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Donor lymphocyte infusion has been used in the management of relapsed hematologic malignancies after allogeneic hematopoietic cell transplantation. It can eradicate minimal residual disease or be used to rescue a hematologic relapse, being able to induce durable remissions in a subset of patients. With the increased use of haploidentical hematopoietic cell transplantation, there is renewed interest in the use of donor lymphocytes to either treat or prevent disease relapse post transplant. Published retrospective and small prospective studies have shown encouraging results with therapeutic donor lymphocyte infusion in different haploidentical transplantation platforms. In this consensus paper, finalized on behalf of the Acute Leukemia Working Party of the European Society for Blood and Marrow Transplantation, we summarize the available evidence on the use of donor lymphocyte infusion from haploidentical donor, and provide recommendations on its therapeutic, pre-emptive and prophylactic use in clinical practice.

Early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) develops from very early cells with the potential for both T-cell and myeloid differentiation. The ambiguous nature of leukemic blasts in ETP-ALL may lead to immunophenotypic alterations at relapse. Here, we address immunophenotypic alterations and related classification issues, as well as genetic features of relapsed pediatric ETP-ALL. Between 2017 and 2022, 7518 patients were diagnosed with acute leukemia (AL). In addition to conventional immunophenotyping, karyotyping, and FISH studies, we performed next-generation sequencing of the T-cell receptor clonal repertoire and reverse transcription PCR and RNA sequencing for patients with ETP-ALL at both initial diagnosis and relapse. Among a total of 534 patients diagnosed with T-cell ALL (7.1%), 60 had ETP-ALL (11.2%). Ten patients with ETP-ALL experienced relapse or progression on therapy (16.7%), with a median time to event of 5 months (ranging from two weeks to 5 years). Most relapses were classified as AL of ambiguous lineage (n = 5) and acute myeloid leukemia (AML) (n = 4). Major genetic markers of leukemic cells remained unchanged at relapse. Of the patients with relapse, four had polyclonal leukemic populations and a relapse with AML or bilineal mixed-phenotype AL (MPAL). Three patients had clonal TRD rearrangements and relapse with AML, undifferentiated AL, or retention of the ETP-ALL phenotype. ETP-ALL relapse requires careful clinical and laboratory diagnosis. Treatment decisions should rely mainly on initial examination data, taking into account both immunophenotypic and molecular/genetic characteristics. [ABSTRACT FROM AUTHOR]

Introduction: Multicolor flow cytometry (MFC) has highly reliable and flexible algorithms for diagnosis and monitoring of acute lymphoblastic leukemia (ALL). However, MFC analysis can be affected by poor sample quality or novel therapeutic options (e.g., targeted therapies and immunotherapy). Therefore, an additional confirmation of MFC data may be needed. We propose a simple approach for validation of MFC findings in ALL by sorting questionable cells and analyzing immunoglobulin/T‐cell receptor (IG/TR) gene rearrangements via EuroClonality‐based multiplex PCR. Patients and Methods: We obtained questionable MFC results for 38 biological samples from 37 patients. In total, 42 cell populations were isolated by flow cell sorting for downstream multiplex PCR. Most of the patients (n = 29) had B‐cell precursor ALL and were investigated for measurable residual disease (MRD); 79% of them received CD19‐directed therapy (blinatumomab or CAR‐T). Results: We established the clonal nature of 40 cell populations (95.2%). By using this technique, we confirmed very low MRD levels (<0.01% MFC‐MRD). We also applied it to several ambiguous findings for diagnostic samples, including those with mixed‐phenotype acute leukemia, and the results obtained impacted the final diagnosis. Conclusion: We have demonstrated possibilities of a combined approach (cell sorting and PCR‐based clonality assessment) to validate MFC findings in ALL. The technique is easy to implement in diagnostic and monitoring workflows, as it does not require isolation of a large number of cells and knowledge of individual clonal rearrangements. We believe it provides important information for further treatment. [ABSTRACT FROM AUTHOR]

Introduction: In this study, we aimed to compare the immunophenotype of tumor cells in children with B‐cell precursor acute lymphoblastic leukemia (BCP‐ALL) harboring rearrangements of the CRLF2 gene with that in children without such aberrations with a specific focus on the surface expression of the related protein thymic stromal lymphopoietin receptor (TSLPR). Methods: We examined bone marrow samples from 46 patients with primary BCP‐ALL who had CRLF2 rearrangements detected by FISH (CRLF2(+) cohort). A total of 140 consecutive patients with intact CRLF2 were included in a control CRLF2(−) cohort. TSLPR expression was studied by flow cytometry. Results: The majority of CRLF2(+) patients were conventionally positive (≥20% positive cells) for TSLPR (33 of 46, 71.7%). Among the remaining children in this group, two were completely TSLPR‐negative, seven had less than 10% TSLPR‐positive cells, and four had between 10% and 20% TSLPR‐positive cells. By contrast, the majority of CRLF2(−) patients had no TSLPR‐positive cells (119 of 140, 85.0%), while in 15 cases (10.7%), the percentage of TSLPR‐positive cells was below 10%, and in six cases (4.3%), it was between 10% and 20%. Receiver operator characteristic analysis revealed a threshold of only 1.6% TSLPR‐positive cells for the effective prediction of the presence of CRLF2 rearrangement. Moreover, this threshold retained its predictive value when only children with low TSLPR positivity were studied. Conclusion: When surface TSLPR is detected at the diagnosis of BCP‐ALL, close attention should be given to the search for chromosomal aberrations involving CRLF2 at any level of expression. [ABSTRACT FROM AUTHOR]

The cells of acute myeloid leukemia are defined by clonal growth and heterogenous immunophenotypes. Chimeric antigen receptors (CARs) commonly recognize molecular targets by single-chain antibody fragments (scFvs) specific to a tumor-associated antigen. However, ScFvs may form aggregates, thus stimulating tonic CAR T-cell activation and reducing CAR T-cell functioning in vivo. Harnessing natural ligands as recognition parts of CARs, specific targeting of membrane receptors can be achieved. Previously, we presented ligand-based Flt3-CAR T-cells targeting the Flt3 receptor. The extracellular part of Flt3-CAR consisted of full-size Flt3Lg. Meanwhile, upon recognition, Flt3-CAR may potentially activate Flt3, triggering proliferative signaling in blast cells. Moreover, the long-lasting presence of Flt3Lg may lead to Flt3 downregulation. In this paper, we present mutated Flt3Lg-based Flt3m-CAR ('m'—for 'mutant') T-cells targeting Flt3. The extracellular part of Flt3m-CAR consists of full-length Flt3Lg-L27P. We have determined that ED50 for recombinant Flt3Lg-L27P produced in CHO cells is at least 10-fold higher than for the wild-type Flt3Lg. We show that the mutation in the recognizing domain of Flt3m-CAR did not affect the specificity of Flt3m-CAR T-cells when compared to Flt3-CAR T-cells. Flt3m-CAR T-cells combine the specificity of ligand–receptor recognition with reduced Flt3Lg-L27P bioactivity, leading to potentially safer immunotherapy. [ABSTRACT FROM AUTHOR]

Mixed-phenotype acute leukemia (MPAL), a rare and heterogeneous category of acute leukemia, is characterized by cross-lineage antigen expression. Leukemic blasts in MPAL can be represented either by one population with multiple markers of different lineages or by several single-lineage populations. In some cases, a major blast population may coexist with a smaller population that has minor immunophenotypic abnormalities and may be missed even by an experienced pathologist. To avoid misdiagnosis, we suggest sorting doubtful populations and leukemic blasts and searching for similar genetic aberrations. Using this approach, we examined questionable monocytic populations in five patients with dominant leukemic populations of B-lymphoblastic origin. Cell populations were isolated either for fluorescence in situ hybridization or for clonality assessment by multiplex PCR or next-generation sequencing. In all cases, monocytic cells shared the same gene rearrangements with dominant leukemic populations, unequivocally confirming the same leukemic origin. This approach is able to identify implicit cases of MPAL and therefore leads to the necessary clinical management for patients. [ABSTRACT FROM AUTHOR]

Acute promyelocytic leukemia (APL) pathogenesis is based on RARA gene translocations, which are of high importance in the diagnosis of and proper therapy selection for APL. However, in some cases acute myeloid leukemia (AML) demonstrates APL-like morphological features such as atypical promyelocytes accumulation. This type of AML is characterized by the involvement of other RAR family members or completely different genes. In the present study, we used conventional karyotyping, FISH and high-throughput sequencing in a group of 271 de novo AML with atypical promyelocytes accumulation. Of those, 255 cases were shown to carry a typical chromosomal translocation t(15;17)(q24;q21) with PML::RARA chimeric gene formation (94.1%). Other RARA-positive cases exhibited cryptic PML::RARA fusion without t(15;17)(q24;q21) (1.8%, n = 5) and variant t(5;17)(q35;q21) translocation with NPM1::RARA chimeric gene formation (1.5%, n = 4). However, 7 RARA-negative AMLs with atypical promyelocytes accumulation were also discovered. These cases exhibited TBL1XR1::RARB and KMT2A::SEPT6 fusions as well as mutations, e.g., NPM1 insertion and non-recurrent chromosomal aberrations. Our findings demonstrate the genetic diversity of AML with APL-like morphological features, which is of high importance for successful therapy implementation. [ABSTRACT FROM AUTHOR]

RASopathies are disorders caused by germline mutations in genes that encode components of the RAS/mitogen‐activated protein kinase (MAPK) pathway. These syndromes share features of developmental delay, facial dysmorphisms, and defects in various organs, as well as cancer predisposition. Somatic mutations of the same pathway are one of the primary causes of cancer. It is thought that germline cancer‐causing mutations would be embryonic lethal, as a more severe phenotype was shown in Drosophila and zebrafish embryos with cancer MAP2K1 mutations than in those with RASopathy mutations. Here we report the case of a patient with RASopathy caused by a cancer‐associated MAP2K1 p.Phe53Leu mutation. The postzygotic mosaic nature of this mutation could explain the patient's survival. [ABSTRACT FROM AUTHOR]

Simple Summary: We aimed to develop an antibody panel and data analysis algorithm for multicolor flow cytometry (MFC), which is a reliable method for minimal residual disease (MRD) detection in patients with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) treated with CD19-directed therapy. We have developed a single-tube 11-color panel for MFC-MRD detection, which was adapted for the case of possible CD19 loss. Based on patterns of antigen expression changes and the relative expansion of normal CD19-negative BCPs, guidelines for MFC data analysis and interpretation were established. The suggested approach was tested in comparison with the molecular techniques with a high rate of qualitative concordance obtained. We aimed to develop an antibody panel and data analysis algorithm for multicolor flow cytometry (MFC), which is a reliable method for minimal residual disease (MRD) detection in patients with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) treated with CD19-directed therapy. The development of the approach, which was adapted for the case of possible CD19 loss, was based on the additional B-lineage marker expression data obtained from a study of primary BCP-ALL patients, an analysis of the immunophenotypic changes that occur during blinatumomab or CAR-T therapy, and an analysis of very early CD19-negative normal BCPs. We have developed a single-tube 11-color panel for MFC-MRD detection. CD22- and iCD79a-based primary B-lineage gating (preferably consecutive) was recommended. Based on patterns of antigen expression changes and the relative expansion of normal CD19-negative BCPs, guidelines for MFC data analysis and interpretation were established. The suggested approach was tested in comparison with the molecular techniques: IG/TR gene rearrangement detection by next-generation sequencing (NGS) and RQ-PCR for fusion-gene transcripts (FGTs). Qualitative concordance rates of 82.8% and 89.8% were obtained for NGS-MRD and FGT-MRD results, respectively. We have developed a sensitive and reliable approach that allows MFC-MRD monitoring after CD19-directed treatment, even in the case of possible CD19 loss. [ABSTRACT FROM AUTHOR]

Optimized conformal total body irradiation (OC-TBI) is a highly conformal image guided method for irradiating the whole human body while sparing the selected organs at risk (OARs) (lungs, kidneys, lens). This study investigated the safety and feasibility of pediatric OC-TBI with the helical TomoTherapy (TomoTherapy) and volumetric modulated arc (VMAT) modalities and their implementation in routine clinical practice. This is the first study comparing the TomoTherapy and VMAT modalities in terms of treatment planning, dose delivery accuracy, and toxicity for OC-TBI in a single-center setting. The OC-TBI method with standardized dosimetric criteria was developed and implemented with TomoTherapy. The same OC-TBI approach was applied for VMAT. Standardized treatment steps, namely, positioning and immobilization, contouring, treatment planning strategy, plan evaluation, quality assurance, visualization and treatment delivery procedure were implemented for 157 patients treated with TomoTherapy and 52 patients treated with VMAT. Both modalities showed acceptable quality of the planned target volume dose coverage with simultaneous OARs sparing. The homogeneity of target irradiation was superior for TomoTherapy. Overall assessment of the OC-TBI dose delivery was performed for 30 patients treated with VMAT and 30 patients treated with TomoTherapy. The planned and delivered (sum of doses for all fractions) doses were compared for the two modalities in groups of patients with different heights. The near maximum dose values of the lungs and kidneys showed the most significant variation between the planned and delivered doses for both modalities. Differences in the patient size did not result in statistically significant differences for most of the investigated parameters in either the TomoTherapy or VMAT modality. TomoTherapy-based OC-TBI showed lower variations between planned and delivered doses, was less time-consuming and was easier to implement in routine practice than VMAT. We did not observe significant differences in acute and subacute toxicity between TomoTherapy and VMAT groups. The late toxicity from kidneys and lungs was not found during the 2.3 years follow up period. The study demonstrates that both modalities are feasible, safe and show acceptable toxicity. The standardized approaches allowed us to implement pediatric OC-TBI in routine clinical practice. [ABSTRACT FROM AUTHOR]

Serial evaluation of CD19 surface expression in pediatric B-cell malignancies following CD19-targeted therapy. In contrast to the CD19-negative precursors, the level of CD19-positives increased significantly during the first three months of maintenance therapy, although the continued presence of B-lineage regeneration remained more or less stable (Fig. Keywords: CD19 targeting; minimal residual disease; ALL; flow cytometry; CD19-negative precursors EN CD19 targeting minimal residual disease ALL flow cytometry CD19-negative precursors e6 e9 4 12/27/21 20220101 NES 220101 Targeting the pan-B-lymphocyte antigen CD19 with the bispecific T-cell engager blinatumomab is one of the modern ways of treating acute lymphocytic B-cell precursor leukaemia (BCP-ALL).1,2 This drug has been very successful in treating patients with relapsed/refractory (R/R) disease,3,4 but it has recently been shown that blinatumomab is also effective in eradicating minimal residual disease (MRD) in patients with relapsed or primary BCP-ALL.5,6 Loss of the target molecule is one of the key mechanisms for leukaemia cells to escape the selective pressure of blinatumomab.7,8 The resulting possible CD19 negativity interferes with MRD monitoring by multicolour flow cytometry (MFC), which is usually based on analysis of the CD19-positive compartment.9 On the other hand, the use of other early B-line antigens [CD22, CD24, CD10, intracellular (i) CD79a] instead of CD19 for primary B-cell gating, such as to improve the MFC-MRD technique in the case of CD19-targeted treatment,7,10,11 could lead to errors in the interpretation of the immunophenotype of normal haematopoietic cells. CD19-negative B-cell precursors were gated as described previously.12 All BM samples obtained after completion of blinatumomab were MFC-MRD-negative, including those from patients who were MFC-MRD-positive at EOI ( I n i = 7, median MFC-MRD 0-030%, range 0-001-0-166%). [Extracted from the article]

Total body irradiation (TBI) in combination with chemotherapy is widely used as a conditioning regimen in pediatric and adult hematopoietic stem cell transplantation (HSCT). The combination of TBI with chemotherapy has demonstrated superior survival outcomes in patients with acute lymphoblastic and myeloid leukemia when compared with conditioning regimens based only on chemotherapy. The clinical application of intensity-modulated radiation therapy (IMRT)-based methods (volumetric modulated arc therapy (VMAT) and TomoTherapy) seems to be promising and has been actively used worldwide. The optimized conformal total body irradiation (OC-TBI) method described in this study provides selected dose reduction for organs at risk with respect to the most significant toxicity (lungs, kidneys, lenses). This study included 220 pediatric patients who received OC-TBI with subsequent chemotherapy and allogenic HSCT with TCRαβ/CD19 depletion. A group of 151 patients received OC-TBI using TomoTherapy, and 40 patients received OC-TBI using the Elekta Synergy™ linac with an Agility-MLC (Elekta, Crawley, UK) using volumetric modulated arc therapy (VMAT). Twenty-nine patients received OC-TBI with supplemental simultaneous boost to bone marrow—(SIB to BM) up to 15 Gy: 28 patients (pts)—TomoTherapy; one patient—VMAT. The follow-up duration ranged from 0.3 to 6.4 years (median follow-up, 2.8 years). Overall survival (OS) for all the patients was 63% (95% CI: 56–70), and event-free survival (EFS) was 58% (95% CI: 51–65). The cumulative incidence of transplant-related mortality (TRM) was 10.7% (95% CI: 2.2–16) for all patients. The incidence of early TRM (<100 days) was 5.0% (95% CI: 1.5–8.9), and that of late TRM (>100 days) was 5.7 (95% CI: 1.7–10.2). The main causes of death for all the patients were relapse and infection. The concept of OC-TBI using IMRT VMAT and helical treatment delivery on a TomoTherapy treatment unit provides maximum control of the dose distribution in extended targets with simultaneous dose reduction for organs at risk. This method demonstrated a low incidence of severe side effects after radiation therapy and predictable treatment effectiveness. Our initial experience demonstrates that OC-TBI appears to be a promising technique for the treatment of pediatric patients. [ABSTRACT FROM AUTHOR]