Tobias Schimming

TERT Promoter Mutation Status as an Independent Prognostic Factor in Cutaneous Melanoma

BACKGROUND Recently, TERT promoter mutations were identified at high frequencies in cutaneous melanoma tumor samples and cell lines. The mutations were found to have a UV-signature and to lead to increased TERT gene expression. We analyzed a large cohort of melanoma patients for the presence and distribution of TERT promoter mutations and their association with clinico-pathological characteristics. METHODS 410 melanoma tumor samples were analyzed by Sanger sequencing for the presence of TERT promoter mutations. An analysis of associations between mutation status and various clinical and pathologic variables was performed. RESULTS TERT promoter mutations were identified in 154 (43%) of 362 successfully sequenced melanomas. Mutation frequencies varied between melanoma subtype, being most frequent in melanomas arising in nonacral skin (48%) and melanomas with occult primary (50%), and less frequent in mucosal (23%), and acral (19%) melanomas. Mutations carried a UV signature (C>T or CC>TT). The presence of TERT promoter mutations was associated with factors such as BRAF or NRAS mutation (P < .001), histologic type (P = .002), and Breslow thickness (P < .001). TERT promoter mutation was independently associated with poorer overall survival in patients with nonacral cutaneous melanomas (median survival 80 months vs 291 months for wild-type; hazard ratio corrected for other covariates 2.47; 95% confidence interval [CI] = 1.29 to 4.74; P = .006). CONCLUSIONS UV-induced TERT promoter mutations are one of the most frequent genetic alterations in melanoma, with frequencies varying depending on melanoma subtype. In nonacral cutaneous melanomas, presence of TERT promoter mutations is independently associated with poor prognosis.

Conjunctival Melanomas Harbor BRAF and NRAS Mutations and Copy Number Changes Similar to Cutaneous and Mucosal Melanomas

Purpose: Conjunctival melanoma is a rare but potentially deadly tumor of the eye. Despite effective local therapies, recurrence and metastasis remain frequent. Once the tumor has metastasized, treatment options are limited and the prognosis is poor. To date, little is known of the genetic alterations in conjunctival melanomas. Experimental Design: We conducted genetic analysis of 78 conjunctival melanomas, to our knowledge the largest cohort reported to date. An oncogene hotspot array was run on 38 samples, screening for a panel of known cancer-relevant mutations. Thirty tumors were analyzed for genome-wide copy number alterations (CNA) using array-based comparative genomic hybridization. Sanger sequencing of selected target mutations was conducted in all samples. Results: BRAF mutations were identified in 23 of 78 (29%) tumors. NRAS mutations, previously not recognized as relevant in conjunctival melanoma, were detected in 14 of 78 (18%) tumors. We found CNAs affecting various chromosomes distributed across the genome in a pattern reminiscent of cutaneous and mucosal melanoma but differing markedly from uveal melanoma. Conclusions: The presence of NRAS or BRAF mutations in a mutually exclusive pattern in roughly half (47%) of conjunctival melanomas and the pattern of CNAs argue for conjunctival melanoma being closely related to cutaneous and mucosal melanoma but entirely distinct from uveal melanoma. Patients with metastatic conjunctival melanoma should be considered for therapeutic modalities available for metastatic cutaneous and mucosal melanoma including clinical trials of novel agents. Clin Cancer Res; 19(12); 3143–52. ©2013 AACR.

pHH3 immunostaining improves interobserver agreement of mitotic index in thin melanomas.

Abstract: According to the seventh edition of the American Joint Committee on Cancer guidelines, the TNM staging category in thin cutaneous melanomas depends on the mitotic rate (MR). In this study, we analyze the interobserver agreement of the MR in a series of 92 thin cutaneous melanomas. Serial sections of the tumors were either stained with hematoxylin and eosin or immunohistochemically stained with pHH3, an antibody for phosphohistone H3, and analyzed by 4 observers. Determination of MR with pHH3 immunostaining resulted in higher sensitivity in counting mitosis for all observers. Moreover, interobserver agreement was higher with pHH3. Immunostaining with pHH3 is a sensitive method to detect mitosis in thin cutaneous melanomas, with good reproducibility of MR between independent observers. Further studies are needed to find out if higher sensitivity in the detection of mitosis by pHH3 immunostaining has additional prognostic relevance.

TERT Promoter Mutations Are Frequent in Cutaneous Basal Cell Carcinoma and Squamous Cell Carcinoma

Activating mutations in the TERT promoter were recently identified in up to 71% of cutaneous melanoma. Subsequent studies found TERT promoter mutations in a wide array of other major human cancers. TERT promoter mutations lead to increased expression of telomerase, which maintains telomere length and genomic stability, thereby allowing cancer cells to continuously divide, avoiding senescence or apoptosis. TERT promoter mutations in cutaneous melanoma often show UV-signatures. Non-melanoma skin cancer, including basal cell carcinoma and squamous cell carcinoma, are very frequent malignancies in individuals of European descent. We investigated the presence of TERT promoter mutations in 32 basal cell carcinomas and 34 cutaneous squamous cell carcinomas using conventional Sanger sequencing. TERT promoter mutations were identified in 18 (56%) basal cell carcinomas and in 17 (50%) cutaneous squamous cell carcinomas. The recurrent mutations identified in our cohort were identical to those previously described in cutaneous melanoma, and showed a UV-signature (C>T or CC>TT) in line with a causative role for UV exposure in these common cutaneous malignancies. Our study shows that TERT promoter mutations with UV-signatures are frequent in non-melanoma skin cancer, being present in around 50% of basal and squamous cell carcinomas and suggests that increased expression of telomerase plays an important role in the pathogenesis of these tumors.

Targeted massively parallel sequencing of angiosarcomas reveals frequent activation of the mitogen activated protein kinase pathway

Angiosarcomas are rare malignant mesenchymal tumors of endothelial differentiation. The clinical behavior is usually aggressive and the prognosis for patients with advanced disease is poor with no effective therapies. The genetic bases of these tumors have been partially revealed in recent studies reporting genetic alterations such as amplifications of MYC (primarily in radiation-associated angiosarcomas), inactivating mutations in PTPRB and R707Q hotspot mutations of PLCG1. Here, we performed a comprehensive genomic analysis of 34 angiosarcomas using a clinically-approved, hybridization-based targeted next-generation sequencing assay for 341 well-established oncogenes and tumor suppressor genes. Over half of the angiosarcomas (n = 18, 53%) harbored genetic alterations affecting the MAPK pathway, involving mutations in KRAS, HRAS, NRAS, BRAF, MAPK1 and NF1, or amplifications in MAPK1/CRKL, CRAF or BRAF. The most frequently detected genetic aberrations were mutations in TP53 in 12 tumors (35%) and losses of CDKN2A in 9 tumors (26%). MYC amplifications were generally mutually exclusive of TP53 alterations and CDKN2A loss and were identified in 8 tumors (24%), most of which (n = 7, 88%) arose post-irradiation. Previously reported mutations in PTPRB (n = 10, 29%) and one (3%) PLCG1 R707Q mutation were also identified. Our results demonstrate that angiosarcomas are a genetically heterogeneous group of tumors, harboring a wide range of genetic alterations. The high frequency of genetic events affecting the MAPK pathway suggests that targeted therapies inhibiting MAPK signaling may be promising therapeutic avenues in patients with advanced angiosarcomas.

Melanoma diagnosed in lesions previously treated by laser therapy.

Laser therapy has become a routine procedure in dermatological practice and is frequently also used for pigmented lesions. Few reports exist of melanomas diagnosed in lesions previously treated by laser therapy. Between 2007 and 2014, we identified 11 patients who presented to our department with a melanoma diagnosed in a region previously treated by laser therapy. The course of events until the diagnosis of melanoma was assessed as well as patient outcome including treatment for disease progression. No histological assessment had been performed prior to laser therapy in nine of 11 (82%) cases. Benign melanocytic lesions had been diagnosed by biopsy prior to laser therapy in the other two cases. Time from laser therapy to diagnosis of melanoma ranged from less than 1 to 10 years. Stage of disease at diagnosis varied from stage IA to IIIC. Four patients progressed to stage IV disease, of whom at least one died of melanoma. We conclude that laser treatment of pigmented lesions can complicate the diagnosis of melanoma and lead to diagnosis delay with potentially fatal consequences. Considering this risk, we believe laser therapy for pigmented lesions should either be avoided entirely or at a minimum performed only after prior histological assessment.

SF3B1 and BAP1 mutations in blue nevus-like melanoma

Blue nevi are melanocytic tumors originating in the cutaneous dermis. Malignant tumors may arise in association with or resembling blue nevi, so called ‘blue nevus-like melanoma’, which can metastasize and result in patient death. Identifying which tumors will behave in a clinically aggressive manner can be challenging. Identifying genetic alterations in such tumors may assist in their diagnosis and prognostication. Blue nevi are known to be genetically related to uveal melanomas (eg, both harboring GNAQ and GNA11 mutations). In this study, we analyzed a large cohort (n=301) of various morphologic variants of blue nevi and related tumors including tumors diagnosed as atypical blue nevi (n=21), and blue nevus-like melanoma (n=12), screening for all gene mutations known to occur in uveal melanoma. Similar to published reports, we found the majority of blue nevi harbored activating mutations in GNAQ (53%) or GNA11 (15%). In addition, rare CYSLTR2 (1%) and PLCB4 (1%) mutations were identified. EIF1AX, SF3B1, and BAP1 mutations were also detected, with BAP1 and SF3B1 R625 mutations being present only in clearly malignant tumors (17% (n=2) and 25% (n=3) of blue nevus-like melanoma, respectively). In sequencing data from a larger cohort of cutaneous melanomas, this genetic profile was also identified in tumors not originally diagnosed as blue nevus-like melanoma. Our findings suggest that the genetic profile of coexistent GNAQ or GNA11 mutations with BAP1 or SF3B1 mutations can aid the histopathological diagnosis of blue nevus-like melanoma and distinguish blue nevus-like melanoma from conventional epidermal-derived melanomas. Future studies will need to further elucidate the prognostic implications and appropriate clinical management for patients with tumors harboring these mutation profiles.

Conjunctival Melanomas Harbor BRAF and NRAS Mutations— Response

In our recently published study ([1][1]), we describe genetic similarities between conjunctival and cutaneous melanoma and propose that the treatment rationale for patients with metastasized conjunctival melanoma should be adapted to that for metastasized cutaneous melanoma. Weber and colleagues ([2

Frequent GNAQ, GNA11, and EIF1AX Mutations in Iris Melanoma

Purpose The most common malignant intraocular tumors with a high mortality in adults are uveal melanomas. Uveal melanomas arise most frequently in the choroid or ciliary body (97%) and rarely in the iris (3%). Whereas conjunctival and posterior uveal (ciliary body and choroidal) melanomas have been studied in more detail genetically, little data exist regarding iris melanomas. Methods In our study, we genetically analyzed 19 iris melanomas, 8 ciliary body melanomas, 3 ring melanomas, and 4 iris nevi. A targeted next-generation sequencing approach was applied, covering the mutational hotspot regions of nine genes known to be mutated in conjunctival and uveal melanoma (BRAF, NRAS, KIT, GNAQ, GNA11, CYSLTR2, SF3B1, EIF1AX, and BAP1). Results Activating GNAQ or GNA11 hotspot mutations were detected in a mutually exclusive fashion in 84% (16/19) of iris melanomas. EIF1AX gene mutations also were frequent, detected in 42% (8/19) of iris melanomas. In 4 iris nevi, one GNAQ mutation was identified. GNAQ, GNA11, EIF1AX, and BAP1 mutations were identified at varying frequencies in ciliary body and ring melanomas. Conclusions In this most comprehensive genetic analysis of iris melanomas published to date, we find iris melanomas to be related genetically to choroidal and ciliary body melanomas, frequently harboring GNAQ, GNA11, and EIF1AX mutations. Future studies will need to assess if screening mutation profiles in iris melanomas may be of diagnostic or prognostic value.

Corticosteroids Augment BRAF Inhibitor Vemurafenib Induced Lymphopenia and Risk of Infection

We have previously demonstrated an impact of the BRAF inhibitor vemurafenib on patient lymphocyte counts. In the current study, the extent to which concomitant use of corticosteroids in BRAF inhibitor treated patients affects lymphocyte counts and predisposes to infection was investigated. A cohort of 102 patients receiving either the selective BRAF inhibitor vemurafenib or dabrafenib was analyzed. The amount of patients receiving either medication with or without systemic corticosteroids (dexamethasone) was determined and lymphocyte counts before and under therapy assessed. Additionally, the number and severity of infections occurring in these groups was analyzed. Vemurafenib treatment led to a considerable decrease in lymphocyte cell counts, with 62.3% of patients having lymphopenia. Dabrafenib treated patients only rarely demonstrated lymphopenia (12.5%). Dexamethasone co-administration further diminished lymphocyte counts. Lymphopenias were observed in 84.6% of patients receiving vemurafenib and dexamethasone. In our cohort, infections were noted in 9 patients, 4 of these were severe and 2 eventually fatal. All 9 cases with infections demonstrated lymphopenia, 8 of these had received dexamethasone and 7 of these a therapy with vemurafenib. Our findings demonstrate a significant lymphopenia in patients treated with the BRAF inhibitor vemurafenib, which is further augmented by dexamethasone and predisposes to infection. If validated in other studies, risk of infection should be considered when applying corticosteroids in combination with BRAF inhibitors, in particular vemurafenib.