Small extracellular vesicles loaded with carboplatin effectively enhance the cytotoxicity of drug-resistant cells from Y79 cells-in vitro.

Drug resistance (DR) is one of the challenges in treating retinoblastoma (Rb) that warrants novel approaches. With the emerging evidence on the role of small extracellular vesicles (sEVs) as a drug-delivery carrier system, in this study, we derived the drug-resistant (DR) clones of Y79 cells and evaluated the efficacy of sEVs-loaded with carboplatin (sEVs-CPT) to reverse the chemoresistance. Drug-resistant clones of Y79 cells (DR-Y79) were systematically developed through sequential exposure to carboplatin (CPT), showcasing a sixfold increase in inhibitory concentration when compared to parental Y79 cells (IC50: 41.4 µg/mL and 6.2 µg/mL) (P<0.0001). These DR-Y79 cells show higher expression of ABCG2 and higher expression of DR genes than parental Y79 cells (P<0.0001). The sEVs were isolated from the conditioned media of Y79 cells using ultracentrifugation (UC) and characterized. Further, the sEVs were loaded with CPT and achieved higher encapsulation efficiency at one hour, and drug release of sEVs-CPT was highest at ∼80% at pH 5.0. The cytotoxicity of sEVs-CPT on Y79 cells and DR-Y79 was higher when compared to the CPT (IC50: 3.5 µg/mL vs 6.2 µg/mL; 23.1 µg/mL vs 41.2 µg/mL) (p<0.0001). This study demonstrates that sequential exposure to CPT generates DR clones of Y79 cells, which could serve as an appropriate model to evaluate the efficacy of drugs. The sEVs-CPT were highly effective in enhancing cytotoxicity in DR-Y79 cells, and appear to hold promise as a novel complimentary drug delivery system.

Identifying Treatment Resistance Related Pathways by Analyzing Serum Extracellular Vesicles of Patients With Resistant Versus Regressed Retinoblastoma.

Purpose: To identify the genes and pathways responsible for treatment resistance (TR) in retinoblastoma (RB) by analyzing serum small extracellular vesicles (sEVs) of patients with TR active RB (TR-RB) and completely regressed RB (CR-RB). Methods: Serum-derived sEVs were characterized by transmission electron microscopy and nanoparticle tracking analysis. sEV transcriptome profiles of two TR-RB and one CR-RB with good response (>20 years tumor free) were compared to their age-matched controls (n = 3). Gene expression data were analyzed by the R Bioconductor package. The CD9 protein and mRNA expression of CD9, CD63, and CD81 were studied in five RB tumors and two control retinae by immunohistochemistry and quantitative reverse transcription-polymerase chain reaction. Results: The isolated serum sEVs were round shaped and within the expected size (30-150 nm), and they had zeta potentials ranging from -10.8 to 15.9 mV. The mean ± SD concentrations of sEVs for two adults and four children were 1.1 × 1012 ± 0.1 and 5.8 × 1011 ± 1.7 particles/mL. Based on log2 fold change of ±2 and P < 0.05 criteria, there were 492 dysregulated genes in TR-RB and 184 in CR-RB. KAT2B, VWA1, CX3CL1, MLYCD, NR2F2, USP46-AS1, miR6724-4, and LINC01257 genes were specifically dysregulated in TR-RB. Negative regulation of apoptotic signaling, cell growth, and proton transport genes were greater than fivefold expressed only in TR-RB. CD9, CD63, and CD81 mRNA levels were high in RB tumors versus control retina, with increased and variable CD9 immunoreactivity in the invasive areas of the tumor. Conclusions: Serum sEVs could serve as a potential liquid biopsy source for understanding TR mechanisms in RB.

Human Lacrimal Gland Derived Mesenchymal Stem Cells - Isolation, Propagation, and Characterization.

Purpose: The existing treatment options for dry eye disease (DED) due to lacrimal gland (LG) dysfunction are mainly palliative. Mesenchymal stem cells (MSCs) based therapies and 3D-LG organoids have been explored as a curative option for LG regeneration in animal models. Human LG epithelial cultures are previously established and, here, we aim to isolate and characterize the spindle-shaped cells obtained from primary human LG cultures in order to unveil its MSC property. Methods: Normal human lacrimal glands were obtained from individuals undergoing LG debulking surgery. The conditions for human LG-MSC culture were standardized to obtain pure population of LG-MSCs at passage 3. Population doubling time (PDT), expression of phenotypic markers, tri-lineage differentiation, colony forming potential, and gene expression analysis were carried out to assess the phenotypic and genotypic characteristics compared to bone marrow-MSCs (BM-MSCs). Results: Our data show that these spindle-shaped cells exhibit similar phenotypic expression, colony-forming ability, and trilineage differentiation like BM-MSCs. Moreover, the gene expression also did not show any significant difference, except for increased IL1-ß in LG-MSCs. The LG-MSCs do not express any lacrimal epithelial markers unlike LG tissue. Conclusions: This study reveals the first-time evidence for the presence of MSC population within the human LGs, and these cells might play a role in maintaining healthy microenvironment within normal LG and repair in diseased LGs.

Newer approaches to dry eye therapy: Nanotechnology, regenerative medicine, and tissue engineering.

Definitive treatment of dry eye disease (DED), one of the commonest ocular surface disorders, has remained elusive despite several recent advances in better diagnostics and the introduction of newer therapeutic molecules. The current treatment paradigms rely heavily on lubricating eye drops and anti-inflammatory agents that may need to be used long-term and are mainly palliative. Research is ongoing not only for a curative treatment option but also to improve the potency and efficacy of existing drug molecules through better formulations and delivery platforms. In the past two decades, significant advancement has been made in terms of preservative-free formulations, biomaterials such as nanosystems and hydrogels, stem cell therapy, and creation of a bioengineered lacrimal gland. This review comprehensively summarizes the newer approaches to DED treatment, which are biomaterials such as nanosystems, hydrogels, and contact lenses for drug delivery, cell and tissue-based regenerative therapy for damaged lacrimal gland and ocular surface, and tissue engineering for developing artificial lacrimal gland. Also, their potential efficacies in animal models or in vitro studies and possible limitations are discussed. The ongoing research looks promising and needs to be supported with clinical efficacy and safety studies for human use.

Specific location of ocular adnexal lymphoma and mortality: an international multicentre retrospective study.

AIMS: To examine whether the specific location of ocular adnexal lymphoma (OAL) and the American Joint Committee on Cancer (AJCC) TNM tumour stage are prognostic factors for mortality in the main OAL subtypes. METHODS: Clinical and survival data were retrospectively collected from seven international eye cancer centres. All patients from 1980 to 2017 with histologically verified primary or secondary OAL were included. Cox regression was used to compare the ocular adnexal tumour locations on all-cause mortality and disease-specific mortality. RESULTS: OAL was identified in 1168 patients. The most frequent lymphoma subtypes were extranodal marginal zone B-cell lymphoma (EMZL) (n=688, 59%); follicular lymphoma (FL) (n=150, 13%); diffuse large B-cell lymphoma (DLBCL) (n=131, 11%); and mantle cell lymphoma (MCL) (n=89, 8%). AJCC/TNM tumour-stage (T-stage) was significantly associated with disease-specific mortality in primary ocular adnexal EMZL and increased through T-categories from T1 to T3 disease. No associations between AJCC/TNM T-stage and mortality were found in primary ocular adnexal FL, DLBCL, or MCL. EMZL located in the eyelid had a significantly increased disease-specific mortality compared with orbital and conjunctival EMZL, in both primary EMZL and the full EMZL cohort. In DLBCL, eyelid location had a significantly higher disease-specific mortality compared with an orbital or lacrimal gland location. CONCLUSION: Disease-specific mortality is associated with AJCC/TNM T-stage in primary ocular adnexal EMZL patients. Lymphoma of the eyelid has the highest disease-specific mortality in primary EMZL, and in the full cohort of EMZL and DLBCL patients.

Using the Chick Embryo Model to Examine the Effects of Hypoxia Pre-conditioning of Uveal Melanoma Cells on Tumor Growth and Metastasis.

PURPOSE: Highly dynamic oxygen gradients occur within tumors that can result in a hypoxic response, contributing to tumor progression and metastasis. Evidence in uveal melanoma (UM) suggests an upregulated hypoxia response in some poor prognosis UM characterized by HIF1α signaling. We aimed to investigate the effects of exposure to hypoxia on tumor growth and dissemination in the chick embryo chorioallantoic membrane (CAM) model. METHODS: UM cell lines (MP41, 92.1, MP46, and OMM1) were grown in two-dimensional culture and pre-exposed to hypoxic (1% O2) conditions for 72 h. The effects of this hypoxia pre-conditioning on cell number and clonogenicity as compared with 21% O2 ("normoxia") were investigated prior to transplantation of the cells onto the CAM. Nodule-forming efficiency (NFE), nodule size, and the presence/absence of tumor cell dissemination were determined macroscopically and histologically. RESULTS: Exposure of UM cell lines to hypoxia upregulated HIF1α expression compared to cells cultured in normoxia. A 72-h pre-exposure to hypoxia significantly reduced cell number and clonogenicity in the MP41 and OMM1 cell lines while it had little effect in 92.1 and MP46 cells. When 72-h hypoxia pre-conditioned cells were grown in three-dimensions on the CAM, a reduction in NFE and nodule size was observed when compared with normoxic UM cells. All nodules were composed of proliferating (Ki-67+) Melan-A + cells and displayed chick blood vessel recruitment. Spread of UM cells into the adjacent CAM was observed; however, dissemination to the chick liver was only seen with 92.1 cells grown under normoxia. CONCLUSIONS: Hypoxia pre-conditioning does not appear to drive a metastatic phenotype in UM; however, further understanding of how oxygen dynamics within the tumor microenvironment regulates HIF1 signaling is needed to determine whether inhibitors of HIF signaling represent a therapeutic option in metastatic UM.

Emerging therapeutic targets for retinoblastoma.

INTRODUCTION: Retinoblastoma (Rb) is an early childhood intraocular tumor of the retina and is managed by multimodal therapeutic approaches. Recent advanced targeted delivery of chemotherapeutic drugs to the eye has improved the possibility of globe salvage. However, enucleation is inevitable for advanced and recurrent Rb. The cumulative knowledge of identification of newer molecular biology tools, exosomal cargo, role of cancer stem cells (CSCs), and its microenvironment in the progression of the diseases warrants a relook at the traditional treatment protocol and explore the feasibility of targeted therapies. AREAS COVERED: This review covers Rb pathobiology, novel molecular-targeted therapeutics, and strategies targeting Rb CSCs and provides an update on potential therapeutic targets such as second messengers and exosomal cargo. EXPERT OPINION: The emergence of early diagnosis and multimodality treatment protocols have significantly improved the clinical outcome of children with advanced Rb; however, the problem of tumor recurrence has not yet been overcome. Improved understanding of the molecular pathways, identification, and characterization of CSCs opens up new targeted therapy approaches. The contemporary evidence from other fields shows promising evidence that combining conservative treatment modalities with targeting therapies specific for CSCs in clinical practice is essential for achieving high globe salvage rate in Rb patients.

Comprehensive Analysis of Serum Small Extracellular Vesicles-Derived Coding and Non-Coding RNAs from Retinoblastoma Patients for Identifying Regulatory Interactions.

The present study employed nanoparticle tracking analysis, transmission electron microscopy, immunoblotting, RNA sequencing, and quantitative real-time PCR validation to characterize serum-derived small extracellular vesicles (sEVs) from RB patients and age-matched controls. Bioinformatics methods were used to analyze functions, and regulatory interactions between coding and non-coding (nc) sEVs RNAs. The results revealed that the isolated sEVs are round-shaped with a size < 150 nm, 5.3 × 1011 ± 8.1 particles/mL, and zeta potential of 11.1 to −15.8 mV, and expressed exosome markers CD9, CD81, and TSG101. A total of 6514 differentially expressed (DE) mRNAs, 123 DE miRNAs, and 3634 DE lncRNAs were detected. Both miRNA-mRNA and lncRNA-miRNA-mRNA network analysis revealed that the cell cycle-specific genes including CDKNI1A, CCND1, c-MYC, and HIF1A are regulated by hub ncRNAs MALAT1, AFAP1-AS1, miR145, 101, and 16-5p. Protein-protein interaction network analysis showed that eye-related DE mRNAs are involved in rod cell differentiation, cone cell development, and retinol metabolism. In conclusion, our study provides a comprehensive overview of the RB sEV RNAs and regulatory interactions between them.

Exosomes in the visual system: New avenues in ocular diseases.

Exosomes are a subgroup of membrane-bound extracellular vesicles secreted by all cell types and present virtually in all biological fluids. The composition of exosomes in the same cell type varies in healthy and disease conditions. Hence, exosomes research is a prime focus area for clinical research in cancer and numerous age-related metabolic syndromes. Functions of exosomes include crucial cell-to-cell communication that mediates complex cellular processes, such as antigen presentation, stem cell differentiation, and angiogenesis. However, very few studies reported the presence and role of exosomes in normal physiological and pathological conditions of specialized ocular tissues of the eye and ocular cancers. The eye being a protected sense organ with unique connectivity with the rest of the body through the blood and natural passages, we believe that the role of exosomes in ocular tissues will significantly improve our understanding of ocular diseases and their interactions with the rest of the body. We present a review that highlights the existence and function of exosomes in various ocular tissues, their role in the progression of some of the neoplastic and non-neoplastic conditions of the eyes.

Lacrimal gland regeneration: The unmet challenges and promise for dry eye therapy.

Dry eye disease (DED) is a common multifactorial disease of the tear film and the ocular surface. The problem of DED has gained attention globally, with millions of people affected by the disorder. Although the treatment strategies for DED have significantly evolved over time, most of the existing modalities fall under the category of standard palliative care when viewed from a long-term perspective. To address these limitations, different approaches have been explored by various groups to uncover alternative treatment strategies that can contribute to a full regeneration of the damaged lacrimal gland, which is responsible for producing the major aqueous component of the tear film. For this, multiple groups have investigated the role of lacrimal gland cells in DED based on their regenerating, homing, and differentiating capabilities. In this review, we discuss in detail the therapeutic mechanisms and regenerative strategies that can potentially be applied for lacrimal gland regeneration as well as their therapeutic applications. This review mainly focuses on aqueous deficiency dry eye disease (ADDE) caused by lacrimal gland dysfunction and possible future treatment strategies. The current key findings from cell and tissue-based regenerative therapy modalities that could be utilised to achieve lacrimal gland tissue regeneration are summarized. In addition, this review summarises the available literature from in vitro to in vivo studies, their limitations in relation to lacrimal gland regeneration and the possible clinical applications. Finally, current issues and unmet needs of cell-based therapies in providing complete lacrimal gland tissue regeneration are discussed.

A short-term chick embryo in vivo xenograft model to study retinoblastoma cancer stem cells.

Purpose: Cancer stem cells (CSCs) reported in various tumors play a crucial role in tumorigenesis and metastasis of retinoblastoma (Rb). Following the efforts to reduce, replace, and refine the use of mammalian models, we aimed to establish a short-term xenograft for Rb to evaluate the CSC properties of CD133- Rb Y79 cells, using the well-established chick embryo chorioallantoic membrane (CE-CAM) assay. Methods: Y79 cells were cultured, labeled with two different dyes (CM-Dil Y79 and enhanced green fluorescent protein (eGFP)) and sorted for CD133- and CD133 + subsets. Two million cells from each of the labeled groups were transplanted onto the abraded CAM on embryonic day 7 (E7). On E14, the tumor nodule formation on CAM and spontaneous metastasis to the embryos were evaluated by confocal microscopy, in vivo imaging, and histology. Results: Y79 cells formed pink-white raised perivascular nodules with feeder vessels on the CAM with both the types of labeled CD133- cells. CD133- cells, when compared to CD133 + cells, demonstrated significantly larger tumor volume (40.45 ± 7.744 mm3 vs 3.478 ± 0.69 mm3, P = 0.0014) and higher fluorescence intensity (CM-Dil: AUF = 6.37 × 107 ± 7.7 × 106 vs 1.08 × 107 ± 1.6 × 106; P < 0.0001; eGFP: AUF = 13.94 × 104 ± 2.54 × 104 vs AUF = 1.39 × 104 ± 0.4 × 104; P = 0.0003). The metastatic potential of CD133- cells was also observed to be higher as noted by in vivo imaging and histopathology. Conclusion: This study highlights that CE-CAM is a feasible alternative nonmammalian model for evaluating tumorigenicity and metastatic potential of Y79 CSCs. Increased tumorigenicity and metastatic potential of CD133- subset of tumor cells substantiate their CSC properties.

Does Multimodal Treatment Improve Eye and Life Salvage in Adenoid Cystic Carcinoma of the Lacrimal Gland?

PURPOSE: To evaluate the efficacy of multimodal treatment in adenoid cystic carcinoma (ACC) of the lacrimal gland. METHODS: A retrospective comparative case series of 40 consecutive patients with ACC of the lacrimal gland without systemic metastasis at the initial presentation and primarily managed by one of the 3 treatment protocols-surgery + external beam radiotherapy (EBRT) (group 1), surgery + EBRT + adjuvant chemotherapy (group 2), and neoadjuvant chemotherapy + surgery + EBRT + adjuvant chemotherapy (multimodal treatment) (group 3) at a tertiary care ocular oncology center. Local tumor control, eye salvage, and systemic metastasis were the primary outcome measures. RESULTS: The age ranged from 11 to 72 (mean ± SD, 36 ± 13; median, 36) years with 26 (65%) male and 14 (35%) female patients. Twelve (30%) patients belonged to group 1, 8 (20%) to group 2, and 20 (50%) to group 3. Primary surgery included tumor excision in 36 (90%) and orbital exenteration in 4 (10%). Chemotherapy composed of cisplatin + 5 fluorouracil (5FU) for 6 cycles in 28 (70%) patients. Extended-field stereotactic EBRT with a dose of 5,000-6,000 cGy included the entire pretreatment extent of the tumor with a 10-mm margin all around, superior orbital fissure, inferior orbital fissure, cavernous sinus, and temporal fossa. Mean duration of follow up after completion of treatment was 58 ± 26 (range, 29-180; median, 60) months. In all, local tumor recurrence occurred in 10 (25%) patients at a mean of 38 ± 23 (range, 12-120; median, 24) months. Local tumor recurrence was noted in 5 (42%) patients in group 1, 2 (25%) in group 2, and 3 (15%) in group 3. Overall, eye salvage was possible in 34 (85%) patients, with visual acuity >20/40 in 28 (82%). Systemic metastasis occurred in 10 (25%) patients at a mean of 53 ± 28 (range, 12-120; median, 43) months. Eight (67%) patients in group 1, 1 (13%) in group 2, and 1 (5%) in group 3 developed systemic metastasis. Six (15%) overall, 5 (42%) in group 1 and 1 (13%) in group 2, died with systemic metastasis. CONCLUSIONS: Multimodal treatment with sequential neoadjuvant chemotherapy, followed by surgery, extended-field stereotactic EBRT, and adjuvant chemotherapy seems relatively more effective in providing local tumor control and eye salvage and in minimizing the risk of systemic metastasis in ACC of the lacrimal gland.

Identification of novel RB1 genetic variants in Retinoblastoma patients and their impact on clinical outcome.

BACKGROUND: Retinoblastoma (RB) is an intraocular childhood cancer develops due to inactivation of RB1 gene. Identification of RB1 genetic variants, correlating and confirming genetic test results with clinical outcomes are crucial for effective RB management. METHODS: Retrospective study of 62 RB patients and 14 family members who underwent genetic testing either by next generation sequencing (NGS) or multiplex ligation-dependent probe amplification (MLPA) or by both for screening RB1 germline mutations present in peripheral blood. Mutational outcomes were correlated with clinical outcomes evaluated over a follow-up period of 12 months. RESULTS: Of the 62 patients, 35 (56%) had bilateral RB and 27 (44%) had unilateral RB. Out of 24 (52%) variants detected by NGS, 9 (37.5%) were novel and 15 (62.5%) were known in 46 probands. Six (18%) gross deletions were detected by MLPA in 34 probands. The mutation detection rate by NGS and MLPA in unilateral cases was 15% (n = 4) and 74% (n = 26) in bilateral cases. In patients with RB1 genetic mutations versus those without, the rate of primary enucleation (7 (12%) vs 18 (44%) eyes; p = .0008) was inversely proportional to tumor recurrence (25 (45%) vs 6 (15%) eyes; p = .002). There was no difference in the rate of globe salvage and metastasis, over a mean follow-up period of 12 months. CONCLUSION: The mutations screening is important for risk assessment in future siblings and offspring of RB patients and most important in unilateral RB for determining if hereditary or not hereditary RB. Its role in predicting clinical outcomes is yet to be determined.

Carboplatin- and Etoposide-Loaded Lactoferrin Protein Nanoparticles for Targeting Cancer Stem Cells in Retinoblastoma In Vitro.

Purpose: Cancer stem cells (CSCs) are known to contribute to tumor relapses by virtue of their chemoresistance. With the knowledge that nanoformulations can overcome drug resistance, we evaluated the efficacy and cytotoxicity of clinical-grade carboplatin (CPT)- and etoposide (ETP)-loaded lactoferrin nanoparticles (Lf-Nps) on total, CD133-enriched (non-CSC), and CD133-depleted (CSC) populations of retinoblastoma (Rb) Y79 cells. Methods: Physicochemical properties of drug-loaded Lf-Nps were measured with transmission electron microscopy and attenuated total reflectance-Fourier transform infrared. The encapsulation efficiency, uptake, and release of drug-loaded Lf-Nps were measured using high-performance liquid chromatography and a UV-visible spectrophotometer. Cytotoxicity of the standard and drug-loaded Lf-Nps was evaluated by the MTT assay. Results: The mean (SD) size and encapsulation efficiency of Lf-CPT and Lf-ETP were 61.2 (3.94) nm, 60% and 45.15 (5.85) nm, 38%, respectively, and the drug release efficiency was highest at pH 6. The increased drug uptake and lower release of drug-loaded Lf-Nps were observed in CSC and non-CSC populations compared to their standard forms. The relative increase of drug uptake and sustained intracellular retention of the drug-loaded Lf-Nps compared to standard drugs showed an enhanced cytotoxicity up to 50%, especially in Rb Y79 CSCs (IC50: CPT, 230.3; Lf-CPT, 118.2; ETP, 198.1; and Lf-ETP, 129) compared to non-CSCs. Conclusions: Our study documents an increase in drug uptake, retention, and cytotoxicity of Lf-CPT and Lf-ETP on Y79 CSCs and non-CSCs as compared to their standard drugs in vitro. The reversal of chemoresistance in the CSC population by nanoformulation appears promising with the potential to pave the way for improved targeted therapy and better clinical outcomes.

Ocular Features in a Large Cohort of Indians With Xeroderma Pigmentosum.

PURPOSE: Xeroderma pigmentosum (XP) is an extreme hypersensitivity to sunlight causing skin freckling and pigmentary changes because of defective DNA repair mechanisms. The purpose of this article is to evaluate the spectrum of ocular and systemic features in XP at a tertiary eye care center in India over 32 years. METHODS: Data from 418 eyes of 209 patients diagnosed with XP from 1987 to 2018 were reviewed retrospectively for demographics, complaints, ocular features, systemic associations, and their management. RESULTS: Median age at diagnosis was 2 years (interquartile range, 0.5-5 years). A total of 124 patients (59.3%) were men. There was parental consanguinity in 74.4% cases. Common ocular complaints were photophobia (47.1%), ocular discomfort (45%), defective vision (36.6%), redness (13.4%), tissue growth (12%), white spot (11.2%), and pain (10.5%). At presentation, 43.5% had corneal scars (45.5% were located inferiorly and 70.9% covered visual axis). Corneal vascularization and limbal stem cell deficiency were noted in 37.4%. A total of 56% of patients had at least 1 tumor, and 6.7% had neurological abnormalities. At least 1 ocular surgery was performed in 37.8% of patients. Ophthalmic surgical interventions included tumor excision (23%), keratoplasty (13.4%), and nontumor ocular surface surgery (3.4%). CONCLUSIONS: XP is a disorder that has high ocular morbidity in Indian patients. The recognition of common signs and symptoms and relative frequency of various ocular complications with time trends will help in managing and reducing the sequelae of this otherwise untreatable and progressive disease.

Overexpression of metastasis-associated in colon cancer 1 in retinoblastoma.

INTRODUCTION: Metastasis-associated in colon cancer 1 (MACC1), one of the prognostic markers for colonic and other tumours was noted to be overexpressed in retinoblastoma (Rb) Y79 cancer stem cells. This prompted us to evaluate its expression in primary Rb tumour and serum samples with clinicopathologic correlation. The interacting partner, c-MET was also evaluated in primary tumour tissues to explore the activation of MACC1 signaling. METHODOLOGY: This study was done following institutional review board approval from participating institutes. Semiquantitative gene expression for MACC1 was evaluated using formalin-fixed paraffin-embedded sections and unfixed tumour samples from primary Rb cases (n = 44). Immunolocalization for MACC1 was assessed in primary Rb tumours (n = 22), bone marrow aspirates with metastasis (n = 3), and c-MET expression was also assessed in Rb tumours (n = 17). Serum MACC1 levels were analysed using enzyme-linked immunosorbent assay in samples collected from Rb patients undergoing enucleation (n = 31), Rb patients with proven clinical metastasis (n = 3), and compared to appropriate controls. Clinicopathologic correlation of MACC1 expression was analysed using the medical records with specific reference to histologic risk factors (HRF) for metastasis and differentiation. RESULTS: High expression of MACC1 gene was noted in all the tumour samples (n = 44), more so in cases with versus without HRF (p < 0.0001). In cases with HRF, MACC1 and c-MET showed diffuse nuclear and cytoplasmic staining whereas it was predominantly cytoplasmic in cases without HRF. Mean immunoreactivity score of MACC1 and c-MET tissue immunolocalization revealed that cases with HRF showed significantly higher expression compared to cases without HRF (p < 0.05). Unlike the findings in colonic tumours, serum levels of MACC1 were lower in patients compared to normal controls. CONCLUSION: Overexpression of MACC1 and c-MET in retinoblastoma tissues, specifically those with risk factors for metastasis, suggests its role in proliferation and possibly in invasion. However, the current data do not support it to be a clinical prognostic marker in retinoblastoma tumours. The inverse serum expression is an intriguing finding, which warrants further studies especially in retinoblastoma.

Immunomodulatory properties of bone marrow mesenchymal stem cells.

Bone marrow mesenchymal stem cells (BM-MSCs) are multipotent progenitor cells of mesodermal origin possessing multilineage differentiation potential and ease of expansion in vitro. Over the years, these cells have gained attention owing to their potential in cell-based therapies in treating various diseases. In particular, the wide spectrum of immunoregulatory/immunomodulatory role of MSCs in various clinical conditions has gained immense attention. The immunomodulatory properties of BM-MSCs are mediated by either cell-cell contact (interactions with various immune cells in a context-dependent manner), paracrine mode of action or extracellular vesicles, making them a potential option as immunosuppressants/immunomodulators in treating various clinical conditions. A plethora of studies have demonstrated that MSCs do so by exhibiting a profound effect on various immune cells for example they can inhibit the proliferation of T cells, B cells, and natural killer cells; modulate the activities of dendritic cells and induce regulatory T cells both in vitro and in vivo. In this review we aim at briefly elucidating the characteristics of BM-MSCs, specifically addressing the current understanding on the hypoimmunogeneticity and immunomodulatory properties of the same with specific reference to their interactions with B cells, T cells, Dendritic cells and natural killer cells. We also aim at reviewing the secretory profile and their role in some clinical conditions that have shown promising outcomes.

Lymphoma of the Lacrimal Gland - An International Multicenter Retrospective Study.

PURPOSE: To characterize the clinical features of subtype-specific lacrimal gland lymphoma and their effect on patient survival. DESIGN: Multicenter retrospective interventional case series. METHODS: Patient data were collected from 6 international eye cancer centers from January 1, 1980, through December 31, 2017. All patients with histologically verified primary or secondary lymphoma of the lacrimal gland were included. Primary endpoints were overall survival (OS) and disease-specific survival (DSS). RESULTS: A total of 260 patients with lacrimal gland lymphoma were identified. The median age was 58 years and 52% of patients were men. Non-Hodgkin B-cell lymphomas constituted 99% (n = 258) and T-cell lymphomas constituted 1% (n = 2). The most frequent lymphoma subtypes were extranodal marginal zone B-cell lymphoma (EMZL) (n = 177, 68%), follicular lymphoma (FL) (n = 26, 10%), diffuse large B-cell lymphoma (DLBCL) (n = 25, 10%), and mantle cell lymphoma (MCL) (n = 17, 7%). Low-grade lymphomas (EMZL and FL) were most commonly treated with external beam radiotherapy (EBRT), whereas high-grade lymphomas (DLBCL and MCL) were treated with chemotherapy in combination with rituximab and/or EBRT. The prognosis was relatively good with a 5-year OS and DSS of 73.8% and 87.5%, respectively. Lymphoma subtype was a statistically significant predictor for DSS, with EMZL (5-year DSS: 93.4%) having the best prognosis and DLBCL (5-year DSS: 52.6%) having the poorest. CONCLUSIONS: This is the largest reported collection of data of subtype-specific lacrimal gland lymphoma. The subtype distribution of lacrimal gland lymphoma resembles that of the ocular adnexa. Prognosis is good and the histologic subtype is a significant predictor for disease-specific survival.