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Molecular Biology and Genetics |
Departments of Pathology [A. K. V., S. M., A. F. G.] and Internal Medicine and Pharmacology [J. D. M.], Hamon Center for Therapeutic Oncology Research [I. I. W., C. B., A. K. V., G. M., L. G., A. F. G., J. D. M.] and McDermott Center for Human Growth and Development and the Center for Biomedical Inventions, University of Texas Southwestern Medical Center [J. W. F., H. R. G.], Dallas, Texas 75390; Department of Pathology, Pontificia Universidad Catolica de Chile, Santiago, Chile [I. I. W.]; British Columbia Cancer Agency, Vancouver, British Columbia, V5Z 355 Canada [S. L.]; Department of Paediatrics and Child Health, University of Birmingham, Birmingham B15 2TT, United Kingdom [F. L.]; Laboratory of Immunobiology, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, Maryland 21702 [M. I. L.]; and Department of Pathology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030 [B. M.]
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INTRODUCTION |
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LOH involving markers on the short arm of chromosome 3 is one of the most frequent acquired genetic changes occurring in the pathogenesis of lung cancer (7) . This phenomenon was first detected in lung cancers by cytogenetic analysis showing 3p deletions (8, 9, 10) and was later confirmed by allele loss and comparative genome hybridization studies (11, 12, 13, 14) . These LOH sites are candidates to contain TSGs. Chromosome 3p allele loss has been detected in nearly all SCLCs and in about three-fourths of NSCLCs (7) . Thus far, several distinct 3p regions have been identified as showing frequent allele loss in lung cancer including 3p25–26, 3p21.3–22, 3p14, and 3p12 (15) , suggesting that there are probably several different TSGs located in the 3p region. However, the identities of such genes remain elusive despite intensive investigation. In addition, 3p allele loss has been detected as the most frequent and earliest genetic change in the multistage development of lung cancer, with 3p allele loss occurring in histologically normal bronchial epithelium in patients with cancer and in the epithelium of smokers without lung cancer (16, 17, 18, 19, 20) .
Several homozygous deletions, presumably encompassing TSG locations,
have been found in lung cancer cell lines in the 3p21.3
(21, 22, 23)
, 3p14.2 FHIT/FRA3B
(24, 25, 26)
, and 3p12 (U2020) regions (27
, 28)
.
Homozygous deletions of the 3p21.3 region have also been found in
uncultured lung tumors (29)
. Currently, two distinct
3p21.3 regions are under study because of the existence of multiple
homozygous deletion in lung cancer cell lines. One region is defined by
homozygous deletions present in the breast cancer cell line HCC1500 and
the SCLC cell lines NCI-H740, NCI-H1450, and GLC20 with a 120-kb
minimum common deleted region (21, 22, 23
, 30
, 31)
. The other
region, 
800 kb in size and probably telomeric to the first region,
is defined by a homozygous deletion that has thus far been found only
in a Japanese lung cancer cell line (32)
. Many genes have
been identified in the first 3p21.3 homozygous deletion region,
although none of them have been shown to have frequent mutations in
lung cancer (23
, 31
, 33, 34, 35, 36, 37)
. Using the genomic DNA
sequence of a 600-kb (cosmid and P1 phage) clone contig covering the
120-kb deletion overlap region at 3p21.3 and a new computational system
we developed for the prediction of polymorphic loci from human genomic
sequences, 22 new loci were found to be polymorphic (38)
.
These markers provide for very high density allelotyping studies in
this 3p21.3 region.
To understand and clarify the role of chromosome 3p allele loss
in the pathogenesis of lung cancer and to more precisely identify
additional targets for positional cloning efforts, we performed a
detailed allelotyping analysis of the entire chromosome 3p arm using 28
microsatellite markers. We studied 216 samples including lung cancer
cell lines; microdissected archival primary lung tumors; histologically
normal epithelium; histologically identified areas of hyperplasia,
dysplasia, and CIS from lung cancer patients and from smokers without
lung cancer; and respiratory epithelium of never smokers. We
found at least eight different chromosome 3p regions that undergo
allele loss in lung cancer and histologically normal and abnormal
epithelium and thus may harbor TSGs. These 3p allele losses are
frequent, often discontinuous in nature, and appear to be early events
in the pathogenesis of lung cancer, especially those in the 600-kb
3p21.3 homozygous deletion region. In this region, along with a
600-kb region in the 3p14.2 (FHIT/FRA3B region) and the
6-Mb 3p12 (U2020/DUTT1 homozygous deletion region), a
high density of breakpoints is localized (tumor-specific junctions
between LOH and retention of heterozygosity), representing either
deletions and/or hot spots of mitotic recombination. Finally, 3p allele
loss was very frequently detected in the bronchial epithelium of
smokers without cancer but was not found in the bronchi of never
smokers.
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MATERIALS AND METHODS |
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; Fig. 3
; Ref. 39
). All of the lung cancer cell lines as
well as most of the B lymphoblastoid lines were initiated by the
authors or co-workers at the National Cancer Institute (Bethesda, MD;
NCI-Hxxxx cell lines) or Hamon Center for Therapeutic Oncology Research
(Dallas, TX; HCCxxxx cell lines). The NSCLC lines consisted of 12
adenocarcinomas, 2 large cell carcinomas, 2 squamous cell carcinomas, 1
adenosquamous carcinoma, and 1 carcinoma with neuroendocrine features.
They were deposited at the American Type Culture Collection (Manassas,
VA). Cells were grown in RPMI 1640 with 5% FCS, and DNA was prepared
by standard methods (39)
.
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. Serial
5-µm sections were cut from archival, formalin-fixed,
paraffin-embedded tissue. All slides were stained with H&E, and one of
the slides was coverslipped. The coverslipped slide was used as a guide
to localize regions of interest for microdissection of the other
slides.
Normal Epithelium and Histologically Abnormal Lesions
Accompanying Resected Squamous Cell Lung Cancer.
We selected archival surgical specimens from 10 of the resected
primary squamous cell lung carcinoma cases (Table 1)
that also
contained multiple foci of histologically varied changes. The
microslides were examined by two pathologists (A. F. G. and
I. I. W.) and scored using published criteria for the histological
identification of epithelial lesions of the lung (40)
.
Histopathological diagnoses were categorized as follows: (a)
normal respiratory epithelium; (b) hyperplasia (goblet cell
or basal cell type) or simple squamous metaplasia without dysplasia (we
use the term "mildly abnormal" throughout this report for this
histological category); (c) dysplasia (we did not divide
dysplasias into mild, moderate, and severe categories); and
(d) CIS. Besides the 10 cancer specimens, we identified 54
histologically discrete foci in these resected specimens, each of which
contained at least 800 cells. They included samples from 19
hyperplasias or squamous metaplasias (mildly abnormal lesions) and 15
dysplastic lesions, 12 CIS lesions, and 8 samples of histologically
normal epithelium. One or more foci of hyperplasia/squamous metaplasia
were present in all 10 cases, one or more samples of histologically
normal epithelium were analyzed in seven cases, and one or more
discrete dysplasia and CIS lesions were identified in nine cases. All
nontumoral specimens were obtained from centrally located large bronchi
(lobar, segmental, and subsegmental).
Bronchial Biopsy Specimens from Smokers.
We studied 47 biopsy specimens obtained by fluorescence bronchoscopy as
described previously (41)
from 28 subjects, 13 current
smokers (mean, 2 samples/subject) and 15 former smokers (mean, 1.5
samples/subject; Table 1
). All subjects were studied by S. L. at the
British Columbia Cancer Agency (Vancouver, British Columbia, Canada) as
part of an institutional review board-approved clinical trial to study
the effect of smoking on the respiratory epithelium. All participants
gave written informed consent. Subjects were categorized according to
smoking status as described previously (19)
. All smokers
had exposure histories of more than 20 pack-years, except for one
subject (10 pack-years). Most former smokers (11 of 15 former smokers;
73%) had ceased smoking for 5 years or longer (mean, 22 years). Other
relevant subject information is presented in Table 1
. Pathological
diagnoses were categorized as stated previously. The samples included 2
histologically normal epithelial specimens, 13 mildly abnormal
epithelial specimens (hyperplasia and squamous metaplasia), 29
dysplasias, and 3 CIS lesions.
Bronchial Epithelium Specimens from Never Smokers.
We obtained normal (n = 4) and hyperplastic
(n = 14) bronchial epithelial specimens from
surgical specimens from seven never smokers (six women and one man;
median age, 52 years; age range, 32–87 years) who underwent lung
resection (lobectomy) for carcinoid lung tumor (two patients), lung
adenocarcinoma (two patients), granulomatous inflammation of the lung
(two patients), and metastasis of renal cell carcinoma (one patient).
Archival Specimen Microdissection and DNA Extraction.
Microdissection from archival paraffin-embedded tissues was performed
either by laser capture microdissection (PixCell apparatus; Arcturus
Engineering, Inc., Mountain View, CA; Ref. 42
) or by
manually using a micromanipulator (16)
on multiple
microslides of each sample. DNA extraction was performed as described
previously (16)
. Dissected lymphocytes or stromal cells
from the same slides were used as a source of constitutional DNA from
each case. After DNA extraction, 5 µl of the proteinase K-digested
samples containing DNA from at least 100 cells were used for each
multiplex PCR reaction.
Polymorphic DNA Markers and PCR-LOH Analysis.
To evaluate LOH, we used primers flanking dinucleotide and
multinucleotide microsatellite repeat polymorphisms spanning the entire
chromosome 3p arm (Fig. 1
). In lung cancer cell line analysis, all 54 polymorphic markers were
examined, including 19 polymorphic markers located in the 600-kb 3p21.3
region (D3S4597/P1.5, D3S4598/P1.4,
D3S4600/P1.2, D3S4602/Luca20.2, D3S4604/Luca19.1,
D3S4606/Luca17.2, D3S4608/Luca13.4, D3S4610/Luca11.4,
D3S4611/Luca11.2, D3S4612/Luca11.1,
D3S4613/Luca8.3, D3S4614/Luca8.2,
D3S4615/Luca8.1, D3S4617/Luca7.1,
D3S4622/Luca4.1, D3S4623/Luca2.2,
D3S4624/Luca2.1, D3S4625/Luca1.3, and
D3S4627/Luca1.1). In addition, 13 markers in this 600-kb
3p21.3 region (D3S4595/P1.7, D3S4596/P1.6,
D3S4599/P1.3, D3S4601/Luca22,
D3S4605/Luca17.4, D3S4607/Luca17.1,
D3S4609/Luca12.1, D3S4616/Luca7.2,
D3S4618/Luca6.3, D3S4619/Luca4.4,
D3S4620/Luca4.3, D3S4621/Luca4.2, and
D3S4626/Luca1.2) were not informative in any cell line
specimen, and they are not shown in Fig. 3
; however, they were used to
screen for small homozygous deletions. In microdissected archival
tumors and precursor lesions and normal epithelium samples, a subset of
28 polymorphic markers was used (Fig. 1
, bold markers).
Because of the limited material from bronchial biopsies of smokers
without lung cancer and never smokers, only the six markers within the
600-kb region at 3p21.3 (D3S4597/P1.5,
D3S4604/Luca19.1, D3S4614/Luca8.2,
D3S4622/Luca4.1, D3S4623/Luca2.2, and
D3S4624/Luca2.1) suitable for paraffin-embedded tissues were
used to assess this region. Primer sequences can be obtained from the
Genome Database for all of these markers, and those microsatellite
markers in the 3p21.3 region identified using the POMPOUS system have
been published previously (38)
. For cell lines, PCR-LOH
analysis was performed directly on genomic DNA as described previously
(39)
, whereas for microdissected samples, a two-round PCR
strategy was used as described previously (43)
. LOH was
scored by visual detection of complete absence of one allele in
autoradiographs (Fig. 2
). All of the data were put into an Excel spreadsheet and analyzed with
our own software constructed in Microsoft Visual Basic (available on
request), which was designed to facilitate color formatting and
visualization of the data as shown in Figs. 3
and 4
.
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RESULTS |
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, and Fig. 2
shows
examples of data from our allelotype studies of multiple 3p sites in
samples from microdissected tumors, microdissected respiratory
epithelial samples from patients with cancer, or bronchoscopy biopsy
specimens of current and former smokers without lung cancer. We first
tested DNA samples from lung cancer cell lines using 41 microsatellite
polymorphic markers distributed across chromosome 3p (Fig. 1
); an
additional 13 rarely polymorphic markers (labeled with an
asterisk in the 600-kb 3p21.3 deletion region in Fig. 1
)
from the 600-kb homozygous deletion 3p21.3 region were also tested to
look for small homozygous deletions, but none were detected (data not
shown; Ref. 38
). Allelic losses involving one or more 3p
regions were detected in nearly all (30 of 31) SCLC and NSCLC cell
lines (Fig. 3
; Table 2
). In all 13 SCLC cell lines and 8 of 18 (44%) NSCLC cell lines, all or
most of the 3p arm was deleted. Nine of the 10 remaining NSCLC cell
lines demonstrated more localized 3p deletions affecting predominantly
the telomeric 3p22–p25 and/or centromeric 3p12–p14.2 regions or more
localized 3p21 regions but not the 600-kb 3p21.3 region (Fig. 3
). The
previously described homozygous deletions in SCLC NCI-H1450 and NSCLC
HCC95 involving 3p21.3 markers D3S1573 to
D3S4597/LucaP1.5 (23)
and 3p14.2 marker
D3S4103 (FHIT; Ref. 25
),
respectively, were detected, as was another smaller 3p21.3 homozygous
deletion in SCLC NCI-H1399 at D3S4627/Luca1.1 (data not
shown).
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,
bold markers). Allele loss at one or more 3p regions was
very frequent in the three major types of lung cancer (100% of 22
SCLCs, 100% of 23 squamous cell cancers, and 90% of 21
adenocarcinomas; Fig. 4A,
left panel; Table 2
).
We found no correlation between chromosome 3p allelic loss and amount
of smoking exposure, clinicopathological stage, sex, or age. As with
lung cancer cell lines, allele loss of most of the 3p arm was found in
SCLCs (91%) and squamous cell carcinomas (95%; Fig. 4A
).
In contrast, the sizes of the 3p allele loss regions in adenocarcinomas
were much smaller. Of note, in nearly every primary tumor, there was
discontinuous LOH with several regions of 3p allele loss (Fig. 4A
, red boxes) separated by intervening regions of
heterozygosity (Fig. 4A
, green boxes). For purposes of
discussion, we refer to the junctions between the red and
green boxes as "breakpoints." These junctions could
occur by physical deletion and/or by mitotic recombination. We
emphasize that our use of the term "breakpoint" does not imply a
mechanism but refers only to the detection of juxtaposed regions of
allele retention and allele loss (see "Discussion").
Chromosome 3p Allele Loss Frequently Occurs in Histologically
Normal and Abnormal Epithelium Accompanying Lung Cancers.
We microdissected 54 discrete foci of histologically normal epithelia,
mildly abnormal (hyperplasia or squamous metaplasia) epithelia, or more
abnormal lesions (dysplasia and CIS) from the surgically resected
squamous cell lung cancer specimens. This analysis was limited to foci
accompanying squamous cell lung carcinoma because the sequence of
histological changes accompanying this cancer has been well
established. The same panel of 28 highly informative microsatellite
markers spanning the entire 3p arm previously used to examine
microdissected lung tumors was also used in allelotyping the CIS,
dysplasia, hyperplasia/metaplasia lesions, and histologically normal
epithelial samples (Fig. 4A
, right panel). The present
findings confirm and extend our previously published data
(18)
, indicating that small chromosome 3p allelic losses
were detected in histologically normal and mildly abnormal epithelium,
as well as in dysplasias and CIS (Fig. 4A
; Table 2
).
Increasing severity of histological change (from mildly abnormal to
dysplasia to CIS) was, in general, also characterized by increasing
frequency and size of allelic loss in the chromosome 3p regions (Fig. 4A
). Thus, each 3p region of allele loss detected in normal
and mildly abnormal epithelium affected only one to three 3p
polymorphic markers. By contrast, half of dysplasias had 6–13 markers
affected, and 80% of CIS lesions had 
10 markers lost. We found no
correlation between chromosome 3p allelic loss in histologically normal
epithelium or histologically identified precursor lesions and the
amount of smoking exposure, clinical stage, sex, or age. As we have
noted previously, for any particular marker showing allele loss, we
often found the same parental allele to be lost in the lung cancer and
in the various respiratory epithelial lesions, a phenomenon we call
allele-specific loss or allele-specific mutation (16
, 18 , 19)
. We determined the frequencies of allele-specific loss
(comparing parental allele lost in the preinvasive foci and invasive
tumor) in the 42 foci demonstrating one or more sites of allelic loss
in all 10 cancer patients. For all 28 microsatellite markers tested on
these 42 foci, this gave 244 possible comparisons. For all 244
comparisons, the same parental allele was lost in 215 (88%
concordance). The possibility of this concordance happening by chance
is extremely remote, as tested by the cumulative binomial test
(P = 1.2 x 10-36).
Definition of Multiple Chromosome 3p Regions Demonstrating LOH in
Lung Cancer.
Analysis of the 3p allelotyping data pooled together from all of the
specimens and sorted by number of markers with allele loss showed that
nearly all of the 28 markers exhibited frequent allele loss, and the
LOH patterns were complex, with many chromosome 3p breakpoints (and
thus discontinuous regions of LOH), indicating the presence of multiple
separate 3p regions with high-frequency allele loss (Fig. 4B
; Table 3
). In fact, several markers demonstrated discrete allele loss or were
located in at least one flanking end of an allele loss area (Table 3)
.
The results from the normal epithelium and histologically abnormal
epithelial lesions associated with lung cancers especially helped to
define small regions of allele loss (Fig. 4, A and B
). From examining the data in Fig. 4B
, it is
clear that there is no single region on 3p that could account for the
various patterns we found in the different tumors and respiratory
epithelial lesions. Because we ultimately want to identify potential
TSGs on 3p, we needed to define the multiple different sites. To do
this, we first asked which of the markers individually, in pairs, or in
triplets represented consistent sites of local allele loss. In this
analysis we only counted (using the data in Fig. 4B
) allele
loss for a single marker, doublet, or triplet of markers when the two
contiguous flanking markers were informative and exhibited retention of
heterozygosity. After considering all of the data for groups of triplet
markers, the following nine regions had 10 examples with allele loss
in one or more of the triplet members but retention of heterozygosity
of both flanking markers: (a) D3S1111-D3S2432;
(b) D3S1293-D3S1537; (c)
D3S1537-D3S1612; (d) D3S4604-D3S4622;
(e) D3S4624-D3S1582; (f)
D3S1234-D3S1300; (g) D3S4103-D3S1284;
(h) D3S1300-D3S1274; and (i)
D3S1284-D3S1577. The following six regions had 6 examples
with allele loss in one or both of the doublet markers but retention of
heterozygosity of both flanking markers: (a)
D3S4604-D3S4614; (b) D3S4614-D3S4622;
(c) D3S4103-D3S1300; (d)
D3S1300-D3S1284; (e) D3S1284-D3S1274;
and (f) D3S1274-D3S1277. Finally, the following
four regions had 3 or 4 examples of isolated single marker allele loss
with retention of heterozygosity of the flanking markers:
(a) D3S1537;(b) D3S4624;
(c) D3S1766; and (d)
D3S1300. In addition, both of the end telomeric and
centromeric markers D3S1597 and D3S1511 had
multiple examples of LOH alone or as part of doublets or triplets. From
these analyses, the data were most consistent with the following sites
of localized allelic loss: (a) telomere-D3S1597;
(b) D3S1111-D3S2432; (c)
D3S2432-D3S1537; (d) D3S1537;
(e) D3S1537-D3S1612; (f)
D3S4604-D3S4622; (g) D3S4624;
(h) D3S4624-D3S1582; (i)
D3S1766; (j) D3S1234-D3S1300
(FHIT/FRA3B region centered on D3S1300;
Refs.24
and 25
); (k)
D3S1284-D3S1577 (U2020/DUTT1 region centered on
D3S1274); and (l) D3S1511-centromere
(45)
.
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; Table 2
). Using a
subset of six microsatellite markers extending over the 600-kb 3p21.3
region, a very high incidence of allele loss in this small region was
detected in primary resected SCLCs (20 of 20; 100%) and squamous cell
carcinomas (22 of 23; 96%; Fig. 4A
; Table 2
). In lung
adenocarcinomas, the allele loss in this 3p21.3 region was less
frequent (10 of 20; 50%) and often involved only one or two 3p21.3
markers (Fig. 4A
). The 600-kb 3p21.3 region was the most
frequently detected site of 3p allele loss in normal and histologically
abnormal epithelium lesions associated with squamous cell lung cancer
(Table 2
; Fig. 4A
). Progression in the LOH frequency and
size of 3p21.3 allele loss regions also occurred with increasing
histological severity (for example, compare normal and mildly abnormal
epithelium to dysplasia and CIS in Fig. 4A
).
We next studied 47 biopsy specimens obtained by fluorescence
bronchoscopy from 28 subjects (13 current and 15 former smokers) and 18
samples from 7 never smokers (Tables 1
and 2)
. Because of the limited
material, only the six polymorphic markers within the 600-kb 3p21.3
region were used. We found 3p21.3 allele loss in 49% of the lesions
studied, representing 59% of the subjects (Table 2)
. By contrast, we
found no lesions with 3p21.3 allele loss in 7 never smokers (Fig. 2
;
Table 2
). The frequencies of 3p21.3 allele loss detected in bronchial
samples from smokers without lung cancer were lower in the dysplasia
and CIS categories than in the same histological lesions found in
squamous cell lung cancer patients (compare data in Table 2
). We also
tested for allele-specific loss when two or more biopsies from the same
subject demonstrated losses of the same marker(s). Although the numbers
of comparisons are very small, allele-specific loss was noted in 13 of
15 (87%) comparisons involving the six 3p21.3 markers. By the
cumulative binomial test, the probability of this finding happening by
chance is P = 0.003.
We then pooled all of the data on 3p21.3 markers from 162
specimens of tumor cell lines, tumors, bronchial epithelium associated
with cancer, and bronchial epithelium from smokers without cancer (Fig. 4C
). For this analysis, we only included the bronchial
epithelial samples showing some example of 3p allele loss. In the case
of the lesions associated with cancer, this included the 42 of 54
lesions (78%) with loss at any of the 28 markers; for the epithelial
samples from smokers, this represented 23 of the 47 specimens (49%)
showing loss at one of the six 3p21.3 markers. Analysis of
histologically normal epithelium and precursor specimens demonstrating
loss of one or more 3p21.3 markers revealed that markers
D3S4604/Luca19.1, D3S4622/Luca4.1, and
D3S4624/Luca2.1 showed the highest frequencies of LOH in
both cancer patients and smokers. Although the number of persons
studied was small, higher frequencies of 3p21.3 allelic losses were
detected in current smokers than in former smokers (data not shown).
However, we found no statistically significant differences in the
frequencies and patterns of LOH between the two groups, and,
anecdotally, allele loss in this region was found in biopsy specimens
from a former smoker who had quit smoking 48 years ago.
We analyzed the combined 3p21.3 LOH data from all of 162 samples
(including 91 samples showing breakpoints within the 3p21.3 region) to
try to define one area of shortest region of overlap (Fig. 4C
). The vast majority of the samples (125 of 136) showing
any 3p21.3 allele loss were consistent with a subregion defined by
D3S4622/Luca4.1 extending through
D3S4604/Luca19.1 and centered on D3S4614/Luca8.2.
However, in a few cases (11 of 136), separate subregions beginning at
D3S4624/Luca2.1, beginning at D3S4597/P1.5, or
centered at D3S4604/Luca19.1 were seen (Fig. 4C
).
Chromosome 3p Breakpoint Patterns at 3p21.3, 3p14.2
(FHIT), and 3p12 (U2020).
As the result of the detailed allelotyping analysis performed in lung
cancer and bronchial epithelial specimens, we identified multiple
breakpoints throughout the 3p arm in several samples (Fig. 4, B and C
). In each sample, we compared the
presence of breakpoints in the 600-kb 3p21.3 region with those in the
similar size (600 kb) 3p14.2 (D3S1300-D3S1234 portion of
the FHIT/FRA3B region) region and the larger 3p12 (6 Mb)
portion of the U2020 region (Table 4)
. Although there were differences in the breakpoint pattern
frequencies between tumor cell lines, tumors, and bronchial epithelial
lesions, overall 25% had no change in any of the three regions,
60% had breakpoints in one of the three regions, and the remainder had
breakpoints in two or more regions simultaneously (Table 4)
. The
breakpoints in these three regions appeared to occur independently of
one another.
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DISCUSSION |
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50% of these preinvasive clones are clonally independent of the
tumor occurring in the same patient (18)
. Thus, this
intriguing phenomenon merits additional study including study in other
tumors such as breast cancer that also have preinvasive lesions with
allele loss in the accompanying epithelium. Whereas frequent allele loss at several chromosomal 3p regions has been previously detected in lung carcinoma and other neoplasms, few genes have strong evidence supporting their candidacy as a TSG in lung cancer (15 , 45) . One candidate in the 3p25 region is the Von Hippel-Lindau (VHL) TSG; however, VHL is only rarely mutated or is not expressed in lung carcinomas (51) . Another candidate gene, FHIT, which spans the FRA3B fragile site at 3p14.2, is associated with homozygous deletions in lung cancer and with cancer-related FHIT cDNA splicing aberrations but is only rarely associated with small mutations altering one or a few amino acids (24 , 25) . However, several studies have shown loss of expression of Fhit protein in 50–75% of lung cancers (52, 53, 54) . Whereas FHIT abnormalities may only represent alterations in the FRA3B fragile site (25 , 55) , several groups including our own have demonstrated that introduction of a wild-type FHIT gene suppresses tumorigenicity and induces apoptosis, although other groups have not found tumor-suppressing activity (53 , 56 , 57) . Recently, a new candidate TSG, DUTT1, has been cloned residing in the U2020 3p12 deletion region and crossing a small (<100 kb) lung cancer homozygous deletion at D3S1274, the center of our allele loss in this region (45) . However, its tumor-suppressing activity and protein expression patterns in tumors are unknown.
A higher frequency of allelic loss and more extensive regions of chromosome 3p allele loss were detected in centrally arising SCLCs and squamous cell cancers compared to peripherally arising adenocarcinomas. Other differences in the allelic loss and mutation patterns have been reported previously between squamous and adenocarcinomas, suggesting that more genetic changes accumulate during tumorigenesis in squamous cell carcinomas than in adenocarcinomas (58) . These differences may be related to differences in tumorigenic mechanisms, including smoking damage (59) . However, no correlation between 3p allelic losses and amount of smoking exposure was detected in our resected lung carcinoma cases.
As we described previously (18) , chromosome 3p allelic losses present in bronchial epithelium were the earliest changes beginning in normal and hyperplastic epithelium. However, our present findings indicate that within 3p, the 600-kb 3p21.3 region is the region most frequently undergoing allelic loss in normal and histologically abnormal epithelium associated with lung cancer, indicating that this region plays an important role in the early development of this neoplasm. We examined the frequency and pattern of LOH at very high resolution in this particular 3p21.3 region in normal and abnormal bronchial epithelia of current and former smokers without cancer. Allelic losses at one or more 3p21.3 markers were frequently detected in the bronchial epithelia of smokers (49%) and were detected even in normal and mildly abnormal histological categories. Of great interest, no deletions at this region were detected in the 18 bronchial epithelial samples obtained from 7 never smokers. Our findings suggest that chromosome 3p21.3 allelic losses may be useful markers in smoking-related damaged epithelium for risk assessment and for monitoring the efficacy of chemopreventive regimens.
A high density of markers in the 600-kb 3p21.3 region was chosen to
assess this location for very small areas of allele loss because we
have focused on this region to identify putative TSGs (7
, 30
, 34
, 35
, 60)
. There are currently 19 different protein coding
candidate TSGs in this region under study. One 120-kb subregion
within the 600-kb area contains eight genes (GenBank deposits
2
2 Ca2+ channel/AF040709,
PL6/U09584, 101F6/AF040704,
Gene21/AF040707, BLU/U70880,
123F2/AF040703, Fus1/AF055479, and
LUCA2/HYAL2/U09577) and has the gene
LUCA1/HYAL1/U03056, AF173154 on its telomeric border
(21, 22, 23
, 31)
. The 120-kb region, which was tested in this
report with markers D3S4614/Luca8.2 and
D3S4622/Luca4.1, is defined by overlapping homozygous
deletions present in the SCLC cell lines NCI-H740, NCI-H1450, and GLC20
and in breast cancer cell line HCC1500 (21, 22, 23
, 30
, 31)
.
Another subregion, which was defined in this report by marker
D3S4604/Luca19.1, is a more telomeric site at the
SEMA3F/Semaphorin IV (U38276, U33920) gene locus (23
, 34
, 61)
. We analyzed the combined LOH data within the 600-kb
region for all 162 samples (tumors, tumor cell lines, and bronchial
epithelium from smokers and patients with cancer) to try to define one
area of shortest region of overlap (Fig. 4C
). This included
91 samples showing breakpoints within the 3p21.3 region. The majority
of the 136 examples of 3p21.3 allele losses were consistent with the
presence of TSG(s) at both the nine gene-containing 120-kb site
(92% consistent) and the SEMA3F site (85% consistent),
whereas the others were consistent with a more centromeric site
bordered by the marker D3S4624/Luca2.1 that contains
3pk/U09578 and a telomeric site bordered by
D3S4597/P1.5 that contains gene15 (U23946) and
gene16 (U50839). These data are consistent with the
hypothesis that several different TSGs may exist even in the 600-kb
3p21.3 region.
As a result of the detailed allelotyping analysis, we were able to identify multiple areas of discontinuous LOH and thus multiple breakpoints throughout the 3p arm in many tumor and bronchial epithelial samples. We compared the frequency of breakpoints in the 3p21.3 region with those obtained in the 3p12 (U2020 homozygous deletion, DUTT1 gene) and the 3p14.2 (FHIT gene) regions in lung tumors and associated bronchial epithelial specimens. The 3p14.2 FHIT region harbors the most common known aphidicolin-inducible fragile site in the genome (FRA3B; Ref. 55 ). Of interest, breakpoints occurring in the very restricted 3p21.3 region were as common or more common than those occurring at the 3p14.2 or 3p12 regions, which also had frequent breakpoints. In addition, these three regions also had breakpoints that appeared to occur independently of one another. We have used the term "breakpoint" to identify juxtaposed regions where one region retains heterozygosity and the other region loses heterozygosity. How the multiple regions of 3p allele loss in individual tumor samples occur is a major question. Also, do these changes represent alteration of only one parental chromosome or both parental chromosomes? These transitions in allele loss could occur by a physical deletion and/or represent a mitotic recombination event. Whereas we and others have described cytogenetic evidence of 3p deletions, the frequency of discontinuous LOH makes the possibility of mitotic recombination highly likely. This has been found to be a very frequent mechanism of LOH in sporadic and inherited retinoblastomas (62) . Also, in model systems selecting for an inactive Aprt allele either spontaneously in vivo or after an oxidative mutagenic insult, discontinuous LOH generated by mitotic recombination was also common (63 , 64) . Turker et al. (64) noted that such discontinuous LOH was present in several human tumors at a variety of chromosomal loci, and they hypothesized that discontinuous LOH was a "signature" mutational pattern for oxidative damage that is widespread in human cancer. For example, discontinuous LOH has been seen for chromosome region 9p in SCLC and head and neck cancers and at 11q in carcinoids (65, 66, 67) . Therefore, it is likely that several chromosomal regions may show discontinuous LOH when studied with multiple markers. Our detailed 3p allelotyping data support these hypotheses and raise the question of whether the multiple 3p sites harbor TSGs or only reflect a mutational signature. Given the occurrence of 3p allele loss, especially 3p21.3 breakpoints in preinvasive clones with and without histological changes, it will be important to resolve these issues in future studies.
Whatever the mechanism, all these data suggest that the 3p12, 3p14.2, and 3p21.3 sites represent highly unstable regions that undergo frequent allele loss associated with breakpoints after smoking exposure. In a recent study, using a fluorescence in situ hybridization probe centered on D3S4604/Luca19.1, aberrations in this 3p21.3 region were shown to be significantly more frequent in peripheral blood lymphocytes of lung cancer patients compared to controls without lung cancer when exposed to BPDE, an active metabolic product of tobacco smoke (68) . Thus, the phenotype of large numbers of BPDE-induced 3p21.3 breaks in lymphocytes may become useful as a new risk assessment marker for lung cancer. It will be of great importance to directly test the quantitative correlation between BPDE-induced breaks in lymphocytes and smoking-induced breaks in the target respiratory epithelial tissue in the same persons, as well as testing these same tissues for breaks at the 3p14.2 and 3p12 sites.
Although at least 19 genes have been identified in this 600-kb 3p21.3 region, none of them have been shown to have frequent mutations in lung cancer. Do the frequent hemizygous 3p21.3 allele losses reflect only the fragility of this region with a predisposition to inter- and intrachromosomal recombination events (69) , or do they also indicate the presence of an underlying TSG(s), perhaps resulting in tumorigenicity because of haplo-insufficiency? Additional studies including functional tests of the genes in this region are needed to resolve this issue. Because 3p deletions at several sites including 3p21.3 have been detected as frequent events in the pathogenesis of several other human carcinomas, including cervical, breast, and renal cancer (30 , 55 , 70 , 71) , future studies need to be conducted to determine whether similar patterns of deletions are detected in the multistage development of these neoplasms.
|
FOOTNOTES |
|---|
1 Supported by Grant CA71618, Specialized Program
of Research Excellence Grant P50-CA70907, and USPHS Contract N01CN45580
from the NIH (Bethesda, MD). 
2 To whom requests for reprints should be
addressed, at Hamon Center for Therapeutic Oncology Research,
University of Texas Southwestern Medical Center, 5323 Harry Hines
Boulevard, Dallas, TX 75390-8593. Phone: (214) 648-4900; Fax: (214)
648-4940; E-mail: minna{at}simmons.swmed.edu
3 The abbreviations used are: SCLC, small cell
lung cancer; TSG, tumor suppressor gene; NSCLC, non-small cell lung
cancer; LOH, loss of heterozygosity; CIS, carcinoma in
situ; BPDE, benzo(a)pyrene diol epoxide.
Received 9/27/99. Accepted 2/ 3/00.
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I. Kuzmin, L. Liu, R. Dammann, L. Geil, E. J. Stanbridge, S. P. Wilczynski, M. I. Lerman, and G. P. Pfeifer Inactivation of RAS Association Domain Family 1A Gene in Cervical Carcinomas and the Role of Human Papillomavirus Infection Cancer Res., April 15, 2003; 63(8): 1888 - 1893. [Abstract] [Full Text] [PDF]
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 A. Augustin, C. Spenlehauer, H. Dumond, J. Menissier-de Murcia, M. Piel, A.-C. Schmit, F. Apiou, J.-L. Vonesch, M. Kock, M. Bornens, et al. PARP-3 localizes preferentially to the daughter centriole and interferes with the G1/S cell cycle progression J. Cell Sci., April 15, 2003; 116(8): 1551 - 1562. [Abstract] [Full Text] [PDF]
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 U. Novak, E. Oppliger Leibundgut, J. Hager, D. Muhlematter, M. Jotterand, C. Besse, N. Leupin, D. Ratschiller, J. Papp, G. Kearsey, et al. A high-resolution allelotype of B-cell chronic lymphocytic leukemia (B-CLL) Blood, August 13, 2002; 100(5): 1787 - 1794. [Abstract] [Full Text] [PDF]
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Y. Tomizawa, T. Kohno, H. Kondo, A. Otsuka, M. Nishioka, T. Niki, T. Yamada, A. Maeshima, K. Yoshimura, R. Saito, et al. Clinicopathological Significance of Epigenetic Inactivation of RASSF1A at 3p21.3 in Stage I Lung Adenocarcinoma Clin. Cancer Res., July 1, 2002; 8(7): 2362 - 2368. [Abstract] [Full Text] [PDF]
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L. Shivakumar, J. Minna, T. Sakamaki, R. Pestell, and M. A. White The RASSF1A Tumor Suppressor Blocks Cell Cycle Progression and Inhibits Cyclin D1 Accumulation Mol. Cell. Biol., June 15, 2002; 22(12): 4309 - 4318. [Abstract] [Full Text] [PDF]
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 I. I. Wistuba, R. Ashfaq, A. Maitra, H. Alvarez, E. Riquelme, and A. F. Gazdar Fragile Histidine Triad Gene Abnormalities in the Pathogenesis of Gallbladder Carcinoma Am. J. Pathol., June 1, 2002; 160(6): 2073 - 2079. [Abstract] [Full Text] [PDF]
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L. Ji, M. Nishizaki, B. Gao, D. Burbee, M. Kondo, C. Kamibayashi, K. Xu, N. Yen, E. N. Atkinson, B. Fang, et al. Expression of Several Genes in the Human Chromosome 3p21.3 Homozygous Deletion Region by an Adenovirus Vector Results in Tumor Suppressor Activities in Vitro and in Vivo Cancer Res., May 1, 2002; 62(9): 2715 - 2720. [Abstract] [Full Text] [PDF]
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J. A. O'Shaughnessy, G. J. Kelloff, G. B. Gordon, A. J. Dannenberg, W. K. Hong, C. J. Fabian, C. C. Sigman, M. M. Bertagnolli, S. P. Stratton, S. Lam, et al. Treatment and Prevention of Intraepithelial Neoplasia: An Important Target for Accelerated New Agent Development : Recommendations of the American Association for Cancer Research Task Force on the Treatment and Prevention of Intraepithelial Neoplasia Clin. Cancer Res., February 1, 2002; 8(2): 314 - 346. [Abstract] [Full Text] [PDF]
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A. H. Banham, N. Beasley, E. Campo, P. L. Fernandez, C. Fidler, K. Gatter, M. Jones, D. Y. Mason, J. E. Prime, P. Trougouboff, et al. The FOXP1 Winged Helix Transcription Factor Is a Novel Candidate Tumor Suppressor Gene on Chromosome 3p Cancer Res., December 1, 2001; 61(24): 8820 - 8829. [Abstract] [Full Text] [PDF]
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T. Tsujiuchi, Y. Sasaki, N. Murata, M. Tsutsumi, Y. Konishi, and D. Nakae FHIT alterations in lung adenocarcinomas induced by N-nitrosobis(2-hydroxypropyl)amine in rats Carcinogenesis, December 1, 2001; 22(12): 2017 - 2022. [Abstract] [Full Text] [PDF]
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J. Xian, K. J. Clark, R. Fordham, R. Pannell, T. H. Rabbitts, and P. H. Rabbitts Inadequate lung development and bronchial hyperplasia in mice with a targeted deletion in the Dutt1/Robo1 gene PNAS, November 29, 2001; (2001) 251407098. [Abstract] [Full Text] [PDF]
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Y. Tomizawa, Y. Sekido, M. Kondo, B. Gao, J. Yokota, J. Roche, H. Drabkin, M. I. Lerman, A. F. Gazdar, and J. D. Minna Inhibition of lung cancer cell growth and induction of apoptosis after reexpression of 3p21.3 candidate tumor suppressor gene SEMA3B PNAS, November 20, 2001; 98(24): 13954 - 13959. [Abstract] [Full Text] [PDF]
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Y. Aoyagi, T. Yokose, Y. Minami, A. Ochiai, T. Iijima, Y. Morishita, T. Oda, K. Fukao, and M. Noguchi Accumulation of Losses of Heterozygosity and Multistep Carcinogenesis in Pulmonary Adenocarcinoma Cancer Res., November 1, 2001; 61(21): 7950 - 7954. [Abstract] [Full Text] [PDF]
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 A Martinez, R A Walker, J A Shaw, S J Dearing, E R Maher, and F Latif Chromosome 3p allele loss in early invasive breast cancer: detailed mapping and association with clinicopathological features Mol. Pathol., October 1, 2001; 54(5): 300 - 306. [Abstract] [Full Text] [PDF]
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D.-S. Byun, M.-G. Lee, K.-S. Chae, B.-G. Ryu, and S.-G. Chi Frequent Epigenetic Inactivation of RASSF1A by Aberrant Promoter Hypermethylation in Human Gastric Adenocarcinoma Cancer Res., October 1, 2001; 61(19): 7034 - 7038. [Abstract] [Full Text] [PDF]
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M.-G. Lee, H.-Y. Kim, D.-S. Byun, S.-J. Lee, C.-H. Lee, J. I. Kim, S.-G. Chang, and S.-G. Chi Frequent Epigenetic Inactivation of RASSF1A in Human Bladder Carcinoma Cancer Res., September 1, 2001; 61(18): 6688 - 6692. [Abstract] [Full Text] [PDF]
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S. Toyooka, H. I. Pass, N. Shivapurkar, Y. Fukuyama, R. Maruyama, K. O. Toyooka, M. Gilcrease, A. Farinas, J. D. Minna, and A. F. Gazdar Aberrant Methylation and Simian Virus 40 Tag Sequences in Malignant Mesothelioma Cancer Res., August 1, 2001; 61(15): 5727 - 5730. [Abstract] [Full Text] [PDF]
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A. Maitra, I. I. Wistuba, C. Washington, A. K. Virmani, R. Ashfaq, S. Milchgrub, A. F. Gazdar, and J. D. Minna High-Resolution Chromosome 3p Allelotyping of Breast Carcinomas and Precursor Lesions Demonstrates Frequent Loss of Heterozygosity and a Discontinuous Pattern of Allele Loss Am. J. Pathol., July 1, 2001; 159(1): 119 - 130. [Abstract] [Full Text] [PDF]
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D. G. Burbee, E. Forgacs, S. Zochbauer-Muller, L. Shivakumar, K. Fong, B. Gao, D. Randle, M. Kondo, A. Virmani, S. Bader, et al. Epigenetic Inactivation of RASSF1A in Lung and Breast Cancers and Malignant Phenotype Suppression J Natl Cancer Inst, May 2, 2001; 93(9): 691 - 699. [Abstract] [Full Text] [PDF]
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S. Zöchbauer-Müller, K. M. Fong, A. Maitra, S. Lam, J. Geradts, R. Ashfaq, A. K. Virmani, S. Milchgrub, A. F. Gazdar, and J. D. Minna 5' CpG Island Methylation of the FHIT Gene Is Correlated with Loss of Gene Expression in Lung and Breast Cancer Cancer Res., May 1, 2001; 61(9): 3581 - 3585. [Abstract] [Full Text]
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I. I. Wistuba, M. Tang, A. Maitra, H. Alvarez, P. Troncoso, F. Pimentel, and A. F. Gazdar Genome-wide Allelotyping Analysis Reveals Multiple Sites of Allelic Loss in Gallbladder Carcinoma Cancer Res., May 1, 2001; 61(9): 3795 - 3800. [Abstract] [Full Text]
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N. Rosenberg New transformation tricks from a barnyard retrovirus: Implications for human lung cancer PNAS, April 10, 2001; 98(8): 4285 - 4287. [Full Text] [PDF]
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R. Dammann, G. Yang, and G. P. Pfeifer Hypermethylation of the CpG Island of Ras Association Domain Family 1A (RASSF1A), a Putative Tumor Suppressor Gene from the 3p21.3 Locus, Occurs in a Large Percentage of Human Breast Cancers Cancer Res., April 1, 2001; 61(7): 3105 - 3109. [Abstract] [Full Text]
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T. Liloglou, P. Maloney, G. Xinarianos, M. Hulbert, M. J. Walshaw, J. R. Gosney, L. Turnbull, and J. K. Field Cancer-specific Genomic Instability in Bronchial Lavage: A Molecular Tool for Lung Cancer Detection Cancer Res., February 1, 2001; 61(4): 1624 - 1628. [Abstract] [Full Text]
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M. I. Lerman and J. D. Minna The 630-kb Lung Cancer Homozygous Deletion Region on Human Chromosome 3p21.3: Identification and Evaluation of the Resident Candidate Tumor Suppressor Genes Cancer Res., November 1, 2000; 60(21): 6116 - 6133. [Abstract] [Full Text]
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A. K. Virmani, A. Rathi, S. Zochbauer-Muller, N. Sacchi, Y. Fukuyama, D. Bryant, A. Maitra, S. Heda, K. M. Fong, F. Thunnissen, et al. Promoter Methylation and Silencing of the Retinoic Acid Receptor-{beta} Gene in Lung Carcinomas J Natl Cancer Inst, August 16, 2000; 92(16): 1303 - 1307. [Abstract] [Full Text] [PDF]
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J. Xian, K. J. Clark, R. Fordham, R. Pannell, T. H. Rabbitts, and P. H. Rabbitts Inadequate lung development and bronchial hyperplasia in mice with a targeted deletion in the Dutt1/Robo1 gene PNAS, December 18, 2001; 98(26): 15062 - 15066. [Abstract] [Full Text] [PDF]
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