Citations

Abstract The terminology for human papillomavirus (HPV)–associated squamous lesions of the lower anogenital tract has a long history marked by disparate diagnostic terms derived from multiple specialties. It often does not reflect current knowledge of HPV biology and pathogenesis. A consensus process was convened to recommend terminology unified across lower anogenital sites. The goal was to create a histopathologic nomenclature system that reflects current knowledge of HPV biology, optimally uses available biomarkers, and facilitates clear communication across different medical specialties. The Lower Anogenital Squamous Terminology (LAST) Project was cosponsored by the College of American Pathologists and the American Society for Colposcopy and Cervical Pathology and included 5 working groups; 3 work groups performed comprehensive literature reviews and developed draft recommendations. Another work group provided the historical background and the fifth will continue to foster implementation of the LAST recommendations. After an open comment period, the draft recommendations were presented at a consensus conference attended by LAST work group members, advisors, and representatives from 35 stakeholder organizations including professional societies and government agencies. Recommendations were finalized and voted on at the consensus meeting. The final, approved recommendations standardize biologically relevant histopathologic terminology for HPV-associated squamous intraepithelial lesions and superficially invasive squamous carcinomas across all lower anogenital tract sites and detail the appropriate use of specific biomarkers to clarify histologic interpretations and enhance diagnostic accuracy. A plan for disseminating and monitoring recommendation implementation in the practicing community was also developed. The implemented recommendations will facilitate communication between pathologists and their clinical colleagues and improve accuracy of histologic diagnosis with the ultimate goal of providing optimal patient care.

Background— Despite several advances in postresuscitation care over the past decade, population-based mortality rates for patients hospitalized with cardiac arrest in the United States have not been studied over this time period. The aim of this study was to determine the annual in-hospital mortality rates of patients with cardiac arrest from 2001 to 2009. Methods and Results— The US mortality rates for hospitalized patients with cardiac arrest were determined using the 2001 to 2009 US National Inpatient Sample, a national hospital discharge database. Using the International Classification of Diseases , 9 th Edition, code 427.5, we identified patients hospitalized in the United States with cardiac arrest from 2001 to 2009. The main outcome measure was in-hospital mortality. A total of 1 190 860 patients were hospitalized with a diagnosis of cardiac arrest in the United States from 2001 to 2009. The in-hospital mortality rate decreased each year from 69.6% in 2001 to 57.8% in 2009. In multivariable analysis, when controlling for age, sex, race, and comorbidities, earlier year was a strong independent predictor of in-hospital death. The mortality rate declined across all analyzed subgroups, including sex, age, race, and stratification by comorbidity. Conclusions— The in-hospital mortality rate of patients hospitalized with cardiac arrest in the United States decreased by 11.8% from 2001 to 2009.

Abstract Microarray gene expression profiling has facilitated identification of molecular subtypes which can be associated with disease outcome and treatment response. However current platforms lack disease focus, potentially missing vital information contained in patient tissue samples. Moreover, a wide range of FFPE sample cohorts are available but their use is limited by the mRNA degradation inherent to the sample fixation process. By combining the Breast Cancer DSA® with powerful data analysis methodology, we reproduced the five major molecular subtypes in breast cancer based on a cohort of 107 FFPE samples. Furthermore, we showed that the established subtypes do not extend to BRCA1 and BRCA2 mutant tumours, necessitating expansion of the original breast cancer subtypes definition. Following RMA pre-processing of the 107 raw profiles, the data was transformed to overcome the association between profile quality and expression variability. If left untreated, this technical variability, often observed with FFPE tissues, can impair the study of inherent biological variations. Using the intrinsic list of 277 genes previously identified as differentiating breast cancer subtypes in fresh frozen samples [Sorlie et al., 2003], initially the sporadic samples were examined using hierarchical agglomerative clustering. All five original subtypes were identified, verifying the ability to detect subtypes in FFPE samples using the breast cancer DSA®. When the intrinsic list was applied to all 107 samples including BRCA1 and BRCA2 mutant samples, the subtypes were still distinguishable, although 31% of the mutant tumors were unclassified suggesting that specific biological processes defining BRCA1/BRCA2 mutant tumours are not captured by the original breast cancer molecular classification. We have successfully overcome the limitation imposed by FFPE sample microarray profiling by using the Breast Cancer DSA® and advanced data manipulation and exploration to reproduce molecular subtypes previously identified using fresh frozen samples. Further investigation to extend the previously identifed subtypes will progress the investigation of the heterogeneous biology underlying breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 326. doi:10.1158/1538-7445.AM2011-326

Abstract MUC1 is over expressed and aberrantly glycosolated in >60% of pancreatic ductal adenocarcinomas. Over expression of MUC1 in pancreatic cancer is known, however the functional role has yet to be fully elucidated due to a dearth of appropriate models. In the present study, we have generated mouse models that spontaneously develop pancreatic ductal adenocarcinoma (KC), which are either Muc1 null (KCKO) or express human MUC1 as a self antigen (KCM). We show that PDA mice lacking Muc1 have significantly slower tumor progression, with tumor weight reaching up to a third of the KCM tumor and half of KC tumor. One out of 10 KCKO mice developed secondary metastasis versus 6 out of 10 and 3 out of 10 KCM and KC respectively. Cell lines derived from KCKO tumors have significantly lower tumorigenic capacity compared to cells derived from KCM tumors. Therefore, mice with KCKO tumors had a significant survival benefit compared to mice bearing the KCM tumors. In vitro, the KCKO cells have reduced proliferation and invasion compared to KCM cells and failed to respond to exogenous epidermal growth factor (EGF), platelet-derived growth factor (PDGF), or matrix metalloproteinase-9 (MMP9) stimuli. Further, significantly fewer KCKO cells entered the G2M phase of the cell cycle compared to the KCM cells, consequently more KCKO cells were observed at the S-phase of the cell cycle. Proteomics and western blotting analysis revealed a complete loss of cdc-25c expression, phosphorylation of MAPK, as well as a significant decrease in Nestin and Tubulin α-2 chain expression in KCKO cells. Treatment of the KCM cells with a MEK1/2 inhibitor, U0126, completely abrogated the enhanced proliferation of the KCM cells but had minimal effect on KCKO cells, strongly suggesting that MUC1 is necessary for MAPK activity and downstream oncogenic signaling. This is the first study to utilize a Muc1-null PDA mouse in order to fully elucidate the oncogenic role of MUC1, both in vivo and in vitro. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1070. doi:10.1158/1538-7445.AM2011-1070

Abstract Motivation: KRAS is commonly mutated in a variety of cancers including lung cancer. The KRAS gene is frequently mutated at codons 12 and 13 in lung adenocarcinomas in patients with a history of smoking. Tumors harboring an activating KRAS mutation are aggressive and are often resistant to available therapies. In the present study, we set out to identify pathways that are specifically altered in lung cancer patients whose tumors harbor a KRAS mutation using next generation sequencing technology. Methods: We performed 50nt paired-end RNA-sequencing in 15 lung adenocarcinoma samples (8 and 7 samples with and without KRAS mutation). Reads were aligned to genome and exon junctions using Illumina's alignment tool ELAND_RNA. CASAVA and Genome Studio data analysis software were used to obtain read counts for genes, exons and exon junctions. We applied a variety of computational methods (Casper R package, Bowtie, TopHat) and softwares (Partek, R statistical software, JMP, Ingenuity Pathway Anlaysis, DAVID) to carry out our analyses. Results: Differential gene expression and splicing analysis were performed between the two groups. We identified 115 genes that were differentially expressed and 112 genes that consist of splicing variants with at least 2 fold changes and p-value < 0.01. We randomly selected 6/15 samples and performed real-time qRT-PCR for 6 differentially expressed genes. Significant correlations were observed ranging from 0.44 to 0.83, when qRT-PCR results were compared with RNA-sequencing expression data. Pathway analysis with 115 differentially expressed genes and 112 splicing variants revealed that the most significant pathway is composed entirely of NFκB focus genes, suggesting that there is a direct connection between oncogenic KRAS and activation of the NFκB signaling pathway. Conclusions: NFκB has been implicated in KRas-mediated formation of early stage lung adenomas in the LSL-Kas mouse model. Our data indicate that this connection also occurs in human lung cancer and establish NFκB activation as a key manifestation of KRAS mutation in human lung cancer. Hence drugs targeting NFκB and NFκB related genes may potentially be helpful for the treatment of patients with oncogenic KRAS mutations. (Supported in part by grants from the 26.2 with Donna Foundation and the Florida Department of Health Bankhead/Coley program.) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4975. doi:10.1158/1538-7445.AM2011-4975

Abstract Background: Estrogen receptor (ER) expression in breast caner is an important biomarker for targeted therapy and outcome prediction. Nevertheless, the molecular mechanism that controls the phenotypic difference is little known. We used deep sequence technology to profile the transcriptome, gene copy number, and CpG island methylation simultaneously in eight commonly used breast cell lines to develop a model for how these genomic features are integrated in ER+ and ER- breast cancer. Materials and methods: Total mRNA sequence (mRNA-seq), gene copy number (DNA-seq), and genomic CpG island methylation (Methyl-seq) were carried out using the Illumina Genome Analyzer. Sequences were mapped to the human genome (hg18) to obtain digitized gene expression data, DNA copy number in reference to the non-tumor cell line (MCF10A), and methylation status of 21,570 CpG islands; differentially expressed genes between ER+ and ER- cell lines were selected and then correlated with methylation status of these genes’ CpG islands within 5kb of transcript start or copy number changes. The genes that appeared controlled by methylation or copy number changes in cell lines were further evaluated for their expression in the dataset of 129 primary breast tumors. Results: ER+ breast cancer cells had very different gene expression pattern from ER- cancer cells. The two cell line types formed two distinct clusters in unsupervised cluster and 1,873 genes were differentially expressed by moderated t statistics. We identified 149 differentially expressed genes that exhibited differential methylation of one or more CpG islands within 5kb of the 5’ end of the gene and for which mRNA abundance was inversely correlated with CpG island methylation status. Eighty nine of these 149 genes were also differentially expressed in the primary tumor samples and 84 of them were consistent with cell line gene expression and methylation data. The set of 149 genes were significantly enriched in the ER+ tumors by Gene Set Enrichment Analysis. Part of chromosome 8 was deleted in all ER- cells and part of chromosome 17 amplified in all ER+ cells. These loci encoded 30 genes that were overexpressed in ER+ cells; however, only 9 of these genes were overexpressed in ER+ tumors. Many of the methylation or CNA affected genes were functionally significant and some are known to be associated with breast cancer outcome such as GATA3 and LYN. Conclusions: A global pattern of differential CpG island methylation influences expression of a cohort of genes that contribute to the transcriptome landscape of ER+ and ER- breast cancer cells and tumors. The role of gene amplification/deletion in defining the transcriptome landscape of ER+ and ER- cells appears to more modest, although several potentially significant genes appear to be globally regulated by copy number aberrations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4908. doi:10.1158/1538-7445.AM2011-4908

Abstract Rare germline mutations in the breast and ovarian cancer predisposition genes BRCA1 and BRCA2 are present in roughly 5 percent of ovarian cancer patients. Both genes play key roles in DNA damage repair but appear to have distinct, although often complementary, functions. The risks of breast and ovarian cancer have been shown to differ between BRCA1 and BRCA2 carriers, and mutation-specific effects have also been suggested for both groups. Published reports comparing the survival of ovarian cancer patients with and without BRCA1/2 mutations generally show a survival advantage among mutation carriers. This is thought to be related to an improved response of carriers to platinum-based therapies. Since it is not known whether BRCA1 and BRCA2 carriers show similar survival patterns, we have performed a large, multicenter study to investigate the impact of germline BRCA1 and BRCA2 mutations on ovarian cancer survival. 3,531 invasive epithelial ovarian cancer cases (1,178 BRCA1, 367 BRCA2 and 1,986 BRCA-negative) with survival time data from twenty-four studies in the U.S.A, Europe, Israel and Asia were included. In our main analysis, we excluded patients who were known or who were likely to have not received platinum-based therapy. Compared to non-carriers, BRCA1 and BRCA2 carriers were more likely to present with advanced stage (BRCA1; p=2×10−4, BRCA2; p=4×10−6), high grade (BRCA1; p=4×10−9, BRCA2; p=1×10−4), serous disease (BRCA1; p=3×10−6, BRCA2; p=0.003). BRCA1 carriers were younger at diagnosis (p=2×10−9) and BRCA2 slightly older at diagnosis (p=0.002) than non-carriers. BRCA1 and BRCA2 carriers did not differ in terms of tumor stage, grade or histology but did show differences in the age at diagnosis (p=2×10−14). In an unadjusted analysis, neither BRCA1 nor BRCA2 carriers differed from non-carriers in overall survival (BRCA1: HR=1.02, p=0.77, BRCA2: HR=0.89, p=0.36). After adjusting for stage, grade, histology and age at diagnosis, BRCA1 carriers showed a modest, non-significant, survival advantage (HR=0.84, p=0.12) while BRCA2 carriers showed a marked improvement (HR=0.57, p=6×10−4) compared to non-carriers. This survival advantage was also seen when comparing BRCA2 carriers to BRCA1 carriers after adjustment for age at diagnosis (HR=0.69, p=5×10−4). Similar results were obtained when we restricted the analysis to high grade, advanced stage, serous cases. The survival differences we observed between BRCA1 and BRCA2 carriers could be related to differences in tumor biology or chemo-sensitivity. Additional analyses investigating the later will be presented in addition to mutation-specific effects within BRCA1 and BRCA2. In this study, we provide strong evidence for genetic heterogeneity of survival effects in ovarian cancer patients with BRCA mutations. This could be applied to both clinical prediction and to aid our understanding of the complex biology of BRCA mutations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2752. doi:10.1158/1538-7445.AM2011-2752

Abstract Background: Based upon preclinical synergy and prior phase 1 study results, the clinical efficacy of flavopiridol combined with cisplatin was assessed in patients with recurrent ovarian and primary peritoneal cancers. Methods: A two cohort phase 2 trial of cisplatin (60 mg/m2 IV) followed by flavopiridol (100 mg/m2 IV, 24 h continuous infusion; 21 day cycles) was undertaken in patients with recurrent platin-sensitive or platin-resistant ovarian/primary peritoneal cancers (defined by disease progression > vs. <6 months following platin-based therapy). Measurable disease (RECIST criteria) – or evaluable disease plus CA125 >2X the post-treatment nadir – was required, as was ECOG performance <2 and exposure to only one prior treatment regimen. Results: Forty-five patients were enrolled between April 20, 2004 and March 4, 2010 – 40 platin-resistant patients (Group 1), and 5 platin-sensitive patients (Group 2). In Group 1, the median number of treatment cycles was 3 (range 2-12); 39 of the 40 eligible patients have now discontinued treatment. While only 10% of all patients incurred grade 4 toxicities, grade 3 toxicities were seen in the majority (65%). The most frequent grade 3 and 4 toxicities were neutropenia (all grade 3, 17.5%); nausea (12.5%); vomiting, fatigue, thrombosis, anemia (10% each). Sensory neuropathy, grade 1 or 2, was observed in 75% of all patients – with grade 3 and 4 neuropathy not observed primarily due to pre-specified aggressive dose reductions. Six patients (15%) in Group 1 achieved a confirmed response (1 CR, 5 PR), with a median response duration of 119 days (range 84-212). Ten additional Group 1 patients (32.5%) experienced maintained stable disease. Median Group 1 overall time to progression was 3.7 months; overall survival was 17.2 months. Pilot assessment of attained ascites flavopiridol level and sensitivity of patient ascitic tumor cells to flavopiridol confirmed that patient flavopiridol levels were consistent with observed clinical antitumor efficacy. In Group 2, although 2 of 5 patients also responded (40%; 2 PR), the cohort was closed due to poor accrual. Conclusions: The combination of flavopiridol and cisplatin has promising clinical activity in both platin-sensitive and platin-resistant ovarian and primary peritoneal cancers. Supported in part by NCI CA097129, CA15083 and CM62205; clinicaltrials.gov identifier NCT00083122 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4712. doi:10.1158/1538-7445.AM2011-4712

Abstract The role of fusion gene transcripts such as BRC-ABL1 has been long appreciated in hematopoietic malignancies. Recent evidence from next generation sequencing projects has suggested that gene fusion events and the resultant chimaeric transcripts may be expressed in solid tumors. We have developed a novel bioinformatics analytical pipeline to detect fusion gene transcripts from paired-end mRNA-seq data. The bioinformatics tool used for fusion transcript discovery employs multiple steps of false positive filtering and nominates the fusion transcript candidates with high confidence (approaching 100%). The screening and validation of the fusion candidates were quickly carried out using the recommended primer design regions as one of the outputs of the SnowShoes-FD pipeline. This pipeline was used to analyze the transcriptome of 22 breast cancer cell lines and 9 non-transformed breast cell populations. Fifty-four fusion candidates were nominated, all of which were validated using reverse transcription PCR and Sanger sequencing. Five fusion products were predicted to have a second fusion junction point between two fusion partners. These 54 fusion transcripts consist of 103 partner genes that are involved in cell cycle and/or nuclear receptor signaling. No fusion transcripts were identified from the non-transformed breast cell lines. We subsequently analyzed a panel of primary breast tumors, 8 each HER2+, triple negative, and ER+. Fifteen novel fusion transcript candidates have been nominated. Validation and functional analysis of these candidates is in progress. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-276. doi:10.1158/1538-7445.AM2011-LB-276

Abstract Myeloproliferative diseases (MPDs) are a subset of myeloid malignancies that are characterized by the expansion of a multipotent hematopoietic stem cell. Chronic MPDs can be classified into two categories, those harboring the BCR-ABL oncogene and those that are negative. This later category of neoplasms encompasses polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). Recent discovery of activating mutations in the tyrosine kinase gene, JAK2 and constitutive activation of JAK2-STAT pathway, in large number of MPD patients has ignited considerable interest in MPD and has highlighted JAK2 as a therapeutic intervention point for drug discovery efforts. However, high-sequence homology with other JAK family members has posed a major challenge to design selective JAK2 inhibitors. Given that other JAK family members are involved in the regulation of immune function, it is important to maintain selectivity for JAK2 over these family members in order to mitigate the risks associated with undesired immunosuppression. Several JAK2 inhibitors with varying selectivity profiles are currently being evaluated in preclinical testing as well as in clinical trials for the treatment of MPD. Additionally, emerging genetic and pharmacologic evidence suggest that inhibition of the JAK2-STAT pathway may be an important therapeutic intervention point in other hematological malignancies as well as in certain solid tumors. We report here the discovery and characterization of BMS-911543, a functionally selective small molecule inhibitor of the Janus kinase family (JAK) member, JAK2. BMS-911543 is a potent and reversible inhibitor of JAK2 with a biochemical Ki of 0.48 nM. It has over 65-, 74- and 350-fold selectivity against the other JAK family members, TYK2, JAK3 and JAK1, respectively. Importantly, examination of > 450 other kinases in competition binding assays and in selected biochemical kinase assays did not reveal significant inhibitory activity for this JAK2 inhibitor, highlighting its high degree of biochemical selectivity for JAK2. Functionally, BMS-911543 displayed potent antiproliferative and pharmacodynamic (PD) effects in mutated JAK2-expressing cell lines dependent upon JAK2-STAT signaling and had little activity in cell types dependent upon other pathways such as JAK1 and JAK3. Further, single agent antiproliferative activity was not observed for BMS-911543 in a variety of solid tumor cell lines dependent upon other signaling pathways. In contrast, BMS-911543 was evaluated in colony growth assays using primary progenitor cells isolated from patients with JAK2V617F-positive myeloproliferative disease (MPD) and resulted in an increased antiproliferative response in MPD cells as compared with those from healthy volunteers. Similar to these in vitro observations, BMS-911543 was also highly active in in vivo models of JAK2-pSTAT signaling in multiple species with durable and potent pathway suppression observed after a single oral dose. Additionally, BMS-911543 was evaluated for effects in a JAK2V617F-expressing SET-2 xenograft model system and displayed a minimally effective dose of <2 mg/kg on pSTAT5 pathway suppression, which lasted up to 8 hours. To test the hypothesis that a JAK2 selective inhibitor would have less effect on immune system function, BMS-911543 was compared to pan-JAK inhibitors in a mouse model of immunosuppression. At low dose levels active in JAK2-dependent PD models, no effects were observed on antigen-induced IgG and IgM production for BMS-911543 whereas a pan-JAK family inhibitor showed pronounced effects at all dose levels tested. The mechanistic selectivity of BMS-911543 to pan-JAK family inhibitors was extended through comparative analysis of these inhibitors in whole genome gene expression profiling experiments performed in sensitive and resistant cell types. In this comparison, BMS-911543 modulated a distinct subset of transcriptional changes as compared to pan-JAK inhibitors in clinical testing, thereby defining a minimal set of transcriptional changes underlying the pharmacologic effects of JAK2 inhibition. Collectively these results define the mechanistic basis for a differential therapeutic index between selective JAK2 and pan-JAK family inhibition pre-clinically and suggest a therapeutic rationale for the further characterization of BMS-911543 in patients with MPD and in other malignancies reliant upon constitutively active JAK2 signaling. References: Levine, R.L., et al. Role of JAK2 in the pathogenesis and therapy of myeloproliferative disorders (2007). Nature Rev. Cancer, 7, 673-683. Atallah, E. and Verstovsek, S. Prospect of JAK2 inhibitor therapy in myeloproliferative neoplasms. (2009). Expert Rev. Anticancer Ther. 9, 663-670. Ghoreschi, K., et al. Janus kinases in immune cell signaling. (2009). Immunol. Rev.,228, 273-287. Mesa, R.A. and Tefferi, A. Emerging drugs for the therapy of primary and post essential thrombocythemia, post polycythemia vera myelofibrosis (2009). Expert Opin. Emerging Drugs, 14, 1-9. Roll, J.D. and Reuther, G.W. CRLF2 and JAK2 in B-progenitor acute lymphoblastic leukemia: a novel association in oncogenesis. (2010) Cancer Res, 70, 7347-7352. Rui et al., Cooperative epigenetic modulation by cancer amplicon genes (2010). Cancer Cell, 18, 590-605. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr DDT01-03. doi:10.1158/1538-7445.AM2011-DDT01-03