Vol. 1, 359-365, March 1995 Clinical Cancer Research 359
3 The abbreviations used are: NSCLC, non-small cell lung carcinomas;RFLP, restriction fragment length polymorphism.
Mutations of K-ras Oncogene and Absence of H-ras Mutations in
Squamous Cell Carcinomas of the Lung1
JiItI Vachtenheim,2 Irena Hor#{225}kov#{225},
Hana Novotn#{225}, Petr Op#{225}lka,
and Helena Roubkov#{225}
Laboratory of Molecular Biology [J. V., I. H., H. N.] and Department
of Clinical Oncology [P. 0., H. R.], Institute of Chest Diseases,18071 Prague 8, Czech Republic
ABSTRACT
Mutations of the K-ras gene have been implicated in the
pathogenesis of human lung adenocarcinomas. In most stud-
ies published so far, squamous cell lung cancers harboredras mutations only exceptionally or no mutations were de-
tected at all. We have examined 141 lung tumor DNA sam-ples for mutations in codons 12, 13, and 61 of K-ras and
H-ras oncogenes. A large panel of 118 squamous cell carci-
nomas was included in the study. For K-ras codon 12, weused a sensitive two-step PCR-restriction fragment length
polymorphism method which detects < 1 % of mutated DNAin the sample. K-ras mutations were found in 17 tumors(12%; 14 in codon 12 and 3 in codon 13). Among 19 adeno-carcinomas, mutation was revealed in 7 samples (37%). Of
these, one sample harbored two point mutations in codon 12.
Nine mutational events were found in squamous cell carci-
nomas (8%, one adenosquamous carcinoma included, all incodon 12). Of four large cell carcinomas, one contained amutation. Mutant-enriched PCR products harboring muta-tions were directly sequenced. Fifteen mutational events
were G-�T transversions or G-+A transitions, one was a
G-+C transition, and one sample revealed a frameshift de-letion of one G from codon 12. Similar mutational spectrum
was found in both squamous cell carcinomas and adenocar-cinomas, suggesting similar carcinogenic pathways in both
histological types of the tumor. The presence of mutationsdid not correlate with the stage of the disease. Moreover, weanalyzed all samples for mutations in codons 12, 13, and 61of the H-ras gene. We found only one mutation in codon 12.
Thus, H-ras mutations apparently play an inferior role inlung carcinogenesis. We conclude that mutations of the K-ras oncogene can play a role in the development of not onlylung adenocarcinomas but also of a subset (about 8%) ofsquamous cell carcinomas.
INTRODUCTION
The ras genes code for small membrane-bound proteins
(p21’�) that bind and hydrolyze GTP and participate, through a
cascade of protein kinases, in transmitting signals into the flu-
cleus. Activating point mutations in genes of the ras gene family
that convert these proto-oncogenes into their transforming forms
are well documented to occur in many human tumors. Most
frequently, mutations of the c-Ki-ras-2 (K-ras) oncogene were
detected in 70-95% of pancreatic cancers (1-4), in 30-60% of
colon and rectal tumors (5-8), and in about 45% of ovarian
tumors (9). K-ras was also reported to be activated in the
neoplasms of the lung and other organs. Most of these mutations
occur in codon 12 and only a minority of mutational events is
located in codons 13 and 61. In many tumor cell types, K-ras
mutations have been suggested to play a role in the multistep
molecular pathogenesis of neoplastic transformation. For exam-
ple, in carcinomas of the colon, K-ras appears to become
mutated during the adenoma growth. Recently, although at
lower frequencies, the mutations were detected even in normal
colonic mucosa from patients suffering from the tumor (10, 1 1).
Both borderline and invasive ovarian carcinomas were also
shown mutated in K-ras (9), implying that borderline tumors
may represent a continuum between benign and invasive ovar-
ian tumors. Also, K-ras mutations were found in 2 of 16 atypical
endometrial hyperplasias, which are precursors of endometrial
carcinomas (12). Pancreatic precancerous lesions (mucous cell
hyperplasias), the presumed precursors of the pancreatic carci-
noma, have been also shown to contain K-ras mutations at high
frequencies similar to those documented for ductal type of
pancreatic carcinomas (13). All of these findings indicate that
K-ras mutations may be relatively early genetic events in the
formation of many human neoplasms.
About 80% of lung cancers are histologically classified as
NSCLC,3 comprising the two most frequent types, adenocarci-
noma and squamous cell carcinoma. It has been well docu-
mented that K-ras is mutated in a subset of lung adenocarcino-
mas. The frequency of mutations varied from 15 to 60% (14-
25). Squamous cell carcinomas of the lung were reported to
contain mutated K-ras only exceptionally. Of the 21 squamous
cell lung tumors, 2 showed activated K-ras (24) and 2 of 6 cell
lines established from squamous cell lung carcinomas contained
K-ras mutations in codon 12 (21). In earlier studies, no muta-
tions of K-ras in this type oftumor were detected (14, 17). Also,
two mutations of H-ras, both in codon 61, were detected among
36 lung squamous cell carcinomas (19). In a recent study,
however, the authors described an unusual distribution of K-ras
activating mutations in lung neoplasms, the squamous cell tu-
mors being mutated more frequently (8’38) than adenocarcino-
mas (3’22; Ref. 26). ras mutations are absent from small cell
lung carcinomas (21) and neuroendocrine neoplasms of the lung
(27). The clinical significance of K-ras mutations in lung car-
Received 9/21/94; accepted 10/21/94.t This work was supported by Grants 0072-3, 0952-3, and 2290-3 from
the Internal Granting Agency, Ministry of Health, Czech Republic (J. V.).2 To whom requests for reprints should be addressed.
Research. on August 19, 2018. © 1995 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
360 Mutations of K-ras in Lung Cancer
cinomas has also been investigated. Tumors harboring these
mutations were found to have a generally worse prognosis and
were associated with shortened survival (18, 26, 28).
In this work, we have undertaken the study on a large panel
of squamous cell lung carcinomas to analyze the mutations in
K-ras and H-ras oncogenes. In most studies carried out to date,
activating mutations were detected by methods that do not
enable revelation of the mutation if present in less than about
5-10% of cells (oligonucleotide hybridization, single-strand
conformation polymorphism, and direct sequencing). Here we
used a modified two-step mismatch, PCR method (29, 30) in
which the detection of mutation in <1% of the tumor cell
population is reliably accomplishable. In this method, the arti-
ficial restriction site is introduced by a mismatched primer. The
normal allele is destroyed after the first PCR step by restriction
enzyme digestion, enabling only the mutant allele to be ampli-
fled in the second PCR step (29, 30). We used this approach to
screen changes in codon 12 of the K-ras gene, which is the most
frequent mutational hot spot of ras genes in lung carcinomas
and in most of other human solid tumors. For codons 13 and 61
of K-ras and all three codons (codons 12, 13, and 61) of H-ras,
a single-step mismatch PCR assay was used. To determine the
type of mutation, the mutant-enriched PCR products were di-
rectly sequenced.
MATERIALS AND METHODS
Tissue Samples, Preparation of DNA, and Control CellLines. Fresh tumor samples were obtained from surgically
removed lungs immediately after resection. The patients were
treated at the Institute of Chest Diseases (Prague, Czech Repub-
lic) and did not receive chemotherapy or radiotherapy before
surgical resection. A fragment of tumor tissue adjacent to that
used for histopathological diagnosis was frozed in liquid nitro-
gen and stored at -75#{176}C. High molecular weight DNA was
prepared by standard techniques using proteinase K digestion
and phenol/chloroform extraction.
DNA extracted from cell lines (American Type Culture
Collection) with known mutations served as controls. SW 480
cell line contains a homozygous mutation in codon 12 of K-ras
(GGT-�GTT). In lines Calu-1 and A-427, one allele is mutated
at the same codon (GGT-�TGT and GGT-+GAT, respective-
ly). LoVo cell line harbors GGC-�GAC mutation in codon 13
of K-ras and NCI-H460 has a CAA-�CAT mutation in codon
61 of K-ras. pT24C3 plasmid was a control for the screening of
the H-ras (codon 12) mutations.
PCR. Genomic DNA (0.2 or 0.4 p.g) was amplified in
20- or 40-pA volumes in a reaction containing primers (Genset),
dNTPs (see below), 50 mM KC1, 10 mtvt Tris-HC1 (pH 8.3),
0.01% (w/v) gelatin, and 1.5 m� MgC12. For codons 12 and 13
of the K-ras, the concentration of MgC12 was 2 mr�t. Table 1
gives the primers used in amplification reactions. Final concen-
tration of primers in the reaction was 0.5 p�M except for codon
13 H-ras where the concentration 1 JiM was used. dNTPs were
at the following final concentrations: 50 p.M (K-ras, codons 12
and 13), 100 p.M (K-ras, codon 61; H-ras, codon 61), or 200 p.M
(H-ras, codons 12 and 13). One unit of Taq polymerase (Perkin
Elmer’Cetus) was added to the 40-pA reaction in hot start set-
tings. Amplification mixtures were cycled 30 or 35 cycles in a
thermal cycler (Perkin Elmer/Cetus), each cycle at 94#{176}Cfor 1
mm followed by 1 or 2 mm at the annealing temperature and 1
or 2 mm at 72#{176}C.For codon 12 of K-ras, samples were ampli-
fled 16 cycles in the first step and then diluted 400 times in the
second-step amplification. For single-step PCR screening of
K-ras codon 12 mutations, primers for the second step were
used (Table 1). The annealing temperatures were as follows:
55#{176}Cfor K-ras, codon 12 (both steps); 52#{176}Cfor K-ras, codons13 and 61; 60#{176}Cfor H-ras, codons 12 and 13; and 40#{176}Cfor
H-ras, codon 61.
The template for codon 13 of K-ras amplification was a
176-base pair PCR product amplified from genomic DNA by
use of non-mismatched primers (K125 and K12A; Ref. 21) and
diluted 10,000 times in the second amplification. For screening
of codon 61 of H-ras, an approach described by the same
authors was used: PCR product obtained by amplification of
genomic DNA with primers 5’-ATGAGAGGTACCAGG�A-
GAG and 5’-TCACGGGGTflTCACCTGTACT served as a tem-
plate for amplification.
Restriction Enzyme Cleavage and Gel Electrophoresis.After amplification, reaction products were incubated with the
appropriate restriction enzyme (Table 1) for a minimum of 4 h
(3 units enzyme/sample) and electrophoresed through native 8%
polyacrylamide gel or 3% NuSieve 3:1 agarose gel (FMC),
stained in ethidium bromide, and photographed. Restriction
enzymes were purchased from Fermentas (MvaI, BsuRI, BclI,
and MspI), Sigma (HphI and A1wNI), and New England Biolabs
(Earl). All samples showing mutant-specific band were pro-
cessed twice to exclude a possible error introduced by Taq
polymerase.
DNA Sequencing. For sequencing, the mutant allele-
enriched PCR amplification products with mutations in codon
12 of the K-ras gene were purified by Magic PCR Preps kit
(Promega) without restriction enzyme cleavage. The samples
with mutations in codon 13 of K-ras were prepared for sequenc-
ing as follows: 176-base pair template (above) was first ampli-
fled with the antisense primer as in Table 1 and a sense primer
(5’-TATFATAAGGCGTGCTGAAAATGA) having one mis-
match in order to destroy the native BsuRI site, cleaved with
BsuRI, diluted and amplified again with primers shown in Table
1 for K-ras, codon 13. The PCR product was then purified on a
Minicon 30 (Amicon) microconcentrator.
Double-stranded PCR products were sequenced by Cir-
cumVent (New England Biolabs) orfinol (Promega) sequencing
kits according to the conditions specified by the suppliers.
32P-labeled antisense primer (second step, Table 1) and the
sense primer (Table 1) were used for sequencing PCR products
to detect mutations in codon 12 and codon 13 of the K-ras gene,
respectively.
Statistical Methods. Two-tailed Fisher’s exact test was
used to examine the correlation of data (stage of the disease,
histology, and presence or absence of mutations).
RESULTS
Mismatch PCR.RFLP Method for the Detection of Mu-tations in K-ras and H-ras Genes. A RFLP created through
mismatched primers is a rapid and efficient method to detect
point mutations and has been applied to the study of activation
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- N � 1� �ri ‘0 N 00 C’s 2 � � � Z
a
b
--- - �$#{149}‘ H’-� -� �
4- Uncut4- Mutant
4- Wild
b...� � � ‘-� �J � \ . .
�“e � -‘.�tS’d�led�.a#{224}�”1
Clinical Cancer Research 361
Table 1 Summary of PCR primers, amplification products, restriction enzymes, and diagnostic restriction fragments for the K-ras and H-ras
genes
Primers
PCRproduct
(6p)(’
Restriction
enzyme
Mutant-specific
fragment (bp)
Wild allele-specific
fragment (bp)
K-ras
Codon 12 s:
a:
5 :
a:
5�ACTGAATATAAACTTGTGGTAGTTGGACCTt�
5’-TCAAAGAATGGTCCTGCACC (first step)
5’-ACTGAATATAAACTTGTGGTAGTTGGACCT
5’-TCAAAGAATGGTCCTGGACC (second step) 157 MvaI 142 113
Codon 13 5:
a:5’-TATTATAAGGCCTGCTGAAAATGA
5’-TATCGTCAAGGCACTCTTGCCTAGG 83 BsuRI 73 48
Codon 6 1 5 :
a:5:
a:
5 ‘ -CTTGGATATTCTCGACACAGCTGAT
5’-AATTACTCCTTAATGTCAGC
5’-GGATATTCTCGACACAGCAGGTGA
5’-AATTACTCCTTAATGTCAGC
179
176
BclI
Earl
179
176
155
157
H-ras
Codon 12 5: 5’-GAGACCCTGTAGGAGGACCC 312 MspI 291 235
a : 5 ‘ -GGGTGCTGAGACGAGGGACT
Codon 13 5:
a:5’-CCTCCTTGGCAGGTGAGGCAGGA
5’-GGTCAGCGCACTCTTGCCCTCA 125 HphI 101 90
Codon 6 1 5 :
a:
5 ‘ -ATGAGAGGTACCAGGCAGAG
5’-CGCATGGCGCTGTACAGCTC 177 A1wNI 157 139
a bp, base pairs; s, sense primer; a, antisense primer.b Mismatched nucleotides are bold.
of the ras gene group (21, 31). For codon 12 of K-ras, the
primers used here in the two-step screening were the same as
described (11). Sequences of all primers are summarized in
Table 1. In most assays, a further mismatch is introduced into
the primers to create a control restriction site. Because of this the
sizes of PCR products differ from those obtained after restric-
tion enzyme digestion enabling thus to distinguish between the
low level of the mutant allele and possible incomplete digestion
by a restriction enzyme.
When the positive control DNA (SW 480, homozygously
mutated K-ras codon 12) was diluted with normal DNA and
processed by a two-step assay, the mutant allele-specific band
was still discernible in the 1:333 dilution (not shown).
Frequency of Mutations in NSCLC and Single-Step
versus Double-Step Assay for K-ras Codon 12. The non-
small cell lung tumor samples analyzed in our study comprised
1 17 cases of squamous cell carcinomas, 1 adenosquamous cell
carcinoma, 19 adenocarcinomas, and 4 large cell carcinomas.
All tumors have been subjected to the two-step PCR-RFLP
analysis to detect mutations in codon 12 of K-ras. Of 141
tumors 14 (10%) were found mutated in this codon (Fig. la).
Four of these mutations were detected among I 9 adenocarcino-
mas. Nine of 118 squamous cell carcinomas (1 adenosquamous
carcinoma included) and 1 of 4 large cell tumors harbored the
mutation. The 14 samples were also screened by a less sensitive
one-step procedure. As shown in Fig. lb, the band intensity of
mutated allele varied. In several samples, the mutated allele-
specific band was very weak and in two samples (Fig. la, Lanes
4 and 5), the mutation was just at the limit of detection. It means
that, in mutation-positive tumors, at least 5% of the cells were
mutated. This detection limit for the single-step method was
estimated by a titration experiment with mutated (SW480) and
control DNA (not shown). Of the 14 samples with K-ras codon
12 mutation, 1 mutation was found to be homozygous (Fig. 1,
4- Uncut4- Mutant
4- Wild
Fig. I Mutation of K-ras codon 12 in NSCLC. Two-step (a) and
single-step (b) PCR-RFLP assay in 14 tumor samples. Order of samplesin a and b is the same and the numbers correspond to those in Table 2
and in Fig. 3a. PCR-RFLP conditions are described in “Materials and
Methods’ ‘. Two or three independent amplifications were performed to
verify the mutations; the same results were obtained.
Lane 2). In this sample, either the normal allele was lost or both
alleles had the same type of mutation. Normal allele-specific
band was detectable in all other samples, along with the mutated
one (Fig. lb).
We screened codons 13 and 61 of the K-ras gene further
for point mutations by a one-step PCR-RFLP procedure and
found three mutations in codon 13 (Fig. 2). All of them occurred
in adenocarcinomas. In the titration experiment for codon 13,
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362 Mutations of K-ras in Lung Cancer
%r� � S �,- - - 0
‘E- Uncut
�- Mutant
�- Wild
Fig. 2 K-ras mutations in codon 13 detected by a single-step PCR-RFLP method. Three tumors show a mutant-specific band of the samesize as positive control DNA (LoVo cell line). Numbers of tumorscorrespond to those in Table 2 and in Fig. 3b. The sizes of fragments arein Table 1 . The assay was repeated three times with the samples shownand the same results were obtained.
mutation was seen until the dilution 1:20 (LoVo cell line DNA
diluted in a normal DNA, not shown). For K-ras codon 61, no
artificial restriction site could be designed to detect point mu-
tations in all three nucleotides in a single assay. The mutations
in the two first bases and the mutation in the third position were
therefore analyzed separately as described (21). No mutation
was found in this codon. In the control DNA (NCI-H460), a
clear mutant-specific band was seen (not shown).
Mutations of H-ras were rarely detected in NSCLC. Since
v-Ha-ras is capable of transforming normal human bronchial
epithelial cells in culture (32), we were interested in determining
if there are any mutations in any of the three codons of H-ras in
our samples. Only 1 mutation in codon 12 was observed among
141 NSCLC samples analyzed. This mutant-specific band was
very weak, indicating that only a minor portion of tumor cell
population (<5%) was mutated (not shown; this sample was not
sequenced). Thus, H-ras mutations do not play a role in the
development of human NSCLC. In the codon 12 of H-ras,
however, G-�C change in the second position cannot be de-
tected by a PCR-RFLP assay because this mutation creates a
new MspI restriction site just three bases downstream, resulting
in the PCR product which is indiscernible from the wild one on
the gel.
Correlation of Clinical Parameters with the MutationalFrequency. When clinical parameters were compared to the
finding of a mutation, no statistically significant correlation
between the stage of the disease (ThM classification I-IV) and
the presence or absence of K-ras mutation was found. However,
all three codon 13 mutations were in stages III or IV. Although
the K-ras mutations were found about two times more fre-
quently in NSCLC cases with node metastases, no statistically
significant correlation exists between these two groups. Further-
more, while the occurrence of mutations in codon 12 was
common to both types of lung cancer studied, codon 13 muta-
tions of K-ras were absent from lung squamous cell carcinomas.
Spectrum of K-ras Mutations in NSCLC. The histo-
logical type of individual tumors, the mutational events, and the
stages of the disease are summarized in Table 2. The mutations
were determined by direct sequencing of mutant-enriched PCR
products after the second step of PCR (‘ ‘Materials and Meth-
ods’ ‘). Fig. 3a shows examples of sequences around the codon
12 of K-ras in mutated NSCLC tumors and Fig. 3b depicts an
example of mutations found in the three adenocarcinomas mu-
tated in K-ras codon 13. Since mutant-enriched PCR products
were sequenced, only the mutated nucleotide bands or more
prominent mutated bands besides residual normal bases are seen
on sequence autoradiograms (Fig. 3). All of the mutational
events, except for one, are G-T transversions (10 events) or
G-+A transitions (6 cases). One G-t’C transversion in the
second position of codon 12 has been observed in a squamous
cell carcinoma. In codon 12, the point mutations resulted in the
following amino acid changes: from glycine (wild type) to
cysteine (three samples), aspartic acid (four samples), valine
(two samples), serine (two samples), alanine (one sample), and
phenylalanine (two nucleotide changes, one sample). In addi-
tion, a single nucleotide deletion of one G in codon 12 of K-ras
was detected in another squamous cell carcinoma (Fig. 3a, Lane
14). This mutation causes a frameshift resulting in a stop codon
several bases downstream. The translation product of the tran-
scribed RNA should therefore result in a short truncated peptide
having apparently no transforming activity because most of the
carboxy part of the protein should not be synthesized. The
frameshift mutation in this sample was further confirmed by
several independent amplifications of genomic DNA and se-
quencing both the antisense and sense strands (not shown). In
codon 13, all three cases revealed a substitution from glycine to
cysteine. Concerning both codons 12 and 13, nine- and eight-
point mutations were in the first and the second position, re-
spectively. Control cell lines were also processed by the same
procedures as tumor DNAs and corresponding mutations were
detected (not shown).
The overall mutational frequency was 17 (12%) of 141 in
all NSCLC samples, 7 (37%) of 19 in adenocarcinomas, and 9
(8%) of 118 in squamous cell carcinomas (including 1 adeno-
squamous carcinoma), and 1 (25%) of 4 in large cell carcino-
mas. Therefore, it can be concluded that K-ras mutations occur
not only in lung adenocarcinomas but also, at lower frequencies,
in squamous cell carcinomas of the lung.
DISCUSSION
The two-step PCR-RFLP method was shown to be a reli-
able procedure to detect K-ras codon 12 point mutations present
in only a small percentage of the tumor cells (4, 10, 1 1, 30).
Here we screened 141 NSCLC DNA samples using this assay.
All mutations were clearly recognized as prominent bands on
the gel (Fig. 1). This method could be used in the screening of
samples such as sputum, bronchoalveolar lavage, pancreatic
juice, or feces, into which tumor cells are often shed from the
tumor. Mutations of K-ras have been detected in sputum from
patients with lung cancer and in pancreatic juice from patients
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b
Fig. 3 Direct sequencing of mutant-enriched PCR products resulting from two-step amplification procedures. Examples of all types of mutations
found in codon 12 (a) and codon 13 (b) of the K-ras gene are presented. For codon 12, antisense strand is shown (normal codon 12 sequence is ACC).
The sense strand was synthesized in codon 13 sequencing reactions (normal codon 13 sequence is GGC). The numbers of tumor samples correspondto the numbers in Figs. I and 2 and in Table 2. Arrowheads, mutated bases. Normal codon triplet (sense strand) is also displayed under each scquencc.
Mismatched nucleotide is underlined in the sequence displayed on the left for each codon. Each sample was sequenced twice to verify the mutation.
Clinical Cancer Research 363
Table 2 Types of K-r as mutations in NSCLC
Patient Sex, age Histological diagnosis Codon Mutation ThM stage
1 M, 64 Squamous cell carcinoma 12 GGT-*TGT (Cys) II
2 M, 46 Squamous cell carcinoma 12 GGT-*GAT (Asp)” III3 M, 65 Squamous cell carcinoma 12 GGT-*AGT (5cr) II
4 M, 59 Adenocarcinoma 12 GGT-*GAT (Asp) I
5 M, 51 Squamous cell carcinoma 12 GGT-O.GTI’ (Val) I6 M, 56 Adenosquamous carcinoma 12 GGT-+TGT (Cys) I
7 F, 65 Adenocarcinoma 12 GGT-t.GAT (Asp) II
8 M, 52 Squamous cell carcinoma 12 GGT-*GCT (Ala) III9 M, 68 Large cell carcinoma 12 GGT-*GTT’ (Val) III
10 F, 62 Squamous cell carcinoma 12 GGT-*AGT (5cr) I
1 1 F, 54 Adenocarcinoma 12 GGT-*TGT (Cys) III
12 M, 64 Adenocarcinoma 12 GGT-*1TF (Phe)” I
13 M, 46 Squamous cell carcinoma 12 GGT-OGAT (Asp) II
14 M, 39 Squamous cell carcinoma 12 GGT-*deletion of one G III
15 F, 48 Adenocarcinoma 13 GGC-’TGC (Cys) III16 M, 68 Adenocarcinoma 13 GGC-*TGC (Cys) III17 F, 47 Adenocarcinoma 13 GGC-*TGC (Cys) IV
a Wild-type allele has not been present.h double mutation in codon 12.
G\TCGA TCGA TCGA TCGA TCGA TCGA TCGA
all r�n’n
�� I Control No.2 No.7 No.3 No.5 No.6 No.8
V GGT(GIy) GAT(Asp) GAT(Asp) AGT(Ser) GTT(Val) TGT(Cys) GCT(Ala)
TCGA TCGA
No.12 No.14m(Phe) GGT>GT
with pancreatic cancer by another sensitive assay, namely, mu-
tant-allele-specific amplification (33, 34). Very recently, mu-
tated p.53 and ras genes have been detected in archival sputum
samples obtained before the clinical diagnosis of lung cancer
(35). Also, K-ras mutations were found in the DNA isolated
from the stool of patients with mutation-positive colorectal
tumors (36) and suggested to be useful for early detection of
colorectal tumors. In the case of bronchogenic carcinoma, pre-
TCGA�TCC
00T00I bC -
0- 1.-4
CI��)I
GGC(Gly)
TCGA
No. 16TGC(Cys)
invasive stages persist for years and classical screening methods
are inefficient to detect the tumor at this stage (reviewed in Ref.
37). It remains to be elucidated the timing of appearance of
K-ras mutations during the carcinogenesis of NSCLC and to test
the diagnostic value of possible mutations of K-ras and other
genes detected in sputum or bronchoscopic material.
Although all of the mutations of K-ras were well above the
detection limit of the sensitive two-step PCR-RFLP method,
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364 Mutations of K-ras in Lung Cancer
several samples were near or at the detection limit of the
one-step assay. In other samples, most tumor cells were mu-
tated. It can be therefore inferred that K-ras mutations appear
early during the progression of the tumor. However, if K-ras
mutation occurs as an early event during the development of the
squamous cell lung carcinoma, it probably does not confer a
growth advantage to cells at least in a subset of mutated tumors.
In a recent report, K-ras mutations were suggested to be ac-
quired early in lung adenocarcinomas (38).
In this work, the pattern of K-ras mutations in NSCLC was
similar to that reported previously. Among the samples ana-
lyzed, one revealed G-�C transition. G-�A transitions and
G-+T transversions were detected in all other mutated DNAS.
These two types of mutations arise as a result of treatment by
specific carcinogens in animal model systems. G-t’A transitions
are detected at high frequency on application of methylating
N-nitroso compounds such as methylnitrosourea, presumably
through the formation of 06-methyldeoxyguanosine. Benzo
(a)pyrene, forming hydrophobic DNA adducts, produces mostly
G-+T transversions. Both the benzo(a)pyrene and derivatives of
N-nitroso compounds are constituents of the cigarette smoke. As
all but one patient with K-ras mutation in our study were current
or former smokers, K-ras mutations detected here might have
been formed as a consequence of action of these groups of
carcinogens.
We found here a codon-specific distribution of K-ras mu-
tations in NSCLC tumors. Codon 12 mutations occurred in all
histological types of non-small cell lung cancer. On the other
hand, mutations in codon 13 were absent in squamous cell
carcinomas. In articles reporting the mutations of K-ras in
squamous cell lung cancers (fresh tumors or cell lines, 12
samples total), no codon 13 mutations were described (19, 21,
24, 26). Either codon 12 or codon 61 mutations were present.
Thus, our results, based on a large group of squamous cell lung
tumors, further suggest that there is an absence of the mutations
in codon 13 of K-ras in this type of tumor. Besides the K-ras
codon 12 mutations, Rosell et al. (26) observed mutations in
codon 61. Here, on the contrary, we were unable to detect any
mutation in this codon among 141 NSCLC samples. K-ras
mutations were reported to occur also in large cell lung carci-
nomas (21, 26). We found one mutation (codon 12) out of four
of these tumors examined.
In more than a one-half of the NSCLC samples analyzed in
this work, we have previously studied the methylation status of
the specific MspI/HpaII sites at the 3’ end of H-ras gene and
loss of heterozygosity at the H-ras locus (39). We hypothesized
that hypomethylation may contribute to allelic loss at the H-ras
locus (39). Neither H-ras allelic loss nor H-ras hypomethylation
(the latter change found in about one-third of tumor samples)
correlated with K-ras mutations. An interesting fact, however,
has been noted because the only sample with the loss of the
shorter H-ras allele having no methylation change (five other
DNAS with shorter allele loss were hypomethylated) displayed
the homozygous mutation of K-ras described here (Fig. 1 and
Table 2, patient 2).
We have shown that the same types of mutations appear in
both adenocarcinomas and squamous cell carcinomas of the
lung. We have demonstrated on an extended number of squa-
mous cell tumors that this histological type also harbors K-ras
mutations, although at a frequency four times lower than that in
adenocarcinomas. All mutations found in squamous cell tumors
clustered in codon 12 of K-ras. One mutation was also noted in
codon 12 of H-ras (squamous cell carcinoma) in a minority of
tumor cells. H-ras mutations, therefore, do not have any impor-
tance in the formation of NSCLC. It remains to be investigated
if screening of mutations in codon 12 of K-ras by a sensitive
mutant allele-enriched method might become a contribution to
an early diagnosis of NSCLC.
ACKNOWLEDGMENTS
We thank Dr. Fiala and Dr. Hytych for help in collecting the tumor
samples.
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