Lata Singh | IGIB - Academia.edu (original) (raw)
Papers by Lata Singh
JCO global oncology, Aug 1, 2023
153 Background: High melanin content could lead to high metastatic potential in Uveal Melanoma (U... more 153 Background: High melanin content could lead to high metastatic potential in Uveal Melanoma (UM). In the Asian population, Microphthalmia-Associated Transcription Factor (MITF) regulates the melanogenesis pathway and activates Silver Protein (SILV) for eumelanin synthesis. MITF also stimulates the hypoxia–inducible factor 1-alpha (HIF-1α) gene that regulates hypoxia around the tumor microenvironment. Although their oncogenic potential has been observed in a variety of malignancies, MITF and SILV have not been investigated in UM in the Asian population. Therefore, our study aimed to detect the prognostic significance of MITF, SILV, and HIF1-α gene/protein expression levels in UM patients. Methods: Immunohistochemistry of MITF, SILV & HIF-1 α was performed on 82 UM tissue samples. Western blot of all three markers was carried out on representative cases. The mRNA expression level was done by qRT-PCR in 70 fresh UM cases. Cox proportional hazard model and log-rank test were used to determine the prognostic outcome of these markers. Results: MITF and SILV proteins were expressed in 56% and 70% of cases, respectively. High pigmentation, BAP1 loss, and HIF-1α expression were statistically correlated with MITF and SILV protein expression. Upregulation of both genes was found in more than 50% of cases at the mRNA level. This was also statistically significant with high pigmentation and HIF-1α mRNA expression. High expression of MITF protein showed reduced disease-free survival. Conclusions: Our study highlighted the fact that MITF and SILV could be potential biomarkers in UM development. Hence, it might play a key role in future therapeutic target.
Annals of Oncology, Oct 1, 2019
Background Uveal melanoma is the most common primary intraocular malignancy in adults. Almost hal... more Background Uveal melanoma is the most common primary intraocular malignancy in adults. Almost half of patients with uveal melanoma may die from metastatic disease and the most frequent site of metastases is the liver. It is considered as an inflammatory phenotype. NFκB plays a connecting link between the inflammation and cancer. c-Rel and p50 are together considered as a heterodimer (c-Rel/p50) which plays an important role in inflammation and cancer progression. Therefore, the aim of the study is to detect c-Rel/p50 heterodimer expression in the tumour microenvironment of uveal melanoma and its prognostic significance. Methods Evaluation of c-Rel/p50 heterodimer expression was performed in 75 formalin fixed uveal melanoma tissues by using immunohistochemistry. Transcriptional analysis was performed on 58 fresh tissues by real-time PCR (q-PCR). Co-Immunoprecipitation (Co-IP) was done on 10 representative cases to assess the presence of c-Rel/p50 heterodimer and validation of immunohistochemistry results was performed by western blotting. Results were then correlated with clinicopathological parameters. Results Immunoexpression of c-Rel+/p50+ is localized to the cytoplasm in normal choroid whereas both cytoplasmic and nuclear expression was seen in 33.33% cases. At the transcriptional level, upregulation of c-Rel+/p50+ heterodimer was shown in 43.10%. Expression of both cytoplasmic and nuclear c-Rel+/p50+ heterodimer were found to have significant correlation with cases having tumour infiltrating lymphocytes, macrophages (CD68+) and high pigmentation. There was a statistically significant difference in the overall survival of patients with nuclear and cytoplasmic expression of c-Rel+/p50+ heterodimer (p
Investigative Ophthalmology & Visual Science, Apr 30, 2014
Investigative Ophthalmology & Visual Science, Jun 16, 2013
Therapeutic advances in ophthalmology, 2022
Background: The definitive diagnosing of ocular tuberculosis (TB) is difficult; therefore, there ... more Background: The definitive diagnosing of ocular tuberculosis (TB) is difficult; therefore, there is a need of better understanding of investigating TB DNA in presumed ocular TB patients. Objectives: The aim of this study is to correlate tubercular DNA PCR of aqueous/vitreous and blood in cases of presumed ocular TB. Design: A prospective study. Methods: DNA was extracted from aqueous of cases of choroidal tuberculoma (group 1) and serpiginous choroiditis (group 2) and from vitreous of cases of vasculitis (group 3) and macular hole/retinal detachment (group 4). Gel-based PCR and real-time PCR amplification were performed using IS6110 primer on ocular fluids. The same was also performed on the blood samples of cases in which tubercular DNA was detected in the ocular fluids. Results: Overall, 31 cases were analysed in our study. Tubercular DNA was detected in ocular fluids of seven cases: group 1, two cases (67%); group 2, one case (17%); group 3, four cases (27%); and no case of group 4. Blood samples of six of these seven patients were positive for tubercular DNA. Of these six patients, four had evidence of systemic TB and were on ATT. Two cases had no evidence of active systemic TB, yet PCR was positive from blood and ocular fluids. Conclusion: Tubercular DNA detected from ocular fluids may possibly be due to bystander DNA and may not indicate primary ocular tubercular infection. Thus, caution must be exercised prior to labelling a case of uveitis as being tubercular based on the results of molecular assays on ocular fluids alone. The results of PCR on ocular fluids should be correlated with PCR on blood and systemic findings.
Clinical & Translational Oncology, May 23, 2018
Purpose Uveal melanoma, although a rare form of cancer, is the most common primary malignancy of ... more Purpose Uveal melanoma, although a rare form of cancer, is the most common primary malignancy of the eye in adults. Nuclear factor-κB (NF-κB) is a transcription factor that transactivates genes involved in the regulation of cell growth, apoptosis, angiogenesis, and metastasis, but the molecular mechanisms that negatively regulate NF-κB activation are not fully understood. NF-κB can also be activated by DNA damage pathway through NEMO protein. Therefore, the objective of this study is to elucidate the role of NEMO/IKKγ protein in uveal melanoma patients. Methods Seventy-five formalin-fixed paraffin-embedded prospective tissues of uveal melanoma were included in the present study. These cases were reviewed and investigated for the expression of NEMO/IKKγ protein by immunohistochemistry and validated by western blotting along with the qRT-PCR for mRNA expression. Expression levels were correlated with the clinicopathological parameters and patients' outcome. Results Immunohistochemistry showed cytoplasmic expression of NEMO/IKKγ expression in only 22 out of 75 (29.33%) cases. This result was confirmed by western blotting, and correlated well with the immunohistochemical expression of NEMO/IKKγ protein (48 kDa). In addition, downregulation of this gene was found in 87.93% of the cases when compared with the normal tissues. On statistical analysis, loss of NEMO/IKKγ protein was correlated with neovascularization, high mitotic count, and presence of vascular loop (p < 0.05). There was less overall survival rate with low expression of NEMO/ IKKγ protein in patients with uveal melanoma. Conclusion This was the first study suggesting the relevant role of NEMO/IKKγ protein, and highlights the prognostic significance with outcome in uveal melanoma patients. This protein might be used as a screening biomarker in these patients after large-scale validation and translational studies.
Investigative Ophthalmology & Visual Science, Jul 22, 2019
British Journal of Ophthalmology, Apr 10, 2020
BackgroundImmune checkpoint blockade strategies have gained attention in the treatment/prognosis ... more BackgroundImmune 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).MethodsThe 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.ResultsThe 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.ConclusionOur 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.
Investigative Ophthalmology & Visual Science, Apr 30, 2014
Annals of Oncology, Dec 1, 2019
Background The goal of this study is to identify the pathological findings and expression of immu... more Background 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 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 was 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 reduced in cases of PD-L1 (80.76%) expressed primary retinoblastoma, and PD-1 (63.28%) expressed chemoreduced retinoblastoma. Conclusion This is the first of its kind study predicting a relevant role of the immune checkpoint markers in 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. Legal entity responsible for the study The authors. Funding Has not received any funding. Disclosure All authors have declared no conflicts of interest.
Clinical & Translational Oncology, Nov 25, 2019
Purpose Uveal melanoma (UM) is the most common intraocular cancer with a high mortality rate that... more Purpose Uveal melanoma (UM) is the most common intraocular cancer with a high mortality rate that requires new research in the field of prevention and treatment. c-REL is a member of the nuclear factor κB (NF-κB) transcription factor family and an emerging regulator of tumorigenesis. Therefore, the objective of the study is to evaluate the constitutive expression of c-REL in uveal melanoma patients and its prognostic significance. Methods Detection of c-REL expression was carried out by immunohistochemistry in all 75 patients, and qRT-PCR performed on 58 fresh cases of uveal melanoma along with IL-6 status. Immunoblot was performed to validate immunohistochemistry results. Expression of c-REL protein correlated with clinicopathological parameters and overall survival of patients. Results Immunohistochemistry results revealed nuclear expression of the c-REL protein (56%) in our cases. Out of 75 cases, 31 cases showed nuclear expression, and 11 cases had cytoplasmic expression. qRT-PCR showed upregulation of the REL gene in 56.89% cases at the transcriptional level. There was a statistically significant difference in the overall survival of patients with c-REL nuclear immunopositivity (p = 0.0048). On multivariate analysis, scleral invasion and c-REL nuclear expression found to be an independent prognostic factor (p < 0.05) Conclusions To the best of our knowledge, this was the first study reporting the expression of the c-REL protein in uveal melanoma. Strong nuclear immunoexpression of c-Rel suggests NFκB pathway activation which might be involved in the progression of the disease. Differential expression of c-REL protein may be used as an attractive target for the development of anticancer strategies.
Pediatric Hematology Oncology Journal, 2022
Annals of Oncology, Oct 1, 2018
Annals of Oncology, Nov 1, 2019
Abstract Background Uveal melanoma (UM) is an intraocular malignancy commonly arising from the ch... more Abstract Background Uveal melanoma (UM) is an intraocular malignancy commonly arising from the choroid. There is a lack of effective therapy to detect and treat early metastatic spread in these patients. Therefore, it is important to find new biomarkers that help in early detection of metastasis. ATR is localized at position 23 on the same chromosome 3 where BAP1 is located at position 21.1. Loss of BAP1 is a known poor prognostic marker of uveal melanoma. There is no current study available on UM with respect to ATR protein that induces DNA damage response. Therefore, the aim of the study is to detect the expression of ATR protein in the UM patients and correlate its expression with BAP1 loss. Methods Expression of nuclear ATR was investigated on sixty-nine UM patients which were divided into two groups on the basis of BAP1 expression. Formalin-fixed paraffin embedded choroidal melanoma samples were taken to evaluate the expression of ATM and BAP1 by immunohistochemistry and validated by semi-quantitative PCR. Results were then correlated with clinicopathological parameters. To determine the prognostic significance, Kaplan–Meier and multivariate analysis were performed. Results Patients with BAP1 loss also showed ATR loss in 62% of the cases which was statistically significant with high tumor pigmentation, LTD >10mm and cell type. At transcription level, down-regulation of ATR gene was found in 45% of cases and these results were in line with the IHC results. On multivariate analysis, advanced tumor staging and loss of ATR protein found to be independent prognostic factors. Conclusions Our study emphasized the fact that loss of ATR could be a potential biomarker to detect the early metastasis in uveal melanoma. Hence, it could have a key role for therapeutic purpose. However, further studies are required in a larger cohort of patients with longer follow up and translational validation needs to be performed. Legal entity responsible for the study The authors. Funding Has not received any funding. Disclosure All authors have declared no conflicts of interest.
British Journal of Ophthalmology, Mar 14, 2023
BackgroundResponse rate of PD-1/PD-L1 immunotherapeutic blockade agents in uveal melanoma (UM) is... more 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.MethodsThe 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.ResultThe 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).ConclusionOur 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.
Annals of Oncology, Dec 1, 2021
Experimental and Molecular Pathology, Dec 1, 2019
This is a PDF file of an article that has undergone enhancements after acceptance, such as the ad... more This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Clinical & Translational Oncology, Mar 2, 2020
Background The role of DNA damage response (DDR) proteins is poorly understood in uveal melanoma.... more Background The role of DNA damage response (DDR) proteins is poorly understood in uveal melanoma. ATR belongs to one of those proteins that induce DDR by arresting the cell cycle which leads to DNA repair. ATR is localized at position 23 on the same chromosome 3 where BAP1 is located at position 21.1 which is a known poor prognostic marker of UM. The aim of our study is to detect the expression of ATR at the protein and RNA levels and determine its prognostic significance. Methods Expression of nuclear ATR was investigated on sixty-nine UM patients. Formalin-fixed paraffin-embedded choroidal melanoma samples were taken to evaluate the expression of ATR. Fifty samples were also validated by real-time PCR. Results of both protein and mRNA were then correlated with clinicopathological parameters. To determine the prognostic significance, Kaplan-Meier and multivariate analyses were performed. Results Loss of ATR protein was seen in 72% cases which was statistically significant with epithelioid cell type (p = 0.005), tumor thickness (p = 0.016), mitotic figures (p = 0.001) and BAP1 loss (p < 0.001). At the transcriptional level loss of ATR was seen in 76% cases which were statistically significant with metastasis (p = 0.046), staging (0.044) and loss of BAP1 (p = 0.022). On multivariate analysis loss of ATR and tumor staging came out to be independent prognostic parameters. Conclusion Our data suggest that ATR might serve as a potential prognostic marker in UM patients and could serve as a potential therapeutic target.
International Journal of Molecular Sciences
This study uses personalized chronic lymphoblastic leukemia (CLL) cybrid cells to test various dr... more 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-1β, TNFα, and TGFβ) were measured using qu...
JCO global oncology, Aug 1, 2023
153 Background: High melanin content could lead to high metastatic potential in Uveal Melanoma (U... more 153 Background: High melanin content could lead to high metastatic potential in Uveal Melanoma (UM). In the Asian population, Microphthalmia-Associated Transcription Factor (MITF) regulates the melanogenesis pathway and activates Silver Protein (SILV) for eumelanin synthesis. MITF also stimulates the hypoxia–inducible factor 1-alpha (HIF-1α) gene that regulates hypoxia around the tumor microenvironment. Although their oncogenic potential has been observed in a variety of malignancies, MITF and SILV have not been investigated in UM in the Asian population. Therefore, our study aimed to detect the prognostic significance of MITF, SILV, and HIF1-α gene/protein expression levels in UM patients. Methods: Immunohistochemistry of MITF, SILV & HIF-1 α was performed on 82 UM tissue samples. Western blot of all three markers was carried out on representative cases. The mRNA expression level was done by qRT-PCR in 70 fresh UM cases. Cox proportional hazard model and log-rank test were used to determine the prognostic outcome of these markers. Results: MITF and SILV proteins were expressed in 56% and 70% of cases, respectively. High pigmentation, BAP1 loss, and HIF-1α expression were statistically correlated with MITF and SILV protein expression. Upregulation of both genes was found in more than 50% of cases at the mRNA level. This was also statistically significant with high pigmentation and HIF-1α mRNA expression. High expression of MITF protein showed reduced disease-free survival. Conclusions: Our study highlighted the fact that MITF and SILV could be potential biomarkers in UM development. Hence, it might play a key role in future therapeutic target.
Annals of Oncology, Oct 1, 2019
Background Uveal melanoma is the most common primary intraocular malignancy in adults. Almost hal... more Background Uveal melanoma is the most common primary intraocular malignancy in adults. Almost half of patients with uveal melanoma may die from metastatic disease and the most frequent site of metastases is the liver. It is considered as an inflammatory phenotype. NFκB plays a connecting link between the inflammation and cancer. c-Rel and p50 are together considered as a heterodimer (c-Rel/p50) which plays an important role in inflammation and cancer progression. Therefore, the aim of the study is to detect c-Rel/p50 heterodimer expression in the tumour microenvironment of uveal melanoma and its prognostic significance. Methods Evaluation of c-Rel/p50 heterodimer expression was performed in 75 formalin fixed uveal melanoma tissues by using immunohistochemistry. Transcriptional analysis was performed on 58 fresh tissues by real-time PCR (q-PCR). Co-Immunoprecipitation (Co-IP) was done on 10 representative cases to assess the presence of c-Rel/p50 heterodimer and validation of immunohistochemistry results was performed by western blotting. Results were then correlated with clinicopathological parameters. Results Immunoexpression of c-Rel+/p50+ is localized to the cytoplasm in normal choroid whereas both cytoplasmic and nuclear expression was seen in 33.33% cases. At the transcriptional level, upregulation of c-Rel+/p50+ heterodimer was shown in 43.10%. Expression of both cytoplasmic and nuclear c-Rel+/p50+ heterodimer were found to have significant correlation with cases having tumour infiltrating lymphocytes, macrophages (CD68+) and high pigmentation. There was a statistically significant difference in the overall survival of patients with nuclear and cytoplasmic expression of c-Rel+/p50+ heterodimer (p
Investigative Ophthalmology & Visual Science, Apr 30, 2014
Investigative Ophthalmology & Visual Science, Jun 16, 2013
Therapeutic advances in ophthalmology, 2022
Background: The definitive diagnosing of ocular tuberculosis (TB) is difficult; therefore, there ... more Background: The definitive diagnosing of ocular tuberculosis (TB) is difficult; therefore, there is a need of better understanding of investigating TB DNA in presumed ocular TB patients. Objectives: The aim of this study is to correlate tubercular DNA PCR of aqueous/vitreous and blood in cases of presumed ocular TB. Design: A prospective study. Methods: DNA was extracted from aqueous of cases of choroidal tuberculoma (group 1) and serpiginous choroiditis (group 2) and from vitreous of cases of vasculitis (group 3) and macular hole/retinal detachment (group 4). Gel-based PCR and real-time PCR amplification were performed using IS6110 primer on ocular fluids. The same was also performed on the blood samples of cases in which tubercular DNA was detected in the ocular fluids. Results: Overall, 31 cases were analysed in our study. Tubercular DNA was detected in ocular fluids of seven cases: group 1, two cases (67%); group 2, one case (17%); group 3, four cases (27%); and no case of group 4. Blood samples of six of these seven patients were positive for tubercular DNA. Of these six patients, four had evidence of systemic TB and were on ATT. Two cases had no evidence of active systemic TB, yet PCR was positive from blood and ocular fluids. Conclusion: Tubercular DNA detected from ocular fluids may possibly be due to bystander DNA and may not indicate primary ocular tubercular infection. Thus, caution must be exercised prior to labelling a case of uveitis as being tubercular based on the results of molecular assays on ocular fluids alone. The results of PCR on ocular fluids should be correlated with PCR on blood and systemic findings.
Clinical & Translational Oncology, May 23, 2018
Purpose Uveal melanoma, although a rare form of cancer, is the most common primary malignancy of ... more Purpose Uveal melanoma, although a rare form of cancer, is the most common primary malignancy of the eye in adults. Nuclear factor-κB (NF-κB) is a transcription factor that transactivates genes involved in the regulation of cell growth, apoptosis, angiogenesis, and metastasis, but the molecular mechanisms that negatively regulate NF-κB activation are not fully understood. NF-κB can also be activated by DNA damage pathway through NEMO protein. Therefore, the objective of this study is to elucidate the role of NEMO/IKKγ protein in uveal melanoma patients. Methods Seventy-five formalin-fixed paraffin-embedded prospective tissues of uveal melanoma were included in the present study. These cases were reviewed and investigated for the expression of NEMO/IKKγ protein by immunohistochemistry and validated by western blotting along with the qRT-PCR for mRNA expression. Expression levels were correlated with the clinicopathological parameters and patients' outcome. Results Immunohistochemistry showed cytoplasmic expression of NEMO/IKKγ expression in only 22 out of 75 (29.33%) cases. This result was confirmed by western blotting, and correlated well with the immunohistochemical expression of NEMO/IKKγ protein (48 kDa). In addition, downregulation of this gene was found in 87.93% of the cases when compared with the normal tissues. On statistical analysis, loss of NEMO/IKKγ protein was correlated with neovascularization, high mitotic count, and presence of vascular loop (p < 0.05). There was less overall survival rate with low expression of NEMO/ IKKγ protein in patients with uveal melanoma. Conclusion This was the first study suggesting the relevant role of NEMO/IKKγ protein, and highlights the prognostic significance with outcome in uveal melanoma patients. This protein might be used as a screening biomarker in these patients after large-scale validation and translational studies.
Investigative Ophthalmology & Visual Science, Jul 22, 2019
British Journal of Ophthalmology, Apr 10, 2020
BackgroundImmune checkpoint blockade strategies have gained attention in the treatment/prognosis ... more BackgroundImmune 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).MethodsThe 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.ResultsThe 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.ConclusionOur 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.
Investigative Ophthalmology & Visual Science, Apr 30, 2014
Annals of Oncology, Dec 1, 2019
Background The goal of this study is to identify the pathological findings and expression of immu... more Background 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 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 was 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 reduced in cases of PD-L1 (80.76%) expressed primary retinoblastoma, and PD-1 (63.28%) expressed chemoreduced retinoblastoma. Conclusion This is the first of its kind study predicting a relevant role of the immune checkpoint markers in 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. Legal entity responsible for the study The authors. Funding Has not received any funding. Disclosure All authors have declared no conflicts of interest.
Clinical & Translational Oncology, Nov 25, 2019
Purpose Uveal melanoma (UM) is the most common intraocular cancer with a high mortality rate that... more Purpose Uveal melanoma (UM) is the most common intraocular cancer with a high mortality rate that requires new research in the field of prevention and treatment. c-REL is a member of the nuclear factor κB (NF-κB) transcription factor family and an emerging regulator of tumorigenesis. Therefore, the objective of the study is to evaluate the constitutive expression of c-REL in uveal melanoma patients and its prognostic significance. Methods Detection of c-REL expression was carried out by immunohistochemistry in all 75 patients, and qRT-PCR performed on 58 fresh cases of uveal melanoma along with IL-6 status. Immunoblot was performed to validate immunohistochemistry results. Expression of c-REL protein correlated with clinicopathological parameters and overall survival of patients. Results Immunohistochemistry results revealed nuclear expression of the c-REL protein (56%) in our cases. Out of 75 cases, 31 cases showed nuclear expression, and 11 cases had cytoplasmic expression. qRT-PCR showed upregulation of the REL gene in 56.89% cases at the transcriptional level. There was a statistically significant difference in the overall survival of patients with c-REL nuclear immunopositivity (p = 0.0048). On multivariate analysis, scleral invasion and c-REL nuclear expression found to be an independent prognostic factor (p < 0.05) Conclusions To the best of our knowledge, this was the first study reporting the expression of the c-REL protein in uveal melanoma. Strong nuclear immunoexpression of c-Rel suggests NFκB pathway activation which might be involved in the progression of the disease. Differential expression of c-REL protein may be used as an attractive target for the development of anticancer strategies.
Pediatric Hematology Oncology Journal, 2022
Annals of Oncology, Oct 1, 2018
Annals of Oncology, Nov 1, 2019
Abstract Background Uveal melanoma (UM) is an intraocular malignancy commonly arising from the ch... more Abstract Background Uveal melanoma (UM) is an intraocular malignancy commonly arising from the choroid. There is a lack of effective therapy to detect and treat early metastatic spread in these patients. Therefore, it is important to find new biomarkers that help in early detection of metastasis. ATR is localized at position 23 on the same chromosome 3 where BAP1 is located at position 21.1. Loss of BAP1 is a known poor prognostic marker of uveal melanoma. There is no current study available on UM with respect to ATR protein that induces DNA damage response. Therefore, the aim of the study is to detect the expression of ATR protein in the UM patients and correlate its expression with BAP1 loss. Methods Expression of nuclear ATR was investigated on sixty-nine UM patients which were divided into two groups on the basis of BAP1 expression. Formalin-fixed paraffin embedded choroidal melanoma samples were taken to evaluate the expression of ATM and BAP1 by immunohistochemistry and validated by semi-quantitative PCR. Results were then correlated with clinicopathological parameters. To determine the prognostic significance, Kaplan–Meier and multivariate analysis were performed. Results Patients with BAP1 loss also showed ATR loss in 62% of the cases which was statistically significant with high tumor pigmentation, LTD >10mm and cell type. At transcription level, down-regulation of ATR gene was found in 45% of cases and these results were in line with the IHC results. On multivariate analysis, advanced tumor staging and loss of ATR protein found to be independent prognostic factors. Conclusions Our study emphasized the fact that loss of ATR could be a potential biomarker to detect the early metastasis in uveal melanoma. Hence, it could have a key role for therapeutic purpose. However, further studies are required in a larger cohort of patients with longer follow up and translational validation needs to be performed. Legal entity responsible for the study The authors. Funding Has not received any funding. Disclosure All authors have declared no conflicts of interest.
British Journal of Ophthalmology, Mar 14, 2023
BackgroundResponse rate of PD-1/PD-L1 immunotherapeutic blockade agents in uveal melanoma (UM) is... more 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.MethodsThe 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.ResultThe 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).ConclusionOur 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.
Annals of Oncology, Dec 1, 2021
Experimental and Molecular Pathology, Dec 1, 2019
This is a PDF file of an article that has undergone enhancements after acceptance, such as the ad... more This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Clinical & Translational Oncology, Mar 2, 2020
Background The role of DNA damage response (DDR) proteins is poorly understood in uveal melanoma.... more Background The role of DNA damage response (DDR) proteins is poorly understood in uveal melanoma. ATR belongs to one of those proteins that induce DDR by arresting the cell cycle which leads to DNA repair. ATR is localized at position 23 on the same chromosome 3 where BAP1 is located at position 21.1 which is a known poor prognostic marker of UM. The aim of our study is to detect the expression of ATR at the protein and RNA levels and determine its prognostic significance. Methods Expression of nuclear ATR was investigated on sixty-nine UM patients. Formalin-fixed paraffin-embedded choroidal melanoma samples were taken to evaluate the expression of ATR. Fifty samples were also validated by real-time PCR. Results of both protein and mRNA were then correlated with clinicopathological parameters. To determine the prognostic significance, Kaplan-Meier and multivariate analyses were performed. Results Loss of ATR protein was seen in 72% cases which was statistically significant with epithelioid cell type (p = 0.005), tumor thickness (p = 0.016), mitotic figures (p = 0.001) and BAP1 loss (p < 0.001). At the transcriptional level loss of ATR was seen in 76% cases which were statistically significant with metastasis (p = 0.046), staging (0.044) and loss of BAP1 (p = 0.022). On multivariate analysis loss of ATR and tumor staging came out to be independent prognostic parameters. Conclusion Our data suggest that ATR might serve as a potential prognostic marker in UM patients and could serve as a potential therapeutic target.
International Journal of Molecular Sciences
This study uses personalized chronic lymphoblastic leukemia (CLL) cybrid cells to test various dr... more 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-1β, TNFα, and TGFβ) were measured using qu...