Keywords: Breast cancer, chromosome 11q, loss of heterozygosity. ..... We are grateful to the Cancer Research Campaign for support. 1 Keldysh PL, Dragani TA, ...
4 Clin Pathol 1995;48:424-428 424
Loss of heterozygosity breast cancer
on
chromosome 11 q in
I P M Tomlinson, J E Stickland, A S G Lee, L Bromley, M F Evans, J Morton,
J O'D McGee
Abstract Aims-Chromosome llq23 seems to be a site of frequent mutation in cancer. It also contains loci such as ataxia telangiectasia with possible importance in the pathogenesis of breast tumours. The short arm of chromosome 11 has been studied extensively in breast cancer, but the long arm, in particular the distal part, has been studied less frequently. Cytogenetic analysis has shown possible involvement of chromosome llq in breast tumours. Chromosome transfer experiments have also implicated chromosome llq in breast cancer. A high frequency of mutations might therefore be expected to occur on chromosome llq in breast cancers. Methods-Using restriction fragment analysis, the primary tumours of 41 patients with breast cancer were screened for mutations at five loci on chromosome llq (DllZl, INT2 (FGF3), DRD2, NCAM, and
DllS29).
Nuffield Department of Pathology and Bacteriology, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DU I P M Tomlinson J E Stickland A S G Lee L Bromley M F Evans J Morton J O'D McGee Correspondence to: Professor JO'D McGee. Accepted for publication 26 September 1994
Results-Allelic loss occurred at a high frequency (59%) at DllS29. At NCAM, novel alleles were frequently seen on autoradiographs. Relatively low frequencies of mutation were detected at the other loci. A11elic loss at DllS29 was associated with the presence of lymph node metastases, but this may be a chance association. Conclusions-The frequency of allelic loss at the DSllS29 locus is high. The significance of novel alleles at NCAM and their relation to allelic loss at DllS29 are unclear. The results presented here do not permit fine mapping of a region of allelic loss, but suggest that the region of greatest loss lies distal to DRD2. The results provide further evidence for the importance of gene(s) near 1lq23 in the pathogenesis of breast cancer, and of tumours in general. (J7 Clin Pathol 1995;48:424-428)
Many cancers have mutations at or near chromosome band 11 q23. Particular examples include haematological malignancies, colorectal tumours, ovarian cancer,' and malignant melanoma.3 Chromosome 1 1 q23 also carries several genes, such as the ataxia telangiectasia (AT) complementation groups and Fanconi anaemia (FA) complementation group D,4 that may be involved in cancer predispostion. In breast cancer chromosome lip has been studied extensively, with mapping of a putative p The tumour suppressor gene to band 1pl5.5.5 long arm of chromosome 11 has been analysed less frequently, especially the distal part. Cytogenetic analysis, however, has shown possible involvement of chromosome 11 q in breast tumours, especially in patients with metastatic disease.i' Chromosome transfer experiments have also implicated 11 q in breast cancer.' We have used restriction fragment analysis to search for mutations in this region of the genome in 41 cases of sporadic breast carcinoma. We report the frequency of mutations at the NCAM, DRD2, INT2 (FGF3), D11S29, ETS1, and DllZl loci (table 1). Each patient's age at presentation was known, and tumour stage, grade and origin were determined histologically. Associations between mutations and the clinical data have also been determined.
Methods
DNA was extracted by standard techniques from the peripheral blood and primary tumours of each patient, providing paired samples of constitutional and tumour DNA. For each locus, 8jtg of each DNA was digested with the restriction endonuclease shown in table 1. Restricted DNAs were electrophoresed on a 0-8-1 -0% agarose gel for 24-48 hours to ensure complete separation of fragments. DNA loading was estimated by visual inspection of ethidium bromide stained agarose gels, or by the Keywords: Breast cancer, chromosome 11 q, loss of use of control probes when studying the INT2 and ETS 1 oncogene loci. DNA was transferred heterozygosity.
Table 1 Loci studied and gene probes used Map position Locus symbol Locus name
11 cen
DllZl
11ql3
INT2 (FGF3) Centromeric repeat Murine mammary tumour virus integration sequence site 2 homologue Probe DllZl SS6 Source Oncor Inc. G. Peters, C. Dickson Restriction enzyme Xbal BamHl Constant bands (kb) None Multiple 8-4 [0-63] Polymorphic bands (kb) Multiple 5-6, 2-8 [0-37] [and reported frequency] Loci shown in map order; kb, kilobase.
llq23 3 11q23 1 l1q23 1 D11S29 DRD2 NCAM Dopamine receptor Neural cell adhesion Anonymous D2 molecule
HD2G1 ATCC Taql 10-5, 2-3 6-6 [0-24] 3-7, 2-9 [0 76]
pMl.3 F. Walsh BamHl 2-4 9-8 [0-77] 6-0, 3-8 [0-23]
L7 ATCC Taql None 13-9 [0-24] 10-9 [0-76]
llq23 3 ETS1 Avian erythroblastosis virus E26 oncogene homologue pHE5.4 ATCC BamHl 0 5, 10-0 None
Loss of heterozygosity on chromosome llq in breast cancer
425
Table 2 Allelic loss, amplification and rearrangement at the six loci studied Heterozygotes Locus
No. cases studied
No.
heterozygotes
Heterozygotes with LOH n(°o/)
DllS29 NCAM DRD2 DIIZI INT2 ETS1
41 36 33 13 34 32
17 14 10 4
10(59) 2(14) 2(20) 1(25)
with
rearrangements
nf(0%)
No. homozygotes
0(0) 3(21) 0(0) 0(0)
24 22 23 9
Homozygotes with
rearrangements
n('o)
Cases with
Cases with rearrangements n(0o)
amplification n(%o)
0(0) 3(14) 1(4) 1(11)-
-9(26) 3
0()
(9)
0(0)
LOH, loss of heterozygosity, allelic loss.
A
N
T
13.9 kb 10.9 kb B(i)
968kb-
N
T
I
6.0kb--
_
B(iii)
BOii)
N
N
T
I
I
,
-4-
4-
t
T _
2-4kb-
2 4kb-
3-8 kb-
3.8 kb-
3.8 kb-
6-0 kb 6 0 kb-
2.4 kb B(v)
24 4k
9-8 kb
4.. N
T
9-8 kbN
b.
..::.
.,£
3.8 kb .o:.
6.0 kb
a_
.%....
9.8 kb-
-
imp
.w
Figure 1 A: Loss of the 10 9 kilobase allele at the D11S29 locus. Sizes of the bands (in kilobases(kb)) are shown. Allelic loss is arrowed. B: Each of the six cases with allelic rearrangements at the NCAM locus. Sizes of the bands (kb) are shown in all cases. Novel alleles are arrowed. (i) Novel allele of about 8 kb in constitutional heterozygote. Note the loss of the 3-8 kb band here, seen very faintly in the tumour, suggesting that the new alele arose from this band by duplication, translocation or loss of a restriction site. (ii) Novel alleles of about 1 5 kb and 5 5 kb in constitutional heterozygote, together with loss of the 3-8kb and 6-Okb bands. It is likely that the new alleles arose from the chromosome carrying the 3 8/60 kb bands. (iii) Novel alleles of about 1 5 kb and 5-0 kb in constitutional heterozygote, together with loss of the 3-8 kb and 6-0 kb bands. Again, it is probable that the new alleles arose from the chromosome carrying the 3 816 0 kb bands. (iv) Novel alleles of about S kb in two constitutional homozygotes and novel allele of about 4 5 kb in another constitutional homozygote (plus a probable further novel allele, slightly larger than the 5 0 kb band and just visible). A normal heterozygote is shown in the centre in order to permit companison between the sizes of the novel alleles seen in the tumours and the normal 6-0 kb and 3-8 kb polymorphic alleles (seen faintly).
to Hybond nylon membranes (Amersham, Little Chalfont, UK) in 25 mM phosphate
buffer and bound by ultraviolet cross-linking
(1200 mj).
Gene probes to each locus of interest were obtained from the sources shown in table 1 and isolated from their vectors. Using the random priming method, probes were labelled with
Tomlinson, Stickland, Lee, Bromley, Evans, Morton,
426
Table 3 Association between LOH at DIIS29 and tumour stage 10 1 11
0 6 6
al
had an allelic rearrangement at NCAM. Patients presented at a mean age of 53 2 years (SD = 11 1). All cases had undergone axillary node sampling: 20 cases were axillary node positive on histopathological grounds, 20 were node negative, while the nodal status of one was unknown. None of the patients had distant metastases. Thirty four patients had tumours of ductal origin: of these, two were scored as grade 1, 17 as grade 2 and 15 as grade 3. Six tumours were of lobular origin and there was one case of ductal carcinoma in situ. Unfortunately, examination of the latter carcinoma did not provide much useful information: it showed no amplification at INT2 and ETS 1, did not show allelic loss at patients
No. patients node + ve No. patients node -ve Total LOH at DllS29 No LOH at DllS29 Total
et
10 7 17
LOH, loss of heterozygosity; X2= 10-1 (Yates' correction), p
