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).     

Impulse cytophotometric DNA analysis in pituitary adenomas

Flow cytometric DNA analysis was carried out on 32 microsurgically removed pituitary adenomas. Additionally, the histograms of tumor cell nuclei of 7 patients were compared with those of the cultured cells from the same tumor samples.The tumors were classified into 3 groups according to the proliferation index (PI) of the flow cytometric results: 1) tumors with DNA patterns of slow proliferation (PI under 10), to which the majority of the examined pituitary adenomas belonged; 2) pituitary adenomas with diploid karyograms and PI values from 10 to 15; 3) diploid or aneuploid karyograms with PI values above 15.The third group were characterized histologically by increased chromatin content, nuclear polymorphism, mitoses, and extrapituitary infiltration of the tumor cells, and were, therefore, no longer benign. However, there was no direct relationship between the intensity of hormone secretory activity of the tumors and DNA ploidy. Cultured adenoma cells examined by flow cytometry remained stable in all cases but one.     

Smooth muscle tumors of the gastrointestinal tract. Flow cytometric quantitation of DNA and nuclear antigen content and correlation with histologic grade

Simultaneous flow cytometric quantitation of DNA content and the proliferation-associated nuclear antigen p105 was performed on 41 gastrointestinal smooth muscle neoplasms and the results were correlated with histologic features. Aneuploid DNA stemlines were found in 17 cases (41%), including four of 15 (21%) tumors of unknown malignant potential, eight of 17 (47%) low-grade leiomyosarcomas, and five of seven (71%) high-grade leiomyosarcomas. In 10 of the 17 aneuploid tumors, an aneuploid peak was clearly identified on the single parameter DNA histogram, with a mean DNA index of 1.36. In the other seven aneuploid cases, a near-diploid, aneuploid population (mean DNA index, 1.08) was identified only by simultaneous immunofluorescence for p105. Clinical follow-up information was available for 14 patients. Mean survival of 10 patients with aneuploid tumors was 32 months, whereas mean survival of four patients with diploid tumors was 51 months. Of the seven patients who died within 1 year of diagnosis, six had aneuploid leiomyosarcomas. These findings demonstrate that DNA aneuploidy is common in high-grade gastrointestinal leiomyosarcomas and may be associated with shortened survival.     

The distribution of nuclear DNA from human brain-tumor cells.

Flow cytometry (FCM) is a technique that measures the quantity of DNA contained in individual nuclei and records a frequency distribution of the DNA content per nucleus in the sampled cell population. Nuclei from a variety of human brain-tumor types were isolated by means of tissue grinding, purified by centrifugation through 40% sucrose (15 minutes at 4000 rpm), fixed with 10% formalin, stained with acriflavin-Feulgen, and analyzed by FCM. Profiles of DNA distribution in histologically benign tumors, such as meningiomas, pituitary adenomas, neuroblastomas, and low-grade astrocytomas, revealed a large diploid population (2C) with a few nuclei in DNA synthesis, as well as a small premitotic population (G2 cells) that contains a 4C DNA complement. In contrast, malignant gliomas, including glioblastomas, consist of more cells in DNA synthesis; these tumor cells show a highly variable distribution of ploidy consisting not only of diploid, and/or aneuploid, but also of triploid, tetraploid, and possibly octaploid populations. Also, a large variability between different regions of each tumor was always observed. In contrast, metastatic brain tumors, despite the fact that they contain a considerable number of cells undergoing DNA synthesis, demonstrate little variability within each individual tumor. The ability to rapidly characterize the cell populations of human brain tumors with FCM may enhance the effectiveness of their clinical management.     

Relationships among DNA Index, S-Phase, and invasive behavior in anterior pituitary adenomas. A cytometric study of 61 cases with Feulgen-positive DNA analysis

BACKGROUND: Pituitary adenomas are usually well differentiated neoplasms, although in about 1/3 of cases they invade the surrounding dura mater and bone, as confirmed by surgical findings, resulting in a long-term possibility of relapse.METHODS: To identify the cellular growth rate and to correlate it with surgical evidence of invasiveness, we performed the analysis of DNA with static cytometric quantitation on fresh surgical specimens, using a computer-assisted image processor. The DNA index and the percentage of cells in S-phase (%SPh) were obtained in 61 pituitary tumors consecutively operated on. In relation to surgically verified infiltration of dura and bone, we identified 39 noninvasive and 22 invasive adenomas. The cavernous sinus (CS) was infiltrated in 13 cases. On the basis of immunohistochemical staining and endocrine activity we recognized 27 nonsecreting and 34 secreting adenomas.RESULTS: The DNA content was aneuploid in 33 cases (11 nonfunctioning, 22 functioning; p = 0.05); there was no correlation with the invasive behavior of the adenomas. The DNA index ranged between 0.93 and 2.50 (median 1.13); the range of %SPh was 0-12.00% (median 2.54%). In invasive adenomas the mean DNA index was 1.33 (p not significant) and the mean %SPh was 4.03% (p = 0.05). In CS-infiltrating pituitary adenomas, the mean DNA index was 1.44 (p = 0.04) and the mean %SPh was 4.52% (p = 0.05).CONCLUSIONS: Our preliminary results seem to reveal a correlation between DNA index, %SPh, and invasive behavior of pituitary adenomas, encouraging the use of DNA analysis in the prognostic evaluation of these tumors.     

Proliferative activity assessed on the basis of DNA ploidy patterns in primary lung cancer

Flow cytometric cellular DNA-RNA content analysis by acridine orange staining was conducted on surgical fresh specimens of primary lung carcinomas from 66 patients (31 squamous cell carcinomas, 34 adenocarcinomas and 1 large cell carcinoma). The frequency of aneuploid tumors was 84.6% among the tumors. RNA content (RNA Index) in the DNA aneuploid tumor much more significantly (p less than 0.05) increased than the DNA diploid tumor. Tumor doubling time in the DNA aneuploid tumor was significantly (p less than 0.05) shorter than in the DNA diploid tumor. In the patients with lung cancers that recurred within 1 years, recurrence of the DNA aneuploid tumor was higher than the DNA diploid tumor. It is evident from the above results that proliferative activity in the DNA aneuploid tumor increases much more than in the DNA diploid tumor. This in turn may induce early recurrence in patients with lung cancer.     

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.     

DNA pattern and cytological findings in fine-needle aspirates of untreated prostatic tumors. A flow-cytofluorometric study

The cellular DNA content in fine-needle prostatic aspirates from 500 untreated patients was determined by flow cytofluorometry. According to the DNA patterns diploid, tetraploid, and non-tetraploid aneuploid cases were identified. In 301 cytologically benign cases more than 90% showed diploid DNA patterns. Among 166 carcinomas the incidence of aneuploid DNA values increased with the degree of anaplasia, ie, 44% in well-differentiated, 78% in moderately differentiated, and 97% in poorly differentiated tumors. In aneuploid cases of well-differentiated carcinomas almost exclusively tetraploid DNA patterns were observed, while in poorly differentiated carcinomas about 80% showed non-tetraploid aneuploid DNA distributions. Among aneuploid cases of moderately differentiated carcinomas 2/3 were tetraploid and 1/3 non-tetraploid aneuploid. Morphologically similar tumors may thus be separated by the DNA profiles. The biological significance of these results must be further evaluated by clinical follow-up of the patients.     

Tumor DNA content in resectable, primary colorectal carcinoma.

Tumor DNA content was measured in patients with colorectal carcinoma in order to determine whether tumor ploidy was a prognostic indicator independent of standard clinical and pathologic characteristics. One hundred forty-seven patients were analyzed who had their primary resectable colorectal carcinomas resected with curative intent from 1974 to 1981. Aneuploid colorectal cancers (i.e., tumors with abnormal DNA content) tended to be less well-differentiated, to invade the serosa or extend beyond, and to have lymph node metastases rather than diploid tumors (i.e., tumors with normal DNA content). A significantly increased rate of recurrent disease was demonstrated in patients with aneuploid tumors as opposed to those with diploid tumors (46.7% vs. 4.8%, respectively [p less than 0.001]). In addition, patients with aneuploid tumors exhibited a significantly decreased disease-free and overall survival in comparison with patients with diploid colorectal carcinomas. A Cox regression analysis demonstrated that tumor DNA content was the single most important factor in predicting recurrence or death from colorectal carcinoma.     

Immunohistochemical staining of DNA topoisomerase IIalpha in human gliomas.

OBJECT: The enzyme DNA topoisomerase IIalpha (Topo IIalpha) was tested as a measure of cell proliferation in gliomas. METHODS: Immunostaining for the Topo IIalpha and for the Ki-67 antigen (MIB-1 antibody) was performed in paraffin-embedded tissue sections obtained from 25 resected human gliomas. Additionally, cultured human glioma cells were subjected to simultaneous flow cytometry to determine Topo IIalpha and DNA content. Using flow cytometric analysis, the authors found that the Topo IIalpha antibody labeled cells in the S, G2, and M phases of the cell cycle and also those in some parts of the G0 and G1 phases. In histological sections, Topo IIalpha showed more distinct staining than MIB-1, particularly in older archival cases. The proliferative indices (PIs) based on cells staining for MIB-1 and Topo IIalpha correlated highly with one another (r = 0.96). The Topo IIalpha PI immunopositivity was seen in 4.07% of cells in the low-grade astrocytoma group, 11.97% in the anaplastic astrocytoma group, and 13.84% in the glioblastoma multiforme group, representing significant differences between low-grade astrocytoma and both anaplastic astrocytoma and glioblastoma. A Topo IIalpha PI less than 5% predicted longer patient survival (p = 0.003). CONCLUSIONS: Immunostaining for Topo IIalpha represents a useful alternative to MIB-1 as a proliferative index in human gliomas.     

High-grade gliomas in children

High-grade gliomas (HGGs) are malignant tumors and typically include glioblastoma multiforme and anaplastic astrocytoma subtypes. Brainstem gliomas and ependymomas are separate entities with respect to clinical presentation, treatment, prognosis, and outcome in comparison with supratentorial HGGs. In children, these tumors account for 3% to 7% of newly diagnosed brain tumors and 20% of all diagnoses of pediatric supratentorial brain tumors. These neoplasms are highly proliferative and mitotically active and of glial origin. This article reviews clinical, diagnostic, and pathologic features of HGG and current treatments and potential future therapies specific to pediatric patients with HGGs.     

Cellular DNA profiles of benign and malignant adrenocortical tumors

We evaluated the DNA content of 43 adrenocortical neoplasms by flow cytometry and related it to histopathologic criteria of malignancy and survival. Tumor tissue was selected from paraffin blocks and processed by a modification of the Hedley technique. The tumors were classified as adenomas and carcinomas by the criteria of Weiss. Aneuploid stem-lines were identified in nine of 13 (69%) of the carcinomas and in six of 30 (20%) of the adenomas. Five of the six patients with aneuploid adenomas are alive and well (mean follow-up, 59 months); the sixth was lost to follow-up. Although there was a significant correlation between ploidy and histologic diagnosis (p = 0.041), the sensitivity and specificity of aneuploidy for predicting clinical outcome were only 56% and 65%, respectively. In addition, there was no significant difference in survival between patients with diploid versus aneuploid tumors, despite a highly statistically significant difference in survival between patients with a histologic diagnosis of adenoma versus carcinoma (p = 0.00080). We found correlations between ploidy and tumor size, mitotic rate, and nuclear grade (p = 0.0033, p = 0.0017, and p = 0.018, respectively). There was also a significant correlation between the proliferation fraction of a tumor and its nuclear grade (p = .0093), but not its mitotic count or clinical outcome. Because both adrenal adenomas and carcinomas may contain abnormal DNA stem-lines, ploidy alone is not a reliable discriminator in individual cases.     

Prognostic importance of DNA ploidy in medulloblastoma of childhood

The deoxyribonucleic acid (DNA) content of 53 medulloblastomas was analyzed by means of flow cytometry and compared with the clinical and histological findings in the host patients. Analysis of DNA showed that about half of the tumors were diploid and the other half were aneuploid. More diploid tumors were found among patients of a young age, but the difference was without statistical significance. Cellular differentiation of the tumor did not correlate with DNA ploidy. No correlation was found between Chang's T staging system and the DNA ploidy, whereas the M staging correlated with the ploidy; diploid medulloblastomas had a greater tendency to metastasize than aneuploid medulloblastomas (p = 0.0003). Four-year survival was compared with the extent of resection and DNA ploidy. The patients with total resection and aneuploid medulloblastoma had a better prognosis than those with subtotal resection and diploid tumor (p = 0.001). There was only one survivor among eight patients with subtotally resected diploid medulloblastomas, while all of the seven patients with totally resected aneuploid medulloblastomas survived. Comparison of the G0/G1 phase fraction and S phase fraction in the surviving group and the deceased group offered no significant information.     

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.     

Cytochemical assessments of the nuclear DNA distribution pattern by means of image and flow cytometry in thyroid neoplasms and in non-neoplastic thyroid lesions. A comparative methodological study

The two most prevalent techniques for cytochemical DNA assessment of the nuclear DNA distribution pattern in neoplastic cells are image cytometry (ICM) and flow cytometry (FCM). The aim of the present study was to compare the results of nuclear DNA assessments, obtained by means of these two methods, in fresh surgical biopsy specimens from the thyroid gland, both in neoplasms and in nonneoplastic lesions. Material for DNA analysis was taken preoperatively by fine needle aspiration (FNA) biopsy from 13 papillary thyroid carcinomas and analyzed by the two methods. Surgical specimens were taken from 48 papillary thyroid carcinomas (one of which showed low differentiated papillary and anaplastic giant cell formations at autopsy), 2 follicular carcinomas, 66 follicular adenomas, 7 medullary carcinomas as well as from the nodules of 17 non-toxic colloid goitres and 19 specimens from the diffusely hyperplastic thyroid parenchyma in patients with hyperthyroidism. For the ICM assessments, FNA biopsies or imprints were made from the macroscopically identified fresh thyroid biopsy specimens; for the FCM assessments FNA specimens from the same region were used. In 155 out of the 159 specimens the results obtained by means of the two methods were the same. The DNA distribution pattern in 106 of the neoplasms and in all the 36 non-neoplastic lesions were of the "euploid" type (i.e. "diploid" or diploid/tetraploid"), whereas that of 17 neoplasms were of the "aneuploid" type. Fifteen of the histopathologically benign follicular adenomas showed a cytochemical DNA distribution pattern that by means of FCM was of the "aneuploid" type.(ABSTRACT TRUNCATED AT 250 WORDS)     

Clinicopathological study of diffuse type brainstem gliomas: analysis of 40 autopsy cases

Diffuse type brainstem glioma is one of the most malignant types of brain tumors and the prognosis is extremely poor. The proliferative potential of these tumors is presumed to be very high, but there is little information about the cell kinetics of brainstem glioma because surgical resection is rarely performed. The histological grade, tumor spread, growth potential, and prognosis were evaluated in 40 autopsy cases of diffuse type brainstem glioma. To quantify the growth potentials of individual tumors, the proliferating cell indices of Ki-67 (MIB-1) and proliferating cell nuclear antigen (PCNA) monoclonal antibodies were measured. Mean MIB-1 and PCNA proliferating cell indices were 20.4% (24 cases) and 37.0% (28 cases), respectively, in 34 glioblastomas. The median survival time was 40 weeks in 22 treated patients. The mean PCNA proliferating cell index was 10.8% in four of five anaplastic astrocytomas and the median survival time in four treated patients was 91 weeks. The MIB-1 and PCNA proliferating cell indices of one astrocytoma were 2.9% and 20.3%, respectively, and the survival time was 56 weeks. The overall median survival time was 32 weeks. There was a significant difference in PCNA proliferating cell indices between glioblastomas and anaplastic astrocytomas (p < 0.05) and there was a significant difference in survival time between glioblastomas (40 weeks) and anaplastic astrocytomas plus astrocytoma (74 weeks) among the treated patients (p < 0.05). Supratentorial extension was more frequent in glioblastomas than in anaplastic astrocytomas (p < 0.05). Our results suggest that the majority of diffuse type brainstem gliomas are glioblastoma and the proliferative potential is probably as high as that of adult supratentorial glioblastoma. Supratentorial extension and dissemination are relatively frequent in the advanced stage. Anaplastic astrocytoma or astrocytoma is rarer and less infiltrative and proliferative, and carries a slightly better prognosis than glioblastoma.     

DNA in meningioma tissues and explant cell cultures. A flow cytometric study with clinicopathological correlates

Flow cytometry was performed on stored frozen tissues and explant cell cultures from 39 meningiomas using ethidium bromide and mithramycin in a selective staining technique for deoxyribonucleic acid (DNA). The ploidy index and percentage of cells in the G0/G1, S, and G2/M phases were calculated for each specimen. The results were compared with the age and sex of the patients; the site, the histological subtype, and mitotic rate of the neoplasms; and the estrogen- and progesterone-receptor levels assayed in cytosol-enriched supernatants from cryostat-cut sections. Sixteen neoplasms (41%) were aneuploid. These included two recurrent neoplasms, seven of the eight neoplasms from patients with multiple meningiomas, and three clinically aggressive neoplasms (one hemangiopericytic and two anaplastic meningiomas). Significant correlations were found between values for the ploidy index (r = 0.75, p less than 0.01), the percentage of S-phase cells (r = 0.82, p less than 0.01), and the percentage of G2/M-phase cells (r = 0.69, p less than 0.05) in vivo and in vitro. The results support the suggestion that flow cytometry for DNA in meningiomas may be of value in predicting the behavior of these neoplasms, and indicate that under controlled conditions explant cell cultures may provide a useful model for the proliferative characteristics of meningiomas in vivo.     

大肠癌ras p21表达和DNA含量定量分析

应用流式细胞术和细胞免疫荧光染色技术，对４６例大肠肿瘤细胞ｒａｓ ｐ２１表达和ＤＮＡ含量进行了宣分析。结果：大肠部的ｒａｓ ｐ２１表达阳性率为６５．７％，ＤＮＡ异倍体率为７４．３％，大肠部细胞ｒａｓ ｐ２１表达明显高于腺癌和正常粘膜；ＤＮＡ倍体和细胞增殖指数值与ｒａｓ ｐ２１表达量密切相关。本实验结果提示：测定大肠癌的ｒａｓ ｐ２１表达和ＤＮＡ含量将有助于其预后判断。     

Analysis of flow cytometric nuclear DNA content of the thymoma and its relationship to malignant intensity

Determination was made of the nuclear DNA content of paraffin-embedded specimens of resected thymoma using flow cytometry in 39 patients. Relationships among nuclear DNA content, clinicopathological findings and prognosis were studied. The frequency of DNA aneuploid tumors was 30.8%, 6.7% in stage I (15 patients), 28.6% in stage II (7 patients), 44% in stage III (9 patients) and 62.5% in stage IV (8 patients) according to Masaoka's classification, indicating more in increase with advancing stage and significant (p less than 0.05) more increase invasive thymoma than in noninvasive thymoma. The 5 year and 10 year survival rates of DNA diploid tumors were each 94%, while DNA aneuploid tumors, 75% and 45%. Patients with DNA aneuploid tumors showed less favorable prognosis than those with DNA diploid tumors. Similar results were found in patients with invasive thymoma and in those on whom subtotal or partial resection were performed. The present results indicate DNA aneuploid tumors to have greater malignant intensity than DNA diploid tumors in resected thymoma. Flow cytometric nuclear DNA content analysis provides useful biological data and new indices for evaluating the malignancy of resected thymoma.