Introduction
Activating mutations of the epidermal growth factor
receptor (EGFR) gene are specific to non-small cell lung
cancer (NSCLC), particularly adenocarcinoma (1), indicating its diagnostic value for
lung tumor. The status of EGFR mutation is also
therapeutically significant for the selection of patients for
treatment with EGFR tyrosine kinase inhibitors (EGFR-TKIs)
(2). Approximately 90% of
EGFR mutations are composed of either in-frame deletions in
exon 19 or a specific missense mutation in exon 21 (L858R)
(1–3). Direct DNA sequencing is the most
accurate detection assay for EGFR mutations, although we and
others have developed a number of highly sensitive assays capable
of detecting extremely low numbers of EGFR mutant alleles
among numerous wild-type alleles (4–7). Of
note, EGFR mutations were previously detected in
histologically normal respiratory epithelium surrounding
histologically malignant regions (8).
Computed tomography (CT) imaging enables small lung
tumors to be identified (9).
CT-guided lung needle biopsy (CTNB) is useful for the pathological
diagnosis of such tumors (10,11).
However, correct diagnosis of tumors is occassionally impossible
since there are no malignant cells in the CTNB specimens,
particularly when materials are insufficient. CTNB specimens may
also be used for molecular analysis, including those for genetic
mutations (4,5). We previously demonstrated that
EGFR mutations are capable of being detected in CTNB
specimens even when the proportion of cancer cells is low,
suggesting that the detection of EGFR mutations in the cells
aids the diagnosis of NSCLC (4,7).
In this study, the EGFR mutational status of
pathologically non-malignant CTNB specimens were examined using a
modified mutant-enriched polymerase-chain reaction(PCR) assay and
compared with the EGFR mutational status of surgically
resected tumors that had a pathological diagnosis of NSCLC.
Materials and methods
Materials
Between April 2000 and August 2008, CTNB for lung
tumors was performed in 1,109 patients at Okayama University
Hospital (Japan). Histological diagnosis was determined using
hematoxylin and eosin (H&E)-stained sections of CTNB specimens
and surgically resected tumors. Of these tumors, 24 tumors were
diagnosed as non-malignant, but the clinical findings suggested a
high likelihood of malignancy. Finally, 15 of the 24 tumors were
diagnosed as primary lung cancer. Twelve of the tumors were
available for further study. The clinical data of these 12 cases
are shown in Table I. DNA was
extracted from the paraffin-embedded specimens of the CTNB and the
surgically resected tumors without micro-dissection using the
QIAamp® DNA FFPE tissue kit (Qiagen Inc., Valencia, CA,
USA) according to the manufacturer’s instructions.
 | Table IClinical characteristics of 12
computed tomography-guided lung needle biopsy specimens diagnosed
as non-malignant. |
Table I
Clinical characteristics of 12
computed tomography-guided lung needle biopsy specimens diagnosed
as non-malignant.
| Case | Age | Gender | Smoking status | ap-Stage | Histology of resected
specimens | Size (mm) | EGFR
mutation |
|---|
| | | | | | |
|
|
|---|
| | | | | | | Tumor | CTNB |
|---|
| 1 | 61 | F | Never | IA | AD | 11 | ex19 del. | ex19 del. |
| 2 | 77 | M | Ever | IA | AD | 13 | ex19 del. | WT |
| 3 | 75 | M | Ever | IA | AD | 22 | ex19 del. | WT |
| 4 | 76 | F | Never | IA | AD | 12 | WT | WT |
| 5 | 70 | M | Ever | IA | AD | 14 | WT | WT |
| 6 | 76 | M | Ever | IA | SQ | 15 | WT | WT |
| 7 | 57 | M | Ever | IB | AD | 40 | WT | WT |
| 8 | 75 | M | Ever | IA | AD | 30 | WT | WT |
| 9 | 72 | F | Never | IA | AD | 12 | WT | WT |
| 10 | 77 | M | Ever | IA | AD | 22 | WT | WT |
| 11 | 58 | M | Ever | IA | AD | 13 | WT | WT |
| 12 | 73 | M | Ever | IA | AD | 10 | WT | WT |
Methods
EGFR gene mutations in exons 19 and 21 were
determined using mutant-enriched PCR, a two-step PCR with
intermittent restriction digestion to selectively eliminate
wild-type alleles and to enrich mutated alleles (4,7). Our
previously developed mutant-enriched PCR assay was initially used
on all 12 CTNB samples. However, amplification was not achieved in
some CTNB samples, particularly when they were small specimens.
Therefore, we developed a modified mutant-enriched PCR to detect
exon 19 deletions in which the amplicon was shorter than that of
the previous version (79 bp versus 138 bp). Primer sequences were
as follows: 5′-AAGATAAAATTCCCGTCGCT-3′ (forward primer) and
5′-CTCACATCGAGGATTTCCTTG-3′ (reverse primer), and PCR conditions
were the same as those of our previous study. The first round of
amplification used 5–100 ng sample DNA, 150 μmol/l deoxynucleotide
triphosphate, 2 pmol of each primer and 0.25 units HotStarTaq DNA
polymerase (Qiagen, Inc.). Intermittent restriction digestion of 2
μl first-round PCR products was carried out with 10 IU MseI
at 37°C for 6 h. An aliquot of first-round PCR product was used as
a template for second-round amplification under the same conditions
as the first-round PCR. The product of the second amplification was
analyzed by ethidium bromide staining of 12% polyacrylamide gel
electrophoresis and reproducibility was confirmed in two
independent experiments.
Results
Clinical characteristics
The histological diagnoses of CTNB specimens and
surgically resected specimens were re-evaluated to confirm that the
CTNB samples of all 12 cases showed no malignancy, whereas all of
the pathological diagnoses of corresponding resected specimens were
NSCLCs. The characteristics of the cases are shown in Table I.
Mutations
We determined the EGFR mutational status of
12 surgically resected specimens, and identified three deletions in
exon 19 (25%), but no EGFR exon 21 L858R mutations. The
mutational status of CTNB samples was blindly evaluated using the
modified mutant-enriched PCR assay, with the finding that one
sample harbored an EGFR exon 19 deletion (8.3%). This result
was confirmed in two independent experiments. The histological
images of CTNB and the resected tumor of Case 1 are shown in
Fig. 1. The pathological findings
of the Case 1 tumor revealed a clear separation of malignant and
non-malignant cell areas (Fig. 1A).
Of note, the same type of EGFR exon 19 deletion (E746 to
A750) was present in the CTNB specimen and the resected tumor of
Case 1 (Fig. 2, sequencing data is
not shown). In addition, the EGFR mutation was not present
in the distant peripheral normal lung tissue of Case 1. No
EGFR mutation was found in the CTNB samples of Cases 2 and
3.
Discussion
The detection of EGFR mutations is crucial
for the diagnosis of NSCLC as well as for selecting patients for
EGFR-TKI treatment (2,12,13).
Sensitive assays have been developed to detect EGFR
mutations in small clinical samples that are assumed to contain
numerous non-malignant cells, including CTNB specimens, pleural
effusion and circulating tumor cells (4–7,14). In
the present study, EGFR mutations were detected in
pathologically non-malignant CTNB specimens using our modified
mutant-enriched PCR assay. This finding may be explained in two
ways: i) the presence of a small portion of malignant cells only in
DNA-extracted sections and not in the H&E-stained section, ii)
the presence of EGFR mutations in the lung tissue of
normal-appearing surrounding tumor tissue harboring EGFR
mutations. The latter is supported by a previous study by Tang
et al (8). These authors
determined EGFR mutational status in 21 lung adenocarcinoma
cases and their adjacent and distant normal lung tissues to find
that EGFR mutations were present in 43% of adenocarcinomas,
24% of adjacent normal tissues and none of the distant tissues
(8). These authors also reported
that the mutational patterns in the tumors and corresponding
adjacent, non-malignant specimens were identical. Of note, more
frequent mutations affecting normal epithelium were found in exon
19 (54%) compared with exon 21 (7%). The mutational type of the
mutant case in this study in a non-malignant CTNB specimen was also
an exon 19 deletion. Our results support the hypothesis that a
localized type of field effect phenomenon may exist for EGFR
mutations in the lung respiratory epithelium surrounding lung
adenocarcinomas (8).
Our results also have clinical implications. The
administration of EGFR-TKIs is recommended for patients with
EGFR mutation and is exclusively found in NSCLC (15). Thus, detection of an EGFR
mutation from lung tumors may be considered to be NSCLC. As shown,
sensitive assays may be capable of detecting EGFR mutations
from pathologically non-malignant CTNB specimens, resulting in
avoidance of additional invasive procedures for patients. Our study
suggests that the EGFR mutation should be analyzed even in
biopsied specimens with non-malignant diagnosis to determine
whether patients should be treated with EGFR-TKI when they are
clinically suspected as having NSCLC.
Acknowledgements
We would like to thank Ms. Fumiko Isobe, General
Thoracic surgery and Mr. Tetsushi Iwasa, Central Research
Laboratory, Okayama University Graduate School of Medicine,
Dentistry and Pharmaceutical Sciences for their excellent technical
support.
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