DOCTORAL THESIS (SUMMARY)
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1 Translation from Romanian UNIVERSITY OF MEDICINE AND PHARMACY CRAIOVA THE FACULTY OF MEDICINE DOCTORAL THESIS (SUMMARY) Scientific coordinator Prof. Dr. Laurentiu MOGOANTA PhD student, Dr. Madalin IONILA CRAIOVA
2 UNIVERSITY OF MEDICINE AND PHARMACY CRAIOVA THE FACULTY OF MEDICINE DOCTORAL THESIS HISTOLOGICAL AND IMMUNOHISTOCHEMICAL STUDY OF COLLOID COLON CARCINOMA Scientific coordinator Prof. dr. Laurentiu MOGOANTA PhD student, Dr. Madalin IONILA CRAIOVA
3 INTRODUCTION The colon cancer is a major cause of morbidity and mortality around the world, occupying, in terms of incidence, the fourth place both for men and for women (Parkin DM, Bray F, Ferlay J, Pisani P. 2005). With the introduction of some new methods for early detection of colorectal cancer, respectively the screening tests, the incidence of this cancer during the last two decades has diminished in the United States from 66.3 cases annually per 100,000 inhabitants in 1985 to 45.5 cases per 100,000 inhabitants in 2006, which represents an average decrease in annual incidence of 3.0% per year for men and 2,2% annually for women. Unfortunately, in developing countries, the colorectal cancer presented increased levels of incidence for the last two decades (Parkin DM, Bray F, Ferlay J, Pisani P. 2005). Regarding the mortality, the colorectal cancer is the third leading cause of death by cancer for both sexes. Only in the United States, approximately 56,300 patients die annually from colorectal cancer (American Cancer Society, 2010). It is estimated that, annually, over 50% of patients with colorectal cancer may survive if their disease would be prevented or at least diagnosed earlier, during a curable stage. Regarding the mucinous carcinoma of the colon, the dates of the specialized literature indicate a frequency that varies between 5% and 30.51%. Starting from these dates from the specialized literature, we have proposed to observe the epidemiological profile of the colloid colon carcinomas, their microscopic aspects, the relationship between epithelial and stoma elements. HISTOLOGICAL STUDY OF COLLOID COLON CARCINOMA The studied material was represented by 573 surgical resection specimens from patients with ages between 25 and 89 years, hospitalized in the Clinical General Surgery I, II and III of the Clinical Hospital of Emergency, no. 1, Craiova, who presented the clinical diagnosis of colon tumor. From the Anatomo-pathological Registries of registering the casuistry, we collected the dates concerning the histopathological diagnosis of the condition and the macroscopic aspects of the tumors for the cases studied retrospectively. From the total of 573 resection specimens, we selected a number of 149 cases of mucinous carcinoma. In the immunohistochemical study, the studied material was represented by a number of 42 cases of mucinous adenocarcinomas representing the clinicopathological perspective for the 149 cases histopathologically investigated. In the morphological study we used the classical histological technique by paraffin inclusion. As coloration methods were used: hematoxylin-cosine (HE), Masson trichrome, Mucicarmine, Alcian blue - periodic acid Schiff (PAS-AA), Alcian blue - colloidal iron (High Iron Diamine) and silver impregnation. In the immunohistochemical study we haveused as a working method, the ABC / HRP technique (Avidin complexed with biotinylated peroxide). The antibodies used: Antibody Clone Antigenic exposure Dilution Positive control MUC2 Ccp58 citrate ph 6 1:800 colon mucosa MUC 5AC 1-13M1 citrate ph 6 1:800 gastric mucosa Tag 72 citrate ph 6 1:1000 gastric adenocarcinoma Caspase 3 citrate ph 6 1:100 invasive ductal
4 carcinoma p 53 DO-7 EDTA, ph 9 1:50 tonsil Ki-67 MIB 1 EDTA, ph 9 1:100 tonsil E-cadherin NCH 38 EDTA, ph 9 1:50 Gl. mammary The positive external control has been performed on normal tissues containing the antigen target investigated target (positive sections). These have been treated under the same conditions as the investigated tumor. The interpretation of immunohistochemical reactions observed the expression of the markers at primary tumor mass, metastases and at adjacent colonic mucosa level. The interpretation of immunohistochemical reactions envisaged the illustration of the chromogene at the antigenic targets levels. THE RESULTS OF THE HISTOLOGICAL STUDY OF THE COLON COLLOIDS CARCINOMA The Histopathological examination in accordance with the WHO classification (2000) of the surgical removal parts of the 573 hospitalized patients with the clinical diagnosis of colon tumor in The General Surgery Clinics in the five years of study, has shown the existence of the following morphological varieties: adenocarcinoma (469), mucinous adenocarcinoma (3), signet-cell carcinoma (24), adenosquamous carcinoma (49) and undifferentiated carcinoma (11). The analysis of the dates of the table above indicate the predominance of adenocarcinoma with 401 cases, followed by 149 cases with mucinous adenocarcinoma, signet-cell carcinoma with 11 cases, adenosquamous carcinoma with 6 cases and respectively the varieties of the pure squamous carcinoma and undifferentiated carcinoma with 3 cases each. The distribution on age groups shows a prevalence number of cases of conventional colon adenocarcinoma, especially in the fourth decade while for the cases of mucinous adenocarcinoma, the greatest frequency was in the fifth decade. The topographic distribution of cases of colon carcinoma shows a predisposition to the variant pure adenocarcinoma in the left colon and for the variant of mucinous adenocarcinoma, the casuistry was relatively uniformly distributed in the two colonic segments, existing a small prevalence cases regarding the location in the left colon. In the pure mucinous adenocarcinoma, the carcinomatous proliferation was observed in following aspects: - Acinar-like structures floating in lakes of mucus, which involve agglomeration of carcinoma cells with eosinophilic cytoplasm and pyknotic nucleus; - Unicellular or multi-layered cords with carcinoma cells delimiting mucus lakes with varying sizes and shapes; - Isolated carcinoma cells floating in lakes of mucus. In mixed adenocarcinoma, excepting the described aspects, were presented aspects of conventional adenocarcinoma with various grades of differentiation: - well differentiated in which carcinomatous proliferation presents a glandular pattern or of neoplastic tubes, the prolific structures being uniform in shape and size, recalling the morphology of the normal glands of the colonic mucosa. - Moderately differentiated in which the carcinoma proliferations have an appearance of simple neoplastic tubes with a single or complex cito-architecture of different shapes and sizes, lined with a neoplastic epithelium single-or multi-layered in which the nucleus has lost his basal polarity. - Poorly differentiated in which the carcinoma proliferations present a pattern like the cellular compact layers without forming glandular lumens and with pronounced atypies, cytological and nuclear.
5 The results of the histochemical study of mucus secreted from carcinomatous proliferations: The PAS / Alcian blue coloration has illustrated the overwhelming prevalence of cases with mucinous adenocarcinoma of mixed secretion of acidic and neutral mucins (> 61%). A qualitative assessment of these cases has shown the prevalence mostly of the acidic mucins (> 89% of mixed types). On the histopathological sub-variant of mucinous adenocarcinoma, it was found that: - In pure mucinous type predominate markedly the acidic mucins (> 90% of these cases); - In the type with a mucinous component> 50% predominate the acidic mucins (> 58%); - In the type with a mucinous component <50% exists a balance between the two types of mucins secreted; The stroma of the mucoid adenocarcinoma investigated by us was generally reduced. It was represented by thin septa of collagen fibers that separate the carcinomatous proliferations and lakes of mucus. Inflammatory elements- lymphocytes and plasma cells type- were rare and were regularly present around the acinar-like carcinoma proliferations. The distribution of the cases of mucinous adenocarcinoma studied according to the ptnm stadialisation. Most of the investigated cases are placed in the stage III, 45% of cases, followed closely by the second stage with 42% of cases. Based on the histopathologic subtype, we observed the prevalence of pure mucinous adenocarcinoma and those with a mucinous component between 50-80% from the tumor volume in the ptnm III stage, respectively 52% and 56.5% of the total cases. The distribution of lymphoganglionary metastasis: 57% of the investigated cases showed no lymphoganglionary invasion. For the remaining cases, the lymphoganglionary spread was approximately proportional, respectively in 22% of cases were detected up to 3 locoregional lymphogangliones and more of 4 such invaded lymphogangliones. II. THE RESULTS OF THE IMMUNOHISTOCHEMICAL STUDY OF COLON CARCINOMA COLLOIDS The immune response to mucin MUC2 was present in all investigated cases of mucinous adenocarcinoma. Quantitatively scores 2 and 3 predominated, the pure mucinous subtype having the highest scores quantitatively and qualitatively. Regarding the small scores, the mucinous variant had less than 50% extracellular mucin. The immune response to the mucin MUC5AC was present in only 60% of total cases of investigated mucinous adenocarcinoma. Quantitatively, the score 2 was predominant, the subtype with extracellular mucin, varying between 50-80% from the tumor volume with the largest quantity and quality scores. In other histological subtypes, we recorded a relatively even distribution of the two scores of the casuistry. The evaluation of the immunolabeling with the anti TAG72 glycoprotein. On a normal mucosa far from the neoplastic process, the immune response to 2 TAG was negative but was present but with a reduced intensity in the adjacent mucosa to the carcinomatous proloferations and with a moderate or even high level of transitional mucosal (with dysplastic lesions) in the vicinity of the tumor process. The immune reaction to the TAG72 glycoprotein was present in 81% from the total cases of the investigated mucinous adenocarcinoma. Quantitatively, the score 2 predominated, the subtype with pure mucinous having the highest scores quantitatively and qualitatively, the maximum intensity of the reaction being present at the lakes of the mucosa. The subtype with less than 50% extracellular mucin was in last place, 33% of these cases being negative at this marking. The evaluation of the immunohistochemical reaction at anti Caspase-3 antibodies. On a normal mucosa adjacent to carcinoma proliferation we have not observed the presence of a positive
6 immune response. In the tumor tissue, the pattern of the reaction was nuclear. The immune response to caspase-3 was present in only 76% of the total cases of investigated mucinous adenocarcinoma. Quantitatively, the score 2 predominated, the subtype with pure mucinous having the highest scores quantitatively and qualitatively, the subtype with less than 50% of tumor volume was in last place, half of these cases being negative at this marking. The evaluation of the immunohistochemical reaction to the anti protein p53. In tumor tissue, the pattern of the reaction was nuclear. The immune response to p53 was present in only 68% of total cases of investigated mucinous adenocarcinoma. Quantitatively, the score 1 predominated; the subtype with extracellular mucin varies between 50-80% from the tumor volume with the largest scores qualitatively and quantitatively. The pure mucinous subtype has recorded more than a third of cases with a negative immune response. The semi-quantitative and qualitative evaluation of the immunolabeling to anti Ki-67 antibody. On normal mucosa adjacent to carcinoma proliferation we observed a rare nuclear immunolabeling in the basal cells from the crypts of the glands Liberkuhn. In the tumor tissues, the immune response to Ki-67 was present in 69% of total cases of investigated mucinous adenocarcinoma. Quantitatively, the score 1 predominated, the subtype with less than 50% extracellular mucin had the highest scores. DISCUSSIONS In the colonic localisation, on the casuistry investigated by us, the mucinous adenocarcinoma was ranked second with frequency as approximately 26% after the conventional adenocarcinoma located in the first place with 70%. The dates from the specialized literature indicate for mucinous colon carcinoma a frequency between 5-15% [Nozoe si colab., 2009 ; Du si colab., 2004]. In Asian countries, the percentage of these tumors is less than 5.5% [Song si colab., 2009]. In our country the dates on the incidence of the colon mucinous adenocarcinoma are inconsistent with values ranging from 6.52% [Cipaian, 2003] to 29.29% [Gurzu, 2008] and respectively 30.51% [Crisan, 2001]. Regarding the distribution of the investigated casuistry concerning the age and gender groups, our study has shown that in case of mucinous adenocarcinoma, not taking into account the histological subtype, the most part were diagnosed in the fifth decade, these by developing with almost 10 years faster than the conventional adenocarcinoma. The dates of the literature indicate for colon colloid adenocarcinoma an increased incidence in patients under 40 years [Ordone si colab., 1982 ; Torsello si colab., 2008]. These studies show for colloid carcinoma an incidence ranging from 8-17% for patients with an age over 45 years and respectively a frequency of 30% (with limits ranging from 19-88%) between patients with an age under 45 years. Regarding the gender distribution, our study has not shown significant statistical differences between mucinous adenocarcinoma and conventional adenocarcinoma. Regarding the tumor topography we have noted a prevalence of the pure adenocarcinoma especially in the left colon while the mucinous adenocarcinomas had a relatively uniform distribution, but correlated with the histologic subtype, we have noted a predominance of pure mucinous forms and of those mixed ones with more than 50% mucinous component especially in the right colon. Regarding the degree of differentiation, we have noted a greater frequency of good and modestly differentiated forms which together accounted for approximately 85% of total investigated mucinous adenocarcinomas. Slightly differentiated forms represented 15% and belonged mainly to the pure mucinous subtype (55%). The results are congruent with those in the literature indicating the prevalence of the well-differentiated forms of 'colon mucinous adenocarcinoma [Negri si colab., 2005 ; Safaee si colab., 2010].
7 Regarding the biochemical type of mucins that prevaled in our casuistry, we noted the prevalence of cases with mixtures of acidic mucins and neutral mucins (approximately 62% of cases), the acidic mucins being those that prevailed quantitatively, as shown especially in mucinous pure forms (> 90% of cases). With respect to the variant with the extracellular mucin under 50%, we noted a positive correlation of age groups with the acidic mucin / neutral mucins content. Concerning the stadialisation, the study undertaken by us has shown that many of the colon adenocarcinomas mucinous were developed in the third stage (445%) and the second stage (42%), pur histological subtypes and the one with a mucinous component between 50-80% from the tumor volume were diagnosed primarily in the third stage (52% and 56.5%). Regarding the degree of tumor invasion, we noted the overwhelming prevalence of the studied casuistry in the invasion phase of the muscular coating of the colon (93%). Several studies undertaken in the colon mucinous carcinoma have shown that it has a tendency to be locally invasive, with a local dissemination and peritoneal seeding, the hematogenous spread is not a characteristic of this type of cancer [Kanemitsu si colab., 2003 ; As si colab., 2004 ; Longo si colab., 2006 ; Song si colab., 2009]. CONCLUSIONS - The epidemiological profile of the mucinous adenocarcinomas studied indicates the prevalence of these in persons that are in the fourth decade of life, especially among men (M / F = 1.4 / 1 to 1.6 / 1) and in the location of the right colon (especially for the purely mucinous). - Were predominated the mixed adenocarcinomas in which the extracellular mucinous component has achieved <50% tumor volume (52.50%), - From the biochemical point of view, the cases mixtures of acidic mucins and neutral mucins (62%) have dominated, acidic mucins being the best represented, as shown especially in the purely mucinous forms (> 90%). In addition, our study has shown the prevalence of sialo-mucins (68%) which predominated in the purely mucinous subtype (77%). - Mucinous colon adenocarcinoma were developed in the third stage (45%) and the second stage (42%), the mucin-rich forms being detected especially in the third stage. In over 90% of investigated cases, we noted the invasion of the muscular coating of the colon, the pure mucinous subtype being diagnosed only in this phase (93%). - The metastasis rate was 43%, histopathologically the most frequent metastases being the mixed cases with a mucinous component varying between 50-80% (56.5%), followed by pure mucinous subtype (46%). - The immunoreactivity for mucin showed us the positivity of MUC2 almost exclusively intracytoplasmatically compared with MUC5AC, also present extra cellularly. Regarding the histopathological subtype, the purely mucinous variant presented high scores especially for MUC2; for MUC5AC, the reactivity was the most reduced. Statistically only for MUC5AC, we noted a decrease in reactivity with decreasing in the degree of differentiation, not taking into account the histological type. - For the glycoprotein TAG-72, the immunolabeling was most evident at the purely mucinous variant. In addition, we noted a significant correlation of this marker with the degree of differentiation and depth of invasion, the immunohiostochimical scores being obtained in the most differentiated forms and in the course and T3-T4, not taking into consideration the histopatological form. - The highest degree of apoptosis investigated by means of caspase-3 was obtained in pure mucinous forms, slightly differentiated. The marker presented a statistically significant correlation with the degree of inverse and direct differentiation with depth of invasion, not taking into consideration the histopatological type. The immunolabeling for the p53 onco -
8 protein was still shown in the subtype with extracellular mucin varying between 50-80% from the tumor volume and was inversely correlated with the depth of tumor invasion and the degree of differentiation. - The rate of cell proliferation through the investigated marker Ki-67 has shown a decrease in the proliferative activity with a reduction in the level of differentiation and an increase in the amount of mucin.
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