Proc. Nati. Acad. Sci. USA Vol. 86, pp. 7186-7190, September 1989 Medical Sciences Loss of distinct regions on the short arm of chromosome 17 associated with tumorigenesis of human astrocytomas (tumor suppressor genes/antioncogenes/brain tumors/neurofibromatosis/colon cancer) M. EL-AzOUZI*, R. Y. CHUNG*, G. E. FARMER*, R. L. MARTUZA*, P. McL. BLACKt, G. A. ROULEAUt, C. HETTLICH*, E. T. HEDLEY-WHYTE§, N. T. ZERVAS*, K. PANAGOPOULOS*, Y. NAKAMURA¶, J. F. GUSELLAt, AND B. R. SEIZINGER*tII *Molecular Neurooncology Laboratory, Neurosurgery Service, tMolecular Neurogenetics Laboratory, and §Neuropathology Laboratory, Massachusetts General Hospital, and Harvard Medical School, Boston, MA 02114; *Department of Neurosurgery, Brigham and Women's Hospital, and Harvard Medical School, Boston, MA 02115; and lHoward Hughes Medical Institute, and University of Utah, Salt Lake City, UT 84132 Communicated by Richard L. Sidman, June 28, 1989 (received for review February 2, 1989) ABSTRACT Astrocytomas, including glioblastoma multi- differentiated astrocytomas and the glioblastoma multiforme. forme, represent the most frequent and deadly primary neo- Although some patients with anaplastic astrocytoma respond plasms of the human nervous system. Despite a number of well to chemotherapy and/or radiotherapy, other patients do previous cytogenetic and oncogene studies primarily focusing not (2). Anaplastic astrocytomas, therefore, may be com- on malignant astrocytomas, the primary mechanism of tumor posed of several distinct biological subgroups, which cannot initiation has remained obscure. The loss or inactivation of be detected by standard histopathological techniques (6, 7). "tumor suppressor" genes are thought to play a fundamental Thus, alternative diagnostic tools, such as genetic markers, role in the development ofmany human cancers. Thus, we have could be useful in further subclassifying glial tumors and analyzed astrocytomas of various histological malignancy leading to more accurate grades with polymorphic DNA markers to search for specific clinical prognosis and more appro- chromosomal deletions potentially pointing to loci containing priate choice of therapies. tumor suppressor genes. Loss of constitutional heterozygosity In the present study, we have analyzed astrocytomas of indicating chromosomal loss or deletions was most frequently various malignancy grades with polymorphic DNA markers seen for markers on the short arm of chromosome 17 in 50% to search for the loss of chromosomal regions containing of the informative tumors (5 of 10 informative cases) and, to a tumor suppressor genes (for review, see ref. 8). This tech- lesser extent, for markers on chromosomes 1 and 10. Deletions nique is particularly powerful for uncovering chromosomal on chromosome 17p were seen in both low-grade and high- deletions in benign solid tumors (9-11); karyotyping in these grade manant astrocytomas, suggesting that this chromo- tumors is often unsuccessful due to their low mitotic index. some may contain a tumor suppressor gene associated with the Moreover, molecular probes may detect small (interstitial) early events in tumorigenesis. The common region of deletions deletions and mitotic recombinations not detectable by stan- on the short arm of chromosome 17 is, therefore, dearly dard cytogenetic techniques. Markers for chromosomes 17 distinct from the gene causing von Recklinghausen neurofibro- and 22 were chosen because these chromosomes contain the matosis (NF1), a tumor syndrome associated with glial tumors two types of neurofibromatosis, NF1 and NF2, respectively that maps to the long arm of chromosome 17. The search for (9-14). Both forms of neurofibromatosis are associated with progressively smaller deletions on chromosome 17p in astro- certain types ofglial tumors (for review, see refs. 15 and 16). cytomas may be the way to clone and characterize this locus, Chromosomes 1, 7, and 10 were selected because ofprevious thus leading to insights into normal and abnormal growth and reports on their frequent numerical and structural aberrations differentiation of gial cells. in glial tumors (17-21). Gliomas are the most common primary tumors of the human MATERIALS AND METHODS central nervous system (1). The majority of brain tumors are astrocytomas (1), which are most commonly divided into DNA of high relative molecular weight was isolated from 14 three histopathological malignancy groups (2-4). However, astrocytomas and corresponding normal tissues (peripheral alternative classification systems are in use, including the leukocytes) as described (9, 10). Tumors A3, A6, A13, and classical four-tiered system by Kernohan (2-4), and even A14 are recurrent tumors; tumors A3 and A6 were irradiated experienced neuropathologists may disagree in their assign- before the tumor specimens analyzed in this study were ments of astrocytomas to a precise malignancy grade. Ac- removed. All other specimens represent primary tumors cording to the three-tiered classification, the first and least removed before any chemotherapy or radiotherapy. Tumor malignant type is the well-differentiated astrocytoma (astro- A14 was from a patient with NF1; all other cases represent cytoma grade I). These tumors are often manageable by sporadic tumors from patients without any history of NF1 or surgery, although recurrence is frequent. The recurrent tu- any other hereditary tumor syndrome. mors are often less differentiated and of higher grade, sug- Representative samples of each tumor were histopatho- gesting that they are derived from the less malignant forms in logically analyzed by two experienced neuropathologists at a progressive fashion. Glioblastoma multiforme (astrocy- the Massachusetts General Hospital, using a three-tiered toma grade III) is the most malignant type and is virtually classification system of malignancy, according to that estab- always lethal, despite the best available surgery, radiation, lished by the World Health Organization, modified by Zulch and chemotherapy (5). The anaplastic astrocytomas (astro- (4) (Table 1). cytoma grade II) are intermediate between the well- Abbreviations: NF1 and -2, neurofibromatosis type 1 and 2, respec- The publication costs of this article were defrayed in part by page charge tively. payment. This article must therefore be hereby marked "advertisement" 'To whom correspondence and reprint requests should be ad- in accordance with 18 U.S.C. §1734 solely to indicate this fact. dressed. 7186 Downloaded by guest on September 29, 2021 Medical Sciences: El-Azouzi et al. Proc. Natl. Acad. Sci. USA 86 (1989) 7187 Table 1. Loss of heterozygosity for loci on chromosome 17p in astrocytomas Chromosome (probe and RFLP enzyme) 1 2 3 7 10 10 10 10 (pYNZ2 (pYNH24 (H3H2 (pJCZ67 (pEFD75 (5-1 (OAT (OAT Patient Tumor type Msp I) Msp I) HindIII) Rsa I) Taq I) Taq I) Msp I) Rsa I) Al Astro II 12 12 12 A2 Astro I-II* 12 12 A3 Astro III 12 12 12 A4 Astro III 12 A5 Astro II 2 12 12 A6 Astro III 2 12 12 12 12 12 A7 Astro I-II* 12 12 12 12 A8 Astro I-II* 12 12 A9 Astro I 12 A10 Astro III 12 12 12 12 12 All Astro II-III* 12 A12 Astro III 12 A13 Astro I 12 12 12 A14 Astro III 12 1 Chromosome (probe and RFLP enzyme) 17 17 17 17 17 17 17 17 17 22 22 22 (pYNZ22 (pYNH37.3 (pHF12-1 (EW301 (EW203 (EW203 (EW206 (EW207 (pTHH59 (p22/34 (pMS3-18 (W13E Patient Rsa I) Taq I) Msp I) Bgl II) Bgl II) Taq I) Msp I) HindIII) Taq I) Taq I) Bgl II) Taq I) Al 12 12 12 12 A2 2 12 - - 12 12 A3 12 12 12 12 A4 12 A5 12 - 12 - 12 12 A6 12 12 - 12 12 12 A7 2 1 12 12 12 12 A8 12 - 12 12 12 12 12 12 12 A9 12 12 12 AlO 2 2 12 12 12 12 12 12 All A12 2 1 2 2 12 A13 - - 12 12 12 12 A14 2 1 2 Tumor DNA and normal DNA from astrocytoma patients was analyzed with polymorphic DNA markers for loss of constitutional heterozygosity as described. Phenotype of the tumor tissue is shown for every case where blood DNA displayed heterozygosity. 12, Heterozygosity (even though different allele pairs may be present for certain multiallele markers); 1, continued presence of the larger allelic restriction fragment and loss ofthe smaller allelic fragment relative to normal tissue DNA; 2, continued presence ofthe smaller allelic restriction fragment and loss of the larger fragment. A minus sign indicates that normal DNA was tested but proved uninformative because the DNA did not display heterozygosity. Absence ofan entry indicates that a marker was not tested or did not give a readable result for that particular patient. All astrocytomas were independently graded by the two neuropathologists. Tumors A2, A7, A8, and All (marked with an asterisk) were classified by one neuropathologist as grades I, I, I, and III, respectively; the other neuropathologist classified all four tumors as grades II. However, both agreed that tumors A2, A7, and A8 represent low-grade astrocytomas, whereas All is of higher-grade malignancy. For probe definitions, see Materials and Methods. Astro, astrocytoma. Approximately 5 ,g of normal and tumor DNA were gration. To determine whether loss of one allele for chromo- digested to completion with an appropriate restriction en- some 17 in the tumor tissue was associated with duplication zyme, fractionated by agarose gel electrophoresis, trans- of the remaining allele, the hybridization signals for chromo- ferred to nylon membrane, and hybridized to 32P-labeled some 17 probes were normalized to those obtained when the probe DNA (9, 10). The following probes known to reveal same Southern blots were rehybridized with probes for loci restriction fragment length polymorphism in human