CTNNB1 exon 3 mutations in metastatic solid pseudopapillary neoplasm of the pancreas.

BACKGROUND AND OBJECTIVES: Solid pseudopapillary neoplasm (SPN) of the pancreas demonstrates an indolent disease course; however, some patients present with a "malignant" phenotype, including distant metastases resistant to chemotherapy. This analysis identifies molecular drivers of metastatic SPN using the world's largest clinicogenomics database. METHODS: The American Association for Cancer Research Project Genomics Evidence Neoplasia Information Exchange was queried for primary and metastatic SPN samples. Sample-level genomic alterations were compared. A pan-pancreatic cancer analysis assessed relevant mutations among all metastatic pancreatic malignancies. RESULTS: Among 28 SPN samples identified (n = 17 primary, n = 11 metastatic), the most commonly mutated gene was CTNNB1, (24/28 samples; 85.7%). Most mutations were missense (21/24; 87.5%) or in-frame deletions (3/24; 12.5%). The most common CTNNB1 mutations in primary SPN were exon 3 S37F/C missense mutations (6/16 profiled patients, 37.5%), contrasting exon 3 D32N/Y/H missense mutations in metastatic samples (6/11 profiled patients, 54.5%). Metastatic SPN had higher rates of CTNNB1 mutations than metastases from pancreatic ductal adenocarcinoma (72.7% vs. 1.1%; q < 0.0001), pancreatic neuroendocrine tumor (72.7% vs. 2.5%; q < 0.0001), and pancreatic acinar cell carcinoma (72.7% vs. 11.5%; q = 0.0254). CONCLUSIONS: Missense mutations along exon 3 of CTNNB1 predominate metastatic SPN, differentiating these patients from those with metastases from analogous pancreatic malignancies.

Lymph node metrics following neoadjuvant therapy to refine patient selection for adjuvant chemotherapy in resected pancreatic cancer: A multi-institutional analysis.

BACKGROUND: In patients with localized pancreatic ductal adenocarcinoma (PDAC) undergoing neoadjuvant therapy (NAT) and resection, selection of adjuvant chemotherapy (AC) is typically guided by high-risk features on histopathologic examination. We evaluated the interaction between post-NAT lymph node metrics and AC receipt on survival. METHODS: Patients who received NAT followed by pancreatectomy (2010-2020) at seven centers were reviewed. Overall survival (OS) in patients receiving AC or not was stratified by lymph node positivity (LNP) or lymph node ratio (LNR) dichotomized at 0.1. Cox models evaluated the independent association between these nodal metrics, AC receipt, and OS. RESULTS: Of 464 patients undergoing NAT and resection, 264 (57%) received AC. Patients selected for AC were younger (median 63 vs. 67 years; p < 0.001), received shorter duration of NAT (2.8 vs. 3.2 months; p = 0.01), had fewer postoperative complications (Clavien-Dindo grade > 3: 1.2% vs. 11.7%; p < 0.001), and lower rates of pathologic complete response (4% vs. 11%; p = 0.01). The median number of nodes evaluated was similar between cohorts (n = 20 in both; p = 0.9). Post-NAT LNP rates were not different, and median LNR was 0.1, in AC and non-AC cohorts. Both LNP (hazard ratio [HR]: 2.1, p < 0.001) and LNR (0 < LNR ≤ 0.1: HR: 1.98, p = 0.002; LNR > 0.1: HR 2.46, p < 0.001) were independently associated with OS on Cox modeling, although receipt of AC was not associated with improved OS (median 30.6 vs. 29.4 months; p = 0.2). In patients with LNR > 0.1, receipt of AC was associated with significantly longer OS compared to non-AC (24 vs. 20 months, respectively; p = 0.04). CONCLUSIONS: LNR following NAT, not simply nodal positivity, may be useful to refine selection of AC in resected PDAC.

Leveraging real-world data to predict cancer cachexia stage, quality of life, and survival in a racially and ethnically diverse multi-institutional cohort of treatment-naïve patients with pancreatic ductal adenocarcinoma.

Introduction: Cancer-associated cachexia (CC) is a progressive syndrome characterized by unintentional weight loss, muscle atrophy, fatigue, and poor outcomes that affects most patients with pancreatic ductal adenocarcinoma (PDAC). The ability to identify and classify CC stage along its continuum early in the disease process is challenging but critical for management. Objectives: The main objective of this study was to determine the prevalence of CC stage overall and by sex and race and ethnicity among treatment-naïve PDAC cases using clinical, nutritional, and functional criteria. Secondary objectives included identifying the prevalence and predictors of higher symptom burden, supportive care needs, and quality of life (QoL), and examining their influence on overall survival (OS). Materials and methods: A population-based multi-institutional prospective cohort study of patients with PDAC was conducted between 2018 and 2021 by the Florida Pancreas Collaborative. Leveraging patient-reported data and laboratory values, participants were classified at baseline into four stages [non-cachexia (NCa), pre-cachexia (PCa), cachexia (Ca), and refractory cachexia (RCa)]. Multivariate regression, Kaplan Meier analyses, and Cox regression were conducted to evaluate associations. Results: CC stage was estimated for 309 PDAC cases (156 females, 153 males). The overall prevalence of NCa, PCa, Ca, and RCa was 12.9%, 24.6%, 54.1%, and 8.4%, respectively. CC prevalence across all CC stages was highest for males and racial and ethnic minorities. Criteria differentiated NCa cases from other groups, but did not distinguish PCa from Ca. The most frequently reported symptoms included weight loss, fatigue, pain, anxiety, and depression, with pain significantly worsening over time. The greatest supportive care needs included emotional and physical domains. Males, Black people, and those with RCa had the worst OS. Conclusions: Using clinical, nutritional, and functional criteria, nearly one-quarter of the PDAC cases in our diverse, multi-institutional cohort had PCa and 62.5% had Ca or RCa at the time of diagnosis. The PCa estimate is higher than that reported in prior studies. We recommend these criteria be used to aid in CC classification, monitoring, and management of all incident PDAC cases. Findings also highlight the recommendation for continued emotional support, assistance in alleviating pain, and supportive care needs throughout the PDAC treatment journey.

Cell-specific nanoengineering strategy disrupts tolerogenic signaling from myeloid-derived suppressor cells to invigorate antitumor immunity in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by intratumoral abundance of neutrophilic/polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) which inhibit T-cell function through JAK2/STAT3-regulated arginase activity. To overcome limitations of systemic inhibition of PMN-MDSCs in cancer-bearing patients-i.e., neutropenia and compensatory myelopoietic adaptations-we develop a nanoengineering strategy to target cell-specific signaling exclusively in PMN-MDSCs without provoking neutropenia. We conjugate a chemically modified small-molecule inhibitor of MDSC-surface receptor CXCR2 (AZD5069) with polyethylene glycol (PEG) and chemically graft AZD5069-PEG constructs onto amphiphilic polysaccharide derivatives to engineer CXCR2-homing nanoparticles (CXCR2-NP). Cy5.5 dye-loaded CXCR2-NP showed near-exclusive uptake in PMN-MDSCs compared with PDAC tumor-cells, cancer-associated fibroblasts, and macrophages. Encapsulation of JAK2/STAT3i Ruxolitinib (CXCR2-NP Ruxo ) resulted in more durable attenuation in STAT3-regulated arginase activity from PMN-MDSCs and induction of cytolytic T-cell activity vs. free Ruxolitinib in-vitro and in-vivo . Cell-specific delivery of payloads via CXCR2-homing immunonanoparticles represents a novel strategy to disrupt MDSC-mediated immunosuppression and invigorate antitumor immunity in PDAC.

The Pancreatic Cancer Early Detection (PRECEDE) Study is a Global Effort to Drive Early Detection: Baseline Imaging Findings in High-Risk Individuals.

BACKGROUND: Pancreatic adenocarcinoma (PC) is a highly lethal malignancy with a survival rate of only 12%. Surveillance is recommended for high-risk individuals (HRIs), but it is not widely adopted. To address this unmet clinical need and drive early diagnosis research, we established the Pancreatic Cancer Early Detection (PRECEDE) Consortium. METHODS: PRECEDE is a multi-institutional international collaboration that has undertaken an observational prospective cohort study. Individuals (aged 18-90 years) are enrolled into 1 of 7 cohorts based on family history and pathogenic germline variant (PGV) status. From April 1, 2020, to November 21, 2022, a total of 3,402 participants were enrolled in 1 of 7 study cohorts, with 1,759 (51.7%) meeting criteria for the highest-risk cohort (Cohort 1). Cohort 1 HRIs underwent germline testing and pancreas imaging by MRI/MR-cholangiopancreatography or endoscopic ultrasound. RESULTS: A total of 1,400 participants in Cohort 1 (79.6%) had completed baseline imaging and were subclassified into 3 groups based on familial PC (FPC; n=670), a PGV and FPC (PGV+/FPC+; n=115), and a PGV with a pedigree that does not meet FPC criteria (PGV+/FPC-; n=615). One HRI was diagnosed with stage IIB PC on study entry, and 35.1% of HRIs harbored pancreatic cysts. Increasing age (odds ratio, 1.05; P<.001) and FPC group assignment (odds ratio, 1.57; P<.001; relative to PGV+/FPC-) were independent predictors of harboring a pancreatic cyst. CONCLUSIONS: PRECEDE provides infrastructure support to increase access to clinical surveillance for HRIs worldwide, while aiming to drive early PC detection advancements through longitudinal standardized clinical data, imaging, and biospecimen captures. Increased cyst prevalence in HRIs with FPC suggests that FPC may infer distinct biological processes. To enable the development of PC surveillance approaches better tailored to risk category, we recommend adoption of subclassification of HRIs into FPC, PGV+/FPC+, and PGV+/FPC- risk groups by surveillance protocols.

Tumor Cell-Intrinsic p38 MAPK Signaling Promotes IL1α-Mediated Stromal Inflammation and Therapeutic Resistance in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a KRAS-driven inflammatory program and a desmoplastic stroma, which contribute to the profoundly chemoresistant phenotype. The tumor stroma contains an abundance of cancer-associated fibroblasts (CAF), which engage in extensive paracrine cross-talk with tumor cells to perpetuate protumorigenic inflammation. IL1α, a pleiotropic, tumor cell-derived cytokine, plays a critical role in shaping the stromal landscape. To provide insights into the molecular mechanisms regulating IL1A expression in PDAC, we performed transcriptional profiling of The Cancer Genome Atlas datasets and pharmacologic screening in PDAC cells and identified p38α MAPK as a key positive regulator of IL1A expression. Both genetic and pharmacologic inhibition of p38 MAPK significantly diminished IL1α production in vitro. Chromatin- and coimmunoprecipitation analyses revealed that p38 MAPK coordinates the transcription factors Sp1 and the p65 subunit of NFκB to drive IL1A overexpression. Single-cell RNA sequencing of a highly desmoplastic murine PDAC model, Ptf1aCre/+; LSL-KrasG12D/+; Tgfbr2flox/flox (PKT), confirmed that p38 MAPK inhibition significantly decreases tumor cell-derived Il1a and attenuates the inflammatory CAF phenotype in a paracrine IL1α-dependent manner. Furthermore, p38 MAPK inhibition favorably modulated intratumoral immunosuppressive myeloid populations and augmented chemotherapeutic efficacy to substantially reduce tumor burden and improve overall survival in PKT mice. These findings illustrate a cellular mechanism of tumor cell-intrinsic p38-p65/Sp1-IL1α signaling that is responsible for sustaining stromal inflammation and CAF activation, offering an attractive therapeutic approach to enhance chemosensitivity in PDAC. SIGNIFICANCE: Inhibition of p38 MAPK suppresses tumor cell-derived IL1α and attenuates the inflammatory stroma and immunosuppressive tumor microenvironment to overcome chemotherapeutic resistance in pancreatic cancer.

Malignant Solid Pseudopapillary Neoplasm of the Pancreas: An Orthogonal Analysis.

BACKGROUND: Pancreatic solid pseudopapillary neoplasms (SPN) are generally indolent; however, some patients present with "malignant" SPN. An orthogonal analysis of multiple datasets was performed to investigate the utility of complete surgical resection (CSR) for malignant SPN. METHODS: A systematic review was performed for cases of malignant SPN, defined as T4, N1, and/or M1. Malignant SPN was analyzed within the National Cancer Database (NCDB) and compared with T1-3N0M0 SPN. Predictors of malignant SPN were assessed, and treatments were analyzed by using survival analysis. RESULTS: The systematic review yielded 164 cases of malignant SPN. Of 31 children, only one died due to malignant SPN. Among adults, CSR was associated with improved disease-specific survival (DSS) (P = 0.0002). Chemotherapy did not improve malignant SPN DSS, whether resected (P = 0.8485) or not (P = 0.2219). Of 692 adults with SPN within the NCDB, 93 (13.4%) had malignant SPN. Pancreatic head location (odds ratio [OR] 2.174; 95% confidence interval [CI] 1.136-4.166; P = 0.0186) and tumor size (OR 1.154; 95% CI 1.079-1.235; P < 0.0001) associated with the malignant phenotype. Malignant SPN predicted decreased overall survival (OS) compared with T1-3N0M0 disease (P < 0.0001). Resected malignant SPN demonstrated improved OS (P < 0.0001), including resected stage IV malignant SPN (P = 0.0003). Chemotherapy did not improve OS for malignant SPN, whether resected (P = 0.8633) or not (P = 0.5734). Within a multivariable model, resection was associated with decreased hazard of death (hazard ratio 0.090; 95% CI 0.030-0.261; P < 0.0001). CONCLUSIONS: Approximately 13% of patients with SPN present with a malignant phenotype. Pediatric cases may be less aggressive. Resection may improve survival for malignant SPN, which does not appear chemosensitive.

Multiomic Characterization Reveals a Distinct Molecular Landscape in Young-Onset Pancreatic Cancer.

PURPOSE: Using a real-world database with matched genomic-transcriptomic molecular data, we sought to characterize the distinct molecular correlates underlying clinical differences between patients with young-onset pancreatic cancer (YOPC; younger than 50 years) and patients with average-onset pancreatic cancer (AOPC; 70 years and older). METHODS: We analyzed matched whole-transcriptome and DNA sequencing data from 2,430 patient samples (YOPC, n = 292; AOPC, n = 2,138) from the Caris Life Sciences database (Phoenix, AZ). Immune deconvolution was performed using the quanTIseq pipeline. Overall survival (OS) data were obtained from insurance claims (n = 4,928); Kaplan-Meier estimates were calculated for age- and molecularly defined cohorts. Significance was determined as FDR-corrected P values (Q) < .05. RESULTS: Patients with YOPC had higher proportions of mismatch repair-deficient/microsatellite instability-high, BRCA2-mutant, and PALB2-mutant tumors compared with patients with AOPC, but fewer SMAD4-, RNF43-, CDKN2A-, and SF3B1-mutant tumors. Notably, patients with YOPC demonstrated significantly lower incidence of KRAS mutations compared with patients with AOPC (81.3% v 90.9%; Q = .004). In the KRAS wild-type subset (n = 227), YOPC tumors demonstrated fewer TP53 mutations and were more likely driven by NRG1 and MET fusions, whereas BRAF fusions were exclusively observed in patients with AOPC. Immune deconvolution revealed significant enrichment of natural killer cells, CD8+ T cells, monocytes, and M2 macrophages in patients with YOPC relative to patients with AOPC, which corresponded with lower rates of HLA-DPA1 homozygosity. There was an association with improved OS in patients with YOPC compared with patients with AOPC with KRAS wild-type tumors (median, 16.2 [YOPC-KRASWT] v 10.6 [AOPC-KRASWT] months; P = .008) but not KRAS-mutant tumors (P = .084). CONCLUSION: In this large, real-world multiomic characterization of age-stratified molecular differences in pancreatic ductal adenocarcinoma, YOPC is associated with a distinct molecular landscape that has prognostic and therapeutic implications.

Remodeling of Stromal Immune Microenvironment by Urolithin A Improves Survival with Immune Checkpoint Blockade in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a significant contributor to cancer-related morbidity and mortality, and it is known for its resistance to conventional treatment regimens, including chemotherapy and immune checkpoint blockade (ICB)-based therapies. We have previously shown that Urolithin A (Uro A), a gut microbial metabolite derived from pomegranates, can target and inhibit KRAS-dependent PI3K/AKT/mTOR signaling pathways to overcome therapeutic resistance and improve survival in PDAC. However, the effect of Uro A on the tumor immune microenvironment and its ability to enhance ICB efficacy has not been explored. This study demonstrates that Uro A treatment reduces stromal fibrosis and reinvigorates the adaptive T-cell immune response to overcome resistance to PD-1 blockade in a genetically engineered mouse model (GEMM) of PDAC. Flow cytometric-based analysis of Uro A-treated mouse tumors revealed a significant attenuation of immunosuppressive tumor-associated M2-like macrophages with a concurrent increase in the infiltration of CD4+ and CD8+ T cells with memory-like phenotype along with reduced expression of the exhaustion-associated protein, PD-1. Importantly, the combination of Uro A treatment with anti-PD-1 immunotherapy promoted enhancement of the antitumor response with increased infiltration of CD4+ Th1 cells, ultimately resulting in a remarkable improvement in overall survival in GEMM of PDAC. Overall, our findings provide preclinical evidence for the potential of Uro A as a novel therapeutic agent to increase sensitivity to immunotherapy in PDAC and warrant further mechanistic exploration in preclinical and clinical studies. Significance: Immunotherapeutic agents are ineffective against pancreatic cancer, mainly due to the immunosuppressive tumor microenvironment and stromal desmoplasia. Our current study demonstrates the therapeutic utility of a novel gut microbial metabolite, Uro A, to remodel the stromal-immune microenvironment and improve overall survival with anti-PD-1 therapy in pancreatic cancer.

Multi-omic characterization reveals a distinct molecular landscape in young-onset pancreatic cancer.

Purpose: Using a real-world database with matched genomic-transcriptomic molecular data, we sought to characterize the distinct molecular correlates underlying clinical differences between young-onset pancreatic cancer (YOPC; <50-yrs.) and average-onset pancreatic cancer (AOPC; ≥70-yrs.) patients. Methods: We analyzed matched whole-transcriptome and DNA sequencing data from 2430 patient samples (YOPC, n=292; AOPC, n=2138) from the Caris Life Sciences database (Phoenix, AZ). Immune deconvolution was performed using the quanTIseq pipeline. Overall survival (OS) data was obtained from insurance claims (n=4928); Kaplan-Meier estimates were calculated for age-and molecularly-defined cohorts. Significance was determined as FDR-corrected P -values ( Q )<0.05. Results: YOPC patients had higher proportions of mismatch repair-deficient (dMMR)/microsatellite instability-high (MSI-H), BRCA2 -mutant, and PALB2 -mutant tumors compared with AOPC patients, but fewer SMAD4-, RNF43-, CDKN2A- , and SF3B1- mutant tumors. Notably, YOPC patients demonstrated significantly lower incidence of KRAS mutations compared with AOPC patients (81.3% vs. 90.9%; Q =0.004). In the KRAS- wildtype subset (n=227), YOPC tumors demonstrated fewer TP53 mutations and were more likely driven by NRG1 and MET fusions, while BRAF fusions were exclusively observed in AOPC patients. Immune deconvolution revealed significant enrichment of natural killer (NK) cells, CD8 + T cells, monocytes, and M2 macrophages in YOPC patients relative to AOPC patients, which corresponded with lower rates of HLA-DPA1 homozygosity. There was an association with improved OS in YOPC patients compared with AOPC patients with KRAS -wildtype tumors (median 16.2 [YOPC- KRAS WT ] vs. 10.6 [AOPC- KRAS WT ] months; P =0.008) but not KRAS -mutant tumors ( P =0.084). Conclusion: In this large, real-world multi-omic characterization of age-stratified molecular differences in PDAC, YOPC is associated with a distinct molecular landscape that has prognostic and therapeutic implications.

Cell-Autonomous Cxcl1 Sustains Tolerogenic Circuitries and Stromal Inflammation via Neutrophil-Derived TNF in Pancreatic Cancer.

We have shown that KRAS-TP53 genomic coalteration is associated with immune-excluded microenvironments, chemoresistance, and poor survival in pancreatic ductal adenocarcinoma (PDAC) patients. By treating KRAS-TP53 cooperativity as a model for high-risk biology, we now identify cell-autonomous Cxcl1 as a key mediator of spatial T-cell restriction via interactions with CXCR2+ neutrophilic myeloid-derived suppressor cells in human PDAC using imaging mass cytometry. Silencing of cell-intrinsic Cxcl1 in LSL-KrasG12D/+;Trp53R172H/+;Pdx-1Cre/+(KPC) cells reprograms the trafficking and functional dynamics of neutrophils to overcome T-cell exclusion and controls tumor growth in a T cell-dependent manner. Mechanistically, neutrophil-derived TNF is a central regulator of this immunologic rewiring, instigating feed-forward Cxcl1 overproduction from tumor cells and cancer-associated fibroblasts (CAF), T-cell dysfunction, and inflammatory CAF polarization via transmembrane TNF-TNFR2 interactions. TNFR2 inhibition disrupts this circuitry and improves sensitivity to chemotherapy in vivo. Our results uncover cancer cell-neutrophil cross-talk in which context-dependent TNF signaling amplifies stromal inflammation and immune tolerance to promote therapeutic resistance in PDAC. SIGNIFICANCE: By decoding connections between high-risk tumor genotypes, cell-autonomous inflammatory programs, and myeloid-enriched/T cell-excluded contexts, we identify a novel role for neutrophil-derived TNF in sustaining immunosuppression and stromal inflammation in pancreatic tumor microenvironments. This work offers a conceptual framework by which targeting context-dependent TNF signaling may overcome hallmarks of chemoresistance in pancreatic cancer. This article is highlighted in the In This Issue feature, p. 1275.

Racial Disparity in Pathologic Response following Neoadjuvant Chemotherapy in Resected Pancreatic Cancer: A Multi-Institutional Analysis from the Central Pancreatic Consortium.

BACKGROUND: Major pathologic response (MPR) following neoadjuvant therapy (NAT) in pancreatic ductal adenocarcinoma (PDAC) patients undergoing resection is associated with improved survival. We sought to determine whether racial disparities exist in MPR rates following NAT in patients with PDAC undergoing resection. METHODS: Patients with potentially operable PDAC receiving at least 2 cycles of neoadjuvant FOLFIRINOX or gemcitabine/nab-paclitaxel ± radiation followed by pancreatectomy (2010-2019) at 7 high-volume centers were reviewed. Self-reported race was dichotomized as Black and non-Black, and multivariable models evaluated the association between race and MPR (i.e., pathologic complete response [pCR] or near-pCR). Cox regression evaluated the association between race and disease-free (DFS) and overall survival (OS). RESULTS: Results of 486 patients who underwent resection following NAT (mFOLFIRINOX 56%, gemcitabine/nab-paclitaxel 25%, radiation 29%), 67 (13.8%) patients were Black. Black patients had lower CA19-9 at diagnosis (median 67 vs. 204 U/mL; P = 0.003) and were more likely to undergo mild/moderate chemotherapy dose modification (40 vs. 20%; P = 0.005) versus non-Black patients. Black patients had significantly lower rates of MPR compared with non-Black patients (13.4 vs. 25.8%; P = 0.039). Black race was independently associated with worse MPR (OR 0.26, 95% confidence interval [CI] 0.10-0.69) while controlling for NAT duration, CA19-9 dynamics, and chemotherapy modifications. There was no significant difference in DFS or OS between Black and non-Black cohorts. CONCLUSIONS: Black patients undergoing pancreatectomy appear less likely to experience MPR following NAT. The contribution of biologic and nonbiologic factors to reduced chemosensitivity in Black patients warrants further investigation.

Neutrophil-mediated fibroblast-tumor cell il-6/stat-3 signaling underlies the association between neutrophil-to-lymphocyte ratio dynamics and chemotherapy response in localized pancreatic cancer: A hybrid clinical-preclinical study.

Background: Partial/complete pathologic response following neoadjuvant chemotherapy (NAC) in pancreatic cancer (PDAC) patients undergoing pancreatectomy is associated with improved survival. We sought to determine whether neutrophil-to-lymphocyte ratio (NLR) dynamics predict pathologic response following chemotherapy in PDAC, and if manipulating NLR impacts chemosensitivity in preclinical models and uncovers potential mechanistic underpinnings underlying these effects. Methods: Pathologic response in PDAC patients (n=94) undergoing NAC and pancreatectomy (7/2015-12/2019) was dichotomized as partial/complete or poor/absent. Bootstrap-validated multivariable models assessed associations between pre-chemotherapy NLR (%neutrophils÷%lymphocytes) or NLR dynamics during chemotherapy (ΔNLR = pre-surgery-pre-chemotherapy NLR) and pathologic response, disease-free survival (DFS), and overall survival (OS). To preclinically model effects of NLR attenuation on chemosensitivity, Ptf1aCre/+; KrasLSL-G12D/+;Tgfbr2flox/flox (PKT) mice and C57BL/6 mice orthotopically injected with KrasLSL-G12D/+;Trp53LSL-R172H/+;Pdx1Cre(KPC) cells were randomized to vehicle, gemcitabine/paclitaxel alone, and NLR-attenuating anti-Ly6G with/without gemcitabine/paclitaxel treatment. Results: In 94 PDAC patients undergoing NAC (median:4 months), pre-chemotherapy NLR (p<0.001) and ΔNLR attenuation during NAC (p=0.002) were independently associated with partial/complete pathologic response. An NLR score = pre-chemotherapy NLR+ΔNLR correlated with DFS (p=0.006) and OS (p=0.002). Upon preclinical modeling, combining NLR-attenuating anti-Ly6G treatment with gemcitabine/paclitaxel-compared with gemcitabine/paclitaxel or anti-Ly6G alone-not only significantly reduced tumor burden and metastatic outgrowth, but also augmented tumor-infiltrating CD107a+-degranulating CD8+ T-cells (p<0.01) while dampening inflammatory cancer-associated fibroblast (CAF) polarization (p=0.006) and chemoresistant IL-6/STAT-3 signaling in vivo. Neutrophil-derived IL-1ß emerged as a novel mediator of stromal inflammation, inducing inflammatory CAF polarization and CAF-tumor cell IL-6/STAT-3 signaling in ex vivo co-cultures. Conclusions: Therapeutic strategies to mitigate neutrophil-CAF-tumor cell IL-1ß/IL-6/STAT-3 signaling during NAC may improve pathologic responses and/or survival in PDAC. Funding: Supported by KL2 career development grant by Miami CTSI under NIH Award UL1TR002736, Stanley Glaser Foundation, American College of Surgeons Franklin Martin Career Development Award, and Association for Academic Surgery Joel J. Roslyn Faculty Award (to J. Datta); NIH R01 CA161976 (to N.B. Merchant); and NCI/NIH Award P30CA240139 (to J. Datta and N.B. Merchant).

Combined MEK and STAT3 Inhibition Uncovers Stromal Plasticity by Enriching for Cancer-Associated Fibroblasts With Mesenchymal Stem Cell-Like Features to Overcome Immunotherapy Resistance in Pancreatic Cancer.

BACKGROUND & AIMS: We have shown that reciprocally activated rat sarcoma (RAS)/mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK) and Janus kinase/signal transducer and activator of transcription 3 (STAT3) pathways mediate therapeutic resistance in pancreatic ductal adenocarcinoma (PDAC), while combined MEK and STAT3 inhibition (MEKi+STAT3i) overcomes such resistance and alters stromal architecture. We now determine whether MEKi+STAT3i reprograms the cancer-associated fibroblast (CAF) and immune microenvironment to overcome resistance to immune checkpoint inhibition in PDAC. METHODS: CAF and immune cell transcriptomes in MEKi (trametinib)+STAT3i (ruxolitinib)-treated vs vehicle-treated Ptf1aCre/+;LSL-KrasG12D/+;Tgfbr2flox/flox (PKT) tumors were examined via single-cell RNA sequencing (scRNAseq). Clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeats associated protein 9 silencing of CAF-restricted Map2k1/Mek1 or Stat3, or both, enabled interrogation of CAF-dependent effects on immunologic remodeling in orthotopic models. Tumor growth, survival, and immune profiling via mass cytometry by time-of-flight were examined in PKT mice treated with vehicle, anti-programmed cell death protein 1 (PD-1) monotherapy, and MEKi+STAT3i combined with anti-PD1. RESULTS: MEKi+STAT3i attenuates Il6/Cxcl1-expressing proinflammatory and Lrrc15-expressing myofibroblastic CAF phenotypes while enriching for Ly6a/Cd34-expressing CAFs exhibiting mesenchymal stem cell-like features via scRNAseq in PKT mice. This CAF plasticity is associated with M2-to-M1 reprogramming of tumor-associated macrophages, and enhanced trafficking of cluster of differentiation 8+ T cells, which exhibit distinct effector transcriptional programs. These MEKi+STAT3i-induced effects appear CAF-dependent, because CAF-restricted Mek1/Stat3 silencing mitigates inflammatory-CAF polarization and myeloid infiltration in vivo. Addition of MEKi+STAT3i to PD-1 blockade not only dramatically improves antitumor responses and survival in PKT mice but also augments recruitment of activated/memory T cells while improving their degranulating and cytotoxic capacity compared with anti-PD-1 monotherapy. Importantly, treatment of a patient who has chemotherapy-refractory metastatic PDAC with MEKi (trametinib), STAT3i (ruxolitinib), and PD-1 inhibitor (nivolumab) yielded clinical benefit. CONCLUSIONS: Combined MEKi+STAT3i mitigates stromal inflammation and enriches for CAF phenotypes with mesenchymal stem cell-like properties to overcome immunotherapy resistance in PDAC.

Distinct mechanisms of innate and adaptive immune regulation underlie poor oncologic outcomes associated with KRAS-TP53 co-alteration in pancreatic cancer.

Co-occurrent KRAS and TP53 mutations define a majority of patients with pancreatic ductal adenocarcinoma (PDAC) and define its pro-metastatic proclivity. Here, we demonstrate that KRAS-TP53 co-alteration is associated with worse survival compared with either KRAS-alone or TP53-alone altered PDAC in 245 patients with metastatic disease treated at a tertiary referral cancer center, and validate this observation in two independent molecularly annotated datasets. Compared with non-TP53 mutated KRAS-altered tumors, KRAS-TP53 co-alteration engenders disproportionately innate immune-enriched and CD8+ T-cell-excluded immune signatures. Leveraging in silico, in vitro, and in vivo models of human and murine PDAC, we discover a novel intersection between KRAS-TP53 co-altered transcriptomes, TP63-defined squamous trans-differentiation, and myeloid-cell migration into the tumor microenvironment. Comparison of single-cell transcriptomes between KRAS-TP53 co-altered and KRAS-altered/TP53WT tumors revealed cancer cell-autonomous transcriptional programs that orchestrate innate immune trafficking and function. Moreover, we uncover granulocyte-derived inflammasome activation and TNF signaling as putative paracrine mediators of innate immunoregulatory transcriptional programs in KRAS-TP53 co-altered PDAC. Immune subtyping of KRAS-TP53 co-altered PDAC reveals conflation of intratumor heterogeneity with progenitor-like stemness properties. Coalescing these distinct molecular characteristics into a KRAS-TP53 co-altered "immunoregulatory program" predicts chemoresistance in metastatic PDAC patients enrolled in the COMPASS trial, as well as worse overall survival.