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4.
Leukemia ; 36(7): 1879-1886, 2022 07.
Artículo en Inglés | MEDLINE | ID: mdl-35676453

RESUMEN

Several studies have reported that chronic myeloid leukaemia (CML) patients expressing e14a2 BCR::ABL1 have a faster molecular response to therapy compared to patients expressing e13a2. To explore the reason for this difference we undertook a detailed technical comparison of the commonly used Europe Against Cancer (EAC) BCR::ABL1 reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) assay in European Treatment and Outcome Study (EUTOS) reference laboratories (n = 10). We found the amplification ratio of the e13a2 amplicon was 38% greater than e14a2 (p = 0.015), and the amplification efficiency was 2% greater (P = 0.17). This subtle difference led to measurable transcript-type dependent variation in estimates of residual disease which could be corrected by (i) taking the qPCR amplification efficiency into account, (ii) using alternative RT-qPCR approaches or (iii) droplet digital PCR (ddPCR), a technique which is relatively insensitive to differences in amplification kinetics. In CML patients, higher levels of BCR::ABL1/GUSB were identified at diagnosis for patients expressing e13a2 (n = 67) compared to e14a2 (n = 78) when analysed by RT-qPCR (P = 0.0005) but not ddPCR (P = 0.5). These data indicate that widely used RT-qPCR assays result in subtly different estimates of disease depending on BCR::ABL1 transcript type; these differences are small but may need to be considered for optimal patient management.


Asunto(s)
Proteínas de Fusión bcr-abl , Leucemia Mielógena Crónica BCR-ABL Positiva , Proteínas de Fusión bcr-abl/genética , Humanos , Mesilato de Imatinib , Leucemia Mielógena Crónica BCR-ABL Positiva/diagnóstico , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Neoplasia Residual/genética , Reacción en Cadena en Tiempo Real de la Polimerasa
5.
Mol Ther Methods Clin Dev ; 23: 119-127, 2021 Dec 10.
Artículo en Inglés | MEDLINE | ID: mdl-34631931

RESUMEN

The piggyBac transposon system provides a non-viral alternative for cost-efficient and simple chimeric antigen receptor (CAR) T cell production. The generation of clinical-grade CAR T cells requires strict adherence to current good manufacturing practice (cGMP) standards. Unfortunately, the high costs of commonly used lentiviral or retroviral vectors limit the manufacturing of clinical-grade CAR T cells in many non-commercial academic institutions. Here, we present a manufacturing platform for highly efficient generation of CD19-specific CAR T cells (CAR19 T cells) based on co-electroporation of linear DNA transposon and mRNA encoding the piggyBac transposase. The transposon is prepared enzymatically in vitro by PCR and contains the CAR transgene flanked by piggyBac 3' and 5' arms. The mRNA is similarly prepared via in vitro transcription. CAR19 T cells are expanded in the combination of cytokines interleukin (IL)-4, IL-7, and IL-21 to prevent terminal differentiation of CAR T cells. The accurate control of vector copy number (VCN) is achieved by decreasing the concentration of the transposon DNA, and the procedure yields up to 1 × 108 CAR19 T cells per one electroporation of 1 × 107 peripheral blood mononuclear cells (PBMCs) after 21 days of in vitro culture. Produced cells contain >60% CAR+ cells with VCN < 3. In summary, the described manufacturing platform enables a straightforward cGMP certification, since the transposon and transposase are produced abiotically in vitro via enzymatic synthesis. It is suitable for the cost-effective production of highly experimental, early-phase CAR T cell products.

6.
Br J Haematol ; 194(1): 53-60, 2021 07.
Artículo en Inglés | MEDLINE | ID: mdl-34114218

RESUMEN

Measurement of BCR activator of RhoGEF and GTPase -ABL proto-oncogene 1, non-receptor tyrosine kinase (BCR-ABL1) mRNA levels by reverse transcription quantitative polymerase chain reaction (RTqPCR) has been critical to treatment protocols and clinical trials in chronic myeloid leukaemia; however, interlaboratory variation remains a significant issue. Reverse transcriptase droplet digital PCR (RTddPCR) has shown potential to improve testing but a large-scale interlaboratory study is required to definitively establish this. In the present study, 10 BCR-ABL1-positive samples with levels ranging from molecular response (MR)1·0 -MR5·0 were tested by 23 laboratories using RTddPCR with the QXDX BCR-ABL %IS kit. A subset of participants tested the samples using RTqPCR. All 23 participants using RTddPCR detected BCR-ABL1 in all samples to MR4·0 . Detection rates for deep-response samples were 95·7% at MR4·5 , 78·3% at MR4·7 and 87·0% at MR5·0 . Interlaboratory coefficient of variation was indirectly proportional to BCR-ABL1 level ranging from 29·3% to 69·0%. Linearity ranged from 0·9330 to 1·000 (average 0·9936). When results were compared for the 11 participants who performed both RTddPCR and RTqPCR, RTddPCR showed a similar limit of detection to RTqPCR with reduced interlaboratory variation and better assay linearity. The ability to detect deep responses with RTddPCR, matched with an improved linearity and reduced interlaboratory variation will allow improved patient management, and is of particular importance for future clinical trials focussed on achieving and maintaining treatment-free remission.


Asunto(s)
Proteínas de Fusión bcr-abl/sangre , Ensayos de Aptitud de Laboratorios , Leucemia Mielógena Crónica BCR-ABL Positiva/sangre , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Asia , Biomarcadores de Tumor/sangre , Europa (Continente) , Células HL-60/química , Humanos , Células K562/química , Laboratorios Clínicos , Modelos Lineales , América del Norte , Juego de Reactivos para Diagnóstico , Reproducibilidad de los Resultados
8.
Leukemia ; 34(8): 2113-2124, 2020 08.
Artículo en Inglés | MEDLINE | ID: mdl-32472084

RESUMEN

This work investigated patient-specific genomic BCR-ABL1 fusions as markers of measurable residual disease (MRD) in chronic myeloid leukaemia, with a focus on relevance to treatment-free remission (TFR) after achievement of deep molecular response (DMR) on tyrosine kinase inhibitor (TKI) therapy. DNA and mRNA BCR-ABL1 measurements by qPCR were compared in 2189 samples (129 patients) and by digital PCR in 1279 sample (62 patients). A high correlation was found at levels of disease above MR4, but there was a poor correlation for samples during DMR. A combination of DNA and RNA MRD measurements resulted in a better prediction of molecular relapse-free survival (MRFS) after TKI stop (n = 17) or scheduled interruption (n = 25). At 18 months after treatment cessation, patients with stopped or interrupted TKI therapy who were DNA negative/RNA negative during DMR maintenance (green group) had an MRFS of 80% and 100%, respectively, compared with those who were DNA positive/RNA negative (MRFS = 57% and 67%, respectively; yellow group) or DNA positive/RNA positive (MRFS = 20% for both cohorts; red group). Thus, we propose a "traffic light" stratification as a TFR predictor based on DNA and mRNA BCR-ABL1 measurements during DMR maintenance before TKI cessation.


Asunto(s)
Proteínas de Fusión bcr-abl/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Reacción en Cadena de la Polimerasa/métodos , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Adulto , Anciano , Femenino , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/mortalidad , Masculino , Persona de Mediana Edad , Neoplasia Residual , ARN Mensajero/análisis , Inducción de Remisión , Privación de Tratamiento
9.
J Cancer Res Clin Oncol ; 145(6): 1645-1650, 2019 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-30941573

RESUMEN

PURPOSE: The advent of tyrosine kinase inhibitor (TKI) therapies has revolutionized the treatment of chronic myeloid leukemia (CML). The European LeukemiaNet (ELN) recommends quantification of BCR-ABL1 transcripts by real-time quantitative PCR every 3 months during TKI treatment. Since a proportion of patients in deep molecular response (DMR: MR4, MR4.5, MR5) maintain remission after treatment stop, assessment of DMR is crucial. However, systematically collected molecular data, monitored with sensitive standardized assays, are not available outside clinical trials. METHODS: Data were collected on the standardized assessment of molecular response in the context of real-life practice. BCR-ABL1 transcript levels after > 2 years of TKI therapy were evaluated for DMR by local laboratories as well as standardized EUTOS laboratories. Since standardized molecular monitoring is a prerequisite for treatment discontinuation, central surveillance of the performance of the participating laboratories was carried out. RESULTS: Between 2014 and 2017, 3377 peripheral blood samples from 1117 CML patients were shipped to 11 standardized reference laboratories in six European countries. BCR-ABL1 transcript types were b3a2 (41.63%), b2a2 (29.99%), b2a2/b3a2 (3.58%) and atypical (0.54%). For 23.72% of the patients, the initial transcript type had not been reported. Response levels (EUTOS laboratory) were: no MMR, n = 197 (6.51%); MMR, n = 496 (16.40%); MR4, n = 685 (22.64%); MR4.5, n = 937 (30.98%); MR5, n = 710 (23.47%). With a Cohen's kappa coefficient of 0.708, a substantial agreement between EUTOS-certified and local laboratories was shown. CONCLUSIONS: Multicenter DMR assessment is feasible in the context of real-life clinical practice in Europe. Information on the BCR-ABL1 transcript type at diagnosis is crucial to accurately monitor patients' molecular response during or after TKI therapy.


Asunto(s)
Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mieloide de Fase Crónica/tratamiento farmacológico , Leucemia Mieloide de Fase Crónica/genética , Inhibidores de Proteínas Quinasas/administración & dosificación , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Cohortes , Europa (Continente) , Humanos , Laboratorios/normas , Laboratorios/estadística & datos numéricos , Leucemia Mielógena Crónica BCR-ABL Positiva/sangre , Leucemia Mieloide de Fase Crónica/sangre , Persona de Mediana Edad , Sistema de Registros , Adulto Joven
11.
Pediatr Transplant ; 11(4): 441-7, 2007 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-17493228

RESUMEN

We present an infant with cDGS overlapping with CHARGE syndrome, who suffered from T-cell deficiency treated with screened healthy DLI from an unrelated donor (8/10 match). The first dose of DLI (1.1 x 10(6) CD3+/kg) was administered at the age of six months, the second one (0.9 x 10(6) CD3+/kg) 36 days later. No conditioning was employed, GvHD prophylaxis consisting of CsA was used only during the second infusion. Since day+10 after the first DLI, split chimerism showing T-cell engraftment has been documented. Proliferative response to PHA was detected on day+145. The treatment was complicated by severe acute GvHD (grade II-III) after the first DLI and prolonged chronic liver cholestatic GvHD developing after the second DLI. Vigorous EBV proliferation four wk after the second DLI was accompanied by peripheral expansion of CD8+ donor cells. The patient, 26-months old, is clinically well and has slowly started to gain his developmental milestones. We believe that infusions of small doses of DLI from an unrelated donor represent a potentially helpful therapeutic option in patients with cDGS/CHARGE phenotype.


Asunto(s)
Coloboma/terapia , Síndrome de DiGeorge/terapia , Transfusión de Linfocitos/métodos , Ano Imperforado/terapia , Síndrome de DiGeorge/sangre , Oído/anomalías , Enfermedades del Oído/congénito , Enfermedades del Oído/terapia , Estudios de Seguimiento , Genitales Masculinos/anomalías , Enfermedad Injerto contra Huésped/etiología , Enfermedad Injerto contra Huésped/prevención & control , Trastornos del Crecimiento/terapia , Cardiopatías Congénitas/terapia , Humanos , Recién Nacido , Discapacidad Intelectual/terapia , Transfusión de Linfocitos/efectos adversos , Masculino , Linfocitos T
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