Untreated Tumors Research Articles

Abstract 1952: Co-alterations in PIK3CA and ARID1A lead to enhanced sensitivity to PI3K inhibition

Abstract Background: There is increasing interest in developing precision medicine strategies for cancers with PIK3CA mutations. Co-alterations in PIK3CA and ARID1A (AD) occur in ~1% of all cancers. Previously, patients with co-alterations in PIK3CA and AD have shown both an improved objective response rate and improved progression free survival following copanlisib (cop) treatment compared to cancers with PIK3CA mutations and wild-type AD. Here we evaluate potential mechanisms for this enhanced clinical benefit. Methods: AD was knocked out in SW48 and SW48PIK3CA-H1047R (SW48PK) human 2D colorectal cancer cell lines using CRISPR (SW48AD; SW48PKAD). Knockouts were confirmed using PCR genotyping, amplicon sequencing, and western blot (WB). A cell proliferation assay (WST-1) was performed at increasing doses of cop for 48hrs to determine cell viability. WBs were performed at 6hrs and 24hrs after cop treatment to determine levels of phosphorylation of AKT, S6, and 4EBP1 and levels of apoptosis-related proteins (MCL-1, BCL-xL, BCL-2, PARP). Athymic nude mice were flank injected with SW48PK and SW48PKAD and treated with cop (10mg/kg in PEG400/acidified water (0.1N HCl, pH 3.5; 20/80, v/v) or vehicle for 25 days or until moribund. Results: WST-1 assay showed highest sensitivity to cop in SW48PKAD cells compared to SW48, SW48PK and SW48AD. At 10nM (p=0.01), there was a significant difference in cell viability between SW48PK and SW48PKAD. No significant difference was seen in the phosphorylation (p) of AKT, S6, or 4EBP1 between SW48PK and SW48PKAD. Expression of MCL-1 protein decreased in SW48PKAD compared to SW48PK. No difference was seen in levels of BCL-xL and BCL-2. On treatment with cop, there was a greater reduction in pS6 in SW48PKAD at 24 hours compared to SW48PK, but no significant difference in pAKT and p4EBP1. There was a gradual increase in MCL-1, BCL-xL, and cleaved PARP in SW48PKAD overtime following cop treatment compared to SW48PK. In vivo, untreated SW48PKAD tumors grew more quickly than the SW48PK tumors (8-fold increase in tumor size in SW48PKAD compared to 5-fold increase in SW48PK by day 25, p=0.06). SW48PKAD mice had significant tumor reduction (p=0.002) on cop treatment by day 25; however, SW48PK mice did not. Conclusions: ARID1A loss along with PIK3CA mutation leads to greater PI3K inhibition response most likely due to enhanced induction of apoptosis. Further confirmation of this enhanced sensitivity is required with better in vitro models such as patient-derived cancer organoids. Citation Format: Shirsa Udgata, Jordan N. Stoecker, Anna L. Lippert, Cheri A. Pasch, Dustin A. Deming. Co-alterations in PIK3CA and ARID1A lead to enhanced sensitivity to PI3K inhibition [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 1952.

Abstract 700: Impact of heterogeneity for mismatch repair activity on colon tumor development and therapeutic response

Abstract Background: Colorectal cancers (CRC) that are deficient for DNA mismatch repair (dMMR) proteins have high levels of genetic instability and consequently high mutational burden, creating numerous neoantigens that can elicit an immune response. Therefore, dMMR CRC are the best candidates for immune checkpoint inhibition (ICI) compared to proficient DNA mismatch repair (pMMR) CRC. Early clinical trials revealed that only 30-55% metastatic dMMR CRC responded to ICI. Recent studies utilizing allograft models have demonstrated that tumor immune landscape can be primed with radiopharmaceutical therapy (RPT). Although early results have been quite promising, allograft models often fail to accurately predict efficacy for many reasons, but most often are due to a lack of heterogeneity. Methods: To better understand how heterogenous MMR expression impacts tumor development and response to ICI alone or in combination with RPT, we developed a new mouse model that develops three tumor types: (1) homotypic dMMR tumors that express green fluorescent protein, (2) homotypic pMMR tumors that express red fluorescent protein, and (3) heterotypic tumors with a mixture of dMMR and pMMR cells. Surveillance bright field and fluorescent colonoscopies allow us to assess treatment response by measuring tumor size and proportion of dMMR and pMMR tumor cells in real-time. These outcomes are verified through ex vivo imaging and histological analysis. Results: Tumor response to anti-PD-L1 ICI was dependent on MMR status. Homotypic dMMR tumors exhibited the strongest response: significantly smaller in treated mice than controls, exhibited areas of vascular congestion and increased CD8+ cytotoxic T-cells infiltration. Response of heterotypic MMR tumors varied depending on percentage of dMMR cells; tumors with a high percentage of dMMR cells remained small, whereas tumors with a low percentage of dMMR had an outgrowth of pMMR cells. Treated and untreated homotypic pMMR tumors were indistinguishable. To enhance the response of all three tumor types, mice were treated with dual ICI treatment, anti-PD-L1 and anti-CTLA-4, or RPT. Animals administered the tri-therapy (dual ICI + RPT) survived significantly longer with fewer tumors than animals treated with either dual ICI or RPT alone. Conclusions: Our innovative mouse model allows for the highly detailed characterization of tumor response to therapies. Simplistic allograft models may not adequately account for the dynamic effects that neighboring cell death may have on radio-resistant cells, on tumor vascular perfusion and oxygenation, or on tumor immune infiltration and adaptive immune recognition of remaining cells. We begin to fill these critical gaps in knowledge and therefore the results from our studies will help guide the translation and integrated use of ICIs and RPTs in clinic. Citation Format: Santina M. Snow, Dawn Albrecht, Paul A. Clark, Caroline P. Kerr, Joseph J. Grudzinski, Justin J. Jeffery, Hansel Comas Rojas, Reinier Hernandez, Kristina A. Matkowskyj, Jamey P. Weichert, Zachary S. Morris, Richard B. Halberg. Impact of heterogeneity for mismatch repair activity on colon tumor development and therapeutic response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 700.

Abstract 1226: Genomically encoded lineage plasticity drives resistance to EGFR targeted therapy in lung adenocarcinoma

Abstract EGFR-mutant lung adenocarcinoma (LUAD) represents 20% of all non-small cell lung carcinomas, with most patients presenting with incurable metastatic disease. Treatment with mutant-selective EGFR tyrosine kinase inhibitor (TKI) therapies such as osimertinib, the current standard of care for metastatic EGFR-mutant LUAD, results in greater overall and progression-free survival. However, all patients eventually relapse. Uncovering the mechanisms driving primary and acquired resistance to osimertinib is therefore critical to improving disease management. Here, we use clinical genomic sequencing (n=47) and single-nucleus RNA (snRNA) sequencing (n=62) to profile a cohort of unmatched treatment-naïve (n=26), on-treatment (n=6), and post-treatment relapsed (n=30) EGFR-mutant LUADs treated with osimertinib. Relapsed tumors are marked by increased tumor mutation burden, along with elevated rates of genome doubling and loss-of-heterozygosity. We derived expression-based signatures of LUAD, lung squamous cell carcinoma (LUSC), and small cell lung carcinoma (SCLC) and applied them to tumor cells across our cohort. Relapsed tumors had a marked decrease in LUAD gene signature expression compared to untreated tumors and accompanying increases in epithelial-mesenchymal transition (EMT) and stemness markers, consistent with dedifferentiation. Notably, tumors harboring secondary mutations to EGFR, MET, or other MAPK pathway genes and showing sustained MAPK signaling retained a more differentiated state. Patients lacking these secondary on-target or bypass mutations were more likely to experience relapse with histological transdifferentiation, marked by complete loss of LUAD signatures, and accompanying increases in LUSC or SCLC signatures. Phenotype matching of tumor cells to normal cells from the developing fetal lung revealed enrichment of airway progenitor populations in non-histologically transformed treatment-resistant patients, in contrast to a proportion of late bud-tip and club-like epithelial cells which predominated in treatment-naïve samples. In rare cases, we observed subpopulations of basal stem-like progenitors in otherwise well-differentiated pre-treatment tumors. In summary, single-cell mapping of histological and developmental phenotypes has revealed divergent evolutionary trajectories during the acquisition of resistance to osimertinib, defined by MAPK signaling reactivation or by loss of LUAD identity and histological transformation respectively. Citation Format: Matthew Zatzman, Alvaro Quintanal-Villalonga, Sohrab Salehi, Christina Falcon, Nicholas Ceglia, Sohrab P. Shah, Charles M. Rudin, Helena A. Yu. Genomically encoded lineage plasticity drives resistance to EGFR targeted therapy in lung adenocarcinoma [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 1226.

Abstract 6181: Digital pathology workflow: Building a bridge for translational biomarker development in humanized mice

Abstract The introduction of immune checkpoint inhibitors (ICIs) directed against programmed death protein 1 (PD-1), as well as cytotoxic T lymphocyte antigen 4 (CTLA-4), has revolutionized the treatment of non-small cell lung cancer (NSCLC) in the last decade. These therapies have shown significant success in a subset of NSCLC patients, leading to durable response and improved overall survival. However, clear predictive biomarkers for response are still yet to be defined. Tumor-infiltrating lymphocytes (TILs) as one important part of the tumor microenvironment (TME) have proven to be predictive biomarkers in other tumor types such as melanoma, whereas not fully understood in NSCLC. The assessment of TILs typically involves techniques such as flow cytometry (FC) or immunohistochemistry (IHC). While the former has the advantage of being quantitative and relatively easy to standardize it lacks translational value as most clinical samples will be examined using IHC. The current study aims to compare both methods at terminal end point and for treatment outcome, using a panel of 14 NSCLC PDX models, implanted in humanized NSG mice and treated with anti-CTLA4, anti-PD1 and the combination thereof. Based on FC analyses of the untreated tumor tissue the PDX models were stratified into seven hot (>5% TILs) and seven cold (<5% TILs) tumors. The IHC samples were analyzed by digital pathology for TIL infiltration by making use of automated segmentation of the three main tissue classes: tumor, stroma and necrosis. Small number of IHC slides were manually annotated to form a training set, which were then used to train a deep learning model using Visiopharm, a commercial AI platform for image analysis. By quantification of TILs in a spatial context of the tumor tissue, across all models, tumor was the most prominent tissue class. The tissue class distribution as well as the TIL rate (FC or IHC measure) is a model immanent feature. Under treatment, the ratio was stable in case of the cold tumors. In the hot tumors the stroma compartment increased while tumor tissue decreased. Treatment sensitive models displayed an increase of TILs confirmed by FC and IHC. The latter revealed that the TILs specifically increased in the tumor tissue whereas decreased or was stable in the stroma. This observation was significantly more pronounced in cold tumors compared to hot tumors (Oneway ANOVA, p < 0.001). Furthermore, a positive correlation between FC and IHC results on CD45 positive cells in the tumors (p< 0.002) could be confirmed. In this study, we proofed the feasibility of software-based image analysis as translational relevant read-out in preclinical animal experiments. The possibility to reproducibly quantify tissue classes, immune cell markers and including spatial information paves the way towards discovery of novel features predicting response in translational immuno-oncology research. Citation Format: Matti Hakkarainen, Timmo Bragge, Jussi Rytkönen, Eva Oswald, Julia B. Schueler. Digital pathology workflow: Building a bridge for translational biomarker development in humanized mice [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 6181.

Novel PET Imaging Probe for Quantitative Detection of Senescence In Vivo.

Real-time detection of cellular senescence remains a clinical challenge. Here, we aimed to develop a positron emission tomography (PET) imaging probe targeting senescence-associated β-galactosidase (SA-β-Gal), the most widely used biomarker of cellular senescence, and investigate its performance for real-time in vivo quantitative detection of cellular senescence. A stable PET imaging agent [68Ga]Ga-BGal was obtained with a high labeling yield (90.0 ± 4.3%) and a radiochemical purity (>95%). [68Ga]Ga-BGal displayed high sensitivity and specificity for β-Gal both in vitro and in vivo. The reaction and uptake of the probe correlated with the β-Gal concentration and reaction time. In PET imaging, high β-Gal-expressing CT26.CL25 tumors and doxorubicin-treated HeLa tumors showed high signals from [68Ga]Ga-BGal, while a low signal was observed in CT26.WT and untreated HeLa tumors. In summary, we showcased successful PET imaging of senescence in preclinical models using probe [68Ga]Ga-BGal. This finding holds the potential for translating senescence imaging into clinical applications.

Histoplasty Modification of the Tumor Microenvironment in a Murine Preclinical Model of Breast Cancer

PurposeTo develop a noninvasive therapeutic approach able to alter the biophysical organization and physiology of the extracellular matrix (ECM) in breast cancer. Materials and MethodsIn a 4T1 murine model of breast cancer, histoplasty treatment with a proprietary 700-kHz multielement therapy transducer using a coaxially aligned ultrasound (US) imaging probe was used to target the center of an ex vivo tumor and deliver subablative acoustic energy. Tumor collagen morphology was qualitatively evaluated before and after histoplasty with second harmonic generation. Separately, mice bearing bilateral 4T1 tumors (n = 4; total tumors = 8) were intravenously injected with liposomal doxorubicin. The right flank tumor was histoplasty-treated, and tumors were fluorescently imaged to detect doxorubicin uptake after histoplasty treatment. Next, 4T1 tumor-bearing mice were randomized into 2 treatment groups (sham vs histoplasty, n = 3 per group). Forty-eight hours after sham/histoplasty treatment, tumors were harvested and analyzed using flow cytometry. ResultsHistoplasty significantly increased (P = .002) liposomal doxorubicin diffusion into 4T1 tumors compared with untreated tumors (2.12- vs 1.66-fold increase over control). Flow cytometry on histoplasty-treated tumors (n = 3) demonstrated a significant increase in tumor macrophage frequency (42% of CD45 vs 33%; P = .022) and a significant decrease in myeloid-derived suppressive cell frequency (7.1% of CD45 vs 10.3%; P = .044). Histoplasty-treated tumors demonstrated increased CD8+ (5.1% of CD45 vs 3.1%; P = .117) and CD4+ (14.1% of CD45 vs 11.8%; P = .075) T-cell frequency. ConclusionsHistoplasty is a nonablative focused US approach to noninvasively modify the tumor ECM, increase chemotherapeutic uptake, and alter the tumor immune microenvironment.

IL-12-Overexpressed Nanoparticles Suppress the Proliferation of Melanoma Through Inducing ICD and Activating DC, CD8+ T, and CD4+ T Cells.

The drug resistance and low response rates of immunotherapy limit its application. This study aimed to construct a new nanoparticle (CaCO3-polydopamine-polyethylenimine, CPP) to effectively deliver interleukin-12 (IL-12) and suppress cancer progress through immunotherapy. The size distribution of CPP and its zeta potential were measured using a Malvern Zetasizer Nano-ZS90. The morphology and electrophoresis tentative delay of CPP were analyzed using a JEM-1400 transmission electron microscope and an ultraviolet spectrophotometer, respectively. Cell proliferation was analyzed by MTT assay. Proteins were analyzed by Western blot. IL-12 and HMGB1 levels were estimated by ELISA kits. Live/dead staining assay was performed using a Calcein-AM/PI kit. ATP production was detected using an ATP assay kit. The xenografts in vivo were estimated in C57BL/6 mice. The levels of CD80+/CD86+, CD3+/CD4+ and CD3+/CD8+ were analyzed by flow cytometry. CPP could effectively express EGFP or IL-12 and increase ROS levels. Laser treatment promoted CPP-IL-12 induced the number of dead or apoptotic cell. CPP-IL-12 and laser could further enhance CALR levels and extracellular HMGB1 levels and decrease intracellular HMGB1 and ATP levels, indicating that it may induce immunogenic cell death (ICD). The tumors and weights of xenografts in CPP-IL-12 or laser-treated mice were significantly reduced than in controls. The IL-12 expression, the CD80+/CD86+ expression of DC from lymph glands, and the number of CD3+/CD8+T or CD3+/CD4+T cells from the spleen increased in CPP-IL-12-treated or laser-treated xenografts compared with controls. The levels of granzyme B, IFN-γ, and TNF-α in the serum of CPP-IL-12-treated mice increased. Interestingly, CPP-IL-12 treatment in local xenografts in the back of mice could effectively inhibit the growth of the distant untreated tumor. The novel CPP-IL-12 could overexpress IL-12 in melanoma cells and achieve immunotherapy to melanoma through inducing ICD, activating CD4+ T cell, and enhancing the function of tumor-reactive CD8+ T cells.

Contemporary preclinical mouse models for pediatric rhabdomyosarcoma: from bedside to bench to bedside.

Rhabdomyosarcoma (RMS) is the most common pediatric soft-tissue malignancy, characterized by high clinicalopathological and molecular heterogeneity. Preclinical in vivo models are essential for advancing our understanding of RMS oncobiology and developing novel treatment strategies. However, the diversity of scholarly data on preclinical RMS studies may challenge scientists and clinicians. Hence, we performed a systematic literature survey of contemporary RMS mouse models to characterize their phenotypes and assess their translational relevance. We identified papers published between 01/07/2018 and 01/07/2023 by searching PubMed and Web of Science databases. Out of 713 records screened, 118 studies (26.9%) were included in the qualitative synthesis. Cell line-derived xenografts (CDX) were the most commonly utilized (n = 75, 63.6%), followed by patient-derived xenografts (PDX) and syngeneic models, each accounting for 11.9% (n = 14), and genetically engineered mouse models (GEMM) (n = 7, 5.9%). Combinations of different model categories were reported in 5.9% (n = 7) of studies. One study employed a virus-induced RMS model. Overall, 40.0% (n = 30) of the studies utilizing CDX models established alveolar RMS (aRMS), while 38.7% (n = 29) were embryonal phenotypes (eRMS). There were 20.0% (n = 15) of studies that involved a combination of both aRMS and eRMS subtypes. In one study (1.3%), the RMS phenotype was spindle cell/sclerosing. Subcutaneous xenografts (n = 66, 55.9%) were more frequently used compared to orthotopic models (n = 29, 24.6%). Notably, none of the employed cell lines were derived from primary untreated tumors. Only a minority of studies investigated disseminated RMS phenotypes (n = 16, 13.6%). The utilization areas of RMS models included testing drugs (n = 64, 54.2%), studying tumorigenesis (n = 56, 47.5%), tumor modeling (n = 19, 16.1%), imaging (n = 9, 7.6%), radiotherapy (n = 6, 5.1%), long-term effects related to radiotherapy (n = 3, 2.5%), and investigating biomarkers (n = 1, 0.8%). Notably, no preclinical studies focused on surgery. This up-to-date review highlights the need for mouse models with dissemination phenotypes and cell lines from primary untreated tumors. Furthermore, efforts should be directed towards underexplored areas such as surgery, radiotherapy, and biomarkers.

Abstract B024: A histological data-driven analysis of the hypoxic microenvironment of preclinical murine bladder tumors

Abstract Hypoxia is common in many solid tumors, including bladder cancer (BC). Preclinical murine models that mimic relevant molecular and phenotypical characteristics of clinical BC were used to analyze the immune cell composition within the hypoxic tumor microenvironment. The experimental protocols involve intravesical installations, adoptively transferred T cells, and chemotherapy and were designed to recreate therapeutic procedures used in a recently open feasibility clinical trial of intravesical adoptive cell therapy (ACT) for BC patients. We analyzed 18 murine BC tissues from a treatment protocol that included intravesical installment of MB49-OVA tumor cells on day 0, gemcitabine treatment on day 10, and/or intravesical transfer of OT-I transgenic T cells on day 14, in addition to untreated controls and normal bladder tissues. The OT-I cells mimic the T cells used in clinical ACT. The collected tissues were sliced, and consecutive sections were stained for vasculature, tumor, and immune cells: CD8+, CD4+, and myeloid-derived suppressor cells (MDSCs). Subsequently, these histology images were co-registered, segmented, and preprocessed to identify the tumor boundaries, select viable vasculature, resolve cell overlaps, and annotate bladder cavities. A multi-cell off-lattice hybrid agent-based model that combines discrete cells and vasculature with continuous oxygen kinetics was developed to recreate the differential oxygenation in the tumor microenvironment. In all tissues, the hypoxic microenvironment was more prevalent in the tumor regions than in the nontumor regions due to differences between the irregular tumor vasculature vs. normal vasculature. The T cell and MDSC infiltration was increased in the tumors treated with gemcitabine (as mono- or combination therapy). Despite the significant hypoxia level in these tumors, only small proportions of CD8+s and CD4+s were hypoxic, as the majority resided in the well-oxygenated portions of the tumor. High infiltration of CD8+ cells was observed in tumors treated with OT-I cells compared to the controls. However, only a small proportion were hypoxic, and most CD8+s and MDSCs were well-oxygenated. The CD8+s were located far from the intravesical source, with some cells near the vasculature. Hypoxic native CD8+s in untreated tissues were observed inside the tumors, and most well-oxygenated CD8+s were near the tumor borders. The CD8+s also resided in the well-oxygenated regions in the normal tissues. Our reconstructed tissue oxygenation based on histology images revealed that the hypoxic tumor microenvironment hinders T cell accumulation in treated and untreated tumors. Such quantitative analyses may encourage designing more effective hypoxia-mediated therapies. Citation Format: Awino Maureiq E. Ojwang, Anjun Hu, McKenzie Williams, Sarah Bazargan, Joseph O. Johnson, Shari Pilon-Thomas, Katarzyna A. Rejniak. A histological data-driven analysis of the hypoxic microenvironment of preclinical murine bladder tumors [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr B024.

Abstract B059: Epigenetically defined sub-clonal heterogeneity drives therapy resistance in triple-negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) is a highly aggressive and heterogeneous disease with an average survival of less than 50%. Although immunotherapy is showing promising results, many patients do not respond, and chemotherapy remains the main treatment. High TNBC recurrence rates are in part due to its molecular heterogeneity. Here, we sought to characterise the sub-clonal and spatial diversity of chemotherapy resistant TNBC to identify the epigenetic hallmarks of lethal sub-populations of cells that may induce cell state transition, therapy resistance and ultimately metastatic progression. Methods: Sixteen primary untreated triple-negative breast tumours (eight chemotherapy sensitive (ChemoS) and eight chemotherapy resistant (ChemoR, who died of their disease within three years)) were subjected to cell-matched high-depth multiome single nuclei RNA- and ATAC-sequencing. Four tumours were subject to spatial RNA and ATAC profiling. Fourteen TNBC PDX models generated from patients with residual disease post NAC were subjected to single nuclei sequencing and used for validation. Meta-clustering of epithelial cells from the RNA profiles was used to identify sub-populations of cells harbouring distinct epigenetic and transcriptomic features enriched in ChemoR patients. Findings were validated in the BrighTNess TNBC NAC clinical trial and in chemotherapy treated TNBC patients from the SCAN-B study. Results: Unsupervised meta-clustering (MC) of epithelial cells in G1 phase of the cell cycle identified nine MC’s across the dataset, including a distinct immune-like MC. Two MCs were dominated by cells from ChemoR patients and were defined by hypoxia, EMT, and extra-cellular matrix signalling. Sub-populations of cells in the ChemoR dominant MCs were underpinned by both promoter and distal differential chromatin accessibility, that mapped to H3K27ac enhancer sites, suggesting a role of enhancer regulation of MC transcription. Spatial ATAC and RNA profiling of a subset of tumours identified spatially epigenetically defined regions that showed distinct chromatin profiles, suggesting that the epigenetic regulation of genes that define the MCs are spatially distinct. ChemoR clusters were independently reproduced in a cohort of fourteen residual disease TNBC PDXs. Genes pertaining to the immune-like MC were associated with a good prognosis in independent cohorts of contemporary treated TNBC (SCAN-B) and with response to NAC in the BrighTNess trial. Marker genes from ChemoR dominant MCs were associated with poor prognosis and significant levels of residual disease following NAC. Conclusions: Our multi-modal integrative analysis reveals unprecedented insight into the role of epigenetically defined spatially distinct, sub-clonal populations that pre-exist in treatment naïve TNBC and predict therapy response. This suggests pre-existing populations of cells have already acquired the epigenetic footprints and hence transcriptomic features to allow them to evade therapy. Citation Format: Fatemeh Ahmadimoghari, Yu Zhang, Ioanna Mavrommatis, Shafali Thakur, Chris Starling, Ioannis Roxanis, Syed Haider, Rachael Natrajan. Epigenetically defined sub-clonal heterogeneity drives therapy resistance in triple-negative breast cancer [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 B059.

Abstract B025: MHC class II and CD1d expressing conventional dendritic cells with immunoglobulin therapy drives anti-tumor abscopal effect by recruiting Natural Killer T cells to the tumor microenvironment

Abstract Background Dendritic cells (DC) are the primary antigen presenting cells in the body responsible for processing and presenting MHC Class I and II antigens to initiate adaptive T cell response against cancer. In addition to MHC, DC also express non-classical receptors such as CD1d to activate invariant TCR α chain natural killer T (iNKT) cells. The conventional type-I dendritic cells (cDC1) presenting MHC class-II tumor associated antigens within tumor microenvironment (TME) have shown robust efficacy in mouse models of breast cancer (BC). Here, we have extended prior knowledge to deliver DC-based therapeutics, co-presenting class II antigens and CD1d directly to TME to show the potential of co-stimulation of multiple effector T cells in combination of tumor specific antibody (IgG1) mediating antibody dependent cellular cytotoxicity (ADCC). We hypothesize that merger of adaptive and innate responses will attain greater pathologic response in pre-clinical and clinical settings to design futuristic therapeutics against BC. Methods Present study uses variety of pre-clinical mouse models of BC and other types of cancer to test the therapeutic efficacy of intratumoral delivery of mouse bone marrow-derived conventional type I (CD103+ CD1d+) polarized DC pulsed with MHC class II HER2-derived peptides (HER2-DC1) in combination with systemic delivery of tumor specific IgG1 (aSEMA4D) to evoke ADCC. Cellular mechanism of response was evaluated using downstream methods including multiplex immunofluorescence, flow cytometry, and transcriptomics assays. Results We exhibit that specifically intratumoral combination therapy of HER2-DC1 expressing CD1d and aSEMA4D primes both innate and adaptive immune responses to completely regress tumor in 75-80% of mice with significant survival advantage in comparison to mice that received no therapy or monotherapies. The early CD4 Th1 response through interferon gamma combined with ADCC played a critical role in the decrease of pro-tumorigenic myeloid cells and increase in anti-tumor effector cells with a major contribution from classical CD4+ NKT cells within the TME. Interestingly, intratumoral combination therapy intensified DC1 migration from primary tumor to secondary tumor sites along with dynamic changes in subpopulations of DC with an associated increase in NKT cells at untreated tumor sites to display abscopal effect. Likewise, early data from phase I/II clinical trial illustrated great potential of cDC1 and IgG1 combination therapy to recruit anti-tumor effector cells such CD8 and NKT cells to mediate cancer eradication in HER2+ BC setting. Conclusions Specifically, intratumoral delivery of HER2-DC1 in combination with anti-tumor antibody mediating ADCC shows great promise for future BC clinical trials and improved patient outcomes. Furthermore, present study highlighted the role of NKT cells in favorable anti-tumor response. Our future studies are focused on understanding the crosstalk between DC and NKT to modulate the function of various cell types in TME to design improved cancer immunotherapies. Citation Format: Saurabh K Garg, Colin Snyder, Ganesan Ramamoorthi, Elizabeth E Evans, Amy L Aldrich, Namrata Gautam, Doris Wiener, Marie C Lee, Hyo S Han, Koski K Gary, Brian J Czerniecki. MHC class II and CD1d expressing conventional dendritic cells with immunoglobulin therapy drives anti-tumor abscopal effect by recruiting Natural Killer T cells to the tumor microenvironment [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 B025.

Dkk1 as a Prognostic Marker for Neoadjuvant Chemotherapy Response in Breast Cancer Patients.

To investigate the role of Dkk1 as a predictor of response to NACT in BC patients. This retrospective monocentric study included 145 women who had undergone NACT followed by breast surgery. Dkk1 protein expression was assessed using immunohistochemistry staining in core needle biopsies and mammary carcinoma specimens. Dkk1 levels were lower in treated BC tumours than in untreated tumours. The outcomes of 68 matched pre- and post-therapy tissues showed that Dkk1 levels in mammary carcinoma tissues were significantly predicted by levels in core needle biopsies and that Dkk1 expression was reduced in 83% of cases. Smaller cT stage, positive Her2 expression, and decreased Dkk1-IRS in core needle biopsy tissues were all independent predictors of regression grade (R4), according to Sinn. However, the percentage of Dkk1 expression differences prior to and following NACT had no effect on PFS or OS. In this study, we demonstrated for the first time that Dkk1 could be identified as an independent predictor of NACT response in BC patients, particularly those with TNBC. Further research with a multicentric expanded (pre-/post-therapy) sample set and better-defined populations in terms of molecular subtypes, therapy modality, and long-term follow-up is recommended to obtain more solid evidence.

Abstract A036: Quantitative molecular imaging of pancreatic tumor fibrosis for evaluation of response to neoadjuvant therapy: Pre-clinical and first-in-human application of collagen PET/MRI

Abstract Purpose: One of the major challenges in the management of pancreatic ductal adenocarcinoma (PDAC) is the lack of a reliable imaging tool to monitor tumor response to treatment. A high degree of fibrosis, mainly collagen type I, has been recognized as the hallmark of PDAC, which further increases in response to neoadjuvant chemoradiotherapy (CRT). This study introduces an image-guided paradigm for monitoring treatment response using the collagen type I specific PET imaging probe, 68Ga-CBP8 in mouse models and patients with PDAC. Methods: Subcutaneous mouse models of human PDAC were generated using FOLFIRNOX-sensitive (PANC1 and PDAC6) and FOLFIRINOX-resistant (SU8686) PDAC cells in nude mice (n=88). Mice were randomized into 2 groups of treatment with FOLFIRINOX or vehicle i.v. twice a week. Animals underwent PET/MRI with 68Ga-CBP8 (3.7-11.1 MBq) prior to, and at 7- and 15-days post-treatment (n=4-8/group). To assess the specificity of the probe, additional PANC1 tumor-bearing mice (n=4) were imaged with 68Ga-CBP8, and a collagen non-binding probe, 68Ga-CNBP (3.7-11 MBq). PET values were correlated with tumor growth over 21 days and quantification of fibrosis by Picrosirius red and IHC for collagen I, α-SMA, FAP-α, collagen rtPCR, and hydroxyproline assay. 8 patients (49-65 yrs.) with newly diagnosed PDAC underwent 68Ga-CBP8 PET (122-281 MBq) and standard MRI. Thus far, 3/8 subjects have undergone repeat PET/MRI after completion of neoadjuvant CRT. The pre-and post-CRT PET parameters for tumor and pancreas were compared and correlated with tumor size change, histology, and spatial analyses for PDAC and fibroblasts. Results: PET/MRI of PANC1 tumors showed significantly higher 68Ga-CBP8 uptake compared to control 68Ga-CNBP at each time point (p < 0.05), demonstrating the specificity of the probe in targeting collagen. PET values significantly increased over time in FOLFIRINOX-sensitive tumors (PANC1 %ID/cc: 0.8 ± 0.2 vs. 1.1 ± 0.3 vs. 1.9 ±0.2, p < 0.05 in day-0 vs. day-7 vs. day-15, respectively, p = 0.0005), whereas no significant change was seen in the probe uptake in the vehicle or resistant tumors (p > 0.05). There was a significant tumor growth reduction in responders compared to vehicle and resistant tumors. Histology showed a continuous increase of collagen in responding tumors (% collagen proportion area (CPA) in PANC1: 5.2 ± 3.8 vs. 16.2 ± 8.7 vs. 46.9 ± 6.0, p < 0.05, at Day-0 vs. Day-7 vs. Day-15, respectively, p < 0.0001), no change in resistant SU8686 (CPA: 33.0 ± 8.9 vs. 30.0 ± 9.7 vs. 28.1 ± 7.2, at Day-0 vs. Day-7 vs. Day-15, respectively, p = 0.66) and unchanged minimal collagen in pancreas. PET/MRI of patients showed a significantly higher SUVmean in the tumor compared to pancreas (2.4 ± 0.37 vs. 1.9 ± 0.31, p = 0.01), and a significantly higher uptake in the treated compared to untreated tumors (SUVmean: 2.8 ± 0.33 vs. 1.9 ± 0.56, p = 0.04). Response to treatment was confirmed on histology. Conclusion: Collagen PET could be used as a reliable non-invasive tool for monitoring response to neoadjuvant therapy in PDAC. Citation Format: Shadi A. Esfahani, Hua Ma, Shriya Krishna, Sergey Shuvaev, Arvin Hajmirzaian, Iris Zhou, Ciprian Catana, Mozhdeh Sojoodi, Eric Abston, David Ting, Pedram Heidari, Michael Lanuti, Kenneth Tanabe, Motaz Qadan, Carlos Fernandez de-Castillo, Aparna Parikh, Colin Weekes, Theodore Hong, Peter Caravan. Quantitative molecular imaging of pancreatic tumor fibrosis for evaluation of response to neoadjuvant therapy: Pre-clinical and first-in-human application of collagen PET/MRI [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A036.

Loss of the epithelial marker CDX1 predicts poor prognosis in early-stage CRC patients

BackgroundWe have previously shown that non-curative chemotherapy imposes fetal conversion and high metastatic capacity to cancer cells. From the set of genes differentially expressed in Chemotherapy Resistant Cells, we obtained a characteristic fetal intestinal cell signature that is present in a group of untreated tumors and is sufficient to predict patient prognosis. A feature of this fetal signature is the loss of CDX1. MethodsWe have analyzed transcriptomic data in public datasets and performed immunohistochemistry analysis of paraffin embedded tumor samples from two cohorts of colorectal cancer patients. ResultsWe demonstrated that low levels of CDX1 are sufficient to identify patients with poorest outcome at the early tumor stages II and III. Presence tumor areas that are negative for CDX1 staining in stage I cancers is associated with tumor relapse. ConclusionsOur results reveal the actual possibility of incorporating CDX1 immunostaining as a valuable biomarker for CRC patients.

TLR9 Monotherapy in Immune-Competent Mice Suppresses Orthotopic Prostate Tumor Development.

Prostate cancer is ranked second in the world for cancer-related deaths in men, highlighting the lack of effective therapies for advanced-stage disease. Toll-like receptors (TLRs) and immunity have a direct role in prostate cancer pathogenesis, but TLR9 has been reported to contribute to both the progression and inhibition of prostate tumorigenesis. To further understand this apparent disparity, we have investigated the effect of TLR9 stimulation on prostate cancer progression in an immune-competent, syngeneic orthotopic mouse model of prostate cancer. Here, we utilized the class B synthetic agonist CPG-1668 to provoke a TLR9-mediated systemic immune response and demonstrate a significant impairment of prostate tumorigenesis. Untreated tumors contained a high abundance of immune-cell infiltrates. However, pharmacological activation of TLR9 resulted in smaller tumors containing significantly fewer M1 macrophages and T cells. TLR9 stimulation of tumor cells in vitro had no effect on cell viability or its downstream transcriptional targets, whereas stimulation in macrophages suppressed cancer cell growth via type I IFN. This suggests that the antitumorigenic effects of CPG-1668 were predominantly mediated by an antitumor immune response. This study demonstrated that systemic TLR9 stimulation negatively regulates prostate cancer tumorigenesis and highlights TLR9 agonists as a useful therapeutic for the treatment of prostate cancer.

Collagen type I PET/MRI enables evaluation of treatment response in pancreatic cancer in pre-clinical and first-in-human translational studies.

Pancreatic ductal adenocarcinoma (PDAC) is an invasive and rapidly progressive malignancy. A major challenge in patient management is the lack of a reliable imaging tool to monitor tumor response to treatment. Tumor-associated fibrosis characterized by high type I collagen is a hallmark of PDAC, and fibrosis further increases in response to neoadjuvant chemoradiotherapy (CRT). We hypothesized that molecular positron emission tomography (PET) using a type I collagen-specific imaging probe, 68Ga-CBP8 can detect and measure changes in tumor fibrosis in response to standard treatment in mouse models and patients with PDAC. Methods: We evaluated the specificity of 68Ga-CBP8 PET to tumor collagen and its ability to differentiate responders from non-responders based on the dynamic changes of fibrosis in nude mouse models of human PDAC including FOLFIRNOX-sensitive (PANC-1 and PDAC6) and FOLFIRINOX-resistant (SU.86.86). Next, we demonstrated the specificity and sensitivity of 68Ga-CBP8 to the deposited collagen in resected human PDAC and pancreas tissues. Eight male participant (49-65 y) with newly diagnosed PDAC underwent dynamic 68Ga-CBP8 PET/MRI, and five underwent follow up 68Ga-CBP8 PET/MRI after completing standard CRT. PET parameters were correlated with tumor collagen content and markers of response on histology. Results: 68Ga-CBP8 showed specific binding to PDAC compared to non-binding 68Ga-CNBP probe in two mouse models of PDAC using PET imaging and to resected human PDAC using autoradiography (P < 0.05 for all comparisons). 68Ga-CBP8 PET showed 2-fold higher tumor signal in mouse models following FOLFIRINOX treatment in PANC-1 and PDAC6 models (P < 0.01), but no significant increase after treatment in FOLFIRINOX resistant SU.86.86 model. 68Ga-CBP8 binding to resected human PDAC was significantly higher (P < 0.0001) in treated versus untreated tissue. PET/MRI of PDAC patients prior to CRT showed significantly higher 68Ga-CBP8 uptake in tumor compared to pancreas (SUVmean: 2.35 ± 0.36 vs. 1.99 ± 0.25, P = 0.036, n = 8). PET tumor values significantly increased following CRT compared to untreated tumors (SUVmean: 2.83 ± 0.30 vs. 2.25 ± 0.41, P = 0.01, n = 5). Collagen deposition significantly increased in response to CRT (59 ± 9% vs. 30 ± 9%, P=0.0005 in treated vs. untreated tumors). Tumor and pancreas collagen content showed a positive direct correlation with SUVmean (R2 = 0.54, P = 0.0007). Conclusions: This study demonstrates the specificity of 68Ga-CBP8 PET to tumor type I collagen and its ability to differentiate responders from non-responders based on the dynamic changes of fibrosis in PDAC. The results highlight the potential use of collagen PET as a non-invasive tool for monitoring response to treatment in patients with PDAC.

Analysis of translesion polymerases in colorectal cancer cells following cetuximab treatment: A network perspective.

Adaptive mutagenesis observed in colorectal cancer (CRC) cells upon exposure to EGFR inhibitors contributes to the development of resistance and recurrence. Multiple investigations have indicated a parallel between cancer cells and bacteria in terms of exhibiting adaptive mutagenesis. This phenomenon entails a transient and coordinated escalation of error-prone translesion synthesis polymerases (TLS polymerases), resulting in mutagenesis of a magnitude sufficient to drive the selection of resistant phenotypes. In this study, we conducted a comprehensive pan-transcriptome analysis of the regulatory framework within CRC cells, with the objective of identifying potential transcriptome modules encompassing certain translesion polymerases and the associated transcription factors (TFs) that govern them. Our sampling strategy involved the collection of transcriptomic data from tumors treated with cetuximab, an EGFR inhibitor, untreated CRC tumors, and colorectal-derived cell lines, resulting in a diverse dataset. Subsequently, we identified co-regulated modules using weighted correlation network analysis with a minKMEtostay threshold set at 0.5 to minimize false-positive module identifications and mapped the modules to STRING annotations. Furthermore, we explored the putative TFs influencing these modules using KBoost, a kernel PCA regression model. Our analysis did not reveal a distinct transcriptional profile specific to cetuximab treatment. Moreover, we elucidated co-expression modules housing genes, for example, POLK, POLI, POLQ, REV1, POLN, and POLM. Specifically, POLK, POLI, and POLQ were assigned to the "blue" module, which also encompassed critical DNA damage response enzymes, for example. BRCA1, BRCA2, MSH6, and MSH2. To delineate the transcriptional control of this module, we investigated associated TFs, highlighting the roles of prominent cancer-associated TFs, such as CENPA, HNF1A, and E2F7. We found that translesion polymerases are co-regulated with DNA mismatch repair and cell cycle-associated factors. We did not, however, identified any networks specific to cetuximab treatment indicating that the response to EGFR inhibitors relates to a general stress response mechanism.

Genetic and transcriptomic analyses of diffuse large B-cell lymphoma patients with poor outcomes within two years of diagnosis

Despite the improvements in clinical outcomes for DLBCL, a significant proportion of patients still face challenges with refractory/relapsed (R/R) disease after receiving first-line R-CHOP treatment. To further elucidate the underlying mechanism of R/R disease and to develop methods for identifying patients at risk of early disease progression, we integrated clinical, genetic and transcriptomic data derived from 2805 R-CHOP-treated patients from seven independent cohorts. Among these, 887 patients exhibited R/R disease within two years (poor outcome), and 1918 patients remained in remission at two years (good outcome). Our analysis identified four preferentially mutated genes (TP53, MYD88, SPEN, MYC) in the untreated (diagnostic) tumor samples from patients with poor outcomes. Furthermore, transcriptomic analysis revealed a distinct gene expression pattern linked to poor outcomes, affecting pathways involved in cell adhesion/migration, T-cell activation/regulation, PI3K, and NF-κB signaling. Moreover, we developed and validated a 24-gene expression score as an independent prognostic predictor for treatment outcomes. This score also demonstrated efficacy in further stratifying high-risk patients when integrated with existing genetic or cell-of-origin subtypes, including the unclassified cases in these models. Finally, based on these findings, we developed an online analysis tool (https://lymphprog.serve.scilifelab.se/app/lymphprog) that can be used for prognostic prediction for DLBCL patients.

Transferrin receptor in primary and metastatic breast cancer: Evaluation of expression and experimental modulation to improve molecular targeting.

Conjugation of transferrin (Tf) to imaging or nanotherapeutic agents is a promising strategy to target breast cancer. Since the efficacy of these biomaterials often depends on the overexpression of the targeted receptor, we set out to survey expression of transferrin receptor (TfR) in primary and metastatic breast cancer samples, including metastases and relapse, and investigate its modulation in experimental models. Gene expression was investigated by datamining in twelve publicly-available datasets. Dedicated Tissue microarrays (TMAs) were generated to evaluate matched primary and bone metastases as well as and pre and post chemotherapy tumors from the same patient. TMA were stained with the FDA-approved MRQ-48 antibody against TfR and graded by staining intensity (H-score). Patient-derived xenografts (PDX) and isogenic metastatic mouse models were used to study in vivo TfR expression and uptake of transferrin. TFRC gene and protein expression were high in breast cancer of all subtypes and stages, and in 60-85% of bone metastases. TfR was detectable after neoadjuvant chemotherapy, albeit with some variability. Fluorophore-conjugated transferrin iron chelator deferoxamine (DFO) enhanced TfR uptake in human breast cancer cells in vitro and proved transferrin localization at metastatic sites and correlation of tumor burden relative to untreated tumor mice. TfR is expressed in breast cancer, primary, metastatic, and after neoadjuvant chemotherapy. Variability in expression of TfR suggests that evaluation of the expression of TfR in individual patients could identify the best candidates for targeting. Further, systemic iron chelation with DFO may upregulate receptor expression and improve uptake of therapeutics or tracers that use transferrin as a homing ligand.

Deep learning approach for brain tumor classification using metaheuristic optimization with gene expression data

AbstractThis study addresses the critical challenge of accurately classifying brain tumors using artificial intelligence. Early detection is crucial, as untreated tumors can be fatal. Despite advances in AI, accurately classifying tumors remains a challenging task. To address this challenge, we propose a novel optimization approach called PSCS combined with deep learning for brain tumor classification. PSCS optimizes the classification process by improving Particle Swarm Optimization (PSO) exploitation using Cuckoo search (CS) algorithm. Next, classified gene expression data of brain tumor using Deep Learning (DL) to identify different groups or classes related to a particular tumor along with the PSCS optimization technique. The proposed optimization technique with DL achieves much better classification accuracy than other existing DL and Machine learning models with the different evaluation matrices such as Recall, Precision, F1‐Score, and confusion matrix. This research contributes to AI‐driven brain tumor diagnosis and classification, offering a promising solution for improved patient outcomes.