Pediatric Soft Tissue Sarcoma in Limb-Girdle Muscular Dystrophy: Molecular Findings and Clinical Implications

Introduction

Limb-girdle muscular dystrophy recessive 1 (LGMDR1, MIM #253600), previously known as LGMD2A, is an autosomal recessive disorder characterized by progressive weakness of the proximal leg and shoulder girdle muscles [1]. Based on public sequencing databases, the disease has an estimated prevalence of 0.02 to 27.0 per million individuals, highly dependent on the geographical location [2]. The debut of symptoms is very variable, ranging from early childhood to over 40 years of age [3]. The disease-causing gene, CAPN3, encodes a calcium-dependent cysteine protease with roles in muscle formation, turnover, and repair and calcium release/uptake in the sarcomere [4]. Loss of CAPN3 activity in dystrophic muscle leads to calcium imbalances, oxidative damage, mitochondrial alterations, sarcomere disorganization, impaired muscle adaptation, and abnormal muscle regeneration [5].

Soft tissue sarcomas (STS) are rare malignant tumors arising from soft tissue. Pediatric presentations have an incidence of 12 in 100 000 children, accounting for 6.7% of all childhood malignancies [6]. STS are a heterogeneous group of tumors, with distinct clinical behavior, histology, and tumor biology [7]. Although there is no clear cause, different factors associated with the development of STS have been suggested, including genetic predisposition, oncogenic viruses, immunodeficiency, chemical carcinogens, and chronic inflammation [8].

Desmoplastic small round cell tumors (DSRCT) are aggressive malignant STS, which often develop in the abdominal cavity, commonly metastasizing to the peritoneum, liver, and lymphatic system [9,10]. DSRCT are extremely rare. An epidemiological study in the United States calculated an age-adjusted incidence of 0.3 cases per million with a 3.6 male to female ratio, and a peak age at diagnosis of 20 to 24 years [11]. Molecularly, DSRCT are characterized by the EWSR1::WT1 gene fusion, usually due to the recurrent chromosomal translocation t(11;22)(p13;q12), which combines the transactivation region of EWSR1 and the DNA binding domain of WT1 [10,12]. The chimeric protein acts as an oncogenic transcription factor, regulating the expression of several growth and transcription factors involved in carcinogenesis, including, for instance, PDGFRα, IGF1R, EGFR, MYC, PAX2, and WT1 [10]. DSRCT diagnosis includes a positive dot-like Desmin staining, often positive cytokeratin, and negative staining for skeletal muscle-associated markers Myogenin and MyoD1. Detection of the EWSR1::WT1 fusion transcript is desirable [13]. The current management for DSRCT is multimodal, including a combination of chemotherapy, radiotherapy, and cytoreductive surgery. The prognosis remains poor, as recurrences are common, and the survival rate is low [10].

Independent studies have demonstrated that mouse models of different muscular dystrophies (MD), including LGMDR1, are susceptible to STS, with varied penetrance and age-at-onset depending on the muscular disease, mouse strain, and genetic variant modelled [14–19]. The study from Schmidt et al included a small cohort of Capn3 −/− mice, of which 1/19 (5%) developed STS. The STS rate was increased in Dmd −/− Capn3 −/− double-knockout mice, with 24/55 (44%) mice affected [14]. Seven reports of cancer in individuals with LGMD have been published in the literature [20–22]. Only one of them had an STS; a patient with concomitant liposarcoma and LGMD2B [21].

Here, we delineate the clinical, molecular, and genetic characteristics of a 17-year-old male patient with LGMDR1 who developed a DSRCT and discuss possible pediatric cancer associations.

Case Report

TUMOR AND GERMLINE WHOLE GENOME SEQUENCING RESULTS:

Upon diagnosis of the primary DSRCT, the patient was enrolled in the national Genomic Medicine Sweden pediatric cancer project, as part of which matched genomic DNA from peripheral blood and DNA and RNA from fresh frozen tumor tissue were extracted at Clinical Genomics, Stockholm. Whole genome sequencing was then performed via paired Illumina sequencing with 30× depth for germline DNA and 90× for tumor DNA, as previously described [24–26], and RNA sequencing with the Illumina stranded paired-end mRNA method, as previously described [24].

Manual inspection of germline whole genome sequencing results did not identify any pathogenic variants in 189 known childhood cancer predisposition genes [26]. RNA sequencing analyses of both the primary and relapsed tumor on the scalp confirmed the EWSR1::WT1 (t(11;22)(p13;q12) fusion transcript (Figure 1E). Furthermore, both the primary and relapsed tumors exhibited a near triploid karyotype and displayed multiple shared numerical and segmental chromosomal aberrations, indicative of a clonal relationship. Nonetheless, new rearrangements emerged in distinct chromosomal regions, suggesting genomic instability during tumor evolution (Figure 2). No potentially damaging single nucleotide variants or insertions/deletions were discovered in either tumor.

Discussion

We present the clinical and genetic characteristics of a patient with LGMDR1 who developed a DSRCT. To the best of our knowledge, this is the first reported case of DSRCT or any type of pediatric cancer in LGMD. The patient’s LGMDR1 was caused by compound heterozygous variants in the CAPN3 gene, namely NP_000061.1: p.(Thr184ArgfsTer36) and NP_000061.1:p.(Arg448Cys), while the tumor was diagnosed as a DSRCT with an EWSR1::WT1 (t(11;22)(p13;q12)) fusion transcript. We suggest a possible association between the development of DSRCT and LGMD. However, we cannot rule out the possibility that carcinogenesis in this patient was fortuitous.

The patient described in this case report developed DSRCT, a very rare STS [11]. The diagnosis was confirmed by positive dot-like Desmin staining, negative staining of the skeletal muscle markers Myogenin and MyoD1, and the presence of a EWSR1::WT1 fusion transcript, detected by RT-PCR in the primary tumor. In addition, RNA sequencing in the primary and relapse tumors confirmed the fusion transcript. Although the WT1 C-terminal antibody, generally used to diagnose DSRCT, was not available in our laboratory, 3 independent assays confirmed the chromosomal translocation t(11;22)(p13;q12), leading to the definitive diagnosis of DSRCT. Of note, a positive cytokeratin staining is usually expected in DSRCT [13]. However, the tumor in our patient presented a negative staining. Although the absence of pan cytokeratin expression is unusual, previous reports have been published in the literature of pan cytokeratin-negative DSRCT [27–29]. The detection of the EWSR1::WT1 fusion is therefore desirable for DSRCT diagnosis [28].

It is well known that DSRCT are highly aggressive pediatric tumors [10,30]. The severe clinical course of the DSRCT observed in our patient, including multiple metastasis at diagnosis and poor treatment response, is similar to what has been reported in other individuals with this tumor type [30,31]. Although the patient was treated with multimodal therapy, as suggested for DSRCTs [10], the response was poor, and the patient has now been put on palliative care. This is also in line with observations on other individuals with DSRCT, in which the prognosis is poor, with a 5-year survival rate below 25% [32,33]. Finally, as in most DSRCT diagnoses [11,30,31], our patient was young and male. All in all, the clinical course of the DSRCT in our patient resembled that of other individuals with DSRCT not diagnosed with LGMD.

Previous studies on MD mice models report an increased risk of mixed sarcomas in aged animals, including in LGMD [14–19]. CAPN3 knockout mice models develop mixed sarcomas with characteristics of rhabdomyo-, fibro-, and liposarcomas [14], whereas the patient presented in this report was diagnosed with a sarcoma of uncertain differentiation, according to the classification of tumors from the World Health Organization [13].

In addition, 7 patients with concomitant LGMD and cancer have been reported in the literature [20–22]. Apart from 1 single individual with liposarcoma [21], 3 patients with melanoma [20], and 3 with myeloma [22] and LGMD have been described. Sarcoma presentations in patients with Duchenne MD have also been described [34–40]. Finally, a recent Swedish population-based epidemiological study conducted by our group, in which 2355 patients with MD were included, showed an increased risk of pediatric central nervous system tumors and gliomas and adult pancreatic and non-thyroid endocrine tumors in individuals with MD. An increased risk of sarcoma was not observed. However, it was not possible to evaluate the cancer risk and risk spectrum in individuals with LGMD exclusively [41]. Thus, the cancer incidence in this specific patient group remains unclear and needs further exploration.

Multiple theories exist about the events that could possibly lead to cancer development in dystrophic muscle. Schmidt et al showed that there is genetic instability, including DNA damage, aneuploidies, and increased double strand break repair in muscles from mice models and patients with MD [14]. This instability was observed as early as in myoblasts, in line with recent results suggesting that in MD mice, mixed sarcomas arise from muscle stem cells [19]. It has been proposed that conflicting differentiation signals given to myoblasts in MD are permissive for tumor formation [42]. Chronic inflammation and a tumor suppressor role in some MD disease-causing genes are additional proposed mechanisms for carcinogenesis in MD [42]. Understanding the risk of DSRCT and cancer in general in individuals with LGMD could have important implications for patients, including surveillance and improved genetic counseling [26]. However, before changes in clinical practice for patients with LGMD can be considered, further studies are needed to better understand their DSRCT risk and to delineate their cancer risk spectrum.

Conclusions

We describe the clinical, genetic, and molecular characteristics of a patient with LGMDR1 who developed a DSRCT. Very limited information is available about the incidence of cancer in this patient group. Therefore, increased awareness, further case reports, and epidemiological studies are warranted to better understand the link between pediatric cancer and LGMD.