Cytogenetic Investigations on a Cell Line Derived from a Carcinoma Arising in a Salivary Gland Pleomorphic Adenoma Jorn Bullerdiek, Karl-Josef Hutter, Giinther Brandt, Margot Weinberg, Gazanfer Beige, and Sabine Bartnitzke

ABSTRACT: This article discusses the results of cytogenetic im (~stigzztiuus of a c¢~ll line derived h'om u mulignanI t u m o r arising in a benign salivm'v glzmd pleomurphic (ulenom(L Initial km'yutypic studies bez:ame possible with (:ells of the second in xitro passage und rm'e(ded the existence ¢lt hy'pertetraploidy. Furthermore, (~ marked p(zlysomy 7 and (I tr(mslo(:ution involvh~g 12(t 13-15, (z breukpz)int a/szz j'requently seen in benign ple(m~orphi(: (nlenomos, were n(~t~wurthy', l)urin~ long term (:ulture, the number o l c h r o m o s o m e s per (:ell (leerease(I (zs well (is th(~ mun/)l!r of

copies o lchrzzmosume 7. Cell tumorigenicilS" was przzved by hcterotr(msphnltz*tion t(z nude mi(:c. The resulting t u m o r s were kzH'5"(zts"pe(t ugain. N() signi(ic(mt (:h(mges (zf'the chrom(zsome n u m b e r ()r of the degree of polo'sorer 7 were lzztmd (:()mlmred to the (:ells be(ore heterutrans;dmduti(m. In (:ontr(lst, ttle (:ells from the nude mh:e tumurs showed (x rem(irkat)le mmlber of dit(erent isl)z:hrom(zsomes przzbably hldit:ating an u n k n o w n ~(l(:tor supporting the generation o~ is(zchromz)sumes. The consistent/)resen(:e of the t(t2:?)lql 3 15:?) makes the (:ell line well suited for m o l e c u l a r studies of this breakpoint region,

INTRODUCTION For analysis of cytogenetic changes accompanying or even causing malignant transformation of human tumors, malignant tumors originating from benign stages are of significant interest. Examples of tumors belonging to this category are carcinnmas developing in salivary gland pleomorphic adenomas, in contrast to primary malignant [1] salivary gland tumors, to the best of our knowledge, no results of cytogenetic investigations on these tumors offering insight into the chromosonlal mechanisins of their nlalignant transformation have been published so far. Here we present cytogenetic and flow-karyotypic data on a carcinoma ex pleomorphic adenoma with special reference to a comparison of the karyotype before and after heterotransplantation to nude mice. The relationship between the chromosomal aberrations observed and cytogenetic changes in benign pleomorphic adenomas [2-5] will be discussed as well as the significance of karyotypic heterogeneity for tumor progression and metastazation.

From the Centre of Ituman Gcneli¢:s, thlivcrsity of Brenmn. Brenmn, F.R.G. (]. B., M. W., G. B., S. B.I, German Cancer Research Center, th~idHberg, F.R.G. {K.-]. lid, and Institute for Pathcdogy, Zentralkrankenhaus St,-lurgen-Str.. Bremen, F.R.(;. (G. ILl.

Address reprint requests to: Dr. 16rn Bullerdiek, (?entre u) H u m ( m Genetics and Geneti(: C o m l s e l l i n g , University of Bremen, Leobener Str. NW2, 1)-2800 Bremen 33, West Germany. Received Mareh 27, 1989; accepted July 28. t989.

253 :c, 1990 Elsevier Science l~ublishing Co., inc. 655 Avenue of the Americas, New York. NY 191110

Cancer (;(,'net Cyh~gcnel 44:253 262 (1990) 0165-4608/90/$03.50

254

J. Bullerdiek et al.

Figure 1 Carcinoma in a pleomorphi(: adenoma and remnants of the, pleomorphi(: adenolna (hyaline-fibrous stroma). H&E × 70.

CASE REPORT A 77-year-old male came to the hospital for surgery of a parotid gland tumor. He had first noticed the rapidly growing tumor 5 months before. The tumor, which measured 21 x 18 x 30 mm was removed. Histological examination of the partly encapsulated tumor revealed a poorly differentiated carcinoma arising in a pleomorphic adenoma (Fig. 1). Fifteen months after the operation, when the patient was last seen, there was no evidence of recurrent disease.

MATERIALS AND METHODS Cell Cultures A small piece of tumor tissue was set up for cell culture. One part was treated enzymatically to start cell cultures as recently described J4], the other part was mechanically minced to get small fragments allowing establishment of " s a n d w i c h " cultures in Leighton-tubes. The culture m e d i u m was TC 199 s u p p l e m e n t e d with 20% fetal calf serum and antibiotics (0.5 U/ml penicillin, 0.5 tzg/ml streptomycin).

C h r o m o s o m e Analysis Harvesting of cells for chromosome preparations was done according to routine methods (2 hours Colcemid to a final concentration of 0.1 t,g/ml, hypotonic treatment; methanol: glacial acetic acid 3 : 1 fixation; air drying). All chromosome preparations were G-banded. G-banding of chromosomes obtained after flow-sorting was performed using a modified protocol recently outlined in detail [6J.

255

Cytogenetic Investigations on a Cell Line

Table 1

S u m m a r y of the chromosome numbers"

Passage 2nd 5th 9th 14th 2nd after nu\m-mouse transplantation

Number of metaphases studied

Chromosome number/cell (main range)

100 12 19 50 20

91-126 91-123 98-118 85-114 87-109

" Metaphase found at different in vitro passa~4esand in one nude mouse tumor issueFi~. 4).

Isolation of Metaphase Chromosomes, Flow Cytometric Analysis, and Sorting After Colcemid-arrest (10 hours) mitotic cells were detached by mechanit:al shock, collected in 10 ml hypotonic solution (0.075 mol/L KCI) and incubated at 37°C for 10 minutes. After centrifugation (100xg, 10 minutes) the pellet was resuspended in 1 ml of chromosome isolation buffer (25 mM Tris-HCl, 0.75 M hexylenglycol, 0.5 mM CaCl z, 10 mM MgCIa.6H20, pH 3.2) s u p p l e m e n t e d with 1% (vol/vol) glacial acetic acid. Chromosomes are released through ultrasonic treatment and stained with DAPI according to Stoehr et al. [7]. Flow cytometric analysis and sorting was carried out using the HEIFAS equipment [8].

Heterotransplantation to Nu\nu-mice For heterotransplantation, cells from the 15th in vitro passage were used. After trypsinization, the cells were suspended in Hanks' solution to a final concentration of about 1.6 × 1(# cells/0.2 ml. Aliquots (2 × 0.2 ml) of this s u s p e n s i o n were subcutaneously injected per animal.

RESULTS Cytogenetic Findings In both types of cultures, cells attached to the bottom of the vessel were frst seen after 2 - 3 days. The cells had a fibroblast-like morphology. A 25-cm 2 culture flask could be harvested for chromosome preparation after 19 days. A total of 10 metaphases were fully karyotyped. The analysis revealed a normal male karyotype at about 400 bands per haploid set stage. On the 8th day after initiation of the cultures, cells with an epithelial-like morphology growing out from the explants were observed in two Leighton-tubes. The cells were characterized by a high proliferative capacity allowing them to overgrow the sparse fibroblastoid monolayer. First chromosome analysis of these cells was possible 41 days after setting up the cultures. A hypertetraploid chromosome n u m b e r distribution was found. For 89% of the metaphases (89/100), the chromosome n u m b e r ranged from 90 to 130 chromosomes (Table 1). The remaining cells had chromosome numbers, which were the result of doubling of the original chromosome number. Furthermore, 15 G-banded metaphases were fully karyotyped. G-banding analysis revealed a polysomy 7 with a range of 5 to 9 chromosomes per cell (average:6.2). A high n u m b e r of

256

1. B u l l e r d i e k et al.

£

0 g

8 ~ 1-~--~1 ~ ~~,,.,-- ,., ~ •~ IpI It F

6--

E o

~0-~-11-|-~;e~l-:

11 . .~ . ." ....

" " "~'~ ~

12 I

5 5

13--

r

E 0 C >,

19

~-~-~

20-~- -~ -'~-'~-

21-~-.-..-.~-~.-~-- 22---~%---,'

E 13..

8-

/ .

m

o

8-~!.....

9-~--

1O- - -

11- ~ ' -

12--~--

t.) • r;:i'I:';;-

-

13

E

F i g u r e 2 Repre, sentative G-banded karyolype of a cell afler 41 days in vitro growth. Large characteristic marker (:hromos(m]es a r e t l t l i ] ] b e , l ' e d . Markers no. 2. no. 6. a l / d IlO, 9 s h o w rearrange,ments of chromosome 12 with a 12(tl 3-14 breakl)oint. (]orresponding marker (:hromosomes from other metaphases are marke(t by arrows (for marker no. 2. s e e Fig. 3). Other aberralions identified art, t(6;13)(6qte.r---,6p21::13q12~13qter) (marker no. 1), marker no. 3: t(6;?)(6qter~6p23::?), mark(;r no. 4: 1(6;?)(6pt(;r---,Bql3::HSR(?)), marker no. 7: t(13:?)(131)ler~13q34::? ), marker no. 8: t(15;?j(15qter~-15q11::?) and marker no. 11: i(18q).

s t r u c t u r a l l y altered marker ( ' h r o m o s o m e s was a c o n s i s t e n t finding (Fig. 2). A m o n g these, three marker c h r o m o s o m e s w e r e p a r t i c u l a r l y n o t e w o r t h y because they w e r e f o u n d to be the result of c h r o m o s o m a l r e a r r a n g e m e n t s i n v o l v i n g 1 2 q l 3 - 1 5 , w h i c h is the b r e a k p o i n t region f r e q u e n t l y i n v o l v e d in p l e o m o r p h i c a d e n o m a s as well. T h r e e c o p i e s of o n e of these m a r k e r c h r o m o s o m e s c o n t a i n i n g the 1 2 q 1 3 - 1 5 ~ 1 2 q t e r s e g m e n t w e r e present in all (:ells of the first and two to three c o p i e s in all further p r e p a r a t i o n s of the (:ell line. For exact d e t e r m i n a t i o n of the breakpoint, p r o m e t a p h a s e - c h r o m o s o m e s of this marker w e r e a n a l y s e d (Fig. 3). A l t h o u g h it was not possible to u n a m b i g u ously i d e n t i f y the s e c o n d part of the m a r k e r c h r o m o s o m e , the s e g m e n t of c h r o m o s o m e 12 i n v o l v e d was 12qter ~ 12q13 or 14. During long term culture, the c h r o m o s o m e n u m b e r per cell d e c r e a s e d [Table 1, Fig. 4) as well as the degree of p o l y s o m y 7. Fifty m e t a p h a s e s d e r i v e d from the 14th passage w e r e c h e c k e d for the degree of p o l y s o m y 7, ranging from 2 - 7 c o p i e s per cell w i t h an average of 4.1 per cell. The cells h a v e now passed the 29th passage in vitro. This is an e x c e p t i o n a l l y long t i m e for cells from benign salivary gland tumors. We therefore s u p p o s e that the cells d e r i v e d from the m a l i g n a n t part of the t u m o r and are i m m o r t a l i z e d .

257

Cytngenetic Investigations on a (Jell Line I

I

m

I

m

I

t I

II III

a

!

I

Figure 3

Marke,r no. 2 (see, Fig. 2) from five metaphases showing different stages of band resolulion. The long arm of the marker comprises the segment 12qter~12q13orq14 (dishd I)arl) and an unidentified (:hromosomal segment which is dupli(:aled (proximal parl).

Heterotransplantation to Nude Mice Six n u d e mice were injected. Three months later, five of these had several tnmors. The animals were killed and the tumors removed. Their largest diameter was about 1 cm. Cell cultures were set up froln four tumors derived from two animals. From ea(:h culture, 20 metaphases were fully karyotyped. All cell cultures revealed characteristi(: cytogenetic features of the original (:ell line but new marker chromosomes and a higher instability were noticed. However, no evidence for clonal karyotypic differences distinguishing i n d i v i d u a l tumors was found. Particular attention was fo(:used on the polysomy 7 now ranging from 3 to 6 chromosomes/cell (average: 4.2J. Also, in all (:ultures derived from the four tumors, a high n u m b e r ()f different is()chromosomes was found (Fig. 5). Except for an i[lq) present in four metaphases from one individual tnmor, none of them was proved to be clonal.

Flow Cytometry The rapid proliferation and the high mitotic index of the cells allowed flow karyotyping at the 8th passage. Due to the marker chromosolnes, tile resolution was rather low. However, a distinct peak among the chromosomes with middle DNA content was observed (Fig. 6). Flow sorting of this peak and G-banding of the chromnsolnes revealed that the peak corresponded to the polysomy 7 in this cell line. Attempts to isolate the n u m b e r 2 marker described above by flow sorting did not result in a frequency of more than 18% in the corresponding fraction. Flow karyotyping was repeated using cells established from the n u d e mice tumors now showing a further decreased resolution of chromosomal fractions corresponding to the enhanced (:ytogenetic instability of the cell line after heterotransplantation.

DISCUSSION The ability of pleomorphic adenomas to undergo malignant transformation particularly after long periods of latency or in recurrences makes carcinomas arising ill these tnmors an interesting object for chromosomal studies. In this paper, the cytogenetic

258

J. B u l l e r d i e k et al.

45

40-

35

30-

25

20

1:5-

|/

10-

WIIIIAI

71 --BO

I

!

I vn-1~I I B1-90

I

I v11:r~ I WHIA I

I~

I vH11~

91-100

I01-110111-120121-130131-140141-150151-160

> 160

91-100

101-110111-120121-130131-140141-150151-160

>160

60

50-

4¢1

30-

20~

10-

I 71-80

81-90

F i g u r e 4 Distribution of the n u m b e r of c h r o m o s o u l e s per m e t a p h a s e . T h e cells from the 2nd in vitro passage (upper part, n - 100 m e t a p h a s e s ) are c o m p a r e d with t h o s e from the 14th in vitro p a s s a g e (lower part, 11 50 m e t a p h a s e s ) . Abscissa: n u m b e r of c h r o m o s o m e s / c e l l ; ordinate: percentage of cells in the different groups.

259

Cytogenetic Investigations on a Cell Line

~

~

2-

4 i ~

I/1

E O

g E o -5

5 -~-~-~,.,

6-- 7- ~ - ~ - ~ - ~

E O C

19. . . . . . ~ - -

20-,~-~-~-~'~ -

21-,,,,-~,~-~--

22-~ .... ~-,,i

Eo 13 O-

8-

X--~-~

--

7-

8 - - ~- ~--

mW

Y-~'--

~

Q

E O 2

.~

10-

-

11m

12--

m

N

Figure 5 Representative G-banded metaphase of a nude mouse tumor. The numbered marker chromosmnes correspond to those shown in Fig. 2. Inset: lsochromosomes from two other metaphases. investigations of a cell line derived from a carcinoma arising in a pleomorphic adenoma allow the reconstruction of different steps of chromosomal aberrations. Accordingly, the changes will be discussed herein following a hypothetical temporal order. About 20% of pleomorphic adenomas karyotyped in our laboratory are characterized by structural clonal abnormalities involving chromosome 12q13-15 [5]. Translocation chromosomes with a similar or even identical breakpoint were also observed in the cell line described here. Although the karyotype of the originally benign tumor cannot be presupposed, the malignant tumor may probably have been originated from a pre-existing pleomorphic adenoma belonging to the 12ql 3-15 rearrangement group, whereas the normal metaphases, which were seen first, most likely represent cells derived from s u r r o u n d i n g normal tissue. The chromosome n u m b e r distribution of the tumor cells was in the hypertetraploid range. Therefore, we have assumed that the chromosome n u m b e r was the result of a duplication of the whole chromosomal c o m p l e m e n t after the 12q13-15 rearrangement and some secondary aberrations had originated. Accordingly, the remarkable polysomy 7 observed can be explained by tri- or tetrasomy 7 preexisting before the duplication. Tri- or tetrasomy 7 as the sole abnormality or among other chromosomal changes is often found in h u m a n malignant or premalignant neoplasms [9-11] indicating a role of this chromosome for neoplastic transformation. It is of considerable interest that Mark and Dahlenfors [12] have

260

J. Bullerdiek et al.

N= RBgRO~

COUNTS

,~

1050

Y 0 I

0

50

I

qO0

q50

1

I

200

250

CHANNEL NUMBER ( RELo

LOG

)

Figure 6 How karyotype of the cell line before heh;rotrallsphmtation to nude mice. l)ue to the high number of structurally altered marker (:hrolnosonle,s, th(~ resolution is rather low. The peak indicated by an arrow corresponds to the I)olysolny 7 as revealed by I'lm,v sorting and

G-banding of the chromosomes o1' this t'racli¢m.

described gains of number 7 in metaphases from eight nut of 100 plenmnrphic adenomas. Next to gains of number 8, trisomy 7 is the second mnst frequent chromosonlal gain in this benign tumnr. Akin to trisomy 8, it usually characterizes small sidelines of the tumors probably in a bnrderli ne stage between benign growth and malignancy. Since the mean number of chrnnmsomes 7 per metaphase decreased during serial subcultivation of the cell line, even though metaphases with the initial high numbers were still present, we have xenografted the cell line. Recently, evidence for a role of c h r o m o s o m e 7 for malignant transformation was presented by Collard et al. [131 who were able to show that in interspecies hybrids between mouse and hunmn cells, tumorigenicity of the cells depends on the presence of one h u m a n c h r o m o s o m e 7. Unexpectedly, in our experiments, the mean number of copies of c h r o m o s o m e 7 per cell did not increase again after heterntransplantation. However, this does not necessarily offer e v i d e n c e against a role of polysomy 7 for metastatic spread or a more aggressive behavior of the tumors. Although the underlying molecular m e c h a n i s m s are unknown, a simple explanation is a pre-existing heterozygotic situation with one number 7 carrying an activated allele of a gene important for tumor d e v e l o p m e n t and one normal number 7. The polysomy may be due to a duplication of the activated number 7, whereas during further subcultivation, a subsequent loss of the normal number 7 has occurred not altering or even further enhancing the tumorigenic potential of the cells. Furthermore, corresponding to a decreasing c h r o m o s o m e number during long-term cultivation the cells established from the nude mice tumors showed a reduced c h r o m o s o m e number. The decreasing c h r o m o s o m e number may offer evidence that the peritriploid or hypotetraploid c h r o m o s o m e numbers frequently seen in malignant solid tumors can be the result of a duplication of the whole c h r o m o s o m e

C y t o g e n e t i c I n v e s t i g a t i o n s on a Cell Line

261

c o m p l e m e n t f o l l o w e d by loss of c h r o m o s o m e s rather than of a s t e p w i s e d u p l i c a t i n n of single c h r o m o s o m e s . A n o t h e r interesting finding after h e t e r o t r a n s p l a n t a t i o n was the f r e q u e n t o c c u r r e n c e of different i s o c h r o m o s o n r e s . H o w e v e r , e x c e p t for i s o ( : h r o m o s o m e (lq), a f r e q u e n t se(:nndary marker in o t h e r m a l i g n a n c i e s as well, n o n e of t h e m was found to be oh)hal. Thus, certain e n v i r o n m e n t a l factors or a particular type of genetic instability have s e e m i n g l y favored the g e n e r a t i o n of i s o c h r o m o s o m e s , and o n l y th()se g i v i n g the (:ells a p r o l i f e r a t i v e a d v a n t a g e persisted in stable p o p u l a t i o n s . In s u m m a r y , w e a s s o m e that, a l t h o u g h already c h a r a c t e r i z e d by c o m p l e x karyntypic r e a r r a n g e m e n t s , the cell line was e s t a b l i s h e d relatively sh()rtly after the oc(:urf e n c e of essential steps for m a l i g n a n t t r a n s f o r m a t i o n w i t h i n a p l e o m o r p h i c a d e n o m a . C u l t i v a t i o n of the cells in vitro and in particular h e t e r o t r a n s p l a n t a t i n n r e v e a l e d further steps of k a r y o t y p i c e v o l u t i o n a l h ) w i n g insights as to how, step by step, (:ells from a p l e o m o r p h i c a d e n o m a cell p o p u l a t i o n may turn to a m a l i g n a n t t u m o r and i n c r e a s i n g m a l i g n a n c y . In additi()n, the p r e s e n c e of the i m m b e r 12 markers m a k e s the cell line suited for m o l e c u l a r studies, since it is the first i m m o r t a l i z e d salivary gland t u m o r cell line s h o w i n g the 12q 13-15 r e a r r a n g e m e n t . C h r o n m s o m a l r e a r r a n g e m e n t s w i t h b r e a k p o i n t s in this region h a v e also been f o u n d in l i p o m a s [14, 15[, l i p o s a r c o m a s 116], and l e i o m y o m a s [17]. E s t a b l i s h e d cell lines of these tuinors will be helpful for c o m p a r a t i v e m o l e c u l a r studies of the b r e a k p o i n t regions i n v o l v e d .

We thank Mrs. Kerstin Me,yer for her excellent tc(:huical assistance, Suuported by a grant from lhe Deutsche l:nrschungsgemeinschafl.

REFERENCES 1. Stemnan G, Mark ], Sandros ] (1989): Cytogenetic and molecular analyses on truman salivary ghmd tumors. 111:Abstracts, 3rd lnteruational Workshop on Chromosomes in Solid Tnmors, February 26-28, 1989. Tucson, Arizona. 2. Mark 1, Dahleufors R, Ekedabl C, Stenman G {1980}: The mixed salivary glan(I tumor a normally benign human neoplasm frequently showing specific cbromnsomal abnormalities. Cancer Genet Cytogene, t 2:231-241. 3. Mark I. Dahlenfors R, Eke(tahl C, Stenman G (1982): C h r o m o s n n m l patterns in a haman benign neoplasm, the mixed salivary ghmd tulnor. Here(titas 96:141 148. 4. Bullerdiek J, B6schen C, Bartnitzke S {19871: Aberrations of chronmsome 8 in mixed saliw~ry gland tumors cytogeuetic findings on seven cases. Cancer Genet (]ytogenet 24:205 212. 5. Bullerdiek I, Bartnitzke S. Weinberg M, Chilla R, Haubrich 1, Schloot W {1987}: Rearrangements of chromosome region 12(t13-15 in pleomorphic adenomas of the human salivary gland (PSA). Cytogenet Cell Genet 45:187 190. 6. Rommel B, Hutter KI, Bullerdiek l, Bartnitzke S, Goerttler K, Schloot W (19881: Identification of flow-sorted chromosomes by G-banding and in situ hybridization. Cytometry 9:504-507. 7. Stoehr M. Hurter JK, Frank M, Goerttler K {1982): A reliable preparation of monodispersed chromosome suspensions for flow cytometry. Histochemistry 74:305-313, 8. Stoehr M, Gnerttler K {1979): The Heidelberg flow analyzer and sorter (HEIFAS) approach on the prescreeningof uterine(:ancer.] Histocbem Cytochem 27:564 566. 9. Lee IS, Pathak S, Hopwood V, Tomasovic B, Mullins TD, Baker FL, Spitzer G, Neidhart ]A (1987): Involvement of chromosome 7 in primary hmg tumor and nonmalignant normal lung tissue. Cancer Res 47:6349-6352. 10. Babu RV, Lutz MD, Miles BJ, Farah RN, Weiss L, van Dyke DL (1987): Tumor behavior in transitional (:ell carcinoma of the bladder in relation to c h r o m o s o m a l markers and histopathology. Cancer Res 47:6800-6805. 11. de long B, Oosterhuis JW, ldenburg VlS, Castedo SMMI, Dam A, Mensink HIA 11988): Cytogenetics of 12 cases of renal adenocarcinoma. Cancer Genet Cytogenet 30:53-61. 12. Mark ], Dahlenfors R {1986]: Cytogenetical observations in 100 hmnan benign pleomorphic

262

J. B u l l e r d i e k et al.

adenomas: Specificity of the chromosomal aberrations and their relationship to sites of localized oncogenes. Anticancer Res 6:299-308. 13. Collard ]G, van de Poll M, Scheffer A, Roos E, Hopman AHM, van Kessel AHMG, van Dongen JJM (1987): Location of genes involved in invasion and metastasis on human chromosome 7. Cancer Res 47:6666-6670. 14. Turc-Carel C, Dal Cin P, Rao U, Karakousis C, Sandberg AA (1986): Cytogenetic studies of adipose tissue tumors: 1. Benign lipoma with reciprocal translocation t(3:12)(q28;q141. Cancer Genet Cytogenet 23:283 289. 15. Mandahl N, Heim S, Arheden K, Rydholm A, Willen H, Mitehnan F (1988): Three major cytogenetic subgroups can be identified among chromosomally abnormal solitary lipomas. Human Genet 79:203-208. 16. Turc-Carel (', Limon J, Dal Cin P, Rao U, Karakousis C, Sandberg AA {1986): Cytogenetic studies of adipose tissue tumors: II. Recurrent reciprocal translocation t(12;161(q13;p111 in myxoid liposarcomas. Cancer Genet Cytogenet 23:291-299. 17. Vanni R, Lecca (1988): Involvement of the long arm of chromosome 12 in chromosome rearrangements of uterine leiomyoma. Cancer Genet Cytogenet 32:33-34.