Publications by authors named "Peter T Simpson"

85 Publications

Epigenome erosion and SOX10 drive neural crest phenotypic mimicry in triple-negative breast cancer.

NPJ Breast Cancer 2022 May 2;8(1):57. Epub 2022 May 2.

The University of Queensland Faculty of Medicine, UQ Centre for Clinical Research, Herston, QLD, Australia.

Intratumoral heterogeneity is caused by genomic instability and phenotypic plasticity, but how these features co-evolve remains unclear. SOX10 is a neural crest stem cell (NCSC) specifier and candidate mediator of phenotypic plasticity in cancer. We investigated its relevance in breast cancer by immunophenotyping 21 normal breast and 1860 tumour samples. Nuclear SOX10 was detected in normal mammary luminal progenitor cells, the histogenic origin of most TNBCs. In tumours, nuclear SOX10 was almost exclusive to TNBC, and predicted poorer outcome amongst cross-sectional (p = 0.0015, hazard ratio 2.02, n = 224) and metaplastic (p = 0.04, n = 66) cases. To understand SOX10's influence over the transcriptome during the transition from normal to malignant states, we performed a systems-level analysis of co-expression data, de-noising the networks with an eigen-decomposition method. This identified a core module in SOX10's normal mammary epithelial network that becomes rewired to NCSC genes in TNBC. Crucially, this reprogramming was proportional to genome-wide promoter methylation loss, particularly at lineage-specifying CpG-island shores. We propose that the progressive, genome-wide methylation loss in TNBC simulates more primitive epigenome architecture, making cells vulnerable to SOX10-driven reprogramming. This study demonstrates potential utility for SOX10 as a prognostic biomarker in TNBC and provides new insights about developmental phenotypic mimicry-a major contributor to intratumoral heterogeneity.
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http://dx.doi.org/10.1038/s41523-022-00425-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9061835PMC
May 2022

Whole genome deep sequencing analysis of cell-free DNA in samples with low tumour content.

BMC Cancer 2022 Jan 20;22(1):85. Epub 2022 Jan 20.

Institute for Molecular Bioscience, University of Queensland, St Lucia, Brisbane, Australia.

Background: Circulating cell-free DNA (cfDNA) in the plasma of cancer patients contains cell-free tumour DNA (ctDNA) derived from tumour cells and it has been widely recognized as a non-invasive source of tumour DNA for diagnosis and prognosis of cancer. Molecular profiling of ctDNA is often performed using targeted sequencing or low-coverage whole genome sequencing (WGS) to identify tumour specific somatic mutations or somatic copy number aberrations (sCNAs). However, these approaches cannot efficiently detect all tumour-derived genomic changes in ctDNA.

Methods: We performed WGS analysis of cfDNA from 4 breast cancer patients and 2 patients with benign tumours. We sequenced matched germline DNA for all 6 patients and tumour samples from the breast cancer patients. All samples were sequenced on Illumina HiSeqXTen sequencing platform and achieved approximately 30x, 60x and 100x coverage on germline, tumour and plasma DNA samples, respectively.

Results: The mutational burden of the plasma samples (1.44 somatic mutations/Mb of genome) was higher than the matched tumour samples. However, 90% of high confidence somatic cfDNA variants were not detected in matched tumour samples and were found to comprise two background plasma mutational signatures. In contrast, cfDNA from the di-nucleosome fraction (300 bp-350 bp) had much higher proportion (30%) of variants shared with tumour. Despite high coverage sequencing we were unable to detect sCNAs in plasma samples.

Conclusions: Deep sequencing analysis of plasma samples revealed higher fraction of unique somatic mutations in plasma samples, which were not detected in matched tumour samples. Sequencing of di-nucleosome bound cfDNA fragments may increase recovery of tumour mutations from plasma.
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http://dx.doi.org/10.1186/s12885-021-09160-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8772083PMC
January 2022

Blood-Derived Extracellular Vesicle-Associated miR-3182 Detects Non-Small Cell Lung Cancer Patients.

Cancers (Basel) 2022 Jan 5;14(1). Epub 2022 Jan 5.

Tumour Microenvironment Laboratory, QIMR Berghofer Medical Research Institute, Herston, QLD 4006, Australia.

With five-year survival rates as low as 3%, lung cancer is the most common cause of cancer-related mortality worldwide. The severity of the disease at presentation is accredited to the lack of early detection capacities, resulting in the reliance on low-throughput diagnostic measures, such as tissue biopsy and imaging. Interest in the development and use of liquid biopsies has risen, due to non-invasive sample collection, and the depth of information it can provide on a disease. Small extracellular vesicles (sEVs) as viable liquid biopsies are of particular interest due to their potential as cancer biomarkers. To validate the use of sEVs as cancer biomarkers, we characterised cancer sEVs using miRNA sequencing analysis. We found that miRNA-3182 was highly enriched in sEVs derived from the blood of patients with invasive breast carcinoma and NSCLC. The enrichment of sEV miR-3182 was confirmed in oncogenic, transformed lung cells in comparison to isogenic, untransformed lung cells. Most importantly, miR-3182 can successfully distinguish early-stage NSCLC patients from those with benign lung conditions. Therefore, miR-3182 provides potential to be used for the detection of NSCLC in blood samples, which could result in earlier therapy and thus improved outcomes and survival for patients.
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http://dx.doi.org/10.3390/cancers14010257DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8750562PMC
January 2022

Correction: McCart Reed et al. The Genomic Landscape of Lobular Breast Cancer. 2021, , 1950.

Cancers (Basel) 2021 Aug 9;13(16). Epub 2021 Aug 9.

Centre for Clinical Research, The University of Queensland, Herston, Brisbane, QLD 4029, Australia.

The authors wish to make the following corrections to this paper [...].
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http://dx.doi.org/10.3390/cancers13164010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8392523PMC
August 2021

Characterization of Immune Cell Subsets of Tumor Infiltrating Lymphocytes in Brain Metastases.

Biology (Basel) 2021 05 11;10(5). Epub 2021 May 11.

UQ Centre for Clinical Research, Faculty of Medicine, University of Queensland, Herston, QLD 4029, Australia.

The heterogeneity of tumor infiltrating lymphocytes (TILs) is not well characterized in brain metastasis. To address this, we performed a targeted analysis of immune-cell subsets in brain metastasis tissues to test immunosuppressive routes involved in brain metastasis. We performed multiplex immunofluorescence (mIF), using commercially available validated antibodies on formalin-fixed paraffin embedded whole sections. We quantitated the subsets of immune-cells utilizing a targeted panel of proteins including PanCK, CD8, CD4, VISTA and IBA-1, and analyzed an average of 15,000 cells per sample. Classifying tumors as either high (>30%) or low (<30%) TILs, we found that increased TILs density correlated with survival. Phenotyping these TILs we found tumors with low TILs had significantly higher expression of the immune-checkpoint molecule VISTA in tumor cells ( < 0.01) as well as in their microenvironment ( < 0.001). Contrastingly, the tumors with high TILs displayed higher levels of microglia, as measured by IBA-1 expression. Low TILs-tumors displayed CD8+ T-cells that co-express VISTA ( < 0.01) significantly more compared to high TILs group, where CD8+cells significantly co-express IBA-11 ( < 0.05). These results were supported by RNA analysis of a publicly available, independent cohort. Our work contributes to a growing understanding of the immune surveillance escape routes active in brain metastasis.
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http://dx.doi.org/10.3390/biology10050425DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8150725PMC
May 2021

The Genomic Landscape of Lobular Breast Cancer.

Cancers (Basel) 2021 Apr 18;13(8). Epub 2021 Apr 18.

Centre for Clinical Research, The University of Queensland, Herston, Brisbane, QLD 4029, Australia.

Invasive lobular carcinoma (ILC) is the second most common breast cancer histologic subtype, accounting for approximately 15% of all breast cancers. It is only recently that its unique biology has been assessed in high resolution. Here, we present a meta-analysis of ILC sequencing datasets, to provide a long-awaited ILC-specific resource, and to confirm the prognostic value and strength of association between a number of clinico-pathology features and genomics in this special tumour type. We consider panel ( = 684), whole exome ( = 215) and whole genome sequencing data ( = 48), and review histology of The Cancer Genome Atlas cases to assign grades and determine whether the ILC is of classic type or a variant, such as pleomorphic, prior to performing statistical analyses. We demonstrate evidence of considerable genomic heterogeneity underlying a broadly homogeneous tumour type (typically grade 2, estrogen receptor (ER)-positive); with genomes exhibiting few somatic mutations or structural alterations, genomes with a hypermutator phenotype, and tumours with highly rearranged genomes. We show that while (E-cadherin) and mutations do not significantly impact survival, overall survival is significantly poorer for patients with a higher tumour mutation burden; this is also true for grade 3 tumours, and those carrying a somatic mutation (and these cases were more likely to be ER-negative). Taken together, we have compiled a meta-dataset of ILC with molecular profiling, and our analyses show that the genomic landscape significantly impacts the tumour's variable natural history and overall survival of ILC patients.
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http://dx.doi.org/10.3390/cancers13081950DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8073944PMC
April 2021

Invasive lobular carcinoma of the breast: the increasing importance of this special subtype.

Breast Cancer Res 2021 01 7;23(1). Epub 2021 Jan 7.

UQ Centre for Clinical Research, The University of Queensland, Herston, Brisbane, Australia.

Invasive lobular carcinoma (ILC) is the most common of the breast cancer special types, accounting for up to 15% of all breast cancer cases. ILCs are noted for their lack of E-cadherin function, which underpins their characteristic discohesive growth pattern, with cells arranged in single file and dispersed throughout the stroma. Typically, tumours are luminal in molecular subtype, being oestrogen and progesterone receptor positive, and HER2 negative. Since last reviewing the lobular literature (McCart Reed et al., Breast Cancer Res 17:12, 2015), there has been a considerable increase in research output focused on this tumour type, including studies into the pathology and management of disease, a high-resolution definition of the genomic landscape of tumours as well as the evolution of several potential therapeutic avenues. There abounds a huge amount of new data, which we will review herein.
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http://dx.doi.org/10.1186/s13058-020-01384-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7792208PMC
January 2021

Phenotypic drift in metastatic progression of breast cancer: A case report with histologically heterogeneous lesions that are clonally related.

Clin Case Rep 2020 Dec 27;8(12):2725-2731. Epub 2020 Aug 27.

UQ Centre for Clinical Research, Faculty of Medicine The University of Queensland Brisbane Qld Australia.

Breast cancer metastasis to the stomach is rare; invasive lobular carcinoma has a predilection to spread to the gastrointestinal system and is morphologically similar to primary diffuse gastric carcinoma. This case highlights heterogeneous metastatic progression and that documentation of heterogeneity is important for informing future treatment strategies and prognostication.
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http://dx.doi.org/10.1002/ccr3.3257DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7752647PMC
December 2020

Integrin alpha-2 and beta-1 expression increases through multiple generations of the EDW01 patient-derived xenograft model of breast cancer-insight into their role in epithelial mesenchymal transition in vivo gained from an in vitro model system.

Breast Cancer Res 2020 12 4;22(1):136. Epub 2020 Dec 4.

Invasion and Metastasis Unit, St. Vincent's Institute, Melbourne, VIC, Australia.

Background: Breast cancers acquire aggressive capabilities via epithelial to mesenchymal transition (EMT), in which various integrins/integrin-linked kinase signalling are upregulated.

Methods: We investigated this in two patient-derived xenografts (PDXs) developed from breast-to-bone metastases, and its functional significance in a breast cancer cell line system. ED03 and EDW01 PDXs were grown subcutaneously in immunocompromised SCID mice through 11 passages and 7 passages, respectively. Tumour tissue was assessed using immunohistochemistry (IHC) for oestrogen receptor (ER)-alpha, E-cadherin, vimentin, Twist1, beta-catenin, P120-RasGAP, CD44, CD24 and Ki67, and RT-qPCR of EMT-related factors (CDH1, VIM, CD44, CD24), integrins beta 1 (ITGB1), alpha 2 (ITGA2) and ILK. Integrin and ILK expression in epidermal growth factor (EGF)-induced EMT of the PMC42-ET breast cancer cell line was assessed by RT-qPCR and Western blotting, as were the effects of their transient knockdown via small interfering RNA +/- EGF. Cell migration, changes in cell morphology and adhesion of siRNA-transfected PMC42-ET cells to various extracellular matrix (ECM) substrates was assessed.

Results: The ED03 (ER+/PR-/HER2-/lobular) and EDW01 (ER+/PR-/HER2-/ductal) PDXs were both classified as molecular subtype luminal A. ED03 xenografts exhibited mutated E-cadherin with minimal expression, but remained vimentin-negative across all passages. In EDW01, the hypoxic indicator gene CAIX and Twist1 were co-ordinately upregulated at passages 4-5, corresponding with a decrease in E-cadherin. At passages 6-7, VIM was upregulated along with ITGB1 and ITGA2, consistent with an increasing EMT. The ED03 PDX displayed minimal change over passages in mice, for all genes examined. ILK, ITGB1 and ITGA2 mRNAs were also increased in the EGF-induced EMT of PMC42-ET cells (in which CDH1 was downregulated) although siRNA against these targets revealed that this induction was not necessary for the observed EMT. However, their knockdown significantly reduced EMT-associated adhesion and Transwell migration.

Conclusion: Our data suggest that despite an increase in ITGA2 and ITGB1 gene expression in the EMT exhibited by EDW01 PDX over multiple generations, this pathway may not necessarily drive the EMT process.
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http://dx.doi.org/10.1186/s13058-020-01366-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7716465PMC
December 2020

Clinicopathologic significance of nuclear HER4 and phospho-YAP(S) in human breast cancers and matching brain metastases.

Ther Adv Med Oncol 2020 31;12:1758835920946259. Epub 2020 Jul 31.

The University of Queensland Faculty of Medicine, UQ Centre for Clinical Research, Herston, Qld, Australia.

Background: Human epidermal growth factor receptor-4 (HER4) and yes-associated protein-1 (YAP) are candidate therapeutic targets in oncology. YAP's transcriptional coactivation function is modulated by the HER4 intracellular domain (HER4-ICD) , but the clinical relevance of this has not been established. This study investigated the potential for targeting the HER4-YAP pathway in brain metastatic breast cancer.

Methods: We performed immuno-phenotypic profiling of pathway markers in a consecutive breast cancer series with 25 years of clinical follow up ( = 371), and patient-matched breast and metastatic brain tumours ( = 91; 30 pairs).

Results: Membrane localisation of phospho-HER4 [pHER4(Y)] was infrequent in primary breast cancer, but very frequent in brain metastases (5.9% 75% positive), where it was usually co-expressed with pHER3(Y) ( < 0.05). The presence of YAP in tumour cell nuclei was associated directly with nuclear pERK5(T/Y) ( = 0.003). However, relationships with disease-specific survival depended on oestrogen receptor (ER) status. Nuclear pYAP(S) was associated with smaller, good prognostic ER+ breast tumours (log-rank hazard-ratio 0.53;  = 9.6E), but larger, poor prognostic triple-negative cancers (log-rank hazard-ratio 2.78;  = 1.7E), particularly when co-expressed with nuclear HER4-ICD ( = 0.02). This phenotype was associated with stemness and mitotic instability markers (vimentin, SOX9, ID1, SPAG5, TTK, geminin;  < 0.05). YAP expression in brain metastases was higher than matched primary tumours; specifically, nuclear pYAP(S) in ER-negative cases ( < 0.05). Nuclear YAP was detected in ~70% of ER-negative, HER4-activated brain metastases.

Discussion: Our findings suggest that the canonical-mechanism where Hippo pathway-mediated phosphorylation of YAP ostensibly excludes it from the nucleus is dysfunctional in breast cancer. The data are consistent with pYAP(S) having independent transcriptional functions, which may include transducing neuregulin signals in brain metastases. Consistent with mechanistic studies implicating it as an ER co-factor, nuclear pYAP(S) associations with breast cancer clinical outcomes were dependent on ER status.

Conclusion: Preclinical studies investigating HER4 and nuclear YAP combination therapy strategies are warranted.
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http://dx.doi.org/10.1177/1758835920946259DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7517995PMC
July 2020

Metaplastic breast cancers frequently express immune checkpoint markers FOXP3 and PD-L1.

Br J Cancer 2020 11 17;123(11):1665-1672. Epub 2020 Sep 17.

Faculty of Medicine, UQ Centre for Clinical Research, The University of Queensland, Herston, Brisbane, QLD, Australia.

Background: Metaplastic breast carcinoma encompasses a heterogeneous group of tumours with differentiation into squamous and/or spindle, chondroid, osseous or rhabdoid mesenchymal-looking elements. Emerging immunotherapies targeting Programmed Death Ligand 1 (PD-L1) and immune-suppressing T cells (Tregs) may benefit metaplastic breast cancer patients, which are typically chemo-resistant and do not express hormone therapy targets.

Methods: We evaluated the immunohistochemical expression of PD-L1 and FOXP3, and the extent of tumour infiltrating lymphocytes (TILs) in a large cohort of metaplastic breast cancers, with survival data.

Results: Metaplastic breast cancers were significantly enriched for PD-L1 positive tumour cells, compared to triple-negative ductal breast cancers (P < 0.0001), while there was no significant difference in PD-L1 positive TILs. Metaplastic breast cancers were also significantly enriched for TILs expressing FOXP3, with FOXP3 positive intra-tumoural TILs (iTILs) associated with an adverse prognostic outcome (P = 0.0226). Multivariate analysis identified FOXP3 iTILs expression status as an important independent prognostic factor for patient survival.

Conclusions: Our findings indicate the clinical significance and prognostic value of FOXP3, PD-1/PD-L1 checkpoint and TILs in metaplastic breast cancer and confirm that a subset of metaplastics may benefit from immune-based therapies.
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http://dx.doi.org/10.1038/s41416-020-01065-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7686342PMC
November 2020

Using whole-genome sequencing data to derive the homologous recombination deficiency scores.

NPJ Breast Cancer 2020 7;6:33. Epub 2020 Aug 7.

Centre for Clinical Research, Faculty of Medicine, The University of Queensland, Brisbane, QLD Australia.

The homologous recombination deficiency (HRD) score was developed using whole-genome copy number data derived from arrays as a way to infer deficiency in the homologous recombination DNA damage repair pathway (in particular or deficiency) in breast cancer samples. The score has utility in understanding tumour biology and may be indicative of response to certain therapeutic strategies. Studies have used whole-exome sequencing to derive the HRD score, however, with increasing use of whole-genome sequencing (WGS) to characterise tumour genomes, there has yet to be a comprehensive comparison between HRD scores derived by array versus WGS. Here we demonstrate that there is both a high correlation and a good agreement between array- and WGS-derived HRD scores and between the scores derived from WGS and downsampled WGS to represent shallow WGS. For samples with an HRD score close to threshold for stratifying HR proficiency or deficiency there was however some disagreement in the HR status between array and WGS data, highlighting the importance of not relying on a single method of ascertaining the homologous recombination status of a tumour.
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http://dx.doi.org/10.1038/s41523-020-0172-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7414867PMC
August 2020

Association of Sperm-Associated Antigen 5 and Treatment Response in Patients With Estrogen Receptor-Positive Breast Cancer.

JAMA Netw Open 2020 07 1;3(7):e209486. Epub 2020 Jul 1.

Department of Clinical Oncology, Nottingham University Hospitals NHS Trust, Nottingham, United Kingdom.

Importance: There is no proven test that can guide the optimal treatment, either endocrine therapy or chemotherapy, for estrogen receptor-positive breast cancer.

Objective: To investigate the associations of sperm-associated antigen 5 (SPAG5) transcript and SPAG5 protein expressions with treatment response in systemic therapy for estrogen receptor-positive breast cancer.

Design, Settings, And Participants: This retrospective cohort study included patients with estrogen receptor-positive breast cancer who received 5 years of adjuvant endocrine therapy with or without neoadjuvant anthracycline-based combination chemotherapy (NACT) derived from 11 cohorts from December 1, 1986, to November 28, 2019. The associations of SPAG5 transcript and SPAG5 protein expression with pathological complete response to NACT were evaluated, as was the association of SPAG5 mRNA expression with response to neoadjuvant endocrine therapy. The associations of distal relapse-free survival with SPAG5 transcript or SPAG5 protein expressions were analyzed. Data were analyzed from September 9, 2015, to November 28, 2019.

Main Outcomes And Measures: The primary outcomes were breast cancer-specific survival, distal relapse-free survival, pathological complete response, and clinical response. Outcomes were examined using Kaplan-Meier, multivariable logistic, and Cox regression models.

Results: This study included 12 720 women aged 24 to 78 years (mean [SD] age, 58.46 [12.45] years) with estrogen receptor-positive breast cancer, including 1073 women with SPAG5 transcript expression and 361 women with SPAG5 protein expression of locally advanced disease stage IIA through IIIC. Women with SPAG5 transcript and SPAG5 protein expressions achieved higher pathological complete response compared with those without SPAG5 transcript or SPAG5 protein expressions (transcript: odds ratio, 2.45 [95% CI, 1.71-3.51]; P < .001; protein: odds ratio, 7.32 [95% CI, 3.33-16.22]; P < .001). Adding adjuvant anthracycline chemotherapy to adjuvant endocrine therapy for SPAG5 mRNA expression in estrogen receptor-positive breast cancer was associated with prolonged 5-year distal relapse-free survival in patients without lymph node involvement (hazard ratio, 0.34 [95% CI, 0.14-0.87]; P = .03) and patients with lymph node involvement (hazard ratio, 0.35 [95% CI, 0.18-0.68]; P = .002) compared with receiving 5-year endocrine therapy alone. Mean (SD) SPAG5 transcript was found to be downregulated after 2 weeks of neoadjuvant endocrine therapy compared with pretreatment levels in 68 of 92 patients (74%) (0.23 [0.18] vs 0.34 [0.24]; P < .001).

Conclusions And Relevance: These findings suggest that SPAG5 transcript and SPAG5 protein expressions could be used to guide the optimal therapies for estrogen receptor-positive breast cancer. Retrospective and prospective clinical trials are warranted.
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http://dx.doi.org/10.1001/jamanetworkopen.2020.9486DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7341179PMC
July 2020

Proteogenomic analysis of Inhibitor of Differentiation 4 (ID4) in basal-like breast cancer.

Breast Cancer Res 2020 06 11;22(1):63. Epub 2020 Jun 11.

The Kinghorn Cancer Centre and Cancer Research Division, Garvan Institute of Medical Research, Darlinghurst, NSW, 2010, Australia.

Background: Basal-like breast cancer (BLBC) is a poorly characterised, heterogeneous disease. Patients are diagnosed with aggressive, high-grade tumours and often relapse with chemotherapy resistance. Detailed understanding of the molecular underpinnings of this disease is essential to the development of personalised therapeutic strategies. Inhibitor of differentiation 4 (ID4) is a helix-loop-helix transcriptional regulator required for mammary gland development. ID4 is overexpressed in a subset of BLBC patients, associating with a stem-like poor prognosis phenotype, and is necessary for the growth of cell line models of BLBC through unknown mechanisms.

Methods: Here, we have defined unique molecular insights into the function of ID4 in BLBC and the related disease high-grade serous ovarian cancer (HGSOC), by combining RIME proteomic analysis, ChIP-seq mapping of genomic binding sites and RNA-seq.

Results: These studies reveal novel interactions with DNA damage response proteins, in particular, mediator of DNA damage checkpoint protein 1 (MDC1). Through MDC1, ID4 interacts with other DNA repair proteins (γH2AX and BRCA1) at fragile chromatin sites. ID4 does not affect transcription at these sites, instead binding to chromatin following DNA damage. Analysis of clinical samples demonstrates that ID4 is amplified and overexpressed at a higher frequency in BRCA1-mutant BLBC compared with sporadic BLBC, providing genetic evidence for an interaction between ID4 and DNA damage repair deficiency.

Conclusions: These data link the interactions of ID4 with MDC1 to DNA damage repair in the aetiology of BLBC and HGSOC.
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http://dx.doi.org/10.1186/s13058-020-01306-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7291584PMC
June 2020

Digital spatial profiling application in breast cancer: a user's perspective.

Virchows Arch 2020 Dec 26;477(6):885-890. Epub 2020 May 26.

Centre for Clinical Research, University of Queensland, Herston, Brisbane, Australia.

The disciplines of oncology and pathology are at present experiencing a wave of changes as precision medicine becomes embedded as standard-of-care. Consequently, the need to assess increasing numbers of biomarkers simultaneously has become more urgent and recognising the vast intra-tumoural heterogeneity, including within the microenvironment, requires a complex dimensional understanding of the localisation of the biomarker expression. Digital spatial profiling (DSP; nanoString™) technology spatially resolves and digitally quantifies proteins in a highly multiplexed assay, underpinned by the nCounter® barcoding platform. We present the application of this technology to breast cancer samples. Applying the 'off the shelf' cancer panel and a custom-conjugated E-cadherin antibody, we quantify vast intra-tumoural heterogeneity in immunological and tumour markers, and demonstrate a need for focussed selection of target cell populations. The technology offers enormous potential not only for making research advances but also for improving standard operating procedures in diagnostic applications.
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http://dx.doi.org/10.1007/s00428-020-02821-9DOI Listing
December 2020

Morphologic and Genomic Heterogeneity in the Evolution and Progression of Breast Cancer.

Cancers (Basel) 2020 Mar 31;12(4). Epub 2020 Mar 31.

UQ Centre for Clinical Research, Faculty of Medicine, The University of Queensland, Herston, Brisbane 4029, Australia.

Breast cancer is a remarkably complex and diverse disease. Subtyping based on morphology, genomics, biomarkers and/or clinical parameters seeks to stratify optimal approaches for management, but it is clear that every breast cancer is fundamentally unique. Intra-tumour heterogeneity adds further complexity and impacts a patient's response to neoadjuvant or adjuvant therapy. Here, we review some established and more recent evidence related to the complex nature of breast cancer evolution. We describe morphologic and genomic diversity as it arises spontaneously during the early stages of tumour evolution, and also in the context of treatment where the changing subclonal architecture of a tumour is driven by the inherent adaptability of tumour cells to evolve and resist the selective pressures of therapy.
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http://dx.doi.org/10.3390/cancers12040848DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7226487PMC
March 2020

LobSig is a multigene predictor of outcome in invasive lobular carcinoma.

NPJ Breast Cancer 2019 27;5:18. Epub 2019 Jun 27.

1UQ Centre for Clinical Research, Faculty of Medicine, The University of Queensland, Herston, QLD 4029 Australia.

Invasive lobular carcinoma (ILC) is the most common special type of breast cancer, and is characterized by functional loss of E-cadherin, resulting in cellular adhesion defects. ILC typically present as estrogen receptor positive, grade 2 breast cancers, with a good short-term prognosis. Several large-scale molecular profiling studies have now dissected the unique genomics of ILC. We have undertaken an integrative analysis of gene expression and DNA copy number to identify novel drivers and prognostic biomarkers, using in-house ( = 25), METABRIC ( = 125) and TCGA ( = 146) samples. Using in silico integrative analyses, a 194-gene set was derived that is highly prognostic in ILC ( = 1.20 × 10)-we named this metagene 'LobSig'. Assessing a 10-year follow-up period, LobSig outperformed the Nottingham Prognostic Index, PAM50 risk-of-recurrence (Prosigna), OncotypeDx, and Genomic Grade Index (MapQuantDx) in a stepwise, multivariate Cox proportional hazards model, particularly in grade 2 ILC cases ( ,  = 9.0 × 10), which are difficult to prognosticate clinically. Importantly, LobSig status predicted outcome with 94.6% accuracy amongst cases classified as 'moderate-risk' according to Nottingham Prognostic Index in the METABRIC cohort. Network analysis identified few candidate pathways, though genesets related to proliferation were identified, and a LobSig-high phenotype was associated with the TCGA proliferative subtype ( ,  < 8.86 × 10). ILC with a poor outcome as predicted by LobSig were enriched with mutations in , , , and . LobSig has the potential to be a clinically relevant prognostic signature and warrants further development.
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http://dx.doi.org/10.1038/s41523-019-0113-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6597578PMC
June 2019

Diff-Quik Cytology Smears from Endobronchial Ultrasound Transbronchial Needle Aspiration Lymph Node Specimens as a Source of DNA for Next-Generation Sequencing Instead of Cell Blocks.

Respiration 2019;97(6):525-539. Epub 2019 Feb 7.

Centre for Clinical Research, Faculty of Medicine, The University of Queensland, Brisbane, Queensland, Australia.

Background: Next-generation sequencing (NGS) in lung cancer specimens from endobronchial ultrasound transbronchial needle aspiration (EBUS-TBNA) is usually performed on formalin-fixed paraffin-embedded cell block material.

Objectives: Since DNA can be damaged by this process, we investigated the potential of using DNA extracted from Diff-Quik cytology smears made for rapid on-site evaluation during EBUS-TBNA.

Methods: In a prospective study, 67 patients undergoing diagnostic EBUS-TBNA were ana-lysed. We compared cell blocks and smears for DNA yields and sequencing (TruSeq Amplicon Cancer Panel) outcomes. Smears were also evaluated for tumour cell fraction and overall cellularity (cell count).

Results: Primary lung cancer was diagnosed in 64 patients and metastatic malignancy in 3 patients. The DNA yield from smears was significantly higher than that obtained from matched cell blocks (mean 1,740 vs. 434 ng; p = 0.001). For 33 cases with matched smears and cell blocks the mutation profiles were similar. Smears with abundant malignant cells (using a cut-off of > 25% tumour cell fraction and > 1,000 cells) accurately predicted high (> 50 ng) DNA yield and therefore success in triaging samples to sequencing. In terms of tissue workflow, using only smears as source DNA for sequencing was an improvement in the use of only cell blocks (54/67 [80.6%] vs. 41/67 [61.2%]); however, the use of cell blocks when smears were not available or did not yield sufficient DNA further improved the success rate to 62/67 (92.5%) cases.

Conclusion: We recommend smears in laboratory workflows as the primary source of DNA for NGS following an EBUS procedure.
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http://dx.doi.org/10.1159/000495661DOI Listing
September 2020

The circular RNome of primary breast cancer.

Genome Res 2019 03 28;29(3):356-366. Epub 2019 Jan 28.

The Netherlands Cancer Institute, 1066CX Amsterdam, the Netherlands.

Circular RNAs (circRNAs) are a class of RNAs that is under increasing scrutiny, although their functional roles are debated. We analyzed RNA-seq data of 348 primary breast cancers and developed a method to identify circRNAs that does not rely on unmapped reads or known splice junctions. We identified 95,843 circRNAs, of which 20,441 were found recurrently. Of the circRNAs that match exon boundaries of the same gene, 668 showed a poor or even negative ( < 0.2) correlation with the expression level of the linear gene. In silico analysis showed only a minority (8.5%) of circRNAs could be explained by known splicing events. Both these observations suggest that specific regulatory processes for circRNAs exist. We confirmed the presence of circRNAs of , , and in an independent pool of primary breast cancers. We identified circRNA profiles associated with subgroups of breast cancers and with biological and clinical features, such as amount of tumor lymphocytic infiltrate and proliferation index. siRNA-mediated knockdown of was shown to significantly reduce viability of the breast cancer cell lines MCF-7 and BT-474, further underlining the biological relevance of circRNAs. Furthermore, we found that circular, and not linear, levels are predictive for progression-free survival time to aromatase inhibitor (AI) therapy in advanced breast cancer patients, and found that is detectable in cell-free RNA from plasma. We showed that circRNAs are abundantly present, show characteristics of being specifically regulated, are associated with clinical and biological properties, and thus are relevant in breast cancer.
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http://dx.doi.org/10.1101/gr.238121.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6396421PMC
March 2019

Author Correction: Landscape of somatic mutations in 560 breast cancer whole-genome sequences.

Nature 2019 02;566(7742):E1

Wellcome Trust Sanger Institute, Hinxton, Cambridge, CB10 1SA, UK.

In the Methods section of this Article, 'greater than' should have been 'less than' in the sentence 'Putative regions of clustered rearrangements were identified as having an average inter-rearrangement distance that was at least 10 times greater than the whole-genome average for the individual sample. '. The Article has not been corrected.
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http://dx.doi.org/10.1038/s41586-019-0883-2DOI Listing
February 2019

Breast cancer metastasis to gynaecological organs: a clinico-pathological and molecular profiling study.

J Pathol Clin Res 2019 01 22;5(1):25-39. Epub 2018 Oct 22.

Centre for Clinical Research, Faculty of Medicine, The University of Queensland, Brisbane, Australia.

Breast cancer metastasis to gynaecological organs is an understudied pattern of tumour spread. We explored clinico-pathological and molecular features of these metastases to better understand whether this pattern of dissemination is organotropic or a consequence of wider metastatic dissemination. Primary and metastatic tumours from 54 breast cancer patients with gynaecological metastases were analysed using immunohistochemistry, DNA copy-number profiling, and targeted sequencing of 386 cancer-related genes. The median age of primary tumour diagnosis amongst patients with gynaecological metastases was significantly younger compared to a general breast cancer population (46.5 versus 60 years; p < 0.0001). Median age at metastatic diagnosis was 54.4, time to progression was 4.8 years (range 0-20 years), and survival following a diagnosis of metastasis was 1.95 years (range 0-18 years). Patients had an average of five involved sites (most frequently ovary, fallopian tube, omentum/peritoneum), with fewer instances of spread to the lungs, liver, or brain. Invasive lobular histology and luminal A-like phenotype were over-represented in this group (42.8 and 87.5%, respectively) and most patients had involved axillary lymph nodes (p < 0.001). Primary tumours frequently co-expressed oestrogen receptor cofactors (GATA3, FOXA1) and harboured amplifications at 8p12, 8q24, and 11q13. In terms of phenotype conversion, oestrogen receptor status was generally maintained in metastases, FOXA1 increased, and expression of progesterone receptor, androgen receptor, and GATA3 decreased. ESR1 and novel AR mutations were identified. Metastasis to gynaecological organs is a complication frequently affecting young women with invasive lobular carcinoma and luminal A-like breast cancer, and hence may be driven by sustained hormonal signalling. Molecular analyses reveal a spectrum of factors that could contribute to de novo or acquired resistance to therapy and disease progression.
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http://dx.doi.org/10.1002/cjp2.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6317061PMC
January 2019

Characterization of a novel breast cancer cell line derived from a metastatic bone lesion of a breast cancer patient.

Breast Cancer Res Treat 2018 Jul 21;170(1):179-188. Epub 2018 Feb 21.

QIMR Berghofer Medical Research Institute, Royal Brisbane Hospital, Locked Bag 2000, Brisbane, QLD, 4029, Australia.

Purpose: We aimed to generate and characterize a novel cell line from a breast cancer bone metastasis to better study the progression of the disease.

Methods: The cell line, P7731, was derived from a metastatic bone lesion of a breast cancer patient and assessed for marker expression. P7731 was analyzed for DNA copy number variation, somatic mutations, and gene expression and was compared with the primary tumor.

Results: P7731 cells are negative for estrogen receptor alpha (ERα), progesterone receptor (PR), and HER2 (triple-negative); strongly express vimentin (100% of cells positive) and also express cytokeratins 8/18 and 19 but at lower frequencies. Flow cytometry indicates P7731 cells are predominantly CD44/CD49f/EpCAM, consistent with a primitive, mesenchymal-like phenotype. The cell line is tumorigenic in immunocompromised mice. Exome sequencing identified a total of 45 and 76 somatic mutations in the primary tumor and cell line, respectively, of which 32 were identified in both samples and included mutations in known driver genes PIK3CA, TP53, and ARID1A. P7731 retains the DNA copy number alterations present in the matching primary tumor. Homozygous deletions detected in the cell line and in the primary tumor were found in regions containing three known (CDKN2A, CDKN2B, and CDKN1B) and 23 putative tumor suppressor genes. Cell line-specific gene amplification coupled with mRNA expression analysis revealed genes and pathways with potential pro-metastatic functions.

Conclusion: This novel human breast cancer-bone metastasis cell line will be a useful model to study aspects of breast cancer biology, particularly metastasis-related changes from breast to bone.
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http://dx.doi.org/10.1007/s10549-018-4719-9DOI Listing
July 2018

Expression of MAGE-A and NY-ESO-1 cancer/testis antigens is enriched in triple-negative invasive breast cancers.

Histopathology 2018 Jul 14;73(1):68-80. Epub 2018 Apr 14.

Faculty of Medicine, The University of Queensland, The Royal Brisbane and Women's Hospital, Herston, Queensland, Australia.

Aims: A better understanding of the expression of cancer/testis antigens (CTAs) in breast cancer might enable the identification of new immunotherapy options, especially for triple-negative (TN) tumours, which lack expression of the conventional therapeutic targets oestrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2. The aim of this study was to quantify the expression of MAGE-A and NY-ESO-1 CTAs in breast cancer, and relate this to known clinicopathological parameters.

Methods And Results: We surveyed MAGE-A and NY-ESO-1 expression in an unselected cohort of 367 breast tumours (of which 65 were TN), with accompanying clinical follow-up data, by using immunohistochemical analysis of tissue microarrays. Relevant to their potential as vaccine targets in breast cancer, MAGE-A was expressed in 13% of cases, and NY-ESO-1 in 3.8%, with the majority of tumours showing fairly homogeneous staining within individual tissue cores (~85% of cases with staining in >75% of tumour cells). Most NY-ESO-1-positive cases also expressed MAGE-A (P = 2.06 × 10 ), and both were strongly associated with the TN phenotype (P < 0.0001), with the most proliferative and poorly differentiated cases, in paticular, showing genomic instability. This was characterised by coexpression of c-Kit and TTK, and overexpression of p53.

Conclusions: MAGE-A and NY-ESO-1 are frequently expressed in TN breast cancer (~47% and 17% of TN cases, respectively), suggesting that targeting them could be feasible in this patient group. Expression is reasonably homogeneous in positive cases, suggesting that immunohistochemical analysis of tissue biopsies would be a reliable companion biomarker.
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http://dx.doi.org/10.1111/his.13498DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6635746PMC
July 2018

Corrigendum to "Molecular signatures in breast cancer" [Methods 131 (2017) 135-146].

Methods 2019 Jul 3;164-165:120. Epub 2018 Feb 3.

The University of Queensland, Centre for Clinical Research, Faculty of Medicine, Herston, QLD 4029, Australia. Electronic address:

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http://dx.doi.org/10.1016/j.ymeth.2018.01.007DOI Listing
July 2019

Mixed ductal-lobular carcinomas: evidence for progression from ductal to lobular morphology.

J Pathol 2018 04 9;244(4):460-468. Epub 2018 Mar 9.

Centre for Clinical Research, Faculty of Medicine, The University of Queensland, Brisbane, Australia.

Mixed ductal-lobular carcinomas (MDLs) show both ductal and lobular morphology, and constitute an archetypal example of intratumoural morphological heterogeneity. The mechanisms underlying the coexistence of these different morphological entities are poorly understood, although theories include that these components either represent 'collision' of independent tumours or evolve from a common ancestor. We performed comprehensive clinicopathological analysis of a cohort of 82 MDLs, and found that: (1) MDLs more frequently coexist with ductal carcinoma in situ (DCIS) than with lobular carcinoma in situ (LCIS); (2) the E-cadherin-catenin complex was normal in the ductal component in 77.6% of tumours; and (3) in the lobular component, E-cadherin was almost always aberrantly located in the cytoplasm, in contrast to invasive lobular carcinoma (ILC), where E-cadherin is typically absent. Comparative genomic hybridization and multiregion whole exome sequencing of four representative cases revealed that all morphologically distinct components within an individual case were clonally related. The mutations identified varied between cases; those associated with a common clonal ancestry included BRCA2, TBX3, and TP53, whereas those associated with clonal divergence included CDH1 and ESR1. Together, these data support a model in which separate morphological components of MDLs arise from a common ancestor, and lobular morphology can arise via a ductal pathway of tumour progression. In MDLs that present with LCIS and DCIS, the clonal divergence probably occurs early, and is frequently associated with complete loss of E-cadherin expression, as in ILC, whereas, in the majority of MDLs, which present with DCIS but not LCIS, direct clonal divergence from the ductal to the lobular phenotype occurs late in tumour evolution, and is associated with aberrant expression of E-cadherin. The mechanisms driving the phenotypic change may involve E-cadherin-catenin complex deregulation, but are yet to be fully elucidated, as there is significant intertumoural heterogeneity, and each case may have a unique molecular mechanism. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
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http://dx.doi.org/10.1002/path.5040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5873281PMC
April 2018

Multidimensional phenotyping of breast cancer cell lines to guide preclinical research.

Breast Cancer Res Treat 2018 01 9;167(1):289-301. Epub 2017 Sep 9.

Faculty of Medicine, The University of Queensland, Herston, QLD, Australia.

Purpose: Cell lines are extremely useful tools in breast cancer research. Their key benefits include a high degree of control over experimental variables and reproducibility. However, the advantages must be balanced against the limitations of modelling such a complex disease in vitro. Informed selection of cell line(s) for a given experiment now requires essential knowledge about molecular and phenotypic context in the culture dish.

Methods: We performed multidimensional profiling of 36 widely used breast cancer cell lines that were cultured under standardised conditions. Flow cytometry and digital immunohistochemistry were used to compare the expression of 14 classical breast cancer biomarkers related to intrinsic molecular profiles and differentiation states: EpCAM, CD24, CD49f, CD44, ER, AR, HER2, EGFR, E-cadherin, p53, vimentin, and cytokeratins 5, 8/18 and 19.

Results: This cell-by-cell analysis revealed striking heterogeneity within cultures of individual lines that would be otherwise obscured by analysing cell homogenates, particularly amongst the triple-negative lines. High levels of p53 protein, but not RNA, were associated with somatic mutations (p = 0.008). We also identified new subgroups using the nanoString PanCancer Pathways panel (730 transcripts representing 13 canonical cancer pathways). Unsupervised clustering identified five groups: luminal/HER2, immortalised ('normal'), claudin-low and two basal clusters, distinguished mostly by baseline expression of TGF-beta and PI3-kinase pathway genes.

Conclusion: These features are compared with other published genotype and phenotype information in a user-friendly reference table to help guide selection of the most appropriate models for in vitro and in vivo studies, and as a framework for classifying new patient-derived cancer cell lines and xenografts.
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http://dx.doi.org/10.1007/s10549-017-4496-xDOI Listing
January 2018

Molecular signatures in breast cancer.

Methods 2017 12 29;131:135-146. Epub 2017 Jun 29.

The University of Queensland, Centre for Clinical Research, Faculty of Medicine, Herston, QLD 4029, Australia. Electronic address:

The use of molecular signatures to add value to standard clinical and pathological parameters has impacted clinical practice in many cancer types, but perhaps most notably in the breast cancer field. This is, in part, due to the considerable complexity of the disease at the clinical, morphological and molecular levels. The adoption of molecular profiling of DNA, RNA and protein continues to reveal important differences in the intrinsic biology between molecular subtypes and has begun to impact the way patients are managed. Several bioinformatic tools have been developed using DNA or RNA-based signatures to stratify the disease into biologically and/or clinically meaningful subgroups. Here, we review the approaches that have been used to develop gene expression signatures into currently available diagnostic assays (e.g., OncotypeDX® and Mammaprint®), plus we describe the latest work on genome sequencing, the methodologies used in the discovery process of mutational signatures, and the potential of these signatures to impact the clinic.
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http://dx.doi.org/10.1016/j.ymeth.2017.06.032DOI Listing
December 2017

HRDetect is a predictor of BRCA1 and BRCA2 deficiency based on mutational signatures.

Nat Med 2017 Apr 13;23(4):517-525. Epub 2017 Mar 13.

Equipe Erable, INRIA Grenoble-Rhône-Alpes, Montbonnot-Saint Martin, France.

Approximately 1-5% of breast cancers are attributed to inherited mutations in BRCA1 or BRCA2 and are selectively sensitive to poly(ADP-ribose) polymerase (PARP) inhibitors. In other cancer types, germline and/or somatic mutations in BRCA1 and/or BRCA2 (BRCA1/BRCA2) also confer selective sensitivity to PARP inhibitors. Thus, assays to detect BRCA1/BRCA2-deficient tumors have been sought. Recently, somatic substitution, insertion/deletion and rearrangement patterns, or 'mutational signatures', were associated with BRCA1/BRCA2 dysfunction. Herein we used a lasso logistic regression model to identify six distinguishing mutational signatures predictive of BRCA1/BRCA2 deficiency. A weighted model called HRDetect was developed to accurately detect BRCA1/BRCA2-deficient samples. HRDetect identifies BRCA1/BRCA2-deficient tumors with 98.7% sensitivity (area under the curve (AUC) = 0.98). Application of this model in a cohort of 560 individuals with breast cancer, of whom 22 were known to carry a germline BRCA1 or BRCA2 mutation, allowed us to identify an additional 22 tumors with somatic loss of BRCA1 or BRCA2 and 47 tumors with functional BRCA1/BRCA2 deficiency where no mutation was detected. We validated HRDetect on independent cohorts of breast, ovarian and pancreatic cancers and demonstrated its efficacy in alternative sequencing strategies. Integrating all of the classes of mutational signatures thus reveals a larger proportion of individuals with breast cancer harboring BRCA1/BRCA2 deficiency (up to 22%) than hitherto appreciated (∼1-5%) who could have selective therapeutic sensitivity to PARP inhibition.
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http://dx.doi.org/10.1038/nm.4292DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5833945PMC
April 2017
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