Joel K. Greenson

One-year intense nutritional counseling results in histological improvement in patients with nonalcoholic steatohepatitis : A pilot study

BACKGROUND AND AIM:In individuals with biopsy-proven non-alcoholic steatohepatitis (NASH), short-term weight loss has been shown to improve biochemical abnormalities; however, its effect on liver histology is largely unknown. The aim of the article is to determine if dietary intervention is effective in improving histological features of steatohepatitis in patients with biopsy-proven NASH.METHODS:Twenty-three patients (11M/12F) with BMI >25 kg/m2 and biopsy-proven NASH received standardized nutritional counseling aimed at reducing insulin resistance (IR) and weight. Blood tests were checked at baseline and every 1–4 months, and liver biopsy was repeated at month 12. IR was assessed by the homeostasis model assessment (HOMA). Liver biopsies were scored according to modified Brunt criteria for NASH. “Histologic response” was defined as a reduction in total NASH score of ≥2 points with at least one point being in the non-steatosis component.RESULTS:Sixteen patients (8M/8F) completed 12 months of dietary intervention, and 15 underwent repeat liver biopsies. At month 12, mean weight decreased from 98.3 to 95.4 kg. Mean waist circumference, visceral fat, fasting glucose, IR, triglycerides, AST, ALT, and histologic score were all reduced but the difference was not significant. Nine patients had a histologic response, six had stable scores, and none had a worsened score. Compared to patients with unchanged histologic scores, patients with improved scores had significantly greater reduction in weight, waist circumference, AST, ALT, steatosis grade, and total NASH score.CONCLUSION:Among patients who successfully completed 1 yr of intense dietary intervention, nine of 15 patients with NASH displayed histologic improvement. This pilot study suggests that dietary intervention can be effective in improving histology in patients with biopsy-proven NASH.

Organ-Specific Molecular Classification of Primary Lung, Colon, and Ovarian Adenocarcinomas Using Gene Expression Profiles

Molecular classification of tumors based on their gene expression profiles promises to significantly refine diagnosis and management of cancer patients. The establishment of organ-specific gene expression patterns represents a crucial first step in the clinical application of the molecular approach. Here, we report on the gene expression profiles of 154 primary adenocarcinomas of the lung, colon, and ovary. Using high-density oligonucleotide arrays with 7129 gene probe sets, comprehensive gene expression profiles of 57 lung, 51 colon, and 46 ovary adenocarcinomas were generated and subjected to principle component analysis and to a cross-validated prediction analysis using nearest neighbor classification. These statistical analyses resulted in the classification of 152 of 154 of the adenocarcinomas in an organ-specific manner and identified genes expressed in a putative tissue-specific manner for each tumor type. Furthermore, two tumors were identified, one in the colon group and another in the ovarian group, that did not conform to their respective organ-specific cohorts. Investigation of these outlier tumors by immunohistochemical profiling revealed the ovarian tumor was consistent with a metastatic adenocarcinoma of colonic origin and the colonic tumor was a pleomorphic mesenchymal tumor, probably a leiomyosarcoma, rather than an epithelial tumor. Our results demonstrate the ability of gene expression profiles to classify tumors and suggest that determination of organ-specific gene expression profiles will play a significant role in a wide variety of clinical settings, including molecular diagnosis and classification.

Impaired gastric acid secretion in gastrin-deficient mice

To further understand the role of the peptide hormone gastrin in the development and function of the stomach, we have generated gastrin-deficient mice by gene targeting in embryonic stem cells. Mutant mice were viable and fertile, without obvious visible abnormalities. However, gastric function was severely affected by the loss of gastrin. Basal gastric acid secretion was abolished and could not be induced by histamine, carbachol, or gastrin. Histological analysis revealed alterations in the two cell types primarily involved in acid secretion, parietal and enterochromaffin-like (ECL) cells. Parietal cells were reduced in number with an accumulation of immature cells lacking H(+)-K(+)-adenosinetriphosphatase (H(+)-K(+)-ATPase). ECL cells were positioned closer to the base of the gastric glands, with markedly lower expression of histidine decarboxylase. Gastrin administration for 6 days reversed the effects of the gastrin deficiency, leading to an increase in the number of mature, H(+)-K(+)-ATPase-positive parietal cells and a partial restoration of acid secretion. The results show that gastrin is critically important for the function of the acid secretory system.To further understand the role of the peptide hormone gastrin in the development and function of the stomach, we have generated gastrin-deficient mice by gene targeting in embryonic stem cells. Mutant mice were viable and fertile, without obvious visible abnormalities. However, gastric function was severely affected by the loss of gastrin. Basal gastric acid secretion was abolished and could not be induced by histamine, carbachol, or gastrin. Histological analysis revealed alterations in the two cell types primarily involved in acid secretion, parietal and enterochromaffin-like (ECL) cells. Parietal cells were reduced in number with an accumulation of immature cells lacking H+-K+-adenosinetriphosphatase (H+-K+-ATPase). ECL cells were positioned closer to the base of the gastric glands, with markedly lower expression of histidine decarboxylase. Gastrin administration for 6 days reversed the effects of the gastrin deficiency, leading to an increase in the number of mature, H+-K+-ATPase-positive parietal cells and a partial restoration of acid secretion. The results show that gastrin is critically important for the function of the acid secretory system.

Mouse Model of Colonic Adenoma-Carcinoma Progression Based on Somatic Apc Inactivation

Mutations in the adenomatous polyposis coli (APC) gene are pivotal in colorectal tumorigenesis. Existing mouse intestinal tumor models display mainly small intestinal lesions and carcinomas are rare. We defined human CDX2 sequences conferring colon epithelium-preferential transgene expression in the adult mouse. Mice carrying a CDX2P-NLS Cre recombinase transgene and a loxP-targeted Apc allele developed mainly colorectal tumors, with carcinomas seen in 6 of 36 (17%) of mice followed for 300 days. Like human colorectal lesions, the mouse tumors showed biallelic Apc inactivation, beta-catenin dysregulation, global DNA hypomethylation, and aneuploidy. The predominantly distal colon and rectal distribution of tumors seen in mice where one Apc allele was inactivated in epithelial cells from distal ileum to rectum suggests that regional differences in the intestinal tract in the frequency and nature of secondary genetic and epigenetic events associated with adenoma outgrowth have a contributing role in determining where adenomas develop. The presence of large numbers of small intestine tumors seemed to inhibit colorectal tumor development in the mouse, and gender-specific effects on tumor multiplicity in the distal mouse colon and rectum mimic the situation in humans where males have a larger number of advanced adenomas and carcinomas in the distal colon and rectum than females. The mouse model of colon-preferential gene targeting described here should facilitate efforts to define novel factors and mechanisms contributing to human colon tumor pathogenesis, as well as work on tumor-promoting environmental factors and agents and strategies for cancer prevention and treatment.

IL-17+ Regulatory T Cells in the Microenvironments of Chronic Inflammation and Cancer

Foxp3+CD4+ regulatory T (Treg) cells inhibit immune responses and temper inflammation. IL-17+CD4+ T (Th17) cells mediate inflammation of autoimmune diseases. A small population of IL-17+Foxp3+CD4+ T cells has been observed in peripheral blood in healthy human beings. However, the biology of IL-17+Foxp3+CD4+ T cells remains poorly understood in humans. We investigated their phenotype, cytokine profile, generation, and pathological relevance in patients with ulcerative colitis. We observed that high levels of IL-17+Foxp3+CD4+ T cells were selectively accumulated in the colitic microenvironment and associated colon carcinoma. The phenotype and cytokine profile of IL-17+Foxp3+CD4+ T cells was overlapping with Th17 and Treg cells. Myeloid APCs, IL-2, and TGF-β are essential for their induction from memory CCR6+ T cells or Treg cells. IL-17+Foxp3+CD4+ T cells functionally suppressed T cell activation and stimulated inflammatory cytokine production in the colitic tissues. Our data indicate that IL-17+Foxp3+ cells may be “inflammatory” Treg cells in the pathological microenvironments. These cells may contribute to the pathogenesis of ulcerative colitis through inducing inflammatory cytokines and inhibiting local T cell immunity, and in turn may mechanistically link human chronic inflammation to tumor development. Our data therefore challenge commonly held beliefs of the anti-inflammatory role of Treg cells and suggest a more complex Treg cell biology, at least in the context of human chronic inflammation and associated carcinoma.

Phenotype of microsatellite unstable colorectal carcinomas: Well-differentiated and focally mucinous tumors and the absence of dirty necrosis correlate with microsatellite instability.

&NA; The phenotypic markers of colorectal carcinomas with microsatellite instability have been widely studied and include mucinous or poor differentiation, prominent host response, a circumscribed growth pattern, histologic heterogeneity, and right‐sided location. As part of a population‐based case–control study of colorectal cancer in northern Israel, we reviewed the pathology and microsatellite status of 528 consecutively diagnosed colorectal cancers. Phenotypic analysis was performed by one pathologist (J.K.G.) and included assessment of grade, mucinous histology (>50%, or focal), histologic heterogeneity, growth pattern, necrosis, and host response. Microsatellite status was determined on microdissected portions of formalin‐fixed, paraffin‐embedded tissue using a panel of 5 NCI consensus primers. Fifty‐two of 528 colorectal carcinomas were microsatellite unstable (9.85%). Multivariate analysis found that >2 tumor infiltrating lymphocytes per high power field (p <0.0001), the lack of dirty necrosis (p = 0.0054), a Crohns‐like host response (p = 0.0064), right‐sided location (p = 0.032), well or poor differentiation (p = 0.037), and any mucinous differentiation (p = 0.039) were independent predictors of microsatellite instability. Tumor infiltrating lymphocytes were the single best histologic predictor of microsatellite instability. The absence of dirty necrosis and the presence of well‐differentiated tumors and tumors with only focal mucinous differentiation were also important markers for microsatellite instability that have not been emphasized previously. The combination of >2 tumor infiltrating lymphocytes per high power field and/or any mucinous differentiation and/or the absence of dirty necrosis identified all MSI‐H tumors in this study.

Regenerating islet-derived 3-alpha is a biomarker of gastrointestinal graft-versus-host disease

There are no plasma biomarkers specific for GVHD of the gastrointestinal (GI) tract, the GVHD target organ most associated with nonrelapse mortality (NRM) following hematopoietic cell transplantation (HCT). Using an unbiased, large-scale, quantitative proteomic discovery approach to identify candidate biomarkers that were increased in plasma from HCT patients with GI GVHD, 74 proteins were increased at least 2-fold; 5 were of GI origin. We validated the lead candidate, REG3α, by ELISA in samples from 1014 HCT patients from 3 transplantation centers. Plasma REG3α concentrations were 3-fold higher in patients at GI GVHD onset than in all other patients and correlated most closely with lower GI GVHD. REG3α concentrations at GVHD onset predicted response to therapy at 4 weeks, 1-year NRM, and 1-year survival (P ≤ .001). In a multivariate analysis, advanced clinical stage, severe histologic damage, and high REG3α concentrations at GVHD diagnosis independently predicted 1-year NRM, which progressively increased with higher numbers of onset risk factors present: 25% for patients with 0 risk factors to 86% with 3 risk factors present (P < .001). REG3α is a plasma biomarker of GI GVHD that can be combined with clinical stage and histologic grade to improve risk stratification of patients.

Metabolic Syndrome Is Associated with Greater Histologic Severity, Higher Carbohydrate, and Lower Fat Diet in Patients with NAFLD

OBJECTIVES:Nonalcoholic fatty liver disease (NAFLD) is considered as the hepatic manifestation of metabolic syndrome. Insulin resistance (IR) is a key component of metabolic syndrome. The aim was to determine the dietary composition, physical activity, and histologic severity between NAFLD patients with and without metabolic syndrome.METHODS:Ninety-one patients with NAFLD completed the Block Food Frequency Questionnaire and the Paffenbarger Physical Activity Questionnaire. IR was assessed by the homeostasis model assessment (HOMA) index. Metabolic syndrome was defined by the ATP III clinical definition. Nonalcoholic steatohepatitis (NASH) Clinical Network Scoring System was used to determine the histologic severity of NAFLD.RESULTS:Thirty-one patients (34%) had metabolic syndrome. Patients with metabolic syndrome had a higher HOMA index (7.66 vs 4.45, p = 0.04), and consumed more carbohydrates (51% vs 45%, p = 0.03) and less fat (34% vs 40%, p = 0.01) compared with those without metabolic syndrome; total daily calorie, protein consumption, and physical activity were similar between the two groups. Patients with metabolic syndrome had higher scores for steatosis (2.0 ± 0.8 vs 1.37 ± 1, p = 0.02), NASH activity (4.13 ± 1.4 vs 3.13 ± 1.7, p = 0.004), and global NASH score (5.9 ± 1.7 vs 4.4 ± 2.3, p = 0.0006) compared with those without metabolic syndrome. When controlled for other factors including dietary composition and physical activity, the presence of metabolic syndrome was a significant risk factor for global NASH severity in addition to HOMA index and female gender.CONCLUSION:Metabolic syndrome in patients with NAFLD is associated with a diet containing more carbohydrate and less fat and greater histologic severity. The role of a carbohydrate-restricted diet in decreasing the risk for metabolic syndrome and histologic severity should be assessed in patients with NAFLD.

Cyclooxygenase-2 expression in gastric antral mucosa before and after eradication of Helicobacter pylori infection

Objective:Helicobacter pylori (H. pylori) causes chronic gastritis. The inducible prostaglandin synthetase cyclooxygenase 2 (COX-2) plays an important role in inflammatory conditions. We hypothesized that H. pylori-associated chronic gastritis would express COX-2 protein. Our aim was to evaluate the effect of eradication of H. pylori infection on COX-2 expression in the antral mucosa of patients before and after antibiotic therapy.Methods:Tissues were obtained from patients with nonulcer dyspepia undergoing H. pylori eradication. Ten patients with proven H. pylori infection and subsequent successful eradication were studied. Three biopsies of antral mucosa were evaluated before and after H. pylori eradication. The amount of acute and chronic inflammation was quantitated. Immunohistochemical staining for COX-2 was expressed as a percentage of the total number of cells and correlated with the degree of chronic inflammation.Results:Specific immunostaining for COX-2 was observed in antral mucosa of patients infected with H. pylori. Patchy cytoplasmic staining was seen in surface epithelial cells and strong cytoplasmic staining for COX-2 was seen in parietal cells. Spotty cytoplasmic staining for COX-2 was also seen in lamina propria plasma cells, as well as there being macrophages present in the germinal centers of lymphoid aggregates. COX-2 expression could be detected both before and after eradication of H. pylori. The mean percentage of cells staining for COX-2 was significantly higher in H. pylori-infected mucosa, compared with mucosa after successful H. pylori eradication (33.4%± 5.4 vs 18.9%± 3.3, p= 0.038). COX-2 immunostaining correlated best with the chronic inflammation score (r2= 0.78, p < 0.001). There was a strong correlation for those subjects who were H. pylori infected, as well as for those who had successful H. pylori eradication.Conclusions:H. pylori associated acute and chronic antral inflammation was associated with immunohistochemical detection of COX-2 protein in epithelial cells, in addition to associated mononuclear cells and parietal cells. Expression was reduced, but not eliminated, in the epithelium after successful eradication of H. pylori. Despite the reduction in COX-2 expression after H. pylori eradication, expression of COX-2 in epithelial cells remained and strongly correlated with the extent of the chronic inflammatory cell infiltrate. The clinical implications of H. pylori-associated induction of COX-2 expression for patients on selective COX-2 inhibitors, in addition to the role of COX-2 in gastric carcinogenesis, deserve further study.

CEACAM1, a novel serum biomarker for pancreatic cancer

Objectives: Serum biomarkers for early diagnosis of pancreatic adenocarcinoma are not currently available. We recently observed elevated expression of CEACAM1 in pancreatic adenocarcinomas and sought to determine whether serum CEACAM1 levels were elevated in pancreatic cancer patients. Methods: CEACAM1 messenger RNA levels were measured in pancreatic tissue samples using quantitative reverse transcription-polymerase chain reaction. CEACAM1 was localized by immunohistochemistry in adenocarcinomas and in pancreatic intraductal neoplasia lesions. CEACAM1 serum levels were assessed by a double determinant enzyme-linked immunosorbent assay and compared with serum levels of CA19-9. Results: CEACAM1 had higher expression levels in pancreatic adenocarcinomas compared with noncancerous pancreas (P < 0.0001) and was localized to neoplastic cells (95% (45/47) of adenocarcinomas and 85% (17/20) of pancreatic intraductal neoplasia 3 lesions. CEACAM1 was expressed in the sera of 91% (74/81) of pancreatic cancer patients, 24% (15/61) of normal patients, and 66% (35/53) of patients with chronic pancreatitis, with a sensitivity and specificity superior to CA19-9. The combination of CEACAM1 and CA19-9 had significantly higher diagnostic accuracy than CA19-9. Conclusions: CEACAM1 is expressed in pancreatic adenocarcinoma, and serum levels of CEACAM1 serve as a useful indicator for the presence of pancreatic cancer. Additional validation studies on the use of serum CEACAM1 as a diagnostic marker in pancreatic cancer are warranted.Abbreviations: AUC - area under the curve, ROC - receiver operator characteristics