Characterization of the 12q13-15 amplicon in soft tissue tumors

ELSEVIER

Characterization of the 12q13-15 Amplicon in Soft Tissue Tumors Mef Nilbert, Anders Rydholm, Felix Mitelman, Paul S. Meltzer, and Nils Mandahl

Amplification of the genes MDM2, SAS, and CDK4, all located on the long arm of chromosome 12, has recently been demonstrated in human soft tissue tumors. To determine the extent of the amplification unit, we examined 16 soft tissue tumor samples, including pleomorphic liposarcoma, malignant fibrous histiocytoma (MFH), and atypical lipoma, by Southern blot analysis using 13 chromosome 12 probes. All tumors had previously been shown to have 3- to 20-fold amplification of MDM2. In five samples, all MFH, only MDM2 was amplified, whereas in the remaining 11 samples, two to five additional genes were amplified. The amplicon included markers both proximal and distal to MDM2, but was in all but one atypical lipoma confined to the chromosome region 12q13-15. Discontinuous amplicons were found in two of the tumors. This study indicates that MDM2, or possibly an as-yet-unidentified gene in its proximity, is the target gene of the 12q13-15 amplification in soft tissue tumors. ABSTRACT:

INTRODUCTION Oncogene amplification has been demonstrated in several tumor types, e.g., MYCN in neuroblastoma; MYC, MYCL, and MYCN in small cell lung carcinoma, RAS in bladder carcinoma, and ERBB2 in breast and ovarian carcinomas [1]. Functional analyses and clinical observations support the hypothesis that amplification of cellular oncogenes contributes to the tumorigenic phenotype or to tumor progression [2]. Gene amplification is sometimes associated with double minute chromosomes (dmin) or homogeneously staining regions (hsr). Three genes of potential pathogenetic importance for bone and soft tissue tumor development in humans, all located in chromosome bands 12q13-14, have recently been identified: the sarcoma amplified sequence (SAS), the human homolog of the murine double minute type 2 gene (MDM2), and the cyclin-dependent kinase gene (CDK4), All three genes have been shown to be amplified in various histopathologic tumor types, including malignant fibrous histiocytoma (MFH), fibrosarcoma, liposarcoma, malignant schwannoma, rhabdomyosarcoma, hemangiopericytoma, osteosarcoma, and Ewing's sarcoma [3-8]. SAS was isolated from a large

From the Departments of Clinical Genetics (M. N., F. M., N. M.) and Orthopedics (A. R.), Lund University Hospital, Lund, Sweden; and the Laboratory of Cancer Genetics (P. S. M.), National Center for Human Genome Research, National Institutes of Health, Bethesda, Maryland. Address reprints to: Dr. M. Nilbert, Department of Clinical Genetics, Lund University Hospital, S-221 85 Lurid, Sweden. Received September 1, 1994; accepted January 17, 1995. Cancer Genet Cytogenet83:32-36 (1995) © Elsevier ScienceInc., 1995 655 Avenueof the Americas, New York, NY 10010

marker chromosome with abnormally banding regions found in an MFH tumor biopsy using in gel renaturation combined with PCR [8]. The functional product of the SAS gene is a transmembrane protein [9]. mdm-2 was originally isolated from a tumorigenic murine cell line with dmin. Overexpression of mdm-2 in murine cell lines is associated with increased tumorigenicity [10]. MDM2 presumably functions as a cellular regulator and mediator of the tumor suppressor protein TP53, similar to the action of other TP53-associated proteins such a s the Wilms' tumor protein, WT1, the recently identified replication protein A, and several viral oncoproteins [11]. Thus, increased levels of MDM2 may functionally inactivate TP53 [12]. Support for this hypothesis comes from studies showing that one-third of MFH and liposarcomas had MDM2 amplification and one-third had mutations of TP53, including homozygous loss, rearrangements, and point mutations [13, 14]. TP53 mutation and MDM2 amplification seem to be mutually exclusive events, which both interfere with TP53 binding, and thus represent alternative pathways whereby tumor cells escape normal regulation of cell growth [14]. CDK4 encodes a cyclin-dependent kinase, which acts as a catalytic subunit of the D-type cyclins during the G1 phase of the cell cycle. A defective interaction between CDKs and D-type cyclins is believed to contribute to aberrant cell proliferation in human cancer, and overexpression of cyclins has been demonstrated in, e.g., breast carcinomas and squamous cell carcinomas of the head and neck [6]. In a recent study of 94 mesenchymal tumors, we found 3- to 20-fold amplification of MDM2 in 20 tumors: one pleomorphic liposarcoma, 12 MFH, and seven lipomas (four of which were classified as atypical lipomas) [15]. We also found

0165-4608/95/$09.50 SSDI0165-4608(95)00016-I

12q13-15 A m p l i c o n in Soft Tissue Tumors

Table 1

33

T u m o r material, a m p l i f i c a t i o n level, a n d ring c h r o m o s o m e s

Tumor sample

Diagnosis°

1 2 3 4 5 6 7a 7b 8 9 10 11 12 13 14 15

PL MFH MFH MFH MFH MFH MFH MFH MFH MFH MFH MFH AL AL AL AL

Copy number

Ring(s)

20 5 4 10 20 4 5 10 3 5 10 10 3 3

+ + + + + + + +

4

+

5

+

o PL, pleomorphic liposarcoma; MFH, malignant fibrous histiocytoma; AL, atypical lipoma.

a strong correlation between MDM2 a m p l i f i c a t i o n and the p r e s e n c e of marker ring chromosomes, w h i c h r e p r e s e n t recurrent karyotypic features of both MFFI a n d atypical lipomas, suggesting that the rings harbor genetic material derived from c h r o m o s o m e 12. To determine the extent of the a m p l i f i c a t i o n u n i t (amplicon) a n d p o s s i b l y the essential gene, we have further investigated by S o u t h e r n blot analysis 16 of the 20 t u m o r s a m p l e s w i t h MDM2 amplification. Thirteen markers from c h r o m o s o m e 12 were studied, i n c l u d i n g several genes k n o w n to be rearranged or a m p l i f i e d in different t u m o r types, such as INT/, GLI, CHOP, SAS, and CDK4 [4, 6, 16-18].

c o r r e s p o n d to tumors 15-17], were s t u d i e d (Table 1). All tumors were located in the extremities or in the trunk wall, and no patient h a d received cytotoxic treatment prior to surgery. The tumors were classified histopathologically according to Enzinger and Weiss [19]. Of the 16 t u m o r samples, four (cases 2, 6, 7a, and 7b) were locally recurrent MFH; t u m o r s a m p l e s 7a and 7b were local recurrences that occurred w i t h i n four months in the same patient. All tumors had been cytogenetically characterized, and for some of t h e m the detailed karyotypes have been reported: t u m o r 3 by C)rndal et el. [20] (case 3); tumors 4, 6, a n d 8 by M a n d a h l et al. [21] (cases 4, 26, and 17); t u m o r 7 by O r n d a l et al. [22] (case 2); t u m o r 12 by M a n d a h l et el. (23) (case 15); and tumors 13-15 by M a n d a h l et al. [24] (cases 61, 43, and 96). Ring chromosomes were found in 10 tumors (Table 1). Southern blot analysis was performed essentially as described [18]. High-molecular weight DNA was extracted from frozen t u m o r tissue s a m p l e s adjacent to those used for cytogenetic analysis. DNA was restricted with EcoRI and 5 I~g of DNA was size-separated on 0.8% agarose gels. Transfer was performed u n d e r alkaline conditions. The probes u s e d (Table 2) were r a d i o l a b e l e d w i t h 32p-dCTP After hybridization and washing, the filters were automdiographed, and the level of amplification was d e t e r m i n e d by serial dilutions. To verify equal loading, the blots were reprobed also w i t h the probe H3.25, derived from 13q22 [32].

MATERIALS AND METHODS Sixteen soft tissue t u m o r samples obtained from 15 patients, i n c l u d i n g one p l e o m o r p h i c liposarcoma, 11 MFH, and four atypical lipomas, all of w h i c h h a d MDM2 amplification [reference 15: tumors 1-12 have the same numbers, tumors 13-15

Table 2

RESULTS A m p l i f i c a t i o n , in a d d i t i o n to MDM2, was detected in 11 tum o r samples for one or m o r e of the markers SAS, CDK4, RAPIB, D12S8,PTPB, D12S7, and D12S52. In tumors with two or more a m p l i f i e d markers, the amplification level was the same in each t u m o r (Table 1). The a m p l i c o n extended, in relation to MDM2, towards both the centromere and the telomere. Coamplification of D12S8, BAPIB, SAS, a n d CDK4, found in nine, eight, seven, and seven tumors, respectively, was most frequent. In one MFH (tumor 4) and one atypical lipoma (tumor 15) discontinuous amplicons were found (Figure 1). No difference c o u l d be detected between the extension of the amplicons in tumors with and without rings. None of the tumors showed amplification of KBAS2, INT1, GLI, CHOP, or ALDH.

Data on the c h r o m o s o m e 12 markers used

Locus

Location

Probe

Source

Reference

KRAS2 INT1 GLI CHOP SAS CDK4 MDM2 RAPIB D12S8 PTPB D12S7 D12S52 ALDH

p12.1 q13 q13 q13.3 q13-14 q13-14 q13-14 q14 q14 q15 q22 q22 q24.2q

pSW11-1 pAL1 pKK36P1 CHOP 10 pSJP2 CDK4 MDM2 RAPIB p7G11 PTPRB pDL32B pR7 ALDHJ

JCRB R. Nusse B. Vogelstein P. ,~man P. Meltzer P. Meltzer B. Vogelstein V. Pizon ATTC F. Jirik ATTC R. Rimokh ATTC

[25] [26] [27] [18] [8] Personal communication [3] [28] [29] [30] [29] Pesonal communication [31]

34

M. Nilbert et al. Tumor sample

Locus

KRAS2 INT~ GEl CHOP SAS CDK4 MDMZ

RAP1B D12S8 PTPB D12S7 DlZSSZ ALDH HS.ZS

1

;~

3

4

5

6

7a 7b

8

9

13

• • •

0 0 I 0 0 0 0 0 0 Q • O 0 ~ ~ 0 O • 0 O ~ • 0 • • • • • • 0 • • •

0 0 0 0 • 0 0 0 • • • •

• 0

0 0

• 0

0 O •

•

0 0 0 0

0 0 0 0

• • 0 0

0 0 0 0

0 0 0 0

0 0 0 0

0 0 0 0

0 0 0 O 0 0

0 0 0 0 0 0

0 0 0 0 0 0

• 0 0 Q 0 0

0 •

O

O O O 0 O O 0 0 0 0 0 • 0 0 0 0

• 0

0 0 0 0

0 0 0 0

D

0

0 •

0 0 0 0

• 0 Q 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 O O 0

• •

0 0 0 0

0 0

A BC

14 15

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 O 00

0 ~

0 0 0 0 0 0 0 0 0 0 0 0 0 0 O O 0 0 0 0

11 12

0 0 0 O

0 0

0

10

l~gurel ~pl~tion~m16so~tmsue~or~ples. Sampie 1 is a pleomorphic liposarcoma, 2-11 are malignant fibrous histiocytomas, and 12-15 are atypical lipomas. Filled circles represent amplified markers, and unfilled circles nonamplified markers.

SAS

CDK4

MDM2

DISCUSSION A m p l i f i c a t i o n in m a m m a l i a n cells often involves DNA segments larger t h a n one gone [2]. A m o n g the 16 t u m o r samples w i t h MDM2 amplification, we found coamplification of a d d i t i o n a l markers from 12q13-q22 in 11 samples. In five M F H o n l y MDM2 was amplified, w h i c h might argue that MDM2, or a gene in its proximity, is the target of the 12q amplification event in MFH. We s t u d i e d two local M F H recurrences (samples 7a and 7b) from one patient. The first recurrence showed a 5-fold a m p l i f i c a t i o n of o n l y MDM2 (Figure 2). In the second recurrence, w h i c h o c c u r r e d four m o n t h s later, the level of MDM2 amplification had increased to 10-fold, and, in addition, there was a 10-fold a m p l i f i c a t i o n also of SAS, CDK4, RAPIB, and D12S8. The a m p l i c o n h a d t h u s extended to i n c l u d e markers both p r o x i m a l a n d distal to MDM2. The fact that there was a 10-fold a m p l i f i c a t i o n for all markers involved in 7b suggests that the a m p l i f i c a t i o n of the a d d i t i o n a l four markers occurred in one step, rather than t h r o u g h a sequential extension of markers flanking MDM2. Based on the cytogenetic findings in the two recurrences, (3rndal et al. [22] argued that the clone (R2), found in the second recurrence, was not a direct c o n t i n u a t i o n of the clone (R1) in the first recurrence, b u t that the two clones h a d a c o m m o n hypothetical ancestor (Re), If this interpretation is correct, it is possible that Re d i d not contain any a m p l i f i c a t i o n w i t h i n the 12q13-14 segment, b u t that different cells in the Re population i n d e p e n d e n t l y u n d e r w e n t amplification: one comprising only MDM2 gave rise to the R 1 lineage, the other including also the flanking markers SAS, CDK4, RAP1B, and D12S8 to R2. If so, the a m p l i f i c a t i o n w o u l d r e p r e s e n t a secondary, progression-related event. In two tumors, one MFH (tumor 4) a n d one atypical lip o m a (tumor 15), both of w h i c h h a d ring chromosomes, a n o n a m p l i f i e d segment containing the BAS-related protein gene, RAP1B, was flanked by a m p l i f i e d regions (Fig. 1).

D1288

Figure 2 Southern blot analysis using probes for SAS, CDK4, MDM2, and D12S8. Lane A corresponds to tumor 7a, lane B to tumor 10, and lanes C and D are nonamplified controls. All lanes conrain 5 ~g of DNA. W h e t h e r this finding reflects separate amplicons, each with its own target gene, or deleted sequences w i t h i n one amplified unit, is u n k n o w n . As both tumors w i t h d i s c o n t i n u o u s a m p l i c o n s contained ring chromosomes, a possible explanation might be that ring c h r o m o s o m e formation is a complex event that can involve interstitial rearrangements, e.g., inversions, insertions, or deletions, w h i c h may change the order of the genes w i t h i n the segment. In t u m o r 15 the amplified segment included the anonymous markers D12S7 and D12S52 in 12q22. No marker was available for the region between q15 a n d q22, and therefore this finding could also reflect the presence of an additional, i n d e p e n d e n t a m p l i c o n in 12q22. In a series of 98 bone and soft tissue tumors (including fibrosarcoma, liposarcoma, osteosarcoma, malignant schwannoma, rhabdomyosarcoma, and hemangiopericytoma), Fetus et el. [5], studied gene amplification in the 12q13-14 region using probes for the genes A2MB, GLI, CHOP (also referred to as GADD153), SAS, a n d MDM2, a n d found amplification in two, two, four, ten, and nine tumors, respectively. They also demonstrated that the extension of the

12q13-15 A m p l i c o n i n Soft Tissue Tumors

a m p l i c o n s varied between tumors, a n d detected a discont i n u o u s a m p l i c o n in one liposarcoma, w h i c h showed amplification of MDM2, SAS, and part of A2MB [located proximal to GLI), but not of GLI and CHOP. Based on the f i n d i n g that o n l y MDM2 was amplified i n another liposarcoma, whereas SAS and CHOP, but not MDM2, were amplified i n two osteosarcomas a n d one rhabdomyosarcoma, this suggested that the target of the amplification event in 12q13-14 may be a gene located between SAS a n d MDM2. Similar results were obtained by Khatib et el. [6], who studied amplification of CDK4, MDM2, and GLI i n 14 sarcoma cell lines. Two of them showed amplification; all three genes were amplified in one osteosarcoma cell line, a n d GLI and CDK4, but not MDM2, were amplified i n one rhabdomyosarcoma cell line. Coamplification of SAS a n d MDM2, but no amplification of CHOP, GLI, a n d A2MB were detected by Pedeutour et al., i n six well-differentiated liposarcomas [33]. The possibility remains, however, that the target gene differs among various histopathologic types of bone and soft tissue tumors. The amplification of markers within 12q13-22 in bone and soft tissue tumors shows similarities to the coamplification of syntenic markers in 11q13 i n several tumor types. Amplification involving a n o n y m o u s markers and several potential tumor-relevant genes, i n c l u d i n g HST, INT2, and CYCD1, has been observed i n various t u m o r types, such as carcinomas of breast, bladder, esophagus, larynx, and hypopharynx [34]. Interestingly, amplification discontinuities resulting i n discrete a m p l i c o n s have also been detected with markers i n 11q13 [34]. We thank the colleagues mentioned in Table I for generously sharing with us their chromosome 12 probes. This study was financially supported by grants from the Swedish Cancer Society, the Swedish Work EnvironmentFund, and the Lund University Medical Faculty.

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9.

10.

11. 12.

13.

14.

15.

16.

1Z

18.

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