Long-term expanding human airway organoids for disease modeling.

Organoids are self-organizing 3D structures grown from stem cells that recapitulate essential aspects of organ structure and function. Here, we describe a method to establish long-term-expanding human airway organoids from broncho-alveolar resections or lavage material. The pseudostratified airway organoids consist of basal cells, functional multi-ciliated cells, mucus-producing secretory cells, and CC10-secreting club cells. Airway organoids derived from cystic fibrosis (CF) patients allow assessment of CFTR function in an organoid swelling assay. Organoids established from lung cancer resections and metastasis biopsies retain tumor histopathology as well as cancer gene mutations and are amenable to drug screening. Respiratory syncytial virus (RSV) infection recapitulates central disease features, dramatically increases organoid cell motility via the non-structural viral NS2 protein, and preferentially recruits neutrophils upon co-culturing. We conclude that human airway organoids represent versatile models for the in vitro study of hereditary, malignant, and infectious pulmonary disease.

Non-functional pancreatic neuroendocrine tumours: ATRX/DAXX and alternative lengthening of telomeres (ALT) are prognostically independent from ARX/PDX1 expression and tumour size.

OBJECTIVE: Recent studies have found aristaless-related homeobox gene (ARX)/pancreatic and duodenal homeobox 1 (PDX1), alpha-thalassemia/mental retardation X-linked (ATRX)/death domain-associated protein (DAXX) and alternative lengthening of telomeres (ALT) to be promising prognostic biomarkers for non-functional pancreatic neuroendocrine tumours (NF-PanNETs). However, they have not been comprehensively evaluated, especially among small NF-PanNETs (≤2.0 cm). Moreover, their status in neuroendocrine tumours (NETs) from other sites remains unknown. DESIGN: An international cohort of 1322 NETs was evaluated by immunolabelling for ARX/PDX1 and ATRX/DAXX, and telomere-specific fluorescence in situ hybridisation for ALT. This cohort included 561 primary NF-PanNETs, 107 NF-PanNET metastases and 654 primary, non-pancreatic non-functional NETs and NET metastases. The results were correlated with numerous clinicopathological features including relapse-free survival (RFS). RESULTS: ATRX/DAXX loss and ALT were associated with several adverse prognostic findings and distant metastasis/recurrence (p<0.001). The 5-year RFS rates for patients with ATRX/DAXX-negative and ALT-positive NF-PanNETs were 40% and 42% as compared with 85% and 86% for wild-type NF-PanNETs (p<0.001 and p<0.001). Shorter 5-year RFS rates for ≤2.0 cm NF-PanNETs patients were also seen with ATRX/DAXX loss (65% vs 92%, p=0.003) and ALT (60% vs 93%, p<0.001). By multivariate analysis, ATRX/DAXX and ALT status were independent prognostic factors for RFS. Conversely, classifying NF-PanNETs by ARX/PDX1 expression did not independently correlate with RFS. Except for 4% of pulmonary carcinoids, ATRX/DAXX loss and ALT were only identified in primary (25% and 29%) and NF-PanNET metastases (62% and 71%). CONCLUSIONS: ATRX/DAXX and ALT should be considered in the prognostic evaluation of NF-PanNETs including ≤2.0 cm tumours, and are highly specific for pancreatic origin among NET metastases of unknown primary.

Colonoscopic-Assisted Laparoscopic Wedge Resection for Colonic Lesions: A Prospective Multicenter Cohort Study (LIMERIC-Study).

OBJECTIVE: The aim of this study was to evaluate the safety and efficacy of a modified CAL-WR. SUMMARY BACKGROUND DATA: The use of segmental colectomy in patients with endoscopically unresectable colonic lesions results in significant morbidity and mortality. CAL-WR is an alternative procedure that may reduce morbidity. METHODS: This prospective multicenter study was performed in 13 Dutch hospitals between January 2017 and December 2019. Inclusion criteria were (1) colonic lesions inaccessible using current endoscopic resection techniques (judged by an expert panel), (2) non-lifting residual/recurrent adenomatous tissue after previous polypectomy or (3) an undetermined resection margin after endoscopic removal of a low-risk pathological T1 (pT1) colon carcinoma. Thirty-day morbidity, technical success rate and radicality were evaluated. RESULTS: Of the 118 patients included (56% male, mean age 66 years, standard deviation ± 8 years), 66 (56%) had complex lesions unsuitable for endoscopic removal, 34 (29%) had non-lifting residual/recurrent adenoma after previous polypectomy and 18 (15%) had uncertain resection margins after polypectomy of a pT1 colon carcinoma. CAL-WR was technically successful in 93% and R0 resection was achieved in 91% of patients. Minor complications (Clavien-Dindo i-ii) were noted in 7 patients (6%) and an additional oncologic segmental resection was performed in 12 cases (11%). Residual tissue at the scar was observed in 5% of patients during endoscopic follow-up. CONCLUSIONS: CAL-WR is an effective, organ-preserving approach that results in minor complications and circumvents the need for major surgery. CAL-WR, therefore, deserves consideration when endoscopic excision of circumscribed lesions is impossible or incomplete.

Pancreatic cancer organoids recapitulate disease and allow personalized drug screening.

We report the derivation of 30 patient-derived organoid lines (PDOs) from tumors arising in the pancreas and distal bile duct. PDOs recapitulate tumor histology and contain genetic alterations typical of pancreatic cancer. In vitro testing of a panel of 76 therapeutic agents revealed sensitivities currently not exploited in the clinic, and underscores the importance of personalized approaches for effective cancer treatment. The PRMT5 inhibitor EZP015556, shown to target MTAP (a gene commonly lost in pancreatic cancer)-negative tumors, was validated as such, but also appeared to constitute an effective therapy for a subset of MTAP-positive tumors. Taken together, the work presented here provides a platform to identify novel therapeutics to target pancreatic tumor cells using PDOs.

Extension of early esophageal squamous cell neoplasia into ducts and submucosal glands and the role of endoscopic ablation therapy.

BACKGROUND AND AIMS: Early esophageal squamous cell neoplasia (ESCN) is preferably treated with en-bloc endoscopic resection. Ablation might be an alternative for flat ESCN, but ESCN extension along the epithelial lining of ducts and submucosal glands (SMGs) might jeopardize ablation efficacy. Clinical studies suggest that local recurrence might arise from such buried ESCN niches after ablation. We studied human endoscopic resection specimens of ESCN to quantify ESCN extension into ducts/SMGs and performed a prospective porcine study to evaluate the depth of radiofrequency ablation (RFA) and CryoBalloon ablation (CBA) into ducts/SMGs. METHODS: Endoscopic submucosal dissection specimens of flat-type ESCN from a Japanese (n = 65) and Dutch cohort (n = 14) were evaluated for presence and neoplastic involvement of ducts/SMGs. Twenty-seven pigs were treated with circumferential RFA (c-RFA; n = 4), focal CBA (n = 20), and focal RFA (n = 3) with 4, 60, and 9 treatment areas, respectively. After prespecified survival periods (0 hours, 8 hours, 2 days, 5 days, and 28 days), treatment areas were evaluated for uniformity and depth of ablation and affected SMGs. RESULTS: Neoplastic extension in ducts/SMGs was observed in most lesions: 58% (38/65) in the Japanese and 64% (9/14) in the Dutch cohort. In the animal study, 33% of SMGs (95% confidence interval, 28-50) were not affected after c-RFA, although the overlying epithelium was ablated. Focal RFA and CBA resulted in uniform ablations with effective treatment of all SMGs. CONCLUSIONS: ESCN extends into ducts/SMGs in most patients. In an animal model, focal RFA and CBA effectively ablated SMGs, whereas c-RFA inadequately ablated SMGs. Given this potential reason for recurrence, endoscopic resection should remain the standard of care.

Sequential cancer mutations in cultured human intestinal stem cells.

Crypt stem cells represent the cells of origin for intestinal neoplasia. Both mouse and human intestinal stem cells can be cultured in medium containing the stem-cell-niche factors WNT, R-spondin, epidermal growth factor (EGF) and noggin over long time periods as epithelial organoids that remain genetically and phenotypically stable. Here we utilize CRISPR/Cas9 technology for targeted gene modification of four of the most commonly mutated colorectal cancer genes (APC, P53 (also known as TP53), KRAS and SMAD4) in cultured human intestinal stem cells. Mutant organoids can be selected by removing individual growth factors from the culture medium. Quadruple mutants grow independently of all stem-cell-niche factors and tolerate the presence of the P53 stabilizer nutlin-3. Upon xenotransplantation into mice, quadruple mutants grow as tumours with features of invasive carcinoma. Finally, combined loss of APC and P53 is sufficient for the appearance of extensive aneuploidy, a hallmark of tumour progression.

Genetic Analysis of Small Well-differentiated Pancreatic Neuroendocrine Tumors Identifies Subgroups With Differing Risks of Liver Metastases.

OBJECTIVE: The aim of this study was to investigate the key molecular alterations in small primary pancreatic neuroendocrine tumors (PanNETs) associated with the development of liver metastases. BACKGROUND: Well-differentiated PanNETs with small size are typically indolent; however, a limited subset metastasize to the liver. METHODS: A total of 87 small primary PanNETs (<3 cm), including 32 metastatic cases and 55 nonmetastatic cases after a 5-year follow-up, were immunolabeled for DAXX/ATRX and analyzed for alternative lengthening of telomeres (ALT) by Fluorescence In Situ Hybridization. A subset of these cases, 24 that metastasized and 24 that did not metastasize, were assessed by targeted next-generation sequencing and whole-genome copy number variation. RESULTS: In the entire cohort, high Ki-67 (OR 1.369; 95% CI 1.121-1.673; P = 0.002), N-stage (OR 4.568; 95% CI 1.458-14.312; P = 0.009), and ALT-positivity (OR 3.486; 95% CI 1.093-11.115; P = 0.035) were independently associated with liver metastases. In the subset assessed by next-generation sequencing and copy number variation analysis, 3 molecular subtypes with differing risks of liver metastases were identified. Group 1 (n = 15; 73% metastasized) was characterized by recurrent chromosomal gains, CN-LOH, DAXX mutations, and ALT-positivity. Group 2 (n = 19; 42% metastasized, including 5 G1 tumors) was characterized by limited copy number alterations and mutations. Group 3 (n = 14; 35% metastasized) were defined by chromosome 11 loss. CONCLUSIONS: We identified genomic patterns of small PanNETs associated with a different risk for liver metastases. Molecular alterations, such as DAXX mutations, chromosomal gains, and ALT, are associated with an increased risk of metastasis in small PanNETs. Therefore, targeted sequencing and/or ALT analysis may help in the clinical decisions for these small PanNETs.

Tumor Seeding During Colonoscopy as a Possible Cause for Metachronous Colorectal Cancer.

BACKGROUND AND AIMS: In patients who have undergone surgery for colorectal cancer (CRC), 3% have recurrence of (metachronous) CRC. We investigated whether tumor seeding during colonoscopy (iatrogenic implantation of tumor cells in damaged mucosa) increases risk for metachronous CRC. METHODS: In a proof of principle study, we collected data from the Dutch National Pathology Registry for patients with a diagnosis of CRC from 2013 through 2015, with a second diagnosis of CRC within 6 months to 3.5 years after surgery. We reviewed pathology reports to identify likely metachronous CRC (histologically proven adenocarcinoma located elsewhere in the colon or rectum from the surgical anastomosis). For 22 patients fulfilling the inclusion criteria, we ascribed the most likely etiology to tumor seeding when endoscopic manipulations, such as biopsies or polypectomy, occurred at the location where the metachronous tumor was subsequently detected, after endoscopic manipulation of the primary tumor. We collected clinical data from patients and compared molecular profiles of the primary and metachronous colorectal tumors using next-generation sequencing. We then examined the source of seeded tumor. We tested whether tumor cells stay behind in the working channel of the endoscope after biopsies of colorectal tumors, and whether these cells maintain viability in organoid cultures. RESULTS: In total, tumor seeding was suspected as the most likely etiology of metachronous CRC in 5 patients. Tumor tissues were available from 3 patients. An identical molecular signature was observed in the primary and metachronous colorectal tumors from all 3 patients. In 5 control cases with a different etiology of metachronous CRC, the molecular signature of the primary and metachronous tumor were completely different. Based on review of 2147 patient records, we estimated the risk of tumor seeding during colonoscopy to be 0.3%-0.6%. We demonstrated that the working channel of the colonoscope becomes contaminated with viable tumor cells during biopsy collection. Subsequent instruments introduced through this working channel also became contaminated. These cells were shown to maintain their proliferative potential. CONCLUSIONS: In an analysis of primary and secondary tumors from patients with metachronous CRC, we found that primary tumor cells might be seeded in a new location after biopsy of the primary tumor. Although our study does not eliminate other possibilities of transmission, our findings and experiments support the hypothesis that tumor seeding can occur during colonoscopy via the working channel of the endoscope. The possibility of iatrogenic seeding seems low. However, our findings compel awareness on this potentially preventable cause of metachronous CRC.

Associations of non-pedunculated T1 colorectal adenocarcinoma outcome with consensus molecular subtypes, immunoscore, and microsatellite status: a multicenter case-cohort study.

Advanced colorectal cancer (CRC) consensus molecular subtype 4 (CMS4) or CRC with a low immunoscore is associated with shorter survival times. Non-metastatic CRC with microsatellite instability (MSI) is associated with a lower risk of recurrence. We evaluated outcome (lymph node metastases [LNM] or cancer recurrence) in these tumor subtypes in patients with surgically-removed non-pedunculated T1 CRC by performing a multicenter case-cohort study. We included all patients in 13 hospitals in the Netherlands from 2000-2014 (n = 651). We randomly selected a subgroup of patients (n = 223) and all patients with LNM or recurrence (n = 63), and median follow-up of 44 months. We centrally reviewed tumor-slides, and constructed and immunostained tissue microarrays determining MSI, CMS (MSI/CMS1, CMS2/3, or CMS4), and immunoscore (I-low/I-high). We used weighted Cox proportional hazard models to evaluate the association of MSI, CMS, and immunoscore with LNM or recurrence, adjusting for conventional histologic risk factors. In the randomly selected subgroup of patients, 7.1% of tumors were MSI/CMS1, 91.0% CMS2/3, 1.8% CMS4, and 25% I-low. In the case-cohort, patients with CMS4 tumors had an increased risk for LNM or recurrence compared with patients with tumors of other CMSs (adjusted hazard ratio [HR], 3.97; 95% CI, 1.12-14.06; P = 0.03). Albeit not significant, tumors with MSI had a lower risk for LNM or recurrence than other tumor subtypes (adjusted HR, 0.52; 95% CI, 0.12-2.30; P = 0.39), whereas tumors with a low immunoscore had an increased risk for LNM or recurrence (adjusted HR, 1.30; 95% CI, 0.68-2.48; P = 0.43). In conclusion, in a case-cohort study of patients with non-pedunculated T1 CRC, MSI, and immunoscore were not significantly associated with adverse outcome after surgery. CMS4 substantially increased the risk of adverse outcome. However, CMS4 is rare in T1 CRCs, limiting its value for determining the risk in patients.

Why is pancreatic cancer so deadly? The pathologist's view.

The remarkable aggressiveness of pancreatic cancer has never been fully explained. Although clearly multifactorial, we postulate that venous invasion, a finding seen in most pancreatic cancers but not in most cancers of other organs, may be a significant, underappreciated contributor to the aggressiveness of this disease. Copyright © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Genetic dissection of colorectal cancer progression by orthotopic transplantation of engineered cancer organoids.

In the adenoma-carcinoma sequence, it is proposed that intestinal polyps evolve through a set of defined mutations toward metastatic colorectal cancer (CRC). Here, we dissect this adenoma-carcinoma sequence in vivo by using an orthotopic organoid transplantation model of human colon organoids engineered to harbor different CRC mutation combinations. We demonstrate that sequential accumulation of oncogenic mutations in Wnt, EGFR, P53, and TGF-ß signaling pathways facilitates efficient tumor growth, migration, and metastatic colonization. We show that reconstitution of specific niche signals can restore metastatic growth potential of tumor cells lacking one of the oncogenic mutations. Our findings imply that the ability to metastasize-i.e., to colonize distant sites-is the direct consequence of the loss of dependency on specific niche signals.

Efficacy and Safety of Curcumin in Treatment of Intestinal Adenomas in Patients With Familial Adenomatous Polyposis.

BACKGROUND & AIMS: Familial adenomatous polyposis is an autosomal dominant disorder characterized by the development of hundreds of colorectal adenomas and eventually colorectal cancer. Oral administration of the spice curcumin has been followed by regression of polyps in patients with this disorder. We performed a double-blinded randomized trial to determine the safety and efficacy of curcumin in patients with familial adenomatous polyposis. METHODS: This study included 44 patients with familial adenomatous polyposis (18-85 years old) who had not undergone colectomy or had undergone colectomy with ileorectal anastomosis or ileal anal pouches, had at least 5 intestinal adenomatous polyps, and had enrolled in Puerto Rico or the United States from September 2011 through November 2016. Patients were randomly assigned (1:1) to groups given 100% pure curcumin (1,500 mg orally, twice per day) or identical-appearing placebo capsules for 12 months. The number and size of lower gastrointestinal tract polyps were evaluated every 4 months for 1 year. The primary outcome was the number of polyps in the curcumin and placebo groups at 12 months or at the time of withdrawal from the study according to the intention-to-treat principle. RESULTS: After 1 year of treatment, the average rate of compliance was 83% in the curcumin group and 91% in the placebo group. After 12 weeks, there was no significant difference in the mean number of polyps between the placebo group (18.6; 95% CI, 9.3-27.8) and the curcumin group (22.6; 95% CI, 12.1-33.1; P = .58). We found no significant difference in mean polyp size between the curcumin group (2.3 mm; 95% CI, 1.8-2.8) and the placebo group (2.1 mm; 95% CI, 1.5-2.7; P = .76). Adverse events were few, with no significant differences between groups. CONCLUSIONS: In a double-blinded randomized trial of patients with familial adenomatous polyposis, we found no difference in the mean number or size of lower intestinal tract adenomas between patients given curcumin 3,000 mg/day and those given placebo for 12 weeks. Clinicaltrials.gov ID NCT00641147.

Histologic Factors Associated With Need for Surgery in Patients With Pedunculated T1 Colorectal Carcinomas.

BACKGROUND & AIMS: Most patients with pedunculated T1 colorectal tumors referred for surgery are not found to have lymph node metastases, and were therefore unnecessarily placed at risk for surgery-associated complications. We aimed to identify histologic factors associated with need for surgery in patients with pedunculated T1 colorectal tumors. METHODS: We performed a cohort-nested matched case-control study of 708 patients diagnosed with pedunculated T1 colorectal tumors at 13 hospitals in The Netherlands, from January 1, 2000 through December 31, 2014, followed for a median of 44 months (interquartile range, 20-80 months). We identified 37 patients (5.2%) who required surgery (due to lymph node, intramural, or distant metastases). These patients were matched with patients with pedunculated T1 colorectal tumors without a need for surgery (no metastases, controls, n = 111). Blinded pathologists analyzed specimens from each tumor, stained with H&E. We evaluated associations between histologic factors and patient need for surgery using univariable conditional logistic regression analysis. We used multivariable least absolute shrinkage and selection operator (LASSO; an online version of the LASSO model is available at: http://t1crc.com/calculator/) regression to develop models for identification of patients with tumors requiring surgery, and tested the accuracy of our model by projecting our case-control data toward the entire cohort (708 patients). We compared our model with previously developed strategies to identify high-risk tumors: conventional model 1 (based on poor differentiation, lymphovascular invasion, or Haggitt level 4) and conventional model 2 (based on poor differentiation, lymphovascular invasion, Haggitt level 4, or tumor budding). RESULTS: We identified 5 histologic factors that differentiated cases from controls: lymphovascular invasion, Haggitt level 4 invasion, muscularis mucosae type B (incompletely or completely disrupted), poorly differentiated clusters and tumor budding, which identified patients who required surgery with an area under the curve (AUC) value of 0.83 (95% confidence interval, 0.76-0.90). When we used a clinically plausible predicted probability threshold of ≥4.0%, 67.5% (478 of 708) of patients were predicted to not need surgery. This threshold identified patients who required surgery with 83.8% sensitivity (95% confidence interval, 68.0%-93.8%) and 70.3% specificity (95% confidence interval, 60.9%-78.6%). Conventional models 1 and 2 identified patients who required surgery with lower AUC values (AUC, 0.67; 95% CI, 0.60-0.74; P = .002 and AUC, 0.64; 95% CI, 0.58-0.70; P < .001, respectively) than our LASSO model. When we applied our LASSO model with a predicted probability threshold of ≥4.0%, the percentage of missed cases (tumors mistakenly assigned as low risk) was comparable (6 of 478 [1.3%]) to that of conventional model 1 (4 of 307 [1.3%]) and conventional model 2 (3 of 244 [1.2%]). However, the percentage of patients referred for surgery based on our LASSO model was much lower (32.5%, n = 230) than that for conventional model 1 (56.6%, n = 401) or conventional model 2 (65.5%, n = 464). CONCLUSIONS: In a cohort-nested matched case-control study of 708 patients with pedunculated T1 colorectal carcinomas, we developed a model based on histologic features of tumors that identifies patients who require surgery (due to high risk of metastasis) with greater accuracy than previous models. Our model might be used to identify patients most likely to benefit from adjuvant surgery.

Pedunculated Morphology of T1 Colorectal Tumors Associates With Reduced Risk of Adverse Outcome.

BACKGROUND & AIMS: Risk stratification for adverse events, such as metastasis to lymph nodes, is based only on histologic features of tumors. We aimed to compare adverse outcomes of pedunculated vs nonpedunculated T1 colorectal cancers (CRC). METHODS: We performed a retrospective study of 1656 patients diagnosed with T1CRC from 2000 through 2014 at 14 hospitals in The Netherlands. The median follow-up time of patients was 42.5 months (interquartile range, 18.5-77.5 mo). We evaluated the association between tumor morphology and the primary composite end point, adverse outcome, adjusted for clinical variables, histologic variables, resection margins, and treatment approach. Adverse outcome was defined as metastasis to lymph nodes, distant metastases, local recurrence, or residual tissue. Secondary end points were tumor metastasis, recurrence, and incomplete resection. RESULTS: Adverse outcome occurred in 67 of 723 patients (9.3%) with pedunculated T1CRCs vs 155 of 933 patients (16.6%) with nonpedunculated T1CRCs. Pedunculated morphology was independently associated with decreased risk of adverse outcome (adjusted odds ratio [OR], 0.59; 95% CI, 0.42-0.83; P = .003). Metastasis, incomplete resection, and recurrence were observed in 5.8%, 4.6%, and 3.9% of pedunculated T1CRCs vs 10.6%, 8.0%, and 6.6% of nonpedunculated T1CRCs, respectively. Pedunculated morphology was independently associated with a reduced risk of metastasis (adjusted OR, 0.62; 95% CI, 0.41-0.94; P = .03), incomplete resection (adjusted OR, 0.57; 95% CI, 0.36-0.91; P = .02), and recurrence (adjusted hazard ratio, 0.52; 95% CI, 0.32-0.85; P = .009). Metastasis, incomplete resection, and recurrence did not differ significantly between low-risk pedunculated vs nonpedunculated T1CRCs (0.8% vs 2.9%, P = .38; 1.5% vs 0%, P = .99; 1.5% vs 0%; P = .99). However, incomplete resection and recurrence were significantly lower for high-risk pedunculated vs nonpedunculated T1CRCs (6.5% vs 12.5%; P = .007; 4.4% vs 8.6%; P = .03). CONCLUSIONS: In a retrospective study of patients with T1CRC, we found pedunculated morphology to be associated independently with a decreased risk of adverse outcome in a T1CRC population at high risk of adverse outcome. Incorporating morphologic features of tumors in risk assessment could help predict outcomes of patients with T1CRC and help identify the best candidates for surgery.

Immunolabeling of Cleared Human Pancreata Provides Insights into Three-Dimensional Pancreatic Anatomy and Pathology.

Visualizing pathologies in three dimensions can provide unique insights into the biology of human diseases. A rapid and easy-to-implement dibenzyl ether-based technique was used to clear thick sections of surgically resected human pancreatic parenchyma. Protocols were applicable to both fresh and formalin-fixed, paraffin-embedded tissue. The penetration of antibodies into dense pancreatic parenchyma was optimized using both gradually increasing antibody concentrations and centrifugal flow. Immunolabeling with antibodies against cytokeratin 19 was visualized using both light sheet and confocal laser scanning microscopy. The technique was applied successfully to 26 sections of pancreas, providing three-dimensional (3D) images of normal pancreatic tissue, pancreatic intraepithelial neoplasia, intraductal papillary mucinous neoplasms, and infiltrating pancreatic ductal adenocarcinomas. 3D visualization highlighted processes that are hard to conceptualize in two dimensions, such as invasive carcinoma growing into what appeared to be pre-existing pancreatic ducts and within venules, and the tracking of long cords of neoplastic cells parallel to blood vessels. Expanding this technique to formalin-fixed, paraffin-embedded tissue opens pathology archives to 3D visualization of unique biosamples and rare diseases. The application of immunolabeling and clearing to human pancreatic parenchyma provides detailed visualization of normal pancreatic anatomy, and can be used to characterize the 3D architecture of diseases including pancreatic intraepithelial neoplasia, intraductal papillary mucinous neoplasm, and pancreatic ductal adenocarcinomas.

Generation of an inducible colon-specific Cre enzyme mouse line for colon cancer research.

Current mouse models for colorectal cancer often differ significantly from human colon cancer, being largely restricted to the small intestine. Here, we aim to develop a colon-specific inducible mouse model that can faithfully recapitulate human colon cancer initiation and progression. Carbonic anhydrase I (Car1) is a gene expressed uniquely in colonic epithelial cells. We generated a colon-specific inducible Car1CreER knock-in (KI) mouse with broad Cre activity in epithelial cells of the proximal colon and cecum. Deletion of the tumor suppressor gene Apc using the Car1CreER KI caused tumor formation in the cecum but did not yield adenomas in the proximal colon. Mutation of both Apc and Kras yielded microadenomas in both the cecum and the proximal colon, which progressed to macroadenomas with significant morbidity. Aggressive carcinomas with some invasion into lymph nodes developed upon combined induction of oncogenic mutations of Apc, Kras, p53, and Smad4 Importantly, no adenomas were observed in the small intestine. Additionally, we observed tumors from differentiated Car1-expressing cells with Apc/Kras mutations, suggesting that a top-down model of intestinal tumorigenesis can occur with multiple mutations. Our results establish the Car1CreER KI as a valuable mouse model to study colon-specific tumorigenesis and metastasis as well as cancer-cell-of-origin questions.

Discordance Among Pathologists in the United States and Europe in Diagnosis of Low-Grade Dysplasia for Patients With Barrett's Esophagus.

BACKGROUND & AIMS: There is suboptimal inter-observer agreement, even among expert gastrointestinal pathologists, in the diagnosis of low-grade dysplasia (LGD) in patients with Barrett's esophagus (BE). We analyzed histopathologic criteria required for a diagnosis of LGD using the new subcategories of LGD with inflammatory and dysplastic features. We categorized each diagnosis based on the level of confidence and assessed inter-observer agreement among gastrointestinal pathologists from 5 tertiary centers in the United States and Europe. METHODS: In the first phase of the study, 3 pathologists held a consensus conference at which they discussed the diagnostic criteria for LGD. In the second phase, 79 slides from patients with BE (23 samples of non-dysplastic BE, 22 samples of LGD, and 34 samples of high-grade dysplasia) were identified, randomly assigned to 7 pathologists (4 from the United States and 3 from Europe), and interpreted in a blinded fashion. κ Values were calculated for inter-observer agreement. We performed multinomial logistic regression analysis to assess the weighting of histologic features with the diagnosis. RESULTS: The overall κ value for diagnosis was 0.43 (95% confidence interval [CI], 0.42-0.48). When categorized based on degree of dysplasia, the κ value was 0.22 (95% CI, 0.11-0.29) for non-dysplastic BE, 0.11 (95% CI, 0.004-0.15) for LGD, and 0.43 (95% CI, 0.36-0.46) for high-grade dysplasia. When all pathologists made a diagnosis with high confidence, the inter-observer agreement was substantial among the US pathologists (κ, 0.63; 95% CI, 0.61-0.66) and European pathologists (κ, 0.80; 95% CI, 0.74-0.97). The κ values for all diagnoses made by European pathologists were higher than those made by US pathologists. CONCLUSIONS: In an analysis of criteria used in histopathologic diagnosis of LGD, we did not observe improvement in level of agreement among experienced pathologists, even after accounting for inflammation. The level of inter-observer agreement increased with level of pathologist confidence. There was also a difference in reading of histopathology samples of BE tissues between US and European pathologists.

Patients With Barrett's Esophagus and Confirmed Persistent Low-Grade Dysplasia Are at Increased Risk for Progression to Neoplasia.

BACKGROUND & AIMS: For patients with Barrett's esophagus, the diagnosis of low-grade dysplasia (LGD) is subjective, and reported outcomes vary. We analyzed data from a multicenter study of endoscopic therapy to identify factors associated with progression to high-grade dysplasia (HGD) or esophageal adenocarcinoma (EAC) in patients with LGD of the esophagus. METHODS: We performed a retrospective analysis of data from 255 patients with a primary diagnosis of LGD (78% men; mean age, 63 years) who participated in a randomized controlled trial of surveillance vs radiofrequency ablation in Europe. Three expert pathologists independently reviewed baseline and subsequent LGD specimens. The presence and degree of dysplasia was separately recorded for each biopsy and classified according to the Vienna Classification system. The primary end point was development of HGD or EAC. We performed univariate logistic regression analyses to assess the association between outcomes and factors such as number of pathologists confirming LGD, multifocality of LGD, and persistence of LGD over time. RESULTS: Of the 255 patients, 45 (18%) developed HGD or EAC during a median 42-month follow-up period (interquartile range, 25-61 months); patients were examined by a median 4 endoscopies (interquartile range, 3-6 endoscopies). The number of pathologists confirming LGD was strongly associated with progression to neoplasia; risk for progression increased greatly when all 3 pathologists agreed on LGD (odds ratio, 47.14; 95% confidence interval, 13.10-169.70). When LGD was detected at baseline and confirmed by a subsequent endoscopy, the odds for progression to neoplasia also increased greatly (odds ratio, 9.28; 95% confidence interval, 4.39-19.64). Multifocal LGD was not significantly associated with progression to neoplasia. CONCLUSIONS: The number of pathologists confirming LGD and persistence of LGD over time increase risk for development of HGD or EAC in patients with Barrett's esophagus and LGD. These simple, readily available variables can help stratify risk and select patients for prophylactic ablation therapy.

Whole-exome sequencing of duodenal neuroendocrine tumors in patients with neurofibromatosis type 1.

Neurofibromatosis type 1 (NF1) is a hereditary cancer predisposition syndrome characterized by frequent cutaneous and nervous system abnormalities. Patients with NF1 also have an increased prevalence of multiple gastrointestinal and peripancreatic neoplasms-neuroendocrine tumors of the ampulla that express somatostatin are particularly characteristic of NF1. In this study, we characterize the genetic alterations of a clinically well-characterized cohort of six NF1-associated duodenal neuroendocrine tumors using whole-exome sequencing. We identified inactivating somatic mutations in the NF1 gene in three of six tumors; the only other gene altered in more than one tumor was IFNB1. Copy number analysis revealed deletion/loss of heterozygosity of chromosome 22 in three of six patients. Analysis of germline variants revealed germline deleterious NF1 variants in four of six patients, as well as deleterious variants in other tumor suppressor genes in two of four patients with deleterious NF1 variants. Taken together, these data confirm the importance of somatic inactivation of the wild-type NF1 allele in the formation of NF1-associated duodenal neuroendocrine tumors and suggest that loss of chromosome 22 is important in at least a subset of cases. However, we did not identify any genes altered in the majority of NF1-associated duodenal neuroendocrine tumors that uniquely characterize the genomic landscape of this tumor. Still, the genetic alterations in these tumors are distinct from sporadic neuroendocrine tumors occurring at these sites, highlighting that unique genetic alterations drive syndromic tumors.

Pancreatic undifferentiated carcinoma with osteoclast-like giant cells is genetically similar to, but clinically distinct from, conventional ductal adenocarcinoma.

Undifferentiated carcinoma of the pancreas with osteoclast-like giant cells (UCOGC) is currently considered a morphologically and clinically distinct variant of pancreatic ductal adenocarcinoma (PDAC). In this study, we report clinical and pathological features of a series of 22 UCOGCs, including the whole exome sequencing of eight UCOGCs. We observed that 60% of the UCOGCs contained a well-defined epithelial component and that patients with pure UCOGC had a significantly better prognosis than did those with an UCOGC with an associated epithelial neoplasm. The genetic alterations in UCOGC are strikingly similar to those known to drive conventional PDAC, including activating mutations in the oncogene KRAS and inactivating mutations in the tumor suppressor genes CDKN2A, TP53, and SMAD4. These results further support the classification of UCOGC as a PDAC variant and suggest that somatic mutations are not the determinants of the unique phenotype of UCOGC. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.