Objective: To investigate the application of environmentally-sustainable reagents in ophthalmic tissue pathology techniques. Methods: We prospectively collected 190 surgical specimens submitted to the pathology department of Zhongshan Ophthalmic Center between June 2023 and March 2024. Each specimen was divided into two sets, one treated with environmentally-sustainable reagents and the other with conventional reagents for fixation, dehydration, clearing, infiltration, staining, and mounting. The quality of histological sections from both groups was evaluated and compared. Results: Compared to the traditional reagent group, environmentally-sustainable reagent group showed good dehydration performance, moderately soft and hard tissue sections with intact cut surfaces and no cracks. H&E staining sections prepared with environmentally-sustainable reagents exhibited clear nuclear structures, distinct nuclear-cytoplasmic contrast, and clean backgrounds. The excellent section rates were 97.9% and 98.4% for the two, respectively. There was no significant difference in section quality between the two groups (Z=-0.645, P=0.519). Tissue antigenicity is well-preserved during immunohistochemical procedures, with strong positive signals and accurate localization without background staining. Conclusions: The use of environmentally-sustainable reagents as a replacement for conventional reagents in the dehydration and staining of ophthalmic tissue yields satisfactory results, meeting the daily requirements for routine histological sectioning and staining. The entire process is xylene-free, environmentally-sustainable, cost-effective and worth being applied widely.

The topoisomerase I inhibitor topotecan is an effective chemotherapeutic agent for retinoblastoma; however, treatment resistance remains a major clinical challenge, and its mechanisms remain elusive. Using genome-wide CRISPR-Cas9 knockout screening, we identified ELF2 as a key gene involved in topotecan resistance. Here, we show that surviving retinoblastoma cells exposed to topotecan showed progressively decreased ELF2 expression, accompanied by reduced apoptosis. In a mouse xenograft model, ELF2 disruption diminished the antitumor efficacy of topotecan, with ELF2-knockout cells exhibiting reduced topotecan-induced apoptosis. RNA sequencing further revealed that the MT-CYB pathway, associated with ATP synthesis, contributes to ELF2-mediated resistance. Importantly, clinical analysis demonstrated a correlation between ELF2 expression and tumor volume in retinoblastoma patients treated with topotecan. Together, these findings interrogate the mechanisms underlying topotecan resistance in retinoblastoma and suggest ELF2 as a potential therapeutic target to overcome drug resistance.

PURPOSE. This study aimed to investigate the role of minichromosome maintenance complex component 6 (MCM6), a DNA replication licensing factor, in retinoblastoma progression and its impact on melphalan chemosensitivity. METHODS. MCM6 expression patterns were analyzed using single-cell RNA sequencing (scRNA-seq) of retinoblastoma and validated in patient tumors, including specimens obtained after failed melphalan therapy. Stable MCM6 knockdown cell lines were established for proliferation and cell-cycle assays, DNA damage analyses, and chemosensitivity testing. In vivo xenograft models were employed to evaluate the therapeutic efficacy of MCM6 knockdown combined with melphalan. RESULTS. The scRNA-seq revealed that MCM6 was highly expressed in retinoblastoma cells and embedded in a proliferation-associated gene network. Elevated expression was also confirmed in human retinoblastoma, particularly in tumors from patients with failed melphalan therapy. MCM6 knockdown suppressed cell proliferation and cellcycle progression while enhancing melphalan-induced DNA damage, thereby sensitizing retinoblastoma cells to melphalan. In vivo, MCM6 depletion synergized with melphalan to significantly inhibit intraocular tumor growth. CONCLUSIONS. MCM6 acts as a critical regulator of retinoblastoma growth and modulates response to melphalan. Targeting MCM6 may offer a therapeutic approach to improve outcomes of chemotherapy in retinoblastoma.

Without histological analysis, the diagnosis and grading of ocular surface squamous neoplasia (OSSN) are challenging in clinical differentiation between malignant and premalignant lesions. This study examined EZH2 expression in corneal and conjunctival epithelial benign hyperplasia, intraepithelial neoplasia (CIN) and squamous cell carcinoma (SCC), and evaluated its relationship with clinicopathological features for diagnostic value. Immunohistochemistry was used to detect EZH2 protein expression in 13 cases of benign corneal and conjunctival epithelial benign hyperplasia, 66 CIN cases, and 26 SCC cases. Statistical analysis was performed to assess the relationship between EZH2 expression and clinicopathological features including age, gender, laterality, tumor diameter and histopathological differentiation. The mean age of all patients was 58.4 ± 16.56 years, with a predominance of males. A positive EZH2 expression rate as high as 96.2

Amyloidosis involving the lacrimal sac is extremely rare. In this study, we demonstrated a rare case of localized light chain amyloidosis in the lacrimal sac region. The lacrimal sac lesion presented as infiltrative with bony erosion. Given the slow growth of the lesion and the absence of a blood flow signal inside, we concluded that the lesion was less likely to be malignant. Complete removal of the lacrimal sac lesion combined with simultaneous lacrimal passage reconstruction was performed. The diagnosis of light chain amyloidosis was confirmed by histology. The surgical results were favorable, and no recurrence was observed over one-year follow-up. Our case report enriches the understanding of amyloid deposition in the ocular adnexa.

Introduction: The enhancer of zeste homolog 2 (EZH2) is a member of the polycomb repressive complex 2 (PRC2) and is important in cell-cycle regulation. Increased expression of EZH2 has been reported in retinoblastoma (RB). The aim of the study was to determine EZH2 expression, compare this with clinicopathological parameters in RB, and assess its relationship with tumor cell proliferation. Methods: Ninety-nine retrospective cases of enucleated RB were included in the present study. Expression of EZH2 and the marker of cell proliferation, Ki67, were investigated by immunohistochemistry. Results: Among the 99 cases of RB in this study, EZH2 was found highly expressed (positive expression rate ≥70%) in 92 cases. EZH2 was expressed in tumor cells but absent in normal retinal tissues. The expression of EZH2 was positively linked to Ki67 expression (r = 0.65, p < 0.001). Conclusion: Elevated EZH2 expression was found in most RB cases, indicating that EZH2 could be a potential therapeutic target for RB.

Aging is the strongest risk factor for cardiovascular disease, with progressive decline in the function of vascular endothelial cells (ECs) with age. Systematic analyses of the effects of aging on different cardiac EC types remain limited. Here, we constructed a scRNA atlas of EC transcriptomes in young and old mouse hearts. We identified 10 EC subclusters. The multidimensionally differential genes (DEGs) analysis across different EC clusters shows molecular changes with aging, showing the increase in the overall inflammatory microenvironment and the decrease in angiogenesis and cytoskeletal support capacity of aged ECs. And we performed an in-depth analysis of 3 special ECs, Immunology, Proliferating and Angiogenic. The Immunology EC seems highly associated with some immune regulatory functions, which decline with aging at different degrees. Analysis of two types of neovascular ECs, Proliferating, Angiogenic, implied that Angiogenic ECs can differentiate into multiple EC directions after initially originating from proliferating ECs. And aging leads to a decrease in the ability of vascular angiogenesis and differentiation. Finally, we summarized the effects of aging on cell signaling communication between different EC clusters. This cardiac EC atlas offers comprehensive insights into the molecular regulations of cardiovascular aging, and provides new directions for the prevention and treatment of age-related cardiovascular disease.

Purpose: To investigate the relationship between epidermal growth factor receptor (EGFR) expression and clinicopathological characteristics in sebaceous carcinoma (SbC) of the eyelid. Methods: Clinical records and microscopic slides of 102 cases of SbC in the eyelid were reviewed. An immunohistochemical antibody for EGFR was employed. Differentiation, pagetoid spread, and mitosis were evaluated. Results: Of the 102 patients, 46 (45.1%) cases were male and 56 (54.9%) cases were female (male:female, 1:1.2). The mean age of the patients was 57.32 +/- 13.23 years (range, 26-85 years). Fifty-two (51%) cases occurred in the right eye and 50 (49%) cases in the left eye. The stage T1 and stage T2 cases were 71 (69.6%) and 31 (30.4%), respectively. There were 69 (67.6%) cases with pagetoid spread and 33 (32.4%) cases without pagetoid spread. There were 15 (14.7%) well-differentiated cases, 33 (32.4%) moderately differentiated cases, and 54 (52.9%) poorly differentiated cases. There was 1 (1%) case of 0 to 1/ high power field (HPF) mitosis, 46 (45.1%) cases of 2 to 5/HPF mitoses, and 55 (53.9%) cases of >5/HPF mitoses, respectively. The EGFR positivity of SbCs was 97.1% (99 cases) with 2% (2 cases) weak expression, 46.1% (47 cases) moderate expression, and 49% (50 cases) strong expression. While EGFR was weakly positive only in a few conjunctival epithelial cells and basal cells of the sebaceous glands. The EGFR expression of SbCs was related to the clinic T category statistically (P = 0.048) but not related to age, gender, differentiation, nuclear mitosis, and pagetoid spread of these tumors statistically (P > 0.05). And the differentiation of these SbCs was related to the mitosis of these tumors statistically (P < 0.001). Conclusions: The EGFR expression of SbCs was related to the tumor stage statistically, which implied that EGFR might be used as a prognostic marker of SbCs. EGFR is expressed in most SbC cases, which implied that it might act in the tumorigenesis mechanisms of SbC and could be a therapeutic target in the treatment of SbC for some metastatic cases.

目的:探索用细胞块制备试剂盒对眼内玻璃体液微量细胞制备细胞块的成功率,苏木素-伊红(hematoxylin-eosin,HE)染色效果及技术要点.方法:收集中山大学中山眼科中心临床病理科2020年9月至2021年1月由临床送检的25例玻璃体液(含玻璃体切割液),应用细胞块制备试剂盒制备细胞块后,常规固定、脱水、包埋、切片,随后进行HE染色,观察染色效果.结果:25例玻璃体细胞蜡块制作成功率达到100％,制片后HE染色效果好,背景干净,细胞形态清晰,核质对比分明.结论:应用细胞块制备试剂盒能将眼内玻璃体液微量细胞制成蜡块,极大提高了标本的利用率,为后续的病理研究提供丰富的材料.

目的:探讨眼眶黏膜相关淋巴组织(mucosa-associated lymphoid tissue,MALT)结外边缘区B细胞淋巴瘤的临床表现、影像学特征及病理学特点,以期减少眼眶淋巴瘤的误诊,提高生存率.方法:对2020年1月至2020年12月中山大学中山眼科中心诊治的71例眼眶MALT结外边缘区B细胞淋巴瘤的临床、影像及病理学资料进行回顾性分析.结果:71例患者中,男38例,女33例;左侧眼眶31例,右侧眼眶34例,双侧眼眶6例;原发病例67例,复发病例4例;年龄23～84岁,病程为3个月～15年.最常见的临床表现是眼部肿块和不同程度的眼球活动受限;磁共振成像(magnetic resonance imaging,MRI)检查见密度均匀的软组织影,呈"铸造样",眼球内未见侵犯;组织病理检查可见肿瘤由小至中等大小淋巴样细胞组成,瘤细胞呈弥漫或片状生长,核小到中等、不规则,核仁不明显,部分细胞呈单核样淋巴瘤细胞改变,其中9例可见浆细胞样分化,伴浆细胞分化的病例kappa与lambda的表达不对称.结论:眼眶MALT结外边缘区B细胞淋巴瘤有多种临床表现,影像学检查具有一定的特征,可辅助术前诊断.病理学检查可用于术后的准确诊断及分型,据此制定合适的治疗方案,提高疗效.

Intraocular lymphoma (IOL) is a rare lymphocytic malignancy which contains two main distinct forms. Primary intraocular lymphoma (PIOL) is mainly a sub-type of primary central nervous system lymphoma (PCNSL). Alternatively, IOL can originate from outside the central nervous system (CNS) by metastasizing to the eye. These tumors are known as secondary intraocular lymphoma (SIOL). The IOL can arise in the retina, uvea, vitreous, Bruch’s membrane and optic nerve. There are predominantly of B-cell origin; however there are also rare T-cell variants. Diagnosis remains challenging for ophthalmologists and pathologists, due to its ability to masquerade as noninfectious or infectious uveitis, white dot syndromes, or occasionally as other metastatic cancers. Laboratory tests include flow cytometry, immunocytochemistry, interleukin detection (IL-10: IL-6, ratio >1), and polymerase chain reaction (PCR) amplification. Methotrexate-based systemic chemotherapy with external beam radiotherapy and intravitreal chemotherapy with methotrexate are useful for controlling the disease, but the prognosis remains poor. Therefore, it is important to make an early diagnose and treatment. This review is focused on the clinical manifestations, diagnosis, treatment and prognosis of the IOL.

Purpose. In this study, we aimed to investigate the presence of Epstein–Barr virus (EBV) in the eyelid sebaceous gland carcinoma (SGC) and its association with the clinicopathologic features. Methods. One hundred and thirty paraffin-embedded SGC specimens were retrieved from the Clinical Pathology Department of Zhongshan Ophthalmic Center. Epstein–Barr virus-encoded RNA (EBER) was detected with in situ hybridization (ISH) using the Leica BOND system autostainer. The age and gender distributions of all patients were analyzed and compared with earlier reports. Pearson’s χ2 and Fisher’s exact tests were used to determine the association between clinicopathological features such as age, gender, laterality eye, tumor basal dimension, degree of tumor differentiation, and EBER positivity. Likewise, the relationship between the grade and tumor basal dimension in EBER-positive SGC of the eyelid was analyzed. Results. Thirty-four out of one hundred and thirty (26.2%) eyelid SGC specimens were positively stained for EBER. The age range of highest incidence was 46–75 years, and the female to male ratio was 1 : 0.9. No significant correlation was found between EBER-positivity and age (p=0.5370), gender (p=0.4758), and degree of tumor differentiation (p=0.7787). However, EBV positivity was strongly correlated with the right eye (p=0.0287), the tumor basal dimension (p=0.0001). EBV positivity grade presented statistically associated with tumor size (p=0.0329). Conclusion. We conclude that ISH is a sensitive method to identify EBV in SGC of the eyelid. A possible causal association of EBV in SGC patients is suggested by high frequency of EBER-ISH positivity and its association with the clinicopathologic features.

AIMTo discuss the prognostic significant of autophagy related proteins (ARPs) in retinoblastoma (RB) and to find the molecular marker to distinguish retinocytoma (RC) and RB by investigating the different expression profiling of microtubule-associated protein light chain 3 (LC3B) and other ARPs in RC and RB.METHODSSpecimens with retinocytoma region (RCR) or mainly composed with Flexner-Winterstein rosettes (FWR) were screen out from 219 paraffin-embedded RB samples and respectively taken as RCR group and FWR group. Others were taken as undifferentiated (UD) group. Immunochemistry (IHC) of LC3B and electronic microscopy was used to identify autophagy. The IHC scores of LC3B and other ARPs, such as Beclin, PTEN, p27, p16(INK4a), mTOR and BCL-2 were compared and correlation analysis was applied to find potential proteins which may involve in autophagy regulation. The prognostics significance of LC3B was evaluated by comparing the high risk features (HRFs) in 3 groups of total 219 samples.RESULTSTwenty-one specimens with RCR and 36 specimens mainly composed with FWR were screen out. RCR cell had a high level of LC3B and lots of autophagic vacuoles. Beclin, PTEN, p27 had positive correlation with LC3, and p16(INK4a) had negative correlation, while the expression of mTOR and BCL-2 in RCR and RB region did not show any difference. Cases with RCR had lower rate of HRFs than undifferentiated cases.CONCLUSIONARPs had different expression pattern between RCR and other pathological types of RB, and could be ideal markers to distinguish RC from RB. Our finding indicated cases with RCR had favorable prognosis just like those with FWR.

PURPOSE:To compare the merits and limitations of hematoxylin-eosin (HE) and methyl violet staining for displaying ghost cells from vitreous or aqueous humor.METHODS:A specimen containing ghost cells was adjusted to five different concentrations: (12 x 10(4), 10 x 10(4), 8 x 10(4), 6 x 10(4) and 4 x 10(4) cells/ml) and subjected to smearing and methyl violet and HE staining. The staining results were observed by light microscopy.RESULTS:The ghost cells were readily observed at a cell density of > 8 x 10(4) cells/ml with methyl violet staining, but only a few cells were occasionally seen at lower cell densities. In contrast, ghost cells were seen at all cell densities with HE staining.CONCLUSION:Methyl violet staining is more rapid and simpler for the identification of ghost cells, but its staining color more readily fades, the slides cannot be stored, and it is only effective at a cell density of > 8 x 10(4) cells/ml. In contrast, HE staining is more time-consuming but it can display cell morphology and distinguish cell components more explicitly and slides can be permanently stored. HE staining has advantages over methyl violet staining in detecting the ghost cells when the concentration is < 8 x 10(4) cells/ml.

PURPOSE To find the better method of eosinophilic granulocytes staining by comparison between HE staining and Giemsa staining METHODS Twenty conjunctivitis patients(40 eyes) were studied. The infected palpabral conjunctiva of both eyes were scroped and stained separately with Giemsa staining and HE staining. RESULT Under microscope, eosinophilic granulocytes were more clear by Giemsa staining than HE staining. CONCLUSION In showing eosinophilic granulocytes, Giemsa staining was better than HE staining.