Flow cytometric minimal residual disease measurement accounting for cytogenetics in children with non-high-risk acute lymphoblastic leukemia treated according to the ALL-MB 2008 protocol.

BACKGROUND: Quantitative measurement of minimal residual disease (MRD) is the "gold standard" for estimating the response to therapy in childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Nevertheless, the speed of the MRD response differs for different cytogenetic subgroups. Here we present results of MRD measurement in children with BCP-ALL, in terms of genetic subgroups with relation to clinically defined risk groups. METHODS: A total of 485 children with non-high-risk BCP-ALL with available cytogenetic data and MRD studied at the end-of-induction (EOI) by multicolor flow cytometry (MFC) were included. All patients were treated with standard-risk (SR) of intermediate-risk (ImR) regimens of "ALL-MB 2008" reduced-intensity protocol. RESULTS AND DISCUSSION: Among all study group patients, 203 were found to have low-risk cytogenetics (ETV6::RUNX1 or high hyperdiploidy), while remaining 282 children were classified in intermediate cytogenetic risk group. For the patients with favorable and intermediate risk cytogenetics, the most significant thresholds for MFC-MRD values were different: 0.03% and 0.04% respectively. Nevertheless, the most meaningful thresholds were different for clinically defined SR and ImR groups. For the SR group, irrespective to presence/absence of favorable genetic lesions, MFC-MRD threshold of 0.1% was the most clinically valuable, although for ImR group the most informative thresholds were different in patients from low-(0.03%) and intermediate (0.01%) cytogenetic risk groups. CONCLUSION: Our data show that combining clinical risk factors with MFC-MRD measurement is the most useful tool for risk group stratification of children with BCP-ALL in the reduced-intensity protocols. However, this algorithm can be supplemented with cytogenetic data for part of the ImR group.

Laboratory characterization of the pediatric B/T subtype of mixed-phenotype acute leukemia: Report of a case series.

OBJECTIVES: Mixed-phenotype acute leukemia (MPAL) is a rare disease associated with difficulties in the correct lineage assignment of leukemic cells. One of the least common subtypes within this category is characterized by the simultaneous presence of B- and T-lineage-defining antigens. Each case of suspected B/T MPAL should be considered in light of all available laboratory and clinical data to avoid misdiagnosis. METHODS: In this study, we describe 6 pediatric patients who presented with leukemic blasts bearing B- and T-lineage antigens at diagnosis, including their clinical, immunophenotypic, morphologic, and cytogenetic characteristics. RESULTS: In 3 patients, more or less distinct populations of B- and T-lymphoid origin were found; the other 3 patients had a single mixed-phenotype blast population. All cases fulfilled the World Health Organization criteria, but not all of them turned out to be bona fide cases of B/T MPAL according to the available clinical and laboratory data. Found genetic lesions were helpful for the confirmation of MPAL instead of 2 concomitant tumors, but for a general B/T MPAL diagnosis, genetic studies provided the only descriptive data. CONCLUSIONS: The accurate diagnosis of B/T MPAL requires a multidisciplinary approach combining high-tech laboratory methods and close cooperation between treating physicians and pathologists.

A Single Dose of PEG-Asparaginase at the Beginning of Induction Not Only Accelerates MRD Clearance but Also Improves Long-Term Outcome in Children with B-Lineage ALL.

This report presents the results of the assessment of MRD response by multicolor flow cytometry (MFC) with regard to the randomized use of pegylated asparaginase (PEG). In this study, PEG was randomly administered at a dose of 1000 U/m2 on day 3 of induction therapy in children with B-lineage ALL. METHODS: Conventional induction therapy consisted of dexamethasone, vincristine, and daunorubicin. MRD data was available in 502 patients who were randomized at the start of induction therapy, standard-risk (SR) patients into three (conventional induction without PEG, induction with additional PEG and with PEG but without daunorubicin) and intermediate-risk (ImR) patients into two groups (with additional PEG and without PEG). RESULTS: The single administration of PEG resulted in a significantly higher proportion of rapid responders, in SR patients even when no anthracyclines were used for induction. In the SR group, the event-free survival of the MFC-MRD fast responders was similar in the PEG- and PEG+ arms (92.0 ± 3.1% vs. 96.2 ± 1.5%, respectively), and the same unfavorable trend was observed for MFC-MRD slow responders (57.5 ± 12.3% vs. 66.7 ± 15.7%, respectively). Results were similar in ImR patients: (94.3 ± 3.2% vs. 95.1 ± 2.4%, for fast responders and 63.3 ± 7.6% vs. 78.1 ± 7.9%, for slow responders in PEG- and PEG+ arms, respectively). However, there is a large difference between the proportion of MFC-MRD slow responders in the PEG- and PEG+ groups (18.3% vs. 5.2% for the SR group and 44.2% vs. 25.0% for the ImR group). CONCLUSIONS: Therefore, early use of PEG-ASP not only leads to an accelerated reduction of blasts, but also to an excellent outcome in a significantly larger proportion of patients in both risk groups.

Flow cytometry for comprehensive assessment of platelet functional activity in response to ADP stimulation.

OBJECTIVES: Flow cytometry with adenosine diphosphate (ADP) allows to characterize molecular changes of platelet function caused by this physiologically important activation, but the methodology has not been thoroughly investigated, standardized and characterized yet. We analyzed the influence of several major variables and chose optimal conditions for platelet function assessment. METHODS: For activation, 2.5 µM CaCl2 , 5 µM ADP and antibodies were added to diluted blood and incubated for 15 min. We analyzed kinetics of antibody binding and effects of their addition sequence, agonist concentration, blood dilution, exogenous calcium addition and platelet fixation. RESULTS: We tested our protocol on 11 healthy children, 22 healthy adult volunteers, 9 patients after a month on dual antiplatelet therapy after percutaneous coronary intervention (PCI), 7 adult patients and 14 children with immune thrombocytopenia (ITP). We found that our protocol is highly sensitive to ADP stimulation with low percentage of aggregates formation. The assay is also sensitive to platelet function inhibition in post-PCI patients. Finally, platelet preactivation with ITP plasma was stronger and caused increase in activation response to ADP stimulation compared to preactivation with low dose of ADP. CONCLUSIONS: Our assay is sensitive to antiplatelet therapy and platelet preactivation in ITP patients under physiological conditions with minimal percentage of aggregates formation.

Unusual Presentation of SET::NUP214-Associated Concomitant Hematological Neoplasm in a Child-Diagnostic and Treatment Struggle.

Simultaneous multilineage hematologic malignancies are uncommon and associated with poorer prognosis than single-lineage leukemia or lymphoma. Here, we describe a concomitant malignant neoplasm in a 4-year-old boy. The child presented with massive lymphoproliferative syndrome, nasal breathing difficulties, and snoring. Morphological, immunocytochemical, and flow cytometry diagnostics showed coexistence of acute myeloid leukemia (AML) and peripheral T-cell lymphoma (PTCL). Molecular examination revealed a rare t(9;9)(q34;q34)/SET::NUP214 translocation as well as common TCR clonal rearrangements in both the bone marrow and lymph nodes. The disease showed primary refractoriness to both lymphoid and myeloid high-dose chemotherapy as well as combined targeted therapy (trametinib + ruxolitinib). Hence, HSCT was performed, and the patient has since been in complete remission for over a year. This observation highlights the importance of molecular techniques for determining the united nature of complex SET::NUP214-positive malignant neoplasms arising from precursor cells with high lineage plasticity.

Comparison of platelet proteomic profiles between children and adults reveals origins of functional differences.

BACKGROUND: Platelets are blood cells responsible for the prevention of blood loss upon vessel wall disruption. It has been demonstrated that platelet functioning differs significantly between adult and pediatric donors. This study aimed to identify potential differences between the protein composition of platelets of pediatric, adolescent, and adult donors. METHODS: Platelet functional testing was conducted with live cell flow cytometry. Using a straightforward approach to platelet washing based on the sequential platelets centrifugation-resuspension, we were able to obtain stable and robust proteomics results, which corresponded to previously published data. RESULTS: We have identified that pediatric donors' platelets have increased amounts of proteins, responsible for mitochondrial activity, proteasome activity, and vesicle transport. Flow cytometry analysis of platelet intracellular signaling and functional responses revealed that platelets of the pediatric donors have diminished granule secretion and increased quiescent platelet calcium concentration and decreased calcium mobilization in response to ADP. We could explain the observed changes in calcium responses by the increased mitochondria protein content, and the changes in granule secretion could be explained by the differences in vesicle transport protein content. CONCLUSIONS: Therefore, we can conclude that the age-dependence of platelet functional responses originates from the difference in platelet protein content. IMPACT: Platelets of infants are known to functionally differ from the platelet of adult donors, although the longevity and persistivity of these differences are debatable. Pediatric donor platelets have enhanced amounts of mitochondrial, proteasomal, and vesicle transport proteins. Platelets of the pediatric donors had increased cytosolic calcium in the resting state, what is explained by the increased numbers of mitochondrial proteins. Infants had decreased platelet granule release, which resolved upon adolescence. Thus, platelets of the infants should be assessed differently from adult platelets. Differences in platelet proteomic contents persisted in adolescent groups, yet, no significant differences in platelet function were observed.

Case Report: Two clinical cases of Wilms tumor comorbid to gingival fibromatosis in young children with constitutionally mutated REST.

Introduction: Nephroblastoma (Wilms tumor (WT)) is an embryonal tumor accounting for >90% of pediatric renal cancers. About 10% of WTs harbor pathogenic germline mutations. The REST gene, classified as a putative tumor suppressor, is affected in 2% of WTs. High-throughput molecular methods facilitate advanced diagnostics of cancer. In addition to this, germline mutations in REST are also associated with familial gingival fibromatosis (GFM). Reciprocally, none of the articles on RESTmut WT mentions GFM as a comorbid condition. This report provides unique evidence on the WT-GFM comorbidity in RESTmut carriers. Case presentation: Patient 1 (a 5-year-old boy with unilateral WT) is a proband, who has two healthy siblings. Patient 2 (a 4-year-old girl with bilateral WT) is a proband from in vitro fertilization (IVF) triplets, with a sister and brother without WT. We analyzed probands' DNA extracted from peripheral blood leucocytes with a custom-targeted next-generation sequencing (NGS)-198 gene panel. The detected variants were checked in family members by Sanger sequencing. Patient 1 had a pathogenic germline mutation in REST: c.1035_1036insTA, p.(E346*), as did his mother and both brothers. There were two other WT cases in this family (proband's maternal uncles). Patient 2 had a pathogenic germline variant in REST: c.2668_2671del, p.(E891Pfs*6), as well as her sister. The mutation was probably inherited from their deceased father, as he had gingival fibromatosis. Family members with REST mutations from both families had gingival fibromatosis. A somatic REST c.663C>A p.C221* mutation was identified in one patient with WT. At the moment both patients with WT are under dynamic observation without signs of the disease. Conclusion: Here, we describe two clinical cases of WT in nonrelated young children with germline-inactivating REST variants identified by next-generation sequencing. Both patients present with familial gingival fibromatosis, regarded as clinically useful comorbidity indicative of the tumor predisposition syndrome. The two cases illustrate Wilms tumor-gingival fibromatosis comorbidity in carriers of germline-inactivated REST alleles previously identified as a predisposition factor for both conditions.

Platelet functional abnormalities in pediatric patients with kaposiform hemangioendothelioma/Kasabach-Merritt phenomenon.

Kaposiform hemangioendothelioma (KHE) is a rare vascular tumor of infancy that is commonly associated with a life-threatening thrombocytopenic condition, Kasabach-Merritt phenomenon (KMP). Platelet CLEC-2, tumor podoplanin interaction is considered the key mechanism of platelet clearance in these patients. Here, we aimed to assess platelet functionality in such patients. Three groups of 6 to 9 children were enrolled: group A with KHE/KMP without hematologic response (HR) to therapy; group B with KHE/KMP with HR; and group C with healthy children. Platelet functionality was assessed by continuous and end point flow cytometry, low-angle light scattering analysis (LaSca), fluorescent microscopy of blood smears, and ex vivo thrombi formation. Platelet integrin activation in response to a combination of CRP (GPVI agonist) and TRAP-6 (PAR1 agonist), as well as calcium mobilization and integrin activation in response to CRP or rhodocytin (CLEC-2 agonist) alone, were significantly diminished in groups A and B. At the same time, platelet responses to ADP with or without TRAP-6 were unaltered. Thrombi formation from collagen in parallel plate flow chambers was also noticeably decreased in groups A and B. In silico analysis of these results predicted diminished amounts of CLEC-2 on the platelet surface of patients, which was further confirmed by immunofluorescence microscopy and flow cytometry. In addition, we also noted a decrease in GPVI levels on platelets from group A. In KHE/KMP, platelet responses induced by CLEC-2 or GPVI activation are impaired because of the diminished number of receptors on the platelet surface. This impairment correlates with the severity of the disease and resolves as the patient recovers.

Accelerated death of megakaryocytes from Wiskott-Aldrich syndrome patients.

Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder caused by WAS gene mutations resulting in haematopoietic/immune cell defects. Recent studies report accelerated death of WAS platelets and lymphocytes. Data on megakaryocyte (MK) maturation, viability and their possible role in thrombocytopenia development in WAS are limited. In this study we evaluate the MK viability and morphology in untreated, romiplostim-treated WAS patients compared with normal controls. The study included 32 WAS patients and 17 healthy donors. MKs were captured from bone marrow aspirates by surface-immobilized anti-GPIIb-IIIa antibody. Viability (by phosphatidylserine [PS] externalization), distribution by maturation stages and size of MK were determined by light microscopy. MK distribution by maturation stages in patients differed from controls. 40 ± 22% of WAS MKs versus 23 ± 11% of normal MKs were at maturation stage 3 (p = 0.02), whereas 24 ± 20% in WAS and 39 ± 14% in controls had megakaryoblast morphology (p = 0.05). Romiplostim treatment changed the MK maturation stages distribution close to normal. PS-positive (PS+) MK in WAS was significantly higher (21 ± 21%) than in healthy controls (2 ± 4%, p < 0.01). WAS patients with more damaging truncating mutations and higher disease score had higher PS+ MK fraction (Spearman r = 0.6, p < 0.003). We conclude that WAS MKs have increased cell death tendency and changes in maturation pattern. Both could contribute to thrombocytopenia in WAS patients.

Vemurafenib combined with cladribine and cytarabine results in durable remission of pediatric BRAF V600E-positive LCH.

Langerhans cell histiocytosis (LCH) is a disorder with a variety of clinical signs. The most severe forms affect risk organs (RO). The established role of the BRAF V600E mutation in LCH led to a targeted approach. However, targeted therapy cannot cure the disease, and cessation leads to quick relapses. Here, we combined cytosine-arabinoside (Ara-C) and 2'-chlorodeoxyadenosine (2-CdA) with targeted therapy to achieve stable remission. Nineteen children were enrolled in the study: 13 were RO-positive (RO+) and 6 RO-negative (RO-). Five patients received the therapy upfront, whereas the other 14 received it as a second or third line. The protocol starts with 28 days of vemurafenib (20 mg/kg), which is followed by 3 courses of Ara-C and 2-CdA (100 mg/m2 every 12 h, 6 mg/m2 per day, days 1-5) with concomitant vemurafenib therapy. After that, vemurafenib therapy was stopped, and 3 courses of mono 2-CdA followed. All patients rapidly responded to vemurafenib: the median disease activity score decreased from 13 to 2 points in the RO+ group and from 4.5 to 0 points in the RO- group on day 28. All patients except 1 received complete protocol treatment, and 15 of them did not have disease progression. The 2-year reactivation/progression-free survival (RFS) for RO+ was 76.9% with a median follow-up of 21 months and 83.3% with a median follow-up of 29 months for RO-. Overall survival is 100%. Importantly, 1 patient experienced secondary myelodysplastic syndrome after 14 months from vemurafenib cessation. Our study demonstrates that combined vemurafenib plus 2-CdA and Ara-C is effective in a cohort of children with LCH, and the toxicity is manageable. This trial is registered at www.clinicaltrials.gov as NCT03585686.

Novel SLFN14 mutation associated with macrothrombocytopenia in a patient with severe haemorrhagic syndrome.

BACKGROUND: Platelet-type bleeding disorder 20 (BDPLT20), as known as SLFN14-related thrombocytopenia, is a rare inherited thrombocytopenia (IT). Previously, only 5 heterozygous missense mutations in the SLFN14 gene have been reported. METHODS: A comprehensive clinical and laboratory examination of a 17-year-old female patient with macrothrombocytopenia and severe mucocutaneous bleeding was performed. Examination was carried out using standardized questionnaires to assess bleeding, high-throughput sequencing (Next Generation Sequencing), optical and fluorescence microscopy, flow cytometry with activation and analysis of intracellular calcium signaling of platelets, light transmission aggregometry and thrombus growth in the flow chamber. RESULTS: Analysis of the patient's genotype revealed a previously undescribed c.655 A > G (p.K219E) variant in the hotspot of the SLFN14 gene. Immunofluorescence and brightfield examination of platelets in the smear showed heterogeneity in cells size, including giant forms over 10 µm (normal size 1-5) in diameter, with vacuolization and diffuse distribution of ß1-tubulin and CD63. Activated platelets showed impaired contraction and shedding/internalization of GPIb. GP IIb/IIIa clustering was increased at rest and attenuated upon activation. Intracellular signalling study revealed impaired calcium mobilization upon TRAP 35.97 nM (reference range 180 ± 44) and CRP-XL 10.08 nM (56 ± 30) stimulation. Aggregation with ADP, collagen, TRAP, arachidonic acid and epinephrine was impaired in light transmission aggregometry; agglutination with ristocetin persisted. In the flow chamber with a shear rate of 400 s-1 platelet adhesion to collagen and clot growth were impaired. CONCLUSION: The revealed disorders of phenotype, cytoskeleton and intracellular signaling explain the nature of SLFN14 platelet dysfunction and the patient's severe hemorrhagic syndrome.

Recognizing Minor Leukemic Populations with Monocytic Features in Mixed-Phenotype Acute Leukemia by Flow Cell Sorting Followed by Cytogenetic and Molecular Studies: Report of Five Exemplary Cases.

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.

Flow cell sorting followed by PCR-based clonality testing may assist in questionable diagnosis and monitoring of acute lymphoblastic leukemia.

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.

Molecular Heterogeneity of Pediatric AML with Atypical Promyelocytes Accumulation in Children-A Single Center Experience.

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.

A simple procedure to identify children with B-lineage acute lymphoblastic leukemia who can be successfully treated with low or moderate intensity: Sequential versus single-point minimal residual disease measurement.

Sequential monitoring of minimal residual disease (MRD) by molecular techniques or multicolor flow cytometry (MFC) has emerged over the past two decades as the primary tool to optimize treatment in pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL). The aim of our study was to compare the prognostic power of repeated MFC-MRD measurement with single-point MRD assessment in children with BCP-ALL treated with the reduced-intensity protocol ALL-MB 2008. Data from consecutive MFC-MRD at day 15 and day 36 (end of induction, EOI) were available for 507 children with Philadelphia-negative BCP-ALL. They were stratified into standard risk (SR, n = 265), intermediate risk (ImR, n = 211), and high risk (HR, n = 31) according to the initial clinical characteristics defined in the ALL-MB 2008 protocol. Quantitative (relative to quantitative thresholds) and kinetic (logarithmic reduction) assessments of MFC-MRD at both time points effectively separated patients into three groups with different risk of recurrence. On the other hand, starting with low (for the SR group) and moderate (for the ImR group) induction therapy, a single MFC-MRD measurement at EOI proved sufficient to unequivocally identify patients in whom this therapy is highly effective and distinguish them from those who cannot be successfully treated with such therapy. Therefore, initiating treatment with low or moderate treatment from the start, together with careful consideration of initial clinical risk factors and just one EOI-MFC-MRD measurement is simple, inexpensive, and entirely sufficient for treatment optimization. Furthermore, for a large proportion of patients, this approach allows better adjustment, in particular also reduction of therapy intensity than sequential MRD measurements.

Correlation of the surface expression of thymic stromal lymphopoietin receptor with the presence of CRLF2 gene rearrangements in children with B-lineage acute lymphoblastic leukemia.

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.

Genetic characteristics and treatment outcome in infants with KMT2A germline B-cell precursor acute lymphoblastic leukemia: Results of MLL-Baby protocol.

The aim of this study was to present the diagnostic and outcome characteristics of infants with germline status of KMT2A gene (KMT2A-g) B-cell precursor acute lymphoblastic leukemia (BCP-ALL) treated consistently according to the MLL-Baby protocol, a moderate-intensity protocol. Of the 139 patients enrolled in the MLL-Baby study, 100 (71.9%) carried different types of rearranged KMT2A (KMT2A-r), while the remaining 39 infants (28.1%) had KMT2A-g. KMT2A-g patients were generally older (77% older than 6 months), less likely to have a very high white blood cell count (greater than 100 × 109 /L), less likely to be central nervous system (CNS)-positive, and more likely to be CD10-positive. The 6-year event-free survival and overall survival rates for all 39 patients were 0.74 (standard error [SE] 0.07) and 0.80 (SE 0.07), respectively. Relapse was the most common adverse event (n = 5), with a cumulative incidence of relapse (CIR) of 0.13 (SE 0.06), while the incidence of a second malignancy (n = 1) and death in remission (n = 3) was 0.03 (SE 0.04) and 0.08 (SE 0.04), respectively. None of the initial parameters, including genetics and the presence of recently described fusions of NUTM1 and PAX5 genes, was able to distinguish patients with different outcomes. Only rapidity of response, measured as minimal residual disease (MRD) by flow cytometry, showed a statistically significant impact. Moderate-intensity therapy, as used in the MLL-Baby protocol in infants with KMT2A-g BCP-ALL, yields results comparable to other infant studies. Patients with a slow multicolor flow cytometry (MFC)-MRD response should be subjected to advanced therapies, such as targeted or immunotherapies.