bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 339: Heterozygous germline mutation in ABRAXAS causes BRCA1 mislocalization and DNA damage response defects
Authors: Winqvist Robert , Bose Muthiah , Sachsenweger Juliane , Laurila Niina , Parplys Ann Christin , Willmann Jonas , Eshraghi Leila , Dunlop Thomas W. , Groth Marco , Rapakko Katrin , Nieminen Pentti , Friedl Thomas W. , Heiserich Lisa , Meyer Felix , Tuppurainen Hanna , Devarajan Ganapathy Raman , Peltoketo Hellevi , Nevanlinna Heli , Pylkäs Katri , Borgmann Kerstin , Wiesmuller Lisa , Pospiech Helmut
Published: August 2018

Whilst heterozygous germline mutations in the ABRAXAS gene have been associated with hereditary breast cancer predisposition, their initial effect on promoting tumorigenesis at the cellular level has not been explored. Here, we demonstrate in patient-derived cells that the Finnish ABRAXAS founder mutation (c.1082G>A, Arg361Gln), even in the heterozygous state leads to decreased BRCA1 protein levels as well as reduced nuclear localization and foci formation of BRCA1 and CtIP. This causes disturbances in basal BRCA1-A complex localization, which is reflected by a restraint in error-prone DNA double-strand break (DSB) repair pathway usage, attenuated DNA damage response, deregulated G2-M checkpoint control and apoptosis. Most importantly, mutation carrier cells display a change in their transcriptional profile, which we attribute to the reduced nuclear levels of BRCA1. The current study clearly demonstrates how the Finnish ABRAXAS founder mutation acts in a dominant-negative manner on BRCA1 to promote genome destabilisation in heterozygous carrier cells.

Citation Format: Robert Winqvist, Muthiah Bose, Juliane Sachsenweger, Niina Laurila, Ann Christin Parplys, Jonas Willmann, Leila Eshraghi, Thomas W. Dunlop, Marco Groth, Katrin Rapakko, Pentti Nieminen, Thomas W. Friedl, Lisa Heiserich, Felix Meyer, Hanna Tuppurainen, Ganapathy Raman Devarajan, Hellevi Peltoketo, Heli Nevanlinna, Katri Pylkäs, Kerstin Borgmann, Lisa Wiesmuller, Helmut Pospiech. Heterozygous germline mutation in ABRAXAS causes BRCA1 mislocalization and DNA damage response defects [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 339.
Abstract 3393: Characterizing recurrent high-grade serous ovarian cancer through whole-genome sequencing
Authors: Christie Elizabeth L. , Pattnaik Swetansu , Fereday Sian , Study Australian Ovarian Cancer , Thorne Heather , Bild Andrea , Bowtell David D.
Published: August 2018

Acquired drug resistance is the major obstacle in controlling high-grade serous ovarian cancer (HGSC) and leads to poor overall survival. Comparatively little massively parallel sequencing data exist from HGSC patients with recurrent disease who have been extensively treated with chemotherapy or newer targeted agents, such as anti-angiogenics or PARP inhibitors (PARPi). We previously identified four mechanisms of acquired resistance from whole-genome sequencing (WGS) of 23 HGSC patients with recurrent disease; a resistance mechanism of any kind was only identified in half of the patients. Since 2012, we have collected tumor samples from 16 HGSC patients through our rapid-autopsy program. On average, we collect 17 tumor sites per patient during the autopsy, and to date we have performed WGS on 28 autopsy samples from 8 patients. These data are allowing us to understand HGSC at end-stage, by examining the complete catalog of resistance mechanisms within an individual patient and reconstructing the natural history of HGSC. One acquired resistance mechanism we identified in HGSC involves transcriptional fusion of the drug efflux pump ABCB1 to an upstream gene that causes its overexpression. ABCB1 encodes the multidrug resistance transporter MDR1, also known as P-glycoprotein. Through WGS and targeted RNA sequencing on end-stage tumor samples as well as recurrent ascites samples, we identified multiple fusion partners to ABCB1, with more than 15% of recurrent HGSC patients harboring ABCB1 transcriptional fusions. A number of HGSC patients carry multiple ABCB1 fusions, demonstrating convergent evolution within patients and the strong selective advantage of ABCB1 overexpression by gene fusion. Extending our analysis to recurrent and autopsy samples from breast and prostate cancer patients also identified ABCB1 transcriptional fusions as a resistance mechanism in these cancers. Our findings suggest that ABCB1 transcriptional fusions are a common mechanism of acquired chemotherapy resistance in ovarian, breast and prostate cancer patients, and that characterizing end-stage disease is particularly informative for understanding resistance mechanisms.

Citation Format: Elizabeth L. Christie, Swetansu Pattnaik, Sian Fereday, Australian Ovarian Cancer Study, Heather Thorne, Andrea Bild, David D. Bowtell. Characterizing recurrent high-grade serous ovarian cancer through whole-genome sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3393.
Abstract 3399: Comparative genomic analyses of osteosarcoma etiology reveal a chromosomal structural rationale for the increased incidence of osteosarcoma in dogs
Authors: Sarver Aaron L. , Mills Lauren , Temiz Nuri , Scott MIlcah , Sarver Anne , Spector Logan , Wang Jinhua , Breen Mathew , Subramanian Subbaya , Moriarity Branden , Modiano Jaime , Largaespada David
Published: August 2018

Risk of osteosarcoma is significantly higher in large and giant breed dogs than in any other animal, including humans. To identify the reason for this observation, we used a comparative genomic approach to identify aberrations responsible for osteosarcoma etiology in a Sleeping Beauty transposon-accelerated mouse model, in human patients, and in naturally occurring canine tumors, using RNA-Seq and exome paired tumor normal analyses. Fusions identified in Sleeping Beauty-mutagenized tumors revealed a role for Cdkn2a disruption in Trp53 signaling, and for numerous genes which cooperate with disrupted TrpP53 signaling, indicating the existence of many diverse routes to osteosarcoma tumor formation. Similarly, human tumors showed TP53 pathway disruption associated with a high level of diversity of additional driver mechanisms, supporting multiple independent routes to tumor formation. However, in the majority of canine tumors, observed TP53 pathway aberrations co-occurred with loss of both copies of the region containing the PTEN tumor suppressor. In human osteosarcoma, only heterozygous PTEN loss was observed, and in both humans and mice, PTEN aberration was observed in a much smaller percentage of the tumor population. We hypothesize that increased osteosarcoma incidence in dogs is partly due to a syntenic rearrangement of the peri-PTEN locus in the canine genome. The PTEN gene is part of a small synteny block that localized in the distal end of canine chromosome 26 (CFA 26) during evolution. We hypothesize that this location change creates a high risk of loss in the context of cytogenetic instability caused by disruption of TP53, thereby providing a structural genetic rational for the higher incidence of osteosarcoma in dogs. Consistent with these results and with the powerful nature of PTEN as a tumor suppressor, canine osteosarcomas with homozygous loss of PTEN are associated with worse outcomes than canine osteosarcomas with intact PTEN. These results suggest that engineering genomes to minimize cancer risk may be a realistic approach to the prevention of cancer.

Citation Format: Aaron L. Sarver, Lauren Mills, Nuri Temiz, MIlcah Scott, Anne Sarver, Logan Spector, Jinhua Wang, Mathew Breen, Subbaya Subramanian, Branden Moriarity, Jaime Modiano, David Largaespada. Comparative genomic analyses of osteosarcoma etiology reveal a chromosomal structural rationale for the increased incidence of osteosarcoma in dogs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3399.
Abstract 34: Identification of a phosphorylation cascade triggering Wnt/planar cell polarity signaling in breast cancer cells
Authors: Borg Jean-Paul
Published: August 2018

The developmental Wnt/planar cell polarity (PCP) pathway is the most recently described branch of Wnt signaling strongly implicated in cancer development at early and late stages. Upregulation of Wnt/PCP components and signaling is indeed observed in many cancers, this event being mostly associated with cancer progression. However, how the function of this signaling is remains presently an open question. Recent work in our laboratory found that Prickle1 and Vangl2, two core Wnt/PCP components, are overexpressed in triple negative breast cancers and associated with poor prognosis. Prickle1 is a cytoplasmic PET/LIM-containing protein phosphorylated by the serine/threonine kinase Mink1 which triggers Prickle1 localization at the plasma membrane and regulates its activity. Activation of the Prickle1-Mink1 axis promotes breast cancer cell motility and metastatic spreading in the mouse by promoting β1-integrin turn-over, focal adhesion dynamics and cytoskeleton remodeling. However, Prickle1 is probably not the unique substrate of Mink1 in breast cancer cells. Through a combination of protein purification and mass spectrometry analysis, we identified novel partners of the Prickle1-Mink1 complex which includes LL5β, a PH domain containing protein involved in β1-integrin turn-over. In parallel, by an independent approach looking for Mink1 substrates in breast cancer cells, we identified LL5β as a potential substrate for this poorly described protein kinase. Using recombinant LL5β and Prickle1, we confirmed that Mink1 can directly phosphorylate both proteins in in vitro kinase assays. We also defined that, in breast cancer cells, Mink1 associates with and phosphorylates LL5β, controlling its subcellular localization, a key step for cancer cell motility. Our data provide novel insights on the regulation of the prometastatic Wnt/PCP pathway by characterizing an yet unknown multi-step phosphorylation cascade with promising targeting potential.

Citation Format: Jean-Paul Borg. Identification of a phosphorylation cascade triggering Wnt/planar cell polarity signaling in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 34.
Abstract 3405: A mutational signature associated with alcohol consumption and prognostically mutated driver genes in esophageal squamous cell carcinoma
Authors: Li Xiangchun , Wang Mengyao , Yang Meng , Dai Hongji , Zhang Baifeng , Wang Wei , Chu Xinlei , Wang Xin , Zheng Hong , Niu Ruifang , Zhang Wei , Chen Kexin
Published: August 2018

Aim: Esophageal squamous cell carcinoma (ESCC) is often diagnosed at advanced and incurable stage. Information on driver genes and prognosticators in ESCC remains incomplete. Herein, we elucidated significantly mutated genes (SMGs), mutational signatures, and prognosticators in ESCC.

Methods: Three MutSig algorithms (i.e. MutSigCV, MutSigCL and MutSigFN) and “20/20” ratio-metric were employed to identify SMGs. Nonnegative matrix factorization was used to decipher mutational signatures. Kaplan-Meier survival analysis, multivariate Cox and logistic regression models were applied to analyze association between mutational features and clinical parameters.

Results: We identified 26 SMGs, including 8 novel (NAV3, TENM3, PTCH1, TGFBR2, RIPK4, PBRM1, USP8 and BAP1) and 18 previously reported. Three mutational signatures were identified to be prevalent in ESCC including clocklike C>T at CpG, APOBEC overactive C>T at TpCp[A/T], and a signature featured by T>C substitution. The T>C mutational signature was significantly correlated with alcohol consumption (OR: 3.59; 95% CI: 2.30-5.67; P < 0.001). This alcohol consumption signature was also observed in liver cancer and head and neck squamous cell carcinoma, and its mutational activity was substantially higher in samples with mutations in TP53. Survival analysis revealed that TENM3 mutations (HR: 5.54; CI: 2.68-11.45; P < 0.001) and TP53 hotspot mutation p.R213* (HR: 3.37; CI: 1.73-8.06; P < 0.001) were significantly associated with shortened survival outcome. The association remained statistically significant after controlling for age, gender, TNM stage and tumor grade.

Conclusions: We have uncovered several new SMGs in ESCC and defined an alcohol consumption related mutational signature. TENM3 mutation and TP53 hotspot mutation p.R213* are independent prognosticators for poor survival in ESCC.

Citation Format: Xiangchun Li, Mengyao Wang, Meng Yang, Hongji Dai, Baifeng Zhang, Wei Wang, Xinlei Chu, Xin Wang, Hong Zheng, Ruifang Niu, Wei Zhang, Kexin Chen. A mutational signature associated with alcohol consumption and prognostically mutated driver genes in esophageal squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3405.
Abstract 3408: Characteristics of circulating tumor DNA in lung cancer patients
Authors: Abe Tomonori , Nakashima Chiho , Sato Akemi , Sueoka Eisaburo , Kimura Shinya , Aragane Naoko
Published: August 2018

Liquid biopsy using circulating tumor DNA (ctDNA) has been spread world-wide. We established fully automatic sensitive mutation detection system, mutation-biased PCR and quenched probe system (MBP-QP) method, and accomplished multicenter prospective study to investigate the utility of ctDNA in lung cancer patients who acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKI). The results of the clinical study showed that ctDNA was frequently detected in lung cancer patients with distant metastasis, and detection of ctDNA was associated with poor prognosis. In metastatic animal model using immunodeficient mice, NOJ mice, the amount or ctDNA was associated with tumor progression such as tumor burden and metastasis. Based on these evidences, we hypothesized that ctDNA has some biological effects on tumor progression. In order to examine that, we analyzed biological and clinical characteristics of ctDNA. ctDNA was extracted from 1000μl plasma by automated DNA extraction system using cellulose magnetic beads. Compared to circulating free DNA (cfDNA) isolated from healthy volunteers, size distribution of ctDNA or cfDNA using a capillary electrophoresis system was different, that is one peak around the size of 170 bp in healthy individuals, and two peaks, 170 bp and 5 kb in advanced lung cancer patients. We next examined 130 plasma samples from 92 lung cancer patients in addition to 18 benign pulmonary disease patients and 20 healthy individuals at Saga University Hospital. The DNA concentration quantified by Quantus®, the fluorescent measurement of dsDNA intercalated dye, was higher in lung cancer patients compared to those in benign pulmonary disease patients and healthy individuals. Among lung cancer patients, DNA concentration was increased in those with advanced stages, especially in presence of metastasis. In addition, 5 kb fragments were significantly increased in these cases compared to 170 bp fragments. To investigate which fragment contained tumor-derived DNA, 170 bp and 5 kb fragments were separately isolated, and EGFR mutation, L858R was examined. L858R was detected in both ctDNA fragments, 170 bp and 5 kb, indicating that both sized DNA fragments contain tumor-derived DNA. Although the 170 bp short fragments of ctDNA are well known as an apoptotic product, the origin of 5 kb long fragments has not been clarified. We have examined whole ctDNA sequence using next generation sequencing, and compared two fragments. In addition, origins of each fragment have been investigated, and biological effects on tumor progression will be analyzed.

Citation Format: Tomonori Abe, Chiho Nakashima, Akemi Sato, Eisaburo Sueoka, Shinya Kimura, Naoko Aragane. Characteristics of circulating tumor DNA in lung cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3408.
Abstract 3410: Copy number estimation from whole-exome sequencing in tumors
Authors: Anderson Shawn , Che Zhiwei , Keshavan Raja , O'Hara Andrea , Lin Dong , Wang Yuzhuo , Collins Colin , Shams Soheil
Published: August 2018

At the Vancouver Prostate Centre (VPC), a personalized medicine approach is the focus to the understanding and treatment of prostate and other cancers. As a part of this approach, the xenograft program is used to identify and validate mechanisms and drivers of progress and therapeutic targets. VPC frequently uses Next-Generation Sequencing (NGS) technologies for copy number (CN) analysis. Many CN from NGS algorithms require a matched normal reference for CN estimation, however, a matched normal may not be possible for many reasons including unavailability of patient blood sample and poor quality of the matched sample in question. The correct CN algorithm for NGS data is critical to overcome these obstacles. Here we will share our results from analysis of Whole Exome Sequencing (WES) of prostate cancer patient-derived xenograft (PC PDX) samples using Nexus Copy Number software with BioDiscovery's BAM MSR algorithm. A pooled reference can be created using unmatched normal samples; however, when normal samples are not available, tumor samples from within the batch and/or run can be used to create a reference. BAM MSR derives CN and B-Allele Frequency (BAF) from WES, whole-exome sequencing (WGS), and targeted panels using a pooled reference. Due to an absence of normal samples, randomly selected tumor samples were used to build a reference file, which was used for baseline CNV results. By using a recursive refinement method, which incorporated evaluation of B-allele frequency (BAF) patterns among the pooled samples, a subset of tumor samples was selected to create a refined pooled reference using BAM MSR. All tumor samples were then subjected to CN estimation, which included adjustment based on tumor ploidy. While creating a normal reference including PC PDX tumor samples may seem counter intuitive due to enrichment of cancer cells and replacement of stromal component with mouse origin (e.g. fibroblasts, endothelium and immune cells), and high early recurrent copy number signatures, the BAM MSR algorithm was able to get results comparable to previous aCGH results. This is of special importance because the lack of normal tissues is commonplace in cancer research.

Citation Format: Shawn Anderson, Zhiwei Che, Raja Keshavan, Andrea O'Hara, Dong Lin, Yuzhuo Wang, Colin Collins, Soheil Shams. Copy number estimation from whole-exome sequencing in tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3410.
Abstract 3412: Androgen receptor binding sites are highly mutated in prostate cancer
Authors: Morova Tunc , Gonen Mehmet , Gursoy Attila , Keskin Ozlem , Lack Nathan A.
Published: August 2018

Androgen receptor (AR) signaling is essential to nearly all prostate cancer cells. Any alterations to AR-mediated transcription can have a profound effect on prostate carcinogenesis and tumor growth. While the AR protein has been extensively studied, little is know about those mutations to non-coding regions where AR binds to DNA. Using whole genome sequencing from 197 primary prostate cancer patients, we demonstrate that AR binding sites have a dramatically increased rate of mutations that is greater than any other transcription factor. This is not due to the specific chromosomal region as increased mutation were only observed in prostate cancer. The rate of mutations correlated with AR occupancy and did not cluster with any histone mark or transcription factor. Suggesting that this may occur with other lineage-specific transcription factors, we observed that estrogen receptor binding sites also had an elevated rate of mutations in breast cancer. Interestingly, the mutations at AR binding sites had a high frequency of TpG->ApG. These uncommon purine transversions have been shown to occur when abasic sites are incorrectly repaired. Based on the mutations observed at the binding site of AR and other related transcription factors, we propose that AR occupancy impairs access of base excision repair enzymes to endogenous DNA damage. Overall, this work demonstrates that non-coding AR binding sites are frequently mutated in prostate cancer and may potentially act as driver mutations.

Citation Format: Tunc Morova, Mehmet Gonen, Attila Gursoy, Ozlem Keskin, Nathan A. Lack. Androgen receptor binding sites are highly mutated in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3412.
Abstract 3421: Genomic correlates of patient age in diffuse-type gastric cancer
Authors: Kim Hark K. , Cho Soo Y.
Published: August 2018

Background: Gastric cancer is increasing in incidence among young white populations in the US. Gastric cancer in young patients is notable for its enrichment of diffuse histology as well as female predominance and aggressive clinical course, whereas gastric cancer is generally more frequent in males. Thus, clinicopathological characteristics of diffuse-type gastric cancer differ according to patient age, but the molecular mechanisms for early-onset gastric cancer's unique clinicopathological characteristics have not been elucidated. While the incidence of intestinal-type gastric cancer is decreasing worldwide, that of diffuse-type gastric cancer remained constant. Nonetheless, relatively small numbers of diffuse-type gastric cancers have been represented in whole exome sequencing studies such as TCGA project. We therefore investigated germline and somatic mutation profiles of diffuse-type gastric cancers with regards to patient age.

Methods: We conducted whole exome and targeted sequencing and SNP6.0 array analyses of resected tumor tissue and blood samples collected from young (45 years old or younger) Korean patients with diffuse-type gastric cancer, and compared the genomic data with those from older Korean patients with diffuse-type gastric cancer. Genomic data of Korean diffuse-type gastric cancers were also compared with TCGA diffuse-type gastric cancers, 70% of which were from Caucasians.

Results: Among 84 young Korean patients with DGC, 7 patients (8.3%) harbored germline mutations in TP53, CDH1, ATM, RAD51D, or PALB2. Three tumors with strong mutation signatures for inherited DNA repair defects harbored germline mutations in either RAD51D or PALB2, with loss-of-heterozygosity in the tumors. The most significant somatic mutations in young patients with diffuse gastric cancer were CDH1, TP53, ARID1A, KRAS, PIK3CA, ERBB3, TGFBR1, FBXW7, RHOA, and MAP2K1. Within the Korean population and within populations of other ethnicities, mutations in CDH1 (42.2%) or TGFBR1 (7.3%) were more frequent in younger patients than in older patients (17.4% and 0.9%, respectively). In contrast, the RHOA mutation (9.2%) was less frequent in younger patients than in older patients (19.1%). CDH1 alterations, but not RHOA mutations, were associated with poor prognosis (hazard ratio, 3.4 (95% CI, 1.5-7.7)), which may be associated with the aggressive clinical course of diffuse-type gastric cancers in young patients.

Conclusions: Our largest-ever genomic analysis of diffuse-type gastric cancer reveals young age-specific mutation profiles (supported by National Cancer Center Grant 1710809 and Multi-omic Research Program).

Citation Format: Hark K. Kim, Soo Y. Cho. Genomic correlates of patient age in diffuse-type gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3421.
Abstract 3425: Exome-sequencing derived mutations of endocrine treated ER-positive early breast cancer
Authors: Schroth Werner , Kandabarau Siarhei , Fritz Peter , Mürdter Thomas , Bacchus Liza , Group German Tamoxifen Study , Schwab Matthias , Brauch Hiltrud
Published: August 2018

Anti-hormonal treatment of ER-positive early breast cancer with tamoxifen and/or aromatase inhibitors (AI) significantly improves patient outcome, however there is a lack of markers that predict the one third of treated patients who relapse or die. Although ER positive (luminal) tumors are known to be genetically highly diverse, the relevance of somatic mutations with regards to endocrine treatment outcome and course of the patients' disease is not fully understood. We conducted a prospective observational clinical trial for the investigation of treatment outcome determinants including 1219 tamoxifen and/or AI-treated postmenopausal ER-positive early breast cancer patients (DRKS00000605, recruitment between 2005-2010, median follow up 5.1 years). Of these, 40 tumors divided into 20 patients each with and without disease recurrence matched by prognostic variables (age, tumor size, grade, nodal status, treatment) were analyzed for their mutation pattern. We performed whole-exome sequencing of tumor and normal DNA (Agilent SureSelect, Illumina) for the identification of single nucleotide variants and small indels. Somatic mutations were identified following subtraction of germline single nucleotide polymorphisms (SNP), filtering based on raw-read quality parameters, in silico predicted biological function, and disease relatedness using the Ingenuity variant analysis software package (Qiagen). Clinical outcome associations were tested either on gene or on variant level. Somatic mutations with predicted deleterious effects were identified in 18 out of 40 known mutation-driver genes including PIK3CA, GATA3, MAP3K1, and TP53 (45%). Thirteen patients carried a mutation in the most frequently affected PIK3CA (32.5%). Of note, three out of four patients with a E542K mutation belonged to the breast cancer recurrence group, while 5 of 6 tumors with the H1047R mutation originated from patients without recurrence suggesting differential effects of these PIK3CA hotspot mutations. Common germline SNPs at IL17RB, ANK2, POM121, PGR, PTPRB, RAD51D, RFPLA4, and SUMO3 provided first hints for a possible association with relapse status (p<0.05). We will report on the extended genotype-outcome-correlations to further evaluate the relevance of somatic mutations for endocrine treatment outcome of early ER-positive breast cancer.

Citation Format: Werner Schroth, Siarhei Kandabarau, Peter Fritz, Thomas Mürdter, Liza Bacchus, German Tamoxifen Study Group, Matthias Schwab, Hiltrud Brauch. Exome-sequencing derived mutations of endocrine treated ER-positive early breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3425.
Abstract 3428: Development and validation of the plasma monitor test system
Authors: Kelly Kevin J. , Uvalic Jasmina , Bergeron Daniel , Burns Shelbi , Soucy Melissa , Ananda Guruprasad , Hesse Andrew , Selvam Pavalan Panneer , Reddi Honey V.
Published: August 2018

Introduction: Non-invasive monitoring of variants in a cancer patient for minimal residual disease, recurrence and/or resistance has tremendous clinical utility and warrants the development of a cell-free circulating tumor DNA panel, which involves the use of a simple blood draw. Towards this end, JAX has validated a new liquid biopsy assay called the Plasma MonitorTM that focuses on 84 clinically significant hotspots across 14 genes to complement our current clinical test menu, with a focus on comprehensive profiling of cancer.

Methods: Post development and optimization of a custom amplicon panel, which included running multiple batches of samples through the assay to determine appropriate extraction methods, input DNA QC metrics, sequencing batch size, and sequencing loading concentrations, clinical validation of The Plasma MonitorTM was initiated. Validation included the evaluation of 20 uncharacterized plasma samples and 14 known controls and was executed in 5 phases: (1) Sample Processing for Validation Parameter determination; (2) LOD, sensitivity, specificity and accuracy (3) inter-assay concordance; (4) intra-assay concordance; (5) clinical validity in terms of interpretation and reporting of variants identified.

Results: The final clinical protocol was developed using an input of 10ng of ctcfDNA quantified and qualified by a custom qPCR assay. Wet lab results of the first validation batch can be seen in Table 1. Using samples containing variants with known allele frequencies and a droplet digital PCR (ddPCR) based confirmation of novel variants identified by our assay, we established the assay's limit of detection (LOD) to be 0.9%. At this LOD, sensitivity, specificity, and accuracy were found to be 96.6%, 100%, and 98% respectively.

Conclusion: Based on the results of this validation, the Plasma Monitor assay will be incorporated into the JAX clinical test menu. This new assay allows for comprehensive profiling and monitoring of cancer progression, response to therapy and minimal residual disease, and a significant benefit to both patients and clinicians.

Citation Format: Kevin J. Kelly, Jasmina Uvalic, Daniel Bergeron, Shelbi Burns, Melissa Soucy, Guruprasad Ananda, Andrew Hesse, Pavalan Panneer Selvam, Honey V. Reddi. Development and validation of the plasma monitor test system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3428.
Abstract 3434: Digital spatial profiling platform allows for spatially resolved, high-plex quantification of mRNA distribution and abundance on FFPE and fresh frozen tissue sections
Authors: Zollinger Daniel , Sorg Kristina , McKay-Fleisch Jill , Barker Kristi , Nguyen Karen , Merritt Chris , Beechem Joseph
Published: August 2018

Characterization of spatial mRNA expression across multiple targets in a single tissue section has proven difficult to accomplish. To address this unmet need, we have developed a novel imaging platform, Digital Spatial Profiling (DSP), designed to simultaneously analyze 10s to 100s of RNAs (or proteins) from discrete regions by detecting oligo-conjugated probes. We show that this system can resolve mRNA expression panels in a highly multiplexed and automated manner on human FFPE and fresh frozen tissues. DSP RNA probes consist of antisense target-recognition sequences and UV-cleavable tags that can be released in a precise manner by the DSP instrument and digitally counted using nCounter® reagents. For this, probes are first hybridized overnight in situ to human FFPE or fresh frozen tissue sections. After hybridization, tags are released by nondestructive exposure of UV light, automatically collected by the DSP Instrument, and quantified using nCounter® technology. The nCounter PlexSet™ reagents are used to enable the analysis of 96 regions of interest with a single nCounter run. To demonstrate the regional specificity of RNA detection on DSP, we profiled distinct regions in human tonsillitis tissues using a 48-plex of RNA target probes. Our results show that genes are detected in the expected region-specific manner. Using an orthogonal in situ hybridization (ISH) approach, we validate the RNA DSP results in tonsil across multiple targets. To determine the regionally specific RNA expression in tumor area and nontumor area in non-small cell lung cancer (NCSLC), we used a high-plex probe mix of relevant Immuno-oncology RNA targets. These results show that the DSP platform can be used to obtain high-plex, spatial RNA expression data on tissue sections. The protein profiling assay on DSP, presented elsewhere, depends upon FFPE-compatible antibodies, which can be difficult to generate for some targets. Utilizing spatial mRNA profiling provides an alternative for cases where the necessary antibodies do not exist. In all, simultaneous, high-plex RNA and protein detection with DSP on serial sections could provide a novel method that can bridge the gap between translational research discovery and clinical applications.

Citation Format: Daniel Zollinger, Kristina Sorg, Jill McKay-Fleisch, Kristi Barker, Karen Nguyen, Chris Merritt, Joseph Beechem. Digital spatial profiling platform allows for spatially resolved, high-plex quantification of mRNA distribution and abundance on FFPE and fresh frozen tissue sections [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3434.
Abstract 3439: GATA3 mutation of luminal-type breast cancer patients in Taiwan
Authors: Tsai Shih Feng
Published: August 2018

To investigate the mutation landscape of luminal-type breast cancer in Taiwan, we have conducted whole-genome sequencing on 20 cases of breast cancer, including 15 cases of those positive for estrogen receptor and 5 cases negative for estrogen receptor. Specifically, we searched for mutations in TP53, GATA3, and ESR1 genes. Additionally, we designed an amplificon sequencing scheme to interrogate the GATA3 coding sequences. GATA3 mutation has been previously reported to be correlated with suppression of proliferation upon aromatase inhibitor treatment. Also, the prevalence and clinical significance of GATA3 mutation-positive breast cancer differed between the TCGA cohort and the Fudan cohort. In our current series that focuses on breast cancer patients of Han Chinese living in Taiwan, we have identified 21 GATA3 mutations among 72 breast cancer tumor specimens screened by the amplicon sequence, and another 3 samples with GATA3 mutation by whole-genome sequencing. Notably, Pro409Alafs mutation occurred in six cases and this truncation mutation showed an average VAF of 0.155. A specific antibody was raised against the additional c-terminal amino acid sequence for the detection of the abnormally expressed proteins in these cases. As GATA3 is required for epithelial cell differentiation and as both normal breast luminal epithelium and estrogen-receptor-positive tumors showed high GATA3 expression, we are conducting further studies to demonstrate the clinical relevance of GATA3 mutation and the potential value of the Pro409Alafs as an outcome predictor for luminal-type breast cancer.

Citation Format: Shih Feng Tsai. GATA3 mutation of luminal-type breast cancer patients in Taiwan [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3439.
Abstract 3443: Full-length characterization of transcript isoforms to investigate cancer-associated mutations
Authors: Tang Alison
Published: August 2018

Mutations in the splicing factor SF3B1 in various cancers have been associated with characteristic alterations in splicing. SF3B1 is one of the most frequently mutated genes in chronic lymphocytic leukemia and is associated with poor patient prognosis. While alternative splicing patterns caused by mutations in SF3B1 on a junction level have been observed, these patterns have not been systematically examined on an isoform level. Isoform-level analyses are imperative for building a better understanding of altered splicing in a cell; exon connectivity is best resolved using long reads. As such, we have resequenced cDNA from CLL samples with and without K700E mutation in SF3B1, as well as a normal B cell, with nanopore sequencing technology. By converting changes in current caused by blockage of DNA threading through a nanopore into sequence, nanopore sequencing can sequence complete molecules of DNA that exceed 200kb in length. We have developed a novel workflow to perform isoform-level differential splicing analyses leveraging the full-length transcript data that nanopore affords. We report results from nanopore sequencing data that are concordant with known SF3B1 biology from short read sequencing as well as altered splicing patterns that are only observed definitively using long reads. Splicing analysis of nanopore reads between the SF3B1WT and SF3B1K700E identifies a bias toward increased alternative 3' splice sites than alternative 5'. In addition, we observe a relative decrease in the expression of genes with intron retention events in CLL samples compared to B cell. We also discover a previously unreported finding of enrichment in intron retention events in SF3B1WT relative to SF3B1K700E and no enrichment between CLL SF3B1MT and B cell, suggesting that mutated SF3B1 in CLL modifies the intron retention landscape to be more similar to a normal phenotype. Gene ontology analyses reveal enrichment in nuclear export in genes associated with intron retention events, and we postulate that many of these are detained introns of transcripts that are kept in the nucleus. With full-length cDNA sequence, we are also able to better detect premature termination codons and estimate the proportion of transcripts subject to nonsense-mediated mRNA decay. As nanopore sequencing has yet to become a routine tool for characterization of the transcriptome, our work demonstrates the utility of nanopore sequencing for cancer and splicing research.

Citation Format: Alison Tang. Full-length characterization of transcript isoforms to investigate cancer-associated mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3443.
Abstract 3455: Biologic functions of BMX/BTK in non-small cell lung cancer
Authors: Huang Wen-Chien
Published: August 2018

Non-small cell lung cancer (NSCLC), a leading cause of tumor-related deaths in Taiwan and worldwide, remains not only a health care but also a social burden in Taiwan. EGFR and KRAS mutations and deregulation of the EGFR/KRAS pathways are critical for lung tumorigenesis and progression; however, our knowledge of the genes responsible for the heterogeneity of lung carcinoma is inconclusive. Despite increasing documentation of the role of the Bruton's tyrosine kinase (BMX/BTK) in tumorigenesis, as well as increasing use of BMX/BTK inhibitors as new cancer drugs, to the best of our knowledge, this is the first study on the identification and characterization of BMX/BTK in lung cancer. Our preliminary data show increased expression of BMX/BTK protein in lung cancer cells compared to their nontumor counterparts, using immunohistochemical (IHC) staining and Western blot. Further, using clinicopathologic analyses of large prognoscan database cohorts, we established a negative correlation between BMX/BTK expression and patients' outcome. However, this bioinformatics-based clinical significance of BMX/BTK and its functional mechanisms in the tumorigenesis of NSCLC remains to be verified. In this project, we will comprehensively investigate the role of the BMX/BTK gene in lung tumorigenesis and progression, with its underlying molecular mechanisms. We will also develop and evaluate the therapeutic effect of in-house small-molecule inhibitors of BMX/BTK in lung cancer using animal model. In aim 1, we will explore the expression of BMX/BTK and its related upstream regulators and downstream effectors in lung cancer using IHC staining of primary NSCLC tissue samples. These data will be collated and statistically correlated with patients' clinical data. In aim 2, we will investigate the role of BMX/BTK in lung cancer cell proliferation, epithelial-to-mesenchymal (EMT), and cancer stem cell phenotype, as well as determine the underlying molecular mechanisms, using loss and gain of BMX/BTK function, and functional assays such as the colony formation, invasion, migration and spheroid formation assays in lung cancer tissues and cell lines. In the aim, we will validate the in vitro findings by examining BMX/BTK activities in vivo, evaluate the therapeutic effect of novel BMX/BTK inhibitors and investigate its clinical significance, using tumor xenograft NOD-SCID mice models. We anticipate that this current study will demystify the role and molecular mechanisms of BMX/BTK in the tumorigenesis of lung cancer and provide relevant information for effective anti-lung cancer therapeutic strategy, including combined targeted therapy.

Citation Format: Wen-Chien Huang. Biologic functions of BMX/BTK in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3455.
Abstract 346: Targeting DNA repair with the combination of p97 and HDAC6 inhibitors in mantle cell lymphoma
Authors: Vekaria Pratikkumar , Vallurupalli Anusha , Subramaniam Dharmalingam , Schoenen Frank , McGuirk Joseph , Rao Rekha M.
Published: August 2018

p97 is an ATPase which is involved in remodeling multi-subunit protein complexes. It plays a pivotal role in the retrotranslocation and proteasomal degradation of misfolded ubiquitinated proteins in the endoplasmic reticulum. In association with cytosolic histone deacetylase 6 (HDAC6), p97 promotes the autophagic degradation of polyubiquitylated proteins and damaged organelles. More recently p97 has been implicated in regulating cell division and in facilitating DNA repair by promoting the recruitment of DNA repair proteins to the site of DNA damage. In this study, we determined the molecular mechanisms through which HDAC6 promotes p97 function in mantle cell lymphoma (MCL) cells. We report that treatment of MCL cells with the p97 inhibitors, DBeQ, ML240, NMS-873 and CB-5083 induces ER stress markers GRP78 and CHOP and resulted in dose-dependent apoptosis. Treatment of Z138C xenografts in NSG mice with CB-5083, the first in-class p97 inhibitor, resulted in increased accumulation of polyubiquitylated proteins and improved survival in vivo compared to control mice. Co-treatment with CB-5083 and the HDAC6-selective inhibitor ACY-1215, resulted in accumulation of polyubiquitylated proteins, impaired DNA double-stranded break repair and resulted in the partial depletion of BRCA1 and Cyclin D1 in MCL cells compared to either agent alone. We also demonstrate that shRNA-mediated knockdown of HDAC6 induced enhanced accumulation of H2AX-γ under basal conditions and following treatment with CB-5083 in cultured and primary MCL cells. Finally, treatment of MCL cells with p97 inhibitors and ACY-1215 results in synergistic apoptotic cell death in MCL cells. Collectively our studies create a strong rationale to test efficacy of the combination of p97 inhibitors in combination with HDAC6 inhibitors in MCL.

Citation Format: Pratikkumar Vekaria, Anusha Vallurupalli, Dharmalingam Subramaniam, Frank Schoenen, Joseph McGuirk, Rekha M. Rao. Targeting DNA repair with the combination of p97 and HDAC6 inhibitors in mantle cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 346.
Abstract 3471: Expression of the DEK oncogene in breast cancer cells promotes M2 polarization of tumor associated macrophages
Authors: Shephard Miranda S. , Pease Nicholas A. , Cheek Jon , Vinnedge Lisa M. Privette
Published: August 2018

Breast cancer is the most common cancer and the second leading cause of cancer deaths among women. DEK is a known oncoprotein found to be highly expressed in more than 60% breast cancers and is found to be an independent marker of poor prognosis. However, the molecular mechanisms by which DEK promotes tumor progression are poorly understood. To elucidate the oncogenic functions of DEK, we performed RNA-seq analysis on isogenic Dek-knockout and complemented murine breast cancer cells, which indicated dysregulation of immune signaling. Among the target genes identified and confirmed was an upregulation of thymic stromal lymphopoietin (TSLP) in Dek expressing breast cancer cells. TSLP was previously shown by Han et al to amplify the alternative (M2) activation of macrophages. M2-like macrophages are tumor promoting, they recycle iron for cell growth and encourage tissue remodeling and repair activities like angiogenesis. Classically (M1) activated macrophages are tumor suppressing and are typically associated with response to infection. To test the immune modulating functions of Dek-expressing breast cancer cells, we treated bone marrow derived macrophages (BMDM) with tumor cell conditioned media. We found that in vitro, Dek expressing cancer cells induced an M2-like polarization of macrophages, as determined by the expression of M2-associated genes, enhanced migration, and iron recycling phenotypes, which was accompanied by inhibited ERK1/2 sighnaling. We found this phenotypic trend to be true in vivo, as well. In sectioned mammary tumors from MMTV-RontgDek+/+ and MMTV-RontgDek-/- mice, we see lower levels of iron staining in Dek+/+ tumors than in Dek-/- tumors using Prussian blue staining that co-localized with F4/80 macrophage marker staining. This suggested that Dek expression in the tumors induced an iron recycling, M2-like phenotype in tumor associated macrophages. These findings suggest that tumor Dek expression may promote breast cancer progression by inducing M2-like macrophage polarization in a murine breast cancer model.

Citation Format: Miranda S. Shephard, Nicholas A. Pease, Jon Cheek, Lisa M. Privette Vinnedge. Expression of the DEK oncogene in breast cancer cells promotes M2 polarization of tumor associated macrophages [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3471.