Potential Marker Research Articles

MiR-301b-3p can be Used as a Potential Marker for the Diagnosis of Lung Adenocarcinoma.

The involvement of aberrantly expressed miR-301b-3p has been discovered in diverse human tumors. Our study was primarily centered around the role of miR-301b-3p in diagnosing lung adenocarcinoma (LUAD). We used the TCGA database to download the TCGA-LUAD dataset and selected miR-301b-3p as the object of our study by differential expression analysis of miRNAs combined with previous studies. The LUAD diagnostic model was constructed utilizing machine learning based on miR-301b-3p expression. The predictive performance of the diagnostic model was found to be excellent by ROC curves combined with the clinical information of the dataset samples. GSEA, GO, and KEGG enrichment analyses demonstrated that miR-301b-3p may mediate the cell cycle by regulating the expression of hormones. Subsequently, combined with tumor immunity and mutation analysis, it was found that patients in the low-expression group had better immune infiltration, indicating that their response rate to immunotherapy may be relatively high. Finally, a mouse xenograft model was constructed to verify how miR-301b-3p affected LUAD progression in mice. The results illustrated that overexpressed miR-301b-3p could cause faster tumor growth in mice. On the contrary, the growth of LUAD could be impeded by the downregulated miR-301b-3p expression. It was suggested that miR-301b-3p had a crucial part in LUAD progression. Overall, the diagnostic performance of the LUAD diagnostic model constructed based on miR-301b-3p is great, and the model can be used as a potential diagnostic marker for LUAD to provide new ideas for clinical diagnosis.

Chemical constitutents and chemotaxonomic research of the deep-sea-derived fungus Talaromyces minioluteus W19

Talaromyces minioluteus W19 was isolated from a deep-sea sediment sample of the western Pacific Ocean. A systematic investigation of its chemical constituents led to the isolation of nine compounds, including one new macrolactin (1), three citrinin (2−4), one sesquiterpene ester (5), one steroid (6), two fatty acid analogues (7 and 8), and one glyceride (9). Compounds 2, 4, and 6−9 were isolated from the genus Talaromyces for the first time. Fusarimone A (3) could be ideal chemotaxonomic marker for T. minioluteus. Drimane sesquiterpenoid-amino acid hybrid is potent chemotaxonomic marker for the genus Talaromyces.

Investigation of thrombin concentration at the time of clot formation in simultaneous thrombin and fibrin generation assays

Thrombin generation (TG) and fibrin clot formation represent the central process of blood coagulation. Up to 95% of thrombin is considered to be generated after the clot is formed. However, this was not investigated in depth. In this study, we conducted a quantitative analysis of the Thrombin at Clot Time (TCT) parameter in 5758 simultaneously recorded TG and clot formation assays using frozen plasma samples from commercial sources under various conditions of activation. These samples were supplemented with clotting factor concentrates, procoagulant lipid vesicles and a fluorogenic substrate and triggered with tissue factor (TF). We found that TCT is often close to a 10% of thrombin peak height (TPH) yet it can be larger or smaller depending on whether the sample has low or high TPH value. In general, the samples with high TPH are associated with elevated TCT. TCT appeared more sensitive to some procoagulant phenotypes than other commonly used parameters such as clotting time, TPH or Thrombin Production Rate (TPR). In a minority of cases, TCT were not predicted from TG parameters. For example, elevated TCT (above 15% of TPH) was associated with either very low or very high TPR values. We conclude that clotting and TG assays may provide complementary information about the plasma sample, and that the TCT parameter may serve as an additional marker for the procoagulant potential in plasma sample.

Self-Rated Health in Middle Age and Risk of Hospitalizations and Death: Recurrent Event Analysis of the ARIC Study.

Self-rated health is a simple measure that may identify individuals who are at a higher risk for hospitalization or death. To quantify the association between a single measure of self-rated health and future risk of recurrent hospitalizations or death. Atherosclerosis Risk in Communities (ARIC) study, a community-based prospective cohort study of middle-aged men and women with follow-up beginning from 1987 to 1989. We quantified the associations between initial self-rated health with risk of recurrent hospitalizations and of death using a recurrent events survival model that allowed for dependency between the rates of hospitalization and hazards of death, adjusted for demographic and clinical factors. Of the 14,937 ARIC cohort individuals with available self-rated health and covariate information, 34% of individuals reported "excellent" health, 47% "good," 16% "fair," and 3% "poor" at study baseline. After a median follow-up of 27.7years, 1955 (39%), 3569 (51%), 1626 (67%), and 402 (83%) individuals with "excellent," "good," "fair," and "poor" health, respectively, had died. After adjusting for demographic factors and medical history, a less favorable self-rated health status was associated with increased rates of hospitalization and death. As compared to those reporting "excellent" health, adults with "good," "fair," and "poor" health had 1.22 (1.07 to 1.40), 2.01 (1.63 to 2.47), and 3.13 (2.39 to 4.09) times the rate of hospitalizations, respectively. The hazards of death also increased with worsening categories of self-rated health, with "good," "fair," and "poor" health individuals experiencing 1.30 (1.12 to 1.51), 2.15 (1.71 to 2.69), and 3.40 (2.54 to 4.56) times the hazard of death compared to "excellent," respectively. Even after adjusting for demographic and clinical factors, having a less favorable response on a single measure of self-rated health taken in middle age is a potent marker of future hospitalizations and death.

Alpha Calcitonin Gene-related Peptide, Neuropeptide Y, and Substance P as Biomarkers for Diagnosis and Disease Activity and Severity in Multiple Sclerosis.

Alpha calcitonin gene-related peptide (aCGRP), neuropeptide Y (NPY), and substance P (SP) are neuropeptides that have emerged recently as potent immunomodulatory factors with potential as novel biomarkers and therapeutic targets in multiple sclerosis (MS). The study aimed to detect serum levels of aCGRP, NPY, and SP in MS patients versus healthy controls and their association with disease activity and severity. Serum levels were measured in MS patients and age and sex-matched healthy controls using ELISA. We included 67 MS patients: 61 relapsing-remitting MS (RR-MS) and 6 progressive MS (PR-MS), and 67 healthy controls. Serum NPY level was found to be lower in MS patients than in healthy controls (p < 0.001). Serum aCGRP level was higher in PR-MS compared to RR-MS (p = 0.007) and healthy controls (p = 0.001), and it positively correlated with EDSS (r = 0.270, p = 0.028). Serum NPY level was significantly higher in RR-MS and PR-MS than in healthy controls (p < 0.001 and p = 0.001, respectively), and it was lower in patients with mild or moderate/severe disease than in healthy controls (p < 0.001). Significant inverse correlations were found between SP level and MS disease duration (r = -0.279, p = 0.022) and duration of current DMT (r = -0.315, p = 0.042). Lower serum levels of NPY were revealed in MS patients compared to healthy controls. Since serum levels of aCGRP are significantly associated with disease activity and severity, it is a potential disease progression marker.

Diagnostic Performance of Carotid Ring Sign on CT-Angiography in Internal Carotid True Occlusion.

To differentiate between pseudo occlusion (PO) and true occlusion (TO) of internal carotid artery (ICA) is important in thrombectomy treatment planning for patients with acute ischemic stroke. Although delayed contrast filling has been differentiated carotid PO from TO, its application has been limited by the implementations of multiphasic computed tomography angiography. In this study, we hypothesized that carotid ring sign, which is readily acquired from single-phasic CTA, can sufficiently differentiate carotid TO from PO. One thousand four hundred and twenty patients with anterior circulation stroke receiving endovascular therapy were consecutively recruited through a hospital- and web-based registry. Two hundred patients with nonvisualization of the proximal ICA were included in the analysis after a retrospective screening. Diagnosis of PO or TO of the cervical segment of ICA was made based on digital subtraction angiography. Diagnostic performances of carotid ring sign on arterial-phasic CTA and delayed contrast filling on multiphasic computed tomography angiography were evaluated and compared. One-hundred twelve patients had ICA PO and 88 had TO. Carotid ring sign was more common in patients with TO (70.5% versus 6.3%; P<0.001), whereas delayed contrast filling was more common in PO (94.9% versus 7.7%; P<0.001). The sensitivity and specificity of carotid ring sign in diagnosing carotid TO were 0.70 and 0.94, respectively, whereas sensitivity and specificity of delayed contrast filling was 0.95 and 0.92 in judging carotid PO. Carotid ring sign is a potent imaging marker in diagnosing ICA TO. Carotid ring sign could be complementary to delayed contrast filling sign in differentiating TO from PO, in particular in centers with only single-phasic CTA.

492 Novel BH3 Mimetic Bcl-2 Inhibitor Promotes Autophagic Cell Death and Reduces in Vivo Glioblastoma Tumor Growth

INTRODUCTION: Anti-apoptotic members of the Bcl-2 family proteins play central roles in the regulation of cell death in glioblastoma, the most malignant type of brain tumor. Despite the advances in GBM treatment, there is still an urgent need for new therapeutic approaches. METHODS: In silico studies resulted in 500,000 molecules being screened with computer-aided drug design methods and the molecule with the highest binding affinity was selected and named BAU-243. Cell viability, IC50 value, tumor formation potential, tumor spheroid formation potential, tumor invasion studies were investigated following BAU-243 application. The molecular changes caused by Bcl-2 inhibition were demonstrated by quantitative PCR, western blot and immunofluorescence methods. In in vivo studies, glioma tumors were transplanted intracranially into Nod Scid Gamma(NSG) mice using the mouse xenograft model. RESULTS: Here, we report a novel 4-thiazolidinone derivative BH3 mimetic, BAU-243 that binds to Bcl-2 with a high affinity. BAU-243 effectively reduced overall glioblastoma cell proliferation including a subpopulation of cancer-initiating cells in contrast to the selective Bcl-2 inhibitor ABT-199. While ABT-199 successfully induces apoptosis in high BCL2-expressing neuroblastoma SHSY-5Y cells, BAU-243 triggered autophagic cell death rather than apoptosis in A172 cells, indicated by the upregulation of BECN1, ATG5, and MAP1LC3B expression. Lc3b-II, a potent autophagy marker, was significantly upregulated following BAU-243 treatment. Moreover, BAU-243 significantly reduced tumor growth in vivo in orthotopic brain tumor models when compared to the vehicle group, and ABT-199 treated animals. To elucidate the molecular mechanisms of action of BAU-243, we performed computational modeling simulations that were consistent with in vitro results. CONCLUSIONS: Results indicated that BAU-243 activates autophagic cell death by disrupting Beclin 1:Bcl-2 complex, and may serve as a potential small molecule for the treatment of glioblastoma.

DNA Ploidy as a Potential Adjunct Prognostic Marker of Low-Risk Prostate Cancer Progression after Radical Prostatectomy.

Post prostatectomy PSA kinetics and General Grade Groups (GGG) are the strongest prognostic markers of biochemical recurrence (BCR) and prostate cancer (PCa)-specific mortality after radical prostatectomy. Despite having low-risk PCa, some patients will experience BCR, for some, clinically significant BCR. There is a need for an objective prognostic marker at the time of prostatectomy to improve risk stratification within this population. In this study, we investigated the prognostic potential of DNA ploidy. Prostatectomy samples from 97 patients with GGG1 and GGG2 with a low-risk CAPRA-S score were included in this study. PCa tissue with the worst Gleason pattern underwent tissue disaggregation, cell isolation and staining with a DNA stoichiometric stain. Using image cytometry, DNA ploidy was measured and a Ploidy Score (PS) was generated. Among the 97 patients, 79 had no BCR, 18 experienced BCR, of which 14 had a PSA doubling time (PSA-DT) >1 year (low-risk group) and 4 had a PSA-DT of <1 year (high-risk group). Using Logistic regression analysis, only pathological T stage (pT) and PS independently predicted BCR with PS being the most significant (p = 0.001). The number of aneuploid cells was significantly higher in the high-risk group compared to the other groups (p = 1.7x10-11). PS combined with GGG diagnosis further stratified risk groups of biochemical recurrence free survival within CAPRA-S low-risk cohort. DNA ploidy is an independent prognostic marker of BCR in low-risk PCa after radical prostatectomy, which could early on identify potentially aggressive PCa recurrences and introduce a more personalized approach to salvage treatments.

Abstract 1086: ZFP36L2 is expected to be a potential prognostic marker in IL-1β+ osteosarcoma patients

Abstract Background: Osteosarcoma (OS) is the most common bone malignancy affect children and young adults. For decades, the long-term survival for patients with recurrences and metastases remains ~30%. There’s an urgent need for new prognostic marker and therapeutic options. ZFP36L2, a member of the tristetraprolin (TTP) family of CCCH zinc finger proteins, stands out for its pivotal role in post transcriptional modifications and modify tumor microenvironment (TME). Notably, ZFP36L2 contributes to the recruitment of macrophages, specifically the M1 subtype, which are known for their ability to secrete IL1β and exhibit anti-tumor functions. Our study reveals that, ZFP36L2 can be used as a predictor of prognosis in OS patients with enriched IL1B expression. This study aims to elucidate the interaction between ZFP36L2 and IL1B in modification of TME and shed light on its potential of prognosis prediction and therapeutic in IL1β positive OS patients. Methods: Firstly, we conducted a comprehensive exploration of ZFP36L2 expression across TCGA cohorts. Then, we performed IHC staining to confirm the evaluated expression of ZFP36L2 in cancerous tissues. Immune infiltration analysis, including QUANTISEQ, TIMER, and CIBERSORT, was conducted. KM plot was employed to analyze the prognosis of OS patients. Finally, qPCR and western blot were used to validate transcriptional and translational changes of key molecules across OS cell lines. Results: Our findings demonstrate that ZFP36L2 exhibits specific expression across various cancer types, with a notable elevation in sarcoma tissues, as confirmed by IHC. Importantly, this high expression of ZFP36L2 in sarcoma tissues is correlated with metastasis. We found ZFP36L2 significantly correlates with tumor metastasis but failed in patient overall survival. Immunoinfiltration analysis indicates a significant correlation between ZFP36L2 and macrophages, as well as T cells, suggesting a role in the TME. Prognostic analysis reveals that ZFP36L2 can serve as a predictive marker for IL1β positive patients. The wetlab experiments demonstrated that ZFP36L2 leads to IRGM mRNA degradation, ultimately affecting IL1β+ macrophages. Conclusion: This study delineates the tumor-specific elevated expression of ZFP36L2 and its potential as a prognostic marker in IL1β+ OS patients. We hypothesize that ZFP36L2 affects IL1β+ macrophages through IRGM degradation. Besides, IRGM also has an impact on cytotoxic T cells. Therefore, the ZFP36L2-IRGM axis plays an important role in remodeling OS TME. These results indicated ZFP36L2, not only serves as a marker, but also can be a therapeutic target in IL1β enriched patients. Citation Format: Peiyao Hao, Piaopiao Luo, Zhenhua Ren, Shenglin Xu, Jijun Han, Xiang Nan. ZFP36L2 is expected to be a potential prognostic marker in IL-1β+ osteosarcoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1086.

Abstract 5140: ITGB6 as a predictive biomarker for overall prognosis and PD-(L)1 immune checkpoint blockade response in various cancer types

Abstract Our previous work studying the effects of integrin αvβ6 knockdown in cancer cells demonstrated αvβ6 as a method of T cell suppression in a co-culture model. ITGB6, the gene encoding the β6 subunit of αvβ6, is a potent prognostic marker across multiple cancer types. As a major activator of latent TGFβ, αvβ6, and consequently, ITGB6, has considerable therapeutic implications due to the immunosuppressive effect that activated TGFβ has on the tumor microenvironment. A Human Protein Atlas search of ITGB6 expression surveyed the upregulation of ITGB6 across cancer types. Cancer ITGB6 expression was then compared to expression in paired adjacent normal tissue using the TNMplot tool (Bartha, 2021) on a concatenated set of GEO, GTex, TCGA, and TARGET RNA-seq databases. Kaplan-Meier curves were generated using TCGA mRNA data through the cBio portal, splitting the cohorts according to an ITGB6 mRNA expression threshold of one standard deviation above the mean (z-score ± 1.0). ITGB6 expression and immune checkpoint blockade (ICB) response were evaluated with the Kaplan-Meier Plotter tool (Kovacs, 2023). ITGB6 expression levels between ICB responders and non-responders were quantified using the ROC Plotter tool (Fekete, 2023), and the figures were generated using Matplotlib in Python. Lung, bladder, head and neck, pancreatic, and cervical cancer all demonstrated high expression of ITGB6. Head and neck squamous cell carcinoma (median fold change 2.70, p&amp;lt;0.0001), bladder urothelial carcinoma (FC 2.41, p&amp;lt;0.001), cholangiocarcinoma (FC 5.33, p&amp;lt;0.01), and esophageal carcinoma (FC 2.72, p&amp;lt;0.03) showed strong upregulation of ITGB6 compared to normal tissue. Interestingly, lung adenocarcinoma (FC 0.77, p&amp;lt;0.04) and lung squamous cell carcinoma (FC 0.55, p&amp;lt;0.0001) showed downregulation of ITGB6 compared to normal tissue. Survival data revealed that ITGB6 was a potent marker of a poor prognosis in pancreatic adenocarcinoma (high ITGB6 median survival 16.79 mos, vs. low ITGB6 21.88 mos, p&amp;lt;0.004) and head and neck squamous cell carcinoma (high ITGB6 30.06 mos vs. low ITGB6 57.42 mos, p&amp;lt;0.03). However, no significant prognostic difference was observed in non-small cell lung cancers. Finally, high pan-cancer expression of ITGB6 led to poorer response to αPD-1 (high ITGB6 Hazard Ratio 1.37, p&amp;lt;0.05) and αPD-L1 (high ITGB6 HR 1.50, p&amp;lt;0.001). In vitro and in vivo experiments to validate the benefit of ITGB6 as a biomarker and as a therapeutic target are underway. Citation Format: William J. MacDonald, Praveen R. Srinivasan, Maximilian Pinho-Schwermann, Vida Tajiknia, Connor Purcell, Lindsey Carlsen, Wafik S. El-Deiry. ITGB6 as a predictive biomarker for overall prognosis and PD-(L)1 immune checkpoint blockade response in various cancer types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5140.

Abstract 6265: FLCN variants and their role in parathyroid cancer and atypical parathyroid tumors

Abstract Parathyroid carcinoma (PC) is rare, poses challenges in definitive diagnosis and treatment, and carries a high rate of recurrence and mortality. Atypical parathyroid tumors (APT) have histopathologic features of malignancy but lack unequivocal evidence of invasion and are of uncertain malignant potential. Analysis of genetic alterations that may play a role in PC development, particularly those that might serve as markers of malignant potential, is important for further understanding this disease. FLCN is a gene found on chromosome 17 that encodes folliculin, a ubiquitously expressed protein with roles in multiple cellular processes such as apoptosis and cell signaling. Inactivating FLCN mutations cause Birt-Hogg-Dubé syndrome (BHD), an autosomal dominant condition characterized by benign fibrofolliculomas, pulmonary cysts and spontaneous pneumothorax, and increased risk of renal cancer. Benign parathyroid tumors had previously been reported in a few BHD patients and recently one study has reported a link between FLCN mutations and parathyroid carcinoma. Germline frameshift mutations in FLCN, accompanied by somatic loss of the normal allele, were reported in two unrelated PC patients, both with clinical features suggestive of BHD, but no established diagnosis of BHD. An identical somatic frameshift FLCN mutation was reported in one PC and one APT. Interestingly, the authors noted low coverage of FLCN on whole exome sequencing, resulting in the failure to detect variants identified by Sanger sequencing, thus raising the possibility that prior studies may have missed FLCN variants. To better understand the frequency of germline or somatic FLCN mutations in PC and APT, we performed Sanger sequence analysis of the entire coding region of the FLCN gene on available tumor DNA from 11 PCs and 15 APTs. We identified no inactivating FLCN mutations in any of the PC or APT samples examined. A germline missense variant, resulting in a p.Gly325Val change, predicted to be benign/tolerated by in silico analyses, was seen in one PC and a synonymous variant (c.1233G&amp;gt;A) was seen in one APT. The absence of pathogenic mutations in our series parathyroid carcinoma and atypical parathyroid tumors suggests that FLCN mutations are rare in these parathyroid tumors. Nevertheless, germline FLCN testing and/or additional screening for BHD-related lesions in parathyroid carcinoma patients merits further consideration and study. Citation Format: Callie Burke, Justin Bellizzi, Jessica Costa-Guda, Andrew Arnold. FLCN variants and their role in parathyroid cancer and atypical parathyroid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6265.

Abstract 78: Superior resolution of intracellular markers with CyTOF reveals greater functional diversity of human T cells

Abstract Measuring functional signatures of immune cells in a comprehensive manner, spanning the inflammatory (Th1/Th17) and immunosuppressive (Th2/Treg) lineages, would provide key insights into several facets of cancer research and therapy. Determining mechanisms underlying success/failure of checkpoint blockade, defining immunosuppressive activity of the tumor-resident cell subsets, and identifying immunological biomarkers that predict clinical outcomes, could all be achievable if detection of cytokines spanning many lineages among individual cells was in reach. To achieve this goal, powerful cytometric research tools are required. Cell phenotyping panels are expanding and often now include measurement of intracellular markers of functional potential, such as cytokines, phosphorylation events, and transcription factors. Many of these readouts, including cytokines of the Th2/Treg lineages (IL-5, IL-10, and IL-13), have been notoriously difficult to detect in human cells using fluorescence-based cytometry. Large panels with multiplex intracellular markers are subject to challenges with autofluorescence and spillover spread, thus limiting fluorochrome capacity and reducing resolving power for rare subsets even with full spectrum flow cytometers. For such applications, mass cytometry may overcome such limitations and enable better signal resolution. To address this, we evaluated three small (13-plex) panels using a full spectrum flow cytometer and the CyTOF XT™ mass cytometer. Each panel was comprised of surface and intracellular analytes (cytokines, phospho-epitopes, or transcription factors) and designed to minimize potential impact of spectral overlap on the resolution of spectral flow data. PBMCs were stimulated, split, and stained with either fluorochrome- (Cytek® Aurora) or metal-conjugated antibodies (CyTOF XT). Data sets were analyzed by PhenoGraph clustering and visualized with opt-SNE to determine cellular functional diversity. Overall, data collected on the CyTOF XT demonstrated superior resolution for many intracellular readouts, including cytokines IL-5, IL-10, and IL-13. Also, when better signal/noise enabled accurate clustering, a higher number of immune cell clusters possessing distinct functional signatures resulted from mass cytometry datasets as compared with fluorescent counterparts. In summary, the CyTOF XT mass cytometry platform offers superior signal resolution compared to spectral flow for a range of intracellular targets. Use of mass cytometry translates to the identification of more diverse functional subsets and thus a clearer understanding of the immune signatures present in a sample in the context of unbiased analysis. Our findings indicate that the CyTOF XT platform could serve as a catalyst for seminal discoveries in immune profiling to drive therapeutic design and advanced disease monitoring in cancer. Citation Format: Erika L. Smith-Mahoney, Michael J. Cohen, Anna C. Belkina, Thirumahal Selvanantham, David King, Christina Loh, Amedeo J. Cappione, Jennifer E. Snyder-Cappione. Superior resolution of intracellular markers with CyTOF reveals greater functional diversity of human T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 78.

Abstract 1038: Identification of diagnostic biomarkers for colon adenocarcinoma by analyzing proteins in human-derived extracellular vesicles and particles

Abstract There is an unmet medical need for circulating biomarkers to improve early cancer detection in colon adenocarcinoma (COAD). We investigated the proteomic profile of extracellular vesicles and particles (EVPs) from the colon tissue explants and plasmas in COAD. The research aims to overcome the limitations of traditional serum Carcinoembryonic Antigen (CEA) testing and invasive procedures like colonoscopy. We analyzed EVPs sourced from tumor and adjacent non-tumor tissues and preoperative and postoperative plasma in COAD patients to identify potential EVP-based tumor diagnostic markers. In the Discovery phase with 50 COAD patients, we conducted a proteomic analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS), identifying approximately 50 markers each from tissue explants and plasma. These markers were validated in an independent cohort of 104 participants, including 84 COAD patients and 20 healthy controls. We utilized ELISA to quantify 11 selected EVP proteins. Notably, seven of these proteins exhibited significant differential expression in preoperative and postoperative plasma, highlighting their diagnostic relevance. These seven proteins also showed marked elevation in preoperative plasma compared to healthy controls, reinforcing their potential as diagnostic markers. The study extended to evaluate the diagnostic accuracy of these seven proteins, referred to as "EV-7 levels", across various stages of COAD. Their performance was compared with serum CEA levels and plasma cfDNA mutation rates, as detected by an FDA-approved liquid biopsy assay. The EV-7 levels demonstrated superior detection rates in the early stages of COAD, particularly in Stages I and II, in contrast to the serum CEA method. Among the 84 patients, 73 (86.9%) exhibited EV-7 levels exceeding the established cut-off. In the validation cohort, EV-7 levels were more accurate across different COAD stages than serum CEA levels and cfDNA mutation rates. CEA levels above 5 ng/ml were 27%, 45%, 42%, and 64% for Stages I through IV. cfDNA somatic mutation rates were 60%, 80%, 75%, and 75%. EV-7 levels exceeding the cut-off were 73%, 100%, 88%, and 84%, respectively, with Stages I and II having the highest detection rate among the three methods. ROC curve analysis for EV-7 levels in Stages I to IV showed statistically significant, with AUC values of 0.913, 1.000, 0.985, and 0.984, respectively. Our findings indicate that the seven EVP proteins are significantly present in the plasma of early-stage COAD patients. Integrating these markers with current diagnostic approaches could improve early cancer detection, showing promise for practical application in clinical settings. Citation Format: Yura Seo, Yoon Dae Han, Linda Bojmar, Kyung-A Kim, Yurin Seo, David Lyden, Han Sang Kim. Identification of diagnostic biomarkers for colon adenocarcinoma by analyzing proteins in human-derived extracellular vesicles and particles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1038.

Abstract 3790: HtrA3 promotes malignant progression of colorectal cancer via PI3K-AKT-FOXO1 signaling pathway

Abstract High-temperature requirement protease A3 (HtrA3) is known to contribute to the progression of various carcinomas through tissue-specific actions, yet its precise role in colorectal cancer (CRC) remains unclear. Our study revealed that HtrA3 is significantly upregulated in CRC at both mRNA and protein levels. TCGA database analysis showed that higher HtrA3 expression in colon adenocarcinoma is linked to poorer patient survival. Moreover, increased HtrA3 expression in HCT116 cells boosted their proliferation, migration, and invasion, while decreasing HtrA3 in SW480 cells had the reverse effect. In HCT116 cells with high HtrA3 levels, there was an increase in p-PI3K, p-AKT, and p-FOXO1, which reduced upon treatment with the PI3K inhibitor LY294002. Conversely, in SW480 cells with lowered HtrA3, these molecular levels decreased and rose again after treatment with the PI3K activator 740Y-P. HtrA3 also promoted cell proliferation, migration, and invasion in the presence of LY294002 and 740Y-P. In vivo experiments demonstrated that HtrA3 overexpression accelerated xenograft tumor growth. This study confirms that HtrA3’s overexpression in CRC cells promotes their proliferation, migration, and invasion by activating the PI3K-AKT-FOXO1 signaling pathway. Therefore, HtrA3 could be a significant factor in the advancement of CRC and may serve as a potential prognostic marker for guiding targeted CRC therapies. Citation Format: Jing Zheng, Xianhua Huang, Xian-e Peng, Yunli Wu. HtrA3 promotes malignant progression of colorectal cancer via PI3K-AKT-FOXO1 signaling pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3790.

Abstract 1914: Amivantamab efficacy in wildtype EGFR NSCLC tumors correlates with levels of ligand expression

Abstract Amivantamab (RYBREVANT™) is an FDA-approved, low-fucose IgG1 bispecific antibody targeting EGF and Met receptor tyrosine kinases, with proven clinical activity against EGFR mutant non-small cell lung cancer (NSCLC). Preclinical studies demonstrated that three context-dependent mechanisms of action (MOA) contribute to amivantamab efficacy: immune cell-mediated killing by antibody-dependent cellular cytotoxicity and trogocytosis, receptor internalization and degradation, and inhibition of ligand binding to both EGF and Met receptors. In tumors lacking known cancer-driver genetic aberrations, ligand binding to wildtype EGFR (EGFRWT) and/or MetWT may activate downstream signaling pathways, inducing pro-tumorigenic processes. Among the EGFR ligands, we demonstrated that amphiregulin (AREG) is highly expressed in EGFRWT NSCLC primary tumors, with significantly higher circulating protein levels in NSCLC patients than in healthy volunteers, and induces EGFR phosphorylation, promoting downstream signaling and increasing cell proliferation. Importantly, treatment of AREG-stimulated EGFRWT cells/tumors with amivantamab, or an AREG-targeting antibody, inhibits ligand-induced signaling and cell/tumor proliferation/growth. Across 10 EGFRWT NSCLC patient-derived xenograft mouse models, amivantamab efficacy correlated with AREG RNA levels, suggesting AREG as a potential predictive marker for amivantamab activity in this population. Interestingly, in these xenograft models, amivantamab anti-tumor activity was independent of amivantamab's Fc engagement with immune cells, suggesting that, in this context, the amivantamab’s ligand-blocking function is sufficient for its efficacy. Juxtaposedly, we previously demonstrated that low fucose amivantamab, active Fc domain, was necessary for maximal anti-tumor activity against tumors with EGFR or Met activating mutations. Altogether, these results demonstrated amivantamab context-dependent MOA for efficacy. In conclusion, these data 1) highlight EGFR ligand AREG as a driver of tumor growth in some EGFRWT NSCLC models, 2) illustrate the preclinical efficacy of amivantamab in ligand-driven EGFRWT NSCLC, and 3) identify AREG as a potential predictive biomarker for amivantamab against anti-EGFRWT therapies. Citation Format: Ricardo Rivera-Soto, Benjamin Henley, Marian Pulgar, Stacey Lehman-Tenuto, Himanshu Gupta, Megan Weindorfer, Smruthi Vijayaraghavan, Tsun-Wen Yao, Sylvie Laquerre, Sheri Moores. Amivantamab efficacy in wildtype EGFR NSCLC tumors correlates with levels of ligand expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1914.

Abstract 5296: Enhancing the strength and durability of vaccine-induced anti-tumor immunity with CD27 agonism of CD4+ T cells

Abstract Despite encouraging early phase clinical trials, decades of research have yet to yield effective cancer vaccine immunotherapies. To determine potential markers for success, we performed a follow-up study of a HER2-pulsed dendritic cell cancer vaccine Phase I trial performed in advanced HER2+ cancer patients. Surprisingly, we found that all treated patients were alive at over 18 years post-treatment. These patients still possessed significant HER2-specific memory T cell responses, typified by CD27 expression. The expansion of HER2-specific CD27+ memory T cells was also observed in patients from a separate clinical trial, who received a HER2-expressing viral vaccine. To validate these clinical responses, we vaccinated human CD27 transgenic mice with a HER2 viral vector again observing HER2-specific CD27+ memory T cells, suggesting the significance of this axis in eliciting and maintaining effective vaccine-specific memory T cell responses. We hypothesized that CD27 expression was not just a marker of memory T cells, but represented a critical activation pathway during memory formation. To determine the potential impact of CD27 agonism in vaccination, we combined HER2 vaccination with an anti-CD27 agonist antibody (Varlilumab). The combination treatment elicited a robust increase in HER2-specific CD4+ memory T cell responses that was maintained for at least 300 days post-vaccination. Using therapeutic HER2 vaccination in combination with anti-CD27 of HER2+ tumor bearing mice, we show significantly increased anti-tumor efficacy and further enhancement in combination with PD1 inhibition. The anti-tumor response was triggered by anti-CD27 antibody during the vaccine priming phase, which enhanced HER2-specific CD4+ T cells. Critically, we found that HER2 specific CD4+ T cell responses allowed for anti-tumor responses that persisted after CD8+ T cell depletion, indicating a direct role for antigen-specific CD4+ T cells independent of CD8+ T cells. Collectively, our results support a central role for antigen-specific CD27+ CD4+ T cells in cancer vaccination. These data support the addition of CD27 agonism to improve the therapeutic index of cancer vaccines and potentially enhance responses to immune checkpoint blockade in refractory cancers. Citation Format: Bin-Jin Hwang, Erika Crosby, Timothy Trotter, Li-Chung Tsao, Tao Wang, Congxiao Liu, Xiao Yang, Gangjun Lei, Junping Wei, Xingru Ma, Bushanqing Liu, Amy Hobeika, Michael Morse, Tibor Keler, Li-Zhen He, Herbert Kim Lyerly, Zachary Hartman. Enhancing the strength and durability of vaccine-induced anti-tumor immunity with CD27 agonism of CD4+ T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5296.

Abstract 6440: Novel N-myristoyltransferase immunohistochemistry-based predictive and prognostic tests for breast cancer

Abstract Breast cancer (BC) is the most common cancer worldwide, surpassing lung cancer incidence for the first time in 2020, and is the most common cancer diagnosed in American women. Despite the relative success of endocrine therapies in treating hormone receptor-positive (HR+) BC, de novo and developed resistances to these therapies (endocrine resistance) are still significant concerns. Furthermore, triple-negative breast cancer has a poor prognosis and remains one of the most challenging cancers to treat. We have discovered that the expression and localization of N-myristoyltransferase 2 (NMT2) could be prognostic and predictive markers for metastatic BC. We determined the expression patterns of NMT2 by immunohistochemical (IHC) analyses on primary tumor samples from treatment naïve BC patients. This retrospective study was blinded, and the tumor tissues in duplicate were stained with NMT2 monoclonal/polyclonal antibodies on an autostainer (Leica Bond). Stained slides were scored, and an "H" score representing the intensity and percent positive cells was given to each sample. In a combined cohort of BC represented by ER+, Triple Positive, and Triple Negative (n= 598) cases, we determined the expression and localization of NMT2. The localization pattern of NMT2 turned out to be a significant prognostic and predictive molecular signature. The nuclear localization serves as both prognostic and predictive markers. In a cohort of 447 ER+ BC cases that included 18 triple positive cases, the majority of the cases showed negative staining for nuclear NMT2 (n=394) and positive nuclear staining with differential expression in 53 cases. Whereas, in a cohort of 151 TNBC cases, nuclear NMT2 staining was positive in most cases (n= 116) and negative in 20 cases (undetermined; n= 15). The most important finding was the dichotomy of the nuclear NMT2 staining in predicting treatment response and prognosis. Both high cytoplasmic H score (&amp;gt;180) and positive nuclear staining (&amp;gt;0) predicted significant association with worse clinical outcomes for OS (HR = 1.72, P = .0284, 95% CI 1.06 to 2.81 for cytoplasmic NMT2 and HR = 1.74, P = 0.0006, CI 1.27 to 2.39 for the nuclear NMT2). Nuclear NMT2 status alone predicted a significant association with worse clinical outcomes to RFS (HR = 1.56, P = 0.0027, CI 1.17 to 2.09). The data from our study indicate that NMT2 could be a potential marker for predicting the likelihood of recurrence of BC and become part of routine first-line prognostic and predictive tests. Citation Format: Anuraag Shrivastav, Dean Reddick, Abinash Meher, Shailly Varma Shrivastav, Sahil Mittal, Vijayakrishna K. Gadi, Leigh Murphy. Novel N-myristoyltransferase immunohistochemistry-based predictive and prognostic tests for breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6440.

Abstract 3689: Optimizing CTC isolation techniques for molecular characterization of circulating tumor cells in clear cell renal cell carcinoma: A comparative study of EpCAM-based and density-based methods

Abstract Introduction: Renal cell carcinoma (RCC) belongs to the most invasive cancers with the high molecular intrapatient heterogeneity. Circulating tumor cells (CTCs) are cells that detach from the tumor, reflecting the activity of the metastatic procedure. The most popular CTCs isolation methods are based on EpCAM signal detection on epithelial tumor cells. The clear cell RCC (ccRCC) originate from epithelial entity, since that is the EpCAM-based method suitable to isolate CTCs in this cancer. However, the prone of CTCs to EMT is known, which may in consequence downregulate the EpCAM expression and change the cell profile (hybrid phenotype). This is one of the points challenging the CTCs isolation by RCC disease. In our study, we compare the effectivity of antibody-based and density-based isolations technics in ccRCC patients to detect potential markers in CTCs. Purpose: Our aim was to find a precise and costs effective method to isolate and characterize CTCs from ccRCC patients. Materials and Methods: Prospective blood samples (3mL) from pulmonary and/or osseous metastatic ccRCC patients were collected during the immunotherapy and from the healthy donors. CTCs were isolated using EpCAM beads (RCC n=13; healthy n=15) or density-based (RCC n=13; healthy n=15) method. Expression of potential RCC biomarkers (mucin-1 (Muc1), androgen receptor (AR)) was identified using qPCR (Real-Time PCR) followed by dPCR (digital PCR), the results status received from both methods was evaluated. Summary: Based on our prior results, we first isolated CTCs based on EpCAM beads. Using qPCR we analyzed expression of genes, which we previously associated to the RCC (Muc1, PD-L1, AR). The results revealed differences in Muc1 (40% of tested patients positive but none of the healthy donors), when PD-L1 did not differed and AR was not detected. Applying more precise dPCR did neither show any expression. We continued our investigations with isolation of CTCs using density-based method. We confirmed more frequent expression of Muc1 by RCC Patients than by donors, as in our EpCAM cohort. PD-L1 was similar in both groups. Results of AR obtained using qPCR were confusing, showing deviations within same patients. The use of dPCR displayed significant differences in expression between RCC patients (77%; median [range] 0.28 [0.00 - 0.62] and donors (60%; 0.04 [0.00 - 0.51] confirming dPCR as optimal tool for molecular CTC characterization. Conclusions: Based on our results we demonstrate higher expression of Muc1 and AR in CTCs of metastatic ccRCC patients. The isolation technic should be adapted to the disease status and treatment choice. Further investigations confirming Muc1 and AR as ccRCC biomarkers are required. Citation Format: Joanna Bialek, Anne Muthe, Stefan Yankulov, Felix Kawan, Georgios Gakis, Gerit Theil. Optimizing CTC isolation techniques for molecular characterization of circulating tumor cells in clear cell renal cell carcinoma: A comparative study of EpCAM-based and density-based methods [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3689.

An urban image in an urbanized landscape: measuring the visual impact of Tibur's amphitheater

Abstract Though infrequently used and largely superfluous, amphitheaters were often the most physically imposing and ideologically charged structures in a Roman city. The preponderance of extramural amphitheaters in Italy and their appearance in visual culture confirm they were potent markers of urban life and civic status. This paper contextualizes Tibur's imperial amphitheater within the Roman suburbium's persistent urban sprawl and villas, especially Hadrian's Villa, using a novel GIS visibility analysis. Its apparent size from various points in the surrounding landscape is quantified within empirical and qualitative scales developed for modern visual impact assessments. The results demonstrate the amphitheater's suburban location did more than integrate Tibur's extramural growth into the older urban center. It emphasized the city's urban appearance, even from long distances, and monumentalized alternate routes to the city used by the villa-owning elite, countering the ambiguous status of a liminal city that was both Rome's annex and an autonomous municipium.

Necroptosis pathway emerged as potential diagnosis markers in spinal cord injury.

The present research focused on identifying necroptosis-related differentially expressed genes (NRDEGs) in spinal cord injury (SCI) to highlight potential therapeutic and prognostic target genes in clinical SCI. Three SCI-related datasets were downloaded, including GSE151371, GSE5296 and GSE47681. MSigDB and KEGG datasets were searched for necroptosis-related genes (NRGs). Differentially expressed genes (DEGs) and NRGs were intersected to obtain NRDEGs. The MCC algorithm was employed to select the first 10 genes as hub genes. A protein-protein interaction (PPI) network related to NRDEGs was developed utilizing STRING. Several databases were searched to predict interactions between hub genes and miRNAs, transcription factors, potential drugs, and small molecules. Immunoassays were performed to identify DEGs using CIBERSORTx. Additionally, qRT-PCR was carried out to verify NRDEGs in an animal model of SCI. Combined analysis of all datasets identified 15 co-expressed DEGs and NRGs. GO and KEGG pathway analyses highlighted DEGs mostly belonged to pathways associated with necroptosis and apoptosis. Hub gene expression analysis showed high accuracy in SCI diagnosis was associated with the expression of CHMP7 and FADD. A total of two hub genes, i.e. CHMP7, FADD, were considered potential targets for SCI therapy.