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Molecular Oncology, Markers, Clinical Correlates |
Departments of Cytopathology [P. V., F. D. N., F. M., S. F., R. P. D.] and Surgery [C. B.], Regina Elena National Cancer Institute of Rome, 00161 Rome, and Department of Experimental Medicine and Pathology, University of Rome "La Sapienza" [P. L. A., G. T., U. D. T.], Rome, Italy
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Histological classification was carried out on H&E-stained slides. Adenomas (colonscopic polypectomies) were classified according to the WHO classification (1) : growth type (tubular, n = 18; tubular-villous, n = 71; villous, n = 11); and (2) and grade of dysplasia (low, n = 43; moderate, n = 40; high, n = 17). All of the tumors were adenocarcinomas (surgical specimens from colectomy); 31 tumors were localized in the right colon, and 69 in the left colon or rectum. Stage was defined on the surgical specimens according to the International Union Against Cancer tumor-node-metastasis (UICC-TNM 1997) classification. The study was performed on cases with tumor stages pT1 (n = 7), pT2 (n = 30), pT3 (n = 57), and pT4 (n = 6) and with lymph node involvement pN1 (n = 26) and pN2 (n = 13). No case revealed distant metastasis, so that all of the cases were classified as pM0. Six cases were well differentiated (G1), 50 were moderately differentiated (G2),and 44 were poorly differentiated (G3) adenocarcinomas. For each tumor, ANNM at a distance of 1 cm from neoplasia was analyzed. Ten cases of histologically normal colon mucosa from patients with diverticulosis were chosen as controls.
Patient Follow-Up Techniques
All of the patients were followed postoperatively for five years until July 1998. OS rates were calculated from the time of surgery to the date of death of disease. Forty-five patients died of cancer during this period, with a median follow-up of 36 months.
Immunohistochemical Assay
Sections (2–3 µm thick) were cut from the paraffin blocks and mounted on Super-Frost/Plus microscope slides (BDH Laboratory Supplies, Menzel, Germany). The immunoperoxidase method (14)
, using an avidin-biotinylated horseradish peroxidase complex (Vectastain Elite ABC; Vector Laboratories, Burlingame, CA), was used. Sections were incubated with nonimmune rabbit serum 1:100 dilution in Tris buffered saline (pH 7.6) at 37°C for 30 min, and, after intervening washes, with each primary antibody at concentrations ranging from 10 to 50 µg/ml for 60 min at 25°C in a moist chamber. To block endogenous peroxidase activity, sections were subsequently incubated with 3% hydrogen peroxidase in methanol for 30 min. After incubation with biotynlated rabbit antimouse antibody for 1 h at 25°C, immunoenzymatic reaction was developed using avidin-biotinylated horseradish peroxidase complex and 3'-3' diaminobenzidine (Biomeda Univeral Kit, Foster City, CA) as chromogen substrate. Other sections were incubated with an alkaline phosphatase-labeled reagent in buffer bath and developed using the chromogen system Fast Red (Dako Corporation, Carpinteria, CA). Nuclear counterstaining was obtained using Meyer’s hematoxylin. The anti-p53 was a mouse monoclonal antibody, clone DO-7 (Dako, Glostrup, Denmark); the anti-ki67 was the MIB-1 mouse monoclonal antibody (Immunotech, Hamburg, Germany); the anti-bcl-2 was clone 124 (Dako); the anti-c-myc was the clone 9E11 (YLEM, Rome, Italy) synthetic peptide representing residues 408–420 of the human c-myc protein; the anti-p-21-ras was clone NCC-RAS-001 (Dako Corporation, Carpinteria, CA).
Immunohistochemical Expression of FAS.
Expression of FAS was evaluated according to Rashid et al. (13)
. Tissue staining in coded sections was semiquantitatively graded for intensity as negative/weak, moderate, and strong. Staining distribution and intensity were determined by two observers independently (G. T., P. V.). Cases were deemed positive when moderate-to-strong granular cytoplasmic staining was present without observable nuclear staining.
Scoring Expression of p53, bcl-2, p21-ras, Ki67, and c-myc.
p53 immunostaining was evaluated as negative/weak, moderate, and strong, based on the intensity of the nuclear staining; all of the cases with a moderate or strong staining were considered positive for statistic evaluation. The immunohistochemical results for bcl-2, p21-ras, Ki67, and c-myc were expressed as a percentage of the positive number of cells counted on 10 microscopic cellular areas (x40) of adenoma, adenocarcinoma, and ANNM specimen. Positive cells were quantified and expressed as a percentage of the number of cells and were assigned to one of four categories—1: 0–5%; 2: 6–10%; 3: 11–20%; 4: >20%. bcl-2 was regarded as positive when more than 10% of the cells examined showed cytoplasmic staining. p21-ras was regarded as positive when more than 20% of the cells examined showed staining of the cytoplasm. c-myc was regarded as positive when more than 20% of the cells showed cytoplasmic and/or nuclear staining. Ki67 was regarded as positive when more than 20% of the cells examined showed nuclear staining. The cutoff value of 20% for p21-ras, c-myc, and ki67 and the cutoff value of 10% for bcl-2 were chosen arbitrarily for statistical requirements according to literature data (2
, 5
, 15, 16, 17, 18, 19)
. Because we had seen that bcl-2 expression had in all of the cases (normal mucosa, ANNM, adenomatous and adenocarcinomatous mucosa) a lesser percentage of cell expression compared with the other markers, and because it seemed important to evaluate it in adenomas in which, differently from the other markers, it was constantly expressed, we deemed it important for a correct statistical evaluation to lower the value of cutoff to 10%.
Statistical Analysis
The intensity of staining and the percentage of cells stained were compared with clinicopathological features using the 
2 of Fisher’s exact test when appropriate. The primary statistical outcome in the study of prognosis was the OS measured from the date of surgery. Univariate survival analysis was conducted using the Kaplan-Meier method, and the difference between survival curves was evaluated by the log-rank test. Multivariate analysis was performed using the Cox proportional hazard model with the stepwise forward selection of independent variables based on the likelihood ratio. To verify the prognostic impact of tumor-related and ANNM-related pathological parameters, two different multivariate analyses were performed. In the first model, p53, c-myc, bcl-2, FAS, and Ki67 status of colorectal adenocarcinoma was compared with tumor population details previously acquired (see "Materials and Methods"), which included patient age, tumor site, histological grade, size (pT stage), and nodal involvement (pN stage). In the second model, p53, c-myc, bcl-2, and FAS status of colorectal ANNM was compared with patient age, tumor site, histological grade, pT stage, and pN stage. The end point of both statistical evaluations was OS. Statistical significance was considered at a value of P < 0.05. All of the analyses were conducted using the BMDP software statistical package (20)
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Only 1 of 100 ANNM specimens showed a uniformly moderate FAS expression; the corresponding adenocarcinoma was FAS-positive. All of the other 99 cases of ANNM were negative.
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. Positive FAS immunostaining were seen in 7 (17%) of 40 cases of adenomas with moderate-grade dysplasia, in 9 (53%) of 17 cases of adenomas with high-grade dysplasia, and in 81% of adenocarcinomas (P < 0.0001; Fig. 1B
).
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Adenomas and Adenocarcinomas.
A positive p53 immunostaining was expressed in 21 (52.2%) of 40 adenomas with moderate-grade dysplasia, in all (100%) of the 17 with high-grade dysplasia (Fig. 1C)
, and in 63% of adenocarcinomas (P = 0.0001; Fig. 1D
). No adenoma with low-grade dysplasia revealed strong p53 immunostaining.
bcl-2 Expression
Normal Mucosa and ANNM.
Nine (90%) of 10 normal colon mucosa specimens expressed bcl-2 staining in a range of 6–10% in the cytoplasm of basal epithelial cells of the colorectal crypts, and, therefore, the cases were considered as negative with regard to the normal expression of bcl-2 in mucosa. The expression was uniformly low in case controls, whereas it seemed to increase for a number of cases (38%) and for a percentage of cells stained (range, 11–20%) in ANNM. Twenty-one (55.2%) of 38 cases of ANNM-bcl-2-positive demonstrated positive expression of bcl-2 in the corresponding adenocarcinomas.
Adenomas and Adenocarcinomas.
bcl-2 immunopositivity was more frequently detected in adenomas with low-grade dysplasia (63%; Fig. 1E
) compared with adenomas with moderate-grade dysplasia (39%) and adenomas with high-grade dysplasia (41%; P = 0.009). No significant difference in bcl-2 expression was observed between adenocarcinomas and adenomas with moderate or high-grade dysplasia.
c-myc Expression
Normal Mucosa and ANNM.
All of the 10 cases of normal mucosa revealed staining for c-myc-related protein, but less than 20% of the cells were positive. c-myc-positive cells were detected in the proliferative zone of crypts. Fifty-two cases of ANNM revealed more than 20% of the epithelial cells stained (P = 0.005; Fig. 1F
). Fifty (92%) of 52 cases of ANNM-c-myc-positive showed c-myc-positivity in the corresponding adenocarcinomas.
Adenomas and Adenocarcinomas.
Twenty-three (43%) of 43 adenomas with low-grade dysplasia, 22 (55%) of 40 adenomas with moderate-grade of dysplasia, and 11 (65%) of the 17 cases with high-grade dysplasia revealed more than 20% of epithelial cells stained for c-myc. Staining was confined to the nucleus in the majority of adenomas with low-grade dysplasia. Cytoplasmic staining occurred only in the epithelial cells of the adenomas with high-grade dysplasia. No correlation was found between c-myc immunostaining and the degree of dysplasia. Seventy-eight per cent of adenocarcinomas were c-myc-positive for cytoplasmic staining.
p21-ras Expression
Normal Mucosa and ANNM.
All of the 10 normal colon mucosa were stained for p21-ras, but less than 20% of the cells were positive. ANNM showed consistently enhanced immunostaining in 38 of 100 cases that revealed p21-ras expression in more than 20% of the epithelial cells (P = 0.01). Thirty-seven (97%) of 38 cases of ANNM-p21-ras-positive showed a p21-ras-positivity in the corresponding adenocarcinomas (Fig. 1G)
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Adenomas and Adenocarcinomas.
All of the adenomas showed diffuse and enhanced staining. Eighteen (42%) of the 43 adenomas with low-grade dysplasia showed more than 20% of the cells stained. Thirty (75%) of 40 cases with moderate-grade dysplasia and 14 (82%) of 17 cases with high-grade dysplasia were p-21-ras positive (P = 0.001).
Eighty-two adenocarcinomas showed immunostaining in more than 20% of the cells (Fig. 1H)
. Only one case expressed p21-ras in less than 5% of the neoplastic cells.
Ki67 Expression
Normal Mucosa and ANNM.
Ki67 expression was observed in all of the 10 cases of the normal mucosa in a range of 0–5% of the cells stained. A positive Ki67 immunoreaction was seen in the proliferative zone of the crypts in less than 5% of cells in all of the cases of ANNM.
Adenomas and Adenocarcinomas.
Ki67 was expressed more commonly in the colon of patients with adenomas having low-grade dysplasia; however, in these patients, positive immunostaining was detected in only less than 20% of the cells; therefore, the cases were considered negative. Conversely, with the increase of dysplasia there was an increase in the number of the cells stained (P = 0.02). Ninety per cent of adenocarcinomas showed more than 20% of the cells positive (P = 0.0001).
Relationship between Immunohistochemical Results and Clinical/Pathological Variables
A positive correlation in the primary adenocarcinomas was observed between pT stage and p53 expression (P = 0.003) and between pT stage and c-myc expression (P = 0.006). In ANNM, a significant correlation was seen between pT stage of the primary adenocarcinoma and c-myc (P = 0.003).
Survival Analysis
Univariate Analysis.
Univariate analysis of pathological/immunohistochemical parameters of the adenocarcinomas and OS revealed pT stage (P = 0.0001) and pN stage (P = 0.0001) to be statistically significant (Table 2)
. Other significant parameters were the degree of differentiation (P = 0.004) and p53 (P = 0.0007) expression in primary adenocarcinomas and p53 (P = 0.004; Fig. 2
), c-myc (P = 0.001; Fig. 3
), and p21-ras (P = 0.01) expression in ANNM.
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included the following variables: tumor site, histological grade, pT stage, pN stage, p21-ras, and p53 in the primary adenocarcinoma. The results indicate pT stage (P = 0.00001), pN stage (P = 0.00001), and p21-ras (P = 0.012) to be independent predictors of OS. The second model (Table 4)
included the following variables: tumor site, histological grade, pT stage, pN stage, p21-ras, c-myc, and p53 in the ANNM. A significant independent correlation with OS was demonstrated for pT stage (P = 0.00001), pN stage (P = 0.00001), c-myc (P = 0.013), and p21-ras (P = 0.009) in ANNM.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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FAS expression has already been studied in carcinomas of the breast, endometrium, and prostate; its overexpression has been associated with poor prognosis (10 , 11 , 21 , 22) . In these organs, FAS is regulated by sex steroid hormones. In fact, in hormone-dependent endometrial cells, FAS expression is a part of the estrogen-driven cellular response that leads to proliferation. Its linkage to proliferation is such that FAS expression is shown in proliferating cells of endometrial carcinomas that acquire hormone independence. In this study, we have demonstrated that FAS expression was correlated to the late phase of colon carcinogenesis but not to prognosis. Our data are in accord with Rashid et al. (13) , who demonstrated that FAS expression in colon carcinomas was correlated to high pT stage, lymph-node metastasis, and aggressive histological grade but did not seem to be an independent prognostic factor of OS. The mechanism of FAS expression in steroid receptor-negative tumors, such as colorectum carcinomas, is unclear. Probably, dysregulation of an unknown transcription factor or, alternatively, an increase of demand for fatty acid for membrane synthesis caused by dysregulated cell proliferation, could produce an increase in FAS expression and synthesis. Moreover, recent data have shown that the inhibition of fatty acid synthesis inflicts rapid lethal injury to carcinoma cells via activation of the cell death program, apoptosis (23) .
Apoptosis, or programmed cell death, represents a mechanism by which cells possessing DNA damage can be deleted. The regulation of apoptosis may be a complex process with many genes involved; some of these are c-myc, p53, and bcl-2. bcl-2 and c-myc have been assumed to cooperate in colorectal carcinogenesis. The c-myc protein is primarily involved in cell cycle progression, and its protein expression or overexpression, in the absence of proliferating factors, found in the G1 phase of cell cycle, induces the cell to undergo apoptosis (15 , 24 , 25) . Elevated levels of c-myc occur in up to 70–80% of colorectal cancer (4) . Our study regarding the c-myc expression demonstrated that c-myc immunoreactivity was associated with histologically normal ANNM. In this context, c-myc expression in ANNM may be regarded as a marker of early undetectable carcinogenesis rather than a marker of neoplastic progression, and it may be an important tool with regard to prognostic evaluation. However, we cannot exclude two possible explanations for expression of c-myc in ANNM. One is that the tissue directly adjacent to the tumor is not normal but has molecular alteration (an expansion of a predisposing genetic lesion). The other possibility is that the tumors secrete some transforming growth factors that could activate c-myc in adjacent mucosa without neoplasia (4) . To our knowledge, the innovative finding of our work is that the prognostic value of c-myc expression in ANNM has never been evaluated. p53 gene mutation is a common event in colorectal cancers and seems to be inactivated in up to 60% of colorectal carcinomas (16) . This inactivation eliminates the growth-suppressive function and can result in an overexpression of the protein. The p53 protein can cause cell cycle arrest but can also stimulate apoptosis (6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 , 20, 21, 22, 23, 24, 25) . p53 expression is strongly correlated to neoplastic progression.
We, in accord with other investigators, found a progressive increase of p53 in the adenomas-with-low-grade-dysplasia/adenomas-with-high-grade-dysplasia/carcinoma sequence (17 , 18) . Our data revealed also that p53 expression in the neoplastic cells and in the ANNM were correlated to prognosis, which confirmed previous data (19) .
The bcl-2 proto-oncogene is a known inhibitor of apoptosis that may allow the accumulation and propagation of cells with genetic alterations (26, 27, 28) . bcl-2 expression is up-regulated in most colorectal adenomas but only in some carcinomas; it has a role in oncogenesis, probably in the early phases of the adenoma-carcinoma sequence. Our data, in accord with Baretton et al. (17) , indicate that bcl-2 expression is characteristic of the early phase of colorectal oncogenesis, being overexpressed in ANNM. bcl-2 immunoreactivity in adenomas was inversely correlated with the degree of dysplasia. A negative correlation was found between OS and bcl-2 expression. This probably demonstrates that a low bcl-2 expression in carcinomas can be associated with a better clinical course.
Mutational activation of the ras oncogene has been estimated to occur in 10–20% of human tumors. Increased expression of ras protein (p21) expression has been demonstrated in a broad spectrum of human tumors (29 , 30) . Enhanced p21-ras expression can play a role in colon tumor progression. Our study, in accord with Jansson et al., reveals that p21-ras expression is an early event of carcinogenesis, in general, and of the colorectum, in particular (31) . p21-ras seems correlated, as is p53, with the neoplastic progression. A progressive increase of p21-ras expression was found in all of the phases of the sequence of colorectal carcinogenesis. Moreover, p21-ras seems significantly expressed in ANNM and seems correlated to prognosis (P = 0.009).
Surgery and chemotherapy are the elective therapies for colorectal carcinoma. The knowledge of proliferative markers may be useful to support these options or to create alternative tools (5) . Recent studies indicate that neoplastic colon cells that express high levels of muted bcl-2 and p53 are associated with the multidrug resistance to chemotherapy and particularly to fluorouracil (5) . Moreover it has been demonstrated that cerulenin, a potent inhibitor of FAS, induces apoptosis in neoplastic cells with high FAS expression (32 , 33) . Evaluation of FAS, bcl-2, and p53 in colorectal carcinoma may give important information about the progression of the disease and the optimal treatment.
Our data indicate, for the first time, that c-myc and p21ras expressed in ANNM are important prognosticators of colon carcinoma recurrence. Increased levels of c-myc, p21-ras, bcl-2 expression in ANNM may also reveal important information about neoplastic progression. Therefore, abnormal tissue, although found to be histologically normal, may reflect a "field type of effect" occurring in the early phases of tumorigenesis.
The study of ANNM can lead to important conclusions: (a) indication of more aggressive drug treatment for low-stage carcinoma; (b) systematic check-ups of all patients at risk for developing colon carcinoma; and (c) correct follow-up for patients with previous colon carcinoma.
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FOOTNOTES |
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1 To whom requests for reprints should be addressed, at Regina Elena National Cancer Institute, Department of Cytopathology, Viale Regina Elena, 291, 00161 Rome, Italy. Phone: 0039-06-49852050; Fax: 0039-06-49852342. 
2 The abbreviations used are: FAS, fatty acid synthase; ANNM, adjacent nonneoplastic mucosa; OS, overall survival.
Received 2/16/99; revised 9/10/99; accepted 9/27/99.
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