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Clinical Cancer Research Vol. 9, 5660-5665, November 15, 2003
© 2003 American Association for Cancer Research

Molecular Oncology, Markers, Clinical Correlates

Immunohistochemical and Mutational Analyses of ß-catenin, Ki-ras, and p53 in Two Subtypes of Colorectal Mucinous Carcinoma

Satoshi Ikeda1, Yosuke Shimizu1, Masahiko Fujimori, Yasuyo Ishizaki, Takeshi Kurihara, Yasutomo Ojima, Masazumi Okajima and Toshimasa Asahara

Department of Surgery, Division of Frontier Medical Science, Programs for Biomedical Research, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Purpose: The adenoma-carcinoma sequence theory is accepted in carcinogenesis of colorectal carcinoma. To elucidate the nature and genetic alterations in colorectal mucinous carcinoma (MC), we analyzed clinical and pathological characteristics of colorectal MC and nonmucinous carcinoma (NMC), and, furthermore, we compared the prognoses and the statuses of the Wnt signaling pathway, Ki-ras, and p53 in these two subtypes.

Experimental Design: Samples of colorectal MC obtained by surgical resections from 41 patients during the period from 1980 to 1999 were classified into fixed (FIX) type and floating (FLO) type (22 and 19 cases, respectively). The statuses of the Wnt signaling pathway and p53 protein were estimated by immunohistochemistry of ß-catenin and p53 proteins, respectively. The mutations in the Ki-ras gene were examined by direct sequencing.

Results: The prognosis of colorectal MC was poorer than that of NMC at both stage II and stage III (P = 0.0037 and <0.0001, respectively). The survival rate of patients with the FLO type of MC was lower than that of patients with the FIX type (P = 0.021). Although the results of immunohistochemistry of ß-catenin and mutational analysis of the Ki-ras gene in the two subtypes were not significantly different; the rate of positive nuclear staining of p53 was lower in the FLO type than in the FIX type (P = 0.04).

Conclusions: Colorectal MC, particularly the FLO type, has a more aggressive nature than does colorectal NMC. The FLO type of colorectal MC may develop through different mechanisms from those through which NMC and the FIX type develop.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Colorectal MC2 is one of the subsets of colorectal malignant tumors and is defined as an adenocarcinoma in which a substantial amount of mucin (>50% of the tumor) is retained within the tumor according to the WHO definition (1) . Although colorectal MC is a well-recognized entity, it is still not clear whether patients with colorectal MC have a poorer prognosis than do those with NMC (2, 3, 4, 5, 6) .

In colorectal carcinogenesis, the adenoma-carcinoma sequence theory, in which a variety of genes, including the APC, Ki-ras, and p53 genes, are mutated as the tumor progresses, is widely accepted (7) . However, the genetic mechanisms by which the mucinous phenotype arises is not well understood. It has been reported that the frequency of mutations in the Ki-ras gene in colorectal MC is higher than that in colorectal NMC (8) . In contrast, low frequency of mutations in the p53 gene or overexpression of p53 protein and loss of heterozygosity in the DCC gene in colorectal MC have been reported (9, 10, 11, 12) . In addition, MUC2, which is one of the glycosylated proteins, was reported to be overexpressed in colorectal MC, but not in NMC (12 , 13) . These findings suggest that MC develops through different genetic mechanisms from those through which NMC develops.

According to the WHO definition, colorectal MC shows two types of growth pattern (1) . In this study, we designated these two types of MC as the FIX type and FLO type, respectively. We compared the prognosis of colorectal MC with that of colorectal NMC. In addition, we compared the prognoses between the two subtypes of MC and analyzed the frequencies of alterations in the Wnt signaling pathway and nuclear accumulation of p53 by immunohistochemistry and the mutations in the Ki-ras gene by direct sequence analysis.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients and Pathological Classification of Colorectal MC.
We analyzed 813 samples from patients with colorectal NMC and 41 samples from those with colorectal MC who had undergone operations in the Department of Surgery II, Hiroshima University Hospital, during the period from 1980 to 1999. Informed consent was obtained from the patients. All cases of MC did not fulfill the Amsterdam criteria for clinical hereditary nonpolyposis colorectal cancer diagnosis. The 41 cases of colorectal MC were pathologically classified into two subtypes: FIX and FLO types (22 and 19 cases, respectively). According to the WHO definition, the FIX type was defined as glands lined by a columnar mucus-secreting epithelium together with interstitial mucin, and the FLO type was defined as chains or irregular clusters of cells surrounded by mucus (1) . The histological stages were determined according to the Union International Contre Cancer Tumor-Node-Metastasis Classification (14) .

Immunohistochemistrical Analysis of ß-Catenin and p53.
Four micrometers of each formalin-fixed and paraffin-embedded tumor section were deparaffinized and subjected to antigen retrieval by microwaving in 10 mM of sodium citrate (pH 6.0) for 30 min. The sections were incubated with the anti-ß-catenin monoclonal antibody (Transduction Laboratories, Lexington, KY) at a dilution of 1:250 and with the anti-p53 monoclonal antibody (DO-7; Novocastra Laboratories, Newcastle, United Kingdom) at a dilution of 1:100 for 12–16 h at 4°C and stained by the avidin-biotin method using a Histofine kit (Nichirei, Tokyo, Japan). The ß-catenin and p53 immunostaining results for the nucleus were evaluated by comparing the staining intensities of adjacent nontumor cells. Immunostaining was defined as positive when >10% of tumor cells were stained more strongly than were nontumor cells (15 , 16) .

DNA Extraction and Sequencing Analysis of the Ki-ras Gene.
Genomic DNA was extracted from each tumor of 10-µm-thick formalin-fixed and paraffin-embedded tumor section as described previously (17) . Extracted DNA was amplified by PCR in the regions containing codons 12, 13, and 61 of the Ki-ras gene. The primers used for the PCR were as follows: 5'-TTTTTATTATAAGGCCTGCTGAA-3' and 5'-CATATTCGTCCACAAAATGA-3' for codon 12 and 13 and 5'-ACCTGTCTCTTGGATATTCT-3' and 5'-TGATTTAGTATTATTTATGGCA-3' for codon 61 of Ki-ras. The PCR products were purified from agarose gel, and sequence analysis was performed using BigDye Terminator Cycle Sequencing FS Reaction kits with an ABI PRISM 310 sequencer (Applied Biosystems, Forester, CA).

Statistical Analyses.
Clinicopathological data were statistically analyzed using the ÷2 test. Student’s t test was used for comparison of means. Survival was analyzed using the Kaplan-Meier method, and differences between curves were tested using the log-rank test. P = 0.05 was accepted as significant. All of statistical analyses were performed using Statview software (Version 5.0; Abacus Concepts, Berkeley, CA).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Patients.
The clinicopathological characteristics of the 854 patients with colorectal carcinoma are shown in Table 1Citation . Forty-one patients (4.8%) were diagnosed pathologically as having colorectal MC. There were no significant differences between the characteristics of patients with MC and those with NMC, except for stage of disease.


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  		Table 1 Clinicopathological characters of MC and NMC

 
Comparison of Prognoses of MC and NMC.
The overall survival rates of the 41 patients with colorectal MC and the 813 patients with colorectal NMC were compared by Kaplan-Meier survival analysis. The overall 5-year survival rate of patients with colorectal MC was 43.1%, and that of patients with colorectal NMC was 79.4% (P < 0.0001; Fig. 1ACitation ). Furthermore, the prognoses of colorectal NMC and MC were compared at stage II and stage III. Survival curves of patients with colorectal MC and NMC in stage II and stage III indicated that colorectal MC had a significantly poorer prognosis than did NMC in either stage (P = 0.0037 and P < 0.0001, respectively; Fig. 1, B and CCitation ).



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  		Fig. 1. Kaplan-Meier survival plots for patients with colorectal NMC and those with MC. Survival rates of patients with all stages (A), stage II (B), and stage III (C) are shown. The survival rate of patients with MC was significantly worse than that of patients with NMC in each category (all stages, P < 0.0001; stage II, P = 0.0037; stage III, P < 0.0001).

 
Comparison of Prognoses of the FIX and FLO Types of MC.
Colorectal MC was classified into two subtypes, FIX type (22 cases) and FLO type (19 cases). In H&E staining, the FIX type showed differentiated carcinoma cells surrounding regularly along mucin lakes and the FLO type showed single cells or clusters of small numbers of cells, often with signet ring cells, in mucin lakes (Fig. 2, A and B)Citation . There were no significant differences between the clinicopathological characteristics of patients with the FIX type and patients with the FLO type (Table 2)Citation . The 5-year survival rates were 68.4% for the patients with the FIX type of colorectal MC and 21.4% for the patients with the FLO type of colorectal MC (P = 0.021; Fig. 3Citation ).



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  		Fig. 2. Results of immunohistochemistry of ß-catenin and p53 in the FIX and FLO types of colorectal MC. A, H&E staining of the FIX type showed a mucin lake surrounded by differentiated carcinoma cells. B, H&E staining of the FLO type showed a cluster of cells floating in a mucin lake. Nuclear positive staining of ß-catenin was observed in all cases of the FIX type (C) and in 94.7% of the cases of the FLO type (D). Accumulation of p53 in the nucleus was detected in 63.6% of the cases of FIX type (E) and in 31.6% of the cases of FLO type (F). Scale bar, 50 µm.

 

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  		Table 2 Clinicopathological characters of the FIX type and the FLO type of MC

 


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  		Fig. 3. Kaplan-Meier survival plots for patients with the FIX and FLO types of colorectal MC. Five-year survival rates of patients with the FIX type were 68.4% and 21.4%, respectively (P = 0.021).

 
Immunohistochemistry of ß-Catenin.
We performed immunohistochemistry of ß-catenin to detect the alterations in the Wnt signaling pathway in colorectal MC. Nuclear positive staining of ß-catenin was observed in 40 (97.6%) of the 41 cases of colorectal MC, whereas ß-catenin staining was localized to the cell membranes of adjacent normal epithelial cells in all cases of colorectal MC. All of the 22 cases of FIX type and 18 of the 19 cases of FLO type (94.7%) showed nuclear positive staining of ß-catenin (P = 0.92; Fig. 2, C and DCitation , and Table 3Citation ).


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  		Table 3 Status of ß-catenin immunohistochemistry, Ki-ras mutations and p53 immunohistochemistry in MC

 
Mutational Analysis of the Ki-ras Gene.
Mutational analysis of codons at 12, 13, and 61 in the Ki-ras gene by direct sequencing analysis showed mutations in 12 (29.3%) of the 41 cases of colorectal MC (Table 4)Citation . All of these nucleotide alterations were accompanied by changes in the amino acid sequence at each codon. Eight cases (36.4%) of the FIX type and 4 cases (21.1%) of the FLO type had mutations in the Ki-ras gene (P = 0.46; Table 3Citation ).


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  		Table 4 Mutations in the Ki-ras gene of MC

 
Immunohistochemistry of p53.
Nuclear accumulation of p53 was observed in 20 (48.8%) of the 41 cases of colorectal MC (Fig. 2, E and F)Citation , and accumulation of p53 was detected in 14 (63.6%) of the FIX type cases and in 6 (31.6%) in the FLO type cases (P = 0.04; Table 3Citation ).


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we assessed the clinicopathological and genetic features of colorectal MC, and we classified colorectal MC into two subtypes: the FIX type and FLO type, according to the WHO definition (1) . The FIX type of colorectal MC have glands lined by a columnar mucus-secreting epithelium, together with interstitial mucin, and the FLO type of colorectal MC has chains or irregular clusters of cells surrounded by mucus. We also analyzed frequencies of alteration in the Wnt signaling pathway, the Ki-ras gene mutations and p53 overexpression in these two subtypes of colorectal MC.

A comparison of the prognoses of colorectal MC and NMC showed that the 5-year survival rate of patients with MC was worse than that of patients with NMC (43.1% and 79.4%, respectively; P < 0.0001). This result is consistent with previously reported results (2, 3, 4) . It has been suggested that the poorer prognosis of colorectal MC is related to the stage of the disease and to the extent of spread, but not to the mucinous histological type (3 , 4) . Indeed, the patients with colorectal MC examined in the present study received surgery at a more advanced stage than did patients with NMC. However, our results showing a poorer prognosis of MC than that of NMC at both stage II and stage III suggests that MC has a more aggressive nature than does NMC. In the present study, we also compared the prognosis of the two subtypes of colorectal MC. The prognosis of the FLO type of colorectal MC was significantly poorer than that of the FIX type, even though there was no significant difference between their clinicopathological characteristics. Taken together, the results suggest that colorectal MC, particularly the FLO type, has a higher potential of biologically aggressive behavior than does colorectal NMC. Interestingly, the 5-year survival rate of patients with the FIX type of colorectal MC was similar to that of patients with colorectal NMC (68.4% versus 79.4%, respectively; P = 0.1447).

The genetic background of tumorigenesis in colorectal MC has not been elucidated. In this study, we analyzed dysfunction in the Wnt signaling pathway, the Ki-ras gene mutations, and accumulation of p53, which are key molecules in the colorectal adenoma-carcinoma sequence theory (7) . In the adenoma-carcinoma sequence theory of colorectal tumorigenesis, the APC gene is defined as a gatekeeper because mutations and loss of heterozygosity in the APC gene are observed in 70–80% of cases of colorectal carcinoma, even in an early step of the colorectal carcinogenesis (7) . APC is a component of the Wnt signaling pathway, which regulates tissue development and cell growth (18) . APC and Axin promote GSK-3ß-dependent phosphorylation of ß-catenin, resulting in regulation of ß-catenin stability (19 , 20) . Activating mutations in the ß-catenin gene and inactivation of APC and Axin lead to accumulation of ß-catenin in the cytoplasm, and the accumulated ß-catenin translocates to the nucleus, thereby promoting the activity of T-cell factor/lymphoid enhancer binding factor family through interaction with each other (18) . The activation of T-cell factor/lymphoid enhancer binding factor family members results in an increase in transcription of cyclin D1 and c-myc (21 , 22) . In the present study, we analyzed dysfunction of the Wnt signaling pathway by immunohistochemistry of ß-catenin. Nuclear accumulation of ß-catenin protein was observed in 100% and 94.7% of the cases of FIX and FLO types of colorectal MC, respectively, showing similar frequency to that of colorectal NMC (90% of NMC; data not shown). This result indicates the possibility that both two types of colorectal MC, as well as colorectal NMC, arise through dysfunction in the Wnt signaling pathway, perhaps including mutations in the APC gene or ß-catenin gene. We also performed sequencing analysis of the Ki-ras gene. Mutations at codons 12, 13, and 61 in Ki-ras, which lead to an oncogenic activation of Ki-ras (23) , were observed in 36.4% and 21.1% of the FIX type and FLO type cases, respectively. The frequency of the Ki-ras gene mutations in the FIX type cases was similar to that reported previously in NMC cases (24) , whereas the frequency of the Ki-ras gene mutations in the FLO types cases was lower, but the difference was not statistically significant. Further analysis of the Ki-ras gene using many colorectal MC samples will be needed to determine whether there is a different frequency between the two subtypes of colorectal MC. Furthermore, accumulation of p53 in the nucleus was observed less frequently in the FLO type than in the FIX type by immunohistochemistry (31.6% and 63.6%, respectively; P = 0.04). Although nuclear accumulation of p53 is thought to generally indicate the existence of dysfunction of p53, including mutations in the p53 gene, we did not perform sequencing analysis of the p53 gene in this study. In previous studies, mutations in the p53 gene or accumulation of p53 was observed in 50–70% of colorectal carcinomas, a similar frequency to that observed in the FIX type of colorectal MC (9 , 25 , 26) . The finding in the present study that the FLO type showed a poorer prognosis and a lower frequency of p53 abnormality is interesting in view of the fact that there have been descriptions in many reports of an association between p53 mutations and tumor aggressive behavior in several types of malignant tumor, including colorectal cancer (25 , 26) . The frequencies of abnormalities in the Wnt signaling pathway, Ki-ras, and p53 in the FIX type of colorectal MC are similar to those in colorectal NMC, suggesting that the FIX type of colorectal MC develops through mechanisms similar to those through which colorectal NMC develops. On the other hand, the frequency of alterations in p53 in the FLO type of colorectal MC was lower than that in NMC, suggesting that the FLO type occurs through a different pathway from that through which NMC occurs. It is necessary to note that we compared the frequency of abnormalities in the Ki-ras gene and p53 with the previously reported data, because we did not perform the analyses of NMC in this study. Further study is needed to understand the mechanisms by which the FLO type of colorectal MC develops.

Finally, the most interesting findings in this study are that the FLO type showed a poorer prognosis than did the FIX type and NMC, simultaneously showing a different frequency of alteration of p53.


   ACKNOWLEDGMENTS
 
We thank the staff of Research Center for Molecular Medicine, Hiroshima University Faculty of Medicine for the use of their facilities.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Dr. Satoshi Ikeda, Department of Surgery, Division of Frontier Medical Science, Programs for Biomedical Research, Graduate School of Biomedical Sciences, Hiroshima University, 1–2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan. Phone: 81-82-257-5222; Fax: 81-82-257-5224; E-mail: sikeda{at}hiroshima-u.ac.jp

1 These authors contributed equally to this work. Back

2 The abbreviation used are: MC, mucinous carcinoma; NMC, nonmucinous carcinoma; APC, adenomatous polyposis coli; FIX, fixed; FLO, floating. Back

Received 3/17/03; revised 8/11/03; accepted 8/14/03.


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 RESULTS
 DISCUSSION
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Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
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