Sir Peter MacCallum Department of Oncology - Research Publications

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    Detection of clinically relevant early genomic lesions in B-cell malignancies from circulating tumour DNA using a single hybridisation-based next generation sequencing assay
    Blombery, PA ; Ryland, GL ; Markham, J ; Guinto, J ; Wall, M ; McBean, M ; Jones, K ; Thompson, ER ; Cameron, DL ; Papenfuss, AT ; Prince, MH ; Dickinson, M ; Westerman, DA (WILEY, 2018-10)
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    Characterization of a novel venetoclax resistance mutation (BCL2 Phe104Ile) observed in follicular lymphoma
    Blombery, P ; Birkinshaw, RW ; Nguyen, T ; Gong, J-N ; Thompson, ER ; Xu, Z ; Westerman, DA ; Czabotar, PE ; Dickinson, M ; Huang, DCS ; Seymour, JF ; Roberts, AW (WILEY, 2019-09)
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    Quantitation of CMV Specific T-Cell Expansion Using T Cell Receptor Beta Locus Deep Sequencing to Identify Patients at Risk of Viral Complications
    Kuzich, JA ; Kankanige, Y ; Guinto, J ; Ryland, G ; McBean, M ; Thompson, E ; Wong, E ; Koldej, R ; Collins, J ; Westerman, D ; Ritchie, DS ; Blombery, P (ELSEVIER SCIENCE INC, 2020-03)
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    Inotuzumab ozogamicin resistance associated with a novel CD22 truncating mutation in a case of B-acute lymphoblastic leukaemia
    Ryland, GL ; Barraclough, A ; Fong, CY ; Fleming, S ; Bajel, A ; Hofmann, O ; Westerman, D ; Grimmond, S ; Blombery, P (WILEY, 2020-10)
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    Validation of a modified pre-lysis sample preparation technique for flow cytometric minimal residual disease assessment in multiple myeloma, chronic lymphocytic leukemia, and B-non Hodgkin lymphoma
    Bayly, E ; Nguyen, V ; Binek, A ; Piggin, A ; Baldwin, K ; Westerman, D ; Came, N (WILEY, 2020-09)
    BACKGROUND: Minimal residual disease (MRD) assessment of hematopoietic neoplasia below 10-4 requires more leukocytes than is usually attainable by post-lysis preparation. However, not all laboratories are resourced for consensus Euroflow pre-lysis methodology. Our study aim was to validate a modified pre-lysis protocol against our standard post-lysis method for MRD detection of multiple myeloma (MM), chronic lymphocytic leukemia (CLL), and B-non Hodgkin lymphoma (B-NHL), to meet demand for deeper MRD assessment by flow cytometry. METHOD: Clinical samples for MRD assessment of MM, CLL, and B-NHL (50, 30, and 30 cases, respectively) were prepared in parallel by pre and post-lysis methods for the initial validation. Total leukocytes, MRD, and median fluorescence intensity of antigen expression were compared as measures of sensitivity and antigen stability. Lymphocyte and granulocyte composition were compared, assessing relative sample processing stability. Sensitivity of the pre-lysis assay was monitored post validation for a further 18 months. RESULTS: Pre-lysis achieved at least 10-4 sensitivity in 85% MM, 81% CLL, and 90% B-NHL samples versus 24%, 48%, and 26% by post-lysis, respectively, with stable antigen expression and leukocyte composition. Post validation over 18 months with technical expertise improving, pre-lysis permitted 10-5 MRD assessment in 69%, 86%, and 82% of the respective patient groups. CONCLUSION: This modified pre-lysis procedure provides a sensitive, robust, time efficient, and relatively cost-effective alternative for MRD testing by MFC at 10-5 , facilitating clinically meaningful deeper response assessment for MM, CLL, and B-NHL. This method adaptation may facilitate more widespread adoption of highly sensitive flow cytometry-based MRD assessment.
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    ASXL1 c.1934dup;p.Gly646Trpfs*12-a true somatic alteration requiring a new approach
    Yannakou, CK ; Jones, K ; McBean, M ; Thompson, ER ; Ryland, GL ; Doig, K ; Markham, J ; Westerman, D ; Blombery, P (NATURE PUBLISHING GROUP, 2017-12-20)
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    Quantitative threefold allele-specific PCR (QuanTAS-PCR) for highly sensitive JAK2 V617F mutant allele detection
    Zapparoli, GV ; Jorissen, RN ; Hewitt, CA ; McBean, M ; Westerman, DA ; Dobrovic, A (BMC, 2013-04-24)
    BACKGROUND: The JAK2 V617F mutation is the most frequent somatic change in myeloproliferative neoplasms, making it an important tumour-specific marker for diagnostic purposes and for the detection of minimal residual disease. Sensitive quantitative assays are required for both applications, particularly for the monitoring of minimal residual disease, which requires not only high sensitivity but also very high specificity. METHODS: We developed a highly sensitive probe-free quantitative mutant-allele detection method, Quantitative Threefold Allele-Specific PCR (QuanTAS-PCR), that is performed in a closed-tube system, thus eliminating the manipulation of PCR products. QuantTAS-PCR uses a threefold approach to ensure allele-specific amplification of the mutant sequence: (i) a mutant allele-specific primer, (ii) a 3'dideoxy blocker to suppress false-positive amplification from the wild-type template and (iii) a PCR specificity enhancer, also to suppress false-positive amplification from the wild-type template. Mutant alleles were quantified relative to exon 9 of JAK2. RESULTS: We showed that the addition of the 3'dideoxy blocker suppressed but did not eliminate false-positive amplification from the wild-type template. However, the addition of the PCR specificity enhancer near eliminated false-positive amplification from the wild-type allele. Further discrimination between true and false positives was enabled by using the quantification cycle (Cq) value of a single mutant template as a cut-off point, thus enabling robust distinction between true and false positives. As 10,000 JAK2 templates were used per replicate, the assay had a sensitivity of 1/10(-4) per replicate. Greater sensitivity could be reached by increasing the number of replicates analysed. Variation in replicates when low mutant-allele templates were present necessitated the use of a statistics-based approach to estimate the load of mutant JAK2 copies. QuanTAS-PCR showed comparable quantitative results when validated against a commercial assay. CONCLUSIONS: QuanTAS-PCR is a simple, cost-efficient, closed-tube method for JAK2 V617F mutation quantification that can detect very low levels of the mutant allele, thus enabling analysis of minimal residual disease. The approach can be extended to the detection of other recurrent single nucleotide somatic changes in cancer.
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    Negative selection of chronic lymphocytic leukaemia cells using a bifunctional rosette-based antibody cocktail
    Essakali, S ; Carney, D ; Westerman, D ; Gambell, P ; Seymour, JF ; Dobrovic, A (BMC, 2008-01-29)
    BACKGROUND: High purity of tumour samples is a necessity for accurate genetic and expression analysis and is usually achieved by positive selection in chronic lymphocytic leukaemia (CLL). RESULTS: We adapted a bifunctional rosette-based antibody cocktail for negative selection of B-cells for isolating CLL cells from peripheral blood (PB). PB samples from CLL patients were split into aliquots. One aliquot of each sample was enriched by density gradient centrifugation (DGC), while the other aliquot of each sample was incubated with an antibody cocktail for B-cell enrichment prior to DGC (RS+DGC). The purity of CLL cells after DGC averaged 74.1% (range: 15.9 - 97.4%). Using RS+DGC, the purity averaged 93.8% (range: 80.4 - 99.4%) with 23 of 29 (79%) samples showing CLL purities above 90%. RNA extracted from enriched CLL cells was of appropriately high quality for microarray analysis. CONCLUSION: This study confirms the use of a bifunctional rosette-based antibody cocktail as an effective method for the purification of CLL cells from peripheral blood.
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    A multicentre retrospective comparison of central nervous system prophylaxis strategies among patients with high-risk diffuse large B-cell lymphoma
    Cheah, CY ; Herbert, KE ; O'Rourke, K ; Kennedy, GA ; George, A ; Fedele, PL ; Gilbertson, M ; Tan, SY ; Ritchie, DS ; Opat, SS ; Prince, HM ; Dickinson, M ; Burbury, K ; Wolf, M ; Januszewicz, EH ; Tam, CS ; Westerman, DA ; Carney, DA ; Harrison, SJ ; Seymour, JF (NATURE PUBLISHING GROUP, 2014-09-09)
    BACKGROUND: Central nervous system (CNS) relapse in diffuse large B-cell lymphoma (DLBCL) is a devastating complication; the optimal prophylactic strategy remains unclear. METHODS: We performed a multicentre, retrospective analysis of patients with DLBCL with high risk for CNS relapse as defined by two or more of: multiple extranodal sites, elevated serum LDH and B symptoms or involvement of specific high-risk anatomical sites. We compared three different strategies of CNS-directed therapy: intrathecal (IT) methotrexate (MTX) with (R)-CHOP 'group 1'; R-CHOP with IT MTX and two cycles of high-dose intravenous (IV) MTX 'group 2'; dose-intensive systemic antimetabolite-containing chemotherapy (Hyper-CVAD or CODOXM/IVAC) with IT/IV MTX 'group 3'. RESULTS: Overall, 217 patients were identified (49, 125 and 43 in groups 1-3, respectively). With median follow-up of 3.4 (range 0.2-18.6) years, 23 CNS relapses occurred (12, 10 and 1 in groups 1-3 respectively). The 3-year actuarial rates (95% CI) of CNS relapse were 18.4% (9.5-33.1%), 6.9% (3.5-13.4%) and 2.3% (0.4-15.4%) in groups 1-3, respectively (P=0.009). CONCLUSIONS: The addition of high-dose IV MTX and/or cytarabine was associated with lower incidence of CNS relapse compared with IT chemotherapy alone. However, these data are limited by their retrospective nature and warrant confirmation in prospective randomised studies.
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    Reproducible diagnosis of chronic lymphocytic leukemia by flow cytometry: An European Research Initiative on CLL (ERIC) & European Society for Clinical Cell Analysis (ESCCA) Harmonisation project
    Rawstron, AC ; Kreuzer, K-A ; Soosapilla, A ; Spacek, M ; Stehlikova, O ; Gambell, P ; McIver-Brown, N ; Villamor, N ; Psarra, K ; Arroz, M ; Milani, R ; de la Serna, J ; Teresa Cedena, M ; Jaksic, O ; Nomdedeu, J ; Moreno, C ; Rigolin, GM ; Cuneo, A ; Johansen, P ; Johnsen, HE ; Rosenquist, R ; Niemann, CU ; Kern, W ; Westerman, D ; Trneny, M ; Mulligan, S ; Doubek, M ; Pospisilova, S ; Hillmen, P ; Oscier, D ; Hallek, M ; Ghia, P ; Montserrat, E (WILEY, 2018-01)