Rhabdomyosarcoma

Abstract

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children and represents a high-grade neoplasm of skeletal myoblast-like cells. Decades of clinical and basic research have gradually improved our understanding of the pathophysiology of RMS and helped to optimise clinical care. The two major subtypes of RMS, originally characterized based on light microscopic features, are driven by fundamentally different molecular mechanisms and pose distinct clinical challenges. Curative therapy depends on control of the primary tumor, which can arise at many distinct anatomic sites, as well as controlling disseminated disease that is known or assumed to be present in every case. Sophisticated risk-stratification for children with RMS incorporates a variety of clinical, pathological, and molecular features, and that information is used to guide the application of multi-faceted therapy. Such therapy has historically included cytotoxic chemotherapy as well as surgery, ionizing radiation, or both. This Primer describes our current understanding of RMS epidemiology, disease susceptibility factors, disease mechanisms, and elements of clinical care including diagnostics, risk-based care of newly diagnosed and relapsed disease, and the prevention and management of late effects in survivors. We also outline potential opportunities to further translate new biological insights into improved clinical outcomes.

Introduction

Soft tissue sarcoma accounts for ~7% of cancers in children and 1% of cancers in adults¹. Approximately half of the population of pediatric patients with soft tissue sarcoma have rhabdomyosarcoma (RMS), which is a high-grade, malignant neoplasm in which cancer cells have a propensity for myogenic differentiation. Two major RMS subtypes exist, ‘alveolar’ RMS (ARMS) and ‘embryonal’ RMS (ERMS), which are driven by fundamentally different mechanisms. Both subtypes pose substantial clinical challenges because achieving a cure requires controlling the primary tumor, which may arise in a wide variety of anatomical sites, by surgical resection and/or ionizing radiation and eradicating systemic metastatic disease using intensive chemotherapy. The past 30 years have witnessed dramatic improvements in survival for many children with RMS, as evidenced by the development and implementation of sequential clinical trials conducted nationally or internationally as cooperative groups spanning North America and Europe ^2-7. Moreover, advancements in molecular biology and genetics have also enabled more sophisticated understanding of RMS pathogenesis. Those approaches continue to provide a platform to improve diagnosis, disease classification, patient risk-stratification and management strategies.

ARMS and ERMS have emerged as the two major RMS subtypes based on light microscopic features of cells distributed around an open central space ⁸ or cells resembling immature skeletal myoblasts ⁹. That distinction was supported by the recognition that ARMS was often associated with balanced chromosomal translocations involving chromosomes 2 or 1 and chromosome 13 (referred to here as t(2;13) and t(1;13)), originally detected by cytogenetics ^10-12. As described in detail below, a small but substantial fraction of patients with ARMS do not harbor one of these translocations, and those tumors are biologically and clinically similar to ERMS.

The World Health Organization (WHO) also recognizes two rarer RMS subtypes. Pleomorphic RMS is a morphological variant of RMS that typically occurs in adults ¹³. Like ERMS, unifying molecular genetic aberrations in pleomorphic RMS are not yet clear. In children, a spindle cell/sclerosing RMS variant is seen; those tumors arising in the head/neck region seem to be more likely to carry specific somatic mutations and have a poorer prognosis¹³.

Disease classification of RMS subtypes has been further refined by the identification of ‘fusion positive’ (FP) and ‘fusion negative’ (FN) RMS. Molecular biology approaches and next-generation DNA and RNA sequencing have shown ARMS-associated translocations to generate novel fusion proteins involving paired box protein PAX3 or PAX7 and forkhead box protein O1 (FOXO1) ^14,15,16. Excluding pleomorphic RMS that occurs in adults, most experts consider childhood RMS to be best described based on fusion status. We adhere to that convention in this Primer, but we use ARMS and ERMS as descriptors when describing pathology reports and earlier research based on those classifiers.

Despite many advances, the chance of cure for children with widely-metastatic and recurrent disease remains very low. Moreover, patients experience months of intensive, multifaceted therapies that can bring life-threatening acute toxicities and, in some cases, life-changing late effects.

In this Primer, focusing on pediatric RMS, we reveal new light being shed on RMS biology through the use of next-generation nucleic acid sequencing and the employment of whole organism-based models of disease. We also discuss how the application of functional approaches for therapeutic target identification and validation should propel further clinical advances.

Epidemiology

Global disease burden

Although a rare disease, RMS is a fairly common form of childhood cancer and is the most common soft tissue sarcoma in children. The overall incidence rate of RMS is approximately 4.5 patients per million individuals aged <20 years¹⁷. In the United States, this equates to ~350 new cases per year. On the basis of data from the Surveillance, Epidemiology and End Results (SEER) Program, we know that the incidence of RMS differs both by age and histology¹⁸ (Figure 1). Moreover, the incidence of RMS in Europe appears to be similar to that in the United States. For example, a 2016 report from Sweden indicates an overall annual incidence of 4.9 patients with RMS per million individuals aged <15 years¹⁹. Interestingly, the incidence of RMS appears to be lower in parts of Asia with just over 2 patients per million individuals reported in Japanese, Indian, and Chinese populations ²⁰.

Figure 1: RMS incidence varies with age and subtype.

(A) Graph showing the relative proportion of each RMS subtypes presenting in various age groups. Spindle cell/sclerosing and botryoid forms not shown owing to their relative rarity. Note that relative frequency of PAX7-FOXO1 and PAX3-FOXO1 ARMS across the age spectrum has not been well studied and is captured by SEER. (B) Graph showing how the incidence of each major RMS subtype has changed or remained stable over the past ~30 years. The apparent increased incidence of ARMS since the 1990s, but that may, in part, relate to evolving diagnostic definitions, as described in the main text. Data are from the Surveillance, Epidemiology, and End Results (SEER) April 2008 release. Figure has been reproduced with permission from¹⁸. ARMS, alveolar rhabdomyosarcoma; ERMS, embryonal rhabdomyosarcoma.

Influence of age and sex

RMS incidence rates are influenced by several factors intrinsic to the tumor and to individual patients. For example, a recent analysis of SEER data showed the diagnosis of ERMS to be ~2.5-fold more frequent than ARMS¹⁸. However, the exact frequency of these two forms is affected by evolving diagnostic criteria (discussed below), especially in North America ^13,18. Age also influences RMS incidence. ERMS is the most common form in early childhood (Figure 1A), but some data suggests a second peak in early adolescence for ERMS (Supplemental Figure 1) ²¹. Bimodal peaks are not evident in children with ARMS; incidence of ARMS remains constant throughout childhood and adolescence ¹⁷. We also note that one report showed the median age of children with PAX7–FOXO1-positive disease to be younger than those with PAX3–FOXO1 RMS (age 6 versus 13 years)²², but that conclusion from a small, retrospective analysis of a limited number of US institutions has uncertain significance. In contrast to ERMS and ARMS, WHO-classified pleomorphic RMS primarily occurs in adult males in their 6^th decade of life ²³. “Pleomorphic” RMS in children is typically classified as ERMS with diffuse anaplasia¹³.

RMS incidence also varies by sex, as male children have a higher incidence of ERMS compared to female children (male:female ratio of 1.51, 95% confidence interval (CI): 1.27–1.80) ²¹. On the basis of an analysis of pooled cancer registry data from five US states, which focused on the risk of RMS by parental ethnicity, overall notable differences in incidence by ethnicity have not been observed²⁴. The only exception was that the risk of RMS in children was significantly lower when both parents were of Hispanic ethnicity (odds ratio of 0.65, 95% CI: 0.48–0.88).

Using data from SEER, several groups have reported the incidence of ERMS for the period 1975–2005 to have remained relatively stable ^18,25. In contrast, a significant increase in the incidence of ARMS (annual percentage change of 4.20%, 95% CI: 2.60–5.82) was evident over the same period (Figure 1B). This apparent increase may be related to fluctuations in diagnostic criteria, such as the proportion of the tumor required to display alveolar features in order to diagnose ARMS. Although the use of a more objective RMS classification scheme based on presence or absence of the t(2;13) or t(1;13) translocations or expression of PAX3–FOXO1 or PAX7–FOXO1 fusion transcript may clarify this matter, that information has not been captured by SEER or many other large cancer registries.

Risk factors

As opposed to osteosarcoma ²⁶ and Ewing sarcoma ²⁷ (two other fairly common childhood soft tissue sarcomas), there has not been a published genome-wide associations study for RMS. Moreover, whole-exome and whole-genome sequencing has identified somatic mutations in RMS ^28-30, but few studies have characterized the role of germline DNA on disease susceptibility. It remains challenging to define risk factors in a rare cancer type with an incidence of 4–5 patients per million individuals. However, a great deal of literature exists to support the hypothesis that genetic susceptibility and environmental factors play a part in RMS development.

Genetic risk factors.

Numerous reports highlight that children with certain genetic disorders develop RMS more frequently than unaffected peers. Syndromes that are most commonly seen in children with ERMS include Li-Fraumeni syndrome (germline mutation of TP53, a tumour suppressor)³¹; Neurofibromatosis type I (deletions in the NF1 gene) ^32,33; Costello syndrome (HRAS mutation) ^34,35; Noonan syndrome (germline genetic variants activating RAS–MAPK pathways) ³⁴; Beckwith-Wiedemann syndrome ³⁶, and DICER1 syndrome (germline DICER1 mutations) ³⁷ (Table 1). However, on the basis of smaller clinic-based studies, only ~5% of patients with RMS are thought to have co-morbid germline susceptibility syndromes ³⁸. Interestingly, cancer predisposition syndromes appear to be more frequent in patients with ERMS as compared to those with ARMS ^33-35,39. This finding seems to contrast experimental studies showing that germline loss of specific tumor suppressors facilitates PAX3–FOXO1-driven neoplasia in genetically-engineered mouse models ⁴⁰. Importantly, large population-based studies are required to systematically characterize mutations (Table 1) in children with RMS and to evaluate differences by fusion-protein status.

Table 1:

Heritable syndromes associated with an increased risk of RMS

Syndrome	Phenotype	Associated gene(s)	References
Li-Fraumeni	Cancer susceptibility syndrome	TP53	31
Neurofibromatosis type 1	Systemic effects	NF1	32,33
DICER1	Cancer susceptibility syndrome	DICER1	37
Costello	Systemic effects	HRAS	34,35
Noonan	Systemic effects	BRAF; KRAS; NRAS; PTPN11; RAF1; SOS1	34
Beckwith-Wiedemann syndrome	Overgrowth disorder	IGF2; CDKN1C; H19; KCNQ1OT1	36

Environmental risk factors.

Several environmental exposures and other factors have been implicated in RMS risk in children. Many published reports are based on a large epidemiological case-control study of RMS that was enabled through the former Intergroup Rhabdomyosarcoma Study Group (IRSG) and current Children’s Oncology Group (COG), who drive therapeutic studies for 80–85% of all children with RMS in North America ⁴¹. In that study, 322 patients with RMS aged ≤20 at the time of diagnosis and 322 control individuals matched by sex, age and ethnicity were enrolled between April 1982 to July 1988. Key findings include prenatal X-ray exposures²⁵, parental recreational drug use ⁴², and several other factors^43-45, correlating with increased risk for RMS (Table 2).

Table 2:

Environmental and other risk factors associated with RMS development

Risk factor	OR (95% CI)	Referencea
Birth defects	2.4 (0.9–6.5)	33
Prenatal X-ray exposure	1.9 (1.1–3.4)	25
Maternal drug use	3.1 (1.4–6.7)	42
Paternal drug use	2.0 (1.3–3.3)	42
Childhood allergies	0.6 (0.4–0.9)	44
Use of fertility medications	0.7 (0.2–2.3)	43
Vaginal bleeding during pregnancy	1.8 (1.1–2.7)	43
Premature birth	2.5 (0.7–8.5)	43
First–degree relative with ERMS	2.4 (1.5–3.9)b	45
First-degree relative with ARMS	1.0 (0.3–3.5)	45
Paternal exposure to Agent Orange	1.7 (0.6–5.4)	234

^aThis table summarizes findings from a series of case-control studies of Intergroup Rhabdomyosarcoma Study III in which environmental exposure and other clinical factors are correlated with risk for RMS development.

^bCombined with data from the Utah Population Database

ARMS, alveolar RMS; ERMS, embryonic RMS; OR, odds ratio; CI, confidence interval; RMS, rhabdomyosarcoma. Ranges that are significant are highlighted by bold.

Knowledge gaps

In contrast to research into osteosarcoma and Ewing sarcoma ^46,47, the epidemiology of RMS has been understudied. Although clusters of cases have been reported in the literature ^48,49, it remains unclear if these patterns are indicative of specific aetiologies or due to chance alone. A molecular basis for the apparent influence of male sex or Hispanic ethnicity on RMS diagnosis also remains unknown. Additional studies are needed to describe the global distribution of RMS across the age spectrum and to identify risk factors for this childhood cancer, as prevention strategies are not well developed or routinely implemented. Future studies should be conducted in a large scale setting because some associations identified in smaller analyses have not been confirmed in independent assessments.

Mechanisms/pathophysiology

The two major subtypes of RMS, ARMS and ERMS, were originally recognized based on light microscopic features. However, the pathogenesis of those two subtypes is distinct, as ARMS tumour cells usually contain a balanced chromosomal translocation generating an oncogenic “fusion protein” that is absent in ERMS. As the fusion protein has considerable biological and clinical implications and not all ARMS cases harbor a fusion protein, most experts feel RMS is better classified as FP and FN disease. Many insights into RMS pathogenesis stemmed initially from analyses of tumor-derived DNA and RNA. Genomics approaches are being complemented by functional approaches to study disease mechanisms in a variety of models (Box 1).

Box 1: Functional genomic insights into RMS.

Functional genomics approaches are increasingly used to explore the importance of genes implicated by next-generation sequencing and to identify new candidate disease drivers that were not apparent based on mutations. These approaches include organism-level model systems and higher throughput, cell culture-based genomics studies.

Cultured cells and genetically-engineered mouse models are used to study fusion positive (FP) and fusion negative (FN) rhabdomyosarcoma (RMS). For example, expression of the alveolar RMS (ARMS) associated PAX3–FOXO1 fusion protein in a variety of cell types, including human fetal skeletal muscle cells ²¹⁷ and mouse mesenchymal stem cells ²²⁴, promotes a malignant in vitro phenotype, which can be increased with other genetic manipulations of tumour suppressors, such as expression of a dominant negative form of p53 or knock-down of INK4A ^50,224. Similarly, stable ectopic expression of oncoproteins RAS^V12G, SV40 T/t Ag, and hTERT in human skeletal muscle progenitor cells fosters the formation of embryonic RMS (ERMS)-like xenografts in immunocompromised mice ²¹⁷.

Mouse models have been generated that conditionally express the mouse Pax3–Foxo1 allele only in skeletal muscle cells⁴⁰. These animals develop ARMS-like tumors at a very low tumor incidence, which increases when bred into p53 or Ink4a/Arf deficient backgrounds⁴⁰.

Several models of FN RMS have been established in mice and zebrafish. A heterozygous knockout of Ptch1 (encoding a receptor for sonic hedgehog) in mice causes a high frequency of RMS²²⁵. This finding suggested that sonic hedgehog deregulation might be sufficient for RMS, which is further evidenced as this pathway is activated in non-myogenic progenitors of RMS ^51,226. This finding also suggests that RMS does not necessarily arise from cells destined to form skeletal muscle. Another FN mouse model was established by broad transgenic expression of Scatter Factor/Hepatocyte Growth Factor (SF/HGF) using the mouse metallothionein I promoter, which leads to high frequency RMS when bred into Cdkn2a (encoding a negative regulator of cell proliferation) knock-out animals ²¹⁸.

A RAS-driven zebrafish model of ERMS has also been established ²¹³. Microarray analysis identified a RAS-induced gene signature in common between the model and human ERMS – an interesting finding as activating mutations in RAS genes are among the most highly recurrent mutations in RMS ^28,29,30. These models should enable discovery of cooperating genetic and gene expression changes in RMS ¹³⁰ and be amenable to high throughput screening approaches.

It should be noted that, although all subtypes of RMS resemble skeletal myoblasts, the cell of origin is not well characterized. Indeed, elegant genetic models indicate that RMS-like tumors can form when certain oncogenic proteins are expressed in cells from the skeletal myoblast lineage ^40,50, but also in non-myogenic cells ⁵¹. It is conceivable that RMS driven by different oncogenic changes or at different anatomic sites may originate in different types of cells that are programmed during tumor formation to express a complement of skeletal myocyte genes.

Fusion-positive RMS

Fusion proteins generated by translocations.

Cytogenetic studies identified recurrent chromosomal translocations, including a frequent t(2;13)(q35;q14) or a variant t(1;13)(p36;q14), in the majority of patients with ARMS^52,53 (Figure 2). These translocations juxtapose PAX3 on chromosome 2 or PAX7 on chromosome 1 with FOXO1 on chromosome 13 ^14,15,54. PAX3 and PAX7 encode highly related members of the paired box transcription factor family, which are expressed in skeletal muscle progenitors, whereas FOXO1 encodes a widely-expressed member of the fork head transcription factor family. The t(2;13) and t(1;13) translocations respectively generate PAX3–FOXO1 and PAX7–FOXO1 fusion genes that are expressed as fusion transcripts and translated into neomorphic fusion proteins. These fusion genes encode chimeric transcription factors containing an N-terminal PAX3 or PAX7 region with an intact DNA binding domain and a carboxyl-terminal FOXO1 region containing an intact transcriptional activation domain. In contrast to wild-type PAX3 and PAX7 proteins, the PAX3–FOXO1 and PAX7–FOXO1 fusion proteins have enhanced transcriptional activity, which is attributed to decreased sensitivity of the FOXO1 transactivation domain to the inhibitory effects of N-terminal PAX3 or PAX7 domains ^55,56. In addition to the functional effects, there is higher expression of PAX3–FOXO1 and PAX7–FOXO1 mRNA and protein relative to the wild-type products ⁵⁷. The high level of PAX3–FOXO1 expression results from a copy number-independent increase in transcription of the fusion gene whereas the high level of PAX7–FOXO1 expression results from increased copy number due to in vivo amplification of the fusion gene⁵⁷.

Figure 2: PAX–FOXO1 fusion gene drives RMS formation.

FP RMS is defined by balanced translocations between PAX3, residing in the Giemsa band 35 on the long (q) arm of chromosome 2 (2q35) or PAX7 on chromosome 1 (1q36) with the FOXO1 gene on chromosome 13 (13q14) generating fusion proteins. These balanced translocations generate two derivative (der) chromosomes (left side panel), only one of which encodes for the PAX3 (or PAX7)-FOXO1 fusion mRNA and protein (right side of panel). These fusion proteins contain the amino terminal portion of either PAX3 or, less commonly, PAX7 and the carboxyl portion of FOXO1. The amino terminus of the fusion protein includes motifs needed for DNA binding from the respective PAX gene, and the carboxyl terminus of the fusion protein is felt to alter the transcriptional activation domain of the oncogenic transcription factor. Note that the alternate derivative chromosomes are not known to contribute to RMS pathogenesis. Chrom, chromosome; FP, fusion positive; RMS rhabdomyosarcoma; der; derivative chromosome; DBD, DNA binding domain; TAD, transcriptional activation domain; PB, paired-box domain; HB, homeobox domain; FH, Forkhead-related domain; FKHR, forkhead homolog in RMS (original designation of FOXO1 gene).

Post-translational modifications.

The expression and/or function of the PAX3–FOXO1 fusion protein, the most well-studied of RMS-associated fusion proteins, is also influenced at the post-translational level. Within the C-terminal FOXO1 region, there are multiple phosphorylation and acetylation sites. Some of these sites, such as AKT kinase-dependent phosphorylation sites at S437 and S500, regulate the subcellular localization and degradation of wild-type FOXO1 but not PAX3–FOXO1 ⁵⁸. However, the stability of the fusion protein is influenced by post-translational modifications at other sites. Expression of the PAX3–FOXO1 fusion protein is stabilized by S503 phosphorylation mediated by the serine/threonine protein kinase PLK1 and by K426 and K429 acetylation mediated by the histone acetyltransferase KAT2B. Interestingly, pharmacological inhibition of PLK1 or KAT2B in ARMS-derived tumor cells results in ubiquitin-mediated degradation of PAX3–FOXO1, an observation which could lead to ultimately lead to a way to therapeutically target this oncogenic fusion protein^59,60. In addition, studies with the pharmacological kinase inhibitor PKC412 revealed a regulatory role for phosphorylation of the N-terminal PAX3 region on DNA binding and transcriptional function of the fusion protein ⁶¹. As multiple phosphorylation sites appear to be involved, the specific proteins mediating these events have not been identified.

Transcriptional and epigenetic effects.

As a transcription factor, the PAX3–FOXO1 fusion protein usually functions in ARMS tumour cells as an activator that increases the expression of downstream target genes by binding to PAX3 binding sites near these genes. ChIP-Seq studies demonstrated that most regions bound by PAX3–FOXO1 are situated >2.5 kb distal to the nearest transcriptional start site and were associated with active enhancer chromatin marks, such as acetylation of histone 3 K27 ^62,63. Furthermore, these regions often contain E-box DNA binding motifs in addition to PAX3 binding sites. PAX3–FOXO1 binds to these regions along with the E-box-specific transcription factor N-Myc (encoded by MYCN), and myogenic basic helix-loop-helix transcription factors, MyoD1 and Myogenin (encoded by MYOD1 and MYOG), and generates super enhancers near a subset of target genes, including ALK (encoding Anaplastic Lymphoma Kinase), FGFR4 (encoding fibroblast Growth Factor Receptor 4), and MYCN, MYOD1 and MYOG. In addition, PAX3–FOXO1 also interacts directly or indirectly with chromatin-related proteins, including chromatin remodeling proteins Bromodomain Containing 4 (BRD4) and the Chromodomain Helix DNA binding protein CHD4 ^63,64. By interacting with these transcription factors and chromatin-related proteins, PAX3–FOXO1 likely reprograms the chromatin landscape and establishes super-enhancers that associate with target gene promoters by three-dimensional looping. The co-dependence of ARMS cells on the fusion protein as well as these other collaborating proteins is demonstrated by the high sensitivity of ARMS cells to siRNAs and small molecules targeting each of these proteins ^63,64.

Oncogenic effects.

The PAX3–FOXO1 and PAX7–FOXO1 fusion proteins function as oncoproteins by dysregulating multiple cellular pathways (Table 3). Following gene transfer into several mammalian cell types, exogenous PAX3–FOXO1 expression is associated with transforming activity, including loss of contact inhibition of proliferation and gain of anchorage independence^65,66. Repression of PAX3–FOXO1 expression in patient-derived human ARMS cell lines with siRNA or shRNA constructs results in decreased proliferation and survival, increased differentiation, and decreased motility and invasion ^67,68. Moreover, a mouse knock-in model that conditionally expresses a Pax3–Foxo1 fusion in cells of the myogenic lineage develops ARMS-like tumors, and tumor susceptibility is greatly increased in animals lacking tumor suppressors encoded by either the p53 or Cdkn2a genes ⁴⁰. The oncogenic effect of these fusion proteins on growth, survival, differentiation and other pathways is mediated through activation of numerous downstream target genes such as the aforementioned ALK, FGFR4, MYCN, and MYOD1 as well as genes encoding the C-X-C motif chemokine receptor 4 (CXCR4) and the MET proto-oncogene receptor tyrosine kinase (MET) ⁶².

Table 3:

Aberrant gene expression in RMS

Cause of aberrant gene expression	Gene	Effect	Invasion/ Migration	Proliferation	Transformation	Survival
PAX3/7–FOXO1 fusion protein	CDH3/P-Cadherin	Upregulation	++	++	−	−
	CNR1	Upregulation	++	−	−	−
	CXCR4	Upregulation	++	++	−	−
	FGFR4	Upregulation	−	++	−	−
		Mutation	−	++	++	−
	IGF2	Upregulation and/or loss of imprinting	−	++	++	++
	MET	Upregulation	++	++	−	++
	MYCN	Upregulation and/or amplification	−	++	++	−
	TFAP2B	Upregulation	++	−	−	++
Somatic mutation	TP53	Loss of Function	−	−	−	++
12q13-15	MDM2	Amplification	−	−	−	++
	CDK4	Amplification	−	++	−	−
13q31-32	miR-17-92	Amplification	−	++	−	++
	GPC5	Amplification	−	++	−	−

++, process is upregulated; −, no effect.

Data from table 3 was originally presented in Ref. ⁵².

Molecular differences between subtypes

RMS-specific fusion genes can be detected in clinical biopsy material by RT-PCR and fluorescent in situ hybridization (FISH) assays. These assays have revealed that 60% of patients with ARMS express PAX3–FOXO1, 20% express PAX7–FOXO1, and 20% are FN ⁶⁹. A small subset of patients with ARMS lack detectable PAX3-FOXO1 or PAX7–FOXO1 fusion proteins, but have novel variants, such as PAX3–FOXO4 or PAX3–NCOA1 (NCOA1 encodes nuclear receptor coactivator 1) ^70,71. The clinical or biological consequences of these variants are not clear. Nucleic acid sequencing has shown that patients with FN ARMS do not express fusion proteins but instead show genetic changes in tumour cells that are similar to ERMS tumors, such as whole chromosome gains, recurrent point mutations, and 11p15.5 allelic loss ^28,30,72-74. In addition, studies of genome-wide mRNA expression revealed that ERMS and FN ARMS tumors have very similar expression profiles, which are distinct from the expression profiles in PAX3–FOXO1-positive and PAX7–FOXO1-positive ARMS tumors ^71,72,75. These studies thus provide genetic evidence for the combination of ERMS and FN ARMS tumors into a single FN RMS subset and the combination of PAX3–FOXO1- and PAX7–FOXO1-positive ARMS tumors into a distinct FP RMS subset.

Aberrant signalling pathways

In FN RMS, various mutations have been identified that largely converge on a limited number of pathways. Interestingly, these pathways are also perturbed in FP RMS through upregulation of downstream targets of the fusion proteins and/or genomic amplification, indicating some commonality in the molecular driving forces in RMS (Figure 3).

Figure 3: Key functional pathways are perturbed in RMS.

Key processes of apoptosis, cell proliferation, cellular differentiation, and epigenetic homeostasis are deregulated by mutation or gene copy-number and/or gene expression alterations in fusion negative (FN) or fusion positive (FP) rhabdomyosarcoma (RMS). In FP RMS, chromosomal translocations result in PAX3–FOXO1 or PAX7–FOXO1 fusion genes. The aberrant PAX3–FOXO1 fusion protein can synergize with loss of p16 or p53 functionality that is associated with CDKN2A gene loss and/or promoter methylation and TP53 mutation. The stability and subcellular localization of the PAX3–FOXO1 protein is dependent on phosphorylation of specific sites and it works in a complex that can include BRD4. The PAX3-FOXO1 containing complex acts as pioneer factor and drives expression of other transcription factors such as MYCN and MYOD1 via super-enhancers that lead to reprogramming of the transcriptional and epigenetic landscape of tumors. The genes encoding MYCN and MYOD1 transcription factors may themselves be genetically amplified or mutated, likely contributing to RMS formation or progression in a subset of cases, respectively. The fusion protein also drives expression of specific receptor tyrosine kinases (RTKs). Overexpression and activating mutations of genes encoding the same RTKs, and mutation of genes encoding downstream signaling components, are seen in FN RMS. Together this leads to frequent activation of PI3K and RAS pathway signaling in FP and FN RMS, which likely contribute to disease pathogenesis by altering cell proliferation, apoptosis, and other metabolic pathways in ways that are not yet precisely defined. Next-generation DNA sequencing and other molecular genetics tools have demonstrated deleterious mutations in genes encoding certain proteins involved in RMS pathogenesis (*). Exactly how these pathways driven RMS pathogenesis is not clear.

Mutations in RAS–PI3K pathway components.

Aberrations in various genes associated with RAS pathway signaling are predominant in FN RMS. Approximately one third of patients with FN RMS are reported to have activating mutations in key components of the RAS pathway, including mutations in NRAS, KRAS, and HRAS. NRAS mutations are predominant in adolescents and KRAS and HRAS mutations are more frequent in infants aged <1 year ^28,74,76. Mutations in genes encoding proteins associated with RAS pathway intracellular signaling, such as protein tyrosine phosphatase, non-receptor type 11 (PTPN11), NF1, the B-Raf proto-oncogene, BRAF (encoding a serine/threonine kinase) and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA, protein is also known as PI3Kα), are also described. Overall, >50% of patients with FN RMS harbor a mutation expected to impact RAS–RAF–MAPK (mitogen-activated protein kinase 1) and/or PI3K–AKT (serine–threonine kinase 1)–mTOR (mammalian target of rapamycin) pathways ^{28,30,74,77,76}. Activation of these pathways in a large fraction of FN RMS is also supported by gene expression analyses ^78-80. These findings are further supported by immunohistochemistry analyses of phosphorylated AKT, MAPK and ribosomal protein S6 kinase B1 (known as S6 Kinase) that show >80% of RMS biopsy samples show activation of the PI3K pathway, with co-activation of the MAPK pathway in over a third of ARMS biopsy samples and nearly half of ERMS biopsy samples⁸¹. Various cell surface receptor tyrosine kinases (RTKs) can signal through these pathways, including RTKs that are induced by the fusion protein ⁶², and these RTKs are implicated in RMS development and progression.

Receptor tyrosine kinase signaling.

Activating mutations in FGFR4 (encoding a fibroblast growth factor receptor), occur in ~7% of patients with FN RMS. The K535 and E550 FGFR mutants activate RAS and STAT (signal transducer and activator of transcription) signaling pathways and induce tumor growth and metastatic behavior in mouse tumor cells expressing the human protein ⁸². In FP RMS, expression of wild-type FGFR4 and FGFR2 is also elevated as these genes are downstream transcriptional targets of PAX3–FOXO1 ^62,63, and they contribute to tumor behavior ^83-85 (Shipley, unpublished). Similarly, insulin growth factor receptor 1 (IGF1R) is highly expressed in FP RMS, occasionally via genomic amplification ⁸⁶. Very frequent high expression of the ligand IGF2 also occurs in RMS, resulting from loss of heterozygosity or loss of imprinting of the 11p15.5 locus that encodes the IGF2 and H19 genes. This molecular lesion is more common in patients with FN RMS ²⁸. IGF signaling is pro-survival and anti-apoptotic in RMS tumour cells and increases tyrosine phosphorylation of the insulin receptor substrate-1 (IRS-1) ⁸⁷. In poor prognosis patients with RMS (that is, those with advanced or metastatic disease), IRS-1 activation appears refractory to normal negative feedback mediated by increased phosphorylated mTOR and S6 Kinase ⁸⁸, and inhibiting IGF1R activity can promote compensatory regulatory activity of the SRC family kinase, YES ⁸⁹. The epidermal growth factor receptor tyrosine kinase ERBB2 is frequently expressed ^90,91 and upregulated with associated MAPK signalling in response to IGF1R inhibition ⁹². Furthermore, knockdown of PIK3CA leads to increased expression of PIK3CA isoforms and elevated RAS pathway signaling in cell line models ⁸¹. Changes in feedback loops and dynamic signaling after targeting specific pathway components suggests that RMS may need to be treated with combination therapeutic strategies

Other RTKs implicated in RMS development include Ephrin receptors ⁹³. High PAX7 expression sustains migration and invasiveness in ERMS cells by upregulating the Ephrin receptors A3 (EPHA3) and A1 (EFNA1) ⁹⁴. Moreover, ERMS cell lines deficient in ephrin receptors EPHA2 and EPHB2 have reduced migratory capacity and are induced to differentiate into a myogenic-like phenotype⁹⁵. Similarly, increased signalling via MET in FN RMS contributes to invasive tumour growth^96,97 and PAX3–FOXO1-driven MET expression in FP RMS promotes motility, mainly through extracellular signal-regulated kinase ERK2 ⁹⁸, and blocks myogenic differentiation ^99,100. Platelet-derived growth factor receptor alpha (PDGFRA) gene expression is driven by the fusion protein in FP RMS, and PDGFRA is occasionally mutated in FN RMS, whereas the PDGFRB is primarily expressed in the vascular stroma of RMS ^28,62,101. Together, PDGFR activity regulates cancer cell stemness, differentiation, senescence, and apoptosis, with the stromal compartment providing a supportive role ¹⁰².

Loss of PTEN, TP53 and CDKN2A.

Mutation or promoter methylation of the tumour suppressor PTEN can occur in FN RMS and negatively regulates PI3K signaling ³⁰. In a recent targeted re-sequencing study of tumour cells from 631 patients with FN RMS, TP53 mutations were identified at a higher incidence of 12% than had been previously reported ²⁸,¹⁰³. High expression of MDM2 occurs in 10% of patients with RMS ^28,30, and negatively regulates p53 (encoded by TP53). In addition, promoter methylation, allelic loss and mutation of tumour suppressor cyclin dependent kinase inhibitor 2A (CDKN2A) is frequent. The finding of p53 pathway disruption or loss of CDKN2A in RMS is concordant with model systems in which these changes are synergistic and critical for increasing penetrance in PAX3–FOXO1 transgenic mouse models (Box 1) ^{30,40,104,105}.

Involvement of developmental pathways.

The Catenin Beta 1 gene, CTNNB1, is commonly mutated in FN RMS^28,30,74, and β-Catenin and other proteins associated with canonical WNT signaling (a key pathway involved in development) are expressed in a high proportion of FN and FP RMS tumors ¹⁰⁶. Notably, inhibition of Wnt Family Member 3a (Wnt3a) protein or Glycogen Synthase Kinase 3 Beta (GSK3B) in cultured RMS cells results in the nuclear translocation and transcriptional activation of β-Catenin in RMS cells, followed by decreased proliferation and the induction of differentiation, which suggests a potential therapeutic approach^106,107. Analyses of primary tumors, cell lines, and mouse models supports other developmental pathways contributing to RMS development and progression including Hedgehog (Box 1) ^51,108-110, Notch ¹¹¹ and Hippo signaling pathways ^79,112,113. Cross-talk between these developmental pathways and RAS signalling ¹¹⁴ creates an integrated signalling network that supports the development of RMS ^111,115.

Myogenic regulation and epigenetics

Mutations in MYOD1 occurring together with mutations in genes of the PI3K–AKT pathway genes define a particularly aggressive form of FN RMS in children and adults ¹¹⁶. Interestingly, DNA binding sites of L122R mutant MYOD1 are similar to those of the MYC proto-oncogene protein¹¹⁷, potentially explaining a switch from differentiation to proliferation. In addition, mutations may lead to decreased MYOD1 binding adjacent to critical myogenic genes, which is thought to contribute to reduced expression of the myogenic program in RMS ¹¹⁸. In FP RMS, the histone methyltransferase KMT1A is highly expressed and associates with MYOD1, thereby adding repressive marks to histone H3K9 residues at MYOD1-regulated target genes, thus blocking differentiation ¹¹⁹. These findings add to our understanding of how MYOD1, which usually promotes the differentiation of skeletal myocytes, contributes to the undifferentiated phenotype of RMS. Similarly, PAX3–FOXO1-driven expression of JARID2 (a regulator of histone methyltransferase complexes) recruits PRC2 (a repressive histone methyltransferase) via EZH2 (the catalytic subunit of PRC2) to the MYOG and MYLH genes, also contributing to failed myogenic differentiation ^120,121. Patients with RMS may have mutations in BCOR that inactivate the BCL6 Corepressor, which interacts with histone deacetylases ^28,122. Mutations also occur in the gene encoding ARID1A, a protein involved in the SWI/SNF chromatin remodelling complex ²⁸. Together with global and specific changes in DNA methylation and histone marks, and expression of microRNAs and other noncoding RNAs ^30,63,123, there is increasing evidence for epigenetic regulation shaping RMS development and progression.

Cellular and physiological processes

From a physiological and cellular level, metastasis is particularly important and often the lethal component of RMS ¹²⁴. Early studies of FP RMS suggested that presence of the PAX3–FOXO1 fusion transcript correlated with greater metastasis risk than PAX7–FOXO1⁶⁹, but that initial finding has not been consistently validated in clinical trials. Certain experimental models implicate the cannabinoid receptor 1 (Cnr1) or the cytoskeleton-associated protein Ezrin as potential mediators of metastasis induced by the PAX3–FOXO1 fusion protein¹²⁵ or the Sine Oculis gene SIX1¹²⁶, respectively. While PAX3-FOXO1 is clearly important in the human disease, whether SIX1 represents a potentially actionable vulnerability or biomarker for metastasis in RMS is not clear. In many epithelial cancers, the epithelial-to-mesenchymal transition (EMT: a complex series of molecular and cellular changes enabling epithelial cells to mobilize during normal organism development or in healing of certain epithelial wounds) plays a critical role to enhance cancer cell mobility and drive metastasis^127,128. As a mesenchymal cancer, it remains unclear whether RMS undergoes an EMT-like process. Interestingly, genes well-known to induce EMT, SNAIL ^129,130 and SNAIL2^131,132, have been implicated as oncogenic drivers for RMS. Expression of SNAIL and SNAIL2 derails the myogenic differentiation program ^129,133, which is associated with cell proliferation arrest. However, the underlying cellular mechanisms for metastasis control in RMS remain to be elucidated.

Evading immune surveillance is also widely-recognized as a key process in human cancer^134-136. The immune landscape of RMS is understudied. Indeed, little is known about interactions between RMS cells and the tumor stroma and non-stromal elements of the tumor microenvironment, including immune cells. Large next generation-sequencing studies of RMS have focused on characterizing tumor cell-intrinsic features, like single nucleotide variants and gene copy-number alterations, not the tumor microenvironment or stroma ^28,29. Similarly, transcriptome profiling identified gene expression signatures correlating with RMS subtype and prognosis ^75,78,137, but did not reveal substantial insight into stromal elements, immune surveillance escape mechanisms, or metastasis. Interestingly, gene expression analysis of RMS occurring on the extremities ¹³⁸ indicated that higher expression of immune genes (for example, β2-microglobulin, complement genes, major histocompatibility complex genes) in the primary tumor correlated with regional lymph node metastasis risk. Defining the tumor stroma and immune cell landscape in RMS may reveal new insights into disease biology. The potential for immune-based therapeutics could still be limited by the relatively low mutational burden in RMS ^28,29. Obviously, the fusion proteins driving FP RMS represent neoantigens that, in principle, could be amenable to immune targeting. That possibility is supported by results from a small pilot study suggesting improved survival for children with FP RMS (and also Ewing sarcoma) who received post-cytoreductive consolidation using dendritic cells pulsed with peptides derived from the relevant fusion protein ¹³⁹.

Diagnosis, screening and prevention

RMS is a global problem and many factors exist that influence delivery of care to childhood cancer patients, including those with RMS (Box 2). The following section focuses on how the disease presents and is managed in what could be construed as an idealized setting.

Box 2: Global Variation in RMS Management.

Little effort has been made to investigate RMS as a global disease. Hints exist that RMS incidence varies across certain ethnic groups, suggesting that population-based genetic variants can influence disease susceptibility and, perhaps disease biology²²⁷. Also, global variations in treatment approaches are likely to exist and influence outcomes, as witnessed between European and North American groups ^1,177. When considering RMS diagnosis and treatment in low-income and middle-income countries, many factors may limit the delivery of effective childhood cancer therapy, including availability and training of physicians, nurses, and other medical staff; laboratory and pathology services; diagnostic imaging capabilities; availability and security of chemotherapy and blood products; and surgical and radiation oncology facilities and expertise ²²⁸. A study of children with RMS and other forms of soft tissue sarcoma in Guatemala highlighted that delayed diagnosis leads to increased numbers of children developing advanced stage disease, and treatment abandonment likely also contributes to poor outcomes²²⁹. Conceivably, rapidly improving capabilities of web-based telecommunications/telemedicine capabilities and “twinning” programs could help to realize improved global outcomes.

Clinical presentation

The diagnosis of RMS has been traditionally based on recognizing the features of skeletal myoblast-like tumor cells using light and, in some cases, electron microscopy, and the use of immunohistochemical (IHC) staining for skeletal muscle proteins^13,140. RMS can arise in virtually any anatomic site, however, the most common sites depend on the histological subtype: ERMS most commonly arises in the head and neck, including the eye socket, or in genitourinary sites; ARMS typically arises at extremity sites, with a smaller fraction arising in the head and/or neck or torso¹⁷. Radiographic or clinical evidence for distant metastatic disease is present in ~20% of children at diagnosis¹. Metastases arise by both lymphatic and hematogenous routes, and spread to lung, bone, and bone marrow are relatively common ¹⁴¹.

Few comprehensive studies exist of presenting signs or symptoms in children with RMS. Signs and symptoms are typically associated with soft tissue mass and are often described as painless masses found in the extremities, or head and/or neck region ^142,143; they can also be associated with signs and symptoms due to mass effect on adjacent organs or neurovascular tissues, or associated with a visible mass protruding from an orifice. For example, a ‘grape-like’ mass in botryoid RMS of the vagina^142,144,145. Orbital primary sites typically present as a unilateral, space occupying lesion with proptosis ¹⁴⁶.

Pathological assessment

The diagnosis of RMS requires the direct analysis of tumor tissue from either an incisional or excisional biopsy or core needle biopsy and subjected to a series of histology and molecular pathology studies. The World Health Organization had recognized three histological variants of RMS – ARMS, ERMS, and pleomorphic RMS – with ARMS and ERMS being the most frequent childhood forms. A recent update from the WHO now includes spindle cell/sclerosing RMS as a distinct entity ^13,147. Morphologically, RMS cells are of heterogeneous shapes ranging from undifferentiated and round cells, ovoid cells, ‘tadpole-like’ cells, spindle-shaped cells and fully differentiated rhabdomyoblasts. ^148,149

As previously mentioned, RMS cells usually display some evidence for skeletal muscle lineage specification and/or differentiation evident by light or electron microscopy and/or IHC or molecular evidence for expression of skeletal muscle gene products like muscle-specific actin and myosin, desmin, myoglobin, Z-band protein, and MyoD1 or myogenin. Notably, a diagnosis of RMS can be made even if only a minority of tumor cells display detectable expression of skeletal muscle proteins. Such a finding reflects the failed terminal differentiation that is central to RMS. However, cellular morphology and architecture must also be considered for accurate diagnosis because myogenic proteins can be expressed in other childhood neoplasms, such as Wilms tumor and malignant triton tumor ¹⁴⁹.

ARMS and ERMS.

ARMS and ERMS can be subdivided further based on histological or molecular features. For example, recognized histological variants include botryoid RMS and spindle cell/sclerosing RMS, which are generally associated with a superior prognosis ¹³. In addition, ARMS can be sub-divided based on the presence or absence of the PAX3–FOXO1 or PAX7–FOXO1 fusion transcript ⁷². ARMS is typically composed of densely packed, small, round cells lining septations that are reminiscent of pulmonary alveoli, whereas ERMS comprises immature “rhabdomyoblasts” in a less dense, stroma-rich background without an alveolar pattern (Figure 4)¹⁵⁰. It should be noted that some RMS specimens can display intra-tumor heterogeneity with a “mixed” phenotype of both alveolar and embryonal features; fusion gene detection in tumor classification is particularly valuable in this context ^151,152.

Figure 4: ERMS and ARMS can be distinguished based on histopathology features.

Representative photomicrographs of embryonal RMS (ERMS; left panels) and alveolar RMS (ARMS; right panels) following staining with hematoxylin and eosin (H&E; top panels) or with primary antibodies to detect Myogenin (middle panels) or Desmin (bottom panels) to mark the skeletal muscle lineage. ARMS often, but not always, displays loosely associated tumor cells in clusters resembling pulmonary alveoli and robust immunohistochemical staining for Myogenin; however, confirmation by analysis of PAX3–FOXO1 or PAX7–FOXO1 fusion is required to confirm FP state. Original magnification: 400x (Image provided by D. Rakheja, University of Texas Southwestern Medical Center)(contact information: dinesh.rakheja@utsouthwestern.edu)

Pleomorphic RMS.

Pleomorphic RMS is a rare adult variant of RMS that has distinct histological features (Box 3), which is similarly composed of cells displaying evidence of skeletal muscle lineage commitment or differentiation. Pathological review of biopsy samples from 38 primary tumors revealed three histological sub-types: classic, round cell, and spindle cell, based on general morphology. The pleomorphic rhabdomyoblasts that define this subtype express a range of skeletal muscle proteins detected by immunohistochemistry, which helps discriminate it from adult pleomorphic sarcoma^23,153. Pleomorphic RMS generally arises in older adults, more commonly in males, and often involving the lower extremities¹⁵⁴. Owing to its rarity, little is known about the pathogenesis of pleomorphic RMS. For example, one report describes very complex cytogenetic and/or chromosomal abnormalities, similar to that seen in children with FN RMS¹⁵⁵. Defining molecular abnormalities are not clear for this disease.

Box 3: Adults with RMS.

Although RMS is typically a childhood cancer, it can rarely occur in adults. Scant information is available on the clinical and biological findings of adult RMS. All studies, however, highlight a poorer outcome than in children, with overall survival in the range of 20–50% ^230,231. Using data from the Surveillance, Epidemiology and End Results (SEER) program, comparing the clinical features and outcome of 1,071 adults (age > 19 years) and 1,529 children (age ≤ 19 years), one group found that adults were more likely to have adverse prognostic variables (alveolar histology, unfavorable anatomic site like the extremity) and confirmed that adults do have a worse prognosis (5-year overall survival: 26.6% versus 60.5% in children), apparently independent of known prognostic variables ²³⁰. Different outcomes may be driven partly by different therapeutic approaches, which is supported by a study analyzing 171 patients aged >18 years with ERMS and ARMS; this team showed a poor 5-year overall survival of 40%, and they noted that only 39% of patients were treated in line with pediatric protocols²³¹. In the patients treated in a manner that parallels childhood RMS therapy, the 5-year overall survival was 61%. In contrast, outcomes for adults with pleomorphic RMS are generally much poorer, with median overall survival of only 12.8 months for those with localized disease ¹⁵⁴.

Although fundamental differences in disease mechanisms may exist between children and adults, these reports suggest that pediatric-style chemotherapy is active in adults with RMS, and adults with RMS may have better outcomes if treated with more intensive therapy. However, barriers to developing adult therapy exist. For example, older adults may simply not tolerate intensive treatments originally developed for children ²⁰³. A lack of physician familiarity with this disease or awareness of clinical trial opportunities in adults with RMS may also contribute. North American RMS studies often had upper age limits of age 50, but adults were rarely enrolled – a general problem for the adolescent and young adult cancer population ²³². Continued efforts to foster communication between adult and pediatric cooperative groups in study trial design and implementation may begin to break down the barriers to management in adults.

Molecular diagnostics

The advent of tools for molecular diagnostics has greatly aided the identification of RMS. In particular, ARMS is more precisely diagnosed as FP RMS based on detecting the presence of PAX–FOXO1 fusion in tumour cells, observed using FISH, or by detecting the fusion transcript by RT-PCR assays^156,157 (Figure 5). Systematic application of molecular diagnostic tests to biopsy samples from patients with pathologically diagnosed ARMS reveals that approximately 20% of these patients are negative for the fusion transcript ¹⁵⁰. This finding is particularly important because FN ARMS has molecular features reminiscent of ERMS and the clinical outcome of children with FN ARMS parallels that of children with ERMS ^158,159.

Figure 5: PAX–FOXO1 translocation can be detected by FISH.

The clinical importance of determining PAX–FOXO1 fusion status in RMS has led to the development of clinical-grade molecular assays, such as fluorescence in situ hybridization (FISH). Here, FOXO1 (13q14) and PAX3 (2q36.1) break-apart probe sets (left and right, respectively) illustrate the splitting of the normally juxtaposed orange and green signals within the neoplastic interphase nuclei of alveolar rhabdomyosarcoma cells, indicating rearrangements of these loci. (Images provided by J. Bridge, University of Nebraska Medical Center)(contact information: jbridge@unmc.edu)

A molecular diagnosis based on fusion status clarifies some confusing issues regarding routine pathology of RMS. For example, the fusion transcript can be detected in many patients with ARMS tumours that have a solid morphology, with relatively few alveolar spaces and in some cases with ‘mixed’ alveolar and embryonal features, clarifying the diagnosis¹⁵⁰. With the exception of pleomorphic RMS in adults, all patients with FN RMS are felt to have an equivalent disease to ERMS. However, spindle/sclerosing RMS and botryoid subtypes of ERMS disease can have distinct clinical features, and may also carry distinct molecular defects, such as MYOD1 mutation. Finally, trisomy of chromosome 8 and loss of heterozygosity (LOH) at 11p15 are also relatively common in FN RMS ^28,29. Because these chromosomal aberrations are found in other childhood cancers (including some patients with FP RMS), they are not routinely employed to make a diagnosis of FN RMS.

The clinical relevance of fusion status over histology underlines the importance of evaluating fusion gene status to complement traditional pathology assessment, which will enable the diagnosis of FP RMS to be made using a molecular assay. A recent study evaluated the potential for FP and FN RMS to be distinguished by a panel of IHC stains, rather than FISH, a more accessible approach for smaller clinical programs. Strong IHC staining for myogenin, AP2β, and NOS-1 can identify FP status while HMGA2 expression is consistent with FN RMS, and the diagnosis may be made using an algorithmic approach¹⁶⁰. On-going COG trials are incorporating fusion gene status into patient risk stratification (https://clinicaltrials.gov/ct2/show/NCT02567435), however, diagnosis only using fusion gene detection has not yet risen to routine practice, nor has the use of IHC staining as a surrogate for evaluating fusion status.

Surveillance and prevention

Surveillance strategies for RMS, like most childhood cancers, are still in early stages of development, and preventive strategies are not clear. In principle, the recognition of genetic syndromes that predispose to RMS (and other cancers) should provide a foundation to focus RMS surveillance strategies. Indeed, at least one report shows the potential value of biochemical and imaging-based surveillance for children with Li-Fraumeni syndrome ¹⁶¹. This research led to a consensus statement advocating for cancer surveillance in children with germline p53 variants ¹⁶², as well as recognition of the need for further study ¹⁶³. Conceivably, advances in next-generation sequencing enabling sensitive detection of circulating, cell-free tumor DNA may lead to affordable and minimally-invasive surveillance for RMS in at-risk populations ¹⁶⁴.

Management

General overview

Over >30 years of study in large, cooperative group clinical trials, the 5-year overall survival of pediatric RMS has improved substantially, such that it now exceeds 70% ^{2,5-7,165-167}(Figure 6a). Notably, management and outcome for adults with RMS considerably differs (Box 3). Several factors contribute to this improved survival including: the use of multifaceted therapies that typically include surgical resection of the primary tumor, ionizing radiation to the primary tumor, and multi-agent, intensive chemotherapy; the development of clinical and pathological staging systems enabling risk-adapted therapy; and the systematic evaluation of new therapeutic approaches in multi-institutional clinical trials conducted on a national or international scale.

Figure 6: Survival in children with RMS.

A. A graph showing Kaplan-Meier plots for proportion of children surviving when treated according to successive Intergroup Rhabdomyosarcoma Study Group (IRS) studies from 1972-1997. Overall survival improves in successive RMS clinical trials. Multiple factors contributed to the improved survival, as detailed in the text. B. Clinical outcomes in data from IRS-III and IRS-IV clinical trials, showing failure free survival (FFS) in children with RMS. Note that survival for Intermediate Risk group represents average of survival for children with Stage 2 or 3, Group III ERMS (73%) and those with Stage 1-3, Group I-III ARMS (65%).

Figure 6 part a reproduced from Ref ²³³. Data in figure 6 part b are from ref ¹⁶⁸.

The three main cooperative groups dedicated to RMS are the COG Soft Tissue Sarcoma Committee in North America ^168,1, the European pediatric Soft Tissue Sarcoma Study Group (EpSSG), which involves many European countries as well as Argentina, Brazil, and Israel ¹⁶⁹, and Cooperative Weichteilsarkom Studiengruppe der GPOH (CWS) group, which includes predominantly German-speaking countries in Europe ¹⁷⁰. Cooperative group trials of different childhood soft tissue sarcomas often include the majority of children with that specific disease. For example, a recent EpSSG study compared the number of children enrolled in EpSSG clinical trials with the expected RMS incidence between 2008–2015 showed that 77% of patients with RMS aged <14 years were enrolled ¹⁶⁹. Notably, the rate of enrollment for older adolescents and young adults was somewhat lower, which highlights an opportunity to focus future trials on this population.

Curing RMS first requires eradication of the gross primary tumor (that is, local disease), which is often accomplished using a combination of surgery and/or external beam ionizing radiation. Curative therapy has also included systemic chemotherapy to eradicate disseminated disease that is assumed or proven to exist in most children with RMS. That most, if not all, patients have disseminated disease is based, in part, on observations from the 1960s and 1970s, including a Children’s Cancer Study Group A (CCSGA) trial in the US showing common regional and distant recurrence in eight of 15 children treated without chemotherapy ¹⁷¹. Further, sensitive molecular biology tools like reverse transcriptase-polymerase chain reaction has been reported to detect RMS cells in peripheral blood or bone marrow in 12 of 16 children with RMS, even those with grossly localized disease ¹⁷². The COG Soft Tissue Sarcoma Committee (and its predecessors, the CCSGA and IRSG) developed and systematically tested a wide variety of approaches to achieve both goals (reviewed in Ref. ^1,168) over more than 40 years, and some differences in approaches have evolved over that time. The current standards of care – where they exist – and on-going research goals in the field are outlined in more detail below.

Risk stratification

A major advancement in RMS management is the capacity define risk groups, based on clinical, pathological and, increasingly, molecular features ^1,173. Improvements in risk stratification have enabled tailored therapy. The IRSG first introduced the concept that RMS disease status can be described in terms of both “stage” and clinical “group” ¹⁶⁸ (Table 4). RMS Stage, from 1 to 4, depends on the anatomic site of the primary tumor (with involvement of the bladder and/or prostate, or the extremities indicating a more advanced stage), tumor size, presence or absence of regional lymph node involvement, and presence or absence of distant metastasis ¹⁷⁴. Clinical Group, from I to IV, applies surgical and/or pathological features, including the degree of resection of localized or regionally-spread tumor and presence or absence of distant metastasis ¹⁷⁵. Recent recognition that molecularly-defined RMS (that is, FP or FN RMS) ^152,158,159 and more detailed consideration of clinical features, including the number of metastatic sites ¹⁴¹, improves our ability to predict outcome, may enable further refined stratification. Further, a number of “metagene” expression signatures may have prognostic value, especially in FN RMS ^137,158. The most robust of these is a 5-gene metagene (MG5) signature, which was first defined as a prognostic variable in FN RMS in a European cohort, and has been validated using additional cohorts, including one from the COG ¹⁷⁶. Although promising, the MG5 signature has not yet been widely-incorporated in clinical practice.

Table 4:

Staging and clinical group classification systems for RMS

Stage	Sites	Invasiveness	Size	Nodes	Metastasis
1	Orbit, H/N (non-PM), GU (non-BP), Biliary	T1 or T2	a or b	N0, N1 or Nx	M0
2	BP, Ext, PM, other	T1 or T2	a	N0 or Nx	M0
3	BP, Ext, PM, other	T1 or T2	a	N1	M0
			b	N0, N1 or Nx
4	Any	T1 or T2	a or b	N0, N1 or Nx	M1
Clinical group	Definition
I	Localized disease, completely resected
II	Total gross resection with evidence for regional spread
II A	Grossly resected tumour with microscopic residual
II B	Involved regional LNs, completely resected with no microscopic residual
II C	Involved regional LNs, grossly resected with microscopic residual
III	Biopsy only or partial resection with gross residual
IV	Distant metastatic disease

Abbreviations: H/N, Head and Neck; PM, parameningeal; BP, bladder/prostate; Ext, extremity; T1, primary tumor confined to anatomic site of origin; T2, primary tumor with extension and/or fixation to surrounding tissue; Size a: primary tumor ≤ 5 cm; Size b: primary tumor > 5 cm; N0, regional nodes not involved; N1, regional nodes involved; Nx, status of regional nodes not known; M0, no distant metastasis; M1, metastasis present (includes positive CSF, pleural or peritoneal cytology).

Risk stratification is used to determine treatment allocation in clinical trials, however, the details of risk stratification differ in North American and European groups. For example, a recent EpSSG protocol (RMS 2005) defined low, standard, high, and very high risk groups for children with RMS, based on tumor histology, anatomic site and degree of surgical resection of the primary tumor, and presence or absence of detectable metastatic disease ¹⁷⁷. By contrast, the most recent series of COG trials (ARST0331, ARST0431, and ARST0531) incorporated only three risk groups ¹ (Table 5; Figure 6). The overlap between the EpSSG high risk and COG intermediate risk groups is particularly noteworthy because children with histologically-defined ARMS and regional lymph node involvement are classified as very high risk by EpSSG but intermediate risk by COG ¹. Thus, developing informatics infrastructure to enable comparisons across these two cooperative groups represents a challenge and an opportunity to establish a global resource for collection, storage, and sharing of RMS clinical trial.

Table 5:

Risk stratification for patients with RMS

COG risk group	Stage	Group	Histology	COG study	Therapy
Low, subset 1	1	I or II	ERMS	ARST0331	VACx4, VAx4; 24 weeks
	1	III (site of primary tumour: orbit)
	2	I or II
Low, subset 2	1	III (site of primary tumour: non-orbit)	ERMS	ARST0331	VACx4, VAx12; 48 weeks
	3	I or II
Intermediate	2 or 3	III	ERMS	ARST0531	VAC vs VAC/VI; 42 weeks
	1, 2, or 3	I, II, or III	ARMS
High	4	IV	ERMS or ARMS	ARST08P1	VI/VDC/IE/VAC with Cixutumumab or Temozolomide

Abbreviations: VAC, vincristine, actinomycin, cyclophosphamide; ab, antibody; VA, vincristine, actinomycin; VI, vincristine, irinotecan; VDC, vincristine, doxorubicin, cyclophosphamide; IE, ifosfamide, etoposide.

Intermediate and high risk disease

Systemic therapy for RMS continues to be based on a foundation of intensive, alkylating-based, multi-agent chemotherapy administered at intervals over 6–9 months. In North America, combination of vincristine, actinomycin D, and cyclophosphamide (known as VAC) is the standard backbone therapy for RMS, whereas in Europe the standard is ifosfamide, vincristine, actinomycin D (known as IVA). It should be noted that a randomized comparison of either VAC or IVA as the initial therapy (followed by VAC for all patients) showed similar outcomes².

The EpSSG RMS 2005 trial evaluated two randomized questions. First, the efficacy and safety of doxorubicin in patients with grossly-localized, but otherwise high risk RMS was studied after an initial pilot study demonstrated activity and safety of IVA plus doxorubicin (IVADo)¹⁷⁸. However, the EpSSG RMS 2005 study failed to show that the addition of doxorubicin to the IVA backbone improved event-free survival ¹⁷⁷. Second, this group studied the efficacy of 24 weeks of maintenance therapy with low-dose, continuous chemotherapy of oral cyclophosphamide and vinorelbine in children with localized RMS who achieved a complete radiographic response after 27 weeks of intensive therapy. Previous evaluation of the maintenance therapy in a pilot study had demonstrated activity in patients with recurrent RMS ¹⁷⁹. In the EpSSG RMS 2005 trial, the addition of maintenance therapy improved overall survival (87.3% in maintenance arm vs 77.4% in standard treatment arm at three-years post randomization, p=0.011), with a marginal improvement in disease-free survival (78.4% vs 72.3%, p=0.061) ¹⁸⁰.

In North America, the COG conducted trials to test the addition of a camptothecin drug to the VAC backbone in randomized studies of children with intermediate risk disease. The D9803 trial, conducted between 1999–2005, failed to show improved event free survival with the addition of topotecan ⁵. The subsequent ARST0531 trial compared standard VAC versus VAC alternating with vincristine-irinotecan, based on the particularly high activity of vincristine-irinotecan in a Phase II “window” study ¹⁸¹; and the 4-year event free survival and overall survival were similar in the two arms ¹⁸². Presently, the VAC backbone for RMS therapy remains a commonly accepted, North American standard for those with intermediate risk disease, whereas IVA is the European standard for those with localized disease, which includes patients similar to the COG intermediate risk stratum ^177,183 (Table 5).

It should be emphasized that the outcome for children with metastatic disease is very poor, with overall survival rates at 3 years of ~25–30%, despite intensive systemic therapy, even therapy including high dose chemotherapy and autologous stem cell rescue ^1,141,184. In a departure from the traditional approach, the COG recently conducted a Phase II study (ARST0431) testing whether an “interval compression” strategy, in which the interval between chemotherapy doses was decreased, utilizing doxorubicin, ifosfamide, etoposide, and irinotecan to the VAC backbone. ARST0431 showed an improved event free survival for a subset of children with metastatic RMS as compared to historical control individuals¹⁸⁵.

Low risk disease

In contrast to children with metastatic disease, outcomes are excellent for children with low risk disease, generally considered to be those with localized, histologically confirmed ERMS (in favorable anatomic sites; localized and grossly resected ERMS; and ERMS localized to the orbit only (Table 4) ¹⁷³. The most recent low risk study in the COG, ARST0331, investigated two strategies to reduce the burden of therapy without compromising survival. In one subset of patients with Stage 1 or 2–Group I or II ERMS, or Stage 1–Group III orbital ERMS, therapy was shortened to 24 weeks and total cyclophosphamide dose was decreased ¹⁸⁶. This less intensive therapy resulted in excellent three year failure free survival (survival without relapse of disease) and overall survival (89% and 98%, respectively). The second subset of patients included those with Stage 1–Group III, non-orbital or Stage 3–Group I or II ERMS; in these patients total cyclophosphamide dose was decreased but VA chemotherapy was kept at a standard duration of 48 weeks. Notably, radiation therapy was also omitted for female patients with vaginal tumors achieving a complete response with or without surgical resection ¹⁸⁷. Although the three-year overall survival was still very good (92%), the failure free survival was 70% overall and 57% for girls with genital tract tumors. COG investigators concluded that these failure free survival rates are sub-optimal, if the primary goal is to prevent disease recurrence and avoid the even more intensive therapy likely needed to achieve ultimate cure.

It is worth noting that the ‘philosophy’ in the European cooperative groups has been primarily on reducing morbidities associated with local control while retaining excellent overall survival. As such, a higher incidence of local disease recurrence has often been tolerated, with the understanding that a more intensive therapy may need to be employed at recurrence, but that the majority of individuals would not be exposed to that therapy ⁶. Studying the relative benefits and costs of the North American and European approaches would be valuable, especially for those in which chance of survival is high, therefore, concern for acute and late toxicities and cost effectiveness become more important.

Special management considerations

Local disease control.

Local control of RMS depends on surgical resection and/or use of ionizing radiation. RMS represents a cancer that is initially sensitive to cytotoxic chemotherapy and radiation, therefore surgical approaches are generally limited to those that are not form- or function-compromising ^188,189. Nevertheless, the improved survival seen in patients with Group I and II versus Group III disease (Figure 6b) supports the importance of primary tumor resection when possible ^3,175,190. Historically, ionizing radiation was classified a crucial adjunct to surgical resection to optimize local disease control. However, in current practice, the dose is adjusted based on degree of previous surgical resection to reduce radiation-related sequelae, such as skeletal muscle and/or soft tissue changes, joint stiffness, axial and appendicular skeletal growth problems, and secondary malignancy^191-193. In European trials, radiation therapy is omitted in certain circumstances in an attempt to spare the majority of patients from adverse treatment effects as long as effective salvage therapy is available.

RMS in very young children.

Children aged <3 years, especially infants up to 12 months old, pose a management challenge because their outcome is often worse than in older children^194,195. Reports suggest that the worse outcome might arise in part from hesitancy to offer the same local disease control or chemotherapy dose intensity that is typically applied to older children owing to toxicity concerns. Also conceivable is the fact that the pathophysiology of RMS may differ according to age. Indeed, a recent report showed variant gene fusions involving VGLL2 fusion to either CITED2 or NCOA2 in infants with sclerosing/spindle cell RMS ¹⁹⁶. As next generation sequencing analyses move forward, correlation of molecular genotype with clinical prognostic features, like anatomic site, tumor stage and clinical group, and age, may provide additional evidence to support this possibility.

Management of recurrent RMS

Recurrent RMS has a very poor prognosis, and a standard of care has not been widely accepted. Overall survival following RMS relapse depends on several factors including the stage of disease at original diagnosis, the site of relapse, and tumor histology. In general, patients undergoing more intensive therapy at original diagnosis had worse outcomes following relapse than patients receiving less intensive therapy at the time of original diagnosis. For example, individuals with relapse after treatment for Stage 1–Group I ERMS disease displayed a 5-year event-free survival after relapse of 52% compared to ~20% for those with Stage 3–Group III disease and ~12% for those with Stage 4 disease at original diagnosis¹⁹⁷. Also, children originally diagnosed with Stage 1–Group I disease who had a local recurrence also had a better prognosis than those with regional or distant recurrence¹⁹⁷. Unfortunately, post-relapse treatment data were not included in this analysis.

Given the bleak outlook, cooperative groups in North America have been studying novel therapeutic approaches in children with relapsed RMS (and other sarcomas). Phase II trials of promising agents, such as the anti-IGF1 receptor antibody R1507 ¹⁹⁸ and the multi-RTK inhibitor sorafenib ¹⁹⁹, were recently conducted within the COG, but unfortunately neither study showed evidence for significant, single-agent activity. The addition of the mTOR inhibitor temsirolimus to the anti-IGF1 receptor antibody cixutumumab also failed to significantly improve outcomes despite promising preclinical data ²⁰⁰. The most recent COG trial for relapsed RMS (ARST0921) employed a backbone of vinorelbine and cyclophosphamide, taking a cue from a previous European study showing these agents had some activity as low-dose, maintenance therapies¹⁷⁹. To that backbone, ARST0921 randomized the addition of either the vascular endothelial growth factor inhibitor bevacizumab or temsirolimus in a Phase II pilot study to select one of the two agents for further study ¹⁶⁸. This pilot study showed relatively more benefit from temsirolimus²⁰¹ and informed the COG Soft Tissue Sarcoma Committee’s decision to study it in a randomized, Phase III study of children with intermediate risk RMS (ARST1431). Because overall survival was still poor for those children with relapsed RMS, despite the use of temsirolimus, investigators continue to study new therapeutic approaches in children with relapsed RMS.

Quality of life

For almost 40 years, the focus of North American and European clinical trials groups has been on improving survival. Some consideration has been given to Quality of Life (QOL) issues, such as the general philosophy to avoid form-compromising or function-compromising surgical interventions, however, few QOL data have been collected on those trials. As the majority of children with RMS will be long-term, disease-free survivors, greater consideration should be given to QOL data during and after RMS therapy.

Children with cancer are known to experience high levels of suffering and ongoing symptom burden related to their cancer and its treatment ²⁰². Toxicity reporting in pediatric cooperative group studies relies on symptom descriptions documented by health-care providers, and then extracted from medical records by researchers. As such, these data may not reflect the experiences of patients. Prospective collection of patient-reported outcomes (PROs) could be used to explore symptom experience by children, from diagnosis and throughout initial therapy. Owing to the wide range of patient ages, and the widely-ranging sites of disease collection of PROs is a challenge with current clinical trial models.

Despite the historical challenges, some studies suggest progress in defining symptom and toxicity data and in examining age-based comparisons. For example, on the COG ARST0431 study of children with high-risk RMS, adolescents were less likely to complete therapy (63% of individuals aged greater than 13 years vs. 76% of children aged 13 or less) and more likely to have unplanned dose modifications outside of protocol guidelines (23% vs. 2.7%) compared to younger children²⁰³. Furthermore, nausea and vomiting (17% vs. 4%) and pain (20% vs. 6%) were more likely to be reported in adolescents compared to younger patients²⁰³. These findings should be interpreted with caution, as symptom reporting may be more effective in adolescents than younger children. It remains unclear whether PRO collection will overcome that obstacle. In settings where clinical balance exists, differences in PROs may help guide treatment decisions for an individual patient.

QOL assessments in survivors of RMS therapy are important, yet knowledge gaps still exist. Genitourinary and sexual and/or reproductive health are two areas where issues may arise in this population, yet those QOL data are mostly from small series studies. For example, one study of the effects of proton therapy in bladder and/or prostate disease was described in only five children cured of their disease with problems ranging from none to enuresis ²⁰⁴. In another small study of four patients >14 years of age and previously treated with multimodality therapy for prostate or bladder RMS, sexual function was assessed using a modified International Erectile Function Index and found that two of the four patients had moderate or good quality scores each, respectively, after pelvic radiation therapy and cystectomy ²⁰⁵. In a study of 13 patients (median age 20 years) who had survived pelvic RMS, erectile dysfunction (assessed by Self-Estimation Index of Erectile Function-No Sexual Intercourse (SIEF-NS) and the Erection Hardness Scale) was present in ten men, three of whom had radiation therapy, whereas three men without radiation therapy had no evidence of erectile dysfunction ²⁰⁶. In a separate study, 13 of 18 survivors who previously underwent partial cystectomy and/or partial prostatectomy, followed by low dose-rate interstitial brachytherapy, were found to have normal QOL, assessed by a questionnaire derived from the International Workshop on bladder and/or prostate RMS ²⁰⁷. In this study, although all pubertal patients reported having normal erections, the validated SIEF-NS tool was not used. In the largest review to date, medical records of 164 individuals who had survived bladder and/or prostate RMS were retrospectively reviewed to identify 44 who had undergone partial urinary bladder cystectomy and had medical record data regarding urinary continence. Of these 44 patients, 12 were described to have had urinary incontinence, frequency, or urgency²⁰⁸. How best to target ionizing radiation to large fields, such as the pelvis for those with bladder–prostate RMS, continues to be highly debated ²⁰⁹. If survival is similar with and without radiation therapy ^138,210, prospective collection of data on bladder and sexual function becomes particularly important in order to guide treatment decisions in those likely to be cured of their disease.

Outlook

Basic research

Over the last thirty years, our understanding of the pathophysiology of RMS has become increasingly sophisticated, and these insights have enabled more robust clinical diagnostics and prognostic assessments, which have contributed to the development of more precise therapeutic approaches. These intensive or experimental approaches are aimed at improving the outcome of patients with a poor prognosis and also attempt to reduce harsh acute and lasting treatment-associated effects in individuals likely to be cured by standard approaches.

The next step toward precision medicine entails the development of robust, objective and accessible biomarkers that are highly predictive of response to targeted therapy. One major challenge for RMS is highlighted above: the paucity of highly-recurrent and targetable protein coding genes, especially in fusion-driven RMS ^28-30. Current efforts for targeted re-sequencing of primary RMS biopsy samples will more precisely define the frequency of previously-identified somatic variants; correlation of these variants with clinical features, such as age, anatomic site, and outcome, should lead to better understanding of tumor biology and enable better risk stratification. However, it seems likely that integrative computational analyses, especially those considering gene expression changes driven by epigenetic reprogramming, will be required to find targetable oncogenic drivers that were not unmasked by nucleic acid sequencing.

In parallel, as the field progresses more clearly into a post-genomics era (Box 1), functional approaches and high-throughput screening also represent important laboratory-based paths toward better therapy. CRISPR–Cas9-based gene editing provides an opportunity for targeted (for example, kinome-wide) or completely unbiased searches for vulnerabilities in RMS ²¹¹, especially if utilized in well-characterized patient-derived xenograft models that are increasingly available ²¹². Organism-based models in zebrafish ^213,214 and drosophila ²¹⁵ are particularly intriguing ways to conduct high throughput chemical or genetic screens for targetable cancer drivers, especially for fusion oncogene-driven disease. Those non-mammalian systems have potential pitfalls, but findings there could be confirmed in the elegant mammalian models already employed (Box 1)^40,216-218.

Another incompletely evaluated approach toward “personalized” RMS therapy includes immunotherapy. Immunotherapy in which the patient’s dendritic cells are purified, exposed in vitro to neoantigens from the patient’s tumor, and returned to the patient has shown some promise for high risk sarcoma ^139,219. However, because immune checkpoint inhibitors such as ipilimumab ²²⁰ have demonstrated little single-agent activity in RMS, research should focus on identifying predictive biomarkers. In addition, researchers should investigate combination therapies, including coupling checkpoint blockade with ionizing radiation to increase neoantigen presentation and have a potential abscopal effect on systemic disease ²²¹.

Clinical and translational research

Clinical and translational research efforts should continue to enable the development of personalized medical approaches. One short term goal should be to prospectively validate improved prognostic signatures, such as prospectively validating the 5-gene metagene signature, MG5, which has already been applied to several independent, retrospective cohorts ^158,176. Moreover, efforts should be made to develop an MG5 score that could be used in treatment assignment for individual patients with RMS to further aid in risk stratification. In addition, translational studies should focus on whether prognostic biomarker signatures can be enhanced by additional molecular characteristics, such as presence or absence of RAS gene mutations. These mutations are relatively common and potentially a marker for more aggressive disease course in those with FN RMS ²⁹.

Another direction for future research should be into molecularly targeted therapies, which may further improve outcomes, especially in children with metastatic or recurrent disease. The outcomes for these children remain bleak, and numerous attempts to intensify therapy by the addition of new cytotoxic agents or by interval compression have failed. One on-going COG trial (ARST1431) for children with intermediate risk disease is a randomized, Phase III study, adding the mTOR inhibitor temsirolimus to the VAC/VI therapy backbone used in North America. This trial is based on a previous study that found that the addition of temsirolimus to vinorelbine and cyclophosphamide improved survival in children with relapsed RMS compared to the addition of bevacizumab to vinorelbine and cyclophosphamide²⁰¹. As new, molecularly-targeted therapies are identified, they could be studied in the context of a ‘basket’, such as the Pediatric MATCH trial, supported in North America by the COG and the National Cancer Institute ²²². This ‘basket’ trial provides a mechanism to test a number of agents – such as larotrectinib, selumetinib, and pablociblib, targeting TRK, MEK, and CDK4/6, respectively – in parallel and in a number of different types of tumors, including RMS. This type of trial may identify an agent active against RMS, which could then be pursued in a more focused study.

Finally, effort should be devoted to building improved informatics systems, to better capture, assimilate, and curate the ever-increasing quantity of clinical data arising from cooperative group studies. Moreover, these clinical data can be paired with banked biopsy samples, pathology descriptions and/or actual digital photomicrographic images of pathology specimens, radiographic images, and molecular genetic, epigenetic and proteomic data could greatly facilitate efforts to improve outcomes for children with RMS. Building such a data commons for childhood cancer – or even just for RMS – could enable clinical and translational research discovery in many ways, especially by taking advantage of ever increasing artificial intelligence capabilities ²²³. Extending this type of effort across North America and Europe will greatly enable trans-Atlantic (or even global) analyses of what will likely become progressively smaller, clinically and molecularly-defined RMS subgroups. In addition, expanding the scope of such work to include QOL and PRO analyses may be progressively more important as the likelihood of patient survival continues to improve.