Download Loss of heterozygosity (LOH) at 17q and 14q in human lung

Oncogene (1998) 17, 3029 ± 3033
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Loss of heterozygosity (LOH) at 17q and 14q in human lung cancers
Pataer Abujiang1, Tomoharu J Mori1, Takashi Takahashi2, Fumihiko Tanaka3, Ippei Kasyu1,
Shigeki Hitomi3 and Hiroshi Hiai1
1
Department of Pathology and Biology of Diseases, Graduate School of Medicine, Kyoto University, Yoshida-Konoe-cho, Sakyoku, Kyoto 606-8051; 2Laboratory of Ultrastructure Research, Aichi Cancer Center Research Institute, 1-1 Kanokoden, Chikusa-ku,
Nagoya 464; 3Department of Thoracic Surgery, Chest Disease Research Institute, Kyoto University, Shogoin-Kawahara-cho,
Sakyo-Ku, Kyoto 606, Japan
Our recent linkage study of urethane-induced pulmonary
adenomas in SMXA RI strains of mouse revealed two
host resistance genes, Par1 (chromosome 11) and Par3
(chromosome 12). The map positions of Par1 and Par3
correspond to human 17q11-23 and 14q11-24, based on
synteny between mouse and human. In this study, we
examined the loss of heterozygosity (LOH) in these two
homologous human chromosomal regions in 30 primary
lung adenocarcinoma samples with matched normal
DNA. Using 15 highly polymorphic markers, two
commonly deleted regions were identi®ed on human
chromosomes 14 and 17, respectively. At 17q21, nine
(53%) of 17 informative tumors showed LOH between
D17S588 and D17S518. On the other hand, at 14q11-12,
seven (32%) of 22 informative tumors showed LOH at
loci between D14S261 and D14S80. Subsequently, we
examined 25 squamous cell carcinomas (SQ) and 24
small cell carcinomas (SCC). At 14q11-12, six (38%) of
16 informative SQ and ®ve (42%) of 12 informative
SCC showed LOH. In contrast, at 17q11-23, one (7%)
of 15 informative SQ and two (14%) of 14 SCC showed
LOH. Therefore, the gene on 17q seemed to a€ect
selectively adenocarcinomas, whereas the other gene on
14q, all three types of lung carcinomas. These
observations indicate that a comparative genetic analysis
provides a promising approach to survey genes involved
in multifactorial process of human lung carcinogenesis.
Keywords: pulmonary adenoma; lung cancer, LOH,
tumor suppressor gene
Introduction
Lung cancer is one of most frequent malignancies in
industrialized countries. Lung cancer develops through
a multistep process in which genetic alterations are
accumulated to promote tumorigenesis. Inactivation of
tumor suppressor genes plays an important role in this
process (Carbone et al., 1992). Loss of heterozygosity
(LOH) is a frequent observation in such tumors. In
human lung cancers, LOHs are found on chromosomes
1q, 2q, 3p, 5q, 8q, 9p, 13q, 11p, and 17p and
localizations of tumor suppressor genes have been
con®rmed or suggested (Tsuchiya et al., 1992).
Urethane-induced pulmonary adenoma (PA) in mice
is an excellent model of human lung adenocarcinoma
Correspondence: H Hiai
Received 9 March 1998; revised 18 June 1998; accepted 19 June 1998
since both tumors share a number of morphological
and molecular biological properties (Malkinson et al.,
1992). Mutations in kras2 in mouse PA are as
prevalent as in human lung cancers and its allelotype
is the primary determinant of disease susceptibility
(Dragani et al., 1995). The involvement of multiple
host genes in lung carcinogenesis has been studied
extensively in mouse models (Festing et al., 1994).
Recently, we reported two autosomal dominant
resistance loci to urethane-induced PA in the SMXA
recombinant inbred strain of mice, Par1 and Par3
(Abujiang et al., 1997). From the synteny map, the
regions of Par1 and Par3 are homologous to human
17q11-23 and 14q11-24, respectively. Here, we analysed
human lung adenocarcinoma for LOH in homologous
regions and found two minimally deleted regions.
Subsequent examination of squamous cell carcinomas
(SQ) and small cell carcinomas (SCC) for LOH in
these regions revealed that 17q contained a gene
a€ecting selectively adenocarcinomas and 14q, another
gene a€ecting all three histological types of lung
cancers.
Results
Most of urethane-induced lung tumors in mice are
adenomas in histology. Considering the possibility that
the function of the Par1 or Par3 homolog may be
somehow selective to target cells of tumorigenesis, we
®rst selected 30 primary lung adenocarcinomas and
examined their genotypes at 15 highly polymorphic
microsatellite marker loci distributed in 14q11-23 and
17q11-24 regions. Figure 1 lists the marker loci in
descending order from centromere to telomere, and
shows their patterns and frequencies of LOH in the
adenocarcinomas examined. Two minimal regions of
deletion were identi®ed by this analysis. The ®rst
region was bounded by D14S261 proximally and by
D14S80 distally. These marker loci have been
cytogenetically mapped to chromosome 14q11 and
14q21, respectively. The second region was bounded
by D17S588 proximally and by D17S518 distally, and
these marker loci have been cytogenetically mapped to
chromosome 17q21 and 17q23, respectively. LOH was
observed most frequently at D17S588 and D14S261.
Nine (53%) of 17 informative tumors showed LOH at
marker locus D17S588. At locus D14S261, seven (32%)
of 22 informative tumors showed LOH. Representative
cases of LOH analysis at several marker loci are shown
in Figure 2. At D14S261, the patient 9 did not show
LOH, since DNAs from both tumor and non-tumor
tissue retained two alleles, whereas the patient 22
showed LOH as one allele was missing in the tumor
Loss of heterozygosity in human lung cancers
P Abujiang et al
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Figure 1 Deletion mapping of chromosomes 14 and 17. Fifteen microsatellite markers spanning 14q11-23 and 17q11-24, were used
to study 30 human lung adenocarcinomas for evidence of allelic loss. Patient numbers are shown above the map. Percentage of
informative cases with LOH (%LOH) is shown in the right column
DNA. Similar LOH was observed in the patients 3 and
10 at D17S588 and D17S855, respectively.
To examine whether LOHs in these regions are
speci®c to adenocarcinomas or observed also in other
types of lung cancers, we further studied 25 cases of SQ
and 24 cases of SCC. Figure 3A,B schematically shows
the patterns of LOH and their frequencies in these
tumors. Comparing with lung adenocarcinomas, LOHs
at 17q21 were less frequent in SQ and SCC as
summarized in Table 1. Therefore, the LOH on 17q
seemed to be associated with adenocarcinomas
(P50.05). On the other hand, LOHs at 14q11-12 in
SQ (30%) and SCC (42%) were as frequent as in
adenocarcinomas (32%) (Table 1), indicating the LOHs
on 14q were prevalent to all types of lung cancers.
To further characterize the role of the putative
tumor suppressor gene in this region in lung cancer, we
examined the possible associations of LOHs on 14q
and 17q with clinicopathological parameters including
sex, degree of di€erentiation, smoking history, TNM
stage and lymph node metastasis. However, no positive
association was found (Table 2).
Discussion
Figure 2 Representative data of detection of loss of heterozygosity. PCR products for the microsatellite locus from normal
lung (N) and tumor DNA were compared. Patient numbers are
shown at left. The upper boxed ®gure indicates the length of the
fragment, and the lower boxed ®gure shows the peak area
Comparative genetic analysis is one of promising
approaches to characterize complicated diseases in
human. It is increasingly evident that there is an
extensive syntenic conservation of genomic organization among species, and comparative genetic maps
have been used to predict the locations of genes in
other species. The present study aimed to extend the
genetic information obtained in chemical-induced lung
tumors in the mouse to survey genetic changes in
human lung cancers. Analysis of 79 cases of human
lung cancer revealed frequent LOHs in two distinct
chromosomal regions. One of locus at 17q21 seem to
play signi®cant role in lung adenocarcinomas. On the
other hand, another locus at 14q11-12 seemed to play
Loss of heterozygosity in human lung cancers
P Abujiang et al
signi®cant role in all types of lung cancers. These
results suggest the existence of putative tumor
suppressor genes in these regions. LOHs in 17q21
have been reported in a variety of malignancies
including breast (Cropp et al., 1993), ovary
(Wertheim et al., 1996) and prostate cancers (Gao et
Figure 3 Deletion map of (A) 25 squamous cell carcinomas and (B) 24 small cell carcinomas. Patient numbers are shown above the
map
Table 1 Frequency of LOH on chromosomes 14 and 17 in lung cancer
Human
chromosome
Allelic loss/heterozygosity (% LOH)
Adenocarcinoma
Squamous (SQ)
Small (SCC)
Marker locusa
17q21
14q11±12
D17S588
D14S261
9/17 (53%)
7/22 (32%)
1/15 (7%)
3/10 (30%)
2/14 (14%)
5/12 (42%)
a
The markers with the highest frequency of LOH on 17q and 14q
Table 2
Correlation between allelic loss and clinicopathological features
Cases with Cases without
17q LOH
17q LOH
Sex
Male
Female
P value
Cases with Cases without
14 q LOH 14q LOH
P value
14
3
22
8
0.48
16
5
20
20
0.08
Di€erentiation
Well
Mod, poorly
3
8
4
16
0.64
5
9
9
12
0.67
Smoking history
Non-smokers
Smokers
6
8
5
23
0.08
7
14
8
22
0.6
TNM
I
II, III
7
5
9
14
0.27
7
11
9
15
0.92
Metastasis
Without
With
6
5
15
8
0.54
6
8
10
11
0.78
3031
Loss of heterozygosity in human lung cancers
P Abujiang et al
3032
al., 1995a). The tumor suppressor genes prohibitin
(Cliby et al., 1993a), BRCA1 (Gao et al., 1995b) and
NME1 (Niederacher et al., 1997) are mapped in this
region. A large panel of human cancers of the breast,
ovary, liver, and lung have been examined for somatic
mutations in the prohibitin gene. Although mutations
are observed in a few sporadic breast cancers, none is
identi®ed in any other cancers (Sato et al., 1993).
Recent positional cloning of the BRCA1 from that
region and its mutation screening support that the
BRCA1 is responsible for inherited breast (Jandrig et
al., 1996) and ovarian cancer (Merajver et al., 1995).
It would be interesting to screen for BRCA1
mutations in tumor samples which demonstrate LOH
at the BRCA1 locus. This will provide evidence to
either prove or disprove the hypothesis that the
BRCA1 gene is indeed involved in lung cancer.
Fong et al. (1995) also showed 45% of LOH in lung
adenocarcinomas and 29% of LOH in SQ overall 17q.
They used Southern blot analysis of RFLPs (Fong et
al., 1995), so that it is dicult to compare their results
with ours. Whether or not these observations direct
the deletion of the same critical locus will require
further study.
Cancers of colorectal tract (Chang et al., 1995),
kidney (Thrash-Bingham et al., 1995), endometrium
(Chang et al., 1995), bladder (Chang et al., 1995),
ovary (Cliby et al., 1993b) and neuroblastoma
(Takayama et al., 1992) are solid cancers associated
with a signi®cant degree of 14q LOH. The proximal
end of the region de®ned in our study overlaps with the
region de®ned in the ovarian carcinoma study.
Assuming that the deletion observed at 14q11-12 in
bladder, ovarian and lung cancers are driven by the
same gene, the identi®cation and characterization of
this tumor suppressor locus would be expected to
represent a substantial advance in the ®eld of cancer
molecular genetics. Although these regions are not
small enough to plausibly indicate a positional cloning
e€ort, it is possible to narrow down the minimal
regions of deletion through the identi®cation of
additional microsatellite markers to saturate these
regions and to perform LOH analysis of a larger
series of tumors with them. We concentrated on lung
cancers, because the primary aim of this study was to
seek common genetic events among species in
carcinogenesis of homologous type tumors.
In murine urethane-induced PA, four resistance genes
(Par1-4) have been identi®ed (Abujiang et al., 1997;
Manenti et al., 1997). Par2 is mapped on chromosome
18 close to the putative tumor suppressor gene DCC.
Although we did not examine the Par2 homologous
region, frequent LOH of DCC in human lung cancers
has been reported (Zhang et al., 1995). Par4 was
recently mapped to chromosome 6 (Manenti et al.,
1997), but its syntenic region has not been examined. To
date, therefore, 3 of 4 mouse PA resistance genes have
syntenic human chromosomal regions bearing hot spots
for LOH in lung adenocarcinomas. These observations
suggest the presence of shared genetic steps in lung
carcinogenesis across species.
Materials and methods
Samples and DNA extraction.
Lung tumors and corresponding nontumorous tissue were
obtained at surgery from 79 patients. Types of tumors were
con®rmed histologically. Tissues were frozen immediately
and stored at 7808C. DNA was extracted from frozen
tissue according to the methods described previously
(Abujiang et al., 1996).
Fluorescent microsatellite analysis.
We chose 15 microsatellite markers along 14q and 17q arms.
Map positions of these loci were based on the Human
Genetic Map (Cold Spring Harbor Laboratory Press 1993,
Stephen J O'Brien) and CEPH/CHLC databases. The
oligonucleotides were labeled with 6-FAM ¯uorescent dye,
and the TAMRA dye was reserved for the size standard.
PCR for ¯uorescent markers was carried out in a volume of
9 ml and included 106PCR bu€er, 50 ng of DNA, 250 mM
of each dNTP, 0.5 units of Dynazyme Taq polymerase and
10 pmol of each primer. The thermal pro®le was as follows:
948C for 2 min; 35 cycles of 948C for 30 s, 538C for 40 s,
and 728C for 1 min; and a ®nal extension of 728C for
10 min. One ml of products were added to 2.5 ml of
formamide, 0.5 ml of blue dextran and 0.5 ml of TAMRA
500 size standard (Applied Biosystems), loaded on 4%
polyacrylamide 6-M urea gels, and run for 2 h in a 377A
Automated Sequencer (Applied Biosystems). The data were
collected automatically and analysed using GeneScan
software (Applied Biosystems); ®nally, the Genotyper
software (Applied Biosystems) and Exel software were
used for allele scoring and assessment of LOH. In the case
of constitutional heterozygotes, two alleles are detected in
normal tissue, and if one is absent in the tumor the result is
LOH. The program checks for LOH by calculating the ratio
of the constitutional alleles (Canxian et al., 1996).
Statistical analysis.
The X2 test was performed to compare LOH frequencies in
respect of histopathological type, stage, and grade. P50.05
was considered to indicate statistical signi®cance.
Abbreviations
Par, pulmonary adenoma resistant; Pas, pulmonary
adenoma susceptible; PA, pulmonary adenoma; RI,
recombinant inbred; LOH, loss of heterozygosity; SQ,
squamous cell carcinoma; SCC, Small cell carcinoma.
Acknowledgements
We thank Drs Hiromi Wada (Department of Thoracic
Surgery, Chest Disease Research Institute, Kyoto University) for valuable discussions. This study was supported by
grants from the Ministry of Education, Culture, and
Science, from the Ministry of Health and Welfare, Japan
and from the Japanese Owner's Association.
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