Putative Preneoplastic Lesions Research Articles

Morphological, immunohistochemical and molecular features of inflammatory bowel disease associated colorectal carcinoma and associated mucosal lesions – Single institution experience

BackgroundPatients with inflammatory bowel disease (IBD) – ulcerative colitis (UC) and Crohn’s disease (CD) have an elevated risk of developing colorectal carcinoma (CRC). Major risk factor in IBD patients is the continuous chronic inflammation leading to development of dysplasia and carcinoma. Nevertheless, other types of non-conventional but suspicious mucosal changes serrated change/dysplasia, NOS and villous hypermucinous change, have also been reported in IBD patients. Preneoplastic potential of these lesions is still not well elucidated. AimsThe aim of this study was identification of IBD-associated CRCs focusing on finding related precursor lesions in the surgical specimen or in archival biopsy samples followed by a detailed morphological, immunohistochemical and molecular evaluation. For the purpose of the study the mucosal lesions were divided into conventional IBD-associated dysplasia and non-conventional lesions that were merged under a provisory term of putative preneoplastic lesions (PPL). MethodsA total of 309 consecutive IBD colectomy specimens diagnosed during a 10-year period were reviewed. Detailed morphological evaluation, immunohistochemical analysis of mismatch repair (MMR) proteins, p53 and O6-methylguanine DNA methyltransferase (MGMT) expression and molecular analysis for KRAS, NRAS and BRAF gene mutation were performed in the retrieved CRC cases as well as in the detected dysplasia and PPLs of these patients. ResultsWe identified 11 cases of morphologically heterogenous IBD-associated CRCs, occurring in 5 males and 6 females, aged 26–79 years (mean 44 years). A total of 22 mucosal lesions were revealed in 8 CRC patients comprising conventional IBD-associated dysplasia (4 lesions), PPLs as serrated change/dysplasia NOS (11 lesions), villous hypermucinous change (5 lesions), and two true serrated lesions (one sessile serrated adenoma and one traditional serrated adenoma). More than one type of lesion was found in 6 patients. Seven CRC cases harbored mutation of KRAS/NRAS and one case of BRAF. Two patients with KRAS-mutated CRC showed the same mutation in PPL in the same specimen (one serrated change NOS and one TSA with high-grade dysplasia). Similarly, one BRAF-mutated carcinoma case presented the same mutation in serrated change/dysplasia, NOS in the same specimen. Of the CRCs, two showed deficient MMR system profile, six presented with loss of MGMT expression, and six showed aberrant p53 expression. PPLs showed deficient MGMT expression (14 cases) and aberrant p53 (10 cases) as well. ConclusionIBD-associated CRCs are very heterogeneous entities. Besides conventional IBD-related dysplasia, other types of mucosal lesions may be associated with long lasting IBD and CRC e.g. villous hypermucinous change and serrated change/dysplasia, NOS. Since these lesions share certain genetic or immunohistochemical changes with the related CRC, a suspicion is raised that these lesions may also have preneoplastic potential. Awareness of these changes is necessary to prevent their missing and under-reporting, and further studies of these lesions should be carried out.

Prediction of carcinogenic potential of chemicals using repeated-dose (13-week) toxicity data

Sub-chronic toxicity studies of 163 non-genotoxic chemicals were evaluated in order to predict the tumour outcome of 24-month rat carcinogenicity studies obtained from the EFSA and ToxRef databases. Hundred eleven of the 148 chemicals that did not induce putative preneoplastic lesions in the sub-chronic study also did not induce tumours in the carcinogenicity study (True Negatives). Cellular hypertrophy appeared to be an unreliable predictor of carcinogenicity. The negative predictivity, the measure of the compounds evaluated that did not show any putative preneoplastic lesion in de sub-chronic studies and were negative in the carcinogenicity studies, was 75%, whereas the sensitivity, a measure of the sub-chronic study to predict a positive carcinogenicity outcome was only 5%. The specificity, the accuracy of the sub-chronic study to correctly identify non-carcinogens was 90%. When the chemicals which induced tumours generally considered not relevant for humans (33 out of 37 False Negatives) are classified as True Negatives, the negative predictivity amounts to 97%. Overall, the results of this retrospective study support the concept that chemicals showing no histopathological risk factors for neoplasia in a sub-chronic study in rats may be considered non-carcinogenic and do not require further testing in a carcinogenicity study.

Oral 5-fluorouracil colon-specific delivery through in vivo pellet coating for colon cancer and aberrant crypt foci treatment

In situ coating of 5-fluorouracil pellets by ethylcellulose and pectin powder mixture (8:3 weight ratio) in capsule at simulated gastrointestinal media provides colon-specific drug release in vitro. This study probes into pharmacodynamic and pharmacokinetic profiles of intra-capsular pellets coated in vivo in rats with reference to their site-specific drug release outcomes. The pellets were prepared by extrusion-spheronization technique. In vitro drug content, drug release, in vivo pharmacokinetics, local colonic drug content, tumor, aberrant crypt foci, systemic hematology and clinical chemistry profiles of coated and uncoated pellets were examined against unprocessed drug. In vivo pellet coating led to reduced drug bioavailability and enhanced drug accumulation at colon (179.13 μg 5-FU/g rat colon content vs 4.66 μg/g of conventional in vitro film-coated pellets at 15 mg/kg dose). The in vivo coated pellets reduced tumor number and size, through reforming tubular epithelium with basement membrane and restricting expression of cancer from adenoma to adenocarcinoma. Unlike uncoated pellets and unprocessed drug, the coated pellets eliminated aberrant crypt foci which represented a putative preneoplastic lesion in colon cancer. They did not inflict additional systemic toxicity. In vivo pellet coating to orally target 5-fluorouracil delivery at cancerous colon is a feasible therapeutic treatment approach.

Abstract B7: Molecular signaling in the human fallopian tube: Insights into proliferation and p53 expression

Abstract Ovarian cancer is the most lethal gynecological malignancy in women. This is primarily due to patients presenting in late stage disease when the cancer has already metastasized. Contributing to this delayed diagnosis is uncertainty regarding the origin of ovarian cancer and what causes initial transformation. It has recently been hypothesized that the distal epithelia of the fallopian tube is the origin of transformation leading to ‘ovarian’ cancer, particularly the serous histotype. A potential precursor lesion to serous cancer, known as the ‘p53 signature’, has been identified in the fallopian tube. However, little is known about the normal human fallopian tube and what factor(s) might lead this potential precursor lesion to develop into serous ovarian cancer. In order to examine the role of the fallopian tube in the development of serous cancer, as well as potential initiation factors that might be involved in early disease, a novel three-dimensional (3D) culture method was utilized. This 3D culture supports growth within the normal tissue architecture and retains epithelia in contact with its stromal environment using alginate hydrogels. This system was utilized for examination of normal human fallopian tissue collected from women undergoing salpingectomy for both benign and malignant conditions. Donated tissues were treated with factors hypothesized to have a role in the onset of serous cancer for 2 and 7 days in culture. Insulin (5 ug/ml) was used to stimulate glycolysis, hydrogen peroxide (H2O2, 1 mM) was used as an oxidative stress mimetic, and the dominant female steroid hormones estrogen (10 nM) and progesterone (100 nM) were utilized to investigate hormone controlled cell growth. Resulting cultured tissues retained normal fallopian morphology as demonstrated by immunohistochemistry for cytokeratin-8 and E-cadherin. Additionally, both tubal epithelial sub-types (secretory and ciliated) were retained in culture (denoted by PAX8 and acetylated-tubulin staining, respectively). Fimbriae cultures were further analyzed for changes in proliferation, DNA damage, and importantly, induction of p53 expression, which is thought to be linked to the onset of serous cancer. Initial findings reveal ex vivo inducible p53 expression can be acquired. These hotspots of p53 expression are secretory cell enriched similarly to those seen in vivo, but are not always expressed concomitantly with γH2AX, a marker of DNA damage. Additionally, it was noted that oxidative stress (H2O2) and insulin significantly increased the rate of proliferation as compared to vehicle control. Conversely, the effect of the steroid hormones on proliferation was less notable, with progesterone treatment consistent with possible promotion of fallopian epithelial cell death. Characterization of biological function within early putative preneoplastic lesions is integral to ovarian cancer research. Without identifying the tissue of origin, current research may be focusing on genetic or protein changes that are different only because they are compared to the wrong normal tissue of origin. Further, targeted therapies might fail due to identification of targets that are not in the correct progenitor cell population. In order to evaluate the p53 signature as a potential precursor lesion to serous cancer, we must understand what induces p53 expression in the fallopian tube and why. Citation Format: Sharon L. Eddie, Suzanne M. Quartuccio, Jessica A. Shepherd, Rajul Kothari, Joanna E. Burdette. Molecular signaling in the human fallopian tube: Insights into proliferation and p53 expression. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr B7.

Histologic changes in type A chronic atrophic gastritis indicating increased risk of neuroendocrine tumor development: the predictive role of dysplastic and severely hyperplastic enterochromaffin-like cell lesions

The role of putative preneoplastic enterochromaffin-like cell lesions, either hyperplastic or dysplastic, in the genesis of type 1 enterochromaffin-like cell neuroendocrine tumors associated with type A chronic atrophic gastritis, their actual neoplastic risk, and their precise histogenetic mechanism deserve further clarification by specific histopathologic studies coupled with patient follow-up. A total of 100 patients with severe type A chronic atrophic gastritis, enterochromaffin-like cell hyperplasia, and antral G-cell hyperplasia were endoscopically and histologically followed up for a median of 90.1 months (total of 9118 person-months). Preneoplastic enterochromaffin-like cell lesions and newly developed neuroendocrine tumors were investigated histologically and histochemically, in parallel with enterochromaffin-like cell lesions found in nontumor mucosa of another 32 well-characterized and previously reported type 1 neuroendocrine tumors. Both neuroendocrine and nonneuroendocrine mucosa changes were analyzed and statistically evaluated. During follow-up, 7 of 100 patients developed neuroendocrine tumors: 5 were in a group of 20 cases with previous enterochromaffin-like cell dysplasia and 2 were among 80 cases showing only enterochromaffin-like cell hyperplasia throughout the study (hazard ratio, 20.7; P < .001). The severity of enterochromaffin-like cell hyperplasia at first biopsy, with special reference to linear hyperplasia with 6 chains or more per linear millimeter, also increased the risk of neuroendocrine tumor development during follow-up (hazard ratio, 13.0; P < .001). Enterochromaffin-like cell microinvasive dysplastic lesions arising at the epithelial renewal zone level, in connection with immature proliferating mucous-neck cells, were found to be linked to early intramucosal neuroendocrine tumor histogenesis. Both enterochromaffin-like cell dysplasia and severe hyperplasia indicate increased risk of neuroendocrine tumor development in type A chronic atrophic gastritis with hypergastrinemia/G-cell hyperplasia.

Follicular epithelial dysplasia of the thyroid: morphological and immunohistochemical characterization of a putative preneoplastic lesion to papillary thyroid carcinoma in chronic lymphocytic thyroiditis

In chronic lymphocytic thyroiditis (CLT), the follicular epithelial cells display cytological atypia resembling papillary thyroid carcinoma (PTC), and epidemiological studies have suggested an increased risk of PTC in patients with this condition. While reactive atypia is observed diffusely in CLT-affected thyroid parenchyma, it is not unusual to find microscopic foci morphologically distinct from the surrounding parenchyma, exhibiting more pronounced cytological and architectural atypia. These small atypical lesions, which we term "follicular epithelial dysplasia" (FED), are particularly prominent in cases of severe CLT, yet lack invasive growth, papillary architecture, or intranuclear pseudoinclusions. To gain further insight into their biological significance, we constructed a tissue microarray of 70 cases of CLT, comprised of morphologically normal thyroid, thyroid with reactive atypia, FED, follicular nodular disease (nodular hyperplasia or follicular adenoma), and PTC. Immunohistochemical staining was performed for a marker panel including PTC (HBME-1, cytokeratin 19, galectin-3, and cyclin-D1) as well as TTF-1, thyroglobulin, and p63. Slides were digitally scanned and immunohistochemical staining evaluated using automated image analysis software. FED lesions were positive for TTF-1 and thyroglobulin (50/50, 100%), though some (13/50, 26%) also expressed p63. Similar to PTC, strong diffuse staining was observed for HBME-1 (43/50, 86%), cytokeratin 19 (48/50, 96%), galectin-3 (20/50, 40%) and cyclin-D1 (38/50, 76%). In contrast, normal thyroid, reactive atypia, and follicular nodular disease were negative, or at most, exhibited focal weak staining for HBME-1, cytokeratin 19, and galectin-3. The results of this study demonstrate the presence of atypical microscopic lesions in CLT with an immunohistochemical profile similar to PTC, supporting the concept of a premalignant lesion preceding PTC, arising in the context of severe chronic inflammation.

Inflammatory Pathogenesis of Prostate Cancer and Celecoxib

proliferative inflammatory atrophy (PIA), a putative preneoplastic lesion, and described the immunophenotype of the inflammatory stroma that invariably accompanied PIA as largely consisting of CD3 T cells, but also CD20 B lymphocytes and CD-68 macrophages. Luo et al 2 have reported that RANKL-expressing T lymphocytes and macrophages in the inflammatory microenvironment may activate IKK in prostate cancer cells, repressing maspin expression and thereby promoting metastatic behavior of prostate cancer cells, largelyunlinkedfromproliferativebehavior.Inthisimportantpreoperative study of the anti-inflammatory agent celecoxib in low and intermediate-gradeadenocarcinomaoftheprostatebyAntonarakiset al, 3 tissueeffectsofcelecoxibcouldnotbedetectedwithbiomarkersof oxidative damage, prostaglandin concentrations, COX-1 and 2, and Ki-67 expression. Given that peripheral blood lymphocytes and monocyteslikelytrafficcontinuallythroughtheinflammatorymicroenvironment of the preneoplastic and neoplastic prostate, one questions whether biomarkers of celecoxib effect 4 could be detected in the

Abstract PL02-05: Aberrant crypt foci as surrogate markers for chemoprevention

Abstract Aberrant crypt foci (ACF) are putative preneoplastic lesions that occur in the colons of rodent tumor models and are abundant in the colorectum of humans. Although their presence within the colonic mucosa is incontrovertible, their clinical significance remains a source of controversy. In the following presentation, we will review the history of ACF from their first identification in rodent carcinogen models to their identification and characterization in human colons. In order to better define ACF, one must consider their histopathology, and, here, the difference between rodent and human is striking. Whereas in the rodent a single crypt can acquire dysplastic features, such is not the case in human where ACF as large as 50–100 crypts across are rarely dysplastic. In over 1500 lesions we have examined from approximately 500 patients, we observe that if endoscopic criteria are strictly adhered to, ACF are almost always hyperplastic lesions, with either a “distended” or “serrated” appearance when studied en face. We and others have clearly identified a number of colon cancer risk factors that also associate with at least the density of ACF in the distal 20 cm of colorectum. These risk factors include central obesity, increased bioavailability of IGF-1, prior or synchronous colon neoplasia, family history of colonic neoplasia and cigarette smoking. In fact, a history of 20 pack-years or more of smoking is associated with an increased likelihood (adjusted OR=3.45; 95% CI 1.93–6.18) of having more than the median number of ACF (≥ 15) compared to non-smokers (Anderson et al., under review). Considering their prevalence in the colons of cancer patients, their corresponding association with a panel of key colon cancer risk factors and their cancer-related molecular aberrations, we argue that these lesions may provide an important signal of one or more risk factors, and are likely part of the ‘field effect’ within the colonic mucosa. If a clearer association can be established with high-grade adenomas, we believe ACF will ultimately be of great utility as surrogate markers for cancer prevention studies. Citation Information: Cancer Prev Res 2010;3(1 Suppl):PL02-05.

Dietary flavonoids suppress azoxymethane-induced colonic preneoplastic lesions in male C57BL/KsJ- db/ db mice

Obesity is known to be a risk factor for colon carcinogenesis. Although there are several reports on the chemopreventive abilities of dietary flavonoids in chemically induced colon carcinogenesis, those have not been addressed in an obesity-associated carcinogenesis model. In the present study, the effects of 3 flavonoids (chrysin, quercetin and nobiletin) on modulation of the occurrence of putative preneoplastic lesions, aberrant crypt foci (ACF), and β-catenin-accumulated crypts (BCACs) in the development of colon cancer were determined in male db/ db mice with obesity and diabetic phenotypes. Male db/ db mice were given 3 weekly intraperitoneal injections of azoxymethane (AOM) to induce the ACF and BCAC. Each flavonoid (100 ppm), given in the diet throughout the experimental period, significantly reduced the numbers of ACF by 68–91% and BCAC by 64–71%, as well as proliferation activity in the lesions. Clinical chemistry results revealed that the serum levels of leptin and insulin in mice treated with AOM were greater than those in the untreated group. Interestingly, the most pronounced suppression of development of preneoplastic lesions and their proliferation were observed in the quercetin-fed group, in which the serum leptin level was lowered. Furthermore, quercetin-feeding decreased leptin mRNA expression and secretion in differentiated 3T3-L1 mouse adipocytes. These results suggest that the present dietary flavonoids are able to suppress the early phase of colon carcinogenesis in obese mice, partly through inhibition of proliferation activity caused by serum growth factors. Furthermore, they indicate that certain flavonoids may be useful for prevention of colon carcinogenesis in obese humans.

Gastric carcinogenesis by N‐Methyl‐N‐Nitrosourea is enhanced in db/db diabetic mice

In 2005, a Japanese epidemiological study showed that increase in plasma glucose levels is a risk factor for gastric cancer. However, no animal model has hitherto shown any association between diabetes mellitus and neoplasia in the stomach. Diabetic (db/db) mice have obese and diabetic phenotypes, including hyperglycemia, because of disruption of the leptin receptor. In the present study, effects of hyperglycemia and/or hyperinsulinemia on the development of proliferative lesions were therefore examined in db/db mice given N-methyl-N-nitrosourea (MNU). A total of 120 mice were assigned to four groups: Group A, 40 db/db mice with MNU; Group B, 40 + /db mice with MNU; Group C, 30 misty (wild-type) mice with MNU; Group D, 10 db/db mice without MNU. MNU was given at 60 ppm in drinking water for 20 weeks. Subgroups of animals were sacrificed at weeks 21 and 30 and blood samples were collected to measure glucose, insulin, leptin, and adiponectin concentrations. The removed stomachs were fixed in formalin, and embedded in paraffin for histological examination and immunohistochemistry. At week 30 in Groups A, B, C and D, hyperplasia was observed in 100, 79, 57, and 0%, and dysplasia in 91, 43, 71, and 0%, respectively. Adenocarcinomas and pepsinogen-altered pyloric glands (PAPG), putative preneoplastic lesions, were observed only in Group A, at an incidence of 45%. The serum levels of insulin and leptin were also elevated in Group A. Gastric carcinogenesis by MNU was enhanced in db/db mice, possibly in association with hyperinsulinemia and hyperleptinemia.

Possible contribution of rubiadin, a metabolite of madder color, to renal carcinogenesis in rats

Madder color (MC) has been shown to exert carcinogenic potential in the rat kidney in association with degeneration, karyomegaly, increased cell proliferation of renal tubule cells and increased renal 8-OHdG levels. To clarify the causal relationship of components and metabolites of MC to renal carcinogenesis, male F344 rats were fed lucidin-3- O-primeveroside (LuP) or alizarin (Alz), and the genotoxic LuP metabolites lucidin (Luc) or rubiadin (Rub) for up to 26 weeks. After one week and four weeks, Luc did not induce any renal changes. In contrast, after one week, cortical tubule degeneration was apparent in the Alz and LuP groups, and cytoplasmic swelling with basophilic change and karyomegaly in the outer medulla was observed only in the Rub group. LuP and Rub increased the proliferative activity of tubule cells in the outer medulla, and Alz and LuP increased renal 8-OHdG levels. After 26 weeks, Rub but not Alz induced atypical tubules, a putative preneoplastic lesion, and karyomegaly in the outer medulla. These results indicate that Rub may be a potent carcinogenic metabolite of MC, targeting proximal tubule cells in the outer medulla, although oxidative stress increased by Alz or LuP might also be involved in renal carcinogenesis by MC.

Matrix metalloproteinase‐7 controls pancreatic acinar cell transdifferentiation by activating the notch signaling pathway

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer death in the United States, demonstrating the lowest five‐year survival rate of any cancer. An important observation made in cases of PDAC is the consistent presence of putative preneoplastic lesions, known as metaplastic ducts, which arise from a process known as acinar‐to‐ductal metaplasia. Molecular dissection of this process in vitro has shown that primary acinar cells, in response to EGF receptor ligands, can transdifferentiate into duct‐like epithelia in a Notch pathway dependent manner. Here we show that in vitro acinar transdifferentiation is dependent on Matrix Metalloproteinase‐7 (MMP‐7), a proteinase expressed in most metaplastic epithelia in vivo. MMP‐7 was found to be required for Notch activation, leading to dedifferentiation of acinar cells to a nestin‐positive transitional cell. We also showed that ADAM‐10, but not ADAM‐17, may be necessary for MMP‐7's effect on Notch activity. MMP‐7 activity was also found to be sufficient to induce the process, indicating that molecular signals capable of initiating MMP‐7 expression also have the potential to induce formation of metaplastic epithelia in the pancreas.

A Molecular Signature of Gastric Metaplasia Arising in Response to Acute Parietal Cell Loss

Loss of gastric parietal cells is a critical precursor to gastric metaplasia and neoplasia. However, the origin of metaplasia remains obscure. Acute parietal cell loss in gastrin-deficient mice treated with DMP-777 leads to the rapid emergence of spasmolytic polypeptide/trefoil factor family 2 (TFF2)-expressing metaplasia (SPEM) from the bases of fundic glands. We now sought to characterize more definitively the pathway for emergence of SPEM. Emerging SPEM lineages in gastrin-deficient mice treated with DMP-777 were examined for immunolocalization of TFF2, intrinsic factor, and Mist1, and morphologically with electron microscopy. Emerging SPEM was isolated with laser-capture microdissection and RNA was analyzed using gene microarrays. Immunohistochemistry in mouse and human samples was used to confirm up-regulated transcripts. DMP-777-induced SPEM was immunoreactive for TFF2 and the differentiated chief cell markers, Mist1 and intrinsic factor, suggesting that SPEM derived from transdifferentiation of chief cells. Microarray analysis of microdissected SPEM lineages induced by DMP-777 showed up-regulation of transcripts associated with G1/S cell-cycle transition including minichromosome maintenance deficient proteins, as well as a number of secreted factors, including human epididymis 4 (HE4). HE4, which was absent in the normal stomach, was expressed in SPEM of human and mouse and in intestinal metaplasia and gastric cancer in human beings. Although traditionally metaplasia was thought to originate from normal mucosal progenitor cells, these studies indicate that SPEM evolves through either transdifferentiation of chief cells or activation of a basal cryptic progenitor. In addition, induction of metaplasia elicits the expression of secreted factors, such as HE4, relevant to gastric preneoplasia.

The Expression of Transforming Growth Factor-α in Cirrhosis, Dysplastic Nodules, and Hepatocellular Carcinoma

The emergence of hepatocellular carcinoma (HCC) is thought to be a stepwise process, with high-grade dysplastic nodules (HGDN) representing premalignant lesions arising in a background of cirrhosis. Earlier studies have revealed altered expression of transforming growth factor-alpha (TGF-alpha) (a mitogen capable of inducing hepatocarcinogenesis in mice) in HCC and its surrounding parenchyma. DNA topoisomerase II-alpha (Topo II-alpha) is a nuclear protein targeted by several chemotherapeutic agents and is overexpressed in HCC. The expression of both TGF-alpha and Topo II-alpha in putative preneoplastic hepatocytic lesions, however, has not been extensively studied. We examined the patterns of TGF-alpha and Topo II-alpha expression in noncirrhotic liver, liver cirrhosis, low-grade dysplastic nodules (LGDN), HGDN, and HCC to define the possible relationships of these markers to tumor progression. Paraffin sections from formalin-fixed material were immunostained with antibodies against TGF-alpha, Topo II-alpha, and Ki-67. Forty-six HCC, 17 HGDN, and 12 low-grade dysplastic nodules were identified in 52 cirrhotic livers from explanted or resected specimens. Nuclear staining for Ki-67 and Topo II-alpha was significantly increased in the progression from cirrhosis, through HGDN, to HCC, whereas the scores for TGF-alpha in these lesions showed an inverse relationship. In comparison with 18 HCC arising in noncirrhotic livers, the expression of TGF-alpha is significantly stronger in cirrhotic liver than in noncirrhotic parenchyma and its expression is also stronger in HCC arising in cirrhosis than in HCC arising in noncirrhotic parenchyma. The increased expression of Topo II-alpha and Ki-67 from HGDN to HCC, when compared with cirrhosis, suggests that HGDN is a precursor lesion in hepatocarcinogenesis. The inverse relationship between these proliferative markers and TGF-alpha expression in these lesions and stronger expression of TGF-alpha in HCC arising in cirrhosis suggest that TGF-alpha may play an important role in the early events of liver carcinogenesis.

Novel mucilage fraction of Sinapis alba L. (mustard) reduces azoxymethane-induced colonic aberrant crypt foci formation in F344 and Zucker obese rats

Seeds of Sinapis alba Linn. (commonly called yellow or white mustard) and their components have been reported to possess anticancer properties. In this study, we evaluated the efficacy of a novel mucilaginous fraction of mustard seeds in inhibiting colonic preneoplastic changes in animal models of sporadic and obesity-associated colon cancer. In two separate studies, male Sprague-Dawley or female Zucker obese rats, injected with azoxymethane (15 or 10 mg/kg body wt. once a week for 2 weeks, respectively), were fed AIN-93G diets with or without 5% mustard mucilage (MM) (w/w) for 8 weeks. Our aim was to measure the ability to modulate the number of aberrant crypt foci (ACF), putative preneoplastic lesions of the colon. The data were classified into total numbers of ACF and large ACF (crypt multiplicity of 4 or more). We report here that 5% MM significantly (p<0.05) decreased the number of total (approximately 21% inhibition) and large (approximately 50% inhibition) ACF in the colons of Sprague-Dawley rats compared to that in untreated controls. In addition, 5% MM supplemented diet significantly lowered (p<0.05) the number of total (approximately 63% inhibition) and large (approximately 60% inhibition) colonic ACF in Zucker obese rats compared to untreated obese rats, and had no effect on fasting plasma cholesterol or triglyceride levels. These results demonstrate the possible role of MM as a functional food against sporadic and obesity-associated colon cancer, and provide impetus to conduct research to understand the underlying mechanism(s) of action.

Tumor Prevalence in Mummichogs from the Delaware Estuary Watershed.

Mummichogs Fundulus heteroclitus were collected in 2002-2004 from six locations within the Delaware Estuary watershed and examined histopathologically. The objective was to compare the prevalence of skin and liver tumors in fish from locations with a range of sediment polynuclear aromatic hydrocarbons (PAHs) based on available data. Fish were collected from (1) three locations within the industrialized Christina River subwatershed (Hershey Run, Newport Marsh (upriver and downriver)); (2) one location in the Motiva Enterprises refinery discharge canal to the Delaware River; (3) one location in the St. Jones River; and (4) one location in Blackbird Creek. Adult mummichogs (≥70 mm; n = 21-30 fish per location) were seined and held for necropsy. No neoplastic skin lesions were diagnosed in any fish. No liver tumors were diagnosed in the St. Jones, Motiva, Newport Marsh upriver, or Newport Marsh downriver collections. One of 30 Blackbird Creek mummichogs had foci of hepatocellular alteration (a putative preneoplastic lesion) and one had hepatocellular carcinoma. There was a significant difference in hepatocellular carcinoma prevalence in Hershey Run mummichogs in 2002 (9 of 21 fish, or 43%) and in 2003 (3 of 29 fish, or 10%) compared with all other locations pooled (1 of 145 fish, or 0.7%). Hershey Run is contaminated with creosote from an adjacent Superfund site, where sediments were frequently reported to have total PAH concentrations greater than 100 mg of total PAH/kg and a maximum of 13,300 mg/kg. No other locations had reports of total PAHs higher than 11 mg/kg. In conclusion, liver neoplasia was associated with exposure to sediment containing high concentrations of PAHs derived from creosote. Studies that include tumor prevalence, biomarkers (e.g., DNA adducts and PAH metabolites in bile), and sediment and/or tissue chemistry are recommended to develop a weight of evidence for specific chemical classes.

Carcinogenicity of 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin in experimental models

The contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a prototype compound of a whole class of halogenated aromatic hydrocarbons termed 'dioxinlike' contaminants present in food, human tissue, mothers milk, and environmental samples. Among the various adverse effects caused by TCDD in animal experiments, its carcinogenic effects caused particular concern. In rodents, long-term TCDD treatment leads to the development of tumors of the liver, thyroid, lung, skin, oral cavity and other sites. The occurrence of liver tumors mainly observed in female rats has been used as a basis for quantitative cancer risk assessment for TCDD. TCDD does not behave like a 'complete carcinogen', i. e. no DNA binding of the parent compound or metabolites thereof could be detected. However, enhanced oxidative damage of hepatic DNA was observed, probably resulting from a dramatic induction of cytochrome P450 enzymes, which are under the regulatory, transcriptional control of the TCDD-activated aryl hydrocarbon receptor. The marked enhancement of TCDD-related oxidative liver DNA damage in rats by estrogens warrants further mechanistic investigation. Furthermore, TCDD acts as a tumor promoter, i. e. it facilitates the growth of putative preneoplastic hepatic lesions after initiation with a complete carcinogen. The mechanisms underlying this effect may be related to altered intracellular signaling involving pronounced changes in the phosphorylation pattern of proteins regulating growth and apoptosis. These effects are thought to result in an enhanced survival of preneoplastic cells, some of which can undergo further steps on the way to malignancy. In summary, a better understanding of the mechanisms of the carcinogenicity of TCDD is mandatory to provide a rational basis for a better inter-species extrapolation. The final aim of these efforts is a more reliable risk assessment for the carcinogenic potency of the class of dioxinlike contaminants in humans.

Gastric-and-intestinal mixed-type intestinal metaplasia: aberrant expression of transcription factors and stem cell intestinalization

Helicobacter pylori plays a causative role in the development of chronic atrophic gastritis, intestinal metaplasia (IM), and stomach cancer. Although IM has long attracted attention as a putative preneoplastic lesion for stomach cancers, its clinicopathologic significance has yet to be clarified in detail. Using gastric and intestinal epithelial cell markers, IM was here divided into two major types: a gastric-and-intestinal (GI) mixed type and a solely intestinal (I) type. In the former, gastric and intestinal phenotypic markers appeared not only at the glandular but also at the cellular level. Furthermore, neuroendocrine cells also showed intestinalization along with their exocrine counterparts. In animal models, GI-type IM was found to appear first, followed by the solely I type. Summarizing these data, it was suggested that IM might be caused by the gradual intestinalization of stem cells from the GI to the I type. The molecular mechanisms of IM include the ectopic expression of CDX1, CDX2, OCT-1, and members of the Erk pathway. Suppression of the expression of gastric transcription factors such as SOX2, genes that are involved in the Sonic hedgehog pathway, and RUNX3, a tumor suppressor gene, could be additional relevant alterations. The expression of PDX1 may also be associated with pseudopyloric gland metaplasia and IM. Detailed analysis of gene regulation may shed light on the molecular bases of gastric lesions, leading to strategies for chemoprevention.

Aged Garlic Extract Inhibits Development of Putative Preneoplastic Lesions in Rat Hepatocarcinogenesis

A unique garlic preparation, aged garlic extract (AGE), was examined for its modifying effect on diethylnitrosamine (DEN)-induced neoplasia of the liver in male F344 rats, using the medium-term bioassay system based on the 2-step model of hepatocarcinogenesis. Carcinogenic potential was scored by comparing the numbers and areas of induced glutathione S-transferase placental form (GST-P)-positive hepatocellular foci. GST-P-positive foci were significantly decreased in rats treated with AGE at doses of 2, 5, and 10 mL/kg, i.g., 5 times per week during the promotion phase. In addition, to clarify the mechanism underlying the inhibitory effect of AGE, the effect of AGE on hepatocellular proliferation was evaluated using partially hepatectomized rats as a liver-regeneration model. The bromodeoxyuridine-labeling indices in the livers of the AGE group were significantly lower than those in the control group at 24 h, the maximum proliferation period after partial hepatectomy. These findings indicate that AGE inhibited the development of putative preneoplastic lesions in rat hepatocarcinogenesis, involving a slowing in the proliferation rate of liver cells after partial hepatectomy.

Potent Protection against Aflatoxin-Induced Tumorigenesis through Induction of Nrf2-Regulated Pathways by the Triterpenoid 1-[2-Cyano-3-,12-Dioxooleana-1,9(11)-Dien-28-Oyl]Imidazole

Synthetic triterpenoid analogues of oleanolic acid are potent inducers of the phase 2 response as well as inhibitors of inflammation. We show that the triterpenoid, 1-[2-cyano-3-,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO-Im), is a highly potent chemopreventive agent that inhibits aflatoxin-induced tumorigenesis in rat liver. The chemopreventive potency of CDDO-Im was evaluated by measuring inhibition of formation of putative preneoplastic lesions (glutathione S-transferase P positive foci) in the liver of rats exposed to aflatoxin B1. CDDO-Im produces an 85% reduction in the hepatic focal burden of preneoplastic lesions at 1 micromol/kg body weight and a >99% reduction at 100 micromol/kg body weight. CDDO-Im treatment reduces levels of aflatoxin-DNA adducts by approximately 40% to 90% over the range of 1 to 100 micromol/kg body weight. Additionally, changes in mRNA levels of genes involved in aflatoxin metabolism were measured in rat liver following a single dose of CDDO-Im. GSTA2, GSTA5, AFAR, and EPHX1 transcripts are elevated 6 hours following a 1 micromol/kg body weight dose of CDDO-Im. Microarray analysis using wild-type and Nrf2 knockout mice confirms that many phase 2 and antioxidant genes are induced in an Nrf2-dependent manner in mouse liver following treatment with CDDO-Im. Thus, low-micromole doses of CDDO-Im induce cytoprotective genes, inhibit DNA adduct formation, and dramatically block hepatic tumorigenesis. As a point of reference, oltipraz, an established modulator of aflatoxin metabolism in humans, is 100-fold weaker than CDDO-Im in this rat antitumorigenesis model. The unparalleled potency of CDDO-Im in vivo highlights the chemopreventive promise of targeting Nrf2 pathways with triterpenoids.