Heterogeneous Expression Pattern Research Articles

Heterogeneous pattern of gene expression driven by TTN mutation is involved in the construction of a prognosis model of lung squamous cell carcinoma.

To investigate the impact that TTN mutation had on the gene heterogeneity expression and prognosis in patients with lung adenocarcinoma. In this study, the Cancer Genome Atlas (TCGA) dataset was used to analyze the TTN mutations in lung adenocarcinoma. Lung adenocarcinoma data was collected from the TCGA database, clinical information of patients was analyzed, and bioinformatics statistical methods were applied for mutation analysis and prognosis survival analysis. The results were verified using the GEO dataset. The incidence of TTN mutations in lung adenocarcinoma was found to be 73%, and it was related to the prognosis of lung adenocarcinoma. Ten genes were screened with significant contributions to prognosis. A prognosis model was constructed and verified by LASSO COX analysis in the TCGA and GEO datasets based on these ten beneficial factors. The independent prognostic factor H2BC9 for TTN mutation-driven gene heterogeneity expression was screened through multi-factor COX regression analysis. Our data showed that the gene heterogeneity expression, which was driven by TTN mutations, prolonged the survival of lung adenocarcinoma patients and provided valuable clues for the prognosis of TTN gene mutations in lung adenocarcinoma.

Unique Patterns of Heterogeneous Mismatch Repair Protein Expression in Colorectal Cancer Unveil Different Degrees of Tumor Mutational Burden and Distinct Tumor Microenvironment Features

Mismatch repair (MMR) protein expression in colorectal cancer (CRC) cells is usually homogeneously retained or lost. Rare lesions may show a heterogeneous pattern of MMR protein expression. We evaluated MMR protein expression (MLH1, MSH2, MSH6, and PMS2) in 200 CRCs, identifying 3 groups with proficient MMR protein expression (MMRp), deficient MMR protein expression (MMRd), and heterogeneous MMR protein expression (MMRh). MMRh tumors were microdissected on the basis of the expression of the heterogeneous marker. DNA was extracted and subjected to targeted sequencing. RNA was purified from bulk tumors of all MMRh cases and in a control series of 15 MMRp and 10 MMRd CRCs and analyzed using the PanCancer IO 360 Panel (NanoString Technologies). Twenty-nine of the 200 cases (14.5%) were MMRd. Nine cases (4.5%) showed a heterogeneous pattern of MMR expression, with 6 tumors harboring concomitant loss of one of the other MMR proteins, thus featuring areas with double loss at immunohistochemistry (IHC) testing (MMRh double-loss cases). Four of the 6 MMRh double-loss cases were suitable for a separate sequence variant analysis of IHC double-negative and IHC single-negative components of the tumor. In all lesions, both components exhibited a high tumor mutation burden (TMB). Nevertheless, a significant increase in TMB in the double-negative components was observed (mean TMB: negative, 70 mut/Mb vs positive, 59 mut/Mb) because of a higher number of subclonal variants compared with the other component. Comparative gene expression analyses among MMRd, MMRp, and MMRh CRCs highlighted differential gene expression patterns and an increased number of tumor-infiltrating lymphocytes in MMRh lesions, which is also characterized by a substantial population of exhausted CD8+ lymphocytes. We describe a unique subgroup of CRCs showing heterogeneous expression of MMR proteins in a background of concomitant loss of one of the other markers.

Comprehensive Characterization of HATs and HDACs in Human Cancers Reveals Their Role in Immune Checkpoint Blockade.

Histone acetylation that controlled by two mutually antagonistic enzyme families, histone acetyl transferases (HATs) and histone deacetylases (HDACs), as one of major epigenetic mechanisms controls transcription and its abnormal regulation was implicated in various aspects of cancer. However, the comprehensive understanding of HDACs and HATs in cancer is still lacking. Systematically analysis through 33 cancer types based on next-generation sequence data reveals heterogeneous expression pattern of HDACs and HATs across different cancer types. In particular, HDAC10 and HDAC6 show significant downregulation in most cancers. Principal components analysis (PCA) of pan-cancer reveals significant difference of HDACs and HATs between normal tissues and normal tissue adjacent to the tumor. The abnormal expression of HDACs and HATs was partially due to CNV and DNA methylation in multiple types of cancer. Prognostic significance (AUC reached 0.736) of HDACs and HATs demonstrates a five-gene signature including KAT2A, HAT1, KAT5, CREBBP and SIRT1 in KIRC. Analysis of NCI-60 drug database reveals the cytotoxic effect of several drugs are associated with dysregulated expression of HDACs and HATs. Analysis of immune infiltration and immunotherapy reveals that KAT2B and HDAC9 are associated with immune infiltration and immunotherapy. Our analysis provided comprehensive understanding of the regulation and implication of HDACs and HATs in pan-cancer. These findings provide novel evidence for biological investigating potential individual HDACs and HATs in the development and therapy of cancer in the future.

A spatiotemporally resolved single-cell atlas of the Plasmodium liver stage.

Malaria infection involves an obligatory, yet clinically silent liver stage1,2. Hepatocytes operate in repeating units termed lobules, exhibiting heterogeneous gene expression patterns along the lobule axis3, but the effects of hepatocyte zonation on parasite development at the molecular level remain unknown. Here we combine single-cell RNA sequencing4 and single-molecule transcript imaging5 to characterize the host and parasite temporal expression programmes in a zonally controlled manner for the rodent malaria parasite Plasmodium berghei ANKA. We identify differences in parasite gene expression in distinct zones, including potentially co-adaptive programmes related to iron and fatty acid metabolism. We find that parasites develop more rapidly in the pericentral lobule zones and identify a subpopulation of periportally biased hepatocytes that harbour abortive infections, reduced levels of Plasmodium transcripts and parasitophorous vacuole breakdown. These 'abortive hepatocytes', which appear predominantly with high parasite inoculum, upregulate immune recruitment and key signalling programmes. Our study provides a resource for understanding the liver stage of Plasmodium infection at high spatial resolution and highlights the heterogeneous behaviour of both the parasite and the host hepatocyte.

Characterization of Intercalated Cell Markers KIT and LINC01187 in Chromophobe Renal Cell Carcinoma and Other Renal Neoplasms.

Introduction. Chromophobe renal cell carcinoma (chromophobe RCC) is the third major subcategory of renal tumors after clear cell RCC and papillary RCC, accounting for approximately 5% of all RCC subtypes. Other oncocytic neoplasms seen commonly in surgical pathology practice include the eosinophilic variant of chromophobe RCC, renal oncocytoma, and low-grade oncocytic unclassified RCC. Methods. In our recent next-generation sequencing based study, we nominated a lineage-specific novel biomarker LINC01187 (long intergenic non-protein coding RNA 1187) which was found to be enriched in chromophobe RCC. Like KIT (cluster of differentiation 117; CD117), a clinically utilized chromophobe RCC related biomarker, LINC01187 is expressed in intercalated cells of the nephron. In this follow-up study, we performed KIT immunohistochemistry and LINC01187 RNA in situ hybridization (RNA-ISH) on a cohort of chromophobe RCC and other renal neoplasms, characterized the expression patterns, and quantified the expression signals of the two biomarkers in both primary and metastatic settings. Results. LINC01187, in comparison to KIT, exhibits stronger and more uniform expression within tumors while maintaining temporal and spatial consistency. LINC01187 also is devoid of intra-tumoral heterogeneous expression pattern, a phenomenon commonly noted with KIT. Conclusions. LINC01187 expression can augment the currently utilized KIT assay and help facilitate easy microscopic analyses in routine surgical pathology practice.

Matrix stiffness-dependent STEAP3 coordinated with PD-L2 identify tumor responding to sorafenib treatment in hepatocellular carcinoma

BackgroundFerroptosis have been implicated in tumorigenesis, tumor progression, and chemo- and immuno-therapy in cirrhotic hepatocellular carcinoma (HCC), indicating its association with matrix stiffness and clinical benefit of targeting drugs or immune checkpoint inhibitor. Here, we postulated that increased matrix stiffness reduces ferroptosis and impairs tumor immunity by regulating the expression of ferroptosis- and immune-related genes in HCC, which might be a robust predictor of therapeutic efficacy.MethodsUsing publicly available tissue microarray datasets, liver cancer rat model, and clinical specimen, ferroptosis-related differential genes in HCV-infected cirrhotic HCC and its mechanical heterogeneous pattern of expression were screened and identified. Further investigation on the underlying mechanism of matrix stiffness-regulated ferroptosis and the expression of immune mediator were performed. Finally, threshold analysis of HCC cases with sorafenib treatment revealed the value of clinical applications of these potential predictors.ResultsSTEAP3 was identified as the ferroptosis-related differential genes in HCV-infected cirrhotic HCC. Stiffer matrix decreased STEAP3 in the invasive front area of HCC and the liver cirrhotic tissue. Contrarily, softer matrix induced STEAP3 in the central area of HCC and the normal liver tissue. Immunological correlation of STEAP3 in cirrhotic HCC showed that STEAP3-mediated immune infiltration of CD4+ T and CD8+ T cells, macrophages, neutrophils, and dendritic cells and HCC prognosis, predicting to regulate immune infiltration. Overexpression of STEAP3 induced ferroptosis and inhibited the expression of immune mediator of PD-L2 on a stiff matrix. Especially, the ferroptosis- and immune-related gene predictive biomarker (FIGPB), including STEAP3 and PD-L2, predicts better clinical benefit of sorafenib in HCC patients.ConclusionsThis finding identifies matrix stiffness impairs ferroptosis and anti-tumor immunity by mediating STEAP3 and PD-L2. More importantly, coordinated with PD-L2, matrix stiffness-dependent STEAP3 could be applied as the independent predictors to favorable sorafenib response, and thus targeting it could be a potential diagnosis and treatment strategy for HCC.

Expression analysis of HIF-3α as a potent prognostic biomarker in various types of human cancers: a case of meta-analysis.

Hypoxia-inducible factors (HIFs) are transcription factors that get activated and stabilized in the heterodimerized form under hypoxic conditions. many studies have reported the importance of the HIF-1α and HIF-2α activity in biological pathways of hypoxic cancer cells. However, the importance of HIF-3α in a variety of cancers remains unknown. The expression profile of 13 different types of cancer samples from the Cancer Genome Atlas (TCGA) database were subjected to normalization, and differential gene expression analysis was performed using computational algorithms by R programming. Receiver operating characteristic tests and survival analyses were carried out for HIF-α subunits in different cancers. The expression status of HIF-3α was notably less in all cancer samples in contrast to their adjacent normal tissues. The expression degree of HIF-1α varied among distinct types of cancer and the expression degree of HIF-2α was lower in nearly all types of cancers. HIF-3α had very weak diagnostic potential, while HIF-2α had better diagnostic potential in most types of cancers compared to HIF-1α. Patients who had a higher level of HIF-3α had better survival, while the higher expression level of HIF-1α and HIF-2α were associated with worse survival in many types of cancers. Our findings showed that each HIF-α subunit had a unique heterogeneous expression pattern in different classes of cancers. The expression level of each HIF-α subunit correlated differently with the stages, tumor sizes, and survival rate of patients from different classes of TCGA cancers.

Profiling of extracellular vesicles of metastatic urothelial cancer patients to discover protein signatures related to treatment outcome.

The prognosis of metastatic urothelial carcinoma (mUC) patients is poor, and early prediction of systemic therapy response would be valuable to improve outcome. In this exploratory study, we investigated protein profiles in sequential plasma‐isolated extracellular vesicles (EVs) from a subset of mUC patients treated within a Phase I trial with vinflunine combined with sorafenib. The isolated EVs were of exosome size and expressed exosome markers CD9, TSG101 and SYND‐1. We found, no association between EVs/ml plasma at baseline and progression‐free survival (PFS). Protein profiling of EVs, using an antibody‐based 92‐plex Proximity Extension Assay on the Oncology II® platform, revealed a heterogeneous protein expression pattern. Qlucore bioinformatic analyses put forward a protein signature comprising of SYND‐1, TNFSF13, FGF‐BP1, TFPI‐2, GZMH, ABL1 and ERBB3 to be putatively associated with PFS. Similarly, a protein signature from EVs that related to best treatment response was found, which included FR‐alpha, TLR 3, TRAIL and FASLG. Several of the markers in the PFS or best treatment response signatures were also identified by a machine learning classification algorithm. In conclusion, protein profiling of EVs isolated from plasma of mUC patients shows a potential to identify protein signatures that may associate with PFS and/or treatment response.

Epidermal growth factor induces a trophectoderm lineage transcriptome resembling that of human embryos during reconstruction of blastoids from extended pluripotent stem cells.

This study aims to optimize the human extended pluripotent stem cell (EPSC) to trophectoderm (TE)-like cell induction with addition of EGF and improve the quality of the reconstructing blastoids. TE-like cells were differentiated from human EPSCs. RNA-seq data analysis was performed to compare with TE-like cells from multiple human pluripotent stem cells (hPSCs) and embryos. A small-scale compound selection was performed for optimizing the TE-like cell induction and the efficiency was characterized using TE-lineage markers expression by immunofluorescence stanning. Blastoids were generated by using the optimized TE-like cells and the undifferentiated human EPSCs through three-dimensional culture system. Single-cell RNA sequencing was performed to investigate the lineage segregation of the optimized blastoids to human blastocysts. TE-like cells derived from human EPSCs exhibited similar transcriptome with TE cells from embryos. Additionally, TE-like cells from multiple naive hPSCs exhibited heterogeneous gene expression patterns and signalling pathways because of the incomplete silencing of naive-specific genes and loss of imprinting. Furthermore, with the addition of EGF, TE-like cells derived from human EPSCs enhanced the TE lineage-related signalling pathways and exhibited more similar transcriptome to human embryos. Through resembling with undifferentiated human EPSCs, we elevated the quality and efficiency of reconstructing blastoids and separated more lineage cells with precise temporal and spatial expression, especially the PE lineage. Addition of EGF enhanced TE lineage differentiation and human blastoids reconstruction. The optimized blastoids could be used as a blastocyst model for simulating early embryonic development.

High nuclear ABCG1 expression is a poor predictor for hepatocellular carcinoma patient survival

BackgroundATP-binding cassette transporter G1 (ABCG1) regulates cellular cholesterol homeostasis and plays a significant role in tumor immunity. But, for hepatocellular carcinoma (HCC), the role of ABCG1 has not been investigated. Thus, the aim of this study was to evaluate the prognostic value and clinicopathological significance of ABCG1 in HCC. MethodsOne hundred and four adult patients with HCC were enrolled, and ABCG1 expression in paired HCC specimens was determined by immunohistochemistry. All these patients were stratified by ABCG1 expression, Kaplan-Meier analysis was used to compare the overall survival (OS) and recurrence-free survival (RFS), and Cox regression analysis was used to determine independent predictors of tumor recurrence. ResultsUpregulation of ABCG1 was observed in HCC samples compared to matched tumor-adjacent tissues. Patients with high nuclear ABCG1 expression had lower OS and RFS (P = 0.012 and P = 0.020, respectively). High nuclear ABCG1 expression was related to larger tumor size (P = 0.004) and tumor recurrence (P = 0.027). Although ABCG1 was expressed in the cytoplasm, cytosolic expression could not predict the outcome in patients with HCC. A new stratification pattern was established based on the heterogenous ABCG1 expression pattern: high risk (Highnucleus/Lowcytosol), moderate risk (Highnucleus/Highcytosol or Lownucleus/Lowcytosol), and low risk (Lownucleus/Highcytosol). This ABCG1-based risk stratification could distinguish the different OS and RFS in patients with HCC. Multivariate Cox regression analysis indicated that ABCG1 high risk was an independent predictor of poor RFS (P = 0.015). ConclusionsHigh nuclear ABCG1 expression indicates poor prognosis in patients with HCC. Asymmetric distribution of ABCG1 in the nucleus and cytoplasm may have an important role in tumor recurrence.

Abstract 5419: A unique pattern of heterogeneous MMR protein expression in colorectal cancer unveils different degrees of tumor mutational burden and distinct tumor microenvironment features

Abstract Introduction: Mismatch repair (MMR) protein expression in colorectal cancer (CRC) is usually homogeneously retained or lost. However, rare CRC lesions may show a heterogeneous pattern of MMR protein expression. We aimed to evaluate the molecular effect of a heterogeneous expression of the MMR proteins in a complex context of concomitant loss of one of the other effectors of the DNA repair mechanism. Experimental procedures: We evaluated MMR protein expression (by immunohistochemistry -IHC- for MLH1, MSH2, MSH6 and PMS2) in 200 CRCs, identifying 3 groups with proficient (MMRp), deficient (MMRd) and heterogeneous (MMRh) MMR. MMRh tumors were micro-dissected based on the expression of the heterogeneous marker, DNA was extracted and subjected to targeted sequencing (TSO500 panel, Illumina). RNA was purified from bulk tumors of all MMRh cases and in a control series of 10 MMRp and 10 MMRd CRCs and analyzed using the IO360 panel (NanoString). The degree of tumor infiltrating lymphocytes (TILs) was calculated on H&E slides and on IHC sections stained with antibodies raised against CD8. Results: Twenty-nine out of 200 cases were MMRd (14.5%). Nine cases (4.5%) showed a heterogeneous pattern of MMR expression, with 6 tumors harboring concomitant loss of one of the other MMR proteins, resulting in a microsatellite instable (MSI) phenotype. Five out of the 9 cases were suitable for separate mutational analysis of IHC-positive and IHC-(double)negative components of the tumor. Both components of all the lesions exhibited a high tumor mutation burden (mean TMB: 59 mut/Mb) in line with the MSI status, nevertheless a significant increase in TMB in the double-negative components was observed (mean TMB: neg. 67 mut/Mb vs pos. 53 mut/Mb), due to a higher number of sub-clonal, non-synonymous variants compared to the positive component. Comparative gene expression analyses between MMRd, MMRp and MMRh CRCs highlighted differential gene expression patterns, with MMRh tumors displaying a strong activation of angiogenesis, MAPK and PI3K-AKT axes. Moreover, these tumors showed the highest number of TILs, characterized by a substantial population of exhausted CD8+ lymphocytes. Conclusions: We describe a unique subgroup of CRCs showing heterogeneous expression of MMR proteins, in a background of concomitant loss of one of the other markers. This heterogeneity is associated with i) an increase of TMB and of unstable loci in the negative component, with additional markedly sub-clonal variants, ii) a high number of TILs, mostly exhausted CD8+ lymphocytes, and iii) a differential activation of signaling pathways. Taken together these data provide indirect evidence that heterogeneous loss of MMR proteins may impact on the biology of CRCs. Whether this pattern may influence response to immune checkpoint inhibition remains to be determined. Citation Format: Enrico Berrino, Maria Costanza Aquilano, Emanuele Valtorta, Vito Amodio, Giovanni Germano, Marco Gusmini, Ivana Sarotto, Anna Sapino, Silvia Marsoni, Andrea Sartore-Bianchi, Alberto Bardelli, Salvatore Siena, Emanuela Bonoldi, Caterina Marchio. A unique pattern of heterogeneous MMR protein expression in colorectal cancer unveils different degrees of tumor mutational burden and distinct tumor microenvironment features [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5419.

Abstract 2780: Loss of tumor HLA-A and B2M expression and association with biomarkers and clinical outcome

Abstract Loss of MHC-I and/or Beta 2 microglobulin (B2M) expression is regarded as a frequent resistance mechanism to immunotherapies. Indeed, downregulation of MHC-I expression and/or defects in the pathways of antigen presentation machinery are commonly reported in human cancers. Despite many reports published, available tools lack the robustness, specificity and sensitivity needed for systematic integration and analysis in clinical trials. In order to explore loss of antigen presentation as one of the major resistance mechanisms to checkpoint inhibitor (CPI) therapy treatment and to enable identification of patients with tumors harboring antigen presentation defects, we have developed immunohistochemistry (IHC)-based robust prototype assays to quantify HLA-A and B2M proteins in patient tissue samples. We have determined the prevalence of loss of HLA-A and B2M in banked primary tumor blocks as well as in 329 tumor metastatic biopsies collected during Roche-sponsored Phase 1 clinical trials investigating novel immunotherapies as single agents or in combination with CPI. Below are the key findings and preliminary interpretations:We confirm a significant prevalence of HLA-A and B2M loss (up to 65%) depending on indication, revealing heterogeneous expression patterns across patients and indications.We describe an increase of HLA-A and B2M loss from primary to metastatic lesions, which may reflect a selection of MHC-I-low/negative clones in spreading cancer cells (and maybe lines of therapies).We show that high on-treatment B2M expression is positively associated with clinical outcome (RECIST), while high baseline B2M expression only shows a trend (but no statistical significance) towards positive association with response. If the former (on-treatment expression) could be seen as a consequence of the drug effect (induction of inflammation, IFN-signaling and subsequent induction of MHC-I expression), the latter (baseline expression) would have been the foundation for enriching patient populations based on MHC-I expression. Based on these data, the absence of strong correlation between baseline expression and response does not support a cross-CIT enrichment approach at this stage.We see a treatment-induced increase of MHC-I expression in most of the patients. Preliminary conclusions could be that 1/MHC-I loss can be rescued in most of the cases we analyzed (few hard losses), 2/Since we only see few responders, MHC-I loss is not the dominant resistance mechanism or the drug(s) did not induce a strong-enough MHC-I expression to rescue it.This investigation reveals a highly dynamic expression of HLA-A and B2M in tumors affected by key variables such as indication, metastatic status, immunophenotype and immunotherapy treatment. We plan to explore MHC-I expression in larger cohorts of CPI-treated patients in order to confirm and broaden our preliminary findings. Citation Format: Bernhard Reis, Sebastian Dziadek, Jan Attig, Nico Graefe, Astrid Heller, Natascha Rieder, Bruno Gomes. Loss of tumor HLA-A and B2M expression and association with biomarkers and clinical outcome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2780.

A Novel Somatic Mutation of CACNA1H p.V1937M in Unilateral Primary Hyperaldosteronism.

BackgroundSomatic mutations for excess aldosterone production have been frequently identified as important roles in the pathogenesis of unilateral primary hyperaldosteronism (uPA). Although CACNA1H mutation represents a minor etiology in primary aldosteronism, it plays a significant role in causing uPAs in sporadic cases.ObjectiveTo identify novel somatic CACNA1H mutation in patients with uPA and investigate the pathophysiological, immunohistological, and clinical characteristics of the variant.MethodsWe applied a customized and targeted gene panel next-generation sequencing approach to detect mutations from the uPA cohort in Taiwan Primary Aldosteronism Investigation study group. Information from pre-diagnostic to postoperative data was collected, including past history, medications, blood pressure readings, biochemical data, and image studies. The functional role of the variant was confirmed by in vitro studies, demonstrating aldosterone production in variant-transfected human adrenal cell lines.ResultsWe identified a novel somatic CACNA1H mutation c.5809G>A (p.Val1937Met) in a uPA case. The CACNA1H gene encodes the pore-forming alpha-1H subunit of the voltage-dependent T-type calcium channel Cav3.2. This somatic CACNA1H p.V1937M variant showed excellent clinical and biochemical outcomes after ipsilateral adrenalectomy. The functional effect of somatic CACNA1H p.V1937M variant results in increased CYP11B2 expression and aldosterone biosynthesis in HAC15 cells. A distinct heterogeneous foamy pattern of CYP11B2 and CYP17A1 expression was identified in immunohistological staining, supporting the pathological evidence of aldosterone synthesis.ConclusionsThe somatic mutation of CACNA1H p.V1937M might be a pathogenic driver in aldosterone overproduction. This study provides new insight into the molecular mechanism and disease outcomes of uPA.

Gene Expression Profiling in Abdominal Aortic Aneurysms.

Gene expression profiling of abdominal aortic aneurysms (AAA) indicates that chronic inflammatory responses, active matrix metalloproteinases, and degradation of the extracellular matrix components are involved in disease development and progression. This study investigates intra- and interpersonal RNA genome-wide expression profiling differences (Illumina HumanHT-12, BeadCHIP expression) of 24 AAA biopsies from 12 patients using a single gene and pathway (GeneOntology, GO enrichment) analysis. Biopsies were collected during open surgical AAA repair and according to prior finite element analysis (FEA) from regions with the highest and lowest wall stress. Single gene analysis revealed a strong heterogeneity of RNA expression parameters within the same and different AAA biopsies. The pathway analysis of all samples showed significant enrichment of genes from three different signaling pathways (integrin signaling pathway: fold change FC 1.63, p = 0.001; cholecystokinin receptor pathway: FC 1.60, p = 0.011; inflammation mediated by chemokine signaling pathway: FC 1.45, p = 0.028). These results indicate heterogeneous gene expression patterns within the AAA vascular wall. Single biopsy investigations do not permit a comprehensive characterization of activated molecular processes in AAA disease.

DNA Damage Response Inhibitor and Anti-PD-L1 Therapy for Prostate Cancer: Development of Predictive Biomarkers

Despite significant benefit for other cancer subtypes, immune checkpoint therapy (ICT) has not yet been shown to significantly improve outcomes for men with castrationresistant prostate cancer (CRPC). Previous studies have shown that DNA damage response (DDR) deficiency, via genetic alteration and/or pharmacologic induction using DDR inhibitors (DDRi), may improve ICT response in prostate cancer and potentially oither solid tumors, in part due to the induction of innate immune activation and regulation of the tumor cell– and/or immune cell–expressed B7 immune checkpoint protein PD-L1, which can be targeted by anti-PD-L1 agents. With this information and orientation, development of PD-L1 expression as a potential predictive biomarker for clinical response to ICT therapy has received widespread attention. However, the complex heterogeneous expression patterns of tumor cell–expressed PD-L1 and its related posttranslational modification in tumors substantially complicate detection and interpretation of PD-L1 in clinical samples using IHC and currently available PD-L1 antibodies. We discuss the potential roles of PD-L1 in prostate cancer and potential approaches to more accurately detect its expression in clinical tumor samples. Additional understanding of PD-L1 functions and development of improved methods to detect PD-L1 as a predictive biomarker for prostate cancer immunotherapy will likely benefit patients with advanced prostate cancer.

Nitric Oxide (NO) Differentially Modulates the Ascorbate Peroxidase (APX) Isozymes of Sweet Pepper (Capsicum annuum L.) Fruits.

Nitric oxide (NO) is a free radical which modulates protein function and gene expression throughout all stages of plant development. Fruit ripening involves a complex scenario where drastic phenotypical and metabolic changes take place. Pepper fruits are one of the most consumed horticultural products worldwide which, at ripening, undergo crucial phenotypical and biochemical events, with NO and antioxidants being implicated. Based on previous transcriptomic (RNA-Seq), proteomics (iTRAQ), and enzymatic data, this study aimed to identify the ascorbate peroxidase (APX) gene and protein profiles in sweet peppers and to evaluate their potential modulation by NO during fruit ripening. The data show the existence of six CaAPX genes (CaAPX1–CaAPX6) that encode corresponding APX isozymes distributed in cytosol, plastids, mitochondria, and peroxisomes. The time course expression analysis of these genes showed heterogeneous expression patterns throughout the different ripening stages, and also as a consequence of treatment with NO gas. Additionally, six APX isozymes activities (APX I–APX VI) were identified by non-denaturing PAGE, and they were also differentially modulated during maturation and NO treatment. In vitro analyses of fruit samples in the presence of NO donors, peroxynitrite, and glutathione, showed that CaAPX activity was inhibited, thus suggesting that different posttranslational modifications (PTMs), including S-nitrosation, Tyr-nitration, and glutathionylation, respectively, may occur in APX isozymes. In silico analysis of the protein tertiary structure showed that residues Cys32 and Tyr235 were conserved in the six CaAPXs, and are thus likely potential targets for S-nitrosation and nitration, respectively. These data highlight the complex mechanisms of the regulation of APX isozymes during the ripening process of sweet pepper fruits and how NO can exert fine control. This information could be useful for postharvest technology; NO regulates H2O2 levels through the different APX isozymes and, consequently, could modulate the shelf life and nutritional quality of pepper fruits.

Implications of Heterogeneity of Epithelial-Mesenchymal States in Acromegaly Therapeutic Pharmacologic Response.

Acromegaly is caused by excess growth hormone (GH) produced by a pituitary tumor. First-generation somatostatin receptor ligands (SRLs) are the first-line treatment. Several studies have linked E-cadherin loss and epithelial-mesenchymal transition (EMT) with resistance to SRLs. Our aim was to study EMT and its relationship with SRLs resistance in GH-producing tumors. We analyzed the expression of EMT-related genes by RT-qPCR in 57 tumors. The postsurgical response to SRLs was categorized as complete response, partial response, or nonresponse if IGF-1 was normal, had decreased more than 30% without normalization, or neither of those, respectively. Most tumors showed a hybrid and variable EMT expression profile not specifically associated with SRL response instead of a defined epithelial or mesenchymal phenotype. However, high SNAI1 expression was related to invasive and SRL-nonresponsive tumors. RORC was overexpressed in tumors treated with SRLs before surgery, and this increased expression was more prominent in those cases that normalized postsurgical IGF-1 levels under SRL treatment. In conclusion, GH-producing tumors showed a heterogeneous expression pattern of EMT-related genes that would partly explain the heterogeneous response to SRLs. SNAI1 and RORC may be useful to predict response to SRLs and help medical treatment decision making.

Abstract PD6-03: Spatio-molecular dissection of the breast cancer metastatic microenvironment

Abstract Metastatic breast cancer (MBC) remains incurable due to inevitable development of therapeutic resistance. Although tumor cell intrinsic mechanisms of resistance in MBC are beginning to be elucidated by bulk sequencing studies, the roles of the tumor microenvironment and intratumor heterogeneity in therapeutic resistance remain underexplored due to both technological barriers and limited availability of samples. To comprehensively capture these characteristics we have adapted a research biopsy protocol to collect tissue for an array of single-cell and spatio-molecular assays whose performance we have optimized for MBC, including single-cell and single-nucleus RNA sequencing, Slide-Seq, Multiplexed Error-Robust FISH (MERFISH), Expansion Sequencing (ExSEQ), Co-detection by Indexing (CODEX) and Multiplexed Ion Beam Imaging (MIBI). To date, we have successfully performed single-cell or single-nucleus RNAseq in 67 MBC biopsies and generated detailed accompanying clinical annotations for each. These samples provide a representation of the clinicopathological diversity of MBC including different breast cancer subtypes (44 HR+/HER2-, 3 HR-/HER2+, 3 HR+/HER2+, 16 TNBC, 1 unknown), common anatomic sites of metastasis (37 liver, 9 axilla, 7 breast, 5 bone, 3 chest wall, 3 neck, 1 brain, 1 lung, 1 skin), metastatic presentations (53 recurrent, 14 de novo) and histologic subtypes in the breast (45 IDC, 7 ILC, 6 mixed, 3 DCIS, 1 mucinous, 5 unknown/NA). Following optimization, both single-cell and single-nucleus RNA seq perform well in these MBC biopsies recovering all expected cell types including the malignant, stromal (e.g. fibroblasts, endothelial cells), myeloid (e.g. monocytes, macrophages) and lymphoid compartments (e.g. T cells, B cells, NK cells) as well as relevant oncogenic programs (e.g. cell cycle programs in all compartments; EMT-like and ER signaling programs in the malignant compartment, immune checkpoint programs in the lymphoid compartment; and fibroblast activation and vascular homeostasis programs in the stromal compartment). In addition to differences between the two techniques, these data demonstrate substantial intratumor heterogeneity in cell type composition. For example in liver biopsies the average number of cells per sample compartment by single nucleus RNA-seq was 6745 malignant (56%, SD 4216), 4637 stromal (41%, SD 3727), 1196 lymphoid (8%, SD 1617) and 874 myeloid (6%, SD 852); in breast biopsies the average number of cells per compartment by single nucleus RNA-seq was 6421 malignant (70%, SD 3497), 1628 stromal (24%, SD 117), 333 lymphoid (4%, SD 170) and 213 myeloid (3%, SD 117). Additionally, we find both inter- and intra-tumor heterogeneity in expression patterns and programs including, for example, expression of ER, PR and HER2 within clinical receptor subtypes (log normalized counts for ER expression in tumor cells by single cell RNA-seq: HR+/HER2- 0.921 (SD 0.714); HR+/HER2+ 0.768 (SD 0.624); HR-/HER2+ 0.018 (SD 0.122); and HR-/HER2- 0.005 (SD 0.066). For a subset of 13 biopsies we are also completing the spatiomolecular characterization methods on serial sections of a single adjacent biopsy. This unique experimental setup was designed to enable efficient comparison and integration of these assays. In spite of differences between experimental techniques and readouts, cell typing can be approached by annotation transfer from matching single cell or single nucleus RNAseq data, enabling exploratory analyses including evaluation of spatial phenotypes and cell type colocalization. Overall, these single cell and spatial data afford a comprehensive atlas including cell types, cell states/programs, cell interactions and spatial organization in MBC lesions. Future analyses will include serial biopsies over time and integration of clinicopathologic data including therapeutic response and resistance. Citation Format: Daniel L Abravanel, Johanna Klughammer, Timothy Blosser, Yury Goltsev, Sizun Jiang, Yunjao Bai, Evan Murray, Shahar Alon, Yi Cui, Daniel R Goodwin, Anubhav Sinha, Ofir Cohen, Michal Slyper, Orr Ashenberg, Danielle Dionne, Judit Jané-Valbuena, Caroline BM Porter, Asa Segerstolpe, Julia Waldman, Sébastien Vigneau, Karla Helvie, Allison Frangieh, Laura DelloStritto, Miraj Patel, Jingyi We, Kathleen Pfaff, Nicole Cullen, Ana Lako, Madison Turner, Isaac Wakiro, Sara Napolitano, Abhay Kanodia, Rebecca Ortiz, Colin MacKichan, Stephanie Inga, Judy Chen, Aaron R Thorner, Asaf Rotem, Scott Rodig, Fei Chen, Edward S Boyden, Garry P Nolan, Xiaowei Zhuang, Orit Rozenblatt-Rosen, Bruce E Johnson, Aviv Regev, Nikhil Wagle. Spatio-molecular dissection of the breast cancer metastatic microenvironment [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD6-03.

English

BACKGROUND Early diagnosis of colorectal cancer is associated with a good prognosis and uncovering the biological mechanisms underlying pathogenesis and progression of this cancer form the recent focus of research. E-cadherin is one of the strong intercellular adhesion molecules in epithelial cells and one of the best characterized markers of epithelial-mesenchymal transition. The objectives of this study were to assess the pattern of expression of E-cadherin in colorectal cancer in patients from a medical college in Kerala, India and also determine the association between the expression of E-cadherin and different morphological parameters as well as the grade and stage of the tumour. METHODS All specimens of histologically diagnosed colorectal cancers received in the department were included in the study. Immunohistochemical staining of formalinfixed paraffin-embedded tissue was done. The intensity of E-cadherin staining was scored and analyzed for different tissues. Frequency tables of different variables were analyzed using the chi-square test. The significance of the correlation between different variables was assessed using Fischer’s exact test. P-value < 0.05 was considered statistically significant. RESULTS A total of 43 specimens of histologically diagnosed colorectal cancers were analyzed for the study. The mean age of the participants was 58 years. Majority of the tumours were < 5 cm in size. Both homogenous (55.8 %) and heterogeneous staining (44.2 %) were noted in the E-cadherin staining pattern among the participants. In patients with low E-cadherin index, 12 of them showed a heterogeneous pattern of expression. E-cadherin expression was not statistically associated with tumour differentiation in the patients. CONCLUSIONS There is a significant difference in E-cadherin expression between low and highgrade tumours of colorectal cancer. High-grade tumours showed a more heterogeneous staining pattern correlating with increased invasiveness of the tumour. The heterogeneous pattern of staining in E-cadherin among adjacent cells of tumour results from reduced expression of the protein in the tumour. KEY WORDS E cadherin, Colorectal Cancer, Cancer Marker.

Gap junctions mediate glucose transfer to promote colon cancer growth in three-dimensional spheroid culture

In vivo tumor growth is characterized by a necrotic core generated by oxygen and nutrients gradients, which is replicated by in vitro three-dimensional (3D) tumor spheroids but not traditional two-dimensional cell monolayers. Gap junctions provide direct communication between adjacent cells and play a critical role in cancer development, but their effects are still debatable. In this study, we found that connexin 43 (Cx43) reduced the area of necrotic core in colon cancer 3D spheroids, thus providing a growth advantage. This impact is dependent on gap junction channel functions, as the channel blocker carbenoxolone or connexin channel death mutant reverses this effect. Additionally, enhanced glucose uptake was detected in Cx43-overexpressing spheroids, along with upregulated mTOR, downregulated AMPK signaling, increased ATP content, and enhanced oxygen consumption rate. Furthermore, the xenograft mouse model confirmed the growth advantage of Cx43 in vivo. RNAseq data and clinical information from The Cancer Genome Atlas (TCGA) database indicated a more heterogeneous expression pattern of Cx43 in colon cancer compared to normal colon tissue, and higher Cx43 level is associated with worse clinical outcomes. Our data suggest a novel function of connexin in tumor growth, that gap junctions may provide nutrients transmitting routes in lieu of vasculature to meet the increasing metabolic requirement of solid tumors.