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.

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.

[Differential diagnoses of benign eyelid tumors in children and adolescents]. / Differenzialdiagnosen benigner Lidtumoren bei Kindern und Jugendlichen.

BACKGROUND: The clinical diagnosis as well as the treatment approach of periocular tumors in childhood and adolescence can be challenging. Knowledge of the most important differential diagnoses and their clinicopathological correlation is helpful for the treatment approach. OBJECTIVE: The clinical and histological characteristics of various eyelid tumors in childhood and adolescence are presented taking the excision frequencies into consideration. MATERIAL AND METHODS: The frequencies and clinicopathologic correlation of the most important eyelid tumors (n = 485) are presented based on the data of the ophthalmopathology laboratory of the University Eye Hospital Bonn from 1998-2023. RESULTS: The most frequent tumor in childhood and adolescence is chalazion (57.3%), followed by dermoid cysts (16.7%) and molluscum contagiosum (9.6%). Other lesions of childhood and adolescence include pilomatrixoma (2.1%), hemangioma and other vascular malformations (4.7%) and rare differential diagnoses, such as subcutaneous calcifying nodules and xanthogranuloma. Guidance on the approach in different age groups is presented in the form of a decision tree. CONCLUSION: Tumors in children and adolescents are mostly benign, yet there are important indications for excision. A histological examination of any excised tissue in childhood and adolescence is obligatory because unexpected findings are not uncommon and the spectrum of lesions also differs from that in adulthood. Knowledge of the histological picture can be very helpful in the preoperative clinical classification and for planning further procedures.

Histopathological Changes in Corneal Ectasia. / Histopathologische Veränderungen bei kornealen Ektasien.

Corneal ectasia comprises keratoconus, keratoglobus, pellucid marginal degeneration, and iatrogenic keratectasia. In all forms of corneal ectasia, there is a thinning of the cornea, usually accompanied by steepening of the cornea, leading to irregular astigmatism. Here, we provide an overview of histopathologic alterations of the various corneal ectasias. Furthermore, histologic changes after surgical procedures that are performed in severe cases of corneal ectasia, such as corneal cross-linking and penetrating keratoplasty (pKPL), as well as refractive surgical procedures that may lead to postsurgical ectasia are presented. In addition to a literature review, histopathologic archival material was reviewed and examined to illustrate the specific histologic changes.

PPAR-Responsive Elements Enriched with Alu Repeats May Contribute to Distinctive PPARγ-DNMT1 Interactions in the Genome.

BACKGROUND: PPARγ (peroxisome proliferator-activated receptor gamma) is involved in the pathology of numerous diseases, including UM and other types of cancer. Emerging evidence suggests that an interaction between PPARγ and DNMTs (DNA methyltransferase) plays a role in cancer that is yet to be defined. METHODS: The configuration of the repeating elements was performed with CAP3 and MAFFT, and the structural modelling was conducted with HDOCK. An evolutionary action scores algorithm was used to identify oncogenic variants. A systematic bioinformatic appraisal of PPARγ and DNMT1 was performed across 29 tumor types and UM available in The Cancer Genome Atlas (TCGA). RESULTS: PPAR-responsive elements (PPREs) enriched with Alu repeats are associated with different genomic regions, particularly the promotor region of DNMT1. PPARγ-DNMT1 co-expression is significantly associated with several cancers. C-terminals of PPARγ and DNMT1 appear to be the potential protein-protein interaction sites where disease-specific mutations may directly impair the respective protein functions. Furthermore, PPARγ expression could be identified as an additional prognostic marker for UM. CONCLUSIONS: We hypothesize that the function of PPARγ requires an additional contribution of Alu repeats which may directly influence the DNMT1 network. Regarding UM, PPARγ appears to be an additional discriminatory prognostic marker, in particular in disomy 3 tumors.

Choroidal melanocytes: subpopulations of different origin?

BACKGROUND: The human choroid derives from the mesectoderm, except the melanocytes originating from the neuroectoderm. To date, it is unclear whether all choroidal melanocytes share the same origin or might have different origins. The purpose of this study was to screen immunohistochemically for mesenchymal elements in the adult healthy human choroid, in the malignant melanoma of the choroid, as well as in the developing human fetal choroid. METHODS: Human choroids were obtained from cornea donors and prepared as flat whole mounts for paraffin- and cryoembedding. Globes enucleated for choroidal melanoma and eyes from human fetuses between 11 and 20 weeks of gestation were also embedded in paraffin. Sections were processed for immunohistochemistry of the mesenchymal marker vimentin, the melanocyte marker Melan-A, and the macrophage marker CD68, followed by light-, fluorescence-, and confocal laser scanning-microscopy. RESULTS: The normal choroid contained 499 ± 139 vimentin, 384 ± 78 Melan-A, and 129 ± 57 CD68 immunoreactive cells/mm2. The vimentin immunopositive cell density was significantly higher than the density of Melan-A and CD68 immunopositive cells (p < 0.001, respectively). By confocal microscopy, 24 ± 8% of all choroidal melanocytes displayed vimentin immunoreactivity. In choroidal melanomas, numerous melanoma cells of the epithelioid and spindle cell type revealed immunopositivity for both vimentin and Melan-A. The intratumoral density of vimentin immunoreactive cells was 1758 ± 106 cells/mm2, significantly higher than the density of Melan-A and CD68 immunopositive cells (p < 0.001, respectively). Comparing to healthy choroidal tissue, the choroidal melanomas revealed significantly higher densities of vimentin, Melan-A, and CD68 immunoreactive cells (p < 0.001, respectively). In the developing human fetal choroid, numerous vimentin and Melan-A immunopositive cells were detected not before the 16th week of gestation, with some of them showing colocalization of vimentin and Melan-A. CONCLUSIONS: The adult healthy human choroid is endowed with a significant number of vimentin immunopositive mesenchymal structures, including a subpopulation of vimentin immunoreactive choroidal melanocytes. These vimentin immunopositive melanocytic cells are also present in choroidal melanomas as well as in the developing human fetal choroid. Therefore, different embryologic origins can be considered for choroidal melanocytes.

Wound Healing of Descemet Membrane After Penetrating Keratoplasty and Its Relevance for Descemet Membrane Endothelial Keratoplasty Surgeons.

ABSTRACT: Compared with penetrating keratoplasty (PK), Descemet membrane endothelial keratoplasty (DMEK) is characterized as lower risk for complications such as immunological graft reaction and faster and better postoperative visual recovery. In patients with endothelial graft failure after PK, DMEK can be used to regenerate PK graft transparency. The surgical technique for DMEK in this specific situation is still under debate, particularly regarding stripping of Descemet membrane (DM) from the failed PK and diameter of the DMEK graft. Here we report a case of a 75-year-old female patient with a failed graft 16 years after PK for Fuchs endothelial dystrophy, who underwent uneventful DMEK surgery. Stripping of DM in this particular case was performed outside the failed PK and demonstrated a biomechanically stable junction between the PK donor and the host DM. Histopathologic analysis of the excised DM showed continuous extracellular matrix connecting the host and donor DM, indicating primary intention wound healing after PK at this tissue level. This case demonstrates that after PK, a biomechanically stable and histologically continuous DM can enable Descemetorhexis outside the failed graft and transplantation of a DMEK graft larger than the previous PK. This may provide more endothelial cells for transplantation.

Epigenetic Regulatory Enzymes: mutation Prevalence and Coexistence in Cancers.

Epigenetic regulation is an important layer of transcriptional control with the particularity to affect the broad spectrum of genome. Over the years, largely due to the substantial number of recurrent mutations, there have been hundreds of novel driver genes characterized in various cancers. Additionally, the relative contribution of two dysregulated epigenomic entities (DNA methylation and histone modifications) that gradually drive the cancer phenotype remains in the research focus. However, a complex scenario arises when the disease phenotype does not harbor any relevant mutation or an abnormal transcription level. Although the cancer landscape involves the contribution of multiple genetic and non-genetic factors, herein, we discuss specifically the mutation spectrum of epigenetically-related enzymes in cancer. In addition, we address the coexistence of these two epigenetic entities in malignant human diseases, especially cancer. We suggest that the study of epigenetically-related somatic mutations in the early cellular differentiation stage of embryonic development might help to understand their later-staged footprints in the cancer genome. Furthermore, understanding the co-occurrence and/or inverse association of different disease types and redefining the general definition of "healthy" controls could provide insights into the genome reorganization.

Validation of the Newly Proposed World Health Organization Classification System for Conjunctival Melanocytic Intraepithelial Lesions: A Comparison with the C-MIN and PAM Classification Schemes.

PURPOSE: We sought to compare the sensitivity, specificity, accuracy, and interobserver agreement of the two most commonly used classification systems for conjunctival melanocytic intraepithelial lesions with the new World Health Organization (WHO) classification. DESIGN: Retrospective case series and evaluation of classification systems. METHODS: We reviewed the pathology and medical records of all patients who underwent a primary biopsy procedure for conjunctival primary acquired melanosis (PAM) at Wills Eye Hospital between 1974 and 2002 who had ≥36 months of follow-up. Data collected included age, sex, clinical findings, recurrence, and progression to melanoma. Twelve ophthalmic pathologists analyzed scanned hematoxylin and eosin-stained virtual microscopic slides using 3 classification systems: PAM, conjunctival melanocytic intraepithelial neoplasia, and the WHO 4th edition classification of conjunctival melanocytic intraepithelial lesions. Observer agreement, sensitivity, specificity, and diagnostic accuracy of each classification system were assessed. RESULTS: There were 64 patients who underwent 83 primary excisions with cryotherapy for conjunctival PAM who had adequate tissue for histopathologic evaluation. The interobserver agreement in distinction between the low- and high-grade lesions was 76% for PAM, 67% for conjunctival melanocytic intraepithelial neoplasia, and 81% for WHO classification system. Low-grade lesions provided the greatest interpretative challenge with all 3 classification systems. The 3 classification systems had comparable accuracy of 81%-83% in their ability to identify lesions with potential for recurrence. CONCLUSIONS: This study highlights the comparable strengths and limitations of the 3 classification systems for conjunctival melanocytic intraepithelial lesions and suggests that the simplified WHO classification scheme is appropriate for evaluation of these lesions.

[The history of an eye-Investigation of enucleated eyes]. / Die Geschichte eines Auges ­ Untersuchung enukleierter Bulbi.

BACKGROUND: A variety of findings can be obtained from enucleated eyes, which provide the ophthalmologist with information with respect to the effectiveness, improvement options and of course also the possible complications of surgical interventions. Representative for many important findings, relevant clinical and ophthalmopathological findings are presented and discussed on the basis of three enucleated eyeballs. METHODS: Clinical histopathological correlation of three enucleated eyeballs. RESULTS: Enucleated eyeballs typically exhibit advanced changes in different tissues making it sometimes difficult to draw direct conclusions about the original surgical interventions. Nevertheless, many findings on the pathophysiology of different diseases, on wound healing and also on the surgical procedures can be deduced, as shown here in three different cases of epithelial invasion, historical retinal surgery and an endogenous fungal endophthalmitis. CONCLUSION: Each enucleated eye should be histopathologically examined and under consideration of the clinical history in order to understand the course of the disease and to use the knowledge gained for the treatment of future patients.

Ubiquitin Carboxyl-Terminal Hydrolases (UCHs): Potential Mediators for Cancer and Neurodegeneration.

Emerging evidence suggests an inverse association between cancer and neurodegenerative diseases (NDD). Although phenotypically different, both diseases display a significant imbalance in the ubiquitination/deubiquitination processes. Therefore, we particularly investigated the expression of ubiquitin C-terminal hydrolases (UCHs: UCH-L1, UCH-L3, UCH-L5 and BAP1), a subfamily of deubiquitinating enzymes (DUBs), using publically available datasets (GTEx, TCGA) and observed altered expression of UCH-L1, UCH-L3, UCH-L5 in 17 cancer types. Interestingly, UCH-L1 (known to be enriched in neurons and interacting with the Parkinson's disease-associated protein α-synuclein) appeared to be a prognostic indicator of unfavorable outcome in endometrial and urothelial cancer, while increased expression of UCH-L3 and UCH-L5 was associated with poor survival in liver and thyroid cancer, respectively. In normal tissues, UCH-L1 was found to be strongly expressed in the cerebral cortex and hypothalamus, while UCH-L3 expression was somewhat higher in the testis. The occurrence of mutation rates in UCHs also suggests that BAP1 and UCH-L5 may play a more dominant role in cancers than UCH-L1 and UCH-L3. We also characterized the functional context and configuration of the repeat elements in the promoter of DUBs genes and found that UCHs are highly discriminatory for catabolic function and are mainly enriched with LINE/CR1 repeats. Regarding the thesis of an inverse association between cancer and NDD, we observed that among all DUBs, UCHs are the one most involved in both entities. Considering a putative therapeutic potential based on presumed common mechanisms, it will be useful to determine whether other DUBs can compensate for the loss of UCH activity under physiological conditions. However, experimental evidence is required to substantiate this argument.

[Ophthalmic Pathology - Still the Gold Standard?] / Goldstandard Ophthalmopathologie ­ noch zeitgemäß?

Ophthalmic pathology has a long tradition in Germany. And, like in general pathology, there is continuous progress due to new technologies and the improvement of molecular biology techniques. Ophthalmic pathology cannot be disregarded, particularly in the context of basic research but also as a medium for understanding pathophysiologic interrelationships and evaluating innovative surgical techniques. By means of various examples, the "four columns" of ophthalmic pathology shall be illustrated: diagnostics, clinicopathologic correlation, evaluation of new surgical and medical techniques and finally research. Ophthalmic pathology is not a discipline of the past but is rather one of the future. It develops and improves together with medical and ophthalmological progress and serves, at the same time, as a critical evaluation tool. Clinicopathologic correlations are of paramount importance for a lasting quality in ophthalmology, and we should not risk depriving ourselves of this instrument by carelessly saving at the wrong end and closing our laboratories. Ophthalmic pathology was, is and will further be the gold standard in many aspects of ophthalmology.

Regression of Periocular Basal Cell Carcinoma: A Report of Four Cases with Clinicopathologic Correlation.

OBJECTIVES: To describe the spectrum of clinical and histopathological features of a case series of basal cell carcinoma (BCC) with spontaneous regression and to discuss this phenomenon. METHOD: Four cases of BCC with complete/substantial regression were retrospectively identified. Patients' records were analyzed for demographic data, clinical appearance, and the postoperative course. Formalin-fixed, paraffin-embedded specimens were routinely processed and stained with hematoxylin and eosin and periodic acid Schiff. RESULTS: Complete (n = 1) or partial (n = 3) regression of BCC was observed in 4 patients. Two lesions at the medial canthus were histologically diagnosed as nodular BCC with significant regression. One lesion at the lower eyelid exhibited a complete regression which did not require surgical intervention. The other lesion at the lower eyelid presenting with ulceration and madarosis was excised. Scar tissue without evidence for a neoplasm was present histologically. Subsequently, the patient developed a recurrence with a histologically proven micronodular BCC. CONCLUSIONS: BCC can show spontaneous substantial or complete regression. Histological tumor absence in lesions which are clinically suspicious for a neoplasm can be a hint for a regressive BCC. Recurrences may develop from remaining tumor islands warranting periodical clinical visits in cases of clinically as well as histologically suspected regressive BCC.

Genome organization in proximity to the BAP1 locus appears to play a pivotal role in a variety of cancers.

Cancer studies primarily focus on the characterization of the key driver genes and the underlying pathways. However, the contribution of other cancer-associated genes located in the genomic neighborhood of the driver genes could help to understand further aspects of cancer progression. Given the frequent involvement of chromosome 3 in multiple human cancers, in particular in the form of the prognostically highly relevant monosomy 3 in uveal melanoma (UM), we investigated the cumulative impact of cancer-associated genes on chromosome 3. Our analysis showed that these genes are enriched with repetitive elements with genes surrounded by distinctive repeats (MIR, hAT-Charlie, ERVL-MaLR, LINE-2, and simple/low complexity) in the promoter being more precisely associated with cancer-related pathways than the ones with major transposable elements (SINE/Alu and LINE-1). Additionally, these genes showed strong intrachromosomal chromatin interactions in 3D nuclear organization. Further investigations revealed a genomic hotspot in the vicinity of BAP1 locus, which is affected in 27 types of different cancers and contains abundant noncoding RNAs that are often expressed in a tissue-specific manner. The cross-species comparison of these cancer-associated genes revealed mostly a shared synteny in closer primates. However, near to the BAP1 locus signs of chromosomal inversions were observed during the course of evolution. To our knowledge, this is the first study to characterize the entire genomic neighborhood of cancer-associated genes located on any single chromosome. Based on our results, we hypothesize that monosomy of chromosome 3 will have important clinical and molecular consequences in the respective diseases and in particular in UM.

Mutational Landscape of the BAP1 Locus Reveals an Intrinsic Control to Regulate the miRNA Network and the Binding of Protein Complexes in Uveal Melanoma.

The BAP1 (BRCA1-associated protein 1) gene is associated with a variety of human cancers. With its gene product being a nuclear ubiquitin carboxy-terminal hydrolase with deubiquitinase activity, BAP1 acts as a tumor suppressor gene with potential pleiotropic effects in multiple tumor types. Herein, we focused specifically on uveal melanoma (UM) in which BAP1 mutations are associated with a metastasizing phenotype and decreased survival rates. We identified the ubiquitin carboxyl hydrolase (UCH) domain as a major hotspot region for the pathogenic mutations with a high evolutionary action (EA) score. This also includes the mutations at conserved catalytic sites and the ones overlapping with the phosphorylation residues. Computational protein interaction studies revealed that distant BAP1-associated protein complexes (FOXK2, ASXL1, BARD1, BRCA1) could be directly impacted by this mutation paradigm. We also described the conformational transition related to BAP1-BRCA-BARD1 complex, which may pose critical implications for mutations, especially at the docking interfaces of these three proteins. The mutations affect - independent of being somatic or germline - the binding affinity of miRNAs embedded within the BAP1 locus, thereby altering the unique regulatory network. Apart from UM, BAP1 gene expression and survival associations were found to be predictive for the prognosis in several (n = 29) other cancer types. Herein, we suggest that although BAP1 is conceptually a driver gene in UM, it might contribute through its interaction partners and its regulatory miRNA network to various aspects of cancer. Taken together, these findings will pave the way to evaluate BAP1 in a variety of other human cancers with a shared mutational spectrum.

Melanocytoma of the Conjunctiva: Clinicopathologic Features of Three Cases.

BACKGROUND: Melanocytoma (magnocellular nevus) is a jet-black benign lesion histologically composed of polygonal tumor cells with small, inconspicuous nuclei and abundant cytoplasm. Melanocytomas in general are rare. Most cases occur in the optic nerve head. Conjunctival melanocytoma (magnocellular nevus) is extremely rare, and only 3 lesions of the ocular surface have been reported. OBJECTIVES: To describe the clinical and histological spectrum of conjunctival melanocytoma and discuss differential diagnoses of this rare lesion. METHOD: Four heavily pigmented conjunctival lesions were excised for slight tumor growth and histologically processed. The specimens were routinely stained with hematoxylin and eosin and periodic acid-Schiff. Sections were bleached and immunohistochemical stains were performed for CD68, HMB-45, S100, melanin, and Ki-67. RESULTS: Histological examination revealed findings of a conjunctival melanocytoma in 3 cases. The fourth case was diagnosed histologically as a combined melanocytic lesion with a compound nevus and an inverted type A nevus. None of the lesions exhibited transition towards malignancy. The differential diagnoses included conjunctival melanoma, granular cell nevus, compound nevus with reactive changes, and blue nevus. CONCLUSIONS: Conjunctival melanocytic lesions suspicious for melanocytoma should be bleached to evaluate their cytologic features. CD68 can be helpful in identifying heavily pigmented melanomacrophages which may mimic a melanocytoma. As conjunctival melanocytomas are extremely rare, their pathogenesis may be different from that of other conjunctival nevi.