Improved overall survival in patients with high-grade serous ovarian cancer is associated with CD16a+ immunologic neighborhoods containing NK cells, T cells and macrophages.

Background: For patients with high grade serous carcinoma of the ovary (HGSC), survival rates have remained static for the last half century. Despite the presence of tumor mutations and infiltration of immune cells, existing immunotherapies have achieved little success against HGSC. These observations highlight a gap in the understanding of how the immune system functions and interacts within HGSC tumors. Methods: We analyzed duplicate core samples from 939 patients with HGSC to understand patterns of immune cell infiltration, localization, and associations with clinical features. We used high-parameter immunohistochemical/Opal multiplex, digital pathology, computational biology, and multivariate analysis to identify immune cell subsets and their associations with HGSC tumors. Results: We defined six patterns of cellular infiltration by spatially restricted unsupervised clustering of cell subsets. Each pattern was represented to some extent in most patient samples, but their specific distributions differed. Overall (OS) and progression-free survival (PFS) corresponded with higher infiltration of CD16a+ cells, and their co-localization with macrophages, T cells, NK cells, in one of six cellular neighborhoods that we defined with our spatial assessment. Conclusions: Immune cell neighborhoods containing CD16a+ cells are associated with improved OS and PFS for patients with HGSC. Patterns of immunologic neighborhoods differentiate patient outcomes, and could inform future, more precise approaches to treatment.

PAXgene Fixation for Pancreatic Cancer: Implications for Molecular and Surgical Pathology.

Genomic profiling of pancreatic cancer using small core biopsies has taken an increasingly prominent role in precision medicine. However, if not appropriately preserved, nucleic acids (NA) from pancreatic tissues are known to be susceptible to degradation due to high intrinsic levels of nucleases. PAXgene fixation (PreAnalytix, Switzerland) represents a novel formalin-free tissue preservation method. We sought to compare the NA and histomorphological preservation of pancreatic cancer tissues preserved with PAXgene-fixed paraffin-embedding (PFPE) and formalin-fixed paraffin-embedding (FFPE). Tissues from 19 patients were obtained prospectively from pancreaticoduodenectomy specimens and evaluated by four gastrointestinal pathologists. The extracted NA were quantified by Nanodrop and Qubit and assessed for quality by qPCR, targeted next-generation sequencing (NGS) assay, and RNA-sequencing. Our results demonstrated that, when assessed blindly for morphological quality, the four pathologists deemed the PFPE slides adequate for diagnostic purposes. PFPE tissues enable greater yields of less fragmented and more amplifiable DNA. PFPE tissues demonstrated significantly improved quality control (QC) metrics in a targeted NGS assay including Median Absolute Pair-wise Difference (MAPD) scores. Our results support the use of PAXgene fixative for the processing of specimens from pancreatic cancers with the potential benefits of improved yields for more amplifiable DNA in low-yield biopsy specimens and its ideal use for amplicon-based NGS assays.

A Case of Whipple Disease With Cutaneous Manifestations.

ABSTRACT: Whipple disease (WD) is a rare bacterial infectious disease that is classically characterized by years of arthralgia, followed by malabsorption, diarrhea, and weight loss. However, WD may manifest in virtually any organ system, and patients with WD rarely develop subcutaneous erythema nodosum-like lesions. We report a case of a 51-year-old man diagnosed with WD who subsequently developed widely distributed erythematous subcutaneous nodules after 5 months of antibiotic therapy.

Deep learning for computer-assisted diagnosis of hereditary diffuse gastric cancer.

BACKGROUND: Patients with hereditary diffuse gastric cancer often undergo prophylactic gastrectomy to minimize cancer risk. Because intramucosal poorly cohesive carcinomas in this setting are typically not grossly visible, many pathologists assess the entire gastrectomy specimen microscopically. With 150 or more slides per case, this is a major time burden for pathologists. This study utilizes deep learning methods to analyze digitized slides and detect regions of carcinoma. METHODS: Prophylactic gastrectomy specimens from seven patients with germline CDH1 mutations were analyzed (five for training/validation and two for testing, with a total of 133 tumor foci). All hematoxylin and eosin slides containing cancer foci were digitally scanned, and patches of size 256×256 pixels were randomly extracted from regions of cancer as well as from regions of normal background tissue, resulting in 15,851 images for training/validation and 970 images for testing. A model with DenseNet-169 architecture was trained for 150 epochs, then evaluated on images from the test set. External validation was conducted on 814 images scanned at an outside institution. RESULTS: On individual patches, the trained model achieved a receiver operating characteristic (ROC) area under the curve (AUC) of 0.9986. This enabled it to maintain a sensitivity of 90% with a false-positive rate of less than 0.1%. On the external validation dataset, the model achieved a similar ROC AUC of 0.9984. On whole slide images, the network detected 100% of tumor foci and correctly eliminated an average of 99.9% of the non-cancer slide area from consideration. CONCLUSIONS: Overall, our model shows encouraging progress towards computer-assisted diagnosis of hereditary diffuse gastric cancer.

Urachal Mucinous Cystic Tumor of Low Malignant Potential with Concurrent Sigmoid Colon Adenocarcinoma.

Urachal mucinous tumors are rare neoplasms with behaviour that can range from relatively benign to malignancy that can spread distantly or throughout the peritoneum as pseudomyxoma peritonei or peritoneal carcinomatosis. Here we describe a unique case of urachal mucinous cystic tumor of low malignant potential confined to an intact cyst at the dome of the urinary bladder, without rupture or peritoneal spread. The urachal mucinous tumor was an incidental finding on a staging CT scan performed for sigmoid colon adenocarcinoma. We believe that this case illustrates a potential diagnostic pitfall which could have prognostic and therapeutic implications. Due to the intestinal phenotype of these neoplasms, a urachal tumor of low malignant potential could be mistaken for metastatic spread from a colonic adenocarcinoma in the rare situation such as this case, where the two neoplasms occur concurrently.

Ciliated columnar epithelium in the esophagus and gastroesophageal junction: A different perspective from study of a North American population.

An index case of ciliated columnar epithelium in a gastroesophageal (GE) junction biopsy identified in routine surgical pathology practice struck us as highly unusual. However, pathology literature, mainly from Asian populations, reports ciliated columnar epithelium in up to 40% of tissue samples from the upper GI tract. This was inconsistent with our pathology practice experience, so we initiated a local review of cases at our Canadian centre. 1048 consecutive tissue samples from the esophagus and GE junction were reviewed retrospectively and no ciliated epithelium was identified. This review included 1000× oil immersion microscopy of 22 cases with "multilayered epithelium". In 971 cases verified in prospective surgical pathology practice following identification of the index case, 3 additional cases of ciliated columnar epithelium were identified. The index case had ciliated pseudostratified columnar epithelium, resembling respiratory epithelium, and had strong, diffuse expression of TTF-1 by immunohistochemistry. In the other 3 cases, the cilia were located on the surface of a pseudostratified columnar epithelium, a multilayered epithelium, or a low columnar epithelium, all TTF-1 negative. Over a year later, the index case proved to have arisen from a bronchial-esophageal fistula. The other cases were not associated with a fistula. Our conclusion is that ciliated columnar epithelium is rare in Canadian adults (<0.5% of patients). Ciliated epithelium due to a bronchial-esophageal fistula is exceptional, but something to consider if there is a suspicious clinical picture and TTF-1 expression. Other cases might represent a rare metaplastic phenomenon or remnant from fetal development.

Safety and Feasibility of Using Magnetic Resonance Imaging Criteria to Identify Patients With "Good Prognosis" Rectal Cancer Eligible for Primary Surgery: The Phase 2 Nonrandomized QuickSilver Clinical Trial.

IMPORTANCE: Chemoradiotherapy (CRT), followed by surgery, is the recommended approach for stage II and III rectal cancer. While CRT decreases the risk of local recurrence, it does not improve survival and leads to poorer functional outcomes than surgery alone. Therefore, new approaches to better select patients for CRT are important. OBJECTIVE: To conduct a phase 2 study to evaluate the safety and feasibility of using magnetic resonance imaging (MRI) criteria to select patients with "good prognosis" rectal tumors for primary surgery. DESIGN, SETTING, AND PARTICIPANTS: Prospective nonrandomized phase 2 study at 12 high-volume colorectal surgery centers across Canada. From September 30, 2014, to October 21, 2016, a total of 82 patients were recruited for the study. Participants were patients newly diagnosed as having rectal cancer with MRI-predicted good prognosis rectal cancer. The MRI criteria for good prognosis tumors included distance to the mesorectal fascia greater than 1 mm; definite T2, T2/early T3, or definite T3 with less than 5 mm of extramural depth of invasion; and absent or equivocal extramural venous invasion. INTERVENTIONS: Patients with rectal cancer with MRI-predicted good prognosis tumors underwent primary surgery. MAIN OUTCOMES AND MEASURES: The primary outcome was the proportion of patients with a positive circumferential resection margin (CRM) rate. Assuming a 10% baseline probability of a positive CRM, a sample size of 75 was estimated to yield a 95% CI of ±6.7%. RESULTS: Eighty-two patients (74% male) participated in the study. The median age at the time of surgery was 66 years (range, 37-89 years). Based on MRI, most tumors were midrectal (65% [n = 53]), T2/early T3 (60% [n = 49]), with no suspicious lymph nodes (63% [n = 52]). On final pathology, 91% (n = 75) of tumors were T2 or greater, 29% (n = 24) were node positive, and 59% (n = 48) were stage II or III. The positive CRM rate was 4 of 82 (4.9%; 95% CI, 0.2%-9.6%). CONCLUSIONS AND RELEVANCE: The use of MRI criteria to select patients with good prognosis rectal cancer for primary surgery results in a low rate of positive CRM and suggests that CRT may not be necessary for all patients with stage II and III rectal cancer. TRIAL REGISTRATION: ISRCTN.com identifier: ISRCTN05107772.

ACTH-Secreting Neuroendocrine Carcinoma of the Cecum: Case Report and Review of the Literature.

BACKGROUND: Approximately 30% of neuroendocrine tumors (NETs) present with secretory syndromes or develop one during the course of the disease. Cushing syndrome caused by a gastrointestinal tract NET is rare, with limited published information. We describe a patient with florid Cushing syndrome due to ectopic adrenocorticotropic hormone (ACTH) from a NET of colonic origin. A literature review was conducted to describe the spectrum of this clinical and pathologic entity as reported in the scientific literature. PATIENT AND METHODS: Next-generation sequencing and microsatellite instability testing was carried out on the tumor from our case. A preliminary PubMed search was conducted using the following terms under the publication type "Case Reports": "Cushing" AND "colon," "neuroendocrine" AND "colon" and "neuroendocrine AND Cushing AND "colon." A manual search was performed to review all references for inclusion and relevant clinical, biochemical and pathologic data was abstracted. RESULTS: Mutations in BRAF V600E and TP53 were detected in our case. We retrieved 18 previously reported cases of Cushing syndrome associated with a NET of colonic origin, none of which had next-generation sequencing performed. Median age at diagnosis was 54.5 years (range, 24-74 years), with equal gender distribution. ACTH was detected by immunohistochemistry in the primary tumor and/or metastatic lesion in 61.5%. Review of the reports suggested that ectopic ACTH secretion from a colonic tumor might be more common in mixed glandular and NETs, including mixed adenocarcinoma-neuroendocrine carcinoma. Among studies reporting outcomes, the unadjusted mortality rate was 77.7%, with median overall survival from presentation of 63 days (range, 17-380 days). CONCLUSION: Cushing syndrome associated with ectopic ACTH from tumors of colonic origin is a rare phenomenon with poor outcomes and can be associated with pure NETs, adenocarcinomas, and mixed-phenotype tumors, including mixed adenocarcinoma-neuroendocrine carcinoma.

Goblet cell carcinoid of the appendix - An interobserver variability study using two proposed classification systems.

Goblet cell carcinoid (GCC) is an uncommon tumor of the vermiform appendix. Due to a broad spectrum of morphological differentiation, subclassification and grading of GCCs remains an area of controversy. Two separate systems have proposed classifying GCC tumors into three (classical GCC; adenocarcinoma ex-GCC, signet ring cell type; adenocarcinoma ex-GCC, poorly differentiated carcinoma type) OR two subgroups (low and high grade GCC) based on morphological criteria. We independently compared the inter-observer variability associated with each classification system. Overall, both systems had moderate interobserver agreement, with the two-tiered system (κ=0.54) performing slightly better than the three-tiered system (κ=0.42). GI-specialist pathologists had substantial agreement for both two and three-tiered systems (κ=0.65 vs. 0.65). Non-GI trained pathologists had lower overall agreement than GI trained pathologists, but their agreement was better using the two-tiered system (κ=0.44) than the three-tiered system (κ=0.22). A sub-analysis of 6 cases with a high rate of discordant classification revealed several challenges that exist in applying current criteria, including differentiating "goblet" vs. "signet ring" cell morphology, applying a 1 mm2 criteria to multifocal non-contiguous glandular and single infiltrating cell architecture, differentiating fibro-inflammatory stroma from desmoplastic stroma, and solid architecture in cases with abundant extracellular mucin, and distinguishing "reactive" nuclear atypia from true "cytologic atypia". Despite these challenges, the study identified better agreement among GI pathologists than non-GI trained pathologists. While GI pathologist review may be helpful, further research on objective classification criteria remains an area of interest.

Validation of a Cytotechnologist Manual Counting Service for the Ki67 Index in Neuroendocrine Tumors of the Pancreas and Gastrointestinal Tract.

CONTEXT: - Pathologists routinely assess Ki67 immunohistochemistry to grade gastrointestinal and pancreatic neuroendocrine tumors. Unfortunately, manual counts of the Ki67 index are very time consuming and eyeball estimation has been criticized as unreliable. Manual Ki67 counts performed by cytotechnologists could potentially save pathologist time and improve accuracy. OBJECTIVE: - To assess the concordance between manual Ki67 index counts performed by cytotechnologists versus eyeball estimates and manual Ki67 counts by pathologists. DESIGN: - One Ki67 immunohistochemical stain was retrieved from each of 18 archived gastrointestinal or pancreatic neuroendocrine tumor resections. We compared pathologists' Ki67 eyeball estimates on glass slides and printed color images with manual counts performed by 3 cytotechnologists and gold standard manual Ki67 index counts by 3 pathologists. RESULTS: - Tumor grade agreement between pathologist image eyeball estimate and gold standard pathologist manual count was fair (κ = 0.31; 95% CI, 0.030-0.60). In 9 of 20 cases (45%), the mean pathologist eyeball estimate was 1 grade higher than the mean pathologist manual count. There was almost perfect agreement in classifying tumor grade between the mean cytotechnologist manual count and the mean pathologist manual count (κ = 0.910; 95% CI, 0.697-1.00). In 20 cases, there was only 1 grade disagreement between the 2 methods. Eyeball estimation by pathologists required less than 1 minute, whereas manual counts by pathologists required a mean of 17 minutes per case. CONCLUSIONS: - Eyeball estimation of the Ki67 index has a high rate of tumor grade misclassification compared with manual counting. Cytotechnologist manual counts are accurate and save pathologist time.

Primary large cell neuroendocrine carcinoma of the skin: An under-recognized entity and a mimic of metastatic disease.

Primary large cell neuroendocrine carcinomas of the skin are exceptionally rare and can be diagnosed only when a metastasis from another organ has been excluded. We report the case of a 62-year-old woman with a cutaneous papule on the mid-chest which generated a differential diagnosis of vascular lesion and basal cell carcinoma. Following excision, microscopic evaluation revealed a dermal large cell undifferentiated carcinoma, with a brisk mitotic rate and focal geographic necrosis. Mucin production was absent. On immunohistochemistry, the lesion expressed CK7, AE1AE3, CK8/18, chromogranin, synaptophysin, CD56, calcitonin (patchy) and TTF-1 (minimal focal). Stains for neurofilament, CK20, CK5/6, p40, p63, SOX10, MART-1, EMA, CEA, ER/PR, GATA3, GCDFP, mammoglobin, PAX-8, CDX2, napsin, ERG and MCPyV proved negative. The histopathological diagnosis was of a large cell neuroendocrine carcinoma, probably metastatic. The patient underwent comprehensive clinical, laboratory and radiographic investigations and no underlying primary carcinoma was detected. During a 20-month follow-up period with an oncologist, the patient remains well and free of any apparent carcinoma. This suggests a primary large cell neuroendocrine carcinoma of skin. To date, 3 such cases have been reported in Japanese patients. This is the first in a Caucasian resident of North America.

Fat-Containing Liver Lesions on Imaging: Detection and Differential Diagnosis.

OBJECTIVE: The purpose of this article is to review how fat is detected on imaging and to discuss the differential diagnosis of fat-containing liver lesions. CONCLUSION: Fat is a highly useful feature in characterizing liver lesions on imaging. Although a variety of liver lesions can show fat on cross-sectional imaging, the presence of fat usually indicates that the lesion is of hepatocellular origin. Less commonly, nonhepatocellular fatty lesions may be distinguished by ancillary clinical and imaging features.

Ramipril-associated cholestasis in the setting of recurrent drug-induced liver injury.

Aim: Angiotensin-converting enzyme inhibitors (ACEIs) are commonly used to treat hypertension. Although generally well tolerated, the adverse effects of ACEIs include hypotension, cough, acute kidney injury and hyperkalemia. Rare reports of ACEI-induced hepatotoxicity have been described, most notably a cholestatic pattern of injury related to captopril. A 67-year-old male presented to the emergency department with a three-week history of jaundice, pruritis and weakness. Eight weeks before, he began taking ramipril and clopidogrel. His past medical history was significant for previous acute cholestatic liver injury approximately 20 years earlier, which was attributed to methimazole. Abnormal blood work demonstrated aspartate aminotransferase (AST) 47 U/L, alanine aminotransferase (ALT) 46 U/L, total bilirubin 230 µmol/L, direct bilirubin 176 µmol/L, and alkaline phosphatase (ALP) 470 U/L. Abdominal ultrasound and magnetic resonance cholangiopancreatography showed no bile duct obstruction. Further work-up was negative for infectious, autoimmune, or other causes. Percutaneous liver biopsy showed marked cholestasis. With discontinuation of ramipril, the patient demonstrated prolonged cholestasis with partial biochemical improvement and was discharged after six weeks in hospital. This case represents the first described cross reactivity between ramipril and methimazole, illustrating the complex and poorly understood nature of DILI. Despite the relatively few instances of ACEI-induced liver hepatotoxicity, consideration should be given to discontinuation of ramipril in situations of unknown liver damage.

Biliary Adenofibroma of Liver: Morphology, Tumor Genetics, and Outcomes in 6 Cases.

Biliary adenofibroma is a rare primary hepatic neoplasm, recognized in the World Health Organization classification, although only 14 cases have been reported to date. This series includes extended follow-up from 2 of the early case reports and 4 novel cases. Clinical history and histology were reviewed in all 6 cases. Tumor DNA was analyzed for point mutations by multiplex polymerase chain reaction and copy number alterations by array comparative genomic hybridization. The patients included 4 females and 2 males presenting between 46 and 83 years of age, with tumors ranging from 7 to 16 cm in diameter. The tumors had similar morphology, with tubules and cysts lined mainly by bland to mildly atypical cuboidal epithelium embedded in fibrous stroma. Multiplex polymerase chain reaction did not identify mutations in 4 tumors tested. Three tumors tested by array comparative genomic hybridization showed chromosomal copy number alterations, including 1 with amplifications of CCND1 and ERBB2. Three patients underwent resection with no recurrence at 21, 20, and 3 years of follow-up. One patient is alive after 14 months with no resection. Two patients with margin-positive resections had local recurrence at 1 and 6 years after surgery. No patient had distant metastasis. The distinct morphology and multiple clonal cytogenetic alterations in biliary adenofibromas indicate that the lesions are neoplastic. Amplifications of CCND1 and ERBB2 are not typical of benign neoplasms, and suggest that these tumors may have the ability to behave aggressively. However, the clinical outcomes in these patients suggest the neoplasms are only slowly progressive.

Duodenal Villous Atrophy in a TTG-Negative Patient Taking Olmesartan: A Case Report and Review of the Literature.

Olmesartan, an angiotensin II receptor antagonist used to treat hypertension, is associated with few adverse effects. Here, a case of severe sprue-like enteropathy and acute kidney injury is described in a 68-year-old male taking olmesartan for 3-4 years. He presented to hospital with a five-week history of diarrhea, vomiting, and a 20 lb weight loss. Anti-TTG was negative with a normal IgA. Biopsies of the distal duodenum and duodenal cap revealed marked blunting of the villi with near complete villous atrophy of the biopsies from the bulb. There was an increase in intraepithelial lymphocytes as well as neutrophils in the surface epithelium. The patient's diarrhea improved upon discontinuation of olmesartan and he returned to his previous weight. Repeat endoscopy four months later demonstrated complete resolution of inflammatory change with normal villous architecture. Long-term olmesartan use is associated with severe sprue-like enteropathy. The mechanism of intestinal injury is unknown. Duodenal biopsy results may mimic other enteropathies such as celiac disease. Physicians should consider medications as potential etiologies of enteropathy.

Spontaneous colonic perforation: a rare complication of collagenous colitis.

Collagenous colitis is a clinicopathologic syndrome characterized by chronic watery diarrhea and unique histopathologic features. Spontaneous colonic perforation in the setting of collagenous colitis is a highly unusual complication, with only three cases reported in the literature to date. We present a fourth case and propose a potential pathologic mechanism for acute colonic perforation in this patient population.

Ciliated Foregut Cyst of the Common Hepatic Duct: An Unusual Mimic of a Type II Choledochal Cyst.

Ciliated foregut cysts typically arise in the liver and pancreas, with rare cases reported to arise in the gallbladder wall. Herein, we describe an unusual case of a patient with a ciliated foregut cyst arising from the wall of the common hepatic duct, which has not been reported previously. As a result of its unique anatomical location, the cyst was mistaken for a type II choledochal cyst on preoperative imaging. The cyst caused intermittent biliary obstruction that resolved following surgical resection.

Prevalence of oesophageal epidermoid metaplasia in 1048 consecutive patients and 58 patients with squamous neoplasms.

AIMS: Oesophageal epidermoid metaplasia is defined by a dense granular layer with overlying hyperorthokeratosis, resembling the epidermis of skin. A possible association between epidermoid metaplasia, squamous dysplasia and squamous cell carcinoma has been proposed. The aim of this study was to compare the prevalence of epidermoid metaplasia in patients with oesophageal squamous neoplasms with that in a control cohort. METHODS AND RESULTS: Medical records and slides from 1048 consecutive oesophageal biopsies and resections for any indication and 58 patients with oesophageal squamous neoplasms were reviewed. Two cases (0.19%) of epidermoid metaplasia were identified in the 1048-patient control group. The prevalence of epidermoid metaplasia was significantly higher (P < 0.05) in the 58 patients with oesophageal squamous neoplasms, two of whom (3.5%) had concurrent epidermoid metaplasia (odds ratio 18.1, 95% confidence interval 2.5-131). One case was associated with a verrucous carcinoma and the other with a well-differentiated, superficial (pT1), exophytic squamous cell carcinoma. No patients had epidermoid metaplasia in a biopsy prior to the diagnosis of squamous neoplasia. CONCLUSIONS: The increased prevalence of epidermoid metaplasia observed in patients with squamous neoplasms provides some additional support for the proposed association. The hypothesis that epidermoid metaplasia is a precursor to squamous neoplasms remains unproven.

Ileitis associated with Meckel's diverticulum.

AIMS: Histological features of chronic active ileitis in the small intestine neighbouring a Meckel's diverticulum raise the possibility of concurrent Crohn's disease. Several studies have reported a significant association between Meckel's diverticulum and Crohn's disease, whereas some case reports have proposed that the ileitis is attributable to acid-secreting gastric heterotopia. The aim of this study was to evaluate the incidence, histomorphology and clinical follow-up of Meckel's diverticulum-associated ileitis. METHODS AND RESULTS: Medical records and slides from 48 consecutive surgical resections performed for Meckel's diverticulum were reviewed. Nine of 48 adults had significant inflammatory changes in the small intestine neighbouring the diverticulum. Two were transmural ulcers attributable to ingestion of a sharp object. Two patients had established Crohn's disease, both with long segments (>95 mm) of transmural inflammation located >100 mm from the diverticulum. The remaining five patients had inflammatory changes (ulceration, pseudopyloric metaplasia, submucosal fibrosis, and muscularis mucosa hyperplasia) confined to a short segment (<20 mm) of mucosa/submucosa within 50-60 mm of the diverticulum. Two had gastric heterotopia in the diverticulum. None of these five patients used non-steroidal anti-inflammatory drugs (NSAIDs). On follow-up, none had symptoms, imaging or pathology suggestive of Crohn's disease. CONCLUSIONS: Ileitis affecting a short segment of mucosa and submucosa in the small intestine near a Meckel's diverticulum is relatively common, and is not necessarily related to Crohn's disease.