit from this chimeric monoclonal antibody therapy. As the antibody targets the epidermal growth factor receptor (EGFR), cell surface expression by immunohistochemistry was hypothesized to be a biomarker of clinical efficacy; subsequent clinical trials have shown that this was not the case. Tumor KRAS mutation (the most frequently observed site is at codon 12) has been shown to be a negative biomarker (ie, a marker of cetuximab resistance); since 2008, treatment of patients with cetuximab has been restricted to those whose tumors do not harbor a KRAS mutation. There is considerable heterogeneity of KRAS mutations, and studies are ongoing to determine whether cetuximab resistance extends to those patients whose tumors have a KRAS codon 13, 61, and 164, mutation. EGFR gene copy, or more precisely a lack of increase in EGFR gene copy number, has been demonstrated to be a negative biomarker of EGFR efficacy; currently, it is not in routine use as a clinical standard of care. Tumor BRAF status, NRAS status, and PIK3CA mutation status are being evaluated as additional potential negative biomarkers of treatment efficacy. High expression of the receptor ligands epiregulin and amphiregulin has been shown to be a positive biomarker for treatment efficacy and is continuing to be studied clinically. After almost 10 years following the widespread introduction of cetuximab into the clinic as a treatment for metastatic colorectal cancer, the story of identifying suitable biomarkers of efficacy is still evolving. The tremendous tumor heterogeneity at the molecular level and the cell complexity of signaling pathways suggest that there is still a significant amount of work to be done to optimally define those patients most likely to benefit from this form of therapy. Keywords: biomarkers, epidermal growth factor receptor, EGFR, KRAS mutation

Personalized therapy for metastatic colorectal cancer in Colombia (ONCOLGroup).

e14635 Background: Targeted therapies have improved the survival of patients with metastatic colorectal cancer (mCRC). Data about expression of EGFR, KRAS, NRAS, and BRAF mutation status, and PI3K/...

Intermittent chemotherapy plus either intermittent or continuous cetuximab for first-line treatment of patients with KRAS wild-type advanced colorectal cancer (COIN-B): a randomised phase 2 trial

SummaryBackgroundAdvanced colorectal cancer is treated with a combination of cytotoxic drugs and targeted treatments. However, how best to minimise the time spent taking cytotoxic drugs and whether molecular selection can refine this further is unknown. The primary aim of this study was to establish how cetuximab might be safely and effectively added to intermittent chemotherapy.MethodsCOIN-B was an open-label, multicentre, randomised, exploratory phase 2 trial done at 30 hospitals in the UK and one in Cyprus. We enrolled patients with advanced colorectal cancer who had received no previous chemotherapy for metastases. Randomisation was done centrally (by telephone) by the Medical Research Council Clinical Trials Unit using minimisation with a random element. Treatment allocation was not masked. Patients were assigned (1:1) to intermittent chemotherapy plus intermittent cetuximab or to intermittent chemotherapy plus continuous cetuximab. Chemotherapy was FOLFOX (folinic acid and oxaliplatin followed by bolus and infused fluorouracil). Patients in both groups received FOLFOX and weekly cetuximab for 12 weeks, then either had a planned interruption (those taking intermittent cetuximab) or planned maintenance by continuing on weekly cetuximab (continuous cetuximab). On RECIST progression, FOLFOX plus cetuximab or FOLFOX was recommenced for 12 weeks followed by further interruption or maintenance cetuximab, respectively. The primary outcome was failure-free survival at 10 months. The primary analysis population consisted of patients who completed 12 weeks of treatment without progression, death, or leaving the trial. We tested BRAF and NRAS status retrospectively. The trial was registered, ISRCTN38375681.FindingsWe registered 401 patients, 226 of whom were enrolled. Results for 169 with KRAS wild-type are reported here, 78 (46%) assigned to intermittent cetuximab and 91 (54%) to continuous cetuximab. 64 patients assigned to intermittent cetuximab and 66 of those assigned to continuous cetuximab were included in the primary analysis. 10-month failure-free survival was 50% (lower bound of 95% CI 39) in the intermittent group versus 52% (lower bound of 95% CI 41) in the continuous group; median failure-free survival was 12·2 months (95% CI 8·8–15·6) and 14·3 months (10·7–20·4), respectively. The most common grade 3–4 adverse events were skin rash (21 [27%] of 77 patients vs 20 [22%] of 92 patients), neutropenia (22 [29%] vs 30 [33%]), diarrhoea (14 [18%] vs 23 [25%]), and lethargy (20 [26%] vs 19 [21%]).InterpretationCetuximab was safely incorporated in two first-line intermittent chemotherapy strategies. Maintenance of biological monotherapy, with less cytotoxic chemotherapy within the first 6 months, in molecularly selected patients is promising and should be validated in phase 3 trials.FundingUK Medical Research Council, Merck KGaA.

Current approaches for predicting a lack of response to anti-EGFR therapy in KRAS wild-type patients.

Targeting epidermal growth factor receptor (EGFR) has been one of the most effective colorectal cancer strategies. Anti-EGFR antibodies function by binding to the extracellular domain of EGFR, preventing its activation, and ultimately providing clinical benefit. KRAS mutations in codons 12 and 13 are recognized prognostic and predictive biomarkers that should be analyzed at the clinic prior to the administration of anti-EGFR therapy. However, still an important fraction of KRAS wild-type patients do not respond to the treatment. The identification of additional genetic determinants of primary or secondary resistance to EGFR targeted therapy for further improving the selection of patients is urgent. Herein, we review the latest published literature highlighting the most important genes that may predict resistance to anti-EGFR monoclonal antibodies in colorectal cancer patients. According to the available findings, the evaluation of BRAF, NRAS, PIK3CA, and PTEN status could be the right strategy to select patients who are likely to respond to anti-EGFR therapies. In the future, the combination of those biomarkers will help establish consensus that can be introduced into clinical practice.

Influence of BRAF and NRAS Mutations on Distant Intracranial Recurrence and Survival in Metastatic Melanoma Following Radiosurgery

The clinical course of patients with metastatic melanoma with brain metastases can be quite varied. This study aims to compare outcomes based on mutational status of BRAF and NRAS following stereotactic radiosurgery (SRS). A prospectively maintained database of patients receiving SRS between November 2002 and December 2012 was queried to determine the rate of new brain metastasis development in patients with metastatic melanoma following SRS. Patients receiving whole brain radiation therapy as first line treatment were excluded. BRAF and NRAS mutational status was determined by Sanger sequencing. The impact of mutation status on risk of distant intracranial recurrence and overall survival (OS) from brain metastasis diagnosis was determined using Poisson regression with robust estimation of errors and Cox proportional hazards analyses, respectively. Other factors including systemic disease control and intracranial disease burden were explored as potential predictors of recurrence and OS in a similar fashion. One hundred eight patients were identified with a mean age of 63 years (range, 24-90 years), of which 68% were male and 94.4% were white. A NRAS mutation was found in 9 patients (8.3%) and BRAF mutation in 24 (22.2%). Median OS from brain metastasis diagnosis was 312 days (range, 228-471 days). A larger number of brain metastases at diagnosis and uncontrolled systemic disease were found to be univariate predictors of worsened OS (HR 1.04, p = 0.012; HR 2.52, p = 0.001, resp). Only uncontrolled systemic disease was a significant predictor of OS on multivariate analysis (HR 10.63, p = 0.022). Mutation status for BRAF and NRAS did not appear to influence survival in our cohort. NRAS-mutated patients were found to be at significantly higher risk of distant intracranial recurrence as compared to wild type patients (IRR for wild type compared to NRAS-mutated, 0.27, p = 0.011). No other variables were found to be significant predictors of recurrence. Although metastatic melanoma follows a highly variable clinical course, NRAS mutations appear to influence the number of intracranial recurrences experienced by patients. Other disease factors including systemic disease control and number of brain metastases at diagnosis seem to influence OS.

Lack of KRAS, NRAS, BRAF and TP53 mutations improves outcome of elderly metastatic colorectal cancer patients treated with cetuximab, oxaliplatin and UFT

There is conflicting evidence on the predictive role of KRAS status when cetuximab is added to oxaliplatin-based regimens. This study investigated the impact of KRAS, NRAS, BRAF, PI3KCA and TP53 status on outcome of elderly metastatic colorectal cancer patients enrolled in TEGAFOX-E (cetuximab, oxaliplatin and oral uracil/ftorafur--UFT) phase II study. Twenty-eight patients were enrolled and all were evaluable for safety and activity. Twenty-three specimens were analysed for KRAS, BRAF, NRAS, PI3KCA and TP53 mutational status by means of polymerase chain reaction and correlated with objective response, progression-free survival and overall survival. An evident increase of response rate was noted in KRAS/NRAS wild-type cases (70 versus 33%, P = 0.198). KRAS/NRAS wild-type status showed an independent association with a longer progression-free survival (44 versus 9 weeks, P = 0.009). Considering the combined assessment of BRAF, KRAS/NRAS and TP53, a trend towards an increase of response rate was noted in patients without mutations (83 versus 33%, P = 0.063). Moreover, patients with all wild-type genes had significantly longer progression-free survival than patients with any mutation (48 versus 10 weeks, P = 0.007). As a single biomarker, only KRAS/NRAS proteins maintained an independent value for outcome prediction. Patients with KRAS/NRAS, BRAF and TP53 wild-type tumours could derive the maximal benefits from treatment with cetuximab, oxaliplatin and UFT.

CRM1 and BRAF Inhibition Synergize and Induce Tumor Regression in BRAF-Mutant Melanoma

Resistance to BRAF inhibitor therapy places priority on developing BRAF inhibitor-based combinations that will overcome de novo resistance and prevent the emergence of acquired mechanisms of resistance. The CRM1 receptor mediates the nuclear export of critical proteins required for melanoma proliferation, survival, and drug resistance. We hypothesize that by inhibiting CRM1-mediated nuclear export, we will alter the function of these proteins resulting in decreased melanoma viability and enhanced BRAF inhibitor antitumoral effects. To test our hypothesis, selective inhibitors of nuclear export (SINE) analogs KPT-185, KPT-251, KPT-276, and KPT-330 were used to induce CRM1 inhibition. Analogs PLX-4720 and PLX-4032 were used as BRAF inhibitors. Compounds were tested in xenograft and in vitro melanoma models. In vitro, we found CRM1 inhibition decreases melanoma cell proliferation independent of BRAF mutation status and synergistically enhances the effects of BRAF inhibition on BRAF-mutant melanoma by promoting cell-cycle arrest and apoptosis. In melanoma xenograft models, CRM1 inhibition reduces tumor growth independent of BRAF or NRAS status and induces complete regression of BRAF V600E tumors when combined with BRAF inhibition. Mechanistic studies show that CRM1 inhibition was associated with p53 stabilization and retinoblastoma protein (pRb) and survivin modulation. Furthermore, we found that BRAF inhibition abrogates extracellular signal-regulated kinase phosphorylation associated with CRM1 inhibition, which may contribute to the synergy of the combination. In conclusion, CRM1 inhibition impairs melanoma survival in both BRAF-mutant and wild-type melanoma. The combination of CRM1 and BRAF inhibition synergizes and induces melanoma regression in BRAF-mutant melanoma.

Phase II trial of cetuximab (Cmab) plus irinotecan (CPT-11) for chemorefractory Japanese patients with advanced and/or metastatic colorectal cancer (mCRC), to evaluate the efficacy and safety based on KRAS, BRAF, PIK3CA, NRAS, and AKT1 mutation status (T-CORE0801).

e14597 Background: Mutations in the KRAS gene have been identified as negative predictors of response to anti-epidermal growth factor receptor (EGFR) antibody (mAb) therapy among patients with mCRC based on Caucasian studies. To date, few studies have examined the relationship between KRAS status and response to Cmab therapy in Asian populations. Therefore, this prospective study investigated the relationship between the mutation status of KRAS and other EGFR-related genes along with clinical outcomes in response to Cmab + CPT-11 therapy in Japanese patients with mCRC. Methods: Samples that had been collected from 43 chemo-refractory patients with mCRC, who had undergone Cmab + CPT-11 therapy at 11 medical centers in Japan, were subjected to direct DNA sequencing to determine their KRAS, BRAF, PIK3CA, NRAS, and AKT1 status. The response rate (RR) was the primary endpoint along with safety, median progression free survival (mPFS), and median overall survival (mOS) as secondary endpoints. Results: The clinical outcomes in response to Cmab + CPT-11 therapy was observed to be better in the wild-type (WT) KRAS subgroup than in the mutant (MT) KRAS subgroup (RR: 17.9% vs. 0%; mPFS: 3.7 vs. 1.6 months (M) ; mOS: 10.3 vs. 7.5 M). The differences in clinical outcomes were more remarkable between patients with both WT KRAS and BRAFand those with MT of either of these genes (RR: 19.2% vs. 0%; mPFS: 5.2 vs. 1.6 M; mOS: 11.8 vs. 7.4 M), as well as between patients with all 5 WT genes and those with MT of either of these genes (RR: 26.3% vs. 0%; mPFS: 5.2 vs. 1.8 M; mOS: 11.8 vs. 7.4 M). Conclusions: Despite identification of a lower than expected RR to treatment among the WT KRAS subgroup, KRAS mutation status appeared to be a useful predictive marker of response to Cmab + CPT-11 therapy in Japanese patients with mCRC . Combined analysis of KRAS and BRAF improved both the sensitivity and specificity of the predictive biomarker. However, the combined analysis of all 5 genes did not result in any considerable improvement compared with that achieved with the KRAS and BRAF analysis alone. Clinical trial information: UMIN000001668.

Low BRAF and NRAS expression levels are associated with clinical benefit from DTIC therapy and prognosis in metastatic melanoma

Metastatic melanoma is characterized by a poor response to chemotherapy. Furthermore, there is a lack of established predictive and prognostic markers. In this single institution study, we correlated mutation status and expression levels of BRAF and NRAS to dacarbazine (DTIC) treatment response as well as progression-free and overall survival in a cohort of 85 patients diagnosed with advanced melanoma. Neither BRAF nor NRAS mutation status correlated to treatment response. However, patients with tumors harboring NRAS mutations had a shorter overall survival (p < 0.001) compared to patients with tumors wild-type for NRAS. Patients having a clinical benefit (objective response or stable disease at 3 months) on DTIC therapy had lower BRAF and NRAS expression levels compared to patients progressing on therapy (p = 0.037 and 0.003, respectively). For BRAF expression, this association was stronger among patients with tumors wild-type for BRAF (p = 0.005). Further, low BRAF as well as NRAS expression levels were associated with a longer progression-free survival in the total population (p = 0.004 and <0.001, respectively). Contrasting low NRAS expression levels, which were associated with improved overall survival in the total population (p = 0.01), low BRAF levels were associated with improved overall survival only among patients with tumors wild-type for BRAF (p = 0.013). These findings indicate that BRAF and NRAS expression levels may influence responses to DTIC as well as prognosis in patients with advanced melanoma.Electronic supplementary materialThe online version of this article (doi:10.1007/s10585-013-9587-4) contains supplementary material, which is available to authorized users.

GNA11 and N‐RAS mutations: alternatives for MAPK pathway activating GNAQ mutations in primary melanocytic tumours of the central nervous system

Primary melanocytic tumours are uncommon neoplasms of the central nervous system. Although similarities with uveal melanomas have been hypothesized, data on their molecular features are limited. In this study, we investigated the mutational status of BRAF(V600E) , KIT, GNAQ, GNA11, N-RAS and H-RAS in a series of 19 primary melanocytic tumours of the central nervous system (CNS). We identified six cases harbouring mutations in the hotspot codon 209 of the GNAQ gene and two cases with mutations in the hotspot codon 209 of the GNA11 gene. Two mutations in codon 61 of N-RAS were also found. In the single strand conformation polymorphism (SSCP) analysis, no shifts corresponding to BRAF(V600E) mutations or suggesting activating mutations in the KIT gene were observed. In primary melanocytic tumours of the CNS, GNA11 and N-RAS mutations represent a mechanism of MAPK pathway activation alternative to the common GNAQ mutations. On the other hand, BRAF(V600E) mutations and activating KIT mutations seem to be absent or very rare in these tumours.