INTRODUCTION
Oral cancer is a major health problem in the Indian subcontinent where it ranks among the top three types of cancer in the country (Elangoet al., 2006)(1). Age- adjusted rates of oral cancer in India arehigh that is, 20 per 100,000 population and accounts for over 30% of all cancers in the country (Sankaranarayananet al., 2005)(2). Increase in prevalence of oral cancer is observed due to cultural practices such as betel-quid chewing, and varying patterns of tobacco and alcohol use are risk factors that predispose to cancer of the oral cavity(Mamta Agarwal et al.2012,Sree Vidya Krishna Raoet al.,2013)(3). Smoking and smokeless tobacco usage are identified as the major risk factors(Sadeq Ali Al-Maweriet al.,2014)(4). Infection with high risk HPV genotypes 16 & 18 is also a risk factor for the development of Oral squamous cell carcinomas (Suyamindra S Kulkarni et al.,2011)(5).Oral cancer is often preceded by premalignant lesions such as oral submucous fibrosis, leukoplakia and lichen planus. An early recognition and timely intervention is the key to successful management of oral cancer and its prevention from attaining a malignant stage.Although, histopathology is the gold standard yet a number of diagnostic tools including clinical assessment,
ERA’S JOURNAL OF MEDICAL RESEARCH, VOL.4 NO.1
Accepted on : 15-06-2017
Address for correspondence
Dr. Nishi Tandon
Associate Professor,
Department of Pathology,
Era’s Lucknow Medical College &
Hospital, Lucknow-226003.
Email:drnishitandon@gmail.com Contact no. : +91-9452296953
REVIEW ARTICLE
DOI:10.24041/ejmr2017.16
SALIVARY EGFR AS BIOMARKER FOR ORAL CANCER & PRE-CANCER :A
CASE CONTROL STUDY
Sumaiya Irfan*, Nishi Tandon*, Mohammad Iqbal**, A.N. Srivastava*,Nirupma Lal*, Vijay Kumar***, Department of Pathology*, Otorhinolarngology**, Neurosurgery**, Surgical oncology***,
Era's Lucknow Medical College & Hospital, Lucknow, U.P., India-226003.*
Sanjay Gandhi Institute of Medical Science, Rae Bareli Road, Lucknow, U.P., India-226014.**
King George Medical University, Lucknow, UP, India.-226003***
Received on : 06-06-2017
ABSTRACT
VOL.4 NO.1

ERA’S JOURNAL OF MEDICAL RESEARCH

Oral cancer is one of the leading cause of cancer in India, new diagnostic modalities for early diagnosis and treatment will increase the survival of the patients. The present study was carried out with an aim to evaluate salivary levels of EGFR in oral cancer and pre-cancer as tumor marker. For this purpose an observational case-control study was carried out in which a total of 72 subjects were enrolled.Of these 24 (33.3%) were patients of premalignant oral lesions and 24 (33.3%) were patients of malignant oral lesions& 24 (33.3%) subjects were normal healthy controls. Demographic information and clinical data was obtained, estimation for EGFR was

performed in saliva. In premalignant cases, buccal mucosa was the most common site involved (87.50%) whereas in malignant cases tongue was the

most common site involved (n=10;41.67%). Mean salivary EGFR levels

were higher in malignant cases (0.23±0.17 pg/ml) and low in controls (0.10±0.19 pg/ml). Mean EGFR levels in premalignant cases were 0.12±0.22 pg/ml. statistically, this difference was not significant (p=0.052). Statistically, no significant difference in mean EGFR levels among different TNM stages could be seen (p=0.145). EGFR levels showed a potential to discriminate between malignant and premalignanat cases but this difference was statistical insignificant due to lower sample size.

KEYWORD: Epidermal Growth Factor, Squamous Cell Carcinoma, Salivary Biomarker.

biochemical and laboratory assessments are performed with variable accuracy.

Saliva as a biofluid is historically well studied biochemically & physiologically, has entered the postgenomic era where its proteomics, genomics & m i c r o b i o m i c c o n s t i t u e n t s h a v e b e e n comprehensively deciphered. An increasing number of systemic diseases and conditions, amongst them oral cancer, have been shown to be reflected diagnostically in saliva. Moreover, using saliva as a diagnostic fluid meets the demands for inexpensive, noninvasive, and accessible diagnostic methodology (Markopoulos et al., KMK Masthanet al. ,2010)6,7.

Significant difference in level of various proteins ,mRNA, enzymes is seen in saliva of OSCC and control patients these could help in early detection of disease,(8).

Saliva has been found to contain constituents that reflect the diseased or physiological state of the human body, and hence could be utilized for diagnostic purposes (Wong, 2006; Wong, 2006; Castagnolaet al., 2011)(9-11), Salivary biomarkers, whether produced by healthy individuals or by individuals affected by specific disease, are sentinel molecules that could be used to scrutinize health and disease surveillance.

Page: 21

SALIVARY EGFR AS BIOMARKER FOR ORAL CANCER & PRE-CANCER :A CASE CONTROL STUDY

Saliva has been long proposed and used as a diagnostic medium (Malamud, 1992; Strekfus and Bigler, 2002)(12,13), because it is easily accessible and its collection is non-invasive, not time-consuming and inexpensive, requires minimal training and can be used for the mass screening of large population samples.

Various biomarkers have been associated with the pathway included with oral cancer like IL-6, IL-8, TNF-α, MMP, Cyclin D1, Ki-67, Maspin, Endothelin- 1, EGFR, Her-2, EGF (Balicki et al., 2005; Pickering et al., 2007; Shpitzeret al., 2009; Bernardeset al., 2010; Cheng et al., 2011; Cheng et al., 2014;Rajkumar Krishnan et al.,2014)(14,-19). Epidermal growth factor receptor (EGFR) is a 170-kilodalton (kD) trans membrane cell-surface receptor. It is a tyrosine kinase (TK) receptorthat is commonly altered in epithelial tumors.

The present study was planned with an aim to evaluate salivary levels of EGFR in oral cancer and pre-cancer as tumor markers.

MATERIALAND METHODS

The present study was undertaken at the Department of Pathology Era's Lucknow Medical College, Lucknow. A total of 72 subjects were enrolled for the study. 24 cases of premalignant oral lesions, 24 cases of squamous cell carcinoma & 24 healthy age and gender matched controls. The samples taken were saliva from cases and controls. Tissue biopsy from cases for confirmation of diagnosis. With the consent of the patient's whole, unstimulated saliva was collected from cases and controls (Salimetrics, LLC; 2009),(20).

The subjects were asked to accumulate saliva in the floor of the mouth before spitting in a vial kept on ice every 60 seconds. Approximately 5ml of saliva was collected for analysis.Following collection, saliva samples were centrifuged at 2600g for 15 minutes at 4 degree Celsius. Proteinase inhibitor cocktail (P2714 Sigma Aldrich) was added to each milliliter of the supernatant to prevent protein degradation. All samples were stored at -80 degree Celsius until further used.

Tissue biopsy was also taken from the same patient after collection of saliva. Once the biopsy was taken it was further subjected to routine H & E staining procedures by taking appropriate steps on formalin fixed paraffin embedded tissue. Once the procedure was carried out, their results were compared and noted.

Salivary EGFR was analyzed with ELISA using kit (SEA757Hu) Uscn life Sciences Inc .Results were tabulated and subjected to analysis by SPSS 15 software.

RESULTS

Age of cases ranged from 13 to 70 years. Majority of cases were aged 40 years and above (n=27; 56.3%).Buccal mucosa was found to be the affected site in higher proportion of premalignant cases (87.50%). Tongue was affected in higher proportion of SCC cases (41.67%)

Expression of EGFR(Table 1.0,Graph 1.0) was relatively much lower in control group and ranged between 0 and 0.64pg/ml. EGFR values of premalignant cases ranged between 0 and 0.66 pg/ml while range of EGFR in SCC cases was between 0 and

0.88pg/ml. Mean EGFR values of controls (0.10+0.19 pg/ml) and premalignant cases (0.11+0.23 pg/ml) was found to be lower than that of SCC cases (0.23+0.17 pg/ml) and difference in EGFR values of controls, premalignant and SCC cases was not found to be statistically significant (p=0.052).An overlap of EGFR values of premalignant and controls was found. Extreme and outlier values were also found SCC cases and outlier values were observed in premalignant and Controls.

Among 24 cases of SCC, Salivary EGFR values was found to be lowest for T1N1M0 stage (0.17+0.21 pg/ml) and maximum for T1N0M0 (0.66+0.31 pg/ml) and T2N1 0 (0.66+0.24 pg/ml), (Table 1.1,Graph 1.1)difference in Salivary EGFR values of SCC cases of different TNM stages was not found to be statistically significant (p=0.145).

 

0.5

 

 

 

0.4

 

 

 

0.3

 

 

Mean±SD

0.2

 

 

0.1

 

 

 

0

 

 

 

Premalignant Cases

SCC Cases

Controls

 

-0.1

 

 

 

-0.2

 

 

Graph 1.0: Comparison of Salivary EGFR

levels in Premalignant Cases, Squamous

Cell Carcinoma Cases and Controls

ERA’S JOURNAL OF MEDICAL RESEARCH, VOL.4 NO.1

Page: 22

Jul - Dec 2017

ERA’S JOURNAL OF MEDICAL RESEARCH

VOL.4 NO.1

 

1.2

 

 

 

 

 

1

 

 

 

 

 

0.8

 

 

 

 

Mean±SD

0.6

 

 

 

 

0.4

 

 

 

 

 

 

 

 

 

 

0.2

 

 

 

 

 

0

 

 

 

 

 

T1N0M0

T1N1M0

T2N0M0

T2N1M0

T2N2M0

 

-0.2

 

 

 

 

Graph 1.1: Comparison of Salivary EGFR

values in different TNM Stage of Squamous

Cell Carcinoma Cases

DISCUSSION

Of various biomarkers available, we evaluated and compared the expression of salivary EGFR with an objective to differentiate normal, precancerous and cancerous lesions. For this purpose a total of 72 salivary and tissue specimen were obtained from 24 cases of premalignant oral lesions, 24 cases of squamous cell carcinoma and 24 healthy volunteers.

Age of cases ranged from 13 to 70 years. Majority of cases were aged 40 years and above (n=27; 56.3%). There were only 2 (4.17%) cases in age group <20 years. Mean age of cases was 40.83±11.85 years. Similar to results in present study, Gambhiret al. (2011)21, found 63.0% of their patients to be above 40 years of age. However, Mathew et al. (2008)22, in their series, also found majority of oral cancerous and precancerous lesions in subjects aged >40 years. Oral lesions most commonly occurs in middle-aged and older individuals, although a disturbing number of these malignancies is also being documented in younger adults in recent years (Chen et al., 1990; Llewellyn et al., 2001; Schantz and Yu, 2002)23, 24,

25.Oral lesions in young adolescents and children have also been reported in literature. Mathew et al.

(2008)21,in their study found nearly 9% of their cases to be below 20 years of age. In the series of Gambhiret

al. (2011)20, 18.4% of patients were aged <20 years. In present study, only 1 ( 2.1%) case was below 20 years of age. The increasing prevalence of oral lesions in younger age groups might be attributed to increasing prevalence of adverse oral and dietary habits (poor oral hygiene, pan masala, tobacco, smoking, spicy food consumption) (Ranaet al., 2009; Jha and Parmar, 2011)26, 27.

In present study, all the malignant cases had oral squamous cell carcinoma. Oral squamous cell carcinoma is the most common type of oral cancer and generally has involvement of tongue (Mirboud and Ahing, 2000; Epstein et al., 2008)28, 29. In a retrospective as well as prospective study of changing pattern of oral lesions carried out at Allahabad (India), Misraet al. (2009)30, also reported tongue to be more commonly involved in squamous cell carcinoma cases whereas buccal mucosa to be more commonly involved in premalignant cases in their prospective series. Similar observations have been reported in other studies too (Neville et al., 2009; Shivakumaret al., 2010)31, 32.

Yi Fanet al. did a study to analyze the survival rate in oral cancer patients and found out that disease free survival rate were significantly higher in patients with early-stage disease than with advanced stage(Yi Fanet al. ,2014)(33).

In present study, it was observed that mean EGFR levels were minimum in Control (0.10±0.19 pg/ml) and premalignant groups (0.12±0.22 pg/ml) and maximum in SCC cases (0.23±0.17 pg/ml). None of the between group differences were significant (p>0.05). The relationship between EGFR levels and oral malignancy has yielded mixed results. Inoet al. (1993)34, in their study showed that salivary EGF levels were markedly low in patients with oral inflammations (stomatitis aphthous, or peritonsillar abscess) or head and neck tumors (squamous cell carcinoma of the tongue, oral cavity, hypopharynx or larynx). Bernards et al. (2010)16, in their study did not find a significant difference in salivary EGFR levels between OSCC cases and healthy controls before the surgical procedure. However, in their study they found mean EGFR levels of OSCC cases to be significantly increased by showing a multifold increase after the surgery. The finding thus suggests that post-surgery EGFR levels were influenced by removal of tumor. However, the authors could not provide a justifiable explanation for such fall. IHC expression of salivary EGFR has been shown to be significantly increased between pre-operative OSCC and healthy controls and showed a significant difference. Similar findings were made in our study using different estimation criteria but we could not

ERA’S JOURNAL OF MEDICAL RESEARCH, VOL.4 NO.1

Page: 23

SALIVARY EGFR AS BIOMARKER FOR ORAL CANCER & PRE-CANCER :A CASE CONTROL STUDY

achieve a statistically significant association.

We also attempted to evaluate the association of TNM staging with EGFR levels and found a random but significant relationship between tumor staging/nodal involvement and EGFR levels (p=0.003). Although this relationship was statistically significant yet owing to fewer numbers of cases with each TNM stage, it would be hastier to generalize these results. For trend, the lone case of T2N2M0 stage had EGFR expression of 22.98 pg/ml while the range in other TNM stages ranged from 2.57 to 15.43 and mean values in different TNM stages ranged from 5.42±2.48 pg/ml to 7.96±2.29 pg/ml. Given the stage sample sizes ranging from 1 to 8 samples, it is difficult to find out any linear relationship.

With respect to salivary EGFR levels there are limited studies using the technique and methodology like ours. The study closest to our study was that of Inoet al. (1993)(33), and Bernardes et al. (2010)(16), but both had different end points. Serum EGFR levels have shown to have mixed outcome as far as significance of differences between SCC patients and controls is considered. Feng et al. (2010)(35), reported a significant difference while Gokhaleet al. (2005)(36),did not find a significant difference between two. YasamanSardariet al (2012)(37), did a study to evaluate the salivary level and tissue expression of HER2/neu (a member of EGFR family) in patients with head and neck squamous cell carcinoma and observed that there was no overexpression of HER2/neu.

One of the reasons for such discrepancies is the fact that the level of research on this issue is in preliminary phase and the samples included in the study are fewer in number, lack a uniform estimation method and are exploratory in nature rather than being conclusive.

With respect to GFR levels, the trends obtained in present study indicates that malignant cases generally had higher mean values as compared to premalignant and controls yet the association could not be proven statistically. High variability in range of EGFR levels was observed within each group, thus indicating that the EGFR levels are also affected by some confounding variables, however, the impact of removal of confounding effect by strict criteria could not result in exclusion of confounding effect.

On discussing the issue of high within group variability and possible impact of time of collection, posture at which collection was made, comorbidities and processing conditions.Multiple sampling might be a resolution for some of these issues. It is noteworthy to mention here that for markers that have a high within group variation in expression, the strategy of

averaging the levels obtained for multiple specimens could be a better alteration that could be explored. However, in present study this proposition could not be tested and hence it is one of the recommendations of the present study for such estimations in future

The findings in present study, thus indicate a possible role of EGFR levels in differentiation of normal, premalignant and malignant oral lesions which is based on a sound theoretical basis but has been addressed using different methodologies and has limited human studies. The present study adds to the pool of knowledge on the issue and makes some recommendations based on the observations in the present study. The findings of the present study substantiate the observation that salivary EGFR has the potential to discriminate between oral tumors of different types. However, it is disappointing to see that there are limited studies on the issue and as such the work done so far is of pilot level, pinpointing the deficiencies in each study and providing stepping stones for further assessment. In view of high within group variability and exploration of the potential factors responsible for led us to assume that in order to neutralize effect of these potential factors multiple specimen taken at a suitable time apart could be the option. In view of the observations made in the present study, we recommend further studies on improved methodology and on a larger sample size.

CONCLUSION

EGFR levels showed a potential to discriminate between malignant& premalignant oral lesions but were not corroborated statistically. Standardization of estimation assays and multiple sampling is suggested as a possible improvement to yield more consistent and discriminatory outcomes. Further studies on these recommendations are suggested.

REFERENCES

1.Elango JK, Gangadharan P, Sumithra Set al. Trends of head and neck cancers in urban and rural India, Asian Pacific Journal of Cancer Prevention.2006; 7: 108–112.

2.Sankaranarayanan R, Ramadas K, Thomas G, et al. Effect of screening on oral cancer mortality in Kerala, India: a cluster-randomised controlled trial. The Lancet.2005; 9475: 1927–1933.

3.SreeVidya Krishna Rao, Gloria Mejia, Kaye Roberts-Thomson,etal.Epidemiology of Oral Cancer in Asia in the Past Decade- An Update

(2000-2012). Asian Pac J Cancer Prev.2013; 14: 5567-5577.

4.Sadeq Ali Al-Maweri, AbdallahAddas, BasselTarakji, et al.Public Awareness and

ERA’S JOURNAL OF MEDICAL RESEARCH, VOL.4 NO.1

Page: 24

Jul - Dec 2017

ERA’S JOURNAL OF MEDICAL RESEARCH

VOL.4 NO.1

Knowledge of Oral Cancer in Yemen.Asian Pac J Cancer Prev.2014;15: 10861-10865.

5.Suyamindra S Kulkarni, Sujayendra S Kulkarni, Priyanka P Vastrad, et al. Prevalence and

 

D i s t r i b u t i o n o f H i g h R i s k H u m a n

 

Papillomavirus (HPV) Types16 and 18 in

 

Carcinoma of Cervix, Saliva of Patients with

 

Oral Squamous Cell Carcinoma and in the

 

General Population in Karnataka, India. Asian

 

Pacific J Cancer Prev.2011; 12: 645-648.

6.

M a r k o p o u l o s A K , M i c h a i l i d o u E Z ,

 

Tzimagiorgis G. Salivary Markers for Oral

 

Cancer Detection. The Open Dentistry Journal

 

.2010; 4: 172-178.

7.KMK Masthan, N AravindhaBabu, Kailash Chandra et al .Advanced Diagnostic Aids in Oral Cancer. Asian Pacific J Cancer Prev.2102; 13: 3573-3576.

8.R.S Bedi, GauriPande, Jaipal Singh Patel, Zeeshan Khan and Nivedita Chauhan. Oral cancer: a review. Era's journal of medical research.2017;2(1):22-14.

9Wong DT. Salivary diagnostics for oral cancer. J Calif Dent Assoc.2006; 34: 303–8.

10.Wong D. Salivary diagnostics powered by nanotechnologies, proteomics and genomics. J Am Dent Assoc.2006; 137:313–21

11.Castagnola M, Picciotti PM, Messana I, et al. Potential applications of human saliva as

diagnostic fluid. Acta Otorhinolaryngol Ital.2011; 31: 347–57.

12.Malamud D. Saliva as a diagnostic fluid. Br Med J.1992;8: 207-8.

13.Streckfus CF, Bigler L. Saliva as a diagnostic fluid. Oral Dis.2002;8:69-76.

14.Balicki R, Grabowska SZ, Citko A. Salivary epidermal growth factor in oral cavity cancer. Oral Oncol.2005;41:48-55.

15.Pickering V, Jordan RC, Schmidt BL. Elevated salivary endothelin levels in oral cancer patients a pilot study. Oral Oncol.2007; 43: 37–41.

16.Shpitzer T, Hamzany Y, Bahar G, et al. Salivary analysis of oral cancer biomarkers. British Journal of Cancer .2009; 101: 1194-1198.

17.Bernardes VF, Gleber-Netto F, Sousa SF, et al. Clinical significance of EGFR, Her-2 and EGF in oral squamous cell carcinoma: a case control study. Journal of Experimental&,Clinical Cancer Research. 2010;29: 1-7.

18.Cheng Y-SL, Rees T, Wright J. A review of research on salivary biomarkers for oral cancer

detection. Clinical and Translational Medicine. 2014; 3:3.

19.Rajkumar Krishnan, Dinesh Kumar Thayalan, Rajashree Padmanaban, et al .Association of Serum and Salivary Tumor Necrosis Factor-α with Histological Grading in Oral Cancer and its Role in Differentiating Premalignant and Malignant Oral Disease.Asian Pac J Cancer Prev.2014; 15: 7141-7148.

20.Salimetrics, LLC.(2009).

21.Gambhir RS, Veeresha KL, Sohi R, et al. The prevalence of oral mucosal lesions in the patients visiting a dental school in Northern India in relation to sex, site and distribution: A retrospective study. J ClinExp Dent. 2011;3: 10-7.

22.Mathew AL, Pai KM, Sholapurkar AA, et al . The prevalence of oral mucosal lesions in

patients visiting a dental school in Southern India. Indian J Dent es.2008;19:99-103.

23.Chen JK, Katz V, Krutchkoff DJ. Intraoral squamous cell carcinoma. Epidemiologic patterns in Connecticut from 1935 to 1985. Cancer 1990;66:1288-1296.

24.Llewellyn CD, Johnson NW, Warnakulasuriya KA. Risk factors for squamous cell carcinoma of the oral cavity in young people — a comprehensive literature review. Oral Oncol. 1992;37:401-418.

25SchantzSP,Yu GP. Head and neck cancer incidence trends in young Americans, 1973- 1997, with a special analysis for tongue cancer. A r c h O t o l a r y n g o l H e a d N e c k S u rg . 2002;128:268-274.

26.Rana ZA, Khoso NA, Bajaj DR, et al. Risk Factors for Precancerous Lesions of Oral Mucosa. Ann. Pak. Inst. Med. Sci. 2009; 5: 220-223.

27.Jha R, Parmar DP. A study of precancerous lesions for oral cancer in Jamnagar city. J. Ind. Acad. Oral Med. Radiol.2011; 23: S333-335.

28.Mirbod SM, Ahing SL. Tobacco-Associated

Lesions of the Oral Cavity: Part II. Malignant Lesions. J Can Dent Assoc.2000; 66:308-11.

29.Epstein JB, Gorsky M, CabayRJ,et al . Screening for and diagnosis of oral premalignant lesions and oropharyngeal squamous cell carcinoma. Can Fam Physician.2008;54:870-5.

30.Misra V, Singh PA, Lal N, et al. Changing Pattern of Oral Cavity Lesions and Personal Habits Over a Decade: Hospital Based Record Analysis from Allahabad. Indian JCommunity Med. 2009;34: 321–325.

ERA’S JOURNAL OF MEDICAL RESEARCH, VOL.4 NO.1

Page: 25

SALIVARY EGFR AS BIOMARKER FOR ORAL CANCER & PRE-CANCER :A CASE CONTROL STUDY

31.Neville BW, DammDD,Allen CM, et al. Oral & maxillofacial pathology. Third ed. Phila., PA: Saunder.2009;425-433.

32.Shivakumar GC, SahanaS(2010). Correlation between the Functional and Histological Staging of Oral Submucous Fibrosis. J. Ind. Acad. Oral Med. Radiol. 2010; 22:133-135.

33.Yi Fan, Lei Zheng, Ming-Hui Mao, et al . Survival Analysis of Oral Squamous Cell Carcinoma in a Subgroup of Young Patients. Asian Pac J Cancer Prev.2014; 15: 8887-8891.

34.Ino M, Ushiro K, Ino C, et al. Kinetics of epidermal growth factor in saliva. Acta Otolaryngol Suppl.1993;500:126-30.

35.Feng XY, Li JH, Li JZ, et al. Serum SCCA, Cyfra

21-1, EGFR and Cyclin D1levels in patients with oral squamous cell carcinoma. Int J Biol Markers.2010; 25:93-8.

36.Gokhale AS, Haddad RI, Cavacini LA, et al. Serum concentrations of interleukin-8, vascular endothelial growth factor, and epidermal growth factor receptor in patients with squamous cell cancer of the head and neck. Oral Oncology. 2005; 41:70–76.

37.Yasaman Sardari,SoheilPardis, Azadeh Andisheh Tadbir, et al.HER2/neu Expression in Head and Neck Squamous Cell Carcinoma Patients is not Significantly Elevated. Asian Pacific J Cancer Prev.2012; 13: 2891-2896

▄ ▄

How to cite this article : Irfan S., Tandon N,. Iqbal M., Srivastava A.N., Lal N., Kumar V., Salivary egfr as biomarker for oral cancer &

pre-cancer :a case control study, Era's Journal of Medical Research.2017;4(1):21-26.

ERA’S JOURNAL OF MEDICAL RESEARCH, VOL.4 NO.1

Page: 26