J. Pathol. 187: 403–409 (1999)

WIDESPREAD CHROMOSOMAL ABNORMALITIES INHIGH-GRADE DUCTAL CARCINOMA IN SITU OF THEBREAST. COMPARATIVE GENOMIC HYBRIDIZATION

STUDY OF PURE HIGH-GRADE DCIS

1, 1, 2, 3 . 1*1Department of Pathology, Mater Misericordiae Hospital and Biotechnology Centre, University College Dublin, Ireland

2Department of Pathology, Withington Hospital, Manchester, U.K.3Department of Surgery, Mater Misericordiae Hospital, University College Dublin, Ireland

SUMMARY

For a variety of technical reasons it is rarely possible to study cytogenetic abnormalities in ductal carcinoma in situ (DCIS) usingtraditional techniques. However, by combining molecular biology and computerized image analysis it is possible to carry out cytogeneticanalyses on formalin-fixed, paraffin-embedded tissue, using comparative genomic hybridization (CGH). The purpose of this study wasto identify the prevalence of chromosomal amplifications and deletions in high-grade DCIS and to look specifically for unique orconsistent abnormalities in this pre-invasive cancer. Twenty-three cases of asymptomatic, non-palpable, screen-detected, high-gradeDCIS were examined using CGH on tumour cells obtained from histology slides. All cases showed chromosomal abnormalities. A widevariety of amplifications and deletions were spread across the genome. The most frequent changes were gains of chromosomes 17 (13 of23), 16p (13 of 23), and 20q (9 of 23) and amplifications of 11q13 (22 of 23), 12q 24.1–24.2 (12 of 23), 6p21.3 (11 of 23), and 1q31-qter(6 of 23). The most frequent deletions were on 13q 21.3–q33 (7 of 23), 9p21 (4 of 23), and 6q16.1 (4 of 23). These findings indicate thathigh-grade DCIS is, from a cytogenetic viewpoint, an advanced lesion. There was no absolutely consistent finding in every case, butamplification of 11q13 was found in 22 of the 23 cases. The precise significance of this is unknown at present. This region of chromosome11q harbours a number of known oncogenes, including cyclin D1 and INT2. It is likely that many of these findings are the result ofaccumulated chromosomal abnormalities, reflecting an unstable genome in established malignancy. Copyright ? 1999 John Wiley &Sons, Ltd.

KEY WORDS—breast; DCIS; in situ carcinoma; CGH; chromosomes

INTRODUCTION

Ductal carcinoma in situ (DCIS) is an early clinicaland pathological stage of breast cancer. Pathologically,it is classified into high, intermediate, and low grade, or,alternatively, into high- and non-high-grade DCIS.High-grade DCIS is biologically more aggressive thannon-high-grade; it is more likely to recur and becomeinvasive.1 High-grade DCIS nuclei are large, pleomor-phic, and often have macronucleoli. It seems reasonableto assume that such morphological abnormalities reflectprofound underlying DNA abnormalities. Studies show-ing that such nuclei are often aneuploid or aneusomicsupport this assumption.2,3

Recent DCIS studies have reported loss of hetero-zygosity (LOH) at various chromosomal locations, someof which contain known cancer genes, such as p53 on17p, Rb on 13q, and DCC on 18q.4–12 Other studiesusing fluorescence in situ hybridization (FISH) andimmunohistochemistry have shown amplification oroverexpression of c-erbB-2.13–16 The standard practiceof fixing breast biopsy specimens without palpablelesions eliminates the availability of fresh DCIS tumourfor tissue culture and karyotype studies. Comparativegenomic hybridization (CGH) is a new technique that

CCC 0022–3417/99/040403–07$17.50Copyright ? 1999 John Wiley & Sons, Ltd.

examines, in a single experiment, each pair of chromo-somes for genetic amplification and deletions.17,18 Itcan substitute, to some extent, for karyotyping and itcan be performed on formalin-fixed, paraffin-embeddedtissue.19,20 In combination with microdissection, CGHanalysis can be directed at small groups of tumour cells.

In the present study, we examined 23 cases of high-grade DCIS to identify the frequency of chromosomalamplifications and deletions, to find previouslyunknown abnormalities, and to look for specific non-random changes common to all or most high-gradeDCIS.

*Correspondence to: Professor Peter A. Dervan, Department ofPathology, Mater Hospital, Eccles Street, Dublin 7, Ireland. E-mail:pdervan@mater.ie.

MATERIALS AND METHODS

One hundred and four randomly selected casesof asymptomatic, non-palpable, screen-detected (bymammography) high-grade DCIS were selected initially.The criteria for final selection were (a) the cases shouldbe pure DCIS with no invasive components; (b) theyshould be high grade; and (c) there should be enoughtumour present in the chosen histology sections andparaffin blocks to allow extraction of good quality DNA(see below). Only 23 cases yielded good quality DNA.Two experienced surgical pathologists with a specialinterest in breast pathology reviewed all cases (PAD,JC). Previously published grading criteria were used.1,21

Received 19 February 1998Revised 15 August 1998

Accepted 18 November 1998

robustness of these thresholds.

404 E. MOORE ET AL.

Microdissection and DNA extraction

Histology sections were used to identify ducts contain-ing tumour cells. Tumour cells were microdissected,using the technique of Pan et al.22 Where a tissue sectioncontained DCIS surrounded by considerable stromawith stromal fibroblasts, endothelial cells or inflamma-tory cells, we microdissected tumour cells from the ductsto avoid contamination by non-neoplastic nuclei. Thesespecimens required DOP-polymerase chain reaction(PCR) to produce adequate amounts of DNA. Whenapproximately 90 per cent or greater of the histologysection was occupied by tumour-filled ducts, 50 ìmsections cut directly from the block were used. Elevenof the 23 cases analysed required microdissectionand DOP-PCR amplification. The number of ductsexamined depended on the number of available tumour-filled ducts and this ranged from 2 to 22. In some cases,we successfully analysed DCIS with only two ductsavailable. In other cases, despite an abundance oftumour cells, we failed to get successful analyses becauseof DNA degradation.

Probe preparation

Test DNA was DOP-PCR amplified and biotin-labelled by PCR according to Kuukasjarvi et al.23

Normal control DNA was prepared from peripheralblood lymphocytes from a healthy female donor. Thiswas subsequently DOP-PCR amplified (to counteractpossible amplification artefacts) and labelled withdigoxigenin by nick translation.

Hybridization and detection

Hybridizations were carried out as described byBrinkschmidt et al.24 with slight modifications; 1 ìg ofbiotin-labelled tumour DNA, 1 ìg digoxigenin (DIG)-labelled reference DNA, and 50 ìg of COT I DNA wereco-hybridized to normal metaphase spreads. Hybridizedtumour DNA was detected using a single layer ofavidin-fluorescein isothiocyanate (FITC), 1:150 andhybridized normal reference DNA was detected usingmouse anti-digoxin, 1:500 followed by goat anti-mouseTRITC, 1:800. Metaphases were counterstained withDAPI for chromosomal identification.

Digital image analysis

CGH hybridizations were analysed using an OlympusBX50> fluorescence microscope and a cooled CCDcamera attached to an Applied Imaging CytovisionSystem>. Data for an average of 10 metaphase spreadsper hybridization were combined and an average ratioprofile for each chromosome was determined. Gainswere identified by a profile shift of an entire chromo-some or a chromosome arm. Amplifications were ident-ified by a similar shift of a localized region within anarm. Gains and amplifications were defined by a greaterthan 1·25 green to red fluorescence ratio, and deletionsby a less than 0·75 green to red ratio. These thresholdsare defined arbitrarily on the assumption that 50 per

Copyright ? 1999 John Wiley & Sons, Ltd.

cent of the cells in a diploid tumour population carrychromosmal imbalance. For a monosomy the theoreticalratios are 0·75, and for trisomy 1·25. Although thelimitations of this methodology are acknowledged,previous investigators have shown the usefulness and

25,26

Controls

(1) biotin-labelled peripheral blood DNA co-hybridizedwith DIG-labelled peripheral blood DNA;

(2) biotin-labelled DOP-PCR-amplified blood DNAco-hybridized with DIG-labelled blood DNA;

(3) biotin-labelled DOP-PCR-amplified blood DNAco-hybridized with DIG-labelled DOP-PCR-amplified blood DNA;

(4) biotin-labelled DOP-PCR-amplified ‘normal’ DNAfrom a cosmetic reduction mammoplasty co-hybridized with DIG-labelled DOP-PCR-amplifiedblood DNA;

(5) biotin-labelled MCF7 cell line DNA co-hybridizedwith DIG-labelled blood DNA;

(6) biotin-labelled DOP-PCR-amplified MCF7 cell lineDNA co-hybridized with DIG-labelled blood DNA;

(7) biotin-labelled DOP-PCR-amplified MCF7 cell lineDNA co-hybridized with DIG-labelled DOP-PCR-amplified blood DNA.

Controls 1, 2, 3, and 4 gave a flat profile and con-trols 5, 6, and 7 gave the same expected profiles, thusvalidating the use of DOP-PCR-amplified DNA forCGH. Each CGH experiment included metaphasesco-hybridized with peripheral blood/peripheral bloodDNA as a negative control (control 3) (Fig. 1a) andMCF7 cell line/blood DNA as a positive control(control 7) (Fig. 1b).

RESULTS

Normal (negative) controls produced a ‘flat’ profile,as expected, with no amplifications or deletions on anychromosomes. Cells from an MCF cell line withknown chromosomal aberrations showed the expectedamplifications.

DNA sequence copy number changes were present inall 23 cases. The abnormalities involved complete chro-mosomes, entire chromosomal arms, or discrete regionscorresponding to chromosomal bands on conventionalkaryotypes. The most frequent changes were gains ofchromosomes 17 (12 of 23), 16p (13 of 23), and 20q (10of 23) and amplifications of 11q13 (22 of 23), 12q24.1–24.2 (10 of 23), 6p21.3 (11 of 23), and 1q31-qter (6of 23). Deletions of 13q 21.3–q33 (7 of 23), 9p21 (4 of23), and 6q16.1 (4 of 23) were most frequently observed.Additional amplifications were present at 1p (18 of 23),chromosome 19 (18 of 23), and 22q (12 of 23). Mostinvestigators find that these latter three regions givespurious profiles due to the large numbers of GC-richregions here. A complete CGH analysis of a single caseis shown in Fig. 2. The regions involved in each individ-ual case are detailed in Table I. Figure 3 summarizes the

J. Pathol. 187: 403–409 (1999)

Fig. 1—(a) Negative control. Profile obtained using DOP-PCR-amplified normal peripheral blood DNA, biotin-labelled, co-hybridized withDOP-PCR-amplified normal peripheral blood DNA, DIG-labelled. The green number under each chromosome identifies the chromosome. Thered ‘n= ’ beneath the centre of the profile indicates the average number of chromosomes examined to produce the profile. As explained in theMaterials and Methods section, the following regions are not examined: 1p32-pter, 1q12, 9q12, 16q12 and chromosomes 19, 22, X and Y. Theratio of these two normal DNA samples produces a flat profile, with minimal ratio fluctuation. (b) Positive control. Profile obtained usingMCF7 cell line DNA, biotin-labelled, co-hybridized with normal peripheral blood reference DNA, DIG-labelled. This profile shows regionalcopy number increases at 1qcen-q32, 3p14, 8q21-qter, 16q23–q24, 17q22–q24, and 20q13. These chromosomal regions are known to be

amplified in this cell line

Copyright ? 1999 John Wiley & Sons, Ltd. J. Pathol. 187: 403–409 (1999)

406 E. MOORE ET AL.

Fig. 2—Profile obtained from case 55 using DOP-PCR-amplified tumour DNA, biotin-labelled, co-hybridized with DOP-PCR-amplified normal peripheral blood reference DNA, DIG-labelled. This profile shows amplifications at 1q32.1, 3p21.3, 6p21.3,9q34.1-qter, 11q13, 12q24.2-qter, 16q22-qter, and 21q22.1-qter and gains of 16p, 17, and 20q

Table I—Amplifications and deletions seen in each case

Case No. Most commonly seen aberrations

1 +7qcen–q11.23,+11q13,+17q21.32 +3p21.3,+6p21.3,+11–q13,+12q24.1–q24.2,+173 +6p21.3,+11q13,+16p,+17qcen–q21.3,+20q4 +1q41-qter,+9q34.1-qter,+11q13,+16p,+17p,+17q21.3,+18pcen–p11.25 +7qcen–q11.23,+11q13,+12q24.1–q24.27 +11q13

10 +11q13,+17q21.311 +11q13,+17q21.314 +11q13,+17pcen–p1219 +3p21.3,+7qcen–q21.3,+11qcen–q13.4,+17q24-qter25 +6p21.3,+9q34.1-qter,+11q13,+16,+17,+20,+21q22.1-qter36 "1p21–p22.3,+1q31-qter,"q32.1–32.3,"6qcen–q22.3,"q21.3,+16p,+17,+18p+20qcen–q1238 +11q1350 "2q32.1–32.3,"3p12,+6p21.3,"6qcen–22.3,+11–q13,"13q21.3–22,+16p,+1752 +1q41,+3p21.3,+6p21.3,+9q34.1-qter,+11q13,+12q24.2-qter,+16p,+17,+20+21q22.1-qter53 "1p31.1,+6p21.3,"6q16.1,+11q13,+12q23.2-qter,"13q21.3–q33,+16p+16q22-qter,+17,+2054 +6p21.3–p25,+11q13,+12q23-qter,"13q21.3–q33,+16p,+17,+20,+21q22.1-qter55 +1q32.1,+3p21.3,+6p21.3,+9q34.1-qter,+11q13,+12q24.2-qter,+16p,+16q22-qter,+17,+20q,+21q22.1-qter63 "q16.1,"9p21,+11q13,+16p,+17,+18pcen–11.287 "9p21-pter,+11q13,+12q24.2-qter,"13q21.3–p33,+16p,+17p,+18,+20

135 "1p21–p22.3,+1q32.1-qter,"2q32.1–q32.2,+3p21.3,+6p21.1–p22.1,"6qcen–q22.3,"9p21-pter,+11q13,+12q23-qter,"13q21.3–q22,+16p,+17,+20,+21q22.1-qter

136 "3p12,+6p21.3,+9q34.1-qter,+11q13,+12q24.2-qter,"13q21.3,+17,+18pcen–p11.2,+20q,+21q22.1-qter137 +1q31-qter,+6p21.3–p25,"9p21-pter,+9q,+11q13,+12q23-qter,+16p,+17,+20,+21q22.1-qter

Copyright ? 1999 John Wiley & Sons, Ltd. J. Pathol. 187: 403–409 (1999)

407CGH ANALYSIS OF HIGH-GRADE DCIS

location, frequency, and type of abnormality for allcases, apart from rare abnormalities detected on singleindividual cases.

Fig. 3—Schematic representation of the most frequently found aberrations. The banding pattern of the chromosomeideograms is broadly similar to that of G banding. Green lines on the right of each chromosome ideogram representamplifications and red lines on the left represent deletions. The length of these lines indicate the size of the chromosomalregion involved. Broad lines represent five cases and fine lines represent one case

DISCUSSION

This study identified chromosomal aberrations at lociwhich harbour known oncogenes, including cyclin D1(CCND1), INT2, EMS1, and hst1 at 11q13;27,28

c-erbB-2 at 17q11.2–q12; and the tumour suppressornm23 at 17q24. Amplification of 11q13 was present in 22of the 23 cases. Other genetic aberrations detected, someoccurring at high frequency (16p, 20q, 12q24.1–q24.2,6p21.3, 13q21.3–q33), may contain previously unknowngenes whose altered expression may contribute to thedevelopment or progression of DCIS. High-grade DCIScontains numerous large and apparently random alter-ations involving most chromosomes. The significance ofmany of these changes, however, is poorly understood.They may result from genetic instability, and reflect acascade of genetic damage in the tumourigenic process.It is possible that such alterations contribute to tumourprogression. However, it is also possible (or probable)that many are a consequence of tumourigenesis—mereepiphenomena.29 Nevertheless, the identified geneticchanges involve genes known to influence the cell cycleand cell growth, such as those located at 11q13. It iswidely accepted that alterations of some or many ofthese genes influence tumour growth and progression.

Copyright ? 1999 John Wiley & Sons, Ltd.

Our DCIS findings are broadly similar to CGHchanges reported in invasive breast cancer30–33 andshow overlapping similarities with those reported byKuukasjarvi et al.23 They studied nine cases of pureDCIS (four of which were high grade) and two cases ofDCIS mixed with invasive carcinoma. Many of thechanges in invasive carcinoma were also in the DCIS.They found 11q13 amplification in one case. James et al.also studied five cases of pure DCIS and four casesassociated with small foci of microinvasion.11 Theirhigh-grade lesions had widespread abnormalities. Bothstudies found abnormalities in low- and intermediate-grade DCIS. In general, lower-grade lesions had fewerabnormalities. It is clear from our findings, and fromprevious studies, that high-grade DCIS harboursextensive genomic alterations.

The main conclusion from many studies (often usingdifferent methodologies) is that invasive breast carci-nomas contain chromosomal abnormalities that fre-quently involve chromosomes and chromosome arms 1,2q, 3p, 5, 6q, 8, 11, 12, 13q, 14, 16, 17, and 18. The mostcommon cytogenetic abnormalities are rearrangementsof chromosome 1.34,35

The logistics of harvesting, growing, and karyotypingtumour cells from pure DCIS are overwhelminglydifficult and for practical purposes almost impossible.Nevertheless, the literature contains a couple of DCISkaryotypic studies.36 These do not state if the cases werehigh or low grade, or a combination of grades, or if theywere pure DCIS. Because these studies antedate the

J. Pathol. 187: 403–409 (1999)

408 E. MOORE ET AL.

widespread use of screening mammography, it is likelythat they were carried out on clinically palpabletumours. DCIS lends itself more readily to PCR-basedstudies, such as those looking at LOH, or in situstudies of specific gene amplifications or translocations.Previous DCIS studies found mutations and changesconsistent with loss of tumour suppressor genes, oramplification of growth-promoting genes.16,37,38 A fewstudies have shown chromosomal aneusomy using in situhybridization techniques. We have shown frequentchromosome 1 aneusomy in high-grade DCIS.39

CGH is relatively rapid and overcomes the need toculture and karyotype tumour cells. For pathologists,one of its most attractive advantages is that it can beused on archival tissue. Unlike blind tissue extractiontechniques, it allows the investigator to examine specificareas within a tumour or, as in this study, specific smallgroups of tumour cells. It distinguishes between chromo-somal amplification and loss (unlike LOH studies whichidentify allelic imbalance only23) and allows these alter-ations to be mapped to specific chromosomes and tospecific areas on each chromosome. CGH, when com-bined with target-directed microdissection, may alsobecome a powerful technique for the cytogeneticanalysis of premalignant lesions.

The main theoretical disadvantages relate to itsrelative lack of sensitivity. CGH is limited in the size ofdeletions and amplifications that it can identify. A 50 percent increase in copy number should be detectable if theregion is 2 MB or larger. An amplified region of 250 kbwould need a 40 per cent copy number increase to enableCGH detection. Detection depends on copy numberchange and the size of the amplicon. It cannot be used toidentify mutations or balanced translocations. Becauseof the relative lack of CGH sensitivity, small amplifica-tions are likely to occur more frequently than our resultsshow. High quality DNA is necessary for successfulCGH experiments. Tumour samples should contain aslittle contaminating normal DNA as possible. If morethan 50 per cent of the nuclei in a tumour section arenormal (e.g. normal epithelial, stromal or inflammatorycells), inaccurate CGH profiles will be obtained, due tothe dilution effect of normal DNA.

We started with 104 cases. After the application ofrigorous selection and quality assurance criteria wecould use only 23 cases for complete CGH analysis. Anumber of factors possibly contributed to our low yield.Our starting material came from two separate hospitalsand was not fixed under standardized conditions.Biopsies fixed in formalin for a short time (4 h) yieldexcellent DNA.20 This yield drops off with longerfixation. Screen-detected breast specimens, with theirhigh content of adipose tissue, often require prolongedfixation. Formalin fixation causes extensive cross-linkingof (nuclear) proteins, tight complexes between proteinsand DNA, and DNA fragmentation. Smooth hybridiz-ation requires that the DNA probe fragment size be inthe region of 300–1000 bp, which requires startingmaterial of fragment size greater than 3 kb.20 Anotherfactor contributing to the problem of obtaining goodquality DNA is necrosis, which is often extensive inhigh-grade DCIS.

Copyright ? 1999 John Wiley & Sons, Ltd.

From a chromosomal viewpoint, high-grade DCIS isalready an ‘advanced’ lesion. Chromosomal and (byimplication) genetic changes in high-grade DCIS areextensive. These alterations are broadly similar to thoseoccurring in invasive breast cancer. However, there mustbe genetic differences between high-grade DCIS andinvasive cancer, changes that code for invasion andmetastasis. For the present these remain elusive. Perhapssome of these alterations reside in the amplificationsidentified at 11q13.

REFERENCES1. Schwarz GF, Lagios MD, Carter D, et al. Consensus conference on the

classification of ductal carcinoma in situ. Hum Pathol 1997; 28: 1221–1225.2. Crissman JD, Visscher DW, Kubus J. Image cytophotometric DNA analy-

sis of atypical hyperplasias and intraductal carcinomas of the breast. ArchPathol Lab Med 1990; 114: 1249–1253.

3. Killeen JL, Namiki H. DNA analysis of ductal carcinoma in situ of thebreast: a comparison with histologic features. Cancer 1991; 68: 2602–2607.

4. Zhuang Z, Merino MJ, Chuaqui R, Liotta LA, Emmert Buck MR. Identicalallelic loss on chromosome 11q13 in microdissected in situ and invasivehuman breast cancer. Cancer Res 1995; 55: 467–471.

5. Radford DM, Fair KL, Phillips NJ, et al. Allelotyping of ductal carcinomain situ of the breast: deletion of loci on 8p, 13q, 16q, 17p and 17q. CancerRes 1995; 55: 3399–3405.

6. Munn KE, Walker RA, Menasce L, Varley JM. Mutation of the TP53 geneand allelic imbalance at chromosome 17p13 in ductal carcinoma in situ.Br J Cancer 1996; 74: 1578–1585.

7. Munn KE, Walker RA, Menasce L, Varley JM. Allelic imbalance in theregion of the BRCA1 gene in ductal carcinoma in situ of the breast. Br JCancer 1996; 73: 636–639.

8. Munn KE, Walker RA, Varley JM. Frequent alterations of chromosome 1in ductal carcinoma in situ of the breast. Oncogene 1995; 10: 1653–1657.

9. Yaremko ML, Kutza C, Lyzak J, Mick R, Recant WM, Westbrook CA.Loss of heterozygosity from the short arm of chromosome 8 is associatedwith invasive behavior in breast cancer. Genes Chromosomes Cancer 1996;16: 189–195.

10. Chappell SA, Walsh T, Walker RA, Shaw JA. Loss of heterozygosity atchromosome 6q in preinvasive and early invasive breast carcinomas. Br JCancer 1997; 75: 1324–1329.

11. James LA, Mitchell EL, Menasce L, Varley JM. Comparative genomichybridisation of ductal carcinoma in situ of the breast: identification ofregions of DNA amplification and deletion in common with invasive breastcarcinoma. Oncogene 1997; 14: 1059–1065.

12. Chuaqui RF, Zhuang Z, Emmert-Buck MR, Liotta LA, Merino MJ.Analysis of loss of heterozygosity on chromosome 11q13 in atypical ductalhyperplasia and in situ carcinoma of the breast. Am J Pathol 1997; 150:297–303.

13. De PC, Schelfhout AM, Verbeeck P, et al. neu overexpression correlateswith extent of disease in large cell ductal carcinoma in situ of the breast.Hum Pathol 1995; 26: 601–606.

14. Poller DN, Silverstein MJ, Galea M, et al. Ideas in pathology. Ductalcarcinoma in situ of the breast: a proposal for a new simplified histologicalclassification association between cellular proliferation and c-erbB-2 proteinexpression. Mod Pathol 1994; 7: 257–262.

15. Leal CB, Schmitt FC, Bento MJ, Maia NC, Lopes CS. Ductal carcinomain situ of the breast. Histologic categorization and its relationship to ploidyand immunohistochemical expression of hormone receptors, p53, andc-erbB-2 protein. Cancer 1995; 75: 2123–2131.

16. Liu E, Thor A, He M, Barcos M, Ljung BM, Benz C. The HER2 (c-erbB-2)oncogene is frequently amplified in in situ carcinomas of the breast.Oncogene 1992; 7: 1027–1032.

17. Hermsen MA, Meijer GA, Baak JP, Joenje H, Walboomers JJ. Compara-tive genomic hybridization: a new tool in cancer pathology. Hum Pathol1996; 27: 342–349.

18. Kallioniemi A, Kallioniemi OP, Sudar D, et al. Comparative genomichybridization for molecular cytogenetic analysis of solid tumors. Science1992; 258: 818–821.

19. Speicher MR, du Manoir S, Schrock E, et al. Molecular cytogenetic analysisof formalin-fixed, paraffin-embedded solid tumors by comparative genomichybridization after universal DNA-amplification. Hum Mol Genet 1993; 2:1907–1914.

20. Isola J, DeVries S, Chu L, Ghazvini S, Waldman F. Analysis of changesin DNA sequence copy number by comparative genomic hybridizationin archival paraffin-embedded tumor samples. Am J Pathol 1994; 145:1301–1308.

21. Lagios MD. Duct carcinoma in situ. Pathology and treatment. Surg ClinNorth Am 1990; 70: 853–871.

J. Pathol. 187: 403–409 (1999)

409CGH ANALYSIS OF HIGH-GRADE DCIS

22. Pan LX, Diss TC, Peng HZ, Isaacson PG. Clonality analysis of definedB-cell populations in archival tissue sections using microdissection and thepolymerase chain reaction. Histopathology 1994; 24: 323–327.

23. Kuukasjarvi T, Tanner M, Pennanen S, Karhu R, Kallioniemi OP, Isola J.Genetic changes in intraductal breast cancer detected by comparativegenomic hybridization. Am J Pathol 1997; 150: 1465–1471.

24. Brinkschmidt C, Christiansen H, Terpe HJ, et al. Comparative genomichybridization (CGH) analysis of neuroblastomas—an important methodo-logical approach in paediatric tumour pathology. J Pathol 1997; 181:394–400.

25. Piper J, Rutovitz D, Sudar D, et al. Computer image analysis of compara-tive genomic hybridization. Cytometry 1995; 19: 10–26.

26. du Manoir S, Schrock E, Bentz M, et al. Quantitative analysis of compara-tive genomic hybridization. Cytometry 1995; 19: 27–41.

27. Hui R, Campbell DH, Lee CS, et al. EMS1 amplification can occurindependently of CCND1 or INT-2 amplification at 11q13 and mayidentify different phenotypes in primary breast cancer. Oncogene 1997; 15:1617–1623.

28. Theillet C, Le RX, De LO, et al. Amplification of FGF-related genesin human tumors: possible involvement of HST in breast carcinomas[published erratum appears in Oncogene 1989; 4: 1537]. Oncogene 1989; 4:915–922.

29. Rubin H. Mutations and oncogenes—cause or effect. Nature 1984; 309: 518.30. Nishizaki T, DeVries S, Chew K, et al. Genetic alterations in primary breast

cancers and their metastases: direct comparison using modified comparativegenomic hybridization. Genes Chromosomes Cancer 1997; 19: 267–272.

Copyright ? 1999 John Wiley & Sons, Ltd.

31. Courjal F, Theillet C. Comparative genomic hybridization analysis of breasttumors with predetermined profiles of DNA amplification. Cancer Res 1997;57: 4368–4377.

32. Nishizaki T, Chew K, et al. Genetic alterations in lobular breast cancer bycomparative genomic hybridization. Int J Cancer 1997; 74: 513–517.

33. Barlung M, Tirkkonen M, Forozan F, Tanner MM, Kallioniemi O,Kallioniemi A. Increased copy number at 17q22–q24 by CGH in breastcancer is due to high-level amplification of two separate regions. GenesChromosomes Cancer 1997; 20: 372–376.

34. Heim S, Mitelman F. Tumors of the breast. In: Anonymous CancerCytognetics. 2nd edn. New York: Wiley-Liss, 1995: 369–388.

35. Mitchell EL, Santibanez-Koref MF. 1p13 is the most frequently involvedband in structural chromosomal rearrangements in human breast cancer.Genes Chromosomes Cancer 1990; 2: 278–289.

36. Nielsen KV, Blichert Toft M, Andersen J. Chromosome analysis of in situbreast cancer. Acta Oncol 1989; 28: 919–922.

37. Bishop JM, Weinberg RA. Scientific American Molecular Oncology. NewYork: Scientific American Medicine, 1996.

38. O’Malley FP, Vnencak Jones CL, Dupont WD, Parl F, Manning S, PageDL. p53 mutations are confined to the comedo type ductal carcinoma in situof the breast. Immunohistochemical and sequencing data. Lab Invest 1994;71: 67–72.

39. Harrison M, Magee HM, O’Loughlin J, Gorey TF, Dervan PA. Chromo-some 1 aneusomy, identified by interphase cytogenetics, in mammographi-cally detected ductal carcinoma in situ of the breast. J Pathol 1995; 175:303–309.

J. Pathol. 187: 403–409 (1999)