Patient-matched Primary Tumors Research Articles

Osteopontin is a therapeutic target that drives breast cancer recurrence

Recurrent breast cancers often develop resistance to standard-of-care therapies. Identifying targetable factors contributing to cancer recurrence remains the rate-limiting step in improving long-term outcomes. In this study, we identify tumor cell-derived osteopontin as an autocrine and paracrine driver of tumor recurrence. Osteopontin promotes tumor cell proliferation, recruits macrophages, and synergizes with IL-4 to further polarize them into a pro-tumorigenic state. Macrophage depletion and osteopontin inhibition decrease recurrent tumor growth. Furthermore, targeting osteopontin in primary tumor-bearing female mice prevents metastasis, permits T cell infiltration and activation, and improves anti-PD-1 immunotherapy response. Clinically, osteopontin expression is higher in recurrent metastatic tumors versus female patient-matched primary breast tumors. Osteopontin positively correlates with macrophage infiltration, increases with higher tumor grade, and its elevated pathway activity is associated with poor prognosis and long-term recurrence. Our findings suggest clinical implications and an alternative therapeutic strategy based on osteopontin’s multiaxial role in breast cancer progression and recurrence.

Characterisation of the tumour microenvironment in primary and recurrent glioblastomas.

Glioblastoma patients have a dismal prognosis, due to inevitable tumour recurrence and respond poorly to immunotherapy. Tumour-associated microglia/macrophages (TAMs) dominate the glioblastoma tumour microenvironment and have been implicated in tumour progression and immune evasion. Early recurrent glioblastomas contain focal reactive regions with occasional fibrosis, chronic inflammation, TAMs and tumour cells. Surgical specimens from these tumours are rare and provide crucial insights into glioblastoma recurrence biology. This study aimed to characterise TAM- and lymphocyte phenotypes in primary vs early- and late-recurrent glioblastomas. Patient-matched primary and recurrent glioblastomas were compared between patients with early recurrences (n = 11, recurrence ≤6months) and late recurrences (n = 12, recurrence after 12-19 months). Double-immunofluorescence stains combining Iba1 with HLA-DR, CD14, CD68, CD74, CD86, CD163, CD204 and CD206 along with stains for CD20, CD3, CD8 and FOXP3 were quantified with software-based classifiers. Reactive regions in early recurrent tumours contained more TAMs (31.4% vs 21.7%, P = 0.01), which showed increased expression of CD86 (59.4% vs 38.4%, P = 0.04), CD204 (48.5% vs 28.4%, P = 0.03), CD206 (25.5% vs 14.4%, P = 0.04) and increased staining intensity for CD163 (86.4 vs 57.7 arbitrary units, P = 0.02), compared to late recurring tumours. Reactive regions contained more B-lymphocytes compared to patient-matched primary tumours (0.71% vs 0.40%, P = 0.04). Fractions of total, cytotoxic and regulatory T-lymphocytes did not differ. Early recurrent glioblastomas showed enrichment for TAMs, expressing both pro- and anti-inflammatory markers and B-lymphocytes. This may indicate a time-dependent response to immunotherapy explained by time-dependent alterations in the immune-microenvironment in recurrent glioblastomas.

AI/ML-derived mechanistically interpretable whole-genome biomarkers of patient survival in pre-treatment primary neuroblastoma tumors and whole blood.

10043 Background: Prediction, understanding, and management, of neuroblastoma (NBL) outcomes, from spontaneous regression to relapse and death, remain limited, and rely mostly on the International Neuroblastoma Staging System (INSS) stage, age, and the one-gene test for MYCN amplification. The entire multi-ome affects every aspect of the disease. But typical artificial intelligence and machine learning (AI/ML) cannot discover effective biomarkers from real-life, small-cohort, multi-omic, and noisy data. Methods: We use our AI/ML to identify two whole-genome biomarkers from open-source pre-treatment NBL patient-matched primary tumor and whole blood DNA copy numbers, and primary tumor mRNA expression. Our data-agnostic, unsupervised algorithms extend the mathematics that underlies quantum mechanics to overcome the limitations of typical AI/ML [1,2]. Our platform- and reference genome-agnostic biomarkers in astrocytoma, including glioblastoma, and lung, ovarian, and uterine adenocarcinomas, were mathematically discovered, and computationally and experimentally validated, repeatedly, in federated, imbalanced datasets from as few as 50–100 patients [3]. Results: We discover the biomarkers in “skinny” datasets of minimally preprocessed ≈3M-bin whole-genome sequences from tumors and blood of a set of 101 patients. Blindly, i.e., label-free, we separate both biomarkers from the normal X chromosome-number variation, demonstrating sex-agnostic learning. Combined, the biomarkers are statistically independent of all standard-of-care indicators, with the univariate Cox hazard ratio of 4.0 (log-rank P-value=2.3×10–5) within the 95% confidence intervals of the bivariate ratios. The risk that the tumor’s whole genome confers upon outcome, as is reflected by the bivariate ratios, is greater than that conferred by all standard-of-care indicators, including DNA ploidy, the Children’s Oncology Group (COG) risk, and the mitosis-karyorrhexis index (MKI), except for histopathology. We validate the biomarkers in ≈10K-bin target-capture sequences of a mutually-exclusive set of 419 patients, demonstrating generalizability as well as site-, platform-, and protocol-agnostic transfer learning. At 73–80% concordance, in both the tumor and blood profiles of both the discovery and validation sets, the biomarkers combined are more accurate than MYCN. We show that the biomarkers identify known and previously unrecognized disease mechanisms and druggable gene alterations, including co-amplification of MYCN with genes encoding for extra-embryonic transcripts, as well as the hijacking of embryonic development toward aneuploidy, which can spontaneously regress via embryonic self-correction. Conclusions: Our AI/ML is uniquely suited for personalized medicine. 1. doi: 10.1063/1.5099268 2. doi: 10.1073/pnas.0530258100 3. doi: 10.1063/1.5142559.

429 Spatial Investigation of the Extracellular Matrix Metastatic Niche in Invasive Breast Cancer by Mass Spectrometry Imaging

OBJECTIVES/GOALS: Metastasis to regional areas decreases invasive breast cancer (IBC) survival rate by 13%. Despite the clinical importance of lymph node involvement, the role of extracellular matrix (ECM) remodeling in metastases is unknown. We hypothesize that the spatial dysregulation of the collagen proteome facilitates pro-tumorigenic immune infiltration. METHODS/STUDY POPULATION: Lymph node metastases were compared to patient-matched primary tumor and normal lymph nodes using tissue microarrays (TMA) from 31 generational South Carolina women with IBC (black women, BW n=10, white women, WW n=21) and lumpectomies from 5 triple-negative breast cancer (TNBC) patients (BW n=3; WW n=2) by ECM-targeted mass spectrometry imaging. RESULTS/ANTICIPATED RESULTS: Between metastatic and normal lymph nodes, 10% of peptides, primarily from fibrillar collagens, were significantly different by area under the receiver operating curve (AUROC>70%; p-value< 0.01) within the TMAs. In a subsequent preliminary study of the TNBC metastatic niche, a segmentation analysis of 152 putatively identified peptides and 117,909 pixels revealed 10 uniquely localized proteomic groups. 12 peptides were found to have significantly decreased relative peak intensities in lymph node metastases compared to the primary tumor and normal lymph nodes by a one-way ANOVA test (p< 0.05). 7 peptides could discriminate between metastatic and normal lymph nodes, while 22 peptides could discriminate between metastatic lymph nodes and the primary tumor (AUROC>0.70; p-value < 0.05). DISCUSSION/SIGNIFICANCE: Our preliminary interrogation highlights emerging differences between lymph node metastases, the primary tumor, and normal lymph nodes. Future work is needed to connect these discrete ECM proteomes to immune infiltration alterations, which could contribute to disparate patient outcomes.

Islands of genomic stability in the face of genetically unstable metastatic cancer.

Metastatic cancer affects millions of people worldwide annually and is the leading cause of cancer-related deaths. Most patients with metastatic disease are not eligible for surgical resection, and current therapeutic regimens have varying success rates, some with 5-year survival rates below 5%. Here, we test the hypothesis that metastatic cancer can be genetically targeted by exploiting single base substitution mutations unique to individual cells that occur as part of normal aging prior to transformation. These mutations are targetable because ~10% of them form novel tumor-specific "NGG" protospacer adjacent motif (PAM) sites targetable by CRISPR-Cas9. Whole genome sequencing was performed on five rapid autopsy cases of patient-matched primary tumor, normal and metastatic tissue from pancreatic ductal adenocarcinoma decedents. CRISPR-Cas9 PAM targets were determined by bioinformatic tumor-normal subtraction for each patient and verified in metastatic samples by high-depth capture-based sequencing. We found that 90% of PAM targets were maintained between primary carcinomas and metastases overall. We identified rules that predict PAM loss or retention, where PAMs located in heterozygous regions in the primary tumor can be lost in metastases (private LOH), but PAMs occurring in regions of loss of heterozygosity (LOH) in the primary tumor were universally conserved in metastases. Regions of truncal LOH are strongly retained in the presence of genetic instability and, therefore, represent genetic vulnerabilities in pancreatic adenocarcinomas. A CRISPR-based gene therapy approach targeting these regions may be a novel way to genetically target metastatic cancer.

Limiting mitochondrial plasticity by targeting DRP1 induces metabolic reprogramming and reduces breast cancer brain metastases.

Disseminated tumor cells with metabolic flexibility to utilize available nutrients in distal organs persist, but the precise mechanisms that facilitate metabolic adaptations remain unclear. Here we show fragmented mitochondrial puncta in latent brain metastatic (Lat) cells enable fatty acid oxidation (FAO) to sustain cellular bioenergetics and maintain redox homeostasis. Depleting the enriched dynamin-related protein 1 (DRP1) and limiting mitochondrial plasticity in Lat cells results in increased lipid droplet accumulation, impaired FAO and attenuated metastasis. Likewise, pharmacological inhibition of DRP1 using a small-molecule brain-permeable inhibitor attenuated metastatic burden in preclinical models. In agreement with these findings, increased phospho-DRP1 expression was observed in metachronous brain metastasis compared with patient-matched primary tumors. Overall, our findings reveal the pivotal role of mitochondrial plasticity in supporting the survival of Lat cells and highlight the therapeutic potential of targeting cellular plasticity programs in combination with tumor-specific alterations to prevent metastatic recurrences.

Abstract 4831: Mitochondrial plasticity enables metabolic reprogramming and metastatic latency

Abstract Introduction: Metastatic relapses are common in epidermal growth factor receptor 2 (HER2) amplified breast cancer patients considered disease-free after primary diagnosis and treatment. Stem-like disseminated tumor cells with specialized metastatic traits adapt and survive as latent metastases in distal organs by overcoming oxidative stress, nutrient limitation, microenvironmental and immune defenses. These latent subclinical metastatic cells are responsible for late recurrences. Understanding the traits and vulnerabilities of these cells is critical for developing strategies to prevent metastatic relapse. Method: Here, we employed mouse models and patient samples along with RNA sequencing, Steady-state metabolite analysis, Lipid profiling, C13-isotope tracing, C12-BODIPY pulse chase, Lentiviral shRNA Knockdown, Transmission electron microscopy etc. to investigate how disseminated latent metastatic cells meet their cellular energetic demands and initiate overt metastasis in the lipid-rich brain microenvironment. Result: The ability to utilize available nutrients in distal organs and rewire metabolism is critical for survival of disseminated tumor cells. We report that latent metastatic cells in the brain uptake, store, and utilize fatty acids secreted by tumor-associated reactive astrocytes to meet their cellular energetic needs. Fatty acid oxidation (FAO) and dynamin-related protein 1(DRP1) driven punctate mitochondria enabled utilization of fatty acids and promote survival of latent cells by maintaining cellular bioenergetics and redox homeostasis. Attenuating fatty acid oxidation by genetic and pharmacological inhibition disrupted mitochondrial dynamics and limits metastatic incidence. Likewise, depleting DRP1 altered mitochondrial dynamics that results in reduced FAO, increased reactive oxygen species and attenuated metastatic latency and relapse in HER2+ breast cancer brain metastatic models. Furthermore, comparison with patient-matched primary tumor and brain metastasis identified increased pDRP1 expression in brain metastatic lesions. Moreover, pharmacological inhibition of DRP1 reduced brain metastatic burden in these preclinical models. Conclusion: Despite significant advances in targeted therapies, HER2+ breast cancer patients often develop brain metastasis and their prevention and management remain an unmet need. Our findings in latent HER2+ breast cancer brain metastatic models demonstrate mitochondrial plasticity and altered fatty acid metabolism are key determinants of metastatic adaptation to the brain and establish DRP1 as a promising therapeutic target for treating brain metastasis. Key Words: Breast Cancer Brain Metastasis, Mitochondrial Plasticity, DRP1, Metastatic Latency, Redox Homeostasis. Citation Format: Pravat Kumar Parida, Srinivas Malladi. Mitochondrial plasticity enables metabolic reprogramming and metastatic latency. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4831.

Abstract 5545: Assessing LGR5 expression levels on colorectal cancer tissue samples for use in LGR5-targeting CAR-T cell therapy clinical trial

Abstract Leucine-rich repeat containing G-protein coupled receptor (LGR5) is stem cell marker that potentiates canonical signaling in the Wnt/β-catenin signaling pathway (Barker et al. 2007). LGR5 is known to be expressed on several tissues; particularly the stem cells within intestinal crypts and the stem cell niche at the base of hair follicles (Barker et al. 2007 and Jaks et al. 2008). The plasticity and stem phenotype of LGR5+ cells has led to their identification as a cancer stem cell marker for some solid tumors including colorectal cancer (CRC) (Barker et al. 2009). This presents LGR5 as a potential target molecule for CAR-T cell directed specific tumor cell killing. For treatment of CRC with LGR5-directed CAR-T cells, it is critical to gain an understanding of the level of LGR5 expression on normal tissue compared with cancerous tissue. The aim of this study was to develop a screening technique for patient selection, and importantly identify potential responders to LGR5-targeting CAR-T cell therapy.Utilizing commercially purchased tissue microarrays a cohort of patient primary tumor biopsies (n=472) were assessed for LGR5 expression; ~60% displayed moderate to high level LGR5 positivity, with a trend toward higher grade, later stage tumors having higher levels of LGR5. LGR5 expression levels on lymph node metastases showed a positive correlation to LGR5 expression on patient-matched primary tumor tissue. Seven out of ten liver biopsies from metastatic colon cancer lesions showed a medium to high level LGR5 staining, indicating this may be used as a screening tool for metastatic CRC patients. We also observed restricted expression of LGR5 across the many normal tissues, suggesting limited off-target effects of LGR5-targeting CAR-T cells.The data obtained in this study provides evidence that LGR5 IHC of biopsies could be used as a diagnostic screening tool of CRC patients for inclusion into a Phase1a/2 clinical trial. Barker N, et al., 2007, Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature 449: 1003–1007 Barker N. et al. 2009, Crypt stem cells as the cells-of-origin of intestinal cancer. Nature 457: 608–611Jaks V. et al. 2008, Lgr5 marks cycling, yet long-lived, hair follicle stem cells. Nat Genet. Nov;40(11):1291-9. Citation Format: Emma J. Thompson, Veronika Bandara, Batjargal Gundsambuu, Silvana Napoli, Stuart Mills, Jade Foeng, Dylan McPeake, Timona Tyllis, Caitlin Abbott, Allison Cowin, Timothy Sadlon, Shaun McColl, Carmela Ricciardelli, Simon C. Barry, Claudine S. Bonder. Assessing LGR5 expression levels on colorectal cancer tissue samples for use in LGR5-targeting CAR-T cell therapy clinical trial. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5545.

Ciliated Cells in Ovarian Cancer Decrease with Increasing Tumor Grade and Disease Progression.

Ciliated cell markers expressed in epithelial ovarian cancers (EOC) are associated with improved survival. We examined the distribution of cells expressing ciliated cell markers in various EOC histologies and stages. Immunohistochemistry and/or multiplex immunofluorescence were used to determine the expression of FOXJ1 and/or CAPS (ciliated cell markers) in tissue microarrays including 4 normal fallopian tubes, 6 normal endometria, 16 cystadenomas, 25 borderline tumors, 21 low-grade carcinomas, and 118 high-grade carcinomas (HGSOC) (46 serous, 29 endometrioid, 30 clear cell, 13 mucinous). CAPS+ cells were observed in normal fallopian tubes and endometria and in ~85% of serous benign and borderline tumors and low-grade carcinomas but only in <40% of HGSOC. mRNA data from an independent cohort showed higher FOXJ1 and CAPS expression in serous borderline tumors and low-grade carcinomas compared to HGSOC. In HGSOC, ciliated cell-positive markers were observed in 52% primary tumors compared to 26% of patient-matched synchronous metastases, and 24% metachronous metastases (p = 0.009). mRNA data from an independent HGSOC cohort showed lower levels of CAPS in metastases than in primary tumors (p = 0.03). Overall, the study revealed that ciliated cells were less common in mucinous EOC, the percentage of ciliated cell marker-positive cases decreased with increasing grade, and the percentage of ciliated cells decreased in HGSOC metastases compared to patient-matched primary tumors.

CNSC-22. IDENTIFICATION OF MECHANISMS INVOLVED IN THE RECURRENCE OF GLIOBLASTOMA BY TRANSCRIPTOMIC, PROTEOMIC AND SPATIAL PROFILING ANALYSIS

Abstract BACKGROUND Glioblastoma (GBM) is the most aggressive and common brain tumor. Despite multimodal therapy which consists of complete tumor removal, chemo- and radiation therapy in most patients, the tumor recurs, and the overall survival of only 14 months remains very poor. To identify tumor-induced immune evasion and treatment resistance mechanisms induced during GBM recurrence, we aimed at characterizing the immunological/molecular changes in patient-matched treatment-naïve and recurrent GBM samples. MATERIAL AND METHODS Proteins and total RNA were extracted from 16 patient-matched treatment-naïve (primary) and recurrent GBM fresh frozen tumor samples. The expression of genes involved in brain immune responses were compared using Nanostring panels encompassing Cancer and Neuroinflammation genes. Proteomic was conducted using 6 patient-matched primary and recurrent tumor samples. Immunostaining was performed on a tissue microarray containing matched primary and recurrent tumor samples to identify tumor cells and microglia in order to assess spatial transcriptome of these specific cell types in situ using GeoMX technology. Moreover, correlation analysis of differentially genes and proteins with time to relapse was performed. RESULTS Genes significantly differentially expressed between primary and recurrent tumors revealed genes overexpressed in recurrent tumors belonging to “FCGR-mediated Phagocytosis” and “synapse pruning” pathways. Proteomic confirmed the synaptic signalling considering overexpressed proteins. Correlation studies with the time to relapse indicated that higher expression of FCG receptors, complement molecules and synaptic components correlate with shorter time to relapse, sharing similarities with neurodegenerative/cognitive disorders such as schizophrenia and Alzheimer disease. The source for these molecules is under evaluation with the spatial transcriptomic analysis. CONCLUSION Upregulation of specific proteins such as FCG receptors, complement and synaptic components may result in more favourable conditions for tumor regrowth and may therefore be identified as negative predictors patient outcome. Interfering with these molecules may represent novel therapeutic options after initial GBM surgery.

P17.10.B Identification of mechanisms involved in the recurrence of glioblastoma by transcriptomic, proteomic and spatial profiling analysis

Abstract Background Glioblastoma (GBM) is the most aggressive and common brain tumor. Despite multimodal therapy which consists of complete tumor removal, chemo- and radiation therapy in most patients, the tumor recurs, and the overall survival of only 14 months remains very poor. To identify tumor-induced immune evasion and treatment resistance mechanisms induced during GBM recurrence, we aimed at characterizing the immunological/molecular changes in patient-matched treatment-naïve and recurrent GBM samples. Material and Methods Proteins and total RNA were extracted from 16 patient-matched treatment-naïve (primary) and recurrent GBM fresh frozen tumor samples. The expression of genes involved in brain immune responses were compared using Nanostring panels encompassing Cancer and Neuroinflammation genes. Proteomic was conducted using 6 patient-matched primary and recurrent tumor samples. Immunostaining was performed on a tissue microarray containing matched primary and recurrent tumor samples to identify tumor cells and microglia in order to assess spatial transcriptome of these specific cell types in situ using GeoMX technology. Moreover, correlation analysis of differentially genes and proteins with time to relapse was performed. Results Genes significantly differentially expressed between primary and recurrent tumors revealed genes overexpressed in recurrent tumors belonging to “FCGR-mediated Phagocytosis” and “synapse pruning” pathways. Proteomic confirmed the synaptic signalling considering overexpressed proteins. Correlation studies with the time to relapse indicated that higher expression of FCG receptors, complement molecules and synaptic components correlate with shorter time to relapse, sharing similarities with neurodegenerative/cognitive disorders such as schizophrenia and Alzheimer disease. The source for these molecules is under evaluation with the spatial transcriptomic analysis. Conclusion Upregulation of specific proteins such as FCG receptors, complement and synaptic components may result in more favourable conditions for tumor regrowth and may therefore be identified as negative predictors patient outcome. Interfering with these molecules may represent novel therapeutic options after initial GBM surgery.

Systematic study of tissue factor expression in solid tumors.

Elevated tissue factor (TF) expression, although restricted in normal tissue, has been reported in multiple solid cancers, and expression has been associated with poor prognosis. This manuscript compares TF expression across various solid tumor types via immunohistochemistry in a single study, which has not been performed previously. To increase insight in the prevalence and cellular localization of TF expression across solid cancer types, we performed a detailed and systematic analysis of TF expression in tumor tissue obtained from patients with ovarian, esophageal, bladder, cervical, endometrial, pancreatic, prostate, colon, breast, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), and glioblastoma. The spatial and temporal variation of TF expression was analyzed over time and upon disease progression in patient-matched biopsies taken at different timepoints. In addition, TF expression in patient-matched primary tumor and metastatic lesions was also analyzed. TF expression was detected via immunohistochemistry (IHC) using a validated TF-specific antibody. TF was expressed in all cancer types tested, with highest prevalence in pancreatic cancer, cervical cancer, colon cancer, glioblastoma, HNSCC, and NSCLC, and lowest in breast cancer. Staining was predominantly membranous in pancreatic, cervical, and HNSCC, and cytoplasmic in glioblastoma and bladder cancer. In general, expression was consistent between biopsies obtained from the same patient over time, although variability was observed for individual patients. NSCLC biopsies of primary tumor and matched lymph node metastases showed no clear difference in TF expression overall, although individual patient changes were observed. This study shows that TF is expressed across a broad range of solid cancer types, and expression is present upon tumor dissemination and over the course of treatment.

Abstract 60: Functional mapping reveals widespread remodelling and unrecognized pathway dependencies in recurrent glioblastoma

Abstract Glioblastoma is a highly fatal brain cancer. The underlying functional drivers of treatment resistance and disease recurrence are unclear. By applying a genome-wide CRISPR-Cas9 library to patient-derived glioblastoma stem cell models, we systematically map genetic dependencies in patient-matched pre-treatment primary and post-treatment recurrent tumor cells. These insights reveal a large-scale remodelling of genetic dependency profiles at disease recurrence, arming recurrent tumor cells with newly-acquired genetic drivers and further loss of tumor suppressors. These analyses support parallel tumor-intrinsic mechanisms of treatment resistance which rely on acquisition of immunosuppressive capacity, including a defective mismatch repair pathway, ablation of PTEN activity, and a novel combination of de novo mutations in SWI/SNF components. We map a multilayered genetic and functional response to drive tumor recurrence, identifying protein tyrosine phosphatase 4A2 (PTP4A2) as a novel driver of self-renewal, proliferation and tumorigenicity at glioblastoma recurrence. Mechanistically, genetic perturbation and a small molecule inhibitor of PTP4A2 results in greater survival and reduced tumor growth in patient-derived models of recurrent glioblastoma. Citation Format: Chirayu R. Chokshi, Kevin Brown, Chitra Venugopal, Jason Moffat, Sheila K. Singh. Functional mapping reveals widespread remodelling and unrecognized pathway dependencies in recurrent glioblastoma [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 60.

Abstract 5123: Establishing circulating tumor cell-derived xenografts following a period of in vitro culture

Abstract Cancer metastasis—the spread of cancer cells from a primary node to a distant site—is responsible for as much as 90% of all cancer-associated deaths due to treatment resistance and increased tumor burden. Once established, metastatic nodules oftentimes exhibit distinct molecular phenotypes from their original primary tumor, undermining gold-standard methods for determining treatments for patients, which typically rely on molecular characterization of the primary tumor. Unfortunately, a lack of robust, scalable, and appropriate models for studying differences in patient-derived primary and metastatic tumors has hindered progress in identifying metastasis-specific therapies. Previously, we reported the ability to establish in vitro cultures of circulating tumor cells (CTCs), a unique precursor population of cells that exhibits an ability to escape the primary tumor but not yet fully complete metastasis. We have since expanded the application of this GU-CTC platform to culture CTCs from the blood of patients diagnosed with a variety of metastatic cancers, resulting in the successful culture of 31 unique patient-derived CTC cell lines. Furthermore, following an initial short-term culture period, we also performed subcutaneous injection of mixed CTC cultures into immunocompromised mice, resulting in not only primary tumor formation, but also systemic metastatic nodule formation in as short as 3 months. These resulting CTC-derived xenografts (CDXs) have been demonstrated using metastatic pancreatic, colorectal, and lung adenocarcinoma-derived CTCs. Encouragingly, all CDX models also exhibited replicable metastatic patterns upon reimplantation, reinforcing the important role of CTCs as precursors to metastasis. Bulk RNA-sequencing of patient-matched whole blood, cultured CTCs, and CDX-derived primary tumor and metastatic tissues further identified enrichment of an epithelial-mesenchymal transition as well as several cancer-specific gene signatures consistent with an invasive phenotype. This extended GU-CTC platform therefore enables not only the robust and rapid expansion of CTCs through both in vitro and in vivo methods, but also offers an avenue for personalized drug screens in the effort to target cancer metastasis. Citation Format: Jerry Xiao, Richard Schlegel, Seema Agarwal. Establishing circulating tumor cell-derived xenografts following a period of in vitro culture [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 5123.

3D patient-derived tumor models to recapitulate pediatric brain tumors In Vitro

Brain tumors are the leading cause of cancer-related deaths in children. Tailored therapies need preclinical brain tumor models representing a wide range of molecular subtypes. Here, we adapted a previously established brain tissue-model to fresh patient tumor cells with the goal of establishing3D in vitro culture conditions for each tumor type.Wereported our findings from 11 pediatric tumor cases, consisting of three medulloblastoma (MB) patients, three ependymoma (EPN) patients, one glioblastoma (GBM) patient, and four juvenile pilocytic astrocytoma (Ast) patients. Chemically defined media consisting of a mixture of pro-neural and pro-endothelial cell culture medium was found to support better growth than serum-containing medium for all the tumor cases we tested. 3D scaffold alone was found to support cell heterogeneity and tumor type-dependent spheroid-forming ability; both properties were lost in 2D or gel-only control cultures. Limited in vitro models showed that the number of differentially expressed genes between in vitro vs. primary tissues, are 104 (0.6%) of medulloblastoma, 3,392 (20.2%) of ependymoma, and 576 (3.4%) of astrocytoma, out of total 16,795 protein-coding genes and lincRNAs. Two models derived from a same medulloblastoma patient clustered together with the patient-matched primary tumor tissue; both models were 3D scaffold-only in Neurobasal and EGM 1:1 (v/v) mixture and differed by a 1-mo gap in culture (i.e., 6wk versus 10wk). The genes underlying the in vitrovs. in vivo tissue differences may provide mechanistic insights into the tumor microenvironment. This study is the first step towards establishing a pipeline from patient cells to models to personalized drug testing for brain cancer.

Abstract PD14-05: Portraying tumor evolution of lobular breast cancer through phylogenetic analysis

Abstract Background: Despite therapeutic advancements, a substantial number of patients with treated early-stage invasive lobular carcinoma (ILC) of the breast will still relapse as late as 20 years after the initial tumor diagnosis, thus making follow-up a challenging task. Here, using patient-matched primary and metastatic tumors from patients with ILC, we aimed to decipher the evolution of lobular breast cancer through time and explore metastatic dissemination events and underlying heterogeneity. Materials and Methods: We retrospectively identified 38 patients with metastatic ILC, of which 6 with de novo metastatic disease. In total, 99 formalin-fixed paraffin embedded tissue samples of primary tumor and metastasis, together with well-annotated clinicopathological data, were available. Low coverage whole-genome sequencing was performed to infer somatic copy number aberrations (CNA). Phylogenetic reconstruction was performed with Integer Linear Program for the Copy-Number Tree Problem algorithm. Results: Reconstruction of the phylogenetic trees showed different patterns of dissemination. For patients with multiple metastatic samples sequenced (4/4 patients), the metastases were found to be seeded from a common metastatic precursor. For a subset of patients with multiple primaries (5/10 patients), distant metastases were seeded from specific clones inside the primary tumor. Detailed analysis of known driver genes showed that alterations in common driver genes such as TP53, MYC and CCND1 were shared between primary tumor and metastases in a large proportion of the patients (47%, 32% and 21% of patients respectively) whilst CNA in other driver genes including FGFR2, PTEN or AKT2 among others, were frequently private in the metastases. Interestingly, we observed that in approximately 30% of the patients, the metastases harbored less CNA than the matched primary tumor, resulting in shorter metastatic branches and possibly indicating a decelerated tumor progression. Further analysis of the genomic and clinicopathological characteristics showed that these patients were mostly de novo metastatic (p-value=0.002), metastasizing primarily to the liver or bone and had significantly more aberrations in NF1, AKT1 and FGFR (p-value=0.04, 0.01 and 0.03 respectively), when compared to the rest of the cohort. Finally, most of the patients (63%) had a lower number of CNA accumulated after the metastatic lineage separation compared to the common trunk, implying a late dissemination event. Conclusions: In this study, we described different metastatic dissemination events taking place in ILC. Our analyses allowed us to identify a subset of tumors characterized by slow tumor progression, indicating potential tumor dormancy, as well as evidence of late dissemination evidence for most of the patients, thus highlighting the importance of early detection through cancer screening. These findings further shed light on how ILCs evolves through time and could potentially influence the clinical management of invasive lobular breast cancer. Citation Format: Danai Fimereli, David Venet, Mattia Rediti, David N Brown, Bram Boeckx, Marion Maetens, Samira Majjaj, Ghizlane Rouas, Maria Capra, Giuseppina Bonizzi, Federica Contaldo, Christine Galant, Martine Piccart, Giancarlo Pruneri, Denis Larsimont, Diether Lambrechts, Christine Desmedt, Françoise Rothé, Christos Sotiriou. Portraying tumor evolution of lobular breast cancer through phylogenetic analysis [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 PD14-05.

Prognostic signatures associated with high infiltration of Tregs in bone metastatic prostate cancer.

Metastatic cancer especially bone metastasis (BM) is the lethal end-stage of castration-resistant prostate cancer (CRPC). To understand the possible molecular mechanisms underlying the development of the distant metastasis is of potential clinical value. We sought to identify differentially expressed genes between patient-matched primary and bone metastatic CRPC tumors. Functional enrichment, protein-protein interaction networks, and survival analysis of DEGs were performed. DEGs with a prognostic value considered as candidate genes were evaluated, followed by genetic analysis of tumor infiltrating immune cells based on Wilcoxon test and immunofluorescence identification. Expression profiles analysis showed that 381 overlapping genes were screened as differentially expressed genes (DEGs), of which 16 DEGs were randomly selected to be validated and revealed that most of these genes showed a transcriptional profile similar to that seen in the datasets (Pearson’s r = 0.76). Six core genes were found to be involved in regulation of extracellular matrix receptor interaction and chemotactic activity, and four of them were significantly correlated with the survival of PCa patients with bone metastases. Immune infiltration analysis showed that the expressions levels of COL3A1, RAC1, FN1, and SDC2 in CD4+T cells were significantly higher than those in tumor cells, especially regulatory T cell infiltration was significantly increased in BM tumors. We analyzed gene expression signatures specifically associated with the development of bone metastases of CRPC patients. Characterization of genes associated with BM of mCRPC is critical for identification of predictive biomarkers and potential therapeutic targets.

Prognostic relevance of the hexosamine biosynthesis pathway activation in leiomyosarcoma

Metabolic reprogramming of tumor cells and the increase of glucose uptake is one of the hallmarks of cancer. In order to identify metabolic pathways activated in leiomyosarcoma (LMS), we analyzed transcriptomic profiles of distinct subtypes of LMS in several datasets. Primary, recurrent and metastatic tumors in the subtype 2 of LMS showed consistent enrichment of genes involved in hexosamine biosynthesis pathway (HBP). We demonstrated that glutamine-fructose-6-phosphate transaminase 2 (GFPT2), the rate-limiting enzyme in HBP, is expressed on protein level in a subset of LMS and the expression of this enzyme is frequently retained in patient-matched primary and metastatic tumors. In a new independent cohort of 327 patients, we showed that GFPT2 is associated with poor outcome of uterine LMS but not extra-uterine LMS. Based on the analysis of a small group of patients studied by 18F-FDG-PET imaging, we propose that strong expression of GFPT2 in primary LMS may be associated with high metabolic activity. Our data suggest that HBP is a potential new therapeutic target in one of the subtypes of LMS.

Identification of PDE7B as a Potential Core Gene Involved in the Metastasis of Clear Cell Renal Cell Carcinoma.

BackgroundMetastasis is the main cause of treatment failure in various cancer, including ccRCC. However, the key genes involved in ccRCC metastasis remain largely unknown.PurposeThe identification of the aberrant gene expression patterns associated with metastatic traits is of great clinical significance. The aim of this study was to investigate the clinical significance and function of PDE7B in ccRCC.Materials and MethodsExpression profiling data for patient-matched primary and metastatic ccRCC tumors were obtained from GEO Dataset. Limma package was used to identify differentially expressed genes (DEGs) between the metastatic and the primary groups. Gene Ontology, Kyoto Encyclopedia of Genes Genomes (KEGG), and PPI network analysis were used to study the interacting activities and the interconnection of the DEGs. CCK-8 assays and Transwell assays were performed to detect the proliferation and migration of renal cancer cells.ResultsWe obtained 163 DEGs, including 132 that were upregulated and 31 that were downregulated in metastatic ccRCC tissues. Both Gene Ontology function and KEGG pathway analysis showed that DEGs were involved in extracellular matrix (ECM) organization and cell adhesion. After utilizing PPI network to explore the interconnection among the DEGs, 22 genes were selected as the hub genes. Subsequently, survival analysis revealed that seven hub genes (SFN, NKX2-1, HP, MAPT, EPHA4, KCNAB1, and PDE7B) were significantly associated with overall survival disease-specific survival, and progression-free interval in ccRCC. Moreover, the low expression of PDE7B was found in clinical ccRCC samples and correlated with TNM stage and histologic grade. We further showed that knockdown of PDE7B increased cell growth and migration of renal cancer cells.ConclusionOur results implicated that PDE7B may play a key role in the development of metastatic RCC.

Tradeoff between metabolic i-proteasome addiction and immune evasion in triple-negative breast cancer

In vitro studies have suggested proteasome inhibitors could be effective in triple-negative breast cancer (TNBC). We found that bortezomib and carfilzomib induce proteotoxic stress and apoptosis via the unfolded protein response (UPR) in TNBC cell lines, with sensitivity correlated with expression of immuno-(PSMB8/9/10) but not constitutive-(PSMB5/6/7) proteasome subunits. Equally, the transcriptomes of i-proteasome-high human TNBCs are enriched with UPR gene sets, and the genomic copy number landscape reflects positive selection pressure favoring i-proteasome activity, but in the setting of adjuvant treatment, this is actually associated with favorable prognosis. Tumor expression of PSMB8 protein (β5i) is associated with levels of MHC-I, interferon-γ-inducible proteasome activator PA28β, and the densities of stromal antigen-presenting cells and lymphocytes (TILs). Crucially, TILs were protective among TNBCs that maintain high β5i but did not stratify survival amongst β5i-low TNBCs. Moreover, β5i expression was lower in brain metastases than in patient-matched primary breast tumors (n = 34; P = 0.007), suggesting that suppression contributes to immune evasion and metastatic progression. Hence, inhibiting proteasome activity could be counterproductive in the adjuvant treatment setting because it potentiates anti-TNBC immunity.