bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 175: Determining the genetic alterations in initiation and progression of high-grade serous ovarian carcinoma
Authors: Phuong Daryl J. , Armstrong Amanda P. , Flesken-Nikitin Andrea , Nikitin Alexander Y. , Schimenti John C.
Published: April 2023

High grade serous carcinoma (HGSC) is the most common and deadly form of ovarian cancer. Although HGSC can arise from cells in the ovarian surface epithelium (OSE) or fallopian (oviductal) tubal epithelium (TE), the cell state and genetic alterations for HGSC initiation and progression remains unclear. Previous studies with cell type-specific mouse knockouts found inconsistencies in tumorigenesis and chemoresistance. These data can be explained if specific gene signatures in specific cell types have varied tumorigenic potential and chemoresistance. To determine specific gene combinations necessary for OSE and TE malignant transformation, we designed a CRISPR knock out (KO) mini-library against commonly mutated genes of HGSC. Our previous results revealed efficient gene combinations for ex vivo OSE transformation events and a preference for OSE stem cell transformation. To build on our previous studies, we extended our screening methods into TE cells. Our studies utilize an organoid approach to identify the most cancer-prone cell populations. They also test if cell differentiation state determines a specific set of genetic alterations necessary for HGSC initiation. Our screening platform has the potential to determine whether specific genetic alterations in specific cell types are required for malignant transformation. Additionally, our results may improve on previous models of HGSC initiation and progression.

Citation Format: Daryl J. Phuong, Amanda P. Armstrong, Andrea Flesken-Nikitin, Alexander Y. Nikitin, John C. Schimenti. Determining the genetic alterations in initiation and progression of high-grade serous ovarian carcinoma [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 175.
Abstract 1750: Removal of mucin glycans by Bifidobacterium bifidum enhances chemotherapy-mediated cell death in mucinous adenocarcinomas
Authors: Stripe Leah K. , Engevik Amy C. , Engevik Mindy A.
Published: April 2023

Background: Mucinous colorectal adenocarcinoma, a subtype of colorectal cancer, is characterized by mucin production and increased resistance to several chemotherapeutic interventions; resulting in a poorer prognosis compared to non-mucus secreting cancers. Advances in treatment are stymied by the limited number of efficacious chemotherapeutic agents. The most common drug used to treat colorectal cancer is 5-Fluorouracil (5-FU), but less than 10% of mucinous tumors respond to this drug. The production of mucus has been theorized to limit the uptake of chemotherapeutic agents via charge repulsion and size exclusion, thereby promoting chemo-resistance and driving poor outcomes.

Hypothesis: We predict that mucin-degrading microbes secrete glycosyl hydrolase enzymes which cleave mucin glycans and promote the uptake of chemotherapeutic agents and enhance cell death.

Methods & Results: We queried the human gut microbiota and identified multiple diverse microbes which encode enzymes to degrade mucin glycans. Among the mucin-degrading bacteria, we found that Bifidobacterium bifidum harbored one of the most extensive repertoires of mucin-degrading enzymes. B. bifidum was grown in a chemically defined medium with or without porcine intestinal mucus and the secreted products were then added to mucus-producing adenocarcinoma cell lines: HT29-MTX and T84 cells. Supernatant from cultures with mucus removed sialic acid residues and enhanced 5-FU uptake after 48 hrs in both cell lines. Supernatant without mucus also promoted 5-FU mediated cell death; although to a lesser degree than supernatant from cultures incubated with mucus. Gene expression analysis and immunostaining revealed decreased MUC13 gene expression and protein in B. bifidum metabolite treated cell lines compared to media controls. In contrast, we found that secreted products from B. dentium, which is unable to degrade mucus, had no effect on 5-FU induced cell death.

Conclusions: Our data suggests that B. bifidum elicits chemo-sensitivity via the production of mucin-degrading enzymes, which cleave negatively charged sialic acid, increasing the activity of 5-FU in these cancers as well as by lowering the expression of adherent mucins. This work points to a novel role of mucin-degrading microbes to promote 5-FU penetration of mucinous colorectal adenocarcinoma. This strategy has the potential to provide unique treatment strategies for patients with mucinous colorectal cancer.

Citation Format: Leah K. Stripe, Amy C. Engevik, Mindy A. Engevik. Removal of mucin glycans by Bifidobacterium bifidum enhances chemotherapy-mediated cell death in mucinous adenocarcinomas [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 1750.
Abstract 1752: Darolutamide reverses osimertinib resistance in non-small cell lung cancer
Authors: Schmitz John C. , Zhang Guojing , Liu Zhentao , Huang Yufei , Sica Gabriel , Sun Shi-Yong , Owonikoko Taofeek K.
Published: April 2023

Targeted therapies, such as EGFR inhibitors, have improved survival for patients with non-small cell lung cancer (NSCLC). Overall cure rates remain low due to eventual development of resistance. Osimertinib is a third-generation EGFR inhibitor that overcomes T790M-mediated resistance to earlier generation EGFRi. However, resistance to osimertinib has also emerged through mechanisms that are EGFR-dependent and EGFR-independent. To uncover novel strategies to overcome osimertinib resistance, we explored androgen receptor (AR) antagonists in combination with osimertinib as recent studies have demonstrated AR inhibition enhanced TKI-targeted therapy. For these experiments, we utilized lab-derived osimertinib-resistant PC9-AR and HCC827-AR NSCLC cell lines and matching parental lines in both in vitro and in vivo model systems. Clonogenic assays revealed that darolutamide in combination with osimertinib synergistically suppressed clonal growth in resistant cell lines. Similar results were observed with the combination of enzalutamide and osimertinib. Given this result, we next wanted to establish whether the promising in vitro activity of this combination translates into antitumor efficacy in vivo. Mice bearing either HCC827AR or PC9AR xenografts were treated orally daily with both drugs. At the doses administered, neither drug alone had antitumor activity. However, the combination significantly delayed tumor growth by more than 60%. We confirmed this ability to reverse resistance as a class effect using a different AR antagonist, enzalutamide in PC9AR-bearing mice. Significant tumor growth inhibition was observed with the combination of enzalutamide and osimertinib. To investigate the mechanism by which this combination may reverse osimertinib-resistance, RNA-seq studies were performed following drug exposure in vitro using parental and resistant cell lines under various experimental conditions. Comparison of gene expression profiles between parental and resistant cell lines under different therapeutic pressure and pathway enrichment analysis revealed important differentially expressed genes pointing plausible activated pathways that underline osimertinib resistance. AR antagonists have the potential to reverse EGFR inhibitor resistance in treatment of EGFR-mutant NSCLC.

Citation Format: John C. Schmitz, Guojing Zhang, Zhentao Liu, Yufei Huang, Gabriel Sica, Shi-Yong Sun, Taofeek K. Owonikoko. Darolutamide reverses osimertinib resistance 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 1752.
Abstract 1820: Efficacy and safety of the TLR7 agonist micelles MBS8(1V270) tested in pre-clinical models
Authors: Jensen Simon Skjøde , Christensen Esben , Winther Martin Bak , Hansen Anders Elias , Henriksen Jonas Rosager , Andersen Thomas Lars , Petersen Morten Just , Panina Svetlana
Published: April 2023

TLR7 agonists have shown efficacy in pre-clinical cancer models, however, their clinical use is restricted to topical treatment due to a high systemic toxicity. We have developed a micellar formulation of the TLR7 agonist MBS8(1V270) which demonstrated low toxicity and high efficacy in multiple murine cancer models when administered intravenously as monotherapy. In combination treatment, MBS8(1V270) showed a synergistic effect with PD-1 and PD-L1 inhibitors. Moreover, in several models resistant to anti-PD-1 treatment, MBS8(1V270) co-administration restored sensitivity to anti-PD-1 thus indicating potential for combination treatment in clinical setting. GLP toxicology studies in SD rats and cynomolgus monkeys did not reveal significant toxicities up to the human equivalent dose level of ~1.3 mg/kg which is above anticipated therapeutic dose. Additionally, ex vivo stimulation of human whole blood demonstrated significantly lower IL-6 induction by MBS8(1V270) as compared to the benchmark drug resiquimod. Tissue distribution and excretion of MBS8(1V270) was investigated in SD rats. No signs of drug accumulation were observed in any of the analyzed organs with nearly complete clearance of the drug 24 hours post-end of infusion. The major route of the MBS8(1V270) elimination was via faeces. Cellular uptake of MBS8(1V270) by human blood cells was investigated ex vivo using whole blood from healthy donors. The main cell populations taking up the drug were monocytes and B cells followed by NK cells. No MBS8(1V270) was detected in neutrophils or T cells.In conclusion, MBS8(1V270) was well tolerated in rodents and cynomolgus monkeys at dose levels above therapeutic effective doses identified in mice, thus providing a good therapeutic window. It showed significant anti-cancer activity in multiple murine tumor models when administered either alone or in combination with PD-1/PD-L1 inhibitors. Together, these data provide a rationale for clinical studies of MBS8(1V270) as monotherapy and in combination with immune checkpoint inhibitors. MBS8(1V270) is currently under clinical investigation in the clinical trial MBS8-101 (ClinicalTrials.gov identifier NCT04855435).

Citation Format: Simon Skjøde Jensen, Esben Christensen, Martin Bak Winther, Anders Elias Hansen, Jonas Rosager Henriksen, Thomas Lars Andersen, Morten Just Petersen, Svetlana Panina. Efficacy and safety of the TLR7 agonist micelles MBS8(1V270) tested in pre-clinical models [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 1820.
Abstract 1881: Pre-clinical evaluation of a novel antibody drug conjugate (ADC) LM-306 targeting IL-13Rα2 in immuno-oncology
Authors: Huang Wentao , Liu Zhifang , Li Yifan , Pan Heng , Li Yuan , Qin Xia , Fei Da , Li Runsheng
Published: April 2023

Interleukin-13 receptor alpha 2 (IL-13Rα2) is overexpressed in various types of cancers including glioma and melanoma and is known to be associated with increased tumor invasiveness and metastases. Here, we present the preclinical efficacy and toxicity results for a novel, anti-IL-13Rα2 ADC-LM-306. In vitro binding activity and internalization of LM-306 and LM-106 (unconjugated antibody of LM-306) were assessed in human IL-13Rα2 overexpressing and endogenous cell lines using flow cytometry. The antibody-dependent cell-mediated cytotoxicity (ADCC) and direct cytotoxicity were assessed using viability assays. Cross-species reactivity of drug was assessed in cells expressing cynomolgus, rat and mouse IL-13Rα2. In vivo A375 CDX model was used to assess the effect of LM-306 anti-tumor activity. The toxicity of LM-306 was evaluated in repeated-dose intravenous dose range-finding (DRF) toxicity and single intravenous maximum tolerated dose (MTD) study in cynomolgus monkeys. Both LM-106 and LM-306 showed concentration dependent binding affinity to IL-13Rα2 in various cell lines (Table 1). In addition, LM-106 also showed a dose dependent internalization and ADCC and LM-306 showed significant cytotoxicity in all cell lines (Table 1). Furthermore, LM-306 showed comparable binding affinity to human IL-13Rα2 to its unconjugated counterpart LM-106 (KD:7.43E-10 vs 1.77E-9). LM-306 was also found to be cross-reactive toward cynomolgus IL-13Rα2. In vivo, LM-306 showed tumor growth inhibition at a dose of 1, 3, 10 mg/kg given intravenously once weekly, indicating its strong anti-tumor activity. The highest non-severely toxic dose of LM-306 was determined to be 9 mg/kg in the DRF study and MTD was determined to be 18mg/kg in MTD study for male monkey. In summary, LM-306 showed high binding affinity to human IL13Rα2 expressing cells with potent cytotoxicity in vitro and significant in vivo inhibition of tumor growth, suggesting its therapeutic benefit in immuno-oncology.

Summary of parameters of LM-106 and LM-306 binding affinity, internalization, ADCC and cytotoxicity Assay Antibody EC50 (nM) HEK293/H_IL-13Rα2 U251 A375 SKMEL5 Binding affinity LM-106 0.99 0.21 0.57 0.1 LM-306 1.47 0.33 0.8 0.12 Internalization LM-106 2.22 - 0.43 - ADCC LM-106 0.16 - 0.03 - LM-306 0.31 - 0.03 - Cytotoxicity (IC50) LM-306 0.03 0.05 0.01 0.005

Citation Format: Wentao Huang, Zhifang Liu, Yifan Li, Heng Pan, Yuan Li, Xia Qin, Da Fei, Runsheng Li. Pre-clinical evaluation of a novel antibody drug conjugate (ADC) LM-306 targeting IL-13Rα2 in immuno-oncology [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 1881.
Abstract 1907: Unique and novel deleterious MSH6 mutation among African American patients with colorectal cancer
Authors: Rashid Mudasir , Rashid Rumaisa , Varma Sudhir , Sherif Zaki , Carethers John , Brim Hassan , Ashktorab Hassan
Published: April 2023

Background: Colorectal cancer (CRC) incidence and mortality in African Americans (AA) is higher compared to Caucasians (CAU). It has also been reported that the disparity is due to differences in the mutation spectrum particularly germline mutations in the DNA mismatch repair (MMR) genes (such as hMSH6). Aim: We aimed to determine MSH6 mutation profile in AAs with CRC.

Methods: We performed RNA sequencing in 8 paired CRC samples (AA, n=4 and CAU, n=4) to identify the unique transcript and novel pathogenic mutation in AA compared to CAU by comparing CRC matched to corresponding normal epithelia. In silico analysis of TCGA COAD RNA sequencing data was performed.

Results: We found non-synonymous germline mutations (p.G1139S) in exon 5 of MSH6 gene (one of the MMR genes) in AA CRC samples. Interestingly, this mutation is present in the active site of ATP binding (1135 to 11442 residue) motif of MSH6 protein. In silico analysis was used to determine the novelty and pathogenicity of this mutation by comparison to various publicly available databases. Also, the deleterious effect of p.G1139S missense mutation on the structure of MSH6 protein revealed that p.G1139S is destabilizing MSH6 protein. Based on Ramachandran plot analysis, we found lot of clash and outliers in the mutant type compared to wild type MSH6 protein. Structural analysis revealed that serine substitution results in a wide ATP binding site compared to glycine present in wild-type MSH6. In addition, we found a loss of hydrogen bonding or gain of ionic interaction between wild-type and mutant MSH6 protein.

Conclusion: Based on our in silico analysis, the deleterious effect of the detected missense mutation in the active site of ATP binding motif of MSH6 affects the structure and function of MSH6. This may result in the accumulation of mono-, or di-nucleotide repeats instability in the genome, leading to genomic alterations that associate with carcinogenesis in African American CRC patients.

Citation Format: Mudasir Rashid, Rumaisa Rashid, Sudhir Varma, Zaki Sherif, John Carethers, Hassan Brim, Hassan Ashktorab. Unique and novel deleterious MSH6 mutation among African American patients with colorectal 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 1907.
Abstract 191: CTC-derived organoids from liver and pancreatic cancer patients for personalized therapy
Authors: Galeano-Garces Catalina , Smith Kaylee J. , Heller Nicholas , Ahmadi Mahdi , Hong Jiarong , Parthasarathy Jayant , Drake Justin M.
Published: April 2023

Organoids are a widely used 3D culture system that has the potential to recapitulate the profile and phenotype of physiological cells. The culture of circulating tumor cells (CTCs) in this 3D system not only mimics the tumor physiological environment but allows researchers to test therapeutic responses to certain drugs based on a patient's own cancer phenotype. The purpose of our study is to perform an in vitro evaluation of patient-derived CTC organoids to more precisely identify personalized therapeutic options based on the response of these CTCs to each specified inhibitor. Using Astrin Bioscience’s proprietary CTC enrichment method, collected CTCs are plated as organoid cultures. The performance of our proprietary enrichment method has been assessed using both spiked cancer cell lines (in whole blood) and patient CTCs. We have successfully grown organoids from four prostate cancer cell lines and patient CTCs across multiple cancer types including pancreatic and liver adenocarcinoma. RNA sequencing of liver CTC organoids expressed EPCAM, a marker of CTC epithelial cells and coincided with data from a prostate epithelial cell line, LNCaP. This serves as robust evidence that our patient derived organoids truly originated as CTCs. Functional assays using different chemotherapies and targeted therapies will be applied to our organoid culture system. Drugs are uniquely selected for each patient based on cancer type, cancer stage and previous failed therapies. Efficacy of therapy is evaluated using imaging and cell viability assays to determine sensitivity or resistivity to each therapy. Transcriptomic and proteomic analyses will also be performed on these samples to aid in the identification of other predictive biomarkers and future novel therapy testing. Thus, this platform creates a pre-clinical drug screening tool to create a personalized guide to potential therapies for each patient.

Citation Format: Catalina Galeano-Garces, Kaylee J. Smith, Nicholas Heller, Mahdi Ahmadi, Jiarong Hong, Jayant Parthasarathy, Justin M. Drake. CTC-derived organoids from liver and pancreatic cancer patients for personalized therapy [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 191.
Abstract 1915: Cancer disparities in the University of Chicago Comprehensive Cancer Center Catchment Area
Authors: Zhang Stephanie , Ssepuuya Joel , Pierce Brandon
Published: April 2023

Disparities in cancer incidence and mortality have not been described for the University of Chicago Comprehensive Cancer Center’s patient catchment area (CA), the region from which the center draws its patients. The purpose of this research was to describe incidence and mortality rates (2013-2017) for leading cancers in the CA, which consists of Cook County, IL; DuPage County, IL; Lake County, IL; Will County, IL; and Lake County, IN. Age-adjusted rates (per 100,000) were calculated for leading cancers by sex for each county, the CA, U.S., and IL. Racial disparities were also examined by comparing Black rates to White rates. Incidence rates were higher in the CA compared to the U.S for all cancers data was available for: all sites combined, breast, and prostate. For males, mortality rates of prostate, colorectal, pancreatic, and stomach cancers were higher in the CA compared to the U.S. For females, mortality rates of breast, colorectal, myeloma, uterine, stomach, and bladder cancers, as well as all sites combined were higher in the CA compared to the U.S. By county, males experienced incidence rates 8% to nearly 2.5 times higher and mortality rates 3% to over 1.5 times higher compared to the U.S. for various cancers. By county, females experienced incidence rates 5% to over 4 times higher and mortality rates 4% to over 1.5 times higher compared to the U.S. for various cancers. Lake County, IN generally had the highest incidence and mortality rates. Racial disparities in incidence and mortality were present for most cancers in the CA overall and by county. For males, racial disparities in mortality for prostate, stomach, and larynx are of greatest concern. For females, racial disparities in mortality for myeloma, cervical, and uterine are of greatest concern. This research highlights important health inequities and priorities for the UChicago Cancer Center.

Citation Format: Stephanie Zhang, Joel Ssepuuya, Brandon Pierce. Cancer disparities in the University of Chicago Comprehensive Cancer Center Catchment Area [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 1915.
Abstract 1937: Racial differences of epigenome in normal breast tissue reveal biological pathways implicated in racial disparities in breast cancer
Authors: Lin Nan , Castle James , Liu Jinpeng , He Chunyan
Published: April 2023

Racial disparity in breast cancer is well recognized. Compared to European American (EA) women, African American (AA) women are more often diagnosed with breast cancer at younger ages, have a more advanced or aggressive disease, and have poorer outcomes. Increasing evidence suggests that biological differences between EA and AA women, particularly differences in epigenetic changes of DNA methylation (DNAm), may contribute to the observed cancer disparity. We hypothesize that racial differences in DNAm in normal breast tissue may reflect distinct genomic susceptibility to breast cancer in different racial groups. Therefore, we investigated epigenome-wide differences of DNAm in normal breast tissue between 178 EA and 272 AA women using the Illumina TruSeq Methyl Capture EPIC library and NGS technology. We identified 17,340 differentially methylated CpG loci (DMLs) between EA and AA women, of which 7,744 were hypermethylated in AA women and 9,596 were hypermethylated in EA women. Pathway analyses of genes annotated to these racial DMLs suggested enrichment of biological processes including cell movement, cell motility, and cell migration, features that are likely linked to the aggressiveness and metastatic potentials of tumor cells. We further examined racial differences in DNAm of breast cancer-related candidate genes and GWAS-identified breast cancer risk loci. Approximately 30% of candidate genes were found to have racial differences in DNAm at more than three CpG sites and these candidate genes were enriched for pathways related to response to stimulus and anatomic structure development. Most interestingly, racial DMLs were found to be enriched for breast cancer risk loci that are associated with risk of estrogen-receptor negative (ER-) breast cancer, treatment response to aromatase inhibitor, and breast cancer-specific morality, phenotypes of which we observed differences between EA and AA breast cancer patients. Our results suggest that racial differences in DNAm in normal breast tissue may induce different biological processes that lead to distinct clinical features of breast tumors observed in EA and AA women and provide an epigenetic mechanism underlying the observed racial disparities. Further research is need to validate the findings of this study.

Citation Format: Nan Lin, James Castle, Jinpeng Liu, Chunyan He. Racial differences of epigenome in normal breast tissue reveal biological pathways implicated in racial disparities in 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 1937.
Abstract 195: Establishment and validation of pheochromocytoma organoids for high-throughput drug screening
Authors: Calucho Maite , Cheng ZiMing , Nguyen Huyen Thi-Lam , Shihabi Ahmad Al , Gonzalez-Cantu Hector , Guo Qianjin , Thaker Maneesha , Bechmann Nicole , Eisenhofer Graeme , Ding Yanli , Dahia Patricia , Soragni Alice
Published: April 2023

Pheochromocytomas and paragangliomas (PPGL) are rare catecholamine-secreting neuroendocrine tumors known for their genetic diversity. They can be categorized in discrete molecular subgroups, including tumors presenting with pseudohypoxia or kinase signaling activation. Metastatic and recurrent PPGLs have few therapeutic options, in part due to the lack of appropriate study models. Here, we report progress on the establishment of viable organoids from n=10 distinct sporadic or familial PPGLs spanning varios genotypes. We also perform detailed histological, molecular, biochemical, and functional characterization. The ten PPGLs were obtained from patients with distinct clinical history and diverse ethnic and genetic backgrounds. We show how we can generate PPGL organoids from fresh and frozen tissue in a format compatible with histologic characterization and high-throughput drug screening (mini-rings, Phan et al, 2019, Al Shihabi et al, 2022). Our mini ring approach uses fewer cells with no need for expansion in vitro, and is amenable to implement automation for facile high-throughput studies. We can quantify PPGL growth in culture using a machine learning-based pipeline (Al Shihabi et al, 2022). All PPGL organoids for which sufficient number of cells could be obtained grew, regardless of the genotype and molecular subtype. PPGL organoids could be cultured short term (6 days) as well as long term (4 weeks). We investigated cellular composition by staining for the neuroendocrine marker chromogranin A, the sustentacular cell marker S100, and the vascular marker CD34. We also analyzed the expression of catecholamines in the cell culture supernatant by LC/MS/MS. We confirmed that secreted catecholamines matched the primary tumor pattern. Lastly, we present the results of pilot drug screening on both short and long term cultures. Drug sensitivity profiles pinpoint tumor-specific responses. In conclusion, we can establish PPGL organoids that largely recapitulate features of the tumor of origin. These models will provide the ability to investigate tumor initiation and progression for various genetic backgrounds and may reveal novel patterns of drug sensitivity and resistance.

Citation Format: Maite Calucho, ZiMing Cheng, Huyen Thi-Lam Nguyen, Ahmad Al Shihabi, Hector Gonzalez-Cantu, Qianjin Guo, Maneesha Thaker, Nicole Bechmann, Graeme Eisenhofer, Yanli Ding, Patricia Dahia, Alice Soragni. Establishment and validation of pheochromocytoma organoids for high-throughput drug 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 195.
Abstract 1988: FAP-targeted non-internalising drug conjugates: A comparative evaluation between small molecules, antibodies and peptides
Authors: Zana Aureliano , Moreno Claudia Puig , Gilardoni Ettore , Neri Dario , Cazzamalli Samuele
Published: April 2023

Conventional cancer chemotherapy relies on the use of cytotoxic drugs which are not designed and capable of selective accumulation to neoplastic lesions. The conjugation to tumour-targeting delivery vehicles has been proposed as a strategy to enhance the therapeutic index of potent cytotoxic drugs. To date, fifteen Antibody-Drug Conjugate products have received marketing approval for the treatment of solid and liquid human malignancies. We present an in vivo comparative evaluation of ADCs against products based on small organic delivery vehicles (Small Molecule-Drug Conjugates, or SMDCs) or on tumour-targeting peptides (Peptide-Drug Conjugates, or PDCs). All products are designed to target and to be activated by Fibroblast Activation Protein in the tumour microenvironment. Our results reveal that both targeted ADC and SMDC products mediate potent anti-cancer activity and complete tumour remission in a FAP-positive preclinical murine model of cancer, when administered at the same molar dose. Non-targeted albumin and antibody conjugates included in our comparative studies delayed tumour growth but presented suboptimal anti-cancer efficacy in the same cancer model. Our results indicate that both FAP-targeted ADCs and SMDCs are promising product candidates for the treatment of multiple human malignancies.

Citation Format: Aureliano Zana, Claudia Puig Moreno, Ettore Gilardoni, Dario Neri, Samuele Cazzamalli. FAP-targeted non-internalising drug conjugates: A comparative evaluation between small molecules, antibodies and peptides [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 1988.
Abstract 1992: P-selectin-targeted delivery of intracranial therapeutics across an intact BBB in Sonic Hedgehog medulloblastoma
Authors: Heller Daniel A. , Tylawsky Daniel , Raju G. Praveen
Published: April 2023

Medulloblastoma (MB) is the most common malignant pediatric brain tumor with ~30% mediated by Sonic hedgehog (SHH) signaling. While SHH effector Smoothened inhibition is promising, therapeutic index is reduced by high systemic doses required for sufficient intracranial drug concentrations. Treatment-related toxicities associated with radiation therapy result in lifelong morbidity, and SHH pathway inhibition in children notably results in permanent bone defects. Here, we found that nanoparticle-mediated P-selectin targeting on tumor vasculature induces caveolin-1-dependent transcellular passage across the blood-brain barrier. A vismodegib nanocarrier that targets P-selectin exhibited striking efficacy and markedly reduced both bone-related toxicities and drug exposure to healthy brain. These findings demonstrate a potent strategy and novel mechanism for targeted intracranial pharmacodelivery that overcomes the restrictive endothelial barrier to achieve enhanced tumor-selective penetration and has therapeutic implications for other diseases within the central nervous system.

Citation Format: Daniel A. Heller, Daniel Tylawsky, G. Praveen Raju. P-selectin-targeted delivery of intracranial therapeutics across an intact BBB in Sonic Hedgehog medulloblastoma [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 1992.
Abstract 2036: Distinct mutational processes shape selection of MHC class I and class II mutations across primary and metastatic tumors
Authors: Mumphrey Michael , Hosseini Noshad , Parolia Abhijit , Geng Jie , Zou Weiping , Raghavan Malini , Chinnaiyan Arul , Cieslik Marcin
Published: April 2023

Disruption of antigen presentation via loss of MHC expression is a strategy whereby cancer cells escape immune surveillance and develop resistance to immunotherapy. We developed the personalized genomics algorithm Hapster and accurately called somatic mutations within the MHC genes of 10,001 primary and 2,199 metastatic tumors, creating a catalog of 1663 nonsynonymous mutations that provide key insights into MHC mutagenesis. We found that MHC-I genes are among the most frequently mutated genes in both primary and metastatic tumors, while MHC-II mutations are more restricted. Recurrent deleterious mutations are found within haplotype and cancer-type specific hotspots associated with distinct mutational processes. Functional classification of MHC residues revealed significant positive selection for mutations disruptive to the B2M, peptide, and T-cell binding interfaces, as well as MHC chaperones. At the cohort level, all cancers with positive selection for MHC mutations are responsive to immune checkpoint inhibitors, underscoring the translational relevance of our findings.

Citation Format: Michael Mumphrey, Noshad Hosseini, Abhijit Parolia, Jie Geng, Weiping Zou, Malini Raghavan, Arul Chinnaiyan, Marcin Cieslik. Distinct mutational processes shape selection of MHC class I and class II mutations across primary and metastatic tumors [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 2036.
Abstract 2041: Research strategies for rare germline variants and cancer study
Authors: Kim Seokhyeon , Koh Youngil , Yoon Sung-Soo
Published: April 2023

Exploration of rare germline variants in cancer is the fields of ongoing research. So, we would like to suggest several considerations for the rare pathogenic germline variants studies. We confirmed the pathogenicity of each variant using ClinVar (https://www.ncbi.nlm.nih.gov/clinvar). In addition, we utilized in silico prediction tools such as Polyphen and SIFT. Even for the same variant, the report of the variant may vary depending on the database used. We conducted rare germline variants screening using ICGC (International Cancer Genome Consortium) PCAWG (Pan-Cancer Analysis of Whole Genomes) data. we filtered allele frequencies using the gnomAD (v2.1.1) exome database. We utilized several ClinVar versions to check the clinical significance of germline variants. Next, we have investigated some in silico tools that predict ClinVar pathogenic and benign variants well. We observed inconsistent variant reports across versions of ClinVar. Although the information on variants may change over time, there have been inconsistent changes in the clinical significance of variants reported as P (Pathogenic) or LP (Likely pathogenic). The clinical significance of the variants reported in the previous version was P or LP, but those in the updated version were changed to “Conflicting interpretations of pathogenicity”. It was confirmed that the variant report changes when at least one “Uncertain Significance” review is added. Among various prediction tools, we found that the predictive power of a tool called “MetaRNN” was useful. Compared with several tools, we confirmed that MetaRNN’s AUC (Area Under the ROC Curve) value was 0.985, which is the best performance. But, it is not enough to judge the pathogenicity of a variant only by the prediction result of the tool. And, prediction tools are also known to be more efficient in predicting missense variants than others. Therefore, it is necessary to examine the pathogenicity of germline variants using both the prediction tool and the clinical significance of ClinVar. In summary, for rare germline variants in cancer studies, it is necessary to observe variants compared to the previous versions of ClinVar. Moreover, if prediction tools are appropriately used, researchers will increase the possibility of finding a variant or gene with more crucial clinical significance to reveal the association between the rare germline variants and cancer development.

Citation Format: Seokhyeon Kim, Youngil Koh, Sung-Soo Yoon. Research strategies for rare germline variants and cancer study [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 2041.
Abstract 2183: Absolute quantification of PD-L1 gene expression in single cell on tissue for accurately predicting response to ICIs
Authors: Gerstenberg Amandine , Ponzoni Adele , Ramos Corinne , Stauber Jonathan
Published: April 2023

Background PD-L1 immunohistochemistry (IHC) has been traditionally used for predicting clinical responses to immune checkpoint inhibitors (ICIs). However, there are at least 4 different assays and antibodies used for PD-L1 IHC, each developed with a different ICI. Spatial transcriptomic analysis at the single cell level can be helpful for deeply characterize the complexity and heterogeneity of tumors. We set to test if in-situ hybridization-based RNA scope coupled to single cell quantification is a robust method to determine PD-L1 mRNA expression levels in each cell and furthermore efficacy of predicting response to ICIs as compared to routinely used standardized IHC procedures. Method FFPE sections from retrospectively collected surgically resected NSCLCs (adenocarcinoma and squamous cell carcinoma) tumor with progression disease or complete response from adjuvant pembrolizumab therapy were used. Target genes CD274 (PD-L1) and GZMB (Granzyme B) were assessed. For fluorescent detection, the label probe was conjugated to Opal 570 and 620 respectively. Tissues cells were counterstained with DAPI to detect the nuclei. A Digital pathology software QuPath was used for cell segmentation and dot’s detection for gene expression quantification and further classification. Results Difference in expression levels were seen between PD-L1 and GZMB across the tumor with PD-L1 staining mostly located in the tumoral area and Granzyme B in stromal area of the NSCLC tissue. This is consistent with the expected expression of PD-L1 on tumor epithelial cells and Granzyme B on T cells. This heterogeneity in the signal distribution specific to the analyzed tissue correspond to intra-tissue cells metabolism heterogeneity between cell types. This heterogeneity of expression was captured though a digital workflow capable of quantifying the number of cells expressing PD-L1 and GZMB. Among the cells expressing the markers, 96% were positive for PD-L1 for the tumor that did not respond to therapy with the threshold of positivity being set at 1 of dots, that corresponds to 1 transcript, per cell.

Conclusion Our results demonstrate that PD-L1 was highly expressed in a tumor context that was not responding to PD-1 ICI inhibitor. Measurement of mRNA expression at the single cell level on tissue provides another layer of PD-L1 detection which can be exploited to predict tumor response to ICI suggesting that moving forward with this technology is a viable approach for this dynamic biomarker.

Citation Format: Amandine Gerstenberg, Adele Ponzoni, Corinne Ramos, Jonathan Stauber. Absolute quantification of PD-L1 gene expression in single cell on tissue for accurately predicting response to ICIs [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 2183.
Abstract 2192: Potential of galectin-7 as differentiating biomarker of lung cancer subtypes
Authors: Funkhouser Avery T. , Shealy Jonah C. , Kesic Alexandra E. , Blair Bailey B. , Martin Julie C. , Arthur Connie M. , Funk Christopher R. , Edenfield W. Jeffery , Blenda Anna V.
Published: April 2023

Background. Lung cancer is the leading cause of cancer-related death in both men and women in the United States. Non-small Cell Lung Cancer is the most prevalent and includes adenocarcinoma (AD), and squamous cell lung cancers. The unique cellular biology of these cancers could potentially allow for serological detection and identification via tissue specific protein markers. Galectins are a group of β-galactoside binding proteins which are known to be alternatively expressed in cancers and are being studied for their biomarker potential. Galectin-7 is expressed in epithelial tissues as well as in all layers of the epidermis as it is normally highly expressed by squamous cells. Purpose. We are evaluating potential of galectin-7 to be used as a serum biomarker and delineator of several lung cancer subtypes.

Methods. The serum concentrations of galectin-7 were measured in 96 cancer patients using ELISA. Twenty-eight samples were from adenocarcinoma lung cancer patients, 25 were from squamous cell lung carcinomas patients, 19 were from all other types of lung cancer, and 28 were from other squamous cell carcinomas of non-pulmonary origin. The serum galectin-7 concentrations in cancer patients were compared amongst themselves by their histological subtypes. One-way analysis was performed using Student’s t-test for pairwise comparisons.

Results. Galectin-7 concentrations were significantly elevated in serum samples from squamous cell lung cancer patients (n=25; mean, 1.91 ng/mL) relative to adenocarcinoma lung cancer patients (n=24; mean, 1.02; p-value, 0.0149). Additionally, levels of galectin-7 in samples of squamous cell lung carcinomas were higher than samples of squamous cell carcinomas of non-pulmonary origin (n=28; mean, 0.33 ng/mL, p-value, <0.0001). These results show that serum galectin-7 is increased in patients with squamous cell lung cancer compared to adenocarcinoma lung cancer.

Conclusion. Potential clinical applications of this study include the measurement of galectin-7 in blood samples of cancer patients to aid in cancer screening and tumor type identification. Further explorations comprise measuring serum concentrations of galectin-7 by stage of squamous cell lung cancer and histological subtype (keratinizing, non-keratinizing, and basaloid) to determine a more nuanced understanding of the tumor molecular biology.

Citation Format: Avery T. Funkhouser, Jonah C. Shealy, Alexandra E. Kesic, Bailey B. Blair, Julie C. Martin, Connie M. Arthur, Christopher R. Funk, W. Jeffery Edenfield, Anna V. Blenda. Potential of galectin-7 as differentiating biomarker of lung cancer subtypes [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 2192.
Abstract 2276: Spatiotemporal analysis of metastatic melanoma reveals mechanisms of resistance to immune checkpoint blockade
Authors: Wei Shiyou , Lee Jinho , Labrie Marilyne , Betts Courtney , Liu Lunxu , Coussens Lisa , Herlyn Meenhard , Boland Genevieve , Mills Gordon , Zhang Gao
Published: April 2023

The treatment of metastatic melanoma has been revolutionized with the introduction of immune checkpoint blockade (ICB) therapies in the last decade. Despite durable responses exhibited by a subgroup of patients, most of patients do not respond to ICB therapies and the vast majority (60%-90%) of patients experience disease progression within five years. Molecular mechanisms of primary and acquired resistance to ICB are still widely unknown, which warrants further mechanistic investigation. Towards that goal, we used a holistic approach driven by integrated genomic tools to molecularly profile 35 longitudinal tumor specimens at pre-, on-, and post-treatment time points which were derived from seven patients with metastatic melanoma who progressed on sequential ICB therapies, including three responders and four non-responders. We integrated analyses of data generated from RNA sequencing, Whole-exome Sequencing (WES), NanoString nCounter vantage 3D, Cyclic Immunofluorescence (CyCIF), NanoString Digital Spatial Profiling (DSP) and Multiplex Immunohistochemistry (mIHC) platforms to elucidate evolutionary trajectories that occurred in both the tumor and the tumor immune microenvironment (TiME) that may have orchestrated response and resistance to ICB therapies. Simultaneous analyses conducted on genomic, transcriptomic and proteomic levels identified the re-activation of the MAPK pathway as a potential mechanism of resistance to ICB therapies. Spatially-resolved, image-based immune monitoring analysis at single cell resolution revealed that response to ICB is associated with infiltration of immune cells accompanied by induced activities of myeloid, T cells and macrophages in the TiME. In summary, integrated analyses allow us not only to reveal the dynamic co-revolution of both tumor and immune cells in TiME following sequential ICB therapies but also to provide a comprehensive perspective to molecular mechanisms of response and resistance to ICB therapies. A deeper understanding and elucidation of the evolution of mechanisms of response and resistance to ICB therapies will point us to generate hypothesis-driven, potential salvage therapies to overcome resistance to ICB therapies.

Citation Format: Shiyou Wei, Jinho Lee, Marilyne Labrie, Courtney Betts, Lunxu Liu, Lisa Coussens, Meenhard Herlyn, Genevieve Boland, Gordon Mills, Gao Zhang. Spatiotemporal analysis of metastatic melanoma reveals mechanisms of resistance to immune checkpoint blockade [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 2276.