Cytogenetic Analysis of a Parachordoma

Cytogenetic Analysis of a Parachordoma Frédérique Tihy, Patrick Scott, Pierre Russo, Martin Champagne, Jean-Claude Tabet, and Nicole Lemieux

ABSTRACT: We report the cytogenetic and histopathological findings in a 7-year-old female child with an intranasal tumor that is most consistent with a parachordoma. Karyotypic analysis of the tumor revealed clonal numerical and structural chromosome abnormalities. Seven cells displayed recurrent changes: der(2)t(2;4), del(3q), and the loss of chromosomes 9, 10, 20, and 22. Four cells showed a loss of chromosome 17. To the best of our knowledge, these are the first clonal chromosome abnormalities described in parachordoma. © Elsevier Science Inc., 1998 INTRODUCTION Parachordoma is a rare tumor of the soft tissues, described by Laskowski in 1955 [1] as a chordoma periphericum and reviewed by Dabska in 1977 [2]. It grows very slowly and can develop after a trauma in the area. Because it is very infrequent, its histogenesis is still uncertain. Its light microscopic, ultrastructural features and immunostaining properties are similar to those of a chordoma. It is located adjacent to osseous structures but also close to tendons or synovia. Parachordoma is less aggressive compared with chordoma, can be totally removed in most cases, and never shows metastasis. The prognosis of parachordoma is therefore very good. On the other hand, chordoma is locally aggressive, and its location often makes it difficult to remove totally, so its recurrence rate is high [3]. Metastases also can develop. Here we report the clinical and cytogenetic findings of a parachordoma in a 7-year-old female child. To the best of our knowledge, only one cytogenetic study has been done on a parachordoma [4], and only a few classical forms of chordoma have been described cytogenetically [5–8]. In those cases, although 7 of 13 displayed recurrent chromosome abnormalities, the remaining 6 were normal or failed to reveal any cytogenetic information. We herein

From the Service de Génétique (F. T.), the Département de Pathologie et Centre de Recherche Pédiatrique (P. R., N. L.), the Département d’Hématologie (M. C.), and the Département d’Otorhino-Laryngologie (J.-C. T.), Hôpital Sainte-Justine, Montréal, Québec, Canada; and the Département de Pathologie, Faculté de Médecine, Université de Montréal (P. S., N. L.), Montréal, Québec, Canada. Address reprint requests to: Dr. Nicole Lemieux, Département de Pathologie, Faculté de Médecine, Université de Montréal, C.P. 6128, Succursale Centre-ville, Montréal (Québec) H3C 3J7, Canada. Received September 8, 1997; accepted December 4, 1997. Cancer Genet Cytogenet 105:14–19 (1998)  Elsevier Science Inc., 1998 655 Avenue of the Americas, New York, NY 10010

present cytogenetic results in a tumor and compare them with the findings of the previously described chordomas.

CASE HISTORY The patient is a 7-year-old female child first seen in September 1993 with left nasal obstruction and an associated edema of the left eye. Clinically, there was tumefaction of the nose with a bulging mass from the left nostril and ipsilateral proptosis. Ophthalmic examination was normal. Imaging showed a 7 3 3.8 3 3.5 cm3 mass destroying the left nasal space, with invasion of the left maxillary sinus and orbit with extension into the superior nasopharynx. There was no intracerebral extension. Biopsy specimens were submitted for electron microscopy, flow cytometry (ploidy), and cytogenetics. The patient received one cycle of vincristine, actinomycin, and cyclophosphamide followed by complete surgical resection and radiotherapy (6,000 cGy). After 3.5 years of follow-up, she remains free of recurrent disease.

MATERIALS AND METHODS The specimens for studies were obtained at surgery. Sections for light microscopy were fixed in 10% formalin, postfixed in Bouin’s solution, embedded in paraffin, processed, and stained for routine histology by using hematoxylin phloxine saffran (HPS) as well as periodic acid-schiff reaction (PAS) and alcian blue stains. For immunocytochemistry, the following antibodies were used on deparaffinized slides with an avidin-biotin peroxidase technique at the indicated dilutions: cytokeratin (MNF116) (1/200, Dako), epithelial membrane antigen (1/100, Dako), desmin (1/20, Dako), vimentin (1/20, Dako), neuron-specific enolase (1/500, Dako), chromogranin A (1/20, Enzo Diagnostics), carcinoembryonic antigen (1/200, Dako), protein S-100 (1/400, Dako), and synaptophysin (1/20, Boehringer Mannheim).

0165-4608/98/$19.00 PII S0165-4608(97)00481-0

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Cytogenetics of a Parachordoma

Figure 1 Most of the tumor was composed of ribbons and trabeculae of cells with an abundant clear cytoplasm in a loose, hypocellular myxoid matrix (HPS, 3800).

Tissue submitted for electron microscopy was fixed in 2.5% glutaraldehyde, postfixed in 1% osmium tetroxide (OsO4), dehydrated, and embedded in Epon 812. Ultrathin sections were stained with uranyl acetate and lead citrate and examined with a Philips 812 electron microscope. The tumor sample submitted for cytogenetic analysis was carefully hand cut into a cell suspension and processed as reported in [9]. A suspension (1 mL) of small clumps of cells and of single cells was seeded into culture flasks (Falcon, 25 cm) covered with type I collagen (Nitrogen 100 TM). The cells were cultured in DMEM medium supplemented with 10% of fetal bovine serum. Cultures were incubated for 4–7 days at 37oC in a constant atmosphere of 5% CO2. Subcultures were done and 106 cells were seeded into new bottles. Chromosome analysis was performed on parachordoma tumor cells after primary cultures and subcultures without synchronization. The criteria used to define a clone are based on guidelines reported by Knutsen et al. [10]. GTGbanding [11] was made on metaphase chromosomes, and karyotypes were prepared according to the International System for Human Cytogenetic Nomenclature [12]. RESULTS By light microscopy, the tumor presented two patterns. The most frequent consisted of cohesive nests or ribbons of cells with an abundant clear cytoplasm and a high nu-

clear–cytoplasmic ratio set in a loose myxoid background (Fig. 1). The second pattern consisted of chondroid areas, comprising 15–20% of the tumor area, composed of clear cells set in a cartilagenous matrix that stained prominently with alcian blue (Fig. 2). Nuclear atypia was mild to moderate, and mitoses were rare. Positive immunohistochemical staining was observed in 25% to 50% of the tumor cells; all the reactive cells exhibited positivity for cytokeratin, epithelial membrane antigen, vimentin, protein S-100, and neuron-specific enolase. Staining with synaptophysin, chromogranin, desmin, and carcinoembryonic antigen was negative. By electron microscopy, tumor cells formed small acini with a central lumen (Fig. 3). The cell cytoplasm was surrounded by interrupted basal lamina, and numerous welldeveloped intercellular junctions with occasional desmosome-like structures were noted (Fig. 4). The cytoplasm contained large pools of glycogen; there was abundant dilated endoplasmic reticulum to which mitochondria were intimately apposed. Microvili were noted at the apical surface of some of the cells. Twenty-six GTG-banded metaphases were analyzed (Table 1) and four karyotypes were done. Twelve cells were karyotypically normal, and three others were tetraploid with no other chromosome anomaly. In the remaining cells, two abnormal clones were detected, one clone consisted of seven cells with two structural rearrangements involving chromosomes 2 and 4, chromosome 3,

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Figure 2 Dense cluster of tumor cells with focal area of cartilaginous differentiation (HPS, 3500). Figure 3 Low-power electron micrograph revealing a cohesive nest of tumor cells surrounded by a basal lamina (uranyl-lead, 310,000).

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Figure 4 The tumor cells contain abundant glycogen; they are surrounded by a well-formed basement membrane and connected by junctional complexes (uranyl-lead, 321,000)

and the loss of chromosomes 9, 10, 20, and 22 (Fig. 5). One of the seven cells was polyploid with the same abnormalities, and another showed the structural rearrangement involving chromosomes 2 and 4 and the loss of chromosomes 4, 9, 10, 17, and 20. The other clone consisted of four cells with the numerical loss of chromosome 17. DISCUSSION Parachordoma is a benign tumor of uncertain histogenesis. It might derive from an ectopic notochord [13], like chordomas or Schwann or other neuron-related cells [14]. The immunohistology is often the same as that of a chordoma, but some variations can be observed, depending on the tumor. Our findings correspond to previously described cases with a positive reaction for antibodies to S-100, neu-

ron-specific enolase, vimentin, epithelial membrane antigen, and cytokeratin [13, 14], whereas other published cases were negative for epithelial membrane antigen and keratin [15]. Carcinoembryonic antigen was negative in the present tumor and was positive in some other cases [14]. It was difficult to compare our cytogenetic findings with others because only one other study has been reported in the literature [4]. That study revealed mostly normal chromosomes, but there was often deviation from the diploid state, the aneuploid metaphases resulting from chromosomal gains. Structural chromosomal aberrations, other than the ones that we describe, were also observed, but they were not clonal. Even the cytogenetic data on chordomas are few. Only 7 cases of 13 displayed recurrent chromosome abnormalities [5–8].

Table 1 Chromosome findings in cultured parachordoma cells No. of mitoses 12 3 4 5 1 1

Karyotype 46,XX 92,XXXX 41z45,XX,217 42z43,XX,t(2;4)(p25;q25),del(3)(q13.1),29,210,220,222 41,XX,t(2;4)(p25;q25),24,29,210,217,220 85,XXXX,t(2;4)(p25;q25)32,del(3)(q13.1)32,29,210,210,220,220,222,222

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Figure 5 Representative GTG-banded karyotype of one parachordoma cell clone: 42,XX,t(2;4)(p25;q25), del(3)(q13.1),29,210,220,222.

Mertens et al. [8] reviewed the cytogenetic literature of chordomas and showed that only numerical changes were recurrent. These changes involve chromosomes 3 and 4 and, to a lesser extent, chromosomes 10 and 13. In the case reported here, the cytogenetic study revealed that the tumor cells displayed two clonal chromosome aberrations (Table 1). The bands involved in the translocation, (2;4)(p25;q25), observed here are neither involved in the known cases of chordomas nor are frequently rearranged in other neoplasms. The deletion del(3)(q13.1) can be related to the loss of the whole chromosome 3 in some chordomas and to the loss of chromosomes 4 and 10 in some of our cells. We did not observe the involvement of chromosome 13, as is the case for chordomas. The monosomies for chromosomes 9, 10, 20, and 22 do not seem to be characteristic chromosomal abnormalities of chordomas, be-

cause only chromosomes 20 and 22 are seen, once each, in the seven cases with deviations studied thus far. The presence of a clone with the loss of chromosome 17 as the sole abnormality is intriguing, but its significance is unknown. Interestingly, monosomy for chromosome 17 was seen (one cell) in the other clone. Chromosomal studies have added, in a variety of ways, to our understanding of tumor biology, and, in some cases, the cytogenetic detection of a specific alteration has proved useful in the diagnosis and even the prognosis of a tumor. The translocations can be particularly interesting, as in the example of chronic myelogenous leukemia with the Philadelphia chromosome [16]. Our results are neither similar nor totally divergent from the chordoma findings. The number of cases studied is still too small to furnish conclusive information. More cytogenetic analyses of

Cytogenetics of a Parachordoma parachordomas and chordomas should provide information useful in ascertaining precise genetic aberrations of these uncommon neoplasms and facilitate their diagnosis. The authors thank E.L. Barnes for slide review. The authors also acknowledge the skilled technical help of Diane Lachance and the excellent photographic work of Jean Léveillé.

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19 7. DeBoer JM, Neff JR, Bridge JA (1992): Cytogenetics of sacral chordoma. Cancer Genet Cytogenet 64:95–96. 8. Mertens F, Kreichbergs A, Rydholm A, Willen H, Carlen B, Mitelman F, Mandami N (1994): Clonal chromosome aberrations in three sacral chordomas. Cancer Genet Cytogenet 73:147–151. 9. Lemieux N, Leung TK, Michaud J, Milot J, Richer CL (1990): Neuronal and photoreceptor differentiation of retinoblastoma in culture. Ophthalmic Paediatr Genet 11:109–120. 10. Knutsen T, Bixenman HA, Lawce H, Martin PK (1991): Chromosome analysis guidelines preliminary report. Cancer Genet Cytogenet 52:11–17. 11. Seabright M (1971): A rapid banding technique for human chromosomes. Lancet 2:971–972. 12. ISCN (1995): An International System for Human Cytogenetic Nomenclature. F Mitelman, ed. S. Karger, Basel. 13. Shin HJC, Mackay B, Ichinose H, Ayala AG, Romsdahl MM (1994): Parachordoma. Ultrastruct Pathol 18:249–256. 14. Hirokawa M, Manabe T, Sugihara K (1994): Parachordoma of the buttock: an immunohistochemical case study and review. Jpn J Clin Oncol 24:336–339. 15. Wiebe BM, Jensen K, Laursen H (1995): Parachordoma of the sacrococcygeal region: a neuroepithelial tumor. Clin Neuropathol 14:343–346. 16. Nowell PC, Hungerford DA (1960): A minute chromosome in human chronic granulocytic leukemia. Science 132:1497.