Publications by authors named "Sarah-Jane Dawson"

60 Publications

Detection of cell-free microbial DNA using a contaminant-controlled analysis framework.

Genome Biol 2021 06 23;22(1):187. Epub 2021 Jun 23.

Peter MacCallum Cancer Centre, Melbourne, Australia.

Background: The human microbiome plays an important role in cancer. Accumulating evidence indicates that commensal microbiome-derived DNA may be represented in minute quantities in the cell-free DNA of human blood and could possibly be harnessed as a new cancer biomarker. However, there has been limited use of rigorous experimental controls to account for contamination, which invariably affects low-biomass microbiome studies.

Results: We apply a combination of 16S-rRNA-gene sequencing and droplet digital PCR to determine if the specific detection of cell-free microbial DNA (cfmDNA) is possible in metastatic melanoma patients. Compared to matched stool and saliva samples, the absolute concentration of cfmDNA is low but significantly above the levels detected from negative controls. The microbial community of plasma is strongly influenced by laboratory and reagent contaminants introduced during the DNA extraction and sequencing processes. Through the application of an in silico decontamination strategy including the filtering of amplicon sequence variants (ASVs) with batch dependent abundances and those with a higher prevalence in negative controls, we identify known gut commensal bacteria, such as Faecalibacterium, Bacteroides and Ruminococcus, and also other uncharacterised ASVs. We analyse additional plasma samples, highlighting the potential of this framework to identify differences in cfmDNA between healthy and cancer patients.

Conclusions: Together, these observations indicate that plasma can harbour a low yet detectable level of cfmDNA. The results highlight the importance of accounting for contamination and provide an analytical decontamination framework to allow the accurate detection of cfmDNA for future biomarker studies in cancer and other diseases.
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http://dx.doi.org/10.1186/s13059-021-02401-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8220693PMC
June 2021

HIV is associated with an increased risk of age-related clonal hematopoiesis among older adults.

Nat Med 2021 06 7;27(6):1006-1011. Epub 2021 Jun 7.

Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia.

People with human immunodeficiency virus (HIV) have higher rates of certain comorbidities, particularly cardiovascular disease and cancer, than people without HIV. In view of observations that somatic mutations associated with age-related clonal hematopoiesis (CH) are linked to similar comorbidities in the general population, we hypothesized that CH may be more prevalent in people with HIV. To address this issue, we established a prospective cohort study, the ARCHIVE study (NCT04641013), in which 220 HIV-positive and 226 HIV-negative participants aged 55 years or older were recruited in Australia. Demographic characteristics, clinical data and peripheral blood were collected to assess the presence of CH mutations and to identify potential risk factors for and clinical sequelae of CH. In total, 135 CH mutations were identified in 100 (22.4%) of 446 participants. CH was more prevalent in HIV-positive participants than in HIV-negative participants (28.2% versus 16.8%, P = 0.004), overall and across all age groups; the adjusted odds ratio for having CH in those with HIV was 2.16 (95% confidence interval 1.34-3.48, P = 0.002). The most common genes mutated overall were DNMT3A (47.4%), TET2 (20.0%) and ASXL1 (13.3%). CH and HIV infection were independently associated with increases in blood parameters and biomarkers associated with inflammation. These data suggest a selective advantage for the emergence of CH in the context of chronic infection and inflammation related to HIV infection.
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http://dx.doi.org/10.1038/s41591-021-01357-yDOI Listing
June 2021

Evolution of late-stage metastatic melanoma is dominated by aneuploidy and whole genome doubling.

Nat Commun 2021 03 4;12(1):1434. Epub 2021 Mar 4.

Peter MacCallum Cancer Centre, Melbourne, VIC, Australia.

Although melanoma is initiated by acquisition of point mutations and limited focal copy number alterations in melanocytes-of-origin, the nature of genetic changes that characterise lethal metastatic disease is poorly understood. Here, we analyze the evolution of human melanoma progressing from early to late disease in 13 patients by sampling their tumours at multiple sites and times. Whole exome and genome sequencing data from 88 tumour samples reveals only limited gain of point mutations generally, with net mutational loss in some metastases. In contrast, melanoma evolution is dominated by whole genome doubling and large-scale aneuploidy, in which widespread loss of heterozygosity sculpts the burden of point mutations, neoantigens and structural variants even in treatment-naïve and primary cutaneous melanomas in some patients. These results imply that dysregulation of genomic integrity is a key driver of selective clonal advantage during melanoma progression.
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http://dx.doi.org/10.1038/s41467-021-21576-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7933255PMC
March 2021

Towards Routine Implementation of Liquid Biopsies in Cancer Management: It Is Always Too Early, until Suddenly It Is Too Late.

Diagnostics (Basel) 2021 Jan 11;11(1). Epub 2021 Jan 11.

Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne Centre for Cancer Research, Parkville, VIC 3000, Australia.

Blood-based liquid biopsies are considered a new and promising diagnostic and monitoring tool for cancer. As liquid biopsies only require a blood draw, they are non-invasive, potentially more rapid and assumed to be a less costly alternative to genomic analysis of tissue biopsies. A multi-disciplinary workshop ( = 98 registrations) was organized to discuss routine implementation of liquid biopsies in cancer management. Real-time polls were used to engage with experts' about the current evidence of clinical utility and the barriers to implementation of liquid biopsies. Clinical, laboratory and health economics presentations were given to illustrate the opportunities and current levels of evidence, followed by three moderated break-out sessions to discuss applications. The workshop concluded that tumor-informed assays using next-generation sequencing (NGS) or PCR-based genotyping assays will most likely provide better clinical utility than tumor-agnostic assays, yet at a higher cost. For routine application, it will be essential to determine clinical utility, to define the minimum quality standards and performance of testing platforms and to ensure their use is integrated into current clinical workflows including how they complement tissue biopsies and imaging. Early health economic models may help identifying the most viable application of liquid biopsies. Alternative funding models for the translation of complex molecular diagnostics, such as liquid biopsies, may also be explored if clinical utility has been demonstrated and when their use is recommended in multi-disciplinary consensus guidelines.
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http://dx.doi.org/10.3390/diagnostics11010103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7826562PMC
January 2021

Circulating Tumor DNA Predicts Outcome from First-, but not Second-line Treatment and Identifies Melanoma Patients Who May Benefit from Combination Immunotherapy.

Clin Cancer Res 2020 11 16;26(22):5926-5933. Epub 2020 Oct 16.

School of Medical and Health Sciences, Edith Cowan University, Perth, Australia.

Purpose: We evaluated the predictive value of pretreatment ctDNA to inform therapeutic outcomes in patients with metastatic melanoma relative to type and line of treatment.

Experimental Design: Plasma circulating tumor DNA (ctDNA) was quantified in 125 samples collected from 110 patients prior to commencing treatment with immune checkpoint inhibitors (ICIs), as first- ( = 32) or second-line ( = 27) regimens, or prior to commencing first-line BRAF/MEK inhibitor therapy ( = 66). An external validation cohort included 128 patients commencing ICI therapies in the first- ( = 77) or second-line ( = 51) settings.

Results: In the discovery cohort, low ctDNA (≤20 copies/mL) prior to commencing therapy predicted longer progression-free survival (PFS) in patients treated with first-line ICIs [HR, 0.20; 95% confidence interval (CI) 0.07-0.53; < 0.0001], but not in the second-line setting. An independent cohort validated that ctDNA is predictive of PFS in the first-line setting (HR, 0.42; 95% CI, 0.22-0.83; = 0.006), but not in the second-line ICI setting. Moreover, ctDNA prior to commencing ICI treatment was not predictive of PFS for patients pretreated with BRAF/MEK inhibitors in either the discovery or validation cohorts. Reduced PFS and overall survival were observed in patients with high ctDNA receiving anti-PD-1 monotherapy, relative to those treated with combination anti-CTLA-4/anti-PD-1 inhibitors.

Conclusions: Pretreatment ctDNA is a reliable indicator of patient outcome in the first-line ICI treatment setting, but not in the second-line ICI setting, especially in patients pretreated with BRAF/MEK inhibitors. Preliminary evidence indicated that treatment-naïve patients with high ctDNA may preferentially benefit from combined ICIs.
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http://dx.doi.org/10.1158/1078-0432.CCR-20-2251DOI Listing
November 2020

Non-genetic mechanisms of therapeutic resistance in cancer.

Nat Rev Cancer 2020 12 8;20(12):743-756. Epub 2020 Oct 8.

Peter MacCallum Cancer Centre, Melbourne, VIC, Australia.

Therapeutic resistance continues to be an indominable foe in our ambition for curative cancer treatment. Recent insights into the molecular determinants of acquired treatment resistance in the clinical and experimental setting have challenged the widely held view of sequential genetic evolution as the primary cause of resistance and brought into sharp focus a range of non-genetic adaptive mechanisms. Notably, the genetic landscape of the tumour and the non-genetic mechanisms used to escape therapy are frequently linked. Remarkably, whereas some oncogenic mutations allow the cancer cells to rapidly adapt their transcriptional and/or metabolic programme to meet and survive the therapeutic pressure, other oncogenic drivers convey an inherent cellular plasticity to the cancer cell enabling lineage switching and/or the evasion of anticancer immunosurveillance. The prevalence and diverse array of non-genetic resistance mechanisms pose a new challenge to the field that requires innovative strategies to monitor and counteract these adaptive processes. In this Perspective we discuss the key principles of non-genetic therapy resistance in cancer. We provide a perspective on the emerging data from clinical studies and sophisticated cancer models that have studied various non-genetic resistance pathways and highlight promising therapeutic avenues that may be used to negate and/or counteract the non-genetic adaptive pathways.
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http://dx.doi.org/10.1038/s41568-020-00302-4DOI Listing
December 2020

Circulating tumour DNA in metastatic breast cancer to guide clinical trial enrolment and precision oncology: A cohort study.

PLoS Med 2020 10 1;17(10):e1003363. Epub 2020 Oct 1.

Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia.

Background: Metastatic breast cancer (mBC) is a heterogenous disease with increasing availability of targeted therapies as well as emerging genomic markers of therapeutic resistance, necessitating timely and accurate molecular characterization of disease. As a minimally invasive test, analysis of circulating tumour DNA (ctDNA) is well positioned for real-time genomic profiling to guide treatment decisions. Here, we report the results of a prospective testing program established to assess the feasibility of ctDNA analysis to guide clinical management of mBC patients.

Methods And Findings: Two hundred thirty-four mBC patients (median age 54 years) were enrolled between June 2015 and October 2018 at the Peter MacCallum Cancer Centre, Melbourne, Australia. Median follow-up was 15 months (range 1-46). All patient samples at the time of enrolment were analysed in real time for the presence of somatic mutations. Longitudinal plasma testing during the course of patient management was also undertaken in a subset of patients (n = 67, 28.6%), according to clinician preference, for repeated molecular profiling or disease monitoring. Detection of somatic mutations from patient plasma was performed using a multiplexed droplet digital PCR (ddPCR) approach to identify hotspot mutations in PIK3CA, ESR1, ERBB2, and AKT1. In parallel, subsets of samples were also analysed via next-generation sequencing (targeted panel sequencing and low-coverage whole-genome sequencing [LC-WGS]). The sensitivity of ddPCR and targeted panel sequencing to identify actionable mutations was compared. Results were discussed at a multidisciplinary breast cancer meeting prior to treatment decisions. ddPCR and targeted panel sequencing identified at least 1 actionable mutation at baseline in 80/234 (34.2%) and 62/159 (39.0%) of patients tested, respectively. Combined, both methods detected an actionable alteration in 104/234 patients (44.4%) through baseline or serial ctDNA testing. LC-WGS was performed on 27 patients from the cohort, uncovering several recurrently amplified regions including 11q13.3 encompassing CCND1. Increasing ctDNA levels were associated with inferior overall survival, whether assessed by ddPCR, targeted sequencing, or LC-WGS. Overall, the ctDNA results changed clinical management in 40 patients including the direct recruitment of 20 patients to clinical trials. Limitations of the study were that it was conducted at a single site and that 31.3% of participants were lost to follow-up.

Conclusion: In this study, we found prospective ctDNA testing to be a practical and feasible approach that can guide clinical trial enrolment and patient management in mBC.
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http://dx.doi.org/10.1371/journal.pmed.1003363DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7529214PMC
October 2020

Clinical implications of prospective genomic profiling of metastatic breast cancer patients.

Breast Cancer Res 2020 08 18;22(1):91. Epub 2020 Aug 18.

Division of Research, Peter MacCallum Cancer Centre, Melbourne, Australia.

Background: Metastatic breast cancer remains incurable. Next-generation sequencing (NGS) offers the ability to identify actionable genomic alterations in tumours which may then be matched with targeted therapies, but the implementation and utility of this approach is not well defined for patients with metastatic breast cancer.

Methods: We recruited patients with advanced breast cancer of any subtype for prospective targeted NGS of their most recent tumour samples, using a panel of 108 breast cancer-specific genes. Genes were classified as actionable or non-actionable using the European Society of Medical Oncology Scale for Clinical Actionability of Molecular Targets (ESCAT) guidelines.

Results: Between February 2014 and May 2019, 322 patients were enrolled onto the study, with 72% (n = 234) of patients successfully sequenced (n = 357 samples). The majority (74%, n = 171) of sequenced patients were found to carry a potentially actionable alteration, the most common being a PIK3CA mutation. Forty-three percent (n = 74) of patients with actionable alterations were referred for a clinical trial or referred for confirmatory germline testing or had a change in therapy outside of clinical trials. We found alterations in AKT1, BRCA2, CHEK2, ESR1, FGFR1, KMT2C, NCOR1, PIK3CA and TSC2 to be significantly enriched in our metastatic population compared with primary breast cancers. Concordance between primary and metastatic samples for key driver genes (TP53, ERBB2 amplification) was > 75%. Additionally, we found that patients with a higher number of mutations had a significantly worse overall survival.

Conclusion: Genomic profiling of patients with metastatic breast cancer can have clinical implications and should be considered in all suitable patients.
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http://dx.doi.org/10.1186/s13058-020-01328-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7436992PMC
August 2020

Combining liquid biopsies and PET-CT for early cancer detection.

Nat Med 2020 07;26(7):1010-1011

Peter MacCallum Cancer Centre, Melbourne, Australia.

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http://dx.doi.org/10.1038/s41591-020-0970-9DOI Listing
July 2020

Longitudinal Monitoring of ctDNA in Patients with Melanoma and Brain Metastases Treated with Immune Checkpoint Inhibitors.

Clin Cancer Res 2020 08 22;26(15):4064-4071. Epub 2020 Apr 22.

Department of Biomedical Science, Macquarie University, Sydney, New South Wales, Australia.

Purpose: Brain involvement occurs in the majority of patients with metastatic melanoma. The potential of circulating tumor DNA (ctDNA) for surveillance and monitoring systemic therapy response in patients with melanoma brain metastases merits investigation.

Experimental Design: This study examined circulating , and mutations in patients with melanoma with active brain metastases receiving PD-1 inhibitor-based therapy. Intracranial and extracranial disease volumes were measured using the sum of product of diameters, and response assessment performed using RECIST. Longitudinal plasma samples were analyzed for ctDNA over the first 12 weeks of treatment (threshold 2.5 copies/mL plasma).

Results: Of a total of 72 patients, 13 patients had intracranial metastases only and 59 patients had concurrent intracranial and extracranial metastases. ctDNA detectability was 0% and 64%, respectively, and detectability was associated with extracranial disease volume ( < 0.01). Undetectable ctDNA on-therapy was associated with extracranial response ( < 0.01) but not intracranial response. The median overall survival in patients with undetectable ( = 34) versus detectable ( = 38) ctDNA at baseline was 39.2 versus 10.6 months [HR, 0.51; 95% confidence interval (CI), 0.28-0.94; = 0.03] and on-therapy was 39.2 versus 9.2 months (HR, 0.32; 95% CI, 0.16-0.63; < 0.01).

Conclusions: ctDNA remains a strong prognostic biomarker in patients with melanoma with brain metastases, especially in patients with concurrent extracranial disease. However, ctDNA was not able to detect or monitor intracranial disease activity, and we recommend against using ctDNA as a sole test during surveillance and therapeutic monitoring in patients with melanoma.
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http://dx.doi.org/10.1158/1078-0432.CCR-19-3926DOI Listing
August 2020

Selective targeting of BD1 and BD2 of the BET proteins in cancer and immunoinflammation.

Science 2020 04 19;368(6489):387-394. Epub 2020 Mar 19.

Cellzome GmbH, Functional Genomics R&D, GlaxoSmithKline, Heidelberg, Germany.

The two tandem bromodomains of the BET (bromodomain and extraterminal domain) proteins enable chromatin binding to facilitate transcription. Drugs that inhibit both bromodomains equally have shown efficacy in certain malignant and inflammatory conditions. To explore the individual functional contributions of the first (BD1) and second (BD2) bromodomains in biology and therapy, we developed selective BD1 and BD2 inhibitors. We found that steady-state gene expression primarily requires BD1, whereas the rapid increase of gene expression induced by inflammatory stimuli requires both BD1 and BD2 of all BET proteins. BD1 inhibitors phenocopied the effects of pan-BET inhibitors in cancer models, whereas BD2 inhibitors were predominantly effective in models of inflammatory and autoimmune disease. These insights into the differential requirement of BD1 and BD2 for the maintenance and induction of gene expression may guide future BET-targeted therapies.
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http://dx.doi.org/10.1126/science.aaz8455DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7610820PMC
April 2020

Coding and noncoding drivers of mantle cell lymphoma identified through exome and genome sequencing.

Blood 2020 07;136(5):572-584

Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC, Canada.

Mantle cell lymphoma (MCL) is an uncommon B-cell non-Hodgkin lymphoma (NHL) that is incurable with standard therapies. The genetic drivers of this cancer have not been firmly established, and the features that contribute to differences in clinical course remain limited. To extend our understanding of the biological pathways involved in this malignancy, we performed a large-scale genomic analysis of MCL using data from 51 exomes and 34 genomes alongside previously published exome cohorts. To confirm our findings, we resequenced the genes identified in the exome cohort in 191 MCL tumors, each having clinical follow-up data. We confirmed the prognostic association of TP53 and NOTCH1 mutations. Our sequencing revealed novel recurrent noncoding mutations surrounding a single exon of the HNRNPH1gene. In RNA-seq data from 103 of these cases, MCL tumors with these mutations had a distinct imbalance of HNRNPH1 isoforms. This altered splicing of HNRNPH1 was associated with inferior outcomes in MCL and showed a significant increase in protein expression by immunohistochemistry. We describe a functional role for these recurrent noncoding mutations in disrupting an autoregulatory feedback mechanism, thereby deregulating HNRNPH1 protein expression. Taken together, these data strongly imply a role for aberrant regulation of messenger RNA processing in MCL pathobiology.
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http://dx.doi.org/10.1182/blood.2019002385DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7440974PMC
July 2020

RET Solvent Front Mutations Mediate Acquired Resistance to Selective RET Inhibition in RET-Driven Malignancies.

J Thorac Oncol 2020 04 24;15(4):541-549. Epub 2020 Jan 24.

Kolling Institute of Endocrinology, Royal North Shore Hospital, and the University of Sydney, Sydney, Australia.

Introduction: Novel rearranged in transfection (RET)-specific tyrosine kinase inhibitors (TKIs) such as selpercatinib (LOXO-292) have shown unprecedented efficacy in tumors positive for RET fusions or mutations, notably RET fusion-positive NSCLC and RET-mutated medullary thyroid cancer (MTC). However, the mechanisms of resistance to these agents have not yet been described.

Methods: Analysis was performed of circulating tumor DNA and tissue in patients with RET fusion-positive NSCLC and RET-mutation positive MTC who developed disease progression after an initial response to selpercatinib. Acquired resistance was modeled preclinically using a CCDC6-RET fusion-positive NSCLC patient-derived xenograft. The inhibitory activity of anti-RET multikinase inhibitors and selective RET TKIs was evaluated in enzyme and cell-based assays.

Results: After a dramatic initial response to selpercatinib in a patient with KIF5B-RET NSCLC, analysis of circulating tumor DNA revealed emergence of RET G810R, G810S, and G810C mutations in the RET solvent front before the emergence of clinical resistance. Postmortem biopsy studies reported intratumor and intertumor heterogeneity with distinct disease subclones containing G810S, G810R, and G810C mutations in multiple disease sites indicative of convergent evolution on the G810 residue resulting in a common mechanism of resistance. Acquired mutations in RET G810 were identified in tumor tissue from a second patient with CCDC6-RET fusion-positive NSCLC and in plasma from patients with additional RET fusion-positive NSCLC and RET-mutant MTC progressing on an ongoing phase 1 and 2 trial of selpercatinib. Preclinical studies reported the presence of RET G810R mutations in a CCDC6-RET patient-derived xenograft (from a patient with NSCLC) model of acquired resistance to selpercatinib. Structural modeling predicted that these mutations sterically hinder the binding of selpercatinib, and in vitro assays confirmed loss of activity for both anti-RET multikinase inhibitors and selective RET TKIs.

Conclusions: RET G810 solvent front mutations represent the first described recurrent mechanism of resistance to selective RET inhibition with selpercatinib. Development of potent inhibitor of these mutations and maintaining activity against RET gatekeeper mutations could be an effective strategy to target resistance to selective RET inhibitors.
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http://dx.doi.org/10.1016/j.jtho.2020.01.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7430178PMC
April 2020

HBO1 is required for the maintenance of leukaemia stem cells.

Nature 2020 01 11;577(7789):266-270. Epub 2019 Dec 11.

Cancer Therapeutics CRC, Melbourne, Victoria, Australia.

Acute myeloid leukaemia (AML) is a heterogeneous disease characterized by transcriptional dysregulation that results in a block in differentiation and increased malignant self-renewal. Various epigenetic therapies aimed at reversing these hallmarks of AML have progressed into clinical trials, but most show only modest efficacy owing to an inability to effectively eradicate leukaemia stem cells (LSCs). Here, to specifically identify novel dependencies in LSCs, we screened a bespoke library of small hairpin RNAs that target chromatin regulators in a unique ex vivo mouse model of LSCs. We identify the MYST acetyltransferase HBO1 (also known as KAT7 or MYST2) and several known members of the HBO1 protein complex as critical regulators of LSC maintenance. Using CRISPR domain screening and quantitative mass spectrometry, we identified the histone acetyltransferase domain of HBO1 as being essential in the acetylation of histone H3 at K14. H3 acetylated at K14 (H3K14ac) facilitates the processivity of RNA polymerase II to maintain the high expression of key genes (including Hoxa9 and Hoxa10) that help to sustain the functional properties of LSCs. To leverage this dependency therapeutically, we developed a highly potent small-molecule inhibitor of HBO1 and demonstrate its mode of activity as a competitive analogue of acetyl-CoA. Inhibition of HBO1 phenocopied our genetic data and showed efficacy in a broad range of human cell lines and primary AML cells from patients. These biological, structural and chemical insights into a therapeutic target in AML will enable the clinical translation of these findings.
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http://dx.doi.org/10.1038/s41586-019-1835-6DOI Listing
January 2020

An Evolutionarily Conserved Function of Polycomb Silences the MHC Class I Antigen Presentation Pathway and Enables Immune Evasion in Cancer.

Cancer Cell 2019 10 26;36(4):385-401.e8. Epub 2019 Sep 26.

Peter MacCallum Cancer Centre, 305 Grattan Street, Melbourne, VIC 3000, Australia; Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, VIC 3052, Australia; Centre for Cancer Research, University of Melbourne, Parkville, Australia. Electronic address:

Loss of MHC class I (MHC-I) antigen presentation in cancer cells can elicit immunotherapy resistance. A genome-wide CRISPR/Cas9 screen identified an evolutionarily conserved function of polycomb repressive complex 2 (PRC2) that mediates coordinated transcriptional silencing of the MHC-I antigen processing pathway (MHC-I APP), promoting evasion of T cell-mediated immunity. MHC-I APP gene promoters in MHC-I low cancers harbor bivalent activating H3K4me3 and repressive H3K27me3 histone modifications, silencing basal MHC-I expression and restricting cytokine-induced upregulation. Bivalent chromatin at MHC-I APP genes is a normal developmental process active in embryonic stem cells and maintained during neural progenitor differentiation. This physiological MHC-I silencing highlights a conserved mechanism by which cancers arising from these primitive tissues exploit PRC2 activity to enable immune evasion.
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http://dx.doi.org/10.1016/j.ccell.2019.08.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6876280PMC
October 2019

Targeting enhancer switching overcomes non-genetic drug resistance in acute myeloid leukaemia.

Nat Commun 2019 06 20;10(1):2723. Epub 2019 Jun 20.

Cancer Research Division, Peter MacCallum Cancer Centre, Melbourne, VIC, Australia.

Non-genetic drug resistance is increasingly recognised in various cancers. Molecular insights into this process are lacking and it is unknown whether stable non-genetic resistance can be overcome. Using single cell RNA-sequencing of paired drug naïve and resistant AML patient samples and cellular barcoding in a unique mouse model of non-genetic resistance, here we demonstrate that transcriptional plasticity drives stable epigenetic resistance. With a CRISPR-Cas9 screen we identify regulators of enhancer function as important modulators of the resistant cell state. We show that inhibition of Lsd1 (Kdm1a) is able to overcome stable epigenetic resistance by facilitating the binding of the pioneer factor, Pu.1 and cofactor, Irf8, to nucleate new enhancers that regulate the expression of key survival genes. This enhancer switching results in the re-distribution of transcriptional co-activators, including Brd4, and provides the opportunity to disable their activity and overcome epigenetic resistance. Together these findings highlight key principles to help counteract non-genetic drug resistance.
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http://dx.doi.org/10.1038/s41467-019-10652-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6586637PMC
June 2019

Circulating Tumor DNA in HER2-Amplified Breast Cancer: A Translational Research Substudy of the NeoALTTO Phase III Trial.

Clin Cancer Res 2019 06 12;25(12):3581-3588. Epub 2019 Mar 12.

Medical Oncology Department, Institut Jules Bordet, Université Libre de Bruxelles, Bruxelles, Belgium.

Purpose: In the neoadjuvant treatment (NAT) setting, dual HER2-targeted therapy is associated with increased pathologic complete response (pCR) rates compared with each therapy alone. Biomarkers allowing to predict treatment response during NAT are needed. We aim to evaluate whether circulating tumor DNA (ctDNA) is associated with response to anti-HER2-targeted therapy.

Experimental Design: Plasma DNA collected before NAT, at week 2, and before surgery from patients enrolled in the NeoALTTO trial was assessed using digital PCR for and mutation detection.

Results: A total of 69 of 455 (15.2%) patients had a and/or mutation detected in the baseline tumor sample and evaluable ctDNA results from baseline samples. CtDNA was detected in 41%, 20%, and 5% patients before NAT, at week 2, and before surgery, respectively. ctDNA detection before NAT was significantly associated with older age and ER-negative status. ctDNA detection before NAT was associated with decreased odds of achieving pCR (OR = 0.15; 95% CI, 0.034-0.7; = 0.0089), but not with event-free survival (EFS). Analyses for EFS were underpowered. Interestingly, the patients with HER2-enriched subtype tumors and undetectable ctDNA at baseline had the highest pCR rates. In contrast, patients with persistent ctDNA detection at baseline and week 2 had the lowest rate of pCR.

Conclusions: ctDNA detection before neoadjuvant anti-HER2 therapies is associated with decreased pCR rates. Interestingly, patients with HER2-enriched tumors and undetectable ctDNA at baseline had the highest pCR rates, therefore appearing as the best candidates for treatment deescalation strategies.
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http://dx.doi.org/10.1158/1078-0432.CCR-18-2521DOI Listing
June 2019

The clinical use of circulating tumor cells (CTCs) enumeration for staging of metastatic breast cancer (MBC): International expert consensus paper.

Crit Rev Oncol Hematol 2019 Feb 19;134:39-45. Epub 2018 Dec 19.

Women Cancer Center, Azienda Socio Sanitaria Territoriale di Cremona, University of Trieste, Italy.

Background: The heterogeneity of metastatic breast cancer (MBC) necessitates novel biomarkers allowing stratification of patients for treatment selection and drug development. We propose to use the prognostic utility of circulating tumor cells (CTCs) for stratification of patients with stage IV disease.

Methods: In a retrospective, pooled analysis of individual patient data from 18 cohorts, including 2436 MBC patients, a CTC threshold of 5 cells per 7.5 ml was used for stratification based on molecular subtypes, disease location, and prior treatments. Patients with ≥ 5 CTCs were classified as Stage IV, those with < 5 CTCs as Stage IV Survival was analyzed using Kaplan-Meier curves and the log rank test.

Results: For all patients, Stage IV patients had longer median overall survival than those with Stage IV (36.3 months vs. 16.0 months, P < 0.0001) and similarly for de novo MBC patients (41.4 months Stage IV vs. 18.7 months Stage IV, p < 0.0001). Moreover, patients with Stage IV disease had significantly longer overall survival across all disease subtypes compared to the aggressive cohort: hormone receptor-positive (44 months vs. 17.3 months, P < 0.0001), HER2-positive (36.7 months vs. 20.4 months, P < 0.0001), and triple negative (23.8 months vs. 9.0 months, P < 0.0001). Similar results were obtained regardless of prior treatment or disease location.

Conclusions: We confirm the identification of two subgroups of MBC, Stage IV and Stage IV, independent of clinical and molecular variables. Thus, CTC count should be considered an important tool for staging of advanced disease and for disease stratification in prospective clinical trials.
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http://dx.doi.org/10.1016/j.critrevonc.2018.12.004DOI Listing
February 2019

A Phase Ib Dose-Escalation and Expansion Study of the BCL2 Inhibitor Venetoclax Combined with Tamoxifen in ER and BCL2-Positive Metastatic Breast Cancer.

Cancer Discov 2019 03 5;9(3):354-369. Epub 2018 Dec 5.

The Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria, Australia.

Venetoclax, a potent and selective BCL2 inhibitor, synergizes with endocrine therapy in preclinical models of ER-positive breast cancer. Using a phase Ib 3 + 3 dose-escalation and expansion study design, 33 patients with ER and BCL2-positive metastatic disease (mean prior regimens, 2; range, 0-8) were treated with daily tamoxifen (20 mg) and venetoclax (200-800 mg). Apart from uncomplicated "on-target" lymphopenia, no dose-limiting toxicities or high-grade adverse events were observed in the escalation phase (15 patients), and 800 mg was selected as the recommended phase II dose (RP2D). In the expansion phase (18 patients), few high-grade treatment-related adverse events were observed. For 24 patients treated at the RP2D, the confirmed radiologic response rate was 54% and the clinical benefit rate was 75%. Treatment responses were preempted by metabolic responses (FDG-PET) at 4 weeks and correlated with serial changes in circulating tumor DNA. Radiologic responses (40%) and clinical benefit (70%) were observed in 10 patients with plasma-detected mutations. SIGNIFICANCE: In the first clinical study to evaluate venetoclax in a solid tumor, we demonstrate that combining venetoclax with endocrine therapy has a tolerable safety profile and elicits notable activity in ER and BCL2-positive metastatic breast cancer. These findings support further investigation of combination therapy for patients with BCL2-positive tumors...
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http://dx.doi.org/10.1158/2159-8290.CD-18-1151DOI Listing
March 2019

Dynamic molecular monitoring reveals that SWI-SNF mutations mediate resistance to ibrutinib plus venetoclax in mantle cell lymphoma.

Nat Med 2019 01 19;25(1):119-129. Epub 2018 Nov 19.

Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia.

Ibrutinib plus venetoclax is a highly effective combination in mantle cell lymphoma. However, strategies to enable the evaluation of therapeutic response are required. Our prospective analyses of patients within the AIM study revealed genomic profiles that clearly dichotomized responders and nonresponders. Mutations in ATM were present in most patients who achieved a complete response, while chromosome 9p21.1-p24.3 loss and/or mutations in components of the SWI-SNF chromatin-remodeling complex were present in all patients with primary resistance and two-thirds of patients with relapsed disease. Circulating tumor DNA analysis revealed that these alterations could be dynamically monitored, providing concurrent information on treatment response and tumor evolution. Functional modeling demonstrated that compromise of the SWI-SNF complex facilitated transcriptional upregulation of BCL2L1 (Bcl-xL) providing a selective advantage against ibrutinib plus venetoclax. Together these data highlight important insights into the molecular basis of therapeutic response and provide a model for real-time assessment of innovative targeted therapies.
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http://dx.doi.org/10.1038/s41591-018-0243-zDOI Listing
January 2019

Effects of Collection and Processing Procedures on Plasma Circulating Cell-Free DNA from Cancer Patients.

J Mol Diagn 2018 11 28;20(6):883-892. Epub 2018 Aug 28.

Cancer Research UK Cambridge Institute, Li Ka Shing Centre, Robinson Way, University of Cambridge, Cambridge, United Kingdom; Cancer Research UK Major Centre, Cambridge, United Kingdom. Electronic address:

Circulating tumor DNA (ctDNA) offers new opportunities for noninvasive cancer management. Detecting ctDNA in plasma is challenging because it constitutes only a minor fraction of the total cell-free DNA (cfDNA). Pre-analytical factors affect cfDNA levels contributed from leukocyte lysis, hence the ability to detect low-frequency mutant alleles. This study investigates the effects of the delay in processing, storage temperatures, different blood collection tubes, centrifugation protocols, and sample shipment on cfDNA levels. Peripheral blood (n = 231) from cancer patients (n = 62) were collected into KEDTA or Cell-free DNA BCT tubes and analyzed by digital PCR, targeted amplicon, or shallow whole-genome sequencing. To assess pre-analytic effects, plasma was processed under different conditions after 0, 6, 24, 48, 96 hours, and 1 week at room temperature or 4°C, or using different centrifugation protocols. Digital PCR showed that cfDNA levels increased gradually with time in KEDTA tubes, but were stable in BCT tubes. KEDTA samples stored at 4°C showed less variation than room temperature storage, but levels were elevated compared with BCT. A second centrifugation at 3000 × g gave similar cfDNA yields compared with higher-speed centrifugation. Next-generation sequencing showed negligible differences in background error or copy number changes between KEDTA and BCT, or following shipment in BCT. This study provides insights into the effects of sample processing on ctDNA analysis.
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http://dx.doi.org/10.1016/j.jmoldx.2018.07.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6197164PMC
November 2018

Circulating tumor DNA for disease monitoring in the era of CAR T-cell therapy.

Leuk Lymphoma 2019 02 20;60(2):279-280. Epub 2018 Aug 20.

a Peter MacCallum Cancer Centre , Melbourne , Victoria , Australia.

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http://dx.doi.org/10.1080/10428194.2018.1485916DOI Listing
February 2019

Characterizing the Cancer Genome in Blood.

Cold Spring Harb Perspect Med 2019 04 1;9(4). Epub 2019 Apr 1.

Divisions of Cancer Medicine and Research, Peter MacCallum Cancer Centre, Melbourne 3000, Australia; Centre for Cancer Research, University of Melbourne, Melbourne 3010, Australia.

Cell-free circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs) can be found in the bloodstream of individuals with cancer and are increasingly being explored as biomarkers in various aspects of cancer management. The application of next-generation sequencing (NGS) technologies to ctDNA and CTC analysis are providing new opportunities to characterize the cancer genome from a simple blood test and can facilitate the ease with which tumor-specific genomic changes can be followed over time. The serial analysis of ctDNA and CTCs has enormous potential to provide insights into intratumor heterogeneity and clonal evolution during disease progression, and may ultimately allow noninvasive molecular disease monitoring to guide therapeutic decisions and improve patient outcomes.
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http://dx.doi.org/10.1101/cshperspect.a026880DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6444693PMC
April 2019

Ibrutinib plus Venetoclax for the Treatment of Mantle-Cell Lymphoma.

N Engl J Med 2018 03;378(13):1211-1223

From the Peter MacCallum Cancer Centre, Melbourne, VIC (C.S.T., M.A.A., R.A., S.H., R.J.H., K.B., G.T., J.D.I., M.B., D.W., S.L., S.-J.D., M.A.D., J.F.S., A.W.R.), and the Victorian Comprehensive Cancer Centre (C.S.T., M.A.A., R.A., S.H., R.J.H., K.B., D.W., S.-J.D., M.A.D., J.F.S., A.W.R.), the Faculty of Medicine (C.S.T., R.J.H., S.-J.D., M.A.D., J.F.S., A.W.R.) and Centre for Cancer Research (S.-J.D., M.A.D.), University of Melbourne, the Department of Clinical Haematology and Bone Marrow Transplantation, the Royal Melbourne Hospital (C.S.T., M.A.A., K.B., J.F.S., A.W.R.), and the Division of Cancer and Haematology, Walter and Eliza Hall Institute of Medical Research (M.A.A., A.W.R.), Parkville, VIC - all in Australia; and the University Hospital of Schleswig-Holstein, Kiel (C.P.), and Klinikum der Universität, Ludwig-Maximilian University of Munich, Munich (M.D.) - both in Germany.

Background: Both the BTK inhibitor ibrutinib and the BCL2 inhibitor venetoclax are active as monotherapy in the treatment of mantle-cell lymphoma. Complete response rates of 21% have been observed for each agent when administered as long-term continuous therapy. Preclinical models predict synergy in combination.

Methods: We conducted a single-group, phase 2 study of daily oral ibrutinib and venetoclax in patients, as compared with historical controls. Patients commenced ibrutinib monotherapy at a dose of 560 mg per day. After 4 weeks, venetoclax was added in stepwise, weekly increasing doses to 400 mg per day. Both drugs were continued until progression or an unacceptable level of adverse events. The primary end point was the rate of complete response at week 16. Minimal residual disease (MRD) was assessed by flow cytometry in bone marrow and by allele-specific oligonucleotide-polymerase chain reaction (ASO-PCR) in blood.

Results: The study included 24 patients with relapsed or refractory mantle-cell lymphoma (23 patients) or previously untreated mantle-cell lymphoma (1 patient). Patients were 47 to 81 years of age, and the number of previous treatments ranged from none to six. Half the patients had aberrations of TP53, and 75% had a high-risk prognostic score. The complete response rate according to computed tomography at week 16 was 42%, which was higher than the historical result of 9% at this time point with ibrutinib monotherapy (P<0.001). The rate of complete response as assessed by positron-emission tomography was 62% at week 16 and 71% overall. MRD clearance was confirmed by flow cytometry in 67% of the patients and by ASO-PCR in 38%. In a time-to-event analysis, 78% of the patients with a response were estimated to have an ongoing response at 15 months. The tumor lysis syndrome occurred in 2 patients. Common side effects were generally low grade and included diarrhea (in 83% of the patients), fatigue (in 75%), and nausea or vomiting (in 71%).

Conclusions: In this study involving historical controls, dual targeting of BTK and BCL2 with ibrutinib and venetoclax was consistent with improved outcomes in patients with mantle-cell lymphoma who had been predicted to have poor outcomes with current therapy. (Funded by Janssen and others; AIM ClinicalTrials.gov number, NCT02471391 .).
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http://dx.doi.org/10.1056/NEJMoa1715519DOI Listing
March 2018

The value of cell-free DNA for molecular pathology.

J Pathol 2018 04 12;244(5):616-627. Epub 2018 Mar 12.

Marie-José and Henry R. Kravis Center for Molecular Oncology, Memorial Sloan Kettering Cancer Center, New York, NY, USA.

Over the past decade, advances in molecular biology and genomics techniques have revolutionized the diagnosis and treatment of cancer. The technological advances in tissue profiling have also been applied to the study of cell-free nucleic acids, an area of increasing interest for molecular pathology. Cell-free nucleic acids are released from tumour cells into the surrounding body fluids and can be assayed non-invasively. The repertoire of genomic alterations in circulating tumour DNA (ctDNA) is reflective of both primary tumours and distant metastatic sites, and ctDNA can be sampled multiple times, thereby overcoming the limitations of the analysis of single biopsies. Furthermore, ctDNA can be sampled regularly to monitor response to treatment, to define the evolution of the tumour genome, and to assess the acquisition of resistance and minimal residual disease. Recently, clinical ctDNA assays have been approved for guidance of therapy, which is an exciting first step in translating cell-free nucleic acid research tests into clinical use for oncology. In this review, we discuss the advantages of cell-free nucleic acids as analytes in different body fluids, including blood plasma, urine, and cerebrospinal fluid, and their clinical applications in solid tumours and haematological malignancies. We will also discuss practical considerations for clinical deployment, such as preanalytical factors and regulatory requirements. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
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http://dx.doi.org/10.1002/path.5048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6656375PMC
April 2018

Circulating Tumor DNA Guides Prognosis in Metastatic Triple-Negative Breast Cancer.

J Clin Oncol 2018 02 3;36(6):523-524. Epub 2018 Jan 3.

Andjelija Zivanovic Bujak and Sarah-Jane Dawson, Peter MacCallum Cancer Centre and University of Melbourne, Melbourne, Victoria, Australia.

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http://dx.doi.org/10.1200/JCO.2017.76.5461DOI Listing
February 2018

CMTM6 maintains the expression of PD-L1 and regulates anti-tumour immunity.

Nature 2017 09 16;549(7670):101-105. Epub 2017 Aug 16.

Cancer Research Division, Peter MacCallum Cancer Centre, Melbourne, Victoria 3000, Australia.

Cancer cells exploit the expression of the programmed death-1 (PD-1) ligand 1 (PD-L1) to subvert T-cell-mediated immunosurveillance. The success of therapies that disrupt PD-L1-mediated tumour tolerance has highlighted the need to understand the molecular regulation of PD-L1 expression. Here we identify the uncharacterized protein CMTM6 as a critical regulator of PD-L1 in a broad range of cancer cells, by using a genome-wide CRISPR-Cas9 screen. CMTM6 is a ubiquitously expressed protein that binds PD-L1 and maintains its cell surface expression. CMTM6 is not required for PD-L1 maturation but co-localizes with PD-L1 at the plasma membrane and in recycling endosomes, where it prevents PD-L1 from being targeted for lysosome-mediated degradation. Using a quantitative approach to profile the entire plasma membrane proteome, we find that CMTM6 displays specificity for PD-L1. Notably, CMTM6 depletion decreases PD-L1 without compromising cell surface expression of MHC class I. CMTM6 depletion, via the reduction of PD-L1, significantly alleviates the suppression of tumour-specific T cell activity in vitro and in vivo. These findings provide insights into the biology of PD-L1 regulation, identify a previously unrecognized master regulator of this critical immune checkpoint and highlight a potential therapeutic target to overcome immune evasion by tumour cells.
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http://dx.doi.org/10.1038/nature23643DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5706633PMC
September 2017

Click chemistry enables preclinical evaluation of targeted epigenetic therapies.

Science 2017 06 15;356(6345):1397-1401. Epub 2017 Jun 15.

Cancer Research Division, Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia.

The success of new therapies hinges on our ability to understand their molecular and cellular mechanisms of action. We modified BET bromodomain inhibitors, an epigenetic-based therapy, to create functionally conserved compounds that are amenable to click chemistry and can be used as molecular probes in vitro and in vivo. We used click proteomics and click sequencing to explore the gene regulatory function of BRD4 (bromodomain containing protein 4) and the transcriptional changes induced by BET inhibitors. In our studies of mouse models of acute leukemia, we used high-resolution microscopy and flow cytometry to highlight the heterogeneity of drug activity within tumor cells located in different tissue compartments. We also demonstrate the differential distribution and effects of BET inhibitors in normal and malignant cells in vivo. This study provides a potential framework for the preclinical assessment of a wide range of drugs.
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http://dx.doi.org/10.1126/science.aal2066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5865750PMC
June 2017

Wet or Dry? Do Liquid Biopsy Techniques Compete with or Complement PET for Disease Monitoring in Oncology?

J Nucl Med 2017 06 27;58(6):869-870. Epub 2017 Apr 27.

Peter MacCallum Cancer Center, Melbourne, Victoria, Australia; and

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http://dx.doi.org/10.2967/jnumed.117.190116DOI Listing
June 2017
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