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Clinical Cancer Research Vol. 12, 159-168, January 2006
© 2006 American Association for Cancer Research

Cancer Therapy: Clinical

Prognostic and Predictive Effects of Immunohistochemical Factors in High-Risk Primary Breast Cancer Patients

Nicolaus Kröger1, Karin Milde-Langosch2, Sabine Riethdorf3, Claudia Schmoor4, Martin Schumacher5, Axel R. Zander1 and Thomas Löning2

Authors' Affiliations: 1 Department of Bone Marrow Transplantation, Transplant Center; 2 Department of Pathology; and 3 Institute of Tumor Biology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; 4 Center of Clinical Trials and 5 Institute of Medical Biometry and Medical Informatics, University Hospital Freiburg, Freiburg, Germany

Requests for reprints: Nicolaus Kröger, Bone Marrow Transplantation, University Hospital Hamburg-Eppendorf, Martinistrasse 52, D-20246 Hamburg, Germany. Phone: 49-40-42803/5864; Fax: 49-40-42803/3795; E-mail: nkroeger{at}uke.uni-hamburg.de.


   Abstract
Top
 Abstract
Patients and Methods
 Results
 Discussion
 References
 
Purpose: To analyze prognostic and predictive effects of immunohistochemical factors within a randomized study of high-dose versus standard-dose chemotherapy in high-risk breast cancer with >10 involved lymph nodes.

Experimental Design: Histopathologic specimens in 188 of 302 patients were analyzed for Ki-67, p16, maspin, Bcl-2, Her2/neu, and p53.

Results: In a univariate analysis after adjustment for therapy, tumor size, and estrogen receptor, Her2/neu positivity (P = 0.001) was a negative and Bcl2 positivity (P = 0.003) was a positive prognostic factor for event-free survival. In a multivariate analysis, Her2/neu positivity (hazard ratio, 3.68; 95% confidence interval, 2.01-6.73; P = 0.0001) had a negative influence on event-free survival, whereas p53 positivity (hazard ratio, 0.57; 95% confidence interval, 0.34-0.95; P = 0.03) and Bcl2 positivity (hazard ratio, 0.35; 95% confidence interval, 0.19-0.64; P = 0.0006) were associated with a better event-free survival. Analyzing the predictive effect of the immunohistochemical factors, an interaction between p53 and treatment could be shown (P = 0.005). The hazard ratio for high-dose chemotherapy versus standard chemotherapy is estimated as 2.3 (95% confidence interval, 0.67-7.92) in p53-negative patients and as 0.46 (95% confidence interval, 0.2-1.07) in p53-positive patients, which indicates a superiority of high-dose chemotherapy in p53-positive patients and an inferiority in p53-negative patients. No interactive effect could be shown for the other factors.

Conclusions: Her2/neu and Bcl-2 are prognostic but not predictive factors in patients with high-risk primary breast cancer; p53-positive patients might benefit more from high-dose chemotherapy than from standard chemotherapy, and p53-negative patients might benefit more from standard chemotherapy than from high-dose therapy.

Despite the completion of several randomized high-dose chemotherapy studies in high-risk breast cancer, no clear recommendation concerning its use outside of clinical studies can be given. Although in several reports an at least numerical decrease in recurrence in the high dose–treated patients was reported, no significant benefit in overall survival after a relative short follow-up could be shown (1–7). Because of the low number of patients included in most trials, only major differences in outcome can be detected with a sufficient statistical power. In some studies, subgroups, such as young patients, estrogen receptor–negative tumors, G3 tumors, or negativity of Her2/neu, are in favor for high-dose chemotherapy (5, 7, 8).

Several molecular markers detected by immunohistochemical analysis, such as estrogen receptor and progesterone receptor, p53, Her2/neu, or Bcl-2, are found to be of prognostic value in breast cancer regarding outcome irrespectively of therapy, and, more recently, also as predictive factors for outcome after conventional or high-dose chemotherapy (9–12). In particular, most of the literature suggested that overexpression of Her2/neu and p53 are of predictive value for breast cancer patients treated with standard chemotherapy or high-dose chemotherapy regimens (8, 13–17). The Her2/neu receptor belongs to the epidermal growth factor receptor family of receptor tyrosine kinases, which are important mediators of cell proliferation and differentiation, and is expressed in ~20% to 30% of breast cancer patients (18). The tumor suppressor gene p53 is an important factor for cell cycle regulations and, moreover, it induces apoptosis after a toxic damage (19, 20). Other immunohistochemical factors that are of prognostic importance in breast cancer patients are Ki-67, p16, maspin, and Bcl-2. The Ki-67 protein is tightly regulated and depends on the proliferative status of a cell. It is present in the nuclei of proliferating cells but absent in resting cells (21). Maspin is a member of the serpin family of protease inhibitors, which inhibits tumor cell motility and invasion in cell culture and tumor growth and metastases in animal models. Only few reports about the prognostic effect in human breast cancer patients are available (22). p16 is a tumor suppressor protein, which avoids retinoblastoma protein hyperphosphorylation and therefore entry into S-phase by inhibition of the G1-cyclin D–dependent kinases, cdk4, and cdk6 (23, 24). Interestingly, p16 INK4a protein expression is associated with poor survival of breast cancer patients after CMF therapy (25). Bcl-2 is a member of a family of cytoplasmic proteins whose transcription is regulated by p53 and whose activity is regulated by phosphorylation by tyrosine kinase. Bcl-2 inhibits apoptosis by inhibiting the release of cytochrome c and apoptosis-inducing factor (12, 17, 18, 26, 27).

In the current study, we analyzed the prognostic and predictive value of these immunohistochemical factors as determined by immunohistochemistry of the primary tumors in 188 high-risk primary breast cancer patients with >10 involved lymph nodes who were treated within the randomized German Adjuvant Breast Cancer study comparing high-dose chemotherapy (CTM) with standard chemotherapy (7). This is one of the first randomized studies between high-dose chemotherapy and standard-dose chemotherapy investigating the prognostic and predictive effect of immunohistochemical markers.


   Patients and Methods
Top
 Abstract
 Patients and Methods
Results
 Discussion
 References
 
The German Adjuvant Breast Cancer study
Histopathologic specimens of 188 of 302 patients treated within a randomized study comparing high-dose chemotherapy study with standard-dose chemotherapy between 1993 and 2000 were reviewed for the immunohistochemical factors Ki-67, p16, maspin, Bcl-2, p53, and Her2/neu to evaluate their prognostic and predictive effects. The pathologic institutes of the participating centers were asked to send specimen for central pathologic review and for further histochemical analysis. No selection of the material was done and 188 specimens (62%) were evaluable for analysis. For the remaining 114 patients, no tissue was available. The study was a prospective randomized multicenter trial for patients with primary breast cancer with ≥10 positive axillary lymph nodes to investigate the effect of high-dose chemotherapy followed by autologous stem cell transplantation compared with standard chemotherapy. The study was reviewed by the ethics committee of each participating center and a written informed consent was obtained from all patients.

The details of the study are described elsewhere (7).

Immunohistochemical analysis
Immunohistochemistry. For detection of estrogen receptor, progesterone receptor, Ki-67, p16, p53, Bcl-2, maspin, and Her2/neu, automatic immunostaining on the DAKO Autostainer (DakoCytomation, Glostrup, Denmark) was done using the following antibodies and concentrations:

For estrogen receptor, progesterone receptor, p16, Bcl-2, erbB2, and Ki-67, antigen retrieval occurred by microwave treatment for 10 to 20 minutes in citrate buffer (10 mmol/L, pH 6.0). Blocking of endogenous peroxidase activity and application of the primary antibody were followed by the incubation with biotinylated goat anti-mouse/rabbit immunoglobulins and streptavidin conjugated to horseradish peroxidase (ChemMate Peroxidase/DAB Detection kit, DakoCytomation). 3,3'-Diaminobenzidine chromogen solution and a substrate buffer containing hydrogen peroxide served as substrate system. Tissue sections were counterstained by hematoxylin and were permanently mounted. As positive control, a tumor with known positive immunostaining for the analyzed protein was used, whereas the primary antibody was omitted for negative controls.

Microscopic semiquantitative evaluation. Evaluation of immunohistochemistry was done on serial sections by two investigators independently. In cases of interobserver variability, the slides were reexamined and a third person was consulted, if necessary. Only the invasive parts of the tumor were used for evaluation of immunostaining.

For estrogen receptor, progesterone receptor, p53, Bcl-2, maspin, and p16, every tumor was given a score in which the intensity of the staining (no staining = 0; low level = 1; medium staining = 2; strong staining = 3) and the percentage of stained cells (0% = 0; under 10% = 1; 10-50% = 2; 51-80% = 3; over 80% = 4) were multiplied. Using this system, the maximum score is 12 (with over 80% of the cells showing strong staining). For p16, nuclear and cytoplasmic staining was regarded as p16INK4a-specific staining and was evaluated (24). For estrogen receptor, progesterone receptor, p53, and Ki-67, only nuclear staining was regarded as positive. Maspin and Bcl-2 immunohistochemistry resulted in mainly cytoplasmic staining. In the case of Ki-67, only the percentage of positive nuclei in the tumor was determined, the intensity being always strong.

Scoring of Her2/neu results was done as suggested for the HercepTest (DAKO) using the following categories: 0, negative result or membrane staining in <10% tumor cells; 1+, weak and incomplete membrane staining in >10% tumor cells; 2+, weak or moderate, complete membrane staining in >10% tumor cells; 3+, strong, complete membrane staining in >10% tumor cells. Representative results of Bcl-2, Her2/neu, and p53 immunostaining are shown in Fig. 1.



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  		Fig. 1. Representative results of BCI-2 (A and B), Her-2/neu (C and D), and p53 (E and F) immunostaining in mammary carcinomas (x200). A, negative BCI-2 staining in tumor cells with positive adjacent lymphocytes (score: 0). B, strong cytoplasmic BCI-2 immunoreactivity in tumor cells (score: 12). C, weak membranous HER2/neu staining in tumor cells (because large areas of these carcinomas were HER2 negative; score: 1+). D, strong membrane staining in tumor cells (score: 3+). E, weak nuclear p53 immunoreactivity in <50% of the tumor cells (score: 2). F, strong p53 staining in >80% tumor cell nuclei (score: 12). T, tumor; L, lymphocytes.

 
Statistics
The end point for all analyses was event-free survival time. Event-free survival time was defined as time from primary surgery to the first event of failure (locoregional recurrence, distant metastases, and second cancer) or death without recurrence. For patients who did not experience the event of interest during following up, the time from primary surgery to the last documented follow-up was used as censored observation. The event-free survival rates were calculated according to the Kaplan-Meier method. The hazard ratio between different groups defined by treatment or prognostic factors with corresponding confidence intervals was determined by the Cox regression model. A check for assumption of proportional hazards in the Cox model has been done.

Data storage and analysis were done using the Statistical Analysis System. All data analyses were carried out according to a prespecified analysis plan. The categorization of the immunohistochemical factors in subgroups was predefined independently of results of analyses. The categorization of the standard prognostic factors—number of positive lymph nodes, degree of lymph node involvement, tumor size, tumor grade, and estrogen and progesterone receptor status—was predefined as used in the primary analysis and report of this study (7). In contrast to that report, estrogen receptor status and progesterone receptor status, as determined centrally, were used. All analyses are based on 157 patients for whom all immunohistochemical factors are completely documented. In a first step, the effects of the standard factors were analyzed in this patient population in a multivariate analysis. In a second step, it was analyzed whether the immunohistochemical factors have prognostic effects additionally to the standard prognostic factors in this patient population. In these analyses, the effects of the immunohistochemical factors were adjusted for treatment and for those standard factors, which had shown an effect in the former multivariate analysis at the 15% level. First, univariate analyses of the immunohistochemical factors were done. Then, a multivariate analysis of those immunohistochemical factors was done, which had shown an effect in the univariate analyses at the 10% level. In this analysis, the categories "weakly positive" and "strongly positive" were combined into one category "positive."

For investigating the predictive effect of the immunohistochemical factors, interactions between treatment and the factors Her2/neu, Ki-67, p53, p16, maspin, and Bcl-2 were examined. This was done using a separate Cox regression model for each of these factors, including the respective factor and separate treatment effects for both values (negative and positive) of the interesting factor, respectively. The interaction was tested by a Wald test of equality of the treatment effects in the resulting groups. Because of multiple testing, a significance level of 1% was used for these tests. Additionally, the corresponding multiplicative interactive effects were estimated with 99% confidence intervals. For a quantification of treatment effects in the subgroups defined by each of the factors, the hazard ratios between the treatment groups were estimated with 99% confidence intervals.

For p53, additional analyses were done, which were not prespecified in the analysis plan. For a further exploration of the results, a different categorization into p53-negative and p53-positive tumors was additionally analyzed.


   Results
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 Abstract
 Patients and Methods
 Results
Discussion
 References
 
Patient characteristics. For 157 patients, a complete data set of all investigated immunohistochemical factor was evaluable. The characteristics of the patients included in the current immunohistochemical study are listed in Table 1. Prognostic factors in patients with complete immunohistochemical analysis and in patients with incomplete immunohistochemical analysis are rather balanced, showing that patients included in this analysis were not selected because of their prognostic characteristics.


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  		Table 1. Characteristics of patients with complete and with incomplete immunohistochemical analysis (n = 157)

 
Table 2 shows the distribution of patients to the different categories of the immunohistochemical factors.


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  		Table 2. Immunohistochemical factors investigated and categorizing according to scoring

 
Effect of standard prognostic factors. The same statistical model as used for the multivariate analysis of the treatment effect adjusted for standard prognostic factors in the primary analysis and report of this study (7) was fitted to the patient population with complete immunohistochemical factors. In contrast to the original study (7), hormone receptors were determined by a central laboratory. In this population, only tumor size and estrogen receptor status show a significant effect on event-free survival in this multivariate analysis (results not shown in detail).

Univariate analysis of immunohistochemical factors. In univariate analyses of the immunohistochemical factors adjusted for the standard factors, such as tumor size, estrogen receptor status, and therapy (standard versus high-dose), Her2/neu, Bcl-2, and p53 showed a significant influence at the 10% level on the event-free survival (see Table 3; Fig. 2).


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  		Table 3. Univariate analyses of immunohistochemical factors, adjusted for therapy, tumor size, and estrogen receptor status (n = 157, 68 events)

 


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  		Fig. 2. A, effect of Her2/neu expression on tumor cells on event-free survival. B, effect of Bcl-2 expression on event-free survival. C, effect of p53 expression on event-free survival.

 
Multivariate analysis of immunohistochemical factors. The results of a multivariate analysis of those immunohistochemical factors showing an effect at the 10% significance level in univariate analyses, and that included the standard factors tumor size, estrogen receptor status, and treatment for adjustment, are displayed in Table 4. Her2/neu positivity and p53 and Bcl-2 negativity were associated with a significantly worse event-free survival.


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  		Table 4. Multivariate analysis of immunohistochemical factors adjusted for treatment, tumor size, and estrogen receptor status (n = 157, 68 events)

 
The result of the prognostic effect of p53 seems to be in contradiction to results in the literature, where usually either no prognostic effect of p53 is reported (8) or patients with p53-positive tumors show a worse prognosis than patients with p53-negative tumors (16, 28). An explanation for this result may be the scoring of p53 positivity in our study. Using a score, which includes the intensity of the staining and the percentage of stained cells, we found 30% negative and 24% strongly positive tumors. Furthermore, scores between 1 and 4 were noted as "weakly positive" and account for 46% of the patients. Combining the patients with weak and strong p53 immunoreactivity, the 70% prevalence of p53 accumulation in our study was higher than the prevalence of 20% to 40% reported in the literature (8, 16, 28–33). Therefore, an additional analysis of p53 defining only strong-positive tumors with a score larger than 4 as p53 positive (24%) was conducted. Using the same model as presented in Table 4, now patients with p53-positive tumors (score >4) and patients with p53-negative tumors (score ≤4) had a similar prognosis (hazard ratio, 0.98; 95% confidence interval, 0.54-1.80; P = 0.95), indicating overall no prognostic effect of p53 using this categorization.

Analysis of heterogeneous treatment effects in subgroups defined by the immunohistochemical factors (interactions). Table 5 shows the results of the analyses regarding the predictive effect of the different immunohistochemical factors (i.e., it was analyzed whether the treatment effect of high-dose chemotherapy versus standard-dose chemotherapy on event-free survival is heterogeneous in prognostic subgroups of patients defined by these factors). For a quantification of treatment effects in the subgroups defined by each of the factors, the hazard ratios between the treatment groups with 99% confidence intervals are displayed in Table 5. Additionally, the event-free survival rates in different subgroups are shown in Figs. 2, 3, 4, and 5.


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  		Table 5. Effect of treatment (high-dose versus standard chemotherapy) in subgroups defined by immunohistochemical factors (n = 157, 68 events)

 


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  		Fig. 3. A, effect of treatment (high-dose versus standard-dose chemotherapy) in patients with p53-negative tumors (negative = score 0). B, effect of treatment (high-dose versus standard chemotherapy) in patients with p53-positive tumors (positive = weak or strong positive, score 1-12). C, effect of treatment (high-dose versus standard chemotherapy) in patients with p53-negative tumors (negative = negative or weak positive, score 0-4). D, effect of treatment (high-dose versus standard chemotherapy) in patients with p53-positive tumors (positive = strong positive, score 5-12).

 


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  		Fig. 4. A, effect of treatment (high-dose versus standard chemotherapy) in patients with Her2/neu–negative tumors. B, effect of treatment (high-dose versus standard chemotherapy) in patients with Her2/neu–positive tumors.

 


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  		Fig. 5. A, effect of treatment (high-dose versus standard chemotherapy) in patients with Bcl-2–negative tumors. B, effect of treatment (high-dose versus standard chemotherapy) in patients with Bcl-2–positive tumors.

 
p53 is the only factor for which a significant interactive effect with treatment on event-free survival can be shown. The hazard ratio of high-dose chemotherapy versus standard-dose chemotherapy is estimated as 2.30 (99% confidence interval, 0.67-7.92) in p53-negative patients (score = 0) and as 0.46 (99% confidence interval, 0.20-1.07) in p53-positive patients (score = 1-12). The test for interaction resulted in P = 0.005. As also shown in Fig. 3A and B, this indicates a superiority of high-dose chemotherapy in p53-positive patients and an inferiority in p53-negative patients. However, it has to be mentioned that both confidence intervals are large and include the value 1 of equality of treatments.

Figure 3A and B shows that in the treatment group with high-dose chemotherapy, patients with p53-negative tumors seem to have a worse prognosis than patients with p53-positive tumors, again being contradictory to some studies in the literature. For further exploration, this analysis was also done using the other categorization mentioned above, defining only strong-positive tumors with a score larger than 4 as p53 positive (24 %). With this definition, there was still an interactive effect between p53 and treatment, although it is less pronounced with a P value of 0.06. This result is also displayed in Table 4 and Figs. 3C and D. Now, in the treatment group with standard-dose chemotherapy, patients with p53-positive tumors (score >4) had a worse prognosis than patients with p53-negative tumors (score ≤4; hazard ratio, 2.10; 95% confidence interval, 1.00-4.41; P = 0.05), and in the treatment group with high-dose chemotherapy, patients with p53-positive tumors (score >4) and patients with p53-negative tumors (score ≤4) had a similar prognosis (hazard ratio, 0.71; 95%, confidence interval, 0.29-1.76; P = 0.46).

Although Her2/neu positivity was a negative factor for the whole study population, no difference in the outcome could be observed between the different treatment approaches resulting in a worse outcome of Her2/neu–positive patients after standard chemotherapy and after high-dose chemotherapy (see Fig. 4).

Similar results were obtained for Bcl-2, which, if it was negative, resulted in a significant lower event-free survival for the whole study population. No difference, however, could be observed for Bcl-2 negativity between the treatment arms (see Fig. 5).


   Discussion
Top
 Abstract
 Patients and Methods
 Results
 Discussion
References
 
After completion of several randomized studies comparing high-dose chemotherapy with standard-dose chemotherapy in high-risk breast cancer, only a few have shown an advantage for high-dose chemotherapy, especially in those patients with >10 involved lymph nodes (4, 5, 34). The analysis presented in this article was undertaken to evaluate immunohistochemical factors, which might predict the outcome after standard or high-dose chemotherapy in these high-risk breast cancer patients, and, therefore, improve an individualization of the patient management.

In the first step of our analysis, we investigated the prognostic effect of the immunohistochemical factors Her2/neu, Bcl-2, p53, p16, maspin, and Ki-67 on event-free survival in a patient population, which was treated in a randomized German study protocol (7). In a univariate analysis after adjustment for treatment and the standard prognostic factors tumor size and estrogen receptor, only Bcl-2 and Her2/neu showed a statistically significant influence on event-free survival. It could be shown that patients with Her2/neu–positive or Bcl-2–negative tumors had a significant higher probability of relapse/death during follow-up. For immunohistochemical accumulation of p53, only a borderline significance (P = 0.07) could be detected, with patients with p53 weak-positive tumors having a better prognosis than patients with p53-negative or p53 strong-positive tumors. For other factors like Ki-67, p16, and maspin, no influence on event-free survival could be shown.

As prespecified in the statistical analysis plan, the categories weak positive and strong positive were combined into one category—"positive"—in the multivariate analysis of the immunohistochemical factors. In this analysis, Her2/neu, p53, and Bcl-2 were independent prognostic factors for event-free survival, showing that Her2/neu positivity and p53 and Bcl-2 negativity were associated with a significant worse event-free survival. The results for Bcl-2 and Her2/neu are in line with data of several other studies reporting a prognostic effect of these factors for breast cancer patients (9, 11). The result for the prognostic effect of p53 seems to be in contradiction to some studies in the literature (16, 31). Therefore, an additional exploratory analysis of p53 was conducted, defining only strong-positive tumors as p53 positive, which yields a p53-positive rate of 24% being more in line with the literature. Using this categorization, overall no prognostic effect of p53 could be shown.

The second aim of our study was to investigate whether the immunohistochemical factors have predictive effects (i.e., if treatment effects of high-dose chemotherapy followed by autologous stem cell transplantation compared with standard chemotherapy are different in subgroups defined by the immunohistochemical factors). Schrama et al. (35) investigated in a small randomized study of 81 patients the prognostic effects of Her2/neu, p53, Ki-67, and Bcl-2 and found no influence of these factors. Rodenhuis et al. (5) investigated in a randomized study in 885 patients the predictive effect of a range of factors. They found an interactive effect between Her2/neu and treatment, showing a beneficial effect of high-dose chemotherapy compared with standard-dose chemotherapy in Her2/neu–negative tumors but not in Her2/neu–positive tumors. They further investigated predictive effects of patient and tumor characteristics in the subgroup of 620 patients with Her2/neu–negative tumors. Subgroups of Her2/neu–negative patients for which high-dose chemotherapy may offer a benefit over standard-dose chemotherapy are younger patients, patients with positive progesterone receptor status, patients with p53-positive tumors, patients with tumors having a low mitotic activity index, and a low histologic tumor grade. However, these results may only be interpreted in an explorative sense because multiple tests have been done in unplanned subgroup analyses.

Her2/neu overexpression as a possible predictor for sensitivity to anthracycline-based chemotherapy has been shown by the Cancer and Leukemia Group B and National Surgical Adjuvant Breast and Bowel Project studies (13, 14). Our study confirmed the negative effect of Her2/neu overexpression in high-risk breast cancer patients who underwent high-dose chemotherapy and autologous stem cell transplantation as it has been recently shown by several investigators (8, 16, 17); however, this was also true for patients treated with standard chemotherapy. The interactive effect between Her2/neu and treatment found in the above mentioned randomized study by Rodenhuis et al. (5) was not detected in our study, although identical cut points for definition of Her2/neu–negative and Her2/neu–positive tumors have been used.

Only few reports are published about the prognostic and predictive value of Bcl-2 in breast cancer treatment. Van Slooten et al. (36) found some prognostic effects of Bcl-2 but no predictive value for neoadjuvant chemotherapy. The same was true in our study where we found a strong prognostic effect for Bcl-2, but no predictive value for high-dose chemotherapy. In Bcl-2–negative and in Bcl-2–positive patients, the event-free-survival rates of patients treated with high-dose and those treated with standard chemotherapy did not differ.

The major finding of our study was a predictive value of p53 on treatment strategies. Patients with tumors negative in p53 immunohistochemistry showed a higher event-free survival with standard chemotherapy compared with high-dose chemotherapy, whereas, in contrast, patients with p53-positive tumors had a higher event-free survival rate after treatment with high-dose chemotherapy. Our results obtained for the patients with high-dose chemotherapy are in line with data obtained by Bertheau et al. (37) who reported complete responses to high-dose chemotherapy in 8 of 14 patients with tumors containing p53 mutations. As a reason for the absence of response in the other six patients with p53 mutations, the authors suggested intratumorous heterogeneity of p53 status. However, these results of a predictive value of p53 on the effect of high-dose chemotherapy are in contrast to some other studies reported in the literature. Nieto et al. (8) reported on 146 patients who underwent high-dose chemotherapy according to the STAMP protocol, and they found no correlation between p53 mutation and relapse-free survival. In contrast, Somlo et al. (16) reported a negative influence of p53 positivity on overall survival in a multivariate analysis, including 115 high-risk breast cancer patients who underwent high-dose chemotherapy. Similar results were also reported by Hensel et al. (38) in 149 patients.

Heterogeneous results in different studies may be due to several sources. Applying different experimental protocols may influence the results: In our study, we used the DO-1 antibody, whereas, in other studies, mainly the DO-7 clone was used. However, recent studies have shown that DO-1 and DO-7 are comparable and seem to be superior to the rest of the p53 antibodies (39). Another important factor may be the definition of p53 positivity. Therefore, for analyzing interactive effects, the different categorization of p53 was also explored, defining only strong-positive tumors as p53 positive (24%). With this definition, there was still an interactive effect between treatment and p53, indicating a tendency of a superiority of high-dose chemotherapy only in p53-positive patients; however, this was less pronounced with a P value of 0.06. In the treatment group with standard-dose chemotherapy, patients with p53-positive tumors (score >4) had a worse prognosis than patients with p53-negative tumors (score ≤4). On the other hand, in the treatment group with high-dose chemotherapy, patients with p53-positive tumors (score > 4) and patients with p53-negative tumors (score ≤ 4) had a similar prognosis.

It is known that immunohistochemically assessed p53 overexpression is not optimal for the detection of p53 mutations because there are a few immunohistochemically negative cases carrying mutations and some others accumulating p53 protein independent of p53 mutations. For the two studies, which had shown a predictive value of p53 for conventional chemotherapy, DNA sequencing was used (24, 37).

Another issue for different results in different studies may be the drug composition of different chemotherapy regimens. Recently, it has been questioned whether tumor cell death from apoptosis after anticancer therapy requires the presence of wild-type p53. Several studies have shown that the loss of function of p53 does not necessarily alter sensitivity to DNA-damaging agents (32, 40). Therefore, it has been suggested that the effect of chemotherapy depends on p53 modulating activity: either directly by triggering apoptosis or by blocking cell cycle, or by both (23, 41). Thus, if p53 mutations prevent cells from inducing p53-triggered arrest of cell cycle, it might be possible that the chemotherapeutic drugs will rather target cycling cells (37). Whereas cells without p53 mutations are sensitive to anthracycline chemotherapy in vitro, in our study p53 mutations might prevent mitoxantrone from p53-triggered arrest of cell cycle, which allows targeting of high-dose chemotherapy to cycling cells. This might explain the increased sensitivity of p53-positive tumors to high-dose therapy in our study.

We conclude that Her2/neu and Bcl-2 are prognostic factors in patients with high-risk primary breast cancer. However, both factors have no predictive value on event-free survival after high-dose or standard chemotherapy. In contrast, using the monoclonal DO-1 antibody and the scoring system described above, p53-negative patients benefit most from standard chemotherapy, whereas p53-positive patients benefit more from high-dose chemotherapy followed by stem cell transplantation. To compare results between different laboratories, an adequate standardization of the reagents used for the assessment of p53 and careful reports about the chemotherapy regimens used in the study are mandatory. This study also shows the need to include biological factors prospectively within clinical treatment studies.


   Footnotes
 
Grant support: Erich und Gertrud Roggenbuck Foundation, Hamburg; German Krebshilfe; and Federal Ministry of Education and Research (The Center of Clinical Trials, University Hospital Freiburg).

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.

Received 6/21/05; revised 9/23/05; accepted 10/ 6/05.


   References
Top
 Abstract
 Patients and Methods
 Results
 Discussion
 References
 

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