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Relationship between MUC5AC and altered expression of MLH1 protein in mucinous and non-mucinous colorectal carcinomas
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Pathology – Research and Practice 200 (2004) 371–377
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ORIGINAL ARTICLE
Relationship between MUC5AC and altered expression of MLH1 protein in mucinous and non-mucinous colorectal carcinomas Lorena Losia,*, Alessandra Scarsellib, Piero Benattib, Maurizio Ponz de Leonb, Luca Roncuccib, Monica Pedronib, Francesca Borghib, Igor Lambertib, Giuseppina Rossib, Massimiliano Marinob, Giovanni Pontib, Giovanni Zangardib, Mirco Menigattib, Carmela Di Gregorioc a
Department of Pathology, University of Modena and Reggio Emilia, Italy Department of Internal Medicine, University of Modena and Reggio Emilia, Italy c Service of Pathology, Hospital of Carpi, Italy b
Received 3 November 2003; accepted 7 January 2004
Abstract The main purpose of this study was to examine the expression of mucins and mismatch repair proteins in colorectal carcinomas. The immunohistochemical distribution of apomucins MUC2, MUC5AC, and the expression of MLH1 and MSH2 proteins were examined in 76 mucinous and 60 non-mucinous colorectal carcinomas. MUC2 was noted in all mucinous carcinomas, whereas MUC5AC was present in 41 cases only (54%). In non-mucinous carcinomas, MUC2 was expressed in 61.7% of the tumors; by contrast, MUC5AC was present in 20% of the cases. The expression level of apomucins was significantly different in mucinous and non-mucinous lesions (po0:001). Twenty-seven (35.5%) of the mucinous carcinomas showed no MLH1 expression, whereas 11 (18.3%) of the non-mucinous tumors did. This difference was statistically significant (po0:005). Altered expression of MSH2 protein was never observed. The lack of MLH1 expression was considerably more frequent in carcinomas with secretion of MUC5AC (po0:005). Our study has demonstrated this close relationship by immunohistochemical methods. In summary, our data show: (1) differences in the expression of mucins between mucinous and non-mucinous tumors; (2) a high frequency of altered MLH1 protein expression (35.5%) in mucinous carcinomas; (3) a significant relationship between the presence of MUC5AC and the altered expression of MLH1 protein in colorectal carcinomas. r 2004 Elsevier GmbH. All rights reserved. Keywords: Mucinous carcinoma; MUC2; MUC5AC; MLH1 mismatch repair protein; MSH2 mismatch repair protein
Introduction Mucins are synthesized and secreted from specialized cells of the intestinal mucosa [1]. Chemically, mucins *Corresponding author. Dipartimento di Anatomia Patologica e di Medicina Legale, Sezione di Anatomia Patologica, Universit"a di Modena e Reggio Emilia, Via del Pozzo 71, 41100 Modena, Italy. Tel.: 059-4222092-4224819; fax: 059-4224820. E-mail address: [email protected] (L. Losi). 0344-0338/$ - see front matter r 2004 Elsevier GmbH. All rights reserved. doi:10.1016/j.prp.2004.01.008
represent a family of high-molecular weight glycoproteins that are widely expressed by epithelial tissues. They have been identified both in the cell surface and in fully released forms [8,9], and their encoding genes are all clustered at chromosome 11p15.5 [25]. A subfamily of released mucins (‘‘gel-forming’’ mucins) includes MUC2, MUC5AC, MUC5B, and MUC6, which constitute the major macromolecular components of mucus [12]. MUC2 is normally expressed in colorectal mucosa, and can be overexpressed in mucinous carcinomas
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in stage II, 26 in stage III, and 12 in stage IV; the corresponding figures for non-mucinous lesions were 3, 29, 20, and 8, respectively.
[7,13]. By contrast, MUC5AC, 5B and 6 are strongly expressed in the upper gastrointestinal tract [14,28], and MUC5AC shows de novo expression in colorectal adenomas [2,3,20]. Mucinous carcinomas are characterized by an extracellular production of mucins, which, according to the WHO definition, affect up to 50% of the evaluated sample or more [17]. Mucinous carcinomas are rather frequent in hereditary non-polyposis colorectal cancer (HNPCC) [22], as well as in sporadic colorectal neoplasms showing microsatellite instability (MSI) [11,15,23,24]. It has been demonstrated immunohistochemically that MSI colorectal carcinomas frequently show loss of mismatch repair protein expression [5,6]. Both the altered expression of mucins and the impairment of the DNA mismatch repair system are significant events in colorectal cancer development [3,16,18]; therefore, in this study, we evaluated the relationship between the expression of two mucins, MUC2 and MUC5AC, and the immunohistochemical evaluation of the two main proteins (MLH1 and MSH2) involved in DNA repair mechanisms.
Monoclonal antibodies The following monoclonal mouse antibodies were used: anti human MLH1 (Clone 168-15) and anti human MSH2 (Clone G129-1129) (Pharmingen, San Diego, California) for the study of mismatch repair protein expression. MUC2 Ab-2 (clone M53), which recognizes a single protein of 520 KDa, identified as mucin 2 glycoprotein, and MUC5AC, gastric mucin Ab-1 (clone 45M1), which recognizes the core peptide of gastric mucin M1, recently identified as Mucin 5AC (NeoMarkers, Fremont, CA), for studying mucin expression.
Immunohistochemistry Formalin-fixed, paraffin-embedded samples of colorectal mucinous carcinomas were cut (3–4 mm), and the slides were deparaffined and rehydrated using graded ethanol. Immunoperoxidase staining using Diaminobenzidine as chromogen was carried out with the Nexes Automatic Staining System (Ventana, Strasburg, France). For the detection of MLH1 and MSH2 protein expression, the sections were immersed in 10 mM sodium citrate buffer, pH 6.0, and subjected to heatinduced antigen retrieval (350 W for 30 min). MLH1 and MSH2 monoclonal antibodies were used at 1:40 dilution. Definite nuclear staining of adjacent nonneoplastic epithelial and lymphocytes served as an internal positive control. For the analysis of MUC2, the sections were incubated in 10 mM citrate buffer, pH 6.0, and boiled at 98 C for 20 min; prediluted monoclonal antibody was applied at room temperature for 30 min. Pretreatment was not necessary for the analysis of MUC5AC, and the prediluted monoclonal antibody was used at room temperature for 1 hour. Histologically, normal mucosa from margins of the specimens was used as a positive control for MUC2, and gastric biopsies for MUC5AC. As a negative control, the sections were incubated with PBS alone, with omission of the primary antibody. The presence of immunoreactive cancer cells stained was graded as follows: O (negative); 1+(o25% of stained cells); 2+(26–50%); 3+(51–75%); 4+(>75%) [13].
Materials and methods Patients A total of 136 tumor specimens were analyzed. Of these, 76 were of the mucinous histological type, i.e., they had a mucin share of over 50% in a standard section, and 60 were non-mucinous. The two series were taken from the files of a colorectal cancer registry established in 1984 [26]. All patients underwent surgery for malignancies of the large bowel, and tissue samples were analyzed and classified at the Department of Pathology (University of Modena and Reggio Emilia), following the guidelines of the International Classification of Diseases for Oncology [17]. Mucinous tumors were selected consecutively during a 5-year registration period; non-mucinous tumors were matched to mucinous lesions, so that their distribution in the three main colorectal subsites (right colon, left colon, rectum) was similar (Table 1). Cases showing clinical features suggestive of HNPCC were excluded from the analysis; thus, the study focused on sporadic neoplasms. Tumors were staged according to the TNM classification [10]. Regarding mucinous tumors, four were in stage I, 34
Table 1. Main clinical features of mucinous and non-mucinous carcinomas Age (years)
Mucinous (76) Non-mucinous (60)
Sex
Location
Mean
Range
M
F
Right
Left
Rectum
69.0 68.4
(39–89) (47–85)
39 31
37 29
48 38
17 15
11 7
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The statistical significance of differences in protein expression between mucinous and non-mucinous lesions was evaluated using the w2 test. The analysis was carried out with the Statistical Package for Social Sciences software.
Results Immunohistochemical localization of MUC2 and MUC5AC MUC2 was focally expressed in the perinuclear cytoplasm of normal goblet cells. In neoplastic tissues, MUC2 was present in the cytoplasm of cells assumed to be of goblet lineage, while the extracellular mucin remained unstained (Fig. 1). In normal cells, focal expression of MUC5AC was seen in the cytoplasm and mucous droplets of goblet cells in a minor proportion of crypts. In neoplastic cells, staining of MUC5AC was similar to that of MUC2, although it was more often restricted to cells with an evident goblet-cell theca. The extracellular mucin was stained focally (Figs. 2 and 3A).
Mucin expression, clinicopathological features and mismatch repair protein expression MUC2 was expressed in all mucinous carcinomas (100%). The percentage of positive malignant cells in
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the tumors was variable, but usually elevated: 4+ in 40 cases (53%), 3+ in 21 cases (28%), 2+ in 10 cases (13%); five carcinomas showed 1+ of positive cancer cells (6%). MUC5AC was expressed in 41 of the 76 carcinomas (54%); only few cancer cells were positive in the majority of cases: 1+ in 21 cases (51%), 2+ in 8 cases (20%), 3+ in 9 cases (22%), and 4+ in 3 cases only (7%). The presence of the mucinous phenotype (MUC2+ and MUC5AC+ expression) was not significantly related to age at diagnosis, sex, tumor localization, and stage. In the non-mucinous carcinomas, MUC2 was focally expressed in 37 of 60 carcinomas (61.7%), and the percentage of positive neoplastic cells was usually low (1+ in 16 cases, 2+ in 21 cases). MUC5AC was expressed in 12 samples (20%); of these, 10 showed associated MUC2 positivity (16.7%). Usually, there was a low number of positive neoplastic cells. The expression of mucins was not significantly related to age at diagnosis or to other clinical variables. Differences in mucin expression in the mucinous and non-mucinous carcinomas are summarized in Table 2. MLH1 protein expression (Fig. 3B) was absent in 38 of 136 carcinomas (27.9%), whereas altered expression of MSH2 was never observed. There was no correlation between mismatch repair protein expression and age at diagnosis, sex, tumor localization, and stage. Twentyseven (35.5%) of the 76 mucinous and 11 (18.3%) of the
Fig. 1. Immunohistochemistry of MUC2 expression in the cytoplasm of neoplastic cells of mucinous carcinoma (20 ).
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Fig. 2. Immunohistochemistry of MUC5AC expression in the cytoplasm of neoplastic cells of mucinous carcinoma (20 ).
Fig. 3. (A) MUC5AC expression in colorectal mucinous carcinoma (20 ). (B) Magnification (40 ) of the inset of (A) showing lack of MLH1 expression in neoplastic cells of mucinous carcinoma. The nuclei of lymphocytes and stromal cells represented a positive internal control (arrows).
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Table 2. Mucin and protein expression in mucinous and non-mucinous colorectal carcinomas Mucin expression
Mucinous carcinomas
Non-mucinous carcinomas
p
MUC2+ MUC5AC+ MUC2+/MUC5AC+
76/76 (100%) 41/76 (54%) 41/76 (54%)
37/60 (61.7%) 12/60 (20%) 10/60 (16.7%)
o0.001 o0.001 o0.001
Mismatch repair proteins MLH1 MSH2
27/76 (35.5%) 0/76 (0%)
11/60 (18.3%) 0/60 (0%)
o0.005
Table 3. Correlation between MUC5AC and MLH1 expression in colorectal mucinous carcinomas MUC5AC MLH1
0
1+
2+
3+
4+
Expressed Not expressed Total
26 (74%) 9 (26%) 35 (100%)
13 (62%) 8 (38%) 21 (100%)
5 (62%) 3 (37%) 8 (100%)
4 (44%) 5 (56%) 9 (100%)
1 (33%) 2 (67%) 3 (100%)
non-mucinous carcinomas showed no MLH1 protein expression (po0:005). Table 3 gives a comparison between the expression of MUC5AC and that of MLH1 protein. In the 27 mucinous carcinomas without MLH1 protein expression, 18 (67%) showed the combined mucinous phenotype (MUC2+/MUC5AC+), while the remaining 9 expressed only MUC2 mucin. Moreover, a relationship was found between the number of cells positive for mucin MUC5AC and the absence of MLH1 protein expression. In particular, the lack of MLH1 protein expression increased from 38% in the cases with 1+ and 2+ to 67% for the cases with 4+ mucin expression. Of the 11 non-mucinous carcinomas without MLH1 expression, 2 cases were negative for mucin expression, 3 showed only MUC2 expression, and 6 showed the combined mucinous phenotype (MUC2+/MUC5AC+) (54.5%). There was a significant correlation between the absence of MLH1 expression and the presence of MUC5AC (po0:005); this relationship was also noted when all cases (mucinous and non-mucinous) were considered together.
Discussion Mucinous carcinomas frequently occur in patients with HNPCC, and are over-represented in sporadic carcinomas showing MSI [11,15,22,24]. It has been reported [23] that colorectal mucinous carcinomas show MSI more frequently than non-mucinous carcinomas, suggesting that these tumors represent a subset sepa-
rated from other histological types. Moreover, numerous studies have shown a good correlation between MSI and altered immunohistochemical expression of the mismatch repair proteins (MLH1 and MSH2) [5,6]. Mucinous carcinomas are characterized by frequent and strong MUC2 protein expression [4,13,21]. De novo expression of MUC5AC in adenomas of the colon and rectum has also been reported [2,20]. These studies suggest that as the degree of villous architecture and dysplasia increases, the level of MUC5AC expression decreases. De novo expression of MUC5AC may occur in colorectal carcinomas, either in the mucinous or in the non-mucinous type, but with stronger expression in the mucinous histological type [4,19,27]. Moreover, increased expression of secretory mucins MUC2 and MUC5AC was observed in sporadic cancers with MSI [4]. As reported in other experiments [4,13,14,21], our study shows an overexpression of MUC2 in all colorectal mucinous carcinomas. In non-mucinous colorectal cancers, MUC2 expression was significantly lower, thus suggesting that the development of mucinous lesions is associated with an enhancement of MUC2 expression. The overexpression of mucin proteins may be due to an increased mRNA level [14]. By contrast, other studies showed normal mRNA levels in cancer tissues [27]. Finally, recent observations suggest that the reduction of MUC2 may play a role in intestinal carcinogenesis [29]. MUC5AC expression was noted in 53 out of 136 tumors analyzed (39%). This ‘‘gastric’’ mucin was aberrantly expressed in mucinous carcinoma (54% vs. 20%); this is in accordance with other reports that demonstrated a high frequency of MUC5AC expression in the mucinous cancers analyzed [4,19].
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In our study, the absence of expression of mismatch repair proteins in mucinous tumors was significantly higher (35.5%) than in the non-mucinous lesions (18.3%), although we did not observe any association with the localization of tumors in the large bowel, as reported in a previous investigation [24]. No previous studies have been conducted to correlate the expression of mucins (MUC2 and MUC5AC) and mismatch repair proteins using immunohistochemical methods. In our study, the lack of MLH1 expression was significantly more frequent in carcinomas that showed the combined expression of MUC2 and MUC5AC (po0:005). An increased expression of the secretory mucins MUC2 and MUC5AC in sporadic MSI-positive colorectal cancers has been reported by others [4]. In addition, our observations suggest the existence of a trend towards an increased number of MUC5ACpositive cancer cells and the absence of MLH1 protein expression. The mechanisms that account for the up-regulation of the mucinous phenotype in MSI carcinomas or in neoplasms lacking mismatch repair protein are unknown. There is no evidence that a reduced half-life of mRNA, or an alteration in the efficiency of translation, may vary in colorectal cancers according to the microsatellite status. The difference in the detection of core proteins related to the actual mucin expression changes can be associated with secretory lineage differentiation [4]. In conclusion, our studies give a further contribution to elucidating the complex relationship between mucin expression and DNA mismatch repair proteins, two factors that are closely associated with the development of a subgroup of colorectal malignancies, i.e., those showing the mucinous histological type. Thus, we confirm the increased expression of MUC5AC in mucinous colorectal neoplasms compared with nonmucinous lesions. Moreover, the lack of MLH1 protein expression was rather common in mucinous tumors, whereas the expression of the other major DNA mismatch repair protein (MSH2) is normal. Finally, the correlation between the extent of MUC5AC expression and the lack of MLH1 protein at the immunohistochemical level might suggest a functional association between the two proteins. Further studies should clarify whether these observations are of relevance to colorectal tumorigenesis.
Acknowledgements We thank Paola Manni, Emanuela Duzzi and Luca Fabbiani for their excellent technical assistance. This work was supported by grants ex 40%: Progetti di Ricerca avanzata.
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ORIGINAL ARTICLE
Relationship between MUC5AC and altered expression of MLH1 protein in mucinous and non-mucinous colorectal carcinomas Lorena Losia,*, Alessandra Scarsellib, Piero Benattib, Maurizio Ponz de Leonb, Luca Roncuccib, Monica Pedronib, Francesca Borghib, Igor Lambertib, Giuseppina Rossib, Massimiliano Marinob, Giovanni Pontib, Giovanni Zangardib, Mirco Menigattib, Carmela Di Gregorioc a
Department of Pathology, University of Modena and Reggio Emilia, Italy Department of Internal Medicine, University of Modena and Reggio Emilia, Italy c Service of Pathology, Hospital of Carpi, Italy b
Received 3 November 2003; accepted 7 January 2004
Abstract The main purpose of this study was to examine the expression of mucins and mismatch repair proteins in colorectal carcinomas. The immunohistochemical distribution of apomucins MUC2, MUC5AC, and the expression of MLH1 and MSH2 proteins were examined in 76 mucinous and 60 non-mucinous colorectal carcinomas. MUC2 was noted in all mucinous carcinomas, whereas MUC5AC was present in 41 cases only (54%). In non-mucinous carcinomas, MUC2 was expressed in 61.7% of the tumors; by contrast, MUC5AC was present in 20% of the cases. The expression level of apomucins was significantly different in mucinous and non-mucinous lesions (po0:001). Twenty-seven (35.5%) of the mucinous carcinomas showed no MLH1 expression, whereas 11 (18.3%) of the non-mucinous tumors did. This difference was statistically significant (po0:005). Altered expression of MSH2 protein was never observed. The lack of MLH1 expression was considerably more frequent in carcinomas with secretion of MUC5AC (po0:005). Our study has demonstrated this close relationship by immunohistochemical methods. In summary, our data show: (1) differences in the expression of mucins between mucinous and non-mucinous tumors; (2) a high frequency of altered MLH1 protein expression (35.5%) in mucinous carcinomas; (3) a significant relationship between the presence of MUC5AC and the altered expression of MLH1 protein in colorectal carcinomas. r 2004 Elsevier GmbH. All rights reserved. Keywords: Mucinous carcinoma; MUC2; MUC5AC; MLH1 mismatch repair protein; MSH2 mismatch repair protein
Introduction Mucins are synthesized and secreted from specialized cells of the intestinal mucosa [1]. Chemically, mucins *Corresponding author. Dipartimento di Anatomia Patologica e di Medicina Legale, Sezione di Anatomia Patologica, Universit"a di Modena e Reggio Emilia, Via del Pozzo 71, 41100 Modena, Italy. Tel.: 059-4222092-4224819; fax: 059-4224820. E-mail address: [email protected] (L. Losi). 0344-0338/$ - see front matter r 2004 Elsevier GmbH. All rights reserved. doi:10.1016/j.prp.2004.01.008
represent a family of high-molecular weight glycoproteins that are widely expressed by epithelial tissues. They have been identified both in the cell surface and in fully released forms [8,9], and their encoding genes are all clustered at chromosome 11p15.5 [25]. A subfamily of released mucins (‘‘gel-forming’’ mucins) includes MUC2, MUC5AC, MUC5B, and MUC6, which constitute the major macromolecular components of mucus [12]. MUC2 is normally expressed in colorectal mucosa, and can be overexpressed in mucinous carcinomas
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in stage II, 26 in stage III, and 12 in stage IV; the corresponding figures for non-mucinous lesions were 3, 29, 20, and 8, respectively.
[7,13]. By contrast, MUC5AC, 5B and 6 are strongly expressed in the upper gastrointestinal tract [14,28], and MUC5AC shows de novo expression in colorectal adenomas [2,3,20]. Mucinous carcinomas are characterized by an extracellular production of mucins, which, according to the WHO definition, affect up to 50% of the evaluated sample or more [17]. Mucinous carcinomas are rather frequent in hereditary non-polyposis colorectal cancer (HNPCC) [22], as well as in sporadic colorectal neoplasms showing microsatellite instability (MSI) [11,15,23,24]. It has been demonstrated immunohistochemically that MSI colorectal carcinomas frequently show loss of mismatch repair protein expression [5,6]. Both the altered expression of mucins and the impairment of the DNA mismatch repair system are significant events in colorectal cancer development [3,16,18]; therefore, in this study, we evaluated the relationship between the expression of two mucins, MUC2 and MUC5AC, and the immunohistochemical evaluation of the two main proteins (MLH1 and MSH2) involved in DNA repair mechanisms.
Monoclonal antibodies The following monoclonal mouse antibodies were used: anti human MLH1 (Clone 168-15) and anti human MSH2 (Clone G129-1129) (Pharmingen, San Diego, California) for the study of mismatch repair protein expression. MUC2 Ab-2 (clone M53), which recognizes a single protein of 520 KDa, identified as mucin 2 glycoprotein, and MUC5AC, gastric mucin Ab-1 (clone 45M1), which recognizes the core peptide of gastric mucin M1, recently identified as Mucin 5AC (NeoMarkers, Fremont, CA), for studying mucin expression.
Immunohistochemistry Formalin-fixed, paraffin-embedded samples of colorectal mucinous carcinomas were cut (3–4 mm), and the slides were deparaffined and rehydrated using graded ethanol. Immunoperoxidase staining using Diaminobenzidine as chromogen was carried out with the Nexes Automatic Staining System (Ventana, Strasburg, France). For the detection of MLH1 and MSH2 protein expression, the sections were immersed in 10 mM sodium citrate buffer, pH 6.0, and subjected to heatinduced antigen retrieval (350 W for 30 min). MLH1 and MSH2 monoclonal antibodies were used at 1:40 dilution. Definite nuclear staining of adjacent nonneoplastic epithelial and lymphocytes served as an internal positive control. For the analysis of MUC2, the sections were incubated in 10 mM citrate buffer, pH 6.0, and boiled at 98 C for 20 min; prediluted monoclonal antibody was applied at room temperature for 30 min. Pretreatment was not necessary for the analysis of MUC5AC, and the prediluted monoclonal antibody was used at room temperature for 1 hour. Histologically, normal mucosa from margins of the specimens was used as a positive control for MUC2, and gastric biopsies for MUC5AC. As a negative control, the sections were incubated with PBS alone, with omission of the primary antibody. The presence of immunoreactive cancer cells stained was graded as follows: O (negative); 1+(o25% of stained cells); 2+(26–50%); 3+(51–75%); 4+(>75%) [13].
Materials and methods Patients A total of 136 tumor specimens were analyzed. Of these, 76 were of the mucinous histological type, i.e., they had a mucin share of over 50% in a standard section, and 60 were non-mucinous. The two series were taken from the files of a colorectal cancer registry established in 1984 [26]. All patients underwent surgery for malignancies of the large bowel, and tissue samples were analyzed and classified at the Department of Pathology (University of Modena and Reggio Emilia), following the guidelines of the International Classification of Diseases for Oncology [17]. Mucinous tumors were selected consecutively during a 5-year registration period; non-mucinous tumors were matched to mucinous lesions, so that their distribution in the three main colorectal subsites (right colon, left colon, rectum) was similar (Table 1). Cases showing clinical features suggestive of HNPCC were excluded from the analysis; thus, the study focused on sporadic neoplasms. Tumors were staged according to the TNM classification [10]. Regarding mucinous tumors, four were in stage I, 34
Table 1. Main clinical features of mucinous and non-mucinous carcinomas Age (years)
Mucinous (76) Non-mucinous (60)
Sex
Location
Mean
Range
M
F
Right
Left
Rectum
69.0 68.4
(39–89) (47–85)
39 31
37 29
48 38
17 15
11 7
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The statistical significance of differences in protein expression between mucinous and non-mucinous lesions was evaluated using the w2 test. The analysis was carried out with the Statistical Package for Social Sciences software.
Results Immunohistochemical localization of MUC2 and MUC5AC MUC2 was focally expressed in the perinuclear cytoplasm of normal goblet cells. In neoplastic tissues, MUC2 was present in the cytoplasm of cells assumed to be of goblet lineage, while the extracellular mucin remained unstained (Fig. 1). In normal cells, focal expression of MUC5AC was seen in the cytoplasm and mucous droplets of goblet cells in a minor proportion of crypts. In neoplastic cells, staining of MUC5AC was similar to that of MUC2, although it was more often restricted to cells with an evident goblet-cell theca. The extracellular mucin was stained focally (Figs. 2 and 3A).
Mucin expression, clinicopathological features and mismatch repair protein expression MUC2 was expressed in all mucinous carcinomas (100%). The percentage of positive malignant cells in
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the tumors was variable, but usually elevated: 4+ in 40 cases (53%), 3+ in 21 cases (28%), 2+ in 10 cases (13%); five carcinomas showed 1+ of positive cancer cells (6%). MUC5AC was expressed in 41 of the 76 carcinomas (54%); only few cancer cells were positive in the majority of cases: 1+ in 21 cases (51%), 2+ in 8 cases (20%), 3+ in 9 cases (22%), and 4+ in 3 cases only (7%). The presence of the mucinous phenotype (MUC2+ and MUC5AC+ expression) was not significantly related to age at diagnosis, sex, tumor localization, and stage. In the non-mucinous carcinomas, MUC2 was focally expressed in 37 of 60 carcinomas (61.7%), and the percentage of positive neoplastic cells was usually low (1+ in 16 cases, 2+ in 21 cases). MUC5AC was expressed in 12 samples (20%); of these, 10 showed associated MUC2 positivity (16.7%). Usually, there was a low number of positive neoplastic cells. The expression of mucins was not significantly related to age at diagnosis or to other clinical variables. Differences in mucin expression in the mucinous and non-mucinous carcinomas are summarized in Table 2. MLH1 protein expression (Fig. 3B) was absent in 38 of 136 carcinomas (27.9%), whereas altered expression of MSH2 was never observed. There was no correlation between mismatch repair protein expression and age at diagnosis, sex, tumor localization, and stage. Twentyseven (35.5%) of the 76 mucinous and 11 (18.3%) of the
Fig. 1. Immunohistochemistry of MUC2 expression in the cytoplasm of neoplastic cells of mucinous carcinoma (20 ).
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Fig. 2. Immunohistochemistry of MUC5AC expression in the cytoplasm of neoplastic cells of mucinous carcinoma (20 ).
Fig. 3. (A) MUC5AC expression in colorectal mucinous carcinoma (20 ). (B) Magnification (40 ) of the inset of (A) showing lack of MLH1 expression in neoplastic cells of mucinous carcinoma. The nuclei of lymphocytes and stromal cells represented a positive internal control (arrows).
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Table 2. Mucin and protein expression in mucinous and non-mucinous colorectal carcinomas Mucin expression
Mucinous carcinomas
Non-mucinous carcinomas
p
MUC2+ MUC5AC+ MUC2+/MUC5AC+
76/76 (100%) 41/76 (54%) 41/76 (54%)
37/60 (61.7%) 12/60 (20%) 10/60 (16.7%)
o0.001 o0.001 o0.001
Mismatch repair proteins MLH1 MSH2
27/76 (35.5%) 0/76 (0%)
11/60 (18.3%) 0/60 (0%)
o0.005
Table 3. Correlation between MUC5AC and MLH1 expression in colorectal mucinous carcinomas MUC5AC MLH1
0
1+
2+
3+
4+
Expressed Not expressed Total
26 (74%) 9 (26%) 35 (100%)
13 (62%) 8 (38%) 21 (100%)
5 (62%) 3 (37%) 8 (100%)
4 (44%) 5 (56%) 9 (100%)
1 (33%) 2 (67%) 3 (100%)
non-mucinous carcinomas showed no MLH1 protein expression (po0:005). Table 3 gives a comparison between the expression of MUC5AC and that of MLH1 protein. In the 27 mucinous carcinomas without MLH1 protein expression, 18 (67%) showed the combined mucinous phenotype (MUC2+/MUC5AC+), while the remaining 9 expressed only MUC2 mucin. Moreover, a relationship was found between the number of cells positive for mucin MUC5AC and the absence of MLH1 protein expression. In particular, the lack of MLH1 protein expression increased from 38% in the cases with 1+ and 2+ to 67% for the cases with 4+ mucin expression. Of the 11 non-mucinous carcinomas without MLH1 expression, 2 cases were negative for mucin expression, 3 showed only MUC2 expression, and 6 showed the combined mucinous phenotype (MUC2+/MUC5AC+) (54.5%). There was a significant correlation between the absence of MLH1 expression and the presence of MUC5AC (po0:005); this relationship was also noted when all cases (mucinous and non-mucinous) were considered together.
Discussion Mucinous carcinomas frequently occur in patients with HNPCC, and are over-represented in sporadic carcinomas showing MSI [11,15,22,24]. It has been reported [23] that colorectal mucinous carcinomas show MSI more frequently than non-mucinous carcinomas, suggesting that these tumors represent a subset sepa-
rated from other histological types. Moreover, numerous studies have shown a good correlation between MSI and altered immunohistochemical expression of the mismatch repair proteins (MLH1 and MSH2) [5,6]. Mucinous carcinomas are characterized by frequent and strong MUC2 protein expression [4,13,21]. De novo expression of MUC5AC in adenomas of the colon and rectum has also been reported [2,20]. These studies suggest that as the degree of villous architecture and dysplasia increases, the level of MUC5AC expression decreases. De novo expression of MUC5AC may occur in colorectal carcinomas, either in the mucinous or in the non-mucinous type, but with stronger expression in the mucinous histological type [4,19,27]. Moreover, increased expression of secretory mucins MUC2 and MUC5AC was observed in sporadic cancers with MSI [4]. As reported in other experiments [4,13,14,21], our study shows an overexpression of MUC2 in all colorectal mucinous carcinomas. In non-mucinous colorectal cancers, MUC2 expression was significantly lower, thus suggesting that the development of mucinous lesions is associated with an enhancement of MUC2 expression. The overexpression of mucin proteins may be due to an increased mRNA level [14]. By contrast, other studies showed normal mRNA levels in cancer tissues [27]. Finally, recent observations suggest that the reduction of MUC2 may play a role in intestinal carcinogenesis [29]. MUC5AC expression was noted in 53 out of 136 tumors analyzed (39%). This ‘‘gastric’’ mucin was aberrantly expressed in mucinous carcinoma (54% vs. 20%); this is in accordance with other reports that demonstrated a high frequency of MUC5AC expression in the mucinous cancers analyzed [4,19].
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In our study, the absence of expression of mismatch repair proteins in mucinous tumors was significantly higher (35.5%) than in the non-mucinous lesions (18.3%), although we did not observe any association with the localization of tumors in the large bowel, as reported in a previous investigation [24]. No previous studies have been conducted to correlate the expression of mucins (MUC2 and MUC5AC) and mismatch repair proteins using immunohistochemical methods. In our study, the lack of MLH1 expression was significantly more frequent in carcinomas that showed the combined expression of MUC2 and MUC5AC (po0:005). An increased expression of the secretory mucins MUC2 and MUC5AC in sporadic MSI-positive colorectal cancers has been reported by others [4]. In addition, our observations suggest the existence of a trend towards an increased number of MUC5ACpositive cancer cells and the absence of MLH1 protein expression. The mechanisms that account for the up-regulation of the mucinous phenotype in MSI carcinomas or in neoplasms lacking mismatch repair protein are unknown. There is no evidence that a reduced half-life of mRNA, or an alteration in the efficiency of translation, may vary in colorectal cancers according to the microsatellite status. The difference in the detection of core proteins related to the actual mucin expression changes can be associated with secretory lineage differentiation [4]. In conclusion, our studies give a further contribution to elucidating the complex relationship between mucin expression and DNA mismatch repair proteins, two factors that are closely associated with the development of a subgroup of colorectal malignancies, i.e., those showing the mucinous histological type. Thus, we confirm the increased expression of MUC5AC in mucinous colorectal neoplasms compared with nonmucinous lesions. Moreover, the lack of MLH1 protein expression was rather common in mucinous tumors, whereas the expression of the other major DNA mismatch repair protein (MSH2) is normal. Finally, the correlation between the extent of MUC5AC expression and the lack of MLH1 protein at the immunohistochemical level might suggest a functional association between the two proteins. Further studies should clarify whether these observations are of relevance to colorectal tumorigenesis.
Acknowledgements We thank Paola Manni, Emanuela Duzzi and Luca Fabbiani for their excellent technical assistance. This work was supported by grants ex 40%: Progetti di Ricerca avanzata.
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