Download Homozygous Deletion Map at 18q21.1 in

[CANCER RESEARCH56, 490-494, February 1. 19961
Advances in Brief
Homozygous Deletion Map at 18q21.1 in Pancreatic Cancer1
Stephan A. Hahn, A T M. Shamsul Hoque, Christopher A. Moskaluk, Luis T da Costa, Mieke Schutte,
Ester
Rozenbium,
Albert
B. Seymour,
Craig
L. Weinstein,
Charles
J- Yeo, Ralph
H. Hruban,
and Scott E. Kern2
DepartmentsofPathologyfS.A.H.,A.T.M.S.H.,
CAM.,
MS., E.R.,A.B.S.,
C.LW.,
R.H.H.,
Graduate Program oflluman Genetics IL T. d. CI. The Johns Hopkins University School ofMedicine,
Abstract
Absolute genetic differences between neoplastic and nonneoplastic cells
can be discerned
at sites of homozygous
deletions.
These deletions are of
critical interest because they might be useful in the identification of
defective biochemical pathways in neoplastic cells, and subsequently for
the development of new treatment strategies in human cancer.
We identified an area at 18q21.1 involved by homozygous deletions in
30% of pancreatic
carcinomas.
To characterize
the homozygous
deletions,
we constructed a detailed physical map of nearly 2 Mb, containing yeast
artificial chromosomes, P1-derived artificial chromosomes, cosmids and
24 sequence-tagged sites. The homozygously
candidate tumor-suppressor
gene (DPC4).
deleted area contained
a new
To date, 23 (64%) of 35 pancreatic carcinomas carry at least one
homozygous
deletion
at a published
locus.
The
study
of the total
gene
content of these loci, facilitated by the sequence-tagged site markers and
maps
of these
regions,
should
help
to reveal
the
absolute
biochemical
S.E.K.J, Oncology(R.H.H.,
Baltimore. Maryland 21205
S.E.K.J,
andSurgeryfC.J.Y.J,
and The
by RDA,3 termed DPC (for deleted in pancreatic carcinoma; loci 1
and 2; Ref. 2). These loci were found to colocalize at I3q12, the
region of the BRCA2 gene. Mapping of this region placed DPC1 and
DPC2 within a single contiguous homozygous deletion spanning less
than 250 kb (3). A third locus identified as homozygously deleted was
at 9q21, targeting the pitS gene (4, 5).
In an allelotype study, chromosome 18 was identified as having one
of the highest rates of allelic loss seen in pancreatic cancer (6). A more
detailed analysis of 18q was initiated with the aim to identify possible
homozygous deletions. A combination of deletion mapping and phys
ical mapping revealed a region at 18q21.l that was homozygously
deleted in 30% of pancreatic carcinomas. Within this region, a novel
candidate tumor suppressor gene, DPC4 (7), was identified. The map
presented here will help to establish the boundaries and gene content
of these common deletions in the DPC4 region.
differences between neoplastic and nonneoplastic cells for a common
human tumor.
Materials
Introduction
pancreas cancers was typed in a PCR-based assay using I I commercially
available microsatellite markers (Research Genetics, Huntsville, AL; Ref. 8;
and Methods
Microsatellite Analysis. GenomicDNA prepared from 31 xenografted
A major goal of tumor biology is to identify the difference between
neoplastic and nonneoplastic cells. One way to ascertain key distinc
tions is through the genetic differences between these cells. Some of
the most interesting types of variance are those involving a complete
absence of genes or their function. Such genetic alterations might
unambiguously indicate an absolute biochemical difference between
neoplastic and nonneoplastic cells. This knowledge might be directly
useful
in the development
of therapeutic
strategies
Fig.
Cancer,
NIH
Krebshilfe (S. A. H.), and JNICI' Scholarship BD1508/9l
McDonnell Foundation Scholar.
2 To whom
requests
for reprints
should
be addressed,
Grant
CA62924,
Fax: (410)
were
Grand Island, NY) with 0.25 ,@MPCR primers and 0.5
amide8 Murea gel, and autoradiographywas performed.
Identification
ofYACs and Pi/PACs.
Microsatellite markers D18S46 and
D18S363 were used in PCR to screen the Généthon
megaYAC library (Re
search Genetics). Additional YACs were identified by hybridization data from
the on-line Infoclone service at Généthon.
The DuPont Merck P1 phage library
(DMPC-HFF#l) was screened (by Genome Systems, SL Louis, MO) using
STSs D18S474 and D18S46. A second and third screen were performed by
hybridization
to human
PAC library
filters (purchased
from Genome
Systems;
Ref. 9) using random primer-labeled PCR products derived from STSs p0960F5, p1210-ClO,
and p128-N21, to gridded PAC library filters.
Preparation of a Region-Specific Cosmid Library. Partial NdeII-di
gested (Boebringer Mannheim, Indianapolis, IN) YAC DNA (average frag
ment size, 50 kb) from the region spanning YACs y747B 11, y9l7C8 and
y899E8 was subcloned into a BamHI-digested and dephosphorylated Super
Cos-I vector (Stratagene, La Jolla, CA), packaged in phage A (MaxPlax;
Epicentre Technologies, Madison, WI), and used to infect MR-l cells (Strat
agene). Colonies with human derived inserts were detected by hybridizing
random primer-labeled human Cot-l DNA (Bio-Rad, Hercules, CA) to filter
lifts. Individual cosmid DNAs (25 ng each) were spotted on Zeta-Probe GT
membranes
(Bio-Rad).
Region-specific
cosmids
were isolated
by hybridization
of end-labeled STS-specific oligonucleotides, and positive clones were con
Deutsche
(L. T. d. C.). S. E. K. is a
at Department
of Oncology,
3 The
The
abbreviations
artificial chromosome;
site.
Johns Hopkins University School of Medicine, Baltimore, MD 21205-2196. Phone: (410)
614-3314;
for microsatellites
5-mn extension at 72°C.The products were separated on a 6.0% polyacryl
Received I 2/28/95; accepted I 2/28/95.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
in Gastrointestinal
18q. PCRs
(Hybaid, Omnigene, Middlesex, UK) using microtiter plates, followed by a
with a high spec
pendent loss of a considerably smaller area.
We are currently engaged in a comprehensive evaluation of ho
mozygous deletions in pancreatic carcinoma. Two loci were identified
SPORE
arm
unit Taq DNA Polymerase (GIBCO-BRL). DNA was amplified for 35 cycles
of 95°Cfor 15 s, 55°Cfor 30 s, and 72°Cfor 30 s in a temperature cycler
two events, the loss of a larger chromosomal region and the inde
by the
for chromosomal
buffer (GIBCO-BRL,
ificity for the tumor cells.
Genetically, the absence of a gene or its function can occur through
biallelic inactivation (1). One mode of biallelic inactivation involves
the combination of an intragenic mutation of one allele, together with
the loss of a relatively large chromosomal region containing the
second allele. This loss of one copy of a chromosomal region or a
gene is called allelic loss or loss of heterozygosity. A second mech
anism for biallelic inactivation is a combination of two inactivating
point mutations targeting a specific gene. A third involves homozy
gous deletions. Homozygous deletions are thought to be the result of
I Supported
1) specific
performed under the following conditions: [email protected] volume; 1.5 mM
MgCI2; 2.0% (v/v) DMSO; 200 iM dATP, dGTP, and dTFP; 5.0 @tM
dCFP,
0.2 @tl[a-32P]dCTP (NEN DuPont: 800 Ci/mmol, 10 @aCi/pi)in a 1X PCR
614-0671.
used
are:
RDA,
representational
difference
PAC, P1-derived artificial chromosome;
analysis;
YAC,
yeast
STS, sequence-tagged
490
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ABSOLUTE DIFFERENCES IN PANCREATIC CANCERS
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Fig. I . Chromosome 18 deletion map. •,homozygous deletion;
xenograft, followed by an identification number.
firmed
by PCR. Restriction
digest
analysis
indicated
@,
loss of heterozygosity; El, retention of heterozygosity; empty spaces, uninformative marker loci. PX, pancreatic
an average
insert size of
30 kb.
Generation of STSs. YAC ends were isolated by an inverse PCR tech
nique
( 10) using
a panel
of 5 restriction
endonucleases
(NlaIII,
BstUI,
EcoOlO9l, HaeIII, TaqI, and BanII; New England Biolabs, Beverly, MA).
Once amplified, the ligation fragments were sequenced by cycle sequencing
(SequiTerm; Epicentre) and 20-mer oligonucleotide pairs for STSs were de
signed.
P1/PAC
(Sequilerm;
end sequences
Epicentre)
were
generated
or by a PCR-based
either
by direct
sequencing
amplification
technique
using slow
ramping of the annealing temperature (1l).@Selected cosmid insert ends were
sequenced
(SequiTerm;
Epicentre)
The localization to chromosome
using primers
specific
for vector sequences.
18 of all STSs was ensured by PCR analysis
of monochromosomal somatic cell hybrid DNA (NIGMS mapping panel 2,
Coriell Cell Repositories). Suspected chimeric YACs were excluded from the
contig
based
on these
data
and the hybridization
data
from
the Généthon
database. The primer sequences are given in Table 1.
Homozygous Deletion Mapping by STSs. STSs were amplifiedusing 40
ng of genomic
DNA
in 67 mtvi Tris-HCI
(pH 8.8); 4 mM MgC12;
16 mM
(NH4)2S04; 10 mM2-mercaptoethanol; 100 g/ml bovine serum albumin; 200
M each dATP,
polymerase
dCTP,
dGTP,
(GIBCO-BRL)
and dTFP;
1 j.LMeach primer;
in a final reaction
was added after a preheating
volume
and 2 units of Taq
of 15 M1, The enzyme
step of 2 mm at 94°C. Thirty
30 s, 58°C for 1 mm, and 72°C for 1 mm were followed
cycles
of 94°C for
by a final extension
of 5 mm at 72°C.A homozygous deletion was defined as the absence of a PCR
product
from a carcinoma
DNA template,
when compared
to a strong product
from the paired constitutional
DNA template from the same patient. All PCR
reactions were repeated at least three times and confirmed by a second primer
pair designed
on nearby
sequences
to exclude
the possibility
of a primer
site
polymorphism. The quality of the DNA was further assured by the successful
amplification
of a I .8-kb fragment
sets for microsatellite
(exons 5—9ofp53)
and of numerous
primer
markers.
Results
Allelic Loss Analysis and the Identification of Homozygous
Deletions. Allelic loss at 18q was identified in 28 (91 %) of 31
pancreatic cancers (Fig. I ). The smallest consensus of allelic boss
4 L. T.
da
Costa,
unpublished
among the 31 cases mapped between markers D18S364 and D18S68.
Two markers, D18S46 and D18S363, were homozygously deleted in
four pancreatic cancer xenografts (PX16, PX61, PX92, and PX94).
The homozygous deletions were confirmed by multiplex PCR and by
Southern blot analysis (data not shown). Marker D18S46 had been
localized centromeric to DCC by radiation hybrid mapping (12),
whereas marker D18S363 was not unambiguously placed in relation
to D18S46 nor to DCC at that time. The microsatellite marker DCC
within the DCC gene (at nucleotide 1432; Ref. 13) was not homozy
gously deleted in any of the cases. To conclusively exclude the DCC
region, we further analyzed the four cases having a homozygous
deletion with markers SSAV, D18S523, D18S526, and DJ8SJOJ, all
known to map centromenc to DCC (12) and tebomeric to D8S46. All
four cases were shown not to involve the DCC region, confirming the
localization of the new locus centromeric to DCC. The locus was
termed DPC4 for deleted in pancreatic carcinoma, locus 4.
Isolation and Analysis of YAC Clones from the DPC4 Region.
To generate a physical map from the DPC4 region, the Géndthon
megaYAC library was screened with markers D18S46 and D18S363.
Seven YAC clones were identified (y957Bl 1, y747El, y953Gl2,
y945B 11, y9l7C8, y899E8, and y747A6). All YACs except y953Gl2
and y747El were positive for both markers, establishing the proximity
of the markers D18S46 and D18S363. Two additional linking YACs
(y779Al0 and y790A2) were identified from the Généthon
on-line
database (Infoclone). The initial physical map comprised the nine
YAC-end STSs, microsatellite markers mapping to these YACs as
given in the Généthon
database (D18S474 and D18S479), and the
above-mentioned markers localizing centromeric to DCC. The com
posite YAC contig map is shown in Fig. 2. YAC y747El appeared to
be chimenc because neither of its end sequences mapped to chromo
some 18. The centromeric end of YAC y953Gl2 similarly could not
be placed on chromosome 18. The YACs y779A10, y790A2, and
y747A6 were positive for marker SSAV but were negative for addi
tional markers mapping between the SSAV locus and the DCC locus
(D18S523,
observations.
D18S526,
DJ8SJOJ),
thus establishing
the telomeric
491
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ABSOLUTE DIFFERENCES IN PANCREATIC CANCERS
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Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1996 American Association for Cancer Research.
ABSOLUTE DIFFERENCES IN PANCREATIC CANCERS
STSsPrimer
SizePrimer
(L@,p)y945Bl
IDfLocus0
Table 1
sequence sense
(5'3')
AAT
TGA
CAT
GCc
AGA
CAG
TTG
TGG
111y945Bl
lR
123y9l7C8R
1L
150y917C8L
141y899E8R
GAG
AAT
GCA
AGG
136y899E8L
ATT GGT TTC
139y747A6R
ATT ATG GTG GTT TAA AGA CAT G
GTG CAT AAT GCC GAA TGT TC
103y747A6L
CAT
TAR
137y953G12R
AAA TCA GTT GTA TTT CTA TTC AC
CTC
ATT
AGG
GCA
GAT
CAG
ATG
GCA
TCT
TAT
GTG
AGT
ATC
GAA
Primer sequence antisense
(5'—3')
A@
AG
TAG
AG
AGT
ATG
CTC
ATG
@ccAGA GTT TTG
TGA
ATT
TGA
GAA
TAC CCT
TGT
@cc
@TT GCC
CAG GCC
GTT
CAG
TTA
CTT
GTG
AAG
AAG
c@c
CTA
GAA
AAT
AGA
TC
AG
CAG
c
TTG TTC CTC TCT CAT GAT TTG
AG
GAT
CTT
TTC
ATT
GGA
TTA
TG
TCT TGA CTT TTT CAG AAG TGT TG
180p263-ElS
GTG GTC TGG AGA GTC TAA AC
TAC CTT GGC TGC CAA ACA TC
96p1210-ClO
ATG
GGC
TTA
TAA
CTG
TGA
TAG
CTT
ACA
ACA
ATG
CTA
GTA
AGA
88pl28-N21
161p224-J22
102p313-N14
84p0630-H5-SP6
122p063O-H5-T7
93p0960-F5
TCC
GCT
CTG
TTA
CCC
CCG
CCT
TTG
CAG
TAC
TGC
TAC
TCA
TAA
CTC
CAA
AAC
CTG
CAG
CTT
TGT
TTA
ACA
GCC
CTA
GCT
GAG
TGG
CAA
AGA
GCA
TTG
ATG
GAA
TTT
AGT
AG
ATT C
TCA
CTG
TCA
AAT
GAG
ATG
TTT
GCT
CTC
TAA
GGC
ACA
TCG
AAA
CTT
AAT
AGG
TGA
CAC
CTT
ACT
TCA
AlA
TGG
AGG
CCC
AGC
CAA
GGA
TGA
CAA
AAA
TAT
CAA
ART
TGA
AC
TG
G
TAG TG
AC
TTT G
121c917-46-T3
TCC
CAA
AGT
GCT
GGG
ATT
TC
GTG
AGT
TGC
TOG
GAT
TAG
AG
GTT
GTC
TGA
TGG
GAA
GCT
AAT
TGT
AGA
ATA
CTG
CAC
ACT
GGT
TAG
TCA
GCC
CTG
AAC
CAT
CAG
GTA
AAT
GTT
CAG
TTC
CAG
AGC
ACC
GGC
TGA
TCC
AGC
TAC
GAC
TCT
CAG
ATT
TTG
TTT
AAT
ATC
CAA
CAG
CTA
CAC
AGT
GTG
CTG
CCT
TGG
AAC
GAA
CAT
TCA
TGG
G
CAT
GAT
GGG
TG
AG
CT
C
AAG AGC
AT
ACA
CTT
AAT
TGG
CAG
TTG
AGT
AAG
CAG
CCT
GCA
ACT
CTA
CTT
ATT
GGA
GGT
CCT
TTT
CCG
GTT
TTG
CCT
TCA
AAG
ACT
TTG
CCC
CTG
CTG
TGG
AAA
TGC
ATG
TGA
GCT
ACA
ATC
TCT
TGA
GAC
TGG
TAG
TCA
AAA
CTA
GAG
CAA
GCT
CGG
AAG
ATG
GAA
GAA
CAG
CAT
AGT
TGT
AGG
TGT
TG
TTC AG
T
GG
CAT ATG TG
TC
GC
TC
AG
CCA
YAC
derived
63c917-46-T7
86D18S6
900D18S474
19-139D18S46
129-153D18S363
177-247D18S479
294—304DPC4
201DPC4
exonl
262SSAV
exoni 1
430a
and“c―
DI8S
markers
and
SSAV
marker
are
described
in Refs.
12,
8, and
19,
ACA G
AC
C
AG
TG
CAG
respectively.
Suffixes
used
for
markers
were
“y―
for
markers,
I
“p―
for
P1/PAC
markers
2.derfor cosmid markers. Physical localization of all markers is shown in Fig.
of the contig. The depth of the contig at the region of interest was
five(p224-J22).Isolation
YACs (excluding chimeric YAC y747El).
and Analysis of P1/PAC Clones from the DPC4 Re-
consensus of deletion were, thus, P224-J22 and p1210-ClO, narrow
ing the consensus to the size of one PAC
Isolation of Cosmids from the DPC4 Region and Gene Identi
wereof
gion. All STSs from the YAC contig
fication.
were applied
to map the extent
FourFurther,
the four identified homozygous deletions relative to the contig.
2).homozygous
the markers within and closest to the consensus region of
retainedtumors deletions were used to screen an “extended―
panel of
c9l7-46.nomas,
(41 xenografts derived from primary pancreatic adenocarciallxenografts
10 pancreatic cell lines, 22 breast cancer cell lines, and
consensuscers).
of 4 primary biliary cancers and 2 primary bladder canofcentromeric
An additional 10 homozygous deletions were identified. The
ofdefined
end of the consensus of homozygous deletions was now
a2).
by marker D18S474 and the telomeric end by D18S46 (Fig.
(7).the
These two STSs were used to initiate a P1 walk from both sites of
isp0630-H5,
contig. Seven P1 clones were identified (p129-G19, p263-E15,
thePls
p128-N2l, p1 l53-D6, p1210-ClO, p0960-F5). Selected
andmapping
were used to generate eight end sequence-specific STSs. The
of these STSs to the contig and another round of tumor
panela
screening identified STS pl2lO-Cl0 and p0960-F5 as the borders of
deletionswith
new consensus of homozygous deletions. A second walk performed
ofestablishing
these STSs found two linking PACs (p224-J22 and p313-N14)
beD18S46.
a complete contig between markers D18S474 and
thecoverage
A third PAC library screen was performed to increase the
1.N14
at the area of the link between PACs p224-J22 and p313and to exclude large interstitial deletions (cloning artifacts) as the
reason
homozygouslywere
for the link. Two additional PACs (p103-K3 and p227-K7)
atSTSsthus identified, confirming the contig. End sequence-specific
were again used to screen the tumor panel. One STS (p128-N21)
was found to be deleted in all cases. The markers flanking the new
These three STSs (p128-N2l,
p224-J22,
and pl2l0-C1O)
used to screen filters of the cosmid mini-library of the region.
cosmids were found, mapped, and used to generate new STSs (Fig.
The STS specific for the telomeric end of cosmid c917-46 was
in PX9 and PX19. PX1 15 retained the centromeric end STS of
The 5Th p128-N2l, located within cosmid c917-46, was deleted in
cases including the three mentioned above, thus localizing the
ofhomozygous cDNA deletion to this cosmid. Through a combination
exon amplification, cDNA library screening, 5' rapid amplification
cDNA ends and BLAST thtabase searches of dbEST, we identified
novel candidate tumor suppressor gene, DPC4, on cosmid c917-46
STS c917-46-T7 is located in exon 8 of DPC4 and STS p128-N21
located within an intron ofthe DPC4 gene 5' to exon 7. The study of
ESTs derived from DPC4 exons 1 and 11, and STS c9l7-46-T7
p128-N21, identified 25 (30%) of 84 pancreatic carcinomas (41 pancre
atic xenografts and 10 pancreatic cancer cell lines of the extended
and 33 additional pancreatic xenografts) to have homozygous
involving the DPC4 gene. The identification and sequence analysis
DPC4 is detailed in a separate paper (7). Together, 24 STSs could
unambiguously ordered within the DPC4 region. A complete list of
515 primer sequences is given in Table
Summarized Homozygous Deletion StatUs in Pancreatic Carci
nomas. There are now three published loci known to be
deleted in pancreatic carcinoma, DPC1,2 at 13q12 (2), p16 (DPC3)
9p21 (5), and DPC4 at 18q21.l (7). In a series of 36 pancreatic carcino
mas studied for all three loci, thepl6 and DPC4 loci revealed the highest
Fig. 2. Physical map and homozygous deletion boundaries of the DPC4 region at chromosome 18q2l.l. Shaded area, DPC4 gene region. Physical map: the STSs, including DJ8S
markers, are positioned based on the data from the YAC, Pi/PAC, and cosmid clones and the mapping data from the homozygous deletions in pancreatic carcinomas. Sizes of clones
are not in scale, and the relative distance of the STS markers is arbitrary, reflecting relative position. Small vertical ticks, on the clones, presence of the corresponding STS. STS content
of YAC and P1/PAC clones was tested only for selected markers. chim, chimeric YAC; grey shaded YAC ends, chimeric YAC clone ends. Deletion map:
, areas without
homozygous deletion, all corresponding markers of the map being present; —
—
—
—,area of homozygous deletion. cen, direction to centromere; tel, direction to telomere. PX, pancreatic
cancer xenografts, except PX1 15 is a carcinoma of the distal common bile duct arising in the pancreas, and CFPAC1 and HS766T are pancreatic carcinoma cell lines. Only cases that
were important to the mapping of STSs or for the definition of the consensus of homozygous deletions are shown. For STS primer sequences, refer to Table I.
493
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ABSOLUTE DIFFERENCES IN PANCREATIC CANCERS
7). Homozygous deletions at any of the three loci were identified in 23
(64%) of 36 pancreatic cancers (Table 2).
fore, represents the biochemical difference of certain tumor cells. Indeed,
the total absence of functional copies of a gene contained within a
homozygous deletion might provide a more tumor-specific approach to
chemotherapy through the use of agents that are toxic to the tumor cells
while remaining nontoxic to normal cells. The here-presented regional
map provides a second significant region of frequent homozygous debe
tions in which to search for tumor-specific therapeutic targets. Together,
64% of pancreatic carcinomas are known to harbor a homozygous
deletion at either thepl6 or DPC4 loci. The search for additional hotspots
of homozygous deletions, and the characterization of their gene contents,
should aid translational applications of molecular genetics to patient care.
Discussion
References
carcinomas―One
Table 2 Homozygous deletions in pancreatic
(44%)Two
known homozygous deletion16
known homozygous deletions7
(19%)One
or more homozygous deletions23
36(total)
a Summarized
are 36 pancreatic
carcinomas
(64%) of
studied
for homozygous
deletions
at three
loci: DPCJ,2IBRCA2, pitS gene (9p2l), and DPC4 (18q21.l).
frequencies
of homozygous
deletions
(42 and 39%, respectively;
Refs. 4,
This report presents an example of a directed search for homozygous
deletions, with the goal to identify candidate loci for tumor suppressor
genes. The search was performed on a chromosomal arm known to have
a high frequency of allelic loss in pancreatic cancer. A locus of frequent
homozygous deletion, termed DPC4, was identified at 18q21.l. From
this locus, an integrated high-resolution physical map was constructed.
The map of this region, described here, contains 24 STSs derived from
YAC, P1/PAC, and cosmid end sequences, including 4 microsatellites.
The STSs were localized in an unambiguous order over an approximately
2-Mb region. The identification of a novel candidate tumor suppressor
gene, DPC4, within this region is reported elsewhere (7).
Although the initial purpose of the physical map is fulifiled, the
generated STSs and their localizations are important for the definition of
the boundaries and gene content of the homozygous deletions in pancre
atic carcinoma and other tumor types. Homozygous deletions inactivate
the targeted tumor suppressor genes, as well as neighboring genes within
the deleted region, resulting in clones of cells lacking functional copies of
these genes. In other words, homozygous deletions and inactivated tumor
suppressor
genes
might
help to establish
some
absolute
biochemical
differences (except in the cases of redundant protein function) between
neoplastic
and nonneoplastic
cells. The knowledge
of these differences
in
various cancer types could be instrumental in devising therapeutic strat
egies that are highly specific for the neoplastic cells.
The general applicability of this approach in human tumors will
eventually
depend on the actual frequency
of homozygous
deletions
and
chromosomal
region, is the technique
A.
Hahn,
S.
E.
Kern,
and
0.
Olopade,
unpublished
of a homozygous deletion in pancreatic carcinoma that lies within the BRCA2 region.
Proc. Natl. Acad. Sci. USA, 92: 5950—5954, 1995.
3. Schutte, M., Rozenblum, E., Moskaluk, C. A., Guan, X., Hoque, A. T. M. S., Hahn,
S. A., da Costa, L. T., de Jong, P. J., and Kern, S. E. An integrated high-resolution
physical map of the DPC/BRCA2 region at chromosome 13ql2. Cancer Res., 55:
4570—4574, 1995.
4. Caldas, C., Hahn, S. A., da Costa, L. T., Redston, M. S., Schuue, M., Seymour, A. B.,
Weinstein, C. L., Hruban, R. H., Yeo, C. J., and Kern, S. E. Frequent somatic
mutations and homozygous deletions of the pitS (MTSI) gene in pancreatic adeno
carcinoma. Nat. Genet., 8: 27—32,1994.
5. Kamb, A., Gruis, N. A., Weaver-Feldhaus, J., Liu, Q. Y., Harshman, K., Tavtigian,
S. V., Stockert, E., Day, R. S., Johnson, B. E., and Skolnick, M. H. A cell cycle
regulator potentially involved in genesis of many tumor types. Science (Washington
DC), 264: 436—440,1994.
6. Hahn, S. A., Seymour, A. B., Hoque, A. T. M. S., Schutte, M., da Costa, L. T., Reston,
M. S., Caldas, C., Weinstein, C. L., Fischer, A., Yeo, C. J., Hruban, R. H., and Kern,
S. E. Allelotype of pancreatic adenocarcinoma using xenograft enrichment. Cancer
Res., 55: 4670—4675, 1995.
7. Hahn, S. A., Schuue, M., Hoque, A. T. M. S., Moskaluk, C. A., da Costa, L. T.,
Rozenblum, E., Weinstein, C. L., Fischer, A., Yeo, C., Hruban, R. H., and Kern, S. E.
DPC4. a candidate tumor-suppressor gene at 18q21 .1. Science (Washington DC), in
press, 1996.
8. Weber, J. L., and May, P. E. Abundant class of human DNA polymorphisms which
can be typed using the polymerase chain reaction. Am. J. Hum. Genet., 44: 388—396,
1989.
9. Ioannou, P. A., Amemiya, T., Games, J., Kroisel, P. M., Hiroaki, S., Chen, C., Batzer,
M. A., and de Jong, P. J. A new bacteriophage P1- derived vector for the propagation
of large human DNA fragments. Nat. Genet., 6: 84—89, 1994.
10. Silverman, G. A. Isolating vector-insert junctions from yeast artificial chromosomes.
11. Liu, Y-G., and Whittier, R. F. Thermal asymetric interlaced PCR: automatable
amplification and sequencing of insert end fragments from P1 and YAC clones for
chromosomal walking. Genomics, 25: 674—681, 1995.
12. Francke, U., Chang, E., Comeau, K., Geigl, E-M., Giacalone, J., Li, X., Luna, J.,
Moon, S., Welch, S., and Wilgenbus, P. A radiation hybrid map of human chromo
some 18. Cytogenet. Cell Genet., 66: 196—213,1994.
13. Risinger, J. I., and Boyd, J. Dinucleotide repeat polymorphism in the human DCC
gene at chromosome l8q21. Hum. Mol. Genet., 1: 657, 1992.
14. Dryja, T. P., Rapaport, J. M., Joyce, J. M., and Peterson, R. A. Molecular detection
of deletions involving band q14 of chromosome 13 in retinoblastoma. Proc. Nail.
Acad. Sci. USA, 83: 7391—7394, 1986.
15. Fearon, E. R., Cho, K. R., Nigro, J. M., Kern, S. E., Simons, J. W., Ruppert, J. M.,
Hamilton, S. R., Preisinger, A. C., Thomas, G., Kinzler, K. W., and Vogelstein, B.
Identification of a chromosome l8q gene that is altered in colorectal cancers. Science
(Washington DC), 247: 49—56,1990.
16. Lisitsyn, Ni., Lisitsyn, Na., and Wigler, M. Cloning the difference between two
complex genomes. Science (Washington DC), 259: 946—951, 1993.
17. Lisitsyn, N. A., Lisitsina, N. M., Dalbagni, G., Barker, P., Sanchez, C. A., Gnarra, J.,
Linehan, W. M., Reid, B. J., and Wigler, M. H. Comparative genomic analysis of
tumors: detection of DNA losses and amplification. Proc. NatI. Acad. Sci. USA, 92:
of RDA (16). A num
ber of candidate loci have been identified to date by RDA, including one
that aided the localization of the BRCA2 gene (2, 17).
An example of a potential therapeutic target is the MTAP (methyl
thioadenosine phosphorylase) gene, localized near the p16 gene locus
at 9p2l (18). The pitS locus is a hotspot of homozygous deletions in
various tumor types. It is homozygously deleted in 40% of pancreatic
cancers (4), and one-half of these deletions include the MTAP gene.5 The
absence versus presence of this purine salvage pathway member, there
5 5.
Hruban, R. H., and Kem, S. E. Identification by representational difference analysis
PCR Methods AppI., 3: 141—150,
1993.
the effectiveness of strategies to identify them. Our knowledge of the
frequency of homozygous deletions is very limited. They generally
involve small regions of a chromosome, and probably most deletions are
smaller than 2 Mb in size. Two techniques are currently available for
direct searches of homozygous deletions. One technique is the screening
of a preselected chromosomal area with markers (used in this study). The
dramatic increase of the number of STSs assigned to individual human
chromosomes in recent years, makes this approach increasingly procluc
tive. Examples for the successful application of this technique are the
discovery of the RBJ, DCC, p16, and DPC4 genes (5, 14, 15). An elegant
way to detect homozygous deletions, without the prior limitation of a
preselected
1. Knudson, J. A. G. Hereditary cancer, oncogenes, and antioncogenes. Cancer Res., 45:
1437—1443,1985.
2. Schutte, M., da Costa, L. T., Hahn, S. A., Moskaluk, C., Hoque, A. T. M. S.,
Rozenblum, E., Weinstein, C. L., Bittner, M., Melzer, P. 5., Trent, J. M., Yeo, C. J.,
151—155,
1995.
18. Olopade, 0. I., Pomykala, H. M., Hagos, F., Sveen, L. W., Espinosa, R., III, Dreyling,
M. H., Gursky, S., Stadler, W. M., La Beau, M. M., and Bohlander, S. K. Construc
tion of a 2.8-megabase yeast artificial chromosome contig and cloning of the human
methylthioadenosine phosphorylase gene from the tumor suppressor region on 9p2l.
Proc. NaIl. Acad. Sci. USA, 92: 6489—6493, 1995.
19. Gyapay, G., Morisette, J., Vignal, A., Dib, C., Fizames, C., Millasseau, P., Marc, S.,
Bernardi, G., Lathorp, M., and Weissenbach, J. The 1993—1994Généthon
human
genetic linkage map. Nat. Genet.. 7: 246—249, 1994.
data.
494
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Homozygous Deletion Map at 18q21.1 in Pancreatic Cancer
Stephan A. Hahn, A. T. M. Shamsul Hoque, Christopher A. Moskaluk, et al.
Cancer Res 1996;56:490-494.
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