Detection of pretreatment circulating tumor DNA as a biomarker of poor outcome in intermediate-risk rhabdomyosarcoma

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children, representing about 3% of pediatric malignancies. Approximately 350 new cases are diagnosed in children and adolescents per year in the United States with almost two-thirds of cases presenting in patients younger than 6 years of age with another peak in adolescents (1). The tumor can occur in any site of the body. There are two main and distinct histologic subtypes of RMS, alveolar and embryonal. The alveolar subtype is often associated with recurring translocations involving the PAX3 or PAX7 gene on chromosome 2 or 1 with the FOXO1 gene on chromosome 13. Because not all RMS with features of alveolar subtype histology harbor these translocations and behave more like the favorable embryonal subtype, the disease classification of RMS has been further refined to FOXO1 fusion-positive RMS (FP-RMS) and FOXO1 fusion-negative RMS (FN-RMS) (2,3). The treatment for RMS is multimodal, including chemotherapy, surgery and/or radiation therapy, and is tailored based on risk group assignment. The Children’s Oncology Group risk group assignments are designated as very low, low, intermediate, and high and are based on clinical, pathological, and more recently molecular features. Patients with very low or low risk disease have excellent outcomes of over 90% survival. Those with high-risk disease have a dismal prognosis of less than 20% survival. Patients with intermediate-risk (IR) disease represent the largest and most heterogenous group with survival ranging from 50–70% (4,5).

The article by Abbou et al. (6) published in the Journal of Clinical Oncology entitled “Circulating Tumor DNA Is Prognostic in Intermediate-Risk Rhabdomyosarcoma: A Report From the Children’s Oncology Group” explores the detection of circulating tumor DNA (ctDNA) in pretreatment serum samples in patients with newly diagnosed IR RMS as a potential biomarker of prognosis in IR RMS, including FP-RMS and FN-RMS. FP-RMS is defined by a PAX3/7 gene fusion and less frequent copy number alterations (CNAs) and FN-RMS is defined by aneuploidy in nearly every tumor, and single nucleotide variants (SNVs) in a small number of genes (7). Due to this heterogeneity of molecular alterations, the authors used two methods, ultralow passage whole genome sequencing (ULP-WGS) for detecting CNAs and an RMS-specific hybrid capture assay (Rhabdo-Seq) to identify translocations and SNVs in 22 RMS-relevant genes. The lower level of detection of ctDNA for these assays is reported to be 3% of the total of cell-free DNA for ULP-WGS and 0.4% for the Rhabdo-Seq assay.

A total of 124 patient samples from patients enrolled on the Children’s Oncology Group biology study D9902 who had clinical characteristics similar to patients with IR RMS treated on the Children’s Oncology Group (COG) trial ARST0531 (8) were analyzed, 75 from patients with FN-RMS and 49 from FP-RMS. Tumor tissue was available for 69 and 35 patients, respectively. Tumor tissue testing is relevant for studies evaluating ctDNA because in general, if the tumor does not have CNAs, then ULP-WGS is uninformative. Similarly, if the specific SNVs or translocations assessed by Rhabdo-Seq are not present in the tumor, the assay is uninformative. However, as with the current study, there are circumstances when ctDNA may be detectable when the tumor was not informative. This situation is likely due to the heterogeneity of the tumor, such that the molecular abnormality may not have been present or present at below the assay level of detection in the area of the tumor that was sampled for testing. The yield of potentially detecting a more comprehensive assessment of the genetic landscape of a tumor may be higher if multiple sections from histologically diverse are included for assessment.

In patients with FN-RMS (n=75), the authors report the detection of ctDNA in 23 patients (31%) by either assay, 13 patients (17%) by ULP-WGS and 18 patients (24%) by Rhabdo-Seq. Molecular profiling of the available tumor tissue in FN-RMS showed detectable CNAs in all 69 tumors tested by ULP-WGS, and SNVs in one or more targeted genes in 40 of 57 tumors (70%) by Rhabdo-Seq. In patients with FP-RMS (n=49), ctDNA was detected in 28 patients (57%) by either assay, 8 patients (16%) by ULP-WGS and in 27 patients (55%) by Rhabdo-Seq. Molecular profiling of the available tumor tissue in the FP-RMS cases revealed segmental CNAs in 25 of 35 tumors (71%) detected by ULP-WGS and a PAX/FOXO1 translocation in 28 of 30 tumors (93%). Two patients were reported to have FOXO1 fusion by fluorescence in situ hybridization, but the authors were unable to confirm this finding.

The authors next assessed whether the detection of ctDNA in pretreatment samples was correlated with clinicopathologic features and outcome. For the entire cohort of IR-RMS, the detection of ctDNA was an independent prognostic factor for poor 5-year event-free survival and overall survival. In the FN-RMS patients, detection of ctDNA was significantly correlated in univariable analysis with disease stage, tumor size and poor outcome. In multivariable analysis, detection of ctDNA was an independent prognostic factor for outcome for this cohort. For FP-RMS patients, pretreatment unresectable tumors (Clinical Group III), T2 stage (invasiveness) and poor outcome were associated with detection of ctDNA. However, unlike FN-RMS, detection of ctDNA was not an independent prognostic factor for outcome in multivariable analysis. The smaller number of patients in the FP-RMS cohort may be one reason for this finding. Another reason could be that the higher sensitivity of Rhabdo-Seq assay to detect the ctDNA, thus fusion gene, suggesting that high risk clinical features are as good at predicting outcome as detection of ctDNA.

Although reports are emerging of assessing ctDNA in RMS (9-12), the study by Abbou et al. is one of the first publications to report on ctDNA detection at diagnosis in a large cohort of RMS patients with localized disease. The findings suggest that the detection of pretreatment ctDNA, at least in the IR FN-RMS, may be a useful biomarker to augment therapy for this group of patients. Prior to implementing this strategy in clinical practice or clinical trials, there are several important limitations of the study that should be considered. First, the findings are based on analysis of samples collected retrospectively in a cohort of patients that were not uniformly treated and not all patients had corresponding tumor samples for analysis. Validation of these findings in a prospective study with a larger cohort of patients is warranted. Second, the technology for detecting ctDNA, including the way specimens are collected and processed, is continuing to evolve. As the sensitivity of these methods evolve, it is possible that most patients will have detectable ctDNA in the blood and that the presence of ctDNA alone may no longer have prognostic significance. Perhaps the addition of methylation status, the presence of circulating tumor cells and assays based on RNA or protein extracted from extracellular vesicles will provide better biomarkers from liquid biopsies. As the authors propose, perhaps the promise of liquid biopsies in RMS will not only be in refining risk-stratification but also in monitoring the kinetics of the ctDNA during therapy, both to assess efficacy, detect early relapse and resistance, and inform duration of therapy as has been shown in adult cancers (13-15) and being explored in other pediatric tumors, including sarcomas (16-20).

Despite the unanswered questions, the authors should be commended for their leading efforts in evaluating ctDNA in RMS as well as other pediatric solid tumors (21-24). We eagerly await the results of the analysis on the serial, prospective collection of samples from patients enrolled on the recently completed COG ARST1431 trial for IR RMS. In ARST1431, patients with IR RMS were randomized to treatment with or without temsirolimus with 42 weeks of vincristine, dactinomycin and cyclophosphamide alternating with vincristine and irinotecan with local control at week 13 followed by 24 weeks (6 cycles) of maintenance therapy with vinorelbine and cyclophosphamide. Liquid biopsy samples were collected at 6 timepoints and at the time of relapse, if applicable, with the goal of estimating the frequency of patients with detectable ctDNA at diagnosis and subsequent time-points.

As we consider incorporating detection of ctDNA into clinical practice in the context of the results of these studies, attention should be paid to the generalizability of the methodologies with efforts toward standardization. As an example, we and others have developed plasma-based liquid biopsy assays for pediatric solid tumors (10,11,25,26). It remains to be seen if the sensitivity and specificity of the results are comparable across platforms. Challenges in feasibility of performing multiple assays and accessibility will need to be addressed as well as the turnaround time for the results.

In summary, ctDNA as a form of liquid biopsy is emerging as a powerful noninvasive diagnostic, prognostic and monitoring tool for many adult and pediatric cancers. ctDNA can also provide insight into tumor heterogeneity and tumor resistance and inform potential targeted therapies. It is exciting to consider that ctDNA as a potential biomarker for prognosis in RMS is just the tip of the iceberg for where this methodology can be utilized to improve the care of patients and our understanding of the disease.

Acknowledgments

Funding: None.

Footnote

Provenance and Peer Review: This article was commissioned by the editorial office, Translational Pediatrics. The article has undergone external peer review.

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Conflicts of Interest: Both authors have completed the ICMJE uniform disclosure form (available at https://tp.amegroups.com/article/view/10.21037/tp-24-7/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring the questions related to the accuracy and integrity of any part of the work are appropriately investigated and resolved.

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