Flow cytometric analysis of DNA content in human ovarian cancers

A total of 155 samples from 101 patients with ovarian cancer were investigated using flow cytometry to evaluate the DNA index and the percentage of cells in the various cell cycle phases. Thirty-four samples were DNA diploid tumours, while the other 121 were DNA aneuploid tumours. The DNA index was very stable in different sites and over time in the same patient. Tumour stage and ploidy were significantly associated: stages III and IV tumour stage were more likely to be DNA aneuploid. Patients with residual tumour size at first surgery greater than 2 cm had a significantly larger number of DNA aneuploid than DNA diploid tumours. The DNA index was also related to the degree of differentiation of the tumours. The percentage of cells in the S phase of the cell cycle was significantly higher in DNA aneuploid and in poorly differentiated tumours than DNA diploid and well differentiated tumours. Multivariate analysis using the Cox model showed that the DNA index and the percentage of cells in S phase were not independent prognostic variables in this study. Prospectively collected data should be accumulated before assigning the DNA index an important role as a biological prognostic factor in ovarian cancer.     

Molecular genetic analysis of flow-sorted ovarian tumour cells: improved detection of loss of heterozygosity.

Detection of loss of heterozygosity (LOH) is usually performed on homogenised tumour specimens. In this type of analysis samples with a low percentage of tumour cells have to be excluded and possible intra-tumour heterogeneity is obscured. In this study we report the application of polymerase chain reaction (PCR)-driven LOH detection with in total 22 microsatellite markers for chromosome 1q, 3p, 3q, 4p, 6p, 6q, 11p, 11q, 17p, 17q, 18p, 18q, Xp and Xq on flow-sorted cells from fresh and paraffin-embedded ovarian tumour tissue. Titration experiments showed that LOH can be detected with as few as 100 cell equivalents of DNA. Clear examples of LOH could be detected in the sorted aneuploid fractions from one unilateral and two bilateral ovarian tumours from three patients. In two samples the sorted fraction was less than 10% of the total sample. The bilateral tumours from the same patient showed loss of identical alleles for one marker (case OV64) and two markers (case OV69), indicative of their monoclonal origin. Multiparameter flow cytometry using two different ovarian tumour markers (MOv18 and BMA180), an anti-cytokeratin monoclonal antibody (MAb) (M9), an anti-vimentin MAb (V9) and a MAb against the panepithelial antigen 17-1A on the fresh ascites cells of the fourth ovarian cancer patient was used to investigate possible intra-tumour heterogeneity. We showed the presence of at least three phenotypically different populations, of which the diploid, keratin-positive, vimentin-negative population showed a similar LOH pattern as the aneuploid population (DNA index = 1.7), indicative of its neoplastic origin. The same LOH pattern was shown in an omentum metastasis from this patient also having the same aneuploid DNA index of 1.7. The sharing of the same LOH pattern by the diploid and aneuploid tumour cell populations suggests that the observed allele loss events occurred before the development of aneuploidy. PCR on flow-sorted cells is thus an important tool to study clonal diversity in tumours.     

Flow-cytometric DNA ploidy analysis in primary and metastatic canine thyroid carcinomas.

DNA ploidy was measured by flow cytometry in 36 primary malignant thyroid neoplasms (including 6 bilateral tumours which were considered as separate neoplasms) from 30 dogs. In addition, DNA ploidy was determined in local recurrences in 3 dogs, and in 18 metastatic sites from 14 dogs. Aneuploidy was found in 21 of 36 (58%) primary sites. Eighteen of the 21 (86%) aneuploid tumours contained hypodiploid cell populations, with 12 having single hypodiploid peaks, and 6 being multiploid. Three other tumours had single aneuploid peaks with a DNA index (DI) greater than 1.0. The DIs in local recurrences were identical to those in the original neoplasms. Ploidy status (diploid vs. aneuploid) was identical in primary and metastatic sites in 10 out of the 14 dogs. Aneuploidy was more frequent in carcinomas from dogs with distant metastases (78%) than from dogs with less advanced stages of disease (53%), although this difference was not significant. There was no significant correlation between DNA ploidy and histopathological variables. From the strikingly high frequency of hypodiploidy in canine tumours, it is concluded that ploidy evolution in canine neoplasms may differ from that in human tumours.     

Flow cytometric DNA analysis and chromosomal aberrations in malignant glioblastomas.

In this study we combined flow cytometry with fluorescence in situ hybridization to detect numerical aberrations in chromosomes. Fifty-nine human malignant gliomas were examined by flow cytometry for DNA-content and cell cycle analysis and for numerical aberrations of chromosome 1 by in situ hybridization using a chromosome specific centromere probe. Of the gliomas analysed, 42% were diploid and 58% showed aneuploid tumour cell populations. The DNA index was heterogeneous ranging from 1.0 to 2.3. The S-phase analysis showed proliferation activity from a very low range of 0.7% up to 17.0%. In general, diploid gliomas exhibited a lower S-phase activity than aneuploid gliomas. Of the aneuploid gliomas, 15% showed a peridiploid pattern with a DNA index mean of 1.1. In these peridiploid tumours a trisomy of chromosome 1 could be detected by fluorescence in situ hybridization (FISH). The frequency of trisomic chromosome 1 in malignant gliomas reflects a very slight increase in DNA index from diploid to peridiploid (DNA index 1.1). Comparison of chromosome numbers and DNA content gave good correlation. Also important, the results reflects the cell cycle, specifically the extent of S-phase activity. In general, cell proliferation of diploid and peridiploid gliomas is much less than in higher aneuploid gliomas. The analysis of DNA content may thus yield results with respect to the biological behaviour of tumours in general.     

Flow cytometric analysis of DNA and cell proliferation in ovarian tumors

DNA content in tumor cells from 50 patients with ovarian tumors was analysed by flow cytometry (FCM). Solid tissue samples were processed to obtain monodispersed cells. Staining for DNA analysis was achieved with ethidium bromide and mithramycin. Peripheral blood lymphocytes were used as reference diploid cell population. All benign ovarian tumors exhibited only diploid cells. DNA aneuploid cell lines were found 66.6% of serous carcinomas and in 80% of malignant granulosa cell tumors. The S-phase fraction of DNA diploid cells in benign ovarian tumors (S = 2.4 +/- 1.2%) was smaller than those of malignant tumors (S = 8.2 +/- 5.2%). DNA aneuploid cell populations in serous carcinomas display a higher S-phase fraction (S = 19.2 +/- 9.3%) than DNA diploid cells (S = 11.7 +/- 3.2%). No major differences were obtained between primary ovarian tumors and their metastases, as far as degree of aneuploidy and S-phase fraction are concerned. A high degree of correlation was established between the grade of differentiation of ovarian tumors and the DNA ploidy abnormalities.     

Selection of tumour cell subpopulations occurs during cultivation of human tumours in soft agar. A DNA flow cytometric study

To examine whether selection of tumour cell subpopulations occurs during cultivation in soft agar, we compared in 23 human tumours of different histological types the DNA content of cells from colonies formed in soft agar (method of Courtenay and Mills, 1978) with that of the original tumour cells. The ploidy as well as the fraction of cells in S phase were determined from DNA histograms after staining of the nuclei with a propidium-iodide procedure and flow cytometric recordings. In 8 of 17 aneuploid tumours analysed, specific aneuploid subpopulations disappeared during cultivation or new aneuploid populations, not demonstrable in the original cell suspensions, appeared in the colonies. In 9 cases identical aneuploid populations were found in the colonies and the tumours. In one of 6 diploid tumours examined, aneuploid cell populations not revealed in the original cell suspension, were found in addition to diploid cells, whereas 5 tumours gave rise to colonies containing a purely diploid population. The results show that in a variety of human malignant tumours cultivation in soft agar may select specific aneuploid tumour cell populations.     

Progression of diploid tumor cells in aneuploid head and neck squamous cell carcinomas

The development of aneuploid clones from diploid progenitor cells is a regular characteristic of head and neck squamous cell carcinoma progression. While the significance of aneuploidy formation for the acquisition of invasive and metastatic behavior is well documented, little is known about the contribution of diploid tumor cells after aneuploid clones have emerged. To distinguish diploid cells of epithelial origin from benign cellular components, we applied multiparameter flow cytometry of DNA content and cytokeratin (CK) expression to 36 primary tumors. Twenty-seven carcinomas accommodated aneuploid cell lines that stained positive for CK. All diploid cell populations obtained from aneuploid carcinomas contained CK-positive subpopulations as did all of nine tumors that consisted exclusively of diploid cells. The proportions of CK-positive diploid cells ranged between 6% and 80%, independent of whether they were achieved from entirely diploid or from aneuploid carcinomas. CK-gated diploid and aneuploid cell populations showed largely identical S-phase fractions. These results emphasize that diploid tumor cells regularly persist after the development of aneuploid clones and significantly contribute to local tumor progression. Despite the presence of diploid epithelial cells in aneuploid primary tumors, exclusively the aneuploid clones of eight corresponding lymph node metastases were CK-positive. This provides further evidence of a largely reduced metastatic potential of diploid tumor cells.     

2-DE protein expression in endometrial carcinoma

The objective of this study was to explore the protein expression pattern in normal endometrial mucosa (n = 5) and endometrial carcinoma (n = 15) of low (diploid) and high (aneuploid) malignancy potential by two-dimensional gel electrophoresis (2-DE). The specimens were evaluated for histopathologic subtype, stage and grade in relation to DNA ploidy. A match-set consisting of five samples from normal endometrium, eight diploid and seven aneuploid tumours was created. All the diploid and three of the aneuploid tumours were of endometrioid subtype, while the remaining four were of uterine seropapillary type. There were 192 protein spots differentiating diploid tumours from normal endometrium and 238 protein spots were separating aneuploid tumours from normal endometrium (p < 0.01). A cluster analysis based on 52 significantly deviating protein spots within the groups showed clustering and separation of the normal endometrium, diploid and aneuploid tumours. In conclusion this study showed significant differences in protein expression between normal endometrium and endometrial carcinoma as well as between endometrial carcinoma of low and high malignancy potential. In future studies these results may provide useful in finding new sensitive prognostic markers for endometrial cancer.     

Effect of tamoxifen upon cell DNA analysis by flow cytometry in primary carcinoma of the breast

The effect of tamoxifen upon cellular DNA ploidy in carcinoma of the breast was assessed by flow cytometry (FCM), in a prospective group of 77 patients with primary operable disease. Each had a needle biopsy at the outpatient visit for diagnosis and FCM analysis, and definitive surgery was performed a median of 8 days later. Forty received tamoxifen during this period - 40 mg qds loading dose for 24 h, followed by 20 mg daily until the day of operation: 37 patients received no therapy. The DNA histogram from the needle biopsy was compared with that obtained from the resected tumour for each individual. There was little change between the pair of histograms from tumours from the untreated patients. In those who had received tamoxifen the most consistent effect was a marked reduction in the magnitude of the 'tetraploid' peak in tetraploid or near-tetraploid tumours with DNA indices 1.8-2.0. There was little change in diploid or 'other DNA-aneuploid' tumours. In tetraploid tumours (DNA index of 2.0) the percentage of nuclei in the diploid S phase was significantly related to the percentage of nuclei in the diploid G2 + M/tetraploid G1 peak (P less than 0.003, unpaired t test). These data suggest that an effect of tamoxifen can be demonstrated by FCM upon tumours exhibiting a tetraploid or near-tetraploid DNA content. It is possible that tetraploid or near-tetraploid human mammary tumours may be a distinct group of endocrine responsive tumours within the overall group of aneuploid tumours, and that the majority are probably derived from the diploid population rather than being a true aneuploid population.     

Survival of large bowel carcinoma patients with different DNA ploidy

One hundred patients operated for large bowel carcinoma were divided into a distinct aneuploid group of 63, and a near diploid one of 37. Flow cytometry was used for determination of the DNA ploidy pattern. All tumours in the aneuploid group contained one or more aneuploid cell populations. All patients were followed clinically from 3.5 to 7.8 years. The corrected 5 year survival was 64% and 49% for patients with near diploid and aneuploid tumours, respectively (not significant). Significant differences in corrected survival time were not observed for Dukes' stages A, B, and C patients pooled, nor for Dukes' stage D patients. However, for Dukes' stage C patients alone, there was a tendency (P = 0.10) for patients with near diploid tumours to show a better survival. A highly significant predominance of aneuploid tumours was seen in males, in contrast to an equal distribution of aneuploid and near diploid tumours in females. A slight predominance of aneuploid tumours in the left colon and rectum was seen. Both these findings indicate the influence of environmental factors (hormonal, anatomical, phenotypical) on the development of tumours with a particular DNA ploidy pattern.     

Flow cytometric analysis of human uterine sarcomas and cell lines

Flow cytometric techniques were used to characterize multiple human uterine sarcomas and cell lines derived from some of these tumors. Analysis of DNA content showed that 9 of the 11 uterine sarcomas investigated were composed of at least one aneuploid population as well as a distinct diploid population. These data indicate that aneuploidy, as measured by flow cytometry, is a characteristic more common to uterine sarcomas than that previously reported for uterine adenocarcinomas. Unlike the original tumors, the cell lines established from three of the sarcomas contained predominantly diploid populations with only minor aneuploid populations. Treatment of one of the sarcoma cultures with tumor promoters did not result in an increase in the aneuploid populations. Tumors which arose in nude mice upon transplantation of two of the sarcomas did not contain the same distribution of tumor subpopulations as found in the original sarcomas. Apparently, the in vitro culture and and in vivo nude mouse conditions were not appropriate for maintaining the original equilibrium between the aneuploid and diploid subpopulations but instead provided a selective environment that resulted in the preferential growth of only certain tumor populations. Dual-parameter analysis of DNA content and alkaline phosphatase levels of one of the sarcomas were useful for distinguishing the aneuploid from the diploid population coexisting in this tumor. Our data suggest that flow cytometry is a valuable tool to analyze the characteristics of the tumor populations residing in primary uterine sarcomas as well as to determine which of these tumor subpopulations survive in culture and transplantation to nude mice.     

Cell DNA content--correlation with clonogenicity in the human tumour cloning system (HTCS)

Thirty-six ovarian and renal-cell tumours were analyzed by flow cytometry (FCM) for DNA content and in parallel were assayed for colony formation in a human tumour cloning system (HTCS). While 15/19 (79%) tumours with an abnormal (aneuploid) DNA stemline formed colonies in the HTCS, only 2/17 (12%) of diploid tumours formed colonies. All samples contained tumour cells as assessed by routine cytological examination. The capacity to form colonies in the HTCS was not correlated in these tumours with grade or stage of disease or tumour type. The level of aneuploidy expressed as the FCM DNA index did not correlate with the cloning efficiency in HTCS. These findings suggest that tumour growth in the HTCS reflects a biologically important potential, related in at least some tumours to an abnormal DNA stemline.     

DNA aneuploidy and low S-phase fraction as favourable prognostic signs in metastatic melanoma

The prognostic value of cellular DNA content in melanoma metastases was investigated by flow cytometric analysis of fresh or paraffin-embedded tumour blocks from 95 consecutive patients referred to the Helsinki University Central Hospital Melanoma Team. Thirty-three per cent of the tumours were DNA diploid and 67% DNA aneuploid. S-phase fractions were lower in DNA diploid than in DNA aneuploid tumours (10.7% and 17.6%). Tumour ploidy and S-phase fraction were shown by multivariate Cox model analysis to be independent prognostic variables and major determinants of survival after first recurrence. Surprisingly, patients with DNA aneuploid tumours and with tumours with low SPF survived significantly longer than those with DNA diploid or high SPF tumours. This exceptional finding of favourable prognosis for DNA aneuploid tumours was more prominent among patients receiving intensive systemic therapy and among patients with stage IV disease, probably indicating a tendency for DNA aneuploid tumours to have higher sensitivity to systemic therapy.     

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

Sequential fine-needle aspirates (FNAs) for cytodiagnosis and flow cytometry were taken from 21 patients with primary breast carcinoma at intervals ranging from 1 to 3 months after the commencement of first-line tamoxifen therapy. Nine patients achieved a sustained complete or near complete response over a 3-9 month period. The tumour cells from seven out of nine of these patients were initially aneuploid, while the remaining two patients had diploid tumours. An analysis of sequential FNAs showed that, in three out of the seven aneuploid tumours, only benign epithelial cells could be detected by cytology in the post-tamoxifen sample. In the remaining six cases, including the two diploid tumours, there was no change in ploidy but a reduction in S-phase fraction (SPF) to approximately 50% of the pretreatment level. In all cases, these changes in ploidy or SPF were seen with a mean lead time of 4 months before the tumour had reached clinical complete remission. None of these patients have relapsed after a mean follow-up period of 18 months. The tumours of 12 patients achieved no more than a temporary partial response to primary tamoxifen therapy. In seven out of eight of these cases, which were all initially aneuploid, sequential FNAs during tamoxifen therapy revealed either an increase or no change in the SPF with the tumour remaining aneuploid. In the remaining four cases the tumours were all recorded as being diploid in the pretreatment sample. However, although three of these cases had a temporary partial response to tamoxifen, an aneuploid component was picked up in repeat sequential FNAs with a mean lead time of 5 months before clinical confirmation of eventual disease progression. We conclude that changes in ploidy and SPF detected by flow cytometry may predict initial response and the likelihood of relapse of breast tumours to tamoxifen before clinical changes become evident. These data justify a larger study.     

Multiparameter flow cytometry for simultaneous assessment of p53 protein expression and cellular DNA content in oral squamous cell carcinomas: evidence for the development of aneuploid clones from p53-deficient diploid progenitor cells

Diploid tumour cells regularly continue to progress after the development of aneuploid cell populations in head and neck squamous cell carcinomas. The coexistence of aneuploid clones with their diploid progenitor cells provides a unique opportunity to study the order of appearance of p53 mutation and aneuploidy in the same tumour. Multiparameter flow cytometry was therefore applied to 22 oral squamous cell carcinomas to simultaneously assess cellular DNA content and p53 protein expression on a single-cell basis. Concurrent measurements of cytokeratin expression served to identify tumour cells of epithelial origin. One of 5 diploid and 2 of 17 aneuploid carcinomas were p53-negative. For 15 p53-positive aneuploid tumours, overexpression of p53 protein was identified for the aneuploid clones as well as for coexisting diploid tumour cell populations in 14 cases. On the understanding that coexisting diploid and aneuploid tumour cell populations have a common clonal origin, these results provide evidence that aneuploid tumour clones typically develop from p53-deficient diploid progenitor cells. Loss of wild-type p53 function may therefore contribute to the development of aneuploidy in head and neck cancer.     

DNA aneuploidy and cell proliferation in breast tumors

The cellular DNA content of 30 benign and 180 malignant breast tumors was analyzed by means of flow cytometry (FCM). All benign tumors exhibited a normal DNA content (diploid), whereas 65% of the malignant tumors showed an abnormal DNA content (aneuploid). The ploidy distribution of malignant tumors was bimodal with an increasing frequency near diploid DNA index (DI), and a second group had a DI ranging from triploid to tetraploid. In estimating the degree of malignancy eight independent histomorphologic and cytologic criteria were introduced. A good correlation was observed between DNA content abnormalities and the grade of differentiation of breast carcinomas. The percentage of S-phase cells of DNA aneuploid cell lines was significantly higher than in the diploid ones. The highly differentiated breast carcinomas (Grade 1) indicated lower S-phase values as compared to the undifferentiated (Grade 3) ones. S-phase values estimated by FCM were about two times higher than the 3H-thymidine labeling index (LI) obtained by an in vitro procedure. The data estimated in this study showed that DNA determinations as an adjunct to conventional histopathologic assessment may provide objective clinically relevant information with respect to the degree of malignancy and prognosis of patients with breast carcinoma.     

Nuclear DNA Distribution in Neuroendocrine Gastroenteropancreatic Tumors Before and During Treatment

The nuclear DNA contents of tumor cells in 73 patients with endocrine gastrointestinal tumors, 19 patients with endocrine pancreatic tumors (EPT) and 54 patients with malignant carcinoid tumors were determined before and after treatment. The DNA profiles were divided into diploid and aneuploid. In untreated patients, 9 out of 10 (90%) primary EPT and all 9 primary malignant carcinoid tumors (100%) were diploid. Tumor cell imprints from liver metastases of patients with untreated EPT showed aneuploidy in 5 of 11 cases, but only in 7 out of 46 DNA records from patients with untreated carcinoid liver metastases. DNA alteration from diploid to aneuploid profiles occurred in 2 patients with endocrine pancreatic tumors who had received chemotherapy. A change from diploid to aneuploid records was also seen in 7/23 (30%) carcinoid tumors after treatment. The DNA patterns before and after treatment did not show any correlation with survival or treatment response.     

DNA analysis by flow cytometry, response to endocrine treatment and prognosis in advanced carcinoma of the breast

The relationship between DNA content of mammary cancer and subsequent response to endocrine therapy was studied in 136 patients with advanced disease. All were treated with tamoxifen or ovarian ablation as first-line systemic therapy after relapse and were evaluable for response according to UICC criteria. DNA characterisation by flow cytometry was used on formalin fixed paraffin-embedded samples of tumour. Tumours were grouped according to DNA index into diploid (n = 52, 38%), 'tetraploid' (n = 46, 34%) and 'other DNA-aneuploid' (n = 38, 28%). The highest proportion of oestrogen receptor positive tumours (ER + ve) was found in the 'tetraploid' tumours (38/46, 85%, Chi-square = 8.53, P less than 0.02), and response rates, (SD + PR + CR), were 26/52 (50%), 34/46 (74%), and 15/38 (39%) respectively (Chi-square = 10.88, P less than 0.005). Patients with diploid or 'tetraploid' tumours survived longer and stayed in remission longer than those with 'other DNA-aneuploid' tumours. We suggest that 'tetraploid' or 'near tetraploid' human mammary tumours may comprise a distinct group of endocrine responsive tumours within the overall group of aneuploid tumours. The conventional interpretation of DNA histograms, grouping into diploid and aneuploid, may be masking important features of some tumour groups.     

The prognostic significance of nuclear DNA content in malignant epithelial tumors of the ovary

Recent studies have indicated that the nuclear DNA content of certain malignant neoplasms can be used as an adjunct in predicting their biologic behavior. The DNA content of 99 ovarian carcinomas was determined by flow cytometric analysis of nuclei obtained from paraffin-embedded tissue. Of the 99 tumors, 51 were diploid and 48 showed one or more aneuploid peaks. The 5-year survival for patients with diploid tumors (50%) was significantly higher than for patients with aneuploid tumors (22%) (P less than 0.01). Other factors which significantly affected survival were clinical stage (P less than 0.001), tumor pattern grade (P less than 0.01), DNA index (P less than 0.01), the presence of ascites (P less than 0.001), peritoneal carcinomatosis (P less than 0.0001), and residual tumor at second-look laparotomy (P less than 0.05). Diameter of the primary ovarian tumor, diameter of the largest peritoneal implant before debulking, and the percent S-phase had no significant correlation with survival. Of 16 patients with aneuploid tumors who underwent second-look laparotomy, nine (56%) had residual tumor, compared to six of 22 of patients with diploid tumors (27%). Of seven patients with aneuploid tumors and a negative second-look laparotomy, four (57%) died from recurrent tumor. By comparison, of 16 patients with diploid tumors and a negative second-look laparotomy, only four (25%) died from recurrent tumor. The determination of DNA ploidy in ovarian carcinomas may be used as an adjunct in predicting tumor behavior, response to chemotherapy, and late recurrence of disease.     

Flow-cytometric analysis of colorectal cancer with hepatic metastases and its relationship to metastatic characteristics and prognosis

Studying the DNA ploidy patterns of 52 primary tumors, diploid tumors accounted for 48.1% and aneuploid tumors for 51.9%. Out of 31 patients with liver metastases, 35.5% had diploid tumors and 64.5%, aneuploid tumors. Heterogeneity (difference in DNA ploidy pattern between the primary lesion and liver metastases) was found in 20% of the patients examined. In 28 of the patients, the liver metastases were unresectable, and their prognoses were such that the 1- and 2-year survival rates from the diploid tumors were 42.9 and 14.3%, respectively, while 1-year survivors from aneuploid tumors died within 2 years. In resected cases of hepatic metastases, the DNA ploidy pattern of the metastatic lesions did not correlate with the metastasis period, extent of spread or number of lesions. The recurrence rate of aneuploid tumors in the residual livers was 50%, which was slightly higher than the rate of 36.4% for diploid tumors. The prognoses in patients with diploid tumors were significantly better than those in patients with aneuploid tumors: 5-year survival was 71.1% in diploid tumor patients, compared with 21% in aneuploid tumor patients.