Whole-exome sequencing in eccrine porocarcinoma indicates promising therapeutic strategies.

Malignant sweat gland tumours are rare, with the most common form being Eccrine porocarcinoma (EP). To investigate the mutational landscape of EP, we performed whole-exome sequencing (WES) on 14 formalin-fixed paraffin-embedded samples of matched primary EP and healthy surrounding tissue. Mutational profiling revealed a high overall median mutation rate. This was attributed to signatures of mutational processes related to ultraviolet (UV) exposure, APOBEC enzyme dysregulation, and defective homologous double-strand break repair. All of these processes cause genomic instability and are implicated in carcinogenesis. Recurrent driving somatic alterations were detected in the EP candidate drivers TP53, FAT2, CACNA1S, and KMT2D. The analyses also identified copy number alterations and recurrent gains and losses in several chromosomal regions including that containing BRCA2, as well as deleterious alterations in multiple HRR components. In accordance with this reduced or even a complete loss of BRCA2 protein expression was detected in 50% of the investigated EP tumours. Our results implicate crucial oncogenic driver pathways and suggest that defective homologous double-strand break repair and the p53 pathway are involved in EP aetiology. Targeting of the p53 axis and PARP inhibition, and/or immunotherapy may represent promising treatment strategies.

Delayed skin reaction after mRNA-1273 vaccine against SARS-CoV-2: a rare clinical reaction.

BACKGROUND: The coronavirus disease 2019 (COVID-19) is associated with a wide clinical spectrum of skin manifestations, including urticarial, vesicular, vasculitic and chilblain-like lesions. Recently, delayed skin reactions have been reported in 1% individuals following mRNA vaccination against SARS-CoV-2. The exact pathophysiology and the risk factors still remain unclear. PATIENTS AND METHODS: 6821 employees and patients were vaccinated at our institutions between February and June 2021. Every patient received two doses of the mRNA-1273 vaccine in our hospitals, and reported back in case of any side effects which were collected in our hospital managed database. RESULTS: Eleven of 6821 vaccinated patients (0.16%) developed delayed skin reactions after either the first or second dose of the mRNA-1273 vaccine against SARS-CoV-2. Eight of 11 patients (73%) developed a rash after the first dose, while in 3/11 (27%), the rash occurred after the second dose. More females (9/11) were affected. Four of 11 patients required antihistamines, with two needing additional topical steroids. All the cutaneous manifestations resolved within 14 days. None of the skin reactions after the first dose of the vaccine prevented the administration of the second dose. There were no long-term cutaneous sequelae in any of the affected individuals. CONCLUSION: Our data suggests that skin reactions after the use of mRNA-1273 vaccine against SARS-CoV-2 are possible, but rare. Further studies need to be done to understand the pathophysiology of these lesions.

Integrative Genomic Analyses of Patient-Matched Intracranial and Extracranial Metastases Reveal a Novel Brain-Specific Landscape of Genetic Variants in Driver Genes of Malignant Melanoma.

BACKGROUND: Development of brain metastases in advanced melanoma patients is a frequent event that limits patients' quality of life and survival. Despite recent insights into melanoma genetics, systematic analyses of genetic alterations in melanoma brain metastasis formation are lacking. Moreover, whether brain metastases harbor distinct genetic alterations beyond those observed at different anatomic sites of the same patient remains unknown. EXPERIMENTAL DESIGN AND RESULTS: In our study, 54 intracranial and 18 corresponding extracranial melanoma metastases were analyzed for mutations using targeted next generation sequencing of 29 recurrently mutated driver genes in melanoma. In 11 of 16 paired samples, we detected nucleotide modifications in brain metastases that were absent in matched metastases at extracranial sites. Moreover, we identified novel genetic variants in ARID1A, ARID2, SMARCA4 and BAP1, genes that have not been linked to brain metastases before; albeit most frequent mutations were found in ARID1A, ARID2 and BRAF. Conclusion: Our data provide new insights into the genetic landscape of intracranial melanoma metastases supporting a branched evolution model of metastasis formation.

Hidradenitis Suppurativa is Associated with Symptoms of Keratoconjunctivitis Sicca.

AIM OF THE STUDY: Hidradenitis suppurativa (HS) and psoriasis vulgaris (PSO) are chronic inflammatory dermatoses in which proinflammatory cytokines, such as IL-17, play a central role. The prevalence of keratoconjunctivitis sicca (KCS) is commonly higher in PSO than in healthy individuals. This study was thus set up to investigate the prevalence of KCS among patients with HS. MATERIALS AND METHODS: In a cross-sectional study standardized tear film parameters and symptom-oriented questionnaires (OSDI, SPEED) were analyzed in a total of 71 subjects (HS n = 20, PSO n = 20, healthy controls n = 31). Additionally, IL-17 and MMP-9 in the tear film were analyzed. These parameters were correlated to the clinical severity of the skin disease. PSO patients served as inflammatory control group. RESULTS: There were statistically significant differences in OSDI (p = .003) and SPEED (p ≤ 0.001) between HS and the control group, but not between PSO and controls. For HS, there was a statistically significant correlation between symptoms (OSDI) and the severity of HS according to Hurley stage (p = .023). Tear film concentrations showed significantly increased levels of IL-17 (p = .018), but not MMP-9, in PSO alone compared to the control group. CONCLUSION: Data show that subjective complaints of KCS may be associated with HS and correlate with the severity of the respective Hurley stage, but do not involve alterations of tear film MMP-9 and IL-17. Clinicians should remain mindful that ocular complications in HS are often more vague than in psoriatic patients, but dry eye symptoms might be detrimental for the patients' quality of life.

Macrophages/Microglia Represent the Major Source of Indolamine 2,3-Dioxygenase Expression in Melanoma Metastases of the Brain.

The manifestation of brain metastases in patients with advanced melanoma is a common event that limits patient's survival and quality of life. The immunosuppressive properties of the brain parenchyma are very different compared to the rest of the body, making it plausible that the current success of cancer immunotherapies is specifically limited here. In melanoma brain metastases, the reciprocal interplay between immunosuppressive mediators such as indoleamine 2, 3-dioxygenase (IDO) or programmed cell death-ligand 1 (PD-L1) in the context of neoplastic transformation are far from being understood. Therefore, we analyzed the immunoreactive infiltrate (CD45, CD3, CD8, Forkhead box P3 [FoxP3], CD11c, CD23, CD123, CD68, Allograft Inflammatory factor 1[AIF-1]) and PD-L1 with respect to IDO expression and localization in melanoma brain metastases but also in matched metastases at extracranial sites to correlate intra- and interpatient data with therapy response and survival. Comparative tissue analysis identified macrophages/microglia as the major source of IDO expression in melanoma brain metastases. In contrast to the tumor infiltrating lymphocytes, melanoma cells per se exhibited low IDO expression levels paralleled by cell surface presentation of PD-L1 in intracranial metastases. Absolute numbers and pattern of IDO-expressing cells in metastases of the brain correlated with recruitment and localization of CD8+ T cells, implicating dynamic impact on the regulation of T cell function in the brain parenchyma. However, paired analysis of matched intra- and extracranial metastases identified significantly lower fractions of cytotoxic CD8+ T cells in intracranial metastases while all other immune cell populations remain unchanged. In line with the already established clinical benefit for PD-L1 expression in extracranial melanoma metastases, Kaplan-Meier analyses correlated PD-L1 expression in brain metastases with favorable outcome in advanced melanoma patients undergoing immune checkpoint therapy. In summary, our data provide new insights into the landscape of immunosuppressive factors in melanoma brain metastases that may be useful in the implication of novel therapeutic strategies for patients undergoing cancer immunotherapy.

Mandible handling in the surgical treatment of oral squamous cell carcinoma: lessons from clinical results after marginal and segmental mandibulectomy.

OBJECTIVE: The aim of this retrospective, single-center study was to analyze long-term results after marginal and segmental mandibulectomies in patients with oral squamous cell carcinoma (OSCC). STUDY DESIGN: The study included 259 patients treated for OSCC with mandibulectomy between 1996 and 2010. Data acquisition consisted of analysis of operation reports, re-evaluation of histologic bone specimens, and collection of clinical follow-up data. RESULTS: Of the included patients, 86.5% had received segmental and 13.5% marginal mandibulectomies. Patients who received segmental mandibulectomy generally displayed a higher TNM (tumor-node-metastasis) stage; 47% of patients who received segmental mandibulectomy and 14% of those receiving marginal mandibulectomy showed bone infiltration (pT4 a). Of all patients with bone infiltration, 49% showed an invasive histologic infiltration pattern, and 35% showed an erosive histologic infiltration pattern. We found healthy residual crestal bone height in 43% of all segmental mandibulectomies. Only 8% of all patients were prosthodontically rehabilitated. With regard to prognostic parameters, there was no significant difference between patients receiving marginal mandibulectomy and those receiving segmental mandibulectomy. CONCLUSIONS: Because healthy residual crestal bone height was found in 43% of all patients who had received segmental mandibulectomies, it is conceivable that a significant number of patients would profit from marginal mandibulectomy, at least in cases of absent or erosive bone infiltration pattern, because the residual crestal bone is functionally stable.

Immune dysregulation in patients with PTEN hamartoma tumor syndrome: Analysis of FOXP3 regulatory T cells.

BACKGROUND: Patients with heterozygous germline mutations in phosphatase and tensin homolog deleted on chromosome 10 (PTEN) experience autoimmunity and lymphoid hyperplasia. OBJECTIVES: Because regulation of the phosphoinositide 3-kinase (PI3K) pathway is critical for maintaining regulatory T (Treg) cell functions, we investigate Treg cells in patients with heterozygous germline PTEN mutations (PTEN hamartoma tumor syndrome [PHTS]). METHODS: Patients with PHTS were assessed for immunologic conditions, lymphocyte subsets, forkhead box P3 (FOXP3)+ Treg cell levels, and phenotype. To determine the functional importance of phosphatases that control the PI3K pathway, we assessed Treg cell induction in vitro, mitochondrial depolarization, and recruitment of PTEN to the immunologic synapse. RESULTS: Autoimmunity and peripheral lymphoid hyperplasia were found in 43% of 79 patients with PHTS. Immune dysregulation in patients with PHTS included lymphopenia, CD4+ T-cell reduction, and changes in T- and B-cell subsets. Although total CD4+FOXP3+ Treg cell numbers are reduced, frequencies are maintained in the blood and intestine. Despite pathogenic PTEN mutations, the FOXP3+ T cells are phenotypically normal. We show that the phosphatase PH domain leucine-rich repeat protein phosphatase (PHLPP) downstream of PTEN is highly expressed in normal human Treg cells and provides complementary phosphatase activity. PHLPP is indispensable for the differentiation of induced Treg cells in vitro and Treg cell mitochondrial fitness. PTEN and PHLPP form a phosphatase network that is polarized at the immunologic synapse. CONCLUSION: Heterozygous loss of function of PTEN in human subjects has a significant effect on T- and B-cell immunity. Assembly of the PTEN-PHLPP phosphatase network allows coordinated phosphatase activities at the site of T-cell receptor activation, which is important for limiting PI3K hyperactivation in Treg cells despite PTEN haploinsufficiency.

Chronic UVB-irradiation actuates perpetuated dermal matrix remodeling in female mice: Protective role of estrogen.

Chronic UVB-exposure and declined estradiol production after menopause represent important factors leading to extrinsic and intrinsic aging, respectively. Remodeling of the extracellular matrix (ECM) plays a crucial role in both responses. Whether the dermal ECM is able to recover after cessation of UVB-irradiation in dependence of estradiol is not known, however of relevance when regarding possible treatment options. Therefore, the endogenous sex hormone production was depleted by ovariectomy in female mice. Half of the mice received estradiol substitution. Mice were UVB-irradiated for 20 weeks and afterwards kept for 10 weeks without irradiation. The collagen-, hyaluronan- and proteoglycan- (versican, biglycan, lumican) matrix, collagen cleavage products and functional skin parameters were analyzed. The intrinsic aging process was characterized by increased collagen fragmentation and accumulation of biglycan. Chronic UVB-irradiation additionally augmented the lumican, versican and hyaluronan content of the dermis. In the absence of further UVB-irradiation the degradation of collagen and accumulation of biglycan in the extrinsically aged group was perpetuated in an excessive matter. Whereas estradiol increased the proteoglycan content, it reversed the effects of the perpetuated extrinsic response on collagen degradation. Suspension of the intrinsic pathway might therefore be sufficient to antagonize UVB-evoked long-term damage to the dermal ECM.

DMBA/TPA treatment is necessary for BCC formation from patched deficient epidermal cells in Ptch(flox/flox)CD4Cre(+/-) mice.

The development of basal cell carcinoma (BCC), the most frequently diagnosed tumor among persons with European ancestry, is closely linked to mutations in the Hedgehog (Hh) receptor and tumor suppressor Patched1 (Ptch). Using Ptch(flox/flox)CD4Cre(+/-) mice, in which Ptch was ablated in CD4Cre-expressing cells, we demonstrate that the targeted cells can give rise to BCC after treatment with DMBA (7,12-dimethylbenz(a)anthracene)/TPA (12-O-tetradecanoylphorbol-13-acetate), but not after wounding of the skin. In addition, in this model, BCC are not caused by malfunctioning of Ptch-deficient T cells, as BCC did not develop when bone marrow (BM) of Ptch(flox/flox)CD4Cre(+/-) mice was transplanted into Ptch wild-type mice. Instead, lineage-tracing experiments and flow cytometric analyses suggest that the tumors are initiated from rare Ptch-deficient stem cell-like cells of the epidermis that express CD4. As DMBA/TPA is a prerequisite for BCC development in this model, the initiated cells need a second stimulus for expansion and tumor formation. However, in contrast to papilloma, this stimulus seems to be unrelated to alterations in the Ras signaling cascade. Together, these data suggest that biallelic loss of Ptch in CD4(+) cells does not suffice for BCC formation and that BCC formation requires a second so far unknown event, at least in the Ptch(flox/flox)CD4Cre(+/-) BCC mouse model.

Estradiol protects dermal hyaluronan/versican matrix during photoaging by release of epidermal growth factor from keratinocytes.

Hyaluronan (HA) and versican are key components of the dermis and are responsive to ultraviolet (UV)B-induced remodeling. The aim of this study was to explore the molecular mechanisms mediating the effects of estrogen (E(2)) on HA-rich extracellular matrix during photoaging. Hairless skh-1 mice were irradiated with UVB (three times, 1 minimal erythema dose (80 mJ/cm(2)), weekly) for 10 weeks, and endogenous sex hormone production was abrogated by ovariectomy. Subcutaneous substitution of E(2) by means of controlled-release pellets caused a strong increase in the dermal HA content in both irradiated and nonirradiated skin. The increase in dermal HA correlated with induction of HA synthase HAS3 by E(2). Expression of splice variant 2 of the HA-binding proteoglycan versican was also increased by E(2). In search of candidate mediators of these effects, it was found that E(2) strongly induced the expression of epidermal growth factor (EGF) in UVB-irradiated epidermis in vivo and in keratinocytes in vitro. EGF in turn up-regulated the expression of HAS3 and versican V2 in dermal fibroblasts. HAS3 knockdown by shRNA caused inhibition of fibroblast proliferation. Furthermore, HAS3 and versican V2 induction by E(2) correlated positively with proliferation in vivo. In addition, the accumulation of inflammatory macrophages, expression of inducible cyclooxygenase 2, as well as proinflammatory monocyte chemotactic protein 1 were decreased in response to E(2) in the dermis. Collectively, these data suggest that E(2) treatment increases the amount of dermal HA and versican V2 via paracrine release of EGF, which may be implicated in the pro-proliferative and anti-inflammatory effects of E(2) during photoaging.

Nos2 inactivation promotes the development of medulloblastoma in Ptch1(+/-) mice by deregulation of Gap43-dependent granule cell precursor migration.

Medulloblastoma is the most common malignant brain tumor in children. A subset of medulloblastoma originates from granule cell precursors (GCPs) of the developing cerebellum and demonstrates aberrant hedgehog signaling, typically due to inactivating mutations in the receptor PTCH1, a pathomechanism recapitulated in Ptch1(+/-) mice. As nitric oxide may regulate GCP proliferation and differentiation, we crossed Ptch1(+/-) mice with mice lacking inducible nitric oxide synthase (Nos2) to investigate a possible influence on tumorigenesis. We observed a two-fold higher medulloblastoma rate in Ptch1(+/-) Nos2(-/-) mice compared to Ptch1(+/-) Nos2(+/+) mice. To identify the molecular mechanisms underlying this finding, we performed gene expression profiling of medulloblastomas from both genotypes, as well as normal cerebellar tissue samples of different developmental stages and genotypes. Downregulation of hedgehog target genes was observed in postnatal cerebellum from Ptch1(+/+) Nos2(-/-) mice but not from Ptch1(+/-) Nos2(-/-) mice. The most consistent effect of Nos2 deficiency was downregulation of growth-associated protein 43 (Gap43). Functional studies in neuronal progenitor cells demonstrated nitric oxide dependence of Gap43 expression and impaired migration upon Gap43 knock-down. Both effects were confirmed in situ by immunofluorescence analyses on tissue sections of the developing cerebellum. Finally, the number of proliferating GCPs at the cerebellar periphery was decreased in Ptch1(+/+) Nos2(-/-) mice but increased in Ptch1(+/-) Nos2(-/) (-) mice relative to Ptch1(+/-) Nos2(+/+) mice. Taken together, these results indicate that Nos2 deficiency promotes medulloblastoma development in Ptch1(+/-) mice through retention of proliferating GCPs in the external granular layer due to reduced Gap43 expression. This study illustrates a new role of nitric oxide signaling in cerebellar development and demonstrates that the localization of pre-neoplastic cells during morphogenesis is crucial for their malignant progression.

Autoimmunity, intestinal lymphoid hyperplasia, and defects in mucosal B-cell homeostasis in patients with PTEN hamartoma tumor syndrome.

The Phosphatase And Tensin Homolog Deleted On Chromosome 10 (PTEN) regulates the phosphoinositol-3-kinase (PI3K)-AKT signaling pathway. In a series of 34 patients with PTEN mutations, we described gastrointestinal lymphoid hyperplasia, extensive hyperplastic tonsils, thymus hyperplasia, autoimmune lymphocytic thyroiditis, autoimmune hemolytic anemia, and colitis. Functional analysis of the gastrointestinal mucosa-associated lymphoid tissue revealed increased signaling via the PI3K-AKT pathway, including phosphorylation of S6 and increased cell proliferation, but also reduced apoptosis of CD20(+)CD10(+) B cells. Reduced activity of PTEN therefore affects homeostasis of human germinal center B cells by increasing PI3K-AKT signaling via mammalian target of rapamycin as well as antiapoptotic signals.

Antitumoral effects of calcitriol in basal cell carcinomas involve inhibition of hedgehog signaling and induction of vitamin D receptor signaling and differentiation.

Activation of the Hedgehog (Hh)-signaling pathway due to deficiency in the Hh receptor Patched1 (Ptch) is the pivotal defect leading to formation of basal cell carcinoma (BCC). Recent reports provided evidence of Ptch-dependent secretion of vitamin D(3)-related compound, which functions as an endogenous inhibitor of Hh signaling by repressing the activity of the signal transduction partner of Ptch, Smoothened (Smo). This suggests that Ptch-deficient tumor cells are devoid of this substance, which in turn results in activation of Hh-signaling. Here, we show that the application of the physiologically active form of vitamin D(3), calcitriol, inhibits proliferation and growth of BCC of Ptch mutant mice in vitro and in vivo. This is accompanied by the activation of the vitamin D receptor (Vdr) and induction of BCC differentiation. In addition, calcitriol inhibits Hh signaling at the level of Smo in a Vdr-independent manner. The concomitant antiproliferative effects on BCC growth are stronger than those of the Hh-specific inhibitor cyclopamine, even though the latter more efficiently inhibits Hh signaling. Taken together, we show that exogenous supply of calcitriol controls the activity of 2 independent pathways, Hh and Vdr signaling, which are relevant to tumorigenesis and tumor treatment. These data suggest that calcitriol could be a therapeutic option in the treatment of BCC, the most common tumor in humans.

Analysis of the functional integrity of the p53 tumor-suppressor gene in malignant melanoma.

Derogation of the p53 pathway is a hallmark in human malignancies but its implication in melanomas remains unclear. p53 is frequently accumulated in melanomas despite protein stabilizing mutations being rare. For a panel of six melanoma cell lines we performed transcript sequence analysis of the entire coding region and determined p53 protein stability and messenger RNA stability by western blot experiments and quantitative reverse-transcription-PCR, respectively. Transcript levels of p53 modifying genes as well as p53 target genes were investigated after ultraviolet irradiation, interferon-α-2b, and chemotherapy (cisplatin or dacarbazine) by quantitative reverse-transcription-PCR. Transcript sequence analysis identified three aberrations in three of six melanomas. Four of six melanomas showed high-constitutive p53 protein levels. p53 transcripts remained stable in four of six melanomas. All p53-expressing melanomas displayed high p53 protein stability. Constitutively, and after ultraviolet irradiation, mouse double min-2 expression was reduced in melanomas. We detected high homeodomain-interacting protein kinase-2 level in melanomas-expressing mutant p53. Most experimental conditions resulted in lower expression of p21, GADD45A, and PUMA, and a higher expression of CDC2 in melanomas. Altogether, accumulation of p53 protein is due to posttranslational modification or aberrant expression of p53 modifiers. p53 is functionally disrupted although the p53 upstream signaling pathway remains inducible.

Requirement of CCL17 for CCR7- and CXCR4-dependent migration of cutaneous dendritic cells.

Chemokines are known to regulate the steady-state and inflammatory migration of cutaneous dendritic cells (DCs). The beta-chemokine CCL17, a ligand of CCR4, is inducibly expressed in a subset of DCs and is strongly up-regulated in atopic diseases. Using an atopic dermatitis model, we show that CCL17-deficient mice develop acanthosis as WT mice, whereas dermal inflammation, T helper 2-type cytokine production, and the allergen-specific humoral immune response are significantly decreased. Notably, CCL17-deficient mice retained Langerhans cells (LCs) in the lesional skin after chronic allergen exposure, whereas most LCs emigrated from the epidermis of allergen-treated WT controls into draining lymph nodes (LNs). Moreover, CCL17-deficient LCs showed impaired emigration from the skin after exposure to a contact sensitizer. In contrast, the absence of CCR4 had no effect on cutaneous DC migration and development of atopic dermatitis symptoms. As an explanation for the major migratory defect of CCL17-deficient DCs in vivo, we demonstrate impaired mobility of CCL17-deficient DCs to CCL19/21 in 3D in vitro migration assays and a blockade of intracellular calcium release in response to CCR7 ligands. In addition, responsiveness of CCL17-deficient DCs to CXCL12 was impaired as well. We demonstrate that the inducible chemokine CCL17 sensitizes DCs for CCR7- and CXCR4-dependent migration to LN-associated homeostatic chemokines under inflammatory conditions and thus plays an important role in cutaneous DC migration.

Tumor stroma-derived Wnt5a induces differentiation of basal cell carcinoma of Ptch-mutant mice via CaMKII.

Basal cell carcinoma (BCC) is the most common skin tumor in humans. Although BCCs rarely metastasize, they can cause significant morbidity due to local aggressiveness. Approximately 20% of BCCs show signs of spontaneous regression. The understanding of molecular events mediating spontaneous regression has the potential to reduce morbidity of BCC and, potentially, other tumors, if translated into tumor therapies. We show that BCCs induced in conditional Ptch(flox/flox)ERT2(+/-) knockout mice regress with time and show a more differentiated phenotype. Differentiation is accompanied by Wnt5a expression in the tumor stroma, which is first detectable at the fully developed tumor stage. Coculture experiments revealed that Wnt5a is upregulated in tumor-adjacent macrophages by soluble signals derived from BCC cells. In turn, Wnt5a induces the expression of the differentiation marker K10 in tumor cells, which is mediated by Wnt/Ca(2+) signaling in a CaMKII-dependent manner. These data support a role of stromal Wnt5a in BCC differentiation and regression, which may have important implications for development of new treatment strategies for this tumor. Taken together, our results establish BCC as an easily accessible model of tumor regression. The regression of BCC despite sustained Hedgehog signaling activity seems to be mediated by tumor-stromal interactions via Wnt5a signaling.