Immune recovery and the role of recent thymic emigrated T lymphocytes after pediatric hematopoietic stem cell transplantation.

BACKGROUND AIMS: Adequate re-establishment of thymopoiesis is critical for long-term immune reconstitution after hematopoietic cell transplantation (HCT), potentially impacting patient survival rates. This study aimed to evaluate immune reconstitution in pediatric HCT recipients by quantifying recent thymic emigrants (RTEs), specifically CD3+CD31+CD45RA+ cells. METHODS: We conducted a retrospective analysis of 186 pediatric patients transplanted between 2013 and 2020, undergoing their first allogeneic HCT, who were alive in the first 100 days after transplantation with immune recovery evaluation at three time points: day 100, day 180 and day 360 after HCT. We analyzed the distribution of peripheral blood subsets of T, B and natural killer lymphocytes and assessed the impact of underlying disease, HCT type, stem cell source, recipient age, conditioning regimen, graft-versus-host disease (GVHD) occurrence and cytomegalovirus (CMV) reactivation on immune recovery. RESULTS: At day 100, patients under 10 years exhibited higher RTE CD4+ and CD8+CD31+CD45RA+ counts compared with older patients (5.3 versus 2.2 cells/µL, P = 0.022 and 48 versus 72.8 cells/µL, P = 0.049, respectively). Patients with haploidentical HCT had lower RTE CD4+ counts compared with those with unrelated or related donors (2.4 versus 4.4 versus 7.9 cells/µL, P = 0.024). Administration of rabbit anti-thymocyte globulin negatively impacted RTE CD4+ production (median, 6.5 versus 2.4 cells/µL, P = 0.007). At day 180, the presence of GVHD had a negative influence on RTE production (11.7 versus 56.8 cells/µL, P < 0.001), particularly higher-grade acute GVHD (without, 56.8 cells/µL, grade 1-2, 28.1 cells/µL, grade 3-4, 6.0 cells/µL, P < 0.001). Patients with CMV reactivation had higher CD8+CD31+CD45RA+ compared with those without reactivation (median, 204.6 versus 100.2 cells/µL, P = 0.022). At day 360, no variables significantly affected RTE recovery. Overall survival at 5-year follow-up was 87.7%, with a median of 1170 days (range, 122-3316). Multivariate analysis showed that age >10 years (P = 0.038), negative CMV donor serology (P = 0.0029) and acute GVHD (P = 0.0026) had a negative impact on survival. CONCLUSIONS: This study highlights variations in RTE production based on patient age, donor type and immunosuppression regimen employed.

The Role of Molecular or Cytogenetic Response as a Favorable Prognostic Factor Before Hematopoietic Stem Cell Transplantation for Chronic Myeloid Leukemia.

Tyrosine kinase inhibitors (TKIs) have revolutionized therapy for patients with chronic myeloid leukemia (CML) over the last two decades. However, some patients still do not achieve an adequate response to these drugs, and hematopoietic stem cell transplantation (HSCT) is indicated in this scenario. We present the results of a 20-year follow-up study of 70 patients who underwent transplantation after TKI failure. The primary objective of this study was to evaluate overall survival (OS) and the secondary objective was to evaluate the outcomes of relapse-free survival (RFS), GVHD-free, relapse-free survival (GFRS) and the incidences of relapse (RI), non-relapse mortality (NRM), acute and chronic GVHD. Median survival was 11 years, with a 1-year OS of 70% (57.8 to 79.3) and a 5-year OS of 57.7% (45.1 to 68.5). The estimated 5-year OS was not different for CP1 (60%) versus advanced stages (45%); P = .60. The degree of response immediately before transplantation was directly associated with worse outcomes [HR 5.89 (1.19-29.16); P = .03] for patients with only a hematological response compared with patients with a cytogenetic or molecular response. This study corroborates the potential of HSCT in the scenario of therapeutic failure and highlights the role of molecular or cytogenetic response as a potential target to be achieved before transplantation.

Colonization by multidrug-resistant bacteria in hematological patients undergoing hematopoietic stem cell transplantation and clinical outcomes: A single-center retrospective cohort study.

BACKGROUND: Bloodstream infections are a leading cause of death in patients who undergo hematopoietic stem cell transplantation (HSCT) and are more severe when caused by multidrug-resistant (MDR) bacteria. This study proposed to investigate if colonization by MDR bacteria negatively affects the clinical outcomes in hematological patients after HSCT, as well as to evaluate possible risk factors for death due to bacteremia by the same colonizing agent. METHODS: A single-center retrospective cohort study was conducted with 405 hematological patients submitted to a single HSCT procedure between 2015 and 2021. Patients were classified as colonized (n = 132) or noncolonized (n = 273) based on the surveillance cultures from D-30 to D+30 of transplantation, and their relevant clinical and laboratory data were collected until D+100. RESULTS: Colonization by MDR bacteria increased blood culture positivity by all micro-organisms and also specifically by MDR bacteria, with a more pronounced effect when caused by carbapenemase-producing Klebsiella pneumoniae. Patients colonized with carbapenem-resistant K. pneumoniae had increased overall mortality (HR = 4.07, 95% CI 1.85-8.91, P = .0005) and had prolonged hospital length of stay in the context of autologous transplantation. Risk factors for death due to bacteremia by the same colonizing agent were neutropenia, colonization by carbapenem-resistant K. pneumoniae and use of high-dose total body irradiation in conditioning. CONCLUSION: Hematological patients colonized by MDR bacteria presented a higher incidence of bloodstream infections, and colonization by carbapenemase-producing K. pneumoniae was associated with reduced overall survival.

Donor-Specific HLA Antibodies Are Associated with Graft Failure and Delayed Hematologic Recovery after Unrelated Donor Hematopoietic Cell Transplantation.

Graft failure (GF) is one of the major concerns after allogeneic hematopoietic cell transplantation (allo-HCT) and remains a significant cause of morbidity and mortality. Although previous reports have associated the presence of donor-specific HLA antibodies (DSAs) with an increased risk of GF after unrelated donor allo-HCT, recent studies have failed to confirm this association. We sought to validate the presence of DSAs as a risk factor for GF and hematologic recovery in the unrelated donor allo-HCT setting. We retrospectively evaluated 303 consecutive patients who underwent their first unrelated donor allo-HCT at our institution between January 2008 and December 2017. DSA evaluation was performed using 2 single antigen bead (SAB) assays; DSA titration with 1:2, 1:8, and 1:32 dilutions; C1q-binding assay; and absorption/elution protocol to assess possible false-positive DSA reactivity. The primary endpoints were neutrophil and platelet recovery and GF, and the secondary endpoint was overall survival. Multivariable analyses were performed using Fine-Gray competing risks regression and Cox proportional hazards regression models. The median patient age was 14 years (range, 0 to 61 years), 56.1% were male, and 52.5% underwent allo-HCT for nonmalignant disease, Eleven patients (3.63%) were DSA-positive, including 10 with preexisting DSAs and 1 with post-transplantation de novo DSAs. Nine patients had 1 DSA, 1 patient had 2 DSAs, and 1 patient had 3 DSAs, with a median mean fluorescent intensity (MFI) of 4334 (range, 588 to 20,456) and 3581 (range, 227 to 12,266) in LABScreen and LIFECODES SAB assays, respectively. Overall, 21 patients experienced GF, including 12 with primary graft rejection, 8 with secondary graft rejection, and 1 with primary poor graft function. The cumulative incidence of GF was 4.0% (95% confidence interval [CI], 2.2% to 6.6%) at 28 days, 6.6% (95% CI, 4.2% to 9.8%) at 100 days, and 6.9% (95% CI, 4.4% to 10.2%) at 365 days. In the multivariable analyses, DSA-positive patients had significantly delayed neutrophil recovery (subdistribution hazard ratio [SHR], .48; 95% CI, .29 to .81; P = .006) and platelet recovery (SHR, .51; 95% CI, .35 to .74; P = .0003) compared to patients without DSAs. In addition, only DSAs were significant predictors of primary GF at 28 days (SHR, 2.78; 95% CI, 1.65 to 4.68; P = .0001). The Fine-Gray regression also demonstrated that the presence of DSAs was strongly associated with a higher incidence of overall GF (SHR, 7.60; 95% CI, 2.61 to 22.14; P = .0002). DSA-positive patients with GF had significantly higher median MFI values than DSA-positive patients who achieved engraftment in the LIFECODES SAB assay using neat serum (10,334 versus 1250; P = .006) and in the LABScreen SAB at 1:32 dilution (1627 versus 61; P = .006). All 3 patients with C1q-positive DSAs failed to engraft. DSAs were not predictive of inferior survival (HR, .50; 95% CI, .20 to 1.26; P = .14). Our results validate the presence of DSAs as a significant risk factor for GF and delayed hematologic recovery after unrelated donor allo-HCT. Careful pretransplantation DSA evaluation may optimize unrelated donor selection and improve allo-HCT outcomes.

Investigation of mutations in Fanconi anemia genes and malignancy predisposition in Brazilian patients.

INTRODUCTION: This study proposed to identify Fanconi anemia (FA) mutations in Brazilian patients and to investigate their impact on clinical manifestations and malignancies onset. METHODS: A total of 116 patients were screened for nine mutations in FANCA, FANCC, FANCG. Those with no mutations were investigated by multiplex ligation-dependent probe amplification (MLPA) and Sanger sequencing for FANCA, FANCC, FANCE, FANCF, FANCG, FANCD1/BRCA2. RESULTS: Genetic subtype was identified in 107/116 (78 FA-A, 8 FA-C, 13 FA-G, 8 FA-E), with only one mutation in 1/116, and no mutations in 9/116 patients. Before hematopoietic cell transplantation (HCT), malignancies were detected in 16/116 patients (14/78 FA-A, 01/08 FA-C, 01/08 FA-E), and 12 of them were hematological. Observed to expected ratio (O/E) of hematologic malignancy was 303.7 (95% CI = 148.6-458.7). CONCLUSION: This study allowed the identification of biallelic mutations in 91.4% of patients. FANCG and FANCC mutations had significantly earlier bone marrow failure onset, and FANCG severe cytopenia at diagnosis. Despite the inherent limitations of the small number of malignancy events in each genetic subtype, the hematologic malignancies O/E ratio was very high. Cumulative incidence of malignancy before HCT was higher in the third and fourth decades of life, considering HCT and death as competing risks. The cumulative incidence of HCT increased during the first decade, competing with malignancy development.

Immune reconstitution after allogenic stem cell transplantation: An observational study in pediatric patients.

INTRODUCTION: The immune reconstitution (IR) after the allogenic hematopoietic stem cell transplantation (allo-HSCT) is a progressive process intrinsically correlated to the therapeutic success. It is essential to understand the interfering factors in IR to prevent the HSCT-related mortality. METHODS: We retrospectively evaluated the clinical outcomes, absolute lymphocyte counts (ALCs) and lymphocyte subtypes at different time-points of 111 pediatric patients with allogeneic HSCT for malignant and non-malignant diseases from 2013 to 2018. RESULTS: The ALCs gradually increased on D+30, D+100, and D+180 (medians 634/µL, 1022/µL and 1541/µL, respectively). On D+100, the CD3+CD8+ achieved the highest recovery rate (68%), followed by the CD16+CD56+ (47%), CD3+CD4+ (39%) and CD19+ (8%). The adequate ALC recovery was associated with age < 8 years, bone marrow grafts, myeloablative conditioning, non-use of serotherapy and non-haploidentical donors. The ALC and CD3+CD8+ on D+100 counts were higher in patients with the cytomegalovirus infection. The CD3+CD4+ recovery was associated with an age < 8 years, a non-malignant disease and a lower incidence of acute graft-versus-host disease ≥ grade 2. Furthermore, the ALC recovery on D+100 resulted in a higher overall survival, regardless of the disease type (HR 3.65, 1.05 - 12.71, p = 0.04). CONCLUSION: Several factors influenced the IR after the allo-HSCT. The ALC ≥ 500/µL on D+100 was a simple IR predictor of survival, easily available to resource-limited centers.

Immune reconstitution after allogenic stem cell transplantation: An observational study in pediatric patients

Abstract Introduction The immune reconstitution (IR) after the allogenic hematopoietic stem cell transplantation (allo-HSCT) is a progressive process intrinsically correlated to the therapeutic success. It is essential to understand the interfering factors in IR to prevent the HSCT-related mortality. Methods We retrospectively evaluated the clinical outcomes, absolute lymphocyte counts (ALCs) and lymphocyte subtypes at different time-points of 111 pediatric patients with allogeneic HSCT for malignant and non-malignant diseases from 2013 to 2018. Results The ALCs gradually increased on D+30, D+100, and D+180 (medians 634/μL, 1022/μL and 1541/μL, respectively). On D+100, the CD3+CD8+ achieved the highest recovery rate (68%), followed by the CD16+CD56+ (47%), CD3+CD4+ (39%) and CD19+ (8%). The adequate ALC recovery was associated with age < 8 years, bone marrow grafts, myeloablative conditioning, non-use of serotherapy and non-haploidentical donors. The ALC and CD3+CD8+ on D+100 counts were higher in patients with the cytomegalovirus infection. The CD3+CD4+ recovery was associated with an age < 8 years, a non-malignant disease and a lower incidence of acute graft-versus-host disease ≥ grade 2. Furthermore, the ALC recovery on D+100 resulted in a higher overall survival, regardless of the disease type (HR 3.65, 1.05 - 12.71, p= 0.04). Conclusion Several factors influenced the IR after the allo-HSCT. The ALC ≥ 500/μL on D+100 was a simple IR predictor of survival, easily available to resource-limited centers.

Untreated Donor-Specific HLA Antibodies Are Associated With Graft Failure and Poor Survival After Haploidentical Transplantation With Post-Transplantation Cyclophosphamide in Pediatric Patients With Nonmalignant Disorders.

Donor-specific HLA antibodies (DSAs) have been recognized as a major risk factor for graft failure (GF) in adult patients with malignancies undergoing haploidentical transplantation with post-transplantation cyclophosphamide (haplo-PTCy). However, the impact of DSAs after pediatric haplo-PTCy for nonmalignant disorders (NMDs) has been poorly reported. We sought to investigate whether preexisting DSAs adversely affect pediatric haplo-PTCy outcomes. We retrospectively analyzed 59 pediatric patients (≤21 years) who received their first haplo-PTCy for NMDs from January 2008 to December 2017. DSA testing was performed using single antigen beads, and mean fluorescence intensity (MFI) >1000 was considered positive, and MFI <1000 and >500 was considered potentially positive, based on HLA epitope reactivity patterns. Primary endpoints were neutrophil and platelet recovery and GF, whereas secondary endpoints included event-free and overall survival. Multivariable analyses were performed using Fine-Gray competing risk regression or Cox proportional hazards regression models. The median age was 10 years, and 66.1% were male. Main indications for haplo-PTCy were Fanconi anemia (n = 33) and severe aplastic anemia (n = 11). All patients received bone marrow as the graft source, and most patients (91.5%) received fludarabine-based conditioning. Overall, 15 patients (25.4%) had DSAs >500 MFI. Four patients had false-positive DSAs with median MFI of 1762. Of the 11 patients with true-positive DSA reactivity, 5 had 1 DSA, 5 had 2 DSAs, and 1 had 3 DSAs, with median MFI of 2372 (range 527-24,200). Four patients received desensitization therapy with rituximab and plasmapheresis, whereas 7 patients were untreated. All patients with treated DSAs achieved donor engraftment. In the multivariable analyses, untreated DSAs were associated with lower neutrophil recovery (subdistribution hazard ratio [SHR] = 0.15; 95% confidence interval [CI], 0.03-0.63; P = .001), increased GF (SHR = 20.57; 95% CI, 6.57-64.43; P < .001), inferior event-free survival (hazard ratio [HR] = 10.09; 95% CI, 3.37-30.22; P < .001), and poor overall survival (HR 5.56; 95% CI, 1.92-16.12; P = .002). Both treated DSAs (SHR = 0.26; 95% CI, 0.10-0.68; P = .006) and untreated DSAs (SHR = 0.13; 95% CI, 0.04-0.37; P < .001) adversely affected platelet recovery. Our results indicate that the presence of DSAs is an independent predictor of poor outcomes after pediatric haplo-PTCy for NMDs. Therefore DSA-positive donors should be avoided whenever possible, and when a DSA-negative donor is unavailable, desensitization therapy must be performed to enhance the likelihood of donor engraftment and improve transplantation outcomes.

Transplantation for Fanconi anaemia: lessons learned from Brazil.

Fanconi anaemia is a challenging disease to manage, and haematopoietic stem-cell transplantation (HSCT) is the treatment of choice for the haematological complications related to this disease. Over these past two decades, we have observed a substantial improvement in survival outcomes after matched related and unrelated donor HSCT, even for patients living in low-income and middle-income countries. Long-term overall survival is still suboptimal because of the risk of malignancies and other disease-related complications. For patients without well matched donors, alternative donor transplantation using mismatched related donors is an option but is historically associated with a high incidence of graft failure and graft-versus-host disease (GVHD). Herein we discuss the development of a HSCT programme for Fanconi anaemia in our centre in Curitiba, Brazil. Because ex vivo, T-cell depletion is unavailable in our country, we adapted the haploidentical donor transplantation platform using post-HSCT cyclophosphamide to overcome graft failure and GVHD associated with HLA-mismatched donor transplantation. The withdrawal of pre-HSCT cyclophosphamide reduced the severity of mucositis and did not interfere with engraftment. The addition of serotherapy improved overall survival by decreasing the incidence of severe acute and chronic GVHD. Although we have improved overall survival and expanded access to HSCT for Fanconi anaemia, our patients face many challenges, especially viral reactivation and GVHD disease, that merit attention. We acknowledge that there is a learning curve to adopt the haploidentical approach for Fanconi anaemia to low-resourced settings, and this Brazilian experience might require further modifications along with national and international collaborations to be implemented in other countries.

The impact of donor-specific anti-human leukocyte antigen antibodies in salvage haploidentical hematopoietic cell transplantation with posttransplant cyclophosphamide in patients with nonmalignant disorders.

The presence of donor-specific anti-human leukocyte antigen (HLA) antibodies (DSAs) has been recognized as a major risk factor for graft failure (GF) after haploidentical hematopoietic cell transplantation with posttransplant cyclophosphamide (haplo-PTCy). However, the role of DSAs in salvage haplo-PTCy for rescuing patients with nonmalignant disorders (NMDs) has not yet been reported. The present study retrospectively analyzed 22 patients with NMDs who underwent salvage haplo-PTCy from January 2008 to December 2017. The median age at the time of the rescue haplo-PTCy was 9 years (range, 1-26 years). Median time from the first transplant to second haplo-PTCy was 56 days (range, 37-591 days). Among all patients, six (27.3%) had DSAs, with a median DSA strength (mean fluorescence intensity [MFI]) of 5201 (range, 1412-11,543) in the first DSA testing. In addition, the median DSA MFI was 2672 (range, 832-10,498) before the bone marrow infusion. Overall, GF occurred in 5 (25%) of the 20 assessable patients. Three of four (75%) patients with DSAs experienced GF versus 2 of 16 (12.5%) DSA-negative patients (P = 0.032). The median DSA MFI for patients with GF was 6437 (range, 1412-10,498) versus 1845 (range, 832-2672) for those who engrafted or had early death (P = 0.030). One-year event-free survival was significantly lower in DSA-positive patients than in those without DSAs (16.7% vs. 62.5%, P = 0.002). DSA-negative patients had an acceptable 1-year survival of 62.5%. In conclusion, this study suggests that DSAs may be associated with deleterious outcomes after salvage haplo-PTCy in patients with NMDs.