Digital Spatial Profiling Research Articles

EPCO-48. RESOLVING INTRATUMORAL TRANSCRIPTIONAL HETEROGENEITY ACROSS THE CORE-EDGE SPATIAL AXIS IN GLIOBLASTOMA

Abstract Intratumoral heterogeneity promotes tumor progression, treatment resistance, and recurrence in glioblastoma (GBM), and the cellular tumor core and infiltrating edge represent transcriptionally distinct cellular ecosystems. In this study, we characterize and validate the molecular differences across the core-edge spatial axis in GBM by applying whole-transcriptome GeoMx Digital Spatial Profiling (DSP) to 5 IDH-wildtype tumors. Three 3-micron tissue cores were extracted from each tumor and arranged into a tissue microarray (TMA). Core and edge regions of interest (ROIs) were chosen through coregistration of H&E and immunofluorescent TMA scans. Following DSP, data for 44 ROIs (30 core and 14 edge) and 8530 RNA targets were bioinformatically analyzed. Single-sample gene set enrichment analysis aligned the DSP core and edge regional signatures to those of publicly annotated datasets, substantiating our profiling methodology. Unsupervised analyses revealed ROI clusters unique to each tumor specimen for both pre- and post-normalized data. To mitigate a potential patient-level effect in subsequent differential gene expression and pathway analyses, we leveraged a linear mixed-effect model using core versus edge as a fixed effect and tumor specimen as a random effect. Our analyses showed upregulation of epigenomic markers in core ROIs and neuronal markers in edge ROIs. Further, core and edge signatures were incongruent with the canonical GBM subtypes (Mesenchymal, Classical, Proneural) and malignant cell states (OPC-like, NPC-like, AC-like, MES-like). Cellular deconvolution analysis highlighted differential enrichment of neoplastic cells in core ROIs and neuroglia (astrocytes and oligodendrocytes) in edge ROIs. In summary, we leverage spatial profiling to further validate the geographic molecular heterogeneity of GBM, which is characterized by divergent neuronal programs within the infiltrating edge.

Digital spatial profiling for pathologists.

The advent of "omics" technologies for high-depth tumor profiling has provided new information regarding cancer heterogeneity. However, a bulk omics profile can only partially reproduce tumor complexity, and it does not meet the preferences of pathologists used to perform an in situ assessment of marker expression, for instance, with immunohistochemistry. The NanoString GeoMx® Digital Spatial Profiler (DSP) is a platform for morphology-guided multiplex profiling of tissue slides, which allows the digital quantification of target analytes in different neoplastic settings. To illustrate the feasibility and opportunities offered by DSP from a pathologist's perspective, we applied DSP in three different representative neoplastic settings: breast carcinoma, thyroid anaplastic carcinoma, and biphasic mesothelioma. Because of the perfect overlap between the hematoxylin-eosin-stained slide and the GeoMx areas of interest, in breast carcinoma, two different antibodies allowed the distinction of the tumor cells from the surrounding tumor microenvironment. In biphasic mesothelioma, we could distinguish the epithelioid from the sarcomatoid neoplastic component, and in the thyroid, we easily separated the anaplastic areas from the well-differentiated carcinoma. DSP is a promising tool that combines traditional histological evaluation, allowing spatial assessment of a tumor and its surroundings, and innovative in situ digital profiling. Pathologists should not miss the opportunity to combine morphological and genomic analyses and be at the forefront of investigating the progression of dysplasia/neoplasia, low-grade or high-grade, epithelial/mesenchymal, and, more in general, overcoming the concept of in situ vs. bulk genomic methods.

Spatially resolved gene expression profiles of fibrosing interstitial lung diseases

Fibrosing interstitial lung diseases (ILDs) encompass a diverse range of scarring disorders that lead to progressive lung failure. Previous gene expression profiling studies focused on idiopathic pulmonary fibrosis (IPF) and bulk tissue samples. We employed digital spatial profiling to gain new insights into the spatial resolution of gene expression across distinct lung microenvironments (LMEs) in IPF, chronic hypersensitivity pneumonitis (CHP) and non-specific interstitial pneumonia (NSIP). We identified differentially expressed genes between LMEs within each condition, and across histologically similar regions between conditions. Uninvolved regions in IPF and CHP were distinct from normal controls, and displayed potential therapeutic targets. Hallmark LMEs of each condition retained distinct gene signatures, but these could not be reproduced in matched lung tissue samples. Based on these profiles and unsupervised clustering, we grouped previously unclassified ILD cases into NSIP or CHP. Overall, our work uniquely dissects gene expression profiles between LMEs within and across different types of fibrosing ILDs.

Induced collagen type-I secretion by hepatocytes of the melanoma liver metastasis is associated with a reduction in tumour-infiltrating lymphocytes.

Overall patients with melanoma liver metastasis (MLiM) have a dismal prognosis and poor responses to the standard of care treatment. Understanding the role of the tumour microenvironment (TME) is critical for discovering better strategies to overcome intrinsic therapy resistance in MLiM. The aim was to understand the crosstalk signalling pathways between hepatocytes and metastatic melanoma cells in the TME of MLiM. Hepatocytes and melanoma tumour cells of MLiM were assessed using transcriptomic NanoString GeoMx digital spatial profiling (NGDSP) assay. Functional assays were performed using normal hepatocytes and MLiM-derived cell lines. Validation was performed using multiplex immunofluorescence. In NGDSP analysis adjacent normal hepatocytes (ANH) had higher CXCR4 and COL1A1/2 levels than distant normal hepatocytes (DNH), while melanoma cells had higher TNF-α levels. In vitro, MLiM cell lines released TNF-α which upregulated CXCR4 and CXCL12 levels in ANH. CXCL12 activated CXCR4, which triggered AKT and NFκB signalling pathways. Consequently, AKT signalling induced the upregulation of collagen type I. MLiM were significantly encircled by a shield of collagen, whereas other liver metastases showed reduced levels of collagen. Of all the liver metastasis analyzed, the presence of collagen in melanoma liver metastasis was associated with a reduction in tumour-infiltrating lymphocytes. MLiM modified ANH to increase collagen production and created a physical barrier. The collagen barrier was associated with a reduction of immune cell infiltration which could potentially deter MLiM immune surveillance and treatment responses. Spatial analyses of melanoma liver metastasis show that adjacent normal hepatocytes have increased collagen-type I levels. Melanoma liver metastases tumour cells secrete enhanced levels of TNF-α to stimulate CXCR4/CXCL12 upregulation in adjacent normal hepatocytes. Activation of CXCR4 promotes AKT and NF-κB signalling pathways to promote collagen-type I secretion in adjacent normal hepatocytes. Elevated collagen levels were associated with reduced tumour-infiltrating lymphocytes.

Spatial Transcriptomic Signatures of Early Acute T Cell-mediated Rejection in Kidney Transplants.

Kidney transplantation significantly improves the quality of life for those with end-stage renal failure, yet allograft rejection resulting from immune cell interactions remains a persistent challenge. Although T cell-directed immunosuppressive drugs effectively contain graft rejection in most patients, a notable proportion still experiences acute T cell-mediated rejection (TCMR). Despite an emphasis on suppressing T cell-mediated immune responses, successful control over TCMR is not always achieved, suggesting the potential involvement of factors beyond T cells. Biopsy samples from suspicious (borderline) for acute TCMR (borderline TCMR) and non-TCMR patients were obtained 9 d postsurgery, and spatial transcriptomics profiling was conducted using the GeoMx Digital Spatial Profiler platform. Regions of interest in the glomerulus and interstitium were selected on the basis of immunohistochemistry staining anti-CD3 to identify areas with T-lymphocyte infiltration. Differential gene expression analysis was performed using unpaired t tests. Unbiased clustering of transcriptional profiles across all regions of interest showed distinct transcriptional profiles between glomeruli and interstitium in non-TCMR samples, whereas borderline TCMR samples displayed no distinct transcriptional profiles between these regions. Contrary to the prevailing T cell-centric view, we observed pathways and genes associated with innate immunity-related inflammatory conditions expressed in glomerular regions of borderline TCMR biopsies. Immunofluorescence staining for CD68 confirmed the presence of macrophages in the glomeruli of the post-TCMR sample in a validation cohort, indicating macrophage involvement in the glomerular response after TCMR. Activation of the innate immune response in borderline TCMR appears to impact not only the interstitium but also the glomerulus. Glomerulus-specific immune signatures suggest the role of the innate immune system in rejection. This nuanced understanding proposes the necessity for tailored therapeutic interventions targeting both innate and adaptive immune pathways to enhance transplant outcomes.

Amyloid-β (Aβ) immunotherapy induced microhemorrhages are linked to vascular inflammation and cerebrovascular damage in a mouse model of Alzheimer’s disease

BackgroundAnti-amyloid-β (Aβ) immunotherapy trials have revealed amyloid-related imaging abnormalities (ARIA) as the most prevalent and serious adverse events linked to pathological changes in cerebral vasculature. Recent studies underscore the critical involvement of perivascular macrophages and the infiltration of peripheral immune cells in regulating cerebrovascular damage. Specifically, Aβ antibodies engaged at cerebral amyloid angiopathy (CAA) deposits trigger perivascular macrophage activation and the upregulation of genes associated with vascular permeability. Nevertheless, further research is needed to understand the immediate downstream consequences of macrophage activation, potentially exacerbating CAA-related vascular permeability and microhemorrhages linked to Aβ immunotherapy.MethodsThis study investigates immune responses induced by amyloid-targeting antibodies and CAA-induced microhemorrhages using RNA in situ hybridization, histology and digital spatial profiling in an Alzheimer's disease (AD) mouse model of microhemorrhage.ResultsIn the present study, we have demonstrated that bapineuzumab murine surrogate (3D6) induces profound vascular damage, leading to smooth muscle cell loss and blood–brain barrier (BBB) breakdown. In addition, digital spatial profiling (DSP) reveals that distinct immune responses contribute to vascular damage with peripheral immune responses and perivascular macrophage activation linked to smooth muscle cell loss and vascular fibrosis, respectively. Finally, RNA in situ hybridization identifies two distinct subsets of Trem2+ macrophages representing tissue-resident and monocyte-derived macrophages around vascular amyloid deposits. Overall, these findings highlight multifaceted roles of immune activation and vascular damage in driving the development of microhemorrhage.ConclusionsIn summary, our study has established a significant link between CAA-Aβ antibody immune complex formation, immune activation and vascular damage leading to smooth muscle cell loss. However, the full implications of this cascade on the development of microhemorrhages requires further exploration. Additional investigations are warranted to unravel the precise molecular mechanisms leading to microhemorrhage, the interplay of diverse immune populations and the functional roles played by various Trem2+ macrophage populations in response to Aβ immunotherapy.

Rigor and Reproducibility of Digital Spatial Profiling on Clinically Sourced Human Tissues.

Spatial transcriptomic profiling enables precise quantification of gene expression with simultaneous localization of expression profiles onto tissue structures. This new technology promises to improve our understanding of the disease mechanisms. Therefore, there is intense interest in applying these methods in clinical trials or as laboratory developed tests to aid in diagnosis of disease. Before these technologies can be more broadly deployed in clinical research and diagnostics, it is necessary to thoroughly understand their performance in real world conditions. In this study, we vet technical reproducibility, data normalization methods and assay sensitivity focusing predominantly on one widely used spatial transcriptomic methodology, digital spatial profiling. Using clinically sourced human tissue specimens, we find that digital spatial profiling exhibits high rigor and reproducibility. Our approach lays the foundation for incorporation of digital spatial profiling methods into clinical workflows.

Immunoproteomics Reveal Different Characteristics for the Prognostic Markers of Intratumoral-Infiltrating CD3+ T Lymphocytes and Immunoscore in Colorectal Cancer

Tumor-infiltrating lymphocytes (TILs) and immunoscoring based on densities of CD3+ and CD8+ TILs are both favorable prognostic markers in colorectal cancer (CRC). However, determination of the molecular features of TILs, particularly their immunoproteomic signatures would require the development of large scale in situ spatiotemporal technologies. Recently, a multiplex in situ digital spatial proteomic profiling (DSP) tool GeoMx DSP has been applied to identify biomarkers predictive of therapeutic responses and to understand disease mechanisms and progression. Taking advantage of this tool, we simultaneously characterized the spatial distribution and interactions of 42 immune proteins in tumor cells (TCs), CD3+ T stromal TILs (sTILs), and CD20+ B sTILs using tissue microarrays, and further studied their associations with CD3+ T TILs and immunoscores in CRC. First, our data showed that well-known immune checkpoints, such as PD-L1, PD-L2, and LAG3, were expressed at low levels, whereas some other immune proteins, such as CD11c, CD68, STING, and CD44, were highly expressed. Second, 8 spatial interactions were identified, including 5 interactions between TC and CD20+ B sTILs, 2 interactions between CD3+ T sTILs and CD20+ B sTILs, and 1 interaction among TC, CD3+ T sTILs, and CD20+ B sTILs. Third, the differential immune microlandscape in the spatial compartments was identified in tissues with positive CD3+ T intratumoral TILs and high immunoscores. Collectively, to our knowledge, our study is the first to provide in situ spatial immune characteristics at the proteomic level. Moreover, our findings provide direct evidence supporting the infiltration of CD3+ T sTILs from stoma to TC and shed important insights into better understanding and treating CRC patients related to different immune prognostic markers.

RUNX2 as a novel biomarker for early identification of patients progressing to advanced-stage mycosis fungoides.

The majority of patients with mycosis fungoides (MF) have an indolent disease course, but a substantial fraction (20-30%) of patients progress to advanced stages - usually with a grave prognosis. Early differentiation between indolent and aggressive types of MF is important for the choice of treatment regimen and monitoring of the individual patient. Good biomarkers are therefore desired. Here, we used spatial transcriptomics on skin samples at time-of-diagnosis to enable prediction of patients who later progressed to advanced stages of MF. Formalin-fixed, paraffin-embedded skin biopsies at time of diagnosis from six patients with MF who progressed to advanced stages of disease within 4 months to 12 years after diagnosis, and nine patients who remained in early-stage disease over 9 to 27 years were analyzed using the GeoMx Digital Spatial Profiler to capture spatially resolved high-plex RNA gene expression data. Five different regions of interest (the epidermis, the basal layer of epidermis, CD4+ T-cells and neighboring cells, and Pautrier's microabscesses) were profiled for further assessment. Interestingly, RUNX2, SHMT2, and MCM7 were upregulated in the enriched population of malignant T-cells in Pautrier's microabscesses in patients who later developed advanced stages of disease. Expression of RUNX2, SHMT2 and MCM7 in malignant T-cells was confirmed in a subset of patients in MF skin using scRNA-seq datasets across multiple studies and correlating with stage of disease. Taken together, we provide first evidence that RUNX2 has potential as a biomarker to identify MF patients progressing to advanced stage disease. As RUNX2 has not previously been linked to MF, our data also shows the analytical strength of combining spatial transcriptomics with scRNA-seq analysis.

Multi-omics analysis and response prediction of PD-1 monoclonal antibody containing regimens in patients with relapsed/refractory diffuse large B-cell lymphoma

Patients with relapsed/refractory (r/r) diffuse large B-cell lymphoma (DLBCL) show varied responses to PD-1 monoclonal antibody (mAb) containing regimens. The mechanisms and predictive biomarkers for the efficacy of this regimen are unclear. This study retrospectively collected r/r DLBCL patients who received PD-1 mAb and rituximab regimens as salvage therapy. Clinical and genomic features were collected, and mechanisms were explored by multiplex immunofluorescence and digital spatial profiling. An artificial neural network (ANN) model was constructed to predict the response. Between October 16th, 2018 and May 4th, 2023, 50 r/r DLBCL patients were collected, 29 were response patients and 21 were non-response patients. CREBBP (p = 0.029) and TP53 (p = 0.015) alterations were statistically higher in non-response patients. Patients with PD-L1 CPS ≥ 5 were correlated with a longer overall survival (OS) than those with PD-L1 CPS < 5 (median OS: not reached vs. 9.7 months, hazard ratio [HR]: 3.8, 95% confidence interval [CI] 0.64–22.44, p = 0.016). Immune-related pathways were activated in response patients. The proportion and spatial organization of tumor-infiltrating immune cells affect the response. PD-L1 CPS level, age, and alterations of TP53, MYD88, CREBBP, EP300, GNA13 were used to build an ANN predictive model that showed high prediction efficiency (training set area under curve [AUC] of 0.97 and test set AUC of 0.94). The proportion and spatial distribution of tumor-infiltrating immune cells may be related to the function of immune-related pathways, thereby influencing the efficacy of PD-1 mAb containing regimens. The ANN predictive model showed potential value in predicting the responses of r/r DLBCL patients received PD-1 mAb and rituximab regimens.

Spatial profiling of METex14-altered NSCLC under tepotinib treatment: Shifting the immunosuppressive landscape

MET inhibitors have demonstrated efficacy in treating patients with non-small cell lung cancer (NSCLC) harboring METex14 skipping alterations. Advancements in spatial profiling technologies have unveiled the complex dynamics of the tumor microenvironment (TME), a crucial factor in cancer progression and therapeutic response.This study uses spatial profiling to investigate the effects of the MET inhibitor tepotinib on the TME in a case of locally advanced NSCLC with a METex14 skipping alteration. A patient with resectable stage IIIB NSCLC, unresponsive to neoadjuvant platinum-based chemotherapy, received tepotinib following the detection of a METex14 skipping alteration. Paired pre- and post-treatment biopsies were subjected to GeoMx Digital Spatial Profiling using the Cancer Transcriptome Atlas and immune-related protein panels to evaluate shifts in the immune TME.Tepotinib administration allowed for a successful lobectomy and a pathological downstaging to stage IA1. The TME was transformed from an immunosuppressive to a more permissive state, with upregulation of antigen-presenting and pro-inflammatory immune cells. Moreover, a marked decrease in immune checkpoint molecules, including PD-L1, was noted. Spatial profiling identified discrete immune-enriched clusters, indicating the role of tepotinib in modulating immune cell trafficking and function. Tepotinib appears to remodel the immune TME in a patient with METex14 skipping NSCLC, possibly increasing responsiveness to immunotherapy.Our study supports the integration of genetic profiling into the management of early and locally advanced NSCLC to guide personalized, targeted interventions. These findings underscore the need to further evaluate combinations of MET inhibitors and immunotherapies.

Spatial resolution of the head and neck cancer tumor microenvironment to identify tumor and stromal features associated with therapy response.

Head and neck cancer (HNC) is the seventh most common cancer globally, resulting in 440 000 deaths per year. While there have been advancements in chemoradiotherapy and surgery, relapse occurs in more than half of HNCs, and these patients have a median survival of 10 months and a 2-year survival of < 20%. Only a subset of patients displays durable benefits from immunotherapies in metastatic and recurrent HNC, making it critical to understand the tumor microenvironment (TME) underpinning therapy responses in HNC. To recognize biological differences within the TME that may be predictive of immunotherapy response, we applied cutting-edge geospatial whole-transcriptome profiling (NanoString GeoMx Digital Spatial Profiler) and spatial proteomics profiling (Akoya PhenoCycler-Fusion) on a tumor microarray consisting of 25 cores from 12 patients that included 4 immunotherapy-unresponsive (8 cores) and 2 immunotherapy-responsive patients (5 cores), as well as 6 immunotherapy naïve patients (12 cores). Through high-plex, regional-based transcriptomic mapping of the tumor and TME, pathways involved with the complement system and hypoxia were identified to be differentially expressed in patients who went on to experience a poor immunotherapy response. Single-cell, targeted proteomic analysis found that immune cell infiltration of the cancer cell mass and interactions of CD8 Tcells with tumor and other immune cells were associated with positive immunotherapy response. The relative abundance of specific tumor phenotypes and their interactions with various immune cells was identified to be different between response groups. This study demonstrates how spatial transcriptomics and proteomics can resolve novel alterations in the TME of HNC that may contribute to therapy sensitivity and resistance.

Spatial Profiling of Ovarian Clear Cell Carcinoma Reveals Immune-Hot Features

Ovarian clear cell carcinoma (OCCC) has a high incidence in Asia, with a frequent occurrence at an early stage, but without sufficient data on molecular stratification for high-risk patients. Recently, immune-hot features have been proposed as indicators of poor prognosis in early stage OCCC. Specific patterns of intratumoral heterogeneity associated with immune-hot features must be defined. NanoString Digital Spatial Profiling technology was used to decipher the spatial distribution of the 18-plex protein panel. Regions of interest (ROIs) were collected based on the reference hematoxylin and eosin–stained morphology. Areas of illumination (AOIs) were defined according to the ROI segmentation using the fluorescence signals of the visualization markers pan-cytokeratin (PanCK), CD45, or DNA. Unsupervised hierarchical clustering of 595 AOIs from 407 ROIs showed that the PanCK segments expressed different combinations of immune markers, suggestive of immune mimicry. The following 3 immune-hot clusters were identified: granzyme B–high, immune signal-high , and immune-like cells; the following 2 immune-cold clusters were identified: fibronectin-high and immune checkpoint–high cells. In tumor samples at the International Federation of Gynecology and Obstetrics stage IC1/2 experiencing recurrence, there was an increased occurrence of PanCK+ AOIs with immune signal-high and immune-like cell groups in the papillary morphology surrounded by macrophage lineage tumor-infiltrating immune cells (TIIs). In contrast, for tumor samples at the International Federation of Gynecology and Obstetrics stage IC3/II with recurrence, PanCK+ AOIs were prevalent in the fibronectin-high group, particularly in those with a tubulocystic morphology surrounded by lymphoid lineage non-TIIs. Our study on the spatial profiling of early stage OCCC tumors revealed that the immune mimicry of tumor cells, presence of TIIs, and morphologic patterns were associated with recurrence, which switched during tumor progression.

Identifying High Recurrence Risk in Breast Carcinoma Patients Through Spatial Transcriptomic Analysis.

Comparing gene expression profiles according to recurrence risk using spatially resolved transcriptomic analysis has not been reported. This study aimed to identify distinct genetic features of breast carcinoma associated with a high Oncotype DX Recurrence Score (ORS). Patients were categorized into two groups, ORS-high (ORS-H; two patients) and ORS-non-high (ORS-NH; five patients). We performed digital spatial profiling and bioinformatic analyses to investigate the spatial transcriptomic profiles. Lysozyme (LYZ), complement C1q C chain (C1QC), and complement C1q B chain (C1QB) exhibited the highest fold changes in the stromal compartment of the ORS-H group. Gene ontology enrichment analysis of the ORS-H group revealed significant up-regulation of genes associated with immune response in the stromal compartment, including lymphocyte-mediated immunity, adaptive immune response related to the immunoglobulin superfamily, and leukocyte-mediated immunity. Gene set enrichment analysis showed significant positive enrichment of gene sets associated with interferon (IFN) response and complement pathways in the stromal compartment. This study highlights significant differences in gene expression profiles and spatially resolved transcriptional activities between ORS-H and ORS-NH breast carcinomas. The significant up-regulation of genes and pathways associated with cell-mediated immunity, IFN response, and complement C1q in the stromal compartment of the ORS-H group warrants further evaluation with larger population cohorts.

Abstract PR004: Differential gene expression analysis comparing Black and White patients with HPV-positive oropharyngeal squamous cell carcinoma reveals suppressed immune response and higher rates P16/HPV DNA discordance in Black patients

Abstract Background-Human papilloma virus (HPV-positive (+)) oropharynx cancer (OPC) are a distinct neoplastic entity associated with a significant survival advantage compared to HPV- OPCs. The survival advantage is so great that the presence of regional cervical lymph node metastasis is no longer considered an advanced stage III/IV cancer. The survival advantage associated with HPV+ OPC is not the same for Black patients compared to all other races and emerging data now suggests Black patients with HPV+ OPC have survival outcomes similar to White patients with HPV- OPC. We conducted a differential gene expression analysis and high-risk HPV serotyping comparing genomic difference between treatment naïve Black and White patients with HPV+ OPC. Methods: Surgical samples were obtained from Duke, the University of Texas Medical Branch, and the University of Mississippi, through an IRB approved protocol. HPV status determined by p16 immunohistochemistry (IHC). We performed digital spatial profiling and conducted a differential gene expression analysis in 14 Black and 13 White patients using the Nanostring™ Whole Transcriptome Atlas. Morphology markers included CD45, PANCK, and alpha SMA to label lymphocytes, epithelium, and stroma, respectively. A component of the bioinformatics pipeline included conducing a KEGG pathway analysis using pre-ranked test statistics from self-reported Black and White patients HPV+ OPC. The presence of high-risk oncogenic HPV was determined in parallel by RT-PCR (Creative Biogene). P16, CD68 and CD3 expression was measured by IHC using standard techniques. Results: There was a significant decrease in antigen processing and presentation and T-cell receptor signaling pathways in CD45+ lymphocytes (p&amp;lt;0.0002) and B-Cell receptor and chemokine signaling pathways in PANCK+ tumor epithelial cells (p&amp;lt;0.03) in Black patients compared to White patients with HPV+ OPC. There was a significant difference in p16 expression and the absence of HPV DNA in Black patients compared to White patients (p&amp;lt;0.017). 32% of Black patients with p16+ OPC were HPV DNA negative. There was no p16/HPV16 DNA discordance observed in the White patient cohort. Three patients were co-infected with other carcinogenic HPV serotypes (45, 51, and 59) in addition to HPV16 (two Black and one White). CD68 expression, associated with M1-like anti-tumor macrophages, was significantly decreased in Black patients with p16+/HPV16+ OPC, compared to White patients (p&amp;lt;0.017), suggesting alterations in tumor immune response may be important in cancer disparities. Conclusion: The favorable prognosis associated with HPV+ OPC, including recruitment in currently ongoing radiation de-escalation clinical trials cannot be widely applied to all patients with HPV+ OPC and studies addressing racial disparities, HPV/p16 discordance, and predictors of poor response represent a significant health equity gap in the field of OPC disparities. Citation Format: Tammara L. Watts, Katerine Gonzalez, Xiangfeng Shen, Layne Rogers, Nosa Osazuwa-Peters, Gina D. Jefferson, Cecilia G. Clement, Danielle Range, Russel R Kahmke, Walter T Lee, Daniel J. Rocke, Trinitia Y Cannon, Yvonne Mowery, Kouros Owzar. Differential gene expression analysis comparing Black and White patients with HPV-positive oropharyngeal squamous cell carcinoma reveals suppressed immune response and higher rates P16/HPV DNA discordance in Black patients [abstract]. In: Proceedings of the 17th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2024 Sep 21-24; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2024;33(9 Suppl):Abstract nr PR004.

Abstract C029: Window-of-opportunity trial of metastatic pancreatic cancer reveals potential mechanisms of response to targeting RAS/MEK signaling

Abstract KRAS is mutated in ∼85% of pancreatic ductal adenocarcinoma (PDAC) patient's tumors, suggesting that it is a key node of PDAC targeted therapy. RAF/MEK/ERK signaling is downstream of KRAS signaling. Preclinical studies targeting MEK showed promises but clinical trials failed to show the benefit compared to Standard-of-Care treatment chemotherapy. To elucidate the mechanism of action and resistance to several targeted therapies including MEK inhibition in human PDAC, we conducted a Window-Of-Opportunity trial for Metastatic pancreatic cancer (WOOM trial). In this trial, metastatic PDAC patients received 10 days of Cobimetinib, an FDA approved MEK inhibitor, therapy and specimens of pre- and post- treatment were obtained by biopsy to evaluate the response to targeted treatment using deep multi-omic analytics including DNA-seq, RNA-seq, multiplex IHC, cyclic IF and Digital Spatial Profiling (DSP). Four of 13 patient’s tumors were defined as Responsive tumors by using CA19- 9 and KI67 as a readout. Genomic sequence showed that KRAS amplifications were frequently seen in non-responsive tumors and KrasG12R mutations were seen with higher frequency in Responsive tumors. RNA-seq analysis showed that these Responsive tumors had lower KRAS activity and were more likely to be classical-like subtype compared to non-responsive tumors. DSP analysis confirmed the downregulation of ERK phosphorylation with paradoxical phospho- MEK activation after treatment in all examined cases which is consistent with effective target inhibition. Phospho-MYC protein expression and MYC transcriptomic activity were significantly decreased in responsive tumors, which suggest MYC downregulation could contribute to an active response to MEK inhibition. Single cell RNA-seq and spacial analysis showed tumor microenvironment changes after Cobimetinib treatment including the increase of CD8 T cell infiltration in responsive tumors. Furthermore, PDX from patient’s biopsy recapitulated patient's response to Cobimetinib and faithfully metastasized to the liver. Interestingly, Cobimetinib resistant PDX was also resistant to KRAS inhibitor. This model could be a great tool for testing new therapeutic strategies targeting the KRAS/MEK pathway. Here, we summarize the multi- omics data and discuss potential mechanisms of response and resistance to targeting KRAS signaling and related microenvironmental changes. Citation Format: Motoyuki Tsuda, Colin Daniel, Xiaoyan Wang, Carl Peltz, Hayley Zimmny, Alexander Smith, John Muschler, Xi Li, Tugba Yildiran Ozmen, Furkan Ozmen, Dove Keith, Christopher Corless, Koei Chin, Jonathan Brody, Charles Lopez, Gordon Mills, Rosalie Sears. Window-of-opportunity trial of metastatic pancreatic cancer reveals potential mechanisms of response to targeting RAS/MEK signaling [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C029.

Abstract A030: Modulation of lymphocyte adhesion and migration by iCAF and myCAF fibroblasts in PDAC

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) poses significant challenges in treatment due to its complex interaction with the tumour microenvironment (TME). Fibroblasts, a crucial component of the TME, influence tumour progression and therapeutic responses. This study explores the diversity of cancer-associated fibroblast (CAF) populations in PDAC tumours versus adjacent normal fibroblasts (ANFs), focussing on myofibroblasts (myCAFs) and inflammatory fibroblasts (iCAFs). These fibroblast subtypes exhibit distinct characteristics and localisation within the tumour. The research examines the impact of CAFs and ANFs on immune cell behaviour, particularly in relation to adhesion and migration. Methods: Tumour and juxta-tumoral tissue from patients were digested into single cell suspensions. Cells were stained with a 22-plex antibody panel by flow cytometry. Pseudo-emperipolesis assays were assessed by co-culture of lymphocytes with CAF and ANF fibroblasts. Furthermore, these were performed with or without stimulation of fibroblasts with by TGF-b or TNF-a/IFN-g, to promote myCAF or iCAF phenotypes respectively. Gene expression analysis of tumour-derived fibroblast subtypes was assessed by single-cell RNA sequencing using the 10X Genomics platform. The transcriptional profile of spatially resolved fibroblasts which were proximal or distant to tumour epithelium was determined by the NanoString GeoMx Digital Spatial Profiler (DSP) platform. Results: Flow cytometry analysis revealed that iCAFs predominated within the tumour stroma compared to myCAFs, although iCAF populations were less prevalent within ANF populations. Upon stimulation with inflammatory cytokines (TNF-α/IFN-γ), CAFs promoted lymphocyte migration in contrast to ANFs. However, TGF-β stimulation of CAF downregulated the migration of lymphocytes. As such, TGF-β or TNF-α/IFN-γ stimulation exhibited antagonistic effects on lymphocyte adhesion and migration in CAFs. Tumour-proximal fibroblasts exhibited myCAFs characteristics expressing α-SMA and PDPN, as well as ECM molecules such as collagen and integrins, suggesting a barrier to lymphocyte migration. Conversely, distal fibroblasts displayed an inflammatory profile, expressing cytokines and chemokines including CCL2, CXCL9, CXCL12, IL6, IL11 and LIF, thereby contributing to the inflammatory milieu within the tumour microenvironment. Additionally, iCAFs demonstrated higher transcription levels of hyaluronan synthase (Has1) and fibulin 2 (FBLN2) which are crucial for ECM formation and remodelling. This study identifies a differential role for iCAFs and myCAFs in the modulate of lymphocyte adhesion and migration within the PDAC microenvironment, highlighting the complex interplay between fibroblast subtypes and immune cell dynamics. Citation Format: Fouzia Zayou, Hayden Pearce, Wayne Croft, Samantha Nicol, Sandra Margielewska-Davies, Sarah Powell-Brett, Rachel Brown, Jianmin Zuo, Keith Roberts, Helen M. McGettrick, Paul Moss. Modulation of lymphocyte adhesion and migration by iCAF and myCAF fibroblasts in PDAC [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A030.

A Comprehensive Proteogenomic and Spatial Analysis of Innate and Acquired Resistance of Metastatic Melanoma to Immune Checkpoint Blockade Therapies.

This study paves the way for the creation of innovative therapeutic strategies, aimed at subverting resistance to immune checkpoint blockade (ICB) therapies in metastatic melanoma patients. By unraveling the specific molecular mechanisms underlying resistance, scientists can design effective alternative treatments that target pathways such as pathways associated with cell cycle dysregulation and c-MYC signaling. Furthermore, through the application of advanced immune monitoring techniques such as NanoString Digital Spatial Profiling (DSP) and Cyclic Immunofluorescence (CyCIF), this study has significantly enriched our understanding of the tumor microenvironment. This enhanced characterization facilitates the discovery of potential biomarkers that may forecast a patient's response to ICB treatment. Ultimately, these advancements could potentially refine patient outcomes and foster the development of more personalized cancer treatments in the future.

Abstract B073: Organ-specific diversity of tumor immune microenvironment in metastatic pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) that is refractory to treatment frequently leads to extensive systemic and multi-organ metastases. PDAC is characterized as an immune “cold” cancer; however, the persistence of this “cold” tumor immune microenvironment (TIME) across distant metastases, and the mechanisms by which metastases influence their respective TIME, remain poorly understood. In this study, we conducted spatial protein expression profiling of the TIME using the Nanostring GeoMx Digital Spatial Profiling (DSP) platform. We employed a 43- plex panel on 54 tissue samples, including 14 primary tumors and 40 matched metastases from organs commonly affected by PDAC metastases (liver, lung, and peritoneum), obtained through a rapid (“warm”) autopsy program. We quantified protein expression in three cellular compartments: PanCK+ epithelial cells, α-SMA+ fibroblasts, and CD45+ immune cells, across 181 regions of interest (ROIs). Our analysis revealed a significant enrichment of CD45+ immune cells in lung metastases compared to other organ sites, with a notably lower presence of these cells in liver metastases. Focusing on the immune cell compartment, we analyzed the CD45 segments for further investigation. After normalization using housekeeping proteins, including Histone H3, GAPDH, and ribosomal protein S6, we found that the immune profiles were most distinct between liver and lung metastases. Specifically, liver metastases exhibited the greatest deviation in immune profile compared to primary PDAC, lung, and peritoneal metastases. We scored protein expression signatures for various immune cell subsets based on specific marker expressions. When comparing liver to lung metastases, scores for T cells, myeloid cells, checkpoint markers, macrophages, and interferon signaling were significantly lower in the liver, underscoring the immune “coldness” of the liver metastasis environment. Our cohort included six untreated patients and nine patients treated with chemotherapies. We compared protein expressions in each organ site between untreated and treated patients. Notably, treatment induced divergent expression patterns of multiple proteins between liver and lung metastases. While treatment significantly reduced the expression of several immune markers, including CD45 (total immune), CD3 (T cell), CD4 (T helper cell), CD45RO (memory T cell), CD163 (M2 macrophage), and B7-H3 (checkpoint marker), in lung metastases, these markers were significantly upregulated in liver metastases of treated patients. This finding indicates a pronounced divergence in TIME modulation in response to treatment between liver and lung metastases, potentially fostering a treatment-resistant environment in PDAC patients with multi- organ metastasis. Consequently, this urges the need to develop tailored therapeutic strategies for PDAC metastasis, taking into account the distinct immune landscapes of different metastasis sites. Citation Format: Jimin Min, Vittorio Branchi, Kimal I Rajapakshe, Nisha Narayanan, Guangsheng Pei, Linghua Wang, Jean L Grem, Thomas C Caffrey, Paul M Grandgenett, Michael A Hollingsworth, Paola A Guerrero, Anirban Maitra. Organ-specific diversity of tumor immune microenvironment in metastatic pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B073.

Abstract B023: Unravelling fibroinflammatory and immune signatures for pancreatic cancer early detection

Abstract Pancreatic cancer is associated with late-stage diagnosis and poor patient prognosis. The disease is characterised by chronic dysregulated immunity and inflammation. Moreover, chronic pancreatitis is linked with an increased risk of pancreatic cancer. Tissue remodelling during disease progression involves a complex network of cells and a crosstalk between cancer cells, stromal components and immune cells, but it remains poorly explored. Thus, to improve the outcome of patients, there is a need for better understanding of biological changes associated with the initiation and progression of pancreatic cancer at the molecular and spatial levels. Using samples obtained from our Accelerated Diagnosis of neuroEndocrine and Pancreatic TumourS (ADEPTS) biobank, we have evaluated blood samples for the discovery of non- invasive biomarkers to discriminate patients with pancreatic cancer from symptomatic patients without cancer. In this pilot study, the transcriptome of peripheral blood mononuclear cells (PBMCs), isolated using a ficoll gradient was explored to identify PDAC-associated mRNA signatures. Moreover, the Olink® Explore Inflammation and Oncology panels were performed in a cohort of 40 patients with early stage (I-II) PDAC, 40 chronic pancreatitis samples and 40 control patients with benign symptomatic upper GI diseases. To validate and enrich these results, a panel of inflammatory and immune markers including immunoglobulins, cytokines and chemokines was also run using multiplex techniques. Symptoms observed in our cohort were also analysed together with clinical (e.g diabetic status, biochemistry profile) and demographic data (age, sex, ethnicity, post code etc). We have identified mRNA transcriptome signatures and differentially expressed proteins associated with inflammatory and immunological processes in PDAC compared to benign symptomatic samples. Our results offer potential diagnostic utility for early-stage disease. Additionally, our unique cohort of patient samples comprising of those with confirmed PDAC and those presenting to clinic with benign disease and similar non-specific symptoms, allows for the development of non-invasive diagnostic tests with better translational applicability. Future work will involve the validation of these findings in a larger patient cohort and the use of NanoString GeoMx® Digital Spatial Profiler to perform whole transcriptome and proteome profiling in human chronic pancreatic and pancreatic cancer tissue samples. Citation Format: Kyra Fraser, Maria Rosado, Stephen P Pereira, Pilar Acedo. Unravelling fibroinflammatory and immune signatures for pancreatic cancer early detection [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B023.