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ORIGINAL ARTICLE
Ahead of print publication  

Molecular profiling and utility of cell-free DNA in nonsmall carcinoma of the lung: Study in a tertiary care hospital


1 Department of Pathology, IPGME&R, Kolkata, West Bengal, India
2 Department of Chest Medicine, IPGME&R, Kolkata, West Bengal, India
3 Department of Radiotherapy, IPGME&R, Kolkata, West Bengal, India

Date of Submission23-Jan-2020
Date of Decision07-Oct-2020
Date of Acceptance21-Dec-2020
Date of Web Publication05-Aug-2021

Correspondence Address:
Chhanda Das,
31 Eastern Park, First Road, Santoshpur, Kolkata - 700 075, West Bengal
India
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jcrt.JCRT_99_20

 > Abstract 


Background: Lung carcinoma accounts to the most common cause of cancer globally. Optimal management of nonsmall cell lung carcinoma (NSCLC) requires prognostic biomarkers that help in targeted therapy and identification of tumor subsets with a distinctive molecular profile that can foretell response to therapy. Quantitative analysis of circulating cell-free DNA is considered as a possible aid for lung cancer screening.
Aims and Objectives: The main aim of our study was detection of the clinicopathological spectrum of NSCLC, immunohistochemical (IHC) study of lung adenocarcinoma with epidermal growth factor receptor (EGFR), anaplastic lymphoma kinase (ALK), and molecular expression of EGFR mutation using Formalin fixed paraffin embedded tissue (FFPE) and cell-free DNA (cfDNA) from blood samples.
Materials and Methods: It was a prospective and observational study conducted in the Department of Pathology in association with the Department of Chest Medicine in a tertiary care hospital for 18 months, done on 50 patients. Histological subtyping of lung carcinomas was done, followed by IHC analysis using P40, thyroid transcription factor (TTF1), EGFR, and ALK. Molecular analysis for EGFR mutation was done using FFPE and cfDNA from the patient's blood samples.
Results and Analysis: On histological subtyping, majority (66%) of the cases were found to be adenocarcinoma. All adenocarcinoma (66%) cases show TTF1 positivity and all squamous cell carcinoma (32%) cases show P40 positivity. All the ALK-positive (6%) cases were never smokers and histologically diagnosed as adenocarcinoma. About 58% of the NSCLC cases were found to be EGFR IHC positive. Formalin-fixed paraffin tissue (FFPE) showed EGFR mutation in 32% cases, of which majority were deletion (19, 28%) and rest (4%) of the cases involving mutation in exon 21. From cfDNA, mutations were noticed in 16% of the cases where majority involved deletion 19 (12%), whereas the rest of the cases were positive for missense mutation in exon 21 of the EGFR gene (2%) and compound heterozygous mutation involving deletion 19 and missense mutation for exon 21 (2%). On correlation of EGFR mutation studies from FFPE with that of cfDNA analysis, the study was statistically significant (P = 0.000).
Conclusion: This study reports clinicopathological, immunochemical, and molecular analysis of EGFR among NSCLC cases. EGFR mutation detection from cfDNA has its advantage of being a noninvasive technique to avoid rebiopsy in cases of the progressive disease to detect resistance to a drug and emergence of a newer mutation. Mutation detection from FFPE samples still remains the gold standard for targeted therapy using EGFR tyrosine kinase inhibitors. ALK rearrangement detection using IHC serves as an adjunct to EGFR diagnosis.

Keywords: Adenocarcinoma, cell-free DNA, mutation



How to cite this URL:
Ghosh M, Mukhopadhyay M, Das C, Chatterjee S, Naskar BG. Molecular profiling and utility of cell-free DNA in nonsmall carcinoma of the lung: Study in a tertiary care hospital. J Can Res Ther [Epub ahead of print] [cited 2021 Dec 5]. Available from: https://www.cancerjournal.net/preprintarticle.asp?id=323176




 > Introduction Top


Lung carcinoma accounts to the most common cause of cancer globally (2.09 million) and cancer-related death worldwide with a burden of 1.76 million deaths (World Health Organization [WHO] 2018 statistics).[1] It is the most common cancer in men internationally having an age-standardized rate (ASR; per 100000) of 34.2 with an incidence of 1.2 million, and the fourth commonest among women (ASR 13.6; incidence 0.6 million) after breast, colorectum, and cervical carcinoma.[2] Lung cancers are largely divided into small cell lung carcinoma (SCLC) and nonsmall cell lung carcinoma (NSCLC). SCLC is the most aggressive subtype and comprises 15% of lung cancer worldwide, whereas 85% are represented by NSCLCs.[3] NSCLC is further divided into – adenocarcinoma, squamous cell carcinoma, and large cell carcinoma, of which 40% are adenocarcinoma making it the most common subtype.[4],[5],[6] Different driver mutations play a significant role in tumorigenesis, and inactivation of these driver mutations results in the death of these cancer cells. Epidermal growth factor receptor (EGFR), anaplastic lymphoma kinase (ALK), and Kirsten Rous sarcoma virus (KRAS) are the exemplary driver mutations found in lung carcinoma in all-purpose. EGFR, ALK are most important that help in targeted therapy and identification of tumor subsets in optimal management of Nsclc.[7],[8],[9]

Mutations associated with enhanced sensitivity to EGFR tyrosine kinase inhibitors (TKIs) are found in exons 18–21 of the TK domain of EGFR.[10] The prevalence of EGFR mutations differs according to ethnicity; approximately 10%–12% of non-Asian patients and 30%–40% of Asian patients.[11],[12]

Two types of mutation – short in-frame deletions in exon 19, clustered around the amino-acid residues 747–750, and a specific exon 21 point mutation (L858R) – have been reported to comprise up to 90% of all activating EGFR mutations. In a prior published study, it has been seen that EGFR mutation-positive NSCLC patients have a longer progression-free survival (11.5 months) as compared to the EGFR mutation-negative patients.[13] The EGFR mutations also have a close association with clinical characteristics of lung carcinoma patients and mainly seen in nonsmoking female patients with adenocarcinomas.[14],[15]

The oncogenic role of the ALK fusion oncogene provides a potential pathway for therapeutic intervention. EML4 ALK fusion protein can be subdued by ALK inhibitors and c met inhibitors (Crizotinib) with more than 60% response rates.[16],[17]

Quantitative analysis of circulating cell-free DNA (cfDNA) by liquid biopsy is considered as a possible aid for lung cancer screening.[18] Liquid biopsy is a minimally invasive test to detect circulating tumor cells and tumor DNA fragments and is helpful in assisting in the initial diagnosis and can also aid in disease monitoring detecting resistance mutation recurrence of tumor and screening purposes. The precision of this test is facilitated to a great extent by the advancement of molecular techniques such as polymerase chain reaction (PCR)-based methods, DNA sequencing, digital PCR, and next-generation sequencing technologies.[19]

The main aim of our study was detection and correlation of the immunohistochemical status of EGFR with the mutation status using FFPE and cfDNA samples and ALK rearrangement study with immunohistochemical (IHC) of NSCLC in a tertiary care hospital.


 > Materials and Methods Top


The study was performed after obtaining the approval from the ethics committee. It was a prospective and observational study conducted in the Department of Pathology in association with the Department of Chest medicine in a tertiary care hospital. The study was conducted from January 2018 to June 2019 (18 months). The study population (n = 50 cases) consisted of patients with a confirmed diagnosis of lung carcinoma on histopathology. After routine hematoxylin and eosin staining, IHC staining was done for P40, thyroid transcription factor (TTF1), EGFR, and ALK. EGFR expression was studied from IHC and correlated with its molecular expression from FFPE and cfDNA samples. Only NSCLC cases were selected for molecular testing. The PCR-based study was done for EGFR-mutation testing and IHC-testing was done to see ALK expression. In case of the IHC study, the primary antibody was the mouse monoclonal p40 antibody and TTF1. P40: Dilution: 1:100, TTF: Dilution: 1:100. TTF-1 and P40 markers were scored as 0, 1+, 2+, 3+ by percentage of immunoreactive tumour cells with 0-weakly positive/negative <10% positive cells-1+, 10%–50%+ as 2+ and ≥50% positive cells as 3+. The intensity was recorded as 1+ (< normal cells), 2+ (same as normal cells), and 3+ (more intensity than normal cells).[20] EGFR: Dilution: 1: 100. EGFR IHC scoring was based on membranous and/or cytoplasmic staining: 0 (no/faint staining in <10% of tumor cells); 1+ (weak staining in ≥10% of tumor cells); 2+ (moderate staining in ≥10% of tumor cells); and 3+ as more intense staining in ≥10% of tumor cells.[21] ALK-Dilution: 1: 250, strong granular brown cytoplasmic staining was considered positive, whereas weak cytoplasmic staining was considered as negative.[22]

Subsequently, molecular tests were performed for detection of EGFR mutations by extracting cfDNA from the blood samples using the Diatech method and by detection of EGFR mutation from formalin-fixed paraffin-embedded (FFPE) block of the same patients with EGFR XL strip assay of Vienna lab for EGFR.

Statistical analysis

All data processed through Microsoft Excel. For quantitative variables, mean and median values with standard deviation were assessed. The Chi-square test was conducted which included the clinicopathologic parameters. The statistical analyses were performed using SPSS software version 20.0 (IBM, Armonk, New York, USA). P < 0.05 was considered statistically significant and all the P values were two tailed.


 > Results and Analysis Top


During the study period, the patient age ranged from 44 to 78 years, the mean age of patient was 58.56 ± 10.16 years. 58% of cases of lung carcinoma presented between the age of 41 and 60 years and (42%) presented between 61 and 78 years. The patients most commonly presented with cough (66%), followed by chest pain and dyspnoea (44%) and hemoptysis (42%). About 52% of the cases presented with a right-sided involvement. Majority of the NSCLC cases involved the peripheral part of the lung. The most common lobe involved was the upper lobe (66%) followed by the lower (32%) and middle lobe respectively (7%). 52% presented with cavitary lesions and 48%with lymph nodes involvement. [Table 1] shows the clinicopathological parameters. On histological subtyping, majority (66%) of the cases were adenocarcinoma [Figure 1]. For confirmation, histological examinations were followed by IHC studies with TTF1 and P40. About 32% of NSCLC showed positivity for P40 [Figure 2]. Immunohistochemistry for TTF1 [Figure 3] showed positivity in 66% of cases. On correlation of TTF1 with histological diagnosis, there was a significant correlation (P < 0.001). ALK-rearrangement study was performed with ALK IHC testing using Ventana ALK (D5F3) CDx assay [Figure 4]. Only three cases came out to be positive for ALK, hence clinicopathologic correlation was not found to be significant for ALK testing (P = 0.773) (however, all the positive cases were never smokers and histologically diagnosed as adenocarcinoma). Immunohistochemistry for EGFR was performed in all cases from the paraffin-embedded blocks and 58% of the cases were found to be positive for EGFR IHC [Figure 5].
Table 1: Comparison of clinicopathological parameters with EGFR expression

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Figure 1: Histopathology showing glandular formation along with mucin suggestive of nonsmall cell lung carcinoma favoring adenocarcinoma (H and E, ×400)

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Figure 2: Immunohistochemistry of lung adenocarcinoma showing thyroid transcription factor 1 positivity (×100)

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Figure 3: Immunohistochemistry of lung squamous cell carcinoma showing P40 positivity (×100)

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Figure 4: Immunohistochemistry of lung adenocarcinoma showing anaplastic lymphoma kinase positivity (×400)

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Figure 5: Immunohistochemistry of lung adenocarcinoma showing epidermal growth factor receptor positivity (×400)

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[Table 2] shows the immunochemical analysis among different histological subtypes.

Molecular analysis of the EGFR mutation was done in all the cases. EGFR mutation status from formalin-fixed paraffin tissue (FFPE) showed normal gene sequence in 68% of cases, whereas mutation was found majority in deletion 19 (28%) and rest (4%) of the cases involving mutation in exon 21 [Figure 6].
Table 2: Immunohistochemical expression in histological subtypes

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Figure 6: Diagram of epidermal growth factor receptor XL strip with mutation lines and control lines

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On mutation status analysis from cfDNA, 16% showed mutations of which majority was deletion 19 (12%) 1 case was positive for deletion 19 and T790M mutation of the EGFR gene whereas the rest of the cases were positive for compound heterozygous mutation of deletion 19 and missense mutation for exon 21 [Figure 7] and [Figure 8].
Figure 7: QC-cell free DNA Agilent Tapestation 4200

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Figure 8: Interpretation of mutations in cell-free DNA by real-time polymerase chain reaction

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On clinicopathologic correlation, it was found that among EGFR positive cases (29/50), there were 75.86% male patients and 24.13% female patients. There was a significant correlation between female nonsmokers having adenocarcinoma (P < 0.05).

On histological correlation, out of a total of 29 EGFR positive cases, 96.13% were histologically diagnosed as adenocarcinoma and only 3.4% were with nonsmall cell carcinoma NOS on histology. The IHC correlation with histopathology findings was found to be highly significant (P < 0.001) [Table 3] and [Table 4].
Table 3: Molecular expression of EGFR from FFPE in histopathology subtypes

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Table 4: Comparative analysis of molecular expression of EGFR from FFPE with EGFR IHC

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On correlation of mutation studies from cfDNA samples with histopathology, it was found that 8 cases were positive for EGFR mutations and all of these 8 cases were histologically diagnosed as adenocarcinoma. The results were found to be highly significant (P < 0.001) [Table 5]. It was seen that 32% of the total cases showed EGFR mutation status from FFPE, whereas EGFR mutation was found in only 16% of the total cases by analysis using cfDNA. On correlation of EGFR mutation studies of cfDNA analysis with that of FFPE, the study was found to be highly significant with P < 0.01 [Table 6]. EGFR mutation studies of cfDNA analysis were less sensitive than that from FFPE.
Table 5: Molecular expression of EGFR from CFDNA in histopathology subtypes

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Table 6: Correlation of egfr molecular analysis from ffpe and CFDNA

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 > Discussion Top


From the review of different updated and allied publications, two main constitutive activations of EGFR mutations were found which were strongly associated with females, never smokers and East-Asian ethnicity which are as follows -in -frame deletions of exon 19 and L858R and point mutation in exon 21 (L861Q).[23],[24] These EGFR mutant codons are targeted by TKI like gefitinib and erlotinib or by monoclonal antibody (e.g., Cetuximab).[25],[26] The EGFR TKIs may undergo resistance due to different mechanisms either primary (de novo) or acquired. Thereafter, rebiopsy should be well thought-out to assess progression and detection of resistance. Mutation in the EGFR gene at exon 20, which encodes T790M is resistant to small-molecule EGFR TKIs (50% cases).[27] The second commonest mechanism of resistance is MET amplification (5%–20%) which leads to EGFR inhibition.[28],[29] EGFR mutation serves as a prognostic factor and is receptive to treatment with EGFR TKI.[30],[31],[32]

ALK rearrangement is also related to a specific population, majority being younger, nonsmoker patients with adenocarcinoma.[33],[34],[35] Resistance might develop to crizotinib in cases with ALK-rearranged NSCLC due to ALK amplification, upregulation of EGFR/HER1, HER2, and HER3, cKIT amplification, and other ALK mutations.[36],[37],[38],[39],[40]

EGFR mutation analysis from FFPE is a widely accepted method nowadays, but it has certain limitations as the tissue samples represent a single snapshot in time thereby leading to selection bias with regard to tumor heterogeneity.[41] Liquid biopsies are minimally invasive tests to detect circulating tumor cells, nucleic acids, exosomes.[42] Preanalytical steps are the most important in these methods, of which blood collection and handling are major steps for optimizing detection of mutations.

In our study, our main aim was to expansively review the substantiality of using the circulating cfDNA as a method of detection of mutation in lung carcinoma patients with expression of EGFR mutations and ALK rearrangements.

As we compare our study with prior published cases, significant concordance was found. In a study by Laufer-Geva et al., 116 patients with advanced NSCLC were retrospectively analyzed with cfDNA; 66% were diagnosed as adenocarcinoma, whereas 32% of cases were squamous cell carcinoma.

Our study reported that EGFR mutation status from formalin-fixed paraffin tissue (FFPE) showed normal gene sequence in 68% of cases, whereas mutation was found majority in deletion 19 (28%) and rest (4%) of the cases involving mutation in exon 21. On mutation status analysis from cfDNA, mutations were noticed in 8 cases (16%) of the cases where majority involved deletion 19 (12%), 1 case was positive for deletion 19 and T790M mutation of the EGFR gene, whereas the rest of the cases were positive for missense mutation in exon 21 of the EGFR gene and compound heterozygous mutation, deletion 19 and missense mutation for exon 21. All these results were found to be concordant with the previous studies as described below.

Laufer-Geva et al. showed 25.9% to be EGFR testing positive.[43] In the study by Macías et al., 52% were never smokers. EGFR mutations were detected in 12 patients by this cfDNA, 2 patients were diagnosed with a T790 M mutation. Exon 19 mutations presented in three patients who did not have a previous mutation.[44]

In a study by Seki et al., the correlation between patient characteristics and EGFR mutations detection was done from FFPE and cfDNA and were compared.[45] Multiple targeted EGFR mutation analysis in cfDNA can be helpful in those patients with already detected EGFR mutations in tissue.[46] From our study, tumor tissue was found to be more sensitive in mutation analysis. However, cfDNA analysis at progressive diseases has led to the detection of previously detected mutations as well as new mutations were also found out. Thus, it came out to be a helpful adjunct in progressive diseases where newer mutations could even be detected. It is a useful complementary technique in monitoring of the disease processes.

Owing to the different mechanisms between cfDNA with other tumor markers, cfDNA assay might be useful in clinical practice to complement the properties of conventional biomarkers. Thus, it might be a helpful adjunct in the detection of mutations. The discrimination power is low, when used alone. The results of cfDNA should be interpreted along with other conventional tests including clinical examination, imaging, cytological, and histological examination. EGFR cfDNA analysis might help as a complementary diagnostic approach in cases where biopsy has not been recently done or not available, thereby helping in the detection of the mutational information.[47] cfDNA can also be used in monitoring the treatment, as the mutation load decreases when the patient responds to the respective therapy and elevates with disease progression. Therefore, cfDNA can be helpful in the patients with already detected EGFR mutations by tissue biopsy. It also helps in evaluating the mutational status, where a recent biopsy is not feasible. The sequential and repeated sampling also allows the monitoring of the effectiveness of therapy and the detection of various resistance mutations.


 > Conclusion Top


In an era of molecular analysis, cancer management paradigms have shifted toward the basis of personalized and therapeutic approaches. Therefore, an accurate and unbiased molecular categorization becomes a necessity to delineate the therapeutic strategy of NSCLC combating multiple driver alterations. This study reports clinicopathological immunochemical and molecular analysis of EGFR, ALK among NSCLC cases. CfDNA has its advantage of being a noninvasive technique holds an upper hand to avoid rebiopsy in cases of progressive disease, to detect resistance to a drug (e.g., crizotinib), and emergence of a newer mutation. Mutation detection from FFPE samples still remains the gold standard. Detection of ALK rearrangement detection using IHC techniques has led to a sensitive, specific, and cost-effective assay. This serves as an adjunct to EGFR diagnosis and detects specific driver mutations, thereby improving diagnostic accuracy and to detect all patients harboring an ALK-rearranged lung carcinoma. A larger, population-based trial with more number of study samples is required to define prevalence, detailed clinicopathological features, and determining the prognosis of the patients. Standardization of IHC procedures using surrogate markers is required to come to a uniform and cost-effective system that can be used worldwide. Molecular markers need to be standardized and more widely accepted worldwide to provide benefits of targeted therapy to the patients. More advancement in the detection of cfDNA or ctDNA should be sought after so as to use liquid biopsy as a proper screening method even in cases of upfront lung carcinoma and to detect lung carcinoma at its earliest.

Limitations

The main limitation of our study was its short sample size (50 cases) and short duration. The limitations regarding the IHC approaches were procedural reproducibility and subjective interpretation. Molecular tests needed proper setup and keen, sincere, and proper knowledge-based skilled approaches in every step.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
 > References Top

1.
Ferlay J, Colombet M, Soerjomataram I, Mathers C, Parkin DM, Piñeros M, et al. Estimating the global cancer incidence and mortality in 2018: GLOBOCAN sources and methods. Int J Cancer 2019;144:1941-53.  Back to cited text no. 1
    
2.
Ferlay J, Soerjomataram I, Dikshit R, Eser S, Mathers C, Rebelo M, et al. Cancer incidence and mortality worldwide: Sources, methods and major patterns in GLOBOCAN 2012. Int J Cancer 2015;136:E359-86.  Back to cited text no. 2
    
3.
Früh M, De Ruysscher D, Popat S, Crinò L, Peters S, Felip E; ESMO Guidelines Working Group. Smallcell lung cancer (SCLC): ESMO Clinical Practice Guidelines for diagnosis, treatment and followup. Ann Oncol 2013;24 Suppl 6:vi99105.  Back to cited text no. 3
    
4.
WHO Classification of Tumours of the Lung, Pleura, Thymus and Heart; Molecular Testing for Treatment Selection in Lung Cancer. 4th ed. Lyon, France: International Agency for Research on Cancer; 2015.  Back to cited text no. 4
    
5.
Youlden DR, Cramb SM, Baade PD. The international epidemiology of lung cancer: Geographical distribution and secular trends. J Thorac Oncol 2008;3:819-31.  Back to cited text no. 5
    
6.
Couraud S, Zalcman G, Milleron B, Morin F, Souquet PJ. Lung cancer in never smokers – A review. Eur J Cancer 2012;48:1299-311.  Back to cited text no. 6
    
7.
Thunnissen E, van der Oord K, den Bakker M. Prognostic and predictive biomarkers in lung cancer. A review. Virchows Arch 2014;464:347-58.  Back to cited text no. 7
    
8.
Novello S, Barlesi F, Califano R, Cufer T, Ekman S, Levra MG, et al. Metastatic non-small-cell lung cancer: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol 2016;27:v1-27.  Back to cited text no. 8
    
9.
Lee JK, Hahn S, Kim DW, Suh KJ, Keam B, Kim TM, et al. Epidermal growth factor receptor tyrosine kinase inhibitors vs conventional chemotherapy in non-small cell lung cancer harboring wild-type epidermal growth factor receptor: A meta-analysis. JAMA 2014;311:1430-7.  Back to cited text no. 9
    
10.
Sharma SV, Bell DW, Settleman J, Haber DA. Epidermal growth factor receptor mutations in lung cancer. Nat Rev Cancer 2007;7:169-81.  Back to cited text no. 10
    
11.
Cortes-Funes H, Gomez C, Rosell R, Valero P, Garcia-Giron C, Velasco A, et al. Epidermal growth factor receptor activating mutations in Spanish gefitinib-treated non-small-cell lung cancer patients. Ann Oncol 2005;16:1081-6.  Back to cited text no. 11
    
12.
Yoshida K, Yatabe Y, Park JY, Shimizu J, Horio Y, Matsuo K, et al. Prospective validation for prediction of gefitinib sensitivity by epidermal growth factor receptor gene mutation in patients with non-small cell lung cancer. J Thorac Oncol 2007;2:22-8.  Back to cited text no. 12
    
13.
Shaw AT, Yeap BY, Solomon BJ, Riely GJ, Iafrate AJ, Shapiro G, et al. Journal of Clinical Oncology 2011 29:15_suppl, 7507-750.  Back to cited text no. 13
    
14.
Paez JG, Jänne PA, Lee JC, Tracy S, Greulich H, Gabriel S, et al. EGFR mutations in lung cancer: Correlation with clinical response to gefitinib therapy. Science 2004;304:1497-500.  Back to cited text no. 14
    
15.
Mitsudomi T, Kosaka T, Yatabe Y. Biological and clinical implications of EGFR mutations in lung cancer. Int J Clin Oncol 2006;11:190-8.  Back to cited text no. 15
    
16.
Han JY, Park K, Kim SW, Lee DH, Kim HY, Kim HT, et al. First-SIGNAL: First-line single-agent iressa versus gemcitabine and cisplatin trial in never-smokers with adenocarcinoma of the lung. J Clin Oncol 2012;30:1122-8.  Back to cited text no. 16
    
17.
Dong-Seok K, Ahn MJ, Yongliang S, Pas TM, Yang PC, Riely GJ. Results of a Global Phase II Study with Crizotinib in Advanced ALK-positive Non-small Cell Lung Cancer (NSCLC). J Clin Oncol (2012) 30.  Back to cited text no. 17
    
18.
Zhang R, Shao F, Wu X, Ying K. Value of quantitative analysis of circulating cell free DNA as a screening tool for lung cancer: A meta-analysis. Lung Cancer 2010;69:225-31.  Back to cited text no. 18
    
19.
Cheung AH, Chow C, To KF. Latest development of liquid biopsy. J Thorac Dis 2018;10:S1645-51.  Back to cited text no. 19
    
20.
Lilo MT, Allison D, Wang Y, Ao M, Gabrielson E, Geddes S, et al. Expression of P40 and P63 in lung cancers using fine needle aspiration cases. Understanding clinical pitfalls and limitations. J Am Soc Cytopathol 2016;5:123-32.  Back to cited text no. 20
    
21.
Kim CH, Kim SH, Park SY, Yoo J, Kim SK, Kim HK. Identification of EGFR mutations by immunohistochemistry with EGFR mutation-specific antibodies in biopsy and resection specimens from pulmonary adenocarcinoma. Cancer Res Treat 2015;47:653-60.  Back to cited text no. 21
    
22.
Wynes MW, Sholl LM, Dietel M, Schuuring E, Tsao MS, Yatabe Y, et al. An international interpretation study using the ALK IHC antibody D5F3 and a sensitive detection kit demonstrates high concordance between ALK IHC and ALK FISH and between evaluators. J Thorac Oncol 2014;9:631-8.  Back to cited text no. 22
    
23.
Yatabe Y, Borczuk AC, Powell CA. Do all lung adenocarcinomas follow a stepwise progression? Lung Cancer 2011;74:7-11.  Back to cited text no. 23
    
24.
Mounawar M, Mukeria A, Le Calvez F, Hung RJ, Renard H, Cortot A, et al. Patterns of EGFR, HER2, TP53, and KRAS mutations of p14arf expression in non-small cell lung cancers in relation to smoking history. Cancer Res 2007;67:5667-72.  Back to cited text no. 24
    
25.
Westra WH. Early glandular neoplasia of the lung. Respir Res 2000;1:163-9.  Back to cited text no. 25
    
26.
Carr LL, Chung JH, Duarte Achcar R, Lesic Z, Rho JY, Yagihashi K, et al. The clinical course of diffuse idiopathic pulmonary neuroendocrine cell hyperplasia. Chest 2015;147:415-22.  Back to cited text no. 26
    
27.
Yatabe Y, Mitsudomi T, Takahashi T. TTF-1 expression in pulmonary adenocarcinomas. Am J Surg Pathol 2002;26:767-73.  Back to cited text no. 27
    
28.
Tang X, Shigematsu H, Bekele BN, Roth JA, Minna JD, Hong WK, et al. EGFR tyrosine kinase domain mutations are detected in histologically normal respiratory epithelium in lung cancer patients. Cancer Res 2005;65:7568-72.  Back to cited text no. 28
    
29.
Nakanishi K, Kawai T, Kumaki F, Hiroi S, Mukai M, Ikeda E. Survivin expression in atypical adenomatous hyperplasia of the lung. Am J Clin Pathol 2003;120:712-9.  Back to cited text no. 29
    
30.
Sequist LV, Bell DW, Lynch TJ, Haber DA. Molecular predictors of response to epidermal growth factor receptor antagonists in nonsmall-cell lung cancer. J Clin Oncol 2007;25:587-95.  Back to cited text no. 30
    
31.
Ladanyi M, Pao W. Lung adenocarcinoma: Guiding EGFR-targeted therapy and beyond. Mod Pathol 2008;21 Suppl 2:S16-22.  Back to cited text no. 31
    
32.
Pao W, Ladanyi M. Epidermal growth factor receptor mutation testing in lung cancer: Searching for the ideal method. Clin Cancer Res 2007;13:4954-5.  Back to cited text no. 32
    
33.
Li J, Wang L, Mamon H, Kulke MH, Berbeco R, Makrigiorgos GM. Replacing PCR with COLD-PCR enriches variant DNA sequences and redefines the sensitivity of genetic testing. Nat Med 2008;14:579-84.  Back to cited text no. 33
    
34.
Ogino S, Kawasaki T, Brahmandam M, Yan L, Cantor M, Namgyal C, et al. Sensitive sequencing method for KRAS mutation detection by Pyrosequencing. J Mol Diagn 2005;7:413-21.  Back to cited text no. 34
    
35.
Dogan S, Shen R, Ang DC, Johnson ML, D'Angelo SP, Paik PK, et al. Molecular epidemiology of EGFR and KRAS mutations in 3,026 lung adenocarcinomas: Higher susceptibility of women to smoking-related KRAS-mutant cancers. Clin Cancer Res 2012;18:6169-77.  Back to cited text no. 35
    
36.
Huang YS, Yang JJ, Zhang XC, Yang XN, Huang YJ, Xu CR, et al. Impact of smoking status and pathologic type on epidermal growth factor receptor mutations in lung cancer. Chin Med J (Engl) 2011;124:2457-60.  Back to cited text no. 36
    
37.
Sahoo R, Harini VV, Babu VC, Patil Okaly GV, Rao S, Nargund A, et al. Screening for EGFR mutations in lung cancer, a report from India. Lung Cancer 2011;73:316-9.  Back to cited text no. 37
    
38.
Chougule A, Prabhash K, Noronha V, Joshi A, Thavamani A, Chandrani P, et al. Frequency of EGFR mutations in 907 lung adenocarcioma patients of Indian ethnicity. PLoS One 2013;8:e76164.  Back to cited text no. 38
    
39.
Doval DC, Azam S, Batra U, Choudhury KD, Talwar V, Gupta SK, et al. Epidermal growth factor receptor mutation in lung adenocarcinoma in India: A single center study. J Carcinog 2013;12:12.  Back to cited text no. 39
[PUBMED]  [Full text]  
40.
Aggarwal S, Patil S, Minhans S, Pungliya M, Soumitra N. A study of EGFR mutation in nonsmoker NSCLC: Striking disparity between north and south India patients. Journal of Clinical Oncology 2012 30:15_suppl, e18041-e18041.  Back to cited text no. 40
    
41.
Diaz LA Jr., Bardelli A. Liquid biopsies: Genotyping circulating tumor DNA. J Clin Oncol 2014;32:579-86.  Back to cited text no. 41
    
42.
Kanikarla-Marie P, Lam M, Menter DG, Kopetz S. Platelets, circulating tumor cells, and the circulome. Cancer Metastasis Rev 2017;36:235-48.  Back to cited text no. 42
    
43.
Laufer-Geva S, Rozenblum AB, Twito T, Grinberg R, Dvir A, Soussan-Gutman L, et al. The clinical impact of comprehensive genomic testing of circulating cell-free DNA in advanced lung cancer. J Thorac Oncol 2018;13:1705-16.  Back to cited text no. 43
    
44.
Macías M, Alegre E, Alkorta-Aranburu G, Patiño-García A, Mateos B, Andueza MP, et al. The Dynamic Use of EGFR Mutation Analysis in Cell-Free DNA as a Follow-Up Biomarker during Different Treatment Lines in Non-Small-Cell Lung Cancer Patients”, Disease Markers 2019;2019:7. Article ID 7954921.  Back to cited text no. 44
    
45.
Seki Y, Fujiwara Y, Kohno T, Yoshida K, Goto Y, Horinouchi H, et al. Circulating cell-free plasma tumour DNA shows a higher incidence of EGFR mutations in patients with extrathoracic disease progression. ESMO Open 2018;3(2):e000292.  Back to cited text no. 45
    
46.
Macías M, Alegre E, Alkorta-Aranburu G, Patiño-García A, Mateos B, Maria P et al. “The Dynamic Use of EGFR Mutation Analysis in Cell-Free DNA as a Follow-Up Biomarker during Different Treatment Lines in Non-Small-Cell Lung Cancer Patients”, Disease Markers, 2019:7Article ID 7954921.  Back to cited text no. 46
    
47.
Wan JC, Massie C, Garcia-Corbacho J, Mouliere F, Brenton JD, Caldas C, et al. Liquid biopsies come of age: Towards implementation of circulating tumour DNA. Nat Rev Cancer 2017;17:223-38.  Back to cited text no. 47
    


    Figures

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    Tables

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    -  Mukhopadhyay M
    -  Das C
    -  Chatterjee S
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