Correlation of Content and Histology in Prognosis of Astrocytomas STEPHEN W. COONS, M.D., JOHN R. DAVIS, M.D., AND DENNIS L. WAY, B.S. Thirty-two cases of astrocytoma were analyzed for content and cell-cycle proliferation features by flow cytometry using paraffin-embedded tissue. The findings were correlated with histologic grading and survival. (aneiiploidy or elevated G-M fraction %) was present in cases (%). Glioblastoma multiforme () had of (%), anaplastic astrocytomas () of (%), and low-grade () neoplasms of cases with abnormal content. Short-term survival (^ months) occurred in all patients with (%), of patients with (%), and of patients with neoplasms (%). Seventeen of patients (%) with abnormal content were short-term survivors (P <.). content was found in of short-term survivors (%), whereas histologic grading identified of such cases (%). A combination of grading and abnormal content identified of (%) of the poor survival cases. content was most useful in the anaplastic group. Six of seven cases (%) with abnormal content had short survival (P < -), and three of four (%) with normal content had long survival. analysis combined with histologic grading improves prognosis designation. (Key words: Flow cytometry; ; Cell-cycle; Astrocytoma prognosis; Glioblastoma multiforme) Am J Clin Pathol ;:- DESPITE RECENT MODIFICATIONS, histologic grading of astrocytomas remains an imperfect method of establishing prognosis. In the past, both three- and four-tiered systems gave weight to a variety of histologic features (including the presence or absence of necrosis, cellularity, nuclear pleomorphism, and endothelial proliferation) in order to assign astrocytomas into prognostically significant categories. However, a recent review of, cases by Burger and colleagues showed that the only factor that reliably indicates a poor prognosis is the presence of necrosis. Their study resulted in a revised three-tiered system. Necrosis is the sine qua non of glioblastoma multiforme (); anaplastic astrocytomas (s) demonstrate moderate cellularity and moderate nuclear pleomorphism; and (low-grade) () astrocy- Received November, ; received revised manuscript and accepted for publication February,. The results were presented in part at the American Association of Neuropathologists meeting in Charleston, S.C., June -,. Address reprint requests to Dr. Coons: Barrow Neurological Institute, St. Joseph's Hospital and Medical Center, W. Thomas Road, Phoenix, Arizona -. Department of Pathology, University of Arizona Health Sciences Center, Tucson, Arizona tomas show mild hypercellularity and mild nuclear pleomorphism. The two extreme categories correlate well with prognosis, with having a uniformly poor prognosis (-% survival) and tumors having a relatively favorable prognosis. The central category is less satisfactory, with some anaplastic tumors behaving as and some allowing long survival. Nuclear content serves as a useful prognostic factor in a variety of neoplasms. y (abnormal nuclear content) has been correlated with a poorer prognosis in a number of human tumors. ", Other flow cytometry studies have shown a relationship between the histologic grade and nuclear content of astrocytomas, with aneuploidy more frequently seen in higher grade ("malignant") gliomas than lower grade tumors., However, the early flow cytometry studies tended to have a low degree of resolution and lacked a uniform grading system. More recent studies have failed to show a positive correlation between aneuploidy and behavior in astrocytomas. BrDU arid H-labeling of astrocytomas have shown a correlation between histologic grade and the S-phase fraction, with higher values associated with higher grade tumors.,, The S-phase fraction reflects the level of synthesis and is presumably a good measure of proliferative potential. Most of these studies were limited to fresh material with insufficient follow-up intervals and therefore could not provide prognostic information. In order to gain information regarding the relationship of content and histologic grade in astrocytomas, flow cytometry was used to analyze the of cases of astrocytoma by use of archival paraffin-embedded material. Materials and Methods Study cases were limited to pure astrocytomas: mixed gliomas, gangliogliomas, and gliosarcomas were ex- Downloaded from https://academic.oup.com/ajcp/article-abstract//// on December
COONS, DAVIS, AND WAY A.J.C.P. September eluded. Histologic characteristics were graded according to the revised three-tiered system described by Burger, yielding cases of, s, and astrocytomas. Only cases in which the initial tissue diagnosis was made in our hospital were included. Patient ages at diagnosis ranged from to years. Medical records and tumor registry data were reviewed to establish disease course and follow-up. Follow-up for survivors was a minimum of months. For analysis, samples of neoplasm were retrieved from paraffin blocks and prepared for flow cytometry according to the method of Schutte and associates, which was modified to provide greater cellular disaggregation. Hematoxylin and eosin-stained sections were used to direct retrieval in order to maximize tumor purity. Briefly, the method involves manually removing thick sections from the paraffin blocks, mincing the slices, followed by standard dewaxing in xylene and rehydration through graded alcohol concentrations. Our modification used a novel enzymatic disaggregation. The tissue fragments were incubated for hours in ml Dulbecco's phosphate buffered saline (PBS-A) with g/l trypsin :,, U/L collagenase type IV, and g/l edetate (EDTA). The friable fragments were further disaggregated with a Dounce (Wheaton Scientific, Millville, N.J.) homogenizer until finely dispersed. The samples were filtered through a -/im mesh, washed, and resuspended in ml PBS-A containing mg RNase and g/l NP for minutes. A drop of the cell suspension was stained with % alcoholic thionine to establish the presence and concentration of free nuclei. Before analysis all samples received ng propidium iodide and immediately before, X " freshly thawed chicken red blood cells were added. content was measured by use of a Coulter EPICS V argon laser flow cytometer. Ten thousand cells were analyzed in each case. Cell-cycle components were calculated from the histograms by the PARA analysis program (Coulter). Assessment of content highlighted the G-M component rather than the S-phase or a combination of the two, as has been done for other tumors. This was done for two reasons. First, technical limitations inherent in PARA- analysis and related to the presence of necrosis or background signal were felt to more greatly affect measurement of the S-phase. Second, a disproportionately high S-phase component was seen in some astrocytomas, consistent with the BrDU data from Hoshino." A neoplasm was classified as aneuploid if a peak was present with a index outside the range of.-. times the index of the normal G- peak. No attempt was made to distinguish between tetraploidy and G-M phase. After flow cytometric analysis, the cases were reviewed to determine if significant differences in the extent of necrosis, mitotic rate, or pleomorphism were present between the aneuploid and diploid neoplasms. Results High-grade astrocytomas have an -% two-year survival rate. Analysis of our data suggested that the break between aggressive and less aggressive neoplasms occurred at months. Therefore, patients dead of disease in less than months were designated short-term survivors, and those alive longer than months, longterm survivors. The survival data, histologic grading, and patterns of the short-term survivors and long-term survivors are listed in Table. The mean G-M component for the short-term survivors was.%, with a standard deviation of.. The mean G-M component for the long-term survivors was.%, with a standard deviation of.. (Because case was believed to represent unusually long survival in a patient with a high-grade neoplasm, it was not used in determining the mean.) Analysis of the means by use of student's /-test showed significance at the % level. For the long-term survivors, the upper limit of the % confidence interval for G-M was.%; therefore, a G-M component greater than % was designated (in addition to frank aneuploidy) as abnormal content. With the use of these criteria, cases had abnormal and cases had normal. Six combinations of and histologic results were identified: astrocytoma with normal ; with normal ; anaplastic with nonaneuploid abnormal ; with nonaneuploid abnormal ; with aneuploidy; and with normal. The short-term survivors with nonaneuploid abnormal had a mean G-M component of.%. Student's Mest showed that the difference in the mean G-M components of the short-term survivors with abnormal and the long-term survivors was significant at the % level. y was present in of cases of (%) and of cases overall (%). was found in of (%) short-term survivors and of (%) cases overall. Seven of (%) short-term survivors had normal patterns. The complete distribution is shown in Table. Sixteen of (%) short-term survivors had histologic diagnosis of and were therefore classified correctly as short-term survivors by histologic results alone. content was found in of ( %) short-term survivors, including not identified by histologic results, and in only of long-term survivors Downloaded from https://academic.oup.com/ajcp/article-abstract//// on December
Vol. No. BRIEF SCIENTIFIC REPORTS Table. Histology, Content, and Clinical Data Case No. Histology* Ploidy % G-M Interpretation Status Survival (months) t -* > cytoma. glioblastoma multiforme; = anaplastic astrocytoma: = low-grade astro- t Dead of disease. % Uncertain. {P <.). was associated with shortterm survival in of cases (P <.). The two methods combined identified of short-term survivors with only the single false positive result from the analysis. Sensitivity, specificity, and predictive values for each method are shown in Table. Mean survival data for all patients is classified by histologic results and pattern in Table. The mean survival rate of months in with abnormal includes the single long-term survivor, which skews the mean. If this outlying value is excluded, the mean survival is eight months, equivalent to that of the population. Table. Correlation of Histologic Results and Content with Short-Term Survival Table. Correlation of Content and Histologic Results Content Method Sensitivity Specificity A. All histologic grades ( patients) Histology % % / / PV+* % / PV- % / Histologic Results ly High G-M / (%) / (%) / (%) / (%) / (%) / (%) / (%) / (%) / (%) % % / / Histology + % % / / B. Anaplastic astrocytomas ( patients) % % / / Predictive value. % / % / % / % / % / % / Downloaded from https://academic.oup.com/ajcp/article-abstract//// on December
COONS, DAVIS, AND WAY A.J.C.P.'September Table. Mean Survival Months Related to Histologic Results and Content Mean Survival (months) Histology All Cases Nonaneuploid / = aneuploid or G-M greater than or equal to %. Review of the cases did not demonstrate significant histologic differences between the aneuploid and diploid groups. Specifically, there were no correlations between tumor ploidy and pleomorphism, mitotic rate, or extent of tumor necrosis. Discussion The data showed a definite relationship between content and behavior of astrocytomas with short survival in of patients with abnormal (P <.). Both aneuploidy and elevated G-M peaks (an index of proliferation) were associated with aggressive neoplasms. The latter confirms the findings of Hoshino and colleagues, who tied proliferation to prognosis using H-labeled tissue ; however, almost all of the highly proliferative neoplasms in that study were with only two s. The clinically relevant setting for analysis is primarily for s; the behavior of is predicted by histologic characteristics alone. In this regard it should be noted that because all six study cases with aneuploidy were, the identification of aneuploidy did not aid in our identification of high-risk cases. However, aneuploidy has been reported in s and was seen in a case we excluded from our study because of inadequate follow-up. Our data showed a trend toward shorter survival in cases with aneuploidy. There was no consistent correlation for neoplasms that "appeared aneuploid," with a high degree of pleomorphism and obvious bizarre nuclei and the finding of aneuploidy by flow cytometry, nor was there a correlation between histologic appearance and survival within the aneuploid group, which ranged from to months. The degree of necrosis in the cases ranged from rare microscopic foci to approximately % of the histologic sections. No relationship was found between tumor ploidy or survival and extent of necrosis. Similarly, there was no correlation between aneuploidy and mitotic rate; if anything, there was a trend toward a higher mitotic rate in diploid neoplasms and in neoplasms demonstrating relatively little pleomorphism. This would be consistent with the observations of Giangasparro that a higher proliferation rate is seen among the small anaplastic cells than the large pleomorphic cells in malignant astrocytomas. Thus, the significance of aneuploidy and ploidy analysis per se is not clear for astrocytomas. With short survival times (comparable to those in the population) in six of seven s with abnormal (P <.), our findings suggested that analysis can identify a subpopulation of anaplastic tumors that behave more aggressively. However, because of the limited number of cases, the data are of borderline statistical significance. The bases for this identification are probably twofold. In addition to a more aggressive population of true s, it is likely that with the sampling problems inherent in biopsy material, some of the cases were (appropriately) misclassified as anaplastic astrocytomas because of the absence of necrosis in the biopsy material. The use of histologic results and analysis together enhanced the designation of prognosis. Histologic results and analysis alone identified % and % of the aggressive neoplasms, respectively; used together, % were identified. Our study confirms the relationship of ploidy and cell-cycle distribution with histologic grade and behavior of astrocytomas. The tentative identification of a subpopulation of more aggressive s suggests that analysis of ploidy and cellular proliferation has clinical value and can enhance the prediction of tumor behavior. Thesefindingswarrant the study of additional cases to establish firm statistical significance. Also, they indicate the need for improved technology for cell-cycle analysis by flow cytometry and continued development of other measures of proliferation. References. Bigner SH, Bjerkvig R, Laerum OD: content and chromosomal composition of malignant human gliomas. Neurology Clinics ;:-.. Broggi G, Franzini A, Costa A, Melcame A, Allegranza A: Cell kinetics of neuroepithelial tumors in serial stereotactic biopsies. A new combined approach. Appl Neurophysiol ;:-.. Burger PC, Vogel FS, Green SB, Strike TA: Glioblastoma multiforme and anaplastic astrocytoma: pathologic criteria and prognostic implications. Cancer ;:-. Downloaded from https://academic.oup.com/ajcp/article-abstract//// on December
Vol. No. BRIEF SCIENTIFIC REPORTS. Christov K, Zapryanov Z: Flow cytometry in brain tumors. I. Ploidy abnormalities. Neoplasma ;:-.. Davis JR, Aristizabal S, Way DL, Weiner SA, Hicks MJ, Hagaman RM: ploidy, grade and stage in prognosis of uterine cervical cancer. Gynecol Oncol (in press).. Ferenc MJ, Nans GJ: Predictive value of flow cytometric content analysis of paraffin-embedded tissue in carcinoma of the breast. Lab Invest ;:A.. Frankfurt OS, Chin JL, Englander LS, Greco WR, Pontes JE, Rustum YM: Relationship between ploidy, glandular differentiation and tumor spread in human prostate cancer. Cancer Res ;:-.. Friedlander ML, Hedley DW, Taylor IW, Russell P, Coates AS, Tattersall MHN: Influence of cellular content on survival in advanced ovarian cancer. Cancer Res ;:-.. Giangasparo F, Chieco P, Lisigroli G, Burger PC: Comparison of cytologic composition with microfluorometric analysis of the glioblastoma multiforme and anaplastic astrocytoma. Cancer ;:-.. Hedley DW, Friedlander ML, Taylor IW: Application of flow cytometry to paraffin-embedded archival material for the study of aneuploidy and its clinical significance. Cytometry ;:-.. Hoshino T, Nagashima T, Murovic JA, et al: In situ cell kinetics on human neuroectodermal tumors with bromodeoxyuridine labeling. J Neurosurg ;:-.. Hoshino T, Townsend JJ, Muradea I, Wilson CB: An autoradiographic study of human gliomas: growth kinetics of anaplastic astrocytoma and glioblastoma multiforme. Brain ; : -.. Hoshino T, Wilson CB: Cell kinetic analyses of human malignant brain tumors (gliomas). Cancer ;:-.. Hoshino T, Wilson CB, Knebel RD, Gray JW: The distribution of nuclear from human brain tumor cells:flowcytometric studies. J Neurosurg ;:-.. Kawamoto K, Herz F, Wolley RC, Hirano A, Koss : Flow cytometric analysis of the content in cultured human brain tumor cells. Virchows Arch [Cell Pathol] ;:-.. Kokal W, Sheibani K, Terz J, Harada JR: Tumor content in the prognosis of colorectal carcinoma. JAMA ;:-.. McComb RO, Burger PC: Pathologic analysis of primary brain tumors. Neurologic Clinics ;:-.. Schutte B, Reynders MMJ, Rosman FT, Blijham GH: Flow cytometric determination of ploidy level in nuclei isolated from paraffin-embedded tissue. Cytometry ;:-. Plasma Fibronectin Values in Patients with Acquired Immunodeficiency Syndrome (AIDS) and AIDS-Related Complex DARYL M. OGDEN, M.S., HARALD E. FISCHER, M.D., PH.D., FRANK J. LIU, M.D., ADAN RIOS, M.D., AND BENJAMIN LICHTIGER, M.D., PH.D. The authors studied the circulating fibronectin concentrations in the plasma of patients with acquired immunodeficiency syndrome (AIDS) or AIDS-related complex (ARC) and of age- and sex-matched healthy blood donors. They adapted a commercially available turbidimetric immunoassay for use with a centrifugal analyzer. The assay showed within-run precision of.%,.%,.%, and.%, and an accuracy of %, %, %, and % at fibronectin concentrations of mg/l, mg/l, mg/l, and mg/l, respectively. Betweenrun precision was %, %, and % for mg/l, mg/l, and mg/l concentrations, respectively. Plasma fibronectin values obtained from the healthy blood donors were in good agreement with those values reported by other investigators using various methods. No significant differences between the plasma fibronectin values of the patient population (mean ± Received December, ; received revised manuscript and accepted for publication March,. Presented in part at the Fall Meeting of the American Society of Clinical Pathologists/College of American Pathologists, New Orleans, October. Address reprint requests to Mr. Ogden: Division of Laboratory Medicine, U.T.M.D. Anderson Hospital, Holcombe Boulevard, Box, Houston, Texas. Division of Laboratory Medicine and Division of Medical Oncology, The University of Texas M.D. Anderson Hospital and Tumor Institute at Houston, Houston, Texas SD = mg/l ± mg/l) and of the control group (mean ± SD = mg/l ± mg/l) were noted. The authors conclude that the measurement of fibronectin concentrations in patients with AIDS or ARC does not contribute significantly to the diagnosis and therapeutic management of these patients. (Key words: Fibronectin; AIDS; Immunoassay) Am J Clin Pathol ;:- PLASMA FIBRONECTIN (FN) is a dimeric serum glycoprotein, M r = kd, that acts as a nonantibody, non-complement-dependent opsonin. It is present in soluble form in the blood and tissuefluidsand in insoluble form in basement membranes, extracellular matrices, and connective tissue. ' ' ', Although most circulating FN is probably synthesized by the liver, cultured cells ' ' ' as well as macrophages, ' have been shown to secrete this protein. In fact, blood monocytes Downloaded from https://academic.oup.com/ajcp/article-abstract//// on December