Paraffin-embedded Skin Biopsies Research Articles

Diagnostic Utility of C4d by Direct Immunofluorescence in Bullous Pemphigoid.

Bullous pemphigoid (BP) is an autoimmune blistering disease that commonly affects elderly patients. Direct immunofluorescence (DIF) for immunoglobulin G (IgG) and C3c on frozen skin biopsies is the gold standard for the diagnosis of BP. In a minority of cases, IgG and/or C3c are found negative, and in these situations, there is a need for a more stable diagnostic marker of BP. C4d is biologically inactive, but has a long half-life, rendering it a long-lived marker for antibody-mediated complement activation. Previous studies already demonstrated that C4d was diagnostically useful in formalin-fixed paraffin-embedded skin biopsies of patients with BP. We hypothesized that C4d detected by DIF could also be a promising diagnostic marker for BP, particularly in IgG and/or C3c DIF-negative cases. In this single-center retrospective study, 69 cases of BP were analyzed for linear deposition of C4d; of the 69 cases, n = 26 were IgG+/C3c-, n = 10 IgG+/C3c+, and n = 33 IgG-/C3c-. Results were compared with n = 39 negative controls. Seven of the 26 (27%) IgG+/C3c- and 3 of the 33 (9%) IgG-/C3c- BP cases were positive for C4d. All 10 IgG+/C3c+ cases were also C4d positive. In the negative control group, 2 of the 39 (5%) were found positive for C4d. In conclusion, the current study shows that C4d is a more sensitive but not a 100% specific marker of BP. We conclude that C4d by DIF could be an interesting diagnostic adjunct for BP, particularly in IgG-/C3c- double negative cases.

Evaluation of the cutaneous inflammatory cells in dogs with leishmaniosis and in dogs without the disease that were naturally infected by Leishmania infantum (syn. L. chagasi).

Canine leishmaniosis (CanL) is associated with an aberrant cutaneous immune response. Few studies have compared cutaneous immune cells among dogs with leishmaniosis or infected without leishmaniosis, and noninfected dogs. Evaluate the number of neutrophils, histiocytes, T lymphocytes, T-bet-positive, GATA3-positive, FoxP3-positive and interleukin (IL)-17-positive cells, in lesional (Group A) and normal-looking (Group B) skin of nine dogs with stage II/III leishmaniosis; in normal-looking PCR-positive (Group C; n=6) or PCR-negative (Group D; n=6) skin of infected dogs; and in the normal-looking skin of 12 noninfected dogs (Group E). Diagnosis of CanL considered clinical signs, clinicopathological abnormalities, detection of Leishmania amastigotes in lymph nodes, and/or bone marrow and positive serological results. Paraffin-embedded skin biopsies were processed for routine immunofluorescence and positive cells were identified using commercially available anti-canine antibodies. In Group A, there was a significantly higher number of neutrophils (P<0.001), histiocytes (P=0.012), T lymphocytes (P=0.011), GATA3-positive (P=0.02) and IL-17A-positive (P=0.002) cells compared to Group E. In Group B, there was a significantly higher number of histiocytes (P=0.02), T lymphocytes (P=0.004), GATA3-positive (P=0.006) and FoxP3-positive (P=0.028) cells compared to Group E. There was no difference in between groups A and B and between groups C or D and E. In the lesional and/or normal-looking skin of dogs with moderate/severe CanL there is an infiltration of neutrophils, histiocytes, T lymphocytes, GATA3-, FoxP3- and IL17A-positive cells. By contrast, the number of these cells is not increased in the normal-looking skin of infected dogs without CanL compared to the skin of noninfected dogs.

Diagnostic application of cyclin D1 fluorescent in situ hybridization for histologically undetermined early lesions of acral melanoma in situ: A case series

Histologically undetermined early acral melanoma in situ (HUAMIS) is rare but a diagnostic challenge, being clinically and dermoscopically MIS (late onset, a large size (>7 mm), parallel ridges pattern) but microscopically without recognizable cytological atypia. Cyclin D1 (CCND1) gene amplification is a genetic aberration occurring in the early radial growth phase of AMs and could thus help determine malignancy for this disease.We determine the value of CCND1 amplification by FISH as a diagnostic marker for HUAMIS. CCND1 amplification was examined in paraffin-embedded skin biopsies and excisions using a dual-probes fluorescence in situ hybridization (FISH) (11q13 and CEP11). One FISH-negative case 6 was additionally examined by Mypath Melanoma (qRT-PCR). Seventeen cases (12 dysplastic nevi, 3 AMIS, and 2 invasive AM) were served as negative controls for FISH.All six patients (4 females and 2 males) were Hispanic. Pigment lesions were on the left plantar foot (4), right third finger palm (1), and right thumb subungual (1). All cases showed similar clinical and dermoscopical characteristics, including late onset (50 to 74 years old), long duration (from 2 to 15 years), large-sized pigments (from 16 to 40 mm), and a parallel ridge pattern. Junctional melanocytes with no or minimal atypia from five cases showed CCND1 amplifications. Four of 5 cases were received 1st or/and 2nd wide excisions, which demonstrated foci of histologically overt MIS. One FISH-negative case 6 demonstrated “likely malignancy” scores (>2) by Mypath Melanoma (qRT-PCR). None of negative controls showed the amplification.We propose here a simple CCND1 FISH is a practical diagnostic test to determine the malignancy of the very early progression phase of AM preceding histopathologically defined MIS. Cases presented here could be an indolent subtype of AMIS characterized by carrying a long latent radial growth phase without vertical growth, mimicking lentigo maligna.

TCL-302: Immunohistochemical Characterization of Anaplastic Large Cell Lymphoma Using Tissue Microarray

Context: Anaplastic Large Cell Lymphoma (ALCL) is a type of mature T-cell lymphomas with systemic and primary cutaneous (PCALCL) subtypes. Systemic ALCL includes anaplastic lymphoma kinase (ALK)+ and ALK-. ALK+ usually has the NPM/ALK gene, which has favorable prognosis compared to ALK-. PCALCL is usually indolent. DUSP22 rearrangement in ALK-ALCL has a favorable outcome like ALK+, indicating alternate pathways of ALCL. Objective: Our primary aim was to compare protein expression levels of selected signaling molecules in oncogenic pathways of ALCL (JAK1, STAT3, DUSP22, ERB4, BLIMP1, SOCS3) among the three subtypes. We also correlated expression levels to ORR, PFS, DFS, and OS. Design: This is a retrospective study correlating protein expression levels of various antibodies across 27 patient tissue samples. Setting: The study was set at H. Lee Moffitt Cancer Center and Research Institute, an academic referral center. Patients or other participants: We identified 50 ALCL patients from 2000–2019. Twenty-seven tissue samples (6 ALK+, 10 ALK-, 8 PCALCL, 3 controls) met eligibility criteria, which included pathologist-reviewed diagnosis of ALCL subtypes (ALK+, ALK-, PCALCL) and consent to the Tissue Core database. Interventions: Replicate cores were taken and TMAs were constructed from formalin-fixed, paraffin-embedded lymph node or skin biopsies. IHC was performed with Abs to selected proteins. Stained TMA was scanned using Aperio™ ScanScope XT2 with a 200×/0.8NA objective lens at 5 min/slide via Basler tri-linear array detection. TMAs were analyzed using Aperio Nuclear V9 Default algorithm with thresholds for stain intensity: Strong = 130, Moderate = 188, Weak = 162 to determine the % positive biomarker stain within each core. Main outcome measures: The primary study outcome measurement was comparison of protein expression levels based on specific pathways known or hypothesized to be involved in ALCL pathogenesis. We hypothesized that these protein expression levels would correlate to time to event outcomes Results: PCALCL, ALK+, and ALK- subgroups demonstrated a significant difference in SOCS3 expression with mean and std respectively: 42.4% (0.153), 17.4% (0.089), and 21.0% (0.155);p=0.008. Among all pairs, ALK+ and PCALCL were statistically different (p=0.011). Patients with high BLIMP1 expression (% larger than median, 28.5%) regardless of ALCL subtype were associated with better mOS (not reached) compared to patients with low ( Conclusions: We identified a significant difference in expression of two Abs among three subtypes of ALCL. Increased BLIMP1 was associated with favorable prognosis and could potentially be a positive prognostic marker. Increased SOCS3 appears more prevalent in PCALCL than systemic ALCL, suggesting a potential utility in DDx. We plan to validate our results in a larger sample. Adding secondary markers like Bcl-2 can be used in the future to correlate expression levels and response to targeted agents, such as venetoclax. Funding: University of South Florida Graduate Medical Education Grant ($7500).

Identification of Leishmania species by high-resolution DNA dissociation in cases of American cutaneous leishmaniasis

BackgroundAmerican cutaneous leishmaniasis is an infectious dermatosis caused by protozoa of the genus Leishmania, which comprises a broad spectrum of clinical manifestations depending on the parasite species involved in the infections and the immunogenetic response of the host. The use of techniques for amplification of the parasites DNA based on polymerase chain reaction polymerase chain reaction and the recent application of combined techniques, such as high-resolution DNA dissociation, have been described as a viable alternative for the detection and identification of Leishmania spp. in biological samples. ObjectivesTo identify the Leishmania species using the polymerase chain reaction high-resolution DNA dissociation technique in skin biopsies of hospital-treated patients, and compare with results obtained by other molecular identification techniques. MethodsA retrospective study assessing patients with suspected American cutaneous leishmaniasis seen at a hospital in São Paulo/Brazil was conducted. The paraffin blocks of 22 patients were analyzed by polymerase chain reaction high-resolution DNA dissociation to confirm the diagnosis and identify the species. ResultsOf the 22 patients with suspected American cutaneous leishmaniasis, the parasite was identified in 14, comprising five cases (35.6%) of infection by L. amazonensis, four (28.5%) by L. braziliensis, two (14.4%) by L. amazonensis+L. infantum chagasi, two (14.4%) by L. guyanensis, and one (7.1%) by Leishmania infantum chagasi. In one of the samples, in which the presence of amastigotes was confirmed on histopathological examination, the polymerase chain reaction high-resolution DNA dissociation technique failed to detect the DNA of the parasite. Study limitationsThe retrospective nature of the study and small number of patients. ConclusionsThe method detected and identified Leishmania species in paraffin-embedded skin biopsies with a sensitivity of 96.4% and could be routinely used in the public health system.

Diagnostic 2-Gene Classifier in Early-Stage Mycosis Fungoides: A Retrospective Multicenter Study

Introduction Mycosis fungoides (MF) is the most common type of cutaneous T-cell lymphoma and represents more than 50% of all primary cutaneous lymphomas. It is typically an indolent disorder with limited patches and plaques. One third, however, experience progression with ulcerating tumors and possible further systemic dissemination. Currently, the diagnosis of MF is based on clinical and histological examinations, but this has proven, especially in early-stage disease, to be challenging due to similarities with several benign skin conditions such as psoriasis, pityriasis lichenoides chronica, and dermatitis. Despite intensive research, reliable diagnostic biomarkers for early-stage MF are still needed. This study aims to identify a diagnostic classifier that could support the diagnostic workup leading to an exact diagnosis in the early-stage of this complex and potentially lethal disease. Methods We analyzed 43 formalin-fixed and paraffin-embedded (FFPE) skin biopsies from 36 patients with early-stage MF. Seven patients had 2 longitudinal biopsies available for analysis. These were compared with FFPE skin biopsies from patients with unspecified dermatitis (n=29) and healthy skin (n=12). All samples were collected from the archives of the Department of Pathology, Region Zealand, Denmark in the period 1990-2016. The histological samples were revised and clinical records were reviewed to establish the diagnosis and stage for each patient. Total RNA was extracted from ten 10-μm sections of FFPE tissue, and 50-100 ng of RNA was analyzed on the NanoString nCounter platform by applying the Myeloid Innate Immunity Panel, which quantifies the expression of 800 immune related genes. Differentially expressed genes (DEG, 2-fold change, p&lt;0.05), were assessed by ANOVA, and a Support Vector Machine (SVM) diagnostic classifier was built based on 2-10 DEG and evaluated by 10-fold cross-validation. The classifier was tested on an independent, early-stage MF patient cohort (n=27). Protein expression was validated with immunohistochemistry and digitally analyzed by applying a specifically designed algorithm with the Leica Tissue IA 2.0 software. Double immunofluorescence staining protocols were developed to identify subtypes of TRAF1 positive cells in combination with various macrophage/dendritic cell markers (CD168, CD63, CD11c, CD1a, CD14, and S100) as well as T-cell (CD3) and B-cell (CD20) markers. Results A diagnostic classifier consisting of TOX and TRAF1 was able to distinguish early-stage MF from dermatitis with an overall accuracy of 85% in the discovery cohort and 80% in an independent validation cohort. TOX and TRAF1 protein levels were significantly elevated in early-stage MF compared to the dermatitis group (p &lt; 0.0001). TOX and TRAF1 were also significantly increased in the progression from early-stage MF to tumor stage MF (p=0.003 and p=0.004, respectively). Subtypes of TRAF1-positive dendritic cells in early-stage MF consisted primarily of S100+ cells in both the epidermal and dermal compartment. A few TRAF1+ cells in the Pautrier microabscesses stained double positive with CD11c. In tumor stage MF the majority of TRAF1+ dendritic cells counterstained with CD1a and CD11c. Both neoplastic and reactive T-cells (CD3+) expressed TRAF1 in a minor degree in early-stage MF, while T-cells in tumor stage MF expressed TRAF1 in a much higher degree and the majority of the neoplastic T-cells were TRAF1 positive. No macrophages (CD68+ or CD163+) or B-cells double stained with TRAF1. Conclusion In the present study, we developed a two gene mRNA diagnostic classifier discriminating early-stage MF from dermatitis. The protein expression level of TOX and TRAF1 confirmed our gene expression levels and identified a highly significant difference between early-stage MF and dermatitis, which can prove useful in diagnostics of early-stage MF. Disclosures Andersen: Novo Nordisk: Other: Holds stock in Novo Nordisk; Hologic Deutchland GmbH: Research Funding. Odum:Micreos human Health B.V: Consultancy. Litman:Leo Pharma A/S: Research Funding. Gjerdrum:Nanostring Technologies: Other: Recieves founding from NanoString regarding a B-cell lymphoma research project., Research Funding; Celgene: Other: Celgene has funded the participation in ASH 2019.

Investigating the presence of neutrophil extracellular traps in cutaneous lesions of different subtypes of lupus erythematosus

Neutrophil extracellular traps (NETs) are implicated in the pathogenesis of systemic lupus erythematosus (SLE). However, little is known about the implication of NETs in cutaneous lupus. In this case series of 30 patients, we compared the amounts of neutrophils producing NETs in cutaneous lesions of different subtypes of lupus (5 with discoid lupus or DLE, 5 with subacute cutaneous lupus or SCLE, 11 with acute cutaneous SLE, 7 with lupus panniculitis and 2 with chilblains). Immunofluorescence was performed on formalin-fixed paraffin-embedded skin biopsies using antibodies against neutrophilic granules (elastase, myeloperoxidase, PR-3 proteins and citrullinated histone 3). Dihydroethidium staining was performed to detect reactive oxygen species (ROS), known inducers of NETs. NETs were detected in the different subtypes of cutaneous lupus as well as in cutaneous lesions of SLE. The amounts of neutrophils producing NETs were significantly higher in lupus panniculitis (49%), acute cutaneous SLE (41%) and DLE (32%), in comparison with SCLE (5%) and chilblains (0%). This suggests that NETs might be associated with more tissue damage and scarring. ROS were observed in the different cutaneous subtypes of lupus independent of NETs.

072 Activation status of cutaneous and blood eosinophils in Drug Reaction with Eosinophilia and Systemic Symptoms (DRESS)

DRESS is a drug-induced hypersensivity syndrom that can lead to severe organs failure. The role of cutaneous (c-Eo) and blood eosinophils (b-Eo) in tissue damage remains unknown. We aimed to analyze their activation status in DRESS. We collected retrospectively paraffin-embedded skin biopsies for patients from a French University hospital, who fulfilled criteria for drug-induced maculopapular exanthema (EMP) or DRESS. 1) in 38 patients (18 EMP and 20 DRESS), c-Eo and extracellular eosinophilic granules (ECP, MBP) were identified by immunohistochemical staining and quantified by an original semi-automatic method according to the color deconvolution and circularity parameters. 2) Fluorescence Activated Cell Sorting (FACS) was performed for activation markers of b-Eo in 4 DRESS and 4 healthy subjects. The number of c-Eo was significantly higher in DRESS patients (with anti-MBP staining, p=0,044). Analysis restricted to patients with Eo infiltrate in skin showed that granules were more frequent in the DRESS group according to the number of degranulation areas (p=0,027) and area of degranulation (p=0,02) suggesting that c-Eo from DRESS patients were activated. Analysis of activation markers of b-Eo by FACS showed lower expression of tissue-homing markers like CRTH2 (p=0,018) or activation markers like CD63 (p=0,029) and HLA-DR (p=0,029) in patients with DRESS suggesting a down-regulation of surface markers on b-Eo correlated with their activated status in the skin. Further study will be necessary to better understand the mechanisms of Eo recruitment in DRESS.

Nested Polymerase Chain Reaction and Cutaneous Tuberculosis.

The role of Mycobacterium tuberculosis in the etiology and pathogenesis of cutaneous tuberculosis is controversial because of the difficulties associated with demonstrating the presence of these mycobacteria in tuberculid cutaneous lesions by routinely available microbiological and histological techniques. In this study, we aimed to demonstrate the presence of M. tuberculosis in cutaneous tuberculosis. Multiple polymerase chain reaction (PCR) followed by nested PCR was used to amplify genomic fragments from 3 different mycobacteria species. DNA was isolated from 30 paraffin-embedded skin biopsies. Samples were selected randomly from patients with a clinical and histopathological diagnosis of the most frequent groups of cutaneous tuberculosis in Mexico as follows: 5 cases of scrofuloderma tuberculosis; 2 cases of lupus vulgaris tuberculosis; and 5 cases of tuberculosis verrucosa cutis. The other cases denominated tuberculids in some countries such as Mexico and included the following: 7 cases of rosacea-like tuberculosis; one case of papulonecrotic tuberculosis; and 10 cases of erythema induratum of Bazin. Four normal skin biopsies were included as controls. M. tuberculosis DNA was amplified successfully by nested PCR in 80% of the samples (24 of the 30 samples) assayed. Mycobacterial DNA was not detected in the normal skin biopsies used as controls. Detection of M. tuberculosis DNA in 80% of cutaneous tuberculosis analyzed implicates this mycobacterium in the pathogenesis of multiple clinical forms of cutaneous tuberculosis.

Dysregulation of the TOX-RUNX3 pathway in cutaneous T-cell lymphoma.

Studies have examined gene expression changes in Sézary syndrome (SS), but disease pathogenesis remains largely unknown, and diagnosis and treatment are difficult. TOX is a transcription factor involved in CD4+ T-cell development with downstream effects on RUNX3, a known tumor suppressor gene. We sought to identify genes involved in SS disease pathogenesis with the potential to enable diagnosis and treatment. We utilized previously reported transcriptome sequencing data to construct a list of candidate genes, which was narrowed using pathway analysis. qRT-PCR confirmed TOX upregulation (>7 fold increase) in SS (n = 5), as well as two established markers, PLS3 and KIRD3DL2. We also evaluated expression of members of the TOX-RUNX3 pathway and confirmed downregulation of RUNX3 (0.59 fold decrease) and upregulation of GATA3 (2 fold increase). Moreover, TOX and RUNX3 expression were significantly inversely proportional. Using siRNA to suppress TOX, we demonstrated that TOX knockdown rescues RUNX3 expression and reduces cell viability. We evaluated TOX protein expression in paraffin-embedded skin biopsies with immunohistochemistry, showing nuclear staining of CTCL infiltrates, suggesting it is a candidate diagnostic biomarker. Further studies validating our findings and evaluating the TOX-RUNX3 pathway and the role of TOX as a disease marker and therapeutic target are warranted.

Contribution of Immunohistochemistry in Revealing S100A7/JAB1 Colocalization in Psoriatic Epidermal Keratinocyte.

The application of immunohistological methods provides an invaluable contribution in revealing the protein colocalization, which may reflect the occurrence of molecular interaction processes.This chapter describes comprehensive protocols for detection of S100A7/JAB1 colocalization by immunohistochemistry in archival formalin-fixed and paraffin-embedded skin biopsies from healthy and psoriatic subjects. In addition, we provide a protocol for immunocytochemical detection of S100A7/JAB1 colocalization in S100A7 CRISPR-activated human keratinocyte cell line.

Clinical and diagnostic aspects of feline cutaneous leishmaniosis in Venezuela

Venezuela is an endemic area for human and canine leishmaniosis due to Leishmania infantum and parasites of the Leishmania braziliensis and L. mexicana complexes. Limited data are available on feline leishmaniosis (FeL) in this region. The aim of this study was to describe clinical and diagnostic aspects of FeL in Venezuela. Thirty-one domestic cats from urban areas of Lara State in Venezuela were enrolled. Twenty-five were healthy. Six other cats had solitary or multiple nodular lesions, which were located on the nose, ears, tail and lower limbs. Cutaneous lesions were characterized by diffuse pyogranulomatous infiltrate in all sick cats with numerous intracellular and extracellular amastigotes, and immunohistochemistry was positive for Leishmania in five sick cats. All healthy cats were seronegative for L. infantum and L. braziliensis antigens by ELISA. Two out of five sick cats yielded a positive ELISA result to both Leishmania antigens with higher antibody levels to L. braziliensis compared to L. infantum. Significantly higher antibody levels by ELISA as well as a higher number of bands by Western blot (WB) were found for L. braziliensis when compared to L. infantum antigens in all sera from Venezuelan sick and healthy cats. All healthy cats were blood Leishmania spp. qPCR negative while three out of six sick cats were blood qPCR positive. All paraffin-embedded skin biopsies (n = 4) as well as cutaneous cytology (n = 3) were positive by Leishmania spp. qPCR in sick cats. Leishmania speciation was obtained only from the cutaneous lesion samples from cytological preparations of two out of three sick cats which were identified as infected with L. mexicana or a closely related specie. Feline leishmaniosis should be included in the differential diagnosis list of nodular-ulcerative lesions. The most reliable diagnostic technique in sick cats is cytological or histopathological examination along with immunohistochemistry, since blood PCR and serology by ELISA might be negative. WB appears to be more sensitive in detecting infection. Cats with leishmaniosis from Venezuela are most likely infected with species of L. mexicana or a closely related species or the L. braziliensis species complex and not with L. infantum.

Evaluation of tissue levels of glutathione S-transferases (GST) isoenzymes in patients with discoid lupus erythematosus

Aim: Cutaneous lupus erythematosus (CLE) has a multifactorial pathogenesis involving genetic and environmental triggers and congenital and acquired immune response. The aim of this study was to investigate the effects of Glutathione S-Transferases (GST) isoenzymes including GSTT1, GSTM1, and GSTP1 in the CLE patients with an etiology of solar radiation exposure. Material and Methods: Paraffin-embedded skin biopsy sections from the patients were stained by immunohistochemical methods. The results were evaluated under a light microscope by a pathologist. The pattern, localization, and distribution of the immunohistochemical staining were recorded for each patient. Staining of the nucleus or cytoplasm was considered as positive staining. The accuracy of staining was determined based on the intensity and percentage of staining. Results: No significant difference was found between the patient and control groups regarding staining intensity. In terms of staining percentage, the prevalence of GSTP1-3 genotype was significantly lower in the patient group compared to the control group (25% vs. 63.33%) (p=0.002). Conclusion: No significant difference was observed in the staining intensity of GSTP1, GSTT1, and GSTM1 between the patient and control groups and the staining percentage in some genotypes was even higher in the control group compared to the patient group.

Treponema pallidum ssp. pallidum identification by real-time PCR targetting the polA gene in paraffin-embedded samples positive by immunohistochemistry.

Syphilis is a systemic and sexually transmitted infection caused by Treponema pallidum ssp. pallidum. This spirochete causes different clinical and subclinical stages depending on the duration of infection and immune status of the host. Several tests have been developed for diagnosis, and are classified into direct and indirect methods. The first one includes dark field microscopy, direct fluorescent antibody test in fluids or tissue, and molecular biology techniques. In the indirect method (serologic), the routine tests are used, and are divided in two categories: non-treponemal and treponemal ones. The objective of this work was to identify T. pallidum ssp. pallidum in paraffin-embedded skin biopsies positive by immunohistochemistry, using conventional polymerase chain reaction (PCR) and quantitative real time PCR (qPCR). We included a sample of 17 paraffin-embedded biopsies. DNA was extracted and processed by conventional PCR and real-time PCR with a TaqMan® probe to identify the polA gene. Using PCR, 11 tested positive (64.7%) and 6 (35.3%) were negative. With qPCR and TaqMan® probe, 100% of samples tested positive. The minimum number of spirochetes detected in each sample was 2. With this work, we can conclude that qPCR is a fast and very accurate method for diagnosis of syphilis in tissue specimens.

Folliculocentric Herpes: A Clinicopathological Study of 28 Patients.

The cutaneous manifestations of herpes infection are primarily in the context of active infection and of the post-herpetic zosteriform eruption. The former manifests cytopathic alterations diagnostic of herpes. The latter includes lichen planus-like and granuloma annulare-like eruptions and lymphocytoma cutis. We encountered skin biopsies from 28 patients whose acute or chronic herpetic or post-herpetic zosteriform lesions manifested folliculocentricity. The clinical appearance of the lesions was correlated with the histopathologic and immunohistochemical features of paraffin-embedded skin biopsies to determine the specific viral etiology. A history of underlying medical disease was noted if present. There were 16 men and 12 women with a folliculocentric eruption occurring after a known herpetic eruption or manifesting cytopathic changes and/or immunohistochemical findings compatible with herpes virus in lesional skin biopsies. Underlying immune dysregulatory states were present in most cases, namely, malignancy, anticonvulsant or antidepressant therapy, diabetes mellitus, psoriasis, Crohn disease, and other conditions. All biopsies demonstrated dense lymphohistiocytic infiltrates in or around hair follicles with variable necrosis, while active infections also showed cytopathic and/or immunohistochemical evidence of herpetic alterations, most commonly varicella zoster. Other features included interfollicular interface dermatitis, lymphocytic eccrine hidradenitis, neuritis, and folliculocentric vasculitis. Cutaneous herpetic eruptions can evoke a predominantly folliculocentric mononuclear cell reaction and vasculitis; there is an association with underlying endogenous and/or iatrogenic immune dysregulation. Most cases are secondary to reactivation of varicella zoster. The histomorphology suggests a role for cell-mediated immunity. Antigenic homology of an endogenous 72-kd heat shock protein in follicles with that of a herpetic heat shock protein, in concert with an intrafollicular proliferative response of γ-δ T lymphocytes, may explain the follicular localization and composition of the infiltrate.

Localized parakeratotic hyperkeratosis in sixteen Boston terrier dogs.

Although zinc responsive dermatosis is typically a disorder of Arctic breed dogs, this study identifies similar cutaneous lesions on the face and pressure points of Boston terrier dogs. To document the clinical and histological features of localized parakeratotic hyperkeratosis of Boston terrier dogs, to determine if the lesions respond to zinc supplementation and to determine whether tissue zinc levels were decreased in affected versus unaffected dogs. Sixteen Boston terrier dogs with similar gross and histological findings were identified retrospectively from two institutions. Follow-up information for nine dogs from one institution was obtained from referring veterinarians using a questionnaire. Tissue zinc levels were measured from formalin-fixed paraffin-embedded skin biopsy samples of affected and unaffected dogs using inductively coupled plasma mass spectrometry. Mild to severe parakeratotic hyperkeratosis with follicular involvement was present in all 16 cases. Of the nine dogs for which follow-up information was available, five dogs received oral zinc supplementation and four dogs had documented clinical improvement or resolution of dermatological lesions. The median skin zinc levels were not significantly different between affected and unaffected dogs. To the best of the authors' knowledge this is the first report of localized parakeratotic hyperkeratosis in Boston terrier dogs, some of which improved with oral zinc supplementation. Prospective studies in Boston terrier dogs are warranted to document potential zinc deficiency (serum and/or tissue levels, pre- and post-treatment) and to objectively assess response to zinc supplementation and other therapies.

Genomic imbalances and microRNA transcriptional profiles in patients with mycosis fungoides.

Mycosis fungoides is the most common type of primary cutaneous T cell lymphoma. We have evaluated CDKN2A losses and MYC gains/amplifications by FISH analysis, as well as expression of miR-155 and members of the oncogenic cluster miR-17-92 (miR17, miR18a, miR19b, and miR92a) in MF patients with advanced disease. Formalin-fixed paraffin-embedded skin biopsies from 36 patients at diagnosis, 16 with tumoral MF (T-MF), 13 in histological transformation to a large T cell lymphoma (TR-MF), and 7 cases with folliculotropic variant (F-MF), were studied. Twenty cases showed genomic alterations (GAs): 8 (40%) had CDKN2A deletion, 7 (35%) showed MYC gain, and 5 (25%) exhibited both alterations. GAs were more frequently observed in F-MF (p=0.004) and TR-MF (p=0.0001) than T-MF. GAs were significantly higher in cases presenting lesions in head, neck, and lower extremities compared to those observed in trunk and upper extremities (p=0.03), when ≥25% neoplastic cells were CD30 positive (p=0.016) as well as in cases with higher Ki-67 proliferation index (p=0.003). Patients with GAs showed bad response to treatment (p=0.02) and short survival (p=0.04). Furthermore, MF patients showed higher miRNA expression compared to controls (p≤0.0223). T-MF showed higher miR17 and miR-18a expression compared to F-MF and TR-MF (p≤0.0387) while miR19b, miR92a, and miR-155 showed increased levels in F-MF and TR-MF with respect to T-MF (p ≤ 0.0360). Increased expression of miR17 and miR19b in GA group compared to cases without alterations (p≥0.0307) was also detected. Our results add new information about genomic imbalances in MF patients, particularly in F-MF, and extend the present view of miRNA deregulation in this disease.

Molecular and histological tools to diagnose an imported case of American cutaneous leishmaniasis in Cuba

Leishmaniasis represents a polymorphous group of diseases caused by around 20 different species of Leishmania parasite. Increases in the number of cases of leishmaniasis reported as a consequence of the growth in travel and migration are of concern to epidemiologists and are diagnostically challenging in non-endemic areas. Molecular and histological analyses of a paraffin-embedded skin biopsy were used in parallel to detect Leishmania parasites in a Cuban woman with suspicious lesions arriving in Cuba from Venezuela. Primers based on the 18S fragment of ribosomal ribonucleic acid (rRNA) and heat shock protein 70 genes (hsp70) were used for molecular detection. Histological studies detected the presence of the parasite. A small fragment of Leishmania DNA was amplified by polymerase chain reaction (PCR) targeting the 18S fragment using, for the first time, nucleic acid obtained from paraffin-embedded tissue as a template. Amplification of a larger fragment from the hsp70 gene did not occur. The detection of Leishmania DNA from paraffin-embedded tissue by means of 18S-targeted PCR is a feasible approach to diagnosis. In combination with classical methods such as histology, the molecular detection of the parasite was demonstrated to be useful in confirming Leishmania infection in a traveler.

Differential expression of TOX by skin-infiltrating T cells in Sézary syndrome and erythrodermic dermatitis.

The histopathologic differentiation between Sézary syndrome (SS) and erythrodermic dermatitis may be extremely difficult. In this immunohistochemical study, it was investigated if thymocyte selection-associated high mobility group box protein (TOX) and C-MYC can be used as additional diagnostic markers to differentiate between SS and erythrodermic dermatitis. Paraffin-embedded skin biopsies from 15 SS patients and 17 erythrodermic dermatitis patients were stained and scored for TOX or C-MYC expression. Strong nuclear staining for TOX in more than 50% of skin-infiltrating T cells was observed in 13 of 15 (87%) SS cases, whereas erythrodermic dermatitis cases showed weak nuclear staining in 11-50% (median: 25%) of the T cells; strong nuclear staining as found in SS was never observed in erythrodermic dermatitis. No significant differences in C-MYC expression between SS and erythrodermic dermatitis were found. In most patients of both groups, percentages of C-MYC positive-cells varied between less than 10 and 25% of skin-infiltrating T cells. Our results suggest that strong expression of TOX in more than 50% of skin-infiltrating T cells in erythrodermic skin is a useful marker in the differentiation between SS and erythrodermic dermatitis, whereas staining for C-MYC does not contribute to differential diagnosis.

Immunohistochemical localization of basement membrane laminin 5 and collagen IV in adult linear IgA disease.

Linear immunoglobulin A disease (LAD), also known as linear IgA bullous dermatosis, is an autoimmune disorder characterized by subepidermal bullae caused by IgA autoantibodies directed against several antigens located in the basement membrane zone of the skin. Laminin 5 (laminin-332) is considered a key component of the lamina lucida/lamina densa interface, which provides stable attachment of the epidermis to the dermis. Meanwhile, collagen IV is a major component of the lamina densa. Laminin 5 and collagen IV bind to the cell membrane and induce cytoskeletal rearrangements, which contribute to the basement membrane's final mat-like structure. The study aimed to evaluate the immunohistochemical staining of laminin 5 and collagen IV and to identify the site of blister formation in formalin-fixed, paraffin-embedded skin biopsies from adults with LAD. Skin biopsies from 20 adult patients with LAD were subjected to routine hematoxylin-eosin as well as immunohistochemical staining of collagen IV and laminin 5. Linear staining was positive on the floor of the blister for laminin 5 in 65% and collagen IV in 90% of biopsies denoting that the site of separation in most cases of LAD is above the lamina densa. The use of laminin 5 and collagen IV immunohistochemistry can be considered as an adjuvant diagnostic tool and may aid in the identification of the site of blister formation in routine skin biopsies in adults with LAD.