Larger Tumor Size and Elevated Serum Chromogranin A Levels Predict Metastatic Disease on DOTATATE Imaging in Patients with Gastroenteropancreatic Neuroendocrine Tumors.

PURPOSE: DOTATATE PET/CT (DOTATATE) is superior to conventional imaging in detecting metastasis for gastroenteropancreatic neuroendocrine tumors (GEP-NETs). However, limited availability, high-cost, and additive radiation exposure necessitate guidelines for its use. This study seeks to investigate the relationship between clinical characteristics and metastasis on DOTATATE. METHODS: This was a retrospective analysis of 815 patients who underwent DOTATATE at UCLA from 2014 to 2022. After applying inclusion and exclusion criteria, the study cohort consisted of 163 patients with pathologically diagnosed GEP-NETs, who either underwent primary tumor resection within 1-year prior, or had not undergone resection at the time of DOTATATE imaging. The presence of metastasis was determined using DOTATATE. Fisher's exact test, chi-squared test, and Mann-Whitney test were conducted to compare intergroup difference. Multivariate analysis was performed to identify clinical characteristics associated with metastasis on DOTATATE. RESULTS: Of patients with GEP-NETs, 40.5% (n = 66) were diagnosed with metastases by using DOTATATE. Those with metastatic disease were more likely to exhibit a larger primary tumor size (median 3.4 vs. 1.2, cm, P < 0.001), elevated serum chromogranin A level (CgA, median 208 vs. 97, mg/ml, P = 0.005), and higher tumor grade (P < 0.001). Primary tumor size ≥2 cm and serum CgA level ≥150 ng/mL for metastatic disease had a sensitivity and specificity of 64% and 89%, and 72% and 59%, respectively. Multivariate analysis demonstrated that primary tumor size (≥2/<2, cm, odds ratio [OR] 47.90, P < 0.001), tumor functionality (functional/nonfunctional, adjusted OR 10.17 P = 0.008), serum CgA level (≥150/<150, ng/ml, OR 6.25, P = 0.005), and tumor grade G2 (G2/G1, OR 9.6, P < 0.001) were independently associated with metastases on DOTATATE. CONCLUSIONS: Among patients with GEP-NETs, primary tumor size ≥2 cm, serum CgA level ≥150 ng/mL, and tumor grade G2 are associated with an increased risk of metastases on DOTATATE, and these predictors may be helpful to identify patients where DOTATATE is indicated for complete staging.

Sendai virus is robust and consistent in delivering genes into human pancreatic cancer cells.

Background: Pancreatic ductal adenocarcinoma (PDAC) is a highly intratumorally heterogeneous disease that includes several subtypes and is highly plastic. Effective gene delivery to all PDAC cells is essential for modulating gene expression and identifying potential gene-based therapeutic targets in PDAC. Most current gene delivery systems for pancreatic cells are optimized for islet or acinar cells. Lentiviral vectors are the current main gene delivery vectors for PDAC, but their transduction efficiencies vary depending on pancreatic cell type, and are especially poor for the classical subtype of PDAC cells from both primary tumors and cell lines. Methods: We systemically compare transduction efficiencies of glycoprotein G of vesicular stomatitis virus (VSV-G)-pseudotyped lentiviral and Sendai viral vectors in human normal pancreatic ductal and PDAC cells. Results: We find that the Sendai viral vector gives the most robust gene delivery efficiency regardless of PDAC cell type. Therefore, we propose using Sendai viral vectors to transduce ectopic genes into PDAC cells.

Clinical and pathological factors associated with survival in patients with pancreatic cancer who receive adjuvant therapy after neoadjuvant therapy: A retrospective multi-institutional analysis.

BACKGROUND: Neoadjuvant therapy is being increasingly used for patients with pancreatic cancer. The role of adjuvant therapy in these patients is unclear. The purpose of this study was to identify clinical and pathologic characteristics that are associated with longer overall survival in patients with pancreatic cancer who receive adjuvant therapy after neoadjuvant therapy. METHODS: This study was conducted using multi-institutional data. All patients underwent surgery after at least 1 cycle of neoadjuvant therapy for pancreatic cancer. Patients who died within 3 months after surgery and were known to have distant metastasis or macroscopic residual disease were excluded. Mann-Whitney U test, χ2 analysis, Kaplan-Meier plot, and univariate and multivariate Cox regression analysis were performed as statistical analyses. RESULTS: In the present study, 529 patients with resected pancreatic cancer after neoadjuvant therapy were reviewed. For neoadjuvant therapy, 177 (33.5%) patients received neoadjuvant chemotherapy, and 352 (66.5%) patients received neoadjuvant chemoradiotherapy. The median duration of neoadjuvant therapy was 7.0 months (interquartile range, 5.0-8.7). Patients were followed for a median of 23.0 months after surgery. Adjuvant therapy was administered to 297 (56.1%) patients and was not associated with longer overall survival for the entire cohort (24 vs 22 months, P = .31). Interaction analysis showed that adjuvant therapy was associated with longer overall survival in patients who received less than 4 months neoadjuvant therapy (hazard ratio 0.40; 95% confidence interval 0.17-0.95; P = .03) or who had microscopic margin positive surgical resections (hazard ratio 0.56; 95% confidence interval 0.33-0.93; P = .03). CONCLUSION: In this retrospective study, there was a survival benefit associated with adjuvant therapy for patients who received less than 4 months of neoadjuvant therapy or had microscopic positive margins.

MEK Inhibition Sensitizes Pancreatic Cancer to STING Agonism by Tumor Cell-intrinsic Amplification of Type I IFN Signaling.

PURPOSE: Stimulator of interferon genes (STING) agonists are currently in development for treatment of solid tumors, including pancreatic ductal adenocarcinoma (PDAC). Response rates to STING agonists alone have been promising yet modest, and combination therapies will likely be required to elicit their full potency. We sought to identify combination therapies and mechanisms that augment the tumor cell-intrinsic effect of therapeutically relevant STING agonists apart from their known effects on tumor immunity. EXPERIMENTAL DESIGN: We screened 430 kinase inhibitors to identify synergistic effectors of tumor cell death with diABZI, an intravenously administered and systemically available STING agonist. We deciphered the mechanisms of synergy with STING agonism that cause tumor cell death in vitro and tumor regression in vivo. RESULTS: We found that MEK inhibitors caused the greatest synergy with diABZI and that this effect was most pronounced in cells with high STING expression. MEK inhibition enhanced the ability of STING agonism to induce type I IFN-dependent cell death in vitro and tumor regression in vivo. We parsed NFκB-dependent and NFκB-independent mechanisms that mediate STING-driven type I IFN production and show that MEK signaling inhibits this effect by suppressing NFκB activation. CONCLUSIONS: Our results highlight the cytotoxic effects of STING agonism on PDAC cells that are independent of tumor immunity and that these therapeutic benefits of STING agonism can be synergistically enhanced by MEK inhibition.

Generation of liver metastases in a mouse model using ultrasound-guided intravenous injection.

Here, we present a protocol to generate a murine model of liver metastasis by directly injecting tumor cells into the portal vein under ultrasound guidance. We describe steps for animal and cell preparation and two techniques for injecting tumor cells. One technique is freehand, while the other technique is device-assisted using a 3D-printed prototype device. Finally, we describe tumor surveillance with bioluminescent imaging.

T-cell Dysfunction upon Expression of MYC with Altered Phosphorylation at Threonine 58 and Serine 62.

As a transcription factor that promotes cell growth, proliferation, and apoptosis, c-MYC (MYC) expression in the cell is tightly controlled. Disruption of oncogenic signaling pathways in human cancers can increase MYC protein stability, due to altered phosphorylation ratios at two highly conserved sites, Threonine 58 (T58) and Serine 62 (S62). The T58 to Alanine mutant (T58A) of MYC mimics the stabilized, S62 phosphorylated, and highly oncogenic form of MYC. The S62A mutant is also stabilized, lacks phosphorylation at both Serine 62 and Threonine 58, and has been shown to be nontransforming in vitro. However, several regulatory proteins are reported to associate with MYC lacking phosphorylation at S62 and T58, and the role this form of MYC plays in MYC transcriptional output and in vivo oncogenic function is understudied. We generated conditional c-Myc knock-in mice in which the expression of wild-type MYC (MYCWT), the T58A mutant (MYCT58A), or the S62A mutant (MYCS62A) with or without expression of endogenous Myc is controlled by the T-cell-specific Lck-Cre recombinase. MYCT58A expressing mice developed clonal T-cell lymphomas with 100% penetrance and conditional knock-out of endogenous Myc accelerated this lymphomagenesis. In contrast, MYCS62A mice developed clonal T-cell lymphomas at a much lower penetrance, and the loss of endogenous MYC reduced the penetrance while increasing the appearance of a non-transgene driven B-cell lymphoma with splenomegaly. Together, our study highlights the importance of regulated phosphorylation of MYC at T58 and S62 for T-cell transformation. IMPLICATIONS: Dysregulation of phosphorylation at conserved T58 and S62 residues of MYC differentially affects T-cell development and lymphomagenesis.

Reprogramming of nucleotide metabolism by interferon confers dependence on the replication stress response pathway in pancreatic cancer cells.

We determine that type I interferon (IFN) response biomarkers are enriched in a subset of pancreatic ductal adenocarcinoma (PDAC) tumors; however, actionable vulnerabilities associated with IFN signaling have not been systematically defined. Integration of a phosphoproteomic analysis and a chemical genomics synergy screen reveals that IFN activates the replication stress response kinase ataxia telangiectasia and Rad3-related protein (ATR) in PDAC cells and sensitizes them to ATR inhibitors. IFN triggers cell-cycle arrest in S-phase, which is accompanied by nucleotide pool insufficiency and nucleoside efflux. In combination with IFN, ATR inhibitors induce lethal DNA damage and downregulate nucleotide biosynthesis. ATR inhibition limits the growth of PDAC tumors in which IFN signaling is driven by stimulator of interferon genes (STING). These results identify a cross talk between IFN, DNA replication stress response networks, and nucleotide metabolism while providing the rationale for targeted therapeutic interventions that leverage IFN signaling in tumors.

High-content single-cell combinatorial indexing.

Single-cell combinatorial indexing (sci) with transposase-based library construction increases the throughput of single-cell genomics assays but produces sparse coverage in terms of usable reads per cell. We develop symmetrical strand sci ('s3'), a uracil-based adapter switching approach that improves the rate of conversion of source DNA into viable sequencing library fragments following tagmentation. We apply this chemistry to assay chromatin accessibility (s3-assay for transposase-accessible chromatin, s3-ATAC) in human cortical and mouse whole-brain tissues, with mouse datasets demonstrating a six- to 13-fold improvement in usable reads per cell compared with other available methods. Application of s3 to single-cell whole-genome sequencing (s3-WGS) and to whole-genome plus chromatin conformation (s3-GCC) yields 148- and 14.8-fold improvements, respectively, in usable reads per cell compared with sci-DNA-sequencing and sci-HiC. We show that s3-WGS and s3-GCC resolve subclonal genomic alterations in patient-derived pancreatic cancer cell lines. We expect that the s3 platform will be compatible with other transposase-based techniques, including sci-MET or CUT&Tag.

Leukocyte Heterogeneity in Pancreatic Ductal Adenocarcinoma: Phenotypic and Spatial Features Associated with Clinical Outcome.

Immunotherapies targeting aspects of T cell functionality are efficacious in many solid tumors, but pancreatic ductal adenocarcinoma (PDAC) remains refractory to these treatments. Deeper understanding of the PDAC immune ecosystem is needed to identify additional therapeutic targets and predictive biomarkers for therapeutic response and resistance monitoring. To address these needs, we quantitatively evaluated leukocyte contexture in 135 human PDACs at single-cell resolution by profiling density and spatial distribution of myeloid and lymphoid cells within histopathologically defined regions of surgical resections from treatment-naive and presurgically (neoadjuvant)-treated patients and biopsy specimens from metastatic PDAC. Resultant data establish an immune atlas of PDAC heterogeneity, identify leukocyte features correlating with clinical outcomes, and, through an in silico study, provide guidance for use of PDAC tissue microarrays to optimally measure intratumoral immune heterogeneity. Atlas data have direct applicability as a reference for evaluating immune responses to investigational neoadjuvant PDAC therapeutics where pretherapy baseline specimens are not available. SIGNIFICANCE: We provide a phenotypic and spatial immune atlas of human PDAC identifying leukocyte composition at steady state and following standard neoadjuvant therapies. These data have broad utility as a resource that can inform on leukocyte responses to emerging therapies where baseline tissues were not acquired.This article is highlighted in the In This Issue feature, p. 1861.

Acidic fibroblast growth factor underlies microenvironmental regulation of MYC in pancreatic cancer.

Despite a critical role for MYC as an effector of oncogenic RAS, strategies to target MYC activity in RAS-driven cancers are lacking. In genetically engineered mouse models of lung and pancreatic cancer, oncogenic KRAS is insufficient to drive tumorigenesis, while addition of modest MYC overexpression drives robust tumor formation, suggesting that mechanisms beyond the RAS pathway play key roles in MYC regulation and RAS-driven tumorigenesis. Here we show that acidic fibroblast growth factor (FGF1) derived from cancer-associated fibroblasts (CAFs) cooperates with cancer cell-autonomous signals to increase MYC level, promoter occupancy, and activity. FGF1 is necessary and sufficient for paracrine regulation of MYC protein stability, signaling through AKT and GSK-3ß to increase MYC half-life. Patient specimens reveal a strong correlation between stromal CAF content and MYC protein level in the neoplastic compartment, and identify CAFs as the specific source of FGF1 in the tumor microenvironment. Together, our findings demonstrate that MYC is coordinately regulated by cell-autonomous and microenvironmental signals, and establish CAF-derived FGF1 as a novel paracrine regulator of oncogenic transcription.

Innate αß T Cells Mediate Antitumor Immunity by Orchestrating Immunogenic Macrophage Programming.

Unconventional T-lymphocyte populations are emerging as important regulators of tumor immunity. Despite this, the role of TCRαß+CD4-CD8-NK1.1- innate αß T cells (iαßT) in pancreatic ductal adenocarcinoma (PDA) has not been explored. We found that iαßTs represent ∼10% of T lymphocytes infiltrating PDA in mice and humans. Intratumoral iαßTs express a distinct T-cell receptor repertoire and profoundly immunogenic phenotype compared with their peripheral counterparts and conventional lymphocytes. iαßTs comprised ∼75% of the total intratumoral IL17+ cells. Moreover, iαßT-cell adoptive transfer is protective in both murine models of PDA and human organotypic systems. We show that iαßT cells induce a CCR5-dependent immunogenic macrophage reprogramming, thereby enabling marked CD4+ and CD8+ T-cell expansion/activation and tumor protection. Collectively, iαßTs govern fundamental intratumoral cross-talk between innate and adaptive immune populations and are attractive therapeutic targets. SIGNIFICANCE: We found that iαßTs are a profoundly activated T-cell subset in PDA that slow tumor growth in murine and human models of disease. iαßTs induce a CCR5-dependent immunogenic tumor-associated macrophage program, T-cell activation and expansion, and should be considered as novel targets for immunotherapy.See related commentary by Banerjee et al., p. 1164.This article is highlighted in the In This Issue feature, p. 1143.

A Stromal Lysolipid-Autotaxin Signaling Axis Promotes Pancreatic Tumor Progression.

Pancreatic ductal adenocarcinoma (PDAC) develops a pronounced stromal response reflecting an aberrant wound-healing process. This stromal reaction features transdifferentiation of tissue-resident pancreatic stellate cells (PSC) into activated cancer-associated fibroblasts, a process induced by PDAC cells but of unclear significance for PDAC progression. Here, we show that PSCs undergo a dramatic lipid metabolic shift during differentiation in the context of pancreatic tumorigenesis, including remodeling of the intracellular lipidome and secretion of abundant lipids in the activated, fibroblastic state. Specifically, stroma-derived lysophosphatidylcholines support PDAC cell synthesis of phosphatidylcholines, key components of cell membranes, and also facilitate production of the potent wound-healing mediator lysophosphatidic acid (LPA) by the extracellular enzyme autotaxin, which is overexpressed in PDAC. The autotaxin-LPA axis promotes PDAC cell proliferation, migration, and AKT activation, and genetic or pharmacologic autotaxin inhibition suppresses PDAC growth in vivo. Our work demonstrates how PDAC cells exploit the local production of wound-healing mediators to stimulate their own growth and migration. SIGNIFICANCE: Our work highlights an unanticipated role for PSCs in producing the oncogenic LPA signaling lipid and demonstrates how PDAC tumor cells co-opt the release of wound-healing mediators by neighboring PSCs to promote their own proliferation and migration.See related commentary by Biffi and Tuveson, p. 578.This article is highlighted in the In This Issue feature, p. 565.

MYC regulates ductal-neuroendocrine lineage plasticity in pancreatic ductal adenocarcinoma associated with poor outcome and chemoresistance.

Intratumoral phenotypic heterogeneity has been described in many tumor types, where it can contribute to drug resistance and disease recurrence. We analyzed ductal and neuroendocrine markers in pancreatic ductal adenocarcinoma, revealing heterogeneous expression of the neuroendocrine marker Synaptophysin within ductal lesions. Higher percentages of Cytokeratin-Synaptophysin dual positive tumor cells correlate with shortened disease-free survival. We observe similar lineage marker heterogeneity in mouse models of pancreatic ductal adenocarcinoma, where lineage tracing indicates that Cytokeratin-Synaptophysin dual positive cells arise from the exocrine compartment. Mechanistically, MYC binding is enriched at neuroendocrine genes in mouse tumor cells and loss of MYC reduces ductal-neuroendocrine lineage heterogeneity, while deregulated MYC expression in KRAS mutant mice increases this phenotype. Neuroendocrine marker expression is associated with chemoresistance and reducing MYC levels decreases gemcitabine-induced neuroendocrine marker expression and increases chemosensitivity. Altogether, we demonstrate that MYC facilitates ductal-neuroendocrine lineage plasticity in pancreatic ductal adenocarcinoma, contributing to poor survival and chemoresistance.

Deep learning based Nucleus Classification in pancreas histological images.

Tumor specimens contain a variety of healthy cells as well as cancerous cells, and this heterogeneity underlies resistance to various cancer therapies. But this problem has not been thoroughly investigated until recently. Meanwhile, technological breakthroughs in imaging have led to an explosion of molecular and cellular profiling data from large numbers of samples, and modern machine learning approaches including deep learning have been shown to produce encouraging results by finding hidden structures and make accurate predictions. In this paper, we propose a Deep learning based Nucleus Classification (DeepNC) approach using paired histopathology and immunofluorescence images (for label), and demonstrate its classification prediction power. This method can solve current issue on discrepancy between genomic- or transcriptomic-based and pathology-based tumor purity estimates by improving histological evaluation. We also explain challenges in training a deep learning model for huge dataset.

Quantitative analysis of histological tissue image based on cytological profiles and spatial statistics.

The cellular heterogeneity and complex tissue architecture of most tumor samples is a major obstacle in image analysis on standard hematoxylin and eosin-stained (H&E) tissue sections. A mixture of cancer and normal cells complicates the interpretation of their cytological profiles. Furthermore, spatial arrangement and architectural organization of cells are generally not reflected in cellular characteristics analysis. To address these challenges, first we describe an automatic nuclei segmentation of H&E tissue sections. In the task of deconvoluting cellular heterogeneity, we adopt Landmark based Spectral Clustering (LSC) to group individual nuclei in such a way that nuclei in the same group are more similar. We next devise spatial statistics for analyzing spatial arrangement and organization, which are not detectable by individual cellular characteristics. Our quantitative, spatial statistics analysis could benefit H&E section analysis by refining and complementing cellular characteristics analysis.

The activities of MYC, MNT and the MAX-interactome in lymphocyte proliferation and oncogenesis.

The MYC family of proteins plays essential roles in embryonic development and in oncogenesis. Efforts over the past 30 years to define the transcriptional activities of MYC and how MYC functions to promote proliferation have produced evolving models of MYC function. One picture that has emerged of MYC and its partner protein MAX is of a transcription factor complex with a seemingly unique ability to stimulate the transcription of genes that are epigenetically poised for transcription and to amplify the transcription of actively transcribed genes. During lymphocyte activation, MYC is upregulated and stimulates a pro-proliferative program in part through the upregulation of a wide variety of metabolic effector genes that facilitate cell growth and cell cycle progression. MYC upregulation simultaneously sensitizes cells to apoptosis and activated lymphocytes and lymphoma cells have pro-survival attributes that allow MYC-driven proliferation to prevail. For example, the MAX-interacting protein MNT is upregulated in activated lymphocytes and was found to protect lymphocytes from MYC-dependent apoptosis. Here we review the activities of MYC, MNT and other MAX interacting proteins in the setting of T and B cell activation and oncogenesis. This article is part of a Special Issue entitled: Myc proteins in cell biology and pathology.

A critical role for Mnt in Myc-driven T-cell proliferation and oncogenesis.

Mnt (Max's next tango) is a Max-interacting transcriptional repressor that can antagonize both the proproliferative and proapoptotic functions of Myc in vitro. To ascertain the physiologically relevant functions of Mnt and to help define the relationship between Mnt and Myc in vivo, we generated a series of mouse strains in which Mnt was deleted in T cells in the absence of endogenous c-Myc or in the presence of ectopic c-Myc. We found that apoptosis caused by loss of Mnt did not require Myc but that ectopic Myc expression dramatically decreased the survival of both Mnt-deficient T cells in vivo and Mnt-deficient MEFs in vitro. Consequently, Myc-driven proliferative expansion of T cells in vitro and thymoma formation in vivo were prevented by the absence of Mnt. Consistent with T-cell models, mouse embryo fibroblasts (MEFs) lacking Mnt were refractory to oncogenic transformation by Myc. Tumor suppression caused by loss of Mnt was linked to increased apoptosis mediated by reactive oxygen species (ROS). Thus, although theoretically and experimentally a Myc antagonist, the dominant physiological role of Mnt appears to be suppression of apoptosis. Our results redefine the physiological relationship between Mnt and Myc and requirements for Myc-driven oncogenesis.

The role of senescence and prosurvival signaling in controlling the oncogenic activity of FGFR2 mutants associated with cancer and birth defects.

Mutations in fibroblast growth factor receptors (FGFRs) cause human birth defect syndromes and are associated with a variety of cancers. Although forced expression of mutant activated FGFRs has been shown to oncogenically transform some immortal cell types, their activity in primary cells remains unclear. Here, we show that birth defect and cancer-associated FGFR2 mutants promote DNA-damage signaling and p53-dependent senescence in primary mouse and human cells. Senescence promoted by FGFR mutants was associated with downregulation of c-Myc and forced expression of c-Myc facilitated senescence escape. Whereas c-Myc expression facilitated senescence bypass, mutant FGFR2 signaling suppressed c-Myc-dependent apoptosis and led to oncogenic transformation. Cells transformed by coexpression of a constitutively activated FGFR2 mutant plus c-Myc appeared to be become highly addicted to FGFR-dependent prosurvival activities, as small molecule inhibition of FGFR signaling resulted in robust p53-dependent apoptosis. Our data suggest that senescence-promoting activities of mutant FGFRs may normally limit their oncogenic potential and may be relevant to their ability to disrupt morphogenesis and cause birth defects. Our results also raise the possibility that cancers originating through a combination of constitutive FGFR activation and deregulated Myc expression may be particularly sensitive to small molecule inhibitors of FGF receptors.