S3 guideline: Diagnosis and treatment of epidermal necrolysis (Stevens-Johnson syndrome and toxic epidermal necrolysis) - Part 1: Diagnosis, initial management, and immunomodulating systemic therapy.

Stevens-Johnson syndrome and toxic epidermal necrolysis (SJS/TEN) are rare, predominantly drug-induced, acute, life-threatening diseases of skin and mucosae. SJS and TEN are nowadays considered variants of one disease entity with varying degrees of severity called epidermal necrolysis (EN). EN is associated with high morbidity and mortality and constitutes a major disease burden for affected patients. The guideline "Diagnosis and treatment of epidermal necrolysis (Stevens-Johnson syndrome and toxic epidermal necrolysis)" was developed under systematic consideration of existing scientific literature and in a formal consensus process according to regulations issued by the Association of Scientific Medical Societies in Germany (AWMF) to establish an evidence-based framework to support clinical decision-making. The interdisciplinary guideline commission consisted of representatives from various specialist societies and patient representatives. The guideline is aimed at specialists in the fields of dermatology, ophthalmology, plastic surgery, intensive care, and pediatrics in hospitals and offices, as well as other medical speciallved in the diagnosis and treatment of EN. The guideline is also aimed at patients, their relatives, insurance funds, and policymakers. This first part focuses on the diagnostic aspects, the initial management as well as the immunomodulating systemic therapy.

HIF1α and HIF2α immunoreactivity in epithelial tissue of primary and recurrent pterygium by immunohistochemical analysis.

PURPOSE: Hypoxia-inducible factors (HIFs) are considered to play a significant role in the pathogenesis of pterygium. The aim of this study was to investigate the relative expression or immunoreactivity of HIF1α and HIF2α in the epithelium of primary pterygium, recurrences and healthy conjunctiva. METHODS: Immunohistochemical staining was performed with antibodies against HIF1α and HIF2α, respectively, on 55/84 primary pterygium specimens, 6/28 recurrences and 20/20 control tissues (healthy conjunctiva). RESULTS: Immunohistochemical staining revealed lower epithelial immunoreactivity of HIF1α and HIF2α in both primary pterygium (11% and 38%) and recurrences (18% and 21%) when compared to healthy conjunctival tissue (46% and 66%). Differences between immunoreactivity of HIF1α and of HIF2α in primary pterygium and controls were each highly significant (p < .001). Within the group of primary pterygium, epithelial immunoreactivity of HIF2α (38%) was significantly higher than that of HIF1α (11%). In recurrent pterygium and healthy conjunctiva, immunoreactivity levels of HIF2α were higher than those of HIF1α as well; however, differences between both isoforms were not significant. CONCLUSION: Our study shows evidence that the higher expressed epithelial HIF2α, rather than HIF1α, and the balance between both HIF isoforms might be relevant factors associated with pathogenesis of primary pterygium. Modulation of HIF2α levels and activity may thus offer a new therapeutic approach to the treatment of advancing pterygium where the initial stage with its HIF1-peak has already passed.

Merkel Cell Carcinoma of the Upper Eye Lid with Atypical Clinical Appearance. / Merkel-Zell-Karzinome des Oberlides mit ungewöhnlichem klinischem Erscheinungsbild.

Merkel cell carcinoma (MCC) is a rare aggressive neuroendocrine skin tumor that occurs mainly in the sun-exposed head and neck region, but rarely in the eyelid region. Early lymphogenic spread often leads to locoregional metastases, which is why early diagnosis is crucial. Classically, periocular MCC presents as a reddish-livid nodule in elderly patients, a visual diagnosis. However, given its low incidence and variable appearance, diagnosis can also be challenging. In both cases presented here, the MCC presented as a skin-colored swelling. In patient 1, the tumor showed a partially deep subaponeurotic localization and mimicked a B-cell lymphoma histopathologically, whereas in patient 2 it imitated a diffuse chalazion clinically. After immunohistochemical characterization and exclusion of metastases, the initial clinically benign appearing lesions in both patients were classified as CK20 negative MCC. The extensive upper eyelid defects were reconstructed by Cutler-Beard plastic surgery, a particular challenge in patient 1 given the oculus unicus situation. Our two cases demonstrate that Merkel cell carcinomas not only manifest as deep red cherry-shaped tumors, but can be skin-colored and mimic benign changes when atypical in location or infiltration pattern.

3' MACE RNA-sequencing allows for transcriptome profiling in human tissue samples after long-term storage.

This study aims to compare the potential of standard RNA-sequencing (RNA-Seq) and 3' massive analysis of c-DNA ends (MACE) RNA-sequencing for the analysis of fresh tissue and describes transcriptome profiling of formalin-fixed paraffin-embedded (FFPE) archival human samples by MACE. To compare MACE to standard RNA-Seq on fresh tissue, four healthy conjunctiva from four subjects were collected during vitreoretinal surgery, halved and immediately transferred to RNA lysis buffer without prior fixation and then processed for either standard RNA-Seq or MACE RNA-Seq analysis. To assess the impact of FFPE preparation on MACE, a third part was fixed in formalin and processed for paraffin embedding, and its transcriptional profile was compared with the unfixed specimens analyzed by MACE. To investigate the impact of FFPE storage time on MACE results, 24 FFPE-treated conjunctival samples from 24 patients were analyzed as well. Nineteen thousand six hundred fifty-nine transcribed genes were detected by both MACE and standard RNA-Seq on fresh tissue, while 3251 and 2213 transcripts were identified explicitly by MACE or RNA-Seq, respectively. Standard RNA-Seq tended to yield longer detected transcripts more often than MACE technology despite normalization, indicating that the MACE technology is less susceptible to a length bias. FFPE processing revealed negligible effects on MACE sequencing results. Several quality-control measurements showed that long-term storage in paraffin did not decrease the diversity of MACE libraries. We noted a nonlinear relation between storage time and the number of raw reads with an accelerated decrease within the first 1000 days in paraffin, while the numbers remained relatively stable in older samples. Interestingly, the number of transcribed genes detected was independent on FFPE storage time. RNA of sufficient quality and quantity can be extracted from FFPE samples to obtain comprehensive transcriptome profiling using MACE technology. We thus present MACE as a novel opportunity for utilizing FFPE samples stored in histological archives.

HIF-1α, HIF-2α, and ProExC: diagnostic or prognostic relevance in conjunctival intraepithelial neoplasia?

PURPOSE: The aim of this study was to investigate HIF-1α, HIF-2α, and ProExC expression in conjunctival intraepithelial neoplasia (CIN), to differentiate between metaplasia and dysplasia, and to access their value as diagnostic and prognostic immunohistochemical markers. Recurrence and progression into SCC (squamous cell carcinoma) were defined as endpoints. METHODS: Forty-three specimens including CIN I (2), CIN II (9), CIN III (29), with and without metaplasia, and metaplasia alone (3), as well as 21 conjunctival control specimens, were stained with antibodies against HIF-1α, HIF-2α, and ProExC. The percentage of positively stained cells were calculated and used for further analysis. RESULTS: The mean percentages of HIF-1α and HIF-2α were not increased in CIN. In comparison, the expressions of these markers were even significantly elevated in control specimens (p < 0.001). Upper epithelial cells in CIN were more often ProExC-positive compared with normal conjunctiva or metaplasia (p = 0.06 and p = 0.07). Cox proportional-hazards analysis was performed for characterization of factors influencing the combined endpoint and showed a significant elevated hazard ratio for staining with ProExC (p = 0.04) compared with HIF-1α (p = 0.26) and HIF-2α (p = 0.49). CONCLUSION: Our study shows that HIF-1α and HIF-2α do not serve as diagnostic or prognostic markers in CIN. ProExC seems to be a potential indicator for CIN, but not a reliable diagnostic marker. However, control specimens occasionally also display a high percentage of ProExC-positive cells and staining over the entire epithelial layer.

Conjunctival HLA-DR and CD8 expression detected by impression cytology in ocular graft versus host disease.

PURPOSE: To assess the expression of human leucocyte antigen (HLA)-DR in epithelial cells and cluster of differentiation (CD8)-positive lymphocytes as possible markers of chronic ocular graft versus host disease (cGvHD) after hematological stem cell transplantation (HSCT). METHODS: Twenty-seven consecutive patients with dry-eye symptoms following HSCT (24 [89%] with peripheral blood stem cell transplantation and 3 [11%] with bone marrow transplants; 17 [63%] familiar allogenic grafts) and 19 age-matched controls were included. Conjunctival impression cytology specimens were stained for HLA-DR, cytokeratin 19, and CD8. Oxford grading scale, blinking frequency, Schirmer test, tear film break-up time (TBUT), and Ocular Surface Disease Index (OSDI) were also recorded. Wilcoxon nonparametric testing was used to compare controls and HSCT recipients and to assess HSCT recipient subgroups with and without clinical cGVHD. RESULTS: Eighteen patients showed clinical signs of ocular cGVHD. TBUT and Schirmer test scores were significantly lower in patients, while Oxford grades and OSDI were significantly higher than in controls. Epithelial HLA-DR expression was generally higher in HSCT recipients than in controls, but it did not correlate with ocular cGVHD status. CD8-positive lymphocytes were identified in five patients with ocular cGvHD and one control. CONCLUSIONS: A strong HLA-DR expression as detected by impression cytology appears to indicate a general HSCT response and fails to predict ocular cGVHD. However, the detection of CD8-positive lymphocytes using impression cytology was frequently associated with ocular cGvHD. Our data warrant further evaluation of CD8 expression in impression cytology, along with comparison to conjunctival biopsies and brush cytology, as impression cytology may offer a less invasive strategy for assessing cGVHD status.

MACE RNA sequencing analysis of conjunctival squamous cell carcinoma and papilloma using formalin-fixed paraffin-embedded tumor tissue.

Recent advances in the field of biomedical research allow for elucidation of the transcriptional signature of rare tumors such as conjunctival squamous cell carcinoma (SCC). In this study we compare its expression profile to conjunctival papilloma (Pap) and healthy conjunctival tissue (Ctrl) and develop a classification tool to differentiate these entities. Seven conjunctival SCC, seven Pap and ten Ctrl were formalin-fixed and paraffin-embedded (FFPE) and analyzed using Massive Analysis of cDNA Ends (MACE) RNA sequencing. Differentially expressed genes (DEG) and gene ontology (GO) clusters were explored and the abundance of involved cell types was quantified by xCell. Finally, a classification model was developed to distinguish SCC from Pap and Ctrl. Among the most prominent DEG in SCC a plethora of keratins were upregulated when compared to Pap and Ctrl. xCell analysis revealed an enrichment of immune cells, including activated dendritic cells and T-helper type 1 cells (Th1), in SCC when compared to Ctrl. The generated classification model could reliably discriminate between the three entities according to the expression pattern of 30 factors. This study provides a transcriptome-wide gene expression profile of rare conjunctival SCC. The analysis identifies distinct keratins, as well as dendritic and Th1 cells as important mediators in SCC. Finally, the provided gene expression classifier may become an aid to the conventional histological classification of conjunctival tumors in uncertain cases.

Transcriptional characterization of conjunctival melanoma identifies the cellular tumor microenvironment and prognostic gene signatures.

This study characterizes the transcriptome and the cellular tumor microenvironment (TME) of conjunctival melanoma (CM) and identifies prognostically relevant biomarkers. 12 formalin-fixed and paraffin-embedded CM were analyzed by MACE RNA sequencing, including six cases each with good or poor clinical outcome, the latter being defined by local recurrence and/or systemic metastases. Eight healthy conjunctival specimens served as controls. The TME of CM, as determined by bioinformatic cell type enrichment analysis, was characterized by the enrichment of melanocytes, pericytes and especially various immune cell types, such as plasmacytoid dendritic cells, natural killer T cells, B cells and mast cells. Differentially expressed genes between CM and control were mainly involved in inhibition of apoptosis, proteolysis and response to growth factors. POU3F3, BIRC5 and 7 were among the top expressed genes associated with inhibition of apoptosis. 20 genes, among them CENPK, INHA, USP33, CASP3, SNORA73B, AAR2, SNRNP48 and GPN1, were identified as prognostically relevant factors reaching high classification accuracy (area under the curve: 1.0). The present study provides new insights into the TME and the transcriptional profile of CM and additionally identifies new prognostic biomarkers. These results add new diagnostic tools and may lead to new options of targeted therapy for CM.

Histologic safety margin in basal cell carcinoma of the eyelid: correlation with recurrence rate.

OBJECTIVE: To examine the correlation between the minimum histologic safety margin (HSM) and recurrence rate of periorbital basal cell carcinomas (BCC). DESIGN: Cohort study. PARTICIPANTS: One hundred one patients with 101 BCCs treated surgically between 1997 and 1999 at the eye hospital in Freiburg were enrolled in this study. Mean follow-up was 7 years (range, 104 days to 9.7 years). METHODS: The tumors' minimum HSM was measured retrospectively in photographs of hematoxylin and eosin-stained paraffin slides using the digital picture analysis system AnalySIS of Soft Imaging System Inc, and/or calculated according to the tumor-free section number. Statistical analysis was performed using the Kaplan-Meier method and the log-rank test. MAIN OUTCOME MEASURES: Histologic margins of solid and fibrous BCC and recurrence rate. RESULTS: Seven of the 101 patients experienced tumor recurrence (6.93%) after a mean follow-up of 34.7 months (range, 3-83) according to Kaplan-Meier analysis. The patients were assigned to 1 of 3 groups: (I) those without HSM (n = 11), 3 recurrences (27.27%); (II) those with HSM <0.2 mm (n = 18), 3 recurrences (16.67%); and (III) those with HSM >0.2 mm (n = 72), 1 recurrence (1.39%). The difference in recurrences between those groups with HSM and HSM = 0, as well as between the HSM <0.2 mm-group and HSM >0.2 mm-group were statistically significant (P = 0.01; P = 0.03). CONCLUSIONS: Extremely small HSMs are likely to prevent recurrences. At critical and visually easily accessible tumor sites (e.g., adjacent to the lacrimal puncta) a re-resection in solid BCCs with tumor-positive margins may not be mandatory, provided the surgical site is clinically inspected regularly. This conclusion does not apply to fibrous BCC.

Cicatricial ectropion in progressive skin diseases.

PURPOSE. To report the clinical course, patient care and treatment of cicatricial ectropion in patients with progressive skin diseases. METHOD. Review and photo series of three typical cases, which were followed for up to 10 years. RESULTS. In certain severe progressive skin diseases, tissue shrinkage may progress permanently. The soft lid tissue cannot withstand the forces of vertical lid traction. As a result, recurrent ectropion occurs. Patients with lamellar ichthyosis and with eruptive Grzybowski-type keratoacanthoma were followed for up to 10 years. Free skin grafts of severely involved donor skin were repeatedly transplanted to the lids. The lid margins were fixed by traction sutures in order to spread out the wound and to allow rapid vascular ingrowth and undisturbed healing. In this way, early wound contracture could be prevented. Nevertheless, follow-up revealed progressive shrinkage of the transplanted lid skin. Eversion of the lacrimal punctum was the first sign of progression. Epiphora was the leading complaint of the patients. Bacterial superinfection of the deepened lacrimal lake was more frequent in advanced ectropion. CONCLUSION. Patients should understand the natural history of their disease in order to accept multiple surgical procedures. Ectropion should be re-operated in time in order to reduce epiphora, to prevent corneal complications, and to avoid metaplasia and keratinization of the conjunctiva and thickening of the lid margin. The elasticity of the lid skin should be improved pharmacologically and by increasing the relative humidity of the home environment, especially in winter. Consistent vertical lid massage can delay recurrence.

Histological and immunohistochemical characterisation of conjunctival graft vs host disease following haematopoietic stem cell transplantation.

BACKGROUND: Conjunctival graft vs host disease (cnGvHD) is a complication of haematopoietic stem cell transplantation, in most cases as part of systemic GvHD. Diagnostic biopsies are commonly collected from bulbar conjunctiva only. The aims of our study were to evaluate whether additional biopsies from the tarsal conjunctiva increase sensitivity upon histopathologic evaluation and to investigate the staining profile for common immunohistochemical markers in cnGvHD. We additionally propose an adaptive histological classification for cnGvHD analogous to Lerner's GvHD skin classification for predicting patient survival. METHODS: Formalin-fixed and paraffin-embedded conjunctival specimens from 23 post-mortem control eyes and 42 patients after haematopoietic stem cell transplantation (HSCT) were stained with haematoxylin and eosin (HE), periodic acid-Schiff (PAS) stain and with antibodies against CD1a, CD4, CD8, CD25, CD45RO, CD68, Fas ligand, TIA-1, HLA-DRalpha by means of immunohistochemistry. Cell counting took place in ten representative fields at 64.4 microm (length) x 21.2 microm (width). Multifactorial analysis of variance was performed to assess any influence of cnGvHD on the staining pattern for the immunohistochemical markers. Survival times were estimated by the Kaplan-Meier method. RESULTS: All 42 specimens and none of the controls were diagnosed as cnGvHD. The bulbar specimens were staged according to the modified Lerner classification: grade (G) I: 0; G II: 17 (tarsal with GII, 8); G III: 12 (tarsal with GIII: 1); G IV: 12 (tarsal with G

Systemic mycophenolate mofetil avoids immune reactions in penetrating high-risk keratoplasty: preliminary results of an ongoing prospectively randomized multicentre study.

Recently, in a monocentre study mycophenolate mofetil (MMF) was demonstrated to be efficacious and safe in penetrating high-risk keratoplasty. Here, preliminary results of a randomized multicentre trial are presented. To date, 86 of 140 scheduled patients undergoing high-risk penetrating keratoplasty have already been randomized into the two study groups: 48 into the MMF group and 38 into the control group. All 86 patients received fluocortolon 1 mg/kg body weight/day, tapered within 3 weeks, and topical prednisolone acetate 1% tapered within 5 months. MMF was administered at a daily oral dose of 2 x 1000 mg for the first 6 postoperative months. Thereafter, MMF was tapered within 2 weeks. The proportion of grafts with immune reactions and side-effects were the main outcome measures. Within an average follow up of 9.2 +/- 6.6 months two patients developed reversible endothelial immune reactions in the MMF group after cessation of MMF application. In the control group, five reversible and three irreversible immune reactions were observed within an average follow up of 10.1 +/- 7.6 months. According to Kaplan and Meier analysis, the ratio of grafts without immune reactions was estimated 89% 1 year postoperatively in the MMF group, in contrast to only 67% in the control group (P = 0.03; log-rank test). Fifteen patients experienced side-effects, especially gastroenterotoxicity, tachycardia, arthralgia or systemic infections. All attributable side-effects were reversible. Systemic MMF may be an effective and safe immune modulating drug in the prophylaxis of immune reactions after penetrating high-risk keratoplasty.