Measurable residual disease monitoring by ddPCR in the early posttransplant period complements the traditional MFC method to predict relapse after HSCT in AML/MDS: a multicenter retrospective study.

BACKGROUND: Droplet digital PCR (ddPCR) is widely applied to monitor measurable residual disease (MRD). However, there are limited studies on the feasibility of ddPCR-MRD monitoring after allogeneic hematopoietic stem cell transplantation (allo-HSCT), especially targeting multiple molecular markers simultaneously. METHODS: Our study collected samples from patients with acute myeloid leukemia (AML) or high-risk myelodysplastic syndrome (MDS) in complete remission after allo-HSCT between January 2018 and August 2021 to evaluate whether posttransplant ddPCR-MRD monitoring can identify patients at high risk of relapse. RESULTS: Of 152 patients, 58 (38.2%) were MRD positive by ddPCR within 4 months posttransplant, with a median variant allele frequency of 0.198%. The detectable DTA mutations (DNMT3A, TET2, and ASXL1 mutations) after allo-HSCT were not associated with an increased risk of relapse. After excluding DTA mutations, patients with ddPCR-MRD positivity had a significantly higher cumulative incidence of relapse (CIR, 38.7% vs. 9.7%, P < 0.001) and lower rates of relapse-free survival (RFS, 55.5% vs. 83.7%, P < 0.001) and overall survival (OS, 60.5% vs. 90.5%, P < 0.001). In multivariate analysis, ddPCR-MRD positivity of non-DTA genes was an independent adverse predictor for CIR (hazard ratio [HR], 4.02; P < 0.001), RFS (HR, 2.92; P = 0.002) and OS (HR, 3.12; P = 0.007). Moreover, the combination of ddPCR with multiparameter flow cytometry (MFC) can further accurately identify patients at high risk of relapse (F+/M+, HR, 22.44; P < 0.001, F+/M-, HR, 12.46; P < 0.001 and F-/M+, HR, 4.51; P = 0.003). CONCLUSION: ddPCR-MRD is a feasible approach to predict relapse after allo-HSCT in AML/MDS patients with non-DTA genes and is more accurate when combined with MFC. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT06000306. Registered 17 August 2023 -Retrospectively registered ( https://clinicaltrials.gov/study/NCT06000306?term=NCT06000306&rank=1 ).

The effectiveness of blinatumomab in clearing measurable residual disease in pediatric B-cell acute lymphoblastic leukemia patients detected by next-generation sequencing.

BACKGROUND: Blinatumomab improved survival outcomes in B-cell acute lymphoblastic leukemia (B-ALL) patients with measurable residual disease (MRD) <10-4 . However, data on blinatumomab clearing MRD with high sensitivity of 10-6 remain scarce. This study evaluates the effectiveness of blinatumomab in eradicating extremely low level (up to <10-6 ) of MRD, as detected by next-generation sequencing (NGS), in children with B-ALL. METHODS: Patients (n = 19) whose MRD was undetectable by multiparameter flow cytometry (MFC) (sensitivity of 10-4 ) but detectable by NGS after chemotherapy and followed by blinatumomab consolidation were included retrospectively. RESULTS: After one course of blinatumomab, 13/19 patients (68%) successfully achieved NGS-MRD clearance (undetectable). With a median follow-up of 13.3 months, three of patients who were NGS-MRD positive relapsed within 1.8 months, while another three remained complete remission. CONCLUSIONS: Our study was the first to demonstrate that blinatumomab could further eradicate MRD after patients achieve MFC-MRD undetectable in B-ALL patients.

Minimal residual disease detection by next-generation sequencing of different immunoglobulin gene rearrangements in pediatric B-ALL.

While the prognostic role of immunoglobulin heavy chain locus (IGH) rearrangement in minimal residual disease (MRD) in pediatric B-acute lymphoblastic leukemia (B-ALL) has been reported, the contribution of light chain loci (IGK/IGL) remains elusive. This study is to evaluate the prognosis of IGH and IGK/IGL rearrangement-based MRD detected by next-generation sequencing in B-ALL at the end of induction (EOI) and end of consolidation (EOC). IGK/IGL rearrangements identify 5.5% of patients without trackable IGH clones. Concordance rates for IGH and IGK/IGL are 79.9% (cutoff 0.01%) at EOI and 81.0% (cutoff 0.0001%) at EOC, respectively. Patients with NGS-MRD < 0.01% at EOI or <0.0001% at EOC present excellent outcome, with 3-year event-free survival rates higher than 95%. IGH-MRD is prognostic at EOI/EOC, while IGK-MRD at EOI/EOC and IGL-MRD at EOI are not. At EOI, NGS identifies 26.2% of higher risk patients whose MRD < 0.01% by flow cytometry. However, analyzing IGK/IGL along with IGH fails to identify additional higher risk patients both at EOI and at EOC. In conclusion, IGH is crucial for MRD monitoring while IGK and IGL have relatively limited value.

Ruxolitinib Treatment of Steroid-Refractory Graft-versus-Host Disease in Children: A Case Series and Review of the Literature.

BACKGROUND: Ruxolitinib has been increasingly used in the treatment of steroid-refractory graft-versus-host disease (SR-GVHD) in allogeneic hematopoietic stem cell transplantation (allo-HSCT) patients. However, there are limited data on the use of ruxolitinib in children. OBJECTIVE: This study aimed to assess the efficacy and toxicity of ruxolitinib in the treatment of SR-GVHD in children. PATIENTS AND METHODS: Data of patients who suffered from SR-GVHD after allo-HSCT and received ruxolitinib treatment between June 2018 and December 2020 at our center were analyzed retrospectively. The characteristics of patients, the dosage of ruxolitinib, the response, toxicity, and the survival data were collected. RESULTS: A total of 14 pediatric patients were diagnosed with SR-GVHD after allo-HSCT and received ruxolitinib. The age of the patients ranged from 3 months to 12 years old. The dosage of ruxolitinib ranged from 2.5 mg twice daily to 7.5 mg twice daily, mainly according to patient weight. The total overall response rate (ORR) was 64.3% (9/14), with 63.6% (7/11) in aGVHD and 67% (2/3) in cGVHD. Of the 14 patients, adverse effects were observed in 9 patients (64.3%), including cytopenia, infection, and elevated alanine aminotransferase. In addition, seven reports on the treatment of SR-GVHD in children with ruxolitinib were included for systematic analysis, with the ORR ranging from 45 to 87% in aGVHD and 70-91% in cGVHD. CONCLUSION: Given its effectiveness and safety, ruxolitinib could be used to treat SR-GVHD in children after HSCT.

[Clinical and laboratory characteristics of chronic active Epstein-Barr virus infection in children].

OBJECTIVE: To study the clinical and laboratory characteristics of chronic active Epstein-Barr virus (EBV) infection (CAEBV) in children and to provide a basis for the diagnosis and treatment of CAEBV. METHODS: The clinical data of 13 children with CAEBV, as well as 15 cases of acute EBV infection (AEBV) as controls, were analyzed, including clinical manifestations, EBV antibodies, EBV DNA, and peripheral blood lymphocyte subsets. RESULTS: Both groups of patients had infectious mononucleosis-like symptoms such as fever, hepatomegaly, splenomegaly, and lymphadenectasis, but CAEBV patients had a longer course of disease and continuous and recurrent symptoms. Compared with the AEBV group, the CAEBV group had a significantly higher EBV DNA load in peripheral blood (P<0.05), a significantly higher VCA-IgG titer (P<0.05), and significantly lower numbers of white blood cells, lymphocytes, B cells, total T cells, CD4+ T cells, and CD8+ T cells in peripheral blood (P<0.05). Among 13 CAEBV patients followed up, 8 cases died, 2 cases showed an improvement, 2 cases had a recurrence, and 1 case was lost to follow-up after being transferred to another hospital. All the AEBV patients were cured and had no recurrence during the one-year follow-up. CONCLUSIONS: The clinical manifestations of CAEBV vary in children. It is difficult to distinguish CAEBV from AEBV early. More attention should be paid to CAEBV because of its severe complications, poor prognosis, and high mortality. Measurement of EBV DNA load, VCA-IgG titer, and lymphocyte subsets in peripheral blood may be helpful in the diagnosis and differential diagnosis of CAEBV.

[Diagnostic significance of Th1/Th2 cytokine pattern in childhood hemophagocytic lymphohistiocytosis].

OBJECTIVE: To illustrate the diagnostic value of Th1/Th2 cytokine pattern in childhood hemophagocytic lymphohistiocytosis (HLH) and its diagnostic accuracy. METHOD: The BD(TM) CBA Human Th1/Th2 Cytokine Kit II was used to measure the serum Th1 and Th2 cytokines, including Interferon-gamma (IFN-γ), tumor necrosis factor (TNF), interleukin (IL)-10, IL-6, IL-4 and IL-2 in 50 patients with de novo HLH admitted to our hospital from Oct. 2005 to Aug. 2009. The above cytokine levels were also determined in 250 healthy volunteers and 235 patients with sepsis as controls. RESULT: The primary features of these patients were prolonged high-grade fever (50/50), hepatomegaly (44/50), splenomegaly (38/50), hemocytopenia (47/50), hyperferritinemia (49/50), coagulopathy (44/50), hemophagocytosis in bone marrow (42/50), liver dysfunction (42/50) and hypertriglyceridemia (42/50). The IFN-γ, TNF, IL-10, IL-6, IL-4 and IL-2 levels for healthy children were (4.6 ± 1.8) ng/L, (4.0 ± 1.2) ng/L, (6.5 ± 1.3) ng/L, (6.0 ± 1.5) ng/L, (2.9 ± 0.8) ng/L and (2.6 ± 0.7) ng/L, while the median levels of them in acute phase of HLH children were 1138.5 (49.2 - 5000.0) ng/L, 3.4 (1.0 - 25.1) ng/L, 740.5 (26.5 - 5000.0) ng/L, 66.1 (3.9 - 4472.6) ng/L, 3.9 (1.0-32.8) ng/L and 4.0 (1.0 - 51.1) ng/L, respectively. The cytokine levels decreased to 9.1 (1.9 - 180.1) ng/L, 2.9 (1.0 - 11.0) ng/L, 11.4 (2.9 - 184.2) ng/L, 6.5 (1.0 - 44.8) ng/L, 2.7 (1.0 - 6.5) ng/L and 4.1 (1.0 - 12.0) ng/L respectively after remission. The IFN-γ, IL-10 and IL-6 levels in acute phase were significantly higher than those after remission and those of the healthy control (P all < 0.001). IL-4, IL-2 and TNF slightly elevated or at normal range in acute phase of HLH. The patients with sepsis showed a different cytokine pattern, with an extremely high level of IL-6 (median: 251.3 ng/L, range: 8.4- > 5000.0 ng/L) and moderately elevated level of IL-10 (median: 46.5 ng/L, range: 3.1 - 5000.0 ng/L), whereas IFN-γ was only slightly elevated (median: 9.2 ng/L, range: 1.3 - 498.8 ng/L). When the criteria for HLH set as the following: IFN-γ > 100 ng/L, IL-10 > 60 ng/L and the concentration of IFN-γ higher than that of IL-6, the specificity reached as high as 98.7% and the sensitivity was 88.0% for the diagnosis of HLH among patients with HLH and sepsis. Meanwhile, the positive predictive value (PPV) and negative predictive value (NPV) could reach 93.6% and 97.5%, respectively. CONCLUSION: The significant increase of IFN-γ and IL-10 with slightly increased level of IL-6 is a sensitive and specific cytokine pattern for childhood HLH, which is helpful for its diagnosis and differential diagnosis.