BRAF Wild-type Tumors Research Articles

Paradoxical Activation of T Cells via Augmented ERK Signaling Mediated by a RAF Inhibitor

RAF inhibitors selectively block ERK signaling in BRAF-mutant melanomas and have defined a genotype-guided approach to care for this disease. RAF inhibitors have the opposite effect in BRAF wild-type tumor cells, where they cause hyperactivation of ERK signaling. Here, we predict that RAF inhibitors can enhance T cell activation, based upon the observation that these agents paradoxically activate ERK signaling in BRAF wild-type cells. To test this hypothesis, we have evaluated the effects of the RAF inhibitor BMS908662 on T cell activation and signaling in vitro and in vivo. We observe that T cell activation is enhanced in a concentration-dependent manner and that this effect corresponds with increased ERK signaling, consistent with paradoxical activation of the pathway. Furthermore, we find that the combination of BMS908662 with CTLA-4 blockade in vivo potentiates T cell expansion, corresponding with hyperactivation of ERK signaling in T cells detectable ex vivo. Lastly, this combination demonstrates superior anti-tumor activity, compared to either agent alone, in two transplantable tumor models. This study provides clear evidence that RAF inhibitors can modulate T cell function by potentiating T cell activation in vitro and in vivo. Paradoxical activation of ERK signaling in T cells offers one mechanism to explain the enhanced antitumor activity seen when RAF inhibitors are combined with CTLA-4 blockade in preclinical models.

RNA Sequencing Identifies Multiple Fusion Transcripts, Differentially Expressed Genes, and Reduced Expression of Immune Function Genes in BRAF (V600E) Mutant vs BRAF Wild-Type Papillary Thyroid Carcinoma

The BRAF V600E mutation (BRAF-MUT) confers an aggressive phenotype in papillary thyroid carcinoma, but unidentified additional genomic abnormalities may be required for full phenotypic expression. RNA sequencing (RNA-Seq) was performed to identify genes differentially expressed between BRAF-MUT and BRAF wild-type (BRAF-WT) tumors and to correlate changes to patient clinical status. BRAF-MUT and BRAF-WT tumors were identified in patients with T1N0 and T2-3N1 tumors evaluated in a referral medical center. Gene expression levels were determined (RNA-Seq) and fusion transcripts were detected. Multiplexed capture/detection and digital counting of mRNA transcripts (nCounter, NanoString Technologies) validated RNA-Seq data for immune system-related genes. BRAF-MUT patients included nine women, three men; nine were TNM stage I and three were stage III. Three (25%) had tumor infiltrating lymphocytes. BRAF-WT included five women, three men; all were stage I, and five (62.5%) had tumor infiltrating lymphocytes. RNA-Seq identified 560 of 13 085 genes differentially expressed between BRAF-MUT and BRAF-WT tumors. Approximately 10% of these genes were related to MetaCore immune function pathways; 51 were underexpressed in BRAF-MUT tumors, whereas 4 (HLAG, CXCL14, TIMP1, IL1RAP) were overexpressed. The four most differentially overexpressed immune genes in BRAF-WT tumors (IL1B; CCL19; CCL21; CXCR4) correlated with lymphocyte infiltration. nCounter confirmed the RNA-Seq expression level data. Eleven different high-confidence fusion transcripts were detected (four interchromosomal; seven intrachromosomal) in 13 of 20 tumors. All in-frame fusions were validated by RT-PCR. BRAF-MUT papillary thyroid cancers have reduced expression of immune/inflammatory response genes compared with BRAF-WT tumors and correlate with lymphocyte infiltration. In contrast, HLA-G and CXCL14 are overexpressed in BRAF-MUT tumors. Sixty-five percent of tumors had between one and three fusion transcripts. Functional studies will be required to determine the potential role of these newly identified genomic abnormalities in contributing to the aggressiveness of BRAF-MUT and BRAF-WT tumors.

Analysis of PTEN, BRAF and PI3K status for determination of benefit from cetuximab therapy in metastatic colorectal cancer patients refractory to chemotherapy with wild-type KRAS

We investigated predictive values of BRAF, PI3K and PTEN in cetuximab responses in KRAS wild-type (+) chemotherapy refractory, metastatic colorectal cancer (CRC) patients. Primary tumour tissues of 41 KRAS wild-type mCRC patients receiving cetuximab-based chemotherapy were investigated for PI3K, PTEN, KRAS and BRAF mutations. Progression-free survival (PFS) and overall survival (OS) periods were calculated with Kaplan-Meier method and the Cox proportional hazards model was used. PTEN and PI3K expressions were 63 and 42 %, respectively. BRAF mutation was observed as 9.8 % among patients. Tumours with BRAF mutation had statistically lower response rates (RR) for cetuximab-based treatment than tumours with BRAF wild type (0 vs. 58 %, p = 0.02). PTEN expressing tumours had statistically higher RR for cetuximab-based treatment than tumours with PTEN loss (42 vs. 12 %, p = 0.04). PI3K expression had worse significant effect on cetuximab RR than PI3K non-expressed tumours (15 vs. 44 %, p = 0.023). Median PFS was significantly longer in patients with PTEN expression (14 months) than in patients with PTEN loss (5 months) (HR, 0.4; p = 0.028). Median PFS was significantly longer in patients with PI3K non-expression (15.2 months) than in patients with PI3K expression (4.1 months) (HR, 0.31; p = 0.001). Significant difference in PFS and OS between patients with BRAF mutated and BRAF wild-type tumours was not detected. However, patients with PTEN expression had significantly longer OS (15.1 months) than patients with PTEN loss tumour (9.9 months) (HR, 0.34; p = 0.008). Patients without PI3K expression had significantly longer OS (18.2 months) than patients with PI3K expression (10.1 months) (HR, 0.27; p = 0.001). Multivariate analyses revealed that PTEN expression (HR, 0.48; p = 0.02) and absence of PI3K expression (HR, 0.2; p = 0.001) were independent prognostic factors for increased PFS. Similarly, PTEN overexpression (HR, 0.62; p = 0.03) and absence of PI3K expression (HR, 0.27; p = 0.005) were independent prognostic factors for increased OS. In PTEN loss, PI3K expression may be used as biomarkers to further select KRAS wild-type patients undergoing anti-epidermal growth factor receptor treatment.

Differential Inhibition of Ex-Vivo Tumor Kinase Activity by Vemurafenib in BRAF(V600E) and BRAF Wild-Type Metastatic Malignant Melanoma

BackgroundTreatment of metastatic malignant melanoma patients harboring BRAF(V600E) has improved drastically after the discovery of the BRAF inhibitor, vemurafenib. However, drug resistance is a recurring problem, and prognoses are still very bad for patients harboring BRAF wild-type. Better markers for targeted therapy are therefore urgently needed.MethodologyIn this study, we assessed the individual kinase activity profiles in 26 tumor samples obtained from patients with metastatic malignant melanoma using peptide arrays with 144 kinase substrates. In addition, we studied the overall ex-vivo inhibitory effects of vemurafenib and sunitinib on kinase activity status.ResultsOverall kinase activity was significantly higher in lysates from melanoma tumors compared to normal skin tissue. Furthermore, ex-vivo incubation with both vemurafenib and sunitinib caused significant decrease in phosphorylation of kinase substrates, i.e kinase activity. While basal phosphorylation profiles were similar in BRAF wild-type and BRAF(V600E) tumors, analysis with ex-vivo vemurafenib treatment identified a subset of 40 kinase substrates showing stronger inhibition in BRAF(V600E) tumor lysates, distinguishing the BRAF wild-type and BRAF(V600E) tumors. Interestingly, a few BRAF wild-type tumors showed inhibition profiles similar to BRAF(V600E) tumors. The kinase inhibitory effect of vemurafenib was subsequently analyzed in cell lines harboring different BRAF mutational status with various vemurafenib sensitivity in-vitro.ConclusionsOur findings suggest that multiplex kinase substrate array analysis give valuable information about overall tumor kinase activity. Furthermore, intra-assay exposure to kinase inhibiting drugs may provide a useful tool to study mechanisms of resistance, as well as to identify predictive markers.

Clinical, Pathologic, and Biologic Features Associated with BRAF Mutations in Non–Small Cell Lung Cancer

BRAF mutations are found in a subset of non-small cell lung cancers (NSCLC). We examined the clinical characteristics and treatment outcomes of patients with NSCLC harboring BRAF mutations. Using DNA sequencing, we successfully screened 883 patients with NSCLC for BRAF mutations between July 1, 2009 and July 16, 2012. Baseline characteristics and treatment outcomes were compared between patients with and without BRAF mutations. Wild-type controls consisted of patients with NSCLC without a somatic alteration in BRAF, KRAS, EGFR, and ALK. In vitro studies assessed the biologic properties of selected non-V600E BRAF mutations identified from patients with NSCLC. Of 883 tumors screened, 36 (4%) harbored BRAF mutations (V600E, 18; non-V600E, 18) and 257 were wild-type for BRAF, EGFR, KRAS, and ALK negative. Twenty-nine of 36 patients with BRAF mutations were smokers. There were no distinguishing clinical features between BRAF-mutant and wild-type patients. Patients with advanced NSCLC with BRAF mutations and wild-type tumors showed similar response rates and progression-free survival (PFS) to platinum-based combination chemotherapy and no difference in overall survival. Within the BRAF cohort, patients with V600E-mutated tumors had a shorter PFS to platinum-based chemotherapy compared with those with non-V600E mutations, although this did not reach statistical significance (4.1 vs. 8.9 months; P = 0.297). We identified five BRAF mutations not previously reported in NSCLC; two of five were associated with increased BRAF kinase activity. BRAF mutations occur in 4% of NSCLCs and half are non-V600E. Prospective trials are ongoing to validate BRAF as a therapeutic target in NSCLC.

KIT, NRAS, BRAF and PTEN mutations in a sample of Swedish patients with acral lentiginous melanoma

BackgroundAcral lentiginous melanoma (ALM) accounts for <10% of all melanomas in Caucasians. Although the involvement of KIT, NRAS and BRAF mutations is well known in ALM, the impact of these mutations on clinicopathological features has not been established. ObjectiveTo define the KIT, NRAS, BRAF and PTEN mutation frequencies in Swedish patients with ALM and to evaluate the impact of mutation status on patient and tumor characteristics. MethodsTumor cells were microdissected from 88 primary ALMs and 16 paired metastases and analyzed for KIT, NRAS and BRAF mutations. A subset of 25 ALMs was also evaluated for PTEN mutations. ResultsBRAF mutations were identified in 17% of the primary ALMs. Both NRAS and KIT mutations were found at a similar frequency of 15%. Only one of the ALMs that were screened for PTEN harbored a mutation (4%). The KIT, NRAS and BRAF mutation status in paired primary and metastatic ALMs was identical. Patients with BRAF mutated tumors were significantly younger (57 years) than those with BRAF wild-type tumors (73 years, p=0.028). BRAF mutations were significantly more common in females (p=0.011) and more often found in tumors located on the feet (p=0.039). Anatomical site was an independent prognostic factor for overall survival; patients with ALMs on the hands or under fingernails had a better prognosis than those with tumors on the feet or under toenails (p=0.025). ConclusionOur results confirm the presence of KIT, NRAS and BRAF mutations in ALM and provide evidence that mutations in these genes occur at similar frequencies. Our results also show that PTEN is mutated in a small subset of ALM tumors.

Frequent Somatic TERT Promoter Mutations in Thyroid Cancer: Higher Prevalence in Advanced Forms of the Disease

TERT encodes the reverse transcriptase component of telomerase, which adds telomere repeats to chromosome ends, thus enabling cell replication. Telomerase activity is required for cell immortalization. Somatic TERT promoter mutations modifying key transcriptional response elements were recently reported in several cancers, such as melanomas and gliomas. The objectives of the study were: 1) to determine the prevalence of TERT promoter mutations C228T and C250T in different thyroid cancer histological types and cell lines; and 2) to establish the possible association of TERT mutations with mutations of BRAF, RAS, or RET/PTC. TERT promoter was PCR-amplified and sequenced in 42 thyroid cancer cell lines and 183 tumors: 80 papillary thyroid cancers (PTCs), 58 poorly differentiated thyroid cancers (PDTCs), 20 anaplastic thyroid cancers (ATCs), and 25 Hurthle cell cancers (HCCs). TERT promoter mutations were found in 98 of 225 (44%) specimens. TERT promoters C228T and C250T were mutually exclusive. Mutations were present in 18 of 80 PTCs (22.5%), in 40 of 78 (51%) advanced thyroid cancers (ATC + PDTC) (P = 3 × 10(-4) vs PTC), and in widely invasive HCCs (4 of 17), but not in minimally invasive HCCs (0 of 8). TERT promoter mutations were seen more frequently in advanced cancers with BRAF/RAS mutations compared to those that were BRAF/RAS wild-type (ATC + PDTC, 67.3 vs 24.1%; P < 10(-4)), whereas BRAF-mutant PTCs were less likely to have TERT promoter mutations than BRAF wild-type tumors (11.8 vs 50.0%; P = .04). TERT promoter mutations are highly prevalent in advanced thyroid cancers, particularly those harboring BRAF or RAS mutations, whereas PTCs with BRAF or RAS mutations are most often TERT promoter wild type. Acquisition of a TERT promoter mutation could extend survival of BRAF- or RAS-driven clones and enable accumulation of additional genetic defects leading to disease progression.

Differential Effects of Aspirin Before and After Diagnosis of Colorectal Cancer

ALTHOUGH MORBIDITY AND MORTALITY FROM COLOrectal cancer have steadily decreased over the past 3 decades, colorectal cancer remains the third most common form of cancer and the third most common cause of cancer death in the United States. Early disease detection through increases in screening that can identify and allow removal of precancerous polyps is thought to be the major reason for the decrease in colorectal cancer incidence. Additionally, new chemotherapy regimens and targeted therapies have improved the outcome of patients with locally advanced and metastatic colorectal cancer. Several risk factors for colorectal cancer have been identified in epidemiologic studies. These include obesity, 50 years or older, personal and family history of polyps or colorectal cancer, diabetes, and black race/ethnicity. Similarly, several factors are associated with decreased colorectal cancer risk. For example, observational data suggest that physical activity and increased dietary calcium intake are associated with decreased risk of colorectal cancer. There are no generally accepted chemopreventive recommendations for individuals at average risk of colorectal cancer but several medications have been shown to have a cancer preventive effect in averageand high-risk populations. The most compelling evidence comes from observational and interventional trials showing that aspirin is protective against the development of colorectal cancer, colorectal cancer metastases, and disease recurrence after diagnosis of the disease. In addition, the protective effect of aspirin also has been demonstrated in patients with hereditary colorectal cancer. The mechanism of action of the colorectal cancer chemopreventive and anticancer effects of aspirin is not fully understood, but it has been attributed to the antiinflammatory effects of the drug, specifically inhibition of prostaglandin-endoperoxide synthase 2 (PTGS2, formerly named cyclooxygenase 2 or COX-2). PTGS2 is the ratelimiting step for the conversion of arachidonic acid to prostaglandins, and it is overexpressed in the majority of colorectal cancers. In the past few years, several studies have attempted to dissect aspirin’s mechanism of action with respect to colorectal cancer development and progression to identify which subtypes of colorectal cancer aspirin most effectively prevents both before and after diagnosis. In 2009, Chan et al demonstrated that regular aspirin intake after diagnosis of colorectal cancer in patients whose primary tumors overexpressed PTGS2 as assessed by immunohistochemistry was associated with a 60% decreased risk of colorectal cancer– specific mortality. In contrast, there was no association between aspirin use and lower mortality risk among patients whose primary tumors had weak or absent PTGS2 expression. In 2012, Liao et al tested the hypothesis that mutations in the PIK3CA gene, one of the most commonly mutated genes in colorectal cancer, might influence the effect of aspirin in patients with a diagnosis of colorectal cancer. This study showed that the decreased risk of colorectal cancer recurrence associated with the use of aspirin was restricted to patients with tumors harboring a mutated PIK3CA gene. PIK3CA belongs to the PI3K-AKT signaling pathway, which is inappropriately activated in many cancers, including colorectal cancer. The BRAF gene belongs to the RAS-RAF-MEK-ERK signaling pathway and is mutated in approximately 14% of colorectal cancers leading to uncontrolled activation of the pathway. Furthermore, there is evidence that somatically acquired mutations of the BRAF gene portend a poor prognosis. Whether the BRAF gene acts as a mediator of the effect of aspirin prior to and after diagnosis of colorectal cancer is unknown. Importantly, both pathways are directly activated by the epidermal growth factor receptor (EGFR), an established therapeutic target in advanced colorectal cancer. In this issue of JAMA, Nishihara et al analyzed tumors from 1226 patients with a diagnosis of colorectal cancer for mutations within the BRAF gene. When compared to nonusers, aspirin users had a 27% lower risk of having tumors with an intact BRAF gene. In contrast, aspirin use was not associated with any change in risk for BRAF-mutated tumors. Use of aspirin after diagnosis of colorectal cancer did not modify the outcome for patients with either BRAFmutated or BRAF–wild-type tumors.

PD-0005 - Prognostic Biomarkers in a Series of Stage II Colon Cancer

Background: Different clinico-pathological factors are used to define a group of patients with high-risk stage II colon cancer that may benefit from adjuvant chemotherapy. Moreover, several molecular markers, such as microsatellite instability (MSI) and BRAF, have been widely investigated as putative prognostic factors. Recently a high stroma component (tumors with mesenchymal phenotype consistent with epithelial to mesenchymal transition) has also been associated with poor outcome.Methods: Between 1996 and 2006 data from patients diagnosed with colorectal cancer at Hospital Universitari Bellvitge (HUB) and its referral comprehensive cancer center, Institut Català d'Oncologia (ICO)/ L'Hospitalet, were prospectively included in a database. Formalin-fixed paraffin-embedded tissue samples from 432 stage II colon cancer patients operated at HUB were included in the molecular analyses. MSI status, BRAF V600E mutation and tumor-stroma ratio in tumor samples were analyzed. MSI status was assessed by the analysis of 5 mononucleotide repeat markers (BAT-25, BAT-26, NR-21, NR-24 and MONO-27). BRAF V600E mutation was analyzed by single strand conformation polymorphism technique. Tumor-stroma ratio was analyzed by immunohistochemistry. Associations between molecular factors and clinical features were assessed by Chi-Squared (X2) tests. A Cox regression model was used to evaluate the Relapse Free Survival (RFS) and the Colon Cancer specific Survival.Results: MSI status could be determined by genotyping in 350 tumor samples. 48 patients (14%) had MSI high (MSI-H) tumors and 302 (86%) had microsatellite stable (MSS) tumor. BRAF status could be determined in 380 tumor samples. 58 cases (15%) were BRAF mutated tumors and 322 (85%) were BRAF wild type tumors. Tumor-stroma ratio was analyzed in 407 cases. 176 tumors (43%) had more than 50% intra-tumor stroma and were classified as stroma-high. MSI-H tumors were significantly located in the right colon (X2 p-value = 1.03e-5), were poorly differentiated (X2 p-value = 0.003) and had more than 12 lymph nodes resected (X2 p-value = 0.037). MSI-H tumors were associated with BRAF mutation (X2 p-value = 0.02) and stroma-low (X2 p-value = 0.0005). When a molecular prognostic risk classification was performed, patients with MSI-H and stroma-low suggested a low risk of relapse comparing to MSI-H/stroma-high, MSS/stroma-low and MSS/stroma-high (Hazard Ratio = 3.15; 95% CI, 0.76 - 13.1, p-value = 0.0602).Conclusion: MSI-H tumors are typically poorly differentiated, located in the right colon and have BRAF mutation as well as low intra-tumor stroma. Our results suggest that low-stroma could partly explain prognosis in patients with MSI-H stage II tumors. Background: Different clinico-pathological factors are used to define a group of patients with high-risk stage II colon cancer that may benefit from adjuvant chemotherapy. Moreover, several molecular markers, such as microsatellite instability (MSI) and BRAF, have been widely investigated as putative prognostic factors. Recently a high stroma component (tumors with mesenchymal phenotype consistent with epithelial to mesenchymal transition) has also been associated with poor outcome. Methods: Between 1996 and 2006 data from patients diagnosed with colorectal cancer at Hospital Universitari Bellvitge (HUB) and its referral comprehensive cancer center, Institut Català d'Oncologia (ICO)/ L'Hospitalet, were prospectively included in a database. Formalin-fixed paraffin-embedded tissue samples from 432 stage II colon cancer patients operated at HUB were included in the molecular analyses. MSI status, BRAF V600E mutation and tumor-stroma ratio in tumor samples were analyzed. MSI status was assessed by the analysis of 5 mononucleotide repeat markers (BAT-25, BAT-26, NR-21, NR-24 and MONO-27). BRAF V600E mutation was analyzed by single strand conformation polymorphism technique. Tumor-stroma ratio was analyzed by immunohistochemistry. Associations between molecular factors and clinical features were assessed by Chi-Squared (X2) tests. A Cox regression model was used to evaluate the Relapse Free Survival (RFS) and the Colon Cancer specific Survival. Results: MSI status could be determined by genotyping in 350 tumor samples. 48 patients (14%) had MSI high (MSI-H) tumors and 302 (86%) had microsatellite stable (MSS) tumor. BRAF status could be determined in 380 tumor samples. 58 cases (15%) were BRAF mutated tumors and 322 (85%) were BRAF wild type tumors. Tumor-stroma ratio was analyzed in 407 cases. 176 tumors (43%) had more than 50% intra-tumor stroma and were classified as stroma-high. MSI-H tumors were significantly located in the right colon (X2 p-value = 1.03e-5), were poorly differentiated (X2 p-value = 0.003) and had more than 12 lymph nodes resected (X2 p-value = 0.037). MSI-H tumors were associated with BRAF mutation (X2 p-value = 0.02) and stroma-low (X2 p-value = 0.0005). When a molecular prognostic risk classification was performed, patients with MSI-H and stroma-low suggested a low risk of relapse comparing to MSI-H/stroma-high, MSS/stroma-low and MSS/stroma-high (Hazard Ratio = 3.15; 95% CI, 0.76 - 13.1, p-value = 0.0602). Conclusion: MSI-H tumors are typically poorly differentiated, located in the right colon and have BRAF mutation as well as low intra-tumor stroma. Our results suggest that low-stroma could partly explain prognosis in patients with MSI-H stage II tumors.

Impact of age on treatment of primary melanoma patients.

9054 Background: Although patient age at diagnosis is not currently included in guidelines for treatment of primary melanoma, several lines of evidence suggest that patient age is an important, yet understudied, factor when considering treatment options. Here, we attempt to address the limited knowledge of the impact of age on primary melanoma treatment. Methods: In a prospectively enrolled and followed-up cohort of melanoma patients at NYU, we used logistic regression models to evaluate the association between patient age at diagnosis, tumor baseline characteristics, including BRAF and NRAS mutation status, and likelihood of receiving and responding to adjuvant therapy. We examined adjuvant therapy effectiveness using recurrence and melanoma-specific survival as endpoints. Results: 444 primary melanoma patients were included in the study (median follow-up: 6.3 years; age range: 19-95 years). Age was categorized into three groups spanning the range of age at presentation: younger (19-45 years; 24%), middle (46-70 years; 50%), and older (71-95 years; 26%). Older patients were significantly more likely to have advanced stage, nodular subtype (P &lt; 0.01, both variables), and BRAF wildtype tumors (P = 0.04). Controlling for these factors as well as gender, older patients experienced a higher risk of recurrence (HR older vs. younger 3.34, 95% CI 1.53-7.25; P &lt; 0.01). Of the 128/444 (29%) patients who were eligible for adjuvant treatment (clinical stage ≥ IIB), only 67/128 (52%) received treatment. Using a propensity score that accounts for stage at presentation, patients in the middle age group were more likely to receive adjuvant therapy than those in the older group (OR 2.61, 95% CI 1.12-6.08; P = 0.03). In addition, a trend suggesting benefit from adjuvant therapy (defined as longer melanoma-specific survival) was observed only in the middle age group (P = 0.07). Conclusions: Our data suggest that older melanoma patients, despite having a significantly worse prognosis, are less likely to receive and benefit from adjuvant therapy. Further work is needed to understand the biological variables contributing to the limited response to treatment in elderly primary melanoma.

Discovering new targeted therapies for BRAF mutant-like colorectal cancers.

3623 Background: Approximately 10% of colorectal cancers (CRCs) harbor a BRAF mutation (BRAFm). Patients with BRAFm tumors have poor prognosis and are a therapeutic challenge. A BRAFm gene expression signature has been communicated (Popovici et al, JCO 2012), which can identify BRAFm tumors as well as BRAF wild-type tumors that display a similar expression pattern. Collectively, these tumors are termed BRAFm-like. Our goal was to validate this signature using next-generation sequencing and to discover novel therapies for BRAFm-like CRCs using a systems biology approach. Methods: We developed a semi-automated workflow that integrates publicly available tools named the Cancer In-silico Drug Discovery (CIDD). To validate the BRAFm-like signature, we used CIDD to analyze the CRC dataset from the The Cancer Genome Atlas Network (TCGA). Samples were stratified on BRAFm status using exome-sequencing, and expression profiles were inferred from RNA-sequencing. We matched expression profiles with drug-induced signatures inferred from the Connectivity Map (CMap) – a systems biology tool that contains expression data of cell lines treated with 1,500 compounds. CIDD statistically ranks candidate compounds and annotates them to pathways using public databases. Results: When applied to TCGA RNA-sequencing data, a classifier based on the BRAFm-like signature resulted in 93.3% sensitivity and 83.5% specificity for detecting BRAFm samples. When applied to Agilent gene expression data, this resulted in 80% sensitivity and 91.1% specificity. 41% of KRAS-mutated samples and 14% of double wild-type samples were predicted to be BRAFm-like. 100% of MSI-high and 18% of MSS samples were predicted to be BRAFm-like. Compounds near the top of our drug rankings include Gefitinib and MG-262 a proteasome inhibitor. Conclusions: We have validated the BRAFm-like signature using RNA-sequencing and Agilent expression data from the TCGA, and showed a high degree of robustness across technologies. We have identified EGFR and proteasome inhibitors as potential compounds to target BRAFm-like CRCs.

Assessment of clinical parameters associated with mutational status in metastatic malignant melanoma: a single-centre investigation of 141 patients

Inhibitors of the mutated, constitutively activated BRAF protein have shown efficacy in the treatment of metastatic melanoma in clinical trials. Mutation analysis especially of the BRAF, NRAS and KIT genes is essential to identify patients suitable for targeted therapies and has been introduced into routine patient care. To correlate mutational status with clinical parameters including age, skin type, number of melanocytic naevi, primary tumour location, chronic sun damage and exposure to ultraviolet (UV) irradiation. The overall aim was to define subgroups with an increased or decreased likelihood of gene mutations. Additionally, the impact of activating BRAF mutations on clinical course was investigated. In a single-centre, retrospective approach, mutation analysis was performed on patients with metastatic malignant melanoma. Clinical parameters were correlated with molecular findings. The total sun-burden score was assessed using a validated standardized questionnaire. The analysis included 141 patients with metastatic melanoma. Forty-four per cent of patients had activating BRAF mutations and were significantly younger than patients with wild-type BRAF or with NRAS mutations. KIT mutations were detected in only 3% of the patients. BRAF-mutated melanomas developed preferentially in intermittently sun-exposed areas of the body, and patients had significantly more melanocytic naevi. Once patients had progressed into stage IV disease, survival times were identical for those with BRAF-mutated and BRAF wild-type tumours. Mutations of the BRAF gene are correlated with younger age, a higher number of melanocytic naevi and a tumour location in intermittently UV-exposed skin. Signs of chronic photodamage are not indicative of mutational status. Patients with metastatic melanoma with BRAF mutations showed a nonsignificant tendency to progress later to stage IV disease, but once metastases were present the prognosis was identical to that with BRAF wild-type tumours.

MEK162 for patients with advanced melanoma harbouring NRAS or Val600 BRAF mutations: a non-randomised, open-label phase 2 study

Patients with melanoma harbouring Val600 BRAF mutations benefit from treatment with BRAF inhibitors. However, no targeted treatments exist for patients with BRAF wild-type tumours, including those with NRAS mutations. We aimed to assess the use of MEK162, a small-molecule MEK1/2 inhibitor, in patients with NRAS-mutated or Val600 BRAF-mutated advanced melanoma. In our open-label, non-randomised, phase 2 study, we assigned patients with NRAS-mutated or BRAF-mutated advanced melanoma to one of three treatment arms on the basis of mutation status. Patients were enrolled at university hospitals or private cancer centres in Europe and the USA. The three arms were: twice-daily MEK162 45 mg for NRAS-mutated melanoma, twice-daily MEK162 45 mg for BRAF-mutated melanoma, and twice-daily MEK162 60 mg for BRAF-mutated melanoma. Previous treatment with BRAF inhibitors was permitted, but previous MEK inhibitor therapy was not allowed. The primary endpoint was the proportion of patients who had an objective response (ie, a complete response or confirmed partial response). We report data for the 45 mg groups. We assessed clinical activity in all patients who received at least one dose of MEK162 and in patients assessable for response (with two available CT scans). This study is registered with ClinicalTrials.gov, number NCT01320085, and is currently recruiting additional patients with NRAS mutations (based on a protocol amendment). Between March 31, 2011, and Jan 17, 2012, we enrolled 71 patients who received at least one dose of MEK162 45 mg. By Feb 29, 2012 (data cutoff), median follow-up was 3·3 months (range 0·6-8·7; IQR 2·2-5·0). No patients had a complete response. Six (20%) of 30 patients with NRAS-mutated melanoma had a partial response (three confirmed) as did eight (20%) of 41 patients with BRAF-mutated melanoma (two confirmed). The most frequent adverse events were acneiform dermatitis (18 [60%] patients with NRAS -mutated melanoma and 15 [37%] patients with the BRAF-mutated melanoma), rash (six [20%] and 16 [39%]), peripheral oedema (ten [33%] and 14 [34%]), facial oedema (nine [30%] and seven [17%]), diarrhoea (eight [27%] and 15 [37%]), and creatine phosphokinase increases (11 [37%] and nine [22%]). Increased creatine phosphokinase was the most common grade 3-4 adverse event (seven [23%] and seven [17%]). Four patients had serious adverse events (two per arm), which included diarrhoea, dehydration, acneiform dermatitis, general physical deterioration, irregular heart rate, malaise, and small intestinal perforation. No deaths occurred from treatment-related causes. To our knowledge, MEK162 is the first targeted therapy to show activity in patients with NRAS -mutated melanoma and might offer a new option for a cancer with few effective treatments. Novartis Pharmaceuticals.

Progression-free survival in metastatic, BRAF-mutated colorectal cancer.

458 Background: BRAF mutations occur in 5-8% of metastatic colorectal cancers and are associated with a significantly worse overall prognosis relative to patients with BRAF wild-type tumors. However, the outcomes with standard chemotherapy of a large cohort of patients with BRAF mutant tumors have not been described. Methods: We searched the MD Anderson Cancer Center databases for colorectal cancer patients with identified BRAF mutations between December 2003 and May 2012. Patients were analyzed for clinical characteristics, progression-free survival, and chemotherapeutic agents used. Survival was estimated according to the Kaplan-Meier method. Results: Among the 1567 patients tested for BRAF mutations at our institution, 127 (8.1%) had tumors with BRAF mutations. The average age was 59.6 years at the time of diagnosis, and 65/127 patients (51.2%) were male. The 71 patients who presented with metastatic disease received a median of 2 lines of chemotherapy. For the first three lines of chemotherapy, median progression-free survival was 5.6 months (n=69 patients), 3.9 months (n=58), and 3.4 months (n=31), respectively. Median PFS was not affected by the backbone chemotherapeutic agent in the first-line setting, whether oxaliplatin-based or irinotecan-based (6.1 months vs. 5.1 months, respectively, p-value = 0.78). Conclusions: Progression-free survival is expectedly poor for patients with BRAF-mutated metastatic colorectal cancer. Despite the ascertainment bias present in this cohort (with testing preferentially performed in patients suitable for clinical trials in refractory disease), fewer than half of patients with metastatic disease received more than 2 lines of therapy. This data not only provides historic controls suitable for future study design in this population but also supports the idea that novel therapeutic options are essential for this group of patients.

BRAF mutation-specific promoter methylation of FOX genes in colorectal cancer

BackgroundCancer-specific hypermethylation of (promoter) CpG islands is common during the tumorigenesis of colon cancer. Although associations between certain genetic aberrations, such as BRAF mutation and microsatellite instability, and the CpG island methylator phenotype (CIMP), have been found, the mechanisms by which these associations are established are still unclear. We studied genome-wide DNA methylation differences between colorectal tumors carrying a BRAF mutation and BRAF wildtype tumors.ResultsUsing differential methylation hybridization on oligonucleotide microarrays representing 32,171 CpG-rich regions, we identified 1,770 regions with differential methylation between colorectal tumor and paired normal colon. Next, we compared the tumor/normal methylation ratios between different groups of patients. Related to CIMP, we identified 749 differentially methylated regions, of which 86% had a higher tumor/normal methylation ratio in the CIMP-positive group. We identified 758 regions with a BRAF mutation-specific methylation change, of which 96% had a higher tumor/normal methylation ratio in the BRAF mutant group. Among the genes affected by BRAF mutation-specific methylation changes, we found enrichment of several cancer-related pathways, including the PI3 kinase and Wnt signaling pathways. To focus on genes that are silenced in a tumor-specific rather than a lineage-specific manner, we used information on the epigenetic silencing mark H3K27me3 in embryonic stem (ES) cells. Among the genes showing BRAF mutation-specific promoter methylation but no H3K27me3 mark in ES cells were forkhead box (FOX) transcription factors associated with the PI3 kinase pathway, as well as MLH1 and SMO. Repression of FOXD3 gene expression in tumors could be related to its promoter hypermethylation.ConclusionsWe identified new BRAF mutation-specific methylation changes in colorectal cancer. Epigenetic downregulation of these targets may contribute to mutationally active BRAF-driven tumorigenesis, explaining its association with aberrant DNA methylation.

Targeted Therapy; From Advanced Melanoma to the Adjuvant Setting

Targeted Therapy; From Advanced Melanoma to the Adjuvant Setting

NRAS mutant melanoma: biological behavior and future strategies for therapeutic management

The recent years have seen a significant shift in the expectations for the therapeutic management of disseminated melanoma. The clinical success of BRAF targeted therapy suggests that long-term disease control may one day be a reality for genetically defined subgroups of melanoma patients. Despite this progress, few advances have been made in developing targeted therapeutic strategies for the 50% of patients whose melanomas are BRAF wild-type. The most well-characterized subgroup of BRAF wild-type tumors is the 15-20% of all melanomas that harbor activating NRAS (Neuroblastoma Rat Sarcoma Virus) mutations. Emerging preclinical and clinical evidence suggests that NRAS mutant melanomas have patterns of signal transduction and biological behavior that is distinct from BRAF mutant melanomas. This overview will discuss the unique clinical and prognostic behavior of NRAS mutant melanoma and will summarize the emerging data on how NRAS-driven signaling networks can be translated into novel therapeutic strategies.

Oncogenic BRAF(V600E) Promotes Stromal Cell-Mediated Immunosuppression Via Induction of Interleukin-1 in Melanoma

In this study, we assessed the specific role of BRAF(V600E) signaling in modulating the expression of immune regulatory genes in melanoma, in addition to analyzing downstream induction of immune suppression by primary human melanoma tumor-associated fibroblasts (TAF). Primary human melanocytes and melanoma cell lines were transduced to express WT or V600E forms of BRAF, followed by gene expression analysis. The BRAF(V600E) inhibitor vemurafenib was used to confirm targets in BRAF(V600E)-positive melanoma cell lines and in tumors from melanoma patients undergoing inhibitor treatment. TAF lines generated from melanoma patient biopsies were tested for their ability to inhibit the function of tumor antigen-specific T cells, before and following treatment with BRAF(V600E)-upregulated immune modulators. Transcriptional analysis of treated TAFs was conducted to identify potential mediators of T-cell suppression. Expression of BRAF(V600E) induced transcription of interleukin 1 alpha (IL-1α) and IL-1β in melanocytes and melanoma cell lines. Further, vemurafenib reduced the expression of IL-1 protein in melanoma cell lines and most notably in human tumor biopsies from 11 of 12 melanoma patients undergoing inhibitor treatment. Treatment of melanoma-patient-derived TAFs with IL-1α/β significantly enhanced their ability to suppress the proliferation and function of melanoma-specific cytotoxic T cells, and this inhibition was partially attributable to upregulation by IL-1 of COX-2 and the PD-1 ligands PD-L1 and PD-L2 in TAFs. This study reveals a novel mechanism of immune suppression sensitive to BRAF(V600E) inhibition, and indicates that clinical blockade of IL-1 may benefit patients with BRAF wild-type tumors and potentially synergize with immunotherapeutic interventions.

Prognostic Value of CD133 Caused by Mutant K-Ras and B-Raf—Letter

Kemper and colleagues (1) provided evidence that the expression of the cancer stem cell (CSC) marker CD133 is upregulated in colorectal tumors that have a hyperactivated Ras–Raf–MEK–ERK pathway and is, therefore, related to mutations in K-Ras or B-Raf. Thus, CD133 expression is not indicative for CSC numbers but rather for the mutation or activity status of the Ras–Raf pathway.In our opinion, some key points should be discussed in more detail. First of all, the choice to focus on stage II colon cancer is valuable, as adjuvant treatment is a matter of debate in this subset. However, Kemper and colleagues do not include all the clinical variables with already known prognostic value, particularly lymphovascular invasion and the number of examined nodes. Therefore, it is difficult to argue for the additive value of CD133 evaluation in the prognostic stratification of stage II patients beyond conventional clinicopathologic features.More importantly, CD133 levels seem to retain more prognostic information than that enclosed in the K-Ras/B-Raf mutational status. In fact, neither K-Ras nor B-Raf mutations [as well as microsatellite instability (MSI) status] are associated with relapse-free survival (RFS) at univariate analysis, whereas an analysis comprising all the examined variables is not provided. As reviewed in the work of Sinicrope and Sargent (2), K-Ras and B-Raf mutations do not predict RFS, whereas MSI status is convincingly associated with tumor recurrence in stage II colon cancer. The prognostic value of B-Raf mutation in colorectal cancer seems confined to the metastatic setting, as shown by the association with overall survival rather than RFS in larger series of stage II–III cases (2). Unfortunately, Kemper and colleagues do not evaluate the role of MSI in B-Raf mutant patients, as the literature suggests that prognosis may be particularly poor in MSI-low or stable B-Raf mutant tumors (2).From a biologic perspective, presented data indicate that K-Ras and B-Raf mutations contribute to the regulation of CD133 expression, but they are probably not the only actors. Additional investigation, therefore, is needed to reach the conclusion that the link between CD133 levels and prognosis is only dependent on the Ras–Raf axis. Moreover, factors other than somatic mutations contribute to the regulation of the Ras–Raf pathway, as proven by the recent identification of a subpopulation of B-Raf wild-type tumors with poor prognosis and gene expression patterns similar to those of B-Raf mutant tumors (independently of the K-Ras status; ref. 3). Furthermore, it is unlikely that a single marker (such as CD133) covers all the complexity and dynamics of CSCs (4). For example, Huang and colleagues showed that only the CD133+/CD44+ fraction predicts metastatic risk in colorectal cancer (5). We suggest that CSC-specific signaling pathways, rather than putative CSC surface markers, may be more useful prognostic and predictive biomarkers.In conclusion, factors other than K-Ras/B-Raf mutational status may be involved in CD133 regulation and, more intriguingly, additional CSC parameters may strengthen the value of CD133 in clinics beyond already available prognostic factors. We are therefore indebted to the authors for their report, but this is just a glimpse into the dark mysteries of CSCs in colorectal cancer.See the Response, p. 4474No potential conflicts of interest were disclosed.

Selective BRAF inhibition decreases tumor-resident lymphocyte frequencies in a mouse model of human melanoma

The development of targeted therapies and immunotherapies has markedly advanced the treatment of metastasized melanoma. While treatment with selective BRAFV600E inhibitors (like vemurafenib or dabrafenib) leads to high response rates but short response duration, CTLA-4 blocking therapies induce sustained responses, but only in a limited number of patients. The combination of these diametric treatment approaches may further improve survival, but pre-clinical data concerning this approach is limited. We investigated, using Tyr::CreERT2PTENF−/−BRAFF-V600E/+ inducible melanoma mice, whether BRAFV600E inhibition can synergize with anti-CTLA-4 mAb treatment, focusing on the interaction between the BRAFV600E inhibitor PLX4720 and the immune system. While PLX4720 treatment strongly decreased tumor growth, it did not induce cell death in BRAFV600E/PTEN−/− melanomas. More strikingly, PLX4720 treatment led to a decreased frequency of tumor-resident T cells, NK-cells, MDSCs and macrophages, which could not be restored by the addition of anti-CTLA-4 mAb. As this effect was not observed upon treatment of BRAF wild-type B16F10 tumors, we conclude that the decreased frequency of immune cells correlates to BRAFV600E inhibition in tumor cells and is not due to an off-target effect of PLX4720 on immune cells. Furthermore, anti-CTLA-4 mAb treatment of inducible melanoma mice treated with PLX4720 did not result in enhanced tumor control, while anti-CTLA-4 mAb treatment did improve the effect of tumor-vaccination in B16F10-inoculated mice. Our data suggest that vemurafenib may negatively affect the immune activity within the tumor. Therefore, the potential effect of targeted therapy on the tumor-microenvironment should be taken into consideration in the design of clinical trials combining targeted and immunotherapy.