0 1995 Wiley-Liss, Inc. Cytometry 2195-100 (1995)

Detection of Chromosome Aneuploidy in Breast Lesions With Fluorescence In Situ Hybridization:

Comparison of Whole Nuclei to Thin Tissue Sections and Correlation With Flow Cytometric DNA Analysis

Daniel W. Visscher, Tracie Wallis, and Carole A. Ritchie Department of Pathology, Hdrper Hospital and Wayne State University, Detroit, Michigan

Received for publicdtion Januar) 1 1995 accepted Mdrch 28, 1995

We compared flow-cytometric DNA histogram pat- tern to counts of 4 fluorescent-labelled centromeric probes (chromosomes 1,7,8, and 17) in whole nu- clei (WN) and in nuclei from the corresponding for- malin-fixed deparaffinized thin tissue section (TS) in 25 breast lesions (9 invasive carcinomas, 1 duct carcinoma-in-situ, 5 fibroadenomas, 10 fibrocystic change). In benign lesions, signal gains (i.e., tri- somic nuclei) were never observed in greater than 10% of nuclei from either WN or TS preparations. Loss of signal in benign breast lesions, however, varied considerably (043%) between individual case and between chromosome probes. The mean incidence of signal loss in WN of benign lesions ranged from 8.9% (chromosome 7) to 14.4 % (chro- mosome 1) of nuclei. These signal loss frequencies exceeded those of benign lymphoid control cells. In three benign lesions, signal loss in WN (with one probe) was observed in at least 25% of nuclei. Signal Losses in benign TS, on average, were 50-150% greater than in matched WN preparations (chromo- some 1-21.7%, chromosome 7-21.5%). Malignant lesions generally, but not always, displayed fewer

monosomic nuclei and more trisomic nuclei in WN compared to TS, compatible with a slicing (i.e., nu- clear truncation) artifact. Signal counts in carci- nomas correlated well with flow cytometric DNA index; however, they were also characterized by ev- idence of genetic instability, manifest as signal gains in a subset of nuclei (10-25%) with individual probes in diploid range cases, as well as intratu- moral heterogeneity, reflected as discrepancies in probe counts between WN and TS samples.

We conclude that signal losses with centromeric probes are largely, but not entirely, explained by nuclear slicing. The miniium signal loss threshold for establishment of monosomy using interphase cytogenetics is thus unclear, even in WN. Signal gains indicative of trisomy, in contrast, are reliably associated with malignancy and may reflect gross DNA aneuploidy as well as genetic instability. 0 1995 Wiley-Liss, Inc.

Key terms: Interphase cytogenetics, breast carci- noma, proliferative breast disease

With fluorescent-labelled, centromere-specific DNA probes it is possible to assess chromosome copy number without metaphase spreads. This technology, interphase cytogenetics, may be adapted to intact nuclei, as in touch preparations and fine needle aspirates, or to cells in par- affin-embedded tissue sections. Evaluation of cytogenetic profile in archival paraffin blocks offers the advantages not only of selectivity for potentially uncommon lesions of interest, but also of the capability of studying cell pop- ulations in the context of precise microanatomical distri- bution (1) . It would be possible, for example, to assess the pre-invasive component of a breast carcinoma or a dedifferentiated focus in a sarcoma.

One major issue with enumerating chromosome copy number in thin tissue sections (TS) is the artifact induced by nuclear slicing. Trisomic nuclei in TS would thus be

underrepresented and many disomic nuclei would be falsely represented as monosomic. Since aneuploid chro- mosome gain is considered pathological in some settings, nuclear slicing would also impose a theoretical reduction in sensitivity. In the case of monosomy, however, speci- ficity becomes an issue, since signal loss may reflect true deletion, inefficient probe hybridization or nuclear slic- ing. This is especially problematic if only a portion of the population at interest has lost a chromosome copy.

Presented in part at thc Annudl Meeting of the United States and Ca- nadian Academy of Pathology, San Francisco, California, March 12-18,

Address reprint requests to Daniel W. Visscher, M.D., Department of 1994.

Pathology, Harper Hospital, 3990 John K. Detroit, MI 48201.

96 VISSCHER ET AL.

Previous studies in our laboratory, employing inter- phase cytogenetics, have suggested that chromosomal losses may occur in certain forms of proliferative breast disease (7). These data were noteworthy in view of meta- phase analyses, which clearly show that chromosome losses accompany the progression of malignant breast neopiasia ( 1-3,5,8). Since tissue dissociation renders evaluation of focal, microscopic proliferative breast le- sions quite difficult and since metaphase analyses of be- nign breast epithelial populations are generally unsuc- cessful, interphase cytogenetic analyses is a potentially useful method of studying pro1 iferative breast disease. The objective of this study was to carefully define the extent of artifacts associated with detection of chromo- somal numerical alterations in tissue sections of breast lesions using interphase cytogenetics.

MATERIALS AND METHODS Case Selection and Tissue Handling

Breast tissues were obtained prospectively from the Surgical Pathology and Histology Laboratory at Harper Hospital (Detroit, MI). They represented diagnostic spec- imens (mastectomy and biopsy) received fresh for patho- logic evaluation. Following dissection, a representative slice of lesional tissue was selected and utilized to make 6 whole nuclear (WN) imprint preparations by touching to polylysine coated slides. These slides were fixed im- mediately by immersion in methanoVacetic acid. One of the imprint slides was stained with hematoxylin and eosin and examined to ensure adequate cellularity and representation. The corresponding tissue slice was then fixed in neutral buffered formalin and embedded in par- affin; the hematoxylin and eosin stained sections, cut with minimal trim, were then used to select a represen- tative area’(i.e., corresponding to imprints) for interphase cytogenetic analysis. This was performed on 4 pm sec- tions applied to silanized slides. In malignant cases a mir- ror image tissue slice was utilized for flow cytometric DNA analysis, performed as described previously (10).

A total of 15 benign biopsies were evaluated. Among these were 4 fibroadenomas, 1 tubular adenoma, and 10 cases of fibrocystic change (2 non-proliferative, 1 adeno- sis, 4 mild-moderate duct epithelial hyperplasia, 3 florid duct epithelial hyperplasia). One example of non-prolif- erative fibrocystic change was obtained from benign tis- sue in a mastectomy specimen (i.e., in a quadrant remote from a previous biopsy site). The malignant lesions in- cluded 9 invasive and 1 in situ carcinomas. Flow cyto- metric DNA indices, available on the invasive tumors, included 4 diploid range tumors, 1 hypodiploid tumor, 4 hyperdiploid tumors, 1 tetraploid tumor and 1 hypertet- raploid tumor. For normal tissue control, hybridizations and counts were performed on a benign lymph node from which imprints and tissue sections had been prepared in a similar manner.

Probe Selection and Hybridization Fluorescent-labelled (digoxygenin or FITC) centro-

meric probes and most reagents necessary for hybridiza-

tion were purchased from Oncor (Gaithersburg, MD). Four probes (chromosomes 1, 7 , 8 , 17) were selected for analysis based on their strong signals and the relative frequency with which these chromosomes are numeri- cally altered in karyotypes of breast carcinomas.

Hybridizations were performed essentially per instruc- tions which accompany the probes, with minor modif- cation. Briefly, following slide dehydration with graded ethanol washes, denaturation was performed by incuba- tion in formamide solution at 70°C. Hybridization with labelled probes was carried out in a humidified chamber ( 4 16-h incubations at 43°C). After washes with forma- mide and then phosphate buffer detergent solutions, rho- damine anti-digoxigenidFITC avidin was applied to slides (45 pl, 15 min, 37°C). The nuclei were counter- stained with DAPI.

Hybridization of intact tissue sections differed from im- prints in the following manner: 1) The slides were de- paraffinized in xylene and incubated with sodium met- abisulfite powder (37”C, 15-30 min). 2) The sections were digested with proteinase K (Oncor) (43”C, 10-50 min) to expose target DNA via removal of nuclear inem- brane and matrix proteins in formalin-fixed cells. 3) Posthybridization washes were carried out at low strin- gency compared to imprints.

Signal Enumeration and Analysis Only signals in non-overlapped, apparently intact nu-

clei were counted. In both TS and WN slides, nuclei ap- parently corresponding to spindle cells or inflammatory populations were avoided. All cases were counted by one individual (TW) and, in each sample, 200 nuclei were counted. Percentages of disomic ( 2 signals), monohulli- somic (<2 signals), and trisomic ( > 2 signals) nuclei were calculated for each sample and direct comparisons were made between imprints and the corresponding thin tissue section. Although we employed lymph nodes as a “control“ sample, it should be recognized that lymphoid and breast tissues are not completely analogous with re- spect to factors which may affect probe hybridization and signal detection (i.e., typdamount of stroma, nuclear size, and cell orientation). We thus view lymphoid control tissue, especially imprints, primarily as a measure of in- herent probe hybridization efficiency. For these reasons, conservative criteria were employed as thresholds for un- equivocal aneusomy; > lo% cells with signal gain for trisomy and >50% of cells with signal loss for mono- somy. We acknowledge that “partial” monosomy (i.e., chromosome loss in minority of the population counted) would not necessarily be accounted for using this threshold.

RESULTS Control and Benign Tissues

In benign lymphoid control tissue, the frequency of trisomic nuclei for all probes was less than 7% (TS and WN preparations). The frequency of monosomy was less than 10% in imprints and less than 20% in tissue sec- tions.

CHROMOSOME ANEUPLOIDY I N BREAST LESIONS 97

Table 1 Incidence (%) of Chromosome Signal Losses in Whole Nuclei

(WNj us, Corresponding Thin Tissue Section (TS) of Benign Cases

Chromosome 1 7 a 17

Case WN TS WN TS WN TS WN TS - 1 2 3 4 5 6 7 8 9

10 1 1 12

NA 17 15 12 26 17 7

NA 10 20

5 15

NA 27 15 40 28 23 26 NA 19 25 14 15

10 3

16 14 12 3

15 12 2 3

15 10

20 23 32 30 19 28 15 23 21 15 16 43

5 28 14 1 1 15 10 3

16 5

10 10 13

12 12 13 15 13 22 15 14 22 16 20 5 12 14 30 19 15 17 17 10 14 5 26 25

29 31 35 40 22 41 22 37 16 14 13 26

Table 2 Incidence (%) of Signal Gains in Benign Breast Cases

Chromosome (sample type) Caseldiagnosis 1 7 8 17

1 NPa NA lO(WN) 0 0 2 T A ~ 0 0 0 0 3 FAc 0 0 0 0 4 FA 5 FA 6 FA 7 NP

9 PBD 10 PBD 1 1 PBD 12 PBD 13 PBD 14 PBD 15 PBD

a P B D ~

0

0 0 NA 2.5 (WN) 3.5 (WN) 0

2 (TS)

4 (WN) 9 (WN) 3 (TS) NA

0

0 0 0 0 0 0 0 0 0 0

2 (TS)

13 19 1 2 2 7 8 2 1 2 7 "NP = non-proliferative fibrocystic changes. 14 10 20 12 31 10 16 17 37 15 NA NA 4 0 4 25 8 27 'FA = fibroadenoma. Average (%) 14.4 22 8.9 21.5 10.8 18.1 13.4 26.5 dPBD = proliferative breast disease.

bTA = tubular adenoma.

The chromosome counts for each of the 15 benign breast lesions are summarized in Tables 1 and 2. For whole nuclei, the incidence of signal loss (ix., apparent monosomy and nullisomy) varied from 2 to 28%, and in the corresponding thin tissue section it varied from 0 to 43%. The average signal loss (combined monosomy and nullisomy) in whole nuclei was 8.9% for chromosome 7, 10.8% for chromosome 8, 13.4% for chromosome 17, and 14.4% for chromosome 1. This signal loss was largely accounted for by cells having one signal. Average fre- quency of nullisomy in whole nuclei was 1.6% for chro- mosome 1, 0.6% for chromosome 7, 1.4% for chromo- some 8, and 1.3% for chromosome 17 (range 0-9%). Average percent signal losses in corresponding TS sam- ples were significantly greater: 22% for chromosome 1, 21.5% for chromosome 7 , 18.1% for chromosome 8, and 26.5% for chromosome 17. Comparison of WN vs. TS counts for individual cases revealed disparities in degree of signal loss which could generally be accounted for by nuclear slicing artifact (see Fig. 1). The WN signal loss frequency exceeded the TS signal loss frequency in orJy 5 (out of 5 7 ) pairs and in only one of these 5 was it more than 10% greater.

Signal gains were detected in 10 of the 15 benign cases (67% ), and were always observed in 10% or fewer nuclei (see Table 2). Case one exhibited 10% of whole nuclei with four signals (tetrasomy) for chromosome 7. Four of the cases displayed signal gain with two or more probes. Signal gain was evenly distributed between intact nuclear preparations and tissue sections. There was no clear as- sociation between presence of trisomic nuclei and either the degree of signal loss or the histologic features. Gains were more frequent with probes for chromosomes 1 and 8 (6 cases each) than chromosome 7 (4 cases) or 17 (one case).

Malignant Tumors

Table 3 summarizes the percentages of signal gain and loss by chromosome, type of sample (i.e., WN vs. TS), and DNA Index in the carcinomas. As a group, the carcinomas are considerably more heterogeneous with respect to aneusomy than the benign lesions. This reflects not only the greater degree and complexities of signal gains and losses, but also less concordance between whole nuclear and tissue section preparations.

The five flow-cytometrically near-diploid tumors (cases 1-5 in Table 2) typify the heterogeneity associ- ated with malignant lesions. In three of these five, signal losses compatible with unequivocal monosomy were ap- parent, albeit with only one probe (cases 1 and 2-chro- mosome 17; case 5-chromosome 7). In four of the five near-diploid tumors, moreover, signal gains exceeding 10% of nuclei were observed. However, these signal gains uniformly involved less than 25% of counted nuclei and were identified in onlj one type of sample (i.e., WN vs. TS) with only one probe. One near-diploid tumor (case 3) was essentially norma1,as was the single exam- ple of duct carcinoma-in-situ (case 6) .

All of the flow-cytometrically DNA hyperdiploid tu- mors (cases 7-10) exhibited unequivocal signal gain with at least two chromosome probes. In three of these four, moreover, signal gains exceeded 50% of enumer- ated nuclei. None of the hyperdiploid carcinomas dis- played unequivocal signal loss for the probes tested. Gen- erally, tissue sections displayed higher frequencies of signal loss and lower frequencies of gain than correspond- ing touch preparations. This relationship, however, was less predictable than in the benign cases. Four of the 34 WN/TS pairs (12% ) had significant disparities (i.e., dif- ferences which would result in different interpretations)

FIG. 1 .

CHROMOSOME ANEUPLOIDY IN BREAST LESIONS 99

Table 3 Incidence of Chromosome Signal Losses and Gains in Whole Nuclei (WN) us. Thin

Tissue Sections (TS) of Malignant Breast Tumorf

Chromosome (% with loss/% with gain) 1 7 8 17

DNA index WN TS WN TS WN TS WN TS 0.9 3/78 - 8/16’ 3/5 22/0 19/0 62/0 76/0 1 .o 24/4 25/4 16/0 25/0 15/4 4/13 66/0 56/0 1.0 20/0 22/2 11/0 24/0 11/0 26/0 28/0 18/0 1.0 - - 118 3710 2111‘ 29/23 5/8 2710 1.0 5/21 18/15 10b/6 51/0 7/20 23/10 10/10 27/0 NA 20/0 9/0 11/0 910 15/0 3210 1910 3310 1.6 0/76 - 0182 OR4 4/74 28/58 0183 O f 7 1 1.7 - - 0/60 14/56 7/17 13/7 713 23/0 2.0 - - 20/vb 21/23 8/24 20/29 36/8 22/0 3.0 - - 0/67 0/65 4/5 2013 0167 OR7

“Boldface = examples with unequivocal aneusomy. Case 6 = carcinoma-in-situ. bDiscordant WN-TS pairs.

in degree of signal loss or gain for one or more probes (see Table 2).

DISCUSSION Our data show that in thin tissue sections, a significant

proportion of signal loss with fluorescent-labeled centro- meric probes (approximately 30-50% of total) is likely an artifact of nuclear slicing. Nevertheless, whole nuclear touch preps of benign lesions may demonstrate signal loss frequencies substantially greater than the control 5-10% rate observed with imprints of benign lymphoid tissue. Although in most such cases this frequency was consid- erably less than the 50-80% rate of monosomy observed in near-diploid or hypodiploid carcinomas, it equalled or exceeded 20% (with at least one probe) in whole nuclei from 15 (33%) benign cases. Further, this occurred de- spite a low average frequency of nullisomy ( < 2 % ). We have thus failed to exclude nuclear slicing and hybridiza- tion inefficiency as the sole causes of centromere signal losses in benign breast lesions.

It would be incorrect to imply that nuclear truncation and hybridization inefficiency (i.e., inherent to each probe) were the only artifacts associated with our use of interphase cytogenetic technology in this study. Since the touch preparations were not exposed to deparaffiniza- tion, or protease digestion, and were fixed differently than the tissue sections, they may have had different pro- cedure related hybridization efficiencies. It is unclear, though, whether these differences would be expected to impair hybridization relative to TS. Second, although a section from the face of a tissue block may represent the

FIG. 1. Comparison of TS and WN preparations of breast carcinoma. Top: Low magnification photomicrograph of an intact TS, showing re- tention of tissue architecture following protease digestion. Middle: Same section hybridized to centromeric probe for chromosome 8 showing many sliced nuclei with rare cells exhibiting three signals. Bottom: Corresponding WN, hybridized with same probe, showing signal gain in all nuclei.

best approximation of a corresponding whole nuclear sample, it is not perfect. Some degree of trimming is in- evitable during slide preparation, which may expose ge- netically different populations. A degree of morphologic, and likely genetic, heterogeneity may even be observed within the populations present in a single tissue section. This factor may have accounted for discrepant analyses among the malignant tumors, which are notoriously het- erogeneous with respect to chromosome aneusomies (8). Finally, manual counting of fluorescent signals intro- duces biases into enumeration of chromosome counts. It is not always possible to distinguish nuclei of lesional cells in touch preparations from those of normal epithe- lial or stromal derivation, particularly following process- ing. In both tissue sections and imprints, moreover, signal overlapping may occur. A variety of other selection arti- facts may also occur, depending on the experience and diligence of the individual performing the counts. Be- cause of these considerations, TS and WN preparations should probably be viewed as complimentary, with each having inherent limitations.

Despite these and other limitations of interphase cyto- genetics, we believe that, in at least some cases, signal losses in less than 50% of nuclei may indeed reflect “par- tial” monosomy, or chromosome loss in a subset of cells. Loss of whole chromosomes has been demonstrated in benign breast lesions, including fibroadenomas (4). De- gree of signal loss in some cases, moreover, was analo- gous to that observed in some malignant lesions, where toss of chromosomes in karyotypes is also frequent within varying proportions of neoplastic cells.

What the minimum threshold of signal loss frequency should be, even in whole nuclear preparations, in order to establish “true” monosomy is not entirely clear. Ex- periments utilizing tumors with known karyotype would be quite useful in this respect but likely would not re- solve the issue of intratumoral genetic heterogeneity. Based on our data we would argue that signal losses in greater than 25% of whole nuclei, or in greater than 40% of nuclei from thin tissue sections, are highly suspicious

100 VISSCHER ET AL.

of monosomy involving a subset of nuclei. Further, we would view over 30% signal loss in WN and 50% signal loss in TS as very convincing evidence of monosomy. These cutoffs are admittedly arbitrary, and therefore not applicable to other studies, especially in view of the vari- ability associated with tissue type, probe characteristics, and hybridization conditions between laboratories.

Derivation of criteria for genetic deletions involving whole or partial chromosomes using DNA in situ hybrid- ization technology is not a trivial undertaking. Whole chromosome losses, as presumably detected in our study, are one of many cellular level genetic aberrations in breast carcinomas, and their clinical relevance remains undetermined. With use of locus-specific sequence com- plementary probes, however, it has become possible to enumerate the copy number of tumor suppressor genes, losses of which are putatively important to neoplastic progression. Obviously, the ability to perform and inter- pret such analyses in the context of extremely focal (or uncommon) archival lesions will have considerable prac- tical importance. Use of thick tissue sections has been advocated by some as a methodologic improvement to refine interphase cytogenetic technology (9).

The results of this study also have implications con- cerning the interpretation and meaning of signal gains. Trisomic nuclei never constituted greater than 10% of the count in the benign tissues. Although our sample size is small, making generalizations difficult, these data seem to suggest that significant trisomy (i.e., with these probes) is either uncommon in benign breast lesions or an event which is closely associated with morphologic evidence of malignant transformation. We are uncertain whether to interpret rare trisomic nuclei in benign tis- sues as artifactual (due to inadvertent counting of over- lapped nuclei or “false” hybridization) or real (i.e., bio- logical). Others have reported trisomy in benign epithelial populations; however, these cells were present in tissues which harbored either pre-invasive or invasive carcinomas (6). It is noteworthy that one of our benign cases with signal gain was derived from a mastectomy specimen, in an area which was histologically negative for malignant neoplasm.

Significant trisomy (i.e., >lo% of nuclei), in contrast, was observed in most of the malignant tumors, including

the diploid range cases. It may be noted from Table 3 that some trisomies observed in DNA hyperdiploid or hyper- tetraploid tumors are apparently clonal, since they were present within a majority of the counted cells. Trisomic cells in diploid range tumors, however, were focal in dis- tribution. This may also be observed in karyotype analy- ses of breast carcinomas, in which chromosome counts generally diverge in a normal distribution from a modal number ( 2 ) . Such phenomena have been interpreted as a reflection of “genetic instability,” or inability of the tis- suekumor to maintain a consistent DNA content and structure (6). These “partial” trisomies also highlight the relative insensitivity of cytophotometric DNA analysis as a measure of genetic deviation in neoplasia, since they were also present in flow cytometrically diploid range cases.

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