Loss of ATG4B and ATG4A results in two-stage cell cycle defects in pancreatic ductal adenocarcinoma cells.

Pancreatic ductal adenocarcinoma (PDAC) exhibits elevated levels of autophagy, which promote tumor progression and treatment resistance. ATG4B is an autophagy-related cysteine protease under consideration as a potential therapeutic target, but it is largely unexplored in PDAC. Here, we investigated the clinical and functional relevance of ATG4B expression in PDAC. Using two PDAC patient cohorts, we found that low ATG4B mRNA or protein expression is associated with worse patient survival outcomes, poorly differentiated PDAC tumors and a lack of survival benefit from adjuvant chemotherapy. In PDAC cell lines, ATG4B knockout reduced proliferation, abolished processing of LC3B (also known as MAP1LC3B), and reduced GABARAP and GABARAPL1 levels, but increased ATG4A levels. ATG4B and ATG4A double knockout lines displayed a further reduction in proliferation, characterized by delays in G1-S phase transition and mitosis. Pro-LC3B accumulated aberrantly at the centrosome with a concomitant increase in centrosomal proteins PCM1 and CEP131, which was rescued by exogenous ATG4B. The two-stage cell cycle defects following ATG4B and ATG4A loss have important therapeutic implications for PDAC.

Standardizing Randomized Controlled Trials in Celiac Disease: An International Multidisciplinary Appropriateness Study.

BACKGROUND & AIMS: There is a need to develop safe and effective pharmacologic options for the treatment of celiac disease (CeD); however, consensus on the appropriate design and configuration of randomized controlled trials (RCTs) in this population is lacking. METHODS: A 2-round modified Research and Development/University of California Los Angeles Appropriateness Method study was conducted. Eighteen gastroenterologists (adult and pediatric) and gastrointestinal pathologists voted on statements pertaining to the configuration of CeD RCTs, inclusion and exclusion criteria, gluten challenge, and trial outcomes. Two RCT designs were considered, representing the following distinct clinical scenarios for which pharmacotherapy may be used: trials incorporating a gluten challenge to simulate exposure; and trials evaluating reversal of histologic changes, despite attempted adherence to a gluten-free diet. Each statement was rated as appropriate, uncertain, or inappropriate, using a 9-point Likert scale. RESULTS: For trials evaluating prevention of relapse after gluten challenge, participants adherent to a gluten-free diet for 12 months or more with normal or near-normal-sized villi should be enrolled. Gluten challenge should be FODMAPS (fermentable oligosaccharides, disaccharides, monosaccharides, and polyols) free, and efficacy evaluated using histology with a secondary patient-reported outcome measure. For trials evaluating reversal of villus atrophy, the panel voted it appropriate to enroll participants with a baseline villus height to crypt depth ratio ≤2 and measure efficacy using a primary histologic end point. Guidance for measuring histologic, endoscopic, and patient-reported outcomes in adult and pediatric patients with CeD are provided, along with recommendations regarding the merits and limitations of different end points. CONCLUSIONS: We developed standardized recommendations for clinical trial design, eligibility criteria, outcome measures, gluten challenge, and disease evaluations for RCTs in patients with CeD.

Reliability and Responsiveness of Histologic Indices for the Assessment of Crohn's Disease Activity.

BACKGROUND & AIMS: The operating properties of histologic indices for evaluating Crohn's disease (CD) activity are poorly characterized. We assessed the reliability and responsiveness of existing histologic indices/items used in CD and ulcerative colitis (UC), in addition to 3 novel items, and developed exploratory ileal, colonic, and colonic-ileal CD instruments. METHODS: Blinded central readers independently reviewed paired baseline and week 12 image sets from the EXTEND trial. Disease activity was scored using 4 indices (the Global Histologic Activity Score, Geboes Score, Nancy Histological Index, and Robarts Histopathology Index) and 3 items identified by an expert panel (mucin depletion, basal plasmacytosis, and ileal pyloric gland metaplasia). Reliability and responsiveness were quantified using the intraclass correlation coefficient (ICC) and area under the receiver operating curve (AUC), respectively. Exploratory indices were developed using backward stepwise linear regression analysis. Candidate independent variables were items with an inter-rater ICC ≥0.40 and AUC ≥0.56. The dependent variable was histologic disease activity measured by a 100-mm visual analogue scale. RESULTS: Paired image sets were available from 55 patients. Substantial to almost perfect inter-rater reliability (ICC, 0.63-0.87) and some responsiveness (AUC, 0.57-0.94) were observed for all existing indices regardless of whether individual colonic and ileal segments, combined colonic segments, or combined colonic and ileal segments were assessed and the calculation method used. Five items were tested as candidate items, and exploratory colonic, ileal, and colonic-ileal indices were developed. CONCLUSIONS: CD and UC indices were similarly reliable and responsive in measuring histologic CD activity. Exploratory index development did not offer benefit over current histologic instruments.

Quantitative Pathologic Analysis of Digitized Images of Colorectal Carcinoma Improves Prediction of Recurrence-Free Survival.

BACKGROUND & AIMS: To examine whether quantitative pathologic analysis of digitized hematoxylin and eosin slides of colorectal carcinoma (CRC) correlates with clinicopathologic features, molecular alterations, and prognosis. METHODS: A quantitative segmentation algorithm (QuantCRC) was applied to 6468 digitized hematoxylin and eosin slides of CRCs. Fifteen parameters were recorded from each image and tested for associations with clinicopathologic features and molecular alterations. A prognostic model was developed to predict recurrence-free survival using data from the internal cohort (n = 1928) and validated on an internal test (n = 483) and external cohort (n = 938). RESULTS: There were significant differences in QuantCRC according to stage, histologic subtype, grade, venous/lymphatic/perineural invasion, tumor budding, CD8 immunohistochemistry, mismatch repair status, KRAS mutation, BRAF mutation, and CpG methylation. A prognostic model incorporating stage, mismatch repair, and QuantCRC resulted in a Harrell's concordance (c)-index of 0.714 (95% confidence interval [CI], 0.702-0.724) in the internal test and 0.744 (95% CI, 0.741-0.754) in the external cohort. Removing QuantCRC from the model reduced the c-index to 0.679 (95% CI, 0.673-0.694) in the external cohort. Prognostic risk groups were identified, which provided a hazard ratio of 2.24 (95% CI, 1.33-3.87, P = .004) for low vs high-risk stage III CRCs and 2.36 (95% CI, 1.07-5.20, P = .03) for low vs high-risk stage II CRCs, in the external cohort after adjusting for established risk factors. The predicted median 36-month recurrence rate for high-risk stage III CRCs was 32.7% vs 13.4% for low-risk stage III and 15.8% for high-risk stage II vs 5.4% for low-risk stage II CRCs. CONCLUSIONS: QuantCRC provides a powerful adjunct to routine pathologic reporting of CRC. A prognostic model using QuantCRC improves prediction of recurrence-free survival.

An International Consensus to Standardize Integration of Histopathology in Ulcerative Colitis Clinical Trials.

BACKGROUND & AIMS: Histopathology is an emerging treatment target in ulcerative colitis (UC) clinical trials. Our aim was to provide guidance on standardizing biopsy collection protocols, identifying optimal evaluative indices, and defining thresholds for histologic response and remission after treatment. METHODS: An international, interdisciplinary expert panel of 19 gastroenterologists and gastrointestinal pathologists was assembled. A modified RAND/University of California, Los Angeles appropriateness methodology was used to address relevant issues. A total of 138 statements were derived from a systematic review of the literature and expert opinion. Each statement was anonymously rated as appropriate, uncertain, or inappropriate using a 9-point scale. Survey results were reviewed and discussed before a second round of voting. RESULTS: Histologic measurements collected using a uniform biopsy strategy are important for assessing disease activity and determining therapeutic efficacy in UC clinical trials. Multiple biopsy strategies were deemed acceptable, including segmental biopsies collected according to the endoscopic appearance. Biopsies should be scored for architectural change, lamina propria chronic inflammation, basal plasmacytosis, lamina propria and epithelial neutrophils, epithelial damage, and erosions/ulcerations. The Geboes score, Robarts Histopathology Index, and Nancy Index were considered appropriate for assessing histologic activity; use of the modified Riley score and Harpaz Index were uncertain. Histologic activity at baseline should be required for enrollment, recognizing this carries operational implications. Achievement of histologic improvement or remission was considered an appropriate and realistic therapeutic target. Current histologic indices require validation for pediatric populations. CONCLUSIONS: These recommendations provide a framework for standardized implementation of histopathology in UC trials. Additional work is required to address operational considerations and areas of uncertainty.

Genomic Features and Classification of Homologous Recombination Deficient Pancreatic Ductal Adenocarcinoma.

BACKGROUND AND AIMS: Homologous recombination deficiency (HRD) in pancreatic ductal adenocarcinoma (PDAC), remains poorly defined beyond germline (g) alterations in BRCA1, BRCA2, and PALB2. METHODS: We interrogated whole genome sequencing (WGS) data on 391 patients, including 49 carriers of pathogenic variants (PVs) in gBRCA and PALB2. HRD classifiers were applied to the dataset and included (1) the genomic instability score (GIS) used by Myriad's MyChoice HRD assay; (2) substitution base signature 3 (SBS3); (3) HRDetect; and (4) structural variant (SV) burden. Clinical outcomes and responses to chemotherapy were correlated with HRD status. RESULTS: Biallelic tumor inactivation of gBRCA or PALB2 was evident in 43 of 49 germline carriers identifying HRD-PDAC. HRDetect (score ≥0.7) predicted gBRCA1/PALB2 deficiency with highest sensitivity (98%) and specificity (100%). HRD genomic tumor classifiers suggested that 7% to 10% of PDACs that do not harbor gBRCA/PALB2 have features of HRD. Of the somatic HRDetecthi cases, 69% were attributed to alterations in BRCA1/2, PALB2, RAD51C/D, and XRCC2, and a tandem duplicator phenotype. TP53 loss was more common in BRCA1- compared with BRCA2-associated HRD-PDAC. HRD status was not prognostic in resected PDAC; however in advanced disease the GIS (P = .02), SBS3 (P = .03), and HRDetect score (P = .005) were predictive of platinum response and superior survival. PVs in gATM (n = 6) or gCHEK2 (n = 2) did not result in HRD-PDAC by any of the classifiers. In 4 patients, BRCA2 reversion mutations associated with platinum resistance. CONCLUSIONS: Germline and parallel somatic profiling of PDAC outperforms germline testing alone in identifying HRD-PDAC. An additional 7% to 10% of patients without gBRCA/PALB2 mutations may benefit from DNA damage response agents.

Responsiveness of a Histologic Scoring System Compared With Peak Eosinophil Count in Eosinophilic Esophagitis.

INTRODUCTION: The optimal instrument for assessing histologic disease activity in patients with eosinophilic esophagitis (EoE) is unclear. We assessed the responsiveness of the EoE Histologic Scoring System (EoE-HSS) when compared with that of the peak eosinophil count (PEC). METHODS: Histopathology slides were obtained from patients with EoE at baseline and after 8 weeks of treatment with swallowed topical budesonide or elimination diet. Two blinded gastrointestinal pathologists scored biopsies on the EoE-HSS, PEC, and 100-mm visual analog scale (VAS) of overall histologic severity. Change was defined as an improvement by ≥0.5 SD in baseline VAS. Responsiveness was quantified using the standardized effect size (SES) and the probability that the index distinguishes a patient with improvement from a patient without improvement, which is the area under the receiver operating characteristic curve (AUC). Longitudinal validity was assessed using Pearson correlations between changes in EoE-HSS and both PEC and VAS. RESULTS: The EoE-HSS grade (SES 2.18 [95% confidence interval, CI: 1.46-2.88]; AUC 0.73 [95% CI: 0.57-0.84]) and stage (SES 2.07 [95% CI: 1.37-2.77]; AUC 0.73 [95% CI: 0.58-0.84]) were highly responsive, similar to PEC (SES 1.44 [95% CI: 0.80-2.07]; AUC 0.73 [95% CI: 0.58-0.84]). The EoE-HSS grade and stage were more highly correlated with changes in VAS (grade 0.92 [95% CI: 0.86-0.95]; stage 0.89 [95% CI: 0.81-0.94]) than with changes in PEC (grade 0.74 [95% CI: 0.58-0.85]; stage 0.66 [95% CI: 0.47-0.80]). DISCUSSION: The EoE-HSS is highly responsive, performs similarly to PEC, and is better correlated with changes in overall histologic activity in patients with EoE.

Serrated Lesion Detection in a Population-based Colon Screening Program.

BACKGROUND: Serrated lesions give rise to 15% to 30% of all colorectal cancers, driven predominantly by the sessile serrated polyp (SSP). Fecal immunochemical test (FIT), has low sensitivity for SSPs. SSP detection rate (SSPDR) is influenced by performance of both endoscopists and pathologists, as diagnosis can be subtle both on endoscopy and histology. GOALS: To evaluate the SSPDR in a population-based screening program, and the influence of subspecialty trained pathologists on provincial reporting practices. STUDY: The colon screening program database was used to identify all FIT-positive patients that received colonoscopy between January 2014 and June 2017. Patient demographics, colonoscopy quality indicators, pathologic diagnoses, and FIT values were collected. This study received IRB approval. RESULTS: A total of 74,605 colonoscopies were included and 26.6% had at least 1 serrated polyp removed. The SSPDR was 7.0%, with 59% of the SSPs detected having a concurrent conventional adenoma. The mean FIT value for colonoscopies with only serrated lesions was less than that for colonoscopies with a conventional adenoma or colorectal cancer (P<0.0001). Centers with a gastrointestinal subspecialty pathologist diagnosed proportionally more SSPs (P<0.0001), and right-sided SSPs than centers without subspecialists. CONCLUSIONS: Serrated lesions often occur in conjunction with conventional adenomas and are associated with lower FIT values. Knowledge of the characteristics of SSPs is essential for pathologists to ensure accurate diagnosis of SSPs.

Modelling hereditary diffuse gastric cancer initiation using transgenic mouse-derived gastric organoids and single-cell sequencing.

Hereditary diffuse gastric cancer (HDGC) is a cancer syndrome caused by germline variants in CDH1, the gene encoding the cell-cell adhesion molecule E-cadherin. Loss of E-cadherin in cancer is associated with cellular dedifferentiation and poor prognosis, but the mechanisms through which CDH1 loss initiates HDGC are not known. Using single-cell RNA sequencing, we explored the transcriptional landscape of a murine organoid model of HDGC to characterize the impact of CDH1 loss in early tumourigenesis. Progenitor populations of stratified squamous and simple columnar epithelium, characteristic of the mouse stomach, showed lineage-specific transcriptional programs. Cdh1 inactivation resulted in shifts along the squamous differentiation trajectory associated with aberrant expression of genes central to gastrointestinal epithelial differentiation. Cytokeratin 7 (CK7), encoded by the differentiation-dependent gene Krt7, was a specific marker for early neoplastic lesions in CDH1 carriers. Our findings suggest that deregulation of developmental transcriptional programs may precede malignancy in HDGC. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Clinical and genomic characterisation of mismatch repair deficient pancreatic adenocarcinoma.

OBJECTIVE: To describe the clinical, pathological and genomic characteristics of pancreatic cancer with DNA mismatch repair deficiency (MMRD) and proficiency (MMRP). DESIGN: We identified patients with MMRD and MMRP pancreatic cancer in a clinical cohort (N=1213, 519 with genetic testing, 53 with immunohistochemistry (IHC)) and a genomic cohort (N=288 with whole-genome sequencing (WGS)). RESULTS: 12 out of 1213 (1.0%) in the clinical cohort were MMRD by IHC or WGS. Of the 14 patients with Lynch syndrome, 3 (21.4%) had an MMRP pancreatic cancer by IHC, and 4 (28.6%) were excluded because tissue was unavailable for testing. MMRD cancers had longer overall survival after surgery (weighted HR after coarsened exact matching 0.11, 95% CI 0.02 to 0.78, p=0.001). One patient with an unresectable MMRD cancer has an ongoing partial response 3 years after starting treatment with PD-L1/CTLA-4 inhibition. This tumour showed none of the classical histopathological features of MMRD. 9 out of 288 (3.1%) tumours with WGS were MMRD. Despite markedly higher tumour mutational burden and neoantigen loads, MMRD cancers were significantly less likely to have mutations in usual pancreatic cancer driver genes like KRAS and SMAD4, but more likely to have mutations in genes that drive cancers with microsatellite instability like ACV2RA and JAK1. MMRD tumours were significantly more likely to have a basal-like transcriptional programme and elevated transcriptional markers of immunogenicity. CONCLUSIONS: MMRD pancreatic cancers have distinct clinical, pathological and genomic profiles. Patients with MMRD pancreatic cancer should be considered for basket trials targeting enhanced immunogenicity or the unique genomic drivers in these malignancies.

Stroma vs epithelium-enhanced prognostics through histologic stratification in pancreatic ductal adenocarcinoma.

The mixture of epithelial and stromal components in pancreatic ductal adenocarcinoma (PDAC) may confound sequencing-based studies of tumor gene expression. Virtual microdissection has been suggested as a bioinformatics approach to segment the aforementioned components, and subsequent prognostic gene sets have emerged from this research. We examined the prognostic signature from the epithelial gene set of one such study using laser capture microdissected (LCM) epithelial samples. We also examined this gene set in matched stromal samples to determine whether prognostic findings were specific to the epithelium. LCM samples from 48 long-term and 48 short-term PDAC survivors were obtained. The resultant epithelial and stromal components were subjected to direct mRNA quantification using a 49 gene published PDAC classifier. Component-specific unsupervised hierarchical clustering was used to derive groups and survival differences were quantified. Immunohistochemical validation of particular genes was performed in an independent cohort. Clustering in the epithelial component yielded prognostic differences in univariable analysis (P = .02), but those differences were not significant when controlled for other clinicopathologic covariates (P = .06). Clustering in the stromal component yielded prognostic differences that persisted in the presence of other clinicopathologic covariates (P = .0005). Validation of selected genes in the epithelium (KRT6A-negative prognostic [P = .004]) and stroma (LY6D-improved prognostic [P = .01] and CTSV-negative prognostic [P = .0002]) demonstrated statistical independence in multivariable analysis. Although the genes used in this study were originally identified as being representative of the epithelial component of PDAC, their expression in the stroma appears to provide additional information that may aid in improved prognostication.

Colorectal adenocarcinomas diagnosed following a negative faecal immunochemical test show high-risk pathological features in a colon screening programme.

AIMS: The faecal immunochemical test (FIT) is used every 2 years to screen average-risk British Columbians aged 50-74 years, with follow-up colonoscopy for positive results. Non-screen-detected colorectal adenocarcinomas are defined as those detected within 25 months following a negative FIT. We aimed to more clearly characterise these malignancies. METHODS AND RESULTS: A medical chart and focused pathology review of colorectal malignancies from 926 individuals who completed FIT in the British Columbia Colon Screening Program in 2014, and whose pathology reports were available for review, was conducted. This cohort was divided into two groups: individuals with colorectal adenocarcinomas diagnosed following a positive FIT (screen-detected) and individuals with colorectal adenocarcinoma diagnosed within 25 months of a negative FIT (FIT-interval cancers). Rates of clinically relevant pathological parameters, as outlined in the American Joint Committee on Cancer (AJCC), 8th edition, were compared between the screen-detected and FIT-interval cancer groups. A total of 876 screen-detected and 50 FIT-interval cancers were identified. FIT-interval cancers exhibited higher rates of high-grade differentiation (including poorly differentiated and undifferentiated cases; P < 0.01) and aggressive histotype (signet ring cell and mucinous carcinomas; P < 0.01) than did screen-detected cancers after Bonferroni correction. Colorectal adenocarcinoma diagnosed after a negative FIT may therefore be associated with worse prognostic determinants than screen-detected cancers. CONCLUSION: FIT-interval cancers are associated with high-risk pathological features; the possibility that more aggressive, fast-growing lesions which arise in the interval after truly negative FITs cannot be ruled out. Further study of a larger cohort of FIT-interval cancers controlling for interaction among the different pathologic parameters will be undertaken.

Development and initial validation of a deep learning algorithm to quantify histological features in colorectal carcinoma including tumour budding/poorly differentiated clusters.

AIMS: To develop and validate a deep learning algorithm to quantify a broad spectrum of histological features in colorectal carcinoma. METHODS AND RESULTS: A deep learning algorithm was trained on haematoxylin and eosin-stained slides from tissue microarrays of colorectal carcinomas (N = 230) to segment colorectal carcinoma digitised images into 13 regions and one object. The segmentation algorithm demonstrated moderate to almost perfect agreement with interpretations by gastrointestinal pathologists, and was applied to an independent test cohort of digitised whole slides of colorectal carcinoma (N = 136). The algorithm correctly classified mucinous and high-grade tumours, and identified significant differences between mismatch repair-proficient and mismatch repair-deficient (MMRD) tumours with regard to mucin, inflammatory stroma, and tumour-infiltrating lymphocytes (TILs). A cutoff of >44.4 TILs per mm2 carcinoma gave a sensitivity of 88% and a specificity of 73% in classifying MMRD carcinomas. Algorithm measures of tumour budding (TB) and poorly differentiated clusters (PDCs) outperformed TB grade derived from routine sign-out, and compared favourably with manual counts of TB/PDCs with regard to lymphatic, venous and perineural invasion. Comparable associations were seen between algorithm measures of TB/PDCs and manual counts of TB/PDCs for lymph node metastasis (all P < 0.001); however, stronger correlations were seen between the proportion of positive lymph nodes and algorithm measures of TB/PDCs. Stronger associations were also seen between distant metastasis and algorithm measures of TB/PDCs (P = 0.004) than between distant metastasis and TB (P = 0.04) and TB/PDC counts (P = 0.06). CONCLUSIONS: Our results highlight the potential of deep learning to identify and quantify a broad spectrum of histological features in colorectal carcinoma.

Dataset for the reporting of carcinoma of the exocrine pancreas: recommendations from the International Collaboration on Cancer Reporting (ICCR).

AIMS: Current guidelines for pathology reporting on pancreatic cancer differ in certain aspects, resulting in divergent reporting practices and a lack of comparability of data. Here, we report on a new international dataset for pathology reporting on resection specimens with cancer of the exocrine pancreas (ductal adenocarcinoma and acinar cell carcinoma). The dataset was produced under the auspices of the International Collaboration on Cancer Reporting (ICCR), which is a global alliance of major (inter)national pathology and cancer organisations. METHODS AND RESULTS: According to the ICCR's rigorous process for dataset development, an international expert panel consisting of pancreatic pathologists, a pancreatic surgeon and an oncologist produced a set of core and non-core data items based on a critical review and discussion of current evidence. Commentary was provided for each data item to explain the rationale for selecting it as a core or non-core element and its clinical relevance, and to highlight potential areas of disagreement or lack of evidence, in which case a consensus position was formulated. Following international public consultation, the document was finalised and ratified, and the dataset, which includes a synoptic reporting guide, was published on the ICCR website. CONCLUSIONS: This first international dataset for cancer of the exocrine pancreas is intended to promote high-quality, standardised pathology reporting. Its widespread adoption will improve the consistency of reporting, facilitate multidisciplinary communication, and enhance the comparability of data, all of which will help to improve the management of pancreatic cancer patients.

Regulation of pH by Carbonic Anhydrase 9 Mediates Survival of Pancreatic Cancer Cells With Activated KRAS in Response to Hypoxia.

BACKGROUND & AIMS: Most pancreatic ductal adenocarcinomas (PDACs) express an activated form of KRAS, become hypoxic and dysplastic, and are refractory to chemo and radiation therapies. To survive in the hypoxic environment, PDAC cells upregulate enzymes and transporters involved in pH regulation, including the extracellular facing carbonic anhydrase 9 (CA9). We evaluated the effect of blocking CA9, in combination with administration of gemcitabine, in mouse models of pancreatic cancer. METHODS: We knocked down expression of KRAS in human (PK-8 and PK-1) PDAC cells with small hairpin RNAs. Human and mouse (KrasG12D/Pdx1-Cre/Tp53/RosaYFP) PDAC cells were incubated with inhibitors of MEK (trametinib) or extracellular signal-regulated kinase (ERK), and some cells were cultured under hypoxic conditions. We measured levels and stability of the hypoxia-inducible factor 1 subunit alpha (HIF1A), endothelial PAS domain 1 protein (EPAS1, also called HIF2A), CA9, solute carrier family 16 member 4 (SLC16A4, also called MCT4), and SLC2A1 (also called GLUT1) by immunoblot analyses. We analyzed intracellular pH (pHi) and extracellular metabolic flux. We knocked down expression of CA9 in PDAC cells, or inhibited CA9 with SLC-0111, incubated them with gemcitabine, and assessed pHi, metabolic flux, and cytotoxicity under normoxic and hypoxic conditions. Cells were also injected into either immune-compromised or immune-competent mice and growth of xenograft tumors was assessed. Tumor fragments derived from patients with PDAC were surgically ligated to the pancreas of mice and the growth of tumors was assessed. We performed tissue microarray analyses of 205 human PDAC samples to measure levels of CA9 and associated expression of genes that regulate hypoxia with outcomes of patients using the Cancer Genome Atlas database. RESULTS: Under hypoxic conditions, PDAC cells had increased levels of HIF1A and HIF2A, upregulated expression of CA9, and activated glycolysis. Knockdown of KRAS in PDAC cells, or incubation with trametinib, reduced the posttranscriptional stabilization of HIF1A and HIF2A, upregulation of CA9, pHi, and glycolysis in response to hypoxia. CA9 was expressed by 66% of PDAC samples analyzed; high expression of genes associated with metabolic adaptation to hypoxia, including CA9, correlated with significantly reduced survival times of patients. Knockdown or pharmacologic inhibition of CA9 in PDAC cells significantly reduced pHi in cells under hypoxic conditions, decreased gemcitabine-induced glycolysis, and increased their sensitivity to gemcitabine. PDAC cells with knockdown of CA9 formed smaller xenograft tumors in mice, and injection of gemcitabine inhibited tumor growth and significantly increased survival times of mice. In mice with xenograft tumors grown from human PDAC cells, oral administration of SLC-0111 and injection of gemcitabine increased intratumor acidosis and increased cell death. These tumors, and tumors grown from PDAC patient-derived tumor fragments, grew more slowly than xenograft tumors in mice given control agents, resulting in longer survival times. In KrasG12D/Pdx1-Cre/Tp53/RosaYFP genetically modified mice, oral administration of SLC-0111 and injection of gemcitabine reduced numbers of B cells in tumors. CONCLUSIONS: In response to hypoxia, PDAC cells that express activated KRAS increase expression of CA9, via stabilization of HIF1A and HIF2A, to regulate pH and glycolysis. Disruption of this pathway slows growth of PDAC xenograft tumors in mice and might be developed for treatment of pancreatic cancer.

Improved structural variant interpretation for hereditary cancer susceptibility using long-read sequencing.

PURPOSE: Structural variants (SVs) may be an underestimated cause of hereditary cancer syndromes given the current limitations of short-read next-generation sequencing. Here we investigated the utility of long-read sequencing in resolving germline SVs in cancer susceptibility genes detected through short-read genome sequencing. METHODS: Known or suspected deleterious germline SVs were identified using Illumina genome sequencing across a cohort of 669 advanced cancer patients with paired tumor genome and transcriptome sequencing. Candidate SVs were subsequently assessed by Oxford Nanopore long-read sequencing. RESULTS: Nanopore sequencing confirmed eight simple pathogenic or likely pathogenic SVs, resolving three additional variants whose impact could not be fully elucidated through short-read sequencing. A recurrent sequencing artifact on chromosome 16p13 and one complex rearrangement on chromosome 5q35 were subsequently classified as likely benign, obviating the need for further clinical assessment. Variant configuration was further resolved in one case with a complex pathogenic rearrangement affecting TSC2. CONCLUSION: Our findings demonstrate that long-read sequencing can improve the validation, resolution, and classification of germline SVs. This has important implications for return of results, cascade carrier testing, cancer screening, and prophylactic interventions.

Loss of switch/sucrose non-fermenting complex protein expression in undifferentiated gastrointestinal and pancreatic carcinomas.

AIMS: Undifferentiated carcinoma refers to an epithelial malignancy that lacks morphological evidence of differentiation. Recent studies have implicated the loss of constitutively expressed switch/sucrose non-fermenting (SWI/SNF) complex subunits in undifferentiated carcinomas of the gastrointestinal tract and other sites. In this study we examine the expression of SWI/SNF and mismatch repair (MMR) proteins in a series of undifferentiated carcinomas from the gastrointestinal tract and the pancreas. METHODS AND RESULTS: We searched pathology databases from four Canadian health centres for primary undifferentiated carcinoma from gastrointestinal and pancreatic resection specimens. Upon review of 31 cases, 19 were confirmed to be undifferentiated carcinomas (eight colonic, six gastric, three pancreatic, one appendiceal and one duodenal). Immunohistochemical analysis of SMARCA4, SMARCA2, SMARCB1, ARID1A, ARID1B, MSH2, MSH6, MLH1 and PMS2 was performed on whole sections. Five of 19 (26%) showed loss of core SWI/SNF proteins (two loss of SMARCA4, one loss of SMARCB1 and two concurrent loss of ARID1A and ARID1B). SMARCA4, SMARCB1, or ARID1A/ARID1B-deficient undifferentiated carcinoma consistently exhibited sheet-like growth pattern, with cellular discohesion and rhabdoid morphology. Nine of 17 undifferentiated carcinomas tested were MMR-deficient by immunohistochemistry. In comparison, none of the 12 poorly differentiated carcinomas that were originally diagnosed as undifferentiated carcinomas showed loss of SMARCA4, SMARCA2, SMARCB1 or ARID1B. CONCLUSIONS: Undifferentiated gastrointestinal/pancreatic carcinomas show frequent loss of expression of SWI/SNF complex proteins. The loss of these core components of SWI/SNF complex may contribute to the arrest of cellular differentiation, resulting in the undifferentiated histology and aggressive clinical behaviour.

Should you repeat mismatch repair testing in cases of tumour recurrence? An evaluation of repeat mismatch repair testing by the use of immunohistochemistry in recurrent tumours of the gastrointestinal and gynaecological tracts.

AIMS: The role of mismatch repair (MMR) testing has evolved from identifying Lynch syndrome patients to predicting response to immune checkpoint inhibitors. This has led to requests from clinicians to retest recurrences of MMR-proficient primary tumours in the hope that the recurrence may show a different MMR status and qualify the patient for treatment. We aimed to determine whether repeat testing is warranted. METHODS AND RESULTS: We evaluated recurrent tumours (local recurrences or metastases) from 137 patients with MMR-proficient primary tumours of the gastrointestinal and gynaecological tracts. The local recurrences and metastases all occurred at least 30 days after resection of the primary tumour. We used a combination of a tissue microarray and whole slide staining to perform immunohistochemistry (IHC) for PMS2, MLH1, MSH2, and MSH6, and compared the results with the MMR status of the primary tumour. Three of 137 (2%) initially showed a discordant staining pattern. However, further investigation showed that these discordances were attributable to some of the known pitfalls associated with MMR IHC interpretation - post-radiotherapy loss of MSH6 expression and subclonal loss of MLH1 staining. We did not identify any cases with a genuine discordance in MMR status. CONCLUSION: We conclude that repeat MMR IHC testing of recurrences is not warranted, as MMR status does not change relative to that of the primary tumour.

Cell of origin affects tumour development and phenotype in pancreatic ductal adenocarcinoma.

OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive tumour thought to arise from ductal cells via pancreatic intraepithelial neoplasia (PanIN) precursor lesions. Modelling of different genetic events in mice suggests both ductal and acinar cells can give rise to PDAC. However, the impact of cellular context alone on tumour development and phenotype is unknown. DESIGN: We examined the contribution of cellular origin to PDAC development by inducing PDAC-associated mutations, KrasG12D expression and Trp53 loss, specifically in ductal cells (Sox9CreER;KrasLSL-G12D;Trp53flox/flox ('Duct:KPcKO ')) or acinar cells (Ptf1aCreER;KrasLSL-G12D;Trp53flox/flox ('Acinar:KPcKO ')) in mice. We then performed a thorough analysis of the resulting histopathological changes. RESULTS: Both mouse models developed PDAC, but Duct:KPcKO mice developed PDAC earlier than Acinar:KPcKO mice. Tumour development was more rapid and associated with high-grade murine PanIN (mPanIN) lesions in Duct:KPcKO mice. In contrast, Acinar:KPcKO mice exhibited widespread metaplasia and low-grade as well as high-grade mPanINs with delayed progression to PDAC. Acinar-cell-derived tumours also had a higher prevalence of mucinous glandular features reminiscent of early mPanIN lesions. CONCLUSION: These findings indicate that ductal cells are primed to form carcinoma in situ that become invasive PDAC in the presence of oncogenic Kras and Trp53 deletion, while acinar cells with the same mutations appear to require a prolonged period of transition or reprogramming to initiate PDAC. Our findings illustrate that PDAC can develop in multiple ways and the cellular context in which mutations are acquired has significant impact on precursor lesion initiation, disease progression and tumour phenotype.

Loss of Pten and Activation of Kras Synergistically Induce Formation of Intraductal Papillary Mucinous Neoplasia From Pancreatic Ductal Cells in Mice.

BACKGROUND & AIMS: Intraductal papillary mucinous neoplasias (IPMNs) are precancerous cystic lesions that can develop into pancreatic ductal adenocarcinomas (PDACs). These large macroscopic lesions are frequently detected during medical imaging, but it is unclear how they form or progress to PDAC. We aimed to identify cells that form IPMNs and mutations that promote IPMN development and progression. METHODS: We generated mice with disruption of Pten specifically in ductal cells (Sox9CreERT2;Ptenflox/flox;R26RYFP or PtenΔDuct/ΔDuct mice) and used PtenΔDuct/+ and Pten+/+ mice as controls. We also generated KrasG12D;PtenΔDuct/ΔDuct and KrasG12D;PtenΔDuct/+ mice. Pancreata were collected when mice were 28 weeks to 14.5 months old and analyzed by histology, immunohistochemistry, and electron microscopy. We performed multiplexed droplet digital polymerase chain reaction to detect spontaneous Kras mutations in PtenΔDuct/ΔDuct mice and study the effects of Ras pathway activation on initiation and progression of IPMNs. We obtained 2 pancreatic sections from a patient with an invasive pancreatobiliary IPMN and analyzed the regions with and without the invasive IPMN (control tissue) by immunohistochemistry. RESULTS: Mice with ductal cell-specific disruption of Pten but not control mice developed sporadic, macroscopic, intraductal papillary lesions with histologic and molecular features of human IPMNs. PtenΔDuct/ΔDuct mice developed IPMNs of several subtypes. In PtenΔDuct/ΔDuct mice, 31.5% of IPMNs became invasive; invasion was associated with spontaneous mutations in Kras. KrasG12D;PtenΔDuct/ΔDuct mice all developed invasive IPMNs within 1 month. In KrasG12D;PtenΔDuct/+ mice, 70% developed IPMN, predominately of the pancreatobiliary subtype, and 63.3% developed PDAC. In all models, IPMNs and PDAC expressed the duct-specific lineage tracing marker yellow fluorescent protein. In immunohistochemical analyses, we found that the invasive human pancreatobiliary IPMN tissue had lower levels of PTEN and increased levels of phosphorylated (activated) ERK compared with healthy pancreatic tissue. CONCLUSIONS: In analyses of mice with ductal cell-specific disruption of Pten, with or without activated Kras, we found evidence for a ductal cell origin of IPMNs. We also showed that PTEN loss and activated Kras have synergistic effects in promoting development of IPMN and progression to PDAC.