Analysis of an EGFR mutation by PNA clamping method in lung carcinoid tumors

Jong In Kim

doi:10.7180/kmj.2015.30.2.141

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Kim: Analysis of an EGFR mutation by PNA clamping method in lung carcinoid tumors

Original Article

Kosin Medical Journal 2015;30(2):141-147.

Published online: 20 January 2015

DOI: https://doi.org/10.7180/kmj.2015.30.2.141

Analysis of an EGFR mutation by PNA clamping method in lung carcinoid tumors

Jong In Kim

Department of Thoracic and Cardiovascular Surgery, College of Medicine, Kosin University, Busan, Korea.

Corresponding Author: Jong In Kim, Department of Thoracic and Cardiovascular Surgery, College of Medicine, Kosin University, 262, Gamcheon-ro, Seo-gu, Busan 49267, Korea. TEL: +82-51-990-6253 FAX: +82-51-990-3994 E-mail: schoi@ns.kosinmed.or.kr

Received 21 October 20143 March 2015 Accepted 1 April 2015

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

Pulmonary carcinoid tumors consisting of typical carcinoid tumors (TC) and atypical carcinoid tumors (AC) are rare, accounting for 2–5% of all lung tumors. TC is considered a low-grade tumor with a rate of distant metastasis up to 12%. In contrast, ACs are more aggressive tumors, displaying a metastatic rate up to 70%. Surgery is the treatment of choice; however, the current treatment outcomes of metastatic lung carcinoids are discouraging. This study aimed to investigate the EGFR mutation using the PNA-mediated clamping method and to provide basic data for using EGFR-TK1 and its clinical implications.

Materials and Methods

A total of 14 cases that underwent surgery were diagnosed as carcinoid tumors and pathologically classified as TC and AC. The paraffin-embedded tissues were analyzed for EGFR mutations using the PNA-mediated PCR clamping technique. The mutant type was noted in the cases with a^△Ct greater than 2.0.

Result

Of 14 cases, eight were AC and six cases were TC. No known EGFR mutation was detected with a^△Ct less than 2.0.

Conclusion

The EGFR genotype determined using the PNA-mediated PCR clamping method was wild-type in all pulmonary carcinoid tumors. Therefore, the application of EGFR-TK1 is limited in pulmonary carcinoid tumors.

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Keywords: carcinoid, EGFR mutation, Lung, PNA clamping

References

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Fig. 1.

The four steps of the PCR cycle in PNA Clamp^TM

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Fig. 2.

The wild type was detected in PNA-mediated clamping real-time PCR for EGFR.

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Table 1.

The clinical and pathological characteristics of 14 cases of pulmonary carcinoid tumors

Case	Dx	Age	Gender	Tumor Size (cm)	No of mitosis events (/10HPF)	Necrosis	Lymph node metastasis	Neuroendocrine markers
1	T	63	F	2.8	0	–	–	+
2	T	41	F	3	1	–	–	+
3	T	48	M	5	0	–	–	+
4	T	53	M	3.5	1	–	–	+
5	T	31	M	1.8	0	–	–	+
6	T	59	M	1.2	0	–	–	+/-
7	A	34	M	4	6	–	–	+
8	A	55	M	5	7	–	+	+
9	A	47	M	4.8	5	–	–	+
10	A	61	M	4.5	10	+	–	+
11	A	54	M	3.3	4	–	+	+
12	A	72	M	5	4	+	–	+
13	A	62	M	2.3	3	–	–	+
14	A	57	M	0.5	5	–	–	+

A: atypical carcinoid, T: typical carcinoid, M: male, F: female, +: present, -: absent

Table 2.

The results of analysis of EGFR PNA-mediated clamping using real-time PCR

Sample No(Ct)	G719X (①)	E19 del. (②)	T790M (③)	S768I (④)	Ins. 3 dup (⑤)	Ins.3 (⑥)	L858R & L861Q(⑦)	Non-PNA (⑧)
15	39.11	38	38	33.24	39.02	36.5	38	28.63
Clamping control	38	36.16	39.79	31.82	30.42	35.92	34.42	23.66
standard Ct	36	33	33	30	27	30	33
△Ct	–2	–0.06	–0.37	–3.91	–3.45	–8	–6.62
determine	wild	wild	wild	wild	wild	wild	Wild

Criterion: EGFR mutation is noted when the △Ct value, standard Ct minus sample Ct, is 2.0 or above. [△Ct = standard ct – sample ct]

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Case	Dx	Age	Gender	Tumor Size (cm)	No of mitosis events (/10HPF)	Necrosis	Lymph node metastasis	Neuroendocrine markers
1	T	63	F	2.8	0	–	–	+
2	T	41	F	3	1	–	–	+
3	T	48	M	5	0	–	–	+
4	T	53	M	3.5	1	–	–	+
5	T	31	M	1.8	0	–	–	+
6	T	59	M	1.2	0	–	–	+/-
7	A	34	M	4	6	–	–	+
8	A	55	M	5	7	–	+	+
9	A	47	M	4.8	5	–	–	+
10	A	61	M	4.5	10	+	–	+
11	A	54	M	3.3	4	–	+	+
12	A	72	M	5	4	+	–	+
13	A	62	M	2.3	3	–	–	+
14	A	57	M	0.5	5	–	–	+

Case	Dx	Age	Gender	Tumor Size (cm)	No of mitosis events (/10HPF)	Necrosis	Lymph node metastasis	Neuroendocrine markers
1	T	63	F	2.8	0	–	–	+
2	T	41	F	3	1	–	–	+
3	T	48	M	5	0	–	–	+
4	T	53	M	3.5	1	–	–	+
5	T	31	M	1.8	0	–	–	+
6	T	59	M	1.2	0	–	–	+/-
7	A	34	M	4	6	–	–	+
8	A	55	M	5	7	–	+	+
9	A	47	M	4.8	5	–	–	+
10	A	61	M	4.5	10	+	–	+
11	A	54	M	3.3	4	–	+	+
12	A	72	M	5	4	+	–	+
13	A	62	M	2.3	3	–	–	+
14	A	57	M	0.5	5	–	–	+

Case	Dx	Age	Gender	Tumor Size (cm)	No of mitosis events (/10HPF)	Necrosis	Lymph node metastasis	Neuroendocrine markers
1	T	63	F	2.8	0	–	–	+
2	T	41	F	3	1	–	–	+
3	T	48	M	5	0	–	–	+
4	T	53	M	3.5	1	–	–	+
5	T	31	M	1.8	0	–	–	+
6	T	59	M	1.2	0	–	–	+/-
7	A	34	M	4	6	–	–	+
8	A	55	M	5	7	–	+	+
9	A	47	M	4.8	5	–	–	+
10	A	61	M	4.5	10	+	–	+
11	A	54	M	3.3	4	–	+	+
12	A	72	M	5	4	+	–	+
13	A	62	M	2.3	3	–	–	+
14	A	57	M	0.5	5	–	–	+