Abstract
Objective: The aim of the study was to investigate the mutation status of multiple driver genes by RT-qPCR and their significance in advanced lung adenocarcinoma using cytological specimens. Materials and Methods: 155 cytological specimens that had been diagnosed with lung adenocarcinoma in the Fourth Hospital of Hebei Medical University were selected from April to November 2019. The cytological specimens included serous cavity effusion and fine-needle aspiration biopsies. Among cytological specimens, 108 cases were processed by using the cell block method (CBM), and 47 cases were processed by the disposable membrane cell collector method (MCM) before DNA/RNA extraction. Ten drive genes of EGFR, ALK, ROS1, BRAF, KRAS, NRAS, HER2, RET, PIK3CA, and MET were combined detected at one step by the amplification refractory mutation system and ABI 7500 RT-qPCR. Results: The purity of RNA (p = 0.005) and DNA (p = 0.001) extracted by using the MCM was both significantly higher than that extracted by using the CBM. Forty-seven cases of fresh cell specimens processed by the MCM all succeeded in multigene detections, while of 108 specimens processed by the CBM, 6 cases failed in multigene detections. Among 149 specimens, single-gene mutation rates of EGFR, ALK, ROS1, RET, HER2, MET, KRAS, NRAS, BRAF, and PIK3CA mutations were 57.71%, 6.04%, 3.36%, 2.68%, 2.01%, 2.01%, 1.34%, 0.67%, 0% and 0% respectively, and 6 cases including 2 coexistence mutations. We found that mutation status was correlated with gender (p = 0.047), but not correlated with age (p = 0.141) and smoking status (p = 0.083). We found that the EGFR mutation status was correlated with gender (p = 0.003), age (p = 0.015) and smoking habits (p = 0.007), and ALK mutation status was correlated with age (p = 0.002). Conclusion: Compared with the CBM, the MCM can improve the efficiency of DNA/RNA extraction and PCR amplification by removing impurities and enriching tumor cells. And we speculate that the successful detection rate of fresh cytological specimens was higher than that of paraffin-embedded specimens. EGFR, ALK, and ROS1 mutations were the main driver mutations in patients with advanced lung adenocarcinoma. We speculate that EGFR and ALK are more prone to concomitant mutations, respectively. Targeted therapies for patients with coexisting mutations need further study.
References
1.
Gu
Y
, Zhang
J
, Zhou
Z
, Liu
D
, Zhu
H
, Wen
J
, Metastasis patterns and prognosis of octogenarians with NSCLC: a population-based study
. Aging Dis
. 2020
;11
(1
):82
–92
.2.
Wang
K
, Chen
R
, Feng
Z
, Zhu
YM
, Sun
XX
, Huang
W
, Identification of differentially expressed genes in non-small cell lung cancer
. Aging
. 2019
;11
(23
):11170
–85
. .3.
Ettinger
DS
, Wood
DE
, Aggarwal
C
, Aisner
DL
, Akerley
W
, Bauman
JR
, NCCN guidelines insights: non–small cell lung cancer, version 1.2020
. J Natl Compr Canc Netw
. 2019
;17
(12
):1464
–72
.4.
Ellison
G
, Donald
E
, McWalter
G
, Knight
L
, Fletcher
L
, Sherwood
J
, A comparison of ARMS and DNA sequencing for mutation analysis in clinical biopsy samples
. J Exp Clin Cancer Res
. 2010
;29
(1
):132
. .5.
Shi
X
, Wu
H
, Lu
J
, Duan
H
, Liu
X
, Liang
Z
. Screening for major driver oncogene alterations in adenosquamous lung carcinoma using PCR coupled with next-generation and Sanger sequencing methods
. Sci Rep
. 2016
;6
(1
):22297
–306
. .6.
Gao
J
, Wu
H
, Shi
X
, Huo
Z
, Zhang
J
, Liang
Z
. Comparison of next-generation sequencing, quantitative PCR, and sanger sequencing for mutation profiling of EGFR, KRAS, PIK3CA and BRAF in clinical lung tumors
. Clin Lab
. 2016
;62
(4
):689
–96
. .7.
Shaozhang
Z
, Ming
Z
, Haiyan
P
, Aiping
Z
, Qitao
Y
, Xiangqun
S
. Comparison of ARMS and direct sequencing for detection of EGFR mutation and prediction of EGFR-TKI efficacy between surgery and biopsy tumor tissues in NSCLC patients
. Med Oncol
. 2014
;31
(5
):926
. .8.
Guo
Y
, Cao
R
, Zhang
X
, Huang
L
, Sun
L
, Zhao
J
, Recent progress in rare oncogenic drivers and targeted therapy for non-small cell lung cancer
. Onco Targets Ther
. 2019
;12
:10343
–60
. .9.
NCCN
. Clinical practice guidelines in oncology. Non-small cell lung cancer
. https://www.nccn.org/professionals/physician_gls/default.aspx Accessed 10 May 2019.10.
Ji
X
, Che
N
, Lin
R
, Chen
J
, Wu
X
. Efficient ten-gene analysis of NSCLC tissue samples by next-generation sequencing
. Pathol Res Pract
. 2019
;215
(5
):1066
–70
. .11.
Kuo-Hsuan
H
, Chao-Chi
H
, Te-Chun
H
, Tseng
JS
, Su
KY
, Wu
MF
, Identification of five driver gene mutations in patients with treatment-native lung adenocarcinoma in Taiwan
. Plos One
. 2015
;10
(3
):120852
–866
.12.
Wen
S
, Dai
L
, Wang
L
, Wang
W
, Wu
D
, Wang
K
, Genomic signature of driver genes identified by target next‐generation sequencing in Chinese non‐small cell lung cancer
. The Oncol
. 2019
;24
(11
):1070
–81
. .13.
Ying
M
, Zhu
XX
, Zhao
Y
, Li
DH
, Chen
LH
. KRAS mutation as a biomarker for survival in patients with non-small cell lung cancer, a meta-analysis of 12 randomized trials
. Asian Pac J Cancer Prev
. 2015
;16
(10
):4439
–45
.14.
Campos-Parra
AD
, Zuloaga
C
, Manriquez
ME
, Avilés
A
, Borbolla-Escoboza
J
, Cardona
A
, KRAS mutation as the biomarker of response to chemotherapy and EGFR-TKIs in patients with advanced non-small cell lung cancer: clues for its potential use in second-line therapy decision making
. Am J Clin Oncol
. 2015
;38
(1
):33
–40
.15.
Shaw
AT
, Kim
DW
, Nakagawa
K
, Seto
T
, Crinó
L
, Ahn
MJ
, Crizotinib versus chemotherapy in advanced ALK-positive lung cancer
. N Engl J Med
. 2013
;368
(25
):2385
–94
. .16.
Shaw
AT
, Ou
SH
, Bang
YJ
, Camidge
DR
, Solomon
BJ
, Salgia
R
, Crizotinib in ROS1-rearranged non-small-cell lung cancer
. N Engl J Med
. 2014
;371
(21
):1963
–71
. .17.
Drilon
A
, Wang
L
, Hasanovic
A
, Suehara
Y
, Lipson
D
, Stephens
P
, Response to cabozantinib in patients with RET fusion-positive lung adenocarcinomas
. Cancer Discov
. 2013
;3
(6
):630
–5
. .18.
Yang
JJ
, Zhang
XC
, Su
J
, Xu
CR
, Zhou
Q
, Tian
HX
, Lung cancers with concomitant EGFR mutations and ALK rearrangements: diverse responses to EGFR‐TKI and crizotinib in relation to diverse receptors phosphorylation
. Clin Cancer Res
. 2014
;20
(5
):1383
–92
.19.
Won
JK
, Keam
B
, Koh
J
, Cho
HJ
, Jeon
YK
, Kim
TM
, Concomitant ALK translocation and EGFR mutation in lung cancer: a comparison of direct sequencing and sensitive assays and the impact on responsiveness to tyrosine kinase inhibitor
. Ann Oncol
. 2015
;26
(2
):348
–54
. .20.
Lou
NN
, Zhang
XC
, Chen
HJ
, Zhou
Q
, Yan
LX
, Xie
Z
, Clinical outcomes of advanced non-small-cell lung cancer patients with EGFR mutation, ALK rearrangement and EGFR/ALK co-alterations
. Oncotarget
. 2016
;7
(40
):65185
–95
. .21.
Planchard
D
, Besse
B
, Groen
HJM
, Souquet
PJ
, Quoix
E
, Baik
CS
, Dabrafenib plus trametinib in patients with previously treated BRAF(V600E)-mutant metastatic non-small cell lung cancer: an open-label, multicentre phase 2 trial
. Lanet Oncology
. 2016
;17
(7
):984
–93
.22.
Odogwu
L
, Mathieu
L
, Blumenthal
G
, Larkins
E
, Goldberg
KB
, Griffin
N
, FDA approval summary: dabrafenib and trametinib for the treatment of metastatic non-small cell lung cancers harboring BRAF V600E mutations
. Oncologist
. 2018
;23
(6
):740
–5
.23.
Ohashi
K
, Sequist
LV
, Arcila
ME
, Lovly
CM
, Chen
X
, Rudin
CM
, Characteristics of lung cancers harboring NRAS mutations
. Clin Cancer Res
. 2013
;19
(9
):2584
–91
. .24.
Reungwetwattana
T
, Liang
Y
, Zhu
V
, Ou
SI
. The race to target MET exon 14 skipping alterations in non-small cell lung cancer: the why, the how, the who, the unknown, and the inevitable
. Lung Cancer
. 2017
;103
:27
–37
.25.
Heist
RS
, Shim
HS
, Gingipally
S
, Mino-Kenudson
M
, Le
L
, Gainor
JF
, MET exon 14 skipping in non-small cell lung cancer
. Oncologist
. 2016
;21
(4
):481
–6
. .26.
Engelman
JA
, Zejnullahu
K
, Mitsudomi
T
, Song
Y
, Hyland
C
, Park
JO
, MET amplification leads to gefitinib resistance in lung cancer by activating ERBB3 signaling
. Science
. 2007
;316
(5827
):1039
–43
. .27.
De Grève
J
, Teugels
E
, Geers
C
, Decoster
L
, Galdermans
D
, De Mey
J
, Clinical activity of afatinib (BIBW 2992) in patients with lung adenocarcinoma with mutations in the kinase domain of HER2/neu
. Lung Cancer
. 2012
;76
(1
):123
–7
.28.
Schuler
M
, Wu
YL
, Hirsh
V
, O'Byrne
K
, Yamamoto
N
, Mok
T
, First-line afatinib versus chemotherapy in patients with non-small cell lung cancer and common epidermal growth factor receptor gene mutations and brain metastases
. J Thorac Oncol
. 2016
;11
(3
):380
–90
. .29.
Sequist
LV
, Waltman
BA
, Dias-Santagata
D
, Digumarthy
S
, Turke
AB
, Fidias
P
, Genotypic and histological evolution of lung cancers acquiring resistance to EGFR inhibitors
. Sci Transl Med
. 2011
;3
(75
):75ra26
–75
. .30.
Drilon
A
, Wang
L
, Hasanovic
A
, Suehara
Y
, Lipson
D
, Stephens
P
, Response to cabozantinib in patients with ret fusion-positive lung adenocarcinomas
. Cancer Discov
. 2013
;3
(6
):630
–5
. .31.
Zhuang
X
, Zhao
C
, Li
J
, Su
C
, Chen
X
, Ren
S
, Clinical features and therapeutic options in non-small cell lung cancer patients with concomitant mutations of EGFR, ALK, ROS1, KRAS or BRAF
. Cancer Med
. 2019
;8
(6
):2858
–66
.32.
Lin
E
, Li
L
, Guan
Y
, Soriano
R
, Rivers
CS
, Mohan
S
, Exon array profiling detects EML4-ALK fusion in breast, colorectal, and non-small cell lung cancers
. Mol Cancer Res
. 2009
;7
(9
):1466
–76
. .33.
Choi
YL
, Takeuchi
K
, Soda
M
, Inamura
K
, Togashi
Y
, Hatano
S
, Identification of novel isoforms of the EML4-ALK transforming gene in non-small cell lung cancer
. Cancer Res
. 2008
;68
(13
):4971
–6
. .34.
Ettinger
DS
, Akerley
W
, Bepler
G
, Blum
MG
, Chang
A
, Cheney
RT
, Non–small cell lung cancer, version 4.2015
. J Natl Compr Canc Netw
. 2014
;12
(12
):1738
–61
.35.
Yao
Y
, Peng
M
, Shen
Q
, Hu
Q
, Gong
H
, Li
Q
, Detecting EGFR mutations and ALK/ROS1 rearrangements in non-small cell lung cancer using malignant pleural effusion samples
. Thorac Cancer
. 2019
;10
(2
):193
–202
. .36.
Zhao
JY
, Wang
XY
, Zeng
HY
, The comparison of EGFR mutation detection in clinical biopsy samples of lung cancer by ARMS and direct sequencing
. Zhongguo Ai Zheng Za Zhi
. 2013
;23
(2
):106
–13
.37.
Suciu
BA
, Pap
Z
, Denes
L
, Brînzaniuc
K
, Copotoiu
C
, Pávai
Z
. Allele-specific PCR method for identification of EGFR mutations in non-small cell lung cancer: Formalin-fixed paraffin-embedded tissue versus fresh tissue
. Rom J Morphol Embryol
. 2016
;57
(2
):495
–500
.38.
Silke
VA
, Andreas
M
, Klaus
B
, Schlumpberger
M
. Determinants of RNA quality from FFPE samples
. Plos One
. 2007
;2
(12
):e1261
.39.
Yakovleva
A
, Plieskatt
JL
, Jensen
S
, Humeida
R
, Lang
J
, Li
G
, Fit for genomic and proteomic purposes: sampling the fitness of nucleic acid and protein derivatives from formalin fixed paraffin embedded tissue
. Plos One
. 2017
;12
(7
):e0181756
.40.
Lewis
F
, Maughan
NJ
, Smith
V
, Hillan
K
, Quirke
P
. Unlocking the archive–gene expression in paraffin-embedded tissue
. J Pathol
. 2001
;195
(1
):66
–71
. .41.
Gilda
DCS
, Saieg
MA
. Preanalytic specimen triage: smears, cell blocks, cytospin preparations, transport media, and cytobanking
. Cancer Cytopathology
. 2017
;125
(S6
):455
–64
.42.
Huang
M
, Shuanzeng
S
. Overview of molecular testing of cytology specimens
. Acta cytologica
. 2020
;64
(1–2
):136
–46
.43.
Liu
L
, Liu
J
, Shao
D
, Deng
Q
, Tang
H
, Liu
Z
, Comprehensive genomic profiling of lung cancer using a validated panel to explore therapeutic targets in East Asian patients
. Cancer Sci
. 2017
;108
(12
):2487
–94
. .© 2021 S. Karger AG, Basel
2021
Copyright / Drug Dosage / Disclaimer
Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.
You do not currently have access to this content.
