Multiple Unrelated Chromosome Abnormalities in a Metastatic Mucoepidermoid Carcinoma of the Parotid Gland Anders Nordkvist, Staffan Edstr6m, Joachim Mark, and G6ran Stenman

ABSTRACT: We describe cytogenetic findings in a poorly differentiated, metastatic mucoepidermoid carcinoma of the parotid gland. The t u m o r was characterized by multiple, unrelated chromosome abnormalities. Except for two small aberrant clones showing t(1;7) and t(2;15), respectively, all other abnormal cells showed unique, mostly structural rearrangements peculiar to each cell. No less than 34 different abnormal karyotypes were observed. A similar karyotypic heterogeneity was also described recently in s q u a m o u s cell carcinomas of the head and neck.

INTRODUCTION Malignant tumors of the salivary glands constitute a morphologically and clinically heterogeneous group of neoplasms. Clinically, the m u c o e p i d e r m o i d and acinic cell tumors present a special p r o b l e m because both benign (lowgrade) and malignant (high-grade) variants of these tumors exist [1, 2]. Most m u c o e p i d e r m o i d tumors are well-differentiated tumors w h i c h behave in a c o m p l e t e l y benign fashion, but, about 1 5 - 2 0 % of the m u c o e p i d e r m o i d tumors are aggressive poorly differentiated tumors w h i c h metastasize and show a high recurrence rate. These tumors have a poor prognosis, with a 5-year survival rate of less than 40% [2]. We describe the cytogenetic findings in a tumor of the latter type, i.e., a poorly differentiated, metastatic mucoepiderm o i d carcinoma of the parotid gland.

MATERIALS AND METHODS Case Report A 63-year-old p r e v i o u s l y healthy man sought medical help in A p r i l 1986 for a t u m o r mass in the left parotid gland. A fine-needle aspiration b i o p s y i n d i c a t e d a malignant epithelial tumor. The tumor, measuring 1.0 × 1.5 cm, was removed by a parotidectomy. Light-microscopic examination of the surgical s p e c i m e n s h o w e d a poorly differentiated m u c o e p i d e r m o i d carcinoma. A n extended local resection

From the Laboratory of Cytogenetics, Departments of Oral Pathology (A. N., G. S.), and Otorhinolaryngology (A. N., S. E.), University of G6teborg, G6teborg, and Department of Pathology (J. M.), Central Hospital, Sk6vde, Sweden. Address reprint requests to: Dr. G6ran Stenman, Laboratory of Cytogenetics, Department of Oral Pathology, University of G6teborg, Medicinaregatan 12, S-413 90 G6teborg, Sweden. Received December 13, 1991; accepted February 6, 1992. 158 Cancer Genet Cytogenet 61:158 161 (1992) 0165-4608/92/$05.00

and a neck dissection were performed. The patient also received postoperative radiation t h e r a p y (60 Gy). Examination in January 1988 s h o w e d m u l t i p l e t u m o r n o d u l e s on the left side of the neck in the s u p r a c l a v i c u l a r region. The nodules were removed together w i t h the overlying skin, and the w o u n d was covered w i t h a split skin graft. The largest nodule, measuring 0.5 × 1.2 cm, was taken for cytogenetic analysis. A 0.5-cm 2 b i o p s y was also taken from irradiated skin about 5 cm a w a y from the t u m o r n o d u l e s in an area clinically free of t u m o r tissue. Histopathologic examination s h o w e d that tumor n o d u l e s r e p r e s e n t e d cutaneous metastases of the m u c o e p i d e r m o i d c a r c i n o m a (Fig. 1). In the next 12 months the patient d e v e l o p e d several additional cutaneous metastases as well as m u l t i p l e lung metastases. Interferon treatment was initiated with no effect. Palliative r a d i o t h e r a p y was given to the left side of the neck and the thoracic region. C h e m o t h e r a p y (5-fluorouracil and methotrexate) was also initiated in June 1989 and was a d m i n i s t e r e d in rounds until March 1990. The patient res p o n d e d poorly to this treatment as well, and the lung and cutaneous metastases increased in n u m b e r and size. The patient deteriorated and died of respiratory failure in December 1990.

Cytogenetic Analysis Fresh tumor tissue from a cutaneous metastasis was m i n c e d into small pieces and digested in a collagenase solution (1,000 U / m l ) for 45 m i n u t e s [3]. The resulting tumor cell s u s p e n s i o n was seeded in 25-cm 2 flasks in Dulbecco's m i n i m u m essential m e d i u m (DMEM)/Ham's F12 (1:1) s u p p l e m e n t e d with mitogenic substances as previously described [3]. The skin b i o p s y was cut in small pieces and e x p l a n t e d in 25-cm 2 flasks in Eagle's c~-MEM s u p p l e m e n t e d with 10% fetal calf serum (FCS), 5 /~g streptomycin/mL, 200 U b e n s y l p e n i c i l l i n - K / m l , and 200 mM L-glutamine. © 1992 Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas, New York, NY 10010

C y t o g e n e t i c H e t e r o g e n e i t y in a m u c o e p i d e r m o i d C a r c i n o m a

159

T h e m e t h o d s for c h r o m o s o m e p r e p a r a t i o n and G - b a n d i n g w e r e as p r e v i o u s l y d e s c r i b e d [4]. M a g n i f i e d p h o t o g r a p h s w e r e u s e d for k a r y o t y p e analysis. T h e n o m e n c l a t u r e f o l l o w s that of the I n t e r n a t i o n a l S y s t e m for H u m a n Cytogenetic N o m e n c l a t u r e (ISCN)[5].

RESULTS

F i g u r e 1 Histologic appearance of a cutaneous metastasis of the mucoepidermoid carcinoma. Growth of mainly poorly differentiated epidermoid cells and occasional mucus-secreting cells. (H & E, original magnification x 320.)

Table 1

Cells f r o m the m u c o e p i d e r m o i d c a r c i n o m a w e r e karyot y p e d after 16 days in vitro. T h e cells had an e p i t h e l i a l in vitro g r o w t h m o r p h o l o g y . Less t h a n 5% of the cells had a fibroblastic a p p e a r a n c e . T h e c h r o m o s o m a l f i n d i n g s in the 44 cells k a r y o t y p e d are s h o w n in Table 1. All cells h a d c h r o m o s o m e c o u n t s in the d i p l o i d - h y p o d i p l o i d region. Exc e p t for n i n e cells w i t h a n o r m a l d i p l o i d k a r y o t y p e , all cells had an aberrant karyotype. S t r u c t u r a l r e a r r a n g e m e n t s p r e d o m i n a t e d and w e r e o b s e r v e d in 29 cells, w h e r e a s five cells had o n l y n u m e r i c a l d e v i a t i o n s . T h e k a r y o t y p i c variability a m o n g the aberrant cells w a s c o n s i d e r a b l e ; all but four c h r o m o s o m e types w e r e i n v o l v e d in s t r u c t u r a l rearrangements. No less t h a n 23 u n i q u e t r a n s l o c a t i o n s and n i n e d e l e t i o n s w e r e r e c o r d e d (Table 1), b u t o n l y t w o of the

K a r y o t y p i c characteristics of 44 a n a l y z e d cells f r o m the m u c o e p i d e r m o i d c a r c i n o m a

Karyotype 42,XY,del(2)(p21),- 5,del(7)(q11.1-11.2),- 8 , - 1 1 , - 12,del(12)(q12) 42,XY,der(6)t(6;?)(q12-14;?) - 9 , - 16, - 17,der(18)t(18;?)(q23;?),- 2 0 , - 22, + 2mar 43,XY,der(1)t(1;7)(p13;q11.2),- 7 , - 10, - 11 43,X, - Y,der(3)del(3)(p14)del(3)(q11.2-12), - 8, - 15 44,XY, - 6, - 10 45,X - Y,t(1;14)(p22;q13) 46,XY + 2,der(4)t(4;8)(q21;qll - 12),del(5)(q21),- 8 45,XY,t(2;15)(q35;q15),- 16 45,XY,der(9)t(9;9)(p12;q12),- 16, + 17 45,X - Y,t(6;7)(p25;p12) 45,XY,t(7;16)(q21;p12),- 22 45,XY, - 9 45,XY,- 15,der(17),t(15;17)(q22;q24) 45,XY, - 22 46,XY,t(1;7)(p13;q11.2) 46,X,-Y,+ 2 46,XY,del(2)(q11.2), + 5, + 7,t(7;17)(q31 ;q12),- 8 , - 9,del(9)(q12),- 16 46,XY,del(2)(q14.1-14.3) 46,XY,t(2;15)(q35;q15) 45,Y, - X,t(2;15)(q35;q15), + 4,t(9;12)(p12;q12), - 19 46,XY,t(3;12)(p21;q13) 46,XY,inv(3)(p24q13.1) 46,XY,t(4;17)(p14;q11.2) 46,XY, - 4,der(4)t(4;15?)(p16;q15?), - 7, + 9, + 10, + 14, + 14, - 15, - 21 46,XY,t(4;17)(q21;q12) 46,XY,t(7;12)(p12-13;q22) 46,XY,t(9;12)(q21;q22) 46,XY,t(9;12;16)(q12;q13;p13.3),del(10)(q24) 46,XY,t(10;13)(p12;q21) 46,XY,t(10;21)(q22;q22) 46,XY, + 14, - 15 46,Y,t(X;3)(q23-25;q13.3) 46,Y,t(X;7)(p21;p12) 46,Y,t(X;19)(q13 ;q13.3-13.4) 46,XY

No. of cells

160

A. Nordkvist et al. Table 2

S u m m a r y of karyotypic abnormalities in six previously p u b l i s h e d m u c o e p i d e r m o i d tumors

Case

Chromosomal findings

Reference

9 22 23 24 25

46,XY/41-50,XY,del(3)(q21),del(11)(q22) + other abnormalities 49,XX,+ 3,+ 7 , + 13,+ 15,- 21/46,XX 45,X, Y,t(11;17)(q22;p11)/46,XY,t(11;17)(q22;p11)/46,XY 46,XY/46,XY,del(6)(q25)/45,X, - Y 46,XX/47,XX, + 8 46,XX,t(3;8)(p21;q12),del(5)(q22)

[15] [3] [31 [3] [3] [16]

translocations were recurrent, i.e., a t(1;7)(p13;q11.2) and a t(2;15)(q35;q15) in three cells each. No other cells with the same karyotype were observed. Despite this heterogeneity, a preferential involvement of certain chromosomes in the structural variation could be discerned. Chromosome 7 was involved in seven different rearrangements, chromosomes 9, 12, and 15 were involved in five rearrangements each, and chromosomes 4 and 17 were involved in four each. Recurrent numerical changes were much less common than the structural changes and i n c l u d e d two cells with trisomy 2, two cells with gain of one or two chromosomes 14, and three or more cells with loss of chromosomes 8, 9, 15, 16, 22, and Y, respectively. Twenty fibroblast cells derived from the skin b i o p s y were also karyotyped. Eighteen of these had a normal karyotype, i n c l u d i n g a few cells with r a n d o m losses of single chromosomes. There were also two near-triploid cells, one of w h i c h contained two marker chromosomes; the other showed del(3)(q13.3) and del(3)(q24), and two markers. DISCUSSION

We describe the cytogenetic findings in a poorly differentiated, metastatic m u c o e p i d e r m o i d carcinoma of the parotid gland. The tumor was characterized by m u l t i p l e unrelated c h r o m o s o m e abnormalities, except for two small aberrant clones [three cells each with t(1;7) and t(2;15)]; all other abnormal cells s h o w e d unique, mostly structural rearrangements peculiar to each cell. In our previous series of more than 250 benign and malignant salivary gland tumors [6; u n p u b l i s h e d observations] a similar heterogeneous picture was observed in only one case of pleomorphic a d e n o m a [7]. The polyclonal pattern observed in that tumor was believed to be due to radiographic treatment of tuberculous l y m p h a d e n i t i s of the neck during childhood. A similar p o l y c l o n a l pattern was also detected in fibroblasts from a b i o p s y taken from irradiated skin adjacent to the tumor of the same patient. No normal cells were detected in the skin biopsy. Our patient had no previous history of radiation therapy before d e v e l o p m e n t of the carcinoma, but 1½ years before removal of the metastatic lesion described in this report, the patient received radiation therapy to the left side of the head and neck (60 Gy). Cytogenetic analysis of the biopsy taken from clinically tumor-free irradiated skin showed, with the exception of two cells, only normal karyotypes. Because the patient had m u l t i p l e cutaneous metastases in the region, the two a n e u p l o i d cells detected might well

represent microscopic spread of the neoplastic process. These findings suggest that the rearrangements observed in the tumor cells are probably related to the neoplastic process rather than being radiation i n d u c e d , but we cannot rule out the possibility that some of the m a n y rearrangements detected in the t u m o r were i n d u c e d during irradiation because that radiation induces an increased frequency of c h r o m o s o m e breaks [8]. That we could not detect any clonal abnormalities c o m m o n to most or all of the cells suggests that the tumor might have originated with a grossly normal karyotype subjected to s u b m i c r o s c o p i c changes at one or more loci and that the heterogeneous pattern of rearrangements detected in the tumor reflects a p r o n o u n c e d chromosomal instability in a dedifferentiated, highly malignant metastastic carcinoma. An alternative e x p l a n a t i o n of our findings w o u l d be that the t u m o r actually has a multicellular origin. Our k n o w l e d g e about the chromosomal patterns in benign and malignant salivary gland tumors do not support the concept of a p o l y c l o n a l origin of salivary gland tumors, however. A similar karyotypic heterogeneity was also observed p r e v i o u s l y in several other types of e p i d e r m o i d tumors, i n c l u d i n g squamous cell carcinomas and p a p i l l o m a s of the skin, larynx, and nasal mucosa [9-12]. The characteristic findings in these tumors were multiple, often complex, unrelated clonal chromosome abnormalities. The lack of a c o m m o n cytogenetic d e n o m i n a t o r in squamous cell tumors argues that these tumors might be of multiclonal origin or alternatively that the primary change is a s u b m i c o s c o p i c lesion (9). Whether this remarkable cytogenetic heterogeneity and c o m p l e x i t y is an inherent characteristic of several types of epithelial tumors remains to be clarified. Despite the p r o n o u n c e d karyotypic heterogeneity in the present m u c o e p i d e r m o i d tumor we c o u l d recognize a clustering of breakpoints at certain c h r o m o s o m e regions. Thus, the c h r o m o s o m e bands or segments l p 1 3 , 2q35, 7q11.2, 9p12-q12, 12q12-13, and 15q15 were each involved in three or m o r e arrangements. The 9 p 1 2 - 2 2 and 12q13-15 regions are also involved in structural rearrangements in p l e o m o r p h i c a d e n o m a s of the salivary glands [6, 13, 14]. A subgroup of these tumors has a reciprocal t(9;12)(p12-22;q13-15). An a p p a r e n t l y identical t(9;12) was also observed in one cell in the present m u c o e p i d e r m o i d carcinoma. Few m u c o e p i d e r m o i d tumors have been s t u d i e d cytogenetically. The c h r o m o s o m a l findings in five cases described by Stenman et al. [15] and Sandros et al. [3] and in one case described by Bnllerdiek et al. [16] are s u m m a r i z e d in Table

C y t o g e n e t i c H e t e r o g e n e i t y in a m u c o e p i d e r m o i d C a r c i n o m a

2. In all five cases, c l o n a l c h r o m o s o m e a b n o r m a l i t i e s p e c u liar to e a c h case w e r e o b s e r v e d . T h e o n l y r e c u r r e n t abnorm a l i t i e s o b s e r v e d w e r e loss of the Y c h r o m o s o m e and structural r e a r r a n g e m e n t s of b a n d 11q22 in t w o t u m o r s each. T h e p a t t e r n in the p r e s e n t m u c o e p i d e r m o i d t u m o r is also quite different f r o m that in the five p r e v i o u s l y r e p o r t e d tumors. F u r t h e r s t u d i e s are n e e d e d , h o w e v e r to establish w h e t h e r this g r o u p of t u m o r s a c t u a l l y s h o w s any c o m m o n chromosome abnormalities. This study was supported by grants from the GOteborg Medical Society, The Swedish Medical Society, and the Swedish Cancer Society.

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7. Mark J, Ekedahl C (1987): Polyclonal chromosomal evolution in a benign mixed salivary gland tumor. Cancer Genet Cytogenet 28:237-243. 8. Therman E (1985): Human Chromosomes--Structure, Behavior, Effects, 2nd Ed Springer Verlag, New York, pp. 79-80, 83. 9. Helm S, Mertens F, Jin Y, Mandahl N, Johansson B, Bi6rklund A, Wennerberg J, Jonsson N, Mitelman F (1989): Diverse chromosome abnormalities in squamous cell carcinomas of the skin. Cancer Genet Cytogenet 39:69-76. 10. Jin Y, Helm S, Mandahl N, Bi6rklund A, Wennerberg J, Will6n R, Mitelman F(1989): Two unrelated clonal chromosome rearrangements in a nasal papilloma. Cancer Genet Cytogenet 39:29-34. 11. Jin Y, Helm S, Mandahl N, Bi6rklund A, Wennerberg J, Mitelman F (1990): Multiple clonal chromosome aberrations in squamous cell carcinomas of the larynx. Cancer Genet Cytogenet 44:209-216. 12. Mertens F, Helm S, Jin Y, Johansson B, Mandahl N, Bi6rklund A, Wennerberg J, Jonsson N, Mitelman F (1989): A benign epithelial skin tumor with multiple cytogenetic clones. Cancer Genet Cytogenet 37:235-239. 13. Bullerdiek J, Chilla R, Haubrich J, Meyer K, Bartnitzke S (1987): Rearrangements of chromosome region 12q13-q15 in pleomorphic adenomas of the human salivary gland (PSA). Cytogenet Cell Genet 45:187-190. 14. Sahlin P, Mark J, Stenman G (1992): The INT1 and GLI genes are not rearranged or amplified in benign pleomorphic adenomas with chromosome abnormalities of 12q13-15. Cancer Genet Cytogenet 58:85-88. 15. Stenman G, Sandros J, Dahlenfors R, Juberg-Ode M, Mark J (1986): 6 q - and loss of the Y chromosome--two common deviations in malignant human salivary gland tumors. Cancer Genet Cytogenet 22:283-293. 16. Bullerdiek J, Vollrath M, Wittekind C, Caselitz J, Bartnitzke S (1990): Mucoepidermoid tumor of the parotid gland showing a translocation (3;8)(p21;q12) and a deletion (5)(q22)as sole chromosome abnormalities. Cancer Genet Cytogenet 50: 161-164.