Pancreatic Ductal Adenocarcinoma Lesions Research Articles

Abstract PR02: Matched metastatic pancreatic ductal adenocarcinoma biopsies and organoid models reveal tumor cell transcriptional plasticity and subtype-specific microenvironmental crosstalk

Abstract The majority of patients with pancreatic ductal adenocarcinoma (PDAC) present with metastatic disease at diagnosis and have median survival times of less than 12 months. Recent studies have demonstrated that PDAC tumors with distinct transcriptional signatures are associated with different clinical outcomes, and that the tumor microenvironment may contribute to PDAC pathogenesis. In parallel, there are ongoing efforts to understand if patient-derived organoid models can be used as functional surrogates for an individual patient’s disease. However, it remains unclear if patient transcriptional phenotypes are preserved in their matched organoid models. Here, we describe a pipeline that permits both direct characterization of the PDAC liver metastatic niche via single-cell RNA-sequencing and functional assessment of PDAC tumor biology in patient-matched organoid models. Starting from core needle biopsies of metastatic PDAC lesions containing 50-100k viable cells, we simultaneously perform (1) low-input single-cell RNA-sequencing using Seq-Well and (2) three-dimensional organoid culture generation. We have applied this approach to profile 21 patients and their matched early passage organoid models. Our pipeline yields high-quality single-cell measurements across diverse cell types—both tumor and nontumor stromal—enabling a principled dissection of tumor intrinsic and extrinsic factors. Evaluation of clinically relevant transcriptional signatures (e.g., basal-like vs. classical) revealed extensive heterogeneity at the single-cell level and identified new, hybrid expression states. We also observed evidence of significant subtype-specific crosstalk between immune populations and tumor cells—specifically between T cells and tumor cells originating from basal-like tumors. Serial sampling at different stages of treatment revealed transcriptional shifts in tumor cells suggestive of significant plasticity. We similarly found that organoids derived from basal-like tumors exhibited considerable plasticity in vitro and had decreased fitness in standard organoid culture conditions, suggesting the need for distinct environments to support specific transcriptional subtypes. Overall, our approach provides actionable insights into the heterogeneity and plasticity of human PDAC, as well as a pipeline and framework for the analysis of PDAC and other cancers. This abstract is also being presented as Poster A50. Citation Format: Peter S. Winter, Srivatsan Raghavan, Andrew W. Navia, Hannah Williams, Jennyfer Galvez-Reyes, Radha Kalekar, Ashir Borah, Alan DenAdel, Manisha Raghavan, Kristen Lowder, Nolawit Mulugeta, Junning Wang, Emma Reilly, Lauren Brais, Lorin Crawford, James McFarland, James M. Cleary, Jonathan Nowak, Brian M. Wolpin, Andrew J. Aguirre, William C. Hahn, Alex K. Shalek. Matched metastatic pancreatic ductal adenocarcinoma biopsies and organoid models reveal tumor cell transcriptional plasticity and subtype-specific microenvironmental crosstalk [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr PR02.

Chronic inflammatory changes and oxidative stress in the background of "pancreatic ductal adenocarcinoma concomitant with intraductal papillary mucinous neoplasm".

Cases of "pancreatic ductal adenocarcinoma (PDAC) concomitant with intraductal papillary mucinous neoplasm" (IPMN) have multiple PDAC lesions more frequently than cases of "PDAC without IPMN". However, the mechanism of carcinogenesis in this former disease category remains unknown. The main objective of this work was thus to investigate the effects of chronic inflammation on carcinogenesis in PDAC cases. We selected 31 "PDAC concomitant with IPMN" patients and 58 "PDAC without IPMN" patients and pathologically evaluated their background pancreatic parenchyma. Fibrosis and inflammation scores of background pancreas were higher in "PDAC concomitant with IPMN" than in "PDAC without IPMN" (P < 0.0001 and P < 0.0001, respectively), whereas the fatty infiltration score of background pancreas was high in "PDAC without IPMN" (P = 0.0024). Immunohistochemically, the expression of 8-hydroxy-2'-deoxyguanosine (8-OHDG), an oxidative stress marker, in the background pancreas was high in "PDAC concomitant with IPMN" compared with that in "PDAC without IPMN" (P < 0.0001). Chronic inflammation activates oxidative stress in tissue throughout the pancreas and probably confers susceptibility to tumorigenesis in "PDAC concomitant with IPMN".

Laser-assisted 3D bioprinting of exocrine pancreas spheroid models for cancer initiation study

Pancreatic ductal adenocarcinoma (PDAC) is the most common malignancy of the pancreas. It has shown a poor prognosis and a rising incidence in the developed world. Other pathologies associated with this tissue include pancreatitis, a risk condition for pancreatic cancer. The onset of both pancreatitis and pancreatic cancer follows a common pattern: exocrine pancreatic acinar cells undergo a transdifferentiation to duct cells that triggers a 3D restructuration of the pancreatic tissue. However, the exact mechanism underlying this process remains partially undefined. Further understanding the cellular events leading to PDAC could open new avenues in the development of novel therapeutic approaches. Since current 2D cell culture models fail to mimic the tridimensional complexity of the pancreatic tissue, new in vitro models are urgently needed. Here, we generated 3D pancreatic cell spheroid arrays using laser-assisted bioprinting and characterized their phenotypic evolution over time through image analysis and phenotypic characterization. We show that these bioprinted spheroids, composed of both acinar and ductal cells, can replicate the initial stages of PDAC development. This bioprinted miniaturized spheroid-based array model should prove useful for the study of the internal and external factors that contribute to the formation of precursor PDAC lesions and to cancer progression, and may therefore shed light on future PDAC therapy strategies.

Abstract B39: Regulation and function of IL-1b in immune modulation of pancreatic cancer

Abstract Pancreatic intraepithelial neoplasms (PanINs) are well-defined precursor lesions of pancreatic ductal adenocarcinoma (PDA). The initiation and progression of PanINs is marked by dynamic stromal reconstruction and localized acute inflammation. Using an orthotopic transplantation model of early and advanced pancreatic neoplasia in immunocompetent recipient mice, we have uncovered a crucial role for the cytokine IL-1b in KRasG12D-driven PanIN evolution. Tumor cell-derived IL-1b¢ was found to be critical for shaping the protumorigenic PanIN microenvironment by promoting activation of quiescent pancreatic stellate cells and establishing an immunosuppressive milieu mediated by M2 macrophages, MDSCs and CD1dhiCD5+ regulatory B cells. Furthermore, loss of IL-1 signaling in tumor stroma activates the cytotoxic T-cell immune response, significantly attenuates PanIN growth and confers survival advantage to PDAC-bearing mice. Tumor cell expression of IL-1b in vivo is driven by upregulation of TLR4 signaling and activation of the NLRP3 inflammasome. KrasG12D enhances surface TLR4 expression, sensitizing transformed cells to TLR4 ligands in the microenvironment, including the pancreatic microbiome. Collectively, our study describes a complex tumor-stromal interplay orchestrated by the TLR4-NLRP3-IL-1b signaling axis, which is critical for shaping the tumor microenvironment and driving pancreatic tumorigenesis. Citation Format: Shipra Das, Sandra Vogt, Christina Hadju, Dafna Bar-Sagi. Regulation and function of IL-1b in immune modulation of pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B39.

Differential impact of the ERBB receptors EGFR and ERBB2 on the initiation of precursor lesions of pancreatic ductal adenocarcinoma

Earlier diagnosis of pancreatic ductal adenocarcinoma (PDAC) requires better understanding of the mechanisms driving tumorigenesis. In this context, depletion of Epidermal Growth Factor Receptor (EGFR) is known to impair development of PDAC-initiating lesions called acinar-to-ductal metaplasia (ADM) and Pancreatic Intraepithelial Neoplasia (PanIN). In contrast, the role of v-erb-b2 erythroblastic leukemia viral oncogene homolog 2 (ERBB2), the preferred dimerization partner of EGFR, remains poorly understood. Here, using a mouse model with inactivation of Erbb2 in pancreatic acinar cells, we found that Erbb2 is dispensable for inflammation- and KRasG12D-induced development of ADM and PanIN. A mathematical model of EGFR/ERBB2-KRAS signaling, which was calibrated on mouse and human data, supported the observed roles of EGFR and ERBB2. However, this model also predicted that overexpression of ERBB2 stimulates ERBB/KRAS signaling; this prediction was validated experimentally. We conclude that EGFR and ERBB2 differentially impact ERBB signaling during PDAC tumorigenesis, and that the oncogenic potential of ERBB2 is only manifested when it is overexpressed. Therefore, the level of ERBB2, not only its mere presence, needs to be considered when designing therapies targeting ERBB signaling.

Proteome alterations in pancreatic ductal adenocarcinoma.

Proteins are the essential functional biomolecules profoundly implicated in all aspects of pancreatic tumorigenesis and its progression. While common genomic factors, such as KRAS, TP53, SMAD4, and CDKN2A have been well recognized in association of pancreatic ductal adenocarcinoma (PDAC), our understanding of functional changes at the proteome level merits further investigation. Malignance associated proteome alterations can be attributed to the convoluted outcomes from genetic, epigenetic and environmental factors in initiating and progressing PDAC, and may reflect on changes in protein expressional level, structure, localization, as well as post-translational modifications (PTMs) status. The study of localized or systemic proteome alterations in PDAC, as well as its precursor lesions, such as pancreatic intraepithelial neoplasia (PanIN) and mucinous pancreatic cystic neoplasm, would provide unique perspectives in elucidating functional molecular events underlying PDAC. While efforts have been made, challenges still exist to comprehensively integrate much of the proteomic discovery to the perspectives gained from genomic studies in the context of biomarker discovery. Novel approaches and data from well-defined longitudinal clinical studies and experimental models are needed to facilitate the study of PDAC and precursor lesions for early detection and intervention.

Surveillance for pancreatic cancer in high‐risk individuals

BackgroundSurveillance of individuals at high risk of pancreatic ductal adenocarcinoma (PDAC) and its precursors might lead to better outcomes. The aim of this study was to determine the prevalence and outcomes of PDAC and high‐risk neoplastic precursor lesions among such patients participating in surveillance programmes.MethodsA multicentre study was conducted through the International CAncer of the Pancreas Screening (CAPS) Consortium Registry to identify high‐risk individuals who had undergone pancreatic resection or progressed to advanced PDAC while under surveillance. High‐risk neoplastic precursor lesions were defined as: pancreatic intraepithelial neoplasia (PanIN) 3, intraductal papillary mucinous neoplasia (IPMN) with high‐grade dysplasia, and pancreatic neuroendocrine tumours at least 2 cm in diameter.ResultsOf 76 high‐risk individuals identified in 11 surveillance programmes, 71 had undergone surgery and five had been diagnosed with inoperable PDAC. Of the 71 patients who underwent resection, 32 (45 per cent) had PDAC or a high‐risk precursor (19 PDAC, 4 main‐duct IPMN, 4 branch‐duct IPMN, 5 PanIN‐3); the other 39 patients had lesions thought to be associated with a lower risk of neoplastic progression. Age at least 65 years, female sex, carriage of a gene mutation and location of a lesion in the head/uncinate region were associated with high‐risk precursor lesions or PDAC. The survival of high‐risk individuals with low‐risk neoplastic lesions did not differ from that in those with high‐risk precursor lesions. Survival was worse among patients with PDAC. There was no surgery‐related mortality.ConclusionA high proportion of high‐risk individuals who had surgical resection for screening‐ or surveillance‐detected pancreatic lesions had a high‐risk neoplastic precursor lesion or PDAC at the time of surgery. Survival was better in high‐risk individuals who had either low‐ or high‐risk neoplastic precursor lesions compared with that in patients who developed PDAC.

Molecular characterization of early precursor lesions of pancreatic ductal adenocarcinoma

Molecular characterization of early precursor lesions of pancreatic ductal adenocarcinoma

1126 ENDOSCOPIC ULTRASOUND FEATURES IN THE SOJOURN PHASE OF PANCREATIC DUCTAL ADENOCARCINOMA

Pancreatic ductal adenocarcinoma (PDAC) has an 8.5% 5-year survival rate. Early detection, increasing use of neoadjuvant therapy and the opportunity for oncologic surgery is the best chance for cure. EUS is considered to be the superior imaging modality to detect small (<20mm) PDAC lesions and their precursors. We aimed to identify suspect lesions on EUS performed before the eventual diagnosis of PDAC.

Pathology of pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy and estimated to become the second leading cause of cancer-related deaths by 2030. Although overall 5-year survival rates have constantly remained below 10% for the last decades, several key points important for accurate patient stratification have emerged during recent years. These key points include a highly standardized gross examination of PDAC resection specimens, using an axial slicing technique and inking of the circumferential resection margin (CRM), as well as a meticulous microscopic examination, taking into account the prognostic relevance of factors such as the exact resection status (R0 vs. R1 1-mm vs. R1 resection), histopathological tumor grading and the so-called lymph node ratio (LNR). With increasing use of neoadjuvant therapy in PDAC, tumor regression grading (TRG) for PDAC is currently rising in relevance in order to stratify and manage pre-operatively treated PDAC patients. As all current TRG systems for PDAC are unsatisfactory, new standardized international protocols are urgently needed. Several morphological subtypes of PDAC exist, some of which share the same molecular background with classical PDAC, while others are characterized by a distinct molecular pathogenesis. While some show a prognosis similar to classical PDAC, other subtypes stand out due to a better or even worse prognosis than classical PDAC. Prognostic relevant molecular subtypes of PDAC have been proposed as well, however, limitations of used cohorts and the lacking correlation of molecular subtypes with histomorphological subtypes limit the translation of these findings into valuable clinical applications. Lastly, several macroscopic and microscopic precursor lesions of PDAC have been described in genetically engineered mouse models (GEMM) and humans in recent times, providing further insight into PDAC carcinogenesis. In addition, improved diagnosis of PDAC precursors represents a chance to select patients for resection before invasive PDAC is present.

Expression and prognostic value of NSD1 and SETD2 in pancreatic ductal adenocarcinoma and its precursor lesions

Epigenetic regulation is emerging as a critical mechanism for pancreatic ductal adenocarcinoma (PDA) development. Histone methylation is an important regulatory mechanism, altering chromatin structure and promoter accessibility and causing aberrant gene expression. NSD1 and SETD2 genes encoding two histone H3K36 methyltransferases, are mutated or altered in 8-10% of PDA cases. However, whether there is altered protein expression of NSD1 or SETD2 in PDA and its precursors, and whether they have diagnostic or prognostic utility is unknown. Tissue microarrays composed of a total of 190 and 192 duplicated cases of PDA (n=74 and 75), metastatic PDA (n=17 and 18), pancreatic intraepithelial neoplasia (PanIN; n=19 and 24), intraductal papillary mucinous neoplasm (IPMN; n=36), mucinous cystic neoplasm (MCN; n=12) and benign pancreatic tissues (n=27 and 32) were analysed for expression of NSD1 and SETD2 by immunohistochemistry. We assessed intensity and percentage of positive cells. NSD1 expression was significantly increased in metastatic PDA compared to benign ducts, primary PDA, and all other lesions combined (p=0.03, 0.02, and 0.03 respectively). Additionally, significantly decreased SETD2 protein expression was found in metastatic PDA and PanIN lesions compared to benign ducts (p=0.04 and 0.007, respectively). High NSD1 expression was associated with clinical stage III/IV disease (p=0.026), tumour grade 2 (p=0.022), use of neoadjuvant therapy (p=0.037), and overall higher clinical stage (p=0.022). There is no significant difference in overall and progression-free survival between NSD1/SETD2 high and low PDA. Expression of NSD1 and SETD2 is specifically altered in metastatic PDA and some of the PDA precursor lesions, supporting their important role in PDA development and metastasis. In addition, increased NSD1 expression is significantly associated with higher clinical stage and neoadjuvant therapy, suggesting that NSD1 may be a useful prognostic marker.

Oncogenic ERBB2 aberrations and KRAS mutations cooperate to promote pancreatic ductal adenocarcinoma progression.

Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with few therapeutic options, representing one of the great challenges in oncology. Activating KRAS mutation, occurring in >90% PDACs, is present in pancreatic intraepithelial neoplasia lesions, the precursor ductal lesions of PDAC, indicating additional genetic alterations contribute to the pathogenesis of PDAC. PDAC sequencing projects identify recurrent genomic ERBB2 alterations, mutations and amplifications, in 8.5% of PDAC patients, ranking as the top hit among the 100 receptor tyrosine kinases-encoding genes. Introduction of the ERBB2 mutations encoding protein variants S310F, S423R, R678Q, Q679L, E717D, L755S, V777L and V842I into human pancreatic epithelial cells causes oncogenic transformation, increasing ERBB2 signaling, anchorage-independent cell growth and tumor xenograft growth in nude mice, demonstrating that they are activating mutations. Interestingly, in many PDACs, mutations in ERBB2 and KRAS occur together. ERBB2 activating mutants facilitate KRAS-driven oncogenic properties. Introduction of ERBB2 mutations into KRAS-mutant PDAC cells activates ERBB2 signaling, promotes tumor growth and attenuates KRAS dependency. In contrast, a CRISPR-mediated knockout (KO) of ERBB2 in ERBB2-amplified PDAC cells inhibits ERBB2 signaling, colony formation, anchorage-independent growth and tumor xenograft formation. Finally, oncogenic ERBB2 aberrations can be abrogated by treatment with small-molecule inhibitors. ERBB2 and KRAS inhibition cooperate to suppress PDAC cell growth in vitro and to promote tumor regression in nude mice, providing a rationale for testing an anti-ERBB2 drug in combination with a KRAS inhibitor in ERBB2-mutant PDAC patients that are currently untreatable.

Single-Cell Transcriptomics of Pancreatic Cancer Precursors Demonstrates Epithelial and Microenvironmental Heterogeneity as an Early Event in Neoplastic Progression.

Early detection of pancreatic ductal adenocarcinoma (PDAC) remains elusive. Precursor lesions of PDAC, specifically intraductal papillary mucinous neoplasms (IPMNs), represent a bona fide pathway to invasive neoplasia, although the molecular correlates of progression remain to be fully elucidated. Single-cell transcriptomics provides a unique avenue for dissecting both the epithelial and microenvironmental heterogeneities that accompany multistep progression from noninvasive IPMNs to PDAC. Single-cell RNA sequencing was performed through droplet-based sequencing on 5,403 cells from 2 low-grade IPMNs (LGD-IPMNs), 2 high-grade IPMNs (HGD-IPMN), and 2 PDACs (all surgically resected). Analysis of single-cell transcriptomes revealed heterogeneous alterations within the epithelium and the tumor microenvironment during the progression of noninvasive dysplasia to invasive cancer. Although HGD-IPMNs expressed many core signaling pathways described in PDAC, LGD-IPMNs harbored subsets of single cells with a transcriptomic profile that overlapped with invasive cancer. Notably, a proinflammatory immune component was readily seen in low-grade IPMNs, composed of cytotoxic T cells, activated T-helper cells, and dendritic cells, which was progressively depleted during neoplastic progression, accompanied by infiltration of myeloid-derived suppressor cells. Finally, stromal myofibroblast populations were heterogeneous and acquired a previously described tumor-promoting and immune-evading phenotype during invasive carcinogenesis. This study demonstrates the ability to perform high-resolution profiling of the transcriptomic changes that occur during multistep progression of cystic PDAC precursors to cancer. Notably, single-cell analysis provides an unparalleled insight into both the epithelial and microenvironmental heterogeneities that accompany early cancer pathogenesis and might be a useful substrate to identify targets for cancer interception.See related commentary by Hernandez-Barco et al., p. 2027.

Optimal respiratory-gated [18F]FDG PET/CT significantly impacts the quantification of metabolic parameters and their correlation with overall survival in patients with pancreatic ductal adenocarcinoma

PurposeMetabolic parameters are increasingly being used to characterize tumors. Motion artifacts due to patient respiration introduce uncertainties in quantification of metabolic parameters during positron emission tomography (PET) image acquisition. The present study investigates the impact of amplitude-based optimal respiratory gating (ORG) on quantification of PET-derived image features in patients with pancreatic ductal adenocarcinoma (PDAC), in correlation with overall survival (OS).MethodsSixty-nine patients with histologically proven primary PDAC underwent 2′-deoxy-2′-[18F]fluoroglucose ([18F]FDG) PET/CT imaging during diagnostic work-up. Standard image acquisition and reconstruction was performed in accordance with the EANM guidelines and ORG images were reconstructed with a duty cycle of 35%. PET-derived image features, including standard parameters, first- and second-order texture features, were calculated from the standard and corresponding ORG images, and correlation with OS was assessed.ResultsORG significantly impacts the quantification of nearly all features; values of single-voxel parameters (e.g., SUVmax) showed a wider range, volume-based parameters (e.g., SUVmean) were reduced, and texture features were significantly changed. After correction for motion artifacts using ORG, some features that describe intra-tumoral heterogeneity were more strongly correlated to OS.ConclusionsCorrection for respiratory motion artifacts using ORG impacts the quantification of metabolic parameters in PDAC lesions. The correlation of metabolic parameters with OS was significantly affected, in particular parameters that describe intra-tumor heterogeneity. Therefore, interpretation of single-voxel or average metabolic parameters in relation to clinical outcome should be done cautiously. Furthermore, ORG is a valuable tool to improve quantification of intra-tumoral heterogeneity in PDAC.

Expression of SNAIL in accompanying PanIN is a key prognostic indicator in pancreatic ductal adenocarcinomas.

Pancreatic ductal adenocarcinoma (PDAC) is the most lethal cancer, mainly because of its invasive and metastatic characteristics. Pancreatic intraepithelial neoplasia (PanIN) is one of the major precursor lesions of PDAC. Although epithelial‐to‐mesenchymal transition (EMT) is known to play an important role for these malignant behaviors, the association between PanIN and EMT has not been clearly understood. Therefore, we explored possible molecules for regulation of EMT immunohistochemically. Using surgically resected specimens from 71 PDAC patients, expressions of SNAIL, SLUG, TWIST1, and ZEB1 were investigated in high‐grade PanIN (HG‐PanIN) and PDAC. Results demonstrated that PDAC accompanied by SNAIL‐positive HG‐PanIN showed a significantly better relapse‐free survival (RFS) (median survival time (MST) of 11.3 months vs 4.4 months, P < 0.001) and overall survival overall survival (OS) (MST of 25.2 months vs 13.6 months, P < 0.001). In PDAC accompanied by SLUG‐positive HG‐PanIN, RFS and OS (P = 0.09 and P = 0.05) tended to have a better prognosis. In contrast, we could not find any significant prognostic benefits in the expression of TWIST1 or ZEB1 in PDAC accompanied by HG‐PanIN. Our present results suggest that (1) EMT may play an important role in the development of PDAC from HG‐PanIN, and (2) SNAIL may predict a distinct subgroup that shows a better prognosis.

Abstract A096: The potential role of fibroblast activation protein as a natural killer cell immune checkpoint

Abstract Fibroblast activation protein (FAP) is a type II transmembrane serine protease that functions as both a dipeptidyl peptidase and an endopeptidase. FAP is minimally expressed in normal pancreas but overexpressed in 90% of pancreatic ductal adenocarcinoma (PDAC) specimens. A meta-analysis of PDAC studies demonstrated elevated tumor FAP expression is associated with worse clinical outcomes. While immunotherapy offers remarkable results for certain cancer types, it has been largely ineffective in PDAC. This lack of efficacy may be attributed to the dense stromal fibrosis, comprised largely of pancreatic stellate cells (PSCs), that is characteristic of PDAC lesions. Here we demonstrate that human NK cell line (NK92) is activated by and kill PSCs. Upon direct contact with PSCs, NK92 cells upregulate FAP. FAP expression by NK92 cells is associated with an inactivation phenotype. Talabostat, a non-specific inhibitor of FAP, enhances NK92 killing of PSCs in vitro and enhances PDAC tumor clearance in vivo. This suggests that FAP may be a novel NK cell immune checkpoint that can be pharmacologically modulated to enhance NK cell antitumor activity. Citation Format: Allison O'Connell, Shangzi Wang, Louis M. Weiner. The potential role of fibroblast activation protein as a natural killer cell immune checkpoint [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A096.

Expression of Chosen Carcinoembryonic-Related Cell Adhesion Molecules in Pancreatic Intraepithelial Neoplasia (PanIN) Associated with Chronic Pancreatitis and Pancreatic Ductal Adenocarcinoma (PDAC)

Aims: Carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) are members of the glycosylphosphatidylinositol (GPI)-linked immunoglobulin (Ig) superfamily and take part in regulation of cell adhesion, tumor suppression and angiogenesis. Overexpression of CEACAM 1, 5 and 6 is widely described in several gastrointestinal epithelial tumors. The aim of study was to evaluate the expression of CEACAM 1, CEACAM 5 and CEACAM 6 in the most common precursor lesions of pancreatic ductal adenocarcinoma -pancreatic intraepithelial neoplasia (PanIN).Methods and results: The study group consisted of 32 patients treated for chronic pancreatitis and 38 patients with pancreatic ductal adenocarcinoma who also had pancreatic intraepithelial neoplasia. The expression of CEACAM was performed by immunohistochemical method and evaluated using 3-point scale: 0 - lack of positive reaction in pancreatic intraepithelial neoplasia, 1 (weak and moderate) - reaction present in 1-30% epithelial cells in PanIN and 2 (strong) - reaction present in >30% epithelial cells in PanIN. Expression of CEACAM 1, 5 and 6 increased with increasing degree of advancement of PanIN. Differences in expression of CEACAM 1, 5 and 6 between normal pancreatic ducts and different degrees of PanIN were statistically significant (p<0.001). We observed relationship between CEACAM1 expression and localization of PanIN in different parts of the pancreas.Conclusions: CEACAM 1, CEACAM 5 and CEACAM 6 expression appears to be an early event in pancreatic carcinogenesis. Moreover, expression of CEACAM 1, 5 and 6 may represent a useful biomarker that may aid in the identification of precancerous lesions in the pancreas.

Subtyping of intraductal papillary mucinous neoplasms - pitfalls of MUC1 immunohistochemistry.

Intraductal papillary mucinous neoplasms (IPMNs) are precursor lesions of pancreatic ductal adenocarcinoma (PDAC). Current edition of WHO Classification of Tumors of the Digestive System recognizes four different subtypes (gastric, intestinal, pancreatobiliary, and oncocytic) and recommends analysis of mucin expression (MUC1, MUC2, MUC5AC, MUC6) as well as evaluation of architectural and cell differentiation patterns for correct classification. However, there is no consensus on MUC1 expression of IPMN-lesions in the literature. Current recommendations are based on studies where antibodies against the core MUC1 protein or sialylated MUC1 (tumor associated MUC1), not the fully glycosylated MUC1 were used. We have recently reported that MUC1 is strongly expressed in both gastric and intestinal types IPMN specimens from the cystic wall, obtained by endoscopic ultrasound guided microbiopsy procedure. We have used a commercial MUC1 antibody, validated and recommended for diagnostic use, which recognizes fully glycosylated MUC1. Based on the above, we propose a revision of the WHO Classification, specifying that antibodies against tumor associated MUC1 should be used for IPMN subtyping.

140P - Oral cavity immune response in pancreatic ductal adenocarcinoma (PDAC)

Background: We studied the humoral immune response in patients with pancreatic ductal adenocarcinoma (PDAC) using mouthwashes from the oral cavity. We found lower immune response against recall antigens, fusobacterium nucleatum and tumor associated antigens before immunotherapy treatment. We also found better prognosis when the antibodies against tested antigens got higher. Finally, we found significant differences in the humoral immune response in the oral cavity against serum. Methods: Antigens from fusobacterium nucleatum, recall and tumor associated antigens were tested by IgG + IgM + IgA ELISA in both oral cavity and serum of PDAC patients n = 50. We provided 5 ml of saline solution to all the patients and controls to rinse the oral cavity hardly for 5 minutes and deposit the content in 15 ml tube. Sera were obtained. All was done after the approval of the local IRB committee. Results: All the evaluated antigens were lower in the oral cavity of PDAC patients compared with controls (p = 0.001). Serum responses for all antigens were significantly different than oral cavity (p = 0.03). At the end of the immunotherapy treatment consisting in a combination of immunomodulation, multi peptide antigen specific active immunotherapy and immunogenic chemotherapy with doxorubicin and oxaliplatin the levels of antibodies were increased significantly. For example, FAP (p = 0.0001), EGFR (p = 0.005), Fascin-1 (p = 0001), VCP (P = 001). Also, the immunotherapy combination significantly increased antibody immune response against fusobaterium lysate (p = 0001). Conclusions: PDAC is a challenge disease especially in late stages. PDAC patients had low antibody titters against several antigens in comparison with controls and before treatment the oral cavity. Serum antibodies were lower than oral cavity even after treatment. Oral cavity antibodies may be a useful early biomarker in patients in high risk of PDAC. This data also suggest that antibodies get low when the cancer or premalignant lesions of PDAC are present. Legal entity responsible for the study: Centro de Investigacion de Cancer en Sonora (CICS). Funding: CICS USA. Disclosure: All authors have declared no conflicts of interest.

Abstract 3121: Characterization of immune profiling of pancreatic intraductal papillary mucinous neoplasm using multiplex immunofluorescence and image analysis approaches

Abstract Pancreatic intraductal papillary mucinous neoplasms (IPMN) are cystic mucin-secreting tumors that arise within the pancreatic ductal system and are considered precursor lesions of pancreatic ductal adenocarcinoma. Profiling the immune infiltrate of IPMNs is essential to understand the immune mechanisms associated with the progression of IPMN. The aim of this study was to characterize the immune profiling of IPMN. We examined 31 formalin-fixed and paraffin-embedded tumor samples from surgically resected IPMN. The diagnostic slides were histologically reviewed by 2 expert pathologists in pancreas and classified as low-grade IPMN (low to intermediate grade dysplasia, N=11), high-grade IPMN (high grade dysplasia, N=18) and IPMN with invasive adenocarcinoma (N=2). Multiplex immunofluorescence (mIF) was used to characterize the immune profiling using two panels: Panel 1: PD-L1, PD1, CD3 (pan T cells), CD8 (T-cytotoxic), CD68 (macrophages), CK (AE1/AE3), DAPI. Panel 2: CD20 (B cells), CD45RO (T-memory), CD57 (NK), Granzyme B (NK and cytotoxic), FOXP3 (T reg), CK (AE1/AE3), DAPI. The slides were scanned using the Vectra multispectral microscope and analyzed by the InForm software (PerkinElmer). We quantified and co-localized immune phenotypes (epithelial cells PD-L1+; Total T-cell lymphocytes; T-cells antigen-experienced; Cytotoxic T-cells; Total macrophages; Macrophages PD-L1+; Total B-cell lymphocytes; Memory T cells; Granzyme B+ cells; Regulatory T cells; NK cells) in low grade dysplasia (LGD), high grade dysplasia (HGD), and invasive carcinoma areas in epithelial and stromal compartments. The Wilcoxon rank sum test or the Wilcoxon singed-rank test was used to test the equality of medians between two independent groups or paired samples, respectively. In all patients, HGD areas showed significantly higher density of macrophages (P= 0.041) and lower density of B-cell lymphocytes (P= 0.028) compared with LGD areas in the epithelial/stromal and epithelial compartments, respectively. Similar differences were observed when patients with high-grade IPMN (n=18) were analyzed for immune infiltrates in the HGD areas compared with LGD areas in the same tumor samples: HGD areas showed significantly higher density of macrophages (P= 0.035) and lower density of B-cell lymphocytes (P= 0.031). Patients with high-grade IPMN had significantly lower densities of B-cell lymphocytes (P= 0.011), T-cell lymphocytes (P= 0.034), and memory T-cells (P= 0.031) in the HGD areas when compared with LGD areas from patients with low-grade IPMN, in the epithelial compartment. No significant differences were found between LGD areas from patients who had high-grade IPMN and LGD areas from patients with low-grade IPMN. In conclusion, we characterized the immune landscape of IPMN and identified immune cell biomarkers associated with progression of IPMN. Citation Format: Luisa M. Solis, Naohiro Uraoka, Edwin Roger Parra, Yu Shen, Wei Wei, Mei Jiang, Barbara Mino, Kajsa Affolter, Courtney L. Scaife, Michele T. Yip-Schneider, C. Max Schmidt, Matthew Firpo, Sean Mulvihill, Eugene J. Koay,, Huamin Wang, Ignacio I. Wistuba, Anirban Maitra, Subrata Sen. Characterization of immune profiling of pancreatic intraductal papillary mucinous neoplasm using multiplex immunofluorescence and image analysis approaches [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3121.