A novel, minimally invasive implant to assist in repeated intraocular drug delivery.

The standard of care for posterior segment disorders such as wet age-related macular degeneration, diabetic macular oedema and retinal vascular occlusions is pharmacotherapy by intravitreal drug delivery. Since the therapeutic effect of these drugs lasts only around 4 to 8 weeks, repeated intravitreal injections are required. Pain is experienced by the patients during injection as the needle courses through the sclera and choroid. The current work describes the design and development of a novel anodized titanium alloy implant that allows for intravitreal injections through the implant so that the needle transverses only the conjunctiva, thus minimizing discomfort to the patient. Both ex-vivo testing of the implant in enucleated goat's eye as well as in-vivo validation in rabbit eyes was carried out. The implant was placed through pars plana via a minor surgical procedure and was sutured to the sclera and covered with conjunctiva. Subsequent intravitreal injections were administered under topical anaesthesia with a 30-gauge needle through the implant thus delivering the drug into the vitreous cavity. Repeated intravitreal injections were administered every 2 weeks via the implant for 3 months in 4 rabbits. Apart from cataract in 1 rabbit, no complications were observed. There was no evidence of intra-ocular inflammation or infection at final follow-up. Histopathological analysis did not reveal any inflammation or necrosis around the area of implant. The implants were subsequently removed at 5 months and scleral wound was closed with a single suture. The sclera and overlying conjunctiva healed well and no intraocular complications were observed after removal.

Vascular endothelial growth factor (VEGF) and hypoxia inducible factor-1 alpha (HIF-1ɑ) in lacrimal gland Adenoid cystic carcinoma: Correlation with clinical outcome.

PURPOSE: VEGF and HIF-1α are important regulators of angiogenesis, overexpressed in various tumors. Lacrimal gland Adenoid cystic carcinoma (ACC) is a malignant tumor whose angiogenic properties remain unexplored. This study was designed to evaluate the expression of HIF-1α and VEGF in lacrimal gland ACC. METHODS: VEGF and HIF-1α immunoexpression was undertaken in 30 lacrimal gland ACC cases. mRNA expression of VEGF and HIF-1α was analysed in 17 cases by quantitative real time PCR. The results obtained were correlated with clinicopathological features and survival of the patients to determine the prognostic significance. RESULTS: Immunoexpression of HIF-1α and VEGF was seen in 36.6% and 46.6% ACC cases. HIF-1α expression showed significant association with advanced T-stage (P = 0.001) and VEGF with intracranial extension (P = 0.014) and solid histological pattern (P = 0.045). HIF-1α mRNA expression was seen in 29.4% cases and showed significant association with perineural invasion (P = 0.027). Recurrence occurred in 60%, distant metastasis in 20% and death in 20% cases. Survival analysis revealed that patients with HIF-1α, VEGF immunoexpression, solid histological pattern, perineural invasion, bone erosion, intracranial extension, metastasis, advanced T-stage, and exenteration had poor survival. On multivariate analysis VEGF immunoexpression (hazard ratio, 16.785; 95% confidence interval, 1.872-150.495; P = 0.012) was the most significant poor prognostic factor. CONCLUSIONS: This study demonstrates that VEGF is a potential predictor for poor clinical outcome in lacrimal gland Adenoid cystic carcinoma.

Comparison of Serum Nucleosomes and Tissue Inhibitor of Metalloproteinase1 (TIMP1) in Predicting Mortality in Adult Critically Ill Patients in Sepsis: Prospective Observational Study.

Background: Sepsis is a life-threatening organ dysfunction due to dysregulated host response to infection. Timely identification is important for risk reduction and better outcomes in critically ill patients. Nucleosomes and tissue inhibitors of metalloproteinase1 (TIMP1) are the biomarkers whose validity and utility in predicting organ dysfunction and mortality in sepsis have been proven. However, which biomarker among these two has better predictive value in elucidating disease severity, organ dysfunction, and mortality in sepsis is yet to be answered, and further studies are needed. Methods: Eighty patients with sepsis/septic shock, aged between 18 and 75 years admitted in intensive care unit (ICU) were recruited in this prospective observational trial. Quantification of serum nucleosomes and TIMP1 was done using enzyme linked immunosorbent assay (ELISA) within 24 hours of diagnosis of sepsis/septic shock. The primary outcome was to compare the predictability of nucleosomes and TIMP1 in estimating sepsis mortality. Results: The area under the receiver operating characteristic curve (AUROC) for TIMP1 and nucleosomes to discriminate between survivors and non-survivors were 0.70 [95% Confidence interval (CI), 0.58-0.81] and 0.68 (0.56-0.80), respectively. Although independent, TIMP1 and nucleosomes have statistically significant capacity to discriminate between survivors and non-survivors (p = 0.002 and p = 0.004, respectively), superiority of one biomarker over the other in discriminating between survivors and non-survivors was not observed. Conclusion: The median values of each biomarker showed statistically significant differences between survivors and non-survivors, superiority of one biomarker over other in predicting mortality was not observed. However, this was an observational study and larger studies are needed in the future to validate the findings of this study. How to cite this article: Rai N, Khanna P, Kashyap S, Kashyap L, Anand RK, Kumar S. Comparison of Serum Nucleosomes and Tissue Inhibitor of Metalloproteinase1 (TIMP1) in Predicting Mortality in Adult Critically Ill Patients in Sepsis: Prospective Observational Study. Indian J Crit Care Med 2022;26(7):804-810.

Prognostic significance of PD-1/PD-L1 expression in uveal melanoma: correlation with tumor-infiltrating lymphocytes and clinicopathological parameters.

BACKGROUND: To understand how to improve the effect of immune checkpoint inhibitors in uveal melanoma (UM), we need a better understanding of the expression of PD-1 and PD-L1, their relation with the presence of tumor-infiltrating lymphocytes (TILs), and their prognostic relevance in UM patients. MATERIALS AND METHODS: Expression of PD-1 and PD-L1 was assessed in 71 UM tissue samples by immunohistochemistry and quantitative real-time PCR (qRT-PCR), and further validated by western blotting. The effect of interferon gamma (IFN-γ) on PD-1/PD-L1 expression was determined on four UM cell lines. RESULTS: Immunoreactivity of PD-1 was found in 30/71 cases and of PD-L1 in 44/71 UM samples. Tumor-infiltrating lymphocytes were found in 46% of UM tissues. PD-1 was expressed on TILs while tumor cells expressed PD-L1. UM with and without TILs showed expression of PD-1 in 69% and 18% cases, respectively (p = 0.001). Similarly, PD-L1 was found in 75% of UM with TILs and in 50% of cases without TILs, respectively (p = 0.03). DFS rate were lower in patients with TILs with expression of PD-1 and PD-L1, but the rate of DFS was higher with expression of PD-L1 in patients without TILs. After treatment of UM cell lines with IFN-γ, PD-1 expression was induced in all UM cell lines whereas PD-L1 expression was found at a lower level in untreated cells, while expression also increased following treatment with IFN-γ. CONCLUSION: Our study suggests that increased infiltration with TILs promotes the aggressive behavior and suppresses the immune response of UM cells, thereby inhibiting immunotherapy.

An unusual presentation of conjunctival aspergillosis as a polyp in an immunocompetent child.

Fungi are a part of normal ocular flora and usually do not cause clinical infection in the absence of predisposing factors. We report a 7-year-old healthy boy from a rural area of India, who presented with a gradually increasing mass in the left eye. Excisional biopsy of the mass was performed, and pathological examination revealed multiseptate hyphae with acute-angle branching consistent with aspergillosis.

Clinical relevance of the comparative expression of immune checkpoint markers with the clinicopathological findings in patients with primary and chemoreduced retinoblastoma.

PURPOSE: The goal of this study is to identify the pathological findings and expression of immune checkpoint marker (PD-1, PD-L1, and CTLA-4) in the tumor microenvironment of both primary and chemoreduced retinoblastoma and correlate them with clinicopathological parameters and patient outcome. METHODS: Total of 262 prospective cases was included prospectively in which 144 cases underwent primary enucleation and 118 cases received chemotherapy/radiotherapy before enucleation (chemoreduced retinoblastoma). Immunohistochemistry, qRT-PCR and western blotting were performed to evaluate the expression pattern of immune checkpoint markers in primary and chemoreduced retinoblastoma. RESULTS: Tumor microenvironment were different for both primary and chemoreduced retinoblastoma. Expression of PD-1 was found in 29/144 (20.13%) and 48/118 (40.67%) in primary and chemoreduced retinoblastoma, respectively, whereas PD-L1 was expressed in 46/144 (31.94%) and 22/118 (18.64%) in cases of primary and chemoreduced retinoblastoma, respectively. Expression pattern of CTLA-4 protein was similar in both groups of retinoblastoma. On multivariate analysis, massive choroidal invasion, bilaterality and PD-L1 expression (p = 0.034) were found to be statistically significant factors in primary retinoblastoma, whereas PD-1 expression (p = 0.015) and foamy macrophages were significant factors in chemoreduced retinoblastoma. Overall survival was reduced in cases of PD-L1 (80.76%) expressed primary retinoblastoma, and PD-1 (63.28%) expressed chemoreduced retinoblastoma. CONCLUSIONS: This is the first of its kind study predicting a relevant role of the immune checkpoint markers in both groups of primary and chemoreduced retinoblastoma with prognostic significance. Differential expression of these markers in both group of retinoblastoma is a novel finding and might be an interesting and beneficial target for chemoresistant tumors.

Expression of BAP1 and ATM proteins: Association with AJCC tumor category in uveal melanoma.

BACKGROUND: Our aim is to detect the association of BAP1 with ATM protein with AJCC tumor category and its prognostic significance. METHODS: Based on AJCC tumor category, 69 patients samples were categorized into group A (LBD > 15 mm & tumor thickness ≥ 8 mm) and group B (LBD ≤ 15 mm & tumor thickness < 8 mm) subjected to immunohistochemistry to assess the nuclear expression of ATM and BAP1 proteins. Mutational analysis of BAP1 was performed on five samples from each group. RESULTS: Group A tumors showed insertion mutation of BAP1 gene while there was no mutation seen in group B tumor. At translational level loss of ATM and BAP1 was found in 65% and 66% of cases respectively. Loss of ATM with BAP1 was seen in 55% of cases which was more frequent in group A which was statically significant with metastasis (p = 0.006), advanced tumor staging (p = 0.021) and reduced metastasis-free survival (p = 0.048). On multivariate analysis loss of ATM along with BAP1 came out to be an independent prognostic marker (p = 0.035). CONCLUSION: Our data suggest that loss of BAP1 along with ATM might serve as a potential prognostic indicator in patients with an advanced AJCC tumor category, which leads to an increased risk of metastasis.

Clinicopathological relevance of NFκB1/p50 nuclear immunoreactivity and its relationship with the inflammatory environment of uveal melanoma.

PURPOSE: To analyze the activation of NFκB1/p50 in the inflammatory and non-inflammatory environment of uveal melanoma and its association with clinicopathological factors and patient outcome. METHODS: Activation of NFκB1/p50 was evaluated in 75 cases of uveal melanoma by immunohistochemistry. mRNA expression in 58 fresh UM specimen was measured by quantitative reverse-transcriptase PCR (qRT-PCR). Western blotting was performed to validate the immunohistochemistry results in representative cases. RESULTS: Forty-five cases showed both cytoplasmic and nuclear immunoreactivity of NFκB1/p50. Increased level of NFκB1/p50 activation was more frequent in the inflammatory environment group as compared to non-inflammatory environment group at both transcriptional and translational level. In multivariate analysis, infiltrating macrophages and nuclear immunoreactivity of NFκB1/p50 (p < .05) in tumor cells were found to be an independent prognostic factor for poor survival. CONCLUSION: Our results suggest that nuclear immunoreactivity NFκB1/p50 may serve as a useful marker in assessing the prognosis of uveal melanoma patients.

Prognostic relevance of ATM protein in uveal melanoma and its association with clinicopathological factors.

PURPOSE: Uveal melanoma (UM) is an intraocular malignancy commonly arising from choroid which can cause visual loss or metastasis. Ataxia-telangiectasia mutated (ATM) protein is an activator of DNA damage response and its role in uveal melanoma (UM) is still unexplored. Therefore, the study aims to detect the expression and localization of ATM protein and its association with clinicopathological parameters METHODS: Expression of nuclear ATM (nATM) was investigated on 69 formalin fixed paraffin embedded choroidal melanoma samples by immunohistochemistry and validated by western blotting. Results were then correlated with clinical and histopathological parameters. Prognostic significance was determined by the Kaplan-Meier analysis and the multivariate analysis by Cox's hazard proportional method. RESULTS: Loss of nATM was observed in 65% of cases, which was statistically significant with the reduced disease-free survival (p = 0.042). This loss was more frequently found in cases with high-risk histopathological factors like epithelioid cell type, tumor infiltrating lymphocytes and high pigmentation which might help in the progression of melanoma. On multivariate analysis, extraocular spread and loss of nATM were found to be independent prognostic factors (p < 0.05). CONCLUSION: Our data suggest that loss of nATM protein might serve as a poor prognostic marker in the pathogenesis of uveal melanoma which may lead to increased risk of metastasis.

A study of changes in levator muscle in congenital ptosis.

PURPOSE: To study microscopic and ultrastructural changes of levator palpebrae superioris (LPS) muscle in congenital ptosis. METHODS: In this prospective observational study, LPS muscle was studied in 77 eyelids with congenital ptosis; 35-simple congenital ptosis (SCP), 12-Marcus Gunn jaw winking phenomenon (MGJWP), and 30-blepharophimosis epicanthus inversus syndrome (BPES). Light microscopy, enzyme histochemistry, immunohistochemistry and electron microscopy were performed, and results were analyzed. RESULTS: Muscle fibers were detected in 83.33% of MGJWP, 22.86% of SCP and 16.67% of BPES eyelids. Fibers were detected significantly more in individuals with moderate ptosis, LPS action > 4 mm, present eyelid crease and eyelid fold. Severe endomysial and perimysial fibrosis was seen significantly more in individuals with MGJWP. Fat infiltration and nuclei internalization were seen in all three groups. The absence of degenerating or regenerating fibers and inflammatory cells, normal staining pattern on immunohistochemistry and absence of accumulation of any abnormal substance were found in all three groups. Abnormal mitochondrial staining pattern was seen occasionally in three groups. On electron microscopy, muscle was detected in 1 SCP eyelid and 8 MGJWP eyelids out of which 4 had myofibrillary disruption. All other eyelids where muscle fibers were not detected had only fibrocollagenous tissue. CONCLUSION: Fibrocollagenous tissue predominated in all the cases, and muscle fibers detected correlated inversely with the severity of ptosis. The absence of degenerating, regenerating fibers and inflammatory cells supported the theory of dysgenesis of muscle. However, internalization of nucleus seen in all the subtypes is a feature favoring dystrophy.

Vitreous Amyloidosis: Ocular, Systemic, and Genetic Insights.

PURPOSE: To report the unique clinical and surgical characteristics encountered in eyes with vitreous amyloidosis. Systemic evaluation and visual outcome after vitrectomy are discussed. A novel mutation in the transthyretin gene (TTR) in Indian patients with familial amyloid polyneuropathy (FAP) is described. DESIGN: Retrospective, observational study. PARTICIPANTS: Ten eyes of 5 patients from 2 pedigrees with a diagnosis of vitreous amyloidosis. METHODS: Detailed history, pedigree charting, systemic and ocular examination of 10 eyes (5 patients from 2 pedigrees) were carried out. Tests were performed to rule out vitreitis, retinal vasculitis, vitreous hemorrhage, and systemic amyloidosis. Genetic analysis to identify the mutation was performed in 1 patient. Vitreous biopsy, followed by 25-gauge pars plana vitrectomy, was performed in the same sitting in all cases. Samples were sent for Congo red staining and polarized microscopy. Patients were followed up on days 1, 7, and 28 and then every 2 months. Visual acuity assessment, intraocular pressure measurement, and fundus examination were performed each time. MAIN OUTCOME MEASURES: Mutations in TTR and postoperative visual acuity. RESULTS: Mean age at presentation was 32 years, with a 3:2 male-to-female distribution. Family history was positive in all patients. Nine eyes had pseudopodia lentis, whereas all 10 had glass wool-like vitreous. Glaucoma developed in 1 patient (2 eyes). Waxy paper-like vitreous with firm vitreous adhesions beyond major arcades and along retinal vessels was noted during surgery in all eyes. Congo red staining and apple green birefringence demonstrated vitreous amyloidosis. The mean preoperative best-corrected visual acuity (BCVA) was 1.39±0.64 logarithm of the minimum angle of resolution (logMAR), whereas the postoperative BCVA improved to 0.17±0.07 logMAR (P = 0.004). Gene sequencing revealed a phenylalanine→isoleucine mutation in the 33rd position of exon 2 of TTR in 1 patient of 1 pedigree, confirming the diagnosis of FAP. Two patients subsequently were found to have sensorimotor autonomic neuropathy, whereas 2 others had subclinical autonomic dysfunction. CONCLUSIONS: The clinical clues, management strategy, surgical characteristics, vitrectomy outcomes, and significance of systemic evaluation in vitreous amyloidosis are highlighted. A novel single mutation (Phe33Ile) in a case of FAP with vitreous amyloidosis from India is reported.

Invasive Ocular Surface Squamous Neoplasia Masquerading as Nodular Scleritis.

The authors report a rare case of ocular surface squamous neoplasia with intraocular involvement that had an initial masquerade presentation of recurrent anterior nodular scleritis. A 35-year-old male patient presented with right eye recurrent anterior nodular scleritis for which a lamellar patch graft was done. Two months later, the patient presented with recurrence of symptoms. Histopathology review revealed the presence of well-differentiated squamous cell malignancy. A high index of suspicion for malignancy is required in such cases when they do not respond to conventional therapy.

Ultrastructural changes of mitochondria in human retinoblastoma: correlation with tumor differentiation and invasiveness.

Retinoblastoma still represents a challenge for pediatric tumors. Mitochondria have been implicated in tumor progression, cell differentiation, and apoptotic pathways. Electron microscopy allows the study of mitochondrial morphology and it is still debated in human retinoblastoma. Demographic, clinical, and histopathological parameters were recorded in 17 enucleated retinoblastoma specimens. Hematoxylin and eosin staining was performed to study tumor characteristics and the extent of invasion in ocular structures. The aim of this study was to describe and analyze the mitochondrial morphology in human retinoblastoma by transmission electron microscopy (TEM). There was a male preponderance in our study. Ages ranged from 2 to 78 months. Histopathological analysis revealed that 15 (88.2 %) tumors were poorly differentiated retinoblastomas. Massive choroidal invasion was the most frequent histopathological high-risk factor among the others. Histopathological high-risk factors were found in 7/17 (41.1 %) cases. Tumor samples of all patients were examined by means of TEM. All cases showed tumor cells with high nucleocytoplasmic ratio. Poorly differentiated retinoblastoma cases showed fewer mitochondria, scant cytoplasm, disorganized organelles (mitochondria), and necrosis, whereas well-differentiated retinoblastomas had larger number of mitochondria and more organized organelles. However, there was no significant difference in mitochondrial changes between invasive and noninvasive tumors. Our study observed that cristolysis and swollen mitochondria were more frequent in retinoblastoma tumors. Understanding the structural and functional characteristics of mitochondria in retinoblastoma might be essential for the design of future therapeutic strategies. The authors have no proprietary or commercial interest in any materials discussed in this article.

Chlamydia and ocular adnexal lymphomas: An Indian experience.

CHLAMYDIA AND OCULAR ADNEXAL LYMPHOMAS: AN INDIAN EXPERIENCE: Ocular adnexal lymphomas (OALs) are a heterogeneous group of malignancies, majority being extranodal mucosa-associated lymphoid tissue (MALT) type. Different geographical regions have reported association of Chlamydia with OALs (MALT type). In India, role of Chlamydia in OALs remains unexplored. The aim of this study was to detect Chlamydia and to correlate with clinicopathological features of OALs in India. The clinicopathological features of 41 OAL cases were studied prospectively. Chlamydia DNA was detected by genus specific PCR amplifying major outer membrane protein (MOMP) gene followed by DNA sequencing. Chlamydia immunoexpression was evaluated by immunofluorescence and immunohistochemistry. The results were correlated with clinicopathological features including follow-up and survival. Chlamydia genome was detected in 3/41 (7.3%) OAL cases by PCR. Direct sequencing revealed C. trachomatis in 3 positive cases. Immunofluorescence and immunohistochemistry showed Chlamydia antigen in 5/41 and 1/41 cases respectively. Immunofluorescence demonstrated higher sensitivity than immunohistochemistry. A significant association was observed between Chlamydia positivity and orbital location (P=0.05). Follow-up revealed relapse in 2 Chlamydia positive cases (P=0.056). Our results demonstrate for the first time presence of C. trachomatis genome in 7.3% OAL cases in India. As no other reports are documented, more detailed studies from different regions within India are needed to explore status of Chlamydia in OALs.

Prognostic significance of NADPH oxidase-4 as an indicator of reactive oxygen species stress in human retinoblastoma.

BACKGROUND: Reactive oxygen species (ROS) have been shown to enhance the proliferation of cancer cells. NADPH oxidases (NOX4) are a major intracellular source of ROS and are found to be associated with cancer and tumor cell invasion. Therefore, the purpose of this study is to evaluate the expression of NOX4 protein in human retinoblastoma. METHODS: Immunohistochemical expression of NOX4 protein was analyzed in 109 specimens from prospective cases of retinoblastoma and then correlated with clinicopathological parameters and patient survival. Western blotting confirmed and validated the immunoreactivity of NOX4 protein. RESULTS: In our study we found a male preponderance (55.9 %), and 25/109 (22.9 %) were bilateral. Massive choroidal invasion was the histopathological high-risk factor (HRF) most frequently observed, in 42.2 % of the cases. NOX4 protein was expressed in 67.88 % (74/109) of primary retinoblastoma cases and was confirmed by Western blotting. NOX4 was statistically significant with massive choroidal invasion and pathological TNM staging. There was a statistically significant difference in overall survival in patients with NOX4 expression (p = 0.0461). CONCLUSION: This is the first study to show the expression of NOX4 protein in retinoblastoma tumors. Hence, a retinoblastoma tumor may exhibit greater ROS stress. This protein may prove to be useful as a future therapeutic target for improving the management of retinoblastoma.

Uveal melanoma.

Uveal melanoma is the most common primary intraocular malignancy in adults. It is associated with a high rate of distant tumor spread and consequent mortality. Unlike retinoblastoma, for which treatment advances over the last few decades have resulted in a dramatic improvement in survival, outcomes for patients with uveal melanoma remain unchanged. Despite improvement in local control of this tumor, roughly 50% of patients develop metastatic disease within 15 years. Delays in diagnosis and marked vascularity of this tumor may underlie that situation. Tumor size, location, histopathologic appearance, cytogenetic abnormalities, and molecular profiling are used in prognostication. The revised 7th edition of the American Joint Committee on Cancer (AJCC) manual has presented new information that may improve that process as well. Herein, we review current knowledge on uveal melanoma.

Orbital medulloepithelioma in an adult patient: Radiation-induced second neoplasia?

Second cancers in survivors of hereditary retinoblastoma occur much more commonly than in the general population. This can be attributed both to the germline mutation of the RB gene and chemoradiation used for treatment of this paediatric cancer. Medulloepithelioma is an uncommon tumor of neuroectodermal origin, seen largely in the paediatric population and rarely reported in adults. Though the incidence of second malignancies is common in retinoblastoma, medulloepithelioma as a second malignancy in retinoblastoma survivors is rare, with only one case reported so far. Herein, we present a case of a 29-year-old patient presenting with medulloepithelioma of the right orbit, arising in the radiation field of previously treated retinoblastoma. This case was also peculiar in that though the origin of tumor was in the eyeball it had a very aggressive clinical course.

Association of ZEB1 and TCF4 rs613872 changes with late onset Fuchs endothelial corneal dystrophy in patients from northern India.

PURPOSE: Fuchs endothelial corneal dystrophy (FECD) results in loss of vision associated with progressive corneal edema and loss of corneal transparency. The aim of the study was to evaluate changes in ZEB1, COL8A2, SLC4A11, and TCF4 rs613872 and correlate them with clinical findings. METHODS: Eighty-two patients with clinically diagnosed FECD and 143 controls were recruited during the period 2007-2012. Clinical details, pedigree information up to three generations, and 5 ml of blood samples were collected. Histopathological and transmission electron microscopy studies were performed on host corneal buttons from patients who underwent keratoplasty. Genomic DNA from blood was processed for PCR amplification followed by direct sequencing to screen genetic changes in the candidate genes. The pathogenic nature of the genetic variants was assessed using Sorting Intolerant From Tolerant (SIFT) and MutationTaster. RESULTS: The mean age at the onset of symptoms was 59.14±1.41years, the male to female ratio was 1:1.5, and the mean specular count (endothelial cell density) was 1629±93.62 cells/mm(2) with a mean central corneal thickness (CCT) of 617.30±15.73 µm. ZEB1 showed a novel variant IVS2+276 C/T in 14% of the cases, a novel nonsense p.Leu947stop mutation in one patient, two novel missense mutations (p.Glu733Lys, p.Ala818Val) in one patient each, and one novel synonymous variation (p.Ser234Ser) in two patients. Reported mutation p.Gln840Pro and five polymorphisms were also identified. The TCF4 single nucleotide polymorphism (SNP) rs613872 was significantly higher in patients with FECD. CONCLUSIONS: This is the first report of genetic variations in ZEB1 and TCF4 SNP rs613872 in patients with FECD from northern India that suggests a possible role in disease pathogenesis and the regulation of endothelial cell density.

Expression of pro-apoptotic Bax and anti-apoptotic Bcl-2 proteins in human retinoblastoma.

BACKGROUND: Regulation of apoptosis is a complex process that involves a number of genes, including Bcl-2, Bcl-x, Bax and other Bcl-2 family members. The aim of the present study is to assess the expression of Bcl- 2 and Bax in retinoblastoma, and correlate them with clinical and histopathological parameters. METHODS: The expression of Bcl-2 and Bax proteins were examined using immunohistochemistry, Western blotting and reverse transcriptase-polymerase chain reaction in a series of 60 prospective cases of primary retinoblastoma tissues. RESULTS: Immunohistochemistry showed expression of Bcl-2 in 40/60 (66.6%), whereas Bax expression was found only in 18/60 (30%) cases, and these correlated with mRNA expression. The Western blotting results also correlated well with the immunohistochemical expression of Bcl-2 (25 kDa) and Bax (21 kDa) proteins. Bcl-2 was expressed in 96% (24/25) of invasive tumours and in 45.7% (16/35) of non-invasive tumours. Expression of Bcl-2 significantly correlated with tumour invasiveness (P = 0.0274) and poor differentiation (P = 0.0163), whereas loss of Bax correlated with massive choroidal invasion and Pathological Tumor-Node-Metastasis (pTNM) (P = 0.0341). However, no correlation was found between Bax and Bcl-2 expression. CONCLUSIONS: Our findings suggest that these apoptotic regulatory proteins may serve as poor prognostic markers and can be used as a therapeutic target for the treatment of invasive retinoblastoma. Further functional studies are required to explore the role of Bax and Bcl-2 in retinoblastoma.

Prognostic significance of polo-like kinases in retinoblastoma: correlation with patient outcome, clinical and histopathological parameters.

BACKGROUND: Retinoblastoma is evolving, but it is still a therapeutic challenge for pediatric oncologists. Polo-like kinases (PLKs) plays an important role in cell cycle events. They play a crucial role in cell proliferation which may lead to tumour formation. The objective of this study is to investigate the role of PLK1 and PLK3 proteins in human retinoblastoma tissues. DESIGN: Non-randomized, prospective study was performed in the Dr R. P. Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi, India. PARTICIPANTS: This study included 74 primary enucleated retinoblastoma tissues. METHODS: Expression of PLK1 and PLK3 protein were assessed in primary enucleated retinoblastoma tissues by immunohistochemistry and western blotting. MAIN OUTCOME MEASURES: Expression of PLK1 and PLK3 protein were correlated with clinical and histopathological parameters, tumour staging and overall survival of patients. RESULTS: Immunohistochemical results revealed expression of PLK1 in 47/74 (63.51%) cases and PLK3 in 31/74 (41.89%) cases. Western blotting confirmed the immunoreactivity results. Expression of PLK1 showed correlation with poor differentiation and tumour invasion. In addition, PLK1 was statistically significant with massive choroidal invasion, whereas PLK3 did not correlate with any of the clinical or histopathological parameters. There was no statistical correlation in the overall survival of patients with PLK1 and PLK3 expression. CONCLUSIONS: PLK1 expression was associated with poor tumour differentiation and histopathological high-risk factors. These proteins may be involved in tumorigenesis and progression of disease. These results suggest that PLK1 may act as a potential therapeutic target and a promising marker for developing potent small molecule inhibitors of PLK isoforms in retinoblastoma.