RESUMEN
Allogeneic hematopoietic stem cell transplantation (HSCT) is still needed for many children with very high-risk acute leukemia. An HLA-haploidentical family donor is a suitable option for those without an HLA-matched donor. Here we present outcomes of a novel HLA-haploidentical HSCT (haplo-HSCT) strategy with adoptive immunotherapy with thymic-derived CD4+CD25+ FoxP3+ regulatory T cells (Tregs) and conventional T cells (Tcons) performed between January 2017 and July 2021 in 20 children with high-risk leukemia. Median age was 14.5 years (range, 4-21), 15 had acute lymphoblastic leukemia, 5 acute myeloid leukemia. The conditioning regimen included total body irradiation (TBI), thiotepa, fludarabine, cyclophosphamide. Grafts contained a megadose of CD34+ cells (mean 12.4 × 106/Kg), Tregs (2 × 106/Kg) and Tcons (0.5-1 × 106/Kg). All patients achieved primary, sustained full-donor engraftment. Only one patient relapsed (5%). The incidence of non-relapse mortality was 15% (3/20 patients). Five/20 patients developed ≥ grade 2 acute Graft versus Host Disease (aGvHD). It resolved in 4 who are alive and disease-free; 1 patient developed chronic GvHD (cGvHD). The probability of GRFS was 60 ± 0.5% (95% CI: 2.1-4.2) (Fig. 6), CRFS was 79 ± 0.9% (95% CI: 3.2-4.9) as 16/20 patients are alive and leukemia-free. The median follow-up was 2.1 years (range 0.5 months-5.1 years). This innovative approach was associated with very promising outcomes of HSCT strategy in pediatric patients.
Asunto(s)
Enfermedad Injerto contra Huésped , Trasplante de Células Madre Hematopoyéticas , Leucemia Mieloide Aguda , Humanos , Niño , Adolescente , Inmunoterapia Adoptiva/efectos adversos , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Enfermedad Injerto contra Huésped/etiología , Leucemia Mieloide Aguda/complicaciones , Células Madre Hematopoyéticas , Acondicionamiento Pretrasplante/efectos adversosRESUMEN
For patients with hematologic malignancies at high risk of relapse who do not have matched donors, a suitable alternative stem cell source is the HLA-haploidentical 2- or 3-loci mismatched family donor who is readily available for nearly all patients. Transplantation across the major HLA barrier is associated with strong T-cell alloreactions, which were originally manifested as a high incidence of severe GVHD and graft rejection. The present overview of the 7th symposium on haplidentical transplantation that took place at the Weizmann Institute on February 2014, shows how these obstacles to successful transplantation can now be overcome. The review also discusses the advantages and drawbacks of current options for full haplotype-mismatched transplantation and highlights innovative approaches for rebuilding immunity, reducing leukemia relapse and improving survival after transplantation. In addition, new modalities for immune tolerance induction following nonmyeloablative conditioning are discussed, showing new options for treatment of elderly patients who cannot tolerate myeloablative conditioning protocols, as well as novel strategies for immune tolerance and chimerism induction as a platform for cell therapy and organ transplantation.
Asunto(s)
Enfermedad Injerto contra Huésped/prevención & control , Antígenos HLA/inmunología , Trasplante de Células Madre Hematopoyéticas/métodos , Leucemia/terapia , Acondicionamiento Pretrasplante/métodos , Aloinjertos , Congresos como Asunto , Supervivencia sin Enfermedad , Enfermedad Injerto contra Huésped/etiología , Enfermedad Injerto contra Huésped/inmunología , Humanos , Leucemia/inmunología , Leucemia/mortalidad , Leucemia/patología , Recurrencia , Tasa de Supervivencia , Linfocitos T/inmunología , Linfocitos T/patologíaRESUMEN
Relapse is still the major cause of failure of allogeneic stem cell transplantation in high-risk acute leukemia patients. Indeed, whoever the donor and whatever the transplantation strategy, post-transplant relapse rates are ~30%, which is hardly satisfactory. The present phase 2 study analyzed the impact of adoptive immunotherapy with naturally occurring FoxP3+ T-regulatory cells (2 × 10(6) per kg) and conventional T lymphocytes (1 × 10(6) per kg) on prevention of GvHD and leukemia relapse in 43 high-risk adults undergoing full-haplotype mismatched transplantation without any post-transplant immunosuppression. Ninety-five percent of patients achieved full-donor type engraftment. Only 6/41 patients (15%) developed ⩾ grade II acute GvHD. Specific CD4(+) and CD8(+) for opportunistic pathogens emerged significantly earlier than after standard T-cell-depleted haplo-transplantation. The probability of disease-free survival was 0.56. At a median follow-up of 46 months (range 18-65 months), only 2/41 evaluable patients have relapsed. The cumulative incidence of relapse was significantly lower than in historical controls (0.05 vs 0.21; P = 0.03). These results demonstrate that the immunosuppressive potential of Tregs can be used to suppress GvHD without loss of the benefits of GvL activity. Humanized murine models provided insights into the mechanisms underlying separation of GvL from GvHD.
Asunto(s)
Traslado Adoptivo , Enfermedad Injerto contra Huésped , Antígenos HLA , Trasplante de Células Madre Hematopoyéticas , Leucemia , Linfocitos T Reguladores/trasplante , Enfermedad Aguda , Adolescente , Adulto , Anciano , Aloinjertos , Supervivencia sin Enfermedad , Femenino , Estudios de Seguimiento , Enfermedad Injerto contra Huésped/mortalidad , Enfermedad Injerto contra Huésped/prevención & control , Humanos , Leucemia/mortalidad , Leucemia/terapia , Masculino , Persona de Mediana Edad , Recurrencia , Tasa de SupervivenciaRESUMEN
In adults, one-haplotype-mismatched haematopoietic SCT (haploidentical HSCT) is associated with slow immune recovery due to decaying thymic function and extensive T-cell depletion of the graft. Although essential for preventing GVHD, T-cell depletion underlies the major reasons for transplant failure: leukemia relapse and infections, with infection-related mortality accounting for about 40% of non-leukemic deaths. Adoptive T-cell therapy would be helpful for these patients but to administer it without causing GVHD, alloreactive T cells need to be eliminated from donor T lymphocytes before infusion. In a preclinical study, to address this problem, we determined the efficacy of photodynamic purging of alloreactive T cells, by investigating combinations of parameters in order to achieve maximum allodepletion, preservation of T-regulatory cells and of pathogen and leukemia-specific T-cell responses in donor-vs-recipient MLR. We also needed to identify an optimal method to quantify the Ag-specific T-cell repertoires. Optimal procedures were identified. In particular, we compared limiting-dilution analyses (LDA) of proliferating T cells with H(3)-thymidine incorporation by bulk T cells and with flow cytometry CD25 expression, which is accepted as a T-cell activation marker. This study demonstrated that LDA is a reliable, predictable and sensitive method for measuring alloreactive, pathogen- and leukemia-specific T-cell frequencies.
Asunto(s)
Trasplante de Células Madre Hematopoyéticas/métodos , Inmunoterapia Adoptiva/métodos , Activación de Linfocitos/inmunología , Depleción Linfocítica/métodos , Estudios de Cohortes , Epítopos de Linfocito T/inmunología , Haplotipos/inmunología , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Humanos , Fotoquimioterapia/métodos , Linfocitos T/inmunologíaRESUMEN
Although CD4(+)/CD25(+) T regulatory cells (T(regs)) are a potentially powerful tool in bone marrow transplantation, a prerequisite for clinical use is a cell-separation strategy complying with good manufacturing practice guidelines. We isolated T(regs) from standard leukapheresis products using double-negative selection (anti-CD8 and anti-CD19 monoclonal antibodies) followed by positive selection (anti-CD25 monoclonal antibody). The final cell fraction (CD4(+)/CD25(+)) showed a mean purity of 93.6% +/- 1.1. Recovery efficiency was 81.52% +/- 7.4. The CD4(+)/CD25(+bright) cells were 28.4% +/- 6.8. The CD4(+)/CD25(+) fraction contained a mean of 51.9% +/- 15.1 FoxP3 cells and a mean of 18.9% +/- 11.5 CD127 cells. Increased FoxP3 and depleted CD127 mRNAs in CD4(+)CD25(+)FoxP3(+) cells were in line with flow cytometric results. In Vbeta spectratyping the complexity scores of CD4(+)/CD25(+) cells and CD4(+)/CD25(-) cells were not significantly different, indicating that T(regs) had a broad T cell receptor repertoire. The inhibition assay showed that CD4(+)/CD25(+) cells inhibited CD4(+)/CD25(-) cells in a dose-dependent manner (mean inhibition percentages: 72.4 +/- 8.9 [ratio of T responder (T(resp)) to T(regs), 1:2]; 60.8% +/- 20.5 (ratio of T(resp) to T(regs), 1:1); 25.6 +/- 19.6 (ratio of T(resp) to T(regs), 1:0.1)). Our study shows that negative/positive T(reg) selection, performed using the CliniMACS device and reagents, enriches significantly CD4(+)CD25(+)FoxP3(+) cells endowed with immunosuppressive capacities. The CD4(+)CD25(+)FoxP3(+) population is a source of natural T(reg) cells that are depleted of CD8(+) and CD4(+)/CD25(-) reacting clones which are potentially responsible for triggering graft-versus-host disease (GvHD). Cells isolated by means of this approach might be used in allogeneic haematopoietic cell transplantation to facilitate engraftment and reduce the incidence and severity of GvHD without abrogating the potential graft-versus-tumour effect.
Asunto(s)
Linfocitos T CD4-Positivos/citología , Factores de Transcripción Forkhead/inmunología , Subunidad alfa del Receptor de Interleucina-2/inmunología , Adulto , Linfocitos T CD4-Positivos/inmunología , Factores de Transcripción Forkhead/genética , Humanos , Separación Inmunomagnética , Inmunofenotipificación/métodos , Subunidad alfa del Receptor de Interleucina-2/análisis , Subunidad alfa del Receptor de Interleucina-5/análisis , Subunidad alfa del Receptor de Interleucina-7/genética , Leucaféresis/métodos , Persona de Mediana Edad , Fenotipo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Cariotipificación Espectral/métodosAsunto(s)
Western Blotting/métodos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Animales , Anticuerpos , Especificidad de Anticuerpos , Humanos , Mutación , Proteínas Nucleares/inmunología , Nucleofosmina , ConejosAsunto(s)
Duplicación de Gen , Genoma Humano , Leucemia Mieloide Aguda/genética , Mutación/genética , Señales de Exportación Nuclear , Proteínas Nucleares/genética , Biología Computacional , Citoplasma/metabolismo , Humanos , Técnicas para Inmunoenzimas , Leucemia Mieloide Aguda/patología , Nucleofosmina , Fragmentos de Péptidos/fisiologíaAsunto(s)
Linfocitos B/metabolismo , Leucemia Mieloide/genética , Mutación , Proteínas Nucleares/genética , Linfocitos T/metabolismo , Transporte Activo de Núcleo Celular , Enfermedad Aguda , Linfocitos B/patología , Biopsia , Médula Ósea/metabolismo , Linaje de la Célula , Citoplasma/metabolismo , Técnica del Anticuerpo Fluorescente , Humanos , Técnicas para Inmunoenzimas , Leucemia Mieloide/diagnóstico , Células Mieloides , Nucleofosmina , Fosfoproteínas/genética , Fracciones Subcelulares , Linfocitos T/patologíaAsunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Cromosomas Humanos Par 12/genética , Cromosomas Humanos Par 5/genética , Leucemia Monocítica Aguda/genética , Proteínas del Tejido Nervioso/genética , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/genética , Translocación Genética , Adulto , Humanos , Hibridación Fluorescente in Situ , Leucemia Monocítica Aguda/diagnóstico , Leucemia Monocítica Aguda/terapia , MasculinoAsunto(s)
Inestabilidad Genómica , Hepatitis C/complicaciones , Linfoma no Hodgkin/genética , Adulto , Anciano , Anciano de 80 o más Años , Aberraciones Cromosómicas , Femenino , Humanos , Hibridación Fluorescente in Situ , Linfoma no Hodgkin/virología , Masculino , Persona de Mediana Edad , Hibridación de Ácido NucleicoAsunto(s)
Cromosomas Humanos Par 16 , Cromosomas Humanos Par 5 , Fusión Génica , Leucemia Mielomonocítica Crónica/genética , Síndrome de Noonan/genética , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/genética , Metiltransferasa de ADN de Sitio Específico (Adenina Especifica)/genética , Transcripción Genética , Adulto , Secuencia de Bases , Femenino , Humanos , Hibridación Fluorescente in Situ , Translocación GenéticaRESUMEN
We investigated genetically affected leukemic cells in FIP1L1-PDGFRA+ chronic eosinophilic leukemia (CEL) and in BCR-ABL1+ chronic myeloid leukemia (CML), two myeloproliferative disorders responsive to imatinib. Fluorescence in situ hybridization specific for BCR-ABL1 and for FIP1L1-PDGFRA was combined with cytomorphology or with lineage-restricted monoclonal antibodies and applied in CML and CEL, respectively. In CEL the amount of FIP1L1-PDGFRA+ cells among CD34+ and CD133+ cells, B and T lymphocytes, and megakaryocytes were within normal ranges. Positivity was found in eosinophils, granulo-monocytes and varying percentages of erythrocytes. In vitro assays with imatinib showed reduced survival of peripheral blood mononuclear cells but no reduction in colony-forming unit growth medium (CFU-GM) growth. In CML the BCR-ABL1 fusion gene was detected in CD34+/CD133+ cells, granulo-monocytes, eosinophils, erythrocytes, megakaryocytes and B-lymphocytes. Growth of both peripheral blood mononuclear cells and CFU-GM was inhibited by imatinib. This study provided evidence for marked differences in the leukemic masses which are targeted by imatinib in CEL or CML, as harboring FIP1L1-PDGFRA or BCR-ABL1.
Asunto(s)
Proteínas de Fusión bcr-abl/análisis , Síndrome Hipereosinofílico/patología , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Células Madre Neoplásicas/enzimología , Proteínas de Fusión Oncogénica/análisis , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/análisis , Factores de Escisión y Poliadenilación de ARNm/análisis , Antígeno AC133 , Antígenos CD/análisis , Antígenos CD34/análisis , Antineoplásicos/uso terapéutico , Benzamidas , Linaje de la Célula , Enfermedad Crónica , Células Clonales/enzimología , Resistencia a Medicamentos , Eosinófilos/enzimología , Eritrocitos/enzimología , Proteínas de Fusión bcr-abl/antagonistas & inhibidores , Glicoforinas/análisis , Glicoproteínas/análisis , Granulocitos/enzimología , Células Madre Hematopoyéticas/enzimología , Humanos , Síndrome Hipereosinofílico/tratamiento farmacológico , Síndrome Hipereosinofílico/enzimología , Síndrome Hipereosinofílico/genética , Mesilato de Imatinib , Inmunofenotipificación , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/enzimología , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Subgrupos Linfocitarios/enzimología , Megacariocitos/enzimología , Monocitos/enzimología , Células Mieloides/enzimología , Proteínas de Fusión Oncogénica/antagonistas & inhibidores , Péptidos/análisis , Piperazinas/uso terapéutico , Inhibidores de Proteínas Quinasas/uso terapéutico , Pirimidinas/uso terapéutico , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/antagonistas & inhibidores , Ensayo de Tumor de Célula Madre , Inactivación del Cromosoma X , Factores de Escisión y Poliadenilación de ARNm/antagonistas & inhibidoresRESUMEN
In haplo-identical hematopoietic transplantation, donor vs. recipient natural killer (NK) cell alloreactivity derives from a mismatch between donor NK clones bearing inhibitory killer cell Ig-like receptors (KIR) for self-HLA class I molecules and their HLA class I ligands (KIR ligands) on recipient cells. When faced with mismatched allogeneic targets, these NK clones sense the missing expression of self-HLA class I alleles and mediate alloreactions. KIR ligand mismatches in the GvH direction trigger donor vs. recipient NK cell alloreactions, which improve engraftment, do not cause GvHD and control relapse in AML patients . The mechanism whereby alloreactive NK cells exert their benefits in transplantation has been elucidated in mouse models. The infusion of alloreactive NK cells ablates (i) leukemic cells, (ii) recipient T cells that reject the graft and (iii) recipient DC that trigger GvHD, thus protecting from GvHD.
Asunto(s)
Trasplante de Células Madre Hematopoyéticas/normas , Antígenos de Histocompatibilidad Clase I/inmunología , Reacción Huésped-Injerto , Células Asesinas Naturales/inmunología , Trasplante Homólogo/inmunología , Animales , Células Clonales , Modelos Animales de Enfermedad , Enfermedad Injerto contra Huésped , Haploidia , Antígenos de Histocompatibilidad Clase I/genética , Humanos , Inmunoterapia Adoptiva , RatonesAsunto(s)
Fusión Génica , Síndrome Hipereosinofílico/genética , ARN Mensajero/genética , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/genética , Tropomiosina/genética , Cromosomas Humanos Par 1 , Cromosomas Humanos Par 10 , Cromosomas Humanos Par 5 , Enfermedad Crónica , Cartilla de ADN , Humanos , Síndrome Hipereosinofílico/patología , Hibridación Fluorescente in Situ , Datos de Secuencia Molecular , Neoplasias de la Tiroides/genéticaRESUMEN
Fluorescence in situ hybridization and comparative genomic hybridization characterized 6p rearrangements in eight primary and in 10 secondary myeloid disorders (including one patient with Fanconi anemia) and found different molecular lesions in each group. In primary disorders, 6p abnormalities, isolated in six patients, were highly heterogeneous with different breakpoints along the 6p arm. Reciprocal translocations were found in seven. In the 10 patients with secondary acute myeloid leukemia/myelodysplastic syndrome (AML/MDS), the short arm of chromosome 6 was involved in unbalanced translocations in 7. The other three patients showed full or partial trisomy of the 6p arm, that is, i(6)(p10) (one patient) and dup(6)(p) (two patients). In 5/7 patients with unbalanced translocations, DNA sequences were overrepresented at band 6p21 as either cryptic duplications (three patients) or cryptic low-copy gains (two patients). In the eight patients with cytogenetic or cryptic 6p gains, we identified a common overrepresented region extending for 5-6 megabases from the TNF gene to the ETV-7 gene. 6p abnormalities were isolated karyotype changes in four patients. Consequently, in secondary AML/MDS, we hypothesize that 6p gains are major pathogenetic events arising from acquired and/or congenital genomic instability.
Asunto(s)
Cromosomas Humanos Par 6/genética , Leucemia Mieloide/genética , Síndromes Mielodisplásicos/genética , Neoplasias Primarias Secundarias/genética , Translocación Genética/genética , Enfermedad Aguda , Adulto , Anciano , Anciano de 80 o más Años , Análisis Citogenético , Femenino , Humanos , Hibridación Fluorescente in Situ , Leucemia Mieloide/diagnóstico , Masculino , Persona de Mediana Edad , Síndromes Mielodisplásicos/diagnóstico , Neoplasias Primarias Secundarias/diagnóstico , Sensibilidad y EspecificidadRESUMEN
Mutations in exon 12 of the nucleophosmin (NPM1) gene occur in about 60% of adult AML with normal karyotype. By exploiting a specific feature of NPM1 mutants, that is insertion at residue 956 or deletion/insertion at residue 960, we developed highly sensitive, real-time quantitative (RQ) polymerase chain reaction (PCR) assays, either in DNA or RNA, that are specific for various NPM1 mutations. In all 13 AML patients carrying NPM1 mutations at diagnosis, cDNA RQ-PCR showed >30 000 copies of NPM1-mutated transcript. A small or no decrease in copies was observed in three patients showing partial or no response to induction therapy. The number of NPM1-mutated copies was markedly reduced in 10 patients achieving complete hematological remission (five cases: <100 copies; five cases: 580-5046 copies). In four patients studied at different time intervals, the number of NPM1 copies closely correlated with clinical status and predicted impending hematological relapse in two. Thus, reliable, sensitive RQ-PCR assays for NPM1 mutations can now monitor and quantify MRD in AML patients with normal karyotype and NPM1 gene mutations.