The acquisition of resistance to protein kinase inhibitors is a growing problem in cancer treatment. We modeled acquired resistance to the MEK1/2 (mitogen-activated or extracellular signal-regulated protein kinase kinases 1 and 2) inhibitor selumetinib (AZD6244) in colorectal cancer cell lines harboring mutations in BRAF (COLO205 and HT29 lines) or KRAS (HCT116 and LoVo lines). AZD6244-resistant derivatives were refractory to AZD6244-induced cell cycle arrest and death and exhibited a marked increase in ERK1/2 (extracellular signal-regulated kinases 1 and 2) pathway signaling and cyclin D1 abundance when assessed in the absence of inhibitor. Genomic sequencing revealed no acquired mutations in MEK1 or MEK2, the primary target of AZD6244. Rather, resistant lines showed a marked up-regulation of their respective driving oncogenes, BRAF(600E) or KRAS(13D), due to intrachromosomal amplification. Inhibition of BRAF reversed resistance to AZD6244 in COLO205 cells, which suggested that combined inhibition of MEK1/2 and BRAF may reduce the likelihood of acquired resistance in tumors with BRAF(600E). Knockdown of KRAS reversed AZD6244 resistance in HCT116 cells as well as reduced the activation of ERK1/2 and protein kinase B; however, the combined inhibition of ERK1/2 and phosphatidylinositol 3-kinase signaling had little effect on AZD6244 resistance, suggesting that additional KRAS effector pathways contribute to this process. Microarray analysis identified increased expression of an 18-gene signature previously identified as reflecting MEK1/2 pathway output in resistant cells. Thus, amplification of the driving oncogene (BRAF(600E) or KRAS(13D)) can drive acquired resistance to MEK1/2 inhibitors by increasing signaling through the ERK1/2 pathway. However, up-regulation of KRAS(13D) leads to activation of multiple KRAS effector pathways, underlining the therapeutic challenge posed by KRAS mutations. These results may have implications for the use of combination therapies.

Tyrosine kinase growth factor receptors and Ras/Raf/MEK/MAPK signalling have been implicated in the suppression as well as augmentation of programmed cell death. In addition, a Ras-independent role for Raf as a suppressor of programmed cell death has been suggested by the recent finding that Craf1 interacts with members of the Bcl-2 family at mitochondrial membranes. However, genetic studies of C. elegans and Drosophila, as well as the targeted mutagenesis of the murine Araf gene, have failed to support such a role. Here we show that mice with a targeted disruption in the Braf gene die of vascular defects during mid-gestation. Braf -/- embryos, unlike Araf -/- or Craf1 -/- embryos (L.W. et al., unpublished), show an increased number of endothelial precursor cells, dramatically enlarged blood vessels and apoptotic death of differentiated endothelial cells. These results establish Braf as a critical signalling factor in the formation of the vascular system and provide the first genetic evidence for an essential role of Raf gene in the regulation of programmed cell death.

The protein kinase BRAF is a key component of the RAS-RAF signaling pathway which plays an important role in regulating cell proliferation, differentiation, and survival. Mutations in BRAF at codon 600 promote catalytic activity and are associated with 8% of all human (solid) tumors, including 8% to 10% of colorectal cancers (CRC). Here, we report the preclinical characterization of vemurafenib (RG7204; PLX4032; RO5185426), a first-in-class, specific small molecule inhibitor of BRAF(V600E) in BRAF-mutated CRC cell lines and tumor xenograft models. As a single agent, vemurafenib shows dose-dependent inhibition of ERK and MEK phosphorylation, thereby arresting cell proliferation in BRAF(V600)-expressing cell lines and inhibiting tumor growth in BRAF(V600E) bearing xenograft models. Because vemurafenib has shown limited single-agent clinical activity in BRAF(V600E)-mutant metastatic CRC, we therefore explored a range of combination therapies, with both standard agents and targeted inhibitors in preclinical xenograft models. In a BRAF-mutant CRC xenograft model with de novo resistance to vemurafenib (RKO), tumor growth inhibition by vemurafenib was enhanced by combining with an AKT inhibitor (MK-2206). The addition of vemurafenib to capecitabine and/or bevacizumab, cetuximab and/or irinotecan, or erlotinib resulted in increased antitumor activity and improved survival in xenograft models. Together, our findings suggest that the administration of vemurafenib in combination with standard-of-care or novel targeted therapies may lead to enhanced and sustained clinical antitumor efficacy in CRCs harboring the BRAF(V600E) mutation.

BACKGROUND

The approval of vemurafenib in the US 2011 and in Europe 2012 improved the therapy of not resectable or metastatic melanoma. Patients carrying a substitution of valine to glutamic acid at codon 600 (p.V600E) or a substitution of valine to leucine (p.V600K) in BRAF show complete or partial response. Therefore, the precise identification of the underlying somatic mutations is essential. Herein, we evaluate the sensitivity, specificity and feasibility of six different methods for the detection of BRAF mutations.

METHODS

Samples harboring p.V600E mutations as well as rare mutations in BRAF exon 15 were compared to wildtype samples. DNA was extracted from formalin-fixed paraffin-embedded tissues by manual micro-dissection and automated extraction. BRAF mutational analysis was carried out by high resolution melting (HRM) analysis, pyrosequencing, allele specific PCR, next generation sequencing (NGS) and immunohistochemistry (IHC). All mutations were independently reassessed by Sanger sequencing. Due to the limited tumor tissue available different numbers of samples were analyzed with each method (82, 72, 60, 72, 49 and 82 respectively).

RESULTS

There was no difference in sensitivity between the HRM analysis and Sanger sequencing (98%). All mutations down to 6.6% allele frequency could be detected with 100% specificity. In contrast, pyrosequencing detected 100% of the mutations down to 5% allele frequency but exhibited only 90% specificity. The allele specific PCR failed to detect 16.3% of the mutations eligible for therapy with vemurafenib. NGS could analyze 100% of the cases with 100% specificity but exhibited 97.5% sensitivity. IHC showed once cross-reactivity with p.V600R but was a good amendment to HRM.

CONCLUSIONS

Therefore, at present, a combination of HRM and IHC is recommended to increase sensitivity and specificity for routine diagnostic to fulfill the European requirements concerning vemurafenib therapy of melanoma patients.

Mitochondrial division is essential for mitosis and metazoan development, but a mechanistic role in cancer biology remains unknown. Here, we examine the direct effects of oncogenic RAS(G12V)-mediated cellular transformation on the mitochondrial dynamics machinery and observe a positive selection for dynamin-related protein 1 (DRP1), a protein required for mitochondrial network division. Loss of DRP1 prevents RAS(G12V)-induced mitochondrial dysfunction and renders cells resistant to transformation. Conversely, in human tumor cell lines with activating MAPK mutations, inhibition of these signals leads to robust mitochondrial network reprogramming initiated by DRP1 loss resulting in mitochondrial hyper-fusion and increased mitochondrial metabolism. These phenotypes are mechanistically linked by ERK1/2 phosphorylation of DRP1 serine 616; DRP1(S616) phosphorylation is sufficient to phenocopy transformation-induced mitochondrial dysfunction, and DRP1(S616) phosphorylation status dichotomizes BRAF(WT) from BRAF(V600E)-positive lesions. These findings implicate mitochondrial division and DRP1 as crucial regulators of transformation with leverage in chemotherapeutic success.

BACKGROUND

Fine-needle aspiration cytology (FNAC) is the gold standard for the differential diagnosis of thyroid nodules but has the limitation of inadequate sampling or indeterminate lesions.

OBJECTIVE

We aimed to verify whether search of thyroid cancer-associated protooncogene mutations in cytological samples may improve the diagnostic accuracy of FNAC.

METHODS

One hundred seventy-four consecutive patients undergoing thyroid surgery were submitted to FNAC (on 235 thyroid nodules) that was used for cytology and molecular analysis of BRAF, RAS, RET, TRK, and PPRgamma mutations. At surgery these nodules were sampled to perform the same molecular testing.

RESULTS

Mutations were found in 67 of 235 (28.5%) cytological samples. Of the 67 mutated samples, 23 (34.3%) were mutated by RAS, 33 (49.3%) by BRAF, and 11 (16.4%) by RET/PTC. In 88.2% of the cases, the mutation was confirmed in tissue sample. The presence of mutations at cytology was associated with cancer 91.1% of the times and follicular adenoma 8.9% of the time. BRAF or RET/PTC mutations were always associated with cancer, whereas RAS mutations were mainly associated with cancer (74%) but also follicular adenoma (26%). The diagnostic performance of molecular analysis was superior to that of traditional cytology, with better sensitivity and specificity, and the combination of the two techniques further contributed to improve the total accuracy (93.2%), compared with molecular analysis (90.2%) or traditional cytology (83.0%).

CONCLUSIONS

Our findings demonstrate that molecular analysis of cytological specimens is feasible and that its results in combination with cytology improves the diagnostic performance of traditional cytology.

BACKGROUND

Molecular genetic analyses of lung adenocarcinoma have recently become standard of care for treatment selection. The Lung Cancer Mutation Consortium was formed to enable collaborative multi-institutional analyses of 10 potential oncogenic driver mutations. Technical aspects of testing and clinicopathologic correlations are presented.

METHODS

Mutation testing in at least one of the eight genes (epidermal growth factor receptor [EGFR], KRAS, ERBB2, AKT1, BRAF, MEK1, NRAS, and PIK3CA) using SNaPshot, mass spectrometry, Sanger sequencing+/- peptide nucleic acid and/or sizing assays, along with anaplastic lymphoma kinase (ALK) and/or MET fluorescence in situ hybridization, were performed in six labs on 1007 patients from 14 institutions.

RESULTS

In all, 1007 specimens had mutation analysis performed, and 733 specimens had all 10 genes analyzed. Mutation identification rates did not vary by analytic method. Biopsy and cytology specimens were inadequate for testing in 26% and 35% of cases compared with 5% of surgical specimens. Among the 1007 cases with mutation analysis performed, EGFR, KRAS, ALK, and ERBB2 alterations were detected in 22%, 25%, 8.5%, and 2.4% of cases, respectively. EGFR mutations were highly associated with female sex, Asian race, and never-smoking status; and less strongly associated with stage IV disease, presence of bone metastases, and absence of adrenal metastases. ALK rearrangements were strongly associated with never-smoking status and more weakly associated with presence of liver metastases. ERBB2 mutations were strongly associated with Asian race and never-smoking status. Two mutations were seen in 2.7% of samples, all but one of which involved one or more of PIK3CA, ALK, or MET.

CONCLUSIONS

Multi-institutional molecular analysis across multiple platforms, sample types, and institutions can yield consistent results and novel clinicopathological observations.

OBJECTIVE

The management of pancreatic cysts poses challenges to both patients and their physicians. We investigated whether a combination of molecular markers and clinical information could improve the classification of pancreatic cysts and management of patients.

METHODS

We performed a multi-center, retrospective study of 130 patients with resected pancreatic cystic neoplasms (12 serous cystadenomas, 10 solid pseudopapillary neoplasms, 12 mucinous cystic neoplasms, and 96 intraductal papillary mucinous neoplasms). Cyst fluid was analyzed to identify subtle mutations in genes known to be mutated in pancreatic cysts (BRAF, CDKN2A, CTNNB1, GNAS, KRAS, NRAS, PIK3CA, RNF43, SMAD4, TP53, and VHL); to identify loss of heterozygozity at CDKN2A, RNF43, SMAD4, TP53, and VHL tumor suppressor loci; and to identify aneuploidy. The analyses were performed using specialized technologies for implementing and interpreting massively parallel sequencing data acquisition. An algorithm was used to select markers that could classify cyst type and grade. The accuracy of the molecular markers was compared with that of clinical markers and a combination of molecular and clinical markers.

RESULTS

We identified molecular markers and clinical features that classified cyst type with 90%-100% sensitivity and 92%-98% specificity. The molecular marker panel correctly identified 67 of the 74 patients who did not require surgery and could, therefore, reduce the number of unnecessary operations by 91%.

CONCLUSIONS

We identified a panel of molecular markers and clinical features that show promise for the accurate classification of cystic neoplasms of the pancreas and identification of cysts that require surgery.

This study investigated the sensitivity and specificity of immunohistochemical (IHC) analysis using an anti-BRAF antibody to detect the presence of the BRAF V600E mutation in patients with metastatic melanoma. A total of 100 patients with American Joint Committee on Cancer stage IIIC unresectable or stage IV melanoma and who underwent tumor DNA BRAF mutation testing were selected. Paraffin-embedded, formalin-fixed melanoma biopsies were analyzed for the BRAF mutation status by independent, blinded observers using both conventional DNA molecular techniques and IHC with the novel BRAF V600E mutant-specific antibody, VE1. The antibody had a sensitivity of 97% (37/38) and a specificity of 98% (58/59) for detecting the presence of a BRAF V600E mutation. Of the BRAF-mutated cases, none of the non-V600E cases (including V600K) stained positive with the antibody (0/11). There were 5 cases with discordant BRAF mutation results. Additional molecular analysis confirmed the immunohistochemically obtained BRAF result in 3 cases, suggesting that the initial molecular testing results were incorrect. Two of these patients would not have received a BRAF inhibitor on the basis of the initial false-negative mutation testing result. Two cases remained discordant. The reported IHC method is an accurate, rapid, and cost-effective method for detecting V600E BRAF mutations in melanoma patients. Clinical use of the V600E BRAF antibody should be a valuable supplement to conventional mutation testing and allow V600E mutant metastatic melanoma patients to be triaged rapidly into appropriate treatment pathways.

BRAF mutations occur in approximately 10% of colorectal cancers. Although RAF inhibitor monotherapy is highly effective in BRAF-mutant melanoma, response rates in BRAF-mutant colorectal cancer are poor. Recent clinical trials of combined RAF/EGFR or RAF/MEK inhibition have produced improved efficacy, but patients ultimately develop resistance. To identify molecular alterations driving clinical acquired resistance, we performed whole-exome sequencing on paired pretreatment and postprogression tumor biopsies from patients with BRAF-mutant colorectal cancer treated with RAF inhibitor combinations. We identified alterations in MAPK pathway genes in resistant tumors not present in matched pretreatment tumors, including KRAS amplification, BRAF amplification, and a MEK1 mutation. These alterations conferred resistance to RAF/EGFR or RAF/MEK combinations through sustained MAPK pathway activity, but an ERK inhibitor could suppress MAPK activity and overcome resistance. Identification of MAPK pathway reactivating alterations upon clinical acquired resistance underscores the MAPK pathway as a critical target in BRAF-mutant colorectal cancer and suggests therapeutic options to overcome resistance.

CONCLUSIONS

RAF inhibitor combinations represent promising approaches in clinical development for BRAF-mutant colorectal cancer. Initial characterization of clinical acquired resistance mechanisms to these regimens identified several MAPK pathway alterations driving resistance by reactivating MAPK signaling, highlighting the critical dependence of BRAF-mutant colorectal cancers on MAPK signaling and offering potential strategies to overcome resistance.

The PI3K/AKT/mTOR and RAS/RAF/MEK/ERK pathways are two of the most frequently dysregulated kinase cascades in human cancer. Molecular alterations in these pathways are implicated in tumorigenesis and resistance to anticancer therapies. The PI3K/AKT/mTOR and RAS/RAF/MEK/ERK pathways are known to interact with each other at several nodes, and mounting evidence suggests that dual blockade of both pathways may be required to achieve anticancer effects in certain contexts. This may include tumor types with a high frequency of RAS/RAF/MEK/ERK pathway activation, or situations in which dual pathway strategies may be required to overcome resistance to current targeted therapies. Several clinical studies are currently evaluating the combination of PI3K and MEK inhibitors in a variety of different cancers with certain types of molecular alterations. This review will summarize existing knowledge of the PI3K/AKT/mTOR and RAS/RAF/MEK/ERK pathways, the cross-talk between them, and the current generation of PI3K and MEK inhibitors that target them. The preclinical rationale for dual pathway inhibition will be discussed within the context of the major tumor types currently being explored in ongoing clinical trials, namely malignant melanoma with BRAF or NRAS mutations, and colorectal, ovarian, pancreatic, and basal-like breast cancers. The emerging clinical profile of PI3K and MEK inhibitor combinations, as reported in Phase I trials, will also be discussed.

OBJECTIVE

NRAS and BRAF mutations are common in cutaneous melanomas, although rarely detected mutually in the same tumor. Distinct clinical correlates of these mutations have not been described, despite in vitro data suggesting enhanced oncogenic effects. This study was designed to test the hypothesis that primary human cutaneous melanomas harboring mutations in NRAS or BRAF display a more aggressive clinical phenotype than tumors wild type at both loci.

METHODS

Microdissection of 223 primary melanomas was carried out, followed by determination of the NRAS and BRAF mutational status. Genotypic findings were correlated with features known to influence tumor behavior including age, gender, Breslow depth, Clark level, mitotic rate, the presence of ulceration, and American Joint Committee on Cancer (AJCC) staging.

RESULTS

Breslow depth and Clark level varied significantly among the genotypes, with NRAS mutants showing the deepest levels and wild-type tumors the least depth. Ulceration also differed significantly among the genotypes, with BRAF mutants demonstrating the highest rate. In addition, tumors with mutated NRAS were more likely to be located on the extremities. Patients whose tumors carried either mutation presented with more advanced AJCC stages compared with patients with wild-type tumors, and specifically, were more likely to have stage III disease at diagnosis. Overall survival did not differ among the 3 groups.

CONCLUSIONS

Distinct clinical phenotypes exist for melanomas bearing NRAS and BRAF mutations, whether considered together or separately, and are associated with features known to predict aggressive tumor behavior. The impact of these mutations is most evident at earlier stages of disease progression.

The effect of NRAS mutations on the pathological features and clinical outcomes in patients with cutaneous melanoma was compared with that of tumors containing BRAF(V600E) mutations and tumors wild type for both (WT). Clinical outcome data were obtained from a prospective cohort of 249 patients. Mutations involving NRAS and BRAF(V600E) were detected by PCR and were sequence verified. Cox proportional hazards regression was performed to relate NRAS and BRAF mutations to clinical outcome. Seventy-five percentage of NRAS mutations occurred in tumors >1 mm thick (BRAF(V600E) 40%, WT 34%); 75% of NRAS mutations had >1 mitosis/mm(2) (BRAF(V600E) 40%, WT 55%). When compared to WT, multivariate analysis of melanoma-specific survival (MSS) identified NRAS mutations as an adverse prognostic factor [hazard ratio (HR) 2.96; P = 0.04] but not BRAF(V600E) mutations (HR 1.73; P = 0.23). NRAS mutations were associated with thicker tumors and higher rates of mitosis when compared to BRAF(V600E) and WT melanoma and independently of this, with shorter MSS.

Accumulating evidence suggests that colorectal cancer (CRC) should be viewed as a heterogeneous disease, with proximal and distal CRCs showing multiple biological and clinical differences. The aim of this study was to develop a clinicopathological, molecular and protein profile for CRCs based on their region and thus providing insight into their heterogeneity. CRC patients (n=399) were evaluated for clinicopathologic and molecular features including K-RAS, BRAF and MSI status. Tumors were also screened for expression of 50 immunohistochemical markers linked to major signaling pathways involved in tumor-progression or immune response. Proximally located tumors show significantly larger tumor size, higher T-stage, higher tumor grade and more frequent mucinous histologic subtype compared to the distal colon and rectum. The frequency of BRAF mutation and MSI-high phenotype were significantly higher in proximal colon cancers. There is a significant difference in regional expression of 10 tumor-associated markers (CDX2, CD44v6, CD44s, TOPK, nuclear beta-catenin, pERK, APAF-1, E-cadherin, p21 and bcl2) and 4 immune response markers (CD68, CD163, FoxP3 and TIA-1). In multivariate analysis CD44s, CD44v6, nuclear beta-catenin and CD68 expression was found to best discriminate left- versus right-sided colon cancers. Tumor diameter, pT stage and MSI status best distinguish right-sided colon cancers from rectal cancers and pT stage and E-cadherin best discriminate left-sided colon cancers and rectal cancers. These data along with existing evidence for the presence of distinct regional embryological origin and gene expression profile are highly supportive of the concept that proximal and distal CRCs are distinct clinicopathologic entities.

BRAF mutations play a well-established role in melanomagenesis; however, without additional genetic alterations, tumor development is restricted by oncogene-induced senescence (OIS). Here, we show that mutations in the NF1 tumor suppressor gene cooperate with BRAF mutations in melanomagenesis by preventing OIS. In a genetically engineered mouse model, Nf1 mutations suppress Braf-induced senescence, promote melanocyte hyperproliferation, and enhance melanoma development. Nf1 mutations function by deregulating both phosphoinositide 3-kinase and extracellular signal-regulated kinase pathways. As such, Nf1/Braf-mutant tumors are resistant to BRAF inhibitors but are sensitive to combined inhibition of mitogen-activated protein/extracellular signal-regulated kinase kinase and mTOR. Importantly, NF1 is mutated or suppressed in human melanomas that harbor concurrent BRAF mutations, NF1 ablation decreases the sensitivity of melanoma cell lines to BRAF inhibitors, and NF1 is lost in tumors from patients following treatment with these agents. Collectively, these studies provide mechanistic insight into how NF1 cooperates with BRAF mutations in melanoma and show that NF1/neurofibromin inactivation may have an impact on responses to targeted therapies.

ERK signaling requires RAS-induced RAF dimerization and is limited by feedback. Activated BRAF mutants evade feedback inhibition of RAS by either of two mechanisms. BRAF V600 mutants are activated monomers when RAS activity is low; all other activating BRAF mutants function as constitutive RAS-independent dimers. RAF inhibitors effectively inhibit mutant monomers, but not dimers; their binding to one site in the dimer significantly reduces their affinity for the second. Tumors with non-V600E BRAF mutants are insensitive to these drugs, and increased expression of BRAF V600E dimers causes acquired resistance. A compound that equally inhibits both sites of mutant RAF dimers inhibits tumors driven by either class of mutants or those BRAF V600E tumors with dimer-dependent acquired resistance to monomer-specific inhibitors.

OBJECTIVE

This study investigated the utility of BRAF mutation testing of thyroid fine-needle aspiration biopsy (FNAB) specimens for preoperative risk stratification in papillary thyroid cancer (PTC).

METHODS

We assessed the T1799A BRAF mutation status in thyroid FNAB specimens obtained from 190 patients before thyroidectomy for PTC and its association with clinicopathologic characteristics of the tumor revealed postoperatively.

RESULTS

We observed a significant association of BRAF mutation in preoperative FNAB specimens with poorer clinicopathologic outcomes of PTC. In comparison with the wild-type allele, BRAF mutation strongly predicted extrathyroidal extension (23% v 11%; P = .039), thyroid capsular invasion (29% v 16%; P = .045), and lymph node metastasis (38% v 18%; P = .002). During a median follow-up of 3 years (range, 0.6 to 10 years), PTC persistence/recurrence was seen in 36% of BRAF mutation-positive patients versus 12% of BRAF mutation-negative patients, with an odds ratio of 4.16 (95% CI, 1.70 to 10.17; P = .002). The positive and negative predictive values for preoperative FNAB-detected BRAF mutation to predict PTC persistence/recurrence were 36% and 88% for overall PTC and 34% and 92% for conventional PTC, respectively.

CONCLUSIONS

Preoperative BRAF mutation testing of FNAB specimens provides a novel tool to preoperatively identify PTC patients at higher risk for extensive disease (extrathyroidal extension and lymph node metastases) and those who are more likely to manifest disease persistence/recurrence. BRAF mutation, as a powerful risk prognostic marker, may therefore be useful in appropriately tailoring the initial surgical extent for patients with PTC.

BACKGROUND

The prognostic potential of individual clinical and molecular parameters in stage II/III colon cancer has been investigated, but a thorough multivariable assessment of their relative impact is missing.

METHODS

Tumors from patients (N = 1404) in the PETACC3 adjuvant chemotherapy trial were examined for BRAF and KRAS mutations, microsatellite instability (MSI), chromosome 18q loss of heterozygosity (18qLOH), and SMAD4 expression. Their importance in predicting relapse-free survival (RFS) and overall survival (OS) was assessed by Kaplan-Meier analyses, Cox regression models, and recursive partitioning trees. All statistical tests were two-sided.

RESULTS

MSI-high status and SMAD4 focal loss of expression were identified as independent prognostic factors with better RFS (hazard ratio [HR] of recurrence = 0.54, 95% CI = 0.37 to 0.81, P = .003) and OS (HR of death = 0.43, 95% CI = 0.27 to 0.70, P = .001) for MSI-high status and worse RFS (HR = 1.47, 95% CI = 1.19 to 1.81, P < .001) and OS (HR = 1.58, 95% CI = 1.23 to 2.01, P < .001) for SMAD4 loss. 18qLOH did not have any prognostic value in RFS or OS. Recursive partitioning identified refinements of TNM into new clinically interesting prognostic subgroups. Notably, T3N1 tumors with MSI-high status and retained SMAD4 expression had outcomes similar to stage II disease.

CONCLUSIONS

Concomitant assessment of molecular and clinical markers in multivariable analysis is essential to confirm or refute their independent prognostic value. Including molecular markers with independent prognostic value might allow more accurate prediction of prognosis than TNM staging alone.

The clinically important melanoma diagnostic antibodies HMB-45, melan-A, and MITF (D5) recognize gene products of the melanocyte-lineage genes SILV/PMEL17/GP100, MLANA/MART1, and MITF, respectively. MITF encodes a transcription factor that is essential for normal melanocyte development and appears to regulate expression of several pigmentation genes. In this report, the possibility was examined that MITF might additionally regulate expression of the SILV and MLANA genes. Both genes contain conserved MITF consensus DNA sequences that were bound by MITF in vitro and in vivo, based on electrophoretic mobility shift assay and chromatin-immunoprecipitation. In addition, MITF regulated their promoter/enhancer regions in reporter assays, and up- or down-regulation of MITF produced corresponding modulation of endogenous SILV and MLANA in melanoma cells. Expression patterns were compared with these factors in a series of melanoma cell lines whose mutational status of the proto-oncogene BRAF was also known. SILV and MLANA expression correlated with MITF, while no clear correlation was seen relative to BRAF mutation. Finally, mRNA expression array analysis of primary human melanomas demonstrated a tight correlation in their expression levels in clinical tumor specimens. Collectively, this study links three important melanoma antigens into a common transcriptional pathway regulated by MITF.

The RAS/RAF/MEK pathway is activated in more than 30% of human cancers, most commonly via mutation in the K-ras oncogene and also via mutations in BRAF. Several allosteric mitogen-activated protein/extracellular signal-regulated kinase (MEK) inhibitors, aimed at treating tumors with RAS/RAF pathway alterations, are in clinical development. However, acquired resistance to these inhibitors has been documented both in preclinical and clinical samples. To identify strategies to overcome this resistance, we have derived three independent MEK inhibitor-resistant cell lines. Resistance to allosteric MEK inhibitors in these cell lines was consistently linked to acquired mutations in the allosteric binding pocket of MEK. In one cell line, concurrent amplification of mutant K-ras was observed in conjunction with MEK allosteric pocket mutations. Clonal analysis showed that both resistance mechanisms occur in the same cell and contribute to enhanced resistance. Importantly, in all cases the MEK-resistant cell lines retained their addiction to the mitogen-activated protein kinase (MAPK) pathway, as evidenced by their sensitivity to a selective inhibitor of the ERK1/2 kinases. These data suggest that tumors with acquired MEK inhibitor resistance remain dependent on the MAPK pathway and are therefore sensitive to inhibitors that act downstream of the mutated MEK target. Importantly, we show that dual inhibition of MEK and ERK by small molecule inhibitors was synergistic and acted to both inhibit the emergence of resistance, as well as to overcome acquired resistance to MEK inhibitors. Therefore, our data provide a rationale for cotargeting multiple nodes within the MAPK signaling cascade in K-ras mutant tumors to maximize therapeutic benefit for patients.

MicroRNAs (miRNA) are small non-coding RNAs involved in post-transcriptional gene regulation that have crucial roles in several types of tumors, including papillary thyroid carcinoma (PTC). miR-146b-5p is overexpressed in PTCs and is regarded as a relevant diagnostic marker for this type of cancer. A computational search revealed that miR-146b-5p putatively binds to the 3' untranslated region (UTR) of SMAD4, an important member of the transforming growth factor β (TGF-β) signaling pathway. The TGF-β pathway is a negative regulator of thyroid follicular cell growth, and the mechanism by which thyroid cancer cells evade its inhibitory signal remains unclear. We questioned whether the modulation of the TGF-β pathway by miR-146b-5p can contribute to thyroid tumorigenesis. Luciferase reporter assay confirmed the direct binding of miR-146b-5p on the SMAD4 3'UTR. Specific inhibition of miR-146b-5p with a locked nucleic acid-modified anti-miR-146b oligonucleotide significantly increased SMAD4 levels in the human papillary carcinoma cell lines, TPC-1 and BCPAP. Moreover, suppression of miR-146b-5p increased the cellular response to the TGF-β anti-proliferative signal, significantly decreasing the proliferation rate. The overexpression of miR-146b-5p in normal rat follicular PCCL3 cells decreased SMAD4 levels and disrupted TGF-β signal transduction. MiR-146b-5p overexpression in PCCL3 cells also significantly increased cell proliferation in the absence of thyroid-stimulating hormone and conferred resistance to TGF-β-mediated cell-cycle arrest. Additionally, the activation of thyroid most common oncogenes RET/PTC3 and BRAF in PCCL3 cells upregulated miR-146b-5p expression. Our results confirm the oncogenic role of miR-146b-5p in thyroid follicular cells and contribute to knowledge regarding the modulation of TGF-β signal transduction by miRNAs in PTCs.

Tissue hypoxia commonly occurs in tumors. Hypoxia- inducible factor (HIF)-1 and HIF-2, which are essential mediators of cellular response to hypoxia, regulate gene expression for tumor angiogenesis, glucose metabolism, and resistance to oxidative stress. Their key regulatory subunits, HIF1A (HIF-1alpha) and endothelial PAS domain protein 1 (EPAS1; HIF-2alpha), are overexpressed and associated with patient prognosis in a variety of cancers. However, prognostic or molecular features of colon cancer with HIF expression remain uncertain. Among 731 colorectal cancers in two prospective cohort studies, 142 (19%) tumors showed HIF1A overexpression, and 322 (46%) showed EPAS1 overexpression by immunohistochemistry. HIF1A overexpression was significantly associated with higher colorectal cancer-specific mortality in Kaplan-Meier analysis (log-rank test, P < 0.0001), univariate Cox regression (hazard ratio = 1.84; 95% confidence interval, 1.37 to 2.47; P < 0.0001) and multivariate analysis (adjusted hazard ratio = 1.72; 95% confidence interval, 1.26 to 2.36; P = 0.0007) that adjusted for clinical and tumoral features, including microsatellite instability, TP53 (p53), PTGS2 (cyclooxygenase-2), CpG island methylator phenotype, and KRAS, BRAF, PIK3CA, and LINE-1 methylation. In contrast, EPAS1 expression was not significantly associated with patient survival. In addition, HIF1A expression was independently associated with PTGS2 expression (P = 0.0035), CpG island methylator phenotype-high (P = 0.013), and LINE-1 hypomethylation (P = 0.017). EPAS1 expression was inversely associated with high tumor grade (P = 0.0017) and obesity (body mass index>> or = 30 kg/m2) (P = 0.039). In conclusion, HIF1A expression is independently associated with poor prognosis in colorectal cancer, suggesting HIF1A as a biomarker with potentially important therapeutic implications.

OBJECTIVE

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.

METHODS

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.

RESULTS

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.

CONCLUSIONS

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.

OBJECTIVE

Mutations affecting the KRAS gene are established predictive markers of outcome with anti-epithelial growth factor receptor (EGFR) antibodies in advanced colorectal cancer (CRC). The relevance of these markers for anti-vascular endothelial growth factor (VEGF) therapy is controversial. This analysis was performed to assess the predictive and prognostic impact of KRAS and BRAF gene mutation status in patients receiving capecitabine with bevacizumab (CG) or capecitabine without bevacizumab in the phase III AGITG MAX (Australasian Gastrointestinal Trials Group MAX) study.

METHODS

Mutation status was determined for 315 (66.9%) of the original 471 patients. Mutation status was correlated with efficacy outcomes (response rate, progression-free survival [PFS], and overall survival [OS]), and a predictive analyses was undertaken.

RESULTS

Mutations in KRAS and BRAF genes were observed in 28.8% and 10.6% of patients, respectively. KRAS gene mutation status (wild type [WT] v mutated [MT]) had no prognostic impact for PFS (hazard ratio [HR], 0.89; CI, 0.69 to 1.14) or OS (HR, 0.97; CI, 0.73 to 1.28). BRAF mutation status (WT v MT) was not prognostic for PFS (HR, 0.80; CI, 0.54 to 1.18) but was prognostic for OS (HR, 0.49; CI, 0.33 to 0.73; P = .001). By using the comparison of capecitabine versus capecitabine and bevacizumab (CB) and CB plus mitomycin (CBM), KRAS gene mutation status was not predictive of the effectiveness of bevacizumab for PFS or OS (test for interaction P = .95 and 0.43, respectively). Similarly, BRAF gene mutation status was not predictive of the effectiveness of bevacizumab for PFS or OS (test for interaction P = .46 and 0.32, respectively).

CONCLUSIONS

KRAS gene mutation status was neither prognostic for OS nor predictive of bevacizumab outcome in patients with advanced CRC. BRAF gene mutation status was prognostic for OS but was not predictive of outcome with bevacizumab.