bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract LB-125: The identification of FOXA1/ERα pathway modulators for the treatment of hormone-based therapy resistant breast cancer
Authors: roffey jon , Lejeune Aurore , Barnard Michelle , Stock Julie , Wong Ai Ching , McKelvie Jenny , Holmes Kelly , Carroll Jason , Myatt Stephen
Published: August 2015

Estrogen receptor alpha (ERα) is expressed in ∼75% of breast cancers where it binds and regulates specific target genes which drive cancer cell proliferation. Hormone-based treatments such as selective estrogen receptor modulators (SERMs) and aromatase inhibitors prevent estrogen signalling through the ERα, but in many cases resistance to these therapies develops. FOXA1 is a pioneer factor for ERα that is required to facilitate ERα binding to its target genes and its expression is considered to be a defining gene that characterizes ERα positive luminal breast cancers. Here we show that FOXA1 is an essential gene in ERα positive, tamoxifen resistant and aromatase-resistant breast cancer cell lines suggesting inhibitors that target the FOXA1/ERα pathway could have broad utility in these settings. To this end we performed a cell-based screen to identify novel small molecule regulators of FOXA1/ERα pathway activity. Our novel cell-based assay allowed us to screen over 180, 000 discrete compounds in a high through-put manner. Counter screens and phenotypic profiling in FOXA1 positive and negative breast cancer cell lines were used to remove non-specific compounds and direct ERα modulators. Second batches of confirmed actives were profiled through the screening cascade and by using ChIP assays we have observed a reduction in FOXA1 binding at the chromatin that is commensurate with a similar loss of ERα binding, demonstrating for the first time pharmacological modulation of FOXA1/ERα occupancy at the chromatin level.

Citation Format: jon roffey, Aurore Lejeune, Michelle Barnard, Julie Stock, Ai Ching Wong, Jenny McKelvie, Kelly Holmes, Jason Carroll, Stephen Myatt. The identification of FOXA1/ERα pathway modulators for the treatment of hormone-based therapy resistant breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-125. doi:10.1158/1538-7445.AM2015-LB-125
Abstract LB-131: In vivo chromosomal engineering with the CRISPR/Cas9 model: a new frontier for mouse modeling
Authors: Maddalo Danilo
Published: August 2015

Introduction: Chromosomal rearrangements are often detected in several types of cancer and result in expression of aberrant genes with oncogenic functions. In spite of their importance in tumor formation, modeling such rearrangements has been proven challenging and requires heavy and long germline manipulation.

Purpose of the study: in this study we propose to induce chromosomal rearrangements in vivo by delivering the CRISPR/Cas9 system making use of viral vectors.

Experimental procedures: for in vitro studies we transfected NIH/3T3 cells with plasmids expressing the Cas9 protein and the specific sgRNA. For in vivo delivery of the CRISPR/Cas9 system to the lung of adult mice we made use of Adenoviral vectors containing the Cas9 and two sgRNAs expressed under the control of two independent U6 promoters. Imaging of the lungs was performed with μCT scan.

Results: two sgRNAs to direct the endonuclease Cas9 to the intron 19 of Alk and the intron 14 of Eml4 to induce the Eml4-Alk inversion were designed to induce Eml4-Alk inversion, detected in a subset of patients with non-small cell lung cancer (NSCLC). Co-delivery of the sgRNAs and the Cas9 resulted in the expected rearrangements, indicating that two sgRNAs and the endonuclease are sufficient to induce chromosomal rearrangements in vitro. For in vivo delivery to the lung of adult mice, we generated an adenoviral system for the concomitant delivery of the Cas9 and the two sgRNAs (Ad-EA) or the Cas9 alone (Ad-Cas9). Within 8 weeks post-infection with Ad-EA, mice developed lung adenomas while mice infected with Ad-Cas9 were tumor free. All the tumors carried the genomic inversion Eml4-Alk and were negative for p63 and positive for TTF-1 resembling the histological feature of NSCLC. Finally we tested the sensitivity of the tumors to the ALK/MET inhibitor crizotinib, used in therapy for the treatment of ALK+ NSCLC. Radiological and histological analysis showed in all cases tumor regression, indicating that our CRISPR-induced mouse model closely resembles human NSCLC.

Conclusion: In conclusion the strategy here presented expands significantly our possibility of modeling cancers driven by chromosomal rearrangements and represents an opportunity to test novel targeted therapies and thee mechanisms leading to drug resistance.

Citation Format: Danilo Maddalo. In vivo chromosomal engineering with the CRISPR/Cas9 model: a new frontier for mouse modeling. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-131. doi:10.1158/1538-7445.AM2015-LB-131
Abstract LB-141: Specific and potent silencing of K-Ras by asymmetric silencing RNA (aiRNA) reveals addiction of cancer stem cells to mutant K-Ras amplification
Authors: Oishi Jun , Umehara Hiroki , Jesuraj Nithya , Barbulovic Jelena , Sun Xiangao , Li Chiang J.
Published: August 2015

K-Ras, the first oncogene identified in human cancer, is mutated in about 30% of human solid tumors. K-Ras protein, a small membrane-bound GTP-binding protein, acts as a molecular switch to transduce cell proliferation signals. Activating mutations of K-Ras lock the Ras protein into the hyper-active GTP-bound state, resulting in the activation of numerous signaling pathways that control cell survival and proliferation. Ras is also an important oncoprotein in many cancers where it is not mutated since Ras can be functionally activated through aberrant activation of other signal transduction elements. Activated K-Ras proteins are, therefore, found in a large proportion of all human cancers, and occupy a central position of interest.

Hypermalignant cancer cells, termed cancer stem cells (CSCs), that are highly tumorigenic and metastatic have been isolated from cancer patients with a variety of tumor types and found to have high stemness properties. These stemness-high cancer cells are hypothesized to be fundamentally responsible for cancer metastasis and relapse. Furthermore, a number of stemness genes, such as beta-catenin, nanog, Sox2, Oct3/4 have been implicated in cancer cell stemness. The role of oncogenes such as K-Ras in cancer cell stemness, however, is not clear.

To elucidate the role of K-Ras in the maintenance of cancer cell stemness, we employed asymmetric silencing RNA technology (aiRNA) which is able to silence target genes with high potency and precision. Moreover, aiRNA technology can be readily applied to CSCs. Here we report, to our surprise, that CSCs are not simply addicted to activating mutations of K-Ras, or activation of downstream regulators of the Ras pathway. However, CSCs with amplified mutant K-Ras are highly sensitive to K-Ras silencing. Moreover, the DNA copy number of the mutant K-Ras directly predicts the sensitivity of CSCs to K-Ras silencing. Our studies suggest that amplified mutated K-Ras is required for the maintenance of malignancy and cancer cell stemness, which may have significant implications for understanding the connections between oncogenes and cancer cell stemness, and for developing cancer stem cell inhibitors.

Citation Format: Jun Oishi, Hiroki Umehara, Nithya Jesuraj, Jelena Barbulovic, Xiangao Sun, Chiang J. Li. Specific and potent silencing of K-Ras by asymmetric silencing RNA (aiRNA) reveals addiction of cancer stem cells to mutant K-Ras amplification. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-141. doi:10.1158/1538-7445.AM2015-LB-141
Abstract LB-144: Derivation of a model of cancer stem cell from human induced pluripotent stem cells
Authors: Kasai Tomonari , Hoshikawa Kenta , Takejiri Shuto , Ikeda Masashi , Kumon Kazuki , Sanchez Calle Anna , Vaidyanath Arun , Mizutani Akifumi , Ling Chen , Seno Masaharu
Published: August 2015

The existence of cancer stem cell (CSC) has been considered as one of the important reason as to why patients have a poor prognosis. However, heterotopic transplantation of embryonic stem cells and induced pluripotent stem cells has been shown to form teratoma, but not malignant teratoma. Since the microenvironment niche is playing a significant role for the proper differentiation of stem cells, the cancerous niche should drive stem cells into malignant cells in vivo. According to this hypothesis, we tried to generate cancer cells from human induced pluripotent stem (hiPS) cells. For the conversion into CSC, the conditioned medium from different human cancer cell lines was collected from confluent dishes and filtered using 0.22 micrometer filter. Then, hiPS cells, without MEF feeder cells, were maintained in the conditioned medium (CM) in the ratio of 1:1. The medium was changed every day with CM for 4 weeks. hiPS cells with the complete medium were used as control. For transplantation studies, 10^4 cells were suspended in HBSS and were xenotransplantated into NOD-SCID mice. After 3 months, tumors were excised and fixed in 10% neutral formalin buffer solution, or subjected to primary culture. The converted cells and primary cultured cells formed spheroids in suspension culture, and had tumorigenicity in vivo. The stemness of living cells was checked under fluorescent microscopy observation with rBC2LCN-FITC staining. The RNAs were extracted from converted cells and microarray analysis was perfomed. The RNA expression patterns of cell lines were visualized by sphered self-organizing map (sSOM) analysis. The sSOM analysis perfomed based upon various parameters shows the converted CSCs can be characterized into various cell types. Utilizing this method, we successfully established two different hiPS-CSC lines using CM from A172 and RERF-LC-KJ.

The comprehensive understanding of cancer could be realized as the heterogeneity of cancer tissues is clarified and their component cells are identified. This study will lead to the development of the true personalized therapy of cancer in the future.

Citation Format: Tomonari Kasai, Kenta Hoshikawa, Shuto Takejiri, Masashi Ikeda, Kazuki Kumon, Anna Sanchez Calle, Arun Vaidyanath, Akifumi Mizutani, Chen Ling, Masaharu Seno. Derivation of a model of cancer stem cell from human induced pluripotent stem cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-144. doi:10.1158/1538-7445.AM2015-LB-144
Abstract LB-149: A novel strategy to inhibit CpG island hypermethylation and restore BRCA1 expression
Authors: Han Yoo Jeong , Zhang Jing , Prat Aleix , Yoshimatsu Toshio , Gomez-Vega Maria , Kwon John , Perou Charles M. , Kannanganattu Prasanth , Olopade Olufunmilayo I.
Published: August 2015

BRCA1 promoter methylation is observed in 20-60% of sporadic triple negative breast cancer (TNBC) and may be an important mechanism contributing to the loss of BRCA1 function in sporadic TNBC and other cancers with low BRCA1 expression. Demethylation and consequent reactivation of tumor suppressor genes are rational approaches being used in the treatment of cancer. However, currently available nucleoside-based DNMT inhibitors affect genome-wide DNA methylation and cannot specifically target tumor suppressor genes like BRCA1. Here, we describe a novel approach to modulate local DNA methylation by silencing a neighboring long non-coding RNA (lncRNA) which tethers DNMT1 at the BRCA1 genomic locus.

The human genomic region encompassing the BRCA1 gene is complex and includes two protein coding genes (BRCA1 and NBR1), a non-coding RNA gene (NBR2), and a pseudogene of BRCA1 (BRCA1P1), within a ∼170kb region of chromosome 17q21. The BRCA1 gene on the minus strand is located head-to-head with NBR2 on the plus strand, whereas BRCA1P1 on the minus strand is located head-to-head with NBR1 on the plus strand. The promoter between the BRCA1 and NBR2 genes and that between the BRCA1P1 and NBR1 genes are bidirectional, expressing transcripts in opposite directions through convergent transcription. The BRCA1P1 pseudogene was generated by a recent evolutionary event: partial duplication of BRCA1 gene followed by insertion of a processed pseudogene of RPLP1. It expresses a chimeric lncRNA retained in nuclei. Interference with the nuclear expression of BRCA1P1 lncRNA using a specific anti-sense oligonucleotide (ASO) decreased the promoter methylation of BRCA1 and increased BRCA1 expression. RNA immunoprecipitation (RIP) assays revealed DNMT1 interactions with BRCA1 mRNA and BRCA1P1 lncRNA at the locus. Chromosome conformation capture (3C) will assess whether there is a long-range interaction between the BRCA1 and BRCA1P1 promoters through DNMT1 and mediators. Our data support a long-range cis-regulation of BRCA1 expression by neighboring BRCA1P1 lncRNA through an interaction with DNMT1 at the locus. Depleting BRCA1P1 with ASO could be developed as a therapeutic method to inhibit BRCA1 promoter methylation and restore BRCA1 expression.

Citation Format: Yoo Jeong Han, Jing Zhang, Aleix Prat, Toshio Yoshimatsu, Maria Gomez-Vega, John Kwon, Charles M. Perou, Prasanth Kannanganattu, Olufunmilayo I. Olopade. A novel strategy to inhibit CpG island hypermethylation and restore BRCA1 expression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-149. doi:10.1158/1538-7445.AM2015-LB-149
Abstract LB-151: Methylation-mediated silencing of TIMP-3 facilitates oral cancer metastasis by modulating binding of SP1 transcription factor
Authors: Yang Shun-Fa , Su Chun-Wen , Lin Chiao-Wen
Published: August 2015

Tissue inhibitor of metalloproteinase (TIMP)-3, a member of the TIMP family, is the only substance that can bind with the ECM and its main role is regulating the cell cycle and cancer cell progression. However, little is known about whether abnormal expression and promoter methylation of TIMP-3 facilitates oral cancer metastasis. In this study, DNA methylation levels of the TIMP-3 CpG islands were assessed using bisulfite DNA sequencing and methylation-specific PCR. The results shown that expression of TIMP-3 is decreased in most oral cancer tissues compared with adjacent normal tissues (p<0.001). DNA methylation analysis showed a hypermethylation of TIMP-3 gene in oral cancer cell lines and in oral cancer tumors. Furthermore, suppression of TIMP-3 transcription by DNA methylation involves inhibition of transcription factor SP1 binding to the TIMP-3 promoter. Functional analyses revealed that the stable overexpression of TIMP-3 reduced the migration ability in oral cancer cells (p<0.001). Moreover, the expression levels of the epithelial markers E-cadherin were increased, while the expression levels of the mesenchymal markers vimentin and fibronectin were decreased in cells overexpressing TIMP-3. In conclusion, these results suggest that suppression of TIMP-3 by DNA methylation contributes to oral cancer metastasis.

Citation Format: Shun-Fa Yang, Chun-Wen Su, Chiao-Wen Lin. Methylation-mediated silencing of TIMP-3 facilitates oral cancer metastasis by modulating binding of SP1 transcription factor. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-151. doi:10.1158/1538-7445.AM2015-LB-151
Abstract LB-163: Pan-cancer analysis of the causes and consequences of Intra-tumor heterogeneity
Authors: Andor Noemi , Graham Trevor A. , Aktipis Athena C. , Petritsch Claudia , Ji Hanlee P. , Maley Carlo C.
Published: August 2015

Tumors are typically mosaics of mutant clones that have evolved from a common ancestral cell. This intra-tumor heterogeneity (ITH) is thought to drive both neoplastic progression and acquired therapeutic resistance. Availability of just one sample per tumor and moderate sequencing depth have limited systematic analysis of ITH during previous TCGA pan-cancer analyses, confining the study of ITH to a small numbers of tumor samples and cancer types. The molecular and histopathological causes of ITH and its prognostic significance have thus far remained uncertain.

To overcome these limitations, we used an analysis method called EXPANDS that estimates the proportion of cells harboring specific mutations from exome sequencing data, as well as other methods that quantify ITH. We extrapolate the number of genetically diverse clonal subpopulations in 1,165 primary tumors among 12 different cancer types from TCGA and investigate mechanisms underpinning ITH as well as the correlation of ITH and genomic instability with prognosis. Lastly we validate the prognostic significance of genomic instability in an independent, high-density SNP-array dataset consisting of 2010 tumor samples, across seven additional cancer types.

We found evidence of ITH in the vast majority of tumors. Mutations in driver genes tended to have a characteristic clone size, suggesting differential fitness effects of those mutations. The detection of a mutation in a driver gene that typically appears in a small clone was a predictor of poor survival. Driver gene mutations, tumor microenvironment composition and prevalence of copy number gains were significantly associated with increased ITH. In general, the prevalence of copy number alterations was a universal biomarker of prognosis: tumors with intermediate copy number burden (50 to 75% of the genome affected by copy number alterations) progressed faster than tumors with higher copy number burden, independent of cancer type. Chemo-radiation therapy administration was more efficient in decelerating tumor progression among patients with intermediate copy number burden than among patients with low or high copy number burden. These results were validated and confirmed in the independent SNP-array dataset.

This study suggests a tradeoff exists between the costs and benefits of genomic instability that impacts both the evolvability and fitness of the tumor cell population. In the future, this tradeoff might be exploited to improve survival.

Citation Format: Noemi Andor, Trevor A. Graham, Athena C. Aktipis, Claudia Petritsch, Hanlee P. Ji, Carlo C. Maley. Pan-cancer analysis of the causes and consequences of Intra-tumor heterogeneity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-163. doi:10.1158/1538-7445.AM2015-LB-163
Abstract LB-170: Single cell sequencing identifies clonal stasis and punctuated copy number evolution in triple-negative breast cancer patients
Authors: Gao Ruli , Davis Alexander , Sei Emi , Shi Xiuqing , Unruh Anna , Waters Jill , Zhang Amy (Hong) , Meric-Bernstam Funda , Navin Nicholas
Published: August 2015

Intratumor heterogeneity is widely reported in many human tumors including breast cancer. However most studies to date have been limited to genomic analysis of bulk tumor tissues and cytogenetic analysis. Here, we applied single cell copy number profiling to delineate the clonal substructure of triple-negative (ER-, PR-, Her2-) breast cancer (TNBC). We applied Single Nucleus Sequencing (SNS) to profile genomic copy number in 1000 single cells from 11 TNBC patients. Our data show that copy number profiles within the tumor are highly stable, belonging to a few (N = 1-3) major clonal subpopulations that share similar amplifications and deletions,. In addition to the clonal cells, we also identified diverse copy number profiles that belong to two classifications: 1) replicating cells, or 2) apoptotic cells. We also find evidence of rare diploid cells that harbor non-clonal amplification and deletions and may play an important role in aneuploidy initiation. Our data suggests a model of punctuated genome evolution and clonal stasis, in which aneuploidy occurs early in tumor evolution, followed by one or more stable clonal expansion to form the tumor mass. These data challenge the paradigm of gradual copy number evolution and extensive copy number diversity in triple-negative breast cancer, and suggest that that amplifications and deletions are ideal markers for personalized cancer therapy.

Citation Format: Ruli Gao, Alexander Davis, Emi Sei, Xiuqing Shi, Anna Unruh, Jill Waters, Amy (Hong) Zhang, Funda Meric-Bernstam, Nicholas Navin. Single cell sequencing identifies clonal stasis and punctuated copy number evolution in triple-negative breast cancer patients. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-170. doi:10.1158/1538-7445.AM2015-LB-170
Abstract LB-171: Collaborating mutations in gastrointestinal stromal tumor
Authors: Waterfall Joshua , Killian J. Keith , Zhu Yuelin Jack , Lau C. Christopher , Miettinen Markku , Helman Lee J. , Meltzer Paul S.
Published: August 2015

Gastrointestinal stromal tumor (GIST), the most common mesenchymal tumor of the gastrointestinal tract, is driven most commonly by activating mutations in the receptor tyrosine kinases KIT or PDGFRA. Alternatively, inactivating mutations of genes for the succinate dehydrogenase complex (SDH) constitute a distinct oncogenic mechanism in GIST. Using integrated genomic analyses we have recently reported that SDH deficiency in GIST may also arise from epigenomic inactivation of SDHC, reflected by coupled DNA methylation and expression silencing (1). SDHC epimutation is particularly frequent in, though not restricted to, patients with the cancer predisposition syndrome Carney Triad. It is well established that these primary driver mechanisms correlate with unique clinical, molecular, and epidemiological profiles. In order to identify collaborating oncogenic pathways associated with specific GIST subtypes we have extended our integrated genomic profiling of over 70 tumors enriched for SDH deficient cases. The primary focus is sequencing a panel of 200 cancer related genes for both somatic and inherited variants. We also include RNA expression arrays, DNA methylation arrays, and SNP arrays in this analysis. We have identified mutations in several known cancer associated genes including PTEN, KRAS, ARID1A, and TP53. In conclusion, collaborating mutations and deregulated pathways are identifiable in GIST and can increase our understanding of the underlying cancer biology as well as suggesting treatment possibilities for this malignancy. We acknowledge the contributions of the NIH Wild-Type GIST clinic investigators to this project.

References

1. Killian JK, Miettinen M, Walker RL, Wang Y, Zhu YJ, Waterfall JJ, et al. Recurrent epimutation of SDHC in gastrointestinal stromal tumors. Sci Transl Med. 2014;6:268ra177.

Citation Format: Joshua Waterfall, J. Keith Killian, Yuelin Jack Zhu, C. Christopher Lau, Markku Miettinen, Lee J. Helman, Paul S. Meltzer. Collaborating mutations in gastrointestinal stromal tumor. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-171. doi:10.1158/1538-7445.AM2015-LB-171
Abstract LB-172: Novel candidate oncogenic drivers in pineoblastoma
Authors: Snuderl Matija , Kannan Kasthuri , Aminova Olga , Dolgalev Igor , Heguy Adriana , Faustin Arline , Zagzag David , Gardner Sharon L. , Allen Jeffrey C. , Wisoff Jeffrey H. , Capper David , Hovestadt Volker , Ahsan Sama , Eberhart Charles , Pfister Stefan M. , Jones David T. w. , Karajannis Matthias A.
Published: August 2015

INTRODUCTION: Pineoblastoma (PB) is one of the rarest and most aggressive brain tumors of childhood. PB is considered a “primitive neuroectodermal tumor” (PNET) based on histology, and commonly treated using treatment protocols developed for medulloblastoma; however the survival remains poor. A subset of PBs may occur in the setting of germline mutations involving DICER1 or RB1, but no next-generation sequencing studies have been published on PB to date, and the genetic drivers of sporadic PB remain unknown. METHODS: 21 tumor samples with a histological diagnosis of PB (including recurrent/metastatic samples) from 15 patients were included in this study. Matching germline DNA was available from 2 patients. We performed genome-wide methylation array profiling (Illumina Infinium 450k) on all samples, as well as whole-genome (for samples with matching germline DNA) or whole-exome sequence analysis. Fluorescence in situ hybridization (FISH) and digital droplet PCR (ddPCR) was performed to confirm select focal somatic gains.

RESULTS: 14/18 samples from 9/13 patients analyzed by 450k profiling had a methylation signature similar to previously profiled PBs from a reference cohort. Samples from 4 patients were found to be more consistent with a diagnosis of embryonal tumor with multilayered rosettes (ETMR) - like tumor (non 19q amplified), papillary tumor of the pineal region, or pineal parenchymal tumor of intermediate differentiation, respectively. No mutations in DICER1 or RB1 were found. Homozygous deletions in DROSHA were found in tumors from 3 PB patients. In addition, we identified novel recurrent somatic gains involving chromosomal region 1q21 that were confirmed by FISH and ddPCR in 4/5 PB patients.

CONCLUSION: Our studies revealed multiple candidate drivers of oncogenesis in PB. We identified novel homozygous deletions in DROSHA, a nuclease involved in microRNA processing. We also identified novel, highly recurrent somatic focal gains involving chromosomal region 1q21, which has been linked to brain growth, autism and schizophrenia, but not previously associated with cancer.

Citation Format: Matija Snuderl, Kasthuri Kannan, Olga Aminova, Igor Dolgalev, Adriana Heguy, Arline Faustin, David Zagzag, Sharon L. Gardner, Jeffrey C. Allen, Jeffrey H. Wisoff, David Capper, Volker Hovestadt, Sama Ahsan, Charles Eberhart, Stefan M. Pfister, David T. w. Jones, Matthias A. Karajannis. Novel candidate oncogenic drivers in pineoblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-172. doi:10.1158/1538-7445.AM2015-LB-172
Abstract LB-191: Association of anti-estrogen therapy in breast cancer patients and subsequent risk of rheumatoid arthritis: An electronic health record study
Authors: Breitenstein Matthew K. , Ho Ming-Fen , Weinshilboum Richard M. , Cerhan James R. , Pathak Jyotishman , Bongartz Tim , Wang Liewei , Ingle James N.
Published: August 2015

Background: The use of anti-estrogen (AE) therapies (selective estrogen receptor modulators [SERMs], aromatase inhibitors [AIs]) in breast cancer (BC) patients was recently associated with risk of developing rheumatoid arthritis (RA) (J Rheum 2015;42:55-9). We attempted to replicate and extend these results using electronic health record (EHR) enhanced cancer registry data that assessed specific AE therapies and also accounted for potential confounding by age, smoking status, and chemotherapy use.

Methods: Using cancer registry and EHR data, we identified a cohort of BC patients who were newly diagnosed and treated at Mayo Clinic Rochester from 1998-2011 and had no previous diagnosis of RA. Smoking status at diagnosis, BC treatments and RA at diagnosis and during follow-up were identified using validated EHR-based algorithms. Hazard ratios (HRs) and 95% confidence intervals (CI) from a multivariate Cox model were used to estimate the association of AE use with risk of RA, controlling for age, smoking status at BC diagnosis, and BC chemotherapy use.

Results: The analytic cohort of 9,244 newly diagnosed BC patients treated and followed at Mayo Clinic had a median age at diagnosis of 59 years (range 18-97); 32% were smokers; and 29% were treated with chemotherapy. During a median follow-up of 49 months (range 1-191), 19 patients developed RA. Compared to BC patients not receiving any AE therapy, those receiving AI monotherapy, but not SERM monotherapy or both SERMs and AIs, were at significantly increased risk of developing RA (Table).

AE Therapy# of RA Cases / # patientsHR* (95% CI)P-valueNo AE therapy6/4,0711.00 (reference)SERMs only5/2,5371.20 (0.36 - 3.95)0.77AIs only5/7126.37 (1.77 - 22.92)0.005SERMs then AIs3/1,8750.90AI then SERMs0/490.91 (0.21 - 3.86)*Adjusted for age, smoking status and use of chemotherapy

Conclusions: Our findings indicate that the use of AIs for BC therapy, but not SERMs or SERMs followed by AIs, is associated with increased risk for development of RA, when controlling for smoking status and use of chemotherapy. While needing replication, these findings suggest a specific role for AIs in RA development and suggest pathways that could be targeted to prevent this potential treatment complication.

Citation Format: Matthew K. Breitenstein, Ming-Fen Ho, Richard M. Weinshilboum, James R. Cerhan, Jyotishman Pathak, Tim Bongartz, Liewei Wang, James N. Ingle. Association of anti-estrogen therapy in breast cancer patients and subsequent risk of rheumatoid arthritis: An electronic health record study. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-191. doi:10.1158/1538-7445.AM2015-LB-191
Abstract LB-204: Reprogramming inflammatory monocytes to mediate anti-tumor activity in pancreatic carcinoma
Authors: Long Kristen B. , Gladney Whitney L. , Tooker Graham M. , Beatty Gregory L.
Published: August 2015

In cancer, increased mobilization of inflammatory monocytes from the bone marrow into the peripheral blood correlates inversely with patient survival. Peripheral blood inflammatory monocytes, which express CCR2, are recruited to tumor tissue by the chemokine CCL2 where they then differentiate into macrophages and support tumor development, growth, and metastasis. While neutralization of CCL2 can block inflammatory monocyte recruitment and inhibit metastasis, this approach has recently been shown to be at risk for lethal outcomes if therapy is interrupted. An alternative approach to inhibiting inflammatory monocyte recruitment to tumors is to reprogram monocytes with anti-tumor activity. Using the KrasG12D/+; Trp53R172H/+; Pdx-1 Cre (KPC) genetically engineered mouse model of pancreatic ductal adenocarcinoma (PDAC), we report that systemic immune activation induced with an agonist CD40 monoclonal antibody can redirect Gr-1+ inflammatory monocytes to infiltrate the tumor microenvironment and degrade tumor-associated fibrosis leading to tumor regression. Extra-tumoral macrophages were found to be necessary for mobilization of inflammatory monocytes from the bone marrow into the peripheral blood. In addition, systemic IFN-γ released in response to anti-CD40 therapy was necessary for reprogramming inflammatory monocytes with anti-tumor activity. Inflammatory monocytes responding to IFN-γ displayed a distinct matrix metalloproteinase gene expression profile necessary for selective degradation of extracellular matrix proteins, including type I collagen and fibronectin, which define fibrosis in PDAC. Therefore, although inflammatory monocytes are commonly associated with pro-tumor activity, our findings demonstrate that they can also be reprogrammed with potent anti-tumor properties.

Citation Format: Kristen B. Long, Whitney L. Gladney, Graham M. Tooker, Gregory L. Beatty. Reprogramming inflammatory monocytes to mediate anti-tumor activity in pancreatic carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-204. doi:10.1158/1538-7445.AM2015-LB-204
Abstract LB-214: DNAJB1-PRKACA chimera increases Aurora kinase A expression in fibrolamellar hepatocellular carcinoma
Authors: Lim Irene Isabel P. , Greene-Colozzi Emily A. , Murphy Jennifer M. , Heaton Todd E. , Simon Sanford M. , LaQuaglia Michael P.
Published: August 2015

Purpose: Treatment of fibrolamellar hepatocellular carcinoma (FL-HCC), a rare liver cancer affecting the pediatric and adolescent population, is limited to surgical resection with no agents or therapeutic modalities available for unresectable disease. There has been recent interest in using Aurora kinase A (AURKA) inhibitors, with a clinical trial evaluating its efficacy in FL-HCC pending. Previously, we described a consistent single deletion of approximately 400 kB in chromosome 19 that results in a chimera between the heat shock protein DNAJB1 and the catalytic subunit of protein kinase A, PRKACA, in all FL-HCC tumor tissue studied. Furthermore, comparative ribonucleic acid sequence analysis has demonstrated that expression of AURKA, a regulator of cytokinesis and mitosis, is increased in FL-HCC tumor tissue compared with adjacent normal tissue. We hypothesize that the DNAJB1-PRKACA chimera is responsible for changes in AURKA expression in FL-HCC.

Methods: The DNAJB1-PRKACA chimera was cloned into a lentiviral vector with a fluorescent reporter. Two stable cell lines (HeLa and a hepatocyte cell line, Huh-7) were transduced with a lentivirus containing either an empty multicloning site (control) or the DNAJB1-PRKACA chimera. Transduction was confirmed by the presence of a positive epifluorescent signal and immunoblots showing expression of the chimeric protein. Microarray data were evaluated for changes in AURKA expression while immunoblots were analyzed for the presence of AURKA in all transduced cells.

Results: Microarray analysis showed an increase in AURKA transcripts in cells stably expressing the DNAJB1-PRKACA chimera. Compared to cells transduced with the control lentivirus, HeLa and Huh-7 cells transduced with the DNAJB1-PRKACA chimera demonstrated an increase in AURKA on immunoblots. Levels of a loading control, actin, were equivalent, indicating equal levels of protein examined between samples.

Conclusion: Expression of the DNAJB1-PRKACA chimera results in an increase in AURKA. Upregulation of AURKA, in turn, may be responsible for subsequent tumor formation in FL-HCC. This finding further supports the DNAJB1-PRKACA chimera's role in the pathogenesis of FL-HCC and provides a potential therapeutic target for a disease that has no effective treatment besides surgery.

Citation Format: Irene Isabel P. Lim, Emily A. Greene-Colozzi, Jennifer M. Murphy, Todd E. Heaton, Sanford M. Simon, Michael P. LaQuaglia. DNAJB1-PRKACA chimera increases Aurora kinase A expression in fibrolamellar hepatocellular carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-214. doi:10.1158/1538-7445.AM2015-LB-214
Abstract LB-217: PD-1 suppresses antibody responses to Tn+ tumors
Authors: Leyva Marcela A. , Haas Karen M.
Published: August 2015

Cancer cells often have aberrant glycosylation of surface molecules leading to glycan antigens that are not typically found on normal cells. These antigens are referred to as tumor associated carbohydrate antigens (TACAs) and are a promising target for anti-cancer vaccines. One such TACA is the Thomsen-nouvelle (Tn) antigen, which is expressed by up to 90% of adenocarcinomas. The mechanisms regulating the B cell response to Tn and other TACAs are not well understood. This knowledge is essential for the development of vaccines to elicit protective immune responses to tumors bearing TACAs.

PD-1 (programmed cell death 1) is a member of the B7:CD28 superfamily of receptors found on antigen-activated B and T cells. It negatively regulates antigen receptor signaling and thereby limits adaptive immune responses, including the anti-tumor T cell response. However, virtually nothing is known about the role of PD-1 in regulating B cell responses to tumors. In this study, we tested the hypothesis that PD-1 suppresses B cell production of Tn-specific antibodies and thereby suppresses protective humoral immunity against Tn+ tumors. Our results show that PD-1−/− mice immunized with a Tn-bearing mucin (desialyated bovine submaxillary mucin) have increased Tn+ tumor-reactive IgM and IgG antibodies, relative to wild type mice. PD-1−/− mice immunized with the Tn+ mucin had significantly increased survival relative to wild type mice following challenge with a Tn+ bearing mammary tumor (TA3-Ha). Survival in PD-1−/− mice was dependent on the presence of B cells. Therefore, our results show that PD-1 plays a critical role in suppressing the protective anti-tumor B cell response elicited by Tn-bearing antigen immunization.

This work was supported by NIH/NCI F31 CA183567-01 fellowship to M.A.L. and an American Cancer Society Research Scholar Grant (RSG-12-170-01-LIB) to K.M.H.

Citation Format: Marcela A. Leyva, Karen M. Haas. PD-1 suppresses antibody responses to Tn+ tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-217. doi:10.1158/1538-7445.AM2015-LB-217
Abstract LB-233: Blockade of TGF-beta1 and 2 without TGF-beta3 blockade is sufficient to facilitate tumor vaccine efficacy
Authors: Terabe Masaki , Robertson Faith , Kato Shingo , De Ravin Emma , Clark Katharine , Mizra Amer M. , Berzofsky Jay A.
Published: August 2015

TGF-beta is one of the most potent immunosuppressive cytokines involved in the regulation of tumor immunity. TGF-beta has three isoforms, TGF-beta1, 2 and 3. It has been demonstrated in multiple mouse tumor models that blockade of all three isoforms of TGF-beta facilitates natural tumor immunosurveillance as well as tumor vaccine efficacy. Most studies on the roles of TGF-beta in immunology are on TGF-beta1, which has been demonstrated to induce Treg cells, IL-17-producing T cells and MDSCs. Although some studies reported immunosuppressive activities of TGF-beta2, the role of TGF-beta3 in immune regulation is not well understood. In this study we asked whether it is necessary to inhibit TGF-beta3 to enhance tumor immunity induced by a tumor vaccine in a syngeneic TC1 tumor model. When the tumor reached at least 5 mm in diameter, the mice were given a peptide-based vaccine targeting HPV16 E7, which is an oncogene expressed in TC1 cells. Although the vaccine alone had minimal effects on tumor growth, combination with an anti-TGF-beta antibody that neutralizes all three isoforms significantly delayed tumor growth. A similar effect was obtained with an antibody that neutralizes only TGF-beta1 and 2 but not TGF-beta3. Thus, it is not necessary to block TGF-beta3 to overcome immune suppression. Flow cytometric analysis of immune cells in tumor draining lymph nodes and tumors showed that there was no difference in the number of Treg and MDSCs between mice with/without treatment. The vaccine significantly increased the number of tumor antigen-specific CD8+ T cells and IFN-gamma producing T cells in both lymph nodes and tumors, but there was no further increase in combination with anti-TGF-beta. The vaccine also induced a significant number of T-bet-expressing T cells (both CD4 and CD8) in both tumor draining lymph nodes and tumors, and anti-TGF-beta, regardless of TGF-beta3 blockade, further increased the number of these cells. Together the results suggested that blockade of TGF-beta1 and 2 alone is sufficient to enhance therapeutic tumor vaccine efficacy by facilitating the induction of Th1 type T cells. With the notion that TGF-beta3 might be beneficial for patients in some cancers, developing TGF-beta antagonists that do not inhibit TGF-beta3 may be lead to better outcomes.

Citation Format: Masaki Terabe, Faith Robertson, Shingo Kato, Emma De Ravin, Katharine Clark, Amer M. Mizra, Jay A. Berzofsky. Blockade of TGF-beta1 and 2 without TGF-beta3 blockade is sufficient to facilitate tumor vaccine efficacy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-233. doi:10.1158/1538-7445.AM2015-LB-233
Abstract LB-237: Vaccination increases the breadth and diversity of melanoma neoantigen-specific T cells in humans
Authors: Carreno Beatriz M. , Magrini Vincent , Becker-Hapak Michelle , Kaabinejadian Saghar , Hundal Jasreet , Petti Allegra A. , Ly Amy , Lie Wen-Rong , Hildebrand William H. , Mardis Elaine R. , Linette Gerald P.
Published: August 2015

Melanoma genomes harbor somatic mutations that are caused by exposure to mutagens such as UV light. Tumor missense mutations (MM), translated into amino acid substitutions (AAS), may provide a form of non-self that elicits tumor specific T cell immunity. Indeed, T cell immunity directed against tumor encoded AAS has been reported in humans with cutaneous melanoma, thus implicating MM as a source of patient specific (private) neoantigens. However, a systematic evaluation of these putative neoantigens as validated targets is lacking and it is unknown whether the immune response directed against tumor encoded AAS can be augmented by vaccination. Here we show that vaccination against melanoma AAS-encoding peptides augments naturally occurring T cell immunity and reveals new HLA class I restricted private neoantigens in patients with advanced melanoma. Neoantigen specific T cells recognized naturally processed and presented antigen but failed to recognize wild type (non-mutated) antigen. The presentation of neoantigens by HLA-A*02:01 in human melanoma was confirmed by mass spectrometry. As determined by TCRβ CDR3 DNA sequencing, vaccination promotes a repertoire of neoantigen-specific T cells that is highly diverse in terms of both TCRβ usage and clonal composition. No evidence of autoimmunity was apparent after vaccination. Our results demonstrate that tumor MM can be targeted as neoantigens and vaccination directed at tumor AAS somatic mutations broadens the antigenic breadth and clonal diversity of anti-tumor immunity. These findings provide a new paradigm for private neoantigen identification in neoplasia and may form the basis of personalized cancer immunotherapies targeting somatic mutations.

Citation Format: Beatriz M. Carreno, Vincent Magrini, Michelle Becker-Hapak, Saghar Kaabinejadian, Jasreet Hundal, Allegra A. Petti, Amy Ly, Wen-Rong Lie, William H. Hildebrand, Elaine R. Mardis, Gerald P. Linette. Vaccination increases the breadth and diversity of melanoma neoantigen-specific T cells in humans. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-237. doi:10.1158/1538-7445.AM2015-LB-237
Abstract LB-247: Querying the RAS genomic network with siRNAs and and flow cytometry: Automatic, multidimensional phenotyping of 135 cancer cell lines by Gaussian mixture fitting and expectation maximization
Authors: Amzallag Arnaud , Yuan Tina L. , Bagni Rachel , Yi Ming , Stephens Robert , Ramawamy Sridhar , McCormick Frank , Benes Cyril H.
Published: August 2015

To discover novel therapeutic modalities and genomic predictors of response, large screen utilizing small molecules or sh/siRNA are performed on increasingly large collections of cancer cell lines. However these screens suffer from two main limitations: 1) the off-target effects of the probes 2) the coarse measurement of the cellular response that cannot distinguish between different outcomes such as proliferation block and apoptosis.

Here we profile 50 lung cancer cell lines using highly specific combinations of siRNAs against effector nodes of KRAS, and measured several characteristics of phenotypic response by flow cytometry including viability, level of reactive oxygen species and cell membrane integrity.

For each assay [node-cell line], typically 25,000 events were measured. We often observed multi modal distributions following node silencing. We used the expectation maximization algorithm to fit the different cell populations induced by the gene silencing. This allows us to automatically extract the proportion of dying cells, and also provides estimates of the cell growth impairment. Assessment of replicates shows that our results are highly reproducible, provide an accurate estimate of the proportion of dying cells, and reveal the complexity of multi-modal response of KRAS cancer cell lines to perturbation of key signaling nodes. Using the genomic profiling and pharmaceutical screen of the same cell lines, we reveal the association of specific node silencing with 1) the basal genomic state of the cell lines 2) the activity of a panel of drugs with the goal of identifying new targeting modalities and patient selection strategies for KRAS mutant cancers.

Citation Format: Arnaud Amzallag, Tina L. Yuan, Rachel Bagni, Ming Yi, Robert Stephens, Sridhar Ramawamy, Frank McCormick, Cyril H. Benes. Querying the RAS genomic network with siRNAs and and flow cytometry: Automatic, multidimensional phenotyping of 135 cancer cell lines by Gaussian mixture fitting and expectation maximization. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-247. doi:10.1158/1538-7445.AM2015-LB-247