Publications by authors named "Huei San Leong"

14 Publications

  • Page 1 of 1

Suspected clonal hematopoiesis as a natural functional assay of TP53 germline variant pathogenicity.

Genet Med 2021 Nov 30. Epub 2021 Nov 30.

Department of Genetics and Computational Biology, QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia. Electronic address:

Purpose: Some variants identified by multigene panel testing of DNA from blood present with low variant allele fraction (VAF), often a manifestation of clonal hematopoiesis. Research has shown that the proportion of variants with low VAF is especially high in TP53, the Li-Fraumeni syndrome gene. Based on the hypothesis that variants with low VAF are positively selected as drivers of clonal hematopoiesis, we investigated the use of VAF as a predictor of TP53 germline variant pathogenicity.

Methods: We used data from 260,681 TP53 variants identified at 2 laboratories to compare the distribution of pathogenic and benign variants at different VAF intervals.

Results: Likelihood ratios toward pathogenicity associated with a VAF < 26% equated to the American College of Medical Genetics/Association of Molecular Pathology strong strength level and were applicable for 1 in 5 variants of unknown significance.

Conclusion: In conclusion, detection of variants with low VAF in blood can be considered an in vivo functional assay to aid assessment of TP53 variant pathogenicity.
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http://dx.doi.org/10.1016/j.gim.2021.10.018DOI Listing
November 2021

Development and Validation of the Gene Expression Predictor of High-grade Serous Ovarian Carcinoma Molecular SubTYPE (PrOTYPE).

Clin Cancer Res 2020 10 17;26(20):5411-5423. Epub 2020 Jun 17.

Department of Gynaecological Oncology, Westmead Hospital, Sydney, New South Wales, Australia.

Purpose: Gene expression-based molecular subtypes of high-grade serous tubo-ovarian cancer (HGSOC), demonstrated across multiple studies, may provide improved stratification for molecularly targeted trials. However, evaluation of clinical utility has been hindered by nonstandardized methods, which are not applicable in a clinical setting. We sought to generate a clinical grade minimal gene set assay for classification of individual tumor specimens into HGSOC subtypes and confirm previously reported subtype-associated features.

Experimental Design: Adopting two independent approaches, we derived and internally validated algorithms for subtype prediction using published gene expression data from 1,650 tumors. We applied resulting models to NanoString data on 3,829 HGSOCs from the Ovarian Tumor Tissue Analysis consortium. We further developed, confirmed, and validated a reduced, minimal gene set predictor, with methods suitable for a single-patient setting.

Results: Gene expression data were used to derive the predictor of high-grade serous ovarian carcinoma molecular subtype (PrOTYPE) assay. We established a standard as a consensus of two parallel approaches. PrOTYPE subtypes are significantly associated with age, stage, residual disease, tumor-infiltrating lymphocytes, and outcome. The locked-down clinical grade PrOTYPE test includes a model with 55 genes that predicted gene expression subtype with >95% accuracy that was maintained in all analytic and biological validations.

Conclusions: We validated the PrOTYPE assay following the Institute of Medicine guidelines for the development of omics-based tests. This fully defined and locked-down clinical grade assay will enable trial design with molecular subtype stratification and allow for objective assessment of the predictive value of HGSOC molecular subtypes in precision medicine applications..
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http://dx.doi.org/10.1158/1078-0432.CCR-20-0103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7572656PMC
October 2020

CNspector: a web-based tool for visualisation and clinical diagnosis of copy number variation from next generation sequencing.

Sci Rep 2019 04 23;9(1):6426. Epub 2019 Apr 23.

Department of Pathology, Peter MacCallum Cancer Centre, Parkville, VIC, Australia.

Next Generation Sequencing is now routinely used in the practice of diagnostic pathology to detect clinically relevant somatic and germline sequence variations in patient samples. However, clinical assessment of copy number variations (CNVs) and large-scale structural variations (SVs) is still challenging. While tools exist to estimate both, their results are typically presented separately in tables or static plots which can be difficult to read and are unable to show the context needed for clinical interpretation and reporting. We have addressed this problem with CNspector, a multi-scale interactive browser that shows CNVs in the context of other relevant genomic features to enable fast and effective clinical reporting. We illustrate the utility of CNspector at different genomic scales across a variety of sample types in a range of case studies. We show how CNspector can be used for diagnosis and reporting of exon-level deletions, focal gene-level amplifications, chromosome and chromosome arm level amplifications/deletions and in complex genomic rearrangements. CNspector is a web-based clinical variant browser tailored to the clinical application of next generation sequencing for CNV assessment. We have demonstrated the utility of this interactive software in typical applications across a range of tissue types and disease contexts encountered in the context of diagnostic pathology. CNspector is written in R and the source code is available for download under the GPL3 Licence from https://github.com/PapenfussLab/CNspector . A server running CNspector loaded with the figures from this paper can be accessed at https://shiny.wehi.edu.au/jmarkham/CNspector/index.html .
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http://dx.doi.org/10.1038/s41598-019-42858-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6478945PMC
April 2019

Profound MEK inhibitor response in a cutaneous melanoma harboring a GOLGA4-RAF1 fusion.

J Clin Invest 2019 05 5;129(5):1940-1945. Epub 2019 Mar 5.

Department of Pathology, and.

BRAF and CRAF are critical components of the MAPK signaling pathway which is activated in many cancer types. In approximately 1% of melanomas, BRAF or CRAF are activated through structural arrangements. We describe here a metastatic melanoma with a GOLGA4-RAF1 fusion and pathogenic variants in CTNNB1 and CDKN2A. Anti-CTLA4/anti-PD1 combination immunotherapy failed to control tumor progression. In the absence of other actionable variants the patient was administered MEK inhibitor therapy on the basis of its potential action against RAF1 fusions. This resulted in a profound and clinically significant response. We demonstrated that GOLGA4-RAF1 expression was associated with ERK activation, elevated expression of the RAS/RAF downstream co-effector ETV5, and a high Ki67 index. These findings provide a rationale for the dramatic response to targeted therapy. This study shows that thorough molecular characterization of treatment-resistant cancers can identify therapeutic targets and personalize management, leading to improved patient outcomes.
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http://dx.doi.org/10.1172/JCI123089DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6486352PMC
May 2019

SDH-deficient renal cell carcinoma associated with biallelic mutation in succinate dehydrogenase A: comprehensive genetic profiling and its relation to therapy response.

NPJ Precis Oncol 2018 20;2. Epub 2018 Mar 20.

1Department of Pathology, Peter MacCallum Cancer Centre, Melbourne, Victoria, 3000 Australia.

Succinate dehydrogenase (SDH)-deficient renal cell carcinoma (RCC) is a rare RCC subtype that is caused by biallelic mutation of one of the four subunits of the SDH complex (, , , and ) and results in inactivation of the SDH enzyme. Here we describe a case of genetically characterized SDH-deficient RCC caused by biallelic (germline plus somatic) mutations. pathogenic variants were detected using comprehensive genomic profiling and SDH absence was subsequently confirmed by immunohistochemistry. Very little is known regarding the genomic context of SDH-deficient RCC. Interestingly we found genomic amplifications commonly observed in RCC but there was an absence of additional variants in common cancer driver genes. Prior to genetic testing a PD-1 inhibitor treatment was administered. However, following the genetic results a succession of tyrosine kinase inhibitors were administered as targeted treatment options and we highlight how the genetic results provide a rationale for their effectiveness. We also describe how the genetic results benefited the patient by empowering him to adopt dietary and lifestyle changes in accordance with knowledge of the mechanisms of SDH-related tumorigenesis.
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http://dx.doi.org/10.1038/s41698-018-0053-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5871886PMC
March 2018

Genome-wide binding and mechanistic analyses of Smchd1-mediated epigenetic regulation.

Proc Natl Acad Sci U S A 2015 Jul 19;112(27):E3535-44. Epub 2015 Jun 19.

The Walter and Eliza Hall Institute of Medical Research, Melbourne, VIC 3052, Australia; University of Melbourne, Melbourne, VIC 3010, Australia;

Structural maintenance of chromosomes flexible hinge domain containing 1 (Smchd1) is an epigenetic repressor with described roles in X inactivation and genomic imprinting, but Smchd1 is also critically involved in the pathogenesis of facioscapulohumeral dystrophy. The underlying molecular mechanism by which Smchd1 functions in these instances remains unknown. Our genome-wide transcriptional and epigenetic analyses show that Smchd1 binds cis-regulatory elements, many of which coincide with CCCTC-binding factor (Ctcf) binding sites, for example, the clustered protocadherin (Pcdh) genes, where we show Smchd1 and Ctcf act in opposing ways. We provide biochemical and biophysical evidence that Smchd1-chromatin interactions are established through the homodimeric hinge domain of Smchd1 and, intriguingly, that the hinge domain also has the capacity to bind DNA and RNA. Our results suggest Smchd1 imparts epigenetic regulation via physical association with chromatin, which may antagonize Ctcf-facilitated chromatin interactions, resulting in coordinated transcriptional control.
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http://dx.doi.org/10.1073/pnas.1504232112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4500281PMC
July 2015

Whole-genome characterization of chemoresistant ovarian cancer.

Nature 2015 May;521(7553):489-94

Victorian Institute of Forensic Medicine, Southbank, Victoria 3006, Australia.

Patients with high-grade serous ovarian cancer (HGSC) have experienced little improvement in overall survival, and standard treatment has not advanced beyond platinum-based combination chemotherapy, during the past 30 years. To understand the drivers of clinical phenotypes better, here we use whole-genome sequencing of tumour and germline DNA samples from 92 patients with primary refractory, resistant, sensitive and matched acquired resistant disease. We show that gene breakage commonly inactivates the tumour suppressors RB1, NF1, RAD51B and PTEN in HGSC, and contributes to acquired chemotherapy resistance. CCNE1 amplification was common in primary resistant and refractory disease. We observed several molecular events associated with acquired resistance, including multiple independent reversions of germline BRCA1 or BRCA2 mutations in individual patients, loss of BRCA1 promoter methylation, an alteration in molecular subtype, and recurrent promoter fusion associated with overexpression of the drug efflux pump MDR1.
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http://dx.doi.org/10.1038/nature14410DOI Listing
May 2015

Why weight? Modelling sample and observational level variability improves power in RNA-seq analyses.

Nucleic Acids Res 2015 Sep 29;43(15):e97. Epub 2015 Apr 29.

Molecular Medicine Division, The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria 3052, Australia Department of Medical Biology, The University of Melbourne, Parkville, Victoria 3010, Australia School of Mathematics and Statistics, The University of Melbourne, Parkville, Victoria 3010, Australia

Variations in sample quality are frequently encountered in small RNA-sequencing experiments, and pose a major challenge in a differential expression analysis. Removal of high variation samples reduces noise, but at a cost of reducing power, thus limiting our ability to detect biologically meaningful changes. Similarly, retaining these samples in the analysis may not reveal any statistically significant changes due to the higher noise level. A compromise is to use all available data, but to down-weight the observations from more variable samples. We describe a statistical approach that facilitates this by modelling heterogeneity at both the sample and observational levels as part of the differential expression analysis. At the sample level this is achieved by fitting a log-linear variance model that includes common sample-specific or group-specific parameters that are shared between genes. The estimated sample variance factors are then converted to weights and combined with observational level weights obtained from the mean-variance relationship of the log-counts-per-million using 'voom'. A comprehensive analysis involving both simulations and experimental RNA-sequencing data demonstrates that this strategy leads to a universally more powerful analysis and fewer false discoveries when compared to conventional approaches. This methodology has wide application and is implemented in the open-source 'limma' package.
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http://dx.doi.org/10.1093/nar/gkv412DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4551905PMC
September 2015

Efficient molecular subtype classification of high-grade serous ovarian cancer.

J Pathol 2015 Jul 30;236(3):272-7. Epub 2015 Apr 30.

The Peter MacCallum Cancer Centre, East Melbourne, Victoria, Australia.

High-grade serous carcinomas (HGSCs) account for approximately 70% of all epithelial ovarian cancers diagnosed. Using microarray gene expression profiling, we previously identified four molecular subtypes of HGSC: C1 (mesenchymal), C2 (immunoreactive), C4 (differentiated), and C5 (proliferative), which correlate with patient survival and have distinct biological features. Here, we describe molecular classification of HGSC based on a limited number of genes to allow cost-effective and high-throughput subtype analysis. We determined a minimal signature for accurate classification, including 39 differentially expressed and nine control genes from microarray experiments. Taqman-based (low-density arrays and Fluidigm), fluorescent oligonucleotides (Nanostring), and targeted RNA sequencing (Illumina) assays were then compared for their ability to correctly classify fresh and formalin-fixed, paraffin-embedded samples. All platforms achieved > 90% classification accuracy with RNA from fresh frozen samples. The Illumina and Nanostring assays were superior with fixed material. We found that the C1, C2, and C4 molecular subtypes were largely consistent across multiple surgical deposits from individual chemo-naive patients. In contrast, we observed substantial subtype heterogeneity in patients whose primary ovarian sample was classified as C5. The development of an efficient molecular classifier of HGSC should enable further biological characterization of molecular subtypes and the development of targeted clinical trials.
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http://dx.doi.org/10.1002/path.4536DOI Listing
July 2015

Immune response to RB1-regulated senescence limits radiation-induced osteosarcoma formation.

J Clin Invest 2013 Dec 15;123(12):5351-60. Epub 2013 Nov 15.

Ionizing radiation (IR) and germline mutations in the retinoblastoma tumor suppressor gene (RB1) are the strongest risk factors for developing osteosarcoma. Recapitulating the human predisposition, we found that Rb1+/- mice exhibited accelerated development of IR-induced osteosarcoma, with a latency of 39 weeks. Initial exposure of osteoblasts to carcinogenic doses of IR in vitro and in vivo induced RB1-dependent senescence and the expression of a panel of proteins known as senescence-associated secretory phenotype (SASP), dominated by IL-6. RB1 expression closely correlated with that of the SASP cassette in human osteosarcomas, and low expression of both RB1 and the SASP genes was associated with poor prognosis. In vivo, IL-6 was required for IR-induced senescence, which elicited NKT cell infiltration and a host inflammatory response. Mice lacking IL-6 or NKT cells had accelerated development of IR-induced osteosarcomas. These data elucidate an important link between senescence, which is a cell-autonomous tumor suppressor response, and the activation of host-dependent cancer immunosurveillance. Our findings indicate that overcoming the immune response to senescence is a rate-limiting step in the formation of IR-induced osteosarcoma.
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http://dx.doi.org/10.1172/JCI70559DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3859382PMC
December 2013

Polycomb repressive complex 2 (PRC2) suppresses Eμ-myc lymphoma.

Blood 2013 Oct 27;122(15):2654-63. Epub 2013 Aug 27.

The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia; and.

Deregulation of polycomb group complexes polycomb repressive complex 1 (PRC1) and 2 (PRC2) is associated with human cancers. Although inactivating mutations in PRC2-encoding genes EZH2, EED, and SUZ12 are present in T-cell acute lymphoblastic leukemia and in myeloid malignancies, gain-of-function mutations in EZH2 are frequently observed in B-cell lymphoma, implying disease-dependent effects of individual mutations. We show that, in contrast to PRC1, PRC2 is a tumor suppressor in Eµ-myc lymphomagenesis, because disease onset was accelerated by heterozygosity for Suz12 or by short hairpin RNA-mediated knockdown of Suz12 or Ezh2. Accelerated lymphomagenesis was associated with increased accumulation of B-lymphoid cells in the absence of effects on apoptosis or cell cycling. However, Suz12-deficient B-lymphoid progenitors exhibit enhanced serial clonogenicity. Thus, PRC2 normally restricts the self-renewal of B-lymphoid progenitors, the disruption of which contributes to lymphomagenesis. This finding provides new insight regarding the functional contribution of mutations in PRC2 in a range of leukemias.
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http://dx.doi.org/10.1182/blood-2013-02-484055DOI Listing
October 2013

Epigenetic regulator Smchd1 functions as a tumor suppressor.

Cancer Res 2013 Mar 26;73(5):1591-9. Epub 2012 Dec 26.

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

SMCHD1 is an epigenetic modifier of gene expression that is critical to maintain X chromosome inactivation. Here, we show in mouse that genetic inactivation of Smchd1 accelerates tumorigenesis in male mice. Loss of Smchd1 in transformed mouse embryonic fibroblasts increased tumor growth upon transplantation into immunodeficient nude mice. In addition, loss of Smchd1 in Eμ-Myc transgenic mice that undergo lymphomagenesis reduced disease latency by 50% relative to control animals. In premalignant Eμ-Myc transgenic mice deficient in Smchd1, there was an increase in the number of pre-B cells in the periphery, likely accounting for the accelerated disease in these animals. Global gene expression profiling suggested that Smchd1 normally represses genes activated by MLL chimeric fusion proteins in leukemia, implying that Smchd1 loss may work through the same pathways as overexpressed MLL fusion proteins do in leukemia and lymphoma. Notably, we found that SMCHD1 is underexpressed in many types of human hematopoietic malignancy. Together, our observations collectively highlight a hitherto uncharacterized role for SMCHD1 as a candidate tumor suppressor gene in hematopoietic cancers.
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http://dx.doi.org/10.1158/0008-5472.CAN-12-3019DOI Listing
March 2013
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