Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
ICANCERRESEARCH 56.855-859.February 15.19961 Karyotypic Comparisons of Multiple Tumorous and Macroscopically Normal Surrounding Tissue Samples from Patients with Breast Cancer1 ManuelR. Teixeira,NikosPandis,GeorgiaBardi, JohanA. Andersen,and SverreHeim2 Department of Genetics, The Norwegian Radium Hospital and Institute for Cancer Research, Montebello, N-0310 Oslo, Norway (M. R. T., S. H.); Departments of Medical Genetics (M. R. T., N. P., G. B., S. H.] and Pathology If. A. A.]. Odense University and University Hospital, DK-5tX1O Odense, Denmark; Department of Genetics, Saint Savas Hospital. GR-11522 Athens, Greece [N. P., G. B.J; and Department of Clinical Genetics, University Hospital, 5-22185 Lund, Sweden (N. P., G. B., S. H.J ABSTRACT the moreremarkablebecause these analyses probablyseverely under estimate the actual karyotypic variability present; the single, often Many tumor tissuesare madeup of geneticallydifferentcell popula small, sample routinely examined is unlikely to be representative of all tumor areas (15). The spatial distribution of cytogenetically dis parate tumor cell subpopulations and their possible relation to zonal phenotypic heterogeneity are issues that have received practically no lions, and the study of the causes and consequences of this heterogeneity must play a central role in cancer research. We haveStudiedbreast cancer clonal heterogeneity by cytogenetic analysis of 4123 cells from 52 success fully short-term-culturedtumorous,metastatic,andmacroscopically nor madbreasttissuesamplesfrom 6 womenwith thisdisease.All 7 carcino attention in the past. We have recently reported a preliminary study of cytogenetic heterogeneity in breast cancer, in which we demonstrated the local ization of karyotypically distinct cell populations to separate intratu morous domains (16). In this report, we extend that investigation by the detailed analysis of 58 tumorous, metastatic, and macroscopically normal breast tissue samples from another 6 patients with breast cancer. mas (one woman had bilateral disease) contained 1 to 9 karyotypically related as well as unrelated clones, unevenly distributed among the tumor quadrants. Two clonal chromosomeabnormalities were recurrent: inter stitial 3p deletionswere found in 5 carcinomas,whereasdel(l)(q42) was detected in another 2 tumors. Both successfully analyzed metastatic le sions (one axillary lymph node and one metastasisin the subcutls) con tamed only one of several clones present in the primary tumor, thus exemplifyinga reductionin overallkaryotypiccomplexityduring card noma spreading. In the case with the cytogenetically abnormal lymph node,another karyotypically unrelated clone was found to Invadelocally in the surrounding breast; also, histological evidenceof carcinoma infil tration was seenin thesetissuesamples.In none of the other caseswere clonalkaryotypicchangesfoundin macroscopically normal,extratumor ousbreasttissue.We concludethat a largeproportionof breastcarcino mas are polyclonal with cytogenetically panding within separate domains distinct cell subpopulations of the growing tumor. MATERIALS AND METHODS Sevenconsecutivebreastcarcinomas(Table 1) with a diameterlargerthan20 mm were included in this study. None of the 6 women (cases VI and VII were from a patientwith bilateraldisease)hadanyknownhereditarypredisposition to cancer.Four samplesfrom each tumor (1 from each imaginary tumor quadrant)wereobtainedandlabeledclockwiselA—D.In addition,samplesof ex Karyotypically disparate neoplastic cells may have different capacities to display malig nancy-specific features (e.g., to grow invasively and set up distant metas macroscopically tases). It is presumed that their synergetic action is required for the full-blown carcinoma phenotype. samples. Only when fat tissue dominated, normal breast tissue were taken from at least 10 mm outside the outer limits of the tumor and labeled 2A—D,in analogy with the tumor so that no normal breast tissue was visible in a given surrounding area, was no sample taken. A total number of 28 tumor samples,24 macroscopicallynormal breasttissuesamples,1 sample from a subcutaneous metastasis(labeled3), andS samplesfrom lymph node INTRODUCTION metastases(labeled 4) were obtained for cytogenetic analysis. The histopatho It is now generally accepted that tumor tissues are heterogeneous not only across the parenchyma-stroma borderline, but also among subpopulations of truly neoplastic parenchymatous cells (1, 2). Anal yses of tumor behavior and biology must take into account the causes and consequences of this phenotypic heterogeneity starting point the central tenet of the somatic mutation theory of cancer (5, 6), have provided evidence that genetic mechanisms are crucially involved in the generation of cell-to-cell and clone-to-clone variation in breast tumors (7—1 1). In contrast to most chemistry-based investigative methods, which yield pictures of an idealized average tumor cell, cytogenetic techniques reveal the karyotypic constitution of individual cells and thus are uniquely well suited to shed light on the question of intratumor genetic heterogeneity (12). Multiple karyo typically related, as well as unrelated, clones have indeed been de tected in a high proportion of breast carcinomas (13, 14). This is all Received 9/28/95; accepted I 2/1 1/95. The costsof publicationof this article weredefrayedin part by the paymentof page charges.This article mustthereforebe herebymarkedadvertisementin accordancewith work was supported by grants from the Norwegian, Danish, and Swedish Cancer 2 To whom requests for reprints should be addressed. and the resulting cells were plated out in 25-cm2 Primaria flasks or Vitrogen-coated slide flasks. The cultures were fed an appropriate medium plasms (3, 4). Several lines of research, all of them taking as their I This of slides from tissue imme accordancewith WHO recommendations (17),withoutprior knowledgeof the cytogeneticfindings. disaggregated, standing of the mechanisms of tumorigenesis is to be achieved. Breast carcinomas are among the most heterogeneoushuman neo Societies. M. R. T. is the recipient of fellowship BD/3109/94 from Programa PRAXIS XXI. which included examination Cells were short-term cultured and analyzed cytogenetically as described previously (18). Briefly, all samples were mechanically and enzymatically if a full under 18 U.S.C. Section 1734 solely to indicate this fact. logical classification, diately adjacent to the tissue processed for chromosome analysis, was made in thatfacilitatesepithelialgrowthandwereharvestedafter5 to 8 days.Thecells were exposed to demecolcine, dislodged by trypsinization, subjected to hypo tonic shock in 0.05 M KCI, and fixed in methanol:acetic acid (3:1). Some culturesin slideflaskswereharvestedin situ asdescribedby Mandahl(19).G bandingwasobtainedwith Wright stain.Theclonalitycriteriaandthedescrip tion of karyotypesfollowed the recommendations of the ISCN (20). The one samplesign testappliedto the differencesbetweenpairedobser vations (21) was used to compare quantitatively the chromosomal findings in tumors and grossly normal breast tissue areas. RESULTS Of the 58 specimensobtained for cytogenetic analysis, all 28 tumor samples, 22 of 24 macroscopically normal surrounding breast tissue samples(only samples2A and 2B from caseI did not grow in culture), 1 of S lymph node metastases(only in case VI was the outgrowth epithelial; the fibroblastic cells growing from the other samples had a 46,XX karyotype), and the locally metastatic lesion were successfully cultured. A total number of 4123 metaphases were analyzed (2354 855 Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1996 American Association for Cancer Research. KARYOTYPIC HETEROGENEITYIN BREAST CARCINOMAS dataClinico-pathological Table 1 Clinico-pathological normalbreast [del(1)(q42)] that was quantitatively dominant in the tumor were detected. Microscopically, these samples were shown to contain both in situ and invasive lobular carcinoma areas. In all the other cases, no clonal chromosome abnormalities were found in the surrounding data on 58 tumorous,metastatic,and macroscopically cases.Case tissue samples obtained for cytogenetic analysis from 7 breast cancer Age Tumor size Histology―1-153/94 no. (yr) (mm) Nodal status 59 + 30 Samples@' breast tissue. Except for 2A—D of case VII, none of these samples DC2A-B IA—D N2C-D EH4 contained microscopic evidence of carcinoma spreading. The fre quencyof nonclonalchanges in the grossly normalbreasttissue varied M11-182/94 47 23 — 42 from 2.2% to 5.7% (median, 3.3%), and hence no statistically signif icant difference was found in the paired comparison of tumorous versus nontumorous samples in this regard (P = 0.7). IDC' + IDC+DCISID DCIS2A, 2C-D DC2A, 1A—D EH3 2C-D M4 N111-242/94 40 DCISlC lA—B + DISCUSSION MIV-250/94 54 36 + Five of the 7 breast carcinomas were shown to contain a del(3)(p12—13pl4--21) in 2—4tumor samples (Table 2). In casesI, III, CDC2A 1A-D EH2B-D N4 and VII, this deletion was the sole chromosomal abnormality of the clone, but in the remaining two cases (IV and V), extensive clonal MV-271/94 77 25 — DCIS2A, 1A—D 44 45 + 2D ILC2A-D 1A-D EHVI-257/94 IDC' + LCIS4 NC+ ILC + MVll-258/94 44 55 + ILC2A-D lA-D LCIS4 a Samples IA—D originate from tumor mass, 2A—D from NC+ ILC + M macroscopically normal surrounding breast tissue, 3 from a subcutaneous metastasis, and 4 from axillary lymph node metastases. b DC, ductal carcinoma; IDC, invasive ductal carcinoma; DCIS, ductal carcinoma in studies; using RFLPs, Sato et a!. (24) detected loss of heterozygosity situ; CDC, comedo type ductal carcinoma; ILC, invasive lobular carcinoma; LCIS, lobular carcinoma in situ; EH, epithelial hyperplasia; N, normal breast tissue; M, metastasis. C Quantitatively dominant histologic evolution had occurred. These results are in keeping with our earlier findings in sporadic breast cancers (13, 22) as well as in breasts of women with a hereditary predisposition to this disease(23); the results support our previous conclusion that interstitial deletions of the short arm of chromosome 3, with 3pl3—l4as a minimally common deleted segment, are consistent primary changes in breast tumorigenesis. Recent evidence that a tumor suppressor gene important in carcino mas of the breast maps to this region has come also from molecular at 3pl3—l4.3 in 47% of the tumors they examined. Chen et al. (25), who combined fluorescence in situ hybridization and RFLP analyses, concluded that the dominant mechanism of allelic loss at 3p in breast finding. from the tumors, 1720 from grossly normal breast tissue, and 49 from the lymph node of case VI and the subcutaneousmetastasis of case III). The complete karyotypic data are presented in Table 2. All 7 breast carcinomas were shown to contain clonal chromosome abnormalities in 3—50% (median 26%) of the cells analyzed; 86% of cancer was physical deletion. Buchhagen et a!. (26), on the other hand, demonstrated three mechanisms by which the putative tumor suppressor gene in 3pl4—2lcould be inactivated: homozygous dde tion, rearrangement, and hypermethylation. The secondchromosomal anomaly detected in more than one tumor in our study was del(l)(q42), which was found in several samples of the tumor samples (24 of 28) were karyotypically abnormal. Non clonal changes were found in 1.1—5.1% (median 3.5%) of the tumor cases II and VI (Fig. 3). Similar terminal lq deletions have been cells in each case. One to 9 clones were found in each tumor, unevenly distributed among the tumor quadrants (Fig. 1 gives an idea of how that this aberration must be accepted as identifying a ninth karyotypic the various clones were spatially arranged in cases III and VI). Two to 7 cytogenetically unrelated clones were detected in 4 of the carci nomas; related clones were found, in addition, in 2 of them. Complex effect of del(1)(q42) is unknown,but loss of a tumorsuppressorlocus could be the relevantoutcome. clones were detected in case IV only, where they coexisted with other simple and cytogenetically unrelated clones. In all other reported previously both by us (27) and others (28), and it is now clear subgroup in breast carcinomas (13). The fundamental pathogenetic The patient with synchronous bilateral disease had the two recur rent cytogenetic abnormalities mentioned above in her right-sided (case VII) carcinomas, the clones detected were near-diploid or, in most instances, pseudodiploid. Two clonal chromosome abnormalities were recurrent. Interstitial 3p deletions spanning bands p12—21were detected in 2—4samples of S carcinomas (minimal common deleted segment 3pl3—l4; Table 2). Extensive clonal evolution of the clone with 3p—was seenin casesIV and V. In case III, two different 3p deletions (Fig. 2) were seen in the tumor samples, and one of them, del(3)(pl3pl4), was also found in the subcutaneous metastasis. The second recurrent chromosome ab normality was the del(l)(q42) detected in several samples of casesH and VI (Fig.3). In addition, band 1q42 was also rearranged in the t(l ; 12) found in case V as one of many clonal chromosome abnor malities. The woman with bilateral breast carcinoma had a del(3)(pl2pl4) as the sole clonal aberrationin her right tumor (case VII) and a del(l)(q42) as the quantitatively dominant clonal abnor mality in her left tumor (case VI). Clonal chromosome abnormalities were found in the macroscopi and left-sided breast tumors (case VI), respectively. Be cause no karyotypicaberrationwas common to the two carcinomas,it would seemthat they arose independently as two primary tumors. We have shown previously that this is not always the case in bilateral breast cancer; the cytogenetic evidence indicates that the disease process spreads from one breast to the other in some instances (27). Carcinoma cells with highly complex cytogenetic abnormalities were detected only in case IV; all the other carcinomas were karyo typically abnormal but had pseudo- or near-diploid clones. Presum ably, these aberrations gave the cells a proliferative advantage. Ap proximately 30 tissue massdoublings are required for a tumor to reach clinical detectability (29), and the fact that many of the karyotypic abnormalities were detected in many cells in several tumor samples indicates that they must have been acquired at an early disease stage. With the methods we use, only about 20% of karyotypically abnormal breast carcinomas are seen to contain highly complex, non-near diploid clones (13), even when a detailed analysis of multiple tumor cally normaltissue surroundingthe breastcarcinomain case VI; in all samples is performed(Ref. 16 and present report). Flow cytometry studies indicate that about half of all breastcarcinomashave diploid 4 samples DNA histograms (30). The combined data are in keeping with theo (2A—D), cells carrying the same karyotypic aberration 856 Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1996 American Association for Cancer Research. KARYOTYPIC HETEROGENEITYIN BREASTCARCINOMAS Table2 Karyotypicdata Summaryof cytogeneticfindings in 52 successfullyculturedtumorous,metastatic,and mascroscopicallynormalbreasttissuesamplesfrom 7 breastcancercases. Case Sample'@ III IA 46,XX[30] IB 1C ID 2C 2D 46,XX,del(3)(p13p14)[2)/46,XX[421 46.XX[451 46,XX,del(3)(p13p14)[41/46,XX[68l 46,XX[62] 46,XX[35] lA 46,XX,del(1)(q42)[3J/46,XX[30] lB 1C 46,XX,del(1)(q42)[91/46,XX[29] 46,XX(50] ID 46,XX,del(l)(q42)[21/46,XX[l7] 2A 2C 46,XX[62] 46,XX[43] 2D 46,XX[40] IA lB IC 46,XX,del(3)(p13p14)[9J/46,XX[54] 46,XX,t(l;19)(p22;p13)[51/46,XX[50l 46,XX,del(3)(p13p14)[45J/92,XXXX,idemx2[31/46,XX,t(l;2)(p36.3;p21)[421/92,XXXX,t(l;2)(p36.3;p21) lD 2A 2C 2D 46,XX,del(3)(p12p14)[19]/46,XX,del(3)(p13p14)[4Y46,XX,t(1;2)(p36.3;p21)[ll/46,XX[73] 46,XX[l07] 46,XX[73] 46,XX[75] x2[5]/46,XX,del(3@pl2pl4)[9J/47,XX,+i(l)(ql0)[2]/46,XX[l38] IV 3 46,XX,del(3)(p13p14)[41/46,XX[25] IA 62—64,XX,—X,add(1)(pll),der(l;13)(qlO;qlO),der(2)t(1;2)(p13;p25),der(3)del(3)(p13p21)inv(3)(p2.3p26),—4,—5,+6, —l4,—l4,der(14)(qter--+p1?::hsr::8q11--s8qter),—15,del(l6)(ql3),der(17)del(17@pl1)t(7;l7@q22;q25),—2O,add(2l)(pl1),—22, —22,+3—Smar[cp2OJ/46,XX[24] lB (qter—øpl?::hsr::$qll--$qter),—lS,del(16)(q13),der(17)del(17)(pll)t(7;17)(q22;q25),—20,add(21)(pll),—22,—22,+3—Smar[cp2.5] /58—62,idem,der(9;15)(qlo;qlO),der(lO)t(l0;l5)(q23;q15),+ l6[cp3]/124—128,idemx2[cp2l/46,XX,add(2)(p24)[4] /46,XXI(l;7)(p36;p13)[2]/46,XXt(3;6)(q29;q16)[2]/46,XX[70] lC ::hsr::Sqll—.Sqter),—15,del(16)(q13),der(17)del(17)(pll)t(7;17)(q22;q25),—20,add(2lXpll),—22,—22,+3—Smar(cp3J/46,XX[15] 46,XX[25] 46,XX[l25] 46,XX[108] 46,XX[82] 46,XX[86] ID 2A 2B 2C 2D V 1A lB IC 46,XX,del(3)(p12p14)[41/46,XX[73] 48,XX,+7+der(16)t(8;16)(ql l;ql3)X2,—l6[4l/46,XX,del(3)(p12p14)[31/46,XX[68] (q22;q32),der(l9)t(3;19)(pl2;p13)del(3)(pl2pl4)[I 1]/46,X,der(X)t(X;2)(p22;p23),der(l)t(l;2)(q24;p14)t(X;2)(p22;p23),der(2) t(l;2)(q24;pl4)[l8]/46,XX,t(4;7)(q23;p2l)[l l}/46,XX,t(I;16)(q21;ql3)[lO]/46,XX,der(l)t(1;12)(q42;q24),t(2;5)(q14;pl3), der( I 2)qter—@q24. 1::pl3—@q24. 1:: 1q42—+lqter)[6]/46,XX[1281 VI VII a Samples ID 46,XXt(5;l2)(q13;qls)[3I1/46,XX,del(3)(p12p14)[61/46,XX[l23] 2A 2D 46,XX[95] 46,XX[l03] IA 46,XX,del(1)(q42)[11V46,XX,t(l;12)(plO;qlO)[81/46,XX,del(1)(q12)[3]/46,XX[46] lB IC 1D 2A 2B 2C 2D 4 IA lB 46,XXdel(1)(q42)[541/92,XXXX,idemx2[6]/46,XX,del(1)(q12)[31/46,XX[151] 46,XX,del(1)(q42)[13J/46,XX,del(1)(q12)[4]/46,XX[73] 46,XX,del(l)(q42)[3]/46,XX[22] 46,XX,inv(l l)(p15q23)[26]/46,XX,del(l)(q42)[41/46,XX,t(l;9)(p21;p2l)[21/46,XX[l26] 46,XX,add(I l)(q14)[5l/46,XX,del(1)(q42)[1l/46,XX[34] 46,XX,del(l)(q42)[21/46,XX[32] 46,XX,del(l)(q42)[1J/46,XX[23] 46,XX,del(l)(q12)[41/46,XX[12] 46,XX,del(3)(p12p14)[91/46,XX[29] 46,XX,del(3)(p12p14)[1531/92,XXXX,idemx2[61/46,XX[79] 1C 1D 46,XX,del(3)(p12p14)[7]/46,XX[28] 46,XX[50) 2A 2B 2C 2D 46,XX[50] 46,XXI147] 46,XX[76] 46,XX[45] 1A—D originate from the tumor mass, 2A—D from the macroscopically normal surrounding breast tissue, 3 from a subcutaneous metastasis, and 4 from an axillary lymph node metastasis. b Clones present in more than one sample in each case are shown in bold. retical scenarios stipulating that whereas some neoplasms show ex- unevenly distributed within the tumor mass (Fig. 1). This demon tensive genetic divergence during tumor progression, others remain strates that cytogenetically distinct cellular subpopulationsexpand genetically stable throughout, or may actually become less diverse as within separatedomains in the growing tumor, as was also indicated the disease goes from bad to worse (31). The actual testing of how by the results of our pilot study (16). Similar regional karyotypic these assumptions apply to breast cancer would require longitudinal heterogeneity was recently shown in a detailed investigation of a monitoring of the neoplastic process with multiple samplings at sev- low-grade glioma (32). The asymmetric clonal distribution highlights eral points in time, which is hardly feasible. the need to sample multiple sites if one wants to obtain a truly Multiple clones were detected in 4 breast carcinomas, always representative, balanced picture of the tumor's genetic characteristics. 857 Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1996 American Association for Cancer Research. KARYOTYPIC HETEROGENEfl@Y IN BREAST CARCINOMAS Much the same conclusion has also been reached via other investiga tive approaches. For example, because the DNA index is underesti mated, the prognostic significance of flow cytometry data in breast cancer has been found to be unnecessarily low when only one sample 11 del(1)(q42) 3 = Fig. 3. Partialkaryotypesandideogramillustratingthe identical lq terminaldeletions in cases II (right) and VI (left). is analyzed; at least 4 tumor samples must be examined to detect all stemlines with a larger than 90% probability (33). It seemsclear that analysesof the genomic constitution of breast carcinomas (be it at the total DNA, chromosomal, or genic level) that do not take this factor into consideration should be interpreted with caution. More often than not, multiclonality in the form of cytogenetically unrelated clones is seen in breast carcinomas (13, 14). In this series, 4 of the carcinomas contained 2—7 independent clones. Is this type of intratumor karyotypic heterogeneity compatible with the most com mon interpretation of the somatic mutation theory of carcinogenesis (5), which says that the process begins with a single mutated, and hence transformed, cell? One of the possible explanations that have to be taken into account is that the unrelated clones might reflect somatic cell mosaicism in breast tissue existing prior to the appearance of any @ 2C neoplasia. No histologically surroundings, nonneoplastic 2B Fig. 1. Spatialrelationshipof the different aberrantclonesfound in the variousintra andextratumoroussamples,aswell asin the metastaticlesions,of casesifi (top) andVI genetically unrelated clones detected in several independent cultures (bottom). Samples 1A—Dwere from each quadrant of the tumor mass, samples 2A—Dwere from the macroscopically normal surrounding breast tissue, sample 3 was from a subcu taneous metastasis, and sample 4 was from an axillary lymph node metastasis. Different graphic texture patterns represent cytogenetically unrelated clones (see Table 2 for detailed karyotypic information). Similar patterns in each case represent the same or a cytogenetically related clone. ‘}F 1! liii t(1 ;2)(p36.3;p21) J(@ del(3)(p12p14) clonal chromosome abnormalities were detected in the benign breast tissue samples taken from the tumor however, nor did previous cytogenetic analyses of breasts reveal such changes (34—37).The large, cyto del(3Xp13p14) from separate tumor areas but not elsewhere in the breast evidenfly possessa proliferative edge; to doubt that they are part of the neo plastic parenchyma in the face of the evidence now at hand seems illogical to us. The conclusion that the near- or pseudodiploid clones really have a malignant potential gains further credibility from the findings in the metastatic lesions. In case VI, a del(l)(q42) was detected in all tumor samples as well as in the 4 surrounding areas, where small foci of in situ and invasive lobular carcinoma were found microscopically. Another clone, characterized by a del(1)(q12), was present in 3 of the tumor samples and was the only one to be detected in the axillary lymph node metastasis. It appears that whereas cells carrying the del(1)(q42) were able to invade locally within the breast, the karyo typically unrelated cells with del(l)(q12) were the ones to metastasize via the lymphatic system (the karyotypic relationship between breast carcinomas and their lymph node metastasesin this and other cases will be dealt with more extensively by Pandis et al.3). A similar situation had occurred in case HI: only 1 of the 7 clones present in the tumor (3 of them detected in multiple samples; Fig. 2) was found in the subcutaneous metastatic lesion. Finally, the frequency of non clonal changes in tumor and surrounding tissue cultures was similar, arguing against the hypothesis that the karyotypically unrelated clones with relatively minor chromosomal aberrations should be a conse quence of some kind of clastogenic effect exerted by the actual neoplastic cells on neighboring stromal or nonneoplastic epithelial cells. Fig. 2. Partialkaryotypesandideogramsof thethreecytogeneticallyunrelatedclones detected in more than one tumor sample in case III. 3 Pandis et aL, manuscript in preparation. 858 Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1996 American Association for Cancer Research. KARYOTYPIC HETEROGENEITYIN BREAST CARCINOMAS Although it seemsunavoidable to conclude that the various karyo typically unrelated clones are part of the neoplastic parenchyma, the question still remains whether they are truly independent or have arisen as a consequence of clonal evolution, in which case they must be supposed to share a submicroscopic tumorigenic mutation that preceded and presumably induced the chromosome-level changes. Whenever multiple tumor samples were analyzed (Ref. 16 and herein), the proportion of breast carcinomas with this type of multiclonality could be seen to be as high as 70%. The hypothesis of a unifying submicroscopic mutation in most breast carcinomas does not explain why karyotypic multiclonality is so much more common here than in, e.g., hematological (38) or mesenchymal (39) neoplasms; evidently, a polyclonal model of carcinogenesis would be the one that best accommodates the cytogenetic data. We underscore that the finding of monoclonality in polymerase chain reaction-based analyses of X-inactivation patterns in breast carci nomas (40) does not make our position untenable. That technique identifies a tumor as monoclonal whenever a cell subpopulation makes up 50% or more of the total, and the presence of additional, independent, smaller clones therefore cannot be ruled out on the primarybreastcarcinomas:identificationof eightkaryotypicsubgroups.GenesChro mosomes & Cancer, 12: 173—185,1995. 14. Heim, S., and Mitelman, F. Tumors of the breast. In: Cancer Cytogenetics, Ed. 2, pp. 369—388. New York: Wiley-Liss, 1995. 15. Pandis, N., Bardi, G., and Heim, S. Interrelationship between methodological choices and conceptualmodelsin solid tumor cytogenetics.CancerGenet.Cytogenet.,76: 77—84, 1994. 16. Teixeira, M. R., Pandis, N., Barth, G., Andersen, J. A., Mitelman, F., and Heim, S. Clonal heterogeneityin breastcancer:karyotypiccomparisonsof multiple intra-and extratumoroussamplesfrom threepatients.Int. J. Cancer,63: 63—68, 1995. 17. Sobin, L. H. Histological Typing of Breast Tumors, Ed. 2. Geneva: WHO, 1981. 18. Pandis, N., Heim, S., Bardi, G., Limon, J., Mandahi, N., and Mitelman, F. Improved technique for short-term culture and cytogenetic analysis of human breast cancer. GenesChromosomes& Cancer,5: 14—20, 1992. 19. Mandahl, N. Methods in tumor cytogenetics.In: D. E. Rooney and B. H. Czepulkowski (eds.), Human Cytogenetics: A Practical Approach, Ed. 2, p. 177. New York: IRl@Press,1992. 20. Mitelman, F. (ed). ISCN (1991). Guidelines for Cancer Cytogenetics, Supplement to an International System for Human Cytogenetic Nomenclature. Basel: S. Karger AG, 1991. 21. Altman, D. G. Comparing groups-continuous data. In: Practical Statistics for Medical Research,pp. 186—187. London:Chapman& Hall, Ltd., 1991. 22. Pandis, N., Jin, Y., Limon, J., Barth, G., Idvall, I., Mandahl, N., Mitelman, F., and Heim, S. Interstitial deletion of the short arm of chromosome3 as a primary chromosomeabnormality in carcinomasof the breast. GenesChromosomes& Cancer,6: 151—155, 1993. 23. Teixeira, M. R., Pandis, N., Genies, A-M., Dietrich, C. U., Barth, G., Andersen, 3. A., Graversen,H. P.,Mitelman,F., andHeim, S. Cytogeneticabnormalitiesin an in situ ductal carcinomaand five prophylacticallyremovedbreastsfrom membersof a basis of the X-inactivation results. Whichever explanation for the emergence of cytogenetic poly family with hereditary breast cancer. Breast Cancer Res. Treat., in press, 1996. 24. Sato, T., Akiyama, F., Sakamoto, G., Kasumi, F., and Nakamura, Y. Accumulation of clonality in breast carcinomas will eventually be found to be the most accurate, the fact remains that these neoplasms exhibit a higher degree of intratumor genetic heterogeneity than one has become accustomed to expect in other types of neoplasia. Further more, this heterogeneity seems to have an anatomical correlate in the sense that different clones dominate in different domains of the tumor. The findings we have described also provide evidence that different neoplastic cell subpopulations may have different capac genetic alterationsand progressionof primary breast cancer. Cancer Res., 51: 5794—5799, 1991. 25. Chen, L-C., Matsumura, K., Deng, G., Kurisu, W., Ljung, B-M., Lerman, M. I., Waldman, F. M., and Smith, H. S. Deletion of two separate regions on chromosome 3p in breast cancers. Cancer Res., 54: 3021—3024,1994. 26. Buchhagen, D. L., Qiu, L., and Etkind, P. Homozygous delection, rearrangement and hypermethylationimplicate chromosomeregion 3p14.3—3p21.3 in sporadicbreast cancerdevelopment.Int. J. Cancer,57: 473—479, 1994. 27. Pandis, N., Teixeira, M. R., Gerdes, A-M., Limon, J., Barth, G., Andersen, J. A., Idvall, I., Mandahl,N., Mitelman, F., and Heim, S. Chromosomeabnormalitiesin bilateral breastcarcinomas:cytogeneticevaluationof the clonal origin of multiple primary tumors.Cancer(Phila.), 76: 250—258, 1995. ities to display malignancy-specific features (e.g., to grow inva sively and set up distant metastases). Presumably, the total tumor phenotype is expressed as the integrated functional result of the influence of all the separate cell populations of which 28. Mitelman, F. Catalog of Chromosome Aberrations in Cancer, Ed. 5. New York: Wiley-Liss, 1994. 29. Spratt, J. S., and Spratt, J. A. What is breast cancer doing before we can detect it? J. Surg. Oncol., 30: 156—160,1985. 30. Wenger, C. R., Beardsiee, S., Owens, M. A., Pounds, G., Oldaker, T., Vendely, P., Pandian, M. R., Harrington, D., Clark, G. M., and McGuire, W. L. DNA ploidy, it consists acting in concert (41, 42). Such clonal synergism in tumor devel opment (43) could also explain why the cytogenetic heterogeneity seemed to surpass the histopathologic variability in some of the tumors we examined. S-phase,and steroidreceptorsin more than 127,000breastcancerpatients.Breast CancerRes.Treat.,28: 9—20, 1993. REFERENCES 1. Heppner, G. H. Tumor heterogeneity. Cancer Rca., 44: 2259—2265,1984. 2. Wolman,S. R., andHeppner,G. H. Geneticheterogeneityin breastcancer.J. Natl. CancerInst., 84: 469—470, 1992. 3. Patchefsky,A. S., Schwartz,0. F., Finkelstein,S. D., Prestipino,A., Sohn,S. E., Singer, J. S., and Feig, S. A. Heterogeneity of intraductal carcinoma of the breast. Cancer(Phila.),63: 731—741, 1989. 4. Lennington, W. J., Jensen,R. A., Dalton, L W., and Page,D. L Ductal carcinoma in situ of the breast. Heterogeneity of individual lesions. Cancer (Phila.), 73: 118—124, 1994. 5. Nowell, P. C. The clonal evolution of tumor cell populations. Science (Washington DC), 194: 23—28,1976. 31. Heim, S., Mandahl, N., and Mitelman, F. Genetic convergence and divergence in tumor progression. Cancer Res., 48: 5911—5916,1988. 32. Coons, S. W., Johnson, P. C., and Shapiro, J. R. Cytogenetic and flow cytometry DNA analysisof regional heterogeneityin a low grade human glioma. Cancer Res., 55: 1569—1577, 1995. 33. Beerman, H., Smit, V. T. H. B. M., Kluin, P. M., Bonsing, B. A., Hermans, 3., and Cornelisse, C. J. Flow cytometric analysis of DNA stemline heterogeneity in primary and metastaticbreastcancer.Cytometry,12: 147—154, 1991. 34. Wolman, S. R., Smith, H. S., Stampfer, M., and Hackett, A. J. Growth of diploid cells from breast cancers. Cancer Genet. Cytogenet., 16: 49—64,1985. 35. Zhang, R., Wiley, J., Howard, S. P., Meisner, L. F., and Gould, M. N. Rare clonal karyotypicvariantsin primaryculturesof humanbreastcarcinomacells.CancerRes. 49: 444—449, 1989. 36. Geleick, D., Muller, H., Matter, A., Torhorst, J., and Regenass, U. Cytogenetics of breastcancer.CancerGenet.Cytogenet.,46: 217—229, 1990. 6. Heim, S. Tumor progression:karyotypickeysto multistagepathogenesis. In: 0. H. 37. Petersson,C., Johansson,B., Pandis, N., Gorunova, L, Ingvar, C., Idvall, I., Mandahl, N., and Mitelman, F. Clonal chromosome aberrations in fibrocystic diseaseassociated Iversen (ed), New Frontiers in Cancer Causation, pp. 247—259. Washington: Taylor & Francis, 1993. 7. Meyer, J. S., and Wiuliff, J. L. Regional heterogeneity in breast carcinoma: with increasedrisk of cancer.list. J. Oncol.,5: 1207—1210, 1994. thymidine labelling index, steroid hormone receptors, DNA ploidy. Int. J. Cancer, 47: 213—220, 38. Heim, S., and Mitelman, F. Cytogenetically unrelated clones in hematological neo plasms. Leukemia (Baltimore), 3: 6—8,1989. 1991. 39. Orndal, C., Rydholm, A., Willén,H., Mitelman, F., and Mandahl, N. Cytogenetic 8. Fuhr, J. E., Frye, A., Kattine, A. A., and van Meter, S. Flow cytometric determination intratumor heterogeneity in soft tissue tumors. Cancer Genet. Cytogenet., 78: of breast tumor heterogeneity. Cancer (Phila.), 67: 1401—1405,1991. 127—137, 1994. 9. Bonsing,B. A., Beerman,H., Kuipers-Dijkshoorn,N., Fleuren,G. J.,andComelisse, C. J. High levelsof DNA index heterogeneityin advancedbreastcarcinomas.Cancer (Phila.), 71: 382—391,1993. 10. Chen,L-C., Kurisu,W., Ljung, B-M., Goldman,E. S.,Moore,D., II, andSmith,H. S. Heterogeneity for allelic loss in human breast cancer. J. Nail. Cancer Inst., 84: 506—510,1992. I 1. LOnn,U., Lonn, S., Nilsson, B., and Stenkvist,B. Intratumoralheterogeneityfor amplified genes in human breast carcinoma. tat. J. Cancer, 58: 40—45,1994. 12. Heim, S. Is cancer cytogenetics reducible to the molecular genetics of cancer cells? Genes Chromosomes & Cancer, 5: 188—196,1992. 13. Pandis, N., un, Y., Gorunova, L., Petersen, C., Bardi, G., Idvall, I., Johansson, B., Ingvar, C., Mandahl, N., Mitelman, F., and Heim, S. Chromosome analysis of 97 40. Noguchi, S., Motomura, K., Inaji, H., Imaoka, S., and Koyama, H. Clonal analysis of humanbreastcancerby meansof the polymerasechain reaction.CancerRes.,52: 6594—6597, 1992. 41. Heppner,G. H., Miller, B. E., andMiller, F. R. Tumor subpopulationinteractionsin neoplasms. Biochim. Biophys. Acta, 695: 215—226,1983. 42. Alexander, P. Do cancers arise from a single transformed cell or is monoclonality of tumoursa lateeventin carcinogenesis? Br. J. Cancer,51: 453—457, 1985. 43. Teixeira, M. R., Pandis, N., Barth, G., Andersen, J. A., Mandahl, N., Mitelman, F., Heim, S. Cytogeneticanalysisof multifocal breastcarcinomas:detectionof karyo typically unrelated clones as well as clonal similarities between tumour foci. Br. J. Cancer,70: 922—927, 1994. 859 Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1996 American Association for Cancer Research. Karyotypic Comparisons of Multiple Tumorous and Macroscopically Normal Surrounding Tissue Samples from Patients with Breast Cancer Manuel R. Teixeira, Nikos Pandis, Georgia Bardi, et al. Cancer Res 1996;56:855-859. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/56/4/855 Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1996 American Association for Cancer Research.