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Background Copy Number Alterations (CNAs) represent changes in the copy number of genomic segments of somatic cells due to chromosomal instability. CNAs include gene amplifications or deletions and can be involved in tumorigenesis. We analyzed CNAs data in pre- and post-treatment (ttm) tumors from patients (pts) with early breast cancer (BC) in the neoadjuvant trials GEICAM/2006-03 and GEICAM/2006-14, with the aim to identify CNAs in particular genomic regions (genetic entropy) associated with treatment response. Methods GEICAM/2006-03 (NCT00432172) HER2-negative pts were selectively treated according to clinical subtypes: triple negative (TN) pts were treated with standard anthracycline/taxane -based chemotherapy (AT-CT) +/- carboplatin, while luminal patients were randomized to AT-CT vs. hormonotherapy; GEICAM/2006-14 (NCT00841828) HER2+ pts received AT-CT plus anti-HER2 therapy.Shallow-whole genome Illumina sequencing DNA data from 204 paraffin-embedded tumors (100 pre- and 104 post-ttm) were segmented to obtain CNAs and recurrent altered genomic regions were defined. We used Wilcoxon test to analyze the frequency of altered regions and logistic regression analyses to explore their association with tumor response, in terms of pathological complete response (pCR) in breast and axilla. Validation of altered genes associated with therapy response was performed in the microarray gene expression-based Hatzis dataset (GSE25066) from pts receiving neoadjuvant AT-CT (1). Results A total of 672 regions covering the whole genome were identified upon analysis of CNAs data. Regions were categorized according to their alteration status as amplified, normal and lost. Comparative analysis of alterations revealed 11 regions significantly different (p Conclusions According to our results, neoadjuvant therapy can modulate genomic aberrations landscape in BC. Our data suggest that amplification of specific genes in the genomic locus (21q22.12) is involved in the neoadjuvant therapy response in early BC. (1): Hatzis et al., JAMA 2011, 305(18) 1873-81 Citation Format: Alba E, Rueda OM, Lluch A, Albanell J, Chin S-F, Chacón JI, Calvo L, De la Haba-Rodriguez J, Bermejo B, Ribelles N, Sánchez-Rovira P, Plazaola A, Barnadas A, Cirauqul B, Ramos M, Arcusa A, Carrasco E, Herranz J, Chiesa M, Caballero R, Santonja A, Rojo F, Caldas C. Genome copy number entropy as predictor of response for neoadjuvant therapy in early breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-12-03.

Background: Recent studies have demonstrated that women with early stage ER-positive (ER+) and HER2-negative (HER2-) breast cancer have a persistent risk of recurrence and cancer related death up to 20 years post diagnosis, highlighting the chronic nature of ER+ breast cancer and critical need to identify tumor characteristics that are more predictive of risk of recurrence than standard clinical covariates. However, progress in delineating the dynamics of breast cancer relapse and biomarkers of late recurrence has been hindered by the lack of large cohorts with long-term clinical follow-up and molecular information. Methods: We report the results of a cohort of 3,240 breast cancer patients from the United Kingdom and Canada with 20 years of follow-up (median 9.75 years), including 1,980 with accompanying molecular data from the primary breast tumor. Information for each patient on loco-regional recurrence (LR), distant recurrence (DR), and site(s) of metastases was collected. We developed a non-homogenous Markov chain model that accounted for different clinical endpoints and timescales, as well as competing risks of mortality and the distinct baseline hazards that characterize different molecular subgroups. This approach enabled robust analysis of the spatio-temporal dynamics of breast cancer recurrence across the clinical subgroups, PAM50 subgroups and the integrative clusters, while also enabling individual risk of relapse predictions. Results: We employed our multistate model to compute the probability of experiencing a LR or DR, as well as the baseline transition probabilities from surgery, LR or DR at various time intervals for average individuals in each of the clinical/molecular subgroups. These analyses reveal four late-recurring ER+ (predominantly HER2-) subgroups, together accounting for 26% of all ER+ tumors, with high (median 42-55%) risk of recurrence up to 20 years post-diagnosis. Each of these four subgroups maps to one of the Integrative Clusters, defined based on genomic copy number alterations and gene expression, and is enriched for a characteristic copy number amplification events: 11q13 (CCND1, RSF1), 8p12 (FGFR1, ZNF703), 17q23 (RPS6KB1) and 8q24 (MYC). These four molecular subgroups are superior in predicting late DR than standard clinical variables. Conclusions: A detailed understanding of the rates and routes of metastasis and their variability across the distinct molecular subtypes is essential for devising personalized approaches to breast cancer care. We describe a molecularly characterized breast cancer cohort with long-term clinical follow-up and a statistical modeling framework, enabling delineation of the dynamics of breast cancer recurrence at unprecedented resolution. These analyses reveal four late recurring ER+ subgroups and accompanying biomarkers that collectively define the quarter of ER+ cases at highest risk of recurrence. Our findings highlight opportunities for improved patient stratification and biomarker-driven clinical trials directed at the subset of breast cancer patients with persistent risk of recurrence. Citation Format: Curtis C, Rueda OM, Sammut S-J, Chin S-F, Caswell-Jin JL, Seoane JA, Callari M, Batra R, Pereira B, Bruna A, Ali HR, Provenzano E, Liu B, Parisien M, Gillett C, McKinney S, Green A, Murphy L, Purushotham A, Ellis I, Pharoah P, Rueda C, Aparicio S, Caldas C. Dynamics of breast cancer relapse reveal molecularly defined late recurring ER-positive subgroups: Results from the METABRIC study [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr GS3-06.

Background: Understanding to what extent a breast tumor's genetic composition may change over the course of a few months of neoadjuvant therapy has implications for optimal therapeutic approach. However, genomic changes observed across treatment may result from either treatment-induced clonal evolution or geographically disparate sampling of a heterogeneous tumor. We sought to characterize the geographic heterogeneity in primary breast tumors, and to incorporate this information into analysis of clonal evolution with neoadjuvant therapy. Methods: We assembled the largest cohort to date of multi-region (n=2-3) whole-exome sequenced (WES) or whole-genome sequenced untreated primary breast tumors with matched normal and adequate tumor purity for analysis: four tumors with data generated for this study and five tumors compiled from three previous studies. We also generated the first cohort of multi-region (n=2-6) WES breast tumors post-neoadjuvant HER2-targeted therapy and chemotherapy, sequencing one region from a pre-treatment diagnostic specimen, multiple regions from the post-treatment surgical specimen, and matched normal for five HER2+ breast tumors that did not achieve a pathologic complete response. We used an agent-based model of spatial tumor growth to investigate whether the mutational patterns we observed with treatment were consistent with pre-existing heterogeneity or treatment-induced selection. Results: In untreated primary breast tumors, on average 30% (range 1-70%) of apparently clonal mutations from a single region were absent or rare in a second, spatially disparate region (high-frequency regional, or HFR). Intra-tumor heterogeneity was similar post-treatment (HFR 28%, range 10-54%), and was higher in breast tumors than in previously analyzed colon, brain, lung, and esophageal tumors. Simulation studies confirmed that with high heterogeneity as observed in breast tumors, analysis of one pre-treatment and one post-treatment region could not distinguish treatment-induced clonal evolution from pre-existing heterogeneity; however, obtaining at least two post-treatment regions allowed for detection of clonal shifts with treatment. Analysis of multi-region data revealed that clonal replacement occurred with neoadjuvant therapy in two of the five tumors. Candidate causes of therapeutic resistance included amplifications in CCND1, ERBB4, and MYC in one subclone, and functional protein-altering mutations in ERCC2, SMO, and WT1 in another. Mathematical modeling suggested that these putative resistant subclones comprised 0.02-12.5% of the overall pre-treatment cell population, substantially larger than previous estimates of resistant tumor clone size. Conclusions: WES data from multiple regions of untreated and treated primary breast tumors revealed considerable heterogeneity that remained present throughout treatment with chemotherapy and HER2-targeted therapy, even while major clonal sweeps took place in a minority of tumors. Obtaining at least two samples for analysis from breast tumors post-neoadjuvant therapy may reveal the tumor's evolutionary path and, especially as increasing numbers of molecular and immune therapeutic targets are identified, inform new clinical strategies. Citation Format: Caswell-Jin JL, McNamara K, Reiter JG, Sun R, Hu Z, Ma Z, Suarez CJ, Tilk S, Raghavendra A, Forte V, Chin S-F, Bardwell H, Provenzano E, Caldas C, Lang J, West R, Tripathy D, Press MF, Curtis C. Clonal evolution and heterogeneity in breast tumors treated with neoadjuvant HER2-targeted therapy [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-06-01.

Invasive lobular carcinoma (ILC) is the second most common type of breast cancer after invasive ductal carcinoma (IDC), accounting for approximately 10-15% of all breast tumors. ILC is characterized by inactivation of E-Cadherin and neoplastic cells that invade the stroma in a "single-file" pattern. Women with ILC are usually older, have used hormone replacement therapy and are more likely to have hormone receptor–positive disease. ILCs have similar survival to IDCs at both five and 10 years, but despite this, the clinical course is distinct: ILCs are three times more likely to metastasize to the peritoneum, gastrointestinal tract, and ovaries and are more frequently bilateral. Therefore, tailored therapeutic options for this distinct, hard-to-treat subtype of breast cancer are required. As part of the RATHER FP7 HEALTH consortium (www.ratherproject.com), we carried out RNA-Seq analysis of 61 primary ILC samples and identified that high expression of the BET family protein Brd3 was associated with poor recurrence free survival (p=0.03, HR 8.63, CI 1.22-60.85). This observation was further validated in the independent METABRIC cohort (n=99), where again, high Brd3 expression was associated with poor recurrence-free survival (p With a number of BET inhibitors now entering clinical trials, the data described here suggest that BET inhibition is a rational therapeutic option for some ILC cases, and for those that do not respond, combination with venetoclax may be a suitable therapeutic strategy. In our cell line models, baseline Bcl-2 expression was sufficient to predict induction of apoptosis in response to JQ1 and could be used to guide therapeutic choice. These results should now be investigated in vivo before a prospective clinical trial. "This material is based upon works supported by the Irish Cancer Society Collaborative Cancer Research Centre BREAST-PREDICT Grant CCRC13GAL". Citation Format: O'Connor DP, Walsh L, Tarrant F, Chin S-F, Schouten P, Linn S, Bernards R, Caldas C, Gallagher WM, ni Chonghaile T. Bromodomain inhibitors represent a rational therapeutic option for the treatment of invasive lobular carcinoma [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-11-06.

Background: Circulating tumor DNA (ctDNA) analysis holds potential for minimal residual disease (MRD) detection in early stage breast cancer. However, sensitivity for MRD is limited due to low ctDNA levels in early stage patients and limited blood volumes. Loss of input DNA during library preparation, limited multiplexing or low sensitivity of current molecular methods further limit accuracy. To address this gap, we have developed TARgeted DIgital Sequencing (TARDIS), a novel method for simultaneous analysis of multiple patient-specific mutations in plasma DNA. Methods: Using tumor exome sequencing, we identify and prioritize somatic founder mutations, design nested primers and evaluate them for multiplex performance. Using 5-10 ng input plasma DNA, we perform 1) targeted linear pre-amplification to improve downstream molecular conversion, 2) single-stranded adapter ligation to incorporate unique molecular identifiers (UMIs) and 3) targeted PCR to prepare sequencing-ready libraries. The resulting sequencing reads have fixed target-specific ends and variable ligation ends. We utilize fragment size and UMIs to group sequencing reads into read families. To ensure specificity, we require targeted mutations are supported by 2 or more read families. Results: To assess analytical performance, we targeted 8 mutations in cell-free DNA reference samples with 0.25%-2% mutation allele fractions (AFs). Precision across 7-16 replicates at each AF level agreed with expectations of Poisson distribution, demonstrating effective analysis of ˜70% of input DNA. At 2%, 1%, 0.5% and 0.25% AFs, variant-level sensitivity was 96.4%, 96.4%, 91.1% and 65.8%, approaching the theoretical limit given input DNA. At 0.25% AF, 3-7 mutations were detected per sample, achieving 100% sample-level sensitivity. In 16 wild-type replicates, no targeted mutations were called (100% specificity). Averaging multiple mutations improved precision in sample-level AF estimates. Mean AFs from 8 mutations for the 2% sample were 2.34%-2.80% (5.8% CV). In 6 patients with breast cancer treated with neoadjuvant therapy (NAT), we analyzed 8-18 patient-specific mutations (mean 11.8). Before treatment, ctDNA was detected in 5/6 patients at mean AFs of 0.02%-1.19% (mean 0.40%), supported by 2-10 mutations (mean 5.6). Of these 5 patients, 4 had residual disease after NAT and ctDNA was detected pre-operatively or during NAT in 3/4 patients. 1 patient achieved pathological Complete Response and ctDNA was undetectable after NAT. Conclusions: Preliminary results suggest TARDIS enables accurate MRD detection after neoadjuvant therapy in patients with early stage breast cancer. On-going work is expanding this analysis to include additional patients and investigate the clinical validity of peri-operative ctDNA monitoring. Summary of clinical resultsPatientPre-NAT Stage (TNM)SubtypeNo. of Mutations TargetedBaseline ctDNA (AF%, No. of Mutations)ctDNA after or during NAT (AF%, No. of Mutations)Residual Tumor (TNM)1T3 N1ER+ PR+ HER2-8+ (0.02%, 2)-T2 N12T3 N0TNBC12+ (0.29%, 6)+ (0.01%, 1)T1a N03T2 N1TNBC18+ (1.19%, 10)+ (0.01%, 1)T1mi N04T3 N1TNBC10+ (0.02%, 3)+ (0.05%, 3)T3 N15T2 N0TNBC9+ (0.46%, 7)-pathCR6T1c N1TNBC14--pathCR Citation Format: McDonald BR, Contente-Cuomo T, Sammut S-J, Ernst B, Odenheimer-Bergman A, Perdigones N, Chin S-F, Farooq M, Cronin PA, Anderson KS, Kosiorek H, Northfelt D, McCullough A, Patel B, Caldas C, Pockaj B, Murtaza M. Multiplexed targeted digital sequencing of circulating tumor DNA to detect minimal residual disease in early and locally advanced breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-01-21.

Background: Copy-number variations (CNVs) are DNA changes that result in regions of the genome being either duplicated (copy number gains) or deleted (copy number losses). CNVs have been identified in 12% of the human genome. Although CNVs do not appear to have a major role in disease susceptibility, it is known that they are involved in drug metabolism, toxicity and response. Infection is a common dose and/or treatment limiting chemotherapy-related toxicity. Infections can be a major cause of morbidity and mortality. Although a limited number of studies have investigated the association of single nucleotide polymorphisms (SNPs) with severity of infection, no studies so far have identified CNV associations with chemotherapy-related infection. In a non-oncological setting, CNVs have been noted to be associated with altered risk of certain types of infections and severe sepsis. This study aims to evaluate whether CNV are associated with the risk of chemotherapy-related infection in a genome wide association study (GWAS) of breast cancer patients recruited to clinical trials and treated with chemotherapy regimens including paclitaxel, epirubicin, cyclophosphamide, gemcitabine, 5fluorouracil and methotrexate. Method: DNA from blood and saliva samples were collected as part of the pharmacogenetics GWAS, PGSNPS. Chemotherapy-related infection was assessed using the NCI Common Toxicity Criteria (CTC). Patients (n = 1921 samples) were classified as cases (NCI CTC grades 2–4) or as controls (NCI CTC grades 0–1). The Affymetrix SNP6.0 array, which contains more than 946,000 probes for the detection of CNVs and more than 906,600 SNPs, was used as the copy number estimation platform. DNA was segmented using DNAcopy (circular binary segmentation). CNVs were called based on their median and standard deviation. Log-ratios larger than the median plus 2 times the standard deviation were considered gains and log-ratios with values smaller than 2.5 times the standard deviation were considered as losses. Only regions with a copy number alteration present in at least a 10% of the samples were considered for the analysis. Fisher's exact test was performed on each of these regions and the variable ‘infection’. 2-side p-values were obtained and adjusted using Benjamin-Hochberg correction. Results: One CNV region (frequency of gain 0.16) showed an association with increased risk of infection (Odds Ratio=1.79, 95% Confidence Interval [1.34–2.40], P = 6.37 × 10−5). Five other CNV regions demonstrated copy number gains and three CNV regions showed copy number losses that were associated with an increased risk of infection (P = 10−4). All regions had a frequency of gain or loss > 0.10 and were therefore not rare variants. Conclusion: These data suggest that CNVs may play a role in increased risk of chemotherapy-related toxicity. The most significant CNVs associated with infection are currently undergoing validation using an independent cohort of adjuvant breast cancer patients with similar exposure to chemotherapy and comparable documentation of chemotherapy-related infection. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P3-08-05.

A previously healthy eleven month old male Malay infant presented with fever, upper respiratory tract infection and right knee swelling. Pallor, bilateral proptosis, hepatosplenomegaly, multiple scalp swellings and a right cheek swelling were observed. Investigations revealed that he had acute monoblastic leukemia or FAB M5a. Immunophenotyping by flow cytometry showed that the blast cells were positive for CD45, CD13, CD33, HLA-DR, CDllc, CD71, EMA, and Cytokeratin. They were negative for CD34, CD19, CD10, CD22, CD2, CD3, CD4, CD7, CD8, CD61, NK, Glycophorin A, and CD14. The monoblasts were used to evaluate anti-EMA and anti-cytokeratin. They were unexpectedly found to be positive. Acute monoblastic leukaemias are well known to show extramedullary infiltration and this may be their primary mode of presentation. Thus, in immunochemostry, when using EMA and cytokeratin expression in the differential diagnosis of neoplastic diseases, it is important to consider that monoblasts may express these markers as illustrated by this case.

A coiled quartz tubular reactor has been designed to measure the intrinsic reaction kinetics for homogeneous reactions at high temperatures up to 1100°C. Actual gas residence times were less than 100 ms. A simple and well-studied test reaction (i.e., the decomposition of nitrous oxide, N2O), with published intrinsic kinetics, was used to verify the operation of the experimental reactor. For this system, Peclet numbers (Pe = uL/DL) computed from experimental conversion data were greater than 1000, indicating that the plug flow assumption could be used with this reactor system to determine intrinsic rate expressions with errors of less than 5% for the conditions studied. On a concu un reacteur tubulaire a Serpentins en quartz afin de mesurer les cinetiques de reactions intrinseques pour des reactions homogenes a des temperatures elevees allant jusqu'a 1100°C. Les temps de sejour reels du gaz sont inferieurs a 100 ms. On a utilise une reaction d'essai simple et bien etudie (a savoir la decomposition de l'oxyde nitreux, N2O), ainsi qu'une des cinetiques intrinseques publiees, pour verifier le fonctionnement du reacteur experimental. Pour ce Systeme, les nombres de Peclet (Pe = uL/DL) calcules par ordinateur a partir de donnees de conversions experimentales sont superieurs a 1000, ce qui indique que l'hypothese de l'ecoulement piston pourrait etre utilisee avec ce Systeme de reacteur pour determiner les expressions de vitesses intrinseques avec des erreurs inferieures a 5% pour les conditions etudiees.

Experimental kinetic data are reported for key side reactions occurring in the front end [i. e. the reaction furnace (RF) and the waste heat boiler (WHB)] of modified Claus plants used for sulfur recovery from the sour gases evolved in the treatment of natural gas. An extensive experimental study was conducted in a high temperature tubular reactor system for two important homogenous gas-phase reactions. Firstly, experiments were carried out to study the oxidation of hydrogen sulfide and methane mixtures in the presence of oxygen. Secondly, the reaction between methane and sulfur dioxide was investigated experimentally. These results showed that methane was much less competitive for oxygen than hydrogen sulfide. Hence, in a partially oxidizing environment of a RF, data showed that methane reacted significantly with other major sulfur containing species, as secondary reactions, to form COS and especially CS2. This is highly problematic from an environmental point of view. Des donnees cinetiques experimentales sont presentees pour les principales reactions secondaires survenant dans la partie frontale [c.-a-d. le four de reaction (RF) et la chaudiere de recuperation des chaleurs perdues (WHB)] d'usines Claus modifiees pour la recuperation du soufre a partir des gaz acides produits dans le traitement du gaz naturel. Une etude experimental approfondie a ete menee dans un reacteur tubulaire a temperatures elevees pour deux reactions en phase gazeuse homogenes. On a d'abord mene des experiences afin d'etudier l'oxydation des melanges d'hydrogene sulfure et de methane en presence d'oxygene. On a ensuite etudie experimentalement la reaction entre le methane et le dioxyde de soufre. Les resultats montrent que le methane est beaucoup moins competitif pour l'oxygene que pour l'hydrogene sulfure. En consequence, dans un environnement partiellement oxydant d'un RF, les donnees montrent que le methane reagit de maniere importante avec les autres especes principales contenant du soufre, comme reactions secondaires, pour former du COS et particulierement du CS2.Ceciest hautement problematique d'un point de vue environnemental.

Triple-negative breast cancer (TNBC) is defined by absent expression of estrogen receptor (ER), progesterone receptor (PR) and non-overexpression of human epidermal growth factor receptor 2 (HER2), representing a heterogeneous subgroup of breast cancer with substantial genotypic and phenotypic diversity. TNBC patients commonly exhibit poor prognosis and high relapse rates at early stages after conventional treatments. Currently, there is a lack of biomarkers and targeted therapies for the management of TNBC. During tumour development and progression, alterations in cellular behaviour are frequently linked with kinase expression and activity. Here, we aimed to identify novel kinase targets that may play a pivotal role in the progression of TNBC and, thus, offer new therapeutic vantage points. We initially focused on identifying kinases correlated with differential outcome. Using publicly available transcriptomic data from a collated set of TNBC patients (n = 483), we identified 9 kinases that were significantly associated with survival at the mRNA level. From this in silico screen, CDK7 (cyclin-dependent kinase 7) was found to be correlated with poor recurrence-free survival. CDK7's trait as a marker of poor prognosis was further validated within another TNBC cohort (n=109) via assessment of a tissue microarray generated as part of the RATHER Consortium (www.ratherproject.com). At the protein level, high CDK7 expression was associated with poor breast cancer-specific, recurrence-free and distant recurrence-free survival. To evaluate CDK7 as a therapeutic target in TNBC, two TNBC cell lines (BT-549 and MDA-MB-231) were selected to evaluate phenotypic alterations post shRNA-mediated CDK7 knockdown. CDK7 silencing led to decreased cell proliferation, colony formation and migration in vitro. CDK7 down-regulation also increased TNBC cell sensitivity to doxorubicin. BS-181 and THZ1, two highly specific CDK7 inhibitors, attenuated TNBC tumour growth by inducing G2/M phase cell cycle arrest and apoptosis, as well as down-regulation of RNAPII phosphorylation, an indication of global RNA transcription inhibition. Moreover, the covalent CDK7 inhibitor THZ1 demonstrated 1000-fold higher potency than BS-181. Inhibition of global RNA transcription preferentially affects proteins with short half-lives. Accordingly, we detected a reduction in the expression of the anti-apoptotic protein MCL-1 in both cell lines. Next, we assessed anti-apoptotic dependence in MDA-MB-231 cells following treatment with THZ1 via BH3 profiling technology, and observed an increased response to the BAD and HRK peptides, inferring an elevated survival dependence on BCL-2/BCL-XL. We subsequently evaluated the combination of the BCL-2/BCL-XL inhibitor ABT-263 with THZ1 and discovered a synergistic inhibition of cell growth and apoptosis. Resulting combination index (CI) values demonstrated that synergistic cell death occurred following combined treatment with THZ1 and ABT-263/ABT-199 at various doses in both TNBC cell lines tested. Our data implicate high CDK7 expression as a promising biomarker of poor prognosis in TNBC. Moreover, these findings suggest that targeting CDK7, combined with the BCL-2/BCL-XL inhibitor ABT-263, may be a useful therapeutic strategy for TNBC. Citation Format: Gallagher WM, Li B, Ni Chonghaile T, Fan Y, Klinger R, O'Connor AE, Conroy E, Tarrant F, O'Hurley G, Mallya Udupi G, Gaber A, Chin S-F, Schouten PC, Dubois T, Linn S, Jirstrom K, Caldas C, Bernards R, O'Connor DP. CDK7: A marker of poor prognosis and tractable therapeutic target in triple-negative breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr PD3-01.

The screening of a small focused library of rhodanine derivatives as inhibitors of Bcl-2 proteins led to the discovery of two structurally related compounds with different binding profiles against the Bcl-XL and the Mcl-1 proteins. Subsequent NMR studies with mutant proteins and in silico docking studies provide a possible rationale for the observed specificity.

We compared whole genomic changes in cell lines generated from esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC). To do so, we investigated chromosomal DNA copy number changes in four EAC cell lines and three ESCC cell lines using comparative genomic hybridization (CGH). Frequent gains of chromosome 5p, 8q, and 20q occurred in both ESCC and EAC cell lines, but gains of 3q, 5q, and 9q were mainly seen in ESCC cell lines; gain of chromosome 10q25-qtel was mainly seen in EAC cell lines. It was noticeable that 18q12 loss existed in 2 EAC and 1 ESCC cell lines in our study. The chromosomal abnormalities common to all of the cell lines may help to identify candidate genes related to both EAC and ESCC. The chromosome aberrations mainly seen in either EAC or ESCC cell lines are in keeping with their known different etiology and may lead to the identification of genes important for disease specific pathogenesis.

Background: The Rational Therapy for Breast Cancer (RATHER) Consortium aims to identify novel kinase targets for therapy in poor-prognosis subtypes of breast cancer for which there are currently no targeted therapies available. In this project, the focus is on invasive lobular carcinomas (ILC), which represent 10% of breast tumors. The main strength of the RATHER project is the unique combination of comprehensive molecular data together with detailed clinical information, which enables translation of state-of-the art genomics analyses to the clinic. Our main goal is to identify and validate novel kinase targets for breast cancer therapy in a comprehensive way using large-scale and complementary genomics data (DNA and RNA sequence, copy number variation, gene expression and protein expression). Integrated analysis of these molecular data is performed to define molecular subtypes of ILC with differential clinical outcome. Methods: One hundred and fifty ILC samples (fresh frozen) with >5 years follow-up were collected from two institutes (Cambridge, Netherlands Cancer Institute). All samples were processed following one standard operating protocol to isolate RNA, DNA and protein of high quality. We used a five-pronged approach to identify and validate novel kinase targets for therapy in ILC, namely i) direct re-sequencing of the kinome of 150 ILC breast tumors, ii) determination of abundance and activation status of kinases in these tumors by reverse phase protein lysate array (RPPA) technology, iii) determination of copy number variation (CNV) by genome-wide SNP arrays, iv) mRNA quantitation of both genome and kinome using DNA microarrays and v) RNAseq of a subset of ILC tumors. ILC are compared to triple negative, which allows us to highlight differences and/or similarities between these subtypes and provide clues for therapeutic targets. Results: Data from these independent genome-scale technologies were integrated, yielding a prioritized list of potential kinase targets for therapy in ILC breast cancer. Deep sequencing of the kinome has revealed somatic mutations characteristic of ILC, which are currently being validated via mass spectrometry-based genotyping technology and their possible effects confirmed with gene expression, protein expression and phosphorylation changes. In addition, on a subset of the ILC samples, RNA sequencing was performed to confirm expression of particular mutants. Known gene mutations in ILC such as loss of CDH1 were confirmed. Moreover, the PI3K pathway is found to be frequently altered (50% of the samples). Gene expression analysis, as well as integrative analysis of CNV and gene expression data revealed subsets of ILCs that significantly regulate alternate biological processes and show differential clinical outcome. Such biological subsets are currently being validated with clinical and follow-up data. Updated results will be presented at the meeting. Conclusion: The RATHER project aims to deliver proof-of-concept for novel therapeutic interventions, together with companion molecular diagnostic assays for patient stratification, for up to 10% of breast cancer patients, where current treatment options are unsatisfactory. Ongoing validation of a number of potential targets proves to be promising. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-04-02.

The extensive chromosome rearrangements of breast carcinomas must contribute to tumour development, but have been largely intractable to classical cytogenetic banding. We report here the analysis by 24-colour karyotyping and comparative genomic hybridization (CGH) of 19 breast carcinoma cell lines and one normal breast epithelial cell line, which provide model examples of karyotype patterns and translocations present in breast carcinomas. The CGH was compared with CGH of 106 primary breast cancers. The lines varied from perfectly diploid to highly aneuploid. Translocations were very varied and over 98% were unbalanced. The most frequent in the carcinomas were 8;11 in five lines; and 8;17, 1;4 and 1;10 in four lines. The most frequently involved chromosome was 8. Several lines showed complex multiply-translocated chromosomes. The very aneuploid karyotypes appeared to fall into two groups that evolved by different routes: one that steadily lost chromosomes and at one point doubled their entire karyotype; and another that steadily gained chromosomes, together with abnormalities. All karyotypes fell within the range seen in fresh material and CGH confirmed that the lines were broadly representative of fresh tumours. The karyotypes provide a resource for the cataloguing and analysis of translocations in these tumours, accessible at http://www.path.cam.ac.uk/~pawefish. © 2000 Cancer Research Campaign

This paper describes deaths of American workers involving forklifts during the 15-year period from January 1, 1980 to December 31, 1994.Death certificate data were obtained from the National Institute for Occupational Safety and Health's (NIOSH's) National Traumatic Occupational Fatality (NTOF) surveillance system. The narrative fields on the death certificate were searched for keywords indicating that a powered industrial vehicle (PIV) or forklift was involved in the death. This study examined the circumstances of the forklift-related deaths, the nature of the injury, and the decedent's age, gender, race, occupation, and industry. Average annual employment data from the Bureau of the Census were used to calculate civilian fatality rates by age, gender, industry, and occupation.A total of 1,021 deaths were identified. The average age of the fatally injured worker was 38 years; the 1,021 forklift-related deaths resulted in a total of 27,505 years of productive life lost. The three most common circumstances of the fatalities were forklift overturns (22%), pedestrian struck by forklifts (20%), and worker crushed by forklift (16%). The greatest proportion of the fatalities (37%) occurred to workers in Manufacturing, followed by Transportation, Communication, and Public Utilities, (TCPU), (17%), Construction (16%), Wholesale Trade (8%), and Agriculture, Forestry, and Fishing (AFF) (7%). The highest forklift-related fatality rates per ten million workers occurred among transport operatives (34.0) and laborers (32.0).Many of the fatalities resulting from forklift "overturns" might have been prevented if the operator had been restrained with a lap/shoulder belt. Careful consideration should be given to separating pedestrian and forklift traffic, and restricting the use of forklifts near time clocks, exits, and other areas where large numbers of pedestrians pass through an area in a short time. Additionally, systematic traffic control, including rules for pedestrian and forklift traffic, will be necessary to reduce the enormous injury and death toll associated with forklifts. Am. J. Ind. Med. 36:504-512, 1999. Published 1999 Wiley-Liss, Inc.

We studied the effect of conjugated linoleic acid (CLA) on rat development and growth. Primigravid female Fischer rats were fed control or CLA-supplemented (0.25% or 0.5% CLA) diets during gestation and/or lactation. Conjugated linoleic acid was incorporated into milk fat and tissue lipids proportional to the level of CLA fed and the duration of CLA feeding. Conjugated linoleic acid was incorporated into fetal and neonatal tissues; it did not affect litter size nor induce apparent abnormalities. To the contrary, feeding CLA to the dams during gestation and lactation improved the postnatal body weight gain of pups (P0.05), measured on d 10 of lactation. Pups that continued to receive the CLA-supplemented diet after weaning had significantly greater body weight gain and improved feed efficiency relative to control animals (P0.05).

Several fixation and permeabilization techniques that enable the flow cytometric analysis of the cell contents have been introduced in recent years. These methods allow sensitive detection of intracellular antigens that facilitates the diagnosis of certain diseases. We have undertaken in this study to evaluate a simple method of fixation and permeabilization using 2% paraformaldehyde and Tween 20. Intracellular antigens in three different leukaemia cases were analysed. We found that the method was reliable and easy. Intracellular kappa light chains were found in abundance in a case of plasma cell leukaemia. CD3 and CD22 were found in greater amount intracellularly than on the surface in pre-T-ALL and pre-pre B-ALL respectively.

Conjugated dienoic derivative of linoleic acid (CLA) is a collective term which refers to a mixture of positional and geometric isomers of linoleic acid. It is a naturally occurring substance in food and is present at higher concentrations in products from animal sources. The present study reports that synthetically prepared CLA is an effective agent in inhibiting the development of mammary tumors induced by dimethylbenz(a)anthracene. Rats were fed either the AIN-76A basal diet or the same diet supplemented with 0.5, 1, or 1.5% CLA by weight. These diets were started 2 weeks before carcinogen administration and continued until the end of the experiment. The total number of mammary adenocarcinomas in the 0.5, 1, and 1.5% CLA groups was reduced by 32, 56, and 60%, respectively. The final tumor incidence and cumulative tumor weight were similarly diminished in rats fed the CLA-containing diets. In general, there appeared to be a dose-dependent protection at levels of 1% CLA and below, but no further beneficial effect was evident at levels above 1%. Chronic feeding of up to 1.5% CLA produced no adverse consequences in the animals. Analysis of the phospholipid fraction from liver and mammary tumor extracts showed that only the c9,t11 isomer of CLA was incorporated and that the level of incorporation increased with dietary intake. An interesting property of CLA is its ability to suppress peroxide formation from unsaturated fatty acid in a test-tube model (Cancer Res., Ha et al. 50: 1097-1101, 1990). In view of this information, the amount of thiobarbituric acid-reactive substances (lipid peroxidation products) present endogenously in liver and mammary gland was quantitated. The feeding of CLA (for either 1 or 6 months) resulted in a decrease in the extent of lipid peroxidation in the mammary gland, but such a suppressive effect was not detected in the liver. It should be noted that maximal antioxidant activity was observed with only 0.25% CLA in the diet, whereas maximal tumor inhibition was achieved at about 1% CLA. Hence there is a discrepancy between the antioxidant efficacy of CLA and its anticarcinogenic potency, suggesting that some other mechanisms might be involved in cancer protection. Unlike the stimulatory effect of linoleic acid in carcinogenesis (Cancer Res., Ip et al., 45: 1997-2001, 1985), the reaction of CLA in cancer prevention is specific, and CLA is more powerful than any other fatty acid in modulating tumor development.

Conjugated dienoic derivatives of linoleic acid (referred to by the acronym CLA) constitute a newly recognized class of anticarcinogenic fatty acids. Of the eight major CLA isomers, the cis-9, trans-11 isomer alone is incorporated into phospholipid and may be the most biologically relevant isomer. CLA exhibits potent antioxidant activity; evidence is presented indicating that CLA acts both as an in vitro and in vivo antioxidant. The formation of CLA in foods, and its possible biological significance in cell membranes, is discussed.

A 64-wk experiment was conducted in which the effects of ad libitum vs. restricted feeding were studied during the pullet-layer transition and laying periods. Broiler breeder females were reared through 23 wk of age according to the breeder's recommendation. At 24 wk of age the following four treatments were imposed: T1, fed restricted amounts of feed according to the breeder's management guide; T2, feed restricted from 24 to 32 wk of age (as in T1), then fed ad libitum; T3, fed ad libitum from 24 to 64 wk of age; and T4, fed ad libitum from 24 to 32 wk of age, then restricted to 85% of the average 24 to 32-wk consumption. Birds fed ad libitum during the pullet-layer transition period (T3 + T4) consumed an average of 37 g more feed per day and reached sexual maturity 14 days earlier than restricted birds (T1 + T2). Live body weight, carcass weight, carcass weight corrected to zero fat content, and percentage carcass fat were all significantly higher at first egg in ad libitum vs. restricted birds, but these differences were small. Birds fed ad libitum during lay (T2 + T3) produced more eggs, achieved a higher peak percentage hen-day production, consumed more feed, and contained more body fat at last egg than hens restricted during lay (T1 + T4). However, neither mortality, feed efficiency, egg weight, egg fertility, nor egg specific gravity was affected by treatment.