A Multicenter Study Validates the WHO 2022 Classification for Conjunctival Melanocytic Intraepithelial Lesions With Clinical and Prognostic Relevance.

Several nomenclature and grading systems have been proposed for conjunctival melanocytic intraepithelial lesions (C-MIL). The fourth "WHO Classification of Eye Tumors" (WHO-EYE04) proposed a C-MIL classification, capturing the progression of noninvasive neoplastic melanocytes from low- to high-grade lesions, onto melanoma in situ (MIS), and then to invasive melanoma. This proposal was revised to the WHO-EYE05 C-MIL system, which simplified the high-grade C-MIL, whereby MIS was subsumed into high-grade C-MIL. Our aim was to validate the WHO-EYE05 C-MIL system using digitized images of C-MIL, stained with hematoxylin and eosin and immunohistochemistry. However, C-MIL cases were retrieved from 3 supraregional ocular pathology centers. Adequate conjunctival biopsies were stained with hematoxylin and eosin, Melan-A, SOX10, and PReferentially expressed Antigen in Melanoma. Digitized slides were uploaded on the SmartZoom platform and independently scored by 4 ocular pathologists to obtain a consensus score, before circulating to 14 expert eye pathologists for independent scoring. In total, 105 cases from 97 patients were evaluated. The initial consensus diagnoses using the WHO-EYE04 C-MIL system were as follows: 28 benign conjunctival melanoses, 13 low-grade C-MIL, 37 high-grade C-MIL, and 27 conjunctival MIS. Using this system resulted in 93% of the pathologists showing only fair-to-moderate agreement (kappa statistic) with the consensus score. The WHO-EYE05 C-MIL system (with high-grade C-MIL and MIS combined) improved consistency between pathologists, with the greatest level of agreement being seen with benign melanosis (74.5%) and high-grade C-MIL (85.4%). Lowest agreements remained between pathologists for low-grade C-MIL (38.7%). Regarding WHO-EYE05 C-MIL scoring and clinical outcomes, local recurrences of noninvasive lesions developed in 8% and 34% of the low- and high-grade cases. Invasive melanoma only occurred in 47% of the cases that were assessed as high-grade C-MIL. This extensive international collaborative study is the first to undertake a comprehensive review of the WHO-EYE05 C-MIL scoring system, which showed good interobserver agreement and reproducibility.

Real-life data of adjuvant IFN-α2b and MMC in conjunctival melanocytic lesions.

PURPOSE: We herein compare topical interferon alpha 2b (IFN-α2b) to topical mitomycin C (MMC) in the adjuvant management after excision of primary acquired melanosis with atypia (PAM) and melanoma of the conjunctiva/cornea (CM). METHODS: We included 25 tumors from 25 patients (six with PAM and 19 with CM). After surgical excision, four patients started with adjuvant IFN-α2b (two in combination with radiotherapy), 19 with MMC, and two with radiotherapy alone. Five patients were switched from initial MMC/radiotherapy to IFN-α2b during follow-up. Efficacy was assessed via time to tumor recurrence and initial therapy response. RESULTS: With initial IFN-α2b, three patients (3/4, two with additional radiotherapy) showed complete remission (follow-up: 1478-1750 days) and one recurrence (1/4) was noted after 492 days. With initial MMC, no recurrence was recorded in 15 of the 19 patients (follow-up: 99-4732 days). Five patients were switched from MMC or radiotherapy to IFN-α2b: two patients showed complete remission (2/5), while another two (2/5) experienced recurrences and remained without recurrence after repeated courses of IFN-α2b (follow-up: 1798 and 1973 days). Only one patient showed incomplete response. Adverse effects were recorded in five patients, all received MMC. CONCLUSION: Topical IFN-α2b (arguably together with radiotherapy) may be a viable alternative to MMC in PAM and CM. We observed fewer side effects at similar response rates. However, when response to MMC was poor, IFN-α2b may also be of limited utility.

The Human Eye Transcriptome Atlas: A searchable comparative transcriptome database for healthy and diseased human eye tissue.

The applications of deep sequencing technologies in life science research and clinical diagnostics have increased rapidly over the last decade. Although fast algorithms for data processing exist, intuitive, portable solutions for data analysis are still rare. For this purpose, we developed a web-based transcriptome database, which provides a platform-independent, intuitive solution to easily explore and compare ocular gene expression of 100 diseased and healthy human tissue samples from 15 different tissue types collected at the Eye Center of the University of Freiburg. To ensure comparability of expression between different tissues, reads were normalized across all 100 samples. Differentially expressed genes were calculated between each tissue type to determine tissue-specific genes. Unsupervised analysis of all 100 samples revealed an accurate clustering according to different tissue types and a high tissue specificity by analyzing known tissue-specific marker genes. Bioinformatic cell type deconvolution using xCell provided detailed insights into the cellular profiles of each tissue type. Several new tissue-specific marker genes were identified. These genes were involved in tissue- or disease-specific processes, such as myelination for the optic nerve, visual perception for retina, keratinocyte differentiation for conjunctival carcinoma, as well as endothelial cell migration for choroidal neovascularization membranes. The results are accessible at the Human Eye Transcriptome Atlas website at https://www.eye-transcriptome.com. In summary, this searchable transcriptome database enables easy exploration of ocular gene expression in healthy and diseased human ocular tissues without bioinformatics expertise. Thus, it provides rapid access to detailed insights into the molecular mechanisms of various ocular tissues and diseases, as well as the rapid retrieval of potential new diagnostic and therapeutic targets.

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.

Transcriptomic Characterization of Human Choroidal Neovascular Membranes Identifies Calprotectin as a Novel Biomarker for Patients with Age-Related Macular Degeneration.

Recent studies deciphering the transcriptional profile of choroidal neovascularization (CNV) in body donor eyes with neovascular age-related macular degeneration are limited by the time span from death to preservation and the associated 5'-RNA degradation. This study therefore used CNV and control specimens that were formalin-fixed and paraffin-embedded immediately after surgical extraction and analyzed them by a 3'-RNA sequencing approach. Transcriptome profiles were analyzed to estimate content of immune and stromal cells and to define disease-associated gene signatures by using statistical and bioinformatics methods. This study identified 158 differentially expressed genes (DEGs) that were significantly increased in CNV compared with control tissue. Cell type enrichment analysis revealed a diverse cellular landscape with an enrichment of endothelial cells, macrophages, T cells, and natural killer T cells in the CNV. Gene ontology enrichment analysis found that DEGs contributed to blood vessel development, extracellular structure organization, response to wounding, and several immune-related terms. The S100 calcium-binding proteins A8 (S100A8) and A9 (S100A9) emerged among the top DEGs, as confirmed by immunohistochemistry on CNV tissue and protein analysis of vitreous samples. This study provides a high-resolution RNA-sequencing-based transcriptional signature of human CNV, characterizes its compositional pattern of immune and stromal cells, and reveals S100A8/A9 to be a novel biomarker and promising target for therapeutics and diagnostics directed at age-related macular degeneration.

Imaging mass cytometry for high-dimensional tissue profiling in the eye.

BACKGROUND: Imaging mass cytometry (IMC) combines the principles of flow cytometry and mass spectrometry (MS) with laser scanning spatial resolution and offers unique advantages for the analysis of tissue samples in unprecedented detail. In contrast to conventional immunohistochemistry, which is limited in its application by the number of possible fluorochrome combinations, IMC uses isoptope-coupled antibodies that allow multiplex analysis of up to 40 markers in the same tissue section simultaneously. METHODS: In this report we use IMC to analyze formalin-fixed, paraffin-embedded conjunctival tissue. We performed a 18-biomarkers IMC analysis of conjunctival tissue to determine and summarize the possibilities, relevance and limitations of IMC for deciphering the biology and pathology of ocular diseases. RESULTS: Without modifying the manufacturer's protocol, we observed positive and plausible staining for 12 of 18 biomarkers. Subsequent bioinformatical single-cell analysis and phenograph clustering identified 24 different cellular clusters with distinct expression profiles with respect to the markers used. CONCLUSIONS: IMC enables highly multiplexed imaging of ocular samples at subcellular resolution. IMC is an innovative and feasible method, providing new insights into ocular disease pathogenesis that will be valuable for basic research, drug discovery and clinical diagnostics.

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.

Expression of the COVID-19 receptor ACE2 in the human conjunctiva.

SARS-CoV-2 is assumed to use angiotensin-converting enzyme 2 (ACE2) and other auxiliary proteins for cell entry. Recent studies have described conjunctival congestion in 0.8% of patients with laboratory-confirmed severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), and there has been speculation that SARS-CoV-2 can be transmitted through the conjunctiva. However, it is currently unclear whether conjunctival epithelial cells express ACE2 and its cofactors. In this study, a total of 38 conjunctival samples from 38 patients, including 12 healthy conjunctivas, 12 melanomas, seven squamous cell carcinomas, and seven papilloma samples, were analyzed using high-throughput RNA sequencing to assess messenger RNA (mRNA) expression of the SARS-CoV-2 receptor ACE2 and its cofactors including TMPRSS2, ANPEP, DPP4, and ENPEP. ACE2 protein expression was assessed in eight healthy conjunctival samples using immunohistochemistry. Our results show that the SARS-CoV-2 receptor ACE2 is not substantially expressed in conjunctival samples on the mRNA (median: 0.0 transcripts per million [TPM], min: 0.0 TPM, max: 1.7 TPM) and protein levels. Similar results were obtained for the transcription of other auxiliary molecules. In conclusion, this study finds no evidence for a significant expression of ACE2 and its auxiliary mediators for cell entry in conjunctival samples, making conjunctival infection with SARS-CoV-2 via these mediators unlikely.

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.

Molecular mechanism of CHRDL1-mediated X-linked megalocornea in humans and in Xenopus model.

Chordin-Like 1 (CHRDL1) mutations cause non-syndromic X-linked megalocornea (XMC) characterized by enlarged anterior eye segments. Mosaic corneal degeneration, presenile cataract and secondary glaucoma are associated with XMC. Beside that CHRDL1 encodes Ventroptin, a secreted bone morphogenetic protein (BMP) antagonist, the molecular mechanism of XMC is not well understood yet. In a family with broad phenotypic variability of XMC, we identified the novel CHRDL1 frameshift mutation c.807_808delTC [p.H270Wfs*22] presumably causing CHRDL1 loss of function. Using Xenopus laevis as model organism, we demonstrate that chrdl1 is specifically expressed in the ocular tissue at late developmental stages. The chrdl1 knockdown directly resembles the human XMC phenotype and confirms CHRDL1 deficiency to cause XMC. Interestingly, secondary to this bmp4 is down-regulated in the Xenopus eyes. Moreover, phospho-SMAD1/5 is altered and BMP receptor 1A is reduced in a XMC patient. Together, we classify these observations as negative-feedback regulation due to the deficient BMP antagonism in XMC. As CHRDL1 is preferentially expressed in the limbal stem cell niche of adult human cornea, we assume that CHRDL1 plays a key role in cornea homeostasis. In conclusion, we provide novel insights into the molecular mechanism of XMC as well as into the specific role of CHRDL1 during cornea organogenesis, among others by the establishment of the first XMC in vivo model. We show that unravelling monogenic cornea disorders like XMC-with presumably disturbed cornea growth and differentiation-contribute to the identification of potential limbal stem cell niche factors that are promising targets for regenerative therapies of corneal injuries.

Connection of histological corneal endothelial cell count with endothelial cell density before penetrating keratoplasty.

PURPOSE: Corneal endothelial cell density and the integrity of the monolayer are essential for maintenance of a clear cornea. In 1992, Williams et al. introduced a method to estimate the endothelial cell density in histopathologic examination. It would enable an evaluation of the corneal host endothelium, even if preoperative measurement was not possible. The goal of this study is to evaluate the accuracy of the Williams equation in corneal buttons obtained from penetrating keratoplasties. METHODS: High power field (HPF) photographs and histological endothelial cell counts were made from the corneal endothelial cells of each corneal histopathological cross-section. We then compared the calculated endothelial cell density using the Williams equation with the preoperative measured endothelial cell density. A bivariate regression analysis of the histological HPF cell counts and the preoperative endothelial cell density count was also performed. RESULTS: The equation of Williams et al. overestimates the endothelial density in all of our patients. Linear regression showed a strong relation between the central histological HPF count and the preoperative endothelial cell density. The regression formula for the endothelial cell density is 59.66 + (272.447 × HPF count); p < 0.001, R 2 = 0.901. CONCLUSION: This study confirms the relation between the corneal endothelial cell density, measured with specular microscopy, and the histopathological endothelial cell count in a HPF. However, the equation of Williams et al. provides an overestimation of the endothelial cell density. To proper utilize the histopathological endothelial cell count, a calibration of the equation coefficients in the local setting is necessary to prevent systematic errors.

Identification of two novel mutations in the cornea-specific TGFBI gene causing unique phenotypes in patients with corneal dystrophies.

The purpose of this study was to report on two novel missense mutations of the cornea-specific TGFBI gene in one single patient and in two generations of a family diagnosed with unique corneal dystrophy (CD) phenotypes. Ophthalmologic examination, in several cases ocular coherence tomography of the anterior segment (AS-OCT), was performed in 21 affected patients and in two unaffected members of one affected family. Coding regions of the TGFBI gene were direct sequenced in all 23 individuals. The two novel mutations were verified by RFLP analysis. A novel mutation c.1640T > G (p.Phe574Cys) in exon 12 of the TGFBI gene was detected in one single patient with recurrent granular intrastromal deposits comparable to a type of granular dystrophy. In AS-OCT, the deposit pattern reached up to the Descemet's layer. A further novel mutation c.393G > T(p.Glu131Asp) in exon 4 of the TGFBI gene was detected in all three affected members of one family with superficial cloud- and honeycomb-like opacifications, comparable to a Schnyder corneal dystrophy. Two unaffected members did not carry this alteration. The two identified novel mutations add other two phenotypes in patients suffering from TGFBI-linked CD to those reported so far. In one case, clinical finding indicates a Schnyder corneal dystrophy-like phenotype due to its superficial crystalline shape, and in the second one, granular deposits who reach Descemet's layer indicate a granular CD subtype. Molecular genetic analysis may help to distinguish those subtypes and to decide for specific treatment in time of a wide variation of corneal surgical techniques.

Analysis of filaggrin mutations and expression in corneal specimens from patients with or without atopic dermatitis.

BACKGROUND: Filaggrin is expressed in the epidermis and is essential for the maintenance of the epidermal barrier. Null mutations within the filaggrin gene (FLG) lead to a disturbed epidermal barrier and are associated with a significantly increased risk of atopic dermatitis (AD). The association of AD with ocular surface disorders prompted us to speculate that common FLG mutations may be particularly prevalent in AD patients with ocular comorbidities. METHODS: Corneal buttons and biopsies from AD patients with ocular involvement (n = 11) and from non-atopic patients (n = 9) with a histological diagnosis of keratitis were included in the study. DNA samples obtained from paraffin-embedded corneal specimens were genotyped for the two most common FLG mutations (R501X and 2282del4). Filaggrin protein expression was analysed by immunohistochemistry. RESULTS: Normal skin and corneal specimens (n = 6) were positive for filaggrin, which could be detected in the stratum corneum of the skin and in the basal epithelial layer of the cornea. Interestingly, all AD corneal specimens as well as the specimens from keratitis patients without AD were negative for filaggrin expression. Genotyping of the FLG mutations R501X and 2282del4 revealed wild-type alleles in all analysed samples. CONCLUSIONS: The lack of filaggrin expression observed in the analysed corneal specimens from AD patients is not due to the two most common FLG mutations (R501X, 2282del4) but is most likely secondary to inflammation, as all keratitis specimens of non-AD patients showed lack of filaggrin expression as well.

Progesterone and estrogen receptors in conjunctival melanoma and nevi.

BACKGROUND: Since it has been observed that melanocytic lesions can alter their appearance during pregnancy, we analyzed whether hormone receptors are expressed in conjunctival nevi as well as conjunctival melanoma. We further analyzed whether the number of estrogen (ER) or progesterone receptors (PR) might be associated with the disease course in conjunctival melanoma. METHODS: Twenty-seven paraffin-embedded samples of conjunctival nevi and 27 conjunctival melanoma specimens were examined using immunohistological analysis with antibodies against PR and ER. The percentage of stained cells were analyzed, taking into account patient gender and age. Out of the melanoma group, all patients with complete data for tumor thickness, tumor localization, age at diagnosis, gender, and follow-up including recurrence, metastasis and tumor-related death were included in the second part of the study (n = 15), where hormone receptor rates were associated with tumor outcome, regarding recurrences, metastasis or death. Written consent was received from all included patients. RESULTS: Both nevi and melanomas showed high rates of PR- and ER-positive cells. In Nevi, 64 ± 25 % of cells stained positive for PR and 35 ± 34 % for ER. In melanoma specimens, 68 ± 30 % showed PR and 44 ± 34 % ER expression. Differences between men and women in expression rates were not statistically significant. Out of 15 melanoma patients (nine female, six male), 53 % (five women and three men) experienced 1-4 recurrences, and four patients developed metastases. The median estimated survival time was 12.2 years. A multivariate survival model taking into account known risk factors for prognosis in conjunctival melanoma confirmed tumor location to be an important predictive factor for outcome (p = 0.05). The rate of PR or ER did not show a statistically significant correlation with the disease course in our cohort. CONCLUSIONS: We observed that conjunctival melanocytic lesions express hormone receptors, which could explain why these tumors can alter their appearance under hormonal changes. Regarding the prognosis of conjunctival melanoma, no statistically significant correlation between hormone receptor expression and event-free survival was found in this analysis.

Updates on eyelid cancers.

In this review, we aim to provide an overview of the five most common malignant eyelid tumors with current treatment recommendations based on international guidelines. Particular attention is paid to the clinicopathological correlation and the update with regard to adequate treatment. Newer systemic therapies enrich the existing treatment options, of which complete tumor excision remains the most important therapeutic measure.

Mass Spectrometry-Based Profiling of Histone Post-Translational Modifications in Uveal Melanoma Tissues, Human Melanocytes, and Uveal Melanoma Cell Lines - A Pilot Study.

Purpose: Epigenetic alterations in uveal melanoma (UM) are still neither well characterized, nor understood. In this pilot study, we sought to provide a deeper insight into the possible role of epigenetic alterations in the pathogenesis of UM and their potential prognostic relevance. To this aim, we comprehensively profiled histone post-translational modifications (PTMs), which represent epigenetic features regulating chromatin accessibility and gene transcription, in UM formalin-fixed paraffin-embedded (FFPE) tissues, control tissues, UM cell lines, and healthy melanocytes. Methods: FFPE tissues of UM (n = 24), normal choroid (n = 4), human UM cell lines (n = 7), skin melanocytes (n = 6), and uveal melanocytes (n = 2) were analyzed through a quantitative liquid chromatography-mass spectrometry (LC-MS) approach. Results: Hierarchical clustering showed a clear separation with several histone PTMs that changed significantly in a tumor compared to normal samples, in both tissues and cell lines. In addition, several acetylations and H4K20me1 showed lower levels in BAP1 mutant tumors. Some of these changes were also observed when we compared GNA11 mutant tumors with GNAQ tumors. The epigenetic profiling of cell lines revealed that the UM cell lines MP65 and UPMM1 have a histone PTM pattern closer to the primary tissues than the other cell lines analyzed. Conclusions: Our results suggest the existence of different histone PTM patterns that may be important for diagnosis and prognosis in UM. However, further analyses are needed to confirm these findings in a larger cohort. The epigenetic characterization of a panel of UM cell lines suggested which cellular models are more suitable for epigenetic investigations.

Transcriptional Profiling Identifies Prognostic Gene Signatures for Conjunctival Extranodal Marginal Zone Lymphoma.

This study characterizes the transcriptional profile and the cellular tumor microenvironment of conjunctival extranodal marginal zone lymphoma (EMZL) and identifies prognostically relevant biomarkers. Ten formalin-fixed and paraffin-embedded conjunctival EMZL and eight healthy conjunctival specimens were analyzed by Massive Analysis of cDNA Ends (MACE) RNA sequencing. The 3417 upregulated genes in conjunctival EMZL were involved in processes such as B cell proliferation and Rac protein signaling, whereas the 1188 downregulated genes contributed most significantly to oxidative phosphorylation and UV protection. The tumor microenvironment, as determined by deconvolution analysis, was mainly composed of multiple B cell subtypes which reflects the tumor's B cell lineage. However, several T cell types, including T helper 2 cells and regulatory T cells, as well as innate immune cell types, such as anti-inflammatory macrophages and plasmacytoid dendritic cells, were also strongly enriched in conjunctival EMZL. A 13-biomarker prognostic panel, including S100A8 and S100A9, classified ocular and extraocular tumor recurrence, exceeded prognostic accuracy of Ann Arbor and American Joint Committee on Cancer (AJCC) staging, and demonstrated prognostic value for patient survival in 21 different cancer types in a database of 12,332 tumor patients. These findings may lead to new options of targeted therapy and may improve prognostic prediction for conjunctival EMZL.

Enhanced TKTL1 expression in malignant tumors of the ocular adnexa predicts clinical outcome.

PURPOSE: Malignant tumors metabolize glucose to lactate even in the presence of oxygen via the pentose-phosphate pathway. The metabolic switch from oxidative glycolysis to nonoxidative fermentation in tumors has been associated with overexpression of the transketolase-like-1-gene (TKTL1), which encodes an essential and rate-limiting enzyme in the nonoxidative part of the pentose-phosphate pathway. This study investigates the role of TKTL1 in ocular adnexal tumors and analyzes how its expression correlates with the clinical outcomes against the background of tumor thickness and mitotic rate. DESIGN: Comparative case studies. PARTICIPANTS: We included 89 subjects with malignant tumors of the ocular adnexa (44 squamous cell carcinomas, 26 lymphomas, 19 malignant melanomas) who had been treated at the University Eye Hospital Freiburg from 1994 to 2008. Sixteen subjects with conjunctival nevi, 19 with conjunctival papilloma, and 2 with conjunctival-reactive lymphoid hyperplasia were included as controls. METHODS: TKTL1 expression was assessed by reverse transcriptase-polymerase chain reaction and immunohistochemistry and semiquantitatively analyzed using an established immunoreactive score (IRS). The tumor recurrence rate, metastasis occurrence, and survival time of each patient were assessed retrospectively and correlated with the TKTL IRS using Kaplan-Meier and Cox regression analyses. MAIN OUTCOME MEASURES: TKTL1 expression, mitotic rate within the tumor mass, and tumor thickness and its association with clinical outcome. RESULTS: We identified increased TKTL1 protein levels in malignant conjunctival tumors compared with control samples and detected an average IRS of 1.78 (standard deviation [SD], ± 0.46) for melanomas, 1.3 for lymphomas (SD, ± 0.79), and 1.22 for squamous cell carcinomas (SD, ± 0. 97) compared with 0.86 for conjunctival nevi (SD, ± 0.57) and 0.5 for conjunctival papilloma (SD, ± 0.83). Multifactorial survival analysis showed that TKTL1 overexpression correlated with the patient outcomes in malignant tumors (P = 0.045). In the squamous cell carcinomas, tumor thickness and mitotic rate correlated more strongly with prognosis compared with TKTL1 overexpression (P = 0.0061, P = 0.015, and P = 0.061, respectively). CONCLUSIONS: TKTL1 is dysregulated in malignant tumors of the ocular adnexa, and enhanced expression seems to predict clinical outcome, especially the tumor recurrence rate.

What is the significance of the conjunctiva as a potential transmission route for SARS-CoV-2 infections?

Recent studies have described conjunctivitis in approximately 1% of COVID-19 patients and speculated that SARS-CoV­2 can be transmitted via the conjunctiva. In this article we recapitulate the molecular mechanisms of host cell entry of SARS-CoV­2 and discuss the current evidence for a potential conjunctival transmission of SARS-CoV­2. The current body of evidence indicates that SARS-CoV­2 requires the membrane-bound angiotensin-converting enzyme 2 (ACE2) and the membrane-bound serine protease TMPRSS2 to enter cells. Recent studies suggest that COVID-19 patients rarely exhibit viral RNA in tear film and conjunctival smears and that, ACE2 and TMPRSS2 are only expressed in small amounts in the conjunctiva, making conjunctival infection with SARS-CoV­2 via these mediators unlikely. Nevertheless, we consider the current evidence to be still too limited to provide a conclusive statement and recommend appropriate protective measures for healthcare personnel who are in close contact with suspected and confirmed COVID-19 patients.