Autoantibody profiling of alveolar rhabdomyosarcoma patients unveils tumor-associated antigens with diagnostic and prognostic significance

Abstract

Alveolar rhabdomyosarcoma (ARMS) is a highly aggressive subtype of childhood cancer for which efficacious treatments are needed. Immunotherapy represents a new therapeutic opportunity to pursue, but it requires the identification of worthwhile tumor antigens. Herein, we exploited the capacity of ARMS autoantibodies to recognize tumor self-antigens, probing human protein microarrays with plasma from ARMS patients and healthy subjects. We assessed the autoantibody response in ARMS, validated data with independent techniques, and estimated autoantibodies diagnostic and prognostic significance by receiver-operator characteristic curves (ROC), uni-and multivariate analysis. Of the 48 tumor antigens identified, General Transcription Factor II-I (GTF2i) and Protocadherin Gamma Subfamily C5 (PCDHGC5) were selected as candidate targets to validate tumor-restricted antigen expression and autoantibody reactivity through an independent technique and wider cohort of cases. GTF2i and PCDHGC5 overexpression was observed in tumor tissues compared to normal counterparts, and anti-GTF2i and -PCDHGC5 autoantibodies were found able to distinguish ARMS patients from healthy subjects as well as cases with different histology. Moreover, low levels of PCDHGC5 autoantibodies characterized patients with worse event-free survival and proved to be an independent negative prognostic factor. This approach provided the first comprehensive autoantibody profile of ARMS, gave novel insights into the immune response of this malignancy and paved the way toward novel potential antibody-based therapeutic applications suitable to improve the survival of ARMS patients.

Immunoreactivity against fibroblast growth factor 8 in alveolar rhabdomyosarcoma patients and its involvement in tumor aggressiveness

“…To identify abnormally regulated proteins in very high-risk RMS patients, we cross-checked tumor antigens, previously identified in metastatic PAX3-FOXO1 -positive (PF+) ARMS by means of immune response binding profiling (IRBP) analysis, 31 with genes under the regulatory control of PAX3-FOXO1 fusion protein. 32 Among the 55 antigens showing significant difference of immunoreactivity compared to healthy subjects ( Figure 1a and Table S3), six were direct targets of PAX3-FOXO1 ( Figure 1b ).…”

“…(a) Left , experimental workflow followed for the identification of autoantibodies in ARMS patients using plasma samples and ProtoArray TM technology. 31 The immune response profile was obtained from 10 metastatic ARMS patients and 15 healthy subjects (HS), probing protein microarray chips with plasma. Reactivity of 9374 spotted antigens was evaluated after signal detection, filtration and normalization using robust linear model (RLM).…”

“…By employing human proteomic chips for antibody screening and patients plasma profiling we identified 55 autoantibodies capable of clustering sick children from healthy ones. 31 Among them, six were reactive against known PAX3-FOXO1 target genes, including FGF8, a growth factor that has been recently found to support RMS recurrence in mice after PAX3/FOXO1 oncogene deinduction. 46 With the aim of identifying tumor antigens involved in RMS dissemination and recurrence we selected FGF8 for further investigation and validation, since it plays a role in early myogenesis during embryonic development, while it has been shown to be expressed at a low level in normal adult epithelial cells and at an increased level in cancer cells.…”

Immune Microenvironment in Childhood Cancers: Characteristics and Therapeutic Challenges

“…M2-type TAMs are associated with various other types of pediatric tumors, including osteosarcoma, rhabdomyosarcoma, leukemia, lymphoma, and brain tumors. These TAMs express M2-phenotype markers such as CD163, CD204, and CD206, which have been shown to prevent cytotoxic T lymphocytes (CTL) infiltration into the tumor core [ 29 , 30 , 31 , 32 , 33 ].…”