Swapna Vemula

Mutations in GNA11 in Uveal Melanoma

BACKGROUND Uveal melanoma is the most common intraocular cancer. There are no effective therapies for metastatic disease. Mutations in GNAQ, the gene encoding an alpha subunit of heterotrimeric G proteins, are found in 40% of uveal melanomas. METHODS We sequenced exon 5 of GNAQ and GNA11, a paralogue of GNAQ, in 713 melanocytic neoplasms of different types (186 uveal melanomas, 139 blue nevi, 106 other nevi, and 282 other melanomas). We sequenced exon 4 of GNAQ and GNA11 in 453 of these samples and in all coding exons of GNAQ and GNA11 in 97 uveal melanomas and 45 blue nevi. RESULTS We found somatic mutations in exon 5 (affecting Q209) and in exon 4 (affecting R183) in both GNA11 and GNAQ, in a mutually exclusive pattern. Mutations affecting Q209 in GNA11 were present in 7% of blue nevi, 32% of primary uveal melanomas, and 57% of uveal melanoma metastases. In contrast, we observed Q209 mutations in GNAQ in 55% of blue nevi, 45% of uveal melanomas, and 22% of uveal melanoma metastases. Mutations affecting R183 in either GNAQ or GNA11 were less prevalent (2% of blue nevi and 6% of uveal melanomas) than the Q209 mutations. Mutations in GNA11 induced spontaneously metastasizing tumors in a mouse model and activated the mitogen-activated protein kinase pathway. CONCLUSIONS Of the uveal melanomas we analyzed, 83% had somatic mutations in GNAQ or GNA11. Constitutive activation of the pathway involving these two genes appears to be a major contributor to the development of uveal melanoma. (Funded by the National Institutes of Health and others.).

KIT as a Therapeutic Target in Metastatic Melanoma

CONTEXT Some melanomas arising from acral, mucosal, and chronically sun-damaged sites harbor activating mutations and amplification of the type III transmembrane receptor tyrosine kinase KIT. We explored the effects of KIT inhibition using imatinib mesylate in this molecular subset of disease. OBJECTIVE To assess clinical effects of imatinib mesylate in patients with melanoma harboring KIT alterations. DESIGN, SETTING, AND PATIENTS A single-group, open-label, phase 2 trial at 1 community and 5 academic oncology centers in the United States of 295 patients with melanoma screened for the presence of KIT mutations and amplification between April 23, 2007, and April 16, 2010. A total of 51 cases with such alterations were identified and 28 of these patients were treated who had advanced unresectable melanoma arising from acral, mucosal, and chronically sun-damaged sites. INTERVENTION Imatinib mesylate, 400 mg orally twice daily. MAIN OUTCOME MEASURES Radiographic response, with secondary end points including time to progression, overall survival, and correlation of molecular alterations and clinical response. RESULTS Two complete responses lasting 94 (ongoing) and 95 weeks, 2 durable partial responses lasting 53 and 89 (ongoing) weeks, and 2 transient partial responses lasting 12 and 18 weeks among the 25 evaluable patients were observed. The overall durable response rate was 16% (95% confidence interval [CI], 2%-30%), with a median time to progression of 12 weeks (interquartile range [IQR], 6-18 weeks; 95% CI, 11-18 weeks), and a median overall survival of 46.3 weeks (IQR, 28 weeks-not achieved; 95% CI, 28 weeks-not achieved). Response rate was better in cases with mutations affecting recurrent hotspots or with a mutant to wild-type allelic ratio of more than 1 (40% vs 0%, P = .05), indicating positive selection for the mutated allele. CONCLUSIONS Among patients with advanced melanoma harboring KIT alterations, treatment with imatinib mesylate results in significant clinical responses in a subset of patients. Responses may be limited to tumors harboring KIT alterations of proven functional relevance. Trial Registration clinicaltrials.gov Identifier: NCT00470470.

Loss-of-function mutations in Notch receptors in cutaneous and lung squamous cell carcinoma

Squamous cell carcinomas (SCCs) are one of the most frequent forms of human malignancy, but, other than TP53 mutations, few causative somatic aberrations have been identified. We identified NOTCH1 or NOTCH2 mutations in ∼75% of cutaneous SCCs and in a lesser fraction of lung SCCs, defining a spectrum for the most prevalent tumor suppressor specific to these epithelial malignancies. Notch receptors normally transduce signals in response to ligands on neighboring cells, regulating metazoan lineage selection and developmental patterning. Our findings therefore illustrate a central role for disruption of microenvironmental communication in cancer progression. NOTCH aberrations include frameshift and nonsense mutations leading to receptor truncations as well as point substitutions in key functional domains that abrogate signaling in cell-based assays. Oncogenic gain-of-function mutations in NOTCH1 commonly occur in human T-cell lymphoblastic leukemia/lymphoma and B-cell chronic lymphocytic leukemia. The bifunctional role of Notch in human cancer thus emphasizes the context dependency of signaling outcomes and suggests that targeted inhibition of the Notch pathway may induce squamous epithelial malignancies.

Frequent Mutations in the MITF Pathway in Melanoma

Microphthalmia‐associated transcription factor (MITF) is involved in melanocyte cell development, pigmentation and neoplasia. To determine whether MITF is somatically mutated in melanoma, we compared the sequence of MITF from primary and metastatic lesions to patient‐matched normal DNA. In the 50 metastatic melanoma tumor lines analysed, we discovered four samples that had genomic amplifications of MITF and four that had MITF mutations in the regions encoding the transactivation, DNA binding or basic, helix‐loop‐helix domains. Sequence analysis for SOX10, a transcription factor, which both acts upstream of MITF and synergizes with MITF, identified an additional three samples with frameshift or nonsense mutations. Microphthalmia‐associated transcription factor and SOX10 were found to be mutated in a mutually exclusive fashion, possibly suggesting disruption in a common genetic pathway. Taken together we found that over 20% of the metastatic melanoma cases had alterations in the MITF pathway. We show that the MITF pathway is also altered in primary melanomas: 2/26 demonstrated mutations in MITF and 6/55 demonstrated mutations in SOX10. Our findings suggest that altered MITF function during melanomagenesis can be achieved by MITF amplification, MITF single base substitutions or by mutation of its regulator SOX10.

Recurrent BRAF kinase fusions in melanocytic tumors offer an opportunity for targeted therapy.

BRAF is the most prevalent oncogene and an important therapeutic target in melanoma. In some cancers, BRAF is activated by rearrangements that fuse its kinase domain to 5′ partner genes. We examined 848 comparative genomic hybridization profiles of melanocytic tumors and found copy number transitions within BRAF in 10 tumors, of which six could be further characterized by sequencing. In all, the BRAF kinase domain was fused in‐frame to six N‐terminal partners. No other mutations were identified in melanoma oncogenes. One of the seven melanoma cell lines without known oncogenic mutations harbored a similar BRAF fusion, which constitutively activated the MAP kinase pathway. Sorafenib, but not vemurafenib, could block MAP kinase pathway activation and proliferation of the cell line at clinically relevant concentrations, whereas BRAFV600E mutant melanoma cell lines were significantly more sensitive to vemurafenib. The patient from whom the cell line was derived showed a durable clinical response to sorafenib.

Activating MET kinase rearrangements in melanoma and Spitz tumours

Oncogenic gene fusions have been identified in many cancers and many serve as biomarkers or targets for therapy. Here we identify six different melanocytic tumors with genomic rearrangements of MET fusing the kinase domain of MET in-frame to six different N-terminal partners. These tumors lack activating mutations in other established melanoma oncogenes. We functionally characterize two of the identified fusion proteins (TRIM4-MET and ZKSCAN1-MET) and find that they constitutively activate the mitogen-activated protein kinase (MAPK), phosphoinositol-3 kinase (PI3K), and phospholipase C gamma 1 (PLCγ1) pathways. The MET inhibitors cabozantinib (FDA-approved for progressive medullary thyroid cancer) and PF-04217903 block their activity at nanomolar concentrations. MET fusion kinases thus provide a potential therapeutic target for a rare subset of melanoma for which currently no targeted therapeutic options currently exist.

Molecular analysis of a case of nevus of ota showing progressive evolution to melanoma with intermediate stages resembling cellular blue nevus.

Nevus of Ota is a variant of congenital nevus, which is morphologically paucicellular and resembles a common blue nevus. Although nevus of Ota is a risk factor for uveal melanoma in white people, the development of cutaneous melanoma within nevus of Ota is a very rare occurrence with only a few reported cases. We present a case of a long-standing nevus of Ota, with radiologic imaging demonstrating a large retro-orbital mass and a biopsy showing melanoma. The histopathology of the eye exenteration specimen illustrated various stages of melanocytic progression including areas resembling a nevus of Ota, blue nevus, cellular blue nevus, and melanoma. There was heterogeneity in the overtly malignant sections with some areas displaying expansile nodules of blander appearing spindled cells, whereas other areas were composed of epithelioid cells with higher mitotic counts and zones of necrosis. The extensive lesion also infiltrated the soft tissue and bone. We performed gene mutation analysis for GNAQ, BRAF, NRAS, and KIT and fluorescence in situ hybridization (FISH) targeting commonly altered chromosomal loci in melanoma and comparative genomic hybridization (CGH). Copy number changes typical of melanoma were identified by both FISH and CGH in the morphologically malignant areas illustrating the relationship of tumor progression and the progressive acquisition of genetic aberrations.

Ambiguous Melanocytic Tumors with loss of 3p21

Germline loss-of-function mutations in BAP1 are associated with the development of cutaneous melanocytic tumors with some histopathologic characteristics seen in Spitz nevi. Similar melanocytic tumors occurring in a sporadic setting have been demonstrated to have biallelic loss of BAP1. In some of these sporadic tumors, loss of BAP1 occurs through mutation of 1 allele and genomic loss of the other. We screened our database of comparative genomic hybridization profiles of ambiguous melanocytic tumors to identify cases with a single genomic event involving loss of the BAP1 locus. The prevalence of tumors with a single genomic event involving loss of BAP1 was 6.7% in our study population. We further characterized the BAP1 status in 17 of these tumors with available additional material, confirming loss of BAP1 in all cases. We describe BAP1 loss in a blue nevus–like melanoma and further expand the histopathologic spectrum of spitzoid melanocytic neoplasms with BAP1 loss.

Transcription restores DNA repair to heterochromatin, determining regional mutation rates in cancer genomes

Somatic mutations in cancer are more frequent in heterochromatic and late-replicating regions of the genome. We report that regional disparities in mutation density are virtually abolished within transcriptionally silent genomic regions of cutaneous squamous cell carcinomas (cSCCs) arising in an XPC(-/-) background. XPC(-/-) cells lack global genome nucleotide excision repair (GG-NER), thus establishing differential access of DNA repair machinery within chromatin-rich regions of the genome as the primary cause for the regional disparity. Strikingly, we find that increasing levels of transcription reduce mutation prevalence on both strands of gene bodies embedded within H3K9me3-dense regions, and only to those levels observed in H3K9me3-sparse regions, also in an XPC-dependent manner. Therefore, transcription appears to reduce mutation prevalence specifically by relieving the constraints imposed by chromatin structure on DNA repair. We model this relationship among transcription, chromatin state, and DNA repair, revealing a new, personalized determinant of cancer risk.

Clinical, histopathologic, and genomic features of Spitz tumors with ALK fusions.

Activating kinase fusions have recently been described as early oncogenic events that are mutually exclusive with HRAS and BRAF mutations in Spitz tumors. Here, we report a series of 32 Spitz tumors with ALK fusions (6 Spitz nevi, 22 atypical Spitz tumors, and 4 spitzoid melanomas) in patients ranging from 5 months to 64 years (median=12 y) of age. The tumors typically presented as exophytic papules on the extremities and were occasionally darkly pigmented. In addition to ALK fusions previously described in other tumor types (NPM1-ALK, TPR-ALK), we identified 2 novel ALK fusions (CLIP1-ALK and GTF3C2-ALK) in our cohort of Spitz tumors. Array comparative genomic hybridization of 19 of these tumors demonstrated a high frequency of chromosome 2 aberrations (where ALK resides, 63%) and chromosome 1p loss in 37% of the cases. Spitz tumors with ALK fusions demonstrated unique histopathologic features. Clefts and small vesicle-like spaces were arrayed between plump spindled melanocytes with fibrillar cytoplasm and enlarged nuclei. These melanocytes were typically arrayed in elongated and fusiform nests with radial orientation. The tumors often had extension into the dermis or subcutis with a wedge-shaped or bulbous lower border (45% and 17%, respectively). An infiltrative growth pattern was often present at the periphery of the tumor and was highlighted by ALK immunohistochemistry. In conclusion, Spitz tumors with ALK rearrangement show distinct histopathologic features that should aid in improving classification of these diagnostically challenging tumors.