Histochemical Detection of Collagen Fibers by Sirius Red/Fast Green Is More Sensitive than van Gieson or Sirius Red Alone in Normal and Inflamed Rat Colon

Collagen detection in histological sections and its quantitative estimation by computer-aided image analysis represent important procedures to assess tissue localization and distribution of connective fibers. Different histochemical approaches have been proposed to detect and quantify collagen deposition in paraffin slices with different degrees of satisfaction. The present study was performed to compare the qualitative and quantitative efficiency of three histochemical methods available for collagen staining in paraffin sections of colon. van Gieson, Sirius Red and Sirius Red/Fast Green stainings were carried out for collagen detection and quantitative estimation by morphometric image analysis in colonic specimens from normal rats or animals with 2,4-dinitrobenzenesulfonic acid (DNBS) induced colitis. Haematoxylin/eosin staining was carried out to assess tissue morphology and histopathological lesions. Among the three investigated methods, Sirius Red/Fast Green staining allowed to best highlight well-defined red-stained collagen fibers and to obtain the highest quantitative results by morphometric image analysis in both normal and inflamed colon. Collagen fibers, which stood out against the green-stained non-collagen components, could be clearly appreciated, even in their thinner networks, within all layers of normal or inflamed colonic wall. The present study provides evidence that, as compared with Sirius Red alone or van Gieson staining, the Sirius Red/Fast Green method is the most sensitive, in terms of both qualitative and quantitative evaluation of collagen fibers, in paraffin sections of both normal and inflamed colon.     

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.     

Chromoendoscopy with a Standard-Resolution Colonoscope for Evaluation of Rectal Aberrant Crypt Foci

Colorectal cancer (CRC) is the second most common cause of death worldwide. According to the theory by Vogelstein, colorectal carcinogenesis involves a series of successive changes in the normal colonic mucosa, starting with excessive proliferation and focal disorders of intestinal crypts, followed by adenoma and its subsequent malignant transformation. The first identifiable changes in CRC carcinogenesis are aberrant crypt foci (ACF). ACF are invisible during routine colonoscopy yet are well identifiable in chromoendoscopy using methylene blue or indigo carmine. High-resolution colonoscopes are used for assessment of ACF. The aim of the present study was to evaluate the usefulness of standard-resolution colonoscopy for identification of rectal ACF. The following parameters were evaluated: duration of chromoendoscopy of a given rectal segment, type of ACF, sensitivity and specificity of endoscopy combined with histopathological evaluation. The mean duration of colonoscopy and chromoendoscopy was 26.8 min. In the study population, typical ACF were found in 73 patients (p = 0.489), hyperplastic ACF in 49 (p = 0.328), and dysplastic ACF in 16 patients (p = 0.107). Mixed ACF were observed in 11 individuals (p = 0.073). The sensitivity of the method was found to be 0.96 whereas its specificity 0.99.Identification of rectal ACF using standard-resolution colonoscopy combined with rectal mucosa staining with 0.25% methylene blue is characterised by high sensitivity and specificity.     

Reduction of sialic acid O-acetylation in human colonic mucins in the adenoma-carcinoma sequence

The oligo-O-acetylation of sialic acids found in normal colonic mucins is greatly reduced in colorectal cancer. Mucins prepared from cancer tissue in adenocarcinoma showed this reduction, while normal O-acetylation was detected in resection margin and control cases and total mucin sialic acid content was significantly decreased in cancer vs control samples. A reduction of the O-acetyl transferase activity catalysing the O-acetylation reaction was also found. A series of cultured human colorectal cell lines derived from the same premalignant adenomatous line, and representative of the adenoma-carcinoma sequence were examined and revealed a depletion of oligo-O-acetylation in the original diploid premalignant line, re-expression in a further premalignant line and reduction in malignant mucinous and adenocarcinoma cell lines. Reduction of sialic acid O-acetylation appears as an early event in the process of malignant transformation in human colorectal cancer.     

Chicken egg yolk anti-asialoGM1 immunoglobulin (IgY): an inexpensive glycohistochemical probe for localization of T-antigen in human colorectal adenocarcinomas.

A egg yolk polyclonal IgY has been prepared by immunization of white leghorn chickens with small unilamellar liposomal asialoGM1. The newly prepared anti-asialoGM1 IgY has been characterized to be specific toward the terminal carbohydrate moiety of asialoGM1, and has no cross reactivity to its sialylated counterpart (ganglioside, GM1) as evidenced by immunochromatographic studies. General glycohistochemical methods along with antigen specific lectin and immunohistochemical staining using anti-asialoGM1 IgY were used to study the expression of Thomsen-Friedenreich (T-) disaccharide antigen in human colorectal adenocarcinoma tissues. The expression of T-antigen in colon cancer tissue was detected by two T-disaccharide specific probes, chicken anti-T-yolk antibody (IgY) and Artocarpus integrifolia lectin (AIL) and was found to be more pronounced in both the secreted mucin as well as the cytoplasmic mucin deposits. These immunochemical detection methods for T-antigen showed a weaker correlation with other glycostaining methods using, alcian-blue/periodic acid-Schiff (AB-PAS) and high iron diamine (HID). However, a general enzymatic staining for galactose and galactosamine containing glycoconjugates, by galactose oxidase-Schiff method, showed a good correlation with T-antigen detection. While the T-beta specific anti-asialoGM1 could localize T-antigen in 11 of 13 (84%) human colorectal adenocarcinoma tissue sections tested, the T-alpha specific AIL could localize the T-antigen in only 6 of the tissues (46%). These observations confirm previously reported findings, of the prevalence of T-beta conformation in colon cancer, that binds significantly more with the anti-asialoGM1 IgY than with the T-alpha specific AIL. Hence, both anti-T IgY and the AIL immunohistochemical probes may have useful diagnostic value because of the ease of preparation and cost effectiveness, but the T-beta specific anti-asialoGM1 probe (IgY) would have a better prognostic value in colon adenocarcinomas.     

The principles and advances of quantitative pathology

This article gives an overview of the quantitative pathologic techniques used today, with special emphasis on interactive morphometry and its application in the clinical setting. At present, stereologic calculations may be necessary, but in a diagnostic setting have only rarely proved to be essential. The reproducibility of the measurements is discussed in relation to the definition of the particles and staining methods. A number of technical factors that cause random errors are mentioned, such as quality of the slides, magnification, definition of the particles to be measured and measuring protocol. Large-scale experiments have revealed that the means of nuclear morphometric and certain stereologic features are reproducible, such as in the volume percentage of epithelium, the surface densities of glands and the mitotic activity index. The diagnostic applications of the standard deviation and shape factors of nuclear quantitative features require additional precautions, however. Having quantified cell and tissue features, multivariate analysis may result in a better discrimination of two or more groups under study. The quantitative pathologic examination of cells and tissues can provide important diagnostic and prognostic information. Quantitation in pathology is especially useful in so-called continuous lesions, in which interobserver and intraobserver disagreement is considerable. An important requirement of diagnostic morphometry is object selection by a skilled pathologist; the use of morphometry as a black box can result in dramatic errors. The criteria used for a morphometric classification rule that can be used for clinical applications are summarized. Quality control of the whole measuring system is essential. Application of these techniques for more than six years in diagnostic pathology has repeatedly corrected previous qualitative diagnoses.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chemopreventive Efficacy of Andrographis paniculata on Azoxymethane-Induced Aberrant Colon Crypt Foci In Vivo

Andrographis paniculata is a grass-shaped medicinal herb, traditionally used in Southeast Asia. The aim of this study was to evaluate the chemoprotective effects of A. paniculata on colorectal cancer. A. paniculata ethanol extract was tested on azoxymethane (AOM)-induced aberrant crypt foci (ACF) in vivo and in vitro. A. paniculata treated groups showed a significant reduction in the number of ACF of the treated rats. Microscopically, ACF showed remarkably elongated and stratified cells, and depletion of the submucosal glands of AOM group compared to the treated groups. Histologically, staining showed slightly elevated masses above the surrounding mucosa with oval or slit-like orifices. Immunohistochemically, expression of proliferating cell nuclear antigen (PCNA) and β-catenin protein were down-regulated in the A. paniculata treated groups compared to the AOM group. When colon tissue was homogenized, malondialdehyde (MDA) and nitric oxide (NO) levels were significantly decreased, whereas superoxide dismutase (SOD) activity was increased in the treated groups compared to the AOM group. A. paniculata ethanol extract showed antioxidant and free radical scavenging activity, as elucidated by the measure of oxidative stress markers. Further, the active fractions were assessed against cell lines of CCD841 and HT29 colon cancer cells.     

Quantitative evaluation of immunohistochemical staining in gastrointestinal stromal tumors

OBJECTIVE: To evaluate the differences among pathologists' interpretations in gastrointestinal stromal tumors (GISTs) and to demonstrate the usefulness of quantitative pathology in the assessment of immunohistochemical staining. STUDY DESIGN: Twenty GISTs were separately evaluated by 4 pathologists by the visual estimation method using a 6-antibody panel. Each case was then quantitatively measured with a computer-assisted image analysis system by 2 pathologists. Cohen's kappa test was performed for statistical analysis. RESULTS: All GISTs showed some degree of expression of CD 117, CD34, SMA and Ki-67. No case was immunoreactive for desmin or S-100 protein. There were remarkable differences in the pathologists' visual estimations. Moreover, the discrepancies between visual and quantitative methods were noteworthy. The differences in interpretations showed the greatest variability for Ki-67, which is known to be related to poor prognosis. CONCLUSION: Quantitative pathology in assessment of immunohistochemical staining of GISTs may improve the consistency in the interpretation of staining results and provide some degree of reproducibility.     

Value of morphometric nuclear image analysis using the Feulgen reaction in renal cell carcinoma

OBJECTIVE: To evaluate retrospectively the ability of morphometric nuclear image analysis to predict survival in patients with renal cell carcinoma. STUDY DESIGN: The subjects were 40 patients with previously untreated renal cell carcinoma. Pathologic stage was determined using Robson's stage system. Nuclear grade was assigned according to the criteria of Fuhrman et al. We used the Feulgen staining technique, which has been widely used for the histochemical assessment of nuclear DNA content. A minimum of 300 nuclei were analyzed from each subject. Five variables in morphometric nuclear image analysis were measured: nuclear area, nuclear perimeter, nuclear ellipticity, nuclear regularity and DNA content. Cox's proportional hazard model was applied to identify prognostic usefulness with respect to survival time. RESULTS: All nuclear morphometric variables but nuclear regularity correlated with tumor grade. According to univariate survival analyses, Robson stage and nuclear ellipticity revealed a prognosis on survival with statistical significance. After adjustments for age and sex, nuclear ellipticity remained the only significant prognostic factor related to survival (P < .01). The survival rates were relatively high for patients with nuclear ellipticity > 773 as compared to those with nuclear ellipticity < 773 (P < .05). CONCLUSION: These findings indicate that morphometric nuclear image analysis using the Feulgen reaction is a reliable and efficient technique and that nuclear ellipticity is the most discriminating morphometric variable for predicting the prognosis of renal cell carcinoma patients.     

Early lesions induced in rat colon epithelium by N-methyl-N′-nitro-N-nitrosoguanidine

The development of ACF (aberrant crypt foci), adenoma and cancer following intrarectal administration of the alkylating agent N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) has been described. However, microscopic lesions not previously reported were observed as soon as two weeks following carcinogen treatment. These lesions protrude slightly over the epithelial lining of the colon, with a micropolyp-like appearance. Oriented sections show that the centre of these lesions present pseudo-“cystic” appearance, with disorganized crypts made of normal cells. The chorion of the lesion is invaded by numerous inflammatory cells and some ACF may be present nearby. The epithelium lining the cysts and the distorted crypts shows expression of gastric mucin M1/MUC5AC, an early marker of colonic carcinogenesis which is not present in normal colon. This mucin is retained within the “cysts” together with some inflammatory cells.The micropolyps observed contain in a minute form some histological elements described in ulcerative colitis or short-term radiotherapy (distortion of crypts, crypt abscesses, increase of chorion cellularity, infiltration by immune cells). In addition, the presence of bifid crypts nearby suggests mucosal regeneration.Our hypothesis is that these modifications are steps in a normal healing pathway that may in some cases degenerate into precancerous lesions and cancer.     

Quantitation of lectin binding sites in human colon mucins by use of peanut and wheat germ agglutinins

We have developed a novel method for quantitation of lectin binding sites in mucins derived from colon tissues. Binding of peanut agglutinin and wheat germ agglutinin was measured in extracts from normal and malignant human colon epithelium. Binding of wheat germ agglutinin was used as an estimate of the total mucin present in the tissue extract. Peanut agglutinin was found to bind to mucin from normal colon, but at levels that may be difficult to appreciate by fluorescence microscopy. The yield of mucin extracted from colon cancer was more variable than that from normal colon, and the binding ratio of peanut agglutinin to wheat germ agglutinin was greater in extracts from tumors than in normal tissues. Our findings confirm the histological observation that peanut agglutinin binds more avidly to mucins from colon cancer than to those from normal colon. The finding of peanut agglutinin binding sites in mucins front normal colon was not expected. The quantitative technique may have detected small numbers of binding sites not readily appreciable by fluorescence microscopy. Alternatively, the chromatographic method for measuring lectin binding may be sufficiently sensitive to detect nonspecific binding of the lectin to terminal galactose residues other than the Thomsen-Friedenreich antigen.     

Expression of pim-1 in Tumors,Tumor Stroma and Tumor-Adjacent Mucosa Co-Determines the Prognosis of Colon Cancer Patients

Provirus integration site for Moloney murine leukemia virus (pim-1) is a proto-oncogene that is linked to the development and progression of several cancers. In this study, we evaluated pim-1 expression in tumors, tumor stroma and tumor-adjacent mucosa together as an independent prognostic factor for colon cancer patients. The study included 343 colon cancer patients. Immunohistochemical staining was used to detect pim-1. Multivariate cox regression for disease-free survival (DFS) were used to identify independent prognostic factors. Analytic hierarchy process (AHP) was used to calculate the weight of pim-1 in tumors, tumor stroma and tumor-adjacent mucosa in order to obtain a Pim-1 total score (PTS) for recurrence and survival. Kaplan–Meier DFS curves and OS curves for patients with different pim-1 expression levels were compared using the log-rank test. In this study, four independent prognostic factors were identified for colon cancer patients: pim-1 expression in tumors, tumor stroma, tumor-adjacent mucosa, as well as tumor stage. It has been established that clinical stage is an important prognostic factor for colon cancer patients. However, PTS can identify the patients who are likely to recur not only in the whole radical excision group but also within each stage of this group. Based on the results of this study we can conclude that the PTS combined with clinical staging system may be a better predictor of colon cancer patients’ prognosis than using the clinical stage system alone. Clinical Trials Gov. Number: ChiCTR-PRCH-12002842     

Discrimination between benign and malignant melanocytic skin lesions by multivariate analysis, quantitative S-100 immunohistochemistry, nuclear morphometry and DNA cytometry

OBJECTIVE: To determine whether combined quantitative immunohistochemistry of S-100, nuclear morphometry and DNA image cytometry improves discrimination between benign and malignant melanocytic skin lesions (MSLs). STUDY DESIGN: S-100 protein expression was measured in tissue sections of MSLs using an image cytometry system. Localized areas of high S-100 expression were used to identify regions in sequential, facing sections in which morphometric and cytometric features of nuclei, including DNA ploidy, were also measured. RESULTS: Malignant cases had significantly higher S-100 protein staining intensity, larger nuclei and greater DNA content (P < .05). High staining intensity for S-100 protein weakly correlated with variation in size of the mean nuclear area (P = .04) and DNA content (P = .03). Combining the features of nuclear area and DNA integrated optical density in areas of high-intensity staining for S-100 protein discriminated more accurately between 12 benign and 16 malignant areas than any of the features along (P = .0003). CONCLUSION: Combined multivariate quantitative immunohistochemical, morphometric and DNA cytometric analysis greatly improves discrimination between benign MSLs and malignant melanoma. Larger test sets are required to confirm the promising results of this initial study.     

COX-2 and iNOS, good targets for chemoprevention of colon cancer

Cyclooxygenase (COX)-2 has been suggested to play an important role in colon carcinogenesis. We found that the COX-2 selective inhibitor, nimesulide, reduces azoxymethane (AOM)-induced aberrant crypt foci (ACF) in rats and colon carcinogenesis in mice, as well as formation of intestinal polyps in Min mice. Thus, selective inhibitors of COX-2, which catalyzes the synthesis of prostanoids, could be good candidates as chemopreventive agents against colon cancer. Examination of the effect of prostanoid receptor deficiency and a selective antagonist of prostanoid receptor on the development of AOM-induced ACF in mice revealed the involvement of the EP1 receptor. Moreover, a selective EP1 antagonist reduced the number of intestinal polyps in Min mice. These results suggest that PGE2 contributes to colon carcinogenesis through binding to the EP1 receptor. Nitric oxide synthase (NOS) is known to be overexpressed in colon cancers of humans and rats, and a NOS inhibitor, L-NG-nitroarginine methyl ester, was found to inhibit the development of AOM-induced ACF in rats. Thus, NOS including iNOS could also be a good target for chemoprevention of colon cancer, as in the COX-2 case.     

Standardization of the Papanicolaou stain. I. A comparison of five nuclear stains.

The staining characteristics of five nuclear stains used in a Papanicolaou staining procedure were investigated. Alcohol-fixed cervical smears were stained with a modified Papanicolaou procedure using hematoxylin, alcoholic thionin bromide, alcoholic Victoria blue B, gallocyanin or the thionin Feulgen reagent (thionin-SO2) as the nuclear stain. The same anionic counterstain was used for all slides, and the optical densities of cell nuclei and cytoplasm were measured with the IBAS 2000 image analyzer. Alcoholic thionin gave the most intense nuclear stain, with a very high reproducibility of the staining pattern. Hematoxylin showed the highest coefficient of variation of the staining intensity. Both hematoxylin and gallocyanin gave some nonspecific cytoplasmic staining. Thionin-SO2 allowed a quantitative assessment of DNA, but gave a low staining intensity. Staining with the metal complex dyes interfered with subsequent staining with the pararosaniline Feulgen reagent. Alcoholic thioinin is thus recommended as a nuclear stain for cervical cytology in the Papanicolaou procedure, both for image analysis and for visual microscopy.     

Silver staining of extensively glycosylated proteins on sodium dodecyl sulfate-polyacrylamide gels: enhancement by carbohydrate-binding dyes.

Two methods are described for detecting less than 1 microgram of highly glycosylated proteins, such as mucins, on sodium dodecyl sulfate-polyacrylamide gels. They combine commonly employed periodic acid-Schiff (PAS) and Alcian blue dyes with silver stain. Carbohydrate prestaining renders mucins more cationic and favors greater complexation with ionic silver. Comparisons of different mucin samples stained either with PAS-silver or alcian blue-silver indicate differential staining between the two techniques. Such differences may, in part, be due to an affinity of Alcian blue for sulfated glycoproteins. These two staining protocols when used in conjunction with silver staining alone are particularly valuable for assessing sample purity and for detecting contaminating proteins during mucin purification protocols.     

The Use of Mouse Models of Breast Cancer and Quantitative Image Analysis to Evaluate Hormone Receptor Antigenicity after Microwave-assisted Formalin Fixation

Microwave methods of fixation can dramatically shorten fixation times while preserving tissue structure; however, it remains unclear if adequate tissue antigenicity is preserved. To assess and validate antigenicity, robust quantitative methods and animal disease models are needed. We used two mouse mammary models of human breast cancer to evaluate microwave-assisted and standard 24-hr formalin fixation. The mouse models expressed four antigens prognostic for breast cancer outcome: estrogen receptor, progesterone receptor, Ki67, and human epidermal growth factor receptor 2. Using pathologist evaluation and novel methods of quantitative image analysis, we measured and compared the quality of antigen preservation, percentage of positive cells, and line plots of cell intensity. Visual evaluations by pathologists established that the amounts and patterns of staining were similar in tissues fixed by the different methods. The results of the quantitative image analysis provided a fine-grained evaluation, demonstrating that tissue antigenicity is preserved in tissues fixed using microwave methods. Evaluation of the results demonstrated that a 1-hr, 150-W fixation is better than a 45-min, 150-W fixation followed by a 15-min, 650-W fixation. The results demonstrated that microwave-assisted formalin fixation can standardize fixation times to 1 hr and produce immunohistochemistry that is in every way commensurate with longer conventional fixation methods.     

Consumption evaluation of one apple flesh a day in the initial phases prior to adenoma/adenocarcinoma in an azoxymethane rat colon carcinogenesis model

Colorectal cancer (CRC) is the fourth cancer with the most new cases reported in 2018 worldwide. Consumption of fruit and vegetables is a protective factor against the risk of CRC. Beyond this, flavonoids could orchestrate these healthy effects. Apart from containing the typical apple flavonoids, red-fleshed apples also contain anthocyanins, mainly cyanidin-3-O-galactoside (Cy3Gal). Through an azoxymethane rat carcinogenesis model, a study was carried out in order to assess the possible protective effects of apple polyphenols, with special attention to anthocyanins. In addition, apart from negative and positive controls, a group with chemotherapy with 5-fluorouracil (5FU) was included to compare their performance against the output collected from the animal treatments with white-fleshed apple (WF), red-fleshed apple (RF) and Cy3Gal (AE).Although the 5FU group presented the best performance towards aberrant crypt foci (ACF) inhibition (70.1%), rats fed with white-fleshed apples ('Golden Smoothee') were able to achieve 41.3% ACF inhibition, while none of the challenged treatments (WF, RF and AE) suffered mucin depletion in their colonocytes. Expression changes of 17 genes related to CRC were assessed. In detail, the ACF inhibition phenotype detected in 5FU and WF groups could be explained through the expression changes detected in the apoptosis-related genes of Aurka, p53 and Cox2. Moreover, in the apple consumption groups (WF and RF), a reduced protein expression of matrix metalloproteinases with gelatinase activity (MMP-2 and 9) was detected.Overall, our study suggests an effect of apple polyphenols and apple anthocyanin Cy3Gal against colon carcinogenesis, retarding/diminishing the appearance of the precancerous markers studied.     

Evaluation of improved zirconyl hematoxylin compared to the classical pH 2.5 Alcian blue method for demonstrating acid mucins

Abstract

Acid mucins have diagnostic significance for many pathological conditions, especially in certain tumors. We compared the classical pH 2.5 Alcian blue method to a new, improved zirconyl hematoxylin (IZH) method for demonstrating acid mucins using two fixatives: Bouin`s solution and 10% neutral buffered formalin (NBF). We used rabbit small intestine, large intestine and trachea. Specimens were fixed in Bouin`s solution and NBF. A total of 160 paraffin sections were prepared and stained with pH 2.5 Alcian blue and IZH. The stained acid mucins were assessed using digital image analysis software. Stained mucins were quantified for each staining procedure and fixative. No important differences were observed in acid mucin staining by either method after either fixative. The IZH method provides results as good as pH 2.5 Alcian blue and can be used to obtain reliable staining for acid mucins.