Cell Biology: Research & TherapyISSN: 2324-9293

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Research Article, Cell Biol Res Ther Vol: 3 Issue: 1

Ck AE1/AE3 Tumor Marker Expression in SCC of Head and Neck

Nauman Rauf Khan1*, Amna Nauman Khan2, Ejaz Waris3, Bilquis A. Suleman4 and Ayyaz Ali Khan5
1Oral Pathology, Sharif Medical & Dental College, Pakistan
2Community Dentistry, Sharif Medical & Dental College, Pakistan
3Department of Histopathology, Akhtar Saeed Medical & Dental College, Pakistan
4Department of Histopathology, University of Lahore, Pakistan
5Department of Oral Health Sciences, Shaikh Zayed Post Graduate Medical Complex, Pakistan
Corresponding author : Dr. Nauman Rauf Khan
Oral Pathology, Sharif Medical & Dental College, 397-Q, Model Town, Lahore, Pakistan
E-mail: [email protected]
Received: January 01, 2014 Accepted: March 10, 2014 Published: March 13, 2014
Citation: Khan NR, Khan AN, Waris E, Suleman BA, Khan AA (2014) Ck AE1/AE3 Tumor Marker Expression in SCC of Head and Neck. Cell Biol: Res Ther 3:1. doi:10.4172/2324-9293.1000109

Abstract

Immunostaining is one of the latest and accurate modality for evaluating the diagnosis and prognosis of tumors. It has also become a very important tool of differentiating between the tumors of different origin. Aims of the present study were to assess the frequency, pattern, sensitivity and specificity of CytokeratinAE1/AE3 as an immunohistochemical marker for the Squamous Cell Carcinomas(SCC) of head and neck.

Keywords: Squamous cell carcinoma; Immunostaining; Cytokeratin AE1/AE3; Head and neck cancer; Genotoxic stress; Oligomerization

Keywords

Squamous cell carcinoma; Immunostaining; Cytokeratin AE1/AE3; Head and neck cancer; Genotoxic stress; Oligomerization

Introduction

Cancer is an uncontrolled tissue growth resulting from the imbalance between the cell division and the programmed cell death [1]. This is a cloned growth of the cells that have under gone specific genetic and epigenetic alterations in oncogenes or tumor suppressor genes [2,3]. Genetic alterations are seen at all the stages including the pre-cancerous stage [4]. Pre-cancerous lesions refer to early changes in normal cells that are changing into cancer cells [5,6]. Clinically this represents as erythroplakia and leukoplakia [7] and histopathologically as hyperplasia, dysplasia or carcinoma in situ [8].
Lack of public awareness of cancer is one of a significant factor of its poor survival rate, but this disease could be found at early, highly survivable stages through a simple, painless, five minute examination by a trained medical or dental professional [9]. Routine HE staining of a biopsy is the basic modalities in the diagnosis, and the gold standard for diagnosis remains biopsy [10].
Immunohistochemistry (IHC) techniques are being used to supplement H&E routine stains. Adjunctive use of IHC with H&E sections enhances tissue interpretation, proper diagnosis and spares resection of additional tissue. Polyclonal antibodies used in IHC offer greater sensitivity than routine H&E stains and can easily detect the residual undetected tumor [11].
A good tumor marker must fulfill the criteria of sensitivity, specificity and clinical effectiveness. There has been no serological and biological tumor marker yet been established for the SCC of the head and neck [11].
Cytokeratins (CKs) constitute a family of intermediate filament proteins whose expression is generally confined to epithelia and their neoplasm. At least 20 different CKs have been described, which exhibit a highly diverse expression in various epithelia. These (CKs) are essential intracellular components, underlying or reflecting distinct cellular properties and differentiation stages in epithelial organs. Squamous, stratified epithelium is usually characterized by the expression of CK 5, which is found mainly in the basal cell layers and CK 5 is associated with the proliferative potential of these cells. The intermediary cell layers show an additional expression of CK’s 1 and 10, which are regarded as signs of cellular differentiation. In contrast, glandular epithelia reveal the expression of low-molecular weight cytokeratins CK’s 8/18 and 19 as typical features expression pattern of CK 8/18 is rather uncommon in mature squamous epithelium [12].
Anti-cytokeratin 14 antibody & CytokeratinAE1/AE3 has been reported to more accurately map out tumor cells of SCC within dense inflammatory aggregates. The stringent, but broad, specificity of pooled AE1/AE3 antibody has made this preparation very useful as a general stain for normal and neoplastic cells of epithelial origin [12].
Aim of the present study is to determine the potential significance of CytokeratinAE1/AE3 through determining its sensitivity, specificity, frequency and pattern of expression in the squamous cell carcinomas (SCCs) of the head and neck.

Materials and Methods

Study group comprised of total of 90 patients of which 60 were cases of benign lesions and the remaining 30 of Squamous cell carcinomas both from the head and neck region. It was done at Shaikh Zayed Hospital, Department of Histopathology, on the formalin fixed specimens. The female to male ratio was 1:1.7. The mean age of the participants was 44 ± 19.047 (SD), with age ranging from 3 years to 80 years. The SCC group was a primary sample (previously untreated), with female to male ratio of 1: 4, age averaging of 55 ± 11 (SD) and ranging from 30 -75 years.
The total 90 slides were made i.e., one slide out of each specimen for the Cytokeratin AE1/AE3. The SCC groups were receiving surgical treatment with curative intensions and were previously assessed through routine H&E staining. The control group was from the patient undergoing routine oral surgeries. Four-micrometer serial sections from formalin-fixed, paraffin-embedded blocks of tumor representative areas and representative benign tissue were cut for each case. Only sections containing sufficient epithelium to assess the antibody reactivity with 1000 cells were considered for this study. Immunohistochemistry was then performed on these sections using Ultra Vision LP Detection System, mounted on the slides which were previously coated with Poly-L-lysine (glass adhesive). To evaluate the anti-bodies expression, a mean percentage of positive tumor cells were determined from the percentage of positive stained cells derived from the analysis of 100 cells in 10 random areas at 400X magnification. The positivity for marker was evaluated independently by three authors other histopathologists and were blinded to the clinicopathological data and scored by the intensity of the stain. After Deparaffinization and rehydration the slides were incubated in Hydrogen Peroxide Block for 10 minutes to reduce nonspecific background staining due to endogenous peroxidase, then slides were placed in the Phosphate Buffer Saline PBS and 4 changes were done, each for 5 minutes. Ultra V block was applied and slides were incubated for 5 minutes at room temperature to block non-specific back ground staining. After UV block, washing with PBS was done. After washing primary antibody application was done. Cytokeratin AE1/AE3 was used at the dilution of 1:50. i.e., 1 μL antibody: 50 μL PBS and incubated for 60 minutes at room temperature. After the primary antibody application, slides were again washed in the PBS solution. Primary antibody enhancer was applied and incubation was done for 10 minutes in the incubation chamber. After the primary antibody enhancer application slides were again washed in the PBS solution. HRP polymer was applied and incubation for 15 minutes. Slides were again washed in PBS and dried as and freshly prepared Substrate DAP was applied followed by counter staining which was done with the routinely available hematoxylin. After dehydration and clearing Cover slips were then applied with a mounting medium DPX.
Informed consent was taken prior to all the surgeries. Recorded data was coded and entered using SPSS statistical package version 16.0. Numerical data like the age of the patients was reported as mean and standard deviation. Range was also given along with the minimum and maximum values.
Score of each stain was determined against the different types of tissues used by applying the following criteria:
• If microscopic field has the stained cells equal to 10% or less, they were considered (–ve).
• More than 10% but less than or equal to 30% were considered single positive (+ve).
• More than 30% but less than or equal to 50% were considered double positive (++ve).
• Above 50% were considered to be triple positive (+++ve).
Nominal data like the status of case, gender, site of tissue and result of each stain was reported as frequency and percentages. Chisquare Test was used to test the association between age, gender, site of the tissue, size of the tissue and the grading of the tumor. For all analysis, p value of 0.05 or less was considered significant. Fisher exact test was applied to determine correlation between the grading of tumor and the score of the different stains. 2 x 2 tables were applied to determine the sensitivity and specificity of the markers.

Results

30 cases were of squamous cell carcinoma and 60 controls, both from the head and neck region. The female to male ratio among SCC cases was 1: 4. The mean age among the cases was 55 ± 11 (SD), with minimum age of 30years and a maximum age of 75 years.
The H&E staining of the SCC cases is given in Figure 1 a and b. The scoring of the marker among both cases and control are given among the following tables. In 60 control cases Cytokeratin AE1/ AE3 has cytoplasmic pattern of expression, staining the cytoplasm of the epithelial cells but commutatively staining is seen to be less than 10% of the total cells as shown in Figure 2. However, in SCCs cases different staining pattern has been observed. It has been seen that the pattern of expression differs with the grading of the neoplasm for each marker. In case of well differentiated neoplasm the staining pattern is similar to that of the normal mucosa with positive cell comprising of 10-30% of the total cells as shown in Figure 3. In moderately differentiated SCCs staining has been more intense and diffuses involving the suprabasal layers. In poorly differentiated SCCs, usually stained cells are greater than 50% of the total cells. However, the staining of keratin pearls has been seen to be intense and prominent as shown in Figure 4.
Figure 1a: Photomicrograph showing section of Oro-pharynx reveals feature of well differentiated Squamous cell carcinoma. Multiple keratin pearls are visible. (H&E Stain, 115X).
Figure 1b: Photomicrograph showing H&E staining of section of Oro-pharynx revealing features of well differentiated Squamous cell carcinoma. (H&E stain 462X).
Figure 2: Photomicrograph showing section of Nasal polypoidal tissue revealing features of normal epithelium. Brown cytoplasmic staining can be seen of the entire length of the epithelium with no staining of the underlying connective tissue. (Cytokeratin AE1/AE3 Stain, 115X).
Figure 3: Photomicrograph showing section of Oro-pharynx reveals feature of well differentiated Squamous cell carcinoma. Brown cytoplasmic staining can be seen with unstained oval to round nucleus. (Cytokeratin AE1/AE3 Stain, 115X).
Figure 4: Photomicrograph showing section of Oro-pharynx reveals feature of well differentiated Squamous cell carcinoma. Brown cytoplasmic staining of the keratin pearl can be seen with unstained oval to round nucleus. (Cytokeratin AE1/AE3 Stain, 462X).
The positive and negative scores of Cytokeratin AE1/AE3 are given in the Table 1. Table 2 is used to determine the sensitivity and specificity of the marker. Figure 5 shows trend lines for the marker Cytokeratin AE1/AE3. There is an increasing trend and gradient for poorly differentiated SCC to be more strongly stained as compared to well differentiated and moderately differentiated SCC. However, again there is a positive trend in both moderately and poorly differentiated SCC but the slope of poorly differentiated SCC is more steep and sharp.
Figure 5: Bar chart with trend lines for the Marker cytokeratin AE1/AE3 (X- Axis) Score of the stain. Cytokeratin AE1/AE3. (Y- Axis) Number of cases of SCCs.
Table 1: Positive and negative score of Cytokeratin AE1/AE3.
Table 2: Sensitivity and specificity of Cytokeratin AE1/AE3.
Chi-square Test was applied to test the association between age, gender, size of the tissue and the grading of the tumor with the scores of cytokeratin marker. P values were calculated for the association between score of the stain and age, gender and size of the tissue.
• score of the stain and the age is 0.28.
• score of the stain and gender is 0.292.
• score of the stain and size of tissue is calculated to be 0.484. All the p values calculated were seen to be insignificant.
No association between the score of the stain , the gender of the patient and the size of the tissue was seen with Cytokeratin AE1/ AE3. In addition, to assess the efficacy of the marker for the differential diagnosis of well differentiated and poorly differentiated SCCs, we evaluated the score of the stain (+++) of the marker and the grade1 and 3 tumors by fisher exact test. The result showed that there is a statistically significant association between the grade 3 tumors and score (+++) of the marker with 2-sided P value to be < .0001, with 95.83% specificity and 100% negative predictive value but for well differentiated SCCs , p value > .05, with 62.5% specificity and 71.4% NPV.

Discussion

A tumor marker is any biological marker measureable in tissue or body fluids and is related to the malignancy.13 A good tumor marker must fulfill the criteria of specificity and sensitivity [13]. A large number of markers have been examined in the squamous cell carcinomas of head and neck but their sensitivity and specificity values have always been controversial [14-16]. This study was conducted to assess the sensitivity and specificity of Cytokeratin AE1/ AE3 for diagnosing Squamous cell carcinomas of the head and neck, taking H&E as a gold standard.
In cytokeratin the pattern of expression is seen to be cytoplasmic. In our study, Cytokeratin AE1/AE3 expression was seen to be in the entire mucosa but more prominent and stronger in the basal cell layer which is considered to be the anatomical location of the progenitor cells. However, Ck5/6 and Ck19 are also reported to be expressed mainly in the basilar layer [17-19]. Suprabasal expression of Ck19 seems to be correlated with premalignant transformation in oral epithelium [20] but Ck8 and Ck18 are not expressed or are rather uncommon in mature squamous epithelium [17,18]. In contrast, glandular epithelia reveal expression of low-molecular weight cytokeratins Ck’s 8/18 and 19 as typical features [17,18]. The intermediary cell layers show an additional expression of Ck’s 1 and 10, which are regarded as signs of cellular differentiation [17]. Ck7 and Ck20 is also reported to be more commonly expressed in the glandular epithelium [21]. Tumor markers like p53 and p63 has repeatedly shown its appearance in the Basal cell layer and Suprabasaler layer [22-24]. Although the pattern of expression is different for both Cytokeratin and p53/p63 our study findings were close with investigations like layer [25-27] in which the expression of p63 in both the basal and parabasal layers with gradual decrease in the intensity as we moved to the superficial layers [22,28]. However, our study was in contradiction with studies reporting p63 staining in suprabasal layer [29] and other investigations reporting p63 staining predominantly in basal cell layer [22,23]. This difference can be due to the usage of different clone of p63 antibody in these studies. Stem cells of stratified epithelium have been repeatedly described as the major cellular targets for cancer causing mutations and therefore might give in a long term rise to the development of SCC’s [21,30]. In the context of the existing literature it seems logical that SCC’s retain and are phenotypically characterized by the increase expression of Cytokeratins.
In squamous neoplastic lesions, the numbers of Cytokeratin positive cells were outstandingly increased as compared to the normal mucosa and this is in cohesion with the number of investigations [19,21,30-35]. The number of cells and the layers of cell stained both were remarkably increased. Both the basal and suprabasal layers were intensely stained by Cytokeratin AE1/AE3. Evidence cytokeratin positivity in SCCs has been stated by number of investigations but its comparison with clinico-pathological features has been rarely performed [23]. In our study, we evaluated any association between the score of the stain , the gender of the patient and the size of the tissue and it was seen to be insignificant which is in consistent with the studies conducted by L.L. Muzio et al. and Khan NR et al. showing no relationship of the number of positively stained cells with that of the age, sex and size of the tissue [23,28]. Our study displayed that there is a statically significant association between the percentage of positive cells to that of the grading of neoplasms. The more the grading of the tumor, more strongly was the expression of all markers, which is cohernt with the three investigations [23,28,36], however two of these three investigations also corellated with the perineural infilteration, metastasis and survival rates [28,36]. These factors like nodal involvement, perineural infilteration and survival rates were not included in our current study.
In case Cytokeratin AE1/AE3, sensitivity of 100%, specificity 81.53%, PPV 81.08% and NPV of 100% was seen. Work has been reported to be done on the cervical lymph nodal metastasis of the esophageal squamous cell carcinoma with sensitivity as high as 93%, [37,38] however, no sensitivity and specificity assessment of AE1/ AE3 has been seen in the literature regarding the squamous cell carcimoma of the head and neck. We have seen this marker to be highly sensitive with both the sensitivity and negative perdictive value of 100%. However, different clones of cytokeratin has been used on the squamous cell carcinoma but so far only CK 1, 5/6, 8/18, 10, 14, 19 has been reported to be sensitive but with poor specificity [30]. Many have been seen to be staining more of the malignant salivary gland tumors making them the markers for the glandular tissue [21]. CK 5/6 and CK 7/20 are the most common and significant cytokeratins seen significant in the salivary gland tumors [21]. CK 7 and 20 are seen to be negative in SCCs [21]. A study of p63, CK 5/6 and CK 7 was done on the neuroendocrine tumors and it was seen that both p63 4A4 and cytokeratin 5/6 are highly significant in neuroendocrine tumors but negative in the adenocarcinoma [39], and mesotheliomas [40]. Hence, p63 together with CK 5/6 can be used for the differentiation between squamous cell carcinoma from adenocarcinoma and mesothelioma [39-41].
Different studies has shown wide range in the sensitivity of p63 4A4 for the squamous cell carcinomas ranging from 75% to 86.6% [23,28,39,41], however we report to have higher sensitivity of Cytokeratin AE1/AE3i.e., 100%. Specificity seen for p63 4A4 ranges from 86% to 100% [23,40,41] but our value lies below this range which is 81.53%. Different studies have reported wide variation. These differences can be attributed to the different technical errors which each system may face during the staining procedures [42].
Furthermore, in the present study, as in other investigations, a positive association between Cytokeratin AE1/AE3 expression and the grade of neoplasm differentiation was noticed, supporting the use of Cytokeratoin AE1/AE3 as an additional marker for diagnostic use in H&N SCC.
We also assessed the sensitivity and specificity of our markers for the well differentiated and poorly differentiated SCCs. The specificity was low 62.5% with negative perdictive value (NPV) of 71.4% for well differentiated SCCs which was in cohesion to L.Lo Muzio et al and Khan NR [23,28,35]. For poorly differentiated SCCs the specificity of 95.83% and NPV of 100% was seen which is seen in concordance with LLo Muzio et al. and Khan NR both having NPV of 94.20 [28,35]. These statistics indicate that our markers are more specific for the poorly differentiated SCCs as compared to the well differentiated SCCs.Also if a combination of Cytokeratin AE1/AE3 is established with markers like p63 4A4 can aid in early detection and proper diagnosis.

Conclusion

• Cytokeratin AE1/AE3 plays important role in the normal cellular and carcinogenetic proliferation.
• Cytokeratin AE1/AE3 marker is seen to be significant in both sensitivity and specificity and can be used for a confirmatory diagnosis of the squamous cell carcinoma and is a proliferative marker of poor prognosis.

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