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Abstract Tenosynovial giant cell tumor (TGCT) is the most common benign ..... FH (1997) Malignant giant cell tumour of the tendon sheaths and joints (malignant.
Virchows Arch (2002) 441:475–480 DOI 10.1007/s00428-002-0640-y

O R I G I N A L A RT I C L E

M. Nilsson · M. Höglund · I. Panagopoulos R. Sciot · P. Dal Cin · M. Debiec-Rychter · F. Mertens N. Mandahl

Molecular cytogenetic mapping of recurrent chromosomal breakpoints in tenosynovial giant cell tumors Received: 14 December 2001 / Accepted: 12 February 2002 / Published online: 13 April 2002 © Springer-Verlag 2002

Abstract Tenosynovial giant cell tumor (TGCT) is the most common benign tumor of synovium and tendon sheath. Cytogenetic data indicate that 1p11–13 is the region most frequently involved in structural rearrangements. With the aim of eventually identifying the genes associated with TGCT development, we have investigated 1p11–13 breakpoints using fluorescence in situ hybridization (FISH) analysis, with a panel of yeast artificial chromosome (YAC) probes covering 1p11–21. Twentysix tumors were analyzed by G-banding, and 24 of these showed a breakpoint in 1p11–13. The cytogenetic findings add to previous observations that, among a variety of translocations involving 1p11–13, chromosome 2 is the most common translocation partner, with a breakpoint in 2q35–37. This aberration was found in eight cases. Other recurrent translocation partners, found in two or three cases, were 5q22–31, 11q11–12, and 8q21–22. Material from 21 tumors was available for FISH analysis, which revealed that the breakpoints clustered to one region spanned by two YAC probes, 914F6 and 885F12 located in 1p13.2, in 18 cases. Bacterial artificial chromosome probes were used to map the recurrent breakpoint on chromosome 2. In four of seven cases there was a breakpoint within the sequence covered by probe 260J21, where the RDC1 gene is located, a gene reported to fuse with HMGIC in lipomas with a 2;12 translocation. M. Nilsson (✉) · M. Höglund · I. Panagopoulos · F. Mertens N. Mandahl Department of Clinical Genetics, University Hospital, 221 85 Lund, Sweden e-mail: [email protected] Tel.: +46-46-173398, Fax: +46-46-131061 R. Sciot Department of Pathology, University of Leuven, Leuven, Belgium P. Dal Cin Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA M. Debiec-Rychter Center for Human Genetics, University of Leuven, Leuven, Belgium

Keywords Tenosynovial giant cell tumor · Cytogenetics · FISH · Breakpoint mapping

Introduction Tenosynovial giant cell tumors (TGCTs) involve the fibrous sheaths of tendons. They are benign lesions, with some capacity for local recurrence, but rare malignant forms have been described [1]. TGCTs occur as a localized form, predominantly involving the fingers, and a less-common, diffuse form affecting various sites, primarily the knee, ankle, and foot [3, 23]. These tumors may occur at any age but affect mainly middle-aged persons, and the sex ratio for localized tumors in particular is skewed toward women [3]. To date, 30 cases of TGCT with clonal chromosome aberrations have been reported [15]. All cases have had near- or pseudodiploid karyotypes, mostly with simple structural and/or numerical aberrations. The most common numerical changes are gain of chromosomes 5 and 7, which in some cases occur as the sole anomalies. Among the structural aberrations, the short arm of chromosome 1 is frequently involved, with a clustering of breakpoints in the chromosome segment 1p11–13. Although 1p11–13 has been found to recombine with several other chromosome segments, a recurrent t(1;2) (p11;q35–36) has been identified [2]. Among published cases, there are no karyotypic differences between localized and diffuse tumors, except for trisomies 5 and/or 7 being more common in the diffuse form [22]. It has been debated whether TGCT is a neoplastic lesion or a reactive, proliferative process [3]. Although the observation of polyclonal X-chromosome inactivation in pigmented villonodular synovitis [19] might seem to favor the latter view, the frequent findings of non-random, clonal chromosome aberrations can be taken as strong support for a neoplastic origin [4, 17, 22]. In the present study, we have analyzed TGCTs cytogenetically using chromosome banding. These tumors and those previously analyzed in our laboratories were

476 Table 1 Clinical and karyotypic data. Aberrations involving 1p are shown in bold type Case no.

Age/sex

Type

Localization

Karyotype

1a 2a 3a 4a 5a

45/Female 68/Male 13/Female 27/Female 54/Male

Diffuse Localized Diffuse Localized Localized

Foot Finger Knee Hand Finger

6a 7 8 9a

31/Male 15/Female 45/Male 76/Female

Diffuse Diffuse Localized Diffuse

Hip Foot Finger Thigh

10a 11 12 13a 14a 15a 16a 17a 18a 19a 20 21 22 23 24 25

46/Female 65/Female 33/Male 57/Female 60/Male 88/Female 32/Male 53/Female 41/Female 47/Male 47/Female 30/Female 15/Female 68/Female 56/Male 40/Female

Diffuse Localized Localized Diffuse Localized Localized Diffuse Localized Diffuse Localized Diffuse Diffuse Diffuse Diffuse Localized Diffuse

Knee Hand Toe Knee Finger Hand Toe Finger Knee Finger Knee Knee Knee Knee Finger Knee

26

37/Female

Diffuse

Hip

46,XX,t(1;19)(p11;p12)/47,idem,+12 46,XY,ins(5;1)(q31;p13p34)/46,idem,t(2;4)(p23;q21)/45,X,-Y 46,XX,t(1;2)(p22;q35-37) 46,XX,t(1;11)(p11;q11),t(8;16)(p11;q22) 46,XY,t(1;16)(p22;q24),t(7;15)(p13;p11),-12,+mar/46,XY,del(2)(q31), der(10)t(10;16)(p15;?q?),inv(13)(q12q22),der(16)t(2;16)(?q?;q24) 46,XY,add(1)(p11),add(19)(p13) 46,XX,t(1;11)(p11-12;q12) 46,XY,t(1;2)(p13;q35),t(5;11;10)(q33;q23;p15) 47,X,-X,del(1)(p32),der(3)add(3)(p21)add(3)(q21),add(5)(q31), +der(5)t(5;8)(p15;q22),+i(7)(q10),der(8)t(1;8)(p13;q11), der(13)t(5;13)(q11;p11),add(5)(q31),der(16)add(16)(p11)add(16)(q13), add(21)(q22)/46,idem,-10,add(19)(q13) 48,XX,t(1;12)(p13;q24),+5,+7 46,XX,t(1;2)(p13-22;q35-37)/47,XX,+r 46,XY,t(1;2)(p12;q36) 46,XX,add(1)(q32),add(1)(p11),der(22)t(1;22)(q21;q13)ins(1;?)(q32;?) 46,XY,t(1;2)(p11;q37) 46,XX,t(1;2)(p11;q36) 46,XY,t(1;5)(p11;q22) 46,XX,t(1;2)(p11;q36-37) 46,XX,t(1;11)(p11;q12),t(2;5)(p24;q31) 46,XY,t(1;5)(p11;q31) 46,XX,t(1;9)(p11;q32) 46,XX,t(1;2)(p11;q35) 46,XX,t(1;8)(p12;q22) 46,XX,t(1;12)(p13;q15) 46,XY,t(1;8)(p13;q21) 46,XX,der(1)t(1;6)(p11;q13)t(1;15)(q22;q12),der(3)t(1;3)(p11;q11), der(6)t(6;19)(q13;q11),der(15)del(15)(q12)t(3;15)(q11;p11), der(19)t(15;19)(p11;q11) 46,XX,t(1;2)(p13;q37)

a Karyotype

previously published, see references [14] and [20]

then subjected to fluorescence in situ hybridization (FISH) in order to map the breakpoints in more detail. The aim was to find out whether the breaks were scattered or clustered within a narrow region, which could indicate the presence of a tumor-associated gene.

Materials and methods Patients Tissue samples from 26 cases, 17 women and 9 men, with TGCTs were obtained from orthopedic centra in Lund and Leuven (Table 1). The ages of the patients ranged from 13 years to 88 years (mean 46 years). Fifteen tumors were classified as diffuse lesions and eleven were localized, all of them showing the typical histological features of TGCT. All cases were investigated by means of chromosome banding, and material for FISH analysis was available from 21 cases. Chromosome preparations and G-banding The cells were short-term cultured, harvested and G-banded as described previously [11]. In brief, the tumor tissue was disaggregated mechanically and then enzymatically in collagenase II for 3–5 h. RPMI 1640 medium, supplemented with fetal bovine serum (17%), L-glutamine and antibiotics, was added to the cell suspension. The cells were cultured for 4–10 days in plastic flasks and chamber slides at 37°C in a humidified atmosphere containing 5%

CO2. Colcemid (0.02 µg/ml) was added to arrest dividing cells in metaphase. Prior to banding, chromosome preparations were treated in 2×saline sodium citrate (SSC) at 60°C to remove remaining cytoplasm. The chromosomes were stained according to a G-banding technique using Wright’s stain solution. The description of chromosome aberrations was according to ISCN (1995) [8]. Probes for FISH Whole chromosome painting probes (WCP; Cambio, Cambridge) were used to unequivocally identify derivative chromosomes containing material from chromosome 1 and to identify the translocation partners. Yeast artificial chromosome (YAC) clones were provided by the Centre d’Etude du Polymorphisme Humain (CEPH, Paris). The chromosome 1 YAC probes 665D8, 906D6, 914F6, 885F12, 970D12, 931C5, 953F3, 942H6, 959C4, 802C2, 796D1, 886D7, and 947C9, all located within the chromosome segment 1p11–21, were used for breakpoint mapping at the megabase level (Fig. 1). Bacterial artificial chromosome (BAC) probes 4H21, 205L13, 260J21, 346I14, and 514F21 (BACPAC Oakland) were used for investigation of the breakpoint on chromosome 2 in cases with t(1;2). To find out whether HMGIC was rearranged in a case with t(1;12), probes 27E12 and 142H1 [21] were used. A physical map of the proximal portion of the short arm of chromosome 1 was constructed. The locations of the probes were defined by database searching and by a YAC contig map of chromosome 1, using several different databases (Table 2). Information from these was compared and processed to construct a consensus physical map (Fig. 1). Human DNA from YAC clones was amplified by interAlu PCR, using the primers ILA3′ and ILA5′ [7]. The probe DNA was

477 Fig. 1 Results of fluorescence in situ hybridization (FISH) analyses of chromosome segment 1p11–21 showing the breakpoint distribution in 21 tenosynovial giant cell tumors (TGCTs). YAC clones are represented by horizontal lines and the breakpoints by arrows

Table 2 World Wide Web resources used for localization of YAC probes, bacterial artificial chromosome probes and sequence-tagged sites and construction of the physical map of the involved region on chromosome 1 (status as of 25 May 2001) Database

Uniform resource locator

Entrez Foundation Jean Dausset CEPH Gene map ‘99 The genome database Human physical mapping project at the Whitehead Institute/MIT The Sanger Centre

http://www.ncbi.nlm.nih.gov/Entrez/ http://www.cephb.fr http://www.ncbi.nlm.nih.gov/genemap99/ http://www.gdb.org http://www-genome.wi.mit.edu/ http://www.sanger.ac.uk/HPG/Cytogenetics/Bacset.shtml

labeled with either dUTP or dCTP coupled with biotin-16-dUTP or digoxigenin-11-dUTP (Boehringer Mannheim), dCTP-Cy3 (Amersham) and dethylaminocoumarin (DEAC)-5-dUTP (NEN) using Amersham’s megaprime kit. FISH analyses Hybridization to metaphase chromosomes were as previously described [5]. Posthybridization washing was performed in 0.4×SSC at 72°C for 2 min before detection of indirectly labeled probes. Biotin-labeled probes were detected by 1 µg/ml Cy5-conjugated avidin (Amersham). Digoxigenin was detected by fluorescein isothiocyanate (FITC)-conjugated sheep antidigoxigenin antibodies (Boehringer Mannheim). As an unspecific counterstaining of chromosomes, 0.5 mg/l 4,6-diamino-2-phenyl-indole (DAPI; Boehringer Mannheim) in 2% 1,4-diazabicyclo-[2, 2, 2]-octan (DABCO; Sigma) was used. The signals from the probes were detected in an epifluorescence microscope (Zeiss), coupled to a Cytovision ChromoFluor System (Applied Imaging) and a CCD camera. For each hybridization, 3-25 metaphase cells were analyzed.

Results Cytogenetics All 26 tumors analyzed by means of G-banding showed abnormal karyotypes (Table 1). The chromosome number was diploid in 24 cases and hyperdiploid in two cases. Seemingly balanced karyotypes were found in 21 cases, whereas five cases had partial or total gain and/or loss of chromosomes. A breakpoint in 1p11–13 was found in 24 cases, and four cases had more distal break-

points (1p22–32); cases 2 and 9 had two breaks in 1p. In the 24 cases with a break in 1p11–13, the translocation partner could be identified, except in case 13. Recurrent aberrations were observed. Thus, 1p11–13 recombined with 2q35–37 (eight cases), 5q22–31 (three cases), 11q11–12 (three cases), and 8q21–22 (two cases). In another two cases, these chromosomes were involved in other rearrangements, interpreted as t(1;2)(p22;q35–37) in case 3 and der(8)t(1;8)(p13;q11) in case 9. Fluorescence in situ hybridization In 18 of the 21 tumors available for FISH analysis, the probe signal pattern showed that the chromosome 1 breakpoints clustered to one common region, spanned by the two YAC probes 914F6 and 885F12, hybridizing to sequences in 1p13.2 (Fig. 1). Examples of split signals are shown in Fig. 2. The three remaining cases had breakpoints outside the cluster region, two being more distal (cases 5 and 23) and one more proximal (case 4). In the former cases, signals for all probes used were present on the derivative chromosome 1, whereas in case 4 split signals were seen for probe 802C2. Of the nine cases with a 2q35–37 breakpoint, material for FISH analyses was available from seven. In four cases – 11, 17, 21, and 26 – there was a breakpoint within the sequence covered by probe 260J21 (Table 3). In case 26, a split signal was seen also when using probe 514F21, which partly overlaps 260J21. In case 11, the

478 Table 3 Fluorescence in situ hybridization (FISH) analyses of tumors with t(1;2) by chromosome 2-specific bacterial artificial chromosome probes. split signal present on both der(1) and der(2), der(1) signal only on der(1), der(2) signal only on der(2), n.d. not determined

Probe

260J21 514F21 205L13 4H21 346I14

Signal distribution Case 11

Case 12

Case 14

Case 15

Case 17

Case 21

Case 26

split der(1) der(1) der(1) n.d

der(2) der(2) der(2) der(2) der(2)

der(2) der(2) der(2) n.d n.d

der(2) der(2) der(2) der(2) der(2)

split n.d n.d n.d n.d

split n.d n.d n.d n.d

split split der(1) n.d der(1)

Discussion

Fig. 2 Metaphase fluorescence in situ hybridization (FISH) showing split signals with 1p probes. Case 7 (top) showing a split 885F12 signal (red). Case 20 (bottom) showing a split 914F6 signal (yellow). Whole chromosome painting probe (WCP) signals for chromosomes 1 and 9 are shown in green

Fig. 3 Ideogram showing the breakpoint distribution in tenosynovial giant cell tumor (TGCT) based on all available cytogenetic data. Filled circles indicate breakpoints involved in recombination with 1p11–13 and open circles indicate all other breakpoints



514F21 signal was seen only on the der(1), but remained on der(2) in cases 12, 14, and 15; material was not available for additional analyses in cases 17 and 21. The probes 205L13, 4H21, and 346I14 gave no split signal in any of the cases. The signals remained on the der(2) in cases without split 260J21 signals, but were located on the der(1) in cases with a breakpoint in the 260J21 sequence.

The breakpoint distribution in 31 TGCT with structural aberrations, including the present tumors and cases from other previous reports [2, 14, 16, 18, 22] shows a clearly non-random pattern (Fig. 3). The combined data confirm the previous notion that at least two distinct cytogenetic subgroups of TGCT can be distinguished, one major characterized by rearrangements involving 1p11–13 and a minor subgroup with 16q22–24 aberrations. In both subgroups, these chromosome segments recombine with a variety of other segments, but so far recurrent translocations have involved only 1p11–13. These include 2q35–37, 5q22–31, 8q21–22, and 11q11–12, with t(1;2) being the preferred rearrangement. Similar observations of different cytogenetic subgroups, each characterized by recombination between one chromosome segment and one preferred and many alternative translocation partners, have been made in several other benign solid tumors [15]. The four chromosome segments mentioned above, recurrently recombining with 1p11–13 in TGCT, are also involved in rearrangements in lipoma, uterine leiomyoma, pleomorphic adenoma of the salivary gland, and pulmonary hamartoma, but, apart from a dic(1;11)(p12;q11) in an adenoma [9], never together with 1p11–13 [15]. In particular, 2q35–37 is frequently involved in lipomas and leiomyomas, most often recombining with 12q13–15. In analogy with the involvement of the HMGIC gene in lipomas characterized by 12q13–15 rearrangements, it seems reasonable to suggest that 1p11–13 in TGCT contains a gene that becomes involved in a gene fusion or is dysregulated as a consequence of the translocations and is of significance in TGCT tumorigenesis. Available cytogenetic data indicate that the localization of breakpoints in 1p in TGCT is heterogeneous with a concentration to 1p11, 1p12, or 1p13. However, the FISH results demonstrated a greater uniformity, with a breakpoint clustering to sequences corresponding to the YAC probes 914F6 and 885F12 located in 1p13.2 in 18 of 21 cases. This discrepancy could be explained, at least partly, by the difficulty to identify cytogenetically breakpoints close to the centromere, as this region is variable due to repetitive sequences.

479

480

FISH analysis showed that three cases had breakpoints outside the cluster region. In case 5, the break was more distal (1p22) as determined by both banding and FISH analyses. Cases 4 and 23 had cytogenetic breaks in 1p11 and 1p13, respectively, and the FISH analyses showed the breakpoints to be proximal and distal, respectively, to the cluster region. Whether it is a coincidence or not, that two of these three cases had a rearrangement of 16q22–24 could at present not be determined. If, in these cases, the rearrangement of 16q22–24 is the pathogenetically important event the 1p rearrangements may have been chance secondary aberrations, explaining why the breakpoints were outside the cluster region on 1p. The third case was the only TGCT with a breakpoint in 12q15. However, no involvement of the HMGIC gene could be detected by FISH analyses (data not shown). Bearing in mind that dysregulation of the HMGIC gene has been observed previously in tumors with breakpoints outside the coding sequence [20], it is still conceivable that the translocation resulted in altered HMGIC expression. Recombination between 1p11–13 and 12q13–15 has been reported in one case each of lipoma, uterine leiomyoma, pleomorphic adenoma of the salivary gland, and hamartoma of the lung [10, 12, 13, 24]. An attempt was made to localize the breakpoints in 2q35–37. Split signals were seen in four cases when hybridizing to probe 260J21 and in one case when 514F21 was used. These two overlapping probes span a sequence where the G-protein-coupled receptor gene RDC1 is located [6], a gene that was found to fuse with HMGIC in a subset of lipomas with t(2;12) (Broberg, personal communication). Acknowledgements This work was supported by the Swedish Cancer Society and the Swedish Child Cancer Fund.

References 1. Bertoni F, Unni K, Beabout JW, Sim FH (1997) Malignant giant cell tumour of the tendon sheaths and joints (malignant pigmented villonodular synovitis). Am J Surg Pathol 21:153– 163 2. Dal Cin P, Sciot R, Samson I, De Smet L, De Wever I, Van Damme B, Van Den Berghe H (1994) Cytogenetic characterization of giant cell tumors (nodular tenosynovitis). Cancer Res 54:3986–3987 3. Enzinger FM, Weiss SW (1995) Soft tissue tumors, 2nd edn. Mosby, St Louis 4. Fletcher JA, Henkle C, Atkins L, Rosenberg AE, Morton CC (1992) Trisomy 5 and trisomy 7 are nonrandom aberrations in pigmented villonodular synovitis. Genes Chromosomes Cancer 4:264–266 5. Gisselsson D (2000) Refined characterization of chromosome aberrations in tumors by multicolor banding and electronic mapping resources. In: Sharma AK (ed) Chromosome painting. Kluwer, Dordrecht 6. Heesen M, Berman MA, Charest A, Housman D, Gerard C, Dorf ME (1998) Cloning and chromosome mapping of an orphan chemokine receptor: mouse RDC1. Immunogenetics 47:364–370

7. Höglund M, Sidén T, Åman P, Mandahl N, Mitelman F (1995) Isolation and characterisation of radiation hybrids for human chromosome 12. Cytogenet Cell Genet 69:240–245 8. ISCN (1995) An international system for human cytogenetic nomenclature. Mitelman F (ed) Karger, Basel 9. Jin C, Martins C, Jin Y, Wiegant J, Wennerberg J, Dictor M, Gisselsson D, Strömbeck B, Fonseca I, Mitelman F, Tanke HJ, Höglund M, Mertens F (2001) Characterization of chromosome aberrations in salivary gland tumors by FISH, including multicolor COBRA-FISH. Genes Chromosomes Cancer 30:161–167 10. Kazmierczak B, Meyer-Bolte K, Tran KH, Wockel W, Breightman I, Rosigkeit J, Bartnitzke S, Bullerdiek J (1999) A high frequency of tumors with rearrangements of genes of the HMGI(Y) family in a series of 191 pulmonary chondroid hamartomas. Genes Chromosomes Cancer 26:125–133 11. Mandahl N (2001) Methods in solid tumor cytogenetics. In: Rooney DE (ed) Human cytogenetics, malignancy and acquired abnormalities, 3rd edn. Oxford University Press, Oxford 12. Meloni AM, Surti U, Contento AM, Davare J, Sandberg AA (1992) Uterine leiomyomas: cytogenetic and histologic profile. Obstet Gynecol 80:209–217 13. Mercher S, Marondel I, Pedeutour F, Gaudray P, Kucherlapati R, Turc-Carel C (1997) Identification of new translocation breakpoints at 12q13 in lipomas. Genomics 46:70–77 14. Mertens F, Örndahl C, Mandahl N, Heim S, Bauer HFC, Rydholm A, Tufvesson A, Willén H, Mitelman F (1993) Chromosome aberrations in tenosynovial giant cell tumors and nontumorous synovial tissue. Genes Chromosomes Cancer 6:212–217 15. Mitelman F, Johansson B, Mertens F (eds) (2001) Mitelman database of chromosome aberrations in cancer. http://cgap.nci. nih.gov/Chromosomes/Mitelman 16. Ohjimi Y, Iwasaki H, Ishiguro M, Kaneko Y, Tashiro H, Emoto G, Ogata K, Kikuchi M (1996) Short arm of chromosome 1 aberration recurrently found in pigmented villonodular synovitis. Cancer Genet Cytogenet 90:80–85 17. Ray RA, Morton CC, Lipinsky KK, Corson JM, Fletcher JA (1991) Cytogenetic evidence of clonality in a case of pigmented villonodular synovitis. Cancer 67:121–125 18. Rowlands CG, Roland B, Hwang WS, Sevick RJ (1994) Diffuse-variant tenosynovial giant cell tumor: a rare and aggressive lesion. Hum Pathol 25:423–425 19. Sakkers RBJ, de Jong D, van der Heul RO (1991) X-chromosome inactivation in patients who have pigmented villonodular synovitis. J Bone Joint Surg Am 73:1532–1536 20. Schoenberg Fejzo M, Ashar HR, Krauter KS, Powell WL, Rein MS, Weremowicz S, Yoon S-J, Kucherlapati RS, Chada K, Morton CC (1996) Translocation breakpoints upstream of the HMGIC gene in uterine leiomyomata suggest dysregulation of this gene by a mechanism different from that in lipomas. Genes Chromosomes Cancer 17:1–6 21. Schoenmakers EFPM, Geurts JMW, Kools PFJ, Mols R, Huysmans C, Bullerdiek J, Van den Berghe H, Van de Ven WJM (1995) A 6-Mb yeast artificial chromosome contig and long-range physical map encompassing the region on chromosome 12q15 frequently rearranged in a variety of benign solid tumors. Genomics 29:665–678 22. Sciot R, Rosai J, Dal Cin P, de Wever I, Fletcher CDM, Mandahl N, Mertens F, Mitelman F, Rydholm A, Tallini G, van den Berghe H, Vanni R, Willén H (1999) Analyses of 35 cases of localized and diffuse tenosynovial giant cell tumor: a report from the chromosomes and morphology (CHAMP) study group. Mod Pathol 12:576–579 23. Somerhausen NS, Fletcher CD (2000) Diffuse-type giant cell tumor: clinicopathologic and immunohistochemical analysis of 50 cases with extraarticular disease. Am J Surg Pathol 24:479–492 24. Stern C, Meyer K, Bartnitzke S, Schloot W, Bullerdiek J (1990) Pleomorphic adenomas with unbalanced chromosomal abnormalities have an increased in vitro life time. Cancer Genet Cytogenet 46:55–63