The majority of melanomas show constitutive activation of the RAS-RAF-MAP/ERK kinase (MEK)-mitogen-activated protein kinase (MAPK) pathway. AZD6244 is a selective MEK1/2 inhibitor that markedly reduces tumor P-MAPK levels, but it produces few clinical responses in melanoma patients. An improved understanding of the determinants of resistance to AZD6244 may lead to improved patient selection and effective combinatorial approaches. The effects of AZD6244 on cell growth and survival were tested in a total of 14 Braf-mutant and 3 wild-type human cutaneous melanoma cell lines. Quantitative assessment of phospho-protein levels in the Braf-mutant cell lines by reverse phase protein array (RPPA) analysis showed no significant association between P-MEK or P-MAPK levels and AZD6244 sensitivity, but activation-specific markers in the phosphoinositide 3-kinase (PI3K)-AKT pathway correlated with resistance. We also identified resistant cell lines without basal activation of the PI3K-AKT pathway. RPPA characterization of the time-dependent changes in signaling pathways revealed that AZD6244 produced durable and potent inhibition of P-MAPK in sensitive and resistant Braf-mutant cell lines, but several resistant lines showed AZD6244-induced activation of AKT. In contrast, sensitive cell lines showed AZD6244 treatment-induced upregulation of PTEN protein and mRNA expression. Inhibition of AKT, TORC1/2, or insulin-like growth factor I receptor blocked AZD6244-induced activation of AKT and resulted in synergistic cell killing with AZD6244. These findings identify basal and treatment-induced regulation of the PI3K-AKT pathway as a critical regulator of AZD6244 sensitivity in Braf-mutant cutaneous melanoma cells and the novel regulation of PTEN expression by AZD6244 in sensitive cells, and suggest new combinatorial approaches for patients.

Increased expression of the Microphthalmia-associated transcription factor (MITF) contributes to melanoma progression and resistance to BRAF pathway inhibition. Here we show that the lack of MITF is associated with more severe resistance to a range of inhibitors, while its presence is required for robust drug responses. Both in primary and acquired resistance, MITF levels inversely correlate with the expression of several activated receptor tyrosine kinases, most frequently AXL. The MITF-low/AXL-high/drug-resistance phenotype is common among mutant BRAF and NRAS melanoma cell lines. The dichotomous behaviour of MITF in drug response is corroborated in vemurafenib-resistant biopsies, including MITF-high and -low clones in a relapsed patient. Furthermore, drug cocktails containing AXL inhibitor enhance melanoma cell elimination by BRAF or ERK inhibition. Our results demonstrate that a low MITF/AXL ratio predicts early resistance to multiple targeted drugs, and warrant clinical validation of AXL inhibitors to combat resistance of BRAF and NRAS mutant MITF-low melanomas.

OBJECTIVE

BRAF V600E mutation is seen in 5% to 8% of patients with metastatic colorectal cancer (CRC) and is associated with poor prognosis. Vemurafenib, an oral BRAF V600 inhibitor, has pronounced activity in patients with metastatic melanoma, but its activity in patients with BRAF V600E-positive metastatic CRC was unknown.

METHODS

In this multi-institutional, open-label study, patients with metastatic CRC with BRAF V600 mutations were recruited to an expansion cohort at the previously determined maximum-tolerated dose of 960 mg orally twice a day.

RESULTS

Twenty-one patients were enrolled, of whom 20 had received at least one prior metastatic chemotherapy regimen. Grade 3 toxicities included keratoacanthomas, rash, fatigue, and arthralgia. Of the 21 patients treated, one patient had a confirmed partial response (5%; 95% CI, 1% to 24%) and seven other patients had stable disease by RECIST criteria. Median progression-free survival was 2.1 months. Patterns of concurrent mutations, microsatellite instability status, CpG island methylation status, PTEN loss, EGFR expression, and copy number alterations were not associated with clinical benefit. In contrast to prior expectations, concurrent KRAS and NRAS mutations were detected at low allele frequency in a subset of the patients' tumors (median, 0.21% allele frequency) and were apparent mechanisms of acquired resistance in vemurafenib-sensitive patient-derived xenograft models.

CONCLUSIONS

In marked contrast to the results seen in patients with BRAF V600E-mutant melanoma, single-agent vemurafenib did not show meaningful clinical activity in patients with BRAF V600E mutant CRC. Combination strategies are now under development and may be informed by the presence of intratumor heterogeneity of KRAS and NRAS mutations.

OBJECTIVE

Recently, it was reported that BRAF mutations are frequent in melanoma. Previously, we analyzed a large series of paired primary and metastatic melanomas for NRAS codon 61 mutations and showed that they arise early and are preserved during tumor progression. Here, we have screened the same tumor samples for BRAF mutations.

METHODS

Primary melanomas (n = 71) and corresponding metastases (n = 88) from 71 patients were screened for BRAF exon 11 and exon 15 mutations using single-strand conformational polymorphism and nucleotide sequence analysis

RESULTS

BRAF mutations were found in 42 of 71 patients (59%). Thirty-seven patients had mutations that lead to a Val599Glu change, whereas mutations resulting in Gly468Ser, Val599Arg, Val599Lys, and Lys600Glu changes were detected in one patient each. Furthermore, one patient had a 6-bp insertion between codons 598 and 599, encoding two threonine residues. In most cases, paired primary and metastatic lesions had the same BRAF genotype (i.e., mutations present in the primary tumors were preserved in the corresponding metastases, and mutations did not arise at the metastatic stage if they were not present in the primary lesion). Using laser-capture microdissection, BRAF mutations were found in the radial growth phase of the primary lesions. BRAF mutations occurred exclusively in tumors that were wild type for NRAS, and in total, 89% of the patients analyzed (63 of 71) had mutations in either of these two genes.

CONCLUSIONS

The Ras-Raf-mitogen-activated protein kinase/extracellular signal-regulated kinase-extracellular signal-regulated kinase signaling pathway is activated in the vast majority of melanomas. Activation occurs through either NRAS or BRAF mutations, both of which arise early during melanoma pathogenesis and are preserved throughout tumor progression.

BACKGROUND

Activating mutation of KRAS and BRAF are focused on as potential prognostic and predictive biomarkers in patients with colorectal cancer (CRC) treated with anti-EGFR therapies. This study investigated the clinicopathological features and prognostic impact of KRAS/BRAF mutation in advanced and recurrent CRC patients.

METHODS

Patients with advanced and recurrent CRC treated with systemic chemotherapy (n=229) were analysed for KRAS/BRAF genotypes by cycleave PCR. Prognostic factors associated with survival were identified by univariate and multivariate analyses using the Cox proportional hazards model.

RESULTS

KRAS and BRAF mutations were present in 34.5% and 6.5% of patients, respectively. BRAF mutated tumours were more likely to develop on the right of the colon, and to be of the poorly differentiated adenocarcinoma or mucinous carcinoma, and peritoneal metastasis. The median overall survival (OS) for BRAF mutation-positive and KRAS 13 mutation-positive patients was 11.0 and 27.7 months, respectively, which was significantly worse than that for patients with wild-type (wt) KRAS and BRAF (40.6 months) (BRAF; HR=4.25, P<0.001, KRAS13; HR=2.03, P=0.024). After adjustment for significant features by multivariate Cox regression analysis, BRAF mutation was associated with poor OS (HR=4.23, P=0.019).

CONCLUSIONS

Presence of mutated BRAF is one of the most powerful prognostic factors for advanced and recurrent CRC. The KRAS13 mutation showed a trend towards poor OS in patients with advanced and recurrent CRC.

This portion of the NCCN Guidelines for Colon Cancer focuses on the use of systemic therapy in metastatic disease. Considerations for treatment selection among 32 different monotherapies and combination regimens in up to 7 lines of therapy have included treatment history, extent of disease, goals of treatment, the efficacy and toxicity profiles of the regimens, KRAS/NRAS mutational status, and patient comorbidities and preferences. Location of the primary tumor, the BRAF mutation status, and tumor microsatellite stability should also be considered in treatment decisions.

BACKGROUND

Multiplex digital PCR (dPCR) enables noninvasive and sensitive detection of circulating tumor DNA with performance unachievable by current molecular-detection approaches. Furthermore, picodroplet dPCR facilitates simultaneous screening for multiple mutations from the same sample.

METHODS

We investigated the utility of multiplex dPCR to screen for the 7 most common mutations in codons 12 and 13 of the KRAS (Kirsten rat sarcoma viral oncogene homolog) oncogene from plasma samples of patients with metastatic colorectal cancer. Fifty plasma samples were tested from patients for whom the primary tumor biopsy tissue DNA had been characterized by quantitative PCR.

RESULTS

Tumor characterization revealed that 19 patient tumors had KRAS mutations. Multiplex dPCR analysis of the plasma DNA prepared from these samples identified 14 samples that matched the mutation identified in the tumor, 1 sample contained a different KRAS mutation, and 4 samples had no detectable mutation. Among the tumor samples that were wild type for KRAS, 2 KRAS mutations were identified in the corresponding plasma samples. Duplex dPCR (i.e., wild-type and single-mutation assay) was also used to analyze plasma samples from patients with KRAS-mutated tumors and 5 samples expected to contain the BRAF (v-raf murine sarcoma viral oncogene homolog B) V600E mutation. The results for the duplex analysis matched those for the multiplex analysis for KRAS-mutated samples and, owing to its higher sensitivity, enabled detection of 2 additional samples with low levels of KRAS-mutated DNA. All 5 samples with BRAF mutations were detected.

CONCLUSIONS

This work demonstrates the clinical utility of multiplex dPCR to screen for multiple mutations simultaneously with a sensitivity sufficient to detect mutations in circulating DNA obtained by noninvasive blood collection.

Immune cells in the tumor microenvironment have an important role in regulating tumor progression. Therefore, stimulating immune reactions to tumors can be an attractive therapeutic and prevention strategy. Cancer cells and host cells constantly interact with each other in the tumor microenvironment; thus, cancer immunology is an interdisciplinary area where integrated analysis of both host and tumor factors is needed. Cancer represents a heterogeneous group of diseases with different genetic and epigenetic alterations; therefore, molecular classification of cancer (for example lung, prostate and breast cancers) is an important component in clinical decision making. However, most studies on antitumor immunity and clinical outcome lack analysis of tumor molecular biomarkers. In this Review, we discuss colorectal cancer as a prototypical example of cancer. Common molecular classifiers of colon cancer include KRAS, BRAF and PIK3CA mutations, microsatellite instability, LINE-1 methylation, and CpG island methylator phenotype. Since tumor molecular features and immune reactions are inter-related, a comprehensive assessment of these factors is critical. Examining the effects of tumor-host interactions on clinical outcome and prognosis represents an evolving interdisciplinary field of molecular pathological epidemiology. Pathological immunity evaluation may provide information on prognosis and help identify patients who are more likely to benefit from immunotherapy.

Anaplastic thyroid cancer (ATC) is a rare malignancy. While external beam radiation therapy has improved locoregional control, the median survival of approximately 4 months has not changed in more than half a century due to uncontrolled systemic metastases. The objective of this study was to review the literature in order to identify potential new strategies for treating this highly lethal cancer. PubMed searches were the principal source of articles reviewed. The molecular pathogenesis of ATC includes mutations in BRAF, RAS, catenin (cadherin-associated protein), beta 1, PIK3CA, TP53, AXIN1, PTEN, and APC genes, and chromosomal abnormalities are common. Several microarray studies have identified genes and pathways preferentially affected, and dysregulated microRNA profiles differ from differentiated thyroid cancers. Numerous proteins involving transcription factors, signaling pathways, mitosis, proliferation, cell cycle, apoptosis, adhesion, migration, epigenetics, and protein degradation are affected. A variety of agents have been successful in controlling ATC cell growth both in vitro and in nude mice xenografts. While many of these new compounds are in cancer clinical trials, there are few studies being conducted in ATC. With the recent increased knowledge of the many critical genes and proteins affected in ATC, and the extensive array of targeted therapies being developed for cancer patients, there are new opportunities to design clinical trials based upon tumor molecular profiling and preclinical studies of potentially synergistic combinatorial novel therapies.

The controls that enable melanoblasts and melanoma cells to proliferate are likely to be related, but so far no key regulator of cell cycle progression specific to the melanocyte lineage has been identified. The microphthalmia-associated transcription factor Mitf has a crucial but poorly defined role in melanoblast and melanocyte survival and in differentiation. Here we show that Mitf can act as a novel anti-proliferative transcription factor able to induce a G1 cell-cycle arrest that is dependent on Mitf-mediated activation of the p21(Cip1) (CDKN1A) cyclin-dependent kinase inhibitor gene. Moreover, cooperation between Mitf and the retinoblastoma protein Rb1 potentiates the ability of Mitf to activate transcription. The results indicate that Mitf-mediated activation of p21Cip1 expression and consequent hypophosphorylation of Rb1 will contribute to cell cycle exit and activation of the differentiation programme. The mutation of genes associated with melanoma, such as INK4a or BRAF that would affect either Mitf cooperation with Rb1 or Mitf stability respectively, would impair Mitf-mediated cell cycle control.

In the past decade, major advances have been made in the understanding of melanoma. New predisposition genes have been reported and key somatic events, such as BRAF mutation, directly translated into therapeutic management. Surgery for localised melanoma and regional lymph node metastases is the standard of care. Sentinel-node biopsy provides precise staging, but has not been reported to affect survival. The effect of lymph-node dissection on survival is a topic of investigation. Two distinct approaches have emerged to try to extend survival in patients with metastatic melanoma: immunomodulation with anti-CTLA4 monoclonal antibodies, and targeted therapy with BRAF inhibitors or MEK inhibitors for BRAF-mutated melanoma. The combination of BRAF inhibitors and MEK inhibitors might improve progression-free survival further and, possibly, increase overall survival. Response patterns differ substantially-anti-CTLA4 immunotherapy can induce long-term responses, but only in a few patients, whereas targeted drugs induce responses in most patients, but nearly all of them relapse because of pre-existing or acquired resistance. Thus, the long-term prognosis of metastatic melanoma remains poor. Anti-PD1 and anti-PDL1 antibodies have emerged as breakthrough drugs for melanoma that have high response rates and long durability. Biomarkers that have predictive value remain elusive in melanoma, although emerging data for adjuvant therapy indicate that interferon sensitivity is associated with ulceration of the primary melanoma. Intense investigation continues for clinical and biological markers that predict clinical benefit of immunotherapeutic drugs, such as interferon alfa or anti-CTLA4 antibodies, and the mechanisms that lead to resistance of targeted drugs.

BACKGROUND

Meta-analyses were conducted to characterize patterns of mutation incidence in non small-cell lung cancer (NSCLC).

METHODS

Nine genes with the most complete published mutation coincidence data were evaluated. One meta-analysis generated a 'mutMap' to visually represent mutation coincidence by ethnicity (Western/Asian) and histology (adenocarcinoma [ADC] or squamous cell carcinoma). Another meta-analysis evaluated incidence of individual mutations. Extended analyses explored incidence of EGFR and KRAS mutations by ethnicity, histology, and smoking status.

RESULTS

Genes evaluated were TP53, EGFR, KRAS, LKB1, EML4-ALK, PTEN, BRAF, PIK3CA, and ErbB2. The mutMap highlighted mutation coincidences occurring in ≥5% of patients, including TP53 with KRAS or EGFR mutations in patients with ADC, and TP53 with LKB1 mutation in Western patients. TP53 was the most frequently mutated gene overall. Frequencies of TP53, EGFR, KRAS, LKB1, PTEN, and BRAF mutations were influenced by histology and/or ethnicity. Although EGFR mutations were most frequent in patients with ADC and never/light smokers from Asia, and KRAS mutations were most frequent in patients with ADC and ever/heavy smokers from Western countries, both were detected outside these subgroups.

CONCLUSIONS

Potential molecular pathology segments of NSCLC were identified. Further studies of mutations in NSCLC are warranted to facilitate more specific diagnoses and guide treatment.

Histiocytoses are rare disorders of unknown origin with highly heterogeneous prognosis. BRAF mutations have been observed in Langerhans cell histiocytosis (LCH). We investigated the frequency of BRAF mutations in several types of histiocytoses. Histology from 127 patients with histiocytoses were reviewed. Detection of BRAF(V600) mutations was performed by pyrosequencing of DNA extracted from paraffin embedded samples. Diagnoses of Erdheim-Chester disease (ECD), LCH, Rosai-Dorfman disease, juvenile xanthogranuloma, histiocytic sarcoma, xanthoma disseminatum, interdigitating dendritic cell sarcoma, and necrobiotic xanthogranuloma were performed in 46, 39, 23, 12, 3, 2, 1, and 1 patients, respectively. BRAF status was obtained in 93 cases. BRAF(V600E) mutations were detected in 13 of 24 (54%) ECD, 11 of 29 (38%) LCH, and none of the other histiocytoses. Four patients with ECD died of disease. The high frequency of BRAF(V600E) in LCH and ECD suggests a common origin of these diseases. Treatment with vemurafenib should be investigated in patients with malignant BRAF(V600E) histiocytosis.

OBJECTIVE

To investigate the prognostic value of the BRAF V600E mutation and the recently identified TERT promoter mutation chr5:1,295,228C>T (C228T), individually and in their coexistence, in papillary thyroid cancer (PTC).

METHODS

We performed a retrospective study of the relationship of BRAF and TERT C228T mutations with clinicopathologic outcomes of PTC in 507 patients (365 women and 142 men) age 45.9 ± 14.0 years (mean ± SD) with a median follow-up of 24 months (interquartile range, 8 to 78 months).

RESULTS

Coexisting BRAF V600E and TERT C228T mutations were more commonly associated with high-risk clinicopathologic characteristics of PTC than they were individually. Tumor recurrence rates were 25.8% (50 of 194;77.60 recurrences per 1,000 person-years; 95% CI, 58.81 to 102.38) versus 9.6% (30 of 313; 22.88 recurrences per 1,000 person-years; 95% CI, 16.00 to 32.72) in BRAF mutation-positive versus -negative patients (hazard ratio [HR], 3.22; 95% CI, 2.05 to 5.07) and 47.5% (29 of 61; 108.55 recurrences per 1,000 person-years; 95% CI, 75.43 to 156.20) versus 11.4% (51 of 446; 30.21 recurrences per 1,000 person-years; 95% CI, 22.96 to 39.74) in TERT mutation-positive versus -negative patients (HR, 3.46; 95% CI, 2.19 to 5.45). Recurrence rates were 68.6% (24 of 35; 211.76 recurrences per 1,000 person-years; 95% CI, 141.94 to 315.94) versus 8.7% (25 of 287; 21.60 recurrences per 1,000 person-years; 95% CI, 14.59 to 31.97) in patients harboring both mutations versus patients harboring neither mutation (HR, 8.51; 95% CI, 4.84 to 14.97), which remained significant after clinicopathologic cofactor adjustments. Disease-free patient survival curves displayed a moderate decline with BRAF V600E or TERT C228T alone but a sharp decline with two coexisting mutations.

CONCLUSIONS

Coexisting BRAF V600E and TERT C228T mutations form a novel genetic background that defines PTC with the worst clinicopathologic outcomes, providing unique prognostic and therapeutic implications.

Breast cancer has for long been recognized as a highly diverse tumor group, but the underlying genetic basis has been elusive. Here, we report an extensive molecular characterization of a collection of 41 human breast cancer cell lines. Protein and gene expression analyses indicated that the collection of breast cancer cell lines has retained most, if not all, molecular characteristics that are typical for clinical breast cancers. Gene mutation analyses identified 146 oncogenic mutations among 27 well-known cancer genes, amounting to an average of 3.6 mutations per cell line. Mutations in genes from the p53, RB and PI3K tumor suppressor pathways were widespread among all breast cancer cell lines. Most important, we have identified two gene mutation profiles that are specifically associated with luminal-type and basal-type breast cancer cell lines. The luminal mutation profile involved E-cadherin and MAP2K4 gene mutations and amplifications of Cyclin D1, ERBB2 and HDM2, whereas the basal mutation profile involved BRCA1, RB1, RAS and BRAF gene mutations and deletions of p16 and p14ARF. These subtype-specific gene mutation profiles constitute a genetic basis for the heterogeneity observed among human breast cancers, providing clues for their underlying biology and providing guidance for targeted pharmacogenetic intervention in breast cancer patients.

BRAF and NRAS are commonly mutated in cancer and represent the most frequent genetic events in malignant melanoma. More recently, a subset of melanomas was shown to overexpress KIT and harbor KIT mutations. Although most gastrointestinal stromal tumors (GISTs) exhibit activating mutations in either KIT or PDGFRA, about 10% of the cases lack mutations in these genes. It is our hypothesis following the melanoma model that mutations in BRAF or NRAS may play a role in wild-type GIST pathogenesis. Alterations in RAS/MEK/ERK pathway may also be involved in development of imatinib resistance in GIST, particularly in tumors lacking secondary KIT or PDGFRA mutations. Imatinib-naive wild-type GISTs from 61 patients, including 15 children and 28 imatinib-resistant tumors without secondary KIT mutations were analyzed. Screening for hot spots mutations in BRAF (exons 11 and 15) and NRAS (exons 2 and 3) was performed. A BRAF exon 15 V600E was identified in 3 of 61 GIST patients, who shared similar clinical features, being 49- to 55-years-old females and having their tumors located in the small bowel. The tumors were strongly KIT immunoreactive and had a high risk of malignancy. An identical V600E BRAF mutation was also identified in one of 28 imatinib resistant GIST lacking a defined mechanism of drug resistance. In conclusion, we identified a primary BRAF V600E mutations in 7% of adult GIST patients, lacking KIT/PDGFRA mutations. The BRAF-mutated GISTs show predilection for small bowel location and high risk of malignancy. A secondary V600E BRAF mutation could represent an alternative mechanism of imatinib resistance. Kinase inhibitors targeting BRAF may be effective therapeutic options in this molecular GIST subset.

BRAF is a serine/threonine protein kinase activating the MAP kinase/ERK-signaling pathway. About 50 % of melanomas harbors activating BRAF mutations (over 90 % V600E). BRAFV600E has been implicated in different mechanisms underlying melanomagenesis, most of which due to the deregulated activation of the downstream MEK/ERK effectors. The first selective inhibitor of mutant BRAF, vemurafenib, after highly encouraging results of the phase I and II trial, was compared to dacarbazine in a phase III trial in treatment-naïve patients (BRIM-3). The study results showed a relative reduction of 63 % in risk of death and 74 % in risk of tumor progression. Considering all trials so far completed, median overall survival reached approximately 16 months for vemurafenib compared to less than 10 months for dacarbazine treatment. Vemurafenib has been extensively tested on melanoma patients expressing the BRAFV600E mutated form; it has been demonstrated to be also effective in inhibiting melanomas carrying the V600K mutation. In 2011, both FDA and EMA therefore approved vemurafenib for metastatic melanoma carrying BRAFV600 mutations. Some findings suggest that continuation of vemurafenib treatment is potentially beneficial after local therapy in a subset of patients with disease progression (PD). Among who continued vemurafenib >30 days after local therapy of PD lesion(s), a median overall survival was not reached, with a median follow-up of 15.5 months from initiation of BRAF inhibitor therapy. For patients who did not continue treatment, median overall survival from the time of disease progression was 1.4 months. A clinical phase I/II trial is evaluating the safety, tolerability and efficacy of vemurafenib in combination with the CTLA-4 inhibitor mAb ipilimumab. In the BRIM-7 trial vemurafenib is tested in association with GDC-0973, a potent and highly selective inhibitor of MEK1/2. Preliminary data seem to indicate that an additional inhibitor of mutated BRAF, GSK2118436, might be also active on a wider range of BRAF mutations (V600E-K-D-R); actually, treatment with such a compound is under evaluation in a phase III study among stage III-IV melanoma patients positive for BRAF mutations. Overall, BRAF inhibitors were well tolerated; common adverse events are arthralgia, rash, fatigue, alopecia, keratoacanthoma or cutaneous squamous-cell carcinoma, photosensitivity, nausea, and diarrhea, with some variants between different inhibitors.

BACKGROUND

Differences exist between the proximal and distal colon in terms of developmental origin, exposure to patterning genes, environmental mutagens, and gut flora. Little is known on how these differences may affect mechanisms of tumorigenesis, side-specific therapy response or prognosis. We explored systematic differences in pathway activation and their clinical implications.

METHODS

Detailed clinicopathological data for 3045 colon carcinoma patients enrolled in the PETACC3 adjuvant chemotherapy trial were available for analysis. A subset of 1404 samples had molecular data, including gene expression and DNA copy number profiles for 589 and 199 samples, respectively. In addition, 413 colon adenocarcinoma from TCGA collection were also analyzed. Tumor side-effect on anti-epidermal growth factor receptor (EGFR) therapy was assessed in a cohort of 325 metastatic patients. Outcome variables considered were relapse-free survival and survival after relapse (SAR).

RESULTS

Proximal carcinomas were more often mucinous, microsatellite instable (MSI)-high, mutated in key tumorigenic pathways, expressed a B-Raf proto-oncogene, serine/threonine kinase (BRAF)-like and a serrated pathway signature, regardless of histological type. Distal carcinomas were more often chromosome instable and EGFR or human epidermal growth factor receptor 2 (HER2) amplified, and more frequently overexpressed epiregulin. While risk of relapse was not different per side, SAR was much poorer for proximal than for distal stage III carcinomas in a multivariable model including BRAF mutation status [N = 285; HR 1.95, 95% CI (1.6-2.4), P < 0.001]. Only patients with metastases from a distal carcinoma responded to anti-EGFR therapy, in line with the predictions of our pathway enrichment analysis.

CONCLUSIONS

Colorectal carcinoma side is associated with differences in key molecular features, some immediately druggable, with important prognostic effects which are maintained in metastatic lesions. Although within side significant molecular heterogeneity remains, our findings justify stratification of patients by side for retrospective and prospective analyses of drug efficacy and prognosis.

OBJECTIVE

To examine the prevalence of BRAF mutation among thyroid cancer histologic subtypes and determine the association of BRAF mutation with indicators of poor prognosis for papillary thyroid cancer and patient outcome.

BACKGROUND

The appropriate extent of surgical treatment, adjuvant therapy and follow-up monitoring for thyroid cancer remains controversial. Advances in the molecular biology of thyroid cancer have helped to identify candidate markers of disease aggressiveness. A commonly found genetic alternation is a point mutation in the BRAF oncogene (BRAF V600E), which is primarily found in papillary thyroid cancer and is associated with more aggressive disease.

METHODS

BRAF V600E mutation status was determined in 347 tumor samples from 314 patients with thyroid cancer (245 with conventional papillary thyroid cancer, 73 with follicular thyroid cancer, and 29 with the follicular variant of papillary thyroid cancer). Univariate and multivariate analyses were performed to determine the association of BRAF V600E with clinicopathologic factors and patient outcome.

RESULTS

: The prevalence of BRAF V600E mutation was higher in conventional papillary thyroid cancer (51.0%) than in follicular variant of papillary thyroid cancer (24.1%) and follicular thyroid cancer (1.4%) (P < 0.0001). In patients with conventional papillary thyroid cancer, BRAF V600E mutation was associated with older age (P = 0.0381), lymph node metastasis (P = 0.0323), distant metastasis (P = 0.045), higher TNM stage (I and II vs. III and IV, P = 0.0389), and recurrent and persistent disease (P = 0.009) with a median follow-up time of 6.0 years. Multivariate analysis showed that BRAF V600E mutation [OR (95% CI) = 4.2 (1.2-14.6)] and lymph node metastasis [OR (95% CI) = 7.75 (2.1-28.5)] were independently associated with recurrent and persistent disease in patients with conventional papillary thyroid cancer.

CONCLUSIONS

BRAF V600E mutation is primarily present in conventional papillary thyroid cancer. It is associated with an aggressive tumor phenotype and higher risk of recurrent and persistent disease in patients with conventional papillary thyroid cancer. Testing for this mutation may be useful for selecting initial therapy and for follow-up monitoring.

Hyperactivated extracellular signal-regulated kinase (ERK) signaling is common in human cancer and is often the result of activating mutations in BRAF, RAS, and upstream receptor tyrosine kinases. To characterize the mitogen-activated protein kinase/ERK kinase (MEK)/ERK dependence of lung cancers harboring BRAF kinase domain mutations, we screened a large panel of human lung cancer cell lines (n = 87) and tumors (n = 916) for BRAF mutations. We found that non-small cell lung cancers (NSCLC) cells with both V600E and non-V600E BRAF mutations were selectively sensitive to MEK inhibition compared with those harboring mutations in epidermal growth factor receptor (EGFR), KRAS, or ALK and ROS kinase fusions. Supporting its classification as a "driver" mutation in the cells in which it is expressed, MEK inhibition in (V600E)BRAF NSCLC cells led to substantial induction of apoptosis, comparable with that seen with EGFR kinase inhibition in EGFR mutant NSCLC models. Despite high basal ERK phosphorylation, EGFR mutant cells were uniformly resistant to MEK inhibition. Conversely, BRAF mutant cell lines were resistant to EGFR inhibition. These data, together with the nonoverlapping pattern of EGFR and BRAF mutations in human lung cancer, suggest that these lesions define distinct clinical entities whose treatment should be guided by prospective real-time genotyping. To facilitate such an effort, we developed a mass spectrometry-based genotyping method for the detection of hotspot mutations in BRAF, KRAS, and EGFR. Using this assay, we confirmed that BRAF mutations can be identified in a minority of NSCLC tumors and that patients whose tumors harbor BRAF mutations have a distinct clinical profile compared with those whose tumors harbor kinase domain mutations in EGFR.

OBJECTIVE

Cholangiocarcinoma, the second most common liver cancer, can be classified as intrahepatic cholangiocarcinoma (ICC) or extrahepatic cholangiocarcinoma. We performed an integrative genomic analysis of ICC samples from a large series of patients.

METHODS

We performed a gene expression profile, high-density single-nucleotide polymorphism array, and mutation analyses using formalin-fixed ICC samples from 149 patients. Associations with clinicopathologic traits and patient outcomes were examined for 119 cases. Class discovery was based on a non-negative matrix factorization algorithm and significant copy number variations were identified by Genomic Identification of Significant Targets in Cancer (GISTIC) analysis. Gene set enrichment analysis was used to identify signaling pathways activated in specific molecular classes of tumors, and to analyze their genomic overlap with hepatocellular carcinoma (HCC).

RESULTS

We identified 2 main biological classes of ICC. The inflammation class (38% of ICCs) is characterized by activation of inflammatory signaling pathways, overexpression of cytokines, and STAT3 activation. The proliferation class (62%) is characterized by activation of oncogenic signaling pathways (including RAS, mitogen-activated protein kinase, and MET), DNA amplifications at 11q13.2, deletions at 14q22.1, mutations in KRAS and BRAF, and gene expression signatures previously associated with poor outcomes for patients with HCC. Copy number variation-based clustering was able to refine these molecular groups further. We identified high-level amplifications in 5 regions, including 1p13 (9%) and 11q13.2 (4%), and several focal deletions, such as 9p21.3 (18%) and 14q22.1 (12% in coding regions for the SAV1 tumor suppressor). In a complementary approach, we identified a gene expression signature that was associated with reduced survival times of patients with ICC; this signature was enriched in the proliferation class (P < .001).

CONCLUSIONS

We used an integrative genomic analysis to identify 2 classes of ICC. The proliferation class has specific copy number alterations, activation of oncogenic pathways, and is associated with worse outcome. Different classes of ICC, based on molecular features, therefore might require different treatment approaches.

The identification of oncogenic driver mutations underlying sensitivity to epidermal growth factor receptor and anaplastic lymphoma kinase tyrosine kinase inhibitors has led to a surge of interest in identifying additional targetable oncogenes in non-small-cell lung cancer. A number of new potentially oncogenic gene alterations have been characterized in recent years, including BRAF mutations, HER2 insertions, PIK3CA mutations, FGFR1 amplifications, DDR2 mutations, ROS1 rearrangements, and RET rearrangements. In this review, we will discuss the techniques used to discover each of these candidate oncogenes, the prevalence of each in non-small-cell lung cancer, the preclinical data supporting their role in lung cancer, and data on small molecular inhibitors in development.

The BRAFT1799A mutation is the most common genetic alteration in papillary thyroid carcinomas (PTC). It is also found in a subset of papillary microcarcinomas, consistent with a role in tumor initiation. PTCs with BRAFT1799A are often invasive and present at a more advanced stage. BRAFT1799A is found with high prevalence in tall-cell variant PTCs and in poorly differentiated and undifferentiated carcinomas arising from PTCs. To explore the role of BRAFV600E in thyroid cancer pathogenesis, we targeted its expression to thyroid cells of transgenic FVB/N mice with a bovine thyroglobulin promoter. Two Tg-BRAFV600E lines (Tg-BRAFBRAFBRAFBRAFBRAFBRAFBRAFBRAFV600E induces goiter and invasive PTC, which transitions to poorly differentiated carcinomas. This closely recapitulates the phenotype of BRAF-positive PTCs in humans and supports a key role for this oncogene in its pathogenesis.

Gastrointestinal stromal tumours (GISTs) are mesenchymal neoplasms that arise in the gastrointestinal tract, usually in the stomach or the small intestine and rarely elsewhere in the abdomen. They can occur at any age, the median age being 60-65 years, and typically cause bleeding, anaemia, and pain. GISTs have variable malignant potential, ranging from small lesions with a benign behaviour to fatal sarcomas. Most tumours stain positively for the mast/stem cell growth factor receptor KIT and anoctamin 1 and harbour a kinase-activating mutation in either KIT or PDGFRA. Tumours without such mutations could have alterations in genes of the succinate dehydrogenase complex or in BRAF, or rarely RAS family genes. About 60% of patients are cured by surgery. Adjuvant treatment with imatinib is recommended for patients with a substantial risk of recurrence, if the tumour has an imatinib-sensitive mutation. Tyrosine kinase inhibitors substantially improve survival in advanced disease, but secondary drug resistance is common.