ETV6::RUNX1-like ALL is defined by a gene expression signature similar to that of ETV6::RUNX1-positive ALL and absence of all genetic subtype-defining aberrations, including the ETV6::RUNX1 fusion. Within the International BFM Study Group, we assembled and analyzed a cohort of 100 patients (including 97 children) with ETV6::RUNX1-like ALL. We describe their diverse genetic landscape, centered around ETV6 aberrations with frequent IKZF1 disruptions, as previously shown, but including various rare non-ETV6/non-IKZF1 gene fusions, and rearrangements of CRLF2 (CRLF2r). We show that ETV6 and IKZF1 aberrations do not occur exclusively in this subtype, which hampers its classification based solely on genomic data. We confirm our previous observation of a strong association of the CD27-positive/CD44low-negative immunophenotype with ETV6::RUNX1(-like) subtype. Compared to ETV6::RUNX1-positive ALL, patients with ETV6::RUNX1-like ALL are younger, have higher white blood cell counts at diagnosis, and have an inferior early treatment response. While overall survival is comparable, event-free survival is significantly lower in patients with ETV6::RUNX1-like ALL, with NCI risk, early treatment response, IKZF1 deletions, CRLF2r, and JAK2 mutations having prognostic relevance. Notably, Down syndrome is highly prevalent and associated with a worse outcome in ETV6::RUNX1-like ALL. In conclusion, we provide biological, demographic, and clinical characteristics of the largest ETV6::RUNX1-like cohort presented to date.

e17020 Background: Testicular germ cell tumors (TGCT) are the most common malignancy in young adult men, with a globally increasing incidence. While these tumors are highly curable due to their exceptional sensitivity to cisplatin (cDDP), a proportion of patients develops a resistance to this chemotherapy, which results in disease progression and patient death. The cause of cisplatin resistance in TGCT has not been elucidated yet and is supposed to be multifactorial. Methods: We conducted a CRISPR Knockout (KO) screening in a TGCT cell line (NCCIT) using a lentiviral sgRNAs library (Addgene #101926) targeting 3015 genes involved in cell cycle regulation and cancer development. The screening population was cultured with or without cDDP (1.25 uM) for 7 days. Samples of treated and control cells were harvested before and after cDDP exposure, the sgRNA cassettes were sequenced by massive parallel sequencing, and the differences in sgRNA abundance were determined using the MAGeCK/MAGeCK-Flute pipeline. Results: The CRISPR-KO screening data analysis identified 59 gene KOs that were significantly positively selected and 7 gene KOs that were significantly negatively selected after cDDP exposure. Among the top positive hits were genes involved in the regulation of apoptosis ( BMF, CDIP1 and BAX ), epigenetic modifiers ( HDAC8, SSX2 ), G-protein regulators ( ADCY8, GNG10 ), and others (e.g. MAGEA2, ACADM, LAMP1 ) — inactivation of these genes potentially contributes to the development of cDDP resistance. Among the top negative hits were genes implicated in DNA damage repair ( FANCB , ERCC5 ), sphingolipid metabolism ( KDSR ), and cytokinesis ( SPECC1L ) — inactivation of these genes may increase sensitivity to cDDP. Conclusions: The results highlight several potential mechanisms of cDPP resistance in TGCT. Some of these genes and pathways are well known and have been previously described in association with platinum resistance in malignant tumors, while others — such as the G-protein regulators, sphingolipid synthesis, and SPECC1L — are novel in this context and may reflect the unique biology of TGCT progression. SPECC1L is involved in spindle organization and microtubule stabilization; its paralog SPECC1 is highly expressed in testis. Polyenoic sphingolipids are essential for the completion of meiosis in male germ cells and spermatozoa production. ADCY8 controls the production of cAMP, which is a key signaling molecule in spermatogenesis. They have also been described in association with malignant proliferation and progression. The identified hits will be validated in subsequent experiments.

Background: B cells are central in the pathogenesis of anti-neutrophil cytoplasmic antibody (ANCA) associated vasculitides (AAV). The efficacy of remission induction and maintenance by B cell depleting therapy strongly underlines the importance of B cells in the disease and the use of rituximab (RTX) has successfully been implemented in the treatment of AAV. However, B cell depletion after application of RTX is markedly prolonged in AAV compared to other autoimmune diseases, suggesting an impairment of the B cell compartment. Objectives: Our study aimed to understand the base of the defect in B cell reconstitution after RTX treatment in AAV, and to dissect B cell function and development in AAV. Methods: We recruited 91 AAV patients and performed deep phenotyping of the peripheral B cell compartment by spectral flow cytometry. In a subgroup of patients, we analyzed the bone marrow using flow- as well as mass cytometry. In vitro modeling assays of B lymphopoiesis have been applied to study dynamic of development of AAV B cell precursors. BAFFR involvement into peripheral B cell survival and maturation have been studied by investigating serum BAFF concentration by ELISA, BAFFR expression of isolated B cells was determined by qPCR and Western Blot, and by in vitro survival assays. Results: AAV patients show B-lymphocytopenia. Low transitional B cell numbers in treatment-naive patients indicate an impaired central B cell development. In RTX-treated AAV patients the median time of B cell depletion, defined as less than 5 cells/µL, was 17 months (IQR 9-36). We studied phenotype and development of bone marrow B cells and found a defect in early B cell development in both treatment-naive and RTX-treated AAV patients. Only in a subgroup of RTX-treated patients, transitional B cells were increased, indicative of beginning or ongoing B cell repopulation. The increased amount of transitional B cells did not lead to replenishment of the later stages of peripheral B cell maturation, suggesting a maturation stop in peripheral B cell reconstitution in these patients. We found low BAFFR expression in AAV peripheral B cells, caused by enhanced shedding of BAFFR, that resulted in a reduced B cell survival in response to BAFF. Conclusion: Our data suggest that prolonged depletion of B cells in AAV patients after RTX therapy indicate a B cell defect that is unmasked by treatment. Impaired central B lymphopoiesis associated with disturbed peripheral B cell maturation because of enhanced BAFFR processing and shedding, resulting in reduced immature B cell survival, contribute to a delayed recovery of the peripheral B cell pool after RTX treatment in AAV. Our data point to a severe defect in B cell maturation and development in patients with AAV that may influence the therapeutic use of B cell depleting agents in this disease. In addition, our results emphasize the need for close immune monitoring after B cell depletion in AAV. REFERENCES: NIL. Acknowledgements: NIL. Disclosure of Interests: None declared.

Understanding complex, organ-level single-cell datasets represents a formidable interdisciplinary challenge. This study aims to describe developmental trajectories of thymocytes and mature T cells. We developed tviblindi , a trajectory inference algorithm that integrates several autonomous modules - pseudotime inference, random walk simulations, real-time topological classification using persistent homology, and autoencoder-based 2D visualization using the vaevictis algorithm. This integration facilitates interactive exploration of developmental trajectories, revealing not only the canonical CD4 and CD8 development but also offering insights into checkpoints such as TCRβ selection and positive/negative selection. Furthermore, it allows us to thoroughly characterize thymic regulatory T cells, tracing their development from the negative selection stage to mature thymic regulatory T cells with an extensive proliferation history and an immunophenotype of activated and recirculating cells. tviblindi is a versatile and generic approach suitable for any mass cytometry or single-cell RNA-seq dataset, equipping biologists with an effective tool for interpreting complex data.

Defective FAS (CD95/Apo-1/TNFRSF6) signaling causes autoimmune lymphoproliferative syndrome (ALPS). Hypergammaglobulinemia is a common feature in ALPS with FAS mutations (ALPS-FAS), but paradoxically, fewer conventional memory cells differentiate from FAS-expressing germinal center (GC) B cells. Resistance to FAS-induced apoptosis does not explain this phenotype. We tested the hypothesis that defective non-apoptotic FAS signaling may contribute to impaired B cell differentiation in ALPS. We analyzed secondary lymphoid organs of patients with ALPS-FAS and found low numbers of memory B cells, fewer GC B cells, and an expanded extrafollicular (EF) B cell response. Enhanced mTOR activity has been shown to favor EF versus GC fate decision, and we found enhanced PI3K/mTOR and BCR signaling in ALPS-FAS splenic B cells. Modeling initial T-dependent B cell activation with CD40L in vitro, we showed that FAS competent cells with transient FAS ligation showed specifically decreased mTOR axis activation without apoptosis. Mechanistically, transient FAS engagement with involvement of caspase-8 induced nuclear exclusion of PTEN, leading to mTOR inhibition. In addition, FASL-dependent PTEN nuclear exclusion and mTOR modulation were defective in patients with ALPS-FAS. In the early phase of activation, FAS stimulation promoted expression of genes related to GC initiation at the expense of processes related to the EF response. Hence, our data suggest that non-apoptotic FAS signaling acts as molecular switch between EF versus GC fate decisions via regulation of the mTOR axis and transcription. The defect of this modulatory circuit may explain the observed hypergammaglobulinemia and low memory B cell numbers in ALPS.

Detailed knowledge of the human B-cell development is crucial for proper interpretation of inborn errors of immunity and for malignant diseases. It is of interest to understand the kinetics of protein expression changes during the B cell development, but also to properly interpret the major and possibly alternative developmental trajectories. We have investigated human bone marrow and peripheral blood samples from healthy individuals with the aim to describe all B-cell developmental trajectories across the two tissues. We validated a 30-parameter mass cytometry panel and demonstrated the utility of “ vaevictis ” visualization of B-cell developmental stages. We used our recently developed trajectory inference tool “ tviblindi ” to exhaustively describe all trajectories leading to all developmental ends discovered in the data. Focusing on Natural Effector B cells, we demonstrated the dynamics of expression of nuclear factors (PAX-5, TdT, Ki-67, Bcl-2), cytokine and chemokine receptors (CD127, CXCR4, CXCR5) in relation to the canonical B-cell developmental stage markers (CD34, CD10, sIgM, IgD, CD20, CD27). Lastly, we performed analysis of the expression changes related to developmental branching points (Natural Effector versus Switched Memory B cells, marked by up-regulation of CD73).In conclusion, we developed, validated and presented a comprehensive set of tools for investigation of B-cell development.### Competing Interest StatementThe authors have declared no competing interest.

520 Background: Although highly curable, a proportion of testicular germ cell tumors (TGCT) develop resistance to cisplatin-based chemotherapy with subsequent disease relapses and possible patient death. The causes of disease progression and cisplatin (CDDP) resistance have not yet been well elucidated. Methods: We analyzed 56 samples of 15 patients with advanced and relapsing TGCT including primary tumors, metastases persisting after CDDP-containing chemotherapy, and circulating free tumor DNA (cfDNA) from the disease progression, with gDNA from blood leucocytes used as a background control. The quantity and quality of cfDNA was checked by capillary electrophoresis. All samples were sequenced by whole exome sequencing with SureSelect libraries on Illumina platform. Only mutations that were found in malignant samples or cfDNA with variant allele frequency (VAF) ≥ 5% and at least in 3 reads, not present in gDNA, with a minimum total read depth of 5 quality reads, excluding synonymous variants, were further analyzed. Results: Mutations of 6 genes with a significant role in carcinogenesis and/or testis development were identified in more than one patient: RBMX (in 4 pts), TPTE2 and ANKRD30A (in 3 pts), CDC27, PRAMEF8, and PRDM9 (in 2 pts). All the mutations were missense, with VAF 5 - 86%. They were all detectable in patients’ cfDNA, with increasing VAF during the tumor progression; the VAFs in cfDNA collected at advanced disease stages were even higher than those in primary tumors. PRDM9 mutations were found only in cfDNA from disease progression, but not in the primary testicular tumors. Of the identified genes, only CDC27 mutations have been previously described in TGCT. The genes newly associated with TGCT in this study are involved in genomic stability maintenance ( RBMX), spermiogenesis ( TPTE2), cell proliferation and apoptosis ( PRAMEF8), and several of them show testis-specific expression ( TPTE2, PRAMEF8, ANKRD30A, PRDM9). PRDM9 encodes a zinc-finger protein with histone methyltransferase activity, responsible for H3 methylation during meiosis. As epigenetic changes are supposed to play a crucial role in TGCT pathogenesis and resistance, and aberrant H3 methylation has been previously related to abnormal expression of OCT3/4 transcription factor promoting TGCT development, this gene is a strong candidate for TGCT progression and evolving CDDP resistance. Conclusions: Mutations of 5 novel genes have been identified in association with TGCT progression and cisplatin resistance. Their impact is strongly suggested by their functions in normal and malignant regulatory pathways and testis-specific expression patterns but has to be confirmed in experimental studies. The National Institute for Cancer Research project (Programme EXCELES, ID Project No. LX22NPO5102) - Funded by the European Union - Next Generation EU. Supported by grants MH CZ - DRO (00064190, 00064203).

Detailed knowledge of human B-cell development is crucial for the proper interpretation of inborn errors of immunity and malignant diseases. It is of interest to understand the kinetics of protein expression changes during development, but also to properly interpret the major and possibly alternative developmental trajectories. We have investigated human samples from healthy individuals with the aim of describing all B-cell developmental trajectories. We validated a 30-parameter mass cytometry panel and demonstrated the utility of "vaevictis" visualization of B-cell developmental stages. We used the trajectory inference tool "tviblindi" to exhaustively describe all trajectories leading to all developmental ends discovered in the data. Focusing on Natural Effector B cells, we demonstrated the dynamics of expression of nuclear factors (PAX-5, TdT, Ki-67, Bcl-2), cytokine and chemokine receptors (CD127, CXCR4, CXCR5) in relation to the canonical B-cell developmental stage markers. We observed branching of the memory development, where follicular memory formation was marked by CD73 expression. Lastly, we performed an analysis of two example cases of abnormal B-cell development caused by mutations in RAG-1 and Wiskott-Aldrich syndrome gene in patients with primary immunodeficiency. In conclusion, we developed, validated, and presented a comprehensive set of tools for the investigation of B-cell development in the bone marrow compartment.

Introduction: In several subtypes of acute lymphoblastic leukemia (ALL), lymphoblasts often switch to a myeloid (mostly monocytic) lineage during the early phase of treatment. This unusual but well-documented phenomenon can cause diagnostic problems, particularly in the assessment of response to treatment. The cause of the lineage switch and its dynamics are not yet fully understood. Modern multiomic methods provide a unique opportunity to elucidate the progression of B-to-myeloid transdifferentiation and to clarify how different subtypes of ALL with a propensity for lineage instability differ. Previous studies have focused only on blasts at diagnosis (Dx); in our study, we also incorporated blasts from peripheral blood (PB) during or after the initial corticosteroid prephase of treatment. Methods: We used 10x Genomics' Single Cell 5‘ v2 platform to simultaneously evaluate gene expression and cell surface protein expression at the single-cell level from 3 pediatric patients with different subtypes of ALL prone to phenotypic instability (DUX4 rearrangement, ZNF384 rearrangement, and PAX5 P80R mutation). Blasts from bone marrow (BM) or PB from Dx were analyzed together with PB from day 8 (n=1) or day 5 (n=1) of a BFM-type treatment protocol. The samples were stained with a panel of TotalSeq-C antibody-oligonucleotide conjugates and processed using the Single Cell 5‘ workflow. Sequencing was conducted on an Illumina NextSeq2000 platform. A mass cytometry panel combining myeloid and lymphoid markers was used to measure protein expression in paired samples (Dx and on-treatment sample) from patients with a B-to-myeloid switch with DUX4r (n=3), PAX5 P80R (n=1), and ZNF384r (n=1) subtypes. Both RNA and protein counts were derived with a pipeline based on The Single-cell Pediatric Cancer Atlas project (Hawkins, bioRxiv, doi: 10.1101/2024.04.19.590243) and further data processing, including background removal and doublet detection, was carried out in accordance with current recommendations (Heumos, Nat Rev Genet 2023). Our trajectory inference framework, tviblindi (Stuchly, eLife 2024), was used to infer dynamic processes from the static snapshots provided by single-cell measurements. Results: In all subtypes, we were able to detect an intermediate population connecting B- and switched blasts, favoring a model in which the switch occurs via transdifferentiation. In the PAX5 P80R patient, we observed an abrupt change to a myeloid phenotype on day 5 of treatment. However, we identified the intermediate population at Dx on a transcriptomic level. In the ZNF384r patient, lineage instability was already evident at Dx, with a portion of the blasts showing differentiation towards the myeloid lineage while harboring a clonal immunoglobulin heavy chain (IGH) rearrangement on a DNA level as determined from sorted myeloid blasts. The expression of this IGH rearrangement decreased in myeloid blasts together with IGHM. In the DUX4r patient, B-to-myeloid transdifferentiation was detected on day 8 of the corticoid pre-phase, accompanied by a smaller B lymphoid blast population showing increased protein expression of CD45 and an altered transcriptome distinguishing this cluster from B lymphoid blasts at Dx. The immunophenotype of the intermediate populations was typical of B-blasts, but a shared set of myeloid genes was upregulated in their transcriptomes (e.g., CSF2RA, CEBPD, CD86, CD68, CYBB, S100A4, TYROBP, SRGN). Trajectory inference analysis was consistent with a transdifferentiation path through these intermediate populations in all samples, with an expected lag between transcriptomic and immunophenotypic alterations. Notably, CD371 was upregulated at the onset of the switch, along with other myeloid markers. Summary: Although the dynamics of the switch varied between subtypes, we observed an intermediate cellular population of varying size in patients with DUX4r, ZNF384r, and PAX5 P80R ALL, which was immunophenotypically consistent with B-blasts while sharing a common myeloid-primed transcriptomic signature. Furthermore, pseudotemporal ordering with tviblindi placed these intermediate populations before the emergence of monocytic cells in all samples, supporting our transdifferentiation hypothesis. Supported by NU23-05-00353, NW24-07-0026, NU23J-03-00026, UNCE/24/MED/003, and LX22NPO5102.

OBJECTIVES:B-cell depletion time after rituximab (RTX) treatment is prolonged in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) compared with other autoimmune diseases. We investigated central and peripheral B-cell development to identify the causes for the defect in B-cell reconstitution after RTX therapy. METHODS:We recruited 91 patients with AAV and performed deep phenotyping of the peripheral and bone marrow B-cell compartment by spectral flow and mass cytometry. B-cell development was studied by in vitro modelling and the role of BAFF receptor by quantitative PCR, western blot analysis and in vitro assays. RESULTS:Treatment-naïve patients with AAV showed low transitional B-cell numbers, suggesting impaired B-lymphopoiesis. We analysed bone marrow of treatment-naïve and RTX-treated patients with AAV and found reduced B-lymphoid precursors. In vitro modelling of B-lymphopoiesis from AAV haematopoietic stem cells showed intact, but slower and reduced immature B-cell development. In a subgroup of patients, after RTX treatment, the presence of transitional B cells did not translate in replenishment of naïve B cells, suggesting an impairment in peripheral B-cell maturation. We found low BAFF-receptor expression on B cells of RTX-treated patients with AAV, resulting in reduced survival in response to BAFF in vitro. CONCLUSIONS:Prolonged depletion of B cells in patients with AAV after RTX therapy indicates a B-cell defect that is unmasked by RTX treatment. Our data indicate that impaired bone marrow B-lymphopoiesis results in a delayed recovery of peripheral B cells that may be further aggravated by a survival defect of B cells. Our findings contribute to the understanding of AAV pathogenesis and may have clinical implications regarding RTX retreatment schedules and immunomonitoring after RTX therapy.

Triple‐negative breast cancer (TNBC) is an aggressive and complex subtype of breast cancer that lacks targeted therapy. TNBC manifests characteristic, extensive intratumoral heterogeneity that promotes disease progression and influences drug response. Single‐cell techniques in combination with next‐generation computation provide an unprecedented opportunity to identify molecular events with therapeutic potential. Here, we describe the generation of a comprehensive mass cytometry panel for multiparametric detection of 23 phenotypic markers and 13 signaling molecules. This single‐cell proteomic approach allowed us to explore the landscape of TNBC heterogeneity, with particular emphasis on the tumor microenvironment. We prospectively profiled freshly resected tumors from 26 TNBC patients. These tumors contained phenotypically distinct subpopulations of cancer and stromal cells that were associated with the patient's clinical status at the time of surgery. We further classified the epithelial‐mesenchymal plasticity of tumor cells, and molecularly defined phenotypically diverse populations of tumor‐associated stroma. Furthermore, in a retrospective tissue‐microarray TNBC cohort, we showed that the level of CD97 at the time of surgery has prognostic potential.

Recently, we defined “CML-like” subtype of BCR::ABL1-positive acute lymphoblastic leukemia (ALL), resembling lymphoid blast crisis of chronic myeloid leukemia (CML). Here we retrospectively analyzed prognostic relevance of minimal residual disease (MRD) and other features in 147 children with BCR::ABL1-positive ALL (diagnosed I/2000–IV/2021, treated according to EsPhALL (n = 133) or other (n = 14) protocols), using DNA-based monitoring of BCR::ABL1 genomic breakpoint and clonal immunoglobulin/T-cell receptor gene rearrangements. Although overall prognosis of CML-like (n = 48) and typical ALL (n = 99) was similar (5-year-EFS 60% and 49%, respectively; 5-year-OS 75% and 73%, respectively), typical ALL presented more relapses while CML-like patients more often died in the first remission. Prognostic role of MRD was significant in the typical ALL (p = 0.0005 in multivariate analysis for EFS). In contrast, in CML-like patients MRD was not significant (p values > 0.2) and inapplicable for therapy adjustment. Moreover, in the typical ALL, risk-prediction could be further improved by considering initial hyperleukocytosis. Early distinguishing typical BCR::ABL1-positive ALL and CML-like patients is essential to enable optimal treatment approach in upcoming protocols. For the typical ALL, tyrosine-kinase inhibitors and concurrent chemotherapy with risk-directed intensity should be recommended; in the CML-like disease, no relevant prognostic feature applicable for therapy tailoring was found so far.

BACKGROUND:Testicular germ cell tumors (TGCT) are unique malignancies of young adult men; their biology is, however, underexplored and there has not been much progress in their treatment for decades. Circulating free tumor DNA (cfDNA) analysis represents a promising way of discovering novel diagnostic and treatment options.OBJECTIVE:The study evaluates the clinical value of cfDNA detection in TGCT patients.DESIGN AND METHODS:Total cfDNA concentration and ratio of its 2 main fragments (180 and 360 bp) were evaluated by spectrophotometry, capillary electrophoresis and qPCR in peripheral blood plasma of 96 TGCT patients (173 samples) and 31 normal controls. Non-parametric tests were used for statistical analyses.RESULTS:The total cfDNA concentration was significantly higher in TGCT than in controls (P < 0.0001), with the highest levels at disease progression, but with no clear threshold between malignant and normal samples. Patients with positive tumor markers had higher cfDNA concentrations than those with negative markers (P = 0.01). Longer 360 bp cfDNA fragments were found in 58% of TGCT patients including almost all samples from relapse or disease progression but no normal controls (P < 0.0001).CONCLUSION:Total cfDNA levels are significantly increased in TGCT patients but without a clear threshold separating normal and tumor samples, thus total cfDNA amount itself is not a sensitive enough marker to identify or monitor TGCT. Longer cfDNA fragments have been found exclusively in a proportion of tumors and predominantly at disease progression, representing a novel potential marker for TGCT monitoring that would deserve further exploration.

Recently we showed that in 20-30% of children diagnosed as BCR::ABL1-positive (Ph+) acute lymphoblastic leukemia (ALL) the BCR::ABL1 fusion is present in a wider clone, involving myeloid cells, non-ALL B-cells and T-cells. This leads to discordant minimal residual disease (MRD) levels assessed by the quantification of two available MRD targets - immunoglobulin (IG)/T-cell receptor (TR) genes rearrangements and BCR::ABL1 fusion. As the cases with multilineage BCR::ABL1 involvement resemble lymphoid blast crisis (LBC) of chronic myeloid leukemia (CML) we named these leukemias "CML-like", as distinct from "typical Ph+ ALL". To reveal biological and clinical differences between the two subtypes, we retrospectively analyzed prognostic relevance of MRD and other features in 147 children with BCR::ABL1-positive ALL treated according to EsPhALL (n = 133) or other (n = 14) protocols. MRD was assessed by DNA-based monitoring of BCR::ABL1 genomic breakpoint and of clonal IG/TR rearrangements. Although overall prognosis of CML-like (n = 48) and typical Ph+ ALL (n = 99) was similar (5-year EFS 60% and 49%; 5-year OS 75% and 73%, respectively), typical Ph+ ALL presented more relapses (12/48 vs. 42/99; p = 0.046) while CML-like patients more often died in the first remission (6/48 vs. 3/99; p = 0.059 for CR1 deaths in total and p = 0.01 for CR1 deaths out of the total deaths). Prognostic role of MRD measured at standard treatment timepoints (TP1 - end of induction IA, day 33; TP2 - end of consolidation IB, week 12) was highly significant in the typical Ph+ ALL (p = 0.0005 for EFS). In contrast, in CML-like patients prognostic impact of MRD was not significant and thus inapplicable for therapy adjustment. Similarly, although diagnostic white blood cell counts were similar in the two subgroups, impact of the hyperleukocytosis ≥ 50 x 109/l on outcome was highly significant in the typical Ph+ ALL (and remained significant in multivariate analyses, being apparent even in patients with fast MRD response), but no prognostic impact was found in CML-like patients. While in the typical Ph+ ALL the NCI risk tended to impact 5-year OS (89% vs. 67%; p = 0.03), there was no impact on prognosis in the CML-like patients. The BCR::ABL1 transcript type (minor/p190 vs. Major/p210), IKZF1 deletion, sex, or the age at diagnosis (< 10 vs. ≥ 10 years) did not show any prognostic relevance. Earlier start of continuous tyrosine kinase inhibitors (TKI) treatment (day 15 vs. day 33) was associated with lower MRD levels in the typical Ph+ ALL but not in CML-like patients. However, earlier TKI start tended to result in better 5-year OS in both subtypes (78% vs. 67%; p = 0.082 and 83% vs. 60%; p = 0.066 for typical Ph+ ALL and CML-like, respectively). To shed more light on the nature of the two subtypes, we now collect additional data (including transcriptome profiles, single cell characteristics, genomic breakpoint features) in order to reveal biological background, cell of origin and further potential differences between the subtypes. Such data could help to find optimal treatment strategies, and also to answer the attractive question whether CML-like disease is in fact a regular CML in LBC or whether there is a wider spectrum of BCR::ABL1-positive leukemias. If these studies lead to a conclusion that CML-like is indeed indistinguishable from LBC-CML, it will be necessary to re-define CML as a disease manifesting - at least in childhood - very often in LBC and with the minor-BCR::ABL1/p190 fusion variant. In conclusion, our data unequivocally confirm that although clinically presenting at diagnosis as one entity, there are two biologically distinct subtypes of BCR::ABL1-positive ALL - typical Ph+ ALL and CML-like disease. The different biology is reflected in treatment response and although the overall survival rates of both subtypes are similar on current protocol, the key reasons of treatment failure are different - toxicity of intensive ALL treatment in CML-like patients and relapses in typical Ph+ ALL. Thus, early distinguishing of the two subtypes is essential to enable optimal treatment approach and therapy adjustments in upcoming trials. For the typical Ph+ ALL, TKI and concurrent chemotherapy with risk-directed intensity should be recommended; in the CML-like disease, representing one-fourth to one-third of childhood patients diagnosed as BCR::ABL1-positive ALL, no relevant risk factor applicable for therapy tailoring was found so far.

Fusion of the ZNF384 gene as the 3' partner to several different 5' partner genes occurs recurrently in B-cell precursor acute lymphoblastic and mixed phenotype B/myeloid leukemia. These canonical fusions (ZNF384r) contain the complete ZNF384 coding sequence and are associated with a specific gene expression signature. Cases with this signature, but without canonical ZNF384 fusions (ZNF384r-like cases), have been described previously. Although some have been shown to harbor ZNF362 fusions, the primary aberrations remain unknown in a major proportion. We studied 3 patients with the ZNF384r signature and unknown primary genetic background and identified a previously unknown class of genetic aberration affecting the last exon of ZNF384 and resulting in disruption of the C-terminal portion of the ZNF384 protein. Importantly, in 2 cases, the ZNF384 aberration, indel, was missed during the bioinformatic analysis but revealed by the manual, targeted reanalysis. Two cases with the novel aberrations had a mixed (B/myeloid) immunophenotype commonly associated with canonical ZNF384 fusions. In conclusion, we present leukemia cases with a novel class of ZNF384 aberrations that phenocopy leukemia with ZNF384r. Therefore, we show that part of the so-called ZNF384r-like cases represent the same genetic subtype as leukemia with canonical ZNF384 fusions.

Recently, we described B-cell precursor acute lymphoblastic leukemia (BCP-ALL) subtype with early switch to the monocytic lineage and loss of the B-cell immunophenotype, including CD19 expression. Thus far, the genetic background has remained unknown. Among 726 children consecutively diagnosed with BCP-ALL, 8% patients experienced switch detectable by flow cytometry (FC). Using exome and RNA sequencing, switch was found to positively correlate with three different genetic subtypes: PAX5-P80R mutation (5 cases with switch out of 5), rearranged DUX4 (DUX4r; 30 cases of 41) and rearranged ZNF384 (ZNF384r; 4 cases of 10). Expression profiles or phenotypic patterns correlated with genotypes, but within each genotype they could not identify cases who subsequently switched. If switching was not taken into account, the B-cell-oriented FC assessment underestimated the minimal residual disease level. For patients with PAX5-P80R, a discordance between FC-determined and PCR-determined MRD was found on day 15, resulting from a rapid loss of the B-cell phenotype. Discordance on day 33 was observed in all the DUX4r, PAX5-P80R and ZNF384r subtypes. Importantly, despite the substantial phenotypic changes, possibly even challenging the appropriateness of BCP-ALL therapy, the monocytic switch was not associated with a higher incidence of relapse and poorer prognosis in patients undergoing standard ALL treatment.

Background Acute leukemia is the most common malignancy of childhood. Most of pediatric acute leukemias are derived from B-cell precursor, and about 15% are from T-cell origin and lead to T-cell acute lymphoblastic leukemia (T-ALL). Despite recent progress in treatment, the prognosis of T-ALL patients is worse, and ~30% of cases relapse (Pui CH, Seminars in hematology 2009). Despite the need for targeting molecules to treat resistant and high risk pediatric T-ALLs, in vitro drug testing may not be predictive of efficacy in vivo due to the complexity of the signalling network and to the cell heterogeneity within the individual's sample. High-resolution analysis of signalling profile at single-cell level may represent an innovative tool in the comprehension of signalling network and its role in the response to drugs (Bodenmiller B et al, Science signaling 2010). Currently mass cytometry (CyTOF) technique allows the measurement of more than 30 parameter per single cell (Bendall SC et al, Science 2011), rendering this approach an ideal method to investigate the complex biology of T-ALL (Girardi T et al, Blood. 2017) and to assay drug candidates for cellular targeting. Methods Using single-cell mass cytometry we developed panel of 39 metal-labeled monoclonal antibodies (moAbs) identifying T-ALL blasts and non-malignant T-cells. Using phospho-specific moAbs and moAbs targeting proliferation and apoptosis we detected signal transduction upon in vitro treatment of 17 diagnostic and 5 relapse T-ALL samples with IL-7 (Jak/STAT5 pathway activator) , BEZ-235 (inhibitor of PI3K and mTOR), and Pervanadate (inhibitor of tyrosine phosphatases). We have used sample barcoding by anti-CD45 antibodies to unify sample preparation and acquisition for all treatment conditions and we resolved the cells of interest by manual gating. We evaluated both, the individual change sin p-kinases and the overall changes using dimensionality reduction approach. Results T-ALL cells showed constitutive activation of various signaling pathways as well as proliferation markers compared to residual bone marrow T-cells and T-cells isolated from healthy donors. Up-regulated activity of PI3K-mTOR pathway (p4E-BP1, pAkt, pS6), proliferation rate (pRb, Ki-67), MAPK pathway (p-p38, pErk1/2), translation (pCREB) in T-ALL cells was detected, whereas lower levels of anti-apoptotic protein Bcl2 in T-ALL cells were detected. Interleukin 7 (IL-7) activates three main signalling pathways such as STAT5, PI3K/Akt/mTOR and MEK/Erk, leading to the promotion of leukemia cell viability, cell cycle progression and growth. Thus we interrogated T-ALLs in their ability to respond to IL -7 in vitro . We used an hierarchical clustering analysis (with Euclidean distance metrics and an average linkage) and we were able to divide T-ALL samples in IL-7 responder (6 out of 17) and IL-7 non responder (11 out of 17). Of note, no significant differences in IL7Ra (CD127) expression was observed between the two groups. Interestingly IL-7 responders had higher levels of pRb and Ki-67 proliferation markers as compared to both IL-7 non-responders and non-malignant T-cells. Moreover IL-7 non-response correlate with poor response in vivo to prednisone and higher level of minimal residual disease (MRD) at day15 of remission induction treatment. Finally T-ALL cells were treated ex vivo with PI3K/AkT/mTOR dual inhibitor BEZ-235. Of the 17 T-ALL patients tested, 9 responded to BEZ-235 but not to IL-7 , by contrast 4 did not respond to BEZ-235 being IL-7 responders, two patients responded both to IL-7 activation and BEZ-235 inhibition. One single patient did not respond neither to IL-7 nor to BEZ-235 Conclusions In summary we characterized pediatric T-ALL samples demonstrating the feasibility of CyTOF-based single-cell profiling of signal transduction pathways in this setting. We detected constitutively active pathways in T-ALL blasts as compared to residual non-malignant T-cells. Importantly we identified by functional read outs distinct clusters of IL-7 and BEZ-235 T-ALL responders patients, supporting the notion of a mutual exclusivity between JAK-STAT (or Ras) pathway genomic alterations and PI3K-AKT pathway alterations (Liu et al. Nat Genet. 2017). Our observation can contribute to the better understanding of the complex signalling network governing T-ALL behaviour and response to therapy. Disclosures No relevant conflicts of interest to declare.