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(Radiology. 2001;218:881-885.)
© RSNA, 2001

Technical Developments

Stereotactic Core Biopsy Breast and Blood Cell By-products: A Source of Material for Molecular Genetics Research—Initial Experience1

Paul C. Stomper, MD, Carleton C. Stewart, PhD and Daniel L. Stoler, PhD

1 From the Departments of Diagnostic Imaging (P.C.S.), Flow Cytometry (C.C.S.), and Experimental Pathology (D.L.S.), Roswell Park Cancer Institute, School of Medicine and Biomedical Sciences, SUNY, Elm and Carlton Sts, Buffalo, NY 14263. Received November 18, 1999; revision requested December 28; final revision received July 11, 2000; accepted August 15. Address correspondence to P.C.S. (e-mail: Paul.Stomper@roswellpark.org).


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 Materials and Methods
 Results
 Discussion
 REFERENCES
 
Stereotactic core biopsy washings and blood drop samples, routinely discarded by-products, provide satisfactory fresh cellular material for flow cytometry and molecular genetics microsatellite polymerase chain reaction (PCR) analysis for detection of loss of DNA alleles (loss of heterozygosity). Cytokeratin-positive (epithelial) cells from the core biopsy washings were sorted by means of flow cytometry prior to PCR analysis. DNA allele loss was detected in benign breast epithelial cells in three (20%) of 15 patients.

Index terms: Breast, biopsy, 00.1267 • Breast, diseases, 00.72 • Breast neoplasms, 00.30 • Genes and genetics • Molecular analysis • Specimens


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
Materials and Methods
 Results
 Discussion
 REFERENCES
 
The widespread acceptance and increased use of stereotactic core breast biopsy (1–5) provide new opportunities for translational research between those in the mammography clinic and those in basic science laboratories who are interested in the study of breast disease. Molecular geneticists often require fresh nonfixed cell samples from both breast tissue and blood to assess and compare DNA for somatic changes or loss of heterozygosity in the study of breast cancer pathogenesis.

Stereotactic core breast biopsy washings or rinses, as well as several blood drops obtained during the initial skin scalpel nick performed during the procedure, are both routinely discarded by-products that may provide geneticists with a unique source of fresh breast cell and blood samples for the evaluation of somatic DNA alterations in the breasts of a broad population of women. Flow cytometry techniques in which an anticytokeratin antibody is used for epithelial cell discrimination can be applied to sort the breast epithelial (cytokeratin-positive) and nonepithelial (cytokeratin-negative) cell fractions for specific genetic analysis of the breast epithelium (6,7).

The polymerase chain reaction (PCR) has enabled the detection of maternally and paternally inherited microsatellite markers with minute quantities of DNA. These markers are short, highly polymorphic, repetitive DNA sequences mapped to specific sites throughout the genome. The PCR products from each allele can be resolved as distinct clusters of bands by means of gel electrophoresis. Loss or alteration of one band or allele is an indication of a mutation in that particular region of the genome of a given lesion.

We describe our initial experience in the evaluation of the suitability of using cells from stereotactic core biopsy washes and blood drop samples, material otherwise routinely discarded, for molecular genetics PCR analysis for the loss of heterozygosity of epithelial cells sorted with flow cytometry.


   Materials and Methods
TOP
 ABSTRACT
 INTRODUCTION
 Materials and Methods
Results
 Discussion
 REFERENCES
 
Thirty-nine consecutive patients (median age, 55 years; age range, 37–87 years) without prior breast cancer who were undergoing stereotactic core biopsy at the Cancer Institute mammography center signed informed consent for genetics research during this study. Retrieval of stereotactic core biopsy washings and blood drop samples was attempted in 39 patients, and samples were sent for flow cytometric cytokeratin sorting for epithelial cells followed by microsatellite PCR analysis. Blood drops were not obtained in three (8%) patients due to lack of bleeding at the initial skin scalpel nick site.

The initial 19 patient samples were used for the development of the sequential techniques. Of the last 17 consecutive patient blood and core washings submitted for flow cytometry, 15 (88%) yielded cytokeratin-positive (epithelial) cells during sorting; two (12%) yielded few or no cytokeratin-positive cells for undetermined reasons and were not submitted for molecular genetics analysis. Findings in 15 consecutive stereotactic core biopsy samples submitted for molecular genetics analysis are described in this article.

Immediately prior to the stereotactic core biopsy procedures, the mammographer (P.C.S.) explained the nature of the procedure and research to each patient, and the patients were required to sign a one-page stereotactic core biopsy written informed consent and an optional three-page written informed consent addressing genetics research and patient confidentiality issues of this institutional review board–approved protocol.

Stereotactic core biopsy was performed in the usual manner by using a stereotactic table with digital imaging (LoRad, Danbury, Conn) and a directional vacuum-assisted biopsy device (Mammotome; Biopsys Medical, Irvine, Calif) with 14- or 11-gauge needles. With this biopsy device, a single biopsy needle pass is made into the mammographic lesion, and multiple tissue cores are extracted from the breast lesion site and retracted without reinserting the outer needle to minimize possible contamination with cells from other regions of the breast. After the initial skin nick was made with a scalpel, several drops of blood from the skin nick site were collected by placing a test tube containing heparin below the site under the breast and behind the metal biopsy aperture plate. Immediately after procurement, core biopsy specimens were placed in a container filled with normal saline. Cores of lesions associated with microcalcifications were then placed on a film and radiographed to confirm adequate microcalcification sampling.


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  		Findings at Pathologic Examination of Stereotactic Core Biopsy Samples and DNA Allele Loss Analysis
 
After the procedure, all core specimens were placed in formalin and submitted for pathologic evaluation by cancer institute pathologists. The core biopsy washings container was then labeled with a code to protect patient anonymity and submitted for flow cytometry for epithelial cell segregation by means of cytokeratin sorting. The sampling and labeling tasks for this research required approximately 5 minutes to perform for each patient.

Flow Cytometric Sorting
The washings sent for flow cytometric analysis were fixed in 2 mL of 70% ethanol and stored at -20°C. The cytokeratin antibody with specificity for CK8 and CK18 (Becton Dickinson Biosciences, San Jose, Calif) was directly conjugated with fluorescein isothiocyanate and used at the recommended concentration.

For analysis, cells were centrifuged at 1,500 x g for 3 minutes and washed with 1 mL of phosphate-buffered saline. The pellet was resuspended in 20 µL of antibody and incubated for 30 minutes at 20°C. Then, 1.0 mL of phosphate-buffered saline was added, and the suspension was incubated for 10 minutes to allow unbound antibody to diffuse out of the cells before they were centrifuged at 1,500 x g for 3 minutes. Cells were resuspended in 300 µL of phosphate-buffered saline for sorting.

A flow cytometer (FACSVantage; BDIS, San Jose, Calif) was used to obtain all measurements and sort the cells. For excitation of fluorescein isothiocyanate, an air-cooled argon laser operating at 488 nm was used. Although only fluorescein isothiocyanate was measured, the instrument compensation was adjusted by using normal blood gated on lymphocytes stained in separate tubes with CD45 fluorescein isothiocyanate, or CD4PE (Becton Dickinson Biosciences).

All data were acquired by using logarithmic amplification. A template containing regions that defined the position of each cell population was used each day to ensure that the processed cells were found in the same position by requiring that their mean channel fluorescence be ±40 channels at a resolution of 1,024. A detailed description of instrument quality assurance procedures has been previously reported (8). One author (C.C.S.) performed all flow cytometric analysis and sorting of cytokeratin-positive cells from the breast and blood samples of each patient. Sorted cells were then submitted for molecular genetics PCR analysis. Cytologic analysis of the flow-sorted material was not performed.

DNA Extraction
Fifteen core biopsy washing specimens were sorted into cytokeratin-positive and cytokeratin-negative cell populations. DNA was extracted by using the High Pure PCR Template Preparation Kit (Boehringer Mannheim, Indianapolis, Ind) according to the manufacturer’s directions. DNA from blood was obtained by using a modification of the method of Grimberg et al (9) in which isolated nuclei are digested with proteinase K (1 µg/mL) for 3 hours at 65°C, followed by ribonuclease A (0.5 µg/mL) for 1 hour at 37°C, and phenol-chloroform-isoamyl alcohol extraction and ethanol precipitation.

Microsatellite PCR
The 15 sorted specimens from core biopsy washings were analyzed by using microsatellite PCR. PCR was performed in a 20-µL volume containing 20 ng of DNA from blood or 10 µL (2–10 ng) of DNA from cytokeratin-positive or cytokeratin-negative cells along with 10 ng of three arbitrarily selected PCR primers, one of which was phosphorous 32 end-labeled with polynucleotide kinase. PCR was carried out as previously described (10). The loci and primers used were as follows: D3S 1029 [5'-ATACTCTGGACCCAGATTGATTAC-3' and 5'-TAATTCCCAAA-TGGTTTAGGGGAG-3'], D5S 346 [5'-ACTCACTCTAGTGATAAATCGGG-3' and 5'-AGCAGATAA-GACAAGTATTACTAGTT-3'], and D2S 123 [5'-AAACAGGATGCCTTTA-3' and 5'-GGACTTTCCACCTAT-GGGAC-3']. These markers were chosen for use in our laboratory because they have been described and used for other ongoing genetic analyses. PCR products were separated on 6.6% polyacrylamide denaturing gels. The gels were dried and placed on film for 16-hour exposure or were placed on a phosphoimager screen for 16 hours and analyzed by using a phosphoimager system (Molecular Dynamics; Sunnyvale, Calif) for additional sensitivity.

Normal DNA, such as that obtained from blood or normal tissue, possesses both a maternal and paternal copy (allele) of each of the gene sequences amplified by the polymerose chain reaction. Blood is preferred as a control when possible because blood leukocytes are less likely than the cytokeratin-negative cells to harbor genetic alterations consistent with breast cancer. Since blood cells harbor only hereditary mutations and since these patients were not presumed to be familial high-risk patients, there was no reason to presume that the blood cells would harbor any genetic changes. Blood has traditionally been used as a negative control for genetic analyses of sporadic cancers. Loss of heterozygosity was determined by visually comparing the PCR products from blood (or cytokeratin-negative cells when used as a control) with the PCR product of the sorted cytokeratin-positive (epithelial) cells at each locus.


   Results
TOP
 ABSTRACT
 INTRODUCTION
 Materials and Methods
 Results
Discussion
 REFERENCES
 
The 15 consecutive stereotactic core biopsy samples that were examined at flow cytometric and molecular genetics analyses consisted of a median of eight 11-gauge core samples (range, five to 15 samples) per lesion examined at biopsy (Fig 1). Eight (53%) lesions were microcalcifications, and seven (47%) were soft-tissue abnormalities at mammography. The pathologic diagnoses of the stereotactic core biopsy samples, the rinse cell counts of the flow cytometry–sorted core samples, and the findings of DNA allele loss analysis are summarized in the Table. The median number of sorted cytokeratin-positive (epithelial) cells per sample was 51,998 (range, 4,303–217,277 cells). Final pathologic diagnoses included four (27%) invasive carcinomas, two (13%) ductal carcinomas in situ, one (7%) benign proliferative lesion with atypical ductal hyperplasia or lobular carcinoma in situ, three (20%) benign proliferative lesions without atypia, and five (33%) benign nonproliferative lesions.



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  		Figure 1. Stereotactic core biopsy specimen radiograph confirms sampling of mammographic calcifications (arrows) in several cores.

 
During molecular genetics analysis, extraction yielded sufficient DNA to perform PCR analyses with breast and blood cell samples with all three primers. No DNA was recovered from two patients’ blood samples due to unknown technical reasons. In these patients (patients 3 and 11), the cytokeratin-negative breast cells were used as a source of control DNA. Examples of autoradiographs of PCR products resolved with gel electrophoresis from study patients 1–3 and 11 are shown in Figure 2. Loss of heterozygosity was observed at three loci in the DNA of cytokeratin-positive cells obtained from three patients (patients 2, 3, 11) with benign pathologic findings without atypia (Fig 2). These losses occurred at the D3S 1029 locus in patient 2, the D5S 346 locus in patient 3, and the D2S 123 locus in patient 11.



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  		Figure 2a. PCR products resolved with gel electrophoresis show DNA allele loss (loss of heterozygosity) in cells obtained from core needle biopsy washings sorted with flow cytometry into cytokeratin-positive (+) and cytokeratin-negative (-) populations. Normal alleles are indicated by square brackets. PCR products in a and b were detected at autoradiography. PCR products in c were detected by using a phosphoimager. Blood (B in a) DNA or cytokeratin-negative cells were used as a source of control DNA to demonstrate the normal pattern of maternally and paternally derived allelic bands. (a) The microsatellite marker is D3S 1029. In patient 1, normal alleles in blood DNA and cytokeratin-positive cell DNA overlap in a ductal carcinoma in situ lesion (indicated by overlapping square brackets). Cytokeratin-negative cells exhibit an abnormal second allele (indicated by the curly bracket). In patient 2, the upper allele is lost in the cytokeratin-positive cells. (b) The microsatellite marker is D5S 346. In patient 3, the upper allele is lost in the cytokeratin-positive cells. (c) The microsatellite marker is D2S 123. In patient 11, the upper allele is lost in the cytokeratin-positive cells.

 


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  		Figure 2b. PCR products resolved with gel electrophoresis show DNA allele loss (loss of heterozygosity) in cells obtained from core needle biopsy washings sorted with flow cytometry into cytokeratin-positive (+) and cytokeratin-negative (-) populations. Normal alleles are indicated by square brackets. PCR products in a and b were detected at autoradiography. PCR products in c were detected by using a phosphoimager. Blood (B in a) DNA or cytokeratin-negative cells were used as a source of control DNA to demonstrate the normal pattern of maternally and paternally derived allelic bands. (a) The microsatellite marker is D3S 1029. In patient 1, normal alleles in blood DNA and cytokeratin-positive cell DNA overlap in a ductal carcinoma in situ lesion (indicated by overlapping square brackets). Cytokeratin-negative cells exhibit an abnormal second allele (indicated by the curly bracket). In patient 2, the upper allele is lost in the cytokeratin-positive cells. (b) The microsatellite marker is D5S 346. In patient 3, the upper allele is lost in the cytokeratin-positive cells. (c) The microsatellite marker is D2S 123. In patient 11, the upper allele is lost in the cytokeratin-positive cells.

 


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  		Figure 2c. PCR products resolved with gel electrophoresis show DNA allele loss (loss of heterozygosity) in cells obtained from core needle biopsy washings sorted with flow cytometry into cytokeratin-positive (+) and cytokeratin-negative (-) populations. Normal alleles are indicated by square brackets. PCR products in a and b were detected at autoradiography. PCR products in c were detected by using a phosphoimager. Blood (B in a) DNA or cytokeratin-negative cells were used as a source of control DNA to demonstrate the normal pattern of maternally and paternally derived allelic bands. (a) The microsatellite marker is D3S 1029. In patient 1, normal alleles in blood DNA and cytokeratin-positive cell DNA overlap in a ductal carcinoma in situ lesion (indicated by overlapping square brackets). Cytokeratin-negative cells exhibit an abnormal second allele (indicated by the curly bracket). In patient 2, the upper allele is lost in the cytokeratin-positive cells. (b) The microsatellite marker is D5S 346. In patient 3, the upper allele is lost in the cytokeratin-positive cells. (c) The microsatellite marker is D2S 123. In patient 11, the upper allele is lost in the cytokeratin-positive cells.

 
In the remaining reactions in the 15 patients’ samples, there was either a pattern of bands consistent with amplification from both a maternal and paternal allele or an uninformative electrophoretic pattern in which only apparently comigrating PCR products were produced. With these latter findings, it was impossible to determine whether loss of heterozygosity had occurred. With a single exception (Fig 2, patient 1), both patterns of amplification generated from sorted cells were identical to those observed in the patient’s blood. No cytokeratin-positive cells were found in the blood samples.

One of the alleles amplified with the D3S 1029 primers from patient 1’s cytokeratin-negative (nonepithelial) cell DNA migrated with slower mobility on the gel relative to that of the products from cytokeratin-positive cells and blood; this finding suggested that at least a large segment of the cytokeratin-negative cells contained a common abnormal allele. It can be presumed that this abnormality is harbored by stromal tissue or a population of cytokeratin-negative epithelial cells, as blood and cytokeratin-positive cells share a common pair of PCR-amplified products. It also cannot be ruled out that some tumor cells may not express cytokeratin at detectable levels.


   Discussion
TOP
 ABSTRACT
 INTRODUCTION
 Materials and Methods
 Results
 Discussion
REFERENCES
 
Molecular analysis of benign and early malignant breast lesion biopsy samples is often restricted by the primary need for pathologic diagnosis with these specimens and the lack of fresh cells or tissue for molecular genetics studies. Fresh tissue is often preferred to paraffin-embedded tissue for flow cytometric and molecular genetics studies because the fixation process may render the analysis less accurate (11–14). Microsatellite PCR is a standard tool for the measurement of loss of heterozygosity and microsatellite instability (10,15–21).

The development of flow cytometric sorting techniques to discriminate and sort epithelial (cytokeratin-positive) cells from tissue or cell samples containing a mixture of epithelial and stromal cells, as well as blood elements, that form both benign and malignant breast lesions and adjacent normal breast tissue enables analysis of the pure epithelial component (6,7). This sorting technique can be applied to samples with as few as 1,000 cells and provides an excellent source of material for analysis.

Flow cytometry also serves as a screening test for adequate cytokeratin-positive samples for further molecular genetics analysis. This screening enables more efficient use of more time-consuming genetic analysis. Subsequent to the development of the techniques in our initial experience, 15 (88%) of 17 core biopsy by-product samples submitted for flow cytometry were suitable for further molecular genetics analysis. While the epithelial cells in these samples were derived from the mammographic lesion and often from the adjacent normal breast tissue during core biopsy, it is not known whether any underlying genetic changes would be localized to a single light microscopic epithelial pathologic finding, the adjacent epithelium, or a field of breast tissue, as well. This is a topic of current investigation (22).

The complete loss of alleles demonstrated in Figure 2 suggests that the vast majority of the cytokeratin-positive cells from each core biopsy sample showed the same genetic lesion. If a substantial fraction of the cytokeratin-positive cells contained the second allele, PCR amplification of that allele would have yielded a second band on the gel with an intensity proportional to that fraction of cells. It is possible that if loss of heterozygosity is present in only a small fraction of the cytokeratin-positive cells, the loss of heterozygosity could be masked by the presence of normal allele from the other cytokeratin-positive cells.

While it is not uncommon to find heterogeneous expression of cytokeratin in breast carcinomas, where some cells express more cytokeratin than others, in our flow cytometry laboratory, we have never analyzed a breast carcinoma in which cytokeratin expression was absent (23).

The spectrum of lesions examined at core biopsy during screening mammography (the majority comprising nonproliferative or hyperplastic and atypical benign lesions and the remainder comprising ductal carcinoma in situ and early invasive carcinomas) provides investigators with excellent material with which to study biologic changes associated with the transition between benign and malignant breast lesions. The use of fresh core biopsy samples themselves would preclude pathologic diagnostic evaluation of all the core samples in a patient and would create the risk of missing a cancer or high-risk lesion that is detectable in only one of the cores obtained during a stereotactic core biopsy procedure of small mammographic lesions. Procurement of fresh gross tissue after excision of malignant lesions is not possible in a majority of small clinically occult carcinomas detected at mammography, and subsequent excision is not performed in most benign lesions.

In this study, no comparison was made between the accuracy of core biopsy cell washings and that of paraffin-embedded core biopsy samples or, when available, fresh tissue samples obtained after subsequent excision of malignant lesions. Although there is no standard for genetic analyses of these types of clinically occult breast lesions, a comparison can be made with microdissected samples from histologic sections of paraffin-embedded lesions. This comparison is beyond the scope of this initial experience. Also, sampling of the lesions will not be identical between the two techniques.

Ultimately, larger clinical studies are required to show that any information gained from these techniques is clinically useful and independent of known prognostic or risk factors in a routine clinical setting. Stereotactic core biopsy translational research techniques such as this could be applicable to the large numbers of women undergoing routine clinical stereotactic core biopsy in multicenter clinical trials.

Scanning the genomes of benign lesions and early breast cancers for loss of heterozygosity may provide clues to the identity of genes involved in the earliest stages of tumor progression, stages that are identified as benign at standard pathologic examination as well as identify high-risk benign lesions and provide prognostic information about early breast cancers that potentially alters clinical management. It is not unusual to observe loss of heterozygosity in benign lesions in the breast and other tissue (21,24). Alterations and mutations in normal cell DNA are essential for tumor progression to occur, although the exact number and nature of these changes has not been fully elucidated. Mutations have also been detected in histologically normal epithelium adjacent to a cancer. Frequent loss of heterozygosity at a particular site in the genome has led to the identification of several tumor suppressor genes in a variety of cancers. Microsatellite instability has been associated with loss of function of DNA mismatch repair genes in hereditary nonpolyposis colorectal carcinoma, as well as a small number of sporadic cancers.

Several investigators (25–27) have reported the use of reverse-transcription PCR to assess the expressed levels of various messenger RNA species in breast cancers. These studies have focused on measurements of estrogen-steroid receptor gene expression. Analyses of this type are useful in the creation of expression profiles of tumors and may aid in clinical management decision making. However, these RNA assays do not provide insights into new, as yet undiscovered, cancer genes. With the current level of understanding of the molecular genetics of cancer, we believe that the analyses of tumor DNA alterations and RNA levels are complementary techniques, with the former as a means of gene discovery and the latter as a means of defining the role of identified genes.

While the randomly selected markers used in this study are linked to the hMLH1 gene associated with hereditary nonpolyposis colorectal carcinoma and the APC gene associated with familial adenomatous polyposis (10), it is not established that loss of either of these genes plays any role in the cause of breast cancer, and to our knowledge, no association has been shown previously. It is more likely that these losses represent random events occurring as part of larger genome-wide destabilization. Study of large numbers of breast lesions will allow distinction of random versus breast cancer–related genetic events.

The identification of the loss of heterozygosity in the DNA of benign breast lesion and early carcinoma samples, as compared with the DNA in the control cell samples from the same patients, is the first step in an ongoing study to determine the associations between the number and sites of altered DNA alleles and the pathologic spectrum of benign and malignant breast disease diagnosed in women undergoing core biopsy. Identification of common sites of loss of heterozygosity among patients with specific diseases could lead to specific analyses of the DNA sequence and function at selected allele sites with use of the same DNA extracted for this study.

We conclude that stereotactic core biopsy washings and blood drop samples, routinely discarded by-products, provide satisfactory fresh cellular material for flow cytometry and molecular genetics microsatellite PCR analysis for the detection of loss of DNA heterozygosity in benign and malignant breast epithelium.


   ACKNOWLEDGMENTS
 
We gratefully acknowledge Karin A. Brady for invaluable assistance in the preparation of this manuscript, and Jan L. Hoffman, BS, and David L. Sheedy, BA, for performing the cell staining and sorting.


   FOOTNOTES
 
Abbreviation: PCR = polymerase chain reaction

Author contributions: Guarantors of integrity of entire study, P.C.S., C.C.S., D.L.S.; study concepts and design, P.C.S., C.C.S., D.L.S.; definition of intellectual content, P.C.S., C.C.S., D.L.S.; literature research, P.C.S., C.C.S., D.L.S.; clinical studies, P.C.S.; experimental studies, C.C.S., D.L.S.; data acquisition and analysis, P.C.S., C.C.S., D.L.S.; manuscript preparation, editing, and review, P.C.S., C.C.S., D.L.S.; manuscript final version approval P.C.S.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 Materials and Methods
 Results
 Discussion
 REFERENCES
 

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D. L. Stoler, C. C. Stewart, and P. C. Stomper
Breast Epithelium Procurement from Stereotactic Core Biopsy Washings: Flow Cytometry-sorted Cell Count Analysis
Clin. Cancer Res., February 1, 2002; 8(2): 428 - 432.
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