Sir Peter MacCallum Department of Oncology - Research Publications
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ItemASXL1 c.1934dup;p.Gly646Trpfs*12-a true somatic alteration requiring a new approach(NATURE PUBLISHING GROUP, 2017-12-20)
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ItemQuantitative threefold allele-specific PCR (QuanTAS-PCR) for highly sensitive JAK2 V617F mutant allele detection(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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ItemA multicentre retrospective comparison of central nervous system prophylaxis strategies among patients with high-risk diffuse large B-cell lymphoma(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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ItemCirculating tumour DNA reflects treatment response and clonal evolution in chronic lymphocytic leukaemia(NATURE PUBLISHING GROUP, 2017-03-17)Several novel therapeutics are poised to change the natural history of chronic lymphocytic leukaemia (CLL) and the increasing use of these therapies has highlighted limitations of traditional disease monitoring methods. Here we demonstrate that circulating tumour DNA (ctDNA) is readily detectable in patients with CLL. Importantly, ctDNA does not simply mirror the genomic information contained within circulating malignant lymphocytes but instead parallels changes across different disease compartments following treatment with novel therapies. Serial ctDNA analysis allows clonal dynamics to be monitored over time and identifies the emergence of genomic changes associated with Richter's syndrome (RS). In addition to conventional disease monitoring, ctDNA provides a unique opportunity for non-invasive serial analysis of CLL for molecular disease monitoring.
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ItemA complementary role of multiparameter flow cytometry and high-throughput sequencing for minimal residual disease detection in chronic lymphocytic leukemia: an European Research Initiative on CLL study(NATURE PUBLISHING GROUP, 2016-04)In chronic lymphocytic leukemia (CLL) the level of minimal residual disease (MRD) after therapy is an independent predictor of outcome. Given the increasing number of new agents being explored for CLL therapy, using MRD as a surrogate could greatly reduce the time necessary to assess their efficacy. In this European Research Initiative on CLL (ERIC) project we have identified and validated a flow-cytometric approach to reliably quantitate CLL cells to the level of 0.0010% (10(-5)). The assay comprises a core panel of six markers (i.e. CD19, CD20, CD5, CD43, CD79b and CD81) with a component specification independent of instrument and reagents, which can be locally re-validated using normal peripheral blood. This method is directly comparable to previous ERIC-designed assays and also provides a backbone for investigation of new markers. A parallel analysis of high-throughput sequencing using the ClonoSEQ assay showed good concordance with flow cytometry results at the 0.010% (10(-4)) level, the MRD threshold defined in the 2008 International Workshop on CLL guidelines, but it also provides good linearity to a detection limit of 1 in a million (10(-6)). The combination of both technologies would permit a highly sensitive approach to MRD detection while providing a reproducible and broadly accessible method to quantify residual disease and optimize treatment in CLL.
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ItemDynamic Thromboembolic Risk Modelling to Target Appropriate Preventative Strategies for Patients with Non-Small Cell Lung Cancer(MDPI, 2019-01)Prevention of cancer-associated thromboembolism (TE) remains a significant clinical challenge and priority world-wide safety initiative. In this prospective non-small cell lung cancer (NSCLC) cohort, longitudinal TE risk profiling (clinical and biomarker) was undertaken to develop risk stratification models for targeted TE prevention. These were compared with published models from Khorana, CATS, PROTECHT, CONKO, and CATS/MICA. The NSCLC cohort of 129 patients, median follow-up 22.0 months (range 5.6-31.3), demonstrated a hypercoagulable profile in >75% patients and TE incidence of 19%. High TE risk patients were those receiving chemotherapy with baseline fibrinogen ≥ 4 g/L and d-dimer ≥ 0.5 mg/L; or baseline d-dimer ≥ 1.5 mg/L; or month 1 d-dimer ≥ 1.5 mg/L. The model predicted TE with 100% sensitivity and 34% specificity (c-index 0.67), with TE incidence 27% vs. 0% for high vs. low-risk. A comparison using the Khorana, PROTECHT, and CONKO methods were not discriminatory; TE incidence 17⁻25% vs. 14⁻19% for high vs. low-risk (c-index 0.51⁻0.59). Continuous d-dimer (CATS/MICA model) was also not predictive of TE. Independent of tumour stage, high TE risk was associated with cancer progression (HR 1.9, p = 0.01) and mortality (HR 2.2, p = 0.02). The model was tested for scalability in a prospective gastrointestinal cancer cohort with equipotency demonstrated; 80% sensitivity and 39% specificity. This proposed TE risk prediction model is simple, practical, potent and can be used in the clinic for real-time, decision-making for targeted thromboprophylaxis. Validation in a multicentre randomised interventional study is underway (ACTRN12618000811202).
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ItemFrequent activating STAT3 mutations and novel recurrent genomic abnormalities detected in breast implant-associated anaplastic large cell lymphoma.(Impact Journals, LLC, 2018-11-16)Breast implant-associated anaplastic large cell lymphoma (BIA-ALCL) is a rare form of T-cell lymphoma that occurs after implantation of breast prostheses. We performed comprehensive next generation sequencing based genomic characterization of 11 cases of BIA-ALCL including sequence variant detection on 180 genes frequently mutated in haematological malignancy, genome-wide copy number assessment, structural variant detection involving the T-cell receptor loci and TRB deep-sequencing. We observed sequence variants leading to JAK/STAT activation in 10 out of 11 patients. We also observed germline TP53 mutations in two cases. In addition we detected a recurrent copy number loss involving RPL5 as well as copy number amplifications involving TNFRSF11A [RANK] (in 2 cases), MYC, P2RX7, TMEM119 and PDGFRA. In summary, our comprehensive genomic characterisation of 11 cases of BIA-ALCL has provided insight into potential pathobiological mechanisms (JAK/STAT, MYC and TP53) as well as identifying targets for future therapeutic intervention (TNFRSF11A, PDGFRA) in this rare entity.