Vol. 4, 1543-1547, June 1998 Clinical Cancer Research 1543

Flow Cytometric DNA Analyses of Benign Breast Lesions Detected

by Screening Mammography

Paul C. Stomper,’ James R. DeBloom II,

Rose Marie Budnick, and Carleton C. Stewart

Division of Diagnostic Imaging [P. C. S.l and Department of Flow

Cytometry [R. M. B.. C. C. S.], Roswell Park Cancer Institute. Schoolof Medicine and Biomedical Sciences, State University of New Yorkat Buffalo, and Department of Natural Sciences, Roswell Park

Division of the State University of New York at Buffalo [J. R. D.J,

Buffalo, New York 14263

ABSTRACT

There is little information regarding flow cytometricDNA analyses of benign breast lesions. This prospective

study consists of mammographic and pathological correla-

tion of DNA flow cytometric analyses of specimen mammog-raphy-guided fine-needle aspirates (FNAs) of 189 consecu-tive benign breast lesions and 114 FNAs of adjacent normal

tissue as a control. Clinical follow-up was also performed.

Aneuploidy was detected in 14 of 189 (7%) benign lesion

specimen mammography-guided FNAs and in only 1 of 114

(0.9%) FNAs of adjacent normal tissue (P = 0.01). Aneu-ploidy was detected in two (33%) benign intramammarylymph nodes compared with four (12%) benign lesions withatypia, one benign lesion (3%) with hyperplasia, four benign

lesions (10%) with adenosis, and three (4%) other benign

lesions (P = 0.01). There were no significant associationsbetween DNA content and S-phase percentage and patientage, mammographic appearance, or extent. During a me-

dian follow-up of 40 months (range, 6-84 months), 2 of 13

(15%) patients with aneuploid benign lesions developed ip-

silateral breast carcinoma compared with 5 of 175 (3%)patients with diploid benign lesions (odds ratio, 6.18; 95%

confidence interval, 1.08-35.56). Our data suggest that ane-uploidy, which is detected in a variety of benign breast

lesions, may be associated with a higher risk of development

of breast carcinoma. The combined techniques of specimenmammography-guided fine-needle aspiration and flow cy-tometry provide a practical translational research method

for the study of benign breast disease.

INTRODUCTION

The increased utilization of screening mammography has

led to increased numbers of either excisional or percutaneous

Received 1/5/98; revised 3/20/98: accepted 3/23/98.The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby marked

advertisement in accordance with I 8 U.S.C. Section 1734 solely to

indicate this fact.

I To whom requests for reprints should be addressed, at Division ofDiagnostic Imaging, Roswell Park Cancer Institute, Elm and Carlton

Streets, Buffalo, NY 14263. Phone: (716) 845-5796; Fax: (716) 845-

8759; E-mail: stomper@sc3l02.med.buffalo.edu.

core needle biopsies of clinically occult lesions that subse-

quently prove to be benign. Whereas efforts should continue to

be made to decrease the number of false positive mammograms

and benign biopsies, the study of this biopsy material may lead

not only to further understanding of benign and premalignant

breast disease but also to a possible means of risk assessment for

the subsequent short- or long-term development of breast car-

cinoma for women who undergo benign breast biopsies.

There is little information in the literature regarding ploidy

and S-phase percentage analysis in benign breast lesions (1-6).

There is extensive literature regarding flow cytometrie DNA

analysis of invasive breast carcinoma, and there are several

reports of DNA analysis of ductal carcinoma in situ (5, 7-14).

We have previously reported the use of specimen mammogra-

phy-guided fine-needle aspiration of clinically occult lesions

within excised specimens for DNA analysis by flow cytometry

(15, 16).

This report analyzes a prospective pilot study that was

performed: (a) to determine the feasibility of flow cytometrie

DNA analysis of specimen mammography-guided FNAs2 of

clinically occult lesions detected during screening mammogra-

phy that proved to be benign after wire localization and cxci-

sion; (b) to explore associations with mammographie and path-

ological features of the benign lesions; (c) to explore the

frequency of aneuploidy in benign lesions; and (d) to assess any

subsequent short-term risk of development of breast carcinoma.

MATERIALS AND METHODS

The study group consists of I 88 consecutive patients who

underwent biopsies of 189 clinically occult lesions detected by

screening mammography that showed benign pathology at path-

obogical exam. The median age of the patients in the study group

was 62 years (range, 31-85 years). Eighty-four (44%) of the

patients were less than 50 years old: 105 (56%) were 50 years or

older. Written informed consent was obtained.

Mammograms were obtained with a Senographe 600 1 or

DMR unit (General Electric Medical Systems, Milwaukee, WI)

using Min-R cassettes and Min-R film (Eastman Kodak, Roch-

ester, NY) at a cancer institute mammography center. Each of

the calcified lesions was imaged in vivo with accessory magni-

fication projections. Mammographically guided needle localiza-

tion procedures were performed using the method described by

Kopans et a!. (17). Radiography of the specimen was performed

using the compression and magnification technique (X 1.85) in

each ease with the clinical DMR mammography unit.

The surgical staff was notified immediately of the confir-

mation of the excision of lesions. After the removal of com-

pression, the mammographer performed a 20-gauge needle as-

2 The abbreviations used are: FNA. fine-needle aspirate; LCIS. lobular

carcinoma in situ.

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1544 Flow Cytometric DNA Analyses of Benign Breast Lesions

Table I Flow cytometric DNA analyses of specimen

mammography-guided FNAs of benign breast lesions

No. of lesions 189

No. of aneuploid lesions 14 (7%)

No. of dipboid lesions 175 (93%)

Median S-phase % 2.0Lower 33% S-phase % 1.6Higher 33% S-phase % 4.1

piration within the suspect mammographic lesion in the

operative specimen (16). Needle guidance techniques based on

the radiography of the specimen included: (a) the use of cir-

cumferential coordinates or surface landmarks on the specimen

for larger lesions; and (b) the insertion of a localizing needle and

repeat radiography of the uncompressed specimen for smaller

lesions. Fine-needle aspiration was performed throughout the

mammographically depicted lesion to provide adequate lesion

sampling. The needle aspirate was then sent for flow eytometric

DNA analysis. Exeisional biopsy specimens were sent to the

cancer institute department of pathology.

For I 14 consecutive benign lesions, a second needle was

used to aspirate adjacent normal breast tissue at least 2 cm from

the index lesion within the excised specimen under mammo-

graphic guidance. These samples also underwent flow cytomet-

nc DNA analysis.

The needle aspirates sent for flow cytometric DNA analy-

sis were dispersed by means of repeated aspiration through a

25-gauge needle and then fixed in 70% ethanol and stored at

-20#{176}C.For analysis, the cells were centrifuged, and the pellet

was stained with propidium iodide (50 p.g/ml) in Kristen buffer

[0. 1% sodium citrate, 0.02 mg/mI RNase A, and 0.37% NP4O

(pH 7.4)]. Samples of lysed whole blood from a healthy donor

and chick RBCs processed in identical fashion were also stained

and evaluated to verify the daily reproducible performance of

the cytometer. A minimum of 1000 cells from the needle aspi-

rates were analyzed on a FACSean flow cytometer (Becton

Dickinson, San Jose, CA) with a double t discriminator module

and an excitation beam at 488 nm. Data were collected for the

fluorescence 2 channel, measuring propidium iodide emission

through a 585/42 band-pass filter. Chick RBCs were added to

the samples as an internal standard. The photomultiplier tube

voltage for the fluorescence 2 channel was set so that the cells

in healthy donor blood peaked at channel 200. A threshold of 28

was set on this channel to eliminate cellular debris but include

RBCs. All samples were collected ungated.

Data were first analyzed with the Lysis II program (Beeton

Dickinson) to exclude doublets and debris. The resulting histo-

gram files were further analyzed with Multicycle (Phoenix Fbo

Systems, San Diego. CA). This program calculated the percent-

age of cells in the , 5, and 02 phases for each cycling cell

population present as well as the DNA index of the standard

(chick RBCs) and any aneuploid peak present compared with

that of the diploid peak. The DNA index is the ratio of the peak

0 1 channel of either the standard or the aneupboid cells:the peak

01 channel of dipboid cells. The BAD index and coefficient of

variation for the sample are a measure of the quality of the

sample staining. All specimens exhibited a BAD of less than

25% and a coefficient of variation of less than 6%. The DNA

Table 2 Comparison of DNA content of specimen mammography-

guided FNAs of benign breast lesions and adjacent normal tissue

No. of lesions (%)

DNA content

FNAsample F’ Aneuploid Diploid

Benign lesions 189 ( 100) 14 (7)” 175 (93)

Adjacent normal tissue I 14 (100) I (0.9Y’6 133 (99)

“ P = 0.01.

‘, Aneuploid normal tissue was adjacent to a diploid benign lesion.

index for the sample is a measure of the degree of aneuploidy in

the abnormal population. Without knowledge of mammographic

and pathological findings, coauthor C. C. S. performed all flow

cytometry and interpreted the results.

Cell populations with a DNA index of 1.0 ± 0. 1 (2 SDs)

were considered diploid. All others were considered to have

aneuploidy present. All needle aspirates, including those that

had aneupboid populations, had some dipboid cells present.

When aneupboid cells were present, the aneuploid S-phase per-

centage was used for S-phase percentage analysis rather than the

diploid cell population S-phase value. Cases were then desig-

nated as having either a low, intermediate, or high S-phase

percentage based on their placement within the 33% percentiles

determined for the study group.

Histological material derived from excisional biopsy was

stained with H&E stain. All pathological interpretations were

performed prospectively by the cancer institute department of

pathology staff without knowledge of the flow cytometrie find-

ings. Retrospective pathological review of benign lesions in

patients who subsequently developed ipsilateral breast earci-

noma was also performed. There was no evidence of malig-

nancy in the benign excisional biopsies during each pathological

review. Histological assessment of the entire biopsy specimen

was made with the knowledge of the presence and location of

the mammographie abnormality within the specimen, so that

specific anatomical correlation could be made in each ease. For

calcified lesions, the specimen was sliced and reradiographed to

aid localization during pathological examination.

Benign lesions were grouped into five pathological cate-

gories: (a) atypical hyperplasialLClS; (b) hyperplasia; (c) ad-

enosis; (d) intramammary lymph nodes; and (e) other benign

lesions. These pathological diagnosis categories were then com-

bined as proliferative (categories a-c) and nonproliferative (eat-

egories d and e). Prospective mammographic interpretations

were categorized by appearance: (a) soft tissue mass content or

with caleifications; or (b) mierocaleifleations only. Mammo-

graphic lesion extent was categorized as 1-10, 1 1-20, and

>20 mm.

Clinical follow-up to determine the subsequent develop-

ment of breast carcinoma after the initial benign biopsies was

performed by mammographie and clinical chart review. Statis-

tical analyses were performed with x2 and odds ratio analysis

( 18). Ps < 0.05 were considered significant. The odds ratio was

defined as the relative risk of carcinoma in patients with aneu-

ploid benign lesions relative to that in patients with diploid

benign lesions and was considered significant if the lower limit

of the 95% confidence interval was greater than 1.

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Clinical Cancer Research 1545

Table 3 Associations between clinical mammographic features and flow cytometric DNA analyses of benign lesions”

No. of lesions (%)

DNA analyses

n Aneuploid Diploid Low S-phase % High S-phase %

Patient age (yr)

�49 84(100) 8l0) 76(90) 30(36) 23(27)

�50 105(100) 6(6) 99(94) 34(32) 38(36)

Mammographic appearanceCalcification only 91 ( 100) 8 (9) 83 (91) 32 (35) 31 (34)

Soft tissue mass6 98 (100) 6 (6) 92 (94) 32 (33) 31 (32)Mammographic soft tissue only

lesion extent (mm)

1-10 16(100) 1 (6) 15(94) 3(19) 5(31)

1 1-20 47 ( 100) 2 (4) 45 (96) 16 (34) 14 (30)

>20 19(100) 0(0) 19100) 8(42) 7(37)

Mammographic calcification

lesion extent (mm)

1-10 56(100) 4(7) 52(93) 18(32) 19(34)

11-20 15(100) 213) 13(87) 6(40) 5(33)

>20 20(100) 2(10) 18(90) 8(40) 7(35)

a No significant associations were present.

bThis includes 15 soft tissue masses with calcifications.

Table 4 Associations between histological diagnoses of benign mammographic lesions and flow cytometric DNA analyses

No. of lesions (%)

DNA analyses

Histological diagnoses n Aneuploid Diploid Low_S-phase % High S-phase %

Atypia/LCIS 34(100) 4(12) 30(88) 12(35) 13(38)

Hyperplasia 31 (100) 1 (3) 30 (97) 1 1 (35) 7 (23)

Adcnosis 41 (100) 4(10) 37(90) 12(29) 19(46)

Intrammary lymph nodes 6(100) 2(33)” 4(67) 0(0) 0(0)

Other benign lesions 77 ( 100) 3 (4) 74 (96) 29 (38) 23 (30)

Proliferative” 106(100) 9(8) 97(92) 35(33) 40(38)

Nonproliferative’ 83 (100) 5 (6) 78 (94) 29 (35) 21 (25)

“ P = 0.014 comparing the aneupboid rate of intramammary lymph nodes with all other diagnoses.b Atypia/LCIS, hyperplasia, andC Intramammary lymph nodes and other benign lesions.

RESULTS

Flow cytometrie DNA analysis of specimen mammogra-

phy-guided FNAs was possible on 189 consecutive clinically

occult benign lesions that underwent excision prompted by

screening mammography. Fourteen of 189 (7%) benign lesions

contained ancuploidy. The median S-phase percentage of the

study group was 2.0; the lower 33rd percentile S-phase percent-

age was � 1.6, and the higher 33rd percentile S-phase percent-

age was �4.1 (Table 1).

DNA analysis of specimen mammography-guided FNAs in

adjacent normal mammographic tissue with benign histological

findings showed aneupboidy in only 1 of 1 14 (0.9%) eases

within the study. As shown in Table 2, the aneuploid rate in the

normal tissue control group was significantly lower than that of

the benign lesions (P = 0.01).

Associations between clinical and mammographic features

and DNA analyses of the benign lesions are shown in Table 3.

DNA content and S-phase percentage showed no significant

associations with patient age, mammographie appearance, and

mammographic soft tissue or calcification extent.

Associations between histological diagnoses and DNA

analyses of the benign lesions are shown in Table 4. Benign

histological diagnoses included nonproliferative benign lesions

(77 lesions, 41%), benign intramammary lymph nodes (6 Ic-

sions, 3%), the proliferative changes of adenosis (41 lesions,

22%), and lesions containing hyperplasia (31 lesions, 16%) or

atypia or LCIS (34 lesions, 18%). Aneuploidy was detected in

all categories of benign lesions. The aneupboid rate for benign

intramammary lymph nodes (2 of 6 lesions, 33%) was greater

than the aneupboid rate of all other benign diagnoses combined

(12 of 183 lesions, 6.5%; P = 0.01). There was no significant

association between aneupboid rates and the combined prolifer-

ative histological diagnoses as compared with the nonprolifera-

tive diagnoses. There was no significant association between

high and low S-phase percentage and benign histological diag-

noses.

The risk of development of subsequent breast carcinoma in

patients with aneuploid and diploid benign lesions is shown in

Table 5. During a median follow-up of 40 months (range. 6-84

months), 2 of 13 (15%) patients with aneupboid benign lesions

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1546 Flow Cytometric DNA Analyses of Benign Breast Lesions

Table 5 Subsequent risk of developme nt of ipsilateral breas t carcinoma in p atients with aneupboid and diplo id benign lesions

Clinical and_mammographic_outcome”

No. of patients (%)

Aneupboid Dipboid Low S-phase % High S-phase %

Carcinoma 2(15)” 5(3)” 2(3) 4(7)

No carcinoma I 1 (85)’ 170 (97) 62 (97) 57 (93)

Totals 13 ( 100) 175 (100) 64 (100) 61 (100)“ Median follow-up was 40 months (range. 6-84 months).

6 Odds ratio = 6.18 (95% confidence interval. 1.08-35.56). The odds ratio was defined as the relative risk of carcinoma in patients with

aneupboid benign lesions relative to that of patients with dipboid benign lesions.

‘ Biopsy of a second new microcalcification cluster in one case was benign with atypia and aneuploidy.

developed ipsilateral breast carcinoma. One patient developed a

new mammographic lesion showing duetal carcinoma in situ,

and another patient developed a mammographie lesion showing

infiltrating lobular carcinoma in different quadrants of the same

breast as the index benign biopsies within 8 and 34 months,

respectively. In comparison, 5 of 175 (3%) patients with diploid

benign lesions developed ipsilatcral breast carcinomas. The

calculated odds ratio was 6.18 (95% confidence interval, 1.08-

35.56). No patients developed contralateral breast carcinoma

during the period of the study. One patient with an initial

aneupboid benign lesion did not develop carcinoma but under-

went a subsequent biopsy of benign calcification that contained

atypia and aneuploidy.

DISCUSSION

During this study, DNA flow cytometry was successfully

performed on 189 consecutive specimen mammography-guided

FNAs of clinically occult lesions detected by screening mam-

mography that proved to be benign at histological examination.

Aneuploid DNA content was detected in 7% of the benign

lesions, most commonly in benign intramammary lymph nodes,

but also in benign atypical and hyperplastic lesions, adenosis,

and other nonproliferative benign lesions. The observation that,

in comparison, aneupboidy was detected in only 1 % of FNAs of

adjacent normal tissue (P < 0.01) shows that the detection of

aneuploidy was not a laboratory artifact.

There are little data in the literature regarding DNA flow

cytometry of benign breast lesions (1-6). To our knowledge,

this is the largest series of DNA flow cytometry of benign breast

lesions. The relative lack of aneupboidy in the normal adjacent

breast tissue controls suggests some biological significance of

aneuploidy in benign breast lesions. Whether aneuploidy pre-

dates or is an obligate precursor of light microscopic malig-

nancy is unresolved. Our S-phase percentage data show a wide

range of proliferative activity for benign breast lesions that is

independent of the specific histological diagnoses.

To our knowledge, this is the first study showing the risk of

subsequent development of ipsilateral breast carcinoma in pa-

tients who underwent benign breast biopsies with aneuploidy as

compared to patients who underwent benign breast biopsies

with diploid DNA content. There is one anecdotal report of a

patient who developed ipsilaterab breast cancer 2.5 years after a

benign biopsy showing atypia and ancupboidy (1), but the rela-

tionship of these occurrences and the importance of this isolated

report were uncertain. The patients in our study with aneuploid

benign lesions who have not yet developed malignancy may

represent patients whose potential malignancy was completely

removed by the excisional biopsy or whose follow-up is not yet

long enough. Due to the small numbers and short follow-up in

our pilot study, a study of a larger number and longer follow-up

of patients with benign breast biopsies would be necessary to

validate the risk prediction of aneuploid DNA content for the

subsequent development of ipsilateral breast carcinoma. Deter-

mination of whether the risk associated with aneuploidy in

benign biopsies is ipsilateral or bilateral would also be of

potential clinical significance.

In light of the increasing interest in breast cancer risk

assessment, partially attributed to recent breakthroughs in ge-

netie testing for BRCA1 and BRCA2 mutations (19), there is an

increasing need for intermediate risk markers for the develop-

ment of breast carcinomas, i.e. , markers that would predict the

development of breast carcinoma within 5-10 years rather than

over a lifetime. A study of in vivo FNAs of normal breast tissue

in high- and low-risk women was performed by Fabian et a!.

(20), who performed eight clinically guided fine-needle aspira-

tions/breast. They evaluated the aspirates for overexpression of

estrogen receptor, epidermal growth factor, mutant p53, and

HER-2!neu by immunocytochemistry and for aneuploidy by

image analysis. With their technique and the criteria for ancu-

ploidy, aneupboidy was detected in 32% of high-risk women as

compared with 0% of low-risk women.

With the increasing numbers of benign breast biopsies

performed as a result of screening mammography, the in vitro

specimen mammography-guided fine-needle aspiration tech-

nique is a practical clinical research method that provides mam-

mographic lesion-specific fresh cell samples immediately after

wire localization and excision. Fresh cell samples are preferred

by both our flow cytometry and molecular genetic laboratories

and others (21-23). Core biopsy washings retrieved during

stereotactie core breast biopsies of clinically occult lesions

detected by mammography have also been reported as a source

of fresh cellular material for flow eytometry (24). FNAs and

scrapings of palpable breast carcinoma have also been used for

flow cytometry (4-6, 25), but these techniques are limited to

larger tumors only and exclude smaller clinically occult lesions

detected by screening mammography. We are now performing

combined staining with fluorescein-labeled anticytokeratin an-

tibodies that allows the DNA analysis of epithelial cells to be

separated from that of other elements. The development of

multiple marker immunophenotyping panels by flow cytometry

in breast disease has potential use in risk assessment in cells

obtained from normal breast tissue or benign lesions using

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Clinical Cancer Research 1547

fine-needle aspiration, because multiple tests can be performed

simultaneously on the same low number of cells.

In conclusion, DNA flow cytometry can be performed

successfully on specimen mammography-guided FNAs of mam-

mography-detected lesions that prove to be benign at biopsy.

Our pilot study data suggest that aneuploidy, which is seen in a

variety of benign breast lesions, is associated with a higher

short-term risk of development of ipsilaterab breast carcinoma

than benign lesions with diploidy. The combined techniques of

specimen mammography-guided FNA and flow cytometry pro-

vide a practical translational research method for the further

study and molecular and genetic risk assessment of benign

breast disease.

ACKNOWLEDGMENTS

We gratefully acknowledge Karin Brady for invaluable assistance

in data management and preparation of this manuscript and Les Blu-

menson for statistical analysis.

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1998;4:1543-1547. Clin Cancer Res   P C Stomper, J R DeBloom, 2nd, R M Budnick, et al.   detected by screening mammography.Flow cytometric DNA analyses of benign breast lesions

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