778 Background: Pancreatic ductal adenocarcinoma (PDAC) has a high recurrence rate, with up to 80% of patients experiencing disease recurrence within two years of surgical resection. Standard postoperative monitoring with imaging and serum tumor markers has significant limitations. Here, we evaluated the prognostic value of longitudinal circulating tumor (ct)DNA assessment in an updated cohort of patients with resected PDAC, representing a larger sample size and longer clinical follow-up during post-treatment surveillance. Methods: We retrospectively analyzed 292 banked plasma samples from 43 patients with localized PDAC who underwent surgical resection +/- neoadjuvant and/or adjuvant chemotherapy. Longitudinally collected blood samples during surveillance (from post-definitive treatment to the end of follow-up/recurrence) were used for ctDNA analysis using the personalized, tumor-informed 16-plex PCR-NGS assay (Signatera RUO, Natera, Inc.). The association of ctDNA status with recurrence-free survival (RFS) was evaluated using the Kaplan-Meier method, and comparisons were accomplished using log-rank tests. A multivariable Cox proportional hazards model was used to identify the most significant prognostic factor associated with RFS. Results: Among the 43 patients included in this analysis, 37% (16) had stage I, 30% (13) had stage II, and 33% (14) had stage III disease. The median patient age was 69 years (range: 51-86), and the median follow-up was 25 months (range: 1-52). The median plasma volume was 4.5 mL (range: 0.6-6.8), with ~half of the patient samples with <2 mL of plasma. 32.6% (14/43) received neoadjuvant chemotherapy (NAC) and 62.8% (27/43) received adjuvant chemotherapy (ACT). During follow-up, 72% (31/43) of patients relapsed, 68% (21/31) of whom tested ctDNA-positive at one or more time points post-surgery. In the surveillance window, 32% (10/31) of patients were positive for ctDNA, all of whom relapsed. ctDNA-positivity in the surveillance window was associated with significantly inferior RFS (HR: 16.1, 95% CI: 3.79-68.17, P=0.0002). Upon adjusting for other clinicopathological factors (stage and surgical margin), the multivariate analysis confirmed ctDNA-positivity during surveillance to be the most significant prognostic factor associated with RFS (HR: 8.4, 95%CI: 2.84–24.8, P<0.001). Conclusions: Despite the low sample quality in this cohort, ctDNA showed prognostic value, demonstrating the clinical utility of longitudinal ctDNA monitoring during surveillance.

Table S1: Gene Sets; Table S2: Patient Clinical Information; Table S3: Clustering Results; Table S4: Differential expression analysis between clusters (full dataset); Table S5: GSEA results (full dataset); Table S6: Stromal highly variable genes; Table S7: DE analysis of NL-LGD subset; Table S8: GSEA results (NL-LGD subset); Table S9: Validation dataset clustering results

Figure S1: Hematoxylin and eosin (H&E) stained tissue images from patient p10 corresponding to Figure 1B; Figure S2: Quality control of DSP-RNA data; Figure S3: AOI Filtering Criteria; Figure S4: Gene Filtering Criteria; Figure S5: Normalization of DSP-RNA data; Figure S6: Dimension reduction analysis of DSP-RNA data; Figure S7: Immunofluorescent staining of epithelial marker genes; Figure S8: GSEA analysis comparing IPMN clusters to PDAC molecular subtyping studies; Figure S9: Leading edge analysis of GSEA results; Figure S10: Hallmark pathways associated with IPMN transcriptomic states; Figure S11: Analysis of adjacent stromal (PanCK-, CD3-, CD45-) AOIs; Figure S12: GSEA analyses of NL-LGD AOIs; Figure S13: Quality control of external DSP-RNA datasets incorporated for validation; Figure S14: PCA projection of validation datasets; Figure S15: GSEA and GSVA of the Carpenter and colleagues dataset

Background Gallbladder cancer (GBC) portends a poor prognosis, and guidelines recommend radical cholecystectomy for patients with T1b and greater disease. We evaluated prognostic factors for T2 and T3 GBC among a contemporary cohort. Methods The National Cancer Database (NCDB) was queried for patients with resected pathologic T2 and T3 GBC from 2004 to 2018. Patients were stratified by extent of resection: simple (SC) vs radical cholecystectomy (RC). Overall survival (OS) was compared with Kaplan-Meier and multivariable Cox proportional hazards methods. Results Altogether, 10,107 patients (6,426 [63.6%] T2; 3,681 [36.4%] T3) were identified: 2,203 (21.8%) underwent SC and 7,904 (78.2%) RC. Patients with SC were more likely to have T2 disease, negative nodes, and positive margins. Tumor grade, node positivity, margin positivity, and T3 stage were associated with greater mortality. Treatment at an academic center and receipt of adjuvant chemotherapy were associated with improved survival. Extent of surgical resection was not associated with OS in unadjusted and adjusted analyses (adjusted HR 0.98, 95% CI 0.92-1.05, p=0.62). Discussion Outcomes for patients with resected T2 and T3 GBC remain poor, though these retrospective data suggest RC is not associated with better OS. Multidisciplinary management and prospective investigation are needed to advance outcomes and facilitate selection of patients who may benefit from radical resection.

BACKGROUND:The benefit of adjuvant chemotherapy (AC) for ampullary adenocarcinoma is unclear. The Hidden Genome model classifies prognostic subtypes with greater accuracy than standard histologic classification (intestinal [INT] vs pancreatobiliary [PB]), but its predictive capacity to guide the use of AC remains unstudied. METHODS:We applied the Hidden Genome model to an international cohort of 183 patients with resected ampullary adenocarcinoma who underwent genomic sequencing. The model quantified the predicted cell of origin (colorectal vs pancreas/distal bile duct) in all specimens. Overall survival (OS) was compared using Kaplan-Meier estimates, stratified by AC use versus surgery alone (SA). RESULTS:Most patients (n=128; 69.9%) received AC, which was not associated with a significant improvement in OS (median, 50.9 months [95% CI, 36.5-76.9] vs 53.8 months [95% CI, 32.4-119.0]; P=.816). Histologic subtype was neither associated with prognosis (P=.241) nor predictive of chemotherapy efficacy for INT-subtype (P=.379) or PB-subtype (P=.544) tumors. When stratified by genomic subtype, the colorectal group had a favorable prognosis regardless of AC use (median OS, 74.4 months [95% CI, 33.8-97.8] for AC vs 98.7 months [95% CI, 32.4-140.9] for SA; P=.889). Among patients with pancreas/distal bile duct tumors, those who received AC had longer survival compared with those who underwent SA (78.2 months [9.8-not reached] vs 22.7 months [2.3-not reached], respectively; hazard ratio, 0.17 [95% CI, 0.04-0.80]; P=.024). CONCLUSIONS:AC regimens were not associated with improved survival in histologically defined INT- or PB-subtype ampullary adenocarcinoma. However, genomic classification better stratified risk groups and identified patients more likely to benefit from AC.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by exceedingly high rates of metastatic progression, with the liver representing the most common site of distant spread. Here, we established a platform for multisite immune profiling of human PDAC encompassing the tumor, peripheral circulation, and premetastatic liver, to more comprehensively study how various immune subsets might contribute to patient outcomes. Tumor, liver, and blood samples were obtained from patients undergoing resection for non-metastatic PDAC. Derived immune cells underwent paired single-cell RNA and TCR sequencing. Immune composition, cell-type functional profiles, and T cell clonal expansion patterns were evaluated across tissue sites. In total, 106,539 immune cells were sequenced, of which 85,748 met criteria for analysis. We identified 32 cell populations, of which seven demonstrated significant enrichment within a particular tissue, highlighting that this workflow possesses the granularity needed for identifying potential future biomarkers. Functional profiling revealed tissue-specific differences in cell phenotypes. This included terminally differentiated exhausted CD8 T cells within the tumor, highly active Tregs within the premetastatic liver and tumor, and M1 versus M2 polarization of liver and tumor macrophage populations, respectively. Within the tumor, expanded Treg clones were uniquely abundant, and while expanded clones could be tracked to the blood and premetastatic liver, many of these mapped back to known viral antigens. Leveraging previously validated gene sets, we show how these can be applied to predict the tumor reactivity of intratumoral T cells using transcriptional signatures. We demonstrated a high degree of concordance between multiple independent signatures and tracked high-priority TCRs within the blood and liver. This study demonstrates the feasibility of a platform, which has already been implemented into ongoing clinical protocols, for immune profiling of human PDAC across the sites most relevant to metastatic progression. Future applications of this work can monitor immune populations throughout metastatic progression to build a temporal database of immune phenotypes and track association with clinical outcomes.

PURPOSE:Intraductal papillary mucinous neoplasms (IPMN) occur in 5% to 10% of the population, but only a small minority progress to pancreatic ductal adenocarcinoma (PDAC). The lack of accurate predictors of high-risk disease leads to both unnecessary operations for indolent neoplasms and missed diagnoses of PDAC. Digital spatial RNA profiling (DSP-RNA) provides an opportunity to define and associate transcriptomic states with cancer risk. EXPERIMENTAL DESIGN:We performed whole-transcriptome DSP-RNA profiling on 10 IPMN specimens encompassing the spectrum of dysplastic changes from normal duct to cancer. Epithelial regions within each tissue were annotated as normal duct, low-grade dysplasia, high-grade dysplasia, or invasive carcinoma. The resulting digital gene expression data were analyzed with R/Bioconductor. RESULTS:Our analysis uncovered three distinct epithelial transcriptomic states-"normal-like" (cNL), "low risk" (cLR), and "high risk" (cHR)-which were significantly associated with pathologic grade. Furthermore, the three states were significantly correlated with the exocrine, classical, and basal-like molecular subtypes described in PDAC. Specifically, exocrine function diminished in cHR, classical activation distinguished neoplasia (cLR and cHR) from cNL, and basal-like genes were specifically upregulated in cHR. Intriguingly, markers of cHR were detected in normal duct and low-grade dysplasia regions from specimens with PDAC but not from specimens containing only low-grade IPMN. CONCLUSIONS:DSP-RNA of IPMN revealed low-risk (indolent) and high-risk (malignant) expression programs that correlated with the activity of exocrine and basal-like PDAC signatures, respectively, and distinguished pathologically low-grade specimens from malignant specimens. These findings contextualize IPMN pathogenesis and have the potential to improve risk stratification.

Background:Early-onset colorectal cancer (EO-CRC), defined as being diagnosed before the age of 50 years, is becoming increasingly prevalent. Among these patients, synchronous early-onset colorectal liver metastases (EO-CRLM) have emerged as a leading cause of mortality. This study aims to investigate the epidemiology, clinicopathological characteristics, and survival outcomes related to synchronous EO-CRLM to provide a clearer understanding of the challenges faced by patients with synchronous EO-CRLM. Methods:The National Cancer Database was queried for patients with synchronous CRLM diagnosed from 2010 to 2019. Patients were then stratified by age of onset: EO-CRLM (younger than 50 years old) and late-onset colorectal liver metastases (LO-CRLM) (50 years old or older). The incidence, limited-duration prevalence rates (over a 10-year period), clinicopathological characteristics and overall survival (OS) were assessed. Results:Among 115,422 patients with CRLM, EO-CRLM and LO-CRLM were observed in 17,536 (15.2%) patients and in 97,886 (84.8%) patients, respectively. The annual age-adjusted incidence of EO-CRLM increased from 0.48 to 0.69 per 100,000 population from 2010 to 2019 [average annual percentage change (AAPC): 5.1, 95% confidence interval (CI): 4.1 to 6.1], significantly faster than that of LO-CRLM (AAPC difference: 3.5, 95% CI: 2.5 to 4.5; P<0.001). The 10-year limited-duration prevalence for EO-CRLM increased from 0.0004% in 2010 to 0.0020% in 2019 (AAPC: 15.7, 95% CI: 10.2 to 21.5). A comparison of the clinicopathological features revealed that, compared with LO-CRLM, EO-CRLM patients were more likely to reside in urban areas, have higher rates of high school completion, have higher income, and be covered by private insurance/managed care. Genetic analysis revealed EO-CRLM with microsatellite instability (MSI)-high had the largest increase of AAPC of 28.7 (95% CI: 13.1 to 46.3) compared to MSI-low and microsatellite stability (MSS) (P<0.001). Similarly, EO-CRLM patients had a rapid rise in Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations with AAPC of 10.6 (95% CI: 8.8 to 12.5). Compared with LO-CRLM, EO-CRLM was associated with improved OS in inverse probability of treatment weighting-adjusted Cox proportional hazards regression analysis (hazard ratio: 0.876, 95% CI: 0.838 to 0.915; P<0.001). Conclusions:The incidence of EO-CRLM has increased rapidly, and represents a distinct population in terms of socioeconomic and clinicopathological characteristics. Among this cohort, the prognosis of patients with EO-CRLM was favorable compared to that of patients with LO-CRLM.

Purpose:Intraductal papillary mucinous neoplasms (IPMN) occur in 5-10% of the population, but only a small minority progress to pancreatic ductal adenocarcinoma (PDAC). The lack of accurate predictors of high-risk disease leads both to unnecessary operations for indolent neoplasms as well as missed diagnoses of PDAC. Digital spatial RNA profiling (DSP-RNA) provides an opportunity to define and associate transcriptomic states with cancer risk. Experimental Design:Whole-transcriptome DSP-RNA profiling was performed on 10 IPMN specimens encompassing the spectrum of dysplastic changes from normal duct to cancer. Ductal epithelial regions within each tissue were annotated as normal duct (NL), low-grade dysplasia (LGD), high-grade dysplasia (HGD), or invasive carcinoma (INV). Gene expression count data was generated by Illumina sequencing and analyzed with R/Bioconductor. Results:Dimension reduction analysis exposed three clusters reflecting IPMN transcriptomic states denoted "normal-like" ( cNL ), "low-risk" ( cLR ) and "high-risk" ( cHR ). In addition to specific marker genes, the three states exhibited significant enrichment for the exocrine, classical, and basal-like programs in PDAC. Specifically, exocrine function diminished in cHR , classical activation distinguished neoplasia from cNL , and basal-like genes were specifically upregulated in cHR . Intriguingly, markers of cHR were detected in NL and LGD regions from specimens with PDAC but not low-grade IPMN. Conclusions:DSP-RNA of IPMN revealed low-risk (indolent) and high-risk (malignant) expression programs that correlated with the activity of exocrine and basal-like PDAC signatures, respectively, and distinguished pathologically low-grade from malignant specimens. These findings contextualize IPMN pathogenesis and have the potential to transform existing risk stratification models. Statement of translational relevance:Current consensus guidelines for management of intraductal papillary mucinous neoplasms (IPMN) of the pancreas utilize clinical and radiographic criteria for risk stratification. Unfortunately, the estimated positive predictive value of these criteria for IPMN-associated pancreatic ductal adenocarcinoma (PDAC) is under 50%, indicating that over half of pancreatectomies are performed for benign disease. Moreover, nearly 15% of patients who were deemed "low risk" by the same criteria harbored PDAC. Surgical resection of IPMN has maximal benefit when performed prior to the development of PDAC, as evidence of carcinoma has been associated with a high rate of recurrence and poor overall survival. Thus, the development of molecular diagnostics that improve the accuracy of IPMN risk classification would have immediate relevance for patient care, both in terms of better selecting patients for potentially curative operations, as well as sparing patients with low-risk lesions from invasive procedures.

OBJECTIVE:To sought comprehensively profile tissue and cyst fluid in patients with benign, precancerous, and cancerous conditions of the pancreas to characterize the intrinsic pancreatic microbiome. BACKGROUND:Small studies in pancreatic ductal adenocarcinoma (PDAC) and intraductal papillary mucinous neoplasm (IPMN) have suggested that intrapancreatic microbial dysbiosis may drive malignant transformation. METHODS:Pancreatic samples were collected at the time of resection from 109 patients. Samples included tumor tissue (control, n = 20; IPMN, n = 20; PDAC, n = 19) and pancreatic cyst fluid (IPMN, n = 30; serous cystadenomas, n = 10; mucinous cystic neoplasm, n = 10). Assessment of bacterial DNA by quantitative polymerase chain reaction and 16S ribosomal RNA gene sequencing was performed. Downstream analyses determined the relative abundances of individual taxa between groups and compared intergroup diversity. Whole-genome sequencing data from 140 patients with PDAC in the National Cancer Institute's Clinical Proteomic Tumor Analysis Consortium were analyzed to validate findings. RESULTS:Sequencing of pancreatic tissue yielded few microbial reads regardless of diagnosis, and analysis of pancreatic tissue showed no difference in the abundance and composition of bacterial taxa between normal pancreas, IPMN, or PDAC groups. Low-grade and high-grade dysplasia IPMN were characterized by low bacterial abundances with no difference in tissue composition and a slight increase in Pseudomonas and Sediminibacterium in high-grade dysplasia cyst fluid. Decontamination analysis using the Clinical Proteomic Tumor Analysis Consortium database confirmed a low-biomass, low-diversity intrinsic pancreatic microbiome that did not differ by pathology. CONCLUSIONS:Our analysis of the pancreatic microbiome demonstrated very low intrinsic biomass that is relatively conserved across diverse neoplastic conditions and thus unlikely to drive malignant transformation.

Abstract Even in its earliest stages, pancreatic ductal adenocarcinoma (PDAC) is an essentially incurable disease due to the presence of disseminated, yet radiographically occult cancer cells in the setting of an otherwise “localized” primary tumor. The vast majority (~80%) of patients who undergo curative-intent surgery develop liver metastases, often within six months of surgery, and these hepatic metastases dominate survival outcomes, even in the presence of multisite recurrence. What drives hepatic organotropism remains unknown, in large part due to the exclusion of “normal” liver tissue from PDAC biobanking programs. While recent work in animal models suggests that suppression of the hepatic immune microenvironment may facilitate metastatic progression, confirmatory studies in human patients are lacking, due to a paucity of relevant model systems. To address this deficit, we developed an experimental pipeline to profile hepatic immunity at single-cell resolution vis-à-vis the peripheral and intratumoral immune compartments. We hypothesized that this platform could be leveraged to identify tissue- and patient-specific immunophenotypes associated with hepatic recurrence prior to the development of overt metastases. Patients with localized PDAC at Duke University were enrolled in an IRB-approved (Pro00108288) biobanking program to obtain multisegment liver biopsies, primary tumor specimen, and peripheral blood at the time of curative-intent resection. Immune (CD45+) cells were sorted following enzymatic digestion (tissues) or Ficoll separation (blood) and single-cell gene expression and T cell receptor libraries were created. Data was processed using CellRanger and aligned, filtered, and normalized using R. In total, we identified 32 unique immune cell populations conserved across patients, with both shared and site-specific cell types identified in the T cell, B cell, NK cell, and myeloid compartments. Additionally, we identified expanded T cell clones within the tumor microenvironment and traced a subset of these clonotypes to the premetastatic liver. These tumor-expanded T cells (TTE cells) presumably arise from recognition of a specific tumor antigen, which then instigates activation and proliferation of the clone. Notably, we identified a subset of our patients without known liver metastases who nonetheless demonstrated hepatic infiltration of TTE cells. While the significance of these hepatic TTE cells is still unclear, we hypothesize that this is evidence of a primed immune response to tumor antigens already present in the liver at the time of surgery. This experimental platform has now been implemented within an ongoing window-of-opportunity trial (NCT05634720) testing neoadjuvant hepatic artery chemotherapy in patients undergoing resection of localized PDAC. The correlative scientific objectives will focus on quantifying the variation of hepatic TTE infiltration across patients, determining the function and activation state of hepatic TTE cells, and testing the association between hepatic TTE cell infiltration and clinical outcomes. Citation Format: Elishama N. Kanu, Ashley A. Fletcher, Jiayin Bao, Austin M. Eckhoff, Karrie Comatas, Tao Wang, Bin-Jin Hwang, Michael E. Lidsky, Sabino Zani, Dan G. Blazer, Peter J. Allen, Zhicheng Ji, Daniel P. Nussbaum, Erika J. Crosby. A platform to characterize hepatic immunity reveals variation in hepatic infiltration of tumor expanded T cells in “localized” PDAC [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 B022.

ObjectivePatients with gastrointestinal malignancies represent a heterogenous population, even among those with similar stage and treatment pathways. Here, we used dimensionality reduction in the National Cancer Database (NCDB) to inform unsupervised clustering of patients with three gastrointestinal malignancies and examined outcomes among these computationally-derived groups.MethodsThe NCDB was queried for three cohorts of patients receiving multimodal therapy: stage II/III esophageal cancer, stage II/III gastric cancer, and stage III colon cancer. Multiple correspondence analysis (MCA), a dimensionality reduction technique well-suited for categorical variables such as demographic data in the NCDB, was performed on this cohort with variables including demographic and tumor characteristics. Principal components were analyzed to derive clusters. Outcomes for each cluster were compared using Kaplan-Meier survival methods.ResultsFor esophageal (n=11,399), gastric (n=2,033), and colon (n=72,057) cancer, the same four variables were identified as highly representative. The principal variables were income quartile, education quartile, age quartile, and insurance type. Survival analysis demonstrated significant differences in overall survival between clusters in esophageal (p<0.0001) and colon (p<0.0001) cancer, but not gastric cancer (p=0.56). Clusters defined by high income, high education, younger age, and private insurance fared better.ConclusionsUsing MCA, we identified combinations of 4 demographic variables in the NCDB with stage II/III esophageal cancer, stage II/III gastric cancer, and stage III colon cancer. These groupings had significantly different survival outcomes in colon and esophageal cancer. This work serves as proof-of-concept for the utility of unsupervised clustering for outcomes research in surgical malignancies and identifies at-risk populations.

Importance Ampullary adenocarcinoma (AA) is characterized by clinical and genomic heterogeneity. A previously developed genomic classifier defined biologically distinct phenotypes with greater accuracy than standard histologic classification. External validation is needed before routine clinical use. Objective To test external validity of the prognostic value of the hidden genome classifier of AA. Design, Setting, and Participants This retrospective cohort study took place at 6 international academic institutions. Consecutive patients (n = 192) who underwent curative-intent resection of histologically confirmed AA were included. The data were analyzed from January 2005 through July 2020. Exposures The multilevel meta-feature regression model previously trained on a prospectively sequenced cohort of 3411 patients (1001 pancreatic adenocarcinoma, 165 distal bile duct adenocarcinoma, and 2245 colorectal adenocarcinoma) was applied to AA sequencing data to quantify the relative proportions of parental cell of origin. Main Outcome and Measures Genomic classification was correlated with immunohistologic subtype (intestinal [INT] or pancreatobiliary [PB]) and with overall survival (OS), using the log-rank test and Cox proportional hazard models. Results Among 192 patients with AA (median age, 69.0 [IQR, 60.0-74.0] years and 134 were male [64%]), concordance between immunohistologic and genomic subtypes was 55%. Most INT subtype tumors were categorized into the colorectal genomic subtype (43 of 57 [72.9%]). Of the 114 PB subtype tumors, 29 had a pancreatic genomic profile (25.4%) and 24 had a distal bile duct genomic profile (21.1%). Whereas the standard immunohistologic subtypes were not associated with survival (log rank P = .26), predicted genomic probabilities were correlated with survival probability. Genomic scores with higher colorectal probability were associated with higher survival probability; higher pancreatic and distal bile duct probabilities were associated with lower survival probability. Conclusions and Relevance The AA genomic classifier is reproducible with available molecular testing in a diverse international cohort of patients and improves stratification of the divergent clinical outcomes beyond standard immunohistologic classification. These data provide a molecular classification that may be incorporated into clinical trials for prospective validation.

Abstract Even in its localized stages, pancreatic ductal adenocarcinoma (PDAC) is virtually incurable, characterized by early dissemination and disease recurrence. The liver is the most common site of metastasis, and in most cases hepatic recurrences drive survival outcomes. This underscores the critical need to target and prevent liver metastases. We leveraged a novel biobanking strategy to perform paired single-cell RNA (scRNA-seq) and TCR sequencing (scTCR-seq) to profile multisite immunity in patients with localized PDAC across the tumor, peripheral blood, and premetastatic liver. This analysis of three patients included three tumor, three blood, and nine liver (three per patient from separate segments) samples, and generated 85,748 cells for downstream analysis. We evaluated populations of tumor-expanded T cells as potential indicators of antitumoral immunity, and utilized validated computational approaches to identify tumor-reactive T cells based on key markers of dysfunction and exhaustion. Gene set enrichment analysis was performed on tumor infiltrating lymphocytes (TILs) using signatures specific to tumor-reactive CD4 and CD8 T cells (“NeoTCR4” and “NeoTCR8,” respectively), and scored these using the AUCell package in R. Overall, 153 CD4 and 22 CD8 T cells fit candidate NeoTCR4 and NeoTCR8 signature states, representing 5.6% and 0.8% of the total TIL population respectively. These nominated T cells expressed key marker genes associated with tumor specificity such as CXCL13, TIGIT, PDCD1, ENTPD1, and TOX. Thirteen of these predicted tumor-reactive T cell clones identified within the tumor were also detected within our liver samples, suggestive of a potential antitumoral immune response in the premetastatic liver space. We next performed 10x Xenium in situ spatial transcriptomic analysis of primary tumor, normal adjacent pancreas, and premetastatic liver from two of these patients, using tissue microarrays to incorporate replicate 2mm core biopsies from each of these sites. A custom designed gene panel, guided by our scRNA-seq dataset, targeted tumor-specific mutations (e.g. KRAS G12V, G12D, G12R, and Q61) and immune related genes with a specific focus on markers of T cell activity and exhaustion. Leveraging these latter marker genes, which represent a subset of the NeoTCR4 and NeoTCR8 signatures, we identified a population of exhausted TILs within the tumor microenvironment, representing potentially tumor-reactive T cells whose interactions can now be explored at spatial resolution. Future directions are aimed at experimentally validating the tumor reactivity of the predicted neoantigen-specific TILs within our dataset and characterizing the immune neighborhoods that exist in both the tumor and liver of PDAC patients. This work establishes exciting opportunities for novel T-cell therapy in the treatment of PDAC, and we have already implemented these experimental findings in a forthcoming clinical trial investigating the applicability of this predictive signature score for the generation of TCR-engineered lymphocyte therapy in patients with PDAC. Citation Format: Elishama N Kanu, Ashley A Fletcher, Sri Krishna, Frank J Lowery, Haotian Zhuang, Ethan S Agritelley, Jiayin Bao, Austin M Eckhoff, Karrie Comatas, Tao Wang, Bin-Jin Hwang, Michael E Lidsky, Sabino Zani, Dan G Blazer III, Peter J Allen, Nicholas D Klemen, Zhicheng Ji, Daniel P Nussbaum, Erika J Crosby. Single-cell and spatial analysis of the immune landscape unveils a subset of potentially tumor-reactive T cells in patients with localized 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 B052.