Murine Sarcoma Viral Oncogene Homolog Research Articles

Tumour-to-tumour metastasis from papillary thyroid carcinoma withBRAFmutation to lung adenocarcinoma withEGFRmutation: the utility of mutation-specific antibodies

Tumour-to-tumour metastasis is a rare event. The aim of this study is to demonstrate the utility of mutation-specific antibodies to prove the occurrence of metastatic papillary thyroid cancer donor into lung adenocarcinoma recipient. We report the case of an 80-year-old woman who had a papillary thyroid carcinoma with a v-raf murine sarcoma viral oncogene homologue B1 mutation that metastasized into a lung adenocarcinoma with an epidermal growth factor receptor mutation. Immunohistochemical analysis with mutation-specific antibodies not only clearly revealed two components, but also revealed their gene mutation statuses. As a component of multimodal diagnostic tools, immunohistochemistry can avoid some pitfalls involved in the molecular diagnosis of complicated cases (such as our own) and can help to ensure that patients receive optimal treatments.

Role of methionine adenosyltransferase α2 and β phosphorylation and stabilization in human hepatic stellate cell trans-differentiation.

Myofibroblastic trans-differentiation of hepatic stellate cells (HSCs) is an essential event in the development of liver fibrogenesis. These changes involve modulation of key regulators of the genome and the proteome. Methionine adenosyltransferases (MAT) catalyze the biosynthesis of the methyl donor, S-adenosylmethionine (SAMe) from methionine. We have previously shown that two MAT genes, MAT2A and MAT2B (encoding MATα2 and MATβ proteins respectively), are required for HSC activation and loss of MAT2A transcriptional control favors its up-regulation during trans-differentiation. Hence MAT genes are intrinsically linked to the HSC machinery during activation. In the current study, we have identified for the first time, post-translational modifications in the MATα2 and MATβ proteins that stabilize them and favor human HSC trans-differentiation. Culture-activation of human HSCs induced the MATα2 and MATβ proteins. Using mass spectrometry, we identified phosphorylation sites in MATα2 and MATβ predicted to be phosphorylated by mitogen-activated protein kinase (MAPK) family members (ERK1/2, V-Raf Murine Sarcoma Viral Oncogene Homolog B1 [B-Raf], MEK). Phosphorylation of both proteins was enhanced during HSC activation. Blocking MEK activation lowered the phosphorylation and stability of MAT proteins without influencing their mRNA levels. Silencing ERK1/2 or B-Raf lowered the phosphorylation and stability of MATβ but not MATα2. Reversal of the activated human HSC cell line, LX2 to quiescence lowered phosphorylation and destabilized MAT proteins. Mutagenesis of MATα2 and MATβ phospho-sites destabilized them and prevented HSC trans-differentiation. The data reveal that phosphorylation of MAT proteins during HSC activation stabilizes them thereby positively regulating trans-differentiation.

BRAF inhibitor resistance mediated by the AKT pathway in an oncogenic BRAF mouse melanoma model

BRAF (v-raf murine sarcoma viral oncogene homolog B) inhibitors elicit a transient anti-tumor response in ∼ 80% of BRAF(V600)-mutant melanoma patients that almost uniformly precedes the emergence of resistance. Here we used a mouse model of melanoma in which melanocyte-specific expression of Braf(V618E) (analogous to the human BRAF(V600E) mutation) led to the development of skin hyperpigmentation and nevi, as well as melanoma formation with incomplete penetrance. Sleeping Beauty insertional mutagenesis in this model led to accelerated and fully penetrant melanomagenesis and synchronous tumor formation. Treatment of Braf(V618E) transposon mice with the BRAF inhibitor PLX4720 resulted in tumor regression followed by relapse. Analysis of transposon insertions identified eight genes including Braf, Mitf, and ERas (ES-cell expressed Ras) as candidate resistance genes. Expression of ERAS in human melanoma cell lines conferred resistance to PLX4720 and induced hyperphosphorylation of AKT (v-akt murine thymoma viral oncogene homolog 1), a phenotype reverted by combinatorial treatment with PLX4720 and the AKT inhibitor MK2206. We show that ERAS expression elicits a prosurvival signal associated with phosphorylation/inactivation of BAD, and that the resistance of hepatocyte growth factor-treated human melanoma cells to PLX4720 can be reverted by treatment with the BAD-like BH3 mimetic ABT-737. Thus, we define a role for the AKT/BAD pathway in resistance to BRAF inhibition and illustrate an in vivo approach for finding drug resistance genes.

Application of immunohistochemistry in gastrointestinal and liver neoplasms: new markers and evolving practice.

Diagnosis of primary gastrointestinal and liver neoplasms is usually straightforward. Immunohistochemistry is most helpful to differentiate metastatic carcinomas with morphologic similarity and to resolve tumors of unknown origin. Recently, several new markers highly sensitive and specific for primary liver and gastrointestinal tumors have been discovered. Their potential diagnostic application has not been widely appreciated by general practicing pathologists. In addition, a new trend in immunohistochemistry application has started, focusing on assessing predictive markers (such as human epidermal growth factor receptor 2) and mutation-specific markers (v-raf murine sarcoma viral oncogene homolog B V600E) to directly guide clinical management. Practicing pathologists need to be aware of and prepared for this evolving trend. To summarize the usefulness of several recently discovered immunohistochemical markers in the study of gastrointestinal and liver tumors; to suggest the most current and effective immunohistochemical panels addressing common diagnostic challenges for these tumors; to share practical experience and useful tips for human epidermal growth factor receptor 2 testing in gastric and gastroesophageal junction adenocarcinoma and v-raf murine sarcoma viral oncogene homolog B V600E immunohistochemistry in colorectal carcinoma. Sources include literature review, and authors' research data and practice experience. The cases illustrated are selected from the pathology archives of the Geisinger Medical Center (Danville, Pennsylvania). Application of immunohistochemistry in gastrointestinal and liver tumors continues to evolve. New tumor-specific markers constantly emerge and help pathologists to further improve diagnostic accuracy. Assessment of predictive and prognostic markers by immunohistochemistry in routine pathologic diagnosis is a new trend and will greatly facilitate the advancement of personalized cancer therapy.

BRAF Inhibitor Dabrafenib in Patients with Metastatic BRAF-Mutant Thyroid Cancer

Mutations of v-raf murine sarcoma viral oncogene homolog B (BRAF) are commonly identified in papillary and anaplastic thyroid carcinoma and are associated with worse prognosis compared with the wild type. BRAF inhibition in papillary thyroid carcinoma cell lines and xenografts inhibits proliferation and decreases downstream phosphorylation. Our objectives were to analyze safety and efficacy of the selective BRAF inhibitor dabrafenib in patients with metastatic BRAF-mutant thyroid carcinoma. We present the subset of patients with BRAF-mutant thyroid carcinoma enrolled in a larger phase 1 study, the main results of which are reported elsewhere. Fourteen patients with BRAF(V600E)-mutant thyroid carcinoma were enrolled, of whom 13 (93%) had received prior radioactive iodine. The median duration on treatment was 8.4 months, and seven (50%) patients received treatment for ≥10 months. The most common treatment-related adverse events were skin papillomas (n=8, 57%), hyperkeratosis (n=5, 36%), and alopecia (n=4, 29%), all of which were grade 1. Treatment-related adverse events grade ≥3 included grade 4 elevated lipase and grade 3 elevated amylase, fatigue, febrile neutropenia, and cutaneous squamous cell carcinoma (n=1 for each). Four (29%) partial responses were observed, and nine (64%) patients achieved at least 10% decrease. Only one responder progressed while on the study drug after a response duration of 9.3 months. The other three responders had not progressed, with response duration of 4.6+, 10.4+, and 21.4+ months. With seven (50%) patients showing no progression at the time of study completion, the median progression-free survival was 11.3 months. Dabrafenib was well tolerated and resulted in durable responses in BRAF-mutant differentiated thyroid carcinoma patients.

Correlation between the BRAF V600E mutation status and the clinicopathologic features of papillary thyroid carcinoma.

This study sought to investigate the correlations of V-raf murine sarcoma viral oncogene homolog B1 (BRAF) gene mutations with the clinicopathologic features of papillary thyroid carcinoma and central lymph node metastasis. We retrospectively analyzed the 2-year medical records of patients who underwent surgery for papillary thyroid carcinoma. After screening, the records of 126 patients who met the study requirements were used to assess the characteristics associated with the BRAF V600E gene mutation. The BRAF mutation incidence rate among patients with papillary thyroid carcinoma was 69.0% (87/126). Univariate analysis revealed that the BRAF mutation status was correlated significantly with both tumor size and lymph node metastasis (P < 0.05). Multivariate analysis revealed a significant correlation between lymph node metastasis and BRAF mutation status (P < 0.05). When the tumor diameter was ≤10 mm, the BRAF mutation status had no effect on lymph node metastasis (P > 0.05). When the tumor diameter was >10 mm, the incidence of lymph node metastasis was significantly higher among BRAF mutation-positive patients than among BRAF mutation-negative patients (P < 0.05). BRAF gene mutations independently predicted central lymph node metastasis in patients with papillary thyroid carcinoma. For patients preoperatively diagnosed to be BRAF mutation-positive, the importance of central lymph node dissection should be emphasized because the tumor diameter increases; regional lymphatic and adipose tissue dissection should be routinely conducted. However, in mutation-negative patients with tumor diameters of ≤5 mm, the need for central lymph node dissection should be re-examined.

Prevalence of somatic mutl homolog 1 promoter hypermethylation in Lynch syndrome colorectal cancer.

Colorectal cancers (CRCs) that have microsatellite instability (MSI) and mutL homolog 1 (MLH1) immunoloss are observed in 3 clinical scenarios: Lynch syndrome (LS), sporadic MSI CRC, and Lynch-like syndrome (LLS). v-Raf murine sarcoma viral oncogene homolog B1 (BRAF) mutational analysis is used to differentiate LS from sporadic MSI CRC. The role of MLH1 promoter methylation status for the differential diagnosis of these clinical forms is not well established. The objectives of this study were: 1) to analyze MLH1 promoter methylation in MLH1-deficient CRCs by pyrosequencing, and 2) to assess its role in the differential diagnosis of MLH1-deficient CRCs. In total, 165 CRCs were analyzed, including LS (n = 19), MSI BRAF-mutated CRC (n = 37), MSI BRAF wild-type CRC (n = 60), and a control group of CRCs without MSI (microsatellite stable [MSS] CRC; n = 49). MLH1 promoter methylation status was analyzed by pyrosequencing, and the ability of different strategies to identify LS was assessed. The average ± standard deviation methylation in LS (9% ± 7%) was significantly lower than that in MSI BRAF-mutated CRC (42% ± 17%; P < .001) and in MSI BRAF wild-type CRC (25% ± 19%; P = .002). Somatic MLH1 hypermethylation was detected in 3 patients (15.8%) with LS, in 34 patients (91.9%) with MSI BRAF-mutated CRC, and in 37 patients (61.7%) with MSI BRAF wild-type tumors. Patients with MSI BRAF wild-type, unmethylated tumors (ie, LLS) had a stronger family history of CRC than those who had tumors with MLH1 methylation (P < .05). The sensitivity for ruling out LS was 100% for BRAF analysis, 84.2% for MLH1 methylation analysis, and 84.2% for the combination of both analyses. Somatic MLH1 promoter methylation occurs in up to 15% of LS CRCs. Somatic BRAF analysis is the most sensitive strategy for ruling out LS. Patients who have CRCs with loss of MLH1 protein expression and neither BRAF mutation nor MLH1 methylation resemble patients with LS.

Standard immunohistochemistry efficiently screens for anaplastic lymphoma kinase rearrangements in differentiated thyroid cancer.

The anaplastic lymphoma kinase (ALK) gene is frequently rearranged in various types of cancer and is highly responsive to targeted therapeutics. We developed a system to detect rearrangement of ALK in a large group of Korean thyroid cancer patients. We screened 474 malignant or benign thyroid tumor cases to identify ALK fusions. Expression and translocation of the ALK gene were analyzed by immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), and digital multiplexed gene expression (DMGE) analysis in formalin-fixed paraffin-embedded tissues. Four cases of rearrangement of ALK were detected by IHC, and these cases were validated with FISH on 189 samples. On the other hand, DMGE analysis using Nanostring detected three out of four IHC-positive cases. Two rearrangements of ALK were striatin (STRN)-ALK fusions, which were identified by 5' RACE analysis. Rearrangements of ALK were found exclusively in v-raf murine sarcoma viral oncogene homolog B (BRAF) WT papillary carcinomas. Given the wide availability and accuracy of IHC for detecting ectopic expression of ALK in the thyroid, we suggest that IHC-based screening can be a practical method for identifying patients with ALK rearrangements in differentiated thyroid cancer.

Establishment and biological characteristics of acquired gefitinib resistance in cell line NCI-H1975/gefinitib-resistant with epidermal growth factor receptor T790M mutation.

Non‑small cell lung cancer (NSCLC) cells harboring mutations in the epidermal growth factor receptor (EGFR) gene initially respond well to EGFR tyrosine kinase inhibitors (TKI), including gefitinib. However the tumor cells will invariably develop acquired resistance to the drug. The EGFR T790M mutation is generally considered to be the molecular genetic basis of acquired TKI resistance. The present study aimed to explore how the T790M mutation induces tumor cells to escape inhibition by TKI treatment. An acquired gefitinib‑resistant cell line (NCI‑H1975/GR) was generated from the NCI‑H1975 human NSCLC cell line, which harbors the sensitive L858R and resistant T790M mutations of EGFR. The resistant cell line was established by exposing the cells intermittently to increasing concentrations of gefitinib. The mechanisms by which NSCLC acquires resistance to TKIs based on the T790M mutation, were investigated by detecting the protein expression levels of the EGFR/Kirsten rat sarcoma viral oncogene homolog (KRAS)/v‑Raf murine sarcoma viral oncogene homolog B (BRAF) transduction pathway, and epithelial‑mesenchymal transition (EMT) with immunocytochemistry. The resistance of the NCI‑H1975/GR cells to gefitinib was 2.009‑fold, as compared with the parent cells; however, the protein expression levels of EGFR, KRAS and BRAF were lower in the resistant cells. Some mesenchymal morphology was observed in the NCI‑H1975/GR cells, alongside a decreasing E‑cadherin expression and increasing vimentin expression. These results suggest that the reactivation of the EGFR/KRAS/BRAF transduction pathway was not detected in the NCI‑H1975/GR cells. EMT may have an important role in the development of acquired resistance to EGFR‑TKIs in NSCLC cells with sensitivity and resistance mutations.

BRAF alterations in brain tumours: molecular pathology and therapeutic opportunities.

To summarize the current knowledge on v-RAF murine sarcoma viral oncogene homologue B1 (BRAF) aberrations in tumours of the central nervous system. BRAF alterations are found in variable frequencies across a wide spectrum of diverse central nervous system neoplasms. BRAF V600 point mutations (most commonly of the V600E type) are most common in pleomorphic xanthoastrocytoma (approximately 60% of cases), gangliogliomas (50%), dysembryoplastic neuroepithelial tumours (30%), Langerhans cell histiocytosis (50%), melanoma brain metastases (50%) and papillary craniopharyngiomas (96%) and are also detectable in a fraction of glioblastomas (overall mutation rate of 2-12%, with a higher rate of approximately 50% in epithelioid glioblastomas). BRAF fusions (most commonly KIAA1549: BRAF) are typical for pilocytic astrocytomas and are almost absent from other tumour types. Clinical trials have established tyrosine-kinase inhibitors of BRAF as feasible treatment option in selected patients with mutation-bearing brain metastases of melanoma. Preclinical studies, some case reports and small patient series have documented tumour responses of primary brain tumours with BRAF aberrations to BRAF inhibition. Molecular testing for BRAF alterations in brain tumours may be of clinical relevance for differential diagnostic considerations in some situations or to guide selection of patients for targeted therapy with specific inhibitors. Prospective clinical trials evaluating the efficacy of BRAF inhibitors in central nervous system tumours are strongly supported by the available evidence.

Prospective immunohistochemical analysis of BRAF V600E mutation in melanoma.

The v-raf murine sarcoma viral oncogene homolog B1 (BRAF) V600E mutation is the most common activating genetic alteration of this oncogene and a predictive marker for the therapeutic use of BRAF inhibitors in melanoma. Our aim was to evaluate the performance of BRAF V600E mutation-specific monoclonal antibody (VE1) in a prospective diagnostic setting of melanoma patients (n = 102). All 41 cases (40.2%) that showed a V600E mutation in the cyclic minisequencing analysis of the DNA were also initially scored immunopositive. Two cases that were scored as BRAF V600E mutation positive by immunohistochemistry were negative in the DNA-based mutation analysis and determined to be immunonegative in a repeated staining with more representative specimens. Thus, BRAF V600E mutation detection using immunohistochemistry was 100% sensitive and 96.8% specific, when compared with the analysis of the DNA. None of the BRAF V600K mutations was detected by the VE1 antibody (n = 7). However, the VE1 antibody detected a rare V600E2 mutation. We also studied the role of BRAF V600E mutation in a set of melanoma patients who had been investigated for sentinel node metastasis. Melanoma lymph node metastases were diagnosed in 21.8% (12/55) of the sentinel nodes, and BRAF V600E immunopositivity was detected in 34.5% (19/55) of the cases. BRAF V600E mutation status did not correlate with any clinicopathological parameters. In conclusion, analysis of BRAF V600E mutation in melanoma by immunohistochemistry is a sensitive and specific method, which can be used to identify BRAF inhibitor-sensitive melanoma patients as a first-line method due to its rapid and affordable nature.

Overview of fundamental study of pazopanib in cancer.

Angiogenesis is an indispensible process for tumor growth and metastasis. Anti-angiogenesis based therapy is one of the most promising treatments for inhibiting cancer progression. Through the exploration of inhibitors of vascular endothelial growth factor receptor (VEGFR)-2, deemed as the major angiogenesis pathway, pazopanib was found as a small molecular pan-VEGFR and pan-platelet-derived growth factor receptor (PDGFR) inhibitor, with suitable pharmacodynamic and pharmacokinetic parameters to be an oral drug. In an vitro study, pazopanib exerted anti-tumor effect through mechanisms including the Raf-MAPK/ERK (MEK)-extracellular signal-regulated kinase (ERK) pathway, and directly targeted on v-raf murine sarcoma viral oncogene homolog B (B-raf) as well. It inhibited the proliferation of cell lines, such as DU-145 and HRC-45 in hepatocellular carcinoma, through mechanisms like "cell cycle arrest." In vivo xenograft studies and phase I/II clinical trials revealed a series of plasma cytokine and angiogenic factors, such as interleukin (IL)-6, IL-12, hepatocyte growth factor (HGF), and soluble VEGFR2, which have significant association with clinical curative effect. Pazopanib has been shown to be effective in solid tumors and some hematological malignancies. Future studies should focus on the exploration of biomarkers to screen sensitive patients and concomitant or metronomic dosage with other kinds of medicines.

Melanocytic naevi with globular and reticular dermoscopic patterns display distinct BRAF V600E expression profiles and histopathological patterns.

BRAF (v-raf murine sarcoma viral oncogene homologue B) V600E mutations have been detected with high frequency in melanocytic naevi. Few studies have stratified analyses by naevus dermoscopic pattern. To determine the frequency of BRAF V600E expression and histopathological pattern in acquired melanocytic naevi distinguished by a globular vs. reticular dermoscopic pattern. We retrospectively identified histologically proven melanocytic naevi with banal reticular or globular dermoscopic patterns and evaluated BRAF V600E expression using immunohistochemistry. BRAF V600E expression was detected in 11 of 12 globular naevi vs. four of 13 reticular naevi (91·7% vs. 30·1%, P=0·004). A predominantly dermal growth pattern (P<0·001) and the presence of large junctional nests (P=0·017) were each associated with a globular dermoscopic pattern. The presence of either a predominantly dermal growth pattern or large junctional nests was found in 13 of 15 naevi positive for BRAF V600E and in two of 10 naevi negative for BRAF V600E (86·7% vs. 20%, P=0·002). The frequency of BRAF V600E mutations differs in naevi distinguished by unique dermoscopic structures and microanatomical growth patterns. Globular naevi, which most often histologically correspond to a predominantly dermal growth pattern and/or the presence of large junctional nests, are significantly more likely to express BRAF V600E than reticular naevi. These preliminary results require validation, but may directly inform future studies of naevogenesis and melanoma genesis.

Identification of dysregulated pathways associated with pancreatic cancer by survival analysis.

In order to identify the dysregulated pathways associated with pancreatic cancer, the fourth leading cause of cancer mortality in the United States, tumor and non-tumor samples were systematically analyzed in the present study. Initially, dysregulated genes in pancreatic cancer were identified using paired t-test. Subsequently, dysregulated biological pathways involved in the development of pancreatic cancer were identified by enrichment analysis. Finally, individual survival analysis of the significantly dysregulated functions was conducted at the pathway level. Our results indicated that the pathway named ̔Pathways in cancer was significantly correlated with survival time. In addition, the mean survival time of individual and genetic variation demonstrated a significantly negative correlation, that is, the lower the genetic variation, the longer the survival time. Furthermore, detailed analysis of genes on the pathway named ̔Pathways in cancer denoted that this pathway involved multiple cancer hallmark signals and several dysregulated cancer genes, including tumor protein p53, myelocytomatosis, Kirsten rat sarcoma, phosphatidylinositol 3-kinase, v-raf murine sarcoma viral oncogene homolog B1 and cyclin-dependent kinase inhibitor 2A. According to the DrugBank database, certain oncogenes have been validated to be the targets of drugs, including Sorafenib, Trastuzumab, Imatinib and Paclitaxel or were under investigation. An improved understanding of the pathophysiology of pancreatic cancer has been achieved based on our results and the present study aimed to provide guidance for the development of drugs to treat pancreatic cancer.

Abstract CCOS14-04: Resistance to anti-BRAF and anti-MEK agents: Lost in translation

Abstract The V600E mutation in BRAF (v-raf murine sarcoma viral oncogene homolog B) is the most frequent oncogenic mutation in human cancers, and can be targeted by specific anti-BRAF agents (e.g. vemurafenib, dabrafenib). However, in spite of their proven efficacy, the effect of these drugs is limited by primary, or, more frequently, secondary resistance, that significantly counteracts their clinical benefit and has tempered the initial enthusiasm associated with their use in the clinic. Several resistance mechanisms have been identified. They can lead to reactivation of the initially blocked MAP-kinase pathway (known as ERK-dependent mechanisms) or reactivation of alternative ones, like the PI3K-AKT-mTOR pathway, or inhibition of the apoptotic cascade (ERK-independent). Recent evidence shows that the regulation of mRNA translation plays a pivotal role in gene expression. Furthermore, the complex machinery that regulates mRNA translation is a key element in the interplay between the two critical signalling MAPK and PI3-kinase pathways and the apoptosis cascade. However, it is unknown whether the response to anti-cancer targeted therapies is modulated by the activity of translation factors. By addressing the potential role of the eIF4F eukaryotic translation initiation complex, which regulates the initiation of the cap-dependent translation, in resistance or sensitivity to anti-BRAF and anti-MEK agents in several BRAF mutant cells, xenografts and tumors, it can be demonstrated that the formation of the eIF4F complex is associated with most resistance mechanisms to these targeted therapies. In several melanoma cell lines, resistance to anti-BRAF, anti-MEK or to both agents was consistently associated with persistent binding of the cap binding protein eIF4E to the eIF4G scaffolding protein, forming, together with eIF4A, the eIF4F complex. Conversely, sensitive cell lines and responding tumors from patients exhibited down-regulation of eIF4F complex formation and a concomitant increase in the binding of eIF4E to its inhibitory 4E binding protein (4EBP1). Binding of eIF4E to either eiF4G or 4EBP-1 was also visualized in situ in cell lines and in tumor samples. By exploring the resistance processes involved in the resistant cell lines that we studied, we found that these various mechanisms, independently of their capacity to reactivate or not the MAP-kinase pathway consistently favoured eIF4F complex formation over the eIF4E-4EBP-1 complex. Finally, we tested several eiF4F inhibitors that demonstrated a synergistic effect with anti-BRAF agents on resistant cell lines and in melanoma tumor xenografts. Finally, most mechanisms of resistance to anti-BRAF and /or anti-MEK agents are associated with persistent activation of the eiF4F cap-dependent translation initiation complex that can be explored in tumor samples using a new in situ technology. These findings potentially have important applications in the clinic since, independently of the mechanism leading to its continuous activation, inhibitors of the eiF4F complex synergize with anti-BRAF agents and can thus reverse resistance. The impact of the demonstration of a role for eIF4F in resistance to targeted anti-BRAF therapy might translate into the identification of new biomarkers and might also open new therapeutic avenues. Citation Format: Caroline Robert. Resistance to anti-BRAF and anti-MEK agents: Lost in translation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr CCOS14-04. doi:10.1158/1538-7445.AM2014-CCOS14-04

Ganetespib, a Novel Hsp90 Inhibitor in Patients With KRAS Mutated and Wild Type, Refractory Metastatic Colorectal Cancer

BackgroundHeat shock protein 90 (Hsp90) is a cellular chaperone that is required for the maturation and stability of a variety of proteins that play key roles in colon cancer initiation and progression. The primary objective of the current study was to define the safety and efficacy of ganetespib, a novel, selective small-molecule Hsp90 inhibitor, in patients with refractory metastatic colorectal cancer. Patients and MethodsThe study was a single-arm, Simon 2-stage, phase II trial for patients with chemotherapy-refractory, metastatic colorectal cancer. Patients received ganetespib 200 mg/m2 intravenously. Tumor tissue was collected before treatment and 48 hours after treatment for changes in expression of Hsp90 client proteins and other potential pharmacodynamics markers. V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS), v-Raf murine sarcoma viral oncogene homolog B, and phosphatidylinositol-4, 5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) mutational status was also determined. ResultsSeventeen patients were treated (median age, 58; range, 44-79 years). No patients demonstrated objective regression of disease. Two patients had stable disease of 6.8 and 5.1 months duration. Serious adverse events that were potentially attributable to ganetespib included diarrhea (12%, n = 2), fatigue (17%, n = 3), and increased aspartate aminotransferase/alanine aminotransferase (12%, n = 2) and alkaline phosphatase (6%, n = 1) levels. Of the 17 evaluable patients, 9 (53%) including patients with stable disease as best response, had KRAS-mutant tumors. ConclusionIn this first phase II investigation of an Hsp90 inhibitor in colorectal cancer, ganetespib as a single agent did not demonstrate activity in chemotherapy-refractory metastatic colorectal cancer. However, on the basis of the drug's promising preclinical combination data and the relatively mild toxicity profile, further clinical investigation of this agent in combination with standard cytotoxic agents is planned.

Relationship between BRAFv600E mutation and radioactive iodine uptake in distant metastases from papillary thyroid cancer

Objective To investigate the relationship between V-raf murine sarcoma viral oncogene homolog B1 (BRAF)v600E mutation and radioactive iodine (RAI) uptake in distant metastases from papillary thyroid cancer(PTC).Methods From January 2011 to December 2012,40 PTC patients (21 males,19 females,average age 39.8 years) with distant metastases were recruited and divided into mutation group and wild group according to the BRAFv600E mutation in primary lesions.The clinical,pathological and serological differences were compared between the two groups.The relationship between BRAFv600E mutation and RAI uptake capability in distant metastases from PTC,as well as its relationship with Tg change after 131I treatment were investigated.Statistical analysis was performed with two-sample t test,x2 test or Fisher exact test.Results The BRAFv600E mutation rate was 30.0％ (12/40) in patients with metastases from PTC.There was no significant difference in clinical,pathological and serological features between mutation group (n =12) and wild group (n=28; t:from-0.533 to 1.728,x2:from-1.951 to 1.088,all P＞0.05).Twelve PTC patients had no RAI uptake in the distant metastases,of which 10 belonged to mutation group (83.3％,10/12) and 2 belonged to wild group (7.1％,2/28; x2=19.734,P＜0.05).BRAFv600E mutation group was more likely to have no RAI uptake in the distant metastases.Tg change after 131I treatment in 30 patients were analyzed.In the wild group,Tg level decreased in 66.7％ (14/21) patients,stabilized in 19.0％ (4/21)and increased in 14.3％ (3/21)patients.While there was no decrease of Tg in the mutation group (0/9).Two patients had increased Tg level and 7 patients (with no RAI uptake) kept stable in mutation group.Conclusions Due to poor RAI uptake capability in PTC patients with BRAFv600E mutation,both primary and metastatic sites may have poor response to 131I treatment.Molecular detection of BRAFv600E mutation might be helpful for choosing PTC with distant metastases and predicting the effect of 131 I treatment. Key words: Thyroid neoplasms; Neoplasm metastasis; Genes; Mutation; Radiotherapy; Iodine radioisotopes

TERT promoter mutations and BRAF mutations are rare in sporadic, and TERT promoter mutations are absent in NF1-related malignant peripheral nerve sheath tumors

Hot spot mutations in the promoter region of telomerase reverse transcriptase (TERT promoter mutations) occur frequently in tumors of neuroectodermal origin such as melanoma and glioma. Many of these tumors are of neuroectodermal or ectomesenchymal origin which is suggestive of TERT promoter mutations playing a role in the development of malignant peripheral nerve sheath tumors (MPNSTs). In melanoma a correlation has been suggested between the occurrence of TERT promoter mutations and v-RAF murine sarcoma viral oncogene homolog B1 (BRAF) mutations. We investigated TERT promoter and BRAF mutation frequency in respectively 94 and 86 consecutive MPNST cases from our institute. TERT promoter mutation analysis on DNA from formalin-fixed, paraffin-embedded specimens was performed by SNaPshot analysis. Sequence analysis of BRAF was performed by bidirectional DNA sequencing. We identified TERT C228T or C250T promoter mutations in 10 % (9/94) and BRAF V600E mutations in 3 % (3/86) of MPNSTs. All TERT promoter- and BRAF mutations occurred in NF1 unrelated tumors. One co-occurrence of a TERT promoter- and a BRAF mutation was observed. In comparison with other neuroectodermal derived malignant neoplasms, TERT promoter mutations occur at relatively low frequency in MPNSTs. The observation of TERT promotor and BRAF mutations in sporadic MPNSTs and the absence of TERT promotor and rarity of BRAF mutations in NF1 related tumors may imply an alternative genetic route of tumor progression in both patient groups.

The status of BRAFV600E mutation among Egyptian patients with papillary thyroid carcinoma

The T1799A activating point mutation in v-Raf murine sarcoma viral oncogene homolog B1 (BRAF) is considered to be the most common genetic change in papillary thyroid cancer. It causes a change in the amino acid valine at position 600 to glutamic acid in the BRAF protein kinase, causing abnormal activation of the signaling pathway mitogen-activated protein kinase. Being associated with aggressive clinicopathological outcomes, it is now considered as a strong prognostic molecular marker for poorer prognosis of papillary thyroid carcinoma. The aim of the study was to investigate the frequency of the BRAFV600E mutation among the Egyptian patients with papillary thyroid carcinoma and to correlate with the clinicopathological status of the patients. The study included 50 formalin-fixed paraffin-embedded thyroid tumor tissue samples collected from the Department of Surgical Pathology, Faculty of Medicine, Cairo University, and the Egyptian National Cancer Institute. Patients’ data archived from records included age, sex, multifocality, lymph node metastasis, vascular invasion, and distant metastasis. The V600E mutation was tested by polymerase chain reaction-restriction fragment length polymorphism and direct sequencing. One (2 %) out of the 50 tumor tissue samples was found to be positive for the BRAFV600E mutation. Large-scale studies and inclusion of multiple molecular markers are recommended to elucidate the molecular basis of papillary thyroid carcinoma among Egyptian patients.

Modulation of BRAHMA expression by the mitogen-activated protein kinase/extracellular signal regulated kinase pathway is associated with changes in melanoma proliferation

Brahma (BRM) and Brahma-related gene 1(BRG1) are catalytic subunits of SWItch/sucrose non-fermentable (SWI/SNF) chromatin remodeling complexes. BRM is epigenetically silenced in a wide-range of tumors. Mutations in the v-raf murine sarcoma viral oncogene homolog B1 (BRAF) gene occur frequently in melanoma and lead to constitutive activation of the mitogen-activated protein kinase (MAPK)/extracellular signal regulated kinase (ERK1/2) pathway. We tested the hypothesis that BRM expression is modulated by oncogenic BRAF and phosphorylation of ERK1/2 in melanocytes and melanoma cells. Expression of oncogenic BRAF in melanocytes and melanoma cells that are wild-type for BRAF decreased BRM expression and increased BRG1 expression. Inhibition of mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) or selective inhibition of BRAF in melanoma cells that harbor oncogenic BRAF increased BRM expression and decreased BRG1 expression. Increased BRM expression was associated with increased histone acetylation on the BRM promoter. Over-expression of BRM in melanoma cells that harbor oncogenic BRAF promoted changes in cell cycle progression and apoptosis consistent with a tumor suppressive role. Upon inhibition of BRAF(V600E) with PLX4032, BRM promoted survival. PLX4032 induced changes in BRM function were correlated with increased acetylation of the BRM protein. This study provides insights into the epigenetic consequences of inhibiting oncogenic BRAF in melanoma through modulation of SWI/SNF subunit expression and function.