Cutaneous epithelioid haemangiomas show somatic mutations in the mitogen-activated protein kinase pathway.

BACKGROUND: Epithelioid haemangioma (EH) arising from the skin is a benign vascular tumour with marked inflammatory cell infiltration, which exhibits a high tendency to persist and frequently recurs after resection. So far, the underlying pathogenesis is largely elusive. OBJECTIVES: To identify genetic alterations by next-generation sequencing and/or droplet digital polymerase chain reaction (ddPCR) in cutaneous EH. METHODS: DNA and RNA from an EH lesion of an index patient were subjected to whole-genome and RNA sequencing. Multiplex PCR-based panel sequencing of genomic DNA isolated from archival formalin-fixed paraffin-embedded tissue of 18 patients with cutaneous EH was performed. ddPCR was used to confirm mutations. RESULTS: We identified somatic mutations in genes of the mitogen-activated protein kinase (MAPK) pathway (MAP2K1 and KRAS) in cutaneous EH biopsies. By ddPCR we could confirm the recurrent presence of activating, low-frequency mutations affecting MAP2K1. In total, nine out of 18 patients analysed showed activating MAPK pathway mutations, which were mutually exclusive. Comparative analysis of tissue areas enriched for lymphatic infiltrate or aberrant endothelial cells, respectively, revealed an association of these mutations with the presence of endothelial cells. CONCLUSIONS: Taken together, our data suggest that EH shows somatic mutations in genes of the MAPK pathway which might contribute to the formation of this benign tumour.

Elevated baseline serum PD-1 or PD-L1 predicts poor outcome of PD-1 inhibition therapy in metastatic melanoma.

BACKGROUND: Programmed cell death protein 1 (PD-1) checkpoint inhibition has recently advanced to one of the most effective treatment strategies in melanoma. Nevertheless, a considerable proportion of patients show upfront therapy resistance and baseline predictive biomarkers of treatment outcome are scarce. In this study we quantified PD-1 and programmed death-ligand 1 (PD-L1) in baseline sera from melanoma patients in relation to therapy response and survival. PATIENTS AND METHODS: Sera taken at therapy baseline from a total of 222 metastatic melanoma patients (two retrospectively selected monocentric discovery cohorts, n = 130; one prospectively collected multicentric validation cohort, n = 92) and from 38 healthy controls were analyzed for PD-1 and PD-L1 concentration by sandwich enzyme-linked immunosorbent assay. RESULTS: Melanoma patients showed higher serum concentrations of PD-1 (P = 0.0054) and PD-L1 (P < 0.0001) than healthy controls. Elevated serum PD-1 and PD-L1 levels at treatment baseline were associated with an impaired best overall response (BOR) to anti-PD-1 (P = 0.014, P = 0.041), but not to BRAF inhibition therapy. Baseline PD-1 and PD-L1 serum levels correlated with progression-free (PFS; P = 0.0081, P = 0.053) and overall survival (OS; P = 0.055, P = 0.0062) in patients who received anti-PD-1 therapy, but not in patients treated with BRAF inhibitors. By combining both markers, we obtained a strong discrimination between favorable and poor outcome of anti-PD-1 therapy, with elevated baseline serum levels of PD-1 and/or PD-L1 associated with an impaired BOR (P = 0.037), PFS (P = 0.048), and OS (P = 0.0098). This PD-1/PD-L1 combination serum biomarker was confirmed in an independent multicenter validation set of serum samples prospectively collected at baseline of PD-1 inhibition (BOR, P = 0.019; PFS, P = 0.038; OS, P = 0.022). Multivariable Cox regression demonstrated serum PD-1/PD-L1 as an independent predictor of PFS (P = 0.010) and OS (P = 0.003) in patients treated with PD-1 inhibitors. CONCLUSION: Our findings indicate PD-1 and PD-L1 as useful serum biomarkers to predict the outcome of PD-1 inhibition therapy in melanoma patients and to select patients for PD-1-based versus BRAF-based therapy strategies.

ESMO consensus conference recommendations on the management of locoregional melanoma: under the auspices of the ESMO Guidelines Committee.

The European Society for Medical Oncology (ESMO) held a consensus conference on melanoma on 5-7 September 2019 in Amsterdam, The Netherlands. The conference included a multidisciplinary panel of 32 leading experts in the management of melanoma. The aim of the conference was to develop recommendations on topics that are not covered in detail in the current ESMO Clinical Practice Guideline and where available evidence is either limited or conflicting. The main topics identified for discussion were: (i) the management of locoregional disease; (ii) targeted versus immunotherapies in the adjuvant setting; (iii) targeted versus immunotherapies for the first-line treatment of metastatic melanoma; (iv) when to stop immunotherapy or targeted therapy in the metastatic setting; and (v) systemic versus local treatment of brain metastases. The expert panel was divided into five working groups in order to each address questions relating to one of the five topics outlined above. Relevant scientific literature was reviewed in advance. Recommendations were developed by the working groups and then presented to the entire panel for further discussion and amendment before voting. This manuscript presents the results relating to the management of locoregional melanoma, including findings from the expert panel discussions, consensus recommendations and a summary of evidence supporting each recommendation. All participants approved the final manuscript.

ESMO consensus conference recommendations on the management of metastatic melanoma: under the auspices of the ESMO Guidelines Committee.

The European Society for Medical Oncology (ESMO) held a consensus conference on melanoma on 5-7 September 2019 in Amsterdam, The Netherlands. The conference included a multidisciplinary panel of 32 leading experts in the management of melanoma. The aim of the conference was to develop recommendations on topics that are not covered in detail in the current ESMO Clinical Practice Guideline and where available evidence is either limited or conflicting. The main topics identified for discussion were (i) the management of locoregional disease; (ii) targeted versus immunotherapies in the adjuvant setting; (iii) targeted versus immunotherapies for the first-line treatment of metastatic melanoma; (iv) when to stop immunotherapy or targeted therapy in the metastatic setting; and (v) systemic versus local treatment for brain metastases. The expert panel was divided into five working groups to each address questions relating to one of the five topics outlined above. Relevant scientific literature was reviewed in advance. Recommendations were developed by the working groups and then presented to the entire panel for further discussion and amendment before voting. This manuscript presents the results relating to the management of metastatic melanoma, including findings from the expert panel discussions, consensus recommendations and a summary of evidence supporting each recommendation. All participants approved the final manuscript.

Neoadjuvant immunotherapy with combined ipilimumab and nivolumab in patients with melanoma with primary or in transit disease.

The introduction of new therapeutic agents has revolutionized the treatment of metastatic melanoma. The approval of adjuvant anti-programmed death-1 monotherapy with nivolumab or pembrolizumab, and dabrafenib plus trametinib has recently set a new landmark in the treatment of stage III melanoma. Now, clinical trials have shown that immune checkpoint blockade can be performed in a neoadjuvant setting, an approach established as a standard therapeutic approach for other tumour entities such as breast cancer. Recent studies suggest that a pathological response achieved by neoadjuvant immunotherapy is associated with long-term tumour control and that short neoadjuvant application of checkpoint inhibitors may be superior to adjuvant therapy. Most recently, neoadjuvant ipilimumab plus nivolumab in stage III melanoma was reported. With two courses of dose-optimized ipilimumab (1 mg kg-1 ) combined with nivolumab (3 mg kg-1 ), pathological responses were observed in 77% of patients, while only 20% of patients experienced grade 3 or 4 adverse events. However, the neoadjuvant trials employing combined immune checkpoint blockade conducted so far have excluded patients with in transit metastases, a common finding in stage III melanoma. Here we report four patients with in transit metastases or an advanced primary tumour who have been treated with neoadjuvant ipilimumab plus nivolumab according to the OpACIN-neo trial scheme (arm B). All patients achieved radiological disease control and a pathological response. None of the patients has relapsed so far. Linked Comment: Blankenstein and van Akkooi. Br J Dermatol 2020; 183:421-422.

GNAQ and GNA11 mutant nonuveal melanoma: a subtype distinct from both cutaneous and uveal melanoma.

BACKGROUND: GNAQ and GNA11 mutant nonuveal melanoma represent a poorly characterized rare subgroup of melanoma with a gene mutation profile similar to uveal melanoma. OBJECTIVES: To characterize these tumours in terms of clinical behaviour and genetic characteristics. METHODS: Patients with nonuveal GNAQ/11 mutated melanoma were identified from the prospective multicentre tumour tissue registry ADOREG, Tissue Registry in Melanoma (TRIM) and additional cooperating skin cancer centres. Extensive data on patient, tumour and treatment characteristics were collected retrospectively. Targeted sequencing was used to determine tumour mutational burden. Immunohistochemistry staining was performed for programmed death-ligand 1 and BRCA1-associated protein (BAP)1. Existing whole-exome cutaneous and uveal melanoma data were analysed for mutation type and burden. RESULTS: We identified 18 patients with metastatic GNAQ/11 mutant nonuveal melanoma. Tumours had a lower tumour mutational burden and fewer ultraviolet signature mutations than cutaneous melanomas. In addition to GNAQ and GNA11 mutations (nine each), six splicing factor 3b subunit 1 (SF3B1), three eukaryotic translation initiation factor 1A X-linked (EIF1AX) and four BAP1 mutations were detected. In contrast to uveal melanoma, GNAQ/11 mutant nonuveal melanomas frequently metastasized lymphatically and concurrent EIF1AX, SF3B1 and BAP1 mutations showed no apparent association with patient prognosis. Objective response to immunotherapy was poor with only one partial response observed in 10 treated patients (10%). CONCLUSIONS: Our findings suggest that GNAQ/11 mutant nonuveal melanomas are a subtype of melanoma that is both clinically and genetically distinct from cutaneous and uveal melanoma. As they respond poorly to available treatment regimens, novel effective therapeutic approaches for affected patients are urgently needed. What is already known about this topic? The rare occurrence of GNAQ/11 mutations in nonuveal melanoma has been documented. GNAQ/11 mutant nonuveal melanomas also harbour genetic alterations in EIF1AX, SF3B1 and BAP1 that are of prognostic relevance in uveal melanoma. What does this study add? GNAQ/11 mutant nonuveal melanomas show metastatic spread reminiscent of cutaneous melanoma, but not uveal melanoma. GNAQ/11 mutant nonuveal melanomas have a low tumour mutational burden that is higher than uveal melanoma, but lower than cutaneous melanoma. What is the translational message? Primary GNAQ/11 mutant nonuveal melanomas are a subtype of melanoma that is clinically and genetically distinct from both cutaneous and uveal melanoma. As metastatic GNAQ/11 mutant nonuveal melanomas respond poorly to available systemic therapies, including immune checkpoint inhibition, novel therapeutic approaches for these tumours are urgently needed. Linked Comment: Rafei-Shamsabadi. Br J Dermatol 2020; 183:806-807.

NF1 mutations in conjunctival melanoma.

BACKGROUND: Conjunctival 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. METHODS: A 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. RESULTS: Frequent 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). CONCLUSIONS: Similar 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.

[Molecular and immunohistochemical diagnostics in melanoma]. / Molekulare und immunhistochemische Diagnostik beim Melanom.

To provide appropriate therapy and follow-up to patients with malignant melanoma, proper diagnostics are of critical importance. Targeted therapy of advanced melanoma is based on the molecular genetic analyses of tumor tissue. In addition, sequencing of genes and other genetic approaches can provide insight into the origin of melanocytic tumors and can aid in distinguishing benign from malignant lesions. In this regard, spizoid neoplasms remain a challenging entity. Aside from genetic analyses of tumor tissue, immunohistochemistry remains an essential tool in melanoma diagnostics and TNM classification. With new immunotherapies being approved for advanced melanoma, immunohistochemistry to determine PD-L1 expression has gained clinical interest. While PD-L1 expression is associated with response to PD-1 blockade, a substantial number of patients without PD-L1 expression can still experience tumor remission upon treatment. In this review, current and future developments in melanoma diagnostics with regard to molecular genetics and immunohistochemistry are summarized. The utilization of such analyses in clinical decision making is also discussed.

[Kikuchi-Fujimoto's disease and adult-onset Still's disease. A rare co-occurence]. / Kikuchi-Fujimoto-Syndrom und adulter Morbus Still. Eine seltene Koinzidenz.

Kikuchi-Fujimoto's disease and adult-onset Still's disease are rare inflammatory conditions with overlapping clinical features. Adult-onset Still's disease causes high fevers, a typical salmon-colored rash, and joint pain. The principal symptom of Kikuchi's disease is cervical lymphadenopathy with typical histopathological features including extensive necrosis of the involved lymph nodes. Here, we report on a rare case of concurrent adult-onset Still's disease and Kikuchi-Fujimoto syndrome in a young Caucasian patient.

Differential influence of vemurafenib and dabrafenib on patients' lymphocytes despite similar clinical efficacy in melanoma.

BACKGROUND: Since the majority of melanomas eventually become resistant and progress, combining selective BRAF inhibitors (BRAFi) with immunotherapies has been proposed to achieve more durable treatment responses. Here, we explored the impact of selective BRAFi on the hosts' immune system. PATIENTS AND METHODS: Clinical data, whole blood counts (WBC) and serum lactate dehydrogenase (LDH) of 277 vemurafenib- and 65 dabrafenib-treated melanoma patients were evaluated. The frequency and phenotype of lymphocyte subpopulations were determined by flow cytometry while T cell cytokine secretion was measured by multiplex assays. RESULTS: Progression-free survival (PFS) as well as overall survival (OS) were similar in patients treated with either BRAFi. High pretreatment LDH was associated with shorter PFS and OS in both groups. During therapy, peripheral lymphocytes decreased by 24.3% (median, P < 0.0001) in vemurafenib-treated patients but remained unchanged in dabrafenib-treated patients (+1.2%, P = 0.717). Differentiation of peripheral lymphocytes of vemurafenib-treated patients showed a significant decrease in CD4(+) T cells (P < 0.05). Within CD4(+) T cells obtained during treatment, an increase in CCR7(+)CD45RA(+) (naïve) and a decrease in CCR7(+)CD45RA(-) (central memory) populations were found (P < 0.01 for both). Furthermore, secretion of interferon-γ and interleukin-9 by CD4(+) T cells was significantly lower in samples obtained during vemurafenib treatment compared with baseline samples. CONCLUSION: While both compounds have comparable clinical efficacy, vemurafenib but not dabrafenib decreases patients peripheral lymphocyte counts and alters CD4(+) T cell phenotype and function. Thus, selective BRAFi can significantly affect patients' peripheral lymphocyte populations. Fully understanding these effects could be critical for successfully implementing combinatorial therapies of BRAFi with immunomodulatory agents.

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.

ß-hydroxybutyrate is a metabolic regulator of proteostasis in the aged and Alzheimer disease brain.

Loss of proteostasis is a hallmark of aging and Alzheimer disease (AD). Here, we identify ß-hydroxybutyrate (ßHB), a ketone body, as a regulator of protein solubility in the aging brain. ßHB is a small molecule metabolite which primarily provides an oxidative substrate for ATP during hypoglycemic conditions, and also regulates other cellular processes through covalent and noncovalent protein interactions. We demonstrate ßHB-induced protein insolubility across in vitro, ex vivo, and in vivo mouse systems. This activity is shared by select structurally similar metabolites, is not dependent on covalent protein modification, pH, or solute load, and is observable in mouse brain in vivo after delivery of a ketone ester. Furthermore, this phenotype is selective for pathological proteins such as amyloid-ß, and exogenous ßHB ameliorates pathology in nematode models of amyloid-ß aggregation toxicity. We have generated a comprehensive atlas of the ßHB-induced protein insolublome ex vivo and in vivo using mass spectrometry proteomics, and have identified common protein domains within ßHB target sequences. Finally, we show enrichment of neurodegeneration-related proteins among ßHB targets and the clearance of these targets from mouse brain, likely via ßHB-induced autophagy. Overall, these data indicate a new metabolically regulated mechanism of proteostasis relevant to aging and AD.

Effect of long-term caloric restriction on DNA methylation measures of biological aging in healthy adults from the CALERIE trial.

The geroscience hypothesis proposes that therapy to slow or reverse molecular changes that occur with aging can delay or prevent multiple chronic diseases and extend healthy lifespan1-3. Caloric restriction (CR), defined as lessening caloric intake without depriving essential nutrients4, results in changes in molecular processes that have been associated with aging, including DNA methylation (DNAm)5-7, and is established to increase healthy lifespan in multiple species8,9. Here we report the results of a post hoc analysis of the influence of CR on DNAm measures of aging in blood samples from the Comprehensive Assessment of Long-term Effects of Reducing Intake of Energy (CALERIE) trial, a randomized controlled trial in which n = 220 adults without obesity were randomized to 25% CR or ad libitum control diet for 2 yr (ref. 10). We found that CALERIE intervention slowed the pace of aging, as measured by the DunedinPACE DNAm algorithm, but did not lead to significant changes in biological age estimates measured by various DNAm clocks including PhenoAge and GrimAge. Treatment effect sizes were small. Nevertheless, modest slowing of the pace of aging can have profound effects on population health11-13. The finding that CR modified DunedinPACE in a randomized controlled trial supports the geroscience hypothesis, building on evidence from small and uncontrolled studies14-16 and contrasting with reports that biological aging may not be modifiable17. Ultimately, a conclusive test of the geroscience hypothesis will require trials with long-term follow-up to establish effects of intervention on primary healthy-aging endpoints, including incidence of chronic disease and mortality18-20.

IRX-2, a novel immunotherapeutic, enhances and protects NK-cell functions in cancer patients.

BACKGROUND: IRX-2 is a primary biologic which has been used for the therapy of head and neck squamous cell cancer (HNSCC) with promising clinical results. Since NK-cell function is compromised in HNSCC patients, we tested the effects of IRX-2 on the restoration of human NK-cell functions in vitro. METHODS: Peripheral blood mononuclear cells (PBMC) were isolated from 23 HNSCC patients and 10 normal controls (NC). The NK-cell phenotype and functions were compared before and after culture ± IRX-2 or ± 50 IU/ml rhIL-2. Flow cytometry was used to study the NK-cell phenotype, cytotoxic activity and cytokine expression. RESULTS: Impaired NK-cell cytotoxicity in HNSCC patients was related to lower expression of NKG2D, NKp30 and NKp46 receptors (P < 0.05) and not to a decreased frequency of NK cells. Incubation of patients' NK cells with IRX-2 up-regulated the percentage of receptor-positive NK cells (P < 0.05). It also up-regulated cytotoxicity of patients' NK cells (P < 0.01) more effectively than rhIL-2 (P < 0.01). IRX-2, but not rhIL-2, protected NK cells from suppression mediated by TGF-ß, and it restored (P < 0.05) expression of activating NK-cell receptors and NK-cell cytotoxicity suppressed by TGF-ß. Expression of pSMAD was decreased in NK cells treated with IRX-2 but not in those treated with rhIL-2. CONCLUSIONS: IRX-2 was more effective than IL-2 in enhancing NK-cell cytotoxicity and protecting NK-cell function of HNSCC patients in vitro, emphasizing the potential advantage of IRX-2 as a component of future therapies for HNSCC.

United States university-based officer training and its influence on physical assessment test performance.

INTRODUCTION: Cadets participating in Reserve Officers' Training Corp (ROTC) at US universities undergo both Army Physical Fitness Test (APFT) and Occupational Physical Assessment Test (OPAT) evaluations prior to commissioning. The current study examined the relationship between ROTC physical training (PT) attendance and performance in the APFT and OPAT, and characterised physical activity of cadets outside of PT. METHODS: Twenty-six cadets' (21 males; age=20.81±2.48 years; height=171.16±8.62 cm; body mass=75.49±13.17 kg; body mass index=25.68±3.37 kg/m2) PT attendance, and diagnostic (week 1) and record (week 13) OPAT and APFT scores were documented. Paired samples t-tests evaluated differences in APFT and OPAT scores between the diagnostic and record tests. Pearson correlations were utilised to determine if a relationship existed between PT attendance and test performance. Participating cadets also completed monthly self-reported physical activity questionnaire (September, October, November); findings were reported using descriptive statistics. RESULTS: Cadets attended 87% of PT sessions between OPAT administrations and 85% between APFT administrations. Cadets significantly improved the following test components: standing long jump (p=0.034), seated power throw (p=0.029), shuttle run (p=0.005), sit-ups (p=0.003) and 2-mile run (p=0.045). A significant, positive correlation was found between PT attendance and APFT sit-ups improvements (r=0.473, p=0.015). Cadets' frequently reported additional physical activity days per week (range: 2.8-3.1 aerobic, 3.2-3.8 strength/power, 2.9-3.2 core strength/endurance). CONCLUSIONS: Regular participation in a single semester of ROTC PT was found to significantly increase cadets' scores in some, but not all, components of the APFT and OPAT. Self-reported physical activity results indicate that cadets regularly train outside of organised PT.

Dendritic cell generation and CD4+ CD25high FOXP3+ regulatory t cells in human head and neck carcinoma during radio-chemotherapy.

BACKGROUND: Regulatory T cells (Treg) and dendritic cells (DC) play an important role in tumor immunity and immune escape. However, their interplay and the effects of anti-cancer therapy on the human immune system are largely unknown. METHODS: For DC generation, CD14⊃+ monocytes were enriched by immunomagnetic selection from peripheral blood of advanced head and neck squamous cell carcinoma (HNSCC) patients and differentiated into immature DC using GM-SCF and IL-4. DC maturation was induced by addition of TNFα. The frequency of CD4⊃+CD25⊃highFOXP3⊃+ Treg in HNSCC patients was analyzed before and after radio-chemotherapy (RCT) by four-color flow cytometry. RESULTS: In HNSCC patients, the frequency of Treg (0.33 ± 0.06%) was significantly (p = 0.001) increased compared to healthy controls (0.11 ± 0.02%), whereas RCT had variable effects on the Treg frequency inducing its increase in some patients and decrease in others. After six days in culture, monocytes of all patients had differentiated into immature DC. However, DC maturation indicated by CD83 up-regulation (70.7 ± 5.5%) was successful only in a subgroup of patients and correlated well with lower frequencies of peripheral blood Treg in those patients. CONCLUSION: The frequency of regulatory T cells is elevated in HNSCC patients and may be modulated by RCT. Monocyte-derived DC in HNSCC patients show a maturation deficiency ex vivo. Those preliminary data may have an impact on multimodality clinical trials integrating cellular immune modulation in patients with advanced HNSCC.