Antje Sucker

TERT Promoter Mutations in Familial and Sporadic Melanoma.

Promoter Mutations and Cancer Cancer genome sequencing projects have highlighted the pathogenic role of recurrent mutations within the protein-coding regions of genes. Now, two studies suggest that the scope of mutations in human tumors extends to gene regulatory regions. In a study of 70 melanomas, Huang et al. (p. 957, published online 24 January) found that 71% harbored one of two specific mutations in the promoter region of TERT, the gene coding for the catalytic subunit of telomerase, the enzyme that caps chromosome ends. Independently, Horn et al. (p. 959, published online 24 January) identified a disease-segregating germline mutation in the TERT promoter in a family predisposed to melanoma and found additional TERT promoter mutations in a high percentage of sporadic melanomas and melanoma cell lines. The mutations in both studies generated new binding sites for specific transcription factors and, in reporter assays, caused an increase in transcription. A large fraction of human melanomas harbor mutations in sequences that regulate the expression of telomerase. Cutaneous melanoma occurs in both familial and sporadic forms. We investigated a melanoma-prone family through linkage analysis and high-throughput sequencing and identified a disease-segregating germline mutation in the promoter of the telomerase reverse transcriptase (TERT) gene, which encodes the catalytic subunit of telomerase. The mutation creates a new binding motif for Ets transcription factors and ternary complex factors (TCFs) near the transcription start and, in reporter gene assays, caused up to twofold increase in transcription. We then screened the TERT promoter in sporadic melanoma and observed recurrent ultraviolet signature somatic mutations in 125 of 168 (74%) of human cell lines derived from metastatic melanomas, 45 of 53 corresponding metastatic tumor tissues (85%), and 25 of 77 (33%) primary melanomas. The majority of those mutations occurred at two positions in the TERT promoter and also generated binding motifs for Ets/TCF transcription factors.

Genomic correlates of response to CTLA-4 blockade in metastatic melanoma

Is cancer immunotherapy a private affair? Immune checkpoint blockade, a relatively new cancer treatment, substantially extends the survival of a subset of patients. Previous work has shown that patients whose tumors harbor the largest number of mutations—and thus produce a large number of “neoantigens” recognized as foreign by the immune system—are most likely to benefit. Expanding on these earlier studies, Van Allen et al. studied over 100 patients with melanoma and found a similar correlation (see the Perspective by Gubin and Schreiber). There was no evidence, however, that specific neoantigen sequences were shared by patients who responded. Science, this issue p. 207, see also p. 158 Melanoma patients who respond to immunotherapy do not appear to share common tumor neoantigens. [Also see Perspective by Gubin and Schreiber ] Monoclonal antibodies directed against cytotoxic T lymphocyte–associated antigen-4 (CTLA-4), such as ipilimumab, yield considerable clinical benefit for patients with metastatic melanoma by inhibiting immune checkpoint activity, but clinical predictors of response to these therapies remain incompletely characterized. To investigate the roles of tumor-specific neoantigens and alterations in the tumor microenvironment in the response to ipilimumab, we analyzed whole exomes from pretreatment melanoma tumor biopsies and matching germline tissue samples from 110 patients. For 40 of these patients, we also obtained and analyzed transcriptome data from the pretreatment tumor samples. Overall mutational load, neoantigen load, and expression of cytolytic markers in the immune microenvironment were significantly associated with clinical benefit. However, no recurrent neoantigen peptide sequences predicted responder patient populations. Thus, detailed integrated molecular characterization of large patient cohorts may be needed to identify robust determinants of response and resistance to immune checkpoint inhibitors.

Melanoma genome sequencing reveals frequent PREX2 mutations

Melanoma is notable for its metastatic propensity, lethality in the advanced setting and association with ultraviolet exposure early in life. To obtain a comprehensive genomic view of melanoma in humans, we sequenced the genomes of 25 metastatic melanomas and matched germline DNA. A wide range of point mutation rates was observed: lowest in melanomas whose primaries arose on non-ultraviolet-exposed hairless skin of the extremities (3 and 14 per megabase (Mb) of genome), intermediate in those originating from hair-bearing skin of the trunk (5–55 per Mb), and highest in a patient with a documented history of chronic sun exposure (111 per Mb). Analysis of whole-genome sequence data identified PREX2 (phosphatidylinositol-3,4,5-trisphosphate-dependent Rac exchange factor 2)—a PTEN-interacting protein and negative regulator of PTEN in breast cancer—as a significantly mutated gene with a mutation frequency of approximately 14% in an independent extension cohort of 107 human melanomas. PREX2 mutations are biologically relevant, as ectopic expression of mutant PREX2 accelerated tumour formation of immortalized human melanocytes in vivo. Thus, whole-genome sequencing of human melanoma tumours revealed genomic evidence of ultraviolet pathogenesis and discovered a new recurrently mutated gene in melanoma.

RAS Mutations Are Associated With the Development of Cutaneous Squamous Cell Tumors in Patients Treated With RAF Inhibitors

PURPOSE RAF inhibitors are effective against melanomas with BRAF V600E mutations but may induce keratoacanthomas (KAs) and cutaneous squamous cell carcinomas (cSCCs). The potential of these agents to promote secondary malignancies is concerning. We analyzed cSCC and KA lesions for genetic mutations in an attempt to identify an underlying mechanism for their formation. METHODS Four international centers contributed 237 KA or cSCC tumor samples from patients receiving an RAF inhibitor (either vemurafenib or sorafenib; n = 19) or immunosuppression therapy (n = 53) or tumors that developed spontaneously (n = 165). Each sample was profiled for 396 known somatic mutations across 33 cancer-related genes by using a mass spectrometric-based genotyping platform. RESULTS Mutations were detected in 16% of tumors (38 of 237), with five tumors harboring two mutations. Mutations in TP53, CDKN2A, HRAS, KRAS, and PIK3CA were previously described in squamous cell tumors. Mutations in MYC, FGFR3, and VHL were identified for the first time. A higher frequency of activating RAS mutations was found in tumors from patients treated with an RAF inhibitor versus populations treated with a non-RAF inhibitor (21.1% v 3.2%; P < .01), although overall mutation rates between treatment groups were similar (RAF inhibitor, 21.1%; immunosuppression, 18.9%; and spontaneous, 17.6%; P = not significant). Tumor histology (KA v cSCC), tumor site (head and neck v other), patient age (≤ 70 v > 70 years), and sex had no significant impact on mutation rate or type. CONCLUSION Squamous cell tumors from patients treated with an RAF inhibitor have a distinct mutational profile that supports a mechanism of therapy-induced tumorigenesis in RAS-primed cells. Conceivably, cotargeting of MEK together with RAF may reduce or prevent formation of these tumors.

Lack of clinical efficacy of imatinib in metastatic melanoma

This two-centre phase-II trial aimed at investigating the efficacy of imatinib in metastasised melanoma patients in correlation to the tumour expression profile of the imatinib targets c-kit and platelet-derived growth factor receptor (PDGF-R). The primary study end point was objective response according to RECIST, secondary end points were safety, overall and progression-free survival. In all, 18 patients with treatment-refractory advanced melanoma received imatinib 800 mg day−1. In 16 evaluable patients no objective responses could be observed. The median overall survival was 3.9 months, the median time to progression was 1.9 months. Tumour biopsy specimens were obtained from 12 patients prior to imatinib therapy and analysed for c-kit, PDGF-Rα and -Rβ expression by immunohistochemistry. In four cases, cell lines established from these tumour specimens were tested for the antiproliferative effects of imatinib and for functional mutations of genes encoding the imatinib target molecules. The tumour specimens stained positive for CD117/c-kit in nine out of 12 cases (75%), for PDGF-Rα in seven out of 12 cases (58%) and for PDGF-Rβ in eight out of 12 cases (67%). The melanoma cell lines showed a heterogenous expression of the imatinib target molecules without functional mutations in the corresponding amino-acid sequences. In vitro imatinib treatment of the cell lines showed no antiproliferative effect. In conclusion, this study did not reveal an efficacy of imatinib in advanced metastatic melanoma, regardless of the expression pattern of the imatinib target molecules c-kit and PDGF-R.

NCRs and DNAM-1 mediate NK cell recognition and lysis of human and mouse melanoma cell lines in vitro and in vivo

NK cells use a variety of receptors to detect abnormal cells, including tumors and their metastases. However, in the case of melanoma, it remains to be determined what specific molecular interactions are involved and whether NK cells control metastatic progression and/or the route of dissemination. Here we show that human melanoma cell lines derived from LN metastases express ligands for natural cytotoxicity receptors (NCRs) and DNAX accessory molecule-1 (DNAM-1), two emerging NK cell receptors key for cancer cell recognition, but not NK group 2 member D (NKG2D). Compared with cell lines derived from metastases taken from other anatomical sites, LN metastases were more susceptible to NK cell lysis and preferentially targeted by adoptively transferred NK cells in a xenogeneic model of cell therapy. In mice, DNAM-1 and NCR ligands were also found on spontaneous melanomas and melanoma cell lines. Interference with DNAM-1 and NCRs by antibody blockade or genetic disruption reduced killing of melanoma cells. Taken together, these results show that DNAM-1 and NCRs are critical for NK cell-mediated innate immunity to melanoma cells and provide a background to design NK cell-based immunotherapeutic strategies against melanoma and possibly other tumors.

Atypical Melanocytic Proliferations and New Primary Melanomas in Patients With Advanced Melanoma Undergoing Selective BRAF Inhibition

PURPOSE Selective inhibition of mutant BRAF by using class I RAF inhibitors in patients with metastatic melanoma has resulted in impressive clinical activity. However, there is also evidence that RAF inhibitors might induce carcinogenesis or promote tumor progression via stimulation of MAPK signaling in RAF wild-type cells. We analyzed melanocytic lesions arising under class I RAF inhibitor treatment for dignity, specific genetic mutations, or expression of signal transduction molecules. PATIENTS AND METHODS In all, 22 cutaneous melanocytic lesions that had either developed or considerably changed in morphology in 19 patients undergoing treatment with selective BRAF inhibitors for BRAF-mutant metastatic melanoma at seven international melanoma centers within clinical trials in 2010 and 2011 were analyzed for mutations in BRAF and NRAS genes and immunohistologically assessed for expression of various signal transduction molecules in comparison with 22 common nevi of 21 patients with no history of BRAF inhibitor treatment. RESULTS Twelve newly detected primary melanomas were confirmed in 11 patients within 27 weeks of selective BRAF blockade. In addition, 10 nevi developed of which nine were dysplastic. All melanocytic lesions were BRAF wild type. Explorations revealed that expression of cyclin D1 and pAKT was increased in newly developed primary melanomas compared with nevi (P = .01 and P = .03, respectively). There was no NRAS mutation in common nevi, but BRAF mutations were frequent. CONCLUSION Malignant melanocytic tumors might develop with increased frequency in patients treated with selective BRAF inhibitors supporting a mechanism of BRAF therapy-induced growth and tumorigenesis. Careful surveillance of melanocytic lesions in patients receiving class I RAF inhibitors seems warranted.

Baseline Peripheral Blood Biomarkers Associated with Clinical Outcome of Advanced Melanoma Patients Treated with Ipilimumab.

Purpose: To identify baseline peripheral blood biomarkers associated with clinical outcome following ipilimumab treatment in advanced melanoma patients. Experimental Design: Frequencies of myeloid-derived suppressor cells (MDSC) and regulatory T cells (Treg), serum lactate dehydrogenase (LDH), routine blood counts, and clinical characteristics were assessed in 209 patients. Endpoints were overall survival (OS) and best overall response. Statistical calculations were done by Kaplan–Meier and Cox regression analysis, including calibration and discrimination by C-statistics. Results: Low baseline LDH, absolute monocyte counts (AMC), Lin−CD14+HLA-DR−/low-MDSC frequencies, and high absolute eosinophil counts (AEC), relative lymphocyte counts (RLC), and CD4+CD25+FoxP3+-Treg frequencies were significantly associated with better survival, and were considered in a combination model. Patients (43.5%) presenting with the best biomarker signature had a 30% response rate and median survival of 16 months. In contrast, patients with the worst biomarkers (27.5%) had only a 3% response rate and median survival of 4 months. The occurrence of adverse events correlated with neither baseline biomarker signatures nor the clinical benefit of ipilimumab. In another model, limited to the routine parameters LDH, AMC, AEC, and RLC, the number of favorable factors (4 vs. 3 vs. 2–0) was also associated with OS (P < 0.001 for all pairwise comparisons) in the main study and additionally in an independent validation cohort. Conclusions: A baseline signature of low LDH, AMC, and MDSCs as well as high AEC, Tregs, and RLC is associated with favorable outcome following ipilimumab. Prospective investigation of the predictive impact of these markers following ipilimumab and other treatments, e.g., PD-1 antibodies, is warranted. Clin Cancer Res; 22(12); 2908–18. ©2016 AACR.

Angiopoietin-2 Levels Are Associated with Disease Progression in Metastatic Malignant Melanoma

Purpose: The blood vessel-destabilizing Tie2 ligand angiopoietin-2 (Ang-2) acts in concert with the vascular endothelial growth factor/vascular endothelial growth factor receptor system to control vessel assembly during tumor progression. We hypothesized that circulating soluble Ang-2 (sAng-2) may be involved in melanoma progression. Experimental Design: Serum samples (n = 98) from melanoma patients (American Joint Committee on Cancer stages I-IV), biopsies of corresponding patients, and human melanoma cell lines were analyzed for expression of Ang-2 and S100β. Multiple sera of a subcohort of 33 patients were tested during progression from stage III to IV. Small interfering RNA-based loss-of-function experiments were done to assess effects of Ang-2 on melanoma cells. Results: Circulating levels of sAng-2 correlate with tumor progression in melanoma patients (P < 0.0001) and patient survival (P = 0.007). Analysis of serum samples during the transition from stage III to IV identified an increase of sAng-2 up to 400%. Comparative analyses revealed a 56% superiority of sAng-2 as predictive marker over the established marker S100β. Immunohistochemistry and reverse transcription-PCR confirmed the prominent expression of Ang-2 by tumor-associated endothelial cells but identified Ang-2 also as a secreted product of melanoma cells themselves. Corresponding cellular experiments revealed that human melanoma-isolated tumor cells were Tie2 positive and that Ang-2 acted as an autocrine regulator of melanoma cell migration and invasion. Conclusions: The experiments establish sAng-2 as a biomarker of melanoma progression and metastasis correlating with tumor load and overall survival. The identification of an autocrine angiopoietin/Tie loop controlling melanoma migration and invasion warrants further functional experiments and validate the angiopoietin/Tie system as a promising therapeutic target for human melanomas.

Myeloid-Derived Suppressor Cells Predict Survival of Patients with Advanced Melanoma: Comparison with Regulatory T Cells and NY-ESO-1- or Melan-A–Specific T Cells

Purpose: To analyze the prognostic relevance and relative impact of circulating myeloid-derived suppressor cells (MDSC) and regulatory T cells (Treg) compared with functional tumor antigen–specific T cells in patients with melanoma with distant metastasis. Experimental Design: The percentage of CD14+CD11b+HLA-DR−/low MDSCs, CD4+CD25+FoxP3+ Tregs, and the presence of NY-ESO-1- or Melan-A–specific T cells was analyzed in 94 patients and validated in an additional cohort of 39 patients by flow cytometry. Univariate survival differences were calculated according to Kaplan–Meier and log-rank tests. Multivariate analyses were performed using Cox regression models. Results: NY-ESO-1–specific T cells, the M-category, and the frequency of MDSCs were associated with survival. The absence of NY-ESO-1–specific T cells and the M-category M1c independently increased the risk of death. In a second Cox model not considering results on antigen-specific T cells, a frequency of >11% MDSCs showed independent impact. Its association with survival was confirmed in the additional patient cohort. Median survival of patients with a lower frequency of MDSCs was 13 months versus 8 months for others (P < 0.001, combined cohorts). We observed a strong correlation between high levels of MDSCs and the absence of melanoma antigen–specific T cells implying a causal and clinically relevant interaction. No prognostic impact was observed for Tregs. Conclusions: Circulating CD14+CD11b+HLA-DR−/low MDSCs have a negative impact on survival and inversely correlate with the presence of functional antigen–specific T cells in patients with advanced melanoma. Our findings provide a rationale to investigate MDSC-depleting strategies in the therapeutic setting especially in combination with vaccination or T-cell transfer approaches. Clin Cancer Res; 20(6); 1601–9. ©2013 AACR.