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Objectives 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.

Introduction 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.

With an advancement in the field of molecular diagnostics, there has been a profound evolution in the testing modalities, especially in the field of oncology. In the past decade, sequencing technology has evolved drastically with the advent of high-throughput next-generation sequencing (NGS). Subsequently, the single-gene tests have been replaced by multigene panel-based assays, deep sequencing, massively parallel whole genome, whole-exome sequencing, and so on. NGS has provided molecular diagnostics professionals a wonderful tool to explore and unearth the genetic alterations, underpinning the pathophysiology of the disease. However, this development has posed new challenges which consist of the following; understanding the technology, types of platforms available, various sequencing strategies, bioinformatics and data analysis algorithm, reporting of various variants, and validation of assays and overall for developing NGS assay for clinical utility. The challenges involved sometimes impede development of these high-end assays in laboratories. The present article provides a broad overview of our journey in setting up the NGS assay in a molecular pathology laboratory at a tertiary care oncology center. We hereby describe various important points and steps to be followed while working on the NGS setup, right from its inception to final drafting of the reports, with inclusion of various validation steps. We aim at providing a beginner’s guide to set up NGS assays in the laboratory using recommended best practices and various international guidelines.

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.

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.

The purpose is to highlight the primary intracranial (meningeal-based) occurrence of Ewing sarcoma/primitive neuroectodermal tumor (ES/PNET). 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. 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. 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.

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.

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.

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.

Lately

Isocitrate dehydrogenase (IDH1/IDH2) mutations in gliomas of WHO grade II/III and secondary glioblastoma are almost always heterozygous missense mutations. Here, we report an extremely rare case of homozygous IDH1R132H mutation in a recurrent WHO grade III anaplastic astrocytoma. The authors here also review the relevant literature for the possible metabolic impact of homozygous IDH1/2 mutations in the gliomagenesis.

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. 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. 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. 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.

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.

INTRODUCTION Molecular subgroups of pediatric medulloblastomas are distinctive in infantile and non-infantile age-groups. METHODS Real-time quantitative PCR based GEP of customized 12 protein-coding genes was performed on 206 FFPE childhood medulloblastoma samples. FISH for MYC amplification, monosomy 6 and sequencing for CTNNB1 exon 3 mutation were done in relevant cases. H&E and reticulin-stained slides were used for histological subtyping. p53-protein immunoreactivity pattern was noted. RESULTS Infantile (n=33) comprised 57.6% SHH-activated (desmoplastic: 73.7%; MBEN: 15.8% and classic: 10.5%), 21.2% group 3 (large cell/anaplastic [LCA]: 28.6% and none were desmoplastic) and 12% group 4. 40% of group 3 patients died of disease and 21% of the SHH-activated (all desmoplastic) had subsequent local recurrence. Non-infantile (n=173) comprised 19.4% WNT-activated, 12.9% SHH-activated (15% classic, 30% desmoplastic, 10% paucinodular), 19.4% group 3 (63.3% classic & 26.7% LCA), 48.4% group 4 (73.3% classic, 5.3% desmoplastic, 10.7% paucinodular & 1.4% LCA), and non-WNT/non-SHH (NWNS), NOS (n=14,9%) and unclassified (n=4,2.6%). None of WNT-activated were desmoplastic/LCA histology. Non-infantile WNT-activated and group 3 MBs showed 90% monosomy 6 & CTNNB1 mutation, and 16.7% MYC-amplification respectively. 17.4% (13% spinal, 4.4% local) WNT-activated, 31% (12.5% local, 18.5% distant [spinal: 12.5%, intracranial:6%]) SHH-activated, 27% (18% both spinal and local, 9% spinal) group 3 and 31.5% (7.4% local, 5.5% intracranial, 11.2% spinal, 7.4% both spinal and local) group 4 showed metastases during follow up. CONCLUSIONS SHH-activated and group 3 are the common infantile subgroups but group 4 is not non-existent in infantile age. No desmoplastic (including paucinodular) histological subtype is of WNT- activated and group 3.

BACKGROUND Increasing knowledge on pilocytic astrocytoma (PCA) biology now points towards an aberration in BRAF/MAPK/ERK pathway which has both diagnostic and therapeutic implications. This study was done to note the impact of BRAF aberrations on clinical outcome in childhood PCA. METHODS FFPE tissues of all childhood PCA diagnosed during 2011–2017 were evaluated for BRAFV600E mutation by Sanger sequencing and KIAA1549 fusion transcripts (16–9;15–9;16-11) by reverse transcriptase polymerase chain reaction. Children undergoing gross tumor resection received no adjuvant treatment. Unresectable tumors (only biopsy) and NF-1 associated PCAs, were treated if clinically indicated. Only patients with documented therapy details/followup were included for analysis. STUDY RESULTS Ninety-eight patients (median age-7.7yrs; boy:girl ratio-1.4) were included. Major sites were: Cerebellum-37(38%), 3rd Ventricle-26(27%), Cerebrum-15(15%). While BRAFV600E mutation was noted in 7/89(8%) specimens, BRAF-fusions were found in 34/85(40%). Following surgery/biopsy, 23(24%) and 21(22%) received adjuvant chemotherapy and radiotherapy respectively. The 1-year/3-year/5-year-EFS of the overall cohort was 90.7%/81.3%/67.4% respectively. Cerebellar tumors did better vis-à-vis other sites(5yr-EFS:74.3% v/s 66.4%;p=0.403). The 5yr-EFS of BRAF-fusion positive tumors (34), tumors without any BRAF aberration (40) and BRAFV600E mutant tumors (7) was 84.8%/ 69.6%/ 42.9% (p=0.215). CONCLUSIONS BRAF-fusion and BRAFV600E mutation were associated with good and poor outcomes respectively. Lack of statistical significance could be attributed to use of radiation as planned therapy in patients from earlier years. Data on BRAF aberrations in PCAs aids decision making regarding adjuvant therapy and choosing appropriate salvage-therapy especially in relapsed/refractory PCAs.

BACKGROUND Ependymomas are heterogenous group of tumours with variable clinical course and diverse molecular features. RELA fusion status has been reported to have prognostic impact in ST-EPEN. Our retrospective study analysed the prevalence and clinical impact of RELA fusion in childhood ST-EPEN at our centre. STUDY METHODS FFPE tissues of all childhood ST-EPEN diagnosed during 2011–2017 were evaluated for RELA fusion 1/2 by RT-PCR. Children were treated as per guidelines by the Neuro-oncology multidisciplinary team. Outcomes were correlated with RELA fusion, histological features and immunohistochemical parameters(L1CAM expression and Mib-1 index). Only patients with therapy details were included. RESULTS A total of 37 patients(0–50 years) with ST-EPEN were included(median age-10.2 years; boy:girl ratio-1.4:1)for analysis. Histological grade II, II/III and III was seen in 4(11%),2(5%) and 31(84%) patients respectively. Mib-1 index was assessable in 33 patients of which, 9 patients (24%) had a Mib-1 index >20%. RELA fusion was detected in 13(35%)tumors. The 3-year and 5-year EFS/OS of the overall cohort was 64.2%/83.6% and 60.1%/73.1% respectively. The 3-year/5-year EFS of RELA-positive tumors was inferior compared to RELA-negative tumours (53.8%/36% v/s 62.6%/53.6%; p=0.391). The 3-year/5-year EFS of tumors expressing L1CAM versus negative-expression was comparable (61.1%/55%v/s59.8%/47.9%;p=0.44). Presence of Mib-1>20% correlated with inferior survival (5-year EFS:81.1%vs22.2%; p CONCLUSIONS ST-EPEN with RELA fusion had trend towards increased relapse/progression. High Mib-1 correlated with poor survival. RELA fusion status needs to be studied in a larger cohort prospectively to confirm its clinical impact.

Due to an internal mistake during production the name of one of the co-authors, Aliasgar Moiyadi, was omitted from the original publication. With this Erratum the name of the co-author is included again and the original publication has been modified accordingly. SpringerNature apologizes for this and for any inconvenience associated therewith.

INTRODUCTION: This study was undertaken to evaluate the frequency of RELA fusion 1/2 in supratentorial ependymomas and its L1CAM and cylin D1 expression. MATERIALS AND METHODS: Evaluated for histological features, immunohistochemical parameters (p53 protein, cyclin D1 and L1CAM expression) and rt-PCR for RELA fusion 1/2. The clinical parameters of these cases were also noted. RESULTS: Total 71 cases; age range: 1-76yrs (interquartile range: 6-24yrs). Male (n=46): female (n=25) ratio 1.8: 1. Intraparenchymal-58 (commonly parietal), ventricular-13. Histological subtypes were subependymoma (grade I, n=4), ependymoma (grade II, n=8), anaplastic ependymoma (grade III, n=54) and ependymoma with atypical features (intermediate between grade II and III, n=5). 64.7% (44/68) showed p53 protein over-expression, 79.7% (55/69) cyclin D1 over-expression and 57.7% (41/71) L1CAM positivity. RELA fusion 1/2 positivity was detected in 45% of the cases (27/60; 22 Type 1, 5 Type 2). 63.2% (24/38) were ≤14 years (0-3yrs: 5/8, 4-9yrs: 12/17, 10-14yrs: 7/13). All RELA fusion-positive cases showed p53 protein, cyclin D1 and L1CAM over-expression while none of the cyclin D1 or L1CAM negative cases showed RELA fusion. With RELA fusion by rt-PCR as the reference test, L1CAM IHC showed 100% sensitivity, 59.4% specificity, 66.7% PPV and 100% NPV. CONCLUSIONS: RELA fusions 1/2 are common in younger age (≤ 14yrs) and identified across all histological subtypes except subependymoma. All L1CAM and cyclin D1 negative cases were also negative for RELA fusion, thus combined L1CAM and cyclin D1 negativity can be used as predictive maker for RELA fusion negativity.

INTRODUCTION: This study is correlation of ATRX-loss with histone and IDH-mutations in astrocytic tumours of children and young adults. MATERIALS AND METHODS: Study sample included: 1. High-grade (HGA) and all non-pilocytic astrocytomas, non-BRAF altered low-grade glial tumours (LGANOS) in ≤39 years, 2. EGFR non-amplified/IDH1/2 wild type midline located HGA with loss of ATRX in 40–55 years. ATRX by immunohistochemistry, H3.3, H3.1 and IDH1R132/IDH2R172 mutations by Sanger sequencing was done. RESULTS: Total cases: 365[1st group: 356 (HGA: 346; LGANOS: 10); 2nd group: 9]. 133 (37.4%), 54 (15.2%) and 51 (14.3%) of 1st group showed IDH-mutations, histone-alterations and isolated ATRX-loss respectively. Of the 2nd group, 2 and 3 cases showed histone-alterations and isolated ATRX-loss respectively. Overall 56 were histone gene-altered - H3.3 (K27M=41, K27E=2, G34R=8, G34V=1), H3.1 (HIST3BK27M=3) and frameshift mutation (n=1). Age-range of H3K27M/K27E (n=46): 1–46 years (0–3: 3, 4–6:2, 7–14: 16, 15–18:5, 19–25:8, 26–30:4, 31–39:6, >39: 2) and G34R/G34V: 7–26 years (7–14:4, 15–18:3, 19–25:1, 26–30:1). 11(24%) K27-mutant and 1 (11%) of G34-mutant were of superficial cerebral hemispheric (SCH) location. Four K27M-mutant were low-grade. 28 histone-altered cases [52%; K27M: 19 (43.2%), G34R/V:8 (89%), Frameshift:1] showed ATRX-loss. Interestingly, 54 (14.8%) cases showed isolated ATRX-loss with age-range 4–43 years (4–6:2, 7–14:11, 15–18:2, 19–25:13, 26–30:11, 31–39:12, >39:3). All were high-grade; 4 were midline while the rest were SCH. CONCLUSIONS: Isolated ATRX-loss, a distinct alteration in HGA of children and young adults. H3K27-mutations can be seen in adults; G34-mutations can be seen in young children and rarely both can be uncommon variants (K27E, G34V).

INTRODUCTION: Pilocytic astrocytomas (PCA) are characterised by BRAF fusions and V600E mutation. MATERIAL and METHODS: FFPE tissues of PCA diagnosed during 2011-17 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. RESULTS: 272 cases of PCA of age range 1-46 years (≤14 years:176; 15-25 yrs:76, 26-39yrs:19 and >39yrs:1) formed the sample. BRAFV600E and fusions were mutually exclusive. 25 of 234 (10.7%) were BRAFV600E mutated [cerebellum: 9/90(10%), suprasellar: 5/30(17%), CH: 4/27(15%), brain stem: 2/14(14%) and thalamic: 2/13(15%) 4th ventricle: 1/10 (10%) and 3rd ventricle: 2/7 (28.6%). Whereas 75 of 224 (33.5%) were BRAF fusion [KIAA1549-BRAF 16-9 (n: 52), 15-9 (n:15) & 16-11 (n:8)] positive. 40% of cerebellar (36/91), 37% of suprasellar (10/27), 43% of brain stem (6/14), 36% of spinal (5/14), 21% of CH (5/24), 21% of ON (4/19), 27% of thalamic (3/11), 55.6% of 4th ventricle (5/9) and 50% of pineal (1/2) were fusion positive. None of the 19 ON cases showed BRAFV600E. It was also not detected in 25 yrs age; but was seen in 15.4% (10/65) of cases in 15-25yrs. BRAF fusions were more common in ≤14 yrs (57/145; 39%). Interestingly, none of cases >30 years showed BRAF alterations. Two cases were associated with NF-1 both were negative for BRAF alterations. CONCLUSIONS: BRAF fusions are common than BRAFV600E in PCA across all locations and interestingly both are extremely rare >30years.

Background Glioblastoma (GBM) in children is rare. Pediatric GBM have a distinct molecular profile as compared to adult GBM. There are relatively few studies of pediatric GBMs and no standard of care on adjuvant therapy. We aimed to evaluate the clinical outcome and molecular profile of pediatric GBM. Methods and Materials Between 2004 and 2013, 66 consecutive children with histologically proven GBM were identified from our database. The majority of the children underwent maximal safe resection followed by focal radiotherapy with concurrent and adjuvant temozolomide. Immunohistochemical staining was performed for p53, MIB-1 labeling index, MGMT overexpression, and EGFR amplification and isocitrate dehydrogenase (IDH1) R132H point mutation. Survival and impact of possible prognostic factors on outcomes were analyzed. Result Median survival was 15 months. The overall survival rate at 1 year was 62%, at 2 years was 30%, and at 3 years was 27%. Patients with thalamic tumors (P < .001), incompletely resected tumors (P < .00001), and tumors with MIB-1 labeling index >25% (P < .002) had poor overall survival rates. p53 was overexpressed in 74% of patients, MGMT promoter methylation was seen in 37% of patients, IDH1 mutation was seen in 4% of patients, and no patients had EGFR amplification. MGMT methylation and p53 overexpression did not impact survival. Conclusions Clinical outcome of pediatric GBM is similar to that reported for adult GBM. The frequency of p53 overexpression is higher than in adult GBM, while MGMT methylation, IDH1 mutations and EGFR amplification is lower than in adult GBM. MGMT methylation and p53 expression status do not have any prognostic significance.

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.

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% in5 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.

Longer duration of neoadjuvant (NA) imatinib (IM) used for locally advanced (LA) gastrointestinal stromal tumours (GIST) is not based on biology of the tumour reflected by kit mutation analysis.LA or locally recurrent (LR) GIST treated with NA IM from May 2008 to March 2015 from a prospective database were included in the analysis. Archived formalin-fixed paraffin-embedded tissues (FFPE) were used for testing KIT exons 9, 11, 13 and 17 by PCR.One hundred twenty-five patients with LA or LR GIST were treated with NA IM. Forty-five patients (36 %) had undergone c-kit mutation testing. Exon 11 was seen in 25 patients (55.5 %), 3 with exon 9 (6.7 %) and 2 with exon 13 (4.4 %). Twelve were wild type (26.6 %) and 3 (6.7 %) were declared uninterpretable. Response rate (RR) for the exon 11 mutants was higher than the non-exon 11 mutant group (84 vs. 40 %, p = 0.01). Disease stabilization rate (DSR) rates were also higher in the exon 11 subgroup than non-exon 11 group (92 vs. 75 %). Eighty-four per cent exon 11 and 75 % non-exon 11 mutants were surgical candidates. Patients undergoing surgery had significantly improved event free survival (EFS) (p 0.001) compared to patients not undergoing surgery, with the same trend seen in OS (p = 0.021). Patients with a SD on response to NA IM had a lower EFS (p = 0.076) and OS compared to patients achieving CR/PR. There were no differences between the various exon variants in terms of outcomes and responses CONCLUSION: Upfront evaluation of kit mutation status may help us in delineating separate treatment strategies for potentially biologically different tumours and assessing the correct timing of surgery for this subset of GIST.

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.

Background: The exon 11 KIT mutant gastrointestinal stromal tumors (GIST) is a heterogeneous cohort with variable biological behavior based on different mutational subtypes. Methods: Patients with histologically prove n GIST with KIT exon 11 mutations were selected from a prospectively maintained database, and evaluated for clinical characteristics and event free survival (EFS). Patients were divided into mutations upstream to codon 557 (G1), mutations involving codon 557-558 (G2) and mutation downstream to codon 558 (G3). Results: A total of 90 patients satisfied the inclusion criteria for study. Substitutions, indels and duplications were seen in 23 patients. Deletions were seen in 67 patients, of which 44 patients had large deletions (>6 base pairs), while 23 has small deletions ( vs . 55 months), but this did not achieve statistical significance (univariate analysis: P=0.075). On multivariate analysis, patients undergoing radical intent surgery vs . no surgery (58 vs . 55 months; P=0.005) and G1 or G3 vs . G2 cohort (P=0.058) showed trend towards improved EFS. Conclusions: In patients with GIST exon 11 codon 557-558 mutation subset there is a trend towards an inferior survival even when treated with Imatinib mesylate (IM).

Methylation of O6-methylguanine DNA methyltransferase (MGMT) is one of the predictive and prognostic markers of glioblastomas (GBMs). In this study, GBMs have been evaluated for the same and correlated with other clinicopathological and molecular parameters. MATERIALS AND METHODS: Diagnosed cases of GBM, during the period of June 2014 to December 2014 in the Department of Pathology of our Institute, wherein they have been evaluated for MGMT methylation by methylation specific polymerase chain reaction, IDH1R132H and p53 protein expression by immunohistochemistry and EGFR gene amplification by fluorescence in-situ hybridisation formed the study sample. Statistical correlation was done by Chi-square and Fischer's exact, using IBM SPSS Software 16 version. RESULTS: A total of 97 cases of GBM formed the study sample with age range of 20-75 years (mean age:49.76 years) and male: female ratio of 3.2:1 (M:74;F:23).Cerebral hemispheric location was commonest (frontal:35%; temporal:43 & parietal:19%). 46 (47.4%) cases were MGMT methylated (low methylation: 10) and 51 were unmethylated. 23 out of 84 cases (27.4%) were EGFR amplified; 10 cases (10.3%) were positive for IDH1R132H; 16 showed loss of ATRX protein expression (of which 7 were IDH1R132H positive; while the other 3 IDHR132H positive cases showed retained ATRX protein). Of the 46 MGMT methylated cases, 10 were EGFR amplified (10 out of 23 EGFR amplified cases were MGMT methylated); 5 were IDH1R132H positive and 6 showed loss of ATRX expression. None of the molecular markers showed any correlation for the histological features of calcification and necrosis (neither for pseudopalisading or confluent). CONCLUSIONS: MGMT was methylated in 47.4%of GBMs and had no significant statistical correlation with EGFR gene amplification, IDH1R132H mutation, ATRX protein expression and other histomorphological features. This study did not identify any surrogate marker for MGMT promoter methylation.