Primary Intracranial Germ Cell Tumors without an Isochromosome 12p

Primary Intracranial Germ Cell Tumors without an Isochromosome 12p José Alexandre R. Lemos, José Barbieri-Neto, and Cacilda Casartelli

ABSTRACT: Primary intracranial germ cell tumors are the result of the transformation of primordial cells that are unable to migrate to their normal place. These tumors are rare, accounting for less than 5% of all intracranial tumors and less than 3% of childhood neoplasias. In this paper, we have studied cytogenetically two primary intracranial germ cell tumors, a pineal region germinoma, and a mature teratoma in the right lateral ventricle. Neither tumor presented polyploidy or isochromosome 12p, considered to be characteristic of gonadal germ cell tumors. In the five reports in the literature, only one intracranial germ cell tumor presented i(12p). Perhaps i(12p) negative cases characterize a subtype of germ cell tumors. © Elsevier Science Inc., 1998

INTRODUCTION Extragonadal germ cell tumors probably originate from improper migration of the primordial cells during embryonic development. An early peak of incidence in infancy is mainly due to neoplasms in the sacrococcygeal region. The second peak emerges during puberty with the appearance of ovarian and testicular neoplasms [1]. The biological behavior and aggressiveness of the tumor varies according to site and histological components of the tumor [2]. Cytogenetic analysis of germ cell tumors of individual histologic subtypes and organ origins could provide insights into their pathogenic relationships. Among the different locations of germ cell tumors, the testicular site is the only one cytogenetically well studied. Isochromosome 12p was present in most primary testicular germ cell tumors [3, 4, 5]. Structural abnormalities involving chromosome 1 were also frequently observed. There are some cytogenetic reports on extragonadal germ cell tumors, but chromosome analyses of primary intracranial germ cell tumors are very rare, with only five cases reported in the literature [6–10]. Primary intracranial germ cell tumors are rare, accounting for less than 5% of all intracranial tumors. The tumor usually arises from the pineal or suprasellar region. Histologically, these tumors are very similar to those of testicular origin [11]. Whether they also share similar chromosome From the Departamento de Genética (J. A. R. L., C. C.), Departamento de Patologia (J. B.-N.), Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, São Paulo, Brazil. Address reprint request to: Dr. Cacilda Casartelli, Depto. de Genética, FMRP-USP, Av. Bandeirantes, 3900, 14049-490 Ribeirão Preto-SP-Brazil. Cancer Genet Cytogenet 100:124–128 (1998)  Elsevier Science Inc., 1998 655 Avenue of the Americas, New York, NY 10010

changes is not clear. Several cytogenetic abnormalities were described in these tumors, such as polyploidy, monosomies of chromosome 1 and partial or total trisomies of its short arm; monosomies of chromosomes 4, 7, 9, and 11 with rearrangements involving breakpoint 11q23; i(12p) with rearrangements involving breakpoint 12p11; and monosomies of 13, 16, 18, and 22 [6, 7, 10]. We report here cytogenetic abnormalities detected in two primary intracranial germ cell tumors. MATERIALS AND METHODS Two intracranial germ cell tumors (case 1—germinoma and case 2—mature teratoma) were analyzed cytogenetically after medium-term in vitro culture (one to three weeks). Both patients were males, two and four years old, respectively, and had the tumor in the pineal region. The patients did not receive chemotherapy or radiotherapy before surgery. The tumors were classified morphologically according to the World Health Organization brain tumor classification [12]. Fresh tumor tissue samples collected under sterile conditions were processed promptly. The fragments were first sectioned and then dissociated enzymatically with a 0.4 or 0.8% collagenase solution (type IV) (SIGMA) and transferred to culture flasks containing HAM F-10 medium (SIGMA) supplemented with 20% fetal calf serum, vitamins (DIFCO), and antibiotics. The cultures were maintained at 378C and fed twice a week. Culture time was adjusted individually for each case, depending on mitotic activity. For cytogenetic analysis, cells in the exponential growth phase were first treated with 0.0016% colchicine for at least six hours. Hypotonic 0.075 M KCl was used for

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Chromosomes and Intracranial Germ Cell Tumors Composite Karyotype: 37,42,XY,2X[3],211[3][cp4]

Case 2: Mature Teratoma in the Right Lateral Ventricle Histopathological Diagnosis. The cerebral tissue presented areas of stratified, non-keratinized squamous epithelium seated on weak stroma that supports the mucosal glands and exhibits focal points of mineralized bone tissue and mature adipose tissue (Figure 3).

Figure 1 Germinoma of the pineal region: photomicrograph of neoplastic tissue consisting of germ cells among which lymphoid cells can be observed dispersed throughout vascular-connective stroma.

30 minutes and methanol:acetic acid (3:1) was used as a fixative. Chromosome studies were carried out on primary cultures only. Slides were subjected to GTG banding [13]. The International Standard for Human Cytogenetic Nomenclature [14] was used for karyotypic description. RESULTS Case 1: Germinoma of the Pineal Region Histopathological Diagnosis. The neoplastic tissue showed a solid pattern and was well vascularized. It consisted of fusiform cells with slight cytonuclear polymorphism and low mitotic index. These cells are arranged in coils around blood vessels or are pseudolobular in appearance, with frequent focal points of calcification or hyalinization in the center of the coils. Extensive areas of ischemic necrosis are observed (Figure 1). Cytogenetic Analysis. Chromosome counts were performed on 33 cells. The modal number was 45 chromosomes (15%) (Table 1). Eight cells were analyzed by GTG banding (Table 2). Monosomy of chromosome 11 and loss of chromosome X were clonal numerical alterations. Almost all chromosomes had non-clonal losses. Isochromosomes 2q and two markers (Figure 2) were the only structural abnormalities, although non-clonal, observed in these tumors.

Cytogenetic Analysis. Chromosome counts were performed on 132 cells. The modal number was 46 chromosomes (28%) (Table 3). Seventeen cells were analyzed by GTG banding (Table 4). There was clonal monosomy of chromosomes Y, 4, 8, 9, 12, 17, 18, 19, 20, and 22. Non-clonal losses occurred on all chromosomes, and non-clonal gains occurred on chromosomes 13, 15, 18, and 19. The only structural abnormalities observed (non-clonal) were trisomy 1p due to del(1)(q11) and markers (Figure 4). Composite Karyotype: 35,45,XY,2Y[4],28[4],29[4],212[3], 217[3],218[4],219[3],220[3],222[3][cp11] DISCUSSION In this paper, we have presented an analysis of two primary intracranial germ cell tumors—a germinoma and a teratoma. The germinoma (case 1) was highly hypodiploid (82% of the cells). The hypodiploidy was mainly due to nonclonal losses. The germ cell tumors analyzed in the literature were near-triploid or near-tetraploid [7, 10, 15]. This tumor presented only two clonal chromosome abnormalities, i.e., loss of chromosomes 11 and X found in 75% of the cells analyzed. Total or partial losses of chromosome 11 have been previously described in testis teratoma: 211, del(11)(q23) [16], del(11)(q11) [17], and t(8;11) in a primary intracranial germ cell tumor that involves the breakpoint 11q23 [10]. Loss of heterozygosity on both arms of chromosome 11 was found in testis germ cell tumors [18], suggesting the probable existence of tumor suppressor genes. Monosomy 11 may be playing an important role in the initial malignant phase of the tumor [18]. The germinoma had a non-clonal isochromosome 2q. This alteration was described in testis germinoma by Cast-

Table 2 Detailed karyotypic findings of case 1 Karyotype

Table 1 Distribution of ploidy (case 1) No. of chromosomes Hypohaploidy Hyperhaploidy Hypodiploidy Diploidy Hyperdiploidy Hypotetraploidy Total

,23 24–34 35–45 46 47–57 81–91

No. of cells 05 07 15 03 01 02 29

19,2X,2Y,12,15,26,27,28,19,111,112,113,216,217,218, 219,220 20,Y,2X,11,22,24,26,28,29,216,118,119,220,121,122 31,Y,2X,11,12,14,15,16,17,28,19,211,214,115,116,119, 120,121,222 31,Y,2X,22,13,14,15,17,29,110,211,113,214,116,117,118, 119,120,121 37,2X,2Y,23,27,211,212,213,216,221 37,2X,2Y,26,29,214,218,218,219,222 39,Y,2X,i(2q), 23,25,26,28,29,211,217,221,12mar 42,XY,21,211,216,222

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Figure 2 39,2X,Y,i(2q),23,25,26,28,29,211,217,221,12 mar (case 1: germinoma of the pineal region).

edo et al. [4]; trisomy 2 was found in one germinoma of the ovary [19] and in one teratoma of the testis [16]. This alteration occurring in malignant and non-malignant tumors could have a role in the initiation and/or progression of germ cell tumors. The mature teratoma had a modal chromosome number of 46, with 52% hypodiploid cells and 20% hyperdiploid cells. This tumor presented 10 clonal chromosomal monosomies involving chromosomes Y, 4, 8, 9, 12, 17, 18, 19, 20, and 22 and other non-clonal alterations. The most fre-

Figure 3 Mature teratoma in the lateral right ventricle: photomicrograph representing a lesion consisting of epithelial lining of the respiratory type below which accessory mucosal glands are observed, both types of structures being supported by glial tissue observed in the lower right corner and by adipose tissue observed in the lower left corner.

quent alterations were monosomies of chromosomes 8, 9, 18, and Y (28.6% each). Although non-clonal, some of these alterations could be an initial step for a further clonality, as they were already described in the literature. Some authors described total or partial loss of chromosome 8: isochromosome 8q in testis germinoma [4]; monosomy [16, 20]; and del(8)(p12) and del(8)(p22) [21] in testis teratoma. All these cases point to a preferential loss of the short arm of chromosome 8, possibly indicating the importance of this phenomenon for the initial steps of this tumor. Monosomy of chromosome 9 was found in a testis teratoma [20] and a malignant germinoma of the pineal region [6]. Deletions of 9q were found in a testis teratoma [5]. De Graaff et al. [15] observed deletion 9q12 in a testis teratoma, too, but with a choriocarcinoma component. This abnormality, when clonal, could be related to poor

Table 3 Distribution of ploidy (case 2) No. of chromosomes Hypohaploidy Hyperhaploidy Hypodiploidy Diploidy Hyperdiploidy Hypotriploidy Triploidy Hypertriploidy Hypotetraploidy Tetraploidy Hypertetraploidy Total

,23 24–34 35–45 46 47–57 58–68 69 70–80 81–91 92 93–103

No. of cells 04 22 42 36 02 05 01 02 12 04 02 132

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Chromosomes and Intracranial Germ Cell Tumors Table 4 Detailed karyotypic findings of case 2 Karyotype 24,2X,2Y,11,11,11,13,14,26,27,28,110,116,217,222 34,X,2Y,11,14,15,26,18,111,112,113,116,117,119, 121,122 34,XY,11,16,18,19,114,116,117,118,120,122 35,XY,22,24,28,211,212,214,217,218,219,221,222 36,2X,2Y,24,18,29,210,211,218,219,219,220,220 38,X,2Y,21,22,28,212,217,218,221,222 41,XY,21,23,29,216,219 42,X,2Y,215,118,220,222,222 44,XY,24,28,29,117 44,XY,25,215,218,220 45,X,2Y,1del(1)(q11),212,217,118 45,XY,1X,22,26,28,29,214,13mar 46,XY 47,XY,121 65,XY,2X,1Y,21,21,24,24,25,26,29,211,113,115,119, 220,221 89,XXY,1X,2Y,11,24,29,210,212,216,216,118,119,221 90,XXYY,1X,1X,11,211,212,214,215,217,218,119,119,221

prognosis, since teratomas with this component have a high ability to generate metastases [22]. Monosomy of chromosome 18 in addition to del(18) (p11) was found by some authors [20, 23]. Al-Jehani et al. [18] found loss of heterozygosity in 18q. Monosomies of chromosomes 4, 12, 17, 20, and 22 were already described by other authors in neoplasias of the gonadal sites [16, 20]. The mature teratoma reported here presented deletion 1q11 that resulted in trisomy of the short arm of chromosome 1. This abnormality, when clonal, maybe an important step in the clonal growth of the tumor [24]. This

imbalance between short and long arm may be a significant factor in tumoral pathogeneis [10]. Isochromosome 12p is the most consistent abnormality found in germ cell tumors. It was described in testicular and mediastinal germ cell tumors [25], in dysgerminomas and ovarian yolk sac tumors [19, 26, 27], and in a dysgenetic gonadal tumor [28]. Of the five primary intracranial germ cell tumors reported in the literature, one presented i(12p) [7]. Our cases did not present this abnormality; perhaps 12p negative cases characterize a subtype of germ cell tumor. More studies are necessary to better characterize these tumors. REFERENCES 1. Dehner LP (1983): Gonadal and extragonadal germ cell neoplasia of childhood. Hum Pathol 14:493–511. 2. Ablin A, Isaacs H (1989): Germ cell tumors. In: Principles and Practice of Pediatric Oncology. PA Pizzo, DG Poplack, eds. JB Lippincott, Philadelphia, pp. 713–731. 3. Atkin NB, Baker MC (1983): i(12p): Specific chromosomal marker in seminoma and malignant teratoma of the testis? Cancer Genet Cytogenet 10:199–204. 4. Castedo SMMJ, de Jong B, Oosterhuis JW, Seruca R, Idenburg VJS, Dam A, Te Meerman G, Koopps HS, Sleijfer DT (1989): Chromosome changes in human primary testicular nonseminomatous germ cell tumors. Cancer Res 49:5696–5701. 5. Gibas Z, Prout GR, Pontes E, Sandberg AA (1986): Chromosome changes in germ cell tumors of the testis. Cancer Genet Cytogenet 19:245–252. 6. Albrecht S, Armstrong DL, Mahoney DH, Cheek WR, Cooley LD (1993): Cytogenetic demonstration of gene amplification in primary intracranial germ cell tumor. Genes Chromosom Cancer 6:61–63. 7. De Bruin TWA, Slater RM, Defferrari R, Geurts van Kessel A, Suijkerbuijk RF, Jansen G, de Jong B, Oosterhuis JW (1994): Isochromosome 12p-positive pineal germ cell tumor. Cancer Res 54:1542–1544.

Figure 4 45, XY,22,26,29,13mar (case 2: mature teratoma in the right lateral ventricle).

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