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1.
Mod Pathol ; 34(4): 748-757, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33299109

RESUMEN

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.


Asunto(s)
Anticuerpos Monoclonales/inmunología , Biomarcadores de Tumor/análisis , Inmunohistoquímica , Proteínas de Fusión Oncogénica/análisis , Factores de Transcripción Paired Box/análisis , Rabdomiosarcoma Alveolar/inmunología , Adolescente , Adulto , Animales , Especificidad de Anticuerpos , Niño , Preescolar , Femenino , Células HEK293 , Células HeLa , Humanos , Lactante , Masculino , Ratones , Persona de Mediana Edad , Células 3T3 NIH , Proteínas de Fusión Oncogénica/inmunología , Factores de Transcripción Paired Box/inmunología , Valor Predictivo de las Pruebas , Reproducibilidad de los Resultados , Rabdomiosarcoma Alveolar/patología , Adulto Joven
2.
Nat Commun ; 15(1): 6222, 2024 Jul 23.
Artículo en Inglés | MEDLINE | ID: mdl-39043633

RESUMEN

Chimeric antigen receptor (CAR) T-cells targeting Fibroblast Growth Factor Receptor 4 (FGFR4), a highly expressed surface tyrosine receptor in rhabdomyosarcoma (RMS), are already in the clinical phase of development, but tumour heterogeneity and suboptimal activation might hamper their potency. Here we report an optimization strategy of the co-stimulatory and targeting properties of a FGFR4 CAR. We replace the CD8 hinge and transmembrane domain and the 4-1BB co-stimulatory domain with those of CD28. The resulting CARs display enhanced anti-tumor activity in several RMS xenograft models except for an aggressive tumour cell line, RMS559. By searching for a direct target of the RMS core-regulatory transcription factor MYOD1, we identify another surface protein, CD276, as a potential target. Bicistronic CARs (BiCisCAR) targeting both FGFR4 and CD276, containing two distinct co-stimulatory domains, have superior prolonged persistent and invigorated anti-tumor activities compared to the optimized FGFR4-specific CAR and the other BiCisCAR with the same 4-1BB co-stimulatory domain. Our study thus lays down the proof-of-principle for a CAR T-cell therapy targeting both FGFR4 and CD276 in RMS.


Asunto(s)
Antígenos B7 , Inmunoterapia Adoptiva , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos , Receptores Quiméricos de Antígenos , Rabdomiosarcoma , Ensayos Antitumor por Modelo de Xenoinjerto , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos/metabolismo , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos/genética , Rabdomiosarcoma/terapia , Rabdomiosarcoma/inmunología , Rabdomiosarcoma/genética , Humanos , Animales , Receptores Quiméricos de Antígenos/inmunología , Receptores Quiméricos de Antígenos/metabolismo , Línea Celular Tumoral , Ratones , Inmunoterapia Adoptiva/métodos , Antígenos B7/metabolismo , Antígenos B7/inmunología , Antígenos B7/genética , Proteína MioD/metabolismo , Proteína MioD/genética , Linfocitos T/inmunología , Linfocitos T/metabolismo , Niño , Femenino , Ratones SCID , Ratones Endogámicos NOD
3.
Blood Adv ; 7(15): 4218-4232, 2023 08 08.
Artículo en Inglés | MEDLINE | ID: mdl-36607839

RESUMEN

CD19 chimeric antigen receptor T-cell therapy (CD19-CAR) has changed the treatment landscape and outcomes for patients with pre-B-cell acute lymphoblastic leukemia (B-ALL). Unfortunately, primary nonresponse (PNR), sustained CD19+ disease, and concurrent expansion of CD19-CAR occur in 20% of the patients and is associated with adverse outcomes. Although some failures may be attributable to CD19 loss, mechanisms of CD19-independent, leukemia-intrinsic resistance to CD19-CAR remain poorly understood. We hypothesize that PNR leukemias are distinct compared with primary sensitive (PS) leukemias and that these differences are present before treatment. We used a multiomic approach to investigate this in 14 patients (7 with PNR and 7 with PS) enrolled in the PLAT-02 trial at Seattle Children's Hospital. Long-read PacBio sequencing helped identify 1 PNR in which 47% of CD19 transcripts had exon 2 skipping, but other samples lacked CD19 transcript abnormalities. Epigenetic profiling discovered DNA hypermethylation at genes targeted by polycomb repressive complex 2 (PRC2) in embryonic stem cells. Similarly, assays of transposase-accessible chromatin-sequencing revealed reduced accessibility at these PRC2 target genes, with a gain in accessibility of regions characteristic of hematopoietic stem cells and multilineage progenitors in PNR. Single-cell RNA sequencing and cytometry by time of flight analyses identified leukemic subpopulations expressing multilineage markers and decreased antigen presentation in PNR. We thus describe the association of a stem cell epigenome with primary resistance to CD19-CAR therapy. Future trials incorporating these biomarkers, with the addition of multispecific CAR T cells targeting against leukemic stem cell or myeloid antigens, and/or combined epigenetic therapy to disrupt this distinct stem cell epigenome may improve outcomes of patients with B-ALL.


Asunto(s)
Leucemia-Linfoma Linfoblástico de Células Precursoras , Linfocitos T , Niño , Humanos , Epigenoma , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Antígenos CD19 , Células Madre Hematopoyéticas
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