Flow cytometric analysis of DNA stemline heterogeneity in primary and metastatic breast cancer.

Flow cytometric DNA-ploidy analysis was used to investigate intratumor DNA stemline heterogeneity in primary breast carcinomas and lymph node metastases (LNM). The study was done in tumor specimens from 44 patients 35 of whom had LNM. In all, measurements were done in 214 different samples of primary tumors and 211 lymph nodes. Sixty-one percent (27/44) of the primary tumors were found to have multiple DNA aneuploid stemlines when the data of the separate samples per tumor (mean 4.9) were compared. Only five of 44 (11%) primary tumors were DNA diploid; two of these had DNA aneuploid metastases. Statistical analysis of these results indicated that, on average, four samples are needed for reliable determination of the DNA ploidy status of primary tumors by flow cytometry. In the majority of the cases (26/35), distinct tumor DNA stemlines found in LNM were also present in the primary tumor, which suggests that the generation of DNA ploidy diversity may have taken place prior to metastasis. Multiploidy was not related to tumor size but, particularly for LNM, was significantly correlated with age (r = 0.40, P = 0.02). The results of this study support the view that breast cancer is an extremely heterogeneous disease and that underestimation of this factor might account for the disagreement in literature about the prognostic value of DNA ploidy determinations.     

Flow cytometric DNA measurements in human thyroid tumors

By means of flow cytometry (FCM), DNA distribution pattern and the fraction of cells in the various phases of the cell cycle were studied in 52 samples of normal thyroid tissues, follicular adenomas, follicular carcinomas, medullary carcinoma and fibrosarcomas. In the normal thyroid tissues and follicular adenomas DNA diploid cell populations only were found. Among 20 follicular carcinomas in 13 cases (65%) together with the DNA diploid cells, DNA aneuploid cell lines were also observed. S-phase fraction in follicular adenomas is higher than in the normal thyroid tissues and lower than those in thyroid carcinomas. The percentage of S-phase cells in DNA aneuploid populations is significantly higher (S = 19 +/- 9.3%) than in the diploid cell lines (S = 3.7 +/- 2.6%). DNA aneuploid cell populations were predominantly observed in carcinomas with a high degree of morphological anaplasia.     

DNA ploidy and survival in breast cancer patients

Flow cytometric DNA ploidy measurements using frozen or deparaffinized tumor specimens were performed on 565 primary breast cancers from patients treated in the period 1975-1984. Twenty-nine percent of the cases were diploid, 61% had a single aneuploid stemline, and 10% were multiploid. Aneuploid tumors more often had negative estrogen receptor values than diploid tumors, but no significant correlation was found between ploidy class and TNM stage. Patients with more than ten positive axillary lymph nodes had predominantly aneuploid tumors. Overall and distant relapse-free survival were higher for patients with diploid tumors and low-aneuploid tumors. Stratification of the patients according to degree of lymph node involvement, TNM stage, and menopausal stage showed that the prognostic effect of aneuploidy was apparent predominantly in patients with locally advanced disease. Postmenopausal node-positive patients with diploid tumors had a significantly better prognosis than those with aneuploid tumors, but this difference was not found for the comparable premenopausal group. Multivariate analysis with the Cox proportional hazards model indicated that ploidy is an additional, independent prognostic factor in postmenopausal patients.     

DNA ploidy pattern and amplification of ERBB and ERBB2 genes in human gastric carcinomas

The DNA ploidy pattern and amplification of ERBB and ERBB2 genes were examined in paraffin-embedded tissue from gastric carcinomas using flow cytometry and a slot-blot hybridization technique. The incidence of aneuploidy in well differentiated adenocarcinomas (56%) was significantly higher (p less than 0.05) than that in poorly differentiated adenocarcinomas (21%). The DNA ploidy pattern was not remarkably different between the primary tumors and metastatic deposits in lymph nodes. Of the nine specimens having an aneuploid stem cell line in the primary tumor and/or in metastases, three showed ERBB2 gene amplification and one showed ERBB gene amplification. The incidence of epidermal growth factor (EGF) immunoreactivity in tumor cells showed no difference between diploid and aneuploid tumors. These findings indicate that aneuploidy is frequently associated with amplification of ERBB and ERBB2 genes.     

DNA ploidy pattern and amplification of ERBB and ERBB2 genes in human gastric carcinomas

The DNA ploidy pattern and amplification of ERBB and ERBB2 genes were examined in paraffinembedded tissue from gastric carcinomas using flow cytometry and a slot-blot hybridization technique. The incidence of aneuploidy in well differentiated adenocarcinomas (56%) was significantly higher (p<0.05) than that in poorly differentiated adenocarcinomas (21%). The DNA ploidy pattern was not remarkably different between the primary tumors and metastatic deposits in lymph nodes. Of the nine specimens having an aneuploid stem cell line in the primary tumor and/or in metastases, three showed ERBB2 gene amplification and one showed ERBB gene amplification. The incidence of epidermal growth factor (EGF) immunoreactivity in tumor cells showed no difference between diploid and aneuploid tumors. These findings indicate that aneuploidy is frequently associated with amplification of ERBB and ERBB2 genes.     

Methodologic aspects of nuclear DNA assessment of gliomas with astrocytic and/or oligodendrocytic differentiation. Correlation of image and flow cytometric studies on paraffin-embedded specimens

A total of 239 samples from paraffin-embedded, formalin-fixed astrocytic and/or oligodendrocytic gliomas from 111 patients were deparaffinized and disaggregated for image cytometric (ICM) and flow cytometric (FCM) DNA assessments. Each measurement technique produced evaluable histograms in about 85% of the samples analyzed. In the 10% that could not be analyzed by FCM, the background counts were too high and the coefficients of variation were too broad for precise evaluation. The failures with ICM were due to a shortage of Feulgen-stained tumor cell nuclei after the deparaffinization and disaggregation procedures. The results obtained were identical in 77% of the samples evaluable by both methods and practically identical (i.e., euploid versus aneuploid) in an additional 18%. The reasons for completely divergent DNA ploidy patterns in 5% of the samples could not be clarified. About 80% of the histopathologically highly malignant gliomas were found to consist of neoplastic cells with an aneuploid or tetraploid nuclear DNA distribution pattern. The results show that cytometric DNA assessments can be reliably performed on paraffin-embedded specimens of gliomas with astrocytic and/or oligodendrocytic differentiation by means of FCM and ICM on deparaffinized and disaggregated specimens.     

DNA quantitation of distal bile duct carcinoma measured by image and flow cytometry

OBJECTIVE: To evaluate discrepancies between flow cytometry (FCM) and image cytometry (ICM), ploidy incidence and relation between DNA ploidies and survival in distal bile duct carcinomas (DBDCs). STUDY DESIGN: Forty-four archival tumor samples from patients with DBDC who underwent subtotal pancreatoduodenectomy from 1985 to 1996 were examined for DNA ploidy using FCM and ICM. RESULTS: Overall, 59% (26/44) of the tumors were aneuploid by at least one of the two techniques. We detected more cases of aneuploidy with ICM than FCM in formalin-fixed, paraffin-embedded DBDCs, 62% (21/34) versus 33% (13/40), respectively. When results could be compared, moderate strength of agreement (kappa = .45) was demonstrated. No correlation was found between DNA ploidy by FCM, ICM or combined FCM-ICM and survival time (P = .80, P = .35, and P = .54, respectively). CONCLUSION: Approximately 59% of DNA histograms contained aneuploid cell populations. Although ICM, as compared to FCM, is more sensitive in assessing the ploidy status of DBDC, both methods were complementary. Most discrepancies between FCM and ICM were due to the dilution of aneuploid populations by non-neoplastic diploid cells. DNA ploidy assessment in DBDC did not offer the possibility of improving the ability to predict survival.     

Dual-parameter flow cytometric analysis coupling the measurements of forward-angle light scatter and DNA content of archival ovarian carcinomas of low malignant potential

Paraffin-embedded archival specimens from 45 cases of ovarian carcinoma of low malignant potential (OCLMP) were analyzed by flow cytometry (FCM) using propidium iodide (PI) staining. Since single-parameter FCM analysis is often deficient in the resolution of subtle near-diploid DNA-aneuploid populations, forward-angle light scatter (FALS) was measured as a second parameter. DNA aneuploidy was identified in 15 cases (33%). In 7 of those 15 cases, aneuploidy was resolved with single-parameter FCM; in the remaining 8 cases, DNA aneuploidy was resolved only following dual-parameter analysis coupling DNA content and FALS. In all 15 cases, a single near-diploid aneuploid population was observed (mean DNA index = 1.2); there were no tetraploid aneuploid cases. The proliferative activity for all 45 cases studied ranged from 1.0% to 8.9%, with a mean of 3.5%. No difference in mean proliferative activity was observed between the aneuploid and diploid tumors (P greater than .05). To exclude the possibility that PI staining artifacts caused the observed aneuploidy, five of the eight cases shown to be aneuploid by dual-parameter analysis were further studied using an alternate DNA-binding dye, DAPI, yielding similar results. To exclude the possibility that contaminating stromal and/or inflammatory cells caused the observed aneuploidy, samples from a subset of the dual-parameter cases were sorted, revealing the aneuploid populations to be composed primarily of tumor nuclei.(ABSTRACT TRUNCATED AT 250 WORDS)     

Flow cytometric DNA analysis of renal-cell carcinoma. A study of fine needle aspiration biopsies in comparison with multiple surgical samples

The flow cytometric (FCM) DNA analysis of fine needle aspiration (FNA) biopsy material was compared with the DNA analysis of multiple surgical biopsy material from 44 renal-cell carcinomas. Twenty tumors were heterogeneous with respect to their DNA content. Eleven of the 17 tumors that had both diploid and aneuploid tumor cell clones in the surgical specimens gave a diploid DNA content in the aspiration biopsies; the other 6 cases showed aneuploidy in the aspirate. The fine needle aspirates from 18 homogeneously diploid tumors and 9 tumors with an aneuploid DNA content in all eight surgical samples revealed DNA indices similar to those found in the surgical samples. These findings show that aneuploid clones of renal-cell carcinoma can remain undetected by the use of FNA biopsy as a method for obtaining samples for FCM DNA analysis.     

Flow cytometric DNA content of fresh tumor specimens using keratin-antibody as second stain for two-parameter analysis.

Studies concerning flow cytometric assessed DNA content reveal problems in interpretating DNA histograms of tumor specimens. The main problems are histograms with a broad coefficient of variation in the G0/G1 fraction; a high G2M fraction and samples with a low percentage of tumor cells. Therefore, in the present study, 382 fresh tumor specimens of carcinomas were analysed routinely, double labeled with, on the one hand, propidium-iodide for assessing DNA content and, on the other, a monoclonal keratin-antibody for marking epithelial and tumor cells. Of the 311 tumor samples, using single parameter analysis 165 (54%) were classified as DNA aneuploid and 146 (46%) as DNA "euploid." By double parameter analysis, 224 (72%) samples were keratin positive and 87 (27%) keratin negative and, of the 224 keratin positive tumors, 175 (78%) were DNA aneuploid and 49 (22%) DNA euploid. The DNA histograms of single and double parameter analysis were compared and it was concluded that in 24 cases (11%) keratin labeling was necessary to recognize DNA aneuploidy. In another 23 (10%) cases, keratin labeling was helpful in assessing DNA aneuploidy. Finally when the results of the 311 samples were combined, 215 (68%) were scored as DNA aneuploid and 99 (32%) DNA euploid. Thus the overall gain in assessing DNA aneuploidy using the double labeling technique is 14%. In conclusion, it is shown that keratin labeling on fresh tumor cell suspensions of epithelial tumors is of additional value in establishing DNA content. Because single parameter DNA assessment is adequate in approximately 60% of the tested samples, the double labeling technique can be performed routinely, or after initial single parameter DNA assessment. Histograms having a broad CV and/or a high G2M are good candidates for the double labeling technique. Using this technique, DNA-content assessment becomes more reliable.     

DNA ploidy in colorectal cancer, heterogeneity within and between tumors and relation to survival

Flow cytometry was used to study the incidence of aneuploidy and to determine the significance of multiple sampling from colorectal tumors. DNA ploidy pattern has been proposed as a supplementary prognostic marker, but discrepancies in findings are major. DNA clonal heterogeneity, defined as two or more DNA aneuploid stemlines in the same tumor, is well established. However, most studies have been based on only one biopsy from each tumor. In our study multiple biopsies were taken from 163 patients (88 males and 75 females) electively operated for colorectal cancer. Tumor cells were harvested by fine needle aspiration from fresh frozen biopsies sampled at different sites of each tumor. DNA aneuploidy was detected in tumors from 145 patients (89%), and 18 patients (11%) had a solitary DNA diploid cell population. In a 79 month follow-up period 105 patients had died. Statistical analysis showed that distinction between diploidy and aneuploidy did not predict survival. However, grouping subpopulations into DNA diploid plus near diploid (DNA index (DI) 0. 97-1.15), DNA aneuploid with all aneuploid subpopulations in the interval 1.15-2.06, and DNA aneuploid with subpopulations with DI < 0.97 and/or DI > 2.06, showed a significant difference in survival in a Cox multivariate analysis including Dukes' stage P = 0.049 comparing the second group to the first and P = 0.01 comparing the third group to the first. In 21 (13%) patients only one subpopulation was found, 57 (35%) had two, 44 (27%) had three, and 41 (25%) had four or more different subpopulations. The association of DNA ploidy to survival is shown to be dependent on the number of biopsies analysed.     

DNA ploidy analysis of urinary tract epithelial tumors by laser scanning cytometry

OBJECTIVE: To evaluate a rapid and simple method for DNA content analysis of urinary tract epithelial tumors with laser scanning cytometry (LSC). STUDY DESIGN: The subjects were 25 patients (37 specimens) who underwent surgery for urinary tract epithelial tumors. Tissue specimens of such tumors were frozen immediately after tumor resection and stored at -80 degrees C until used. Touch preparations were made and fixed in ethanol at room temperature. The cell nucleus was stained with propidium iodide solution containing RNase, and DNA ploidy was analyzed by LSC. Nuclear debris and overlapping nuclei were gated out by special statistical filters. In LSC, a normal diploid reference peak was determined by observing lymphocytes morphologically on the computer display of the instrument and/or under the microscope. RESULTS: DNA ploidy could be evaluated in all tumor tissues. The time it took from preparing the tumor specimen to the last measurement was about 40 minutes at the shortest, and measurement of all the specimens was completed within one hour. The coefficient of variation was 2.8-7.8% (mean, 4.4%). All eight specimens (100%) at grade 1 (G1) were DNA diploid, but 20% and 85.7% of the G2 and G3 cells, respectively, were DNA aneuploid. In total, 15 of the 37 specimens were DNA aneuploid. All 17 pTa-pT1 specimens (100%) were DNA diploid, but 100% and 50% of the T2 and T3 tumors, respectively, were DNA aneuploid. CONCLUSION: One can now supplement a morphologic diagnosis with useful information using LSC of touch preparations of tumors obtained at surgery or of imprints of archived, frozen specimens. LSC provides excellent DNA histograms for surgical specimens and has great potential for clinical application in pathology.     

Progression of mammary adenocarcinomas as reflected by nuclear DNA content

In 18 breast cancer patients the DNA histograms observed in the primary tumor at the date of diagnosis were compared with those found in the corresponding local and distant metastases at autopsy up to more than 12 yr later. All patients, except one, exhibited the same type of DNA histogram in both the primary tumor and its metastases. In one patient the DNA histogram changed from an euploid type in the primary breast carcinoma to an aneuploid type in the metastases. The results are interpreted as indicating that mammary adenocarcinoma in general exhibit a high degree of stability of the nuclear DNA content during the history of the disease. It is suggested that in breast cancer progression of the tumor disease is more likely due to a net increase and/or dissemination of tumor cells exhibiting similar genetic properties and malignancy potential than to a progressive dedifferentiation and increase of malignancy of the tumor cells.     

Correlation between automated DNA ploidy measurements of Hürthle-cell tumors and their histopathologic and clinical features

The treatment of Hürthle-cell tumors of the thyroid is controversial because of their rarity and the inconsistent histopathologic criteria for their diagnosis. In order to obtain more objective criteria for the management of Hürthle-cell tumors, the nuclear DNA content of cells from 20 cases was measured with the MicroTICAS system and the correlation between the DNA distribution patterns and the clinical and histopathologic findings was evaluated. Three main DNA patterns were found: euploid, polyploid and aneuploid. The euploid or polyploid Hürthle-cell tumors came from patients who did not develop distant metastases or recurrence whereas the aneuploid variants came from patients who died of their disease and/or developed distant metastases and recurrence. Various correlation analyses were performed between DNA ploidy and age, sex, size of tumor, growth pattern, pleomorphism, invasion and metastases. Our data suggests that an aneuploid DNA pattern or one with a large percentage of aneuploid nuclei with DNA content exceeding 5N may predict eventual metastases or recurrence from Hürthle-cell tumor.     

Characterization of ploidy level in bladder tumors and selected site specimens by flow cytometry

The histopathological grade of atypia was compared with the ploidy level in biopsy specimens from bladder tumors and six grossly normal areas (selected sites). The study included investigation of 303 specimens from 45 patients. The results demonstrated a high correlation between the grade of atypia and the occurrence of aneuploid cell populations. The selected site specimens were diploid in patients with low grade malignant tumors, while high grade malignant tumors were associated with aneuploid selected site specimens in 20% of the cases. The divergence of ploidy level in the tumor and selected site specimens found in some cases clearly demonstrated that the carcinogenic process in the bladder may lead to clonal heterogeneity. Flow cytometric analysis seems suited for mapping of the ploidy levels in different parts of the bladder. The possible importance of the additional information is discussed.     

Improved sensitivity in comparative genomic hybridization analysis of DNA heteroploid cell mixtures after pre-enrichment of subpopulations by fluorescence activated cell sorting.

Cytogenetic analysis of solid tumors with comparative genomic hybridization (CGH) is hampered by the dilution of DNA from individual tumor subpopulations with DNA from other cells. We investigated to what extent this dilution effect can be alleviated using fluorescence activated cell sorting (flow sorting) of experimental DNA heteroploid cell mixtures prior to CGH. From mixtures of normal lymphocytes with triploid K-562 cells the individual components were sorted according to stemline DNA content and processed by CGH in comparison with pure K-562 samples and the original mixtures. Compared with 30 autosome copy number imbalances found in pure K-562 samples, a mixture with 32% K-562 cells showed 16 imbalances, and none were detected in mixtures with 13% or 5% K-562 cells. In contrast, 29, 22 and 23 imbalances were detected in K-562 nuclei sorted from the 32%, 13% and 5% mixtures, respectively. This indicate that CGH analysis of flow sorted DNA aneuploid subpopulations enables a specific cytogenetic analysis of the individual subclones in a DNA heteroploid cell population.     

Comparison of tissue disaggregation techniques of transitional cell bladder carcinomas for flow cytometry and chromosomal analysis

DNA index (DI) measurements and chromosomal analysis of 42 transitional cell carcinomas were done after mechanical and enzymatical disaggregation of the tumor specimens. The results obtained with these different disaggregation techniques were compared in the 33 cases (79%) that showed recognizable chromosomes. The enzymatically obtained cell suspensions could not be used for chromosomal analysis after short-term culture of 24 hours. In four cases, the DI after enzymatical treatment could not be estimated. In most cases, the DI obtained from the tumor cells was similar for both aggregation techniques, with the exception of four cases of enzymatically treated cell suspensions in which the DI could not be estimated. The average DI of the aneuploid tumors was 13% higher than the corresponding chromosome count. In 19% of the aneuploid tumors the proportion of aneuploid cells could not be measured after enzymatical treatment. In the remaining suspensions the proportion of diploid cells was higher after enzymatical disaggregation than after mechanical treatment. It is concluded that for flow cytometric and direct chromosomal analysis of bladder tumors, the mechanical disaggregation technique is most suitable.     

Discrepancies between flow cytometric and cytogenetic studies in the detection of aneuploidy in human solid tumors

Parallel flow cytometric (FCM) cell DNA studies and cytogenetic studies were performed on clinical samples from twenty human solid tumors of various types and on cell lines established in tissue culture from three of these tumors. Six of twenty clinical samples (30%) showed concordance between flow cytometry and cytogenetics with respect to the presence or absence of aneuploidy. Among the fourteen cases with discrepancies between the two methods, 8 (40% of all cases) showed hypodiploidy by cytogenetics and had diploid DNA histograms. Three cases (15%) had prominent discrete peaks in the triploid to tetraploid region by cytogenetics but had only barely discernible corresponding peaks in the DNA histogram. In two cases (10%) cytogenetic studies revealed diffuse aneuploidy. Cytogenetic studies demonstrated near-tetraploidy in three samples, but only one of these was detected by FCM; all three cases exhibited other numerical chromosomal abnormalities. In one case aneuploidy was demonstrated by FCM and not by cytogenetics. Among the tumor cell lines established in culture, the DNA Index was often higher than the cytogenetic index. Overall, 13/20 or 65% of patients with solid tumors in this study had numerical chromosomal abnormalities that were not detected by flow cytometry. Eleven of these patients had distant metastases at the time of tumor sampling, and nine of these died of their disease within 1-11 months of the time of study.     

Clone heterogeneity in diploid and aneuploid breast carcinomas as detected by FISH

We investigated the relationship between DNA ploidy and alterations in chromosomes 1, 8, 12, 16, 17, and 18 in 63 breast carcinoma samples by static cytofluorometry and fluorescence in situ hybridization. Thirty specimens were diploid and 33 were aneuploid. In aneuploid samples, the DNA index value ranged from 1.3 to 3.1, with a main peak near tetraploid values. Diploid clones were present in 21 of 33 aneuploid specimens. Fluorescence in situ hybridization analysis showed a heterogeneous degree of alterations in diploid specimens: one sample was normal, 16 samples had one to three chromosome alterations involving mostly chromosomes 1, 16, and 17, and 13 samples an even higher degree of alterations. The 33 aneuploid specimens showed a very high number of signals (four, five, or more). All the investigated chromosomes were affected in 23 of 33 specimens. Alterations in chromosomes 1 and 17 were detected to a similar percentage in diploid and aneuploid samples, whereas chromosome 16 monosomy was more frequent in diploid samples. Overrepresentation of chromosomes 8, 12, 16, and 18 was significantly higher in aneuploid than in diploid samples. Based on these results, we suggest that diploid and aneuploid breast carcinomas are genetically related. Chromosome 1 and 17 alterations and chromosome 16 monosomy are early changes. Allelic and chromosomal accumulations occur during progression of breast carcinoma by different mechanisms. The high clone heterogeneity found in 17 of 33 aneuploid samples could not be completely explained by endoreduplication and led to the suggestion that chromosomal instability concurs with aneuploidy development. This different evolutionary pathway might be clinically relevant because clone heterogeneity might cause metastasis development and resistance to therapy.