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255 Apoptosis and lmmunohistochemical bcl-2 Expression in Colorectal Adenomas and Carcinomas Aspects of Carcinogenesis and Prognostic Significance Gustavo Bruno Baretton, M.D.' Joachim Diebold, M.D.' Gregor Christoforis, M.D.' Michael Vogt, M.o.' Christian Miiller, M.D.' Karin Dopfer, M.D.' Karin Schneiderbanger, M.D.' Michael Schmidt, M.O? Udo Lohrs, M.D.' ' Institute of Pathology, University of Munich, Munich, Germany Department of Surgery, Klinikum Grosshadern University of Munich, Munich, Germany. Tumor Register, Munich, Germany BACKGROUND. The Ocl-2 oncoprotein confers a survival advantage to cells by inhibiting programmed cell death (PCD) or apoptosis. Overexpression of bcl-2 probably plays a role in colorectal carcinogenesis. The aims of our study were to determine bcl-2 expression and PCD index in colorectal adenomas and carcinonias in correlation with p53 expression, Ki-67 index, and histopathology, and to test their prognostic significance in patients with colorectal carcinomas, METHODS. Immunohistologic staining for bcl-2 (MoAb clone 124), the proliferationassociated Ki-67 antigen (MoAb MIBl), and p53 (MoAb D01) was performed on archival material from 44 colorectal adenomas and 95 adenocarcinomas (TNM classifications pT2 and -3, pNO, and MO). The PCD was visualized by enzymatic detection of DNA fragmentation. RESULTS. bcl-2 was expressed in 86% of the adenomas and 67% of the carcinomas. Mean PCD and Ki-67 rates were 1.7 ? 0.14% and 35 2 13% in adenomas and 1.9 t 0.15% and 28 2 14% in carcinomas, respectively. In carcinomas, Ocl-2 expression was correlated with a low PCD index (< 1.5%; P = 0.005). Furthermore, a high Ki67 index (225%) was associated with a high PCD index (21.5%; P < 0.0001). p53 accumulation was seen in 16% of adenomas and in 42% of carcinomas, and did not correlate with bcl-2 expression or PCD index. In the univariate analyses, significantly longer disease free survival intervals were observed in three groups: all patients with bcl-2-positive carcinomas ( P < 0.05); the subgroup of carcinomas with Ocl-2 expression and low PCD index ( P = 0.037); and the subgroup of OcI-2positive and p53-negative carcinomas ( P = 0.021). In the multivariate analysis, however, only tumor stage and p53 expression were independent risk factors for prognosis. CONCLUSIONS. Our data indicate that Ocl-2 expression is characteristic of the early phase of colorectal carcinogenesis. Its physiologic function as an inhibitor of PCD is preserved in most colorectal carcinomas. whereas p53 is apparently not involved in the regulation of PCD in colorectal neoplasias. bcl-2 expression in colorectal carcinomas is associated with a better clinical course. This correlation became even more evident in the subgroups of patients with carcinomas that also had low PCD index or lacked p53 iminunoreactivity. Cancer 1996; 77955-64. (0 1996 American Caricer Society. This work contains parts of Gregor Christoforis' doctoral thesis. KEYWORDS colon, rectum, carcinoma, adenoma, bcl-2, protooncogene, apoptosis, proliferation, Ki-67, p53, immunohistochemistry, prognosis. Addrlsss for reprints: Dr Gustavo B. Baretton, Pathologisches lnstitut der Universitat, Thalkirchnerstr. 36, [I-80337 Munchen, Germany. T Received June 6, 1995; ievision received September 1, 1995; accepted September 1, 1995. 8 1996 American Cancer Society issue growth depends on both cell proliferation and the rate of cell death.',2Thus it is conceivable that neoplastic growth may be caused or promoted by factors inhibiting cell death. Apoptosis [programmed cell death (PCD)]is defined as a morphologic change culminating in cell death by a process clearly distinct from necrosis.3 The regulation of apoptosis 256 CANCER January 15,1996 I Volume 77 I Number 2 is central to morphogenesis during fetal development and to maintenance of tissue homeostasis during adulthood and seems to be of importance for neoplastic transformation in some bcl-2 is a protooncogene that is involved in the regulation of cell death by inhibiting apoptosis in many cell systems in physiologic and neoplastic condition^.'^^^' Its role as a new kind of oncogene that provides a growth advantage to a tumor cell by blocking PCD has been seen primarily in follicular B-cell lymphomas. In these lymphomas the bcl-2 gene usually located at 18q21 is moved into juxtaposition with powerful enhancer elements in the immunoglobulin heavy chain (IgH) locus at 14q32; the result is overproduction of bcl-2 mRNAs and their encoded proteins.*-“’ Increased bcl-2 expression has recently been reported also in epithelial malignancies, e.g., carcinomas of the lung,””2thyroid,I3 breast,I4 stomach,I5and ovaries.16 Colorectal cancer is one of the most common malignancies in western industrialized countries including the United States” and Germany.’* In these countries colorectal carcinoma is the second leading cause of cancer mortality. Pathogenetically, an adenoma-carcinoma sequence can be postulated for most colorectal carcinomas.’q-2’This concept has been supported by the discovery of molecular genetic changes that were correlated with certain histologic stages of this sequence.”.’3 As one important consequence of these genetic aberrations, cell proliferation is abnormally increased in these premalignant and malignant lesions of the colorectal epithe1ium,24-27 By contrast, only scarce information is available concerning the importance of perturbations in the regulation of cellular loss in colorectal carcinogenesis. In the normal colonic mucosa, cellular shedding is the principal physiologic mechanism of epithelial cell Before mechanical cell elimination takes place, however, apoptosis seems to be triggered in the uppermost nuclei at the edge of the crypts facing the lumen.2HA putative link between the current concept of molecular colonic carcinogenesis and the regulation of apoptosis might be found in the physiologic function of the TP53 tumor suppressor gene. The most important function of wild-type p53 protein is the maintenance of genome integrity by preventing a cell with DNA damage from entering the cell cycle.’”Subsequently, DNA repair can take place, or PCD will be induced if the DNA damage is irreversible. This concept was confirmed by the results of a recent study from our own group showing statistically significant correlations between p53 accumulation and DNA nondiploidy in adenomas and carcinomas, as well as between p53 immunoreactivity and an unfavorable prognosis in nonmetastatic colorectal carcinomas.” Recently, Sinicrope et al.32reported the first data concerning bcZ-2-expression and apoptosis in colorectal tumorigenesis: in 17 of 24 (71%) colonic adenomas and 14 of 21 (67%) adenocarcinomas bcl-2 immunoreactivity could be detected. However, no study has investigated the prognostic importance of bcl-2-expression, PCD index, and proliferation in colorectal carcinomas. By searching for correlations between these parameters and p53 accumulation, a better understanding of growth regulation including apoptosis in colorectal neoplasias might be achieved. We also looked for the prognostic significance of these parameters in nonmetastatic adenocarcinomas of tumor stages pT2 and pT3 according to the Union International Centre le Cancer (UICC) classification.33 MATERIALS AND METHODS Materials Formalin fixed and paraffin embedded tumor tissue from 44 colorectal adenomas (polypectomies) and 95 primary colorectal carcinomas was studied. Histologic classifications were carried out on hematoxylin and eosin (H & Elstained slides. The adenomas were randomly chosen from the files of the Institute of Pathology, University of Munich, and classified according to the following criteria: (1) size [<1 cm, n = 26 (59%),or 2 1 cm, n = 18 (41%)]; (2) histologic growth type according to the World Health Organization (WHO) classification34 [tubular, n = 19 (43%); tubulo-villous, n = 18 (41%); or villous, n = 7 (16%)];and (3) grade of atypia [low, n = 6 (14%);moderate, n = 23 (52%); or severe, n = 15 (34%)],or dysplasia [low, n = 29 (66%), comprising the cases with low and moderate atypia, or high, n = 15 (34%) according to the WHO clas~ification”~1. By applying these criteria, villous growth patterns (in tubulo-villous and villous adenomas) correlated in a statistically significant manner with tumor diameter 2 1 cm ( P < 0.01) and increasing grade of atypia ( P < 0.001) or dysplasia ( P < 0.001). The carcinomas comprised only cases with tumor stages pT2 (n = 49) and pT3 (n = 46) according to UICC with no evidence of lymphogenous or hematogenous metastases (pNO, MO).33All patients had undergone tumor resection at the Department of Surgery, Klinikum Grosshadern, University of Munich, between 1986 and 1988. From all cases clinical follow-up data were available up to 8 years after operation. The mean age of the patients at time of operation was 63.8 2 12.5 years, and the male/female ratio was 1.2:l. Thirty-one tumors were localized in the right colon, and 64 carcinomas in the left colon and rectum. Histologically all tumors were adenocarcinomas. According to the WHO classification the grade of differentiation was high in four cases (Gl), moderate in 75 cases (G2), and low in 16 cases (G3).R4 Methods Detection of apoptotic cells in paraffin sections DNA fragments of apoptotic cells were visualized by an enzymatic reaction using modified protocols of Gavrieli PCD and bcl-2 in Colorectal Neoplasias/Baretton et al. et al.'" and Ansari et al.35Paraffin sections were cut at 34 pm, mounted on silane-coated slides, and dried overnight at 56 "C. After deparaffinization in xylene and ethanols in descending concentrations, the sections were washed in double-distilled water (DDW) two times for 5 minutes. Subsequently, incubation with proteinase K solution containing 400 pI proteinase stock solution 125 mg proteinase K (Sigma, Deisenhofen, Germany) in 2.5 ml DDW] in 200 ml Tris-HC1 buffer (pH 7.0) was performed for 15 minutes at room temperature. After tour washes in DDW for 2 minutes the sections were incubated with terminal transferase mixture under a coverslip for 1 hour at 37 "C. The terminal transferase reaction mixture for 15 slides consisted of 15 pl digoxigenein-dUTP, 2.7 p l dATP, 90 pl terminal transferase reaction buffer containing cacodylate, 30 pl CoC12, 3 pl terminal transferase (all from Boehringer, Mannheim, Germany), and 306.3 p1 DPW. Approximately 25 pI/slide were applied. After lhree washes in TB buffer (300 mM sodium chloride and 30 mM sodium citrate in 1000 ml DDW, pH 7.5) for 5 minutes and one wash in buffer 1 (100 mM TrisHCI and 150 mM NaCl in 2000 ml DDW, pH 7.2) for 5 minutes, the sections were immersed in buffer 2 for 30 minutes. For preparation of this buffer 0.1 g of blocking reagent 0.5% (Boehringer) was mixed for 1 hour at 60 "C with 200 rril of buffer 1. Subsequently, two washes in buffer 1 for 5 minutes were followed by incubation with alkaline phosphataseconjugated Fab fragments of polyclonal sheep antidigoxigenin antibody (Boehringer) at a dilution of 1:ZOOO. Three washes in buffer 1 for 5 minutes and one 5minute wash in a buffer containing 100 mM Tris-HC1, 100 mM NaCI, (and 50 mM MgCI2 in 1000 ml DDW, pH 9.5 (= buffer 3 ) were then followed by incubation with nitroblue tetrazolium (NBT) solution, first for 30 minutes at room temperature in the dark and then overnight at 4 "C in the dark. For preparation of NBT solution 22.5 pl NBT stock solution containing 1 g NBT (Boehringer) in 9.33 ml 70% dimethylformamide solution (Sigma, St. Louis, MO) was mixed with 17.5 pl bromochloroindolylphosphate (Boehringer), 5 m M levamisole (Sigma), and 5000 pl buffer 3. On the next day after a wash in running tap water for 10 minutes the slides were counterstained in nuclear fast red for 10 minutts After two additional washes in DDW the sections were mounted with Aquatex (Merck, Darmstadt, Germany). As a positive control, sections of normal colonic mucosa or lymph nodes were used. Since the enzymatic reaction described labels both apoptotic cells and areas of necrosis, only those labeled cells were regarded as positive that showed additional characteristics of apoptosis, e.g., isolated localization within an intact cell complex without an inflammatory reaction. At least 3000 tumor 257 cells/case were evaluated. The results were expressed as positive cells/1000 tumor cells. As 1.5% represented the median value of all tumorous samples under study, a PCD index < 1.5% was considered low, and a PCD index > 1.5% high. bcl -2 and Ki-67 immunohistochemistty Sections (2-3 pm thick) were cut from the paraffin blocks and mounted on Super-Frost/Plus microscope slides (Menzel, Germany). After deparaffinization and rehydration the slides were placed in a Coplin jar of microwavecompatible plastic (TPX Brand, Germany) that was filled with 10 mM citrate buffer (pH 6.0). The jar was heated in a Panasonic microwave oven ("-4241) at 750 W for 5 minutes. Then, the fluid level was checked and evaporated buffer was supplemented. Two additional heating cycles of 5 minutes followed in the same way. The jar was then allowed to cool for about 1 hour at room temperature. The monoclonal bcl-2-antibody (clone 124) was purchased from Dianova (Hamburg, Germany). The lyophilisate was dissolved in Tris buffer (pH 7.5), and the antibody was used at a dilution of 1:60. The tissue sections were incubated with the primary antibody overnight at 4 "C. For immunohistochemistry of Ki-67, a proliferationassociated antigen, the slides were incubated with the monoclonal antibody MIB 1 (Dianova; dilution 1:15 for 30 minutes at room temperature) after 2x 10-minute microwave pretreatment."," The reactions were visualized by the APAAP-method, i.e., the employment of immune complexes of alkaline phosphatase and monoclonal antialkaline phosphatase.3' For this purpose the sections were incubated with the second rabbit/anti-mouse antibody (Dakopatts, Glostrup, Denmark) at a dilution of 1:25 for 30 minutes at room temperature and subsequently with the APAAP complex (Dakopatts) at a 1:50 dilution for 30 minutes at room temperature. To enhance the intensity of the final staining, these two steps were repeated twice. Fast blue-naphthole AS-MX phosphate (Sigma, Deisenhofen, Germany) served as chromogene. Finally, the slides were counterstained with nuclear fast red and mounted in Aquatex (Merck). Controls were performed by replacing the primary monoclonal antibodies with RPMI. Immunohistologic detection of p53 protein and interpretation of the results were performed as described previously using the MoAb DO 1 (Dianova).3' Analysis of the immunohistochemical reactions bcl-2. The immunohistochemical results for bcl-2 were recorded as positive if the tumor cells showed an unequivocally strong cytoplasmic immunoreaction. Cases 258 CANCER January 15,1996 I Volume 77 I Number 2 1.38%. The PCD index was graded as low in 22 cases (51%) and as high in 21 adenomas (49%). In nearly all cases single apoptotic cells were seen in the surface epithelium of nonneoplastic colonic mucosa. Moreover, scarce apoptotic cells were also detected by terminal transferase reaction in the germinal centers of intramucosal lymphoid follicles. bcl-2 immunohistochemistry FIGURE 1. Tubular colorectal adenoma showing single apoptotic nuclei marked enzymatically by labeling of DNA fragmentation using terminal transferase reaction (arrows). The labeled cells exhibit typical morphologic signs of apoptosis with condensation and margination of nuclear chromatin without associated inflammatory reaction (PCD index 6%; original magnification x400). with only equivocally faint staining were regarded as negative. Ki-67. According to the immunoreaction in at least 300 tumor cells/tumor the percentage of nuclei with a positive immunoreaction was determined (= Ki-67 index). A Ki-67 index was classified as high if it was >25%, which represented the median value in the carcinoma group. 1753. The results of the p53 imniunoreaction were recorded as positive it' a distinct nuclear staining was seen in at least 20% of the tumor cells. Cases with no or only single positive cells were regarded as negative. This classification was used because there is increasing evidence that p53 immunoreactivity in only a few tumor cells or even normal cells does not correlate with a TP53 mutation, but represents enhanced p53 activation due to physiologic DNA repair. Statistical analyses Mean parameter values ? standard error of the mean (SEM)were statistically compared by chi-square analysis. Survival analyses were performed by Kaplan-Meier curves and the Log rank test using the SAS/STAT statistical software (SAS Institute, Cary, NC). Multivariate analysis was carried out by the Cox model. P < 0.05 was regarded as statistically significant. RESULTS Colorectal Adenomas and Adjacent Nonneoplastic Tissues Apoptosis In 43 of 44 adenomas apoptotic cells could be visualized enzymatically (Fig. 1). The mean PCD index was 1.69 & Thirty-eight of 44 adenomas (86%) exhibited a positive cytoplasmatic immunohistochemical reaction for bcl-2 (Fig. 2A). The remaining six cases (14%) were bd-2 negative. In all positive cases more than 30% of the cells were immunolabeled. The pattern of immunostaining was not uniform within the adenomas; clusters of cells that reacted positively alternated with only faint staining areas within an individual case without correlation to tubular or villous architecture or histologic grade of the adenomas. Moreover, variations in staining intensity were also noted from case to case, although the reactivity of lymphocytes within the lamina propria or in the mantle zones of intraniucosal lymphoid follicles was similarly strong in all specimens. A positive cytoplasmatic bcl-2 reaction was also present in nonneoplastic epithelia at the bases of preserved crypts in the adjacent mucosa corresponding with the physiologic proliferative zone and in ganglion cells of the intramural neural plexus of the intestine. Ki-67 The mean Ki-67 index in adenomas was 34.9 2 13%. According to the defined classes of proliferation (see Methods section), in 9 of 44 cases (20%) the Ki-67 index was graded as low and as high in 37 of 44 cases (80%; Fig. 2B). N o statistically significant correlations could be established between Ki-67 index and size or histomorphologic features of the adenomas. In adjacent nonneoplastic intestinal mucosa a positive Ki-67 immunoreaction was seen in the proliferative zone of the crypts (mean Ki-67 index, 16.4 ? 6.7%). The differentiated surface epithelium never showed nuclear staining with the MIBl antibody. Correspondingly, the Ki67 index was graded as low in 90% of the norineoplastic colonic inucosa adjacent to the adenomas or carcinomas. In 10% of the cases, however, a high Ki-67 index with values up to 35% MIBl reactivity was observed in the nonneoplastic mucosa. P53 Thirty-seven of 44 adenomas (84%) were immunohistochemically p53 negative, whereas in the remaining 7 cases (16%) a nuclear p53 accumulation could be observed. In all positive adenomas, however, parts of the neoplasms remained negative. Nonetheless, in the immunoreactive areas staining was quite homogeneous and PCD and bcl-2 in Colorectal Neoplasias/Baretton et al. 259 FIGURE 2. (A) Tubulo-villous colorectal adenoma showing a positive cytoplasrnatic bcl-2 reactivity of different intensity in more than 30% of tumor cells (MoAb clone 124; APAAP, original magnification x100). (B) High Ki-67 index (42%) in the :same case, with nuclear staining for MoAb MI61 (APAAP, original magnification x200). reaction in a colonic adenocarcinorna with high PCD index (8.2%) (terminal traiisferase reai:tion, original magnification x400). FIGURE 4. Strong positive cytoplasmatic staining for bcl-2 in a moderately differentiated colonic adenocarcinorna with interspersed lymphocytes showing also a positive reaction (MoAb clone 124; APAAP. original magnification x400). diffuse. Statistically significant correlations could be observed between p53 expression and occurrence of villous growth pattern in the adenomas ( P <: 0.05), and histologic grade of atypia ( P < 0.01) or dysplasia (P < 0.001). The preserved nonneoplastic intestinal tissues showed no p53 immunoreactivity. tioned above, the PCD index was classified as low in 44 cases (47%) and high in 49 cases (53%; Fig. 3). FIGURE 3. Apoptotic nuclei labeled enzymatically by terminal transferase Colorectal Carcinomas Apoptosis The mean PCD index in the 93 carcinomas suitable for evaluation was 1.9 ? 1.45%. According to the limits men- bcl-2 immunohistochemistry Sixty-four of the 95 colorectal carcinomas analyzed (67%) showed a positive cytoplasmatic immunoreactivity for bcl-2 (Fig. 4). The rate of bcl-2-expressing cases was significantly lower in carcinomas than in adenomas (86%; P < 0.04). As in adenomas, the immunohistological bcl- 260 CANCER January 15,1996 / Volume 77 / Number 2 TABLE 1 bcl-2-ExpressionlPCDRate and Tumor Progression in Colorectal Carcinomas" TABLE 2 bcl-2 Expression and Apoptosis (PCD Rate) in Colorectal Carcinomas" PCD rate No progression Progression Total bcl-2 negiPCD rate 1.5% bcl-2 pos./PCD rate < 1.5% 9 26 13 10 22 (3R) 36 (62) Total 35 (60) 23 (40) 58 (100) Colorectal carcinomas (tumor stages pT2 and pT3, pN0, MO): Statistically significantl) l o w rate of tumor progression in the subgroup of patients harboring tumors with bci-2-expression (MoAb clime 124) and low rate of apoptosis (PCD < 1.5%) compared with patients with carcinomas lacking bcl-2 and hiEh rate of apoptosis IP = 0.0181. Data are numbers or numbers with perceni in parrntheses 'I TABLE 3 Ki-67 Index and PCD Rate in Colorectal Carcinomas PCD rate Total <1.5% >1.5% Ki-67 index < 25% Ki-67 index > 25% 27 16 13 36 40 143) 52 (57) Total 43 (47) 49 (53) 92 (1001 Colorectal carcinonias Itunior stages pT2 and pT3 pNO, MOI. Statistically significanr correlation lietween inimunohistochemical brl-2 expression IMoAb clone 1211 and Ki-6; index IMoAb MlBll IP < 0.0001 I. 2-staining features in carcinomas were heterogeneous. Specific topographic staining patterns, however, were missing. Moreover, there were no correlations between the results of bcl-2 immunostaining and patient age or sex, carcinoma site, tumor stage pT2 or pT3, or grading. Ki-67 The mean value of the Ki-67 index was 28 ? 14% in colorectal carcinomas. In 41 cases (44%) the Ki-67 index was classified as low and as high in 53 cases (56%).One case could not be evaluated. Thus, the rate of carcinomas with a high Ki-67 index (56%)was significantly lower than the rate of adenomas with a high Ki-67 index (80%;P < 0.01). P53 In 40 carcinomas (42%) a nuclear p53 accumulation could be observed. The remaining 55 cases (58%) showed no imniunohistochemical staining for p53. In comparison with adenomas (16%) the rate of aberrant immunohistochemical p53 expression in carcinomas was statistically significantly higher ( P < 0.005). Prognostic significance I n the univariate analysis patients with bcl-2-positive carcinomas showed a statistically significant longer disease Total 4.5% >1.5% bcl-2 negative bcl-2 positive 8 36 22 26 62 (67) Total 44 (48) 48 (52) 92 (100) 30 (33) ' Colorectal carcinomas (tumor stagespT2 and pT3, pNO, MOI. Correlation between immunohistothemical Drl-2 expression (MoAb clone 1211 and rate at apoptotic cells (PCDI determined by enzymatic detection of DNA fragmentation using terminal transferase (P= 0.005). Data are numbers or numbers with percent in parentheses. free time without evidence of tumor progression (no recurrence, no metastasis, no death of disease) than those with bcl-2-negative tumors ( P = 0.047; Fig. 1). In patients with bcl-2-expression and a low PCD index (n = 36), the tumors progressed less frequently (Table 1) compared with patients who had bcl-2-negative tumors and a high PCD index (n = 22). Moreover, these cases and patients harboring bcl-2-positivelp53-negative carcinomas exhibited significantly longer disease free intervals ( P = 0.037 and P = 0.0205; Fig. 5B,C)than the rest of the study group. In the stepwise multivariate analysis using the Cox model and including the parameters age, pT stage, and bcl-2 and p53 expression, however, only tumor stage ( P = 0.0011; risk ratio, 3.1) and p53 expression ( P = 0.0051; risk ratio, 2.6) proved to be independent prognostic risk factors. Correlations Between Results From the Different Methods in Adenomas and Carcinomas Neither in adenomas nor in carcinomas were statistically significant correlations observed between bcl-2 expression, Ki-67 index, or PCD index and morphologic features of the tumors or p53 expression. Whereas in adenomas only a trend toward a negative correlation between bcl2-expression and PCD index could be noted ( P = 0.068), bcl-2-expression in carcinomas was negatively correlated with PCD index in a statistically significant manner (n = 86; P = 0.005; Table 2). Furthermore, in carcinomas the grade of Ki-67 index correlated highly significantly with the grade of PCD ( P < 0.0001; Table 3). DISCUSSION The purpose of our investigation was to look for a possible relation between programmed (PCD) and immunohistochemical bcl-2 expression in colorectal adenomas and carcinomas. Moreover, correlations of these parameters with p53 accumulation and proliferative activity (Ki-67 PCD and bcl-2 in Colorectal Neoplasias/Baretton et al. 100% '1 90% 80% 70% 261 contrast, only scarce data concerning the importance of bcl-2 and apoptosis for gastrointestinal carcinogenesis are available: Lauwers et were able to show t7cl-2 overexpression in 13 of 16 cases (81%)with gastric epithelial dysplasia and 46 of 74 (72%) gastric adenocarcinomas." In gastric cancer bcl-2 expression was highly significantly associated with intestinal p h e n ~ t y p e . ' Sini~ crope et a1" recently described increased bcl-2 expression normal adenoma carcinoma in 17 of 24 (71%) colonic adenomas and 14 of 21 (67%) mucosa adenocarcinomas. To the best of our knowledge, howFIGURE 5. Percent frequencies of immunohistochemical bcl-2-expresever, ours is the first study to investigate systematically sion (>3O%), aberrant p53 imrnunoreactivity (>20%), high Ki-67 index the role of bcl-2 expression and enzymatically determined (>25%), and high rate of apoptosis (PCD index > 1.5%) in nonneoplastic PCD index in colorectal adenomas and carcinomas in colorectal rnucosa, colorectal adenomas, and nonmetastatic colorectal adcorrelation with proliferation (Ki-67 index), p53 accumuenocarcinomas (tumor stages pT2 and pT3). lation, and prognosis. For this purpose, adenomas with different histopathologic features (size, growth pattern, and grade of atypia/dysplasia) and nonmetastatic adenoindex) were to be tested as well as their prognostic sigcarcinomas of tumor stages pT2 and pT3 according to UICC" were investigated. Locally confined tumors were nificance. selected to exclude cases with probably more secondary bcl-2 is a recently recognized protooncogene that bcl-2 expression is a common genetic alterations. In good accordance with the observaregulates cell finding in low grade follicular B-cell lymph om as."^'" In tions published recently by Bosari et al.,4bour results these tumors a 14:18 translocation leads to bcl-2 overexshowed bcl-2 expression in 38 of the 44 (86%) adenomas pression. bcl-2 overexpression has also been found in and 64 of the 95 (67%) adenocarcinomas under study. and epithelial malignancies''-I6 lacking the This may indicate that bcl-2 activation is an early event 14: 18 translocation. The molecular mechanism underlyin colorectal carcinogenesis followed by other genetic ing bcl-2 overexpression in cells without t(14:18), howchanges, e g , p53 accumulation. I n carcinomas a highly ever, is still unknown. bcl-2 must form heterodimers with significant correlation between bcl-2 expression and a low PCD index was found ( P = 0.005; Table 2), whereas in other proteins, e.g., Bax, to carry out its physiologic functliom4"Thus, a disturbance of the posttranslational regulaadenomas only a trend ( P = 0.065) was noted. Thus, our tion of the bcl-2 gene might be present in carcinomas. data assessed by enzymatic labeling of apoptotic cells This theory is further supported by the finding of a dissoconfirm the results published by Sinicrope et al.,j2who ciation of Ocl-2 mRNA level and bd-2 protein expression, determined apoptosis by microscopic examination of which is present in reactive lymphocytes but not in neoconventionally H & E-stained slides. plastic conditions.' 1,39,41 These observations suggest that the physiologic funcIn the gastrointestinal tract the physiologic exprestion of bcl-2 for the regulation of apoptosis is preserved sion of bcl-2 protein is confined to the stem cells and the in most of the colorectal carcinomas under study. Howproliferative zone, i.e., the lower crypt of the intestinal or ever, in 36 of our cases (38%) such a close correlation colonic miicosa."~"' Obviously, the function of bcl-2 here was not recorded, supporting the notion that regulation is to protect the regenerative epithelial compartment of apoptosis is complex and bcl-2 is not the only factor from cell death. Although shedding of differentiated and involved in this process. For example, under certain cirmature cells to i.he luminal surface is the major mechacumstances the proliferation-stimulating oncogen c- myc nism of cellular loss in the gastrointestinal t r a ~ t , ~ " ~ ' is also known to stimulate apoptosis."' Furthermore, actiapoptosis seems to be triggered before mechanical cell vation of a cyclin-dependent kinase at an inappropriate elimination takes place. Thus, apoptosis also contributes time during the cell cycle can have the same physiologically to the normal intestinal cell turnThese observations support the notion that apoptosis over ,2H.:F.-I-I may also be initiated by incompatible or conflicting In the gastrointestinal tract carcinomatous growth growth signal^.^ This is in keeping with the finding of a is frequently preceded by preinvasive or preneoplastic highly significant correlation between a high proliferation epithelial lesions characterized by autonomic hyperprolirate (Ki-67index) and a high PCD index ( P < 0.0001, Table feration.'4-2' This observation led to the concept of the 3) in the carcinomas analyzed. Conversely, although bcladenoma-carcinoma sequence that can be postulated for 2 expression paralleled Ki-67 index, no significant correlamost colorectal Thus, most investigation between these parameters could be established tions have focused on this aspect of tuniorigenesis. By (Fig. 5). . - - CANCER January 15,1996 / Volume 77 / Number 2 262 '"1 btl- L 4c- 50 40 30 hrl-1 nci.lPCD high n = 11 30 20- I 10. 04. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0 8 1 2 1 8 2 4 3 0 3 6 4 2 4 8 5 4 < 8 0 Months A 9op-I, B 0 6 12 18 24 30 36 42 40 54 60 Months % 1W .......... . . . . . . . . . . . . . . . . . . . . . . . . . . . 80 70 T?, 60 ___ , , .....> .. , bcl-2pol.lpIln.l- "-38 ~ _ _ _ . ~ 50 - - 1 40 ,I --_ - - . _ > _ . .z1 ................... '3 30 20 ~ 10 aC.. 0 C 6 . 12 18 24 30 36 42 40 54 60 Months The surprising result of a significantly lower proliferative activity in carcinomas than in adenomas is in contrast to our observations reported previously but may be explained by the selection of only nonmetastatic carcinomas of tumor stages pT2 and pT3 in the present study.'"' Prognostically, patients with bcl-2-positive nonmetastatic colorectal carcinomas showed a better clinical course in the univariate analysis than those with bcl-2-negative tumors ( P < 0.05; Fig. 6A). The difference in disease free interval was even greater between the subgroups bcl-2positivellow PCD index and bcl-2-negativelhigh PCD index ( P = 0.037; Fig. 6B) where the physiologic situation is obviously preserved. These data agree with observations in other malignancies showing that bcl-2 expression is associated with a less malignant behavior. Also, among nonsmall cell lung cancer patients those with tumors expressing bcl-2 had a better prognosis than patients with bcl-2-negative carcinomas,'' and in B-cell lymphomas bcl-2-expression is frequently lost during transformation from low grade into high grade lymphomas.4' Almost all thyroid cancers showing at least poor differentiation are Ocl-2 positive, whereas undifferentiated thyroid carcinomas, which have the worst prognosis, are mostly bcl-2 negative.13 In ovarian carcinomas bcl-2 immunohistochemistry identified a subgroup of patients with p53- and FIGURE 6. Colorectal carcinomas (pT2 and pT3, pNO, MO): Kaplan-Meier curves showing significantly longer disease free survival rates for patients with bcl-2-positive carcinomas versus patients with bcl-2-negative tumors ( P < 0.05) (A); bcl-2-positive tumors with low PCD index versus patients with bcl-2-negative tumors with high PCD index ( P = 0.037) (B); and bcl2-positive and p53-negativetumors versus the rest of the group ( P = 0.021); 1 , bcl-2 positive and p53 positive (n = 28); 2, bcl-2 negative and p53 negative (n = 19); 3, bcl-2 negative and p53 positive ( n = 12). bcl-2-positive tumors who had a significantly better outcome than patients with p53-positive and bcl-2-negative carcinomas." I n the present study, patients with colorectal carcinomas showing bcl-2 expression but lacking p53 immunoreactivity exhibited the best clinical course ( P = 0.0205; Fig. 6C). In the multivariate analysis, however, only tumor stage and p53 expression were independent prognostic risk factors. In contrast to bcl-2 overexpression, which was particularly frequent in adenomas and decreased in carcinomas, p53 immunoreactivity showed an inverse behavior, indicating that loss of TP53 is obviously a later event in the adenoma-carcinoma sequence. In accordance with results from ovarian carcinomas, we found no correlation between bcl-2 and the PCD index on the one hand and p53 accumulation on the other hand in colorectal adenomas and carcinomas." Thus, according to our data there is no evidence for the hypothesis that functional p53 loss is linked to perturbations in the regulation of apoptosis in colorectal neoplasias."' This agrees with the findings of Bosari et aL4' but is contradictory to the data from Sinicrope et al.,3' who report a significantly inverse correlation between bcl-2 and p53 in immunostaining in adenomas. All these findings indicate that apoptosis plays a role PCD and b d 2 in Colorectal NeoplasiadBaretton et al. in tumor progression but that the regulation of apoptosis is complex and cannot simply be reduced to assessment of bcl-2 grne expression or p53 a c c ~ m u l a t i o nThe . ~ coniplete elucidation of these mechanisms remains an issue for further investigation. Furthermore, future research will have to show whether the pathways regulating apoptosis can be used as a target for novel approaches to anticancer therapy. In conclusion, our results clearly show that bcl-2 is strongly expressed in most colorectal adenomas and carcinomas, with the highest incidence in adenomas. bcl-2 expression is evidently characteristic of early stages in colorectal carcinogenesis followed by other genetic (changes like TP53 loss. 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