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6q- and loss of the Y chromosome—Two common deviations in malignant human salivary gland tumors
6 q - and Loss of the Y Chromosome Two Common Deviations in Malignant Human Salivary Gland Tumors G. Stenman, J. Sandros, R. Dahlenfors, M. Juberg-Ode, and J. Mark
ABSTRACT: Nine cases of malignant human salivary gland tumors cultured in vitro were subjected to detailed cytogenetic analysis with G-banding. Together with observations from three earlier published cases, the results of 12 cases were surveyed: five adenoid cystic carcinomas, three acinie cell tumors, three adenocarcinomas, and one mucoepidermoid carcinoma All tumors had stemlines in the diploid-near-diploid mode. The most consistent changes among the adenoid cystic carcinomas were stem lines and/or variant cells with anomalies affecting the terminal part of 6q (i.e., 6q16-25). Deviations affecting the Y chromosome (losses) and, ta a lesser extent, #6 (structural changes) and #8 (gains) characterized the early karyotypic evolution in acinic cell tumors. Two of the three analyzed adenocareinomas showed sfemlines or variant cells with loss of gonosomes. The karyotypic features of the different tumor types, including primary changes, evolutionary characteristics, and progressional pathwaysj are discussed. The cytogenetic relationships between benign and malignant salivary gland tumors also will be considered. INTRODUCTION Extensive chromosomal banding data have been presented recently for more than 80 benign mixed salivary gland tumors (i.e., pleomorphic adenomasl [1, 2]. These studies revealed that: a) pleomorphic adenomas are frequently characterized by abnormal, mostly pseudodiploid stem lines, b) the deviations preferentially consisted of structural rearrangements, in particular, reciprocal translocations, c) the structural changes affected certain regions of especially chromosomes #3, #8, and #12 (i.e., 3p14, 8q12, and 12q13-15), and d) approximately 60% of the cases with abnormal stem lines showed deviations affecting chromosomes or chromosome sites harboring k n o w n oncogenes. A few cases of malignant salivary gland tumors also have been studied cytogenetically [3-5]. The number is too small, however, to allow any conclusions about the Specificity of the deviations in different tumor types. The present report is a continuation of these previous studies and deals with observations by banding in
From the Departments of Oral Pathology and Oral Surgery, University of G6teborg, G~teborg, and the Laboratory of Cytogenetics, Department of Pathology, Central Hospital, Sk6vde, Sweden.
Address requests for reprints to Dr. G. Stenman, Department of Oral Pathology, University of G6teborg, Box 33070, S-400 33, G6teborg, Sweden. Received May 11, 1985; accepted October 15, 1985.
283 © 1986 Elsevier Science Publishing Co., Inc. 52 Vanderbilt Ave,, New York, NY 10017
Cancer Genet Cytogenet22:283--293(19861 0165-4608/66/$03.50
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nine cases of m a l i g n a n t salivary gland tumors. Due to m e t h o d o l o g i c difficulties, in particular a low mitotic index, the use of an in vitro m e t h o d was necessary. The results will furnish a p r e l i m i n a r y v i e w of the c h r o m o s o m a l characteristics of each t u m o r type. The cytogenetic r e l a t i o n s h i p s b e t w e e n benign and malignant salivary gland t u m o r s will also be considered.
MATERIALS AND METHODS Fresh t u m o r s p e c i m e n s from 12 m a l i g n a n t salivary gland tumors, i n c l u d i n g five a d e n o i d cystic carcinomas, three acinic cell tumors, three adenocarcinomas, and one m u c o e p i d e r m o i d carcinoma, were e x p l a n t e d in vitro using a technique described p r e v i o u s l y in detail [6]. Of the 12 tumors, one case each of a d e n o i d cystic carcinoma, acinic cell tumor, a n d a d e n o c a r c i n o m a failed to grow. For the r e m a i n i n g n i n e successful cases the c l i n i c o p a t h o l o g i c data are s u m m a r i z e d in Table 1. Except for case 3, all others were u n t r e a t e d p r i m a r y tumors. Case 3 r e p r e s e n t e d the first recurrence of an a d e n o i d cystic c a r c i n o m a originally excised 4 years ago. So far, clinical e x a m i n a t i o n s have revealed no signs of recurrences or of metastases in cases 1 - 8 (follow-up time varied b e t w e e n 6 m o n t h s a n d 4 years). The patient with the m u c o e p i d e r m o i d c a r c i n o m a (case 9), however, d i e d 4 m o n t h s postoperatively. A u t o p s y revealed a large recurrence as w e l l as lung metastases. The techniques for c h r o m o s o m e preparation, as well as G-banding, were essentially the same as described earlier [5]. Magnified photographs were used for the karyotype analyses. The c h r o m o s o m e s were classified according to ISCN [7].
RESULTS The c h r o m o s o m e counts, as well as the stem- side line karyotypes of the nine analyzed tumors are p r e s e n t e d in Table 2. Table 3 shows the k a r y o t y p i c deviations in d i p l o i d - n e a r - d i p l o i d variant cells (given in relation to the stem- side line karyotypes of each tumor). The derivation of the different marker c h r o m o s o m e s in all p r e p a r a t i o n s of all t u m o r s is specified in Table 4. The m a i n karyotypic findings in each t u m o r are p r e s e n t e d below.
Table 1
Clinicopathologic characteristics of the material
Case
Sex/Age (yr)
Histologic type
1
F/34
2
F/72
3
F/75
4
M/63
5 6 7
M/65 M/36 F/77
Adenoid cystic carcinoma Adenoid cystic carcinoma Adenoid cystic carcinoma Adenoid cystic carcinoma Acinic cell tumor Acinic cell tumor Adenocarcinoma
8 9
F/60 M/74
Adenocarcinoma Mucoepidermoid carcinoma
Duration of symptoms prior to operation (yr)
Tumor size (cm)
Parotid gland
>12
1.5 × 1.5 x 1.5
Soft palate
<1
1.0 × 1.0 × 0.5
Hard palate
<1
1.5 × 1.0 × 1.0
Submandibular gland Parotid gland Parotid gland Submandibular gland Parotid gland Submandibular gland
>1
4.5 x 3.5 × 3.0
>2 >18 >5
1.0 × 0.5 × 0.5 2.5 x 2.0 × 1.5 2.0 x 1.5 x 1.5
>1 >1/2
1.5 x 1.0 × 1.0 5.5 x 3.0 × 3.0
Localization
9 22 12 18 19 27 30 34 37 41 16 17 21 28 32 19 22 31 23 31
2 3 1 2 3 4 la 2c 3d 4b 1 2 I 2 3 1 2 3 1 1
11
1
43
2
33 11
11
2z
22
11 22 53
11
83 11 1l 22 42
32
55 22 11 44 44 5s 95 116
44
33 22
45
11
44 2515 30 zs 2015 2921 2814 2111 147 2213 1812 2713 30 TM 3z 1811 56 ~5 2111 4217 3814 84 169 156 84 7s 22 TM 25 TM 43
46
11
33 33
3~
11
47
11
22
48
1
49
"Superscribed n u m b e r s indicate n u m b e r a n d d i s t r i b u t i o n of k a r y o t y p e d cells. bRoman n u m e r a l s d e n o t e m a r k e r c h r o m o s o m e s . ~I'he letters a through d indicate different areas of the tumor.
S 9
7
6
5
4
3
8
1
2
9 14 15 21
1 2 3 4
1
42
50
51
52
Chromosome counts a
Chromosome counts and stem- side line karyotypes of the tumors
Days Case Preparation c in vitro
Table 2
2
53
41
54
11
87
11
89
11
90
11
91
1
15
11
11
92 29 TM 3227 2015 3123 328 2614 17 I° 2617 1812 33 TM 328 43 2417 634° 3125 5122 5220 117 2214 156 2112 9s 2414 27 TM 196
Total cells 4 6 , X X , 6 q - (1),9p + (II)/46,XX 46,XX,6q-(1),9p+(II)/46,XX 4 6 , X X , 6 q - (1),gp + (II]/46,XX 4 6 , X X , 6 q - (l],9p + (II)/46,XX 46,XX 46,XX 46,XX 46,XX 46,XX 4 6 , X X / 4 6 , X X , 6 q - (1) 46,XX,6q - (1)/46,XX 46,XY 46,XY 46,XY 46,XY 4 6 , X Y / 4 5 , X , - Y / 4 6 , X Y , - 13, + 15 46,XY/45,X, - Y 46,XY/47,XY, + 8 46,XY/47,XY,+8 46,XY 46,XX 46,XX 46,XX 46,XX 46,XY
Stem- side line k a r y o t y p e s b
tO O0
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Table 3
Karyotypic deviations in diploid-near-diploid stem- and side line karyotypes
Case
Preparation b
1
1 2 4
2
1
3
2 3 1 2 3
4
5
v a r i a n t c e l l s i n r e l a t i o n to t h e
Variant cells in relation to stem-(sl) side line (sdl) karyotypes ~ sl sdl 1 sl sdl 1 sl sdl 1 sl
4 la 2c 3d 4b
sl sl sl sl sl sdl 1 sdl 2 sl sl sl sl
1 2
sl sl
6 7
2 1
sl sl
8 9
2 3 1 1
sl sl sl sl
- 6 q - (1)/- 9p + (II), - 1 5 / - 9p + (II), - 9 - 2 1 / - 21 - 18 -21 6 q - {I},- 9p + (II), + 9 / - 2 0 , - X -9 8 q - (I),9q + (II)/8p- (III),21q+ (IV)/- 3 / - 1 2 / - 2 , - 5, - 6, + 8 , - 9(4n = 89)/ - 1 7 , - 1 8 , - 2 2 , - X , - X (4n = 87) + 16, - 17/- 1 2 / - 2 2 / - 22 8 q - (V),Xq+ (VI)/÷ 1 5 / X q - (VII) ( 4 n = 9 2 ) / - 3 , - 1 7 , - X (4n=89)
11q+(II),-8q-/-6/-9/-20 6q - (I) 2 q - (III),17p + (IV)/18q + (V),- 1 5 q - / - 10, + 12, - 1 9 / - 2 0 / - 20 6 q - (I}, - X 9q - (VI)/+ 3, - 8 / - 2 0 / - 20 -22 - 6 / - 9 / - 17/- Y / - Y, - 1 0 / - Y, - 22 - 1 6 / - 1 6 / - 2 2 / - Y/+ 1/+ 7/+ 22 + 1, + 1 0 / - 2 , - 1 6 / 3 q - (I), - Y / - 8, - 1 6 / - 1 6 / - 16, + 2 0 / - 1 2 / - Y / - Y / - Y , +7,+8,+20 2 p - (I)/+ 1 , - 6 , - 7 , + 8
6q-(II)/6q-(II)/-13,+15/-13,+15/-13/-9,-9/-6,-6,+6q-(III), + 6q - (Ill), + 6q + (IV), - 8, - 8, - 16, - 18, + 18q + (V], + 19, + 20(4n = 9 1 ) / - Y, - Y (4n = 90) - X/- Y
-8,-15,-17,-18/12q+(I),-20,-X/-5,-19/-5,-22/-8/-17/-X/ 3q - (II}, + 8/5p - (III) - 6, - 2 2 / 5 q - (IV), - 18
3q-(V},IOq+(VI}/3p+(VII),5p-(VII)/-X/2q+(IX},3q-(X),+8,+20 -13/-6,+9,-X - Y / 6 q - (I),8q- (II),11q- (III),- 1 5 , - 16,17p + (IV),- 18,+ 16p + (V), + 16p
+(V)/+ l,+2,+3q-(VI),+3q-(VI),+ 7,+9,+10,+ llq-(III),-22 °Roman numerals denote marker chromosomes presented in Table 4. ~The letters a through d indicate different areas of the tumor.
Case 1 All preparations from this adenoid cystic carcinoma showed a pseudodiploid stem l i n e c h a r a c t e r i z e d b y a r e c i p r o c a l t(6;9) ( m a r l a n d m a r I I i n T a b l e 4; Fig. 1). A s i d e l i n e (sdl 1) w i t h a n o r m a l k a r y o t y p e w a s also o b s e r v e d i n all p r e p a r a t i o n s . B e t w e e n 1 5 % a n d 3 5 % of t h e a n a l y z e d c e i l s i n t h e d i f f e r e n t p r e p a r a t i o n s b e l o n g e d to s d l 1. I n a d d i t i o n , t h e first a n d s e c o n d p r e p a r a t i o n s also c o n t a i n e d a p s e u d o d i p l o i d s i d e l i n e (sdl 2); it d i f f e r e d f r o m t h e s t e m l i n e b y a loss of m a r I I a n d a g a i n of o n e n o r m a l c h r o m o s o m e # 9 . F u r t h e r m o r e , 10 v a r i a n t c e l l s w i t h n u m e r i c a l a n d / o r s t r u c t u r a l d e v i a t i o n s w e r e r e c o r d e d f r o m t h e f o u r p r e p a r a t i o n s ( T a b l e 3).
Case 2 T h i s a d e n o i d c y s t i c c a r c i n o m a s h o w e d a n o r m a l d i p l o i d s t e m l i n e i n all t h r e e p r e p o r a t i o n s . In a d d i t i o n , all of t h e m c o n t a i n e d s t r u c t u r a l l y or n u m e r i c a l l y c h a n g e d v a r i a n t c e l l s ( T a b l e 3). T h r e e of t h e s e s h o w e d r e c i p r o c a l t r a n s l o c a t i o n s a f f e c t i n g # 8 , w h e r e a s , t w o s h o w e d r e a r r a n g e m e n t s i n v o l v i n g t h e l o n g a r m of t h e X c h r o m o some.
6q- a n d -Y in M a l i g n a n t S a l i v a r y G l a n d T u m o r s
Table 4
D e r i v a t i o n of the d i f f e r e n t m a r k e r c h r o m o s o m e s o b s e r v e d in s t e m lines, side lines, a n d v a r i a n t cells
Case 1
287
Marker
Derivation
I = 6q II = 9p +
t(6;9)(6pter--~6q24::9p23--~9pter) t(6;9)(9qter--~9p23 ::6q24--~6qter)
I=8qII=9q+ IlI = 8p IV=21q+ V=8qVI = Xq + VII = Xq -
t(8;9)(8pter--~cen--~9qter) t(8;9)(9pter-~cen--~8qter} t(8;21)(8qter-~8p21::2 lq22--~2 lqter) t(8;21)(21pter-~21q22::8p21-~8pter) t(X;8}(8pter--~8q12::Xq27--~Xqter) t(X;8}(Xpter--~Xq27::8q12--~8qter) del(X)(q13}
I=6qII = 11q + III = 2 q IV=17p+ V=18q+ VI = 9q -
del(6)(pter-~q16::q22--~qter} t{8?;11){11pter--~11q23::8q12 - 21--~qter) t(2; 17)(2pter-~2q13 ::17p13--~17pter) t(2; 17)(17qter--~ 17p13::2q13--~2qter) t(15;18)(18pter-~18q23::15q13--~15qter) del(9)(q31)
I=3q-
del(3)(q11-12)
I=2pII = 6q III = 6 q IV = 6q + V = 18q +
del(2}(p23) del(6)(q22) i(6p) dic(6)(pter--~q15::q15--~pter) i(18q)
1-12q+
IV = 5q V=3qVI = lOq + VII = 3p + VIII = 5p IX = 2q + X=3q-
t(12;?)(12pter- 12q24::?) i(3p) del(5)(p11) del(5)(q23) t(3;10)(3pter~3q22::10q24-~10qter) t(3;10)(10pter--~10q24::3q22---~3qter) t(3;5)(3qter--~3p27::5p12-)5pter) t(3;5)(Sqter-~5p12::3p27-*3pter) t(2;3)(2pter--~2q36 - 37::3q22--~3qter) t(2;3)(3pter->3q22 ::2q36 - 37--~2qter)
I = 6qII = 8 q III= 1 1 q IV= 17p + V = 16p + ? VI = 3 q -
i(6p) del(8)(q22) del(11}(q22) i(17q) i(16q) del(3)(q21)
II = 3q Ill
-
= 5p
-
Case 3 T h e first three p r e p a r a t i o n s f r o m this r e c u r r e n t a d e n o i d cystic c a r c i n o m a w e r e chara c t e r i z e d b y a n o r m a l d i p l o i d s t e m line. T h e f o u r t h p r e p a r a t i o n , h o w e v e r , s h o w e d a p s e u d o d i p l o i d s t e m l i n e d i s t i n g u i s h e d b y an interstitial l o n g a r m d e l e t i o n of o n e c h r o m o s o m e # 6 (marI in T a b l e 4; Fig. 2). T h i s s t e m l i n e c o u l d be t r a c e d to an i d e n t i c a l p s e u d o d i p l o i d side l i n e (sdl 1) a p p e a r i n g in t h e t h i r d p r e p a r a t i o n a n d c o m p r i s i n g a b o u t 37% of t h e a n a l y z e d cells. F u r t h e r m o r e , in the last p r e p a r a t i o n
288
G. S t e n m a n et al.
S
t2
I$
t4 ¸
19
20
1|
1|
21
22
t7
tl
X
X
Figure 1 Top: G-banded karyotype of a stem line cell of case 1 [46,XX,t(6;9) (q24;p23)]. Bottom; The marker chromosomes in higher magnification showing the reciprocal translocation between 6q and 9p. (Top, x 2600; bottom, x 3700) r e m n a n t s of the former normal stem line formed a side line (sdl 2) comprising about 33% of the analyzed cells. The characteristics of the variant cells in the four preparations are given in Table 3. Case 4 Four different areas from this a d e n o i d cystic carcinoma were studied. At least two preparations from each area were analyzed. In Table 2 the preparations from each area are c o m b i n e d and presented as a single fixation Ca-d). The outgrowth from all four areas was extremely slow. In all areas a normal diploid stem line was found. D i p l o i d - n e a r - d i p l o i d variant cells, however, were recorded in all areas, and espe-
289
6q- and -Y in Malignant Salivary Gland Tumors
XHn ?
z
a 14
11
20
15
I
21
iS
Figure 2 Leftand right: Complete and partial G-banded karyotypes of two stem line cells of case 3; 46,XX, del(6)(q16q22}. (Left, × 2600; right, × 2800} cially in area b (Table 3). Except for one structurally changed cell, all others showed numerical changes, especially losses. An evolutionary change in common for three of the areas was the occurrence of hypodiploid variant cells with a loss of the Y chromosome. Case 5
Both preparations from this acinic cell tumor showed a normal diploid stem line and also a hypodiploid side line (constituting about 25% of the analyzed cells in both preparations). The only deviation in this side line was a loss of the Y chromosome (Fig. 3). The first preparation also contained a small pseudodiploid clone or side line consisting of four cells with monosomy 13 and trisomy 15, Variant cells and clones, with structural changes affecting particularly the long arm of #6 were also seen in both preparations (Table 3). Case 6
All three preparations from this acinic cell tumor showed a normal diploid stem line. Preparations one and two also contained a small hyperdiploid side line characterized by trisomy 8. Only two variant cells were seen and both showed a loss of a gonosome. No markers were observed in any of the analyzed cells. Case 7
A normal stem line was seen in all three preparations from this moderately to poorly differentiated adenocarcinoma. Also, a variety of numerically and/or structurally changed variant cells were found in the different preparations (Table 3). No less than 10 different markers could be identified. Both arms of chromosomes #3 and/or #5 were involved in the formation of most of the markers.
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G. Stenman et al.
Figure 3
Complete G-banded karyotype of a side line cell of case 5; 45,XO, -Y. (× 2800)
Case 8
Due to extremely slow and sparse outgrowth from this poorly differentiated adenocarcinoma it was only possible to obtain one successful preparation. Except for two hypodiploid cells (Table 3), only cells with a normal karyotype were found. No cells with structural changes were observed. Case 9
The growth of this tumor, a poorly differentiated mucoepidermoid carcinoma, was also slow and sparse. Only a few banded metaphases from one preparation could be studied. Available data indicate a bimodal distribution. Except for three normal diploid ceils there were a few hypodiploid and hyperdiploid variant ceils (Table 3). An l l q - marker (marlII in Table 4) was seen both in the hypodiploid and in the hyperdiploid cells indicating a clonal origin of both modal groups. One or two long arm deleted chromosomes #3(marVI in Table 4) were also seen in two further hyperdiploid cells not suitable for complete analyses. DISCUSSION Almost all of the comparatively uncommon malignant tumors of the salivary glands are carcinomas. Most of them are found in the major glands, particularly the parotid gland. Here the most common types are mucoepidermoid tumors, undifferentiated carcinomas, acinic cell tumors, adenocarcinomas, and adenoid cystic carcinomas,
6q- and -Y in Malignant Salivary Gland Tumors
291
in that order of frequency [8]. Almost all of the remaining carcinomas in the major glands are found in the submandibular gland. In this location adenoid cystic, undifferentiated, and epidermoid carcinomas outnumber the other types. The adenoid cystic carcinoma is the predominating type also in minor salivary glands. Except for the undifferentiated and epidermoid carcinomas, all the other types are represented in our series. The observations in this heterogeneous material are primarily best considered separately for each tumor type. In this context comparisons will be made with available, though very scanty, data in the literature. Together with a previously published case of adenoid cystic carcinoma [4] banding data are available from five adenoid cystic carcinomas. Three of these cases have (in one or more preparations) shown pseudodiploid stem lines characterized by one of the following structural changes: t(5;17)(q22;q13), t(6;9)(q24;p23), and del(6)(q16q22). The remaining two cases had normal stem lines. However, these two cases (as those three with an abnormal stem line) contained a great variety of variant cells, clones, and side lines. It should be noted that three of the tumors showed stem lines and/or variant cells with anomalies affecting the terminal part of the long arm of chromosome #6 (i.e., 6q16-25). It is interesting to note that the cmyb oncogene has been localized to this region of #6 (6q22-24) [9]. An interesting evolutionary finding was seen in side lines and variant cells of case 1. Here the 9p + marker, containing the translocated distal segment of #6, was lost and replaced by a normal #9, resulting in monosomy for 6q24-qter and trisomy for 9p23-pter. These observations stress the significance of rearrangements involving the terminal region of the long arm of #6. A feature characteristic of case 2 was the involvement of chromosome #8 and the X chromosome in three and two different rearrangements, respectively. Case 4 showed an unusually high frequency of variant cells with a loss of the Y chromosome. The common involvement of these three chromosome types is of special interest, because they are frequently affected also in other types of salivary gland tumors. Case 3, as well as the previously published case of adenoid cystic carcinoma [4], is informative regarding the influence of prolonged growth in vitro on the chromosomal picture. Thus, in spite of a long in vitro growth period, both cases showed a change from a normal stem line to an abnormal stem- side line in subsequent preparations. The similarities between these "late" deviations and those observed after short periods in vitro argue against the possibility of induced changes in vitro. The most striking observation in both preparations of case 5 (an acinic cell tumor) was a hypodiploid side line with only a loss of the Y chromosome. The other acinic cell tumor (case 6) also showed variant cells with a loss of gonosomes. Furthermore, case 5 showed a small pseudodiploid clone with a long arm deleted #6, as well as a hypotetraploid cell with loss of the two Y chromosomes and extensive structural rearrangements involving both arms of #6. These findings are in agreement with observations from a previously reported case of acinic cell tumor [3], which inter alia showed stem- and side lines characterized by either Y loss or a reciprocal translocation involving chromosome #6 [i.e., (t(6;21)(q13;q22)]. Another interesting observation in case 6 was the finding of a hyperdiploid side line with trisomy 8. In salivary gland tumors this anomaly has been observed earlier in pleomorphic adenomas. Whether or not trisomy 8 is of the same importance for chromosomal progression in acinic cell tumors as in pleomorphic adenomas, however, remains to be clarified. Accordingly, our preliminary observations indicate that deviations affecting the Y chromosome (losses) and, to a lesser extent, #6 (structural changes) and #8 (gains) are the three deviations typical of early karyotypic evolution in acinic cell tumors.
292
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Both a d e n o c a r c i n o m a s (cases 7 and 8) s h o w e d normal stem lines. However, variant cells w i t h n u m e r i c a l and/or structural rearrangements were seen, especially in c a s e 7. Both a r m s of c h r o m o s o m e s # 3 and/or # 5 were i n v o l v e d in most rearrangements in this case. It s h o u l d also be n o t e d that several variant cells with loss of one X c h r o m o s o m e were observed in both cases. This is of interest, as an earlier rep o r t e d case of a d e n o c a r c i n o m a [5] had a stem line with only a gonosomal deviation (loss of the Y chromosome). Even though the present 12 cases of m a l i g n a n t salivary gland tumors constitute a heterogeneous group, e n c o m p a s s i n g no less than four different histopathologic types, t h e y seem to have certain c h r o m o s o m a l characteristics in common. Thus, all types have a p p a r e n t l y originated w i t h a n o r m a l d i p l o i d stem line. During progression they have frequently d e v e l o p e d p s e u d o d i p l o i d or h y p o d i p l o i d stem- and side lines. For each t u m o r type there seemed to exist more than one progressional pathway, and some of the p a t h w a y s might well be shared by more than one t u m o r type. The deviations seen most frequently in the stem- side lines of the 12 cases were different structural changes of the long arm of # 6 and loss of the Y chromosome. Some of the above described features typical of the malignant t u m o r types ar e similar to those observed in benign p l e o m o r p h i c a d e n o m a s i n d i c a t i n g that, to a certain extent, c h r o m o s o m a l progression might follow similar p a t h w a y s in both benign and m a l i g n a n t salivary gland tumors. These observations and ideas s u p p o r t our previously a d v a n c e d h y p o t h e s i s that the characteristics of the c h r o m o s o m e changes in a certain t u m o r t y p e are to a m u c h greater extent d e t e r m i n e d b y tissue specific factors than by the nature of the i n d u c i n g agent[s]. This is also in line w i t h findings in some other t u m o r groups, such as 14q + markers in different l y m p h o i d malignancies [10], and the i n v o l v e m e n t of c h r o m o s o m e # 2 2 in different n e o p l a s m s of the nervous system [11, 12]. Supported by grants from the Swedish Cancer Society and from the Swedish Medical Research Council, project no. 7327.
REFERENCES 1. Mark J, Dahlenfors R, Ekedahl C (1983}: Cytogenetics of the human mixed salivary gland turnout. Hereditas 99:115-129. 2. Stenman G, Mark J, Ekedahl C (1984~: Relationship between chromosomal patterns and proto-oncogenes in human benign mixed salivary gland tumors. Tumour Biol 5:103-117. 3. Mark J, Ekedahl C, Dahlenfors R (1981): Polyclonal chromosomal evolution in a cultured human acinic cell tumour. Anticancer Res 1:45-48. 4. Stenman G, Dahlenfors R, Mark J, Sandberg N (1982): Adenoid cystic carcinoma: A third type of human salivary gland neoplasm characterized cytogenetically by reciprocal translocations. Anticancer Res 2:11-16. 5. Stenman G, Mark J (1983): Loss of the Y chromosome in a cultured human salivary gland adenocarcinoma. J Oral Pathol 12:458-464. 6. Mark J, Dahlenfors R, Ekedahl C, Stenman G (1980): The mixed salivary gland tumor A normally benign human neoplasm frequently showing specific chromosomal abnormalities. Cancer Genet Cytogenet 2:231-241. 7. ISCN (1978): An international system for human cytogenetic nomenclature. Cytogenet Cell Genet 21:309-404. 8. Enaroth CM (1971): Salivary gland tumors in the parotid gland, submandibular gland and the palate region. Cancer 27:1415-1418. 9. Harper ME, Franchini G, Love J, Simon MI, Gallo RC, Wong-Staal F {1983): Chromosomal sublocalization of human c-myb and c-fes cellular onc genes. Nature 304:169-171.
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10. Sandberg, AA (1980): The Chromosomes in Human Cancer and Leukemia. Elsevier-North Holland, NY, pp. 377-412. 11. Mark] (1977): Chromosomal abnormalities and their specificity in human neoplasms--An assessment of recent observations by banding techniques. Adv Cancer Res 24:165-222. 12. Bigner SH, Mark J, Mahaley MS, Bigner DD (1984): Patterns of the early, gross chromosomal changes in malignant human gliomas. Hereditas 101:103-113.
ABSTRACT: Nine cases of malignant human salivary gland tumors cultured in vitro were subjected to detailed cytogenetic analysis with G-banding. Together with observations from three earlier published cases, the results of 12 cases were surveyed: five adenoid cystic carcinomas, three acinie cell tumors, three adenocarcinomas, and one mucoepidermoid carcinoma All tumors had stemlines in the diploid-near-diploid mode. The most consistent changes among the adenoid cystic carcinomas were stem lines and/or variant cells with anomalies affecting the terminal part of 6q (i.e., 6q16-25). Deviations affecting the Y chromosome (losses) and, ta a lesser extent, #6 (structural changes) and #8 (gains) characterized the early karyotypic evolution in acinic cell tumors. Two of the three analyzed adenocareinomas showed sfemlines or variant cells with loss of gonosomes. The karyotypic features of the different tumor types, including primary changes, evolutionary characteristics, and progressional pathwaysj are discussed. The cytogenetic relationships between benign and malignant salivary gland tumors also will be considered. INTRODUCTION Extensive chromosomal banding data have been presented recently for more than 80 benign mixed salivary gland tumors (i.e., pleomorphic adenomasl [1, 2]. These studies revealed that: a) pleomorphic adenomas are frequently characterized by abnormal, mostly pseudodiploid stem lines, b) the deviations preferentially consisted of structural rearrangements, in particular, reciprocal translocations, c) the structural changes affected certain regions of especially chromosomes #3, #8, and #12 (i.e., 3p14, 8q12, and 12q13-15), and d) approximately 60% of the cases with abnormal stem lines showed deviations affecting chromosomes or chromosome sites harboring k n o w n oncogenes. A few cases of malignant salivary gland tumors also have been studied cytogenetically [3-5]. The number is too small, however, to allow any conclusions about the Specificity of the deviations in different tumor types. The present report is a continuation of these previous studies and deals with observations by banding in
From the Departments of Oral Pathology and Oral Surgery, University of G6teborg, G~teborg, and the Laboratory of Cytogenetics, Department of Pathology, Central Hospital, Sk6vde, Sweden.
Address requests for reprints to Dr. G. Stenman, Department of Oral Pathology, University of G6teborg, Box 33070, S-400 33, G6teborg, Sweden. Received May 11, 1985; accepted October 15, 1985.
283 © 1986 Elsevier Science Publishing Co., Inc. 52 Vanderbilt Ave,, New York, NY 10017
Cancer Genet Cytogenet22:283--293(19861 0165-4608/66/$03.50
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nine cases of m a l i g n a n t salivary gland tumors. Due to m e t h o d o l o g i c difficulties, in particular a low mitotic index, the use of an in vitro m e t h o d was necessary. The results will furnish a p r e l i m i n a r y v i e w of the c h r o m o s o m a l characteristics of each t u m o r type. The cytogenetic r e l a t i o n s h i p s b e t w e e n benign and malignant salivary gland t u m o r s will also be considered.
MATERIALS AND METHODS Fresh t u m o r s p e c i m e n s from 12 m a l i g n a n t salivary gland tumors, i n c l u d i n g five a d e n o i d cystic carcinomas, three acinic cell tumors, three adenocarcinomas, and one m u c o e p i d e r m o i d carcinoma, were e x p l a n t e d in vitro using a technique described p r e v i o u s l y in detail [6]. Of the 12 tumors, one case each of a d e n o i d cystic carcinoma, acinic cell tumor, a n d a d e n o c a r c i n o m a failed to grow. For the r e m a i n i n g n i n e successful cases the c l i n i c o p a t h o l o g i c data are s u m m a r i z e d in Table 1. Except for case 3, all others were u n t r e a t e d p r i m a r y tumors. Case 3 r e p r e s e n t e d the first recurrence of an a d e n o i d cystic c a r c i n o m a originally excised 4 years ago. So far, clinical e x a m i n a t i o n s have revealed no signs of recurrences or of metastases in cases 1 - 8 (follow-up time varied b e t w e e n 6 m o n t h s a n d 4 years). The patient with the m u c o e p i d e r m o i d c a r c i n o m a (case 9), however, d i e d 4 m o n t h s postoperatively. A u t o p s y revealed a large recurrence as w e l l as lung metastases. The techniques for c h r o m o s o m e preparation, as well as G-banding, were essentially the same as described earlier [5]. Magnified photographs were used for the karyotype analyses. The c h r o m o s o m e s were classified according to ISCN [7].
RESULTS The c h r o m o s o m e counts, as well as the stem- side line karyotypes of the nine analyzed tumors are p r e s e n t e d in Table 2. Table 3 shows the k a r y o t y p i c deviations in d i p l o i d - n e a r - d i p l o i d variant cells (given in relation to the stem- side line karyotypes of each tumor). The derivation of the different marker c h r o m o s o m e s in all p r e p a r a t i o n s of all t u m o r s is specified in Table 4. The m a i n karyotypic findings in each t u m o r are p r e s e n t e d below.
Table 1
Clinicopathologic characteristics of the material
Case
Sex/Age (yr)
Histologic type
1
F/34
2
F/72
3
F/75
4
M/63
5 6 7
M/65 M/36 F/77
Adenoid cystic carcinoma Adenoid cystic carcinoma Adenoid cystic carcinoma Adenoid cystic carcinoma Acinic cell tumor Acinic cell tumor Adenocarcinoma
8 9
F/60 M/74
Adenocarcinoma Mucoepidermoid carcinoma
Duration of symptoms prior to operation (yr)
Tumor size (cm)
Parotid gland
>12
1.5 × 1.5 x 1.5
Soft palate
<1
1.0 × 1.0 × 0.5
Hard palate
<1
1.5 × 1.0 × 1.0
Submandibular gland Parotid gland Parotid gland Submandibular gland Parotid gland Submandibular gland
>1
4.5 x 3.5 × 3.0
>2 >18 >5
1.0 × 0.5 × 0.5 2.5 x 2.0 × 1.5 2.0 x 1.5 x 1.5
>1 >1/2
1.5 x 1.0 × 1.0 5.5 x 3.0 × 3.0
Localization
9 22 12 18 19 27 30 34 37 41 16 17 21 28 32 19 22 31 23 31
2 3 1 2 3 4 la 2c 3d 4b 1 2 I 2 3 1 2 3 1 1
11
1
43
2
33 11
11
2z
22
11 22 53
11
83 11 1l 22 42
32
55 22 11 44 44 5s 95 116
44
33 22
45
11
44 2515 30 zs 2015 2921 2814 2111 147 2213 1812 2713 30 TM 3z 1811 56 ~5 2111 4217 3814 84 169 156 84 7s 22 TM 25 TM 43
46
11
33 33
3~
11
47
11
22
48
1
49
"Superscribed n u m b e r s indicate n u m b e r a n d d i s t r i b u t i o n of k a r y o t y p e d cells. bRoman n u m e r a l s d e n o t e m a r k e r c h r o m o s o m e s . ~I'he letters a through d indicate different areas of the tumor.
S 9
7
6
5
4
3
8
1
2
9 14 15 21
1 2 3 4
1
42
50
51
52
Chromosome counts a
Chromosome counts and stem- side line karyotypes of the tumors
Days Case Preparation c in vitro
Table 2
2
53
41
54
11
87
11
89
11
90
11
91
1
15
11
11
92 29 TM 3227 2015 3123 328 2614 17 I° 2617 1812 33 TM 328 43 2417 634° 3125 5122 5220 117 2214 156 2112 9s 2414 27 TM 196
Total cells 4 6 , X X , 6 q - (1),9p + (II)/46,XX 46,XX,6q-(1),9p+(II)/46,XX 4 6 , X X , 6 q - (1),gp + (II]/46,XX 4 6 , X X , 6 q - (l],9p + (II)/46,XX 46,XX 46,XX 46,XX 46,XX 46,XX 4 6 , X X / 4 6 , X X , 6 q - (1) 46,XX,6q - (1)/46,XX 46,XY 46,XY 46,XY 46,XY 4 6 , X Y / 4 5 , X , - Y / 4 6 , X Y , - 13, + 15 46,XY/45,X, - Y 46,XY/47,XY, + 8 46,XY/47,XY,+8 46,XY 46,XX 46,XX 46,XX 46,XX 46,XY
Stem- side line k a r y o t y p e s b
tO O0
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Table 3
Karyotypic deviations in diploid-near-diploid stem- and side line karyotypes
Case
Preparation b
1
1 2 4
2
1
3
2 3 1 2 3
4
5
v a r i a n t c e l l s i n r e l a t i o n to t h e
Variant cells in relation to stem-(sl) side line (sdl) karyotypes ~ sl sdl 1 sl sdl 1 sl sdl 1 sl
4 la 2c 3d 4b
sl sl sl sl sl sdl 1 sdl 2 sl sl sl sl
1 2
sl sl
6 7
2 1
sl sl
8 9
2 3 1 1
sl sl sl sl
- 6 q - (1)/- 9p + (II), - 1 5 / - 9p + (II), - 9 - 2 1 / - 21 - 18 -21 6 q - {I},- 9p + (II), + 9 / - 2 0 , - X -9 8 q - (I),9q + (II)/8p- (III),21q+ (IV)/- 3 / - 1 2 / - 2 , - 5, - 6, + 8 , - 9(4n = 89)/ - 1 7 , - 1 8 , - 2 2 , - X , - X (4n = 87) + 16, - 17/- 1 2 / - 2 2 / - 22 8 q - (V),Xq+ (VI)/÷ 1 5 / X q - (VII) ( 4 n = 9 2 ) / - 3 , - 1 7 , - X (4n=89)
11q+(II),-8q-/-6/-9/-20 6q - (I) 2 q - (III),17p + (IV)/18q + (V),- 1 5 q - / - 10, + 12, - 1 9 / - 2 0 / - 20 6 q - (I}, - X 9q - (VI)/+ 3, - 8 / - 2 0 / - 20 -22 - 6 / - 9 / - 17/- Y / - Y, - 1 0 / - Y, - 22 - 1 6 / - 1 6 / - 2 2 / - Y/+ 1/+ 7/+ 22 + 1, + 1 0 / - 2 , - 1 6 / 3 q - (I), - Y / - 8, - 1 6 / - 1 6 / - 16, + 2 0 / - 1 2 / - Y / - Y / - Y , +7,+8,+20 2 p - (I)/+ 1 , - 6 , - 7 , + 8
6q-(II)/6q-(II)/-13,+15/-13,+15/-13/-9,-9/-6,-6,+6q-(III), + 6q - (Ill), + 6q + (IV), - 8, - 8, - 16, - 18, + 18q + (V], + 19, + 20(4n = 9 1 ) / - Y, - Y (4n = 90) - X/- Y
-8,-15,-17,-18/12q+(I),-20,-X/-5,-19/-5,-22/-8/-17/-X/ 3q - (II}, + 8/5p - (III) - 6, - 2 2 / 5 q - (IV), - 18
3q-(V},IOq+(VI}/3p+(VII),5p-(VII)/-X/2q+(IX},3q-(X),+8,+20 -13/-6,+9,-X - Y / 6 q - (I),8q- (II),11q- (III),- 1 5 , - 16,17p + (IV),- 18,+ 16p + (V), + 16p
+(V)/+ l,+2,+3q-(VI),+3q-(VI),+ 7,+9,+10,+ llq-(III),-22 °Roman numerals denote marker chromosomes presented in Table 4. ~The letters a through d indicate different areas of the tumor.
Case 1 All preparations from this adenoid cystic carcinoma showed a pseudodiploid stem l i n e c h a r a c t e r i z e d b y a r e c i p r o c a l t(6;9) ( m a r l a n d m a r I I i n T a b l e 4; Fig. 1). A s i d e l i n e (sdl 1) w i t h a n o r m a l k a r y o t y p e w a s also o b s e r v e d i n all p r e p a r a t i o n s . B e t w e e n 1 5 % a n d 3 5 % of t h e a n a l y z e d c e i l s i n t h e d i f f e r e n t p r e p a r a t i o n s b e l o n g e d to s d l 1. I n a d d i t i o n , t h e first a n d s e c o n d p r e p a r a t i o n s also c o n t a i n e d a p s e u d o d i p l o i d s i d e l i n e (sdl 2); it d i f f e r e d f r o m t h e s t e m l i n e b y a loss of m a r I I a n d a g a i n of o n e n o r m a l c h r o m o s o m e # 9 . F u r t h e r m o r e , 10 v a r i a n t c e l l s w i t h n u m e r i c a l a n d / o r s t r u c t u r a l d e v i a t i o n s w e r e r e c o r d e d f r o m t h e f o u r p r e p a r a t i o n s ( T a b l e 3).
Case 2 T h i s a d e n o i d c y s t i c c a r c i n o m a s h o w e d a n o r m a l d i p l o i d s t e m l i n e i n all t h r e e p r e p o r a t i o n s . In a d d i t i o n , all of t h e m c o n t a i n e d s t r u c t u r a l l y or n u m e r i c a l l y c h a n g e d v a r i a n t c e l l s ( T a b l e 3). T h r e e of t h e s e s h o w e d r e c i p r o c a l t r a n s l o c a t i o n s a f f e c t i n g # 8 , w h e r e a s , t w o s h o w e d r e a r r a n g e m e n t s i n v o l v i n g t h e l o n g a r m of t h e X c h r o m o some.
6q- a n d -Y in M a l i g n a n t S a l i v a r y G l a n d T u m o r s
Table 4
D e r i v a t i o n of the d i f f e r e n t m a r k e r c h r o m o s o m e s o b s e r v e d in s t e m lines, side lines, a n d v a r i a n t cells
Case 1
287
Marker
Derivation
I = 6q II = 9p +
t(6;9)(6pter--~6q24::9p23--~9pter) t(6;9)(9qter--~9p23 ::6q24--~6qter)
I=8qII=9q+ IlI = 8p IV=21q+ V=8qVI = Xq + VII = Xq -
t(8;9)(8pter--~cen--~9qter) t(8;9)(9pter-~cen--~8qter} t(8;21)(8qter-~8p21::2 lq22--~2 lqter) t(8;21)(21pter-~21q22::8p21-~8pter) t(X;8}(8pter--~8q12::Xq27--~Xqter) t(X;8}(Xpter--~Xq27::8q12--~8qter) del(X)(q13}
I=6qII = 11q + III = 2 q IV=17p+ V=18q+ VI = 9q -
del(6)(pter-~q16::q22--~qter} t{8?;11){11pter--~11q23::8q12 - 21--~qter) t(2; 17)(2pter-~2q13 ::17p13--~17pter) t(2; 17)(17qter--~ 17p13::2q13--~2qter) t(15;18)(18pter-~18q23::15q13--~15qter) del(9)(q31)
I=3q-
del(3)(q11-12)
I=2pII = 6q III = 6 q IV = 6q + V = 18q +
del(2}(p23) del(6)(q22) i(6p) dic(6)(pter--~q15::q15--~pter) i(18q)
1-12q+
IV = 5q V=3qVI = lOq + VII = 3p + VIII = 5p IX = 2q + X=3q-
t(12;?)(12pter- 12q24::?) i(3p) del(5)(p11) del(5)(q23) t(3;10)(3pter~3q22::10q24-~10qter) t(3;10)(10pter--~10q24::3q22---~3qter) t(3;5)(3qter--~3p27::5p12-)5pter) t(3;5)(Sqter-~5p12::3p27-*3pter) t(2;3)(2pter--~2q36 - 37::3q22--~3qter) t(2;3)(3pter->3q22 ::2q36 - 37--~2qter)
I = 6qII = 8 q III= 1 1 q IV= 17p + V = 16p + ? VI = 3 q -
i(6p) del(8)(q22) del(11}(q22) i(17q) i(16q) del(3)(q21)
II = 3q Ill
-
= 5p
-
Case 3 T h e first three p r e p a r a t i o n s f r o m this r e c u r r e n t a d e n o i d cystic c a r c i n o m a w e r e chara c t e r i z e d b y a n o r m a l d i p l o i d s t e m line. T h e f o u r t h p r e p a r a t i o n , h o w e v e r , s h o w e d a p s e u d o d i p l o i d s t e m l i n e d i s t i n g u i s h e d b y an interstitial l o n g a r m d e l e t i o n of o n e c h r o m o s o m e # 6 (marI in T a b l e 4; Fig. 2). T h i s s t e m l i n e c o u l d be t r a c e d to an i d e n t i c a l p s e u d o d i p l o i d side l i n e (sdl 1) a p p e a r i n g in t h e t h i r d p r e p a r a t i o n a n d c o m p r i s i n g a b o u t 37% of t h e a n a l y z e d cells. F u r t h e r m o r e , in the last p r e p a r a t i o n
288
G. S t e n m a n et al.
S
t2
I$
t4 ¸
19
20
1|
1|
21
22
t7
tl
X
X
Figure 1 Top: G-banded karyotype of a stem line cell of case 1 [46,XX,t(6;9) (q24;p23)]. Bottom; The marker chromosomes in higher magnification showing the reciprocal translocation between 6q and 9p. (Top, x 2600; bottom, x 3700) r e m n a n t s of the former normal stem line formed a side line (sdl 2) comprising about 33% of the analyzed cells. The characteristics of the variant cells in the four preparations are given in Table 3. Case 4 Four different areas from this a d e n o i d cystic carcinoma were studied. At least two preparations from each area were analyzed. In Table 2 the preparations from each area are c o m b i n e d and presented as a single fixation Ca-d). The outgrowth from all four areas was extremely slow. In all areas a normal diploid stem line was found. D i p l o i d - n e a r - d i p l o i d variant cells, however, were recorded in all areas, and espe-
289
6q- and -Y in Malignant Salivary Gland Tumors
XHn ?
z
a 14
11
20
15
I
21
iS
Figure 2 Leftand right: Complete and partial G-banded karyotypes of two stem line cells of case 3; 46,XX, del(6)(q16q22}. (Left, × 2600; right, × 2800} cially in area b (Table 3). Except for one structurally changed cell, all others showed numerical changes, especially losses. An evolutionary change in common for three of the areas was the occurrence of hypodiploid variant cells with a loss of the Y chromosome. Case 5
Both preparations from this acinic cell tumor showed a normal diploid stem line and also a hypodiploid side line (constituting about 25% of the analyzed cells in both preparations). The only deviation in this side line was a loss of the Y chromosome (Fig. 3). The first preparation also contained a small pseudodiploid clone or side line consisting of four cells with monosomy 13 and trisomy 15, Variant cells and clones, with structural changes affecting particularly the long arm of #6 were also seen in both preparations (Table 3). Case 6
All three preparations from this acinic cell tumor showed a normal diploid stem line. Preparations one and two also contained a small hyperdiploid side line characterized by trisomy 8. Only two variant cells were seen and both showed a loss of a gonosome. No markers were observed in any of the analyzed cells. Case 7
A normal stem line was seen in all three preparations from this moderately to poorly differentiated adenocarcinoma. Also, a variety of numerically and/or structurally changed variant cells were found in the different preparations (Table 3). No less than 10 different markers could be identified. Both arms of chromosomes #3 and/or #5 were involved in the formation of most of the markers.
290
G. Stenman et al.
Figure 3
Complete G-banded karyotype of a side line cell of case 5; 45,XO, -Y. (× 2800)
Case 8
Due to extremely slow and sparse outgrowth from this poorly differentiated adenocarcinoma it was only possible to obtain one successful preparation. Except for two hypodiploid cells (Table 3), only cells with a normal karyotype were found. No cells with structural changes were observed. Case 9
The growth of this tumor, a poorly differentiated mucoepidermoid carcinoma, was also slow and sparse. Only a few banded metaphases from one preparation could be studied. Available data indicate a bimodal distribution. Except for three normal diploid ceils there were a few hypodiploid and hyperdiploid variant ceils (Table 3). An l l q - marker (marlII in Table 4) was seen both in the hypodiploid and in the hyperdiploid cells indicating a clonal origin of both modal groups. One or two long arm deleted chromosomes #3(marVI in Table 4) were also seen in two further hyperdiploid cells not suitable for complete analyses. DISCUSSION Almost all of the comparatively uncommon malignant tumors of the salivary glands are carcinomas. Most of them are found in the major glands, particularly the parotid gland. Here the most common types are mucoepidermoid tumors, undifferentiated carcinomas, acinic cell tumors, adenocarcinomas, and adenoid cystic carcinomas,
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in that order of frequency [8]. Almost all of the remaining carcinomas in the major glands are found in the submandibular gland. In this location adenoid cystic, undifferentiated, and epidermoid carcinomas outnumber the other types. The adenoid cystic carcinoma is the predominating type also in minor salivary glands. Except for the undifferentiated and epidermoid carcinomas, all the other types are represented in our series. The observations in this heterogeneous material are primarily best considered separately for each tumor type. In this context comparisons will be made with available, though very scanty, data in the literature. Together with a previously published case of adenoid cystic carcinoma [4] banding data are available from five adenoid cystic carcinomas. Three of these cases have (in one or more preparations) shown pseudodiploid stem lines characterized by one of the following structural changes: t(5;17)(q22;q13), t(6;9)(q24;p23), and del(6)(q16q22). The remaining two cases had normal stem lines. However, these two cases (as those three with an abnormal stem line) contained a great variety of variant cells, clones, and side lines. It should be noted that three of the tumors showed stem lines and/or variant cells with anomalies affecting the terminal part of the long arm of chromosome #6 (i.e., 6q16-25). It is interesting to note that the cmyb oncogene has been localized to this region of #6 (6q22-24) [9]. An interesting evolutionary finding was seen in side lines and variant cells of case 1. Here the 9p + marker, containing the translocated distal segment of #6, was lost and replaced by a normal #9, resulting in monosomy for 6q24-qter and trisomy for 9p23-pter. These observations stress the significance of rearrangements involving the terminal region of the long arm of #6. A feature characteristic of case 2 was the involvement of chromosome #8 and the X chromosome in three and two different rearrangements, respectively. Case 4 showed an unusually high frequency of variant cells with a loss of the Y chromosome. The common involvement of these three chromosome types is of special interest, because they are frequently affected also in other types of salivary gland tumors. Case 3, as well as the previously published case of adenoid cystic carcinoma [4], is informative regarding the influence of prolonged growth in vitro on the chromosomal picture. Thus, in spite of a long in vitro growth period, both cases showed a change from a normal stem line to an abnormal stem- side line in subsequent preparations. The similarities between these "late" deviations and those observed after short periods in vitro argue against the possibility of induced changes in vitro. The most striking observation in both preparations of case 5 (an acinic cell tumor) was a hypodiploid side line with only a loss of the Y chromosome. The other acinic cell tumor (case 6) also showed variant cells with a loss of gonosomes. Furthermore, case 5 showed a small pseudodiploid clone with a long arm deleted #6, as well as a hypotetraploid cell with loss of the two Y chromosomes and extensive structural rearrangements involving both arms of #6. These findings are in agreement with observations from a previously reported case of acinic cell tumor [3], which inter alia showed stem- and side lines characterized by either Y loss or a reciprocal translocation involving chromosome #6 [i.e., (t(6;21)(q13;q22)]. Another interesting observation in case 6 was the finding of a hyperdiploid side line with trisomy 8. In salivary gland tumors this anomaly has been observed earlier in pleomorphic adenomas. Whether or not trisomy 8 is of the same importance for chromosomal progression in acinic cell tumors as in pleomorphic adenomas, however, remains to be clarified. Accordingly, our preliminary observations indicate that deviations affecting the Y chromosome (losses) and, to a lesser extent, #6 (structural changes) and #8 (gains) are the three deviations typical of early karyotypic evolution in acinic cell tumors.
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Both a d e n o c a r c i n o m a s (cases 7 and 8) s h o w e d normal stem lines. However, variant cells w i t h n u m e r i c a l and/or structural rearrangements were seen, especially in c a s e 7. Both a r m s of c h r o m o s o m e s # 3 and/or # 5 were i n v o l v e d in most rearrangements in this case. It s h o u l d also be n o t e d that several variant cells with loss of one X c h r o m o s o m e were observed in both cases. This is of interest, as an earlier rep o r t e d case of a d e n o c a r c i n o m a [5] had a stem line with only a gonosomal deviation (loss of the Y chromosome). Even though the present 12 cases of m a l i g n a n t salivary gland tumors constitute a heterogeneous group, e n c o m p a s s i n g no less than four different histopathologic types, t h e y seem to have certain c h r o m o s o m a l characteristics in common. Thus, all types have a p p a r e n t l y originated w i t h a n o r m a l d i p l o i d stem line. During progression they have frequently d e v e l o p e d p s e u d o d i p l o i d or h y p o d i p l o i d stem- and side lines. For each t u m o r type there seemed to exist more than one progressional pathway, and some of the p a t h w a y s might well be shared by more than one t u m o r type. The deviations seen most frequently in the stem- side lines of the 12 cases were different structural changes of the long arm of # 6 and loss of the Y chromosome. Some of the above described features typical of the malignant t u m o r types ar e similar to those observed in benign p l e o m o r p h i c a d e n o m a s i n d i c a t i n g that, to a certain extent, c h r o m o s o m a l progression might follow similar p a t h w a y s in both benign and m a l i g n a n t salivary gland tumors. These observations and ideas s u p p o r t our previously a d v a n c e d h y p o t h e s i s that the characteristics of the c h r o m o s o m e changes in a certain t u m o r t y p e are to a m u c h greater extent d e t e r m i n e d b y tissue specific factors than by the nature of the i n d u c i n g agent[s]. This is also in line w i t h findings in some other t u m o r groups, such as 14q + markers in different l y m p h o i d malignancies [10], and the i n v o l v e m e n t of c h r o m o s o m e # 2 2 in different n e o p l a s m s of the nervous system [11, 12]. Supported by grants from the Swedish Cancer Society and from the Swedish Medical Research Council, project no. 7327.
REFERENCES 1. Mark J, Dahlenfors R, Ekedahl C (1983}: Cytogenetics of the human mixed salivary gland turnout. Hereditas 99:115-129. 2. Stenman G, Mark J, Ekedahl C (1984~: Relationship between chromosomal patterns and proto-oncogenes in human benign mixed salivary gland tumors. Tumour Biol 5:103-117. 3. Mark J, Ekedahl C, Dahlenfors R (1981): Polyclonal chromosomal evolution in a cultured human acinic cell tumour. Anticancer Res 1:45-48. 4. Stenman G, Dahlenfors R, Mark J, Sandberg N (1982): Adenoid cystic carcinoma: A third type of human salivary gland neoplasm characterized cytogenetically by reciprocal translocations. Anticancer Res 2:11-16. 5. Stenman G, Mark J (1983): Loss of the Y chromosome in a cultured human salivary gland adenocarcinoma. J Oral Pathol 12:458-464. 6. Mark J, Dahlenfors R, Ekedahl C, Stenman G (1980): The mixed salivary gland tumor A normally benign human neoplasm frequently showing specific chromosomal abnormalities. Cancer Genet Cytogenet 2:231-241. 7. ISCN (1978): An international system for human cytogenetic nomenclature. Cytogenet Cell Genet 21:309-404. 8. Enaroth CM (1971): Salivary gland tumors in the parotid gland, submandibular gland and the palate region. Cancer 27:1415-1418. 9. Harper ME, Franchini G, Love J, Simon MI, Gallo RC, Wong-Staal F {1983): Chromosomal sublocalization of human c-myb and c-fes cellular onc genes. Nature 304:169-171.
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10. Sandberg, AA (1980): The Chromosomes in Human Cancer and Leukemia. Elsevier-North Holland, NY, pp. 377-412. 11. Mark] (1977): Chromosomal abnormalities and their specificity in human neoplasms--An assessment of recent observations by banding techniques. Adv Cancer Res 24:165-222. 12. Bigner SH, Mark J, Mahaley MS, Bigner DD (1984): Patterns of the early, gross chromosomal changes in malignant human gliomas. Hereditas 101:103-113.