Monoclonality of Multifocal Myxoid Liposarcoma: Confirmation by Analysis of TLS-CHOP Or EWS-CHOP Rearrangements

2788 Vol. 6, 2788–2793, July 2000 Clinical Cancer Research

Monoclonality of Multifocal Myxoid Liposarcoma: Confirmation by Analysis of TLS-CHOP or EWS-CHOP Rearrangements

Cristina R. Antonescu, Abul Elahi, myxoid liposarcoma or merely a rare chance occurrence. John H. Healey, Murray F. Brennan, The clinical outcomes observed in this small series suggest Man Yee Lui, Jonathan Lewis, that the prognosis of multifocal myxoid liposarcoma is poor, regardless of its often bland or “low-grade” histological Suresh C. Jhanwar, James M. Woodruff, and appearance. Marc Ladanyi1 Departments of Pathology [C. R. A., M. Y. L., J. M. W., M. L.], INTRODUCTION Human Genetics [A. E., S. C. J., M. L.], and Surgery [J. H. H., 2 M. F. B., J. L.], Memorial Sloan-Kettering Cancer Center, New York, Multifocal STS is a rare and controversial entity, account- New York 10021 ing for about 1% of extremity STS (1). As a general rule, multicentricity in STS is defined as the presence of sarcoma at two or more anatomically separate sites, before the manifesta- ABSTRACT tion of disease in sites where STSs most commonly metastasize, Multifocal presentation, defined as the presence of tu- such as the lungs (1, 2). On the basis of clinical or pathological mor at two or more anatomically separate sites, before the data alone, it is difficult to determine in any specific case manifestation of disease in sites where sarcomas usually whether a tumor arising in a site unusual for metastasis, but metastasize (e.g., lungs) occurs in about 1% of extremity soft common for a primary tumor, is a metastasis or a second tissue sarcomas (STSs). Debate still persists whether multi- primary tumor of the same histological type. The first reported focal STSs represent an unusual pattern of metastasis or case of multifocal or multicentric STS dates to 1934, when multiple separate primary tumors. Among STSs with mul- Siegmund (3) described a patient with multiple fatty tumors, tifocal presentation, myxoid liposarcoma is the predominant which was interpreted as “Lipoblastische Sarkomatose” or a histological type. This subtype of liposarcoma contains the systemic malignant disease of the soft tissue. Since then, fewer specific t(12;16) chromosomal translocation, which results than 50 cases have been reported in the literature, and debate in rearrangement of the TLS and CHOP genes that is clone still persists as to whether this entity represents separate primary specific at the DNA level. We, therefore, sought to address tumors or is simply an unusual pattern of metastasis (4–10). the question of clonality by molecular analysis in six patients A recent study from our institution investigating the prev- who presented with either synchronous or metachronous alence of multifocal sarcoma in a large cohort of extremity STSs multifocal myxoid liposarcoma. In all six cases, adequate identified liposarcoma as the predominant histological type (9 of frozen tumor was available for DNA extraction from at least 16 cases; Ref. 1). Although not stated in the report (1), these two distinct anatomical sites. Southern blot analysis using were all myxoid liposarcomas.3 Myxoid liposarcoma is the most CHOP, TLS, and EWS cDNA probes was performed on common subtype of liposarcoma, accounting for more than half genomic DNA. Five cases contained a TLS-CHOP rearrange- of all cases (2). It has a tendency to recur locally, and about ment, and one case had the variant EWS-CHOP fusion (seen one-third of patients develop distant metastases (11). A propor- in <5% of cases). The size of the rearranged CHOP frag- tion of cases shows histological progression to round cell his- ment differed among the six patients, as expected, but was tology, significantly associated with a poor prognosis (12). The identical in all anatomically separate tumor samples from cytogenetic hallmark of myxoid liposarcoma is the t(12; each patient. Likewise, the sizes of the rearranged bands 16)(q13;p11), which appears highly specific for this tumor type observed with either the TLS or EWS probes supported the and is present by conventional cytogenetics in Ͼ85% of the monoclonality of all cases. Our results confirm the mono- cases. The translocation leads to the fusion of the CHOP and clonal origin of multifocal myxoid liposarcoma, establishing TLS (also called FUS) genes at 12q13 and 16p11, respectively, the metastatic nature of distant soft tissue lesions in these and the generation of a TLS-CHOP hybrid gene encoding an cases. It remains unclear whether this unusual pattern of aberrant transcriptional regulator (13, 14). We have recently metastasis represents an intrinsic property of this subset of confirmed the strong specificity of the TLS-CHOP rearrangement for the entity of myxoid/round cell liposarcoma (15). In four cases of myxoid liposarcoma, a variant chromosomal translocation has been described, t(12;22), resulting in an EWS- Received 1/27/00; revised 4/10/00; accepted 4/14/00. CHOP fusion (16, 17). The costs of publication of this article were defrayed in part by the As in other rearrangements producing specific gene fu- payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 To whom requests for reprints should be addressed, at Department of Pathology, Memorial Sloan-Kettering Cancer Center, 1275 York Ave- 2 The abbreviations used are: STS, soft tissue sarcoma; RT-PCR, reverse nue, New York, NY 10021. Phone: (212) 639-6369; Fax: (212) 717- transcription-PCR. 3515; E-mail: [email protected]. 3 J. M. Woodruff, unpublished observation.

Table 1 Multifocal myxoid liposarcoma: clinicopathologic and molecular data Lung Tumor Local mets Case grade Gene fusion Chronology of recurrence after Follow-up no. Age/sex Site 1 site 1 (all sites tested) Other sites multifocal disease at site 1 diagnosis status 1 68/M Intra-abdominala Low TLS-CHOP Rb thigha Mc - 14 months 10 months DOD 45 months 2 28/M Thigh Low TLS-CHOP Chest wall,a neck, Mc - 14 months 46 months DOD 72 months abd wall,a buttock 3 60/M Thigha Low TLS-CHOP Retroperitoneuma Mc - 67 months 42 months DOD 120 months 4 51/M R thigha/R femur Low TLS-CHOP L femura Sy AWD 12 months 5 33/F R axillaa Low EWS-CHOP R thigh,a, L arm,a Sy 7 months DOD 51 months sternum, intra- Mc - 6 months abdominala 6 25/F L axillaa/L breast High TLS-CHOP R flank, L thigh,a Sy DOD 6 months R thigh, abd. wall; intra-abdominala/ small bowel a Samples analyzed for TLS-CHOP or EWS-CHOP rearrangements. b R, right; L, left; Mc, metachronous; Sy, synchronous; DOD, dead of disease; AWD, alive with disease.

sions, the genomic breakpoints of the t(12;16) are widely dis- York, NY; Ref. 13). The CHOP probe consisted of a 753-bp persed in specific introns of the TLS and CHOP genes and differ PstI-XhoI fragment corresponding to exons 3 and 4. This probe from one tumor to the next. Using the Southern blot pattern of detects essentially all CHOP rearrangements in genomic DNA genomic rearrangements in TLS (or EWS) and CHOP as clone- digested with BamHI or SacI (18, 19). The TLS cDNA probe specific markers, we studied multiple tumors from six patients was a 780-bp XbaI-BglI fragment including exons 1–6 of TLS. with multifocal myxoid liposarcoma to distinguish true multi- According to restriction enzyme site analysis of the complete focal (multiclonal) sarcoma from a metastatic (monoclonal) genomic sequence of TLS (GenBank accession no. AF071213; sarcoma. As part of this study, we also report the fifth case of Ref. 20), this probe covers the entire TLS break point region in myxoid liposarcoma with the variant EWS-CHOP gene fusion. BclI-digested DNA. The EWS probe was a 741-bp PCR-gener- ated partial cDNA probe that hybridizes to exons 6–12 of EWS, MATERIALS AND METHODS covering in EcoRI and HindIII-digested DNA the entire Study Group and Demographic Data. Among 43 con- genomic break point cluster region, as described in detail else- secutive cases of myxoid liposarcoma operated at Memorial where (21). Sloan-Kettering Cancer Center that had confirmatory molecular RT-PCR Analysis. The case showing EWS rearrange- evidence of CHOP gene rearrangement, we identified seven ment was further tested for the presence of an EWS-CHOP patients (16%) who presented with either synchronous or meta- chimeric transcript by RT-PCR. Three micrograms of total RNA chronous multifocal myxoid liposarcoma. Adequate frozen tu- was reverse transcribed (Superscript II; Life Technologies, Inc.) mor for DNA extraction was available from at least two distinct using random hexamers, and the cDNA was subjected to PCR Ј anatomical sites in six of these seven cases. The seventh case using a forward primer in exon 7 of EWS (5 -CTGGATCCTA- Ј had material only from a single site and was, therefore, unin- CAGCCAAGCTCCAAG- 3) and a reverse primer in exon 3 Ј Ј formative for the present analysis. Among the six patients fur- of CHOP (5 -TGTCCCGAAGGAGAAAGGCAATG- 3). The ther analyzed, there were four male and two female patients, and RT-PCR product was identified by agarose gel electrophoresis their ages ranged from 25–68 years (mean, 53). and confirmed by direct automated sequencing. Southern Blot Analysis. Sixteen tumor samples from different anatomical sites were available for DNA extraction RESULTS from these six multifocal myxoid liposarcoma cases (two sam- Histopathological Data and Clinical Course. Among ples in four cases, three samples in one case, and five samples the six patients with multifocal myxoid liposarcoma, three pa- in one case). DNA was isolated from snap-frozen tissue stored tients presented with synchronous multifocal disease and three at Ϫ70°C using a standard organic extraction protocol. In 15 of presented with metachronous lesions (Table 1 and Fig. 1). The 16 tumor samples the extracted DNA was adequate for analysis. histopathological findings revealed low-grade myxoid liposar- Genomic DNA was digested with appropriate restriction en- coma in all anatomical locations examined in two cases, and a zymes and separated by 0.7% agarose gel electrophoresis, trans- high-grade, round cell-type myxoid liposarcoma in at least one ferred onto nylon membranes, and hybridized with radiolabeled of the sites in the remaining four cases (Fig. 2). In five of six probes. BamHI and SacI restriction enzymes were used for the patients, the first site to be operated (site 1) revealed low-grade hybridization with CHOP probe; BclI and PstI for TLS probe; myxoid liposarcoma (Table 1). The number of distinct tumor and EcoRI, BamHI, and HindIII for EWS probe. The CHOP and foci that were clinically evident and surgically removed, exclud- TLS probes were partial cDNAs, derived by restriction enzyme ing local recurrences or lung metastases, ranged from two to six digestion of a full-length TLS-CHOP cDNA clone (LPS41; a (mean, 4). In one case, the multiple tumors were restricted to the gift from D. Ron, New York University Medical Center, New extremities; in the remaining five cases there was also involve-

Fig. 1 Multifocal myxoid liposarcoma in patients 5 and 6. Multiple sites correspond to those described in Table 1. 1, sites synchronous with site 1; 2, metachronous (later) sites. The site shown in light gray in patient 6 was intra-abdominal.

ment of the trunk and intra-abdominal sites (Table 1). Three patients developed local recurrences at the primary site (the anatomical site at which the sarcoma was initially diagnosed). Five patients died of disease, and one (patient 4) was alive Fig. 2 Histopathological heterogeneity of separate tumors in patient 5. with disease 12 months after diagnosis. As per the definition of A, initial excisional biopsy from the right axilla (site 1) showing a multifocal STS and the study criteria, none of the patients had myxoid liposarcoma, entirely low grade and devoid of a more cellular component. Microscopically the tumor shows all of the characteristic lung metastases at diagnosis. Indeed, only one patient (2) de- features of a myxoid liposarcoma: myxoid stroma, prominent delicate veloped pulmonary metastases, which became evident 46 vasculature, and lipoblasts. B, resection of sternal mass showing a months after diagnosis. After his initial presentation, but prior to predominantly high-grade, round cell liposarcoma composed of small to the lung metastases, patient 2 developed myxoid liposarcoma in intermediate hyperchromatic cells, with a high nuclear:cytoplasmic ra- tio, arranged in small nests or diffuse sheets of cells. Tumor foci of both multiple additional foci, including chest wall, at which times histologies contained the same EWS-CHOP rearrangement in this case. imaging studies also failed to detect lung metastases. In four patients, lung metastases were detected neither at presentation, nor at the time of disease-related death. The remaining patient (4) was alive with nonpulmonary disease 12 months after diag- Confirmation of EWS-CHOP Fusion Transcript in Case 5. nosis. To confirm the EWS-CHOP rearrangement in the myxoid lipo- Analysis of CHOP, TLS, and EWS Rearrangements. sarcoma from patient 5, the junction fragment was amplified by In all 15 tumor samples with adequate DNA, available from RT-PCR using EWS forward and CHOP reverse primers (see these six patients with multifocal myxoid liposarcoma, CHOP “Materials and Methods”). Direct sequencing of the 179-bp rearrangement was detected by Southern blotting, using either product showed a chimeric EWS-CHOP cDNA with an in-frame BamHI or SacI digestion. The size of the rearranged bands from junction of exon 7 of EWS to exon 2 of CHOP, identical to that multifocal tumors in each individual patient was identical (Fig. previously reported (16, 17). 3). Rearranged bands were identified with the TLS probe in 11 tumor samples from five patients, using BclI digestion, and the DISCUSSION size of these bands was also constant in different samples from The CHOP gene, a member of leucine zipper transcription each patient (Fig. 4). Tumor samples from patient 5 showed factor family, is implicated in adipocyte differentiation and only germline configuration of TLS. Because EWS has been growth arrest (reviewed in Ref. 22). In myxoid liposarcoma, the reported as an alternative translocation partner for CHOP in rare 3Ј end of the CHOP gene is fused to the 5Ј portion of TLS (for cases of myxoid liposarcoma, we examined EWS in this case translocated in liposarcoma; Ref. 14), also known as FUS (for (four samples), using an EWS cDNA probe. All four tumor fusion; Ref. 13). The hybrid gene encodes a protein that consists samples from patient 5 showed a rearranged EWS band of equal of the 5Ј portion of TLS fused to the entire coding region of size, in HindIII-digested tumor DNA (Fig. 5). CHOP. The TLS-CHOP protein is thought to function primarily

Fig. 5 Monoclonality of EWS rearrangements in case 5. The normal germline band pattern seen in the control lane containing genomic Fig. 3 Monoclonality of CHOP rearrangements by Southern blot anal- placental DNA (Lane PL) consists of three bands in HindIII-digested ysis. The normal germline band pattern seen in the control lane con- DNA. Case 5 contained an EWS rearrangement, absent in three other taining genomic placental DNA (Lane PL), consists of two bands, of 2.1 liposarcomas (left). Four DNA samples from separate sites in case 5 and 9.4 kb, in SacI-digested DNA. Each lane contains DNA from a (right axilla, right thigh, left arm, and intra-abdominal) all showed separate site in each patient (case 4: right thigh and left femur; case 5: the same rearranged EWS band (right). RT-PCR and sequencing right axilla, right thigh, left arm, and intra-abdominal; case 2: abdominal confirmed an EWS-CHOP fusion transcript in this case (see “Re- wall and chest wall). The rearranged CHOP bands are different in each sults”). patient but identical in all sites from each case.

Numerous studies using cytogenetic or RT-PCR-based detection of the t(12;16) have confirmed the concept that round cell liposarcoma represents the high-grade form of myxoid liposarcoma (26–29). In the present study, the initial histopa- thology (at site 1) was low-grade myxoid liposarcoma in five patients, three of whom showed high-grade (round cell) histol- ogy at other synchronous or metachronous sites (Table 1). Anecdotally, the clinical outcome of these five patients seems as poor as that of patients with exclusively high-grade myxoid or round cell liposarcoma, suggesting that the behavior of “low- grade” myxoid liposarcomas with TLS-CHOP may be closer to that of “high-grade” myxoid/round cell liposarcomas than to other types of low-grade liposarcoma. Older clinical studies of Fig. 4 Monoclonality of TLS rearrangements in case 6. The normal liposarcoma have found both grade and histological type to be germline band pattern seen in the control lane containing DNA from a prognostically significant (11), but a systematic analysis of the well-differentiated (nonmyxoid) liposarcoma (Lane C) consists of a prognostic significance of tumor grade in myxoid/round cell single 9.0-kb band in BclI-digested DNA. Three DNA samples from liposarcoma in a uniformly managed series of cases defined by separate sites in case 6 (intra-abdominal, left axilla, and left thigh) all showed the same rearranged TLS band. the presence of TLS-CHOP remains to be done. In our study group, we found that 16% of patients with myxoid liposarcoma presented with either synchronous or metachronous multifocal tumors. This prevalence may be a slight as an aberrant transcriptional regulator that interferes with adi- overestimate because patients with multiple tumors may have pocyte differentiation (Refs. 23 and 24; reviewed in Ref. 22). been more likely to have tumor available for molecular studies. EWS and TLS define a new subfamily of RNA binding proteins, Other authors place the prevalence of apparent multifocal dis- and their extensive structural and sequence similarities suggest ease in liposarcoma at approximately 10% (2). As discussed that they may have originated from a common ancestor gene above, in patients with multifocal myxoid liposarcoma, tumor (25). It is, thus, not entirely surprising that in rare cases of foci from different anatomical locations may have different myxoid liposarcoma the EWS gene at 22q12 is an alternative histological grades (e.g., low-grade myxoid versus high-grade translocation partner of CHOP (16, 17). One of our six patients round cell). Therefore, the clonality of multifocal disease cannot with multifocal myxoid liposarcoma showed EWS rearrange- be deduced from histological features alone. In addition, the ment by Southern blotting analysis in all four tumor samples. clinical details of our group of cases, with only one patient As in the four previously reported cases (16, 17), sequence developing pulmonary metastases, and this only after 4 years analysis of the EWS-CHOP hybrid transcript showed an in- from presentation with multifocal disease, were also not helpful frame fusion of exon 7 of EWS to exon 2 of CHOP. This is, thus, in resolving the issue of monoclonal metastatic versus multi- the fifth case of myxoid liposarcoma with the variant EWS- clonal multifocal disease. Blair et al. (1) attempted to address CHOP gene fusion to be reported. this question by comparing the survival of the patients present-

ing with multifocal STS with patients having solitary tumors ing the TLS-CHOP rearrangement in the vicinity of the with lung metastases. The authors found that the two groups of tumors. We find no evidence for this model of myxoid patients have a similar clinical outcome and consequently con- liposarcoma, insofar as a Southern blot analysis of CHOP in cluded that multifocal STS should be treated as advanced-stage peritumoral normal adipose tissue in three cases (two “uni- disease (1). focal” and one multifocal, not included in the present series) Clonality has been investigated by molecular approaches is negative.4 in various synchronous or metachronous multifocal solid Our present analysis of the genomic rearrangements of tumors, including, among others, carcinomas of the breast TLS, CHOP,orEWS in six patients confirms the monoclonal (30), lung (31), liver (32), bladder, head and neck (33, 34), origin of multifocal myxoid liposarcoma. This unusual clinical ovary and endometrium (35), ovary and appendix (36, 37), phenomenon most likely represents a pattern of presumably and contralateral ovaries (35), as well as in leiomyosarcomas hematogenous metastasis to other soft tissue sites, by tumor arising in immunocompromised individuals (38, 39), gastri- cells seemingly incompetent to seed the lungs. It remains un- nomas (40), and Kaposi’s sarcomas (41). The genetic mark- clear whether this pattern of early metastasis to other soft tissue ers used have included analysis of X inactivation (30, 34, 40, sites (and late or absent lung metastases) represents an intrinsic 41), specific point mutations (e.g., in p53, K-ras, and MEN1; property of this subset of myxoid liposarcomas or merely a rare Refs. 31, 35–37, and 40), viral integration (32), viral episome chance occurrence. structure (38, 39), patterns of allelic loss (34, 40), and nu- merical or structural chromosome abnormalities (33). Certain clonal markers, due to their limited number of possible values ACKNOWLEDGMENTS or states (e.g., X inactivation, allelic loss), may lack the We thank Aime´e Hamelin for technical assistance with the molec- statistical power to convincingly confirm or exclude clonal ular analysis of case 5 and Dr. David Ron for providing the TLS-CHOP cDNA plasmid and for helpful discussions. relationships between separate tumors. In contrast, chromosomal translocations resulting in fusion products provide, at the genomic level, perhaps the most powerful idiotypic clonal REFERENCES marker. In these translocations, the genomic breaks occur 1. Blair, S. L., Lewis, J. J., Leung, D., Woodruff, J. M., and Brennan, within introns separating more proximal and distal exons M. F. Multifocal extremity sarcoma: an uncommon and controversial encoding the functional domains of the respective genes, the entity. Ann. Surg. Oncol., 5: 37–40, 1998. expression and juxtaposition of which is oncogenic in a 2. 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Cristina R. Antonescu, Abul Elahi, John H. Healey, et al.

Clin Cancer Res 2000;6:2788-2793.

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