High prevalence of "non-pathogenic" POLE mutation with poor prognosis in a cohort of endometrial cancer from South India.

OBJECTIVE: The Cancer Genome Atlas (TCGA) project identified favorable prognosis regarding the ultra-mutated endometrial cancer (EC) subtype linked to polymerase epsilon gene (POLE) mutations. This study investigated POLE mutations in EC of Indian patients. METHODS: This retrospective analytical study was conducted between January 2016 and January 2023 at the Government Medical College, Kozhikode, and the MVR Cancer Center, Kozhikode, Kerala. Sanger sequencing of POLE gene exons 9 and 13 in 151 EC patients was carried out to analyze the relationship between mutations and epidemiological factors, clinicopathologic features, and treatment outcomes. RESULTS: Among 151 cases enrolled, 39 were unique POLE-mutated cases. Significant associations were high-grade tumors, myometrial invasion >50%, and Lymph-vascular space invasion (LVSI). The median follow-up was 40 months (95% confidence interval [CI], 34-46). A lower mean disease-specific survival (DSS) of 51.7 months (95% CI, 43.7-59.6) was noted in the POLE-mutated group compared with 72.11 months (95% CI, 67.60-76.62) for the POLE wild-type. A statistically significant hazard ratio (HR) of 2.683 for DSS in the POLE-mutated group was noted. In advanced stages (FIGO stages II-IV), a nine-fold HR for DSS and overall survival (OS) compared with POLE wild-type was identified. After controlling for treatment effects using Cox proportional HR, advanced-stage POLE-mutated tumors had a significantly higher HR of 8.67 for DSS compared with POLE-wild-type tumors of the same stage. CONCLUSION: This study identified a unique set of POLE mutations in Indian EC patients associated with poor prognosis, which were particularly pronounced in advanced stages. Advanced stage of presentation, type of POLE mutations, and possibly ethnicity are predictors of adverse outcomes in POLE-mutated EC. The present study highlights ethnicity as a determinant of phenotypic expression of genetic change.

Plasma Exchange in Patients With Myositis due to Immune Checkpoint Inhibitor Therapy.

ABSTRACT: Immune checkpoint inhibitors used to treat malignancies may lead to various immune-related adverse events (irAEs) including conditions such as myositis and myasthenia gravis (MG). Here, we describe 2 cases of myositis treated effectively with therapeutic plasma exchange (PLEX). A 64-year-old man with thymic cancer developed leg weakness and dyspnea 1 month after the second dose of nivolumab with moderate weakness in proximal and distal muscles, with elevated creatine kinase levels. Another 77-year-old man with Stage IIIB squamous cell carcinoma of the lung developed progressive proximal muscle weakness and became nonambulatory after cycle 2 of durvalumab with persistently high creatine kinase levels despite prednisone treatment. Electrophysiology revealed irritative myopathy without evidence of neuromuscular junction dysfunction and MG antibody testing was nonrevealing. With PLEX, both patients noticed rapid improvement in strength. PLEX in conjunction with other immunosuppressive agents can result in rapid improvement in irAE-myositis even in patients without associated MG.

Is the National Institute of Health Stroke Scale a valid prognosticator of the aftermath in patients with ischemic stroke?

Background: Cerebrovascular disease is the second most common cause of death in people more than 60 years of age. Predicting the outcome of the stroke is a great challenge for physicians. Various risk factors such as age, sex, co-morbidities, smoking and alcohol habits, type of stroke, National Institute of Health Stroke Scale (NIHSS) score, modified Rankin Scale (mRS), and others play the role in the outcome of stoke. Aim: To assess the degree of impact of NIHSS score in comparison to the other traditional risk factors on the functional outcome and 30-day mortality by mRS in the patients with acute ischemic stroke. Materials and Methods: Patients with acute ischemic stroke and age more than 18 years were included. Their admission NIHSS score and the 30-day mRS were analyzed. Patients were divided into two groups as survivors and non-survivors. Results: The mean age of survivors and non-survivors was 59.77 ± 10.99 and 65.58 ± 6.67 years, respectively. The NIHSS score on day 1 for non-survivors was 21.21 ± 8.21, and almost half of this score was seen in survivors. The NIHSS score on day 1 had a significant association with mortality with a relative risk of 0.79 (95% CI = 0.70-0.89). The NIHSS score has 73.7% sensitivity and 74.1% specificity with cutoff value of 15.5 for discriminating the outcome of ischemic stroke. Conclusion: The NIHSS and mRS scales are simple, validated, easily applicable, and reliable tools for assessing the mortality and the functional outcome of ischemic stroke patients.

Randomized Phase II Study of 3 Months or 2 Years of Adjuvant Afatinib in Patients With Surgically Resected Stage I-III EGFR-Mutant Non-Small-Cell Lung Cancer.

For patients with surgically resected disease, multiple studies suggest a benefit of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in delaying cancer recurrence. The necessary duration of therapy for benefit is unknown. MATERIALS AND METHODS: This randomized phase II study enrolled patients with completely resected stage IA-IIIB EGFR-mutant non-small-cell lung cancer (American Joint Committee on Cancer 7th edition) after stage-appropriate standard-of-care adjuvant therapy. Patients were randomly assigned 1:1 to 3 months or 2 years of adjuvant afatinib starting at 30 mg by mouth daily. Computed tomography imaging was performed every 6 months for 3 years and then annually. The primary study end point for this planned 92-patient trial was recurrence rate at 2 years from randomization. A 20% improvement (from 70% with 3 months to 90% with 2 years) was targeted. RESULTS: Forty-six patients enrolled and 45 were treated. The assigned course of afatinib treatment was completed by 96% (22/23) of patients in the 3-month group and only 41% (9/22) in the 2-year group. The 2-year recurrence-free survival (RFS) rates were 70% in the 3-month group and 81% in the 2-year group (P = .55). The median RFS was 42.8 months in the 3-month group and 58.6 months in the 2-year group. Side effects were consistent with those previously described for afatinib. CONCLUSION: Recurrences at 2 years were 11% less common with 2 years versus 3 months of adjuvant afatinib. This difference did not meet the 20% primary study target, likely because of underaccrual and early drug discontinuation on the 2-year group. With the availability of osimertinib with better efficacy and tolerability than earlier-generation agents, the optimal duration of adjuvant EGFR TKI therapy remains an important question.

Role of CAIX Expression in Conventional Renal Cell Carcinomas as a Diagnostic Marker and its Prognostic Importance.

Significant advances in understanding of the biology of renal cell carcinoma (RCC) have been achieved recently, which led to novel targeted therapies, revolutionising the management of patients with advanced disease. To date, there are no molecular markers which can reliably predict RCC outcome. We investigated whether a novel kidney cancer marker, carbonic anhydrase IX (CAIX), is associated with progression and survival. A retrospective study was done on patients diagnosed with renal cell carcinoma over a period of 5 years. Immunohistochemical analysis using a CAIX monoclonal antibody was performed on paraffin-embedded blocks from patients treated with nephrectomy for clear cell RCC. Patients were segregated into two categories based on CA IX expression as CA IX ≤ 85% and CA IX > 85%. A comparison was made based on the survival (from date of diagnosis) with CA IX expression. Correlation of CA IX expression and TNM staging, nuclear grading, tumour volume and age was statistically studied using Student's t test. The association between survival and CA IX was done using Mann-Whitney test. The association of CA IX with rest of the prognostic variables were analysed using Fisher's exact test. In our study, CA IX expression > 85% had longer survival compared with those with lower expression ≤ 85%. A significant statistical association was seen with CAIX and lymphovascular emboli, major vessel, perinephric fat, renal sinus fat involvement and distant metastasis. CAIX reflects significant changes in tumour biology that predicts clinical outcome and identify high-risk patients for adjuvant immunotherapy and CAIX targeted therapies.

Role of DNA Methylation in Cabazitaxel Resistance in Prostate Cancer.

BACKGROUND/AIM: Cabazitaxel is an approved second-line treatment for docetaxel-refractory metastatic castration-resistant prostate cancer. However, the median time to progression on cabazitaxel is 2.8 months. We aimed to determine whether DNA methylation plays a role in cabazitaxel resistance. MATERIALS AND METHODS: DU145 cells, resistant to docetaxel and cabaxitaxel (DU145 10DRCR), were generated from cells resistant to 10 nM docetaxel (DU145 10DR). The effect of pre-treatment with 5-azacytidine was determined with regards to cabazitaxel sensitivity. Gene expression profiling was carried-out on DU145 10DR, DU145 10DRCR and DU145 10DRCR treated with 5-azacytidine. RESULTS: Pre-treatment of cells with 5-azacytidine resulted in enhanced sensitivity to cabazitaxel. Gene expression profiling identified a subset of genes that may be regulated by DNA methylation. CONCLUSION: Our results indicate that DNA methylation of pro-apoptotic and cell-cycle regulatory genes may contribute to cabazitaxel resistance and pre-treatment with 5-azacytidine may restore sensitivity to cabazitaxel in prostate cancer cells.

Phase I/II study of azacitidine, docetaxel, and prednisone in patients with metastatic castration-resistant prostate cancer previously treated with docetaxel-based therapy.

INTRODUCTION: Methylation-mediated silencing of genes contributes to docetaxel resistance in prostate cancer. We propose that azacitidine, a demethylating agent, can reverse docetaxel resistance. PATIENTS AND METHODS: Metastatic castration-resistant prostate cancer (mCRPC) patients, who progressed during or within 6 months of docetaxel chemotherapy, were eligible. Fifteen and 7 patients were treated in phase I and II, respectively. In phase I, azacitidine and docetaxel were alternately escalated in a standard 3 + 3 design. All patients received prednisone 5 mg twice daily continuously. Patients were evaluated for toxicity and efficacy. Growth arrest and DNA damage-inducible alpha (GADD45A) methylation was measured before and after azacitidine treatment in the first cycle in phase I patients. RESULTS: In phase I, no dose-limiting toxicity was observed. At the highest dose (azacitidine 150 mg/m(2) daily for 5 days followed by docetaxel 75 mg/m(2) on day 6), Grade 4 neutropenia was frequent, but infrequent with growth factor. Six patients in the phase II study received the highest dose including growth factor support. The sixth phase II patient died because of neutropenic sepsis. After data and safety monitoring board review, the phase II dose was reduced to azacitidine 75 mg/m(2) daily for 5 days followed by docetaxel 75 mg/m(2) on day 6 with growth factor support. Prostate-specific antigen response was seen in 10 of 19 evaluable patients and objective response was observed in 3 of 10 evaluable patients. Significant demethylation of GADD45A was observed with azacitidine treatment. CONCLUSION: The combination of azacitidine, docetaxel, and prednisone with growth factor support is active in mCRPC patients.

Free circulating DNA as a biomarker of prostate cancer: comparison of quantitation methods.

AIM: To identify a simpler method of free circulating DNA (fcDNA) quantitation that may improve the specificity of the prostate cancer prostate-specific antigen (PSA) screening test. MATERIALS AND METHODS: The patient group consisted of 241 men with elevated PSA/abnormal digital rectal exam (DRE), undergoing prostate biopsy. Serum fcDNA levels were measured by UV absorbance and PicoGreen. Results were compared to previously published quantitative polymerase chain reaction (qPCR) data. RESULTS: We found that levels of fcDNA measured by PicoGreen correlated well with those measured by qPCR (r=0.8552). In the patient group with PSA >4 to 10 ng/ml, those with fcDNA (PicoGreen) >53.1 ng/ml were at increased risk for prostate cancer compared to those with fcDNA ≤ 53.1 ng/ml. Moreover, we found that measuring fcDNA levels by PicoGreen does not compromise the negative predictive value, accuracy or specificity of the qPCR fcDNA test. CONCLUSION: If validated in larger studies, PicoGreen quantitation of fcDNA could serve as a simple method to aid in prostate cancer diagnosis.

5-azacytidine reverses drug resistance in bladder cancer cells.

BACKGROUND: Patients with metastatic and muscle-invasive bladder cancer are commonly treated with cisplatin. A significant proportion of patients develop disease progression after an initial response to chemotherapy. Presently there is no standard of care for such patients. We examined whether pretreatment with an epigenetic agent would result in reversal of drug resistance. MATERIALS AND METHODS: Methylation of proapoptotic and cell cycle genes in bladder cancer cells was examined. Cisplatin- and docetaxel-resistant cells were generated. The effect of target of methylation-induced silencing (TMS1) expression and pretreatment of wild-type and drug-resistant cells with 5-azacytidine on chemosensitivity was determined. RESULTS: Unidirectional crossresistance of cisplatin-resistant UMUC3 cells to docetaxel was observed. Recombinant expression of TMS1 or pre-treatment of wild-type and drug-resistant cells with 5-azacytidine resulted in enhanced sensitivity to cisplatin and docetaxel. CONCLUSION: Our results indicate that epigenetic therapy may restore sensitivity to chemotherapeutic agents in bladder cancer cells.

Methylation-mediated silencing of TMS1 in pancreatic cancer and its potential contribution to chemosensitivity.

BACKGROUND: Resistance to chemotherapeutic agents, resulting in part from epigenetic silencing of pro-apoptotic genes, is one of the causes of treatment failure of pancreatic cancer. We examined whether epigenetic silencing of target of methylation induced silencing 1 (TMS1) contributes to resistance to chemotherapy in pancreatic cancer. MATERIALS AND METHODS: Methylation analysis was performed by methylation-specific PCR (MS-PCR) and gene expression was analyzed by quantitative reverse transcriptase PCR (qRT-PCR). MIA PaCa-2 cells were transfected with pCMV6-XL5/TMS1 plasmid and the effect of TMS1 expression on sensitivity to gemcitabine and docetaxel was determined. Cell viability was measured using Cell Titer Blue assay. RESULTS: TMS1 expression was repressed in MIA PaCa-2 cells by DNA methylation. Up-regulation of TMS1 by recombinant gene expression in MIA PaCa-2 cells or by pre-treatment of these cells with 5-azacytidine resulted in enhanced sensitivity to gemcitabine and docetaxel. CONCLUSION: Our results suggest that TMS1 is a potential therapeutic target in pancreatic cancer.

Serum free circulating DNA is a useful biomarker to distinguish benign versus malignant prostate disease.

BACKGROUND: Free circulating DNA (fcDNA) has been shown to be elevated in serum of prostate cancer patients compared with benign controls. However, studies evaluating the role of fcDNA as a biomarker in a "representative" patient group who have undergone prostate cancer screening are lacking. Our study examined the use of serum fcDNA levels as a biomarker of prostate cancer in such a setting. METHODS: The study included 252 men, with prostate-specific antigen (PSA) levels >4 ng/mL and/or abnormal digital rectal exam. fcDNA levels in serum before prostate biopsy were quantitated by real-time PCR amplification of the glutathione S-transferase, pi, gene. RESULTS: Patients with PSA < or = 10 ng/mL with fcDNA > 180 ng/mL were at increased risk for prostate cancer compared with those with fcDNA < or =180 ng/mL (odds ratio, 4.27; 95% confidence interval, 2.05-8.88; P < 0.001; area under the curve, 0.742). The multivariate model including age, race, PSA, fcDNA, and interaction between fcDNA and PSA yielded a high negative predictive value of 93.1% and increased specificity of 33.1% compared with negative predictive value of 73.3% and specificity of 6.7% in the model excluding fcDNA. CONCLUSIONS: Our results indicate that fcDNA may improve the specificity of prostate cancer screening. IMPACT: Our study shows that adding fcDNA to prostate cancer screening can reduce the number of unnecessary prostate biopsies.

Methylation mediated silencing of TMS1 in breast cancer and its potential contribution to docetaxel cytotoxicity.

BACKGROUND: The tumor suppressor gene TMS1 (target of methylation-induced silencing) has been described in the literature as a pro-apoptotic gene. This study examined the methylation status of TMS1 in breast cancer cells and its potential role in sensitivity to docetaxel chemotherapy. MATERIALS AND METHODS: Methylation of the TMS1 promoter was examined by methylation-specific PCR (MS-PCR) and gene expression was analyzed by reverse transcriptase PCR (RT-PCR). Apoptosis was evaluated by annexin V/propidium iodide staining followed by flow cytometric analysis. RESULTS AND CONCLUSION: The TMS1 promoter was unmethylated in ZR75-1, MB-231 and MCF7 cells which expressed the gene and partially methylated in SKBR3 and Hs578t cells in which TMS1 expression was down-regulated. Treatment of SKBR3 and Hs578t cells with demethylating agents resulted in reactivation of the TMS1 gene. Pretreatment with 5-azacytidine increased sensitivity to docetaxel treatment in SKBR3 and Hs578t cells, indicating that TMS1 reactivation in these cells may contribute to docetaxel sensitivity.

Methylation-mediated repression of GADD45alpha in prostate cancer and its role as a potential therapeutic target.

Defects in apoptotic pathway contribute to uncontrolled proliferation of cancer cells and confer resistance to chemotherapy. Growth arrest and DNA damage inducible, alpha (GADD45alpha) is up-regulated on docetaxel treatment and may contribute to docetaxel-mediated cytotoxicity. We examined the mechanism of regulation of GADD45alpha in prostate cancer cells and the effect of its up-regulation on sensitivity to docetaxel chemotherapy. Expression of GADD45alpha in PC3 cells was higher than that in Du145 and LNCaP cells (17- and 12-fold, respectively; P < 0.05). Although the proximal promoter region was unmethylated in all three cell lines, methylation of a 4 CpG region upstream of the proximal promoter correlated inversely with gene expression levels. Methylation was reversed by treatment of Du145 and LNCaP cells with DNA methyltransferase inhibitors, leading to reactivation of GADD45alpha expression in these cells. The 5' 4 CpG region was also frequently methylated in prostate cancer tissues. Methylation of this region correlated inversely with gene expression in prostate cancer and benign prostate tissues. The methyl binding protein MeCP2 was associated with the methylated 4 CpGs in Du145 cells, and knockdown of MeCP2 in these cells (Du145 MeCP2(-)) led to a significantly increased expression of GADD45alpha (3-fold; P = 0.035) without affecting the methylation status of the gene. Enhanced sensitivity to docetaxel was observed by up-regulation of GADD45alpha in Du145 cells by recombinant expression of GADD45alpha or pretreatment with 5-azacytidine. Our results show that GADD45alpha is epigenetically repressed and is a potential target for treatment of prostate cancer.

The emerging role of epigenetics in urological cancers.

DNA methylation and histone modifications constitute the common epigenetic modifications in vertebrate genomes. The epigenetic changes are early event in the cancer development and are reversible. Over the last decade, the field of epigenetics has made considerable progress both in the diagnosis and treatment of variety of malignancies. Novel epigenetic markers are being studied, which have the potential as sensitive diagnostic and prognostic markers. DNA methylation has been identified as a powerful diagnostic tool in classification, detection and risk assessment of cancers. As DNA methylation is reversible, inhibitors of DNA methyl transferases and histone deacteylases have been designed for use in treatment of a variety of urological malignancies. Variety of drugs targeting epigenetic changes are being studied, which can be effective individually or in combination with other conventional drugs used in cancer therapy. The emerging area of epigenetic therapy holds great promise for novel chemotherapeutic and chemoprevention approaches against cancer.

Methylation-mediated silencing of genes is not altered by selenium treatment of prostate cancer cells.

BACKGROUND: The role of selenium in reducing the risk of multiple cancers has been described in the literature. Although reports have described the antiproliferative and pro-apoptotic function of selenium by up-regulation of genes in these pathways, information is lacking on the target mechanisms of selenium on specific genes. This study examines whether selenium treatment alters the methylation status of epigenetically silenced genes in prostate cancer cells. MATERIALS AND METHODS: Methylation of glutathione sulfotransferase pi (GSTP1) and Ras associated family 1A (RASSF1A) genes was studied using methylation sensitive PCR (MS-PCR). Gene expression was studied using Reverse Transcriptase PCR and Western Blotting. RESULTS AND CONCLUSION: Treatment of prostate cancer cells with selenium did not alter the expression of genes that were silenced by DNA methylation. Furthermore, the methylation status of these genes remained unaltered after treatment with seleno-DL-methionine.

Epigenetic targets in the diagnosis and treatment of prostate cancer.

Prostate cancer (PC) is one of leading cause of cancer related deaths in men. Various aspects of cancer epigenetics are rapidly evolving and the role of 2 major epigenetic changes including DNA methylation and histone modifications in prostate cancer is being studied widely. The epigenetic changes are early event in the cancer development and are reversible. Novel epigenetic markers are being studied, which have the potential as sensitive diagnostic and prognostic marker. Variety of drugs targeting epigenetic changes are being studied, which can be effective individually or in combination with other conventional drugs in PC treatment. In this review, we discuss epigenetic changes associated with PC and their potential diagnostic and therapeutic applications including future areas of research.

Developmentally regulated demethylase activity targeting the betaA-globin gene in primary avian erythroid cells.

Differential expression of globin genes has provided an interesting model system for better understanding commonly inherited diseases such as thalassemia. In the avian beta-type globin cluster (5'-rho-betaH-betaA-epsilon-3'), silencing of the embryonic rho-globin gene occurs concomitantly with the activation of the adult betaA-globin gene during embryonic development. DNA methylation is a dynamic process that regulates gene expression. We observed a progressive loss of methylation of betaA-globin gene, during avian embryonic development that was concurrent with the expression of the gene. The promoter and exon 1 regions of the template strand were completely demethylated, whereas residual methylation was retained in exons 2 and 3. Using a modified methylation-sensitive single-nucleotide primer extension (MS-SNuPE) assay, we observed stage-specific demethylase activity in the nuclear extracts of chicken red cells; activity in 5-, 8-, and 11-day-old erythroid cell nuclear extracts was 6, 76, and 24%, respectively. The demethylase targeted both hemimethylated and fully methylated substrates. Our findings demonstrate stage-specific demethylase activity in nuclear extracts from primary chicken erythroid cells that could target the fully methylated promoter of a developmentally regulated native gene.

Epigenetic targets in the diagnosis and treatment of prostate cancer

Prostate cancer (PC) is one of leading cause of cancer related deaths in men. Various aspects of cancer epigenetics are rapidly evolving and the role of 2 major epigenetic changes including DNA methylation and histone modifications in prostate cancer is being studied widely. The epigenetic changes are early event in the cancer development and are reversible. Novel epigenetic markers are being studied, which have the potential as sensitive diagnostic and prognostic marker. Variety of drugs targeting epigenetic changes are being studied, which can be effective individually or in combination with other conventional drugs in PC treatment. In this review, we discuss epigenetic changes associated with PC and their potential diagnostic and therapeutic applications including future areas of research.

Methylation mediated silencing of TMS1/ASC gene in prostate cancer.

BACKGROUND: Transcriptional silencing associated with aberrant promoter methylation has been established as an alternate pathway for the development of cancer by inactivating tumor suppressor genes. TMS1 (Target of Methylation induced Silencing), also known as ASC (Apoptosis Speck like protein containing a CARD) is a tumor suppressor gene which encodes for a CARD (caspase recruitment domain) containing regulatory protein and has been shown to promote apoptosis directly and by activation of downstream caspases. This study describes the methylation induced silencing of TMS1/ASC gene in prostate cancer cell lines. We also examined the prevalence of TMS1/ASC gene methylation in prostate cancer tissue samples in an effort to correlate race and clinico-pathological features with TMS1/ASC gene methylation. RESULTS: Loss of TMS1/ASC gene expression associated with complete methylation of the promoter region was observed in LNCaP cells. Gene expression was restored by a demethylating agent, 5-aza-2'deoxycytidine, but not by a histone deacetylase inhibitor, Trichostatin A. Chromatin Immunoprecipitation (ChIP) assay showed enrichment of MBD3 (methyl binding domain protein 3) to a higher degree than commonly associated MBDs and MeCP2. We evaluated the methylation pattern in 66 prostate cancer and 34 benign prostatic hyperplasia tissue samples. TMS1/ASC gene methylation was more prevalent in prostate cancer cases than controls in White patients (OR 7.6, p 0.002) while no difference between the cases and controls was seen in Black patients (OR 1.1, p 0.91). CONCLUSION: Our study demonstrates that methylation-mediated silencing of TMS1/ASC is a frequent event in prostate cancer, thus identifying a new potential diagnostic and prognostic marker for the treatment of the disease. Racial differences in TMS1/ASC methylation patterns implicate the probable role of molecular markers in determining in susceptibility to prostate cancer in different ethnic groups.