bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 6024: Precancerous nature of intestinal metaplasia with accelerated DNA methylation along with altered epigenomic landscape
Authors: Takeuchi Chihiro , Yamashita Satoshi , Liu Yu-Yu , Takeshima Hideyuki , Ushijima Toshikazu
Published: April 2023

Objective. The presence of intestinal metaplasia (IM) is a risk factor for gastric cancer. However, it is still controversial whether IM itself is a precancerous or paracancerous lesion. Here, we aimed to explore the precancerous nature of IM in terms of epigenetic alterations.

Design. Genome-wide DNA methylation analysis was conducted by EPIC BeadArray using IM and non-IM crypts isolated by Alcian blue staining. Organoid establishment, ChIP-seq for H3K27ac, and single-cell ATAC-seq were conducted using IM mucosa. NOS2 was induced using a Tet-on gene expression system in normal cells.

Results. IM crypts had a methylation profile unique from that of non-IM crypts, showing extensive DNA hypermethylation in promoter regions with CpG islands, including those of tumor suppressor genes. Also, the IM-specific methylation profile was carried into a fraction of gastric cancers (GC), and the frequency of the IM-type GCs was higher than the fraction of IM cells in the adjacent mucosa, indicating that IM cells have a higher chance of developing into cancer cells than non-IM cells. Moreover, the patients with IM-type GCs showed better overall survival than those without, and IM-type GCs showed good response to chemotherapy. As for the mechanism of accelerated methylation induction, IM organoids had remarkably high endogenous NOS2 expression, and NOS2 induction in normal cells was shown to accelerate aberrant DNA methylation by increasing DNMT activity. IM mucosa underwent dynamic enhancer reprogramming, including the regions involved in abnormal NOS2 expression. Single-cell ATAC-seq also showed NOS2 open chromatin in IM cells but not in gastric cells, and frequent closed chromatin of tumor-suppressor genes in IM cells. NOS2 expression in IM-derived organoids was up-regulated by IL-17A, a cytokine secreted by extracellular bacterial infection.

Conclusions. IM cells were considered to have a strong precancerous nature with an increased chance of converting into cancer cells in terms of the specific methylation profile and the accelerated DNA methylation induction due to abnormal NOS2 expression.

Citation Format: Chihiro Takeuchi, Satoshi Yamashita, Yu-Yu Liu, Hideyuki Takeshima, Toshikazu Ushijima. Precancerous nature of intestinal metaplasia with accelerated DNA methylation along with altered epigenomic landscape [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6024.
Abstract 6048: OGT/CDK5/ACSS2 axis regulates breast cancer brain metastatic growth
Authors: Esquea Emily , Ciraku Lorela , Ming Giang Le , Reginato Mauricio
Published: April 2023

Brain metastases (BMs) in breast cancer patients is considered an end-stage event, with no effective drug treatment and a median survival after diagnosis measured in months. Currently, there are no effective drug treatment for BM patients, thus there is an urgent need to develop novel treatment strategies. Breast cancers that metastasize to the brain must adapt to lack of lipid availability in the brain environment and are highly dependent on fatty acid synthesis for growth and survival. However, the signaling pathways that regulate precursors of fatty acid synthesis and lipid metabolism in breast cancer brain metastatic (BCBM) tumors are not known. Here, we show that BCBM cells can generate acetyl-CoA, a major metabolite feeding lipid biosynthesis, via phosphorylation of acetyl-CoA synthetase 2 (ACSS2) by the cyclin-dependent kinse-5 (CDK5) regulated by the nutrient sensor O-GlcNAc transferase (OGT). Breast cancer cells selected to metastasize to the brain contain higher levels of O-GlcNAcylation, OGT. and ACSS2-Ser267 phosphorylation compared to parental cells, and we show that human breast cancer brain metastatic patient samples contain elevated ACSS2-Ser-267 levels. Additionally, overexpression of the OGT, CDK5, or ACSS2-S267D phospho-mimetic mutant confers a growth advantage of breast cancer cells in the brain in vivo. Importantly, we show that ACSS2 is required for breast cancer growth in the brain but not in the mammary fat pad. Pharmacologically targeting the CDK5 or ACSS2 with respective small molecule inhibitors reduces tumor growth in orthotopic ex vivo brain slice model. These results suggest a crucial role for OGT/CDK5/ACSS2 signaling axis to regulate lipid metabolism in BCBM cells and identify CDK5 and ACSS2 as novel therapeutic targets for treatment of breast cancer brain metastatic growth.

Citation Format: Emily Esquea, Lorela Ciraku, Giang Le Ming, Mauricio Reginato. OGT/CDK5/ACSS2 axis regulates breast cancer brain metastatic growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6048.
Abstract 6069: Genomic and transcriptomic characterization of medullary thyroid cancer
Authors: Lee Seong Eun , Park Seongyeol , Yi Shinae , Lim Joonoh , Choi Na Rae , Kwak JungHak , Koh June-Young , Yi Boram , Koo Jaemo , Chang Jae Won , Ju Young Seok , Koo Bon Seok , Kim Jin Man , Park Young Joo , Shong Minho , Lee Eun Kyung , Won Jae Kyung , Kang Yea Eun , Lee Kyu Eun
Published: April 2023

Medullary thyroid carcinoma (MTC) is a rare thyroid malignancy derived from the parafollicular C cells of thyroid gland. It features relatively aggressive biologic behavior among thyroid cancers, but its genomic landscape has not yet been fully explored. Here, we conducted multi-omics data (whole genome sequencing (WGS), and bulk (bulk RNA-seq) or single nucleus RNA sequencing (snRNA-seq)) on ​​MTC, and found genetic characteristics of MTC and a new gene signature predicting aggressiveness of tumors. WGS was performed for 35 pairs of tumor specimens and normal thyroid tissues from 30 patients. And bulk RNA-seq for 59 samples (20 normal thyroid tissues, 26 primary MTCs, 9 recurred lymph nodes, and 4 metastatic lymph nodes) and snRNA-seq for 3 primary MTCs, and 2 recurred/metastatic lymph nodes were also performed. In WGS results, we verified driver mutations, including RET, RAS, and BRAF. Four patients were classified as germline RET mutations based on familial history. Somatic RET M918T mutations were occurred in 8 patients, 9 patients were observed other RET mutations. Also, we observed other somatic mutations such as HRAS and BRAF. Seven patients occurred somatic HRAS mutations (Q61L, Q61R, G13R, A11 and G15), and somatic BRAF mutations (G469A, K601E) were observed in two patients. We found broad-level copy-number alterations in ~50% of patients, and we also found a patient who shows whole-genome duplication. The copy-number alteration was associated with aggressive phenotype in MTC. In addition, we found complex rearrangements such as microhomology-mediated break-induced replication in five patients. Interestingly, we found intra-tumoral heterogeneity of several genes related to C cell differentiation through snRNA-seq analysis by comparing clusters of tumor cells. Using the genes, we calculated the C cell differentiation score of the samples with bulk RNA-seq by GSVA algorithm. This score was related to poor clinical characteristics such as recurrence. In conclusion, multi-omics profiling of MTC reveals that copy number alterations and C cell differentiation status are the important biomarkers for cancer aggressiveness.

Citation Format: Seong Eun Lee, Seongyeol Park, Shinae Yi, Joonoh Lim, Na Rae Choi, JungHak Kwak, June-Young Koh, Boram Yi, Jaemo Koo, Jae Won Chang, Young Seok Ju, Bon Seok Koo, Jin Man Kim, Young Joo Park, Minho Shong, Eun Kyung Lee, Jae Kyung Won, Yea Eun Kang, Kyu Eun Lee. Genomic and transcriptomic characterization of medullary thyroid cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6069.
Abstract 6083: Extensive epigenetic and transcriptomic donor-specific differences observed in iPSC derived allogenic NK (iNK) cells
Authors: Srivas Rohith , Nariya Snehal , Rojsza Melissa , Morse Barry A. , Manor Ohad , Amin Rupesh , Borges Luis
Published: April 2023

Several autologous CAR-T cell therapies have been approved for the treatment of B cell lymphomas and leukemias and a number of trials are in progress to investigate the use of allogeneic, donor derived CAR-T and CAR-NK cell therapies for multiple tumor indications. However, sourcing cell donors, editing cells and generating large batches of clinical grade allogeneic CAR-T and CAR-NK can be challenging. To overcome these difficulties, we are developing NK and T cell-based therapies derived from induced pluripotent stem cells (iPSCs). iPSCs have the capacity to be genetically engineered, enabling precise edits that can enhance the function of cell therapies, and indefinitely expanded in culture, allowing for the production of highly uniform master cell banks. Yet, an open question of this system is the extent to which the donors’ genetic background shapes the phenotype of the iPSC-derived NK and T cells. To answer this question, we measured the genome-wide landscape of chromatin accessibility and gene expression in iNK cells generated from multiple donors. Donor background was linked to significant changes in chromatin accessibility (~30% of regions tested) and expression (~15% genes). We found accessibility changes at binding sites for transcription factors regulating various aspects of NK cell function, including differentiation (GATA2, EOMES, and ETS1), activation (AP-1, T-bet), and cytokine production (STAT3, STAT5), indicating that the donor background could impact the functionality of iPSC-derived NK cells. In particular, we noted that iNK cells derived from one donor, which had decreased effector functions compared to other cell lines, showed increased accessibility at BACH2 binding sites, a key negative regulator of NK cell function. iNK cells derived from this donor also showed an increase in expression of genes linked to NK cell differentiation and cell development, and a decrease in genes involved in cell cycle, cell proliferation, and inflammatory response pathways. Taken together, these data indicate the importance of assessing the impact of donor background on iPSC derived cell therapies through a variety of functional and genomic assays.

Citation Format: Rohith Srivas, Snehal Nariya, Melissa Rojsza, Barry A. Morse, Ohad Manor, Rupesh Amin, Luis Borges. Extensive epigenetic and transcriptomic donor-specific differences observed in iPSC derived allogenic NK (iNK) cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6083.
Abstract 6104: Prognostic and therapeutic impact of a homologous recombination repair protein TONSL in cancer stem cells
Authors: Lee Hani , Choi SeokGyeong , Ha Sojung , Kim Woo-Young
Published: April 2023

Previously, we found that an important homologous recombination repair protein ‘Tonsoku Like, DNA Repair Protein’ (TONSL) might be required for glioblastoma cancer stem cell (CSC). In this study, we further investigated the role of TONSL in CSC from several cancer cell lines from different tissues. We also analyzed the clinical data available from the public databases to find the clinical impact of TONSL. The higher expression of TONSL is associated with the worse prognosis of lung and gastric cancer. Interestingly, in breast cancer, while its higher expression is also the negative prognostic marker of the luminal A and HER2 positive cancers, that is rather the positive prognostic marker for luminal B and basal cancers. TONSL expression is higher in all 4 subtypes of breast cancer, and ovarian cancer than the corresponding normal tissues. The knockdown of TONSL in an ovarian cancer cell line, OVCAR8, results in the increase of G2/M cells and apoptotic cells in the bulk cultured population while increase also the senescence cells only in the CSC population leading to the severe loss of CSC population. TONSL and the close binding partner in homologous recombination repair (HRR), MMS22L, both were essential for the CSC growth but the bulk cultured cells could survive without those two in a colon cancer cell line, HCT15 suggesting the importance of HRR in colon CSC. Indeed, gemcitabine treatment induced DNA breaks showed a synergistic cytotoxicity with those two HRR factors knockdown in the CSC only. These results suggest that the TONSL and MMS2L mediated HRR is essential for the CSC and while most of bulk cultured cancer cells circumvent the HRR deficit. Collectively, these data showed that the CSCs ultimately depend on the HRR process which requires TONSL and, therefore, CSC population may be more vulnerable to HRR targeting therapeutic approaches.

Citation Format: Hani Lee, SeokGyeong Choi, Sojung Ha, Woo-Young Kim. Prognostic and therapeutic impact of a homologous recombination repair protein TONSL in cancer stem cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6104.
Abstract 6144: Polyploidization enables initial survival of chemotherapy but a progressively increased susceptibility to ferroptosis
Authors: Loftus Luke
Published: April 2023

Once cancer has metastasized it remains incurable owing to evolved resistance to nearly all systemic therapies. Classical views of therapeutic resistance include tumor heterogeneity and genetic instability, but our group and others have identified an unappreciated cancer cell phenotype, the polyaneuploid cancer cell (PACC) state, that provides an alternative model. The PACC state is induced upon exposure to a variety of applied stressors, including multiple different classes of chemotherapy, and is characterized by polyploidization of the aneuploid cancer genome. We have shown that this polyploidization is accomplished through endocycling that leads to an enlarged cell size with increased transcriptomic and protein content, higher oxidative buffer capacity, and increased autophagy. We have demonstrated that PACCs are present in a variety of cell lines, arise in response to multiple stressors in vitro, and are resistant to subsequent treatment with several classes of chemotherapy. Thus, we hypothesize that the PACC state is driving therapeutic resistance to current treatment regimens. Elimination of this overlooked phenotype is critical for sustained anticancer therapy response. Cancer cells, including those in the PACC state, control canonical cell cycle and cell death programs to evade apoptosis from external stressors including chemotherapy, highlighting an opportunity to leverage cell cycle-independent death pathways as an alternative therapeutic strategy1. Ferroptosis is a cell cycle agnostic immunogenic form of cell death that is an ideal pathway for elimination of cells in the PACC state. Characterization of ROS levels, glutathione content, labile iron homeostasis, and lipid content indicate that cells in the PACC state have a mounting susceptibility to ferroptosis. Our data suggests that cells in the PACC state keep ferroptosis in check through elevated levels of reduced glutathione and proficient redox activity, including turn over of GPX4, the sole enzyme counteracting lipid peroxidation. Direct inhibition of GPX4 is highly toxic to cells in the PACC state, particularly at later timepoints post therapy removal when the immediate stress response is largely resolved. These studies will directly lead to development of a therapeutic strategy specifically targeting the PACC state with the intention of eliminating cancer resistance.

1Loftus, L.V.; Amend, S.R.; Pienta, K.J. Interplay between Cell Death and Cell Proliferation Reveals New Strategies for Cancer Therapy. Int. J. Mol. Sci. 2022, 23, 4723. https://doi.org/10.3390/ijms23094723

Citation Format: Luke Loftus. Polyploidization enables initial survival of chemotherapy but a progressively increased susceptibility to ferroptosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6144.
Abstract 6203: CTC1-STN1-TEN1 (CST) complex-independent roles for STN1 in DNA double-strand break repair in pancreatic ductal adenocarcinoma
Authors: Shen Changxian , Yang Linlin , Corrales-Guerrero Sergio , Nair Sindhu , Williams Terence
Published: April 2023

KRAS mutation occurs in 90-95% of pancreatic ductal adenocarcinoma (PDAC) and is a driver of PDAC; however, the underlying molecular mechanisms by which KRAS mutation maintains survival and promotes progression of PDAC need further refinement. We herein report that KRAS mutations increase STN1 (also named OBFC1) expression leading to enhanced repair of DNA double strand breaks (DSBs) in PDAC. Analysis of The Cancer Genome Atlas databases reveal that STN1 is significantly upregulated in PDAC tissue and, is associated with KRAS mutation and poor survival. Silencing KRAS or pharmacologic inhibition of KRAS signaling decreased STN1 expression in PDAC cells. Canonically, STN1 forms a CST (CTC1-STN1-TEN1) complex, which is important for telomere duplication and maintenance. Intriguingly, siRNA-mediated depletion of STN1 (but not CTC1 or TEN1) sensitized PDAC cells to ionizing radiation and chemotherapy as assessed by radiation clonogenic assay and alamarBlue assay, respectively. Consistently, immunoblotting and immunofluorescence analyses of γH2AX (marker of DNA damage) demonstrated that depletion of STN1 (but not CTC1 or TEN1) resulted in DNA damage. To explore the underlying molecular mechanisms, we performed proteomic analysis and found that STN1 physically interacts with many proteins important for DNA repair, replication and cell cycle progression. Of note, we revealed unreported interactions of STN1 with ATM, ATR and DNA-PKcs, key components of DNA damage response signaling. Furthermore, homologous recombination and non-homologous end joining repair reporter assays demonstrated that STN1 silencing reduced both homologous recombination and non-homologous end joining repair of DSBs in U2OS and PDAC cells. Moreover, flow cytometry and immunofluorescence analyses showed that knockdown of STN1 by siRNA impaired cell cycle arrest at S and G2/M phases which was accompanied with mitotic catastrophe in response to ionizing radiation. Our findings have revealed novel CST complex-independent roles for STN1 in DSB repair and suggest STN1 is a novel biomarker for PDAC as well as potentially other cancers.

Citation Format: Changxian Shen, Linlin Yang, Sergio Corrales-Guerrero, Sindhu Nair, Terence Williams. CTC1-STN1-TEN1 (CST) complex-independent roles for STN1 in DNA double-strand break repair in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6203.
Abstract 642: CSF-1R+ macrophages control the gut microbiome-enhanced liver NKT function through IL-18
Authors: Ma Chi , McCallen Justin , McVey John C. , Zhang Qianfei , Ruf Benjamin , Bauer Kylynda , Wang Sophie , Lai Chunwei Walter , Trinchieri Giorgio , Berzofsky Jay A. , Korangy Firouzeh , Greten Tim F.
Published: April 2023

The gut microbiome is an important modulator of the host immune system. Here, we found that altering the gut microbiome by oral vancomycin increases liver NKT cell function. Enhanced NKT cytokine production and activation marker expression were observed in vancomycin-treated mice following both antigen-specific and antigen-independent in vivo NKT stimulations, with a more prominent effect in liver as compared to the spleen. Fecal transplantation studies demonstrated that the NKT functional regulation is mediated by altering the gut microbiome but uncoupled from the modulation of NKT cell population size. Interestingly, when stimulated in vitro, NKT cells from vancomycin-treated mice did not show increased activation, suggesting an indirect regulation. NKT cells expressed high levels of IL-18 receptor, and vancomycin increased the expression of IL-18 in the liver. Blocking IL-18 by neutralizing antibody or using genetically deficient mice attenuated the enhanced NKT activation. Liver macrophages were identified as a major source of IL-18. General macrophage depletion by clodronate abolished this NKT activation. Using anti-CSF-1R depletion or LyzCrexCSF-1RLsL-DTRmice identified CSF-1R+ macrophages as a critical modulator of NKT function. Together, our results demonstrate that the gut microbiome controls liver NKT function via regulating CSF-1R+ macrophages to produce IL-18.

Citation Format: Chi Ma, Justin McCallen, John C. McVey, Qianfei Zhang, Benjamin Ruf, Kylynda Bauer, Sophie Wang, Chunwei Walter Lai, Giorgio Trinchieri, Jay A. Berzofsky, Firouzeh Korangy, Tim F. Greten. CSF-1R+ macrophages control the gut microbiome-enhanced liver NKT function through IL-18 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 642.
Abstract 6505: ColoScape test: a molecular assay to detect early-stage colorectal cancer in plasma cell-free DNA
Authors: Tanaka Hiromi , Shen Shuo , Scimia Mauro , Pastor Larry , Li Jonathan , Fu Andrew Y. , Kim Daniel , Ni Rui , Zhang Aiguo , Sha Michael Y.
Published: April 2023

Colorectal cancer (CRC) is the second most common cause of cancer deaths when men and women are combined in the U.S. Early detection in the precancerous stage is key to reducing the CRC morbidity and mortality rates. Thus, there is a critical need for a cost-effective, time-efficient, and convenient clinical tool for early CRC detection. We have developed a multiplex qPCR assay (ColoScapeTM) to detect CRC-associated genetic and epigenetic changes from liquid biopsy samples (i.e., cell-free DNA) using our proprietary QClamp® XNA technology. The QClamp® XNA technology is very unique and enables the ColoScapeTM assay to selectively amplify the mutant and methylated DNA target sequences by using a synthetic DNA analog XNA (Xenonucleic Acid). This study demonstrates the analytical performance for detecting low-abundant mutated and methylated gene copies, as well as assess the clinical performance using plasma cell-free DNA samples from patients with CRC or advanced adenomas (≥1 cm) and from individuals with normal colonoscopies. The ColoscapeTM test is currently designed to detect mutations in 8 genes with 61 mutations and 7 methylated markers. The test consists of two parts: (i) the detection of mutations in 8 genes (APC, KRAS, BRAF, TP53, CTNNB1, NRAS, SMAD4, and PIK3CA) and (ii) the detection of 7 methylation targeted genes. For the assay analytical performance, the ColoScapeTM test shows that XNA effectively suppressed the wild-type background amplifications and led to amplify mutation and methylation target sequences dominantly, providing a high level of analytical sensitivity and specificity. The ColoScapeTM test is highly reproducible with intra- and inter-assay coefficient of variation of <10% and the cross-reactivity within the assay was limited and negligible. The results for the assay analytical sensitivity indicated that each target could be detected between 0.1% and 0.5% variant allele frequency in 10 ng cfDNA. The preliminary assay clinical specificity and sensitivity were 100% (95% CI: 91.3-100%) and 86% (95% CI: 66-95%) respectively for CRC and 91% specificity (95% CI: 75%-98%) and 60% sensitivity (95% CI: 17%-93%) for advanced adenomas. In summary, the ColoScape test utilizing the XNA-based technology provides high sensitivity and high specificity to CRC and advanced adenomas with a great potential to be used as an early screening test.

Citation Format: Hiromi Tanaka, Shuo Shen, Mauro Scimia, Larry Pastor, Jonathan Li, Andrew Y. Fu, Daniel Kim, Rui Ni, Aiguo Zhang, Michael Y. Sha. ColoScape test: a molecular assay to detect early-stage colorectal cancer in plasma cell-free DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6505.
Abstract 6522: Improved detection of low frequency mutations in ovarian and endometrial cancers by utilizing a highly accurate sequencing platform
Authors: Ragoussis Jiannis , Pezeshkian Nairi , Gilbert Lucy
Published: April 2023

Ovarian and endometrial cancers come within the top-4 for incident cancers as well as deaths in North American women. Cure rates have not improved in 30 years as high-grade subtypes continue to be diagnosed in Stage III/IV. Attempts at early diagnosis have failed because high-grade cancer cells exfoliate and metastasize while the primary cancer is small and undetectable by existing tests based on imaging and blood-based tumour markers. DOvEEgene (Detecting Ovarian and Endometrial cancers Early using genomics) is a genomic uterine pap test developed by a McGill team to screen and detect these cancers while they are confined to the gynecologic organs and curable by surgery. The test identifies pathogenic somatic mutations in uterine brush samples A high sensitivity error-reducing capture technology (DOvEEgene-SureSelectHS) utilizing duplex sequencing interrogates the exons of 23 genes involved in the development of sporadic and hereditary ovarian and endometrial cancers. We apply a combination of germline gene panel testing on saliva samples with deep duplex sequencing to detect somatic mutations at <0.1% VAF, interrogation of microsatellite loci for instability and low coverage WGS for copy number analysis of uterine brush samples. Currently, DOvEEgene is the only test that can discriminate ovarian and endometrial cancers in peri- and postmenopausal women from benign gynecologic diseases common in that age group. This is important because pathogenic somatic driver mutations are also associated with increasing age and benign disease. DOvEEgene incorporates a deep machine-learning derived classifier that can discriminate the mutational signature of these cancers from benign disease aiming for a sensitivity of 70% and a specificity of 100% in a population with high background mutational burden. Here we tested the Onso system, a highly accurate sequencing technology from PacBio in order to potentially increase sensitivity while driving down sequencing costs by reducing required sequencing depth vs the current NGS standard. We sequenced 15 duplex Illumina sequencing libraries produced using the DovEE assay at PE 100bp mode and compared Onso data in non- duplex sequencing mode as well as duplex sequencing mode to the original duplex sequencing method. Here, we present this comparison and highlight the benefits of high accuracy sequencing for the detection of very low frequency (<0.1%) somatic mutations.

Citation Format: Jiannis Ragoussis, Nairi Pezeshkian, Lucy Gilbert. Improved detection of low frequency mutations in ovarian and endometrial cancers by utilizing a highly accurate sequencing platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6522.
Abstract 6540: Multiscale protein networks: de novo aberrant protein interactions and oncogenic regulators in seven cancer types
Authors: Song Won Min , Elmas Abdulkadir , Farias Richard , Xu Peng , Zhou Xianxiao , Hopkins Benjamin , Huang Kuan-lin , Zhang Bin
Published: April 2023

We conducted integrative proteomic network analyses of 687 cases across 7 cancer types including breast cancer (115 tumor samples), clear cell renal carcinoma (100 tumor samples), colorectal cancer (91 tumor samples), hepatocellular carcinoma (101 tumor samples), lung adenocarcinoma (104 tumor samples), stomach adenocarcinoma (80 tumor samples), and uterine corpus endometrial carcinoma (96 tumor samples). Using Multi-scale embedded gene co-expression network analysis (MEGENA), we constructed co-expression protein network for each cancer type, and interrogated the network topology and co-expressed protein modules. For each cancer type, we identified disease-associated pathways as co-expressed protein modules enriched for differentially expressed proteins in tumor. Comparing with respective cancer transcriptome network models, this systematically revealed proteome-specific cancer subnetworks associated with heme metabolism, DNA repair, spliceosome, oxidative phosphorylation and KRAS oncogenic signaling pahways in several cancer types. Cross-cancer comparison identified highly preserved protein modules showing robust pan-cancer interactions and identified endoplasmic reticulum-associated degradation (ERAD) and N-acetyltransferase activity as the central functional axes. Then, we utilized these network models to predict pan-cancer protein network regulators in the up-stream of disease-associated pathways. The predicted pan-cancer regulators were experimentally validated by loss-of-function to confer anti-tumor effects in diverse cancer types: lung (H847), colon (HCT116), fetal kidney (HEK293T) and breast (MDA-MB-231) cancer cells. Overall, the study was designed to provide tractable network models of cancer proteome, and unlock the further potentials to understand oncogenic regulators and mechanisms in different cancer types.

Citation Format: Won Min Song, Abdulkadir Elmas, Richard Farias, Peng Xu, Xianxiao Zhou, Benjamin Hopkins, Kuan-lin Huang, Bin Zhang. Multiscale protein networks: de novo aberrant protein interactions and oncogenic regulators in seven cancer types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6540.
Abstract 6577: CARMEN: A pan-HLA and pan-cancer proteogenomic database on antigen presentation to support cancer immunotherapy
Authors: Kallor Ashwin Adrian , Waleron Michał , Bedran Georges , Eugénio Patrícia , Pesquita Catia , Faria Daniel , Zanzotto Fabio Massimo , Battail Christophe , Rajan Ajitha , Alfaro Javier
Published: April 2023

Cancer immunotherapy has greatly improved the quality of life of cancer patients and it hinges on the discovery of novel cancer antigens that could be targeted to improve disease outcomes. The creation of databases such as IEDB, SysteMHC, TANTIGEN, caAtlas, HLA Ligand Atlas, Cancer Antigenic Peptide Database, SPENCER and IEAtlas support the immunopeptidomics community in understanding the landscape of antigen presentation. We have developed a pan-cancer, pan-HLA, and pan-tissue database containing immunopeptidomics data mapped to transcriptomic, genomic, immunological and biochemical data. The database was generated from 80 different publicly available immunopeptidomics mass spectrometry datasets collected between 2015-2022 (76 cancer and 4 normal datasets), covering 15 different types of cancers and 152 different HLA-I alleles. The peptides contained in our database were obtained by a combination of closed, open and de novo searches using an in-house developed computational pipeline. Following rigorous false discovery rate estimation at 1% and a second-round search to eliminate any false signals that may not have been detected in the previous round of FDR estimation, we obtained a list of 11.2 million peptide-HLA combinations comprising both coding and non-coding regions of the genome as well as bacterial peptides. These peptides have been mapped to chromosomal coordinates to facilitate adoption by the genomics community of this useful resource on antigen presentation. Pathway/biochemical analysis of each peptide was performed using the rWikiPathways package. Finally, mutations associated with each peptide were annotated using COSMIC and dbSNP resources. Our database includes a FAIR knowledge graph which contextualizes and enriches the data to enable clinicians to take effective therapeutic decisions on the appropriate form of treatment for cancer immunotherapy with the case study of clear cell renal cell carcinoma (ccRCC). We will continue to expand our database with new data over the next two years and expand the scope of its applications to facilitate uptake by the larger scientific community.

Citation Format: Ashwin Adrian Kallor, Michał Waleron, Georges Bedran, Patrícia Eugénio, Catia Pesquita, Daniel Faria, Fabio Massimo Zanzotto, Christophe Battail, Ajitha Rajan, Javier Alfaro. CARMEN: A pan-HLA and pan-cancer proteogenomic database on antigen presentation to support cancer immunotherapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6577.
Abstract 6654: Association PD-L1 overexpression with immune checkpoint inhibitor effect in triple-negative breast cancer
Authors: Park A Young , Kim Ju Hee , Lee Sangen , Kim Hong Kyu , Lee Han-Byoel , Han Wonshick
Published: April 2023

Background: Recently, studies on the antitumor effects of PD-L1 inhibitors have gained importance in difficult to treat solid tumors. In particular, the expression of PD-L1 could be an important factor in evaluating the possibility of immunotherapy in triple-negative breast cancer where targeted therapy is not possible and the prognosis is poor. Accordingly, this study investigated the effect of PD-L1 overexpression on the immune checkpoint inhibitor effect in triple-negative breast cancer.

Method: The relationship between T stage and N stage according to PD-L1 mRNA expression was analyzed in the TCGA and METABRIC datasets of breast cancer. To evaluate the role of PD-L1, PD-L1-overexpressing breast cancer cell lines for in vitro and in vivo experiments were established.

Result: Using TCGA and METABRIC datasets, we found that PD-L1 showed the highest expression in breast cancer of the basal subtype, but the correlation between T stage and N stage according to PD-L1 expression was not statistically significant. To determine the functional importance of PD-L1 in breast cancer, we established 4T1-PD-L1-overexpressing breast cancer cell lines. We observed that the overexpression of PD-L1 promoted cell proliferation, migration, and invasion. In the orthotopic tumor mouse model, PD-L1 showed no significant difference in tumor growth compared to the control group, but anti-PD-L1 treatment showed a significant antitumor effect.

Conclusion: We confirmed that overexpression of PD-L1 in an orthotopic breast cancer model did not induce increased tumor growth. Above all, it was confirmed that a high PD-L1 level enhances the antitumor effect of immune checkpoint inhibitors. Our findings show that the expression level of PD-L1 in breast cancer has a significant effect on the immune checkpoint inhibitor effect. We need to further investigate the role of PD-L1 in breast cancer.

Citation Format: A Young Park, Ju Hee Kim, Sangen Lee, Hong Kyu Kim, Han-Byoel Lee, Wonshick Han. Association PD-L1 overexpression with immune checkpoint inhibitor effect in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6654.
Abstract 6656: Expression pattern and clinicopathological implication of B7 family immune checkpoints, VTCN1 and HHLA2, in non-small cell lung cancer
Authors: Lim Sojung , Koh Jaemoon , Song Seung Geun , Yim Jeemin , Han Bogyeong , Kim Young A , Chung Doo Hyun , Jeon Yoon Kyung
Published: April 2023

Background: Efforts are needed to discover novel immunotherapeutic targets in non-responders of current immunotherapies. VTCN1 (B7-H4/B7x/B7S1) and HHLA2 (B7-H7/B7y) are B7 family co-inhibitory molecules reported to be expressed in PD-L1-negative or EGFR-mutant lung adenocarcinoma (LUAD). To elucidate the clinicopathological implications of these two immune checkpoints, we checked their detailed expression patterns including intratumoral heterogeneity.

Methods: VTCN1 and HHLA2 expression were evaluated in public single-cell RNA sequencing (scRNAseq) and The Cancer Genome Atlas (TCGA) dataset. Their immunohistochemistry (IHC) was performed on tissue microarrays of 413 LUAD and 382 lung squamous cell carcinoma (LUSC) specimens. Intratumoral heterogeneity was evaluated by IHC on representative sections of 59 surgically resected LUAD and 49 LUSC specimens.

Results: In LUAD scRNAseq, VTCN1 and HHLA2 were predominantly expressed on epithelial cells and were not co-expressed with PD-L1. However, IHC showed their expression in both tumor and immune cells. In TCGA analysis, there was a negative correlation between VTCN1 and PD-L1 (LUAD, r = -0.094, p = 0.033; LUSC, r = -0.227, p <0.001). VTCN1 and HHLA2 showed positive correlations in both TCGA (LUAD, r = 0.211, p <0.001; LUSC, r = 0.182, p <0.001) and IHC analysis (LUAD, p <0.001; LUSC, p = 0.016). They were more frequently expressed in EGFR-mutant LUAD (52.9% vs. 40.3%, p = 0.015 for VTCN1 in EGFR-mutant vs. wildtype; 59.8% vs. 40.5%, p <0.001 for HHLA2). CD8+ T-cell infiltration was significantly higher in VTCN1- or HHLA2-positive EGFR-mutant LUAD, but there was no significant relationship in EGFR-wildtype. In 60% of cases expressing both VTCN1 and HHLA2, the two showed spatially concordant expression (15 out of 24 LUAD; 3 out of 6 LUSC). However, VTCN1 and PD-L1 showed concordant expression in 37.2% of cases expressing both (6 out of 16 LUAD; 10 out of 27 LUSC). HHLA2 expression was associated with better overall survival in LUSC, but there was no significant difference in survival according to VTCN1 expression.

Conclusions: VTCN1 and HHLA2 are frequently co-expressed in PD-L1-negative NSCLC, especially EGFR-mutant LUAD rich in CD8+ T-cells and can serve as potential novel immunotherapeutic targets.

Citation Format: Sojung Lim, Jaemoon Koh, Seung Geun Song, Jeemin Yim, Bogyeong Han, Young A Kim, Doo Hyun Chung, Yoon Kyung Jeon. Expression pattern and clinicopathological implication of B7 family immune checkpoints, VTCN1 and HHLA2, in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6656.
Abstract 6681: XNA increases assay sensitivity in sanger sequencing, qPCR, NGS and CRISPR mutant screening
Authors: Liu Wei , Brown Robert , Fu Andrew , Shen Shuo , Sha Michael , Zhang Aiguo
Published: April 2023

The proprietary XNA (xeno nucleic acid) technology has wide applications in sensitive detection of rare mutations in different genomic platforms. These target-specific DNA oligo analogs increase assay sensitivity by blocking the polymerase-dependent amplification of wildtype sequence so only amplification of the mutant sequence occurs. Here we show that the XNA technology helps enrich rare somatic mutations in different human specimen types such as FFPE and plasma samples, using technology platforms such as Sanger sequencing, TaqMan qPCR, and NGS. XNA increases assay sensitivity to 0.1% for Sanger sequencing and qPCR. For NGS, XNA allows detection of the mutant sequence at a lower sequencing depth, thus combining XNA technology with NGS for MRD (molecular residual disease) detection could improve cancer management. In addition, We also show CRISPR mutant screening using a target-specific XNA in a SYBR Green qPCR assay. XNA can be used for rapid CRISPR mutant screening for DNA from pool or individual clones generated from gene editing experiments. In summary, XNA technology is a powerful tool when combined with existing genomic mutation detection platforms to screen for rare somatic mutations and for rapid CRISPR mutant screening. The technology is customizable as a service for easy integration with current technology platforms and applications.

Citation Format: Wei Liu, Robert Brown, Andrew Fu, Shuo Shen, Michael Sha, Aiguo Zhang. XNA increases assay sensitivity in sanger sequencing, qPCR, NGS and CRISPR mutant screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6681.
Abstract 6683: Genetic alterations of circulating tumor DNA in advanced pancreatic cancer patients receiving 5-fluorouracil based chemotherapy
Authors: Heo SooBeen , Park Sunhwa , Chun Jung Won , Kim Yun-Hee , Kang Jun-Kyu , Kim Hwang-Phill , Woo Sang Myung , Kong Sun-Young
Published: April 2023

Background: Pancreatic cancer remains one of the most devastating malignancies due to lack of target therapeutic options for advanced disease. Liquid biopsy which analyzes tumor-related biomarkers in blood, has emerged as an innovative technology. The aim of this study was to identify circulating tumor DNA (ctDNA) mutations in pancreatic adenocarcinoma(PDAC) patients using multi-gene panels.

Methods: Total of 45 patients (27 male, median age 63 years) blood samples were obtained at baseline then follow-up after 2 months samples were acquired in 16 patients at National Cancer Center, Korea from November 2021 to April 2022. CtDNA were analyzed by next-generation-sequencing (NGS) panel of 118 genes and the varient allele frequency (VAF) of detected genes were analyzed. Then the association between outcomes of 5FU treated response and survival (overall survival and progression-free survival; OS and PFS) and VAF reduction at follow-up were analyzed.

Results: Total of 35 genes with 114 genetic alterations were detected and frequent mutations were as follows: KRAS (29.9%), TP53 (14.9%), GNAS(4.4%), SMAD4(4.4%), AR(3.5%), RNF434(3.5%), ATM(3.5%), BRCA2(2.6%) and PIK3CA(2.6%). The mean VAF level considering all pathogenic variants of 45 patients was 8.9%(range, 0 - 87.2) including 8 patients who were not detected ctDNA mutations in baseline, and the mean VAF was 4.2% (0 - 50.0) at follow-up in 16 patients. With association of clinical response, patients with partial response (PR) and stable disease (SD) were decreased VAF from 11% at baseline to 10% after 2 months, and three (60%) of patients showed clearance of ctDNA. Progressive disease (PD) patients were also decreased VAF 67% after 2 months, however, clearance of ctDNA was only observed in one patient (12.5%). Reduction of ctDNA more than 50% were associated with longer survival as PFS (> 50% reduction groups vs. no reduction group; 5 vs. 3 months, p value=0.903, OS (undefined vs. 6 months, p value =0.180).

Conclusions: This study showed most common mutation was KRAS with several other mutations of ctDNA in PDAC, and outcomes represented association with ctDNA.

Citation Format: SooBeen Heo, Sunhwa Park, Jung Won Chun, Yun-Hee Kim, Jun-Kyu Kang, Hwang-Phill Kim, Sang Myung Woo, Sun-Young Kong. Genetic alterations of circulating tumor DNA in advanced pancreatic cancer patients receiving 5-fluorouracil based chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6683.
Abstract 6693: Stability of urinary cell-free DNA and detection of T790M variant
Authors: Münch Carolin , Shamkeeva Saikal , Heinemann Mitja , Isermann Berend , Kasimir-Bauer Sabine , Aktas Bahriye , Nel Ivonne
Published: April 2023

Background: The so-called “liquid biopsy” has become a powerful tool for cancer research in the recent years, with circulating cell-free DNA (cfDNA) that originates from tumors as one of the most promising analytes. In contrast to plasma derived cfDNA, only a few studies were performed investigating urinary cfDNA. One reason might be quick degradation and hence relatively low concentrations of urinary cfDNA. This study focused on examining the stability of cfDNA in urine using different ways of preservation under various storage conditions.

Methodology: To mimic patient samples, a pool of healthy male and female urine donors was spiked with a synthetic cfDNA reference standard (fragment size 170 bp) containing the T790M mutation in the EGFR gene and preserved with three different buffers and no buffer over four different storage periods (0; 4; 12; 24 h) at room temperature vs. fridge. Preservatives used were the Urinary Analyte Stabilizer (UAS, Novosanis), the Urine Conditioning Buffer (UCB, Zymo) and a self-prepared buffer called “AlloU”. The cfDNA was extracted with the QIAamp MinElute ccfDNA Mini Kit (Qiagen) and the concentration measured with the Qubit™ 4 fluorometer (Thermo Fisher Scientific). Droplet digital PCR (ddPCR) was used for detection and quantification of the T790M mutation (Bio-Rad)

Results: From the model samples with no preservation buffer, almost no spiked cfDNA could be recovered and the T790M variant could not be detected using ddPCR, indicating that without preservation cfDNA was degraded below the detection limit by the nucleases present in urine. The most effective stabilizing buffer over all storage periods and at room as well as fridge temperature was the UAS, resulting in accurate detection of the T790M variant using ddPCR.

Conclusion: From a technical point of view, urinary cfDNA might be useful for non-invasive disease monitoring when the samples are stabilized and stored adequately during clinical routine.

Citation Format: Carolin Münch, Saikal Shamkeeva, Mitja Heinemann, Berend Isermann, Sabine Kasimir-Bauer, Bahriye Aktas, Ivonne Nel. Stability of urinary cell-free DNA and detection of T790M variant. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6693.