Genomic Classification and Clinical Outcome in Rhabdomyosarcoma: A Report From an International Consortium

Abstract

Purpose: Rhabdomyosarcoma is the most common soft tissue sarcoma of childhood. Despite aggressive therapy, the 5-year survival rate for patients with metastatic or recurrent disease remains poor, and beyond PAX-FOXO1 fusion status, no genomic markers are available for risk stratification. We present an international consortium study designed to determine the incidence of driver mutations and their association with clinical outcome.

Patients and methods: Tumor samples collected from patients enrolled on Children's Oncology Group trials (1998-2017) and UK patients enrolled on malignant mesenchymal tumor and RMS2005 (1995-2016) trials were subjected to custom-capture sequencing. Mutations, indels, gene deletions, and amplifications were identified, and survival analysis was performed.

Results: DNA from 641 patients was suitable for analyses. A median of one mutation was found per tumor. In FOXO1 fusion-negative cases, mutation of any RAS pathway member was found in > 50% of cases, and 21% had no putative driver mutation identified. BCOR (15%), NF1 (15%), and TP53 (13%) mutations were found at a higher incidence than previously reported and TP53 mutations were associated with worse outcomes in both fusion-negative and FOXO1 fusion-positive cases. Interestingly, mutations in RAS isoforms predominated in infants < 1 year (64% of cases). Mutation of MYOD1 was associated with histologic patterns beyond those previously described, older age, head and neck primary site, and a dismal survival. Finally, we provide a searchable companion database (ClinOmics), containing all genomic variants, and clinical annotation including survival data.

Conclusion: This is the largest genomic characterization of clinically annotated rhabdomyosarcoma tumors to date and provides prognostic genetic features that refine risk stratification and will be incorporated into prospective trials.

FIG 1.

Mutations summarized by anatomic location and co-occurrence. (A) Summary of mutations by occurrence within defined risk groups (low, intermediate, or high) or by anatomic location. The reported value is a percentage of the number of cases with a mutation in that gene within each group. (B) Co-occurrence of mutated genes reported as absolute number of cases. GU, genitourinary.

FIG 2.

RAS isoform mutations. (A) Oncoprint of mutations observed in infants < 1 year old (n = 25) showed an enrichment for RAS mutations. (B) Distribution of RAS isoform mutations by age with a distinct peak of HRAS mutant cases discovered in the infant population. (C) HRAS mutations were frequently found to have a higher VAF indicating that the mutation occurred before a loss-of-heterozygosity event on chromosome 11p. VAF, variant allele frequency.

FIG 3.

TP53 mutations and association with survival. (A) KM analysis of EFS within the COG FN cohort (n = 275) by the presence of a TP53 Mut or absence of a TP53 lesion (TP53 WT). (B) KM analysis of EFS within the COG FN cohort by TP53 status and RMS risk category. Total case numbers: low, n = 124; intermediate, n = 126; high, n = 35. (C) KM analysis of EFS within the UK FN cohort (n = 240) by the presence of a TP53 Mut or absence of a TP53 lesion (TP53 WT). (D) KM analysis of EFS within the UK FN cohort by TP53 status and RMS risk category. Total case numbers: low, n = 96; intermediate, n = 113; high, n = 25. Presented P values are log-rank and BH adj. HR with 95% CI. BH adj, Benjamini-Hochberg–adjusted; COG, Children's Oncology Group; EFS, event-free survival; FN, fusion-negative; HR, hazard ratio; KM, Kaplan-Meier; RMS, rhabdomyosarcoma; TP53 Mut, TP53 mutation; TP53 WT, TP53 wild type.

FIG 4.

MYOD1 mutations and survival. (A) All tumors with a mutation in MYOD1 within the entire sequenced cohort (N = 641) are shown to visualize co-occurrence of MYDO1 mutations with other genes. All MYOD1 cases were observed within FN cases. (B) Histology of MYOD1 mutant cases demonstrated the frequent presence of sclerosing or spindle morphology (case 1 and case 2). Cases 3 and 4 show that some cases demonstrated areas more typical of dense embryonal histology. (C) KM analysis of EFS within the COG FN cohort (n = 275) by the presence of a MYOD1 Mut or absence of a MYDO1 lesion (MYOD1 WT). (D) KM analysis of EFS within the COG FN cohort by MYOD1 status and RMS risk category. Total case numbers: low, n = 124; intermediate, n = 126; high, n = 35. (E) KM analysis of EFS within the UK FN cohort (n = 240) by the presence of a MYOD1 Mut or absence of a MYDO1 lesion (MYOD1 WT). (F) KM analysis of EFS within the UK FN cohort by MYOD1 status and RMS risk category. Total case numbers: low, n = 96; intermediate, n = 113; high, n = 25. Presented P values are log-rank and BH adj. HR with 95% CI. BH adj, Benjamini-Hochberg–adjusted; COG, Children's Oncology Group; EFS, event-free survival; FN, fusion-negative; HR, hazard ratio; KM, Kaplan-Meier; MYOD1 Mut, MYOD1 mutation; MYOD1 WT, MYOD1 wild type; RMS, rhabdomyosarcoma.

FIG A1.

Histogram presentation of the age distribution of cases within the cohort (N = 641).

FIG A2.

Survival analysis on the basis of number of called mutations. (A) All FN samples (n = 515) were distributed into groups having no observed mutations (black), one mutation (red), or two or more genes mutated (blue). (B) Kaplan-Meier analysis of EFS from the COG and UK FN cohorts on the basis of the number of observed mutations within a tumor. The COG cohort (n = 275) had 49 patients with no observed mutation, 113 patients with one observed mutation, and 113 patients with two or more observed mutations. The UK cohort (n = 240) had 60 patients with no observed mutation, 100 patients with one observed mutation, and 80 patients with two or more observed mutations. (C) Pairwise testing of the significance of gene-gene interactions was performed to identify interactions that occurred at higher (red and purple) or lower (green) frequencies than expected. COG, Children's Oncology Group; EFS, event-free survival; FN, fusion-negative; HR, hazard ratio.

FIG A3.

CDK4, MYCN, and TP53 mutations in FP RMS. (A) Survival analysis for all three genes within the COG and UK cohorts with and without risk stratification. The log-rank and BH adj P value are presented. (B) Kaplan-Meier analysis of EFS and OS in FP cases within the COG cohort (n = 69) and UK cohort (n = 57) for TP53 Mut or WT. COG cohort has three TP53 mutant cases and UK cohort has two TP53 mutant cases. BH adj, Benjamini-Hochberg–adjusted; COG, Children's Oncology Group; EFS, event-free survival; FP, fusion-positive; HR, hazard ratio; Mut, mutated; OS, overall survival; RMS, rhabdomyosarcoma; WT, wild type.

FIG A4.

RAS isoform and RAS pathway analysis. (A) RAS isoforms and pathway member P values for both EFS and OS. The log-rank P value and the BH adj value are presented. BH adj, Benjamini-Hochberg–adjusted; COG, Children's Oncology Group; EFS, event-free survival; OS, overall survival.

FIG A5.

RAS codon and amino acid usage summary. Absolute number of cases with a mutation in the specified codon by amino acid change and isoform (NRAS, blue; HRAS, red; KRAS, green).

FIG A6.

TP53 mutations. (A) Association of TP53 mutations with other gene mutations across the entire rhabdomyosarcoms cohort. (B) Location and number of mutations across TP53 protein.

FIG A7.

CDKN2A and association with survival. (A) EFS and OS by CDKN2A deletion in the COG cohort (blue) and the UK cohort (orange). (B) EFS and OS by CDKN2A status after removal of any cases with a co-occurring mutation of MYOD1: COG cohort (blue) and UK cohort (orange). (C) EFS within the COG cohort demonstrated that the observed effect of CDKN2A alteration was largely within the intermediate-risk group. BH adj, Benjamini-Hochberg–adjusted; COG, Children's Oncology Group; EFS, event-free survival; HR, hazard ratio; Mut, mutated; OS, overall survival; WT, wild type.