Flow cytometric study of DNA distribution in cytopunctures of benign and malignant breast lesions

One hundred seventy-eight cytopunctures of mammary lesions were obtained for cytologic diagnosis and flow cytometric (FCM) analysis of the nuclear DNA content. All lesions were excised and evaluated histologically; 106 were carcinomas and 72 were benign lesions. The benign lesions showed a diploid DNA content, with one exception. Among the 106 carcinomas, 35 (33%) were diploid, 14 (13%) were tetraploid and 57 (54%) were aneuploid. For 79 carcinomas, the relationship between ploidy and (1) "T" and "N" of TNM staging, (2) the histologic grading of Scarff, Bloom and Richardson, (3) axillary nodal involvement, (4) the presence of estrogen and progesterone receptors, (5) age and (6) menopausal status was investigated. The percentage of aneuploidy was significantly higher (P less than .05) in grade III tumors as compared to grade I tumors. There was no significant relationship between aneuploidy and the other factors. However, a trend was observed between the lack of steroid receptors and a high probability of the tumor being aneuploid. FCM DNA analysis was also carried out on breast carcinomas obtained at surgery in 40 patients for whom FCM DNA analysis had previously been performed on breast cytopuncture specimens. The FCM DNA analyses were found to be best performed on the samples obtained by cytopuncture, which may increase the yield of tumor cells.     

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 and S-phase fraction in carcinoma of the gallbladder related to histopathology, number of gallstones and survival.

Gallstones are a risk factor for the development of gallbladder cancer. We studied DNA ploidy and cell cycle composition by flow cytometry in archival specimens from 52 gall bladder carcinomas in relation to histopathological grade, tumour stage, gallstone number and survival. 69% of the gallbladder carcinomas showed aneuploidy. All tumours with single stones (N=11) were aneuploid while only 61% of tumours with multiple stones (N=41) were aneuploid (p=0.002). DNA aneuploidy was related to increase in T-category (p=0.01), grade (p=0.02), and nuclear pleomorphism (p=0.0005). The distribution of DNA ploidy shifted from tetraploid in low stage towards triploid positions in high stage tumours (p=0.02) combined with higher S-phase values in triploid tumours (p=0.05). S-phase fraction increased during development from normal tissue to dysplasia, cancer in situ and cancer in diploid cases (p=0.0002), and further at the change from diploid to aneuploid (p=0.004). At a median cancer specific survival time of four months patients with diploid tumours had a better survival than those with aneuploid tumours (p=0.02). In multivariate analysis of the tumour characteristic, only T-category and tumour grade were independent prognostic factors.The shift from diploid to aneuploid and the further shift of ploidy within aneuploid tumours are in agreement with the concept of a clonal development of gallbladder cancer. These changes are combined with a stepwise increase in the fraction of S-phase cells. Low frequency of symptoms in single stone patients may be the reason for detection of malignancy at a late stage of tumour development.     

DNA ploidy assessment method applied to primary breast carcinoma FNA biopsies

The goal of our study was to assess suitability of FNA biopsy material as a source of samples (cell suspension) for DNA ploidy assessment in neoplastic tumors using flow cytometry. DNA ploidy is an established prognostic factor in many types of cancers. Aneuploid breast tumors are characterized by increased aggressiveness which manifests itself through rapid local progression and metastatic spread. Investigated specimens were breast cancer FNA biopsy cell suspensions. Measurements were performed using flow cytometry. Material studied comprised 143 cases analyzed in 1999-2000. We found in this group 101 carcinoma cases with aneuploid type and 42 cases of primary breast carcinoma with diploid type of cell cycle. Immunocytochemical assesssment of estrogen receptor and progesterone receptor status was performed in group of 105 cases. DNA ploidy was compared to receptor status of the investigated cells. DNA aneuploidy correlated with weak or no reaction for the presence of estrogen and progesterone receptors. Our study demonstrates the suitability of DNA ploidy assessment method applied to cytological material from FNA biopsies.     

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.     

Comparison of image analysis with flow cytometry for DNA content analysis in pigmented lesions of the skin.

The DNA ploidy of 85 melanocytic skin lesions was determined by flow cytometry (FCM) and interactive image analysis (IA) using nuclear extracts of paraffin-embedded tissue. Of the 85 lesions analyzed, 43 were malignant melanomas in different stages of evolution, 15 were dysplastic nevi, 11 were Spitz nevi, and 16 were other types of nevi. Some of the last had features of congenital nevi. Within the melanoma category, there was 42% aneuploidy by FCM versus 56% by IA. Of those melanomas aneuploid by FCM, all but one were aneuploid by IA. All dysplastic nevi, 10/11 Spitz nevi and 15/16 other nevi were diploid by both methods. One of the 16 nevi from the "other types" category was tetraploid by IA but diploid by FCM. A single Spitz nevus was tetraploid by FCM but diploid by image analysis. While our results suggest that interactive IA is potentially a more sensitive method than FCM for detecting aneuploidy in cutaneous pigmented lesions, it remains to be shown whether this will translate into better prognostic assessment of the biologic behavior of melanocytic neoplasms than provided by flow cytometric ploidy analysis.     

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.     

Flow and image cytometry in thymic neoplasia: correlation with clinical outcome

OBJECTIVE: To compare nuclear DNA by flow (FCM) and image cytometry (ICM) in thymic neoplasms and to relate results to clinical outcome. STUDY DESIGN: DNA ploidy of 44 thymomas and 6 thymic carcinomas was studied by FCM and ICM of single nuclear suspensions from paraffin blocks. RESULTS: By FCM, 33 thymomas (75%) and one thymic carcinoma (17%) were diploid; 6 thymomas (14%) and 4 thymic carcinomas (67%) were aneuploid. By ICM, 36 thymomas (82%) were diploid; 7 thymomas (16%) and 6 thymic carcinomas (100%) were aneuploid. Mean follow-up in 44 cases was 46.2 months (range, 1-162). Ten patients with persistent/recurrent disease included four with thymic carcinoma, who died of the disease (two aneuploid by both techniques, two aneuploid by ICM with unsatisfactory/diploid FCM). Four had invasive thymoma and recurrence after 13-150 months (two diploid and two aneuploid by both methods), one had diploidy and noninvasive thymoma that recurred at 92 months, and one had an epithelial thymoma that recurred at 144 months (aneuploid by FCM, diploid by ICM). CONCLUSION: The results obtained in this preliminary, retrospective study show a high concordance between FCM and ICM; aneuploidy correlated with poor outcome by both methodologies. While these findings are encouraging, larger numbers of cases will be needed to define the role of FCM and ICM in predicting outcome in thymic tumors.     

Proliferative characteristics of primary and metastatic human solid tumors by DNA flow cytometry

The percentage of cells in S-phase (S-index) was calculated from DNA histograms of 453 primary and metastatic human solid tumors (predominantly bladder, breast, colorectal, renal, prostate, ovarian and lung carcinomas, melanomas, and sarcomas). S-indices varied widely among both primary and metastatic tumors (1-48%); there was no significant difference in S-indices between primary and metastatic tumors. The S-indices for aneuploid tumors were significantly higher than for diploid tumors. When data for all aneuploid tumors were analyzed collectively, there was no significant relationship between S-index and DNA ploidy index. However, for colorectal and ovarian carcinomas S-indices increased, and for lung carcinomas S-indices decreased with elevation in the degree of DNA-ploidy. Lung carcinomas had the highest S-indices. Comparison of flow cytometry (FCM) and cytology data indicated that for most diploid tumors S-indices reflect the proportion of S-phase cells among a mixed population of normal and tumor cells. For most aneuploid tumors, the proportion of tumor cells estimated cytologically was similar to the proportion of aneuploid cells estimated by FCM. For a small proportion of aneuploid tumors a comparison of cytology and FCM data indicated the presence of a predominant diploid tumor stemline and a minor stemline with aneuploid DNA content. There was a wide spread in the values of S-indices within tumor groups defined by degree of differentiation and stage of disease at surgery.     

Nasopharyngeal carcinoma heterogeneity of DNA content identified on cytologic preparations.

OBJECTIVE: To evaluate tumor heterogeneity of DNA content in nasopharyngeal carcinoma (NPC) performed on cytologic specimens. STUDY DESIGN: Image cytometric analysis of DNA ploidy status of 40 NPCs was performed on nasopharyngeal brushing smears stained with the Feulgen method after hematoxylin eosin staining. If the DNA distribution pattern from the same tumor exhibited diploid, aneuploid or/and tetraploid peaks or some combination of these patterns, the presence of tumor heterogeneity of DNA content was identified. RESULTS: Thirty-four cases (85%) had a nondiploid DNA pattern among the 40 NPCs. Twenty-eight cases exhibited tumor heterogeneity of DNA content (70%). Of the 28 tumors, 13 (46%) had a combination of diploid and tetraploid patterns, 10 (37%) had a combination of diploid and aneuploid patterns, 3 cases (11%) had a combination of tetraploid and aneuploid patterns, and 2 cases had two aneuploid stem lines. The relationship between DNA ploidy pattern and tumor histologic and cytologic morphology was also examined. CONCLUSION: There is a high incidence of DNA content heterogeneity in NPC. The relevance of tumor heterogeneity to the biologic behavior of NPC awaits further study. DNA quantification with image cytometry on destained cytologic preparations is feasible and reliable.     

Comparison of the intratumor DNA ploidy distribution pattern between differentiated and undifferentiated gastric carcinoma

OBJECTIVE: To compare the differences in intratumor DNA ploidy distribution pattern between differentiated and undifferentiated carcinoma of the stomach. STUDY DESIGN: Two hundred five cases of surgically resected gastric carcinoma were studied, comprising 125 differentiated and 80 undifferentiated carcinomas. DNA ploidy was determined by means of flow cytometry in stepwise sections of the entire tumor. The intratumor DNA ploidy distribution pattern was compared between these two histologic types. RESULTS: Differentiated carcinomas comprised 35 cases of predominantly diploid (28%) and 90 cases of predominantly aneuploid (72%) tumors, while undifferentiated carcinomas comprised 46 cases of predominantly diploid (57.5%) and 34 cases of predominantly aneuploid (42.5%) (P < .01). The frequency of cases showing predominantly aneuploidy among the differentiated carcinomas was significantly higher than among the undifferentiated carcinomas at stages IA and IIIA. The rate of cases showing heterogeneity was lower among the early-stage cases than among the advanced-stage cases in both histologic types. Among the differentiated carcinomas, there were 22 cases that had more than six DNA stemlines in each tumor, whereas among undifferentiated carcinomas, there were six such cases (P < .05). CONCLUSION: There were more cases with high malignant potential among the differentiated carcinoma cases than among the undifferentiated carcinoma cases in the present series.     

Prognostic significance of DNA ploidy determined by high-resolution flow cytometry in breast carcinoma

OBJECTIVE: To assess the prognostic value of DNA ploidy in breast carcinoma and its relation to other established prognostic factors. STUDY DESIGN: We evaluated DNA ploidy in 303 breast carcinoma patients with a median follow-up of 63 months. Flow cytometry was performed on frozen tumor material, yielding histograms with narrow peaks (median coefficient of variation of 2.08). DNA ploidy pattern was classified as either diploid versus nondiploid, euploid (diploid and tetraploid) versus aneuploid or diploid/near-diploid (DNA index < 1.2) versus other, and correlated with relapse-free (RFS) and cancer-specific survival (CSS) along with tumor size, histologic grade and type, axillary lymph node involvement, menopausal and steroid receptor status, age and type of treatment. RESULTS: Seventy-one percent of tumors were DNA nondiploid (14% tetraploid and 57% aneuploid). There was a strong association between DNA ploidy and histologic grade. Histologic grade, lymph node status, tumor size and DNA ploidy (regardless of the classification used) were all significantly associated with RFS and CSS in multivariate analysis. CONCLUSION: These results suggest that DNA ploidy, at least when determined from frozen tumor tissue, is an independent prognostic factor in breast carcinoma; however, its prognostic power seems to be inferior to that of histologic grade, with which it strongly correlates.     

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.     

Modal DNA values of normal and malignant urothelial cells of the bladder in relation to nuclear size

In a study of the correlation between mean nuclear size and DNA content in urinary bladder carcinoma, the modal DNA values of cell suspensions from 125 biopsies, obtained from 86 patients with malignant or normal urinary bladder epithelium, were analyzed by flow cytometry (FCM). Light microscopic measurements of nuclear size were carried out on smears from the same material. The results were correlated to the histopathologic stage and grade. The mean nuclear volumes were significantly larger in diploid tumor cells than in cells of normal epithelium. Aneuploid tumors showed significantly larger nuclei than did diploid tumors. Although there was a significant correlation between increases in the nuclear volume and in the DNA content, there was some overlapping between various grades of malignancy: mean nuclear volumes in aneuploid grade 2 tumors did not differ from those in aneuploid grade 3 tumors. A combination of FCM and morphometry discriminated all but 16% of the tumors from the normal cases. It is concluded that FCM and morphometry are complementary and can be used for the objective characterization of urinary bladder carcinomas.     

Comparison between flow cytometry and image cytometry in ploidy distribution assessments in gynecologic cancer.

The DNA content in 37 tumors from 34 women with gynecological cancer was measured by flow cytometry (FCM) and interactive image cytometry (ICM). Agreement was obtained in 81% of cases as regards ploidy levels, but seven tumors (19%) showed different ploidies. Of these, five were classified as diploid by FCM but either aneuploid (three cases) or polyploid (two cases) by ICM. Two other tumors were aneuploid by ICM but polyploid (one case) and unclassifiable (one case) by FCM. All tumors classified as aneuploid by FCM were also aneuploid by ICM, and all tumors classified diploid by ICM were also diploid by FCM. Of six patients whose tumors were classified as euploid (five diploid and one polyploid) by FCM but classified as aneuploid by ICM, five relapsed, and three of these have died of disease. On the basis of these findings, it is concluded that ICM must be performed in cases classified as diploid by FCM to ensure that small subpopulations of aneuploid tumor cells are not overlooked.     

Prognostic significance of DNA aneuploidy and p21 ras oncoprotein expression in colorectal cancer and their role in the determination of treatment modalities

The purpose of the present study was to investigate the prognostic significance of DNA ploidy, S-phase fraction and p21 ras oncoprotein expression in patients with colorectal cancer and to correlate these factors with the clinical behavior of the tumors and their response to therapy. Of 79 patients with colorectal cancer 57% (45/79) had early stage disease. Forty-one percent (32/79) had aneuploid tumors while 30% (24/79) of the tumors had a high (>10%) S-phase fraction. p21ras oncoprotein expression was detected in 38% (30/79) of tumors. Patients with aneuploid tumors had a worse prognosis than patients with diploid tumors (p=0.0002). Similarly, patients with high S-phase fraction tumors had a shorter survival than those with low S-phase fraction tumors (p=0.005). No such difference was found between p21 raspositive and p21 ras-negative tumor subgroups. In early stage colorectal cancer, aneuploidy was closely correlated with disease outcome (p=0.029). Early stage patients with diploid tumors who received radiotherapy and chemotherapy had a better prognosis than patients with aneuploid tumors. In conclusion, DNA ploidy is a significant and independent prognostic factor in colorectal cancer. Aneuploidy and genetic alteration of the p21 ras oncoprotein are important in determining the biological aggressiveness of colorectal cancer. Furthermore, DNA ploidy may identify those subgroups of patients with early stage disease who may benefit from more aggressive treatment.     

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)     

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.     

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.