Contribuição da citometria de fluxo de alta sensibilidade no estudo da doença residual mensurável em leucemias agudas e seu impacto no transplante de células tronco hematopoiéticas

Abstract

Resumo: A presença de doença residual mensurável (DRM) antes do transplante de células-tronco hematopoiéticas (TCTH) está associada a piores desfechos em leucemias agudas de alto risco. No entanto, os melhores momentos para avaliação pós-TCTH e o impacto clínico de baixos níveis de DRM ainda são pouco definidos. Métodos: Este estudo prospectivo avaliou a DRM por citometria de fluxo multiparamétrica (CFM) de alta sensibilidade antes e após o TCTH (dias +30, +60 e +100), com o objetivo de analisar a cinética de eliminação da doença e sua relação com os desfechos do transplante. Pacientes com leucemia linfoblástica aguda B (LLA-B) foram analisados quanto a características genéticas e fenotípicas, e, nos casos de LLA Philadelphia-positiva (Ph+), a CFM foi comparada à PCR quantitativa (RQ-PCR) para BCR::ABL1. Resultados: Foram incluídos 77 pacientes consecutivos sendo que 27 (45,5%) apresentavam DRM negativa <0,01% (DRM–) e 35 (55,5%) DRM positiva >0,01% (DRM+) no momento imediatamente anterior ao TCTH. A presença de DRM+ associou-se à menor sobrevida global (SG: 87,9% DRM– vs. 54,0% DRM+) e menor sobrevida livre de eventos (SLE: 85,3% DRM– vs. 51,1% DRM+), ambas p= 0,001. A incidência de recidiva da doença foi maior nos pacientes com DRM+ (17,5% vs. 2,6%; p=0,049), assim como a mortalidade não relacionada à recidiva (MNRR: 31,4% DRM+ vs. 12,1% DRM–; p=0,019). Oito pacientes DRM+ (22,9%) e três DRM– (7,1%) recaíram após o TCTH (p=0,02). A DRM foi reavaliada em 30 pacientes no D+30 (38,9%), 27 no D+60 (35,0%) e 60 no D+100 (77,9%). Análises de risco competitivo revelaram que persistência de DRM no D+100 e doença em estágio avançado estavam associadas a pior SG, SLE e maior risco de recidiva. A MNRR também variou por tipo de leucemia, sendo significativamente mais alta nos pacientes DRM+ (LLA: 50% DRM+ vs. 18,9% DRM–; LMA: 21,7% DRM+ vs. 0% DRM–; p=0,0158). Nos casos de LLA Ph+, a CFM apresentou forte correlação com a RQ-PCR (r=0,7801; IC95%: 0,69–0,84; p < 0,001), com concordância em 88% dos casos (?=0,761). A CFM detectou DRM em 82,9% das amostras com transcritos >0,01%. Fenotipicamente, 77,7% dos casos de LLA Ph+ apresentaram blastos CD34+CD38–/fraco vs. 20,2% nos demais subtipos (p<0,0001), além de maior expressão combinada de CD66c, CD73 e CD304 (p < 0,001). Conclusões: A citometria de fluxo de alta sensibilidade demonstrou desempenho comparável ao RQ PCR, consolidando-se como ferramenta complementar na avaliação da DRM na LLA-B. A DRM pré-TCTH foi associada a pior prognóstico, e persistência de DRM no D+100 previu com alta especificidade a recidiva da leucemia. Esses achados reforçam a importância da incorporação da cinética da DRM peri-transplante por citometria de fluxo no manejo das leucemias agudas

Abstract: Measurable residual disease (MRD) monitoring is essential for the management of acute leukemias. While BCR::ABL1 transcript quantification is the gold standard for MRD assessment in Philadelphia-positive acute lymphoblastic leukemia (Ph+ ALL), multiparametric flow cytometry (MFC) is widely used in other genetic subtypes. The presence of MRD before hematopoietic stem cell transplantation (HSCT) is associated with adverse outcomes in high-risk acute leukemia patients, but the clinical relevance of post-transplant MRD remains unclear. Methods: In this prospective real-world study, MRD was evaluated using highly sensitive MFC before and after HSCT (on days +30, +60, and +100) in 77 patients with acute leukemias to investigate MRD kinetics and their impact on outcomes. 106 B-ALL patients were also assessed for genetic subtypes, clinical features, and phenotypic markers. In Ph+ ALL, MFC was compared with BCR::ABL1 quantification by real-time quantitative PCR (RQ-PCR) to validate its local applicability. Results: Pre-transplant MRD was assessed in 77 patients, identifying 42 MRD-negative (MRD-) and 35 MRD-positive (MRD+) individuals. MRD+ status correlated with inferior outcomes: overall survival (OS) was 87.9% in MRD- vs. 54.0% in MRD+ patients, and event-free survival (EFS) was 85.3% vs. 51.1% (p = 0.001). Relapse incidence was higher in MRD+ patients (17.5% vs. 2.6%; p = 0.049), as was non-relapse mortality (NRM: 31.4% vs. 12.1%; p = 0.019). Relapses occurred in eight MRD+ (22.9%) and three MRD- patients (7.1%) post-HSCT (p = 0.02). Post-transplant MRD was assessed in 30 patients on day +30, 27 on day +60, and 60 on day +100. Persistent MRD on day +100 and advanced disease status were associated with lower OS and EFS and higher relapse risk. NRM was also higher in MRD+ cases, both in ALL (50.0% vs. 18.9%) and AML (21.7% vs. 0.0%; p = 0.0158). In Ph+ ALL, there was strong concordance between MFC and RQ-PCR results. Correlation between blast percentages by MFC and BCR::ABL1 transcript levels was high (r = 0.7801; 95% CI: 0.69–0.84; p < 0.001), with 88% agreement (? = 0.761). MFC detected MRD in 82.9% of samples with transcript levels >0.01%. Phenotypically, Ph+ ALL cases frequently exhibited CD34+CD38-/dim blasts (77.7% vs. 20.2% in other B-ALL subtypes; p < 0.0001) and showed higher expression of CD66c/CD73/CD304 combinations (all p < 0.001). Conclusions: High sensitive MFC showed comparable performance to RQ-PCR and can serve as a complementary method for MRD monitoring in acute leukemia, including Ph+ ALL. Pre HSCT MRD effectively identified patients at high risk of poor outcomes, while MRD persistence on day +100 predicted a relapse. These findings support the importance of incorporating peri-transplant MRD kinetics into the routine management of acute leukemias, particularly in low- and middle-income countries