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Chromosome Abnormalities in Bilateral Breast Carcinomas Cytogenetic Evaluation of the Clonal Origin of Multiple Primary Tumors
Nikos Pandis, Ph.D.,*,t Manuel R. Teixeira, M. D.,*'$ Anne-Marie Gerdes, M.D.,* Janusz Limon, M.D.,t Georgia Bardi, Ph.D.,*,t Johan A. Andersen, M.D.,§ Ingrid Idvall, M.D., 11 Nils Mandahl, Ph.D.,t Felix Mitelman, M.D.,t and Sverre Heim, M.D.*,t$
Background. Although acquired somatic mutations presumably are crucial in carcinogenesis, nothing is known about the chromosome aberrations of bilateral breast carcinomas.
Methods. Eighteen specimens from 16 bilateral carci- nomas were analyzed cytogenetically. The banding anal- ysis was supplemented with fluorescence in situ hybrid- ization with painting probes.
Results. In two cases, the finding of the same clonal abnormalities in samples from both breasts indicated that the bilaterality had arisen through a metastatic pro- cess. In the remaining cases, the absence of similarities between the two sides indicated an independent origin of the two carcinomas. Also, in multifocal lesions within the same breast, examples were found both of karyotypi- cally related and unrelated clones. Altogether, multiple clones without similarities were detected in nine speci- mens, sometimes together with other, karyotypically re- lated clones. There was no indication that bilateral carci- nomas of the breast are cytogenetically different from unilateral ones. The following chromosomal abnormali- ties were recurrent: der(l;l6) (qlO;plO), del(1) (qll-n12), del(1) (q42), and del(3) (plZ-nl3pl4-nZ1).
From the Departments of *Medical Genetics and 5Pathology, Odense University and University Hospital, Odense, Denmark; the Departments of tClinica1 Genetics and 11 Clinical Pathology, Univer- sity Hospital, Lund, Sweden; and the $Department of Genetics, The Norwegian Radium Hospital and Institute for Cancer Research, Oslo, Norway.
Supported by grants from the Danish, Swedish, and Norwegian Cancer Societies. Drs. Pandis and Bardi are on leave from the Papan- ikolaou Research Institute, Athens, Greece. Dr. Limon is on leave from the Department of Biology and Genetics, Medical Academy, Gdansk, Poland.
Address for reprints: Nikos Pandis, Ph.D., Department of Clini- cal Genetics, Lund University Hospital, 5-221 85 Lund, Sweden.
Received December 21, 1994; revision received February 21, 1995; accepted March 22, 1995.
Conclusions. Bilateral breast carcinomas have the same cytogenetic aberrations, including evidence of poly- clonality, as unilateral carcinomas. The majority appar- ently arise independently, but some result from a metas- tasis from one breast to the other. In this sense, bilateral breast carcinomas are similar to multifocal breast cancer in general, of which bilateral tumors may represent a spe- cial case. Cancer 1995; 76:250-8.
Key words: cancer, carcinoma, breast, bilateral, cytoge- netics, chromosome, karyotype.
Breast cancers may show a wide range of behaviors. Al- though an explanation for this variability based solely on cellular features would be simplistic, any quest for a more profound understanding of the disease process, including reliable prognostication based on causally im- portant parameters, must involve the acquisition of new knowledge about the inherent differences among tu- mors and tumor cell subpopulations. A central step to- ward this aim is to characterize as accurately as possible the acquired genetic changes that presumably drive the neoplastic process at the chromosomal and DNA levels.
Bilateral breast cancer is relatively common. It may be synchronous or metachronous; the risk of develop- ing a carcinoma in the contralateral breast in the decade after treatment for a first tumor has been estimated at 5- lo%.' One challenge in this context is to predict which patients will develop bilateral carcinomas. Another question, no less intriguing both scientifically and for its clinical implications, is whether one is dealing with two separate primary tumors or the metastasis from one breast to the other.
Many clinicopathologic studies during the last three decades have addressed the bilateral breast cancer
Cytogenetics of Bilateral Breast Cancer/Pandis et aI. 251
problem,',' but none have investigated it using cytoge- netic methods. Among the reasons for the absence of data are the difficulties that have been experienced when attempting to initiate and analyze short term cul- tures from breast turn or^;^,^ the risk of failure multiplies when more than one sample from the same case must be evaluated to obtain informative results. In the cur- rent study we present the first characterization, to the best of our knowledge, of the karyotypic profile of bi- lateral breast carcinomas.
Patient 3
Bilateral breast tumors were detected in this 59-year-old woman. In the right breast, the tumor was a 10-mm, highly differentiated ductal carcinoma (197/92). The cancerous pro- cess in the left breast was multifocal with the two largest foci, both of which exhibited histologic features corresponding to a highly differentiated ductal carcinoma, measuring 10 mm (198/92) and 8 mm. The other foci showed ductal carcinoma in situ (DCIS).
Patient 4 Material and Methods
Sixteen breast carcinomas from eight patients (two from each) were studied. Informed consent was obtained from all of the participants. The samples (the numbers in parentheses in the case reports below refer to sample identification; see Table 1 for corresponding findings) included all bilateral cases successfully analyzed by us from 1990 to 1994. Two and three samples, respec- tively, were taken from clearly separate, macroscopi- cally distinct carcinomatous foci in the left breast in Pa- tients 5 and 7; otherwise, one tumor sample was ob- tained from each breast. All tumors were histopathologically classified in accordance with World Health Organization recommendation^.^ None of the patients belonged to recognized cancer families.
Case Reports
Patient 1
When this woman was 54 years old, she had surgery for right- sided breast cancer (2252/90). Two tumor nodules with the histologic appearance of poorly differentiated ductal carci- noma were found; they measured 20 mm and 35 mm. A pro- nounced intraductal comedo-type tumor component was present within the infiltrating carcinoma.
One year later, cancer was diagnosed in the remaining left breast (1081/91). Again, two foci of poorly differentiated ductal carcinoma were found, both measuring 10 mm in di- ameter. Similar to what was seen in the right-sided breast tu- mor, a prominent intraductal carcinoma component was pres- ent among the areas showing infiltrating growth.
Patient 2
Bilateral, synchronous breast cancer was diagnosed in this 55- year-old woman. In the right breast, a 20-mm in diameter, poorly differentiated ductal carcinoma (669/91) was detected. The left breast contained a 100-mm tumor (668/91) with sim- ilar microscopic features to those seen in the contralateral car- cinoma.
A 57-year-old woman presented with retraction of the right nipple. A 54-mm unifocal, infiltrating lobular carcinoma was found (308/92); the tumor also showed in situ carcinoma growth. In her left breast, a unifocal, highly differentiated, 35- mm ductal carcinoma was found (307/92). Areas with DCIS were present within the infiltrating tumor as well as in other areas of the breast.
Patient 5
A 71-year-old woman had a mammography for diagnosis of what was thought to be fibrocystic breast disease. In the right breast, a 16-mm lobular carcinoma (376/92) with one small focus of lobular carcinoma in situ (LCIS) within the infiltrating tumor was found. The breast also contained numerous areas with DCIS and LCIS outside the main tumor. In the left breast, an 8-mm in diameter infiltrating lobular carcinoma (375A/ 92) was found with LCIS in the tumor area. In addition, one multifocal and three multicentric lobular carcinomas, the largest being 12 mm in diameter (375B/92), were found, as were widespread areas of DCIS and LCIS.
Patient 6
A 60-year-old woman had felt a lump in her right breast. The tumor was shown to be a 13-mm highly differentiated, unifo- cal ductal carcinoma (383/92) with sporadic DCIS formations within the tumor and widespread LCIS outside. In the left breast, a 30-mm highly differentiated ductal carcinoma (382/ 92) was found, with extensive DCIS both within and outside the tumor. LCIS was also seen in many areas.
Patient 7
An 88-year-old woman with senile dementia presented with an ulcerating left-sided breast cancer. The 60-mm tumor con- sisted of a central 15-mm nodular area (50A/93) surrounded by more ordinary-looking carcinoma tissue (50B/93). In addi- tion, five multicentric carcinomas, from 4 to 7 mm (50C/93) in diameter, were detected. All were ductal carcinomas with DCIS in some areas within the tumor. The largest carcinoma was clearly the least differentiated. In the right breast, a uni- focal 60-mm lobular carcinoma (51/93) was detected. Areas
Tab
le 1. C
lonc
al C
ytog
enet
ic A
berr
atio
ns in
Eig
ht B
ilate
ral B
reas
t Car
cino
mas
Pati
ent no.
Lab
. no
Clo
ne
Lef
t bre
ast
Lab
. no
Clo
ne
Rig
ht b
reas
t
1 10
81/9
1
2 66
8/91
3 19
8/92
4
307/
92
5 37
5A/9
2
3758
/92
la
lb
lc
Id
1e
2 3 4 5 6 1 1 1 la
2 3 4 5 6 7 3 8
S2-6
Z,X
,-X,+
add(
l)(
pl3)
drl(
l)(q
42),
+dt.
I( l)
(q24
)~2,
del(
l)
(q32
), dc
r( I)
del(
l)(q
32)t
( l15)
(p36
. ql3)
add(
3)(q
33),
~2,+
2,de
r(3)
t(3,
15)
(q24
;q21
).-4,
der(
4)1(
4; 1
4)(q
33,q
24)a
dd(4
)(p1
6),+
5,t(
6; 1
2)(q
23;q
13),
der(7)t(4;7)(q27.q32),+1(8)(qlO),-9,-IO.-lO,del(l l
)(pl
lp13
).
add(
13)(p13)add(l3)(q32),der(13)t(3:13)(p13,
pl2)
,der
( 14)
t(3;
14)
(pl2
), 1
3- 1
3mar
(cpR
] 52-62,1drm,-add(l)(pl3)del(l)(q42).
drr(
7)t(
4:7)
(q27
,q32
),+d
rr(7
) (4
qtrr
+4q2
7..7
q32-
~ct~
n-7p
l5::
7q11
-7y3
2:.~
q27-
4qt~
~r)[
cpl2
] 52
-62,
idrm
.- ad
d( 1 )
(PI 3
)del
(l)(
q42)
, - dr
r(7)
t(4,
7)(q
27;q
32),
+der
(7)
(4qt
rr+4
y27:
.7q3
2~ct
~r1+
7~15
::7q
11+7
q32:
:4q2
7+4q
tcr)
, T
add(
16)(
q13)
[cp3
] 52
- 62,idc.m,--add(l)(pl3)de1(1)(qJ2).-drr(7)t(4;7)(y27;q32),
r drr
(7)
(4qt
rr-+
4y27
..7q
32~r
i~n~
7pl5
:.7q
ll+7
q32:
.4q2
7-4q
tc.r
),
-add
(l6)
(pl3
).+r
[cp6
] 10
4-1
24,1
dt*m
x2,-a
dd( l)
(pl3
)drl
( l)(q
42)~
2, dc
~(7)
t(4:
7)(q
27;q
32)
~2,rdrr(7)(4qtrr-4i~27:.7y32+rcn+7pl5::7ql 1
+
+7q3
2::-
lq27
~4qt
er)x
Z, r
add(
1 6)(
pl3)
~2,+
rx2[
cp6]
47
,X,-X
,t( I
; l)(
p36;
pl5
),t(
1; h)
(q2 1
; pl l
),t(5
; 15)
(pl3
;q 1 S
),dr1
(5)
(q33
),-1
O,a
dd(l
I)(p
l S),
der(
l6)t
( 10;
16)(
q 1 l;q
22),
tdel
(l7)
(pl l
),-2
1,
dc1(
22)(
ql1)
,+3m
ar[8
] 46
,XX
,dcl
(Z)(
p2l),
t(2; 17
)(q31;y25),add(3)(p2j),dcl(3)(p23),t(6,11)
(p2 I
; q 1
3),a
dd(8
)(~2
3),d
er(l
I)@
, 1 l
j(p2
1; p
l5)t
(3,1
l)(p
23;q
25)[
6]
43,X
X,t(
1; 6)
(pIU
; pl U
),t(2
; 18
)(q2
3; q2
1),d
r1(4
)(y1
2).d
t4(l
lI)(
q22)
, de
r(IO
)t(lO
; 15)
(q2l
;q2l
),t(
11; 1
6)(p
lI ;p
l 1 ),
-13,
drr(
1 S)
t( 13
; 15)
(q
12;
q24)
,der
( 17)
t(4; 1
7)(q
21; q
23)[
6]
46,X
X,t(
l;4)(
q32:
ql2)
,t(3;
13
)(q2
5:q3
4),t
(4;2
0)(q
l2;q
l3),
t(l3
; 22)
46,X
X,t(
1; 7)
(q22
;q2 l
),dc
l(S)
(ql3
q22)
[4]
46,X
X[2
5]
(p2 1
; pl2
),dd
( 1 R
)(pl
1 ), +
19.a
dd( I
9)(q
13)~
2.
-- 2 1
, -22
,add
(22)
(plI
;q11
)151
47,X
X,+
der(
1;16
)(q1
0; pl
O)x
2,-1
6[6]
48
,XX
,+de
l(l)(
ql l)
,+i(
l)(q
lO)[
4]
2252
/90
If
55-59,-X,-X,add(l)(p36),add(l)(pl3),+add(l)(q25),+del
(l)(q32),+2,+der(2)deI(Z)(q35)t(2;
6)(p
2 1; p
Zl)
,add
(3)(
q2l)
,dic
(3; 1
6)
(q13
;~13
),-4,
der(
4)t(
4;14
)(q3
5; q2
4)ad
d(4)
(pl6
),t(6
;12)
(q23
;q13
), +der(7)(4qter-+4q27:7q32-cen-7pl5::7ql1-7q32::4q27+4qter),
-8,-9
,-10,
del(
1 l)(
pl lp
l3),
der(
13)t(
3;13
)(p1
3; p1
2),-
14,d
er( 1
4)
t(3; 14)(p21;p12),-15,add(17)(q23),add(l8)(pl
l),+
add(
19)(
q13)
, -2
l,add
(21)
(pl
l),a
dd(2
2)(p
12),+
8-12
mar
[cp3
] lg
55-59,idem,tadd(10)(q26)x2,+add(16)(pl3)[~pl7]
lh
55-59,idem,+add(lO)(q26)~2.+add(l6)(p13),+r[cp4]
669/
91
2a
2b
3 19
7/92
2
308/
92
2 3 37
6/92
lb
lc
Id
9a
9b
46,X
X,d
el(l)
(q12
)[2]
46
,ide
m,t
der(
12)t
(l ;I
2)(q
12; q
lZ),
der(
18)t(
9;18
)(q1
3; q2
3),-2
1[4]
47
,XX
,+7[
3]
46,X
X,d
e1(3
)(pl
2pl4
)[ 14
1 47
,XX
,+r(
8)[5
] 47
,XX
,+de
r(8;
16)(8qter-8cen::hsr::16cen+16pter),-16[6]
46,X
X,d
er( l
)inv
(l)(
p3 1 q
23)t
( 1; 2
)(p2
1; p2
2)de
l(l)
(p3 1
1, de
r(2)
t(l;
2)(q
23; p
22)[
104]
92
,idem
x2[ 1
21
46,id
em,-X
,t(X
; 3)
(qll
; p26
)[2]
92
,XX
XX
,+de
r(l;
16)(
qlO
;plO
)x2,
-16,
-16[
3]
92,X
XX
X,-
1,-
l,der
(5)t
( 1; 5
)(p3
2; p
l5)t
( 1; 9
)(p3
6; ql
3)xZ
,de1
(9)
(q13
)x2,
tder
(l; 1
6)(q
lO;p
lO)x
2,-1
6,-1
6,ad
d(l7
)(pl
l)x
2,+r
,+r[
3]
Cytogenetics of Bilateral Breast Cancer/Pandis et al. 253
m \
In
m N
\
m m
m m 3
“ ? 0
u
N G. \ N a2 m
m --. m
d Lo
“1
\ m 3 Ln
m
of in situ carcinoma growth were intermingled with areas of infiltration.
Patient 8
A 76-year-old woman had felt a lump in her left breast. The tumor turned out to be a 33-mm unifocal, moderately differ- entiated, infiltrating ductal carcinoma (588/93). DCIS was discovered in the tumor area as well as in the areola, and LCIS was detected in both lower breast quadrants. In the right breast, a unifocal, infiltrating, moderately differentiated duc- tal carcinoma (589/93) with DCIS changes was found.
Cytogenetic techniques. From all the patients, a sample taken from the same area where the histologic characteristics of the tumor had been determined was brought to the cytoge- netics laboratory in a sterile Petri dish. The specimens from all but one tumor (from the right breast of Patient I; this sample was examined before the establishment of the improved tech- nique) were processed for cytogenetic analysis as described by Pandis et al.4 The clonality criteria and description of tumor karyotypes followed the recommendations of the Interna- tional System for Human Cytogenetic Nomenclature (ISCN, 1991).6
Fluorescence in situ hybridization analysis according to the method described by Lichter et al.’ was performed on de- stained, previously mounted slides from the right-sided tumor sample of Patient 4 to identify the homogeneously staining region and ring chromosome that had been detected by band- ing analysis.
Results
Of the 19 specimens taken from the eight patients, clonal chromosome abnormalities were detected in 17 samples from 16 tumors (Table 1). The short term cul- ture from one specimen was a failure. In another, only normal karyotypes were found. In 12 of the samples with chromosomal aberrations, 2 to 7 abnormal clones were detected, corresponding to 56 clones. Cytogeneti- cally unrelated clones, with or without the simulta- neous presence of related ones (subclones), were found in 10 (59%) specimens from 8 (57%) of the 14 karyo- typically abnormal tumors, whereas only related clones were seen in 3 specimens from 3 tumors.
The following rearrangements were recurrent: der( 1; 16)(qlO;plO), del(l)(ql l-12), del( l)(q42), and del(3)(pl2-13pl4-21). Nonclonal changes (not shown) were detected in all the specimens; some of them were the same as the clonal abnormalities seen in other cases [e.g., del(3p) and del(lq)].
Discussion
The structural rearrangements der( 1; 16)(q10; plO), del(l)(qll-12), del(l)(q42), and de1(3)(p12-13p14-21) were detected recurrently (three times each) in our se-
254 CANCER July 1.5, 2995, Volume 76, No. 2
ries of bilateral breast carcinomas. They are the changes most likely to reflect pathogenetically important, com- mon events in mammary carcinogenesis.
The derivative chromosome resulting from the 1; 16 whole-arm translocation was in all three cases found in addition to two normal copies of chromosome 1 and one normal copy of chromosome 16. Hence, cells carry- ing this aberration are effectively trisomic for l q and monosomic for 16q. The der(1; 16) has been repeatedly associated with breast cancer and was the single most common aberration in the two largest series reported until It is unclear whether the gain of l q or the loss of 16q is the functionally crucial outcome of der(1; 16); however, the circumstance that l q gains are common in this tumor type also through other chromo- somal mechanisms might indicate that the former im- balance is more important.
A deletion of all or almost all of the long arm of chromosome 1, del(1) (ql l - l2) , has been described re- peatedly both in complex breast cancer kary~types~-’~ and as the sole abn~rmali ty .~,’~ The smaller del(1) (q42) seen in three of the cases we now present, twice as the only change and once together with other aberrations, has not been as common; only three times was this de- letion found before, and then always as one of several changes in complex karyotypes. 15,17r18 For none of the l q deletions is the essential pathogenetic outcome known, but as always when material is consistently lost, the loss of a tumor suppressor locus is suspected.
An interstitial deletion of the short arm of chromo- some 3, del(3) (p12-13p14-21), was first described in a bilateral breast carcinoma by Zhang et al.” It was later identified as a consistent primary change in breast car- cinogenesis by Pandis et a1.20 The association is strengthened further by the data we now present, which also include an example of clonal evolution (Pa- tient 6, right breast), in which a secondary inv(4) had been added to the primary del(3p). Recent molecular studies have contributed additional information by demonstrating not only that loss of heterozygosity is common from 3p, but also that homozygous deletions, rearrangements, and hypermethylation occur in 3~14.3-3p21.3 during breast cancer development.21 Again, the suspicion is that a tumor suppressor locus is located here and that its inactivation is an important event in mammary carcinogenesis.
Among the findings seen only once in the current series, we call attention to the homogeneously staining region of Patient 4, which was located within a deriva- tive chromosome that, except for the homogeneously staining region, consisted of the long arm of chromo- some 8 and the short arm of chromosome 16. Fluores- cence in situ hybridization with a chromosome 8-spe- cific painting probe (data not shown) revealed that the
homogeneously staining region was made up of mate- rial from this chromosome. In general, 8p is known to be the most common site of homogeneously staining regions in breast carcinoma^.^ The relevant locus whose amplification is the pathogenetically important result of the 8p homogeneously staining region formations is un- known. It is intriguing that the other chromosomally abnormal clone identified in the right breast of Patient 4 had a supernumerary chromosome, identified by banding and fluorescence in situ hybridization as r(8), as the only aberration. Because this too must represent some degree of amplification of chromosome 8 mate- rial, some functional similarity can be said to exist be- tween clones 1 and 2 of that case, despite the fact that they were karyotypically unrelated.
A main finding in our study was that 57% (8 of 14) of the tumors showing chromosomal abnormalities had karyotypically unrelated clones, supporting our previ- ous c o n ~ l u s i o n ~ ~ ~ ~ that a substantial proportion of breast carcinomas are polyclonal. Unrelated clones in breast cancer have also been reported by other investigators, albeit in a smaller proportion of turn or^.^^'"^^ Although the cytogenetically unrelated clones could originate from a single transformed cell and share the same sub- microscopic mutation, this seems unlikely considering the highly disparate nature of the observed changes. As we have argued previou~ly,~~ the finding that preferen- tial X chromosome inactivation occurs in breast carcino- masz5 does not conclusively falsify the polyclonal origin hypothesis. Finally, even if a unifying initial mutation were found to exist, this would not mean that the re- maining genetic abnormalities are unimportant. Carci- nogenesis is a multistage process and also aberrations acquired secondarily influence the tumor’s phenotypic characteristics, features that may include its ability to invade and metastasize.
An important question is whether all the clones or only one of them (or group of related clones) are part of the tumor parenchyma and, thus, represent truly neo- plastic cells. This question is particularly pressing in cases with one complex clone and the simultaneous presence of seemingly unrelated clone(s) with only sim- ple chromosome changes (e.g., case 1). It is not likely that only the complex clone (Fig. 1) reflects the neoplas- tic process, whereas the other(s) exemplify some kind of cytogenetic noise? Indeed, karyotypic complexity has repeatedly been demonstrated in malignant epithelial tumors, including breast carcinomas, and is known to correlate with tumor progression.22,26 However, equally convincing evidence suggests that deceptively simple changes may be tumorigenic in breast cancer as well as in other malignancies. One example is provided by Pa- tient 5 of the current report. The quantitatively domi- nating clone in the left-sided tumor had inv(1) as the
Cytogenetics of Bilateral Breast Cancer/Pandis et al. 255
Figure 1. Karyogram illustrating the complex clone I f from sample 2252/90, Patient 1. See Table 1 for karyotype description. Nonclonal aberrations present in this cell only were der(4)del(4)(pl3)t(4;6)(q27;q21), add(9)(pl l), the gain of the second add(] 9)(q13), and the loss of the second chromosome 15.
sole anomaly (Fig. 2). In the contralateral breast, clonal evolution had taken place (Table 1, Fig. 2). We must conclude that the cells containing inv( 1) were tumori- genic in the left breast and that their descendants had metastasized contralaterally. A second example of tu-
Figure 2. Partial karyotypes from Patient 5. The inv(l)(p31q23) found as the sole anomaly in one clone in the left-sided breast cancer is illustrated to the left. Further rearrangement of the inv(1) led to the formation of a der(l)inv(l)(p31q23)t(1;2)(p21;p22)del(l)(p31) and a der(2)t(l; 2)(q23; p22). These were found in the right-sided breast cancer and are illustrated to the right.
morigenic ability of cells carrying a simple aberration is provided by the finding of a +der( 1; 16), - 16 as the only anomaly in both the primary tumor and an axillary lymph node metastasis in a breast cancer patient.”
Do the available data give any clue as to whether the cytogenetic polyclonality is a feature of early or late stages of breast carcinogenesis? The finding of multiple clones in benign proliferative breast disorder^^^^^^ could be interpreted to indicate that they characterize the ear- liest stages only. However, the detection of unrelated clones in the second and evidently metastatic carcinoma in Patient 1 shows that the polyclonality may well occur later. A similar impression is obtained from Patient 5; the cytogenetic evidence indicates that the carcinoma- tous process in the left breast was primary, but the pre- sumably metastatic right-sided tumor tissue neverthe- less contained at least two seemingly unrelated clones. Karyotypic heterogeneity of this type raises the ques- tion whether the additional clones may perhaps have arisen secondarily also in a causal sense, that they may be a consequence of rather than instrumental in bring- ing about tumor progression. Though we cannot rule out the possibility that new clonal changes are gener- ated as a response to, e.g., the release of mutagens by neighboring cancer cells, we favor the view that the un-
256 CANCER July 25,1995, Volume 76, No. 2
Figure 3. Metaphase plate showing the hyperhaploid clone (n = 29) that was detected in the sample from the left breast in Patient 7. A der(1; 16)(qlO;plO) was the only structural rearrangement.
related clones were probably present already in the pri- mary tumor. Whether a change in the selection pressure made it possible to detect them only after some time had passed or whether we did not see them in the first sample for some unknown stochastic reason is a moot point. The latter possibility is particularly likely in case I, where the specimen from the right-sided carcinoma was examined before our culture techniques had been improved.
From two of the multifocal bilateral carcinomas (Patients 5 and 7), more than one sample was obtained from the lesions in the left breast. The findings in these cases confirm our previous observationz4 that multifocal breast cancers are karyotypically multiclonal with cyto- genetic similarities as well as differences among the foci. The pattern of polyclonality in Patient 7 was particu- larly informative (Table 1). The lb-d subclones found in the A focus are evolutionarily related to the hyper- haploid la subclone (Fig. 3). All of them are cytogenet- ically related to subclones l e and If in the B focus (Fig. 4), which shows that these two foci have evolved as part of the same carcinogenic process, one presumably as the intramammary metastasis of the other. In addi- tion, the B and C foci show karyotypic similarities but of a completely different nature: clone 3a in B is a more complex subclone evolved from clone 3b in C, which had t(1;20) and t(10; 17) as the only chromosomal ab- normalities. We therefore have a chain of interrelated- ness in which A is related to B and B to C, but appar- ently no relation exists between A and C. Peculiar as this pattern may seem, a similar situation was neverthe- less seen aIso in the bilateral and multifocal cancer of
Patient 5 (Fig. 4), in which the clone shared by the two foci sampled in the left breast [3; these cells had del(1) (q42) as the only change] was not related to clone la, the clone that had metastasized to the contralateral breast and evolved into clones lb-d there. We see no way in which a classic Darwinian selection scenario, working from the premise that we are dealing with the malignant transformation of a single cell and its subse- quent monoclonal expansion, can accommodate these data. Maybe early clonal heterogeneity with evolution- ary synergism existing among groups of tumor cells should be invoked to explain or at least describe the dy- namics of the tumor cell p~pulation.~'
Finally, the cytogenetic findings in all informative cases in our series, except for Patients 1 and 5, support the notion that the contralateral carcinoma arose inde- pendently of the first one as a genuinely new primary tumor.' For Patients 1 and 5 , on the other hand, the karyotypic data indicate that spreading of the disease process from one breast to the other had taken place. We can say this with confidence because it is so ex- tremely unlikely that two identical inv( 1) (Patient 5) should occur in the two breasts as the result of indepen- dent mutations, and the same argument applies even more strongly to the finding of the same complex clone bilaterally in Patient 1. We emphasize that at the same time, the traditional histologic parameters indicated that the tumors in both breasts were primary, in as much as widespread formations of in situ carcinoma were found among the areas showing infiltrating g r o ~ t h . ~ ' - ~ ~ The discrepancy between the histologic and cytogenetic conclusions as to tumor origin shows
Cytogenetics of Bilateral Breast Cancer/Pandis et al. 257
Figure 4. Diagram showing the cytogenetic relationships among the clones detected in the bilateral breast cancers in Patients 5 and 7.
CASE V
Left Brew Right Breast
CASE VII
RightBreast 1 Left Breast
that a reevaluation of the criteria used to differentiate between primary and metastatic breast may now be necessary. If in situ carcinoma lesions may arise through a metastatic process, as the cytogenetic data strongly indicate, then their presence can no longer be regarded as a fail-safe criterion of de novo carcinogene- sis.
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