Uwe Hillen

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.

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.

Patch testing with contact allergens

One-year prevalence of contact dermatitis in the general population is 7%. 15 to 20% of the general population are sensitized to one of the common allergens [1, 2]. Contact dermatitis is the subject of a current guideline of the German Dermatologic Society (Deutsche Derma tologische Gesellschaft, DDG) [3]. The patch test is the only instrument suitable for routine diagnostics to prove sensitization to a substance causing an allergic contact dermatitis. Synchronous reproducibility ranges from 60 to 90 %, depend ing on test method and allergen [4, 5].

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.

Panniculitis with arthralgia in patients with melanoma treated with selective BRAF inhibitors and its management.

BACKGROUND Painful lobular panniculitis appears to be a novel cutaneous adverse effect of selective BRAF inhibitors. OBSERVATION We report the clinical course and management in 2 women with metastatic melanomas harboring the BRAF(V600E) mutation, who developed panniculitis with arthralgia during therapy with selective oral BRAF inhibitors. Panniculitis with arthralgia was the acute presenting adverse effect in both patients. Painful, red, nodular lesions were located on the upper and lower extremities. Biopsy specimens of the nodules showed a mild, predominantly lobular neutrophilic panniculitis. Analgesic and anti-inflammatory treatment improved panniculitis and arthralgia in both cases. It was also necessary to reduce the BRAF inhibitor dose in 1 patient. CONCLUSIONS During therapy with selective BRAF inhibitors, panniculitis with arthralgia represents a new adverse effect that can require dose reduction. In case of this adverse effect, treatment with nonsteroidal anti-inflammatory drugs, such as etoricoxib, should be initiated early to keep patients on treatment and to avoid drug discontinuation and tumor progression.

Metastatic status of sentinel lymph nodes in melanoma determined noninvasively with multispectral optoacoustic imaging

Optoacoustic imaging strategies can be used to identify metastasis in excised lymph nodes and to determine SLN status in patients noninvasively. Imaging melanoma metastasis Avoiding invasive biopsy altogether, an imaging technique that relies on endogenous biomolecules to generate acoustic signals could be used to detect metastases in the body. Stoffels et al. devised a multispectral optoacoustic tomography (MSOT) approach that could image the pigment melanin in lymph nodes. Melanin would only be present in the lymph nodes if the primary cancer—melanoma—had spread to distant locations. The authors used handheld MSOT detectors and a near-infrared fluorophore (which pools in lymph nodes) to image metastases in patients, and complemented these optoacoustic images with ultrasound to gain a complete picture of each lymph node’s status. Such a noninvasive approach could reduce the number of patients subjected to sentinel lymph node surgical excision by “ruling out” metastasis. Sentinel lymph node (SLN) excision is included in various cancer guidelines to identify microscopic metastatic disease. Although effective, SLN excision is an invasive procedure requiring radioactive tracing. Novel imaging approaches assessing SLN metastatic status could improve or replace conventional lymph node excision protocols. In our first-in-human study, we used noninvasive multispectral optoacoustic tomography (MSOT) to image SLNs ex vivo and in vivo in patients with melanoma, to determine metastatic status. MSOT significantly improved the tumor metastasis detection rate in excised SLN (506 SLNs from 214 melanoma patients) compared with the conventional EORTC (European Organisation for Research and Treatment of Cancer) Melanoma Group protocol (22.9% versus 14.2%). MSOT combined with the near-infrared fluorophore indocyanine green reliably visualized SLNs in vivo in 20 patients, up to 5-cm penetration and with 100% concordance with 99mTc-marked SLN lymphoscintigraphy. MSOT identified cancer-free SLNs in vivo and ex vivo without a single false negative (189 total lymph nodes), with 100% sensitivity and 48 to 62% specificity. Our findings indicate that a noninvasive, nonradioactive MSOT-based approach can identify and determine SLN status and confidently rule out the presence of metastasis. The study further demonstrates that optoacoustic imaging strategies can improve the identification of SLN metastasis as an alternative to current invasive SLN excision protocols.

A Phase II trial of Belinostat (PXD101) in patients with relapsed or refractory peripheral or cutaneous T-cell lymphoma

Belinostat is a pan‐histone deacetylase inhibitor with antitumour and anti‐angiogenic properties. An open label, multicentre study was conducted in patients with peripheral T‐cell lymphoma (PTCL) or cutaneous T‐cell lymphoma (CTCL) who failed ≥1 prior systemic therapy and were treated with belinostat (1000 mg/m2 intravenously ×5 d of a 21‐d cycle). The primary endpoint was objective response rate (ORR). Patients with PTCL (n = 24) had received a median of three prior systemic therapies (range 1–9) and 40% had stage IV disease. Patients with CTCL (n = 29) had received a median of one prior skin‐directed therapy (range 0–4) and four prior systemic therapies (range 1–9); 55% had stage IV disease. The ORRs were 25% (PTCL) and 14% (CTCL). Treatment‐related adverse events occurred in 77% of patients; nausea (43%), vomiting (21%), infusion site pain (13%) and dizziness (11%) had the highest incidence. Treatment‐related serious adverse events were Grade 5 ventricular fibrillation; Grade 4 thrombocytopenia; Grade 3 peripheral oedema, apraxia, paralytic ileus and pneumonitis; and Grade 2 jugular vein thrombosis. Belinostat monotherapy was well tolerated and efficacious in patients with recurrent/refractory PTCL and CTCL. This trial was registered at www.clinicaltrials.gov as NCT00274651.

TERT promoter mutations in ocular melanoma distinguish between conjunctival and uveal tumours

Background:Recently, activating mutations in the TERT promoter were identified in cutaneous melanoma. We tested a cohort of ocular melanoma samples for similar mutations.Methods:The TERT promoter region was analysed by Sanger sequencing in 47 uveal (ciliary body or choroidal) melanomas and 38 conjunctival melanomas.Results:Mutations of the TERT promoter were not identified in uveal melanomas, but were detected in 12 (32%) conjunctival melanomas. Mutations had a UV signature and were identical to those found in cutaneous melanoma.Conclusion:Mutations of TERT promoter with UV signatures are frequent in conjunctival melanomas and favour a pathogenetic kinship with cutaneous melanomas. Absence of these mutations in uveal melanomas emphasises their genetic distinction from cutaneous and conjunctival melanomas.

An attempt to improve diagnostics of contact allergy due to epoxy resin systems. First results of the multicentre study EPOX 2002

Epoxy resin systems (ERSs) are a frequent cause of occupational allergic contact dermatitis. Sensitization occurs not only to the resins, but also to hardeners and reactive diluents. However, only a fraction of the ERS components currently in use are available for patch testing. With the multicentre study EPOX 2002, we attempted to improve diagnostics in this field by patch testing with components currently used in ERSs. During the first study period (October 2002 to July 2003), in addition to commercially available ERS patch test substances, 16 study substances (1 resin, 9 hardeners and 6 reactive diluents) were patch tested in 70 patients with suspected contact allergy due to ERSs and 22 patients with a prior positive patch test reaction to epoxy resin (ER) in the standard series. Most frequently, allergic reactions to ER based on diglycidyl ether of bisphenol A and F were observed (55.2% and 43.7%, respectively). Agreement between positive reactions to both resins, which can be explained by immunological cross‐sensitization and/or coexposure, was substantial [Cohens kappa 0.65 (95% CI: 0.49–0.80)]. Among the reactive diluents, 1,6‐hexanediol diglycidyl ether (1,6‐HDDGE) and 1,4‐butanediol diglycidyl ether (1,4‐BDDGE) were the most frequent allergens, with 19.5% and 18.4% positive reactions, respectively. Although agreement between positive reactions to 1,6‐HDDGE and 1,4‐BDDGE was even better than with the 2 resins, the sample size is considered too small to decide reliably whether 1,6‐HDDGE alone could serve as a marker allergen for both. Allergic reactions to p‐tert‐butylphenyl glycidyl ether and to phenyl glycidyl ether (PGE) occurred in 11.5% of the patients tested, with only moderate agreement. All patients positive to cresyl glycidyl ether (6.8%) also reacted to PGE. Of the hardeners tested, m‐xylylene diamine was the most frequent allergen (13.8%), followed by isophorone diamine (5.7%). No reactions were observed to several substances, the test concentration of which may have been too low and will be increased in the future.