bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2315: Perceived barriers to early diagnosis of breast cancer in south and southwestern Ethiopia: Qualitative study
Authors: Getachew Sefonias , Addissie Adamu , Tesfaw Aragaw , Taylor Lesley , Kantelhardt Eva
Published: September 2020

Background: Early diagnosis is a key determinant of breast cancer prognosis and survival. However, advanced stage presentation and delays in diagnosis are common problems in Ethiopia. More than half of the cases are diagnosed at advanced stages and the barriers to early diagnosis are not well studied in the country. Hence, this study aims to explore perceived barriers to early diagnosis of breast cancer in South and Southwestern Ethiopia.

Methods: A qualitative study was conducted from March to April 2018 in six selected public hospitals located both in urban and rural settings. Twenty five purposefully selected breast cancer patients and health care providers from these public hospitals were interviewed. An in-depth interview was conducted using a topic guide by qualitative research experts. A thematic analysis was performed using Open Code software version 4.02.

Results: Patient and health system related barriers are two main themes identified for barriers to early diagnosis of breast cancer. Patient related barriers included lack of awareness and knowledge of the disease, beliefs in traditional and religious means of treatments, lack of social and financial support to seek medical care. Health system related barriers included misdiagnosis of cases, long distance referrals due to service inaccessibility, high diagnostic costs, long waiting time, unavailability of screening and diagnostic tests.

Conclusion: Early diagnosis of breast cancer is affected by a multitude of barriers in South and Southwestern Ethiopia. Hence, to increase early diagnosis of breast cancer, awareness campaigns and education about the disease, its prevention, and early detection are needed. Opportunities exist to improve early diagnosis and timely treatment by strengthening referral linkages of health care facilities in rural areas. Efforts are needed to decentralize the tertiary level oncology care and improve capacity in local health facilities.

Citation Format: Sefonias Getachew, Adamu Addissie, Aragaw Tesfaw, Lesley Taylor, Eva Kantelhardt. Perceived barriers to early diagnosis of breast cancer in south and southwestern Ethiopia: Qualitative study [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2315.
Abstract 2328: Polyethnic-1000: Advancing cancer genomics by studying ethnically diverse, underserved patient populations in New York
Authors: Robine Nicolas , Froeling Fieke , Hubert Benjamin , Chu Timothy , Chandler Ashlea , Zody Michael C. , Oschwald Dayna M. , Varmus Harold E. , Sawyers Charles , Tuveson David
Published: September 2020

Advances in sequencing technologies have revolutionized our approaches in medicine. However, our current knowledge about the molecular attributes of cancer has primarily been derived from patients of European descent. The overall aims of Polyethnic-1000 are to study the variability of somatic profiles and the effects of genetic ancestry on cancer biology and to bring genomic innovation to under-represented ethnic groups in the diverse patient population of New York City.A dynamic research platform is being created, involving the New York Genome Center, academic centers and partnering hospitals in the New York City region. In an initial retrospective study, the infrastructure, sample workflow, analysis and data sharing policies within the consortium have been developed. Whole-exome and RNA sequencing has been performed on 146 samples from self-identified non-white cancer patients treated at 14 different centers. Genetic ancestry estimation was performed at the super-population and population level using the 1000Genomes references. Data from over 24 tumor types were shared with our collaborators and the somatic variants were made available through our local installation of cBioPortal. Using the knowledge gained during the first stage, we are designing a prospective study to demonstrate our capacity to obtain samples from patients of varied ethnicities and to allow the formation of cohorts of interest for particular cancer types and particular ethnicities, with uniform consent for germline and somatic sequencing and return of results for potentially actionable mutations.By establishing a collaborative network, Polyethnic-1000 will deepen our understanding of the contributions that ethnicities make to the incidence and biology of cancers, potentially improving outcomes for patients who currently lack access to the most recent advances in medical science.

Citation Format: Nicolas Robine, Fieke Froeling, Benjamin Hubert, Timothy Chu, Ashlea Chandler, Michael C. Zody, Dayna M. Oschwald, Harold E. Varmus, Charles Sawyers, David Tuveson. Polyethnic-1000: Advancing cancer genomics by studying ethnically diverse, underserved patient populations in New York [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2328.
Abstract 2343: The incidence of BRAF mutation in non-small cell lung cancers is low in Korean patients
Authors: Kim Ahrong , Ahn Hyo Yeong , Lee Chang Hun , Kim Young Dae , Lee Min Ki , Jeong Yeon Joo , Lee So Jeong
Published: September 2020

Introduction: BRAF mutation is present in about 8% of all cancer. Oncogenic activity of BRAF V600E mutation was established in lung cancers. Dabrafenib and trametinib combination therapy is preferred first line therapy for advanced or metastatic lung cancer with BRAF V600E mutation. However, there is only little basic knowledge of BRAF mutational status among Korean population. In this study, BRAF mutation, particularly BRAF V600E mutational status was explored among Korean non-small cell lung cancer patients. The clinical and pathologic characteristics of BRAF V600E mutation in non-small cell lung cancers were also investigated.

Materials and Methods: The final cohort was 378 cases of primary non-small cell lung cancers. Fromalin-fixed paraffin embedded (FFPE) tissue blocks were used. PNA-mediated clamping PCR for BRAF V600, BRAF V600E real-time PCR and immunohistochemistry for mutation specific monoclonal antibody of Ventana VE1 were performed on all 378 cases of non-small cell lung cancers. For positive cases, direct sequencing was performed for validation.

Results: BRAF V600 mutation was detected in five cases using PNA clamping method among 378 non-small cell carcinoma cases. By using BRAF real-time PCR, a BRAF V600E mutation was detected in 3 patients among total 378 cohort. There were 2 discordant cases between PNA clamping and real-time PCR. On immunohistochemistry, 3 patients showed positive staining for tumor cytoplasm and all positive cases also had positive results in real-time PCR. Direct Sanger sequencing was done in 5 cases that had any positive results for 3 methods. The results of Sanger sequencing was the same with those of real-time PCR and immunohistochemistry. Direct sequencing using PAN PCR product was performed on 2 negative cases for validation. On sequencing using PNA clamping PCR product, all cases showed mutation for BRAF gene other than V600E genotype. Finally, there were 3 cases (0.8%) of BRAF V600E mutation and 2 cases of BRAF mutation (0.5%) other than V600E genotype. Among BRAF mutated patients, there were 2 never smokers and 3 ever smokers. All patients harboring BRAF mutation had no concomitant EGFR or ALK aberrations.

Conclusion: BRAF mutation incidence is relatively lower than that of Western data set and similar to that of Japanese report. Immunohistochemistry for Ventana VE1 antibody can be a useful screening method to detect BRAF V600E mutation.

Citation Format: Ahrong Kim, Hyo Yeong Ahn, Chang Hun Lee, Young Dae Kim, Min Ki Lee, Yeon Joo Jeong, So Jeong Lee. The incidence of BRAF mutation in non-small cell lung cancers is low in Korean patients [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2343.
Abstract 2361: Mendelian randomization study: The association between metabolic pathways and colorectal cancer risk
Authors: Jung Su Yon , Papp Jeanette , Sobel Eric , Zhang Zuo-Feng
Published: September 2020

Purpose: The roles of obesity-related biomarkers and their molecular pathways in the development of postmenopausal colorectal cancer (CRC) have been inconclusive. We examined insulin resistance (IR) as a major hormonal pathway mediating the association between obesity and CRC risk in a Mendelian randomization (MR) framework.

Methods: We performed MR analysis using individual-level data of 11,078 non-Hispanic white postmenopausal women from our earlier genome-wide association study. We identified four independent single-nucleotide polymorphisms associated with fasting glucose (FG), three with fasting insulin (FI), and six with homeostatic model assessment-IR (HOMA-IR), which were not associated with obesity. We estimated hazard ratios (HRs) for CRC by adjusting for potential confounding factors plus genetic principal components.

Results: Overall, we observed no direct association between the combined 13 IR genetic instruments and CRC risk (HR = 0.96, 95% confidence interval [CI]: 0.78 - 1.17). In phenotypic analysis, genetically raised HOMA-IR exhibited its effects on the increased risk while FG and FI on the reduced risk for CRC, but with a lack of statistical power. Subgroup analyses by physical activity level and dietary fat intake with combined phenotypes showed that genetically determined IR was associated with reduced CRC risk in both physical activity-stratified (single contributor: MTRR rs722025; HR = 0.12, 95% CI: 0.02 - 0.62) and high-fat diet subgroups (main contributor: G6PC2 rs560887; HR = 0.59, 95% CI: 0.37 - 0.94).

Conclusions: Complex evidence was observed for a potential causal association between IR and CRC risk, which may contribute to an additional value of intervention trials to lower IR and reduce CRC risk.

Citation Format: Su Yon Jung, Jeanette Papp, Eric Sobel, Zuo-Feng Zhang. Mendelian randomization study: The association between metabolic pathways and colorectal cancer risk [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2361.
Abstract 2368: Risk factors of pancreatic cancer in Vietnam: A matched case-control hospital-based study
Authors: Dao Tu V. , Tran Thuan V. , Nguyen Dung K. , Bui Oanh T. , Nguyen Quang T. , Tran Huong T.
Published: September 2020

Background: Data about the risk factors and cancer types in developing countries remains limited. We investigate for the first time the role of a number of risk factors (family cancer history, smoking, alcohol consumption, diabetes, hypertension) associated with pancreatic cancer among Vietnamese patients.

Methods: Patients hospitalized at 4 hospitals (K, Bach Mai, Viet Duc, Thai Nguyen) and diagnosed with pancreatic cancer (confirmed by histopathology and/or MRI/CT scan) in the period from January 2017 to May 2019 were included in the study. Some risk factors of the patients were collected and compared with a healthy age- and gender-matched (1:1) control group.

Results: We have identified 196 patients with diagnosis of pancreatic cancer of which 114 males and 82 females. The average age of the patient at the time of diagnosis was 58.28 years (standard deviation of 12.94, ranging from 25 to 87). A positive family history, smoking and hypertension disease of pancreatic cancer significantly reduced the risk (OR and 95% CI were 0.06 [0.02 - 0.18], 0.53 [0.27 - 1.00], 0.1 [0.04 - 0.25], respectively). A past or present history of diabetes significantly increased the cancer risk (OR = 2.76 [1.34 - 6.05]), especially in female (OR = 5.68 [1.49 - 28.69]). Current alcohol drinkers showed a significantly increased risk of pancreatic cancer (OR = 2.23 [1.20 - 4.25]).

Conclusions: Our finding showed that diabetes significantly increased the risk of pancreatic cancer in Vietnam, especially in women. An alcohol intake habit also increased the risk of pancreatic cancer. Further studies are required for fully understood the other risk factors related to this type of cancer.

Citation Format: Tu V. Dao, Thuan V. Tran, Dung K. Nguyen, Oanh T. Bui, Quang T. Nguyen, Huong T. Tran. Risk factors of pancreatic cancer in Vietnam: A matched case-control hospital-based study [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2368.
Abstract 2371: FBXL12 modulates Fanconi anaemia-BRCA signaling under conditions of oncogene-induced replication stress
Authors: Brunner Andrä , Johansson Henrik , Kourtesakis Alexandros , Rantala Juha , Spruck Charles , Wohlschlegel James , Lehtiö Janne , Sangfelt Olle
Published: September 2020

Oncogene-induced replication stress (OIRS) constitutes an early obstacle to cancer progression. De-regulation of cell death or senescence programs contribute to overcoming this barrier. However, even after escaping these fail-safe mechanisms, cancer cells need to handle elevated levels of DNA damage and ensuing genomic instability in order to continue proliferating. How this is achieved is only partially understood.

To advance our understanding of this response and to identify potentially druggable regulators, we screened the family of human F-box proteins (FBP) for effects on replication re-start under conditions of OIRS. FBPs function as interchangeable substrate recognition sub-units of the SKP1-Cullin1-FBP ubiquitin ligase complex. They are required for conferring substrate specificity to the complex and some FBPs have been attributed roles as oncogenes or tumor suppressors while the majority remain largely uncharacterized.

Our screen revealed the relatively unknown FBP FBXL12 as crucial regulator for sustained proliferation and genomic integrity under conditions of cyclin E-induced replication stress. FBXL12-depleted cells exhibit excessive replication fork stalling, high levels of DNA damage signaling and spontaneous senescence in OIRS breast cancer model cell lines. Interestingly, the same effects are not manifest in untransformed cell lines.

Furthermore, we found that FBXL12 interacts with central chromatin-associated components of the Fanconi anemia (FA)-BRCA signaling pathway, ubiquitylating and targeting them for proteasomal degradation, and thereby exerting its effects on stalled replication forks.

The specific nature of FA-BRCA signaling regulation by FBXL12 as well as potential clinical implications for basal-like breast cancers will be discussed.

Citation Format: Andrä Brunner, Henrik Johansson, Alexandros Kourtesakis, Juha Rantala, Charles Spruck, James Wohlschlegel, Janne Lehtiö, Olle Sangfelt. FBXL12 modulates Fanconi anaemia-BRCA signaling under conditions of oncogene-induced replication stress [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2371.
Abstract 2377: Atm-dependent recruitment of brd7 is required for transcriptional repression and repair at dna breaks flanking transcriptional active regions
Authors: Hu Kaishun
Published: September 2020

DNA double-strand break (DSB) is highly cytotoxic and deleterious lesions, causing gross chromosomal rearrangements, genomic instability and tumorigenesis (Meisenberg et al., 2019). In mammalian cells, such damages are mainly repaired through two pathways: non-homologous end joining (NHEJ) and homologous recombination (HR) (San Filippo et al., 2008). To avoid conflicts between ongoing transcription and repair activities, the DNA damage response (DDR) must coordinate the processes of repair signaling and local transcriptional capacity (Adam and Polo, 2014). Cells have evolved the ability of switching off transcription for DSB repair in ATM, DNA-PK and PARP1-dependent manner which is a critical step for maintaining genome-epigenome integrity (Iannelli et al., 2017; Shanbhag et al., 2010; Uckelmann and Sixma, 2017). The mono-ubiquitylation of H2A on K119 is one of the key determinants of transcriptional repression at sites of DNA damage (Kakarougkas et al., 2015; Rona et al., 2018; Shanbhag et al., 2010). However, depletion of these factors did not alleviate completely DSB-induced transcriptional repression, suggesting that additional yet unidentified factors or complexes are involved in the process. Here, we identified that BRD7, another specific subunit of PBAF complex, participated in DNA damage response (DDR) and was recruited to the damaged chromatin via ATM signaling. Mechanistically, BRD7 joined the polycomb repressive complex 2 (PRC2), the nucleosome remodeling and histone deacetylation (NuRD) complex at the damaged DNA and recruited E3 ubiquitin ligase RNF168 to the DSBs. Furthermore, ATM-mediated BRD7 phosphorylation is required for recruitment of the PRC2 complex, DSB sensor complex MRE11-RAD50-NBS1 (MRN) and RNF168 to the active transcription sites at DSBs, resulting in transcriptional repression and DNA repair. Moreover, BRD7 deficiency sensitized cancer cells to PARP inhibition. Collectively, BRD7 is crucial for DNA repair and DDR-mediated transcription repression, which may serve as a therapeutic target. Moreover, our findings have identified the missing link between DNA repair and transcription regulation that maintains genome integrity, as well as highlight the important role of PBAF complex in maintaining genomic integrity.

Citation Format: Kaishun Hu. Atm-dependent recruitment of brd7 is required for transcriptional repression and repair at dna breaks flanking transcriptional active regions [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2377.
Abstract 242: Identification of biomarkers predicting sensitivity to GLS1 inhibition using a CRISPR screen
Authors: Henry Christophe , Berthelot Karine , Lanneau Christophe , Orsini Cécile , Gorge-Bernat Dimitri , Meaux Isabelle , Chassin Dorine , Moll Jürgen , Thiers Berangere , Machnik David , Debussche Laurent
Published: September 2020

The conditional essential amino acid glutamine is utilized by tumors to sustain bioenergetic requirements in a nutrient-poor microenvironment. Based on this specific tumor need, efforts have been made to target the glutamine metabolism using glutaminase inhibitors. This approach has demonstrated activity in preclinical models and in a range of phase I studies. It was shown that a subset of models/patients are benefiting from glutaminase inhibition, however, biomarkers predicting sensitivity to GLS1 inhibitors are lacking. The objective of this study is to identify molecular predictors of the sensitivity towards GLS1 inhibition. Using the cBioportal, we analyzed the most prevalent genetic deficiencies in Triple Negative Breast cancers, Kidney cancers, Acute Myeloid Leukemia and Multiple Myeloma. From this analysis, a library of CRISPR gRNA including the most disease relevant LOF linked-genes was created. Individual cell lines stably expressing Cas9 were used for a focused CRISPR screen in the presence of suboptimal doses of GLS1 inhibitor. The sgRNA library mimicking the major genetic deficiencies identified from the cBioportal datamining was delivered in pool lentiviral particles. Genes deficiencies associated with increased GLS1 sensitivity were identified with sgRNA targeted NGS sequencing of the remaining cell population at treatment completion. We present the setup and validation of an innovative experimental design for the identification of biomarkers predictive to GLS1 inhibition with the aim to identify gene deficiencies associated with increased sensitivity to GLS1 inhibitors that could be used as predictive biomarkers in the clinic.

Citation Format: Christophe Henry, Karine Berthelot, Christophe Lanneau, Cécile Orsini, Dimitri Gorge-Bernat, Isabelle Meaux, Dorine Chassin, Jürgen Moll, Berangere Thiers, David Machnik, Laurent Debussche. Identification of biomarkers predicting sensitivity to GLS1 inhibition using a CRISPR screen [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 242.
Abstract 2428: Emodin enhances efficacy of Oxaliplatin and 5 Fluorouracil in colon cancer by promoting cell cycle arrest
Authors: Saunders Ian T. , Ezeagwu Dexter , Kumar Naresh , Mir Hina , Singh Shailesh
Published: September 2020

Dysregulation of cellular proliferation and evasion of apoptosis are key contributors to colon cancer (CoCa) development. Chemotherapeutic agents such as 5-Fluorouracil (5FU) and Oxaliplatin (OxT) which are frequently offered to treat CoCa are known to target cells in S and G2 phase of cell cycle. Cancer cells often overcome the effects of 5FU and OxT by deregulating cell cycle in response to these drugs. Therefore, to improve the cytotoxic effect of these chemotherapeutic agents it is important to keep CoCa cells in S and G2 phase. Hence, understanding the molecular mechanisms involved in CoCa cell-cycle progression and chemotherapeutic response could provide strategies for developing effective treatment options. Hence, in this study, we determined the effect of natural agent “Emodin” on CoCa cell-cycle regulation and its ability to increase the efficacy of 5FU and OxT by cell cycle regulation. The effects of Emodin, 5FU, and OxT as a single agent or in combination on CoCa (DLD-1, COLO-201, HCT116 p53WT, and HCT116 p53-/-) cell viability were determined by MTT assays. Combination index of Emodin, 5FU, and OxT were evaluated using Calcusyn. Emodin-induced changes in molecules involved in cell-cycle regulation and proliferation were analyzed via RT-PCR and confirmed by western blot (WB) analysis. Cell-cycle distribution with Emodin treatment alone and in combination with 5FU and OxT were assessed via flow cytometry and WB. Collectively, our data shows that Emodin alone was able to decrease cell survival and induced apoptosis in CoCa cells. Furthermore, cytotoxic ability of 5FU and OxT was increased when combined with Emodin as compared to when these chemotherapeutic drugs were given as single agents Emodin-induced cell-cycle regulation resulted in increased expression of tumor suppressor genes such as p21, p27 and p53, modulated cyclin/cyclin-dependent kinase expression, and induced cell-cycle arrest. Emodin also negatively impacted several signaling pathways associated with cell growth and differentiation. Overall, this study demonstrates that Emodin could be a promising adjuvant to either 5-Fluorouracil or Oxaliplatin in the treatment of CoCa.

Citation Format: Ian T. Saunders, Dexter Ezeagwu, Naresh Kumar, Hina Mir, Shailesh Singh. Emodin enhances efficacy of Oxaliplatin and 5 Fluorouracil in colon cancer by promoting cell cycle arrest [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2428.
Abstract 2434: Epigenomic profiling of colorectal cancer to predict patients at high risk of metastasis
Authors: Chatterjee Aniruddha , McCall John , Guilford Parry , Pattison Sharon , Rodger Euan , Ajithkumar Priyadarshana
Published: September 2020

Colorectal cancer (CRC) is one of the biggest cause of death world-wide and the second-highest cause of death by cancer in New Zealand with a mortality rate of approximately four deaths/day and 90% of CRC-associated mortality is caused by metastasis. Development of metastasis and the modifications that occur during various stages in the CRC metastatic cascade remains obscure. Emerging evidence indicates that epigenetic alterations have a greater influence on the development of metastasis than genetic mutations, but the current understanding of epigenetic drivers that cause CRC metastasis is limited. In the current project, as a first step in identifying epigenetic drivers for metastasis, whole-genome methylation and transcriptome profiles from paired primary and metastatic samples from the same NZ patients will be compared. Subsequently, the epigenetic drivers will be validated, and it can potentially be used as CRC metastatic markers. Circulating tumour cells (CTCs) represent the conduit between primary and metastatic tumours. They could be used to identify driver epigenetic changes associated with epithelial-to-mesenchymal transition (EMT), migration and invasion which occur during the metastatic cascade. CTCs are obtained from the blood and would provide a clinically less invasive test rather than requiring tissue samples. The recent development of single-cell sequencing technologies has now made it feasible to identify epigenetic changes in individual CTCs. In the second part of this project, protocols for single-cell DNA methylation and expression analysis will be developed using human colon cancer cell lines as a model. These optimised protocols will then be applied to CTCs from patient blood samples to identify epigenetic changes that predispose patients to metastasis. Identification of epigenetic drivers will help to detect individuals at high risk for metastasis prior to metastasis being clinically apparent, allowing for more personalised treatment and contribute to improving survival.

Citation Format: Aniruddha Chatterjee, John McCall, Parry Guilford, Sharon Pattison, Euan Rodger, Priyadarshana Ajithkumar. Epigenomic profiling of colorectal cancer to predict patients at high risk of metastasis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2434.
Abstract 2445: Epigenetic modulation of tumor cell tolerance to chemotherapy
Authors: Santos Jephesson Alex Alex dos , Santo Carolina Nunes , Pereira Luiza Cherobini
Published: September 2020

Cancer represents a set of diseases that are dynamic and heterogeneous. Their cells are phenotypically and genetically different from each other and, exhibit stochastic fluctuations in their phenotype. Investigations of isogenic populations of tumor cells reveal that tolerance also may emerge through non-genetic mechanisms, often through stochastic fluctuations in key factors in response to drugs. The objective of this work is to measure the dynamics of chemotherapy tolerance phenotype and modulate these dynamics in tumor cells to potentiate the therapy. Glioma cell lines and primary gliomas were plated at low density and after the formation of colonies, cells were treated with Temozolomide (TMZ) for 3 days. Growth rate of individual colonies was measured and the variance of growth rate of colonies with the same size was used to determine the dynamics of the phenotype. We developed a method to measure the dynamics of phenotype called DynaFit and defined a proportion of phenotypic chance in fitness until generation 6 called rCoDyFF (relative Colony DynaFit Factor 6), which is a value between 0% (stable phenotype) and 100% (dynamic phenotype). The untreated glioma cell lines (U251 and A172) and primary gliomas (LS12 and LS17) had rCoDyFF6 of 40%, 33%, 41%, 33% and treatment with 50uM TMZ produced an rCoDyFF6 67% in U251 cells. However, when cells were treated with a combination of epigenetic modulators and chemotherapy agent, 0.5µM Saha plus 3µM Azacytidine, and 50µM TMZ, the rCoDyFF6 result in - 23%, showing a drastic reduction, indicating stabilization of the phenotype. This treatment also increases sensitivity to TMZ. The result demonstrates that epigenetic modulators were able to induce a phenotypically stable state in U251 cells, leading to the copy of the tolerance phenotype of the colony founder cell to all the cells in the colony. Here we showed tumor cells fluctuate their fitness levels in the absence or presence of chemotherapy and this fluctuations can be stabilized by the inhibition of chromatin and DNA modifying enzymes.

Citation Format: Jephesson Alex Alex dos Santos, Carolina Nunes Santo, Luiza Cherobini Pereira. Epigenetic modulation of tumor cell tolerance to chemotherapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2445.
Abstract 2447: Analyses of epigenetic marks and mechanisms in disease: your guide to a successful CUT and RUN assay
Authors: Chen Fang , Comeau Christopher R. , Mason Jillian , Brown Carrie A. , Fry Christopher J.
Published: September 2020

Like the chromatin immunoprecipitation (ChIP) assay, Cleavage Under Targets & Release Using Nuclease (CUT&RUN) is a powerful and versatile technique used for probing protein-DNA interactions within the natural chromatin context of the cell. The CUT&RUN assay can be combined with downstream qPCR or NG-seq to analyze histone modifications and binding of transcription factors, DNA replication factors, or DNA repair proteins at specific target genes or across the entire genome. CUT&RUN provides a rapid, robust, and true low cell number assay for detection of protein-DNA interactions in the cell. Unlike the ChIP assay, CUT&RUN is free from formaldehyde cross-linking, chromatin fragmentation, and immunoprecipitation, making it a much faster and more efficient method for enriching protein-DNA interactions and identifying target genes. CUT&RUN can be performed in as little as one to two days, from live cells to purified DNA, and has been shown to work with as few as 500-1000 cells per assay (1,2). Instead of fragmenting all of the cellular chromatin as done in ChIP, CUT&RUN utilizes an antibody-targeted digestion of chromatin, resulting in much lower background signal than seen in the ChIP assay. As a result, CUT&RUN requires only 1/10th the sequencing depth that is required for ChIP-seq assays. Finally, the inclusion of simple spike-in control DNA allows for accurate quantification and normalization of target-protein binding between samples. This provides for effective normalization of signal between samples and between experiments. I will discuss the basics of the CUT&RUN assay and important factors to consider when setting up your experiment. In addition, I will introduce Cell Signaling Technology's new CUT&RUN Assay Kit and CUT&RUN pAG-MNase enzyme, and present data showing the versatility of this assay for mapping various histone modifications, transcription factor, and transcription cofactor binding across multiple cell types.

Citation Format: Fang Chen, Christopher R. Comeau, Jillian Mason, Carrie A. Brown, Christopher J. Fry. Analyses of epigenetic marks and mechanisms in disease: your guide to a successful CUT and RUN assay [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2447.
Abstract 2466: A novel role for the transcription factor SNAIL in alternative splicing
Authors: Ray Atrayee , Rudraraju Smruthi , Krueger Shawn , kumar Janani , Perley Danielle , Scheidegger Adam , Dhasarathy Archana
Published: September 2020

The transcription factor SNAIL is a master regulator of the Epithelial to Mesenchymal Transition (EMT), a hallmark event during cancer metastasis. SNAIL repressed transcription of several epithelial cell-specific genes such as E-cadherin, leading to a mesenchymal phenotype that is less attached and more migratory. As a DNA-binding transcription factor, SNAIL binds consensus E-box motifs in gene promoters, and recruits epigenetic modifiers to repress or activate gene expression. Previous studies from our lab and others have indicated that SNAIL and its family member SLUG have several gene targets in common but also bind to unique gene targets. Whether SNAIL or SLUG share protein-interacting partners is unknown. To identify interactome partners of SNAIL and SLUG, we performed co-immunoprecipitation followed by iTRAQ mass spectrometry. The protein interactome of SNAIL, but not SLUG, was highly enriched for splicing proteins, of which the top hit was the Serine-Arginine Rich-Splicing Factor, SRSF1. SRSF1 participates in regulation of alternative splicing, a mechanism through which a cell can generate multiple proteins from the same gene. Alternative splicing events play a central role in EMT progression, and SRSF1 is overexpressed in many cancers. We explored the role of SNAIL and SRSF1 in driving alternative splicing events during EMT. First, we confirmed the association of SNAIL and SRSF1 by co-immunofluorescence, PLA assay and co-immunoprecipitation experiments during TGF-beta induction of EMT. RNA-IP (RIP) experiments demonstrated association of SNAIL with mesenchymal specific but not epithelial-specific splice isoforms, further suggesting an RNA binding role for SNAIL. Lastly, RNA-EMSA experiments suggest specificity for some but not all four Zinc fingers of SNAIL in binding to RNA. Based on these observations, we hypothesize that SNAIL regulates alternative splicing events during EMT at least in part through association with SRSF1 on RNA. As transcription and splicing are closely coupled, we predict that the regulation of splicing by transcription factors through RNA binding is universally utilized by other biological systems to alter phenotype in response to external stimuli. We are currently working on identifying the genome-wide changes in alternative splicing regulated by SNAIL, and the mechanism by which SNAIL interacts with the spliceosome to regulate alternative splicing.

Citation Format: Atrayee Ray, Smruthi Rudraraju, Shawn Krueger, Janani kumar, Danielle Perley, Adam Scheidegger, Archana Dhasarathy. A novel role for the transcription factor SNAIL in alternative splicing [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2466.
Abstract 2476: Role of retinoblastoma gene in maintenance of osteoblast function and communication
Authors: Pendleton Elisha , Ketner Anthony , Bodenstine Thomas , Chandar Nalini
Published: September 2020

Retinoblastoma gene (Rb1) loss of function mutations is commonly seen in osteosarcomas resulting in poorly differentiated tumors. In addition to cell cycle regulation, Rb1 function has been shown to be important in cell fate determination. It is known that loss of Rb1 function in mesenchymal stem cells inhibits osteoblast differentiation and predisposes them towards adipocytic lineage. Recent studies have suggested a role for Rb1 in cell adhesion but mechanistic details are missing. As cadherin 11 (Cad11) and gap junctional protein connexin43 (Cx43), play important roles in adhesion and communication in osteoblasts, we studied these proteins in MC3T3-E1 mouse calvaria osteoblast cell line. We compared osteoblasts with reduced Rb1 expression to vector-transfected osteoblasts to assess the relationship between Rb1, Cad11, and Cx43. Decreased RNA and protein expression of Cad11 were noted during differentiation of Rb1 deficient osteoblasts when compared to controls. Gap junctional intercellular communication was also studied and found to be reduced with Rb1 loss. Rb1 deficient cells had a higher steady-state level of adipocyte transcription factors and adipocyte markers when compared to control. Interestingly no changes were observed in Cx43 mRNA or protein expression when comparing the two lines. Immunofluorescence analysis demonstrated that a decrease in Cad11 may have affected its co-localization with Cx43. This, in turn, may have affected the proper formation of intercellular channels and thereby communication. These observations suggest that loss of Rb1 affects adhesion and communication in osteoblasts resulting in the expression of an adipocyte phenotype.

Citation Format: Elisha Pendleton, Anthony Ketner, Thomas Bodenstine, Nalini Chandar. Role of retinoblastoma gene in maintenance of osteoblast function and communication [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2476.
Abstract 2481: Concordance of PAM50 molecular subtyping between oligonucleotide microarray and nanoString nCounter assay for Taiwanese breast cancer
Authors: Huang Chi-Cheng , Tu Chao-Chiang , Tsai Mong-Hsun , Lai Liang-chuan , Huang Ching-Shui
Published: September 2020

Concordance of PAM50 molecular subtyping between oligonucleotide microarray and nanoString nCounter assay for Taiwanese breast cancer

Introduction

Breast cancer is molecularly heterogeneous and at least five molecular subtypes have been defined according to intrinsic genes transcription. Currently microarrays, RT-PCR, and next generation RNA sequencing have been adopted for subtyping in addition to the original nanoString nCounter assay. This study aimed to evaluate the consistency in subtyping Taiwanese breast cancers.

Methods

A total of 109 Taiwanese breast cancers (24 with adjacent normal breast) were assayed with Affymetrix Human Genome U133 plus 2.0 arrays and 96 were assayed with the customized 145-gene nanoString Codeset BCeCSig, of which the academic PAM50 was investigated. Of note, 64 patients were assayed for both platforms. Subtyping with the nearest centroid (single sample prediction) was called for each patient; Spearman's rank correlation coefficients were used, and samples were unclassified if correlation coefficients to all 5 centroids were less than 0.1.

Results

Table 1 showed the distributions of molecular subtypes with clinical IHC results. For 24 matched normal breast tissues, 21 were classified as normal breast-like. For 64 breast cancers (one unclassified) tested for both platforms, 41 (65%) were classified with identical subtype, resulting in a fair Kappa statistic of 0.55. Taking nCounter as gold standard, classification accuracy was 50% (3/6), 72% (13/18), 53% (10/19), 87% (13/15), and 40% (2/5) for basal-like, HER2-enriched, luminal-A, luminal-B, and normal breast-like subtype using Affymetrix microarray. ER-/HER2- phenotype and basal-like subtype experienced worse relapse free survival at 8-year follow-up (55.6% and 63.6% censored).

Table 1.Molecular subtypes and IHC phenotypes (two unclassified and one missing IHC result).Subtype IHCBasal-likeHER2-enrichedLuminal-ALuminal-BNormal breast-likeHR+/HER2+276174HR+/HER2-6442429HR-/HER2+625209HR-/HER2-144003

Conclusions:

Using public domain microarrays as a handy shortcut for breast cancer molecular subtyping is luring in scientific communities. Our experiments showed that fundamental discrepancy existed between distinct approaches, and more cation should be taken without too much extrapolation. On the other hand, adjacent normal breast tissue did display normal breast-like character, and this subtype should be viewed as a continuum of surrounding stroma.

Citation Format: Chi-Cheng Huang, Chao-Chiang Tu, Mong-Hsun Tsai, Liang-chuan Lai, Ching-Shui Huang. Concordance of PAM50 molecular subtyping between oligonucleotide microarray and nanoString nCounter assay for Taiwanese breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2481.
Abstract 249: Wild-type IDH2 is a potential therapeutic target for acute myeloid leukemia
Authors: Zeng Peiting , Huang Peng
Published: September 2020

Isocitrate dehydrogenase 2 (IDH2) is an important mitochondrial enzyme, which catalyzes the metabolic conversion between isocitrate and alpha-ketoglutarate (α-KG) in the TCA cycle. Somatic point mutations in the gene encoding for IDH2 have been identified in a variety of human cancers including certain glioma and acute myeloid leukemia (AML), and are correlated with a favorable prognosis compared with those with wild-type IDH2. However, the role of wild-type IDH2 in cancer development largely remains unknown. Here, we show that wild-type IDH2 is up-regulated in AML, and may play an important role in promoting AML cells survival and proliferation. Database analysis reveals that high expression of IDH2 seems to be correlated with poor survival of AML patients. We show that pharmacological inhibition of wild-type IDH2 in AML cell lines harboring wild-type IDH2 or genetically silencing of IDH2 expression by shRNA could impair cell viability, suppress the proliferation and colony formation capacity of the leukemia cells, and promote apoptosis, leading to significant inhibition of leukemia growth. Metabolic study suggests that disturbance of α-KG metabolism might play a major role in affecting AML cell viability and proliferation. Specifically, suppression of IDH2 caused a significant increase in α-KG and a decrease in isocitrate and citrate, suggesting that the reductive segment of the TCA cycle might be abnormally active in AML cells. Thus inhibition of IDH2 would lead to a severe disruption of the leukemia cellular metabolism due to suppression of α-KG to isocitrate conversion. Furthermore, genetic silencing of the wild-type IDH2 expression in AML cells suppressed in vivo leukemia growth in two xenograft mouse models. Overall, our study has identified wild-type IDH2 as an important molecule that is critical for AML cell survival and proliferation, and suggests that wild-type IDH2 could potentially serve as a new therapeutic target for the potential treatment of acute myeloid leukemia.

Citation Format: Peiting Zeng, Peng Huang. Wild-type IDH2 is a potential therapeutic target for acute myeloid leukemia [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 249.
Abstract 2507: Understanding endometrial cancer heterogeneity at single-cell resolution
Authors: Villarreal Enrique I. Velazquez , Carpten John D. , Craig David W.
Published: September 2020

Endometrial cancer is the fourth most common gynecologic malignancy in the US. Tumor- associated genetic signatures are known to be associated with poorer prognosis of patients with endometrial cancer. In general, frequent gene alterations are enrichments of p53 and KRAS, PTEN, PIK3CA and PI3K/AKT mutations with variable CNV. Considerable evidence for intratumor heterogeneity indicate that bulk genomic sequencing approaches may be insufficient since endometrial cancer is characterized by extensive tumor heterogeneity. Here, to get new insight about endometrial cancer heterogeneity, we applied single cell sequencing since it has the potential to improve our understanding of this genetic feature by providing clonotype and variant information in high resolution. We examined cancer heterogeneity using single cell DNA sequencing Copy Number Variation (sc-DNAseq-CNV) analysis and clonotype detection in three tumors from patients with endometrial cancer. The technology used for processing single cell was 10x ChromiumTM Technology that provides detection of 100 Kb CNV events, calling clonotypes down to 10 of 1000 cell inputs. By processing these samples resulted in the sequencing of around 800 cells. Genome-wide ploidy analysis of the tumor cells showed variable median ploidies per sample. The regional copy number was estimated by processing our data in 20 Kb cases of reads. Multiple sub-clones were identified per sample number one where four clusters of sub-clones characterized cancer heterogeneity. Copy number changes on entire chromosome arms and mutations at variant detection level were detected since the resolution provided by single cell technology applied. Among the variant detection we identified previously reported mutations in POLE, POLE2, TP53, FGFR2, ARID1A, CTNNBI, PIK3CA, PIK3RI, KRAS and SWI/SNF in specific sub clones. Our study addresses the predominant challenge to determine tumor heterogeneity by performing better approaches to detect genomic differences and identifying molecular signatures among women with endometrial cancer. Better understanding of tumor heterogeneity may benefit patients diagnosed with endometrial cancer, or patients with specific molecular characteristics linked to endometrial cancer.

Citation Format: Enrique I. Velazquez Villarreal, John D. Carpten, David W. Craig. Understanding endometrial cancer heterogeneity at single-cell resolution [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2507.
Abstract 2518: Function of microRNA-10a in cholangiocarcinoma
Authors: Spriet Matthieu , Wehrkamp Cody , Henslee Brandon , Mohr Ashley , Kachman Steve , Mott Justin
Published: September 2020

Cholangiocarcinoma, a primary liver cancer of the bile ducts, effects 6,000 patients yearly and incidence has been growing consistently over the past 15 years. Current therapies for cholangiocarcinoma are not curative and patient survival is less than 1 year after diagnosis. Strides toward new treatment rest on better understanding tumor biology. The expression of microRNAs in cholangiocarcinoma cells was analyzed by RNAseq after knockdown of fibroblast growth factor receptor 4 (FGFR4) to identify potential microRNAs regulated by FGFR4. Caspase activity was used to evaluate miR-10a roles in anti-apoptotic signaling. Cell scratch assays were used to test cell migration and cell counting was used to determine proliferation. Quantitative real time PCR was used to measure miR-10a levels in cholangiocarcinoma cell lines Mz-ChA-1, HUCCT-1 and KMCH after FGFR4 knockdown with siRNA. Potential target proteins of miR-10a were evaluated by western blot. Preliminary data showed dozens of microRNAs (miRs) in cholangiocarcinoma cells were modulated by FGFR4 knockdown. Treatment with siRNA to FGFR4 caused increased sensitivity to TRAIL-induced apoptosis, and also reduced miR-10a expression in Mz-ChA-1, HuCCT-1, and KMCH cell lines. Importantly, apoptosis sensitization was reversed by restoring miR-10a expression in cells. miR-10a helped regulate both cell proliferation and cell migration in cholangiocarcinoma cells. When miR-10a was increased in cholangiocarcinoma cells, migration and proliferation increased. When miR-10a was decreased these effects also decreased. Protein targets of miR-10a included the MAP Kinase-related proteins MLK-1 & TAK-1. Other protein targets such as PHLDA1 & AKTIP were also identified. Notably, microRNA binding prediction programs did not include MLK-1 as a target, though our sequence analysis shows multiple stable binding sites. MLK-1 showed the most dramatic response to miR-10a. We investigated the role miR-10a plays for cholangiocarcinoma and the mechanism through which this pathway is regulated. Data supports miR-10a as a key player in cell survival, proliferation and migration. We have identified a set of proteins that are regulated by miR-10a in cholangiocarcinoma cells. Looking forward, identification of the function of these proteins will help elucidate how miR-10a functions in cholangiocarcinoma progression.

Citation Format: Matthieu Spriet, Cody Wehrkamp, Brandon Henslee, Ashley Mohr, Steve Kachman, Justin Mott. Function of microRNA-10a in cholangiocarcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2518.