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Abstract

For subsets of colorectal adenocarcinoma (CRC) patients, nuclear accumulation of p53 (p53^nac) and Bcl-2 expression are prognostic indicators. To understand their role in the progression of CRC we evaluated 90 CRCs and their contiguous adenomatous components (CAdCs) for immunohistochemical expression of these markers. In general, p53^nac and Bcl-2 expression was significantly increased when comparing normal colonic epithelia to CAdCs and CRCs. Thirteen (14%) CAdCs that demonstrated p53^nac continued to express p53^nac in their contiguous CRCs. A similar trend was observed in Bcl-2 expression in that the majority of CAdCs expressing Bcl-2 continued to express it in their matching CRCs (39/44). Patients whose CAdCs and their contiguous CRCs demonstrate p53^nac had shorter median survival (35.9 months) than those patients whose CAdCs and CRCs did not (80.56 months). However, patients whose CAdCs had p53^nac and lacked Bcl-2 expression had the lowest median survival (15.74 months) when compared with patients whose CAdCs did not demonstrate p53^nac but had increased expression of Bcl-2 (71.77 months). These findings suggest that in those adenomas that demonstrate p53^nac but lack Bcl-2 expression, their contiguous CRCs are more likely to be aggressive as they progress.

Keywords

colorectal neoplasia adenomatous component p53 nuclear accumulation Bcl-2 expression

Development of colorectal adenocarcinoma (CRC) principally occurs via the adenoma–carcinoma sequence, a multistep process of tumor progression (Vogelstein et al. 1988). Adenomas are much more common than adenocarcinomas and only a few will become malignant (Burgart 2002). The important predictors of risk of developing invasive cancer from adenomas include the type and size of the adenoma and degree of dysplasia (Hamilton and Aaltonen 2000). However, morphological features of certain adenomas alone may not provide an accurate prediction of their risk of developing into aggressive invasive carcinomas. Therefore, use of molecular biomarkers may further aid in determining risk of developing invasive cancer in these premalignant lesions, thus impacting repeat surveillance decisions.

Some of the genes known to be involved in CRC progression from an adenoma to CRC are APC, β-catenin, K-ras, c-myc, SMAD4, and p53. Of these, p53 mutations (alterations) have been proposed to occur as late carcinogenic events. The wild-type p53 induces apoptosis in response to irreversible DNA damage and to an oncogenic stimulus (Efeyan et al. 2006), whereas mutant p53 inhibits apoptosis and confers an advantage for cell survival (Shaw et al. 1992; Lotem and Sachs 1993). Bcl-2 protooncogene acts as an inhibitor of apoptosis (Flohil et al. 1996). Transfection of p53 into lymphoid cells was shown to downregulate endogenous Bcl-2 expression, whereas simultaneous increase in the expression of Bax, a proapoptotic protooncogene, was observed (Miyashita et al. 1994; Selvakumaran et al. 1994). On the other hand, p53-induced cell death can be prevented by Bcl-2 expression (Hao et al. 1998); specifically the expression of Bcl-2 in p53-deficient mice is enhanced, with accompanying downregulation of Bax expression (Miyashita et al. 1994). These findings suggest that p53 and Bcl-2 may interact and have different roles in carcinogenesis.

Alterations in p53 and Bcl-2 were reported in early stages of CRC development (van den Berg et al. 1989; Purdie et al. 1991; Hague et al. 1994; Bosari et al. 1995; Mosnier et al. 1996; Yao et al. 1999; Krajewska et al. 2005). Furthermore, other studies as well as our own have demonstrated that nuclear accumulation of p53 (p53^nac) and Bcl-2 are important prognostic molecular markers specifically for a subset of patients with CRC (van den Berg et al. 1989; Purdie et al. 1991; Ofner et al. 1995; Mosnier et al. 1996; Manne et al. 1997, 1998, 2000; Kaklamanis et al. 1998; Yao et al. 1999). Although multiple genetic alterations have been reported during the development of colorectal neoplasia, known molecular events that contribute to the aggressive behavior of adenomas are limited. In the present study we attempted to determine the roles of phenotypic expression of p53^nac and Bcl-2 in contiguous adenomatous components (CAdCs) of CRCs to assess the aggressive behavior of their contiguous invasive carcinomas of the colorectum.

Materials and Methods

Patients and Tissues

The institutional review boards of the University of Alabama at Birmingham (UAB) Hospital approved this study. We identified a total of 620 CRC patients from the UAB Hospital who had undergone surgical resection for first primary CRC from 1981 through 1993. We obtained the medical records including surgical pathology reports of these patients, which were reviewed by two of the gastrointestinal (GI) pathologists (CS, NCJ) to ascertain key information. During our initial selection process, those patients who died within a week of their surgery; those who had inflammatory bowel disease; those patients whose archival tissues were not available; those patients with surgical margin involvement, unspecified tumor location, multiple primaries within the colorectum, or multiple malignancies; or those patients with family or personal history of CRC were all excluded from the study population. However, based on the information in patients’ charts, it may have been difficult to identify the familial vs. sporadic nature of the tumors; therefore, our patient populations can be described as ‘consecutive’ populations of CRC patients. To control for treatment bias, we included only those patients who underwent surgery as a therapeutic intervention and excluded patients who received any pre- or postsurgical therapies. Because the use of adjuvant chemotherapy was not widespread during the time frame of this study (1981–1993), we were able to obtain a large number of CRC patients who had not received adjuvant therapy. Only 90/620 cases had CRCs with contiguous adenomatous components. We obtained formalin-fixed paraffin-embedded archival tissue blocks of these cases from the files of The UAB–Surgical Pathology Department.

In our study, two GI pathologists (CS, NCJ) reviewed hematoxylin- and eosin-stained slides of all cases to determine the histomorphological type (tubular, tubulovillous, and villous) and the degree of dysplasia of the CAdCs (Hamilton and Aaltonen 2000). Degree of histological differentiation of CRCs was graded as well, moderate, poor or undifferentiated, as suggested by WHO (Hamilton and Aaltonen 2000). Both pathologists were blinded to the pathological evaluations of the other; however, if there were any discrepancies they were resolved by analyzing cases together to reach a final consensus. Subsequently, we pooled well- and moderately differentiated CRCs into a low-grade group and poor and undifferentiated tumors into a high-grade group (Compton et al. 2000). Pathological staging was performed according to the criteria of the American Joint Commission on Cancer (stages I, II, III, and IV) (Green et al. 2002). The International Classification of Diseases for Oncology (ICD-O) codes were used to specify anatomic location of the tumor (World Health Organization 1990). Anatomic sub-sites were grouped into the proximal colon (cecum, ascending colon, and proximal two thirds of the transverse colon), the distal colon (distal one third of the transverse colon, descending colon, and sigmoid colon), and the rectum.

Patients were followed by The UAB tumor registry until their death or the date of the last documented contact within the study time frame. The tumor registries ascertain outcome (mortality) information directly from patients (or living relatives) and from the physicians of the patients through telephone and mail contacts. This information is further validated against State Death Lists. Tumor registries update follow-up information every 6 months, and follow-up of our cohort ended in April 2007.

Immunohistochemical Analysis

Five-μm tissue sections were cut from paraffin blocks representative of normal and tumor with contiguous CAdC of each case and were mounted on Superfrost/ Plus slides (Fisher Scientific; Pittsburgh, PA). We cut the tissue sections 1–2 days prior to immunostaining to avoid potential problems in antigen recognition due to storage degradation of cut tissue sections on glass slides (Prioleau and Schnitt 1995; Jacobs et al. 1996). Immunostaining was performed as described in our earlier studies of antigen expression in different tissues (Manne et al. 1997, 1998, 2000). In brief, sections were melted and incubated overnight at 37C prior to staining. Tissue sections were then deparaffinized in xylene and subsequently rehydrated in graded alcohols. Sections were then transferred to a Tris-buffer bath (0.05 M Tris base, 0.15 M NaCl, and 0.01% Triton X-100, pH 7.6). Microwave antigen retrieval was performed on the sections for 7 min using citrate buffer 0.01 M, pH 6 (citric acid monohydrate 2.1 g, H₂O 1000 ml, adjusting pH with NaOH) for Bcl-2; however, no antigen retrieval was performed for tissue sections stained for p53^nac (Baas et al. 1996; Manne et al. 1997). Each section was treated with an aqueous solution of 3% H₂O₂ for 5 min to quench endogenous peroxidase activity. Each section was then incubated with 3% goat serum at room temperature for 1 hr to reduce nonspecific immunostaining. Tissue sections were then incubated with anti-human p53 (clone BP53-12.1, dilution 1:80; BioGenex, San Ramon, CA) and anti-human Bcl-2 (clone 124, dilution 1:60; Roche Diagnostics, Indianapolis, IN) primary monoclonal antibodies. Sections on which the primary antibody was not applied were utilized as negative controls.

Secondary detection was accomplished using a multispecies detection system (Signet Laboratories; Dedham, MA). Sections were exposed to biotinylated multispecies antibodies including anti-mouse antibodies for 20 min and then incubated with peroxidase-labeled streptavidin for 20 min. A diaminobenzidine tetrachloride supersensitive substrate kit (BioGenex) was used to visualize the antibody–antigen complex. Each section was then counterstained using hematoxylin, dehydrated using graded alcohols, and soaked in xylene before coverslipping.

The staining evaluation strategy of p53^nac and Bcl-2 expression was described in our earlier studies (Manne et al. 1997, 1998). Slides were independently examined by three pathologists (CS, NCJ, and WEG); however, if there was a discrepancy in individual scores all three pathologists reevaluated together by reaching a consensus agreement before combining the individual scores. As described in our earlier studies (Manne et al. 1997, 1998, 2000), we considered only tumor cells with distinct nuclear immunostaining for p53^nac as positive and considered the tumor positive only if nuclear accumulation was identified in ≥10% of all malignant cells in a tissue section. We chose this cut-off value of 10% positivity because this value showed the highest concordance between immunohistochemical detection of p53^nac and point mutations of the p53 gene as detected by single-strand conformational polymorphism analysis (95% of point mutations) (Grizzle et al. 1998).

A semiquantitative immunostaining score (ISS) for Bcl-2 was obtained as described previously (Manne et al. 1997, 2000; Chatla et al. 2005). In brief, each of the three IHC reviewing pathologists estimated the proportion of cells stained and the intensity of staining in the whole tissue section. Intensity of immunostaining of individual cells was scored onascale from 0(no staining) to 4 (strongest intensity). In addition, each pathologist estimated the proportion of cells stained at each intensity. The percent of cells at each intensity was multiplied by the corresponding intensity value to obtain an ISS that ranged from 0 to 4. Scores of the three investigators were combined to obtain an overall mean ISS. An ISS of ≥0.5 was chosen as a cut-off value for Bcl-2 expression as described earlier (Manne et al. 1997, 2000).

Results

Mean age of patients at the time of surgical resection was 66.5 years (range, 35–86 years). Based on the histopathological features, there were 53 tubular, 14 tubulovillous, and 23 villous types of CAdCs in this study. Further analysis of these histological types based on the degree of dysplasia revealed that 13/53 tubular, 2/14 tubulovillous, and 2/23 villous types were classified as CAdCs with high-grade dysplasia.

Phenotypic expression patterns of p53^nac and Bcl-2 are shown in Figure 1. None of the adjacent normal epithelial mucosa was positive for p53^nac, but Bcl-2 expression was observed in the basal crypts. In the CAdCs and CRCs, p53^nac was localized to the nucleus and Bcl-2 to the cytoplasm. Tubular CAdCs (6/53, 11%) were less positive for p53^nac than tubulovillous (3/14, 21%) and villous CAdCs (4/23, 17%), regardless of degree of dysplasia.

Expression levels of these two makers significantly increased as one moved from adjacent normal-appearing epithelium to CAdC to CRC (Figure 2). Clinicopathological features and expression profiles of p53^nac and Bcl-2 of 90 CRCs and their CAdCs are given in Table 1.p53^nac was observed in 13/90 (14%) CAdCs as compared with 35/90 (39%) of CRCs (Table 1). It is interesting to note that all CAdCs with p53^nac also exhibited p53^nac in their corresponding CRCs.

Increased cytoplasmic expression of Bcl-2 was observed in 44/90 (49%) CAdCs and in 62/90 (69%) CRCs (Table 1). Of 44 Bcl-2-expressing CAdCs, 39 (89%) showed Bcl-2 positivity in their corresponding CRCs. Only 5/90 (5.5%) cases showed loss of Bcl-2 expression in the CRCs with respect to their corresponding contiguous CAdCs, and all these cases were associated with increased expression of p53^nac (data not shown). Individual and/or concomitant expression of Bcl-2 and p53^nac did not show significant correlations with any of the prognostic features of CAdCs like grade of dysplasia, histological subtype, and extent of adenomatous component as well as the stage of their corresponding CRCs, suggesting that expression of both these markers are not dependent on these aggressive tumor features (data not shown).

Table 2 shows patient characteristics and their correlation with possible combinations of p53nac and Bcl-2 expression in CAdCs and in their corresponding CRCs. These correlation analyses suggested that the poor prognostic combination, p53^nac positive plus Bcl-2 negative, was predominantly observed in patients with stage III tumor; however, this association was not statistically significant. However, there was a significant association between the proximal colon site and this poor prognostic combination of p53^nac and Bcl-2 expression (χ² p value, 0.028) (Table 2). Also, the phenotypic expression of p53^nac and lack of Bcl-2 expression in CRCs (11/17, 65%) with adenomatous components were associated with regional lymph node involvement (data not shown).

Figure 1

Immunohistochemical staining of p53^nac and Bcl-2 expression in colorectal lesions. (A, B) p53^nac observed in the adenomatous component (thick arrow), but its adjacent normal colonic epithelium is lacking p53 staining (thin arrow). Note the transition to p53^nac positive in the adenomatous gland in A (thick arrow). (C, E) CRCs of the matching cases of A and B exhibiting p53^nac (thick arrows); p53^nac not present in uninvolved epithelium (thin arrows). (D) CRC exhibiting p53^nac (thick arrow) and its contiguous CAdC lacking p53 staining (thin arrow). (F) Bcl-2 cytoplasmic expression in normal colonic epithelial basal crypts (thick arrow); note the strong staining in lymphocytes (thin arrow). (G, H) CAdC (G) and its contiguous CRC expressing Bcl-2 (thick arrows) (H). (I) CRC lacking Bcl-2 expression (thick arrow), but note the strong staining of Bcl-2 in lymphocytes (thin arrow).

Median survival of patients with p53^nac in both CAdCs and their corresponding CRCs was markedly lower (35.9 months) than those patients whose CAdCs were negative but their CRCs were positive for p53^nac (74.51 months) or to those patients without p53^nac in both CAdCs and CRCs (80.56 months) (Table 3). Bcl-2 expression in these lesions did not show such a significant difference in their median survivals (Table 3); however, when the concomitant expression of p53^nac and Bcl-2 was considered, patients whose CAdCs were positive for p53^nac and lacked Bcl-2 expression had the lowest median survival (15.74 months) when compared with patients whose CAdCs did not demonstrate p53^nac but had increased expression of Bcl-2 (71.77 months) (Table 3). A similar trend in survival was observed in patients whose CRCs were categorized based on these two markers (50.89 vs 77.64 months) (Table 3).

Figure 2

Expression patterns of p53^nac and Bcl-2 in normal colonic epithelia, adenomatous components (CAdCs), adenocarcinomas (CRCs), and concomitantly expressing CAdCs and CRCs (CAdCs + CRCs). None of the adjacent normal colonic epithelium expressed p53^nac.

Discussion

Our study demonstrated that the phenotypic expression levels of p53^nac and Bcl-2 significantly increased in the transition from adjacent normal-appearing epithelia to contiguous CAdCs to CRCs. CAdCs that demonstrated p53^nac continued to express p53^nac in their contiguous CRCs, and a similar pattern was observed for Bcl-2 in that reversal of phenotypes of p53^nac or Bcl-2 expression was rare. Also, even though an abnormality in p53 is considered a late event, p53^nac was present in 14% of CAdCs. A shorter survival period was observed in patients in whom their CAdCs and their contiguous CRCs exhibited p53^nac as compared with those patients with CAdCs negative and CRCs positive for p53^nac or with patients whose CAdCs and CRCs did not demonstrate p53^nac. When CAdCs were immunoreactive for p53^nac but lacked Bcl-2 expression, patients’ tumors were more aggressive than those CAdCs that lacked p53^nac and increased Bcl-2 expression. These results suggest that once p53^nac has been detected and Bcl-2 is downregulated in an adenoma, its contiguous CRC behaves aggressively.

Coexistence of CAdCs in juxtaposition to CRCs supports the now well-accepted hypothesis that a subgroup of adenocarcinomas do develop from adenomas. However, only a small proportion of all adenomas progress to become invasive carcinomas (Burgart 2002). Alterations present at the early stages are likely to play a crucial role in CRC progression. Understanding of the molecular alterations underlying this progression will aid in determining the aggressive behavior of precursor lesions as well as CRCs.

The majority of studies in CRC have demonstrated that abnormal p53, detected by IHC, is a marker of poor patient survival; however, some studies found that p53^nac has limited value in predicting clinical outcome (as reviewed in Grewal et al. 1995; Manne et al. 1997). Specifically, others and our own prior studies have suggested that p53^nac is a poor prognostic indicator for patients with proximal but not distal colon tumors, specifically for non-Hispanic Caucasian patients (Manne et al. 1998; Diez et al. 2000). In this study, 39% of CRC showed p53^nac as compared with 14% of their contiguous CAdCs. A study by Yao et al. (1999) has also demonstrated a similar incidence of p53^nac in independent villous adenomas. Low p53^nac expression in CAdCs, as compared with the juxtaposed CRCs, indicates that p53^nac is a relatively late event in colorectal carcinogenesis as demonstrated by several other studies (van den Berg et al. 1989; Purdie et al. 1991; Hague et al. 1994; Bosari et al. 1995; Mosnier et al. 1996; Yao et al. 1999; Krajewska et al. 2005). All cases with CAdCs demonstrating p53^nac also exhibited p53^nac in their corresponding CRCs. This finding suggests that an adenoma exhibiting p53^nac is more likely to develop into an aggressive (invasive) carcinoma.

Table 1

Clinicopathological characteristics of the study cohort

Variable	Number of patients (%) (n = 90)
Age (years)
<65	36 (40)
≥65	54 (60)
Gender
Female	36 (40)
Male	54 (60)
Ethnicity
African-Americans	35 (39)
Non-Hispanic Caucasians	55 (61)
Tumor stage
I	23 (26)
II	30 (33)
III	28 (31)
IV	9 (10)
Regional lymph node status
Node negative	53 (59)
Node positive	37 (41)
Tumor location
Proximal colon	53 (59)
Distal colorectum	37 (41)
Tumor differentiation
Low grade	78 (87)
High grade	12 (13)
Adenomatous components(CAdCs)
p53^nac positive	13 (14)
Bcl-2 positive	44 (49)
Adenocarcinomas (CRCs)
p53^nac positive	35 (39)
Bcl-2 positive	62 (69)
Both CAdCs and CRCs
p53^nac positive	13 (14)
Bcl-2 positive	39 (42)

Expression of Bcl-2 in CRC has been shown to be a favorable prognostic marker in several studies (Ofner et al. 1995; Manne et al. 1997, 2000; Kaklamanis et al. 1998; Krajewska et al. 2005). Association of this anti-apoptotic protein with patients having a better survival may be due to the ability of Bcl-2 to be converted from a protector to a killer through interactions with other proteins (Lin et al. 2004) or being related to the as yet unexplained role of Bcl-2 in suppressing cell cycle entry (Linette et al. 1996; O'Reilly et al. 1996). As demonstrated in our earlier studies (Manne et al. 1997, 2000), the current study also showed a higher incidence of Bcl-2 expression in CRCs (69%) than in the corresponding CAdCs (49%); however, if Bcl-2 was expressed in the CAdCs it continued to be expressed in the majority of matching CRCs. Because Bcl-2 expression is linked to a favorable prognosis, this suggests that adenomas expressing Bcl-2 may similarly be tied to a favorable outcome even if CRC develops. These results suggest that Bcl-2 oncogenic protein may play a role in colorectal tumorigenesis, possibly in the early phases of the adenoma–carcinoma sequence.

Table 2

Correlation between clinicopathologic features and the concomitant expression patterns of p53 and Bcl-2

	Contiguous adenomatous components (CAdCs) (n = 90)					Colorectal adenocarcinomas (CRCs) (n = 90)
	p53^nac pos + Bcl-2 pos 6 (7%)	p53^nac pos + Bcl-2 neg 7 (8%)	p53^nac neg + Bcl-2 neg 39 (43%)	p53^nac neg + Bcl-2 pos 38 (42%)	χ² p value	p53^nac pos + Bcl-2 pos 18 (20%)	p53^nac pos + Bcl-2 neg 17 (19%)	p53^nac neg + Bcl-2 neg 10 (11%)	p53^nac neg + Bcl-2 pos 45 (50%)	χ² p value
Age (years)
<65	3 (50)	4 (57)	13 (33)	16 (42)		6 (33)	6 (35)	2 (20)	22 (49)
≥65	3 (50)	3 (43)	26 (67)	22 (58)	0.594	12 (67)	11 (65)	8 (80)	23 (51)	0.303
Gender
Female	1 (17)	3 (43)	17 (44)	15 (39)		8 (44)	6 (35)	5 (50)	17 (38)
Male	5 (83)	4 (57)	22 (56)	23 (61)	0.659	10 (56)	11 (65)	5 (50)	28 (62)	0.846
Ethnicity
African-Americans	1 (17)	5 (71)	14 (36)	15 (39)		7 (39)	8 (47)	6 (60)	14 (31)	Caucasians	5 (73)	2 (29)	25 (64)	23 (61)	0.211	11 (61)	9 (53)	4 (40)	31 (69)	0.321
Tumor stage
I	3 (49)	1 (14)	10 (26)	9 (24)		8 (44)	2 (12)	2 (20)	11 (24)
II	1 (17)	2 (29)	16 (41)	11 (29)		5 (28)	4 (24)	3 (30)	18 (40)
III	1 (17)	3 (43)	12 (31)	12 (32)		2 (11)	9 (52)	4 (40)	13 (29)
IV	1 (17)	1 (14)	1 (12)	6 (15)	0.554	3 (17)	2 (12)	1 (10)	3 (7)	0.224
Regional lymph node status
Node negative	4 (67)	3 (43)	26 (67)	20 (53)		13 (72)	6 (35)	5 (50)	29 (64)
Node positive	2 (33)	4 (57)	13 (33)	18 (47)	0.478	5 (28)	11 (65)	5 (50)	16 (36)	0.105
Tumor location
Proximal colon	1 (17)	6 (86)	22 (56)	24 (64)		7 (39)	12 (71)	6 (60)	28 (62)
Distal colon	5 (83)	1 (14)	13 (33)	7 (18)		10 (56)	3 (17)	4 (40)	9 (20)
Rectum	0 (0)	0 (0)	4 (11)	7 (18)	0.028	1 (5)	2 (12)	0 (0)	8 (18)	0.073
Tumor differentiation
Low grade	6 (100)	7 (100)	35 (89)	30 (79)		16 (89)	14 (83)	9 (90)	40 (89)
High grade	0 (0)	0 (0)	4 (11)	8 (21)	0.233	2 (11)	3 (17)	1 (10)	5 (11)	0.899

pos, positive; neg, negative.

Table 3

Patient survival based on the molecular marker expression status of colorectal lesions

Phenotypic marker	Adenomatous components	Carcinoma components	Number of patients	Median survival (months)
Individual markers
p53^nac	Pos	Pos	13	35.90
	Neg	Neg	55	80.56
	Neg	Pos	22	74.51
	Pos	Neg	None	None
Bcl-2 expression	Pos	Pos	39	80.56
	Neg	Neg	22	68.8
	Neg	Pos	24	75.69
	Pos	Neg	5	86.03
Combination of markers
p53^nac and Bcl-2	p53^nac pos + Bcl-2 neg		7	15.74
		p53^nac pos + Bcl-2 neg	17	50.89
	p53^nac neg + Bcl-2 pos		38	71.77
		p53^nac neg + Bcl-2 pos	45	77.64

Pos, positive; Neg, negative.

We conclude that the phenotypic expression of p53 and Bcl-2 progressively increased from adjacent normal-appearing epithelium to CAdC to invasive CRC. The presence of p53^nac in the CAdC is an indicator of aggressive behavior of colonic lesions, and these patients are more likely to develop aggressive invasive cancer. Although the additional prognostic pathological features including tumor budding, host responses (e.g., Crohn-like reaction), extramural invasion, vascular invasion, etc. will significantly influence the clinical outcome, evaluation of a combination of p53 and Bcl-2 expression in CAdCs correlates with patient survival. Therefore, it is of great value to assess the adenomas for the phenotypic expression of these markers, which may aid in patient follow-up and surveillance. Furthermore, these findings may lead to the development of new tools for cancer prevention.

Footnotes

Acknowledgements

This work is supported by the National Institutes of Health/National Cancer Institute (Grant RO1-CA-98932-01) and the Early Detection Research Network (Grant U24-CA-086359).

We thank Harpreet Singh, MD, for assistance in database creation and data processing. We thank the Tissue Procurement Facility of The UAB–Comprehensive Cancer Center for histological services.

References

Baas

van den Berg

Mulder

Clement

Slebos

Hamilton

Offerhaus

(1996) Potential false-positive results with antigen enhancement for immunohistochemistry of the p53 gene product in colorectal neoplasms. J Pathol 178: 264–267.

Bosari

Moneghini

Graziani

Lee

Murray

Coggi

Viale

(1995) bcl-2 oncoprotein in colorectal hyperplastic polyps, adenomas, and adenocarcinomas. Hum Pathol 26: 534–540.

Burgart

(2002) Colorectal polyps and other precursor lesions. Need for an expanded view. Gastroenterol Clin North Am 31: 959–970.

Chatla

Jhala

Katkoori

Alexander

Meleth

Grizzle

Manne

(2005) Recurrence and survival predictive value of phenotypic expression of Bcl-2 varies with tumor stage of colorectal adenocarcinoma. Cancer Biomark 1: 241–250.

Compton

Fielding

Burgart

Conley

Cooper

Hamilton

Hammond

. (2000) Prognostic factors in colorectal cancer. College of American Pathologists Consensus Statement 1999. Arch Pathol Lab Med 124: 979–994.

Diez

Medrano

Muguerza

Ramos

Hernandez

Villeta

Martin

. (2000) Influence of tumor localization on the prognostic value of p53 protein in colorectal adenocarcinomas. Anticancer Res 20: 3907–3912.

Efeyan

Garcia-Cao

Herranz

Velasco-Miguel

Serrano

(2006) Tumour biology: policing of oncogene activity by p53. Nature 443, 159.

Flohil

Janssen

Bosman

(1996) Expression of Bcl-2 protein in hyperplastic polyps, adenomas, and carcinomas of the colon. J Pathol 178: 393–397.

Green

Page

Fleming

Fritz

Balch

Haller

Morrow

(2002) American Joint Committee on Cancer. Cancer Staging Handbook from the AJCC Cancer Staging Manual. 6th ed. New York, Springer-Verlag.

10.

Grewal

Guillem

Klimstra

Cohen

(1995) p53 nuclear overexpression may not be an independent prognostic marker in early colorectal cancer. Dis Colon Rectum 38: 1176–1181.

11.

Grizzle

Myers

Manne

Srivastava

(1998) Immunohistochemical evaluation of biomarkers in prostatic and colorectal neoplasia. In Hanausek

Walaszek

, eds. John Walker's Methods in Molecular Medicine–Tumor Marker Protocols. Totowa, NJ, Humana Press, 143–160.

12.

Hague

Moorghen

Hicks

Chapman

Paraskeva

(1994) BCL-2 expression in human colorectal adenomas and carcinomas. Oncogene 9: 3367–3370.

13.

Hamilton

Aaltonen

(2000) Pathology and Genetics of the Tumors of the Digestive System. Lyon, France, IARC.

14.

Hao

Bishop

Talbot

(1998) Imbalance between proliferation and apoptosis in the development of colorectal carcinoma. Virchows Arch 433: 523–527.

15.

Jacobs

Prioleau

Stillman

Schnitt

(1996) Loss of tumor marker-immunostaining intensity on stored paraffin slides of breast cancer. J Natl Cancer Inst 88: 1054–1059.

16.

Kaklamanis

Savage

Whitehouse

Doussis-Anagnostopoulou

Biddolph

Tsiotos

Mortensen

. (1998) Bcl-2 protein expression: association with p53 and prognosis in colorectal cancer. Br J Cancer 77: 1864–1869.

17.

Krajewska

Kim

Kang

Welsh

Matsuzawa

Tsukamoto

. (2005) Analysis of apoptosis protein expression in early-stage colorectal cancer suggests opportunities for new prognostic biomarkers. Clin Cancer Res 11: 5451–5461.

18.

Lin

Kolluri

Lin

Liu

Han

Cao

Dawson

. (2004) Conversion of Bcl-2 from protector to killer by interaction with nuclear orphan receptor Nur77/TR3. Cell 116: 527–540.

19.

Linette

Roth

Korsmeyer

(1996) Cross talk between cell death and cell cycle progression: BCL-2 regulates NFAT-mediated activation. Proc Natl Acad Sci USA 93: 9545–9552.

20.

Lotem

Sachs

(1993) Regulation by bcl-2, c-myc, and p53 of susceptibility to induction of apoptosis by heat shock and cancer chemotherapy compounds in differentiation-competent and -defective myeloid leukemic cells. Cell Growth Differ 4: 41–47.

21.

Manne

Myers

Moron

Poczatek

Dillard

Weiss

Brown

. (1997) Prognostic significance of Bcl-2 expression and p53 nuclear accumulation in colorectal adenocarcinoma. Int J Cancer 74: 346–358.

22.

Manne

Weiss

Grizzle

(2000) Bcl-2 expression is associated with improved prognosis in patients with distal colorectal adenocarcinomas. Int J Cancer 89: 423–430.

23.

Manne

Weiss

Myers

Danner

Moron

Srivastava

Grizzle

(1998) Nuclear accumulation of p53 in colorectal adenocarcinoma: prognostic importance differs with race and location of the tumor. Cancer 83: 2456–2467.

24.

Miyashita

Krajewski

Krajewska

Wang

Lin

Liebermann

Hoffman

. (1994) Tumor suppressor p53 is a regulator of bcl-2 and bax gene expression in vitro and in vivo. Oncogene 9: 1799–1805.

25.

Mosnier

Perret

Vindimian

Dumollard

Balique

Perpoint

Boucheron

(1996) An immunohistochemical study of the simultaneous expression of bcl-2 and p53 oncoproteins in epithelial tumors of the colon and rectum. Arch Pathol Lab Med 120: 654–659.

26.

Ofner

Riehemann

Maier

Riedmann

Nehoda

Totsch

Bocker

. (1995) Immunohistochemically detectable bcl-2 expression in colorectal carcinoma: correlation with tumour stage and patient survival. Br J Cancer 72: 981–985.

27.

O'Reilly

Huang

Strasser

(1996) The cell death inhibitor Bcl-2 and its homologues influence control of cell cycle entry. EMBO J 15: 6979–6990.

28.

Prioleau

Schnitt

(1995) p53 antigen loss in stored paraffin slides. N Engl J Med 332: 1521–1522.

29.

Purdie

O'Grady

Piris

Wyllie

Bird

(1991) p53 expression in colorectal tumors. Am J Pathol 138: 807–813.

30.

Selvakumaran

Lin

Miyashita

Wang

Krajewski

Reed

Hoffman

. (1994) Immediate early up-regulation of bax expression by p53 but not TGF beta 1: a paradigm for distinct apoptotic pathways. Oncogene 9: 1791–1798.

31.

Shaw

Bovey

Tardy

Sahli

Sordat

Costa

(1992) Induction of apoptosis by wild-type p53 in a human colon tumor-derived cell line. Proc Natl Acad Sci USA 89: 4495–4499.

32.

van den Berg

Tigges

Schipper

den Hartog-Jager

Kroes

Walboomers

(1989) Expression of the nuclear oncogene p53 in colon tumours. J Pathol 157: 193–199.

33.

Vogelstein

Fearon

Hamilton

Kern

Preisinger

Leppert

Nakamura

. (1988) Genetic alterations during colorectal-tumor development. N Engl J Med 319: 525–532.

34.

World Health Organization (1990) International Classification of Diseases for Oncology. 2nd ed. Geneva, World Health Organization.

35.

Yao

Kajiwara

Kouzuki

Iwashita

Tsuneyoshi

(1999) Villous tumor of the colon and rectum with special reference to roles of p53 and bcl-2 in adenoma-carcinoma sequence. Pathol Int 49: 374–382.

p53 Nuclear Accumulation and Bcl-2 Expression in Contiguous Adenomatous Components of Colorectal Adenocarcinomas Predict Aggressive Tumor Behavior

Abstract

Keywords

Materials and Methods

Patients and Tissues

Immunohistochemical Analysis

Results

Discussion

Footnotes

Acknowledgements

References