flow cytometric dna analysis of invasive carcinomas ... · 1790 flow cytometric dna analysis of...

Vol. 4, 1789-1795, July 1998 Clinical Cancer Research 1789

Flow Cytometric DNA Analysis of Invasive Carcinomas Detected by

Screening Mammography: Use of Specimen Mammography-guided

Fine-Needle Aspirates

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

Ellis Levine, Rose Marie Budnick, and

Carleton C. StewartDivision of Diagnostic Imaging [P. C. S.], Department of Medicine

[E. L.], and Department of Flow Cytometry, Division of Pathology[R. M. B., C. C. S.], Roswell Park Cancer Institute, School ofMedicine and Biomedical Sciences, State University of New York atBuffalo, and Department of Natural Sciences, Roswell Park Divisionof State University of New York at Buffalo [J. R. D.], Buffalo, NewYork 14263

ABSTRACTClinical studies of flow cytometric DNA analysis of

breast carcinoma are often limited by the lack of fresh tissue

samples from smaller, nonpalpable carcinomas. In addition,

most studies measuring DNA in the current literature focus

on larger palpable masses that may have less relevance to

the smaller, nonpalpable lesions. A prospective study of flow

cytometric DNA analysis of in vitro specimen mammogra-

phy-guided fine-needle aspirates (FNAs) of 103 consecutive

nonpalpable invasive carcinomas detected by screening

mammography was performed to determine efficacy and

explore associations with mammographic and pathological

features. For 62 (60%) lesions for which DNA analysis on

both FNA and standard tissue incision samples was per-

formed, there was excellent (89%) agreement for ploidy

determinations (K = 0.77) and poor agreement for S-phase

percentage determinations (K = 0.23). Specimen mammog-

raphy-guided FNA analysis detected aneuploidy in 36% of

lesions overall, including 34% of 41 lesions for which stand-

ard tissue procurement was not possible. Mammographic

microcalcifications had a higher aneuploid rate (14 of 28

lesions, 50%) as compared with soft tissue masses (22 of 75

lesions, 29%), P < 0.01. Lobulated masses with indistinct

margins had a higher aneuploid rate (5 of 6 lesions, 83%) as

compared with more irregular, spiculated masses (7 of 27

lesions, 26%), P < 0.01. The aneuploidy rate was independ-

ent of specific histological diagnosis, lesion size, nuclear

grade, or nodal or estrogen receptor status. Flow cytometric

DNA analysis of mammographic lesion-specific, fresh, cel-

lular FNA samples obtained under specimen mammo-

graphic guidance can assess early invasive carcinomas when

gross fresh tissue procurement is not possible. This tech-

nique could be incorporated into larger clinical follow-up

studies to determine the prognostic significance of flow cy-

tometric DNA analysis for these very early breast carcino-

mas.

INTRODUCTIONThe frequency of early invasive breast carcinomas has

increased with the widespread utilization of screening mam-

mography. Recent therapeutic tends toward adjuvant systemic

therapy in node-negative breast cancer patients have increased

the need for better prognostic assessment for these early lesions.

DNA ploidy and S-phase percentage are among the prognostic

markers that are being evaluated for invasive breast carcinomas.

Prior clinical studies suggest that S-phase percentage has greater

prognostic significance than does aneuploidy (1-19). However,

clinical studies of flow cytometric DNA analysis are often

limited by the lack of fresh tissue samples from smaller invasive

carcinomas detected by screening mammography, resulting in a

lack of understanding of the prognostic significance of this test

in an increasingly relevant group of patients.

We have been performing flow cytometric DNA analysis

on specimen mammography-guided FNAs2 of lesions within

excised breast specimens after wire localization and surgical

excision of clinically occult lesions detected by mammography

(20, 21). To our knowledge, there is little information in the

literature regarding flow cytometric DNA analysis of clinically

occult invasive breast carcinomas using this technique of pro-

curement of mammographic lesion-specific fresh cell samples.

There are also little published data regarding mammographic

and pathological associations with flow cytometric DNA anal-

ysis features of screening-detected nonpalpable invasive breast

carcinomas.

This prospective pilot study was undertaken to determine

the DNA ploidy and S-phase percentage of specimen mammog-

raphy-guided FNAs of screening mammography-detected inva-

sive carcinomas by flow cytometry and to explore associations

between mammographic and pathological features with ploidy

and S-phase percentages.

Received 3/5/98; revised 4/7/98; accepted 4/28/98.The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.

I To whom requests for reprints should be addressed, at Division ofDiagnostic Imaging, Roswell Park Cancer Institute, Elm and CantonStreets, Buffalo, NY 14263. Phone: (716) 845-5796; Fax: (716) 845-8796; E-mail: [email protected].

MATERIALS AND METHODS

The study group consists of 103 consecutive patients who

underwent biopsies of clinically occult lesions detected by

screening mammography that showed invasive breast carcinoma

2 The abbreviations used are: FNA, fine-needle aspirate; DCIS, ductal

carcinoma in situ.

Research. on November 28, 2020. © 1998 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

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1790 Flow Cytometric DNA Analysis of Invasive Carcinomas using FNAs

at pathological exam. The median age of the patients in the

study group was 62 years (range, 32-88 years). Twenty-three

(22%) of the patients were less than 50 years old; 80 (78%) of

the patients were 50 years of age or older. All patients had

negative clinical breast examinations performed by breast sur-

geons.

Mammograms were obtained with a Senographe 600 T 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 vito with accessory magni-

fication projections. Mammographically guided needle localiza-

tion procedures were performed with the method described by

Kopans et a!. (22). Radiography of the specimen was performed

with the compression and magnification technique (X 1.85) in

each case with the clinical DMR mammography unit.

The surgical staff was notified immediately of the confir-

mation of the excision of lesions. Subsequently, after the re-

moval of compression, the mammographer performed a 20-

gauge needle aspiration within the suspect mammographic

lesion in the operative specimen. Needle guidance techniques

based on the radiography of the specimen included: (a) use of

circumferential coordinates or surface landmarks on the speci-

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

and repeat radiography of the uncompressed specimen for

smaller lesions. Fine-needle aspiration was performed through-

out the mammographically depicted lesion to provide adequate

lesion sampling (20). The needle aspirate was then sent for flow

cytometric DNA analysis.

Excisional biopsy specimens were sent to the cancer insti-

tute department of pathology. If a palpable mass was present

within the operative specimen, the pathologist obtained a gross

sample of tissue from the mass by incisional biopsy and sub-

milled the tissue sample for flow cytometric analysis.

The needle aspirate sent for flow cytometric DNA analysis

was 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, cells were centrifuged, and the pellet was

stained with propidium iodide (50 p�g/m1) in Kristen buffer

[0. 1% sodium citrate, 0.02 ms/ml 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. Cells (10,000 total) were analyzed on a FACScan flow

cytometer (Becton Dickinson, San Jose, CA) with a doublet

discriminator module and an excitation beam at 488 nm/liter.

Data were collected for the fluorescence 2 channel measuring

propidium iodide emission through a 585/42 band pass filter.

Lysis II software was used only to acquire the data using its

pulse processing capability to reduce aggregates, no data anal-

ysis was performed using this program. 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 the healthy donor blood peaked at channel 200. A threshold

of 28 was set on this channel to eliminate cellular debris. All

samples were collected ungated.

The histogram files were analyzed with Multicycle (Phoe-

nix Flo Systems, San Diego, CA). This program calculated the

percentage of cells in the G0-G , , 5, and G2 phases for each

cycling cell population present as well as the DNA index of the

standard (chick RBCs) and any aneuploid peak present com-

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

of the peak G1 channel for either the standard or the sample

cells:that of diploid cells. The DNA index for the standard is a

measure of the quality of the sample staining. The DNA index

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

abnormal population. Coauthor C. C. S. performed all flow cy-

tometry and interpreted results without knowledge of mammo-

graphic and pathological findings.

Cell populations with a DNA index of 1.0 ± 0.1 (SD) were

considered diploid. All others were considered to have aneu-

ploidy present. All needle aspirates, including those that had

aneuploid populations, had some diploid cells present. When

aneuploid cells were present, the aneuploid S-phase percentage

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

cell population S-phase value. Cases were then designated as

having either a low, intermediate, or high S-phase percentage

based on their placement within the lowest, middle, and highest

thirds determined for the study group.

Histological material derived from excisional biopsy was

stained with H&E. All pathological interpretations were per-

formed prospectively by the cancer institute department of pa-

thology staff without knowledge of flow cytometric findings.

Histological assessment of the entire biopsy specimen was made

with knowledge of the presence and location of the mammo-

graphic abnormality within the specimen so that specific ana-

tomical correlation could be made in each case. For calcified

lesions, the specimen was sliced and reradiographed to aid

localization during pathological examination.

For pathological features, histological diagnosis, invasive

tumor size, nuclear grade, and axillary node status were re-

corded and categorized. The pathological diagnoses were date-

gorized as invasive ductal carcinoma, invasive ductal carcinoma

with greater or less than 90% DCIS, and invasive lobular car-

cinoma. Pathological invasive tumor sizes were categorized as

1-10, 1 1-20, or >20 mm. Nuclear grade I or II invasive

carcinomas were combined for the purpose of this analysis.

Estrogen and progesterone receptor assays for breast carcinoma

were performed with immunohistochemical techniques. For the

immunohistochemical assay, 20% or more nuclear staining was

considered positive.

Lesions were prospectively categorized by mammographic

appearance and size before biopsy. Lesions were categorized as

calcifications only or soft tissue masses (with or without calci-

fications). Mammographic lesions were also subcategorized for

appearance by shape and margin. Mammographic calcifications

were categorized as predominantly linear or predominantly

granular. Soft tissue lesions were categorized as spiculated,

irregular/poorly defined, indistinct lobulated, or indistinct

round/oval masses or as areas of architectural distortion. The

features are based on prior mammographic pathological corre-

lation studies (23, 24). Mammographic measurement of the

greatest dimension of each lesion was categorized as 1-10,

1 1-20, or >20 mm. For spiculated masses, the greatest dimen-

sion of the central mass not including the extent of spiculations

was used.

Statistical analyses were performed with x2 analysis, K

tests, and Wilcoxon signed-rank tests (25). Ps < 0.05 were

considered significant.



Clinical Cancer Research 1791

Table 1 Flow cytometric ploidy agreement in 62 invasive caseswhere both FNA and tissue procurement were performed”

Tissue

FNA Diploid Aneuploid

Diploid 33 6Aneuploid 1 22

a Agreement rate = 89%; K = 0.77 (95% confidence interval,

0.61-0.93); P < 0.0001.

RESULTSFor 62 of the 103 (60%) excisional biopsy specimens in the

study, a gross tissue sample was procured from the lesion by

palpation-guided incision in addition to the specimen mammog-

raphy-guided fine-needle aspiration. These were performed in-

dependently of each other by the mammographer and patholo-

gist. The agreement rate for ploidy between the two

procurement techniques was 89% (Table 1). Using the K test, a

K value of0.77 (95% confidence interval, 0.61-0.93) and a P of

<0.0001 were derived for the ploidy agreement rate between

flow cytometric analysis of gross tissue samples when available

and the FNAs. It is significant that in 41 of our 103 (40%)

invasive carcinomas, no gross tissue could be procured. Only

specimen mammography-guided fine-needle aspiration could be

performed, yielding an aneuploid rate of 34%. The evaluation of

S-phase percentage was also performed for all 62 specimens for

which both gross tissue and FNA were available. The agreement

between flow cytometric analysis of gross tissue samples and

FNAs for S-phase percentage was not significant (K 0.23;

Table 2). Overall, S 1 % of the specimens were in different

percentile groups.

The remainder of the analyses in this study are based on the

FNA flow cytometric data only. For the 103 screening mam-

mography-detected invasive carcinomas in this study, aneu-

ploidy was present in 36% (37 of 103) of the FNAs. The median

S-phase percentage was 4.3% (range, 0-46.7%). The lower

33rd percentile was 2.7%; the higher 33rd percentile was 6.5%.

Associations with Mammographic and Pathological

Features. Mammographic and pathological features with sig-

nificant associations with ploidy in S-phase percentage are

shown in Table 3. For mammographic features, invasive carci-

nomas that manifested themselves as microcalcifications had a

significantly higher rate of aneuploidy (14 of 28, 50%) as

compared with soft tissue abnormalities (23 of 75, 3 1 %), P <

0.01. Among mammographic soft tissue subtypes, lobulated

masses with indistinct margins had a greater association with

aneuploidy (5 of 6, 83%) as compared with either stellate

masses [(7 of 27, 26%) P < 0.01] or round or oval masses with

indistinct margins [(2 of 15, 13%) P < 0.01.]. Lobulated masses

with indistinct margins or lesions manifested by architectural

distortion were also less likely to have low S-phase percentages

[1 of 16 (17%) and 2 of 12 (17%), respectively] as compared

with round or oval masses with indistinct margins (10 of 15,

67%), P < 0.01.

For pathological features, nuclear grade was assessed for

96 of the 103 (93%) invasive carcinomas. Of the seven (7%)

cases not graded, six were pure invasive lobular carcinomas, and

Table 2 Flow cytometric S-phase fraction category agreement in 62invasive cases where both FNA and tissue procurement were

performed”

FNA

Tissue

High Intermediate Low

High 10 5 4

Intermediate 4 8 9Low 8 2 12

a Agreement rate = 49%; K 0.225 (95% confidence interval,

0.019-0.431); P = not significant.

one was a predominately invasive lobular carcinoma for which

nuclear grade was not assessed. Nuclear grade III carcinomas

were more likely associated with aneuploidy (20 of 38, 53%) as

compared with nuclear grade I or II carcinomas (14 of48, 29%),

P < 0.01 . Nuclear grade III carcinomas were more likely

associated with high S-phase percentage ( 1 8 of 38, 47%) as

compared with nuclear grade I or II carcinomas (1 1 or 48, 23%),

P < 0.05. Nuclear grade III carcinomas were also less likely to

be associated with low S-phase percentage (7 of 38, 18%) as

compared with nuclear grade I or II carcinomas (19 of48, 40%),

P < 0.05. Progesterone receptor-negative carcinomas were

more likely to be associated with high S-phase percentage (15 of

30, 50%) as compared with progesterone receptor-positive car-

cinomas (12 of 55, 22%), P < 0.05.

Mammographic and pathological features without signifi-

cant associations with DNA ploidy or S-phase percentage are

shown in Table 4. There were no significant associations be-

tween DNA ploidy or S-phase percentage and patient age, the

pathological diagnoses of invasive ductal carcinoma, mixed

invasive ductal carcinoma and DCIS, or invasive lobular carci-

noma or estrogen receptor status. There were also no significant

associations between DNA ploidy and S-phase percentage and

either mammographic or pathological lesion sizes.

DISCUSSION

Flow cytometric analysis of specimen mammography-

guided FNAs of nonpalpable invasive carcinomas detected by

screening mammography in this study showed an aneuploidy

rate of 36%. This is in the lower range of aneuploidy rates

reported for invasive carcinomas of the breast (1 , I 7). Most

prior studies, some of which required gross tissue samples for

analysis, included or predominately consisted oflarger clinically

palpable carcinomas or tumors that were palpable within the

excised pathological specimen. Also, DNA analysis methodol-

ogy and interpretive criteria vary between studies. Whereas

aneuploidy has not been shown to have major prognostic value

for invasive carcinomas to date, it may have prognostic value for

the earlier invasive carcinomas detected by screening mammog-

raphy. Further clinical evaluation of this DNA analysis tech-

nique is necessary to determine the prognostic significance of

the findings from this pilot study.

For these screening-detected invasive carcinomas, lesions

with the mammographic feature of microcalcifications only

showed significantly greater aneuploidy as compared with soft

tissue lesions. Among soft tissue abnormalities, indistinct lob-

ulated masses with indistinct margins showed significantly




Table 3 Mammographic an d pathologi cal features with significant associations with ploidy or S

detected by screening mammography-ph ase percent age for invasive carc inomas

Low S-phase % High S-phase %Feature n (%) Aneuploidy (%) P no. (%) P no. (%) P

Lesion typeCalcification only 28 (27) 14 (50) 0.006 5 (18) NS� 15 (54) 0.007Soft tissue 75 (73) 23 (31) 27 (36) 19 (25)

Calcification subtypePredominately linear 17 (61) 8 (47) NS 1 (6) 0.040 9 (53) NSGranular 1 1 (39) 6 (55) 4 (36) 6 (55)

Soft tissue subtype

Stellate 27 (40) 7 (26)* 10 (37) 7 (26) NSIrregular, poorly defined 7 (10) 3 (43) * 0.008 2 (29) * 0.009 2 (29)Architectural distortion 12 (18) 5 (42) 1 = 0.002 2 (17)* 0.038 4 (33)

Indistinct round, oval 15 (22) 2 (13)1 10 (67)� 2 (13)Indistinct lobulated 6 (9) 5 (83)�* 1 (l7)1* 3 (50)

Nuclear gradeGrade I or II 52 (54) 15 (29) NS 21 (40) 0.039 12 (23) 0.01 1

Grade III 44 (46) 21 (48) 9 (20) 21 (48)

Progesterone receptor”

Positive 55 (65) 14 (26) 0.009 21 (38) NS 12 (22) 0.015

Negative 30 (35) 17 (57) 6 (29) 15 (50)

a NS, not significant.

!, Receptor assays were not performed on I 8 lesions.

greater aneuploidy and less low S-phase percentages as com-

pared with other soft tissue lesions, including spiculated masses.

The pathological feature of high nuclear grade was also signif-

icantly associated with higher S-phase percentages. No signifi-

cant associations were seen between DNA analysis and patient

age, tumor size, specific histological diagnosis (i.e., lobular

versus ductal carcinoma, associated DCIS), and axillary node or

estrogen receptor status. Due to the small numbers in this pilot

study, these findings are not conclusive.

For these clinically occult carcinomas, the specimen mam-

mography-guided fine-needle aspiration technique enabled

DNA analysis in 40% of lesions for which standard incisional

biopsy tissue procurement could not be performed due to the

lack of palpable tumor within the excised specimen. For the

60% of lesions for which both fine-needle aspiration and mci-

sional biopsy tissue were analyzed, the agreement in ploidy

results was excellent (89%). The level ofagreement is especially

significant due to the fact that each method of procurement was

done blinded and independent of each other. Martelli et a!. (26)

showed an 82% agreement rate (82 of 100) for the detection of

aneuploidy in a study comparing flow cytometric analysis of in

vivo FNAs of larger clinically palpable masses and subsequent

gross palpable tissue incision of the surgical biopsy specimens.

In our study, four of the six cases in which aneuploidy was

detected by gross tissue procurement and not detected in the

FNAs were lesions manifested by larger mammographic abnor-

malities (greater than 2-3 cm) within which a smaller palpable

invasive tumor existed. Because the specimen mammography-

guided FNAs were obtained with imaging guidance only with-

out palpation, it is possible that DCIS adjacent to the palpable

invasive carcinomas was aspirated. Conversely, palpation alone

does not insure adequate sampling of the invasive carcinomas as

seen in the case of aneuploidy detected in the FNA but not in the

tissue sample. A combined imaging and palpation-guided FNA

technique would seem to offer the optimal comparison to gross

fresh tissue procurement.

Because the optimal stratification of lesions into different

groups by S-phase percentage has not been determined, we used

three groups instead of two groups to decrease the chance of

misclassifying tumors with borderline values. The lack of sig-

nificant correlation between the S-phase percentages of FNAs

and gross tissue samples is probably related to the variable

mixtures of tumor cells, benign epithelium, and stromal cells

within different samples. Whether specimen mammography-

guided fine-needle aspiration provides a more accurate assess-

ment of the S-phase percentage of early invasive carcinomas

than gross tissue incision, when possible, is yet to be deter-

mined. We know of no published data comparing S-phase

percentages determined on separate gross incision tissue sam-

pies from the same lesions. We are now performing combined

staining with fluorescein-labeled anticytokeratin antibodies that

allows the DNA content of epithelial cells to be separated from

that of other elements. We are also studying multiple marker

immunophenotyping with flow cytometry. Although there are

no published data showing the benefit of the combined staining

technique, we feel that this will further improve the prognostic

significance of the S-phase percentage in breast carcinoma.

Investigators who have reviewed the clinical utility of flow

cytometric DNA analysis in breast carcinoma note that because

S-phase percentage is a continuous rather than a dichotomous

variable, each laboratory must validate the prognostic signifi-

cance of its own S-phase values (1). We concur that the prog-

nostic significance of S-phase values obtained by this technique

of cell procurement and flow cytometric DNA analysis, like

other methods of procurement and analysis for S-phase deter-

minations, must be validated by larger clinical follow-up stud-

ies. The lack of association between DNA ploidy or S-phase

percentage and size among the screening-detected invasive car-

cinomas in this study suggests that these DNA features of early

breast carcinomas are established at a premammographic phase

and do not change within the screening mammography spec-

trum.




Table 4 Mammographic and pathological fea tures without significant associations with ploidy or S-phase pe

detected by screening mammography

rcentag e for invasive carcin omas

Low S-phase % High S-phase %Feature n (%) Aneuploidy (%) P no. (%) P no. (%) P

Mammographic soft tissue extent (mm)”

1-10 38(57) 13(34) 18(47) 10(36)I 1-20 20 (30) 5 (25) NSb 3 (15) NS 6 (30) NS�2l 9(13) 4(44) 4(44) 2(22)

Mammographic calcification extent (mm)1-10 10 (36) 5 (50) 3 (30) 5 (50)11-20 7(25) 4(57) NS I (14) NS 5(71) NS�2l 11 (39) 5(45) 2(18) 5(45)

Pathological invasive tumor size (mm/

1-10 59 (50) 23 (39) 23 (39) 20 (34)11-20 30(30) 11 (37) NS 9(30) NS 12(40) NS�2l 11 (11) 3(27) 1 (9) 1 (9)

Patient age (yr)<50 23(22) II (48) 4(17) 11 (48)�50 80 (78) 26 (33) NS 30 (38) NS 23 (29) NS

Histological diagnosis

Invasive ductal 46 (45) 16 (35) NS 16 (35) NS I 3 (28) NSInvasive ductal, <90% DCIS 39 (38) 16 (41) 1 1 (28) 14 (36)Invasive ductal, >90% DCIS I I (I I) 4 (36) 3 (27) 6 (55)

Invasive lobular 7 (7) 1 (14) 4 (57) 1 (14)Axillary nodes”

Positive 21 (33) 9 (42) NS 5 (24) NS 9 (43) NSNegative 43(67) 12(28) 16(37) 10(23)

Estrogen receptor’Positive 63 (74) 19 (30) NS 21 (33) NS 19 (30) NSNegative 22 (26) 12 (55) 6 (27) 1 1 (50)

a For stellate masses, the central mass diameter was used.b NS, not significant.C Pathological tumor size was difficult to assess for three lesions.d Values shown are for 64 patients who underwent axillary node dissection.e Receptor assays were not performed on I 8 lesions.

DNA analysis of in vivo stereotactic FNA and in vitro FNA

of frozen tissue samples of nonpalpable breast carcinomas has

been performed using microspectrophotometry (27, 28). DNA

flow cytometry has been performed on paraffin-embedded tis-

sue and fresh tissue samples, needle aspirates, or scrapings from

larger tumors that were palpable at clinical or pathological exam

(5, 18, 20, 29-35). We and other investigators prefer fresh

tissue, rather than paraffin-embedded tissue, because the fixa-

tion process may render the analysis less accurate (36-38).

Aside from the advantages of providing mammographic lesion-

specific fresh cell samples for clinically occult lesions, the

specimen mammography-guided fine-needle aspiration tech-

nique offers several advantages: (a) it is in vitro and does not

directly involve the patient; (b) no significant histological arti-

facts have been reported. This is partially attributable to the lack

of bleeding with the in vitro technique; (c) it takes less than S

mm of the mammographer’s time during the routine and man-

datory performance of specimen radiography after localization

and excision of clinically occult lesions; and (d) for cost-effec-

tiveness in both clinical practice and research, needle aspiration

samples could be stored until a final histological diagnosis of the

lesion is made and analyzed only if a selected diagnosis of

malignancy or specifically invasive carcinoma is made. We

have reported a prior study of the specimen mammography-

guided FNA technique in which a second needle aspirate of each

lesion was sent for cytological examination (21). If cytological

evaluation is also desired, alternatively, a second needle aspirate

could be obtained, and a portion of the pooled cells could be

submitted for cytological evaluation.

The observation that early invasive carcinomas manifested

by mammographic lobulated masses with indistinct margins

have a significantly greater association with aneuploidy and

higher S-phase percentage than spiculated masses should in-

crease caution with regard to these lesions by mammographers.

Whereas lesser circumscription of tumors has been considered a

sign of tumor aggressiveness, this may not always be the case.

Spiculations, associated with greater predictive value for the

presence of malignancy, pathologically represent predominately

fibrosis. The lack of fibrotic spiculations in indistinct lobulated

masses may reflect the aggressive growth of the tumor itself,

precluding the opportunity for development of large fibrotic

spiculations. Whereas larger numbers of lesions must be studied

to validate this finding, careful margin analysis of the more

circumscribed mammographic masses and prompt biopsy of

solid masses with indistinct margins are further supported by

this data.

On the basis of this pilot study data, we conclude that

analysis of specimen mammography-guided FNAs can be used

in prognostic marker evaluations such as flow cytometry DNA

analysis for early invasive carcinomas detected by screening

mammography in which gross tissue procurement and fresh

tissue analysis is often not otherwise possible. The correlations




between DNA analysis and mammographic features increase

our understanding of the mammographic features of early inva-

sive carcinomas. We feel that this technique of mammographic

lesion-specific fresh cell procurement is easy to perform, and

that the flow cytometric DNA analysis data obtained can stratify

early invasive breast carcinomas and could be incorporated into

larger clinical trials to ultimately determine the prognostic sig-

nificance of the findings.

ACKNOWLEDGMENTS

We gratefully acknowledge Karin Brady for invaluable assistance

in data management and preparation of the manuscript and Les Blu-menson for statistical analysis.

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1998;4:1789-1795. Clin Cancer Res P C Stomper, J R DeBloom, 2nd, E Levine, et al. mammography-guided fine-needle aspirates.by screening mammography: use of specimen Flow cytometric DNA analysis of invasive carcinomas detected

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