ORCID Profile
0000-0001-5206-5111
Current Organisation
Peter MacCallum Cancer Centre
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Publisher: The Company of Biologists
Date: 07-2021
DOI: 10.1242/DMM.047431
Abstract: Zebrafish are an important model for studying phagocyte function, but rigorous experimental systems to distinguish whether phagocyte-dependent effects are neutrophil or macrophage specific have been lacking. We have developed and validated transgenic lines that enable superior demonstration of cell-autonomous neutrophil and macrophage genetic requirements. We coupled well-characterized neutrophil- and macrophage-specific Gal4 driver lines with UAS:Cas9 transgenes for selective expression of Cas9 in either neutrophils or macrophages. Efficient gene editing, confirmed by both Sanger and next-generation sequencing, occurred in both lineages following microinjection of efficacious synthetic guide RNAs into zebrafish embryos. In proof-of-principle experiments, we demonstrated molecular and/or functional evidence of on-target gene editing for several genes (mCherry, lamin B receptor, trim33) in either neutrophils or macrophages as intended. These new UAS:Cas9 tools provide an improved resource for assessing in idual contributions of neutrophil- and macrophage-expressed genes to the many physiological processes and diseases modelled in zebrafish. Furthermore, this gene-editing functionality can be exploited in any cell lineage for which a lineage-specific Gal4 driver is available. This article has an associated First Person interview with the first author of the paper.
Publisher: Springer Science and Business Media LLC
Date: 13-04-2008
DOI: 10.1038/NG.117
Publisher: American Association for Cancer Research (AACR)
Date: 15-03-2007
DOI: 10.1158/0008-5472.CAN-06-3597
Abstract: Formalin-fixed, paraffin-embedded (FFPE) material tends to yield degraded DNA and is thus suboptimal for use in many downstream applications. We describe an integrated analysis of genotype, loss of heterozygosity (LOH), and copy number for DNA derived from FFPE tissues using oligonucleotide microarrays containing over 500K single nucleotide polymorphisms. A prequalifying PCR test predicted the performance of FFPE DNA on the microarrays better than age of FFPE s le. Although genotyping efficiency and reliability were reduced for FFPE DNA when compared with fresh s les, closer examination revealed methods to improve performance at the expense of variable reduction in resolution. Important steps were also identified that enable equivalent copy number and LOH profiles from paired FFPE and fresh frozen tumor s les. In conclusion, we have shown that the Mapping 500K arrays can be used with FFPE-derived s les to produce genotype, copy number, and LOH predictions, and we provide guidelines and suggestions for application of these s les to this integrated system. [Cancer Res 2007 (6):2544–51]
Publisher: BMJ
Date: 11-08-2017
DOI: 10.1136/JCLINPATH-2017-204481
Abstract: Massively parallel sequencing (MPS) technology has become routinely available for diagnosis, prognostication and therapeutic decision-making in haematological malignancies. However, increased throughput and wider coverage of genes can have unintended consequences. Germline variants of potential clinical significance (GVPCSs) detected during cancer testing may have implications for patients and families beyond the biological evaluation of a specific tumour. 721 reports generated from MPS panels used in the routine testing of myeloid and lymphoid malignancies were reviewed and variants within genes of potential germline relevance ( TP53 , RUNX1 , GATA2 and WT1 in all contexts and CBL , KRAS and NRAS in the setting of juvenile myelomonocytic leukaemia) were analysed. A variant allele fraction threshold of ≥33.09% for considering germline origin of variants within cancer s les was established. The detection rate of incidental, pathogenic germline variants was 0.42%. Patient education and confirmatory germline s le testing of GVPCSs in appropriate circumstances are recommended.
Publisher: Wiley
Date: 07-09-2017
DOI: 10.1111/BJH.14919
Publisher: Springer Science and Business Media LLC
Date: 28-07-2017
DOI: 10.1038/LEU.2017.243
Abstract: Targeted therapies are frequently combined with standard cytotoxic drugs to enhance clinical response. Targeting the B-cell lymphoma 2 (BCL-2) family of proteins is an attractive option to combat chemoresistance in leukemia. Preclinical and clinical studies indicate modest single-agent activity with selective BCL-2 inhibitors (for ex le, venetoclax). We show that venetoclax synergizes with cytarabine and idarubicin to increase antileukemic efficacy in a TP53-dependent manner. Although TP53 deficiency impaired sensitivity to combined venetoclax and chemotherapy, higher-dose idarubicin was able to suppress MCL1 and induce cell death independently of TP53. Consistent with an MCL1-specific effect, cell death from high-dose idarubicin was dependent on pro-apoptotic Bak. Combining higher-dose idarubicin with venetoclax was able to partially overcome resistance in Bak-deficient cells. Using inducible vectors and venetoclax to differentially target anti-apoptotic BCL-2 family members, BCL-2 and MCL1 emerged as critical and complementary proteins regulating cell survival in acute myeloid leukemia. Dual targeting of BCL-2 and MCL1, but not either alone, prolonged survival of leukemia-bearing mice. In conclusion, our findings support the further investigation of venetoclax in combination with standard chemotherapy, including intensified doses of idarubicin. Venetoclax should also be investigated in combination with direct inhibitors of MCL1 as a chemotherapy-free approach in the future.
Publisher: American Association for Cancer Research (AACR)
Date: 03-2019
DOI: 10.1158/2159-8290.CD-18-1119
Abstract: Why CLL recurs in patients who achieve remission with the BCL2 inhibitor venetoclax has been unknown. We provide the first description of an acquired point mutation in BCL2 arising recurrently and exclusively in venetoclax-treated patients. The mutation reduces venetoclax binding and is sufficient to confer resistance. See related commentary by Thangava el and Byrd, p. 320. This article is highlighted in the In This Issue feature, p. 305
Publisher: Springer Science and Business Media LLC
Date: 24-04-2017
Publisher: Wiley
Date: 03-06-2020
DOI: 10.1111/BJH.16819
Publisher: American Society of Hematology
Date: 18-01-2022
DOI: 10.1182/BLOODADVANCES.2021006211
Abstract: 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 in idual disease context.
Publisher: Ferrata Storti Foundation (Haematologica)
Date: 13-08-2021
Publisher: Wiley
Date: 05-04-2022
DOI: 10.1111/BJH.18178
Publisher: Informa UK Limited
Date: 16-01-2020
Publisher: American Society of Hematology
Date: 20-10-2021
DOI: 10.1182/BLOODADVANCES.2021005083
Abstract: Covalent Bruton tyrosine kinase inhibitors (BTKi’s) and the B-cell lymphoma 2 (BCL2) inhibitor venetoclax have significantly improved outcomes for patients with chronic lymphocytic leukemia (CLL), especially those with biologically adverse disease. Patients with CLL resistant to their first targeted agent (TA) can be effectively treated with the alternative class. However, relapses are expected with second-line TA therapy, and the clinical challenge of double class-resistant disease is now emerging with increasing frequency. To define the characteristics and outcomes of patients with double class-resistant disease, we retrospectively analyzed 17 patients who developed progressive disease (PD) on both TA classes for CLL (venetoclax, then BTKi, n=12 BTKi, then venetoclax, n = 5). The cohort was heavily pretreated (median lines of prior therapy, 4) and enriched for adverse disease genetics (complex karyotype, 12 of 12 tested [100%] del(17p)/TP53 mutations, 15 of 17 [88%]). The median time to progression on prior venetoclax was 24 months (range, 6-94 months) and was 25 months (range, 1-55 months) on prior BTKi. Progression on second-line TA was manifest as progressive CLL in 11 patients and as Richter transformation in 6. The median overall survival after progression on second-line TA was 3.6 months (95% confidence interval, 2-11 months). Patients with double class-resistant CLL have a dismal prognosis, representing a group of high unmet need.
Publisher: Ferrata Storti Foundation (Haematologica)
Date: 19-05-2016
Publisher: Springer Science and Business Media LLC
Date: 12-2017
Publisher: Springer Science and Business Media LLC
Date: 20-12-2017
Publisher: American Society of Hematology
Date: 05-03-2020
Abstract: The BCL2 inhibitor venetoclax has complete response rates of up to 50% in chronic lymphocytic leukemia patients, but secondary resistance reflecting acquired mutations in BCL2 can lead to treatment failure. Blombery et al report that an unexpectedly large number of patients carry multiple BCL2 mutations with subclonal variation in their occurrence.
Publisher: Springer Science and Business Media LLC
Date: 09-01-2018
Publisher: Wiley
Date: 30-04-2023
DOI: 10.1002/JHA2.698
Abstract: SH2B3 is a negative regulator of multiple cytokine receptor signalling pathways in haematopoietic tissue. To date, a single kindred has been described with germline biallelic loss‐of‐function SH2B3 variants characterized by early onset developmental delay, hepatosplenomegaly and autoimmune thyroiditis/hepatitis. Herein, we described two further unrelated kindreds with germline biallelic loss‐of‐function SH2B3 variants that show striking phenotypic similarity to each other as well as to the previous kindred of myeloproliferation and multi‐organ autoimmunity. One proband also suffered severe thrombotic complications. CRISPR‐Cas9 gene editing of zebrafish sh2b3 created assorted deleterious variants in F0 crispants, which manifest significantly increased number of macrophages and thrombocytes, partially replicating the human phenotype. Treatment of the sh2b3 crispant fish with ruxolitinib intercepted this myeloproliferative phenotype. Skin‐derived fibroblasts from one patient demonstrated increased phosphorylation of JAK2 and STAT5 after stimulation with IL‐3, GH, GM‐CSF and EPO compared to healthy controls. In conclusion, these additional probands and functional data in combination with the previous kindred provide sufficient evidence for biallelic homozygous deleterious variants in SH2B3 to be considered a valid gene‐disease association for a clinical syndrome of bone marrow myeloproliferation and multi‐organ autoimmune manifestations.
Publisher: Wiley
Date: 24-06-2019
DOI: 10.1111/BJH.16069
Publisher: Impact Journals, LLC
Date: 16-11-2018
Publisher: Informa UK Limited
Date: 14-08-2021
Publisher: BMJ
Date: 10-2012
DOI: 10.1136/JMEDGENET-2012-101191
Abstract: Recently, rare germline variants in XRCC2 were detected in non-BRCA1/2 familial breast cancer cases, and a significant association with breast cancer was reported. However, the breast cancer risk associated with these variants needs further evaluation. The coding regions and exon-intron boundaries of XRCC2 were scanned for mutations in an international cohort of 3548 non-BRCA1/2 familial breast cancer cases and 1435 healthy controls using various mutation scanning methods. Predictions on functional relevance of detected missense variants were obtained from three different prediction algorithms. The only protein-truncating variant detected was found in a control. Rare non-protein-truncating variants were detected in 20 familial cases (0.6%) and nine healthy controls (0.6%). Although the number of variants predicted to be damaging or neutral differed between prediction algorithms, in all instances these categories were evenly represented among cases and controls. Our data do not confirm an association between XRCC2 variants and breast cancer risk, although a relative risk smaller than two could not be excluded. Variants in XRCC2 are unlikely to explain a substantial proportion of familial breast cancer.
Publisher: Springer Science and Business Media LLC
Date: 23-04-2019
DOI: 10.1038/S41598-019-42858-8
Abstract: Next Generation Sequencing is now routinely used in the practice of diagnostic pathology to detect clinically relevant somatic and germline sequence variations in patient s les. 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 s le 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 lifications, chromosome and chromosome arm level lifications/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 github.com/PapenfussLab/CNspector .
Publisher: Springer Science and Business Media LLC
Date: 17-09-2018
Publisher: Informa UK Limited
Date: 22-04-2020
Publisher: Springer Science and Business Media LLC
Date: 03-02-2009
DOI: 10.1007/S10549-008-0269-X
Abstract: Heterozygous somatic mutations of the transcription factor, GATA-3, have recently been reported in approximately 5% breast of tumors unselected for family history. We sequenced the GATA-3 gene in 55 breast tumors from women with familial breast cancer, and found seven heterozygous somatic mutations, all in non-BRCA1/2 cases in which the frequency was 22%. In contrast, we found mutations of GATA-3 in only 4% of 81 sporadic tumors analysed. It is possible that GATA3 mutations occur earlier in the evolution of BRCAx tumors, compared to BRCA1, BRCA2 or sporadic tumors, and are therefore easier to detect by direct sequencing in the presence of some stromal contamination.
Publisher: Springer Science and Business Media LLC
Date: 13-05-2012
DOI: 10.1007/S10549-012-2088-3
Abstract: KLLN is a p53 target gene with DNA binding function and represents a highly plausible candidate breast cancer predisposition gene. We screened for predisposing variants in 860 high-risk breast cancer families using high resolution melt analysis. A germline c.339_340delAG variant predicted to cause premature termination of the protein after 57 alternative amino acid residues was identified in 3/860 families who tested negative for BRCA1 and BRCA2 mutations and in 1/84 sporadic breast cancer cases. However, the variant was also detected in 2/182 families with known BRCA1 or BRCA2 mutations and in 2/464 non-cancer controls. Furthermore, loss of the mutant allele was detected in 2/2 breast tumors. Our data suggest that pathogenic mutations in KLLN are rare in breast cancer families and the c.339_340delAG variant does not represent a high-penetrance breast cancer risk allele.
Publisher: Wiley
Date: 02-03-2022
DOI: 10.1111/EJH.13755
Abstract: 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. 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. 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. 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.
Publisher: American Association for Cancer Research (AACR)
Date: 08-2019
DOI: 10.1158/2159-8290.CD-18-1455
Abstract: CX-5461 is a first-in-class selective inhibitor of rDNA transcription. This first-in-human study establishes the feasibility of targeting this process, demonstrating single-agent antitumor activity against advanced hematologic cancers with predictable pharmacokinetics and a safety profile allowing prolonged dosing. Consistent with preclinical data, antitumor activity was observed in TP53 wild-type and mutant malignancies. This article is highlighted in the In This Issue feature, p. 983
Publisher: American Association for Cancer Research (AACR)
Date: 15-08-2007
DOI: 10.1158/1078-0432.CCR-07-0502
Abstract: Purpose: Genetic changes in sporadic ovarian cancer are relatively poorly characterized compared with other tumor types. We have evaluated the use of high-resolution whole genome arrays for the genetic profiling of epithelial ovarian cancer. Experimental Design: We have evaluated 31 primary ovarian cancers and matched normal DNA for loss of heterozygosity and copy number alterations using 500K single nucleotide polymorphism arrays. Results: In addition to identifying the expected large-scale genomic copy number changes, & small regions of copy number gain or loss (& kb) were identified among the 31 tumors, including 33 regions of high-level gain (& copies) and 27 homozygous deletions. The existence of such a high frequency of small regions exhibiting copy number alterations had not been previously suspected because earlier genomic array platforms lacked comparable resolution. Interestingly, many of these regions harbor known cancer genes. For ex le, one tumor harbored a 350-kb high-level lification centered on FGFR1 and three tumors showed regions of homozygous loss 109 to 216 kb in size involving the RB1 tumor suppressor gene only. Conclusions: These data suggest that novel cancer genes may be located within the other identified small regions of copy number alteration. Analysis of the number of copy number breakpoints and the distribution of the small regions of copy number change indicate high levels of structural chromosomal genetic instability in ovarian cancer.
Publisher: MDPI AG
Date: 30-09-2021
Abstract: Breast implant-associated lymphoma (BIA-ALCL) is a rare subtype of anaplastic large-cell lymphoma associated with breast prostheses. Most patients present with a localised periprosthetic effusion and are managed with removal of the implant and surrounding capsule. Less commonly, the lymphoma can form a mass associated with the capsule and rarely can present with disseminated disease. Recent series characterising the genomic landscape of BIA-ALCL have led to insights into the mechanisms of lymphomagenesis. Constitutive JAK/STAT pathway activation has emerged as a likely key component while, more recently, aberrancies in epigenetic regulators have been reported. This review describes the genomic characterisation reported to date and the insight these findings have provided into this rare entity.
Publisher: Springer Science and Business Media LLC
Date: 23-06-2020
DOI: 10.1038/S41598-020-67205-0
Abstract: An amendment to this paper has been published and can be accessed via a link at the top of the paper.
Publisher: American Society of Hematology
Date: 24-02-2021
Abstract: The BCL2 inhibitor venetoclax has established therapeutic roles in chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML). As BCL2 is an important determinant of survival of both myeloid progenitor and B cells, we investigated whether clinical and molecular abnormalities arise in the myeloid compartment during long-term continuous venetoclax treatment of CLL in 89 patients (87 with relapsed/refractory CLL). Over a median follow-up of 75 (range 21-98) months, persistent cytopenias (≥1 of neutropenia, thrombocytopenia, anemia) lasting ≥4 months and unrelated to CLL occurred in 25 patients (28%). Of these patients, 20 (80%) displayed clonal hematopoiesis, including 10 with therapy-related myeloid neoplasms (t-MNs). t-MNs occurred exclusively in patients previously exposed to fludarabine-alkylator combination therapy with a cumulative 5-year incidence of 10.4% after venetoclax initiation, consistent with rates reported for patients exposed to fludarabine-alkylator combination therapy without venetoclax. To determine whether the altered myelopoiesis reflected the acquisition of mutations, we analyzed s les from patients with no or minimal bone marrow CLL burden (n = 41). Mutations in the apoptosis effector BAX were identified in 32% (13/41). In cellular assays, C-terminal BAX mutants abrogated outer mitochondrial membrane localization of BAX and engendered resistance to venetoclax killing. BAX-mutated clonal hematopoiesis occurred independently of prior fludarabine-alkylator combination therapy exposure and was not associated with t-MNs. Single-cell sequencing revealed clonal co-occurrence of mutations in BAX with DNMT3A or ASXL1. We also observed simultaneous BCL2 mutations within CLL cells and BAX mutations in the myeloid compartment of the same patients, indicating lineage-specific adaptation to venetoclax therapy.
Publisher: Springer Science and Business Media LLC
Date: 11-06-2021
DOI: 10.1038/S41523-021-00279-9
Abstract: Breast cancer (BC) has a significant heritable component but the genetic contribution remains unresolved in the majority of high-risk BC families. This study aims to investigate the monogenic causes underlying the familial aggregation of BC beyond BRCA1 and BRCA2 , including the identification of new predisposing genes. A total of 11,511 non-BRCA familial BC cases and population-matched cancer-free female controls in the BEACCON study were investigated in two sequencing phases: 1303 candidate genes in up to 3892 cases and controls, followed by validation of 145 shortlisted genes in an additional 7619 subjects. The coding regions and exon–intron boundaries of all candidate genes and 14 previously proposed BC genes were sequenced using custom designed sequencing panels. Pedigree and pathology data were analysed to identify genotype-specific associations. The contribution of ATM , PALB2 and CHEK2 to BC predisposition was confirmed, but not RAD50 and NBN . An overall excess of loss-of-function (LoF) (OR 1.27, p = 9.05 × 10 −9 ) and missense (OR 1.27, p = 3.96 × 10 −73 ) variants was observed in the cases for the 145 candidate genes. Leading candidates harbored LoF variants with observed ORs of 2–4 and in idually accounted for no more than 0.79% of the cases. New genes proposed by this study include NTHL1 , WRN , PARP2 , CTH and CDK9 . The new candidate BC predisposition genes identified in BEACCON indicate that much of the remaining genetic causes of high-risk BC families are due to genes in which pathogenic variants are both very rare and convey only low to moderate risk.
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 03-2019
Publisher: Ferrata Storti Foundation (Haematologica)
Date: 13-02-2021
DOI: 10.3324/HAEMATOL.2019.237693
Abstract: Bone marrow failure (BMF) related to hypoplasia of hematopoietic elements in the bone marrow is a heterogeneous clinical entity with a broad differential diagnosis including both inherited and acquired causes. Accurate diagnostic categorization is critical to optimal patient care and detection of genomic variants in these patients may provide this important diagnostic and prognostic information. We performed real-time, accredited (ISO15189) comprehensive genomic characterization including targeted sequencing and whole exome sequencing in 115 patients with BMF syndrome (median age 24 years, range 3 months - 81 years). In patients with clinical diagnoses of inherited BMF syndromes, acquired BMF syndromes or clinically unclassifiable BMF we detected variants in 52% (12/23), 53% (25/47) and 56% (25/45) respectively. Genomic characterization resulted in a change of diagnosis in 30/115 (26%) including the identification of germline causes for 3/47 and 16/45 cases with pre-test diagnoses of acquired and clinically unclassifiable BMF respectively. The observed clinical impact of accurate diagnostic categorization included choice to perform allogeneic stem cell transplantation, disease-specific targeted treatments, identification of at-risk family members and influence of sibling allogeneic stem cell donor choice. Multiple novel pathogenic variants and copy number changes were identified in our cohort including in TERT, FANCA, RPS7 and SAMD9. Whole exome sequence analysis facilitated the identification of variants in two genes not typically associated with a primary clinical manifestation of BMF but also demonstrated reduced sensitivity for detecting low level acquired variants. In conclusion, genomic characterization can improve diagnostic categorization of patients presenting with hypoplastic BMF syndromes and should be routinely performed in this group of patients.
Publisher: Elsevier BV
Date: 02-2023
DOI: 10.1016/J.PATHOL.2022.07.004
Abstract: FLT3 internal tandem duplication (ITD) quantitation is key to prognostication in acute myeloid leukaemia (AML). One potential source of variability in the allelic ratio (AR) is the number of polymerase chain reaction (PCR) cycles used. Using 30 archived s les of varying ITD lengths and AR, we compared two FLT3-ITD assays (Huang and RATIFY), evaluated the effect of PCR cycle number on each assay, and examined the potential clinical consequences. Huang and RATIFY assays at 35 and 27 PCR cycles, respectively, were highly concordant. A progressive decrease in AR (median 47%) was observed with the RATIFY assay when the PCR cycles were increased from 27 to 35 cycles, potentially impacting risk categorisation in 29% of patients. In contrast, minimal change in AR was observed with the Huang assay. Hence, both FLT3-ITD assays were almost identical using respective standard conditions, but the effect of PCR cycle number is assay-dependent, which may impact risk stratification in AML.
Publisher: Springer Science and Business Media LLC
Date: 17-01-2022
DOI: 10.1038/S41523-021-00373-Y
Abstract: While protein-truncating variants in RAD51C have been shown to predispose to triple-negative (TN) breast cancer (BC) and ovarian cancer, little is known about the pathogenicity of missense (MS) variants. The frequency of rare RAD51C MS variants was assessed in the BEACCON study of 5734 familial BC cases and 14,382 population controls, and findings were integrated with tumour sequencing data from 21 cases carrying a candidate variant. Collectively, a significant enrichment of rare MS variants was detected in cases (MAF 0.001, OR 1.57, 95% CI 1.00–2.44, p = 0.05), particularly for variants with a REVEL score .5 (OR 3.95, 95% CI 1.40–12.01, p = 0.006). Sequencing of 21 tumours from 20 heterozygous and 1 homozygous carriers of nine candidate MS variants identified four cases with biallelic inactivation through loss of the wild-type allele, while six lost the variant allele and ten that remained heterozygous. Biallelic loss of the wild-type alleles corresponded strongly with ER- and TN breast tumours, high homologous recombination deficiency scores and mutational signature 3. Using this approach, the p.Gly264Ser variant, which was previously suspected to be pathogenic based on small case–control analyses and loss of activity in in vitro functional assays, was shown to be benign with similar prevalence in cases and controls and seven out of eight tumours showing no biallelic inactivation or characteristic mutational signature. Conversely, evaluation of case–control findings and tumour sequencing data identified p.Ile144Thr, p.Arg212His, p.Gln143Arg and p.Gly114Arg as variants warranting further investigation.
Publisher: Springer Science and Business Media LLC
Date: 22-08-2202
Publisher: Springer Science and Business Media LLC
Date: 04-11-2011
DOI: 10.1007/S10549-011-1835-1
Abstract: Ductal carcinoma in situ (DCIS) is a non-obligate precursor to invasive ductal carcinoma (IDC). Annotation of the genetic differences between the two lesions may assist in the identification of genes that promote the invasive phenotype. Synchronous DCIS and IDC cells were microdissected from FFPE tissue and analysed by molecular inversion probe (MIP) copy number arrays. Matched IDC and DCIS showed highly similar copy number profiles (average of 83% of the genome shared) indicating a common clonal origin although there is evidence that the DCIS continues to evolve in parallel with the co-existing IDC. Four chromosomal regions of loss (3q, 6q, 8p and 11q) and four regions of gain (5q, 16p, 19q and 20) were recurrently affected in IDC but not in DCIS. CCND1 and MYC showed increased litude of gain in IDC. One region of loss (17p11.2) was specific to DCIS. IDC-specific regions include genes with previous links to breast cancer progression and potential therapeutic targets such as AXL, SPHK1 and PLAUR.
Publisher: Elsevier BV
Date: 12-2016
Publisher: Hindawi Limited
Date: 2005
DOI: 10.1002/HUMU.20220
Abstract: Array-based genotyping platforms, such as the Affymetrix mapping array, have been validated as reliable methods for obtaining high-resolution copy number and allele status information when using DNA derived from fresh tissue sources. However, the suitability of such systems for the examination of DNA derived from formalin-fixed, paraffin-embedded (FFPE) tissues has not been tested. Therefore, we analyzed DNA derived from five matching fresh frozen and FFPE ovarian tumors for gene copy number changes and loss of heterozygosity using the Affymetrix GeneChip Human Mapping 10 K Array Xba 131. The data was analyzed using Affymetrix proprietary software, GeneChip DNA Analysis Software, and Chromosome Copy Number Tool. The average SNP call rate (rate of successful genotype identification) of the fresh s les was 89.44% (range 78.72-96.22%, median 92.72%) and was only slightly lower for the matching FFPE s les at 83.48% (range 76.93-93.17%, median 82.60%). The average concordance (rate of agreement between successful genotype calls) between the fresh and matching FFPE s les was 97.06% (range 92.70-99.41%, median 97.52%). Loss of heterozygosity (LOH) profiles of the fresh and FFPE s les were essentially identical across all chromosomes. Copy number data was also comparable, although the quantification of copy number changes appears overstated in the FFPE s les. In conclusion, we have shown that it is possible to achieve high-performance outcomes using FFPE-derived DNA in the Affymetrix 10 K mapping array. This advance will open up vast archival tissue resources to high-resolution genetic analysis and unlock a wealth of biological information.
Publisher: Archives of Pathology and Laboratory Medicine
Date: 09-02-2018
DOI: 10.5858/ARPA.2017-0229-OA
Abstract: Detection of measurable residual disease after therapy is an important predictor of outcome in acute myeloid leukemia. To investigate the feasibility of using next-generation sequencing (NGS) in the diagnostic laboratory to perform quantitative NPM1 mutation assessment using ultradeep (approximately 300 000×–500 000×) sequencing (NGS-qNPM1) as a method of assessing residual disease burden in patients with acute myeloid leukemia. A flexible NGS-based assay for the detection and quantitation of NPM1 mutations was developed by polymerase chain reaction lification of target DNA sequences, sequencing on an Illumina (San Diego, California) MiSeq, and analyzing data with an in-house–designed bioinformatic pipeline. NGS-qNPM1 was compared with current NPM1 quantitation methods (real-time quantitative-polymerase chain reaction and multiparameter flow cytometry). The NGS-qNPM1 assay had a sensitivity of between 10−4 and 10−5 and showed high concordance and correlation with reference methodologies. Moreover, the NGS-qNPM1 assay was able to be integrated into the laboratory's existing, targeted licon-based sequencing workflow. An NGS-based, quantitative NPM1-mutation assessment can be used to monitor patients with acute myeloid leukemia, and it has some practical advantages over existing modalities.
Publisher: Hindawi Limited
Date: 04-11-2011
DOI: 10.1002/HUMU.21625
Abstract: There is strong evidence that overtly inactivating mutations in RAD51C predispose to hereditary breast and ovarian cancer but the prevalence of such mutations, and whether they are associated with a particular clinical phenotype, remains unclear. Resolving these questions has important implications for the implementation of RAD51C into routine clinical genetic testing. Consequently, we have performed a large RAD51C mutation screen of hereditary breast and ovarian cancer families, and the first study of unselected patients diagnosed with ovarian cancer. Our data confirm a consistent but low frequency (2/335 families) of inactivating RAD51C mutations among families with a history of both breast and ovarian cancer and an absence of mutations among breast cancer only families (0/1,053 families). Our data also provide support for the designation of the missense variant p.Gly264Ser as a moderate penetrance allele.
Publisher: Springer Science and Business Media LLC
Date: 29-04-2005
Publisher: Cold Spring Harbor Laboratory
Date: 04-2011
DOI: 10.1261/RNA.2528811
Abstract: Long noncoding RNAs (lncRNAs) are increasingly recognized to play major regulatory roles in development and disease. To identify novel regulators in breast biology, we identified differentially regulated lncRNAs during mouse mammary development. Among the highest and most differentially expressed was a transcript ( Zfas1 ) antisense to the 5′ end of the protein-coding gene Znfx1 . In vivo, Zfas1 RNA is localized within the ducts and alveoli of the mammary gland. Zfas1 intronically hosts three previously undescribed C/D box snoRNAs (SNORDs): Snord12 , Snord12b , and Snord12c . In contrast to the general assumption that noncoding SNORD-host transcripts function only as vehicles to generate snoRNAs, knockdown of Zfas1 in a mammary epithelial cell line resulted in increased cellular proliferation and differentiation, while not substantially altering the levels of the SNORDs. In support of an independent function, we also found that Zfas1 is extremely stable, with a half-life h. Expression analysis of the SNORDs revealed these were expressed at different levels, likely a result of distinct structures conferring differential stability. While there is relatively low primary sequence conservation between Zfas1 and its syntenic human ortholog ZFAS1 , their predicted secondary structures have similar features. Like Zfas1 , ZFAS1 is highly expressed in the mammary gland and is down-regulated in breast tumors compared to normal tissue. We propose a functional role for Zfas1/ ZFAS1 in the regulation of alveolar development and epithelial cell differentiation in the mammary gland, which, together with its dysregulation in human breast cancer, suggests ZFAS1 as a putative tumor suppressor gene.
Publisher: Wiley
Date: 30-01-2004
DOI: 10.1111/J.1399-0004.2004.00214.X
Abstract: Hereditary non-polyposis colorectal cancer (HNPCC) is an autosomal dominant, inherited condition that is characterized primarily by the development of early-onset colorectal cancer and a number of other epithelial malignancies. The underlying genetic basis of the disease is associated with a breakdown of DNA-mismatch repair. There are many genes involved in DNA-mismatch repair, and five of them have been implicated in HNPCC. Two of the genes (hMSH2 and hMLH1) account for the majority of HNPCC families (approximately 60%), and it is not known what the exact contributions of the remaining three genes (hPMS1, hPMS2, and hMSH6) are in relation to this condition. In addition, a sixth gene (hEXO1) has been associated with a disease phenotype that is consistent with HNPCC. Current estimates suggest that all four of these genes, combined, may account for up to 5% of families. In this report, we examine the contribution of hPMS2 and hEXO1 to a well-defined set of families that fulfill the diagnostic criteria for HNPCC. The genes, hPMS2 and hEXO1, were studied by denaturing high performance liquid chromatography (DHPLC) analysis in 21 families that have previously been determined not to have mutations in hMSH2 or hMLH1. hPMS2 accounts for a small proportion of HNPCC families, and none were deemed to be associated with hEXO1. Mutations in hPMS2 appear to account for a small proportion of families adhering to the Amsterdam II criteria, whereas hEXO1 does not appear to be associated with HNPCC.
Publisher: Oxford University Press (OUP)
Date: 02-04-2012
DOI: 10.1093/BIOINFORMATICS/BTS146
Abstract: Motivation: In light of the increasing adoption of targeted resequencing (TR) as a cost-effective strategy to identify disease-causing variants, a robust method for copy number variation (CNV) analysis is needed to maximize the value of this promising technology. Results: We present a method for CNV detection for TR data, including whole-exome capture data. Our method calls copy number gains and losses for each target region based on normalized depth of coverage. Our key strategies include the use of base-level log-ratios to remove GC-content bias, correction for an imbalanced library size effect on log-ratios, and the estimation of log-ratio variations via binning and interpolation. Our methods are made available via CONTRA (COpy Number Targeted Resequencing Analysis), a software package that takes standard alignment formats (BAM/SAM) and outputs in variant call format (VCF4.0), for easy integration with other next-generation sequencing analysis packages. We assessed our methods using s les from seven different target enrichment assays, and evaluated our results using simulated data and real germline data with known CNV genotypes. Availability and implementation: Source code and s le data are freely available under GNU license (GPLv3) at contra-cnv.sourceforge.net/ Contact: Jason.Li@petermac.org Supplementary information: Supplementary data are available at Bioinformatics online.
No related grants have been discovered for Ella Thompson.