Cindy Franklin

Immunotherapy in melanoma: Recent advances and future directions

Malignant melanoma contributes the majority of skin cancer related deaths and shows an increasing incidence in the past years. Despite all efforts of early diagnosis, metastatic melanoma still has a poor prognosis and remains a challenge for treating physicians. In recent years, improved knowledge of the pathophysiology and a better understanding of the role of the immune system in tumour control have led to the development and approval of several immunotherapies. Monoclonal antibodies against different immune checkpoints have been revolutionizing the treatment of metastatic and unresectable melanoma. Ipilimumab, a monoclonal antibody against the cytotoxic T-lymphocyte antigen 4 (CTLA-4) as well as nivolumab and pembrolizumab which target the programmed cell death protein 1 (PD-1) have been shown to prolong overall survival in patients with advanced melanoma. The latter substances seem to have an increased response rate and more tolerable safety profile compared to ipilimumab. The combination of a CTLA-4 and a PD-1 inhibitor seems to be superior to the monotherapies, especially in patients with PD-L1 negative tumours. Checkpoint inhibitors are currently being tested in the adjuvant setting with initial data for ipilimumab suggesting efficacy in this context. Talimogene laherparepvec (TVEC) is the first oncolytic virus approved in the therapy of metastatic melanoma offering a treatment option especially for patients with limited disease. In this review, data on these recently developed and approved immunotherapies are presented. However, further studies are necessary to determine the optimal duration, sequencing and combinations of immunotherapies to further improve the outcome of patients with advanced melanoma.

Ulcerated necrobiosis lipoidica as a rare cause for chronic leg ulcers: case report series of ten patients.

Necrobiosis lipoidica is a rare granulomatous disorder of the skin. In up to 30% of the affected patients it can lead to ulcerations, which can impair the quality of life and are also very difficult to treat. Its pathogenesis is not fully understood. Only few studies focussing on necrobiosis lipoidica can be found, but none of them focus on ulcerated necrobiosis lipoidica. Therefore, we collected demographic data and comorbidities and assessed treatment options for patients with ulcerated necrobiosis lipoidica. Data of patients who were treated in the wound care centre of the University Hospital of Essen for ulcerated necrobiosis lipoidica over the past 10 years were retrospectively analysed. Hence, data of altogether ten patients (nine women and one man) with ulcerated necrobiosis lipoidica were collected. Of these, 70% of the patients had diabetes mellitus of which 30% had type I diabetes and 40% had type II diabetes; 60% of the patients suffered from arterial hypertension, obesity and hypercholesterolaemia; 40% of the patients suffered from psychiatric disorders such as depression and borderline disorder. Our clinical data demonstrate an association of ulcerated necrobiosis lipoidica and aspects of metabolic syndrome. This leads to a conclusion that ulcerating necrobiosis lipoidica can be seen as part of a generalised inflammatory reaction similar to the inflammatory reaction already known in the pathophysiology of rheumatoid diseases or psoriasis. In patients with clinical atypical painful ulcerations, necrobiosis lipoidica should be considered as a possible differential diagnosis. Therapists should be aware of associated aspects in patients with ulcerated necrobiosis lipoidica who besides diabetes often suffer from other aspects of a metabolic syndrome with increased cardiovascular risk factors. Therefore, these related comorbidities should also be diagnosed and treated.

Multimodal imaging analysis of an orthotopic head and neck cancer mouse model and application of anti-CD137 tumor immune therapy.

Recent technical progress makes sophisticated noninvasive imaging methods available for murine models. For the first time, in this study, we applied fluorodeoxyglucose (FDG)‐positron emission tomography (PET)‐CT and FDG‐PET‐MRI to a murine orthotopic model of head and neck cancer immunotherapy.

Modulation and Apoptosis of Neutrophil Granulocytes by Extracorporeal Photopheresis in the Treatment of Chronic Graft-Versus-Host Disease.

Chronic graft-versus-host disease (cGVHD) is a common side effect of allogeneic stem cell transplantation and a major cause of morbidity and mortality in affected patients. Especially skin, eyes and oral mucosa are affected. This can lead to pain and functional impairment. Extracorporeal photopheresis (ECP) is an effective immunomodulatory therapy with minimal side effects but its mode of action is still largely unknown. The objective of the present study was to examine the effects of ECP on neutrophil granulocytes in patients with cGVHD. Analysis of leukocytes from cGVHD patients obtained from the ECP device during treatment showed that neutrophil granulocytes account for the majority of cells treated during ECP. Neutrophils from healthy donors treated in vitro with 8-methoxypsoralen and UVA light as well as neutrophils from buffy coats of patients with cGVHD treated by ECP showed increased apoptosis and decreased half-life. In remaining non-apoptotic cells chemoirradiation resulted in loss of activation markers and reduced effector functions. This was accompanied by an increase in extracellular arginase-1 activity. Additional comparison of neutrophils isolated from blood of cGVHD patients before and 24h after ECP revealed a decreased half-life and reduction of effector functions of post-ECP neutrophils ex vivo. These observations strongly suggest that ECP induces both apoptosis and physiological changes in neutrophils and that these changes also take place in vivo. This study is the first to show that ECP modulates apoptosis and inflammatory activity in neutrophil granulocytes, indicating that neutrophils may significantly contribute to the overall immunomodulatory effects attributed to this treatment.

Oncogene status as a diagnostic tool in ocular and cutaneous melanoma

The majority of human tumours can be easily and correctly diagnosed based on clinical information and pathological assessment. In some cases however, correct diagnosis can prove difficult. In such cases, molecular approaches can be of significant diagnostic value. In recent years, the understanding of genetic alterations has greatly increased. In cutaneous melanoma, it is now well recognised, that 70-80% of tumours harbour BRAF and NRAS mutations. These mutations never occur in uveal melanoma. On the other hand activating GNAQ and GNA11 mutations are found in ∼90% of uveal melanomas, and are exceptionally rare in other melanomas (<1%). Here, we demonstrate a number of melanoma cases, where distinguishing if a tumour was of cutaneous or ocular origin was not possible based on clinical and pathological assessment. In these cases there was either atypical clinical presentation or metastasis of unclear primary. Histological distinction between uveal and cutaneous melanomas, especially at the stage of metastasis, is not reliable as they can be morphologically very similar. In all cases we present, a simple genetic assessment of oncogene mutation status was able to clearly define the melanoma type. This type of genetic assessment is of great diagnostic value and due to its simplicity could be performed in routine clinical practice even in smaller institutions.

Atypical fibroxanthoma and pleomorphic dermal sarcoma harbor frequent NOTCH1/2 and FAT1 mutations and similar DNA copy number alteration profiles

Atypical fibroxanthomas and pleomorphic dermal sarcomas are tumors arising in sun-damaged skin of elderly patients. They have differing prognoses and are currently distinguished using histological criteria, such as invasion of deeper tissue structures, necrosis and lymphovascular or perineural invasion. To investigate the as-yet poorly understood genetics of these tumors, 41 atypical fibroxanthomas and 40 pleomorphic dermal sarcomas were subjected to targeted next-generation sequencing approaches as well as DNA copy number analysis by comparative genomic hybridization. In an analysis of the entire coding region of 341 oncogenes and tumor suppressor genes in 13 atypical fibroxanthomas using an established hybridization-based next-generation sequencing approach, we found that these tumors harbor a large number of mutations. Gene alterations were identified in more than half of the analyzed samples in FAT1, NOTCH1/2, CDKN2A, TP53, and the TERT promoter. The presence of these alterations was verified in 26 atypical fibroxanthoma and 35 pleomorphic dermal sarcoma samples by targeted amplicon-based next-generation sequencing. Similar mutation profiles in FAT1, NOTCH1/2, CDKN2A, TP53, and the TERT promoter were identified in both atypical fibroxanthoma and pleomorphic dermal sarcoma. Activating RAS mutations (G12 and G13) identified in 3 pleomorphic dermal sarcoma were not found in atypical fibroxanthoma. Comprehensive DNA copy number analysis demonstrated a wide array of different copy number gains and losses, with similar profiles in atypical fibroxanthoma and pleomorphic dermal sarcoma. In summary, atypical fibroxanthoma and pleomorphic dermal sarcoma are highly mutated tumors with recurrent mutations in FAT1, NOTCH1/2, CDKN2A, TP53, and the TERT promoter, and a range of DNA copy number alterations. These findings suggest that atypical fibroxanthomas and pleomorphic dermal sarcomas are genetically related, potentially representing two ends of a common tumor spectrum and distinguishing these entities is at present still best performed using histological criteria.

NF1 mutations in conjunctival melanoma.

BackgroundConjunctival melanoma is a potentially deadly eye tumour. Despite effective local therapies, tumour recurrence and metastasis remain frequent. The genetics of conjunctival melanomas remain incompletely understood.MethodsA large cohort of 63 conjunctival melanomas was screened for gene mutations known to be important in other melanoma subtypes by targeted next-generation sequencing. Mutation status was correlated with patient prognosis.ResultsFrequent mutations in genes activating the MAP kinase pathway were identified. NF1 mutations were most frequent (n = 21, 33%). Recurrent activating mutations were also identified in BRAF (n = 16, 25%) and RAS genes (n = 12, 19%; 11 NRAS and 1 KRAS).ConclusionsSimilar to cutaneous melanomas, conjunctival melanomas can be grouped genetically into four groups: BRAF-mutated, RAS-mutated, NF1-mutated and triple wild-type melanomas. This genetic classification may be useful for assessment of therapeutic options for patients with metastatic conjunctival melanoma

NAMPT signaling is critical for the proangiogenic activity of tumor-associated neutrophils: NAMPT supports neutrophil angiogenic activity

Tumor‐associated neutrophils (TANs) regulate many processes associated with tumor progression, and depending on the microenvironment, they can exhibit pro‐ or antitumor functions. However, the molecular mechanisms regulating their tumorigenicity are not clear. Using transplantable tumor models, we showed here that nicotinamide phosphoribosyltransferase (NAMPT), a molecule involved in CSF3R downstream signaling, is essential for tumorigenic conversion of TANs and their pro‐angiogenic switch. As a result tumor vascularization and growth are strongly supported by these cells. Inhibition of NAMPT in TANs leads to their antitumor conversion. Adoptive transfer of such TANs into B16F10‐tumor bearing mice attenuates tumor angiogenesis and growth. Of note, we observe that the regulation of NAMPT signaling in TANs, and its effect on the neutrophil tumorigenicity, are analogous in mice and human. NAMPT is up‐regulated in TANs from melanoma and head‐and‐neck tumor patients, and its expression positively correlates with tumor stage. Mechanistically, we found that targeting of NAMPT suppresses neutrophil tumorigenicity by inhibiting SIRT1 signaling, thereby blocking transcription of pro‐angiogenic genes. Based on these results, we propose that NAMPT regulatory axis is important for neutrophils to activate angiogenic switch during early stages of tumorigenesis. Thus, identification of NAMPT as the critical molecule priming protumor functions of neutrophils provides not only mechanistic insight into the regulation of neutrophil tumorigenicity, but also identifies a potential pathway that may be targeted therapeutically in neutrophils. This, in turn, may be utilized as a novel mode of cancer immunotherapy.

Granulocytic myeloid-derived suppressor cells in peripheral blood of patients with cutaneous melanoma.

The article published in this issue (p. XX) by Stanojevic et al. (1) describes a myeloid-derived suppressor cell (MDSC) subset that is increased in melanoma patients and shows further accumulation during disease progression. Importantly, these cells were identified and quantified in whole blood. The authors suggest that MDSC measurements in whole blood should be considered as a potential biomarker. Although a very interesting study, several weaknesses warrant further discussion. We would like to point out that it is hard to distinguish conventional neutrophil granulocytes or eosinophils from granulocytic MDSC (grMDSC), especially in whole blood (2). The authors found grMDSC in whole blood to be CD10CD15CD14HLA-DRCD33CD11bCD45CD16 and lineage negative which is a widely used panel for identifying grMDSC in PBMC after density centrifugation (2). Neutrophil granulocytes show the same marker expression but are also positive for CD16. However, in elderly patients reduced CD16 expression on neutrophil granulocytes is a common finding and can contribute to immunesenescence (3). In addition, it is known that neutrophil granulocytes can lose CD16 when they undergo apoptosis (4) and surface expression of CD16 is reduced upon activation (5). Apoptotic downregulation might also explain the lower expression of CD33, CD11b, CD45 and CD10 on those cells considered grMDSC by Stanojevic et al. as compared with regular neutrophils. As discussed by the authors, human eosinophils also share the phenotype attributed by the authors to grMDSC (1). However, distinguishing eosinophils and grMDSC on the basis of low or absent CD16 expression does not appear sufficient in the context of using grMDSC as a biomarker. In their study the authors also compare grMDSC in whole blood and after density gradient centrifugation. Nevertheless, activated and immature granulocytes can show altered buoyancy in density gradients as well, which can result in altered sedimentation properties (2). Finally, human MDSC are better defined by their functional property of suppressing T cell functions than by a phenotypic description. Since such evidence is lacking, we strongly suggest isolating these cells considered to be grMDSC by Stanojevic et al. and assessing their functional properties. We agree that a method able to identify grMDSC in whole blood samples is needed to explore the value of these cells as a biomarker. The manuscript “A subpopulation that may correspond to granulocytic myeloid-derived suppressor cells reflects the clinical stage and progression of cutaneous melanoma” provides preliminary evidence that these cells can be identified in whole blood samples. However, it remains unclear if the approach suggested by Stanojevic et al. is suitable to identify grMDSC in whole blood or if the leukocyte population described might be consistent with immature, activated or apoptotic neutrophil or eosinophil granulocytes.