Reversible chemoresistance of pancreatic cancer grown as spheroids.

Better in vitro models are needed to identify active drugs to treat pancreatic adenocarcinoma (PAC) patients. We used 3D hanging drop cultures to produce spheroids from five PAC cell lines and tested nine FDA-approved drugs in clinical use. All PAC cell lines in 2D culture were sensitive to three drugs (gemcitabine, docetaxel and nab-paclitaxel), however most PAC (4/5) 3D spheroids acquired profound chemoresistance even at 10 µM. In contrast, spheroids retained sensitivity to the investigational drug triptolide, which induced apoptosis. The acquired chemoresistance was also transiently retained when cells were placed back into 2D culture and six genes potentially associated with chemoresistance were identified by microarray and confirmed using quantitative RT-PCR. We demonstrate the additive effect of gemcitabine and erlotinib, from the 12 different combinations of nine drugs tested. This comprehensive study shows spheroids as a useful multicellular model of PAC for drug screening and elucidating the mechanism of chemoresistance.

3D histology reveals that immune response to pancreatic precancers is heterogeneous and depends on global pancreas structure.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal cancer for which few effective therapies exist. Immunotherapies specifically are ineffective in pancreatic cancer, in part due to its unique stromal and immune microenvironment. Pancreatic intraepithelial neoplasia, or PanIN, is the main precursor lesion to PDAC. Recently it was discovered that PanINs are remarkably abundant in the grossly normal pancreas, suggesting that the vast majority will never progress to cancer. Here, through construction of 48 samples of cm3-sized human pancreas tissue, we profiled the immune microenvironment of 1,476 PanINs in 3D and at single-cell resolution to better understand the early evolution of the pancreatic tumor microenvironment and to determine how inflammation may play a role in cancer progression. We found that bulk pancreatic inflammation strongly correlates to PanIN cell fraction. We found that the immune response around PanINs is highly heterogeneous, with distinct immune hotspots and cold spots that appear and disappear in a span of tens of microns. Immune hotspots generally mark locations of higher grade of dysplasia or locations near acinar atrophy. The immune composition at these hotspots is dominated by naïve, cytotoxic, and regulatory T cells, cancer associated fibroblasts, and tumor associated macrophages, with little similarity to the immune composition around less-inflamed PanINs. By mapping FOXP3+ cells in 3D, we found that regulatory T cells are present at higher density in larger PanIN lesions compared to smaller PanINs, suggesting that the early initiation of PanINs may not exhibit an immunosuppressive response. This analysis demonstrates that while PanINs are common in the pancreases of most individuals, inflammation may play a pivotal role, both at the bulk and the microscopic scale, in demarcating regions of significance in cancer progression.

Clinical Relevance of Cancerization of Ducts in Resected Pancreatic Ductal Adenocarcinoma.

OBJECTIVES: Although prevalent in 50%-90% of pancreatic ductal adenocarcinomas, the clinical relevance of "cancerization of ducts" (COD) remains unknown. METHODS: Pathologists retrospectively reviewed slides classifying prevalence of COD. Histopathological parameters, location of first recurrence, recurrence-free survival (RFS), and overall survival (OS) were collected from the institutional pancreatectomy registry. RESULTS: Among 311 pancreatic ductal adenocarcinomas, COD was present in 216 (69.5%) and more prevalent in the cohort that underwent upfront surgery (75.3% vs 63.1%, P = 0.019). Furthermore, COD was associated with female gender (P = 0.040), advanced T stage (P = 0.007), perineural invasion (P = 0.014), lymphovascular invasion (P = 0.025), and R1 margin (P = 0.009), but not N stage (P = 0.401) or tumor differentiation (P = 0.717). In multivariable regression, COD was associated with less liver recurrence (odds ratio, 0.44; P < 0.005). This association was driven by the cohort of patients who had received preoperative treatment (odds ratio, 0.18; P < 0.001). COD was not predictive for RFS or OS. CONCLUSIONS: Cancerization of ducts was not associated with RFS or OS. Currently underrecognized, standardized implementation into histopathological reports may have merit, and further mechanistic scientific experiments need to illuminate its clinical and biologic impact.

Benign Islet Cells Within Peripancreatic Lymph Nodes: A Potential Diagnostic Pitfall.

The presence of epithelial cells within lymph node parenchyma is typically indicative of a metastatic malignancy. However, there are rare instances in which non-neoplastic epithelial or epithelioid cells may be found within lymph nodes, either due to aberrant embryologic migration, mechanical displacement, or physiological trafficking. These can potentially lead to serious potential diagnostic pitfalls, as when such situations are encountered by surgical pathologists, there is substantial risk of overdiagnosing these as metastatic malignancy. Herein, we describe 2 cases of benign pancreatic islet cells within peripancreatic lymph nodes, and underscore the potential for misdiagnosis of this phenomenon as foci of metastatic well-differentiated neuroendocrine tumor. The benign nature of these intranodal islet cells was supported by: (1) the absence of a well-differentiated neuroendocrine tumor in the entirely submitted concomitant pancreatic resection specimen and (2) the presence of an admixture of insulin and glucagon expressing cells by immunohistochemistry in a distribution characteristic of non-neoplastic pancreatic islets. Both cases were incidental microscopic findings in pancreatic resections for intraductal papillary mucinous neoplasms that were previously biopsied and showed associated microscopic areas of fibrosis and chronic pancreatitis and thus this phenomenon may be related to mechanical displacement from prior injury and/or biopsy.

3D genomic mapping reveals multifocality of human pancreatic precancers.

Pancreatic intraepithelial neoplasias (PanINs) are the most common precursors of pancreatic cancer, but their small size and inaccessibility in humans make them challenging to study1. Critically, the number, dimensions and connectivity of human PanINs remain largely unknown, precluding important insights into early cancer development. Here, we provide a microanatomical survey of human PanINs by analysing 46 large samples of grossly normal human pancreas with a machine-learning pipeline for quantitative 3D histological reconstruction at single-cell resolution. To elucidate genetic relationships between and within PanINs, we developed a workflow in which 3D modelling guides multi-region microdissection and targeted and whole-exome sequencing. From these samples, we calculated a mean burden of 13 PanINs per cm3 and extrapolated that the normal intact adult pancreas harbours hundreds of PanINs, almost all with oncogenic KRAS hotspot mutations. We found that most PanINs originate as independent clones with distinct somatic mutation profiles. Some spatially continuous PanINs were found to contain multiple KRAS mutations; computational and in situ analyses demonstrated that different KRAS mutations localize to distinct cell subpopulations within these neoplasms, indicating their polyclonal origins. The extensive multifocality and genetic heterogeneity of PanINs raises important questions about mechanisms that drive precancer initiation and confer differential progression risk in the human pancreas. This detailed 3D genomic mapping of molecular alterations in human PanINs provides an empirical foundation for early detection and rational interception of pancreatic cancer.

Distinctive Pathology Associated With Focal Stenosis of the Main Pancreatic Duct Secondary to Remote Trauma: A Long-term Complication of Seat Belt Pancreatitis.

The radiologic finding of focal stenosis of the main pancreatic duct is highly suggestive of pancreatic cancer. Even in the absence of a mass lesion, focal duct stenosis can lead to surgical resection of the affected portion of the pancreas. We present four patients with distinctive pathology associated with non-neoplastic focal stenosis of the main pancreatic duct. The pathology included stenosis of the pancreatic duct accompanied by wavy, acellular, serpentine-like fibrosis, chronic inflammation with foreign body-type giant cell reaction, and calcifications. In all cases, the pancreas toward the tail of the gland had obstructive changes including acinar drop-out and interlobular and intralobular fibrosis. Three of the four patients had a remote history of major motor vehicle accidents associated with severe abdominal trauma. These results emphasize that blunt trauma can injure the pancreas and that this injury can result in long-term complications, including focal stenosis of the main pancreatic duct. Pathologists should be aware of the distinct pathology associated with remote trauma and, when the pathology is present, should elicit the appropriate clinical history.

Fibroblasts in the Aged Pancreas Drive Pancreatic Cancer Progression.

Pancreatic cancer is more prevalent in older individuals and often carries a poorer prognosis for them. The relationship between the microenvironment and pancreatic cancer is multifactorial, and age-related changes in nonmalignant cells in the tumor microenvironment may play a key role in promoting cancer aggressiveness. Because fibroblasts have profound impacts on pancreatic cancer progression, we investigated whether age-related changes in pancreatic fibroblasts influence cancer growth and metastasis. Proteomics analysis revealed that aged fibroblasts secrete different factors than young fibroblasts, including increased growth/differentiation factor 15 (GDF-15). Treating young mice with GDF-15 enhanced tumor growth, whereas aged GDF-15 knockout mice showed reduced tumor growth. GDF-15 activated AKT, rendering tumors sensitive to AKT inhibition in an aged but not young microenvironment. These data provide evidence for how aging alters pancreatic fibroblasts and promotes tumor progression, providing potential therapeutic targets and avenues for studying pancreatic cancer while accounting for the effects of aging. SIGNIFICANCE: Aged pancreatic fibroblasts secrete GDF-15 and activate AKT signaling to promote pancreatic cancer growth, highlighting the critical role of aging-mediated changes in the pancreatic cancer microenvironment in driving tumor progression. See related commentary by Isaacson et al., p. 1185.

Neoplastic Progression in Macroscopic Precursor Lesions of the Pancreas.

CONTEXT.­: Macroscopic precursor lesions of the pancreas represent a complex clinical management problem. Molecular characterization of pancreatic cysts has helped to confirm and refine clinical and pathologic classifications of these lesions, inform our understanding of tumorigenesis in the pancreas, and provide opportunities for preoperative diagnosis. OBJECTIVE.­: To review the pathologic classification of macroscopic cystic lesions of the pancreas: intraductal papillary mucinous neoplasms (IPMNs), mucinous cystic neoplasms (MCNs), intraductal oncocytic papillary neoplasms (IOPNs), and intraductal tubulopapillary neoplasms (ITPNs), and to describe our current state of understanding of their molecular underpinnings, relationship to invasive carcinomas, and implications for diagnosis and prognostication. DATA SOURCES.­: We assessed the current primary literature and current World Health Organization Classification of Digestive System Tumours. CONCLUSIONS.­: Macroscopic cystic lesions of the pancreas are morphologically and molecularly diverse. IPMNs and MCNs share mucinous cytoplasm with papillae. MCNs are defined by ovarian-type stroma. IOPNs have granular eosinophilic cytoplasm, prominent nucleoli, and complex, arborizing papillae. ITPNs demonstrate complex, back-to-back tubules and anastomosing papillae and lack prominent intracellular mucin. IPMNs and MCNs are characterized by driver mutations in KRAS/GNAS (IPMNs) and KRAS (MCNs), with later driver events in RNF43, CDKN2A, SMAD4, and TP53. In contrast, IOPNs and ITPNs have recurrent rearrangements in PRKACA/PRKACB and MAPK-associated genes, respectively. The recurrent alterations described in cysts provide an opportunity for diagnosis using aspirated cyst fluid. Molecular characterization of IPMNs shows a striking spatial and mutational heterogeneity, challenging traditional models of neoplastic development and creating challenges to interpretation of cyst fluid sequencing results.

Neoadjuvant radioimmunotherapy in pancreatic cancer enhances effector T cell infiltration and shortens their distances to tumor cells.

Radiotherapy is hypothesized to have an immune-modulating effect on the tumor microenvironment (TME) of pancreatic ductal adenocarcinoma (PDAC) to sensitize it to anti-PD-1 antibody (a-PD-1) treatment. We collected paired pre- and posttreatment specimens from a clinical trial evaluating combination treatment with GVAX vaccine, a-PD-1, and stereotactic body radiation (SBRT) following chemotherapy for locally advanced PDACs (LAPC). With resected PDACs following different neoadjuvant therapies as comparisons, effector cells in PDACs were found to skew toward a more exhausted status in LAPCs following chemotherapy. The combination of GVAX/a-PD-1/SBRT drives TME to favor antitumor immune response including increased densities of GZMB+CD8+ T cells, TH1, and TH17, which are associated with longer survival, however increases immunosuppressive M2-like tumor-associated macrophages (TAMs). Adding SBRT to GVAX/a-PD-1 shortens the distances from PD-1+CD8+ T cells to tumor cells and to PD-L1+ myeloid cells, which portends prolonged survival. These findings have guided the design of next radioimmunotherapy studies by targeting M2-like TAM in PDACs.

Immune microenvironment of intimal sarcomas: Adaptive immune resistance with potential therapeutic implications.

OBJECTIVES: Intimal sarcomas are rare, aggressive neoplasms that arise from large blood vessels. Characterization of the tumor immune microenvironment may suggest new treatment strategies. METHODS: Seventeen specimens from 7 patients were labeled by immunohistochemistry for programmed cell death 1 ligand 1 (PD-L1), CD45, CD8, CD4, FOXP3, CD20, CD68, and LAG3. The immune cell density was scored as a percentage of the tumor area (1+ [<5%], 2+ [5%-50%], 3+ [>50%]); PD-L1 expression was scored on tumor cells and on intratumoral immune cells. Immune marker density was quantified using image analysis software. RESULTS: All intimal sarcomas showed immune cell infiltration (41% were 1+, 53% were 2+, 6% were 3+). Tumor and immune cell PD-L1 labeling was seen in 35% and 76% of cases, respectively; PD-L1+ intimal sarcomas had higher CD45+, CD8+, FOXP3+, CD68+, and leukocyte activation gene 3 (LAG3)+ cell densities (P ≤ .01). Similarly, PD-L1 expression on immune cells correlated with higher densities of CD8+ and FOXP3+ cells (P < .04). Higher LAG3+ cell density correlated with higher CD68+ cell density and necrosis (P < .05). One patient with prolonged survival had the highest immune cell density and PD-L1 expression. CONCLUSIONS: These data show that intimal sarcomas have an active tumor microenvironment with an adaptive pattern of PD-L1 expression. Our results suggest that immunotherapy may be an effective treatment option.

Digitize your Biology! Modeling multicellular systems through interpretable cell behavior.

Cells are fundamental units of life, constantly interacting and evolving as dynamical systems. While recent spatial multi-omics can quantitate individual cells' characteristics and regulatory programs, forecasting their evolution ultimately requires mathematical modeling. We develop a conceptual framework-a cell behavior hypothesis grammar-that uses natural language statements (cell rules) to create mathematical models. This allows us to systematically integrate biological knowledge and multi-omics data to make them computable. We can then perform virtual "thought experiments" that challenge and extend our understanding of multicellular systems, and ultimately generate new testable hypotheses. In this paper, we motivate and describe the grammar, provide a reference implementation, and demonstrate its potential through a series of examples in tumor biology and immunotherapy. Altogether, this approach provides a bridge between biological, clinical, and systems biology researchers for mathematical modeling of biological systems at scale, allowing the community to extrapolate from single-cell characterization to emergent multicellular behavior.

Comprehensive DNA Methylation Analysis Indicates That Pancreatic Intraepithelial Neoplasia Lesions Are Acinar-Derived and Epigenetically Primed for Carcinogenesis.

Pancreatic ductal adenocarcinoma (PDAC) is believed to arise from the accumulation of a series of somatic mutations and is also frequently associated with pancreatic intraepithelial neoplasia (PanIN) lesions. However, there is still debate as to whether the cell type-of-origin of PanINs and PDACs in humans is acinar or ductal. As cell type identity is maintained epigenetically, DNA methylation changes during pancreatic neoplasia can provide a compelling perspective to examine this question. Here, we performed laser-capture microdissection on surgically resected specimens from 18 patients to isolate, with high purity, DNA for whole-genome bisulfite sequencing from four relevant cell types: acini, nonneoplastic ducts, PanIN lesions, and PDAC lesions. Differentially methylated regions (DMR) were identified using two complementary analytical approaches: bsseq, which identifies any DMRs but is particularly useful for large block-like DMRs, and informME, which profiles the potential energy landscape across the genome and is particularly useful for identifying differential methylation entropy. Both global methylation profiles and block DMRs clearly implicated an acinar origin for PanINs. At the gene level, PanIN lesions exhibited an intermediate acinar-ductal phenotype resembling acinar-to-ductal metaplasia. In 97.6% of PanIN-specific DMRs, PanIN lesions had an intermediate methylation level between normal and PDAC, which suggests from an information theory perspective that PanIN lesions are epigenetically primed to progress to PDAC. Thus, epigenomic analysis complements histopathology to define molecular progression toward PDAC. The shared epigenetic lineage between PanIN and PDAC lesions could provide an opportunity for prevention by targeting aberrantly methylated progression-related genes. SIGNIFICANCE: Analysis of DNA methylation landscapes provides insights into the cell-of-origin of PanIN lesions, clarifies the role of PanIN lesions as metaplastic precursors to human PDAC, and suggests potential targets for chemoprevention.

Three-dimensional genomic mapping of human pancreatic tissue reveals striking multifocality and genetic heterogeneity in precancerous lesions.

Pancreatic intraepithelial neoplasia (PanIN) is a precursor to pancreatic cancer and represents a critical opportunity for cancer interception. However, the number, size, shape, and connectivity of PanINs in human pancreatic tissue samples are largely unknown. In this study, we quantitatively assessed human PanINs using CODA, a novel machine-learning pipeline for 3D image analysis that generates quantifiable models of large pieces of human pancreas with single-cell resolution. Using a cohort of 38 large slabs of grossly normal human pancreas from surgical resection specimens, we identified striking multifocality of PanINs, with a mean burden of 13 spatially separate PanINs per cm3 of sampled tissue. Extrapolating this burden to the entire pancreas suggested a median of approximately 1000 PanINs in an entire pancreas. In order to better understand the clonal relationships within and between PanINs, we developed a pipeline for CODA-guided multi-region genomic analysis of PanINs, including targeted and whole exome sequencing. Multi-region assessment of 37 PanINs from eight additional human pancreatic tissue slabs revealed that almost all PanINs contained hotspot mutations in the oncogene KRAS, but no gene other than KRAS was altered in more than 20% of the analyzed PanINs. PanINs contained a mean of 13 somatic mutations per region when analyzed by whole exome sequencing. The majority of analyzed PanINs originated from independent clonal events, with distinct somatic mutation profiles between PanINs in the same tissue slab. A subset of the analyzed PanINs contained multiple KRAS mutations, suggesting a polyclonal origin even in PanINs that are contiguous by rigorous 3D assessment. This study leverages a novel 3D genomic mapping approach to describe, for the first time, the spatial and genetic multifocality of human PanINs, providing important insights into the initiation and progression of pancreatic neoplasia.

Cancer-cell-derived sialylated IgG as a novel biomarker for predicting poor pathological response to neoadjuvant therapy and prognosis in pancreatic cancer.

BACKGROUND: Neoadjuvant therapy (NAT) is increasingly applied in pancreatic ductal adenocarcinoma (PDAC); however, accurate prediction of therapeutic response to NAT remains a pressing clinical challenge. Cancer-cell-derived sialylated immunoglobulin G (SIA-IgG) was previously identified as a prognostic biomarker in PDAC. This study aims to explore whether SIA-IgG expression in treatment-naïve fine needle aspirate (FNA) biopsy specimens could predict the pathological response (PR) to NAT for PDAC. METHODS: Endoscopic ultrasonography-guided FNA biopsy specimens prior to NAT were prospectively obtained from 72 patients with PDAC at the Johns Hopkins Hospital. SIA-IgG expression of PDAC specimens was assessed by immunohistochemistry. Associations between SIA-IgG expression and PR, as well as patient prognosis, were analyzed. A second cohort enrolling surgically resected primary tumor specimens from 79 patients with PDAC was used to validate the prognostic value of SIA-IgG expression. RESULTS: SIA-IgG was expressed in 58.3% of treatment-naïve FNA biopsies. Positive SIA-IgG expression at diagnosis was associated with unfavorable PR and can serve as an independent predictor of PR. The sensitivity and specificity of SIA-IgG expression in FNA specimens in predicting an unfavorable PR were 63.9% and 80.6%, respectively. Both positive SIA-IgG expression in treatment-naïve FNA specimens and high SIA-IgG expression in surgically resected primary tumor specimens were significantly associated with shorter survival. CONCLUSIONS: Assessment of SIA-IgG on FNA specimens prior to NAT may help predict PR for PDAC. Additionally, SIA-IgG expression in treatment-naïve FNA specimens and surgically resected primary tumor specimens were predictive of the prognosis for PDAC.

Oncologic resection of pancreatic cancer with isolated liver metastasis: Favorable outcomes in select patients.

BACKGROUND: Patients with pancreatic ductal adenocarcinoma (PDAC) and liver metastasis are treated with palliative chemotherapy, whereas similar patients with metastatic colorectal cancer are considered for aggressive surgery. METHODS: Using an institutional database, PDAC patients undergoing liver resection for isolated metastasis were identified. Their overall survival (OS), treatment factors, and clinicopathological variables associated with survival were also evaluated. RESULTS: Forty-seven patients underwent curative-intent surgery for metastatic PDAC to the liver between 2000 and 2019. Median OS was 21.9 months from diagnosis. Fourteen patients underwent unplanned resection of radiographically occult liver metastasis during pancreatectomy with median OS of 8.7 months. On the other hand, 29 patients received systemic chemotherapy followed by planned resection; this cohort had the most favorable prognosis following aggressive surgery with median OS being 38.1 months from diagnosis and 24.1 months from surgery. Preoperative chemotherapy (HR = 7.1; p = .002) and moderate to well differentiation of the primary tumor (HR = 3.7; p = .003) were associated with prolonged survival in multivariate analysis, whereas lymph node metastases, response to preoperative therapy, number of liver metastasis, and extent of liver surgery were not. CONCLUSIONS: In select patients with PDAC and isolated liver metastasis, curative-intent surgery can result in meaningful survival. This aggressive approach seems most beneficial in patients following induction chemotherapy.

Tailoring Adjuvant Chemotherapy to Biologic Response Following Neoadjuvant Chemotherapy Impacts Overall Survival in Pancreatic Cancer.

BACKGROUND: The role of postoperative chemotherapy in patients with resected pancreatic cancer who receive neoadjuvant treatment is unknown. Clinicians use changes in CA19-9 and histopathologic scores to assess treatment response. We sought to investigate if CA19-9 normalization in response to NAT can help guide the need for postoperative treatment. METHODS: Patients with elevated baseline CA19-9 (CA19-9 > 37U/mL) who received NAT followed by surgery between 2011 and 2019 were retrospectively reviewed. Treatment response was determined by CA19-9 normalization following NAT and histopathologic scoring. The role of postoperative chemotherapy was analyzed in light of CA19-9 normalization and histopathologic response. RESULTS: We identified and included 345 patients. Following NAT, CA19-9 normalization was observed in 125 patients (36.2%). CA19-9 normalization was associated with a favorable histopathologic response (41.6% vs 23.2%, p < 0.001) and a lower ypT (p < 0.001) and ypN stage (p = 0.003). Receipt of adjuvant chemotherapy was associated with improved overall survival in patients in whom CA19-9 did not normalize following NAT (26.8 vs 16.4 months, p = 0.008). In patients who received 5FU-based NAT and in whom CA19-9 did not normalize, receipt of 5FU-based adjuvant chemotherapy was associated with improved OS (p = 0.014). CONCLUSION: CA19-9 normalization in response to NAT was associated with favorable outcomes and can serve as a biomarker for treatment response. In patients where CA19-9 did not normalize, receipt of postoperative chemotherapy was associated with improved OS. These patients also benefited from additional 5FU-based postoperative chemotherapy following 5FU-based NAT.

CODA: quantitative 3D reconstruction of large tissues at cellular resolution.

A central challenge in biology is obtaining high-content, high-resolution information while analyzing tissue samples at volumes relevant to disease progression. We address this here with CODA, a method to reconstruct exceptionally large (up to multicentimeter cubed) tissues at subcellular resolution using serially sectioned hematoxylin and eosin-stained tissue sections. Here we demonstrate CODA's ability to reconstruct three-dimensional (3D) distinct microanatomical structures in pancreas, skin, lung and liver tissues. CODA allows creation of readily quantifiable tissue volumes amenable to biological research. As a testbed, we assess the microanatomy of the human pancreas during tumorigenesis within the branching pancreatic ductal system, labeling ten distinct structures to examine heterogeneity and structural transformation during neoplastic progression. We show that pancreatic precancerous lesions develop into distinct 3D morphological phenotypes and that pancreatic cancer tends to spread far from the bulk tumor along collagen fibers that are highly aligned to the 3D curves of ductal, lobular, vascular and neural structures. Thus, CODA establishes a means to transform broadly the structural study of human diseases through exploration of exhaustively labeled 3D microarchitecture.

Multi-omic analyses of changes in the tumor microenvironment of pancreatic adenocarcinoma following neoadjuvant treatment with anti-PD-1 therapy.

Successful pancreatic ductal adenocarcinoma (PDAC) immunotherapy necessitates optimization and maintenance of activated effector T cells (Teff). We prospectively collected and applied multi-omic analyses to paired pre- and post-treatment PDAC specimens collected in a platform neoadjuvant study of granulocyte-macrophage colony-stimulating factor-secreting allogeneic PDAC vaccine (GVAX) vaccine ± nivolumab (anti-programmed cell death protein 1 [PD-1]) to uncover sensitivity and resistance mechanisms. We show that GVAX-induced tertiary lymphoid aggregates become immune-regulatory sites in response to GVAX + nivolumab. Higher densities of tumor-associated neutrophils (TANs) following GVAX + nivolumab portend poorer overall survival (OS). Increased T cells expressing CD137 associated with cytotoxic Teff signatures and correlated with increased OS. Bulk and single-cell RNA sequencing found that nivolumab alters CD4+ T cell chemotaxis signaling in association with CD11b+ neutrophil degranulation, and CD8+ T cell expression of CD137 was required for optimal T cell activation. These findings provide insights into PD-1-regulated immune pathways in PDAC that should inform more effective therapeutic combinations that include TAN regulators and T cell activators.

The Diagnostic Challenge of Evaluating Small Biopsies from the Pancreatobiliary System.

Examination of fine needle aspirations and small core biopsies of the pancreas can be an extremely difficult and treacherous area for the diagnostic pathologist. The pancreas often yields small and often fragmented specimens, which, in combination with the morphologic overlap between numerous neoplastic and nonneoplastic mimickers, generate multiple potential diagnostic pitfalls. The authors review this challenging topic and provide insight into resolving these pitfalls using morphologic pattern recognition and ancillary testing.

Predictors, Patterns, and Timing of Recurrence Provide Insight into the Disease Biology of Invasive Carcinomas Arising in Association with Intraductal Papillary Mucinous Neoplasms.

OBJECTIVES: To identify predictors, patterns, and timing of recurrence after resection of invasive carcinomas arising in association with an IPMN. BACKGROUND: Postoperative management of an invasive carcinoma arising in association with an intraductal papillary mucinous neoplasm (IPMN), a biologically distinct entity from PanIN-derived pancreatic ductal adenocarcinoma (PDAC), remains largely based on guidelines for PanIN-derived PDAC. To minimize treatment failure and inform disease-specific management, cancer recurrence must be better characterized. METHODS: Patients were identified from a prospectively maintained registry between 1996 and 2018. Predictors of recurrence were evaluated by employing Cox regression models to determine risk-adjusted hazard ratios (HR) with 95% confidence intervals (95%CI). The patterns and timing of recurrence were recognized and compared utilizing a log-rank test, respectively. RESULTS: Of the 213 patients included, 92 (43.2%) recurred with a median RFS of 23.7 months (16.7-30.7). The predominant pattern of recurrence included any systemic (65.2%). The median time to local recurrence was longer than systemic (21.6 versus 11.4 months, p = 0.05). Poor differentiation [HR: 3.01, 95%CI (1.06-8.61)] and nodal disease [N1, HR: 2.23, 95%CI (1.12-4.60); and N2, HR: 5.67 95%CI (2.93-10.99)] emerged as independent predictors of systemic recurrence. For local-specific recurrences, poor differentiation [HR: 3.73, 95%CI (1.04-13.45)] and an R1 margin [high-grade dysplasia or invasive carcinoma; HR: 2.66, 95%CI (1.14-6.21)] emerged as independent predictors. CONCLUSIONS: The predominant pattern of recurrence after resection of invasive carcinomas arising in association with IPMNs is systemic, and occurs earlier than local recurrence. Poor differentiation and nodal disease are associated with systemic recurrence while poor differentiation and an R1 margin are associated with local recurrence. Future studies should investigate the role of systemic (chemotherapy) versus local (radiation) therapies and surveillance strategies in a personalized manner.