Microfluidics-based EGFR mutation detection and its implication in the resource-limited clinical setting.

Management of lung cancer today obligates a mutational analysis of the epidermal growth factor receptor (EGFR) gene particularly when Tyrosine Kinase Inhibitor (TKI) therapy is being considered as part of prognostic stratification. This study evaluates the performance of automated microfluidics-based EGFR mutation detection and its significance in clinical diagnostic settings. Formalin-fixed, paraffin-embedded (FFPE) samples from NSCLC patients (n = 174) were included in a two-phase study. Phase I: Validation of the platform by comparing the results with conventional real-time PCR and next-generation sequencing (NGS) platform. Phase II: EGFR mutation detection on microfluidics-based platform as part of routine diagnostics workup. The microfluidics-based platform demonstrates 96.5% and 89.2% concordance with conventional real-time PCR and NGS, respectively. The system efficiently detects mutations across the EGFR gene with 88.23% sensitivity and 100% specificity. Out of 144 samples analysed in phase II, the platform generated valid results in 94% with mutation detected in 41% of samples. This microfluidics-based platform can detect as low as 5% mutant allele fractions from the FFPE samples. Therefore the microfluidics-based platform is a rapid, complete walkaway, with minimum tissue requirement (two sections of 5 µ thickness) and technical skill requirement. The method can detect clinically actionable EGFR mutations efficiently and can be considered a reliable diagnostic platform in resource-limited settings. From receiving samples to reporting the results this platform provides accurate data without much manual intervention. The study helped to devise an algorithm that emphasizes effective screening of the NSCLC cases for EGFR mutations with varying tumour content. Thus it helps in triaging the cases judiciously before proceeding with multigene testing.

Surgical Tumor Resection Deregulates Hallmarks of Cancer in Resected Tissue and the Surrounding Microenvironment.

Surgery exposes tumor tissue to severe hypoxia and mechanical stress leading to rapid gene expression changes in the tumor and its microenvironment, which remain poorly characterized. We biopsied tumor and adjacent normal tissues from patients with breast (n = 81) and head/neck squamous cancers (HNSC; n = 10) at the beginning (A), during (B), and end of surgery (C). Tumor/normal RNA from 46/81 patients with breast cancer was subjected to mRNA-Seq using Illumina short-read technology, and from nine patients with HNSC to whole-transcriptome microarray with Illumina BeadArray. Pathways and genes involved in 7 of 10 known cancer hallmarks, namely, tumor-promoting inflammation (TNF-A, NFK-B, IL18 pathways), activation of invasion and migration (various extracellular matrix-related pathways, cell migration), sustained proliferative signaling (K-Ras Signaling), evasion of growth suppressors (P53 signaling, regulation of cell death), deregulating cellular energetics (response to lipid, secreted factors, and adipogenesis), inducing angiogenesis (hypoxia signaling, myogenesis), and avoiding immune destruction (CTLA4 and PDL1) were significantly deregulated during surgical resection (time points A vs. B vs. C). These findings were validated using NanoString assays in independent pre/intra/post-operative breast cancer samples from 48 patients. In a comparison of gene expression data from biopsy (analogous to time point A) with surgical resection samples (analogous to time point C) from The Cancer Genome Atlas study, the top deregulated genes were the same as identified in our analysis, in five of the seven studied cancer types. This study suggests that surgical extirpation deregulates the hallmarks of cancer in primary tumors and adjacent normal tissue across different cancers. IMPLICATIONS: Surgery deregulates hallmarks of cancer in human tissue.

BRAF V600E Mutations and Beyond: A Molecular Perspective of Melanoma from a Tertiary Cancer Referral Center of India.

Vaibhavi VengurlekarObjectives Malignant melanoma demonstrates frequently occurring mutations of genes in the serine/threonine kinase pathway, namely BRAF, NRAS, and neurofibromin 1. There is rare documentation of a detailed analysis of these mutations in cases of melanoma among Indian patients. We present molecular features in cases of malignant melanoma, diagnosed at a tertiary cancer referral center in India, over a period of 8 years (2011-2018). Materials and Methods This study was performed on formalin fixed paraffin embedded tissues of 88 histologically confirmed cases of malignant melanoma. BRAF gene alterations were studied by both Sanger sequencing and real-time polymerase chain reaction techniques ( n = 74). Molecular testing for BRAF and NRAS gene alterations was accomplished in 74/88 cases (80%). Molecular test results were correlated with clinicopathological features using IBM SPSS Statistical software 25.0. Results The age ranged from 13 to 79 years (median = 57), with a M:F ratio of 1.4:1. BRAF mutations were observed in 12/74 (16.21%) patients, including V600E ( n = 7), A594T ( n = 1), T599 = ( n = 2), V600K ( n = 1), and Q612P ( n = 1), while NRAS mutations were observed in 6/38 (15.7%) patients. Among various subtypes, nodular melanoma was the most frequent subtype (33%) among cutaneous malignant melanomas. Among non-cutaneous melanomas, mucosal melanomas were observed in 37.5% of cases. Conclusion This constitutes one of the few reports on comprehensive analysis of molecular alterations underlying melanomas in Indian patients. A larger sample size, with more extensive molecular markers, would yield additional information on the disease manifestation.

Risk factors for the development of triple-negative breast cancer versus non-triple-negative breast cancer: a case-control study.

The risk factors for breast cancer have been defined in several studies but there is deficient data for specific subtypes. We report here the pathological characteristics of a breast cancer cohort and risk factors for patients with triple-negative disease. In this case-control study, a prospective breast cancer cohort was evaluated for demographic, reproductive, obesity-related and other risk factors using a validated questionnaire. Tumors were characterized for routine pathological characteristics and immunohistochemical markers of basal-like breast cancer. Patients with triple-negative breast cancer (TNBC) constituted cases and those with non-TNBC were controls. Odds ratios (OR) were calculated for each risk factor and independent associations were tested in an unconditional logistic regression analysis. Between 2011 and 2014, 1146 patients were recruited, of whom 912 [TNBC 266 (29.1%), non-TNBC 646 (70.9%)] with sufficient pathology material were analysed. Reproductive factors of parity, breastfeeding, age-at-menarche, age at first full-term pregnancy and oral contraceptive use were not significantly associated with TNBC. Higher body mass index (BMI > 24.9 vs ≤ 24.9, OR 0.89, 95%CI 0.63-1.24, p = 0.49) was not significantly associated while lesser waist circumference (> 80 cm vs ≤ 80 cm, OR 0.64, 95%CI 0.45-0.9, p = 0.012) and lower waist-to-hip ratio were significantly associated (> 0.85 vs ≤ 0.85, OR 0.72, 95%CI 0.51-1.0, p = 0.056), with TNBC. History of tobacco use was not significantly associated while lower socio-economic status was borderline associated with TNBC (socio-economic category > 5 versus ≤ 5, OR 0.73, 95%CI 0.50-1.06, p = 0.106). No factor was significant after adjustment for covariates. Central obesity seems to be preferentially associated with non-TNBC, and lower socio-economic status with TNBC in India, while most other conventional risk factors of breast cancer show no significant association with TNBC versus non-TNBC.

Targeted molecular profiling of solid tumours-Indian tertiary cancer centre experience.

PURPOSE: Molecular Profiling of solid tumours is extensively used for prognostic, theranostic, and risk prediction. Next generation sequencing (NGS) has emerged as powerful method for molecular profiling. The present study was performed to identify molecular alterations present in solid tumours in Indian tertiary cancer centre. METHODS: Study included 1140 formalin Fixed paraffin embedded samples. NGS was performed using two targeted gene panels viz. Ampliseq Focus panel and Sophia Solid Tumor Plus Solution. Data was analyzed using Illumina's Local Run Manager and SOPHiA DDM software. Variant interpretation and annotations were done as per AMP/ACMG guidelines. RESULTS: Total 896 cases were subjected to NGS after excluding cases with suboptimal nucleic acid quality/quantity. DNA alterations were detected in 64.9% and RNA fusions in 6.9% cases. Among detected variants, 86.7% were clinically relevant aberrations. Mutation frequency among different solid tumours was 70.8%, 67.4%, 64.4% in non-small cell lung (NSCLC), lung squamous cell carcinomas and head neck tumours respectively. EGFR, KRAS, BRAF, ALK and ROS1were commonly altered in NSCLC. Gastrointestinal tumours showed mutations in 63.6% with predominant alterations in pancreatic (88.2%), GIST (87.5%), colorectal (78.7%), cholangiocarcinoma (52.9%), neuroendocrine (45.5%), gall bladder (36.7%) and gastric adenocarcinomas (16.7%). The key genes affected were KRAS, NRAS, BRAF and PIK3CA. NGS evaluation identified co-occurring alterations in 37.7% cases otherwise missed by conventional assays. Resistance mutations were detected in progressive lung tumours (39.5%) against EGFR TKIs and ALK/ROS inhibitors. CONCLUSION: This is the largest Indian study on molecular profiling of solid tumours providing extensive information about mutational signatures using NGS.

Comparative Assessment of DNA Extraction Techniques From Formalin-Fixed, Paraffin-Embedded Tumor Specimens and Their Impact on Downstream Analysis.

OBJECTIVES: Good-quality nucleic acid extraction from formalin-fixed, paraffin-embedded (FFPE) specimens remains a challenge in molecular-oncopathology practice. This study evaluates the efficacy of an in-house developed FFPE extraction buffer compared with other commercially available kits. METHODS: Eighty FFPE specimens processed in different surgical pathology laboratories formed the study sample. DNA extraction was performed using three commercial kits and the in-house developed FFPE extraction buffer. DNA yield was quantified by a NanoDrop spectrophotometer and Qubit Fluorometer, and its purity was measured by the 260/280-nm ratio. A fragment analyzer system was used for accurate sizing of DNA fragments of FFPE DNA. The downstream effects of all extraction methods were evaluated by polymerase chain reaction (PCR) and Sanger sequencing. RESULTS: In comparison with the commercial kits, the in-house buffer yielded higher DNA quantity and quality number (P < .0001). In addition, DNA integrity and fragment size were preserved in a significantly greater number of samples isolated with the in-house buffer (P < .05). The target PCR amplification rate with the in-house buffer extracted samples was also significantly higher, with 98% of the samples showing interpretable sequencing results. CONCLUSIONS: The in-house developed FFPE extraction buffer performed superior to other methods in terms of suitability for downstream applications, time, cost-efficiency, and ease of performance.

Impact of COVID-19 on quality checks of solid tumor molecular diagnostic testing-A surveillance by EQAS provider in India.

BACKGROUND: Molecular tests in solid tumours for targeted therapies call for the need to ensure precision testing. To accomplish this participation in the External Quality Assessment Program (EQAS) is required. This evaluates the consistency of diagnostic testing procedures and offers guidance for improving quality. Outbreak of COVID-19 pandemic led to worldwide lockdown and disruption of healthcare services including participation in EQAS.The present study describes the extended scope of EQAS offered byMPQAP (Molecular Pathology Quality Assurance Program), the first proficiency test provider for solid tumor diagnostics in India. The study surveys the preparedness of molecular testing laboratories in routine diagnostics and participation for quality assessment scheme. METHODS: A documented guideline for measures and precautions to be carried by testing laboratories in performing routine diagnostic tests during the lockdown period were charted and distributed to all MPQAP participant centres. A survey was conducted for MPQAP participants to check whether laboratories were involved in COVID-19 testing and to evaluate the impact of lockdown on the operations of diagnostics procedures. From the acquired response of the survey, 2 cycles out of initially proposed 11 cycles were executed with transformed approach using digital tools and image interpretation modules. FINDINGS: Out of 25 solid tumour testing laboratories registered as participants, 15 consented to participate in survey. The summary of survey conveyed the impact of COVID-19onroutine operations of diagnostics tests such as shortcomings in inventory and human resource management. Thirteen participants showed active willingness and consented to participate in EQAS test scheme. INTERPRETATIONS: The survey findings and assessment of EQAS cycles endorsed the quality testing procedures carried by participating laboratories throughout the lockdown. It highlighted the utility of EQAS participation during pandemic along with emphasis on safety measures for continual improvement in quality of diagnostic services.

Impact of Molecular Tumor Board on the Clinical Management of Patients With Cancer.

PURPOSE: Multidisciplinary molecular tumor boards (MTBs) help in interpreting complex genomic data generated by molecular tumor profiling and improve patients' access to targeted therapies. The purpose of this study was to assess the impact of our institution's MTB on the clinical management of patients with cancer. METHODS: This study was conducted at a tertiary cancer center in India. Cases to be discussed in the MTB were identified by molecular pathologists, scientists, or oncologists. On the basis of the clinical data and molecular test reports, a course of clinical management was recommended and made available to the treating oncologist. We determined the proportion of patients who were recommended a change in the clinical management. We also assessed compliance of the treating oncologists with MTB recommendations. RESULTS: There were 339 discussions for 328 unique patients. The median age of the cohort was 54 years (range 17-87), and the majority of the patients were men (65.1%). Of 339 cases, 133 (39.2%) were recommended continuation of ongoing therapy while the remaining 206 (60.7%) were recommended a change in clinical management. Compliance with MTB recommendations for a change in clinical management was 58.5% (79 of 138 evaluable cases). Compliance and implementation for MTB's recommendation to start a new therapy in 104 evaluable cases were 60.5% and 44.2%, respectively. A total of 248 biopsies had at least one actionable mutation. A total of 646 mutations were identified in the cohort, with EGFR being the most frequently altered gene. CONCLUSION: MTBs help in interpreting results of molecular tests, understanding the significance of molecular abnormalities, and assessing the benefits of available targeted therapies and clinical trials in the management of patients with targetable genetic alterations.

Incidence of SARS-CoV-2 infection among asymptomatic patients undergoing preoperative COVID testing prior to cancer surgery: ASPECT study.

BACKGROUND AND OBJECTIVES: The COVID-19 pandemic, with high rate of asymptomatic infections and increased perioperative complications, prompted widespread adoption of screening methods. We analyzed the incidence of asymptomatic infection and perioperative outcomes in patients undergoing cancer surgery. We also studied the impact on subsequent cancer treatment in those with COVID-19. METHODS: All patients who underwent elective and emergency cancer surgery from April to September 2020 were included. After screening for symptoms, a preoperative test was performed from nasopharyngeal and oropharyngeal swabs before the procedure. Patients were followed up for 30 days postoperatively and complications were noted. RESULTS: 2108 asymptomatic patients were tested, of which 200 (9.5%) tested positive. Of those who tested positive, 140 (70%) underwent the planned surgery at a median of 30 days from testing positive, and 20 (14.3%) had ≥ Grade III complications. Forty (20%) patients did not receive the intended treatment; 110 patients were retested in the Postoperative period, and 41 (37.3%) tested positive and 9(22%) patients died of COVID-related complications. CONCLUSION: Routine preoperative testing for COVID-19 helps to segregate patients with asymptomatic infection. Higher complications occur in those who develop COVID-19 in postoperative period. Prolonged delay in surgery after COVID infection may influence planned treatment.

The Prevalence of BRAF, PIK3CA, and RAS Mutations in Indian Patients with Colorectal Cancer.

Omshree ShettyVikas OstwalIntroduction The present study evaluates the mutation pattern and frequency of BRAF , PIK3CA and RAS in colorectal carcinoma observed in the tertiary cancer center in India. Materials and Methods Consecutive cases of colorectal adenocarcinoma ( n = 330) registered from January 2015 to December 2019 (5-year duration) were selected for the study. Molecular analysis for BRAF . PIK3CA (exon 9 and 20) and RAS ( KRAS & NRAS ) was performed on representative formalin-fixed paraffin-embedded tissues by Sanger sequencing. Results were correlated with clinicopathological features. Patient overall survival (OS) was obtained using Kaplan-Meier method. Results The study cohort was in the age range of 22 to 81 years (median age: 52 years) that included 202 males and 96 females (male: female ratio 2.1:1). BRAF V600E mutation was observed in three cases (1%), while 17 cases (5.7%) had mutations in the PIK3CA gene (exon 9 or exon 20). Mutation analysis for RAS gene ( KRAS & NRAS ) was observed among 42 (15.4%) cases with KRAS mutation and 11 (4%) cases were positive for NRAS mutations. Among RAS, KRAS G12D was the predominant mutation. Median OS with wild-type RAS was 46.6 months (95% confidence interval [CI]: 22.4-70.8), while for RAS mutated patients, it was 25.6 months (95% CI: 16.7-34.5), hazard ratio: 1.7 (95% CI: 1.1-2.7, p = 0.025). Conclusion This study evaluated the prevalence of BRAF, PIK3CA and RAS mutations in the Indian cohort and its impact on clinical behavior. There was lower incidence of BRAF mutations in this cohort and PIK3CA mutation (single) did not impact survival of the patients.

TERT Promoter Mutation in Adult Glioblastomas: It's Correlation with Other Relevant Molecular Markers.

BACKGROUND: Telomerase reverse transcriptase promoter (pTERT) mutation is a dominant altered telomere maintenance mechanism in primary glioblastomas (GBMs). OBJECTIVE: The aim of this study was to correlate pTERT mutations with clinico-histological features and other molecular markers (p53 protein-expression, ATRX protein-expression, IDH mutations, EGFR gene amplification and MGMT methylation) in adult GBMs. MATERIALS AND METHODS: Evaluated for histological patterns, p53 and ATRX protein expression by immunohistochemistry (IHC), IDH mutations by IHC followed by sequencing in IHC negative cases, EGFR gene amplification by fluorescence in situ hybridization, MGMT promoter methylation by methylation-specific PCR and pTERT mutation by sequencing. RESULTS: A total of 155 adult supratentorial GBMs [age-range 20-80 years] formed study cohort. 15.6% were IDH1R132 mutated, none were IDH2R172 mutated and 27% were EGFR amplified. 43% were MGMT methylated and were more common with IDH-mutation (mIDH) than EGFR amplification. 90% of mIDH (but no EGFR amplified) cases showed ATRX-loss. 43.5% were pTERT mutated (C228T was the commonest type) and were mutually exclusive with ATRX-loss. 14% of mIDH and 42% of EGFR amplified cases showed pTERT mutation, the latter was more commonly pMGMT unmethylated (63.6%). CONCLUSIONS: 43.5% of the GBMs showed pTERT mutation (C228T was commonest; 72%). pTERT mutations were mutually exclusive with ATRX protein loss, more commonly associated with IDH wild type and EGFR amplified GBMs.

COVID 19 pandemic testing time - Crisis or opportunity in disguise for India?

The current SARS-CoV-2 infection or the COVID 19 pandemic has taken the world by storm, where the best health care systems in the world seem to be overwhelmed and still this virus is eluding us as we are compelled to explore the preventive and/or therapeutic interventions to control the disease outbreak as well as to prevent deaths. In parallel to clinical services, laboratories have been overwhelmed with task of keeping up with ever increasing demand for testing. Real time PCR detection of COVID19 is the gold standard method, however, has certain shortcomings in terms of availability of infrastructure, reagents, consumables, and technical expertise. All these have paved the way for the alternative testing algorithms and strategies. Countries like United States and Italy have struggled with these issues. India has been criticized for not testing enough and not adopting the right policy, but has been managing the disease within its resource limited health care system to a fair extent. The present review provides the Indian perspective of COVID 19 testing, the journey from not testing enough in the past to a vast expanse and depth of testing in present time.

A Case of Inv(1)(q23q31) TPR-NTRK1 Fusion-Positive Spindle Cell Neoplasm in an Infant-Uncovered by Next-Generation Sequencing: Diagnostic Challenge, Review, and Therapeutic Implications.

Lately, NTRK-positive mesenchymal tumors are being increasingly identified, mostly in pediatric patients, in view of associated treatment implications, especially in recurrent and unresectable tumors. A 1-year-old male child presented with a rapidly growing tumor mass in his cervical region of 2 months duration. Radiologic imaging disclosed a tumor measuring 11 cm in size, almost filing his right neck spaces. Review of biopsy sections revealed a cellular tumor comprising spindle cells arranged in sheets and fascicles with interspersed collagenous strands and areas of adipocytic, myxoid, and hyaline degeneration. Immunohistochemically, tumor cells were diffusely positive for CD34 and S100 protein. Subsequently, on testing the tumor for a solid tumor gene panel by next-generation sequencing, it was found to be positive for inv(1)(q23q31) TPR-NTRK1 fusion. Furthermore, tumor cells displayed NTRK1 gene rearrangement by fluorescence in situ hybridization technique. The patient was offered chemotherapy; however, he had a rapid local progression, leading to respiratory obstruction; he then succumbed to the disease. The present case underpins the value of next-generation sequencing as a useful technique for uncovering NTRK-fusion-positive mesenchymal tumors. Review of similar cases, diagnostic challenge, and treatment implications in such cases are discussed.

Primary intracranial Ewing sarcoma/ peripheral primitive neuroectodermal tumor, an entity of unacquaintance: a series of 8 cases.

PURPOSE: The purpose is to highlight the primary intracranial (meningeal-based) occurrence of Ewing sarcoma/primitive neuroectodermal tumor (ES/PNET). METHODS: This report is a collation of clinicopathological features of eight cases of molecularly and clinicoradiologically confirmed primary (non-metastatic) intracranial (non-osseous) meningeal ES/PNET. RESULTS: The age range was 1 to 33 years with a median age of 9 years. Male to female ratio was 0.6:1. All patients were diagnosed on the debulking surgical material (gross total resection, 2 cases; subtotal resection, 6 cases) and showed primitive embryonal histomorphology with diffuse membranous CD99 immunoexpression and EWSR1 gene rearrangement by fluorescence in situ hybridization. Seven of them showed a typical FISH pattern of split signals with break-apart probe, while one showed an unusual signal pattern of loss of green signals. EFT-2001 adjuvant protocol was followed along with focal radiotherapy (RT) in all cases (except case 8, full course of chemotherapy could not be completed). Two cases had local recurrence-one of them died of disease recurrence before the administration of further treatment. CONCLUSION: This series adds non-osseous intracranial site to the list of uncommon sites of occurrence for ES/PNET and more importantly emphasizes the need to be considered in a differential list of primary intracranial primitive embryonal tumors before embarking as primary central nervous system (CNS) embryonal tumor, NOS.

Comparison between Fluorescence in-situ Hybridization (FISH), Reverse Transcriptase PCR (RT-PCR) and fragment analysis, for detection of t (X; 18) (p11; q11) translocation in synovial sarcomas.

BACKGROUND: Synovial sarcoma (SS) is an aggressive, but a relatively chemosensitive soft tissue sarcoma, characterized by a specific, t (X;18)(p11;q11) translocation, leading to formation of SS18-SSX chimeric transcript. This translocation can be detected by various techniques, such as fluorescence in-situ hybridization (FISH), reverse transcriptase PCR (RT-PCR) and fragment analysis. OBJECTIVES: To compare the results of detection of t (X;18)(p11;q11) translocation, across three different platforms, in order to determine the most optimal and sensitive technique. METHODS: Formalin-fixed paraffin embedded (FFPE) tissue sections of 45 soft tissue sarcomas were analyzed, including 16 cases of SS confirmed by histopathology, immunohistochemistry and molecular technique (s)(Group 1); 13 cases, wherein SS was one of the differential diagnosis, preceding molecular testing (Group 2) and 16 cases of various other sarcomas (Group 3). Various immunohistochemical (IHC) markers studied, including INI1/SMARCB1. All cases were tested for t (X;18) translocation, by fragment Analysis, FISH and RT-PCR. RESULTS: There were 23 cases of SS, including 16 of group 1 and 7 of group 2. By fragment analysis, t (X;18)(p11;q11) translocation was detected in 22/23 cases (95.6%). By FISH, SS18 gene rearrangement was detected in 18/22 cases (78.2%), whereas by RT-PCR, SS18-SSX transcripts were detected in 15/23 cases (65.2%). Immunohistochemically, a unique "weak to absent"/reduced INI1 immunostaining pattern was exclusively observed in 12/13 cases of SS (92.3%). Fragment analysis and FISH were relatively more sensitive techniques. Unique "weak to absent"INI1 immunoexpression significantly correlated with positive t (X;18) translocation results (P = 0.0001). CONCLUSION: The present study constitutes first such study from our subcontinent. Fragment analysis is a promising technique for detection of t (X;18)(p11;q11) translocation. FISH and INI1 immunostaining pattern were also relatively more sensitive, over RT-PCR.

Pilocytic astrocytomas: BRAFV600E and BRAF fusion expression patterns in pediatric and adult age groups.

PURPOSE: Pilocytic astrocytomas (PCAs) are characterized by two dominant molecular alterations of the BRAF gene, i.e., BRAFV600E mutation and KIAA1549-BRAF fusions which show a differential pattern of frequency across different age-groups. METHODS: Formalin-fixed paraffin-embedded tissues of 358 (pediatric 276 and adult 82) consecutive PCAs were evaluated for BRAFV600E mutation by Sanger sequencing and KIAA1549:BRAF fusion transcripts (KIAA1549:BRAF 16-9, KIAA1549:BRAF 15-9, and KIAA1549:BRAF 16-11) by reverse transcriptase polymerase chain reaction, which were correlated with different clinicopathological features. RESULTS: BRAFV600E mutation was detected in 8.9% pediatric and 9.75% adult PCAs, whereas 41.1% and 25.7% of pediatric and adult cases showed KIAA1549-BRAF fusions respectively. BRAFV600E did not show any statistically significant correlation with any of the clinical parameters (age, location, and gender). KIAA1549:BRAF fusions showed a significant statistical association with the pediatric age group and cerebellar location. KIAA1549-BRAF 16-9 was the commonest variant and was predominantly associated with cerebellar location than non-cerebellar whereas fusion variant 15-9 negatively correlated with cerebellar locations. CONCLUSIONS: The present study showed overall frequency of 53.5% and 37.3% BRAF alterations in pediatric and adult PCA cases respectively. BRAF fusion in PCA cases showed a different distribution pattern across age groups and locations; while no such differential pattern was observed for BRAFV600E.

MGMT gene promoter methylation and its correlation with clinicopathological parameters in glioblastomas.

BACKGROUND: MGMT (O6-methyl guanine DNA methyl transferase) promoter hypermethylation is a prognostic and predictive biomarker for glioblastomas (GBM). AIMS: To evaluate the frequency of MGMT methylation status in a single institute series of 134 GBMs and correlate it with clinical (age, sex, location, survival) and other molecular parameters [such as p53 expression, alpha thalassemia/mental retardation syndrome X-linked (ATRX) expression, isocitrate dehydrogenase (IDH) 1R132H mutation, and epidermal growth factor receptor (EGFR) gene amplification]. RESULTS: One hundred and thirty-four GBMs were evaluated by methylation-specific polymerase chain reaction (MSP) for MGMT promoter methylation status. The results were correlated with the above mentioned clinicopathological parameters. MGMT gene promoter methylation was identified in 49.2% (66/134) GBMs, and was significantly associated with IDH1R132H mutation (14/66; 21%; P - value, 0.01) and ATRX loss (15/66; 23%; P - value, 0.01). Confluent necrosis was found to be significantly associated with MGMT unmethylation status (P - value: 0.002). Multivariable logistic regression analysis showed confluent necrosis as a single independent predictor (odds ratio [OR], 2.5; confidence interval [CI], 1.0-5.8; P - value, 0.04) of MGMT unmethylation status among all the parameters studied. CONCLUSIONS: The frequency of MGMT promoter methylation in GBMs was 49.2%, which was significantly associated with IDHR132H mutation and ATRX loss. In addition, the presence of confluent necrosis was significantly associated with MGMT unmethylation and was found to be an independent predictor of the same.

Evidence for the association of Epstein-Barr Virus in breast cancer in Indian patients using in-situ hybridization technique.

Epstein-Barr Virus (EBV) is etiologically linked to Burkitt lymphoma (BL), nasopharyngeal carcinoma, post-transplant lymphomas, Hodgkin disease, and possibly other tumors. However, the association of oncogenic EBV with breast carcinoma (BC) is still controversial and a matter of debate. We aimed to study the presence of EBV genome in BC cases in Indian patients and its association with the clinicopathological features. The formalin fixed paraffin embedded tissues from 83 women with primary invasive BC were studied for the presence of EBV by in-situ hybridization (ISH) technique for Epstein-Barr Virus Encoded RNA (EBER) with appropriate controls. Correlation of EBER-ISH positivity with clinicopathological features was performed using Fisher exact test and P<.05 was considered as significant. Eighty-three BC cases were comprised of 47 (56.5%) triple negative breast cancers (TNBC), 17 (20.5%) hormone positive and 19 (22.9%) HER2 positive cases. Of 83 cases, 25 cases (30.1%) were positive for EBER-ISH test. The positivity was restricted to the tumor cells and not seen in the surrounding breast lobules. EBER-ISH positivity was statistically associated with larger tumor size (52.6% in >5 cm tumors vs 19.3% in ≤5 cm; P=.014) and with TNBCs (21/47 [44.7%] in TNBCs vs 4/36 [11.1%] in non-TNBCs; P=.001). A possible causal association of EBV in BC cases in Indian patients is suggested by high frequency of EBER-ISH positivity noted in our study. This might have therapeutic significance because of the possible role of EBV specific cytotoxic T cells in targeting EBV associated tumor cells and can be considered as a potential targeted therapy. To the best of our knowledge, this is the first study from India to address this issue using EBER-ISH technique.

Impact of WHO 2016 update of brain tumor classification, molecular markers and clinical outcomes in pleomorphic xanthoastrocytoma.

We present outcomes of pleomorphic xanthoastrocytoma (PXA) and correlate the impact of clinical, pathologic and molecular markers. Between 2006 and 2016, 37 patients with histologically verified PXA form the study cohort. All underwent maximal safe resection; those who had good resection and young age were observed. Adjuvant radiotherapy was given in patients with some atypical features such as high MIB-1 index (> 5%), residual disease or at recurrence. Patients with anaplastic PXA were administered adjuvant radiotherapy and systemic therapy. Median age at diagnosis was 20 years (range 4-45). At median follow-up of 33 months, 3-year and 5-year overall survival (OS) was 80.2 and 74% respectively. Patients who underwent GTR (23 cases, 62%) had significantly better 3-year PFS of 85.6% compared to 32.3% (p = 0.001) achieved with STR (13 cases, 35%). PFS was significantly superior in PXA grade II as compared to anaplastic PXA group (3-year estimates 80.2 vs. 32%; p = 0.007). 13 out of 27 patients where BRAFV600E testing was successful showed a mutation (48%). 3-year PFS and OS survival in BRAFV600E mutated patients was 51.9 and 76.9% compared to 73 and 75% in BRAFV600E non-mutated patients, respectively. No patient had IDH1 mutation. This data may provide valuable insights and act as a benchmark for future studies.