Flow cytometric DNA analysis of normal adrenal tissues and adrenal tumors

Flow cytometric DNA analysis (FCM) of adrenal tumors was studied to evaluate whether FCM will be a useful examination for differentiating between benign and malignant adrenal tumors. 10 specimens of surgically resected (for renal cell carcinoma confined within the middle or lower pole) normal adrenal glands and 20 specimens of surgically resected adrenal tumors were submitted for the study. Hyperplastic adrenal cortex as well as normal adrenal gland showed normal diploid pattern. On the other hand, some of the cortical adenomas showed tetraploid patterns, which has been known to be an index of malignancy in most of the flow cytometric intervention to other solid tumors. Conn adenomas were especially apt to show this tendency, in which as much as 86% showed tetraploid pattern. Proliferation Index (PI) (ratio of S + G2 +M cells for the whole population of analyzed cells) were as much as 9.45 +/- 6.97% in normal adrenal cells, whereas it was much higher in cortical adenomas (17.75 +/- 8.53%). As a matter of fact, PI of hyperplastic adrenal cortex was within the same range as that of the normal adrenal glands. In pheochromocytomas, aneuploid pattern, which has been believed to be a definite index of malignancy, was shown in 60% of the cases, tetraploid pattern in 20%, and normal diploid pattern in only 20%. As a matter of fact, a case of non functioning cortical adenocarcinoma and a case of malignant pheochromocytoma were judged to show typical aneuploid pattern. Thus, the application of the flow cytometric diagnosis for adrenal tumors was supposed to require some refinement in understanding the significance of aneuploidy or tetraploidy.     

Flow cytometric analysis of DNA content in adult testicular germ cell tumors]

Flow cytometric DNA analysis was carried out in 54 patients with testicular germ cell tumors (GCTs) experienced at our hospital, to evaluate the clinical relevance of DNA index (DI) and provide some insight into the pathogenesis of testicular GCTs. Histological types with their incidences were seminomas in 31 patients and nonseminomatous germ cell tumors (NSGCTs) in 23 adults. DNA ploidy and DI were analyzed by flow cytometry in 158 paraffin embedded samples; 2.9 samples per case on the average. This study revealed that 52 cases (96%) of evaluable 54 adult GCTs were DNA aneuploid, while DNA diploid tumors were observed in only each one case of NSGCT and seminoma. There was a significant difference (p less than 0.01) between the distribution of DIs in adult NSGCTs (median DI = 1.50) and that in pure seminomas (median DI = 1.85). Although we found no significant correlation between DI and clinical staging of Japanese Urological Association, on the basis of Indiana University staging system, the median DI in NSGCT patients of the advanced extent was lower than those of the other extents. DNA heterogeneity was observed only in 4 of 23 NSGCT patients (17%) and 3 of those 4 patients were assigned to advanced extent. These data suggest that the lower DI and the presence of DNA heterogeneity may have prognostic relevance for NSGCTs.     

Flow cytometric analysis of DNA ploidy using paraffin-embedded germ cell tumors

The DNA content of paraffin-embedded materials was determined retrospectively using flow cytometry (FCM) in 36 germ cell tumors, and related to histological type, clinical staging and tumor marker. These histograms were classified from the basis of mode and variance into a diploid and an aneuploid pattern. We could evaluate the DNA histograms in 20 of 36 specimens (56%). Aneuploid patterns were found in 11 of 20 evaluated cases, but there was no correlation between ploidy patterns and histological types. Aneuploid patterns were demonstrated in 2 of 6 stage I cases (33%), and 4 of 5 stage II cases (80%) in seminomas. The difference between stage I and II cases was not statistically significant. There was no correlation between clinical staging and DNA content in non-seminomas. Of the seminomas with elevated human chorionic gonadotropin (HCG) titers, 3 of 4 cases showed aneuploid patterns. These findings indicate that the determination of DNA ploidy in seminomas may prove to be of prognostic value.     

Flow cytometric determination of nuclear DNA content in benign adrenal pheochromocytomas

Nuclear DNA content of paraffin-embedded tissue from 19 clinically benign adrenal pheochromocytomas and 18 control adrenal glands was analyzed using flow cytometry. All control adrenal glands and 6 pheochromocytomas were diploid. Four tumors were tetraploid. Nine were aneuploid with relative DNA indices in the near diploid range in 2, in the peritriploid range in 5, and in the near tetraploid range in 2. These results indicate that aneuploid DNA content is a frequent occurrence in benign adrenal pheochromocytomas. Aneuploidy per se is not a specific marker of malignancy in these tumors as has been suggested by previous reports.     

Flow cytometric DNA analysis in colorectal cancer

Flow cytometric DNA analysis for assessing malignant potential of colorectal carcinoma was investigated by paraffin-embedded materials. Preservation time of paraffin blocks and formalin fixation time of surgical specimens within 14 days do not influence the nuclear DNA content. There was seen a good correlation between the DNA contents of paraffin-embedded and fresh materials obtained from the same surgical specimens. Using deparaffinized tumor specimens, the nuclear DNA content was measured by flow cytometry in 144 patients with primary colorectal cancer, who had been treated from 1971 to 1985. Forty-four percent of colorectal cancer were diploid and 56% were aneuploid. There was no significant correlation between ploidy pattern and clinicopathological factors. However, the patients with aneuploid tumor had a significantly worse survival than those with diploid tumor (Generalized Wilcoxon test, p less than 0.001). The patients with aneuploid seemed to have an unfavorable survival than those with diploid in the same stage, and had a significantly worse survival in each group of negative nodes, P0 and H0. It is concluded, therefore, that the nuclear DNA content of colorectal cancer may be an important prognostic factor, being independent of pathological stage.     

Flow cytometric DNA analysis in rectal carcinomas

The cellular DNA content was measured with flow cytometry from paraffin-embedded material in 329 patients and metastatic tumors of the liver from the rectum in 11 patients. The classification of the DNA ploidy pattern is as follows: A stem cell peak with a DNA index of 0.9-1.1 is defined as DNA diploid tumor and DNA aneuploid tumor is that with a DNA index greater than or equal to 1.1. There was a good correlation of DNA indices (r = 0.997) obtained from flesh and corresponding paraffin-embedded specimens. It is concluded that accurate determination of DNA index from paraffin-embedded materials is possible in the majority of cases. DNA ploidy of primary tumor cells correlated with clinicopathological findings such as lymphatic invasion, vascular invasion, lymph node metastasis and hepatic metastasis (p less than 0.01), but did not correlate with extramural carcinoma invasion. The cumulative survival rate (Kaplan-Meier) of curatively resected rectal carcinomas was worse in DNA aneuploid than in DNA diploid tumors (p less than 0.01). These observation showed that the determination of DNA ploidy in rectal carcinomas may prove to be of prognostic value.     

Flow cytometric analysis of small renal tumors.

Flow cytometric deoxyribonucleic acid (DNA) content analysis was performed on 26 renal tumors 3.0 cm. or less in maximum diameter. We noted DNA aneuploid cell populations in 8 of 25 tumors (32%) evaluable for DNA ploidy status. DNA aneuploid cells comprised 7 to 59% of the cells in those tumors. In comparison, 12 of 25 tumors (48%) larger than 3.0 cm. had aneuploid cell populations. S phase cell populations were significantly increased in the small aneuploid tumors compared with the small diploid tumors (p < 0.001). We believe that these small tumors have the potential to behave aggressively and, therefore, they should be treated no differently than larger renal neoplasms.     

Flow cytometric DNA analysis of thyroid carcinoma

Abnormal DNA content has been considered as an additional criterion for determining the biological behavior of a tumor. Flowcytometric DNA analysis was done on 121 patients with thyroid carcinoma encountered during the period between 1975 and 1987. Tumor tissues were sampled from paraffin-embedded blocks and the histology of thyroid carcinoma found to consist of 91 papillary, 23 follicular, 2 medullary, 1 squamous cell and 4 anaplastic carcinomas. The incidence of aneuploidy in thyroid carcinoma was 7.4 per cent (9 patients) while that of diploidy was 92.6 per cent (112 patients). The aneuploid specimens consisted of 6 papillary, 1 follicular, 1 medullary and 1 anaplastic carcinomas and, of 4 anaplastic carcinoma patients with subsequent death within 6 months, only 1 was aneuploid. As an indicator of proliferative potential, S-phase fraction (SPF) was also determined by flow cytometry, but this could not be used as an independent prognostic factor. The aneuploid patients showed a significantly decreased survival rate (p<0.01). Thus, although DNA measurement proved useful for predicting the survival of aneuploid patients, there is some discrepancy between DNA content and the biological behavior of the tumor.     

Flow cytometric DNA analysis of thyroid carcinoma

Abnormal DNA content has been considered as an additional criterion for determining the biological behavior of a tumor. Flowcytometric DNA analysis was done on 121 patients with thyroid carcinoma encountered during the period between 1975 and 1987. Tumor tissues were sampled from paraffin-embedded blocks and the histology of thyroid carcinoma found to consist of 91 papillary, 23 follicular, 2 medullary, 1 squamous cell and 4 anaplastic carcinomas. The incidence of aneuploidy in thyroid carcinoma was 7.4 per cent (9 patients) while that of diploidy was 92.6 per cent (112 patients). The aneuploid specimens consisted of 6 papillary, 1 follicular, 1 medullary and 1 anaplastic carcinomas and, of 4 anaplastic carcinoma patients with subsequent death within 6 months, only 1 was aneuploid. As an indicator of proliferative potential, S-phase fraction (SPF) was also determined by flow cytometry, but this could not be used as an independent prognostic factor. The aneuploid patients showed a significantly decreased survival rate (p less than 0.01). Thus, although DNA measurement proved useful for predicting the survival of aneuploid patients, there is some discrepancy between DNA content and the biological behavior of the tumor.     

Flow cytometric bromodeoxyuridine (BrdU)/DNA analysis--study with urogenital tumors

We performed the simultaneous flow cytometric bromodeoxyuridine (BrdU)/DNA analysis in combination with in vitro BrdU labeling method using seven human urogenital tumors. The DNA content and incorporated BrdU content of each tumor cell was analyzed quantitatively using this flow cytometric method. The cell kinetics of each cell line of heterogeneous tumor could be analyzed by this combined method. In the near future, by establishing a procedure decreasing non-specific staining of cells, the development of this flow cytometric two-color analysis in clinical fields is expected.     

Flow cytometric analysis of DNA content in children with neuroblastoma

Flow cytometric DNA content analyses were performed in a retrospective study of archival tumor specimens of 15 patients with diagnosed neuroblastoma treated at a single institution. Survival statistics were positively influenced by the presence of aneuploidy. Kaplan-Meier analysis indicated a significant survival advantage for patients with aneuploid tumors (P = .036). Survivors were significantly younger at the time of diagnosis than nonsurvivors, but age at diagnosis did not correlate with DNA content. We conclude that DNA content analysis may be of value in determining the prognosis for children with neuroblastoma.     

Flow cytometric analysis of the DNA content of thymomas

In this study, the prognostic value of determining the nuclear DNA content of thymomas by flow cytometry was evaluated. Of a total 31 resected thymomas, 10 (32%) showed DNA aneuploidy, the presence of which was significantly correlated with an advanced clinical stage of disease. The patients with an aneuploid tumor had a poorer prognosis than those with a diploid tumor, demonstrating a survival rate of 50% at 7 postoperative years, which was considerably less favorable than that of the patients with a diploid tumor, being 100% in the same period (p<0.05). Moreover, patients with a high DNA index (DI), i.e., a DI1.5, tended to have a poorer prognosis than those with a low DI. These findings indicated that the DNA content can be an important prognostic index in patients with thymomas.     

Flow cytometric analysis of DNA from 98 cases of soft tissue tumors and its clinical significance

The cellular DNA content of 98 soft tissue tumors was determined by means of flow cytometry and the results were discussed in relation to clinical data. It was found that DNA of all 21 benign tumors was diploid, while that of malignant tumors was both diploid and aneuploid. Among 62 sarcomas, 45 were aneuploid (73%). Cell cycle analysis disclosed that diploidy lesions had a low proportion of S and G2 + M cells while most aneuploidy lesions had a high proportion (P less than 0.01). There was a definite correlation between the size of the tumor and the presence of DNA aneuploidy. The results indicate that DNA analysis offers a clinically relevant means of characterizing soft tissue tumors.     

Flow cytometric analysis of DNA ploidy in lymphomas of the thyroid

Four cases of primary non-Hodgkin's lymphoma (NHL) of the thyroid were studied using flow cytometric (FCM) DNA analysis of propidium iodide-stained nuclei retrieved from formalin-fixed, paraffin-embedded tissue. Two of the four cases were aneuploid and two were euploid. In the two euploid cases, both patients are alive and without evidence of recurrent disease after an average of 4 years follow-up. Of the two aneuploid cases, one patient is alive and free of recurrent disease after 1 year. In the other aneuploid case, the patient died of disseminated disease 8 months after presentation despite having a low-grade (follicular, predominantly small cleaved cell type) and low-stage (tumor confined to thyroid at presentation) lymphoma. These data suggest that the DNA ploidy of primary NHL of the thyroid can be determined using fixed, paraffin-embedded tissue. Our results also suggest that a large study to assess the prognostic value of this technique is warranted.     

Flow cytometric analysis of the nuclear DNA content of hepatocellular carcinoma

The nuclear DNA content of 77 resected specimens from 65 cases of hepatocellular carcinoma (HCC) was measured by means of flow cytometry. The DNA index (DI) was calculated and the correlation between the DNA ploidy pattern and clinicopathological findings was studied. In the cases of HCC with a diameter of less than 5 cm, the 3-year survival rate of the aneuploid cases was 44.5 per cent, which was significantly lower than the 91.4 per cent of the diploid cases (p less than 0.001). Serum AFP levels were over 1000 ng/ml in 46.4 per cent of the aneuploid tumors and 18.5 per cent of the diploid tumors (p less than 0.05). The DI's were investigated in several sites of the same tumor and no difference was seen among the different sites in 16 out of 17 tumors. From 8 recurrent cases out of 12 who underwent a second resection, seven did not show any significant differences in DI from their primary tumor. On the other hand, four cases of second primary tumors showed different DI's to those of their first primary tumor. Intra-hepatic metastatic tumors exhibited the same DI's as their primary tumors. Thus, the nuclear DNA ploidy pattern may serve as a stable and valuable marker in predicting the malignant potential and prognosis of HCC.     

Flow cytometric analysis of DNA content in testicular seminomas--correlation to mitotic count

The DNA ploidy of testicular seminomas was studied by flow cytometry using paraffin embedded samples. The mitotic count and DNA index (DI) for 27 seminomas were analyzed in 80 samples with a mean of 3.0 samples per case. Six anaplastic seminomas which were with 3 or more mitoses per a high power field were distinguished from 21 typical seminomas. DNA ploidy pattern was aneuploid in all seminomas except one case of anaplastic seminoma, and clonal heterogeneity in DNA content was found in 3 of 20 (15%) cases of which 2 or more samples were analyzed. Although the DI had no significant difference between those two groups of seminomas classified by mitotic count, the DI in anaplastic seminomas was ranged from 1.5 to 2.0 (median DI = 1.70), otherwise the DI in typical seminomas ranged from 1.5 to 3.5 (median DI = 1.89), particularly 9 cases in 21 (43%) typical seminomas distributed in hypertetraploid region. The median DI of stage I seminomas was 1.88 and that of stages II + III seminomas was 1.75, though there was also no significant correlation between DI and clinical stages. In general, it is postulated that the higher DI is paralleled to the more malignant nature of neoplasms, nevertheless this study suggested that the higher DI in seminomas is not always related to high malignant potentiality determined by histological type and clinical stage.     

Flow cytometric analysis of the nuclear DNA content of hepatocellular carcinoma

The nuclear DNA content of 77 resected specimens from 65 cases of hepatocellular carcinoma (HCC) was measured by means of flow cytometry. The DNA index (DI) was calculated and the correlation between the DNA ploidy pattern and clinicopathological findings was studied. In the cases of HCC with a diameter of less than 5 cm, the 3-year survival rate of the aneuploid cases was 44.5 per cent, which was significantly lower the 91.4 per cent of the diploid cases (p<0.001). Serum AFP levels were over 1000 ng/ml in 46.4 per cent of the aneuploid tumors and 18.5 per cent of the diploid tumors (p<0.05). The DI’s were investigated in several sites of the same tumor and no difference was seen among the different sites in 16 out of 17 tumors. From 8 recurrent cases out of 12 who underwent a second resection, seven did not show any significant differences in DI from their primary tumor. On the other hand, four cases of second primary tumors showed different DI’s to those of their first primary tumor. Intra-hepatic metastatic tumors exhibited the same DI’s as their primary tumors. Thus, the nuclear DNA ploidy pattern may serve as a stable and valuable marker in predicting the malignant potential and prognosis of HCC.     

Flow cytometric analysis of cellular DNA content in human astrocytomas and oligodendrogliomas

This report presents a flow-cytometric analysis of cellular DNA in biopsies and primary cell cultures of 21 human astrocytomas and 19 oligodendrogliomas. A distinct correlation between histological dedifferentiation and pathological DNA distribution was found. Classification was made according to increasing histological anaplasia, corresponding to a four-grade scale and proliferation index (PI). Four types of gliomas were defined according to characteristic DNA patterns and proliferative activities in comparison to their histological grading: 1. purely diploid DNA patterns with low 4C (premitotic) peaks and PI values up to 10 in well-differentiated gliomas; 2. increase of tetraploid cells and PI of 10-16 in tumors with histological grades II or II–III; 3. diploid-tetraploid DNA distribution with PI values up to 30–31 and malignancy grade III; 4. polyploid and aneuploid karyograms with excessive 4C increase, emerging in grade III and especially grade III–IV of these gliomas. Varying DNA distribution during tumor development could be observed in a malignant transformation of an oligodendroglioma I to a glioblastoma after a course of 3 1/2 years. A more detailed subdivision of these tumors according to their DNA content and proliferative activity was achieved. With the exception of occasional variation in karyograms, DNA distribution usually remained stable in primary tissue cultures (PTC).     

Flow cytometric DNA analysis of hepatocellular carcinoma: preliminary report.

Flow cytometric DNA analysis was performed in 50 paraffin-embedded specimens of clinical hepatocellular carcinoma (HCC) after hepatic resections. The DNA distribution pattern was classified in two types, diploid and aneuploid, according to the degree of dispersion on the DNA histogram. The major DNA pattern of HCC in this report proved to be aneuploid (78%), although 22% of tumors revealed a diploid pattern. The serum alpha-fetoprotein level exceeded 40 ng/ml in 86.1% of the aneuploid tumors and in 13.9% of the diploid tumors (p less than 0.05). We found no correlation between DNA distribution and hepatitis B surface antigen positivity, the presence of liver cirrhosis or tumor size. Additionally we noted no significant correlation between the DNA pattern and survival rates in patients with HCC who underwent hepatic resection.