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Liposarcoma is the most common sarcoma in adults. Well-differentiated and dedifferentiated liposarcoma account for 40–45% of all liposarcomas and fall within a biological continuum in which dedifferentiated liposarcoma represents an advanced form of the tumor that has acquired metastatic potential.1 Clinically, dedifferentiated liposarcoma is an aggressive malignancy that is frequently lethal due to multiple recurrences and late metastases, with no effective therapeutic options other than surgery.1, 2, 3 Morphologically, dedifferentiated liposarcoma is usually a non-lipogenic high-grade sarcoma with a wide range of histological appearances, most commonly with spindle cell and pleomorphic cytomorphology, and infrequently inflammatory, round cell, or meningothelial-like patterns, none of which seem to correlate with the clinical outcome.1, 2

Cytogenetic and molecular genetic studies over the last two decades have provided insights into the biology of dedifferentiated liposarcoma. The tumor cells of dedifferentiated liposarcoma contain neochromosomes —ring or giant marker chromosomes— composed of repeated interspersed amplicons from distinct non-contiguous chromosomal regions.4, 5 The structure of dedifferentiated liposarcoma neochromosomes is complex and varies between tumors, between time points in the course of disease, and even between individual cells within a single tumor. Despite such genetic heterogeneity, some general features are observed in dedifferentiated liposarcoma neochromosomes: genetic material from the 12q13-15 chromosomal region is invariably present, usually as small fragments of variable lengths that are amplified at very high levels. Other chromosomes are involved less consistently, such as 1q, 4p, 12q21–22, 13q, and 15q.5, 6, 7, 8 The 12q15 amplicon consistently contains the MDM2 oncogene, which is considered an essential oncogenic driver in dedifferentiated liposarcoma and the main downregulator of the p53 axis in this tumor type.1, 9 Concomitant amplification of HMGA2, CPM, FRS2, and YEATS4, all located at the same chromosomal region, is invariably present along with MDM2, even though the pathogenetic relevance of these genes in dedifferentiated liposarcoma is not well established.5, 10, 11, 12 In addition, the cyclin-dependent kinase CDK4 is amplified in about 90% of dedifferentiated liposarcoma. Although CDK4 is located very close to the main 12q15 amplicon, it belongs to a discontinuous and inconsistent amplicon that includes its locus at 12q14.1 as well as other genes such as TSPAN31.6, 10 CDK4 gene amplification and overexpression very likely have an important role in the tumorigenic process in dedifferentiated liposarcoma, but the contribution of other genes in this amplicon has not been explored.13, 14 In addition to providing greater understanding of the pathogenesis of dedifferentiated liposarcoma, the concurrent amplification and overexpression of MDM2 and CDK4 serve as important diagnostic markers that can be detected by immunohistochemistry or FISH.15 MDM2 and CDK4 also represent attractive potential therapeutic targets—both MDM2 inhibition and CDK4 inhibition have shown promising activity in preclinical models and are being evaluated in clinical trials.3, 16

STAT6 is a member of the STAT family of cytoplasmic transcription factors, which regulate gene expression by transmitting signals to the nucleus and binding to specific DNA promoter sequences in response to extracellular cytokines. Activation of STAT family members has been implicated in physiological and neoplastic cell growth, and can be therapeutically inhibited.17 In mesenchymal neoplasia, recent studies have detected a recurrent intrachromosomal rearrangement in solitary fibrous tumor that results in the formation of a NAB2–STAT6 fusion oncogene.18, 19 The fusion oncoprotein is highly expressed and localizes to the cell nucleus, resulting in a strong nuclear expression of STAT6 that can be detected by immunohistochemistry and serves as a reliable diagnostic marker for solitary fibrous tumor.20, 21 In addition, we have previously reported that a small number of dedifferentiated liposarcomas show STAT6 expression.20 STAT6 is located at chromosomal region 12q13, which contains essential oncogenes amplified in dedifferentiated liposarcoma neochromosomes. The aim of this study was to determine the frequency of STAT6 expression in dedifferentiated liposarcoma and the underlying genetic mechanism, in relationship to the known amplification of 12q13-15 in this sarcoma type.

Materials and methods

The study was approved by the Institutional Review Board at Brigham and Women’s Hospital. Thirty-five previously unpublished cases of dedifferentiated liposarcoma were retrieved from the surgical pathology files of Brigham and Women’s Hospital, Boston, MA, USA. Clinical, histopathological, and immunophenotypical features were reviewed by two surgical pathologists (LAD and AME). All cases showed nuclear expression of MDM2 and CDK4 by immunohistochemistry, and/or cytogenetic features of dedifferentiated liposarcoma—either ring or giant marker chromosomes in the karyotype, MDM2 amplification by FISH, or both. Two solitary fibrous tumors with STAT6 overexpression were selected as controls.

Immunohistochemistry for STAT6 was performed on 4 μm-thick formalin-fixed paraffin-embedded whole-tissue sections following pressure cooker antigen retrieval (0.01 M citrate buffer; pH, 6.0), using a rabbit polyclonal antibody directed against the C-terminus of STAT6 (1:1000; sc-621; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Appropriate positive and negative controls were used throughout. The extent of immunoreactivity was graded according to the percentage of positive tumor cells (0, no staining; 1+, 1–50%; 2+, 51–100%). The intensity of staining was graded as weak, moderate, or strong.

Dual color FISH was performed on 4 μm thick formalin-fixed paraffin-embedded tissue sections for all cases with STAT6 expression, a subset of four dedifferentiated liposarcoma cases without STAT6 expression, and two solitary fibrous tumor controls. Tissue sections were baked overnight at 56 °C, deparaffinized by three consecutive 15 min immersions in xylene, and dehydrated twice in 100% ethanol for 2 min. The slides were then immersed in TRIS–EDTA (100 mM Tris base, 50 mM EDTA, pH 7.0) for 1 h at 95–99 °C and rinsed in PBS for 5 min. Proteolytic digestion of the sections was performed using Digest–All 3 Pepsin solution (Invitrogen, Camarillo, CA, USA) at 37 °C for 10–20 min, twice. The sections were then sequentially dehydrated in alcohol (70, 85, 95, and 100% for 2 min each) and air-dried. The STAT6 probe set consisted of a commercial kit targeting the 12q13 region, which includes a SpectrumOrange probe spanning the STAT6 locus and a telomeric SpectrumGreen probe that spans the CDK4 locus (Vysis, Abbott Molecular Inc, Des Plaines, IL, USA) (Figure 1). One-hundred interphase nuclei were scored per case. The presence of five or more orange signals was considered indicative of STAT6 amplification. Non-neoplastic diploid cells were identified in each case and used as internal controls. The significance of the correlation between immunohistochemistry and FISH was analyzed using a 2 × 2 contingency table, and the exact two-tailed P-value was calculated according to Fisher’s test.

Figure 1
figure 1

Schematic representation of the STAT6 FISH probe set.

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Results

Four of the 35 dedifferentiated liposarcomas (11%) showed STAT6 expression by immunohistochemistry; the clinicopathologic features of these cases are summarized in Table 1. Morphologically, two tumors were high-grade spindle cell and pleomorphic sarcomas (one with focal heterologous smooth muscle differentiation), one was composed of a relatively monotonous population of atypical spindle cells with prominent nuclear palisading, and one was a high-grade spindle-cell sarcoma with hypocellular hyalinized areas characteristic of well-differentiated sclerosing liposarcoma. Three cases showed multifocal (2+) staining of moderate to strong intensity and one (case 4) showed weak focal (1+) staining (Figure 2). STAT6 expression was both cytoplasmic and nuclear in three cases, generally with a stronger nuclear intensity; in case one, although some areas of the tumor predominantly showed nuclear staining, cytoplasmic staining was also present multifocally. STAT6 expression was detected in the dedifferentiated components in all four tumors, and in an area of well-differentiated sclerosing liposarcoma in case four. Interestingly, the tumor with heterologous smooth muscle differentiation (case 2) showed expression of STAT6 only in conventional areas of dedifferentiated liposarcoma; the smooth muscle-like component was negative for STAT6 by immunohistochemistry. Two solitary fibrous tumor control cases showed diffuse strong nuclear STAT6 expression.

Table 1 Clinicopathological features of four cases of dedifferentiated liposarcoma with STAT6 amplification and STAT6 protein expression
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Figure 2
figure 2

High-grade dedifferentiated liposarcoma with pleomorphic and spindle-cell morphology (case 1) (a) showing diffuse expression of STAT6 (b). Dedifferentiated liposarcoma with predominantly spindle cell morphology and prominent nuclear palisading (case 3) (c). The tumor cells showed diffuse nuclear expression and weak cytoplasmic expression of STAT6 (d). Hypocellular hyalinized areas of well-differentiated sclerosing liposarcoma (case 4) (e) showed weak focal expression of STAT6 (f).

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FISH demonstrated STAT6 amplification in all four cases expressing STAT6. The level of amplification was variable between cases, ranging from high-level amplification with innumerable copies of STAT6 per cell (Figure 3a), to 6–10 STAT6 copies present in one case. Within each case, there were also different levels of amplification in different cells, with large atypical cells showing more gene copies than less atypical cells. The smooth muscle-like component present in case two, identified by elongated nuclei arranged in fascicles on the DAPI stain, showed high-level amplification. FISH performed on four dedifferentiated liposarcomas negative for STAT6 expression demonstrated no STAT6 amplification (P=0.0286). All dedifferentiated liposarcomas showed high-level amplification of CDK4 (Figure 3b). CDK4 amplification was consistently higher than STAT6 amplification. Both STAT6-positive solitary fibrous tumors showed just two copies of STAT6 and CDK4 per interphase nucleus.

Figure 3
figure 3

STAT6 amplification detected in dedifferentiated liposarcoma interphase nuclei by FISH. A non-neoplastic cell is shown in each field as an internal control. (a) High-level amplification of STAT6 demonstrated by the presence of multiple red signals, isolated, or overlapping with green signals. CDK4 is highly amplified, as represented by innumerable green signals (case 1). (b) No amplification of STAT6 despite high-level amplification of CDK4 in a dedifferentiated liposarcoma control case, negative for STAT6 by immunohistochemistry.

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Of the four STAT6 amplified dedifferentiated liposarcomas, three patients were males and one was female, ranging in age from 51 to 76 years (Table 1). Tumors were located in the mediastinum (n=2), paratesticular soft tissue (n=1), and perirenal soft tissue (n=1). Three patients received pre-operative chemotherapy +/− radiation therapy before resection.

Discussion

The significance of STAT6 amplification and overexpression in a subset of dedifferentiated liposarcoma must be considered from both a diagnostic and a biological perspective, with potential therapeutic implications. Diagnostically, the specificity of STAT6 immunodetection for the diagnosis of solitary fibrous tumor in the appropriate morphological context is very high.20, 21 Dedifferentiated liposarcoma, however, can resemble solitary fibrous tumor morphologically, and STAT6 expression in a subset of dedifferentiated liposarcoma could complicate this differential diagnosis. The degree of STAT6 expression in solitary fibrous tumor is generally more diffuse and of a stronger intensity than in dedifferentiated liposarcoma, although the range of expression is variable, limiting the value of this observation for the evaluation of individual cases. The predominantly nuclear aberrant localization of STAT6 in solitary fibrous tumor, in contrast to the cytoplasmic and nuclear expression seen in dedifferentiated liposarcoma, may be a helpful discriminatory feature. Nevertheless, expression of STAT6 in dedifferentiated liposarcoma is a potential diagnostic pitfall that must be taken into consideration when evaluating a STAT6-positive spindle cell neoplasm, particularly in small needle biopsies of retroperitoneal or deep lesions. The presence of areas with varied morphologies, including areas of well-differentiated liposarcoma, and the demonstration of MDM2 and CDK4 expression by immunohistochemistry, or amplification by FISH, should allow for a diagnosis of dedifferentiated liposarcoma in the vast majority of cases.11, 15 However, the mere presence of fat formation within the tumor should not prompt a diagnosis of dedifferentiated liposarcoma, given the existence of a well-recognized subgroup of fat-forming solitary fibrous tumor, both benign and malignant.22, 23

Mechanistically, amplification of STAT6 in dedifferentiated liposarcoma is in keeping with the chromosomal location of this gene at 12q13 and the genetic makeup of dedifferentiated liposarcoma. Consistent MDM2 amplification and frequent CDK4 coamplification highlight the relevance of 12q13-15 amplicons in dedifferentiated liposarcoma,4, 10, 24 but the remarkable heterogeneity of these and other amplicons in dedifferentiated liposarcoma is still poorly understood. Ring and giant marker chromosomes in dedifferentiated liposarcoma are topologically complex structures and are thought to result from multiple rounds of sequential breakage, fusion and bridging of donor chromosomes.25 The resulting neochromosomes show a notable degree of plasticity, as demonstrated by the genetic variability between cases, and within each case.4 Some structural considerations have been proposed to explain the composition of certain dedifferentiated liposarcoma amplicons; for example, a fragile site located immediately centromeric to the 5′ end of DDIT3 at 12q14.1 has been proposed to define the centromeric boundary of the CDK4 amplicon, so that chromosomal breakage at this site would have a role in the formation of the non-contiguous amplicons from 12q13-15.6 Nevertheless, several genes centromeric to this breakpoint are coamplified in around 15% of dedifferentiated liposarcoma (including GLI1, DCTN2,6, 14 and as demonstrated in this report, STAT6), indicating that some cases of dedifferentiated liposarcoma contain either larger amplicons extending over this site, or additional discontinuous amplicons from 12q13.

The relevance of STAT6 amplification in the pathogenesis of dedifferentiated liposarcoma remains to be defined. The relatively low frequency of STAT6 amplification in dedifferentiated liposarcoma indicates that it is most likely a passenger genomic event. Nevertheless, STAT6 may contribute substantially to the biology of dedifferentiated liposarcoma. Evidence in support of this interpretation originates from the only study that systematically analyzed the functional relevance of amplified genes in dedifferentiated liposarcoma: shRNA-mediated STAT6 knockdown in three dedifferentiated liposarcoma cell lines significantly reduced cell proliferation, with lethal effects comparable to or greater than those resulting from CDK4 knockdown.14 Despite recent advances in the characterization of dedifferentiated liposarcoma neochromosomes, the functional contribution of each of the genes amplified within dedifferentiated liposarcoma amplicons is still unclear. Although MDM2 is the best studied target of the 12q15 amplicon,11, 16 the consistent presence within the same amplicon of YEATS4 and FRS2 amplification, combined with HMGA2 rearrangement, have also been demonstrated to be biologically essential.6, 12 Abundant experimental evidence also supports the functional relevance of CDK4 in dedifferentiated liposarcoma.13, 14 Less common genomic events have been variably associated with particular biological features, for example, amplification of JUN and dedifferentiation.8 The presence of STAT6 amplification demonstrated in this report, combined with the functional evidence of STAT6 dependency in dedifferentiated liposarcoma,14 highlights the biological and potentially therapeutic relevance of STAT6 in at least a subset of dedifferentiated liposarcoma and provides a basis to explore STAT6-targeting agents17 as a rational therapeutic strategy in dedifferentiated liposarcoma.

In summary, STAT6 amplification occurs in a subset of dedifferentiated liposarcoma, which results in expression of STAT6 that can be detected by immunohistochemistry and may be a potential pitfall in the differential diagnosis of dedifferentiated liposarcoma and solitary fibrous tumor. STAT6 amplification in dedifferentiated liposarcoma further highlights the genomic complexity and heterogeneity of ring and giant marker chromosomes of this tumor type, particularly concerning amplicons originating from the chromosomal region 12q13-15. STAT6 transcriptional activity may be biologically relevant in some cases of dedifferentiated liposarcoma and may provide a therapeutic opportunity.