High Intratumoral Heterogeneity Research Articles

Visual Intratumor Heterogeneity and Breast Tumor Progression

High intratumoral heterogeneity is thought to be a poor prognostic indicator. However, the source of heterogeneity may also be important, as genomic heterogeneity is not always reflected in histologic or ‘visual’ heterogeneity. We aimed to develop a predictor of histologic heterogeneity and evaluate its association with outcomes and molecular heterogeneity. We used VGG16 to train an image classifier to identify unique, patient-specific visual features in 1655 breast tumors (5907 core images) from the Carolina Breast Cancer Study (CBCS). Extracted features for images, as well as the epithelial and stromal image components, were hierarchically clustered, and visual heterogeneity was defined as a greater distance between images from the same patient. We assessed the association between visual heterogeneity, clinical features, and DNA-based molecular heterogeneity using generalized linear models, and we used Cox models to estimate the association between visual heterogeneity and tumor recurrence. Basal-like and ER-negative tumors were more likely to have low visual heterogeneity, as were the tumors from younger and Black women. Less heterogeneous tumors had a higher risk of recurrence (hazard ratio = 1.62, 95% confidence interval = 1.22–2.16), and were more likely to come from patients whose tumors were comprised of only one subclone or had a TP53 mutation. Associations were similar regardless of whether the image was based on stroma, epithelium, or both. Histologic heterogeneity adds complementary information to commonly used molecular indicators, with low heterogeneity predicting worse outcomes. Future work integrating multiple sources of heterogeneity may provide a more comprehensive understanding of tumor progression.

Clinical relevance of circulating tumor DNA in ovarian cancer: current issues and future opportunities.

Ovarian cancer (OC) is the most lethal gynecologic malignancy worldwide. Due to the lack of effective screening and early detection strategies, many patients with OC are diagnosed with advanced disease, where treatment is rarely curative. Moreover, OC is characterized by high intratumor heterogeneity, which represents a major barrier to the development of effective treatments. Conventional tumor biopsy and blood-based biomarkers, such as cancer antigen 125 (CA125), have different limitations. Liquid biopsy has recently emerged as an attractive and promising area of investigation in oncology, due to its minimally invasive, safe, comprehensive, and real-time dynamic nature. Preliminary evidence suggests a potential role of liquid biopsy to refine OC management, by improving screening, early diagnosis, assessment of response to treatment, detection, and profiling of drug resistance. The current knowledge and the potential clinical value of liquid biopsy in OC is discussed in this review to provide an overview of the clinical settings in which its use might support and improve diagnosis and treatment.

Whole exome sequencing analysis of intratumoral heterogeneity, tumor neoantigen burden and human leukocyte antigen loss of heterozygosity in HCC.

e16003 Background: Hepatocellular Carcinoma (HCC) patients has a high postoperative recurrence within 2 years and a low 5-year survival rate. However, molecular mechanisms for early recurrence are poorly understood. Immune checkpoint blockades (ICBs) show clinical benefits in HCC, with the immune microenvironment influencing ICBs therapeutic effects. Exploring the relationship between intratumoral heterogeneity (ITH), tumor neoantigen burden (TNB), and human leukocyte antigen loss of heterozygosity (HLA LOH) can deepen our understanding of the immune evasion mechanisms. Methods: In this study, whole exome sequencing (WES) was employed to analyze tumor specimens and matched peripheral blood controls derived from a cohort of 235 HCC patients. PD-L1 expression levels were assessed in the samples using immunohistochemistry. NetMHCpan was applied to predict neoantigen and quantified the peptide affinity of MHC-I genes measured by IC50. Neoantigen-MHC affinity was categorized as strong (IC50≤50nM), weak (50nM < IC50≤500nM), and negative (IC50 > 500nM). TNB was defined by the number of high affinity neoantigens per megabase in WES. Analysis of LOH was performed on HLA-I class genes and categorized as strong positive, weak positive, and negative groups. We used PyClone to conduct ITH analysis to assess the distribution of multiple subclones within a tumor. The average Cancer Cell Fraction (CCF) for each somatic mutation cluster was calculated, and these clusters were classified into major clones and subclones. Results: In 235 HCC samples, median TMB and TNB were 2.48 Muts/Mb and 0.30 Neos/Mb. Only 6.81% exhibited TMB-H (TMB≥10 Muts/Mb). TMB and TNB are significantly positively correlated (p < 0.0001). HLA LOH occurred in 19.57% HCC patients, with high LOH samples having more high-affinity neoantigens (mean TNB 1.65 Neos/Mb). ITH analysis showed that 83.83% samples harbored subclonal clusters. HCC samples were divided into high (ITH≥0.908) and low (ITH < 0.908) groups according to the median ITH of 0.908. High ITH group had higher TNB (median 0.43 vs 0.20 Neos/Mb) , more HLA LOH (9.24% vs 5.17% strong positive LOH). The high ITH groups exhibited a lower percentage of samples with elevated PD-L1 expression (14.29% vs 25.86%). The Combined Positive Score (CPS) revealed a significant difference between the groups (Fisher's test p = 0.03). Conclusions: In HCC, the high ITH group exhibited a higher proportion of HLA LOH compared to the low ITH group. Moreover, a decreased proportion of PD-L1 positive samples was observed in the high ITH group, suggesting that high ITH may be a key factor influencing treatment failure in immunotherapy. ITH analysis demonstrated a potential multi-subclonal origin of recurrent tumor cells, which is associated with immune escape, tumor malignancy,as well as the development of resistance.

Immune cell infiltration and inflammatory landscape in primary brain tumours

BackgroundPrimary malignant brain tumours are more than one-third of all brain tumours and despite the molecular investigation to identify cancer driver mutations, the current therapeutic options available are challenging due to high intratumour heterogeneity. In addition, an immunosuppressive and inflammatory tumour microenvironment strengthens cancer progression. Therefore, we defined an immune and inflammatory profiling of meningioma and glial tumours to elucidate the role of the immune infiltration in these cancer types.MethodsUsing tissue microarrays of 158 brain tumour samples, we assessed CD3, CD4, CD8, CD20, CD138, Granzyme B (GzmB), 5-Lipoxygenase (5-LOX), Programmed Death-Ligand 1 (PD-L1), O-6-Methylguanine-DNA Methyltransferase (MGMT) and Transglutaminase 2 (TG2) expression by immunohistochemistry (IHC). IHC results were correlated using a Spearman correlation matrix. Transcript expression, correlation, and overall survival (OS) analyses were evaluated using public datasets available on GEPIA2 in Glioblastoma (GBM) and Lower Grade Glioma (LGG) cohorts.ResultsSeven out of ten markers showed a significantly different IHC expression in at least one of the evaluated cohorts whereas CD3, CD4 and 5-LOX were differentially expressed between GBMs and astrocytomas. Correlation matrix analysis revealed that 5-LOX and GzmB expression were associated in both meningiomas and GBMs, whereas 5-LOX expression was significantly and positively correlated to TG2 in both meningioma and astrocytoma cohorts. These findings were confirmed with the correlation analysis of TCGA-GBM and LGG datasets. Profiling of mRNA levels indicated a significant increase in CD3 (CD3D, CD3E), and CD138 (SDC1) expression in GBM compared to control tissues. CD4 and 5-LOX (ALOX5) mRNA levels were significantly more expressed in tumour samples than in normal tissues in both GBM and LGG. In GBM cohort, GzmB (GZMB), SDC1 and MGMT gene expression predicted a poor overall survival (OS). Moreover, in LGG cohort, an increased expression of CD3 (CD3D, CD3E, CD3G), CD8 (CD8A), GZMB, CD20 (MS4A1), SDC1, PD-L1, ALOX5, and TG2 (TGM2) genes was associated with worse OS.ConclusionsOur data have revealed that there is a positive and significant correlation between the expression of 5-LOX and GzmB, both at RNA and protein level. Further evaluation is needed to understand the interplay of 5-LOX and immune infiltration in glioma progression.

Glioblastoma microenvironment-from biology to therapy.

Glioblastoma (GBM) is the most aggressive primary brain cancer. These tumors exhibit high intertumoral and intratumoral heterogeneity in neoplastic and nonneoplastic compartments, low lymphocyte infiltration, and high abundance of myeloid subsets that together create a highly protumorigenic immunosuppressive microenvironment. Moreover, heterogeneous GBM cells infiltrate adjacent brain tissue, remodeling the neural microenvironment to foster tumor electrochemical coupling with neurons and metabolic coupling with nonneoplastic astrocytes, thereby driving growth. Here, we review heterogeneity in the GBM microenvironment and its role in low-to-high-grade glioma transition, concluding with a discussion of the challenges of therapeutically targeting the tumor microenvironment and outlining future research opportunities.

Development of Personalized Strategies for Precisely Battling Malignant Melanoma.

Melanoma is the most severe and fatal form of skin cancer, resulting from multiple gene mutations with high intra-tumor and inter-tumor molecular heterogeneity. Treatment options for patients whose disease has progressed beyond the ability for surgical resection rely on currently accepted standard therapies, notably immune checkpoint inhibitors and targeted therapies. Acquired resistance to these therapies and treatment-associated toxicity necessitate exploring novel strategies, especially those that can be personalized for specific patients and/or populations. Here, we review the current landscape and progress of standard therapies and explore what personalized oncology techniques may entail in the scope of melanoma. Our purpose is to provide an up-to-date summary of the tools at our disposal that work to circumvent the common barriers faced when battling melanoma.

Abstract PO5-14-09: Does transcriptome based DEPTH2 score reflect intratumor heterogeneity in breast cancer patients?

Abstract Background: Intratumor heterogeneity (ITH) refers to the presence of distinct cancer cell populations with different clones within a single tumor mass. Tumors with high ITH are more prone to progression, exhibit resistance to therapies, and are associated with an unfavorable prognosis. Therefore, evaluation of ITH is anticipated to serve as a breakthrough biomarker for prognosis and treatment strategies. Several computational algorithms have been proposed to quantify ITH. However, with the exception of DEPTH2, all of these algorithms require both DNA and RNA-sequence data from the same tumor, which can be challenging. In this study, we investigated whether DEPTH2 which solely analyzes RNA-sequence data, accurately reflects the biological characteristics of ITH. Methods: We analyzed clinicopathological and gene expression data from a total of 6573 breast cancer patients from several large independent cohorts: METABRIC (n = 1903), GSE96058 (n = 3273), GSE21974 (n = 57), GSE28844 (n = 61), GSE87455 (n = 153), GSE180280 (n = 57), and The Cancer Genome Atlas (TCGA, n = 1069). Results: DEPTH2 score was high in Triple Negative Breast Cancer (TNBC) subtype (p < 0.001), but no consistent correlation was observed with lymph node or distal metastasis. Higher DEPTH2 score was associated with cell proliferation, as evidenced by higher Nottingham histological grade and Ki67 gene expression (both p < 0.001). In agreement, high DEPTH2 tumors enriched all cell proliferation-related Hallmark gene sets such as E2F targets, G2M checkpoint, MYC targets v1 and v2, and Mitotic Spindle (FDR < 0.25). Additionally, high DEPTH2 tumors exhibited reduced immune cell infiltrations, such as CD8, CD4, Th1, Th2, B cells, and macrophages, and increased infiltrations of stromal cells, such as adipocytes, fibroblasts, blood and lymphatic endothelial cells (all p < 0.05). In TCGA cohort, DEPTH2 score exhibited a similar trend to intratumoral heterogeneity, homologous recombination deficiency, fraction altered, silent and nonsilent mutations, as well as SNV and Indel neoantigens. However, the Pearson’s correlation coefficient was very weak (p < 0.05). DEPTH2 score decreased in the GSE21974 and GSE87455 cohorts after neoadjuvant chemotherapy (NAC), whereas no change was observed in the GSE28844 and GSE180280 cohorts. DEPTH2 high tumors showed a lower rate of pathologic complete response (pCR) after NAC in estrogen receptor (ER)+HER2- subtype across all cohorts, however, opposite pattern was observed in the HER2+ and TNBC subtypes in all cohorts. Lastly, high DEPTH2 score was associated with lower disease free and overall survival in METABRIC and GSE96058 (p < 0.05), but not in the TCGA cohort. Conclusions: Our findings suggest that DEPTH2 score may not completely capture the biological characteristics associated with intratumor heterogeneity. Citation Format: Kohei Chida, Rongrong Wu, Arya Mariam Roy, Takashi Ishikawa, Kazuaki Takabe. Does transcriptome based DEPTH2 score reflect intratumor heterogeneity in breast cancer patients? [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 PO5-14-09.

Abstract PO2-23-08: High intratumor heterogeneity induced PSME2 confers endocrine resistance via ERα PARylation in hormone receptor-positive and HER2-negative breast cancer

Abstract Background: Hormone receptor-positive and HER2-negative breast cancer, characterized by its significant intratumor heterogeneity (ITH), poses increased recurrence, metastasis risk, and endocrine resistance. A comprehensive understanding of the genomic and biological characteristics underlying ITH is crucial for the development of effective therapeutic strategies. Methods: We leveraged the multiomics data from our large cohort of 381 hormone receptor-positive and HER2-negative breast tumors, supplemented with single-cell sequencing (scRNA-seq) data from 9 individual samples, to investigate ITH and its associated molecular features. Mechanistic investigations were conducted using Stable Isotope Labeling by Amino Acids in Cell culture (SILAC) proteomics, co-immunoprecipitation (Co-IP), Chromatin Immunoprecipitation followed by quantitative PCR (ChIP-qPCR), and dual-luciferase reporter assays. We specifically focused on the role of Proteasome activator subunit beta (PSME2), a potential regulator of drug resistance and metastasis. Results: We quantified the degree of ITH at sample level using multi-omics and scRNA-seq data and observed a correlation between higher ITH levels, increased genomic instability, upregulation of the STING type-I-interferon-dependent pathway, and poor efficacy of endocrine therapy. Both bulk and single-cell data revealed an enrichment of PSME2, associated with type-I interferon, in groups exhibiting high ITH. Patients with elevated PSME2 expression manifested a poorer prognosis and diminished responsiveness to endocrine therapy. Within the context of ITH, we discovered that IFN-I transcriptionally activated PSME2 via STAT1. Further investigation revealed a complex interplay among PSME2, PARP1, and EWS, wherein PSME2-mediated degradation of EWS relieved the transcriptional repression of PARP1, resulting in its overexpression. This series of events, orchestrated by PSME2, led to ERα PARylation and promoted tamoxifen resistance. In subsequent clinical translation studies employing mouse xenograft, patient-derived organoid (PDO), and patient-derived xenograft (PDX) models, we demonstrated that PARP1 inhibitors could effectively mitigate tamoxifen resistance induced by high PSME2 expression, providing new insights into therapeutic strategies. Conclusions: Our study highlights the importance of understanding ITH in hormone receptor-positive and HER2-negative breast cancer and identifies PSME2 as a key player in endocrine resistance and patient prognosis. The application of PARP1 inhibitors shows promise in improving treatment outcomes in tumors with elevated ITH and PSME2 expression. Working Modle Citation Format: Li-Ping Ge, Zi-Yu Wang, Xi Jin, Yi-Zhou Jiang, Zhi-Ming Shao. High intratumor heterogeneity induced PSME2 confers endocrine resistance via ERα PARylation in hormone receptor-positive and HER2-negative breast cancer [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 PO2-23-08.

Abstract LB223: The molecular landscape of injury-induced mammary epithelial cell fate switching

Abstract The most aggressive cancers, such as triple negative basal-like breast cancer (TNBC), have high intratumoral heterogeneity that is often attributed to cell state reprogramming and contributes to cancer progression, treatment resistance, and ultimately cancer death. Tissue injury and microenvironmental changes can cause adult differentiated cells to acquire a plastic “developmentally unstable hybrid cell state” (DUHCS) enabling transdifferentiation into other cell types to effect repair. Yet, the relevant mechanisms that enable cell state reprogramming to arise remain under-explored. Here, we used CRISPR mediated homologous recombination to generate mice in which the mammary basal and luminal cells could be distinguished by their expression of Krt14-mClover-NLS or Krt18-tdTomato-NLS, respectively. We used this novel system to identify mammary epithelial cells undergoing cell state transitions in real time. Using a transplantation paradigm, we found that transplanted basal cells rapidly exhibited a DUHCS resembling mammary embryonic progenitors expressing Sox11, a tightly regulated differentiation factor shut off before birth. We discovered that Sox11 is also expressed in many murine models of TNBC and in single cell transcriptomes from untreated human BRCA1 mutant TNBCs. This suggests that Sox11-expressing cells might be developmentally plastic intermediates in TNBC malignant progression. In a preliminary cohort of patient-derived xenografts from TNBC patients diagnosed at different stages, we detected SOX11-expressing cells in earlier but not in advanced stages. Re-expression of Sox11 in TNBC is consistent with TNBCs being transcriptomically related to fetal mammary stem cells and p53 dysfunction resulting in enhanced cellular plasticity. Taken together, these studies highlight a role for mammary epithelial cell state reprogramming in early breast tumorigenesis, and reveals actionable steps to mitigate tumor heterogeneity by halting tumor progression. Citation Format: Queralt Vallmajó Martín, Nikki K. Lytle, Divya Murali, Sumana Srinivasan, Zhibo Ma, Kavitha Mukund, Christopher Dravis, Liliana Zamora, Ana Peterson, Galina Khramtsova, Funmi Olopade, Shankar Subramaniam, Geoffrey M. Wahl. The molecular landscape of injury-induced mammary epithelial cell fate switching [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB223.

High Ki67 Gene Expression Is Associated With Aggressive Phenotype in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) with high Ki67 protein expression, the most commonly used cell proliferation marker, is associated with an aggressive biologic phenotype; however, conventional immunostaining is hampered by variability in institutional protocol, specific antibody probe, and by assessor subjectivity. To this end, we hypothesized that Ki67 gene (MKi67) expression would identify highly proliferative HCC, and clarify its association with oncologic outcome, tumor progression, and immune cell population in the tumor microenvironment (TME). Furthermore, we sought to identify the cell-cycle gene expression profile that confers this aggressive phenotype. A total of 473 HCC patients with clinicopathological data associated with transcriptome were selected for this study: 358 patients from The Cancer Genome Atlas (TCGA) as the testing cohort, and 115 from GSE76427 as the validation cohort. Each cohort was divided into a highly proliferative group (MKi67-high) and the low MKi67 group (MKi67-low) by the median of Ki67 gene (MKi67) expression levels. MKi67-high HCC patients had worse disease-free survival (DFS), disease-specific survival (DSS), and overall survival (OS) independent of histological grade in the TCGA cohort. MKi67 expression correlated with histological grade and tumor size. MKi67 expression increased throughout the HCC carcinomatous sequence from normal liver, cirrhotic liver, early HCC, and advanced HCC. MKi67-high HCC was associated with higher intratumor heterogeneity, homologous recombination deficiency, and altered fraction as well as intratumoral infiltration of T helper type 1 (Th1) and Th2 cells, but lower interferon-gamma response and M2 macrophage infiltration. Cell proliferation-related gene sets in the Hallmark collection (E2F targets, G2M checkpoint, Myc target v1 and mitotic spindle), MTORC1 signaling, DNA repair, PI3K MTOR signaling, and unfolded protein response were all enriched in the MKi67-high HCC (false discovery rate (FDR) < 0.25). High MKi67 gene expression identified highly proliferative HCC with aggressive biology involving classical pathways in cell cycle regulation and DNA repair, as well as poor overall oncologic outcomes. This suggests potential for personalized treatment strategies, but validation and refinement of these observations require further research to elucidate the underlying mechanisms and validate therapeutic targeting of these pathways in MKi67-high HCC tumors.

Molecular and immune characterization of Chinese early-stage non-squamous non-small cell lung cancer: a multi-omics cohort study.

Albeit considered with superior survival, around 30% of the early-stage non-squamous non-small cell lung cancer (Ns-NSCLC) patients relapse within 5 years, suggesting unique biology. However, the biological characteristics of early-stage Ns-NSCLC, especially in the Chinese population, are still unclear. Multi-omics interrogation of early-stage Ns-NSCLC (stage I-III), paired blood samples and normal lung tissues (n=76) by whole-exome sequencing (WES), RNA sequencing, and T-cell receptor (TCR) sequencing were conducted. An average of 128 exonic mutations were identified, and the most frequently mutant gene was EGFR (55%), followed by TP53 (37%) and TTN (26%). Mutations in MUC17, ABCA2, PDE4DIP, and MYO18B predicted significantly unfavorable disease-free survival (DFS). Moreover, cytobands amplifications in 8q24.3, 14q13.1, 14q11.2, and deletion in 3p21.1 were highlighted in recurrent cases. Higher incidence of human leukocyte antigen loss of heterozygosity (HLA-LOH), higher tumor mutational burden (TMB) and tumor neoantigen burden (TNB) were identified in ever-smokers than never-smokers. HLA-LOH also correlated with higher TMB, TNB, intratumoral heterogeneity (ITH), and whole chromosomal instability (wCIN) scores. Interestingly, higher ITH was an independent predictor of better DFS in early-stage Ns-NSCLC. Up-regulation of immune-related genes, including CRABP2, ULBP2, IL31RA, and IL1A, independently portended a dismal prognosis. Enhanced TCR diversity of peripheral blood mononuclear cells (PBMCs) predicted better prognosis, indicative of a noninvasive method for relapse surveillance. Eventually, seven machine-learning (ML) algorithms were employed to evaluate the predictive accuracy of clinical, genomic, transcriptomic, and TCR repertoire data on DFS, showing that clinical and RNA features combination in the random forest (RF) algorithm, with area under the curve (AUC) of 97.5% and 83.3% in the training and testing cohort, respectively, significantly outperformed other methods. This study comprehensively profiled the genomic, transcriptomic, and TCR repertoire spectrums of Chinese early-stage Ns-NSCLC, shedding light on biological underpinnings and candidate biomarkers for prognosis development.

Metabolomic Differentiation of Tumor Core vs. Edge in Glioma Via Machine Learning

INTRODUCTION: Gliomas exhibit high intra-tumor and inter-patient heterogeneity. Recently, it has been shown that the microenvironment and phenotype differ significantly between glioma core (inner) and edge (invasive) regions. METHODS: Paired glioma core and infiltrating edge samples were obtained from 27 human patients after craniotomy. Liquid-liquid metabolite extraction was performed on the samples, and metabolomic data was obtained via 2DLC-MS/MS. A boosted generalized linear model was employed to predict metabolomic profiles associated with O-6-methylguanine-DNA methyltransferase (MGMT) promoter methylation. RESULTS: A panel of 52 of 160 metabolites were found to significantly differ between the glioma core and edge regions (p = 0.05). Top metabolites with significantly different relative abundances included nicotinamide, creatine, cystathione, D-pantothenic acid, and maleic acid. The machine learning model predicted MGMT promotor methylation status using 5 key metabolic features within the core and edge tissue specimens with AUROCEdge = 0.935 and AUROCCore = 0.959. Top metabolites associated with MGMT status in core included Y-L-glutamyl-L-glutamic acid, D-pantothenic acid, uric acid, and in edge included itaconic acid, N,N-dimethylarginine, cytosine. CONCLUSIONS: Key metabolic differences are identified between core and edge tissue in glioma and can predict MGMT status.

Hypoxia drives shared and distinct transcriptomic changes in two invasive glioma stem cell lines

Glioblastoma (GBM) is the most common primary malignant cancer of the central nervous system. Insufficient oxygenation (hypoxia) has been linked to GBM invasion and aggression, leading to poor patient outcomes. Hypoxia induces gene expression for cellular adaptations. However, GBM is characterized by high intertumoral (molecular subtypes) and intratumoral heterogeneity (cell states), and it is not well understood to what extent hypoxia triggers patient-specific gene responses and cellular diversity in GBM. Here, we surveyed eight patient-derived GBM stem cell lines for invasion phenotypes in 3D culture, which identified two GBM lines showing increased invasiveness in response to hypoxia. RNA-seq analysis of the two patient GBM lines revealed a set of shared hypoxia response genes concerning glucose metabolism, angiogenesis, and autophagy, but also a large set of patient-specific hypoxia-induced genes featuring cell migration and anti-inflammation, highlighting intertumoral diversity of hypoxia responses in GBM. We further applied the Shared GBM Hypoxia gene signature to single cell RNA-seq datasets of glioma patients, which showed that hypoxic cells displayed a shift towards mesenchymal-like (MES) and astrocyte-like (AC) states. Interestingly, in response to hypoxia, tumor cells in IDH-mutant gliomas displayed a strong shift to the AC state, whereas tumor cells in IDH-wildtype gliomas mainly shifted to the MES state. This distinct hypoxia response of IDH-mutant gliomas may contribute to its more favorable prognosis. Our transcriptomic studies provide a basis for future approaches to better understand the diversity of hypoxic niches in gliomas.

Abstract 1488: Spatial characterization of the tumor microenvironment of metastatic triple-negative breast cancer

Abstract Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer associated with high rates of recurrence and metastasis, which contribute to poor overall survival. The challenges in treating TNBC stem from its high intratumor heterogeneity and lack of expression of the estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 druggable targets. The tumor microenvironment (TME) plays a crucial role in disease progression. Within the TME, complex spatial interactions among tumor cells, immune cells, stroma, angiogenesis- and hypoxia-inducing factors collectively contribute to the advancement and spread of TNBC. In-depth understanding of the TME and interactions of the various components within can elucidate newer therapeutic targets, enhancing the prognosis of TNBC patients. Imaging mass cytometry (IMC) enables detailed analysis of cell-cell and cell-matrix interactions within the TME at the single-cell level. In this study, we aim to characterize the spatial relationships among various cell types within the TNBC TME using IMC, while also investigating differences in the TME of TNBC patients who developed distant metastasis (DM) compared to those who remained non-metastatic (NM). IMC was performed on formalin-fixed paraffin-embedded TNBC primary resection tissues (n= 16; DM= 7, NM= 9) from Stavanger University Hospital, Norway, using a panel of 25 metal-conjugated antibodies. Regions of interest were marked by the pathologist and the slides were laser ablated using Hyperion Imaging System. DeepCell was used to segment single cells based on surface markers. Mean intensity values of all markers for each cell were extracted and consolidated in R scripts for unsupervised clustering. Cell clusters were annotated based on phenotypic markers. Normalized expression of markers was compared for each cell type across DM and NM patients, including markers for cell proliferation, angiogenesis, hypoxia, and cell migration. Neighborhood analysis examined spatial associations between different cell types, highlighting differences in DM and NM patients. The IMC panel successfully provided spatial insights into the TME, including the characterization of tumor cells, immune cells (B cells, T cells, macrophages), endothelial cells, and proliferating cells. We identified 18 unique phenotypic cell clusters that were present at different frequencies in DM and NM patients. These clusters demonstrated different expression profiles of functional markers. t-SNE plots revealed distinct patterns of cell type distribution and unique protein expression profiles, highlighting significant differences between the two groups. Our data suggest a distinct spatial organization of the TME in TNBC patients who develop metastasis. This warrants further investigations to determine the role and interplay of TME features in predicting the risk of distant metastasis in TNBC. Citation Format: Mahak Bhargava, Jaspreet Kaur, Geetanjali Saini, Junjun Zheng, Yitian Xu, Shu-Hsia Chen, Ping-Ying Pan, Emma Rewcastle, Umay Kiraz, Emiel Adrianus Janssen, Ritu Aneja. Spatial characterization of the tumor microenvironment of metastatic triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1488.

Abstract 2570: Gastric cancer with high neuron infiltration was associated with enhanced epithelial-to-mesenchymal transition, angiogenesis and poor clinical outcomes

Abstract Introduction: Neurons within the tumor microenvironment of gastric cancer (GC), as identified through immunohistochemistry, have been reported to be associated with worse overall survival (OS) of GC patients. However, histology-based analyses have limitations in quantifying nerve cells and investigating the underlying biology. Thus, we aim to study the clinical significance of intratumoral nerve cells in GC using computational deconvolution of transcriptomes from bulk tumors. Methods: Total of 807 GC patients from two independent cohorts (TCGA; n = 375, GSE84437; n = 432) were included, encompassing both clinical and transcriptomic profiles. The neuron infiltration fraction was determined using the xCell algorithm. Results: GC with high neuron infiltration was associated with high expressions of neurotransmitter receptors; beta-adrenaline, dopamine, muscarinic acetylcholine, and neurokinin receptors in TCGA cohort (all p &amp;lt; 0.02). It was associated with reduced mutation rate and increased intratumoral heterogeneity (all p ≦ 0.002), as well as less immune response; interferon-alpha and interferon-gamma, and with low infiltration of immune cells; CD4 memory, T-helper 1, M1 and M2 macrophages, and NK cells (all p ≦ 0.02). Further, neuron highly infiltrated GC enriched Epithelial-to-Mesenchymal Transition and Angiogenesis gene sets and had higher lymphatic and microvessel endothelial cells and pericyte infiltrations (all p ≦ 0.001). Conversely, GC with high neuron infiltration showed an inverse relationship with cell proliferation-related gene sets; E2F Targets, G2M Checkpoint, and MYC Targets v1 and v2, and had lower Ki67 gene expression (FDR &amp;lt; 0.25 and p &amp;lt; 0.001, respectively). High neuron infiltration in GC was associated with worse patient OS in TCGA cohort, which was validated in GSE84437 cohort (both p &amp;lt; 0.05). It was an independent prognostic factor of OS in both univariate and multivariate Cox-regression analysis in the TCGA cohort (hazard ratio [HR]: 1.46; 95% confidence interval [CI]: 1.01 to 2.12; p = 0.045 and [HR]:1.54; 95% CI: 1.05 to 2.26; p = 0.026). Conclusion: Our research indicates that high infiltration of neurons in GC correlates with high expressions of neurotransmitter receptors, high intratumoral heterogeneity, less immune response and immune cell infiltrations, and less cell proliferation. It enriched EMT and angiogenesis which may explain its association with worse patient survival. Citation Format: Kohei Chida, Masanori Oshi, Arya Mariam Roy, Jun Arima, Pia Sharma, Gabriella Kim Mann, Itaru Endo, Kenichi Hakamada, Kazuaki Takabe. Gastric cancer with high neuron infiltration was associated with enhanced epithelial-to-mesenchymal transition, angiogenesis and poor clinical outcomes [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 2570.

Abstract 2569: VEGFA gene expression is associated with cell proliferation, less immune cell infiltration, and worse survival, but better response to chemotherapy and immunotherapy

Abstract Introduction: Vascular endothelial growth factor-A (VEGFA) is a well-known factor that induces angiogenesis, one of the hallmarks of cancer, which generates new blood vessels in the tumor microenvironment (TME) and provides conduit for cancer to metastasize. Interestingly, an anti-VEGFA drug (Avastin) failed to show benefit in clinical trials for breast cancer (BC). In this study we investigated the clinical relevance of VEGFA gene expression in BC. Methods: A total of 7336 BC patients from eight independent cohorts, TCGA (n= 1073), METABRIC (n= 1903), SCAN-B (GSE96058, n= 3069), GSE20194 (n=278), GSE34138 (n=178), GSE163882 (n=222), GSE25066 (n=508), and ISPY2 (GSE173839, n= 105) were analyzed. These cohorts were divided into high and low VEGFA gene expression groups using the median. Results: High VEGFA expression in BC was associated with worse overall survival in SCAN-B cohort which was validated in METABRIC cohort with overall, disease-specific, and disease-free survival (all p&amp;lt;0.02). VEGFA expression was higher in Triple Negative BC (TNBC) but was lower in BC with lymph node metastasis. High VEGFA BC was associated with significantly high intratumoral heterogeneity, homologous recombination defect, silent and non-silent mutations, and SNV neoantigens in TCGA, and was also associated with higher cell proliferation, evidenced by higher Nottingham histological grade, higher Ki67 gene expression, and enrichment of all the Hallmark cell proliferation-related gene sets: Mitotic spindle, G2M Checkpoint, E2F Targets, MYC Targets v1 and v2, and mTorC1 signaling, consistently, in SCAN-B, METABRIC, and TCGA cohorts. Reflecting its enhanced growth, high VEGFA expression was associated with less infiltration of fibroblasts and adipocytes (all p&amp;lt;0.05). Surprisingly, VEGFA expression did not enrich angiogenesis gene set in any of the cohorts, nor was related with infiltrations of blood or lymphatic vascular endothelial cells, besides pericytes. But high VEGFA BC was associated with significantly less infiltrations of anti-cancer immune cells; CD8, CD4, and dendritic cells were all significantly less, and T helper type 1 cells and regulatory T cells were significantly higher consistently in SCAN-B, METABRIC, and TCGA cohorts. VEGFA expression was significantly higher in BC that achieved pathological complete response after Anthracycline and Taxane based neoadjuvant therapy in both ER+/HER2- and TNBC subtypes in the GSE25066 cohort, and after immunotherapy in ER+/HER2- subtype, but not TNBC, in the ISPY2 clinical trial cohort. This was not validated in GSE20194, GSE34138, GSE163882 cohorts. Conclusion: Our research indicates that high VEGFA BC is associated with high cell proliferation, less immune cell infiltration, and worse survival, but better response to Anthracycline and Taxane based chemotherapy, and immunotherapy. Citation Format: Pia Sharma, Kohei Chida, Kenichi Hakamada, Kazuaki Takabe. VEGFA gene expression is associated with cell proliferation, less immune cell infiltration, and worse survival, but better response to chemotherapy and immunotherapy [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 2569.

High HER2 Intratumoral Heterogeneity Is a Predictive Factor for Poor Prognosis in Early-Stage and Locally Advanced HER2-Positive Breast Cancer.

Breast cancer tumors frequently have intratumoral heterogeneity (ITH). Tumors with high ITH cause therapeutic resistance and have human epidermal growth factor receptor 2 (HER2) heterogeneity in response to HER2-targeted therapies. This study aimed to investigate whether high HER2 heterogeneity levels were clinically related to a poor prognosis for HER2-targeted adjuvant therapy resistance in primary breast cancers. This study included patients with primary breast cancer (n = 251) treated with adjuvant HER2-targeted therapies. HER2 heterogeneity was manifested by the shape of HER2 fluorescence in situ hybridization amplification (FISH) distributed histograms with the HER2 gene copy number within a tumor sample. Each tumor was classified into a biphasic grade graph (high heterogeneity [HH]) group or a monophasic grade graph (low heterogeneity [LH]) group based on heterogeneity. Both groups were evaluated for disease-free survival (DFS) and overall survival (OS) for a median of ten years of annual follow-up. Of 251 patients with HER2-positive breast cancer, 46 (18.3%) and 205 (81.7%) were classified into the HH and LH groups, respectively. The HH group had more distant metastases and a poorer prognosis than the LH group (DFS: p < 0.001 (HH:63% vs. LH:91% at 10 years) and for the OS: p = 0.012 (HH:78% vs. LH:95% at 10 years). High HER2 heterogeneity is a poor prognostic factor in patients with HER2-positive breast cancer. A novel approach to heterogeneity, which is manifested by the shape of HER2 FISH distributions, might be clinically useful in the prognosis prediction of patients after HER2 adjuvant therapy.

Clonal gene signatures predict prognosis in mesothelioma and lung adenocarcinoma

Malignant pleural mesothelioma (MPM) is a rare but lethal pleural cancer with high intratumor heterogeneity (ITH). A recent study in lung adenocarcinoma has developed a clonal gene signature (ORACLE) from multiregional transcriptomic data and demonstrated high prognostic values and reproducibility. However, such a strategy has not been tested in other types of cancer with high ITH. We aimed to identify biomarkers from multi-regional data to prognostically stratify MPM patients. We generated a multiregional RNA-seq dataset for 78 tumor samples obtained from 26 MPM patients, each with one sample collected from a superior, lateral, and inferior region of the tumor. By integrating this dataset with the Cancer Genome Atlas MPM RNA-seq data, we selected 29 prognostic genes displaying high variability across different tumors but low ITH, which named PRACME (Prognostic Risk Associated Clonal Mesothelioma Expression). We evaluated PRACME in two independent MPM datasets and demonstrated its prognostic values. Patients with high signature scores are associated with poor prognosis after adjusting established clinical factors. Interestingly, the PRACME and the ORACLE signatures defined respectively from MPM and lung adenocarcinoma cross-predict prognosis between the two cancer types. Further investigation indicated that the cross-prediction ability might be explained by the high similarity between the two cancer types in their genomic regions with copy number variation, which host many clonal genes. Overall, our clonal signature PRACME provided prognostic stratification in MPM and this study emphasized the importance of multi-regional transcriptomic data for prognostic stratification based on clonal genes.

RAS/RAF Comutation and ERBB2 Copy Number Modulates HER2 Heterogeneity and Responsiveness to HER2-directed Therapy in Colorectal Cancer.

ERBB2-amplified colorectal cancer is a distinct molecular subtype with expanding treatments. Implications of concurrent oncogenic RAS/RAF alterations are not known. Dana-Farber and Foundation Medicine Inc. Colorectal cancer cohorts with genomic profiling were used to identify ERBB2-amplified cases [Dana-Farber, n = 47/2,729 (1.7%); FMI, n = 1857/49,839 (3.7%)]. Outcomes of patients receiving HER2-directed therapies are reported (Dana-Farber, n = 9; Flatiron Health-Foundation Medicine clinicogenomic database, FH-FMI CGDB, n = 38). Multisite HER2 IHC and genomic profiling were performed to understand HER2 intratumoral and interlesional heterogeneity. The impact of concurrent RAS comutations on the effectiveness of HER2-directed therapies were studied in isogenic colorectal cancer cell lines and xenografts. ERBB2 amplifications are enriched in left-sided colorectal cancer. Twenty percent of ERBB2-amplified colorectal cancers have co-occurring oncogenic RAS/RAF alterations. While RAS/RAF WT colorectal cancers typically have clonal ERBB2 amplification, colorectal cancers with co-occurring RAS/RAF alterations have lower level ERRB2 amplification, higher intratumoral heterogeneity, and interlesional ERBB2 discordance. These distinct genomic patterns lead to differential responsiveness and patterns of resistance to HER2-directed therapy. ERBB2-amplified colorectal cancer with RAS/RAF alterations are resistant to trastuzumab-based combinations, such as trastuzumab/tucatinib, but retain sensitivity to trastuzumab deruxtecan in in vitro and murine models. Trastuzumab deruxtecan shows clinical efficacy in cases with high-level ERBB2-amplified RAS/RAF coaltered colorectal cancer. Co-occurring RAS/RAF alterations define a unique subtype of ERBB2-amplified colorectal cancer that has increased intratumoral heterogeneity, interlesional discordance, and resistance to trastuzumab-based combinations. Further examination of trastuzumab deruxtecan in this previously understudied cohort of ERBB2-amplified colorectal cancer is warranted.

Abstract A069: The molecular landscape of injury-induced mammary epithelial cell fate switching

Abstract The most aggressive cancers, such as triple negative basal-like breast cancer (TNBC), have high intratumoral heterogeneity that is often attributed to cell state reprogramming and contributes to cancer progression, treatment resistance, and ultimately cancer death. Tissue injury and microenvironmental changes can cause adult differentiated cells to acquire a plastic “developmentally unstable hybrid cell state” (DUHCS) enabling transdifferentiation into other cell types to effect repair. Yet, the relevant mechanisms that enable cell state reprogramming to arise remain under-explored. Here, we used CRISPR mediated homologous recombination to generate mice in which the mammary basal and luminal cells could be distinguished by their expression of Krt14-mClover-NLS or Krt18-tdTomato-NLS, respectively. We used this novel system to identify mammary epithelial cells undergoing cell state transitions in real time. Using a transplantation paradigm, we found that transplanted basal cells rapidly exhibited a DUHCS resembling mammary embryonic progenitors expressing Sox11, a tightly regulated differentiation factor shut off before birth. We discovered that Sox11 is also expressed in many murine models of TNBC and in single cell transcriptomes from untreated human BRCA1 mutant TNBCs. This suggests that Sox11-expressing cells might be developmentally plastic intermediates in TNBC malignant progression. In a preliminary cohort of patient-derived xenografts from TNBC patients diagnosed at different stages, we detected SOX11-expressing cells in earlier but not in advanced stages. Re-expression of Sox11 in TNBC is consistent with TNBCs being transcriptomically related to fetal mammary stem cells and p53 dysfunction resulting in enhanced cellular plasticity. Taken together, these studies highlight a role for mammary epithelial cell state reprogramming in early breast tumorigenesis, and reveals actionable steps to mitigate tumor heterogeneity by halting tumor progression. Citation Format: Queralt Vallmajo-Martin, Nikki K. Lytle, Divya Murali, Sumana Srinivasan, Zhibo Ma, Kavitha Mukund, Christopher Dravis, Liliana Zamora, Ana Peterson, Galina Khramtsova, Funmi Olopade, Shankar Subramaniam, Geoffrey M. Wahl. The molecular landscape of injury-induced mammary epithelial cell fate switching [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A069.