Advanced molecular pathology for rare tumours: A national feasibility study and model for centralised medulloblastoma diagnostics

Aims: Application of advanced molecular pathology in rare tumours is hindered by low sample numbers, access to specialised expertise/technologies and tissue/assay QC and rapid reporting requirements. We assessed the feasibility of co-ordinated real-time centralised pathology review (CPR), encompassing molecular diagnostics and contemporary genomics (RNA-seq/DNA methylation-array). Methods: This nationwide trial in medulloblastoma (<80 UK diagnoses/year) introduced a national reference centre (NRC) and assessed its performance and reporting to World Health Organisation standards. Paired frozen/formalin-fixed, paraffin-embedded tumour material were co-submitted from 135 patients (16 referral centres). Results: Complete CPR diagnostics were successful for 88% (120/135). Inadequate sampling was the most common cause of failure; biomaterials were typically suitable for methylation-array (129/135, 94%), but frozen tissues commonly fell below RNA-seq QC requirements (53/135, 39%). Late reporting was most often due to delayed submission. CPR assigned or altered histological variant (vs local diagnosis) for 40/135 tumours (30%). Benchmarking/QC of specific biomarker assays impacted test results; fluorescent in-situ hybridisation most accurately identified high-risk MYC/MYCN amplification (20/135, 15%), while combined methods (CTNNB1/chr6 status, methylation-array subgrouping) best defined favourable-risk WNT tumours (14/135; 10%). Engagement of a specialist pathologist panel was essential for consensus assessment of histological variants and immunohistochemistry. Overall, CPR altered clinical risk-status for 29% of patients. Conclusion: National real-time CPR is feasible, delivering robust diagnostics to WHO criteria and assignment of clinical risk-status, significantly altering clinical management. Recommendations and experience from our study are applicable to advanced molecular diagnostics systems, both local and centralised, across rare tumour types, enabling their application in biomarker-driven routine diagnostics and clinical/research studies

Molecular genetic analysis of a constitutional chromosome translocation in a patient with ganglioneuroblastoma

This study aimed to isolate and characterise the breakpoint junction fragment of a constitutional t(1;13)(q22;q12) from DG, a 5 year old boy with ganglioneuroblastoma, to see whether a gene critical to neural development has been disrupted. Somatic cell hybrids were constructed by fusing the lymphoblastoid cell line from DG with mouse 3T3 cells. One subclone contained a derivative chromosome 1 but no other material from chromosomes 1 and 13. Unique probes were isolated by Alu PCR cloning from a somatic cell hybrid containing 13pter to 13q14 as its only human component. Analysis of 180 clones yielded four probes which flanked the DG breakpoint. The probes were mapped by Southern hybridisation, PCR and FISH using cosmids isolated from a chromosome 13-specific cosmid library. All four probes were located in 13q12; 3'Alu66 mapped above and 3'Alu 78,71 and 62 mapped below, the DG breakpoint. The four 3'Alu probes and an STS for the oncogene, FLT1, were used to screen a YAC library. The fourteen YACs identified with these markers were characterised by PFGE, hybridisation and PCR fingerprinting. PCR analysis using STS for the ends of the YAC identified by FLT1, suggested that this YAC was chimeric, which was confirmed by FISH. Cosmid c12.2, mapping 250 kb distal to 3'Alu66 by FISH, has replaced FLT1 as the closest marker distal to the DG breakpoint. Two YACs were identified with 3'Alu66. An STS from the left hand end of one of the YACs (a 1.3 Mb, non-chimeric YAC) was used to isolated five further YACs. The resulting contig of seven YACs will allow high definition mapping of 13q12 and may include clones that contain the DG breakpoint junction fragment and, hence, a gene important in Nb development

Proteomic analysis of Pteropus alecto kidney cells in response to the viral mimic, Poly I:C

BACKGROUND: Bats are recognised as an important reservoir for a number of highly pathogenic zoonotic viruses. While many of these viruses cause severe and often fatal disease in humans, bats are able to coexist with these viruses without clinical signs of disease. The mechanism conferring this antiviral response is not fully understood. Here, we investigated the differential protein expression of immortalised Pteropus alecto kidney cells (PaKiT03) following transfection with the viral mimic, Poly I:C. Two complementary proteomic approaches, difference gel electrophoresis (DIGE) and isobaric tagging for relative and absolute quantitation (iTRAQ) were used to quantify changes in protein expression following Poly I:C stimulation at 4, 8 and 20 hr post treatment (hpt). RESULTS: The expression of ISG54 gene, a known responder to virus infection and Poly I:C treatment, was significantly induced in transfected cells compared with mock-transfected cells. Through iTRAQ analysis we show that Poly I:C up-regulates key glycolytic enzymes at 4 hpt within PaKiT03 cells. In contrast, at 20 hpt PaKiT03 cells down-regulated ribosomal subunit proteins. The analysis with DIGE of Poly I:C transfected PaKiT03 cells showed over 215 individual spots differentially regulated, however only 25 spots could be unambiguously identified by LC-MS/MS. Immunoblotting confirmed the up-regulation of Eno1 and Tpi1 in PaKiT03 cells following Poly I:C transfection. A comparison with human cells (HEK293T and HeLa) and one additional bat cell line (PaLuT02), demonstrated that glycolytic pathways are also induced in these cell types, but at different intensities. CONCLUSION: The two techniques, DIGE and iTRAQ identified largely overlapping sets of differentially expressed proteins, however DIGE unambiguously identified significantly less proteins than iTRAQ. Poly I:C induced a rapid metabolic shift towards glycolysis within the PaKiT03 cells at 4 hpt, presumably as a consequence of increased energy requirements. On the other hand ribosomal subunit proteins were seen as down-regulated by iTRAQ, these proteins may be the limiting factors in the translational machinery available for virus replication. This study provides new insight into the antiviral response of bat cells, highlighting the importance of energy metabolism

Additional file 1: of Proteomic analysis of Pteropus alecto kidney cells in response to the viral mimic, Poly I:C

Full list of identified proteins by LC-MS/MS and their ratio at each replicate time points determined by iTRAQ (Sheet1). A list of the differentially regulated proteins with a mean fold-change of ≥ 1.5 (Sheet2). (XLSX 486 kb

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Hyperfractionated Accelerated Radiotherapy (HART) with maintenance chemotherapy for metastatic (M1-3) Medulloblastoma--a safety/feasibility study

Background and purpose: To evaluate feasibility and toxicity of Hyperfractionated Accelerated Radiotherapy (HART) 1.24 Gy b.i.d. followed by chemotherapy for M1-3 Medulloblastoma (MB). The aim of HART was to use hyperfractionation to improve therapeutic ratio combined with acceleration to minimise tumour cell repopulation during radiotherapy (RT). Materials and methods: Between February 2002 and May 2008, 34 eligible patients (22 male, 12 female) aged 3-15 years (median 7) with metastatic MB (M1-9; M2-3, M3-22) received HART with a craniospinal radiotherapy (CSRT) dose of 39.68 Gy followed by 22.32 Gy boost to the whole posterior fossa and 9.92 Gy metastatic boosts. The 8th and subsequent patients received vincristine (VCR) 1.5 mg/m(2) weekly x 8 doses over 8 weeks starting during the 1st week of RT. Maintenance chemotherapy comprised 8 six-weekly cycles of VCR 1.5 mg/m(2) weekly x 3, CCNU 75 mg/m(2) and cisplatin 70 mg/m(2). Results: Median duration of HART was 34 days (range 31-38). Grade 3-4 toxicities included mucositis (8), nausea (10), anaemia (5), thromboeytopaenia (2), leucopaenia (24). With 4.5-year median follow-up, 3-year EFS and OS were 59% and 71%, respectively. Of 10 relapses, 1 was outside the central nervous system (CNS), 1 posterior fossa alone and 8 leptomeningeal with 3 also associated with posterior fossa. Conclusion: HART with or without VCR was well tolerated and may have a place in the multi-modality management of high-risk MB. (C) 2014 Elsevier Ireland Ltd. All rights reserved