AmericanJournal ofPathology, Vol. 134, No. 4, April 1989 Copyright © American Association ofPathologists Immunocytochemical Study of Transforming Growth Factor Expression in Benign and Malignant Gliomas V. Samuels, J. M. Barrett, S. Bockman, between oncogenes and growth factors has been estab- C. G. Pantazis, and M. B. Allen, Jr. lished for many polypeptide growth factors.4 The autono- From the Departments ofNeurosurgery, Anatomy, and mous production of growth factors has led to the auto- Pathology Medical College ofGeorgia, Augusta, Georgia, crine mechanism for neoplastic conversion. and CoreSCAN, Santa Rosa, California To obtain a more thorough understanding of the ubiq- uitous patterns of polypeptide growth factor production, we performed an immunocytochemical analysis in 20 Immunocytochemical studies usingpolyclonal an- cases of benign and malignant human glial tumors. Glial tibodies to epidermal growth factor (EGF) and tumors were chosen as a model of study because of their transforming growthfactor (TGF) alpha and beta precise morphologic correlation relating to malignant phe- wereperformed on 20 cases ofhuman gliomas. EGF notypes.5 immunoreactive material was detected in both be- nign and malignant glial tumors. In addition, EGF immunoreactive material was detected in normal Materials and Methods brain. TGF-beta was detected in both benign and malignant tumors, but was not detected in normal Twenty human glioma biopsies obtained from archival brain. In contrast, TGF-alpha was highly conserved cases within the Department of Pathology at the Medical in its expression, occurring predominantly in ma- College of Georgia from 1981 to 1984 were examined and lignant compared with benign or normal brain tis- classified by a neuropathologist. Of the 20 specimens, 8 sue (P < 0.0001). In malignant gliomas, glioblas- were grade (benign), 5 were grade 11 (anaplastic), and tomas contained 76% TGF-alpha reactivity (im- 7 were grade IlIl (glioblastoma multiforme). This grading munoreactive product), and anaplastic types system corresponds to that system recommended by the contained 85% reactivity. Benign gliomas con- World Health Organization.6 Anti-TGF-alpha, beta, and tained only 13 % TGF-alpha reactivity. Thesefind- EGF polyclonal antibodies were obtained from Biotope ings support the role of TGF-alpha as an oncopro- Company (Bellevue, WA). These antibodies were gener- tein marker in brain neoplasms. (Am J Pathol ated from synthetically produced polypeptides. The sen- 1989, 134:895-902) sitivity of these polyclonal antibodies was determined by Western blot analysis. The detection limit of these re- agents is between 5 and 10 ng of antigen. EGF does not Transforming growth factors (TGF) are low molecular react with this antisera when examined similarly.7 For weight polypeptides that reversibly induce anchorage-in- these studies, we used an indirect peroxidase-anti-peroxi- dependent growth of nontransformed anchorage-depen- dase method described previously.89 Briefly, sections are dent cells. At least two types of transforming growth fac- deparafinized in 100% xylene, and then rehydrated by tors have been described-alpha and beta. TGF alpha is graded series of ethanols (100 to 50%). The slides con- a M, 6000 single-chain polypeptide that shares 30 to 35% taining 5-it thick sections are then placed in a 0.05 M TRIS, homology with human and rat epidermal growth factor 1.5% saline, pH 7.6 buffer before the start of the proce- (EGF).1 Both EGF and TGF-alpha can compete for the dure. The sections are then blocked by the addition of 5% same membrane receptors.2 In contrast to EGF, TGF-al- powdered milk dissolved in TRIS-saline for 30 minutes. pha secretion is seen primarily in the transformation of This is followed by the addition of 0.3% H202 for 30 min- cells to a malignant phenotype. The release of TGF-alpha by transformed cells is to be the result of thought onco- Accepted for publication January 6, 1989. gene activation rather than a consequence of a cellular Address reprint requests to Dr. Cooley Pantazis, Department of Pa- change during the process of transformation.3 The link thology, Medical College of Georgia, Augusta, GA 30917. 895 896 Samuels et al AJPApril 1989, Vol. 134, No. 4 utes to block endogenous peroxidase. After several The software used for this analysis was developed by rinses in TRIS-saline, the slides are reacted with the anti- one author (SB) and is called "CoreSCAN." CoreSCAN is body (1 :10 dilution in TRIS-saline). Control sections were a color video analysis program. The program compares reacted with goat serum. The antibodies are visualized each scanned pixel to a previously collected list of color by sandwiching with either anti-goat or anti-rabbit linking values. If the color value of the pixel matches the value agents. The labeling agent, peroxidase to anti-peroxidase within the list, the program increments a net pixel count. immunoenzyme complex, is then added. The complex is The net pixel count may be output as a numerical value then visualized by the addition of a chromogenic sub- in operator-specified units. Each matching pixel also is in- strate (amino-ethyl carbazole, AEC). All sections were cluded in a map which may be displayed as an image then counterstained with hematoxylin and mounted with overlay. The operator creates a list of color values for Permount and then analysed. comparison in a controlled collection process. The color- banding feature of CoreSCAN may be used to augment the collection and comparison of color values. Color Color Image Analysis System banding reduces the potential number of unique colors contained in an image, from the theoretical maximum of 32,768 to either 4096, 512, 64, or 8 colors. This effectively Sections of tumor were examined in three high-power coerces similar color values to identical color values, fields, consisting of approximately 100 cells/high power thereby reducing the size of the list of color values used field. Cytoplasmic immunoreactive product was quanti- in comparison and decreasing the comparison time. tated using a Delta Scan Color Image Analyzer (Delta Technologies, Inc., Augusta, GA). This system includes a color video camera mounted on a Zeiss Axiophot micro- Statistical scope. In turn, the video camera is cabled to the input of Analysis an AT&T True Vision Image Capture Board, installed on An analysis of the F ratios of variance was used to deter- an IBM-PC with a hard disk and Microsoft mouse. The mine the acceptable selection of the sample size. De- output of the ICB is directed to a Sony RGB color monitor. scriptive statistics were calculated to determine the mean To maintain constant light intensity conditions, the re- variance of standard deviation and standard error of the corded image was performed in one sitting. Only the fo- means. Statistical differences between the means were cus was changed from specimen to specimen to insure determined by a multiple analysis of variance (MANOVA, that all samples were recorded on the exact magnification followed by Tukey multiple range tests, using StatGraph- and illumination intensity. Each digitized image was then ics [SBSC and Company]). Means deemed significant immediately saved to hard disk for additional analysis. were greater than P < 0.05. Verification of the accuracy of the reaction product was always confirmed by pixel mapping of the localized reac- tion product. Results Demonstration of quantitated immunoreactive product by Color Image Analysis Software color video analysis is illustrated in Figures 1 to 4. Figure 1 is a digitized image of an anaplastic (grade 11) astrocytoma Several investigative methods have been used to quanti- stained with goat anti-TGF-alpha, demonstrating moder- tate immunologic staining in tissue sections.9 10 To obtain ate localization of immunoreactive product. Once the spe- objective quantitation, image processing techniques that cific colors of interest are placed within the color net, Cor- use monochrome optical density for measuring intensity eSCAN will digitize the image and color-pixel map the area have been used.11'12 This approach can analyze both real- of reaction product (Figure 2). Figure 3 shows the digi- time and stored images. Pixel measurements are gener- tized image of a malignant (grade ll) astrocytoma stained ated within an object by high resolution digital sampling. with goat anti-TGF-alpha, with extensive amounts of im- We have developed a video-based microscope system, munoreactive product. Computer analysis of the same im- driven by color algorithms structured in C language. This age of Figure 3 reveals the TGF reaction product that has software system can accurately quantitate by pixel map- been localized and quantitated and indicates the speci- ping digitized images received from the videomicroscope ficity of the color video analysis system (Figure 4). hardware. This system can analyze both real-time and The results of quantitative color video analysis of EGF, stored images. Pixel measurements are generated within TGF-alpha and beta, are illustrated in Figures 5 to 8. EGF an object by high resolution digital sampling. staining patterns were examined in both normal and neo- TGF Expression in Gliomas 897 AJPApril 1989, Vol. 134, No. 4 Figure 1. Digitized image ofanti-TGFalpha staining ofan anaplastic astrocytoma (counterstained with hematoxylin, X400). Figure 2. Colorpixel mapping ofimmunocytochemical reactionproduct ofthe same image in Figure 1. 898 Samuels et al AJPApril 1989, Vol. 134, No. 4 Figure 3. Digitized image ofanti-TGF-alpha staining ofa malignant astrocytoma, grade III (X400). Figure 4. Computer analysis with resultantpixel mapping ofthe same image as in Figure 3. TGF Expression in Gliomas 899 AJPApril 1989, Vol. 134, No. 4 Figure 5. Distribution EGF staining pat- terns of both benign and malignant glio- mas. Results are given as the mean ± stan- EGF dard error ofthe mean (SEM) ofthree ob- 1 nA servationsper group. I %-VVVV 8000 Enus a) -4-Ji E 6000 0 c) a 4000 cn a)u 2000 0 .