Research Article Creative Commons, CC-BY
Characterizing the Molecular Landscape of Fibrosarcoma: An Institutional Experience
*Corresponding author: Rana Naous, Department of Pathology, University of Pittsburgh Medical Center, Pittsburgh, PA, USA.
Received: August 18, 2023; Published: August 24, 2023
Introduction: Fibrosarcoma is a very rare soft tissue sarcoma only diagnosed after excluding other mimicking entities with use of ancillary immunohistochemical and molecular testing.
Objective: Given their rarity, studies addressing the genetic landscape of fibrosarcoma are scarce. We analyzed our institutional fibrosarcoma cases in an attempt to further characterize the molecular landscape of fibrosarcoma and find potential therapeutic targets.
Methods: Targeted next-generation sequencing DNA analysis for characteristic DNA mutations and copy number variation was performed on 8 fibrosarcoma cases involving bone/soft tissue diagnosed at our institution between the years 2000 and present.
Results: Three out of our 8 cohort cases failed testing due to low DNA count. Among the remaining five cases, the most common genetic alteration was CDKN2A/CDKN2B copy number loss (4 cases, 80%), followed by TP53 mutations (3 cases, 60%). NF1 mutation or copy number loss, NOTCH mutations and BAP1 copy number loss had an equal frequency (2 cases each, 40%), and each of SMAD4, TERT, FANCA, FAND2 and PTCH1 alterations occurred at a frequency of 20%. NOTCH, FANCA, FANCD2 and PTCH1 mutations were reported as variants of uncertain significance (VUS).
Conclusion: Our study demonstrated that, similar to most sarcomas, the most common genetic aberrations in fibrosarcoma are CDKN2A/CDKN2B and TP53 alterations. Additional novel alterations detected in our study with potential tumorigenesis role included BAP1 and NF1 copy number loss, TERT mutation, and SMAD4 mutation. Additional large-scale studies are needed to further expand on the molecular landscape and biology of fibrosarcoma in search for potential implications on therapy.
Keywords: Fibrosarcoma, Molecular, Landscape
Sarcomas are extremely rare tumors with less than 15,000 new cases diagnosed each year in the United States . When compared to other tumor types, the predominant genomic aberrations in soft tissue and bone sarcomas are DNA copy number and chromosomal translocations; nonetheless, the number of targetable mutations remains considerably low [2,3]. Fibrosarcoma is a very rare soft tissue sarcoma that is only diagnosed after excluding other mimicking entities with the use of ancillary immunohistochemical and molecular testing . Very few cytogenetic analyses have been reported on fibrosarcoma revealing multiple non-specific complex chromosomal anomalies, including balanced and unbalanced chromosomal translocations and copy number anomalies with no common or characteristic chromosomal changes to date [5- 7]. Given their rarity, studies addressing the genetic landscape of fibrosarcoma are scarce. In our prior 2022 study  we demonstrated a gene fusion (FNDC3B-PIK3CA) of uncertain significance in 1 (10%) of 10 fibrosarcomas diagnosed at our institution in the past 20 years using next-generation sequencing (NGS) RNA fusion analysis while searching for novel gene fusion events, including NTRK-related fusions as described by Yamazaki, et al.,  who had reported the presence of novel neurotrophic receptor tyrosine kinase 3 (NTRK3) fusion in 2 of their fibrosarcoma cases. In the current study the author uses NGS DNA analysis on the corresponding institutional case cohort in search for characteristic DNA mutations and copy number variation in an attempt to further characterize the molecular landscape of fibrosarcoma and find potential therapeutic targets.
Materials and Methods
This study received Institutional Review Board exemption (MOD20080228-002). A detailed description of the case cohort used in this study has been provided in a prior publication . The case cohort included eight pure fibrosarcoma cases involving bone or soft tissue diagnosed between the years 2000 and present with available formalin-fixed paraffin-embedded tissue for molecular testing. All cases demonstrated strict “fibrosarcoma” morphologic criteria as described by the WHO; a monomorphic fascicular spindle cell proliferation with no more than moderate degree of atypia and a non-specific immunophenotype . Tumor grade was either low-grade (6 cases) or intermediate grade (2 cases). Ancillary immunohistochemical and molecular workup was performed to rule out morphologic mimickers. All cases were negative for cytokeratin, S100, desmin, EMA, CD34, and SMA (rare focal staining in 1 case). FISH for SYT gene rearrangement was negative in all cases. The above immunophenotype, along with the absence of SYT gene rearrangement, essentially excluded all morphologic mimics of fibrosarcoma including malignant peripheral nerve sheath tumor, solitary fibrous tumor, synovial sarcoma, biphenotypic sinonasal sarcoma, leiomyosarcoma, Kaposi sarcoma, and sarcomatoid carcinoma.
Targeted next-generation sequencing (NGS) DNA analysis was performed in accordance with standard clinical practice. Briefly DNA was extracted from manually-microdissected surgical FFPE tissue using the DNeasy Blood and Tissue Kit (Qiagen) and quantitated using the Glomax Discover fluorometer (Promega). DNA NGS libraries were generated using the DNA primers of the Oncomine Comprehensive Assay v3 (ThermoFisher Scientific) and quantitated using the TapeStation 4200 (Agilent Technologies) according to the vendor’s protocol. NGS libraries were normalized and pooled for template preparation on the Ion Chef system (ThermoFisher Scientific) and subsequent sequencing on the Ion S5 GeneStudio instrument (ThermoFisher Scientific). Data analysis was performed using Torrent Suite v5.12 (ThermoFisher Scientific) and in-house software for DNA mutation, copy number variation, and tumor mutation burden calculation.
The NGS DNA analysis results are summarized in Table 1. As previously mentioned, all 8 fibrosarcoma cases in our cohort demonstrated a similar morphology with monomorphic fascicular spindle cell proliferation, moderate atypia and variable mitosis (Figure 1A and B). The age range of our patients was 14 to 88 years old (median 60.5), with a male to female ratio of 1.6:1. Tumor location included head, jaw, upper and lower extremities, trunk and penis. Three out of our 8 cohort cases failed testing due to a low DNA count. Among the remaining five cases, the most common genetic alteration was CDKN2A/CDKN2B copy number loss (4 cases, 80%), followed by TP53 mutations (3 cases, 60%). NF1 mutation or copy number loss, NOTCH mutations and BAP1 copy number loss had an equal frequency in our cohort (2 cases each, 40%). Each of SMAD4, TERT, FANCA, FAND2 and PTCH1 alterations occurred at a frequency of 20%. It is worth noting that some of these genetic aberrations may not represent defining events for tumorigenesis especially that some were reported as variants of uncertain significance (VUS), in particular NOTCH, FANCA, FANCD2 and PTCH1 mutations (Table 1).
Figure 1: (A, B) H&E sections of case 5 demonstrating a relatively monomorphic fascicular spindle cell proliferation with moderate atypia set within a collagenous stroma (10x, 20x).
Abbreviations: VUS: Variant of Uncertain Significance; CN: Copy Number; CNR: Copy Number Region; TMB: Tumor Mutation Burden
In our prior publication  case 2 was previously discussed and demonstrated a FNDC3B-PIK3CA gene fusion of uncertain significance, detected via NGS RNA fusion analysis. Of note, DNA/ RNA sequencing performed by an outside institution was also reported on another case from our prior 2022 study cohort whereby BRAF p.G469A gene mutation was detected and mentioned in our prior manuscript with this mutation being most often reported in lung adenocarcinoma with an unknown prevalence in sarcoma. This case was removed from our current cohort.
The most common genes altered in soft tissue sarcomas are TP53 (47%), CDKN2A (22%), RB1 (22%), NF1 (11%), and ATRX (11%) with the majority of alterations in TP53, NF1, and ATRX being point mutations while the predominant alterations in CDKN2A and RB1 being copy number losses . In their study, Bui, et al., tested all genomic alterations for their prognostic significance, and only CDKN2A alterations correlated significantly with prognosis. The study also showed that there was a trend to earlier recurrence with CDKN2A altered patients. In their analysis of 7733 soft tissue sarcoma patients, malignant peripheral nerve sheath tumors (MPNST), myxofibrosarcomas, and undifferentiated pleomorphic sarcomas showed a high prevalence of CDKN2A alterations.
CDKN2A loss has been shown previously to be a defining event for the malignant transformation of neurofibromas into MPNSTs [2,3]. One study  reported a prevalence of 26.3% of CDKN2A aberrations in fibrosarcoma; however, the authors do allude to the fact that their fibrosarcoma cohort had a limitation whereby pathology was not centrally reviewed, hence the fibrosarcoma diagnosis was solely made by the referring physician which may have led to incorrect or outdated diagnoses. CDKN2A (cyclindependent kinase inhibitor 2A) is a tumor suppressor gene that encodes two proteins: p16 and p14arf. The p16 protein is involved in cell cycle and senescence through the regulation of the cyclindependent kinase (CDK) 4/6 and cyclin D complexes, while p14arf activates TP53. Most common alterations are homozygous deletions, followed by inactivating mutations and promoter hypermethylation . Further research is needed into the p16-CDK4-RB1 pathway and its role in targeted therapeutics.
CDKN2B serves as an important inhibitor of cell proliferation and cell cycle as it encodes for CDKN2B protein (p15, INK4B), which belongs to the INK4 class of cell cycle inhibitors . CDKN2B lies adjacent to CDKN2A on human chromosome 9 and the entire CDKN2A-ARF-CDKN2B locus has shown to be frequently mutated or epigenetically silenced in many cancers [13-17]. In a study by Scruggs, et al., , loss of CDKN2B was associated with an increase in myofibroblast differentiation and an increase in expression of myofibroblast-related transcription factors. The authors noticed that such effects were not observed with silencing CDKN2A. Although the actions of CDKN2A and CDKN2B are often considered redundant, being members of the INK4 family of cell cycle inhibitors with similar structural homology and binding affinity to CDK4/6, only CDKN2B appears to play a role in modulating fibroblast biology and fibroblast differentiation. The relationship of this unique CDKN2B role and its contribution in the biology or pathogenesis of fibrosarcoma remains to be elucidated.
TP53 is altered in 18.91% of soft tissue sarcoma patients . This gene is mapped to chromosome 17 and plays a major role in regulating the response of mammalian cells to stresses and damage through the transcriptional activation of genes involved in cell cycle control, DNA repair, senescence, angiogenesis and apoptosis  Inactivation of the p53 pathway may be derived from the p53 mutation itself, or from other alterations, including increased expression of MDM2 thus causing p53 downregulation, or upstream secondary to CDKN2A mutation [21,22]. P53 mutations are seen in most types of human cancer [23,24] and are one of the most prevalent genetic alterations in soft tissue sarcoma . This is restated in our study, whereby the most common genetic alterations detected in our fibrosarcoma cohort were CDKN2A/CDKN2B and TP53 gene alterations.
BAP1 encodes a deubiquitinating hydrolase identified in 1998 that binds to the RING finger domain of the BRCA1 protein. BAP1 is altered in 0.96% of malignant soft tissue neoplasms [19,26]. While not reported so far in fibrosarcoma, BAP1 mutations are associated with many cancer types including uveal melanoma, mesothelioma, cutaneous melanoma, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, meningioma, breast, thyroid, and lung cancer, among others .
NOTCH is altered in 2.1% of soft tissue sarcoma patients . The Notch signaling pathway consists of four trans-membrane receptors, Notch1 to Notch4, that are encoded by homologous genes . NOTCH signaling has been described in synovial sarcoma, Ewing sarcoma and rhabdomyosarcoma . The relationship of NOTCH signaling in fibrosarcoma has not been described to date. NF1 is altered in 5.66% of soft tissue sarcoma patients . NF1 mutations are identified in malignant peripheral nerve sheath tumor, myxofibrosarcoma, rhabdomyosarcoma, undifferentiated pleomorphic sarcoma and pleomorphic liposarcoma . RB1 is altered in 7.14% of fibrosarcoma patients . RB1 gene encodes the cell cycle regulatory retinoblastoma gene protein (pRb), controls cellular differentiation during both embryogenesis and in adult tissues, regulates apoptotic cell death, maintains cell cycle arrest, and preserves chromosome stability. RB1 gene alterations are most commonly seen in leiomyosarcoma. It has been reported to a lesser extent in fibrosarcoma, rhabdomyosarcomas and pleomorphic sarcomas .
Telomerase reverse transcriptase (TERT) promoter gene mutation is identified in very few sarcoma cases including pleomorphic liposarcoma, dedifferentiated liposarcoma, myxoid liposarcomas, solitary fibrous tumor, undifferentiated pleomorphic sarcoma (UPS) and malignant granular cell tumor [19,32,33]. SMAD4 (DPC4) is a tumor suppressor gene located at chromosome 18q21.1 that belongs to the SMAD family, which mediates the TGF beta signaling pathway suppressing epithelial cell growth. Mutation of this gene is seen only rarely in soft tissue sarcomas . Both TERT and SMAD4 mutations have not been described in fibrosarcoma.
FANCA (Fanconi anemia, complementation group A) is a DNA repair gene, when mutated causes hypersensitivity to DNA damage with a greatly increased risk of multiple cancers . FANCA mutation is the most frequent mutation in patients with Fanconi anemia but is less reported in patients with solid tumors . FANCA is altered in 1.17% of soft tissue sarcoma patients . FANCD2 (Fanconi anemia, complementation group D2), similar to FANCA, is also involved in Fanconi anemia, a heterogeneous recessive disorder that causes cytogenetic instability, hypersensitivity to DNA crosslinking agents, increase in chromosomal breakage, and defective DNA repair. FANCD2 is altered in 3.59% of soft tissue sarcoma patients . Both FANCA and FANCD gene mutations have not yet been reported in fibrosarcoma.
PTCH1 is a tumor suppressor gene that is altered in 1.82% of sarcoma patients . PTCH1 alterations have been reported in intimal sarcoma, rhabdomyosarcoma, malignant peripheral nerve sheath tumor, angiosarcoma, extra skeletal osteosarcoma and extra skeletal myxoid chondrosarcoma . Given that the PTCH1 alteration in one of our fibrosarcoma case cohort was reported as a variant of uncertain significance, the relation between PTCH1 aberrations and fibrosarcoma in general has yet to be investigated.
In conclusion, we attempted to characterize the molecular landscape of fibrosarcoma at our institution, and demonstrated that, similar to most sarcomas, the most common genetic aberrations in fibrosarcoma are CDKN2A/CDKN2B and TP53 alterations. Additional novel alterations detected in our study with potential tumorigenesis role included BAP1 and NF1 copy number loss, TERT mutation, and SMAD4 mutation. Overall, our study helps in further characterizing the mutational changes in fibrosarcoma with potential implications on therapy and prognosis. Additional large-scale studies are needed to further expand on the molecular biology of fibrosarcoma.
Declaration of Conflicting Interests
The author declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
The author received no financial support for the research, authorship and/or publication of this article.
This study was deemed to be exempt.
Informed consent was not sought as the study spanned archived tissue from the past 20 years where the patients could not be reached.
This study did not contain any clinical trials.
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