Whole-exome sequencing identification of the risk variants in a middle-aged adult with intracranial Ewing’s sarcoma: A case report

Abstract

BACKGROUND

Ewing’s sarcoma (EWS), which primarily affects young children, and is characterized by rare bone tumors, with occasional metastasis to the brain. However, information on EWS remains limited, particularly concerning gene mutations in adult patients. Consequently, there is a pressing need to identify genetic factors associated with the pathogenesis of EWS to enhance the detection, management, and prevention of this disease.

CASE SUMMARY

Herein, we present the unique case of a middle-aged patient with intracranial EWS. A 51-year-old male was admitted to our hospital for the surgical resection of an intracranial mass. Postoperative pathology confirmed the presence of a small-cell malignant tumor, which was further diagnosed as EWS. Following surgery, the patient was referred to the radiotherapy department to complete his treatment. To identify potential EWS disease-causing gene variants, we performed whole-exome sequencing (WES) analysis using genomic DNA extracted from the patient’s blood and tumor samples. This analysis revealed heterozygous variants in somatic cells, within the neurofibromin 1 [NF1; c.T569A:p.Leu190*(stopgain)] and secreted protein acidic and rich in cysteine (SPARC; c.G764A:p.R255H) genes.

CONCLUSION

WES provided initial evidence supporting the potential involvement of NF1 and SPARC in the pathogenesis of EWS.

Key Words: Ewing’s sarcoma; Intracranial tumor; Whole-exome sequencing; Variants; Case report

Core Tip: Ewing’s sarcoma (EWS) and Ewing’s-like sarcoma represent uncommon and extremely aggressive round cell mesenchymal malignancies. Here, we describe a case of a 51-year-old man with a confirmed diagnosis of EWS. The article introduces the diagnosis and treatment process. The findings from whole-exome sequencing highlighted the plausible impact of neurofibromin 1 and secreted protein acidic and rich in cysteine on EWS progression.

INTRODUCTION

Ewing’s sarcoma (EWS) and Ewing’s-like sarcoma are extremely aggressive and rare round cell mesenchymal tumors that typically affect children and young adults. Overall, EWS constitutes < 1% of all soft tissue sarcomas, and it targets long bone metaphysis in about 80% of cases. Most EWS incidents occur between the first and second years of life[1]. However, it can also impact adults. EWS primarily affects the paravertebral region as well as the deep soft tissues of the proximal lower and upper limbs. Additionally, it also affects visceral sites, specifically the kidney, pancreas, and meninges, as well as the presence of primary skin lesions[2].

EWS pathogenesis is driven by a fusion oncoprotein derived from the translocation of EWSR1 on chromosome 22 with an ETS family member, most commonly FLI1 on the chromosome. Approximately 85% of EWS cases are strongly characterized by this gene translocation[3]. As a pathogenic transcription factor, the EWS/FLI1 fusion protein regulates tumor occurrence and progression[4]. A previous study investigating single-nucleotide polymorphisms (SNPs) within EWS identified CD86, rs9430161 (upstream of TARDBP), rs224278 (upstream of EGR2), and rs4924410 15q15 as being intricately associated with the risk of developing EWS[5]. Additionally, several genetic mutations, such as TP53, Rb1, and STAG2, have been commonly observed among EWS patients[6,7]. However, research on gene mutations related to EWS has been limited to date, and there remains a need to explore novel mutations in EWS in order to improve its diagnosis, prognosis, and to inform treatment strategies.

To the best of our knowledge, only 13 reported cases of intracranial EWS have been reported in individuals over 50 years old[8]. In the present study, we conducted whole-exome sequencing (WES) to analyze the genomes of a middle-aged patient from Liaocheng, China, with intracranial EWS. Through comprehensive analyses, we were able to identify potential genes linked to the pathogenesis of EWS, with great promise as diagnostic indicators for this tumor.

CASE PRESENTATION

Chief complaints

A 51-year-old male sought treatment at our institute for severe headache and nausea. The patient is conscious and has no limb movement disorder.

History of present illness

The patient reported experiencing paroxysmal headache and nausea, which were exacerbated by coughing. He denied experiencing vomiting, an altered mental status, motor impairment, seizures, urinary incontinence, or other related symptoms.

History of past illness

The patient reported no history of surgery, trauma, significant infections, or other notable medical conditions.

Personal and family history

The patient had no personal or family history of any particular medical issues.

Physical examination

The patient exhibited a normal mental status, coherent speech, unimpaired vision, and clear consciousness, and showed no signs of physical movement disorders, convulsions, or incontinence.

Laboratory examinations

Postoperative pathology revealed a tumor located along the blood vessels at a certain distance from the brain parenchyma. Based on hematoxylin and eosin and immunohistochemistry staining (Figure 1), the results were as follows: Vimentin (+), CD99 (+), CK (-), CD56 (+). Considering all the findings and examinations, a small-cell malignancy was diagnosed, with a high-grade neuroepithelial tumor considered. The possibilities of a high-grade sarcoma and metastatic tumors were not excluded.

Figure 1 Immunohistochemical evaluation of the proband pathological sections. A: Hematoxylin and eosin staining; B: Vimentin (+) staining, C: CD99 (+) staining, D: CK (-) staining; E: CD56 (+) staining; F: Fluorescence in situ hybridization imaging. A: Magnification × 100; B-E: Magnification × 200; F: Magnification × 1000.

Imaging examinations

Preoperative cranial magnetic resonance imaging (MRI) was performed and displayed mass-like patchy long and slightly long T1 signals as well as long and slightly long T2 signals in the bilateral frontoparietal lobes and corpus callosum. Also, partial hyperintense signals were observed on the FLAIR sequence, while partial diffusion restriction was noted on diffusion-weighted imaging. Contrast-enhanced scanning revealed a garland-like and patchy enhancement with ill-defined boundaries. The lesion size was approximately 1.7 cm × 2.1 cm × 3.5 cm. Patchy peritumoral edema was detected around the lesion, with mild compression of adjacent brain tissue and bilateral ventricles. No midline shift was found in this case (Figure 2A). Positron emission tomography-computed tomography revealed an increased fluorodeoxyglucose (FDG) uptake (SUV_max = 10.5) only in the operative area of the frontoparietal region and splenium of the corpus callosum. No abnormal lesions or elevated FDG uptake were found in other organs and systemic structures, and no distant metastasis was identified. Relevant baseline laboratory data were collected, including routine blood parameters, liver and renal function, and tumor markers, and revealed: White blood cell count = 8.03 × 10⁹/L, red blood cell count = 5.17 × 10¹²/L, platelet count = 281 × 10⁹/L, and no obvious abnormalities in the liver and kidney function.

Figure 2 Brain magnetic resonance imaging scan. A: Preoperative brain magnetic resonance imaging (MRI) of the patient; B: Postoperative brain MRI; C: Brain MRI following radio and chemotherapy.

MULTIDISCIPLINARY EXPERT CONSULTATION

WES

To screen for pathogenic gene mutation via WES, our radiotherapy department extracted 5 mL peripheral blood samples and pathological sections from the patient in 2023.

To identify potential pathogenic variants, we performed paired-end WES on a NextSeq CN500 instrument (Illumina) using genomic DNA from tumor tissue and peripheral blood leukocytes. After multiple purification and amplification rounds, the final library fragment size was approximately between 250 bps and 350 bps. The mean sequencing depth was 86.3 ×, and the average read number was 9.5 × 10⁷ bps.

Statistical analysis

Raw read quality was assessed using Trimmomatic software (version 0.36), and low-quality sequences or those containing adapters were removed. Burrow-Wheeler Aligner enrichment (v0.7.17) was used for aligning sequences to the human genome assembly (hg38). SNPs and insertions/deletions were screened using GATK-Haplotype Caller (v4.1.5.0), and ANNOVAR was utilized for variant annotation. Variant filtration was performed according to the following criteria: (1) Elimination of variants with a minor allele frequency ≥ 0.01 in the 1000 genomes, ESP, or ExAC databases; (2) Limitation of variants annotated as synonymous single nucleotide variant in the refGene database; (3) Exclusive analysis of variants within the exonic, splicing, UTR5, UTR3, and splicing regions; (4) Variant-mediated protein coding regulation, as predicted by SIFT, LRT, Mutation Assessor, Mutation Taster, FATHMM and PROVEAN; remaining variants were considered deleterious by ≥ 2 software; and (5) Computation of the CADD score for individual variants and deleting variants with CADD_PHRED < 20. Finally, our analysis identified singular heterozygous variants in somatic cells, within the neurofibromin 1 [NF1; c.T569A:p.Leu190*(stopgain)] and secreted protein acidic and rich in cysteine (SPARC; c.G764A:p.R255H) genes, as putative EWS disease-causing mutations (Table 1).

Table 1 Single-nucleotide polymorphisms revealing significantly distinct allele frequencies.

Chr	Gene	Position	Exon	Allele	AAChange.refGene	cytoBand
Chr17	NF1	31169980	5	T>A	c.T569A:p.Leu190* (stopgain)	17q11.2
Chr5	SPARC	151664203	9	G>A	c.G764A:p.R255H	5q33.1

NF1: Neurofibromin 1; SPARC: Secreted protein acidic and rich in cysteine.

We subsequently performed an analysis of amino acid sequence conservation utilizing DNAMAN. Our analysis validated that NF1: L190 and SPARC: R255 were strongly conserved across multiple species (Figure 3A and B). Typically, mutations that occur in highly conserved amino acids are likely to exert a more significant impact on protein function, and so we used Chimera v1.17.3 to visualize the structural alterations caused by the amino acid mutations (Figure 3C and D).

Figure 3 Biological analysis of risk variants. A and B: Neurofibromin 1 Leu 190 (A) and secreted protein acidic and rich in cysteine (SPARC) Arg 255 (B) across species; C and D: Schematic diagrams of the 3D SPARC protein structure prior to and after C > D mutation, which altered the amino acid at position 255 from an arginine to a histidine.

FINAL DIAGNOSIS

After consulting experts from Xuanwu Hospital, Capital Medical University, we performed fluorescence in situ hybridization imaging using the EWSR1 (22q12) gene break probe, upon which separated red/green/yellow signals consistent with EWSR1 rearrangement were observed, the proportion of positive signals is 32%, suggesting that the EWSR1 gene had broken (Figure 1F). Finally, considering all the findings and examinations, the patient was finally diagnosed with EWS.

TREATMENT

The patient underwent craniotomy for resection of an intracranial mass while under general anesthesia. Postoperatively, treatments to improve neural metabolism and reduce intracranial pressure through dehydration were administered.

At the 1-month postoperative follow-up, the patient underwent radiotherapy for residual recurrence. Following radiotherapy, the patient was administered a combination treatment of temozolomide (TMZ) and bevacizumab (BEV). Due to patient concerns about chemotherapy agents and the limitations posed by the blood-brain barrier and other factors in traditional chemotherapy for intracranial EWS, the treatment outcomes were less than optimal. Therefore, combination therapy was considered as the most optimal treatment. In selecting the optimal combination therapy, we reviewed case reports from various regions worldwide and chose BEV and TMZ[9-11], which are the primary agents effective at overcoming the blood-brain barrier.

OUTCOME AND FOLLOW-UP

Following treatment, the patient’s clinical symptoms improved, while the results from the contrast-enhanced MRI scan at the most recent visit also revealed a remarkable improvement compared to the initial visit (Figure 2B and C), indicating effective control of the aforementioned EWS has been achieved. Furthermore, the patient was regularly followed up by telephone until eventual death in July 2025, representing an overall survival (OS) of 26 months. Figure 4 illustrates the patient’s entire treatment course.

Figure 4 Timeline of the patient’s clinical course. PR: Partial response; SD: Stable disease.

DISCUSSION

Primary intracranial EWS is a rare malignant sarcoma with limited representation in the literature beyond the pediatric population. Consistent with most reported cases both domestically and internationally, our patient presented with non-specific intracranial hypertension symptoms (e.g., headache), which are typical initial manifestations of intracranial EWS due to its space-occupying effects. Similar to previously reported cases in the literature, we performed craniotomy for tumor resection to relieve the mass effect of the tumor. However, while most previous cases utilized standard EWS chemotherapy regimens (e.g., VDC/IE) and focal radiotherapy, we chose TMZ + BEV combination therapy (instead of standard chemotherapy) as an individualized decision based on the intracranial location, blood-brain barrier penetration, and patient-specific factors. Our patient’s OS of 26 months is comparable to the mean survival reported in other cases and even exceeds the survival of previous metastatic cases[8]. Although several studies have documented EWS, none have elucidated the underlying the mechanism at the molecular level. In this study, we utilized WES to identify 2 mutations associated with EWS in a middle-aged patient from Liaocheng. These mutations were found in the NF1 gene [c.T569A:p.Leu190*(stopgain)] and the SPARC gene (c.G764A: P.R255H).

The NF1 gene resides in chromosome 17q11.2 and encodes neurofibromin, which is extensively distributed in the cytoplasm of neurons, astrocytes, oligodendrocytes, and Schwann cells. Besides its association with the well-known neurofibromatosis type 1, alterations in the NF1 gene have been observed in approximately 5% of all human sporadic cancers[12]. These alterations are particularly common in various cancer types, including melanomas, acute myeloid leukemias, glioblastomas, breast cancer, and other tumors[13]. As a tumor suppressor gene, NF1 plays a crucial role in inhibiting the RAS/MAPK and PI3K-AKT-mTOR signaling networks. A loss-of-function mutation in the NF1 gene results in the loss of neurofibromin activity, leading to prolonged activation of the RAS/RAF/MAPK signaling pathway. This activation subsequently stimulates the PI3K/AKT/mTOR signaling pathway, ultimately causing uncontrolled cell growth[14]. Previous studies have indicated that there is a strong association between EWS tumors and NF1. Emerging evidence has revealed that the biallelic inactivation of NF1 promotes tumor development, thereby supporting the hypothesis that mutations in the Ras network may act as secondary events in EWS[15]. Additionally, NF1 is recognized as a well-established molecular marker in sarcomas[16]. Alterations in the PI3K/AKT signaling pathway are critical to the pathogenesis of EWS, mediating various oncogenic phenotypes, including cell cycle disruption, aberrant growth, proliferation, and an altered metabolism, increased angiogenesis, and heightened aggressiveness[6].

SPARC, otherwise known as osteonectin or BM-40, is a stromal cell protein that strictly controls cell adhesion, production of the extracellular matrix, growth factor activity, and the cell cycle. SPARC regulates cell adhesion and cell proliferation through different signaling pathways and is a highly conserved extracellular interstitial protein[17]. Previous research has shown that SPARC is closely associated with tumor development and plays a vital role in tumor invasion and metastasis. For instance, SPARC expression was found to be elevated in melanoma, glioma, meningioma and breast cancer[18,19]. In colorectal cancer, SPARC has been identified as a key gene potentiating metastasis[20]. It was also found to activate the PI3K/AKT/PDGFB/PDGFRβ axis, thereby promoting the proliferation, migration, and invasion of oral squamous cell carcinoma[21]. Furthermore, SPARC and p53 regulate cancer cell survival through inverse effects on apoptotic pathways and related signaling mechanisms[22]. In a previous study related to EWS, SPARC expression was shown to prolong the aggregation of albumin-bound paclitaxel, which may have clinical relevance in modulating the chemotherapeutic efficacy[23]. EWS is a biologically and clinically aggressive disease characterized by a high propensity for metastasis. Thus, mutations in SPARC may influence the surrounding extracellular matrix and promote the aggressive and metastatic progression of EWS.

In this study, we identified two unreported variants associated with EWS: NF1 [c.T569A:p.Leu190*(stopgain)] and SPARC (c.G764A:p.R255H). Their nature as seemingly meaningless variations (which may lead to premature protein truncation or conformational changes in proteins) increases the possibility of them having a functional impact, but the lack of functional validation and the inherent limitations of a single-patient case report preclude making definitive conclusions regarding their role in EWS development, leading us to define these only as candidate variants that may be potentially relevant to our patient’s disease. We intend to perform a targeted experimental validation study in future research to explore their true biological significance.

Our patient received TMZ + BEV as an individualized adjuvant therapy after subtotal resection and postoperative radiotherapy. Stable disease was maintained for 14 months after initiating TMZ + BEV treatment, with disease progression occurring again 18 months post-diagnosis. However limited international data exist on TMZ + BEV combination therapy for intracranial EWS. Encouragingly, a 2012 case report documented a case of disseminated intracranial EWS in an adult, wherein, in the advanced phase of treatment, salvage therapy comprising TMZ and BEV was implemented[24]. In another study reporting adult metastatic EWS, a vincristine, irinotecan and TMZ regimen was administered and achieved a 31.3% partial response rate and a median progression-free survival of 8.5 months in patients with brain involvement, further confirming the central nervous system-penetrating potential of TMZ-based combinations[25]. Additionally, BEV’s anti-angiogenic effect has been shown to reduce peritumoral edema and improve symptom control in intracranial sarcomas. This synergistic mechanism may explain the observed efficacy in our case and in the limited literature.

We acknowledge that the efficacy of TMZ + BEV in our case is a preliminary finding and our study is subject to significant limitations that should be noted, specifically: (1) The findings are based on a single patient, which limits their generalizability to all intracranial EWS cohorts; and (2) There is a lack of molecular markers that could predict the responsiveness to TMZ. Furthermore, although our patient’s response was promising, it is crucial to emphasize that TMZ + BEV is generally reserved for recurrent or refractory cases according to standard EWS guidelines. Its potential as a first-line adjuvant therapy requires validation in larger cohorts.

CONCLUSION

This case report describes an intracranial EWS in a middle-aged patient. Using WES, two mutations were identified in NF1 [c.T569A:p.Leu190*(stopgain)] and SPARC (c.G764A:p.R255H). NF1 inactivation promotes tumor development, may activate the RAS/MAPK and PI3K-AKT-mTOR signaling pathways in cells. SPARC, on the other hand, affects the association between cells and the surrounding extracellular matrix, thereby promoting the development of EWS. To sum up, these findings indicate that variants in NF1 and SPARC with molecular alterations may serve as candidate markers for this rare presentation of intracranial EWS. However, further functional validation and additional case studies are necessary to elucidate their pathogenic and therapeutic significance.

ACKNOWLEDGEMENTS

We thank Xuanwu Hospital, Capital Medical University for providing assistance with the molecular diagnosis for this work.