EGFR Mutational Status of Primary Lung Adenocarcinoma in an Indian Cohort Based on 2015 WHO Classification of Lung tumors

Background: Epidermal growth factor receptor (EGFR) mutations have been known to be associated with adenocarcinoma, women, nonsmokers and East-Asian ethnicity. This study was aimed to characterize the frequency of EGFR mutations and their association with histologic subtypes in primary lung adenocarcinoma in an Indian cohort. Methods: Two seventy-four cases were categorized using 2015 WHO classification of lung tumors. The frequency of each histologic subtype and cell type was correlated with EGFR exon sequences in a subset of 120 cases using polymerase chain reaction (PCR) gene sequencing. Results: The predominant biopsy categories in 274 cases were acinar 167(61%), solid 63(23%), mucinous 19(7%), lepidic 11(4%) and others 14(5%). EGFR mutations were detected in 49/120 (40.8%) including 3/5(60%) lepidic, 4/9(44.4%) papillary, 29/68(42.7%) acinar, 10/24(41.7%) solid and 1/13(7.7%) mucinous subtypes and were significantly associated with the cuboidal cell type (p=0.01). These mutations were common in women and non-smokers, although not statistically significant. Exon 19 mutations predominated in 36/49(73.4%). The majority, 213/263 (81%), were thyroid transcription factor 1(TTF-1) positive. The polygonal cell type and the solid subtype were frequent amongst stage IV tumors and smokers. Conclusions: EGFR mutations were most frequently seen with the lepidic and papillary subtypes, not associated with the mucinous subtype, more common in women and non-smokers and significantly associated with the cuboidal cell type.


Introduction
Non-small cell lung cancer (NSCLC) accounts for 75-80% of all lung cancers. Histologically, NSCLC is classified into squamous cell carcinoma, adenocarcinoma, and large cell carcinoma. In India, squamous cell carcinoma has been the most common histological type of NSCLC with a growing predominance of adenocarcinoma [1]. Lung adenocarcinomas are well known to display inter and intra-tumoral heterogeneity with profound implications for exact histological classification by pathologistsaccounting for more deaths than breast, prostate and colon cancer combined. [2] While treatment decisions are determined primarily by stage, therapeutically non small cell lung cancer (NSCLC]. The New International classification (IASLC/ATS/ERS) [3,4] and 2015 World Health Organization (WHO) classification [5] "defined the adenocarcinoma in situ(AIS), minimally invasive adenocarcinoma (MIA) and invasive lepidic adenocarcinoma, replaced the 'mixed subtype category' in 2004 WHO classification [6] with the predominant subtype, included the micropapillary subtype and replaced the term "mucinous bronchioloalveolar carcinoma (BAC)" with invasive mucinous adenocarcinoma.
Over the past decade, introduction of targeted therapy with epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKI) has revolutionized the treatment of adenocarcinoma. EGFR activating mutations are associated with clinical phenotype like females, non-smokers, East-Asian ethnicity, and adenocarcinoma, demonstrating responsiveness to tyrosine kinase inhibitors like gefitinib or erlotinib [7] . Although a histological correlation has been well established between lung adenocarcinoma and EGFR mutations, there is limited information regarding the correlation between the predominant histological subtype of adenocarcinoma and EGFR mutations in an Indian cohort. Therefore, this analytic and descriptive Annals of Pathology and Laboratory Medicine, Vol. 4, Issue 5, September-October, 2017 study was aimed at comprehensive histological subtyping and analysis of the frequency of EGFR mutations in primary lung adenocarcinomas based on 2015 WHO classification of lung tumors and correlating this data with clinicopathological features in an Indian cohort.

Materials and Methods
This retrospective study was conducted in a referral tertiary care center in the Department of Pathology, after the approval from the institutional review board.

Patient Selection and Histologic Analysis:
This study evaluated all cases of primary lung adenocarcinomas from June 2011-December 2012, using the archival stained Haematoxylin and Eosin (H&E) slides and formalin fixed paraffin embedded blocks following strict inclusion and exclusion criteria.
Inclusion Criteria: 1) All cases of primary lung adenocarcinoma biopsied at our institution during June 2011-December 2013.
Exclusion Criteria: 1) Cases with no definite morphological features and/or no immunohistochemical evidence of adenocarcinoma, 2) Squamous cell carcinoma by morphology or by Immunohistochemistry, 3) Non-small cell carcinoma, not otherwise specified (NSCC-NOS); 4) Large cell carcinoma and 5) Inadequate biopsy material for definite categorization.
Cases were categorized using 2015 WHO classification of lung tumors [5]" incorporated from the IASLC/ATS/ ERS classification for small biopsies [3]. D ata regarding the relevant history, clinical diagnosis, and radiological findings were obtained from hospital records. A detailed histopathological analysis was performed independently by two pathologists (A.K and P.R). Individual features including the predominant cell type, presence of mucin (PAS-Diastase), desmoplasia, stromal elastosis, lymphovascular invasion, and necrosis were studied. Comprehensive histologic subtyping was done to assess histologic patterns semi-quantitatively in 5% increments, choosing a single predominant pattern. These features were correlated with one or more immunohistochemical markers namely Thyroid Transcription Factor-1 (TTF-1) (Clone 8G7G3-1, Dako, Glostrup, Denmark), cytokeratin 7(CK7) (Clone OV-TL12/30, Dako, Glostrup, Denmark), and Ep-CAM (BerEp4) (Clone Ber-EP4, Dako, Glostrup, Denmark) where available, using the automated Ventana system. CK7 was used to differentiate between primary and metastatic lung adenocarcinoma, while BerEp4 immunohistochemistry was used to exclude pleural tumors. Details, where available, for stage of disease and smoking status, were included. Patients were staged according to the American Joint Committee on Cancer (AJCC) TNM staging manual, seventh edition [8]. EGFR mutational analyses were performed using polymerase chain reaction (PCR) sequencing of exons 18, 19, 20 and 21.

EGFR Mutational Analysis:
A subset of 120 cases based on sample size calculation were randomly selected from the pool of cases of primary lung adenocarcinoma for EGFR mutational analysis. EGFR mutational analysis was performed by PCR on formalin fixed and paraffin embedded blocks. The area with tumor cellularity of > 50% was identified. The tumor was further manually microdissected and 5 paraffin sections at 3-4 µm thickness were used for DNA extraction. Extraction was carried out using the DNA tissue extraction kit (QIAGEN India Pvt. Ltd, New Delhi, India). The DNA was quantitated using the Nano-drop (Nano-Drop Technologies, Wilmington, Delaware, USA) and the 260/280 ratio was determined.

Molecular Analysis:
The PCR for four exons, namely 18, 19, 20 and 21 was performed using the primer sequences by Lynch et al [9] " (EGFR. All reactions were carried in 25 µl volume. The following thermal cycling profile was used for all PCRs:95 0 C for 8 min, 95 0 C for 30 secs, optimized anneal for 30 secs, 72 0 C for 1 min and final extension of 72 0 C for 10 min. The PCR product was detected using a 1.5% agarose gel. Sequencing of both the sense and antisense strands for all exons was performed with an automated DNA sequencer (ABI PRISM 310 genetic analyzer) using the ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, California, USA). Mutational analysis was performed by comparing the sequence with the wild type and by identifying all known mutations in these exons.
Statistical Analysis: Descriptive statistics for continuous data was analyzed using mean with standard deviation or median with inter-quartile range. Categorical data were described using frequencies and percentages. Histopathological subtypes with EGFR mutational analysis were associated using Fischer's exact test and Chi-square test. For all analyses, a p<0.05 was considered statistically significant. Data were analyzed using the SPSS Statistics software, version 16.
Smoking history was obtained in 165 cases, and a majority of  Figure 3).There were 12(24.5%) cases with exon 21 mutation, all with the L858R mutations.
Although EGFR mutations were more common amongst females 25/52 (48.1%) and non-smokers 30/66 (45.5%), they were also noted amongst smokers 5/18 (27.8%) and males 22/68 (32.4%). There was no statistical association of EGFR mutational status with the geographical distribution of patients, residing in India and outside of India. TTF-1 positivity was significantly associated with EGFR mutational status(p = 0.007). Regression analysis of patients with mutation and TTF-1 positivity showed a relative risk of 5. (Table 3)

Correlation of Cell Type with EGFR Mutations, Smoking Status and Stage of Disease:
Although the polygonal cell type 21/47(44.6%) was more frequent with EGFR mutations, there was no statistical significance between the two. However, there was a significant association between the cuboidal cell type and EGFR mutations (p=0.01). The polygonal cell type was more common in smokers 27/48 (60.4%) and in stage IV disease 112/195(57.4%).

Discussion
The prevalence of EGFR mutations in this study was 40.8% similar to a study published from our center, where a prevalence of 39.6% of EGFR mutations was reported        www.pacificejournals.com/apalm eISSN: 2349-6983; pISSN: 2394-6466 with favorableprogression-free survival response with chemotherapy followed by tyrosine kinase inhibitors (TKI's) [10] .Studies from India have reported the prevalence of EGFR mutations ranging from 16.6%-44% [10][11][12][13] . While this fluctuation in values could be attributed to the technique used and the type of histologic diagnosis selected for the study, the role of genetic heterogeneity within India, contributing to variations in mutation frequencies, cannot be entirely ruled out [14] .
In the present study, there was a predominance of exon 19 mutations in 73.4% cases, with 15 bp del (E746-A750) being the commonest form amongst exon 19 mutations(69.4%). Exon 21 mutations were seen in 24.4%, all being the L858R type. Exon 18 mutation was not seen in any of the cases. Molecular analysis has shown that short in-frame deletions of exon 19 and a point mutation in exon 21 at base pair 2573, resulting in the substitution of leucine by arginine in codon 858 (L858R) make up for ~90% of all mutations. Point mutations in exons 18 and exon 20 account for 5% of EGFR mutations in non-small cell lung cancer [15] . In the present study, there was one case with a combined exon 20(T790M) and 21(L858R) mutation. T790M mutations can occur in 50-60% patients with resistance to TKI's,largely due to a conformational change in the EGFR molecule that prevents drug binding. It is also reported that the T790M mutation may confer primary resistance to TKI's and possibly co-exist with L858R mutations [15] . This finding was noted in the present study. The study did not find any significant association between mutational status and age, gender, smoking status and TTF-1 positivity.
Several studies have shown that EGFR mutations are associated with the lepidic, papillary, micropapillary and acinar subtypes (Table 4). In the present study, EGFR mutations were most common in the lepidic subtype (60%) followed by the papillary (44.4%), and acinar subtypes (42.7%) and were rare with the invasive mucinous subtype (7.7%). These findings are consistent with the reports of Motoi et al [16] , Yoshizawa et al [17] and Sun et al [18] . We did not find an association between EGFR mutations and the micropapillary subtype, probably due to a very small sample number of this particular subtype. Subtyping of tumors in patients undergoing complete resection, using the WHO 2015 classification appears to be very important, as highlighted by Tsao and colleagues since some subtypes like the solid and micropapillary tumors that have a poorer survival would significantly benefit from adjuvant chemotherapy. In contrast, this may not be as beneficial for acinar and papillary predominant tumors, that tend to have a better survival [19] and subtyping tumors might be able to guide therapy.
In this study, EGFR mutations were more common in females (48.1%) and non-smokers (45.5%), although a proportion of smokers (27.8%) and males (32.4%) also were positive for these. Similar results were reported by Sun et al [18] , where the incidence of EGFR mutations in men who smoked with adenocarcinomas was 29.7% (35/118). As per the study by D'Angelo and colleagues in 2,142 patients with lung adenocarcinoma, 57% of EGFR mutations would be missed if testing were restricted to women who never smoked cigarettes [20] . In the present study, we found the solid predominant subtype was more common among smokers, which is similar to the finding by Motoi et al [16] .
The current set guidelines recommend mutational analysis of all lung tumors with a diagnosis of adenocarcinoma or with an adenocarcinoma component, irrespective of the age, gender, ethnicity, smoking status, and histological subtype. In a setting of small biopsies and cytology specimens where an adenocarcinoma component cannot be completely excluded, testing for EGFR mutations can be considered in cases showing a small cell or squamous histology. However, in this scenario, clinical criteria like female gender, the absence of smoking history and age, may be useful indicators in the selection of a subset for testing [21] .
In the present study, EGFR mutations were significantly more common in tumors expressing TTF-1, hence TTF-1 positivity could be used as a predictive marker for EGFR mutations as shown in several other studies [18,22] . By regression analysis, cases that were positive for TTF-1 were 5 times more likely to harbor an EGFR mutation. However, there was no statistical significance established with TTF-1 and any particular histological subtype.
We also compared the predominant cell type with EGFR mutations, smoking status, and stage of the disease.
Although the polygonal cell type was the commonest, the cuboidal cell type was significantly associated with EGFR mutations. A similar study by Okada et al [23] proposed the five-cell type classification and found the hobnail cell type to be significantly associated with EGFR mutations (p<0.001), followed by mixed, columnar/ cuboidal, polygonal and goblet cells. The study also found a higher percentage of smokers to be significantly associated with the cuboidal/ columnar and polygonal cell type, similar to findings in this study where the polygonal cell type was associated with smokers.
Although survival analysis was not done in the present study, it is worthwhile to note that several clinicopathological parameters have been associated with poor disease-free survival, which includes male gender, gross and invasive tumor component, vascular invasion, necrosis and higher stage of disease. The AIS and MIA have a 100% diseasefree survival (DFS) at 5yrs, followed by the lepidic, acinar and papillary subtypes with a 5-year DFS between 83-90%. The solid, micropapillary, colloid and invasive mucinous subtypes that belong to the poor prognostic group have a 5-year DFS between 67-76% [24] .
A limitation of this study is that the histologic and molecular profile have not been correlated with progression-free survival and overall survival, as was done previously for a cohort of 106 cases at our center [9] .

Conclusion
This study has helped to highlight that EGFR mutations were most frequently seen with the lepidic and papillary subtypes, not associated with the mucinous subtype, were more common in women and non-smokers and were significantly associated with the cuboidal cell type. This data will be of value to overcome the lacunae in an Indian setting.