ORIGINAL ARTICLE
Expression of MACC1 and MET in Inflammatory Bowel Disease-associated Colonic Neoplasia Noam Harpaz, MD, PhD,* Sofia Taboada, MD,† Huaibin Mabel Ko, MD,* Jiangzhou Yu, MD, PhD,† Qi Yang, AAS,† Haodong Xu, MD, PhD,‡ and Wenqing Cao, MD†
Background: Metastasis-associated in colon cancer-1 (MACC1), a newly identified regulator of HGF-MET signaling, may participate into the key steps of sporadic colorectal adenocarcinoma development. Given there are many pathogenetic distinctions between colitis-associated colorectal cancer (CAC) and sporadic colorectal adenocarcinomas, the potential roles of MACC1 in CAC carcinogenesis remain unknown. For the first time, we evaluated the expressions of MACC1 and MET in IBD-associated colitis, dysplasia, and adenocarcinoma. Methods: Expression was investigated by immunohistochemistry in tissue microarrays consisting of 13 normal colon, 11 active colitis, 9 dysplasia, 51 conventional CAC, 5 mucinous adenocarcinoma, and 1 signet ring cell adenocarcinoma specimens. The expression level of MACC1 or MET was evaluated with H-score system. Results: MACC1 expression was significantly higher in IBD-associated dysplasia than that in corresponding inflammatory or normal colonic tissue, and its level was further elevated from dysplasia to conventional CAC. Higher MACC1 expression was seen in a patient with CAC who had multifocal dysplasia or synchronous carcinoma. MACC1 overexpression (H-score .100) was seen in 67% of conventional CAC but in 0% of dysplasia and 0% of inflammation or normal colon. There was no difference of MACC1 expression found among well, moderately and poorly differentiated CAC. MET expressions in inflammation, dysplasia, and conventional CAC were statistically similar. No parallel expression of MACC1 and MET was detected in this study. MACC1 and MET expression was not increased in mucinous or signet ring cell carcinoma, 2 distinct variants of CAC.
Conclusions: Stepwise increase of MACC1 expression from IBD-associated colitis to dysplasia to adenocarcinoma suggests that MACC1 is strongly associated with conventional CAC tumorigenesis in a manner independent of MET. MACC1 may serve as a potential marker for early diagnosis of conventional CAC. (Inflamm Bowel Dis 2014;20:703–711) Key Words: metastasis-associated in colon cancer-1, MET, inflammatory bowel disease, dysplasia, colorectal adenocarcinoma
I
nflammatory bowel diseases (IBD)-associated colorectal cancer, also known as colitis-associated colorectal cancer (CAC), accounts for 10% to 15% of deaths in IBD.1 The risk of CAC directly correlates with the severity and longevity of inflammation.2,3 The cumulative probability of CAC in patients with ulcerative colitis (UC) has been shown in meta-analysis to range from 2% after 10 years of disease, up to 18% after 30 years of disease.2,4,5 Patients with Crohn’s disease (CD) also have an increased cumulative risk for CAC, from 2.9% at 10 years to 8.3% after 30 years of disease.3,6 There are distinct differences between CAC and familial or sporadic colorectal adenocarcinoma (CRC) in their biolReceived for publication November 10, 2013; Accepted December 19, 2013. From the *Department of Pathology, Mount Sinai Medical Center, New York, New York; †Department of Pathology and Laboratory Medicine, University of Rochester Medical Center, Rochester, New York; and ‡Department of Pathology and Laboratory Medicine, UCLA Center for the Health Science, Los Angeles, California. The authors have no conflicts of interest to disclose. Reprints: Wenqing Cao, MD, Department of Pathology and Laboratory Medicine, University of Rochester Medical Center, 601 Elmwood Ave, Box 626, Rochester, NY 14642 (e-mail: [email protected]). Copyright © 2014 Crohn’s & Colitis Foundation of America, Inc. DOI 10.1097/01.MIB.0000442679.39804.48 Published online 10 February 2014.
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ogy and underlying mechanisms,7 despite the substantial similarities between them in clinical and pathologic characteristics. The metastasis-associated in colon cancer-1 (MACC1) gene was identified by a genome-wide search for genes differentially expressed by analyzing normal tissues, primary tumors, and metastatic lesions in CRC. Further studies suggest that MACC1 functions as a transcriptional activator for proto-oncogene MET expression.8–10 MACC1 overexpression is associated with crucial steps of transition from adenoma to carcinoma and progression from low stage to high stage CRC.8,11,12 These studies indicate that MACC1 overexpression may serve as a marker for early diagnosis of CRC. In CRC, MET is also considered important for cancer progression and represents a powerful prognostic indicator for early stage invasion and metastasis.13–16 High expression of MET in CRC has been associated with the development of distant metastases and shorter disease-free survival.16 The expression of MET does not parallel with MACC1 expression in CRC progression from adenoma to carcinoma.12 Even though concordance of MACC1 and MET expression has been found in CRC with liver metastasis.8,12 This suggests that MACC1 may contribute to key steps in CRC tumorigenesis in a manner independent of MET. Evidence from the studies on the roles of MACC1 and www.ibdjournal.org |
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MET in CRC initiation, progression, and metastasis may lead to novel biomarkers for diagnosis, prognosis, and targeted therapy. In addition, no study has been done to address the expression patterns of MACC1 and MET in CAC. The histologic features of CRC and CAC are similar except that the incidence of 2 variants of adenocarcinoma, mucinous and signet ring cell carcinoma, are much higher in CAC.17,18 These mucin producing subtypes possess distinct clinical and molecular properties.19 Evaluating MACC1 and MET expression in these subtypes of CAC may provide a better understanding of the molecular characteristics of these tumors and lead to the development of potential diagnostic and prognostic markers and new therapeutic targets. In this study, we investigated the levels of MACC1 and MET proteins in IBD-associated inflammatory tissues, dysplasia, and conventional adenocarcinoma. We further explored the expression of MACC1 and MET in mucinous and signet ring cell carcinoma/differentiation. We found that the expression of MACC1 in conventional CAC showed a very similar pattern to that in CRC. There was no overexpression of MACC1 or MET identified in mucinous or signet ring cell carcinoma/differentiation.
MATERIALS AND METHODS Patients’ Recruitment and Tissue Samples The pathology database at the Mount Sinai Medical Center from 2001 to 2011 and at the University of Rochester Medical Center (URMC) from 2004 to 2011 was searched for IBD, UC, CD, and CRC. A total of 57 patients who had IBD-associated CRC and underwent resection and had histologic specimens in these 2 hospitals were reviewed. The diagnosis of IBD-associated dysplasia or adenocarcinoma for each case was confirmed by 1 of the 2 gastrointestinal pathologists (N.H. and W.C.). In addition, the normal controls were recruited from URMC pathology database by searching diverticulosis and histologic samples that were designated as resection margins. Human tissue specimens were obtained and processed with institutional review board approval from both institutions.
Demographic and Clinical Variables Clinical data were collected on gender, age at CAC, age at IBD diagnosis, type of IBD, and duration of IBD. The histological type of adenocarcinoma was classified as conventional adenocarcinoma, mucinous adenocarcinoma, or signet ring cell carcinoma according to the World Health Organization criteria. The tumor differentiation was graded as well, moderate, or poor. Other features including CAC location, stage, focality, distant dysplasia, and activity of colitis at CAC resection were collected from pathology reports. Not all data were available for all patients. The age at the onset of IBD and duration of colitis when CAC was diagnosed were known for 38 patients. The activity of colitis at CAC resection was reported in 39 patients. The stage of CAC was not specified in 5 patients’ pathology report. The information on focality of neoplasia (distant dysplasia or synchronous CAC) was available for 49 patients.
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Tissue Microarrays Tissue microarrays have constructed from 13 normal colon, 11 IBD colitis, 9 IBD-associated dysplasia, 51 conventional CRC (14 well differentiated, 22 moderately differentiated, and 15 poorly differentiated), 5 mucinous adenocarcinoma, and 1 signet ring cell carcinoma specimens. Two tissue cores (3 mm size) were obtained separately from the selected paraffin blocks.
Immunohistochemical Staining Archival, formalin-fixed, paraffin-embedded tissue blocks from selected patients were procured from the Department of Pathology and Laboratory Medicine at the URMC and the Department of Pathology at the Mount Sinai Medical Center. Tissue microarray sections were immunohistochemically stained with MACC1 and MET antibodies as previously described.12 Briefly, 4 mm paraffin sections were deparaffinized, and this was followed by antigen retrieval in EDTA buffer (pH 9.0) at 988C for 20 minutes. The rest of the procedure was done in a DAKO automated instrument. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide. The slides were incubated with antibodies against MACC1 (HPA020081, 1:600; Sigma-Aldrich, St. Louis, MO) or MET (SC-10, 1:200; Santa Cruz Biotechnology, Santa Cruz, CA) at room temperature for 1 hour and were then incubated for 30 minutes with EnVision+ System horseradish peroxidase-labeled polymer conjugated with biotinylated anti-rabbit secondary antibody and 3,30 -diaminobenzidine substrate. All sections were counterstained with Mayer’s hematoxylin. Colonic adenocarcinomas were used as the positive controls for MACC1 and MET. Negative controls were established by the replacement of primary antibodies with normal serum.
Evaluation of Immunohistochemical Staining As that reported in conventional CRC,12 cytoplasmic and membranous MACC1 and cytoplasmic MET staining were observed, and no positive nuclear staining was found (Fig. 1A). The staining intensity and the percentage of positive MACC1 or MET cells varied not only among the tissue samples but also in the same sample. To more precisely represent the expression level of MACC1 or MET, the H-score system was employed in this study. Two independent pathologists (S.T. and W.C.) evaluated the stained slides as described previously.12,20 The staining intensity for each protein was scored as 0, no staining; 1+, weak; 2+, moderate; and 3+, strong staining. The percentage of tumor cells that stained positive was also estimated (0%–100%). An H-score (range, 0–300) was generated by multiplying the staining intensity score and the percentage of positively stained tumor cells (H-score ¼ 3 · the corresponding positive percentage + 2 · the corresponding positive percentage + 1 · the corresponding positive percentage + 0 · the corresponding positive percentage). The Kappa values for interobserver agreement between the 2 pathologists were 0.86 and 0.81 for MACC1 and MET, respectively.
Statistic Analyses All data were statistically analyzed using the Prism 5 statistical package from GraphPad Software, Inc. (La Jolla, CA). The mean values of H-score of MACC1 and MET in normal
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Expression of MACC1 and MET in IBD
Previous studies have shown low MACC1 expression in normal colonic epithelium. In sporadic colonic adenoma, MACC1 was not increased compared with the corresponding normal tissues.8 Our previous studies on precursor lesions and early stage localized CRC showed significantly increased MACC1 expression in high-grade dysplasia and intramucosal adenocarcinoma suggesting a role of MACC1 in the CRC development.12 In this study, MACC1 expression level was first evaluated in normal colonic epithelium, IBD colitis, and IBD-associated dysplastic lesions. MACC1 expression was significantly increased in IBD-related dysplasia because the mean H-score of MACC1 staining in IBD-related dysplasia was obviously higher than that in normal colonic epithelium or IBD colitis (Table 1; Fig. 1B, left panel). MET expression was not altered among normal colonic epithelium, IBD-associated colitis, and dysplasia (Table 1; Fig. 1B, right panel).
MACC1 Expression Is Further Enhanced from Dysplasia to Adenocarcinoma but Not Associated with the Degree of Tumor Differentiation
FIGURE 1. Expression of MACC1 and MET in normal colonic mucosa (NL), IBD-inflamed mucosa (INF), and IBD-related dysplasia (DYS). A, Immunohistochemistry for MACC1 (a, b, and c) and MET (d, e, and f) (Original magnification, 200·). B, Comparison of H-score. The method for the evaluation of the immunohistochemical studies can be found in the Materials and Methods. *Significant difference between dysplasia and normal and inflamed mucosa (P , 0.05).
colon, inflammation, dysplasia, and different stages of CAC were analyzed by Student’s t test and one-way analysis of variance. P-value ,0.05 was considered statistically significant.
RESULTS Increasing MACC1 but Not MET Expression in IBD-associated Dysplasia The average H-score of each group and the rate of positive cells in different staining intensity are summarized in Table 1. From normal colon to inflammatory colon to dysplasia to adenocarcinoma, the positive cell rate with higher staining intensity of MACC1 and the average H-score seem to increase. However, the trend was not observed with MET expression (Table 1).
MACC1 expression has been found to be increased from adenoma to high-grade dysplasia to CRC in our previous studies.12 In this study, an increasing of MACC1 expression from inflammation to dysplasia was observed. Our data also indicated that the expression of MACC1 was further increased from dysplasia to CAC (mean H-score, 55.6 6 23.5 in dysplasia and 125.0 6 59.1 in CAC, P , 0.05) (Table 1; Fig. 2B, left panel). These data suggested that expression of MACC1 increased stepwise from inflammation to dysplasia to CAC. The mean H-score of MACC1 in well, moderately, or poorly differentiated CAC was also significantly higher than that in dysplasia (Fig. 2B, left panel). However, there was no difference of MACC1 expression detected among well, moderately, and poorly differentiated CAC (Fig. 2 and Table 2). To test if MACC1 could be used as a potential marker to discriminate CAC from benign lesions, we designated H-score greater than 100 as overexpression of MACC1 or MET. We found that the percentage of cases that had MACC1 scored greater than 100 (H-score) was 0% in normal colon, 0% in inflamed colon, 0% in dysplasia, and 67.0% in CAC, respectively (P , 0.05) (Fig. 2C, left panel). In contrast, MET expression was not increased from dysplasia to CAC, and it was not significantly different among well, moderately, and poorly differentiation (Table 1; Table 2; Fig. 2A and 2B, right panel). The percentage of cases with MET score greater than 100 (H-score) was 42.8% in normal colon, 45.4% in inflamed colon, 44.8% in dysplasia, and 45.8% in CAC, respectively (Fig. 2C, right panel). The MET overexpression seems not to be a maker to distinguish CAC from benign lesions.
Expression of MACC1 and MET in IBD-related Conventional Adenocarcinoma and Their Association with Clinicopathologic Characteristics The associations of MACC1 and MET expression in the cohort of conventional CAC with the clinicopathologic characteristics www.ibdjournal.org |
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0 59.2 6 8.0% 6 14.8 94.5 6 0 94.4 6 3.4% 6 6.8 90.3 6
48.0 43.3 56.6 57.0
are summarized in Table 2. Out of 51 conventional CAC patients, 38 (75%) were men. Forty-six patients had history of UC, whereas 5 patients had CD. The median age at the diagnosis of CAC was 53 years (range, 23–67 yr). Neither MACC1 nor MET expression was associated with the patient’s gender or the onset age of CAC. The median duration time of IBD (from diagnosis of IBD to identification of CAC) was 20 years (range, 1–75 yr). Evidence has suggested that the risk of CAC in patients with IBD was significantly elevated after diagnosis of IBD for 8 to 10 years.2,17 In our study, 10 (26%) of 38 patients had CAC develop within 10 years after the diagnosis of IBD. The expression of MACC1 or MET did not show significant difference between the groups of patients who had less than 10 years of IBD and those who had more than 10 years of IBD (Table 2). Furthermore, expression of MACC1 or MET was not dependent on the location and pathologic stage of CAC or the activity of colitis at CAC resection (Table 2). Interestingly, high expression of MACC1 was found in the patients with CAC who had multifocal neoplasia (Table 2). Notably, a previous study indicated that the median time for developing CAC from IBD was shorter in patients who were diagnosed with IBD at older age from about 24.5 years in patients with IBD at age less than 20 years to around 3.5 years in patients with IBD at age greater than 60 years.21 In line with these findings, our data showed that the patients diagnosed with IBD at older age developed CAC in a shorter period of time (Table 3). However, the expression of MACC1 or MET was statistically similar among the patients who had IBD at different ages (Table 3).
57.3% 44.5% 43.3% 43.9%
6 6 6 6
26.4 13.6 22.3 27.4
26.2% 24.5% 18.9% 29.6%
6 6 6 6
22.7 12.1 15.4 25.5
16.5% 23.0% 37.8% 23.3%
6 6 6 6
26.7 15.5 34.9 26.4
3 2 1 0
10.3 19.1 23.5 59.1 6 6 6 6 0 10.2 0 24.8 0 55.6 11.3% 6 18.1 125.0 10.3 17.6 24.5 27.4 6 6 6 6 6 6 6 6 NL (13) INF (11) DYS (9) CA (51)
89.9% 77.1% 51.1% 23.1%
10.3 17.5 19.7 31.6
10.1% 21.2% 42.2% 41.5%
1 0 Group (n)
DYS, dysplasia; INF, inflammation; NL, normal colon.
3 2
No Increasing Expression of MACC1 and MET in Mucinous and Signet Ring Cell Carcinoma/ Differentiation
0 1.8% 6 6.0 6.7% 6 10.0 24.0% 6 21.7
Percentage of Positive Cells in Different Staining Intensities (Mean 6 SD) Percentage of Positive Cells in Different Staining Intensities (Mean 6 SD)
Mean H-score (Mean 6 SD)
MET MACC1
TABLE 1. Distribution of Percentage of Positive Cells in Different Staining Intensities and Mean H-score Among Groups
Mean H-score (Mean 6 SD)
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Mucinous and signet ring cell carcinomas are rare in sporadic CRCs but are significantly more frequent in CAC.17,18 The rarity of the disease leads to less studies of molecular expression in these subtypes of tumors. In this study, 1 case of signet ring cell carcinoma and 4 poorly differentiated CAC with signet ring cell component were included. MACC1 and MET overexpression were not found in signet ring cell carcinoma or signet ring cell component of CAC (Fig. 3A). The immunostaining of MACC1 in signet ring cell component in the 4 CAC cases was similar to that in inflamed mucosa but much lower than in the adjacent conventional adenocarcinoma (Fig. 3A). Expression of MET in signet ring cell carcinoma/component was not only lower than in the adjacent conventional adenocarcinoma but was also lower than in the inflamed mucosa (Fig. 3A). Signet cell carcinoma is considered as a poorly differentiated type of adenocarcinoma and behaves more aggressively than conventional adenocarcinoma.19,22 Moreover, the presence of signet ring cell component in conventional CRC/CAC usually indicates poor differentiation and poor prognosis.22 In agreement with this concept, 100% (4/4) of CACs with signet ring cell differentiation in our study were poorly differentiated. In addition, there were 5 mucinous adenocarcinoma cases, none of them showed elevation of MACC1 or MET expression
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FIGURE 2. Expression of MACC1 and MET in conventional CAC. A, Immunohistochemistry for MACC1 (a, b, and c) and MET (d, e, and f) in well differentiated (WD), moderately differentiated (MD), and poorly differentiated (PD) CAC (Original magnification, 200·). B, Comparison of H-score from DYS, WD, MD, and PD. *Significant difference between adenocarcinoma (WD, MD, and PD) and DYS (P , 0.05). No statistical different expression of MACC1 or MET is found among WD, MD, and PD CAC. C, Percentage of cases that have MACC1 or MET H-score greater than 100 in NL, INF, DYS, or CAC group.
(Fig. 3B). We subsequently evaluated MACC1 and MET expression in 8 CACs with a mucinous component. A similar expression pattern as seen in signet ring cell carcinoma was
found in mucinous differentiation (Fig. 3B). However, only 25% (2 out of 8) of the CAC cases with mucinous component were poorly differentiated. www.ibdjournal.org |
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TABLE 2. Relationship of Expression of MACC1 or MET in Conventional CAC and Clinicopathologic Characteristics MACC1
Age at CAC, yr #53 (n ¼ 27) .53 (n ¼ 24) Gender F (n ¼ 13) M (n ¼ 38) Duration of colitis, yr #10 (n ¼ 10) .10 (n ¼ 28) Activity of colitis at CAC Inactive (n ¼ 13) Active (n ¼ 26) Location of CAC Colon (n ¼ 23) Rectum (n ¼ 27) Grade of CAC Well (n ¼ 14) Moderate (n ¼ 22) Poor (n ¼ 15) Stage of CAC T1-2 (n ¼ 19) T3-4 (n ¼ 27) Focality of neoplasia Multifocal (n ¼ 29) Unifocal (n ¼ 20)
MET
Mean 6 SD
P
Mean 6 SD
P
148.8 6 54.8 132.0 6 64.0
0.431
93.2 6 54.5 87.1 6 60.6
0.709
124.4 6 67.9 125.2 6 56.7
0.966
109.5 6 54.2 83.8 6 57.1
0.162
114.8 6 57.1 137.5 6 65.6
0.337
87.0 6 60.2 95.1 6 61.3
0.720
134.4 6 60.63 127.5 6 64.53
0.753
86.73 6 56.33 91.5 6 63.46
0.783
115.8 6 61.1 135.7 6 61.1
0.557
93.7 6 49.0 88.7 6 58.9
0.923
131.2 6 65.7 121.6 6 62.6 124.2 6 51.1
0.896
95.2 6 61.6 82.4 6 55.6 97.3 6 56.9
0.693
132.3 6 67.7 121.7 6 55.7
0.104
72.4 6 53.4 101.9 6 61.3
0.098
140.0 6 60.1 103.5 6 54.6
0.035a
87.3 6 58.8 101.9 6 53.4
0.370
T1-2, stage T1 and T2 adenocarcinoma; T3-4, stage T3 and T4 adenocarcinoma. Analyzed by Student’s t test or analysis of variance test. a P , 0.05.
TABLE 3. Expression of MACC1 or MET (H-score) in Relation to Age at Diagnosis of IBD and Duration of Colitis to CAC Duration of Colitis, Yrs
Age at IBD diagnosis, yr #30 (n ¼ 21) 31–40 (n ¼ 8) .40 (n ¼ 9) Analyzed by analysis of variance test. a P , 0.05.
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MACC1
MET
Mean 6 SD
P
Mean 6 SD
P
Mean 6 SD
P
27.9 6 16.4 21.9 6 13.5 10.7 6 8.4
0.017a
143.9 6 60.5 105.6 6 81.9 125.8 6 50.5
0.342
108.6 6 53.9 78.4 6 53.4 78.7 6 78.0
0.319
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Expression of MACC1 and MET in IBD
FIGURE 3. Immunohistochemistry for MACC1 and MET in variants of CAC. A, MACC1 and MET expression in signet ring cell carcinoma (a and e, low magnification 40·; b and f, high magnification 200·) and in conventional CAC with signet ring component (c and g, low magnification 40·; d and h, high magnification 200·). Black arrow indicates the signet ring cell component. B, MACC1 and MET expression in mucinous carcinoma (a and e, low magnification 40·; b and f, high magnification 200·) and in conventional CAC with mucinous component (c and g, low magnification 40·; d and h, high magnification 200·). Black arrow indicates the mucinous component.
DISCUSSION We studied MACC1 and MET expression in normal colon, IBD-related inflammatory epithelium, dysplasia, and adenocarcinoma. For the first time, we demonstrated that MACC1 expression was increased in IBD-related dysplasia compared with normal colonic mucosa or IBD colitis. MACC1 expression was further upregulated in conventional CAC compared to dysplasia. Overexpression of MACC1 has been reported in a variety of human malignances, such as colorectal, nasopharyngeal, liver, pancreas, and lung cancer.10,12,23–26 Ours and other studies on MACC1 expression in precursors and invasive CRC have shown that overexpression of MACC1 mainly occurs at several crucial steps in the transition from adenoma to carcinoma.8,12 This study in IBD-related precursor lesions and cancer shows a similar stepwise expression pattern, which suggests MACC1 may be an important gene in CAC development similar to CRC. Interestingly, our study also showed that MACC1 expression was elevated in IBD-related dysplasia (a precursor of CAC), whereas in CRC a significant elevation of MACC1 expression started at the
carcinoma in situ stage,12 suggesting MACC1 may be expressed earlier in CAC development than in CRC development. Aberrant expression of genes at different time frames has been reported in CAC and CRC development. For example, adenomatous polyposis coli mutations, one of the earliest events in CRC pathogenesis, occur much later in the course of CAC cancer development.27–29 In contrast, P53 overexpression is a late event in CRC development but occurs early in the precursor stage (adenoma) in CAC. The earlier upregulation of MACC1 in IBD carcinogenesis provides evidence to further support that there may be different molecular mechanisms underlying the tumorigenesis of CAC and CRC. The exact mechanism underlying early expression of MACC1 in CAC development is unclear and further studies are needed. MACC1 is recently identified as a regulator for MET transcription.8 Thus, to elucidate the role of MACC1 expression in CAC, we investigated the expression of MET. Similar with the findings in conventional CRC that MET is not altered until the late stages of invasive adenocarcinoma,12,30 MET remained consistently www.ibdjournal.org |
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expressed from IBD inflammatory epithelium to dysplasia to adenocarcinoma. No parallel expression of MACC1 and MET in the development of conventional CAC may be related to different regulating mechanisms involving transcription and translation. This further supports our previous postulation that MACC1 may independently function in early tumor development.12 Overexpression of MACC1 seems to be associated with the differentiation in the liver and lung cancers.31 Studies have found that MET expression is not related to differentiation of CRC.13,30 At this time, there is no report on MACC1 expression and differentiation in colon cancer, even though its overexpression has been associated with poor prognosis in CRC. In this study, we found the expression of MACC1 and MET was not statistically different among well, moderately, and poorly differentiated conventional CAC. The data suggest that expression of MACC1 and MET might not contribute to differentiation of conventional CAC. In population-based cohorts, patient with IBD has an increased risk of developing CAC, in which UC increases the risk of CAC by 2.4 fold and CD by 1.9 fold.3,6 Male sex, young age at IBD diagnosis, long duration of IBD, and extensive colitis also increase the risk of CAC.6,32,33 Since only a few cases of CAC resulted from CD in our study, we did not differentiate the expression of MACC1 or MET in CAC developed from UC or CD, respectively. Our data showed that neither the expression of MACC1 nor MET was associated with patient age at the diagnosis of IBD, gender, duration of IBD, CAC location, or stage. These suggest that MACC1 and MET expression may not relate to these clinical characteristics that are associated with high risk of CAC. However, in agreement with the previous report,34 the patients with CAC in our study who were diagnosed with IBD at old age had a significant shorter duration of IBD (Table 3). In comparison with sporadic CRC, CAC has a similar dysplasia-carcinoma sequence. Multifocal dysplasia or widespread neoplasia occurs in the majority of CAC cases, which ranges from 40% to 100%.35–37 Multifocal dysplasia seems not to be associated with most of clinicopathologic characteristics,36 even through one study indicated that multifocal dysplasia was related to the early onset of IBD.34 In our study, 60% (31 of 51) CAC cases possessed multifocal neoplasia, in which MACC1 expression was significantly higher compared with the group without widespread neoplasia. The finding may further support our data from the immunohistochemistry study that MACC1 expression was greatly increased in dysplasia in comparison with inflammatory or normal tissue, indicating the role of MACC1 in CAC tumorigenesis. Mucinous and signet ring cell adenocarcinomas are 2 variants of adenocarcinoma that are more common in IBDrelated CAC.17,18 Evidence suggests that mucinous and signet ring cell carcinomas have distinct molecular characteristics from conventional CRC/CAC. The frequency of BRAF mutation in colorectal signet ring cell carcinoma is higher than in conventional CRC.38 HATH1, MUC2, and SOX2 gene are frequently expressed in signet ring cell carcinoma but rarely expressed in conventional adenocarcinoma.39,40 The frequency of CpG island
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methylation phenotype in signet ring cell carcinoma is significantly increased compared with conventional CRC as detected by quantitative real time polymerase chain reaction.41 Using microarray analysis, Kim et al42 identified 50 classifiers out of 62 differentially expressed genes in mucinous carcinoma compared with non-mucinous adenocarcinoma. In this study, we found that MACC1 and MET expression was not increased in signet ring and mucinous carcinoma cells. The data that MACC1 expression was increased in conventional CAC, but not in mucinous and signet ring cell carcinoma/differentiation, suggest that MACC1 may not play an essential role in the development of mucinous and signet ring cell carcinoma. There are a couple of limitations in this study. It was a retrospective study, which limited the quality and completeness of clinical data. For example, the information on the age of IBD onset and duration of disease when CAC was diagnosed were available only in 75% (38 of 51) patients. Out of these 38 patients, 10 (26%) developed CAC in less than 10 years of IBD. Although this data is similar to previous reports,34,43 our cohort had fewer patients who developed CAC in less than 8 years. One possible explanation might be our patients were diagnosed and followed at a large IBD center so that the delayed diagnosis of IBD was less likely. Another limitation for this study is although we have relatively large study size (total 57 patients with CAC), we had very few Crohn’s-derived CAC (only 5 patients with history of CD); therefore, we were unable to compare MACC1 or MET expression patterns between CAC derived from UC and CD. In conclusion, in this study, we demonstrate that MACC1 and MET expression patterns in conventional CAC are similar to that in conventional CRC, except for high expression of MACC1 in IBD-derived dysplasia. The comparative analysis of gene expression among conventional CAC, mucinous, and signet ring cell carcinoma/differentiation would augment our understanding of factors affecting differentiation in CAC, expand our knowledge on the etiology of these tumors, and possibly establish markers for early detection of CAC. Overexpression of MACC1 may be a potential marker not only for conventional CRC but also for the early diagnosis of conventional CAC.
REFERENCES 1. Munkholm P. Review article: the incidence and prevalence of colorectal cancer in inflammatory bowel disease. Aliment Pharmacol Ther. 2003;18(suppl 2):1–5. 2. Eaden JA, Abrams KR, Mayberry JF. The risk of colorectal cancer in ulcerative colitis: a meta-analysis. Gut. 2001;48:526–535. 3. Jess T, Gamborg M, Matzen P, et al. Increased risk of intestinal cancer in Crohn’s disease: a meta-analysis of population-based cohort studies. Am J Gastroenterol. 2005;100:2724–2729. 4. Feagins LA, Souza RF, Spechler SJ. Carcinogenesis in IBD: potential targets for the prevention of colorectal cancer. Nat Rev Gastroenterol Hepatol. 2009;6:297–305. 5. Westbrook AM, Szakmary A, Schiestl RH. Mechanisms of intestinal inflammation and development of associated cancers: lessons learned from mouse models. Mutat Res. 2010;705:40–59. 6. Canavan C, Abrams KR, Mayberry J. Meta-analysis: colorectal and small bowel cancer risk in patients with Crohn’s disease. Aliment Pharmacol Ther. 2006;23:1097–1104. 7. Grivennikov SI. Inflammation and colorectal cancer: colitis-associated neoplasia. Semin Immunopathol. 2013;35:229–244.
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8. Stein U, Walther W, Arlt F, et al. MACC1, a newly identified key regulator of HGF-MET signaling, predicts colon cancer metastasis. Nat Med. 2009;15:59–67. 9. Stein U, Smith J, Walther W, et al. MACC1 controls Met: what a difference an Sp1 site makes. Cell Cycle. 2009;8:2467–2469. 10. Arlt F, Stein U. Colon cancer metastasis: MACC1 and Met as metastatic pacemakers. Int J Biochem Cell Biol. 2009;41:2356–2359. 11. Shirahata A, Shinmura K, Kitamura Y, et al. MACC1 as a marker for advanced colorectal carcinoma. Anticancer Res. 2010;30:2689–2692. 12. Ren B, Zakharov V, Yang Q, et al. MACC1 is related to colorectal cancer initiation and early-stage invasive growth. Am J Clin Pathol. 2013;140: 701–707. 13. Zeng Z, Weiser MR, D’Alessio M, et al. Immunoblot analysis of c-Met expression in human colorectal cancer: overexpression is associated with advanced stage cancer. Clin Exp Metastasis. 2004;21:409–417. 14. Zeng ZS, Weiser MR, Kuntz E, et al. c-Met gene amplification is associated with advanced stage colorectal cancer and liver metastases. Cancer Lett. 2008;265:258–269. 15. Fujita S, Sugano K. Expression of c-met proto-oncogene in primary colorectal cancer and liver metastases. Jpn J Clin Oncol. 1997;27:378–383. 16. De Oliveira AT, Matos D, Logullo AF, et al. MET Is highly expressed in advanced stages of colorectal cancer and indicates worse prognosis and mortality. Anticancer Res. 2009;29:4807–4811. 17. Ording AG, Horvath-Puho E, Erichsen R, et al. Five-year mortality in colorectal cancer patients with ulcerative colitis or Crohn’s disease: a nationwide population-based cohort study. Inflamm Bowel Dis. 2013; 19:800–805. 18. Redston M. Epithelial neoplasms of the large intestine. In: Goldblum JR, Odze RD, eds. Surgical Pathology of the GI Tract, Liver, Biliary Tract, and Pancreas. 2nd ed. Philadelphia, PA: Saunders; 2009:597–637. 19. Hyngstrom JR, Hu CY, Xing Y, et al. Clinicopathology and outcomes for mucinous and signet ring colorectal adenocarcinoma: analysis from the National Cancer Data Base. Ann Surg Oncol. 2012;19:2814–2821. 20. Zakharov V, Ren B, Ryan C, et al. Diagnostic value of HMGAs, p53 and beta-catenin in discriminating adenocarcinoma from adenoma or reactive atypia in ampulla and common bile duct biopsies. Histopathology. 2013; 62:778–787. 21. Baars JE, Kuipers EJ, van Haastert M, et al. Age at diagnosis of inflammatory bowel disease influences early development of colorectal cancer in inflammatory bowel disease patients: a nationwide, long-term survey. J Gastroenterol. 2012;47:1308–1322. 22. Psathakis D, Schiedeck TH, Krug F, et al. Ordinary colorectal adenocarcinoma vs. primary colorectal signet-ring cell carcinoma: study matched for age, gender, grade, and stage. Dis Colon Rectum. 1999;42:1618–1625. 23. Shimokawa H, Uramoto H, Onitsuka T, et al. Overexpression of MACC1 mRNA in lung adenocarcinoma is associated with postoperative recurrence. J Thorac Cardiovasc Surg. 2011;141:895–898. 24. Wang G, Kang MX, Lu WJ, et al. MACC1: a potential molecule associated with pancreatic cancer metastasis and chemoresistance. Oncol Lett. 2012;4:783–791. 25. Meng F, Li H, Shi H, et al. MACC1 down-regulation inhibits proliferation and tumourigenicity of nasopharyngeal carcinoma cells through Akt/betacatenin signaling pathway. PloS One. 2013;8:e60821.
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26. Zhang Y, Wang Z, Chen M, et al. MicroRNA-143 targets MACC1 to inhibit cell invasion and migration in colorectal cancer. Mol Cancer. 2012;11:23. 27. Redston MS, Papadopoulos N, Caldas C, et al. Common occurrence of APC and K-ras gene mutations in the spectrum of colitis-associated neoplasias. Gastroenterology. 1995;108:383–392. 28. Tarmin L, Yin J, Harpaz N, et al. Adenomatous polyposis coli gene mutations in ulcerative colitis-associated dysplasias and cancers versus sporadic colon neoplasms. Cancer Res. 1995;55:2035–2038. 29. Aust DE, Terdiman JP, Willenbucher RF, et al. The APC/beta-catenin pathway in ulcerative colitis-related colorectal carcinomas: a mutational analysis. Cancer. 2002;94:1421–1427. 30. Baldus SE, Kort EJ, Schirmacher P, et al. Quantification of MET and hepatocyte growth factor/scatter factor expression in colorectal adenomas, carcinomas and non-neoplastic epithelia by quantitative laser scanning microscopy. Int J Oncol. 2007;31:199–204. 31. Gao J, Ding F, Liu Q, et al. Knockdown of MACC1 expression suppressed hepatocellular carcinoma cell migration and invasion and inhibited expression of MMP2 and MMP9. Mol Cell Biochem. 2013;376:21–32. 32. Soderlund S, Granath F, Brostrom O, et al. Inflammatory bowel disease confers a lower risk of colorectal cancer to females than to males. Gastroenterology. 2010;138:1697–1703. 33. Basseri RJ, Basseri B, Papadakis KA. Dysplasia and cancer in inflammatory bowel disease. Expert Rev Gastroenterol Hepatol. 2011;5:59–66. 34. Brackmann S, Andersen SN, Aamodt G, et al. Two distinct groups of colorectal cancer in inflammatory bowel disease. Inflamm Bowel Dis. 2009;15:9–16. 35. Svrcek M, Cosnes J, Beaugerie L, et al. Colorectal neoplasia in Crohn’s colitis: a retrospective comparative study with ulcerative colitis. Histopathology. 2007;50:574–583. 36. Connell WR, Sheffield JP, Kamm MA, et al. Lower gastrointestinal malignancy in Crohn’s disease. Gut. 1994;35:347–352. 37. Hamilton SR. Colorectal carcinoma in patients with Crohn’s disease. Gastroenterology. 1985;89:398–407. 38. Ogino S, Brahmandam M, Cantor M, et al. Distinct molecular features of colorectal carcinoma with signet ring cell component and colorectal carcinoma with mucinous component. Mod Pathol. 2006;19:59–68. 39. Park ET, Oh HK, Gum JR Jr, et al. HATH1 expression in mucinous cancers of the colorectum and related lesions. Clin Cancer Res. 2006; 12:5403–5410. 40. Park ET, Gum JR, Kakar S, et al. Aberrant expression of SOX2 upregulates MUC5AC gastric foveolar mucin in mucinous cancers of the colorectum and related lesions. Int J Cancer. 2008;122:1253–1260. 41. Ogino S, Odze RD, Kawasaki T, et al. Correlation of pathologic features with CpG island methylator phenotype (CIMP) by quantitative DNA methylation analysis in colorectal carcinoma. Am J Surg Pathol. 2006; 30:1175–1183. 42. Kim HS, Kang SH, Park CH, et al. Genome-wide molecular characterization of mucinous colorectal adenocarcinoma using cDNA microarray analysis. Oncol Rep. 2011;25:717–727. 43. Brackmann S, Andersen SN, Aamodt G, et al. Relationship between clinical parameters and the colitis-colorectal cancer interval in a cohort of patients with colorectal cancer in inflammatory bowel disease. Scand J Gastroenterol. 2009;44:46–55.
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Expression of MACC1 and MET in Inflammatory Bowel Disease-associated Colonic Neoplasia Noam Harpaz, MD, PhD,* Sofia Taboada, MD,† Huaibin Mabel Ko, MD,* Jiangzhou Yu, MD, PhD,† Qi Yang, AAS,† Haodong Xu, MD, PhD,‡ and Wenqing Cao, MD†
Background: Metastasis-associated in colon cancer-1 (MACC1), a newly identified regulator of HGF-MET signaling, may participate into the key steps of sporadic colorectal adenocarcinoma development. Given there are many pathogenetic distinctions between colitis-associated colorectal cancer (CAC) and sporadic colorectal adenocarcinomas, the potential roles of MACC1 in CAC carcinogenesis remain unknown. For the first time, we evaluated the expressions of MACC1 and MET in IBD-associated colitis, dysplasia, and adenocarcinoma. Methods: Expression was investigated by immunohistochemistry in tissue microarrays consisting of 13 normal colon, 11 active colitis, 9 dysplasia, 51 conventional CAC, 5 mucinous adenocarcinoma, and 1 signet ring cell adenocarcinoma specimens. The expression level of MACC1 or MET was evaluated with H-score system. Results: MACC1 expression was significantly higher in IBD-associated dysplasia than that in corresponding inflammatory or normal colonic tissue, and its level was further elevated from dysplasia to conventional CAC. Higher MACC1 expression was seen in a patient with CAC who had multifocal dysplasia or synchronous carcinoma. MACC1 overexpression (H-score .100) was seen in 67% of conventional CAC but in 0% of dysplasia and 0% of inflammation or normal colon. There was no difference of MACC1 expression found among well, moderately and poorly differentiated CAC. MET expressions in inflammation, dysplasia, and conventional CAC were statistically similar. No parallel expression of MACC1 and MET was detected in this study. MACC1 and MET expression was not increased in mucinous or signet ring cell carcinoma, 2 distinct variants of CAC.
Conclusions: Stepwise increase of MACC1 expression from IBD-associated colitis to dysplasia to adenocarcinoma suggests that MACC1 is strongly associated with conventional CAC tumorigenesis in a manner independent of MET. MACC1 may serve as a potential marker for early diagnosis of conventional CAC. (Inflamm Bowel Dis 2014;20:703–711) Key Words: metastasis-associated in colon cancer-1, MET, inflammatory bowel disease, dysplasia, colorectal adenocarcinoma
I
nflammatory bowel diseases (IBD)-associated colorectal cancer, also known as colitis-associated colorectal cancer (CAC), accounts for 10% to 15% of deaths in IBD.1 The risk of CAC directly correlates with the severity and longevity of inflammation.2,3 The cumulative probability of CAC in patients with ulcerative colitis (UC) has been shown in meta-analysis to range from 2% after 10 years of disease, up to 18% after 30 years of disease.2,4,5 Patients with Crohn’s disease (CD) also have an increased cumulative risk for CAC, from 2.9% at 10 years to 8.3% after 30 years of disease.3,6 There are distinct differences between CAC and familial or sporadic colorectal adenocarcinoma (CRC) in their biolReceived for publication November 10, 2013; Accepted December 19, 2013. From the *Department of Pathology, Mount Sinai Medical Center, New York, New York; †Department of Pathology and Laboratory Medicine, University of Rochester Medical Center, Rochester, New York; and ‡Department of Pathology and Laboratory Medicine, UCLA Center for the Health Science, Los Angeles, California. The authors have no conflicts of interest to disclose. Reprints: Wenqing Cao, MD, Department of Pathology and Laboratory Medicine, University of Rochester Medical Center, 601 Elmwood Ave, Box 626, Rochester, NY 14642 (e-mail: [email protected]). Copyright © 2014 Crohn’s & Colitis Foundation of America, Inc. DOI 10.1097/01.MIB.0000442679.39804.48 Published online 10 February 2014.
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ogy and underlying mechanisms,7 despite the substantial similarities between them in clinical and pathologic characteristics. The metastasis-associated in colon cancer-1 (MACC1) gene was identified by a genome-wide search for genes differentially expressed by analyzing normal tissues, primary tumors, and metastatic lesions in CRC. Further studies suggest that MACC1 functions as a transcriptional activator for proto-oncogene MET expression.8–10 MACC1 overexpression is associated with crucial steps of transition from adenoma to carcinoma and progression from low stage to high stage CRC.8,11,12 These studies indicate that MACC1 overexpression may serve as a marker for early diagnosis of CRC. In CRC, MET is also considered important for cancer progression and represents a powerful prognostic indicator for early stage invasion and metastasis.13–16 High expression of MET in CRC has been associated with the development of distant metastases and shorter disease-free survival.16 The expression of MET does not parallel with MACC1 expression in CRC progression from adenoma to carcinoma.12 Even though concordance of MACC1 and MET expression has been found in CRC with liver metastasis.8,12 This suggests that MACC1 may contribute to key steps in CRC tumorigenesis in a manner independent of MET. Evidence from the studies on the roles of MACC1 and www.ibdjournal.org |
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MET in CRC initiation, progression, and metastasis may lead to novel biomarkers for diagnosis, prognosis, and targeted therapy. In addition, no study has been done to address the expression patterns of MACC1 and MET in CAC. The histologic features of CRC and CAC are similar except that the incidence of 2 variants of adenocarcinoma, mucinous and signet ring cell carcinoma, are much higher in CAC.17,18 These mucin producing subtypes possess distinct clinical and molecular properties.19 Evaluating MACC1 and MET expression in these subtypes of CAC may provide a better understanding of the molecular characteristics of these tumors and lead to the development of potential diagnostic and prognostic markers and new therapeutic targets. In this study, we investigated the levels of MACC1 and MET proteins in IBD-associated inflammatory tissues, dysplasia, and conventional adenocarcinoma. We further explored the expression of MACC1 and MET in mucinous and signet ring cell carcinoma/differentiation. We found that the expression of MACC1 in conventional CAC showed a very similar pattern to that in CRC. There was no overexpression of MACC1 or MET identified in mucinous or signet ring cell carcinoma/differentiation.
MATERIALS AND METHODS Patients’ Recruitment and Tissue Samples The pathology database at the Mount Sinai Medical Center from 2001 to 2011 and at the University of Rochester Medical Center (URMC) from 2004 to 2011 was searched for IBD, UC, CD, and CRC. A total of 57 patients who had IBD-associated CRC and underwent resection and had histologic specimens in these 2 hospitals were reviewed. The diagnosis of IBD-associated dysplasia or adenocarcinoma for each case was confirmed by 1 of the 2 gastrointestinal pathologists (N.H. and W.C.). In addition, the normal controls were recruited from URMC pathology database by searching diverticulosis and histologic samples that were designated as resection margins. Human tissue specimens were obtained and processed with institutional review board approval from both institutions.
Demographic and Clinical Variables Clinical data were collected on gender, age at CAC, age at IBD diagnosis, type of IBD, and duration of IBD. The histological type of adenocarcinoma was classified as conventional adenocarcinoma, mucinous adenocarcinoma, or signet ring cell carcinoma according to the World Health Organization criteria. The tumor differentiation was graded as well, moderate, or poor. Other features including CAC location, stage, focality, distant dysplasia, and activity of colitis at CAC resection were collected from pathology reports. Not all data were available for all patients. The age at the onset of IBD and duration of colitis when CAC was diagnosed were known for 38 patients. The activity of colitis at CAC resection was reported in 39 patients. The stage of CAC was not specified in 5 patients’ pathology report. The information on focality of neoplasia (distant dysplasia or synchronous CAC) was available for 49 patients.
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Tissue Microarrays Tissue microarrays have constructed from 13 normal colon, 11 IBD colitis, 9 IBD-associated dysplasia, 51 conventional CRC (14 well differentiated, 22 moderately differentiated, and 15 poorly differentiated), 5 mucinous adenocarcinoma, and 1 signet ring cell carcinoma specimens. Two tissue cores (3 mm size) were obtained separately from the selected paraffin blocks.
Immunohistochemical Staining Archival, formalin-fixed, paraffin-embedded tissue blocks from selected patients were procured from the Department of Pathology and Laboratory Medicine at the URMC and the Department of Pathology at the Mount Sinai Medical Center. Tissue microarray sections were immunohistochemically stained with MACC1 and MET antibodies as previously described.12 Briefly, 4 mm paraffin sections were deparaffinized, and this was followed by antigen retrieval in EDTA buffer (pH 9.0) at 988C for 20 minutes. The rest of the procedure was done in a DAKO automated instrument. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide. The slides were incubated with antibodies against MACC1 (HPA020081, 1:600; Sigma-Aldrich, St. Louis, MO) or MET (SC-10, 1:200; Santa Cruz Biotechnology, Santa Cruz, CA) at room temperature for 1 hour and were then incubated for 30 minutes with EnVision+ System horseradish peroxidase-labeled polymer conjugated with biotinylated anti-rabbit secondary antibody and 3,30 -diaminobenzidine substrate. All sections were counterstained with Mayer’s hematoxylin. Colonic adenocarcinomas were used as the positive controls for MACC1 and MET. Negative controls were established by the replacement of primary antibodies with normal serum.
Evaluation of Immunohistochemical Staining As that reported in conventional CRC,12 cytoplasmic and membranous MACC1 and cytoplasmic MET staining were observed, and no positive nuclear staining was found (Fig. 1A). The staining intensity and the percentage of positive MACC1 or MET cells varied not only among the tissue samples but also in the same sample. To more precisely represent the expression level of MACC1 or MET, the H-score system was employed in this study. Two independent pathologists (S.T. and W.C.) evaluated the stained slides as described previously.12,20 The staining intensity for each protein was scored as 0, no staining; 1+, weak; 2+, moderate; and 3+, strong staining. The percentage of tumor cells that stained positive was also estimated (0%–100%). An H-score (range, 0–300) was generated by multiplying the staining intensity score and the percentage of positively stained tumor cells (H-score ¼ 3 · the corresponding positive percentage + 2 · the corresponding positive percentage + 1 · the corresponding positive percentage + 0 · the corresponding positive percentage). The Kappa values for interobserver agreement between the 2 pathologists were 0.86 and 0.81 for MACC1 and MET, respectively.
Statistic Analyses All data were statistically analyzed using the Prism 5 statistical package from GraphPad Software, Inc. (La Jolla, CA). The mean values of H-score of MACC1 and MET in normal
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Previous studies have shown low MACC1 expression in normal colonic epithelium. In sporadic colonic adenoma, MACC1 was not increased compared with the corresponding normal tissues.8 Our previous studies on precursor lesions and early stage localized CRC showed significantly increased MACC1 expression in high-grade dysplasia and intramucosal adenocarcinoma suggesting a role of MACC1 in the CRC development.12 In this study, MACC1 expression level was first evaluated in normal colonic epithelium, IBD colitis, and IBD-associated dysplastic lesions. MACC1 expression was significantly increased in IBD-related dysplasia because the mean H-score of MACC1 staining in IBD-related dysplasia was obviously higher than that in normal colonic epithelium or IBD colitis (Table 1; Fig. 1B, left panel). MET expression was not altered among normal colonic epithelium, IBD-associated colitis, and dysplasia (Table 1; Fig. 1B, right panel).
MACC1 Expression Is Further Enhanced from Dysplasia to Adenocarcinoma but Not Associated with the Degree of Tumor Differentiation
FIGURE 1. Expression of MACC1 and MET in normal colonic mucosa (NL), IBD-inflamed mucosa (INF), and IBD-related dysplasia (DYS). A, Immunohistochemistry for MACC1 (a, b, and c) and MET (d, e, and f) (Original magnification, 200·). B, Comparison of H-score. The method for the evaluation of the immunohistochemical studies can be found in the Materials and Methods. *Significant difference between dysplasia and normal and inflamed mucosa (P , 0.05).
colon, inflammation, dysplasia, and different stages of CAC were analyzed by Student’s t test and one-way analysis of variance. P-value ,0.05 was considered statistically significant.
RESULTS Increasing MACC1 but Not MET Expression in IBD-associated Dysplasia The average H-score of each group and the rate of positive cells in different staining intensity are summarized in Table 1. From normal colon to inflammatory colon to dysplasia to adenocarcinoma, the positive cell rate with higher staining intensity of MACC1 and the average H-score seem to increase. However, the trend was not observed with MET expression (Table 1).
MACC1 expression has been found to be increased from adenoma to high-grade dysplasia to CRC in our previous studies.12 In this study, an increasing of MACC1 expression from inflammation to dysplasia was observed. Our data also indicated that the expression of MACC1 was further increased from dysplasia to CAC (mean H-score, 55.6 6 23.5 in dysplasia and 125.0 6 59.1 in CAC, P , 0.05) (Table 1; Fig. 2B, left panel). These data suggested that expression of MACC1 increased stepwise from inflammation to dysplasia to CAC. The mean H-score of MACC1 in well, moderately, or poorly differentiated CAC was also significantly higher than that in dysplasia (Fig. 2B, left panel). However, there was no difference of MACC1 expression detected among well, moderately, and poorly differentiated CAC (Fig. 2 and Table 2). To test if MACC1 could be used as a potential marker to discriminate CAC from benign lesions, we designated H-score greater than 100 as overexpression of MACC1 or MET. We found that the percentage of cases that had MACC1 scored greater than 100 (H-score) was 0% in normal colon, 0% in inflamed colon, 0% in dysplasia, and 67.0% in CAC, respectively (P , 0.05) (Fig. 2C, left panel). In contrast, MET expression was not increased from dysplasia to CAC, and it was not significantly different among well, moderately, and poorly differentiation (Table 1; Table 2; Fig. 2A and 2B, right panel). The percentage of cases with MET score greater than 100 (H-score) was 42.8% in normal colon, 45.4% in inflamed colon, 44.8% in dysplasia, and 45.8% in CAC, respectively (Fig. 2C, right panel). The MET overexpression seems not to be a maker to distinguish CAC from benign lesions.
Expression of MACC1 and MET in IBD-related Conventional Adenocarcinoma and Their Association with Clinicopathologic Characteristics The associations of MACC1 and MET expression in the cohort of conventional CAC with the clinicopathologic characteristics www.ibdjournal.org |
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0 59.2 6 8.0% 6 14.8 94.5 6 0 94.4 6 3.4% 6 6.8 90.3 6
48.0 43.3 56.6 57.0
are summarized in Table 2. Out of 51 conventional CAC patients, 38 (75%) were men. Forty-six patients had history of UC, whereas 5 patients had CD. The median age at the diagnosis of CAC was 53 years (range, 23–67 yr). Neither MACC1 nor MET expression was associated with the patient’s gender or the onset age of CAC. The median duration time of IBD (from diagnosis of IBD to identification of CAC) was 20 years (range, 1–75 yr). Evidence has suggested that the risk of CAC in patients with IBD was significantly elevated after diagnosis of IBD for 8 to 10 years.2,17 In our study, 10 (26%) of 38 patients had CAC develop within 10 years after the diagnosis of IBD. The expression of MACC1 or MET did not show significant difference between the groups of patients who had less than 10 years of IBD and those who had more than 10 years of IBD (Table 2). Furthermore, expression of MACC1 or MET was not dependent on the location and pathologic stage of CAC or the activity of colitis at CAC resection (Table 2). Interestingly, high expression of MACC1 was found in the patients with CAC who had multifocal neoplasia (Table 2). Notably, a previous study indicated that the median time for developing CAC from IBD was shorter in patients who were diagnosed with IBD at older age from about 24.5 years in patients with IBD at age less than 20 years to around 3.5 years in patients with IBD at age greater than 60 years.21 In line with these findings, our data showed that the patients diagnosed with IBD at older age developed CAC in a shorter period of time (Table 3). However, the expression of MACC1 or MET was statistically similar among the patients who had IBD at different ages (Table 3).
57.3% 44.5% 43.3% 43.9%
6 6 6 6
26.4 13.6 22.3 27.4
26.2% 24.5% 18.9% 29.6%
6 6 6 6
22.7 12.1 15.4 25.5
16.5% 23.0% 37.8% 23.3%
6 6 6 6
26.7 15.5 34.9 26.4
3 2 1 0
10.3 19.1 23.5 59.1 6 6 6 6 0 10.2 0 24.8 0 55.6 11.3% 6 18.1 125.0 10.3 17.6 24.5 27.4 6 6 6 6 6 6 6 6 NL (13) INF (11) DYS (9) CA (51)
89.9% 77.1% 51.1% 23.1%
10.3 17.5 19.7 31.6
10.1% 21.2% 42.2% 41.5%
1 0 Group (n)
DYS, dysplasia; INF, inflammation; NL, normal colon.
3 2
No Increasing Expression of MACC1 and MET in Mucinous and Signet Ring Cell Carcinoma/ Differentiation
0 1.8% 6 6.0 6.7% 6 10.0 24.0% 6 21.7
Percentage of Positive Cells in Different Staining Intensities (Mean 6 SD) Percentage of Positive Cells in Different Staining Intensities (Mean 6 SD)
Mean H-score (Mean 6 SD)
MET MACC1
TABLE 1. Distribution of Percentage of Positive Cells in Different Staining Intensities and Mean H-score Among Groups
Mean H-score (Mean 6 SD)
Harpaz et al
Mucinous and signet ring cell carcinomas are rare in sporadic CRCs but are significantly more frequent in CAC.17,18 The rarity of the disease leads to less studies of molecular expression in these subtypes of tumors. In this study, 1 case of signet ring cell carcinoma and 4 poorly differentiated CAC with signet ring cell component were included. MACC1 and MET overexpression were not found in signet ring cell carcinoma or signet ring cell component of CAC (Fig. 3A). The immunostaining of MACC1 in signet ring cell component in the 4 CAC cases was similar to that in inflamed mucosa but much lower than in the adjacent conventional adenocarcinoma (Fig. 3A). Expression of MET in signet ring cell carcinoma/component was not only lower than in the adjacent conventional adenocarcinoma but was also lower than in the inflamed mucosa (Fig. 3A). Signet cell carcinoma is considered as a poorly differentiated type of adenocarcinoma and behaves more aggressively than conventional adenocarcinoma.19,22 Moreover, the presence of signet ring cell component in conventional CRC/CAC usually indicates poor differentiation and poor prognosis.22 In agreement with this concept, 100% (4/4) of CACs with signet ring cell differentiation in our study were poorly differentiated. In addition, there were 5 mucinous adenocarcinoma cases, none of them showed elevation of MACC1 or MET expression
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FIGURE 2. Expression of MACC1 and MET in conventional CAC. A, Immunohistochemistry for MACC1 (a, b, and c) and MET (d, e, and f) in well differentiated (WD), moderately differentiated (MD), and poorly differentiated (PD) CAC (Original magnification, 200·). B, Comparison of H-score from DYS, WD, MD, and PD. *Significant difference between adenocarcinoma (WD, MD, and PD) and DYS (P , 0.05). No statistical different expression of MACC1 or MET is found among WD, MD, and PD CAC. C, Percentage of cases that have MACC1 or MET H-score greater than 100 in NL, INF, DYS, or CAC group.
(Fig. 3B). We subsequently evaluated MACC1 and MET expression in 8 CACs with a mucinous component. A similar expression pattern as seen in signet ring cell carcinoma was
found in mucinous differentiation (Fig. 3B). However, only 25% (2 out of 8) of the CAC cases with mucinous component were poorly differentiated. www.ibdjournal.org |
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TABLE 2. Relationship of Expression of MACC1 or MET in Conventional CAC and Clinicopathologic Characteristics MACC1
Age at CAC, yr #53 (n ¼ 27) .53 (n ¼ 24) Gender F (n ¼ 13) M (n ¼ 38) Duration of colitis, yr #10 (n ¼ 10) .10 (n ¼ 28) Activity of colitis at CAC Inactive (n ¼ 13) Active (n ¼ 26) Location of CAC Colon (n ¼ 23) Rectum (n ¼ 27) Grade of CAC Well (n ¼ 14) Moderate (n ¼ 22) Poor (n ¼ 15) Stage of CAC T1-2 (n ¼ 19) T3-4 (n ¼ 27) Focality of neoplasia Multifocal (n ¼ 29) Unifocal (n ¼ 20)
MET
Mean 6 SD
P
Mean 6 SD
P
148.8 6 54.8 132.0 6 64.0
0.431
93.2 6 54.5 87.1 6 60.6
0.709
124.4 6 67.9 125.2 6 56.7
0.966
109.5 6 54.2 83.8 6 57.1
0.162
114.8 6 57.1 137.5 6 65.6
0.337
87.0 6 60.2 95.1 6 61.3
0.720
134.4 6 60.63 127.5 6 64.53
0.753
86.73 6 56.33 91.5 6 63.46
0.783
115.8 6 61.1 135.7 6 61.1
0.557
93.7 6 49.0 88.7 6 58.9
0.923
131.2 6 65.7 121.6 6 62.6 124.2 6 51.1
0.896
95.2 6 61.6 82.4 6 55.6 97.3 6 56.9
0.693
132.3 6 67.7 121.7 6 55.7
0.104
72.4 6 53.4 101.9 6 61.3
0.098
140.0 6 60.1 103.5 6 54.6
0.035a
87.3 6 58.8 101.9 6 53.4
0.370
T1-2, stage T1 and T2 adenocarcinoma; T3-4, stage T3 and T4 adenocarcinoma. Analyzed by Student’s t test or analysis of variance test. a P , 0.05.
TABLE 3. Expression of MACC1 or MET (H-score) in Relation to Age at Diagnosis of IBD and Duration of Colitis to CAC Duration of Colitis, Yrs
Age at IBD diagnosis, yr #30 (n ¼ 21) 31–40 (n ¼ 8) .40 (n ¼ 9) Analyzed by analysis of variance test. a P , 0.05.
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MACC1
MET
Mean 6 SD
P
Mean 6 SD
P
Mean 6 SD
P
27.9 6 16.4 21.9 6 13.5 10.7 6 8.4
0.017a
143.9 6 60.5 105.6 6 81.9 125.8 6 50.5
0.342
108.6 6 53.9 78.4 6 53.4 78.7 6 78.0
0.319
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Expression of MACC1 and MET in IBD
FIGURE 3. Immunohistochemistry for MACC1 and MET in variants of CAC. A, MACC1 and MET expression in signet ring cell carcinoma (a and e, low magnification 40·; b and f, high magnification 200·) and in conventional CAC with signet ring component (c and g, low magnification 40·; d and h, high magnification 200·). Black arrow indicates the signet ring cell component. B, MACC1 and MET expression in mucinous carcinoma (a and e, low magnification 40·; b and f, high magnification 200·) and in conventional CAC with mucinous component (c and g, low magnification 40·; d and h, high magnification 200·). Black arrow indicates the mucinous component.
DISCUSSION We studied MACC1 and MET expression in normal colon, IBD-related inflammatory epithelium, dysplasia, and adenocarcinoma. For the first time, we demonstrated that MACC1 expression was increased in IBD-related dysplasia compared with normal colonic mucosa or IBD colitis. MACC1 expression was further upregulated in conventional CAC compared to dysplasia. Overexpression of MACC1 has been reported in a variety of human malignances, such as colorectal, nasopharyngeal, liver, pancreas, and lung cancer.10,12,23–26 Ours and other studies on MACC1 expression in precursors and invasive CRC have shown that overexpression of MACC1 mainly occurs at several crucial steps in the transition from adenoma to carcinoma.8,12 This study in IBD-related precursor lesions and cancer shows a similar stepwise expression pattern, which suggests MACC1 may be an important gene in CAC development similar to CRC. Interestingly, our study also showed that MACC1 expression was elevated in IBD-related dysplasia (a precursor of CAC), whereas in CRC a significant elevation of MACC1 expression started at the
carcinoma in situ stage,12 suggesting MACC1 may be expressed earlier in CAC development than in CRC development. Aberrant expression of genes at different time frames has been reported in CAC and CRC development. For example, adenomatous polyposis coli mutations, one of the earliest events in CRC pathogenesis, occur much later in the course of CAC cancer development.27–29 In contrast, P53 overexpression is a late event in CRC development but occurs early in the precursor stage (adenoma) in CAC. The earlier upregulation of MACC1 in IBD carcinogenesis provides evidence to further support that there may be different molecular mechanisms underlying the tumorigenesis of CAC and CRC. The exact mechanism underlying early expression of MACC1 in CAC development is unclear and further studies are needed. MACC1 is recently identified as a regulator for MET transcription.8 Thus, to elucidate the role of MACC1 expression in CAC, we investigated the expression of MET. Similar with the findings in conventional CRC that MET is not altered until the late stages of invasive adenocarcinoma,12,30 MET remained consistently www.ibdjournal.org |
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expressed from IBD inflammatory epithelium to dysplasia to adenocarcinoma. No parallel expression of MACC1 and MET in the development of conventional CAC may be related to different regulating mechanisms involving transcription and translation. This further supports our previous postulation that MACC1 may independently function in early tumor development.12 Overexpression of MACC1 seems to be associated with the differentiation in the liver and lung cancers.31 Studies have found that MET expression is not related to differentiation of CRC.13,30 At this time, there is no report on MACC1 expression and differentiation in colon cancer, even though its overexpression has been associated with poor prognosis in CRC. In this study, we found the expression of MACC1 and MET was not statistically different among well, moderately, and poorly differentiated conventional CAC. The data suggest that expression of MACC1 and MET might not contribute to differentiation of conventional CAC. In population-based cohorts, patient with IBD has an increased risk of developing CAC, in which UC increases the risk of CAC by 2.4 fold and CD by 1.9 fold.3,6 Male sex, young age at IBD diagnosis, long duration of IBD, and extensive colitis also increase the risk of CAC.6,32,33 Since only a few cases of CAC resulted from CD in our study, we did not differentiate the expression of MACC1 or MET in CAC developed from UC or CD, respectively. Our data showed that neither the expression of MACC1 nor MET was associated with patient age at the diagnosis of IBD, gender, duration of IBD, CAC location, or stage. These suggest that MACC1 and MET expression may not relate to these clinical characteristics that are associated with high risk of CAC. However, in agreement with the previous report,34 the patients with CAC in our study who were diagnosed with IBD at old age had a significant shorter duration of IBD (Table 3). In comparison with sporadic CRC, CAC has a similar dysplasia-carcinoma sequence. Multifocal dysplasia or widespread neoplasia occurs in the majority of CAC cases, which ranges from 40% to 100%.35–37 Multifocal dysplasia seems not to be associated with most of clinicopathologic characteristics,36 even through one study indicated that multifocal dysplasia was related to the early onset of IBD.34 In our study, 60% (31 of 51) CAC cases possessed multifocal neoplasia, in which MACC1 expression was significantly higher compared with the group without widespread neoplasia. The finding may further support our data from the immunohistochemistry study that MACC1 expression was greatly increased in dysplasia in comparison with inflammatory or normal tissue, indicating the role of MACC1 in CAC tumorigenesis. Mucinous and signet ring cell adenocarcinomas are 2 variants of adenocarcinoma that are more common in IBDrelated CAC.17,18 Evidence suggests that mucinous and signet ring cell carcinomas have distinct molecular characteristics from conventional CRC/CAC. The frequency of BRAF mutation in colorectal signet ring cell carcinoma is higher than in conventional CRC.38 HATH1, MUC2, and SOX2 gene are frequently expressed in signet ring cell carcinoma but rarely expressed in conventional adenocarcinoma.39,40 The frequency of CpG island
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methylation phenotype in signet ring cell carcinoma is significantly increased compared with conventional CRC as detected by quantitative real time polymerase chain reaction.41 Using microarray analysis, Kim et al42 identified 50 classifiers out of 62 differentially expressed genes in mucinous carcinoma compared with non-mucinous adenocarcinoma. In this study, we found that MACC1 and MET expression was not increased in signet ring and mucinous carcinoma cells. The data that MACC1 expression was increased in conventional CAC, but not in mucinous and signet ring cell carcinoma/differentiation, suggest that MACC1 may not play an essential role in the development of mucinous and signet ring cell carcinoma. There are a couple of limitations in this study. It was a retrospective study, which limited the quality and completeness of clinical data. For example, the information on the age of IBD onset and duration of disease when CAC was diagnosed were available only in 75% (38 of 51) patients. Out of these 38 patients, 10 (26%) developed CAC in less than 10 years of IBD. Although this data is similar to previous reports,34,43 our cohort had fewer patients who developed CAC in less than 8 years. One possible explanation might be our patients were diagnosed and followed at a large IBD center so that the delayed diagnosis of IBD was less likely. Another limitation for this study is although we have relatively large study size (total 57 patients with CAC), we had very few Crohn’s-derived CAC (only 5 patients with history of CD); therefore, we were unable to compare MACC1 or MET expression patterns between CAC derived from UC and CD. In conclusion, in this study, we demonstrate that MACC1 and MET expression patterns in conventional CAC are similar to that in conventional CRC, except for high expression of MACC1 in IBD-derived dysplasia. The comparative analysis of gene expression among conventional CAC, mucinous, and signet ring cell carcinoma/differentiation would augment our understanding of factors affecting differentiation in CAC, expand our knowledge on the etiology of these tumors, and possibly establish markers for early detection of CAC. Overexpression of MACC1 may be a potential marker not only for conventional CRC but also for the early diagnosis of conventional CAC.
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