Rhonda K. Yantiss

Culture independent analysis of ileal mucosa reveals a selective increase in invasive Escherichia coli of novel phylogeny relative to depletion of Clostridiales in Crohn's disease involving the ileum

Intestinal bacteria are implicated increasingly as a pivotal factor in the development of Crohns disease, but the specific components of the complex polymicrobial enteric environment driving the inflammatory response are unresolved. This study addresses the role of the ileal mucosa-associated microflora in Crohns disease. A combination of culture-independent analysis of bacterial diversity (16S rDNA library analysis, quantitative PCR and fluorescence in situ hybridization) and molecular characterization of cultured bacteria was used to examine the ileal mucosa-associated flora of patients with Crohns disease involving the ileum (13), Crohns disease restricted to the colon (CCD) (8) and healthy individuals (7). Analysis of 16S rDNA libraries constructed from ileal mucosa yielded nine clades that segregated according to their origin (P<0.0001). 16S rDNA libraries of ileitis mucosa were enriched in sequences for Escherichia coli (P<0.001), but relatively depleted in a subset of Clostridiales (P<0.05). PCR of mucosal DNA was negative for Mycobacterium avium subspecies paratuberculosis, Shigella and Listeria. The number of E. coli in situ correlated with the severity of ileal disease (ρ 0.621, P<0.001) and invasive E. coli was restricted to inflamed mucosa. E. coli strains isolated from the ileum were predominantly novel in phylogeny, displayed pathogen-like behavior in vitro and harbored chromosomal and episomal elements similar to those described in extraintestinal pathogenic E. coli and pathogenic Enterobacteriaceae. These data establish that dysbiosis of the ileal mucosa-associated flora correlates with an ileal Crohns disease (ICD) phenotype, and raise the possibility that a selective increase in a novel group of invasive E. coli is involved in the etiopathogenesis to Crohns disease involving the ileum.

Appendiceal mucinous neoplasms: A clinicopathologic analysis of 107 cases

The classification of appendiceal mucinous tumors is controversial and terminology used for them inconsistent, particularly when they lack overtly malignant features but are associated with extra-appendiceal spread. We reviewed 107 appendiceal mucinous neoplasms and classified them as low-grade appendiceal mucinous neoplasm (LAMN) (n = 88), mucinous adenocarcinomas (MACAs) (n = 16), or discordant (n = 3) based on architectural and cytologic features. LAMNs were characterized by a villous or flat proliferation of mucinous epithelium with low-grade atypia. Thirty-nine tumors were confined to the appendix, but 49 had extra-appendiceal tumor spread, including 39 with peritoneal tumor characterized by mucin pools harboring low-grade mucinous epithelium, usually dissecting in a hyalinized stroma. Eight of the 16 MACAs lacked destructive invasion of the appendiceal wall and eight showed an infiltrative pattern of invasion. Extra-appendiceal tumor spread was present in 12 MACAs (four peritoneum, seven peritoneum and ovaries; one ovaries only). In MACAs with an infiltrative pattern, peritoneal tumor consisted of glands and single cells in a desmoplastic stroma. The peritoneal tumor in the remaining cases consisted of mucin pools that contained mucinous epithelium with high-grade atypia and, in some cases, increased cellularity compared with that seen in peritoneal spread in cases of LAMN. Three cases were classified as discordant because the appendiceal tumors were LAMNs but the peritoneal tumors were high-grade. Follow-up was available for 49 LAMNs, 15 MACAs, and 2 discordant cases. None of the patients with LAMNs confined to the appendix experienced recurrence (median follow-up 6 years). LAMNs with extra-appendiceal spread were associated with 3-, 5-, and 10-year survival rates of 100%, 86%, and 45%, respectively. Patients with MACA had 3- and 5-year survival rates of 90% and 44%, respectively (p = 0.04). The bulk of peritoneal disease correlated with prognosis among patients with MACA (p = 0.04) and, to a lesser degree, among patients with LAMNs (p = 0.07). We conclude that: 1) appendiceal mucinous neoplasms can be classified as either low-grade mucinous neoplasms or mucinous adenocarcinoma based on architectural and cytologic features; 2) tumors that can be confidently placed in the low-grade group (which requires rigorous pathologic evaluation of the appendix) and are confined to the appendix are clinically benign in our experience to date; 3) low-grade tumors confined to the appendix are morphologically identical to those with extra-appendiceal spread (except for the usual identification of breach of the wall in the latter cases) and the same designation is appropriate for the appendiceal neoplasia in each situation; 4) the long-term outlook for patients with low-grade tumors and peritoneal spread is guarded with just over half dying of disease after 10 years; 5) appendiceal mucinous tumors with destructive invasion of the appendiceal wall, complex epithelial proliferations, or high-grade nuclear atypia generally pursue an aggressive clinical course and should be classified as mucinous adenocarcinomas; 6) peritoneal tumor can be classified as involvement by LAMN or MACA, and this distinction is of prognostic significance; 7) bulky peritoneal tumor worsens prognosis; and 8) LAMNs associated with high-grade peritoneal tumor behave as adenocarcinoma.

Obesity Is Associated with Inflammation and Elevated Aromatase Expression in the Mouse Mammary Gland

Elevated circulating estrogen levels are associated with increased risk of breast cancer in obese postmenopausal women. Following menopause, the biosynthesis of estrogens through CYP19 (aromatase)-mediated metabolism of androgen precursors occurs primarily in adipose tissue, and the resulting estrogens are then secreted into the systemic circulation. The potential links between obesity, inflammation, and aromatase expression are unknown. In both dietary and genetic models of obesity, we observed necrotic adipocytes surrounded by macrophages forming crown-like structures (CLS) in the mammary glands and visceral fat. The presence of CLS was associated with activation of NF-κB and increased levels of proinflammatory mediators (TNF-α, IL-1β, Cox-2), which were paralleled by elevated levels of aromatase expression and activity in the mammary gland and visceral fat of obese mice. Analyses of the stromal-vascular and adipocyte fractions of the mammary gland suggested that macrophage-derived proinflammatory mediators induced aromatase and estrogen-dependent gene expression (PR, pS2) in adipocytes. Saturated fatty acids, which have been linked to obesity-related inflammation, stimulated NF-κB activity in macrophages leading to increased levels of TNF-α, IL-1β, and Cox-2, each of which contributed to the induction of aromatase in preadipocytes. The discovery of the obesity → inflammation → aromatase axis in the mammary gland and visceral fat and its association with CLS may provide insight into mechanisms underlying the increased risk of hormone receptor-positive breast cancer in obese postmenopausal women, the reduced efficacy of aromatase inhibitors in the treatment of breast cancer in these women, and their generally worse outcomes. The presence of CLS may be a biomarker of increased breast cancer risk or poor prognosis. Cancer Prev Res; 4(3); 329–46. ©2011 AACR.

Endometriosis of the intestinal tract: a study of 44 cases of a disease that may cause diverse challenges in clinical and pathologic evaluation

Endometriosis of the intestinal tract may mimic a number of diseases both clinically and pathologically. The authors evaluated 44 cases of intestinal endometriosis in which endometriosis was the primary pathologic diagnosis, and evaluated them for a variety of gross and histologic changes. Cases with preneoplastic or neoplastic changes were excluded specifically because they were the subject of a previous study. The patients ranged in age from 28 to 56 years (mean age, 44 years), and presenting complaints included abdominal pain (n = 15), an abdominal mass (n = 12), obstruction (n = 8), rectal bleeding (n = 2), infertility (n = 3), diarrhea (n = 2), and increasing urinary frequency (n = 1). The clinical differential diagnoses included diverticulitis, appendicitis, Crohns disease, tubo-ovarian abscess, irritable bowel syndrome, carcinoma, and lymphoma. Forty-two patients underwent resection of the diseased intestine and two patients underwent endoscopic biopsies. In 13 patients there were predominantly mural masses, which were multiple in two patients (mean size, 2.6 cm). In addition, 11 cases had luminal stenosis or strictures, six had mucosal polyps, four had submucosal masses that ulcerated the mucosa (sometimes simulating carcinoma), three had serosal adhesions, one had deep fissures in the mucosa, and one was associated with appendiceal intussusception. Involvement of the lamina propria or submucosa was identified in 29 cases (66%) and, of these, 19 had features of chronic injury including architectural distortion (n = 19), dense lymphoplasmacytic infiltrates (n = 7), pyloric metaplasia of the ileum (n = 1), and fissures (n = 1). Three cases had features of mucosal prolapse (7%), ischemic changes were seen in four (9%), and segmental acute colitis and ulceration were seen in four and six cases (9% and 13%) respectively. In 14 patients, endometriosis formed irregular congeries of glands involving the intestinal surface epithelium, mimicking adenomatous changes. Mural changes included marked concentric smooth muscle hyperplasia and hypertrophy, neuronal hypertrophy and hyperplasia, and fibrosis of the muscularis propria with serositis. Follow-up of 20 patients (range, 1–30 years; mean, 7.8 years) revealed that only two patients had recurrent symptoms. None of the patients developed inflammatory bowel disease. Endometriosis can involve the intestinal tract extensively, causing a variety of clinical symptoms, and can result in a spectrum of mucosal alterations. Because the endometriotic foci may be inaccessible to endoscopic biopsy or may not be sampled because of their focality, clinicians and pathologists should be aware of the potential of this condition to mimic other intestinal diseases.

GASTROINTESTINAL STROMAL TUMOR VERSUS INTRA-ABDOMINAL FIBROMATOSIS OF THE BOWEL WALL: A CLINICALLY IMPORTANT DIFFERENTIAL DIAGNOSIS

Intra-abdominal fibromatosis (IAF) is an uncommon benign neoplasm that usually occurs in the mesentery or retroperitoneum and may, on occasion, mimic a gastrointestinal stromal tumor (GIST). Differentiating between these two entities is important clinically because IAF is a benign tumor whereas GISTs frequently have malignant potential. In this study, the authors identified 13 cases of IAF with prominent involvement of the bowel wall as well as 35 GISTs of the small intestine, colon, or mesentery and analyzed their clinical, gross, histologic, immunophenotypic, and ultrastructural characteristics to identify important distinguishing features. Patients with IAF were younger (mean, 34 yrs) than patients with GIST (mean, 54 yrs). Both types of tumors tended to be large, but GISTs were soft and lobulated with hemorrhage, necrosis, or cystification whereas IAFs were firm, tan, and homogeneous. Histologic features characteristic of GIST included the presence of spindle or epithelioid cells with variable architecture, mitotic activity (range, <1-95 mitoses/50 high-power fields [hpf]; mean, 15 mitoses/50 hpf), nuclear atypia, and myxoid or hyalinized stroma. Necrosis and hemorrhage were seen in 16 and 25 tumors, respectively. In contrast, IAFs were composed of broad, sweeping fascicles of monotonous spindle cells with mitotic activity (range, <3-11 mitoses/50 hpf; mean, 4 mitoses/50 hpf), bland nuclear features, and finely collagenous stroma. Necrosis, hemorrhage, and myxoid degeneration were not seen. Immunohistochemical studies performed on a limited number of GISTs and IAFs demonstrated that cells expressed vimentin (100% GIST and IAF), CD117 (88% GIST and 75% IAF), CD34 (42% GIST and 0% IAF), smooth muscle actin (63% GIST and 75% IAF), muscle actin (75% GIST and 75% IAF), desmin (8% GIST and 50% IAF), and S-100 protein (16% GIST and 0% IAF). Ultrastructural analysis of 21 GISTs revealed incomplete smooth muscle differentiation in some tumors whereas IAFs were shown to have complete myofibroblastic/fibroblastic differentiation. Information regarding clinical outcome was available on 29 patients and revealed that three patients with histologically benign GISTs were alive with no evidence of disease at 5 months to 6 years (mean, 3.5 yrs) and one patient with a histologically benign tumor died of disease after 7 years. Of patients with histologically malignant GIST, one died of surgical complications, 10 were alive without disease at I to 13 years (mean, 5.4 yrs), four were alive with disease at 4 months to 15 years (mean, 3.8 yrs), three had disseminated disease at operation, and seven were dead of disease at 10 months to 3 years (mean, 2.2 yrs). Follow up of eight patients with IAF demonstrated that five were alive without disease at 4 months to 15 years (mean, 5.3 yrs) and three had recurrences at 1 (two patients) and 2 years (one patient). In summary, IAFs can have many features (large size, infiltration of adjacent structures, mitotic activity) that can cause diagnostic confusion with GISTs and, importantly, the degree of mitotic activity present in IAFs may overlap that seen in malignant GISTs. These entities can be distinguished primarily by their light microscopic and ultrastructural features but there is a notable overlap in their immunohistochemical profiles. The distinction between these neoplasms is important because there are important clinical implications for the patient.

Neoplastic and pre-neoplastic changes in gastrointestinal endometriosis: a study of 17 cases.

The clinicopathologic features of neoplasms arising in gastrointestinal endometriosis have not been well characterized. In this series, we report 17 cases of gastrointestinal endometriosis complicated by neoplasms (14 cases) or precancerous changes (three cases). Four patients, one of whom also had hypermenorrhea, presented with chronic abdominal pain and five had obstructive symptoms; one of these also had rectal bleeding. One patient presented with an acute abdomen and fecal peritonitis, one had vaginal bleeding, and one had a progressive change in bowel habits. Nine patients had a long history of endometriosis, 11 patients had had hysterectomies, and eight of these had also received unopposed estrogen therapy. The lesions involved the rectum (6), sigmoid (6), colon, unspecified (2), and small intestine (3), and comprised 8 endometrioid adenocarcinomas (EA), 4 mullerian adenosarcomas (MAS), 1 endometrioid stromal sarcoma (ESS), 1 endometrioid adenofibroma of borderline malignancy (EBA) with carcinoma in situ, 2 atypical hyperplasias (AH), and one endometrioid adenocarcinoma in situ (ACIS). The tumors ranged in size from 2 to 15 cm and all involved the serosa and muscularis propria. Two tumors extended into the mucosa, with mucosal ulceration in one. Follow-up was available in 11 cases. One patient with EA was dead of disease at 1 year, one had two recurrences at 1 and 2 years, and three were alive with no evidence of disease (ANED) at 9 months to 13 years (mean, 68 mos). The patient with the EBA was ANED at 3 months. Two patients with MAS were ANED at 2 and 3 years. The patient with ESS had a recurrence at 3 years and was ANED 6 years after her original diagnosis. One woman with AH was ANED at 60 months and the patient with ACIS was ANED at 16 months. One of the carcinomas was originally misdiagnosed as a primary intestinal adenocarcinoma. The pathologist should be aware of the possibility of a tumor of genital tract type when evaluating intestinal neoplasms in females, particularly if they have a history of endometriosis and have received unopposed estrogen therapy.

KOC (K homology domain containing protein overexpressed in cancer): a novel molecular marker that distinguishes between benign and malignant lesions of the pancreas.

KOC (K homology domain containing protein overexpressed in cancer) is a novel oncofetal RNA-binding protein highly expressed in pancreatic carcinomas. Recently, Corixa Corporation developed a monoclonal antibody specific for KOC that can be used with standard immunohistochemical techniques. The purposes of this study were 1) to assess KOC mRNA expression in pancreatic carcinoma, 2) to determine the pattern of KOC immunoexpression among benign, borderline, and malignant pancreatic epithelial lesions, and 3) to evaluate the utility of the KOC antibody in distinguishing between these entities. mRNA was isolated from fresh pancreatic tissues (19 carcinomas, 2 normal pancreas, 1 chronic pancreatitis) and amplified using standard RT-PCR techniques. Fifteen of 19 (79%) carcinomas overexpressed KOC mRNA relative to non-neoplastic tissue samples and expression increased progressively with tumor stage: the mean copy number of KOC mRNA transcripts was 1.5, 11.1, 31, and 28 for stage I, II, III, and IV carcinomas, respectively, compared with 0.9 and 1 for normal pancreatic tissue and chronic pancreatitis, respectively. Immunostains using the KOC antibody were performed on 50 surgical resection specimens (38 invasive adenocarcinomas, 3 intraductal papillary-mucinous neoplasms, 2 mucinous cystic neoplasms, 7 chronic pancreatitis). KOC staining was present in 37 of 38 (97%) carcinomas: the staining reaction was moderate or strong in 36 of 38 (94%) and present in >50% of the tumor cells in 35 of 38 (92%) cases. Severe dysplasia of the ductal epithelium, present in 19 foci of intraductal papillary mucinous carcinoma, mucinous cystadenocarcinoma, and grade 3 pancreatic intraepithelial neoplasia (PanIN3) showed strong or moderate staining in 15 (79%) cases, whereas foci of mild and moderate dysplasia (intraductal papillary-mucinous neoplasms and mucinous cystic neoplasms with adenoma and/or moderate dysplasia, PanIN1, and PanIN2) were uniformly negative for this marker in 25 and 22 cases, respectively. In the normal pancreas, weak background staining of acini was present in 12 of 50 (24%) cases but was easily distinguishable from the type of staining identified in neoplastic epithelium, and benign ducts and ductules were negative in all cases. Four of 38 (11%) foci of chronic pancreatitis, present in the 7 resections performed for chronic pancreatitis as well as 31 foci of peritumoral chronic pancreatitis, showed weak staining in <10% of the ductules. We conclude that KOC is a sensitive and specific marker for carcinomas and high-grade dysplastic lesions of the pancreatic ductal epithelium. Therefore, immunostains directed against KOC may be of diagnostic utility in the evaluation of pancreatic lesions, particularly when biopsy material is limited.

Effects of Cigarette Smoke on the Human Oral Mucosal Transcriptome

Use of tobacco is responsible for ∼30% of all cancer-related deaths in the United States, including cancers of the upper aerodigestive tract. In the current study, 40 current and 40 age- and gender-matched never smokers underwent buccal biopsies to evaluate the effects of smoking on the transcriptome. Microarray analyses were carried out using Affymetrix HGU133 Plus 2 arrays. Smoking altered the expression of numerous genes: 32 genes showed increased expression and 9 genes showed reduced expression in the oral mucosa of smokers versus never smokers. Increases were found in genes involved in xenobiotic metabolism, oxidant stress, eicosanoid synthesis, nicotine signaling, and cell adhesion. Increased numbers of Langerhans cells were found in the oral mucosa of smokers. Interestingly, smoking caused greater induction of aldo-keto reductases, enzymes linked to polycyclic aromatic hydrocarbon–induced genotoxicity, in the oral mucosa of women than men. Striking similarities in expression changes were found in oral compared with the bronchial mucosa. The observed changes in gene expression were compared with known chemical signatures using the Connectivity Map database and suggested that geldanamycin, a heat shock protein 90 inhibitor, might be an antimimetic of tobacco smoke. Consistent with this prediction, geldanamycin caused dose-dependent suppression of tobacco smoke extract–mediated induction of CYP1A1 and CYP1B1 in vitro. Collectively, these results provide new insights into the carcinogenic effects of tobacco smoke, support the potential use of oral epithelium as a surrogate tissue in future lung cancer chemoprevention trials, and illustrate the potential of computational biology to identify chemopreventive agents. Cancer Prev Res; 3(3); 266–78

Clinical, pathologic, and molecular features of early-onset colorectal carcinoma.

The incidence of colorectal carcinoma has increased among patients <40 years of age for unclear reasons. In this study, we describe the clinical, pathologic, and molecular features of colorectal carcinomas that developed in young patients. We compiled a study group of 24 patients <40 years of age with colorectal carcinoma, and 45 patients ≥40 years of age served as controls. Cases were evaluated for clinical risk factors of malignancy and pathologic features predictive of outcome. The tumors were immunohistochemically stained for O6-methylguanine methyltransferase, MLH-1, MSH-2, MSH-6, β-catenin, chemokine (C-X-C motif) receptor 4, epidermal growth factor receptor, TP53, p16, survivin, and α-methylacyl-CoA racemase; assessed for microsatellite instability and mutations in β-catenin, APC, EGFR, PIK3CA, KRAS, and BRAF; evaluated for micro-RNA expression (miR-21, miR-20a, miR-183, miR-192, miR-145, miR-106a, miR-181b, and miR-203); and examined for evidence of human papillomavirus infection. One study patient each had ulcerative colitis and hereditary nonpolyposis colorectal cancer. Ninety-two percent of tumors from young patients occurred in the distal colon (P=0.006), particularly the rectum (58%, P=0.02), and 75% were stage III or IV. Tumors from young patients showed more frequent lymphovascular (81%, P=0.03) and/or venous (48%, P=0.003) invasion, an infiltrative growth pattern (81%, P=0.03), and α-methylacyl-CoA racemase expression (83%, P=0.02) compared with controls. Carcinomas in this group showed significantly increased expression of miR-21, miR-20a, miR-145, miR-181b, and miR-203 (P≤0.005 for all comparisons with controls). These results indicate that early-onset carcinomas commonly show pathologic features associated with aggressive behavior. Posttranslational regulation of mRNA and subsequent protein expression may be particularly important to the development of colorectal carcinomas in young patients.

Multiple gastrointestinal stromal tumors in type I neurofibromatosis: a pathologic and molecular study.

Multiple gastrointestinal stromal tumors typically occur in familial form associated with KIT receptor tyrosine kinase or platelet-derived growth factor receptor-alpha (PDGFRA) germline mutations, but may also develop in the setting of type 1 neurofibromatosis. The molecular abnormalities of gastrointestinal stromal tumors arising in neurofibromatosis have not been extensively studied. We identified three patients with type 1 neuro-fibromatosis and multiple small intestinal stromal tumors. Immunostains for CD117, CD34, desmin, actins, S-100 protein, and keratins were performed on all of the tumors. DNA was extracted from representative paraffin blocks from separate tumor nodules in each case and subjected to a nested polymerase chain reaction, using primers for KIT exons 9, 11, 13, and 17 and PDGFRA exons 12 and 18, followed by direct sequencing. The mean patient age was 56 years (range: 37–86 years, male/female ratio: 2/1). One patient had three tumors, one had five, and one had greater than 10 tumor nodules, all of which demonstrated histologic features characteristic of gastrointestinal stromal tumors and stained strongly for CD117 and CD34. One patient died of disease at 35 months, one was disease free at 12 months and one was lost to follow-up. DNA extracts from 10 gastrointestinal stromal tumors (three from each of two patients and four from one patient) were subjected to polymerase chain reactions and assessed for mutations. All of the tumors were wild type for KIT exons 9, 13, and 17 and PDGFRA exons 12 and 18. Three tumors from one patient had identical point mutations in KIT exon 11, whereas the other tumors were wild type at this locus. We conclude that, although most patients with type 1 neurofibromatosis and gastrointestinal stromal tumors do not have KIT or PDGFRA mutations, KIT germline mutations might be implicated in the pathogenesis of gastrointestinal stromal tumors in some patients.