Differential Immunoexpression of Inhibitory Immune Checkpoint Molecules and Clinicopathological Correlates in Keratoacanthoma, Primary Cutaneous Squamous Cell Carcinoma and Metastases.

Beyond established anti-programmed cell death protein 1/programmed cell death ligand 1 immunotherapy, T-cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibition motif domain (TIGIT) and its ligand CD155 are promising novel inhibitory immune checkpoint targets in human malignancies. Yet, in cutaneous squamous cell carcinoma, evidence on the collective expression patterns of these inhibitory immune checkpoints is scarce. Complete tumour sections of 36 cutaneous squamous cell carcinoma, 5 cutaneous metastases and 9 keratoacanthomas, a highly-differentiated, squamoproliferative tumour, with disparately benign biologic behaviour, were evaluated by immunohistochemistry for expression of programmed cell death ligand 1 (Tumor Proportion Score, Immune Cell Score), TIGIT, CD155 and CD8+ immune infiltrates. Unlike keratoacanthomas, cutaneous squamous cell carcinoma displayed a strong positive correlation of programmed cell death ligand 1 Tumor Proportion Score and CD115 expression (p < 0.001) with significantly higher programmed cell death ligand 1 Tumor Proportion Score (p < 0.001) and CD155 expression (p < 0.01) in poorly differentiated G3-cutaneous squamous cell carcinoma compared with keratoacanthomas. TIGIT+ infiltrates were significantly increased in programmed cell death ligand 1 Immune Cell Score positive primary tumours (p = 0.05). Yet, a strong positive correlation of TIGIT expression with CD8+ infiltrates was only detected in cutaneous squamous cell carcinoma (p < 0.01), but not keratoacanthomas. Providing a comprehensive overview on the collective landscape of inhibitory immune checkpoint expression, this study reveals associations of novel inhibitory immune checkpoint with CD8+ immune infiltrates and tumour differentiation and highlights the TIGIT/CD155 axis as a potential new target for cutaneous squamous cell carcinoma immunotherapy.

Functionally distinct cancer-associated fibroblast subpopulations establish a tumor promoting environment in squamous cell carcinoma.

Cutaneous squamous cell carcinoma (cSCC) is a serious public health problem due to its high incidence and metastatic potential. It may progress from actinic keratosis (AK), a precancerous lesion, or the in situ carcinoma, Bowen's disease (BD). During this progression, malignant keratinocytes activate dermal fibroblasts into tumor promoting cancer-associated fibroblasts (CAFs), whose origin and emergence remain largely unknown. Here, we generate and analyze >115,000 single-cell transcriptomes from healthy skin, BD and cSCC of male donors. Our results reveal immunoregulatory and matrix-remodeling CAF subtypes that may derive from pro-inflammatory and mesenchymal fibroblasts, respectively. These CAF subtypes are largely absent in AK and interact with different cell types to establish a pro-tumorigenic microenvironment. These findings are cSCC-specific and could not be recapitulated in basal cell carcinomas. Our study provides important insights into the potential origin and functionalities of dermal CAFs that will be highly beneficial for the specific targeting of the cSCC microenvironment.

Patterns of photoprotective behavior, cumulative sun-exposure and skin cancer risk among solid organ transplant recipients: a dermatology transplant clinic experience.

BACKGROUND: The risk for keratinocyte cancer is dramatically increased in solid organ transplant recipients (OTR) with a first post-transplant keratinocyte cancer conferring a high risk for subsequent keratinocyte cancer arising with accelerated dynamics. Despite cumulative ultraviolet radiation (UVR) being the primary responsible environmental carcinogen reduced compliance with photoprotective measures among OTR has been reported. Risk assessment tools could help guide clinical decision-making and targeted prevention strategies for patients at particularly high risk for post-transplant keratinocyte cancer. OBJECTIVES: To evaluate cumulative sun exposure by means of an assigned total sun burden (TSB) score, sunscreen use and associated risk factors for keratinocyte cancer in the post-transplantation phase of OTR. METHODS: A retrospective single-center cohort study analyzing medical records and standardized questionnaires of 290 OTR cared for at a German dermatology transplant clinic. RESULTS: Significantly lower TSB scores were noted in OTR not developing a first keratinocyte cancer compared to OTR developing keratinocyte cancer during their follow-up period ( P = 0.005). Regression analysis assigned a significantly higher risk for the development of first keratinocyte cancer to OTR with TSB scores >10. In total 70.7% of OTR with a history of ≥1 keratinocyte cancer reported intermittent sunscreen use, while daily sunscreen use was overall associated with female gender (21.3%) and age >30 years (17.6%). CONCLUSIONS: The risk of OTR for developing keratinocyte cancer is reflected by their UV-exposure patterns, which may be assessed by the TSB-score, a scored risk assessment tool. Complementing clinical data, the TSB score may help clinicians to identify OTR at particularly high risk for keratinocyte cancer and to endorse intensified prevention strategies and dermato-oncologic care.

Differentiation-related epigenomic changes define clinically distinct keratinocyte cancer subclasses.

Keratinocyte cancers (KC) are the most prevalent malignancies in fair-skinned populations, posing a significant medical and economic burden to health systems. KC originate in the epidermis and mainly comprise basal cell carcinoma (BCC) and cutaneous squamous cell carcinoma (cSCC). Here, we combined single-cell multi-omics, transcriptomics, and methylomics to investigate the epigenomic dynamics during epidermal differentiation. We identified ~3,800 differentially accessible regions between undifferentiated and differentiated keratinocytes, corresponding to regulatory regions associated with key transcription factors. DNA methylation at these regions defined AK/cSCC subtypes with epidermal stem cell- or keratinocyte-like features. Using cell-type deconvolution tools and integration of bulk and single-cell methylomes, we demonstrate that these subclasses are consistent with distinct cells-of-origin. Further characterization of the phenotypic traits of the subclasses and the study of additional unstratified KC entities uncovered distinct clinical features for the subclasses, linking invasive and metastatic KC cases with undifferentiated cells-of-origin. Our study provides a thorough characterization of the epigenomic dynamics underlying human keratinocyte differentiation and uncovers novel links between KC cells-of-origin and their prognosis.

Ablative Fractional Laser-assisted Low-irradiance Photodynamic Therapy for Treatment of Actinic Keratoses in Organ Transplant Recipients: A Prospective, Randomized, Intraindividual Controlled Trial.

Pain and inferior efficacy are major limiting factors of conventional photodynamic therapy for the field treatment of actinic keratoses in immunosuppressed organ transplant recipients. This prospective randomized controlled study evaluates the efficacy and tolerability of ablative fractional laser system pretreatment combined with low-irradiance photodynamic therapy (18.5 mW/cm2) compared with conventional photodynamic therapy (61.67 mW/cm2) in the treatment of actinic keratoses on the face and scalp in organ transplant recipients, using a red light-emitting diode lamp at a total light dose of 37 J/cm2. Low-irradiance photodynamic therapy combined with Er:YAG pretreatment achieved a significantly superior lesion response rate (mean ± standard deviation 77.3 ± 23.6%) compared with conventional photodynamic therapy (61.8 ± 21.4%; p = 0.025) in intra-individual fields at 3 months without negatively impacting pain (p = 0.777) or cosmetic outcome (p = 0.157).

Expression of p53 up-regulated modulator of apoptosis (PUMA) in non-melanoma skin cancer of long-term immunosuppressed solid organ transplant recipients compared to immunocompetent patients.

The risk of UV radiation (UVR)-induced non-melanoma skin cancer (NMSC) is dramatically increased in immunosuppressed organ transplant recipients compared to immunocompetent patients. In the skin, p53 up-regulated modulator of apoptosis (PUMA) is a central regulator of apoptosis in response to UVR damage and immune response regulation. Data on the expression of PUMA in patients with NMSC relative to immune status is limited To study differences in the expression and distribution of PUMA in cutaneous SCC and BCC by immunohistochemistry between immunocompetent patients and organ transplant recipients, and the effect of CsA-containing immunosuppressive maintenance regimens on this expression. PUMA expression in SCC (n = 34) and BCC (n = 20) was analysed comparatively by immunohistochemical staining in matched cohorts of 27 immunocompetent patients and 27 organ transplant recipients SCC and BCC showed unequivocal positive PUMA expression, however, there was no significant difference in NMSC between organ transplant recipients and immunocompetent patients. A 17% reduction in staining score for PUMA in SCC, but not in BCC, of organ transplant recipients treated with a cyclosporin (CsA)-containing regimen was noted compared to organ transplant recipients without chronic CsA intake (p = 0.0381) PUMA expression in SCC, but not BCC, is significantly reduced by CsA-containing therapy, suggesting a disturbance of apoptosis by iatrogenic immunosuppression with a divergent impact on SCC and BCC.

Programmed Death Ligand-1 (PD-L1) Is an Independent Negative Prognosticator in Western-World Gallbladder Cancer.

Inhibition of the programmed cell death protein-1/ligand-1 (PD-1/PD-L1) axis has opened a new era in the treatment of solid cancers. However, there is no data on the expression and relevance of PD-L1 in Western gallbladder cancer (GBC). We assessed PD-L1 immunohistochemically in 131 GBC patients as Tumor Proportion Score (TPS), Immune Cell Score (IC) and Combined Positivity Score (CPS). Tumor cells expressed PD-L1 in a subset of 14.7% GBC patients at a TPS cut-off of 1%. Higher PD-L1 levels above 10% and 25% TPS were reached in 4.7% and 3.1% of GBC cases, respectively. At a 10% cut-off, TPS was associated with distinct histomorphological subtypes and correlated with poor tumor differentiation. Survival analysis revealed a TPS above 10% to be a highly significant and independent negative prognosticator in GBC. PD-L1 expression was associated with increased CD4+, CD8+ and PD-1+ immune cell densities. In 14.8% of the cases, scattered immune cells expressed T-cell immunoreceptor with Ig and ITIM domains (TIGIT), which was correlated to tumoral expression of its ligand CD155. We here show that a high PD-L1 expression confers a negative prognostic value in Western-world GBC and highlight the TIGIT/CD155 immune checkpoint as a potential new target for GBC immunotherapy.

Granulomatous Reaction to Cosmetic Soft Tissue Filler with Late-onset Inflammatory Response.

is missing (Short communication).

Epigenetic deregulation of lamina-associated domains in Hutchinson-Gilford progeria syndrome.

BACKGROUND: Hutchinson-Gilford progeria syndrome (HGPS) is a progeroid disease characterized by the early onset of age-related phenotypes including arthritis, loss of body fat and hair, and atherosclerosis. Cells from affected individuals express a mutant version of the nuclear envelope protein lamin A (termed progerin) and have previously been shown to exhibit prominent histone modification changes. METHODS: Here, we analyze the possibility that epigenetic deregulation of lamina-associated domains (LADs) is involved in the molecular pathology of HGPS. To do so, we studied chromatin accessibility (Assay for Transposase-accessible Chromatin (ATAC)-see/-seq), DNA methylation profiles (Infinium MethylationEPIC BeadChips), and transcriptomes (RNA-seq) of nine primary HGPS fibroblast cell lines and six additional controls, two parental and four age-matched healthy fibroblast cell lines. RESULTS: Our ATAC-see/-seq data demonstrate that primary dermal fibroblasts from HGPS patients exhibit chromatin accessibility changes that are enriched in LADs. Infinium MethylationEPIC BeadChip profiling further reveals that DNA methylation alterations observed in HGPS fibroblasts are similarly enriched in LADs and different from those occurring during healthy aging and Werner syndrome (WS), another premature aging disease. Moreover, HGPS patients can be stratified into two different subgroups according to their DNA methylation profiles. Finally, we show that the epigenetic deregulation of LADs is associated with HGPS-specific gene expression changes. CONCLUSIONS: Taken together, our results strongly implicate epigenetic deregulation of LADs as an important and previously unrecognized feature of HGPS, which contributes to disease-specific gene expression. Therefore, they not only add a new layer to the study of epigenetic changes in the progeroid syndrome, but also advance our understanding of the disease's pathology at the cellular level.

Single-cell transcriptomes of the human skin reveal age-related loss of fibroblast priming.

Fibroblasts are an essential cell population for human skin architecture and function. While fibroblast heterogeneity is well established, this phenomenon has not been analyzed systematically yet. We have used single-cell RNA sequencing to analyze the transcriptomes of more than 5,000 fibroblasts from a sun-protected area in healthy human donors. Our results define four main subpopulations that can be spatially localized and show differential secretory, mesenchymal and pro-inflammatory functional annotations. Importantly, we found that this fibroblast 'priming' becomes reduced with age. We also show that aging causes a substantial reduction in the predicted interactions between dermal fibroblasts and other skin cells, including undifferentiated keratinocytes at the dermal-epidermal junction. Our work thus provides evidence for a functional specialization of human dermal fibroblasts and identifies the partial loss of cellular identity as an important age-related change in the human dermis. These findings have important implications for understanding human skin aging and its associated phenotypes.

Tumor-derived CCL20 affects B16 melanoma growth in mice.

BACKGROUND: Chemokine ligand-20 (CCL20) expressed in the epidermis is a potent impetus for the recruitment of CC-chemokine receptor 6 (CCR6)-expressing subsets of DCs, B-cells and memory T-cells into the skin. CCL20 and CCR6+ immune cells have been detected in chronic inflammatory skin diseases and several malignancies, including melanoma. Yet, the functional contribution of the CCR6/CCL20 axis for melanoma progression remains controversial. OBJECTIVE: The functional contribution of CCR6-expressing immune cell subsets and local CCL20 in the tumor microenvironment for the immune control of melanoma was studied. METHODS: Homeostatic and inducible CCL20 secretion of murine (B16, Ret) and human (A375, C32) melanoma cells was analyzed by ELISA. To assess the functional relevance of CCR6/CCL20 interactions on local tumor progression, prestimulated or retrovirally transduced B16/F1 melanoma cells overexpressing CCL20 (B16-CCL20) were injected subcutaneously into C57BL/6 Wt mice and congenic CCR6-deficient (CCR6-/-) mice. Infiltrating leucocytes were examined by flow cytometry in tumors and draining lymph nodes (DLNs). RESULTS: Melanoma cell lines up-regulate CCL20 secretion upon stimulation with pro-inflammatory cytokines in vitro. While only moderate changes in phenotype and composition of leucocytes were detected in advanced tumors and DLNs, mice injected with CCR6+ B16-CCL20 cells developed smaller tumors compared to B16-Control injected littermates, with CCR6-/- mice displaying the most pronounced reduction in tumor growth and incidence. CONCLUSION: Our results suggest that CCR6/CCL20 interactions and individual independent effects of CCL20 and CCR6 in the microenvironment may be essential for melanoma progression and suggest a decisive role of this chemokine axis for melanoma pathogenesis beyond chemoattraction.

Intracapillary immune complexes recruit and activate slan-expressing CD16+ monocytes in human lupus nephritis.

Lupus nephritis is a major cause of morbidity in patients with systemic lupus erythematosus. Among the different types of lupus nephritis, intracapillary immune complex (IC) deposition and accumulation of monocytes are hallmarks of lupus nephritis class III and IV. The relevance of intracapillary ICs in terms of monocyte recruitment and activation, as well as the nature and function of these monocytes are not well understood. For the early focal form of lupus nephritis (class III) we demonstrate a selective accumulation of the proinflammatory population of 6-sulfo LacNAc+ (slan) monocytes (slanMo), which locally expressed TNF-α. Immobilized ICs induced a direct recruitment of slanMo from the microcirculation via interaction with Fc γ receptor IIIA (CD16). Interestingly, intravenous immunoglobulins blocked CD16 and prevented cell recruitment. Engagement of immobilized ICs by slanMo induced the production of neutrophil-attracting chemokine CXCL2 as well as TNF-α, which in a forward feedback loop stimulated endothelial cells to produce the slanMo-recruiting chemokine CX3CL1 (fractalkine). In conclusion, we observed that expression of CD16 equips slanMo with a unique capacity to orchestrate early IC-induced inflammatory responses in glomeruli and identified slanMo as a pathogenic proinflammatory cell type in lupus nephritis.

Daylight PDT with MAL - current data and practical recommendations of an expert panel.

Photodynamic Therapy (PDT) is one of the standard treatment modalities for actinic keratoses (AKs). Daylight PDT (DL-PDT) with MAL cream is a rather recent development, which, instead of an artificial light source, uses daylight for the activation of the photosensitizer. The present review summarizes available data based on a selective literature search, highlights practical aspects, and reflects the authors' expert knowledge in using DL-PDT. With respect to efficacy, study data shows that DL-PDT is noninferior to conventional PDT (cPDT). However, given that DL-PDT is markedly less painful, it is significantly better tolerated than cPDT. In Europe, DL-PDT can be performed from March to October, on sunny as well as on cloudy days. UV protection of untreated areas of the body should be observed. Outside temperature should not fall below 10°C. On hot days, patients should be advised to stay in the shade if necessary. Representing a useful addition to current therapeutic options, DL-PDT with MAL cream is, among others, suitable for patients with field cancerization and/or those who have experienced severe pain associated with cPDT.

Evaluation of specific humoral and cellular immune responses against the major capsid L1 protein of cutaneous wart-associated alpha-Papillomaviruses in solid organ transplant recipients.

BACKGROUND: Infection with different species of cutaneous human papillomaviruses (cHPV) of genus alpha (cαHPVs) and associated skin disease are highly prevalent in solid organ transplant recipients (OTR), documenting the importance of the immunological control of HPV infection. OBJECTIVES: To investigate the natural course of cαHPV-specific cellular and humoral immune responses during systemic long-term immunosuppression. METHODS: Integrating bead-based multiplex serology and flow cytometry we analyzed natural cαHPV-specific antibodies and T(H) cell responses against the major capsid protein L1 of HPV types 2, 27, 57 (species 4) and 3, 10 and 77 (species 2) in sera and blood of OTR before and after initiation of iatrogenic immunosuppression and in comparison to immunocompetent individuals (IC). RESULTS: Among OTR we observed an overall 42% decrease in humoral L1-specific immune responses during the course of iatrogenic immunosuppression, comparing median values 30 d before and 30 d after initiation of immunosuppressive therapy (p < 0.05). This difference disappeared after long-term (>1 year) immunosuppression. The predominant cellular L1-specific immune response was of type T(H)1 (CD4(+)CD40L(+)IL-2(+)IFN-γ(+)). Consistent with the detected L1-specific antibody titers, L1-specific T(H)1 responses were unchanged in long-term immunosuppressed OTR compared to IC. Notably, cαHPV-L1-specific IL-2(+)/CD40L(+)CD4(+) or IFN-γ(+)/CD40L(+) CD4(+) T(H) cell responses against any of the cαHPV-L1 types were significantly higher in OTR with clinically apparent common warts. CONCLUSION: The systemic humoral immune response against cαHPV may reflect the individual degree of iatrogenic immunosuppression indicating a higher susceptibility for cαHPV infection among OTR during the early phase after organ transplantation. Humoral cαHPV-specific immune responses may show a reconstitution to pre-transplantation levels despite continuous potent immunosuppression.