Infant Leukemia Research Articles

Infant acute lymphoblastic leukemia: treatment strategies and new insights

Acute lymphoblastic leukemia (ALL) in infants, especially KMT2A gene rearranged ALL (KMT2A-rALL), is a rare disease. Its prognosis is extremely poor, with a reported long-term event-free survival rate of ≤50%. In addition, acute and late toxicities caused by intensive treatment remain issues to be resolved. In the context of this background, the introduction of a more appropriate stratification and a novel treatment with minimal toxicities are urgently required. Establishment of evidence-based novel treatment strategies through an international collaborative study is important owing to the rarity of the disease. Currently, an international collaborative study with a European study group, which includes blinatumomab combined therapy, has been proposed. We herein review previous key clinical trials and the latest treatment strategies for infant ALL.

Leukemogenic pathway of infant leukemia with MLL fusion

Acute lymphoblastic leukemia (ALL) in infants remains an intractable and difficult-to-treat leukemia as compared to other pediatric ALLs, for which considerable progress has been achieved in terms of treatment outcomes in recent years. The leukemic cells in infants with ALL frequently carry chromosome translocations involving 11q23, resulting in the rearrangement and fusion of the MLL (KMT2A) gene. Among many MLL fusion genes, MLL-AF4 (KMT2A-AFF1) fusion is characteristically observed in infants with ALL, representing a hallmark of poor prognosis. In MLL-AF4-positive infants with ALL, first leukemic cells with MLL-AF4 were generated in utero. Analysis of several murine and human leukemia models revealed that the target cells for tumorigenesis by MLL-AF4 were not the hematopoietic progenitor cells of the bone marrow, but the early hematopoietic progenitor cells present in the fetal liver during the embryonic period and possibly the undifferentiated cells prior to the commitment to hematopoietic cells in the fetus. Elucidation of the leukemogenic process of infant ALL with MLL-AF4 may lead to early, pre-symptomatic diagnosis of leukemia, resulting in the improvement of prognosis and prevention of the onset of ALL in infants.

MLL-TFE3: a novel and aggressive KMT2A fusion identified in infant leukemia

Key Points A novel KMT2A-rearrangement, MLL-TFE3, was identified in an infant leukemia patient. MLL-TFE3 expression produces aggressive leukemia in a mouse model.

3088 – DISSECTING THE ONTOGENIC REMODELING OF THE PROTEOMIC LANDSCAPE AND FUNCTIONALITY OF HEMATOPOIETIC PROGENITOR CELLS IN NORMAL DEVELOPMENT AND LEUKEMIA

The process of hematopoiesis is subject to extensive ontogenic remodeling that is accompanied by alterations in cellular fate both during normal development and disease. Although the functional differences between fetal and adult hematopoiesis are well established, the responsible molecular mechanisms have long remained largely unexplored on the proteomic level. In our latest work, we characterized and compared the proteomic makeup of fetal and adult HSPCs (Jassinskaja et al., 2017, Cell Reports). Here, we have utilized state-of-the-art mass spectrometry to gain deep coverage of the proteome of 100,000 fetal and adult lymphoid-primed multipotent progenitors (LMPPs), common lymphoid progenitors (CLPs) and granulocyte-monocyte progenitors (GMPs). Our analysis resulted in the identification and quantification of over 4000 proteins, with 200-300 proteins per cell type displaying differential expression between the fetus and the adult. We uncovered ontogenic changes in expression of several transcription factors with critical roles in hematopoiesis. Together with other proteins involved in lineage commitment and differentiation, we show that protein expression in fetal and adult hematopoietic progenitor cells is predictive of differential lineage potential. Our subsequent functional analyses further emphasize that the proteomic differences affect cell fate both in the context of normal differentiation and leukemic hematopoiesis driven by the MLL-ENL fusion oncogene. Collectively, our work represents a significant advancement in the understanding of the molecular programs that govern ontogenic differences in hematopoiesis and provides a solid foundation for future investigation of which factors are responsible for the difference in susceptibility and outcome of different leukemias in infants and in adults.

3051 – IDENTIFYING THE CELL OF ORIGIN IN MLL-AF9 INFANT LEUKAEMIA

MLL rearrangements are the predominant cause of acute infant leukaemias, which initiate in utero within foetal HSPCs and distinctly lack additional cooperating mutations. Strikingly, MLL-AF9 causes an AML or B-ALL, but in adults almost exclusively an AML. Thus, by understanding the contribution from the foetal cell of origin, we can explain differences between patients with respect to disease initiation, progression and outcomes. We utilised inducible MLL-AF9 expression to explore AML and B-ALL initiation from foetal and adult murine HSPCs. MLL-AF9 imparts distinct lineage, proliferation and self-renewal outputs within and between foetal and adult HSPC populations. In contrast to adult HSPCs, MLL-AF9 generated significant increases in lymphoid output under lymphoid conditions across foetal HSPC in vitro, even overriding CMP myeloid bias for B-lymphoid output, highlighting the specific nature of infant biology. HSC/MPP and LMPP consistently gave rise to a pro-B phenotype, suggesting these as cells of origin for the clinically dismal infant pro-B ALL. In AML, transplantation of HSC/MPP causes the most aggressive disease marked by shortest latency and invasive nature. Interestingly, in myeloid assays, foetal HSC/MPP and LMPP display an initial delay in proliferation, unlike CMP and GMP, which proliferate through a foetal-specific phenotype of CD11b+ blasts at first plating. Adult CMP do not replicate the proliferative phenotype of foetal CMP, highlighting the differences in disease initiation between developmental stages. An MLL rearrangement represents the worst prognostic factor for B-ALL, and in AML a description of which foetal cells can initiate disease is lacking. Given the prevalence of unfavourable prognostic factors, defining how foetal blood cell biology shapes the aetiology of infant leukaemia will inform rationale for drug discovery and therapy applications.

The results of allogeneic hematopoietic stem cell transplantation from a matched unrelated and haploidentical donors in children with high-risk infant leukemia in first and second remissions

Aсute myeloid leukemia (AML) in children aged 0–2 years and aсute lymphoid leukemia (ALL) up to 1 year (i.e., infants) frequently characterize high risk and poor prognosis. Аllogeneic hemopoietic stem cell transplantation (аllo-HCST) is a main curative but toxic option for these patients, and choice of allogeneic donor may be one of the important factor for long-term survival. Aim. To evaluate overall survival (OS), relapse free survival (RFS), transplant related mortality (TRM), "graft versus host" disease free/relapse free survival (GRFS) in infant with acute leukemia underwent allo-HCST from MUD vs haplodonor at 1st or 2nd remission. The study was approved by the Independent Ethics Committee and the Scientific Council of the I.P. Pavlov First Saint Petersburg State Medical University. 34 children with infant acute leukemia: 23 pts with AML (68%) and 11 – with ALL (32%) – underwent allo-HSCT from MUD vs haplo at 1st or 2nd remission between 2004–2018 were analyzed. Median age at allo-HCST – 22 months (6 months – 5 y.o.). HSCT was performed from MUD in 19 (56%) pts (group 1), haplo – 15 (44%) pts (group 2). Myeloablative conditioning received 29 (85%) pts. Reduced intensity conditioning received 5 (15%) pts. Posttransplant cyclophosphomyde (PtCy) was used in 10 (53%) pts in the group 1 and 14 (93%) pts. in the group 2 (p = 0.043). Engraftment was identified in 18 pts (95%) of group 1 and 12 pts (80%) of group 2 (p = 0.28). At the median follow up 3.5 years OS is 79% in the group 1 аnd 73% in the group 2 (p = 0.68). RFS is 79% in the group 1 аnd 67% in the group 2 (p = 0.41). GRFS is 39% in the group 1 аnd 47% in the group 2 (p = 0.5). TRM occurred in 2 pts (11%) of group 1 (due to infectious and toxicity) and no one of the group 2 (p = 0.2). Haplo-HSCT with PtCy is a good alternative to MUD with high efficacy and acceptable toxicity in children with infant acute leukemia at 1st or 2nd remission.

CRISPR Gene Editing of Murine Blood Stem and Progenitor Cells Induces MLL-AF9 Chromosomal Translocation and MLL-AF9 Leukaemogenesis.

Chromosomal rearrangements of the mixed lineage leukaemia (MLL, also known as KMT2A) gene on chromosome 11q23 are amongst the most common genetic abnormalities observed in human acute leukaemias. MLL rearrangements (MLLr) are the most common cytogenetic abnormalities in infant and childhood acute myeloid leukaemia (AML) and acute lymphocytic leukaemia (ALL) and do not normally acquire secondary mutations compared to other leukaemias. To model these leukaemias, we have used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing to induce MLL-AF9 (MA9) chromosomal rearrangements in murine hematopoietic stem and progenitor cell lines and primary cells. By utilizing a dual-single guide RNA (sgRNA) approach targeting the breakpoint cluster region of murine Mll and Af9 equivalent to that in human MA9 rearrangements, we show efficient de novo generation of MA9 fusion product at the DNA and RNA levels in the bulk population. The leukaemic features of MA9-induced disease were observed including increased clonogenicity, enrichment of c-Kit-positive leukaemic stem cells and increased MA9 target gene expression. This approach provided a rapid and reliable means of de novo generation of Mll-Af9 genetic rearrangements in murine haematopoietic stem and progenitor cells (HSPCs), using CRISPR/Cas9 technology to produce a cellular model of MA9 leukaemias which faithfully reproduces many features of the human disease in vitro.

Development of embryonic and adult leukemia mouse models driven by MLL-ENL translocation

Rearrangements involving the mixed lineage leukemia gene (MLL) are found in the majority of leukemias that develop within the first year of age, known as infant leukemias, and likely originate during prenatal life. MLL rearrangements are also present in about 10% of other pediatric and adult acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL). These translocations and others occurring in early life are associated with a dismal prognosis compared with adult leukemias carrying the same translocations. This observation suggests that infant and adult leukemias are biologically distinct but the underlying molecular mechanisms for these differences are not understood. In this work, we induced the same MLL chromosomal translocation in the embryo at the time of fetal liver hematopoiesis and in the adult hematopoietic tissues to develop disease models in mice that recapitulate human infant and adult leukemias, respectively. We successfully obtained myeloid leukemia in adult mice after MLL-ENL recombination induction using the interferon inducible Mx1-Cre line. Using this same Cre line, we generated embryonic MLL-ENL leukemias, which were more aggressive than the corresponding adult leukemias. In conclusion, we have developed a novel MLL-ENL embryonic leukemia model in mice that can be used to study some aspects of infant leukemia ontogeny.

The potential underlying mechanism of the leukemia caused by MLL-fusion and potential treatments.

A majority of infant and pediatric leukemias are caused by the mixed-lineage leukemia gene (MLL) fused with a variety of candidates. Several underlying mechanisms have been proposed. One currently popular view is that truncated MLL1 fusion and its associated complex constitutively hijacks super elongation complex, including positive transcription elongation factor b, CDK9, and cyclin T1 complexand DOT1L, to enhance the expression of transcription factors that maintain or restore stemness of leukocytes, as well as prevent the differentiation of hematopoietic progenitor cells. An alternative emerging view proposes that MLL1-fusion promotes the recruitment of TATA binding proteinand RNA polymerase II (Pol II) initiation complex, so as to increase the expression levels of target genes. The fundamental mechanism of both theories are gain of function for truncated MLL1 fusions, either through Pol II elongation or initiation. Our recent progress in transcription regulation of paused Pol II through JMJD5, JMJD6, and JMJD7, combined with the repressive role of H3K4me3 revealed by others, prompted us to introduce a contrarian hypothesis: the failure to shut down transcribing units by MLL-fusions triggers the transformation: loss of function of truncated MLL1 fusions coupled with the loss of conversion of H3K4me1 to H3K4me3, leading to the constitutive expression of transcription factors that are in charge of maintenance of hematopoietic progenitor cells, may trigger the transformation of normal cells into cancer cells. Following this track, a potential treatment to eliminate these fusion proteins, which may ultimately cure the disease, is proposed.

Gene rearrangements of MLL and RUNX1 sporadically occur in normal CD34+ cells under cytokine stimulation.

Gene rearrangements of MLL/KMT2A or RUNX1 are the major cause of therapy‐related leukemia. Moreover, MLL rearrangements are the major cause of infant leukemia, and RUNX1 rearrangements are frequently detected in cord blood. These genes are sensitive to topoisomerase II inhibitors, and various genes have been identified as potential fusion partners. However, fetal exposure to these inhibitors is rare. Therefore, we postulated that even a proliferation signal itself might induce gene rearrangements in hematopoietic stem cells. To test this hypothesis, we detected gene rearrangements in etoposide‐treated or non–treated CD34+ cells cultured with cytokines using inverse PCR. In the etoposide‐treated cells, variable‐sized rearrangement bands were detected in the RUNX1 and MLL genes at 3 hours of culture, which decreased after 7 days. However, more rearrangement bands were detected in the non–treated cells at 7 days of culture. Such gene rearrangements were also detected in peripheral blood stem cells mobilized by cytokines for transplantation. However, none of these rearranged genes encoded the leukemogenic oncogene, and the cells with rearrangements did not expand. These findings suggest that MLL and RUNX1 rearrangements, which occur with very low frequency in normal hematopoietic progenitor cells, may be induced under cytokine stimulation. Most of the cells with gene rearrangements are likely eliminated, except for leukemia‐associated gene rearrangements, resulting in the low prevalence of leukemia development.

H3K79me2/3 controls enhancer\u2013promoter interactions and activation of the pan-cancer stem cell marker PROM1/CD133 in MLL-AF4 leukemia cells

MLL gene rearrangements (MLLr) are a common cause of aggressive, incurable acute lymphoblastic leukemias (ALL) in infants and children, most of which originate in utero. The most common MLLr produces an MLL-AF4 fusion protein. MLL-AF4 promotes leukemogenesis by activating key target genes, mainly through recruitment of DOT1L and increased histone H3 lysine-79 methylation (H3K79me2/3). One key MLL-AF4 target gene is PROM1, which encodes CD133 (Prominin-1). CD133 is a pentaspan transmembrane glycoprotein that represents a potential pan-cancer target as it is found on multiple cancer stem cells. Here we demonstrate that aberrant PROM1/CD133 expression is essential for leukemic cell growth, mediated by direct binding of MLL-AF4. Activation is controlled by an intragenic H3K79me2/3 enhancer element (KEE) leading to increased enhancer–promoter interactions between PROM1 and the nearby gene TAPT1. This dual locus regulation is reflected in a strong correlation of expression in leukemia. We find that in PROM1/CD133 non-expressing cells, the PROM1 locus is repressed by polycomb repressive complex 2 (PRC2) binding, associated with reduced expression of TAPT1, partially due to loss of interactions with the PROM1 locus. Together, these results provide the first detailed analysis of PROM1/CD133 regulation that explains CD133 expression in MLLr ALL.

Pre‐Clinical Evaluation of Drug Induced Proteolysis of MLL‐Fusions in Acute Leukemia

The mixed‐lineage leukemia (MLL) gene is often susceptible to chromosomal rearrangements in pediatric leukemias. The resulting chimeric proteins are a product of reciprocal translocations between the gene and various fusion partners. In MLL‐rearranged leukemia, these fusion proteins drive leukemogenesis by interfering with transcriptional elongation to deregulate target gene expression. MLL fusion proteins, indicative of a more aggressive disease, are responsible for a large proportion of acute infant leukaemias. Therapeutic methods to target oncogenic fusion proteins, such as PML‐RARA, have proven pharmacologically achievable and highly successful. For this purpose, our lab has established a drug screening program using in cellulo bio‐luminescence with the objective of repositioning drugs that cause MLL‐fusion protein degradation. Compounds that produced a 30% reduction in MLL fusion protein expression via two different screening methods, luminescence and western blotting, were considered positive hits. In this present study I validate and characterize one of these positive hits. Upon treatment of MLL‐rearranged cell lines, the compound was capable of ablating MLL fusion protein expression resulting in the down regulation of target gene HOXA10. Furthermore, treatment caused loss of leukaemic stem cell activity via induction of cell death and inhibition of self‐renewal capacity. To further analyze the mechanism of this compound, RNA‐sequencing and Gene Set Enrichment Analysis will be performed to identify gene sets associated with the disease treated phenotype. Overall this study identifies a potential repurposed therapeutic for the treatment of MLL‐rearranged leukaemias.Support or Funding InformationThis work was supported by the UCL Child Health Research Studentship.

Thiotepa Based Conditioning for Haploidentical Stem Cell Transplantation with Post Transplant Cyclophosphamide for Pediatric Acute Leukemia Is Highly Effective

Introduction Haploidentical family donor is universally available and is increasing being used as an alternative donor choice for treating adults with leukaemia needing hematopoietic stem cell transplant (HSCT). Thiotepa is an alkylating agent which has been used successfully as part of conditioning in adult leukemia patients undergoing haploidentical SCT with Post transplant cyclophosphamide (PTCy). But such data is lacking in children. Herein, we describe the experience of treating children with acute leuekmia with thiotepa based conditioning for haploidentical HSCT with PTCy. Methods We retrospectively analysed outcome data of 12 children with acute leukaemia (acute lymphoblastic leukemia-8, acute myeloid leuekmia-3 and Mixed phenotype acute leukemia-1) who underwent haploidentical HSCT with PTCy; median age was 6.5 years (range 1-14 years). All were in complete remission (CR) prior to HSCT (CR1-6, CR2-6). Donors were mobilized with GCSF. The conditioning was myeloablative with Thiotepa 10 mg/kg day-6, Fludarabine 40 mg/m2 day-5 to -2, Cyclophosphamide 14.5 mg/kg on day-5 and -4, Total body irradiation 2 Gray on day-1 in 8 children and Rituximab 100/m2 day-8, Thiotepa 10 mg/kg on day-7, Fludarabine 40 mg/m2 day-6 to -3, Busulfan 3.2 mg/kg day-6 to -3 (TBF) and Rabbit ATG 1.5 mg/kg day-4 to-2 in 4 children. All received PTCy 50 mg/kg on day 3 and 4 as graft vs. host disease (GVHD) prophylaxis along with tacrolimus or cyclosporine and mycophenolate mofetil. A median of 9.5 million of CD34 cells/kg was infused (range 4-20 million/kg). Results All patients engrafted. Median time of neutrophil engraftment was 18 days (range 11-29) and platelet engraftment was 18 days (range 12-26). All achieved fully donor chimerism on day+30. One child with acute myelogenous leukemia died due to steroid refractory grade IV acute gut GVHD on day+38. Child with infant leukemia died on day+330 with chronic GVHD of Lung. One child relapsed 2 years post HSCT. As he had CD20 positive B cell ALL, so he was treated with Rituximab and achieved CR3. He is alive and disease free on monthly rituximab 100 mg/m2 for more than 3 years post his relapse. Chimerism on day +100 and + 180 was fully donor in all patients. Remaining 9 patients are in continued CR. Acute GVHD was seen in 3 patients (grade III-IV in 1) but none in TBF and ATG regimen. Chronic GVHD was seen in four patients (extensive in 3) but only in 1 out of 4 in TBF regimen. Haemorrhagic cystitis with BK virus activation was seen in 4 patients (all 4 received TBF). Viral reactivation of CMV was seen in 5 patients. Overall survival was 83% and event-free survival was 75 % at a median follow-up of 930 days (range 200-1765 days). Transplant related mortality was 17%. Conclusion Thiotepa based myeloablative conditioning is highly effective in children undergoing haploidentical HSCT with PTCy for acute leukemia. Addition of ATG reduced acute and chronic GVHD.

Bioflavonoids cause DNA double-strand breaks and chromosomal translocations through topoisomerase II-dependent and -independent mechanisms

Bioflavonoids have a similar chemical structure to etoposide, the well-characterized topoisomerase II (Top2) poison, and evidence shows that they also induce DNA double-strand breaks (DSBs) and promote genome rearrangements. The purpose of this study was to determine the kinetics of bioflavonoid-induced DSB appearance and repair, and their dependence on Top2. Cells were exposed to bioflavonoids individually or in combination in the presence or absence of the Top2 catalytic inhibitor dexrazoxane. The kinetics of appearance and repair of γH2AX foci were measured. In addition, the frequency of resultant MLL-AF9 breakpoint cluster region translocations was determined. Bioflavonoids readily induced the appearance of γH2AX foci, but bioflavonoid combinations did not act additively or synergistically to promote DSBs. Myricetin-induced DSBs were mostly reduced by dexrazoxane, while genistein and quercetin-induced DSBs were only partially, but significantly, reduced. By contrast, luteolin and kaempferol-induced DSBs increased with dexrazoxane pre-treatment. Sensitivity to Top2 inhibition correlated with a significant reduction of bioflavonoid-induced MLL-AF9 translocations. These data demonstrate that myricetin, genistein, and quercetin act most similar to etoposide although with varying Top2-dependence. By contrast, luteolin and kaempferol have distinct kinetics that are mostly Top2-independent. These findings have implications for understanding the mechanisms of bioflavonoid activity and the potential of individual bioflavonoids to promote chromosomal translocations. Further, they provide direct evidence that specific Top2 inhibitors or targeted drugs could be developed that possess less leukemic potential or suppress chromosomal translocations associated with therapy-related and infant leukemias.

Single-Cell Multi-Omics Reveals Elevated Plasticity and Stem-Cell-Like Blasts Relevant to the Poor Prognosis of <i>KMT2A</i>-Rearranged Leukemia

Infant ALL is a devastating malignancy caused by rearrangements of the KMT2A gene (KMT2A-r) in approximately 70% of patients. The outcome is dismal and younger age at diagnosis is associated with increased risk of relapse. To discover age-specific differences and critical drivers that mediate the poor outcome in KMT2A-r ALL, we subjected KMT2A-r leukemias and normal hematopoietic cells from patients of different ages to multi-omic single cell analysis using scRNA-Seq, scATAC-Seq and snmC-Seq2. We uncovered the following critical new insights: Leukemia cells from infants younger than 6 months have a greatly increased lineage plasticity and contain a hematopoietic stem and progenitor-like (HSPC-like) population compared to older infants. We identified an immunosuppressive signaling circuit between the HSPC-like blasts and cytotoxic lymphocytes in younger patients. Both observations offer a compelling explanation for the ability of leukemias in young infants to evade chemotherapy and immune mediated control. Our analysis also revealed pre-existing lymphomyeloid primed progenitor and myeloid blasts at initial diagnosis of B-ALL. Tracking of leukemic clones in two patients whose leukemia underwent a lineage switch documented the evolution of such clones into frank AML. These findings provide critical insights into KMT2A-r ALL and have potential clinical implications for targeted inhibitors or multitarget immunotherapy approaches. Beyond infant ALL, our study demonstrates the power of single cell multi-omics to detect tumor intrinsic and extrinsic factors affecting rare but critical subpopulations within a malignant population that ultimately determines patient outcome.

Infant Acute Lymphoblastic Leukemia with Atypical Presentation

Infant leukemia is a very rare disease and consists of 5% of all childhood leukemias. Infant leukemia usually presents with high leukocyte counts and numerous extra-hematological features including central nervous system and skin involvement. Herein, we report a 1-month-old girl presented with high phenylalanine level in national newborn screening program for phenylketonuria; and subsequently diagnosed to have acute lymphoblastic leukemia.

In-Depth Proteomic Characterization of Lineage-Biased Hematopoietic Progenitor Cells in the Fetus and Adult

The process of hematopoiesis is subject to extensive ontogenic remodeling that is accompanied by alterations in cellular fate both during normal development and upon malignant transformation. Although the functional differences between fetal and adult hematopoiesis are well established, the responsible molecular mechanisms have long remained largely unexplored at the proteomic level. We hypothesize that an intrinsically programmed proteomic switch in hematopoietic stem and progenitor cells (HSPCs) during ontogeny regulates the outcome of hematopoiesis both during normal development and upon leukemia initiation, and that the proteomic makeup of the leukemia-initiating cell has an instructive role in determining the outcome of the resulting cancer. In our latest work, we utilized quantitative mass spectrometry-based proteomics to characterize and compare the proteomic makeup of fetal and adult Lin- Sca-1+ cKit+ (LSK) HSPCs (Jassinskaja et al., 2017, Cell Reports), representing all of the earliest stem and progenitors in fetal and adult hematopoiesis. We identified differences in several important cellular processes not previously described to play a role in hematopoiesis, highlighting the need for applying proteomic-centric approaches in the field. In order to further increase our understanding of normal and malignant hematopoiesis during ontogeny, we are now continuing this work by focusing on more stringently defined populations of lineage-biased hematopoietic progenitor cells (HPCs). Here, we have utilized encapsulated methods for preparation of microscale samples in combination with state-of-the-art mass spectrometry to gain deep coverage of the proteome of 100,000 fetal (E14.5) and adult lymphoid-primed multipotent progenitors (LMPPs), common lymphoid progenitors (CLPs) and granulocyte-macrophage progenitors (GMPs). Our analysis resulted in the identification and quantification of 4189 proteins, with over 200 proteins per cell type displaying differential expression between the fetus and the adult. Importantly, the differentially expressed proteins were enriched for a broad variety of biological processes. Similar to our previous findings in HSPCs, for all three cell types, proteins higher expressed in the fetus showed a strong enrichment for cell cycle- and translation-related processes, whereas those higher expressed in the adult were enriched for processes related to immune response and redox homeostasis. Our preliminary analysis of hematopoietic cell subset signatures associated with the differentially expressed proteins suggests a stronger lymphoid bias in fetal compared to adult LMPPs as well as CLPs. Surprisingly, the proteomic signature of fetal GMPs suggests a retained megakaryocyte-erythroid potential, which is corroborated by a significantly higher expression of megakaryocyte progenitor marker CD41 on the fetal cells. Upon analyzing expression of transcription factors (TFs) in fetal and adult HPCs, we could confirm differential expression of TFs known to have ontogeny-specific roles in hematopoiesis (e.g. Arid3a and Etv6). Importantly, we also identified several differentially expressed TFs that could represent novel regulators of fetal- and adult-specific features of hematopoiesis, such as Irf8, Btf3, Mndal and Pura. Furthermore, the difference in expression of Irf8 observed here could indicate a previously unknown ontogenic switch in the balance between neutrophil and monocyte production from myeloid-competent progenitors. Lastly, our data shows strong indications of a differential sensitivity towards Rho kinase inhibition between the fetal and the adult HPCs. Collectively, our work represents a significant advancement in the understanding of the molecular programs that govern ontogenic differences in hematopoiesis and provides a solid foundation for future investigation of which factors are responsible for the difference in susceptibility and outcome of different leukemias in infants and in adults. Disclosures No relevant conflicts of interest to declare.

Late Effects in Survivors of Infant Acute Lymphoblastic Leukemia from the 3 Consecutive Japanese Nationwide Clinical Trials

Background Very few studies have reported the incidence of and risk factor for late effects of infant leukemia. In addition, the long-term impact of a preparative regimen for very young children undergoing allogeneic stem cell transplantation (SCT) is not evident. Method To examine the late effects in survivors of infant acute lymphoblastic leukemia (ALL) treated on the Japanese Infant Leukemia Study Group (JILSG) trials MLL96, MLL98 and the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG) trial MLL03, we performed a cross-sectional survey using questionnaires by the attending pediatricians from 2015 to 2017. In all 3 studies, infants with KMT2A gene rearranged (MLL-R) ALL were allocated to receive SCT in their first remission. Choice of preparative regimen was left to the physician's choice in the MLL96/98 studies, while use of non-total body irradiation (TBI) regimen with busulfan (BU) was mandatory in the MLL03 study. Results Seventy out of 102 (69%) eligible subjects were evaluated: 33 were males and 37 were females. 62 patients had an MLL-R ALL. The median age of diagnosis was 5.5 months (range, 0-12m) and 35 (50%) were <6 months of age. At the time of analysis, the median age of the 70 patients was 13.5 years (range, 7-21y) and the median follow-up duration from diagnosis was 12.5 years (range, 7-20y). Fifty-seven (81%) of the 70 patients underwent SCT. Eight patients received the TBI-based regimen, while 35 received BU regimen and one received non-TBI, non-BU regimen. Thirteen patients had SCT twice and twelve patients received both TBI and BU regimen. Relapses occurred in seventeen patients (24%). Only two patients received cranial radiation therapy in 13 patients with central nervous system involved. At least one late effect was observed in 62 of the 68 patients (91%). Identified abnormalities included: underweight (body mass index [BMI]<18.5) (69%), hypogonadism (56%), teeth mal-development (52%), short stature (52%), bone abnormalities (28%), thyroid dysfunctions (27%), skin abnormalities (24%), ophthalmic dysfunctions (24%), pulmonary dysfunctions(22%), neurocognitive dysfunctions (22%), and cardiovascular dysfunctions (14%), gastrointestinal dysfunctions (5%), renal dysfunctions (4%), second malignancy (3%), and overweight (3%). The mean standard deviation (SD) heights at last follow up with TBI regimen were -4.2 and those with multiple SCT (BU+TBI) were -3.8. Both were significantly lower than those with single SCT by BU regimen (-1.4) (TBI vs BU, P=0.014; BU+TBI vs BU, P<0.001). The mean BMI at last follow up was lower in the TBI group (16.8) compared with that in the BU group (17.8) without statistical significance (TBI vs BU, P=0.342), whereas the mean BMI in the BU+TBI group (15.1) was significantly lower than that in the BU group (BU+TBI vs BU, P=0.020). Univariable analysis revealed that the risk of both thyroid dysfunctions (odds ratio [OR]=14.0, P=0.005) and the need of growth hormone (GH) treatment (OR=10.7, P=0.011) were significantly lower in the BU group than those in the TBI group. Hypothyroidism (OR=5.8, P=0.012), teeth mal-development (OR=2.8, P=0.066) and the need of GH treatment (OR=3.5, P=0.084) were more frequently observed in children who were diagnosed at less than 6 months of age. In addition, short stature (OR=23.3, P=0.003), thyroid dysfunctions (OR=4.1, P=0.023), cataract (OR=6.9, P=0.038) and alopecia (OR=6.8, P=0.038) were more frequently observed in relapsed patients. Overall, SCT significantly increased the incidence of short stature (OR=15.5, P=0.012) and underweight (OR=5.3, P=0.018). Conclusion In this study, survivors of infant leukemia had prolonged and various types of late effects. We observed serious growth failure in height and weight in patients treated with SCT and it was further pronounced in the TBI group and in the multiple SCT (BU+TBI) group. More than half of the patients had dental late effects and a third of all the patients had thyroid dysfunctions, which were more frequent in children diagnosed earlier. The number of patients over 20 years was limited in our study, more complications may increase in additional follow-up. Continuous long-term follow up and optimal intervention are crucial for survivors of infant leukemia who are transplanted. Disclosures Kada: Bayer Yakuhin: Other: personal fees for a member of independent data monitoring committee of clinical trials, outside of the submitted work..

Prognostic Value of Minimal Residual Disease Measured By Flow Cytometry in Two Cohorts of Infants with Acute Lymphoblastic Leukemia Treated with MLL-Baby and Interfant Protocols in Large Multicenter Networks

Acute lymphoblastic leukemia (ALL) in infants is a relatively rare disease with peculiar biological features, high frequency of KMT2A gene rearrangements and grim prognosis. Even with new therapeutic approaches, event-free survival (EFS) in infants with ALL does not exceed 50%. Currently large cooperative studies of infant ALL have been promoted by the Interfant and MLL-Baby networks. Minimal residual disease (MRD) monitoring is considered a strong tool for optimizing management of childhood ALL. In contrast to older children in this age group the prognostic impact of MRD detected by multicolor flow cytometry (MFC) is still unclear. Aim of the present study was to evaluate the prognostic value of MFC MRD measurement during induction in infants with ALL treated with Interfant-99 and Interfant-06 protocols in AIEOP centers in Italy as well as MLL-Baby protocol in Russia and Belarus. Patients and methods. Two independent groups of patients were investigated: study cohort of 139 consecutive infants with newly diagnosed ALL enrolled between September 2003 and April 2016 in Russian and Belorussian centers with MLL-Baby protocol and validation group of 146 ones enrolled in AIEOP centers in Interfant trials during the same period. By availability of MFC MRD data obtained at day 15 and/or end of induction (EOI), 81 and 86 patients of study and validation cohorts respectively were selected for outcome evaluation. In 61 MLL-Baby patients (75.3%) and 61 AIEOP cases (70.9%) different types of KMT2A gene rearrangements were identified. All patients were diagnosed as BCP-ALL, except one with cortical T-ALL. Overall, day 15 samples were studied in 64 MLL-Baby patients and 73 AIEOP cases while EOI samples in 75 and 63 cases respectively. MRD detection was performed in Reference Laboratories in Ekaterinburg, Minsk and Padua according the BFM AIEOP FLOW Network SOP. MRD negativity was defined as <0.01% of all bone marrow nucleated cells. Results. Patients were stratified according to the AIEOP-BFM-ALL day 15 stratification usually used for older children into three risk groups: standard risk (SR: MRD<0.1%), intermediate risk (IR: MRD 0.1% to 10%) and high risk (HR: MRD≥10%). Patients' distribution was similar in both study and validation cohorts: 34.4% and 32.9% in SR, 53.1% and 54.5% in ImR, 12.5% and 12.3% in HR respectively. At EOI significant differences in MRD-positive and MRD-negative patients' distribution was observed in different protocols: 44 (58.7%) and 31 (41.3%) cases respectively for MLL-Baby, but 17 (27.0%) and 46 (73.0%) cases respectively for AIEOP group. It was observed that KMT2A-rearranged cases in both trials have slower MRD response compared to the children with wild type KMT2A. In study cohort the 22 SR patients had a 5-year EFS and cumulative incidence of relapse (CIR) significantly better than other ones, thus we considered two major groups of patients with different outcome: SR with 5-year EFS 67.4%, standard error (SE) 10.2; CIR 23.3%, SE 9.4 and non-SR with 5-year EFS 30.8%, SE 7.2; CIR 52.6%, SE 7.9, (p=0.0039 and p=0.0229, respectively). Difference between these groups was observed also in KMT2A-rearranged cases (n=49) both for 5-year EFS (60.0%, SE 12.7 and 23.2%, SE 7.3, p=0.0160) and in 5-year CIR (33.3%, SE 12.7 and 59.2%, SE 8.8, p=0.0881). Analysis of outcome in validation cohort confirmed these data. In study cohort outcome of children being MRD-negative at EOI (n=31, 5-year EFS 60.8%, SE 8.8 and CIR 29.3%, SE 8.4) was significantly better than that of MRD-positive patients (n=44, 5-year EFS 31.1%, SE 7.1 and CIR 57.6%, SE 7.8 with p=0.0153 and p=0.0267, respectively). Outcome by EOI MFC MRD in the validation cohort is generally in keeping with that of the study cohort. Interestingly, in AIEOP cohort MFC data showed a prognostic impact also in KMT2A-rearranged subgroup. In multivariate analysis with KMT2A-status, each MRD time-point data showed independent impact on the risk of relapse. Conclusion. Our data was obtained by well-harmonized MFC MRD monitoring in a large group of infants with ALL treated in a multicenter setting with two different protocols. In spite of differences in therapy, we observed strong and independent prognostic impact of MFC MRD both at day 15 and at EOI regardless the protocol applied. We can conclude that MFC MRD can be used in combination with KMT2A-status to improve treatment allocation in future protocols. Disclosures Parasole: Servier: Honoraria; Baxalta: Honoraria; Eusapharma: Honoraria. Pieters:jazz farmaceuticals: Consultancy; medac: Consultancy.

Novel KMT2A Partner Gene NUTM2A Revealed By Anchored Multiplex PCR in ALL

Objectives Chromosomal rearrangements of the human MLL/KMT2A gene are associated with infant, pediatric and adult acute leukemias. Such rearrangements are more frequent in infants, in whom they are found in 60% - 80% of acute lymphoblastic leukemias (ALL). KMT2A-r group itself is genetically heterogeneous. KMT2A-r can occur with at least 94 partner genes previously described in «THE MLL RECOMBINOME <…> IN 2017». Detection of KMT2A-r is an essential part of AL initial diagnostics. Also, the accurate detection of all KMT2A-r types is crucial in order to perform minimal residual disease (MRD) monitoring, as it is clear now that MRD-based therapy adjustment has a very strong impact on outcome. Here we report a case of novel KMT2A partner in pediatric ALL - NUT family member 2A (NUTM2A). Methods The patient is 8 y.o. girl with T-cell acute lymphoblastic leukemia with pleural effusion. Bone marrow and pleural fluid aspirates were analyzed by G-banded karyotyping and FISH with KMT2A breakapart probe. Pleural fluid aspirates were also analyzed by real-time RT-PCR for 8 most common KMT2A rearrangements screening, long-distance inversed PCR and targeted RNA-seq with FusionPlex Myeloid kit (ArcherDX, CO, USA). Sanger sequencing was used for validation. Results Conventional cytogenetics and FISH showed 47,XX,t(10;11)(q22;q23),+mar[5] karyotype with 100% KMT2A-rearranged nuclei. Gene fusion analysis identified novel fusion KMT2A-NUTM2A with exon 11 - exon 1 breakpoint junction. NUTM2A is a gene at 10q23.2. This gene is not fully described in the literature. Rearrangements of this gene were identified in endometrial stromal sarcomas (ESS) (Cheng-Han Lee et al. 2012) and small round cell sarcoma (SRCS) (Sugita et al. 2017). In case of ESS, this rearrangement results in an in-frame fusion between YWHAE and NUTM2A (or highly homologous gene NUTM2B), and in case of SRCS it results in an in-frame fusion between NUTM2A and CIC. To our knowledge, KMT2A-NUTM2A fusion in our study is the first case demonstrating NUTM2A rearrangement in hematological malignancies. Conclusions Here we for the first time show the novel KMT2A-NUTM2A fusion transcript, which was found in pediatric T-cell acute lymphoblastic leukemia. Anchored multiplex PCR is one of the most sensitive way to detect rare variants of KMT2A rearrangements. It also allows selecting patient-specific primers for further PCR detection of MRD. Disclosures No relevant conflicts of interest to declare.