Stevenson, J.
Barrow-McGee, R.
Yu, L.
Paul, A.
Mansfield, D.
Owen, J.
Woodman, N.
Natrajan, R.
Haider, S.
Gillett, C.
Tutt, A.
Pinder, S.E.
Choudary, J.
Naidoo, K.
(2021). Proteomics of REPLICANT perfusate detects changes in the metastatic lymph node microenvironment. Npj breast cancer,
Vol.7
(1).
show abstract
AbstractIn breast cancer (BC), detecting low volumes of axillary lymph node (ALN) metastasis pre-operatively is difficult and novel biomarkers are needed. We recently showed that patient-derived ALNs can be sustained ex-vivo using normothermic perfusion. We now compare reactive (tumour-free;n = 5) and macrometastatic (containing tumour deposits >2 mm;n = 4) ALNs by combining whole section multiplex immunofluorescence with TMT-labelled LC-MS/MS of the circulating perfusate. Macrometastases contained significantly fewer B cells and T cells (CD4+/CD8+/regulatory) than reactive nodes (p = 0.02). Similarly, pathway analysis of the perfusate proteome (119/1453 proteins significantly differentially expressed) showed that immune function was diminished in macrometastases in favour of ‘extracellular matrix degradation’; only ‘neutrophil degranulation’ was preserved. Qualitative comparison of the perfusate proteome to that of node-positive pancreatic and prostatic adenocarcinoma also highlighted ‘neutrophil degranulation’ as a contributing factor to nodal metastasis. Thus, metastasis-induced changes in the REPLICANT perfusate proteome are detectable, and could facilitate biomarker discovery..
Peck, B.
Bland, P.
Mavrommati, I.
Muirhead, G.
Cottom, H.
Wai, P.T.
Maguire, S.L.
Barker, H.E.
Morrison, E.
Kriplani, D.
Yu, L.
Gibson, A.
Falgari, G.
Brennan, K.
Farnie, G.
Buus, R.
Marlow, R.
Novo, D.
Knight, E.
Guppy, N.
Kolarevic, D.
Susnjar, S.
Milijic, N.M.
Naidoo, K.
Gazinska, P.
Roxanis, I.
Pancholi, S.
Martin, L.-.
Holgersen, E.M.
Cheang, M.C.
Noor, F.
Postel-Vinay, S.
Quinn, G.
McDade, S.
Krasny, L.
Huang, P.
Daley, F.
Wallberg, F.
Choudhary, J.S.
Haider, S.
Tutt, A.N.
Natrajan, R.
(2021). 3D Functional Genomics Screens Identify CREBBP as a Targetable Driver in Aggressive Triple-Negative Breast Cancer. Cancer research,
Vol.81
(4),
pp. 847-859.
Khalique, S.
Pettitt, S.J.
Kelly, G.
Tunariu, N.
Natrajan, R.
Banerjee, S.
Lord, C.J.
(2020). Longitudinal analysis of a secondary
BRCA2
mutation using digital droplet PCR. The journal of pathology: clinical research,
Vol.6
(1),
pp. 3-11.
Khalique, S.
Lord, C.J.
Banerjee, S.
Natrajan, R.
(2020). Translational genomics of ovarian clear cell carcinoma. Seminars in cancer biology,
Vol.61,
pp. 121-131.
Yap, T.A.
O'Carrigan, B.
Penney, M.S.
Lim, J.S.
Brown, J.S.
de Miguel Luken, M.J.
Tunariu, N.
Perez-Lopez, R.
Rodrigues, D.N.
Riisnaes, R.
Figueiredo, I.
Carreira, S.
Hare, B.
McDermott, K.
Khalique, S.
Williamson, C.T.
Natrajan, R.
Pettitt, S.J.
Lord, C.J.
Banerji, U.
Pollard, J.
Lopez, J.
de Bono, J.S.
(2020). Phase I Trial of First-in-Class ATR Inhibitor M6620 (VX-970) as Monotherapy or in Combination With Carboplatin in Patients With Advanced Solid Tumors. J clin oncol,
,
pp. JCO1902404-JCO1902404.
show abstract
PURPOSE: Preclinical studies demonstrated that ATR inhibition can exploit synthetic lethality (eg, in cancer cells with impaired compensatory DNA damage responses through ATM loss) as monotherapy and combined with DNA-damaging drugs such as carboplatin. PATIENTS AND METHODS: This phase I trial assessed the ATR inhibitor M6620 (VX-970) as monotherapy (once or twice weekly) and combined with carboplatin (carboplatin on day 1 and M6620 on days 2 and 9 in 21-day cycles). Primary objectives were safety, tolerability, and maximum tolerated dose; secondary objectives included pharmacokinetics and antitumor activity; exploratory objectives included pharmacodynamics in timed paired tumor biopsies. RESULTS: Forty patients were enrolled; 17 received M6620 monotherapy, which was safe and well tolerated. The recommended phase II dose (RP2D) for once- or twice-weekly administration was 240 mg/m2. A patient with metastatic colorectal cancer harboring molecular aberrations, including ATM loss and an ARID1A mutation, achieved RECISTv1.1 complete response and maintained this response, with a progression-free survival of 29 months at last assessment. Twenty-three patients received M6620 with carboplatin, with mechanism-based hematologic toxicities at higher doses, requiring dose delays and reductions. The RP2D for combination therapy was M6620 90 mg/m2 with carboplatin AUC5. A patient with advanced germline BRCA1 ovarian cancer achieved RECISTv1.1 partial response and Gynecologic Cancer Intergroup CA125 response despite being platinum refractory and PARP inhibitor resistant. An additional 15 patients had RECISTv1.1 stable disease as best response. Pharmacokinetics were dose proportional and exceeded preclinical efficacious levels. Pharmacodynamic studies demonstrated substantial inhibition of phosphorylation of CHK1, the downstream ATR substrate. CONCLUSION: To our knowledge, this report is the first of an ATR inhibitor as monotherapy and combined with carboplatin. M6620 was well tolerated, with target engagement and preliminary antitumor responses observed..
Dalton, W.B.
Helmenstine, E.
Walsh, N.
Gondek, L.P.
Kelkar, D.S.
Read, A.
Natrajan, R.
Christenson, E.S.
Roman, B.
Das, S.
Zhao, L.
Leone, R.D.
Shinn, D.
Groginski, T.
Madugundu, A.K.
Patil, A.
Zabransky, D.J.
Medford, A.
Lee, J.
Cole, A.J.
Rosen, M.
Thakar, M.
Ambinder, A.
Donaldson, J.
DeZern, A.E.
Cravero, K.
Chu, D.
Madero-Marroquin, R.
Pandey, A.
Hurley, P.J.
Lauring, J.
Park, B.H.
(2019). Hotspot SF3B1 mutations induce metabolic reprogramming and vulnerability to serine deprivation. Journal of clinical investigation,
Vol.129
(11),
pp. 4708-4723.
Krasny, L.
Bland, P.
Kogata, N.
Wai, P.
Howard, B.
Natrajan, R.
Huang, P.
(2018). SWATH mass spectrometry as a tool for quantitative profiling of the matrisome. Journal of proteomics,
.
show abstract
Proteomic analysis of extracellular matrix (ECM) and ECM-associated proteins, collectively known as the matrisome, is a challenging task due to the inherent complexity and insolubility of these proteins. Here we present sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH MS) as a tool for the quantitative analysis of matrisomal proteins in both non-enriched and ECM enriched tissue without the need for prior fractionation. Utilising a spectral library containing 201 matrisomal proteins, we compared the performance and reproducibility of SWATH MS over conventional data-dependent analysis mass spectrometry (DDA MS) in unfractionated murine lung and liver. SWATH MS conferred a 15–20% increase in reproducible peptide identification across replicate experiments in both tissue types and identified 54% more matrisomal proteins in the liver versus DDA MS. We further use SWATH MS to evaluate the quantitative changes in matrisome content that accompanies ECM enrichment. Our data shows that ECM enrichment led to a systematic increase in core matrisomal proteins but resulted in significant losses in matrisome-associated proteins including the cathepsins and proteins of the S100 family. Our proof-of-principle study demonstrates the utility of SWATH MS as a versatile tool for in-depth characterisation of the matrisome in unfractionated and non-enriched tissues..
Natrajan, R.
Tutt, A.N.
Lord, C.J.
(2018). Driver Oncogenes but Not as We Know Them: Targetable Fusion Genes in Breast Cancer. Cancer discov,
Vol.8
(3),
pp. 272-275.
show abstract
Two reports in this issue of Cancer Discovery outline how the genomic composition of tumors, including the presence of intragenic gene fusions, could inform the selection of treatment approaches in aggressive forms of the disease. Cancer Discov; 8(3); 272-5. ©2018 AACRSee related article by Matissek et al., p. 336See related article by Liu et al., p. 354..
Khalique, S.
Naidoo, K.
Attygalle, A.D.
Kriplani, D.
Daley, F.
Lowe, A.
Campbell, J.
Jones, T.
Hubank, M.
Fenwick, K.
Matthews, N.
Rust, A.G.
Lord, C.J.
Banerjee, S.
Natrajan, R.
(2018). Optimised ARID1A immunohistochemistry is an accurate predictor ofARID1Amutational status in gynaecological cancers. The journal of pathology: clinical research,
Vol.4
(3),
pp. 154-166.
Piscuoglio, S.
Ng, C.K.
Geyer, F.C.
Burke, K.A.
Cowell, C.F.
Martelotto, L.G.
Natrajan, R.
Popova, T.
Maher, C.A.
Lim, R.S.
Bruijn, I.D.
Mariani, O.
Norton, L.
Vincent-Salomon, A.
Weigelt, B.
Reis-Filho, J.S.
(2017). Genomic and transcriptomic heterogeneity in metaplastic carcinomas of the breast. Npj breast cancer,
Vol.3
(1).
Ng, C.K.
Piscuoglio, S.
Geyer, F.C.
Burke, K.A.
Pareja, F.
Eberle, C.A.
Lim, R.S.
Natrajan, R.
Riaz, N.
Mariani, O.
Norton, L.
Vincent-Salomon, A.
Wen, Y.H.
Weigelt, B.
Reis-Filho, J.S.
(2017). The Landscape of Somatic Genetic Alterations in Metaplastic Breast Carcinomas. Clinical cancer research,
Vol.23
(14),
pp. 3859-3870.
Wilkerson, P.M.
Dedes, K.J.
Samartzis, E.P.
Dedes, I.
Lambros, M.B.
Natrajan, R.
Gauthier, A.
Piscuoglio, S.
Töpfer, C.
Vukovic, V.
Daley, F.
Weigelt, B.
Reis-Filho, J.S.
(2017). Preclinical evaluation of the PARP inhibitor BMN-673 for the treatment of ovarian clear cell cancer. Oncotarget,
Vol.8
(4),
pp. 6057-6066.
Krasny, L.
Paul, A.
Wai, P.
Howard, B.A.
Natrajan, R.C.
Huang, P.H.
(2016). Comparative proteomic assessment of matrisome enrichment methodologies. Biochemical journal,
Vol.473
(21),
pp. 3979-3995.
show abstract
The matrisome is a complex and heterogeneous collection of extracellular matrix (ECM) and ECM-associated proteins that play important roles in tissue development and homeostasis. While several strategies for matrisome enrichment have been developed, it is currently unknown how the performance of these different methodologies compares in the proteomic identification of matrisome components across multiple tissue types. In the present study, we perform a comparative proteomic assessment of two widely used decellularisation protocols and two extraction methods to characterise the matrisome in four murine organs (heart, mammary gland, lung and liver). We undertook a systematic evaluation of the performance of the individual methods on protein yield, matrisome enrichment capability and the ability to isolate core matrisome and matrisome-associated components. Our data find that sodium dodecyl sulphate (SDS) decellularisation leads to the highest matrisome enrichment efficiency, while the extraction protocol that comprises chemical and trypsin digestion of the ECM fraction consistently identifies the highest number of matrisomal proteins across all types of tissue examined. Matrisome enrichment had a clear benefit over non-enriched tissue for the comprehensive identification of matrisomal components in murine liver and heart. Strikingly, we find that all four matrisome enrichment methods led to significant losses in the soluble matrisome-associated proteins across all organs. Our findings highlight the multiple factors (including tissue type, matrisome class of interest and desired enrichment purity) that influence the choice of enrichment methodology, and we anticipate that these data will serve as a useful guide for the design of future proteomic studies of the matrisome..
Showeil, R.
Romano, C.
Valganon, M.
Lambros, M.
Trivedi, P.
Van Noorden, S.
Sriraksa, R.
El-Kaffash, D.
El-Etreby, N.
Natrajan, R.
Foroni, L.
Osborne, R.
El-Bahrawy, M.
(2016). The status of epidermal growth factor receptor in borderline ovarian tumours. Oncotarget,
Vol.7
(9),
pp. 10568-10577.
Campbell, J.
Ryan, C.J.
Brough, R.
Bajrami, I.
Pemberton, H.N.
Chong, I.Y.
Costa-Cabral, S.
Frankum, J.
Gulati, A.
Holme, H.
Miller, R.
Postel-Vinay, S.
Rafiq, R.
Wei, W.
Williamson, C.T.
Quigley, D.A.
Tym, J.
Al-Lazikani, B.
Fenton, T.
Natrajan, R.
Strauss, S.J.
Ashworth, A.
Lord, C.J.
(2016). Large-Scale Profiling of Kinase Dependencies in Cancer Cell Lines. Cell reports,
Vol.14
(10),
pp. 2490-2501.
Miller, R.E.
Brough, R.
Bajrami, I.
Williamson, C.T.
McDade, S.
Campbell, J.
Kigozi, A.
Rafiq, R.
Pemberton, H.
Natrajan, R.
Joel, J.
Astley, H.
Mahoney, C.
Moore, J.D.
Torrance, C.
Gordan, J.D.
Webber, J.T.
Levin, R.S.
Shokat, K.M.
Bandyopadhyay, S.
Lord, C.J.
Ashworth, A.
(2016). Synthetic Lethal Targeting of ARID1A-Mutant Ovarian Clear Cell Tumors with Dasatinib. Molecular cancer therapeutics,
Vol.15
(7),
pp. 1472-1484.
Natrajan, R.
Weigelt, B.
(2016). Risk Stratification and Intrinsic Subtype Classification of Breast Cancer: a Multi-Parameter Test to Rule Them All?. Journal of the national cancer institute,
Vol.108
(9),
pp. djw118-djw118.
Wong, J.P.
Todd, J.R.
Finetti, M.A.
McCarthy, F.
Broncel, M.
Vyse, S.
Luczynski, M.T.
Crosier, S.
Ryall, K.A.
Holmes, K.
Payne, L.S.
Daley, F.
Wai, P.
Jenks, A.
Tanos, B.
Tan, A.-.
Natrajan, R.C.
Williamson, D.
Huang, P.H.
(2016). Dual Targeting of PDGFRα and FGFR1 Displays Synergistic Efficacy in Malignant Rhabdoid Tumors. Cell reports,
Vol.17
(5),
pp. 1265-1275.
Maguire, S.L.
Peck, B.
Wai, P.T.
Campbell, J.
Barker, H.
Gulati, A.
Daley, F.
Vyse, S.
Huang, P.
Lord, C.J.
Farnie, G.
Brennan, K.
Natrajan, R.
(2016). Three‐dimensional modelling identifies novel genetic dependencies associated with breast cancer progression in the isogenic
MCF10
model. The journal of pathology,
Vol.240
(3),
pp. 315-328.
Morrison, E.
Wai, P.
Leonidou, A.
Bland, P.
Khalique, S.
Farnie, G.
Daley, F.
Peck, B.
Natrajan, R.
(2016). Utilizing Functional Genomics Screening to Identify Potentially Novel Drug Targets in Cancer Cell Spheroid Cultures. J vis exp,
(118).
show abstract
The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery of the next generation of novel drug targets. It is becoming apparent that more complex models of cancer are required to fully appreciate the contributing factors that drive tumorigenesis in vivo and increase the efficacy of novel therapies that make the transition from pre-clinical models to clinical trials. Here we present a methodology for generating uniform and reproducible tumor spheroids that can be subjected to siRNA functional screening. These spheroids display many characteristics that are found in solid tumors that are not present in traditional two-dimension culture. We show that several commonly used breast cancer cell lines are amenable to this protocol. Furthermore, we provide proof-of-principle data utilizing the breast cancer cell line BT474, confirming their dependency on amplification of the epidermal growth factor receptor HER2 and mutation of phosphatidylinositol-4,5-biphosphate 3-kinase (PIK3CA) when grown as tumor spheroids. Finally, we are able to further investigate and confirm the spatial impact of these dependencies using immunohistochemistry..
Carvalho, D.
Taylor, K.
Burford, A.
Nowak, I.
Mackay, A.
Vinci, M.
Bjerke, L.
Molinari, V.
Nandhabalan, M.
Ingram, W.
Jury, A.
Natrajan, R.
Jones, D.
Pfister, S.
Shats, L.
Moore, A.
Jacques, T.
Popov, S.
Jones, C.
(2015). HG-09 * INFANTILE GLIOBLASTOMA WITH SARCOMATOUS HISTOLOGY DRIVEN BY ETV6:NTRK3 FUSIONS ARE SENSITIVE TO TRK INHIBITION BY PHA-848125. Neuro-oncology,
Vol.17
(suppl 3),
pp. iii12-iii12.
Maguire, S.L.
Leonidou, A.
Wai, P.
Marchiò, C.
Ng, C.K.
Sapino, A.
Salomon, A.
Reis‐Filho, J.S.
Weigelt, B.
Natrajan, R.C.
(2015). SF3B1
mutations constitute a novel therapeutic target in breast cancer. The journal of pathology,
Vol.235
(4),
pp. 571-580.
Weigelt, B.
Ng, C.K.
Shen, R.
Popova, T.
Schizas, M.
Natrajan, R.
Mariani, O.
Stern, M.-.
Norton, L.
Vincent-Salomon, A.
Reis-Filho, J.S.
(2015). Metastatic breast carcinomas display genomic and transcriptomic heterogeneity. Modern pathology,
Vol.28
(3),
pp. 340-351.
Schouten, P.C.
Grigoriadis, A.
Kuilman, T.
Mirza, H.
Watkins, J.A.
Cooke, S.A.
van Dyk, E.
Severson, T.M.
Rueda, O.M.
Hoogstraat, M.
Verhagen, C.V.
Natrajan, R.
Chin, S.-.
Lips, E.H.
Kruizinga, J.
Velds, A.
Nieuwland, M.
Kerkhoven, R.M.
Krijgsman, O.
Vens, C.
Peeper, D.
Nederlof, P.M.
Caldas, C.
Tutt, A.N.
Wessels, L.F.
Linn, S.C.
(2015). Robust BRCA1-like classification of copy number profiles of samples repeated across different datasets and platforms. Molecular oncology,
Vol.9
(7),
pp. 1274-1286.
Natrajan, R.C.
(2015). Breast cancer heterogeneity: parallel evolution or conscious uncoupling?. The journal of pathology,
Vol.237
(1),
pp. 1-3.
Martelotto, L.G.
De Filippo, M.R.
Ng, C.K.
Natrajan, R.
Fuhrmann, L.
Cyrta, J.
Piscuoglio, S.
Wen, H.-.
Lim, R.S.
Shen, R.
Schultheis, A.M.
Wen, Y.H.
Edelweiss, M.
Mariani, O.
Stenman, G.
Chan, T.A.
Colombo, P.-.
Norton, L.
Vincent-Salomon, A.
Reis-Filho, J.S.
Weigelt, B.
(2015). Genomic landscape of adenoid cystic carcinoma of the breast. The journal of pathology,
Vol.237
(2),
pp. 179-189.
Maley, C.C.
Koelble, K.
Natrajan, R.
Aktipis, A.
Yuan, Y.
(2015). An ecological measure of immune-cancer colocalization as a prognostic factor for breast cancer. Breast cancer research,
Vol.17
(1).
Ng, C.K.
Martelotto, L.G.
Gauthier, A.
Wen, H.-.
Piscuoglio, S.
Lim, R.S.
Cowell, C.F.
Wilkerson, P.M.
Wai, P.
Rodrigues, D.N.
Arnould, L.
Geyer, F.C.
Bromberg, S.E.
Lacroix-Triki, M.
Penault-Llorca, F.
Giard, S.
Sastre-Garau, X.
Natrajan, R.
Norton, L.
Cottu, P.H.
Weigelt, B.
Vincent-Salomon, A.
Reis-Filho, J.S.
(2015). Intra-tumor genetic heterogeneity and alternative driver genetic alterations in breast cancers with heterogeneous HER2 gene amplification. Genome biology,
Vol.16
(1).
Bajrami, I.
Frankum, J.R.
Konde, A.
Miller, R.E.
Rehman, F.L.
Brough, R.
Campbell, J.
Sims, D.
Rafiq, R.
Hooper, S.
Chen, L.
Kozarewa, I.
Assiotis, I.
Fenwick, K.
Natrajan, R.
Lord, C.J.
Ashworth, A.
(2014). Genome-wide profiling of genetic synthetic lethality identifies CDK12 as a novel determinant of PARP1/2 inhibitor sensitivity. Cancer res,
Vol.74
(1),
pp. 287-297.
show abstract
Small-molecule inhibitors of PARP1/2, such as olaparib, have been proposed to serve as a synthetic lethal therapy for cancers that harbor BRCA1 or BRCA2 mutations. Indeed, in clinical trials, PARP1/2 inhibitors elicit sustained antitumor responses in patients with germline BRCA gene mutations. In hypothesizing that additional genetic determinants might direct use of these drugs, we conducted a genome-wide synthetic lethal screen for candidate olaparib sensitivity genes. In support of this hypothesis, the set of identified genes included known determinants of olaparib sensitivity, such as BRCA1, RAD51, and Fanconi's anemia susceptibility genes. In addition, the set included genes implicated in established networks of DNA repair, DNA cohesion, and chromatin remodeling, none of which were known previously to confer sensitivity to PARP1/2 inhibition. Notably, integration of the list of candidate sensitivity genes with data from tumor DNA sequencing studies identified CDK12 deficiency as a clinically relevant biomarker of PARP1/2 inhibitor sensitivity. In models of high-grade serous ovarian cancer (HGS-OVCa), CDK12 attenuation was sufficient to confer sensitivity to PARP1/2 inhibition, suppression of DNA repair via homologous recombination, and reduced expression of BRCA1. As one of only nine genes known to be significantly mutated in HGS-OVCa, CDK12 has properties that should confirm interest in its use as a biomarker, particularly in ongoing clinical trials of PARP1/2 inhibitors and other agents that trigger replication fork arrest..
Natrajan, R.
Wilkerson, P.M.
Marchiò, C.
Piscuoglio, S.
Ng, C.K.
Wai, P.
Lambros, M.B.
Samartzis, E.P.
Dedes, K.J.
Frankum, J.
Bajrami, I.
Kopec, A.
Mackay, A.
A'hern, R.
Fenwick, K.
Kozarewa, I.
Hakas, J.
Mitsopoulos, C.
Hardisson, D.
Lord, C.J.
Kumar-Sinha, C.
Ashworth, A.
Weigelt, B.
Sapino, A.
Chinnaiyan, A.M.
Maher, C.A.
Reis-Filho, J.S.
(2014). Characterization of the genomic features and expressed fusion genes in micropapillary carcinomas of the breast. J pathol,
Vol.232
(5),
pp. 553-565.
show abstract
Micropapillary carcinoma (MPC) is a rare histological special type of breast cancer, characterized by an aggressive clinical behaviour and a pattern of copy number aberrations (CNAs) distinct from that of grade- and oestrogen receptor (ER)-matched invasive carcinomas of no special type (IC-NSTs). The aims of this study were to determine whether MPCs are underpinned by a recurrent fusion gene(s) or mutations in 273 genes recurrently mutated in breast cancer. Sixteen MPCs were subjected to microarray-based comparative genomic hybridization (aCGH) analysis and Sequenom OncoCarta mutation analysis. Eight and five MPCs were subjected to targeted capture and RNA sequencing, respectively. aCGH analysis confirmed our previous observations about the repertoire of CNAs of MPCs. Sequencing analysis revealed a spectrum of mutations similar to those of luminal B IC-NSTs, and recurrent mutations affecting mitogen-activated protein kinase family genes and NBPF10. RNA-sequencing analysis identified 17 high-confidence fusion genes, eight of which were validated and two of which were in-frame. No recurrent fusions were identified in an independent series of MPCs and IC-NSTs. Forced expression of in-frame fusion genes (SLC2A1-FAF1 and BCAS4-AURKA) resulted in increased viability of breast cancer cells. In addition, genomic disruption of CDK12 caused by out-of-frame rearrangements was found in one MPC and in 13% of HER2-positive breast cancers, identified through a re-analysis of publicly available massively parallel sequencing data. In vitro analyses revealed that CDK12 gene disruption results in sensitivity to PARP inhibition, and forced expression of wild-type CDK12 in a CDK12-null cell line model resulted in relative resistance to PARP inhibition. Our findings demonstrate that MPCs are neither defined by highly recurrent mutations in the 273 genes tested, nor underpinned by a recurrent fusion gene. Although seemingly private genetic events, some of the fusion transcripts found in MPCs may play a role in maintenance of a malignant phenotype and potentially offer therapeutic opportunities..
Garcia-Murillas, I.
Sharpe, R.
Pearson, A.
Campbell, J.
Natrajan, R.
Ashworth, A.
Turner, N.C.
(2014). An siRNA screen identifies the GNAS locus as a driver in 20q amplified breast cancer. Oncogene,
Vol.33
(19),
pp. 2478-2486.
show abstract
Poor-prognosis oestrogen receptor-positive breast cancer is characterised by the presence of high-level focal amplifications. We utilised a focused small interfering RNA screen in 14 breast cancer cell lines to define genes that were pathogenic in three genomic regions focally amplified in oestrogen receptor-positive breast cancer, 8p11-12, 11q13 and 20q. Silencing the GNAS locus, that encodes the G protein alpha stimulatory subunit Gαs, specifically reduced the growth of 20q amplified breast cancer cell lines. Examination of a publically available small hairpin RNA data set confirmed GNAS silencing to be selective for 20q amplified cancer cell lines. Cell lines with 20q amplification were found to overexpress specifically the extra long Gαs splice variant (XLαs). Overexpression of XLαs induced cAMP levels to a greater extent than Gαs, suggesting that amplification of the GNAS locus, and overexpression of the XLαs variant in particular, enhanced cAMP signalling. GNAS silencing in amplified cell lines reduced extracellular-signal-regulated kinase 1/2 (ERK1/2) phosphorylation, and conversely overexpression of exogenous XLαs in a non-amplified cell line increased MEK-ERK1/2 phosphorylation, identifying one potential downstream consequence of enhanced cAMP signalling. Our data indicate that amplification of the GNAS locus may contribute to the pathogenesis of breast cancer, and highlight a previously unrecognised role for the GNAS XLαs variant in cancer. .
Dryden, N.H.
Broome, L.R.
Dudbridge, F.
Johnson, N.
Orr, N.
Schoenfelder, S.
Nagano, T.
Andrews, S.
Wingett, S.
Kozarewa, I.
Assiotis, I.
Fenwick, K.
Maguire, S.L.
Campbell, J.
Natrajan, R.
Lambros, M.
Perrakis, E.
Ashworth, A.
Fraser, P.
Fletcher, O.
(2014). Unbiased analysis of potential targets of breast cancer susceptibility loci by Capture Hi-C. Genome res,
Vol.24
(11),
pp. 1854-1868.
show abstract
Genome-wide association studies have identified more than 70 common variants that are associated with breast cancer risk. Most of these variants map to non-protein-coding regions and several map to gene deserts, regions of several hundred kilobases lacking protein-coding genes. We hypothesized that gene deserts harbor long-range regulatory elements that can physically interact with target genes to influence their expression. To test this, we developed Capture Hi-C (CHi-C), which, by incorporating a sequence capture step into a Hi-C protocol, allows high-resolution analysis of targeted regions of the genome. We used CHi-C to investigate long-range interactions at three breast cancer gene deserts mapping to 2q35, 8q24.21, and 9q31.2. We identified interaction peaks between putative regulatory elements ("bait fragments") within the captured regions and "targets" that included both protein-coding genes and long noncoding (lnc) RNAs over distances of 6.6 kb to 2.6 Mb. Target protein-coding genes were IGFBP5, KLF4, NSMCE2, and MYC; and target lncRNAs included DIRC3, PVT1, and CCDC26. For one gene desert, we were able to define two SNPs (rs12613955 and rs4442975) that were highly correlated with the published risk variant and that mapped within the bait end of an interaction peak. In vivo ChIP-qPCR data show that one of these, rs4442975, affects the binding of FOXA1 and implicate this SNP as a putative functional variant. .
Piscuoglio, S.
Ng, C.K.
Martelotto, L.G.
Eberle, C.A.
Cowell, C.F.
Natrajan, R.
Bidard, F.-.
De Mattos-Arruda, L.
Wilkerson, P.M.
Mariani, O.
Vincent-Salomon, A.
Weigelt, B.
Reis-Filho, J.S.
(2014). Integrative genomic and transcriptomic characterization of papillary carcinomas of the breast. Molecular oncology,
Vol.8
(8),
pp. 1588-1602.
Shiu, K.-.
Wetterskog, D.
Mackay, A.
Natrajan, R.
Lambros, M.
Sims, D.
Bajrami, I.
Brough, R.
Frankum, J.
Sharpe, R.
Marchio, C.
Horlings, H.
Reyal, F.
van der Vijver, M.
Turner, N.
Reis-Filho, J.S.
Lord, C.J.
Ashworth, A.
(2014). Integrative molecular and functional profiling of ERBB2-amplified breast cancers identifies new genetic dependencies. Oncogene,
Vol.33
(5),
pp. 619-631.
show abstract
Overexpression of the receptor tyrosine kinase ERBB2 (also known as HER2) occurs in around 15% of breast cancers and is driven by amplification of the ERBB2 gene. ERBB2 amplification is a marker of poor prognosis, and although anti-ERBB2-targeted therapies have shown significant clinical benefit, de novo and acquired resistance remains an important problem. Genomic profiling has demonstrated that ERBB2+ve breast cancers are distinguished from ER+ve and 'triple-negative' breast cancers by harbouring not only the ERBB2 amplification on 17q12, but also a number of co-amplified genes on 17q12 and amplification events on other chromosomes. Some of these genes may have important roles in influencing clinical outcome, and could represent genetic dependencies in ERBB2+ve cancers and therefore potential therapeutic targets. Here, we describe an integrated genomic, gene expression and functional analysis to determine whether the genes present within amplicons are critical for the survival of ERBB2+ve breast tumour cells. We show that only a fraction of the ERBB2-amplified breast tumour lines are truly addicted to the ERBB2 oncogene at the mRNA level and display a heterogeneous set of additional genetic dependencies. These include an addiction to the transcription factor gene TFAP2C when it is amplified and overexpressed, suggesting that TFAP2C represents a genetic dependency in some ERBB2+ve breast cancer cells. .
Wetterskog, D.
Wilkerson, P.M.
Rodrigues, D.N.
Lambros, M.B.
Fritchie, K.
Andersson, M.K.
Natrajan, R.
Gauthier, A.
Di Palma, S.
Shousha, S.
Gatalica, Z.
Toepfer, C.
Vukovic, V.
A'Hern, R.
Weigelt, B.
Vincent-Salomon, A.
Stenman, G.
Rubin, B.P.
Reis-Filho, J.S.
(2013). Mutation profiling of adenoid cystic carcinomas from multiple anatomical sites identifies mutations in the RAS pathway, but no KIT mutations. Histopathology,
Vol.62
(4),
pp. 543-550.
Eccles, S.A.
Aboagye, E.O.
Ali, S.
Anderson, A.S.
Armes, J.
Berditchevski, F.
Blaydes, J.P.
Brennan, K.
Brown, N.J.
Bryant, H.E.
Bundred, N.J.
Burchell, J.M.
Campbell, A.M.
Carroll, J.S.
Clarke, R.B.
Coles, C.E.
Cook, G.J.
Cox, A.
Curtin, N.J.
Dekker, L.V.
dos Santos Silva, I.
Duffy, S.W.
Easton, D.F.
Eccles, D.M.
Edwards, D.R.
Edwards, J.
Evans, D.G.
Fenlon, D.F.
Flanagan, J.M.
Foster, C.
Gallagher, W.M.
Garcia-Closas, M.
Gee, J.M.
Gescher, A.J.
Goh, V.
Groves, A.M.
Harvey, A.J.
Harvie, M.
Hennessy, B.T.
Hiscox, S.
Holen, I.
Howell, S.J.
Howell, A.
Hubbard, G.
Hulbert-Williams, N.
Hunter, M.S.
Jasani, B.
Jones, L.J.
Key, T.J.
Kirwan, C.C.
Kong, A.
Kunkler, I.H.
Langdon, S.P.
Leach, M.O.
Mann, D.J.
Marshall, J.F.
Martin, L.A.
Martin, S.G.
Macdougall, J.E.
Miles, D.W.
Miller, W.R.
Morris, J.R.
Moss, S.M.
Mullan, P.
Natrajan, R.
O’Connor, J.P.
O’Connor, R.
Palmieri, C.
Pharoah, P.D.
Rakha, E.A.
Reed, E.
Robinson, S.P.
Sahai, E.
Saxton, J.M.
Schmid, P.
Smalley, M.J.
Speirs, V.
Stein, R.
Stingl, J.
Streuli, C.H.
Tutt, A.N.
Velikova, G.
Walker, R.A.
Watson, C.J.
Williams, K.J.
Young, L.S.
Thompson, A.M.
(2013). Critical research gaps and translational priorities for the successful prevention and treatment of breast cancer. Breast cancer research,
Vol.15
(5).
Horlings, H.M.
Weigelt, B.
Anderson, E.M.
Lambros, M.B.
Mackay, A.
Natrajan, R.
Ng, C.K.
Geyer, F.C.
van de Vijver, M.J.
Reis-Filho, J.S.
(2013). Genomic profiling of histological special types of breast cancer. Breast cancer research and treatment,
Vol.142
(2),
pp. 257-269.
Natrajan, R.C.
Leonidou, A.
Brough, R.
Frankum, J.
Wai, P.T.
Ng, C.K.
Reis-Filho, J.S.
Lord, C.J.
Ashworth, A.
(2013). Abstract S4-02: Integrated genomic analyses of members of protein kinase C family identifies subtype specific alterations as novel therapeutic targets. General session abstracts,
.
Natrajan, R.
Wilkerson, P.
(2013). From Integrative Genomics to Therapeutic Targets. Cancer research,
Vol.73
(12),
pp. 3483-3488.
Piscuoglio, S.
Ng, C.K.
Martelotto, L.G.
Cowell, C.F.
Natrajan, R.
Bidard, F.-.
Wilkerson, P.M.
Mariani, O.
Vincent-Salomon, A.
Weigelt, B.
Reis-Filho, J.S.
(2013). Abstract P4-04-08: Genomic and transcriptomic characterization of papillary carcinomas of the breast. Poster session abstracts,
.
Patani, N.
Lambros, M.B.
Natrajan, R.
Dedes, K.J.
Geyer, F.C.
Ward, E.
Martin, L.-.
Dowsett, M.
Reis-Filho, J.S.
(2012). Non-existence of caveolin-1 gene mutations in human breast cancer. Breast cancer res treat,
Vol.131
(1),
pp. 307-310.
show abstract
Caveolin-1 is the principal constituent protein of caveolae, which are specialised plasma membrane invaginations with diverse biological roles. Caveolin-1 is suggested to have tumour suppressive functions and CAV1 gene mutations have been reported in 20% of breast cancers. The aim of the present study was to evaluate the frequency of CAV1 mutations in a large cohort of optimally accrued breast cancers. Two independent series of breast cancer samples were analysed: 82 fresh-frozen grade 3 and 158 formalin-fixed paraffin-embedded invasive ductal carcinomas of no special type were consecutively accrued and subjected to microdissection of neoplastic epithelial cells prior to DNA extraction. Thirty-nine human breast cancer cell lines were also included in this study. The trans-membrane region of CAV1 and adjacent sequences, where mutations are reported to cluster, were amplified by PCR, followed by direct sequencing and mutational analysis. None of the reported CAV1 gene mutations, including CAV1 (P132L), were identified in either clinical samples (95% CI: 0-1.5%) or human breast cancer cell lines analysed. One novel non-synonymous germline polymorphism was detected within a reported region of high mutational frequency. This study does not corroborate the reported frequent occurrence of CAV1 gene mutations, including CAV1 (P132L), in primary human breast carcinomas. Our findings demonstrate that if CAV1 mutations do exist, their overall mutational frequency is substantially lower than positive reports have suggested. Taken together with other studies, which have also failed to identify CAV1 mutations, our data call into question the existence and biological and clinical relevance of CAV1 gene mutations in human breast cancer..
Wetterskog, D.
Angeles Lopez-Garcia, M.
Lambros, M.B.
A'Hern, R.
Geyer, F.C.
Milanezi, F.
Cabral, M.C.
Natrajan, R.
Gauthier, A.
Shiu, K.-.
Orr, N.
Shousha, S.
Gatalica, Z.
Mackay, A.
Palacios, J.
S Reis-Filho, J.
Weigelt, B.
(2012). Adenoid cystic carcinomas constitute a genomically distinct subgroup of triple-negative and basal-like breast cancers. Journal of pathology,
Vol.226
(1),
pp. 84-96.
Duprez, R.
Wilkerson, P.M.
Lacroix-Triki, M.
Lambros, M.B.
MacKay, A.
A'Hern, R.
Gauthier, A.
Pawar, V.
Colombo, P.-.
Daley, F.
Natrajan, R.
Ward, E.
MacGrogan, G.
Arbion, F.
Michenet, P.
Weigelt, B.
Vincent-Salomon, A.
Reis-Filho, J.S.
(2012). Immunophenotypic and genomic characterization of papillary carcinomas of the breast. J pathol,
Vol.226
(3),
pp. 427-441.
show abstract
Papillary carcinomas are a special histological type of breast cancer and have a relatively good outcome. We characterized the genomic and phenotypic characteristics of papillary carcinomas to determine whether they would constitute an entity distinct from grade- and oestrogen receptor (ER)-matched invasive ductal carcinomas of no special type (IDC-NSTs). The phenotype of 63 papillary carcinomas of the breast and grade- and ER-matched IDC-NSTs was determined by immunohistochemistry. DNA of sufficient quality was extracted from 49 microdissected papillary carcinomas and 49 microdissected grade- and ER-matched IDC-NSTs. These samples were subjected to high-resolution microarray-based comparative genomic hybridization (aCGH) and Sequenom MassARRAY sequencing analysis of 19 known oncogenes. Papillary carcinomas were predominantly of low histological grade, expressed immunohistochemical markers consistent with a luminal phenotype, and a lower rate of lymph node metastasis and p53 expression than grade- and ER-matched IDC-NSTs. Papillary carcinomas displayed less genomic aberrations than grade- and ER-matched IDC-NSTs; however, the patterns of gene copy number aberrations found in papillary carcinomas were similar to those of ER- and grade-matched IDC-NSTs, including 16q losses. Furthermore, PIK3CA mutations were found in 43% and 29% of papillary carcinomas and grade- and ER-matched IDC-NSTs, respectively. The genomic profiles of encapsulated, solid and invasive papillary carcinomas, the three morphological subtypes, were remarkably similar. Our results demonstrate that papillary carcinomas are a homogeneous special histological type of breast cancer. The similarities in the genomic profiles of papillary carcinomas and grade- and ER-matched IDC-NSTs suggest that papillary carcinomas may be best positioned as part of the spectrum of ER-positive breast cancers, rather than as a distinct entity. Furthermore, the good prognosis of papillary carcinomas may stem from the low rates of lymph node metastasis and p53 expression, low number of gene copy number aberrations and high prevalence of PIK3CA mutations..
Geyer, F.C.
de Biase, D.
Lambros, M.B.
Ragazzi, M.
Lopez-Garcia, M.A.
Natrajan, R.
Mackay, A.
Kurelac, I.
Gasparre, G.
Ashworth, A.
Eusebi, V.
Reis-Filho, J.S.
Tallini, G.
(2012). Genomic profiling of mitochondrion-rich breast carcinoma: chromosomal changes may be relevant for mitochondria accumulation and tumour biology. Breast cancer res treat,
Vol.132
(1),
pp. 15-28.
show abstract
Oncocytic carcinomas are composed of mitochondrion-rich cells. Though recognised by the WHO classification as a histological special type of breast cancer, their status as a discrete pathological entity remains a matter of contention. Given that oncocytic tumours of other anatomical sites display distinct clinico-pathological and molecular features, we sought to define the molecular genetic features of mitochondrion-rich breast tumours and to compare them with a series of histological grade- and oestrogen receptor status-matched invasive ductal carcinomas of no special type. Seventeen mitochondrion-rich breast carcinomas, including nine bona fide oncocytic carcinomas, were profiled with antibodies against oestrogen, progesterone and androgen receptors, HER2, Ki67, GCDFP-15, chromogranin, epithelial membrane antigen, cytokeratin 7, cytokeratin 14, CD68 and mitochondria antigen. These tumours were microdissected and DNA extracted from samples with >70% of tumour cells. Fourteen cases yielded DNA of sufficient quality/quantity and were subjected to high-resolution microarray comparative genomic hybridisation analysis. The genomic profiles were compared to those of 28 grade- and oestrogen receptor status-matched invasive ductal carcinomas of no special type. Oncocytic and other mitochondrion-rich tumours did not differ significantly between themselves. As a group, mitochondrion-rich carcinomas were immunophenotypically heterogenous. Recurrent copy number changes were similar to those described in unselected breast cancers. However, unsupervised and supervised analysis identified a subset of mitochondrion-rich cancers, which often displayed gains of 11q13.1-q13.2 and 19p13. Changes in the latter two chromosomal regions have been shown to be associated with oncocytic tumours of the kidney and thyroid, respectively, and host several nuclear genes with specific mitochondrial function. Our results indicate that in a way akin to oncocytic tumours of other anatomical sites, at least a subset of mitochondrion-rich breast carcinomas may be underpinned by a distinct pattern of chromosomal changes potentially relevant for mitochondria accumulation and constitute a discrete molecular entity..
Hernandez, L.
Wilkerson, P.M.
Lambros, M.B.
Campion-Flora, A.
Rodrigues, D.N.
Gauthier, A.
Cabral, C.
Pawar, V.
Mackay, A.
A'Hern, R.
Marchiò, C.
Palacios, J.
Natrajan, R.
Weigelt, B.
Reis-Filho, J.S.
(2012). Genomic and mutational profiling of ductal carcinomas in situ and matched adjacent invasive breast cancers reveals intra-tumour genetic heterogeneity and clonal selection. J pathol,
Vol.227
(1),
pp. 42-52.
show abstract
The mechanisms underlying the progression from ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC) of the breast are yet to be fully elucidated. Several hypotheses have been put forward to explain the progression from DCIS to IDC, including the selection of a subpopulation of cancer cells with specific genetic aberrations, and the acquisition of new genetic aberrations or non-genetic mechanisms mediated by the tumour microenvironment. To determine whether synchronously diagnosed ipsilateral DCI and IDCs have modal populations with distinct repertoires of gene copy number aberrations and mutations in common oncogenes, matched frozen samples of DCIS and IDC were retrieved from 13 patients and subjected to microarray-based comparative genomic hybridization (aCGH) and Sequenom MassARRAY (Oncocarta v 1.0 panel). Fluorescence in situ hybridization and Sanger sequencing were employed to validate the aCGH and Sequenom findings, respectively. Although the genomic profiles of matched DCI and IDCs were similar, in three of 13 matched pairs amplification of distinct loci (ie 1q41, 2q24.2, 6q22.31, 7q11.21, 8q21.2 and 9p13.3) was either restricted to, or more prevalent in, the modal population of cancer cells of one of the components. Sequenom MassARRAY identified PIK3CA mutations restricted to the DCIS component in two cases, and in a third case the frequency of the PIK3CA mutant allele reduced from 49% in the DCIS to 25% in the IDC component. Despite the genomic similarities between synchronous DCIS and IDC, our data provide strong circumstantial evidence to suggest that in some cases the progression from DCIS to IDC is driven by the selection of non-modal clones that harbour a specific repertoire of genetic aberrations..
Natrajan, R.
Mackay, A.
Lambros, M.B.
Weigelt, B.
Wilkerson, P.M.
Manie, E.
Grigoriadis, A.
A'Hern, R.
van der Groep, P.
Kozarewa, I.
Popova, T.
Mariani, O.
Turaljic, S.
Furney, S.J.
Marais, R.
Rodruigues, D.-.
Flora, A.C.
Wai, P.
Pawar, V.
McDade, S.
Carroll, J.
Stoppa-Lyonnet, D.
Green, A.R.
Ellis, I.O.
Swanton, C.
van Diest, P.
Delattre, O.
Lord, C.J.
Foulkes, W.D.
Vincent-Salomon, A.
Ashworth, A.
Stern, M.H.
Reis-Filho, J.S.
(2012). A whole-genome massively parallel sequencing analysis of BRCA1 mutant oestrogen receptor-negative and -positive breast cancers. J pathol,
Vol.227
(1),
pp. 29-41.
show abstract
BRCA1 encodes a tumour suppressor protein that plays pivotal roles in homologous recombination (HR) DNA repair, cell-cycle checkpoints, and transcriptional regulation. BRCA1 germline mutations confer a high risk of early-onset breast and ovarian cancer. In more than 80% of cases, tumours arising in BRCA1 germline mutation carriers are oestrogen receptor (ER)-negative; however, up to 15% are ER-positive. It has been suggested that BRCA1 ER-positive breast cancers constitute sporadic cancers arising in the context of a BRCA1 germline mutation rather than being causally related to BRCA1 loss-of-function. Whole-genome massively parallel sequencing of ER-positive and ER-negative BRCA1 breast cancers, and their respective germline DNAs, was used to characterize the genetic landscape of BRCA1 cancers at base-pair resolution. Only BRCA1 germline mutations, somatic loss of the wild-type allele, and TP53 somatic mutations were recurrently found in the index cases. BRCA1 breast cancers displayed a mutational signature consistent with that caused by lack of HR DNA repair in both ER-positive and ER-negative cases. Sequencing analysis of independent cohorts of hereditary BRCA1 and sporadic non-BRCA1 breast cancers for the presence of recurrent pathogenic mutations and/or homozygous deletions found in the index cases revealed that DAPK3, TMEM135, KIAA1797, PDE4D, and GATA4 are potential additional drivers of breast cancers. This study demonstrates that BRCA1 pathogenic germline mutations coupled with somatic loss of the wild-type allele are not sufficient for hereditary breast cancers to display an ER-negative phenotype, and has led to the identification of three potential novel breast cancer genes (ie DAPK3, TMEM135, and GATA4)..
Geyer, F.C.
Lacroix-Triki, M.
Colombo, P.-.
Patani, N.
Gauthier, A.
Natrajan, R.
Lambros, M.B.
Khalifeh, I.
Albarracin, C.
Orru, S.
Marchio, C.
Sapino, A.
Mackay, A.
Weigelt, B.
Schmitt, F.C.
Wesseling, J.
Sneige, N.
Reis-Filho, J.S.
(2012). Molecular evidence in support of the neoplastic and precursor nature of microglandular adenosis. Histopathology,
Vol.60
(6B),
pp. E115-E130.
Brough, R.
Bajrami, I.
Vatcheva, R.
Natrajan, R.
Reis-Filho, J.S.
Lord, C.J.
Ashworth, A.
(2012). APRIN is a cell cycle specific BRCA2-interacting protein required for genome integrity and a predictor of outcome after chemotherapy in breast cancer. Embo journal,
Vol.31
(5),
pp. 1160-1176.
Daemen, A.
Wolf, D.M.
Korkola, J.E.
Griffith, O.L.
Frankum, J.R.
Brough, R.
Jakkula, L.R.
Wang, N.J.
Natrajan, R.
Reis-Filho, J.S.
Lord, C.J.
Ashworth, A.
Spellman, P.T.
Gray, J.W.
van't Veer, L.J.
(2012). Cross-platform pathway-based analysis identifies markers of response to the PARP inhibitor olaparib. Breast cancer research and treatment,
Vol.135
(2),
pp. 505-517.
Grigoriadis, A.
Mackay, A.
Noel, E.
Wu, P.J.
Natrajan, R.
Frankum, J.
Reis-Filho, J.S.
Tutt, A.
(2012). Molecular characterisation of cell line models for triple-negative breast cancers. Bmc genomics,
Vol.13.
Mackay, A.G.
Natrajan, R.
Maher, C.
Lambros, M.
Weigelt, B.
Nava-Rodrigues, D.
Wilkerson, P.
CampionFlora, A.
Kozarewa, I.
Fenwick, K.
Campbell, J.
Chen, L.
Lord, C.J.
Gauthier, A.
Pawar, V.
Mariani, O.
Vincent-Salomon, A.
Reis-Filho, J.
(2012). Abstract 5076: Integrative genomic and transcriptomic analysis of metaplastic carcinomas of the breast. Molecular and cellular biology,
.
Ng, C.K.
Gauthier, A.
Mackay, A.
Lambros, M.B.
Rodrigues, D.N.
Arnoud, L.
Lacroix-Triki, M.
Penault-Llorca, F.
Baranzelli, M.C.
Sastre-Garau, X.
Lord, C.J.
Zvelebil, M.
Mitsopoulos, C.
Ashworth, A.
Natrajan, R.
Weigelt, B.
Delattre, O.
Cottu, P.
Reis-Filho, J.S.
Vincent-Salomon, A.
(2012). Abstract PD05-08: Genomic characterisation of invasive breast cancers with heterogeneous HER2 gene amplification. Poster discussion abstracts,
.
Gauthier, A.
Lambros, M.
Rodrigues, D.N.
Arnould, L.
Lacroix-Tikri, M.
Penault-Llorca, F.
Baranzelli, M.-.
Sastre, X.
Natrajan, R.
Delattre, O.
Cottu, P.
Reis-Filho, J.S.
Vincent-Salomon, A.
(2012). Abstract 8: Genomic analyses of heterogeneous HER2 3+ invasive ductal carcinomas of the breast. Molecular and cellular biology,
.
Natrajan, R.
Mackay, A.
Wilkerson, P.M.
Lambros, M.B.
Wetterskog, D.
Arnedos, M.
Shiu, K.-.
Geyer, F.C.
Langerød, A.
Kreike, B.
Reyal, F.
Horlings, H.M.
van de Vijver, M.J.
Palacios, J.
Weigelt, B.
Reis-Filho, J.S.
(2012). Functional characterization of the 19q12 amplicon in grade III breast cancers. Breast cancer res,
Vol.14
(2),
p. R53.
show abstract
INTRODUCTION: The 19q12 locus is amplified in a subgroup of oestrogen receptor (ER)-negative grade III breast cancers. This amplicon comprises nine genes, including cyclin E1 (CCNE1), which has been proposed as its 'driver'. The aim of this study was to identify the genes within the 19q12 amplicon whose expression is required for the survival of cancer cells harbouring their amplification. METHODS: We investigated the presence of 19q12 amplification in a series of 313 frozen primary breast cancers and 56 breast cancer cell lines using microarray comparative genomic hybridisation (aCGH). The nine genes mapping to the smallest region of amplification on 19q12 were silenced using RNA interference in phenotypically matched breast cancer cell lines with (MDA-MB-157 and HCC1569) and without (Hs578T, MCF7, MDA-MB-231, ZR75.1, JIMT1 and BT474) amplification of this locus. Genes whose silencing was selectively lethal in amplified cells were taken forward for further validation. The effects of cyclin-dependent kinase 2 (CDK2) silencing and chemical inhibition were tested in cancer cells with and without CCNE1 amplification. RESULTS: 19q12 amplification was identified in 7.8% of ER-negative grade III breast cancer. Of the nine genes mapping to this amplicon, UQCRFS1, POP4, PLEKHF1, C19ORF12, CCNE1 and C19ORF2 were significantly over-expressed when amplified in primary breast cancers and/or breast cancer cell lines. Silencing of POP4, PLEKHF1, CCNE1 and TSZH3 selectively reduced cell viability in cancer cells harbouring their amplification. Cancer cells with CCNE1 amplification were shown to be dependent on CDK2 expression and kinase activity for their survival. CONCLUSIONS: The 19q12 amplicon may harbour more than a single 'driver', given that expression of POP4, PLEKHF1, CCNE1 and TSZH3 is required for the survival of cancer cells displaying their amplification. The observation that cancer cells harbouring CCNE1 gene amplification are sensitive to CDK2 inhibitors provides a rationale for the testing of these chemical inhibitors in a subgroup of patients with ER-negative grade III breast cancers..
Dedes, K.J.
Natrajan, R.
Lambros, M.B.
Geyer, F.C.
Lopez-Garcia, M.A.
Savage, K.
Jones, R.L.
Reis-Filho, J.S.
(2011). Down-regulation of the miRNA master regulators Drosha and Dicer is associated with specific subgroups of breast cancer. Eur j cancer,
Vol.47
(1),
pp. 138-150.
show abstract
Down-regulation of Drosha and Dicer has been suggested to be of prognostic value in some cancers. The aims of our study were to investigate the down-regulation of Drosha and Dicer in breast cancers and its associations with clinicopathological features, molecular subtypes and outcome. Drosha and Dicer expression was assessed with real-time RT-PCR in 245 patients with breast cancer receiving adjuvant anthracycline-based chemotherapy and compared to expression levels of normal breast tissue. Drosha down-regulation was observed in 18% of cases and was associated with high grade, high Ki-67, lack of Bcl2 expression, HER2 over-expression and gene amplification and TOPO2A gene amplification. Dicer down-regulation was found in 46% of cases and was associated with lack of expression of ER, PR and Bcl2 and with high grade, high Ki-67, triple-negative and basal-like phenotypes. Drosha and Dicer were concurrently down-regulated in 15% of cases and significantly associated with high grade and high Ki-67 index. No significant associations between down-regulation of Drosha and/or Dicer and outcome were observed. Our results suggest that down-regulation of Drosha and/or Dicer is not robustly associated with the outcome of breast cancer patients treated with adjuvant anthracycline-based chemotherapy but preferentially observed in distinct subgroups of breast cancer..
Lambros, M.B.
Wilkerson, P.M.
Natrajan, R.
Patani, N.
Pawar, V.
Vatcheva, R.
Mansour, M.
Laschet, M.
Oelze, B.
Orr, N.
Muller, S.
Reis-Filho, J.S.
(2011). High-throughput detection of fusion genes in cancer using the Sequenom MassARRAY platform. Laboratory investigation,
Vol.91
(10),
pp. 1491-11.
Williams, R.D.
Al-Saadi, R.
Natrajan, R.
Mackay, A.
Chagtai, T.
Little, S.
Hing, S.N.
Fenwick, K.
Ashworth, A.
Grundy, P.
Anderson, J.R.
Dome, J.S.
Perlman, E.J.
Jones, C.
Pritchard-Jones, K.
(2011). Molecular Profiling Reveals Frequent Gain of MYCN and Anaplasia-Specific Loss of 4q and 14q in Wilms Tumor. Genes chromosomes & cancer,
Vol.50
(12),
pp. 982-14.
Geyer, F.C.
Lacroix-Triki, M.
Savage, K.
Arnedos, M.
Lambros, M.B.
MacKay, A.
Natrajan, R.
Reis-Filho, J.S.
(2011). β-Catenin pathway activation in breast cancer is associated with triple-negative phenotype but not with CTNNB1 mutation. Mod pathol,
Vol.24
(2),
pp. 209-231.
show abstract
Aberrant β-catenin expression as determined by assessment of its subcellular localization constitutes a surrogate marker of Wnt signalling pathway activation and has been reported in a subset of breast cancers. The association of β-catenin/Wnt pathway activation with clinical outcome and the mechanisms leading to its activation in breast cancers still remain a matter of controversy. The aims of this study were to address the distribution of β-catenin expression in invasive breast cancers, the correlations between β-catenin expression and clinicopathological features and survival of breast cancer patients, and to determine whether aberrant β-catenin expression is driven by CTNNB1 (β-catenin encoding gene) activating mutations. Immunohistochemistry was performed on a tissue microarray containing 245 invasive breast carcinomas from uniformly treated patients, using two anti-β-catenin monoclonal antibodies. Selected samples were subjected to CTNNB1 exon 3 mutation analysis by direct gene sequencing. A good correlation between the two β-catenin antibodies was observed (Spearman's r >0.62, P<0.001). Respectively, 31 and 11% of the cases displayed lack/reduction of β-catenin membranous expression and nuclear accumulation. Complete lack of β-catenin expression was significantly associated with invasive lobular carcinoma histological type. Subgroup analysis of non-lobular cancers or non-lobular grade 3 carcinomas revealed that lack/reduction of β-catenin membranous expression and/or nuclear accumulation were significantly associated with oestrogen receptor negativity, absence of HER2 gene amplification and overexpression, lack/reduction of E-cadherin expression and tumours of triple-negative and basal-like phenotype. Univariate survival analysis revealed a significant association between β-catenin nuclear expression and shorter metastasis-free and overall survival in the whole cohort; however, β-catenin nuclear expression was not an independent predictor of outcome in multivariate analysis. No CTNNB1 mutations were identified in the 28 selected breast carcinomas analysed. In conclusion, β-catenin/Wnt pathway activation is preferentially found in triple-negative/basal-like breast carcinomas, is associated with poor clinical outcome and is unlikely to be driven by CTNNB1 mutations in breast cancer..
Natrajan, R.
Reis-Filho, J.S.
(2011). Next-generation sequencing applied to molecular diagnostics. Expert rev mol diagn,
Vol.11
(4),
pp. 425-444.
show abstract
Next-generation sequencing technologies have begun to revolutionize the field of cancer genetics through rapid and accurate assessment of a patient's DNA makeup with minimal cost. These technologies have already led to the realization of the inter- and intra-tumor genetic heterogeneity and the identification of novel mutations and chimeric genes, however, several challenges lie ahead. Given the low number of recurrent somatic genetic aberrations in common types of cancer, the identification of 'driver' genetic aberrations has proven challenging. Furthermore, implementation of next-generation sequencing and/or some of its derivatives into routine practice as diagnostic tests will require in-depth understanding of the pitfalls of these technologies and a great degree of bioinformatic expertise. This article focuses on the contribution of next-generation sequencing technologies to diagnosis and cancer prognostication and prediction..
Brough, R.
Frankum, J.R.
Sims, D.
Mackay, A.
Mendes-Pereira, A.M.
Bajrami, I.
Costa-Cabral, S.
Rafiq, R.
Ahmad, A.S.
Cerone, M.A.
Natrajan, R.
Sharpe, R.
Shiu, K.-.
Wetterskog, D.
Dedes, K.J.
Lambros, M.B.
Rawjee, T.
Linardopoulos, S.
Reis-Filho, J.S.
Turner, N.C.
Lord, C.J.
Ashworth, A.
(2011). Functional viability profiles of breast cancer. Cancer discov,
Vol.1
(3),
pp. 260-273.
show abstract
UNLABELLED: The design of targeted therapeutic strategies for cancer has largely been driven by the identification of tumor-specific genetic changes. However, the large number of genetic alterations present in tumor cells means that it is difficult to discriminate between genes that are critical for maintaining the disease state and those that are merely coincidental. Even when critical genes can be identified, directly targeting these is often challenging, meaning that alternative strategies such as exploiting synthetic lethality may be beneficial. To address these issues, we have carried out a functional genetic screen in >30 commonly used models of breast cancer to identify genes critical to the growth of specific breast cancer subtypes. In particular, we describe potential new therapeutic targets for PTEN-mutated cancers and for estrogen receptor-positive breast cancers. We also show that large-scale functional profiling allows the classification of breast cancers into subgroups distinct from established subtypes. SIGNIFICANCE: Despite the wealth of molecular profiling data that describe breast tumors and breast tumor cell models, our understanding of the fundamental genetic dependencies in this disease is relatively poor. Using high-throughput RNA interference screening of a series of pharmacologically tractable genes, we have generated comprehensive functional viability profiles for a wide panel of commonly used breast tumor cell models. Analysis of these profiles identifies a series of novel genetic dependencies, including that of PTEN-null breast tumor cells upon mitotic checkpoint kinases, and provides a framework upon which additional dependencies and candidate therapeutic targets may be identified..
Hungermann, D.
Schmidt, H.
Natrajan, R.
Tidow, N.
Poos, K.
Reis-Filho, J.S.
Brandt, B.
Buerger, H.
Korsching, E.
(2011). Influence of whole arm loss of chromosome 16q on gene expression patterns in oestrogen receptor-positive, invasive breast cancer. The journal of pathology,
Vol.224
(4),
pp. 517-528.
Wilkerson, P.M.
Dedes, K.J.
Wetterskog, D.
Mackay, A.
Lambros, M.B.
Mansour, M.
Frankum, J.
Lord, C.J.
Natrajan, R.
Ashworth, A.
Reis-Filho, J.S.
(2011). Functional characterization of EMSY gene amplification in human cancers. J pathol,
Vol.225
(1),
pp. 29-42.
show abstract
The 11q13-q14 locus is frequently amplified in human cancers, with a complex structure harbouring multiple potential oncogenic drivers. The EMSY gene has been proposed as a driver of the third core of the 11q13-q14 amplicon. This gene encodes a protein reported to be a BRCA2-binding partner, which when over-expressed would lead to impairment of BRCA2 functions and could constitute a mechanism for BRCA2 inactivation in non-hereditary breast and ovarian cancers. We hypothesized that if EMSY amplification abrogates BRCA2 functions, cells harbouring this aberration would be unable to elicit competent homologous recombination DNA repair and, therefore, may have increased sensitivity to genotoxic therapies and potent PARP inhibitors. Microarray-based comparative genomic hybridization of cell lines from distinct tumour sites, including breast, ovary, pancreas, oesophagus, lung and the oral cavity, led to the identification of 10 cell lines with EMSY amplification and 18 without. EMSY amplification was shown to correlate with EMSY mRNA levels, although not all cell lines harbouring EMSY amplification displayed EMSY mRNA or protein over-expression. RNA interference-mediated silencing of EMSY did not lead to a reduction in cell viability in tumour models harbouring EMSY amplification. Cell lines with and without EMSY amplification displayed a similar ability to elicit RAD51 foci in response to DNA damaging agents, and similar sensitivity to cisplatin and olaparib. Taken together, this suggests that EMSY is unlikely to be a driver of the 11q13-q14 amplicon and does not have a dominant role in modulating the response to agents targeting cells with defective homologous recombination..
Lambros, M.B.
Wilkerson, P.M.
Natrajan, R.
Patani, N.
Pawar, V.
Vatcheva, R.
Mansour, M.
Laschet, M.
Oelze, B.
Orr, N.
Muller, S.
Reis-Filho, J.S.
(2011). High-throughput detection of fusion genes in cancer using the Sequenom MassARRAY platform. Laboratory investigation; a journal of technical methods and pathology,
Vol.91
(10),
pp. 1491-1501.
show abstract
Fusion genes have pivotal roles in the development and progression of human cancer and offer potential for rational drug design. Massively parallel sequencing has identified a panoply of in-frame expressed fusion genes, but early reports suggest that the majority of these are present at very low prevalence or are private events. Conventional methods for the identification of recurrent expressed fusion genes in large cohorts of cancers (eg fluorescence in situ hybridization (FISH) and reverse transcriptase PCR (RT-PCR)) are time consuming and prone to artifacts. Here, we describe a novel high-throughput strategy for the detection of recurrent fusion genes in cancer based on the Sequenom MassARRAY platform. Fusion genes were initially identified by massively parallel sequencing of breast cancer cell lines. For each fusion gene, two Sequenom probes were designed. Primary human breast cancers and cancer cell lines were interrogated for 10 fusion genes. Sensitivity, specificity, and predictive values of the MassARRAY method were then determined using FISH and qRT-PCR as the 'gold standard.' By combining two probes per fusion gene, the negative and positive predictive values were 100 and 71.4%, respectively. All fusion genes identified by massively parallel sequencing were accurately detected. No recurrent fusion genes were found. The MassARRAY-based approach described here may, therefore, be employed as a high-throughput screening tool for known fusion genes in human cancer. In keeping with other highly sensitive assays, further refinement of this technique is necessary to reduce the number of false-positive results..
Robinson, D.R.
Kalyana-Sundaram, S.
Wu, Y.-.
Shankar, S.
Cao, X.
Ateeq, B.
Asangani, I.A.
Iyer, M.
Maher, C.A.
Grasso, C.S.
Lonigro, R.J.
Quist, M.
Siddiqui, J.
Mehra, R.
Jing, X.
Giordano, T.J.
Sabel, M.S.
Kleer, C.G.
Palanisamy, N.
Natrajan, R.
Lambros, M.B.
Reis-Filho, J.S.
Kumar-Sinha, C.
Chinnaiyan, A.M.
(2011). Functionally recurrent rearrangements of the MAST kinase and Notch gene families in breast cancer. Nature medicine,
Vol.17
(12),
pp. 1646-U163.
Tan, D.S.
Iravani, M.
McCluggage, W.G.
Lambros, M.B.
Milanezi, F.
Mackay, A.
Gourley, C.
Geyer, F.C.
Vatcheva, R.
Millar, J.
Thomas, K.
Natrajan, R.
Savage, K.
Fenwick, K.
Williams, A.
Jameson, C.
El-Bahrawy, M.
Gore, M.E.
Gabra, H.
Kaye, S.B.
Ashworth, A.
Reis-Filho, J.S.
(2011). Genomic analysis reveals the molecular heterogeneity of ovarian clear cell carcinomas. Clin cancer res,
Vol.17
(6),
pp. 1521-1534.
show abstract
PURPOSE: Ovarian clear cell carcinomas (OCCC) are a drug-resistant and aggressive type of epithelial ovarian cancer. We analyzed the molecular genetic profiles of OCCCs to determine whether distinct genomic subgroups of OCCCs exist. EXPERIMENTAL DESIGN: Fifty pure primary OCCCs were subjected to high-resolution microarray-based comparative genomic hybridization (aCGH). Unsupervised hierarchical clustering using Ward's linkage analysis was performed to identify genomic subgroups of OCCCs. Survival analysis was performed using Kaplan-Meier method and log-rank test. Cox-regression analysis was used to identify independent predictors of outcome. Differentially amplified regions between genomic subgroups of OCCCs were identified using a multi-Fisher's exact test. RESULTS: Hierarchical cluster analysis revealed two distinct clusters of OCCCs with different clinical outcomes. Patients from cluster-1 had a significantly shorter median progression-free survival (PFS) than those from cluster-2 (11 vs. 65 months, P = 0.009), although estimates for ovarian cancer-specific survival (OCS) did not reach statistical significance (P = 0.065). In multivariate analysis, suboptimal debulking surgery and genomic cluster were independently prognostic for PFS. Recurrently amplified genomic regions with a significantly higher prevalence in cluster-1 than cluster-2 OCCCs were identified and validated. HER2 gene amplification and protein overexpression was observed in 14% of OCCCs, suggesting that this may constitute a potential therapeutic target for a subgroup of these tumors. CONCLUSIONS: OCCCs constitute a heterogeneous disease at the genomic level despite having similar histological features. The pattern of genomic aberrations in subgroups of OCCCs is of clinical significance. We have identified recurrently amplified regions that may harbor potential therapeutic targets for subgroups of OCCCs..
Sharpe, R.
Pearson, A.
Herrera-Abreu, M.T.
Johnson, D.
Mackay, A.
Welti, J.C.
Natrajan, R.
Reynolds, A.R.
Reis-Filho, J.S.
Ashworth, A.
Turner, N.C.
(2011). FGFR signaling promotes the growth of triple-negative and basal-like breast cancer cell lines both in vitro and in vivo. Clin cancer res,
Vol.17
(16),
pp. 5275-5286.
show abstract
PURPOSE: The oncogenic drivers of triple-negative (TN) and basal-like breast cancers are largely unknown. Substantial evidence now links aberrant signaling by the fibroblast growth factor receptors (FGFR) to the development of multiple cancer types. Here, we examined the role of FGFR signaling in TN breast cancer. EXPERIMENTAL DESIGN: We examined the sensitivity of a panel of 31 breast cancer cell lines to the selective FGFR inhibitor PD173074 and investigated the potential mechanisms underlying sensitivity. RESULTS: TN breast cancer cell lines were more sensitive to PD173074 than comparator cell lines (P = 0.011), with 47% (7/15) of TN cell lines showing significantly reduced growth. The majority of TN cell lines showed only modest sensitivity to FGFR inhibition in two-dimensional growth but were highly sensitive in anchorage-independent conditions. PD173074 inhibited downstream mitogen-activated protein kinase and PI3K-AKT signaling and induced cell-cycle arrest and apoptosis. Basal-like breast cancer cell lines were found to express FGF2 ligand (11/21 positive) and, similarly, 62% of basal-like breast cancers expressed FGF2, as assessed by immunohistochemistry compared with 5% of nonbasal breast cancers (P < 0.0001). RNA interference targeting of FGF2 in basal-like cell lines significantly reduced growth in vitro and reduced down stream signaling, suggesting an autocrine FGF2 signaling loop. Treatment with PD173074 significantly reduced the growth of CAL51 basal-like breast cancer cell line xenografts in vivo. CONCLUSIONS: Basal-like breast cancer cell lines, and breast cancers, express autocrine FGF2 and show sensitivity to FGFR inhibitors, identifying a potential novel therapeutic approach for these cancers..
Daemen, A.
Wolf, D.M.
Korkola, J.E.
Griffith, O.L.
Frankum, J.R.
Jakkula, L.R.
Wang, N.J.
Natrajan, R.
Reis-Filho, J.S.
Lord, C.J.
Ashworth, A.
Gray, J.W.
Spellman, P.T.
van't Veer, L.
(2011). P1-06-22: Identification of Biomarkers in Breast Cancer for Prediction of Response to PARP Inhibitor Olaparib. Poster session abstracts,
.
Wilkerson, P.
Dedes, K.J.
Wetterskog, D.
Natrajan, R.
Lambros, M.B.
Mackay, A.
Lord, C.J.
Ashworth, A.
Reis-Filho, J.S.
(2011). Abstract 3917: EMSY amplification and overexpression is not associated with defective homologous recombination and does not predict sensitivity to cisplatin or PARP inhibitors. Cellular and molecular biology,
.
Natrajan, R.
Maher, C.A.
Lambros, M.B.
Pawar, V.
Wetterskog, D.
Marchio, C.
Barbashina, V.
Geyer, F.C.
Fenwick, K.
Kozarewa, I.
Mackay, A.
Hakas, J.
Mitsopoulos, K.
Hardisson, D.
Sapino, A.
Vincent-Salomon, A.
Lord, C.J.
Chinniayan, A.
Ashworth, A.
Reis-Filho, J.S.
(2011). Abstract 4829: Massively parallel RNA sequencing analysis of micropapillary carcinomas of the breast. Cellular and molecular biology,
.
Wetterskog, D.
Shiu, K.-.
Chong, I.
Meijer, T.
Natrajan, R.
Lord, C.J.
Ashworth, A.
Reis-Filho, J.S.
(2011). Abstract 4987: Identification of novel genes and pathways involved in resistance to HER2-targeting agents in breast cancer. Cellular and molecular biology,
.
Chong, I.
Wetterskog, D.
Shiu, K.-.
Meijer, T.
Natrajan, R.
Maryou, L.
Reis-Filho, J.
Lord, L.J.
David, C.
Ashworth, A.
(2011). Abstract 3039: Identification of a novel biomarker of resistance to lapatinib common to both breast and oesophagogastric cancer. Cellular and molecular biology,
.
Mackay, A.
Weigelt, B.
Grigoriadis, A.
Kreike, B.
Natrajan, R.
A'Hern, R.
Tan, D.S.
Dowsett, M.
Ashworth, A.
Reis-Filho, J.S.
(2011). Microarray-based class discovery for molecular classification of breast cancer: analysis of interobserver agreement. J natl cancer inst,
Vol.103
(8),
pp. 662-673.
show abstract
BACKGROUND: Breast cancers can be classified by hierarchical clustering using an "intrinsic" gene list into one of at least five molecular subtypes: basal-like, HER2, luminal A, luminal B, and normal breast-like. Five different intrinsic gene lists composed of varying numbers of genes have been used for molecular subtype identification and classification of breast cancers. The aim of this study was to determine the objectivity and interobserver reproducibility of the assignment of molecular subtype classes by hierarchical cluster analysis. METHODS: Three publicly available breast cancer datasets (n = 779) were subjected to two-way average-linkage hierarchical cluster analysis using five distinct intrinsic gene lists. We used free-marginal Kappa statistics to analyze interobserver agreement among five breast cancer researchers for the whole classification and for each molecular subtype separately according to each intrinsic gene list for each breast cancer dataset. RESULTS: None of the classification systems tested produced almost perfect agreement (Kappa ≥ 0.81) among observers. However, substantial interobserver agreement (70.8% to 76.1% of the samples and free-marginal Kappa scores from 0.635 to 0.701) was consistently observed in all datasets for four molecular subtypes (luminal, basal-like, HER2, and normal breast-like). When luminal cancers were subdivided (luminal A, B, and C), none of the classification systems produced substantial agreement (Kappa ≥ 0.61) in all the datasets analyzed. Analysis of each subtype separately revealed that only two (basal-like and HER2) could be reproducibly identified by independent observers (Kappa ≥ 0.81). CONCLUSIONS: Assignment of molecular subtype classes of breast cancer based on the analysis of dendrograms obtained with hierarchical cluster analysis is subjective and shows modest interobserver reproducibility. For the development of a molecular taxonomy, objective definitions for each molecular subtype and standardized methods for their identification are required..
Xu, H.
Yan, M.
Patra, J.
Natrajan, R.
Yan, Y.
Swagemakers, S.
Tomaszewski, J.M.
Verschoor, S.
Millar, E.K.
van der Spek, P.
Reis-Filho, J.S.
Ramsay, R.G.
O'Toole, S.A.
McNeil, C.M.
Sutherland, R.L.
McKay, M.J.
Fox, S.B.
(2011). Enhanced RAD21 cohesin expression confers poor prognosis and resistance to chemotherapy in high grade luminal, basal and HER2 breast cancers. Breast cancer res,
Vol.13
(1),
p. R9.
show abstract
INTRODUCTION: RAD21 is a component of the cohesin complex, which is essential for chromosome segregation and error-free DNA repair. We assessed its prognostic and predictive power in a cohort of in situ and invasive breast cancers, and its effect on chemosensitivity in vitro. METHODS: RAD21 immunohistochemistry was performed on 345 invasive and 60 pure in situ carcinomas. Integrated genomic and transcriptomic analyses were performed on a further 48 grade 3 invasive cancers. Chemosensitivity was assessed in breast cancer cell lines with an engineered spectrum of RAD21 expression. RESULTS: RAD21 expression correlated with early relapse in all patients (hazard ratio (HR) 1.74, 95% confidence interval (CI) 1.06 to 2.86, P = 0.029). This was due to the effect of grade 3 tumors (but not grade 1 or 2) in which RAD21 expression correlated with early relapse in luminal (P = 0.040), basal (P = 0.018) and HER2 (P = 0.039) groups. In patients treated with chemotherapy, RAD21 expression was associated with shorter overall survival (P = 0.020). RAD21 mRNA expression correlated with DNA copy number, with amplification present in 32% (7/22) of luminal, 31% (4/13) of basal and 22% (2/9) of HER2 grade 3 cancers. Variations in RAD21 mRNA expression in the clinical samples were reflected in the gene expression data from 36 breast cancer cell lines. Knockdown of RAD21 in the MDA-MB-231 breast cancer cell line significantly enhanced sensitivity to cyclophosphamide, 5-fluorouracil and etoposide. The findings for the former two drugs recapitulated the clinical findings. CONCLUSIONS: RAD21 expression confers poor prognosis and resistance to chemotherapy in high grade luminal, basal and HER2 breast cancers. RAD21 may be a novel therapeutic target..
Ha, K.C.
Lalonde, E.
Li, L.
Cavallone, L.
Natrajan, R.
Lambros, M.B.
Mitsopoulos, C.
Hakas, J.
Kozarewa, I.
Fenwick, K.
Lord, C.J.
Ashworth, A.
Vincent-Salomon, A.
Basik, M.
Reis-Filho, J.S.
Majewski, J.
Foulkes, W.D.
(2011). Identification of gene fusion transcripts by transcriptome sequencing in BRCA1-mutated breast cancers and cell lines. Bmc med genomics,
Vol.4,
p. 75.
show abstract
BACKGROUND: Gene fusions arising from chromosomal translocations have been implicated in cancer. However, the role of gene fusions in BRCA1-related breast cancers is not well understood. Mutations in BRCA1 are associated with an increased risk for breast cancer (up to 80% lifetime risk) and ovarian cancer (up to 50%). We sought to identify putative gene fusions in the transcriptomes of these cancers using high-throughput RNA sequencing (RNA-Seq). METHODS: We used Illumina sequencing technology to sequence the transcriptomes of five BRCA1-mutated breast cancer cell lines, three BRCA1-mutated primary tumors, two secretory breast cancer primary tumors and one non-tumorigenic breast epithelial cell line. Using a bioinformatics approach, our initial attempt at discovering putative gene fusions relied on analyzing single-end reads and identifying reads that aligned across exons of two different genes. Subsequently, latter samples were sequenced with paired-end reads and at longer cycles (producing longer reads). We then refined our approach by identifying misaligned paired reads, which may flank a putative gene fusion junction. RESULTS: As a proof of concept, we were able to identify two previously characterized gene fusions in our samples using both single-end and paired-end approaches. In addition, we identified three novel in-frame fusions, but none were recurrent. Two of the candidates, WWC1-ADRBK2 in HCC3153 cell line and ADNP-C20orf132 in a primary tumor, were confirmed by Sanger sequencing and RT-PCR. RNA-Seq expression profiling of these two fusions showed a distinct overexpression of the 3' partner genes, suggesting that its expression may be under the control of the 5' partner gene's regulatory elements. CONCLUSIONS: In this study, we used both single-end and paired-end sequencing strategies to discover gene fusions in breast cancer transcriptomes with BRCA1 mutations. We found that the use of paired-end reads is an effective tool for transcriptome profiling of gene fusions. Our findings suggest that while gene fusions are present in some BRCA1-mutated breast cancers, they are infrequent and not recurrent. However, private fusions may still be valuable as potential patient-specific biomarkers for diagnosis and treatment..
Tanas, M.R.
Sboner, A.
Oliveira, A.M.
Erickson-Johnson, M.R.
Hespelt, J.
Hanwright, P.J.
Flanagan, J.
Luo, Y.
Fenwick, K.
Natrajan, R.
Mitsopoulos, C.
Zvelebil, M.
Hoch, B.L.
Weiss, S.W.
Debiec-Rychter, M.
Sciot, R.
West, R.B.
Lazar, A.J.
Ashworth, A.
Reis-Filho, J.S.
Lord, C.J.
Gerstein, M.B.
Rubin, M.A.
Rubin, B.P.
(2011). Identification of a disease-defining gene fusion in epithelioid hemangioendothelioma. Sci transl med,
Vol.3
(98),
p. 98ra82.
show abstract
Integrating transcriptomic sequencing with conventional cytogenetics, we identified WWTR1 (WW domain-containing transcription regulator 1) (3q25) and CAMTA1 (calmodulin-binding transcription activator 1) (1p36) as the two genes involved in the t(1;3)(p36;q25) chromosomal translocation that is characteristic of epithelioid hemangioendothelioma (EHE), a vascular sarcoma. This WWTR1/CAMTA1 gene fusion is under the transcriptional control of the WWTR1 promoter and encodes a putative chimeric transcription factor that joins the amino terminus of WWTR1, a protein that is highly expressed in endothelial cells, in-frame to the carboxyl terminus of CAMTA1, a protein that is normally expressed only in brain. Thus, CAMTA1 expression is activated inappropriately through a promoter-switch mechanism. The gene fusion is present in virtually all EHEs tested but is absent from all other vascular neoplasms, demonstrating it to be a disease-defining genetic alteration. A sensitive and specific break-apart fluorescence in situ hybridization assay was also developed to detect the translocation and will assist in the evaluation of this diagnostically challenging neoplasm. The chimeric WWTR1/CAMTA1 transcription factor may represent a therapeutic target for EHE and offers the opportunity to shed light on the functions of two poorly characterized proteins..
Weigelt, B.
Mackay, A.
Natrajan, R.
Tan, D.S.
Dowsett, M.
Ashworth, A.
Reis-Filho, J.S.
(2010). The importance of gene-centring microarray data Reply. Lancet oncology,
Vol.11
(8),
pp. 720-2.
Turner, N.
Pearson, A.
Sharpe, R.
Lambros, M.
Geyer, F.
Lopez-Garcia, M.A.
Natrajan, R.
Marchio, C.
Iorns, E.
Mackay, A.
Gillett, C.
Grigoriadis, A.
Tutt, A.
Reis-Filho, J.S.
Ashworth, A.
(2010). FGFR1 Amplification Drives Endocrine Therapy Resistance and Is a Therapeutic Target in Breast Cancer. Cancer research,
Vol.70
(5),
pp. 2085-10.
Weigelt, B.
Geyer, F.C.
Natrajan, R.
Lopez-Garcia, M.A.
Ahmad, A.S.
Savage, K.
Kreike, B.
Reis-Filho, J.S.
(2010). The molecular underpinning of lobular histological growth pattern: a genome-wide transcriptomic analysis of invasive lobular carcinomas and grade- and molecular subtype-matched invasive ductal carcinomas of no special type. J pathol,
Vol.220
(1),
pp. 45-57.
show abstract
Invasive lobular carcinoma (ILC) is the most frequent special type of breast cancer. The majority of these tumours are of low histological grade, express hormone receptors, and lack HER2 expression. The pleomorphic variant of ILCs (PLCs) is characterized by atypical cells with pleomorphic nuclei and is reported to have an aggressive clinical behaviour. Expression profiling studies have demonstrated that classic ILCs preferentially display a luminal phenotype, whereas PLCs may be of luminal, HER2 or molecular apocrine subtypes. The aims of this study were two-fold: to determine the transcriptomic characteristics of lobular carcinomas and to define the genome-wide transcriptomic differences between classic ILCs and PLCs. To define the transcriptomic characteristics of ILCs, minimizing the impact of histological grade and molecular subtype on the analysis, we subjected a series of grade- and molecular subtype-matched ILCs and invasive ductal carcinomas (IDCs) to genome-wide gene expression profiling using oligonucleotide microarrays. Hierarchical clustering analysis demonstrated that ILCs formed a separate cluster and a supervised analysis revealed that 5.8% of the transcriptionally regulated genes were significantly differentially expressed in ILCs compared to grade- and molecular subtype-matched IDCs. ILCs displayed down-regulation of E-cadherin and of genes related to actin cytoskeleton remodelling, protein ubiquitin, DNA repair, cell adhesion, TGF-beta signalling; and up-regulation of transcription factors/immediate early genes, lipid/prostaglandin biosynthesis genes, and cell migration-associated genes. Supervised analysis of classic ILCs and PLCs demonstrated that less than 0.1% of genes were significantly differentially expressed between these tumour subtypes. Our results demonstrate that ILCs differ from grade- and molecular subtype-matched IDCs in the expression of genes related to cell adhesion, cell-to-cell signalling, and actin cytoskeleton signalling. However, classic ILCs and PLCs are remarkably similar at the molecular level and should be considered as part of a spectrum of lesions..
Shiu, K.-.
Natrajan, R.
Geyer, F.C.
Ashworth, A.
Reis-Filho, J.S.
(2010). DNA amplifications in breast cancer: genotypic-phenotypic correlations. Future oncology,
Vol.6
(6),
pp. 967-18.
Klingbeil, P.
Natrajan, R.
Everitt, G.
Vatcheva, R.
Marchio, C.
Palacios, J.
Buerger, H.
Reis-Filho, J.S.
Isacke, C.M.
(2010). CD44 is overexpressed in basal-like breast cancers but is not a driver of 11p13 amplification. Breast cancer res treat,
Vol.120
(1),
pp. 95-109.
show abstract
Overexpression and alternative splicing of CD44 have been implicated in tumour progression. Here we describe the identification of a high level amplification of human 11p13, encompassing the CD44 gene, in primary breast cancers and cell lines and test whether CD44 acts as the driver of this amplicon. aCGH analysis revealed 11p13 amplification in 3% (3/100) of primary breast carcinomas and in two cell lines. The minimal region of amplification was 34.38-37.62 Mb. Amplification was confirmed by dual-colour FISH in these cell lines and further validated by CISH in an independent tumour cohort. CD44 expression in primary breast cancers was significantly associated with features of basal-like breast cancer. Detection of CD44 expression in breast cancer cell lines confirmed moderate to high expression in basal-like cell lines and minimal expression in luminal cell lines. In both, primary breast cancers and cell lines, 11p13 amplification was associated with high levels of CD44 mRNA expression. CD44 alternative splicing was detected in four of nine cell lines and in tumour samples, irrespective of the amplification status. RNAi mediated knock down of CD44 failed to reveal an increased dependence on CD44 expression for proliferation or survival in amplified cell lines. Given that expression of CD44 is not an absolute requirement for the survival of cells harbouring CD44 gene amplification, CD44 is unlikely to be a driver of the 11p13 amplicon..
Molyneux, G.
Geyer, F.C.
Magnay, F.-.
McCarthy, A.
Kendrick, H.
Natrajan, R.
Mackay, A.
Grigoriadis, A.
Tutt, A.
Ashworth, A.
Reis-Filho, J.S.
Smalley, M.J.
(2010). BRCA1 basal-like breast cancers originate from luminal epithelial progenitors and not from basal stem cells. Cell stem cell,
Vol.7
(3),
pp. 403-417.
show abstract
Breast cancers in BRCA1 mutation carriers frequently have a distinctive basal-like phenotype. It has been suggested that this results from an origin in basal breast epithelial stem cells. Here, we demonstrate that deleting Brca1 in mouse mammary epithelial luminal progenitors produces tumors that phenocopy human BRCA1 breast cancers. They also resemble the majority of sporadic basal-like breast tumors. However, directing Brca1 deficiency to basal cells generates tumors that express molecular markers of basal breast cancers but do not histologically resemble either human BRCA1 or the majority of sporadic basal-like breast tumors. These findings support a derivation of the majority of human BRCA1-associated and sporadic basal-like tumors from luminal progenitors rather than from basal stem cells. They also demonstrate that when target cells for transformation have the potential for phenotypic plasticity, tumor phenotypes may not directly reflect histogenesis. This has important implications for cancer prevention strategies..
Turner, N.
Pearson, A.
Sharpe, R.
Lambros, M.
Geyer, F.
Lopez-Garcia, M.A.
Natrajan, R.
Marchio, C.
Iorns, E.
Mackay, A.
Gillett, C.
Grigoriadis, A.
Tutt, A.
Reis-Filho, J.S.
Ashworth, A.
(2010). FGFR1 amplification drives endocrine therapy resistance and is a therapeutic target in breast cancer. Cancer research,
Vol.70
(5),
pp. 2085-2094.
show abstract
Amplification of fibroblast growth factor receptor 1 (FGFR1) occurs in approximately 10% of breast cancers and is associated with poor prognosis. However, it is uncertain whether overexpression of FGFR1 is causally linked to the poor prognosis of amplified cancers. Here, we show that FGFR1 overexpression is robustly associated with FGFR1 amplification in two independent series of breast cancers. Breast cancer cell lines with FGFR1 overexpression and amplification show enhanced ligand-dependent signaling, with increased activation of the mitogen-activated protein kinase and phosphoinositide 3-kinase-AKT signaling pathways in response to FGF2, but also show basal ligand-independent signaling, and are dependent on FGFR signaling for anchorage-independent growth. FGFR1-amplified cell lines show resistance to 4-hydroxytamoxifen, which is reversed by small interfering RNA silencing of FGFR1, suggesting that FGFR1 overexpression also promotes endocrine therapy resistance. FGFR1 signaling suppresses progesterone receptor (PR) expression in vitro, and likewise, amplified cancers are frequently PR negative, identifying a potential biomarker for FGFR1 activity. Furthermore, we show that amplified cancers have a high proliferative rate assessed by Ki67 staining and that FGFR1 amplification is found in 16% to 27% of luminal B-type breast cancers. Our data suggest that amplification and overexpression of FGFR1 may be a major contributor to poor prognosis in luminal-type breast cancers, driving anchorage-independent proliferation and endocrine therapy resistance..
Geyer, F.C.
Weigelt, B.
Natrajan, R.
Lambros, M.B.
de Biase, D.
Vatcheva, R.
Savage, K.
Mackay, A.
Ashworth, A.
Reis-Filho, J.S.
(2010). Molecular analysis reveals a genetic basis for the phenotypic diversity of metaplastic breast carcinomas. J pathol,
Vol.220
(5),
pp. 562-573.
show abstract
Cancers may be composed of multiple populations of submodal clones sharing the same initiating genetic lesions, followed by the acquisition of divergent genetic hits. Intra-tumour genetic heterogeneity has profound implications for cancer clinical management. To determine the extent of intra-tumour genetic heterogeneity in breast cancers, and whether the morphological diversity of breast cancers is underpinned by divergent genetic aberrations, we analysed the genomic profiles of microdissected, morphologically distinct components of six metaplastic breast carcinomas, tumours characterized by the presence of morphological areas with divergent differentiation. Each morphologically distinct component was separately microdissected and subjected to high-resolution microarray-based comparative genomic hybridization. Each component was also analysed by immunohistochemistry and in situ hybridization. Clonal relationship between the distinct components was tested by TP53 sequencing and human androgen receptor (HUMARA) X-chromosome inactivation assay. In the majority of cases, all morphologically distinct components from each case were clonal and displayed remarkably similar genetic profiles. In two cases, however, morphologically distinct components harboured specific genetic aberrations. In an adenosquamous carcinoma, the differences were such that only 20% of the genome harboured similar copy number changes. The squamous component displayed EGFR gene amplification, EGFR over-expression and lack of expression of hormone receptors, whereas the lobular component displayed the reverse pattern. The components of a biphasic spindle cell carcinoma harboured similar gains, losses, amplifications of 9p23 and 17q12 (HER2) and identical TP53 mutations, suggesting that these were relatively early events in the development of this tumour; however, each component displayed divergent focal amplifications. Importantly, the metastatic deposit of this case, despite harbouring a TP53 mutation identical to that found in the primary tumour, harboured additional specific focal amplifications. This proof-of-principle study provides direct evidence of intra-tumour genetic heterogeneity in breast cancers, and shows that in some cases morphological diversity may be underpinned by distinct genetic aberrations..
Weigelt, B.
Mackay, A.
A'hern, R.
Natrajan, R.
Tan, D.S.
Dowsett, M.
Ashworth, A.
Reis-Filho, J.S.
(2010). Breast cancer molecular profiling with single sample predictors: a retrospective analysis. Lancet oncol,
Vol.11
(4),
pp. 339-349.
show abstract
BACKGROUND: Microarray expression profiling classifies breast cancer into five molecular subtypes: luminal A, luminal B, basal-like, HER2, and normal breast-like. Three microarray-based single sample predictors (SSPs) have been used to define molecular classification of individual samples. We aimed to establish agreement between these SSPs for identification of breast cancer molecular subtypes. METHODS: Previously described microarray-based SSPs were applied to one in-house (n=53) and three publicly available (n=779) breast cancer datasets. Agreement was analysed between SSPs for the whole classification system and for the five molecular subtypes individually in each cohort. FINDINGS: Fair-to-substantial agreement between every pair of SSPs in each cohort was recorded (kappa=0.238-0.740). Of the five molecular subtypes, only basal-like cancers consistently showed almost-perfect agreement (kappa>0.812). The proportion of cases classified as basal-like in each cohort was consistent irrespective of the SSP used; however, the proportion of each remaining molecular subtype varied substantially. Assignment of individual cases to luminal A, luminal B, HER2, and normal breast-like subtypes was dependent on the SSP used. The significance of associations with outcome of each molecular subtype, other than basal-like and luminal A, varied depending on SSP used. However, different SSPs produced broadly similar survival curves. INTERPRETATION: Although every SSP identifies molecular subtypes with similar survival, they do not reliably assign the same patients to the same molecular subtypes. For molecular subtype classification to be incorporated into routine clinical practice and treatment decision making, stringent standardisation of methodologies and definitions for identification of breast cancer molecular subtypes is needed. FUNDING: Breakthrough Breast Cancer, Cancer Research UK..
Natrajan, R.
Weigelt, B.
Mackay, A.
Geyer, F.C.
Grigoriadis, A.
Tan, D.S.
Jones, C.
Lord, C.J.
Vatcheva, R.
Rodriguez-Pinilla, S.M.
Palacios, J.
Ashworth, A.
Reis-Filho, J.S.
(2010). An integrative genomic and transcriptomic analysis reveals molecular pathways and networks regulated by copy number aberrations in basal-like, HER2 and luminal cancers. Breast cancer res treat,
Vol.121
(3),
pp. 575-589.
show abstract
Breast cancer is a heterogeneous disease caused by the accumulation of genetic changes in neoplastic cells. We hypothesised that molecular subtypes of breast cancer may be driven by specific constellations of genes whose expression is regulated by gene copy number aberrations. To address this question, we analysed a series of 48 microdissected grade III ductal carcinomas using high resolution microarray comparative genomic hybridisation and mRNA expression arrays. There were 5,931 genes whose expression significantly correlates with copy number identified; out of these, 1,897 genes were significantly differentially expressed between basal-like, HER2 and luminal tumours. Ingenuity Pathway Analysis (IPA) revealed that 'G1/S cell cycle regulation' and 'BRCA1 in DNA damage control' pathways were significantly enriched for genes whose expression correlates with copy number and are differentially expressed between the molecular subtypes of breast cancer. IPA of genes whose expression significantly correlates with copy number in each molecular subtype individually revealed that canonical pathways involved in oestrogen receptor (ER) signalling and DNA repair are enriched for these genes. We also identified 32, 157 and 265 genes significantly overexpressed when amplified in basal-like, HER2 and luminal cancers, respectively. These lists include known and novel potential therapeutic targets (e.g. HER2 and PPM1D in HER2 cancers). Our results provide strong circumstantial evidence that different patterns of genetic aberrations in distinct molecular subtypes of breast cancer contribute to their specific transcriptomic profiles and that biological phenomena characteristic of each subtype (e.g. proliferation, HER2 and ER signalling) may be driven by specific patterns of copy number aberrations..
Shiu, K.-.
Natrajan, R.
Geyer, F.C.
Ashworth, A.
Reis-Filho, J.S.
(2010). DNA amplifications in breast cancer: genotypic–phenotypic correlations. Future oncology,
Vol.6
(6),
pp. 967-984.
show abstract
DNA copy number changes in cancer cells, in particular, amplifications, occur frequently, have prognostic impact and are associated with subtypes of breast cancer. Some amplicons contain well-characterized oncogenes, including 11q13 (CCND1) and 17q12 (HER2). HER2 amplification and overexpression defines the HER2+ subgroup of breast cancer patients and is both a prognostic marker for poor outcome and a predictive marker for response to anti-HER2 targeted therapies. Therefore, there is considerable interest in documenting the locations of other recurring amplifications in breast cancers as they may also provide a rich source of new biomarkers and novel therapeutic targets for these subgroups. This article focuses on the genomic profiling of breast cancer, with an emphasis on the characteristics of the amplifications found in subtypes of breast cancer, including luminal (ER+/HER2-), HER2+ and basal-like (ER-/HER2-), and discusses their known or potential roles in cancer biology and their clinical implications. .
Geyer, F.C.
Lambros, M.B.
Natrajan, R.
Mehta, R.
Mackay, A.
Savage, K.
Parry, S.
Ashworth, A.
Badve, S.
Reis-Filho, J.S.
(2010). Genomic and immunohistochemical analysis of adenosquamous carcinoma of the breast. Mod pathol,
Vol.23
(7),
pp. 951-960.
show abstract
Breast adenosquamous carcinomas are rare tumours characterized by well-developed gland formation intimately admixed with solid nests of squamous cells immersed in a highly cellular spindle cell stroma. A low-grade variant has been described that is associated with a better prognosis. Here we studied five cases of adenosquamous carcinomas to determine their genetic profiles and to investigate whether the spindle cell component of these cancers could at least in part stem from the glandular/epithelial components. Five adenosquamous carcinomas of the breast were subjected to (1) immunohistochemical analysis, (2) microdissection and genetic analysis with a high-resolution microarray comparative genomic hybridization platform, and (3) chromogenic in situ hybridization. All cases displayed a triple-negative immunophenotype, consistently expressed 'basal' keratins and showed variable levels of epidermal growth factor receptor expression. Microarray comparative genomic hybridization analysis of two of the cases revealed multiple low-level gains and losses affecting several chromosomal arms. Case 1 displayed gains of the whole of chromosome 7, and case 2 harboured a focal, high-level amplification of 7p12, encompassing the epidermal growth factor receptor gene, which was associated with strong and intense membranous epidermal growth factor receptor expression. Chromogenic in situ hybridization revealed that the genetic features found in the epithelial cells were also present in a minority of the spindle cells of the stromal component, in particular in those near the epithelial clusters, indicating that some of the spindle cells are clonal and derived from the epithelial component of the tumour. In conclusion, breast adenosquamous carcinomas are triple-negative cancers that express 'basal' keratins. These tumours harbour complex genetic profiles. Some of the spindle cells in adenosquamous carcinomas are derived from the epithelial component, suggesting that adenosquamous carcinomas may also be part of the group of metaplastic breast carcinomas with spindle cell metaplastic elements..
Turner, N.
Lambros, M.B.
Horlings, H.M.
Pearson, A.
Sharpe, R.
Natrajan, R.
Geyer, F.C.
van Kouwenhove, M.
Kreike, B.
Mackay, A.
Ashworth, A.
van de Vijver, M.J.
Reis-Filho, J.S.
(2010). Integrative molecular profiling of triple negative breast cancers identifies amplicon drivers and potential therapeutic targets. Oncogene,
Vol.29
(14),
pp. 2013-2023.
show abstract
Triple negative breast cancers (TNBCs) have a relatively poor prognosis and cannot be effectively treated with current targeted therapies. We searched for genes that have the potential to be therapeutic targets by identifying genes consistently overexpressed when amplified. Fifty-six TNBCs were subjected to high-resolution microarray-based comparative genomic hybridization (aCGH), of which 24 were subjected to genome-wide gene expression analysis. TNBCs were genetically heterogeneous; no individual focal amplification was present at high frequency, although 78.6% of TNBCs harboured at least one focal amplification. Integration of aCGH and expression data revealed 40 genes significantly overexpressed when amplified, including the known oncogenes and potential therapeutic targets, FGFR2 (10q26.3), BUB3 (10q26.3), RAB20 (13q34), PKN1 (19p13.12) and NOTCH3 (19p13.12). We identified two TNBC cell lines with FGFR2 amplification, which both had constitutive activation of FGFR2. Amplified cell lines were highly sensitive to FGFR inhibitor PD173074, and to RNAi silencing of FGFR2. Treatment with PD173074 induced apoptosis resulting partly from inhibition of PI3K-AKT signalling. Independent validation using publicly available aCGH data sets revealed FGFR2 gene was amplified in 4% (6/165) of TNBC, but not in other subtypes (0/214, P=0.0065). Our analysis demonstrates that TNBCs are heterogeneous tumours with amplifications of FGFR2 in a subgroup of tumours..
Weigelt, B.
Mackay, A.
Natrajan, R.
Tan, D.S.
Dowsett, M.
Ashworth, A.
Reis-Filho, J.S.
(2010). The importance of gene-centring microarray data – Authors' reply. The lancet oncology,
Vol.11
(8),
pp. 720-721.
Lopez-Garcia, M.A.
Geyer, F.C.
Natrajan, R.
Kreike, B.
Mackay, A.
Grigoriadis, A.
Reis-Filho, J.S.
Weigelt, B.
(2010). Transcriptomic analysis of tubular carcinomas of the breast reveals similarities and differences with molecular subtype-matched ductal and lobular carcinomas. J pathol,
Vol.222
(1),
pp. 64-75.
show abstract
Tubular carcinoma (TC) is an uncommon special type of breast cancer characterized by an indolent clinical course. Although described as part of a spectrum of related lesions named 'low-grade breast neoplasia family' due to immunophenotypical and genetic similarities, TCs, low-grade invasive ductal carcinomas of no special type (IDC-NSTs), and classic invasive lobular carcinomas (ILCs) significantly differ in terms of histological features and clinical outcome. The aim of this study was to investigate whether pure TCs constitute an entity distinct from low-grade IDC-NSTs and from classic ILCs. To define the transcriptomic differences between TCs and IDC-NSTs and ILCs whilst minimizing the impact of histological grade and molecular subtype on their profiles, we subjected a series of grade- and molecular subtype-matched TCs and IDC-NSTs and molecular subtype-matched TCs and classic ILCs to genome-wide gene expression profiling using oligonucleotide microarrays. Unsupervised and supervised analysis revealed that TCs are similar at the transcriptomic level to grade- and molecular subtype-matched IDC-NSTs. However, subtle yet significant differences were detected and validated by quantitative reverse transcriptase-PCR, which may in part explain the reported more favourable outcome of TCs. Transcriptomic differences between TCs and molecular subtype-matched classic ILCs were more overt, predominantly due to lower expression of proliferation and cell cycle genes in TCs and down-regulation of cell adhesion/extracellular matrix-related genes in classic ILCs. Our results support the existence of a 'low-grade breast neoplasia family'; however, the transcriptomes of these lesions display small, yet important differences, which, together with their distinct biological behaviour, warrant their separation as discrete entities..
Lambros, M.B.
Natrajan, R.
Geyer, F.C.
Lopez-Garcia, M.A.
Dedes, K.J.
Savage, K.
Lacroix-Triki, M.
Jones, R.L.
Lord, C.J.
Linardopoulos, S.
Ashworth, A.
Reis-Filho, J.S.
(2010). PPM1D gene amplification and overexpression in breast cancer: a qRT-PCR and chromogenic in situ hybridization study. Mod pathol,
Vol.23
(10),
pp. 1334-1345.
show abstract
PPM1D (protein phosphatase magnesium-dependent 1δ) maps to the 17q23.2 amplicon and is amplified in ∼8% of breast cancers. The PPM1D gene encodes a serine threonine phosphatase, which is involved in the regulation of several tumour suppressor pathways, including the p53 pathway. Along with others, we have recently shown that PPM1D is one of the drivers of the 17q23.2 amplicon and a promising therapeutic target. Here we investigate whether PPM1D is overexpressed when amplified in breast cancers and the correlations between PPM1D overexpression and amplification with clinicopathological features and survival of breast cancer patients from a cohort of 245 patients with invasive breast cancer treated with therapeutic surgery followed by adjuvant anthracycline-based chemotherapy. mRNA was extracted from representative sections of tumours containing >50% of tumour cells and subjected to TaqMan quantitative real-time PCR using primers for PPM1D and for two housekeeping genes. PPM1D overexpression was defined as the top quartile of expression levels. Chromogenic in situ hybridization with in-house-generated probes for PPM1D was performed. Amplification was defined as >50% of cancer cells with >5 signals per nucleus/large gene clusters. PPM1D overexpression and amplification were found in 25 and 6% of breast cancers, respectively. All cases harbouring PPM1D amplification displayed PPM1D overexpression. PPM1D overexpression was inversely correlated with expression of TOP2A, EGFR and cytokeratins 5/6 and 17. PPM1D amplification was significantly associated with HER2 overexpression, and HER2, TOP2A and CCND1 amplification. No association between PPM1D gene amplification and PPM1D mRNA overexpression with survival was observed. In conclusion, PPM1D is consistently overexpressed when amplified; however, PPM1D overexpression is more pervasive than gene amplification. PPM1D overexpression and amplification are associated with tumours displaying luminal or HER2 phenotypes. Co-amplification of PPM1D and HER2/TOP2A and CCND1 are not random events and may suggest the presence of a 'firestorm' genetic profile..
Lacroix-Triki, M.
Suarez, P.H.
MacKay, A.
Lambros, M.B.
Natrajan, R.
Savage, K.
Geyer, F.C.
Weigelt, B.
Ashworth, A.
Reis-Filho, J.S.
(2010). Mucinous carcinoma of the breast is genomically distinct from invasive ductal carcinomas of no special type. J pathol,
Vol.222
(3),
pp. 282-298.
show abstract
Mucinous carcinomas are a rare entity accounting for up to 2% of all breast cancers, which have been shown to display a gene expression profile distinct from that of invasive ductal carcinomas of no special type (IDC-NSTs). Here, we have defined the genomic aberrations that are characteristic of this special type of breast cancer and have investigated whether mucinous carcinomas might constitute a genomic entity distinct from IDC-NSTs. Thirty-five pure and 11 mixed mucinous breast carcinomas were assessed by immunohistochemistry using antibodies against oestrogen receptor (ER), progesterone receptor, HER2, Ki67, cyclin D1, cortactin, Bcl-2, p53, E-cadherin, basal markers, neuroendocrine markers, and WT1. Fifteen pure mucinous carcinomas and 30 grade- and ER-matched IDC-NSTs were microdissected and subjected to high-resolution microarray-based comparative genomic hybridization (aCGH). In addition, the distinct components of seven mixed mucinous carcinomas were microdissected separately and subjected to aCGH. Pure mucinous carcinomas consistently expressed ER (100%), lacked HER2 expression (97.1%), and showed a relatively low level of genetic instability. Unsupervised hierarchical cluster analysis revealed that pure mucinous carcinomas were homogeneous and preferentially clustered together, separately from IDC-NSTs. They less frequently harboured gains of 1q and 16p and losses of 16q and 22q than grade- and ER-matched IDC-NSTs, and no pure mucinous carcinoma displayed concurrent 1q gain and 16q loss, a hallmark genetic feature of low-grade IDC-NSTs. Finally, both components of all but one mixed mucinous carcinoma displayed similar patterns of genetic aberrations and preferentially clustered together with pure mucinous carcinomas on unsupervised clustering analysis. Our results demonstrate that mucinous carcinomas are more homogeneous between themselves at the genetic level than IDC-NSTs. Both components of mixed mucinous tumours are remarkably similar at the molecular level to pure mucinous cancers, suggesting that mixed mucinous carcinomas may be best classified as variants of mucinous cancers rather than of IDC-NSTs..
Dedes, K.J.
Lopez-Garcia, M.-.
Geyer, F.C.
Lambros, M.B.
Savage, K.
Vatcheva, R.
Wilkerson, P.
Wetterskog, D.
Lacroix-Triki, M.
Natrajan, R.
Reis-Filho, J.S.
(2010). Cortactin gene amplification and expression in breast cancer: a chromogenic in situ hybridisation and immunohistochemical study. Breast cancer res treat,
Vol.124
(3),
pp. 653-666.
show abstract
Amplification of 11q13 is found in approximately 15% of breast cancers. Cyclin D1 (CCND1) has been reported to be the 'driver' of this amplicon, however, multiple genes map to the smallest region of amplification of 11q13. Out of these genes, cortactin (CTTN) has been shown to be consistently overexpressed at the mRNA level in tumours harbouring 11q13 amplification. The aims of this study are to define whether CTTN is consistently co-amplified with the main core of the 11q13 amplicon, whether it is consistently overexpressed when amplified and to determine correlations between CTTN amplification and overexpression with clinicopathological features of breast cancers and survival of breast cancer patients. CTTN and CCND1 chromogenic in situ hybridisation (CISH) probes and a validated monoclonal antibody against CTTN were applied to a tissue microarray of a cohort of breast cancers from patients treated with anthracycline-based chemotherapy. CTTN and CCND1 amplifications were found in 12.3 and 12.4% of cases, respectively. All cases harbouring CTTN amplification also displayed CCND1 amplification. High expression of CTTN was found in 10.8% of cases and was associated with CTTN amplification, expression of 'basal' markers and topoisomerase IIα. Exploratory subgroup analysis of tumours devoid of 11q13 amplification revealed that high expression of CTTN in the absence of CTTN gene amplification was associated with lymph node negative disease, lack of hormone receptors and FOXA1, expression of 'basal' markers, high Ki-67 indices, p53 nuclear expression, and basal-like and triple negative phenotypes. CTTN expression and CTTN gene amplification were not associated with disease-, metastasis-free and overall survival. In conclusion, CTTN is consistently co-amplified with CCND1 and expressed at higher levels in breast cancers harbouring 11q13 amplification, suggesting that CTTN may also constitute one of the drivers of this amplicon. CTTN expression is not associated with the outcome of breast cancer patients treated with anthracycline-based chemotherapy..
Dedes, K.J.
Wetterskog, D.
Mendes-Pereira, A.M.
Natrajan, R.
Lambros, M.B.
Geyer, F.C.
Vatcheva, R.
Savage, K.
Mackay, A.
Lord, C.J.
Ashworth, A.
Reis-Filho, J.S.
(2010). PTEN deficiency in endometrioid endometrial adenocarcinomas predicts sensitivity to PARP inhibitors. Sci transl med,
Vol.2
(53),
p. 53ra75.
show abstract
PTEN (phosphatase and tensin homolog) loss of function is the most common genetic aberration in endometrioid endometrial carcinomas. In addition to its well-described role in cell signaling, PTEN is involved in the maintenance of genomic stability. Loss of PTEN function causes defects in repair of DNA double-strand breaks by homologous recombination and, therefore, sensitizes cells to inhibition of the poly(adenosine diphosphate ribose) polymerase (PARP). Here, we determined the PTEN status of eight endometrioid endometrial carcinoma cell lines and correlated it with in vitro sensitivity to the PARP inhibitor KU0058948. PTEN-deficient cells showed a significantly greater sensitivity to KU0058948 than the two endometrioid endometrial carcinoma cell lines with wild-type PTEN. The cell lines lacking PTEN expression were unable to elicit a homologous recombination damage response as assayed by RAD51 focus function (a marker of competent homologous recombination DNA repair) upon irradiation and treatment with PARP inhibitors. PTEN silencing in PTEN wild-type Hec-1b cells resulted in reduced RAD51 foci formation after DNA damage and increased sensitivity to PARP inhibition. PTEN reexpression in PTEN-null cell lines resulted in enhanced RAD51 foci formation and in relative resistance to KU0058948. Given that up to 80% of endometrioid endometrial cancers lack PTEN expression, our results suggest that PARP inhibitors may be therapeutically useful for a subset of endometrioid endometrial cancers..
Dedes, K.J.
Wetterskog, D.
Mendes-Pereira, A.M.
Vatcheva, R.
Natrajan, R.
Lambros, M.B.
Lord, C.J.
Ashworth, A.
Reis-Filho, J.S.
(2010). Preclinical evaluation of PARP inhibition as a treatment for endometrioid endometrial carcinomas. Journal of clinical oncology,
Vol.28
(15).
Tan, D.S.
Lambros, M.B.
Rayter, S.
Natrajan, R.
Vatcheva, R.
Gao, Q.
Marchiò, C.
Geyer, F.C.
Savage, K.
Parry, S.
Fenwick, K.
Tamber, N.
Mackay, A.
Dexter, T.
Jameson, C.
McCluggage, W.G.
Williams, A.
Graham, A.
Faratian, D.
El-Bahrawy, M.
Paige, A.J.
Gabra, H.
Gore, M.E.
Zvelebil, M.
Lord, C.J.
Kaye, S.B.
Ashworth, A.
Reis-Filho, J.S.
(2009). PPM1D is a potential therapeutic target in ovarian clear cell carcinomas. Clin cancer res,
Vol.15
(7),
pp. 2269-2280.
show abstract
PURPOSE: To identify therapeutic targets in ovarian clear cell carcinomas, a chemoresistant and aggressive type of ovarian cancer. EXPERIMENTAL DESIGN: Twelve ovarian clear cell carcinoma cell lines were subjected to tiling path microarray comparative genomic hybridization and genome-wide expression profiling analysis. Regions of high-level amplification were defined and genes whose expression levels were determined by copy number and correlated with gene amplification were identified. The effects of inhibition of PPM1D were assessed using short hairpin RNA constructs and a small-molecule inhibitor (CCT007093). The prevalence of PPM1D amplification and mRNA expression was determined using chromogenic in situ hybridization and quantitative real-time reverse transcription-PCR in a cohort of pure ovarian clear cell carcinomas and on an independent series of unselected epithelial ovarian cancers. RESULTS: Array-based comparative genomic hybridization analysis revealed regions of high-level amplification on 1q32, 1q42, 2q11, 3q24-q26, 5p15, 7p21-p22, 11q13.2-q13.4, 11q22, 17q21-q22, 17q23.2, 19q12-q13, and 20q13.2. Thirty-four genes mapping to these regions displayed expression levels that correlated with copy number gains/amplification. PPM1D had significantly higher levels of mRNA expression in ovarian clear cell carcinoma cell lines harboring gains/amplifications of 17q23.2. PPM1D inhibition revealed that PPM1D expression and phosphatase activity are selectively required for the survival of ovarian clear cell carcinoma cell lines with 17q23.2 amplification. PPM1D amplification was significantly associated with ovarian clear cell carcinoma histology (P = 0.0003) and found in 10% of primary ovarian clear cell carcinomas. PPM1D expression levels were significantly correlated with PPM1D gene amplification in primary ovarian clear cell carcinomas. CONCLUSION: Our data provide strong circumstantial evidence that PPM1D is a potential therapeutic target for a subgroup of ovarian clear cell carcinomas..
Marchio, C.
Iravan, M.
Natrajan, R.
Lambros, M.B.
Geyer, F.C.
Savage, K.
Parry, S.
Tamber, N.
Fenwick, K.
Mackay, A.
Schmitt, F.C.
Bussolati, G.
Ellis, I.
Ashworth, A.
Sapino, A.
Reis-Filho, J.S.
(2009). Mixed micropapillary-ductal carcinomas of the breast: a genomic and immunohistochemical analysis of morphologically distinct components. Journal of pathology,
Vol.218
(3),
pp. 301-15.
Drake, K.M.
Ruteshouser, E.C.
Natrajan, R.
Harbor, P.
Wegert, J.
Gessler, M.
Pritchard-Jones, K.
Grundy, P.
Dome, J.
Huff, V.
Jones, C.
Aldred, M.A.
(2009). Loss of heterozygosity at 2q37 in sporadic Wilms' tumor: putative role for miR-562. Clin cancer res,
Vol.15
(19),
pp. 5985-5992.
show abstract
PURPOSE: Wilms' tumor is a childhood cancer of the kidney with an incidence of approximately 1 in 10,000. Cooccurrence of Wilms' tumor with 2q37 deletion syndrome, an uncommon constitutional chromosome abnormality, has been reported previously in three children. Given these are independently rare clinical entities, we hypothesized that 2q37 harbors a tumor suppressor gene important in Wilms' tumor pathogenesis. EXPERIMENTAL DESIGN: To test this, we performed loss of heterozygosity analysis in a panel of 226 sporadic Wilms' tumor samples and mutation analysis of candidate genes. RESULTS: Loss of heterozygosity was present in at least 4% of cases. Two tumors harbored homozygous deletions at 2q37.1, supporting the presence of a tumor suppressor gene that follows a classic two-hit model. However, no other evidence of second mutations was found, suggesting that heterozygous deletion alone may be sufficient to promote tumorigenesis in concert with other genomic abnormalities. We show that miR-562, a microRNA within the candidate region, is expressed only in kidney and colon and regulates EYA1, a critical gene for renal development. miR-562 expression is reduced in Wilms' tumor and may contribute to tumorigenesis by deregulating EYA1. Two other candidate regions were localized at 2q37.3 and 2qter, but available data from patients with constitutional deletions suggest that these probably do not confer a high risk for Wilms' tumor. CONCLUSIONS: Our data support the presence of a tumor suppressor gene at 2q37.1 and suggest that, in individuals with constitutional 2q37 deletions, any increased risk for developing Wilms' tumor likely correlates with deletions encompassing 2q37.1..
Geyer, F.C.
Kushner, Y.B.
Lambros, M.B.
Natrajan, R.
Mackay, A.
Tamber, N.
Fenwick, K.
Purnell, D.
Ashworth, A.
Walker, R.A.
Reis-Filho, J.S.
(2009). Microglandular adenosis or microglandular adenoma? A molecular genetic analysis of a case associated with atypia and invasive carcinoma. Histopathology,
Vol.55
(6),
pp. 732-12.
Natrajan, R.
Lambros, M.B.
Geyer, F.C.
Marchio, C.
Tan, D.S.
Vatcheva, R.
Shiu, K.-.
Hungermann, D.
Rodriguez-Pinilla, S.M.
Palacios, J.
Ashworth, A.
Buerger, H.
Reis-Filho, J.S.
(2009). Loss of 16q in high grade breast cancer is associated with estrogen receptor status: Evidence for progression in tumors with a luminal phenotype?. Genes chromosomes cancer,
Vol.48
(4),
pp. 351-365.
show abstract
Loss of the long arm of chromosome 16 (16q) is observed in the vast majority of low grade/grade I (GI) invasive ductal carcinomas of no special type (IDC-NSTs), whereas this event is uncommonly seen in high grade/grade III (GIII) IDC-NSTs. Together with data on the pathology and genetics of breast cancer recurrences, this has led to the proposal that GI and GIII breast cancers evolve through distinct genetic pathways and that progression from GI to GIII is an unlikely biological phenomenon. We compared the genomic profiles of GIII-IDC-NSTs with 16q whole arm loss (16qWL) according to estrogen receptor (ER) status. 16qWL was found in 36.5% of cases and was significantly associated with ER expression and luminal phenotype. ER+ GIII-IDC-NSTs with 16qWL displayed significantly higher levels of genomic instability than ER+ IDC-NSTs without 16qWL. Furthermore, ER+ and ER- IDC-NSTs stratified according to the presence of 16qWL harbored distinct patterns of genetic aberrations. Interestingly, ER+/16qWL tumors displayed genetic features usually found in tumors with homologous DNA repair defects and significantly more frequently harbored heterozygous loss of BRCA2 than the remaining ER+ cancers. Our results demonstrate that approximately one third of GIII tumors harbor 16qWL, confirming that progression from low to high grade breast cancer is not found in the majority of breast cancers. 16qWL was significantly more prevalent in ER+/luminal GIII-IDC-NSTs. Given that GI breast cancers harbor a luminal phenotype, our results suggest that if progression from GI to GIII breast cancer does happen, it may preferentially occur in breast cancers of luminal phenotype..
Natrajan, R.
Lambros, M.B.
Rodríguez-Pinilla, S.M.
Moreno-Bueno, G.
Tan, D.S.
Marchió, C.
Vatcheva, R.
Rayter, S.
Mahler-Araujo, B.
Fulford, L.G.
Hungermann, D.
Mackay, A.
Grigoriadis, A.
Fenwick, K.
Tamber, N.
Hardisson, D.
Tutt, A.
Palacios, J.
Lord, C.J.
Buerger, H.
Ashworth, A.
Reis-Filho, J.S.
(2009). Tiling path genomic profiling of grade 3 invasive ductal breast cancers. Clinical cancer research : an official journal of the american association for cancer research,
Vol.15
(8),
pp. 2711-2722.
show abstract
Purpose
To characterize the molecular genetic profiles of grade 3 invasive ductal carcinomas of no special type using high-resolution microarray-based comparative genomic hybridization (aCGH) and to identify recurrent amplicons harboring putative therapeutic targets associated with luminal, HER-2, and basal-like tumor phenotypes.
Experimental design
Ninety-five grade 3 invasive ductal carcinomas of no special type were classified into luminal, HER-2, and basal-like subgroups using a previously validated immunohistochemical panel. Tumor samples were microdissected and subjected to aCGH using a tiling path 32K BAC array platform. Selected regions of recurrent amplification were validated by means of in situ hybridization. Expression of genes pertaining to selected amplicons was investigated using quantitative real-time PCR and gene silencing was done using previously validated short hairpin RNA constructs.
Results
We show that basal-like and HER-2 tumors are characterized by "sawtooth" and "firestorm" genetic patterns, respectively, whereas luminal cancers were more heterogeneous. Apart from confirming known amplifications associated with basal-like (1q21, 10p, and 12p), luminal (8p12, 11q13, and 11q14), and HER-2 (17q12) cancers, we identified previously unreported recurrent amplifications associated with each molecular subgroup: 19q12 in basal-like, 1q32.1 in luminal, and 14q12 in HER-2 cancers. PPM1D gene amplification (17q23.2) was found in 20% and 8% of HER-2 and luminal cancers, respectively. Silencing of PPM1D by short hairpin RNA resulted in selective loss of viability in tumor cell lines harboring the 17q23.2 amplification.
Conclusions
Our results show the power of aCGH analysis in unraveling the genetic profiles of specific subgroups of cancer and for the identification of novel therapeutic targets..
Marchiò, C.
Iravani, M.
Natrajan, R.
Lambros, M.B.
Geyer, F.C.
Savage, K.
Parry, S.
Tamber, N.
Fenwick, K.
Mackay, A.
Schmitt, F.C.
Bussolati, G.
Ellis, I.
Ashworth, A.
Sapino, A.
Reis‐Filho, J.S.
(2009). Mixed micropapillary–ductal carcinomas of the breast: a genomic and immunohistochemical analysis of morphologically distinct components. The journal of pathology,
Vol.218
(3),
pp. 301-315.
Geyer, F.C.
Kushner, Y.B.
Lambros, M.B.
Natrajan, R.
Mackay, A.
Tamber, N.
Fenwick, K.
Purnell, D.
Ashworth, A.
Walker, R.A.
Reis-Filho, J.S.
(2009). Microglandular adenosis or microglandular adenoma? A molecular genetic analysis of a case associated with atypia and invasive carcinoma. Histopathology,
Vol.55
(6),
pp. 732-743.
Natrajan, R.
Lambros, M.B.
Maria Rodriguez-Pinilla, S.
Moreno-Bueno, G.
Tan, D.S.
Marchio, C.
Vatcheva, R.
Rayter, S.
Mahler-Araujo, B.
Fulford, L.G.
Hungermann, D.
Mackay, A.
Grigoriadis, A.
Fenwick, K.
Tamber, N.
Hardisson, D.
Tutt, A.
Palacios, J.
Lord, C.J.
Buerger, H.
Ashworth, A.
Reis-Filho, J.S.
(2009). Tiling Path Genomic Profiling of Grade 3 Invasive Ductal Breast Cancers. Clinical cancer research,
Vol.15
(8),
pp. 2711-12.
Stephens, P.J.
McBride, D.J.
Lin, M.-.
Varela, I.
Pleasance, E.D.
Simpson, J.T.
Stebbings, L.A.
Leroy, C.
Edkins, S.
Mudie, L.J.
Greenman, C.D.
Jia, M.
Latimer, C.
Teague, J.W.
Lau, K.W.
Burton, J.
Quail, M.A.
Swerdlow, H.
Churcher, C.
Natrajan, R.
Sieuwerts, A.M.
Martens, J.W.
Silver, D.P.
Langerød, A.
Russnes, H.E.
Foekens, J.A.
Reis-Filho, J.S.
van 't Veer, L.
Richardson, A.L.
Børresen-Dale, A.-.
Campbell, P.J.
Futreal, P.A.
Stratton, M.R.
(2009). Complex landscapes of somatic rearrangement in human breast cancer genomes. Nature,
Vol.462
(7276),
pp. 1005-1010.
show abstract
Multiple somatic rearrangements are often found in cancer genomes; however, the underlying processes of rearrangement and their contribution to cancer development are poorly characterized. Here we use a paired-end sequencing strategy to identify somatic rearrangements in breast cancer genomes. There are more rearrangements in some breast cancers than previously appreciated. Rearrangements are more frequent over gene footprints and most are intrachromosomal. Multiple rearrangement architectures are present, but tandem duplications are particularly common in some cancers, perhaps reflecting a specific defect in DNA maintenance. Short overlapping sequences at most rearrangement junctions indicate that these have been mediated by non-homologous end-joining DNA repair, although varying sequence patterns indicate that multiple processes of this type are operative. Several expressed in-frame fusion genes were identified but none was recurrent. The study provides a new perspective on cancer genomes, highlighting the diversity of somatic rearrangements and their potential contribution to cancer development..
Iorns, E.
Lord, C.J.
Grigoriadis, A.
McDonald, S.
Fenwick, K.
Mackay, A.
Mein, C.A.
Natrajan, R.
Savage, K.
Tamber, N.
Reis-Filho, J.S.
Turner, N.C.
Ashworth, A.
(2009). Integrated functional, gene expression and genomic analysis for the identification of cancer targets. Plos one,
Vol.4
(4),
p. e5120.
show abstract
The majority of new drug approvals for cancer are based on existing therapeutic targets. One approach to the identification of novel targets is to perform high-throughput RNA interference (RNAi) cellular viability screens. We describe a novel approach combining RNAi screening in multiple cell lines with gene expression and genomic profiling to identify novel cancer targets. We performed parallel RNAi screens in multiple cancer cell lines to identify genes that are essential for viability in some cell lines but not others, suggesting that these genes constitute key drivers of cellular survival in specific cancer cells. This approach was verified by the identification of PIK3CA, silencing of which was selectively lethal to the MCF7 cell line, which harbours an activating oncogenic PIK3CA mutation. We combined our functional RNAi approach with gene expression and genomic analysis, allowing the identification of several novel kinases, including WEE1, that are essential for viability only in cell lines that have an elevated level of expression of this kinase. Furthermore, we identified a subset of breast tumours that highly express WEE1 suggesting that WEE1 could be a novel therapeutic target in breast cancer. In conclusion, this strategy represents a novel and effective strategy for the identification of functionally important therapeutic targets in cancer..
Natrajan, R.
Warren, W.
Messahel, B.
Reis-Filho, J.S.
Brundler, M.-.
Dome, J.S.
Grundy, P.E.
Vujanic, G.
Pritchard-Jones, K.
Jones, C.
(2008). Complex patterns of chromosome 9 alterations including the p16INK4a locus in Wilms tumours. J clin pathol,
Vol.61
(1),
pp. 95-102.
show abstract
BACKGROUND: Previous data implicating genetic and epigenetic events on chromosome 9, including the CDKN2A/2B locus, as molecular predictors of Wilms tumour relapse, have been conflicting. AIMS: To clarify this using genome-wide and focused molecular genetic analysis. METHODS: Microarray-based comparative genomic hybridisation (aCGH) using genome-wide coverage was applied to 76 favourable histology Wilms tumours. Additional investigation of the 9p21 locus was carried out using loss of heterozygosity (LOH) and fluorescence in situ hybridisation (FISH), as well as immunohistochemistry for CDKN2A/p16(INK4a) on a paediatric renal tumour tissue microarray. RESULTS: Approximately half of the tumours were found to show chromosome 9 copy number changes. Those cases which harboured alterations comprised at least four distinct patterns: gain of the entire chromosome, loss of 9p, gain of 9q34, or a more complex combination of gains/losses. None of these tumour groups showed any statistically significant correlation with clinicopathological variables. Deletion mapping of 9p by LOH revealed several regions of overlap, including the CDKN2A/2B locus in 4/34 (11.8%) tumours, which was confirmed to represent hemizygous deletions by FISH. CDKN2A/p16(INK4a) protein expression was predominantly negative in Wilms tumours as assessed by immunohistochemistry on a tissue array, reflecting the expression pattern in normal kidney. However, 38/236 (16.1%) non-anaplastic Wilms tumours, 4/9 (44.4%) anaplastic Wilms tumours, 5/7 (71.4%) rhabdoid tumours of the kidney, and 4/10 (40%) clear cell sarcomas of the kidney showed nuclear CDKN2A/p16(INK4a )immunoreactivity. CONCLUSIONS: These data reveal the complex nature of genetic alterations on chromosome 9 in Wilms tumours, but do not provide evidence for their involvement in or association with treatment failure..
Arriola, E.
Marchio, C.
Tan, D.S.
Drury, S.C.
Lambros, M.B.
Natrajan, R.
Rodriguez-Pinilla, S.M.
Mackay, A.
Tamber, N.
Fenwick, K.
Jones, C.
Dowsett, M.
Ashworth, A.
Reis-Filho, J.S.
(2008). Genomic analysis of the HER2/TOP2A amplicon in breast cancer and breast cancer cell lines. Lab invest,
Vol.88
(5),
pp. 491-503.
show abstract
HER2 and TOP2A are targets for the therapeutic agents trastuzumab and anthracyclines and are frequently amplified in breast cancers. The aims of this study were to provide a detailed molecular genetic analysis of the 17q12-q21 amplicon in breast cancers harbouring HER2/TOP2A co-amplification and to investigate additional recurrent co-amplifications in HER2/TOP2A-co-amplified cancers. In total, 15 breast cancers with HER2 amplification, 10 of which also harboured TOP2A amplification, as defined by chromogenic in situ hybridisation, and 6 breast cancer cell lines known to be amplified for HER2 were subjected to high-resolution microarray-based comparative genomic hybridisation analysis. This revealed that the genomes of 12 cases were characterised by at least one localised region of clustered, relatively narrow peaks of amplification, with each cluster confined to a single chromosome arm (ie 'firestorm' pattern) and 3 cases displayed many narrow segments of duplication and deletion affecting the vast majority of chromosomes (ie 'sawtooth' pattern). The smallest region of amplification (SRA) on 17q12 in the whole series extended from 34.73 to 35.48 Mb, and encompassed HER2 but not TOP2A. In HER2/TOP2A-co-amplified samples, the SRA extended from 34.73 to 36.54 Mb, spanning a region of approximately 1.8 Mb. Apart from HER2 and TOP2A, this region encompassed four additional genes whose expression levels as defined by quantitative real-time PCR are significantly higher in HER2/TOP2A-co-amplified vs HER2-amplified breast cancers: CASC3, CDC6, RARA and SMARCE1. Of the cell lines studied, SKBR3 and UACC812 showed HER2/TOP2A co-amplification. In conclusion, this is the first detailed genome-wide characterisation of HER2/TOP2A-amplified breast cancers; cell lines were identified that can be used to model these cancers in vitro. The 17q12 amplicon is complex and harbours multiple genes that may be associated with breast cancer development and progression, and potentially exploitable as therapeutic targets..
Reis-Filho, J.S.
Natrajan, R.
Vatcheva, R.
Lambros, M.B.
Marchió, C.
Mahler-Araújo, B.
Paish, C.
Hodi, Z.
Eusebi, V.
Ellis, I.O.
(2008). Is acinic cell carcinoma a variant of secretory carcinoma? A FISH study using ETV6'split apart' probes. Histopathology,
Vol.52
(7),
pp. 840-846.
show abstract
AIMS: Acinic cell carcinomas (ACCs) and secretory carcinomas (SCs) of the breast are rare, low-grade malignancies that preferentially affect young female patients. Owing to the morphological and immunohistochemical similarities between these lesions, they have been proposed to be two morphological variants of the same entity. It has been demonstrated that SCs of the breast consistently harbour the t(12;15)ETV6-NTRK3 translocation. The aim was to determine whether ACCs also harbour ETV6 gene rearrangements and are thus variants of SCs. METHODS AND RESULTS: Using the ETV6 fluorescence in situ hybridization DNA Probe Split Signal (Dako), the presence of ETV6 rearrangements in three SCs and six ACCs was investigated. Cases were considered as harbouring an ETV6 gene rearrangement if >10% of nuclei displayed 'split apart signals' (i.e. red and green signals were separated by a distance greater than the size of two hybridization signals). Whereas the three SCs displayed ETV6 split apart signals in >10% of the neoplastic cells, no ACC showed any definite evidence of ETV6 gene rearrangement. CONCLUSIONS: Based on the lack of ETV6 rearrangements in ACCs, our results strongly support the concept that SCs and ACCs are distinct entities and should be recorded separately in breast cancer taxonomy schemes..
Reis-Filho, J.S.
Drury, S.
Lambros, M.B.
Marchio, C.
Johnson, N.
Natrajan, R.
Salter, J.
Levey, P.
Fletcher, O.
Peto, J.
Ashworth, A.
Dowsett, M.
(2008). ESR1 gene amplification in breast cancer: a common phenomenon?. Nat genet,
Vol.40
(7),
pp. 809-810.
Marchiò, C.
Iravani, M.
Natrajan, R.
Lambros, M.B.
Savage, K.
Tamber, N.
Fenwick, K.
Mackay, A.
Senetta, R.
Di Palma, S.
Schmitt, F.C.
Bussolati, G.
Ellis, L.O.
Ashworth, A.
Sapino, A.
Reis-Filho, J.S.
(2008). Genomic and immunophenotypical characterization of pure micropapillary carcinomas of the breast. J pathol,
Vol.215
(4),
pp. 398-410.
show abstract
Pure invasive micropapillary carcinoma (MPC) is a special histological type that accounts for 0.7-3% of all breast cancers. MPC has a distinctive growth pattern and a more aggressive clinical behaviour than invasive ductal carcinomas of no special type (IDC-NSTs). To define the molecular characteristics of MPCs, we profiled a series of 12 MPCs and 24 grade and oestrogen receptor (ER)-matched IDC-NSTs using high-resolution microarray comparative genomic hybridization (aCGH). In addition, we generated a tissue microarray containing a series of 24 MPCs and performed immunohistochemical analysis with ER, PR, Ki-67, HER2, CK5/6, CK14, CK17, EGFR, topoisomerase-IIalpha, cyclin D1, caveolin-1, E-cadherin, and beta-catenin antibodies. In situ hybridization probes were employed to evaluate the prevalence of amplification of HER2, TOP2A, EGFR, CCND1, MYC, ESR1, and FGFR1 genes. aCGH analysis demonstrated that MPCs significantly differed from IDC-NSTs at the genomic level. Gains of 1q, 2q, 4p, 6p, 6q23.2-q27, 7p, 7q, 8p, 8q, 9p, 10p, 11q, 12p, 12q, 16p, 17p, 17q, 19p, 20p, 20q, and 21q, and losses of 1p, 2p, 6q11.1-q16.3, 6q21-q22.1, 9p, 11p, 15q, and 19q were more prevalent in MPCs. High-level gains/amplifications of 8p12-p11, 8q12, 8q13, 8q21, 8q23, 8q24, 17q21, 17q23, and 20q13 were significantly associated with MPCs. A comparison between 24 MPCs and a series of 48 grade and ER-matched IDC-NSTs revealed that high cyclin D1 expression, high proliferation rates, and MYC (8q24) amplification were significantly associated with MPCs. Our results demonstrate that MPCs have distinct histological features and molecular genetic profiles supporting the contention that they constitute a distinct pathological entity..
Marchiò, C.
Natrajan, R.
Shiu, K.K.
Lambros, M.B.
Rodriguez-Pinilla, S.M.
Tan, D.S.
Lord, C.J.
Hungermann, D.
Fenwick, K.
Tamber, N.
Mackay, A.
Palacios, J.
Sapino, A.
Buerger, H.
Ashworth, A.
Reis-Filho, J.S.
(2008). The genomic profile of HER2-amplified breast cancers: the influence of ER status. J pathol,
Vol.216
(4),
pp. 399-407.
show abstract
Expression profiling studies have suggested that HER2-amplified breast cancers constitute a heterogeneous group that may be subdivided according to their ER status: HER2-amplified ER-positive breast carcinomas that fall into the luminal B cluster; and HER2-amplified ER-negative cancers which form a distinct molecular subgroup, known as the erbB2 or HER2 subgroup. ER-negative breast cancer differs significantly from ER-positive disease in the pattern, type, and complexity of genetic aberrations. Here we have compared the genomic profiles of ER-positive and ER-negative HER2-amplified cancers using tiling path microarray-based comparative genomic hybridization (aCGH). Validation of the differentially amplified regions was performed in an independent series of 70 HER2-amplified breast cancers. Although HER2-amplified cancers had remarkably complex patterns of molecular genetic aberrations, ER-positive and ER-negative HER2-amplified breast carcinomas shared most molecular genetic features as defined by aCGH. Genome-wide Fisher's exact test analysis revealed that less than 1.5% of the genome was significantly differentially gained or lost in ER-positive versus ER-negative HER2-amplified cancers. However, two regions of amplification were significantly associated with ER-positive carcinomas, one of which mapped to 17q21.2 and encompassed GJC1, IGFBP4, TNS4, and TOP2A. Chromogenic in situ hybridization analysis of an independent validation series confirmed the association between ER status and TOP2A amplification. In conclusion, although hormone receptor status does not determine the overall genetic profile of HER2-amplified breast cancers, specific genetic aberrations may be characteristic of subgroups of HER2 breast cancers..
Edwards, S.L.
Brough, R.
Lord, C.J.
Natrajan, R.
Vatcheva, R.
Levine, D.A.
Boyd, J.
Reis-Filho, J.S.
Ashworth, A.
(2008). Resistance to therapy caused by intragenic deletion in BRCA2. Nature,
Vol.451
(7182),
pp. 1111-1115.
show abstract
Cells with loss of BRCA2 function are defective in homologous recombination (HR) and are highly sensitive to inhibitors of poly(ADP-ribose) polymerase (PARP), which provides the basis for a new therapeutic approach. Here we show that resistance to PARP inhibition can be acquired by deletion of a mutation in BRCA2. We derived PARP-inhibitor-resistant (PIR) clones from the human CAPAN1 pancreatic cancer cell line, which carries the protein-truncating c.6174delT frameshift mutation. PIR clones could form DNA-damage-induced RAD51 nuclear foci and were able to limit genotoxin-induced genomic instability, both hallmarks of a competent HR pathway. New BRCA2 isoforms were expressed in the resistant lines as a result of intragenic deletion of the c.6174delT mutation and restoration of the open reading frame (ORF). Reconstitution of BRCA2-deficient cells with these revertant BRCA2 alleles rescued PARP inhibitor sensitivity and HR deficiency. Most of the deletions in BRCA2 were associated with small tracts of homology, and possibly arose from error-prone repair caused by BRCA2 deficiency. Similar ORF-restoring mutations were present in carboplatin-resistant ovarian tumours from c.6174delT mutation carriers. These observations have implications for understanding drug resistance in BRCA mutation carriers as well as in defining functionally important domains within BRCA2..
Natrajan, R.
Little, S.E.
Sodha, N.
Reis-Filho, J.S.
Mackay, A.
Fenwick, K.
Ashworth, A.
Perlman, E.J.
Dome, J.S.
Grundy, P.E.
Pritchard-Jones, K.
Jones, C.
(2007). Analysis by array CGH of genomic changes associated with the progression or relapse of Wilms' tumour. J pathol,
Vol.211
(1),
pp. 52-59.
show abstract
Despite aggressive salvage regimens, approximately half of all children who suffer a Wilms' tumour recurrence will die of their disease. Although there are increasing data on molecular genetic prognostic factors present in the tumour at diagnosis, there is little information regarding the molecular events that occur with Wilms' tumour progression and relapse. In the present study, microarray-based comparative genomic hybridization (aCGH) analysis has been carried out on 58 Wilms' tumour samples, which included 38 untreated primary and 20 recurrent tumours. A higher degree of copy number changes was observed in the recurrent tumours (33.0% genomic clones) than in the primary tumour (21.2%). Paired analysis highlighted the acquisition of 15q gain with high levels of IGF1R expression in the tumour recurrence in two cases. The most statistically significant abnormality acquired between diagnosis and relapse was loss of 17p. One case that experienced 17p loss was classified as favourable histology at diagnosis, but exhibited diffuse anaplasia at recurrence and had a homozygous TP53 deletion. Another instructive case with a constitutional 11p13 deletion presented with bilateral tumours and suffered two subsequent recurrences in the left kidney. A somatic WT1 mutation was found only in the right kidney tumour, while the constitutional 11p13 deletion was the only abnormality detected in the initial left kidney tumour by aCGH. The two subsequent relapses in the left kidney contained an accumulation of additional genetic alterations, including an independent WT1 mutation..
Little, S.E.
Bax, D.A.
Rodriguez-Pinilla, M.
Natrajan, R.
Messahel, B.
Pritchard-Jones, K.
Vujanic, G.M.
Reis-Filho, J.S.
Jones, C.
(2007). Multifaceted dysregulation of the epidermal growth factor receptor pathway in clear cell sarcoma of the kidney. Clin cancer res,
Vol.13
(15 Pt 1),
pp. 4360-4364.
show abstract
PURPOSE: Epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase overexpressed in a variety of human malignancies, against which targeted therapies have shown efficacy in lung and brain tumors. Clinical responses to EGFR inhibitors have been found to be highly dependent on the presence of activating mutations, whereas gene amplification, downstream activation of Akt, and abnormalities in PTEN are also reported predictive factors. We sought to evaluate these variables in pediatric renal tumors. EXPERIMENTAL DESIGN: We screened a series of 307 pediatric renal tumors for EGFR expression by immunohistochemistry and gene amplification by chromogenic in situ hybridization. In identifying a striking predilection for certain tumor types, we further analyzed the clear cell sarcomas of the kidney (CCSK) for mutations in EGFR and PTEN. RESULTS: Although only 23 of 177 (13.0%) nonanaplastic Wilms' tumors were EGFR positive, 4 of 11 (36.4%) anaplastic tumors showed receptor overexpression. In addition, 5 of 9 (55.6%) mesoblastic nephromas and 12 of 12 (100%) CCSKs were strongly immunoreactive for EGFR. In studying the CCSKs in more detail, we identified gene amplification in 1 of 12 (8.3%) cases and a somatic T790M EGFR mutation in a further case. These two samples additionally harbored mutations in PTEN. Downstream pathway activation, as assayed by phosphorylated Akt expression, was observed in 8 of 12 (66.7%) cases. CONCLUSIONS: Together, these data show dysregulation of the EGFR pathway at multiple levels in CCSKs. Identification of factors predictive of poor response to targeted therapy, including the drug resistance T790M mutation, may provide a rationale for upfront trials with irreversible inhibitors of EGFR in children with these tumors..
Marchio, C.
Iravani, M.
Natrajan, R.
Lambros, M.
James, M.
Savage, K.
Mackay, A.
Fenwick, K.
Tamber, N.
Schmitt, F.C.
Ashworth, A.
Ellis, I.
Sapino, A.
Bussolati, G.
Reis-Filho, J.S.
(2007). Molecular genetics and immunophenotypical characterization of micropapillary carcinomas of the breast. Virchows archiv,
Vol.451
(2),
pp. 145-2.
Arriola, E.
Lambros, M.
Marchio, C.
Tan, D.
Natrajan, R.
Rodriguez-Pinilla, S.M.
Tamber, N.
Fenwick, K.
Mackay, A.
Jones, C.
Ashworth, A.
Reis-Filho, J.S.
(2007). Her2/TOP2A amplicon in breast: A microarray-based and chromogenic in situ hybridisation analysis. Virchows archiv,
Vol.451
(2),
pp. 141-1.
Natrajan, R.
Williams, R.D.
Grigoriadis, A.
Mackay, A.
Fenwick, K.
Ashworth, A.
Dome, J.S.
Grundy, P.E.
Pritchard-Jones, K.
Jones, C.
(2007). Delineation of a 1Mb breakpoint region at 1p13 in Wilms tumors by fine-tiling oligonucleotide array CGH. Genes chromosomes cancer,
Vol.46
(6),
pp. 607-615.
show abstract
Wilms tumor karyotypes frequently exhibit recurrent, large-scale chromosomal imbalances, among the most common of which are concurrent loss of 1p and gain of 1q. We have previously identified a novel breakpoint at 1p13 by 1 Mb-spaced array CGH, and have now undertaken a fine-tiling oligonucleotide array approach to map the region accurately in four tumors exhibiting rearrangements at this locus. The use of a 10 bp-spaced platform revealed that all four tumors in fact harbored different breakpoints, which targeted intragenic sequences in PHTF1, DCLRE1B, and NRAS, and an intergenic region immediately downstream of TRIM33. All four genes and breakpoints were within the 1.78 Mb intervals identified by the genome-wide BAC arrays. The precise breakpoint interval was in each case mapped to a 200-1,200 bp region and was confirmed for one case to lie within intron 3 of DCLRE1B by quantitative PCR. Analysis of local genome architecture revealed no convincing conservation of repetitive sequences or specific translocation/recombination-associated elements within the breakpoint regions. This study highlights the power of fine-tiling oligonucleotide arrays to delineate breakpoint regions identified by genome-wide screens..
Lambros, M.B.
Natrajan, R.
Reis-Filho, J.S.
(2007). Chromogenic and fluorescent in situ hybridization in breast cancer. Hum pathol,
Vol.38
(8),
pp. 1105-1122.
show abstract
Fluorescent (FISH) and chromogenic (CISH) in situ hybridization have recently become part of the diagnostic armamentarium of breast pathologists. HER2 gene testing by FISH and/or CISH has become an integral part of the diagnostic workup for patients with breast cancer. In this era of high throughput technologies, these techniques have proven instrumental for the validation of results from microarray-based comparative genomic hybridization and for the identification of novel oncogenes and tumor suppressor genes. Furthermore, FISH and CISH applied to tissue microarrays have expedited the characterization of genomic changes associated with specific breast cancer molecular subtypes and the identification of novel prognostic and predictive markers. In this review, we provide in this review a critical assessment of CISH and FISH and the impact of the analysis of amplification of specific oncogenes (eg, HER2, EGFR, MYC, CCND1, and FGFR1) and deletion of tumor suppressor genes (eg, BRCA1 and BRCA2) on our understanding of breast cancer..
Tan, D.S.
Lambros, M.B.
Natrajan, R.
Reis-Filho, J.S.
(2007). Getting it right: designing microarray (and not 'microawry') comparative genomic hybridization studies for cancer research. Lab invest,
Vol.87
(8),
pp. 737-754.
show abstract
The development of high-resolution microarray-based comparative genomic hybridization (aCGH), using cDNA, bacterial artificial chromosome (BAC) and oligonucleotide probes, is providing tremendous opportunities for translational research by facilitating detailed analysis of entire cancer genomes in a single experiment. However, this technology will only fulfil its promise if studies incorporating aCGH are designed with a full understanding of its current limitations and the strategies available to circumvent them. While there have been several excellent reviews on the current status of this technology, there is currently very little guidance available regarding the appropriate design of experiments incorporating aCGH (including the strengths and weaknesses of each platform), and how best to combine the results obtained from aCGH with other 'omic' technologies, including gene expression. In this review, we present the key design issues that need to be considered in order to optimize aCGH studies, including sample selection, the definition of appropriate experimental objectives, arguments for and against the various microarray platforms that are currently available, and methods for data validation and integration. It is envisaged that future well-designed aCGH studies will enhance our understanding of the genetic basis of cancer, and lead to the identification of novel predictive and prognostic cancer biomarkers, as well as molecular therapeutic targets in cancer..
Natrajan, R.
RodriGuez Pinilla, S.M.
Marchio, C.
Vatcheva, R.
Savage, K.
Mackay, A.
Fenwick, K.
Tamber, N.
Palacios, J.
Ashworth, A.
Reis-Filho, J.S.
(2007). Near tiling microarray-based CGH identifies CCNE1 amplification in basal-like breast cancer. Virchows archiv,
Vol.451
(2),
pp. 136-137.
Natrajan, R.
Reis-Filho, J.S.
Little, S.E.
Messahel, B.
Brundler, M.-.
Dome, J.S.
Grundy, P.E.
Vujanic, G.M.
Pritchard-Jones, K.
Jones, C.
(2006). Blastemal expression of type I insulin-like growth factor receptor in Wilms' tumors is driven by increased copy number and correlates with relapse. Cancer res,
Vol.66
(23),
pp. 11148-11155.
show abstract
Most Wilms' tumors are of low stage, favorable histology, and have a high likelihood of cure with current multimodal therapy. Despite this, there remains a group of patients whose tumors recur for whom intensive salvage regimens result in survival of only 50%. Fitting a Cox proportional hazards model to microarray-based comparative genomic hybridization (aCGH) data on 68 Wilms' tumor samples, we identified a significant correlation between increased copy number at chromosome 15q26.3 insulin-like growth factor I receptor (IGFIR) and tumor relapse (adjusted P = 0.014). Wilms' tumors (13%) exhibited a low-level gain corresponding to three to four copies of the gene by aCGH analysis, 9 of 10 of which exhibited high IGFIR mRNA levels. Although IGFIR protein expression was restricted to the epithelial cells of fetal kidney and Wilms' tumors in most cases, 12% of tumors were also found to express IGFIR in the blastemal compartment. Blastemal IGFIR protein expression was associated with an increased copy number and a shorter relapse-free survival time (P = 0.027, log-rank test). In addition to the membrane localization, IGFIR was localized to the perinuclear region of the blastemal cells in 6% of Wilms' tumors. These data provide evidence that an increase in IGFIR gene copy number results in aberrant expression in the blastemal compartment of some Wilms' tumors and is associated with an adverse outcome in these patients. These findings suggest the possibility of use of targeted agents in the therapy of these children..
Lambros, M.B.
Simpson, P.T.
Jones, C.
Natrajan, R.
Westbury, C.
Steele, D.
Savage, K.
Mackay, A.
Schmitt, F.C.
Ashworth, A.
Reis, J.S.
(2006). Unlocking pathology archives for molecular genetic studies: a reliable method to generate probes for chromogenic and fluorescent in situ hybridization. Laboratory investigation,
Vol.86
(4),
pp. 398-11.
Little, S.E.
Vuononvirta, R.
Reis-Filho, J.S.
Natrajan, R.
Iravani, M.
Fenwick, K.
Mackay, A.
Ashworth, A.
Pritchard-Jones, K.
Jones, C.
(2006). Array CGH using whole genome amplification of fresh-frozen and formalin-fixed, paraffin-embedded tumor DNA. Genomics,
Vol.87
(2),
pp. 298-306.
show abstract
The ability to utilize formalin-fixed, paraffin-embedded (FFPE) archival specimens reliably for high-resolution molecular genetic analysis would be of immense practical application in the study of human disease. We have evaluated the ability of the GenomePlex whole genome amplification (WGA) kit to amplify frozen and FFPE tissue for use in array CGH (aCGH). GenomePlex gave highly representative data compared with unamplified controls both from frozen material (Pearson's R(2) = 0.898) and from FFPE (R(2) = 0.883). Artifactual amplification observed using DOP-PCR at chromosomes 1p, 3, 13q, and 16p was not seen with GenomePlex. Highly reproducible aCGH profiles were obtained using as little as 5 ng starting material from FFPE (R(2) = 0.918). This WGA method should readily lend itself to the determination of DNA copy number alterations from small fresh-frozen and FFPE clinical tumor specimens, although some care must be taken to optimize the DNA extraction procedure..
Lambros, M.B.
Simpson, P.T.
Jones, C.
Natrajan, R.
Westbury, C.
Steele, D.
Savage, K.
Mackay, A.
Schmitt, F.C.
Ashworth, A.
Reis-Filho, J.S.
(2006). Unlocking pathology archives for molecular genetic studies: a reliable method to generate probes for chromogenic and fluorescent in situ hybridization. Laboratory investigation,
Vol.86
(4),
pp. 398-408.
Natrajan, R.
Williams, R.D.
Hing, S.N.
Mackay, A.
Reis-Filho, J.S.
Fenwick, K.
Iravani, M.
Valgeirsson, H.
Grigoriadis, A.
Langford, C.F.
Dovey, O.
Gregory, S.G.
Weber, B.L.
Ashworth, A.
Grundy, P.E.
Pritchard-Jones, K.
Jones, C.
(2006). Array CGH profiling of favourable histology Wilms tumours reveals novel gains and losses associated with relapse. J pathol,
Vol.210
(1),
pp. 49-58.
show abstract
Despite the excellent survival of Wilms tumour patients treated with multimodality therapy, approximately 15% will suffer from tumour relapse, where response rates are markedly reduced. We have carried out microarray-based comparative genomic hybridisation on a series of 76 Wilms tumour samples, enriched for cases which recurred, to identify changes in DNA copy number associated with clinical outcome. Using 1Mb-spaced genome-wide BAC arrays, the most significantly different genomic changes between favourable histology tumours that did (n = 37), and did not (n = 39), subsequently relapse were gains on 1q, and novel deletions at 12q24 and 18q21. Further relapse-associated loci included losses at 1q32.1, 2q36.3-2q37.1, and gain at 13q31. 1q gains correlated strongly with loss of 1p and/or 16q. In 3 of 11 cases with concurrent 1p(-)/1q(+), a breakpoint was identified at 1p13. Multiple low-level sub-megabase gains along the length of 1q were identified using chromosome 1 tiling-path arrays. One such recurrent region at 1q22-q23.1 included candidate genes RAB25, NES, CRABP2, HDGF and NTRK1, which were screened for mRNA expression using quantitative RT-PCR. These data provide a high-resolution catalogue of genomic copy number changes in relapsing favourable histology Wilms tumours..
Natrajan, R.
Little, S.E.
Reis-Filho, J.S.
Hing, L.
Messahel, B.
Grundy, P.E.
Dome, J.S.
Schneider, T.
Vujanic, G.M.
Pritchard-Jones, K.
Jones, C.
(2006). Amplification and overexpression of CACNA1E correlates with relapse in favorable histology Wilms' tumors. Clin cancer res,
Vol.12
(24),
pp. 7284-7293.
show abstract
PURPOSE: The most well established molecular markers of poor outcome in Wilms' tumor are loss of heterozygosity at chromosomes 1p and/or 16q, although to date no specific genes at these loci have been identified. We have previously shown a link between genomic gain of chromosome 1q and tumor relapse and sought to further elucidate the role of genes on 1q in treatment failure. EXPERIMENTAL DESIGN: Microarray-based comparative genomic hybridization identified a microamplification harboring a single gene (CACNA1E) at 1q25.3 in 6 of 76 (7.9%) Wilms' tumors, correlating with a shorter relapse-free survival (P = 0.0044, log-rank test). Further characterization of this gene was carried out by measuring mRNA and protein expression as well as stable transfection of HEK293 cells. RESULTS: Overexpression of the CACNA1E transcript was associated with DNA copy number (P = 0.0204, ANOVA) and tumor relapse (P = 0.0851, log-rank test). Immunohistochemistry against the protein product Ca(V)2.3 revealed expression localized to the apical membrane in the distal tubules of normal kidney but not to the metanephric blastemal cells of fetal kidney from which Wilms' tumors arise. Nuclear localization in 99 of 160 (61.9%) Wilms' tumor cases correlated with a reduced relapse-free survival, particularly in cases treated with preoperative chemotherapy (P = 0.009, log-rank test). Expression profiling of stably transfected HEK293 cells revealed specific up-regulation of the immediate early response genes EGR1/EGR2/EGR3 and FOS/FOSB, mediated by activation of the MEK/ERK5/Nur77 pathway. CONCLUSIONS: These data identify a unique genetic aberration with direct clinical relevance in Wilms' tumor relapse and provide evidence for a potential novel mechanism of treatment resistance in these tumors..
Williams, R.D.
Hing, S.N.
Greer, B.T.
Whiteford, C.C.
Wei, J.S.
Natrajan, R.
Kelsey, A.
Rogers, S.
Campbell, C.
Pritchard-Jones, K.
Khan, J.
(2004). Prognostic classification of relapsing favorable histology Wilms tumor using cDNA microarray expression profiling and support vector machines. Genes chromosomes cancer,
Vol.41
(1),
pp. 65-79.
show abstract
Treatment of Wilms tumor has a high success rate, with some 85% of patients achieving long-term survival. However, late effects of treatment and management of relapse remain significant clinical problems. If accurate prognostic methods were available, effective risk-adapted therapies could be tailored to individual patients at diagnosis. Few molecular prognostic markers for Wilms tumor are currently defined, though previous studies have linked allele loss on 1p or 16q, genomic gain of 1q, and overexpression from 1q with an increased risk of relapse. To identify specific patterns of gene expression that are predictive of relapse, we used high-density (30 k) cDNA microarrays to analyze RNA samples from 27 favorable histology Wilms tumors taken from primary nephrectomies at the time of initial diagnosis. Thirteen of these tumors relapsed within 2 years. Genes differentially expressed between the relapsing and nonrelapsing tumor classes were identified by statistical scoring (t test). These genes encode proteins with diverse molecular functions, including transcription factors, developmental regulators, apoptotic factors, and signaling molecules. Use of a support vector machine classifier, feature selection, and test evaluation using cross-validation led to identification of a generalizable expression signature, a small subset of genes whose expression potentially can be used to predict tumor outcome in new samples. Similar methods were used to identify genes that are differentially expressed between tumors with and without genomic 1q gain. This set of discriminators was highly enriched in genes on 1q, indicating close agreement between data obtained from expression profiling with data from genomic copy number analyses..
Natrajan, R.
Louhelainen, J.
Williams, S.
Laye, J.
Knowles, M.A.
(2003). High-resolution deletion mapping of 15q13 2-q21 1 in transitional cell carcinoma of the bladder. Cancer research,
Vol.63
(22),
pp. 7657-7662.
Todd, J.R.
Ryall, K.A.
Vyse, S.
Natrajan, R.C.
Yuan, Y.
Tan, A.C.
Huang, P.H.
Systematic analysis of tumour cell-extracellular matrix adhesion identifies independent prognostic factors in breast cancer. Oncotarget,
.
show abstract
Tumour cell-extracellular matrix (ECM) interactions are fundamental for discrete steps in breast cancer progression. In particular, cancer cell adhesion to ECM proteins present in the microenvironment is critical for accelerating tumour growth and facilitating metastatic spread. To assess the utility of tumour cell-ECM adhesion as a means for discovering prognostic factors in breast cancer survival, here we perform a systematic phenotypic screen and characterise the adhesion properties of a panel of human HER2 amplified breast cancer cell lines across six ECM proteins commonly deregulated in breast cancer. We determine a gene expression signature that defines a subset of cell lines displaying impaired adhesion to laminin. Cells with impaired laminin adhesion showed an enrichment in genes associated with cell motility and molecular pathways linked to cytokine signalling and inflammation. Evaluation of this gene set in the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) cohort of 1,964 patients identifies the F12 and STC2 genes as independent prognostic factors for overall survival in breast cancer. Our study demonstrates the potential of in vitro cell adhesion screens as a novel approach for identifying prognostic factors for disease outcome..
Naidoo, K.
Wai, P.
Maguire, S.
Daley, F.
Haider, S.
Kriplani, D.
Campbell, J.
Mirza, H.
Grigoriadis, A.
Tutt, A.
Moseley, P.
Abdel-Fatah, T.
Chan, S.
Madhusudan, S.
Rhaka, E.
Ellis, I.
Lord, C.
Yuan, Y.
Green, A.
Natrajan, R.
Evaluation of CDK12 Protein Expression as a Potential Novel Biomarker for DNA Damage Response Targeted Therapies in Breast Cancer. Molecular cancer therapeutics,
.
Bajrami, I.
Marlow, R.
van de Ven, M.
Brough, R.
Pemberton, H.
Frankum, J.
Song, F.
Raquif, R.
Konde, A.
Krastev, D.
Menon, M.
Campbell, J.
Gulati, A.
Kumar, R.
Pettitt, S.
Gurden, M.
Cardenosa, M.
Chong, I.
Gazinska, P.
Wallberg, F.
Sawyer, E.
Martin, L.
Dowsett, M.
Linardopoulos, S.
Natrajan, R.
Ryan, C.
Derksen, P.
Jonkers, J.
Tutt, A.
Ashworth, A.
Lord, C.
E-cadherin/ROS1 inhibitor synthetic lethality in breast cancer. Cancer discovery,
.
Read, A.
Natrajan, R.
Splicing dysregulation as a driver of breast cancer. Endocrine-related cancer,
,
pp. ERC-18-0068-ERC-18-0068.
Naidoo, K.
Barrow-McGee, R.
Procter, J.
Gibson, A.
Buus, R.
Holgersen, E.
Natrajan, R.
Haider, S.
Tutt, A.
Real-time ex vivo perfusion of human lymph nodes invaded by cancer (REPLICANT): a feasibility study. Journal of pathology,
.
Emad, A.
Ray, T.
Jensen, T.W.
Parat, M.
Natrajan, R.
Sinha, S.
Ray, P.S.
An epithelial-mesenchymal-amoeboid transition gene signature reveals subtypes of breast cancer progression and metastasis. ,
.
show abstract
AbstractCancer cells within a tumor are known to display varying degrees of metastatic propensity but the molecular basis underlying such heterogeneity remains unclear. We analyzed genome-wide gene expression data obtained from primary tumors of lymph node-negative breast cancer patients using a novel metastasis biology-based Epithelial-Mesenchymal-Amoeboid Transition (EMAT) gene signature, and identified subtypes associated with distinct prognostic profiles. EMAT subtype status improved prognosis accuracy of clinical parameters and statistically outperformed traditional breast cancer intrinsic subtypes even after adjusting for treatment variables. Additionally, analysis of 3D spheroids from an in vitro isogenic model of breast cancer progression reveals that EMAT subtypes display progression from premalignant to malignant and pre-invasive to invasive cancer. EMAT classification is a biologically informed method to assess metastasis risk in early stage, lymph node-negative breast cancer patients..
Barry, P.
Vatsiou, A.
Spiteri Sagastume, I.
Nichol, D.
Cresswell, G.
Acar, A.
Trahearn, N.
Hrebien, S.
Garcia-Murillas, I.
Chkhaidze, K.
Ermini, L.
Cottom, H.
Zabaglo, L.
Koelble, K.
Khalique, S.
Rusby, J.
Muscara, F.
Dowsett, M.
Maley, C.
Natrajan, R.
Yuan, Y.
Schiavon, G.
Turner, N.
Sottoriva, A.
The spatio-temporal evolution of lymph node spread in early breast cancer. Clinical cancer research,
.
Jones, C.
Mackay, A.
Izquierdo, E.
Temelso, S.
Carvalho, D.
Molinari, V.
Burford, A.
Howell, L.
Natrajan, R.
Boult, J.
Robinson, S.
Infant high grade gliomas comprise multiple subgroups characterized by novel targetable gene fusions and favorable outcomes. Cancer discovery,
.
Natrajan, R.
Sailem, H.
Mardakheh, F.K.
Arias Garcia, M.
Tape, C.J.
Dowsett, M.
Bakal, C.
Yuan, Y.
Microenvironmental Heterogeneity Parallels Breast Cancer Progression: A Histology–Genomic Integration Analysis. Plos medicine,
Vol.13
(2),
pp. e1001961-e1001961.
Krasny, L.
Bland, P.
Burns, J.
Lima, N.C.
Harrison, P.T.
Pacini, L.
Elms, M.L.
Ning, J.
Garcia Martinez, V.
Yu, Y.-.
Acton, S.E.
Ho, P.-.
Calvo, F.
Swain, A.
Howard, B.A.
Natrajan, R.C.
Huang, P.H.
A mouse SWATH-MS reference spectral library enables deconvolution of species-specific proteomic alterations in human tumour xenografts. Disease models & mechanisms,
.
show abstract
SWATH-mass spectrometry (MS) enables accurate and reproducible proteomic profiling in multiple model organisms including the mouse. Here we present a comprehensive mouse reference spectral library (MouseRefSWATH) that permits quantification of up to 10,597 proteins (62.2% of the mouse proteome) by SWATH-MS. We exploit MouseRefSWATH to develop an analytical pipeline for species-specific deconvolution of proteomic alterations in human tumour xenografts (XenoSWATH). This method overcomes the challenge of high sequence similarity between mouse and human proteins, facilitating the study of host microenvironment-tumour interactions from ‘bulk tumour’ measurements. We apply the XenoSWATH pipeline to characterise an intraductal xenograft model of breast ductal carcinoma in-situ and uncover complex regulation consistent with stromal reprogramming, where the modulation of cell migration pathways is not restricted to tumour cells but also operate in the mouse stroma upon progression to invasive disease. MouseRefSWATH and XenoSWATH opens new opportunities for in-depth and reproducible proteomic assessment to address wide-ranging biological questions involving this important model organism..
Khalique, S.
Nash, S.
Mansfield, D.
Wampfler, J.
Attygale, A.
Vroobel, K.
Kemp, H.
Buus, R.
Cottom, H.
Roxanis, I.
Jones, T.
von Loga, K.
Begum, D.
Guppy, N.
Ramagiri, P.
Fenwick, K.
Matthews, N.
Hubank, M.
Lord, C.
Haider, S.
Melcher, A.
Banerjee, S.
Natrajan, R.
Quantitative Assessment and Prognostic Associations of the Immune Landscape in Ovarian Clear Cell Carcinoma. Cancers,
Vol.13
(15),
pp. 3854-3854.
show abstract
Ovarian clear cell carcinoma (OCCC) is a rare subtype of epithelial ovarian cancer characterised by a high frequency of loss-of-function ARID1A mutations and a poor response to chemotherapy. Despite their generally low mutational burden, an intratumoural T cell response has been reported in a subset of OCCC, with ARID1A purported to be a biomarker for the response to the immune checkpoint blockade independent of micro-satellite instability (MSI). However, assessment of the different immune cell types and spatial distribution specifically within OCCC patients has not been described to date. Here, we characterised the immune landscape of OCCC by profiling a cohort of 33 microsatellite stable OCCCs at the genomic, gene expression and histological level using targeted sequencing, gene expression profiling using the NanoString targeted immune panel, and multiplex immunofluorescence to assess the spatial distribution and abundance of immune cell populations at the protein level. Analysis of these tumours and subsequent independent validation identified an immune-related gene expression signature associated with risk of recurrence of OCCC. Whilst histological quantification of tumour-infiltrating lymphocytes (TIL, Salgado scoring) showed no association with the risk of recurrence or ARID1A mutational status, the characterisation of TILs via multiplexed immunofluorescence identified spatial differences in immunosuppressive cell populations in OCCC. Tumour-associated macrophages (TAM) and regulatory T cells were excluded from the vicinity of tumour cells in low-risk patients, suggesting that high-risk patients have a more immunosuppressive microenvironment. We also found that TAMs and cytotoxic T cells were also excluded from the vicinity of tumour cells in ARID1A-mutated OCCCs compared to ARID1A wild-type tumours, suggesting that the exclusion of these immune effectors could determine the host response of ARID1A-mutant OCCCs to therapy. Overall, our study has provided new insights into the immune landscape and prognostic associations in OCCC and suggest that tailored immunotherapeutic approaches may be warranted for different subgroups of OCCC patients..
Bliss, J.
Porta, N.
Toms, C.
Banerjee, S.
Stewart, J.
Natrajan, R.
Lord, C.
ATARI trial: ATR inhibitor in combination with olaparib in gynaecological cancers with ARID1A loss or no loss (ENGOT/GYN1/NCRI). International journal of gynecological cancer,
.