Management of Extraocular Retinoblastoma: ICMR Consensus Guidelines.

Retinoblastoma (RB) is the most common intraocular malignancy of childhood. Advanced stage presentation of RB is common in low middle-income countries (LMICs) due to lack of awareness, social taboos associated with enucleation, seeking alternative conservative treatment options, and poor accessibility to health care. Over the last few decades, there have been significant advancements in the management of extraocular RB (EORB) which have improved outcomes and helped in minimizing treatment-related toxicities. The incorporation of multimodality approaches including chemotherapy, surgery, and radiotherapy (RT) has shown promising results; however, prognosis remains poor especially in LMICs. In this article, authors have discussed the ICMR consensus guidelines on the management of EORB, including metastatic RB.

Management of Intraocular Retinoblastoma: ICMR Consensus Guidelines.

Retinoblastoma (RB) is the most common childhood intraocular malignancy. Delayed presentation due to a lack of awareness and advanced intraocular tumors are a common scenario in low-middle income countries (LMICs). Remarkable treatment advances have been made in the past few decades allowing globe salvage in advanced intraocular RB (IORB) including systemic chemotherapy with focal consolidation and targeted treatments like intraarterial chemotherapy and intravitreal chemotherapy. However, a lack of availability and affordability limits the use of such advances in LMICs. External beam radiotherapy, despite risk of second cancers in RB with germline mutations, still remains useful for recalcitrant RB not responding to any other treatment. When choosing conservative treatment for advanced IORB, the cost and long duration of treatment, morbidity from multiple evaluation under anesthesias (EUAs), side effects of treatment and risk of treatment failure need to be taken into account and discussed with the parents. In this article, the authors discuss the ICMR consensus guidelines on the management of IORB.

Epidemiology, Diagnosis and Genetics of Retinoblastoma: ICMR Consensus Guidelines.

Retinoblastoma (RB) is the most common intraocular tumor in childhood. It is mainly caused by mutations in both alleles of the RB1 tumor suppressor gene that is found on chromosome 13 and regulates the cell cycle. Approximately 8000 children are diagnosed with RB globally each year, with an estimated 1500 cases occurring in India. The survival rate of RB has improved to more than 90% in the developed world. Leukocoria and proptosis are the most common presenting features of RB in Asian Indian populations. Most cases of RB are diagnosed by fundus examination followed by ultrasound. The International Classification of Retinoblastoma is the most used scheme for the staging and classification of intraocular RB in India. Prenatal testing and preimplantation genetic testing for RB may be beneficial in high-risk families. Histopathologic risk factors such as massive choroidal invasion and post-laminar optic nerve help in predicting the occurrence of metastasis in children with RB, while presence of microscopic residual disease requires aggressive adjuvant treatment in eyes enucleated for group E RB. The review provides a consensus document on diagnosis and genetics of RB in India.

Prognostic significance of melanogenesis pathway and its association with the ultrastructural characterisation of melanosomes in uveal melanoma.

BACKGROUND: Pigmentation could be a relevant prognostic factor in uveal melanoma (UM) development. Microphthalmia-associated transcription factor (MITF) regulates melanin synthesis by activating tyrosinase-related protein 2 (TYRP2) and silver protein (SILV) that induce the melanogenesis pathway. Although their oncogenic potential has been observed in various malignancies but has not been investigated in UM Asian population. Our aim is to study the ultrastructure of melanosomes and the prognostic significance of pigmentation markers such as TYRP2, MITF and SILV in UM. METHODS: Transmission electron microscopy was performed to compare the ultrastructure of melanosomes in the normal choroid and UM cases. Immunoexpression of TYRP2, SILV and MITF was analysed in 82 UM samples. The mRNA expression level of all genes was measured in 70 UM cases. A statistical correlation was performed to determine the prognostic significance of all markers. RESULTS: Premelanosomes and mature melanosomes undergoing dedifferentiation were observed in high-pigmented UM cases as compared with low-pigmented UM cases. Seventy per cent of UM cases showed high SILV expression while TYRP2 and MITF expression was present in 58% and 56% of cases, respectively. At the mRNA level, upregulation of TYRP2, SILV and MITF markers was seen in around 50% of UM cases, which was statistically significant with high pigmentation. Reduced metastatic-free survival was statistically significant with the MITF protein expression. CONCLUSION: Our results demonstrated that ultrastructural changes in melanosomes and high expression of TYRP2, MITF and SILV could dysregulate the melanogenesis pathway and might be responsible for the aggressive behaviour of UM.

Comparative evaluation of isoflurane and desflurane for postoperative cognitive decline in elderly patients: A prospective observational pilot study.

BACKGROUND: Approximately 16%-62% of patients undergoing non cardiac surgeries develop postoperative cognitive dysfunction. We compared the incidence of postoperative cognitive dysfunction in older adults aged 60-80 years who underwent open abdominal surgeries under general anaesthesia using isoflurane or desflurane and correlated it with the serum concentration of interleukin 1, interleukin 6, tumour necrosis factor alpha, amyloid ß and S100 on postoperative day 3. METHOD: Forty American Association of Anesthesiologists Physical Classification I or II patients were included after acquiring institutional ethics committee approval, registering in the Clinical Trials Registry - India, and informed written consent. They underwent open abdominal surgery under general anaesthesia and epidurals between 2017 and 2019. Patients with substance abuse or any disorder affecting cognition were excluded. Postoperative cognitive dysfunction was assessed by Stroop test, Wisconsin Card Sorting Test, Trail making test - B, Porteus Maze test, PGI memory scale, mini-mental state examination, and Bender Gestalt test the day before surgery and on the third postoperative day along with blood samples. RESULTS: Thirty-seven percent of the patients developed postoperative cognitive dysfunction. The risk was similar to isoflurane in comparison with desflurane (risk ratio: 0.65, 95% confidence interval: 0.30, 1.40). A significant percentage increase in reaction time for Porteus Maze test and Trail making test - B was noted with isoflurane (6.69 (4.20-8.94) and 8.01 (2.08-12.5), respectively) in comparison with desflurane group (13.01 (9.09-17.33), p = 0.003 and 11.62 (7.5-17.5), p = 0.017, respectively). CONCLUSION: Isoflurane and desflurane had a similar impact on the elderly for developing postoperative cognitive dysfunction and no correlation with any of the biomarkers used in the study on postoperative day 3.

Using Human 'Personalized' Cybrids to Identify Drugs/Agents That Can Regulate Chronic Lymphoblastic Leukemia Mitochondrial Dysfunction.

This study uses personalized chronic lymphoblastic leukemia (CLL) cybrid cells to test various drugs/agents designed to improve mitochondrial function and cell longevity. Age-matched control (NL) and CLL cybrids were created. The NL and CLL cybrids were treated with ibrutinib (Ibr-10 µM), mitochondrial-targeted nutraceuticals such as alpha lipoic acid (ALA-1 mM), amla (Aml-300 µg), melatonin (Mel-1 mM), resveratrol (Res-100 µM) alone, or a combination of ibrutinib with nutraceuticals (Ibr + ALA, Ibr + Aml, Ibr + Mel, or Ibr + Res) for 48 h. MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazoliumbromide), H2DCFDA(2',7' Dichlorodihydrofluorescein diacetate), and JC1 assays were used to measure the cellular metabolism, intracellular ROS levels, and mitochondrial membrane potential (∆ψm), respectively. The expression levels of genes associated with antioxidant enzymes (SOD2, GPX3, and NOX4), apoptosis (BAX and CASP3), and inflammation (IL6, IL-1ß, TNFα, and TGFß) were measured using quantitative real-time PCR (qRT-PCR). CLL cybrids treated with Ibr + ALA, Ibr + Aml, Ibr + Mel, and Ibr + Res had (a) reduced cell survivability, (b) increased ROS production, (c) increased ∆ψm levels, (d) decreased antioxidant gene expression levels, and (e) increased apoptotic and inflammatory genes in CLL cybrids when compared with ibrutinib-alone-treated CLL cybrids. Our findings show that the addition of nutraceuticals makes the CLL cybrids more pro-apoptotic with decreased cell survival compared with CLL cybrids exposed to ibrutinib alone.

Implications of LAG3 and CTLA4 immune checkpoints beyond PD-1/PD-L1 as a potential target in determining the prognosis of uveal melanoma patients.

BackgroundResponse rate of PD-1/PD-L1 immunotherapeutic blockade agents in uveal melanoma (UM) is poor. Lymphocyte activation gene 3 (LAG3) and cytotoxic T-lymphocyte-associated protein 4 (CTLA4) are the two promising immune checkpoint targets. Therefore, our aim was to explore at how these proteins were expressed in tumour tissue and serum, as well as their prognostic implications in UM. METHODS: The expression of LAG3, CTLA-4, CD3, CD4, CD8 and FOXP3 was determined by immunohistochemistry in 54 enucleated UM tissue samples. mRNA expression level of LAG3 and CTLA-4 was determined by quantitative real-time PCR and corroborated by western blotting. Furthermore, soluble form of LAG3, CTLA-4 and CCR8 expression in serum was measured in 40 UM patients using ELISA. RESULT: The expression of LAG3, CTLA-4, CD3, CD4, CD8 and FOXP3 was observed in 30%, 33%, 41%, 35%, 50% and 39% of the cases, respectively. Loss of nBAP1 expression was significantly correlated with CD8+expression (p=0.012) but not with tumour infiltrating lymphocytes. LAG3 and CTLA-4 mRNA levels were higher in UM compared with normal uveal tissues. Higher LAG3 expression with CD8+expression was associated with lower metastasis-free survival (MFS) (p=0.049), but not with CTLA-4 in UM patients. MFS rate was reduced in patients having lower levels of CCR8 protein (p=0.050) and increased level of LAG3 protein (p=0.001). CONCLUSION: Our findings suggest that higher levels of LAG3 in UM with histopathologically high-risk parameters predict high metastatic potential and that it could be used as a targeted immunotherapy alone or in combination with PD-1/PD-L1 blockade agents.

Diagnostic utility of immunohistochemistry in concordance with mRNA analysis of PRAME in the stratification of high-risk uveal melanoma patients.

Existing clinical indicators for metastatic risk classification and patient treatment of uveal melanoma (UM) in the Asian population are limited. Preferentially expressed antigen in melanoma (PRAME) has gained attention in the prognosis of cancers and considered as a potential biomarker in many tumors including UM. Therefore, this study investigated the expression of PRAME and its association with loss of nuclear BAP1 (nBAP1) as well as its correlation with clinicopathological parameters and patient outcome. Immunohistochemical expression of PRAME and BAP1 proteins were assessed in 66 prospective cases of UM. mRNA expression level was measured by quantitative real-time PCR. Kaplan-Meier curves and Cox proportional hazard models were used to analyze the correlation of protein expression with clinicopathological parameters, metastasis-free survival and overall survival. Nuclear PRAME (nPRAME) expression and loss of nBAP1 were observed in 24 and 62% cases, respectively. PRAME mRNA expression level was found to be upregulated in 64% (7/11) of metastatic patients. mRNA and immunoexpression of nPRAME were statistically significant with many clinicopathological high-risk factors. On univariate and multivariate analyses, high mitotic activity, extraocular invasion and presence of nPRAME expression were statistically significant (p < 0.05). On Kaplan-Meier survival analysis, patients expressing PRAME had significantly reduced metastasis-free survival (MFS) and overall survival (OS). MFS and OS were also reduced in patients expressing PRAME along with loss of nBAP1. Our data show that nPRAME expression, in combination with loss of nBAP1, could be a useful predictive biomarker in the therapeutic management of UM patients at high risk.

Altered Retrograde Signaling Patterns in Breast Cancer Cells Cybrids with H and J Mitochondrial DNA Haplogroups.

The aim of this study was to determine the role of retrograde signaling (mitochondria to nucleus) in MCF7 breast cancer cells. Therefore, in the present study, MCF7-H and MCF7-J cybrids were produced using the mitochondria from the same H and J individuals that were already used in our non-diseased retinal pigment epithelium (ARPE19) cybrids. MCF7 cybrids were treated with cisplatin and analyzed for cell viability, mitochondrial membrane potential, ROS, and expression levels of genes associated with the cGAS-STING and cancer-related pathways. Results showed that unlike the ARPE19-H and ARPE19-J cybrids, the untreated MCF7-H and MCF7-J cybrids had similar levels of ATP, lactate, and OCR: ECAR ratios. After cisplatin treatment, MCF7-H and MCF7-J cybrids showed similar (a) decreases in cell viability and ROS levels; (b) upregulation of ABCC1, BRCA1 and CDKN1A/P21; and (c) downregulation of EGFR. Cisplatin-treated ARPE19-H and ARPE19-J cybrids showed increased expression of six cGAS-STING pathway genes, while two were increased for MCF7-J cybrids. In summary, the ARPE19-H and ARPE19-J cybrids behave differentially from each other with or without cisplatin. In contrast, the MCF7-H and MCF7-J cybrids had identical metabolic/bioenergetic profiles and cisplatin responses. Our findings suggest that cancer cell nuclei might have a diminished ability to respond to the modulating signaling of the mtDNA that occurs via the cGAS-STING pathway.

Impacts of Bacteriostatic and Bactericidal Antibiotics on the Mitochondria of the Age-Related Macular Degeneration Cybrid Cell Lines.

We assessed the potential negative effects of bacteriostatic and bactericidal antibiotics on the AMD cybrid cell lines (K, U and J haplogroups). AMD cybrid cells were created and cultured in 96-well plates and treated with tetracycline (TETRA) and ciprofloxacin (CPFX) for 24 h. Reactive oxygen species (ROS) levels, mitochondrial membrane potential (ΔψM), cellular metabolism and ratio of apoptotic cells were measured using H2DCFDA, JC1, MTT and flow cytometry assays, respectively. Expression of genes of antioxidant enzymes, and pro-inflammatory and pro-apoptotic pathways were evaluated by quantitative real-time PCR (qRT-PCR). Higher ROS levels were found in U haplogroup cybrids when treated with CPFX 60 µg/mL concentrations, lower ΔψM of all haplogroups by CPFX 120 µg/mL, diminished cellular metabolism in all cybrids with CPFX 120 µg/mL, and higher ratio of dead cells in K and J cybrids. CPFX 120 µg/mL induced overexpression of IL-33, CASP-3 and CASP-9 in all cybrids, upregulation of TGF-ß1 and SOD2 in U and J cybrids, respectively, along with decreased expression of IL-6 in J cybrids. TETRA 120 µg/mL induced decreased ROS levels in U and J cybrids, increased cellular metabolism of treated U cybrids, higher ratio of dead cells in K and J cybrids and declined ΔψM via all TETRA concentrations in all haplogroups. TETRA 120 µg/mL caused upregulation of IL-6 and CASP-3 genes in all cybrids, higher CASP-7 gene expression in K and U cybrids and downregulation of the SOD3 gene in K and U cybrids. Clinically relevant dosages of ciprofloxacin and tetracycline have potential adverse impacts on AMD cybrids possessing K, J and U mtDNA haplogroups in vitro.

Mitochondrial DNA polymorphisms and biogenesis genes in primary and metastatic uveal melanoma cell lines.

PURPOSE: This study was designed to identify mitochondrial (mt) DNA variations in primary and metastatic uveal melanoma (UM) cell lines and their relation with cell metabolism to gain insight into metastatic progression. METHOD: The entire mtDNA genomes were sequenced using Sanger sequencing from two primary UM cell lines (92.1 and MEL270) and two cell lines (OMM2.3 and OMM2.5) derived from liver metastases of the MEL270 patient. The mtDNA copy numbers determined by the ratio of nDNA versus mtDNA. qRT-PCR was used to evaluate expression levels of mitochondrial biogenesis genes. RESULTS: Sequencing showed that cell line MEL270 and metastases-derived OMM2.3 and OMM2.5 cell lines had homoplasmic single nucleotide polymorphisms (SNPs) representing J1c7a haplogroup, whereas 92.1 cells had mtDNA H31a haplogroup. mtDNA copy numbers were significantly higher in primary cell lines. The metastatic UM cells showed down-regulation of POLG, TFAM, NRF-1 and SIRT1 compared to their primary MEL270 cells. PGC-1α was downregulated in 92.1 and upregulated in MEL270, OMM2.3 and OMM2.5. CONCLUSIONS: Our finding suggests that within metastatic cells, the heteroplasmic SNPs, copy numbers and mitochondrial biogenesis genes are modulated differentially compared to their primary UM cells. Therefore, investigating pathogenic mtDNA variants associated with cancer metabolic susceptibility may provide future therapeutic strategies in metastatic UM.

Combined association of massive choroidal and optic nerve invasion as a prognostic relevance in primary retinoblastoma: A 10-year study.

PURPOSE: To determine the significance of both massive choroidal invasion and optic nerve invasion (retrolaminar [(RL]+cut end [CE]) as a criterion for classifying high metastatic potential retinoblastoma and their relationship with other known histopathological high-risk features. METHODS: A retrospective review of 650 eyes diagnosed as retinoblastoma over a 10-year period. In our study, there is male predominance and a higher percentage of the poorly differentiated tumors. The age of most of the patients ranges from 1 month to 8 years with a median age of 2 years. RESULTS: There were 24% of eyes with massive choroidal invasion and 18% of eyes with optic nerve invasion up to the cut end. On performing Cox-proportional hazard analysis, it was found that massive choroidal invasion in association with optic nerve invasion up to the cut end was an independent prognostic parameter. On Kaplan-Meier analysis, overall survival had reduced in patients having both massive choroidal invasion and an optic nerve cut end invasion along with orbital invasion (P < .05). CONCLUSION: The presence of massive choroidal invasion in association with optic nerve cut end invasion (RL+CE) could be used as a better prognostic predictor in assessing retinoblastoma patients with high metastatic potential and need to be kept for longer follow up.

Prognostic significance of immune checkpoints in the tumour-stromal microenvironment of sebaceous gland carcinoma.

BACKGROUND: Immune checkpoint blockade strategies have gained attention in the treatment/prognosis of cancers by targeting the programmed death-1 (PD-1)/programmed death-ligand 1 (PD-L1) pathway alone or in combination with cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) blockade and are currently in clinical trials. The present study investigated the expression of the PD-1, PD-L1, CTLA-4, CD4 and CD8 proteins and their prognostic value in the tumour microenvironment of sebaceous gland carcinoma (SGC). METHODS: The expression levels of PD-1, PD-L1, CTLA-4, CD4 and CD8 proteins were assessed in 52 cases of SGC by immunohistochemistry and validated by western blotting. mRNA expression was measured by quantitative real-time PCR. Kaplan-Meier curves and Cox proportional hazard models were used to analyse the correlation of protein expression with clinicopathological parameters and disease-free survival. RESULTS: The expression of PD-L1 was found to be higher in tumour cells than in stromal cells. In univariate analysis, the expression of PD-1 in tumour-infiltrating lymphocytes (tPD-1) and PD-L1 in tumour cells was associated with reduced disease-free survival, whereas PD-L1 expression in stromal lymphocyte infiltration (sPD-L1) was associated with the increased survival of patients (p<0.05). However, by multivariate analysis, the expression of tPD-1 was found to be an independent prognostic factor for poor survival. CONCLUSION: Our study highlights the prognostic outcome of PD-1 and PD-L1 protein expression in cells of tumour-stromal compartments. These results indicate that the PD-1/PD-L1 pathway mediates important interactions within the tumour microenvironment in SGC.

Notes from the Field: Dynamic Triaging Using Quality Improvement (QI) Methodology to Prevent the Admission of Asymptomatic COVID Positive Obstetric Patients in New Delhi, India.

A single undiagnosed COVID-19 positive patient admitted in the green zone has the potential to infect many Health Care Workers (HCWs) and other patients at any given time with resultant spread of infection and reduction in the available workforce. Despite the existing triaging strategy at the Obstetric unit of a tertiary hospital in New Delhi, where all COVID-19 suspects obstetric patients were tested and admitted in orange zone and non-suspects in green zone, asymptomatic COVID-19 positive patients were found admitted in the green zone. This was the trigger to undertake a quality improvement (QI) initiative to prevent the admission of asymptomatic COVID-19 positive patients in green zones. The QI project aimed at reducing the admission of COVID-19 positive patients in the green zone of the unit from 20% to 10% in 4 weeks' time starting 13/6/2020 by means of dynamic triaging. A COVID-19 action team was made and after an initial analysis of the problem multiple Plan-Do-Study-Act (PDSA) cycles were run to test the change ideas. The main change ideas were revised testing strategies and creating gray Zones for patients awaiting COVID-19 test results. The admission of unsuspected COVID-19 positive cases in the green zone of the unit reduced from 20% to 0% during the stipulated period. There was a significant reduction in the number of HCWs, posted in the green zone, being quarantined or test positive for COVID-19 infection as well. The authors conclude that Quality Improvement methods have the potential to develop effective strategies to prevent spread of the deadly Corona virus.

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.

Prognostic impact of HERC2 protein and pink-eyed dilution protein in uveal melanoma.

Biological understanding of pigmentation and its association with clinicopathological implications in uveal melanoma (UM) risk is still unexplored. HECT and RLD Domain Containing E3 Ubiquitin Protein Ligase 2 (HERC2) and Pink-eyed dilution protein (P-protein) are the important markers that regulate pigmentation in eye. Therefore, our aim of the study was to investigate the expression of HERC2 and P-protein in the UM patients and correlate with patient outcome. Fifty-two formalin-fixed paraffin-embedded UM tissue samples were included to detect the expression of HERC2 and P-protein by immunohistochemistry and validated by western blot. Cox proportional hazard model and log-rank test were used to determine the prognostic potential of these proteins. High pigmentation was seen in 67% of the UM cases. The expression of HERC2 and P-protein was present in 44% and 71% cases, respectively. On statistical analysis, increased pigmentation, epithelioid cell type, and ciliary body invasion were significant with the protein expressions (p < 0.05). Metastasis-free survival was reduced in UM cases which expressed HERC2 and P-protein. On multivariate analysis, P-protein expression was found to be an independent prognostic factor. Our findings suggest that HERC2 and P-protein could be used as novel predictors of high pigmentation in UM cases which have high metastatic potential.

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.

Differential expression of p52 and RelB proteins in the metastatic and non-metastatic groups of uveal melanoma with patient outcome.

PURPOSE: Non-canonical NFκB (NC-NFκB) pathway plays an influential role in metastasis, which promotes cancer proliferation and progression. The aim of the study was to examine the expression of NC-NFκB proteins and their correlation with clinicopathological factors associated with metastatic cases of uveal melanoma (UM) and with the patient outcome. METHOD: Expression of NC-NFκB proteins (p52, RelB, and co-expression of p52/RelB) was evaluated in 75 formalin-fixed cases of uveal melanoma by immunohistochemistry. Validation of nuclear immunoreactivity was done by western blotting. Transcriptional status of NC-NFκB genes was assessed in 60 fresh tumor tissues by quantitative real-time PCR. Co-immunoprecipitation was performed to determine the presence of native p52/RelB heterodimer in UM. Prognostic relevance was determined using Cox proportional hazard and Kaplan-Meier methods. RESULTS: Immunohistochemical expression of p52, RelB, and their co-expression was observed in 81%, 68.7%, 56.2% of metastatic cases, respectively, while their expression was seen only in 38%, 33% and 30% of non-metastatic cases. Loss of BAP-1 was correlated with expression of p52 and RelB proteins. Co-immunoprecipitation assay confirmed the putative interaction of p52 with RelB protein in metastatic cases of uveal melanoma. Co-expression of p52/RelB and expression of p52 protein was significantly correlated with decreased metastasis-free survival (MFS) (p = 0.004; p = 0.002) and overall survival (OS) (p = 0.004; p = 0.032), while the RelB expression only correlated with reduced MFS (p = 0.003). CONCLUSION: Our data showed that non-canonical NFκB proteins were significantly higher in metastatic cases and associated with poor outcome of the patients. Furthermore, the p52 protein could be used as a potential therapeutic biomarker for metastatic cases in uveal melanoma.

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.