Background: Myxofibrosarcoma (MFS) is among the most highly complex sarcoma types. Molecular cytogenetic studies have identified a high level of genomic complexity. Summary: This review provides an update of the current research related to MFS, with particular emphasis on emerging mechanisms of tumorigenesis and their potential therapeutic impact. Many novel possible molecular markers have been identified, not only for prognostication in MFS, but also to serve as possible therapeutic targets, and thereby improve clinical outcomes. However, the molecular pathogenesis of MFS remains incompletely understood. Key Messages: Patients suffering from advanced MFS might benefit from expanded molecular evaluation in order to detect specific expression profiles and identify drug-able targets. Moreover, immunotherapy represents an intriguingly perspective due to the presence of “T-cell inflamed” tumor microenvironment.
Myxofibrosarcoma (MFS) is currently regarded as a distinct fibroblastic/myofibroblastic tumor type defined by cellular pleomorphism, a curvilinear vascular pattern, and a myxoid stromal component .
Genomic instability is a hallmark of all human tumors [2, 3] and can be related to gene mutations, gene copy number alterations, and structural chromosomal abnormalities such as translocations, telomere dysfunction, and whole-chromosome aneuploidy that later results in chromosome instability .
Different studies have shown that MFS is among the most highly complex sarcoma types [4, 5]. Molecular cytogenetic studies have identified a high level of genomic complexity with a recurrent amplification of the chromosome 5p and 7q regions, the biological significance of which is unknown [6, 7].
Clonal heterogeneity may be evident within single samples but can also be observed between different tumor regions within the same primary site (so-called “regional heterogeneity”) or even between primary and metastatic sites . This has recently been highlighted also in MFS by Lohberger et al. .
Many authors attempted to identify novel molecular markers, not only for prognostication in MFS, but also to serve as possible therapeutic targets, and thereby improve clinical outcomes. However, the molecular pathogenesis of MFS remains incompletely understood.
This review provides an update of the current research related to MFS, with particular emphasis on emerging mechanisms of tumorigenesis and their potential therapeutic impact.
Integrin-α10 is overexpressed in primary and metastatic melanoma cells and is associated with melanoma cell migration . In contrast, in other solid tumors, the downregulation of integrin-α10 is associated with loss of phosphorylated retinoblastoma protein (RB) and with disease progression . These results suggest that the role of integrin-α10 in human cancer is context dependent. The role of integrin-α10 in sarcomagenesis remains unknown.
Okada et al.  analyzed the gene expression profiles of 64 primary high-grade MFS and found that expression of ITGA10, which encodes integrin-10, was amplified on chromosome 5p and overexpressed in about 50% of MFS. In addition, 42% MFS possessed coamplification of TRIO and RICTOR and had worse outcomes.
Heitzer et al. , in a series of 25 MFS, observed that while 44% of G3 tumors showed a coamplification of TRIO and RICTOR, the same phenomenon was observed in only one G1 tumor (10%). However, overrepresentation of RICTOR alone was found in 40% of G1 tumors indicating that TRIO amplification is significantly more abundant in G3 tumors and therefore a late event.
Okada et al.  also carried on functional studies in patient-derived MFS cell lines. Knockdown of integrin-10 specifically inhibited growth and decreased RAC and AKT activation in MFS cells but not in normal mesenchymal cells, which suggests that integrin-10 signals through RAC and AKT in a tumor-specific manner. The authors hypothesized that integrin-10 signals through TRIO for the control of RAC and through RICTOR for the control of AKT. Also, cells overexpressing integrin-α10 exhibited greater migration and invasion than the control cells.
Hu et al.  evaluated 61 MFS and undifferentiated pleomorphic sarcoma (UPS). They found that increased ITGA10 and decreased PPP2R2B expression had independent prognostic value. PP2R2B encodes the regulatory B subunit of protein phosphatase 2A. It directly interacts with PDK1 and suppresses its activation. Therefore, both ITGA10 and PPP2R2B might act as upstream regulators of AKT. Somatic mutation was not common in these genes, but DNA methylation showed a profound effect on their expression.
Activation of the AKT/mTORC2 pathway was correlated with histological grade and tumor progression by Takahashi et al.  in a series including 68 primary MFS.
On MFS cell lines, Okada et al.  tested two drugs (NSC27366 and its newly developed derivative EHop-016) which inhibit RAC activation by TRIO [16, 17]. They used selective kinase inhibitor (INK128-MLN0128) to inhibit mTORC2 . Alternatively, the pathway could be targeted with PAK inhibitors, such as IPA-3. Because integrin-α10 exerts its tumor-specific growth signal through both RAC and mTORC2, they hypothesized that inhibitors of RAC and mTORC2 may have cooperative effects in MFS cells.
Integrin-α10 itself is an attractive therapeutic target. Even though no direct inhibitor is currently available, it could plausibly be targeted using chimeric antigen receptor T-cell immunotherapy  or agents that interfere with integrin-α10 ligand binding, such as a ligand-mimetic peptide or a monoclonal antibody against its I domain .
Integrin-mediated signaling is also known to cross-talk with mesenchymal epithelial transition factor (MET) [21, 22].
Mesenchymal Epithelial Transition Factor
MET encodes a transmembrane receptor tyrosine kinase, which constitutes the only known high-affinity receptor of hepatocyte growth factor (HGF). Through combination with HGF, MET could activate the RAS-MAPK or PI3K-AKT signaling pathway to promote cell motility and proliferation . Besides mitogenic and antiapoptotic activities common to many growth factor receptors, enhanced MET activation can stimulate cell-cell detachment, migration, invasiveness, and angiogenesis [23-26].
Lee et al.  first investigated the role of MET in 86 primary localized MFS. Approximately two thirds of MFS displayed MET overexpression at immunohistochemistry (HIC), which correlated with adverse clinicopathological factors (tumor size and mitotic rate) and was independently predictive of shorter survival.
The authors quantified MET transcripts by real-time reverse transcription polymerase chain reaction (RT-PCR) for 16 laser-microdissected tumors and 2 MFS cell lines. Nine (56%) specimens showed apparently upregulated MET transcript, suggesting their frequent upregulation in MFS. Lee et al.  found wild-type MET oncogene in both cell lines. The authors suggested that, as proven in a variety of cancers, MET protein expression might be upregulated by several small noncoding micro-RNAs [27-29].
More recently, Ma et al.  confirmed the relationship between MET and MFS. They used HIC and fluorescence in situ hybridization to detect the MET expression and gene status in 30 MFS. The authors observed MET overexpression in 14 cases (46.7%), with a correlation with FNLCC grade and mitotic rate. They also found a polysomy of chromosome 7 in 11 of these cases, thus suggesting that chromosome 7 polysomy, rather than MET amplification, led to the overexpression of MET protein. Patients with MET overexpression or chromosome 7 polysomy also had a high risk of local recurrence and metastasis.
Ogura et al.  observed also that recurrent mutational targets included genes encoding components of the receptor tyrosine kinase-RAS-PI3K cancer pathway in 31% of the cases.
The findings of the studies [21, 30] strengthen the possible causative function of MET in conferring an aggressive phenotype, implying the potentiality of HGF/MET as an attractive target of therapeutics in MFS [32-34]. Nowadays, there are two approved drugs tested in other cancers, crizotinib  and cabozantinib .
Regarding the NF1 gene, Ogura et al.  found 4 homozygous deletions and 9 somatic mutations in 116 MFS.
These data suggest that a distinctive pattern of NF1 aberrations may play a role in MFS tumorigenesis, similarly to other cancers . Loss-of-function mutations in NF1 gene were also reported by Barretina et al.  in 5 out of 35 MFS. Heitzer et al.  were unable to inform about the NF1 mutation status since they used a hotspot panel for mutation analysis which did not cover NF1. However, loss of NF1 was observed in 2 out of 25 patients.
The use of MEK inhibitors may be a potential therapeutic option in NF1-deficient MFS, as recent studies revealed that tumors harboring NF1 inactivation (inactivating/deleterious NF1 mutations) exhibited activation of the MAPK/ERK pathway and hence are potential targets for MEK inhibitors .
The tyrosine kinase receptor MET and its ligand HGF, splice variants of CD44 and ezrin cooperate .
CD44 are ubiquitously expressed on all cell types, where they act as receptors for hyaluronic acid. They play various roles and are involved in cellular differentiation, cellular migration, and cell-cell contact . Abnormal expression of CD44 may promote cell invasion and was correlated with more aggressive behavior in various cancers, including soft tissue sarcomas (STS) [42-44].
Matuschek et al.  analyzed with PCR 4 variants of CD44 (CD44, CD44s, CD44v6, and CD44v8) in 34 MFS. They found a significant difference in tumor-related survival only for CD44s and CD44v6, with increased CD44s and decreased CD44v6 expression associated with a better prognosis. CD44v6 was shown to be strictly required for MET activation by HGF in rat and human carcinoma cells, in established cell lines as well as in primary keratinocytes . This is consistent with prostate cancer, in which CD44s is a tumor suppressor but certain CD44 variants are oncogenes and promote growth [28, 29, 46].
More recently, Tsuchie et al.  evaluated the HIC expression of CD44s in 44 MFS. The overall expression rate of CD44s in all patients was relatively high. The authors found that high expression of CD44s was associated with local recurrence but did not affect survival.
Ezrin functions as a linker between the plasma membrane and cortical actin cytoskeleton . Increased ezrin levels have been reported to be associated with a high metastatic propensity in different cancers, including rhabdomyosarcoma and osteosarcoma [49-51]. Its underlying mechanistic basis may be ascribed to increased activated ezrin to modulate pleiotropic cellular phenotypes related to cancerous states, such as substrate adhesion, cell survival, cell migration, and formation of cell-cell junctions [50, 51].
Huang et al.  evaluated the expression of ezrin in 78 primary localized MFS with HIC. Ezrin overexpression was present in approximately half of the tumors (49%), and it was significantly associated with important variables related to tumor aggressiveness, including necrosis and FNLCC grading. Additionally, ezrin overexpression was an independent poor prognosticator for survival.
They also measured ezrin mRNA expression levels in 2 MFS cell lines, through real-time RT-PCR and Western blot test [53, 54]. The ezrin mRNA expression level of MFS cell lines was apparently lower than that of normal fibroblasts. However, active ezrin (phosphorylated form at the residue of Thr567) was only detectable in MFS cells, but not in fibroblasts.
α-Methylacyl Coenzyme A Racemase
α-Methylacyl coenzyme A racemase (AMACR) is a peroxisomal and mitochondrial enzyme encoded on chromosome 5p13.3, which acts as a gatekeeper for the β-oxidation of dietary branched-chain fatty acids and bile acid synthesis . It was identified as a protein which drives tumor growth in prostate cancer and other neoplasias, because most malignancies increase the need for fatty acids as an energy source [55-57].
The role of AMACR in MFS has recently been investigated by Li et al.  in a series of 105 primary MFS and in 2 cell lines. AMACR amplification was found in 21% MFS by fluorescence in situ hybridization and was strongly correlated with HIC overexpression. AMACR overexpression was correlated with FNLCC grade and worse survival. However, approximately 40% of AMACR-overexpressing MFS lacked gene amplification. Thus, involvement of alternative regulatory mechanisms was likely in a subset of MFS.
The authors also found that in MFS cell lines and xenografts, AMACR overexpression could increase cyclin D1 expression at the mRNA and protein levels, whereas the mechanisms underlying this regulatory link remain to be elucidated.
Finally, they tested ebselen oxide . Inducing pro-teasome-mediated AMACR degradation and apoptosis, ebselen oxide demonstrates selective cytotoxicity in AMACR-expressing MFS cell lines and dose-dependent inhibition of derived xenografts, suggesting that AMACR is a potential therapeutic target in MFS.
Melanoma-Associated Antigen 3
Melanoma-associated antigen 3 (MAGE-A3) protein was found to be overexpressed in multiple cancers .
MAGE-A3 was overexpressed in 41% of MFS and UPS, significantly higher than in other STS histotypes. High expression was more likely to be seen in recurrences than in primary tumors and was associated with an adverse survival.
Immunotherapies targeting MAGE-A3 have shown both positive and negative results in the treatment of various cancers .
TP53 and Cell Cycle Regulators
Ogura et al.  analyzed a total of 106 MFS. They found frequent alterations in genes related to p53 signaling (51%), along with those associated with the cell cycle checkpoint (43%), including RB1, CDKN2A/CDKN2B, CCND1, and CDK6. Alterations of any of the cell cycle regulators were associated with poorer overall survival. The authors  also identified a novel BRAF fusion gene (SLC37A-BRAF) which could be targeted with anti-BRAF therapies .
Heitzer et al.  evaluated MFS samples from 25 patients. Somatic mutations were identified in only 11 (44%) patients. Grade 3 tumors showed a higher amount of somatic copy number alterations than grade 1. All these 11 patients showed at least one somatic TP53 mutation. TP53 mutations are relatively common in sarcomas with nonspecific genetic aberrations and complex karyotypes compared to sarcomas with reciprocal specific translocations . Moreover, they identified focal amplification/deletion in a variety of known cancer driver genes such as CDKN2A, CCND1, CCNE1, EGFR, EPHA3, EPHB1, FGFR1, JUN, NF1, RB1, or RET, in particular in G3 tumors. In addition, G3 tumor areas of the same tumor showed novel emerged focal amplifications compared to the G1 areas. Many of these, such as BRAF, EGFR, FGFR, KIT, or RET, are indeed actionable targets and are actively being used for precision medicine in different tumor entities.
Ogura et al.  also identified novel recurrently mutated genes such as GNAS (9%), ATRX (9%), KRAS (7%), and JAK1 (4%). The presence of GNAS mutations was significantly associated with local recurrence-free survival .
Amplification of JAK1 could be a therapeutic target in MFS because aberrant activation of the JAK/STAT pathway has been shown to be a promising target in various cancer types [67, 68].
S-Phase Kinase-Associated Protein 2
S-phase kinase-associated protein 2 (SKP2) is a negative regulator of p27kip1 cell-cycle inhibitor. Its overexpression was associated with metastatic propensity in common carcinomas, such as prostatic and esophageal cancers [69, 70].
The role of SKP2 in MFS was evaluated in two consecutive series [71, 72] which included a total of 82 cases. SKP2 gene amplification was detected in 38% of cases. Its amplification strongly correlated with SKP2 overexpression in HIC and with higher FNLCC grades. However, 14% of such tumors and SKP2-expressing cell lines lacked gene amplification. Both SKP2 protein overexpression and gene amplification were highly predictive of worse outcomes.
In addition, pharmacological assays were evaluated both on cell lines and in xenograft models for the therapeutic relevance of bortezomib [73, 74]. The authors showed that bortezomib treatment in SKP2-overexpressing MFS could achieve cytotoxicity in vitro. They also found a remarkably decreased mRNA expression, rather than increased protein degradation, in both MFS cell lines treated. On MFS in vivo studies of MFS xenografts, they observed significant tumor regression with lowered SKP2 labeling, increased p27Kip1 expression, and increased apoptosis .
CD109 and Transforming Growth Factor β
CD109 is a cell surface glycoprotein that is expressed on endothelial cells, activated T lymphocytes, platelets, and a subpopulation of bone marrow CD34 cells. CD109 is a transforming growth factor β (TGF-β) coreceptor that regulates TGF-β receptor endocytosis and degrading, thus inhibiting TGF-β signaling . The roles of TGF-β in remodeling the tumor microenvironment, by suppressing T-cell differentiation and activity, inducing fibrosis and angiogenesis, have been extensively characterized . TGF-β also appears to have a role in chemotherapy resistance, as reported in several tumors including breast cancer and squamous cell carcinoma [78-80].
CD109 has been found to be overexpressed in various cancers, including STS [81, 82]. Emori et al.  evaluated 37 MFS with HIC and found an overexpression of the CD109 protein in 10% of patients, which was significantly associated with decreased survival.
De Vita et al.  analyze 3 patient-derived high-grade MFS cell cultures. HIC analysis showed that tumor cells were strongly positive for CD109. Conversely, CD109 was weakly expressed in normal tissue. Also, higher levels of CD109 were observed with gene expression analysis in all primary MFS cultures with respect to normal tissue.
Over the years, numerous preclinical and clinical studies have sought to leverage these insights and use inhibitors of TGF-β synthesis, receptor binding, or signal transduction in order to inhibit cancer progression .
Programmed death-1 protein (PD-1) is normally expressed on the surface of activated T cells and suppresses unwanted or excessive immune responses, including autoimmune reactions. Its ligand PD-L1 can be expressed by various cells, including macrophages and tumor cells. The PD-1/PD-L1 interaction is a major pathway used by tumors to suppress immune control. Several studies have assessed the expression of PD-L1 in sarcomas .
T-cell infiltration and PD-L1 expression were found to be higher in sarcomas with complex genomics and particularly in UPS than in other STS [87, 88]. Among STS subtypes, UPS/MFS has the highest median macrophage infiltration and the infiltration of immature dendritic cells was positively correlated with survival. Ogura et al.  observed that the average fraction of infiltrated CD8+ T cells was significantly higher in the better prognostic cluster in MFS. Hu et al.  reported that a high level of CD4+ T-cell infiltration was associated with a significantly better relapse-free survival in UPS and MFS. They also found a moderate negative correlation between the 2-gene signature (increased ITGA10 and decreased PPP2R2B) and CD4+ T-cell infiltration. Conley et al.  also found that HLA-A and HLA-DPB1 mRNA expression was significantly higher in UPS/MFS compared to other STS subtypes. Also, the lymphocyte infiltrate in UPS/MFS was significantly higher compared to other histotypes. These findings suggest that UPS and MFS may have immunologically mutated protein targets and thus might respond to immune checkpoint therapy .
The results of successful immunotherapy trials using an anti-PD-1 antibody and combination anti-PD-1/anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) showed that a significant number of UPS experienced a reduction in tumor size. A phase II study of single-agent pembrolizumab (anti-PD-1 antibody) in multiple sarcoma types showed an objective response rate of 40% in UPS [90, 91]. A separate trial involving nivolumab alone and in combination with ipilimumab demonstrated responses in UPS .
Studies in melanoma, non-small-cell lung cancer, and colorectal cancer have identified the presence of a “T-cell inflamed” tumor microenvironment, high tumor mutation burden, microsatellite instability, and PD-L1 expression as biomarkers of response to PD-1/PD-L1 blockade in some patients . A pair of recent papers [94, 95] identified TGF-β signaling in the tumor microenvironment as a determinant of tumor T-cell exclusion and poor response to PD-1/PD-L1 blockade.
In murine models, Mariathasan et al.  demonstrated that combined treatment with antibodies targeting PD-L1 and TGF-β induces CD8+ T-cell infiltration and tumor regression, with complete response rates of 70%, while monotherapy with either agent alone is ineffective. Beyond TGF-β, therapies targeting other oncogenic signaling pathways, including MAPK and AKT-PI3K-mTOR, have also shown promising results in combination with immune checkpoint blockade, at least partly due to their effect on the tumor microenvironment .
Although the outcomes for MFS have not changed, substantial advances in the understanding of the natural history and pathogenesis of this sarcoma have been made. Many possible drug-able targets have been identified. However, MFS harbor a very heterogeneous karyotype with very different clones observed in each patient, but also in distinctive areas of the same tumor. Thus, patients suffering from advanced MFS might benefit from expanded molecular evaluation in order to detect specific expression profiles and identify drug-able targets.
Moreover, promising results have been found with immunotherapy. Future research that combines treatment modalities, including immunotherapy, are warranted.
Statement of Ethics
No ethic approval was needed for reviews.
All the authors have no conflict of interests to declare.
Conceived the paper: A.S., D.M.D., M.D.P. Collected the data: G.B., P.S., M.D.P. Wrote the paper: A.S., G.B., M.D.P., D.M.D. Manuscript final editing: A.S., P.S., M.D.P.