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Ostergaard M.,Aarhus University Hospital | Nyvold C.G.,Aarhus University Hospital | Jovanovic J.V.,Kings College London | Andersen M.T.,Copenhagen University | And 11 more authors.
Leukemia | Year: 2011

Quantitative PCR (qPCR) for detection of fusion transcripts and overexpressed genes is a promising tool for following minimal residual disease (MRD) in patients with hematological malignancies. Its widespread clinical use has to some extent been hampered by differences in data analysis and presentation that complicate multicenter clinical trials. To address these issues, we designed a highly flexible MRD-reporting software program, in which data from various qPCR platforms can be imported, processed, and presented in a uniform manner to generate intuitively understandable reports. The software was tested in a two-step quality control (QC) study; the first step involved eight centers, whose previous experience with the software ranged from none to extensive. The participants received cDNA from consecutive samples from a BCR-ABL chronic myeloid leukemia (CML) patient and an acute myeloid leukemia (AML) patient with both CBFΒ-MYH11 and WT1 target genes, they conducted qPCR on their respective hardware platforms and generated a series of reports with pre-defined features. In step two, five centers used the software to report BCR-ABL MRD in a harmonized manner, applying their recently obtained CML international scale conversion factors. The QC study demonstrated that this MRD-reporting software is suitable for efficient handling of qPCR data, generation of MRD reports and harmonization of MRD data. © 2011 Macmillan Publishers Limited All rights reserved.

Sakellariou S.,Kings College | Sakellariou S.,National and Kapodistrian University of Athens | Al-Hussaini H.,Kings College | Scalori A.,Kings College | And 5 more authors.
Histopathology | Year: 2012

Aims: Glycogen storage disease type I is a metabolic disorder resulting from deficiency of the glucose-6-phosphate complex. Long-term complications include the development of hepatocellular adenoma (HCA). In this retrospective study, our aim was to reclassify according to geno-phenotypic characteristics nodular lesions identified in hepatectomy specimens of such patients transplanted between 1998 and 2008 at our institution. Methods and results: Clinicopathological data of seven consecutive transplanted patients with glycogen storage disease type I were reviewed. Liver nodules were re-examined histologically and by immunohistochemistry. Molecular analysis was performed additionally in a case with specific features. Four patients had multiple tumours. We concluded that 26 of 38 nodules available for study had features of inflammatory hepatocellular adenomas, seven comprised adenomas not otherwise specified and five were found to be focal nodular hyperplasia. Conclusions: Further studies are needed to clarify the pathogenesis of hepatocellular adenomas in glycogen storage disease; in particular to determine whether they share abnormal metabolic pathways with inflammatory adenomas in the general population. Testing for acute phase proteins may be a helpful tool in the early detection of HCA in such patients. Finally, there is a need to further define their risk of malignant transformation, in relation to age and possible cofactors. © 2012 Blackwell Publishing Ltd.

Akiki S.,West Midlands Regional Genetics Laboratory | Akiki S.,University of Birmingham | Dyer S.A.,West Midlands Regional Genetics Laboratory | Dyer S.A.,University of Birmingham | And 15 more authors.
Genes Chromosomes and Cancer | Year: 2013

The cytogenetically cryptic t(5;11)(q35;p15) leading to the NUP98-NSD1 fusion is a rare but recurrent gene rearrangement recently reported to identify a group of young AML patients with poor prognosis. We used reverse transcription polymerase chain reaction (PCR) to screen retrospectively diagnostic samples from 54 unselected pediatric AML patients and designed a real time quantitative PCR assay to track individual patient response to treatment. Four positive cases (7%) were identified; three arising de novo and one therapy related AML. All had intermediate risk cytogenetic markers and a concurrent FLT3-ITD but lacked NPM1 and CEBPA mutations. The patients had a poor response to therapy and all proceeded to hematopoietic stem cell transplant. These data lend support to the adoption of screening for NUP98-NSD1 in pediatric AML without otherwise favorable genetic markers. The role of quantitative PCR is also highlighted as a potential tool for managing NUP98-NSD1 positive patients post-treatment. © 2013 Wiley Periodicals, Inc.

Sakellariou S.,Kings College | Sakellariou S.,National and Kapodistrian University of Athens | Morgan Y.,Molecular Oncology Diagnostics Unit | Heaton N.,Kings College | And 3 more authors.
European Journal of Gastroenterology and Hepatology | Year: 2011

Hepatocellular adenomas (HCAs) containing inactivating HNF1a mutations correspond to a homogenous group of tumors with marked steatosis and no cytological abnormalities or inflammatory infiltrates. We report a case of a 60-year-old woman who was referred with a 13-cm mass in the left lobe of the liver with no history of oral contraception use and no family history of note. Histology revealed a severely steatotic HCA. Immunohistochemistry showed no nuclear staining for β-catenin, limited glutamine synthetase positivity, and slightly attenuated liver-fatty acid binding protein staining. Serum amyloid A2 antibodies produced a coarse granular staining. Mutational screening detected monoallelic partial tandem duplication within exon 4 of TCF1 in tumoral tissue. No mutations in the b-catenin and IL6ST genes were detected. Quantitative reverse transcription PCR showed lower expression levels of FABP1 and uridine glycosyltransferase 2B7 and higher levels of serum amyloid A2 in tumor than in normal hepatocytes. Clinicopathological and molecular investigation of HCA cases with unique features could result in a better understanding of HCAs pathogenesis. © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins.

White H.,National Genetics Reference Laboratory Wessex | White H.,European Commission | Deprez L.,University of Southampton | Corbisier P.,University of Southampton | And 70 more authors.
Leukemia | Year: 2015

Serial quantification of BCR-ABL1 mRNA is an important therapeutic indicator in chronic myeloid leukaemia, but there is a substantial variation in results reported by different laboratories. To improve comparability, an internationally accepted plasmid certified reference material (CRM) was developed according to ISO Guide 34:2009. Fragments of BCR-ABL1 (e14a2 mRNA fusion), BCR and GUSB transcripts were amplified and cloned into pUC18 to yield plasmid pIRMM0099. Six different linearised plasmid solutions were produced with the following copy number concentrations, assigned by digital PCR, and expanded uncertainties: 1.08±0.13 × 10 6, 1.08±0.11 × 10 5, 1.03±0.10 × 10 4, 1.02±0.09 × 10 3, 1.04±0.10 × 10 2 and 10.0±1.5 copies/μl. The certification of the material for the number of specific DNA fragments per plasmid, copy number concentration of the plasmid solutions and the assessment of inter-unit heterogeneity and stability were performed according to ISO Guide 35:2006. Two suitability studies performed by 63 BCR-ABL1 testing laboratories demonstrated that this set of 6 plasmid CRMs can help to standardise a number of measured transcripts of e14a2 BCR-ABL1 and three control genes (ABL1, BCR and GUSB). The set of six plasmid CRMs is distributed worldwide by the Institute for Reference Materials and Measurements (Belgium) and its authorised distributors (https://ec.europa.eu/jrc/en/reference-materials/catalogue/; CRM code ERM-AD623a-f).

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