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From the Laboratory of Cancer Diagnosis and Therapy,*
Saitama Cancer Center Research Institute, Saitama; the Department of
Pathology,
Saitama Cancer Center, Saitama;
the Department of Pathology,§
Cancer Institute,
Tokyo; the Department of Chest Surgery, Cancer
Institute Hospital, Tokyo; and the Department of Thoracic and
Cardiovascular Surgery,
Niigata University
School of Medicine, Niigata, Japan
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mutations in the p53 tumor suppressor gene appear to be important for the genesis of many kinds of tumors, including lung cancers.8,11-14 Their frequency and mutational spectra can be said to reflect carcinogenesis by exogenous or endogenous factors and thus may be helpful for identification of the responsible agents.8,11-13 With lung cancers, tobacco smoke, one of the most important exogenous carcinogenic agents, has been shown to frequently cause p53 mutations, especially G:C to T:A transversions.8,12,15-17 On the other hand, transitions, especially G:C to A:T transitions at CpG sites, are thought to be caused by endogenous mechanisms involved in spontaneous mutations.8,12 Therefore the mutation frequency and spectrum may provide information on the etiological factors for lung cancer.
Working on the hypothesis that different subtypes of adenocarcinoma are caused by different etiological factors, we first subclassified a large series and examined p53 gene mutations in exons 4–8 and 10. As controls, squamous cell carcinomas were also examined. Then the relationships among histological subtypes, p53 mutational status, and smoking history were assessed.
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Materials and Methods |
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We examined 151 non-small-cell lung carcinomas (113
adenocarcinomas and 38 squamous cell carcinomas) that had been resected
consecutively from 1989 to 1993 at Cancer Institute Hospital, Tokyo,
Japan. None of the patients had received chemotherapy or radiotherapy
before surgery, but 79 patients (60 with adenocarcinoma and
19 with squamous cell carcinoma) underwent postoperative adjuvant
therapy. The study population was aged 26–84 (median 62) years and
comprised 98 men and 53 women. Data for other clinicopathological
parameters, differentiation and location of the tumor, pathological
stages, and patient’s smoking status are presented in Table 1
. Differentiation of tumors was
determined according to the 1999 WHO classification of lung
tumors.4
The location of a tumor in the lung was
classified as central when it was considered to have arisen in a main
to segmental bronchus, and peripheral when in a subsegmental or more
distal bronchus.18
The pathological stages (pStages) were
determined using the International Union Against Cancer (UICC) TNM
staging system,19
and statistical difference was
calculated between two groups, pStage I and pStages II–IV. The
patient’s smoking history (number of cigarettes per day, starting age,
and duration of smoking) was obtained from preoperative personal
interviews and expressed as nonsmokers and smokers, the latter
including both patients with a past history of smoking and current
smokers.
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The first
consists of cells with cytoplasmic protrusions or dome formation at
their apices and hobnail- or tadpole-shaped cells. The second is
composed of columnar/cuboidal cells with flat apices. Cytoplasmic mucus
is usually absent, and if it is present it is scanty and is located
near the free cell surface. The third demonstrates a mixture of
hobnail, columnar/cuboidal, and goblet cells or two of these. Most of
this type consists of both the former two cells, in which each cell
type occupies more than 30% of the area. Polygonal cells with or
without mucus in their cytoplasm, proliferating in sheets, are
sometimes observed in tumors of the first three types, but when such
areas made up more than 95% of the tumor, it was diagnosed as the
polygonal cell type. The goblet cell type is composed of columnar or
cuboidal cells with abundant mucus in their cytoplasm. Distribution by
cell type and 1999 WHO classification of adenocarcinomas are presented
in Table 1
. By the WHO classification, more than half of the tumors
were classified as adenocarcinomas with mixed subtypes. There were only
two bronchioloalveolar carcinoma, and no papillary adenocarcinomas
consisting entirely of tall columnar or cuboidal cells were observed.
Almost half of the cells were hobnail type, then columnar, mixed
polygonal, and goblet, in that order. Table 2
shows the relationship between WHO and
the cell type classification of adenocarcinomas. More than half of the
acinar and papillary adenocarcinomas, respectively, were of columnar
and hobnail cell types. The two bronchioloalveolar carcinomas consisted
primarily of hobnail and goblet cells, in one case each, and all of the
solid adenocarcinomas with mucin were of the polygonal cell type. The
adenocarcinomas with mixed subtypes included various cell types, of
which the hobnail type was the most common.
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DNA Preparation, Single-Strand Conformation Polymorphism (SSCP), and DNA Sequencing
Fresh tumor samples paired with corresponding normal tissue were
obtained from all patients, quickly frozen in liquid nitrogen, and
stored at -80°C until DNA extraction analysis, as previously
described.21
Genomic DNAs were prepared, and exons 4–8
and 10 of the p53 gene were analyzed by the polymerase chain
reaction (PCR)–SSCP method.22
Coding sequences including
exon-intron boundaries were amplified by PCR. The sequences of primers
and PCR conditions were described previously.23,24
The 5'
end of each primer was labeled with a fluorescent marker, sense primers
were labeled with 6-carboxyfluorescein, and the antisense primer was
labeled with 4,7,2',7'-tetrachloro-6-carboxyfluorescein (Japan Bio
Service Corp., Asaka, Japan). SSCP using ABI PRISM 377 (Perkin-Elmer
Corp., Norwalk, CT) and fluorescent-labeled primers was
performed at 22°C, loading onto nondenaturing 4% polyacrylamide gels
with 10% glycerol. SSCP data were processed with GeneScan Analysis
2.0.2 computer software (Perkin-Elmer Corp.). When genomic DNA
extracted from tumors showed a SSCP pattern different from that of
corresponding normal lung tissues, both genomic DNAs were amplified
with the primers in the presence of
[
-32P]dCTP to elute the shifted DNA fragment
for sequence analysis. After PCR under the same cycling conditions,
products were electrophoresed in nondenaturing 5% polyacrylamide gels
with 10% glycerol at the most suitable temperature (exon 4, 10°C;
exons 5–7 and 10, 25°C; exon 8, 15°C) and 35 W of constant power
for 2–3 hours. The gels were subjected to drying at 80°C for 1 hour
and autoradiographed at room temperature overnight. Both normal and
abnormal DNA fragments were eluted from the dried gels and reamplified
using the same primers and PCR conditions. To characterize
p53 gene mutations, we sequenced the reamplified DNAs using
a dRhodamine terminator cycle sequencing kit (Applied Biosystems) and
ABI PRISM 377. Some DNA for which mutations could not be identified by
direct sequencing, despite showing abnormal bands in fluorescently
labeled SSCP, were subcloned into plasmid vector pGEM-7Zf(+) (Promega)
and sequenced with an AutoRead sequencing kit (Pharmacia Biotech),
using fluorescently labeled SP6, T7 primers and an A.L.F. DNA Sequencer
II (Pharmacia LKB Biotechnology AB).
Statistical Analysis
To establish any correlations among the p53 gene
mutation status and clinicopathological data, the
2
test or Fisher’s exact probability when
expected values in the 2
test were <5, the
Mann-Whitney U-test and Student’s t-test were
used. Differences were considered to be significant when the
P value was <0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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p53 Mutation
Screening of all tumor samples for p53 mutations in
exons 4–8 and 10, using a fluorescently labeled PCR-SSCP, technique
revealed mutations in 68 of 151 non-small-cell lung carcinomas (45%)
(Tables 1 and 3)
. One case (case no. 17)
had two mutations: a 19-bp deletion in exon 4 and a 1-bp deletion in
exon 8. Of the 68 mutations, four (6%) were located in exon 4, 17
(25%) in exon 5, 9 (13%) in exon 6, 15 (22%) in exon 7, 16 (23%) in
exon 8, four (6%) in exon 10, and 4 (6%) in splicing junctions of
exons. No mutations were found in normal lung tissue samples, except in
patients carrying a polymorphism in exon 4, codon 72.25
Histologically, a trend toward more frequent mutations in squamous cell
carcinomas (58%) than in adenocarcinomas (41%) was found (Table 4
and Figure 2A
), in line with earlier results of a
Japanese study of mutations in exons 2–11 in 115 cases of
non-small-cell lung cancer.26
By the WHO classification,
papillary adenocarcinomas and adenocarcinomas with mixed subtypes
showed the lowest frequency of mutations, although statistically
significant differences from other individual subtypes were not found
(Table 1)
. Using our cytological classification, the highest frequency
of the mutations was observed in the columnar cell type (70%), which
is similar to the finding for for squamous cell lesions, followed by
polygonal (46%), goblet (40%), hobnail (37%), and mixed (11%) cell
types. The differences compared with the latter two were statistically
significant (Table 4
and Figure 2A
). This was also the case for the
squamous cell carcinomas (hobnail, P = 0.048; mixed
cell, P < 0.001; by 2
test).
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Most p53 mutations were transitions (43%) or
transversions (41%), and only 16% were deletions/insertions (Table 4)
. In adenocarcinomas, the frequencies of transitions and
transversions were 46% and 35%, and in squamous cell carcinomas, 36%
and 55%, respectively. No significant differences were observed, in
agreement with a previous Japanese report.26
Comparison of subtypes revealed a significant difference between
hobnail and columnar cell groups: transitions were higher in the former
(65%) than the latter (25%) (Figure 2B)
. Transversions also tended to
be less frequent in the former (20%) than in the latter (50%). With
the WHO subclassification, no significant differences were observed
between subtypes of adenocarcinomas as to the frequencies of
transitions or transversions (data not shown). We did not analyze the
rates of deletions and insertions, because the proportions that
represent changes induced by endogenous versus exogenous
mechanisms remain unclear.27
Next, base substitutions were examined with reference to subtypes
of adenocarcinoma and squamous cell carcinoma (Table 4
and Figure 2C
).
With CpG site transitions, the frequency in the hobnail cell type
(45%) was higher than those for columnar cell type and squamous cell
carcinomas (13% and 23%, respectively), the difference being
statistically significant in the former case. On the other hand, G:C to
T:A transversions tended to be rarer in hobnail cell-type lesions
(15%) than in the other two types (44% and 27%, respectively). With
the WHO subclassification, no such variation was noted.
Strand Bias
It has been hypothesized that mutations induced by exogenous or environmental carcinogens preferentially occur in nontranscribed gene alleles.8,27-29 Therefore evaluating the p53 base substitution for strand bias may also provide clues to suspected carcinogens. In our study, a marked strand bias was observed for G:C to T:A transversions: 17 of the 18 mutations were found on the nontranscribed strand, whereas the 18 G:C to A:T transitions observed in CpG sites were equally distributed on the two strands (9 vs. 9).
Smoking Status in Relation to Histology and p53 Mutation
The percentage of smokers with squamous cell carcinomas was higher
than the percentage of smokers with adenocarcinomas, and the difference
was statistically significant (Figure 2D)
. The percentage of smokers
with columnar cell lesions, 83%, was almost the same as the percentage
of smokers with squamous cell carcinomas, and significantly higher than
the percentages of smokers with the hobnail (44%) or mixed (39%) cell
types.
Mutations were more frequent in lesions observed in smokers (50%) than
in nonsmokers (36%), consistent with previous reports (Table 4)
.8,15
As for the mutational spectra, transitions were
less frequent among smokers (33%) than among nonsmokers (65%), with
statistical significance (P = 0.016, by
2
test). Conversely, transversions were more
common among the former (48%) than the latter (25%), with a
statistical trend (P = 0.068, by the Fisher’s
exact probability test). Furthermore, G:C to A:T transitions at CpG
sites were preferentially found in nonsmokers (35%), and G:C to T:A
transversions more frequently in smokers (29%), although the
differences were not significant.
Relationship between p53 Mutations and Clinicopathological Parameters
As clinicopathological parameters, age, gender, differentiation
status, location, and stage of the tumor were adopted (Table 1)
. There
were no significant differences in p53 status with the first
four of these. The frequency of mutations with pStages II–IV was
significantly higher than that for pStage I for squamous cell
carcinomas (P = 0.038, by Fisher’s exact
probability test) but not for adenocarcinomas.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As for distribution of the subtypes, the hobnail cell type was the most common (48%), almost the same as the figure (39%) for the clara cell type + type II cell in Shimosato’s classification.10 The columnar/cuboidal cell type was the second most frequent (20%), again in the same range as reported earlier (22–34%) for the bronchial surface epithelium type.10,32 The frequency of the mixed type (16%) and those of the other cell types (less than 12%) were also similar for the two classifications.10,32 Therefore, our cases examined can be considered to be representative for surgically resected adenocarcinomas of Japan in terms of subtype distribution.
When the predominant cell type of a tumor occupies around 70% of the tumor area, it is sometimes difficult to classify the cell type. Examination for reproducibility resulted in 17 of 113 cases being classified as other cell types, the concordance rate being 85%. The 17 cases were seven hobnail cell, four mixed, three polygonal, two cuboidal, and one goblet type in the original classification. All of the hobnail cases were changed to mixed, and two of four mixed ones to hobnail. Thus the differentiation between hobnail and mixed cell types was imperfect. However, when the relationship between p53 mutational spectra and the newly classified cell types was examined, the results were the same as originally found.
Exons 4–8 and 10 of the p53 gene were examined for mutations in this study because they encompass more than 98% of the mutations reported in carcinomas so far.26 Epidemiologically, the presence of p53 mutations in lung cancers is closely associated with lifetime tobacco consumption, typically with G:C to T:A transversions and a predominance of guanine residues on the nontranscribed DNA strand.8,12,15,33,34 When lung cancers are classified histologically, an altered p53 mutation status with G:C to T:A transversion is marked in squamous cell carcinomas, which are strongly associated with tobacco smoke, whereas this is less clear for adenocarcinomas, which are only weakly linked with the smoking habit.6-8 Experimentally, compounds included in cigarette smoke, such as benzo[a]pyrene, are reported to produce G:C to T:A transversions.17 Although the same changes can also be induced by endogenous agents like oxygen radicals, a nontranscribed bias has not been reported in such cases.35,36
In our study, when adenocarcinomas were subclassified by cell type, the columnar cell lesion showed high mutation and transversion rates with a nontranscribed bias, and a strong association with smoking, like that for squamous cell carcinomas, was apparent. On the other hand, hobnail cell-type adenocarcinomas showed a significantly weaker association with tobacco smoke, so that other causative factors must be considered. The finding of a high rate of G:C to A:T transitions at CpG sites with no strand bias is interesting in this context. This type of mutation is suspected to be caused mainly by deamination of 5-methylcytosine at CpG sites and occurs spontaneously without exogenous mutagens.37-39 Whether other agents might also play a role remains unclear, but, in at least a subset of hobnail cell-type adenocarcinomas, a contribution of G:C to A:T transitions at CpG sites may be hypothesized. Further studies are now needed to test this.
Hypermethylation of the promoter region of the p16 tumor suppressor and estrogen receptor genes and loss of heterozygosity and exon deletions within the fragile histidine triad (FHIT) gene have been reported to be associated with the smoking habit in lung cancer patients.40 Relationships between these genetic alterations and cell types should be examined to confirm our results.
In conclusion, the present study for the first time clearly showed that lung adenocarcinomas could be subclassified in terms of etiology in addition to morphology. There appear to be two major subtypes, one probably caused by tobacco smoke and the other mainly associated with endogenous, possibly spontaneous mutations. Cell type classification is thus useful for a distinction of differences that extend beyond the morphology level.
For prevention of lung adenocarcinomas, elucidation of what endogenous mechanisms are actually involved is an important next step, in addition to stopping tobacco smoking. In the future, to achieve better clinical control, it will also be necessary to investigate tumor type-specific differences in clinical characteristics, prognosis, and response to chemotherapy, radiotherapy, or innovative therapies.
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Acknowledgements |
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Footnotes |
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Supported by grants from the Ministry of Education, Science, Sports and Culture of Japan, by a research grant from the Ministry of Health and Welfare of Japan, and by the Vehicle Racing Commemorative Foundation.
Accepted for publication August 22, 2000.
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