Merkel cell polyomavirus pan-T antigen knockdown reduces cancer cell stemness and promotes neural differentiation independent of RB1.

Merkel cell carcinoma (MCC) is a highly aggressive skin cancer associated with integration of Merkel cell polyomavirus (MCPyV). MCPyV-encoded T-antigens (TAs) are pivotal for sustaining MCC's oncogenic phenotype, i.e., repression of TAs results in reactivation of the RB pathway and subsequent cell cycle arrest. However, the MCC cell line LoKe, characterized by a homozygous loss of the RB1 gene, exhibits uninterrupted cell cycle progression after shRNA-mediated TA repression. This unique feature allows an in-depth analysis of the effects of TAs beyond inhibition of the RB pathway, revealing the decrease in expression of stem cell-related genes upon panTA-knockdown. Analysis of gene regulatory networks identified members of the E2F family (E2F1, E2F8, TFDP1) as key transcriptional regulators that maintain stem cell properties in TA-expressing MCC cells. Furthermore, minichromosome maintenance (MCM) genes, which encodes DNA-binding licensing proteins essential for stem cell maintenance, were suppressed upon panTA-knockdown. The decline in stemness occurred simultaneously with neural differentiation, marked by the increased expression of neurogenesis-related genes such as neurexins, BTG2, and MYT1L. This upregulation can be attributed to heightened activity of PBX1 and BPTF, crucial regulators of neurogenesis pathways. The observations in LoKe were confirmed in an additional MCPyV-positive MCC cell line in which RB1 was silenced before panTA-knockdown. Moreover, spatially resolved transcriptomics demonstrated reduced TA expression in situ in a part of a MCC tumor characterized by neural differentiation. In summary, TAs are critical for maintaining stemness of MCC cells and suppressing neural differentiation, irrespective of their impact on the RB-signaling pathway.

Incidence and Relative Survival of Patients with Merkel Cell Carcinoma in North Rhine-Westphalia, Germany, 2008-2021.

BACKGROUND: To date, only a few population-representative studies have been carried out on the rare Merkel cell carcinoma (MCC). We provide incidence and survival estimates of MCC, including the conditional relative survival. METHODS: We analyzed data from the cancer registry of North Rhine-Westphalia, Germany, 2008-2021, covering a population of 18 million. We included all newly diagnosed MCCs and calculated age-standardized (old European Standard population) incidence rates and unconditional and conditional relative survival. RESULTS: Our analysis included 2164 MCC patients. The age-standardized incidence of MCC was 5.2 (men) and 3.8 (women) per million person-years. The 5-year relative survival was 58.8% (men) and 70.7% (women). Survival was lower among men than women in all age-sex groups and was highest for MCC of the upper extremity in both men (68.2%) and women (79.3%). The sex difference in survival is particularly due to the better survival of women with MCC of the head and neck. In terms of survival, the first two years are particularly critical. CONCLUSIONS: Our data validate the worse survival among men and highlights a more favorable prognosis for MCCs located on the limbs. The first two years after diagnosis of MCC are the years with the highest excess mortality.

A Prospective Study Investigating Immune Checkpoint Molecule and CD39 Expression on Peripheral Blood Cells for the Prognostication of COVID-19 Severity and Mortality.

In patients with COVID-19, broad panels of immune checkpoint molecules (ICPMs) and the purinergic signaling have not been studied in parallel. We aimed to perform in-depth immunophenotyping of major cell subsets present in human peripheral blood of COVID-19 patients and controls using PD1, TIM3, LAG3, TIGIT, and CD200R, as well as CD39, as markers for the purinergic signaling pathway. We studied 76 COVID-19 patients and 12 healthy controls using peripheral blood mononuclear cells on flow cytometry. Univariable and multivariable statistics were performed. All ICPMs studied were significantly overexpressed on different cell subsets of COVID-19 patients when compared with healthy controls. Elevated lactate dehydrogenase; C-reactive protein; age; and high expression of CD45+, CD39+CD45+, TIM3+CD39+CD4+CD45+, and TIM3+CD39+CD8+CD3+CD4+ cells were significantly associated with severe COVID-19. On multivariable analysis, however, only high expression of CD39+CD45+ (OR 51.4, 95% CI 1.5 to 1763) and TIM3+CD39+CD4+CD3+CD45+ (OR 22.6, 95% CI 1.8 to 277) cells was an independent predictor for severe COVID-19. In conclusion, numerous ICPMs are overexpressed in COVID-19 patients when compared with healthy controls, suggesting a pathophysiological role of these molecules in SARS-CoV-2 infection. However, only TIM3 in co-expression with CD39 remained as a significant independent prognostic ICPM on multivariable analysis. The flow cytometric evaluation of TIM3+CD39+CD4+CD3+CD45+, as well as CD39+CD45+, is a powerful tool for the prognostication of COVID-19 patients on hospital admission.

Current Progress in Vaccines against Merkel Cell Carcinoma: A Narrative Review and Update.

Merkel cell carcinoma is a rare, aggressive skin cancer that mainly occurs in elderly and immunocompromised patients. Due to the success of immune checkpoint inhibition in MCC, the importance of immunotherapy and vaccines in MCC has increased in recent years. In this article, we aim to present the current progress and perspectives in the development of vaccines for this disease. Here, we summarize and discuss the current literature and ongoing clinical trials investigating vaccines against MCC. We identified 10 articles through a PubMed search investigating a vaccine against MCC. From the international clinical trial database Clinical.Trials.gov, we identified nine studies on vaccines for the management of MCC, of which seven are actively recruiting. Most of the identified studies investigating a vaccine against MCC are preclinical or phase 1/2 trials. The vaccine principles mainly included DNA- and (synthetic) peptide-based vaccines, but RNA-based vaccines, oncolytic viruses, and the combination of vaccines and immunotherapy are also under investigation for the treatment of MCC. Although the management of MCC is changing, when compared to times before the approval of immune checkpoint inhibitors, it will still take some time before the first MCC vaccine is ready for approval.

Porocarcinomas with PAK1/2/3 fusions: a series of 12 cases.

AIMS: Porocarcinoma is a malignant sweat gland tumour differentiated toward the upper part of the sweat duct and may arise from the transformation of a preexisting benign poroma. In 2019, Sekine et al. demonstrated the presence of YAP1::MAML2 and YAP1::NUTM1 fusions in most poromas and porocarcinomas. Recently, our group identified PAK2-fusions in a subset of benign poromas. Herein we report a series of 12 porocarcinoma cases harbouring PAK1/2/3 fusions. METHODS AND RESULTS: Five patients were male and the median age was 79 years (ranges: 59-95). Tumours were located on the trunk (n = 7), on the thigh (n = 3), neck (n = 1), or groin area (n = 1). Four patients developed distant metastases. Microscopically, seven cases harboured a benign poroma component and a malignant invasive part. Ductal formations were observed in all, while infundibular/horn cysts and cells with vacuolated cytoplasm were detected in seven and six tumours, respectively. In three cases, the invasive component consisted of a proliferation of elongated cells, some of which formed pseudovascular spaces, whereas the others harboured a predominant solid or trabecular growth pattern. Immunohistochemical staining for CEA and EMA confirmed the presence of ducts. Focal androgen receptor expression was detected in three specimens. Whole RNA sequencing evidenced LAMTOR1::PAK1 (n = 2), ZDHHC5::PAK1 (n = 2), DLG1::PAK2, CTDSP1::PAK1, CTNND1::PAK1, SSR1::PAK3, CTNNA1::PAK2, RNF13::PAK2, ROBO1::PAK2, and CD47::PAK2. Activating mutation of HRAS (G13V, n = 3, G13R, n = 1, Q61L, n = 2) was present in six cases. CONCLUSION: Our study suggests that PAK1/2/3 fusions is the oncogenic driver of a subset of porocarcinomas lacking YAP1 rearrangement.

S1-Guideline Sebaceous Carcinoma.

Sebaceous gland carcinomas are rare malignant cutaneous adnexal tumors with sebocytic differentiation. The typical predilection area is the head and neck region, where sebaceous gland carcinomas are the most common malignant adnexal tumors of the skin. According to their localization a distinction is made between periocular and extraocular sebaceous gland carcinomas. Muir-Torre syndrome (MTS) should always be ruled out if it is suspected. In terms of prognosis, sebaceous gland carcinomas are potentially aggressive tumors with a clear tendency to recur and metastasize. Only small extraocular sebaceous gland carcinomas that have been completely resected have a very good prognosis. Sebaceous gland carcinomas most frequently metastasize lymphogenously to regional or distant lymph nodes; organ metastasis occurs less frequently. Periocular sebaceous gland carcinomas have a higher metastasis rate (up to 15%) than extraocular sebaceous gland carcinomas (up to 2%). Complete micrographically controlled surgery (MCS) of the primary tumor is the therapy of first choice, regardless of periocular or extraocular localization. Adjuvant or therapeutic radiotherapy may be considered. There is currently no established standard therapy for advanced, inoperable, or metastatic sebaceous gland carcinomas. Local procedures and systemic therapies such as chemotherapy or immunotherapy can be considered. The procedure should be determined individually by an interdisciplinary tumor board. Close follow-up care is recommended for these potentially aggressive carcinomas.

Merkel Cell Carcinoma: Integrating Epidemiology, Immunology, and Therapeutic Updates.

Merkel cell carcinoma (MCC) is a rare skin cancer characterized by neuroendocrine differentiation. Its carcinogenesis is based either on the integration of the Merkel cell polyomavirus or on ultraviolet (UV) mutagenesis, both of which lead to high immunogenicity either through the expression of viral proteins or neoantigens. Despite this immunogenicity resulting from viral or UV-associated carcinogenesis, it exhibits highly aggressive behavior. However, owing to the rarity of MCC and the lack of epidemiologic registries with detailed clinical data, there is some uncertainty regarding the spontaneous course of the disease. Historically, advanced MCC patients were treated with conventional cytotoxic chemotherapy yielding a median response duration of only 3 months. Starting in 2017, four programmed cell death protein 1 (PD-1)/programmed cell death-ligand 1 (PD-L1) immune checkpoint inhibitors-avelumab, pembrolizumab, nivolumab (utilized in both neoadjuvant and adjuvant settings), and retifanlimab-have demonstrated efficacy in treating patients with disseminated MCC on the basis of prospective clinical trials. However, generating clinical evidence for rare cancers, such as MCC, is challenging owing to difficulties in conducting large-scale trials, resulting in small sample sizes and therefore lacking statistical power. Thus, to comprehensively understand the available clinical evidence on various immunotherapy approaches for MCC, we also delve into the epidemiology and immune biology of this cancer. Nevertheless, while randomized studies directly comparing immune checkpoint inhibitors and chemotherapy in MCC are lacking, immunotherapy shows response rates comparable to those previously reported with chemotherapy but with more enduring responses. Notably, adjuvant nivolumab has proven superiority to the standard-of-care therapy (observation) in the adjuvant setting.

Eligibility of a novel BW + technology and comparison of sensitivity and specificity of different imaging methods for radiological caries detection.

OBJECTIVES: Bitewing radiography is considered to be of high diagnostic value in caries detection, but owing to projections, lesions may remain undetected. The novel bitewing plus (BW +) technology enables scrolling through radiographs in different directions and angles. The present study aimed at comparing BW + with other 2D and 3D imaging methods in terms of sensitivity, specificity, and user reliability. MATERIALS AND METHODS: Five human cadavers were used in this study. In three cadavers, natural teeth were transplanted post-mortem. BW + , two-dimensional (digital sensors, imaging plates, 2D and 3D bitewing radiographs) and 3D methods (high and low dose CBCT) were taken. Carious lesions were evaluated on 96 teeth at three positions (mesial, distal, and occlusal) and scored according to their level of demineralization by ten observers, resulting in 35,799 possible lesions across all observers and settings. For reference, µCT scans of all teeth were performed. RESULTS: Overall, radiographic evaluations showed a high rate of false-negative diagnoses, with around 70% of lesions remaining undetected, especially enamel lesions. BW + showed the highest sensitivity for dentinal caries and had comparatively high specificity overall. CONCLUSIONS: Within the limits of the study, BW + showed great potential for added diagnostic value, especially for dentinal caries. However, the tradeoff of diagnostic benefit and radiation exposure must be considered according to each patient's age and risk.

T antigen-specific CD8+ T cells associate with PD-1 blockade response in virus-positive Merkel cell carcinoma.

Merkel cell carcinoma (MCC) is a highly immunogenic skin cancer primarily induced by Merkel cell polyomavirus, which is driven by the expression of the oncogenic T antigens (T-Ags). Blockade of the programmed cell death protein-1 (PD-1) pathway has shown remarkable response rates, but evidence for therapy-associated T-Ag-specific immune response and therapeutic strategies for the nonresponding fraction are both limited. We tracked T-Ag-reactive CD8+ T cells in peripheral blood of 26 MCC patients under anti-PD1 therapy, using DNA-barcoded pMHC multimers, displaying all peptides from the predicted HLA ligandome of the oncoproteins, covering 33 class I haplotypes. We observed a broad T cell recognition of T-Ags, including identification of 20 T-Ag-derived epitopes we believe to be novel. Broadening of the T-Ag recognition profile and increased T cell frequencies during therapy were strongly associated with clinical response and prolonged progression-free survival. T-Ag-specific T cells could be further boosted and expanded directly from peripheral blood using artificial antigen-presenting scaffolds, even in patients with no detectable T-Ag-specific T cells. These T cells provided strong tumor-rejection capacity while retaining a favorable phenotype for adoptive cell transfer. These findings demonstrate that T-Ag-specific T cells are associated with the clinical outcome to PD-1 blockade and that Ag-presenting scaffolds can be used to boost such responses.

The RANKL inhibitor denosumab in combination with dual checkpoint inhibition is associated with increased CXCL-13 serum concentrations.

BACKGROUND: Recent evidence suggests additional immunomodulatory properties of RANKL inhibition possibly boosting the clinical efficacy of immune checkpoint inhibitors (ICI). METHODS: We conducted a prospective, multicentre clinical trial in unresectable stage IV melanoma patients with bone metastases who received denosumab in parallel with dual ICI (BONEMET) and performed comprehensive immune monitoring at baseline and 4, 12, and 24 weeks after initiation of therapy. Secondary endpoints included tolerability and efficacy. For comparison, biospecimens from melanoma patients treated with dual ICI without denosumab were analyzed accordingly and served as retrospective reference cohort. RESULTS: In both the BONEMET (n = 16) and the reference cohort (n = 18) serum levels of 17 cytokines, including IFNγ were significantly increased after 4 weeks of treatment. Patients who received ICI and denosumab showed a significantly higher increase in serum CXCL-13 and a significant decrease in VEGFc compared with the reference cohort. While no changes in T cell composition were observed at 4 weeks, patients in the BONEMET cohort showed a significant decrease in the peripheral naïve T-cell population and an increase in CD8+ effector cells after 12 weeks. Treatment-related adverse events occurred with comparable frequency (93.8% in the BONEMET cohort versus 83.3% in the reference cohort). 7/16 patients in the BONEMET cohort and 8/18 patients in the reference cohort achieved disease control. CONCLUSION: Denosumab in combination with dual ICI modulates cytokine expression and T-cell composition in peripheral blood. The upregulation of CXCL-13, a key factor for initiating tertiary lymphoid structures, strengthens the hypothesis that denosumab indeed boost immunological effects.

Is online-only learning as effective as blended learning? A longitudinal study comparing undergraduate students' performance in oral radiology.

INTRODUCTION: Blended learning seems to be an effective teaching concept in oral radiology. During the COVID-19 pandemic, blended learning shifted towards online-only learning. The aim of the present study was to compare the effectiveness of pandemic online-only and pre-pandemic blended learning in three consecutive oral radiology courses (C1, C2 and C3) and to examine whether additional video-based e-learning modules (VBLMs) had a positive impact on undergraduate students' performance during pandemic semesters. MATERIALS AND METHODS: Data from 205 undergraduate dental students participating either in a blended learning or an online-only learning concept were analysed. Pre-pandemic blended learning comprised face-to-face seminars and access to an oral radiology platform (ORP). Pandemic online-only learning comprised online seminars, access to the ORP and additional VBLMs (two VBLMs for C1, four VBLMs for C2 and six VBLMs for C3). Through standardised e-exams at the beginning and end of each semester, performance in final exams and knowledge gain were compared between the two groups. RESULTS: No significant differences in scores in final exams (p = .11) and knowledge gain (p = .18) were found when comparing the pre-pandemic and pandemic groups. On course level, however, students receiving a lower number of VBLMs performed significantly worse in final exams (C1: p < .01, C2: p = .02) and showed inferior knowledge gain (C2: p < .01) during the pandemic. CONCLUSIONS: Within the limitations of the study, the present investigation confirmed that pandemic online-only learning involving VBLMs might be as effective as pre-pandemic blended learning.

Eligibility and efficacy of a CPC- and CHX-based antiviral mouthwash for the elimination of SARS-CoV-2 from the saliva: A randomized, double-blind, controlled clinical trial.

AIM: This study aimed at investigating the efficacy of a 0.05% cetylpyridinium chloride-0.05% chlorhexidine (CPC-CHX) mouthwash in reducing viral load in the saliva as compared with sterile water. MATERIALS AND METHODS: Forty SARS-CoV-2 positive patients were asked to dispense 4 mL of saliva. Half the patients rinsed for 60 s with 15 mL CPC-CHX, and the remaining patients rinsed with sterile water (control). Four millilitres of saliva were collected after 15, 30 and 60 min after rinsing. Quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA) specific for SARS-CoV-2 nucleocapsid protein were performed. For ELISA, the intact (representing the active virus) to total virus load (I/T) was calculated. RESULTS: SARS-CoV-2 copy numbers/mL from RT-qPCR tended to decrease in the control group, whereas in the CPC-CHX group, an increase was observed after T30. However, mixed linear model analysis revealed no statistical differences between groups (p = .124), time points (p = .616) and vaccinated or non-vaccinated patients (p = .953). Similarly, no impact of group (p = .880), time points (p = .306) and vaccination (p = .711) was observed for I/T ratio values. CONCLUSIONS: Within the limitation of this study, there was no evidence that the intervention reduced salivary SARS-CoV-2 viral load during the course of 60 min. Therefore, commonly used pre-procedural rinsing might not be clinically relevant.

Intratumoural and systemic inflammation as predictors for treatment response in BRAF-mutated melanoma patients under targeted therapies.

Intratumoural as well as systemic inflammation in melanoma has thoroughly been studied in the context of patients treated with immune checkpoint inhibitors but not with BRAF/MEK inhibitors (BRAFi/MEKi). We aimed to study whether parameters of intratumoral and systemic inflammation correlate with clinical outcome in patients with BRAF-mutant metastatic melanoma treated with BRAFi/MEKi. We studied 51 CM patients with unresectable stage III or IV who had the indication for BRAFi/MEKi treatment based on confirmed BRAF mutation. Baseline systemic immune-inflammation markers such as the systemic immune-inflammation index (SII) and the expression of intratumoral inflammation markers such as COX-2 protein expression were correlated with clinical outcome measures. On multivariable analyses, lower intratumoral COX-2 expression (OR 33.9, 95% CI 3.2-356.8) and lower SII (OR 6.3, 95% CI 1.1-34.8) proved to be significant independent predictors for objective response to targeted therapy. Elevated S100B (HR 1.2, 95% CI 1.03-1.3) was a significant predictor for progressive disease. Moreover, elevated S100B (HR 1.37, 95% CI 1.14-1.65) and LDH (HR 1.002, 95% CI 1.0001-1.003) were significant independent predictors for melanoma-specific death. In conclusion, the present study indicates that low SII values and low intratumoral COX-2 protein expression are significant independent predictors for treatment response to BRAFi/MEKi.

Is there any sustained effect of the coronavirus disease 2019 pandemic? - A controlled trial comparing pre- and post-pandemic oral radiology courses.

PURPOSE/OBJECTIVES: Due to the coronavirus disease 2019 (COVID-19) pandemic, undergraduate education shifted towards online-only formats from April 2020 until July 2021. Previous research indicated a negative effect on students' competence development, and it remains unclear whether blended learning concepts could provide compensation. Therefore, the present study aimed to compare pre- to post-pandemic students' performance in standardized baseline (BL) and final exams (FE), as well as the associated knowledge gain (KG) in three consecutive undergraduate blended learning-based oral radiology courses (C1-3). METHODS: Ninety-four students participated during two pre-pandemic semesters (October 17-July 18), and ninety-eight students during two post-pandemic semesters (October 21-July 22). Before the pandemic, conventional face-to-face lectures were combined with an oral radiology platform. Two years into the COVID-19 pandemic, additional video-based e-learning modules were adopted from the pandemic online-only curriculum. In each semester, skills and KG were assessed by conducting standardized BL and FE. Students' performance in BL, FE, and the associated KG during pre-pandemic semesters was compared to post-pandemic semesters. RESULTS: In post-pandemic courses, students showed significantly lower BL scores, whereas KG was significantly higher in post- compared to pre-pandemic semesters (27.42% vs. 10.64%, p < 0.001, respectively). FE scores in C1 significantly improved from pre- to post-pandemic semesters. CONCLUSIONS: Within the limitations of our study, the lower BL scores confirmed the negative effect of the pandemic on competence development. Blended learning concepts seem to compensate for this effect and enable students to return to pre-pandemic levels. Future studies are needed to assess the additional impact of video-based e-learning modules.

S-SCAM is essential for synapse formation.

Synapse formation is critical for the wiring of neural circuits in the developing brain. The synaptic scaffolding protein S-SCAM/MAGI-2 has important roles in the assembly of signaling complexes at post-synaptic densities. However, the role of S-SCAM in establishing the entire synapse is not known. Here, we report significant effects of RNAi-induced S-SCAM knockdown on the number of synapses in early stages of network development in vitro. In vivo knockdown during the first three postnatal weeks reduced the number of dendritic spines in the rat brain neocortex. Knockdown of S-SCAM in cultured hippocampal neurons severely reduced the clustering of both pre- and post-synaptic components. This included synaptic vesicle proteins, pre- and post-synaptic scaffolding proteins, and cell adhesion molecules, suggesting that entire synapses fail to form. Correspondingly, functional and morphological characteristics of developing neurons were affected by reducing S-SCAM protein levels; neurons displayed severely impaired synaptic transmission and reduced dendritic arborization. A next-generation sequencing approach showed normal expression of housekeeping genes but changes in expression levels in 39 synaptic signaling molecules in cultured neurons. These results indicate that S-SCAM mediates the recruitment of all key classes of synaptic molecules during synapse assembly and is critical for the development of neural circuits in the developing brain.

Corrigendum: Applications and techniques for fast machine learning in science.

[This corrects the article DOI: 10.3389/fdata.2022.787421.].