Variations associated with the DNA analysis of multiple fine needle aspirates obtained from breast cancer patients

Abstract

Summary The present study was carried out to determine the variation in DNA content between multiple fine needle aspirates (FNA) of the same tumour from patients with breast cancer. Analysis of different aliquots of the same FNA showed good reproducibility in terms of cell cycle distribution and DNA index. Duplicate FNAs taken from different sites in nine of 11 excised tumours showed similar reproducibility. However, two of the aneuploid tumours displayed substantial variations in the distribution of cell populations between the duplicate samples. Sequential FNAs with no intervening therapy were obtained from the same tumour in 17 patients; one at the time of diagnosis and the other at biopsy 1-3 weeks later. Only five cases showed no variation between the sequential FNAs; the remaining 12 displayed different DNA profiles. A further 13 patients were studied before and during systemic therapy. While there was no variation between sequential FNAs in four patients, marked differences in the DNA profile were observed in the remaining nine patients undergoing treatment, the changes not necessarily being associated with clinical response to therapy. It is concluded that the monitoring of cellular changes by DNA analysis of sequential FNAs may be complex and subject to problems associated with heterogeneity.Flow cytometric DNA analysis can provide useful information on the cellular characteristics of breast tumours (Auer et al., 1980;Baildam et al., 1987;Cornelisse et al., 1987;Dowle et al., 1987; Kallioniemi et al., 1987a, b;Owainati et al., 1987). Recently, we demonstrated that fine needle aspiration of most breast cancers can provide sufficient cellular material for meaningful analysis of DNA profiles using flow cytometry (Levack et al., 1987). This use of flow cytometry in the analysis of cellular material from FNAs has potentially wide applications. For example, it may be possible to monitor the effects of treatment by analysing cellular characteristics of sequential tumour FNAs.In this respect it is important to show that analysis of FNAs provides reproducible results. The aim of the present paper was to assess variation in DNA profiles by studying multiple fine needle aspirates taken simultaneously and sequentially from breast tumours. Patients and methods PatientsTumour fine needle aspirates (FNAs) were obtained from patients attending the Professorial Breast Clinics of the University Department of Surgery, Edinburgh. All tumours were shown histologically to be breast cancers and malignant cells were cytologically confirmed to be present in all aspirates. AspiratesThe method of aspiration was that described by Zajicek (1965) and used a 23 gauge needle attached to a 10 ml syringe. After removing an aliquot for cytological confirmation of malignancy (Dixon et al., 1984), the remainder of the aspirate was expelled into 200 dp of citrate buffer and stored at -40°C until analysis (Vindel0v et al., 1983a, b values falling within the 'S' phase fraction of diploid tumour cells.Briefly, frozen cell suspensions were tha...

Breast cancer proliferation measured on cytological samples: a study by flow cytometry of S-phase fractions and BrdU incorporation

“…The second addressed tumour heterogeneity and the representativity of sampling relative to the tumour mass. Repeated samplings were performed on seven patients, as already attempted by others (Mullen & Miller, 1989). Modifications of the DNA histograms were observed in four out of seven, consisting in variable representation of multiple DNA peaks.…”

Measurement of S-phase fraction and ploidy in sequential fine-needle aspirates from primary human breast tumours treated with tamoxifen

“…In two cases (patients 17 and 18 in Table II (Prey et al, 1985;Erhardt & Auer, 1986;Remvikos et al, 1991) which have all shown good correlation between sequential FNAs from the same tumour. Only one study (Mullen & Miller, 1989) has commented on significant variation in DNA analysis caused by intratumoral heterogeneity between two fine-needle aspirates taken from the same tumour. Even here a com-parison of DI between two needle aspirates showed less than 5% variation in 10 out of 11 cases.…”

Response to the letter from Remvikos

“…Sir -The letter from Dr Remvikos makes a number of criticisms of our paper (Mullen & Miller, 1989) which we feel are worth defending. With regard to the originality of the article and the possible omission of other work (Spyratos et al, 1987;Remvikos et al, 1988;Briffod et al, 1989), we feel our work is indeed original.…”

“…Sir -There are a number of points which I believe should be raised with regard to the article by Mullen and Miller (1989) on DNA flow cytometry of breast cancer fine needle aspirates. First, I would point out that the authors have omitted to reference previous contributions on the same topic (Spyratos et al, 1987;Remvikos et al, 1988a, Briffod et al, 1989.…”