Abstract
Background
Identification of molecular events of the recurrent squamous cell carcinoma (SCC) of the larynx and pharynx may aid in refining treatment strategies and improving outcome. The underlying molecular events of these recurrent tumors involve alterations in the tumor suppressor genes (p53) and protooncogenes (Bcl-2). We hypothesize that the development of these recurrent tumors involves alterations of the p53 and Bcl-2 proteins.
Methods
To test this hypothesis, 15 laryngeal and pharyngeal biopsy specimens obtained from 15 patients with recurrent laryngeal or pharyngeal SCC with different grades (II-IV) were immunostained for p53 and Bcl-2 protein expression.
Results
Examination of the percentage of positive cells in the normal mucosa and SCC, respectively, showed significant up-regulation of p53 (0.0 ± 0.0 vs 51.8 ± 5.9, P = .00) and Bcl-2 protein expression (36.5 ± 3.5 vs 74.6 ± 1.9, P = .00).
Conclusions
Alterations of the p53 and Bcl-2 proteins occur during the development of recurrent SCC. Additional studies are needed to confirm and extend our results.
1
Introduction
Squamous cell carcinomas (SCCs) of the larynx and pharynx, common head and neck malignancies, usually have remarkable tendency for recurrence. For example, approximately 15% of laryngeal SCC tumors treated by radiotherapy will prove to be radioresistant, as manifested by tumor recurrence within the original radiotherapy field over the ensuing 12 months. By causing extensive DNA damage, radiotherapy aims to induce apoptosis and tumor regression. Defects in the mechanisms that recognize DNA damage induce cell cycle arrest or control apoptosis, either alone or in combination; and these may be responsible for radioresistance . Although the molecular events underlying recurrence are still unknown, they seem to depend on the disruption of the genomic stability . The latter reflects an interaction among tumor suppressor genes (TSG, p53) and oncogenes (Bcl-2). The p53 gene is a tumor suppressor gene that plays a central role in the protection against DNA damage and other forms of physiologic stress primarily by inducing cell cycle arrest or apoptosis. Therefore, p53 gene represents the guardian of the human genome . Mutation of p53 gene, which is the most frequent genetic alteration detected in human cancers, inactivates these growth regulatory functions and causes a loss of tumor suppressor activity. In some cases, mutation also confers tumor-promoting functions, such as the transcriptional activation of genes involved in cell proliferation, cell survival, and angiogenesis. Consequently, cells expressing some forms of mutant p53 gene show enhanced tumorigenic potential with increased resistance to chemotherapy and radiation .
In SCC, p53 gene is mutated with subsequent overexpression of p53 protein . The Bcl-2 oncogene, a prosurvival molecule, is also involved in SCC . The Bcl-2 gene is an antiapoptotic-membrane–associated molecule that resides in the nuclear envelope and mitochondria. It exerts its antiapoptotic functions by modulating the mitochondrial release of cytochrome c and the interaction of apoptosis activating factors (Apaf-1) with caspase 9, Bax; and c-Myc. As a protooncogene, Bcl-2 seems to be a target of the risk factors (cigarette smoking, alcohol abuse, ionizing radiations) . Several studies have proposed close interactions among p53 and Bcl-2 proteins during carcinogenesis . This proposition is supported by the inhibitory effect of p53 on Bcl-2 through a p53-regulatory domain in the Bcl-2 gene . These observations led us to hypothesize that the development of recurrent laryngeal and pharyngeal SCC involves alterations of the p53 and Bcl-2 proteins. To date, the status of p53 and Bcl-2 proteins in the normal mucosa and recurrent invasive SCC is still largely unknown. To fill this gap in literature and to test our hypothesis, we carried out this work. To accomplish our goal, we examined 15 patients with these lesions using immunoperoxidase methods.
2
Patients and methods
2.1
Patients and tissue samples
This cross-sectional study included 15 laryngeal and pharyngeal biopsy specimens obtained from 15 patients with recurrent laryngeal and pharyngeal SCC. These patients were collected from patients presenting to the ENT and Oncology Departments, Assuit University Hospital, Egypt, during the period between November 2004 and November 2006. Some patients’ samples were drawn from the archives of the Pathology Department, Assuit University Hospital, Assuit, Egypt. Diagnosis of recurrence was based on patient examination using indirect laryngoscopy and fiberoptic nasoendoscopy. Biopsy was taken from any suspicious lesions using rigid endoscopy under general anesthesia in the ENT Department.
2.2
Eligibility criteria
Patients included on the study had these criteria:
- 1.
Pathologically proven SCC of the pharynx (nasopharynx, oropharynx, or hypopharynx) or larynx;
- 2.
Underwent a full endoscopic examination before initial treatment, 1 to 2 months after completion of chemoradiotherapy and on recurrence.
- 3.
Received concurrent chemoradiotherapy as initial treatment.
- 4.
Achievement of a complete clinical remission defined as normalization of radiologic evidence of disease and endoscopic and histologic examinations.
- 5.
Confirmation of recurrence by endoscopic and histologic examinations.
2.3
Chemotherapy
The chemotherapy regimen consisted of cisplatin 20 mg/m 2 once weekly during radiotherapy. All patients received adequate hydration and serotonin antagonist against emesis during cisplatin administration.
2.4
Radiation therapy
Pretreatment computed tomography of the neck was done to assess the extent of the primary tumor, as well as the neck nodes. The treatment volume included the primary tumor site and the neck nodes above the clavicle. The patients were treated with 6 MV photons. The fractional daily dose was 2 Gy with a planned total dose of 60 Gy.
2.5
Pathologic evaluation
Formalin-fixed, paraffin-embedded tissues was obtained from the Department of Pathology, Assuit University Hospitals, Assuit, Egypt. All the specimens entailed variable associations of normal and invasive SCCs (15 cases). The carcinomas were graded as grade I, II, III, or IV according to the severity of nuclear anaplasia. The invasiveness is entailed by the clear-cut violation of the basement membrane with invasion into the underlying subepithelial tissues .
2.6
Immunohistochemical analysis
Immunostaining was carried out as previously described . Briefly, sections mounted on glass slides were deparaffinized and rehydrated through graded alcohols to water. Endogenous peroxidase activity was blocked with 0.6% H 2 O 2 . Sections were then immersed in the retrieval solution (10 mmol/L sodium citrate buffer, pH 6.0) and subjected to heat-induced antigen retrieval for 20 minutes. The slides, in plastic Coplin jars containing retrieval solution, were microwaved in a microwave set at high (∼750 W) for 4 cycles of 5-minute duration each. Nonspecific protein binding was blocked with 10-minute exposure to 10% normal goat serum. Sections were then incubated with mouse monoclonal antibodies for 30 minutes at room temperature (Clone DO1 for p53, obtained from BD Transduction Laboratories, Lexington, KY; Clone 124, IgG1, kappa for Bcl-2, obtained from DAKO Corp, Carpinteria, CA). A catalyzed signal amplification system (K1500, DAKO) was used according to the manufacturer’s instructions. Sections were next treated with peroxidase-labeled streptavidin for 30 minutes at room temperature and incubated with 14-diaminobenzidine and 0.06% H 2 O 2 for 5 minutes. They were counterstained with hematoxylin, dehydrated in alcohol, cleared in xylene, and cover slipped. The slides were independently evaluated by 2 observers.
2.7
Positive controls
Specimens consisting of reactive lymphoid hyperplasia (for Bcl-2 protein) and SCCs (for p53 protein) were used as positive controls.
2.8
Negative controls
Additional sections, running in parallel but with omission of the primary antibody, served as the negative controls .
2.9
Evaluation of p53 and Bcl-2 staining
The percentage of positive cells (PP%) was scored as 0 for less than 5%, 1 for 5% to 25%, 2 for 25% to 50%, 3 for 50% to 75%, and 4 for greater than 75% .
2.10
Evaluation of p53 staining
To evaluate the p53 staining, we adopted the following strategy: (1) the observers independently evaluated the stained slides without knowledge of the clinicopathologic profiles of these lesions, (2) the presence of cells with clear and unequivocal nuclear staining identified positive cases, (3) at least 500 neoplastic cells per sample were analyzed (×400) in at least 5 different areas, and (4) the p53 protein expression values were then compared with those in the controls.
2.11
Evaluation of Bcl-2 staining
The expression of Bcl-2 protein was identified as diffuse golden yellow cytoplasmic staining (sites of the mitochondria). The Bcl-2 expression was considered negative only when more than 95% of the lesional cells were negative with few positive infiltrating lymphocytes .
2.12
Statistical analysis
Analysis of variance and Pearson correlation coefficient with a statistical significance of P < .05 were used.
2
Patients and methods
2.1
Patients and tissue samples
This cross-sectional study included 15 laryngeal and pharyngeal biopsy specimens obtained from 15 patients with recurrent laryngeal and pharyngeal SCC. These patients were collected from patients presenting to the ENT and Oncology Departments, Assuit University Hospital, Egypt, during the period between November 2004 and November 2006. Some patients’ samples were drawn from the archives of the Pathology Department, Assuit University Hospital, Assuit, Egypt. Diagnosis of recurrence was based on patient examination using indirect laryngoscopy and fiberoptic nasoendoscopy. Biopsy was taken from any suspicious lesions using rigid endoscopy under general anesthesia in the ENT Department.
2.2
Eligibility criteria
Patients included on the study had these criteria:
- 1.
Pathologically proven SCC of the pharynx (nasopharynx, oropharynx, or hypopharynx) or larynx;
- 2.
Underwent a full endoscopic examination before initial treatment, 1 to 2 months after completion of chemoradiotherapy and on recurrence.
- 3.
Received concurrent chemoradiotherapy as initial treatment.
- 4.
Achievement of a complete clinical remission defined as normalization of radiologic evidence of disease and endoscopic and histologic examinations.
- 5.
Confirmation of recurrence by endoscopic and histologic examinations.
2.3
Chemotherapy
The chemotherapy regimen consisted of cisplatin 20 mg/m 2 once weekly during radiotherapy. All patients received adequate hydration and serotonin antagonist against emesis during cisplatin administration.
2.4
Radiation therapy
Pretreatment computed tomography of the neck was done to assess the extent of the primary tumor, as well as the neck nodes. The treatment volume included the primary tumor site and the neck nodes above the clavicle. The patients were treated with 6 MV photons. The fractional daily dose was 2 Gy with a planned total dose of 60 Gy.
2.5
Pathologic evaluation
Formalin-fixed, paraffin-embedded tissues was obtained from the Department of Pathology, Assuit University Hospitals, Assuit, Egypt. All the specimens entailed variable associations of normal and invasive SCCs (15 cases). The carcinomas were graded as grade I, II, III, or IV according to the severity of nuclear anaplasia. The invasiveness is entailed by the clear-cut violation of the basement membrane with invasion into the underlying subepithelial tissues .
2.6
Immunohistochemical analysis
Immunostaining was carried out as previously described . Briefly, sections mounted on glass slides were deparaffinized and rehydrated through graded alcohols to water. Endogenous peroxidase activity was blocked with 0.6% H 2 O 2 . Sections were then immersed in the retrieval solution (10 mmol/L sodium citrate buffer, pH 6.0) and subjected to heat-induced antigen retrieval for 20 minutes. The slides, in plastic Coplin jars containing retrieval solution, were microwaved in a microwave set at high (∼750 W) for 4 cycles of 5-minute duration each. Nonspecific protein binding was blocked with 10-minute exposure to 10% normal goat serum. Sections were then incubated with mouse monoclonal antibodies for 30 minutes at room temperature (Clone DO1 for p53, obtained from BD Transduction Laboratories, Lexington, KY; Clone 124, IgG1, kappa for Bcl-2, obtained from DAKO Corp, Carpinteria, CA). A catalyzed signal amplification system (K1500, DAKO) was used according to the manufacturer’s instructions. Sections were next treated with peroxidase-labeled streptavidin for 30 minutes at room temperature and incubated with 14-diaminobenzidine and 0.06% H 2 O 2 for 5 minutes. They were counterstained with hematoxylin, dehydrated in alcohol, cleared in xylene, and cover slipped. The slides were independently evaluated by 2 observers.
2.7
Positive controls
Specimens consisting of reactive lymphoid hyperplasia (for Bcl-2 protein) and SCCs (for p53 protein) were used as positive controls.
2.8
Negative controls
Additional sections, running in parallel but with omission of the primary antibody, served as the negative controls .
2.9
Evaluation of p53 and Bcl-2 staining
The percentage of positive cells (PP%) was scored as 0 for less than 5%, 1 for 5% to 25%, 2 for 25% to 50%, 3 for 50% to 75%, and 4 for greater than 75% .
2.10
Evaluation of p53 staining
To evaluate the p53 staining, we adopted the following strategy: (1) the observers independently evaluated the stained slides without knowledge of the clinicopathologic profiles of these lesions, (2) the presence of cells with clear and unequivocal nuclear staining identified positive cases, (3) at least 500 neoplastic cells per sample were analyzed (×400) in at least 5 different areas, and (4) the p53 protein expression values were then compared with those in the controls.
2.11
Evaluation of Bcl-2 staining
The expression of Bcl-2 protein was identified as diffuse golden yellow cytoplasmic staining (sites of the mitochondria). The Bcl-2 expression was considered negative only when more than 95% of the lesional cells were negative with few positive infiltrating lymphocytes .
2.12
Statistical analysis
Analysis of variance and Pearson correlation coefficient with a statistical significance of P < .05 were used.
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