Organoid models of human and mouse ductal pancreatic cancer.

Pancreatic cancer is one of the most lethal malignancies due to its late diagnosis and limited response to treatment. Tractable methods to identify and interrogate pathways involved in pancreatic tumorigenesis are urgently needed. We established organoid models from normal and neoplastic murine and human pancreas tissues. Pancreatic organoids can be rapidly generated from resected tumors and biopsies, survive cryopreservation, and exhibit ductal- and disease-stage-specific characteristics. Orthotopically transplanted neoplastic organoids recapitulate the full spectrum of tumor development by forming early-grade neoplasms that progress to locally invasive and metastatic carcinomas. Due to their ability to be genetically manipulated, organoids are a platform to probe genetic cooperation. Comprehensive transcriptional and proteomic analyses of murine pancreatic organoids revealed genes and pathways altered during disease progression. The confirmation of many of these protein changes in human tissues demonstrates that organoids are a facile model system to discover characteristics of this deadly malignancy.

Personalized RNA neoantigen vaccines stimulate T cells in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is lethal in 88% of patients1, yet harbours mutation-derived T cell neoantigens that are suitable for vaccines 2,3. Here in a phase I trial of adjuvant autogene cevumeran, an individualized neoantigen vaccine based on uridine mRNA-lipoplex nanoparticles, we synthesized mRNA neoantigen vaccines in real time from surgically resected PDAC tumours. After surgery, we sequentially administered atezolizumab (an anti-PD-L1 immunotherapy), autogene cevumeran (a maximum of 20 neoantigens per patient) and a modified version of a four-drug chemotherapy regimen (mFOLFIRINOX, comprising folinic acid, fluorouracil, irinotecan and oxaliplatin). The end points included vaccine-induced neoantigen-specific T cells by high-threshold assays, 18-month recurrence-free survival and oncologic feasibility. We treated 16 patients with atezolizumab and autogene cevumeran, then 15 patients with mFOLFIRINOX. Autogene cevumeran was administered within 3 days of benchmarked times, was tolerable and induced de novo high-magnitude neoantigen-specific T cells in 8 out of 16 patients, with half targeting more than one vaccine neoantigen. Using a new mathematical strategy to track T cell clones (CloneTrack) and functional assays, we found that vaccine-expanded T cells comprised up to 10% of all blood T cells, re-expanded with a vaccine booster and included long-lived polyfunctional neoantigen-specific effector CD8+ T cells. At 18-month median follow-up, patients with vaccine-expanded T cells (responders) had a longer median recurrence-free survival (not reached) compared with patients without vaccine-expanded T cells (non-responders; 13.4 months, P = 0.003). Differences in the immune fitness of the patients did not confound this correlation, as responders and non-responders mounted equivalent immunity to a concurrent unrelated mRNA vaccine against SARS-CoV-2. Thus, adjuvant atezolizumab, autogene cevumeran and mFOLFIRINOX induces substantial T cell activity that may correlate with delayed PDAC recurrence.

DNA Methylation Profiling Enables Accurate Classification of Nonductal Primary Pancreatic Neoplasms.

BACKGROUND & AIMS: Cytologic and histopathologic diagnosis of non-ductal pancreatic neoplasms can be challenging in daily clinical practice, whereas it is crucial for therapy and prognosis. The cancer methylome is successfully used as a diagnostic tool in other cancer entities. Here, we investigate if methylation profiling can improve the diagnostic work-up of pancreatic neoplasms. METHODS: DNA methylation data were obtained for 301 primary tumors spanning 6 primary pancreatic neoplasms and 20 normal pancreas controls. Neural Network, Random Forest, and extreme gradient boosting machine learning models were trained to distinguish between tumor types. Methylation data of 29 nonpancreatic neoplasms (n = 3708) were used to develop an algorithm capable of detecting neoplasms of non-pancreatic origin. RESULTS: After benchmarking 3 state-of-the-art machine learning models, the random forest model emerged as the best classifier with 96.9% accuracy. All classifications received a probability score reflecting the confidence of the prediction. Increasing the score threshold improved the random forest classifier performance up to 100% with 87% of samples with scores surpassing the cutoff. Using a logistic regression model, detection of nonpancreatic neoplasms achieved an area under the curve of >0.99. Analysis of biopsy specimens showed concordant classification with their paired resection sample. CONCLUSIONS: Pancreatic neoplasms can be classified with high accuracy based on DNA methylation signatures. Additionally, non-pancreatic neoplasms are identified with near perfect precision. In summary, methylation profiling can serve as a valuable adjunct in the diagnosis of pancreatic neoplasms with minimal risk for misdiagnosis, even in the pre-operative setting.

Comprehensive Characterization of Intraductal Oncocytic Papillary Neoplasm of the Pancreas: A Systematic and Critical Review.

Intraductal oncocytic papillary neoplasm (IOPN) of the pancreas is a recently recognized pancreatic tumor. Here, we aimed to determine its most essential features with the systematic review tool. PubMed, Scopus, and Embase were searched for studies reporting data on pancreatic IOPN. The clinicopathologic, immunohistochemical, and molecular data were extracted and summarized. Then, a comparative analysis of the molecular alterations of IOPN with those of pancreatic ductal adenocarcinoma and intraductal papillary mucinous neoplasm from reference cohorts (including The Cancer Genome Atlas) was conducted. The key findings from 414 IOPNs were as follows: 1) The male-to-female ratio was 1.5:1. Pancreatic head was the most common site (131/237; 55.3%), but a diffuse tumor extension involving more than one pancreatic segment was described in about 1 out of 5 cases (49/237; 20.6%). The mean size was 45.5 mm. An associated invasive carcinoma was present in 50% of cases (168/336). In those cases, most tumors were pT1 or pT2 and pN0 (>80%), and vascular invasion was uncommon (20.6%). Regarding survival, more than 90% of patients were alive after surgical resection. 2) Immunohistochemical and molecular features were as follows. The most commonly expressed mucins were MUC5AC (110/112; 98.2%) and MUC6 (78/84; 92.8%). Compared with pancreatic ductal adenocarcinoma and intraductal papillary mucinous neoplasm, the classic pancreatic drivers KRAS, TP53, CDKN2A, SMAD4, and GNAS were less altered in IOPN (P < .01). Moreover, fusions involving PRKACA or PRKACB gene were detected in all of the 68 cases examined, with PRKACB::ATP1B1 being the most common (27/68 cases; 39.7%). These genomic events emerged as an entity-defining molecular alteration of IOPN (P < .01). Thus, such fusions represent a promising biomarker for diagnostic purposes. Recent evidence also suggests their role in influencing the acquisition of oncocytic morphology. IOPN is a distinct pancreatic neoplasm with specific clinicopathologic and molecular features. Considering the clinical or prognostic implications, its recognition is essential for pathologists and, ultimately, patients' management.

SWI/SNF Complex-Deficient Undifferentiated Carcinoma of the Pancreas: Clinicopathologic and Genomic Analysis.

Inactivating alterations in the SWItch/Sucrose NonFermentable (SWI/SNF) Chromatin Remodeling Complex subunits have been described in multiple tumor types. Recent studies focused on SMARC subunits of this complex to understand their relationship with tumor characteristics and therapeutic opportunities. To date, pancreatic cancer with these alterations has not been well studied, although isolated cases of undifferentiated carcinomas have been reported. Herein, we screened 59 pancreatic undifferentiated carcinomas for alterations in SWI/SNF complex-related (SMARCB1 [BAF47/INI1], SMARCA4 [BRG1], SMARCA2 [BRM]) proteins and/or genes using immunohistochemistry and/or next-generation sequencing. Cases with alterations in SWI/SNF complex-related proteins/genes were compared with cases without alterations, as well as with 96 conventional pancreatic ductal adenocarcinomas (PDAC). In all tumor groups, mismatch repair and PD-L1 protein expression were also evaluated. Thirty of 59 (51%) undifferentiated carcinomas had a loss of SWI/SNF complex-related protein expression or gene alteration. Twenty-seven of 30 (90%) SWI-/SNF-deficient undifferentiated carcinomas had rhabdoid morphology (vs 9/29 [31%] SWI-/SNF-retained undifferentiated carcinomas; P < .001) and all expressed cytokeratin, at least focally. Immunohistochemically, SMARCB1 protein expression was absent in 16/30 (53%) cases, SMARCA2 in 4/30 (13%), and SMARCA4 in 4/30 (13%); both SMARCB1 and SMARCA2 protein expressions were absent in 1/30 (3%). Five of 8 (62.5%) SWI-/SNF-deficient undifferentiated carcinomas that displayed loss of SMARCB1 protein expression by immunohistochemistry were found to have corresponding SMARCB1 deletions by next-generation sequencing. Analysis of canonical driver mutations for PDAC in these cases showed KRAS (2/5) and TP53 (2/5) abnormalities. Median combined positive score for PD-L1 (E1L3N) was significantly higher in the undifferentiated carcinomas with/without SWI/SNF deficiency compared with the conventional PDACs (P < .001). SWI-/SNF-deficient undifferentiated carcinomas were larger (P < .001) and occurred in younger patients (P < .001). Patients with SWI-/SNF-deficient undifferentiated carcinoma had worse overall survival compared with patients with SWI-/SNF-retained undifferentiated carcinoma (P = .004) and PDAC (P < .001). Our findings demonstrate that SWI-/SNF-deficient pancreatic undifferentiated carcinomas are frequently characterized by rhabdoid morphology, exhibit highly aggressive behavior, and have a negative prognostic impact. The ones with SMARCB1 deletions appear to be frequently KRAS wild type. Innovative developmental therapeutic strategies targeting this genomic basis of the SWI/SNF complex and the therapeutic implications of EZH2 inhibition (NCT03213665), SMARCA2 degrader (NCT05639751), or immunotherapy are currently under investigation.

Comparison of Ampullary and Pancreatic Adenocarcinomas: Smaller Invasion, Common Adenomatous Components, Resectability, and Histology are Factors for Improved Survival for Patients with Ampullary Adenocarcinoma.

BACKGROUND: The information on the clinicopathologic/outcome differences between ampullary adenocarcinoma (AC) and pancreatic adenocarcinoma (PC) has been conflicting to the extent that it still is questioned whether ACs need to be recognized separately from PCs. METHODS: The characteristics of 413 ACs were compared with those of 547 PCs. RESULTS: The ACs had a better prognosis than the PCs (5-year survival, 57 % vs 23 %; p < 0.001). Even the pancreatobiliary (PB)-type ACs had a better prognosis (5-year survival, 46 % vs 23 %; p < 0.001). Several differences also were identified as contributing factors: (1) the preinvasive adenomatous component often constituted a significant proportion of the mass in ACs (>50 % of the tumor in 16 % vs 1.5 %; p < 0.001); (2) the mean size of the carcinoma was smaller in ACs (2.5 vs 3.2 cm; p < 0.001): when matched for invasion size, the survival advantage of AC was minimized, and when matched for invasion size larger than 2 cm, the survival advantage of AC lost its statistical significance; (3) lymph node (LN) metastases were less common in ACs (49 % vs 71 %; p < 0.001); (4) the definitive R1 rate was lower in ACs (4 % vs 23.5 %; p < 0.001); and (5) non-PB and non-tubular adenocarcinoma types were more common in ACs (17 % vs 3 %; p < 0.001). CONCLUSIONS: Comparatively, ACs have better clinical survival than PCs. Potential contributing factors are the relative abundance of the preinvasive component, smaller invasion, lower LN metastasis rate, higher resectability, and common occurrence of less aggressive histologic phenotypes (intestinal, medullary, mucinous). However, this survival advantage is sustained even in PB-type ACs, highlighting the importance of accurately determining the site of origin.

Subgrading of G2 Pancreatic Neuroendocrine Tumors as 2A (Ki67 3% to < 10%) Versus 2B (10% to ≤ 20%) Identifies Behaviorally Distinct Subsets in Keeping with the Evolving Management Protocols.

BACKGROUND: Grade 1/2 PanNETs are mostly managed similarly, typically without any adjunct treatment with the belief that their overall metastasis rate is low. In oncology literature, Ki67-index of 10% is increasingly being used as the cutoff in stratifying patients to different protocols, although there are no systematic pathology-based studies supporting this approach. METHODS: Ki67-index was correlated with clinicopathologic parameters in 190 resected PanNETs. A validation cohort (n = 145) was separately analyzed. RESULTS: In initial cohort, maximally selected rank statistics method revealed 12% to be the discriminatory cutoff (close to 10% rule of thumb). G2b cases had liver/distant metastasis rate of almost threefold higher than that of G2a and showed significantly higher frequency of all histopathologic signs of aggressiveness (tumor size, perineural/vascular invasion, infiltrative growth pattern, lymph node metastasis). In validation cohort, these figures were as striking. When all cases were analyzed together, compared with G1, the G2b category had nine times higher liver/distant metastasis rate (6.1 vs. 58.5%; p < 0.001) and three times higher lymph node metastasis rate (20.5 vs. 65.1%; p < 0.001). CONCLUSIONS: G2b PanNETs act very similar to G3, supporting management protocols that regard them as potential therapy candidates. Concerning local management, metastatic behavior in G2b cases indicate they may not be as amenable for conservative approaches, such as watchful waiting or enucleation. This substaging should be considered into diagnostic guidelines, and clinical trials need to be devised to determine the more appropriate management protocols for G2b (10% to ≤ 20%) group, which shows liver/distant metastasis in more than half of the cases, which at minimum warrants closer follow-up.

Integrated Genomic and Clinicopathologic Approach Distinguishes Pancreatic Grade 3 Neuroendocrine Tumor From Neuroendocrine Carcinoma and Identifies a Subset With Molecular Overlap.

Distinguishing grade 3 pancreatic neuroendocrine tumor (G3 PanNET) from neuroendocrine carcinoma (PanNEC) is a known diagnostic challenge, and accurate classification is critical because clinical behavior and therapies differ. Although current recommendations suggest that immunohistochemistry for p53, Rb, ATRX, and DAXX can distinguish most cases, some cases remain difficult to classify using this approach. In this study, we reviewed 47 high-grade neoplasms originally diagnosed as pancreatic neuroendocrine neoplasms. In addition to the currently recommended stains, we performed capture-based sequencing of approximately 500 cancer genes and immunohistochemistry for p16 and trypsin or chymotrypsin. Using an integrated molecular and clinicopathologic approach, 42 (89%) of 47 cases had a clear final diagnosis of either G3 PanNET (n = 17), PanNEC (n = 17), or mixed acinar-NEC (n = 8). The 17 G3 PanNETs demonstrated frequent alterations in MEN1 (71%), DAXX (47%), ATRX (24%), TSC2 (35%), SETD2 (42%), and CDKN2A (41%). Contrary to prior reports, TP53 alterations were also common in G3 PanNETs (35%) but were always mutually exclusive with CDKN2A alterations in this group. The 17 PanNECs demonstrated frequent alterations in TP53 (88%), cell cycle genes RB1 (47%), CCNE1/CCND1 (12%), CDKN2A (29%), and in KRAS (53%) and SMAD4 (41%); TP53 was coaltered with a cell cycle gene in 76% of PanNECs. Diffuse strong p16 staining was observed in 69% of PanNECs in contrast to 0% of G3 PanNETs. The 8 acinar-NECs had recurrent alterations in ATM (25%), APC (25%), and STK11 (25%). Five cases remained difficult to classify, 3 of which exhibited overlapping molecular features with alterations in MEN1 with or without ATRX, and RB1 with or without TP53, making it unclear whether to classify as PanNET or PanNEC. Our data demonstrate that molecular profiling and immunohistochemistry for p16 greatly improve the diagnostic accuracy of high-grade pancreatic neuroendocrine neoplasms and identify a subset of rare cases with overlapping features of both PanNET and PanNEC.

Intraductal neoplasms of the pancreatobiliary tract: navigating the alphabet.

Cancers of the pancreatobiliary tract are diseases with unfavourable prognoses. In the last couple of decades, two types of lesions have been described as precursors that precede pancreatobiliary cancers. These include incidental microscopic (flat) lesions known as pancreatic intra-epithelial neoplasia and biliary intra-epithelial neoplasia, and grossly visible, mass-forming lesions (tumoral intra-epithelial neoplasia) including intraductal papillary mucinous neoplasms, intraductal oncocytic papillary neoplasms, intraductal tubulopapillary neoplasms, intraductal papillary neoplasms of the bile duct and intracholecystic papillary neoplasms. Early detection and adequate treatment of these precursor lesions, especially the second group, have the potential to prevent pancreatobiliary cancer or at least improve its prognosis. In this review, we discuss their histopathology and recent updates on molecular profiling of these intraductal neoplasms of the pancreatobiliary tract.

Systematic review of challenging issues in pathology of intraductal papillary mucinous neoplasms.

BACKGROUND: Intraductal papillary mucinous neoplasms (IPMNs) are a cystic precursor to pancreatic cancer. IPMNs deemed clinically to be at high-risk for malignant progression are frequently treated with surgical resection, and pathological examination of the pancreatectomy specimen is a key component of the clinical care of IPMN patients. METHODS: Systematic literature reviews were conducted around eight topics of clinical relevance in the examination of pathological specimens in patients undergoing resection of IPMN. RESULTS: This review provides updated perspectives on morphological subtyping of IPMNs, classification of intraductal oncocytic papillary neoplasms, nomenclature for high-grade dysplasia, assessment of T stage, distinction of carcinoma associated or concomitant with IPMN, role of molecular assessment of IPMN tissue, role of intraoperative assessment by frozen section, and preoperative evaluation of cyst fluid cytology. CONCLUSIONS: This analysis provides the foundation for data-driven approaches to several challenging issues in the pathology of IPMNs.

Identification of unique neoantigen qualities in long-term survivors of pancreatic cancer.

Pancreatic ductal adenocarcinoma is a lethal cancer with fewer than 7% of patients surviving past 5 years. T-cell immunity has been linked to the exceptional outcome of the few long-term survivors, yet the relevant antigens remain unknown. Here we use genetic, immunohistochemical and transcriptional immunoprofiling, computational biophysics, and functional assays to identify T-cell antigens in long-term survivors of pancreatic cancer. Using whole-exome sequencing and in silico neoantigen prediction, we found that tumours with both the highest neoantigen number and the most abundant CD8+ T-cell infiltrates, but neither alone, stratified patients with the longest survival. Investigating the specific neoantigen qualities promoting T-cell activation in long-term survivors, we discovered that these individuals were enriched in neoantigen qualities defined by a fitness model, and neoantigens in the tumour antigen MUC16 (also known as CA125). A neoantigen quality fitness model conferring greater immunogenicity to neoantigens with differential presentation and homology to infectious disease-derived peptides identified long-term survivors in two independent datasets, whereas a neoantigen quantity model ascribing greater immunogenicity to increasing neoantigen number alone did not. We detected intratumoural and lasting circulating T-cell reactivity to both high-quality and MUC16 neoantigens in long-term survivors of pancreatic cancer, including clones with specificity to both high-quality neoantigens and predicted cross-reactive microbial epitopes, consistent with neoantigen molecular mimicry. Notably, we observed selective loss of high-quality and MUC16 neoantigenic clones on metastatic progression, suggesting neoantigen immunoediting. Our results identify neoantigens with unique qualities as T-cell targets in pancreatic ductal adenocarcinoma. More broadly, we identify neoantigen quality as a biomarker for immunogenic tumours that may guide the application of immunotherapies.

Pancreatobiliary Maljunction-associated Gallbladder Cancer Is as Common in the West, Shows Distinct Clinicopathologic Characteristics and Offers an Invaluable Model for Anatomy-induced Reflux-associated Physio-chemical Carcinogenesis.

OBJECTIVE: To determine the associations of pancreatobiliary maljunction (PBM) in the West. BACKGROUND: PBM (anomalous union of common bile duct and pancreatic duct) is mostly regarded as an Asian-only disorder, with 200X risk of gallbladder cancer (GBc), attributed to reflux of pancreatic enzymes. Methods: Radiologic images of 840 patients in the US who underwent pancreatobiliary resections were reviewed for PBM and contrasted with 171 GBC cases from Japan. RESULTS: Eight % of the US GBCs (24/300) had PBM (similar to Japan; 15/ 171, 8.8%), in addition to 1/42 bile duct carcinomas and 5/33 choledochal cysts. None of the 30 PBM cases from the US had been diagnosed as PBM in the original work-up. PBM was not found in other pancreatobiliary disorders. Clinicopathologic features of the 39 PBM-associated GBCs (US:24, Japan:15) were similar; however, comparison with non-PBM GBCs revealed that they occurred predominantly in females (F/M = 3); at younger (<50-year-old) age (21% vs 6.5% in non-PBM GBCs; P = 0.01); were uncommonly associated with gallstones (14% vs 58%; P < 0.001); had higher rate of tumor-infiltrating lymphocytes (69% vs 44%; P = 0.04); arose more often through adenoma-carcinoma sequence (31% vs 12%; P = 0.02); and had a higher proportion of nonconventional carcinomas (21% vs 7%; P = 0.03). Conclusions: PBM accounts for 8% of GBCs also in the West but is typically undiagnosed. PBM-GBCs tend to manifest in younger age and often through adenoma-carcinoma sequence, leading to unusual carcinoma types. If PBM is encountered, cholecystectomy and surveillance of bile ducts is warranted. PBM-associated GBCs offer an invaluable model for variant anatomy-induced chemical (reflux-related) carcinogenesis.

Pancreatic ductal adenocarcinomas associated with intraductal papillary mucinous neoplasms (IPMNs) versus pseudo-IPMNs: relative frequency, clinicopathologic characteristics and differential diagnosis.

The literature is highly conflicted on what percentage of pancreatic ductal adenocarcinomas (PDACs) arise in association with intraductal papillary mucinous neoplasms (IPMNs). Some studies have claimed that even small (Sendai-negative) IPMNs frequently lead to PDAC. Recently, more refined pathologic definitions for mucin-lined cysts were provided in consensus manuscripts, but so far there is no systematic analysis regarding the frequency and clinicopathologic characteristics of IPMN-mimickers, i.e., pseudo-IPMNs. In this study, as the first step in establishing frequency, we performed a systematic review of the pathologic findings in 501 consecutive ordinary PDACs, which disclosed that 10% of PDACs had associated cysts ≥1 cm. While 31 (6.2%) of these were IPMN or mucinous cystic neoplasm (MCN), 19 (3.8%) were other cyst types that mimicked IPMN (pseudo-IPMNs) per recent WHO/consensus criteria. As the second step of the study, we performed a comparative clinicopathologic analysis by also including our entire surgical pathology/consultation databases that was comprised of 60 IPMN-associated PDACs, 30 MCN-associated PDACs and 40 pseudo-IPMN-associated PDACs. We found that 84% of true IPMNs were pre-operatively recognized, whereas IPMN was considered in differential diagnosis of 33% of pseudo-IPMNs. Of the 40 pseudo-IPMNs, there were 15 secondary duct ectasias; 6 large-duct-type PDACs; 5 pseudocysts; 5 cystic tumor necrosis; 4 simple mucinous cysts; 3 groove pancreatitis-associated paraduodenal wall cysts; and 2 congenital cysts. Microscopically, pseudo-IPMNs had at least partial mucinous-lining mimicking IPMN but had smaller cystic (mean = 1.9 cm) and larger PDAC (mean = 3.8 cm) components compared to true IPMNs (cyst = 5.7 cm; PDAC = 2.0 cm). In summary, in this pathologically verified analysis that utilized refined criteria, 10% of PDACs were discovered to have cysts ≥1 cm, about two-thirds of which were IPMN/MCN but about one-third were pseudo-IPMNs. True IPMNs underlying the PDACs are often large and are already diagnosed pre-operatively as having an IPMN component, whereas only a third of the pseudo-IPMNs receive IPMN diagnosis by imaging and their cysts are smaller. At the histopathologic level, pseudo-IPMNs are highly prone to misdiagnosis as IPMN, which presumably accounts for much higher association of IPMNs with PDAC as reported in some studies. The subtle but salient characteristics of pseudo-IPMNs elucidated in this study should be combined with careful radiological/clinical correlation in order to exclude pseudo-IPMNs.

Pancreatoblastomas and mixed and pure acinar cell carcinomas share epigenetic signatures distinct from other neoplasms of the pancreas.

Pancreatic neoplasms are heterogenous and have traditionally been classified by assessing their lines of cellular differentiation using histopathologic methods, particularly morphologic and immunohistochemical evaluation. These methods frequently identify overlapping differentiation along ductal, acinar, and neuroendocrine lines, raising diagnostic challenges as well as questions regarding the relationship of these neoplasms. Neoplasms with acinar differentiation, in particular, frequently show more than one line of differentiation based on immunolabeling. Genome methylation signatures, in contrast, are better conserved within cellular lineages, and are increasingly used to support the classification of neoplasms. We characterized the epigenetic relationships between pancreatoblastomas, acinar cell carcinomas (including mixed variants), pancreatic neuroendocrine tumors, solid pseudopapillary neoplasms, and pancreatic ductal adenocarcinomas using a genome-wide array platform. Using unsupervised learning approaches, pancreatic neuroendocrine tumors, solid pseudopapillary neoplasms, ductal adenocarcinomas, and normal pancreatic tissue samples all localized to distinct clusters based on their methylation profiles, whereas all neoplasms with acinar differentiation occupied a broad overlapping region located between the predominantly acinar normal pancreatic tissue and ductal adenocarcinoma clusters. Our data provide evidence to suggest that acinar cell carcinomas and pancreatoblastomas are similar at the epigenetic level. These findings are consistent with genomic and clinical observations that mixed acinar neoplasms are closely related to pure acinar cell carcinomas rather than to neuroendocrine tumors or ductal adenocarcinomas.

Infiltration pattern predicts metastasis and progression better than the T-stage and grade in pancreatic neuroendocrine tumors: a proposal for a novel infiltration-based morphologic grading.

The advancing edge profile is a powerful determinant of tumor behavior in many organs. In this study, a grading system assessing the tumor-host interface was developed and tested in 181 pancreatic neuroendocrine tumors (PanNETs), 63 of which were <=2 cm. Three tumor slides representative of the spectrum (least, medium, and most) of invasiveness at the advancing edge of the tumor were selected, and then each slide was scored as follows. Well-demarcated/encapsulated, 1 point; Mildly irregular borders and/or minimal infiltration into adjacent tissue, 2 points; Infiltrative edges with several clusters beyond the main tumor but still relatively close, and/or satellite demarcated nodules, 3 points; No demarcation, several cellular clusters away from the tumor, 4 points; Exuberantly infiltrative pattern, scirrhous growth, dissecting the normal parenchymal elements, 5 points. The sum of the rankings on the three slides was obtained. Cases with scores of 3-6 were defined as "non/minimally infiltrative" (NI; n = 77), 7-9 as "moderately infiltrative" (MI; n = 68), and 10-15 as "highly infiltrative" (HI; n = 36). In addition to showing a statistically significant correlation with all the established signs of aggressiveness (grade, size, T-stage), this grading system was found to be the most significant predictor of adverse outcomes (metastasis, progression, and death) on multivariate analysis, more strongly than T-stage, while Ki-67 index did not stand the multivariate test. As importantly, cases <=2 cm were also stratified by this grading system rendering it applicable also to this group that is currently placed in "watchful waiting" protocols. In conclusion, the proposed grading system has a strong, independent prognostic value and therefore should be considered for integration into routine pathology practice after being evaluated in validation studies with larger series.

Histopathological tumour response scoring in resected pancreatic cancer following neoadjuvant therapy: international interobserver study (ISGPP-1).

BACKGROUND: Most tumour response scoring systems for resected pancreatic cancer after neoadjuvant therapy score tumour regression. However, whether treatment-induced changes, including tumour regression, can be identified reliably on haematoxylin and eosin-stained slides remains unclear. Moreover, no large study of the interobserver agreement of current tumour response scoring systems for pancreatic cancer exists. This study aimed to investigate whether gastrointestinal/pancreatic pathologists can reliably identify treatment effect on tumour by histology, and to determine the interobserver agreement for current tumour response scoring systems. METHODS: Overall, 23 gastrointestinal/pancreatic pathologists reviewed digital haematoxylin and eosin-stained slides of pancreatic cancer or treated tumour bed. The accuracy in identifying the treatment effect was investigated in 60 patients (30 treatment-naive, 30 after neoadjuvant therapy (NAT)). The interobserver agreement for the College of American Pathologists (CAP) and MD Anderson Cancer Center (MDACC) tumour response scoring systems was assessed in 50 patients using intraclass correlation coefficients (ICCs). An ICC value below 0.50 indicated poor reliability, 0.50 or more and less than 0.75 indicated moderate reliability, 0.75 or more and below 0.90 indicated good reliability, and above 0.90 indicated excellent reliability. RESULTS: The sensitivity and specificity for identifying NAT effect were 76.2 and 49.0 per cent respectively. After NAT in 50 patients, ICC values for both tumour response scoring systems were moderate: 0.66 for CAP and 0.71 for MDACC. CONCLUSION: Identification of the effect of NAT in resected pancreatic cancer proved unreliable, and interobserver agreement for the current tumour response scoring systems was suboptimal. These findings support the recently published International Study Group of Pancreatic Pathologists recommendations to score residual tumour burden rather than tumour regression after NAT.

Intraductal tubulopapillary neoplasm (ITPN) of the pancreas: a distinct entity among pancreatic tumors.

AIMS: Intraductal tubulopapillary neoplasm (ITPN) of the pancreas is a recently recognized pancreatic tumor entity. Here we aimed to determine the most important features with a systematic review coupled with an integrated statistical approach. METHODS AND RESULTS: PubMed, SCOPUS, and Embase were searched for studies reporting data on pancreatic ITPN. The clinicopathological, immunohistochemical, and molecular data were summarized. Then a comprehensive survival analysis and a comparative analysis of the molecular alterations of ITPN with those of pancreatic ductal adenocarcinoma (PDAC) and intraductal papillary mucinous neoplasm (IPMN) from reference cohorts (including the International Cancer Genome Consortium- ICGC dataset and The Cancer Genome Atlas, TCGA program) were conducted. The core findings of 128 patients were as follows: (i) Clinicopathological parameters: pancreatic head is the most common site; presence of an associated adenocarcinoma was reported in 60% of cases, but with rare nodal metastasis. (ii) Immunohistochemistry: MUC1 (>90%) and MUC6 (70%) were the most frequently expressed mucins. ITPN lacked the intestinal marker MUC2; unlike IPMN, it did not express MUC5AC. (iii) Molecular landscape: Compared with PDAC/IPMN, the classic pancreatic drivers KRAS, TP53, CDKN2A, SMAD4, GNAS, and RNF43 were less altered in ITPN (P < 0.001), whereas MCL amplifications, FGFR2 fusions, and PI3KCA mutations were commonly altered (P < 0.001). (iv) Survival analysis: ITPN with a "pure" branch duct involvement showed the lowest risk of recurrence. CONCLUSION: ITPN is a distinct pancreatic neoplasm with specific clinicopathological and molecular characteristics. Its recognition is fundamental for its clinical/prognostic implications and for the enrichment of potential targets for precision oncology.

Calcifying nested stromal-epithelial tumor: a clinicopathologic and molecular genetic study of eight cases highlighting metastatic potential and recurrent CTNNB1 and TERT promoter alterations.

Calcifying nested stromal-epithelial tumor (CNSET) is a rare hepatic tumor that occurs in children and young adults. With <40 cases in the literature, the mechanism for tumorigenesis and the biological behavior of CNSET remain uncertain. Here, we studied the clinicopathologic and molecular genetic features of eight CNSETs. Six patients (75%) were female, and the median age at presentation was 22.5 years (range 14-34 years). The median tumor size was 14 cm (range 2.7-18 cm). All tumors had fibrous stroma that contained organoid nests of epithelioid to spindled tumor cells with moderate amounts of palely eosinophilic cytoplasm and ovoid, vesicular nuclei. Five tumors showed calcifications, and one showed lymphovascular invasion. Necrosis was absent in all. Immunohistochemistry demonstrated nuclear ß-catenin expression in five of five tested tumors and focal to diffuse nuclear WT-1 positivity in five of seven. Hepatocellular markers (HepPar-1, arginase-1, and albumin in situ hybridization) and neuroendocrine markers (synaptophysin, chromogranin, and INSM1) were uniformly negative. Next-generation sequencing demonstrated CTNNB1 alterations in all seven sequenced tumors. Sanger sequencing demonstrated TERT promoter mutations in all six sequenced tumors. Clinical follow-up was available for seven patients (median duration 4.4 years; range 1.2-6.2 years): four (57%) developed metastatic disease; all four developed lung metastases; and two also had abdominal metastases. All four patients with metastatic disease also had persistent or recurrent liver tumors. Three patients with metastases were alive with disease at the most recent follow-up and one died of disease. The other three patients with available follow-up did not develop metastasis or recurrence. One tumor treated with neoadjuvant chemotherapy showed no response, and another showed 90% tumor fibrosis; the latter patient remained disease-free at 6.2 years of follow-up. Our series demonstrates the presence of TERT promoter mutations and CTNNB1 alterations in all sequenced tumors and suggests that CNSET might perhaps be more aggressive than previously reported.

Amsterdam International Consensus Meeting: tumor response scoring in the pathology assessment of resected pancreatic cancer after neoadjuvant therapy.

Histopathologically scoring the response of pancreatic ductal adenocarcinoma (PDAC) to neoadjuvant treatment can guide the selection of adjuvant therapy and improve prognostic stratification. However, several tumor response scoring (TRS) systems exist, and consensus is lacking as to which system represents best practice. An international consensus meeting on TRS took place in November 2019 in Amsterdam, The Netherlands. Here, we provide an overview of the outcomes and consensus statements that originated from this meeting. Consensus (≥80% agreement) was reached on a total of seven statements: (1) TRS is important because it provides information about the effect of neoadjuvant treatment that is not provided by other histopathology-based descriptors. (2) TRS for resected PDAC following neoadjuvant therapy should assess residual (viable) tumor burden instead of tumor regression. (3) The CAP scoring system is considered the most adequate scoring system to date because it is based on the presence and amount of residual cancer cells instead of tumor regression. (4) The defining criteria of the categories in the CAP scoring system should be improved by replacing subjective terms including "minimal" or "extensive" with objective criteria to evaluate the extent of viable tumor. (5) The improved, consensus-based system should be validated retrospectively and prospectively. (6) Prospective studies should determine the extent of tissue sampling that is required to ensure adequate assessment of the residual cancer burden, taking into account the heterogeneity of tumor response. (7) In future scientific publications, the extent of tissue sampling should be described in detail in the "Materials and methods" section.

Dysplasia and carcinoma of the gallbladder: pathological evaluation, sampling, differential diagnosis and clinical implications.

Pathological evaluation of gallbladder neoplasia remains a challenge. A significant proportion of cases presents as clinically and grossly inapparent lesions, and grossing protocols are not well established. Among epithelial alterations, pseudo-pyloric gland metaplasia is ubiquitous and of no apparent consequence, whereas goblet cell metaplasia and a foveolar change in surface cells require closer attention. Low-grade dysplasia is difficult to objectively define and appears to be clinically inconsequential by itself; however, extra sampling is required to exclude the possibility of accompanying more significant lesions. For high-grade dysplasia ('high-grade BilIN', also known as 'carcinoma in situ'), a complete sampling is necessary to rule out invasion. Designating in-situ or minimally invasive carcinomas limited to muscularis or above as early gallbladder carcinoma (EGBC) helps to alleviate the major geographical differences (West/East) in the criteria for 'invasiveness' to assign a case to pTis or pT1. Total sampling is crucial in proper diagnosis of such cases. A subset of invasive GBCs (5-10%) arise from the intracholecystic neoplasm (ICN, 'adenoma-carcinoma sequence') category. Approximately two-thirds of ICNs have invasive carcinoma. However, this propensity differs by subtype. True 'pyloric gland adenomas' (> 1 cm) are uncommon and scarcely associated with invasive carcinoma. A distinct subtype of ICN composed of tubular, non-mucinous MUC6+ glands [intracholecystic tubular non-mucinous neoplasm (ICTN)] forms a localised pedunculated polyp. Although it is morphologically complex and high-grade, it appears to be invasion-resistant. Some of the invasive carcinoma types in the gallbladder have been better characterised recently with adenosquamous, neuroendocrine, poorly cohesive and mucinous carcinomas often being more advanced and aggressive.