bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1965: Complete remission in advanced epithelial ovarian cancer is predicted by metabolic signatures measured in the media of patient’s primary culture 3D tumor explants: A metabolomic analysis
Authors: Nagourney Adam J. , D'Amora Paulo , Dale Cotrim Guerreiro Silva Ismael , Evans Steven S. , Nagourney Robert A.
Published: June 2022

Ovarian cancer is the leading cause of gynecologic cancer death with over 21,000 new diagnosis and 13,000 deaths each year in the US. Treatment with platinum-based chemotherapy induces remission in up to 80% of patients yet the majority relapse and die from their disease within 5 years. We recently reported the analysis of patients with advanced ovarian cancers using plasma metabolomics to predict response and survival (D’Amora et al Gynecol Oncol, October 2021). As part of that study human tumor explants were submitted for the measurement of platinum resistance by ex vivo analysis of programmed cell death (EVA/PCD࣪). Media obtained from the non-drug exposed control cultures were examined by mass spectrometry for the quantification of 186 individual metabolites using the Biocrates’ p180 platform. Results obtained with the tissue culture media identify biochemical signatures that correlate with the achievement of complete pathologic remission. Among the most discrimination findings were the media concentrations of phosphatidyl choline (PC) aa c42:0 and the ratio of (PC) aa C40:2 divided by (PC) ae C42:1 suggesting either hyperactivity of lipid elongases or defective beta oxidation of very long chain fatty acids in drug refractory patients. When we compared the results of 8 patients who achieved pCR with 3 patients with residual disease the aa C42:0 concentration and the ratio aa C42:0/ae C42:1 provided an AUC= 0.958 (p. < 0.01) The findings suggest that peroxisomal dysfunction leads to the accumulation of very long chain fatty acid lipids. These species are known to induce endoplasmic reticulum and oxidative stress and are associated with the neurodegenerative diseases like Zellweger syndrome. We have reported similar findings in drug refractory breast cancer patients (da Silva et al, Oncotarget, August Aug 2018) suggesting common lipid metabolic changes in drug resistant adenocarcinomas. The results of these analyses could provide prognostic and predictive information for patients diagnosed with advanced epithelial ovarian cancers. Further studies correlating these findings with plasma metabolic signatures are underway as will be reported.

Citation Format: Adam J. Nagourney, Paulo D'Amora, Ismael Dale Cotrim Guerreiro Silva, Steven S. Evans, Robert A. Nagourney. Complete remission in advanced epithelial ovarian cancer is predicted by metabolic signatures measured in the media of patient’s primary culture 3D tumor explants: A metabolomic analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1965.
Abstract 1969: Distinctive profiles on immunotherapy responders versus non responders in Chilean gastric cancer patients
Authors: Pinto Mauricio P. , Medel Matias Muñoz , Delgado Miguel Córdova , Retamal Ignacio N. , Manque Patricio , Berkovits Alejandro , Rodriguez Maria Paz , Rios Juvenal A. , Garcia-Bloj Benjamin , Owen Gareth , Garrido Marcelo
Published: June 2022

Background: Gastric cancer (GC) is the leading cause of cancer death in Chile, claiming >3,000 lives per year. On April 2021, the US Food and Drug Administration approved the combined use of Nivolumab (immunotherapy) plus chemotherapy as frontline treatment for advanced GC. Unfortunately, as occurs with any therapy, a proportion of treated patients are either unresponsive or display poor responses. Our study sought to obtain a profile of GC patients categorized as responders or non-responders to immunotherapy in order to identify better predictors of response and novel targets for treatment refractory patients.

Methods: A total of 50 GC patients were included into our study. Demographic and basic information was obtained from medical records. Additionally, we retrieved information on response to treatment, histological type, PDL1 and HER2 status. Nucleic acid samples were collected from a subset of 45 patients (responders and non-responders) and analyzed by massive analysis of cDNA ends (MACE-seq) assessing 3’ mRNA sequencing.

Results: Patients were predominantly male (76%) and advanced stage (III/IV; 98%). Within this subset, 26 were responders and 24 were non-responders. Among clinical variables, body mass index was significantly higher on responders versus non-responders (p=0.026). Among responders, tumors were predominantly located on the gastro-esophagic junction. As expected, responders also displayed a higher prevalence of HER2- and PDL1+. Our 3’ MACE analysis confirmed distinct profiles on responders compared to non-responders.

Conclusions: Our findings confirm distinctive profiles among Chilean GC patients categorized as responders or non-responders to immunotherapy. These results could deliver novel markers that may serve as predictors of response to immunotherapy (as an alternative to PDL1) or to identify patients more likely to be refractory to treatment. Future studies should further confirm and validate these findings in a larger sample.

Citation Format: Mauricio P. Pinto, Matias Muñoz Medel, Miguel Córdova Delgado, Ignacio N. Retamal, Patricio Manque, Alejandro Berkovits, Maria Paz Rodriguez, Juvenal A. Rios, Benjamin Garcia-Bloj, Gareth Owen, Marcelo Garrido. Distinctive profiles on immunotherapy responders versus non responders in Chilean gastric cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1969.
Abstract 2064: Targeting Cblb-Notch1 axis as a novel strategy for cancer immunotherapy
Authors: Monticone Giulia , Csibi Fred , Leit Silvana , Austin Jermaine E. , Ucar Deniz A. , Hossain Fokhrul M. , Majumder Samarpan , Osborne Barbara A. , Loh Christine , Miele Lucio
Published: June 2022

Background: Tumor-induced immunosuppression is a way in which cancers evade the host immune response. In recent years, a great effort has been made in designing immunotherapies that can boost the immune system response against cancer. However, not all tumors respond to these therapies. Therefore, there remains an unmet need for immunotherapies that are able to circumvent tumor-induced immunosuppression. Adenosine is an immunosuppressive metabolite which is overproduced in hypoxic tumor microenvironments and dampens T-cell anti-tumor immune responses. Adenosine A2A receptor (A2AR) activation was shown to downregulate Notch1, a key regulator of T-cell functions, in CD8+ T-cells, leading to immunosuppression. Notch1 signaling appears to protect T-cells against immunosuppressive signals, as Notch1 overexpressing T-cells were shown to be resistant to tumor-induced immunosuppression mediated by myeloid-derived suppressor cells (MDSC), as well as adenosine. We hypothesize that rescuing Notch1 from downregulation will make T-cells resistant to tumor-induced immunosuppression. We applied several functional assays in primary CD8+ T-cells and tumor-derived organoids to study how Notch1 is regulated by A2AR and explore strategies to target this pathway for cancer immunotherapy.

Results: Our results indicate that the ubiquitin ligase Cbl-b, a negative regulator of T-cell functions, is responsible for the ubiquitination and degradation of Notch1 in CD8+ T-cells. A2AR signaling leads to Notch1 downregulation by promoting Cbl-b-mediated Notch1 degradation. Inhibition of Cbl-b using small molecule compounds restores Notch1 and T-cell functions in CD8+ T-cells in the presence of adenosine, making them resistant to A2AR-mediated immunosuppression. Cbl-b inhibitors show anti-tumor activity in tumor-derived organoids from pre-clinical models and enhance immune-checkpoint immunotherapy, by promoting T-cells anti-cancer responses.

Conclusions: Our work suggests that promoting Notch1 signaling by blocking Cbl-b-mediated degradation results in increased T-cell responses and resistance to immunosuppression. Targeting Cbl-b-Notch1 axis represents a promising novel strategy to boost anti-cancer T-cell responses.

Citation Format: Giulia Monticone, Fred Csibi, Silvana Leit, Jermaine E. Austin, Deniz A. Ucar, Fokhrul M. Hossain, Samarpan Majumder, Barbara A. Osborne, Christine Loh, Lucio Miele. Targeting Cblb-Notch1 axis as a novel strategy for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2064.
Abstract 209: Elucidating the role of NMDA subunit NR2B in non-tumor and tumor-bearing mice
Authors: Castro Jesus , Gaelzer Mariana , Welford Scott
Published: June 2022

Therapeutic options for glioblastoma multiforme (GBM), the most common and fatal brain tumor, have been limited over the course of recent decades, leaving radiation as one of the few effective therapies. While advancements in radiation therapy have improved life expectancy and patient outcome, exposure to radiotherapy causes normal tissue toxicity and damage to the central nervous system, resulting in cognitive impairment and negative patient side effects. In the hippocampus, the memory processing center of the brain, radiotherapy-induced elevated glutamate levels lead to over-excitation of N-methyl-D-aspartate receptors (NMDAR) and thus cell death. To investigate the NMDAR, we repurposed a NMDA subtype specific inhibitor (Ifenprodil) and treated non-tumor mice before radiation. We found that Ifenprodil rescues cell death and cognition by inhibiting the NR2B subunit of NMDA receptors and preventing over-excitement-induced excitotoxicity, despite presence of high glutamate levels. In order to relieve glioma patients of the negative side effects of radiotherapies, we aim to further investigate the critical role of NR2B inhibition in non-tumor and tumor-bearing mice to reduce excitotoxicity and prevent damage to the central nervous system, opening avenues for therapeutic targeting and drug development. To accomplish this, we designed promotor-specific plasmids, only activated when present in their designed neuronal cell types, to initiate CRISPR/Cas9 genome editing and knock out the NR2B subunit of NMDAR. With NR2B knocked out across 5 different cell types, we exposed hippocampal organotypic slice cultures, derived from non-tumor mice, to 2 Gy radiation and ran flow cytometry to reveal which neuronal NR2B subunits play critical roles in excitotoxic phenotypes. To further these findings, we utilized a recently developed in-utero-electroporation mouse model that produces glioblastomas in immunocompetent mice by inactivating select tumor-suppressor genes via CRISPR/Cas9 genome editing at embryonic day 14.5. By applying different CRISPR/Cas9 gRNAs into our tumor-bearing mice, we can not only explore the robust role of NR2B inhibition in tumor brains with varying expressions, but also shed light onto how NR2B inhibition protects normal brain while leaving tumors radiosensitive. In conclusion, this work seeks to further develop our understanding of radiotherapy-induced cytotoxicity in order to prevent cognitive impairment and negative side effects in patients, improving their quality of life. Ultimately, the results from our findings will lead to the advancement of targeted drug development and therapeutic options.

Citation Format: Jesus Castro, Mariana Gaelzer, Scott Welford. Elucidating the role of NMDA subunit NR2B in non-tumor and tumor-bearing mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 209.
Abstract 2090: Humanized IL36R mice provide a preclinical tool for the evaluation of therapeutic drugs targeting IL36R
Authors: Zhou Xiaofei , Chang Jiahui , Frame Jenna , Li Chong , Bai Zhifang , Shen Yuelei
Published: June 2022

The IL36 family of cytokines (IL36a, IL36b, and IL36g) is part of the IL-1 superfamily and triggers proinflammatory signaling through the IL36 receptor complex. This heterodimer complex is formed by the IL36 Receptor (IL1RL2) and IL-1R co-receptor accessory protein (IL-1RAcP). IL36R is present on a diverse variety of cell types, including the surface of dendritic cells, CD4+ T cells, and epithelial cells in various tissues. IL36 signaling can exert widespread inflammation, and its activity is implicated in several inflammatory diseases affecting epithelial tissues, including inflammatory bowel disease, rheumatoid arthritis, and psoriasis. Thus, identification of novel therapeutics targeting this pathway are of significant clinical interest. To provide a preclinical platform for efficacy assessments of novel anti-human IL36R drugs, we generated a humanized hIL36R knock-in mouse model. In this model, a chimeric IL1RL2 coding sequence, which is composed of the human IL1RL2 extracellular region and the mouse Il1rl2 cytoplasmic region, was inserted after the 5’UTR of mouse Il1rl2 gene using CRISPR-Cas9-based gene editing technology. The mouse cytoplasmic region was maintained to ensure intact downstream signaling. Polymerase chain reaction assessment of mRNA isolated from the lung tissue of edited homozygous humanized IL36R mice confirmed the absence of mouse Il1rl2 transcripts. Finally, to confirm the expression of human IL36R protein, immunohistochemistry was performed on the small intestine and skin tissue of humanized mice subjected to a chemical induction of psoriasis. Psoriasis-like symptoms were achieved by application of imiquimod cream on the back skin of the mice for 5 consecutive days. As expected, IL36R protein expression was detected in both tissues isolated from the homozygous humanized mice. These results indicate the successful generation of humanized IL36R mice, and suggest that this model will be an effective tool for future in vivo efficacy evaluation of immunomodulatory drugs targeting human IL36R.

Citation Format: Xiaofei Zhou, Jiahui Chang, Jenna Frame, Chong Li, Zhifang Bai, Yuelei Shen. Humanized IL36R mice provide a preclinical tool for the evaluation of therapeutic drugs targeting IL36R [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2090.
Abstract 2098: Practical cell counting method selection to increase the quality of cell counting results
Authors: Chan Leo , Bell Jordan , Lin Bo
Published: June 2022

The importance of cell counting has increased significantly in the last decade due to the major advances in the fields of cell and gene therapy, biologics production, and regenerative medicine. This has necessitated the development of a standardized approach to cell counting assays. In recent years, the U.S. Food and Drug Administration (FDA), in collaboration with the National Institute of Standards and Technology (NIST) and the International Organization for Standardization (ISO), has launched an effort to standardize cell counting methods to improve the confidence in cell counting measurements. There is a wide range of biological sample types, various formulations, and bioprocessing steps for cell and gene therapy products. Furthermore, there are no ground truth reference materials for live cells, making the determination of accuracy of cell counting difficult defined in the ICH Q2 (R1). Therefore, in order to increase the confidence of cell counting results, it is critical to follow the two recently published ISO cell counting standards to ensure cell counting methods are tailored to the specific sample and purpose.In this work, we will provide insights and guidance for the ISO standards for method selection. The important aspects of the ISO cell counting standards have been distilled to six key factors for selecting the cell counting methods. Attention to these details will allow researchers to more easily adhere to the ISO recommendations to ensure high quality cell counting measurements, and in doing so, sites can meticulously document their rationale, process, and data results.

Citation Format: Leo Chan, Jordan Bell, Bo Lin. Practical cell counting method selection to increase the quality of cell counting results [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2098.
Abstract 2192: HSF1 downregulation of CCL5 reduces CD8 + T cell trafficking in breast cancer
Authors: Jacobs Curteisha LeeAnn , Shan Sakhi , Lu Wen-Cheng , Carpenter Richard
Published: June 2022

Breast cancer is the second leading cause of cancer-related death in women. The presence of cytotoxic immune cells, specifically CD8+ T cells, in breast tumors is associated with better patient outcomes. Understanding the mechanisms that regulate the infiltration of CD8+ T cells into breast tumors could improve the treatment and enhance patient overall survival. Utilizing a novel HSF1 activity gene signature, we observed that HSF1 activity was negatively associated with the presence of CD8+ T cells in breast cancer patients. Both CIBERSORT analysis of TCGA data as well as direct assessment of patient primary specimens demonstrated that patient tumors with high HSF1 activity had lower numbers of CD8+ T cells. To functionally test this relationship, HSF1 was knocked down in 4T1 breast cancer cells and grown orthotopically in BALB/c mice. Tumors with HSF1 knockdown showed lower tumor volumes and increased CD8+ T cell infiltration. We further tested the effect of CD8+ T cells on the growth of HSF1 knockdown tumors by depleting CD8+ T cells in BALB/c mice. HSF1 knockdown tumors were significantly larger with CD8+ T cell depletion suggesting a functional role for HSF1 to inhibit CD8+ T cell infiltration and protect the tumor from immune-mediated killing. To investigate the mechanism by which HSF1 affects CD8+ T cells, we investigated whether HSF1 affects chemotactic cytokines. We found that loss of HSF1 significantly increased secretion of CCL5, a known chemoattractant for CD8+ T cells. Loss of HSF1 also increased mRNA levels of CCL5, suggesting a transcriptional effect on CCL5. To test the importance of CCL5 in the phenotype of HSF1 knockdown, a transwell migration assay was performed wherein T cells from BALB/c mice were placed in the upper chamber of a transwell chamber and the lower chamber contained conditioned medium from 4T1 cells with knockdown of HSF1 with or without knockdown of CCL5. The lower chamber was subjected to flow cytometry for CD3/CD8 to identify CD8+ T cells and we observed that loss of HSF1 increased migration of CD8+ T cells and this was ablated with additional loss of CCL5. These results suggest CCL5 is a significant mediator for the recruitment of CD8+ T cells when HSF1 is lost in cancer cells. We propose the model whereby high HSF1 activity in breast cancer cells suppresses expression and secretion of the chemo-attractant cytokine CCL5 that ultimately leads to a decrease in CD8+ T cells in breast tumors microenvironment.

Citation Format: Curteisha LeeAnn Jacobs, Sakhi Shan, Wen-Cheng Lu, Richard Carpenter. HSF1 downregulation of CCL5 reduces CD8 + T cell trafficking in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2192.
Abstract 2200: Multilineage plasticity in prostate cancer through expansion of stem-like luminal epithelial cells with elevated inflammatory signaling
Authors: Zaidi Samir , Zhao Jimmy , Chan Joseph , Martine Roudier , Wadosky Kristine , Gopalan Anuradha , Karthaus Wouter , Watson Philip , True Lawrence , Nelson Peter , Scher Howard , Morris Michael , Haffner Michael , Goodrich David , Pe'er Dana , Sawyers Charles
Published: June 2022

Lineage plasticity is a well-established mechanism of resistance to targeted therapies in lung and prostate cancer, where tumors transition from adenocarcinoma to small-cell or neuroendocrine carcinoma. Single-cell analysis of a cohort of late stage castration-resistant human prostate cancers (CRPC) revealed a greater degree of plasticity than previously appreciated, with multiple distinct neuroendocrine (NEPC), mesenchymal (EMT-like), and other subpopulations detected within single biopsies. To explore the steps responsible for initiation of this process, we utilized two genetically engineered mouse models of prostate cancer that recapitulate progression from adenocarcinoma to neuroendocrine disease. Time course studies reveal expansion of stem-like luminal epithelial cells (Sca1+, Psca+, called L2) that, based on trajectories, gave rise to at least 4 distinct subpopulations, NEPC (Ascl1+), POU2F3 (Pou2f3+), TFF3 (Tff3+) and EMT-like (Vim+, Ncam1+). Such populations are also seen in human prostate and small cell lung cancers. Furthermore, transformed L2-like cells express stem-like and gastrointestinal endoderm-like transcriptional programs, indicative of reemerging developmental plasticity programs, as well as elevated Jak/Stat, interferon, and FGF pathways. Strikingly pharmacologic inhibition of Jak/Stat and FGFR results in reversal of plasticity states and subsequent sensitivity to androgen receptor inhibitors (ARSIs). In sum, while the magnitude of multilineage heterogeneity, both within and across patients, raises considerable treatment challenges, the identification of highly plastic luminal cells as the likely source of this heterogeneity provides a target for more focused therapeutic intervention.

Citation Format: Samir Zaidi, Jimmy Zhao, Joseph Chan, Roudier Martine, Kristine Wadosky, Anuradha Gopalan, Wouter Karthaus, Philip Watson, Lawrence True, Peter Nelson, Howard Scher, Michael Morris, Michael Haffner, David Goodrich, Dana Pe'er, Charles Sawyers. Multilineage plasticity in prostate cancer through expansion of stem-like luminal epithelial cells with elevated inflammatory signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2200.
Abstract 2212: Racially disparate serum levels of inflammatory cytokines, satiety and stress hormones, and exosomal microRNAs in women with or without a breast cancer diagnosis
Authors: Sudan Sarabjeet Kour , Vikramdeo Kunwar Somesh , Sharma Amod , Deshmukh Sachin Kumar , Srivastava Sanjeev Kumar , Poosarla Teja , Holliday Nicolette P. , Dyess Donna L. , Singh Ajay P. , Singh Seema
Published: June 2022

Breast cancer (BC) is the most common non-cutaneous malignancy and the second leading cause of cancer-related death in American women. Health disparities in incidence and clinical outcome are also reported among different racial groups, most notably African American (AA) women, who are often diagnosed at a young age with aggressive BC and exhibit greater mortality than Caucasian American (CA) women. Since socioeconomic difficulties can have tremendous impact on psychophysiology besides limiting the access to optimal healthcare, we examined the serum levels of stress (cortisol) and satiety (leptin) hormones as well as inflammatory cytokines (resistin and interleukin-6/IL-6) in AA and CA women. To observe a potential epigenomic connection, we also performed profiling of a targeted set of exosomal microRNAs in serum samples. The study was conducted under an Institutional Review Board (IRB) approved protocol. All subjects participated voluntarily, and their consents were obtained. Serum levels of resistin, IL-6, leptin, and cortisol were quantified by Enzyme linked Immunosorbent Assay (ELISA) using commercial kits. Exosomes were isolated using precipitation method and recovered by standard centrifugation. Total RNA was isolated from exosomal preps and subjected to stem-loop RT-PCR for quantitation of a set of inflammation-associated microRNAs. We found that the levels of resistin, leptin, IL-6 and cortisol were higher in women with a BC diagnosis than non-BC subjects. Moreover, AA women with or without BC showed significantly higher levels of these cytokines and hormones in their serum as compared to the CA women with or without a BC diagnosis, respectively. We also observed differential expression of several microRNAs in serum of BC women as compared to their normal counterparts, of which five (miR511, miR33a, miR27a, miR6794, miR143-3p) exhibited highest presence in serum exosomes of AA women with BC. Together, these findings suggest that relatively greater exposure of minority women to social stressors may have epigenomic consequences and may potentially be linked to the observed BC racial health disparities.

Citation Format: Sarabjeet Kour Sudan, Kunwar Somesh Vikramdeo, Amod Sharma, Sachin Kumar Deshmukh, Sanjeev Kumar Srivastava, Teja Poosarla, Nicolette P. Holliday, Donna L. Dyess, Ajay P. Singh, Seema Singh. Racially disparate serum levels of inflammatory cytokines, satiety and stress hormones, and exosomal microRNAs in women with or without a breast cancer diagnosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2212.
Abstract 2221: Molecular analysis finds excision margin width predictive of recurrence in acral melanoma
Authors: Attrill Grace Heloise , Bailey Teresa A. , Lum Trina , Conway Jordan W. , Long Georgina V. , Wilmott James S. , Scolyer Richard A.
Published: June 2022

Surgical excision with histologically clear margins results in cure of most melanoma patients. Involvement of the margins is associated with tumour recurrence and adverse outcomes. Determining whether or not the histological margins are involved by melanoma, particularly in situ melanoma, can be very difficult particularly in acral locations where insitu melanoma can be extremely subtle. This study sought to determine the proximity of acral melanoma in situ to the surgical excision margin using multiple methods including routine histopathological assessment, FISH (CCND1 amplification), and multiplex immunofluorescence (mIF: SOX10, p16, Cyclin D1 and PRAME) in primary acral melanomas with initially histopathologically reported clear excision margins of >0.3mm (n=85). The clear excision margins determined by each modality were compared between those melanomas which recurred (n=47) and those which remained recurrence-free at last follow-up (median = 46 months, range = 1-260 months) (n=35). Histopathologically confirmed excision margin width was smaller in the tumours of patients who recurred (mean = 7.083mm vs 9.964mm, p = 0.0811). Intratumoural CCND1 amplification was increased in recurrence-free patients in comparison to recurrence patients (16.2% vs 7.7%). FISH and mIF confirmed the correlation of intratumoural CCND1 amplification with Cyclin D1 expression SOX10+ melanoma cells (p=0.0347). No correlations between recurrence status and Cyclin D1, PRAME or p16 expression levels in the tumour or epidermis were identified. In histopathologically normal epidermal tissue as assessed on H&E sections,Cyclin D1+, p16- and PRAME+ cells were classified as malignant cells when co-expressed with SOX10. Upon measuring the distance from the tumour margin to the nearest malignant cell, we found that malignant cells were closer to the tumour in recurrence patients in comparison to recurrence-free patients (mean = 7.324mm vs 9.464mm, p = 0.2735). These distances closely correlated with the excision margin width as determined via histopathological assessment (R2 = 0.7424, p<0.0001). In conclusion, histological assessment, FISH and mIF determined excision margins are highly correlative in acral melanomas. Narrower excision margins were associated with local recurrence of melanoma.

Citation Format: Grace Heloise Attrill, Teresa A. Bailey, Trina Lum, Jordan W. Conway, Georgina V. Long, James S. Wilmott, Richard A. Scolyer. Molecular analysis finds excision margin width predictive of recurrence in acral melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2221.
Abstract 2229: Targeted locus amplification based sequencing for mapping viral integration sites in human papillomavirus positive head and neck squamous cell carcinomas
Authors: Demers Imke , Balaji Harini , Feitsma Harma , Sergeeva Irinia , Swennenhuis Joost , Wuerdemann Nora , Wagner Steffen , Kremer Bernd , Huebbers Christian , Klussmann Jens Peter , Speel Ernst-Jan
Published: June 2022

Background: Human papillomavirus (HPV) infections are the principal cause of cervical cancers, subsets of anogenital and head and neck cancers (HNC). During persistent infection, viral DNA integration into the host genome may occur, which is suggested to affect carcinogenesis. One of the most critical limitations of currently used HPV integration detection techniques (PCR-based or NGS-based) is their application to FFPE material, because of DNA fragmentation. The aim of this study was to assess HPV integration in HPV-positive HNSCC cell lines and FFPE tissue comparing the new Targeted Locus Amplification (TLA) technology with previously used PCR technology (APOT/DIPS).

Methods: Seven HPV-positive cell lines and FFPE material of 10 HPV-positive HNSCC were used for HPV integration detection by TLA, a proximity ligation-based next-generation sequencing technique. Crosslinked DNA is digested with restriction enzymes, and re-ligated into chimeric DNA molecules. For cell lines, a PCR based HPV16 target enrichment is performed, and for FFPE material a capture-based target enrichment is performed for HPV16 and HPV18 sequences, both followed by Illumina sequencing.

Results: TLA was able to sequence up to 100 kb around the target, detecting exact HPV integration loci, structural variants, and chromosomal rearrangements. In all cell lines, one or more integration sites were identified, in accordance with APOT/DIPS PCR data and the literature. In the FFPE tissue samples, TLA identified integrated HPV in 6 out of 10 tumors, with simple and complex integration patterns. In general, TLA confirmed PCR data and detected additional integration sites.

Conclusion: TLA provides the opportunity for reliable and robust detection of HPV integration in HNSCC cell lines and FFPE tissue. This new sequencing technology could be a useful tool for further research on HPV integration in disease and patient outcome and eventually in clinical diagnostics.

Citation Format: Imke Demers, Harini Balaji, Harma Feitsma, Irinia Sergeeva, Joost Swennenhuis, Nora Wuerdemann, Steffen Wagner, Bernd Kremer, Christian Huebbers, Jens Peter Klussmann, Ernst-Jan Speel. Targeted locus amplification based sequencing for mapping viral integration sites in human papillomavirus positive head and neck squamous cell carcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2229.
Abstract 2235: Identifying populations at elevated risk of cancer outside those traditionally targeted for screening and prevention
Authors: Patel Alpa V. , Deubler Emily , Teras Lauren R. , Colditz Graham A. , Lichtman Cari J. , Cance William G. , Clarke Christina A.
Published: June 2022

Introduction: Currently, populations are identified for cancer screening interventions one cancer type at a time. With new developments in cancer screening, including multi-cancer early detection tests, it is important and timely to understand the overall cancer burden in populations outside those traditionally targeted for single-cancer screenings. Thus, we sought to identify the most important risk factors for developing any invasive cancer, and to calculate associated 5-year absolute risks, with a particular focus on persons under age 50 years.

Methods: Two large American Cancer Society cohorts followed prospectively for cancer, the Cancer Prevention Study-II Nutrition Cohort (CPS-IINC) enrolled from 1992-1993 and Cancer Prevention Study 3 (CPS-3) enrolled from 2006-2013, were used to examine a broad range of cancer risk factors. Multivariable Cox proportional hazards models were used to estimate hazard ratios (HR) and 95% confidence intervals (CIs) for associations between potential risk factors and subsequent overall risk of any invasive cancer. The Individualized Coherent Absolute Risk Estimation tool used these HRs to estimate absolute risk in 5-year age groups.

Results: 15,226 cancers were identified among 429,991 participants within 5 years of follow-up. Several risk factors including smoking history, obesity, alcohol, family history of cancer, and various chronic medical conditions (e.g., diabetes, hypertension) were associated with a higher risk of cancer. Persons above age 50 years, regardless of other risk factors considered, had at least 2% absolute risk per 5 years. Among persons under age 50 years, smoking status was the most important factor differentiating risk. For men under age 50, absolute risk of any cancer did not exceed 2% except for men ages 45-49 years who currently smoke or recently quit (within 30 years). For women under age 50, absolute risk exceeded 2% starting at age 35-39 years for current or recent former smokers, and all women, even long-term nonsmokers, at age 45-49 had over 2% absolute risk.

Conclusions: Smoking history is important in differentiating absolute risk of any cancer in adults under age 50 years. More research to understand the extent to which other cancer risk factors influence risk for specific populations aged <50 years should be carried out.

Citation Format: Alpa V. Patel, Emily Deubler, Lauren R. Teras, Graham A. Colditz, Cari J. Lichtman, William G. Cance, Christina A. Clarke. Identifying populations at elevated risk of cancer outside those traditionally targeted for screening and prevention [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2235.
Abstract 2262: Associations of plasma carotenes with lung cancer risk in a low-income population in the southeastern united states
Authors: Sun Yan , Wu Jie , Yoon Hyung-Suk , Zheng Wei , Cai Hui , Buchowski Maciej S. , Franke Adrian A. , Blot William J. , Shu Xiao-ou , Cai Qiuyin
Published: June 2022

Lung cancer is the second most common cancer and the leading cause of death from cancer in the U.S. Carotenes, with the properties of antioxidation and immunoregulation, have been demonstrated to have anti-carcinogenic effects. Previous prospective studies showed inverse associations of circulating α-carotene and β-carotene with lung cancer risk among European descendants and Asians. The associations of α-carotene and β-carotene isomers with lung cancer risk have not been investigated, particularly among African Americans (AAs) and low-income populations. We conducted a case-control study including 225 incident lung cancer cases and 429 controls, individually matched on age, race, and sex, nested in the Southern Community Cohort Study, including ~86,000 participants, two-thirds of whom are AAs. Carotene plasma concentrations were determined by a validated assay using high-pressure liquid chromatography with photo-diode array detection. Plasma trans-β-carotene (ORT3 VS. T1=0.54; 95% CI: 0.32-0.90; Ptrend=0.03), cis-β-carotene (ORT3 VS. T1=0.55; 95% CI: 0.32-0.93; Ptrend=0.03), and total β-carotene (ORT3 VS. T1=0.50; 95% CI: 0.30-0.84; Ptrend=0.01) were inversely associated with lung cancer risk. The associations remained significant after excluding cases diagnosed within the first year of follow-up. The inverse associations of plasma cis-β-carotene and total β-carotene with lung cancer risk were only observed among AAs and males. Plasma trans-α-carotene, trans-β-carotene, cis-β-carotene, and total β-carotene were inversely associated with lung cancer risk among those with lower body mass index (<median). In summary, plasma levels of β-carotenes were inversely associated with lung cancer risk in AA and low-income populations. Further studies with a larger sample size are warranted to confirm our findings.

Citation Format: Yan Sun, Jie Wu, Hyung-Suk Yoon, Wei Zheng, Hui Cai, Maciej S. Buchowski, Adrian A. Franke, William J. Blot, Xiao-ou Shu, Qiuyin Cai. Associations of plasma carotenes with lung cancer risk in a low-income population in the southeastern united states [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2262.
Abstract 2274: Identification of predisposition and progression gastric cancer biomarkers in Latinos
Authors: Estrada-Florez Ana Patricia , Lott Paul , Toal Ted , Halmai Nicole , Quino Elizabeth , Poceros-Coba Alma , Guevara Alix , Castro Fabian , Suarez Jhon , Mcpherson John , Venkatesh Rasika , Zhang Hongyong , Vang Apri , Polanco Guadalupe , Guerra Joana , Molina Graciela , Parra Carol , Valle Adriana Della , Castelao Esteban , Teixeira Manuel , Bohorquez Mabel , Torres Javier , Corvalan Alejandro Carvajal , Carvajal-Carmona Luis G.
Published: June 2022

Gastric cancer (GC) disproportionally affects people of Latino ancestry and represent a leading cause of incidence and mortality disparities in this minority populations. When compared to Non-Latino Whites, Latinos are ~2-fold more likely to be diagnosed with and to die of GC. The goal of our research program is to contribute to GC research in patients of Latino ancestry and advance cancer health disparity research. One of the research projects aims at identifying germline variants associated with GC risk. To do so, we are identifying novel gastric cancer genes and detecting mutations in previously reported GC genes, using whole-exome sequencing, in 500 patients who have been diagnosed with early-onset GC and/or who had GC and family history of cancer. Whole exome sequence data collection was recently completed and is being analyzed with the primary goals of estimating the prevalence and penetrance in known cancer genes as well as the identification of new GC genes. Using in-house exome data and publicly available genetic data, we will identify variants with predicted impact in the phenotype to design a panel that will be used for their detection in gastric preneoplasia biopsies. These variants will be tested using duplex sequencing, a highly sensitive method that can detect very rare mutations with ultra-depth sequencing. We will then select the variants with likely functional consequences for tumor initiation and/or progression to generate isogenic gastric organoids to investigate their effect in proliferation, differentiation, and gene expression. We aim to generate a body of work that can be used for risk assessment, prevention, early diagnostic, and that will lead to better outcomes for GC in this important U.S. minority. Dr. Ana Estrada-Florez is the recipient of the AACR Cancer Health Disparities Fellowship.

Citation Format: Ana Patricia Estrada-Florez, Paul Lott, Ted Toal, Nicole Halmai, Elizabeth Quino, Alma Poceros-Coba, Alix Guevara, Fabian Castro, Jhon Suarez, John Mcpherson, Rasika Venkatesh, Hongyong Zhang, Apri Vang, Guadalupe Polanco, Joana Guerra, Graciela Molina, Carol Parra, Adriana Della Valle, Esteban Castelao, Manuel Teixeira, Mabel Bohorquez, Javier Torres, Alejandro Carvajal Corvalan, Luis G. Carvajal-Carmona. Identification of predisposition and progression gastric cancer biomarkers in Latinos [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2274.
Abstract 2282: Efficient sample preparation with Covaris AFA® technology enhances confidence in Illumina® TruSight™Oncology 500 Assay
Authors: Bhattacharyya Debadeep , Pal Kasturi , Daviso Eugenio , Thomann Ulrich
Published: June 2022

High throughput and robust DNA fragmentation is a critical step in Next Generation Sequencing (NGS) for clinical genomics, ultimately ensuring higher quality for the final sequencing data. The typical requirements in NGS workflow (nucleic acid fragmentation, library preparation, sequencing, etc) comprise scalability, efficiency, reliability, and robustness. Failures to achieve confidence in any of the steps can negatively impact sequence data, rendering them unusable for diagnosis. Covaris’ Adaptive Focused Acoustics® (AFA®) technology enables efficient DNA fragmentation regardless of DNA concentration and starting molecular weight, ensuring a tight fragment size distribution, minimal sample loss, and most importantly, desired reliability and reproducibility.Illumina®’s TruSight™ Oncology 500 (TSO 500) protocol describes an enrichment-based approach to convert DNA and RNA extracted from formalin-fixed paraffin embedded (FFPE) tissue samples into libraries enriched for cancer-related genes that can be sequenced on Illumina® sequencing systems. The TSO 500 assay is optimized to prepare libraries from genomic DNA (gDNA) that is sheared to optimized fragment sizes and offers high fidelity determination of DNA variants across 523 cancer relevant genes. The TSO 500 assay is the assay of choice across most cancer centers using a standardized pipeline and therefore is suitable for routine use in precision oncology as a comprehensive genomic profiling solution. This study focuses on demonstrating the optimized DNA fragmentation protocol for TSO 500 assay with parallel 8-sample processing with an ML230 Focused-ultrasonicator.

Citation Format: Debadeep Bhattacharyya, Kasturi Pal, Eugenio Daviso, Ulrich Thomann. Efficient sample preparation with Covaris AFA® technology enhances confidence in Illumina® TruSight™Oncology 500 Assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2282.
Abstract 2307: Sequential treatment with abemaciclib plus endocrine therapy inhibits cell proliferation and triggers apoptosis in cell lines resistant to CDK4/6i
Authors: Zapatero-Solana Elisabet , Ganado Maria P. , Ortiz-Ruiz Maria J. , Mur Cecilia , Litchfield Lacey , Merzoug Farhana , Puig Oscar , Lallena Maria Jsoe
Published: June 2022

CDK4/6 inhibitors combined with endocrine therapy (ET) have shown clinical benefit in HR+, HER2- breast cancer. However, development of resistance highlights the need for treatment strategies to improve outcomes. To study resistance mechanisms, breast cancer cell lines were treated with CDK4/6i (abemaciclib or palbociclib) in combination with 4-OH-tamoxifen (tamoxifen) for 120-144h and sorted for resistant cells defined as geminin positive (GEM+), a marker of S/G2/M cell cycle accumulation. To confirm the resistant phenotype, cell lines were treated with tamoxifen plus the CDK4/6 inhibitor used to drive resistance. To understand if sequential CDK4/6i treatment is effective in controlling cell proliferation, resistant cells generated through treatment with palbociclib + tamoxifen or abemaciclib + tamoxifen were treated with abemaciclib + ET (fulvestrant or tamoxifen) or palbociclib + ET, respectively. Geminin/Ki67, annexin V and colony formation assays were performed to evaluate cell proliferation and viability. Molecular characterization by western blot and RNAseq analysis were utilized to provide insights into mechanisms of resistance and the effects of sequential treatment with CDK4/6i + ET. Cell lines resistant to palbociclib + tamoxifen subsequently treated with abemaciclib + ET showed decreased %GEM+ and colony formation ability, decreased Ki67 levels, and increased apoptosis. These effects were not observed in cell lines resistant to abemaciclib + tamoxifen following subsequent treatment with palbociclib + ET. Western blot analysis showed that palbociclib and abemaciclib-resistant cells had increased CDK6 and pERK levels, compared to control. Importantly, treatment of palbociclib-resistant cells with abemaciclib + ET decreased FOXM1, a key regulator of senescence and apoptosis. Cyclin A, a marker of mitosis, was also decreased, consistent with decreased %GEM+ subpopulation in palbociclib + ET resistant cells. These effects were not observed in abemaciclib-resistant cells treated with palbociclib + ET.In summary, our in vitro data provides mechanistic insights into resistance to combination CDK4/6i + tamoxifen and suggests the potential benefit of sequential treatment with abemaciclib + ET to overcome resistance in breast cancer.

Citation Format: Elisabet Zapatero-Solana, Maria P. Ganado, Maria J. Ortiz-Ruiz, Cecilia Mur, Lacey Litchfield, Farhana Merzoug, Oscar Puig, Maria Jsoe Lallena. Sequential treatment with abemaciclib plus endocrine therapy inhibits cell proliferation and triggers apoptosis in cell lines resistant to CDK4/6i [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2307.
Abstract 2312: Efficacy of targeted osmotic lysis is increased by cell cycle inhibition in M Phase
Authors: Edenfield Samantha , Gould Harry J. , Paul Dennis
Published: June 2022

Voltage-gated sodium channels (VGSCs) are upregulated in aggressive carcinomas, making these channels a target for novel clinical therapies. Targeted Osmotic Lysis (TOL) is a novel cancer therapy that combines pharmacologically blocking Na+,K+, ATPase (sodium pumps) while stimulating VGSCs with a pulsed electric field. The consequent increase in intracellular Na+ causes an osmotic lysis of the cells that overexpress VGSCs. Noncancerous cells, with fewer VGSCs do not lyse. We hypothesized that variability in efficacy of TOL is due to VGSC expression that varies across the cell cycle. We assessed cell surface expression of VGSCs and Na+,K+-ATPase during phases of the cell cycle in which there are single copies of DNA compared with phases in which DNA has doubled. For this, DNA was labeled with either propidium iodide or DAPI and VGSCs were labeled with a pan-specific VGSC antibody while Na+,K+-ATPases were labelled with a pan-specific Na+,K+-ATPase antibody in eight immortalized cancer and non-cancer cell lines. With flow cytometry we showed that VGSC expression doubled during phases of cell division (S-M phases) while Na+,K+-ATPase expression increased by 1.5-fold. To further elucidate the role of VGSCs and Na+,K+-ATPases throughout the cell cycle, S-Trityl-L-cysteine (STLC) was used to suspend the cell cycle in M phase. We hypothesized that treating cancer cells with TOL in the S-M phases would lead to increased cell death, compared to cells that were not suspended in M phase by STLC (G0-G1 phases). Cells were either treated with STLC or media without drug for 24 hours, then exposed to TOL treatment. The efficacy of the treatment was measured using a MTT assay revealing that cell death was greater in STLC-treated cells than control cells. By using cell cycle inhibitors, we may increase the efficacy of TOL for treating advanced carcinomas that overexpress VGSCs.

Citation Format: Samantha Edenfield, Harry J. Gould, Dennis Paul. Efficacy of targeted osmotic lysis is increased by cell cycle inhibition in M Phase [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2312.