Immunohistochemical detection of PAX-FOXO1 fusion proteins in alveolar rhabdomyosarcoma using breakpoint specific monoclonal antibodies.

Alveolar Rhabdomyosarcoma (ARMS) is an aggressive pediatric cancer with about 80% of cases characterized by either a t(1;13)(p36;q14) or t(2;13)(q35;q14), which results in the formation of the fusion oncogenes PAX7-FOXO1 and PAX3-FOXO1, respectively. Since patients with fusion-positive ARMS (FP-RMS) have a poor prognosis and are treated with an aggressive therapeutic regimen, correct classification is of clinical importance. Detection of the translocation by different molecular methods is used for diagnostics, including fluorescence in situ hybridization and RT-PCR or NGS based approaches. Since these methods are complex and time consuming, we developed specific monoclonal antibodies (mAbs) directed to the junction region on the PAX3-FOXO1 fusion protein. Two mAbs, PFM.1 and PFM.2, were developed and able to immunoprecipitate in vitro-translated PAX3-FOXO1 and cellular PAX3-FOXO1 from FP-RMS cells. Furthermore, the mAbs recognized a 105 kDa band in PAX3-FOXO1-transfected cells and in FP-RMS cell lines. The mAbs did not recognize proteins in fusion-negative embryonal rhabdomyosarcoma cell lines, nor did they recognize PAX3 or FOXO1 alone when compared to anti-PAX3 and anti-FOXO1 antibodies. We next evaluated the ability of mAb PFM.2 to detect the fusion protein by immunohistochemistry. Both PAX3-FOXO1 and PAX7-FOXO1 were detected in HEK293 cells transfected with the corresponding cDNAs. Subsequently, we stained 26 primary tumor sections and a rhabdomyosarcoma tissue array and detected both fusion proteins with a positive predictive value of 100%, negative predictive value of 98%, specificity of 100% and a sensitivity of 91%. While tumors are stained homogenously in PAX3-FOXO1 cases, the staining pattern is heterogenous with scattered positive cells only in tumors expressing PAX7-FOXO1. No staining was observed in stromal cells, embryonal rhabdomyosarcoma, and fusion-negative rhabdomyosarcoma. These results demonstrate that mAbs specific for the chimeric oncoproteins PAX3-FOXO1 and PAX7-FOXO1 can be used efficiently for simple and fast subclassification of rhabdomyosarcoma in routine diagnostics via immunohistochemical detection.

Circ-ZNF609 regulates G1-S progression in rhabdomyosarcoma.

Circular RNAs (circRNAs) represent a class of covalently closed RNAs, derived from non-canonical splicing events, which are expressed in all eukaryotes and often conserved among different species. We previously showed that the circRNA originating from the ZNF609 locus (circ-ZNF609) acts as a crucial regulator of human primary myoblast growth: indeed, the downregulation of the circRNA, and not of its linear counterpart, strongly reduced the proliferation rate of in vitro cultured myoblasts. To deepen our knowledge about circ-ZNF609 role in cell cycle regulation, we studied its expression and function in rhabdomyosarcoma (RMS), a pediatric skeletal muscle malignancy. We found that circ-ZNF609 is upregulated in biopsies from the two major RMS subtypes, embryonal (ERMS) and alveolar (ARMS). Moreover, we discovered that in an ERMS-derived cell line circ-ZNF609 knock-down induced a specific block at the G1-S transition, a strong decrease of p-Akt protein level and an alteration of the pRb/Rb ratio. Regarding p-Akt, we were able to show that circ-ZNF609 acts by counteracting p-Akt proteasome-dependent degradation, thus working as a new regulator of cell proliferation-related pathways. As opposed to ERMS-derived cells, the circRNA depletion had no cell cycle effects in ARMS-derived cells. Since in these cells the p53 gene resulted downregulated, with a concomitant upregulation of its cell cycle-related target genes, we suggest that this could account for the lack of circ-ZNF609 effect in ARMS.

Deregulated systemic IL-10/IL-12 balance in advanced and poor prognosis paediatric soft tissue sarcomas.

CONTEXT: The roles of interleukin 10 (IL-10) and IL-12 in regulation of cancer growth and Th1/Th2 immune responses towards cancer are unclear. OBJECTIVE: To establish the prognostic significance of serum IL-10 and IL-12 in paediatric soft tissue sarcomas (STS). MATERIALS AND METHODS: ELISA determinations of cytokines were performed as pre-treatment in 59 children with STS and 30 healthy controls. RESULTS: Elevated IL-10 and decreased IL-12 serum levels correlated with advanced disease, poor response to chemotherapy and poor outcome. IL-10 ≥ 9.5 pg/ml, IL-12 ≤ 65 pg/ml and lymph nodes involvement independently predicted poor overall survival (OS) in multivariate Cox analysis. CONCLUSION: Serum IL-10/IL-12 balance determination may facilitate to assess risk groups and prognosis in childhood STS.

Cetuximab promotes immunotoxicity against rhabdomyosarcoma in vitro.

Multidrug resistance is a common problem in the treatment of childhood rhabdomyosarcoma (RMS). Therefore, novel treatment regimes such as immunotherapy have to be evaluated. The aim of this study was to detect possible targets on RMS cells and to investigate whether corresponding humanized antibodies could be used to treat RMS. Screening for potential targets common for different subtypes of RMS was carried out with Affymetrix mRNA expression arrays on 12 primary RMS samples. Subsequent pathway analysis revealed the epidermal growth factor receptor (EGFR) as a potential target for antibody therapy. Expression of EGFR and binding of its specific antibody Cetuximab to embryonal RMS cell lines RD and A-204 and alveolar RMS Rh30 were monitored by flow cytometry. Cetuximab activity was quantified by proliferation assay on RMS cells, and by antibody dependent cellular cytotoxicity assay with peripheral blood mononuclear cells (PBMCs). Gene expression analysis revealed a high expression of EGFR in all embryonal RMS compared with alveolar RMS. The EGFR specific antibody, Cetuximab binds to Rh30 and to RD but not to A-204 cells. Proliferation of these cells was influenced neither by Cetuximab nor by the growth factor EGF. However, cell dependent cytotoxicity of PBMCs to RMS cells such as Rh30 and RD was enhanced specifically by Cetuximab. These promising Cetuximab effects justify analysis under in vivo conditions using suitable models.

Fusions involving PAX and FOX genes in the molecular pathogenesis of alveolar rhabdomyosarcoma: recent advances.

Rhabdomyosarcoma is the most frequent soft tissue sarcoma in the pediatric population. Two main histopathologic variants have been described, embryonal (ERMS) and alveolar (ARMS), which demonstrate clinical and genetic differences. In particular, most ARMS but not ERMS tumors are characterized by the presence of recurrent chromosomal translocations, which have been cytogenetically defined as t(2;13)(q35;q14) and t(1;13)(p36;q14). These translocations form PAX3-FKHR and PAX7-FKHR gene fusions, which encode chimeric transcription factors. These chimeric proteins are hypothesized to generate a novel transcriptional program in the target cell, thereby contributing to multiple aspects of ARMS tumorigenesis. This review highlights recent advances in numerous areas of biomedical investigation that are providing new insights into the biology, molecular pathology, and translational science of ARMS: the identification of downstream targets of PAX3-FKHR and collaborating events in the process of tumorigenesis and metastasis; generation of animal models based on the gene fusion and collaborating events; development of new assays for diagnosis, prognosis, and detection of minimal disseminated disease; and exploration of immune recognition of this tumor and the fusion protein. These findings highlight the continued importance of the fusion proteins in understanding the biology of this tumor and developing improved diagnostics for this tumor, and have led to the initiation of efforts to explore therapeutic strategies based on the increasing understanding of the biology of these fusion proteins.

Identification and epitope enhancement of a PAX-FKHR fusion protein breakpoint epitope in alveolar rhabdomyosarcoma cells created by a tumorigenic chromosomal translocation inducing CTL capable of lysing human tumors.

Fusion proteins created by chromosomal translocations in tumors can create neoantigenic determinants at the breakpoint, which are unique to the tumor cells but shared by the vast majority of tumors of that histologic type. If the fusion protein is responsible for the malignant transformation, its expression cannot be lost by the tumor to escape immune responses against this tumor antigen. Here, we identify such a fusion protein breakpoint epitope in the PAX-FKHR fusion protein created by the t(2;13) translocation present in 80% of cases of alveolar rhabdomyosarcoma, a highly aggressive pediatric soft-tissue sarcoma. We use autologous dendritic cells pulsed with the RS10 breakpoint fusion peptide to raise a human CTL line from a normal healthy HLA-B7+ blood donor specific for this peptide. These CTLs are CD8+ (CD4-CD56-) and restricted by HLA-B7. These human peptide-specific CTL lyse human HLA-B7+ rhabdomyosarcoma tumor cells. Therefore, the fusion protein is endogenously processed to produce this natural epitope presented by HLA-B7 and thus this peptide is a bone fide human tumor antigen. We also define a substitution that increases the affinity for HLA-B7 without loss of antigenicity. This epitope-enhanced peptide may serve as a candidate cancer vaccine for HLA-B7+ patients with alveolar rhabdomyosarcoma.

Poor outcome of two children with relapsed state stage IV alveolar rhabdomyosarcoma after allogeneic stem cell transplantation.

The authors report on 2 boys, 11(1/2) and 13 years old, who received allogeneic stem cell transplantation (alloSCT) from their HLA-identical sibling after relapse of stage IV alveolar rhabdomyosarcoma. Both patients were transplanted in a non-remission status. After alloSCT both patients experienced disease progression at the primary tumor location sites and died due to the underlying disease 146 and 379 days after transplantation. The authors conclude that an alloSCT derived graft versus tumor effect might not be effective enough to overcome alveolar rhabdomyosarcoma when transplantation is carried out in a nonremission status.

Lack of effective T-lymphocyte response to the PAX3/FKHR translocation area in alveolar rhabdomyosarcoma.

PURPOSE: Alveolar rhabdomyosarcoma (ARMS) frequently contains the fusion transcription factor PAX3/FKHR. Therefore, clinical studies have been initiated to utilize the PAX3/FKHR translocation point area as a peptide vaccine against ARMS. Our study was directed at identifying antigenic T-lymphocyte epitopes at the PAX3/FKHR translocation point area. EXPERIMENTAL DESIGN: The peptide sequence surrounding the PAX3/FKHR translocation point was evaluated by MHC binding algorithms for potential T-lymphocyte antigenic epitopes (class I molecules HLA-A1, -A2 and -A3; class II molecules HLA-DR1, -DR4 and -DR7). Using in vitro techniques, dendritic cells loaded with PAX3/FKHR peptides were used to stimulate naive T-lymphocytes. T-lymphocyte activity was then evaluated by 51Cr release and 3H-thymidine uptake assays. RESULTS: Only one HLA-A3-restricted epitope was predicted by the algorithms. The peptide was prepared and tested for its ability to stimulate naive cytotoxic T-lymphocytes (CTLs). Unfortunately, the peptide was unsuccessful at stimulating naive CTL. However, induction of naive helper T-lymphocytes (HTL) to recognize and respond to the PAX3/FKHR translocation peptide was successful. Yet, this HTL peptide activity did not translate into recognition of PAX3/FKHR-containing ARMS tumor cells. CONCLUSIONS: It appears that the fusion area of PAX3/FKHR may not be a good source of antigenic anti-tumor peptide epitopes. These results raise serious concerns about the success and applicability of future peptide-based vaccine immunotherapy directed at the PAX3/FKHR translocation point.

Pilot trial of tumor-specific peptide vaccination and continuous infusion interleukin-2 in patients with recurrent Ewing sarcoma and alveolar rhabdomyosarcoma: an inter-institute NIH study.

BACKGROUND: Patients with recurrent Ewing sarcoma and alveolar rhabdomyosarcoma have poor prognoses and limited therapeutic options. We have investigated the use of peptide pulsed vaccination in an attempt to immunologically target the breakpoint region of tumor specific fusion proteins expressed in these tumors. PROCEDURE: Sixteen patients with recurrent, translocation positive, Ewing sarcoma, and alveolar rhabdomyosarcoma underwent apheresis for collection of peripheral blood mononuclear cells. Following countercurrent centrifugal elutriation, an apheresis product comprised predominantly of monocytes but containing small numbers of circulating immature dendritic cells was pulsed with peptides derived from the breakpoint region of the fusion proteins. Vaccines were administered intravenously concomitant with continuous intravenous rhIL-2 at 9 x 10(6) IU/m(2)/day. RESULTS: Toxicity was limited to IL-2 related effects and was generally mild. Following vaccination, all patients showed progressive disease, most in a rapid fashion following the first vaccine. One patient showed evidence of an immunologic response and another showed a mixed clinical response. Patients enrolled on this tumor vaccine trial showed significant immunosuppression and large bulky tumors. CONCLUSIONS: Peptide vaccination as administered in this trial did not alter the dismal clinical outcome for patients with recurrent pediatric sarcomas. Future trials of tumor vaccines in this population should target patient populations with improved immune competence and smaller tumor burdens. Furthermore, optimization of the antigen presenting cell populations may be important for inducing immune responses to peptide antigens.

Solid alveolar rhabdomyosarcoma of the thorax in a child.

AIMS: This case illustrates the difficulties and pitfalls of diagnosis of alveolar rhabdomyosarcoma in its solid variant and in an unusual primary location, the mediastinum. CASE DETAILS: A 9-year-old boy presented with a primary thoracic tumour associated with metastasis in the left sacroiliac joint. Bronchial and mediastinal biopsies showed a malignant neoplasm with a solid sheet-like pattern of small round cells with a high nuclear to cytoplasmic ratio associated with little or no fibrosis usually evocative of a peripheral neuroectodermal tumour (PNET) at this age. Immunohistochemical positive staining with vimentin (80% of tumour cells), desmin (20%) and titin (30%) antibodies was suggestive of a rhabdomyosarcoma. In addition, all neural cell adhesion molecule (NCAM) markers tested were positive as well as MIC2, a marker for the Ewing family of sarcomas. There was no rhabdomyoid differentiation at ultrastructural examination. Molecular analysis with RT-PCR amplification of RNA isolated from the tumour demonstrated the presence of a PAX3/FKHR fusion transcript, product of a t(2;13) reciprocal translocation, a genetic marker specific for alveolar rhabdomyosarcoma. CONCLUSIONS: The diagnostic methodology of a small round cell tumour of the child must now include immunohistochemical study and molecular biology to confirm the diagnosis of alveolar rhabdomyosarcoma, in a solid and undifferentiated variant.