We evaluated the relevance of PD-1+CD8+ T-cells in gastric cancer (GC) including prognostic significance, association with chemotherapy and immunotherapy sensitivity and correlations with the tumor microenvironment (TME).Discovery cohort: GC samples were evaluated for AE1/3, CD8, PD-1, Ki-67 and Granzyme-B expression with fluorescence-based multiplex immunohistochemistry (mIHC). Validation cohorts: we analyzed bulk RNAseq GC datasets from TCGA, the "3G" chemotherapy trial and an immunotherapy phase 2 trial. The cox proportional hazards model was used to identify factors that influenced overall survival (OS). To study the TME, we analyzed single-cell RNAseq performed on GCs.In the discovery cohort of 350 GCs, increased PD-1 expression of CD8 T-cells was prognostic for OS (HR 0.822, p = 0.042). PD-1 expression in CD8 T-cells highly correlated with cytolytic [Granzyme-B+] (r = 0.714, p < 0.001) and proliferative [Ki-67+] (r = 0.798, p < 0.001) activity. Analysis of bulk RNAseq datasets showed tumors with high PD-1 and CD8A expression levels had improved OS when treated with immunotherapy (HR 0.117, p = 0.036) and chemotherapy (HR 0.475, p = 0.017). Analysis of an scRNAseq dataset of 152,423 cells from 40 GCs revealed that T-cell and NK-cell proportions were higher (24% vs 18% and 19% vs 15%, p < 0.0001), while macrophage proportions were lower (7% vs 11%, p < 0.0001) in CD8PD-1high compared to CD8PD-1low tumors.This is one of the largest GC cohorts of mIHC combined with analysis of multiple datasets providing orthogonal validation of the clinical relevance of PD-1+CD8+ T-cells being associated with improved OS. CD8PD-1high tumors have distinct features of an immunologically active, T-cell inflamed TME.
Abstract Background Within the tumor microenvironment (TME), the association of B lymphocytes (B cells) with prognosis and therapy response in gastric cancer (GC) remains poorly characterized. We investigated the predictive and prognostic value of B cells, including their spatial organization within the TME, in one of the largest multi-cohort studies to date. Methods Using CD20 immunohistochemistry, we evaluated B cell density in resection specimens from 977 patients with resectable GC across three cohorts, including the randomized phase III Korean CLASSIC trial. The relationship between CD20 density, clinicopathological characteristics, and overall survival (OS) was analyzed. Digital spatial profiling of 1063 regions of interest from 15 patients was performed to characterize B cell distribution within different regions of interest (ROIs) using the NanoString GeoMx platform. Results CD20 density was significantly higher in diffuse-type GC compared to intestinal-type ( p = 0.000012). Patients with CD20-low diffuse-type GC had the shortest OS in the CLASSIC trial (median OS: 49 vs 62 months, HR: 1.9, 95% CI: 1.2–3.0, p = 0.003) and in a Japanese cohort (median OS: 49 vs 67 months, HR: 2.2, 95% CI: 1.2–4.0, p = 0.011). This survival difference was not seen in patients treated with adjuvant chemotherapy (median OS: 62 vs 63 months, HR: 1.8, 95% CI: 0.88–3.5, p = 0.108). Spatial profiling revealed significant B cell enrichment within tumor ROIs compared to the stroma, particularly in diffuse-type GC. Conclusions Low CD20 positivity, especially in diffuse-type GC, is linked to poor prognosis and may identify patients who could benefit from chemotherapy. These findings underscore the role of B cells in GC.
Abstract PURPOSE HER2-positive gastric cancer (HER2+ GC) exhibits significant intra-tumoral heterogeneity and frequent development of resistance to HER2-targeted therapies. This study aimed to characterize the spatial tumor microenvironment (TME) in HER2+ GC and identify mechanisms of resistance to HER2 blockade including trastuzumab and trastuzumab deruxtecan (T-DXd), with the goal of informing novel therapeutic strategies. PATIENTS AND METHODS We performed spatial transcriptomics on pre-and post-treatment samples from patients with HER2+ metastatic GC who received trastuzumab-based therapy. We also established patient-derived organoids (PDOs) to investigate mechanisms of trastuzumab resistance in vitro . RESULTS ERBB2 -high tumor regions were found to be "immune cold", characterized by low PD-L1 expression and reduced lymphocyte infiltration. We identified two distinct mechanisms of acquired trastuzumab resistance: epithelial-mesenchymal transition (EMT) and upregulation of the endoplasmic reticulum-associated protein degradation (ERAD) pathway. EMT-positive tumors showed increased expression of immune checkpoints, including PD-L1 , and the chemokine CCL2 . Non-EMT tumors exhibited upregulation of the ERAD pathway, highlighting it as a potential therapeutic target. Importantly, we observed increased expression of the promising therapeutic target CLDN18.2, in trastuzumab-resistant tumors. Additionally, loss of HLA was identified as a potential mechanism of resistance to trastuzumab deruxtecan (T-DXd). CONCLUSION Our spatial profiling study reveals distinct TME features and resistance mechanisms in HER2+ GC, providing a valuable resource for future research and therapeutic development. The identification of potential therapeutic targets, such as CLDN18.2, may pave the way for novel treatment strategies to overcome resistance and improve outcomes for patients with HER2+ GC.
Intestinal metaplasia (IM) is a pre-malignant condition of the gastric mucosa associated with increased gastric cancer (GC) risk. Analyzing 1,256 gastric samples (1,152 IMs) across 692 subjects from a prospective 10-year study, we identify 26 IM driver genes in diverse pathways including chromatin regulation (ARID1A) and intestinal homeostasis (SOX9). Single-cell and spatial profiles highlight changes in tissue ecology and IM lineage heterogeneity, including an intestinal stem-cell dominant cellular compartment linked to early malignancy. Expanded transcriptome profiling reveals expression-based molecular subtypes of IM associated with incomplete histology, antral/intestinal cell types, ARID1A mutations, inflammation, and microbial communities normally associated with the healthy oral tract. We demonstrate that combined clinical-genomic models outperform clinical-only models in predicting IMs likely to transform to GC. By highlighting strategies for accurately identifying IM patients at high GC risk and a role for microbial dysbiosis in IM progression, our results raise opportunities for GC precision prevention and interception.
Abstract Peritoneal metastases (PM) in gastric cancer (GC) portend a poor prognosis, yet our understanding of tumor microenvironmental (TME) characteristics associated with GCPM remain limited. Here, we analyzed intrinsic genomic alterations and transcriptomic programs predictive of GCPM in a prospective cohort of 248 patients, identifying CDH1 , PIGR , and ELF3 mutations as predictors. By inspecting the spatial dynamics of the TME, we find that tumor compartment infiltration of pro-tumorigenic cell types such as inflammatory cancer-associated fibroblasts (CAFs) predict peritoneal recurrence. Next, in a cross-sectional study of 205 samples from 55 patients, distinct pathways and immune compositions in GCPM relative to liver metastases highlight the TME’s significance in transcoelomic metastases. Notably, several putative therapeutic targets exhibited distinct expression patterns between PTs and PMs. We also observed increased immune infiltration in GCPMs treated with systemic immunotherapy and intraperitoneal chemotherapy. Our findings highlight transcriptomic variations and niche reprogramming in the GCPM peritoneal environment, revealing roles of myeloid dendritic cells, effector memory CD8+ T cells, and CAFs in metastatic progression. Statement of significance Comprehensive molecular profiling of gastric cancer primary and peritoneal tumors unveils crucial insights into the distinct molecular and immune landscape of peritoneal metastases. Identifying predictive markers and therapeutic targets emphasizes the significance of tumor microenvironment alterations in guiding future therapies for gastric cancer peritoneal metastasis.
Abstract Background: The poor prognosis of peritoneal metastasis (PM) in colorectal cancer (CRC) is typically attributed to, and characterized by, resistance to systemic chemotherapy and immunotherapy. The evolutionarily conserved plasma-peritoneal barrier as well as reduced tumor tissue vascularity contributes to poorer responses to systemically administered antineoplastic therapy. Beyond these, we sought to understand transcriptomic variations of important therapeutic targets implicated in peritoneal organotropism and by extension, resistance to systemic regimens in CRC PM. Methodology: FFPE tissue samples from CRC patients were collected during oncological surgical resection of the synchronous PM (cytoreductive surgery [CRS] with or without hyperthermic intra-peritoneal chemotherapy [HIPEC]), or exploratory laparotomy for palliative surgery, with the majority of patients receiving systemic therapy prior to surgery. Samples were sequenced with whole transcriptomic sequencing. Paired and unpaired T-tests between PT and PM samples were undertaken with log2(fragments per kilobase of exon per million mapped [FPKM]) gene expression values. Results: Fifty-six (22 primary tumor [PT], 34 peritoneal metastases [PM]) samples from 35 patients, comprising 21 paired PT-PM samples, were included in this analysis. TEAD1 (unpaired T-test, p=0.087; paired T-test, p=0.041), FGFR1 (unpaired T-test, p=0.003; paired T-test, p<0.001), PIK3CA (unpaired T-test, p=0.034; paired T-test, p=0.003), SMAD4 (unpaired T-test, p=0.095; paired T-test, p=0.010), and HAVCR2 (unpaired T-test, p=0.009; paired T-test, p=0.007) were found to be upregulated in the PM. Conversely, MET was found to be downregulated in PM samples (unpaired T-test, p=0.004; paired T-test, p=0.002). There were no significant differences in gene expression profiles of BRAF, RHOA, CD274, CTLA4, PDCD1 and TIGIT between PT-PM (all p>0.05). Conclusion: Through CRC PT-PM comparisons, we identify potential therapeutic targets implicated in transcoelomic metastasis and provide rationale for development of immunotherapeutic biology and drug discovery for CRC PM. These findings underscore an exigent need to develop intra-peritoneal therapeutic strategies to overcome resistance to systemic regimens. Citation Format: Joseph J. Zhao, Johnny CA Ong, Daryl Kai Ann Chia, Melissa Ching Ching Teo, Qiu Xuan Tan, Gillian Ng, Joey Wee-Shan Tan, Haoran Ma, Xuewen Ong, Su Ting Tay, Patrick Tan, Raghav Sundar. Transcriptional intra-tumoral heterogeneity of putative therapeutic targets in colorectal cancer peritoneal metastases [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 2759.
Abstract Intestinal metaplasia (IM) is a pre-malignant condition of the gastric mucosa associated with increased gastric cancer (GC) risk. We analyzed 1256 gastric samples (1152 IMs) from 692 subjects through a prospective 10-year study. We identified 26 IM driver genes in diverse pathways including chromatin regulation ( ARID1A ) and intestinal homeostasis ( SOX9 ), largely occurring as small clonal events. Analysis of clonal dynamics between and within subjects, and also longitudinally across time, revealed that IM clones are likely transient but increase in size upon progression to dysplasia, with eventual transmission of somatic events to paired GCs. Single-cell and spatial profiling highlighted changes in tissue ecology and lineage heterogeneity in IM, including an intestinal stem-cell dominant cellular compartment linked to early malignancy. Expanded transcriptome profiling revealed expression-based molecular subtypes of IM, including a body-resident “pseudoantralized” subtype associated with incomplete histology, antral/intestinal cell types, ARID1A mutations, inflammation, and microbial communities normally associated with the healthy oral tract. We demonstrate that combined clinical- genomic models outperform clinical-only models in predicting IMs likely to progress. Our results raise opportunities for GC precision prevention and interception by highlighting strategies for accurately identifying IM patients at high GC risk and a role for microbial dysbiosis in IM progression.
Abstract Background: Peritoneal metastases (PM) in gastric cancer (GC) portend a poor prognosis. Yet, biological mechanisms underpinning peritoneal colonization and transcoelomic metastases remain unclear. Utilizing a novel humanized mouse model of GCPM, we seek to understand the evolution of the peritoneum to allow for peritoneal dissemination. The humanized mouse model, defined as mice engrafted with functional human cells or tissues, was selected as the host immune system appears to play a significant role in the mechanisms of transcoelomic metastases. Methodology: Humanized (humice) and NOD-scid Il2rγnull (NSG) mice models were injected with GSU (diffuse type) GC cell lines in the flank (subcutaneous), stomach (orthotopic) and directly into the peritoneum (intra-peritoneal). After 1 month, the mouse models were sacrificed and samples from the primary tumor (PT), PM and adjacent peritoneum were retrieved. The tumors were then harvested, along with normal/adjacent peritoneum and bulk RNA-seq was performed and analyzed. Results: Fifty-six samples including PT, PM were harvested from 10 humice and 11 NSG models and were analysed with whole transcriptome sequencing. All models with orthotopic inoculation were found to create PM while no gross PM were appreciated in models with flank inoculation. Principal component analysis mapping of samples demonstrated transcriptomic distinction between normal peritoneum from the flank humice model and adjacent normal samples in the orthotopic and intra-peritoneal humice model. Compared to adjacent peritoneum of the intra-peritoneal humanized mouse model, the orthotopic model appeared to be more tumorigenic with enriched epithelial mesenchymal transition (EMT), stromal and immune infiltration, inflammatory response (IL2-STAT5, IL6-JAK-STAT3, TGF-β signaling), IFN-γ response signatures. Enrichment scores of these pathways were likewise more pronounced in adjacent normal peritoneal samples in the orthotopic humanized mouse model when contrasted against other sample types. Higher expression of M2 macrophages, B cells and T regs, dendritic cells (activating and resting) was found in adjacent peritoneum in the orthotopic model. Collectively, these describe a phenomenon in which the adjacent uninvolved peritoneum has evolved into a tumorigenic environment to facilitate transcoelomic metastasis. These unique niche changes of the peritoneum were conversely not appreciated in the NSG model. Conclusion: We unveil novel insights that delve into the mechanistic role of the host immune system in orchestrating tumorigenic microenvironmental alterations to support peritoneal organotropism. These provide functional proof alluding to the phenomenon of peritoneal niche conditioning in GC PM. Citation Format: Joseph J. Zhao, Daryl Kai Ann Chia, Zhisheng Her, Haoran Ma, Xuewen Ong, Su Ting Tay, Jimmy Bok Yan So, Qingfeng Chen, Patrick Tan, Raghav Sundar. Humanized mouse model unveils niche conditioning in gastric cancer peritoneal metastasis [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 1497.
218 Background: Peritoneal metastases (PM) in colorectal cancer (CRC) portend a poor prognosis. We sought to elucidate spatially resolved molecular features facilitating transcoelomic dissemination and progression. Methods: 50 PT, 116 PM and 37 primary normal samples from 96 patients were retrieved and profiled with digital spatial profiling (GeoMx DSP, Nanostring Technologies). Through unsupervised clustering, we characterized the microenvironment of tumor-stroma compartments and studied the roles of stromal phenotypes in promulgating tumorigenesis. These findings were orthogonally validated through spatial proteomics (multiplex IHC [mIHC] - COMET, Lunaphore Technologies) of 10 paired PT-PM samples retrieved from 6 patients. Results: A median of 37 region of interests (ROIs) (IQR: 32-38) were selected from a total of 9 tissue microarray (TMA) slides. A total of 269 ROIs (181 tumor, 88 stroma) from PM samples, 45 ROIs (34 tumor, 11 stroma) from PT samples and 28 (all stroma) ROIs from primary normal samples were retrieved and profiled. Through consensus clustering of stroma ROIs, we identify a fibro-collagenous and immune infiltrated stromal phenotype (stromal cluster [SC] 2) characterized by increased cancer associated fibroblasts (CAFs), memory B cells, M2 macrophages and T-cell exhaustion. Patients with SC2 stroma had poorer survival (p=0.036). SC2 stroma was also observed to support adjacent tumor compartments with enriched oncogenic pathways such as TGF-beta, TNF-alpha, hypoxia and JAK-STAT. Through discriminatory gene expression profiles, we developed a 20-gene composite bidirectional signature of SC2. The prognostic significance of the SC2-signature was externally validated through several cohorts including TCGA and MSI-H only cohorts. Next, by inspecting mIHC retrieved immune cell type densities across ROIs, we confirmed a similar phenomenon in which two distinct stromal clusters were found – one being fibroblast infiltrated but T-cell depleted (SC2-like, n=67 ROI) and another which is T-cell infiltrated but fibroblast depleted (SC1-like, n=104 ROI). Diverging spatial distributions of T-cells and fibroblasts between SC1 and SC2-like stroma was appreciated as well. Between both PT and PM, we find close clustering of T cells in SC1-like stromal compartments and fibroblasts in SC2-like stromal compartments. Conclusions: We describe SC2, a pro-tumorigenic stromal phenotype characterized by increased CAFs, T cell exhaustion and is associated with poor prognosis in CRC PM.
Objective Gastric cancer (GC) comprises multiple molecular subtypes. Recent studies have highlighted mesenchymal-subtype GC (Mes-GC) as a clinically aggressive subtype with few treatment options. Combining multiple studies, we derived and applied a consensus Mes-GC classifier to define the Mes-GC enhancer landscape revealing disease vulnerabilities. Design Transcriptomic profiles of ~1000 primary GCs and cell lines were analysed to derive a consensus Mes-GC classifier. Clinical and genomic associations were performed across >1200 patients with GC. Genome-wide epigenomic profiles (H3K27ac, H3K4me1 and assay for transposase-accessible chromatin with sequencing (ATAC-seq)) of 49 primary GCs and GC cell lines were generated to identify Mes-GC-specific enhancer landscapes. Upstream regulators and downstream targets of Mes-GC enhancers were interrogated using chromatin immunoprecipitation followed by sequencing (ChIP-seq), RNA sequencing, CRISPR/Cas9 editing, functional assays and pharmacological inhibition. Results We identified and validated a 993-gene cancer-cell intrinsic Mes-GC classifier applicable to retrospective cohorts or prospective single samples. Multicohort analysis of Mes-GCs confirmed associations with poor patient survival, therapy resistance and few targetable genomic alterations. Analysis of enhancer profiles revealed a distinctive Mes-GC epigenomic landscape, with TEAD1 as a master regulator of Mes-GC enhancers and Mes-GCs exhibiting preferential sensitivity to TEAD1 pharmacological inhibition. Analysis of Mes-GC super-enhancers also highlighted NUAK1 kinase as a downstream target, with synergistic effects observed between NUAK1 inhibition and cisplatin treatment. Conclusion Our results establish a consensus Mes-GC classifier applicable to multiple transcriptomic scenarios. Mes-GCs exhibit a distinct epigenomic landscape, and TEAD1 inhibition and combinatorial NUAK1 inhibition/cisplatin may represent potential targetable options.
'/%3e%3cuse xlink:href='%23a' transform='translate(288.889 -214.211)'/%3e%3cuse xlink:href='%23a' transform='translate(108 342.749)'/%3e%3cuse xlink:href='%23a' transform='translate(469.189 342.749)'/%3e%3c/svg%3e)
