Absence of V599E BRAF mutations in desmoplastic melanomas

BACKGROUND: Desmoplastic melanoma is an uncommon variant of cutaneous melanoma that mimics soft tissue sarcoma both clinically and morphologically. An activating point mutation of the BRAF oncogene has been identified in a high proportion of conventional cutaneous melanomas, but its frequency in the desmoplastic subtype is not known. METHODS: The authors tested 12 desmoplastic melanoma specimens for the thymine (T)-->adenine (A) missense mutation at nucleotide 1796 of the BRAF gene using a newly developed assay that employs a novel primer extension method. They also tested 57 vertical growth phase cutaneous nondesmoplastic melanoma specimens. RESULTS: The 1796 T-->A mutation was detected in 23 of the 57 conventional cutaneous melanoma specimens but in none of the 12 desmoplastic melanoma specimens (40% vs. 0%; P=0.0006, Fisher exact 2-tailed test). CONCLUSIONS: The relative importance of BRAF mutational activation in melanocytic tumorigenesis clearly was not the same across the various subtypes of melanoma, even for melanomas of cutaneous origin that are associated with sun exposure. In contrast to conventional cutaneous melanomas, the desmoplastic variant frequently did not harbor an activating mutation of BRAF. Accordingly, patients with melanomas should not be collectively regarded as a uniform group as new therapeutic strategies are developed that target specific genetic alterations.     

Overexpression of Mcl-1 confers resistance to BRAFV600E inhibitors alone and in combination with MEK1/2 inhibitors in melanoma

Melanoma harboring BRAF mutations frequently develop resistance to BRAF inhibitors, limiting the impact of treatment. Here, we establish a mechanism of resistance and subsequently identified a suitable drug combination to overcome the resistance. Single treatment of BRAF mutant melanoma cell lines with vemurafenib or dabrafenib (BRAF inhibitors) alone or in combination with trametinib (MEK1/2 inhibitor) resulted in overexpression of Mcl-1. Overexpression of Mcl-1 in A375 and SK-MEL-28 by transfection completely blocked BRAF and MEK1/2 inhibitor-mediated inhibition of cell survival and apoptosis. Melanoma cells resistant to BRAF inhibitors showed massive expression of Mcl-1 as compared to respective sensitive cell lines. Silencing of Mcl-1 using siRNA completely sensitized resistant melanoma cells to growth suppression and induction of apoptosis by BRAF inhibitors. In vivo, vemurafenib resistant A375 xenografts implanted in athymic nude mice showed substantial tumor growth inhibition when treated with a combination of vemurafenib and Mcl-1 inhibitor or siRNA. Immunohistochemistry and western blot analyses demonstrated enhanced expression of Mcl-1 and activation of ERK1/2 in vemurafenib-resistant tumors whereas level of Mcl-1 or p-ERK1/2 was diminished in the tumors of mice treated with either of the combination. Biopsied tumors from the patients treated with or resistant to BRAF inhibitors revealed overexpression of Mcl-1. These results suggest that the combination of BRAF inhibitors with Mcl-1 inhibitor may have therapeutic advantage to melanoma patients with acquired resistance to BRAF inhibitors alone or in combination with MEK1/2 inhibitors.     

甲状腺癌中PTEN蛋白表达与BRAFV600E突变的相关性研究

目的：探讨PTEN蛋白在甲状腺乳头状癌（PTC）中的表达情况,并进一步分析此蛋白的表达量与BRAFV600E基因突变的相关性及临床意义,初步揭示两者在PTC发生、发展中的致癌作用。方法：采用免疫组化EnVision两步法检测70例PTC 中PTEN 蛋白的表达水平;采用 PCR法及 DNA 测序法检测上述标本中BRAFV600E突变情况,分析PTEN蛋白表达与BRAFV600E突变的相关性及其与PTC临床病理特征的关系。结果：70例PTC中PTEN阴性率为78．6％（55／70）,与癌旁组织18．6％（13／70）比较,差异有统计学意义（P＜0．05）。PTEN表达下调与性别、肿瘤淋巴结转移、临床分期有关（P＜0．05）;与BRAFV600E突变、年龄、肿瘤直径无关（P＞0．05）。70例PTC中BRAFV600E突变率为60％,BRAFV600E突变与肿瘤淋巴结转移、临床分期密切相关（P＜0．05）;与患者年龄、性别、肿瘤大小无关（P＞0．05）。结论：PTEN蛋白表达与BRAFV600E突变无相关性。两者可能是PTC生物侵袭高的独立危险因素,可能作为评估PTC预后不良的不同肿瘤标记物。两者对甲状腺癌的临床个体化治疗及判定预后有指导意义。     

Potential therapeutic strategies to overcome acquired resistance to BRAF or MEK inhibitors in BRAF mutant cancers

Recent clinical trials with selective inhibitors of the BRAF and MEK kinases have shown promising results in patients with tumors harboring BRAF V600 mutations. However, as has been observed previously with similarly successful targeted therapies, acquired resistance to these agents is an emerging problem that limits their clinical benefit. Several recent studies from our laboratory and others have investigated the causes of acquired resistance to BRAF and MEK inhibitors, and multiple resistance mechanisms have been identified. Here, we review these mechanisms and suggest that they can be broadly grouped into two main classes: ERK-dependent and ERK-independent. We also propose distinct therapeutic strategies that might be employed to overcome each class of acquired resistance..     

Response to BRAF and MEK1/2 inhibition in a young adult with BRAF V600E mutant epithelioid glioblastoma multiforme: A Case Report and Literature Review

Epithelioid glioblastoma multiforme (eGBM) is a rare and aggressive variant of glioblastoma multiforme (GBM) that predominantly affects younger patients and can be difficult to distinguish from other gliomas. Data on how patients with eGBM might be best treated are limited, although genomic analyses have shown that almost half of tumours harbour activating BRAF gene mutations. Here we present the case of a young female with BRAF V600E-mutant eGBM who had a prolonged response to targeted therapy with the BRAF and MEK1/2 inhibitors dabrafenib and trametinib. We review current knowledge about eGBM, including the emerging role for BRAF- ± MEK1/2- targeted therapy.     

Suppression of BRAF/MEK/MAP kinase pathway restores expression of iodide-metabolizing genes in thyroid cells expressing the V600E BRAF mutant.

PURPOSE: The V600E BRAF mutant plays an important role in the pathogenesis of papillary thyroid cancer (PTC) and is associated with loss of expression of thyroid iodide-metabolizing genes. This study was done to investigate the restorability of expression of these genes by suppressing the BRAF/extracellular signal-regulated kinase kinase (MEK)/mitogen-activated protein (MAP) kinase pathway in V600E BRAF-harboring thyroid cells and to explore the mechanisms involved. EXPERIMENTAL DESIGN: We used inducible expression of V600E BRAF, small interfering RNA transfection, and MEK-specific inhibitor to alter the MAP kinase pathway activities and subsequently examined the changes in expression, promoter activities, and methylation status of thyroid genes. RESULTS: MEK inhibitor U0126 or cessation of V600E BRAF expression in PCCL3 cells restored expression of thyroid genes silenced by induced expression of V600E BRAF. U0126 also restored the expression of these genes in V600E BRAF-harboring PTC-derived NPA cells. Knockdown of BRAF by specific small interfering RNA restored expression of some of these genes in NPA cells. Luciferase reporter assay using thyroid-stimulating hormone receptor gene as a model showed that the promoter activity was modulated by the MAP kinase pathway. Promoter methylation in association with DNA methyltransferase expression played a role in gene silencing by MAP kinase pathway in NPA cells. CONCLUSIONS: We showed the restorability of expression of thyroid iodide-metabolizing genes silenced by V600E BRAF, and linked this process to gene methylation in PTC cells. The results provide clinical implications that therapeutic targeting at the BRAF/MEK/MAP kinase pathway may be a good approach in restoring thyroid gene expression for effective radioiodine therapy for BRAF mutation-harboring PTC.     

p53 Reactivation by PRIMA-1Met (APR-246) sensitises V600E/KBRAF melanoma to vemurafenib

Intrinsic and acquired resistance of metastatic melanoma to ^V600E/KBRAF and/or MEK inhibitors, which is often caused by activation of the PI3K/AKT survival pathway, represents a major clinical challenge. Given that p53 is capable of antagonising PI3K/AKT activation we hypothesised that pharmacological restoration of p53 activity may increase the sensitivity of BRAF-mutant melanoma to MAPK-targeted therapy and eventually delay and/or prevent acquisition of drug resistance. To test this possibility we exposed a panel of vemurafenib-sensitive and resistant (innate and acquired) ^V600E/KBRAF melanomas to a ^V600E/KBRAF inhibitor (vemurafenib) alone or in combination with a direct p53 activator (PRIMA-1^Met/APR-246). Strikingly, PRIMA-1^Met synergised with vemurafenib to induce apoptosis and suppress proliferation of ^V600E/KBRAF melanoma cells in vitro and to inhibit tumour growth in vivo. Importantly, this drug combination decreased the viability of both vemurafenib-sensitive and resistant melanoma cells irrespectively of the TP53 status. Notably, p53 reactivation was invariably accompanied by PI3K/AKT pathway inhibition, the activity of which was found as a dominant resistance mechanism to BRAF inhibition in our lines. From all various combinatorial modalities tested, targeting the MAPK and PI3K signalling pathways through p53 reactivation or not, the PRIMA-1^Met/vemurafenib combination was the most cytotoxic. We conclude that PRIMA-1^Met through its ability to directly reactivate p53 regardless of the mechanism causing its deactivation, and thereby dampen PI3K signalling, sensitises ^V600E/KBRAF-positive melanoma to BRAF inhibitors.     

Making sense of MEK1 mutations in intrinsic and acquired BRAF inhibitor resistance

In this issue of Cancer Discovery, Shi and colleagues add further insight into the role of exon 3 MEK1 mutations in BRAF inhibitor resistance by demonstrating the presence of P124SMEK1 and I111SMEK1 mutations concurrently with V600E/KBRAF mutations at baseline in 16% of melanoma specimens. Although the presence of P124SMEK1 or I111SMEK1 mutations did not predict for resistance, and these alleles were not selected for upon BRAF inhibition, other exon 3 MEK1 mutations, such as C121S, did convey resistance, suggesting a role for defined exon 3 MEK1 mutations in acquired BRAF inhibitor resistance.     

BRAF V600E mutations in right-side colon cancer: Heterogeneity detected by liquid biopsy

IntroductionThe prognosis for metastatic colorectal cancer patients (mCRC) with the BRAF^V600E mutation is poor. BRAF^V600E mutation frequency is reportedly low among Asians; however, the frequency of the BRAF^V600E mutation in right-side colon cancer may not be low, even among Asians. In addition, spatial heterogeneity of BRAF^V600E mutations also exists, as for RAS mutations. In this prospective observational study, we evaluated BRAF^V600E mutations in cancer tissue and plasma of Japanese right-side colon cancer patients.Method215 patients with right-side colon cancer were included. BRAF^V600E mutations of cancer tissue and plasma were detected using droplet digital PCR. Blood plasma of patients with BRAF^V600E mutations in cancer tissue or plasma was drawn at intervals throughout chemotherapy, and BRAF^V600E mutations were evaluated.ResultsBRAF^V600E mutations were detected in tissue samples from 35 of 215 patients (16.3%, cecum; 22.4%, ascending colon; 17.8%, and transverse colon; 9.0%). BRAF^V600E mutations were detected in plasma of 10 of 215 (4.7%) patients. Eight of the ten patients had BRAF^V600E mutations in their primary tumours, but two (both were Stage IV) patients did not. Sensitivity of liquid biopsy to detect BRAF ^V600E mutations was 10.3% (3/29) in Stage I-III patients and 83.3% (5/6) in Stage IV patients.ConclusionBRAF^V600E mutations are observed in right-side colon cancer at high frequency, especially in the cecum. BRAF^V600E mutations can be detected in plasma and the detection rate is high in patients with advanced cancer. Spatial heterogeneity was observed using liquid biopsy.     

高分辨率熔解曲线分析技术检测BRAF基因V600E突变方法的建立及初步临床应用

目的建立高分辨率熔解曲线分析技术(high resolution melting,HRM)检测BRAF基因V600E突变的方法,并探讨其在临床检测中的应用价值。方法用所建方法检测16例甲状腺乳头状癌患者超声引导下细针穿刺抽吸活检标本,并与测序法结果进行比较分析。结果所建HRM检测方法 Ct值与Tm的CV值均较小,重复性好。HRM法BRAF基因V600E突变检测标本的结果与测序法相比较,突变检出率分别为43.75%和40.00%,敏感性为100.00%,特异性为90.00%。结论成功建立的HRM法检测BRAF基因V600E突变敏感性高,特异性强,重复性好,操作简便,节约时间,成本低,适合细针穿刺抽吸活检标本检测BRAF基因V600E突变。     

Concurrent loss of the PTEN and RB1 tumor suppressors attenuates RAF dependence in melanomas harboring (V600E)BRAF

Identifying the spectrum of genetic alterations that cooperate with critical oncogenes to promote transformation provides a foundation for understanding the diversity of clinical phenotypes observed in human cancers. Here, we performed integrated analyses to identify genomic alterations that co-occur with oncogenic BRAF in melanoma and abrogate cellular dependence upon this oncogene. We identified concurrent mutational inactivation of the PTEN and RB1 tumor suppressors as a mechanism for loss of BRAF/MEK dependence in melanomas harboring (V600E)BRAF mutations. RB1 alterations were mutually exclusive with loss of p16(INK4A), suggesting that whereas p16(INK4A) and RB1 may have overlapping roles in preventing tumor formation, tumors with loss of RB1 exhibit diminished dependence upon BRAF signaling for cell proliferation. These findings provide a genetic basis for the heterogeneity of clinical outcomes in patients treated with targeted inhibitors of the mitogen-activated protein kinase pathway. Our results also suggest a need for comprehensive screening for RB1 and PTEN inactivation in patients treated with RAF and MEK-selective inhibitors to determine whether these alterations are associated with diminished clinical benefit in patients whose cancers harbor mutant BRAF.     

BRAF V600E mutations in urine and plasma cell-free DNA from patients with Erdheim-Chester disease

Erdheim-Chester disease (ECD) is a rare histiocytosis with a high prevalence of BRAF V600E mutation (>50% of patients). Patients with BRAF-mutant ECD can respond to BRAF inhibitors. Unfortunately, the lack of adequate archival tissue often precludes BRAF testing. We hypothesized that cell-free DNA (cfDNA) from plasma or urine can offer an alternative source of biologic material for testing. We tested for BRAF V600E mutation in cfDNA from the plasma and urine of 6 ECD patients. In patients with available archival tissue, the result of BRAF mutation analysis was concordant with plasma and urine cfDNA results in all 3 patients (100% agreement, kappa 1.00). In all 6 patients, BRAF mutation analysis of plasma and urine cfDNA was concordant in 5 of 6 patients (83% agreement, kappa 0.67). Testing for BRAF V600E mutation in plasma and urine cfDNA should be further investigated as an alternative to archival tissue mutation analysis.     

Kinase domain mutations confer resistance to novel inhibitors targeting JAK2V617F in myeloproliferative neoplasms

The transforming JAK2V617F kinase is frequently associated with myeloproliferative neoplasms and thought to be instrumental for the overproduction of myeloid lineage cells. Several small molecule drugs targeting JAK2 are currently in clinical development for treatment in these diseases. We performed a high-throughput in vitro screen to identify point mutations in JAK2V617F that would be predicted to have potential clinical relevance and associated with drug resistance to the JAK2 inhibitor ruxolitinib (INCB018424). Seven libraries of mutagenized JAK2V617F cDNA were screened to specifically identify mutations in the predicted drug-binding region that would confer resistance to ruxolitinib, using a BaF3 cell-based assay. We identified five different non-synonymous point mutations that conferred drug resistance. Cells containing mutations had a 9- to 33-fold higher EC(50) for ruxolitinib compared with native JAK2V617F. Our results further indicated that these mutations also conferred cross-resistance to all JAK2 kinase inhibitors tested, including AZD1480, TG101348, lestaurtinib (CEP-701) and CYT-387. Surprisingly, introduction of the 'gatekeeper' mutation (M929I) in JAK2V617F affected only ruxolitinib sensitivity (fourfold increase in EC(50)). These results suggest that JAK2 inhibitors currently in clinical trials may be prone to resistance as a result of point mutations and caution should be exercised when administering these drugs.     

Mutant V600E BRAF increases hypoxia inducible factor-1alpha expression in melanoma

Mutations in the BRAF serine/threonine kinase gene are frequently found in cutaneous melanomas. Activation of hypoxia inducible factor-1alpha (HIF-1alpha) in response to both hypoxic stress and oncogenic signals has important implications in cancer development and progression. Here, we report that mutant BRAF(V600E) increases HIF-1alpha expression in melanoma cells. Our microarray profiling data in 35 melanoma and melanocyte cell lines showed that HIF-1alpha gene expression was significantly increased in melanomas harboring BRAF(V600E) mutation. Stable suppression of mutant BRAF(V600E) or both wild-type and mutant BRAF(V600E) by RNA interference in melanoma cells resulted in significantly decreased HIF-1alpha expression. Knockdown of mutant BRAF(V600E) induced significant reduction of cell survival and proliferation under hypoxic conditions, whereas knockdown of both wild-type and mutant BRAF(V600E) resulted in further reduction. The effects of BRAF knockdown can be rescued by reintroducing BRAF(V600E) into tumor cells. Transfection of BRAF(V600E) into melanoma cells with wild-type BRAF induced significantly more hypoxic tolerance. Knockdown of HIF-1alpha in melanoma cells resulted in decreased cell survival under hypoxic conditions. Pharmacologic inhibition of BRAF by BAY 43-9006 also resulted in decreased HIF-1alpha expression. Although HIF-1alpha translational rate was not changed, the protein was less stable in BRAF knockdown cells. In additional, von Hippel-Lindau protein expression was significantly increased in BRAF knockdown cells. Our data show for the first time that BRAF(V600E) mutation increases HIF-1alpha expression and melanoma cell survival under hypoxic conditions and suggest that effects of the oncogenic V600E BRAF mutation may be partially mediated through the HIF-1alpha pathway.     

First-line BRAF/MEK inhibitors versus anti-PD-1 monotherapy in BRAFV600-mutant advanced melanoma patients: a propensity-matched survival analysis

Background Anti-PD-1 antibodies and BRAF/MEK inhibitors are the two main groups of systemic therapy in the treatment of BRAF^V600-mutant advanced melanoma. Until now, data are inconclusive on which therapy to use as first-line treatment. The aim of this study was to use propensity score matching to compare first-line anti-PD-1 monotherapy vs. BRAF/MEK inhibitors in advanced BRAF^V600-mutant melanoma patients.Methods We selected patients diagnosed between 2014 and 2017 with advanced melanoma and a known BRAF^V600-mutation treated with first-line BRAF/MEK inhibitors or anti-PD-1 antibodies, registered in the Dutch Melanoma Treatment Registry. Patients were matched based on their propensity scores using the nearest neighbour and the optimal matching method.Results Between 2014 and 2017, a total of 330 and 254 advanced melanoma patients received BRAF/MEK inhibitors and anti-PD-1 monotherapy as first-line systemic therapy. In the matched cohort, patients receiving anti-PD-1 antibodies as a first-line treatment had a higher median and 2-year overall survival compared to patients treated with first-line BRAF/MEK inhibitors, 42.3 months (95% CI: 37.3-NE) vs. 19.8 months (95% CI: 16.7–24.3) and 65.4% (95% CI: 58.1–73.6) vs. 41.7% (95% CI: 34.2–51.0).Conclusions Our data suggest that in the matched BRAF^V600-mutant advanced melanoma patients, anti-PD-1 monotherapy is the preferred first-line treatment in patients with relatively favourable patient and tumour characteristics.Subject terms: Melanoma, Melanoma, Melanoma