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  Table of Contents  
ORIGINAL ARTICLE
Year : 2015  |  Volume : 52  |  Issue : 3  |  Page : 266-268
 

Dual surrogate markers for rapid prediction of epidermal growth factor receptor mutation status in advanced adenocarcinoma of the lung: A novel approach in resource-limited setting


1 Department of Medical Oncology, K M C, Manipal, Karnataka, India
2 Cancer Institute, Chennai, Tamilnadu, India
3 Department of Pathology, Cancer Institute, Chennai, India

Date of Web Publication18-Feb-2016

Correspondence Address:
K S Udupa
Department of Medical Oncology, K M C, Manipal, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0019-509X.176693

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 » Abstract 

Introduction: Tyrosine kinase inhibitors have revolutionized the treatment of metastatic lung cancer in patients with epidermal growth factor receptor (EGFR) mutations. Amplified refractory mutation system (ARMS)-reverse transcription-polymerase chain reaction (RT-PCR), the current standard for detecting EGFR mutation status is time-consuming and highly expensive. Consequently any surrogate test which are cheaper, faster and as accurate as the PCR method will help in early diagnosis and management of patients with lung cancer, especially in resource-limited settings. Materials and Methods: Eighty-five patients, all of South Indian origin, with adenocarcinoma of lung, registered between October 2009 and January 2013, were evaluated for EGFR mutation status by using scorpion probe based ARMS RT-PCR method. Immunohistochemical (IHC) was performed using the phosphorylated AKT (P-AKT) and thyroid transcription factor-1 (TTF-1) on above patient's sample, and the results were compared with EGFR mutation tests. Results: EGFR mutation was positive in 34 of 85 patients (40%). P-AKT and TTF-1 were positive in 50 (58.8%) and 68 (80%) patients respectively. Both P-AKT and TTF-1 had statistically significant correlation with EGFR mutation status. Positive and negative predictive value of P-AKT in diagnosing EGFR mutation was 58% and 85.5% and that for TTF-1 was 48.5% and 94.1%, respectively. The problem of low positive predictive value can partly be overcome by testing P-AKT and TTF-1 simultaneously. Conclusion: P-AKT and TTF-1 using IHC had statistically significant correlation with EGFR mutation with high negative predictive value. In the case of urgency of starting treatment, EGFR mutation testing may be avoided in those patients who are negative for these IHC markers and can be started on chemotherapy.


Keywords: Epidermal growth factor receptor mutation, lung cancer, phosphorylated AKT, thyroid transcription factor-1


How to cite this article:
Udupa K S, Rajendranath R, Sagar T G, Sundersingh S, Joseph T. Dual surrogate markers for rapid prediction of epidermal growth factor receptor mutation status in advanced adenocarcinoma of the lung: A novel approach in resource-limited setting. Indian J Cancer 2015;52:266-8

How to cite this URL:
Udupa K S, Rajendranath R, Sagar T G, Sundersingh S, Joseph T. Dual surrogate markers for rapid prediction of epidermal growth factor receptor mutation status in advanced adenocarcinoma of the lung: A novel approach in resource-limited setting. Indian J Cancer [serial online] 2015 [cited 2019 Jun 26];52:266-8. Available from: http://www.indianjcancer.com/text.asp?2015/52/3/266/176693



 » Introduction Top


Personalized medicine is the major development of 21st century in the management of lung cancer. Tyrosine kinase inhibitors, the targeted agents against Epidermal growth factor receptor (EGFR) mutation have revolutionized the treatment of lung cancer, resulting in improved survival of patients with metastatic disease who are positive for EGFR mutation.

Epidermal growth factor receptor mutation is seen in 10–15% of the western population and 30–35% of East Asians.[1],[2] Reverse transcription-polymerase chain reaction (RT-PCR) the newer and sensitive method being used worldwide for detection of EGFR mutation by the amplified refractory mutation system (ARMS) techniques is time consuming and expensive for patients in resource poor countries where access for EGFR mutation testing may be limited.[3],[4]

Surrogate tests with high sensitivity and specificity in detecting EGFR mutation, which are less time-consuming and less expensive compared to RT-PCR are the need of the day. We investigated two immunohistochemical (IHC) markers phosphorylated AKT (P-AKT) and thyroid transcription factor-1 (TTF-1) and their correlation to EGFR mutation done through RT-PCR in patients with adenocarcinoma of the lung.


 » Materials and Methods Top


Eighty-five patients, all of South Indian origin with adenocarcinoma of the lung, registered between October 2009 and January 2013, at Cancer Institute, Chennai, India whose tissue sample was adequate to perform EGFR mutation testing by RT-PCR and IHC studies, were included in the study.

Patients were evaluated for EGFR mutation status by using scorpion probe based ARMS RT-PCR method. IHC was performed either from formalin fixed paraffin embedded tissue or fine-needle aspiration cytology obtained from tumor tissue, metastatic nodes or cell blocks of pleural fluid positive for malignant cells.

Exons 18, 19, 20 and 21 of the EGFR gene were screened for hotspots (oncogenic driver mutations) using Scorpions ARMS real-time PCR technology.[5] Twenty-nine somatic mutations that included the resistance mutation T790M were screened for all the samples.

Immunohistochemical was performed on tumor tissue samples using thyroid transcription factor 1 (TTF-1, Leica-product code NCL-TTF-1) and (P-AKT, Cell Signaling Technology, product code#4060P).

Statistical Package for the Social Sciences (SPSS) version 17.0 (SPSS Inc.) was used for statistical analysis. Chi-square test was used to detect a correlation between categorical variables and P < 0.05 was considered statistically significant. Kappa co-efficient was employed to assess the level of agreement between EGFR mutations by RT-PCR with P-AKT, TTF-1 by IHC. Kappa co-efficient >0.75 was considered as excellent, 0.40–0.75 was considered as fair to good, and below 0.40 was considered as poor agreement.


 » Results Top


Eighty-five patients of advanced adenocarcinoma of the lung were enrolled in our study. The baseline characteristics of patients included in the study is shown in [Table 1]. EGFR mutation was positive in 31 patients (40%) and P-AKT was positive in 50 patients (58.8%) [Figure 1]a. The sensitivity and specificity of P-AKT in co-relation to EGFR mutation done through RT-PCR is shown in [Table 2]. The positive predictive value and negative predictive value of the test was 58% and 85.5%, respectively. The kappa co-efficient was 0.409.
Table 1: Base line characteristics of patients

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Figure 1: (a) Tumor cells showing membrane and cytoplasmic positivity to phosphorylated AKT immunohistochemical (IHC, ×40). (b) Tumor cells showing nuclear positivity to thyroid transcription factor-1 (IHC, ×20)

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Table 2: Correlation between EGFR mutation and P-AKT

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TTF-1 was positive in 68 of the 85 eligible patients (80%) [Figure 1]b and was negative in the rest of the 17 patients (20%). The sensitivity and specificity of TTF-1 in relation to EGFR mutation is shown in [Table 3]. The positive predictive value and negative predictive value of the test was 48.5% and 94.1% respectively. The kappa co-efficient was 0.224. In correlation study of P-AKT and TTF-1 with EGFR mutation status, even though negative predictive value was very high, the positive predictive value was very low. This indicates that they had a high incidence of false positivity.
Table 3: Correlation between EGFR mutation status and TTF-1

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A subset of patients with TTF-1 and P-AKT positive compared to subset of patients with TTF-1 positive and P-AKT negative, the sensitivity and specificity in determining EGFR mutation is shown in [Table 4]. The TTF-1 and P-AKT positive group had positive and negative predictive value of 68.6% and 90%, respectively. The Kappa co-efficient was 0.439. The positive predictive value was significantly higher, and false positivity was much lesser compared with TTF-1 and P-AKT assessed separately with EGFR mutation status.
Table 4: Correlation between P-AKT, TTF-1 and EGFR mutation

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 » Discussion Top


In our study, two IHC markers, namely P-AKT and TTF-1 were evaluated, and their correlation with EGFR mutation was performed using RT-PCR. We also evaluated whether these IHC tests could act as surrogate markers in diagnosing EGFR mutation, as the IHC tests are less expensive, less time-consuming and more readily performed by any trained pathologist. P-AKT is a serine-threonine protein kinase that regulates many of the key mechanism responsible for carcinogenesis such as apoptosis, angiogenesis and cell cycle propagation.[6] We found a statistically significant correlation between P-AKT and EGFR mutation. Only five out of 34 EGFR positive patients were P-AKT negative resulting in a negative predictive value of 85.5%. Similar correlation was demonstrated by Ikeda et al.[7]

Thyroid transcription factor-1 is expressed in type 2 pneumocytes and nonciliated bronchial epithelial cells and plays an important role in sustainment of lung cancer.[8] In our study, TTF-1 was positive in 80% of cases. The correlation between EGFR mutation positivity and TTF-1 positivity was statistically significant. Only one out of seventeen TTF-1 negative patients was EGFR mutation positive, accounting for negative predictive value of 94.1%. This high negative predictive value for TTF-1 is consistent with the study by Vincenten et al.[9]

Similar to study by Ikeda et al., in our study IHC markers cannot replace present gold standard in detecting EGFR mutation namely RT-PCR due to low specificity and high false positivity.[7]

In the present study, P-AKT and TTF-1 have high negative predictive values. This means the likelihood of having EGFR mutation positive in patients who are P-AKT and TTF-1 negative is very less. Hence in patients where P-AKT and TTF-1 negative, EGFR mutation testing can be avoided. Such patents can be started on chemotherapy when there is urgency in starting treatment.

The problem of false positivity and low sensitivity with TTF-1 and P-AKT can be partly overcome by testing for above two IHC simultaneously. In a subset of patients who are TTF-1 and P-AKT positive compared with those with TTF-1 positive and P-AKT negative, had sensitivity and specificity was 88.9% and 54.2%, respectively. The false positivity was 31.4% which is much lesser compared with P-AKT and TTF-1 when they were tested separately. Hence, we recommend testing P-AKT and TTF-1 simultaneously and hence that the probability finding EGFR mutation increases in that subset of patients who are positive for both the above-mentioned markers.

In summary, P-AKT and TTF-1 done through IHC had statistically significant correlation with EGFR mutation with high negative predictive value. In the case of urgency of starting treatment, EGFR mutation testing can be avoided in those patients who are negative for these IHC markers and can be started on chemotherapy. The problem of false positivity by using either of these markers can be reduced by testing these markers simultaneously. The likelihood of EGFR mutation significantly increases in the subset patients who are positive both P-AKT and TTF-1.

 
 » References Top

1.
Rosell R, Moran T, Queralt C, Porta R, Cardenal F, Camps C, et al. Screening for epidermal growth factor receptor mutations in lung cancer. N Engl J Med 2009;361:958-67.  Back to cited text no. 1
[PUBMED]    
2.
Tokumo M, Toyooka S, Kiura K, Shigematsu H, Tomil K, Aoe M, et al. The relationship between epidermal growth factor receptor mutations and clinicopathologic features in non-small cell lung cancers. Clin Cancer Res 2005;11:1167-73.  Back to cited text no. 2
    
3.
Newton CR, Graham A, Heptinstall LE, Powell SJ, Summers C, Kalsheker N, et al. Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS). Nucleic Acids Res 1989;17:2503-16.  Back to cited text no. 3
    
4.
Ellison G, Donald E, McWalter G, Knight L, Fletcher L, Sherwood J, et al. A comparison of ARMS and DNA sequencing for mutation analysis in clinical biopsy samples. J Exp Clin Cancer Res 2010;29:132.  Back to cited text no. 4
    
5.
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. 5
    
6.
Kulik G, Klippel A, Weber MJ. Antiapoptotic signalling by the insulin-like growth factor I receptor, phosphatidylinositol 3-kinase, and Akt. Mol Cell Biol 1997;17:1595-606.  Back to cited text no. 6
    
7.
Ikeda S, Takabe K, Inagaki M, Funakoshi N, Suzuki K, Shibata T. Correlation between EGFR gene mutation pattern and Akt phosphorylation in pulmonary adenocarcinomas. Pathol Int 2007;57:268-75.  Back to cited text no. 7
    
8.
Bohinski RJ, Di Lauro R, Whitsett JA. The lung-specific surfactant protein B gene promoter is a target for thyroid transcription factor 1 and hepatocyte nuclear factor 3, indicating common factors for organ-specific gene expression along the foregut axis. Mol Cell Biol 1994;14:5671-81.  Back to cited text no. 8
    
9.
Vincenten J, Smit EF, Vos W, Grünberg K, Postmus PE, Heideman DA, et al. Negative NKX2-1 (TTF-1) as temporary surrogate marker for treatment selection during EGFR-mutation analysis in patients with non-small-cell lung cancer. J Thorac Oncol 2012;7:1522-7.  Back to cited text no. 9
    


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    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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