Showing papers by "Alan Ashworth published in 2009"

Inhibition of Poly(ADP-Ribose) Polymerase in Tumors from BRCA Mutation Carriers

Abstract: Background The inhibition of poly(adenosine diphosphate [ADP]–ribose) polymerase (PARP) is a potential synthetic lethal therapeutic strategy for the treatment of cancers with specific DNA-repair defects, including those arising in carriers of a BRCA1 or BRCA2 mutation. We conducted a clinical evaluation in humans of olaparib (AZD2281), a novel, potent, orally active PARP inhibitor. Methods This was a phase 1 trial that included the analysis of pharmacokinetic and pharmacodynamic characteristics of olaparib. Selection was aimed at having a study population enriched in carriers of a BRCA1 or BRCA2 mutation. Results We enrolled and treated 60 patients; 22 were carriers of a BRCA1 or BRCA2 mutation and 1 had a strong family history of BRCA-associated cancer but declined to undergo mutational testing. The olaparib dose and schedule were increased from 10 mg daily for 2 of every 3 weeks to 600 mg twice daily continuously. Reversible dose-limiting toxicity was seen in one of eight patients receiving 400 mg twice...

Synthetic lethal targeting of PTEN mutant cells with PARP inhibitors

Abstract: The tumour suppressor gene, phosphatase and tensin homolog (PTEN), is one of the most commonly mutated genes in human cancers. Recent evidence suggests that PTEN is important for the maintenance of genome stability. Here, we show that PTEN deficiency causes a homologous recombination (HR) defect in human tumour cells. The HR deficiency caused by PTEN deficiency, sensitizes tumour cells to potent inhibitors of the DNA repair enzyme poly(ADP-ribose) polymerase (PARP), both in vitro and in vivo. PARP inhibitors are now showing considerable promise in the clinic, specifically in patients with mutations in either of the breast cancer susceptibility genes BRCA1 or BRCA2. The data we present here now suggests that the clinical assessment of PARP inhibitors should be extended beyond those with BRCA mutations to a larger group of patients with PTEN mutant tumours.

Does chromosome 17 centromere copy number predict polysomy in breast cancer? A fluorescence in situ hybridization and microarray-based CGH analysis†

Abstract: Approximately 8% of breast cancers show increased copy numbers of chromosome 17 centromere (CEP17) by fluorescence in situ hybridization (FISH) (ie average CEP17 >3.0 per nucleus). Currently, this pattern is believed to represent polysomy of chromosome 17. HER2-amplified cancers have been shown to harbour complex patterns of genetic aberrations of chromosome 17, in particular involving its long arm. We hypothesized that aberrant copy numbers of CEP17 in FISH assays may not necessarily represent true chromosome 17 polysomy. Eighteen randomly selected CEP17 polysomic cases and a control group of ten CEP17 disomic cases, as defined by dual-colour FISH, were studied by microarray-based comparative genomic hybridization (aCGH), which was performed on microdissected samples using a 32K tiling-path bacterial artificial chromosome microarray platform. Additional FISH probes were employed for SMS (17p11.2) and RARA (17q21.2) genes, as references for chromosome 17 copy number. Microarray-based comparative genomic hybridization revealed that 11 out of the 18 polysomic cases harboured gains of 17q with involvement of the centromere, one displayed 17q gain sparing the centromeric region, and only one could be defined as polysomic. The remaining five cases displayed amplification of the centromeric region. Among these, one case, showing score 2+ by immunohistochemistry and 8.5 HER2 mean copy number, was classified as not amplified by HER2/CEP17 ratio and as amplified by HER2/SMS ratio. Our results suggest that true chromosome 17 polysomy is likely to be a rare event in breast cancer and that CEP17 copy number greater than 3.0 in FISH analysis is frequently related to gain or amplification of the centromeric region. Larger studies investigating the genetic profiles of CEP17 polysomic cases are warranted.

Tiling path genomic profiling of grade 3 invasive ductal breast cancers.

Abstract: Purpose: To characterize the molecular genetic profiles of grade 3 invasive ductal carcinomas of no special type using high-resolution microarray-based comparative genomic hybridization (aCGH) and to identify recurrent amplicons harboring putative therapeutic targets associated with luminal, HER-2, and basal-like tumor phenotypes. Experimental Design: Ninety-five grade 3 invasive ductal carcinomas of no special type were classified into luminal, HER-2, and basal-like subgroups using a previously validated immunohistochemical panel. Tumor samples were microdissected and subjected to aCGH using a tiling path 32K BAC array platform. Selected regions of recurrent amplification were validated by means of in situ hybridization. Expression of genes pertaining to selected amplicons was investigated using quantitative real-time PCR and gene silencing was done using previously validated short hairpin RNA constructs. Results: We show that basal-like and HER-2 tumors are characterized by "sawtooth" and "firestorm" genetic patterns, respectively, whereas luminal cancers were more heterogeneous. Apart from confirming known amplifications associated with basal-like (1q21, 10p, and 12p), luminal (8p12, 11q13, and 11q14), and HER-2 (17q12) cancers, we identified previously unreported recurrent amplifications associated with each molecular subgroup: 19q12 in basal-like, 1q32.1 in luminal, and 14q12 in HER-2 cancers. PPM1D gene amplification (17q23.2) was found in 20% and 8% of HER-2 and luminal cancers, respectively. Silencing of PPM1D by short hairpin RNA resulted in selective loss of viability in tumor cell lines harboring the 17q23.2 amplification. Conclusions: Our results show the power of aCGH analysis in unraveling the genetic profiles of specific subgroups of cancer and for the identification of novel therapeutic targets.

Structural basis for recruitment of BRCA2 by PALB2

Abstract: The breast cancer 2, early onset protein (BRCA2) is central to the repair of DNA damage by homologous recombination. BRCA2 recruits the recombinase RAD51 to sites of damage, regulates its assembly into nucleoprotein filaments and thereby promotes homologous recombination. Localization of BRCA2 to nuclear foci requires its association with the partner and localizer of BRCA2 (PALB2), mutations in which are associated with cancer predisposition, as well as subtype N of Fanconi anaemia. We have determined the structure of the PALB2 carboxy-terminal β-propeller domain in complex with a BRCA2 peptide. The structure shows the molecular determinants of this important protein–protein interaction and explains the effects of both cancer-associated truncating mutants in PALB2 and missense mutations in the amino-terminal region of BRCA2.

Methotrexate induces oxidative DNA damage and is selectively lethal to tumour cells with defects in the DNA mismatch repair gene MSH2

Abstract: Mutations in the MSH2 gene predispose to a number of tumourigenic conditions, including hereditary non-polyposis colon cancer (HNPCC). MSH2 encodes a protein in the mismatch repair (MMR) pathway which is involved in the removal of mispairs originating during replication or from damaged DNA. To identify new therapeutic strategies for the treatment of cancer arising from MMR deficiency, we screened a small molecule library encompassing previously utilized drugs and drug-like molecules to identify agents selectively lethal to cells lacking functional MSH2. This approach identified the drug methotrexate as being highly selective for cells with MSH2 deficiency. Methotrexate treatment caused the accumulation of potentially lethal 8-hydroxy-2'-deoxyguanosine (8-OHdG) oxidative DNA lesions in both MSH2 deficient and proficient cells. In MSH2 proficient cells, these lesions were rapidly cleared, while in MSH2 deficient cells 8-OHdG lesions persisted, potentially explaining the selectivity of methotrexate. Short interfering (si)RNA mediated silencing of the target of methotrexate, dihydrofolate reductase (DHFR), was also selective for MSH2 deficiency and also caused an accumulation of 8-OHdG. This suggested that the ability of methotrexate to modulate folate synthesis via inhibition of DHFR, may explain MSH2 selectivity. Consistent with this hypothesis, addition of folic acid to culture media substantially rescued the lethal phenotype caused by methotrexate. While methotrexate has been used for many years as a cancer therapy, our observations suggest that this drug may have particular utility for the treatment of a subset of patients with tumours characterized by MSH2 mutations.

PPM1D Is a Potential Therapeutic Target in Ovarian Clear Cell Carcinomas

Abstract: Purpose: To identify therapeutic targets in ovarian clear cell carcinomas, a chemoresistant and aggressive type of ovarian cancer. Experimental Design: Twelve ovarian clear cell carcinoma cell lines were subjected to tiling path microarray comparative genomic hybridization and genome-wide expression profiling analysis. Regions of high-level amplification were defined and genes whose expression levels were determined by copy number and correlated with gene amplification were identified. The effects of inhibition of PPM1D were assessed using short hairpin RNA constructs and a small-molecule inhibitor (CCT007093). The prevalence of PPM1D amplification and mRNA expression was determined using chromogenic in situ hybridization and quantitative real-time reverse transcription-PCR in a cohort of pure ovarian clear cell carcinomas and on an independent series of unselected epithelial ovarian cancers. Results: Array-based comparative genomic hybridization analysis revealed regions of high-level amplification on 1q32, 1q42, 2q11, 3q24-q26, 5p15, 7p21-p22, 11q13.2-q13.4, 11q22, 17q21-q22, 17q23.2, 19q12-q13, and 20q13.2. Thirty-four genes mapping to these regions displayed expression levels that correlated with copy number gains/amplification. PPM1D had significantly higher levels of mRNA expression in ovarian clear cell carcinoma cell lines harboring gains/amplifications of 17q23.2. PPM1D inhibition revealed that PPM1D expression and phosphatase activity are selectively required for the survival of ovarian clear cell carcinoma cell lines with 17q23.2 amplification. PPM1D amplification was significantly associated with ovarian clear cell carcinoma histology ( P = 0.0003) and found in 10% of primary ovarian clear cell carcinomas. PPM1D expression levels were significantly correlated with PPM1D gene amplification in primary ovarian clear cell carcinomas. Conclusion: Our data provide strong circumstantial evidence that PPM1D is a potential therapeutic target for a subgroup of ovarian clear cell carcinomas.

Expression, mutation and copy number analysis of platelet-derived growth factor receptor A (PDGFRA) and its ligand PDGFA in gliomas

Abstract: Expression, mutation and copy number analysis of platelet-derived growth factor receptor A (PDGFRA) and its ligand PDGFA in gliomas

Integrated Functional, Gene Expression and Genomic Analysis for the Identification of Cancer Targets

Abstract: The majority of new drug approvals for cancer are based on existing therapeutic targets. One approach to the identification of novel targets is to perform high-throughput RNA interference (RNAi) cellular viability screens. We describe a novel approach combining RNAi screening in multiple cell lines with gene expression and genomic profiling to identify novel cancer targets. We performed parallel RNAi screens in multiple cancer cell lines to identify genes that are essential for viability in some cell lines but not others, suggesting that these genes constitute key drivers of cellular survival in specific cancer cells. This approach was verified by the identification of PIK3CA, silencing of which was selectively lethal to the MCF7 cell line, which harbours an activating oncogenic PIK3CA mutation. We combined our functional RNAi approach with gene expression and genomic analysis, allowing the identification of several novel kinases, including WEE1, that are essential for viability only in cell lines that have an elevated level of expression of this kinase. Furthermore, we identified a subset of breast tumours that highly express WEE1 suggesting that WEE1 could be a novel therapeutic target in breast cancer. In conclusion, this strategy represents a novel and effective strategy for the identification of functionally important therapeutic targets in cancer.

Loss of 16q in High Grade Breast Cancer is Associated with Estrogen Receptor Status: Evidence for Progression in Tumors with a Luminal Phenotype?

Abstract: Loss of the long arm of chromosome 16 (16q) is observed in the vast majority of low grade/grade I (GI) invasive ductal carcinomas of no special type (IDC-NSTs), whereas this event is uncommonly seen in high grade/grade III (GIII) IDC-NSTs. Together with data on the pathology and genetics of breast cancer recurrences, this has led to the proposal that GI and GIII breast cancers evolve through distinct genetic pathways and that progression from GI to GIII is an unlikely biological phenomenon. We compared the genomic profiles of GIII-IDC-NSTs with 16q whole arm loss (16qWL) according to estrogen receptor (ER) status. 16qWL was found in 36.5% of cases and was significantly associated with ER expression and luminal phenotype. ER+ GIII-IDC-NSTs with 16qWL displayed significantly higher levels of genomic instability than ER+ IDC-NSTs without 16qWL. Furthermore, ER+ and ER- IDC-NSTs stratified according to the presence of 16qWL harbored distinct patterns of genetic aberrations. Interestingly, ER+/16qWL tumors displayed genetic features usually found in tumors with homologous DNA repair defects and significantly more frequently harbored heterozygous loss of BRCA2 than the remaining ER+ cancers. Our results demonstrate that approximately one third of GIII tumors harbor 16qWL, confirming that progression from low to high grade breast cancer is not found in the majority of breast cancers. 16qWL was significantly more prevalent in ER+/luminal GIII-IDC-NSTs. Given that GI breast cancers harbor a luminal phenotype, our results suggest that if progression from GI to GIII breast cancer does happen, it may preferentially occur in breast cancers of luminal phenotype.

LKB1 is essential for the proliferation of T-cell progenitors and mature peripheral T cells.

Abstract: The serine/threonine kinase LKB1 has a conserved role in Drosophila and nematodes to co-ordinate cell metabolism. During T lymphocyte development in the thymus, progenitors need to synchronize increased metabolism with the onset of proliferation and differentiation to ensure that they can meet the energy requirements for development. The present study explores the role of LKB1 in this process and shows that loss of LKB1 prevents thymocyte differentiation and the production of peripheral T lymphocytes. We find that LKB1 is required for several key metabolic processes in T-cell progenitors. For example, LKB1 controls expression of CD98, a key subunit of the L-system aa transporter and is also required for the pre-TCR to induce and sustain the regulated phosphorylation of the ribosomal S6 subunit, a key regulator of protein synthesis. In the absence of LKB1 TCR-beta-selected thymocytes failed to proliferate and did not survive. LBK1 was also required for survival and proliferation of peripheral T cells. These data thus reveal a conserved and essential role for LKB1 in the proliferative responses of both thymocytes and mature T cells.

Genomic instability and the selection of treatments for cancer

Abstract: A critical link exists between DNA mutation and chromosomal rearrangements (genomic instability) and cancer development. This genomic instability can manifest itself as small changes at the nucleotide level or as gross chromosomal alterations. Mutations in the genes that encode DNA damage response proteins are responsible for a variety of genomic instability syndromes including hereditary non-polyposis colorectal carcinoma, Bloom's syndrome, ataxia-telangiectasia, BRCA-associated breast and ovarian cancers and Fanconi anaemia. Similarly, epigenetic silencing of genes associated with the maintenance of genomic stability have also been implicated in the pathogenesis of cancer. Here, we discuss how different tumours may be classified not only by tumour site but also by the type of underlying genetic instability. This type of classification may assist in the optimization of existing treatment regimens as well as informing the development of new therapeutic approaches.

A high-resolution integrated analysis of genetic and expression profiles of breast cancer cell lines

Abstract: Tumour cell lines derived from breast cancer patients constitute one of the cornerstones of breast cancer research. To characterise breast cancer cell lines at the genetic level, we have developed a full tiling path bacterial artificial chromosome (BAC) array collection for comparative genomic hybridisation (aCGH). This aCGH BAC collection covers 98% of the entire human genome at a resolution of 40-60 kbp. We have used this platform alongside an in-house produced 17 K cDNA microarray set to characterise the genetic and transcriptomic profiles of 24 breast cancer cell lines, as well as cell types derived from non-diseased breast. We demonstrate that breast cancer cell lines have genomic and transcriptomic features that recapitulate those of primary breast cancers and can be reliably subclassified into basal-like and luminal subgroups. By overlaying aCGH and transcriptomic data, we have identified 753 genes whose expression correlate with copy number; this list comprised numerous oncogenes recurrently amplified and overexpressed in breast cancer (e.g., HER2, MYC, CCND1 and AURKA). Finally, we demonstrate that although breast cancer cell lines have genomic features usually found in grade III breast cancers (i.e., gains of 1q, 8q and 20q), basal-like and luminal cell lines are characterised by distinct genomic aberrations.

Mixed micropapillary-ductal carcinomas of the breast: a genomic and immunohistochemical analysis of morphologically distinct components.

Abstract: Micropapillary carcinomas (MPCs) can present as a rare histological special type of breast cancer; however, this histological type is more frequently found admixed with invasive ductal carcinomas of no special type (IDC-NSTs). We have previously demonstrated that pure MPCs constitute a distinct entity at the morphological and genetic levels. Here, we sought to determine whether mixed MPCs have genomic aberrations similar to those found in pure MPCs, and to investigate whether the distinct morphological components of MPCs harbour different genetic aberrations. Using high-resolution microarray comparative genomic hybridization (aCGH), we profiled a series of 10 MPCs of mixed histology and 20 IDC-NSTs matched for grade and oestrogen receptor (ER) status. In addition, we generated tissue microarrays containing a series of 24 pure and 40 mixed MPCs and performed immunohistochemical analysis with ER, progesterone receptor (PR), Ki-67, HER2, cytokeratin (CK) 5/6, CK14, CK17, EGFR, topoisomerase-IIalpha, cyclin D1, caveolin-1 and E-cadherin antibodies. In situ hybridization was employed to evaluate the prevalence of HER2, TOP2A, EGFR, CCND1, MYC and FGFR1 gene amplification. Our results demonstrate that mixed MPCs harbour similar patterns of genomic aberrations and phenotype (82.5% luminal and 17.5% HER2) compared to pure MPCs. A comparison between the distinct morphological components of mixed MPCs in a pairwise fashion revealed that both components harbour strikingly similar genomic profiles. When compared to grade- and ER-matched IDC-NSTs, mixed MPCs significantly more frequently harboured amplification of multiple regions on 8q (adjusted Fisher's p value < 0.05). Furthermore, mixed MPCs displayed higher proliferative rates than grade- and ER-matched IDC-NSTs. Our results suggest that micropapillary differentiation in breast cancer may identify a subgroup of more aggressive ER-positive breast carcinomas, even in those featuring a mixed histology, and that mixed MPCs are more closely related to pure MPCs than to IDC-NSTs.

Targeting Tankyrase 1 as a therapeutic strategy for BRCA-associated cancer

Abstract: The BRCA1 and BRCA2 proteins are involved in the maintenance of genome stability and germ-line loss-of-function mutations in either BRCA1 or BRCA2 strongly predispose carriers to cancers of the breast and other organs. It has been demonstrated previously that inhibiting elements of the cellular DNA maintenance pathways represents a novel therapeutic approach to treating tumors in these individuals. Here, we show that inhibition of the telomere-associated protein, Tankyrase 1, is also selectively lethal with BRCA deficiency. We also demonstrate that the selectivity caused by inhibition of Tankyrase 1 is associated with an exacerbation of the centrosome amplification phenotype associated with BRCA deficiency. We propose that inhibition of Tankyrase 1 could be therapeutically exploited in BRCA-associated cancers.

Parallel RNAi and compound screens identify the PDK1 pathway as a target for tamoxifen sensitization.

Abstract: Tamoxifen is the most commonly used drug to treat breast cancer and acts by blocking ER alpha (oestrogen receptor (alpha) signalling. Although highly effective, its usefulness is limited by the development of resistance. Given this, strategies that limit resistance by sensitizing cells to tamoxifen may be of use in the clinic. To gain insight into how this might be achieved, We used Chemical and genetic screens to identify targets and small-molecule inhibitors that Cause tamoxifen sensitization. A high-throughput genetic Screen, using an RNA interference library targeting 779 kinases and related proteins, identified the PDK1 (phosphoinositide-dependent kinase 1) signalling pathway as It strong determinant of sensitivity to Multiple ER alpha antagonists, including tamoxifen. A chemical screen using existing drugs and known kinase inhibitors also identified inhibitors of the PDK1 pathway, including triciribine and tetrandrine. Aside from identifying novel agents and targets for tamoxifen sensitization, this approach also provides evidence that performing chemical and genetic screens in parallel may be useful.

Pregnancy in the mature adult mouse does not alter the proportion of mammary epithelial stem/progenitor cells

Abstract: In humans, an early full-term pregnancy reduces lifetime breast cancer risk by up to 50% whereas a later pregnancy (>35 years old) can increase lifetime risk. Several mechanisms have been suggested, including changes in levels of circulating hormones, changes in the way the breast responds to these hormones, changes in gene expression programmes which may alter susceptibility to transformation and changes to mammary stem cell numbers or behaviour. Previous studies have shown that the mammary tissue isolated from both virgin and parous mice has the ability to repopulate a cleared mammary fat pad in transplant experiments. Limited dilution transplant assays have demonstrated that early pregnancy (at 5 weeks of age) reduces stem/progenitor cell numbers in the mouse mammary epithelium by twofold. However, the effects on stem/progenitor cell numbers in the mammary epithelium of a pregnancy in older animals have not yet been tested. Mice were put through a full-term pregnancy at 9 weeks of age, when the mammary epithelium is mature. The total mammary epithelium was purified from parous 7-week post-lactation and age-matched virgin mice and analysed by flow cytometry and limiting dilution cleared fat pad transplants. There were no significant differences in the proportions of different mammary epithelial cell populations or numbers of CD24+/Low Sca-1- CD49fHigh cells (stem cell enriched basal mammary epithelial compartment). There was no significant difference in stem/progenitor cell frequency based on limiting dilution transplants between the parous and age-matched virgin epithelium. Although differences between parous and virgin mammary epithelium at later time points post lactation or following multiple pregnancies cannot be ruled out, there are no differences in stem/progenitor cell numbers between mammary epithelium isolated from parous animals which were mated at 9 weeks old and virgin animals. However, a recent report has suggested that animals that were mated at 5 weeks old have a twofold reduction in stem/progenitor cell numbers. This is of interest given the association between early, but not late, pregnancy and breast cancer risk reduction in humans. However, a mechanistic connection between stem cell numbers and breast cancer risk remains to be established.

Family History, Genetic Testing, and Clinical Risk Prediction: Pooled Analysis of CHEK2*1100delC in 1,828 Bilateral Breast Cancers and 7,030 Controls

Abstract: If breast cancers arise independently in each breast the odds ratio (OR) for bilateral breast cancer for carriers of CHEK2 1100delC should be approximately 5.5, the square of the reported OR for a first primary (OR, 2.34). In the subset of bilateral cases with one or more affected relatives, the predicted carrier OR should be approximately 9. We have tested these predictions in a pooled set of 1,828 cases with 2 primaries and 7,030 controls from 8 studies. The second primary OR for CHEK2 1100delC carriers was 6.43 (95% confidence interval, 4.33-9.56; P < 0.0001), significantly greater than the published estimate for a first primary (P < 0.001) but consistent with its square. The predicted increase in carrier OR with increasing numbers of affected relatives was seen using bilateral cases from the UK (P(trend) = 0.0003) and Finland (P(trend) = 0.37), although not using those from the Netherlands and Russia (P = 0.001 for heterogeneity between countries). Based on a standard genetic model, we predict lifetime risks for CHEK2 1100delC carrier and noncarrier daughters of bilateral breast cancer cases of 37% and 18%, respectively. Our results imply that clinical management of the daughter of a woman with bilateral breast cancer should depend on her CHEK2 1100delC carrier status. This and other moderate penetrance breast cancer susceptibility alleles, together with family history data, will thus identify increasing numbers of women at potentially very high risk. Before such predictions are accepted by clinical geneticists, however, further population-based evidence is needed on the effect of CHEK2 1100delC and other moderate penetrance alleles in women with a family history of breast cancer.

Genomic profile of a secretory breast cancer with an ETV6–NTRK3 duplication

Abstract: Background: Secretory breast cancer (SBC) is a rare entity characterised by indolent clinical behaviour, distinctive histological features and the presence of a recurrent chromosomal translocation t(12;15)(p13;q25), leading to the formation of the ETV6–NTRK3 fusion gene. Aim: To describe the molecular genetic features of a case of SBC which harbours a duplication of the t(12;15) translocation. Methods: Tiling path array comparative genomic hybridisation (aCGH) analysis and fluorescence in situ hybridisation (FISH) using in-house-generated probes for ETV6 , NTRK3 and the fusion genes, centromeric probes for chromosomes 12 and 15, and a commercially available split-apart ETV6/NTRK3 probe. Results: FISH revealed the presence of a duplication of the translocation t(12;15), which resulted from the gain of one copy of the derivative chromosome der(15)t(12;15), retention of one normal copy of both ETV6 and NTRK3 genes and deletion of the derivative chromosome der(12)t(12;15). Consistent with FISH findings, aCGH revealed copy number gains of ETV6 and NTRK3 and deletions encompassing the regions centromeric to ETV6 and telomeric to NTRK3 . Additional regions of copy number changes included gains of 10q21, 10q26.3, 12p13.3–p13.31 15q11–q25.3 and 16pq and losses of 6q24.1–q27, 12p13.2–q12 and 15q25.3–q26.3. Conclusions: To the best of our knowledge, this is the first time a carcinoma has been shown to harbour a duplication of the ETV6–NTRK3 translocation. The presence of an additional copy of the derivative chromosome der(15)t(12;15) coupled with deletion of the other derivative der(12)t(12;15) in the modal population of cancer cells suggests that this was either an early phenomenon or conferred additional growth advantage on neoplastic cells.

Tumor Mapping in 2 Large Multigenerational Families With CYLD Mutations: Implications for Disease Management and Tumor Induction

Abstract: Objectives: To comprehensively ascertain the extent and severity of clinical features in affected individuals from 2 large families with proven heterozygous mutations in the CYLD locus and to correlate these findings with the 3 appendageal tumor predisposition syndromes (familial cylindromatosis, Brooke-Spiegler syndrome, and multiple familial trichoepitheliomas) known to be associated with such germline mutations. Design: Interfamilial and intrafamilial observational study. Setting: Tertiary genetic and dermatology referral center. Participants: Thirty-four individuals recruited from 2 large multigenerational families with CYLD mutations. Clinical details, history, and tumor maps were obtained from all participants; in 18, the information was corroborated by detailed clinical examination. Main Outcome Measures: Tumor density, distribution and histologic findings, associated medical conditions, patient symptoms, and impact of disease on quality of life. Results: The severity of penetrance and phenotype varied within families. Although an approximately equal female to male predisposition was noted, 5 women and 1 man (of 26 patients surveyed [23%]) had undergone total scalp removal. The average age at onset was 16 years (range, 8-30 years). Symptoms reported by affected patients included painful tumors (in 12 of 23 patients [52%] who answered the question), conductive deafness, and sexual dysfunction. Of the 26 surveyed patients, tumors were noted on the scalp in 21 (81%), on the trunk in 18 (69%), and in the pubic area in 11 (42%). Tumor mapping provided clinical evidence that correlated with hormonally stimulated hair follicles being particularly vulnerable to loss of heterozygosity and tumor induction. Conclusions: The burden of disease at sites other than the head and neck appears to be underreported in the literature and greatly affects quality of life. Differentiation between the clinical diagnoses has little prognostic or clinical utility in genetic counseling, even within individuals from the same family. Thus, we suggest an encompassing diagnosis of "CYLD cutaneous syndrome." Finally, the clinical distribution of tumors suggests that hormonal factors may play an important role in tumor induction in these patients.

Microglandular adenosis or microglandular adenoma? A molecular genetic analysis of a case associated with atypia and invasive carcinoma

Abstract: Aims: Microglandular adenosis (MGA) is a rare breast lesion, which has long been considered to be hyperplastic. However, atypical forms of MGA (AMGA) and invasive carcinomas arising in the background of MGA are recorded. Recent studies have suggested that MGA may be a non-obligate precursor of invasive carcinomas that are negative for hormone receptors and lack HER-2 overexpression (triple-negative phenotype). The aim of this study was to determine whether MGA is clonal and whether it harbours chromosomal aberrations similar to those found in matched invasive ductal carcinoma of no special type (IDC-NST). Methods and results: We report on a case comprising MGA, AMGA and a high-grade IDC-NST. The three components were separately microdissected and subjected to genetic analysis with high-resolution microarray comparative genomic hybridisation. Identical genetic changes were detected in all components with subsequent acquisition of additional genetic aberrations in the invasive component, suggesting that MGA was the substrate for the development of the invasive carcinoma. Immunohistochemistry revealed concordant profiles across all components, characterized by triple-negative phenotype and variable positivity for basal markers. Conclusions: Similar to adenomas, MGA is, at least in some cases, a clonal lesion and may be a non-obligate precursor of a subgroup of high-grade triple-negative and basal-like breast carcinomas.

Conditional deletion of the Lkb1 gene in the mouse mammary gland induces tumour formation.

Abstract: Heterozygous germline mutations in the LKB1 (STK11) gene cause Peutz-Jeghers syndrome (PJS), an autosomal dominant disorder characterized by hamartomatous polyposis of the gastrointestinal tract and an increased risk of colorectal, breast, and other cancers. To model the role of LKB1 mutation in mammary tumourigenesis, we have used a conditional gene targeting strategy to generate a mouse in which exons encoding the kinase domain of Lkb1 were deleted specifically in the mammary gland. Mammary gland tumours developed in these mice with a latency of 46-85 weeks and occurred in the thoracic or inguinal glands. These tumours were grade 2 invasive ductal carcinomas or solid papillary carcinomas with histological features similar to those described in breast cancers arising in patients with PJS. This mouse model of Lkb1 deficiency provides a potentially useful tool to investigate the role of Lkb1 in tumourigenesis and to guide the development of therapeutic approaches.

Genome‐wide transcriptomic profiling of microdissected human breast tissue reveals differential expression of KIT (c‐Kit, CD117) and oestrogen receptor‐α (ERα) in response to therapeutic radiation

Abstract: The pathogenesis of late normal tissue fibrosis after high-dose ionizing radiation involves multiple cell types and signalling pathways but is not well understood. To identify the molecular changes occurring after radiotherapy, paired normal tissue samples were collected from the non-irradiated breast and from the treated breast of women who had undergone curative radiotherapy for early breast cancer months or years previously. As radiation may induce distinct transcriptional changes in the different components of the breast, laser capture microdissection and gene expression microarray profiling were performed separately for epithelial and stromal components and selected genes were validated using immunohistochemistry. In the epithelial compartment, a reduction of KIT (c-Kit; CD117) and a reciprocal increase in ESR1 (oestrogen receptor-alpha, ERalpha) mRNA and protein levels were seen in irradiated compared to non-irradiated samples. In the stromal compartment, extracellular matrix genes including FN1 (fibronectin 1) and CTGF (connective tissue growth factor; CCN2) were increased. Further investigation revealed that c-Kit and ERalpha were expressed in distinct subpopulations of luminal epithelial cells. Interlobular c-Kit-positive mast cells were also increased in irradiated cases not showing features of post-radiation atrophy. Pathway analysis revealed 'cancer, reproductive system disease and tumour morphology' as the most significantly enriched network in the epithelial compartment, whereas in the stromal component, a significant enrichment for 'connective tissue disorders, dermatological diseases and conditions, genetic disorder' and 'cancer, tumour morphology, infection mechanism' networks was observed. These data identify previously unreported changes in the epithelial compartment and show altered expression of genes implicated in late normal tissue injury in the stromal compartment of normal breast tissue. The findings are relevant to both fibrosis and atrophy occurring after radiotherapy for early breast cancer.

CRK7 modifies the MAPK pathway and influences the response to endocrine therapy.

Abstract: Endocrine therapies, which inhibit estrogen receptor (ER)alpha signaling, are the most common and effective treatment for ER alpha-positive breast cancer. However, the use of these agents is limited by the frequent development of resistance. The cyclin-dependent kinase family member CRK7 (aka CRKRS) was identified from an RNA interference screen for modifiers of tamoxifen sensitivity. Here, we demonstrate that silencing of CRK7 not only causes resistance to tamoxifen but also leads to resistance to additional endocrine therapies including ICI 182780 and estrogen deprivation, a model of aromatase inhibition. We show that CRK7 silencing activates the mitogen-activated protein kinase (MAPK)-signaling pathway, which causes a loss of ER dependence, resulting in endocrine therapy resistance. This study identifies a novel role for CRK7 in MAPK regulation and resistance to estrogen signaling inhibitors.

RNA interference screening demystified

Abstract: Genetic screens, where the effects of modifying gene function on cell behaviour are assessed in a systematic fashion, have for some time provided useful information to those interested in disease pathogenesis and treatment. Genetic screens exploiting the phenomenon of RNA interference (RNAi) are now becoming commonplace. This article explains the different RNAi screen formats and describes some of the applications of RNAi screening that may be pertinent to the research pathologist.

Bringing DNA repair in tumors into focus.

Abstract: Biomarkers are of crucial importance in optimizing cancer therapies. In this edition of Clinical Cancer Research, Banuelos and colleagues assess H2AX phosphorylation as a predictive biomarker of response to DNA damaging agents. We discuss these results as well as the impact that double strand break repair biomarkers may have in cancer therapy.

Integrative Molecular Profiling of Triple Negative Breast Cancers Identifies Potential Therapeutic Targets Including Amplifications of FGFR2.

Abstract: Triple negative breast cancers (TNBCs) have a relatively poor prognosis emphasising the need to identify new subtype specific target therapies. Based on the concept of oncogene addiction, we searched for potential therapeutic targets by identifying genes consistently over-expressed when amplified in TNBC. Fifty six TNBCs were subjected to high resolution tiling path microarray-based comparative genomic hybridisation (aCGH); out of these cases, 24 were also subjected to genome-wide microarray-based mRNA expression analysis. TNBCs showed a high level of genetic instability, with recurrent regions of amplification (>4 copies) included multiple regions on 1q and 8q, 3q25, 10p14, 10q26, 13q34, 15q26 and 19q12-19q13. Integration of aCGH and expression data revealed 38 genes that were significantly overexpressed when amplified. This list includes known oncogenes and potential therapeutic targets, such as MCL1 (1q21.2), FGFR2 (10q26.3), BUB3 (10q26.3), RAB20 (13q34),PKN1 (19p13.12), and NOTCH3 (19p13.12). To validate FGFR2 as a therapeutic target, we screened a panel of cell lines, by western blotting and aCGH, and identified two TNBC cell lines with FGFR2 amplification. In these cell lines FGFR2 was constitutively active in a ligand independent manner, and RNA interference-mediated silencing of FGFR2 selectively decreased survival of cell lines harbouring FGFR2 amplification. Likewise FGFR2 amplified cell lines were highly sensitive to FGFR tyrosine kinase inhibitor PD173074 (IC50 Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3147.

Identifying Modifiers of Tamoxifen Sensitivity Using High-Throughput Genetic and Chemical Screens

Abstract: Endocrine therapies, which inhibit estrogen receptor (ERα) signalling, are the most common and effective treatments for ERα positive breast cancer. However, the utility of these agents is limited by the frequent development of resistance. The precise mechanisms underlying endocrine therapy resistance remain incompletely understood. In our laboratory, an RNA interference (RNAi) screen was used to identify modifiers of sensitivity to the most commonly used endocrine therapy, tamoxifen. The cyclin-dependent kinase 10 (CDK10) gene was identified as an important determinant of resistance and the mechanism whereby this gene modulates sensitivity to tamoxifen was investigated further. Silencing of CDK10 gene expression was shown to activate the MAPK signalling pathway, circumventing the reliance of breast cancer cells upon estrogen signalling. Patients with ERα positive breast tumours that express low levels of CDK10 were shown to relapse early on tamoxifen and methylation of the CDK10 gene promoter was observed in a significant proportion of patients, suggesting a mechanism for loss of CDK10 expression in tamoxifen resistant tumours. By suppressing gene expression RNAi, to a certain extent, models the pharmacological inhibition of a target protein. We performed parallel small molecule screens alongside the RNAi screen to identify compounds that sensitise to tamoxifen. Both the RNAi and small molecule screens identified the PDK1 pathway as a potential target for sensitisation to inhibit the development of endocrine therapy resistance.

Correction: Lkb1 Deficiency Alters Goblet and Paneth Cell Differentiation in the Small Intestine

Abstract: The last line of the Author Contributions is incorrect. It should read: Wrote the paper: BYS JZ AC.