bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2087: Bringing advanced analytics experts to the data: Report of an interdisciplinary machine learning workshop
Authors: Stahl-Toyota Sophia , Glocker Katrin , Perez Analie Pascoe , Knurr Alexander , Denker Alexander , Ückert Frank
Published: September 2020

Gaining insights from medical and molecular omics data with advanced statistical learning methods requires the collaboration of experts with foundational knowledge of the data contents and context as well as computational and statistical background. The purpose of the workshop was to derive the key components that are necessary to apply machine learning methods to non-public real-world clinical, genomic and transcriptomic data from heterogeneous sources in a cross-institutional collaborative effort. Machine learning experts were invited to the premises of the institution that held a comprehensive collection of patient data. During the preparation phase, regular virtual meetings took place in order to discuss decisions regarding the research questions to analyze, transformations of the raw file formats and the technical infrastructure. The interdisciplinary group of experts decided to focus on models that would assign patients matching therapy classes based on their molecular variation profile. During the workshop phase, the experts spent several days at the same location working solely on the analysis in question. Here we present the lessons learned from conducting such an interdisciplinary workshop. The key components can be divided into a preparatory and a workshop phase. The first phase lays the foundation for the collaboration, including the formalities, discussion of the data and the applicable methods. It is especially important to involve all domains in decisions regarding the data preprocessing steps. As an example, a method might have particular requirements considering the data dimensions, which is information that the data experts need to know in order to aid with the meaningful reduction of the degrees of freedom, by e.g. aggregation or filtering. Sharing anonymized sample files that represent the data format in a realistic manner and sample scripts for testing the infrastructure helps all parties to set up the analysis pipelines efficiently. In the second phase, the intense working atmosphere allowed for quick iterations of feature engineering, model training and result evaluation. In the common case that a dataset is not yet publicly sharable, we demonstrate that it is a feasible option to invite analysts to the institution holding the data. Additionally, the focused atmosphere that is created by a limited-time workshop setting is advantageous to the motivation of the collaborators.

Citation Format: Sophia Stahl-Toyota, Katrin Glocker, Analie Pascoe Perez, Alexander Knurr, Alexander Denker, Frank Ückert. Bringing advanced analytics experts to the data: Report of an interdisciplinary machine learning workshop [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 2087.
Abstract 2093: DeepRePath: Finding prognostic features of tumor cell on the histopathologic image of lung cancer using explainable deep convolutional neural network
Authors: Ko Yoon Ho , Hong Soon Auck , Kim Tae-Jung , Hong Ji Hyung , Chun Sang Hoon , Kim Seoree , An Ho Jung , Park Sungsoo , Shim Won Sang
Published: September 2020

Introduction

Histopathological images in high-powered contain quantitative information for tumor cell morphology, but humans could not sufficiently extract prognostic features of tumor cell morphology associated with tumor recurrence of patient. In this study, we developed and trained the DeepRePath model, a deep convolutional neural network (CNN), on histopathological images to predict the recurrence of resected lung adenocarcinoma (LUAD).

Methods

A total of 244 haematoxylin and eosin stained histopathology slides of resected LUAD patients were used to train and validate the DeepRePath model. We manually segmented a cancer region in a LUAD tissue slide and captured to an image. All images were captured at x400 magnification and split into 13,329 image patches. We trained the DeepRePath model by transfer learning using 13,329 image patches from 244 LUAD patients.

Results

95 out of 244 patients developed recurrence after surgical resection. The DeepRePath model showed an average area under the curve (AUC) of 0.78 in 5-fold cross validation on the training cohort. We plan to employ external validation to evaluate the proposed model. In addition, using gradient-weighted class activation mapping (Grad-CAM), we confirmed that DeepRePath identified and used individual tumor cells with atypia to predict the recurrence of the patient.

Conclusions

DeepRePath model using transfer learning and CNN can accurately predict recurrence after curative resection of LUAD based on atypia of tumor cell.

Citation Format: Yoon Ho Ko, Soon Auck Hong, Tae-Jung Kim, Ji Hyung Hong, Sang Hoon Chun, Seoree Kim, Ho Jung An, Sungsoo Park, Won Sang Shim. DeepRePath: Finding prognostic features of tumor cell on the histopathologic image of lung cancer using explainable deep convolutional neural network [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 2093.
Abstract 2109: Amplicon design algorithm for single cell targeted DNA sequencing using machine learning
Authors: Wang Shu , Gulati Saurabh , Kim Dong , Sahu Sombeet , Parikh Saurabh , Li Nianzhen , Manivannan Manimozhi , Beard Nigel
Published: September 2020

Background: To realize the promise of precision medicine for cancer, assessing genetic variation present in rare cells and understanding the role that these cells play in the evolution of tumor progression is essential. High throughput single cell DNA targeted sequencing enables detection of rare mutations in cells and identification of subclones defined by co-occurrence of mutations. The big challenge with multiplex sequencing at single cell level is the non-uniform amplification of the targeted regions during PCR. This results in inadequate coverage of mutations of interest in the panel and hence makes genotyping challenging. To address this challenge, we developed a machine learning engine to optimize amplicon design for uniform amplification by making reliable performance prediction.

Methods: Multiple panels with various sizes were designed with amplicons spanning a wide range of design properties such as amplicon GC, length, secondary structure prediction, primer specificity. These panels were synthesized and processed through Tapestri single cell DNA platform. The tested amplicons are classified into low-performer, OK-performer and high flyer based on their normalized reads-per-cell value. Design properties and property distribution of the amplicons and the panel are the features. We used random forest classifier to calculate feature importance and analyzed the range of the top features for each class and their significance of variance between classes. These ranges were then used as parameters in the assay design pipeline. Next, we train machine learning models with performance data to develop a performance prediction engine.

Results: To test the performance of the design pipeline with new parameters, we designed a small (31), medium (128) and large (287) amplicon panel. Multiple runs were conducted for each panel with different cell types. We were able to achieve high panel performance of 97%, 92% and 88% across the three panels. The new parameters resulted in ~10-20% improvement in panel uniformity. We are working on further optimizing the performance prediction engine by using different ML classification models with K-fold cross validation, training using larger group of amplicons and optimizing features using combinations of properties.

Citation Format: Shu Wang, Saurabh Gulati, Dong Kim, Sombeet Sahu, Saurabh Parikh, Nianzhen Li, Manimozhi Manivannan, Nigel Beard. Amplicon design algorithm for single cell targeted DNA sequencing using machine learning [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 2109.
Abstract 2139: A seven lncRNA based risk score system for predicting the recurrence and prognosis of ovarian cancer patients
Authors: Yang Haojie , Han Changpeng
Published: September 2020

Objective: Ovarian cancer (OC) has the third highest incidence and highest mortality among all gynecological tumors. This study was performed in order to establish a risk score system for OC patients.

Methods: The RNA-seq data of OC (the training set) was extracted from TCGA database, and the GSE32062 (the validation set) data was selected from GEO database. Using the limma package, differentially expressed RNAs between the recurrence and non-recurrence groups were identified. For constructing a risk score system, the independent prognosis-correlated lncRNAs were selected using the survival package. Then, the independent clinical prognostic factors were identified using survival package, and the nomogram survival model was built using the rms package. Furthermore, enrichment analysis was conducted using the DAVID tool, and a lncRNA-mRNA co-expression network was constructed using Cytoscape software.

Results: There were 550 DERs (497 mRNAs and 53 lncRNAs) between the recurrence and non-recurrence samples. After identification of 321 DERs significantly related to survival prognosis, seven independent prognosis-correlated lncRNAs (MORC2-AS1, LINC00189, MYCNOS, SNHG7, DSCR9, TCL6, and RBM26-AS1) were further selected for constructing the risk score system. Subsequently, the nomogram survival model based on independent clinical prognostic factors (including age, tumor recurrence, and RS model status) was established. There were five overlapping pathways (including Jak-STAT and Wnt signaling pathways and other pathways in cancer) between the searched and the enriched pathways. Additionally, the seven independent prognosis-correlated lncRNAs were involved in the co-expression network, which was directly correlated with OC.

Conclusion: The risk score system and nomogram survival model based on lncRNAs (MORC2-AS1, LINC00189, MYCNOS, SNHG7, DSCR9, TCL6, and RBM26-AS1) may be valuable for predicting the prognosis of OC.

Citation Format: Haojie Yang, Changpeng Han. A seven lncRNA based risk score system for predicting the recurrence and prognosis of ovarian cancer 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 2139.
Abstract 2178: Expansion of tumor-specific tumor-infiltrating lymphocytes (TIL) from head and neck tumors
Authors: Nagle Luz , Weber Amy Mackay , Hall MacLean , Beatty Matthew , Wadsworth J. Trad , McMullen Caitlin , Patel Krupal , Vorwald Kathryn , Chung Christine , Pilon-Thomas Shari
Published: September 2020

Background: Until recently, treatment options for patients with head and neck cancer include a combination of surgery, chemotherapy and radiation. Although the combined use of these treatment options modestly improves survival, combination therapy increases toxicity. Overall survival of these treatment regimens for patients with advanced head and neck cancer is just over 10 months. Even with the newly approved and vastly more tolerable use of anti-PD-1 immunotherapy, survival is only extended by 3-5 months. In comparison, adoptive cell therapy (ACT) using tumor-infiltrating lymphocytes (TIL) has improved the median overall survival in patients with metastatic melanoma to 52 months. The goal of this study was to test the feasibility of expanding tumor-reactive TIL from head and neck tumors for the development of a clinical trial.

Methods: Primary tumors were resected from 20 head and neck cancer patients. Tumors were minced into small fragments (1-3 mm3) and plated in high dose IL-2. After 4 weeks, expanded TIL were phenotyped by flow cytometry and tested for tumor reactivity by IFN-gamma production after co-culture of TIL with autologous tumor digest. IFN-gamma levels were determined by ELISA. Reactive TIL were expanded using a rapid expansion protocol (REP).

Results: The mean expansion of TIL per fragment was 1.96 × 106. From 20 primary tumors, seven were contaminated and discarded. TIL were successfully expanded from 11/13 (85%) evaluable tumors. The phenotype of expanded TIL consisted of CD3− CD56+ NK cells and CD3+ T cells. Tumor-specific reactivity was analyzed for seven samples. TIL expanded from three (43%) tumors were predominantly CD8+ and were reactive against autologous tumor as determined by IFN-gamma ELISA. Expansion of TIL during REP resulted in an average 600-fold increase in cell numbers (range of 61 - 1,774 fold) and led to the depletion of NK cells and an increase of CD3+ T cells.

Conclusions: In this study, we have demonstrated the feasibility of growing tumor-reactive TIL from head and neck tumors. This study raises the potential for the implementation of ACT with TIL for the treatment of head and neck cancer patients.

Citation Format: Luz Nagle, Amy Mackay Weber, MacLean Hall, Matthew Beatty, J. Trad Wadsworth, Caitlin McMullen, Krupal Patel, Kathryn Vorwald, Christine Chung, Shari Pilon-Thomas. Expansion of tumor-specific tumor-infiltrating lymphocytes (TIL) from head and neck tumors [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 2178.
Abstract 2190: Engineering off-the-shelf T cell receptor fusion construct (TRuC) T cells
Authors: Donaghey Julie , Kieffer-Kwon Philippe , Patterson Troy , Chan Tiffany , Horton Holly , Tighe Robert , Gutierrez Dario A. , Getts Daniel , Hofmeister Robert
Published: September 2020

The T-Cell Receptor (TCR) can be functionally split into antigen recognition mediated by the TCRα/β heterodimer and signal transduction triggered by the CD3 complex comprising CD3ϵ/γ, CD3ϵ/δ and CD3ζ/ζ dimers. Unlike antibodies, the TCR recognizes its cognate peptide antigen only when presented on human leukocyte antigen (HLA) molecules. Recently, we reported that tethering an antibody-derived binder to one of the TCR subunits redirects T cells to specifically kill tumor cells independent of HLA. Different from CAR-T cells, the T cell receptor fusion constructs (TRuC™) are integrated into the natural TCR and require all TCR subunits for receptor translocation to the cell surface. The development of off-the-shelf TRuC-T cells is desirable to shorten the vein-to-vein production time and reduce manufacturing costs. Here, we describe the generation of TRuC-T cells expressing a fully functional TRuC TCR without alloreactivity. Inactivation of the endogenous TRAC gene employing CRISPR/Cas9 endonuclease disrupts natural TCR formation. Yet, replacing the endogenous TCRα subunit with a human TCRα constant region without variable domain to avoid alloreactivity is insufficient for TRuC TCR expression. A functional TRuC TCR, however, can be created by substituting the following constructs for the inactivated TRAC gene: (i) murine TCRα and β constant domains without variable domains, (ii) chimeric human/murine TCRα and β constant regions or (iii) TCRγ and δ constant domains. Off-the-shelf TRuC-T cells upregulate activation markers, secrete cytokines, and kill tumor cells in an antigen-specific manner. Importantly, the genome-engineered TRuC-T cells lack alloreactivity as demonstrated in mixed lymphocyte reactions and clear tumors in NSG xenograft models without signs of Graft versus host disease (GvHD). Our findings warrant further development of allogeneic TRuC-T cells for cancer therapy.

Citation Format: Julie Donaghey, Philippe Kieffer-Kwon, Troy Patterson, Tiffany Chan, Holly Horton, Robert Tighe, Dario A. Gutierrez, Daniel Getts, Robert Hofmeister. Engineering off-the-shelf T cell receptor fusion construct (TRuC) T cells [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 2190.
Abstract 2266: SAR442085, a next generation anti-CD38 antibody with enhanced antibody-dependent cellular cytotoxicity (ADCC) against multiple myeloma
Authors: Virone-Oddos Angela , Kassem Sahar , Diallo Béré , Tang Alexandre , Fournier Alain , El-Murr Nizar , Carrie-Constantin Nadege , Nicolazzi Céline , Arnould Isabelle , Avet-Loiseau Hervé , Sidhu Sukhvinder S. , Martinet Ludovic , Chiron Marielle
Published: September 2020

CD38 is a cell surface glycoprotein highly expressed in multiple myeloma (MM) cells making it an attractive target for antibody–based therapy. Anti–CD38 treatment represents a major breakthrough in the treatment of relapsing and refractory multiple myeloma (RRMM) patients. However, a number of patients do not respond to anti–CD38 single agent therapy stressing the need for novel approaches. Here we describe SAR442085, a next generation anti–CD38 antibody, with higher affinity for the activating FcγRIIIa and FcγRIIa receptors expressed on effector cells as compared to the approved anti–CD38 antibody daratumumab, resulting in greater effector functions. SAR442085 targets CD38 with high affinity and is able to destroy CD38 expressing tumor cells in vitro through effector function mechanisms (ADCC and antibody dependent cellular phagocytosis, ADCP) as well as by direct apoptosis. SAR442085 exhibits better ADCC activity as compared to daratumumab with more than 8–fold EC50 improvement against RPMI–8226 MM cells. SAR442085 also inhibits CD38 enzymatic activity. Using primary bone marrow aspirates from MM patients, an increased ability of SAR442085 to engage CD16 was demonstrated, resulting in a higher level of NK cell activation and degranulation against primary plasma cells, as compared to daratumumab. In addition, SAR442085 demonstrated potent in vivo single–agent activity against murine tumor cells expressing human CD38, in a transgenic C57BL/6 mouse model humanized for all Fcγ receptors, with 90% mice survival at 10 and 1.25 mg/kg and up to 90 days median survival time. Overall, these data support the current evaluation of this next generation anti–CD38 antibody in phase I clinical trials in patients with relapsed/refractory MM.

Citation Format: Angela Virone-Oddos, Sahar Kassem, Béré Diallo, Alexandre Tang, Alain Fournier, Nizar El-Murr, Nadege Carrie-Constantin, Céline Nicolazzi, Isabelle Arnould, Hervé Avet-Loiseau, Sukhvinder S. Sidhu, Ludovic Martinet, Marielle Chiron. SAR442085, a next generation anti-CD38 antibody with enhanced antibody-dependent cellular cytotoxicity (ADCC) against multiple myeloma [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 2266.
Abstract 227: Maltose enhanced the growth of bladder and colon cancer cells unlike some other disaccharides: Cellobiose, isomaltose, lactose, and sucrose
Authors: Lea Michael A. , desBordes Charles
Published: September 2020

The addiction of most cancer cells to the metabolism of glucose is well known. The metabolism by cancer cells of other saccharides is less well characterized. We have studied the impact on growth of human bladder and colon cancer cells of mono- and disaccharides. The standard RPMI 1640 medium contains 2 mg glucose per ml. Substitution for glucose by other monosaccharides (fructose, galactose and mannose) resulted in similar growth in some cell lines but greatly diminished growth in others. For the Caco-2 and HT29 colon cancer cells, co-incubation with 2-deoxyglucose was more inhibitory for growth with fructose than with glucose as substrate. There was a similar situation with some bladder cancer cell lines (5637, HT1197 and RT4) whereas with other bladder cancer cells (HT1376, T24 and UM-UC-3) 2-deoxyglucose caused greater inhibition with glucose. In those cell lines in which alkaline phosphatase activity could be induced after incubation with butyrate (Caco-2, HT29, 5637, HT1197 and HT1376), induction by butyrate was observed with any of the monosaccharides. Similarly with the disaccharides lactose, maltose and trehalose there was induction of alkaline phosphatase by butyrate. In the course of this work it was apparent that maltose could enhance growth to an extent that was sometimes not significantly different to that seen with glucose and was not seen with cellobiose, isomaltose, lactose or sucrose. HT29 cells were the only cell line to have a substantial increased growth with trehalose. The increased growth with maltose was inhibited by 2-deoxyglucose. In conclusion, maltose increased growth of bladder and colon cancer cells without a period of adaptation.

Citation Format: Michael A. Lea, Charles desBordes. Maltose enhanced the growth of bladder and colon cancer cells unlike some other disaccharides: Cellobiose, isomaltose, lactose, and sucrose [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 227.
Abstract 23: Precision medicine approaches to Fanconi anemia oral cancer personalized prevention and treatment
Authors: Swenson Daniel K. , Wuertz Beverly R. , Ali Mustafa M. , Unger Gretchen M. , Ondrey Frank G.
Published: September 2020

There is an increased risk of oral cavity and other head and neck cancers as Fanconi patients live longer post transplantation. DNA repair defects in Fanconi patients contribute to “field effect” in this population, which causes frequent recurrences and often fatal, second primary oral malignancies. These developments show a need for translation of new basic science concepts for this patient population. We are examining the effects of both Metformin and Pioglitazone, and Polo kinase and Wee Kinase inhibitors in Fanconi anemia oral cancer cell lines, and leukoplakia control cells.

We employed 3 FA oral cancer cell lines for the current study. We performed clonogenic and cell proliferation (MTT) experiments in replicates and repeated those experiments to establish initial dose responses of the inhibitors at biologically relevant concentrations. We used ANOVA analysis with a P<0.05 as our level of significance. Concurrently, we are examining pioglitazone-metformin signaling pathways in biopsies from precancerous oral lesion (non-FA) patients treated with pioglitazone in our multiple chemoprevention clinical trials via confocal fluorescence microscopy.

We found both the Wee Kinase inhibitor and POLO kinase inhibitor resulted in dose dependent decreases in proliferation and clonogenic potential in several experiments in all cell lines. We observed decreased proliferation at 72 hours at clinically achievable serum levels of 75%. However, we also observed cytotoxic effects as well, as judged by MTT reading at 72 hours being lower than the original Day 0 MTT readings. Pioglitazone treatment also resulted in dose dependent decreases in cell proliferation and clonogenic potential at clinically achievable serum levels. Similar effects can be achieved with metformin, however, at higher doses. Additionally, we have identified surrogate endpoints as suitable pharmacodynamic readouts by IHC analysis of the cell lines.

There are precision medicine treatments for FA-associated oral carcinoma and these are feasible, based on earlier promising results we present here. We are attempting to discover new treatment methodologies for these patients, as conventional agents and radiation therapy are often unacceptable, due to the DNA fragility of the patient population. In combination, these efforts support our future goals of chemoprevention in this underserved prevention population.

Citation Format: Daniel K. Swenson, Beverly R. Wuertz, Mustafa M. Ali, Gretchen M. Unger, Frank G. Ondrey. Precision medicine approaches to Fanconi anemia oral cancer personalized prevention and treatment [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 23.
Abstract 2344: Composite score of healthy lifestyle factors and risk of hepatocellular carcinoma: Findings from a prospective cohort study
Authors: Luu Hung N. , Wang Renwei , Behari Jaideep , Adams-Haduch Jennifer , Odegaard Andrew O. , Bee George Goh , Jin Aizhen , Koh Woon-Puay , Yuan Jian-Min
Published: September 2020

Background. While the associations between individual lifestyle factors and risk of hepatocellular carcinoma (HCC) were examined substantially, their combined impact on HCC risk has not been evaluated.

Methods. The association of a composite score of healthy lifestyle factors, including body mass index, alcohol consumption, cigarette smoking, alternative Mediterranean Diet (aMED), and sleep duration, with the risk of developing HCC was examined in the Singapore Chinese Health Study (SCHS), an on-going prospective cohort of 63,257 Chinese men and women aged 45-74 at enrollment in 1993-1998 with up to 25 years of follow-up. Cox proportional hazard regression method was used to estimate hazard ratio (HR) and its 95% confidence interval (CI) of HCC with the composite score after adjustment for multiple potential confounders. The unconditional logistic regression method was used to confirm the association between the composite lifestyle score and HCC risk among hepatitis B surface antigen (HBsAg) negative individuals to eliminate its potential confounding effect.

Results. After a mean follow-up of 17.7 years, 561 participants of the SCHS developed HCC. Individuals with higher composite scores, which represented for healthier lifestyles, were at significantly lower risk of HCC. Compared with the lowest composite score (i.e., 0-4), the HRs (95% CIs) for 5, 6, 7 and 8 were 0.48 (0.36-0.64), 0.44 (0.34-0.67), 0.37 (0.28-0.48), and 0.26 (0.17-0.39), respectively (Ptrend<0.001). A similar inverse association was observed in participants with negative HBsAg serology; HR was 0.12 (95% CI: 0.04-0.38) for the highest versus the lowest category of the composite scores (Ptrend=0.01).

Conclusion. Healthy lifestyles are protective against the development of HCC, especially for individuals without viral infection. This finding highlights the importance of a comprehensive lifestyle modification strategy for primary prevention of HCC.

Funding: The Singapore Chinese Health Study was supported by the National Institutes of Health (NIH) of the United States (grants # R01 CA144034 and UM1 CA182876).

Citation Format: Hung N. Luu, Renwei Wang, Jaideep Behari, Jennifer Adams-Haduch, Andrew O. Odegaard, George Goh Bee, Aizhen Jin, Woon-Puay Koh, Jian-Min Yuan. Composite score of healthy lifestyle factors and risk of hepatocellular carcinoma: Findings from a prospective cohort 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 2344.
Abstract 2356: Novel and recurrent germline mutations in Colombian families with colorectal cancer syndromes
Authors: Rivera-Herrera Ana Lucía , Gómez-Camargo Ana Milena , Serrano-López Martha Lucía , Carvajal-Carmona Luis G. , Sanabria-Salas María Carolina
Published: September 2020

Hereditary colorectal cancer (CRC) syndromes accounts for approximately 5-10% of total CRC cases. Among those, Lynch syndrome (LS) is the most frequent, but the penetrance depends on the mismatch repair gene (MMR) affected. On the other hand, the Familial Adenomatous Polyposis (FAP) syndrome is less frequent, but with penetrance near to 100%. In Colombia we don't know the prevalence of these syndromes. In the National Cancer Institute from Colombia, the largest reference cancer center in the country, we aim to performed germline genetic analyses in Colombian CRC patients with a suspected hereditary cancer syndrome, as part of the Hereditary Cancer Program that seeks to identified high risk families to offer preventive measures and screening recommendations. A total of 66 patients with CRC fulfilling criteria have been so far analyzed with Next Generation Sequencing using a multigene panel. From those, the 32% have negative results, 41% have VUSs and 27% have positive results (meaning a pathogenic or potentially pathogenic genetic variant). The most prevalent CRC syndrome is Lynch (11/18, 61% with MMR mutated genes), followed by polyposis syndromes such as FAP (11% APC) and MAP (1.5% biallelic MUTYH). Most of our Lynch syndrome families fulfilled Bethesda or Amsterdam criteria. Two recurrent mutations in MLH1 were found in Lynch syndrome families (6/11, 54.5%), which are MLH1: c.1918C>T (p.Pro640Ser) and MLH1: c.790+1G>A. Further haplotype analysis will help us to determine if mutation carriers shared a common ancestry.

Citation Format: Ana Lucía Rivera-Herrera, Ana Milena Gómez-Camargo, Martha Lucía Serrano-López, Luis G. Carvajal-Carmona, María Carolina Sanabria-Salas. Novel and recurrent germline mutations in Colombian families with colorectal cancer syndromes [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 2356.
Abstract 2379: Elaborating the mechanism of PARP synthetic lethality in ATM loss
Authors: Muvaffak Asli , Coleman Kevin G.
Published: September 2020

PARP inhibitors have transformed the treatment for ovarian cancer, which is known to have high incidence of homologous recombination (HR) deficiency through the loss of BRCA1 or BRCA2 genes. 30-35% of ovarian cancer patients have defects in BRCA1/2 genes caused by germline, somatic or epigenetic alterations. PARP inhibitors induce cell death in HR-deficient cells through the mechanism of synthetic lethality where cancer cells cannot tolerate the loss of both SSB and DSB repair machinery. Although genetic or epigenetic alterations in other pathways such as Fanconi anemia, or in non-authentic homologous recombination repair (HRR) genes such as PTEN, IDH, and CDK12 have been reported to contribute to HR deficiency, mechanistic understanding of HR loss remains to be determined. Another DNA damage repair (DDR) protein, ATM, a kinase that phosphorylates and activates major DNA damage checkpoints, i.e. CHK2 & BRCA1, has also being increasingly recognized recently as a synthetic lethal partner with PARP inhibitors in multiple tumor indications such as prostate, lung and colorectal cancers. ATM functions in both cell cycle arrest and DNA repair in response to DSBs, however how cells respond to inhibition of PARP in the absence of ATM is not yet fully understood. In this study, we demonstrated that loss of ATM function, i.e. via generating an ATM KO by CRISPR/Cas9 in two genetic backgrounds, i.e. in DLD-1 and HeLa cell lines, induced HR-deficiency indicated by the impairment of Rad51-foci formation and increased phosphorylation of histone H2AX in ATM KO (-/-) cell lines. Furthermore DLD-1 ATM KO (-/-) cell line demonstrated increased sensitivity to niraparib (i.e. 45-fold difference in sensitivity compared to the DLD-1 parental, HR-proficient cell line) in the 14day colony formation assay. In comparison, BRCA2 (-/-) KO in DLD-1 gave a 125-fold increase compared to parental cell line. In DLD-1 ATM KO (-/-) cell line, we have also detected that loss of ATM was associated with low BRCA1 and BRCA2 protein expression suggestive of downstream effect on impairment of HR-mediated DNA checkpoint signaling in ATM loss. Thus, PARPi synthetic lethality phenotype observed in tumors with ATM loss could be regulated through the combination effect coming from the loss of both of ATM & BRCA1/2 and the subsequent effect on both HR gene functions. Our findings highlight that ATM has a critical role in DNA repair for the synergism with PARP inhibitors.

Citation Format: Asli Muvaffak, Kevin G. Coleman. Elaborating the mechanism of PARP synthetic lethality in ATM loss [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 2379.
Abstract 2380: A novel role for PACS-1 in suppression of oncogenic replication stress in cancer cells
Authors: Mani Chinnadurai , Tripathi Kaushlendra , Clark David W. , Thomas Gary , Palle Komaraiah
Published: September 2020

PACS-1 is a protein involved in trafficking, especially transport of proteins like furin and Mannose-6-phosphate into the trans-golgi network. Intriguingly, our studies identified dynamic translocation of PACS-1 during cell division and its association with chromatin in tumor cells. Downregulation of PACS-1 in cancer cell lines attenuated cell growth, induced replication stress mediated DNA damage response (DDR), such as ATM/ATR-mediated checkpoint activation, and formation RPA, FANCD2 and pH2AX foci. Consistent with these observations, live-cell imaging revealed increased nuclear translocation of PACS-1 during S-phase and until the complete cell division. These results point to a novel role for PACS-1 during normal replication or in the suppression of oncogenic replication in tumor cells. However, a direct role for PACS-1 in cancer development or progression is not clear. Analysis of the cancer genome atlas (TCGA) data revealed occurrence of PACS-1 amplifications, mutations and deletions in cancers of different tissues. PACS-1 depletion leads to proteasome-mediated degradation of HDAC2 and HDAC3, compromised chromatin maturation, leads to replication stress and genomic instability. Intriguingly, stable overexpression of PACS-1 in MDA-MB-231 cells enhanced malignant behavior of tumor cells in their ability to form soft agar colonies and matrigel invasion compared to the cells that are expressing vector control. PACS-1 downregulation promotes replication stress in tumor cells suggesting that its upregulation in tumors may be important for replication stress recovery in cancer cells and for an efficient response to chemotherapy induced DNA damage. Furthermore, Prognoscan analysis indicated that PACS-1 expression levels correlates with poor survival in multiple studies, including breast, ovarian and lung cancers, suggesting a role for PACS-1 in tumorigenesis and a biomarker for poor prognosis.

Citation Format: Chinnadurai Mani, Kaushlendra Tripathi, David W. Clark, Gary Thomas, Komaraiah Palle. A novel role for PACS-1 in suppression of oncogenic replication stress in cancer cells [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 2380.
Abstract 2471: Role of FOXO transcription factor dynamics in determining cell fates following EGFR inhibition
Authors: Huynh Julie M. , Aryeh Kayenat S. , Shanks Lisa , Paek Andrew L.
Published: September 2020

Epidermal Growth Factor Receptor (EGFR) tyrosine kinase inhibitors (TKIs) are a first line treatment for patients with advanced non-small cell lung cancer (NSCLC) who have specific hyperactive mutations in EGFR. Though patients have seen improvements in survival after EGFR TKI treatment, resistance commonly develops as not all NSCLC cells die following treatment. Indeed, EGFR inhibitor treatment induces both cell-cycle arrest (allowing for cell survival) and death in cancer cells. The FOXO family of transcription factors have been implicated in both death and arrest responses following EGFR inhibition. This is consistent with FOXOs function in regulating both arrest and apoptotic genes programs. However, it is unknown how the FOXO network ‘decides' between these two oppositional programs to enact following treatment with EGFR inhibitors. Protein dynamics, meaning how the levels or location of a protein changes over time, is a mechanism cells use to encode information about cellular fates. The p53 transcription system is an established example of a dynamically regulated system–it responds to multiple inputs and is capable of enacting multiple outputs (gamma radiation causes oscillations of p53 levels that result in cellular arrest while UV causes a sustained increase of p53 levels that result in cellular death). FOXO's network is similar to the p53 system (multiple input, multiple output), making it a good candidate for dynamic regulation. We have tagged Foxo3a with a fluorescent marker (Venus) at its endogenous locus in PC9 NSCLC cells using CRISPR/Cas9 to determine whether FOXO dynamics dictate cell fate following EGFR TKI treatment. Using live cell microscopy on this line, we have acquired FOXO nuclear cytoplasmic shuttling dynamics as well as cell fate (death/arrest) in single cells. Using time-series analyses we are trying to determine if there are dynamic patterns associated with each fate following EGFR inhibition. Through our studies, we hope to gain a better understanding of this non-genetic mechanism cells use to determine their fate. Our long-term goal is to identify dynamic patterns that push cancer cells to more terminal fates following treatment.

Citation Format: Julie M. Huynh, Kayenat S. Aryeh, Lisa Shanks, Andrew L. Paek. Role of FOXO transcription factor dynamics in determining cell fates following EGFR inhibition [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 2471.
Abstract 2478: Assessing regulatory network rewiring in cancer patients using chromatin accessibility profiles
Authors: Forbes Andre , Xu Duo , Khurana Ekta K.
Published: September 2020

The regulation of gene expression in humans is the result of a complex interplay between DNA, small RNA molecules and a multitude of proteins, particularly histones and transcription factors (TFs). To identify the key TFs regulating gene expression in cancer we are integrating ATAC-Seq data from 371 TCGA patients in conjunction with matching expression data and DNA-binding motifs for 780 TFs and TF families. We infer the regulatory networks in these patients by detecting true TF binding events in individual patients and the regulatory elements that said TFs are bound to using the Protein Interaction Quantification algorithm (PIQ). The inferred networks were then clustered by the similarity of inferred TF binding activity followed by identification of the TFs most likely driving the identity of each cluster. We observe robust clustering by site of origin of disease with canonical TFs such as FOXA1 and ESR1 being putative key TFs in ER+ breast cancer, PAX8 in renal cancer as well as TP63 and SOX2 in multiple squamous cell cancers. To validate these putative super-TFs, we used gene essentiality data from CRISPR-mediated knockout experiments of 18K genes in 625 well characterized cell lines performed by the Broad Institute. We observe statistically significant differences in the essentiality of putative super-TFs in cell lines matching the disease types of clusters.

Citation Format: Andre Forbes, Duo Xu, Ekta K. Khurana. Assessing regulatory network rewiring in cancer patients using chromatin accessibility profiles [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 2478.
Abstract 2525: Identification of mRNA targets associated with PIWIL1 protein complex in lung cancer cells
Authors: Amaar Yousef G. , Reeves Mark E.
Published: September 2020

Introduction: In previous published work we have shown that RASSF1C promotes expression of PIWIL1 and PIWI interacting RNAs (piRNAs) in lung cancer cells, suggesting the hypothesis that that the RASSF1C-PIWI-piRNA pathway may promote lung cancer cell growth and progression. PIWIL1 protein-piRNA complexes have recently been shown to regulate mRNAs in mouse testes, suggesting that PIWIL1 plays an important role in mRNA cleavage to regulate specific targets involved in cellular growth. To investigate this hypothesis, we used a non-small cell lung cancer (NSCLC) cell model to identify candidate mRNAs targeted by the RASSF1C-PIWIL1-piRNA pathway through PIWIL1-protein complex co-immunoprecipitation and RNA sequencing.

Methods: Cell lysates were prepared under non-denaturing conditions from the lung cancer cell line H1299 over-expressing RASSF1C (experimental sample) and H1299 over-expressing the vector backbone (control sample) using a co-immunoprecipitation kit (Pierce). The PIWIL1 2C12 monoclonal antibody (Wako Chemicals Inc., Richmond, VA) was used to pull down PIWIL1 protein using 1 mg of cell lysates from experimental and control samples. The RNA associated with the precipitated PIWIL1 protein complex was isolated using a total RNA isolation kit (Qiagen) and the isolated RNA was subjected to RNA deep sequencing and bioinformatic analysis.

Results: Differential expression analysis based on read counts was carried out using a fold-change threshold of 2.0, maximum p-value of 0.05, and a minimum 1CPM, identified over 5505 genes that are differentially expressed when comparing read counts from H1299 cells over-expressing RASSF1C to H1299 cells over-expressing the vector back bone. Some of the genes identified with reduced mRNA levels correspond to tumor suppressors such as PTEN and p53 and apoptosis-inducing genes such as BAD and CASP3.

Conclusions: Our study was designed to show that PIWIL-protein complexes can be pulled down from lung cancer cell lysates, and that PIWIL-protein complexes are likely to bind and regulate mRNA targets in lung cancer cells. We have identified interesting mRNAs that are potentially silenced by the PIWIL1-piRNA protein complex. Ongoing work is focused on validating some of the potential mRNA targets identified. This paves the way for conducting RNA-sequencing studies to identify specific piRNAs that associate with PIWIL1-protein complexes in the nucleus and cytoplasm.

Citation Format: Yousef G. Amaar, Mark E. Reeves. Identification of mRNA targets associated with PIWIL1 protein complex in lung cancer cells [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 2525.
Abstract 2575: Mechanoactivation of NOX2-generated ROS elicits persistent TRPM8 Ca2+ signals that are inhibited by oncogenic KRas
Authors: Pratt Stephen JP , Lee Rachel M. , Hernandez-Ochoa Erick O. , Ory Eleanor C. , Thompson Keyata N. , Bailey Patrick C. , Mathias Trevor J. , Ju Julia A. , Vitolo Michele I. , Schneider Martin F. , Stains Joseph P. , Ward Christopher W. , Martin Stuart S.
Published: September 2020

Changes in the mechanical microenvironment and mechanical signals are observed during tumor progression, malignant transformation, and metastasis. In this context, understanding the molecular details of mechanotransduction signaling may provide unique therapeutic targets. Here we report that normal breast epithelial cells are mechanically sensitive, responding to mechanical stimuli through a two-part calcium signaling mechanism. We observed an immediate, robust rise in intracellular calcium (within seconds) followed by a persistent extracellular calcium influx (up to 30 minutes). This persistent calcium was sustained via microtubule-dependent mechano-activation of NADPH oxidase 2 (NOX2)-generated reactive oxygen species (ROS), which acted on TRPM8 channels to prolong calcium signaling. In contrast, the introduction of a constitutively-active oncogenic KRas mutation inhibited the magnitude of initial calcium signaling and severely blunted persistent calcium influx. The identification that oncogenic KRas suppresses mechanically-induced calcium at the level of ROS provides a novel mechanism for how KRas could alter cell responses to tumor microenvironment mechanics and may reveal new chemotherapeutic targets for cancer. Moreover, we find that expression changes in both NOX2 and TRPM8 mRNA predicts poor clinical outcome in estrogen receptor (ER) negative breast cancer patients, a population with limited available treatment options. The clinical and mechanistic data demonstrating disruption of this mechanically-activated calcium pathway in breast cancer patients and by KRas activation reveal novel signaling alterations that could influence cancer cell responses to the tumor mechanical microenvironment and impact patient survival.

Citation Format: Stephen JP Pratt, Rachel M. Lee, Erick O. Hernandez-Ochoa, Eleanor C. Ory, Keyata N. Thompson, Patrick C. Bailey, Trevor J. Mathias, Julia A. Ju, Michele I. Vitolo, Martin F. Schneider, Joseph P. Stains, Christopher W. Ward, Stuart S. Martin. Mechanoactivation of NOX2-generated ROS elicits persistent TRPM8 Ca2+ signals that are inhibited by oncogenic KRas [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 2575.
Abstract 2605: Cytokeratin 5 promotes endocytosis to remodel cell adhesions in breast cancer
Authors: McGinn Olivia Frances , Ward Ashley V. , Finlay-Schultz Jessica , Paul Kiran Vinod , Kabos Peter , Sartorius Carol
Published: September 2020

Breast cancer (BC) is clinically classified according to expression of estrogen receptor, progesterone receptor (ER+ BC), HER2 (HER2+), or absence of these markers (triple negative BC). While TNBC has the inferior prognosis in the short term, ER+ breast cancer has an extended risk of recurrence that lasts up to 20 years and accounts for the most deaths overall. Cytokeratin 5 (CK5) is an intermediate filament protein that is expressed in the majority of TNBC cases and 10-50% of ER+ BC. In CK5+/ER+ BC, there is widespread heterogeneity in the amount of CK5+ tumor cells (~1-50%). The mere presence of CK5+ cells is an indicator of poor prognosis across both subtypes of BC; the reasons for this are poorly understood. We have shown that CK5+ cells within ER+ and TNBC cell lines, and heterogeneous PDX tumor models lose membrane localization of adherens junction proteins β-catenin and E-cadherin. Loss of membrane β-catenin and E-cadherin is associated with poor prognosis in breast cancer and is a precursor to cell invasion. CK5+ cells have been reported to have increased invasive potential by us and others. Thus, identifying the mechanism of CK5-dependent loss of adherens proteins at the membrane could lead to development of therapies targeting this process. β-catenin and E-cadherin localization have been reported to be regulated through endocytosis which is mediated through the small GTPase family of Rab proteins. We performed a screen to identify potential CK5 interacting proteins in breast cancer cells and identified several Rab proteins. Thus, we hypothesize that CK5 acts as a scaffold for Rabs to promote endocytosis of β-catenin and E-cadherin to increase invasive potential.To investigate this, we treated CK5 overexpressing ER+ and TNBC cells with the endocytosis inhibitor Dyngo4a and found membrane β-catenin and E-cadherin localization were restored, suggesting that CK5 may be mediating endocytosis of β-catenin and E-cadherin. Rab5 is responsible for trafficking vesicles from the plasma membrane to the early endosomes. CK5 was confirmed to interact with Rab5 by co-IP in TNBC and CK5 overexpressing ER+ cell lines. In ongoing studies, we are testing whether Rab5 knockdown prevents loss of membrane β-catenin and E-cadherin and decreases cell invasiveness. These experiments will collectively determine whether CK5 regulated endocytosis is a targetable feature in breast cancer cells.

Citation Format: Olivia Frances McGinn, Ashley V. Ward, Jessica Finlay-Schultz, Kiran Vinod Paul, Peter Kabos, Carol Sartorius. Cytokeratin 5 promotes endocytosis to remodel cell adhesions in 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 2605.