Investigating druggable kinases for targeted therapy in retinoblastoma.

Retinoblastoma (RB) is a childhood retinal neoplasm and commonly treated with cytotoxic chemotherapeutic agents. However, these therapeutic approaches often lead to diverse adverse effects. A precise molecular therapy will alleviate these side effects and offer better treatment outcomes. Over the years, kinases have become potential drug targets in cancer therapy. Hence, we aimed to investigate genetic alterations of putative kinase drug targets in RB. Targeted exome sequencing was performed on 35 RB tumors with paired blood samples using a gene panel consisting of 29 FDA-approved kinase genes. Single nucleotide variants were analyzed for pathogenicity using an in-house pipeline and copy number variations (CNVs) were detected by a depth of coverage and CNVPanelizer. The correlation between genetic changes and clinicopathological features was assessed using GraphPad Prism. Three somatic mutations, two in ERBB4 and one in EGFR were identified. Two of these mutations (ERBB4 c.C3836A & EGFR c.A1196T) were not reported earlier. CNV analysis revealed recurrent gains of ALK, MAP2K2, SRC, STK11, and FGFR3 as well as frequent losses of ATM, PI3KCA and ERBB4. Notably, nonresponsive tumors had a higher incidence of amplifications in clinically actionable genes such as ALK. Moreover, ALK gain and ATM loss were strongly correlated with optic nerve head invasion. In conclusion, our study revealed genetic alterations of druggable kinases in RB, providing preliminary insights for the exploration of kinase-targeted therapy in RB.

A stepwise strategy for rapid and cost-effective RB1 screening in Indian retinoblastoma patients.

India has the highest number of retinoblastoma (RB) patients among the developing countries owing to its increasing population. Of the patients with RB, about 40% have the heritable form of the disease, making genetic analysis of the RB1 gene an integral part of disease management. However, given the large size of the RB1 gene with its widely dispersed exons and no reported hotspots, genetic testing can be cumbersome. To overcome this problem, we have developed a rapid screening strategy by prioritizing the order of exons to be analyzed, based on the frequency of nonsense mutations, deletions and duplications reported in the RB1-Leiden Open Variation Database and published literature on Indian patients. Using this strategy for genetic analysis, mutations were identified in 76% of patients in half the actual time and one third of the cost. This reduction in time and cost will allow for better risk prediction for siblings and offspring, thereby facilitating genetic counseling for families, especially in developing countries.

Targeted next generation sequencing of RB1 gene for the molecular diagnosis of Retinoblastoma.

BACKGROUND: The spectrum of RB1gene mutations in Retinoblastoma (RB) patients and the necessity of multiple traditional methods for complete variant analysis make the molecular diagnosis a cumbersome, labor-intensive and time-consuming process. Here, we have used targeted next generation sequencing (NGS) approach with in-house analysis pipeline to explore its potential for the molecular diagnosis of RB. METHODS: Thirty-three patients with RB and their family members were selected randomly. DNA from patient blood and/or tumor was used for RB1 gene targeted sequencing. The raw reads were obtained from Illumina Miseq. An in-house bioinformatics pipeline was developed to detect both single nucleotide variants (SNVs) and small insertions/deletions (InDels) and to distinguish between somatic and germline mutations. In addition, ExomeCNV and Cn. MOPS were used to detect copy number variations (CNVs). The pathogenic variants were identified with stringent criteria, and were further confirmed by conventional methods and cosegregation in families. RESULTS: Using our approach, an array of pathogenic variants including SNVs, InDels and CNVs were detected in 85% of patients. Among the variants detected, 63% were germline and 37% were somatic. Interestingly, nine novel pathogenic variants (33%) were also detected in our study. CONCLUSIONS: We demonstrated for the first time that targeted NGS is an efficient approach for the identification of wide spectrum of pathogenic variants in RB patients. This study is helpful for the molecular diagnosis of RB in a comprehensive and time-efficient manner.

Focused cancer pathway analysis revealed unique therapeutic targets in retinoblastoma.

Retinoblastoma (RB) is a pediatric cancer of the eye that occurs in 1/15000 live births worldwide. Albeit RB is initiated by the inactivation of RB1 gene, the disease progression relies largely on transcriptional alterations. Therefore, evaluating gene expression is vital to unveil the therapeutic targets in RB management. In this study, we employed an RT2 Profiler™ PCR array for a focused analysis of 84 cancer-specific genes in RB. An interaction network was built with gene expression data to identify the dysregulated pathways in RB. The key transcript alterations identified in 13 tumors by RT2 Profiler™ PCR array was further validated in 15 tumors by independent RT-qPCR. Out of 84 cancer-specific genes, 68 were dysregulated in RB tumors. Among the 68 genes, 23 were chosen for further analysis based on statistical significance and abundance across multiple tumors. Pathway analysis of altered genes showed the frequent perturbations of cell cycle, angiogenesis and apoptotic pathways in RB. Notably, upregulation of MCM2, MKI67, PGF, WEE1, CDC20 and downregulation of COX5A were found in all the tumors. Western blot confirmed the dysregulation of identified targets at protein levels as well. These alterations were more prominent in invasive RB, correlating with the disease pathogenesis. Our molecular analysis thus identified the potential therapeutic targets for improving retinoblastoma treatment. We also suggest that PCR array can be used as a tool for rapid and cost-effective gene expression analysis.

Towards the Identification and Characterization of Putative Adult Human Lens Epithelial Stem Cells.

The anterior lens epithelium has the ability to differentiate into lens fibres throughout its life. The present study aims to identify and functionally characterize the adult stem cells in the human lens epithelium. Whole mounts of lens epithelium from donor eyes (normal/cataract) were immunostained for SOX2, gap junction protein alpha 1 (GJA1), PAX6, α, ß and γ-crystallins, followed by a confocal analysis. The functional property of adult stem cells was analysed by their sphere forming ability using cultured lens epithelial cells from different zones. Based on marker expression, the lens epithelium was divided into four zones: the central zone, characterized by a small population of PAX6+, GJA1-, ß-crystallin- and γ-crystallin- cells; the germinative zone, characterized by PAX6+, GJA1+, ß-crystallin- and γ-crystallin-; the transitional zone, characterized by PAX6+, GJA1+, ß-crystallin+ and γ-crystallin-; and the equatorial zone, characterized by PAX6+/-, GJA1+, ß-crystallin+, and γ-crystallin+ cells. The putative lens epithelial stem cells identified as SOX2+ and GJA1 membrane expression negative cells were located only in the central zone (1.89 ± 0.84%). Compared to the other zones, a significant percentage of spheres were identified in the central zone (1.68 ± 1.04%), consistent with the location of the putative adult lens epithelial stem cells. In the cataractous lens, an absence of SOX2 expression and a significant reduction in sphere forming ability (0.33 ± 0.11%) were observed in the central zone. The above findings confirmed the presence of putative stem cells in the central zone of the adult human lens epithelium and indicated their probable association with cataract development.

Small RNA Sequencing Reveals a Distinct MicroRNA Signature between Glucocorticoid Responder and Glucocorticoid Non-Responder Primary Human Trabecular Meshwork Cells after Dexamethasone Treatment.

Glucocorticoids (GCs) are known to regulate several physiological processes and are the mainstay in the management of inflammatory eye diseases. The long-term use of GC causes raised intraocular pressure (IOP) or ocular hypertension (OHT) in about 30-50% of the susceptible individuals depending on the route of administration, and can lead to steroid-induced secondary glaucoma. The present study aims to understand the role of microRNAs (miRNAs) in differential glucocorticoid (GC) responsiveness in human trabecular meshwork (HTM) cells using small RNA sequencing. The human organ-cultured anterior segment (HOCAS) model was used to identify whether donor eyes were from GC-responders (GC-R; n = 4) or GC-non-responders (GC-NR; n = 4) following treatment with either 100 nM dexamethasone (DEX) or ethanol (ETH) for 7 days. The total RNA was extracted from cultured HTM cells with known GC responsiveness, and the differentially expressed miRNAs (DEMIRs) were compared among the following five groups: Group #1: ETH vs. DEX-treated GC-R; #2: ETH vs. DEX-treated GC-NR; #3: overlapping DEGs between Group #1 and #2; #4: Unique DEMIRs of GC-R; #5: Unique DEMIRs of GC-NR; and validated by RT-qPCR. There were 13 and 21 DEMIRs identified in Group #1 and Group #2, respectively. Seven miRNAs were common miRNAs dysregulated in both GC-R and GC-NR (Group #3). This analysis allowed the identification of DEMIRs that were unique to GC-R (6 miRNAs) and GC-NR (14 miRNAs) HTM cells, respectively. Ingenuity Pathway Analysis identified enriched pathways and biological processes associated with differential GC responsiveness in HTM cells. This is the first study to reveal a unique miRNA signature between GC-R and GC-NR HTM cells, which raises the possibility of developing new molecular targets for the management of steroid-OHT/glaucoma.

Cytoskeletal drugs prevent posterior capsular opacification in human lens capsule in vitro.

BACKGROUND: To determine whether the cytoskeletal drugs H-7 and Latrunculin B (LAT-B) inhibit posterior capsular opacification (PCO) in the cultured human lens capsular bag. METHODS: Following extracapsular cataract (lens) extraction in human donor eyes, the capsular bag was prepared and cultured by standard techniques. Forty-eight capsular bags were studied, of which 13 were treated with H-7 (50, 100 or 300 µM), 12 with 1% BSS (vehicle of H-7), 11 with LAT-B (2, 5 or 10 µM), and 12 with 0.25% DMSO (vehicle of LAT-B). Forty out of the 48 capsular bags were from paired eyes of 20 donors, with one bag being treated with H-7/LAT-B and the other with BSS/DMSO for each pair, including 20 for the H-7-BSS protocol and 20 for the LAT-B-DMSO protocol. The medium with the cytoskeletal drug/vehicle was replaced every 3-4 days for 4 weeks. PCO was assessed daily using inverted phase-contrast microscopy, and scored on a 4-point scale. RESULTS: In all cultures with BSS or DMSO, residual lens epithelial cells (LECs) on the anterior capsule migrated to and proliferated on the posterior capsule by 3-7 days, and apparent LEC growth on the posterior capsule with severe capsular wrinkling (PCO Grade 3) was seen by 2-3 weeks. When treated continuously with H-7 or LAT-B, the migration and proliferation of LECs and the capsular wrinkling were inhibited in a dose-dependent manner, with the inhibition being complete (PCO Grade 0) in the 300 µM H-7 (n = 8, p < 0.001) or 10 µM LAT-B culture (n = 3, p = 0.002). CONCLUSION: H-7 and LAT-B dose-dependently inhibited PCO formation in the cultured human lens capsular bags, suggesting that cytoskeletal drugs might prevent PCO formation after surgery in the human eye.

A Comparative Genome-Wide Transcriptome Analysis of Glucocorticoid Responder and Non-Responder Primary Human Trabecular Meshwork Cells.

Aim: To investigate genes and pathways involved in differential glucocorticoid (GC) responsiveness in human trabecular meshwork (HTM) cells using RNA sequencing. Methods: Using paired human donor eyes, human organ-cultured anterior segment (HOCAS) was established in one eye to characterize GC responsiveness based on intra ocular pressure (IOP) change and, in the other eye, primary HTM cell culture was established. For RNA sequencing, total RNA extracted from GC-responder (GC-R) and non-responder (GC-NR) cells after dexamethasone (DEX) or ethanol (ETH) treatment for 7d was used. Differentially expressed genes (DEGs) were compared among five groups and validated. Results: In total, 616 and 216 genes were identified as significantly dysregulated in Group #1 and #2 (#1: ETH vs. DEX-treated GC-R; #2: ETH vs. DEX-treated GC-NR), respectively. Around 80 genes were commonly dysregulated in Group #3 (overlapping DEGs between #1 and #2), whereas 536 and 136 genes were uniquely expressed in GC-R (#4) and GC-NR HTM (#5) cells, respectively. Pathway analysis revealed that WNT signaling, drug metabolism cytochrome p450, cell adhesion, TGF-ß signaling, and MAPK signaling were associated with GC responsiveness. Conclusion: This is the first study reporting distinct gene signatures and their associated pathways for GC-R and GC-NR HTM cells. WNT and MAPK signaling are potential therapeutic targets for the management of GC-induced glaucoma.

Short and long-term effect of dexamethasone on the transcriptome profile of primary human trabecular meshwork cells in vitro.

In the quest of identifying newer molecular targets for the management of glucocorticoid-induced ocular hypertension (GC-OHT) and glaucoma (GCG), several microarray studies have attempted to investigate the genome-wide transcriptome profiling of primary human trabecular meshwork (TM) cells in response to dexamethasone (DEX). However, no studies are reported so far to demonstrate the temporal changes in the expression of genes in the cultured human TM cells in response to DEX treatment. Therefore, in the present study, the time-dependent changes in the genome-wide expression of genes in primary human TM cells after short (16 hours: 16 h) and long exposure (7 days: 7 d) of DEX was investigated using RNA sequencing. There were 199 (118 up-regulated; 81 down-regulated) and 525 (119 up-regulated; 406 down-regulated) DEGs in 16 h and 7 d treatment groups respectively. The unique genes identified in 16 h and 7 d treatment groups were 152 and 478 respectively. This study found a distinct gene signature and pathways between two treatment regimes. Longer exposure of DEX treatment showed a dys-regulation of Wnt and Rap1 signaling and so highlighted potential therapeutic targets for pharmacological management of GC-OHT/glaucoma.

Emerging role of tumor microenvironment derived exosomes in therapeutic resistance and metastasis through epithelial-to-mesenchymal transition.

The tumor microenvironment (TME) constitutes multiple cell types including cancerous and non-cancerous cells. The intercellular communication between these cells through TME derived exosomes may either enhance or suppress the tumorigenic processes. The tumor-derived exosomes could convert an anti-tumor environment into a pro-tumor environment by inducing the differentiation of stromal cells into tumor-associated cells. The exosomes from tumor-associated stromal cells reciprocally trigger epithelial-to-mesenchymal transition (EMT) in tumor cells, which impose therapeutic resistance and metastasis. It is well known that these exosomes contain the signals of EMT, but how these signals execute chemoresistance and metastasis in tumors remains elusive. Understanding the significance and molecular signatures of exosomes transmitting EMT signals would aid in developing appropriate methods of inhibiting them. In this review, we focus on molecular signatures of exosomes that shuttle between cancer cells and their stromal populations in TME to explicate their impact on therapeutic resistance and metastasis through EMT. Especially Wnt signaling is found to be involved in multiple ways of exosomal transport and hence we decipher the biomolecules of Wnt signaling trafficked through exosomes and their potential in serving as therapeutic targets.

Human papillomavirus in retinoblastoma: A tertiary eye care center study from South India.

Purpose: This study is aimed to investigate the presence of Human papillomavirus (HPV) DNA in tumors obtained from sporadic retinoblastoma patients. Methods: One hundred six tumor tissues obtained from sporadic RB patients were analyzed for HPV infection by use of both seminested PCR and real-time quantitative PCR. Results: Of 106 RB patients, 55 were male and 51 were female. The mean age at diagnosis was 26.77 ± 15.36 (mean ± Std. dev) months. Almost all patients presented with leukocoria. Molecular investigation by different methods revealed no HPV positivity in any tumor genome. Conclusion: Our study demonstrates no association between HPV and RB, postulating HPV may not be a major risk factor in the etiology of RB.

Assessment of differential intraocular pressure response to dexamethasone treatment in perfusion cultured Indian cadaveric eyes.

The purpose of the present study was to assess the differential intraocular pressure response (IOP) to dexamethasone (DEX) treatment at two dose levels (100 or 500 nM) in perfusion cultured Indian cadaveric eyes to investigate glucocorticoid (GC) responsiveness. In a human organ-cultured anterior segment (HOCAS) set-up, the eye pressure was monitored for every 24 h post DEX infusion (100 or 500 nM) or 0.1% ethanol treatment for 7 days after baseline stabilization. The expression of DEX-inducible proteins such as myocilin and fibronectin in HOCAS-TM tissues was assessed by immunostaining. Elevated IOP was observed in 6/16 eyes [Mean ± SEM (mΔIOP): 15.50 ± 1.96 mmHg; 37.5% responders] and 3/15 eyes (Mean ± SEM mΔIOP: 10 ± 0.84 mmHg; 20% responders) in 100 nM and 500 nM dose groups respectively. Elevated IOP in GC responder eyes was substantiated with a significant increase in myocilin (11.8-fold; p = 0.0002) and fibronectin (eightfold; p = 0.04) expression as compared to vehicle-treated eyes by immunofluorescence analysis. This is the first study reporting the GC responsiveness in Indian cadaveric eyes. The observed GC response rate was comparable with the previous studies and hence, this model will enable us to investigate the relationship between differential gene expression and individual GC responsiveness in our population.

SB772077B (SB77) Alleviated the Aqueous Outflow Resistance Mediated by Cyclic Mechanical Stress in Perfused Human Cadaveric Eyes.

The intraocular pressure lowering property of a new rho kinase inhibitor, SB772077B (SB77) has been previously demonstrated in perfused human cadaveric eyes. In this study, the efficacy of SB77 in alleviating the aqueous outflow resistance mediated by cyclic mechanical stress in perfused human cadaveric eyes was investigated. A human anterior segment perfusion culture model was used to investigate the effect of cyclic intraocular pressure (IOP) on aqueous outflow facility in presence or absence of SB77. The status of RhoA activation and the downstream effector molecule myosin-light chain phosphorylation (p-MLC) was investigated by Western blot. Cyclic mechanical stress resulted in decrease in aqueous outflow facility (-19.79 ± 4.93%; p = 0.019) in perfused human eyes and treatment with SB77 (50 µM) significantly enhanced outflow facility by 15% (p = 0.05). The increase in outflow facility by SB77 was confirmed with the inactivation of RhoA/ROCK signaling and decreased expression of extracellular matrix markers. SB77 effectively reduced the outflow resistance mediated by cyclic IOP and thus may be a potential clinical candidate for the management of glaucoma.

Cancer Stem Cells with Overexpression of Neuronal Markers Enhance Chemoresistance and Invasion in Retinoblastoma.

BACKGROUND: Retinoblastoma is a sight and life-threatening embryonal tumor in children. Though chemotherapy is the main mode of therapy, evolving resistance remains a major obstacle in treatment success. The presence of cancer stem cells (CSC) is frequently reported to be responsible for chemoresistance in multiple tumors. OBJECTIVE: Our study aims to identify the molecular factors that facilitate the chemoresistance through cancer stem cells in retinoblastoma. METHODS: We developed etoposide and carboplatin resistant retinoblastoma (Y79) cell lines by stepwise drug increment treatment, validated with MTT and TUNEL assays. Colony forming and invasive ability were studied by soft-agar colony forming and transwell assays, respectively. Similar analysis in non-responsive retinoblastoma tumors were carried out by histopathology. Finally, expression of CSC/neuronal markers and ABC transporters were examined by quantitative PCR and protein expression of neuronal stem cell markers was confirmed by Western blot. RESULTS: Larger colony size of resistant cells in soft-agar assay provided evidence for increased selfrenewability. Histopathology in non-responsive tumors showed poorly differentiated cells predominantly. Besides, both resistant cell lines and non-responsive tumors showed increased invasion with higher expression of neuronal stem cell markers - SOX2, NANOG, OCT4 and ABC transporters - ABCB1 and ABCC3. Increased self-renewal ability and invasion along with overexpression of stemness markers in resistant cells and tumors provide evidence for stemness driving chemoresistance and invasion in retinoblastoma. CONCLUSION: We have demonstrated Neuronal stem cell/CSC markers that facilitate the maintenance of cancer stem cells. Developing therapies targeting these factors will help in overcoming resistance and improving retinoblastoma treatment.

Smartphone-based application improves the detection of retinoblastoma.

PURPOSE: To improve and validate the smartphone-based leukocoria detection application so that non-ophthalmologists could make use of the smartphone for early detection of Retinoblastoma (RB) in young children without anesthesia and pharmacological dilatation of the pupil. METHODS: Two apps, MDEyeCare and CRADLE, developed for red reflex based leukocoria detection were used in iPhone 6s. MDEyeCare methodology was modified with respect to ambient lighting, the distance between camera and eye and different gazes for better performance. We analyzed totally 34 eyes of 23 RB patients and four normal children. Each of the RB patients was confirmed with clinical examination and radiological investigations. RESULTS: Modification in the methodology of MDEyeCare app could detect the leukocoria in early stages of RB (50% of Group B, 83% of Group C). In late stages (Group D and E), 100% of tumors were detected. The CRADLE app failed to provide adequate leukocoria detection except four late stage RB eyes. Among the 14 normal eyes (6 from unilateral RB and eight from normal children), pseudo-leukocoria was observed in three eyes only at lateral gaze even with MDEyeCare app. CONCLUSION: Improved methodology in smartphone-based app enhanced the detection of RB and this may translate into better outcome after treatment with respect to vision salvage.

Retinoblastoma discordance in families with twins.

Retinoblastoma has an increased inheritance risk of germline RB1 mutations in offspring and siblings, especially twins. Three families, each having one retinoblastoma-affected twin, were selected for genetic analysis and DNA profiling. Germline RB1 mutations were found in all probands. DNA profiling carried on similar-looking twins of families I and II, proved them to be fraternal. This study demonstrates the importance of genetic analysis of RB1 gene for risk prediction in retinoblastoma families. It also emphasizes that DNA profiling is a mandate for genetic screening of families with twins, thus adding a new dimension in counseling of retinoblastoma.

A method to isolate human limbal basal cells enriched for a subset of epithelial cells with a large nucleus/cytoplasm ratio expressing high levels of p63.

The objectives were to develop method of isolating viable human limbal basal cells in order to enrich a subset of small cells with a large Nucleus/Cytoplasm (N/C) ratio expressing high levels of p63, nuclear protein. Limbal tissues were treated with trypsin for 50 min at 37 degrees C in an orbital shaker at 100 rpm with epithelial side down followed by additional 5 min with epithelial side up and then with Dispase II to obtain various epithelial fractions. Isolated cell fractions were assessed for colony forming efficiency and DeltaNp63alpha, connexin (Cx43) mRNA levels. Cytospin smears were double-immunostained for p63 and any one of the stem cell (SC) related markers and analyzed using a laser scanning confocal microscope and advanced image analysis software (Leica Confocal software, 2.61 build 1537 version) for quantification of fluorescence intensity. The isolated limbal basal cells were highly positive for DeltaNp63alpha mRNA but expressing low Cx43 mRNA. They gave rise to higher number of large colonies with compact morphology in contrast to the limbal suprabasal/superficial (LS/S) colonies. Furthermore, a subset with a large N/C ratio expressing high levels of p63 was observed, as much as 25% among the limbal basal cell fraction, in contrast to only about 4% in the total limbal epithelial cells. Such cells were positive for K5 and negative for Ki67, Cx43, and 14-3-3s and were absent in the LS/S fraction. These results collectively substantiate our method of isolation of limbal basal layer cells containing an enriched population of cells with SC phenotype.

SB772077B, A New Rho Kinase Inhibitor Enhances Aqueous Humour Outflow Facility in Human Eyes.

We investigated the effect of a new Rho kinase inhibitor, SB772077B (SB77) on aqueous outflow facility (OF) in human eyes using human organ-cultured anterior segment (HOCAS). IOP was monitored for 24 h post-treatment with either SB77 (0.1/10/50 µM) or vehicle after a stable baseline pressure. The hydrodynamic pattern of aqueous outflow was analysed by labelling outflow pathway with red fluorescent microspheres. The effect of SB77 on cell morphology, actin stress fibers, focal adhesions, ECM, status of RhoA activation and myosin light chain phosphorylation (p-MLC) were evaluated and compared with Y27632, by immunostaining using primary human trabecular meshwork (HTM) cells. Following 24 h treatment, SB77 increased OF by 16% at 0.1 µM (N = 6), 29% at 10 µM (N = 8; p = 0.018) and 39% at 50 µM (N = 8; p = 0.004) in human eyes. There was an overall increase in tracer quantity and in area along inner wall of Schlemm's canal. Treatment with SB77 showed no evidence of cytotoxicity and caused a significant reduction in the expression of fibrotic markers compared to Y27632. The present findings indicate that SB77 treatment was effective in enhancing OF and reducing fibrotic markers in an ex vivo model. Thus SB77 may be a potential clinical candidate for the management of glaucoma.