Introduction
Myxoid liposarcoma (MLS) is the second most common type of liposarcoma (LS) after atypical lipomatous tumor/well-differentiated LS (ALT/WDLS) [
1,
2]. MLS usually presents as a large painless mass within the deep soft tissue of the extremities in young adults and middle-aged individuals. Histologically, MLS is composed of predominantly uniform round- to oval-shaped primitive mesenchymal cells admixed with signet ring or multivacuolated lipoblasts in a prominent myxoid stroma, which is rich in a delicate arborizing “chicken-wire” capillary vasculature. Included in this category are lesions formerly known as round cell LS (RCLS) characterized by hypercellular round cell morphology, which is associated with a significantly poor prognosis [
1‐
3].
Although a variable number of signet ring lipoblasts are found in MLS, extensive mature adipocytic differentiation forming a lipoma-like component is very rare [
4‐
6]. Morphologically, it is often difficult to distinguish MLS with extensive lipoma-like changes (MLSLC) from ALT/WDLS with myxoid changes, but the severe degree of nuclear atypia may help to exclude MLS/MLSLC [
7]. Cytogenetically, most cases of MLS show specific chromosomal translocation t(12;16)(q13;p11) resulting in the formation of a
FUS-DDIT3 (also known as
TLS-CHOP) fusion transcript [
8‐
17], although occasional tumors exhibit the variant t(12;22) (q13;q12), producing an
EWSR1-DDIT3 fusion transcript [
1,
18]. On the other hand, ALT/WDLS is characterized by supernumerary ring chromosome and/or giant markers with amplification of the 12q13 ~ q15 region, which is often associated with overexpression of
CDK4 and
MDM2 [
1,
17,
19‐
23].
Myxoid areas within ALT/WDLS and dedifferentiated LS (DDLS) sometimes possess a prominent plexiform vascularity with thin-walled arborizing capillaries, creating a resemblance to MLS especially when interspersed small fat cells are present [
7,
24‐
26]. On the basis of molecular and immunohistochemical studies, de Vreeze et al. [
24] suggested that apparent primary retroperitoneal MLS/RCLS could be recognized as WDLS/DDLS with morphological features mimicking MLS/RCLS. They considered that finding of the MLS-specific translocations (fusion genes) in a retroperitoneal LS is highly suggestive of metastasis and should prompt search for a primary lesion outside the retroperitoneum.
In addition, rare cases of mixed-type LS composed of an admixture of MLS and WDLS have been reported by some investigators [
17,
27]. More recently, Deyrup et al. [
28] reported a new group of “fibrosarcoma-like lipomatous neoplasm,” which were composed of low-grade spindle cells showing varying degrees of lipoblastic differentiation and sometimes accompanied by abundant myxoid stoma and thin-walled arborizing capillaries similar to those of MLS, but these tumors lacked molecular cytogenetic characteristics of other types of lipomatous tumors.
The development of normal fat cells is considered to be regulated by various factors including peroxisome proliferator-activated receptor-γ (PPARγ) and CCAAT/enhancer-binding protein-α (C/EBPα) [
29]. PPARγ may regulate the expression of lipid droplet-associated proteins including adipophilin and perilipin in normal tissues [
30,
31], but little is known about the mechanism of adipocytic differentiation in MLSLC.
In order to clarify the true nature of rare lipomatous differentiation in MLS, we studied eight cases of MLSLC, by using immunohistochemistry (MDM2, CDK4, PPARγ, C/EBPα, adipophilin, perilipin, and Ki-67), chromosome analysis, fluorescence in situ hybridization (FISH), and reverse transcription polymerase chain reaction (RT-PCR). In addition, ordinary MLS (11 cases), WDLS (4 cases), and DDLS (6 cases) were studied as controls. To our knowledge, this is the first report describing the detailed molecular cytogenetic and clinicopathologic features of MLSLC.
Materials and methods
Tumor material and patient data
The consultation and archival files of molecular cytogenetic analysis of soft tissue tumors in the Department of Pathology, Fukuoka University School of Medicine, between 1987 and 2012 were searched for MLS with or without well-differentiated lipoma-like components. Eight cases of MLSLC were selected for the present study. In addition, 11 cases of ordinary MLS without lipoma-like components, 4 cases of WDLS, and 6 cases of dedifferentiated LS (DDLS) with myxoid changes were studied as controls.
Clinical parameters, including gender, age, location, and macroscopic features, were obtained from medical records. In all cases, histologic sections were available for pathologic review, and the diagnosis was confirmed according to the WHO (2013) classification. Formalin-fixed and paraffin-embedded tumor tissues available in each case were used for immunostaining and molecular analysis. In 16 cases, fresh tumor tissues were utilized for chromosomal analysis as well as molecular study. Follow-up information was obtained from the referring clinicians and from the existing medical records in accord with institutional guidelines.
Immunohistochemical staining
For immunohistochemistry, 3-μm-thick paraffin-embedded tissue sections were mounted on silane-coated glass slides, deparaffinized, and heated in antigen retrieval buffer using a pressure cooker for 10 min or a microwave for 30 min. The following primary antibodies were used: MDM2 (dilution 1/100; Calbiochem, Darmstadt, Germany), CDK4 (dilution 1/200; Invitrogen, Camarillo, CA), PPARγ (dilution 1/100; Perseus Proteomics, Tokyo, Japan), C/EBPα (dilution 1/200; Cell Signaling Technology, Danvers, MA), adipophilin (dilution 1/20; Fitzgerald, Acton, MA), perilipin (dilution 1/200; Cell Signaling Technology, Danvers, MA), and Ki-67 (clone MIB-1, dilution 1/200; Dako, Glostrup, Denmark). Immunohistochemical staining was performed by using the Nichirei Histofine Simple Stain MAX PO (MULTI) (Nichirei Biosciences Inc., Tokyo, Japan). The reactions were visualized with diaminobenzidine, and the sections were counterstained with Mayer’s hematoxylin. For MDM2, CDK4, PPARγ, C/EBPα, adipophilin, and perilipin, the immunoreactivity was graded semiquantitatively as negative (0 %), 1+ (<25 % of neoplastic cells reactive), 2+ (25 to 50 % of neoplastic cells reactive), and 3+ (>50 % of neoplastic cells reactive). The Ki-67 labeling index was obtained as a percentage of positive nuclei by counting 500 neoplastic cells within the areas exhibiting the highest labeling index (hot spots). The Mann-Whitney U test was used to compare the differences of the Ki-67 index.
Cytogenetic analysis
Primary cell cultures, harvesting, and preparation of slides were carried out as previously described [
9,
21,
22]. Chromosome analysis was performed on GTG-banded (Giemsa/trypsin) metaphases, and the karyotypes were described according to the International System for Human Cytogenetic Nomenclature [
32].
FISH analysis for DDIT3 break apart
FISH was performed on paraffin-embedded tissue sections (cases 1–8, 12, 13, 17, 18, 21–25), by using a commercially available
DDIT3 (
CHOP) (12q13) Dual Color, Break Apart Rearrangement Probe (Abbott Molecular Inc.). Briefly, 4-μm-thick paraffin-embedded tissue sections were deparaffinized, dehydrated, and incubated with protease and pretreated by the Vysis Paraffin Pretreatment IV & Post-Hybridization Wash Buffer Kit (Abbott Molecular Inc.) according to the manufacturer’s instructions [
4,
33]. The probe and slides were codenatured at 80 °C for 5 min and incubated at 37 °C overnight in a humidified chamber. Posthybridization washing was performed following standard procedures, and the nuclei were counterstained with DAPI. The slides were examined using a fluorescence microscope with appropriate excitation and emission filters. At least 100 morphologically intact, nonoverlapping nuclei of tumor cells from either myxoid or lipomatous areas were counted. FISH was considered rearranged if more than 10 % of nuclei showed break apart of the dual-color probe signal for the targeted locus.
RT-PCR for FUS-DDIT3
RNA was extracted from fresh-frozen tumor tissues (cases 1, 2, 6–12, 14–21, 23–25, 27–29) as well as formalin-fixed and paraffin-embedded tumor samples (cases 1, 3–8, 13, 22, 26) by using TRIzol® reagent (Life Technologies Japan, Tokyo, Japan) according to the manufacturer’s recommendations. In seven cases of MLSLC (cases 1, 3–8), the paraffin sections were dissected under a microscope, and the samples were taken separately from myxoid and lipoma-like components, which were simultaneously identified by examining the adjacent serial sections stained with H & E.
RNA was reverse transcribed into complementary DNA (cDNA) by PrimScript® II first-strand cDNA Synthesis Kit (Takara Bio, Tokyo, Japan). The PCR to amplify a cDNA that corresponds to
FUS-DDIT3 fusion gene was performed by using KOD-Plus-Ver. 2 (Toyobo, Tokyo, Japan) with the
FUS ex5 and
DDIT3 primer set amplifying a 160-bp (
FUS-
DDIT3 type II) fragment and a 436-bp (
FUS-
DDIT3 type I) fragment or the
FUS ex7 and
DDIT3 primer set amplifying a 129-bp (
FUS-
DDIT3 type I) fragment, according to Powers et al. [
34]. PCR products were analyzed by Microchip Electrophoresis System for DNA/RNA Analysis with DNA-1000 Kit (Shimadzu, Tokyo, Japan).
Discussion
LSs rarely exhibit unusual histologic features with combined patterns of lipoma-like WDLS and MLS [
4,
6,
12,
23]. However, the distinction between MLSLC and WDLS/DDLS with myxoid changes is often difficult because of the morphologic similarities, although the absence of hyperchromatic cells in the adipocytic areas of MLSLC may be a useful diagnostic clue [
7]. The recent advances of molecular cytogenetic techniques including FISH and RT-PCR provide powerful tools for resolving these problems, as shown in the present study.
Our study demonstrated that MLSLCs containing peculiar lipoma-like components actually represent MLS, which is different from either true mixed-type LS or WDLS/DDLS with myxoid changes, on the basis of molecular cytogenetic features. Both lipoma-like and myxoid components of the same MLSLC exhibited the identical abnormality, FUS-DDIT3 specific for MLS, which was confirmed by FISH and RT-PCR. There was a clear cut difference between MLSLC and WDLS/DDLS with myxoid changes; the former was positive for t(12;16)/FUS-DDIT3 and negative for giant marker/ring chromosomes, whereas the latter exhibited converse characteristics.
The occurrence of true mixed-type LS may be very rare, if it can exist as a distinct entity. Meis-Kindblom et al. [
17] described a series of 30 cases of LS including a case of mixed LS, which is composed of WDLS and MLS with a coexistence of ring/giant marker chromosome (characteristic of WDLS) and t(12;16)/
FUS-DDIT3 (specific for MLS). Unfortunately, however, no FISH data from the WDLS component of the tumor was shown in this case. Mentzel et al. [
27] reported another example of mixed-type LS consisting of ALT/WDLS and MLS components. By FISH analysis, they found amplifications of
MDM2 and
CDK4 genes in ALT/WDLS areas and translocations of
DDIT3 and
FUS genes in MLS areas, although their immunohistochemical study failed to demonstrate clear nuclear expressions of MDM2 and CDK4 in the tumor. On the other hand, Antonescu et al. [
16] suggested that in many instances of mixed MLS+WDLS or translocation-negative MLS, the tumors may have represented predominantly myxoid WDLS or pleomorphic LS with myxoid changes, as supported by the cytogenetic data in the respective reports. They considered that the presence of microscopic foci of lipoma-like or sclerosing areas, characteristic of WDLS, constitutes sufficient histologic evidence to exclude the diagnosis of MLS, as supported by the consistent absence of
DDIT3 or
FUS genomic rearrangements in such tumors.
Recently, de Vreeze et al. [
5] analyzed eight cases of LS designated as mixed-type LS with combined patterns of WDLS and MLS. By immunohistochemical and molecular data, they concluded that these mixed-type LSs should not be regarded as collision tumors, but as an extreme variant of morphological entity, explaining biological contradiction of mixed-type LS.
In the present study, FUS-DDIT3 fusion gene was found in distinct lipoma-like components as well as myxoid areas of MLSLC, suggesting that tumor cells of some true MLSs have a potential to differentiate into mature adipocytes producing a lipoma-like masses mimicking WDLS. Conversely, ring/giant marker chromosomes as well as immunohistochemical expressions of CDK4 and MDM2 characteristic of ALT/WDLS were never found in any case of MLS with a lipoma-like component.
Little is known about the mechanism of adipocytic differentiation in MLSLC and ordinary MLS, although various factors have been supposed to be concerned with the development of fat cells. Some molecular studies [
13,
35] suggested that
FUS-DDIT3 prevents the development of adipocytic precursors in MLS by repressing PPARγ and C/EBPα. On the other hand, based on a comparative ultrastructural and RT-PCR analysis of MLS/RCLS, Huang and Antonescu [
36] found that the variation of
FUS-DDIT3 fusion transcript showed no apparent impact on adipogenesis of MLS, and they considered that other factors might be implicated in their level of differentiation.
Normal adipogenesis is thought to occur in two stages: commitment of mesenchymal stem cells to a preadipocyte fate, followed by terminal differentiation to mature adipocytes [
29]. Adipogenic stimuli induce terminal differentiation in committed preadipocytes through the epigenomic activation of PPARγ. The coordination of PPARγ with C/EBP transcription factors maintains adipocyte gene expression.
In our study, both PPARγ and C/EBPα were found in each type of LSs with or without mature lipoma-like components. The results are in accord with the molecular analysis by Tontonoz et al. [
14]. Recent studies suggested that PPARγ may regulate the expression of lipid droplet-associated proteins including adipophilin and perilipin. Adipophilin associates with smaller neutral lipid storage droplets located within many tissues, whereas perilipin is located on the surface of larger triacylglycerol droplets in mature adipocytes and on cholesterol ester droplets in steroidogenic cells [
30,
31]. In the present study, we found that adipophilin was strongly expressed in tiny fat droplets of immature lipoblastic cells of MLSLC and ordinary MLS, whereas mature adipocytes in lipoma-like component of MLSLC exhibited weak expression of this protein. On the other hand, perilipin showed a strong positive staining in large fat vacuoles of signet ring and multivacuolated lipoblasts as well as mature adipocytes in lipoma-like components of MLSLC and WDLS, but myxoid areas of MLSLC and DDLS contained a few cells possessing small fat vacuoles positive for perilipin. The combined immunohistochemical detection of adipophilin and perilipin may provide a useful ancillary tool for identification of lipoblastic cells in soft tissue sarcomas, since the former is localized in less-differentiated lipoblasts, and the latter is confined to more mature lipoblasts and fat cells.
Immunostaining for Ki-67 (MIB-1) demonstrated a lower labeling index of the nuclei in lipoma-like components of MLSLC, when compared with myxoid areas of the same tumor as well as ordinary MLS (p < 0.001). The results suggest that well-differentiated tumor cells resembling mature adipocytes in the lipoma-like components possess a lower level of proliferative activity, whereas undifferentiated or poorly differentiated cells in the myxoid areas of MLS with or without lipoma-like components retain high proliferative activities.
The clinical behavior of LS is highly dependent on the histologic subtype and the location of tumor [
1,
2,
4,
17,
25]. The prognosis of MLS involving the deep soft tissue of the extremities is generally favorable when appropriately treated by a complete wide surgical excision with or without a combined radiation or chemotherapy. In the present study, one (20 %) of five MLSLCs followed up had a recurrent tumor but no metastases, whereas four (44 %) of nine ordinary MLSs showed local recurrences and two (22 %) of these patients suffered from remote metastases. It cannot be denied that MLSLC may be less aggressive in clinical behavior when compared with ordinary MLS, although no appropriate statistical analysis could be applied in the present study because of the insufficient numbers of the patients. A large-scale study with sufficient numbers of patients is required to clarify the biological behavior of MLSLC. On the other hand, the overall poor prognosis of retroperitoneal WDLS may result from the frequent occurrence of dedifferentiation, in addition to late tumor detection, involvement of vital structures, and inability to achieve complete resection [
1,
2,
25].
In conclusion, our molecular cytogenetic study enabled the distinction between MLSLC and WDLS/DDLS with myxoid changes. The recognition of these peculiar conditions is important, because there are considerable differences of clinical behavior and prognosis between these distinct sarcomas presenting similar morphologic features. In addition, the combined immunohistochemical detection of adipophilin and perilipin may provide a useful ancillary tool for identification of lipoblastic cells in soft tissue sarcomas, since the former is localized in less-differentiated lipoblasts, and the latter is chiefly confined to more mature lipoblasts and fat cells.