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.

Effects of fluoroquinolones and tetracyclines on mitochondria of human retinal MIO-M1 cells.

Our goal was to explore the detrimental impacts of ciprofloxacin (CPFX) and tetracycline (TETRA) on human retinal Müller (MIO-M1) cells in vitro. Cells were exposed to 30, 60 and 120 µg/ml of CPFX and TETRA. The cellular metabolism was measured with the MTT assay. The JC-1 and CM-H2DCFDA assays were used to evaluate the levels of mitochondrial membrane potential (MMP) and ROS (reactive oxygen species), respectively. Mitochondrial DNA (mtDNA) copy number, along with gene expression levels associated with apoptotic (BAX, BCL2-L13, BCL2, CASP-3 and CASP-9), inflammatory (IL-6, IL-1ß, TGF-α, TGF-ß1 and TGF-ß2) and antioxidant pathways (SOD2, SOD3, GPX3 and NOX4) were analyzed via Quantitative Real-Time PCR (qRT-PCR). Bioenergetic profiles were measured using the Seahorse® XF Flux Analyzer. Cells exposed 24 h to 120 µg/ml TETRA demonstrated higher cellular metabolism compared to vehicle-treated cells. At each time points, (i) all TETRA concentrations reduced MMP levels and (ii) ROS levels were reduced by TETRA 120 µg/ml treatment. TETRA caused (i) higher expression of CASP-3, CASP-9, TGF-α, IL-1B, GPX3 and SOD3 but (ii) decreased levels of TGF-B2 and SOD2. ATP production and spare respiratory capacity declined with TETRA treatment. Cellular metabolism was reduced with CPFX 120 µg/ml in all cultures and 60 µg/ml after 72 h. The CPFX 120 µg/ml reduced MMP in all cultures and ROS levels (72 h). CPFX treatment (i) increased expression of CASP-3, CASP-9, and BCL2-L13, (ii) elevated the basal oxygen consumption rate, and (iii) lowered the mtDNA copy numbers and expression levels of TGF-B2, IL-6 and IL-1B compared to vehicle-control cells. We conclude that clinically relevant dosages of bactericidal and bacteriostatic antibiotics can have negative effects on the cellular metabolism and mitochondrial membrane potential of the retinal MIO-M1 cells in vitro. It is noteworthy to mention that apoptotic and inflammatory pathways in exposed cells were affected significantly This is the first study showing the negative impact of fluoroquinolones and tetracyclines on mitochondrial behavior of human retinal MIO-M1 cells.

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.

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.

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.

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.

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.

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.

Wnt/ß-Catenin Signaling Pathway in Pediatric Tumors: Implications for Diagnosis and Treatment.

The evolutionarily conserved Wnt signaling has a significant and diverse role in maintaining cell homeostasis and tissue maintenance. It is necessary in the regulation of crucial biological functions such as embryonal development, proliferation, differentiation, cell fate, and stem cell pluripotency. The deregulation of Wnt/ß-catenin signaling often leads to various diseases, including cancer and non-cancer diseases. The role of Wnt/ß-catenin signaling in adult tumors has been extensively studied in literature. Although the Wnt signaling pathway has been well explored and recognized to play a role in the initiation and progression of cancer, there is still a lack of understanding on how it affects pediatric tumors. This review discusses the recent developments of this signaling pathway in pediatric tumors. We also focus on understanding how different types of variations in Wnt signaling pathway contribute to cancer development and provide an insight of tissue specific mutations that lead to clinical progression of these tumors.

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.

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.

Potential Therapeutic Functions of PU-91 and Quercetin in Personalized Cybrids Derived from Patients with Age-Related Macular Degeneration, Keratoconus, and Glaucoma.

The aim of this study is to investigate the therapeutic potential of higher doses of PU-91, quercetin, or in combination on transmitochondrial cybrid cell lines with various mtDNA haplogroups derived from patients with age-related macular degeneration (AMD), glaucoma (Glc), keratoconus (KC), and normal (NL) individuals. Cybrids were treated with PU-91 (P) (200 µM) alone, quercetin (Q) (20 µM) alone, or a combination of PU-91 and quercetin (P+Q) for 48 h. Cellular metabolism and the intracellular levels of reactive oxygen species (ROS) were measured by MTT and H2DCFDA assays, respectively. Quantitative real-time PCR was performed to measure the expression levels of genes associated with mitochondrial biogenesis, antioxidant enzymes, inflammation, apoptosis, and senescence pathways. PU-91(P) (i) improves cellular metabolism in AMD cybrids, (ii) decreases ROS production in AMD cybrids, and (iii) downregulates the expression of LMNB1 in AMD cybrids. Combination treatment of PU-91 plus quercetin (P+Q) (i) improves cellular metabolism in AMD, (ii) induces higher expression levels of TFAM, SOD2, IL6, and BAX in AMD cybrids, and (iii) upregulates CDKN1A genes expression in all disease cybrids. Our study demonstrated that the P+Q combination improves cellular metabolism and mitochondrial biogenesis in AMD cybrids, but senescence is greatly exacerbated in all cybrids regardless of disease type by the P+Q combined treatment.

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.

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.