Diagnostic and prognostic gene expression signatures in 177 soft tissue sarcomas: hypoxia-induced transcription profile signifies metastatic potential

Soft tissue sarcomas (STS) account for ~1% of all malignancies and represent a heterogeneous group of mesenchymal tumors, the clinical management of which is challenging and requires multidisciplinary efforts that take the combined information from clinical investigations, imaging, histopathology, and cytogenetic and molecular genetic analyses into account. STS comprise more than 30 histological subtypes, although malignant fibrous histiocytoma (MFH)/undifferentiated pleomorphic sarcoma (UPS), liposarcoma, leiomyosarcoma, synovial sarcoma (SS), and malignant peripheral nerve sheath tumor (MPNST) account for three-fourth of the tumors [1]. Some STS subtypes are characterized by specific chromosomal translocations causing novel gene fusions e.g. SS18-SSX in SS and TLS-CHOP in myxoid/round-cell liposarcoma. However, a large group of STS, e.g. MFH/UPS, leiomyosarcoma, and dedifferentiated/pleomorphic liposarcoma, lack known specific recurrent alterations and are characterized by a multitude of rearrangements, amplifications, deletions, and somatic alterations, including mutations in TP53, deletions of RB1 and CDKN2A, and amplifications of MDM2 and CDK4 [2, 3].

Gene expression studies have revealed diagnostic profiles and upregulation of specific pathways in sarcomas with type-specific genetic defects, e.g. SS, dermatofibrosarcoma protuberans, clear-cell sarcoma, Ewing sarcoma, rhabdomyosarcoma and gastrointestinal stromal tumors (GIST) [4–14] and have provided potential targets for novel therapies [15–18]. From a diagnostic point of view, the above mentioned tumor types can be identified from their underlying gene fusions, but the expression data reveal novel genes and pathways including multiple downstream targets of the resultant chimeric transcription factors, which provide a basis for the understanding of key biological changes in STS development. In contrast, molecular classification of the predominant, genetically complex STS subgroups, e.g. MFH/UPS, leiomyosarcoma, and dedifferentiated/pleomorphic liposarcoma, has been difficult with extensive pleomorphism that has precluded identification of recurrent profiles [9, 10, 12, 14, 19, 20]. STS are highly malignant and metastases develop unpredictably in one-third of the cases, therefore, new prognostic markers would be of great clinical value. However, there have been only two reports of expression profiles associated with poor outcome in STS, both in leiomyosarcomas [21, 22]. With the aim to establish diagnostic expression profiles for STS and to assess whether gene expression profiling can provide prognostic information, we used 27 k cDNA microarrays to characterize the expression patterns in a mixed series of 177 STS, with particular focus on high-grade pleomorphic tumors.

From a clinical diagnostic perspective, most STS with specific fusion proteins can be correctly classified based on the combined data from histopathology, immunostainings, and cytogenetic and/or molecular genetic analyses. This stands in contrast to the genetically complex STS subtypes, the diagnosis of which is hampered by suboptimal reproducibility due to extensive histopathological heterogeneity, pleomorphism and lack of type-specific genetic defects. Distinct and homogenous expression profiles have been reported in SS, dermatofibrosarcoma protuberans, clear-cell sarcoma, myxoid/round-cell liposarcoma and GIST [4–13]. Furthermore, the discriminatory profiles that often contain genes located downstream of the type-specific pathogenic gene fusions or mutations have provided novel targets for molecular therapy [15–18]. Gene expression profiles have provided clues to the histogenesis of some STS subtypes, suggested similarities between others and allowed distinction between pathologically inseparable lesions, e.g. neural crest origin of SS, close relation between clear-cell sarcoma and melanoma, and distinct expression profiles for well-differentiated and dedifferentiated liposarcomas [9, 11, 27]. Current data from the more pleomorphic STS, however, indicate that their expression profiles are indeed heterogeneous and in these tumor types the primary goal may rather be to identify novel, yet unidentified subgroups, and to clarify deregulated genes and pathways.

Unsupervised cluster analysis of the 177 STS identified two major subclusters; S containing STS with specific fusion genes or mutations, e.g. SS, myxoid/round-cell liposarcoma and GIST, and subcluster C with genetically complex, often pleomorphic STS subtypes, e.g. MFH/UPS, leiomyosarcoma and dedifferentiated/pleomorphic liposarcoma (Figure 1). This is in line with results from previous gene expression and proteomic profiling studies [10, 19, 28].

Discrimination scoring identified genes and pathways differentially regulated in the STS subtypes, with considerable amount of overlap with previously published gene lists despite the differences in tumor material and array platforms [4, 5, 7, 9, 10, 12, 19, 20]. Expression profiles in the distinct subtypes were strong with low FDR whereas the pleomorphic ones had relatively heterogeneous profiles with higher FDR. Identification of diagnostic markers that clearly distinguish different subtypes requires gene lists with very low FDR, but inherent heterogeneity, lack of clear-cut boundaries between subtypes, misdiagnoses and common pathogenic genes and pathways make it rather difficult to generate such strong diagnosis-specific expression profiles in the pleomorphic STS. Some studies chose to exclude samples that did not cluster according to histopathological diagnosis thereby reducing FDR by increasing homogeneity within the subgroups [7, 10]. In the current study, outliers were included but lenient cut-offs were employed allowing some amount of false positives in the gene lists, which were then analyzed using EASE to identify functionally correlated genes. The risk of signifying false positives was reduced by focusing on upregulated pathways and functional groups rather than on individual genes. This approach helped identify biologically relevant genes in dedifferentiated/pleomorphic liposarcoma and fibrosarcoma despite high FDR and upregulation of similar functional groups in SS, GIST, myxoid/round-cell liposarcoma, MPNST and fibrosarcoma.

Although alternative SSX fusions and various lines of differentiation can be demonstrated in SS [29, 30], they display homogenous and distinct expression profiles that enable clear distinction from other STS subtypes [4, 9, 10, 12, 19, 20] and our finding of 4000 differentially expressed genes is in line with these results. In the unsupervised clustering, all but one SS were part of subcluster S, within which 5 formed a tight subcluster, together with 3 MPNST samples on a separate branch, away from the main SS subcluster (Figure 1). Supervised clustering based on the 4000 genes also showed close clustering of SS to MPNST [see Additional file 5]. Previous studies have shown that SS and MPNST share similar patterns of gene expression with upregulation of neuroectodermal differentiation genes thereby suggesting a neural crest origin [4, 7, 9] and several neural differentiation genes were also upregulated in the present study. The SS18-SSX fusion product controls gene expression by association with chromatin remodeling complexes through interactions with SWI/SNF complexes and histones [31–33]. Interestingly, chromatin-remodeling genes including several histones and SMARC genes constituted one of the largest functional groups upregulated in the SS. Moreover, the genes identified herein verified the significance of developmental pathways including the FGF, EGF, TGF-β, Wnt, Notch, retinoic acid and Hh receptor signaling pathways, the targeted inhibition of which may provide novel therapeutic options.

MPNST may morphologically mimic SS [34] and in the unsupervised analysis, 5/8 MPNST clustered within subcluster S, and 3 with a subset of SS. Several developmental, neural differentiation and ribosomal protein genes were found to be overexpressed in both MPNST and SS. Fibrosarcomas, like the MPNST, have been shown to cluster with the SS [7, 10]. The fibrosarcomas in this study were part of subcluster S and a weak expression signature including many developmental genes distinguished these tumors from the remaining STS.

GIST constitute a distinct STS subtype, ~80% of which carry KIT gene activating mutations [35]. These tumors demonstrate distinct expression profiles [5], with upregulation of genes within the KIT signaling pathway. In addition, several genes involved in neurogenesis and neural differentiation were overexpressed, as were muscle-specific genes like smoothelin and myosin, which may reflect the suggested origin of GIST from the interstitial pacemaker cells of Cajal [36].

Liposarcomas account for about 20% of STS and within this subtype, the myxoid/round-cell liposarcomas characterized by the TLS-CHOP or EWS-CHOP fusions [37] formed a distinct cluster closely related to a subset of SS and MPNST within subcluster S with upregulation of several developmental and ribosomal protein genes (Figure 1). One of the top most upregulated genes was the lipid metabolism gene PPARG that regulates adipocyte differentiation and constitutes a potential therapeutic target [38, 39]. The dedifferentiated/pleomorphic liposarcomas contain complex genetic alterations and were scattered among the pleomorphic samples in the unsupervised clustering. Most dedifferentiated liposarcomas are characterized by amplifications of MDM2 and CDK4 as part of the 12q amplicons involved in the formation of ring-chromosomes [40, 41]. A relatively weak discriminatory signature mainly characterized by overexpression of the 12q amplicon genes distinguished a small subset of the dedifferentiated/pleomorphic liposarcomas [see Additional file 17]. PPARA and other lipid metabolism genes were also overexpressed. Comparison between myxoid/round-cell and pleomorphic/dedifferentiated liposarcomas revealed overexpression of ribosomal proteins and developmental genes related to Wnt signaling and neurogenesis in the former group, whereas genes related to cell-cycle, homeostasis, and a greater number of lipid metabolism genes were upregulated in the latter. Skubitz et al. demonstrated separation of myxoid liposarcoma from non-myxoid liposarcoma using a set of ribosomal genes [42].

Our series included a large proportion of pleomorphic, undifferentiated STS with the hope of gaining novel insights into the origin of these genetically complex tumors and establishing a more refined classification. About one-third of the leiomyosarcomas clustered tightly within subcluster C and a 200-gene signature with overexpression of genes related to muscle structure and function was identified in these tumors (Figure 1). This is in line with previous results from gene expression and proteomic profiling demonstrating that while the more pleomorphic leiomyosarcomas cluster with the MFH, another more distinct subset cluster separately [19, 28]. MFH was introduced as a separate diagnostic entity in the 1960s, but constitutes a heterogeneous group of poorly differentiated tumors with poor diagnostic reproducibility [1]. In our series, one-third of the tumors represented MFH/UPS, all but 5 of which fell within subcluster C (Figure 1). A 500-gene signature grouped together about half of the MFH/UPS [see Additional file 15] including several genes from the extracellular matrix and inflammatory response, reflecting the fibrous and histiocytic features of these tumors, as well as others involved in the regulation of cell-cycle, proliferation, adhesion, motility and protein degradation.

Novel prognostic and therapeutic markers would be of great clinical value in STS since risk stratification is currently difficult and adjuvant treatments are toxic and hampered by low efficacy. We identified a 244-gene prognostic signature in 89 primary, high-grade STS mainly representing MFH/UPS and leiomyosarcoma. This signature was characterized by upregulation of several hypoxia-related genes (e.g. HIF1A and its targets) and genes involved in cell proliferation, adhesion and motility in metastasizing STS. Our data are the first to suggest a prognostic profile modulated at least in part by hypoxia in a large series of highly malignant STS of mixed types. The cross-validated classifier predicted metastasis with an accuracy of 64% and provided prognostic information independent of currently used prognostic factors from the SIN-system, namely tumor size, vascular invasion, and necrosis [43]. Gene expression profiles that correlate with poor outcome have previously been recognized in Ewing sarcoma and leiomyosarcoma [21, 22, 44], Ren et al. identified a 92-gene signature in 11 leiomyosarcomas that separated high-grade metastatic tumors from low-grade well-differentiated ones, whereas Lee et al. took a different approach and predicted metastasis in a set of 30 primary leiomyosarcomas and local recurrences using the expression profile of 335 genes that initially distinguished primary leiomyosarcomas from metastases [21, 22]. However, none of the reported genes were among our 244 discriminators, which may not only be because they were established in a set of leiomyosarcomas using different approaches whereas our signature was established in a larger mixed series of pleomorphic primary STS, but also due to the difficulties in identifying prognostic signals which are considerably weaker than the diagnostic ones.

An adverse prognostic impact of hypoxia has been demonstrated in several malignancies and tumor oxygenation studies in STS have suggested an association of hypoxia with tumor grade, presence of mitoses and metastatic development [45–48]. A recent study demonstrated that HIF1A expression was an independent prognostic factor in STS [49]. Hypoxic tumors display high rates of glucose uptake and glycolysis regulated by HIF1 that induces expression of glucose transporters like GLUT1 and glycolytic enzymes like ENO1, HK2, LDHB and PGK1[50], which were overexpressed in the metastasizing tumors. CA9 expression, an intrinsic cellular marker for hypoxia, has been suggested to correlate with poor survival in high-grade STS [51], and GLUT1 expression and enhanced glucose metabolism in STS have been linked to proliferative activity and tumor grade [52–55]. Detwiller et al. demonstrated that the expression pattern of a selection of 107 hypoxia-related genes allowed distinction of mixed STS samples from normal tissue, and several of the overexpressed genes therein were among our discriminators [56].

In summary, diagnostic gene expression profiles were identified for different subtypes with distinct profiles in STS with specific fusion genes or mutations, whereas the diagnostically difficult pleomorphic STS were challenging also with regards to expression profiling. The SS, GIST, myxoid/round-cell liposarcomas, MPNST and fibrosarcomas shared similarities in expression profiles with overrepresentation of developmental genes involved in differentiation and morphogenesis. On the other hand, genes involved in cell-cycle, proliferation, adhesion, motility, protein degradation, homeostasis and immune-response seemed to play an important role in the pleomorphic subtypes. The novel genes and pathways identified provide important information about tumor origin and constitute potential therapeutic targets. Our identification of a prognostic profile in the latter group is highly promising, and provides information independent of the currently used prognosticators. Moreover, it is intriguing that upregulation of hypoxia-related genes predicts metastatic potential in high-grade, pleomorphic and genetically complex STS and calls for further evaluation of HIF1A and its target genes in STS.