Early SARS-CoV-2 Reinfections Involving the Same or Different Genomic Lineages, Spain.

Centers for Disease Control and Prevention guidelines consider SARS-CoV-2 reinfection when sequential COVID-19 episodes occur >90 days apart. However, genomic diversity acquired over recent COVID-19 waves could mean previous infection provides insufficient cross-protection. We used genomic analysis to assess the percentage of early reinfections in a sample of 26 patients with 2 COVID-19 episodes separated by 20-45 days. Among sampled patients, 11 (42%) had reinfections involving different SARS-CoV-2 variants or subvariants. Another 4 cases were probable reinfections; 3 involved different strains from the same lineage or sublineage. Host genomic analysis confirmed the 2 sequential specimens belonged to the same patient. Among all reinfections, 36.4% involved non-Omicron, then Omicron lineages. Early reinfections showed no specific clinical patterns; 45% were among unvaccinated or incompletely vaccinated persons, 27% were among persons <18 years of age, and 64% of patients had no risk factors. Time between sequential positive SARS-CoV-2 PCRs to consider reinfection should be re-evaluated.

Poor graft function after haploidentical stem cell transplantation with post-transplant cyclophosphamide.

This is a retrospective cohort study of consecutive adult patients who received a haploidentical-SCT (haplo-SCT) with post-transplant cyclophosphamide (PT-Cy) in a single centre. Poor graft function (PGF) was defined as the occurrence of either persistent neutropenia (ANC < 0.5 × 109/µL) with poor response to granulocyte colony-stimulating factors (G-CSF) and/or thrombocytopenia (platelets < 20 × 109/L) with transfusion dependence, with complete donor chimerism and without concurrent severe GVHD or underlying disease relapse, during the first 12 months after transplantation. Forty-four (27.5%) out of 161 patients were diagnosed with PGF. Previous CMV reactivation was significantly more frequent in patients with PGF (88.6% versus 73.5%, p = 0.04) and the number of reactivations was also higher in these patients. Besides, early CMV reactivations in the first 6 months post-SCT were also significantly more frequent among patients with PGF (88.6% versus 71.8% p = 0.025). Thirty-two percent of patients with PGF were treated with increasing doses of thrombopoietin-receptor agonists (TRA) and 7 patients were treated with a donor CD34 + selected boost. In total, 93.2% of patients reached adequate peripheral blood counts in a median time of 101 days (range 11-475) after diagnosis. PGF is a frequent complication after haplo-SCT with PT-Cy. CMV reactivation might be the most relevant factor associated to its development. Even when most patients recover peripheral counts with support therapy, there is a group of patients with persistent cytopenias who can effectively be treated with TRA and/or a boost of CD34 + selective cells.

Systematic Genomic and Clinical Analysis of Severe Acute Respiratory Syndrome Coronavirus 2 Reinfections and Recurrences Involving the Same Strain.

Estimates of the burden of severe acute respiratory syndrome coronavirus 2 reinfections are limited by the scarcity of population-level studies incorporating genomic support. We conducted a systematic study of reinfections in Madrid, Spain, supported by genomic viral analysis and host genetic analysis, to cleanse laboratory errors and to discriminate between reinfections and recurrences involving the same strain. Among the 41,195 cases diagnosed (March 2020-March 2021), 93 (0.23%) had 2 positive reverse transcription PCR tests (55-346 days apart). After eliminating cases with specimens not stored, of suboptimal sequence quality, or belonging to different persons, we obtained valid data from 22 cases. Of those, 4 (0.01%) cases were recurrences involving the same strain; case-patients were 39-93 years of age, and 3 were immunosuppressed. Eighteen (0.04%) cases were reinfections; patients were 19-84 years of age, and most had no relevant clinical history. The second episode was more severe in 8 cases.

Allogeneic CD34-selected stem cell boost as salvage treatment of life-threatening infection and severe cytopenias after CAR-T cell therapy.

BACKGROUND: A variable incidence of profound cytopenia has been described in patients receiving chimeric antigen receptor T-cell (CAR-T) therapy for relapsed or refractory (R/R) B-cell acute lymphoblastic leukemia (ALL). This complication leads to severe infection in some cases, especially those who present additional risk factors including prior hematopoietic stem cell transplantation (HSCT). STUDY DESIGN AND METHODS: We report a case of breakthrough invasive fungal infection in a patient with prolonged neutropenia after CAR-T cell therapy administered for relapsed B-cell ALL after allogeneic haploidentical HSCT. RESULTS: After disease progression was discarded, therapy with antifungal agents, G-CSF and thrombopoietin analogue was started. However, no sign of haematological recovery or infection improvement was observed. A fresh mobilized selected CD34-stem cell boost from her haploidentical transplant donor was infused without further conditioning. Within 15 days of mobilized CD34-boost administration the patient showed complete resolution of both the aplasia and fungal infection. DISCUSSION: This case illustrates as proof-of-concept the efficacy and safety of selected CD34-stem cell boost from prior donor as salvage treatment of prolonged cytopenias after CAR-T cell therapy.

Risk prediction of CMV reactivation after allogeneic stem cell transplantation using five non-HLA immunogenetic polymorphisms.

Despite advances in the understanding of the pathophysiology of cytomegalovirus (CMV) infection, it remains as one of the most common infectious complications after allogeneic hematopoietic stem cell transplantation (allo-HSCT). The aim of this study was to determine the genotype of cytokines and chemokines in donor and recipient and their association with CMV reactivation. Eighty-five patients receiving an allo-HSCT from an HLA-identical sibling donor were included in the study. Fifty genes were selected for their potential role in the pathogenesis of CMV infection. CMV DNAemia was evaluated until day 180 after allo-HSCT. CMV reactivation was observed in 51/85 (60%) patients. Of the 213 genetic variants selected, 11 polymorphisms in 7 different genes (CXCL12, IL12A, KIR3DL1, TGFB2, TNF, IL1RN, and CD48) were associated with development or protection from CMV reactivation. A predictive model using five of such polymorphisms (CXCL12 rs2839695, IL12A rs7615589, KIR3DL1 rs4554639, TGFB2 rs5781034 for the recipient and CD48 rs2295615 for the donor) together with the development of acute GVHD grade III/IV improved risk stratification of CMV reactivation. In conclusion, the data presented suggest that the screening of five polymorphisms in recipient and donor pre-transplantation could help to predict the individual risk of CMV infection development after HLA-identical allo-HSCT.

Post-transplant cyclophosphamide for GVHD prophylaxis compared to ATG-based prophylaxis in unrelated donor transplantation.

Post-transplant cyclophosphamide (PTCY) effectively prevents graft-versus-host disease after unmanipulated HLA-haploidentical HSCT. The use of PTCY in the unrelated donor HSCT setting is less explored. We conducted a retrospective study of 132 consecutive patients undergoing a matched or 9/10 mismatched unrelated donor HSCT in 4 centers in Spain, 60 with anti-thymocyte globulin (ATG)-based prophylaxis combined with MTX-CsA, and 72 using a PTCY-based regimen. Peripheral blood stem cells were used as graft in most patients (111 patients, 84%); mMUD donors were balanced between groups. Cumulative incidences of grades II-IV and III-IV acute GVHD at 100 days were lower in the PTCy group (46% vs. 67%, p = 0.008; 3% vs. 34%, p = 0.003), without statistically significant differences in the 2-year cumulative incidence of chronic moderate-severe GVHD. At 2 years, no significant differences were observed in overall survival, event-free survival, cumulative incidence of relapse, and non-relapse mortality. GVHD was the most frequent cause of NRM in the ATG group. No differences were observed between groups in the composite endpoint of GVHD-free and relapse-free survival. In this study, PTCy combined with additional immunosuppression after MUD/mMUD HSCT showed a reduction of aGVHD rate with safety results comparable to those obtained with the ATG-based prophylaxis.

Mechanisms That Contribute to a Profound Reduction of the HIV-1 Reservoir After Allogeneic Stem Cell Transplant.

This article has been corrected. The original version (PDF) is appended to this article as a Supplement. Background: The multifactorial mechanisms associated with radical reductions in HIV-1 reservoirs after allogeneic hematopoietic stem cell transplant (allo-HSCT), including a case of HIV cure, are not fully understood. Objective: To investigate the mechanism of HIV-1 eradication associated with allo-HSCT. Design: Nested case series within the IciStem observational cohort. Setting: Multicenter European study. Participants: 6 HIV-infected, antiretroviral-treated participants who survived more than 2 years after allo-HSCT with CCR5 wild-type donor cells. Measurements: HIV DNA analysis, HIV RNA analysis, and quantitative viral outgrowth assay were performed in blood, and HIV DNA was also measured in lymph nodes, ilea, bone marrow, and cerebrospinal fluid. A humanized mouse model was used for in vivo detection of the replication-competent blood cell reservoir. HIV-specific antibodies were measured in plasma. Results: Analysis of the viral reservoir showed that 5 of 6 participants had full donor chimera in T cells within the first year after transplant, undetectable proviral HIV DNA in blood and tissue, and undetectable replication-competent virus (<0.006 infectious unit per million cells). The only participant with detectable virus received cord blood stem cells with an antithymocyte globulin-containing conditioning regimen, did not develop graft-versus-host disease, and had delayed complete standard chimerism in T cells (18 months) with mixed ultrasensitive chimera. Adoptive transfer of peripheral CD4+ T cells to immunosuppressed mice resulted in no viral rebound. HIV antibody levels decreased over time, with 1 case of seroreversion. Limitation: Few participants. Conclusion: Allo-HSCT resulted in a profound long-term reduction in the HIV reservoir. Such factors as stem cell source, conditioning, and a possible "graft-versus-HIV-reservoir" effect may have contributed. Understanding the mechanisms involved in HIV eradication after allo-HSCT can enable design of new curative strategies. Primary Funding Source: The Foundation for AIDS Research (amfAR).

Donor Cell-Derived Hematologic Neoplasms after Hematopoietic Stem Cell Transplantation: A Systematic Review.

Development of de novo hematologic malignancies in donor cells after allogeneic stem cell transplantation (allo-SCT) provides a useful in vivo model to study the process of leukemogenesis. A systematic analysis of the cases reported in the literature was performed to identify risk factors and mechanisms involved in the pathogenesis of donor cell-derived hematologic neoplasms (DCHN) and leukemogenic transformation. Relevant data were extracted from 137 cases. Cases of DCHN show a wide heterogeneity with regard to recipient/donor age, sex mismatch, and conditioning regimen. Some characteristics, such as the type of primary disease, the type of hematologic malignancy of the DCHN, and the stem cell source used in the transplant procedure, differ from those expected. Mechanisms involved in the pathogenesis of DCHN are complex, and several hypotheses have been proposed, such as pre-existing hematologic neoplasms or premalignant clones in the donor, decreased immune surveillance, and damage to bone marrow microenvironment in the recipient. Most likely several if not all these mechanisms play a role in DCHN development. Novel approaches, such as next-generation sequencing to study consecutive samples after allo-SCT in these patients, appear to be promising to decipher the mechanisms of leukemogenesis.

Busulfan-based myeloablative conditioning regimens for haploidentical transplantation in high-risk acute leukemias and myelodysplastic syndromes.

BACKGROUND: High-risk acute leukemia (AL) and myelodysplastic syndrome (MDS) remain a therapeutic challenge. Unmanipulated haploidentical-related donor transplantation based on a myeloablative conditioning regimen (HAPLO-MAC) and post-transplant cyclophosphamide (PT-Cy) as prophylaxis against graft vs host disease (GvHD) is now a promising rescue strategy that could become universally available. OBJECTIVE: To evaluate the results of HAPLO-MAC with PT-Cy in patients with AL and MDS reported to the Haploidentical Transplantation Subcommittee of the Spanish Group for Hematopoietic Transplantation (GETH). PATIENTS AND METHODS: We report our multicenter experience using an IV busulfan-based HAPLO-MAC regimen and PT-Cy for treatment of 65 adults with high-risk AL and MDS. RESULTS: Engraftment was recorded in 64 patients (98.5%), with a median time to neutrophil and platelet recovery of 16 and 27 days, respectively. The cumulative incidence of grade II-IV acute GvHD and chronic GvHD was 28.6% and 27.5%, respectively. After a median follow-up of 31 months for survivors, the cumulative incidence of non-relapse mortality and relapse at 2 years was 18.8% and 25%, respectively. Estimated 30-month event-free survival and overall survival were 56% and 54.5%, respectively. CONCLUSION: HAPLO-MAC comprising an IV busulfan-based conditioning regimen enabled long-term disease control with acceptable toxicity in high-risk AL and MDS.

Donor CTLA-4 Genotype Modulates the Immune Response to Minor Histocompatibility Antigen Mismatches.

Minor histocompatibility antigen (miHA) mismatches have been related to graft-versus-host disease (GVHD) after allogeneic stem cell transplantation, but this association remains controversial due to the lack of consistency in the results obtained by different groups. The CTLA-4 genotype of the donor has been reported to be relevant in the appearance of acute GVHD. We explored the effect of the donor's CTLA-4 genotype in the incidence of acute GVHD associated with HA-1, HA-8, or H-Y miHA mismatches in a large cohort of 1295 patients receiving an allogeneic transplant from an HLA-identical sibling donor. The incidence of acute GVHD was higher if the donor and recipient were mismatched for HA-1, HA-8, or H-Y, but only when the donor had the CTLA-4 rs231775 AA genotype (hazard ratio [HR], 2.18; 95% confidence interval [CI], 1.27 to 3.75; P = .005; HR, 2.11, 95% CI, 1.06 to 4.18; P = .033; and HR, 1.50; 95% CI, 1.05 to 2.15; P = .025, respectively). In contrast, this increased risk of developing acute GVHD was not found when the donor presented the CTLA-4 rs231775 AG or GG genotypes. We conclude that the immune response to specific miHA mismatches is modulated by the CTLA-4 genotype of the donor.

Inhibitory killer cell immunoglobulin-like receptor (iKIR) mismatches improve survival after T-cell-repleted haploidentical transplantation.

Alloreactivity triggered by interaction between killer cell immunoglobulin-like receptors (KIRs) and natural killer (NK) cells plays a role in the graft-versus-tumor effect after hematopoietic stem cell transplantation (SCT). Our aim in this study was to evaluate this role in the setting of T-cell-repleted haploidentical SCT with postinfusion high-dose cyclophosphamide (PT-Cy). We included 33 patients. Among patient-donor pairs with at least 1 inhibitory KIR (iKIR) gene mismatch, event-free survival (EFS) and cumulative incidence of relapse 1 year after transplant were significantly better (85% vs. 37% [P = 0.008] and 18% vs. 46% [P = 0.041], respectively). A subanalysis in 12 patients with Hodgkin's lymphoma (HL) showed an improvement in EFS 1 year after transplant in those patients with KIR ligand mismatch (100% vs. 25%, P = 0.012), although overall survival (OS) was not affected (85% vs. 80%, P = 0.2). Eight of 12 patient-donors pairs presented iKIR mismatches. Of note, this outcome was better in the small subgroup, both for EFS (100% vs. 25%, P = 0.012) and for OS (100% vs. 37%, P = 0.004). Our data suggest that in the setting of T-cell-repleted haploidentical SCT with PT-Cy, iKIR mismatch is associated with improved survival, with particularly good results for both iKIR and KIR ligand mismatches in patients with HL.

Prognostic impact of minimal residual disease analysis by flow cytometry in patients with acute myeloid leukemia before and after allogeneic hemopoietic stem cell transplantation.

Allogeneic stem cell transplantation (allo-SCT) has become the treatment of choice in patients with intermediate-risk and high-risk acute myeloid leukemia (AML). The quality of response to treatment, assessed in terms of detection of minimal residual disease (MRD), has been consistently associated with prognosis and clinical outcome in patients with AML. The aim of the present study was to evaluate the prognostic impact of analyzing MRD in bone marrow using 4-color multiparametric flow cytometry (MFC) in 29 patients with AML before and after allo-SCT. Eighteen patients who were shown to be MRD-negative [≤0.1% leukemia-associated immunophenotypes (LAIPs)] by MFC at transplantation and underwent allo-SCT had lower rates of relapse (15% vs. 66%, P = 0.045), better overall 1-yr survival (83% vs. 52%, P = 0.021) and a lower cumulative incidence of relapse (P = 0.032) than patients who were MRD-positive (>0.1%). All post-transplant MRD-positive patients underwent a therapeutic intervention after transplant (tapering of immunosuppression, donor lymphocyte infusion, or re-transplant) with the intention of preventing relapse. Disease was controlled and MRD disappeared in five of these patients. Disease recurred in the other seven patients. We can conclude that follow-up with MFC for the detection of MRD in AML before and after SCT is useful for predicting relapse. In the post-transplant setting, monitoring of MRD by MFC could be a key preemptive intervention.

Digital PCR Improves Sensitivity and Quantification in Monitoring CAR-T Cells in B Cell Lymphoma Patients.

Chimeric antigen receptor T cells (CAR-T) has emerged as a promising therapy, over 60% of patients fail to sustain a long-term response. The underlying factors that leads to the effectiveness of this therapy are not completely understood, CAR-T cell persistence and monitoring seems to be pivotal for ensuring a successful response. Various monitoring methods such as multiparametric flow cytometry (MFC) or quantitative PCR (qPCR) have been applied. Our objective is to develop digital PCR (dPCR) assays for detection and quantification of CAR-T cells, comparing them with MFC and qPCR. Samples taken at different follow-up times from 45 patients treated with CAR-T therapy were analyzed to assess the correlation between the different methodologies. dPCR presented a high correlation with MFC and qPCR (r = 0.97 and r = 0.87, respectively), while offering a higher sensitivity (0.01%) compared to MFC (0.1%) and qPCR (1%). dPCR emerged as an alternative and highly sensitivity method for monitoring CAR-T cell dynamics. This technique is well-suited for implementation in clinical practice as a complementary technique to MFC.

Detection of occult cerebrospinal fluid involvement during maintenance therapy identifies a group of children with acute lymphoblastic leukemia at high risk for relapse.

We aimed at assessing the clinical significance of the levels of acute lymphoblastic leukemia (ALL) cells in samples of cerebrospinal fluid (CSF) during therapy. We studied 990 CSF samples from 108 patients, at the time of diagnosis (108) and at each time of intrathecal therapy (882). The proportions of leukemic cells in CSF samples were assessed by flow cytometry (FCM). Patients with central nervous system (CNS) involvement at diagnosis (FCM+) showed predominantly a T-ALL, and higher percentages of known negative prognostic factors: high risk group, higher white blood cell counts, normal karyotype, and the BCR-ABL fusion gene. No differences in relapse free survival (RFS) and overall survival (OS) were observed between FCM+ versus FCM- at diagnosis. Patients with CNS involvement during therapy showed significantly older age, and higher frequencies of T-cell leukemia. We found a significantly higher RFS in patients with FCM+ during therapy. The detection of subclinical CNS disease by FCM during maintenance was associated with significantly lower 3-years RFS and 3-years OS. A sensitive methodology like FCM can be applied for a close follow-up of the levels of ALL in CFS samples, and may identify a group of patients at high risk for relapse.

A polymorphism in the TYMP gene is associated with the outcome of HLA-identical sibling allogeneic stem cell transplantation.

Thymidine phosphorylase (TYMP), an enzyme involved in nucleotide synthesis, has been implicated in critical biological processes such as DNA replication, protection against mutations, and tissue repair. In this work, we retrospectively evaluated the influence of a polymorphism in the TYMP gene (rs112723255; G/A) upon the outcome of 448 patients subjected to allogeneic stem cell transplantation (allo-SCT) from an human leukocyte antigen (HLA)-identical sibling donor. The TYMP genotype of patients correlated with overall survival-carriers of the minor allele (A) being at an increased risk of dying after transplantation (hazard ratio, HR = 1.9; P = 0.004). This effect was mostly due to differences in transplant toxicity-related mortality (HR = 2.5; P = 0.029). In addition, the TYMP genotype of donors was associated with the risk of chronic graft-versus-host disease (GVHD)-carriers of the minor allele being at an increased risk of developing this complication ([HR] = 1.7; P = 0.039). The impact of such polymorphism on the risk of chronic GVHD is limited to patients transplanted in early stage disease (HR = 2.2; P = 0.019). The combination of a donor harboring the minor allele with a patient homozygous for the major allele was associated with the highest risk of chronic GVHD (HR = 2.8; P = 0.008). These findings provide the first evidence of the significant impact of the TYMP genotype upon the clinical outcome of patients treated with HLA-identical sibling allo-SCT.

Systematic genomic analysis of SARS-CoV-2 co-infections throughout the pandemic and segregation of the strains involved.

BACKGROUND: SARS-CoV-2 recombinants involving the divergent Delta and Omicron lineages have been described, and one of them, "Kraken" (XBB.1.5), has recently been a matter of concern. Recombination requires the coexistence of two SARS-CoV-2 strains in the same individual. Only a limited number of studies have focused on the identification of co-infections and are restricted to co-infections involving the Delta/Omicron lineages. METHODS: We performed a systematic identification of SARS-CoV-2 co-infections throughout the pandemic (7609 different patients sequenced), not biassed towards the involvement of highly divergent lineages. Through a comprehensive set of validations based on the distribution of allelic frequencies, phylogenetic consistency, re-sequencing, host genetic analysis and contextual epidemiological analysis, these co-infections were robustly assigned. RESULTS: Fourteen (0.18%) co-infections with ≥ 8 heterozygous calls (8-85 HZs) were identified. Co-infections were identified throughout the pandemic and involved an equal proportion of strains from different lineages/sublineages (including pre-Alpha variants, Delta and Omicron) or strains from the same lineage. Co-infected cases were mainly unvaccinated, with mild or asymptomatic clinical presentation, and most were at risk of overexposure associated with the healthcare environment. Strain segregation enabled integration of sequences to clarify nosocomial outbreaks where analysis had been impaired due to co-infection. CONCLUSIONS: Co-infection cases were identified throughout the pandemic, not just in the time periods when highly divergent lineages were co-circulating. Co-infections involving different lineages or strains from the same lineage were occurring in the same proportion. Most cases were mild, did not require medical assistance and were not vaccinated, and a large proportion were associated with the hospital environment.

Role of Intracellular Drug Disposition in the Response of Acute Myeloid Leukemia to Cytarabine and Idarubicin Induction Chemotherapy.

Despite its often low efficacy and high toxicity, the standard treatment for acute myeloid leukemia (AML) is induction chemotherapy with cytarabine and idarubicin. Here, we have investigated the role of transporters and drug-metabolizing enzymes in this poor outcome. The expression levels (RT-qPCR) of potentially responsible genes in blasts collected at diagnosis were related to the subsequent response to two-cycle induction chemotherapy. The high expression of uptake carriers (ENT2), export ATP-binding cassette (ABC) pumps (MDR1), and enzymes (DCK, 5-NT, and CDA) in the blasts was associated with a lower response. Moreover, the sensitivity to cytarabine in AML cell lines was associated with ENT2 expression, whereas the expression of ABC pumps and enzymes was reduced. No ability of any AML cell line to export idarubicin through the ABC pumps, MDR1 and MRP, was found. The exposure of AML cells to cytarabine or idarubicin upregulated the detoxifying enzymes (5-NT and DCK). In AML patients, 5-NT and DCK expression was associated with the lack of response to induction chemotherapy (high sensitivity and specificity). In conclusion, in the blasts of AML patients, the reduction of the intracellular concentration of the active metabolite of cytarabine, mainly due to the increased expression of inactivating enzymes, can determine the response to induction chemotherapy.