Binucleated Cells in a Human Renal Cell Carcinoma with 34 Chromosomes

Gyula Kovacs, Bisharah Soudah, and Eberhard Hoene

ABSTRACT: Cytogenetic analysis of a human renal cell carcinoma revealed a near-hclploid chromo- some number of 34 with the loss of one chromosome #1, #2, #3, #6, #7, #9. #10, #12, #13, #17, #18, and #21. Binucleated cells were observed in histologic, cytologic, as well as in cytogenetic preparations. The paper briefly discusses the binacleation-polyploidization as a possible compensatory mechanism to maintain the genetic balance in .ear-haploid ceils.

INTRODUCTION

Most h u m a n ma l ignancy cases desc r ibed in the l i tera ture wi th a c h r o m o s o m e num- ber of 34 of fewer c h r o m o s o m e s in the s t eml ine karyotype are acute l y m p h o i d leu- kemias or blast ic crises of ch ron ic m y e l o i d l eukemias (for r ev i ew see Kristoffersson et al. [11). This c h r o m o s o m e pat tern is ex t r eme ly rare in sol id h u m a n tumors . A nea r -hap lo id c b r o m o s o m e n u m b e r of 29 -30 was found in a m e l a n o m a ~21 and in another case of metas ta t ic m e l a n o m a charac te r i sed by a moda l c h r o m o s o m e n u m b e r of 24 131. Visfeldt et al, [4] descr ibed a metas ta t ic m e l a n o m a g rowing in nude mice wi th 32 ch romosomes . Recent ly , Bigner et al. [5] have pub l i shed a t ransp lan tab le h u m a n g l iob las toma l ine marked wi th a nea r -hap lo id s temline .

More than 70 cases of renal cell ca rc inomas (RCCs) ana lyzed in our laboratory usua l ly s h o w e d nea r -d ip lo id c h r o m o s o m a l modes , wi th the lowes t moda l n u m b e r being 41. In one case we observed a moda l c h r o l n o s o m e n u m b e r of 34, i.e., w i th in the hap lo id range. This repor t p resents the cy togene t ic as wel l as the h is topatho- logic and cyto[ogic f indings of the case in ques t ion .

CASE REPORT

A 63~year-old male pa t ient d e v e l o p e d spondy lod i sc i t i s fo l lowing a neurosurg ica l opera t ion in Augus t 1985. Dur ing rehabi l i t a t ion t reatment , a sonography examina- t ion brought to light a mass 5 x 6 cln in size at the u p p e r pole of the left k idney. C o m p u t e d t omography conf i rmed this f inding. There was no sc in t ig raphic sign of bone metastasis , nor did the ches t x-ray give ind ica t ion of p u l m o n a r y metastasis . In February 1986, t r ansper i tonea l t umor n e p h r e c t o m y was carr ied out, wi th adrene lec- tomy and part ial l y m p h a d e n e c t o m y . S ince then, there has been no sign of tunlor progress ion in the course of fo l low-up .

From the Laboratory of Cytogenetics, lnstilute of Pathology, Hannover Medk:al School. l"cderal P, etmtllit: of Germany.

Address requests for reprints to: Dr. G. Karats. Laboratory of C~toge~,etics. lnstitate of Puthology, Hannover Medical School, Ko1~stanty-Gatschow-Str. 8. D-3000 ffalmover 61, F .R .G.

Received Jtlly 14, 1987: accepted September 23. 1987.

211

~9 1988Elsevier Science Publishing Co., Inc. Cancer(;enet Cytogenet 31:211 215 (1988] 52 Vanderbilt Ave., New York, NY 10017 0165-4608/88/$03.50

212 G. Kovacs, B. Soudah, and E. Hoene

Sectioning of the kidney specimen revealed a tumor 5.3 cm in diameter in the upper pole with central necrosis and hemorrhage. Histologic examination showed a RCC with compact growth pattern of relatively large chromophobe cells (Fig. 1). Numerous tumor cells had double nuclei. Cytologic imprint preparations of the fresh tumor tisssue showed a high frequency of binucleated cells in comparison with that found in other RCCs. Two to eight binucleated cells were found per 100 cells analyzed in ten other RCCs, while 25% of the cells of the presented case con- tained two nuclei (p < 0.01) (Fig. 2). Binucleated cells were observed in the short- term culture of the tumor, and a remarkable 50% of the cells in the chromosome preparations showed naked double nuclei after hypotonic treatment (Fig. 2, insert).

CYTOGENETIC STUDIES

Short-term culture and chronlosome preparations using G- and C-banding tech- niques were carried out as described previously [6]. Briefly, small tumor fragments were incubated in 0.1% collagenase (Worthington, CLS III) dissolved in culture me- dium for 60 minutes at 37°C. Then the tissue fragments were washed twice in me- diuln, dispersed vigorously with a Pasteur pipette, and the ensuing small cell clus- ters were seeded in culture. Chromosome analysis was done in the fifth day of culture using GTG and CBG techniques.

For 65 evaluated metaphases, this tumor showed a well-defined mode character- ized by 34 chromosomes counted in 32 cells. Six cells were marked with 33 chro- mosomes, five cells with 32, and one cell with 30 chromosomes. Furthermore, 11 cells showed a chromosome number at 68, 3 cells at 67, 5 cells at 66, and 2 cells at

Figure 1 Microphotography of the tumor, showing numerous binucleated cells (H&E stain, x 160). Insert: high-power magnification of the same tumor areas with hinucleated cells, in- cluding a polyploid cell [asterisk}.

RCC with 34 Chromosomes 213

Figure 2 Binucleated cells occurring in the cytologic specimen as well as in chromosome preparation (insert].

63. Chromosome analysis of 20 G-banded metaphases revealed a near-haploid stem- line karyotype in 15 cells: 3 4 , X Y , - 1 , - 2 , 3 , - 6 , - 7 , 9 , - 1 0 , - 1 2 , 1 3 , - 1 7 , - 1 8 , - 21 (Fig 3). In five addit ional cells random losses of chromosomes was seen. Anal- ysis of seven metaphases using C banding revealed only one chromosome #1, #9, and Y, and two copies of chromosome #16. Among seven G-banded metaphases with higher chromosome number, a double set (68 chromosomes) of stemline chro- mosomes were found in four cells, while the other three cells showed random chro-

mosomal losses. It was a highly remarkable characteristic of this tumor that most metaphases

showed one or more telomeric associations (end-to-end fusion of the entire chro- mosomes). Chromosomes #19, #21p, #20q, and #4p were more frequently in- volved in this phenomenon than other chromosome ends, namely 25, 15, 14, and 10 times, respectively.

C O M M E N T S

Near-haploid clones occurring in human malignancies are extremely rare. Cells with such a low chromosome number seem to have difficulty retaining their growth capacity, but in some cases tumor cells with near-haploid chromosome number were observed, with clonal expansion in vivo (2) as well as in vitro [7]. For the development of severe hypodiploidy or near-haploidy in human tumors, several mechanisms were proposed [8]. The most likely mechanism leading to a chromo- some number lower than 46 is a mult ipolar mitosis of a polyploid cell. An extreme nondis junct ion leading to the loss of several chromosomes from one of the daughter cells is also in the realm of possibility. In the present case, although we cannot

214 c. Kovacs, B. Soudah, and E. Hoene

2 3

6 7 8

4 5

9 10 11 12

a 13

ii

14 15 16 17

19 2 0 21 22 Figure 3 A representative G-banded karyotype of th~ tunlol'.

i 18

XY

prove any of the aforesaid mechanisms responsible for the development of the low chromosome number, it seems to be more likely that the occurrence of telomeric chromosome association led to the near-haploid chromosome number. Telomeric association results in dicentric chromosomes, and such chromosomes are generally unstable at mitosis, rFherefore, telomeric association of the chromosomes may lead to the el iminat ion of the involved chromosomes from the karyotype in the subse- quent cell cycles. This phenomenon is not extremely rare and might perhaps serve for the el iminat ion and also retention of chromosomes, leading to the abnormal tumor karyotype 19-121.

In the case presented here, more than 30% of the metaphases showecl a dupli- cation of the near-haploid stemline karvotype. Although the near-haploid cells showed no nul l isomy in any of the somatic chromosomes, this tendency of dupli- cation of the chromosome set may be regarded a compensatory mechanism to main- tain the genetic balance in tumor cells. The opinion now prevalent is that the same number of gene copies may be achieved by various polyploidization mechanisms. The advantage of polyploidy for the cells is that less time and energy is required for protein and nucleic acid synthesis, and gene transcription may continue unin- terrupted by mitosis and cell division [13].

It is a well-established fact that in many mammalian cell types the first step to higher ploidy levels is that the cells become binucleate [14]. Investigations in vitro have directly demonstrated that a binucleate cell is able to form an uninucleate cell via nuclear fusion [15], thus polyploidization may ohviously be a consequence of

RCC w i t h 34 C h r o m o s o m e s 2 ! 5

b i n u c l e a t i o n . W h e n t he n u c l e i d i v i d e s y n c h r o n o u s l y , the m e t a p h a s e or a n a p h a s e

g roups m a y fuse, l e a d i n g to a p o l y p l o i d cel l [141. In the p r e s e n t case, on the bas i s of cy togene t i c , h i s to log ic , a n d cyto logic f ind ings ,

a f u r t h e r m e c h a n i s m for the c lona l e x p a n s i o n of t u m o r cel ls m i g h t be exp l i ca t ed . N e a r - h a p l o i d cel ls m a y u n d e r g o a n o r m a l mi tos i s , l e ad ing to d a u g h t e r cel ls w i th a set of 34 c h r o m o s o m e s . On the o the r h a n d , some t u m o r cel ls are i n v o l v e d in acy- t ok ine t i c mi tos i s , l e ad i ng to a n u m b e r of b i n u c l e a t e d cells. If the nuc le i of such cel ls fuse, the e n s u i n g cells , w i t h d o u b l e sets of c h r o m o s o m e s , m i g h t u n d e r g o a n o r m a l mi tos i s , r e s u l t i n g in m e t a p h a s e s w i t h 68 c h r o m o s o m e s .

The h igh n u m b e r of b i n u c l e a t e d ce l l s f o u n d in h i s to log ic and cyto logic p repa - r a t i ons in the p r e s e n t case m i g h t t he re fo re be i n t e r p r e t e d as a t r a n s i t i o n a l s tate to po lyp lo idy , a p p r o a c h i n g d i s o m y of c h r o m o s o m e s ca r ry ing genes n e c e s s a r y for the

c lona l e x p a n s i o n of the t u m o r cells.

Supported b?,, a grant from the Deutsche Forschungsgemeinschaft (DFG Ko 841/3 I J.

REFERENCES

1. Kristoffersson U, Olsson H, Kelly D, Akerman M, Mitelman F (1986): Near-haploidy in a case of plasmocytoma. Cancer Genet Cytogenet 19:239 243.

2. Berger R (1968): Sur la rnethodologie de l 'analyse des chromosomes des tumeurs. Thesis, Faculte des Sciences, Paris.

3. Atkin NB, Baker MC (1981): A metastatic malignant melanoma with 24 chromosomes. Hum Genet 58:217 219.

4. Visfeldt 1, Povlsen CO, Rygaard J {1972): Chromosome analyses of human tumours follow- ing heterotransplantation to the mouse mutant nude. Acta path mi(:robiol Scand Section A 80:169-176.

5. Bigner Sit, Mark J, Schold SC, Eng LF, Bigner DD (1985): A serially transplantable human giant (:ell glioblastoma that maintains a near-haploid stem line. Cancer Gene! Cytogenet 18:141 154.

6. Kovacs G, Szfics S, DeRiese W. Baumg/:irtel W (1987): Specific chromosome aberration in buman renal ceil carcinoma, lnt I Cancer 40:171 178,

7. Kohne St, Minowada J, Sandberg AA {1980): Chromosome evolution of near-haploid clones in an established human acute lymphoblastic leukemia cell line (NALM-16). l Nall Cancer Inst 64:485-493.

8. Oshimura M, Freeman At, Sandberg AA (1977): Chromosomes and causation iif human cancer and leukemia. XXllI. Near-haploidyin acute leukemia. Cancer 4(/:1143 1148.

9. Fitzgerald Pit, Morris CM (1984): Telomeric association of chromosomes in B-cell lym- phoid leukenlia. Hum Genet 67:385--390.

10. Mandahl N, Helm S, Kristoffersson 1,1, Mitelman F, R66ser B. Rydholm A, Willen tt (19851: Telomeric association in a malignant fibrous hisiiocytoma. Hum Genet 71:;121 ;124.

11. Morgan R, Jarzabek V. Jaffe JP, Hecht BK, Hecbt F, Sandberg AA (1986): Telomeric fusion in pre-T-cell acute lymphoblastic leukemia. Hun1 Genet 73:260 263.

12. Kovacs G, Mfiller-Brechlin R, Sz{ics S (1987): Telomeric association in two human renal tumors. Cancer Genet Cytogenet 28:363 366.

13. Nagl W (1978): Endoploidy and polyteny in differentiation and evolution. Elsevier North Holland, New York, pp. 283.

14. llgren EB (1981): The initiation and control of trophoblastic growth in the, mouse: binu- cleation and polyploidy. Placenta 2:317 332.

15. Roberts DC, Cole G (1964): Some mechanisms of formation of polyploid and heleroph)id cells in a murine ascites tumor in vitro. J Natl Cancer Inst 32:1023 10:30.