Lung carcinoma is the leading cause of cancer deaths in the world [1]. It is the most common carcinoma among males and is associated with a high mortality rate.The five year survival rate for male patients with lung carcinoma is around 6% to 14% and for female patients with lung carcinoma it ranges between 7% and 18%.

The number of estimated deaths due to lung carcinoma is 1.38 million per year [2]. The carcinoma of lung is broadly classified into Non-Small Cell Lung Carcinoma (NSCLC) which accounts for 85% and Small Cell Lung Carcinomas (SCLC) which accounts for about 15% [3].

Extensive research on the pathophysiology of lung carcinoma has suggested the existence of two distinct molecular pathways, one associated with smoking and activation of the KRAS oncogene and the other not associated with smoking but with activation of epidermal growth factor receptor (EGFR). Introduction of targeted therapy with epidermal growth factor receptors, the tyrosine kinase inhibitors, has revolutionized the treatment of adenocarcinoma. Patients with these tumors survive significantly longer with EGFR tyrosine kinase inhibitor therapy than with conventional chemotherapy [4-8].

Somatic mutations within the tyrosine kinase catalytic domain of EGFR leads to conformational changes that promote permanent active status and are found in approximately 20% of lung adenocarcinomas [9-10]. They are considered to be the most reliable predictors of response to EGFR tyrosine kinase inhibitors [11].

EGFR inhibitors can be mainly categorized into two classes: monoclonal antibodies against the extracellular domain of EGFR, such as cetuximab; and small-molecule tyrosine kinase inhibitors that target the kinase domain, such as erlotinib and gefitinib [12-18]. Sahoo et al observed that 51.8% of the NSCLC population had EGFR mutations in their DNA[19]. This suggested that high prevalence of EGFR positivity is seen in NSCLC in the Indian subpopulation therefore providing opportunities for targeted therapy.

Study setting and study participants: This cross-sectional study was carried out in the

Immunohistochemical staining for total EGFR protein was performed using the Biogenex monoclonal EGFR rabbit antibody; clone (Tyr p1068) prediluted in PBS on both control and test sections according to the 0 manufacturer’s instructions.

Assessment of Immunohistochemical staining: Initially, slides were scanned at 10xmagnification to obtain a general impression of the overall staining pattern of the tumor cells.H score was performed for which 10 fields were chosen randomly at 400 x magnification and the intensity of staining of EGFR was scored on a scale of 0 to 3 based on the staining of the cell membrane of the tumor cells. (Table 1)

Table-1: H score for intensity of staining of EGFR[20].

The total number of 100 cells in each field was counted and the number of cells stained for each level of intensity was counted. A H-score between 0 and 300 was obtained where 300 was equal to 100% of tumour cells stained strongly (3 +). A cutoff Hscore of above 100 was used to categorize tumors as positive,and tumors with scores of 100 and less were categorized as negative [20]. Squamous cell carcinoma of cervix was used as a positive control.

Data analysis: Data was entered and analyzed using SPSS ver 20 software. Chi square test was used to analyze the statistical significance between EGFR expression and clinicopathological parameters. A p value <0.05 was considered statistically significant.

A total of 111 specimens of lung cancer were received during the study period. Among them, majority constituted non small cell carcinoma (95%). Among the non small cell carcinoma, majority of the cases were adenocarcinoma (75) followed by squamous cell carcinoma (27) (Figure 1).

Majority of the participants were > 60years of age in this study. Males were predominant (72%) compared to females. Most tumors were present in the upper lobe (57.3%) followed by lower lobe

Table-4: Association between EGFTable 4: R expression and distant metastasis:

Figure-2: Moderately Differentiated adeno carcinoma 200X

Figure-3: EGFR IHC Staining showing 1+

Figure-6: ALK IHC showing 1+ Positivity 200x

The histological features of the adenocarcinoma are depicted in table 3. Majority of the tumors were moderately differentiated (46%) and were negative for lymph node metastasis (69%). With regards to EGFR positivity, majority of the tumors showed EGFR expression 3+ (57.3%) followed by 2+ (16%). Among the EGFR negative cases, ALK expression was positive in 5% of the cases. (Table 3)

This study showed a statistically significant association between EGFR positivity and presence of distant metastasis (p<0.05) (Table 4) The immunohistochemical staining demonstrating EGFR expression are depicted in Figures 2-6.

Lung carcinoma is the leading cause of cancer related mortality.It accounts for approximately 1.4 million deaths per year worldwide.Increasing Knowledge about the pathogenesis of lung carcinomas especially adenocarcinoma has led to the development of newer therapeutic strategies.In the present study NSCLC accounted for 95% of the lung cancers and adenocarcinoma constituted the major histologic subtype. This was in concordance with worldwide incidence [2]. In the present study the mean age at diagnosis was 62 years.This was similar to the findings of Noronha V et al[2] In the present study,64% of the participants with NSCLS were non smokers. Similar findings were observed in the data reported by Samet JM et al[21]. In the present study the site,laterality and grading had no significance in lung carcinoma.

The assessmentof the EGFR status in adenocarcinomas with pTyr1068 phosphorylated EGFR which is a predictive biomarker for screening the NSCLC with wild type of EGFR was done, as they are responsive to tyrosine kinase inhibitors treatment [22]. The EGFR expression was also correlated with the clinico-pathologic parameters. EGFR positivity was seen in 73% of adenocarcinomas which is in concordance with the study done by Wang et al[22] The EGFR positivity was more common in the 6^th decade and in males (74%).

EGFR was positive in 87% of smokers and 71% ofnon smokers. EGFR positivity was seen 100% in patients who had lesions in the middle lobe and in patients who had lesions in both the lungs.

Ruschoff et al evaluated the inter-observer reprodu-cibility of the EGFR IHC scoring system based on both the tumor cell membrane staining intensity (graded 0 to 3+) and the percentage of cells staining at each level of intensity, as was done in the present study.This allowed a highly reproducible allocation of NSCLCs into clinically relevant high or low EGFR expression groups [26] Determination of H score and a cut off value as suggested by Liang et al was useful in determining clear cut positive and negative groups[20]. In the present study EGFR expression was high (73%), which emphasizes the need and importance for IHC screening of Indian patients with adenocarcinoma lung.

It paves way for targeted therapy when combined with subsequent molecular techniques for specific EGFR mutation analysis. This adds more value to the treatment of lung adenocarcinoma patients.The antibody used in the present study pTyr1068 has been proposed to be a predictive biomarker for screening the population for clinical outcomes of EGFR-TKIs treatment,especially for patients with wild type EGFR [27-30].

It was further studied that the ALK positivity in the EGFR negative patients as EML4-ALK fusion has been observed to be present in adenocarcinoma patients lacking EGFR mutation [31]. It was observed ALK positivity in 5% of the EGFR negative cases, which was similar to the literature published[32-34].The single case which showed ALK positivity had a low level of expression (1+), however was similar to the study done by P. Hofman et al[35].This expression pattern has been explained due to the fact that ALK rearrangements in lung carcinoma are usually associated with low levels of ALK expression in contrast to anaplastic lymphomas with ALK rearrangements where the protein is robustly detected by immunohistochemistry[36].

Limitations

• Treatment details of the study cases have not been analysed.
• Molecular studies have to be performed for specific EGFR mutation

Introduction of targeted therapy with EGFR and ALK TKI has revolutionized the treatment of adeno-carcinoma. Patients with these mutations survive significantly longer with TKI therapy than with conventional chemotherapy.