Aberrant crypt foci in colorectal carcinogenesis. Cell and crypt dynamics

Aberrant crypt foci (ACF) have been identified on the colonic mucosal surface of rodents treated with colon carcinogens and of humans after methylene-blue staining and observation under a light microscope. Several lines of evidence strongly suggest that ACF with certain morphological, histological, cell kinetics, and genetic features are precursor lesions of colon cancer both in rodents and in humans. Thus, ACF represent the earliest step in colorectal carcinogenesis. This paper has the main purpose of reviewing the evidence supporting this view, with particular emphasis on cell and crypt dynamics in ACF. ACF have been used as intermediate biomarkers of cancer development in animal studies aimed at the identification of colon carcinogens and chemopreventive agents. Recently, evidence has also shown that ACF can be effectively employed in chemopreventive studies also in humans

Analysis of single nucleotide polymorphism in the promoter and protein expression of the chemokine Eotaxin-1 in colorectal cancer patients

<p>Abstract</p> <p>Background</p> <p>Previous studies suggest that chemokines (chemotactic cytokines) promote and regulate neoplastic progression including metastasis and angiogenesis. The chemokine eotaxin-1 is a powerful eosinophil attractant but also exerts chemotaxis of other leukocytes. Eotaxin-1 has been implicated in gastrointestinal disorders and may play an important role in colorectal mucosal immunity.</p> <p>Patients and methods</p> <p>The objective of this study was to assess the role of eotaxin-1 in colorectal cancer (CRC). Levels of eotaxin-1 protein in CRC tissues (n = 86) and paired normal mucosa were compared after determination by ELISA. Plasma eotaxin-1 levels from CRC patients (n = 67) were also compared with controls (n = 103) using the same method. Moreover, a TaqMan system was used to evaluate the -384A>G eotaxin-1 gene variant in CRC patients (n = 241) and in a control group (n = 253).</p> <p>Results</p> <p>Eotaxin-1 protein levels in colorectal tumours were significantly (P < 0.0001) higher than in normal tissue. Immunohistochemistry revealed eotaxin-1 expression in stromal cells such as fibroblasts and leukocytes of the CRC tissue. The plasma eotaxin-1 level in CRC patients was lower compared with controls (P < 0.0001). Patients with tumours classified as Dukes' stage B and C had lower levels than patients with tumours in Dukes' stage A. We found no difference in genotype distribution but noted a difference regarding allele distribution (P = 0.036) and a dominance of allele G in rectal cancer patients.</p> <p>Conclusion</p> <p>The up-regulated eotaxin-1 protein expression in cancer tissue may reflect an eotaxin-1 mediated angiogenesis and/or a recruitment of leukocytes with potential antitumourigenic role. We noticed a dominance of the G allele in rectal cancer patients compared with colon cancer patients that was independent of eotaxin-1 expression.</p

Metallothionein crypt-restricted immunopositivity indices (MTCRII) correlate with aberrant crypt foci (ACF) in mouse colon

Metallothionein (MT) crypt-restricted immunopositivity indices (MTCRII) are colonic crypt stem cell mutation markers that may be induced early and in abundance after mutagen treatment. Metallothionein is the endogenous reporter gene for MTCRII, but is not typically implicated in the classical pathway of colorectal tumorigenesis. Hence, the oncological relevance of MTCRII is unclear. This study tests the hypothesis that MTCRII induced by N-methyl-N-nitrosourea (MNU) and lambda carrageenan (λCgN) associate with aberrant crypt foci (ACF) in mouse colon. Undegraded λCgN and MNU were tested alone and in combination against MTCRII and ACF in Balb/c mice, at 20 weeks after the start of treatment. MTCRII were unaffected by λCgN alone. Combined λCgN/MNU treatments induced greater MTCRII (P<0.01) as well as greater number (P<0.001) and crypt multiplicity (P<0.01) of ACF than MNU alone. MTCRII were approximately 10-fold more numerous than ACF, although linear correlations were observed between these parameters (r=0.732; P<0.01). MTCRII are induced by λCgN/MNU interactions in sufficient numbers to provide statistical power from relatively small sample sizes and correlate with ACF formation. MTCRII could thus provide the basis for a novel medium-term murine bioassay relevant to early-stage colorectal tumorigenesis

Mast cells and eosinophils in invasive breast carcinoma

<p>Abstract</p> <p>Background</p> <p>Inflammatory cells in the tumour stroma has gained increasing interest recently. Thus, we aimed to study the frequency and prognostic impact of stromal mast cells and tumour infiltrating eosinophils in invasive breast carcinomas.</p> <p>Methods</p> <p>Tissue microarrays containing 234 cases of invasive breast cancer were prepared and analysed for the presence of stromal mast cells and eosinophils. Tumour infiltrating eosinophils were counted on hematoxylin-eosin slides. Immunostaining for tryptase was done and the total number of mast cells were counted and correlated to the proliferation marker Ki 67, positivity for estrogen and progesterone receptors, clinical parameters and clinical outcome.</p> <p>Results</p> <p>Stromal mast cells were found to correlate to low grade tumours and estrogen receptor positivity. There was a total lack of eosinophils in breast cancer tumours.</p> <p>Conclusion</p> <p>A high number of mast cells in the tumours correlated to low-grade tumours and estrogen receptor positivity. Eosinophils are not tumour infiltrating in breast cancers.</p

Increased variability in ApcMin/+ intestinal tissue can be measured with microultrasound

Altered tissue structure is a feature of many disease states and is usually measured by microscopic methods, limiting analysis to small areas. Means to rapidly and quantitatively measure the structure and organisation of large tissue areas would represent a major advance not just for research but also in the clinic. Here, changes in tissue organisation that result from heterozygosity in Apc, a precancerous situation, are comprehensively measured using microultrasound and three-dimensional high-resolution microscopy. Despite its normal appearance in conventionally examined cross-sections, both approaches revealed a significant increase in the variability of tissue organisation in Apc heterozygous tissue. These changes preceded the formation of aberrant crypt foci or adenoma. Measuring these premalignant changes using microultrasound provides a potential means to detect microscopically abnormal regions in large tissue samples, independent of visual examination or biopsies. Not only does this provide a powerful tool for studying tissue structure in experimental settings, the ability to detect and monitor tissue changes by microultrasound could be developed into a powerful adjunct to screening endoscopy in the clinic

Local and distant recurrences in rectal cancer patients are predicted by the nonspecific immune response; specific immune response has only a systemic effect - a histopathological and immunohistochemical study

BACKGROUND: Invasion and metastasis is a complex process governed by the interaction of genetically altered tumor cells and the immunological and inflammatory host reponse. Specific T-cells directed against tumor cells and the nonspecific inflammatory reaction due to tissue damage, cooperate against invasive tumor cells in order to prevent recurrences. Data concerning involvement of individual cell types are readily available but little is known about the coordinate interactions between both forms of immune response. PATIENTS AND METHODS: The presence of inflammatory infiltrate and eosinophils was determined in 1530 patients with rectal adenocarcinoma from a multicenter trial. We selected 160 patients to analyze this inflammatory infiltrate in more detail using immunohistochemistry. The association with the development of local and distant relapses was determined using univariate and multivariate log rank testing. RESULTS: Patients with an extensive inflammatory infiltrate around the tumor had lower recurrence rates (3.4% versus 6.9%, p = 0.03), showing the importance of host response against tumor cells. In particular, peritumoral mast cells prevent local and distant recurrence (44% versus 15%, p = 0.007 and 86% versus 21%, p < 0.0001, respectively), with improved survival as a consequence. The presence of intratumoral T-cells had independent prognostic value for the occurrence of distant metastases (32% versus 76%, p < 0.0001). CONCLUSIONS: We showed that next to properties of tumor cells, the amount and type of inflammation is also relevant in the control of rectal cancer. Knowledge of the factors involved may lead to new approaches in the management of rectal cancer

Colorectal carcinomas in MUTYH-associated polyposis display histopathological similarities to microsatellite unstable carcinomas

<p>Abstract</p> <p>Background</p> <p>MUTYH-associated polyposis (MAP) is a recessively inherited disorder which predisposes biallelic carriers for a high risk of polyposis and colorectal carcinoma (CRC). Since about one third of the biallelic MAP patients in population based CRC series has no adenomas, this study aimed to identify specific clinicopathological characteristics of MAP CRCs and compare these with reported data on sporadic and Lynch CRCs.</p> <p>Methods</p> <p>From 44 MAP patients who developed ≥ 1 CRCs, 42 of 58 tumours were analyzed histologically and 35 immunohistochemically for p53 and beta-catenin. Cell densities of CD3, CD8, CD57, and granzyme B positive lymphocytes were determined. <it>KRAS2</it>, the mutation cluster region (MCR) of <it>APC, p53</it>, and <it>SMAD4 </it>were analyzed for somatic mutations.</p> <p>Results</p> <p>MAP CRCs frequently localized to the proximal colon (69%, 40/58), were mucinous in 21% (9/42), and had a conspicuous Crohn's like infiltrate reaction in 33% (13/40); all of these parameters occurred at a higher rate than reported for sporadic CRCs. Tumour infiltrating lymphocytes (TILs) were also highly prevalent in MAP CRCs. Somatic <it>APC </it>MCR mutations occurred in 14% (5/36) while 64% (23/36) had <it>KRAS2 </it>mutations (22/23 c.34G>T). G>T tranversions were found in <it>p53 </it>and <it>SMAD4</it>, although the relative frequency compared to other mutations was low.</p> <p>Conclusion</p> <p>MAP CRCs show some similarities to micro-satellite unstable cancers, with a preferential proximal location, a high rate of mucinous histotype and increased presence of TILs. These features should direct the practicing pathologist towards a MAP aetiology of CRC as an alternative for a mismatch repair deficient cause. High frequent G>T transversions in <it>APC </it>and <it>KRAS2 </it>(mutated in early tumour development) but not in <it>P53 </it>and <it>SMAD4 </it>(implicated in tumour progression) might indicate a predominant MUTYH effect in <it>early </it>carcinogenesis.</p

Colorectal Hyperplasia and Dysplasia Due to Human Carcinoembryonic Antigen (CEA) Family Member Expression in Transgenic Mice

CEA and CEACAM6 are immunoglobulin family intercellular adhesion molecules that are up-regulated without structural mutations in approximately 70% of human cancers. Results in in vitro systems showing tumorigenic effects for these molecules suggest that this correlation could indicate an instrumental role in tumorigenesis. To test whether this applies in vivo, transgenic mice harboring 187 kb of the human genome containing four CEA family member genes including the CEA and CEACAM6 genes were created and their copy numbers increased by mating until colonocyte expression levels reached levels seen in human colorectal carcinomas. The colonocyte surface level of integrin α5 and the activation of AKT increased progressively with the expression levels of CEA/CEACAM6. Colonic crypts showed a progressive increase in colonocyte proliferation, an increase in crypt fission, and a strong inhibition of both differentiation and anoikis/apoptosis. All transgenic mice showed massively enlarged colons comprising a continuous mosaic of severe hyperplasia, dysplasia and serrated adenomatous morphology. These results suggest that up-regulated non-mutated adhesion molecules could have a significant instrumental role in human cancer

Detection and Characterization of Oncogene Mutations in Preneoplastic and Early Neoplastic Lesions

While it has been nearly 30 years since its discovery, the ras family of genes has not yet lost its impact on basic and clinical oncology. These genes remain central to the field of molecular oncology as tools for investigating carcinogenesis and oncogenic signaling, as powerful biomarkers for the identification of those who have or are at high risk of developing cancer, and as oncogene targets for the design and development of new chemotherapeutic drugs. Mutational activation of the K-RAS proto-oncogene is an early event in the development and progression of the colorectal, pancreatic, and lung cancers that are the major causes of cancer death in the world. The presence of point mutational "hot spots" at sites necessary for the activation of this proto-oncogene has led to the development of a number of highly sensitive PCR-based methods that are feasible for the early detection of K-RAS oncogene mutations in the clinical setting. In light of these facts, mutation at the K-RAS oncogene has the potential to serve as a useful biomarker in the early diagnosis and risk assessment of cancers with oncogenic ras signaling. This chapter describes a highly sensitive method for detecting mutant K-RAS, enriched PCR, and its application to early detection of alterations in this oncogene in preneoplastic and early neoplastic lesions of the colon and rectum

Identification of Y-Box Binding Protein 1 As a Core Regulator of MEK/ERK Pathway-Dependent Gene Signatures in Colorectal Cancer Cells

Transcriptional signatures are an indispensible source of correlative information on disease-related molecular alterations on a genome-wide level. Numerous candidate genes involved in disease and in factors of predictive, as well as of prognostic, value have been deduced from such molecular portraits, e.g. in cancer. However, mechanistic insights into the regulatory principles governing global transcriptional changes are lagging behind extensive compilations of deregulated genes. To identify regulators of transcriptome alterations, we used an integrated approach combining transcriptional profiling of colorectal cancer cell lines treated with inhibitors targeting the receptor tyrosine kinase (RTK)/RAS/mitogen-activated protein kinase pathway, computational prediction of regulatory elements in promoters of co-regulated genes, chromatin-based and functional cellular assays. We identified commonly co-regulated, proliferation-associated target genes that respond to the MAPK pathway. We recognized E2F and NFY transcription factor binding sites as prevalent motifs in those pathway-responsive genes and confirmed the predicted regulatory role of Y-box binding protein 1 (YBX1) by reporter gene, gel shift, and chromatin immunoprecipitation assays. We also validated the MAPK-dependent gene signature in colorectal cancers and provided evidence for the association of YBX1 with poor prognosis in colorectal cancer patients. This suggests that MEK/ERK-dependent, YBX1-regulated target genes are involved in executing malignant properties