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Loss of Heterozygosity and Microsatellite Instability Are Rare in Sporadic Dedifferentiated Liposarcoma: A Study of 43 Well-Characterized Cases.

Germline mutations in DNA mismatch repair genes, including MLH1, MSH2, MSH6, and PMS2, are seen in Lynch syndrome (hereditary nonpolyposis colon cancer syndrome) and can be detected experimentally by microsatellite instability (MSI). Defects in mismatch repair frequently result in colorectal and endometrial adenocarcinoma. (1,2) However, other malignancies have been demonstrated to arise less frequently in Lynch syndrome and include tumors of the ovary, biliary tract, upper gastrointestinal tract, and urothelium, as well as soft tissue sarcomas. (1-6) Although various sarcomas are associated with Lynch syndrome, including leiomyosarcoma and undifferentiated pleomorphic sarcoma, liposarcoma seems to be the most common. (2-6) In patients with Lynch syndrome, all of these sarcoma subtypes have mutations in mismatch repair (MMR) genes. Further more, a 2003 study (6) showed increased rates of sarcoma in a PMS2-deficient mouse model. Taken together, these findings suggest that defective DNA repair mechanisms may contribute to sarcoma pathogenesis. The role of mismatch repair defects in sporadic sarcomas is poorly understood, although studies (5,7,8) so far suggest that such defects seem to be rare.

Liposarcoma, specifically the well-differentiated and dedifferentiated subgroup, represents the most commonly occurring sporadic (ie, not Lynch syndrome associated) soft tissue sarcoma. Dedifferentiated liposarcoma is defined by the presentation, either synchronous or metachronous, of well-differentiated (ie, lipogenic) and dedifferentiated (ie, nonlipogenic) components. (9) Both components share a reproducible amplification of chromosome subregion 12q13-15. (10-14) Our group recently reported that the matched pairs of lipogenic and nonlipogenic components of dedifferentiated liposarcoma are genetically quite similar. (13) Therefore, large chromosomal gains and losses likely take place early during tumorigenesis. The mechanisms of progression from a lipogenic to a nonlipogenic sarcoma remain poorly characterized but may depend on smaller genetic changes.

Although MSI, resulting from mismatch repair defects, has been rarely reported in a heterogeneous group of sporadic sarcomas, (8,15) its presence in liposarcoma specifically has not been well characterized. Consequently, the role of MSI, if any, in sporadic liposarcoma tumorigenesis and/or progression is unknown. The present study evaluates the presence of MSI in a large, well-categorized group of sporadic dedifferentiated liposarcomas arising from welldifferentiated precursors.

MATERIALS AND METHODS

Case Selection

After approval of this study by our institution's committee on human research, the departmental archives were searched for examples of dedifferentiated liposarcoma. We retrieved 43 tumors from the Department of Pathology database with adequate normal and lesional paraffin-embedded tissue for microdissection.

The diagnosis of well-differentiated liposarcoma required the presence of a lipogenic component and a synchronous or metachronous nonlipogenic component. (9) The dedifferentiated components were graded according to the current World Health Organization criteria, (9) with pleomorphic sarcomas resembling undifferentiated sarcoma (so-called malignant fibrous histiocytoma) considered high grade and uniform spindle cells with mild nuclear atypia, often in a fascicular pattern, considered low grade. As defined herein, the pattern of low-grade dedifferentiation is indistinguishable from "cellular" well-differentiated liposarcoma. (9,16)

We have previously reported on 16 of these cases as demonstrating the 12q13-15 amplification. (13) The remaining 27 cases showed overexpression of MDM2 and CDK4 by immunohistochemistry. All tumors were sporadic, with no documented history of Lynch syndrome.

Microdissection and MSI Testing

From each tumor, the best-preserved nonlipogenic, dedifferentiated component and normal tissue were identified on hematoxylin-eosin-stained slides. Manual microdissection was then performed on unstained slides from formalin-fixed, paraffin-embedded tissue for DNA extraction as previously described (4,17,18) and as typically performed at our institution for MSI testing. The presence of mismatch repair defects was detected using standard fluorescence-based polymerase chain reaction with the Promega MSI Analysis System, version 1.2 (Promega, Madison, Wisconsin) kit, including 5 mononucleotide microsatellite markers (BAT25, BAT26, NR21, NR24, and MONO27), as well as 2 pentanucleotide repeat markers (Penta C and Penta D). Although the pentanucleotide markers are not evaluated for MSI, most individuals are heterozygous at these loci, allowing for detection of loss of heterozygosity (LoH). The mononucleotide repeats are not used in assessment of LoH because most individuals are homozygous at these loci. In cases where MSI or LoH was present, the welldifferentiated component was also tested for both MSI and LoH using the same methods. Microsatellite instability was subcategorized into the following 3 groups: microsatellite stable, defined as the absence of MSI in all 5 mononucleotide markers; MSI-low, defined as MSI at one marker; and MSI-high, defined as MSI at 2 or more loci. (19) Loss of heterozygosity was defined as an apparent loss of 1 of 2 alleles in the lesional tissue compared with normal tissue.

RESULTS

Clinicopathologic and Genomic Features

Most tumors (36 of 43; 84%) showed a lipoma-like liposarcoma (Figure, A) juxtaposed to a high-grade pleomorphic or spindle cell sarcoma in the nonlipogenic component (Figure, B through D). The remaining 7 tumors showed low-grade histology, as defined by the World Health Organization, with uniform spindle cells with mild atypia in a fascicular pattern. (9) All of these latter tumors averaged fewer than 5 mitoses per 10 high-power fields so they may also be considered so-called cellular well differentiated liposarcoma according to the criteria by Evans. (16) None of the tumors demonstrated the recently described "homologous" dedifferentiated phenotype. (20,21) The most common site was the retroperitoneum (25 of 43; 58%). Based on our inclusion criteria, all dedifferentiated liposarcomas demonstrated 12q13-15 amplification (Figure, E) and/or MDM2 and CDK4 overexpression by immunohistochemistry (data not shown).

Microsatellite Instability

Microsatellite instability data are summarized in Table 1, with detailed results showing individual cases and markers given in Table 2. Forty-two of 43 cases (98%) of dedifferentiated liposarcoma were microsatellite stable. All paired lipogenic components separately tested (n = 9) were also microsatellite stable. One case (2%) of dedifferentiated liposarcoma demonstrated MSI at a single locus (NR24) and therefore was classified as MSI-low; this same case also had LoH at Penta D. The case consisted of a high-grade dedifferentiated liposarcoma, 6.5 cm, primary to the trunk, which developed in a 57-year-old man who at initial presentation had metastatic disease to the lung. The paired lipogenic component of this tumor was MSI stable.

Loss of Heterozygosity

Overall, LoH was observed in 8 of 43 dedifferentiated liposarcomas (19%) (Table 1). Loss of heterozygosity was identified at Penta C (3 of 43; 7%), Penta D (3 of 43; 7%), or both (2 of 43; 5%). The dedifferentiated liposarcomas with LoH showed predominantly (7 of 8; 88%) high-grade pleomorphic histology, but the presence of LoH did not correlate with grade (P = .80) or tumor location (P = .40). In 4 tumors (50%), LoH was present only in the nonlipogenic component but was absent in the matched lipogenic component (Table 2).

COMMENT

The association between Lynch syndrome and liposarcomas, as well as rare previous reports of sporadic sarcomas with MSI, raises the possibility that defective mismatch repair genes have roles in the pathogenesis of liposarcoma. (2,5,8,15) However, no previous studies specifically addressing MSI in sporadic liposarcomas have been reported to date. In addition, while dedifferentiated liposarcomas are genetically complex, no known reproducible genomic changes distinguish the lipogenic and nonlipogenic components of these tumors. (13) Our study sought to determine if MSI was present in sporadic cases of dedifferentiated liposarcoma and, if so, to evaluate whether these genetic alterations varied between paired well-differentiated (lipogenic) and dedifferentiated (nonlipogenic) components.

Our results demonstrated largely the absence of MSI, with only one tumor categorized as MSI-low. Therefore, mismatch repair defects likely do not contribute to sporadic dedifferentiated liposarcoma tumorigenesis. The low incidence of MSI, as hypothesized by Suwa et al, (15) suggests that in liposarcoma MSI is a secondary effect of overall genomic loss and alterations, which may affect mismatch repair genes rather than having a causative role in tumorigenesis. The previously observed association between Lynch syndrome and liposarcoma may represent tumorigenesis mechanisms involving mismatch repair defects unique to these patients.

Prior studies examining MSI in sarcomas have focused on morphologically or genetically (ie, pleomorphic undifferentiated sarcoma) heterogeneous groups of sarcomas. Ericson et al (8) examined 209 soft tissue sarcomas with pleomorphic sarcoma-like histology for defects in mismatch repair using both immunohistochemistry (antibodies against MLH1, MSH2, and MSH6) and polymerase chain reaction and demonstrated that rare cases (2 of 209 tumors) have MSI. Suwa et al (15) demonstrated that 3 of 39 sarcomas had MSI, including 1 of 7 liposarcomas. The latter study used different microsatellite loci than are typically used for Lynch syndrome screening and lacked the mononucleotide repeats that have been shown to be more sensitive and specific for detection of defective mismatch repair (22); LoH at the same loci was noted when present. Our study is the first to date to comprehensively examine a large number of dedifferentiated liposarcomas with paired well-differentiated components and more definitively excludes MSI as a tumorigenic mechanism in sporadic liposarcomas.

The present study also demonstrated LoH in a subset (8 of 43) of liposarcomas and showed that LoH occurred more frequently in the dedifferentiated component than in their paired well-differentiated component. Using the same cohort of liposarcoma cases as in the present study, Horvai et al (13) identified complex genetic changes, including confirmation of 12q amplification in both dedifferentiated liposarcoma components. However, no genetic changes were identified that consistently distinguish lipogenic and nonlipogenic components of dedifferentiated liposarcomas. Prior studies using other methods have sought to determine the changes that lead to possible progression in liposarcoma. These include studies (23-25) focused specifically on LoH at the retinoblastoma (Rb) gene, which was identified more frequently in dedifferentiated liposarcomas (54%-100%), with less frequent (0%-8.7%) concurrent loss in paired well-differentiated components and no loss in tumors demon strating only well-differentiated liposarcoma morphology. Given the increased frequency of LoH in dedifferentiated liposarcoma and the role of Rb in cycle cell regulation, it is hypothesized that Rb LoH may contribute to the progression from well-differentiated to differentiated liposarcoma. Suwa et al (15) also identified a single case of liposarcoma with LoH at 2 separate MSI loci. Loss of heterozygosity at other gene loci has not previously been described in liposarcoma, but it could also contribute to tumorigenesis if occurring at loci where genetic alterations would influence tumor suppressors or cycle cell regulatory genes.

The Penta C and Penta D microsatellites are located on chromosomes 9p and 21q, respectively. (26) Our prior work has not shown that these loci are consistently altered in dedifferentiated liposarcoma. (13) Thus, it is unclear whether LoH at Penta C and Penta D represents recurring mutations driving tumorigenesis or passenger mutations. Compared with other pleomorphic sarcomas, dedifferentiated liposarcoma shows few genetic changes, suggesting that early tumorigenesis does not require marked chromosomal instability. (14,27) The dedifferentiated components of liposarcomas, based on the relative enrichment of LoH, may suggest a more generalized genomic unrest during progression of this subgroup of tumors.

In summary, MSI and LoH are rare events in sporadic dedifferentiated liposarcomas. Given that MSI-low only occurred in a single case, mismatch repair defects likely do not contribute to sporadic dedifferentiated liposarcoma tumorigenesis. The results demonstrate that LoH at either of 2 loci is present in a subset of dedifferentiated liposarcoma and less frequently manifests in their paired well-differentiated tumor. Therefore, LoH may contribute to tumorigenesis or may represent passenger mutations during tumorigenesis.

This research was supported by the Residents' Teaching and Research Endowments from the Department of Pathology, University of California, San Francisco (Dr Davis). We thank Martin Powers, MD, for critical review of the manuscript.

Please Note: Illustration(s) are not available due to copyright restrictions.

References

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(14.) Rieker RJ, Weitz J, Lehner B, et al. Genomic profiling reveals subsets of dedifferentiated liposarcoma to follow separate molecular pathways. Virchows Arch. 2010; 456(3):277-285.

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(26.) Bacher J, Schumm JW. Development of highly polymorphic pentanucleotide tandem repeat loci with low stutter. Profiles DNA. 1998; 2(2):3-6.

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Jessica L. Davis, MD; James P. Grenert, MD, PhD; Andrew E. Horvai, MD, PhD

Accepted for publication July 25, 2013.

From the Department of Pathology, University of California, San Francisco.

The authors have no relevant financial interest in the products or companies described in this article.

Presented in part at the 101st Annual Meeting of the United States and Canadian Academy of Pathology; March 19, 2012; Vancouver, British Columbia, Canada.

Reprints: Andrew E. Horvai, MD, PhD, Department of Pathology, University of California, San Francisco, 1600 Divisadero Dr, B220, San Francisco, CA 94115-1656 (e-mail: andrew.horvai@ucsf.edu).

Caption: Representative morphologic and genomic copy number changes in dedifferentiated liposarcoma (hematoxylin-eosin, original magnification X200). A and B, Lipogenic (A) and nonlipogenic (B) components of case 1, which was microsatellite stable and showed no loss of heterozygosity in either component. C, Nonlipogenic component of case 15, which was microsatellite stable but had loss of heterozygosity. D, Nonlipogenic component of case 16, which was the only example to show microsatellite instability E, The presence of 12q13-15 amplification was used to confirm the diagnosis of dedifferentiated liposarcoma.
Table 1. Summary of Loss of Heterozygosity and Microsatellite
Instability (MSI) in 43 Dedifferentiated Liposarcomas

Loss of                 Microsatellite Instability, No. (%)
Heterozygosity,
No. (%)

Penta C   Penta D   Microsatellite   MSI-Low (b)   MSI-High (c)
                      Stable (a)

5 (12)    5 (12)       42 (98)          1 (2)           0

(a) All 5 markers stable.

(b) Instability in 1 of 5 markers.

(c) Instability in at least 2 of 5 markers.

Table 2. Loss of Heterozygosity and Microsatellite Instability (MSI)
Are Uncommon Findings in Dedifferentiated Liposarcomas: Comparison of
Lipogenic (L) Well-Differentiated and Nonlipogenic (NL)
Dedifferentiated Components of Dedifferentiated Liposarcomas

Case        Loss of        Microsatellite Instability
No.     Heterozygosity

       Penta C   Penta D   Microsatellite   MSI-Low (b)
                           Stable (a)

       L   NL    L   NL    L       NL       L     NL

3      +   +     -   -     +       +        -      -
4      -   -     +   +     +       +        -      -
15     +   +     +   +     +       +        -      -
16     -   -     -   +     -       -        -      +
21     -   -     -   +     +       +        -      -
31     -   +     -   +     +       +        -      -
33     +   +     -   -     +       +        -      -
38     -   +     -   -     +       +        -      -

Case   Microsatellite
No.    Instability

       MSI-High (c)

       L      NL

3      -      -
4      -      -
15     -      -
16     -      -
21     -      -
31     -      -
33     -      -
38     -      -

(a) All 5 markers stable.

(b) Instability in 1 of 5 markers.

(c) Instability in at least 2 of 5 markers.
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Author:Davis, Jessica L.; Grenert, James P.; Horvai, Andrew E.
Publication:Archives of Pathology & Laboratory Medicine
Date:Jun 1, 2014
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