Ploidy and proliferative activity of human brain tumors. A flow cytofluorometric study

Ploidy and proliferative characteristics were estimated by flow cytometry of the nuclear DNA content of 92 human brain tumors. Samples were frozen at -20 degrees C immediately after surgery and single cell suspensions were obtained with a mechanical dissociation technique. Propidium iodide was employed for nuclear DNA staining. Human normal brain tissue was used as internal diploid reference standard. 86% of benign tumors had unimodal DNA distribution with a DNA index (DNA I = modal channel of the G0/1 peak of the studied population/modal channel of the G0/1 peak of the normal brain) usually within the diploid or near-diploid range. 14.0% had aneuploidy, with an additional cell peak having a median DNA I of 1.60. Among malignant tumors, these figures were 61.2 and 38.8% (p less than 0.001). The percentage of S phase cells was higher in malignant (median = 3.6) than in benign tumors (median = 2.0, p less than 0.01), without correlation to histological tumor subtype. Flow cytometry appears to be a useful method for evaluating differences in DNA distribution in tumors of the central nervous system.     

DNA content of normal and neoplastic urothelial cells. A comparative cytofluorometric and cytomorphologic study

In search for suitable parameters to detect neoplastic urothelial cells in Acriflavine-Feulgen-SITS stained specimen we compared the cytofluorometric DNA content with the morphology of normal urothelial cells (bladder scrapings) and neoplastic urothelial cells from grade 1, 2, 3 and 4 tumors. An individual normal urothelial cell could not be distinguished from a grade 1 tumor cell, neither morphologically nor fluorometrically. However, the shape of the histograms of DNA measurements of the cell populations of respectively normal bladder scrapings and grade 1 tumors differs. It is postulated that also morphometry of these cell populations may be of some aid to distinguish well-differentiated neoplastic cells from normal urothelial cells. Seventy-one percent of the morphologically malignant cells in the grade 2,3 and 4 tumor samples could be identified by applying the combined parameters: high DNA content (greater than 5 C) and nuclear-cytoplasmic ratio (greater than 0.5) and all grade 2, 3 and 4 tumor samples contained cells which were objectively classified as malignant. Using the same parameters morphologically malignant cells could be distinguished from normal, polyploid umbrella cells, thus these malignant cells are detectable objectively without using chromatin pattern as parameter.     

Pitfalls of liver scan in Wilms' tumors.

Twenty-five patients suffering from nephroblastoma were investigated with a liver scan. The scan results were compared with surgical and pathological findings. This study suggests that different factors (i.e., primary tumor compression, local recurrence and effects of radiotherapy) may determine identical pathological patterns of the scan. Anamnestic information is very important for a correct diagnosis.     

Pleomorphic xanthoastrocytoma. Ultrastructural, immunohistochemical, and DNA cytofluorometric study of a case.

A case of right frontal pleomorphic xanthoastrocytoma that occurred in a 7-year-old boy is reported clinicopathologically. The patient underwent surgery on September 29, 1988. Histologic diagnosis of pleomorphic xanthoastrocytoma was made because, in addition to the unique pleomorphic histologic features, positive glial fibrillary acidic protein in immunohistochemical staining and characteristic ultrastructural features, i.e., cytoplasmic intermediate fibrils and lipid vacuoles, basal lamina, and abundant reticulin networks were demonstrated. The DNA cytofluorometric analysis of the nuclei of the tumor cells disclosed the main mode to be diploid with polyploid classes (4, 8, 16, and 32C) without any aneuploidy. Despite the presence of many pleomorphic nuclei, DNA histogram of the tumor disclosed very few DNA synthetic cells indicating a biologically inactive nature of the tumor. The patient is still alive and totally asymptomatic 20 months postoperatively.     

Improved survival for children with anaplastic Wilms' tumors.

Combined modality treatment has resulted in cure rates of approximately 80% for children with Wilms' tumor. According to the National Wilms' Tumor Studies (NWTS), a group of patients with histologic features of anaplasia or sarcomatous Wilms' tumors (malignant rhabdoid tumors and clear cell sarcomas) were less responsive to vincristine and actinomycin. The survival rate of patients in this group with unfavorable histologic conditions was 54% compared with 90% for those with favorable histologic conditions. We have reviewed 80 consecutive cases of Wilms' tumor treated with a minimum follow-up interval of 5 years. Two pathologists independently reviewed all histologic specimens that were initially classified as having unfavorable histologic conditions and specimens from children with favorable histologic conditions who subsequently relapsed. One of 13 children with favorable histologic conditions had recurrent disease that was found to have unfavorable histologic conditions on rereview. All five patients with sarcomatous Wilms' tumor had a rapidly progressive course. Treatment of eight children with anaplastic Wilms' tumor with vincristine, actinomycin, cyclophosphamide, and abdominal radiation resulted in good disease-free and overall survival rates (5-year survival rate, 87.5%) that were not significantly different from children with tumors having favorable histologic conditions (5-year survival rate, 94%). All children with sarcomatous histologic conditions, however, did not to respond.     

Chromosome analysis of 31 Wilms' tumors

Cytogenetic analysis was done on 31 Wilms' tumors, including 2 renal tumors of clear cell sarcoma type, using short term cultures of primary tumors and/or nude mouse passages. Nonrandom secondary chromosome abnormalities, in particular, were noted as evidence of clonal evolution. Apparently normal karyotypes were found in 5 Wilms' tumors, all in patients less than or equal to 22 months old, and in one clear cell sarcoma. Abnormal karyotypes were seen in 25 tumors (80%); 6 were pseudodiploid, 3 were hypodiploid, and 16 (52%) were hyperdiploid, of which 8 had a modal number of 47-49 and 8 had a modal number of 50-55. Nonrandom structural abnormalities involved 1p/1q, 11p, 7p/7q, 16p/16q, 12q, and 17p/17q. Nonrandom numerical abnormalities included +6, +8, and +18. Trisomy 12 was the most common abnormality, structural or numerical, seen in 52% of tumors (81% of the hyperdiploid). In 2 tumors the +12 was the only apparent abnormality; in 1 other tumor an i(12q) was seen, suggesting that +12 may have special significance in the clonal progression of Wilms' tumor. Informative karyotypes of 68 Wilms' tumors from other reports were reviewed and compared to results in this series.     

DNA cytofluorometric and nuclear morphometric analyses of lung adenocarcinoma

The nuclear DNA content of tumor cells in imprint smears prepared from 72 surgically resected adenocarcinomas of the lung was determined prospectively by means of cytofluorometry, and its relationship to pathologic stage, degree of histologic differentiation, and nuclear atypia represented by the mean nuclear area (MNA) and standard deviation of the nuclear area (SDNA) was studied. The mean nuclear DNA content (MNDC) of poorly differentiated adenocarcinomas was significantly larger than that of well-differentiated adenocarcinomas both in Stages I + II and III + IV (P less than 0.001, each), and the MNDC of Stages III + IV was significantly larger than that of I + II in poorly differentiated adenocarcinomas (P less than 0.01). There was a tendency for histogram Patterns III and IV, in which more aneuploid cells were present than in I and II, to be more common and for the incidence of the aneuploid stem cell line to be higher in less differentiated and more advanced adenocarcinomas, although the differences were not significant statistically. Furthermore, there were significant positive correlations between MNDC and MNA (P less than 0.01) and between MNDC and SDNA (P less than 0.05). The averages of the MNA according to the histogram pattern increased significantly in the increasing order of Patterns I to IV. These results indicate that the nuclear DNA content increases in less differentiated and more advanced adenocarcinomas, and that the nuclear atypia is reflected in abnormal nuclear DNA content (DNA aneuploidy).     

Benign metastasizing giant cell tumors of bone. A DNA flow cytometric study

Approximately 2% of histologically benign giant cell tumors (BGCT) of bone are complicated by lung metastases, which can progress despite their benign histologic appearance. Almost all BGCT studied by DNA flow cytometry (FCM) have been reported to be diploid. However, the very few cases with lung metastases previously analyzed were all aneuploid. To assess the usefulness of DNA FCM in predicting the metastatic potential of BGCT, seven metastasizing BGCT were studied by DNA FCM using paraffin-embedded tissue. Five were purely diploid, one was tetraploid, and one was aneuploid. The primary and the metastasis showed the same DNA distribution in all but the tetraploid case, in which the metastasis was purely diploid. A single patient, who was in the diploid group, had unresectable tumor in the lungs; she remains alive with stable disease at 30 months. The other six patients, who underwent complete resections of their lung metastases, are free of disease. These results suggest that DNA FCM is not a sensitive method for predicting the metastatic potential of BGCT since most metastasizing cases appear to be diploid.     

Surgical treatment of bilateral synchronous Wilms' tumors

Definite progress has been made in the treatment of bilateral Wilms' tumors with marked improvement in the prognosis. This is confirmed in our series of 6 consecutive patients with synchronous tumors. The recent trend toward more conservative surgery, double or triple drug chemotherapy, and avoidance of high-dose radiation therapy has yielded good results.     

Hyaluronic acid-stimulating activity in the pathophysiology of Wilms' tumors

Markedly elevated levels of hyaluronic acid occur in the serum and urine of some patients with Wilms' tumor. We have recently described a glycoprotein factor in fetal serum that stimulates deposition of hyaluronic acid. In a survey of bovine fetal tissue extracts, we have identified the fetal kidney as the source of this circulating activity. Wilms' tumors arise from transformed "rests" of fetal kidney. We demonstrate here that such tumors continue to produce this fetal factor and that the hyaluronic acid-stimulating activity is found in the urine of children with Wilms' tumors. In the three patients with Wilms' tumor who were followed, elevated levels of hyaluronic acid-stimulating activity were found in their urine before treatment. By 2 months after surgical removal of their tumors, these levels had returned to baseline. We propose that hyaluronic acid-stimulating activity is the mechanism for the elevated levels of hyaluronic acid in the sera and urine of these patients. The activity is an oncofetal protein and the first for which a function has been identified. It also is a marker for this common childhood solid tumor and has the potential for identifying children at increased risk.     

The treatment of Wilms' tumor: Results of the national Wilms' tumor study.

The National Wilms' Tumor Study, initiated in 1969, tested competing treatment strategems for patients with tumors ranging from Group (Gp) I (tumors confined to the kidney and totally removed) to Gp IV (remote metastases present at diagnosis). Three hundred and fifty-nine of 606 registered patients were randomized in the trial. Gp I patients under 2 years of age fared well whether postoperative radiation therapy (RT) was or was not added to 15 months' maintenance actinomycin D (AMD). Their prognosis was better than that for older cohorts similarly treated, in whom the difference in relapse rates between treatment groups were suggestive of an RT effect. Combined AMD and vincristine (VCR) gave better results than either agent alone in patients with more advanced tumors (Gps II and III) still confined to the abdomen, all of whom received postoperative RT as well. Preoperative VCR given Gp IV patients in addition to postoperative RT, AMD, and VCR did not improve results. The frequency of mesoblastic nephroma (1%), of bilateral tumors (5%), and of incorrect preoperative diagnosis of Wilms' tumor (5%), the toxicities of the various regimens, and other ancillary data are presented and discussed.     

DNA cytophotometry and prognosis in ovarian tumors of borderline malignancy. A clinicomorphologic study of 80 cases.

Surgical specimens of 80 ovarian tumors of borderline malignancy (OTBM) were investigated by scanning DNA cytophotometry. Diploid or euploid DNA histograms were found for 21 tumors, whereas 59 OTBM showed noneuploid or aneuploid DNA patterns. All patients were followed-up after surgery for at least 3 years (mean observation period, 6.7 years). Follow-up showed 11 cases of recurrent disease and 6 deaths. DNA findings and several other morphologic and clinical details (including patient age, histologic type and stage of disease, and extent of therapy) were correlated to the postoperative course. Statistical analyses disclosed that, of these parameters, only DNA content significantly affected prognosis. Recurrences and deaths resulting from tumor exclusively were observed among patients with noneuploid or aneuploid OTBM, whereas none of the diploid or euploid tumors recurred (P less than 0.05). DNA cytophotometry thus might be regarded as an effective complementary means to assess the prognosis of individual OTBM cases.     

脑膜瘤MR征象与DNA含量的相关性研究

目的:研究脑膜瘤磁共振(magnetic resonance,MR)征象与肿瘤细胞核DNA含量的相关性,从MRI影像学角度评价脑膜瘤的生物学行为及细胞增殖潜能.方法:45例脑膜癌患者,应用细胞图像分析仪,采用吸光度法对脑膜瘤细胞核DNA含量和倍性水平进行检测,所得数据资料与MR征象进行相关性分析.结果:脑膜瘤瘤周水肿、肿瘤外形不规则、瘤脑边界模糊毛糙3种MR征象的DNA含量及倍性水平与无瘤周水肿、肿瘤外形光滑和瘤周边界清晰的脑膜瘤比较,差异均有统计学意义(P均<0.01),这3种征象的MRI得分与DNA含量相关(r=0.696,P=0.000).结论:脑膜瘤MR图像上,有瘤周水肿、不规则肿瘤外形、瘤脑边界模糊毛糙者,其DNA含量及异倍体率较高,可作为预测脑膜瘤增殖潜能和恶性生物学行为的影像学指标.     

RNA expression of the WT1 gene in Wilms' tumors in relation to histology.

BACKGROUND: On the basis of accumulating data, the recently isolated WT1 gene is a Wilms' tumor gene and a putative tumor suppressor gene. These findings include expression in developing fetal kidney, intragenic deletions in tumors, and germline mutations in predisposed individuals. Wilms' tumors, which exhibit a broad range of differentiation, are composed of three cell types: blastema, epithelium, and stroma. PURPOSE: The purpose of this study was to investigate the relationship between WT1 gene expression and histologic composition in Wilms' tumors in an effort to elucidate how the WT1 gene functions in proliferation of these histologic components. METHODS: We used Northern blot hybridization to study WT1 gene expression by messenger RNA (mRNA) accumulation in 20 tumors of varying histology and in adjacent uninvolved kidney tissue. In two patients, tumors were also compared before and after therapy. RESULTS: Tumors that were predominantly blastemal expressed high amounts of WT1 mRNA, whereas predominantly stromal tumors expressed either low or undetectable amounts. Blastemal tumors that were predominantly poorly differentiated expressed WT1 mRNA at higher levels than those that were more well differentiated. Although we expected that a putative tumor suppressor gene like WT1 would generally be expressed at lower levels in tumor than in normal kidney, this was true only in predominantly stromal cells. One of the two patients studied before and after therapy had a dramatic response to therapy accompanied by a decline in WT1 gene expression and disappearance of blastemal and epithelial elements. CONCLUSIONS: A correlation was observed between WT1 gene expression and histology of the tumors. Level of expression was inversely related to the degree of differentiation in blastemal tumors and in the patient with a dramatic response to therapy. These results, in conjunction with the observation that WT1 mRNA is abundant in normal fetal kidney, suggest that WT1 gene expression is related to kidney development, especially in differentiation of blastemal components. IMPLICATIONS: Further studies to search for alterations of the WT1 gene in tumors and to identify regulatory factors in gene expression will increase understanding of the role of this gene in normal development and tumorigenesis.     

Expression and mutation analysis of the Wilms' tumor 1 gene in human neural tumors.

The Wilms' tumor 1 gene, WT1, encodes a zinc-finger protein that is implicated in the development of Wilms' tumor. Mutant or aberrantly expressed WT1 isoforms have also been described in desmoplastic small round cell tumor, acute leukemias, mesothelioma, breast tumors and melanoma. During early development, WT1 is expressed in the brain and spinal cord, however its role in the malignancies that affect these tissues has not been previously investigated. In our study we have examined neural tumors including brain tumors and neuroblastomas for WT1 expression and for mutations affecting the zinc-fingers. Although WT1 expression was detected in gliomas, medulloblastomas and neuroblastomas, neither zinc-finger region mutations nor splicing anomalies affecting the KTS site were detected. We therefore conclude that WT1 does not play a significant role in the etiology of human neural tumors.     

Evaluation of esophageal dysplasia by cytofluorometric analysis.

In order to grade objectively and characterize dysplastic and precancerous esophageal epithelium its DNA content was measured by cytofluormetric methods and compared to normal and cancerous esophageal epithelium. This yielded the following results. With transition of the esophageal eipthelium from mild dysplasia to severe dysplasia and finally to in situ carcinoma, Feulgen-DNA values showed patterns characteristic of a tetraploid population. They lacked prominent peaks which were usually observed with invasive carcinomas. Dominant near-tetraploid population and definite tetraploid-octoploid populations were characteristic of severe dysplasia or carcinoma. The mean Feulgen-DNA values were significantly larger in severe dysplasia than in the lesser grade of dysplasia as well as the normal epithelium. However, this was not the rule in the full blown carcinomas. It would appear that the esophageal cytophotometric patterns are analogous to those previously observed in the skin and uterine cervix.     

Epithelial--stromal interactions in tumors. A morphologic study of fibroepithelial tumors of the breast.

BACKGROUND AND METHODS. The known spatial interaction between normal breast epithelium and its surrounding stroma prompted an investigation of the spatial relationship between stromal mitoses and the epithelial component of fibroepithelial tumors of the breast. The authors applied a novel computerized morphometric technique to routinely processed histologic sections of 23 fibroepithelial tumors (13 fibroadenomas and 10 phyllodes tumors). The proportional area of epithelium in successive concentric annuli surrounding stromal mitoses was measured, and its distribution was compared with that around suitable control points. RESULTS. The authors found that stromal mitotic activity in these tumors was significantly more likely to occur close to rather than remote from the epithelial component, with a significant excess of epithelium around mitoses compared with control points within a range of 79 microns. Essentially similar findings were obtained when randomly identified fibroblast nuclei were used as control points, thus obviating variations in stromal cell density with distance from epithelium as an explanation for the findings. CONCLUSIONS. These findings support the hypothesis that stromal growth in fibroepithelial tumors depends, to a variable extent, on the epithelial component. An interaction in the opposite direction (i.e., the stroma providing the growth support to the epithelium) also may occur, but this was not investigated. It is suggested that there is an interdependence of growth between the epithelial and stromal components in these tumors that explains their complex morphology and that stromal dependence on epithelium is lost with increasing malignancy of the stromal elements.