Lata Singh

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

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.

Expression of CDC25A and CDC25B phosphatase proteins in human retinoblastoma and its correlation with clinicopathological parameters

Background CDC25 proteins play a pivotal role in controlling cell proliferation during development and tumorigenesis. The aim of the study is to elucidate the role of CDC25A and CDC25B proteins in retinoblastoma and their association with the clinical and histopathological parameters. Methods One hundred and nine prospective cases of primary enucleated retinoblastomas were included in the present study. Expression of CDC25A and CDC25B proteins was investigated by immunohistochemistry, western blotting and mRNA expression by reverse-transcriptase PCR. Results Immunohistochemistry showed CDC25A expression in (57/109) 52.29%, whereas CDC25B expressed in (69/109) 63.30% cases. Western blotting confirmed the immunoreactivity results on representative cases. mRNA expression of CDC25A and CDC25B was found in 29/60 (48.33%) and 35/60 (58.33%) cases, respectively. Expression of CDC25A and CDC25B showed significant correlation with poor tumour differentiation and tumour invasion (p<0.05). There was a statistically significant difference in the overall survival of patients with CDC25B expression (p=0.0270). Conclusions Our results suggest that expression of CDC25B may be used as a potential prognostic marker in the pathogenesis of retinoblastoma. These findings demonstrate an important role of CDC25 phosphatase proteins and inhibition of these proteins may have therapeutic potential in retinoblastoma.

Prognostic significance of mitochondrial oxidative phosphorylation complexes: Therapeutic target in the treatment of retinoblastoma

PURPOSE Altered energy metabolism plays an important role in the development and progression of cancer. The objective of this study was to elucidate the role of mitochondrial oxidative phosphorylation complexes and their prognostic significance in retinoblastoma (Rb). METHODS Immunohistochemistry was performed on 109 primary enucleated retinoblastoma tissues for mitochondrial OXPHOS complexes and their expression was confirmed by western blotting. RESULTS Histopathological high risk factors (HRFs) were identified in 42.2% cases. Mitochondrial OXPHOS complexes III, IV and V were expressed in more than 50% of primary retinoblastoma cases each whereas mitochondrial complex I was expressed in only 29/109 (26.60%) cases by immunohistochemistry. Loss of mitochondrial complex I correlated well with poor tumor differentiation and tumor invasion (p < 0.05) whereas expression of mitochondrial complexes III, IV and V was associated with better survival (Kaplan-Meier method). CONCLUSIONS This was the first study predicting a relevant role of mitochondrial OXPHOS complexes and highlights the prognostic significance with patient outcome in retinoblastoma. Loss of mitochondrial complex I immunoexpression could prove to be a useful independent prognostic biomarker to identify high risk retinoblastoma patients. Differential expression of these mitochondrial complexes is a novel finding and may be used as an attractive future anticancer target in primary retinoblastoma tumors. FINANCIAL DISCLOSURE The author(s) have no proprietary or commercial interest in any materials discussed in this article.

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

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.

Prognostic significance of VEGF at baseline in orbital retinoblastoma (IRSS stage II and stage III)

To the Editor: In vitro and in vivo studies have suggested that angiogenesis plays a role in retinoblastoma pathogenesis [1–3]; data correlating angiogenesis with outcome is lacking. We retrospectively evaluated vascular endothelial growth factor (VEGF) expression in International Retinoblastoma Staging System [IRSS] stage II and III retinoblastoma patients who underwent upfront enucleation or exenteration between June 2003 and June 2010, and correlated with outcome, baseline clinical, and pathological parameters. Post surgery, all patients received uniform chemotherapy consisting of vincristine, etoposide and carboplatin, along with radiotherapy [4]. VEGF immunohistochemistry was performed on archived tissue blocks with mouse monoclonal VEGF antibody (VEGF Ab-3, Clone: JH-121, 1:50 dilution) (Neomarkers, Fremont, CA) using streptavidin-biotin peroxidase complex method. For VEGF analysis, tumors were scored by assessment of proportion and intensity of stained tumor cells and then scored by semi-quantitative method. Presence of cytoplasmic staining was considered a positive finding. Positively stained cells were counted in 10 randomly selected fields under 40 magnification. Intensity was graded as negative (0), weak (1þ), moderate (2þ), or intense (3þ), and percentage of stained cells was classified as 50% (grade 1), 51–75% (grade 2), or >75% (grade 3). VEGF staining intensity 1þ was considered positive [3]. Twenty-five patients (10/25 in IRSS stage II and 15/25 in IRSS stage III) were eligible for study. Median age of patients was 3 years with 15 (60%) males and 7/25 (28%) patients with bilateral disease. Enucleation was performed in 19 (76%) and exenteration in 6 (24%) patients. Events were recorded in 10/25 (40%) patients, central nervous system relapse occurred in 4/10 (40%) patients, non-CNS distant relapse in 5 (50%) and 1 (10%) patient had local relapse. Event free survival (EFS) for study patients was 58.3% at median follow-up of 24.37 months. Positive VEGF on immunohistochemistry was observed in 16/ 25 (64%) patients (Table I). EFS for VEGF positive and negative patients was 60.0 and 55.6%, respectively (P 1⁄4 0.945). No significant association was seen between VEGF grade and EFS (P 1⁄4 0.654). No significant association of VEGF expression was seen with sex, stage, bilateral tumor, age, symptom duration, calcification, necrosis, and involvement of choroid, sclera, extrascleral tissue, and optic nerve. Few studies correlated angiogenesis with clinical and pathological parameters in retinoblastoma, but these studies did not report on treatment protocols and outcome; further the enrolled patients have been across various clinical stages and studies have differed in techniques used to quantify angiogenesis [1–3]. Our data on VEGF expression at baseline provides insight on pathology and angiogenesis at presentation in IRSS stage II and III patients which future studies may not provide as more patients are being treated with neoadjuvant chemotherapy (NACT) based protocols. Interestingly, in extraocular retinoblastoma post NACT, intense 3þ VEGF staining intensity was observed in one-third of patients in residual tumor cells which may represent poor response to NACT, potential for local invasion and inferior outcome [5]. To conclude, VEGF expression was observed in 64% patients with stage II and III retinoblastoma at baseline, but it did not correlate with EFS, clinical or pathological parameters.

Role of High-mobility Group Protein A Isoforms and Their Clinicopathologic Significance in Primary Retinoblastoma.

Background: High-mobility group proteins A (HMGA) are more abundant in rapidly dividing and transformed cells. These are a group of proteins regulating tumorigenesis and tumor invasion. Increased expression of HMGA1 and HMGA2 has been reported in various benign and malignant tumors. The aim of the present study was to analyze expression of HMGA1 and HMGA2 proteins in retinoblastoma. Methods: Protein expression of HMGA1 and HMGA2 in 80 formalin-fixed retinoblastoma tissues was performed by immunohistochemistry, and their mRNA expressions were analyzed on 40 fresh primary enucleated retinoblastoma samples by semiquantitative reverse transcription polymerase chain reaction. Results were then correlated with clinicopathologic parameters. Results: Immunohistochemical analysis of HMGA1 and HMGA2 was seen in 56.25% and 58.75% of retinoblastoma cases, respectively. mRNA expressions of HMGA1 and HMGA2 was found to be 57.55% and 62.5%, respectively. The mRNA results correlated well with immunostaining results. Expression of both HMGA1 and HMGA2 was significantly associated with choroidal invasion and poor tumor differentiation. Conclusions: HMGA1 and HMGA2 proteins may contribute to tumorigenesis of Rb. Expression of HMGA1 and HMGA2 predicts poor prognosis and could serve as a therapeutic target in the management of RB. Further experiments are needed to determine the role of these proteins as therapeutic targets in tumorigenesis.

VEGF expression in residual tumor cells in orbital retinoblastoma (IRSS stage III) treated with NACT: a prospective study.

We prospectively evaluated vascular endothelial growth factor (VEGF) expression by immunohistochemistry in 22 consecutive IRSS stage III retinoblastoma patients who underwent enucleation after neoadjuvant chemotherapy (NACT). Positive VEGF expression was observed in 6/22 (27.3%) patients. VEGF expression was associated with local progression on MRI prior to enucleation (P = 0.004), pathological scleral (P = 0.023), and extra‐scleral tumor extension (P = 0.009). EFS for VEGF positive and negative patients was 0% and 56.25%, respectively (P = 0.0002). OS for VEGF positive patients was 33.33% and 54.69% for VEGF negative patients (P = 0.207). Thus, VEGF in residual tumor cells post‐NACT may represent poor response to NACT, potential for local invasion, and inferior outcome. Pediatr Blood Cancer 2012;59:567–569.

Mutational Analysis of the Mitochondrial DNA Displacement-Loop Region in Human Retinoblastoma with Patient Outcome

Alteration in mitochondrial DNA plays an important role in the development and progression of cancer. The Displacement Loop (D-loop) region of mitochondrial DNA (mtDNA) is the regulatory region for its replication and transcription. Therefore, we aimed to characterize mutations in the D-loop region of mitochondrial DNA along with the morphological changes and analyzed their impact on survival in retinoblastoma patients. mtDNA D-loop region was amplified by Nested-Polymerase Chain Reaction (Nested-PCR) and mutations were analyzed in 60 tumor samples from retinoblastoma patients by DNA sequencing. Transmission electron microscopy was performed on 5 retinoblastoma specimens. Mutations were correlated with clinical, histopathological parameters and patient survival. D-loop mutations were found in total of 52/60 (86.6%) patients. The most common mutations were T to C and C to T followed by A to G. There were 5.81% mutations which were not previously reported in the MITOMAP database. A73G (83.33%) were the most frequent mutations found in our cases and it was statistically significant with poor tumor differentiation and age. In addition, this study was further analyzed for morphological changes in retinoblastoma that had disorganized, swollen and less numbers of mitochondria on electron microscopy. This is the first study showing high frequency of mtDNA mutation which might be due to abnormal morphology of mitochondria in retinoblastoma. Our results indicate that pathogenic mtDNA D-loop mutations may be involved in tumorigenesis of retinoblastoma tumor.

Expression of FOXO3a and Correlation With Histopathologic Features in Retinoblastoma.

Forkhead box (FOX) transcription factors are a class of highly conserved proteins, which serve critical cellular functions including cell cycle regulation. The downstream mechanisms of cell cycle regulation involve preservation of retinoblastoma protein function. Its deactivation by phosphorylation and translocation from nucleus to cytoplasm leads to cell proliferation. FOXO3a has been found to be dysregulated in few cancers. However, no study has been reported on role of FOXO3a in retinoblastoma. We assessed the expression of FOXO3a in sections of archived tissue blocks of enucleated/exenterated specimens of retinoblastoma by immunohistochemistry. The histopathologic features were reviewed and correlated with its expression. Effect of FOXO3a expression on survival was assessed. FOXO3a expression was assessed in 100 sections. Six samples did not contain any viable tissue. Retrospective data of 94 patients revealed that median age at presentation was 36 months with male:female ratio of 1.9:1. Fifty-one percent of patients were International Retinoblastoma Staging System stage 1. Of the 94 sections, 68 (72%) showed cytoplasmic expression. Choroidal invasion was associated with cytoplasmic FOXO3a (P=0.04). A trend was also noted in optic nerve cut end involvement (P=0.07). No other histopathologic features were found to be associated with FOXO3a expression. The overall survival and progression-free survival were not found to be affected by FOXO3a expression. Cytoplasmic expression of FOXO3a is frequently found in retinoblastoma and may be involved in pathogenesis. Activation by relocation of FOXO3a to nucleus may activate nonmutated retinoblastoma and may be a potential target of treatment in retinoblastoma.

Sirtuin1 Expression and Correlation with Histopathological Features in Retinoblastoma

Background: Sirtuin1 (Sirt1) is a member of highly conserved proteins and has been implicated as a tumor promoter as well as a tumor suppressor. One of the mechanisms involves deacetylation of retinoblastoma protein, thereby inhibiting the tumor suppressor function. No study has been reported on the expression of Sirt1 in retinoblastoma. Methods: We assessed the expression of Sirt1 in sections of archived tissue blocks of enucleated and exenterated specimens of retinoblastoma patients by immunohistochemistry. The histopathological features were reviewed and correlated with the expression of Sirt1. The effect of Sirt1 expression on survival was also assessed. Results: Retrospective data of 94 patients revealed that the median age at presentation was 36 months, with a male:female ratio of 1.9:1. Fifty-one percent of the patients had International Retinoblastoma Staging System (IRSS) stage 1 disease. Of the 94 sections, 89 (95%) expressed Sirt1. Forty-eight percent of the specimens showed grade 3 staining (>75% of the cells), and the intensity was 3+ in 53%. No association between Sirt1 expression and any histopathological feature was noted. Further, Sirt1 expression did not affect the overall and progression-free survival. Conclusions: Sirt1 was expressed in most of the retinoblastoma samples. However, the degree of Sirt1 expression was not associated with any high-risk histopathological feature or survival.