Aims: To look for correlations between expression of cell cycle regulatory proteins p34cdc2, p21WAF1, and p53 in node negative invasive ductal breast carcinoma, or between these proteins and clinicopathological parameters, and to assess their prognostic value.
Methods: Immunohistochemistry using formalin fixed, paraffin wax embedded sections from 94 breast carcinomas. Adjacent benign epithelial breast tissue was available in 74 cases. Median follow up was 72 months.
Results: Nuclear and cytoplasmic p34cdc2 expression was seen in 80 and 62 tumours, respectively; nuclear expression was seen in adjacent benign epithelium in 12 cases. p21WAF1 and p53 were positive in 48 and 21 tumours, respectively. High expression of p34cdc2 in neoplastic nuclei was associated with higher histological grade and p53 expression, but not with tumour size, steroid receptor status, patient age, menopausal status, recurrence, metastasis, disease free survival (DFS), or overall survival (OS). p34cdc2 in tumour cytoplasm was associated with p34cdc2 nuclear positivity, high tumour grade, and DFS in univariate but not multivariate analysis. In contrast, p34cdc2 expression in benign tissue independently predicted DFS and OS in univariate and multivariate analysis. Expression of p53 was associated with high tumour grade and negative steroid receptors, but not with recurrence, metastasis, DFS, or OS. p21WAF1 expression was not associated with the examined parameters.
Conclusions: p34cdc2, p21WAF1, and p53 expression does not predict outcome in node negative breast carcinoma, although p34cdc2 expression in benign tissue is related to prognosis. The association between p34cdc2 and p53 implicates p53 in G2–M cell cycle checkpoint control, possibly via mediators unrelated to p21WAF1.
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- bn, benign
- CDK, cyclin dependent kinase
- DFS, disease free survival
- ER, oestrogen receptor
- MPF, mitosis or maturation promoting factor
- OS, overall survival
- PR, progesterone receptor
- TBS, Tris buffered saline
- TC, tumour cytoplasm
- TN, tumour nuclei
Breast carcinoma is a heterogeneous disease with variable clinical behaviour. Assessing prognosis is very important for both the prediction of clinical outcome and patient management. Although the lymph node status is a major prognostic parameter,1 30% of those patients with node negative breast carcinoma are estimated to die of their disease without adjuvant treatment.2 Despite the application of valuable prognostic parameters such as tumour size,1,2 grade,3–5 and histological type,6 it is not always feasible to predict the outcome of the disease. Therefore, additional prognostic parameters are needed to identify those patients with node negative breast carcinoma who are more likely to relapse and who might benefit from adjuvant treatment.
Cell cycle deregulation is frequently seen in cancer.7–9 The cell cycle is directly controlled by a series of cyclin dependent kinases (CDKs), cyclins—the CDK positive regulatory subunits—and CDK inhibitors.10,11 Progression of the cell cycle from the G2 to the M phase is controlled by a protein kinase complex, called mitosis or maturation promoting factor (MPF).12,13 MPF consists of two major proteins, the catalytic subunit, p34cdc2,14 and cyclin B1.15 MPF plays an important role in mitotic induction,16 regulating a wide range of mitotic events.17 The complex p34cdc2–cyclin B1 is controlled by the p21WAF1 protein, which is induced by the wild-type p53 protein.18 Neoplastic tissues produce high amounts of p34cdc2, whereas quiescent cells have low or undetectable amounts.19,20 A limited number of studies have investigated the role of p34cdc2 in the prognosis of breast carcinoma.
“Additional prognostic parameters are needed to identify those patients with node negative breast carcinoma who are more likely to relapse and who might benefit from adjuvant treatment”
The p53 tumour suppressor gene is the “cellular gatekeeper for growth and division”.21 p53 not only controls the G1–S transition,10–11,21 but also appears to participate in G2–M cell cycle checkpoint control after DNA damage.18,22–26 p53 inactivation is the most frequent event in human cancer.21,27 The prognostic relevance of p53 abnormalities, detected by immunohistochemistry in node negative breast carcinoma, has been highlighted by certain investigators,28–31 but is still controversial.32–34
The purpose of our study was to investigate the potential correlation between p53 and p34cdc2, the principal participants in the G1–S and the G2–M checkpoints respectively, their relation with p21WAF1, the clinicopathological parameters, and outcome, in addition to the prognostic value of these proteins in node negative invasive ductal breast carcinoma.
Patients and tissue samples
Our study comprised 94 patients with T1–T3, N0, M0 invasive ductal breast carcinoma, for whom paraffin wax embedded tissue blocks and clinical information were available. The administration of neoadjuvant chemotherapy was an exclusion criterion. Information regarding the type of surgery, the greatest tumour diameter, the status of surgical margins, and the number of retrieved axillary lymph nodes was collected from the pathology reports. The clinical records were reviewed for data regarding adjuvant treatment (chemotherapy, hormonal, or radiotherapy) and outcome parameters—that is, the occurrence of recurrence, metastasis or death, disease free survival (DFS), and overall survival (OS).
For each case, representative haematoxylin and eosin stained slides were reviewed to assess tumour grade, according to the Nottingham modification of the Bloom–Richardson grading system,3 histological type, and presence/extent of an in situ component. Information regarding the status of oestrogen and progesterone receptors was obtained from the patients’ records. When such information was not available, the receptor status was examined by immunohistochemistry on paraffin wax embedded tissue.
Formalin fixed, paraffin wax embedded, 5 μm thick sections were dewaxed, rehydrated in graded alcohols, and processed using the streptavidin–biotin–immunoperoxidase method. Briefly, sections were submitted to antigen retrieval by microwave oven treatment for 10 minutes in 0.01 mol/litre citric acid at pH 6.0. This procedure was followed for all antibodies studied. The sections were incubated with 1% hydrogen peroxide for 15 minutes, to block endogenous peroxidase activity, and subsequently with 1% bovine serum albumin diluted in Tris buffered saline (TBS) at pH 7.6 for 20 minutes, to block non-specific binding. The slides were wiped and incubated overnight at 4oC in a humid chamber with appropriately diluted primary antibody. The antibodies used were anti-p53 protein (DO-7) mouse monoclonal antibody (NCL-p53-DO7; Novocastra Laboratories Ltd, Newcastle, Newcastle upon Tyne, UK; 1/50 dilution), anti-cdc2 p34 mouse monoclonal antibody (sc-54; Santa Cruz Biotechnology, Santa Cruz, California, USA; 1/150 dilution), anti-p21WAF1 mouse monoclonal antibody (WAF 1 (Ab-1); Oncogene Research Products/Calbiochem, Cambridge, Massachusetts, USA; 1/20 dilution), anti-oestrogen receptor mouse monoclonal antibody (ER1D5; Immunotech, Marseille, France; 1/50 dilution), and anti-progesterone receptor mouse monoclonal antibody (1A6; Immunotech; 1/30 dilution). The slides were then rinsed three times in TBS and incubated with the reagents of the StrAvigen Multilink HRP concentrated detection kit (Biogenex Laboratories, San Ramon, California, USA; 1/80 dilution), according to the manufacturer’s instructions. After three washes with TBS, the peroxidase reaction was developed in freshly prepared 0.025% diaminobenzidine/0.1% hydrogen peroxide in TBS. Finally, the sections were counterstained with haematoxylin. Tissues previously known to be positive for p34cdc2, p21WAF1, and p53 were used as positive controls. Sections prepared with substitution of the primary antibody by TBS were used as negative controls.
Immunohistochemical evaluation and scoring
Two pathologists (HPK and CDS), blinded to the clinical, pathological, and other immunohistochemical results, independently evaluated the immunohistochemically stained slides. Along with the carcinoma, benign breast tissue was available for immunohistochemical examination, on the same or on a separate histological section, in 74 cases. The non-neoplastic tissue consisted of either terminal duct lobular units present in the periphery of the tumour or normal/ectatic ductal structures entrapped within the tumour. Immunohistochemical expression of the proteins was not evaluated in hyperplastic elements. Each histological section was screened and assessed for the percentage of benign and neoplastic nuclei displaying immunostaining. For p34cdc2, the tumour cytoplasmic positivity was also recorded separately. p34cdc2 immunoreactivity was classified as 1+, 2+, 3+, or 4+ if 0–9%, 10–25%, 26–50%, and 51–100% of the cells, respectively, displayed nuclear or cytoplasmic staining. The p34cdc2 positivity was set at ⩾ 10% nuclear or cytoplasmic p34cdc2 expression. Immunoreactivity for p53 was classified as 0, 1+, 2+, 3+, or 4+ if 0–9%, 10–25%, 26–50%, 51–75%, and 76–100% of the tumour cell nuclei, respectively, were positive. A carcinoma was classified as p53 positive when at least 10% of the nuclei were immunoreactive. Immunoreactivity for p21WAF1 was classified as 1+, 2+, 3+, or 4+ if < 1%, 1–5%, 6–20%, or > 20% of the tumour nuclei, respectively, were positive. A carcinoma was considered p21WAF1 positive when ⩾ 6% of the nuclei were immunoreactive. Oestrogen and progesterone receptor expression was considered positive if seen in ⩾ 10% of the neoplastic nuclei. When evaluation between the observers differed by ⩾ 10% or led to a different stratum of immunoreactivity, the case was re-evaluated until a consensus was achieved.
The associations between the proteins studied immunohistochemically and the clinicopathological parameters were examined by Pearson’s χ2 test. The effect of these factors on clinical outcome was determined in univariate analysis by the log rank test using the Kaplan–Meier method. Multivariate analysis was performed using the Cox proportional hazard model. Survival was measured in months starting from the date of the first pathological diagnosis. Significance was set at p ⩽ 0.05.
Table 1 summarises the clinical and histopathological data of the patients studied.
Table 2 shows the immunohistochemical results for the p34cdc2 protein. Figure 1 shows the expression of p34cdc2 in tumour cells. Expression of p34cdc2 in tumour nuclei (p34cdc2TN) compared with adjacent benign tissue (p34cdc2bn) was higher in 55, lower in four, and equal in 15 patients. Tumour nuclei showed significantly higher immunoreactivity for p34cdc2 (median value, 2+) compared with benign breast epithelium (median value, 1+) (p < 0.0001). Tables 3 , 4, and 5 show the statistical analysis data for p34cdc2TN, p34cdc2 in tumour cytoplasm (p34cdc2TC), and p34cdc2bn, respectively. Tables 6 and 7 and fig 2 show the effect of the examined factors on DFS and OS.
p34cdc2TN was associated with histological grade (p < 0.001), p34cdc2TC (p < 0.001), and p53 expression (p = 0.005), but it did not correlate with patient age, menopausal status, tumour size, steroid receptor status, recurrence, metastasis, DFS, or OS. p34cdc2TC was also associated with grade (p < 0.001) and in univariate analysis with DFS (p = 0.0158). Although not associated with the examined clinicopathological parameters or the proteins studied, p34cdc2bn was associated with longer DFS (p = 0.0030) and OS (p = 0.0046) in univariate analysis, whereas in multivariate analysis p34cdc2bn was the only independent predictor of DFS (p = 0.001).
Table 8 shows the immunohistochemical results for p53 and the statistical analysis data for p53 are shown in table 9. Expression of p53 in the tumour is depicted in fig 3. In all cases, benign breast epithelial cells were negative for p53. The expression of p53 was significantly associated with high tumour grade (p < 0.001) and negative oestrogen (p < 0.001) and progesterone (p = 0.005) receptor status, but there was no correlation with the remaining clinicopathological or outcome parameters.
Table 8 shows the immunohistochemical results for the p21WAF1 protein and the statistical analysis data for p21WAF1 are shown in table 10. Figure 4 depicts the expression of p21WAF1 in the tumour. No cytoplasmic staining was seen for p21WAF1. The expression of this protein did not correlate with the examined clinicopathological or outcome parameters, or the proteins studied.
We found significantly higher expression of p34cdc2 in carcinoma than in adjacent benign breast tissue. p34cdc2 is necessary for the induction of mitosis because it participates in the condensation of chromosomes, the formation of the mitotic spindle, and the breakdown of the nuclear membrane.35 p34cdc2 overexpression in proliferating cells has been reported by several investigators in breast carcinoma19,36,37 and other tumours.20,38–40 Because of its participation in the induction of the M phase of the cell cycle, an excess of p34cdc2 in the neoplastic tissue provides a proliferative advantage and probably facilitates the neoplastic process.
We examined p34cdc2 expression in both tumour nuclei (p34cdc2TN) and cytoplasm (p34cdc2TC), in addition to the adjacent benign breast tissue (p34cdc2bn). An association between p34cdc2TN and p34cdc2TC expression was noted. Both of these parameters were correlated with higher histopathological grade, unlike the results of previous studies,35,37 which did not identify an association between p34cdc2TN and tumour grade. Our results are in partial agreement with those of Winters and co-workers,41 who noted a positive association of grade only with p34cdc2TC. These findings are probably analogous to the association of proliferative index with grade,35,42,43 because p34cdc2 is thought to be an accurate measure of proliferative cellular activity.20 Whether these factors are biologically or even aetiologically associated to produce a certain biological tumour profile remains to be elucidated.
“Benign breast epithelium may express p34cdc2 as a reactive phenomenon, although the protein may be in an inactive state”
No association was seen between the expression of p34cdc2TN or p34cdc2TC and patient’s age, menopausal status, tumour size, p34cdc2bn, or p21WAF1. Wiesener and colleagues35 similarly did not identify an association between p34cdc2 and menopausal status, but noted a correlation of p34cdc2 expression with oestrogen receptor/progesterone receptor negativity, contrary to our results. p34cdc2TN was not associated with DFS or OS, results consistent with recent studies on breast carcinoma19,35,37 and other types of cancer.44,45 In contrast, other studies of breast carcinomas46,47 found p34cdc2 expression to be of independent prognostic significance for disease relapse in multivariate analysis. Although p34cdc2TC was associated with DFS in univariate analysis, in multivariate analysis it failed to remain significant, and did not appear to affect OS. Therefore, p34cdc2TC retention may represent an ineffective mechanism of p34cdc2 inactivation. Contrary to these results, the correlation of p34cdc2 immunoreactivity with Gleason grade, pathological stage, ploidy abnormalities, presence of metastases,48 and disease recurrence49 has been noted in prostatic adenocarcinoma. However in melanoma, although p34cdc2 overexpression has been correlated with mitotic activity, tumour thickness, and Clark’s level, it was not identified as an independent predictor of prognosis.40
Interestingly, in our present study, p34cdc2bn was associated with longer DFS in both univariate and multivariate analyses, whereas p34cdc2bn was the only parameter that affected OS. The reasons for this unexpected finding are unclear, even more so given that p34cdc2bn was not associated with the examined clinicopathological parameters or the examined proteins. Localisation of p34cdc2 in the nucleus may either be associated with inactive p34cdc2 state or may represent a reactive secondary phenomenon to injurious stimuli. This last hypothesis is supported by the observation that G2 arrest after exposure of human cells to ionising radiation may be accompanied by nuclear translocation of p34cdc2.18 Thus, benign breast epithelium may express p34cdc2 as a reactive phenomenon, although the protein may be in an inactive state. The association of p34cdc2 expression in benign epithelium with better survival may be explained by a combination of both hypotheses. Namely, injurious stimuli may result in secondary nuclear translocation of p34cdc2, although additional protective cellular mechanisms (such as phosphorylation or protein binding) inactivate the kinase in both benign and neoplastic cells, thus preventing cellular proliferation. The evaluation of this finding merits further investigation using biochemical methods in larger series of patients.
In general, positive immunohistochemical staining for p53 has been associated with mutant p53 gene status, resulting in a protein product with a longer half life that allows its visualisation using immunohistochemistry. Our study confirmed previous reports50 connecting p53 overexpression with higher grade and negative oestrogen and progesterone receptor status. No association of p53 expression with patient’s age, menopausal status, tumour size, DFS, or OS were identified. Similar results have been reported by others.32,34,50,51 Recently, a consensus statement of the College of American Pathologists included p53 in category II of prognostic factors, indicating “its import needs to be validated further in statistically robust studies”.33 However, it should be noted that the detection of p53 gene mutations has been shown to be of prognostic importance.52–54
p34cdc2TN expression paralleled that of p53, contrary to previous observations.41 Although most carcinomas displayed a p53−/p34cdc2+ phenotype, most p53 negative tumours, assumed to possess wild-type p53, expressed lower amounts of p34cdc2. This probably reflects the fact that intact p53 can cause G2 arrest by reduction of the expression of p34cdc2.26 Our findings associate p53 with the amount of nuclear p34cdc2, a factor crucial for the induction of mitosis, thus associating p53 with G2–M cell cycle checkpoint control. Previous reports have also implicated p53 in G2–M cell cycle checkpoint control.18,22–26
Additional links between p34cdc2 and p53 are proteins that are transcriptionally activated by p53, such as p21WAF1, 14-3-3σ, and GADD45.26 p21WAF1 directly inhibits p34cdc2, and it has been shown that the cyclin B–cdc2 kinase complex is negatively regulated by wild-type p53 mediated transcriptional induction of p21WAF1.18,22,24 In our study, we detected the presence p21WAF1 in the nuclei only. The absence of cytoplasmic staining is probably related to the use of an acidic citrate buffer (pH 6.0) for antigen retrieval.41 p21WAF1 was not associated with the examined clinicopathological parameters, the proteins analysed, or outcome, similar to previous observations.41 Contrary to these findings, the association of high p21WAF1 with high grade and shorter relapse free survival has been previously noted.55 Because of the complex interactions of p21WAF1, in addition to its differing functions according to its stoichiometry (induction of cyclin–cyclin dependent kinase complex formation at low concentration and inhibition of the complex at higher concentrations56), direct or possibly simplistic conclusions regarding the prognostic role of p21WAF1 cannot be made with the use of immunohistochemistry alone.
Two parameters may have adversely affected our results. These are the relatively small number of patients and the administration of adjuvant treatment to all patients studied. This last factor imposes additional difficulties in the identification of those patients who would have relapsed without such treatment. Furthermore, although the median length of the follow up period (72 months) is considered adequate, re-evaluation of the data after extension of the follow up period might provide us with additional information. In the meantime, results concerning DFS and OS should be considered with caution.
Take home messages
Tumour expression of the cell cycle regulators p34cdc2, p21WAF1, and p53 does not predict outcome in node negative breast carcinoma
p34cdc2 expression in benign tissue adjacent to the tumour is related to prognosis
Additional studies in patients with node negative breast carcinoma are needed before any final conclusions can be drawn on the prognostic role of these proteins
The association between p34cdc2 and p53 implicates p53 in G2–M cell cycle checkpoint control, possibly involving mediators unrelated to p21WAF1
“Our findings associate p53 with the amount of nuclear p34cdc2, a factor crucial for the induction of mitosis, thus associating p53 with the G2–M cell cycle checkpoint control”
In conclusion, in our study we found that p34cdc2 was overexpressed in node negative invasive ductal breast carcinoma compared with benign breast tissue, and detected a strong correlation between nuclear and cytoplasmic p34cdc2 overexpression by the tumour and histopathological grade. However, p34cdc2 tumour expression did not affect the patients’ outcome, tumour size, or steroid receptor status. p34cdc2 expression by the benign tissue adjacent to the tumour independently correlated with prognosis. Furthermore, although there was an association of p53 with histopathological grade and negative steroid receptor status, there was no effect of p53 on patient outcome. Similarly, p21WAF1 was not associated with the examined clinicopathological parameters, the proteins analysed, or the clinical outcome. In view of the contradictory results regarding the effect of p34cdc2 and p53 expression on clinical outcome in the literature, it is apparent that additional studies in patients with node negative breast carcinoma are necessary, before drawing any final conclusions on the prognostic role of these proteins. Finally, the relation of p34cdc2 to p53 supports the theories implicating the p53 protein in G2–M cell cycle checkpoint control, thus expanding the complexity of the cellular events involved in cellular homeostasis and neoplastic proliferation. Further studies in patients with breast carcinoma and other neoplasms are needed for a better understanding of the complex cellular mechanisms of cell cycle control.
This work was supported in part by a grant provided by the Greek National Ministry of Health and the Greek Anticancer Organisation.
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