NRAS Q61R and BRAF G466A mutations in atypical melanocytic lesions newly arising in advanced melanoma patients treated with vemurafenib.

BACKGROUND: BRAF inhibition has improved overall survival in patients with BRAF mutant melanoma, but this is associated with a range of known and predictable cutaneous side effects, including squamous cell carcinomas associated with RAS mutations. METHODS: We identified three severely dysplastic nevi, one atypical intraepidermal melanocytic proliferation, and four melanoma in situ lesions, newly arising in four patients undergoing treatment with vemurafenib. To characterize mutations in these atypical melanocytic lesions, we used a custom iPlex panel detecting 74 mutations in 13 genes known to play a role in melanoma pathogenesis. RESULTS: We identified an NRAS mutation at codon 61 (Q61R) and a rare BRAF exon 11 mutation (G466A) in atypical melanocytic lesions that arose in patients treated with vemurafenib. CONCLUSION: There appears to be development or accelerated growth of atypical melanocytic lesions in the setting of BRAF inhibition. Our results underscore the need for careful surveillance for melanocytic lesions in patients on BRAF inhibitor therapy and shed light on potential mechanisms for melanoma pathogenesis in the context of BRAF pathway blockade. Further studies are warranted to show a causal relationship.

Interplay between BRCA1 and RHAMM regulates epithelial apicobasal polarization and may influence risk of breast cancer.

Differentiated mammary epithelium shows apicobasal polarity, and loss of tissue organization is an early hallmark of breast carcinogenesis. In BRCA1 mutation carriers, accumulation of stem and progenitor cells in normal breast tissue and increased risk of developing tumors of basal-like type suggest that BRCA1 regulates stem/progenitor cell proliferation and differentiation. However, the function of BRCA1 in this process and its link to carcinogenesis remain unknown. Here we depict a molecular mechanism involving BRCA1 and RHAMM that regulates apicobasal polarity and, when perturbed, may increase risk of breast cancer. Starting from complementary genetic analyses across families and populations, we identified common genetic variation at the low-penetrance susceptibility HMMR locus (encoding for RHAMM) that modifies breast cancer risk among BRCA1, but probably not BRCA2, mutation carriers: n = 7,584, weighted hazard ratio ((w)HR) = 1.09 (95% CI 1.02-1.16), p(trend) = 0.017; and n = 3,965, (w)HR = 1.04 (95% CI 0.94-1.16), p(trend) = 0.43; respectively. Subsequently, studies of MCF10A apicobasal polarization revealed a central role for BRCA1 and RHAMM, together with AURKA and TPX2, in essential reorganization of microtubules. Mechanistically, reorganization is facilitated by BRCA1 and impaired by AURKA, which is regulated by negative feedback involving RHAMM and TPX2. Taken together, our data provide fundamental insight into apicobasal polarization through BRCA1 function, which may explain the expanded cell subsets and characteristic tumor type accompanying BRCA1 mutation, while also linking this process to sporadic breast cancer through perturbation of HMMR/RHAMM.

A second independent locus within DMRT1 is associated with testicular germ cell tumor susceptibility.

Susceptibility to testicular germ cell tumors (TGCT) has a significant heritable component, and genome-wide association studies (GWASs) have identified association with variants in several genes, including KITLG, SPRY4, BAK1, TERT, DMRT1 and ATF7IP. In our GWAS, we genotyped 349 TGCT cases and 919 controls and replicated top hits in an independent set of 439 cases and 960 controls in an attempt to find novel TGCT susceptibility loci. We identified a second marker (rs7040024) in the doublesex and mab-3-related transcription factor 1 (DMRT1) gene that is independent of the previously described risk allele (rs755383) at this locus. In combined analysis that mutually conditions on both DMRT1 single nucleotide polymorphism markers, TGCT cases had elevated odds of carriage of the rs7040024 major A allele [per-allele odds ratio (OR) = 1.48, 95% confidence interval (CI) 1.23, 1.78; P = 2.52 × 10(-5)] compared with controls, while the association with rs755383 persisted (per allele OR = 1.26, 95% CI 1.08, 1.47, P = 0.0036). In similar analyses, the association of rs7040024 among men with seminomatous tumors did not differ from that among men with non-seminomatous tumors. In combination with KITLG, the strongest TGCT susceptibility locus found to date, men with TGCT had greatly elevated odds (OR = 14.1, 95% CI 5.12, 38.6; P = 2.98 × 10(-7)) of being double homozygotes for the risk (major) alleles at DMRT (rs7040024) and KITLG (rs4474514) when compared with men without TGCT. Our findings continue to corroborate that genes influencing male germ cell development and differentiation have emerged as the major players in inherited TGCT susceptibility.

Application of a BRAF pyrosequencing assay for mutation detection and copy number analysis in malignant melanoma.

Mutations in the BRAF gene are found in the majority of cutaneous malignant melanomas and subsets of other tumors. These mutations lead to constitutive activation of BRAF with increased downstream ERK (extracellular signal-regulated kinase) signaling; therefore, the development of RAF kinase inhibitors for targeted therapy is being actively pursued. A methodology that allows sensitive, cost-effective, high-throughput analysis of BRAF mutations will be needed to triage patients for specific molecular-based therapies. Pyrosequencing is a high-throughput, sequencing-by-synthesis method that is particularly useful for analysis of single nucleotide polymorphisms or hotspot mutations. Mutational analysis of BRAF is highly amenable to pyrosequencing because the majority of mutations in this gene localize to codons 600 and 601 and consist of single or dinucleotide substitutions. In this study, DNAs from a panel of melanocyte cell lines, melanoma cell lines, and melanoma tumors were used to validate a pyrosequencing assay to detect BRAF mutations. The assay demonstrates high accuracy and precision for detecting common and variant exon 15 BRAF mutations. Further, comparison of pyrosequencing data with 100K single nucleotide polymorphism microarray data allows characterization of BRAF amplification events that may accompany BRAF mutation. Pyro-sequencing serves as an excellent platform for BRAF genotyping of tumors from patients entering clinical trial.

Correlation of somatic mutations and clinical outcome in melanoma patients treated with Carboplatin, Paclitaxel, and sorafenib.

PURPOSE: Sorafenib is an inhibitor of VEGF receptor (VEGFR), platelet-derived growth factor receptor (PDGFR), and RAF kinases, amongst others. We assessed the association of somatic mutations with clinicopathologic features and clinical outcomes in patients with metastatic melanoma treated on E2603, comparing treatment with carboplatin, paclitaxel ± sorafenib (CP vs. CPS) EXPERIMENTAL DESIGN: Pretreatment tumor samples from 179 unique individuals enrolled on E2603 were analyzed. Genotyping was performed using a custom iPlex panel interrogating 74 mutations in 13 genes. Statistical analysis was performed using Fisher exact test, logistic regression, and Cox proportional hazards models. Progression-free survival (PFS) and overall survival were estimated using Kaplan-Meier methods. RESULTS: BRAF and NRAS mutations were found at frequencies consistent with other metastatic melanoma cohorts. BRAF-mutant melanoma was associated with worse performance status, increased number of disease sites, and younger age at diagnosis. NRAS-mutant melanoma was associated with better performance status, fewer sites of disease, and female gender. BRAF and NRAS mutations were not significantly predictive of response or survival when treated with CPS versus CP. However, patients with NRAS-mutant melanoma trended toward a worse response and PFS on CP than those with BRAF-mutant or WT/WT melanoma, an association that was reversed for this group on the CPS arm. CONCLUSIONS: This study of somatic mutations in melanoma is the last prospectively collected phase III clinical trial population before the era of BRAF-targeted therapy. A trend toward improved clinical response in patients with NRAS-mutant melanoma treated with CPS was observed, possibly due to the effect of sorafenib on CRAF.

The FBXO4 tumor suppressor functions as a barrier to BRAFV600E-dependent metastatic melanoma.

Cyclin D1-cyclin-dependent kinase 4/6 (CDK4/6) dysregulation is a major contributor to melanomagenesis. Clinical evidence has revealed that p16(INK4A), an allosteric inhibitor of CDK4/6, is inactivated in over half of human melanomas, and numerous animal models have demonstrated that p16(INK4A) deletion promotes melanoma. FBXO4, a specificity factor for the E3 ligase that directs timely cyclin D1 proteolysis, has not been studied in melanoma. We demonstrate that Fbxo4 deficiency induces Braf-driven melanoma and that this phenotype depends on cyclin D1 accumulation in mice, underscoring the importance of this ubiquitin ligase in tumor suppression. Furthermore, we have identified a substrate-binding mutation, FBXO4 I377M, that selectively disrupts cyclin D1 degradation while preserving proteolysis of the other known FBXO4 substrate, TRF1. The I377M mutation and Fbxo4 deficiency result in nuclear accumulation of cyclin D1, a key transforming neoplastic event. Collectively, these data provide evidence that FBXO4 dysfunction, as a mechanism for cyclin D1 overexpression, is a contributor to human malignancy.

Tumor genetic analyses of patients with metastatic melanoma treated with the BRAF inhibitor dabrafenib (GSK2118436).

PURPOSE: Dabrafenib is a selective inhibitor of V600-mutant BRAF kinase, which recently showed improved progression-free survival (PFS) as compared with dacarbazine, in metastatic melanoma patients. This study examined potential genetic markers associated with response and PFS in the phase I study of dabrafenib. EXPERIMENTAL DESIGN: Baseline (pretreatment or archival) melanoma samples were evaluated in 41 patients using a custom genotyping melanoma-specific assay, sequencing of PTEN, and copy number analysis using multiplex ligation amplification and array-based comparative genomic hybridization. Nine patients had on-treatment and/or progression samples available. RESULTS: All baseline patient samples had BRAF(V600E/K) confirmed. Baseline PTEN loss/mutation was not associated with best overall response to dabrafenib, but it showed a trend for shorter median PFS [18.3 (95% confidence interval, CI, 9.1-24.3) vs. 32.1 weeks (95% CI, 24.1-33), P=0.059]. Higher copy number of CCND1 (P=0.009) and lower copy number of CDKN2A (P=0.012) at baseline were significantly associated with decreased PFS. Although no melanomas had high-level amplification of BRAF, the two patients with progressive disease as their best response had BRAF copy gain in their tumors. CONCLUSIONS: Copy number changes in CDKN2A, CCND1, and mutation/copy number changes in PTEN correlated with the duration of PFS in patients treated with dabrafenib. The results suggest that these markers should be considered in the design and interpretation of future trials with selective BRAF inhibitors in advanced melanoma patients.

Acquired resistance to BRAF inhibitors mediated by a RAF kinase switch in melanoma can be overcome by cotargeting MEK and IGF-1R/PI3K.

BRAF is an attractive target for melanoma drug development. However, resistance to BRAF inhibitors is a significant clinical challenge. We describe a model of resistance to BRAF inhibitors developed by chronic treatment of BRAF(V)6°°(E) melanoma cells with the BRAF inhibitor SB-590885; these cells are cross-resistant to other BRAF-selective inhibitors. Resistance involves flexible switching among the three RAF isoforms, underscoring the ability of melanoma cells to adapt to pharmacological challenges. IGF-1R/PI3K signaling was enhanced in resistant melanomas, and combined treatment with IGF-1R/PI3K and MEK inhibitors induced death of BRAF inhibitor-resistant cells. Increased IGF-1R and pAKT levels in a post-relapse human tumor sample are consistent with a role for IGF-1R/PI3K-dependent survival in the development of resistance to BRAF inhibitors.

Phase II Trial of Temozolomide and Sorafenib in Advanced Melanoma Patients with or without Brain Metastases.

PURPOSE: The combination of the oral alkylating agent temozolomide and the oral multikinase inhibitor sorafenib was evaluated in advanced melanoma patients. EXPERIMENTAL DESIGN: Patients with metastatic melanoma (n = 167) were treated on four arms. All patients received sorafenib at 400 mg p.o. twice daily without interruption. Patients without brain metastases or prior temozolomide were randomized between arm A: extended dosing of temozolomide (75 mg/m(2) temozolomide daily for 6 of every 8 weeks) and arm B: standard dosing (150 mg/m(2) temozolomide daily for 5 of every 28 days). Patients previously treated with temozolomide were enrolled on arm C: extended dosing of temozolomide. Patients with brain metastases and no prior temozolomide were assigned to arm D: standard dosing. The primary end point was 6-month progression-free survival (PFS) rate. Secondary end points included response rate, toxicity rates, and the rates of BRAF or NRAS mutations. RESULTS: The 6-month PFS rate for arms A, B, C, and D were 50%, 40%, 11%, and 23%. The median PFS for patients on arm A, B, C, and D was 5.9, 4.2, 2.2, and 3.5 months, respectively. No significant differences were observed between arms A and B in 6-month PFS rate, median PFS, or response rates. Treatment was well tolerated in all arms. No significant differences in toxicity were observed between arms A and B except for more grade 3 to 4 lymphopenia in arm A. CONCLUSION: Temozolomide plus sorafenib was well tolerated and showed activity in melanoma patients without prior history of temozolomide. The activity of this combination regimen warrants further investigation. (Clin Cancer Res 2009;15(24):7711-8).

Common variation in KITLG and at 5q31.3 predisposes to testicular germ cell cancer.

Testicular germ cell tumors (TGCT) have been expected to have a strong underlying genetic component. We conducted a genome-wide scan among 277 TGCT cases and 919 controls and found that seven markers at 12p22 within KITLG (c-KIT ligand) reached genome-wide significance (P < 5.0 x 10(-8) in discovery). In independent replication, TGCT risk was increased threefold per copy of the major allele at rs3782179 and rs4474514 (OR = 3.08, 95% CI = 2.29-4.13; OR = 3.07, 95% CI = 2.29-4.13, respectively). We found associations with rs4324715 and rs6897876 at 5q31.3 near SPRY4 (sprouty 4; P < 5.0 x 10(-6) in discovery). In independent replication, risk of TGCT was increased nearly 40% per copy of the major allele (OR = 1.37, 95% CI = 1.14-1.64; OR = 1.39, 95% CI = 1.16-1.66, respectively). All of the genotypes were associated with both seminoma and nonseminoma TGCT subtypes. These results demonstrate that common genetic variants affect TGCT risk and implicate KITLG and SPRY4 as genes involved in TGCT susceptibility.

HIF-alpha effects on c-Myc distinguish two subtypes of sporadic VHL-deficient clear cell renal carcinoma.

von Hippel-Lindau (VHL) tumor suppressor loss results in hypoxia-inducible factor alpha (HIF-alpha) stabilization and occurs in 70% of sporadic clear cell renal carcinomas (ccRCCs). To determine whether opposing influences of HIF-1alpha and HIF-2alpha on c-Myc activity regulate human ccRCC progression, we analyzed VHL genotype and HIF-alpha expression in 160 primary tumors, which segregated into three groups with distinct molecular characteristics. Interestingly, ccRCCs with intact VHL, as well as pVHL-deficient HIF-1alpha/HIF-2alpha-expressing ccRCCs, exhibited enhanced Akt/mTOR and ERK/MAPK signaling. In contrast, pVHL-deficient ccRCCs expressing only HIF-2alpha displayed elevated c-Myc activity, resulting in enhanced proliferation and resistance to replication stress. These reproducible distinctions in ccRCC behavior delineate HIF-alpha effects on c-Myc in vivo and suggest molecular criteria for selecting targeted therapies.

The Y deletion gr/gr and susceptibility to testicular germ cell tumor.

Testicular germ cell tumor (TGCT) is the most common cancer in young men. Despite a considerable familial component to TGCT risk, no genetic change that confers increased risk has been substantiated to date. The human Y chromosome carries a number of genes specifically involved in male germ cell development, and deletion of the AZFc region at Yq11 is the most common known genetic cause of infertility. Recently, a 1.6-Mb deletion of the Y chromosome that removes part of the AZFc region--known as the "gr/gr" deletion--has been associated with infertility. In epidemiological studies, male infertility has shown an association with TGCT that is out of proportion with what can be explained by tumor effects. Thus, we hypothesized that the gr/gr deletion may be associated with TGCT. Using logistic modeling, we analyzed this deletion in a large series of TGCT cases with and without a family history of TGCT. The gr/gr deletion was present in 3.0% (13/431) of TGCT cases with a family history, 2% (28/1,376) of TGCT cases without a family history, and 1.3% (33/2,599) of unaffected males. Presence of the gr/gr deletion was associated with a twofold increased risk of TGCT (adjusted odds ratio [aOR] 2.1; 95% confidence interval [CI] 1.3-3.6; P = .005) and a threefold increased risk of TGCT among patients with a positive family history (aOR 3.2; 95% CI 1.5-6.7; P = .0027). The gr/gr deletion was more strongly associated with seminoma (aOR 3.0; 95% CI 1.6-5.4; P = .0004) than with nonseminoma TGCT (aOR 1.5; 95% CI 0.72-3.0; P = .29). These data indicate that the Y microdeletion gr/gr is a rare, low-penetrance allele that confers susceptibility to TGCT.