Leukoreduction of red blood cell units decreases dysregulatory micro RNAs during routine storage: An observational study with In-silico analysis.

BACKGROUND: Red Blood cells (RBCs) bring about harmful consequences during storage. MicroRNA (miRNA) dysregulation in stored RBCs could represent potential biomarkers of storage lesions. Although leukoreduction prevents damage to RBCs, it is uncertain whether leukoreduction of RBCs would impact the dysregulation of miRNAs during storage. This study evaluated the potential role of miRNAs for any alteration of leukoreduced (LR) and non-leukoreduced (NLR) RBCs till 21 days of storage. STUDY DESIGN AND METHODS: In this prospective study, thirty male volunteers' blood was equally divided into leukoreduced RBCs (LR) and NLR RBC (NLR) bags and stored till Day 21 at 4-60c. Selected miRNAs were quantified on Days 0 and 21. Further, bioinformatic tools were used to analyze the selected miRNAs and their predicted target genes (mRNAs) and identify the miRNA-mRNA regulatory relationships. RESULTS: A significantly higher fold change values of three miRNAs (miR-96-5p, miR-197-3p, miR-769-3p) were observed in NLR RBCs (p < .05). A significantly higher (p < .05) expression levels of miR-150-5p and miR-197-3p were observed in NLR RBCs till 21 days of storage. Further, the correlation with mRNA quantification confirmed the regulatory role of these miRNAs upon functional pathway enrichment analysis. DISCUSSION: A higher level of dysregulation of miRNAs was observed in NLR RBCs. Validation from In-Silico analysis suggested the regulatory role of miRNAs in cell apoptosis, senescence, and RBC-related signaling pathways. This indicated that stored LR RBCs would likely have better in vivo survival and function following transfusion. However, an in vivo study of miRNA in RBCs is warranted for conclusive evidence.

Holocord metastasis from supratentorial glioblastoma multiforme: an uncommon manifestation of a common tumor.

Intramedullary metastasis from primary glioblastoma multiforme (GBM) is a rare phenomenon with a poor prognosis. The rate of spinal metastasis from intracranial GBM has been variably reported to be 0.4-2%. According to a review by Lawton in 2012, there were only 42 documented cases of primary intracranial GBM with spinal metastasis. We present a unique case of early-onset symptomatic holocord metastasis of GBM in a patient approximately 2 months of detection of primary GBM.

Assessment of Matrix Metalloprotease - 7 (MMP7) Immunohistochemistry in Biliary Atresia and Other Pediatric Cholestatic Liver Diseases.

Background and aims: Biliary atresia (BA) is a progressive fibro-obliterative cholangiopathy. The histopathological diagnosis is often challenging and an immunohistochemical marker is often sought as an adjunct. We evaluated MMP7 immunohistochemistry in BA and other non-BA pediatric cholestatic liver diseases. Materials and methods: MMP7 immunohistochemistry was applied in 5 age-matched normal control, 23 cases of BA and 43 cases of non-BA pediatric cholestasis including 16 cases of choledochal cyst (CC), and a multiplication score was obtained by multiplying the intensity and percentage positivity in the cholangiocytes. Results: BA showed a high mean MMP7 multiplication score which was significantly different from the normal control and other non-BA pediatric cholestatic diseases including CC (p value < 0.001). The sensitivity, specificity, positive, and negative predictive values of MMP7 immunohistochemistry were 91.3%, 93.02%, 87.5%, and 95.2% respectively. Conclusion: MMP7 immunohistochemistry may be an adjunct to histomorphology in BA.

Elevated Expression of DNA Methyltransferases and Enhancer of Zeste Homolog 2 in Helicobacter pylori - Gastritis and Gastric Carcinoma.

AIM: The aim of this study was to study the role of key epigenetic regulators pertaining to DNA methylation and histone-modification systems in Helicobacter pylori (HP)-associated gastritis and gastric carcinogenesis. METHODS: The expression of DNA methyltransferase (DNMT-1, 3A, and 3B) and the catalytic subunit of polycomb repressive complex-2 (enhancer of zeste homolog 2 [EZH2]) in gastric carcinomas (n = 104), mucosa adjacent to carcinoma (n = 104), HP-associated gastritis (n = 95), and histologically normal mucosa (n = 31) was assessed by immunohistochemistry and qRT-PCR. RESULTS: The expression of all 3 DNMTs and EZH2 was significantly higher in HP-associated gastritis and carcinoma cases than in those with adjacent and normal mucosa. The expression of DNMT-1 and 3B was maximum in HP-associated gastritis. DNMT-3A showed higher expression in carcinoma-adjacent mucosa than in normal mucosa. Interestingly, the expression of EZH2 was higher in cases of HP-associated gastritis with metaplasia than in those without metaplasia and also in cases of intestinal type of adenocarcinoma. Significant positive correlation of EZH2 was identified with DNMT-1, DNMT-3A, and DNMT-3B. However, none of these markers was associated with survival outcome. CONCLUSION: This study establishes an important role of the key epigenetic regulators in the pathogenesis of both HP-associated gastritis and gastric carcinoma. Higher expression of all the epigenetic markers in the gastritis and their persistence in the carcinoma point toward their implications in HP-driven gastric carcinogenesis. Further, an inter-relation between the 2 arms of epigenetics, namely, DNA methylation and histone-modification in the pathogenesis of gastric carcinoma, is also documented. Given the reversibility of epigenetic phenomenon, these molecules may be of important therapeutic use.

Cytology of angiofibroma of soft tissue of the inguinal region.

Angiofibroma of the soft tissue is a recently described benign fibroblastic/myofibroblastic tumour. This report describes the cytology of an angiofibroma of soft tissue occurring in a 30-year-old lady which showed bland spindle cells, occasional polygonal cells with nuclear grooving, prominent vessels, frayed stroma around the blood vessels, and scattered lymphocyte-rich inflammatory cells in the background.

Choroid Plexus Carcinoma with Hyaline Globules: An Uncommon Histological Finding.

Background: Choroid plexus carcinoma (CPC) is a predominately pediatric CNS tumor with a variety of histologic features, with hyaline globules only reported once previously. Case report: A two-year-old male child presented with headaches, vomiting, and lower limb weakness. Radiological examination revealed a right temporoparietal intra-axial tumor. On histology, it showed features of CPC containing multiple eosinophilic intracytoplasmic and extracellular hyaline globular structures, which were PAS-positive, diastase resistant, and immunoreactive for alpha-fetoprotein (AFP). Conclusion: CPC can occasionally show AFP immune-positive hyaline globules.

Expression of CD56 is Not Limited to Biliary Atresia and Correlates with the Degree of Fibrosis in Pediatric Cholestatic Diseases.

BACKGROUNDS AND AIMS: CD56 immunostain is used as an adjunct to aid in the preoperative diagnosis of biliary atresia (BA) by liver biopsy. We aimed to study the expression of CD56 in different pediatric cholestatic diseases thereby evaluating its utility in the diagnosis of BA. METHODS: We performed immunohistochemistry for CD56 on 35 cases of pediatric cholestatic diseases and five age-matched controls. CD56 expression was assessed by a multiplication score (percentage positivity x intensity) in the biliary epithelium. RESULTS: The multiplication score between BA and choledochal cyst was not significantly different. High scores were also encountered in other cholestatic disorders. The score showed a significant negative association with serum albumin and a significant positive correlation with the serum ALT level. Very significant positive correlation between the score and portal fibrosis was obtained. CONCLUSION: CD56 expression is an infidel marker for the histological diagnosis of BA and rather provides a clue to the disease status in pediatric cholestatic diseases.

Congenital Acinar Cystic Transformation of the Pancreas with Proximal Jejunal Atresia and Hepatic Iron Overload: An Autopsy Case.

Introduction: Acinar cystic transformation (ACT) of the pancreas is characterized by multiple cysts lined by dual ductal and acinar-type of epithelium. ACT is typically a disease of adulthood and has not been described in a neonate. Case report: Autopsy of this term 3-day old male demonstrated cystic transformation of the entire pancreas measuring 42 mm in its largest dimension. The main pancreatic duct was patent. The numerous variable-sized cysts were lined by both ductal (CK7-positive) and acinar (trypsin-positive) epithelium. Congenital hemochromatosis of the liver, complete proximal jejunal atresia, gangrene of the post-atretic jejunum, and subglottic stenosis were associated features. Discussion/Conclusion: ACT may occur in the neonate in association with other abnormalities.

Neonatal hemochromatosis in a newborn with Down syndrome.

Background: Neonatal hemochromatosis (NH) is a cause of neonatal/pediatric acute liver failure. Liver dysfunction/failure in Down syndrome had been described in relation to increased susceptibility to infection and transient myeloproliferative disease (TMD). The occurrence of NH in Down syndrome is described in only a few case reports. Material and methods: A complete autopsy have been performed in a 79-day-old infant with severe liver dysfunction. TMD was suspected antemortem following a report of peripheral blood leukocytosis with 14% atypical cells. Results: The complete autopsy revealed NH-phenotype to be the cause of liver dysfunction and subsequent death. Conclusion: Though TMD is a common cause of liver dysfunction in Down syndrome, NH should also be considered in its differential.

BRAF V600E-mutated central nervous system tumor with divergent morphological feature - Anaplastic pleomorphic xanthoastrocytoma-like and astroblastoma-like.

Mutational analysis of the BRAF gene (BRAF), especially BRAF V600E, is gaining much importance in neuro-oncology practice due to its diagnostic, prognostic and therapeutic implications. This genetic alteration has been described in a wide morphological spectrum of central nervous system tumors. In the present report we describe a BRAF V600E-mutated tumor with divergent morphological appearance comprising of anaplastic pleomorphic xanthoastrocytoma and astroblastoma. Both of these tumor entities are extremely rare and a combined morphology has not been described till now.

Identification of miR-379/miR-656 (C14MC) cluster downregulation and associated epigenetic and transcription regulatory mechanism in oligodendrogliomas.

INTRODUCTION: Although role of individual microRNAs (miRNAs) in the pathogenesis of gliomas has been well studied, their role as a clustered remains unexplored in gliomas. METHODS: In this study, we performed the expression analysis of miR-379/miR-656 miRNA-cluster (C14MC) in oligodendrogliomas (ODGs) and also investigated the mechanism underlying modulation of this cluster. RESULTS: We identified significant downregulation of majority of the miRNAs from this cluster in ODGs. Further data from The Cancer Genome Atlas (TCGA) also confirmed the global downregulation of C14MC. Furthermore, we observed that its regulation is maintained by transcription factor MEF2. In addition, epigenetic machinery involving DNA and histone-methylation are also involved in its regulation, which is acting independently or in synergy. The post- transcriptionally regulatory network of this cluster showed enrichment of key cancer-related biological processes such as cell adhesion and migration. Also, there was enrichment of several cancer related pathways viz PIK3 signaling pathway and glioma pathways. Survival analysis demonstrated association of C14MC (miR-487b and miR-409-3p) with poor progression free survival in ODGs. CONCLUSION: Our work demonstrates tumor-suppressive role of C14MC and its role in pathogenesis of ODGs and therefore could be relevant for the development of new therapeutic strategies.

Clinicopathological and molecular characteristics of pediatric meningiomas.

Molecular and clinical characteristics of pediatric meningiomas are poorly defined. Therefore, we analyzed clinical, morphological and molecular profiles of pediatric meningiomas. Forty pediatric meningiomas from January 2002 to June 2015 were studied. 1p36, 14q32 and 22q-deletion were assessed by fluorescent in situ hybridization and mutations of most relevant exons of AKT, SMO, KLF4, TRAF and pTERT using sequencing. Expression of GAB1, stathmin, progesterone receptor (PR), p53 along with MIB-1 LI was examined using immunohistochemistry. There were 36 sporadic and four NF2 associated meningiomas. Among sporadic meningiomas, the majority (72.2%) of cases harbored 22q-deletion. Difference in frequency of combined 1p/14q deletion in Grade-I versus Grade-II/III tumors was not significant (13.7% vs 28.5%, P = 0.57). PR immunoreactivity was seen in 65.5% of Grade-I and 14.2% of Grade-II/III tumors (P = 0.03). The majority (97.2%) of meningiomas were immunonegative for p53. Stathmin and GAB co-expression was observed in 58.3% of cases. Notably, AKT, SMO, KLF4, TRAF7 (exon 17) and pTERT mutations were seen in none of the cases analyzed. 1p/14q codeletion was frequent in skull base as compared to non-skull base meningiomas (23% vs 11.1%, P = 0.37). All NF2 meningiomas harbored 22q-deletion and showed GAB and stathmin co-expression while none showed 1p/14q loss. Pediatric meningiomas share certain phenotypic and cytogenetic characteristics with adult counterparts, but GAB and stathmin co-expression in the majority of cases and non-significant difference in frequency of 1p/14q co-deletion between low- and high-grade meningiomas indicate an inherently aggressive nature. Characteristic AKT/SMO, KLF4/TRAF7 and pTERT genetic alterations seen in adults are distinctly absent in pediatric meningiomas.

Genome-wide ChIP-seq analysis of EZH2-mediated H3K27me3 target gene profile highlights differences between low- and high-grade astrocytic tumors.

Enhancer of zeste homolog-2(EZH2) is a key epigenetic regulator that functions as oncogene and also known for inducing altered trimethylation of histone at lysine-27 (H3K27me3) mark in various tumors. However, H3K27me3 targets and their precise relationship with gene expression are largely unknown in astrocytic tumors. In this study, we checked EZH2 messenger RNA and protein expression in 90 astrocytic tumors of different grades using quantitative PCR and immunohistochemistry, respectively. Further, genome-wide ChIP-seq analysis for H3K27me3 modification was also performed on 11 glioblastomas (GBMs) and 2 diffuse astrocytoma (DA) samples. Our results showed EZH2 to be highly overexpressed in astrocytic tumors with a significant positive correlation with grade. Interestingly, ChIP-seq mapping revealed distinct differences in genes and pathways targeted by these H3K27me3 modifications between GBM versus DA. Neuroactive ligand receptor pathway was found most enriched in GBM (P = 9.4 × 10-25), whereas DA were found to be enriched in metabolic pathways. Also, GBM showed a higher enrichment of H3K27me3 targets reported in embryonic stem cells and glioma stem cells as compared with DAs. Our results show majority of these H3K27me3 target genes were downregulated, not only due to H3K27me3 modification but also due to concomitant DNA methylation. Further, H3K27me3 modification-associated gene silencing was not restricted to promoter but also present in gene body and transcription start site regions. To the best of our knowledge, this is the first high-resolution genome-wide mapping of H3K27me3 modification in adult astrocytic primary tissue samples of human, highlighting the differences between grades. Interestingly, we identified SLC25A23 as important target of H3K27me3 modification, which was downregulated in GBM and its low expression was associated with poor prognosis in GBMs.

Retroperitoneal and mediastinal follicular dendritic cell sarcoma: report of 3 cases with review of literature.

Follicular dendritic cell sarcoma (FDCS) is a rare malignant histiocytic proliferation of antigen presenting follicular dendritic cell. It is an uncommon primary malignancy first described by Monda et al. in 1986. Most commonly reported cases are lymph nodal. Occasional cases occur in extra nodal sites. Here, we describe the clinicopathological features, histomorphology and outcome of three patients with extranodal FDCS along with a concise review of literature on the topic. All three patients were adult females. Two patients were in third decade, and one had age of 50 years. Among the three cases, two cases are presented as retroperitoneal mass and one as mediastinal mass. CT scans revealed heterogeneously enhancing masses. All the cases showed ovoid to spindle neoplastic cells arranged predominantly in whorling, fascicular and storiform patterns with inflammatory infiltrate. Immunohistochemically, the tumor cells are positive for CD21, CD23, CD35 and Clustrin. In view of rarity and variable clinical presentation in FDCS, accurate diagnosis is necessary. Copyright © 2015 John Wiley & Sons, Ltd.

Clinicopathological characteristics, molecular subgrouping, and expression of miR-379/miR-656 cluster (C14MC) in adult medulloblastomas.

Medulloblastoma (MB) is a childhood tumor comprising four molecular subgroups: WNT, SHH, group 3 and group 4, with diagnostic and prognostic connotations. Very few studies are available on molecular subgrouping of adult MBs due to their rarity. Recently, loss of chromosome14q has been reported in SHH MBs, with downregulation of miR-379/miR-656 cluster (C14MC) in pediatric SHH MBs. Hence, the present study on adult MBs was undertaken to enumerate clinicopathological characteristics and molecular subgroups, and to analyze expression of C14MC and its transcriptional regulators, MEF2, JUN and ESRRG. Immunohistochemistry for ß-catenin, GAB1 and YAP1 was performed to identify molecular subgroups. MYC amplification was evaluated by FISH. Expression profiling of 47 miRNAs from C14MC was performed using customized Taqman low-density array. Expression of transcriptional regulators was examined using RT-PCR. Seventy-one adult MBs were analyzed. They had male predominance and majority were located laterally (52 %). A significant proportion of cases were of Desmoplastic/nodular histology (32 %); MBEN was not seen. WNT tumors constituted 4.2 %, SHH 62 %, and non-WNT/non-SHH 33.8 %. MYC amplification was identified in 11.1 % cases. Patient outcome was worse in adults. Significant downregulation of C14MC was observed in all MB subgroups, and MEF-2 expression was downregulated. Adult MBs are distinct from childhood MBs in terms of location, histopathological subtypes, molecular subgroups, as well as prognosis. Silencing of C14MC in all MB subgroups suggests its role as a tumor suppressor locus in tumorigenesis. Deregulation of C14MC can possibly be attributed to repression of MEF2.

Expression of DNA methyltransferases 1 and 3B correlates with EZH2 and this 3-marker epigenetic signature predicts outcome in glioblastomas.

This study aims to analyze expression of EZH2 and DNA-methyltransferases (DNMT1, 3A and 3B) in astrocytic tumors and investigate their link as well as their correlation with survival, especially in GBMs. Expression of EZH2 and DNMTs (DNMT1, DNMT3A and DNMT3B) in different grades of astrocytomas (n=93) was assessed by qRT-PCR and immunohistochemistry. GBM-U87MG cell line was used for functional studies. Strong immunopositivity (LI≥25%) for EZH2, DNMT1 and DNMT3B was detected in 52%, 56% and 64% cases of GBMs respectively, which was significantly higher as compared to Grade II/III cases. Similarly, their median fold change of mRNA expression was also significantly higher in GBMs. There was also a significant positive correlation between DNMT1/DNMT3B and EZH2 mRNA and protein expression, which was in concordance with TCGA data set. Inhibition of DNMTs in cell line by Azacytidine resulted in down-regulation of EZH2, while knock-down of EZH2 by siRNA was not associated with any significant alteration of DNMTs, indicating that EZH2 expression in GBMs is possibly regulated by DNMTs, but not the reverse. Strong immunopositivity for EZH2, DNMT1 and DNMT3B were individually associated with significantly shorter survival and showed no correlation with IDH1 mutation status. In addition, the combination of these 3 markers represented an independent prognostic signature with cases having weak/negative expression of all 3 markers being associated with best prognosis. For the first time, the present study describes an epigenetic prognostic signature in GBMs based on immunohistochemical expression of EZH2, DNMT1 and 3B which can be used easily in routine neuropathology practice.

Genome-wide small noncoding RNA profiling of pediatric high-grade gliomas reveals deregulation of several miRNAs, identifies downregulation of snoRNA cluster HBII-52 and delineates H3F3A and TP53 mutant-specific miRNAs and snoRNAs.

Pediatric high-grade gliomas (HGGs) are highly malignant tumors that remain incurable and relatively understudied. The crucial role of noncoding RNAs (ncRNAs) has been reported in various cancers. However, the study on miRNAs in pediatric HGGs is scant and there is no report till date on the status of other small ncRNAs. Genome-wide microarray analysis was performed to investigate small ncRNA expression in pediatric HGG (n = 14) and compared to adult glioblastoma (GBM) signature. The validation of miRNAs and small nucleolar RNAs (snoRNAs) was done by real-time polymerase chain reaction. TP53 and H3F3A mutation-specific miRNA and snoRNA profiles were generated and analyzed. Pediatric HGGs showed upregulation of miR-17/92 and its paralog clusters (miR106b/25 and miR-106a/363), whereas majority of downregulated miRNAs belonged to miR379/656 cluster (14q32). Unsupervised hierarchical clustering identified two distinct groups. Interestingly, Group 2 with downregulated 14q32 cluster showed better overall survival. The miRNAs unique to pediatric HGG as compared to adult GBM were predicted to affect PDGFR and SMAD2/3 pathways. Similarities were seen between pediatric HGG and TP53 mutant miRNA profiles as compared to wild types. Several of H3F3A mutation-regulated genes were found to be the targets of H3F3A mutant-specific miRNAs. Remarkably, a significant downregulation of HBII-52 snoRNA cluster was found in pediatric HGGs, and was specific to H3F3A nonmutants. This is the first genome-wide profiling study on miRNAs and snoRNAs in pediatric HGGs with respect to H3F3A and TP53 mutations. The comparison of miRNA profiles of pediatric HGGs and adult GBM reiterates the overlaps and differences as also seen with their gene expression and methylation signatures.