The Peripheral Lymphatic System Is Impaired by the Loss of Neuronal Control Associated with Chronic Spinal Cord Injury.

Spinal cord injury (SCI) is associated with venous vascular dysfunction below the level of injury, resulting in dysregulation of tissue fluid homeostasis in afflicted skin. The purpose of this study was to determine whether loss of neuronal control in chronic SCI also affects the skin lymphatic system. Morphology of lymphatics was characterized by immunohistochemistry and lymphatic gene expression profiles determined by DNA microarray analysis. In SCI, skin lymphatic function appeared to be impaired, because the ratio of functionally dilated versus collapsed lymphatic vessels was decreased 10-fold compared with control. Consequently, the average lumen area of lymphatic vessels was almost halved, possibly due to the known impaired connective tissue integrity of SCI skin. In fact, collagenases were found to be overexpressed in SCI skin, and dermal collagen structure was impaired. Molecular profiling also suggested an SCI-specific phenotype of increased connective tissue turnover and decreased lymphatic contractility. The total number of lymphatic vessels in SCI skin, however, was doubled, pointing to enhanced lymphangiogenesis. In conclusion, these data show, for the first time, that lymphatic function and development in human skin are under neuronal control. Because peripheral venous and lymphatic vascular defects are associated with disturbed fluid homeostasis, inappropriate wound healing reactions, and impaired skin immunity, they might contribute to the predisposition of afflicted individuals to pressure ulcer formation and wound healing disorders.

Molecular diagnosis of skin infections using paraffin-embedded tissue - review and interdisciplinary consensus.

Nucleic acid amplification techniques (NATs), such as PCR, are highly sensitive and specific methods that have become valuable supplements to culture and serology in the diagnosis of infectious disorders. However, especially when using formalin-fixed and paraffin-embedded tissue, these techniques are associated with both false-negative and false-positive results, a pitfall that is frequently misjudged. Representatives of the German Society of Hygiene and Microbiology (DGHM) and the German Society of Dermatology (DDG) therefore set out to develop a consensus - in the form of a review article - on the appropriate indications for NATs using paraffin-embedded tissue, its contraindications, and the key points to be considered in the pre- and post-analytical phase. Given that fresh, naive tissue is preferably to be used in the workup of a suspected infection, PCR analysis on paraffin sections represents an exception. The latter may be considered if an infection is suspected at a later point in time and fresh tissue has not been preserved or can no longer be obtained. Potential indications include confirmation of histologically suspected infections with Leishmania spp., Bartonella spp., Rickettsia spp., or in case of ecthyma contagiosum. Infections with, for example, mycobacteria or RNA viruses, on the other hand, are not considered useful indications for NATs using paraffin sections. In order to avoid misinterpretation of test results, it is essential that laboratory reports on NATs using paraffin-embedded tissue contain information on the indication/diagnostic circumstances, the required and chosen pre-analytical steps, the limitations of the method, and on diagnostic alternatives.

Molekulare Diagnostik von Hautinfektionen am Paraffinmaterial - Übersicht und interdisziplinärer Konsensus.

Nukleinsäure-Amplifikations-Techniken (NAT), wie die PCR, sind hochsensitiv sowie selektiv und stellen in der mikrobiologischen Diagnostik wertvolle Ergänzungen zur kulturellen Anzucht und Serologie dar. Sie bergen aber gerade bei formalinfixiertem und in Paraffin eingebettetem Gewebe ein Risiko für sowohl falsch negative als auch falsch positive Resultate, welches nicht immer richtig eingeschätzt wird. Daher haben Vertreter der Deutschen Gesellschaft für Hygiene und Mikrobiologie (DGHM) und der Deutschen Dermatologischen Gesellschaft (DDG) einen Konsensus in Form einer Übersichtsarbeit erarbeitet, wann eine NAT am Paraffinschnitt angezeigt und sinnvoll ist und welche Punkte dabei in der Präanalytik und Befundinterpretation beachtet werden müssen. Da bei Verdacht auf eine Infektion grundsätzlich Nativgewebe genutzt werden soll, ist die PCR am Paraffinschnitt ein Sonderfall, wenn beispielsweise bei erst nachträglichaufgekommenem Verdacht auf eine Infektion kein Nativmaterial zur Verfügung steht und nicht mehr gewonnen werden kann. Mögliche Indikationen sind der histologisch erhobene Verdacht auf eine Leishmaniose, eine Infektion durch Bartonellen oder Rickettsien, oder ein Ecthyma contagiosum. Nicht sinnvoll ist oder kritisch gesehen wird eine NAT am Paraffinschnitt zum Beispiel bei Infektionen mit Mykobakterien oder RNA-Viren. Die Konstellation für eine NAT aus Paraffingewebe sollte jeweils benannt werden, die erforderliche Prä-Analytik, die jeweiligen Grenzen des Verfahrens und die diagnostischen Alternativen bekannt sein. Der PCR-Befund sollte entsprechend kommentiert werden, um Fehleinschätzungen zu vermeiden.

A Prognostic Gene Signature Expressed in Primary Cutaneous Melanoma: Synergism With Conventional Staging.

BACKGROUND: Current clinico-pathological American Joint Committee on Cancer (AJCC) staging of primary cutaneous melanoma is limited in its ability to determine clinical outcome, and complementary biomarkers are not available for routine prognostic assessment. We therefore adapted a gene signature, previously identified in fresh-frozen (FF) melanomas and adjacent stroma, to formalin-fixed paraffin-embedded (FFPE) melanomas. The aim was to develop a gene expression profiling (GEP) score to define patient survival probability at the time of first diagnosis. METHODS: Expression of 11 FF melanoma signature genes was quantified by reverse transcription polymerase chain reaction in an FFPE melanoma training cohort (n = 125), corresponding to the combined FF melanoma training and validation cohorts. The resulting GEP score was validated technically and clinically in an independent FFPE melanoma cohort (n = 211). All statistical tests were two-sided. RESULTS: We identified a prognostic eight-gene signature in the tumor area (tumor and adjacent tissue) of AJCC stage I-III melanomas. A signature-based GEP score correlated with melanoma-specific survival (MSS; Kaplan-Meier analysis: P < .0001) was independent of tumor stage (multivariable regression analysis: P = .0032) and stroma content (<5%-90%) and complemented conventional AJCC staging (receiver operating characteristic curve analysis: area under the curve = 0.91). In the clinical validation cohort, the GEP score remained statistically significant (P = .0131) in a multivariable analysis accounting for conventional staging. The GEP score was technically robust (reproducibility: 93%; n = 84) and clinically useful, as a binary as well as a continuous score, in predicting stage-specific patient MSS. CONCLUSIONS: The GEP score is a clinically significant prognostic tool, contributes additional information regarding the MSS of melanoma patients, and complements conventional staging.

Induction of Desmoplastic Trichoepithelioma in a Dermatofibroma.

Induction of follicular germinative structures above a dermatofibroma (DF) is a common finding. Rarely, induction of a trichoblastoma in a DF has been observed. Here, we report the case of a desmoplastic trichoepithelioma induced by a DF. The lesion with clinical and histological appearance of a DF situated on the left dorsal foot showed an associated adnexal proliferation that fulfilled histopathological criteria of desmoplastic trichoepithelioma. Immunohistochemistry (Ber-EP4, Bcl-2, CK17, CK20, CK7, EMA, and Ki67) helped to confirm the diagnosis and to exclude possible differential diagnoses.

An intensified dosing schedule of subcutaneous methotrexate in patients with moderate to severe plaque-type psoriasis (METOP): a 52 week, multicentre, randomised, double-blind, placebo-controlled, phase 3 trial.

BACKGROUND: Methotrexate is one of the most commonly used systemic drugs for the treatment of moderate to severe psoriasis; however, high-quality evidence for its use is sparse and limited to use of oral dosing. We aimed to assess the effect of an intensified dosing schedule of subcutaneous methotrexate in patients with moderate to severe plaque-type psoriasis. METHODS: We did this prospective, multicentre, randomised, double-blind, placebo-controlled, phase 3 trial (METOP) at 16 sites in Germany, France, the Netherlands, and the UK. Eligible patients were aged 18 years or older, had a diagnosis of chronic plaque psoriasis for at least 6 months before baseline, had currently moderate to severe disease, and were methotrexate treatment-naive. Participants were randomly assigned (3:1), via a computer-generated random number sequence integrated into an electronic data capture system, to receive either methotrexate at a starting dose of 17·5 mg/week or placebo for the first 16 weeks, followed by methotrexate treatment of all patients up to 52 weeks (methotrexate-methotrexate vs placebo-methotrexate groups). Dose escalation to 22·5 mg/week was allowed after 8 weeks of methotrexate treatment if patients had not achieved at least a 50% reduction in baseline Psoriasis Area and Severity Index score (PASI), with corresponding volume increases in placebo injections. Treatment was combined with folic acid 5 mg/week. Group allocation was concealed from participants and investigators from the time of randomisation until an interim database lock at week 16, and was open label from week 16 onwards, with no masking of participants or investigators. The primary efficacy endpoint was a 75% reduction in PASI score (PASI 75) from baseline to week 16. We did analysis by modified intention to treat, with non-responder imputation. This study is registered with EudraCT, number 2012-002716-10. FINDINGS: Between Feb 22, 2013, and May 13, 2015, we randomly assigned 120 patients to receive methotrexate (n=91) or placebo (n=29). At week 16, a PASI 75 response was achieved in 37 (41%) patients in the methotrexate group compared with three (10%) patients in the placebo group (relative risk 3·93, 95% CI 1·31-11·81; p=0·0026). Subcutaneous methotrexate was generally well tolerated; no patients died or had serious infections, malignancies, or major adverse cardiovascular events. Serious adverse events were recorded in three (3%) patients who received methotrexate for the full 52 week treatment period. INTERPRETATION: Our findings show a favourable 52 week risk-benefit profile of subcutaneous methotrexate in patients with psoriasis. The route of administration and the intensified dosing schedule should be considered when methotrexate is used in this patient group. FUNDING: Medac.

Mitotic rate in primary melanoma: interobserver and intraobserver reliability, analyzed using H&E sections and immunohistochemistry.

BACKGROUND: In 2009, the AJCC issued a revised melanoma staging system. In addition to tumor thickness and ulceration, the mitotic rate was introduced as the third major prognostic parameter for the classification of primary cutaneous melanoma. Given that, according to the 2009 AJCC classification, the detection of one or more dermal tumor mitoses leads to an upstaging - from stage Ia to Ib - of melanomas with a tumor thickness of ≤ 1.0 mm, we set out to investigate the reproducibility of this new parameter. METHODS: In order to assess interobserver reliability, 17 dermatopathologists und pathologists - all well versed in the diagnosis of cutaneous melanoma - analyzed the mitotic rate in 15 thin primary cutaneous melanomas (mean tumor thickness 0.91 mm) using identical slides. Mitotic rates were determined on H&E and phosphohistone H3 (Ser10)-stained samples. Without knowledge of their previous assessment, five of the aforementioned examiners reevaluated the samples after more than one year in order to ascertain intraobserver reliability. RESULTS: Interobserver reliability of the mitotic rate in thin primary melanomas is disappointing and independent of whether H&E or immunohistochemically stained samples are used (kappa value: 0.088 [H&E], 0.154 [IH], respectively). Kappa values improved to 0.345 (H&E) and 0.403 (IH) when using a cutoff of 0/1 vs. 2+ mitoses. Similarly unsatisfactory, kappa values for intraobserver reliability ranged from 0.18 and 0.348, depending on the individual examiner. DISCUSSION: Given the unsatisfactory reproducibility and large variations in assessing the mitotic rate, it remains a matter of debate whether this diagnostic parameter should play a role in therapeutic decisions.

Mitoserate beim primären Melanom: Interobserver- und Intraobserver-Reproduzierbarkeit am HE-Schnitt und in der Immunhistologie.

HINTERGRUND: Die Melanomklassifikation wurde 2009 durch die AJCC revidiert. Für die Klassifizierung primärer Melanome wurde als dritte Größe neben Tumordicke und Ulzeration die Angabe der Mitoserate neu eingeführt. Gemäß der AJCC-2009-Klassifikation des Melanoms führt der Nachweis nur einer oder mehrerer dermaler Tumormitosen bei Melanomen ≤ 1,0 mm Tumordicke zu einer Umgruppierung des Tumors von T1a nach T1b. Dies erklärt, wie wichtig die Frage nach der Reproduzierbarkeit dieses neuen Parameters ist. METHODEN: Zur Prüfung der Interobserver-Reproduzierbarkeit der Mitoserate haben 17 Dermatopathologen und Pathologen, die in der Befundung des kutanen Melanoms sehr erfahren sind, die Mitoserate in 15 dünnen Melanomen mit einer mittleren Tumordicke von 0,91 mm an demselben Tumorschnitt bestimmt. Die Mitoserate wurde am HE-Schnitt und immunhistologisch (IH) mittels des mitosespezifischen Antikörpers Phospho-Histon-H3 (Ser10) bestimmt. Fünf Befunder wiederholten die Bestimmung nach mehr als einem Jahr ohne Kenntnis ihres Vorbefundes zur Ermittlung der Intraobserver-Reproduzierbarkeit. ERGEBNISSE: Die Interobserver-Reproduzierbarkeit der Mitoserate bei dünnen Melanomen ist unbefriedigend und unabhängig davon, ob die Mitoserate am HE-Schnitt oder am immungefärbten Schnitt bestimmt wird (κ-Werte: 0,088 [HE] bzw. 0,154 [IH]). Bei einer Diskriminationsschwelle von 0/1 vs. 2+ Mitosen verbesserte sich der κ-Wert auf 0,345 (HE) bzw. 0,403 (IH). Die Intraobserver-Reproduzierbarkeit lag mit κ-Werten zwischen 0,18 und 0,348 je nach Befunder ebenfalls im unbefriedigenden Bereich. DISKUSSION: Wegen der unbefriedigenden Reproduzierbarkeit und der großen Variation der Befunde zur Mitoserate bleibt es zweifelhaft, ob dieser Befund als Grundlage für Therapieentscheidungen herangezogen werden kann.

Evidence that a neutrophil-keratinocyte crosstalk is an early target of IL-17A inhibition in psoriasis.

The response of psoriasis to antibodies targeting the interleukin (IL)-23/IL-17A pathway suggests a prominent role of T-helper type-17 (Th17) cells in this disease. We examined the clinical and immunological response patterns of 100 subjects with moderate-to-severe psoriasis receiving 3 different intravenous dosing regimens of the anti-IL-17A antibody secukinumab (1 × 3 mg/kg or 1 × 10 mg/kg on Day 1, or 3 × 10 mg/kg on Days 1, 15 and 29) or placebo in a phase 2 trial. Baseline biopsies revealed typical features of active psoriasis, including epidermal accumulation of neutrophils and formation of microabscesses in >60% of cases. Neutrophils were the numerically largest fraction of infiltrating cells containing IL-17 and may store the cytokine preformed, as IL-17A mRNA was not detectable in neutrophils isolated from active plaques. Significant clinical responses to secukinumab were observed 2 weeks after a single infusion, associated with extensive clearance of cutaneous neutrophils parallel to the normalization of keratinocyte abnormalities and reduction of IL-17-inducible neutrophil chemoattractants (e.g. CXCL1, CXCL8); effects on numbers of T cells and CD11c-positive dendritic cells were more delayed. Histological and immunological improvements were generally dose dependent and not observed in the placebo group. In the lowest-dose group, a recurrence of neutrophils was seen in some subjects at Week 12; these subjects relapsed faster than those without microabscesses. Our findings are indicative of a neutrophil-keratinocyte axis in psoriasis that may involve neutrophil-derived IL-17 and is an early target of IL-17A-directed therapies such as secukinumab.

Increased detection of cutaneous leishmaniasis in Norway by use of polymerase chain reaction.

Cutaneous leishmaniasis (CL) is a parasitic infection and occurs in tropical and subtropical regions worldwide and in the region of the Mediterranean Sea. The diagnosis is based on the clinical appearance and biopsy findings that may be supplemented with polymerase chain reaction (PCR). In this study 20 cases were selected if (i) a histopathological diagnosis of granulomatous dermatitis was made, (ii) CL was taken into consideration or (iii) the diagnosis was CL. PCR analysis with primers specific for leishmania was performed on archived histological specimens and was positive in 6 of the 20 cases. In two cases both the clinical and histopathological diagnosis concurred with CL. In the remaining four cases a clinical diagnosis other than CL was made. In two of these cases the histopathology showed granulomatous dermatitis, and detection of parasites led to consideration of CL. In the last two cases leishmaniasis was not taken into consideration by clinicians or pathologists. Our study shows that CL may occur more often than anticipated in Norway, but clinicians do not consider the diagnosis as often as they should. Pathologists may also fail to diagnose or suggest CL especially when parasites are not visualized in the histopathological specimen.

Histopathological diagnostics of malignant melanoma in accordance with the recent AJCC classification 2009: Review of the literature and recommendations for general practice.

BACKGROUND: TNM classifications are the basis for diagnostic and therapeutic procedures in oncology. Histopathological reports have to enable a proper indexing of tumor specific findings into recent classifications. METHODS: A systematic review of the literature was performed to identify reports dealing with the assessment of mitotic rate and the processing and evaluation of sentinel node biopsies in malignant melanoma. On the basis of this review an expert panel of dermatopathologists and general pathologists discussed and agreed recommendations for general practice. RESULTS: Following recommendations were agreed with a broad consensus (93-100 % agreement): The determination of the mitotic rate in primary melanoma is performed on HE slides. The evaluation of an area of 1 mm(2) is sufficient. Only dermal mitoses are considered. The counted number of mitoses is provided as an integer value. The mitotic rate shall be determined in primary melanomas of ≤1.00 mm vertical tumor thickness according to the hot-spot method and provided as an integer value in relation to an area of 1 mm(2) . The determination of the mitotic rate in the case of thicker primary melanomas is desirable. In general, for the evaluation of each sentinel lymph node, 4 slides should be prepared. For diagnostic purposes, immunohistochemistry (preferably with antibodies against S100ß, Melan A and HMB-45) should be performed in addition to HE staining. The pathology report should provide information about micro-metastases and their longest extension (one-tenth of a millimeter). CONCLUSIONS: These recommendations are suitable for standardizing the histopathological diagnosis of malignant melanoma and for providing a common basis for clinical decisions and scientific research.

Correlation of histopathological patterns in cutaneous melanoma with BRAF mutations / Correlação de padrões histopatológicos de melanomas cutâneos com mutações BRAF

INTRODUCTION: Mutations on BRAF gene located on chromosome 7q are the most frequently found in cutaneous melanomas (60 percent-80 percent). The only study correlating histopathological patterns of cutaneous melanomas with the presence of BRAF mutations was undertaken by Viros et al. in 2008. The authors observed that morphological features of melanomas are associated with BRAF mutations. OBJECTIVES: To correlate histopathological patterns in cutaneous melanoma with the presence of BRAF mutations in order to corroborate the results of the study performed by Viros et al. METHODS: Paraffin embedded surgical specimens of 20 primary cutaneous melanomas with BRAF mutation and 20 specimens without BRAF mutation were evaluated independently by two dermatologists that carried out a blind experiment. The features analyzed were nesting, circumscription, presence of isolated melanocytes in the lesion, size and shape of neoplastic cells, and tumor cell pigmentation. RESULTS: "Nesting" was the most prevalent variable for the determination of melanomas with BRAF mutations according to both observers (r = 0.46; p = 0.04). CONCLUSION: As far as mutational status is concerned, it was not possible to confirm any predictive value for histopathological patterns such as circumscription, presence of isolated melanocytes in the lesion and cytological features. Difficulties in the interpretation of some histological criteria were demonstrated by the variation in the observers' conclusions. It is difficult to state if genetic alterations such as BRAF mutations may serve as biomarkers for melanoma classification.

INTRODUÇÃO: Mutações do gene BRAF localizado no cromossomo 7q são as mais frequentemente encontradas em melanomas cutâneos (60 por cento-80 por cento). O único estudo que correlacionou padrões histopatológicos de melanomas cutâneos com a presença de mutações BRAF foi realizado por Viros et al., em 2008, que observaram que características morfológicas de melanomas estavam associadas a mutações BRAF. OBJETIVOS: Correlacionar padrões histopatológicos de melanomas cutâneos com a presença de mutações BRAF, a fim de confirmar os achados de Viros et al. MÉTODOS: Espécimes em parafina de 20 casos de melanomas cutâneos primários com mutações BRAF e 20 casos sem mutações foram avaliados independentemente por dois dermatologistas sem o conhecimento da presença ou não das mutações. Os padrões analisados foram formação de "ninhos", circunscrição, presença de melanócitos isolados na lesão, tamanho e forma das células neoplásicas e pigmentação das células tumorais. RESULTADOS: A formação de "ninhos" foi a variável com o maior poder de determinação para melanomas com mutações BRAF para ambos os observadores (r = 0,46; p = 0,04). CONCLUSÃO: Não foi possível confirmar nenhum valor preditivo em relação ao status mutacional de um melanoma para os padrões histológicos circunscrição e presença de melanócitos isolados na lesão, bem como para características citológicas. Dificuldades na interpretação de alguns critérios histológicos foram demonstradas pela variação da concordância entre os observadores. É difícil afirmar se alterações genéticas como as mutações BRAF podem servir como biomarcadores para a classificação de melanomas.

Refining criteria for diagnosis of cutaneous infections caused by herpes viruses through correlation of morphology with molecular pathology.

BACKGROUND: Infections of the skin by herpes viruses do not always present themselves in typical fashion. Early diagnosis, however, is crucial for appropriate treatment. Polymerase chain reaction (PCR) allows diagnosis and differential diagnosis of herpes virus infections, but the method is not yet available in large parts of the world, where diagnosis is made based on morphology alone. AIM: To refine criteria for the diagnosis of herpes virus infections of the skin by way of correlation of clinical and histopathologic findings with results of PCR studies. METHODS: We studied 75 clinically diagnosed patients of "zoster," "varicella," and "herpes simplex", to correlate clinical and histopathological findings with results of PCR studies on paraffin embedded biopsy specimens. RESULTS: Clinical suspicion of infection by herpes viruses was confirmed by histopathology in 37% of the cases and by PCR studies in 65% of the cases. Zoster was frequently misdiagnosed as infection with herpes simplex viruses (30%). When diagnostic signs of herpes virus infection were encountered histopathologically, PCR confirmed the diagnosis in 94%. By way of correlation with results of PCR studies, initial lesions of herpes virus infections could be identified to have a distinctive histopathological pattern. Herpetic folliculitis appeared to be a rather common finding in zoster, it occurring in 28% of the cases. CONCLUSION: We conclude that correlation of clinical and histopathological features with results of PCR studies on one and the same paraffin embedded specimen permits identification of characteristic morphologic patterns and helps to refine criteria for diagnosis both clinically and histopathologically.

Herpes incognito most commonly is herpes zoster and its histopathologic pattern is distinctive!

Infections of the skin by herpesviruses do not always present themselves in typical fashion. Conventional microscopy is used routinely to confirm infection by herpesviruses, but sometimes typical signs such as multinucleated epithelial cells or "ghosts" of them are not encountered in a specimen (so-called herpes incognito). We studied 35 patients in whom infection with herpesviruses was differentially diagnosed clinically but in whom a biopsy specimen had been taken for confirmation. Only those patients in whom histopathologic findings had been interpreted as being "not diagnostic" of herpesvirus infection by 2 independent dermatopathologists were included. Clinical and histopathologic findings were correlated with results from polymerase chain reaction studies on formalin-fixed paraffin-embedded tissue. Polymerase chain reaction revealed herpesvirus-specific DNA in 12 of 35 specimens, 10 being varicella zoster virus (VZV) positive, 1 herpes simplex virus (HSV)-2 positive, and 1 HSV-1 positive. Ten of these 12 cases presented themselves in very similar fashion (8 VZV, 1 HSV-1, 1 HSV-2). All lesions were macular or papular and typified mostly by dense perivascular and sparse interstitial superficial and deep infiltrates of lymphocytes, sometimes assuming a patchy lichenoid pattern. Infiltrates were prominent in and around adnexal structures, often peppering follicles, sebaceous glands, and eccrine glands. Lymphocytes were also found in the lower part of the epidermis accompanied by a combination of spongiosis and vacuolar alteration. The papillary dermis was often edematous; extravasated erythrocytes in variable numbers were a common finding. Lymphocytes sometimes had large and polygonal nuclei. Neutrophils and nuclear dust were present occasionally; eosinophils were rare. We conclude that herpes incognito most commonly is herpes zoster and its histopathologic pattern is distinctive.