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Oncogene (2001) 20, 6245 ± 6249
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Loss of heterozygosity analysis de®nes a 3-cM region of 15q commonly
deleted in human malignant mesothelioma
Assunta De Rienzo1, Binaifer R Balsara1, Sinoula Apostolou1, Suresh C Jhanwar2 and
Joseph R Testa*,1
1
Human Genetics Program, Fox Chase Cancer Center, Philadelphia, Pennsylvania, PA 19111, USA; 2Department of Human
Genetics, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA
Previous comparative genomic hybridization and allelic
loss analyses demonstrated frequent deletions from
15q11.1 ± 15 in malignant mesothelioma. Recurrent
losses of 15q11 ± 22 have also been reported in several
other tumor types such as breast and colorectal cancers.
To more precisely map the commonly deleted region, we
have performed a high density loss of heterozygosity
analysis of 46 malignant mesotheliomas, using 26
polymorphic microsatellite markers spanning the entire
long arm of chromsome 15. Allelic loss from 15q was
observed in 22 of 46 (48%) cases. These analyses have
de®ned a minimally deleted region of *3-cM, which was
con®rmed to reside at 15q15 by ¯uorescence in situ
hybridization analysis with yeast arti®cial chromosome
probes. No tumor suppressor genes have been reported
to map to this site. The minimally deleted region
identi®ed in this investigation overlaps those observed
in other kinds of cancer, and is the smallest site of
recurrent 15q loss identi®ed to date in human tumors.
The identi®cation of this commonly deleted site
implicates a putative tumor suppressor gene(s) at
15q15 involved in diverse forms of human neoplasia.
Oncogene (2001) 20, 6245 ± 6249.
Keywords: malignant mesothelioma; LOH; chromosome arm 15q; tumor suppressor gene
Human malignant mesotheliomas (MMs) are highly
aggressive tumors that arise from mesothelial cells
lining the pleural, peritoneal, and pericardial cavities
(Antman, 1981). More than 2000 cases of MM are
diagnosed annually in the USA, and the incidence is
expected to steadily increase worldwide until the year
2020 (Attanoos and Gibbs, 1997). Exposure to asbestos
is known to be a major contributor to the development
of this malignancy (Craighead and Mossman, 1982). It
is unknown whether asbestos ®bers act directly on
mesothelial cells, or indirectly, via formation of
reactive oxygen species and/or increased growth factor
*Correspondence: JR Testa; E-mail: [email protected]
Received 13 April 2001; revised 29 June 2001; accepted 12 July
2001
receptors (Mossman et al., 1996; Pache et al., 1998).
Recent investigations have also implicated simian virus
40 (SV40) in the etiology of some MMs (Testa et al.,
1998). It has been postulated that SV40 and asbestos
could act as co-carcinogens, and that individuals who
are SV40-positive may be at a higher risk of developing
mesothelioma when exposed to asbestos (Carbone et
al., 1999; Testa et al., 1998).
MM is characterized by a long latency from the time
of asbestos exposure to clinical diagnosis (Lynch et al.,
1985), suggesting that an accumulation of multiple
somatic genetic alterations may be required before a
mesothelial cell is converted into a neoplastic cell. The
most common cytogenetic aberrations in MM are
deletions involving discrete regions in chromosome
arms 1p, 3p, 4q, 6q, 9p, 13q, 14q, 15q and 22q (Balsara
et al., 1999; Bell et al., 1997; Bjorkqvist et al., 1997,
1999; De Rienzo et al., 2000; Popescu et al., 1988;
Shivapurkar et al., 1999; Taguchi et al., 1993). The
high frequency of speci®c chromosomal losses suggests
a recessive tumorigenic mechanism in this cancer, and
the inactivation of tumor suppressor genes (TSGs)
residing in these deleted chromosomal regions may
contribute to the development and/or progression of
MM (Taguchi et al., 1993).
Loss of heterozygosity (LOH) is the most frequent
type of genetic alteration found in many kinds of
malignancies. LOH occurs as a consequence of
deletions, whole chromosome losses, or aberrant
mitotic recombination events (Seemayer and Cavenee,
1989; Weinberg, 1993) and can reveal the presence of a
recessive mutation in the remaining allele of a TSG
localized within the a€ected region of the genome
(Knudson, 1989).
Previously, we used comparative genomic hybridization analysis to identify a new common region of
deletion, 15q, in MM (Balsara et al., 1999). LOH
analysis on eight MM cases with 16 microsatellite
markers con®rmed a common region of deletion at 15q
11.1 ± 15 (Balsara et al., 1999). To more precisely
localize the minimal region of 15q loss in MM, we here
report allelic loss studies on a much larger series of
MMs, using 26 polymorphic DNA markers spanning
15q. The location of each marker from the centromere
to the telomere and the corresponding distances in cM
are shown in Figure 1.
15q losses in mesothelioma
A De Rienzo et al
6246
Figure 1 Pattern of LOH in MMs exhibiting deletions in 15q. Thick vertical bar, designated by SRO, indicates the minimal region
of overlapping deletion. Markers, with corresponding cM distances, are shown in the predicted order from the centromere to the
telomere. For each locus, the overall frequency of allelic loss is shown at the right. Primer pairs used for polymerase chain reaction
(PCR) ampli®cation of tetra, tri-, and dinucleotide repeat markers were obtained from Research Genetics. The chromosomal
location of the markers and their relative order were ascertained from either the Genetic Location Database, University of
Southampton (Collins et al., 1996) and the Center of Medical Genetics Database, Marsh®eld Medical Research Foundation
(Broman et al., 1998). PCR was performed in a ®nal volume of 5 ml with 20 ng of genomic DNA, 75 ng of each primer, 50 mM of
each dNTP, 1 unit of AmpliTaq DNA polymerase (Perkin Elmer), and 0.2 mCi [a-32P]-dCTP (DuPont NEN), using a Hybaid OmnE Thermal Cycler. Ampli®cation conditions consisted of an initial denaturation of 5 min at 948C followed by 35 cycles of 1 min at
948C, 1 min at 55 ± 578C and 1 min at 728C, with a ®nal extension of 10 min at 728C. PCR products were diluted 1 : 1 with 95%
formamide gel loading bu€er. Three ml of each PCR product were separated electrophoretically in a 6.5% denaturing (8 M urea)
polyacrylamide gel. Gels were dried at 808C under vacuum and subjected to autoradiography at room temperature for 2 ± 6 h. LOH
was scored when constitutional heterozygosity in control DNA and complete loss of one allelic band in the tumor cell line DNA
was observed
Tumor specimens were surgically resected primary
pleural MMs from newly diagnosed patients. Criteria
for the diagnosis of MM were in accordance with
established guidelines (Antman et al., 1989). Forty-six
tumor cell lines were established from primary MM
cultures as described previously (Taguchi et al., 1993),
and genomic DNA was isolated by standard methods.
Allelic patterns of DNA from 19 tumor cell lines were
compared with those from matched blood samples.
Matched peripheral blood was not available for the
remaining 27 cell lines. In these cases, the constitutional allelic pattern was determined from DNA
obtained from tumor tissue containing a mixture of
tumor and normal cells.
Among the 46 MMs examined, 22 (47.8%) displayed
allelic loss of at least one marker (Figure 1). Nine of
these cases exhibited allelic loss of all informative
markers, presumably because of whole chromosome
loss. Case 17 displayed allelic loss of all markers except
D15S1031. The remaining 12 cases showed interstitial
Oncogene
deletions, 11 of which were overlapping. The exception,
case 7, displayed allelic loss only at GATA153F11,
which may represent a nonspeci®c event because this
locus showed a relatively low overall frequency of
allelic loss.
Analysis of the LOH patterns of the cases with
overlapping interstitial deletions revealed a shortest
region of overlap (SRO) of approximately 3 cM,
located between markers D15S1007 and ACTC at
15q15.3. The proximal boundary was de®ned by case
13, which retained both alleles at D15S1007 (Figure
2A). Case 49 delineated the distal boundary, with
retention of both alleles at marker ACTC (Figure 2B).
The frequencies of LOH in the minimally deleted
region were 42.9% (15 of 35) at D15S1040 and 40.7%
(11 of 27) at GATA50C03. An even higher frequency
of LOH was observed at D15S144, which resides
slightly proximal to the SRO (Figure 1); however, the
number of cases informative for D15S144 was
considerably lower than that for any marker in 15q15.
15q losses in mesothelioma
A De Rienzo et al
6247
Figure 2 Representative autoradiograms showing allelic band
patterns at several loci within 15q. Case numbers are indicated
below each panel. Alleles recognized by microsatellite markers are
depicted by dots (.). Arrowheads: allelic loss. Letters above
panels indicate DNA from tumor cell line (C), primary tumor (T),
and normal peripheral blood (B)
Matched tumor, blood, and cell line DNAs were
available for four of 46 MMs with allelic loss in 15q15.
DNA from the tumor specimens exhibited reduction in
the intensity of the allelic band corresponding to the
one unequivocally lost in the matched cell line (Figure
2B), suggesting that LOH seen in the MM cell lines is
representative of that observed in the corresponding
tumor specimens.
The SRO was con®rmed by two-color ¯uorescence in
situ hybridization (FISH) analysis using YACs containing loci immediately ¯anking or within the SRO. Three
YACs (812-C-4, 908-C-5, and 822-G-2) contained
marker D15S1040, from the SRO, as well as one or
more ¯anking markers (Figure 3). However, each of
these YACs was found to be chimeric and, thus, was
excluded from further analysis. YACs 775-D-2 contained D15S1007 and other markers located proximal
to the SRO, whereas 947-F-7 and 914-A-2 contained
D15S971, which is distal to the SRO. YAC 947-F-7
was excluded because it was chimeric. FISH analysis
was performed with YACs 775-D-2 and 914-A-2 on
cases 13 and 49, which collectively delineated the SRO.
Case 13 showed loss of 914-A-2, while YAC 775-D-2
was retained (Figure 4). FISH analysis for case 49
revealed multiple copies (3 ± 4 each) of two di€erent
marker chromosomes which hybridized to YACs 775D-2 and 914-A-2. One marker exhibited overlapping
signals for each YAC. The other marker showed loss
of 775-D-2, and retention of YAC 914-A-2 (data not
Figure 3 Diagrammatic representation of YAC contig in 15q15.
Markers are shown on the top in the predicted order from the
centromere to the telomere. Horizontal bars represent YACS. We
identi®ed YACs located in the vicinity of the 15q15 SRO using
the Center for Genome Research Database, Whitehead Institute
for Genome Research. Yeast strains carrying the YACs were
obtained from Research Genetics. To verify the positions of these
YACs relative to our SRO, we carried out PCR analysis to
determine which YACs contained the following markers:
D15S1007, D15S1040, ACTC, GAAA11C1, and D15S971.
DNA was puri®ed from yeast strains by standard methods, and
PCR analysis was performed to determine which YACs contained
markers ¯anking or residing within the SRO. Each PCR reaction
was performed in a ®nal volume of 20 ml with 20 ng of yeast
DNA, 100 ng of each primer, 50 mM of each dNTP, and 1.2 units
of AmpliTaq DNA polymerase, using the same ampli®cation
conditions described in Figure 1
shown). Collectively, these results con®rm that the
minimally deleted region resides at 15q15.
MM is characterized by recurrent chromosomal
deletions involving discrete regions in 1p, 3p, 4q, 6q,
9p, 13q, 14q, 15q and 22q (Balsara et al., 1999; Bell et
al., 1997; Bjorkqvist et al., 1997, 1999; De Rienzo et
al., 2000; Seemayer and Cavenee, 1989; Shivapurkar et
al., 1999; Taguchi et al., 1993), suggesting that each of
these regions harbors a putative TSG whose loss/
inactivation may contribute to the pathogenesis of this
malignancy. Thus, far, TSGs within two of these
chromosomal regions, CDKN2A/ARF (encoding the
alternative products p16INK4a and p14ARF) at 9p21 and
NF2 at 22q12, have been implicated in MM. Homozygous deletions appear to be the major mechanism
a€ecting CDKN2A in MM (Cheng et al., 1994).
Inactivating mutations coupled with allelic loss occur
at the NF2 locus (Bianchi et al., 1995; Cheng et al.,
1999; Sekido et al., 1995).
Recently, we identi®ed a minimal region of chromosomal loss in 15q11.1 ± 15 in MM (Balsara et al., 1999).
In this study, we have re®ned the region of
chromosomal loss, de®ning a 3-cM region between
D15S1007 and ACTC, and the location of the SRO
was con®rmed to be at 15q15 by FISH mapping.
Several studies have reported recurrent deletions of 15q
in various types of cancer. In ovarian carcinoma, 15q
losses have been proposed as a late event observed in
most high-grade and in a subset of low-grade tumors
(Dodson et al., 1993). Wick et al. (1996) reported LOH
Oncogene
15q losses in mesothelioma
A De Rienzo et al
6248
Figure 4 Two-color FISH analysis showing heterozygous loss of
YAC 914-A-2 and retention of both copies of YAC 775-D-2 in
MM case 13. Rhodamine (red) signal: YAC 775-D-2; FITC
(green) signal: YAC 914-A-2. Partial metaphase spread shows
single copy of normal chromosome 15 with signals for both YACs
(arrow) and a deleted chromosome 15 with loss of signal for YAC
914-A-2 (arrowhead). Note that overlapping portions of the
adjacent green and red signals in the normal chromosome 15
appear yellow. FISH was performed using biotin-16-dCTP and
digoxigenin-11-dUTP. Probes were labeled by random ocatamer
priming at 378C for 1 h, followed by ethanol co-precipitation with
Cot1 DNA. Hybridization was carried out on metaphase spreads
from phytohemagglutinin-stimulated normal lymphocytes and
MM cells from case 13, as described previously (Altomare et
al., 1996). Hybridization of biotinylated probes was detected with
FITC-avidin (Oncor), and hybridization of digoxigenin-labeled
probes was detected with anti-digoxigenin rhodamine (Boehringer
Mannheim). Chromosomes were counterstained with 4', 6'diamidino-2-phenylindole. Images of chromosomes and hybridization signals were captured separately using a cooled CCD
camera (Photometrics), pseudo-colored, then merged using
computer imaging software from Oncor
from 15q in 42 of 99 (42%) primary and metastatic
breast carcinomas, with the SRO residing between
D15S231 and D15S641, another 15q15 marker, which
resides between D15S971 and D15S118. The frequency
of 15q loss was signi®cantly higher in metastatic cases.
Deletions of 15q have also been reported in 35% of
parathyroid adenomas, with two common regions
being observed, one at 15q11 ± 21 and another at
15q26-qter (Tahara et al., 1996). Kersemaekers et al.
(1998) described a novel site with frequent LOH on
15q in carcinoma of the uterine cervix. They analysed
only three markers from 15q and concluded that a
novel TSG may be present at 15q21. Allelotype
analysis has also demonstrated recurrent losses of 15q
in small cell lung carcinomas, although only a few
markers on this chromosome were evaluated (Stanton
et al., 2000).
To evaluate the possible existence of colorectal
cancer susceptibility genes, a large family with multiple
colorectal adenomas and carcinomas was investigated
by linkage analysis (Tomlinson et al., 1999). All
a€ected individuals shared a haplotype of marker
alleles in a 40-cM interval de®ned by D15S1031 and
Oncogene
D15S153 at 15q14 ± q22. LOH analysis of sporadic
colorectal tumors identi®ed an SRO at 15q21.1 (Park
et al., 2000). However, the region between D15S1040
and D15S971, where our SRO resides, was not
analysed.
Taken collectively, these investigations suggest the
frequent involvement of one or more TSGs located
between 15q14 and 15q22. Our data show that the
SRO in MM is located at 15q15 between the markers
D15S1007 and ACTC. This region overlaps with sites
frequently deleted in other human malignancies and
represents the smallest SRO in 15q reported to date.
One candidate TSG located at 15q14 ± 15 is the
hRAD51 gene, which encodes a member of the RecA/
Rad51 family of proteins. However, this gene resides
4 cM proximal to our SRO. Moreover, in other
cancers with 15q14 ± 15 LOH, neither hRAD51 mutations nor hypermethylation of the hRAD51 promoter
were observed (Schmutte et al., 1999). To date, no
bona ®de TSG has been mapped to our SRO.
Mutations in the actin alpha cardiac muscle (ACTC)
gene, which ¯anks the SRO identi®ed in MM, are
responsible for dilated cardiomyopathy (Olson et al.,
1998). To investigate whether this gene might be
involved in MM, we performed RT ± PCR on normal
mesothelial cells and on several MM cases displaying
LOH in 15q. No di€erences in expression were
detected (data not shown).
A recent report suggested an association between a
rare connective tissue disorder, Marfan's syndrome,
and MM in two a€ected brothers not exposed to
asbestos (Bisconti et al., 2000). The ®brillin gene
(FBN1), which is responsible for Marfan's syndrome,
maps approximately 5-cM distal from the 15q15 SRO.
However, RT ± PCR analysis revealed similar levels of
expression of this gene in normal mesothelial cells and
MM cases with LOH in 15q (data not shown).
In conclusion, we have demonstrated that genomic
losses from 15q are a common occurrence in MM and
have de®ned a discrete region of deletion involving
markers within 15q15. This region overlaps with sites
frequently deleted in several other types of cancer and
is the smallest site of recurrent 15q loss identi®ed to
date in human tumors. Thus, the identi®cation of a
tumor suppressor locus in 15q may be important in
understanding how 15q contributes to the pathogenesis
of MM and other malignancies, as well.
Acknowledgments
This investigation was supported by National Cancer
Institute Grants CA-45745 and CA-06927, by a gift from
the Local #14 Mesothelioma Fund of the International
Association of Heat and Frost Insulators & Asbestos
Workers in memory of Hank Vaughan and Alice Haas,
and by an appropriation from the Commonwealth of
Pennsylvania.
15q losses in mesothelioma
A De Rienzo et al
6249
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