Clinical and histopathological aspects of the most common inflammatory non-infectious skin diseases.

The authors present a didactic overview of the most common inflammatory non-infectious skin diseases. This overview is not exhaustive, but illustrative, especially when regarding the aspect of a systematic approach to the evaluation of skin biopsy with an initial evaluation of the morphological pattern of the inflammatory process. This will subsequently facilitate the diagnosis. Photodocumentation of typical primary skin manifestations is attached to the photomicrograph images. This enables the pathologist to make a basic clinical-pathological correlation, which is of fundamental importance in dermatopathology.

Diagnostic Pitfalls in Dermatopathology.

Dermatopathology is a distinct part of pathology revealing the rich association with soft tissue pathology and hematopathology. Regarding the number and diversity of the skin disorders, dermatopathology is a broad specialty encompassing hundreds of diseases. The diagnostics in dermatopathology contains a range of specific features. The article summarizes several practically important pitfalls in dermatopathology. The adequate timing and locality selection for proper sampling are emphasized. The influence of the topical therapy on the histopathological picture is debated. The frequently used surgical procedures in the skin biopsy are presented. The most frequent incidental findings and artifacts in cutaneous pathology are discussed. Problematics of the alopecia examination and direct immunofluorescence are added. Clinical-pathological correlation performed by the pathologist, and subsequently by the dermatologist, is the essential step in the diagnostic process. The knowledge transcending to the other specialty and reciprocal communication are prerequisite for the right diagnosis.

Stevens-Johnson syndrome and toxic epidermal necrolysis from pathologist's point of view.

Stevens-Johnson syndrome and toxic epidermal necrolysis (Lyell syndrome) are rare diseases characterized by rapid blistering followed by extensive skin and mucosal exfoliation and constitutional symptoms. In most cases, drugs are the main triggers, but the etiopathogenesis of the diseases is not fully understood. Lyell syndrome is associated with a high mortality rate, reported to be around 35%. Therefore, early diagnosis requiring close interdisciplinary cooperation is essential. The diagnosis based on the clinical picture and a detailed pharmacological history should be confirmed by histopathological examination of the skin specimen, including analysis by direct immunofluorescence.

A case of cutaneous collagenous vasculopathy associated with multiple myeloma and with a pathogenic variant of the glucocerebrosidase gene.

Cutaneous collagenous vasculopathy (CCV) is an extremely rare acquired microangiopathy of unknown etiology. The authors describe a case of a 68-year-old man, a carrier of a heterozygous pathogenic variant of the glucocerebrosidase (GBA) gene, who was diagnosed with CCV, revealing uncommon fibrinogen positivity in direct immunofluorescence. The patient was subsequently diagnosed with multiple myeloma. Treatment of the myeloma with combined chemotherapy including bortezomib, followed by autologous stem cell transplantation, led to significant reduction of cutaneous lesions. To the best of the authors' knowledge, this is the first published case of CCV in a carrier of a pathogenic variant of the GBA gene, associated with multiple myeloma and with significant regression of CCV after myeloma treatment. Direct immunofluorescence examination revealed an unusual fibrinogen deposition. Hypothetical causative role of bortezomib treatment was proposed regarding significant regression of CCV.

The Abscopal Effect in the Era of Checkpoint Inhibitors.

Therapy targeting immune checkpoints represents an integral part of the treatment for patients suffering from advanced melanoma. However, the mechanisms of resistance are responsible for a lower therapeutic outcome than expected. Concerning melanoma, insufficient stimulation of the immune system by tumour neoantigens is a likely explanation. As shown previously, radiotherapy is a known option for increasing the production of tumour neoantigens and their release into the microenvironment. Consequently, neoantigens could be recognized by antigen presenting cells (APCs) and subjected to effector T lymphocytes. Enhancing the immune reaction can trigger the therapeutic response also at distant metastases, a phenomenon known as an abscopal effect (from "ab scopus", that is, away from the target). To illustrate this, we present the case of a 78-year old male treated by anti-CTLA-4/ipilimumab for metastatic melanoma. The patient received the standard four doses of ipilimumab administered every three weeks. However, the control CT scans detected disease progression in the form of axillary lymph nodes metastasis and liver metastasis two months after ipilimumab. At this stage, palliative cryotherapy of the skin metastases was initiated to alleviate the tumour burden. Surprisingly, the effect of cryotherapy was also observed in untreated metastases and deep subcutaneous metastases on the back. Moreover, we observed the disease remission of axillary lymph nodes and liver metastasis two months after the cryotherapy. The rarity of the abscopal effect suggests that even primed anti-tumour CD8+ T cells cannot overcome the tumour microenvironment's suppressive effect and execute immune clearance. However, the biological mechanism underlying this phenomenon is yet to be elucidated. The elicitation of a systemic response by cryotherapy with documented abscopal effect was rarely reported, although the immune response induction is presumably similar to a radiotherapy-induced one. The report is a combination case study and review of the abscopal effect in melanoma treated with checkpoint inhibitors.

Cutaneous melanoma dissemination is dependent on the malignant cell properties and factors of intercellular crosstalk in the cancer microenvironment (Review).

The incidence of cutaneous malignant melanoma has been steadily increasing worldwide for several decades. This phenomenon seems to follow the trend observed in many types of malignancies caused by multiple significant factors, including ageing. Despite the progress in cutaneous malignant melanoma therapeutic options, the curability of advanced disease after metastasis represents a serious challenge for further research. In this review, we summarise data on the microenvironment of cutaneous malignant melanoma with emphasis on intercellular signalling during the disease progression. Malignant melanocytes with features of neural crest stem cells interact with non­malignant populations within this microenvironment. We focus on representative bioactive factors regulating this intercellular crosstalk. We describe the possible key factors and signalling cascades responsible for the high complexity of the melanoma microenvironment and its premetastatic niches. Furthermore, we present the concept of melanoma early becoming a systemic disease. This systemic effect is presented as a background for the new horizons in the therapy of cutaneous melanoma.

Serum proteomic analysis of melanoma patients with immunohistochemical profiling of primary melanomas and cultured cells: Pilot study.

The steadily increasing incidence of malignant melanoma (MM) and its aggressive behaviour makes this tumour an attractive cancer research topic. The tumour microenvironment is being increasingly recognised as a key factor in cancer biology, with an impact on proliferation, invasion, angiogenesis and metastatic spread, as well as acquired therapy resistance. Multiple bioactive molecules playing cooperative roles promote the chronic inflammatory milieu in tumours, making inflammation a hallmark of cancer. This specific inflammatory setting is evident in the affected tissue. However, certain mediators can leak into the systemic circulation and affect the whole organism. The present study analysed the complex inflammatory response in the sera of patients with MM of various stages. Multiplexed proteomic analysis (Luminex Corporation) of 31 serum proteins was employed. These targets were observed in immunohistochemical profiles of primary tumours from the same patients. Furthermore, these proteins were analysed in MM cell lines and the principal cell population of the melanoma microenvironment, cancer­associated fibroblasts. Growth factors such as hepatocyte growth factor, granulocyte­colony stimulating factor and vascular endothelial growth factor, chemokines RANTES and interleukin (IL)­8, and cytokines IL­6, interferon­α and IL­1 receptor antagonist significantly differed in these patients compared with the healthy controls. Taken together, the results presented here depict the inflammatory landscape that is altered in melanoma patients, and highlight potentially relevant targets for therapy improvement.

Microenvironment­driven resistance to B­Raf inhibition in a melanoma patient is accompanied by broad changes of gene methylation and expression in distal fibroblasts.

The incidence of malignant melanoma is rapidly increasing and current medicine is offering only limited options for treatment of the advanced disease. For B­Raf mutated melanomas, treatment with mutation­specific drug inhibitors may be used. Unfortunately, tumors frequently acquire resistance to the treatment. Tumor microenvironment, namely cancer­associated fibroblasts, largely influence this acquired resistance. In the present study, fibroblasts were isolated from a patient suffering from acrolentiginous melanoma (Breslow, 4.0 mm; Clark, IV; B­Raf V600E mutated). The present study focused on the expression of structural and functional markers of fibroblast activation in melanoma­associated fibroblasts (MAFs; isolated prior to therapy initiation) as well as in autologous control fibroblasts (ACFs) of the same patient isolated during B­Raf inhibitor therapy, yet before clinical progression of the disease. Analysis of gene transcription was also performed, as well as DNA methylation status analysis at the genomic scale of both isolates. MAFs were positive for smooth muscle actin (SMA), which is a marker of myofibroblasts and the hallmark of cancer stoma. Surprisingly, ACF isolated from the distant uninvolved skin of the same patient also exhibited strong SMA expression. A similar phenotype was also observed in control dermal fibroblasts (CDFs; from different donors) exclusively following stimulation by transforming growth factor (TGF)­ß1. Immunohistochemistry confirmed that melanoma cells potently produce TGF­ß1. Significant differences were also identified in gene transcription and in DNA methylation status at the genomic scale. Upregulation of SMA was observed in ACF cells at the protein and transcriptional levels. The present results support recent experimental findings that tumor microenvironment is driving resistance to B­Raf inhibition in patients with melanoma. Such an activated microenvironment may be viable for the growth of circulating melanoma cells.

Human polyomavirus 6 and 7 are associated with pruritic and dyskeratotic dermatoses.

BACKGROUND: Human polyomavirus (HPyV)6 and HPyV7 are shed chronically from human skin. HPyV7, but not HPyV6, has been linked to a pruritic skin eruption of immunosuppression. OBJECTIVE: We determined whether biopsy specimens showing a characteristic pattern of dyskeratosis and parakeratosis might be associated with polyomavirus infection. METHODS: We screened biopsy specimens showing "peacock plumage" histology by polymerase chain reaction for HPyVs. Cases positive for HPyV6 or HPyV7 were then analyzed by immunohistochemistry, electron microscopy, immunofluorescence, quantitative polymerase chain reaction, and complete sequencing, including unbiased, next-generation sequencing. RESULTS: We identified 3 additional cases of HPyV6 or HPyV7 skin infections. Expression of T antigen and viral capsid was abundant in lesional skin. Dual immunofluorescence staining experiments confirmed that HPyV7 primarily infects keratinocytes. High viral loads in lesional skin compared with normal-appearing skin and the identification of intact virions by both electron microscopy and next-generation sequencing support a role for active viral infections in these skin diseases. LIMITATION: This was a small case series of archived materials. CONCLUSION: We have found that HPyV6 and HPyV7 are associated with rare, pruritic skin eruptions with a distinctive histologic pattern and describe this entity as "HPyV6- and HPyV7-associated pruritic and dyskeratotic dermatoses."

Interleukin-35 is upregulated in systemic sclerosis and its serum levels are associated with early disease.

OBJECTIVES: IL-35 is a member of the IL-12 family consisting of p35/IL-12a and EBI3/IL-27b subunits. IL-35 exerts immunomodulatory activities in experimental and human autoimmune inflammatory conditions. Our aim was to assess IL-35 expression in the skin and circulation of SSc patients and to characterize its potential association with SSc-related features. METHODS: Expression of IL-35 in skin and dermal fibroblasts was quantified by quantitative PCR, immunohistochemistry and immunofluorescence. Serum levels of IL-35 (by ELISA), CRP (by turbidimetry), ANA (by immunofluorescence) and autoantibodies of the ENA complex (by immunoblot) were measured in 40 SSc patients. Serum IL-35 was determined in 40 age- and sex-matched healthy controls. RESULTS: IL-35 expression was increased in SSc skin and dermal fibroblasts in a TGF-ß-dependent manner. IL-35 induced an activated phenotype in resting fibroblasts and enhanced the release of collagen. IL-35 serum levels were increased in patients with SSc compared with healthy controls [median 83.9 (interquartile range 45.1-146.1) vs 36.2 (interquartile range 17.2-49.4) pg/ml, P < 0.0001]. Serum IL-35 was negatively correlated with disease duration (r = -0.4339, P = 0.0052). In line with this finding, serum IL-35 was increased in patients with an early SSc pattern on capillaroscopy assessment compared with those with active and late SSc patterns. CONCLUSION: The present study demonstrates overexpression of IL-35 in SSc skin, dermal fibroblasts and serum. TGF-ß induces IL-35, which in turn activates resting fibroblasts and enhances the release of collagen, thereby contributing to aberrant TGF-ß signalling in SSc. Increased serum IL-35 is associated with early, inflammatory stages of SSc.

Melanoma cells influence the differentiation pattern of human epidermal keratinocytes.

BACKGROUND: Nodular melanoma is one of the most life threatening tumors with still poor therapeutic outcome. Similarly to other tumors, permissive microenvironment is essential for melanoma progression. Features of this microenvironment are arising from molecular crosstalk between the melanoma cells (MC) and the surrounding cell populations in the context of skin tissue. Here, we study the effect of melanoma cells on human primary keratinocytes (HPK). Presence of MC is as an important modulator of the tumor microenvironment and we compare it to the effect of nonmalignant lowly differentiated cells also originating from neural crest (NCSC). METHODS: Comparative morphometrical and immunohistochemical analysis of epidermis surrounding nodular melanoma (n = 100) was performed. Data were compared to results of transcriptome profiling of in vitro models, in which HPK were co-cultured with MC, normal human melanocytes, and NCSC, respectively. Differentially expressed candidate genes were verified by RT-qPCR. Biological activity of candidate proteins was assessed on cultured HPK. RESULTS: Epidermis surrounding nodular melanoma exhibits hyperplastic features in 90% of cases. This hyperplastic region exhibits aberrant suprabasal expression of keratin 14 accompanied by loss of keratin 10. We observe that MC and NCSC are able to increase expression of keratins 8, 14, 19, and vimentin in the co-cultured HPK. This in vitro finding partially correlates with pseudoepitheliomatous hyperplasia observed in melanoma biopsies. We provide evidence of FGF-2, CXCL-1, IL-8, and VEGF-A participation in the activity of melanoma cells on keratinocytes. CONCLUSION: We conclude that the MC are able to influence locally the differentiation pattern of keratinocytes in vivo as well as in vitro. This interaction further highlights the role of intercellular interactions in melanoma. The reciprocal role of activated keratinocytes on biology of melanoma cells shall be verified in the future.

Cultivation-dependent plasticity of melanoma phenotype.

Malignant melanoma is a highly aggressive tumor with increasing incidence and high mortality. The importance of immunohistochemistry in diagnosis of the primary tumor and in early identification of metastases in lymphatic nodes is enormous; however melanoma phenotype is frequently variable and thus several markers must be employed simultaneously. The purposes of this study are to describe changes of phenotype of malignant melanoma in vitro and in vivo and to investigate whether changes of environmental factors mimicking natural conditions affect the phenotype of melanoma cells and can revert the typical in vitro loss of diagnostic markers. The influence of microenvironment was studied by means of immunocytochemistry on co-cultures of melanoma cells with melanoma-associated fibroblast and/or in conditioned media. The markers typical for melanoma (HMB45, Melan-A, Tyrosinase) were lost in malignant cells isolated from malignant effusion; however, tumor metastases shared identical phenotype with primary tumor (all markers positive). The melanoma cell lines also exerted reduced phenotype in vitro. The only constantly present diagnostic marker observed in our experiment was S100 protein and, in lesser extent, also Nestin. The phenotype loss was reverted under the influence of melanoma-associated fibroblast and/or both types of conditioned media. Loss of some markers of melanoma cell phenotype is not only of diagnostic significance, but it can presumably also contribute to biological behavior of melanoma. The presented study shows how the conditions of cultivation of melanoma cells can influence their phenotype. This observation can have some impact on considerations about the role of microenvironment in tumor biology.

Mycobacterium marinum infections in humans and tracing of its possible environmental sources.

The low frequency of nontuberculous mycobacterial infections, nonspecific symptoms for individual mycobacteria, and the lack of specific identification methods could alter correct diagnosis. This study presents a combined microbiology and molecular-based approach for Mycobacterium marinum detection in four aquarists with cutaneous mycobacterial infection. Simultaneously, ecology screening for M. marinum presence in the aquarists' fish tanks was performed. A total of 38 mycobacterial isolates originated from four human patients (n = 20), aquarium animals (n = 8), and an aquarium environment (n = 10). Isolate identification was carried out using 16S rRNA sequence analysis. A microbiology-based approach, followed by 16S rRNA sequence analysis, was successfully used for detection of M. marinum in all four patients. Animal and environmental samples were simultaneously examined, and a total of seven mycobacterial species were isolated: Mycobacterium chelonae , Mycobacterium fortuitum , Mycobacterium gordonae , Mycobacterium kansasii , Mycobacterium mantenii , Mycobacterium marinum , and Mycobacterium peregrinum . The presence of M. marinum was proven in the aquarium environments of two patients. Although M. marinum is described as being present in water, it was detected only in fish.

Mouse 3T3 fibroblasts under the influence of fibroblasts isolated from stroma of human basal cell carcinoma acquire properties of multipotent stem cells.

BACKGROUND INFORMATION: Multipotent mesenchymal stem cells can participate in the formation of a microenvironment stimulating the aggressive behaviour of cancer cells. Moreover, cells exhibiting pluripotent ESC (embryonic stem cell) markers (Nanog and Oct4) have been observed in many tumours. Here, we investigate the role of cancer-associated fibroblasts in the formation of stem cell supporting properties of tumour stroma. We test the influence of fibroblasts isolated from basal cell carcinoma on mouse 3T3 fibroblasts, focusing on the expression of stem cell markers and plasticity in vitro by means of microarrays, qRT-PCR (quantitative real-time PCR) and immunohistochemistry. RESULTS: We demonstrate the biological activity of the cancer stromal fibroblasts by influencing the 3T3 fibroblasts to express markers such as Oct4, Nanog and Sox2 and to show differentiation potential similar to mesenchymal stem cells. The role of growth factors such as IGF2 (insulin-like growth factor 2), FGF7 (fibroblast growth factor 7), LEP (leptin), NGF (nerve growth factor) and TGFß (transforming growth factor ß), produced by the stromal fibroblasts, is established to participate in their bioactivity. Uninduced 3T3 do not express the stem cell markers and show minimal differentiation potential. CONCLUSIONS: Our observations indicate the pro-stem cell activity of cancer-associated fibroblasts and underline the role of epithelial-mesenchymal interaction in tumour biology.

Trichophyton rubrum suppurative tinea of the bald area of the scalp.

An 83-year-old man presented with an approximately 1-year history of an extensive inflammatory purulent crusted lesion in the bald area of the scalp diagnosed as tinea caused by Trichophyton rubrum. The scalp biopsy specimen showed suppurative folliculitis with perifollicular abscesses in upper dermis, and periodic acid-Schiff-positive fungal elements within the hair follicles and in the hyperkeratotic horny layer. The infection probably spread from diseased fingernails. A cure of the scalp lesion was achieved 2 months after starting daily oral treatment with 250 mg terbinafine. To our knowledge, the case presented is the first in which a suppurative abscess-forming T. rubrum infection of the bald area of the scalp in an immunocompetent man has been described.

Causes and risk factors for death in systemic sclerosis: a study from the EULAR Scleroderma Trials and Research (EUSTAR) database.

OBJECTIVES: To determine the causes and predictors of mortality in systemic sclerosis (SSc). METHODS: Patients with SSc (n=5860) fulfilling the American College of Rheumatology criteria and prospectively followed in the EULAR Scleroderma Trials and Research (EUSTAR) cohort were analysed. EUSTAR centres completed a structured questionnaire on cause of death and comorbidities. Kaplan-Meier and Cox proportional hazards models were used to analyse survival in SSc subgroups and to identify predictors of mortality. RESULTS: Questionnaires were obtained on 234 of 284 fatalities. 55% of deaths were attributed directly to SSc and 41% to non-SSc causes; in 4% the cause of death was not assigned. Of the SSc-related deaths, 35% were attributed to pulmonary fibrosis, 26% to pulmonary arterial hypertension (PAH) and 26% to cardiac causes (mainly heart failure and arrhythmias). Among the non-SSc-related causes, infections (33%) and malignancies (31%) were followed by cardiovascular causes (29%). Of the non-SSc-related fatalities, 25% died of causes in which SSc-related complications may have participated (pneumonia, sepsis and gastrointestinal haemorrhage). Independent risk factors for mortality and their HR were: proteinuria (HR 3.34), the presence of PAH based on echocardiography (HR 2.02), pulmonary restriction (forced vital capacity below 80% of normal, HR 1.64), dyspnoea above New York Heart Association class II (HR 1.61), diffusing capacity of the lung (HR 1.20 per 10% decrease), patient age at onset of Raynaud's phenomenon (HR 1.30 per 10 years) and the modified Rodnan skin score (HR 1.20 per 10 score points). CONCLUSION: Disease-related causes, in particular pulmonary fibrosis, PAH and cardiac causes, accounted for the majority of deaths in SSc.

Two case reports of columnar dyskeratosis, an unusual keratinisation disorder.

Keratinisation disorders with distinctive histopathological patterns are few in number. We describe two men with unusual dermatosis, characterised by a distinctive pattern of focal dyskeratosis. Both men suffered from generalised dermatosis formed by verrucous red-brown plaques. Repeated skin biopsies showed the same histopathological pattern with foci of vertically oriented dyskeratotic cells. The dyskeratotic cells on the level of the stratum spinosum and granulosum were positive for AE1/AE3, CK-HW, CK-LS and CK116 immunostaining. PCR for HPV was negative. The similar clinical appearance of skin lesions in both patients together with their identical histopathological pictures seems to represent a unique clinicopathological condition that we believe is best described by the term 'columnar dyskeratosis'.

Immunocyto- and histochemical profiling of nucleostemin expression: marker of epidermal stem cells?

BACKGROUND AND OBJECTIVE: Because the nucleolar protein nucleostemin is present in bone marrow and neuronal stem cells and malignancies originating thereof we monitored its expression in frozen sections from normal human epidermis, basal cell carcinomas, cultured keratinocytes and cells of the squamous carcinoma line FaDu. In addition, probing the value of this protein as a marker of epidermal stem cells was an aim of this study. MATERIALS AND METHODS: To further characterize cell features we added analysis of expression of keratins 10 or 19 as markers of terminal differentiation and Ki67 as marker of proliferating cells as well as three adhesion/growth-regulatory galectins. RESULTS: Immunohistochemical monitoring revealed expression of nucleostemin in cells of both Ki67-positive and -negative nuclei regardless of the K10-expression status. Cultured keratinocytes were positive, when they were prepared from hair follicles and cultured in the presence of feeder cells. A small population of these nucleostemin-positive cells also expressed galectin-1 but not galectins-3 and -9 in their nucleoli. Part of these cells also expressed keratin 19. FaDu cells were strongly positive, illustrating expression in malignant cells which require no feeder layer. Of note, the number of galectin-1-positive nucleoli was reduced in the course of culture. CONCLUSION: Nucleostemin positivity cannot be considered as marker for stem cells in skin sections. In cultured cells, nucleostemin is expressed in a distinct population of the epidermal cells from hair follicle kept in the presence of a feeder layer, intimating an association of nucleostemin expression with this type of epithelio-mesenchymal interaction which is not essential during propagation of malignant cells.

Transient expression of keratin 19 is induced in originally negative interfollicular epidermal cells by adhesion of suspended cells.

Keratin 19 and nuclear reactivity to an endogenous lectin, galectin-1, represent a potential marker of epidermal stem cells. We detected expression of keratin 19 and nuclear binding sites for galectin-1 in adult cells migrating from the hair follicle, where cells expressing keratin 19 are located in the bulge region. The results were compared with the expression of both markers in cells adhering from suspension prepared from the interfollicular epidermis without keratin-19-positive cells and with nuclear binding sites for galectin-1. The results were compared with data from basal cell carcinomas. All cells were analyzed concerning size, as it is known that cell diameter influences the clonogenic potential of keratinocytes. The major result of this study is the observation of transient expression of keratin 19 and nuclear galectin-1 binding sites in originally negative interfollicular epidermal cells induced by adhesion. These cells were very small in size, similar to basal cells of the interfollicular epidermis or the bulge region of the hair follicle. The influence of the suspension regimen on beta1-integrin expression, cell diameter and growth was also monitored. A population of cells highly positive for beta1 integrin of the same diameter as keratin-19-positive cells insensitive to induction of terminal differentiation by lack of anchorage was characterized. Cells of the same size were also observed in the keratin-19-positive cells of basal cell carcinomas. In conclusion, the expression of poor levels of differentiation induced by cell adhesion is transient. Also, keratin 19 expression should not be exclusively regarded as a marker of stem cell activity.