Sir Peter MacCallum Department of Oncology - Research Publications
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ItemEvaluation of T-cell clonality by anti-TRBC1 antibody-based flow cytometry and correlation with T-cell receptor sequencing(Wiley, 2024-03)Flow cytometry (FC) incorporating the T-cell receptor β constant chain-1 (TRBC1) has been recently proposed as a new standard in T-cell clonality assessment. While early studies demonstrated high sensitivity in samples with conspicuous tumour burden, performance in real-world samples, including those with low tumour burden and correlation with molecular methods has been limited. We evaluated TRBC1-FC performance and correlated the results with high-throughput TRB sequencing and a targeted next-generation sequencing gene panel. Our cohort consisted of 90 evaluable samples from 57 patients. TRBC1-FC confirmed T-cell clonality in 37 out of 38 samples (97%) that were involved in a mature T-cell neoplasm (MTCN). T-cell clonality was also identified in nine samples from patients lacking a current or prior diagnosis of MTCN, consistent with the emerging entity T-cell clonality of uncertain significance. TRBC-FC was polyclonal in all samples and negative for disease involvement by standard pathology assessment. However, correlation with TRB sequencing in 17 of these samples identified two cases that harboured the known clonal sequence from index testing, indicating the presence of measurable residual disease not otherwise detected. Our study provides real-world correlative validation of TRBC1-FC, highlighting the strengths and limitations pertinent to its increasing implementation by general diagnostic laboratories.
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ItemVEXAS syndrome: A dermatological perspective(WILEY, 2022-11)VEXAS (Vacuoles, E1 enzyme, X-linked, autoinflammatory and somatic mutation) syndrome is a genetically defined disorder identified in 2020, describing patients with inflammatory syndromes associated with haematological dysfunction. It is a severe, treatment-resistant condition, with estimated mortality between 40% and 63%. A wide range of cutaneous manifestations have been described. Here, we report on two patients with treatment-resistant neutrophilic dermatosis and myelodysplastic syndrome, who were subsequently diagnosed with VEXAS syndrome. Our cases highlight the need for dermatologists' awareness of this novel condition and to initiate early referral to haematologists for appropriate multidisciplinary care.
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ItemNo Preview AvailableEnhancer retargeting of CDX2 and UBTF::ATXN7L3 define a subtype of high-risk B-progenitor acute lymphoblastic leukemia(AMER SOC HEMATOLOGY, 2022-06-16)Transcriptome sequencing has identified multiple subtypes of B-progenitor acute lymphoblastic leukemia (B-ALL) of prognostic significance, but a minority of cases lack a known genetic driver. Here, we used integrated whole-genome (WGS) and -transcriptome sequencing (RNA-seq), enhancer mapping, and chromatin topology analysis to identify previously unrecognized genomic drivers in B-ALL. Newly diagnosed (n = 3221) and relapsed (n = 177) B-ALL cases with tumor RNA-seq were studied. WGS was performed to detect mutations, structural variants, and copy number alterations. Integrated analysis of histone 3 lysine 27 acetylation and chromatin looping was performed using HiChIP. We identified a subset of 17 newly diagnosed and 5 relapsed B-ALL cases with a distinct gene expression profile and 2 universal and unique genomic alterations resulting from aberrant recombination-activating gene activation: a focal deletion downstream of PAN3 at 13q12.2 resulting in CDX2 deregulation by the PAN3 enhancer and a focal deletion of exons 18-21 of UBTF at 17q21.31 resulting in a chimeric fusion, UBTF::ATXN7L3. A subset of cases also had rearrangement and increased expression of the PAX5 gene, which is otherwise uncommon in B-ALL. Patients were more commonly female and young adult with median age 35 (range,12-70 years). The immunophenotype was characterized by CD10 negativity and immunoglobulin M positivity. Among 16 patients with known clinical response, 9 (56.3%) had high-risk features including relapse (n = 4) or minimal residual disease >1% at the end of remission induction (n = 5). CDX2-deregulated, UBTF::ATXN7L3 rearranged (CDX2/UBTF) B-ALL is a high-risk subtype of leukemia in young adults for which novel therapeutic approaches are required.
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ItemNo Preview AvailableSingle-cell multiomics reveal the scale of multilayered adaptations enabling CLL relapse during venetoclax therapy(AMER SOC HEMATOLOGY, 2022-11-17)Venetoclax (VEN) inhibits the prosurvival protein BCL2 to induce apoptosis and is a standard therapy for chronic lymphocytic leukemia (CLL), delivering high complete remission rates and prolonged progression-free survival in relapsed CLL but with eventual loss of efficacy. A spectrum of subclonal genetic changes associated with VEN resistance has now been described. To fully understand clinical resistance to VEN, we combined single-cell short- and long-read RNA-sequencing to reveal the previously unappreciated scale of genetic and epigenetic changes underpinning acquired VEN resistance. These appear to be multilayered. One layer comprises changes in the BCL2 family of apoptosis regulators, especially the prosurvival family members. This includes previously described mutations in BCL2 and amplification of the MCL1 gene but is heterogeneous across and within individual patient leukemias. Changes in the proapoptotic genes are notably uncommon, except for single cases with subclonal losses of BAX or NOXA. Much more prominent was universal MCL1 gene upregulation. This was driven by an overlying layer of emergent NF-κB (nuclear factor kappa B) activation, which persisted in circulating cells during VEN therapy. We discovered that MCL1 could be a direct transcriptional target of NF-κB. Both the switch to alternative prosurvival factors and NF-κB activation largely dissipate following VEN discontinuation. Our studies reveal the extent of plasticity of CLL cells in their ability to evade VEN-induced apoptosis. Importantly, these findings pinpoint new approaches to circumvent VEN resistance and provide a specific biological justification for the strategy of VEN discontinuation once a maximal response is achieved rather than maintaining long-term selective pressure with the drug.
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ItemNo Preview AvailableAcquired mutations in BAX confer resistance to BH3-mimetic therapy in acute myeloid leukemia(AMER SOC HEMATOLOGY, 2023-02-09)Randomized trials in acute myeloid leukemia (AML) have demonstrated improved survival by the BCL-2 inhibitor venetoclax combined with azacitidine in older patients, and clinical trials are actively exploring the role of venetoclax in combination with intensive chemotherapy in fitter patients with AML. As most patients still develop recurrent disease, improved understanding of relapse mechanisms is needed. We find that 17% of patients relapsing after venetoclax-based therapy for AML have acquired inactivating missense or frameshift/nonsense mutations in the apoptosis effector gene BAX. In contrast, such variants were rare after genotoxic chemotherapy. BAX variants arose within either leukemic or preleukemic compartments, with multiple mutations observed in some patients. In vitro, AML cells with mutated BAX were competitively selected during prolonged exposure to BCL-2 antagonists. In model systems, AML cells rendered deficient for BAX, but not its close relative BAK, displayed resistance to BCL-2 targeting, whereas sensitivity to conventional chemotherapy was variable. Acquired mutations in BAX during venetoclax-based therapy represent a novel mechanism of resistance to BH3-mimetics and a potential barrier to the long-term efficacy of drugs targeting BCL-2 in AML.
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ItemEnrichment of BTK Leu528Trp mutations in patients with CLL on zanubrutinib: potential for pirtobrutinib cross-resistance(ELSEVIER, 2022-10-25)The covalent Bruton's tyrosine kinase inhibitors (BTKis) are highly effective for the treatment of chronic lymphocytic leukemia (CLL). The dominant resistance mechanism observed with the BTKi ibrutinib is the development of BTK Cys481 codon mutations. Whether a similar resistance mutation profile exists for the newer-generation, more selective BTKi zanubrutinib is unknown. In samples referred for diagnostic next-generation sequencing in patients with progressive CLL, we observed an enrichment in the kinase-dead BTK Leu528Trp mutation in patients treated with zanubrutinib compared with ibrutinib (54%; 7 of 13 vs 4%; 1 of 24, P = .001). We describe 2 patients with BTK Leu528Trp mutations who showed clinical cross-resistance and progressive enrichment of the BTK Leu528Trp mutation over time when treated with the noncovalent BTKi pirtobrutinib. Both patients subsequently responded to venetoclax-based treatment. In summary, we have identified an enrichment of the BTK Leu528Trp mutation arising in patients treated with zanubrutinib that may impart cross-resistance to the noncovalent inhibitor pirtobrutinib and therefore may have implications for sequencing of these treatments in CLL.
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ItemHealth economic evidence for the use of molecular biomarker tests in hematological malignancies: A systematic review(WILEY, 2022-06)OBJECTIVES: Molecular biomarker tests can inform the clinical management of genomic heterogeneous hematological malignancies, yet their availability in routine care largely depends on the supporting health economic evidence. This study aims to systematically review the economic evidence for recent molecular biomarker tests in hematological malignancies. METHODS: We conducted a systematic search in five electronic databases for studies published between January 2010 and October 2020. Publications were independently screened by two reviewers. Clinical study characteristics, economic methodology, and results were extracted, and reporting quality was assessed. RESULTS: Fourteen studies were identified, of which half (n = 7; 50%) were full economic evaluations examining both health and economic outcomes. Studies were predominantly conducted in a first-line treatment setting (n = 7; 50%) and adopted a non-lifetime time horizon to measure health outcomes and costs (n = 7; 50%). Five studies reported that companion diagnostics for associated therapies were likely cost-effective for acute myeloid leukemia, chronic myeloid leukemia, diffuse large B-cell lymphoma, and multiple myeloma. Four studies suggested molecular biomarker tests for treatment monitoring in chronic myeloid leukemia were likely cost-saving. CONCLUSIONS: Although there is initial confirmation of the promising health economic results, the present research for molecular biomarker tests in hematological malignancies is sparse with many applications of technological advances yet to be evaluated.
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ItemSingle-cell sequencing demonstrates complex resistance landscape in CLL and MCL treated with BTK and BCL2 inhibitors(ELSEVIER, 2022-01-25)The genomic landscape of resistance to targeted agents (TAs) used as monotherapy in chronic lymphocytic leukemia (CLL) is complex and often heterogeneous at the patient level. To gain insight into the clonal architecture of acquired genomic resistance to Bruton tyrosine kinase (BTK) inhibitors and B-cell lymphoma 2 (BCL2) inhibitors in CLL, particularly in patients carrying multiple resistance mutations, we performed targeted single-cell DNA sequencing of 8 patients who developed progressive disease (PD) on TAs (either class). In all cases, analysis of single-cell architecture revealed mutual exclusivity between multiple resistance mutations to the same TA class, variable clonal co-occurrence of multiple mutations affecting different TAs in patients exposed to both classes, and a phenomenon of multiple independent emergences of identical nucleotide changes leading to canonical resistance mutations. We also report the first observation of established BCL2 resistance mutations in a patient with mantle cell lymphoma (MCL) following PD on sequential monotherapy, implicating BCL2 as a venetoclax resistance mechanism in MCL. Taken together, these data reveal the significant clonal complexity of CLL and MCL progression on TAs at the nucleotide level and confirm the presence of multiple, clonally independent, mechanisms of TA resistance within each individual disease context.