Quantitative proteomics identifies and validates urinary biomarkers of rhabdomyosarcoma in children

Abstract

Background Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma with poor prognosis in children. The 5-year survival rate for early RMS has improved, whereas it remains unsatisfactory for advanced patients. Urine can rapidly reflect changes in the body and identify low-abundance proteins. Early screening of tumor markers through urine in RMS allows for earlier treatment, which is associated with better outcomes. Methods RMS patients under 18 years old, including those newly diagnosed and after surgery, were enrolled. Urine samples were collected at the time points of admission and after four cycles of chemotherapy during follow-up. Then, a two-stage workflow was established. (1) In the discovery stage, differential proteins (DPs) were initially identified in 43 RMS patients and 12 healthy controls (HCs) using a data-independent acquisition method. (2) In the verification stage, DPs were further verified as biomarkers in 54 RMS patients and 25 HCs using parallel reaction monitoring analysis. Furthermore, a receiver operating characteristic (ROC) curve was used to construct the protein panels for the diagnosis of RMS. Gene Ontology (GO) and Ingenuity Pathway Analysis (IPA) software were used to perform bioinformatics analysis. Results A total of 251 proteins were significantly altered in the discovery stage, most of which were enriched in the head, neck and urogenital tract, consistent with the most common sites of RMS. The most overrepresented biological processes from GO analysis included immunity, inflammation, tumor invasion and neuronal damage. Pathways engaging the identified proteins revealed 33 common pathways, including WNT/β-catenin signaling and PI3K/AKT signaling. Finally, 39 proteins were confirmed as urinary biomarkers for RMS, and a diagnostic panel composed of 5 candidate proteins (EPS8L2, SPARC, HLA-DRB1, ACAN, and CILP) was constructed for the early screening of RMS (AUC: 0.79, 95%CI = 0.66 ~ 0.92). Conclusions These findings provide novel biomarkers in urine that are easy to translate into clinical diagnosis of RMS and illustrate the value of global and targeted urine proteomics to identify and qualify candidate biomarkers for noninvasive molecular diagnosis.

Proteomic insights into paediatric cancer: Unravelling molecular signatures and therapeutic opportunities

“…MS‐based proteomic analysis of liquid biopsy is currently being investigated in paediatric cancer. For example, in rhabdomyosarcoma, differentially expressed proteins were detected in urine samples using both DDA and DIA by LC‐MS, with a subset of proteins identified as possible biomarkers for non‐invasive molecular diagnoses 92 . In brain cancer, DDA by LC‐MS was used to analyse the proteome of cerebrospinal fluid in control and brain tumour patients.…”

“…For example, in rhabdomyosarcoma, differentially expressed proteins were detected in urine samples using both DDA and DIA by LC-MS, with a subset of proteins identified as possible biomarkers for non-invasive molecular diagnoses. 92 In brain cancer, DDA by LC-MS was used to analyse the proteome of cerebrospinal fluid in control and brain tumour patients. With parallel reaction monitoring for quantification (Table 1), tumour versus non-tumour controls and broad subtypes of brain tumour were differentiated.…”

Wnt/β-Catenin Signaling Pathway in Pediatric Tumors: Implications for Diagnosis and Treatment

“…A group conducted a proteomic analysis to identify certain differential proteins (DPs) as a diagnostic and prognostic biomarker for RMS. Interestingly, the DPs found in this study were mostly involved in the regulation of Wnt/β-catenin signaling, PI3K/AKT signaling, and FAT10 cancer signaling pathways, which modulate the process of cell differentiation and proliferation [ 60 ]. Another study reveals disrupted Wnt signaling genes enhance the growth of ERMS with extremely invasive characteristics in p53/c-fos double mutant mice [ 59 ].…”

“…In a similar way, activation of the classical Wnt/β-catenin pathway via inhibition of GSK3 prevents development and regeneration in ERMS [ 61 ]. There are few studies in the literature which implicate its direct role in invasion, angiogenesis, and metastasis in RMS [ 60 ].…”