Myxoid Variant of Well-Differentiated Liposarcoma Andrew Horvai University of California San Francisco, CA

Clinical History: A 65 year old woman with vague abdominal discomfort was found to have a 10 cm tumor of the retroperitoneum. The tumor was resected. Pathological/Microscopic Findings and any Immunohistochemical or Other Studies: The specimen consisted of an approximately 10 cm relatively well-defined mass with fatty, myxoid and cystic components. Areas of hemorrhage and possible necrosis were noted grossly (Figure 1). At low magnification, the tumor consisted of abundant myxoid stroma and a moderately cellular spindle-cell proliferation. In some areas (Figure 2) the stroma formed pools reminiscent of a so-called "pulmonary edema" pattern. At higher magnification, immature adipocytes (lipoblasts) were admixed with more primitive appearing spindle cells. Some of the lipoblasts showed a small lipid vacuole with an eccentric, scalloped hyperchromatic nucleus suggesting a signet-ring type of morphology (Figure 3). However, further inspection showed transitional areas (Figure 4) where the small signet ring-like lipoblasts contrasted with more cellular pleomorphism. At higher magnification, the latter areas (Figure 5) consisted of pleomorphic spindle cells and lipoblasts with large, bizarre and hyperchromatic nuclei. The tumor cells showed nuclear staining with CDK4 and MDM2 in all areas (Figure 6) as well as amplification of 12q13-15 (Figure 7) Slide 1 Click to view with ImageScope Click to view with a Web-Based Viewer Figure 1 Gross examination demonstrates a fatty and myxoid neoplasm with areas of hemorrhage. Figure 2 Low magnification shows myxoid matrix and areas of cyst formation reminiscent of a so-called "pulmonary edema" pattern. Figure 3 Some areas of tumor demonstrate immature fat cells (lipoblasts), a subset of which are reminiscent of signet ring cells. Figure 4 A transitional area of the tumor shows juxtaposition of myxoid areas with small lipoblasts to cells with higher grade cytomorphology. Figure 5 Higher grade areas have more fibrous stroma and striking nuclear pleomorphism, yet retain adipocytic differentiation Figure 6 Tumor cells show nuclear positivity with antibodies to CDK4 and MDM2 Figure 7 The tumor shows high level amplification of chromosome region 12q13-15 by array comparative genomic hybridization Differential Diagnoses: The differential diagnosis includes: Myxoid / round cell liposarcoma Myxoid / round cell liposarcoma with dedifferentiation Myxoid variant of well-differentiated liposarcoma Mixed type liposarcoma (collision tumor) Final Diagnosis: Myxoid variant of well-differentiated liposarcoma Case Discussion: Summary of adipocyte differentiation Our understanding of adipocyte differentiation from mesenchymal progenitor cells derives from culture data using 3T3-L1 cells. [1] In a markedly simplified paradigm, increased p53 and Rb activity cause cell cycle arrest followed by increased levels of CCAAT/enhancer-binding protein beta (CEBPβ) and CEBPγ . These transcription factors, in turn, induce increased levels of CEBPα and peroxisome proliferator gamma (PPARγ) triggering a feedback loop between the latter two transcription factors. PPARγ , especially, is a master regulator of adipocyte genes such as lipoprotein lipase (LPL), leptin, adipsin and fatty acid synthase. Differential diagnosis and workup of liposarcomas Liposarcomas are classified into three major clinicopathologic families: Liposarcoma group Genetics Well-differentiated / dedifferentiated Amp 12q13-15 Myxoid / round cell t(12;16) FUS;DDIT3 t(12;22) EWS;DDIT3 Pleomorphic Complex The above families of tumors not only differ in clinically and histologically, but also in genetically. Our growing understanding of the genetic abnormalities conserved between liposarcoma subtypes has lead to insights into the pathogenesis of these tumors. Although no established targeted therapy yet exists for liposarcomas, continued efforts to dissect the molecular pathogenesis of these tumors may ultimately lead to effective anticancer drugs. The discussion below focuses on the well-differentiated and myxoid families of liposarcomas because these have been best characterized at the molecular and genetic level. The accurate diagnosis of liposarcoma subtype is clinically important. Whereas well-differentiated liposarcomas essentially never metastasize, their dedifferentiated counterparts as well as myxoid and pleomorphic liposarcomas carry substantial metastatic risk. Consequently, systemic chemotherapy has little role in the treatment of well-differentiated liposarcoma. In most cases, H&E findings are adequate to distinguish between liposarcoma types. In classic examples, well-differentiated liposarcoma consist of adipose tissue with variability of adipocyte size, prominent fibrous septae containing fine, almost powdery, collagen and cytologically atypical spindle cells and atypical adipocytes. The atypia is characterized by nuclear hyperchromasia and in some cases, larger cells with multilobed nuclei. Mitotic activity, necrosis and most importantly, lipoblasts, are not required for the diagnosis. De-differentiated liposarcoma is defined by the presence of two components: well-differentiated liposarcoma and a second, macroscopic, mesenchymal component that lackslipogenic differentiation. Most commonly, both components are present at the time of initial diagnosis, but in about 10% of cases the non-lipogenic component presents years after the diagnosis of well-differentiated liposarcoma. The non-lipogenic component may be a high grade undifferentiated sarcoma (so-called "MFH"), or have heterologous areas including osteosarcoma, chondrosarcoma or rhabdomyosarcoma. [2, 3] The hallmark of myxoid liposarcoma is a reticular pattern of delicate capillaries (so-called chicken wire pattern) in a myxoid matrix. The cellular component consists of variable amounts of short spindle cells with bland nuclei and small, immature adipocytes (so-called signet-ring lipoblasts). The atypical spindle cells and large adipocytes of  well-differentiated liposarcoma are notably absent. Round cell areas, typically at the edges of tumor lobules, are sufficiently dense to have cell-cell contact or even overlap. The cells are round to oval, epithelioid, have amphophilic cytoplasm, central round nuclei sometimes with obvious nucleoli, lack lipid vacuoles but retain the chicken wire vasculature. The metastatic potential of this family of tumors ranges from 20% (Grade I) to 75% (Grade III) based on the proportion of round-cell component. [4, 5] Recent data indicates that any round cell area (even 1%) may confer a worse prognosis. [6] Genetic abnormalities and molecular pathogenesis As previously mentioned, the well-differentiated - dedifferentiated liposarcoma family of tumors share high level amplification of chromosome subregion 12q13-15. Furthermore, this amplification is highly sensitive and specific for well-differentiated and dedifferentiated liposarcoma. [7, 8, 9] The 12q13-15 region is ~10Mb long and encompasses important cell cycle regulatory genes (CDK4 and MDM2) and a gene involved in adipocyte differentiation (HMGA2). In most cases, the region actually encompasses two discontinuous amplicons, one incorporating CDK4 and the other MDM2. [10, 11] The amplification of 12q13-15 can be detected by cytogenetics, fluorescence in-situ hybridization (FISH) or comparative genomic hybridization. Immunohistochemical detection of CDK4 and MDM2 protein overexpression is a highly sensitive and specific surrogate to molecular methods. [12, 13] Though these regulatory proteins have diverse functions, at the simplest level, increased levels of CDK4 and MDMD2 cause down-regulation of Rb and p53, respectively. Therefore, increased CDK4 and MDM2 promote cell proliferation and survival. Dedifferentiated liposarcomas show 12q13-15 amplification but, in addition, have more total amplifications than their well-differentiated counterparts. However, no genetic changes uniformly distinguish between these entities. [11] Through diverse mechanisms, a general increase of AP-1 signaling pathways (through increased C-JUN, ASK-1 and FOS-B) is more common in dedifferentiated tumors. [11, 14] Elevated AP-1 transcription factor activity, in turn, may have an inhibitory effect on adipocyte differentiation by inhibiting both CEBP and PPAR-γ . In contrast, myxoid / round cell liposarcomas uniformly lack 12q13-15 amplification. Rather, these tumors demonstrate t(12;16) or t(12;22) translocations in most cases, resulting in the FUS:DDIT3 or EWS:DDIT3 gene fusions, respectively (in earlier reports, FUS is also known as TLS while DDIT3 is also known as CHOP). In the more common translocation, the N-terminal transactivation domain of FUS is joined to DDIT3, the latter is also a member of the CEBP family of transcription factors. The FUS:DDIT3 fusion product may inhibit adipogenesis by interfering with CEBPβ and PPAR-γ activity based on in vitro and transgenic mouse studies. [15, 16] Of course, this is probably an oversimplified mechanism and other signaling pathways, (e.g. NF kB) may be involved. [17] What about mixed type liposarcoma? As mentioned above, conventional well-differentiated liposarcoma may have myxoid areas, particularly in the retroperitoneum. Further complicating the diagnosis, myxoid areas of well-differentiated liposarcoma may also demonstrate a branching vascular pattern although the vessels tend to be of larger caliber than in true myxoid / round cell liposarcoma. In the past, such retroperitoneal tumors may have been classified as so-called "mixed type liposarcoma" (implying both well-differentiated and myxoid / round cell differentiation) but amplification of 12q13-15 and FUS:DDIT3 translocations appear to be mutually exclusive arguing against this possibility. [18, 19] Furthermore, genetic and clinicopathologic data suggests that true myxoid liposarcoma is exceedingly rare, possibly nonexistent, in the retroperitoneum. [20] The distinction between well-differentiated and myxoid liposarcoma is very significant because the latter has metastatic potential but is also more chemo- and radiosensitive. Review of the Literature/Treatment Options: From biology to Treatments The mainstay of therapy for localized liposarcoma is surgery alone or in combination with radiation therapy. In patients with unresectable, recurrent or metastatic disease, conventional cytotoxic chemotherapy with Adriamycin and ifosphamide based regimens may be used. However, two additional "targeted" modalities, nuclear receptor ligands and Trabectedin, deserve mention. Nuclear receptor ligands As discussed above, PPAR-γ is a transcription factor in the nuclear receptor family that promotes transcription of multiple adipocyte-specific genes. PPAR-γ binds to DNA as a heterodimer with the retinoid X receptor (RXR) and activates transcription. PPAR-γ is expressed in benign adipose tissue and all liposarcoma subtypes. [21] PPAR-γ binds to the thiazolidinedione class of antidiabetic drugs and terminal adipocyte differentiation can be induced in myxoid/round cell and well-differentiated liposarcomas by thiazolidenediones. [22, 23, 24] An initial phase II study of 3 patients with liposarcoma treated with troglitazone showed morphologic and gene expression changes suggesting tumor differentiation. [25] Unfortunately, a second phase II study of nine patients with advanced liposarcoma treated with rosiglitazone did not demonstrate morphologic or gene expression evidence of differentiation. [26] It is important to recognize that such therapies may not be expected to shrink tumors, since differentiation and accumulation of intracellular lipid may actually cause tumors to grow clinically and a decrease in proliferation rate may be difficult to measure in liposarcomas that often have a low proliferative fraction at baseline. Nevertheless, the reasons for the discrepancy between the two studies is unknown but may be related to the tumors, agent used or patients. Stimulation of RXRα with the specific ligand LG268 can also trigger terminal differentiation in vitro [27] but, to date, no clinical trials have investigated the utility of combined RXRα -PPARγ therapy in liposarcoma. Trabectedin Trabectedin (Yondelis, ET-743) is a relatively novel agent derived from the sea squirt Ecteinascidia turbinata and preclinical studies have shown antitumor effects against a number of solid tumors including sarcomas. [28] The suggested mechanisms of action include site specific DNA damage and transcriptional interference. In vitro evidence suggests that Trabectedin promotes differentiation in myxoid liposarcomas by blocking the effects of the FUS:DDIT3 chimeric protein and allowing activation of the CEBPs. Myxoid/ round cell liposarcomas show particular sensitivity to Trabectedin and long term efficacy in a retrospective study. [29] Interestingly, both the clinical and in vitro effects are selective for a subset of FUS:DDIT3 fusions (the shorter, type 1 and 2 fusions). [30] The efficacy of this novel agent in other liposarcoma types remains to be established. Conclusion(s): Understanding of adipocyte differentiation has given insight into the mechanisms of tumorigenesis in liposarcoma. The mainstay of liposarcoma therapy is surgery, but the decision to use adjuvant therapy requires accurate tumor classification. Novel agents targeting nuclear receptors (PPAR-γ, RXR) or FUS:DDIT3 show some promise for future study. References Cowherd RM, Lyle RE, McGehee RE, Jr. Molecular regulation of adipocyte differentiation. Semin Cell Dev Biol 1999; 10:3-10. McCormick D, Mentzel T, Beham A, Fletcher CD. Dedifferentiated liposarcoma. 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