bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2046: Identification of the cuproptosis-related gene signature associated with the tumor environment and prognosis of patients with glioblastoma multiforme (GBM)
Authors: Ahmed Yaman B. , Al-Bzour Ayah N. , Al-Majali Ghayda'a N. , Khalefa Zaid M. , Alzghoul Saja M.
Published: April 2023

Background: Glioblastoma multiforme (GBM) is the most common tumor of the central nervous system with poor prognosis. Cuproptosis is a novel programmed cell death pathway targeting lipoylated tricarboxylic acid cycle proteins. Previous studies have found that it participates in tumor progression, but its role in GBM is still elusive. In this study, we aimed to develop a cuproptosis gene-signature risk score using bioinformatics analysis and machine learning.

Methods: We acquired transcriptomic and clinical information of GBM patients from The Cancer Genome Atlas (TCGA). A total of 2283 differentially expressed genes (DEGs) were obtained from the GEPIA2 database. 26 cuproptosis-related genes (CRGs) were retrieved from literature. A correlation analysis between the 26 CRGs and the DEGs were conducted to retrieve the cuproptosis-related DEGs. Then, a univariate cox analysis was conducted to obtain the prognostic-related DEGs for overall survival (OS). The least absolute shrinkage and selection operator (LASSO) were conducted for regularization and the gene risk score was constructed using the multivariate cox coefficients.

Results: A total of 731 downregulated DEGs were correlated with CRGs, while 68 upregulated DEGs were correlated with CRGs and were further screened for prognostic value using the univariate cox analysis. A total of 70 prognostic related CRGs were identified and were further screened using the LASSO cox analysis. After multivariate cox analysis, a total of seven genes were significantly associated with survival (p-value<0.01). A risk-score gene signature was constructed from the cox coefficients multiplied by the expression of the following genes: -0.0012*DPP10+0.0021*EGR4+0.0015*ITPKA+ 0.0003* PTPRN+ 0.0007* STEAP2+ 0.0006* TENM2+- 0.0017* ZNF540.

Conclusion: Univariate and multivariate Cox regression analyses showed the CRGs-based prognostic signature independently functioned as a risk factor for OS in GBM patients. Furthermore, our results gave a promising understanding of cuproptosis in GBM, as well as a tailored prediction tool for prognosis and immunotherapeutic responses in patients.

Citation Format: Yaman B. Ahmed, Ayah N. Al-Bzour, Ghayda'a N. Al-Majali, Zaid M. Khalefa, Saja M. Alzghoul. Identification of the cuproptosis-related gene signature associated with the tumor environment and prognosis of patients with glioblastoma multiforme (GBM) [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 2046.
Abstract 2194: Performance of a 24-hour turnaround, guideline-complete NSCLC assay, optimized for sparse samples
Authors: Cabrera Kerri , Gole Jeff , Leatham Bryan , Regoli Isabel , Martinez Tiffany , Richards Andrew , Herdt Leah , Enciso Juan , Berroteran Paige , Carolan Heather , Brown Brad
Published: April 2023

Non-Small Cell Lung Cancer (NSCLC) is the most common type of lung cancer with 236,740 new cases diagnosed in 2022 in the United States. In this study, we demonstrate the performance of a digital PCR (dPCR) based assay that reports 15 (NCCN) biomarkers using only 1 slide of FFPE tissue. ChromaCode’s High Definition PCR (HDPCR™) NSCLC Tumor Profiling Panel (Research Use Only) detects guideline biomarkers including variants for EGFR, BRAF, KRAS, ERBB2, ALK, ROS1, RET, MET, and NTRK1-3 with a 24 hour turnaround time and a simple PCR workflow. A total of 34 NSCLC FFPE specimens were evaluated using the HDPCR NSCLC Panel. DNA and RNA were extracted from a single 10 µm section of specimen, with a sister section previously characterized with the Oncomine Precision Assay GX. The DNA/RNA eluates (20ng DNA per well, 20ng RNA) were run with the HDPCR NSCLC Panel on the Qiagen QIAcuity dPCR instrument. Data analysis was conducted using ChromaCloud™, which includes a proprietary algorithm that can detect and quantify 6 targets (5 targets and 1 internal control) for each of the 3 wells. DNA samples discordant with the comparator assay were evaluated using the Archer DNA VariantPlex panel, while RNA discordant samples were analyzed using the Archer RNA fusion plex panel. Of the 34 specimens evaluated, 16 SNV, 13 Insertion/Deletion, and 7 fusion positive specimens were identified using NGS. On the dPCR assay, the results show a high-level of agreement, after discordant resolution, of 95.8% across the panel. The minor allele fraction (MAF), also calculated using ChromaCode Cloud, ranged from 5% to 90%. With 20 ng input (Qubit), the average copy number of amplifiable DNA ranged from 791 copies (~2.3 ng) to 6583 copies (~19.9 ng) with a mean of 2410 copies (~7.43 ng). Here we demonstrate that the HDPCR NSCLC Tumor Profiling Panel provides comparable performance to validated NGS assays with a turnaround that enables adherence to guideline-recommended treatment, and a low sample requirement that maximizes the number of patients for molecular testing. The standard protocols used by the assay and the use of a cloud based analysis pipeline enables easy deployment of this assay in any lab.

Citation Format: Kerri Cabrera, Jeff Gole, Bryan Leatham, Isabel Regoli, Tiffany Martinez, Andrew Richards, Leah Herdt, Juan Enciso, Paige Berroteran, Heather Carolan, Brad Brown. Performance of a 24-hour turnaround, guideline-complete NSCLC assay, optimized for sparse samples [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 2194.
Abstract 2199: Novel urinary marker for diagnosis and prognosis of neuroblastoma
Authors: Hinoki Akinari , Amano Hizuru , Uchida Hiroo , Narita Atsushi , Kumano Shun , Abe Mayumi , Sakairi Minoru , Shirota Chiyoe , Sumida Wataru , Makita Satoshi , Takahashi Yoshiyuki , Osawa Tsuyoshi
Published: April 2023

Background and Aim: Urinary homovanillic acid (HVA) and vanillylmandelic acid (VMA) concentrations are widely used for diagnosis of neuroblastomas, which are catecholamine-secreting tumors. As recent advances in mass spectrometry have facilitated the measurement of metabolites present at extremely low concentrations, urinary metabolites with better diagnostic or prognostic utility for detection of neuroblastomas could be identified. This study utilized quantitative analyses using liquid chromatography-mass spectrometry (LC-MS) to evaluate the diagnostic and prognostic performance of a novel biomarker candidate: 3-methoxytyramine sulfate [3-MTS].

Materials and Methods: The concentrations of the three urinary metabolites (HVA, VMA, and 3-MTS) in urine samples of 41 patients with primary neuroblastoma and urine samples of 155 control participants were measured using LC-MS. The cut-off values for these metabolites were the mean + 2 standard deviation values of the control. Linear discriminant analysis was used to evaluate the association of urinary marker candidates with unfavorable risk factors such as the International Neuroblastoma Pathology Classification and MYCN amplification.

Results: The sensitivity and specificity were as follows: 3-MTS 90.2%, 94.2%; HVA 87.8%, 96.1%; VMA 87.8%, 96.1%; VMA or HVA+3-MTS 97.6%, 92.3%; HVA+VMA 87.8%, 94.8%; respectively. 3-MTS could differentiate between the cases with favorable and unfavorable histology (AUC =0.950). 3-MTS+VMA differentiated between the cases with MYCN amplification and nonamplification (AUC=0.952).

Conclusions: 3-MTS might be a useful urine marker for both diagnosis and prognosis of neuroblastoma.

Citation Format: Akinari Hinoki, Hizuru Amano, Hiroo Uchida, Atsushi Narita, Shun Kumano, Mayumi Abe, Minoru Sakairi, Chiyoe Shirota, Wataru Sumida, Satoshi Makita, Yoshiyuki Takahashi, Tsuyoshi Osawa. Novel urinary marker for diagnosis and prognosis of neuroblastoma [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 2199.
Abstract 2202: Role of FadA secreting Fusobacterium nucleatum in initiation and progression of colorectal cancer
Authors: Huang Angela , Saxena Anjana , Patel Mintoo
Published: April 2023

Colorectal cancer (CRC) is a global disease with poor prognosis. There is an alarming increase in incidence of CRC in younger patients along with well recognized racial disparities. Recent data implicate a role of microbiome in initiation and progression of CRC. Fusobacterium nucleatum (Fn), an oral opportunistic pathogen, has emerged as one of the common pathogens associated with CRC. Fn transformation from commensal to pathogen requires expression of amyloid-like FadA adhesin that plays a key role in its attachment to the colonic mucosal cells resulting in inflammatory, oncogenic, and metastatic changes. CRC initiates as polyps that go on to invade the mucosal layers and metastasize. This study analyzed the significance of Fn as a biomarker in CRC as well as to understand the etiology and metastasis of CRC. Deidentified and delinked formalin fixed paraffin embedded tissue sections from cancerous, precancerous and non-cancerous colorectal biopsies were analyzed for Fn presence and markers in epithelial to mesenchymal transition (EMT) in CRC through immunofluorescence. EMT was studied through biomarkers such as E-cadherin, which is an epithelial cell surface protein, vimentin as an intracellular filament protein normally expressed in mesenchymal cells, and N-cadherin, a cell adhesion molecule that is shown to express aberrantly in some solid tumor cells undergoing EMT. As expected, E-cadherin was abundantly expressed on the surface of epithelial cells while vimentin was found in the stromal cells in the non-cancerous tissue sections. Interestingly, the E-cadherin expression was lower in the cancerous tissue and some cancerous specimens also exhibited the vimentin positive foci within epithelial cells signifying partial EMT. N-cadherin expression was not observed in any of the specimens. The presence of Fn as analyzed through the FadA staining correlated with decline in the E-cadherin expression in some cancer specimens. To further analyze the significance of Fn, a meta-analytic study was conducted from 20 studies that indicated a significant presence of Fn not only in CRC tissues but also in fecal specimens from patients with CRC. A whole genome sequencing analysis corroborated with the abundance of Fn in CRC tissue specimens. These data suggest that analysis for Fn presence offers an attractive opportunity for intervention and targeted treatment for chemo resistant tumors. EMT analysis provides an additional corroboration in early detection of cancer progression which is pivotal in successful treatment of CRC.

Citation Format: Angela Huang, Anjana Saxena, Mintoo Patel. Role of FadA secreting Fusobacterium nucleatum in initiation and progression of 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 2202.
Abstract 2249: Development and validation of a pharmacodynamic (PD) assay for TOS-358, the first covalent inhibitor of PI3Kα in clinical development
Authors: MacDougall John R. , Mak Raymond , Bradley John , Simpson Allison , May Claire , Wei Lan , Ouellette Kendra , Olbrot Marty , Blair Jimmy , Dhawan Neil , Chen Wei
Published: April 2023

TOS-358 is a first-in-class covalent inhibitor of PI3Kα and is currently in clinical development in a variety of solid malignancies. We have developed a fit-for-purpose pharmacodynamic assay to evaluate the target occupancy of TOS-358 in in vitro and in vivo setting at multiple timepoints. The PI3Kα assay allows direct measurement of both occupied and total protein in multiple different samples and tissue types. Signals of occupied and unoccupied PI3Kα proteins can be readily measured in cellular and in vivo samples. The assay was utilized in a large panel of cancer cell lines, revealing a direct correlation between target occupancy and pathway inhibition of markers such as phosphoAKT and phosphoS6. Critically, these correlations revealed the need to achieve near complete suppression of PI3Kα to enable sustained pathway inhibition and cancer cell death. We furthermore utilized this assay to explore the turnover rate of PI3Kα across multiple diverse tumor types, mutations, and tissues. This approach enables highly sensitive target engagement analysis of TOS-358 across multiple formats.

Citation Format: John R. MacDougall, Raymond Mak, John Bradley, Allison Simpson, Claire May, Lan Wei, Kendra Ouellette, Marty Olbrot, Jimmy Blair, Neil Dhawan, Wei Chen. Development and validation of a pharmacodynamic (PD) assay for TOS-358, the first covalent inhibitor of PI3Kα in clinical development [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 2249.
Abstract 2250: Immuno-oncology study to profile the tumor microenvironment in multiple human cancers using high-plex imaging mass cytometry
Authors: Pfister Thomas D. , Lim Liang , Ouladan Shaida , Zabinyakov Nick , Raza Qanber , Loh Christina
Published: April 2023

Immune profiling of tumor tissues has become a key tool in understanding the complexity of the tumor microenvironment (TME), for predictive biomarker discovery, and in cancer treatment. The presence of tumor-infiltrating lymphocytes has been associated with benefit from therapy. Furthermore, the TME also contains immunosuppressive elements that can impede immune response. Imaging Mass Cytometry™ (IMC™) enables detailed assessment of cell phenotype and function using 40-plus markers simultaneously at subcellular resolution on a single slide without spectral overlap or background autofluorescence. High-plex IMC has enabled us to evaluate the TME in different cancer histologies including in highly autofluorescent tissue types like lung, hepatocellular carcinoma, and skin melanoma.

The Maxpar® Human Immuno-Oncology IMC Panel Kit (201508) was customized using antibodies from the Standard BioTools™ catalog to create panels for tissue-based immuno-oncology research. Data acquisition was performed using a Hyperion™ Imaging System. To facilitate cell segmentation, an IMC Cell Segmentation Kit (TIS-00001) was applied to enhance cell membrane boundaries. We applied a pixel classification approach and CellProfiler™ for single-cell segmentation. We used histoCAT™ for single-cell analysis to visualize protein expression in various cancer types via PhenoGraph clustering and t-SNE maps.

Our panels were applied to normal and cancer human tissue microarrays (TMAs) to phenotype and analyze cell populations in these tissues. We provide detailed analysis of the TME by classifying activation state of immune cell populations, epithelial-to-mesenchymal transition (EMT) progression, and composition of the extracellular matrix. In-depth single-cell analysis quantitatively evaluated the cellular makeup and immune cell component in the TME of cancer tissues and identified major tumor, immune, and stromal cell phenotypes. This work demonstrates the capability of IMC for quantitative and spatial identification of multiple immune parameters in the TME on a single slide of cancer patient samples (e.g. tumor microarray).

For Research Use Only. Not for use in diagnostic procedures.

Citation Format: Thomas D. Pfister, Liang Lim, Shaida Ouladan, Nick Zabinyakov, Qanber Raza, Christina Loh. Immuno-oncology study to profile the tumor microenvironment in multiple human cancers using high-plex imaging mass cytometry [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 2250.
Abstract 2281: Dual immune checkpoint blockade induces analogous alterations in the dysfunctional CD8+ T cell and activated Treg compartment
Authors: van der Leun Anne M. , Traets Joleen J. , Vos Joris L. , Elbers Joris B. , Patiwael Sanne , Qiao Xiaohang , Machuca-Ostos Mercedes , Thommen Daniela S. , Haanen John B. , Schumacher Ton N. , Zuur Charlotte L.
Published: April 2023

Immune checkpoint blockade has shown clinical activity in a range of cancer types. To dissect the effect of neoadjuvant PD-1 and CTLA4 blockade on intratumoral T cells in head and neck squamous cell carcinoma, we analyzed immune infiltrates in tumor biopsies from responding and non-responding patients. At baseline, a higher ratio between active (4-1BB+) and inactive regulatory CD4+ T cells was associated with response to therapy. Furthermore, upon therapy, this active Treg population showed a profound decrease in responding patients. In an analogous process, intratumoral dysfunctional CD8+ T cells transitioned to a state of reduced activity and dysfunction in responding patients, while in non-responding patients, NK cells showed an increased cytotoxic transcriptional profile upon treatment. These data reveal the immunological changes in response to dual PD-1 and CTLA4 blockade, including a parallel remodeling of presumed tumor-reactive T cell compartments in responding patients, and indicate that the presence of an activated Treg compartment at baseline may predict response.

Citation Format: Anne M. van der Leun, Joleen J. Traets, Joris L. Vos, Joris B. Elbers, Sanne Patiwael, Xiaohang Qiao, Mercedes Machuca-Ostos, Daniela S. Thommen, John B. Haanen, Ton N. Schumacher, Charlotte L. Zuur. Dual immune checkpoint blockade induces analogous alterations in the dysfunctional CD8+ T cell and activated Treg compartment [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 2281.
Abstract 2314: Development of a 6-color multiplex Crystal Digital PCR assay for the detection of ESR1 and PIK3CA mutations in the plasma of metastatic breast cancer patients
Authors: Quillien Veronique , Corne Julien , Rouge Thibault De la Motte , Du Fanny Le , Godey Florence
Published: April 2023

Blood-based liquid biopsies have become a real asset for patient treatment management in the field of precision oncology over the past few years, as they allow quick and easy access to tumor genetic alterations of interest. The analysis of circulating tumor DNA (ctDNA) from these liquid biopsies is a particularly well-suited strategy for longitudinal monitoring of patients. In patients with HR+/HER2- metastatic breast cancer (MBC), early detection of ESR1 and PIK3CA mutations is a key element to better personalize disease management. Among the technologies currently used for liquid biopsies, digital PCR (dPCR) has the advantage of being among the fastest and least expensive, providing robust results along with unmatched sensitivity as compared to other methods. Until recently, however, dPCR has been previously limited by multiplex capability. The recent availability of the 6-color version of the naica® platform (Stilla Technologies) has allowed us to develop a single-well multiplexed assay that combines an 11 pathogenic mutation ESR1 assay with a 21 mutation PIK3CA assay, for a combined total of 32 ESR1 and PIK3CA mutations. Here we present data on this new high-plex dPCR panel. As ESR1 and PIK3CA analyses are often requested together, this new screening assay represents a significant saving of time and resources for the analysis of our MBC patients with a relative ease of implementation and analysis.

Citation Format: Veronique Quillien, Julien Corne, Thibault De la Motte Rouge, Fanny Le Du, Florence Godey. Development of a 6-color multiplex Crystal Digital PCR assay for the detection of ESR1 and PIK3CA mutations in the plasma of metastatic breast cancer patients [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 2314.
Abstract 2325: IRF1 displays opposing tumor cell-intrinsic and -extrinsic roles in anti-tumor immunity
Authors: Purbey Prabhat Kumar , Paul Manash K. , Iwamoto Keisuke S. , Daly Allison , Seo Joowon , Champhekar Ameya S. , Campbell Katie , Schaue Dorthe , McBride William H. , Dubinett Steven M. , Ribas Antoni , Smale Stephen T. , Scumpia Philip O.
Published: April 2023

Interferons (IFNs), including IFN-α/β and IFN-γ, are multifaceted cytokines critical for antitumor immunity but contribute to T cell exhaustion through immune checkpoints. Recently, tumor cell expression of IRF1 was shown to enhance antitumor immunity, but a comprehensive evaluation of how IRF1 regulates IFN responses in the tumor and host has not been performed. Here, we show that Irf1-/- mice display enhanced tumor growth of WT and Irf1-/- tumors, as they fail to recruit sufficient NK cells and cytotoxic T cell (CTL). However, Irf1-/- tumors in wild-type mice displayed rapid immunogenic control, expansion of intratumoral lymphoid cells with enhanced effector programs and diminished exhaustion programs when compared to WT tumors. Mechanistically, IRF1 positively regulates the expression of distinct subsets of anti-tumor immunity suppression genes including the CD274/PD-L1, TRAIL, and IDO-1 checkpoints but not the IFN-inducible chemokines CXCL9-11. Surprisingly, control of Irf1-/- tumors required host IFN-α/β signaling but not IFN-γ signaling, while PD-L1 overexpression only weakly restored tumor growth. Thus, IRF1 selectively regulates the effects of IFNs on tumor cells and the tumor microenvironment and may be targeted to boost anti-tumor immunity.

Citation Format: Prabhat Kumar Purbey, Manash K. Paul, Keisuke S. Iwamoto, Allison Daly, Joowon Seo, Ameya S. Champhekar, Katie Campbell, Dorthe Schaue, William H. McBride, Steven M. Dubinett, Antoni Ribas, Stephen T. Smale, Philip O. Scumpia. IRF1 displays opposing tumor cell-intrinsic and -extrinsic roles in anti-tumor immunity [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 2325.
Abstract 2331: Loss of LRRC15 in osteosarcoma cells disrupts extracellular matrix integration, intercellular communication, and differentiation
Authors: Mukherjee Sanjit , Jajoo Ankit , Zhu Jack , Pineda Marbin , Walker Robert , Purcell James , Helman Lee J , Meltzer Paul S.
Published: April 2023

Background: Leucine rich repeat containing 15 (LRRC15) is a 581 amino acid type 1 transmembrane protein that contains 15 leucine rich repeats. Cysteine clusters flank the LRR domain that functions as the collagen binding domain. LRRC15 has been found to be upregulated during osteogenic differentiation of mesenchymal stem cells, induced by TGFβ, and highly expressed in multiple solid tumors, notably in osteosarcoma (1). Antibody drug conjugates targeting LRRC15 have shown promising activity in pre-clinical studies (2).

Results: To investigate the function of LRRC15 in the osteosarcoma microenvironment, we generated inducible shRNA knockdown (KD) derivatives targeting 3’UTR and ORF regions of LRRC15 of four osteosarcoma cell lines. Using these models, we characterized the biological properties of LRRC15 knockdown under 2-D and 3-D growth conditions. We found that LRRC15 KD in 2-D culture resulted in reduced adhesion to extracellular matrix (ECM) components and impaired cell migration. In 3-D spheroid culture, LRRC15 KD severely impacted scaffold-free spheroid compaction and invasion of spheroids embedded in Matrigel. Dysregulation of multiple ECM, cell adhesion and communication pathways were revealed by transcriptomic analysis from samples collected at time points during the LRRC15 KD. Of interest, several key transcription factors including RUNX2, CTNNB1, and SP7 were downregulated in LRRC15 deficient cells.

Conclusion: Our comprehensive results establish the importance of LRRC15 as a critical cell-adhesion molecule essential for the maintenance of the osteogenic intercellular communication, invasion and differentiation revealing novel aspects of osteosarcoma cell-biology.

Reference:1.Purcell JW, Tanlimco SG, Hickson J, Fox M, Sho M, Durkin L, et al. LRRC15 Is a Novel Mesenchymal Protein and Stromal Target for Antibody-Drug Conjugates. Cancer Res 2018;78:4059-4072.2.Slemmons KK, Mukherjee S, Meltzer P, Purcell JW, Helman LJ. LRRC15 antibody-drug conjugates show promise as osteosarcoma therapeutics in preclinical studies. Pediatr Blood Cancer 2020:e28771.

Citation Format: Sanjit Mukherjee, Ankit Jajoo, Jack Zhu, Marbin Pineda, Robert Walker, James Purcell, Lee J Helman, Paul S. Meltzer. Loss of LRRC15 in osteosarcoma cells disrupts extracellular matrix integration, intercellular communication, and differentiation [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 2331.
Abstract 2380: Fluorescence labeling of human gastric cancer using novel tumor specific near infrared labeled antibodies
Authors: Cox Kristin E. , Turner Michael A. , Amirfakhri Siamak , Lwin Thinzar M. , Ghosh Pradipta , Obonyo Marygorret , Kelly Kaitlyn M. , Hoffman Robert M. , Yazaki Paul J. , Bouvet Michael
Published: April 2023

Introduction: Gastric cancer has a global incidence of ~1 million cases per year. Currently, complete surgical resection is the only opportunity for cure for patients. Bright labeling of tumor margins with tumor specific markers could improve outcomes. This report demonstrates the advantages of targeting gastric cancer with humanized anti-CEA and TAG-72 antibodies in mouse models.

Methods: A cell line of human gastric cancer, MKN45, and a patient-derived tumor, KG8, were used to create xenograft models. To establish orthotopic models for each, ~1mm tumor fragments were harvested from subcutaneous models of these lines and sutured to the lesser curvature of the stomach. Humanized versions of TAG-72 (huCC44) and CEA (M5A) were conjugated to the near infrared dye IRDye800CW. IgG was conjugated to IRDye800CW and used as a control. For CEA-IR800 and IgG-IR800, mice received 50 µg IV and were imaged after 72 hours. For Tag72-IR800, mice received 75 µg IV and were imaged after 96 hours. Fluorescence imaging was performed on the mice using the LI-COR Pearl Imaging System. A tumor to background ratio (TBR) was calculated by dividing mean fluorescence intensity of the tumor versus normal gastric tissue.

Results: Humanized anti-CEA and TAG-72 brightly labeled multiple mouse models of human gastric cancer (Table 1). The TBR for TAG-72 labeling of KG8 was 2.777. CEA had a superior TBR of 3.528 compared to 0.606 for the control (IgG) in KG8 models. In MKN models labelled with CEA, an excellent average TBR of 7.407 were seen compared to 0.668 for the control.

Conclusion: Humanized anti-CEA and TAG-72 antibodies conjugated to near-infrared fluorescent dyes provide specific labeling of gastric cancers in orthotopic models. These tumor-specific fluorescent antibodies are promising potential clinical tools for visualization of gastric cancer margins at the time of surgical resection and improved surgical outcomes.

Orthotopic Model Antibody Tumor to Background Ratio KG8 anti-TAG-72 2.777 KG8 anti-CEA 3.528 KG8 Control 0.606 MKN anti-CEA 7.407 MKN Control 0.668

Citation Format: Kristin E. Cox, Michael A. Turner, Siamak Amirfakhri, Thinzar M. Lwin, Pradipta Ghosh, Marygorret Obonyo, Kaitlyn M. Kelly, Robert M. Hoffman, Paul J. Yazaki, Michael Bouvet. Fluorescence labeling of human gastric cancer using novel tumor specific near infrared labeled antibodies [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 2380.
Abstract 2485: RAB31 interacts with AGR2 to respond to oxaliplatin resistance and epithelial-mesenchymal transition in colorectal cancer cells via the EGFR-independent pathway
Authors: Chen Chi-Long , Hsiao Michael
Published: April 2023

In the process of colonic epithelial carcinogenesis, the epithelial-mesenchymal transition may occur and bring metastatic ability and drug resistance. These events will affect patient outcomes and clinical treatment strategies. To this end, we investigated differentially expressed genes between several chemotherapy regimens in colon cancer cells through available transcriptomics datasets. We identified a member of the Rab family, RAB31, which is overexpressed after oxaliplatin treatment, while the other option is not (Irinotecan). We confirmed that RAB31 can enhance resistance to oxaliplatin in some overexpression models and that RAB31 has the ability to promote exocytosis and vesicle trafficking. Using shRNA as complementary models, we observed consistent trends when RAB31 function was discarded. Moreover, our evidence determined epithelial-mesenchymal transition (EMT)-related molecules responsive in the RAB31 bidirectional models, including CDH2, SNAI1, SNAI2 and TWIST1. The expression of RAB31 has an independent prognostic value in patients with colon cancer and is significantly positively correlated with mesenchymal markers. Furthermore, the interactome analysis selected RAB31 and AGR2 to form a direct protein interaction. This result differs from previous studies that did not rely on EGFR binding and signaling. Taken together, our study found that RAB31 can modulate oxaliplatin sensitivity and EMT status, which are additional chemotherapeutic strategies and independent prognostic factors for the treatment of colorectal cancer.

Citation Format: Chi-Long Chen, Michael Hsiao. RAB31 interacts with AGR2 to respond to oxaliplatin resistance and epithelial-mesenchymal transition in colorectal cancer cells via the EGFR-independent pathway [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 2485.
Abstract 2487: Metastasis: Cancer cells that have turned on a Yamanaka-like pluripotency program
Authors: Bamdad Cynthia C. , Smagghe Benoit J. , Carter Mark G. , Grant Trevor J. , Reale Laura M. , Nash Michael J. , Walkley Danica M. , Nash Jac-Leen S. , Yi Kevin R. , Stewart Andrew K.
Published: April 2023

Purpose: To develop therapeutics to treat metastasis of solid tumor cancers. To overcome obstacles to developing anti-metastasis treatments by: 1) developing an animal model that reproducibly mimics cancer metastasis and does so within a reasonable timeframe; and 2) figuring out the basic science that drives metastasis.

Experimental: We discovered that all pluripotent human stem cells express a MUC1 cleavage product called MUC1* (muk 1 star). All MUC1 cleavage stops with the onset of differentiation and MUC1 goes back to its quiescent state. Over 75% of solid tumor cancers also express MUC1*. Overexpression of MUC1*, as well as enzymes that cleave MUC1 to MUC1* are predictors of poor prognosis. NME1 is a ligand of MUC1* that is secreted by both embryonic stem cells and cancer cells. As a dimer, NME1 dimerizes the MUC1* extra cellular domain to activate the MAP kinase growth pathway. In an embryo, the more stem cells there are, the more NME1 is secreted and it goes from the active dimer to an inactive hexamer (doesn’t bind MUC1*). The paradox was, “How do stem cells limit self-replication, but cancer cells do not?” Answer: A primitive growth factor, NME7AB, looks like a single chain dimer of NME1, so is always active, is expressed in the earliest, naïve stem cells and in metastatic cancer cells.

Unpublished Results: Growing cancer cells in recombinant NME7AB for 10 days transforms them into metastatic cells: 1) become non-adherent; 2) enter dormancy; 3) upregulate metastatic markers by 200-fold; 4) form sub-cu tumors in mice from as few as 50 cells; iv injection leads to total metastasis in 10 days; 5) the percent of injected tumor cells that were first grown in NME7AB determines the growth of the entire tumor. We developed a monoclonal antibody that blocks interaction of NME7AB and MUC1*. This antibody reverses established metastasis of breast cancers in animals. NME7AB should be turned off early in embryogenesis, but is aberrantly re-activated in metastatic cancers. Yamanaka factors OCT4, SOX2 and NANOG bind to the promoters of NME7, MUC1 and MMP16, an enzyme that cleaves MUC1 to MUC1* and exposes the cryptic binding site for NME7AB.

Conclusions: Primitive growth factor, NME7AB, drives metastasis of solid tumor cancers. Antibody that disrupts NME7AB-MUC1* interaction reverses metastasis in animals.

Citation Format: Cynthia C. Bamdad, Benoit J. Smagghe, Mark G. Carter, Trevor J. Grant, Laura M. Reale, Michael J. Nash, Danica M. Walkley, Jac-Leen S. Nash, Kevin R. Yi, Andrew K. Stewart. Metastasis: Cancer cells that have turned on a Yamanaka-like pluripotency program [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 2487.
Abstract 251: Identification of therapeutic vulnerabilities by genome-wide CRISPR knockout library screening of colon cancer organoids
Authors: Buckhaults Phillip J. , Khalili Sana , Banister Carolyn , Gokare Prashanth R. , Pocalyko Dave , Bachman Kurtis
Published: April 2023

The promise of precision medicine is based on the idea that genetic alterations present only in tumor cells create vulnerabilities that can be targeted for therapeutic intent. In some instances, somatically mutated driver genes such as KRAS, BRAF or PIK3CA can be targeted with small molecule inhibitors specific for mutant oncoproteins. In other cases therapeutic vulnerability is an indirect consequence, such as response to immune checkpoint inhibitors caused by somatic inactivation of mismatch repair genes and subsequent creation of neo-antigens in tumors. Synthetic lethal vulnerabilities dependent on common somatic alterations would be highly specific therapeutic targets applicable to a large number of cancer patients. We sought to identify potential therapeutic vulnerabilities for colon cancer by performing a genome wide CRISPR knockout screen in a colon cancer organoid in both TP53-Wild-Type and TP53-Knockout backgrounds. We identified 1784 gene knockouts with TP53-dependent effects on Darwinian fitness. Examples include MDM2 and PPM1D knockouts, both of which selectively harmed the TP53 WT organoids. 250 gene knockouts selectively harmed the TP53 KO organoids and represent novel avenues for development of TP53 synthetic lethal targeted therapeutics. We also identified ~1000 gene knockouts under significant negative Darwinian selection in both TP53 WT and TP53 KO organoids which are not known to be common essential genes. Several of these organoid model specific vulnerabilities are in pathways that are downstream of the somatic mutations present in the tumor and are therefore candidates for highly-specific targeted therapeutic intervention. Examples include Werner’s pathway dependencies (WRN, EME1, MUS81) which are known to result from mismatch repair deficiency, WNT pathway dependencies (CTNNB1, PORCN) which result from RNF43 inactivating somatic mutation, and BRAF dependency resulting from its own activating oncogenic somatic mutation. In summary, genome-wide CRISPR knockout library screening of human colon cancer organoids can provide a comprehensive overview of dependencies interacting with either natural or engineered mutations and are a promising novel platform for discovering personalized or pathway based therapeutic targets. Future work will focus on target validation and additional primary screens using our biobank of tumor-normal organoid pairs.

Citation Format: Phillip J. Buckhaults, Sana Khalili, Carolyn Banister, Prashanth R. Gokare, Dave Pocalyko, Kurtis Bachman. Identification of therapeutic vulnerabilities by genome-wide CRISPR knockout library screening of colon cancer organoids [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 251.
Abstract 252: A single-tube approach for detection of circulating tumor DNA using customized panels
Authors: Meltzer Robert , Kaur Jessalyn , Kintsurashvili Ekaterina , D'Amato Christopher , Godfrey Tony
Published: April 2023

Circulating tumor DNA (ctDNA) is a promising biomarker for cancer. Applications of ctDNA analysis include screening, diagnosis, treatment selection, minimal residual disease (MRD) detection and recurrence monitoring. While some applications are best served by tumor-agnostic panels covering large regions of the genome, such large panels are not universally appropriate due to limitations in sensitivity, user-friendliness, turn-around time (TAT), and sequencing cost. One application where customized, tumor-informed panels can be preferable is detection of MRD and cancer recurrence, and multiple studies have shown that ctDNA can detect residual/recurrent disease several months earlier than routine clinical imaging. This application requires high sensitivity and in most cases will involve repeat testing of longitudinal samples collected over several months or years. In this scenario, the ideal assay characteristics include tumor-informed, personalized assay panels, with a simple, rapid workflow, and with minimal cost per assay. We have developed the Fluent LBx assay to meet these requirements. Fluent LBx analysis can be performed on-site in any appropriately equipped research or clinical laboratory, avoiding the extra cost and delay associated with external testing. Fluent LBx is a variation on SiMSen-Seq (PMID 27060140) that uses modified target and adapter primer sequences to facilitate barcoding and uniform amplification of multiple targets in a single-tube library construction protocol. Library preparation can be completed in under 1.5 hours with approximately 20 minutes of hands-on time. Using this approach, target DNA molecules are first barcoded and then amplified with indexed adaptors compatible with Illumina sequencers, in a single PCR reaction. After clean-up and sequencing, barcode information is used to compile reads from individual DNA molecules and minimize base calling errors introduced during PCR and sequencing. Here we present data demonstrating uniformity, sensitivity and specificity of Fluent LBx assays using LGC SeraCare quality control standards. Additionally, we present longitudinal clinical demonstration of residual disease detection from ctDNA collected from multiple head and neck and gastrointestinal cancer patients through longitudinal monitoring post therapeutic intervention. The Fluent LBx workflow allows rapid, inexpensive, patient-specific monitoring of MRD and recurrence in multiple cancer indications.

Citation Format: Robert Meltzer, Jessalyn Kaur, Ekaterina Kintsurashvili, Christopher D'Amato, Tony Godfrey. A single-tube approach for detection of circulating tumor DNA using customized panels [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 252.
Abstract 2525: Analyzing the role of MDMX for regulating cell death in prostate cancer cells
Authors: Alsarrani Ahmed , Natarajan Umamaheswari , Alsubhi Samia , Alzahrani Amal , Rathinavelu Appu
Published: April 2023

Prostate cancer (PCa) is the most prominent cancer type and the second leading cause of cancer death among men in the USA. The five-year survival rate for localized PCa is 98% but drops to 31% when PCa spreads to different parts of the body. Various treatment strategies that can induce cell death through different mechanisms are used while treating cancers. In recent years, necroptosis has been established as one of the effective mechanisms of inducing cell death in cancers. Necroptosis can be identified as a regulated form of cell death that can occur independently of executioner caspases. Despite numerous studies on the effects of necroptosis in cancer, it’s role on PCa has not been fully elucidated. In this study, we have evaluated the role of MDMX, a proto-oncogene involved in the negative regulation of p53, in causing cell death through p53-independent mechanisms. We have observed that inhibition of MDMX decreased the cell viability of LNCaP cells in response to 20 µM of NSC (NSC-207895), a MDMX specific inhibitor treatment, in a concentration-dependent manner. MDMX inhibition appears to induce necroptosis in LNCaP prostate cancer cells by elevating the expression of receptor-interacting protein kinase 1 (RIP1), which is a key element of the necroptotic pathway. In general, the necroptosis pathway can trigger two opposing mechanisms in cancer where it can promote tumor migration and metastasis on one hand and can also act as a defensive mechanism on the other hand when apoptosis is disrupted. To further assess the effect of MDMX on cell migration, the LNCaP cells were treated with 20 µM of NSC, which effectively blocked the cancer cell migration for up to 48 h, which was confirmed using the scratch assay. Interestingly, NSC treatment also reduced the expression of Vascular Endothelial Growth Factor A (VEGFA), which is one of the main markers for angiogenesis. Our results show that the p53-independent mechanisms that are activated following MDMX inhibition can regulate both cancer cell migration and angiogenesis. MDMX also seemed to be involved in regulating PCa cell survival through necroptosis. The outcomes of our experiments provide additional knowledge and strategies for making an accurate prognosis and choosing the most suitable therapeutic strategies for offering effective treatments for MDMX-positive PCa. (This research was supported by the Royal Dames of Cancer Research Inc. Ft. Lauderdale, Florida)

Citation Format: Ahmed Alsarrani, Umamaheswari Natarajan, Samia Alsubhi, Amal Alzahrani, Appu Rathinavelu. Analyzing the role of MDMX for regulating cell death in prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2525.
Abstract 2527: Investigation of doxorubicin interference in flow cytometry measurements of apoptosis and necrosis
Authors: Nicoletto Rachel E. , Wilkes Richard , Ofner Clyde M.
Published: April 2023

Background: Doxorubicin (DOX) is frequently used for investigations of breast cancer using flow cytometry with fluorescent stains for apoptosis and necrosis. DOX interference, however, can occur with these fluorescent measurements which is not reported in many published reports. The objectives of this study were to identify such interference and to assess its extent.

Methods: MDA-MB-231 and MCF-7 cells were incubated for 24 h with 0.1, 1, and 10 µM DOX. Cells were then stained for apoptosis and necrosis using two commercial assay kits. Fluorescence was measured with FL1 (apoptosis), FL2 (primary for DOX), and FL3 (necrosis) detectors in cells with DOX alone, with DOX plus stains, and in control cells without DOX. Measurements were repeated two to five times using replicates and trials. Statistics were evaluated by t-tests.

Results: Overall, DOX fluorescence interference increased with concentration in both cell lines. After DOX incubation, cells stained with caspase 3/7 and SYTOX showed fluorescence in the FL1 detector indicating caspase 3/7 activation in apoptosis. But the DOX component of fluorescence with this detector ranged from 11 to 95%. Dox fluorescence in the FL3 detector for SYTOX measurements indicating necrosis ranged from 26 to 96% of total fluorescence. Cells stained with Annexin V (with the FITC label) and propidium iodide (PI) showed fluorescence in the FL1 detector for Annexin V indicating apoptosis. However, the DOX component of fluorescence ranged from 11 to 25%. DOX fluorescence in the FL3 detector for PI indicating necrosis ranged from 87 to 106%.

Conclusions: Without correction for DOX interference, and even at low concentrations, substantial errors can occur for apoptosis and necrosis measurements using flow cytometry with these stains in breast cancer cells. At high DOX concentrations, the interference can erroneously indicate an extensive presence of these cell death processes. Corrections for DOX interference include background subtraction and flow cytometer compensation.

Acknowledgments: The authors gratefully acknowledge financial support from the Agnes Varis Trust for Women in Science (REN), the Milton Lev Memorial Faculty Research Fund (CMO) as well as Saint Joseph’s University support for student research. The authors also thank Bin Chen, PhD, and Richard Howley for assistance with flow cytometry.

Citation Format: Rachel E. Nicoletto, Richard Wilkes, Clyde M. Ofner. Investigation of doxorubicin interference in flow cytometry measurements of apoptosis and necrosis [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 2527.