Expression of DNA mismatch repair proteins in melanoma patients treated with immune checkpoint inhibitors.

PURPOSE: To investigate the protein expression of DNA mismatch repair (MMR) proteins in patients with cutaneous melanoma (CM) under immune checkpoint inhibitor (ICI) therapy. METHODS: Immunohistochemistry was performed on tumor tissue for MMR proteins MLH1, MSH2, MSH6, and PMS2 in 50 metastatic CM patients treated with ICI (ipilimumab, nivolumab, pembrolizumab). RESULTS: Best overall response (BOR) rate was 48% (24/50). Reduced MMR protein expression (nuclear expression in < 80% of tumor cells) was observed in 8 patients (16%). Compared to other clinical parameters, baseline neutrophil/lymphocyte ratio and reduced intratumoral MMR protein expression (P = 0.0033) were determined as the only parameters significantly associated with favorable BOR. However, in this small study population, reduced MMR protein expression did not reach statistical significance in multivariate analysis. CONCLUSION: Reduced MMR protein expression is observed in CM and might predict favorable BOR in patients treated with ICI, as was observed for other entities. However, these findings need to be substantiated in larger patient cohorts.

Pan-immune-inflammation value independently predicts disease recurrence in patients with Merkel cell carcinoma.

PURPOSE: We aimed to determine whether the pan-immune-inflammation value (PIV) of patients with Merkel cell carcinoma (MCC) at primary diagnosis differs from controls and whether it is associated with disease stage and outcome. METHODS: In this retrospective study, we recruited MCC patients with stage I-III. PIV was calculated from absolute complete blood cell counts obtained within one week at MCC diagnosis as follows: [neutrophils (103/mm3) × platelets (103/mm3) × monocytes (103/mm3)]/lymphocytes (103/mm3). As controls, we studied age-gender-matched cutaneous melanoma (CM, stage I-III) patients and healthy controls (HC). Univariate and multivariate statistics were used. RESULTS: The median PIV in MCC patients was significantly increased compared to both CM patients as well as healthy controls. PIV of MCC patients in stage II and III was significantly higher compared to stage I patients. ROC analysis revealed that MCC recurrence was significantly associated with a PIV greater than 372 [p < 0.0001, Youden index 0.58; hazard ratio: 4 (95% confidence interval: 1.7 to 9.2)]. In multivariate analysis, only a PIV greater than 372 and higher MCC stage were determined as independent predictors for disease recurrence. CONCLUSION: We determined, for the first time, the prognostic ability of the promising blood-based biomarker PIV in MCC patients and observed that PIV is increased in MCC patients in dependence on disease stage and independently predicts MCC recurrence.

The pan-immune-inflammation value and systemic immune-inflammation index in advanced melanoma patients under immunotherapy.

PURPOSE: To evaluate the pan-immune-inflammation value (PIV) and systemic immune-inflammation index (SII) in patients with cutaneous melanoma (CM) under immune checkpoint inhibitor (ICI) therapy. METHODS: PIV and SII were calculated before the start of ICI therapy and at time of progression/death in patients with metastatic CM (stage III/IV). Sex-age-matched CM patients in stage I/II and healthy subjects (HC) served as controls. RESULTS: The median PIV of stage III/IV patients was significantly (P = 0.0011) higher than in stage I/II patients and HC. SII was significantly (P = 0.00044) lower in HC than in CM patients. At baseline, PIV and SII did significantly correlate with lactate dehydrogenase (P = 0.045/0.017). However, ROC curve statistics revealed that SII and PIV were not significantly associated with clinical parameters, including best response to ICI treatment (P = 0.87/0.64), progression-free survival (P = 0.73/0.91), and melanoma-specific survival (P = 0.13/0.17). Moreover, there were no significant changes of PIV and SII from baseline to progression/death (P = 0.38/0.52). CONCLUSIONS: Even though both immune-inflammation biomarkers showed some power to differentiate between CM stages and HC, respectively, PIV and SII seem not to be significant predictors for clinical outcome measures of CM patients under ICI therapy.

Comparison of mutation profiles in primary melanomas and corresponding nodal naevi using next-generation sequencing.

BACKGROUND: Nodal naevi (NN) represent aggregates of melanocytes within peripheral lymph nodes. NN are relatively often found in patients with malignant melanoma (MM), and may mimic metastatic disease. AIM: To study mutation profiles in MM and NN to find out whether NN descend from a primary MM. METHODS: Next-generation sequencing was performed on formalin-fixed paraffin-embedded tissue of 26 pairs of primary MM and corresponding NN detected by sentinel lymph node biopsy, and 29 MM-characteristic genes were investigated. RESULTS: In this study, 90% of mutations were detected exclusively in either MM or NN, but not both, in the same patient; the percentage of identical NN and MM mutations in the same individual was only 10%. The most frequently discovered shared mutations were a C>G substitution in the CDKN2A gene and in-frame deletion in ARID1A. Oncogenic driver mutations were frequently observed in MM but only rarely in NN. About three-quarters of mutations in both MM and NN were characterized by C>T or G>A substitutions. The detected rate of ultraviolet (UV)-related C>T base changes was comparably high in both primary MM (35%) and NN (32%). CONCLUSIONS: Based on our data, it seems that NN descend from previously UV-exposed BRAF wildtype cutaneous melanocytes, rather than from primary MM or arrested progenitor cells.

High tumour mutational burden and EGFR/MAPK pathway activation are therapeutic targets in metastatic porocarcinoma.

BACKGROUND: Eccrine porocarcinoma (EPC) is a rare skin cancer arising from the eccrine sweat glands. Due to the lack of effective therapies, metastasis is associated with a high mortality rate. OBJECTIVES: To investigate the drivers of EPC progression. METHODS: We carried out genomic and transcriptomic profiling of metastatic EPC (mEPC), validation of the observed alterations in an EPC patient-derived cell line, confirmation of relevant observations in a large patient cohort of 30 tumour tissues, and successful treatment of a patient with mEPC under the identified treatment regimens. RESULTS: mEPC was characterized by a high tumour mutational burden (TMB) with an ultraviolet signature, widespread copy number alterations and gene expression changes that affected cancer-relevant cellular processes such as cell cycle regulation and proliferation, including a pathogenic TP53 (tumour protein 53) mutation, a copy number deletion in the CDKN2A (cyclin dependent kinase inhibitor 2A) region and a CTNND1/PAK1 [catenin delta 1/p21 (RAC1) activated kinase 1] gene fusion. The overexpression of EGFR (epidermal growth factor receptor), PAK1 and MAP2K1 (mitogen-activated protein kinase kinase 1; also known as MEK1) genes translated into strong protein expression and respective pathway activation in the tumour tissue. Furthermore, a patient-derived cell line was sensitive to EGFR and MEK inhibition, confirming the functional relevance of the pathway activation. Immunohistochemistry analyses in a large patient cohort showed the relevance of the observed changes to the pathogenesis of EPC. Our results indicate that mEPC should respond to immune or kinase inhibitor therapy. Indeed, the advanced disease of our index patient was controlled by EGFR-directed therapy and immune checkpoint inhibition for more than 2 years. CONCLUSIONS: Molecular profiling demonstrated high TMB and EGFR/MAPK pathway activation to be novel therapeutic targets in mEPC.

[Kawasaki syndrome in a 9-year-old boy as a result of COVID-19]. / Kawasaki-Syndrom bei einem 9-jährigen Jungen infolge von COVID-19.

Since the beginning of 2020 an increase in a Kawasaki-like disease has been noted. The WHO assumes a connection to the COVID-19 pandemic and it is defined as the multisystem inflammatory syndrome in children (MIS-C) associated with coronavirus disease 2019.A 9-year-old boy attended the pediatric emergency department with persistent fever and then developed a classical Kawasaki syndrome with affection of the left coronary artery. A specific origin of an infection could not be detected. The SARS-CoV-2 PCR was negative. In due course positive SARS-CoV­2 antibodies were detected. The patient was treated with intravenous immunoglobulins, ASS and a glucocorticoid, which led to an improvement in the clinical and echocardiographic state of the patient.

Response durability after cessation of immune checkpoint inhibitors in patients with metastatic Merkel cell carcinoma: a retrospective multicenter DeCOG study.

BACKGROUND: Immune checkpoint inhibitors (ICI) have led to a prolongation of progression-free and overall survival in patients with metastatic Merkel cell carcinoma (MCC). However, immune-mediated adverse events due to ICI therapy are common and often lead to treatment discontinuation. The response duration after cessation of ICI treatment is unknown. Hence, this study aimed to investigate the time to relapse after discontinuation of ICI in MCC patients. METHODS: We analyzed 20 patients with metastatic MCC who have been retrospectively enrolled at eleven skin cancer centers in Germany. These patients have received ICI therapy and showed as best overall response (BOR) at least a stable disease (SD) upon ICI therapy. All patients have discontinued ICI therapy for other reasons than disease progression. Data on treatment duration, tumor response, treatment cessation, response durability, and tumor relapse were recorded. RESULTS: Overall, 12 of 20 patients (60%) with MCC relapsed after discontinuation of ICI. The median response durability was 10.0 months. Complete response (CR) as BOR to ICI-treatment was observed in six patients, partial response (PR) in eleven, and SD in three patients. Disease progression was less frequent in patients with CR (2/6 patients relapsed) as compared to patients with PR (7/11) and SD (3/3), albeit the effect of initial BOR on the response durability was below statistical significance. The median duration of ICI therapy was 10.0 months. Our results did not show a correlation between treatment duration and the risk of relapse after treatment withdrawal. Major reasons for discontinuation of ICI therapy were CR (20%), adverse events (35%), fatigue (20%), or patient decision (25%). Discontinuation of ICI due to adverse events resulted in progressive disease (PD) in 71% of patients regardless of the initial response. A re-induction of ICI was initiated in 8 patients upon tumor progression. We observed a renewed tumor response in 4 of these 8 patients. Notably, all 4 patients showed an initial BOR of at least PR. CONCLUSION: Our results from this contemporary cohort of patients with metastatic MCC indicate that MCC patients are at higher risk of relapse after discontinuation of ICI as compared to melanoma patients. Notably, the risk of disease progression after discontinuation of ICI treatment is lower in patients with initial CR (33%) as compared to patients with initial PR (66%) or SD (100%). Upon tumor progression, re-induction of ICI is a feasible option. Our data suggest that the BOR to initial ICI therapy might be a potential predictive clinical marker for a successful re-induction.

Expression of Hedgehog signalling molecules in microcystic adnexal carcinoma.

BACKGROUND: Microcystic adnexal carcinoma (MAC) is a rare skin neoplasm that has not been characterized on a molecular basis. AIM: To assess expression profiles of Hedgehog (HH) signalling molecules in MAC and control tumours. METHODS: Immunohistochemistry was performed for Sonic Hedgehog (SHH), Indian Hedgehog (IHH), Patched 1 (PTCH1) and Smoothened (SMO) on patient MAC tissue (n = 26) and control tumour tissue, including syringoma (SyG; n = 11), trichoepithelioma (TE; n = 11) and basal cell carcinoma (BCC; n = 12) tissues. RESULTS: Patched 1 and SMO immunoreactivity was significantly higher in BCC than in SyG, TE or MAC (P < 0.001 and P < 0.03, respectively). The highest IHH expression was observed in BCC and TE compared with SyG and MAC (P < 0.04). Notably, the highest SHH protein expression was observed in SyG compared with MAC, TE and even BCC (P < 0.001). In patients with MAC, SMO immunoreactivity significantly (r = 0.51; P < 0.01) correlated with PTCH1 expression. Further correlation studies did not show significant associations between the HH expression markers assessed (P > 0.05). CONCLUSION: Our results indicate that alterations of the HH signalling are unlikely to play a major role in the pathogenesis of MAC, which is in contrast to the morphologically similar BCC and TE. Our observation provides additional information to the limited molecular pathology knowledge on this rare tumour.

The predictive and prognostic significance of cell-free DNA concentration in melanoma.

BACKGROUND: Melanoma is the leading cause of skin cancer-related deaths worldwide. While there have been significant improvements in the treatment of advanced melanoma in the past decade, biomarker development lagged behind. OBJECTIVES: The majority of liquid biopsy biomarkers rely on the analyses of oncogenic mutations; however, about 20% of melanoma patients are wild type. Therefore, validation of universal predictive and prognostic biomarkers is urgently needed. METHODS: We analysed plasma samples in a discovery cohort (n = 20) and expansion cohort (n = 166) of metastatic melanoma patients and healthy donors (n = 116). Total plasma circulating cell-free DNA (cfDNA) concentrations were measured on the Qubit® platform using assays for single-(ss) and double (ds)-stranded DNA, DNA spectrophotometry and RNase P qPCR. We explored the diagnostic, predictive and prognostic potential of cfDNA concentration by bio-statistical methods and established a cfDNA threshold for risk stratification. RESULTS: Our selected best method was Qubit® dsDNA assay which quantified higher plasma cfDNA concentrations in melanoma patients than in healthy controls (AUC 72%). Measurement of baseline cfDNA concentration revealed that high cfDNA was associated with presence of metastases and higher AJCC stage (P < 0.05). Furthermore, high baseline cfDNA was an indicator of shorter overall survival in patients with oncogenic mutations (HR 2.12, P = 0.0008), and in wild-type patients (HR 5.55, P < 0.0001). CONCLUSIONS: We provide evidence that total cfDNA can be used as a biomarker for melanoma irrespective of the tumour genotype and can provide information on tumour load, risk of progression and risk of death.

["Flipped classroom"-A future concept for student teaching in ophthalmology?] / "Flipped classroom" ­ Ein Zukunftskonzept für die studentische Lehre in der Augenheilkunde?

BACKGROUND: "Flipped classroom" is a didactic teaching concept in which learning contents are prepared by self-study with arranged tools before the classroom session. The concept offers the advantage of a uniform knowledge base for the students at the beginning of the course and also the advantage of a greater theoretical knowledge, which creates more opportunities for practical exercises, application and consolidation in the subsequent joint teaching units. This study describes the establishment and application of such a model in student teaching in ophthalmology and analyzes the student's evaluation. METHODOLOGY: For the winter term 2018/2019, a new teaching module was designed and established in a cooperation between the department of ophthalmology and the Institute for Education and Study Affairs (IfAS) at the medical faculty of the University of Münster. A uniform training of the lecturers as well as a preparation of the students for the restructuring took place. After the course the evaluation of the students was recorded and evaluated using a standardized online evaluation. RESULTS: Between the winter semester 2018/2019 and the winter semester 2019/2020, an average of 112.3 ± 4.0 students were taught with the "flipped classroom" model. Of these 93.7% were able to give an assessment. In the previous semesters with the old teaching concept (summer semester 2015 to summer semester 2018), the average number of students was 115.4 ± 15.1 with an assessment rate of 93.3%. The new teaching concept achieved on average a better assessment than the old module. CONCLUSION: With a "flipped classroom" space and flexibility can be generated for a more individual course preparation and at the same time a higher practical part. Further studies are needed to analyze whether this also enables a sustainable transfer of knowledge.

Does very early timing of lymph node surgery after resection of the primary tumour improve the clinical outcome of patients with melanoma?

BACKGROUND: In patients with cutaneous melanoma (CM), the time span between resection of the primary tumour and sentinel lymph node biopsy (SLNB) as well as the subsequent interval between SLNB and complete lymph node dissection (CLND) varies greatly. AIM: To determine whether very early timing of SLNB after resection of the primary tumour, or timing of CLND after SLNB affect the clinical outcome of patients with CM, compared with longer time intervals. METHODS: We compared the time spans between complete resection of the primary tumour and SLNB, and the interval between SLNB and CLND in a cohort of 896 patients with melanoma who had undergone SLNB. An interval between primary resection and SLNB or between SLNB and CLND of up to 7 days was classified as very early (VE-SLNB and VE-CLND, respectively). This time span was compared with intervals of > 7 days. Univariate and multivariate statistics were performed. RESULTS: VE-SLNB was significantly associated with the presence of micrometastases. However, this was probably due to tumour thickness being significantly higher in patients with VE-SLNB compared with patients with later SLNB. Importantly, VE-SLNB was not significantly associated with disease relapse and VE-CLND was not associated with melanoma-specific death. CONCLUSIONS: VE-SLNB and VE-CLND neither improved nor worsened the clinical outcome of patients. Thus, timing of SLNB and CLND has no influence on the overall clinical outcome of patients with melanoma. Our findings support the rational planning of lymph node surgery after resection of the primary tumour and provide help for effective patient counselling.

Expression of Lefty predicts Merkel cell carcinoma-specific death.

BACKGROUND: Lefty and Nodal are transforming growth factor ß-related proteins, which, beside their role in determination of laterality during embryogenesis, have also been linked with cancer progression. OBJECTIVES: Prompted by the observed significant left-sided laterality of Merkel cell carcinoma (MCC), we addressed whether Lefty and Nodal are expressed in MCC and correlated expression patterns with clinical parameters such as MCC laterality and patient outcome. METHODS: Expression of Lefty and Nodal in primary MCC was assessed in 29 patients by immunohistochemistry. The histology (H-)score was calculated and correlated with clinical parameters. RESULTS: The median (range) H-score of Lefty and Nodal was 17.6 (0-291) and 74.9 (0.7-272), respectively. There was a significant correlation between Lefty expression and Nodal expression (correlation coefficient of 0.60, P = 0.0006). There was no significant correlation between Lefty expression and Nodal expression with either tumour laterality, gender, age, Merkel cell polyomavirus status, disease stage, anatomical localization of primary tumours or disease relapse. On univariate analysis, low Lefty expression and Nodal expression were significantly associated with MCC-specific death (P = 0.010 and P = 0.019, respectively). On univariate analysis, low Lefty expression was the only significant independent predictor for MCC-specific death (P = 0.025) as indicated by an odds ratio of 14 (95% CI: 1.43-137.33). CONCLUSIONS: Lefty and Nodal are frequently expressed in MCC, but not correlated with tumour laterality. Importantly, our data suggest that a low level of Lefty expression in primary MCC is a strong predictor of MCC-specific death.

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.

Search for L5 Earth Trojans with DECam.

Most of the major planets in the Solar System support populations of co-orbiting bodies, known as Trojans, at their L4 and L5 Lagrange points. In contrast, Earth has only one known co-orbiting companion. This paper presents the results from a search for Earth Trojans using the DECam instrument on the Blanco Telescope at CTIO. This search found no additional Trojans in spite of greater coverage compared to previous surveys of the L5 point. Therefore, the main result of this work is to place the most stringent constraints to date on the population of Earth Trojans. These constraints depend on assumptions regarding the underlying population properties, especially the slope of the magnitude distribution (which in turn depends on the size and albedo distributions of the objects). For standard assumptions, we calculate upper limits to a 90% confidence limit on the L5 population of N ET < 1 for magnitude H < 15.5, N ET =60-85 for H < 19.7, and N ET = 97 for H=20.4. This latter magnitude limit corresponds to Trojans ∼300 m in size for albedo 0.15. At H=19.7, these upper limits are consistent with previous L4 Earth Trojan constraints and significantly improve L5 constraints.

Decline of programmed death-1-positive circulating T regulatory cells predicts more favourable clinical outcome of patients with melanoma under immune checkpoint blockade.

BACKGROUND: The role of T regulatory lymphocytes (Tregs) and their immunosuppressive mechanisms in the context of programmed death (PD)-1 blockade is not completely understood. OBJECTIVES: To assess the impact of PD-1-blocking antibody treatment on Treg subpopulations in the blood. METHODS: We studied circulating Treg subpopulations in patients with melanoma under nivolumab or pembrolizumab treatment using flow cytometry and correlated these findings with clinical outcomes. RESULTS: These analyses revealed that the frequency of CD4+  CD25++  CD127-  PD-1+ lymphocytes (PD-1+ Tregs) significantly decreased after the first cycle of immunotherapy (23% vs. 8·6%, P = 0·043). Compared with patients who did not show a significant decline of PD-1+ Tregs after the first treatment, those who did had better clinical outcomes with respect to progression-free survival (PFS, P = 0·022) and melanoma-specific death (MSD, P = 0·0038). Multivariate analysis confirmed that a significant decline of PD-1+ Tregs in peripheral blood after the first treatment cycle is a significant predictor of more favourable PFS and MSD (P = 0·04 and 0·017, respectively). Interestingly, the occurrence of immune-related adverse events was also an independent predictor for decreased risk of MSD (P = 0·047; odds ratio 0·064, 95% confidence interval 0·0042-0·97). CONCLUSIONS: We provide preliminary evidence that circulating PD-1+ Tregs rapidly decline after the initiation of treatment with PD-1-blocking antibodies, which is associated with reduced risk of melanoma progression and MSD. Patients showing no decrease of these PD-1+ Tregs in peripheral blood are characterized by an impaired response to immune checkpoint blockade and worse outcome. What's already known about this topic? Programmed death (PD)-1-blocking antibodies are highly effective in melanoma treatment. However, more than half of patients do not benefit from this therapy and to date it is difficult to predict which patients will respond to it. What does this study add? PD-1-blocking antibody therapy rapidly results in a decline of circulating PD-1+ T regulatory cells (Tregs). What is the translational message? Patients showing a decrease of PD-1+ Tregs appear to have better clinical outcome under PD-1 treatment.