RESUMEN
Autologous stem cell transplantation (SCT) has become standard therapy in high risk stage IV neuroblastoma (NB) patients. Residual NB cells in the bone marrow (BM) shortly before SCT may shape the overall survival.Thus, we sought to thoroughly investigate minimal residual disease (MRD) in BM prior to SCT using conventional and real time RT-PCR for tyrosine hydroxylase (TH) as well as morphology. To avoid influence of residual NB cells in the stem cell harvest, 17 patients transplanted with MRD negative grafts (n=11 CD34-selected and n=6 unmanipulated) are included in the final analysis, only.35% of these patients are alive with a median follow up of 8.6 years. In the BM of 9/17 patients residual NB cells could be detected < 40 d before SCT. These patients had a significant lower overall survival compared to patients without BM involvement based on combined RT-PCR and morphology results (11% vs. 62%, p=0.026) or using RT-PCR, only (p=0.01). In contrast morphology on its own did not lead to a significant discrimination between both groups.Our results obtained in a small cohort of stage IV NB patients suggest that MRD diagnostic in the BM shortly before SCT might be a valuable predictive tool for these patients but requires conformation in a multicenter study.
Asunto(s)
Trasplante de Células Madre Hematopoyéticas , Neuroblastoma/cirugía , Adolescente , Antineoplásicos/uso terapéutico , Biomarcadores de Tumor/análisis , Médula Ósea/patología , Niño , Preescolar , Terapia Combinada , Femenino , Estudios de Seguimiento , Trasplante de Células Madre Hematopoyéticas/mortalidad , Humanos , Lactante , Masculino , Terapia Neoadyuvante , Estadificación de Neoplasias , Neoplasia Residual/mortalidad , Neoplasia Residual/patología , Neoplasia Residual/cirugía , Neuroblastoma/mortalidad , Neuroblastoma/patología , Reacción en Cadena de la Polimerasa , Pronóstico , Reacción en Cadena en Tiempo Real de la Polimerasa , Estudios Retrospectivos , Factores de Riesgo , Tasa de Supervivencia , Resultado del Tratamiento , Tirosina 3-Monooxigenasa/análisis , Adulto JovenRESUMEN
PURPOSE: Real-time reverse-transcriptase PCR (RT-qPCR) or conventional RT-PCR (RT-cPCR) detection of tyrosine hydroxylase (TH) is increasingly used to detect neuroblastoma (NB) cells in clinical samples. However, TH expression in normal tissues can limit its usefulness and make additional diagnostic strategies necessary. METHODS: We analysed TH in 857 tumour, bone marrow aspirate and peripheral blood stem cell samples from 65 NB patients using RT-cPCR, and compared results from 666 samples analysed by RT-qPCR. TH was investigated in 84 samples from patients with other diagnoses and 354 samples from healthy donors as controls, and 132 samples from the entire collection were evaluated for NB cells using 5-colour flow cytometry (FC). RESULTS: Cohen's kappa coefficient demonstrated a substantial agreement between RT-cPCR and RT-qPCR as well as RT-cPCR and FC and a moderate agreement between RT-qPCR and FC. TH expression was also detected in samples from individual patients with Ewing sarcoma, nephroblastoma and rhabdomyosarcoma, but not from healthy donors. FC panels were an effective complementary strategy, detecting as few as 0.002% NB cells, characterised as CD45negCD9+CD81+CD56+ch14:18+GD2+ cells with occasional CD57+CD138+CD166+ expression. CONCLUSION: TH RT-qPCR alone is limited for detection of NB cells because of "false positives" in samples from patients with other diseases. Advanced FC may serve as a complementary method to detect residual NB, but needs further confirmation in larger patient cohorts.
Asunto(s)
Citometría de Flujo , Células Neoplásicas Circulantes/patología , Neuroblastoma/diagnóstico , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tirosina 3-Monooxigenasa/genética , Molécula de Adhesión Celular del Leucocito Activado/genética , Médula Ósea/patología , Línea Celular Tumoral , Niño , Diagnóstico Diferencial , Reacciones Falso Positivas , Estudios de Seguimiento , Ganglioneuroma/diagnóstico , Ganglioneuroma/genética , Perfilación de la Expresión Génica , Marcadores Genéticos/genética , Humanos , Estadificación de Neoplasias , Neoplasia Residual/patología , Neuroblastoma/genética , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
BACKGROUND: Recovery of cytomegalovirus (CMV)-specific T cell mediated immunity after allogeneic hematopoietic stem cell transplantation (SCT) is critical for protection against CMV disease. Tetramer-based technologies have been shown to be a sensitive tool in the enumeration of specific T cells, but have the disadvantage of HLA-restriction of the peptides. PATIENTS AND METHODS: In this pilot study, we tested the feasibility of a panel of 6 CMV-specific tetrameric HLA/CMV-peptide complexes to enumerate CMV-specific CD8 +T cells (CTLs). The reconstitution of CMV-specific CTLs was assessed in 16 children in the first year after allogeneic SCT (median age, 8 years). RESULTS: The presented assay covered more than 85% of our patients transplanted in the last 3 years. During CMV-reactivation, all 4 of the 16 analyzed patients with a high virus-load showed less than 10 CMV-specific CTLs/microl; out of these, three had not any detectable CMV-CTLs. On the other hand, five of the children with less than 10 CMV-specific CTLs/microl did not develop CMV reactivation. When enumeration of T cells was performed by means of different tetrameric HLA/CMV-peptide complexes simultaneously, the numbers of CMV-specific CTLs cells widely differed according to the HLA-type. CONCLUSIONS: Our pilot study suggests that enumeration of CMV-specific T cells by means of a panel of 6 tetramers might be a useful tool in the risk assessment for CMV reactivation in the majority of patients undergoing allogeneic SCT, but future trials have to evaluate whether this method is appropriate in tailoring antiviral therapy in the individual patient.
