RESUMEN
Osteosarcoma (OSA) is a malignant tumor of middle-aged dogs and adolescent humans. The clinical outcome of OSA has not improved over more than three decades, and dogs typically succumb to metastatic disease within 6 months despite tumor resection through limb amputation and adjuvant chemotherapy. Therefore, undetectable tumor cells with potential to form metastases are present at diagnosis. An assay to identify canine immortalized and primary OSA cells through flow cytometric detection of intracellular collagen 1 (Col I) and osteocalcin was optimized, and applied to blood samples from tumor-bearing dogs for detection of circulating tumor cells (CTCs). Spiking variable number of OSA cells into normal dog blood recovered 50-60% of Col I positive cells with high forward and variable side light scatter. An algorithm to exclude nonviable, doublet, and autofluorescent cells was applied to sequential blood samples from three dogs obtained prior to and after limb amputation, and at approximately, triweekly intervals over 121, 142, and 183 days of chemotherapy, respectively. Dogs had >100 CTC/106 leukocytes prior to amputation, variably frequent CTC during chemotherapy, and an increase up to 4,000 CTC/106 leukocytes within 4 weeks before overt metastases or death. Sorted CTCs were morphologically similar to direct tumor aspirates and positive for Col I. Although preliminary, findings suggest that CTCs are frequent in canine OSA, more numerous than carcinoma CTC in humans, and that an increase in CTC frequency may herald clinical deterioration. This assay may enable enumeration and isolation of OSA CTC for prognostic and functional studies, respectively. © 2019 International Society for Advancement of Cytometry.
Asunto(s)
Neoplasias Óseas/veterinaria , Enfermedades de los Perros/diagnóstico por imagen , Citometría de Flujo/métodos , Células Neoplásicas Circulantes/metabolismo , Osteosarcoma/veterinaria , Amputación Quirúrgica , Animales , Neoplasias Óseas/sangre , Neoplasias Óseas/diagnóstico por imagen , Neoplasias Óseas/tratamiento farmacológico , Línea Celular Tumoral , Colágeno/metabolismo , Enfermedades de los Perros/sangre , Enfermedades de los Perros/tratamiento farmacológico , Perros , Procesamiento de Imagen Asistido por Computador , Leucocitos/metabolismo , Células Neoplásicas Circulantes/efectos de los fármacos , Células Neoplásicas Circulantes/patología , Osteocalcina/metabolismo , Osteosarcoma/sangre , Osteosarcoma/diagnóstico por imagen , Osteosarcoma/tratamiento farmacológico , PronósticoRESUMEN
CHARGE syndrome-which stands for coloboma of the eye, heart defects, atresia of choanae, retardation of growth/development, genital abnormalities, and ear anomalies-is a severe developmental disorder with wide phenotypic variability, caused mainly by mutations in CHD7 (chromodomain helicase DNA-binding protein 7), known to encode a chromatin remodeler. The genetic lesions responsible for CHD7 mutation-negative cases are unknown, at least in part because the pathogenic mechanisms underlying CHARGE syndrome remain poorly defined. Here, we report the characterization of a mouse model for CHD7 mutation-negative cases of CHARGE syndrome generated by insertional mutagenesis of Fam172a (family with sequence similarity 172, member A). We show that Fam172a plays a key role in the regulation of cotranscriptional alternative splicing, notably by interacting with Ago2 (Argonaute-2) and Chd7. Validation studies in a human cohort allow us to propose that dysregulation of cotranscriptional alternative splicing is a unifying pathogenic mechanism for both CHD7 mutation-positive and CHD7 mutation-negative cases. We also present evidence that such splicing defects can be corrected in vitro by acute rapamycin treatment.