Primitive Neuroectodermal Tumor Cell Lines: Chromosomal Analysis of Five Cases

Venkateswara R. Potluri, Fred Gilbert, Christiane Helsen, and Lawrence Helson

ABSTRACT: A cytogenetic analysis of primitive neuroectodermal tumor (PNET) cell lines was under- taken. PNET are presumed to be embryologically related to. but clinically and histologically distinct from, other tumors of neuroectodermal origin, including neuroblastoma and retino- blastoma. No single chromosome abnormality was found in all five of the tumors studied. In three of the five cases, however, additional lq material [either as extra chromosome #1 or i(lq)] was found in all cells, and in two of the five, monosomy 13 was noted in all cells; the possible significance of these findings is discussed.

INTRODUCTION

Embryonic neuroectoderm gives rise to a number of cell types in the central and peripheral nervous systems, as well as to certain non-neural tissues (including pig- ment epithelium and portions of the cranium) in the intact organism [1]. The cel- lular derivatives of neuroectoderm may become transformed and among the more common of such tumors arising in children are neuroblastoma and retinoblastoma [2]. A much rarer entity, also presumed to originate from nouroectoderm, is the so- called primitive neuroectodermal tumor (PNET) [3].

PNET can be distinguished from neuroblastoma on clinical and histologic grounds [3, 4]. The latter usually arise within the adrenal medulla or in the auto- nomic ganglia (of the abdominal, mediastinal, or cervical regions), whereas, the former have been reported in peripheral sites (e.g., dorsum of the foot, knee, thigh, testis, ovary, kidney) without evident involvement of the adrenals or autonomic ganglia. In neuroblastoma, the tumor will generally contain ganglion cells in vary- ing stages of maturation (some of which may include neurosecretory granules) and neurofibrils; PNET, on the other hand, have been found to contain primitive neural tubular structures, sheets of small, darkly staining cells with limited cytoplasm (in which neurosecretory granules may be scant or absent), and neuroglial elements and rare ganglion cells, all set in a prominent mesenchymal stroma. In the PNET reported to date, the average age at diagnosis was approximately 7 years (although cases have been reported from birth to 24 years); in neuroblastoma, the median age at diagnosis is less than 2 years (with greater than 70% of cases diagnosed before age 5) [5, 6].

From the Division of Medical Genetics, Mount Sinai School of Medicine (V. R. P., F. G.}; and Memorial- Sloan Kettering Cancer Center (C. H., L. H.), New York, NY.

Address requests for reprints to Dr. Fred Gilbert, Medical Genetics/Annenberg 17-76, Mount Sinai School of Medicine, Fifth Avenue and 100th Street, New York, NY 10029.

Received July 8, 1985; accepted January 6, 1986.

75

© 1987 Elsevier Science Publishing Co., Inc. Cancer Genet Cytogenet 24:75-86(1987) 52 Vanderbilt Ave., New York, NY 10017 0165-4608/87/$03.50

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In a recent review of karyotypes from neuroblastoma, a particular chromosome abnormality [rearrangements resulting in the loss of material from the short arm of chromosome #1 (lp)] was identified in some 70% of the collected cases [7]. In reports of karyotypes from a more primitive tumor of neuroectodermal origin, the so-called peripheral neuroepithelioma,~a particular marker rearrangement, a recip- rocal translocation between chromosomes #11 and #22, has been noted [8]. We now present a cytogenetic analysis of an additional five examples of primitive neu- roectodermal tumors.

MATERIALS AND METHODS

Tumor samples were transported from the operating room in sterile saline or me- dium (RPM1 1640), minced with scissors and scalpel, and allowed to grow in RPM1 1640 medium containing 20% fetal calf serum.

SK-PN-LI is a permanent cell line derived from a tumor in the area of the right scapula, removed September 1979. SK-PN-DW, also a permanent cell line, was es-

8 0 V.R. Potluri et el.

tablished from a presacral mass, excised January 1978. SK-PN-AG is a permanent line established from a chest wall mass, removed in September 1983. SK-PN-WA, a fourth permanent cell line, was established from a thoracic wall tumor, excised in August 1979. Patient BN was initially operated in 1980, at which time a mass ad- jacent to the left ovary, pathologically identified as a PNET, was removed; at reop- eration in 1985, a recurrence at the primary site (designated SK-PN-BW) was re- moved.

Chromosome preparations were made from recent reconstitutions of the perma- nent cell lines from cases LI, DW, AG, and WA, frozen within months of their establishment (the exception for case DW is described in ~SULTS), and from over- night cultures of the recurrent tumor from case BN. The methods of preparation were as previously described [9, 10]. The chromosomes were banded using a mod- ified Seabright technique [11] and karyotyped [12].

RESULTS

No single chromosomal abnormality was found in every cell in all five of the PNET analyzed. As illustrated in Figures 1-5 and Table 1, three of the five tumors con- tained additional lq material [as extra chromosomes #1, extra del(lp) markers, or iso(lq)], and two of the five contained monosomy 13. Each of these abnormalities found in greater than 20% of cells in individual tumors included trisomy 17, iso(17q), extra chromosomes #3, #8, and #14, missing #5, #9, #10, #11, and #18, and structural abnormalities of chromosomes #5, #6, #7, and #12.

In one case (SK-PN-DW), karyotypes were prepared from a reconstituted frozen ampule of the first passage of the tumor (Fig. 2A) and from the permanent line established from the tumor (Fig. 2B). Individual cells of the original tumor had chromosome numbers between 47 and 96, with a mode of 74. There were two cop- ies of chromosome #13 in each hyperdiploid cell, two fewer than the number ex- pected for a tetraploid cell. No consistent deletions or translocations could be iden- tified. Approximately 0-5 double minutes (DM) were noted per cell. In the permanent cell line, after at least 2 years of continuous culture, the chromosome number per cell was reduced to between 37 and 43 per cell, with a mode of 40 to 41. Monosomy 13 was found in 100% of cells; iso(17q) and monosomy 9, 10, 11, and 18 were found in greater than 20% of cells analyzed. In a large proportion of the cells of the permanent line, the DM per cell were too numerous to count (Fig. 2C).

DISCUSSION

Our analysis of karyotypes from primary tumor cells and tumor cell lines from five PNET cases, suggests that one may be able to distinguish between PNET and the embryologically-related neuroblastoma on cytogenetic grounds. None of the five PNET studied contained the marker chromosome abnormality identified in more than 66% of neuroblastomas, namely, deletions of lp (in the absence of trisomy lq) [7].

Cytogenetic analysis also makes it possible to separate the more primitive tumors of neuroectodermal origin that develop in peripheral sites, into two groups: those with a balanced t(11;22) for which we suggest the term "peripheral neuroepithe- lioma" should be reserved [8], and the remainder, with different karyotypic changes (the significance of which is discussed below), which we would designate as PNET. (Whether or not this classification will have additional clinical relevance--e.g., in terms of prognosis, patterns of drug responsiveness, etc.--will become clear as both patient groups are followed over time.)

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Table 1 Chromosome abnormal i t ies in pr imi t ive neuroectodermal tumors

Chromosome abnormalities

PNET Age/Sex Rx d Mode (Range) A (100% cells) B (>20% cells)

SK-PN-LI 3/M CT 46 (45-47) + 1, - 13 SK-PN-DW a 17/M CT 74 (47-96) - 13

SK-PN-DW b 46, 41 (37-43) - 1 3

SK-PN-AG 13/F CT 48 (8 cells) + 8, - 10 65 {1 cell)

SK-PN-WA 19/M No 47 + iso(lq) SK-PN-BN 24C/F CT/RT 47 + lq

+ 5, + 8, i(5q) -9 , -10 , -11, -17,

- 19, DMs - 9, - 10, - 11, i(17q),

-18, DM

t(7;6), 12q+, +14, + marker

aOriginal tumor. bPermanent cell line. COriginal tumor discovered at age 19; recurrence, reoperation at age 24. ~Treatment: CT, chemotherapy; RT, radiotherapy.

Two of the chromosome abnormal i t ies ident if ied in the PNET, extra l q mater ial and DM, however, have been repor ted in neuroblastomas, as well as in many other cancers in humans [7, 13-15]. The lack of specif ici ty of these anomalies for single tumor types suggests that such changes are not required for tumorigenesis . Instead, they are l ikely to p lay a secondary role in tumor development , p resumably by con- ferring a selective growth advantage on the cells in which they are carried [16].

Consistent With this hypothes is was a previous report of neuroblas toma cell karyotypes obtained ser ial ly from the same pat ient [17]. When compared with the original pr imary tumor, karyotypes from mul t ip le metastases sampled 11 months later demonst ra ted a reduct ion in modal chromosome number per cell and t r isomy for a lq marker chromosome.

Case SK-PN-DW is l ikely to represent an in vitro example of the same phenom- enon: When compared with the ini t ial tumor sample, the cont inuous ly growing cell l ine had a sharper and lower modal chromosome number per cell and also con- ta ined more DM per cell.

In all instances s tud ied to date, DM have been shown to represent sites of gene amplif icat ion, containing mul t ip le copies of one or a small number of genes [15]. In most (if not all) pa thologica l ly confirmed neuroblas tomas in which gene amplif ica- t ion has been demonstra ted, a par t icular oncogene, N-myc (mapped to 2p23-24 [18]) has been inc luded in the amplif icat ion unit [19, 20]. In SK-PN-DW, however, an- other member of the myc gene family (c-myc (mapped to 8q24) [21]) is present in mul t ip le copies (H. Rovigatti, personal communicat ion) .

The finding of monosomy 13 in two of the five PNET cases is interest ing in light of the associat ion between abnormal i t ies of chromosome #13 and another neuroec- todermal tumor, ret inoblastoma. Ret inoblastoma has been repor ted in certain pa- t ients who carry dele t ions of 13q14 in all somatic t issues [221. A number of labo- ratories also have repor ted the loss of 13q mater ial (either as delet ions of 13q or monosomy 13) in karyotypes from ret inoblas toma cells from ind iv idua ls whose const i tu t ional karyotypes were normal [23-25]. The actual fraction of ret inoblasto- mas containing monosomy 13 or del(13q) is low: fewer than 20% of all publ i shed cases. However, the combinat ion of cytogenetic data and studies of restr ict ion frag-

Chromosomal Analysis of PNET 8 5

ment length polymorphisms of DNA sequences mapped along 13q has led to the conclusion that the homozygous loss of genetic formation from 13q14 is necessary for tumorigenesis in all cases of retinoblastoma [26].

Monosomy 13 and structural abnormalit ies of chromosome 13q also have been identified in isolated examples of many tumor types [14, 27]. This finding in PNET, is intriguing, however, because it raises the possibility that this segment contains one or more genes that play a role in the differentiation of more than one neuroec- todermal derivative: genes whose loss may affect the proliferative capacity, and contribute to the transformation, of cells other than retinoblasts.

Supported by USPHS Grant CA 36122 (FG).

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