BACKGROUND: Invasive Lobular Carcinoma (ILC) is a morphologically distinct breast cancer subtype that represents up to 15% of all breast cancers. Compared to Invasive Breast Carcinoma of No Special Type (IBC-NST), ILCs exhibit poorer long-term outcome and a unique pattern of metastasis. Despite these differences, the systematic discovery of robust prognostic biomarkers and therapeutically actionable molecular pathways in ILC remains limited. METHODS: Pathway-centric multivariable models using statistical machine learning were developed and tested in seven retrospective clinico-genomic cohorts (n = 996). Further external validation was performed using a new RNA-Seq clinical cohort of aggressive ILCs (n = 48). RESULTS AND CONCLUSIONS: mRNA dysregulation scores of 25 pathways were strongly prognostic in ILC (FDR-adjusted P < 0.05). Of these, three pathways including Cell-cell communication, Innate immune system and Smooth muscle contraction were also independent predictors of chemotherapy response. To aggregate these findings, a multivariable machine learning predictor called PSILC was developed and successfully validated for predicting overall and metastasis-free survival in ILC. Integration of PSILC with CRISPR-Cas9 screening data from breast cancer cell lines revealed 16 candidate therapeutic targets that were synthetic lethal with high-risk ILCs. This study provides interpretable prognostic and predictive biomarkers of ILC which could serve as the starting points for targeted drug discovery for this disease..

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype, characterized by extensive intratumoral heterogeneity, high metastasis, and chemoresistance, leading to poor clinical outcomes. Despite progress, the mechanistic basis of these aggressive behaviors remains poorly understood. Using single-cell and spatial transcriptome analysis, here we discovered basal epithelial subpopulations located within the stroma that exhibit chemoresistance characteristics. The subpopulations are defined by distinct signature genes that show a frequent gain in copy number and exhibit an activated epithelial-to-mesenchymal transition program. A subset of these genes can accurately predict chemotherapy response and are associated with poor prognosis. Interestingly, among these genes, elevated ITGB1 participates in enhancing intercellular signaling while ACTN1 confers a survival advantage to foster chemoresistance. Furthermore, by subjecting the transcriptional signatures to drug repurposing analysis, we find that chemoresistant tumors may benefit from distinct inhibitors in treatment-naive versus post-NAC patients. These findings shed light on the mechanistic basis of chemoresistance while providing the best-in-class biomarker to predict chemotherapy response and alternate therapeutic avenues for improved management of TNBC patients resistant to chemotherapy..

SF3B1 hotspot mutations are associated with a poor prognosis in several tumor types and lead to global disruption of canonical splicing. Through synthetic lethal drug screens, we identify that SF3B1 mutant (SF3B1MUT) cells are selectively sensitive to poly (ADP-ribose) polymerase inhibitors (PARPi), independent of hotspot mutation and tumor site. SF3B1MUT cells display a defective response to PARPi-induced replication stress that occurs via downregulation of the cyclin-dependent kinase 2 interacting protein (CINP), leading to increased replication fork origin firing and loss of phosphorylated CHK1 (pCHK1; S317) induction. This results in subsequent failure to resolve DNA replication intermediates and G2/M cell cycle arrest. These defects are rescued through CINP overexpression, or further targeted by a combination of ataxia-telangiectasia mutated and PARP inhibition. In vivo, PARPi produce profound antitumor effects in multiple SF3B1MUT cancer models and eliminate distant metastases. These data provide the rationale for testing the clinical efficacy of PARPi in a biomarker-driven, homologous recombination proficient, patient population..

Tumour heterogeneity is pervasive amongst many cancers and leads to disease progression, and therapy resistance. In this review, using breast cancer as an exemplar, we focus on the recent advances in understanding the interplay between tumour cells and their microenvironment using single cell sequencing and digital spatial profiling technologies. Further, we discuss the utility of lineage tracing methodologies in pre-clinical models of breast cancer, and how these are being used to unravel new therapeutic vulnerabilities and reveal biomarkers of breast cancer progression. © 2023 The Pathological Society of Great Britain and Ireland..

The emergence of drug-resistant cells, most of which have a mutated TP53 gene, prevents curative treatment in most advanced and common metastatic cancers of adults. Yet, a few, rarer malignancies, all of which are TP53 wild type, have high cure rates. In this Perspective, we discuss how common features of curable cancers offer insights into the evolutionary and developmental determinants of drug resistance. Acquired loss of TP53 protein function is the most common genetic change in cancer. This probably reflects positive selection in the context of strong ecosystem pressures including microenvironmental hypoxia. Loss of TP53's functions results in multiple fitness benefits and enhanced evolvability of cancer cells. TP53-null cells survive apoptosis, and tolerate potent oncogenic signalling, DNA damage and genetic instability. In addition, critically, they provide an expanded pool of self-renewing, or stem, cells, the primary units of evolutionary selection in cancer, making subsequent adaptation to therapeutic challenge by drug resistance highly probable. The exceptional malignancies that are curable, including the common genetic subtype of childhood acute lymphoblastic leukaemia and testicular seminoma, differ from the common adult cancers in originating prenatally from embryonic or fetal cells that are developmentally primed for TP53-dependent apoptosis. Plus, they have other genetic and phenotypic features that enable dissemination without exposure to selective pressures for TP53 loss, retaining their intrinsic drug hypersensitivity..

Mutations and loss of E-cadherin protein expression define the vast majority of invasive lobular carcinomas. In a subset of these cases, the heterogeneous expression of E-cadherin is observed either as wild-type (strong membranous) expression or aberrant expression (cytoplasmic expression). However, it is unclear as to whether the two components would be driven by distinct genetic or epigenetic alterations. Here, we used whole genome DNA sequencing and methylation array profiling of two separately dissected components of nine invasive lobular carcinomas with heterogeneous E-cadherin expression. E-cadherin negative and aberrant/positive components of E-cadherin heterogeneous tumours showed a similar mutational, copy number and promoter methylation repertoire, suggesting they arise from a common ancestor, as opposed to the collision of two independent tumours. We found that the majority of E-cadherin heterogeneous tumours harboured CDH1 mutations in both the E-cadherin negative and aberrant/positive components together with somatic mutations in additional driver genes known to be enriched in both pure invasive carcinomas of no special type and invasive lobular breast cancers, whereas these were less commonly observed in CDH1 wild-type tumours. CDH1 mutant tumours also exhibited a higher mutation burden as well as increased presence of APOBEC-dependent mutational signatures 2 and 13 compared to CDH1 wild-type tumours. Together, our results suggest that regardless of E-cadherin protein expression, tumours showing heterogeneous expression of E-cadherin should be considered as part of the spectrum of invasive lobular breast cancers..

The ARID1A tumour suppressor protein is a component of the SWI/SNF chromatin remodelling complex, which is mutated in approximately 20% of all human cancers. ARID1A mutational status is considered to hold prognostic significance in a range of solid malignancies, yet in endometriosis-related ovarian carcinomas there has been a lack of clarity of its prognostic role. Moreover, the relationship between ARID1A status and immune infiltrate is also poorly understood. In a recent issue of The Journal of Pathology, a large comprehensive study by Heinze, Nazeran et al addressed these areas by reviewing 1,623 endometriosis-associated ovarian carcinomas and correlating ARID1A status using standardised immunohistochemistry to infer mutation status, with comprehensive clinicopathological features, mismatch repair status and CD8+ tumour infiltrating lymphocytes. The study definitively showed that ARID1A status does not provide any independent prognostic value in endometriosis-associated ovarian carcinomas. ARID1A loss was, however, shown to be associated with mismatch repair deficiency and increased CD8+ tumour infiltrating lymphocytes in endometrioid ovarian carcinoma, which may be relevant for future studies. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland..

Invasive lobular carcinoma (ILC) is the second most common histological subtype of breast cancer, and it exhibits a number of clinico-pathological characteristics distinct from the more common invasive ductal carcinoma (IDC). We set out to identify alterations in the tumor microenvironment (TME) of ILC. We used laser-capture microdissection to separate tumor epithelium from stroma in 23 ER+ ILC primary tumors. Gene expression analysis identified 45 genes involved in regulation of the extracellular matrix (ECM) that were enriched in the non-immune stroma of ILC, but not in non-immune stroma from ER+ IDC or normal breast. Of these, 10 were expressed in cancer-associated fibroblasts (CAFs) and were increased in ILC compared to IDC in bulk gene expression datasets, with PAPPA and TIMP2 being associated with better survival in ILC but not IDC. PAPPA, a gene involved in IGF-1 signaling, was the most enriched in the stroma compared to the tumor epithelial compartment in ILC. Analysis of PAPPA- and IGF1-associated genes identified a paracrine signaling pathway, and active PAPP-A was shown to be secreted from primary CAFs. This is the first study to demonstrate molecular differences in the TME between ILC and IDC identifying differences in matrix organization and growth factor signaling pathways..

In 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..

BACKGROUND: ARID1A (AT-rich interactive domain containing protein 1A) loss-of-function mutations have been reported in gynecological cancers, including rarer subtypes such as clear cell carcinoma. Preclinical studies indicate that ARID1A mutant cancers display sensitivity to ATR inhibition while tumors without ARID1A mutations may be sensitive to Ataxia telangiectasia and Rad3 related (ATR) inhibitors in combination with poly-ADP ribose polymerase (PARP) inhibitors. PRIMARY OBJECTIVE: To determine whether the ATR inhibitor, ceralasertib, has clinical activity as a single agent and in combination with the PARP inhibitor, olaparib, in patients with ARID1A 'loss' and 'no loss' clear cell carcinomas and other relapsed gynecological cancers. STUDY HYPOTHESIS: ARID1A deficient clear cell carcinoma of the ovary or endometrium is sensitive to ATR inhibition, while the combination of ATR and PARP inhibition has activity in other gynecological tumors, irrespective of ARID1A status. TRIAL DESIGN: ATARI (ENGOT/GYN1/NCRI) is a multicenter, international, proof-of-concept, phase II, parallel cohort trial assessing ceralasertib activity as a single agent and in combination with olaparib in ARID1A stratified gynecological cancers. Patients with relapsed ovarian/endometrial clear cell carcinoma with ARID1A loss will receive ceralasertib monotherapy (cohort 1A). Relapsed ovarian/endometrial clear cell carcinoma patients with no ARID1A loss (cohort 2) or patients with other histological subtypes (endometrioid, carcinosarcoma, cervical) (cohort 3) will receive combination therapy (olaparib/ceralasertib). Treatment will continue until disease progression. MAJOR INCLUSION/EXCLUSION CRITERIA: Patients with histologically confirmed recurrent clear cell (ovarian, endometrial, or endometriosis related), endometrioid (ovarian, endometrial, or endometriosis related), cervical (adenocarcinomas or squamous), or carcinosarcomas (ovarian or endometrial) are eligible. Patients progressing after ≥1 prior platinum with evidence of measurable (RECIST v1.1) radiological disease progression since last systemic anticancer therapy and prior to trial entry are eligible. Previous ATR or PARP inhibitor treatment is not permissible. PRIMARY ENDPOINT: Best overall objective response rate (RECIST v1.1). SAMPLE SIZE: A minimum of 40 and a maximum of 116. ESTIMATED DATES FOR COMPLETING ACCRUAL AND PRESENTING RESULTS: Accrual is anticipated to be complete by the second quarter of 2022, with reporting of results by the fourth quarter of 2022. Overall accrual targets and reporting timelines are dependent on individual cohort progression to stage 2. TRIAL REGISTRATION NUMBER: NCT0405269..

Triple-negative breast cancers (TNBC) are resistant to standard-of-care chemotherapy and lack known targetable driver gene alterations. Identification of novel drivers could aid the discovery of new treatment strategies for this hard-to-treat patient population, yet studies using high-throughput and accurate models to define the functions of driver genes in TNBC to date have been limited. Here, we employed unbiased functional genomics screening of the 200 most frequently mutated genes in breast cancer, using spheroid cultures to model in vivo-like conditions, and identified the histone acetyltransferase CREBBP as a novel tumor suppressor in TNBC. CREBBP protein expression in patient tumor samples was absent in 8% of TNBCs and at a high frequency in other tumors, including squamous lung cancer, where CREBBP-inactivating mutations are common. In TNBC, CREBBP alterations were associated with higher genomic heterogeneity and poorer patient survival and resulted in upregulation and dependency on a FOXM1 proliferative program. Targeting FOXM1-driven proliferation indirectly with clinical CDK4/6 inhibitors (CDK4/6i) selectively impaired growth in spheroids, cell line xenografts, and patient-derived models from multiple tumor types with CREBBP mutations or loss of protein expression. In conclusion, we have identified CREBBP as a novel driver in aggressive TNBC and identified an associated genetic vulnerability in tumor cells with alterations in CREBBP and provide a preclinical rationale for assessing CREBBP alterations as a biomarker of CDK4/6i response in a new patient population. SIGNIFICANCE: This study demonstrates that CREBBP genomic alterations drive aggressive TNBC, lung cancer, and lymphomas and may be selectively treated with clinical CDK4/6 inhibitors..

Subclonal heterogeneity and evolution are characteristics of breast cancer that play a fundamental role in tumour development, progression and resistance to current therapies. In this review, we focus on the recent advances in understanding the epigenetic and transcriptomic changes that occur within breast cancer and their importance in terms of cancer development, progression and therapy resistance with a particular focus on alterations at the single-cell level. Furthermore, we highlight the utility of using single-cell tracing and molecular barcoding methodologies in preclinical models to assess disease evolution and response to therapy. We discuss how the integration of single-cell profiling from patient samples can be used in conjunction with results from preclinical models to untangle the complexities of this disease and identify biomarkers of disease progression, including measures of intra-tumour heterogeneity themselves, and how enhancing this understanding has the potential to uncover new targetable vulnerabilities in breast cancer..

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..

Development of resistance to platinum and poly(ADP-ribose) polymerase inhibitors via secondary BRCA gene mutations that restore functional homologous recombination has been observed in a number of cancer types. Here we report a case of somatic BRCA2 mutation in a patient with high grade serous ovarian carcinoma. A secondary mutation predicted to restore the BRCA2 open reading frame was detected at low frequency (2.3%) in whole exome sequencing of a peritoneal biopsy at disease progression after treatment that included carboplatin and olaparib. We used digital droplet PCR (ddPCR) to verify the presence and frequency of this mutation in the biopsy sample at progression and also used this approach to assess the presence of the secondary mutation in preceding biopsies at diagnosis and first relapse. We found no evidence for the secondary mutation being present prior to the final progression biopsy, suggesting that this mutation was acquired late in the course of treatment. ddPCR provides a sensitive and specific technique to investigate the presence of low frequency mutations in a time series of biopsies..

Using genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) screens to understand endocrine drug resistance, we discovered ARID1A and other SWI/SNF complex components as the factors most critically required for response to two classes of estrogen receptor-alpha (ER) antagonists. In this context, SWI/SNF-specific gene deletion resulted in drug resistance. Unexpectedly, ARID1A was also the top candidate in regard to response to the bromodomain and extraterminal domain inhibitor JQ1, but in the opposite direction, with loss of ARID1A sensitizing breast cancer cells to bromodomain and extraterminal domain inhibition. We show that ARID1A is a repressor that binds chromatin at ER cis-regulatory elements. However, ARID1A elicits repressive activity in an enhancer-specific, but forkhead box A1-dependent and active, ER-independent manner. Deletion of ARID1A resulted in loss of histone deacetylase 1 binding, increased histone 4 lysine acetylation and subsequent BRD4-driven transcription and growth. ARID1A mutations are more frequent in treatment-resistant disease, and our findings provide mechanistic insight into this process while revealing rational treatment strategies for these patients..

Understanding how breast cancer (BC) grows in axillary lymph nodes (ALNs), and refining how therapies might halt that process, is clinically important. However, modelling the complex ALN microenvironment is difficult, and no human models exist at present. We harvested ALNs from ten BC patients, and perfused them at 37 °C ex vivo for up to 24 h. Controlled autologous testing showed that ALNs remain viable after 24 h of ex vivo perfusion: haematoxylin and eosin-stained histological appearance and proliferation (by Ki67 immunohistochemistry) did not change significantly over time for any perfused ALN compared with a control from time-point zero. Furthermore, targeted gene expression analysis (NanoString PanCancer IO360 panel) showed that only 21/750 genes were differentially expressed between control and perfused ALNs (|log2 FC| > 1 and q < 0.1): none were involved in apoptosis and metabolism, but rather all 21 genes were involved in immune function and angiogenesis. During perfusion, tissue acid-base balance remained stable. Interestingly, the flow rate increased (p < 0.001) in cancer-replaced (i.e. metastasis occupied more than 90% of the surface area on multiple levels) compared to cancer-free nodes (i.e. nodes with no metastasis on multiple sections). CXCL11 transcripts were significantly more abundant in cancer-replaced nodes, while CXCL12 transcripts were significantly more abundant in cancer-free nodes. These cytokines were also detected in the circulating perfusate. Monoclonal antibodies (nivolumab and trastuzumab) were administered into a further three ALNs to confirm perfusion efficacy. These drugs saturated the nodes; nivolumab even induced cancer cell death. Normothermic ALN perfusion is not only feasible but sustains the tumour microenvironment ex vivo for scientific investigation. This model could facilitate the identification of actionable immuno-oncology targets. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland..

Ovarian clear cell carcinomas (OCCC) are rare aggressive, chemo-resistant tumours comprising approximately 13% of all epithelial ovarian cancers, which have distinct clinical and molecular features, when compared to other gynaecological malignancies. At present, there are no specific licensed targeted therapies for OCCC, although a number of candidate targets have been identified. This review focuses on recent knowledge underpinning our understanding of the pathogenesis of OCCC including direct and synthetic-lethal therapeutic strategies in particular focussing on ARID1A deficiency. We also discuss current targeted clinical trials and immunotherapeutic approaches..

Infant high-grade gliomas appear clinically distinct from their counterparts in older children, indicating that histopathologic grading may not accurately reflect the biology of these tumors. We have collected 241 cases under 4 years of age, and carried out histologic review, methylation profiling, and custom panel, genome, or exome sequencing. After excluding tumors representing other established entities or subgroups, we identified 130 cases to be part of an "intrinsic" spectrum of disease specific to the infant population. These included those with targetable MAPK alterations, and a large proportion of remaining cases harboring gene fusions targeting ALK (n = 31), NTRK1/2/3 (n = 21), ROS1 (n = 9), and MET (n = 4) as their driving alterations, with evidence of efficacy of targeted agents in the clinic. These data strongly support the concept that infant gliomas require a change in diagnostic practice and management. SIGNIFICANCE: Infant high-grade gliomas in the cerebral hemispheres comprise novel subgroups, with a prevalence of ALK, NTRK1/2/3, ROS1, or MET gene fusions. Kinase fusion-positive tumors have better outcome and respond to targeted therapy clinically. Other subgroups have poor outcome, with fusion-negative cases possibly representing an epigenetically driven pluripotent stem cell phenotype.See related commentary by Szulzewsky and Cimino, p. 904.This article is highlighted in the In This Issue feature, p. 890..

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 characterize 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 operates in the mouse stroma upon progression to invasive disease. MouseRefSWATH and XenoSWATH open new opportunities for in-depth and reproducible proteomic assessment to address wide-ranging biological questions involving this important model organism..

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..

Cancer-associated mutations in the spliceosome gene SF3B1 create a neomorphic protein that produces aberrant mRNA splicing in hundreds of genes, but the ensuing biologic and therapeutic consequences of this missplicing are not well understood. Here we have provided evidence that aberrant splicing by mutant SF3B1 altered the transcriptome, proteome, and metabolome of human cells, leading to missplicing-associated downregulation of metabolic genes, decreased mitochondrial respiration, and suppression of the serine synthesis pathway. We also found that mutant SF3B1 induces vulnerability to deprivation of the nonessential amino acid serine, which was mediated by missplicing-associated downregulation of the serine synthesis pathway enzyme PHGDH. This vulnerability was manifest both in vitro and in vivo, as dietary restriction of serine and glycine in mice was able to inhibit the growth of SF3B1MUT xenografts. These findings describe a role for SF3B1 mutations in altered energy metabolism, and they offer a new therapeutic strategy against SF3B1MUT cancers..

Disruption of Cyclin-Dependent Kinase 12 (CDK12) is known to lead to defects in DNA repair and sensitivity to platinum salts and PARP1/2 inhibitors. However, CDK12 has also been proposed as an oncogene in breast cancer. We therefore aimed to assess the frequency and distribution of CDK12 protein expression by IHC in independent cohorts of breast cancer and correlate this with outcome and genomic status. We found that 21% of primary unselected breast cancers were CDK12 high, and 10.5% were absent, by IHC. CDK12 positivity correlated with HER2 positivity but was not an independent predictor of breast cancer-specific survival taking HER2 status into account; however, absent CDK12 protein expression significantly correlated with a triple-negative phenotype. Interestingly, CDK12 protein absence was associated with reduced expression of a number of DDR proteins including ATR, Ku70/Ku80, PARP1, DNA-PK, and γH2AX, suggesting a novel mechanism of CDK12-associated DDR dysregulation in breast cancer. Our data suggest that diagnostic IHC quantification of CDK12 in breast cancer is feasible, with CDK12 absence possibly signifying defective DDR function. This may have important therapeutic implications, particularly for triple-negative breast cancers. Mol Cancer Ther; 17(1); 306-15. ©2017 AACR..

Purpose: The most significant prognostic factor in early breast cancer is lymph node involvement. This stage between localized and systemic disease is key to understanding breast cancer progression; however, our knowledge of the evolution of lymph node malignant invasion remains limited, as most currently available data are derived from primary tumors.Experimental Design: In 11 patients with treatment-naïve node-positive early breast cancer without clinical evidence of distant metastasis, we investigated lymph node evolution using spatial multiregion sequencing (n = 78 samples) of primary and lymph node deposits and genomic profiling of matched longitudinal circulating tumor DNA (ctDNA).Results: Linear evolution from primary to lymph node was rare (1/11), whereas the majority of cases displayed either early divergence between primary and nodes (4/11) or no detectable divergence (6/11), where both primary and nodal cells belonged to a single recent expansion of a metastatic clone. Divergence of metastatic subclones was driven in part by APOBEC. Longitudinal ctDNA samples from 2 of 7 subjects with evaluable plasma taken perioperatively reflected the two major evolutionary patterns and demonstrate that private mutations can be detected even from early metastatic nodal deposits. Moreover, node removal resulted in disappearance of private lymph node mutations in ctDNA.Conclusions: This study sheds new light on a crucial evolutionary step in the natural history of breast cancer, demonstrating early establishment of axillary lymph node metastasis in a substantial proportion of patients. Clin Cancer Res; 24(19); 4763-70. ©2018 AACR..

The cell adhesion glycoprotein E-cadherin (CDH1) is commonly inactivated in breast tumors. Precision medicine approaches that exploit this characteristic are not available. Using perturbation screens in breast tumor cells with CRISPR/Cas9-engineered CDH1 mutations, we identified synthetic lethality between E-cadherin deficiency and inhibition of the tyrosine kinase ROS1. Data from large-scale genetic screens in molecularly diverse breast tumor cell lines established that the E-cadherin/ROS1 synthetic lethality was not only robust in the face of considerable molecular heterogeneity but was also elicited with clinical ROS1 inhibitors, including foretinib and crizotinib. ROS1 inhibitors induced mitotic abnormalities and multinucleation in E-cadherin-defective cells, phenotypes associated with a defect in cytokinesis and aberrant p120 catenin phosphorylation and localization. In vivo, ROS1 inhibitors produced profound antitumor effects in multiple models of E-cadherin-defective breast cancer. These data therefore provide the preclinical rationale for assessing ROS1 inhibitors, such as the licensed drug crizotinib, in appropriately stratified patients.Significance: E-cadherin defects are common in breast cancer but are currently not targeted with a precision medicine approach. Our preclinical data indicate that licensed ROS1 inhibitors, including crizotinib, should be repurposed to target E-cadherin-defective breast cancers, thus providing the rationale for the assessment of these agents in molecularly stratified phase II clinical trials. Cancer Discov; 8(4); 498-515. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 371..

ARID1A is a tumour suppressor gene that is frequently mutated in clear cell and endometrioid carcinomas of the ovary and endometrium and is an important clinical biomarker for novel treatment approaches for patients with ARID1A defects. However, the accuracy of ARID1A immunohistochemistry (IHC) as a surrogate for mutation status has not fully been established for patient stratification in clinical trials. Here we tested whether ARID1A IHC could reliably predict ARID1A mutations identified by next-generation sequencing. Three commercially available antibodies - EPR13501 (Abcam), D2A8U (Cell Signaling), and HPA005456 (Sigma) - were optimised for IHC using cell line models and human tissue, and screened across a cohort of 45 gynaecological tumours. IHC was scored independently by three pathologists using an immunoreactive score. ARID1A mutation status was assessed using two independent sequencing platforms and the concordance between ARID1A mutation and protein expression was evaluated using Receiver Operating Characteristic statistics. Overall, 21 ARID1A mutations were identified in 14/43 assessable tumours (33%), the majority of which were predicted to be deleterious. Mutations were identified in 6/17 (35%) ovarian clear cell carcinomas, 5/8 (63%) ovarian endometrioid carcinomas, 2/5 (40%) endometrial carcinomas, and 1/7 (14%) carcinosarcomas. ROC analysis identified greater than 95% concordance between mutation status and IHC using a modified immunoreactive score for all three antibodies allowing a definitive cut-point for ARID1A mutant status to be calculated. Comprehensive assessment of concordance of ARID1A IHC and mutation status identified EPR13501 as an optimal antibody, with 100% concordance between ARID1A mutation status and protein expression, across different gynaecological histological subtypes. It delivered the best inter-rater agreement between all pathologists, as well as a clear cost-benefit advantage. This could allow patients to be accurately stratified based on their ARID1A IHC status into early phase clinical trials..

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. SIGNIFICANCE: The matrisome is a complex network of extracellular matrix (ECM) and ECM-associated proteins that provides scaffolding function to tissues and plays important roles in the regulation of fundamental cellular processes. However, due to its inherent complexity and insolubility, proteomic studies of the matrisome typically require the application of enrichment workflows prior to MS analysis. Such enrichment strategies often lead to losses in soluble matrisome-associated components. In this study, 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. We show that SWATH MS provides a more reproducible coverage of the matrisome compared to data-dependent analysis (DDA) MS. We also demonstrate that SWATH MS is capable of accurate quantification of matrisomal proteins without prior ECM enrichment and fractionation, which may simplify sample handling workflows and avoid losses in matrisome-associated proteins commonly linked to ECM enrichment..

Purpose: Metaplastic breast carcinoma (MBC) is a rare and aggressive histologic type of breast cancer, predominantly of triple-negative phenotype, and characterized by the presence of malignant cells showing squamous and/or mesenchymal differentiation. We sought to define the repertoire of somatic genetic alterations and the mutational signatures of MBCs.Experimental Design: Whole-exome sequencing was performed in 35 MBCs, with 16, 10, and 9 classified as harboring chondroid, spindle, and squamous metaplasia as the predominant metaplastic component. The genomic landscape of MBCs was compared with that of triple-negative invasive ductal carcinomas of no special type (IDC-NST) from The Cancer Genome Atlas. Wnt and PI3K/AKT/mTOR pathway activity was assessed using a qPCR assay.Results: MBCs harbored complex genomes with frequent TP53 (69%) mutations. In contrast to triple-negative IDC-NSTs, MBCs more frequently harbored mutations in PIK3CA (29%), PIK3R1 (11%), ARID1A (11%), FAT1 (11%), and PTEN (11%). PIK3CA mutations were not found in MBCs with chondroid metaplasia. Compared with triple-negative IDC-NSTs, MBCs significantly more frequently harbored mutations in PI3K/AKT/mTOR pathway-related (57% vs. 22%) and canonical Wnt pathway-related (51% vs. 28%) genes. MBCs with somatic mutations in PI3K/AKT/mTOR or Wnt pathway-related genes displayed increased activity of the respective pathway.Conclusions: MBCs are genetically complex and heterogeneous, and are driven by a repertoire of somatic mutations distinct from that of triple-negative IDC-NSTs. Our study highlights the genetic basis and the importance of PI3K/AKT/mTOR and Wnt pathway dysregulation in MBCs and provides a rationale for the metaplastic phenotype and the reported responses to PI3K/AKT/mTOR inhibitors in these tumors. Clin Cancer Res; 23(14); 3859-70. ©2017 AACR..

PURPOSE: To determine if models of ovarian clear cell carcinomas (OCCCs) harbouring defects in homologous recombination (HR) DNA repair of double strand breaks (DSBs) are sensitive to cisplatin and/or PARP inhibition. EXPERIMENTAL DESIGN: The HR status of 12 OCCC cell lines was determined using RAD51/γH2AX foci formation assays. Sensitivity to cisplatin and the PARP inhibitor BMN-673 was correlated with HR status. BRCA1, BRCA2, MRE11 and PTEN loss of expression was investigated as a potential determinant of BMN-673 sensitivity. A tissue microarray containing 50 consecutive primary OCCC was assessed for PTEN expression using immunohistochemistry. RESULTS: A subset of OCCC cells displayed reduced RAD51 foci formation in the presence of DNA DSBs, suggestive of HR defects. HR-defective OCCC cells, with the exception of KOC-7c, had higher sensitivity to cisplatin/ BMN-673 than HR-competent OCCC cell lines (Log10 SF50 -9.4 (SD +/- 0.29) vs -8.1 (SD +/- 0.35), mean difference 1.3, p < 0.01). Of the cell lines studied, two, TOV-21G and KOC-7c, showed loss of PTEN expression. In primary OCCCs, loss of PTEN expression was observed in 10% (5/49) of cases. CONCLUSIONS: A subset of OCCC cells are sensitive to PARP inhibition in vitro, which can be predicted by HR defects as defined by γH2AX/RAD51 foci formation. These results provide a rationale for the testing of HR deficiency and PARP inhibitors as a targeted therapy in a subset of OCCCs..

Metaplastic breast cancer (MBC) is a rare special histologic type of triple-negative breast cancer, characterized by the presence of neoplastic cells showing differentiation towards squamous epithelium and/or mesenchymal elements. Here we sought to define whether histologically distinct subgroups of MBCs would be underpinned by distinct genomic and/or transcriptomic alterations. Microarray-based copy number profiling identified limited but significant differences between the distinct MBC subtypes studied here, despite the limited sample size (n = 17). In particular, we found that, compared to MBCs with chondroid or squamous cell metaplasia, MBCs with spindle cell differentiation less frequently harbored gain of 7q11.22-23 encompassing CLDN3 and CLDN4, consistent with their lower expression of claudins and their association with the claudin-low molecular classification. Microarray-based and RNA-sequencing-based gene expression profiling revealed that MBCs with spindle cell differentiation differ from MBCs with chondroid or squamous cell metaplasia on the expression of epithelial-to-mesenchymal transition-related genes, including down-regulation of CDH1 and EPCAM. In addition, RNA-sequencing revealed that the histologic patterns observed in MBCs are unlikely to be underpinned by a highly recurrent expressed fusion gene or a pathognomonic expressed mutation in cancer genes. Loss of PTEN expression or mutations affecting PIK3CA or TSC2 observed in 8/17 MBCs support the contention that PI3K pathway activation plays a role in the development of MBCs. Our data demonstrate that despite harboring largely similar patterns of gene copy number alterations, MBCs with spindle cell, chondroid and squamous differentiation are distinct at the transcriptomic level but are unlikely to be defined by specific pathognomonic genetic alterations..

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..

The majority of borderline ovarian tumours (BOTs) behave in a benign fashion, but some may show aggressive behavior. The reason behind this has not been elucidated. The epidermal growth factor receptor (EGFR) is known to contribute to cell survival signals as well as metastatic potential of some tumours. EGFR expression and gene status have not been thoroughly investigated in BOTs as it has in ovarian carcinomas. In this study we explore protein expression as well as gene mutations and amplifications of EGFR in BOTs in comparison to a subset of other epithelial ovarian tumours. We studied 85 tumours, including 61 BOTs, 10 low grade serous carcinomas (LGSCs), 9 high grade serous carcinomas (HGSCs) and 5 benign epithelial tumours. EGFR protein expression was studied using immunohistochemistry. Mutations were investigated by Sanger sequencing exons 18-21 of the tyrosine kinase domain of EGFR. Cases with comparatively higher protein expression were examined for gene amplification by chromogenic in situ hybridization. We also studied the tumours for KRAS and BRAF mutations. Immunohistochemistry results revealed both cytoplasmic and nuclear EGFR expression with variable degrees between tumours. The level of nuclear localization was relatively higher in BOTs and LGSCs as compared to HGSCs or benign tumours. The degree of nuclear expression of BOTs showed no significant difference from that in LGSCs (mean ranks 36.48, 33.05, respectively, p=0.625), but was significantly higher than in HGSCs (mean ranks: 38.88, 12.61 respectively, p< 0.001) and benign tumours (mean ranks: 35.18, 13.00 respectively, p= 0.010). Cytoplasmic expression level was higher in LGSCs. No EGFR gene mutations or amplification were identified, yet different polymorphisms were detected. Five different types of point mutations in the KRAS gene and the V600E BRAF mutation were detected exclusively in BOTs and LGSCs. Our study reports for the first time nuclear localization of EGFR in BOTs. The nuclear localization similarities between BOTs and LGSCs and not HGSCs support the hypothesis suggesting evolution of LGSCs from BOTs. We also confirm that EGFR mutations and amplifications are not molecular events in the pathogenesis of BOTs..

BACKGROUND: The intra-tumor diversity of cancer cells is under intense investigation; however, little is known about the heterogeneity of the tumor microenvironment that is key to cancer progression and evolution. We aimed to assess the degree of microenvironmental heterogeneity in breast cancer and correlate this with genomic and clinical parameters. METHODS AND FINDINGS: We developed a quantitative measure of microenvironmental heterogeneity along three spatial dimensions (3-D) in solid tumors, termed the tumor ecosystem diversity index (EDI), using fully automated histology image analysis coupled with statistical measures commonly used in ecology. This measure was compared with disease-specific survival, key mutations, genome-wide copy number, and expression profiling data in a retrospective study of 510 breast cancer patients as a test set and 516 breast cancer patients as an independent validation set. In high-grade (grade 3) breast cancers, we uncovered a striking link between high microenvironmental heterogeneity measured by EDI and a poor prognosis that cannot be explained by tumor size, genomics, or any other data types. However, this association was not observed in low-grade (grade 1 and 2) breast cancers. The prognostic value of EDI was superior to known prognostic factors and was enhanced with the addition of TP53 mutation status (multivariate analysis test set, p = 9 × 10-4, hazard ratio = 1.47, 95% CI 1.17-1.84; validation set, p = 0.0011, hazard ratio = 1.78, 95% CI 1.26-2.52). Integration with genome-wide profiling data identified losses of specific genes on 4p14 and 5q13 that were enriched in grade 3 tumors with high microenvironmental diversity that also substratified patients into poor prognostic groups. Limitations of this study include the number of cell types included in the model, that EDI has prognostic value only in grade 3 tumors, and that our spatial heterogeneity measure was dependent on spatial scale and tumor size. CONCLUSIONS: To our knowledge, this is the first study to couple unbiased measures of microenvironmental heterogeneity with genomic alterations to predict breast cancer clinical outcome. We propose a clinically relevant role of microenvironmental heterogeneity for advanced breast tumors, and highlight that ecological statistics can be translated into medical advances for identifying a new type of biomarker and, furthermore, for understanding the synergistic interplay of microenvironmental heterogeneity with genomic alterations in cancer cells..

New targeted approaches to ovarian clear cell carcinomas (OCCC) are needed, given the limited treatment options in this disease and the poor response to standard chemotherapy. Using a series of high-throughput cell-based drug screens in OCCC tumor cell models, we have identified a synthetic lethal (SL) interaction between the kinase inhibitor dasatinib and a key driver in OCCC, ARID1A mutation. Imposing ARID1A deficiency upon a variety of human or mouse cells induced dasatinib sensitivity, both in vitro and in vivo, suggesting that this is a robust synthetic lethal interaction. The sensitivity of ARID1A-deficient cells to dasatinib was associated with G1-S cell-cycle arrest and was dependent upon both p21 and Rb. Using focused siRNA screens and kinase profiling, we showed that ARID1A-mutant OCCC tumor cells are addicted to the dasatinib target YES1. This suggests that dasatinib merits investigation for the treatment of patients with ARID1A-mutant OCCC. Mol Cancer Ther; 15(7); 1472-84. ©2016 AACR..

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The initiation and progression of breast cancer from the transformation of the normal epithelium to ductal carcinoma in situ (DCIS) and invasive disease is a complex process involving the acquisition of genetic alterations and changes in gene expression, alongside microenvironmental and recognized histological alterations. Here, we sought to comprehensively characterise the genomic and transcriptomic features of the MCF10 isogenic model of breast cancer progression, and to functionally validate potential driver alterations in three-dimensional (3D) spheroids that may provide insights into breast cancer progression, and identify targetable alterations in conditions more similar to those encountered in vivo. We performed whole genome, exome and RNA sequencing of the MCF10 progression series to catalogue the copy number and mutational and transcriptomic landscapes associated with progression. We identified a number of predicted driver mutations (including PIK3CA and TP53) that were acquired during transformation of non-malignant MCF10A cells to their malignant counterparts that are also present in analysed primary breast cancers from The Cancer Genome Atlas (TCGA). Acquisition of genomic alterations identified MYC amplification and previously undescribed RAB3GAP1-HRAS and UBA2-PDCD2L expressed in-frame fusion genes in malignant cells. Comparison of pathway aberrations associated with progression showed that, when cells are grown as 3D spheroids, they show perturbations of cancer-relevant pathways. Functional interrogation of the dependency on predicted driver events identified alterations in HRAS, PIK3CA and TP53 that selectively decreased cell growth and were associated with progression from preinvasive to invasive disease only when cells were grown as spheroids. Our results have identified changes in the genomic repertoire in cell lines representative of the stages of breast cancer progression, and demonstrate that genetic dependencies can be uncovered when cells are grown in conditions more like those in vivo. The MCF10 progression series therefore represents a good model with which to dissect potential biomarkers and to evaluate therapeutic targets involved in the progression of breast cancer. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland..

One approach to identifying cancer-specific vulnerabilities and therapeutic targets is to profile genetic dependencies in cancer cell lines. Here, we describe data from a series of siRNA screens that identify the kinase genetic dependencies in 117 cancer cell lines from ten cancer types. By integrating the siRNA screen data with molecular profiling data, including exome sequencing data, we show how vulnerabilities/genetic dependencies that are associated with mutations in specific cancer driver genes can be identified. By integrating additional data sets into this analysis, including protein-protein interaction data, we also demonstrate that the genetic dependencies associated with many cancer driver genes form dense connections on functional interaction networks. We demonstrate the utility of this resource by using it to predict the drug sensitivity of genetically or histologically defined subsets of tumor cell lines, including an increased sensitivity of osteosarcoma cell lines to FGFR inhibitors and SMAD4 mutant tumor cells to mitotic inhibitors..

Subunits of the SWI/SNF chromatin remodeling complex are mutated in a significant proportion of human cancers. Malignant rhabdoid tumors (MRTs) are lethal pediatric cancers characterized by a deficiency in the SWI/SNF subunit SMARCB1. Here, we employ an integrated molecular profiling and chemical biology approach to demonstrate that the receptor tyrosine kinases (RTKs) PDGFRα and FGFR1 are coactivated in MRT cells and that dual blockade of these receptors has synergistic efficacy. Inhibitor combinations targeting both receptors and the dual inhibitor ponatinib suppress the AKT and ERK1/2 pathways leading to apoptosis. MRT cells that have acquired resistance to the PDGFRα inhibitor pazopanib are susceptible to FGFR inhibitors. We show that PDGFRα levels are regulated by SMARCB1 expression, and assessment of clinical specimens documents the expression of both PDGFRα and FGFR1 in rhabdoid tumor patients. Our findings support a therapeutic approach in cancers with SWI/SNF deficiencies by exploiting RTK coactivation dependencies..

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..

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..

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Mutations in genes encoding proteins involved in RNA splicing have been found to occur at relatively high frequencies in several tumour types including myelodysplastic syndromes, chronic lymphocytic leukaemia, uveal melanoma, and pancreatic cancer, and at lower frequencies in breast cancer. To investigate whether dysfunction in RNA splicing is implicated in the pathogenesis of breast cancer, we performed a re-analysis of published exome and whole genome sequencing data. This analysis revealed that mutations in spliceosomal component genes occurred in 5.6% of unselected breast cancers, including hotspot mutations in the SF3B1 gene, which were found in 1.8% of unselected breast cancers. SF3B1 mutations were significantly associated with ER-positive disease, AKT1 mutations, and distinct copy number alterations. Additional profiling of hotspot mutations in a panel of special histological subtypes of breast cancer showed that 16% and 6% of papillary and mucinous carcinomas of the breast harboured the SF3B1 K700E mutation. RNA sequencing identified differentially spliced events expressed in tumours with SF3B1 mutations including the protein coding genes TMEM14C, RPL31, DYNL11, UQCC, and ABCC5, and the long non-coding RNA CRNDE. Moreover, SF3B1 mutant cell lines were found to be sensitive to the SF3b complex inhibitor spliceostatin A and treatment resulted in perturbation of the splicing signature. Albeit rare, SF3B1 mutations result in alternative splicing events, and may constitute drivers and a novel therapeutic target in a subset of breast cancers..

Metaplastic breast carcinoma is a rare and aggressive histologic type of breast cancer, preferentially displaying a triple-negative phenotype. We sought to define the transcriptomic heterogeneity of metaplastic breast cancers on the basis of current gene expression microarray-based classifiers, and to determine whether these tumors display gene copy number profiles consistent with those of BRCA1-associated breast cancers. Twenty-eight consecutive triple-negative metaplastic breast carcinomas were reviewed, and the metaplastic component present in each frozen specimen was defined (ie, spindle cell, squamous, chondroid metaplasia). RNA and DNA extracted from frozen sections with tumor cell content >60% were subjected to gene expression (Illumina HumanHT-12 v4) and copy number profiling (Affymetrix SNP 6.0), respectively. Using the best practice PAM50/claudin-low microarray-based classifier, all metaplastic breast carcinomas with spindle cell metaplasia were of claudin-low subtype, whereas those with squamous or chondroid metaplasia were preferentially of basal-like subtype. Triple-negative breast cancer subtyping using a dedicated website (http://cbc.mc.vanderbilt.edu/tnbc/) revealed that all metaplastic breast carcinomas with chondroid metaplasia were of mesenchymal-like subtype, spindle cell carcinomas preferentially of unstable or mesenchymal stem-like subtype, and those with squamous metaplasia were of multiple subtypes. None of the cases was classified as immunomodulatory or luminal androgen receptor subtype. Integrative clustering, combining gene expression and gene copy number data, revealed that metaplastic breast carcinomas with spindle cell and chondroid metaplasia were preferentially classified as of integrative clusters 4 and 9, respectively, whereas those with squamous metaplasia were classified into six different clusters. Eight of the 26 metaplastic breast cancers subjected to SNP6 analysis were classified as BRCA1-like. The diversity of histologic features of metaplastic breast carcinomas is reflected at the transcriptomic level, and an association between molecular subtypes and histology was observed. BRCA1-like genomic profiles were found only in a subset (31%) of metaplastic breast cancers, and were not associated with a specific molecular or histologic subtype..

Breast cancers with BRCA1 germline mutation have a characteristic DNA copy number (CN) pattern. We developed a test that assigns CN profiles to be 'BRCA1-like' or 'non-BRCA1-like', which refers to resembling a BRCA1-mutated tumor or resembling a tumor without a BRCA1 mutation, respectively. Approximately one third of the BRCA1-like breast cancers have a BRCA1 mutation, one third has hypermethylation of the BRCA1 promoter and one third has an unknown reason for being BRCA1-like. This classification is indicative of patients' response to high dose alkylating and platinum containing chemotherapy regimens, which targets the inability of BRCA1 deficient cells to repair DNA double strand breaks. We investigated whether this classification can be reliably obtained with next generation sequencing and copy number platforms other than the bacterial artificial chromosome (BAC) array Comparative Genomic Hybridization (aCGH) on which it was originally developed. We investigated samples from 230 breast cancer patients for which a CN profile had been generated on two to five platforms, comprising low coverage CN sequencing, CN extraction from targeted sequencing panels (CopywriteR), Affymetrix SNP6.0, 135K/720K oligonucleotide aCGH, Affymetrix Oncoscan FFPE (MIP) technology, 3K BAC and 32K BAC aCGH. Pairwise comparison of genomic position-mapped profiles from the original aCGH platform and other platforms revealed concordance. For most cases, biological differences between samples exceeded the differences between platforms within one sample. We observed the same classification across different platforms in over 80% of the patients and kappa values of at least 0.36. Differential classification could be attributed to CN profiles that were not strongly associated to one class. In conclusion, we have shown that the genomic regions that define our BRCA1-like classifier are robustly measured by different CN profiling technologies, providing the possibility to retro- and prospectively investigate BRCA1-like classification across a wide range of CN platforms..

Breast cancer is known to display considerable inter- and intra-tumour genetic heterogeneity. It is now widely accepted that no two breast cancers harbour the same complement of genomic alterations, and that both primary and metastatic breast cancers are composed of multiple genetically diverse subclones that evolve under different selective pressures. Recent work published in the Journal of Pathology by Desmedt and colleagues questions the evolutionary dynamics of multi-focal breast cancer with similar pathological features by studying the mutational repertoire of different lesions. Whilst the majority of the lesions showed some common driver alterations, one-third lacked any common mutations, suggesting very early clonal divergence. These and other recent observations underscore the need for a fundamental understanding of the rules governing breast cancer evolution, and highlight the need for in-depth assessment of driver alterations for appropriate patient management and selective treatment..

Adenoid cystic carcinoma (AdCC) is a rare type of triple-negative breast cancer (TNBC) characterized by the presence of the MYB-NFIB fusion gene. The molecular underpinning of breast AdCCs other than the MYB-NFIB fusion gene remains largely unexplored. Here we sought to define the repertoire of somatic genetic alterations of breast AdCCs. We performed whole-exome sequencing, followed by orthogonal validation, of 12 breast AdCCs to determine the landscape of somatic mutations and gene copy number alterations. Fluorescence in situ hybridization and reverse-transcription PCR were used to define the presence of MYB gene rearrangements and MYB-NFIB chimeric transcripts. Unlike common forms of TNBC, we found that AdCCs have a low mutation rate (0.27 non-silent mutations/Mb), lack mutations in TP53 and PIK3CA and display a heterogeneous constellation of known cancer genes affected by somatic mutations, including MYB, BRAF, FBXW7, SMARCA5, SF3B1 and FGFR2. MYB and TLN2 were affected by somatic mutations in two cases each. Akin to salivary gland AdCCs, breast AdCCs were found to harbour mutations targeting chromatin remodelling, cell adhesion, RNA biology, ubiquitination and canonical signalling pathway genes. We observed that, although breast AdCCs had rather simple genomes, they likely display intra-tumour genetic heterogeneity at diagnosis. Taken together, these findings demonstrate that the mutational burden and mutational repertoire of breast AdCCs are more similar to those of salivary gland AdCCs than to those of other types of TNBCs, emphasizing the importance of histological subtyping of TNBCs. Furthermore, our data provide direct evidence that AdCCs harbour a distinctive mutational landscape and genomic structure, irrespective of the disease site of origin..

INTRODUCTION: Abundance of immune cells has been shown to have prognostic and predictive significance in many tumor types. Beyond abundance, the spatial organization of immune cells in relation to cancer cells may also have significant functional and clinical implications. However there is a lack of systematic methods to quantify spatial associations between immune and cancer cells. METHODS: We applied ecological measures of species interactions to digital pathology images for investigating the spatial associations of immune and cancer cells in breast cancer. We used the Morisita-Horn similarity index, an ecological measure of community structure and predator-prey interactions, to quantify the extent to which cancer cells and immune cells colocalize in whole-tumor histology sections. We related this index to disease-specific survival of 486 women with breast cancer and validated our findings in a set of 516 patients from different hospitals. RESULTS: Colocalization of immune cells with cancer cells was significantly associated with a disease-specific survival benefit for all breast cancers combined. In HER2-positive subtypes, the prognostic value of immune-cancer cell colocalization was highly significant and exceeded those of known clinical variables. Furthermore, colocalization was a significant predictive factor for long-term outcome following chemotherapy and radiotherapy in HER2 and Luminal A subtypes, independent of and stronger than all known clinical variables. CONCLUSIONS: Our study demonstrates how ecological methods applied to the tumor microenvironment using routine histology can provide reproducible, quantitative biomarkers for identifying high-risk breast cancer patients. We found that the clinical value of immune-cancer interaction patterns is highly subtype-specific but substantial and independent to known clinicopathologic variables that mostly focused on cancer itself. Our approach can be developed into computer-assisted prediction based on histology samples that are already routinely collected..

BACKGROUND: HER2 is overexpressed and amplified in approximately 15% of invasive breast cancers, and is the molecular target and predictive marker of response to anti-HER2 agents. In a subset of these cases, heterogeneous distribution of HER2 gene amplification can be found, which creates clinically challenging scenarios. Currently, breast cancers with HER2 amplification/overexpression in just over 10% of cancer cells are considered HER2-positive for clinical purposes; however, it is unclear as to whether the HER2-negative components of such tumors would be driven by distinct genetic alterations. Here we sought to characterize the pathologic and genetic features of the HER2-positive and HER2-negative components of breast cancers with heterogeneous HER2 gene amplification and to define the repertoire of potential driver genetic alterations in the HER2-negative components of these cases. RESULTS: We separately analyzed the HER2-negative and HER2-positive components of 12 HER2 heterogeneous breast cancers using gene copy number profiling and massively parallel sequencing, and identified potential driver genetic alterations restricted to the HER2-negative cells in each case. In vitro experiments provided functional evidence to suggest that BRF2 and DSN1 overexpression/amplification, and the HER2 I767M mutation may be alterations that compensate for the lack of HER2 amplification in the HER2-negative components of HER2 heterogeneous breast cancers. CONCLUSIONS: Our results indicate that even driver genetic alterations, such as HER2 gene amplification, can be heterogeneously distributed within a cancer, and that the HER2-negative components are likely driven by genetic alterations not present in the HER2-positive components, including BRF2 and DSN1 amplification and HER2 somatic mutations..

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..

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..

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..

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..

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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..

INTRODUCTION: Breast cancer remains a significant scientific, clinical and societal challenge. This gap analysis has reviewed and critically assessed enduring issues and new challenges emerging from recent research, and proposes strategies for translating solutions into practice. METHODS: More than 100 internationally recognised specialist breast cancer scientists, clinicians and healthcare professionals collaborated to address nine thematic areas: genetics, epigenetics and epidemiology; molecular pathology and cell biology; hormonal influences and endocrine therapy; imaging, detection and screening; current/novel therapies and biomarkers; drug resistance; metastasis, angiogenesis, circulating tumour cells, cancer 'stem' cells; risk and prevention; living with and managing breast cancer and its treatment. The groups developed summary papers through an iterative process which, following further appraisal from experts and patients, were melded into this summary account. RESULTS: The 10 major gaps identified were: (1) understanding the functions and contextual interactions of genetic and epigenetic changes in normal breast development and during malignant transformation; (2) how to implement sustainable lifestyle changes (diet, exercise and weight) and chemopreventive strategies; (3) the need for tailored screening approaches including clinically actionable tests; (4) enhancing knowledge of molecular drivers behind breast cancer subtypes, progression and metastasis; (5) understanding the molecular mechanisms of tumour heterogeneity, dormancy, de novo or acquired resistance and how to target key nodes in these dynamic processes; (6) developing validated markers for chemosensitivity and radiosensitivity; (7) understanding the optimal duration, sequencing and rational combinations of treatment for improved personalised therapy; (8) validating multimodality imaging biomarkers for minimally invasive diagnosis and monitoring of responses in primary and metastatic disease; (9) developing interventions and support to improve the survivorship experience; (10) a continuing need for clinical material for translational research derived from normal breast, blood, primary, relapsed, metastatic and drug-resistant cancers with expert bioinformatics support to maximise its utility. The proposed infrastructural enablers include enhanced resources to support clinically relevant in vitro and in vivo tumour models; improved access to appropriate, fully annotated clinical samples; extended biomarker discovery, validation and standardisation; and facilitated cross-discipline working. CONCLUSIONS: With resources to conduct further high-quality targeted research focusing on the gaps identified, increased knowledge translating into improved clinical care should be achievable within five years..

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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..

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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..

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..

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..

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)..

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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..

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..

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..

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..

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..

A whole chromosome arm loss of 16q belongs to the most frequent and earliest chromosomal alterations in invasive and in situ breast cancers of all common subtypes. Besides E-cadherin, several putative tumour suppressor genes residing on 16q in breast cancer have been investigated. However, the significance of these findings has remained unclear. Thus, other mechanisms leading to gene loss of function (eg haploinsufficiency, or distortion of multiple regulative subnetworks) remain to be tested as a hypothesis. To define the effect on gene expression of whole-arm loss of chromosome 16q in invasive breast cancer, we performed global gene expression analysis on a series of 18 genetically extensively characterized invasive ductal breast carcinomas and verified the results by quantitative real-time PCR (qRT-PCR). The distribution of the differential genes across the genome and their expression status was studied. A second approach by qRT-PCR in an independent series of 30 breast carcinomas helped to narrow down the observed effect. Whole-arm chromosome 16q losses, irrespective of other chromosomal changes, are associated with decreased expression of a number of candidate genes located on 16q (eg CDA08, CGI-128, SNTB2, NQO1, SF3B3, KIAA0174, ATBF1, GABARAPL2, KARS, GCSH, MBTPS1 and ZDHHC7) in breast carcinomas with a low degree of genetic instability. qRT-PCR provided evidence to suggest that the expression of these genes was reduced in a gene dosage-dependent manner. The differential expression of the candidate genes according to the chromosomal 16q-status vanished in genetically advanced breast cancer cases and changed ER status. These results corroborate previous reports about the importance of whole-arm loss of chromosome 16q in breast carcinogenesis and give evidence for the first time that haploinsufficiency, in the sense of a gene dosage effect, might be an important contributing factor in the early steps of breast carcinogenesis..

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..

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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..

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..

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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..

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..

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..

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..

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..

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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..

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..

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..

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..

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..

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..

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..

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..

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..

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..

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..

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..

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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..

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..

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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..

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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..

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..

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..

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..

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..

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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..

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..

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..

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..

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..

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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..

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..

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..

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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..

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..

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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..

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..

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..

Deletions found in several types of human tumor, including carcinomas of the colorectum, breast, and lung, suggest the presence of a potential tumor suppressor gene(s) on chromosome 15. Common regions of deletion in these tumors are at 15q15 and 15q21. Here, we have analyzed loss of heterozygosity (LOH) on chromosome 15 to ascertain its potential involvement in the development and progression of transitional cell carcinoma (TCC) of the bladder. A panel of 26 polymorphic markers, spanning 15q12-15q22, were used to map regions of LOH in 51 TCCs. LOH was found for at least one marker in the region 15q14-15q15.3 in 20 of 51 (39%) tumors. Deletion mapping defined two minimum regions of deletion: a distal region between the markers D15S514 and D15S537 at 15q15.1-15q15.3 (estimated as 3 Mb) and a more proximal region between the markers D15S971 and D15S1042 at 15q14 (estimated as 1.1 Mb). Analysis of a panel of 33 bladder tumor cell lines revealed regions of contiguous homozygosity for markers in 15q15, indicating likely LOH. Fluorescence in situ hybridization analysis demonstrated that mitotic recombination is the predicted mechanism of LOH in two of these. These regions of LOH on 15q may contain tumor suppressor genes the loss or inactivation of which is associated with TCC development. The DNA repair gene RAD51 at 15q15.1 represents a candidate 15q tumor suppressor gene. Expression analysis of rad51 protein in tumor cell lines revealed variable levels of expression but no significant loss of expression in cell lines with likely 15q LOH..

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