Matched Tissues Research Articles

Abstract PO1-02-01: T Cell Receptor Sequencing to Monitor Pelareorep-Induced Expansion of Tumor Infiltrating Lymphocytes

Abstract Background: Tumor infiltrating lymphocytes (TILs) represent a major immunological tumor control mechanism that is associated with better prognosis in breast cancer. We have previously reported the effect of pelareorep (pela), an intravenously delivered, non-engineered oncolytic reovirus, on a composite measurement of TILs and tumor cellularity (CelTIL) from the AWARE-1 window-of-opportunity study in patients with early breast cancer (eBC). These results showed treatment increased in CelTIL scores especially in the atezolizumab group. To confirm and extend these findings we applied T cell receptor sequencing of matched tumor tissue and whole blood pre and post-treatment from the AWARE-1 study to further explore the effects of pela therapy on TILs. Methods: Newly diagnosed HR+/HER2- eBC patients were enrolled into two cohorts: Cohort 1: pela + letrozole (n=10); and Cohort 2: pela + letrozole + atezolizumab (n=10). Pela was administered on days 1, 2 and 8, 9, and atezolizumab was given on day 3. Tumor biopsies (FFPE samples) collected pre-treatment (~D-23) and on day ~21 when tumors were surgically removed. T cell fraction analysis and T cell receptor sequencing were performed by Adaptive Biotechnology (Seattle, Washington) Immunoseq protocol. Results: As was shown for the CelTIL scores, an increase in the tumor T cell fraction, a direct measurement of TILs, was observed at 1-month post-treatment in both cohorts with a mean percent increase of 21% for Cohort 1 and 67% for Cohort 2. Through TCR-sequencing of tumor tissue, we identified TIL clones and tracked their differential abundance in tumor tissue and blood post-treatment. The results of this analysis at Day 21 showed a mean post-treatment expansion of 10 tumor specific clones in the blood for Cohort 1, and a mean post-treatment increase of 40 tumor specific clones for Cohort 2. Conclusions: These results confirm the previously reported CelTIL results from AWARE-1 on pela-induced increases in TILs and demonstrate the expansion of these clones in both tumor and blood as a consequence of pela therapy for HR+/HER2- breast cancer. Citation Format: Richard Trauger, Houra Loghmani, Matt Coffey, Fernando Salvador, Joaquín Gavilá, Luis Manso, Tomás Pascual, Aleix Prat, Thomas Heineman. T Cell Receptor Sequencing to Monitor Pelareorep-Induced Expansion of Tumor Infiltrating Lymphocytes [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-02-01.

Abstract PS11-06: Analysis of HER2 expression changes from breast primary to brain metastases including HER2 Low and impact on overall survival

Abstract Background: Previous studies have shown that breast cancer receptor subtype switching between matched primary and metastatic brain metastases (BrM) is common. HER2 expression at low levels, termed “HER2 Low”, has emerged as a new therapeutic biomarker for highly active antibody drug conjugates with potential intracranial activity. Trastuzumab deruxtecan is FDA approved for the treatment of HER2 Low and HER2 positive breast cancer with emerging evidence of CNS responses and clinical benefit in patients with BrM. This study aimed to investigate the relationship between HER2 expression including HER2-Low between matched breast cancer primary tumors and brain metastases. We also sought to evaluate the impact of low HER2 expression on overall survival in breast cancer brain metastases. Methods: We identified pts with MBC seen at least once at a single, NCI-designated center and had a diagnosis of BrM between 2003 and 2023. Of 1252 MBC pts with a diagnosis of BrM, 265 pts underwent resection/biopsy for BrM with available ER/PR/HER2 status. Only pts with available matched primary and BrM tissue were analyzed. HER2 expression was defined as: HER2+ (3+ or 2+/ISH amplified), HER2-Low (1+, 2+/ISH neg) or HER2-0 [by ASCO-CAP guidelines]. Estrogen receptor (ER) status was defined as ER≥1%. Multivariate overall survival (OS) analyses by Cox proportional hazard models were determined from time of BrM resection to death or last follow-up between HER2+, HER2-Low and HER2-0 patients controlling for ER and age. Results: 197 matched breast cancer primary and BrMs with available ER, PR, and HER2 testing were evaluated. Median age at BrM resection was 51 yrs. ER was positive in 48% (N=95) of BrMs. Of 265 resected BrMs, HER2 expression was found in 72%: 49.8% HER2+ (N=132), 22.2% HER2-Low (N=59), and 27.9% HER2-0 (N=74). HER2 expression by 197 primary tumors was: 57% HER2+ (N=112), 24% HER2-Low (N=48), 19% HER2 0 (N=37). Amongst 112 HER2+ primary tumors, 97% (N=109) had concordant HER2+ BrMs, 3 (2.6%) switched to HER2-Low, with 100% retaining some HER2 expression. Of 37 HER2-0 primaries, 35% (13/37) gained HER2-Low expression and 5.4% (2/37) gained HER2+ expression in the BrM. Amongst 48 HER2-Low primary tumors, half (52%) changed expression to either HER2+ (21%) or HER2-0 (31.2%) in the brain. 66 tumors had extracranial tissue biopsied prior to BrM resection available for HER2 analysis. Of 15 HER2-0 extracranial tumors, 47% gained HER2-Low and 13% gained HER2+ at time of BrMs. After adjusting for age and ER status at the time of BrMs, patients with HER2+ BrM had a statistically significant lower risk of death at time of follow up than those with HER2-Low BrM (HR=0.41, P=0.0006). The risk of death between patients with HER2-0 BrM did not differ from HER2-Low BrM after adjustment for ER and age (HR=0.96, p =0.9). Median survival from BrM resection was 12.4m, 16.1m, 47.6m for HER2-Low, HER2-0 and HER2+ respectively (p = .0004). Conclusions: HER2 expression amongst primary, extracranial and brain metastases is dynamic with frequent change. Over 40% of HER2-0 primary or extracranial tumors gain HER2 expression in the brain. These changes have therapeutic implications given HER2 targeting antibody drug conjugates with improved OS and emerging evidence of CNS activity, highlighting the need for better diagnostics, including those that do not require invasive tissue sampling, to determine HER2 status in the brain. Patients with HER2-Low and HER2-0 BrMs have inferior survival and are a clinical unmet need. Citation Format: Alyssa Pereslete, Melissa Hughes, Alyssa Patterson, Janet Files, Kyleen Nguyen, Lauren Buckley, Ashka Patel, Abigail Moore, Eric Winer, Tianyu Li, Sara Tolaney, Nancy Lin, Sarah Sammons. Analysis of HER2 expression changes from breast primary to brain metastases including HER2 Low and impact on overall survival [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS11-06.

Abstract 1130: Tenascin-C In Arteriovenous Fistula Failure: Unraveling Venous Remodeling Dynamics

Background: AVF are the preferred vascular access for hemodialysis, with superior patency and lower infection rates than grafts or catheters. Nevertheless, AVF patency is reduced by both early thrombosis and NIH, culminating in diminished utilization due to one-year patency rates as low as 40-50%. Tenascin-C (TnC), an ECM glycoprotein with limited adult expression, is transiently present at sites of tissue injury and inflammation. TnC has been implicated in playing a role in the development of NIH. Hypothesis: Sustained TnC expression contributes to NIH, potentially leading to AVF failure. Methods: Female and male wild-type C57Bl/6 mice, aged 9-11 weeks, had sham surgery or AVF creation by puncturing the infrarenal aorta into the inferior vena cava (IVC) with a 25-gauge needle. Doppler ultrasound confirmed AVF presence. Tissue samples were collected for IF, WB, and qPCR. Human matched vein and AVF tissue were obtained from patients undergoing planned two-stage AVF creation surgeries and analyzed with IF. In vitro analysis of human dermal microvascular endothelial cells (HDMEC) was performed to investigate the role of TnC in thrombomodulin expression. Results: TnC expression significantly increased in the AVF by day 7, returning to baseline by day 21 and reaching negligible levels by day 42. The spatial and temporal regulation of TnC expression post-AVF creation correlated with ECM remodeling and inversely with thrombomodulin. Both mice and human studies demonstrated differential regulation of TnC and thrombomodulin in patent and occluded AVF. In HDMEC, TnC knockdown suppressed NF-kB expression and increased thrombomodulin expression, while exogenous TnC had the opposite effect. Conclusion: TnC plays a pivotal role in driving pathological inflammation, with its expression being both temporally and spatially regulated during venous remodeling post-AVF creation. Persistent TnC expression is noted in occluded mouse and human AVF, leading to decreased thrombomodulin expression and heightened thrombogenicity. Sustained TnC expression regulates the shift from physiological to pathological venous remodeling, underscoring the crucial role of the immune response in this process and highlighting potential translational therapeutic strategies.

Exploring Endocrine Signaling between Adipose Tissues and the Heart using Transfer Learning and Induced Pluripotent Stem Cell Models

Background: Because of the amount of evidence pointed toward obesity as a co-morbidity of heart disease, our goal in this study is to use a data-driven method to discover potential cross talk signals between adipose and cardiac tissues, which may reveal a physiological reason for this association. For this study, we hypothesized that adipose-cardiac crosstalk signaling can be found by unexpected co-expression between adipose tissue genes encoding secreted proteins and cardiac tissue genes and vice versa. By discovering new crosstalk signals between these two tissues, we can find new associations between heart disease and obesity. Methods: Cross-tissue correlations of gene expression have been used to identify candidate endocrine signals that mediate tissue crosstalk, which are otherwise diffcult to detect in circulation. However, the identification of significant cross-tissue gene co-expression is hindered by immense multiple-testing burden. Our goal here is to abstract gene expression patterns of tissues into eigengenes and then test their correlation with secreted protein coding genes across tissues in a pairwise fashion. We first used the NIH Common Fund Genotype-Tissue Expression data set (GTEx v8) which contains RNA seq gene expression data from matched tissues of over 17,000 transcriptomes, to learn the latent structure of gene expression across tissues. To do so, we used PLIER (pathway level information extractor), a matrix factorization algorithm to extract the latent targets as eigengenes that map to specific biological pathways in the target tissue (e.g., pathways in the heart in the case of adipose to heart crosstalk). The learned gene expression structure is then transferred to a smaller data set with matching donor adipose and heart transcript expression data, from which a correlation matrix is constructed to find significant relationships between source tissue crosstalk signals and target tissue eigengenes and nominate novel crosstalk signals. Results: Adipose to heart tissue cross talk correlations using PLIER derived latent eigengene targets revealed many adipokines that had strong correlations with pathways found in the heart tissue through similar gene expression. Among these strong correlations were eigengene targets that were wrapped in metabolism pathways particularly those of fatty acid, phospholipid, and protein metabolism. Adipokines that showed high significant and positive correlation with cardiac latent variable expression included Adiponectin (R = 0.32, p = 4.7e−13), Glutathione Peroxidase 3 (R = 0.34, p = 5e−15) and Galectin 9 (R = 0.37, p < 2.2e−16) among others. Based on these results, we nominated new potential relationships between adipose and cardiac tissue. Work is currently under way to validate these candidate crosstalk signals using human induced pluripotent stem cell (iPSC)-derived cardiomyocytes. Future Directions In conclusion, we have found strong evidence in silico to suggest a relationship between known secreted proteins from adipose tissue (adipokine) and the expression of important pathways (such as fatty acid and protein metabolism) in the heart. Based on the findings of these correlations between adipokines and latent eigengene pathways within the heart, our future directions will involved the understanding of potential downstream functional consequences mediated by the adipose secreted proteins in iPSC derived models. This project was supported in part by NIH award R03-OD032666 and the University of Colorado Translational Research Scholar Program to E.L. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The association between the expression level of nuclear paraspeckle assembly transcript 1 and the survival rate of head and neck cancer patients after treatment

Background/purposeThe long non-coding RNA (lncRNA) nuclear paraspeckle assembly transcript 1 (NEAT1) exhibits diverse and complicated functions in cancer progression. Despite reports suggesting both tumor-suppressive and oncogenic effects in various cancers, its specific role in head and neck squamous cell carcinoma (HNSCC) remains unclear. This study aimed to investigate the association between NEAT1 expression levels and survival outcomes in HNSCC patients. Materials and methodsPaired tissue samples of tumor and non-cancerous matching tissues (NCMT) from 92 HNSCC patients were collected. NEAT1 expression was analyzed using RT-qPCR. Clinical characteristics, treatment received, and survival rates of the patients were assessed to determine the correlation with NEAT1 expression and explore its association with alcohol, betel quid, and cigarette use. Additionally, we examined the effect of arecoline on NEAT1 expression in normal human oral keratinocytes (NHOK) and fibroblasts (NHOF). ResultsThe study revealed a significant downregulation of NEAT1 expression in oral cancer tissues compared to NCMT. Meanwhile, arecoline increased NEAT1 expression in NHOK and NHOF cells. However, patients with downregulated NEAT1 expression exhibited higher overall survival rates, particularly in those who did not receive chemotherapy or radiotherapy. ConclusionNEAT1 expression levels are associated with survival outcomes in HNSCC patients, with upregulated expression indicating a worse prognosis, suggesting this lncRNA might contribute to cancer aggressiveness, especially in the absence of active treatment. These findings indicate NEAT1 may serve as a potential prognostic biomarker in HNSCC, but further research is required to elucidate its role in cancer progression and its potential as a therapeutic target.

Up-Regulation of MELK Promotes Cell Growth and Invasion by Accelerating G1/S Transition and Indicates Poor Prognosis in Lung Adenocarcinoma.

Maternal embryonic leucine zipper kinase (MELK) is an oncogene in many tumors, although its contribution to lung adenocarcinoma (LUAD) is unclear. We examined MELK expression in patient LUAD tissue and matched healthy lung tissues. We investigated the connection between MELK expression and tumor differentiation, lymph node metastasis, and patient survival. We downregulated MELK expression using small-hairpin RNA to assess its impact on LUAD cell proliferation, clonogenicity, and invasion. We also investigated the molecular mechanism underlying these effects. MELK expression was significantly heightened in LUAD tissue as opposed to the matching healthy lung tissues. LUAD patients who had MELK overexpression had a worse prognosis. Suppression of MELK hinders proliferation, clonogenicity, and invasion of LUAD cells. The MELK suppression led to the arrest of the cell cycle's G1/S phase by reducing the cyclin E1 and cyclin D expression. Our outcomes manifest that MELK can function as a beneficial prognostic indication and a new therapy target for LUAD. MELK has an essential function in progressing LUAD, manifesting potential as a viable target for therapeutic intervention in this disease management.

Immune cell senescence and exhaustion promote the occurrence of liver metastasis in colorectal cancer by regulating epithelial-mesenchymal transition.

Liver metastasis (LM) stands as a primary cause of mortality in metastatic colorectal cancer (mCRC), posing a significant impediment to long-term survival benefits from targeted therapy and immunotherapy. However, there is currently a lack of comprehensive investigation into how senescent and exhausted immune cells contribute to LM. We gathered single-cell sequencing data from primary colorectal cancer (pCRC) and their corresponding matched LM tissues from 16 mCRC patients. In this study, we identified senescent and exhausted immune cells, performed enrichment analysis, cell communication, cell trajectory, and cell-based in vitro experiments to validate the results of single-cell multi-omics. This process allowed us to construct a regulatory network explaining the occurrence of LM. Finally, we utilized weighted gene co-expression network analysis (WGCNA) and 12 machine learning algorithms to create prognostic risk model. We identified senescent-like myeloid cells (SMCs) and exhausted T cells (TEXs) as the primary senescent and exhausted immune cells. Our findings indicate that SMCs and TEXs can potentially activate transcription factors downstream via ANGPTL4-SDC1/SDC4, this activation plays a role in regulating the epithelial-mesenchymal transition (EMT) program and facilitates the development of LM, the results of cell-based in vitro experiments have provided confirmation of this conclusion. We also developed and validated a prognostic risk model composed of 12 machine learning algorithms. This study elucidates the potential molecular mechanisms underlying the occurrence of LM from various angles through single-cell multi-omics analysis in CRC. It also constructs a network illustrating the role of senescent or exhausted immune cells in regulating EMT.

First insight about the ability of specific glycerophospholipids to discriminate non-small cell lung cancer subtypes.

Introduction: Discrimination between adenocarcinoma (ADC) and squamous cell carcinoma (SCC) subtypes in non-small cell lung cancer (NSCLC) patients is a significant challenge in oncology. Lipidomics analysis provides a promising approach for this differentiation. Methods: In an accompanying paper, we explored oxPCs levels in a cohort of 200 NSCLC patients. In this research, we utilized liquid chromatography coupled with mass spectrometry (LC-MS) to analyze the lipidomics profile of matching tissue and plasma samples from 25 NSCLC patients, comprising 11 ADC and 14 SCC cases. This study builds upon our previous findings, which highlighted the elevation of oxidised phosphatidylcholines (oxPCs) in NSCLC patients. Results: We identified eight lipid biomarkers that effectively differentiate between ADC and SCC subtypes using an untargeted approach. Notably, we observed a significant increase in plasma LPA 20:4, LPA 18:1, and LPA 18:2 levels in the ADC group compared to the SCC group. Conversely, tumour PC 16:0/18:2, PC 16:0/4:0; CHO, and plasma PC 16:0/18:2; OH, PC 18:0/20:4; OH, PC 16:0/20:4; OOH levels were significantly higher in the ADC group. Discussion: Our study is the first to report that plasma LPA levels can distinguish between ADC and SCC patients in NSCLC, suggesting a potential role for LPAs in NSCLC subtyping. This finding warrants further investigation into the mechanisms underlying these differences and their clinical implications.

A Pilot Immunohistochemical Study Identifies Hedgehog Pathway Expression in Sinonasal Adenocarcinoma.

Tumors of the head and neck, more specifically the squamous cell carcinoma, often show upregulation of the Hedgehog signaling pathway. However, almost nothing is known about its role in the sinonasal adenocarcinoma, either in intestinal or non-intestinal subtypes. In this work, we have analyzed immunohistochemical staining of six Hedgehog pathway proteins, sonic Hedgehog (SHH), Indian Hedgehog (IHH), Patched1 (PTCH1), Gli family zinc finger 1 (GLI1), Gli family zinc finger 2 (GLI2), and Gli family zinc finger 3 (GLI3), on 21 samples of sinonasal adenocarcinoma and compared them with six colon adenocarcinoma and three salivary gland tumors, as well as with matching healthy tissue, where available. We have detected GLI2 and PTCH1 in the majority of samples and also GLI1 in a subset of samples, while GLI3 and the ligands SHH and IHH were generally not detected. PTCH1 pattern of staining shows an interesting pattern, where healthy samples are mostly positive in the stromal compartment, while the signal shifts to the tumor compartment in tumors. This, taken together with a stronger signal of GLI2 in tumors compared to non-tumor tissues, suggests that the Hedgehog pathway is indeed activated in sinonasal adenocarcinoma. As Hedgehog pathway inhibitors are being tested in combination with other therapies for head and neck squamous cell carcinoma, this could provide a therapeutic option for patients with sinonasal adenocarcinoma as well.

MiR-451a was selectively sorted into exosomes and promoted the progression of esophageal squamous cell carcinoma through CAB39.

Exosomes are emerging mediators of cell-cell communication, which are secreted from cells and may be delivered into recipient cells in cell biological processes. Here, we examined microRNA (miRNA) expression in esophageal squamous cell carcinoma (ESCC) cells. We performed miRNA sequencing in exosomes and cells of KYSE150 and KYSE450 cell lines. Among these differentially expressed miRNAs, 20 of the miRNAs were detected in cells and exosomes. A heat map indicated that the level of miR-451a was higher in exosomes than in ESCC cells. Furthermore, miRNA pull-down assays and combined exosomes proteomic data showed that miR-451a interacts with YWHAE. Over-expression of YWHAE leads to miR-451a accumulation in the exosomes instead of the donor cells. We found that miR-451a was sorted into exosomes. However, the biological function of miR-451a remains unclear in ESCC. Here, Dual-luciferase reporter assay was conducted and it was proved that CAB39 is a target gene of miR-451a. Moreover, CAB39 is related to TGF-β1 from RNA-sequencing data of 155 paired of ESCC tissues and the matched tissues. Western Blot and qPCR revealed that CAB39 and TGF-β1 were positively correlated in ESCC. Over-expression of CAB39 were cocultured with PBMCs from the blood from healthy donors. Flow cytometry assays showed that apoptotic cells were significantly reduced after CAB39 over-expression and significantly increased after treated with TGF-β1 inhibitors. Thus, our data indicate that CAB39 weakens antitumor immunity through TGF-β1 in ESCC. In summary, YWHAE selectively sorted miR-451a into exosomes and it can weaken antitumor immunity promotes tumor progression through CAB39.

Functional analysis of ESM1 by shRNA-mediated knockdown of its expression in papillary thyroid cancer cells.

Endothelial specific molecule-1 (ESM1) is implicated as an oncogene in multiple human cancers. However, the function of ESM1 in papillary thyroid cancer (PTC) is not well understood. The current study aimed to investigate the effect of ESM1 on the growth, migration, and invasion of PTC to provide a novel perspective for PTC treatment. The expression levels of ESM1 in PTC tissues form 53 tumor tissue samples and 59 matching adjacent normal tissue samples were detected by immunohistochemical analysis. Knockdown of ESM1 expression in TPC-1 and SW579 cell lines was established to investigate its role in PTC. Moreover, cell proliferation, apoptosis, wound healing, and transwell assays were conducted in vitro to assess cell proliferation, migration and invasion. The findings revealed that ESM1 expression was significantly higher in PTC tissues than that found in paraneoplastic tissues (P<0.0001). Knockdown of ESM1 expression inhibited the proliferation, migration, and invasion of TPC-1 and SW579 cells in vitro. Compared with the control group, the mRNA and protein levels of ESM1 in PTC cells were significantly reduced following knockdown of its expression (P<0.01). In addition, ESM1-knockdown cells indicated decreased proliferation and decreased migratory and invasive activities (P<0.01, P<0.01, P<0.001, respectively). ESM1 was identified as a major gene in the occurrence and progression of PTC, which could increase the proliferation, migration, and invasion of PTC cells. It may be a promising diagnostic and therapeutic target gene.

Consistency Analysis of Programmed Death Ligand 1 Expression in Non–Small Cell Lung Cancer Between Pleural Effusion and Matched Primary Lung Cancer Tissues by Immunohistochemical Double Staining

In clinical practice, programmed death ligand 1 (PD-L1) detection is prone to nonspecific staining due to the complex cellular composition of pleural effusion smears. In this study, diaminobenzidine (DAB) and 3-amino-9-ethylcarbazole (AEC) immunohistochemistry double staining was performed to investigate PD-L1 expression in tumor cells from malignant pleural effusion (MPE). MPE was considered as a metastasis in non–small cell lung cancer patients; thus, the heterogeneity between metastatic and primary lung cancer was revealed as well. Ninety paired specimens of MPE cell blocks and matched primary lung cancer tissues from non–small cell lung cancer patients were subjected to PD-L1 and thyroid transcription factor-1(TTF-1)/p63 immunohistochemistry double staining. Two experienced pathologists independently evaluated PD-L1 expression using 3 cutoffs (1%, 10%, and 50%). PD-L1 expression in MPE was strongly correlated with that in matched primary lung cancer tissues (R = 0.813; P < .001). Using a 4-tier scale (cutoffs: 1%, 10%, and 50%), the concordance was 71.1% (Cohen’s κ = .534). Using a 2-tier scale, the concordance was 75.6% (1%, Cohen’s κ = 0.53), 78.9% (10%, Cohen’s κ = 0.574), and 95.6% (50%, Cohen’s κ = 0.754). The rates of PD-L1 positivity in MPE (56.7%) were higher than that in lung tissues (32.2%). All 27 discordant cases had higher scores in MPE. The double-staining method provided superior identification of PD-L1-positive tumor cells on a background with nonspecific staining. In conclusion, PD-L1 expression was moderately concordant between metastatic MPE cell blocks and matched primary lung carcinoma tissues, with variability related to tumor heterogeneity. MPE should be considered to detect PD-L1 when histological specimens are unattainable, especially when PD-L1 expression is >50%. PD-L1 positivity rates were higher in MPE. Double staining can improve PD-L1 detection by reducing false-negative/positive results.

Tubulin alpha-1b chain was identified as a prognosis and immune biomarker in pan-cancer combing with experimental validation in breast cancer

The α-tubulin subtype, Tubulin α-1b chain (TUBA1B), has been shown to influence immune cell infiltration, cancer growth, and survival across various malignancies. However, a comprehensive study has not yet been undertaken examining the immunological and predictive effects of TUBA1B in a pan-carcinoma context. Using data from TCGA, GEO, and other databases, we analyzed TUBA1B expression across various carcinoma types using transcriptional profiling, prognostic implications, genetic and epigenetic alterations, methylation patterns, and immunological significance. To validate our findings, we conducted Western blot analysis to assess TUBA1B protein levels in matched breast cancer tissue samples and performed CCK-8 proliferation assay, flow cytometry, transwell invasion, and migration assays to comprehensively examine the functional impact of TUBA1B on breast cancer cells. Our pan-cancer analysis found TUBA1B upregulation across most tumor types, with varying expression patterns in distinct immune and molecular subtypes. High TUBA1B expression was an independent risk factor and associated with poor prognoses in several cancers, including BRCA, KICH, LGG, LUAD, and MESO. TUBA1B also demonstrates moderate to high diagnostic accuracy in most tumor types. Increased m6A methylation levels were observed in the TUBA1B gene, while its promoter region displayed low methylation levels. TUBA1B's expression impacted some cancers by elevating tumor mutation burden, microsatellite instability, neoantigen formation, immune cell infiltration, and the modulation of immune checkpoints. Functional enrichment analysis highlights TUBA1B’s involvement in important cellular processes such as the cell cycle, p53 signaling, cell senescence, programmed cell death, and the regulation of immune-related pathways. Moreover, our study reveals higher TUBA1B protein expression in breast cancer tissues compared to adjacent tissues. In vitro experiments confirm that TUBA1B deletion reduces breast cancer cell proliferation, invasion, and migration while increasing apoptosis. In conclusion, our study suggests that TUBA1B could potentially serve as a diagnostic marker for predicting cancer immunological profiles and survival outcomes and shed light on the expression and role of TUBA1B in breast cancer, providing a solid foundation for considering it as a promising therapeutic target for breast cancer patient treatment.

Bioenergetic impairment in Gulf War illness assessed via 31P-MRS

Time for post-exercise phosphocreatine-recovery (PCr-R), deemed a robust index of mitochondrial function in vivo, was previously reported to be elevated (signifying impaired ATP production) in veterans with Gulf War illness (GWI). Here we sought to replicate the finding and assess the impact of contravening previous eligibility requirements. The replication sample comprised white males. Cases reported ≥ moderate muscle-weakness to match the organ assessed to an organ affected; controls lacked recent headache or multiple symptoms. The expansion sample added cases without muscle-weakness, controls with recent headache, females, nonwhites. PCr-R, following pedal-depression-exercise, was compared in veterans with GWI versus controls (sample N = 38). In the replication sample, PCr-R results closely matched the prior report: PCr-R veterans with GWI mean(SD) = 47.7(16.5); control mean(SD) = 30.3(9.2), p = 0.017. (Prior-study PCr-R veterans with GWI mean(SD) = 46.1(17.9), control mean(SD) = 29.0(8.7), p = 0.023. Combined replication + prior samples: p = 0.001.) No case–control difference was observed in the expansion sample. In cases, PCr-R related to muscle-weakness: PCr-R = 29.9(7.1), 38.2(8.9), 47.8(15.2) for muscle-weakness rated none/low, intermediate, and high respectively (p for trend = 0.02), validating desirability of matching tissue assessed to tissue affected. In controls, headache/multiple symptoms, sex, and ethnicity each mattered (affecting PCr-R significantly). This study affirms mitochondrial/bioenergetic impairment in veterans with GWI. The importance of careful case/control selection is underscored.

Abstract 5376: Quantitative profiling of HLA class I and class II antigens and neoantigens in tissue biopsy and PBMC samples using an optimized mass spectrometry-based workflow

Abstract Major histocompatibility complex (MHC) molecules play a central role in orchestrating immune responses by presenting antigenic peptides derived from both self and foreign proteins. In the context of cancer, understanding the repertoire of tumor-associated antigens (TAAs) presented by MHC molecules (or HLA molecules in human) is crucial for deciphering how the immune system recognizes and responds to malignant cells. The identification of neoantigens, unique to individual tumors due to somatic mutations, has become a focal point in immunopeptidomic studies. One significant hurdle in systematic immunopeptidomics analysis is the high input material requirement. Here, we present a semi-automated workflow to robustly identify and quantify immunopeptides from reduced amounts of clinical tissue biopsy and peripheral blood mononuclear cell samples. At the core of immunopeptidomics is the enrichment of HLA-associated peptides, followed by identification using mass spectrometry and bioinformatics tools. We optimized the native lysis and a sequential immunoprecipitation workflow for both class I and class II immunopeptides while ensuring scalability and reproducibility. Leveraging the magnetic properties of the beads, 1,000 samples can be processed within a week by a single operator. For both tissue and PBMC samples, we performed a systematic ramping experiment starting with as little as 2.5mg tissue or 5 million PBMCs. In all experiments, The established sample preparation offers high reproducibility and identifications of good quality: 1) Class-I immunopeptides: &amp;gt;60% of the peptides identified are 9-mers, &amp;gt;80% predicted strong binders, and the expected amino acids are enriched at the anchor positions; 2) Class-II immunopeptides, &amp;gt;50% of the peptides identified are 14-to-16-mers, and &amp;gt;50% are predicted strong binders. Furthermore, the pipeline is highly sensitive as we could still identify over 2,800 class-I immunopeptides when processing 2.5 mg fresh frozen tissue and 2,000 - 3,000 class-I immunopeptides when starting from 5 million PBMCs. Furthermore, the developed immunopeptidomics workflow was deployed to profile a cohort of 12 cancerous and matched healthy lung tissue samples. Their Class-I immunopeptidomes clearly displayed a pattern where matched tissues from each subject cluster together, further underlining the fact that the intricate immune-tumor interface is highly personalized. Overall, we established a robust pipeline for for class-I and II immunopeptidome profiling from clinically relevant sample types. Taking advantage of the nature of mass spectrometry-based methods, customized targeted assays can be developed without the need for affinity reagent, allowing specific and absolute quantification of any immunopeptide of interest. Citation Format: Ilja E. Shapiro, Marco Tognetti, Tikira Temu, Oliver M. Bernhardt, Daniel Redfern, Yuehan Feng, Roland Bruderer, Lukas Reiter. Quantitative profiling of HLA class I and class II antigens and neoantigens in tissue biopsy and PBMC samples using an optimized mass spectrometry-based workflow [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 5376.

Abstract 1143: Dissection of melanoma and cutaneous lymphoma spatial molecular architecture by multimodal single-cell and spatial transcriptomics with generative AI

Abstract Advances in single-cell multiomic technologies have revolutionized our understanding of how heterogeneous cell types and cell states shape the tumor microenvironment (TME). However, dissociated single cell profiling lacks spatial context. To resolve the organization, cell-cell communication, and granular structures in the TME, new single-cell spatial transcriptomic (ST) characterizations are needed. Here, we dissected the spatial molecular architecture of the melanoma (30 tumors, 30 patients) and transformed cutaneous T-cell lymphoma (tCTCL, 12 tumors, 6 patients) TMEs by Multiplexed Error-Robust FISH (305 gene MERFISH), with benchmarking scRNAseq data from matched tissues (6 melanoma scFFPEseq, 6 tCTCL scVDJ/RNAseq) for cross-platform validation. We profiled 565,122 cells in melanoma and 92584 cells in tCTCL by MERFISH. We interrogated both datasets using a novel computational framework that includes cell typing by Leiden clustering and marker genes, spatial neighborhood and receptor-ligand (R-L) analyses, and spatial/scRNA imputation via deep learning. In the melanoma dataset, we identified tumor cells, major TME cell types, and rare immune cell types (TCF7+ stem-like T-cells and CD3+TCRα+PAX5+CD79a+ B/T dual-expressor lymphocytes enriched in tertiary lymphoid structures). All cell types were validated in scFFPEseq. As immune R-L interactions are critical therapeutic targets, we developed a novel computation method to quantify spatial R-L interactions by accounting for cell-cell distance, identifying spatially informed R-L pairs that differ from dissociation-based inferences. CNV inference from melanoma scFFPEseq revealed intratumoral heterogeneity (ITH). We therefore adapted the conditional variational autoencoder ENVI to simultaneously incorporate scRNA and MERFISH spatial data into a unified latent embedding. ENVI-ITH successfully learned ITH subgroup information, enabling imputation of phylogenetic lineages on spatial MERFISH. CTCL MERFISH data revealed major TME cell types, yet separation of malignant vs benign T-cells by clustering and marker approaches is hindered by their overlapping gene expression. To overcome this barrier, we deployed ENVI to impute patient-specific malignant and benign TCR clonotypes by training on the matched scVDJ/RNA data, yielding an ENVI-TCR pipeline that can delineate malignant vs benign T-cells in situ. We validated the spatial pattern of malignant T-cells by patient-specific in situ TCR probes. In sum, we have interrogated the melanoma and CTCL TME by MERFISH and presented a novel computational framework for robustly co-embedding scRNA data with imaging-based ST to spatially resolve melanoma ITH and malignant/benign T-cell populations. We believe this approach will be highly impactful for melanoma and CTCL and can be broadly applied to other solid tumors and lymphomas. Citation Format: Zichao Liu, Xiaofei Song, Jiang He, Justin He, Jodi Balasi, Jeffrey H. Chuang, Pei-Ling Chen. Dissection of melanoma and cutaneous lymphoma spatial molecular architecture by multimodal single-cell and spatial transcriptomics with generative AI [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 1143.

Abstract 862: Multi-omic analysis of allelic expression patterns of imprinted and non-imprinted genes in cancer and their association with drug response

Abstract Malignant cells undergo a broad array of epigenetic changes, including changes in imprinting patterns. In normal cells, imprinted genes have parent of origin-specific monoallelic expression, which is controlled via epigenetic mechanisms. Imprinted gene loci are often complex and frequently include multiple isoforms, which have different individual patterns of parent-of-origin monoallelic or biallelic expression in the normal tissues. Many imprinted genes promote cell proliferation and body growth, and their expression is important during embryonic and postnatal development. Dysregulation of imprinting in tumors has been suggested to contribute to growth and proliferation of cancer cells. Previously we reported that chemotherapy responses of cancer cell lines from a variety of tumors and of patient-derived acute myeloid leukemia samples were associated with copy number, expression, or DNA methylation of selected imprinted genes. In the current project, we examined allele-specific patterns of expression (monoallelic vs. biallelic) of imprinted genes and isoforms in tumors and studied their potential effect on response of cancer cells to drug treatment. We analyzed expression patterns at the whole gene, isoform, and exon levels of 59,283 autosomal protein-coding genes and ncRNA genes, with a special focus on 94 imprinted genes and their isoforms. We developed a computational pipeline to evaluate monoallelic and biallelic expression patterns of each gene in cancer cell lines and in tumor samples based on available matched RNA-seq expression data, whole exome sequencing information, and copy number data. Our initial analysis included 108 cell lines from the Cancer Cell Line Encyclopedia (CCLE), which had been derived from 9 pediatric and adult tumor histologies. We observed variation of allelic expression patterns of imprinted genes and their isoforms in tumor cell lines, including differences across and within cancer histologies. We also observed predominantly monoallelic expression of multiple additional genes. Some of them (PRIM2, BCLAF1, MAP2K3, and SEC22B) had been previously reported as potentially imprinted. Monoallelic expression of other genes identified in our analysis (e.g., GRK2, CBX4, and additional genes) was likely caused by molecular mechanisms other than imprinting. Allelic expression patterns and overall expression levels of isoforms of multiple imprinted and non-imprinted monoallelically expressed genes (e.g., CPA4, MAP2K3, and other genes) were associated with in vitro drug response. We also report our ongoing validation of allelic expression patterns of imprinted and non-imprinted genes, isoforms, and exons in primary bladder and breast tumors and in matched solid normal tissue samples using patient data from the Cancer Genome Atlas (TCGA). These results provide new knowledge about epigenetic dysregulation in cancer. Citation Format: Julia Krushkal, Travis L. Jensen, George Wright, Yingdong Zhao. Multi-omic analysis of allelic expression patterns of imprinted and non-imprinted genes in cancer and their association with drug response [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 862.

Abstract 1516: Large scale, transcriptome-based analysis of TCR clonality reveals functional immunity in non-small cell lung cancer

Abstract Tumor-related T-cell activation and subsequent clonal expansion are essential for an effective anti-cancer immune response. Thus, quantifying the clonal distribution of T-cells in cancer tissue from patients might provide prognostic and predictive information, particularly in high immunogenic tumors like non-small cell lung cancer (NSCLC). We evaluated TCR clonality in the tumor microenvironment of NSCLC, its relation to the in situ immune phenotype, and its clinical impact in a cohort of 182 resected NSCLC patients. The α and β TCR clones were determined based on RNA sequencing data, and the Gini index was used to weight individual clonal distribution. We used matched FFPE cancer tissue for multiplex immunofluorescence cell analysis and in-situ sequencing of T-cell clones. The analysis revealed a broad spectrum of TCR clonality patterns, including tissue with high TCR diversity and high evenness (low Gini coefficient) and tissue with clonal dominance with low evenness (high Gini coefficient). Highly expended clones were detected in 15/182 patients, whereas 43/182 revealed no clonal enrichment. A low TCR clone evenness (high Gini coefficient) was significantly more frequent in normal lung areas than in the corresponding tumor areas (n=20, p=0.02). In the cancer tissue, a high Gini index further correlated with high tumor mutational burden, indicating neoantigen-induced T cell clone expansions. Correspondingly, high TCR clonality was associated with an inflamed tumor phenotype associated with PD-1+ immune cells, CD3+, CD8A+, CD163+, and CD138+ cells, as well as higher expression of genes connected to immune activation (PRF1, GZMB, GZMH) and T cell exhaustion (PD-L1, LAG3, TIGIT). Cancer tissue with dominant T-cell clones also showed higher numbers of mature tertiary lymphoid structures with germinal centers. In situ sequencing suggested a close spatial relation between specific T cell clones and tumor cells. Finally, NSCLC patients with dominant T-cell clones showed strong responses and longer survival when treated with immune checkpoint inhibitors. This suggests that TCR clonality analysis is a strong candidate for precision diagnostics. Plausibly, the dominant clones found in NSCLC are responsible for a specific but malfunctioning anti-tumor response that can be unleashed by immune checkpoint inhibition. Citation Format: Hui Yu, Anastasia Magoulopoulou, Masafumi Hori, Amanda Lindberg, Max Backman, Hans Brunström, Millaray Marincevic Zuniga, Johanna Mattsson, Akira Saito, Karin Leandersson, Mats Nilsson, Rose-Marie Amini, Patrick Micke, Carina Strell. Large scale, transcriptome-based analysis of TCR clonality reveals functional immunity in non-small cell lung 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 1516.

Abstract 3759: First clinical evidence of the tissue nanomechanical signature as a novel diagnostic and prognostic tool in patients with non-small-cell lung cancer

Abstract Introduction: Complex structural remodelling of cells and extracellular matrix during cancer initiation and progression elicit substantial biomechanical alterations, which can be measured by Atomic Force Microscopy (AFM) at the nanoscale level. Clinical and translational evidence indicates that tissue nanomechanical signature (NS) can be a useful tool for rapid cancer diagnosis, and assessment of cancer aggressiveness. We demonstrated that the AFM-based Automated and Reliable Tissue Diagnostics (ARTIDIS) platform represents a valid method for rapid diagnosis, prognosis, and treatment response assessment in patients with breast cancer. Here we aimed to use this platform to examine tumour biopsies from non-small cell lung cancer (NSCLC) patients for the first time. Methods: This is an ongoing, single center study at the Hospital Clinic of Barcelona and University of Barcelona (UHB) Spain, aiming to enroll 75 patients by January 2024 in the first phase, followed by 200 patients in the expansion phase. To date, 54 matching tumor and non-tumor fresh tissue biopsies were taken by experienced pathologists from fresh surgical resection specimens from 27 patients with previously diagnosed NSCLC. Tissue biopsies were measured with the ARTIDIS device in a blinded manner, without knowing the malignancy status of the biopsy. Routine clinico-pathologic parameters have been collected. The multiparametric nanomechanical signature has been analyzed in an automated manner using the proprietary AI-based ARTIDISNET software platform together with available clinico-pathologic parameters. Results: In total, the NSCLC tissue samples included 16 squamous cell carcinoma (SCC), 6 Adenocarcinoma (ADC), 1 large cell carcinoma (LCC) and 4 other histological subtypes. Non-tumor tissues exhibited significantly varied composition, including normal lung parenchyma, bronchial tissues, fibrosis and inflammatory changes. Among the 27 patients, 5 patients developed distant progression within 12 months after ARTIDIS measurements. Results analysis showed that the ARTIDIS NS can reliably differentiate malignant NSCLC lesions from non-tumor lung tissue, with a sensitivity of 0.84, specificity of 0.81, positive predictive value (PPV) of 0.79, negative predictive value (NPV) of 0.86 and accuracy of 0.82. In addition, the ARTIDIS NS can differentiate patients with and without distant progression with a sensitivity of 0.62, specificity of 0.81, PPV of 0.42, NPV of 0.90 and accuracy of 0.77. Conclusions: Here we demonstrated first clinical evidence of the ARTIDIS tissue NS as a novel diagnostic and prognostic tool in patients with NSCLC. This study aims to establish the diagnostic, prognostic, and predictive NS in patients NSCLC. In addition to local expansion at the UHB, study results will be further validated in ARTIDIS clinical program at MD Anderson Cancer Centre, USA. Citation Format: Jordi Alcaraz, Héctor Sanz-Fraile, Elba Marin, Paula Gausa Busquets, Kate Neal, Marselina Arshakyan, Marc Rico, Marc Boada, David Sanchez, Reinier Oropesa Nunez, Gitika Srivastava, Ahmed Jizawi, Sara Nizzero, Tobias Appenzeller, Mariam Gachechiladze, Marko Loparic, Marija Plodinec, Noemi Reguart. First clinical evidence of the tissue nanomechanical signature as a novel diagnostic and prognostic tool in patients with non-small-cell lung 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 3759.

Abstract 5668: Long non-coding RNA LINC01614 knockdown decreases carcinogenic behavior of papillary thyroid cancer in vitro

Abstract Papillary thyroid cancer (PTC) accounts for the vast majority of thyroid cancer cases with increasing incidence over the past few decades. PTC has a significantly higher prevalence in females in their childbearing years. Despite high survival rates, disease recurrence and metastasis remain prominent issues, thus highlighting the critical need for molecular biomarker detection. A newer class of molecular regulators, known as long non-coding RNAs (lncRNAs), have demonstrated differential expression patterns in a multitude of cancers. The largest subclass of these RNA molecules in the human genome consists of long intergenic non-coding RNAs (lincRNAs) with many of their functions yet to be identified. Known functional roles of lncRNAs center on their ability to modulate gene expression and drive carcinogenic behavior, deeming them attractive biomarkers and/or therapeutic targets for study. Bioinformatic analysis of RNA sequencing data from our patient sample biobank identified LINC01614 as being significantly upregulated (~12 fold increase) in PTC, when compared to normal, matched thyroid tissue. Further, it was also upregulated ~30-fold in male PTC compared to ~5.5 fold increase in female PTC, highlighting a potential sex correlation for study. Thus, we are studying LINC01614 as a potential PTC biomarker. Gene expression analysis of LINC01614 in vitro was found to be upregulated in various thyroid cancer cell lines, specifically two PTC cell lines: TPC1 (~5 fold; RET/PTC rearrangement; female) and K1 (~2 fold; BRAFV600E; male) when compared to the immortalized “normal” thyroid cell line (Nthy-ori-3-1). For phenotypic evaluation, we utilized the CRISPRi technique to employ transcriptional repression of LINC01614 in both TPC1 and K1. LINC01614 knockdown in TPC1 and K1 resulted in decreased healing capacity (~20%), clonogenicity (~50% and ~34%, respectively), and proliferation (~55% and ~45%, respectively). Studies are currently underway to further assess invasion, morphology, and RNA localization within the cell in vitro, as well as the identification of potential protein interactions and mechanisms of action of LINC01614 in PTC. Studying lncRNAs and their regulatory roles in cancer cell function is critical in the identification of various diagnostic and prognostic biomarkers for therapeutic intervention. Our data support the potential biological role of LINC01614 in driving PTC growth and metastatic potential. Further elucidation of how LINC01614 impacts PTC development and progression is a promising avenue of exploration. Citation Format: Danielle Quaranto, Michelle Carnazza, Nicole R. DeSouza, Sina Dadafarin, Augustine Moscatello, Humayun K. Islam, Raj K. Tiwari, Jan Geliebter. Long non-coding RNA LINC01614 knockdown decreases carcinogenic behavior of papillary thyroid cancer in vitro [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 5668.