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Table of Contents
ORIGINAL ARTICLE
Year : 2010  |  Volume : 12  |  Issue : 1  |  Page : 1-9

EGFR and P21ras expression in Egyptian patients with laryngeal squamous cell carcinoma


1 Department of Otorhinolaryngology, Ain Shams University, Cairo, Egypt
2 Department of Pathology, Ain Shams University, Cairo, Egypt
3 Department of Clinical Pathology, Ain Shams University, Cairo, Egypt

Date of Web Publication24-Dec-2019

Correspondence Address:
MD Samia A Fawaz
52 Sakr Korish,Sheraton Heliopolis, Cairo, postal code is 11361
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1319-8491.273965

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  Abstract 


Background: Despite the advancement in diagnosis and treatment of laryngeal squamous cell carcinoma carcinoma, there has been no significant improvement in the survival rate in the last three decades. Many clinico-pathological factors as well as genetic alterations have been implicated in tumor recurrence and poor patient ‘survival.
Objective: The present study aims to investigate the expression of Epidermal Growth Factor Receptor (EGFR) and P21ras in Egyptian patients with laryngeal squamous cell carcinoma and to correlate the clinico-pathological data to that of the expression of EGFR and P21ras.
Patients and Methods: The present study included 52 patients of whom 30 patients had laryngeal invasive squamous cell carcinoma, 7 patients had laryngeal dysplasia, and 15 patients had benign non-neoplastic laryngeal lesions. Expression of EGFR and P21ras was investigated immunohistochemically in the specimens of all patients using monoclonal mouse antibodies for both EGFR and P21ras.
Results: EGFR positive immunostaining was observed in 96.6% of malignant group with varying degrees of expression, Pearson’s Chi-square test showed a highly significant difference between EGFR expressions in the different three groups. As regard P21ras, positive immunostaining was also observed in 100% of malignant group with varying degrees of expression, and Pearson’s Chi-square test showed a highly significant difference between P21ras expression in the three groups.
Conclusion: We suggest that both markers can be used to select patients with malignant and premalignant laryngeal lesions who might benefit from drugs targeted to them. However, further studies including larger groups of patients and contributing treatment trials with the new therapeutic agents are recommended for evaluation of their effect.

Keywords: EGFR in laryngeal carcinoma, P21 ras oncogene, laryngeal carcinoma


How to cite this article:
El-Hamid OA, Fawaz SA, Yousef TA, El-Maraghi M, Salman MI, El-Fedawi S. EGFR and P21ras expression in Egyptian patients with laryngeal squamous cell carcinoma. Saudi J Otorhinolaryngol Head Neck Surg 2010;12:1-9

How to cite this URL:
El-Hamid OA, Fawaz SA, Yousef TA, El-Maraghi M, Salman MI, El-Fedawi S. EGFR and P21ras expression in Egyptian patients with laryngeal squamous cell carcinoma. Saudi J Otorhinolaryngol Head Neck Surg [serial online] 2010 [cited 2022 Dec 2];12:1-9. Available from: https://www.sjohns.org/text.asp?2010/12/1/1/273965




  Introduction: Top


Cancer is still one of the commonest causes of death worldwide. Head and neck cancer is rated sixth worldwide and constitutes about 5-7% of all malignancies in North America[1],[2],[3] and 1.7% of all malignancies in Egypt[4]. Squamous cell carcinoma of the head and neck (HNSCC) remains a significant cause of morbidity and mortality, with approximately 274 000 new cancer cases and almost 145 000 deaths annually [3]. Patients with identical clinicopathological features may differ widely in the progression of their disease and response to treatment.

The identification of parameters that reflect biological behavior of individual cancer tissues correlating with tumor aggressiveness is a key determinant of prognosis and a fundamental issue for the improvement of cancer therapy[5]. Despite recent progress in defining the molecular mechanisms of cancer development and tumor progression, only a few individual biomarkers providing prognostic information have been identified. Among them, the EGFR pathways attracted the most attention of cancer investigators. [6]. (EGFR), a transmembrane glycoprotein, is a member of the large receptor tyrosine family encoded by a gene located in human chromosome 7p12. EGFR exists in inactive monomer form or in active dimer form. Dimerization can take place between different receptors in order to develop homologue (homodimers) or heterologue (heterodimers) dimers [7]. In either normal or malignant cells, the activation of EGF receptor cascades may have multiple consequences such as cell growth, differentiation, and proliferation. EGF receptor cascades may also promote malignant transformation, angiogenesis, and/or metastatic dissemination [8],[9]. Several cytogenetic and molecular studies have investigated the occurrence of genetic alterations in HNSCC, demonstrating that oncogene activation and tumor suppressor gene inacti-vation are involved in the development and progression of the disease. [10]. The Ras proto-oncogenes encode a group of plasma membrane associated G-proteins that bind guanine nucleotides with high affinity and activate several downstream effector proteins including raf- 1(P21) that are known to activate several distinct signaling cascades that are involved in the regulation of cell survival, proliferation and differentiation in response to extracellular stimuli such as growth factors or hormones [11].

Ras mutations have been identified in less than 5% of the head and neck tumors from Western world patients [12]. A high incidence (35%) of Ha-Ras mutations has been reported in oral squamous cell carcinomas from India [13],[14]. In this case betel quid chewing and reverse smoking are probably initiating agents [13]. The present study aims to evaluate the immunohisto-chemical expression of EGFR and P21ras in Egyptian patients with laryngeal squamous cell carcinoma and correlate the levels of expression with the clinicopatho-logical parameters.


  Patients and Methods Top


The present study was conducted at Ain Shams University Hospitals (Otorhinolaryngology and Pathology departments) between 2005 and 2008 on 52 patients. Patients were divided into three groups:

Group I included 30 patients with laryngeal invasive squamous cell carcinoma.

Group II included 7 patients with various grades of laryngeal dysplasia.

Group III included 15 patients with benign non-neoplastic laryngeal lesions. All patients were subjected to extensive work-up including history, physical examination, neck computed tomography and direct laryngoscopy for biopsy of suspected malignant lesions and excision of benign lesions.

Metastatic work up was done in patients with stage 3 and 4 laryngeal carcinoma.

The planned surgery was done for all the patients with malignancy according to the site and stage of the lesion. Eighteen partial laryngectomies and 12 total laryngec-tomies were performed in laryngeal cancer patients, together with modified neck dissection in 14 patients. All specimens were routinely processed and stained with H&E for routine histopathological examination. Four um sections were cut and mounted on charged slides for immunohistochemical staining. After deparaf-finization endogenous peroxidase activity was blocked using 0.3% H2O2 incubation for 10 minutes, then non specific binding sites were blocked by 3% normal goat serum for 30 minutes .

The slides were incubated with the primary antibodies for EGFR, monoclonal mouse antihuman antibodies (Dako Corporation, Carpentina, CA) diluted 1:100 and for P21ras protein, monoclonal mouse antihuman antibody (Dako Corporation, Carpentina, CA) diluted 1:50. Each primary antibody was incubated at room temperature for 2 hours.

After washing with PBS, the link was added for 45 minutes, which is a biotinylated secondary antibody antimouse IgG (Zymed lab,San Fransisco,CA) Then after wash, a peroxidase conjugated streptavidin was added for 20 minutes followed by wash .The reactions were developed in freshly prepared DAB chromogen for 10 minutes followed by wash and counterstaining with hematoxylin.

Cell membrane and/or cytoplasmic staining were considered positive expression for EGFR and cytoplas-mic staining was considered positive for P21ras. A semi quantitative histochemical score was used for both markers to record their results of staining depending on the percentage of stained cells and the intensity of staining.

0 = negative or focal faint staining.

+= Diffuse mild staining>50% or focal moderate staining <50% of tumor cells

++= Diffuse moderate staining>50% or focal strong staining <50% of tumor cells. +++= Diffuse strong staining >50% of tumor cells. Statistical analysis of the results was performed using the Pearson Chi-square test, by using SPSS software for windows version10.0.


  Results Top


Fifty-two patients were enrolled in the present study.

Group I; included 30 patients with laryngeal invasive squamous cell carcinoma, their ages ranged from 45-74 years (mean=58.9+7.52), they were 26 males and 4 females with a M: F ratio about 6:1, 26 patients were smokers.

Clinical staging according to TNM classification revealed 4 patients (13.3%) with stage T1, 7 patients (23.3%) stage T2, and 19 patients (63.3%) stage T3. 16 patients were N0 and 14 patients were N1 having cervical lymph nodes on the same side. Twelve tumors wereglottic (40%), 11 tumors were trans glottic (36.7%) and 7 tumors were supraglottic (23.3%). All the tumors were invasive squamous cell carcinoma, 5 cases were grade I (16.7%), 13 cases were grade II and 12 cases were grade III (40%).

Group II included 7 patients with age range 32-64 years (mean=49.9-10.6), they were 6 males and one female, 5 patients were smokers. There were 3 cases with dyspla-sia grade I and 4 cases with dysplasia grade II.

Group III included 15 patients whose age ranged from 28 to 64 years (mean=49.9-10.0), they were 8 males and 7 females, 6 patients were smokers. There were 7 cases of laryngeal polyps, 4 cases of laryngeal nodules and 4 cases of vocal fold cysts.

Immunohistochemical Results

EGFR positive immunostaining was observed in 29 out of 30 cases (96.6%) of group I with varying degrees of expression [Figure 1].
Figure 1: Shows Squamous cell carcinoma grade II with moderate cytoplasmic staining of EGFR 2+ (X100)

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In group II only 2 out of 7 cases (28.57%) of grade II dysplasia showed positive staining for EGFR. All cases of group III showed negative staining for EGFR.

Pearson’s Chi-square test showed a highly significant difference between EGFR expressions in the three groups [Table 1].
Table 1: Incidence of EGFR expression in all groups:

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Correlation of EGFR expression with the clinicopatho-logical data of group I is shown in [Table 2]. Over expression of EGFR (++, +++) was highly significantly associated with higher tumor grade (P0.001), advanced tumor stage (P0.002), and with the simultaneous over expression of P21ras (P0.006). Moreover, it was significantly associated with the presence of cervical lymph node metastasis (P0.026). No significant association was found between EGFR expression and the tumor location (P0.3). Moreover, EGFR over expression wasn’t significantly related to age, sex or smoking. P21ras positive immunostaining was observed in all cases (100%) of group I with varying degrees of expres- sion [Figure 2].
Figure 2: Shows Squamous cell carcinoma grade II, with moderate cytoplasmic staining (++) of P21ras (DAB chromogen X 250

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Table 2: Correlation of EGFR expression with the clinicopathological data of group I

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In group II, 6 out of 7 cases (85.71%) showed positive staining for P21ras, 4 of them (57.7%) showed mild staining [Figure 3]. and 2 cases (28.57 %) showed moderate staining.
Figure 3: shows Laryngeal dysplasia with weak (+) staining of cytoplasmic P21 (DAB chromogen X250)

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Figure 3: shows Laryngeal dysplasia with weak (+) staining of cytoplasmic P21 (DAB chromogen X250) Only one case of group III (6.7%) showed mild staining for P21ras. Pearson’s Chi-square test showed a highly significant difference between P21ras expression in the three groups [Table 3].
Table 3: Incidence of P21ras expression in all groups

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Correlation of P21ras expression with the clinicopatho-logical data of group I is shown in [Table 4]. Overexpression of P21ras (++,+++) was highly significantly associated with higher tumor grade (P0.001) and with the presence of cervical lymph node metastasis (P0.007).Moreover, it was highly significantly associated with the tumor location (P0.003) being more in transglottic and supraglottic carcinomas. No significant association was found between P21ras expression and the tumor stage (P0.164), moreover, P21ras over expression wasn’t significantly related to age, sex or smoking. Table IV: Correlation of P21ras expression with the clinico-pathological parameters of group I
Table 4: Correlation of P21ras expression with the clinico-pathological parameters of group I

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  Discussion Top


Squamous cell carcinoma of the head and neck (HNSCC) remains a challenging clinical problem due to the persistence of high rate of local recurrence and distant spread, as well as the occurrence of second primaries [5]. Several cellular oncogenes have been isolated from a variety of human malignant tumors. Their characterization has significantly contributed to better understanding of cancer at the molecular level as well as providing new therapeutic targets [15],[16], and [17]

Epidermal Growth Factor Receptor (EGFR) is a member of the Erb B family of receptors. Its stimulation by endogenous ligands, EGF or TGF-? results in activation of intracellular tyrosine kinase, therefore cell cycle progression [5]. EGFR is frequently amplified and over expressed in many tumors including head and neck tumors [9],[10], and [11].

The present study showed increased frequency of EGFR expression in Egyptian patients with laryngeal invasive squamous cell carcinoma (96.67%), a lesser degree of expression in cases with laryngeal dysplasia (28.57%) and absent expression in the non-neoplastic lesions with a significant difference between the three groups, denoting the association of EGFR expression with the malignant phenotype as well as the premalignant lesions. This finding is parallel to many other studies that concluded that EGFR over expression was a frequent finding in cases of laryngeal carcinoma [13],[14], and [18], Resta et al [19] have also reported positive expression of EGFR in premalignant laryngeal lesions, and suggested the presence of a strong relationship between EGFR and laryngeal carcinogenesis. Moreover , the high frequency of EGFR expression in laryngeal carcinoma cases is encouraging for efforts to use EGFR targeting agents for therapy in such cases, and even the cases of laryngeal dysplasia that show increased EGFR expression may benefit from that therapy to avoid progression to invasive carcinoma [5],[20],[21].

Although patient’s age, sex, smoking and tumor localization didn’t correlate with EGFR expression in the present study, EGFR over expression was highly significantly associated with higher tumor grade, advanced tumor stage as well as with the presence of cervical lymph node metastasis. Since these factors are proven to be of poor prognostic value in laryngeal carcinoma [22],[23], and [24], so EGFR over expression may predict the aggressive biological behavior and poor prognosis in Egyptian patients with laryngeal carcinoma.

Some previous studies have also reported EGFR overexpression in laryngeal tumors of higher grades [14],[25], more advanced tumor clinical stage (14, 18) and in those associated with cervical lymph node metastasis [24] Other studies however didn’t find a significant correlation between EGFR and the prognostic clinical parameters of laryngeal carcinoma as Demiral et al [18], but many of them and others found a strong association between EGFR over expression and reduced patient survival [18],[26],[27], confirming that it is a poor prognostic indicator in laryngeal carcinoma. Moreover, Almadori et al [14] added that over expression of EGFR is associated with an increased degradation of the extracellular matrix by metalloproteinases and cathepsin D, which plays an important role in tumor growth, invasion and metastasis, as well as in tumor-induced angiogenesis [8],[11],[20], and is potentially correlated with invasiveness.

Among the genetic alterations in human malignant tumors is ras mutation which has been identified in around 15% of human solid tumors. The ras proto-oncogenes encode a group of plasma membrane associated G-proteins that bind guanine nucleotides with high affinity and activate several downstream effector proteins including raf-1(P21). [10],[11]. Raf-1(P21) is known to activate several distinct signaling cascades that are involved in the regulation of cell survival, proliferation and differentiation in response to extracellular stimuli such as growth factors or hormones [11],[28],and [29].

At present few reports are available on P21 ras expression in laryngeal squamous cell carcinoma. Reports came mainly from Italy (14, 28), Spain [30], China [29] and Japan [31].Chang and associates [11] and others (12, 13) noticed high proportion of ras mutation in oral cancer in India as compared to UK, and attributed that to the type of tobacco smoked or genetic predisposition in Indians, moreover betel quid chewing and reverse smoking are probably initiating agents [11]. In the present study P21 ras expression was investigated in Egyptian patients as a model for Middle East population, and it showed the high prevalence of p21 expression in the laryngeal carcinoma cases (100%), as well as the laryngeal premalignant lesions (85.71%) as compared to the non neoplastic laryngeal lesions (6.67%) with a highly significant difference .This finding strongly suggests that p21 ras expression is associated with malignant phenotype and therefore may play an essential role in the carcinogenesis and the biology of laryngeal carcinoma in Egyptian patients. Moreover, this high incidence of p21ras expression in Egyptian patients is similar to results of other races in the literature [29],[31]. Because of the evidence of P21ras involvement in laryngeal carcinoma development, interruption of the ras pathway has been a major focus for drug development, these drugs either inhibit ras maturation and thereby membrane localization, or inhibit ras protein expression [32],[33]. Moreover, these drugs may be used to reverse dysplastic lesions that exhibit over expression of P21ras preventing progression to invasive carcinoma.

The present study has also shown no significant association between p21ras expression with age, sex or smoking suggesting that pathways of ras protooncogene activation in HNSCC in Egyptian patients may be different from those in Indian patients. Although the clinical tumor stage didn’t correlate with P21ras expression in the present study, P21ras over expression was highly significantly associated with higher tumor grade, as well as with the presence of cervical lymph node metastasis.

Moreover, P21ras over expression was significantly associated with carcinoma in supraglottic and transglot-tic regions. Since these factors are proven to be of poor prognostic value in laryngeal carcinoma [22],[27], so P21ras over expression may also predict the aggressive biological behavior and poor prognosis in laryngeal carcinoma.

Some previous studies reported similar findings [9],[14], as some found a significant correlation between ras oncogene expression and clinical stage of the tumor especially at T3 andT4 [9]. Moreover, some other studies didn’t find any significant correlation with the clinical parameters [30],[31]. This controversy suggests that the biological behavior of laryngeal carcinoma with ras oncogene mutations may be modulated or aggravated by other factors in different patients. As previously mentioned in the literature that aberrant ras signaling can be activated by over expression of growth factor receptors such as EGFR [33], the current study has shown a highly significant association between EGFR over expression and P21ras over expression in the laryngeal carcinoma cases. This finding suggests that EGFR over expression may play an essential role in the activation of ras-raf signaling pathway in laryngeal carcinoma in Egyptian patients, and since both EGFR and Ras pathways are currently targets for cancer therapy with specific drugs [15],[16], [17] therefore drugs targeting both markers may have a complementary effect in the treatment of laryngeal carcinoma cases especially those with advanced clinical stage or radio-resistant cases.


  Conclusion Top


From this study we can conclude that there is a high incidence of simultaneous over expression of EGFR and P21ras in Egyptian patients with laryngeal Squamous cell carcinoma and both markers are significantly associated with poor prognostic clinicopathological parameters. Therefore we suggest that both markers can be used to select patients with malignant and premalig-nant laryngeal lesions who might benefit from drugs targeted to them. However, further studies including larger groups of patients and contributing treatment trials with the new therapeutic agents are recommended for evaluation of their effect.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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