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Keywords:

  • Laryngeal squamous cell carcinoma;
  • p53, bcl-2, bax;
  • apoptosis;
  • cell proliferation;
  • immunohistochemistry.

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. BIBLIOGRAPHY

Objective The p53, bcl-2, and bax genes are known to be involved in control of cell cycle progression and regulation of apoptotic cell death. Although they are frequently altered in laryngeal squamous cell carcinoma, their clinical relevance is not yet fully understood. In the present study, individual and combined expressions of these genes were related with patient survival as well as with proliferative and apoptotic activity.

Design Retrospective study.

Methods Paraffin-embedded tissue sections of 88 laryngeal squamous cell carcinomas that were diagnosed and treated between 1986 and 1996 were investigated for p53, bcl-2, and bax protein expression by immunohistochemistry. Apoptotic cells were visualized using the nick end labeling method. To assess proliferative activity of tumors, mitotic indices were determined.

Results Age of patients, advanced disease (stages III and IV), high mitotic activity, positive bcl-2 expression, high level of p53 expression, and p53/bcl-2 co-expression were significantly associated with shortened overall survival in univariate analysis. In multivariate analysis, only age and p53/bcl-2 co-expression had independent prognostic value. Other combinations of genes, i.e., bcl-2-to-bax and p53-to-bax ratios, were not associated with patient outcome. A significant positive correlation was found between apoptotic and mitotic activity. However, protein levels of p53, bcl-2, and bax were unrelated to proliferation and apoptosis of tumor cells.

Conclusions The co-expression of p53/bcl-2 was an independent predictor of patient outcome and had a prognostic value superior to both parameters considered separately. The rate of apoptosis mainly counterbalanced proliferative activity but appeared not to be significantly influenced by p53, bcl-2, and bax.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. BIBLIOGRAPHY

Laryngeal squamous cell carcinoma (SCC) is the most frequent tumor of the head and neck region in the Western world. As conventional clinicopathological parameters do not accurately reflect the clinical outcome of patients with this tumor, establishment of additional prognostic factors that may improve understanding of treatment failures is an essential goal.

The genes p53, bcl-2, and bax are known to be involved in cell cycle control and/or regulation of programmed cell death and might have an impact on the biological behavior of tumors. However, most previous studies investigating the clinical relevance of expression levels of these genes in head and neck SCC yielded conflicting results. Although various studies implicated that expression of p53 has no prognostic significance, 1–4 others demonstrated correlations with shortened survival 5–8 and one other with prolonged survival. 9 Regarding bcl-2 overexpression, a majority of studies have found it to be significantly associated with poor prognosis. 1,10,11 Yet opposite results as well as a lack of any clinical relevance have also been published. 8,12 Conversely, high bax expression seemed to correlate with favorable outcome of patients. 13,14 This inconclusiveness of different study results might partly be explained by different methods used to perform immunohistochemistry. Its main reason, however, probably lies in the complexity of death regulation and the slight influence brought about by individual genes on this process. Approaches considering several components together might therefore enable a more reliable prediction of clinical tumor behavior than single parameters alone.

When regulating programmed cell death, the pro-apoptotic p53 gene is closely interrelated to the genes bcl-2 and bax, which act as an inhibitor and an accelerator of apoptosis, respectively. In particular, activation of p53 induces decreases in bcl-2 and increases in bax expression, 15 whereas bcl-2 has been shown to modify p53 function by suppressing p53-dependent gene transcription. 16,17 Despite this functional interweaving of p53, bcl-2, and bax, scarce account has been taken of the question whether concurrent alterations of these genes might have an increased prognostic impact owing to a summation of complementary gene effects. In the present study, protein expression levels of p53, bcl-2, and bax were retrospectively investigated in 88 laryngeal SCCs and were correlated—as individual and combined parameters—with overall survival of patients.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. BIBLIOGRAPHY

Patients and Tumor Tissues

The samples were retrospectively collected from 88 patients who were treated for newly diagnosed laryngeal SCC at the University Hospital of Göttingen between 1986 and 1996. During this period, the treatment concept applied to laryngeal SCC was uniform and aimed at organ preservation in the majority of patients. Inclusion criteria were surgical treatment of the tumor by means of transoral laser microsurgery (82 patients) or laryngectomy (6 patients), complete clinical data, uniformity of histological differentiation throughout the tumor sample, and the availability of sufficient material from the primary tumor for this and other investigations. Patients with previous or synchronous second malignancies or with previous radiation therapy or chemotherapy were excluded from the study. Thirteen patients did additionally undergo selective neck dissection; 12 were irradiated after surgery. The follow-up time ranged from 1 to 144 months (mean, 45.9 mo). Of the 26 patients who died, 10 died of the disease and 3 of second primaries.

The main clinicopathological characteristics of the patients are shown in Tables I and II. Eighty (90.9%) were male, 8 (9.1%) were female. Average age at diagnosis was 62.3 years (median, 62 y; range, 30–94 y). Twenty-five tumors (28.4%) were supraglottic, 47 (53.4%) glottic, 7 (8.0%) subglottic, and 9 (10.2%) transglottic. For staging of the tumors, the TNM classification of the International Union Against Cancer was applied: 18 patients (20.5%) had stage I disease, 31 (35.2%) stage II, 17 (19.3) stage III, and 22 (25.0%) stage IV. Diagnosis and grading were performed by two independent pathologists.

Table Table 1.. Association of Clinicopathological Tumor Parameters With bcl-2, bax, and p53 Expression in 88 Patients with Laryngeal SCC.
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*χ-Squared test.

†Kruskal-Wallis H-test.

‡Mann-Whitney U-test.

§Supra-, sub-, and transglottic tumors.

Table Table 2.. Relation of Variables With Cumulative 5-Year Overall Survival Rates in 88 Patients With Laryngeal Squamous Cell Carcinoma.
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A significance level of P < .1 in the univariate log rank test (third column) was used to select variables for consideration in Cox regression analysis I. Cox analysis II differed from this in that it disregarded the variable “p53/bcl-2 co-expression.” The P values listed in the fourth and fifth columns show the results of the two analyses; bold type indicates inclusion of the respective variable in the calculated model.

*Median cutoff levels.

†p53+/bcl-2, p53−/bcl-2+, and p53−/bcl-2.

Immunohistochemistry

Three- to 4-μm-thick tissue sections were dewaxed in xylene and rehydrated through graded alcohols. Slides were heated in 10-mmol/L sodium citrate buffer (pH 6.0) in a microwave oven (750 W) three times for 5 minutes. Primary antibodies against bcl-2 (mouse monoclonal anti-bcl-2, clone 124, DAKO, Hamburg, Germany, diluted 1:100 in Tris-buffered saline), bax (rabbit polyclonal anti-bax, N-20, Santa Cruz Biotechnology, Heidelberg, Germany, 1:50), and p53 (mouse monoclonal anti-p53, clone DO-1, Immunotech, Marseille, France, 1:20) were applied for 2 hours (bcl-2 and bax) or ½ hour (p53) at room temperature, respectively. After washing in Tris-buffered saline, sections were incubated with the secondary antibodies (rabbit anti-mouse for bcl-2, goat anti-mouse for p53, or mouse anti-rabbit followed by rabbit anti-mouse for bax, all purchased from DAKO, 1:50, 30 min), and then with mouse monoclonal APAAP (DAKO, 1:100, for 50 minutes in darkness). The immunostaining reactions were visualized using new fuchsin (bcl-2 and bax) or fast red (p53) as chromogenic substrates, respectively. Sections were counterstained with hematoxylin and mounted in Glycergel (DAKO).

To confirm immunospecificity, negative controls were performed in each slide run by omission of the primary antibodies. Formalin-fixed, paraffin-embedded sections of breast carcinoma and normal human lymph node served as positive controls for p53, bcl-2, and bax immunohistochemistry, respectively.

Quantification of Immunohistochemistry

All slides were independently reviewed by two investigators in a blinded fashion. Occasional disagreements were subsequently discussed to reach a consensus. In case of persistent differences between the slide readers, the mean of the two opinions was considered.

Immunoreactions for bcl-2 and bax were scored as described previously. 18 In brief, staining intensity of tumor cells was classified as negative (0), weakly positive (1), moderately positive (2), or strongly positive (3), and the fraction of stained tumor cells as 0 (<25%), 1 (25%–75%), or 2 (>75%). Scoring levels were obtained by adding the values of the two parameters. Tumors scored between 0 and 2 were considered negative, those between 3 and 5 positive.

Immunohistochemical staining for p53 was evaluated according to Spafford et al., 8 with minor modifications. As for bcl-2 and bax, staining intensity was assigned to four categories (0–3), whereby the intense immunoreaction detected in breast carcinoma served as control and was designated as strongly positive (3). Percentages of stained tumor cell nuclei were determined by counting of at least 700 nuclei. For each case, the two components were multiplied, resulting in overall staining scores between 0 and 300. To categorize the degree of immunoreactivity, score values below 50 were arbitrarily designated as low, values from 50 to 100 as intermediate, and those exceeding 100 as high.

To investigate combined expressions of the genes, ratios of bcl-2-to-bax and p53-to-bax were determined by dividing the respective scoring levels. The ratios were then categorized into low versus high values by using the respective medians as cutoff points. For combined analysis of p53 and bcl-2, tumors showing coexpression of both genes were compared with the others.

Apoptotic and Mitotic Indices

DNA fragments of apoptotic cells were visualized by the Terminal-deoxynucleotidyl-transferase (TdT)-mediated dUTP nick end labeling (TUNEL) technique using an in situ Cell Death Detection Kit (Boehringer Mannheim, Germany). The labeling procedure was performed following the supplier's instructions. After dewaxing and rehydrating, tissue sections were incubated with proteinase K for 20 minutes at room temperature (Boehringer Mannheim, Germany; 20 μg/mL in 10 mmol/L Tris buffer at pH 7.5). TdT was used to catalyze the addition of fluorescein-conjugated dUTP to 3′-OH ends of DNA fragments. The incorporated fluorescein was then detected by anti-fluorescein antibody Fab fragments from sheep, conjugated with alkaline phosphatase. After color reaction with new fuchsin substrate, the slides were counterstained with hematoxylin and mounted. As positive controls, germinal centers and extrafollicular areas of hyperplastic lymph nodes were also evaluated. To exclude unspecific staining reactions, one specimen of each experimental setup was treated with a fluorescein-dUTP solution lacking the enzyme TdT.

The apoptotic and mitotic indices were determined by microscopic examination of randomly selected high-power fields (× 400). In each specimen, at least 2000 tumor cells were evaluated for the number of apoptotic cells as well as mitotic figures. Because the TUNEL reaction also labels cells in necrotic foci, only those cells were regarded as positive that did additionally show characteristic morphological features of apoptotic cell death, e.g., overall shrinkage and isolated localization within an intact cell complex. The results were expressed in per mil. Specimens containing fewer than 2000 tumor cells were excluded from analysis. The median values of the indices were used to divide tumors into groups of low versus high mitotic and apoptotic activity, respectively.

Statistical Methods

Statistical analyses were performed using SPSS for windows (SPSS Inc., Chicago, IL). The χ2 test was applied to evaluate associations between frequency distributions, i.e., between clinicopathological parameters and immunostaining results for bcl-2 and bax. Mean values were statistically compared by a two-tailed Student t test (normally distributed variables) or by a Mann-Whitney U test (other variables). If more than two samples had to be compared, ANOVA or a Kruskal-Wallis H-test were used, respectively. Correlations between normally distributed variables (mitotic and apoptotic rates) were examined applying Pearson's correlation coefficient analysis; for other variables Spearman's correlation coefficient was determined. Univariate survival analysis was based on the Kaplan-Meier method with the log-rank test. Multivariate analysis considered those variables that reached a significance level of P < .1 in the univariate log-rank test. It was carried out by means of a Cox regression model using a forward stepwise procedure. For all statistical tests, P values of < .05 were considered significant.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. BIBLIOGRAPHY

Immunohistological Staining Reactions

The means of bcl-2 and bax immunoreactive scores amounted to 2.38 (range, 0–5) and 3.86 (range, 3–5), respectively. Forty-eight cases (54.5%) were classified bcl-2 positive (score 3–5) and 40 (45.5%) bcl-2 negative (score 0–2). As essentially all tumor cells expressed bax protein at detectable amounts (fraction of stained cells assigned to category 2), all laryngeal SCCs were immunopositive for bax, with scoring levels ranging between 3 and 5. Intensity of bax expression was inversely correlated with the bcl-2 status (Spearman's correlation coefficient:r = −0.290;P = .0060; coefficient of determination r2 = 0.084).

Nuclear staining reactions for p53 were detectable in 68 cases (77.3%), with an average staining score of 81.3 (range, 1–300). Of the 68 immunoreactive tumors, 30 (44.1%) had low level (score < 50), 16 (23.5%) had intermediate level (score 50–100), and 22 (32.4%) had high level positivity (score > 100). p53 Expression scores did not correlate with bcl-2 or bax immunoreactions (Spearman's r = 0.036;P = .7379, and r = −0.097;P = .3689, respectively).

Associations With Clinicopathological Parameters

As shown in Table I, positive bcl-2 immunostaining was associated with high-grade histology, high T category, regional lymph node metastasis, advanced clinical stage, and supraglottic, subglottic, or transglottic location of tumors. Significant relations between clinicopathologic parameters and intensity of bax expression or p53 scoring levels were not detectable. However, p53 expression in low-grade tumors was preferentially found in the peripheral cells of tumor nodules, whereas staining in most high-grade tumors was diffusely distributed throughout the lesion (Fig. 1). Other features such as age, sex, or smoking habits of the patients were not associated with bcl-2, bax, or p53 (data not shown).

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Figure Fig. 1.. A. Accumulation of p53 immunoreactive cells in the periphery of tumor nodules in a well-differentiated carcinoma. B. Diffuse staining reaction for p53 occurring in a poorly differentiated carcinoma (original magnification ×50).

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Correlation With Mitosis and Apoptosis

Mean and standard deviations of mitotic and apoptotic indices amounted to 2.02 ± 1.89 (median, 1.5) and 2.55 ± 1.57 (median, 2.25), respectively. Pearson's correlation coefficient analysis revealed a significant positive relationship between mitotic and apoptotic indices (r = 0.285;P = .0099) with a coefficient of determination of r2 = 0.081. However, none of the investigated gene expression levels significantly correlated with mitotic or apoptotic activity (Table III). In tumors showing both bcl-2 positivity and high level expression of p53 (score > 100), apoptotic, and mitotic rates were slightly heightened when compared with the others (apoptosis: 3.17 ± 1.74 vs 2.44 ± 1.52;P = .140; mitosis: 3.14 ± 2.93 vs 1.86 ± 1.64;P = .186; means ± SD; Student t test).

Table Table 3.. Spearman's Correlation Coefficient Analysis Investigating the Association of p53, bcl-2, and bax Protein Expression Levels With Mitotic and Apoptotic Rates.
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Correlation With Survival

As shown in Table II, the cumulative 5-year overall survival rate of all patients amounted to 0.69 (SE, 0.06). In univariate analysis, age at diagnosis, advanced clinical stage, high mitotic activity, high level of p53 positivity (score > 100), and positive bcl-2 expression were significantly associated with shortened overall survival. Coexpression of p53 and bcl-2 had a prognostic value superior to both parameters alone (Table II, Fig. 2). p53 Scoring levels below 100, combinations of p53 and bax or of bcl-2 and bax, risk factors, and the mode of therapy did not correlate with survival. Of the variables that reached a significance level of P < .1 in the univariate log-rank test, only age and p53/bcl-2 coexpression were found to have an independent prognostic value in multivariate analysis (Cox analysis I;Table II). When the variable “p53/bcl-2 coexpression” was removed from Cox regression analysis, clinical stage and p53 expression, but not bcl-2 expression, retained statistical significance (Cox analysis II;Table II).

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Figure Fig. 2.. Kaplan-Meier plots of cumulative overall survival in relation to bcl-2 expression (A), p53 expression (B), and p53/bcl-2 co-expression (C).

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. BIBLIOGRAPHY

In the present retrospective study, both p53 and bcl-2 expression were statistically significant predictors of shortened overall survival in laryngeal SCC. Immunoreactivity for bax had no impact on the outcome of patients, although it was inversely related to bcl-2. A concurrent overexpression of p53 and bcl-2 was associated with poorer prognosis than expression of both genes alone and, moreover, had an independent prognostic value beside patient's age in a multivariate Cox regression model. Other gene combinations, i.e., p53-to-bax and bcl-2-to-bax ratios, were unrelated to patient survival. The particular prognostic relevance of a concomitant alteration of p53 and bcl-2 suggests a close interrelationship of these genes within the system of cell death regulation. As mentioned above, p53 and bcl-2 normally act as a positive and a negative modulator of apoptosis, respectively. Thus, the coincidence of p53 inactivation (of which overexpression is suggestive) and bcl-2 upregulation appears to have an additive anti-apoptotic effect, which might account for considerable tumor progression and worsening of patient prognosis. Our findings are in line with a recent study of Pruneri et al., 19 who demonstrated that p53/bcl-2 coexpression had predictive value in laryngeal SCC, while p53 and bcl-2 when considered separately did not. However, in contrast to us, they were unable to prove an independence from other prognostic factors in multivariate analysis.

When disregarding the parameter p53/bcl-2 coexpression in Cox regression analysis (Cox analysis II, Table III), only p53 but not bcl-2 retained statistical significance on the outcome of patients. This superiority of p53 over bcl-2 in multivariate analysis might be explained by their different relationships with clinicopathological parameters found in this and other studies on head and neck SCC. Whereas p53 expression mainly lacked significant associations with grading and staging of tumors, 3–7,9 bcl-2 has frequently been observed to be linked with adverse prognostic parameters, i.e., high histological grade, advanced clinical stage, or both. 10,11,19,20 As stage represents one of the strongest clinical predictors of outcome, it is conceivable that the significance of bcl-2 found in univariate analysis reflects its association with rather than an independent impact on patient survival.

Although p53 scoring levels were not related with histological grades of tumors, p53 immunoreactivity tended to be peripherally located in well-differentiated tumors and diffusely distributed in poorly differentiated tumors (Fig. 1). This staining pattern is in agreement with that reported for oral and laryngeal carcinomas by Piffkó et al. 21 In low grade lesions, it recapitulated that seen in tumor-adjacent normal epithelium, with p53 protein focally restricted to basal, proliferating cell layers, 3,21 while keratinocytic differentiation was obviously accompanied with a loss of p53 immunoreactivity. Hence, the diffuse arrangement of p53 expression as observed in high-grade tumors may partly reflect a decreasing ability of malignant cells to terminally differentiate.

The observation that p53 becomes clinically relevant only when high levels of expression (i.e., score > 100) are considered is in line with a recent study of Jin et al., 6 who found that tumors with more than 75% p53-positive cell nuclei had a worse clinical outcome than those showing lower degrees or a complete lack of p53 immunoreactivity. Thus only a subset of tumors expressing p53 seemed to have an altered biological behavior. This result might emerge from the discrepancies existing between immunohistochemical detectability of p53 and the underlying genetic or epigenetic alterations. The anti-p53 antibody used in the present study (clone DO-1) is known to recognize both wild-type and mutant p53 proteins. Thus apart from gene mutations that account for p53 overexpression in about 60% of cases, 22 positive staining reactions may also be due to irregular complexion of wild-type protein as well as to overproduction of normal protein after DNA damage. 23,24 While the former events are usually associated with inactivation of p53 protein, the latter reflect p53's normal, tumor-suppressing function and could interfere with the relationship supposed between inactivation of p53 and prognosis. p53 Gene analysis as well as investigations on mdm-2, which is capable of forming complexes with p53 and inhibiting its transcriptional activation function, will further clarify this hypothesis.

We finally analyzed the relationships between apoptosis, mitosis, and the expression levels of p53, bcl-2, and bax. The particular prognostic significance of p53/bcl-2 coexpression suggests that a strong suppression of apoptotic cell death might account for the poor outcome of this subset of patients. However, the spontaneous apoptotic rates of tumors did not follow this assumption. On the contrary, tumors that showed both bcl-2 positivity and high-level expression of p53 had even slightly heightened apoptotic rates when compared with the others. Likewise, expression levels of the three genes alone also lacked significant associations with spontaneous apoptosis (Table III). The only significant result was a positive correlation between apoptotic and mitotic rates. This observation points to the regulatory coupling of proliferative and apoptotic pathways that is known to be a basic principle of normal growth control. 18 In malignancies, this coupling mechanism is undoubtedly disturbed, but not completely lost, which is indicated by the positive relations found between apoptosis and proliferative activity in this and other tumor types. 25–27 Thus deregulations of the cell cycle may result in increases of death rates, overriding the effects of p53, bcl-2, and bax within the apoptosis-regulating system.

CONCLUSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. BIBLIOGRAPHY

In this study p53/bcl-2 coexpression proved to be an independent predictor of overall survival in laryngeal SCC and had a prognostic value superior to both parameters considered separately. Although the number of cases underlying this result was limited, the data suggest that analysis of p53 and bcl-2 alterations may supplement conventional clinicopathological parameters in identifying high-risk patients who may benefit from more aggressive therapy or chemoprevention.

BIBLIOGRAPHY

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. BIBLIOGRAPHY