Unusual Cutaneous Manifestation of Acute Lymphoblasic Leukemia in aChild

Pitfalls in the diagnosis of childhood leukaemia.

The role of repeat transplantation of haemopoietic stem cells and adoptive immunotherapy in treatment of leukaemia relapsing following allogeneic transplantation.

Alberto Orfao,* Francisco Ortuno, Maria de Santiago, Antonio Lopez, and Jesus San Miguel Servicio General de Citometria, Universidad de Salamanca, Salamanca, Spain Centro de Investigacion del Cancer y Departamento de Medicina, Universidad de Salamanca, Salamanca, Spain Servicio de Hematologia y Oncologia Medica, Hospital General Universitario J.M. Morales Meseguer, Murcia, Spain Servicio de Hematologia, Hospital Universitario de Salamanca, Salamanca, Spain

In patients with acute leukaemia, studies of minimal (i.e. submicroscopic) residual disease (MRD) should improve measurements of treatment response and enable estimates of the residual leukaemic cell burden during clinical remission, thereby improving the selection of therapeutic strategies and, possibly, long-term clinical outcome. The most useful methods for MRD monitoring currently available are polymerase chain reaction (PCR) amplification of fusion transcripts and rearranged antigen-receptor genes, and flow cytometric detection of aberrant immunophenotypes. Several studies in patients with acute lymphoblastic leukaemia and acute myeloid leukaemia have demonstrated that MRD is a powerful and independent prognostic indicator. The strong association between MRD and risk of relapse was observed in children and adult patients irrespective of the methodology used to detect residual disease. This article discusses the relative advantages and disadvantages of each MRD assay, and reviews the reported correlations between MRD, clinical and biological features of the disease, and outcome. A multitude of factors influence treatment response in patients with acute leukaemia (Lowenberg et al, 1999; Pui et al, 2001). Cell lineage, stage of maturation, karyotype and molecular abnormalities regulate expression of genes that control drug metabolism and apoptosis, and determine the leukaemic cells' capacity to grow and their sensitivity to chemotherapy. Other important factors include size of the tumour burden, dosage of drugs and their interaction, pharmacokinetic and pharmacogenetic variables, and compliance with treatment schedule. These parameters are associated with a varying risk of relapse but their predictive power is far from absolute and their use to make treatment decisions in individual patients is inherently limited. In vivo measurements of leukaemia cytoreduction should reflect the combined effect of clinical and cellular variables; rather than predicting outcome, these measurements provide direct information on the effectiveness of treatment in each patient. However, estimates by conventional morphological techniques have limited sensitivity and accuracy: in most cases, leukaemic cells can be detected in bone marrow with certainty only when they constitute 5% or more of the total cell population. Such a high detection threshold results in the inability to measure fluctuations in the leukaemia tumour mass with accuracy, and resurgent disease can be diagnosed only at its most advanced stages. Methods for detecting MRD (i.e. submicroscopic) are at least 100-times more sensitive than conventional morphological techniques and allow a more stringent definition of 'remission' in patients with acute leukaemia, which is rapidly becoming the standard at many cancer centres (Fig 1). Factors that influence response to treatment in patients with acute leukaemia. An essential premise of MRD studies is that MRD levels provide reliable estimates of the residual leukaemia mass. This assumes that leukaemic cells are homogeneously distributed throughout the bone marrow so that measurements of MRD performed on a small aliquot of bone marrow (perhaps, equivalent to one thousandth of the total active marrow) are representative of the total leukaemia burden. However, observations in patients (Mathe et al, 1966) and in animal models of leukaemia (Martens et al, 1987) indicate that there could be considerable heterogeneity in the distribution of leukaemic cells after treatment and that individual samples may not be informative. Another caveat is that MRD signals may not correspond to viable leukaemic cells with a capacity for cell renewal. Cell viability can only be determined with methods that use intact cells (e.g. flow cytometry) but it is impossible to determine with MRD methods that examine nucleic acid material (e.g. polymerase chain reaction). Moreover, even when viable, leukaemic cells may lack sustained self-renewal capacity, a feature that is not possible to assess on a routine basis. As discussed in this article, most of these reservations have been dispelled by the results of several correlative studies of MRD and treatment outcome. It is now clear that detectable MRD implies the presence of leukaemic stem cells capable of leading to disease recurrence. The first MRD studies in patients with leukaemia were made soon after antibodies for leucocyte differentiation antigens became available. Strong expression of the common acute lymphoblastic leukaemia (ALL) antigen (CD10) and terminal deoxynucleotidyl transferase (TdT) in ALL cells, and apparent absence of these markers on normal peripheral blood cells, suggested the use of these molecules as markers of leukaemia. However, it was soon clear that a proportion of cells in the bone marrow (i.e. B-cell progenitors) expressed both CD10 and TdT (Janossy et al, 1980). The distinction between leukaemic lymphoblasts and normal lymphoid progenitors remains crucial for detecting MRD in patients with B-lineage ALL. In early studies, it was also noted that T-lineage ALL cells (and normal thymocytes) expressed TdT and T-cell markers whereas lymphoid cells in bone marrow and peripheral blood did not (Bradstock et al, 1981). This finding enabled the first productive MRD studies in patients with acute leukaemia, and remains the basis of MRD studies by immunological techniques in T-lineage ALL. Over the following two decades, many methods to study MRD have been tested (Campana & Pui, 1995; Szczepanski et al, 2001). In ALL, the most reliable methods include flow cytometric profiling of aberrant immunophenotypes, polymerase chain reaction (PCR) amplification of fusion transcripts and chromosomal breakpoints, and PCR amplification of antigen-receptor genes. In acute myeloid leukaemia (AML), only the first two of these methods can be applied, as most patients lack antigen-receptor gene rearrangements. Owing to limited sensitivity (approximately 1–5%), conventional karyotyping and fluorescence in situ hybridization (FISH) are occasionally useful for clarifying the nature of morphologically suspicious blast cells, but cannot reliably detect submicroscopic leukaemia (Mancini et al, 2000). However, improvements in image analysis technology, allowing simultaneous visualization of morphological, immunophenotypic and FISH features, may enhance the usefulness of this approach (Bielorai et al, 2002). The success of methods based on differential properties of normal and leukaemic cells in culture has been limited to a few laboratories (Estrov et al, 1986; Uckun et al, 1993). Many comprehensive reviews have addressed in detail the methodological aspects of MRD detection (Campana & Pui, 1995; van Dongen et al, 1999; Foroni et al, 1999; Lo Coco et al, 1999a; Szczepanski et al, 2001; Campana & Coustan-Smith, 2002; Liu & Grimwade, 2002). Table I and the following sections summarize the applicability and the main specific advantages and disadvantages of each technique. Breakpoint fusion regions of chromosomal aberrations, and rearranged immunoglobulin (IG) and T-cell receptor (TCR) genes are leukaemia-specific sequences that have been used with consistent success in molecular studies of MRD. The value of another target, WT-1, is supported by some studies (Cilloni et al, 2002; Ogawa et al, 2003). However, WT-1 is also expressed by normal haematopoietic progenitors (Hosen et al, 2002) and has not been found to be reliable by some investigators (Elmaagacli et al, 2000). Internal tandem duplications (ITD) of the FLT3 gene occur in approximately 20–30% of AML patients and, in principle, could be used as targets for PCR-based MRD studies (Stirewalt et al, 2001). However, FLT3/ITD detected at diagnosis appear to be unstable, often becoming undetectable at relapse (Kottaridis et al, 2002; Shih et al, 2002). With the exception of the TAL1 gene abnormalities, the genomic breakpoints of the most common known leukaemia fusion genes are spread over large distances within each gene locus. PCR analysis starting from DNA would require determination of the exact breakpoint in each patient, which is not practical (van Dongen et al, 1999). As the resulting mRNA is similar in many patients, RNA is the typical starting material for PCR-mediated amplification of breakpoint fusion sequences. After reverse transcription (RT) into cDNA, PCR amplification is applied using primers at opposite sites of the breakpoint fusion region (van Dongen et al, 1999). An advantage of using the breakpoint-fusion regions for MRD studies is their stability during the disease course. However, only 50% or less of ALL and AML cases in children and adults have specific chromosomal aberrations with well-defined breakpoint fusion regions (Look, 1997; Liu & Grimwade, 2002). Therefore, the applicability of this approach is restricted to certain subgroups of leukaemias. The uniqueness of each immunoglobulin (Ig) and TCR molecule depends on the rearrangement and joining of the multiple variable (V), diversity (D) and joining (J) gene segments of the IG and TCR gene loci, on the deletion of nucleotides from the germline sequences of the rearranging gene segments, and on the random insertion of nucleotides at the junctional sites (Foroni et al, 1999; Pongers-Willemse et al, 1999). Thus, the sequences of the junctional regions of rearranged IG and TCR genes are signatures of each lymphoid cell clone, normal or malignant. For MRD studies, the various IG and TCR gene rearrangements must be identified in each patient at diagnosis. The sequence information enables the design of junctional region-specific oligonucleotides, which can be used as primers in the PCR procedure to specifically amplify the rearrangements of the malignant clone (Pongers-Willemse et al, 1999) or as probes to distinguish PCR products derived from leukaemic cells among those that are derived from normal lymphoid cells (Yokota et al, 1991). Virtually all B-lineage ALL patients have rearranged IGH genes (van Dongen & Wolvers-Tettero, 1991). In addition, rearrangements of the IGK deleting element (Kde) occur at a relatively high frequency (approximately 60%) (Beishuizen et al, 1997). Most T-ALL patients have rearranged TCRB, TCRG and/or TCRD genes (van Dongen & Wolvers-Tettero, 1991), and cross-lineage TCR rearrangements are found in many patients with B-lineage ALL (Szczepanski et al, 1999). In the majority (approximately 90%) of B-lineage ALL patients, MRD can be revealed by junctional regions of IGH, IGK-Kde, TCRG and/or TCRD gene rearrangements (Pongers-Willemse et al, 1999), and in most (> 95%) T-ALL patients by TCRB, TCRG and/or TCRD (Kneba et al, 1995; Pongers-Willemse et al, 1999). The need to develop individual probes or primers is one of the main limiting factors in the widespread application of MRD studies by PCR amplification of IG and TCR genes. Some investigators have attempted to bypass this need by identifying the leukaemic DNA on the basis of size and signal intensity after separation by high-resolution gel electrophoresis (Deane & Norton, 1990; Sykes et al, 1997). Polyclonal background levels vary but usually limit the sensitivity of this approach to the detection of one leukaemic cell among 103 normal cells. IG and TCR gene rearrangements in B- and T-lineage ALL are prone to subclone formation and multiple IGH gene rearrangements are already found at diagnosis in 30% to 40% of B-lineage ALL patients (Beishuizen et al, 1994). This creates uncertainty about the prioritization of the clones that should be monitored in some patients. In addition, the emergence of subclones that are not apparent at diagnosis may occur, carrying the risk of false-negative results during MRD monitoring (van Dongen & Wolvers-Tettero, 1991; Beishuizen et al, 1994; Pongers-Willemse et al, 1999). In a recent analysis of 94 patients with B-lineage ALL, studied at diagnosis and relapse, 71% of the potential Ig and TCR targets for MRD analysis identified at diagnosis were preserved at relapse (Szczepanski et al, 2002). The most stable were IGK-Kde rearrangements and the least stable were incomplete TCRD rearrangements. Monoclonal rearrangements were significantly more stable than oligoclonal rearrangement. For these reasons, it has been recommended that at least two PCR targets should be used. In B-lineage ALL, these are available in approximately 70% of children and 50% of adults; in T-lineage ALL, two PCR targets (including TAL1 deletions) can be identified in approximately 90% of children and 85% of adults (Pongers-Willemse et al, 1999). Notably, clonotypic rearrangements of IG and TCR genes are found in only 50% of infants with t(4;11) ALL (Peham et al, 2002). Depending on the uniqueness of the sequence targeted and the quality of the material, PCR can detect one leukaemic cell in 103−106 normal cells. High sensitivity may paradoxically become a problem as it generates a propensity to false-positive results due to contamination, particularly when the same set of primers is applied to different patients. However, investigators are well aware of this potential problem and most take measures to minimize it. A complication of MRD studies by PCR is related to the limited quantitative power of the technique. Nevertheless, under typical conditions and using appropriate techniques, the quantification of leukaemic cells by PCR amplification of single-copy genes (e.g. IGH and TCR genes) can be adequate (Sykes et al, 1992; Cave et al, 1994; Ouspenskaia et al, 1995; Pongers-Willemse et al, 1998; Neale et al, 1999). When RNA is the target molecule, additional potential pitfalls may render the correlation more imprecise. RNA is prone to degradation, and the efficiency of its initial conversion to cDNA by reverse transcriptase may vary. For example, it was reported that, using standard techniques, less than 1000 PML-RARA molecules could be obtained from 1 µg of diagnostic bone marrow RNA derived from approximately 106 acute promyelocytic leukaemia (APL) cells (Seale et al, 1996). Poor yield of PML-RARA cDNA would then lead to low sensitivity of the RT-PCR. Moreover, the number of transcripts per cell is unlikely to be homogeneous in all the leukaemic cell population and is also unlikely to remain stable in cells exposed to chemotherapy. Finally, levels of transcript expression in patients with the same disease may differ considerably (Krauter et al, 1999; Buonamici et al, 2002), which may affect the consistency of MRD measurements in a patient population. MRD is traditionally quantified by comparing the PCR product obtained in the test sample with that of the patient's leukaemic cell DNA or RNA serially diluted into DNA or RNA from normal cells. Efforts to enhance the precision of the assay by competitive PCR or limiting dilution analysis have been effective (Sykes et al, 1992; Cave et al, 1994; Ouspenskaia et al, 1995) but the increased complexity of these approaches may hinder their routine application. Real-time quantitative PCR (RQ-PCR) appears to have solved some of the complications associated with PCR quantification. A fluorescent reporter is used in the PCR, and accumulation of fluorescence during the reaction ('real-time') is measured: the increase in fluorescence is proportional to the amount of target amplicon synthesized. Results are compared with those of serial dilutions of diagnostic material. This methodology can be applied to both breakpoint fusion region (Pallisgaard et al, 1999; Chen et al, 2001; de Haas et al, 2002) and antigen-receptor genes (Pongers-Willemse et al, 1998). In the latter case, to reduce the costs associated with designing fluorescent probes that match patient-specific sequences, probes matching germ line segments, such as V (Donovan et al, 2000; Verhagen et al, 2000), J (Bruggemann et al, 2000) and Kde regions (van der Velden et al, 2002a), and applicable to multiple patients can be used. Alternatively, some investigators have bypassed the requirement for fluoresceinated probes by using the DNA intercalating dye SYBR green I as a fluorescent reporter (Nakao et al, 2000; Li et al, 2002). In patients with T-lineage ALL, MRD can be monitored by searching for cells expressing TdT and CD3 or other cell markers in bone marrow or peripheral blood (Campana & Coustan-Smith, 2002) (Fig 2). In B-lineage ALL and AML, one needs to identify aberrant phenotypes that are not expressed by normal bone marrow or peripheral blood cells (Fig 2). Therefore, MRD studies in these leukaemias are complicated by variations in the cellular composition and immunophenotype of normal bone marrow that occur with age and exposure to various agents. For example, proportions of early lymphoid progenitors (or 'haematogones') are low in the bone marrow of healthy adults and especially low in patients receiving corticosteroids or chemotherapy (Paolucci et al, 1979). In contrast, these cells are found in high proportions in young children and in patients after transplantation or chemotherapy (Lucio et al, 1999; van Lochem et al, 2000; Van Wering et al, 2000; McKenna et al, 2001). These conditions may uncover normal cells expressing phenotypes that are undetectable in studies of healthy individuals. Nevertheless, immunophenotypes that clearly distinguish B-lineage ALL cells from normal lymphoid progenitors and haematogones have been identified (Campana & Coustan-Smith, 2002). Immunophenotypic differences between leukaemia cells and normal bone marrow cells. Flow cytometric dot plots shows expression of markers typically used for detecting MRD in T-lineage ALL, B-lineage ALL and AML (top row), and the expression of the same markers in bone marrow cells from healthy individuals (middle row) and from patients recovering after chemotherapy (bottom row). Dashed circles enclose areas of the dot plot corresponding to leukaemic cells in each case. Immunophenotypic analysis of T-lineage ALL cells was done on CD3+ cells, analysis of B-lineage ALL cells on CD19+ cells and analysis of AML cells on CD33+ and/or CD34+ cells (markers that were expressed in virtually all leukaemic cells at diagnosis). The analysis of the corresponding normal control subjects was done on the same cell subsets. Detection of MRD by flow cytometry in AML presents some specific difficulties. Owing to their immunophenotypic heterogeneity, AML cells usually spread across many areas of the dot plot instead of forming the tight cluster typical of ALL cells (Fig 2). Therefore, with any given marker combination, only a fraction of cells may be phenotypically abnormal. In addition, AML cells often have light scattering properties similar to those of normal cells with high autofluorescence. These features introduce complexity in the analysis, and may reduce the sensitivity of the assay. Nevertheless, sensitive MRD detection in AML is feasible. In a recent study using four-colour flow cytometry, 26 of 54 (48%) children with AML had leukaemia cells expressing immunophenotypes that allowed measurement of MRD with a sensitivity of one leukaemic cell among 104 or more normal cells; another 20 patients (37%) had immunophenotypes that enabled the detection of one leukaemic cell among 103 cells (unpublished observations). In adult AML, the proportion of patients that can be studied with a high degree of sensitivity may be larger. In one study, 46 of 53 patients had phenotypes that were found at frequencies of less than one in 104 cells in normal bone marrow, while seven had phenotypes found in normal bone marrow but at frequencies of less than one in 103 (San Miguel et al, 1997). In another study, 65 of 93 patients had a phenotype for detection of one leukaemic cell in 104 normal cells et al, 2000). The of that enable the analysis of gene expression has to identify markers of leukaemia. The results of one of studies et al, that a of the gene of normal and leukaemic cells identify applicable markers for MRD studies in both ALL and AML, and should allow the design of for reliable and monitoring of MRD. of the main of false-positive MRD results by flow cytometry is the use of markers to distinguish leukaemic cells from normal cells. The of needs to be by studies of bone marrow and peripheral blood cells not only from healthy individuals but also from patients at various of treatment (Campana & Coustan-Smith, 2002). The use of immunophenotypic that only in samples at certain during treatment a high risk of the influence of individual in and treatment compliance on normal is A of false-negative MRD results is the of immunophenotypic et al, 1998; et al, 2001). In the of this on MRD results can be by using multiple of markers in each patient. main influence MRD detection by flow the degree of morphological and between target cells and the cells, and the number of cells that can be As discussed immunophenotypes that not with the normal of leukaemic cells must be The number of cells that can be for each set of markers in clinical samples is usually less than 1 a cluster of at least is to flow cytometric the sensitivity of the assay under these would be (Campana & Coustan-Smith, 2002). Therefore, a sensitivity of (or one leukaemic cell in 104 normal should be during routine MRD even allowing for varying of available cells and immunophenotypes that are not expressed in of leukaemic cells. are various aspects of the procedure that need and have been discussed in detail (Campana & Coustan-Smith, 2002). it to that which may have minimal during routine of leukaemia, may be a of when MRD. signals can from conditions of the sample and of antibodies or to cells (Campana & Coustan-Smith, 2002). flow cytometry and PCR amplification of IGH genes, studied serial dilutions of normal and leukaemic cells and found the two methods to be et al, 1999). then bone marrow samples from children with ALL in clinical et al, 1999). In both techniques detected MRD levels 1 in The of leukaemic cells by the two methods well the had MRD levels 1 in Results were in only two PCR detected two in 104 and in 104 leukaemic cells, whereas the flow cytometric assay was both patients were MRD by both and remain in clinical after of compared the results obtained by using a of antibodies the MRD marker with those of PCR amplification of IGH genes in samples obtained from patients at various during et al, (unpublished In 46 MRD was by PCR analysis and by flow cytometric contrast, leukaemic cells were detected by both methods in and the MRD estimates by the two methods were The two methods results in only of the one was by PCR to have leukaemic cells, but MRD was detected by flow cytometric analysis of the two other samples were by analysis to have leukaemic cells but by PCR to have less than and (unpublished observations). A study comparing the results of flow cytometry and PCR amplification of TCRG and TCRD genes to detect MRD in bone marrow samples from patients with ALL found results in were more during the early of and were to low and presence of PCR et al, 2001). A study comparing flow cytometry to detection of transcripts in bone marrow samples from patients with ALL in observed results in samples et al, 2000). In two samples the assay was while cells were detectable by flow the samples had leukaemic cells by flow cytometry but signal by RT-PCR. also compared the results of flow cytometry with the results of amplification of fusion transcripts and observations). The methods results in of bone marrow samples of children with B-lineage ALL in clinical had MRD and were MRD the two one was by flow cytometry but by the other was by flow cytometry but by PCR patient had MRD that was detectable by both methods in and A similar was performed with samples obtained from 20 children with AML during treatment (unpublished observations). The molecular abnormalities studied were and In a of residual disease of these samples also had residual cells detectable by flow cytometry, whereas one with to levels of residual disease by did leukaemic were not detected by The samples had undetectable leukaemic transcripts or signals corresponding to levels of residual disease than but seven of these with undetectable leukaemic transcripts and with residual disease by had residual disease by flow The variable of MRD detection by flow cytometry in in and by to and the limited quantitative capacity of the conventional used can the observed A correlation between flow cytometric results and PCR detection of WT-1 was in a recent et al, 2002). Several studies in ALL have the of MRD at different during treatment (Fig there are due to differences in chemotherapy these studies have demonstrated the clinical of MRD. In one study, MRD was in patients by a competitive PCR assay junctional sequences of IGH and TCR et al, 1998). The absence or presence and of residual leukaemia during the first of were significantly with the risk of early relapse at each of the with leukaemic cells after the of or those with at had a particularly high risk of Another study monitored MRD in children with ALL to of the (van Dongen et al, 1998). This study also used PCR analysis of IGH and TCR genes as well as TAL1 patients had relapse at than those were MRD at the various at MRD levels at the of treatment and treatment were associated with a relapse when compared with patients with a low degree of MRD, and with a to relapse when compared with patients. MRD information from the first two was particularly allowing the of different risk a of patients with a relapse of a of patients with a relapse of and with a relapse of of MRD during treatment in children with ALL. the of patients, to the studies areas correspond to patients with MRD in each were obtained from et and results for the studies, from Cave et for the for and of study, and from van Dongen et for the used flow cytometry to study MRD in children with diagnosed ALL in a chemotherapy et al, found that detectable MRD (i.e. leukaemic at each of of and and of was significantly associated with a relapse (Fig with high levels of MRD at the of the or at

Bone Marrow Necrosis

Adult acute leukemia

Acute lymphoblastic and myeloblastic leukemias activate lymphotoxin beta receptor in mesenchymal stem cells in the bone marrow to turn off interleukin-7 production and lymphopoiesis and gain competitive advantage.

Acute lymphoblastic and myeloblastic leukemias activate lymphotoxin beta receptor in mesenchymal stem cells in the bone marrow to turn off interleukin-7 production and lymphopoiesis and gain competitive advantage.

Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Methods Data availability References Decision letter Author response Article and author information Metrics Abstract Acute lymphoblastic and myeloblastic leukemias (ALL and AML) have been known to modify the bone marrow microenvironment and disrupt non-malignant hematopoiesis. However, the molecular mechanisms driving these alterations remain poorly defined. Using mouse models of ALL and AML, here we show that leukemic cells turn off lymphopoiesis and erythropoiesis shortly after colonizing the bone marrow. ALL and AML cells express lymphotoxin α1β2 and activate lymphotoxin beta receptor (LTβR) signaling in mesenchymal stem cells (MSCs), which turns off IL7 production and prevents non-malignant lymphopoiesis. We show that the DNA damage response pathway and CXCR4 signaling promote lymphotoxin α1β2 expression in leukemic cells. Genetic or pharmacological disruption of LTβR signaling in MSCs restores lymphopoiesis but not erythropoiesis, reduces leukemic cell growth, and significantly extends the survival of transplant recipients. Similarly, CXCR4 blocking also prevents leukemia-induced IL7 downregulation and inhibits leukemia growth. These studies demonstrate that acute leukemias exploit physiological mechanisms governing hematopoietic output as a strategy for gaining competitive advantage. Editor's evaluation This study investigates a novel pathway by which leukemic cells remodel the bone marrow niche to promote their expansion at the expense of normal hematopoiesis. Feng X, Pereira JP et al. convincingly demonstrate a positive feedback loop between leukemic cells and stromal cells mediated by lymphotoxin produced by cancer cells and its receptor expressed by bone marrow stromal cells. The authors provide compelling evidence suggesting that this pathway disrupts normal blood production and provides a competitive advantage to leukemic cells. https://doi.org/10.7554/eLife.83533.sa0 Decision letter Reviews on Sciety eLife's review process Introduction Blood cell production is a tightly regulated process important for organismal homeostasis. All blood cells develop from a dedicated hematopoietic stem cell (HSC) that colonizes specialized niches in the bone marrow (BM) formed predominantly by mesenchymal stem cells (MSCs) and endothelial cells (ECs) (Morrison and Scadden, 2014; Pinho and Frenette, 2019; Sugiyama et al., 2019). Within these niches, HSCs and hematopoietic progenitors receive critical signals for long-term HSC maintenance and for differentiation into lymphoid, myeloid, and erythroid lineages (Sugiyama et al., 2019; Miao et al., 2020). However, most hematopoietic cytokines act in a short-range manner, and thus hematopoietic stem and progenitor cells rely on localization cues such as CXCL12 for accessing growth factors produced by MSCs and ECs (Noda et al., 2011; Tzeng et al., 2011; Ding and Morrison, 2013; Greenbaum et al., 2013; Cordeiro Gomes et al., 2016). While HSCs and uncommitted hematopoietic progenitors are critically dependent on stem cell factor (SCF, encoded by Kitl), committed progenitors require lineage-specific signals, such as IL7 for lymphocytes, IL15 for NK cells, or M-CSF for monocytes and macrophages. Importantly, most hematopoietic cytokines are produced by MSCs and by a subset of ECs in the BM (Miao et al., 2020; Noda et al., 2011; Cordeiro Gomes et al., 2016; Ding et al., 2012; Baryawno et al., 2019; Comazzetto et al., 2019; Tikhonova et al., 2019). The production of hematopoietic cytokines and chemokines by MSCs and ECs is relatively stable during homeostasis but can change significantly under certain perturbations. For example, systemic inflammation caused by infections enforces the downregulation of multiple hematopoietic cytokines and CXCL12 in the BM (Ueda et al., 2004; Ueda et al., 2005; Manz and Boettcher, 2014). Likewise, acute lymphoblastic and myeloblastic leukemias (ALL and AML) also promote the downregulation of multiple cytokines and CXCL12 produced by MSCs and ECs (Baryawno et al., 2019; Hanoun et al., 2014; Fistonich et al., 2018; Zehentmeier and Pereira, 2019). During systemic infection, the coordinated downregulation of certain cytokines (e.g. IL7) and CXCL12 causes a temporary pause in lymphopoiesis that is necessary for an emergent production of short-lived neutrophils and monocytes (Manz and Boettcher, 2014). In leukemic states, however, the mechanism(s) promoting cytokine and chemokine downregulation are not well defined, and neither is known if these changes are protective or harmful for the host. In humans and in mouse models of B-ALL, leukemic cells use CXCR4 to home to the BM (Juarez et al., 2007; Colmone et al., 2008; van den Berk et al., 2014). However, B-ALL cells do not distribute randomly and seem to reside and proliferate in certain perivascular niches (Colmone et al., 2008; Sipkins et al., 2005). Importantly, CXCL12 production is measurably reduced exclusively in BM niches colonized by B-ALL cells (Colmone et al., 2008; van den Berk et al., 2014). Furthermore, intact CXCR4 signaling presumably in B-ALL cells is required for downregulation of CXCL12 expression in BM niche cells (van den Berk et al., 2014). The fact that CXCR4 expression levels in B-ALL cells inversely correlate with patient outcome suggests that B-ALL-induced changes in the BM microenvironment may favor leukemia progression (van den Berk et al., 2014; Cancilla et al., 2020). The BM microenvironment has also been reported to be severely affected in AML patients and in mouse models of AML. Of note, hematopoietic cytokines and chemokines are significantly downregulated along with the re-programing of MSC and EC transcriptomes (Baryawno et al., 2019; Hanoun et al., 2014; Chandran et al., 2015; Geyh et al., 2016). Although no specific mechanisms have been identified for explaining how AML cells dysregulate MSCs and ECs, some evidence suggests that this may be mediated by direct AML-niche cell interactions (Hérault et al., 2017). Thus, a model emerges where leukemia cells attracted to CXCL12-producing BM niches physically interact and re-program MSCs and ECs to reduce CXCL12 levels, possibly reduce hematopoietic output, and in this way favor leukemic cell expansion. However, the molecular mechanisms utilized by leukemia for MSC and EC re-programing and for reducing non-malignant hematopoiesis remain poorly defined. In this study, we show that ALL and AML cells preferentially turn off lymphopoiesis and erythropoiesis shortly after seeding the BM. We demonstrate that both B-ALL and AML cells express LTα1β2, the membrane-bound ligand of lymphotoxin beta receptor (LTβR), which enforces IL7 downregulation in LTβR-expressing MSCs. Genetic or pharmacological blockade of LTβR signaling in MSCs restores lymphopoiesis but not erythropoiesis at the onset of leukemia, which in turn reduces leukemic cell growth and extends survival of transplant recipients. These studies demonstrate that leukemic cells exploit molecular mechanisms that confer flexibility in blood cell production to suppress normal hematopoiesis. Results ALL inhibits non-leukemic hematopoiesis Although leukemia alters BM niches, whether these changes directly affect hematopoietic cell production has not been carefully studied. To determine if and which hematopoietic cell lineages are affected by leukemia, we transplanted 3 million pre-B-cell precursor ALL cells expressing the BCR-ABL1 oncogene (from here on referred as ALL; BCR-ABL1 reported by YFP expression) into non-irradiated C57Bl6/J mice and analyzed its impact on lymphoid, myeloid, and erythroid cell production over time. As expected, ALL cells expanded rapidly in BM (Figure 1A). Conversely, non-leukemic developing B cells and mature recirculating B cells declined sharply 2 weeks after ALL transplantation (Figure 1B). Monocyte numbers reduced between the first and second weeks by four- to fivefold, but cell numbers recovered to normal levels at 3 weeks (Figure 1C). This contrasted with a moderate twofold decline in neutrophil numbers at 2 weeks that remained stable until 3 weeks (Figure 1D). Changes in immature erythrocytes (Ter119+CD71+ cells) were similar to the reductions seen in B cell progenitors: erythroid cells progressively reduced at 6, 14, and 21 days after ALL transplantation, reaching >10-fold reductions at 3 weeks (Figure 1E). In summary, ALL expansion induces a strong decline in lymphopoiesis and erythropoiesis while their impact on myeloid cell production is modest. Figure 1 Download asset Open asset Kinetics of B-ALL growth and impact on hematopoiesis. (A) B-ALL number. (B) Number of non-malignant developing B cell subsets. (C) Inflammatory monocytes. (D) Neutrophils. (E) Immature (Ter119+CD71+) and mature (Ter119+CD71-) red blood cells. Data in all panels show bone marrow cell numbers obtained from wild-type (WT) mice transplanted with 3×106 BCR-ABL-expressing B-ALL cells. In all panels, X-axis indicates time (days) after B-ALL transplantation. Bars indicate mean, circles depict individual mice. Data are representative of two independent experiments. **p<0.005; ***p<0.0005 unpaired, two-sided, Student’s t test. ###p<0.0005 Mann–Whitney test. ALL, acute lymphoblastic leukemia. ALL induces LTβR signaling in MSCs, downregulates Il7 expression, and modulates lymphopoiesis In previous studies we noted that transplanted ALL cells and Artemis-deficient (pre-leukemic) pre-B cells led to IL7 and CXCL12 downregulation in MSCs (Fistonich et al., 2018), which could explain the negative impact of ALL in non-malignant lymphopoiesis. While the mechanism(s) responsible for IL7 and CXCL12 downregulation remained undefined, earlier studies suggested a role for LTβR signaling in BM stromal cells in development of some lymphoid lineages (Wu et al., 2001; Kim et al., 2014). In recent studies, we found that MSCs express LTβR and that LTβR signaling controls IL7 expression in vivo (Zehentmeier et al., 2022). Furthermore, when mRNA levels of LTB were analyzed in pediatric samples of B-ALL (Children’s Oncology Group Study 9906 for High-Risk Pediatric ALL) and associated with clinical outcome at the time of diagnosis, we noted an inverse correlation between LTB transcript abundance and relapse-free survival that reached statistical significance (Figure 2—figure supplement 1A). These observations led us to hypothesize that leukemic cells express LTβR ligands and induce LTβR signaling in MSCs in vivo. In mice, BCR-ABL1 expressing pre-B ALL cells also express higher LTα/LTβ amounts than non-leukemic pre-B, immature, and mature B cells (Figure 2A and B). The presence of ALL cells in the BM environment did not change LTα and LTβ expression on non-leukemic host pre-B cells (Figure 2A). Importantly, when mouse ALL cells were engineered to over-express LTα/LTβ, these ALLs induced stronger IL7 downregulation in BM MSCs and were lethal more quickly than empty vector transduced ALL cells (Figure 2—figure supplement 1B–E). Combined, these studies suggest a pathogenic role for the LTβR pathway in leukemia progression. Figure 2 with 1 supplement see all Download asset Open asset Lymphotoxin α1β2 expression in B-ALL cells and therapeutic effect of lymphotoxin beta receptor (LTβR) blocking. (A) Histograms of LTα and LTβ expression in B-ALL cells and in pre-B cells. Purple, B-ALL; green, non-malignant pre-B cells (CD19+CD93+IgM-cKit-) in bone marrow (BM) of wild-type (WT) mice transplanted with B-ALL cells; blue, non-malignant pre-B cells in BM of WT mice (no B-ALL); filled gray, non-malignant Ltb-deficient pre-B cells in BM of Ltb-/- mice. (B) LTα and LTβ mean fluorescence intensity (MFI) in developing B cells and ALLs isolated from BM of ALL transplanted mice. (C) Experimental design of data described in panels D–H. (D) Number of non-malignant developing B cell subsets in BM. (E) Immature and mature erythrocyte number. (F) Neutrophils. (G) Monocytes. (H) B-ALL number. Data in panels D–H show BM numbers from WT mice transplanted with 3×106 BCR-ABL-expressing B-ALL cells and treated with HEL-Ig or LTβR-Ig (150 µg/mouse) on day 0 and day 5; mice were analyzed on day 8 post ALL transplantation. (I) Frequency of mouse survival after B-ALL transplantation following pre-treatment with either HEL-Ig or LTβR-Ig (n=5 per group). Mice were treated with HEL-Ig or LTβR-Ig (150 µg/mouse) every 5 days until endpoint. Bars indicate mean, circles depict individual mice. Data are representative of two independent experiments. *p<0.05; **p<0.005; ***p<0.0005 unpaired, two-sided, Student’s t test. ###p<0.0005 Mann–Whitney test. To test if LTβR signaling impacts ALL growth and non-malignant hematopoiesis, we transplanted 3 million ALL cells into WT syngeneic recipient mice (C57BL6/J) treated weekly with a soluble LTβR-Ig decoy (a fusion between LTβR ectodomain and the Fc domain of a mouse IgG1 recognizing Hen Egg Lysozyme) or with control Hel-Ig. Transplanted ALL cells reduced lymphopoiesis significantly, which was reverted with LTβR-Ig treatment (Figure 2C and D). In contrast, LTβR signaling blockade did not restore erythropoiesis or myelopoiesis (Figure 2E–G). Importantly, ALL growth was significantly reduced at 2 weeks (Figure 2H), which reflected in a small but significant extension of mouse survival (Figure 2I). To gain further insight into the mechanisms used by ALL cells for reducing non-malignant hematopoiesis, we analyzed the MSC transcriptome in homeostasis, during ALL expansion, and in mice with ALL but treated with LTβR-Ig. To identify gene expression differences between the three groups, we performed principal component analyses (PCA) on the transcriptome datasets from three to four independent replicates. The first two principal components (PC1 and PC2) represent the main axes of variation within these datasets and explained 46% and 17% of variation, respectively. Samples from control and ALL groups separated by PC1, and within ALL cohorts, samples from LTβR-Ig versus Hel-Ig treated ALL also segregated from each other, thus indicating major transcriptional changes induced by ALL growth in vivo, of which a significant fraction was sensitive to LTβR blocking (Figure 3A). Unsupervised clustering of the top 1000 most variable genes also independently segregated the three groups (Figure 3B). Comparisons between control and ALL treated with Hel-Ig samples revealed 322 differentially expressed genes (DEGs; Padj <0.05, |log2FC|>1), of which 74 were downregulated and 248 were upregulated in MSCs of control mice (Supplementary file 1). Comparisons between the ALL groups (Hel-Ig versus LTβR-Ig) revealed 226 DEGs of which 149 were upregulated and 77 were downregulated in MSCs of mice with ALL and treated with Hel-Ig (Supplementary file 2). Gene set enrichment analyses revealed a strong inflammatory gene signature induced by ALL with a strong statistical significance in interferon α- and γ-induced genes, complement, and cytokines IL2, IL6, and TNFα signaling (Figure 3C). Of note, LTβR blocking further increased the interferon stimulated gene signature, while it reduced the expression of genes associated with TNFα signaling (Figure 3C), consistent with the fact that LTβR is a TNF superfamily member that activates canonical and non-canonical nuclear factor kappa-binding transcription factors (NFκB) (Norris and Ware, 2007). Importantly, of the several hematopoietic cytokines expressed by MSCs, KITL, IL7, IGF1, and CSF1 were significantly downregulated by ALL cells (Figure 3D). These transcriptional changes in MSCs were similar to those described in mice with acute myeloid leukemia (Baryawno et al., 2019). However, of these hematopoietic cytokines, only IL7 downregulation was blocked by LTβR-Ig treatment (Figure 3D). Furthermore, blocking other NFκB-inducing cytokines, such as TNFα and IL1β, did not prevent IL7 downregulation nor did it rescue non-malignant lymphopoiesis or myelopoiesis (Figure 3—figure supplement 1A–C) and did not impact ALL expansion in vivo (Figure 3—figure supplement 1D). Even though ALL cells promoted an interferon-induced gene expression signature in MSCs (Figure 3C), blocking IFNα or IFNγ signaling did not rescue IL7 downregulation and non-malignant hematopoiesis, nor did it reduce ALL growth in vivo (Figure 3—figure supplement 1E–I). Combined, these results show a major impact of ALL expansion in the MSC transcriptome, with a large fraction of DEGs being sensitive to LTβR blocking. Figure 3 with 1 supplement see all Download asset Open asset Lymphotoxin beta receptor (LTβR)-dependent and -independent transcriptomic changes in mesenchymal stem cells (MSCs) induced by B-ALL. (A) Principal component analysis (PCA) distribution plot. (B) Unsupervised hierarchical clustering and heatmap representation of top 1000 differentially expressed genes. (C) GSEA-KEGG pathway alterations in MSCs. (D) Hematopoietic cytokines and chemokine mRNA expression. Data in all panels were generated from analyses of MSC bulk RNA sequencing. *p<0.05; **p<0.005; unpaired, two-sided, Student’s t test. ALL, acute lymphoblastic leukemia. To test if LTβR signaling in MSCs impacts ALL growth, non-malignant hematopoiesis, and mouse survival, we transplanted ALL cells into mice conditionally deficient in Ltbr in MSCs (Ltbrfl/fl; LeprCre/+ mice, from here on referred as LTβR∆) that also report Il7 transcription via GFP expression (Il7GFP/+). We ruled out a role for LTβR signaling in MSCs in promoting ALL homing to the BM by transplanting 3×106 ALL cells into control or LTβR∆ mice (Figure 4A), in agreement with prior studies showing that LTβR signaling in MSCs does not control CXCL12 expression under homeostatic conditions (Zehentmeier et al., 2022). Transplanted ALL cells induced IL7 downregulation in control mice (WT, Lepr+/+; Ltbrfl/fl) but not in LTβR∆ mice (Figure 4B), as expected (Figure 3D). These changes in IL7 production corresponded with reduced lymphopoiesis in WT mice whereas lymphopoiesis was largely unaffected in LTβR∆ mice (Figure 4C–E). In contrast, ALL-induced reductions in myeloid and erythroid lineages were largely independent of LTβR signaling in MSCs (Figure 4G–J). The inability to induce LTβR signaling in MSCs also impacted ALL growth in vivo (Figure 4F and K) such that it extended mouse survival by approximately 1 week (Figure 4L). To further test if ALL cells directly induce LTβR signaling in MSCs, we generated ALL cells genetically deficient in Ltb (Figure 4M). Indeed, Ltb-deficient ALL cells were unable to induce IL7 downregulation in MSCs and to block non-malignant lymphopoiesis (Figure 4N and O). Furthermore, Ltb-deficient ALL cells proliferated significantly less than Ltb-sufficient ALL cells (Figure 4P), which extended mouse survival significantly (Figure 4Q). Finally, to account for reduced Ltb-deficient ALL growth in vivo, we measured changes in Il7 expression in mice transplanted with 3×106 Ltb+/+ ALLs, 3×106 Ltb-/- ALLs, and 9×106 Ltb-/- ALLs (3×Ltb-/-). Importantly, IL7 expression was unchanged even in mice transplanted with threefold higher number of Ltb-deficient ALLs (Figure 4R and S). Combined, these studies show that the ALL-induced IL7 downregulation that we reported in previous studies (Fistonich et al., 2018; Zehentmeier and Pereira, 2019) is mediated by direct delivery of lymphotoxin ligands to LTβR expressed on BM MSCs. Figure 4 with 1 supplement see all Download asset Open asset Effects of mesenchymal stem cell (MSC)-intrinsic lymphotoxin beta receptor (LTβR) signaling in lymphopoiesis and B-ALL growth. (A) Measurements of ALL homing to the bone marrow (BM): 3×106 BCR-ABL ALLs were transferred into control (red) or LTβR∆ (green) mice and allowed to home into the BM for 24 hr. (B) Il7-GFP expression in MSCs. (C–E) Number of non-malignant developing B cell subsets. (C) ProB cells. (D) Pre-B cells. (E) Immature B cells. (F) B-ALL frequency in BM. (G–K) Myeloid and erythroid cell numbers in BM. (G) Neutrophils. (H) Monocytes. (I) Immature RBCs. (J) Mature RBCs. (K) Total ALL number. (L) Probability of wild-type (WT) or LTβR∆ mouse survival after B-ALL transplantation (n=8 mice/group). Mice were transplanted with 3×106 BCR-ABL-expressing B-ALL cells and analyzed on day 8 after transplantation. (M) Histogram of LTβR ligand expression in ALL cells. Green, Ltb-sufficient; brown, Ltb-deficient. (N) Il7-GFP expression in MSCs. (O) Number of non-malignant developing B cell subsets. (P) ALL number. (Q) Frequency of WT mouse survival after Ltb-deficient or Ltb-sufficient ALL transplantation (n=7/group). (R and S) Effects of Ltb-expressing ALLs in MSCs. (R) Ltb+/+ and Ltb-/- B-ALL numbers in BM 3 weeks after transplantation into Il7GFP/+ mice. (S) Il7-GFP expression in MSCs. Gray bar indicates control Il7GFP/+ mice (no ALL); green bar represents Il7GFP/+ mice recipient of 3×106 Ltb+/+ ALLs; brown bar indicates Il7GFP/+ mice recipient of 3×106 Ltb-/- ALLs; red bars depict Il7GFP/+ mice recipient of 9×106 Ltb-/- ALLs (3×Ltb-/-). Bars indicate mean, circles depict individual mice. Data in all panels are representative of two independent experiments. *p<0.05; **p<0.005; ***p<0.0005 unpaired, two-sided, Student’s t test. ALL, acute lymphoblastic leukemia. To test if increased lymphopoiesis due to excess IL7 is directly responsible for reduced ALL growth in vivo, we treated mice transplanted with B-ALL cells with recombinant IL7 complexed with a neutralizing anti-IL7 (aIL7, clone M25) monoclonal antibody (Figure 4—figure supplement 1A), which increases the half-life of recombinant IL-7 in vivo (Boyman et al., 2008). Indeed, mice treated with IL7/aIL7 had significantly higher numbers of developing B cell subsets in the BM (Figure 4—figure supplement of and pre-B cells. Conversely, ALL numbers in BM were significantly which reflected in significant reductions in blood and (Figure 4—figure supplement 1C). Combined, these data demonstrate that the of IL7 production reduces which results in ALL growth. and used an mouse model of Artemis-deficient mice with mice and with mice to study the impact of DNA pathway in B cell In these studies, reported in pre-B cells that could not due to in et al., 2008; et al., 2016). In previous studies this we that Artemis-deficient pre-B cells could also induce in MSCs (Fistonich et al., 2018; Zehentmeier and Pereira, suggesting that the LTβR pathway may also be in with this when the transcriptome of Artemis-deficient and pre-B cells, we noted that Artemis-deficient pre-B cells also expressed significantly higher amounts of LTα and LTβ et al., 2008; et al., 2016). In agreement with these we higher amounts of LTα and LTβ on the cell of pre-B cells than on pre-B cells (Figure supplement 1A). of BM cells into Il7GFP/+ LTβR∆ mice or control revealed Il7 downregulation (Figure supplement which in a increased numbers of Artemis-deficient B cells (Figure supplement 1C). In contrast, did not impact production (Figure supplement and cells unable to which causes a of the DNA damage response pathway and To test if the DNA damage response controls LTα and LTβ expression, we treated ALL cells with a that prevents ALLs upregulated LTα on the cell in an (Figure supplement and The DNA damage response pathway signals of treatment with (a small of significantly reduced lymphotoxin α1β2 expression in ALLs (Figure supplement Combined, these studies demonstrate that LTα and LTβ expression can be by DNA damage response CXCR4 signaling ALL studies have that receptor signaling in cells lymphotoxin α1β2 expression et al., of LTβR expressed on lymphoid stromal cells increases the production of B cell which further increases lymphotoxin α1β2 expression in B cells, thus a loop et al., et al., To test if CXCR4 signaling in ALLs lymphotoxin α1β2 expression, we treated ALLs in with a of CXCL12 and measured Indeed, ALLs upregulated LTα after to CXCL12 (Figure Furthermore, LTα expression was further increased in ALLs treated with CXCL12 and (Figure Although prior studies have also that signaling can promote lymphotoxin α1β2 expression in lymphoid cells et al., in ALL cells signaling was not for LTα even at IL7 even though it promoted (Figure supplement 2A and B). To test if CXCR4 signaling is required for lymphotoxin α1β2 expression in ALLs in vivo, we transplanted 3×106 ALL cells into WT mice for 1 week and treated with or prior to Indeed, lymphotoxin α1β2 expression was significantly reduced in ALLs and in non-leukemic pre-B cells of mice (Figure Conversely, increased CXCR4 expression in ALL and non-leukemic pre-B cells (Figure as expected et al., 2005; et al., 2014). has a half-life in vivo thus it for long-term treatment in vivo et al., 2014; et al., To test if CXCR4 also prevents ALL-induced IL7 downregulation in BM MSCs, we transferred 3×106 ALL cells into Il7GFP/+ mice and treated with an CXCR4 et al., or with by (Figure While mice ALL-induced Il7-GFP downregulation in MSCs, mice treated with CXCR4 IL7 expression within the normal of mice ALL (Figure Similarly, developing B cells were significantly reduced in mice, but their numbers were normal in CXCR4 treated mice (Figure In contrast, ALL numbers were significantly in the BM and of mice treated with CXCR4 (Figure which with extended mouse survival (Figure Figure 5 with 2 see all Download asset Open asset CXCR4 signaling and its impact on acute lymphoblastic leukemia growth in vivo. (A) Histograms of LTα expression in B-ALL cells treated for with CXCL12 at the in (B) Histograms of LTα expression and mean intensity as of cells) in B-ALL cells treated with 1 or in with CXCL12 in and LTα (C) and CXCR4 expression (D) in ALLs and non-leukemic pre-B cells in the bone marrow (BM) of mice transplanted with 3×106 Ltb+/+ ALLs were allowed to in vivo for Mice were treated with or prior to (E) Experimental design of data described in panels Mice were transplanted with 3×106 BCR-ABL-expressing B-ALL cells and treated with CXCR4 on day 2 and until day mice were analyzed on day (F) Il7-GFP expression in mesenchymal stem cells (G) Number of non-malignant developing B cell subsets. (H) Total ALL number in BM and B-ALL in blood (I) Frequency of mouse survival after B-ALL transplantation into mice treated with or CXCR4 (J) CXCR4 expression on developing B cells and ALLs in BM. (K) In of developing B cells and Data in panels and are from BM of mice 1 week after ALL transplantation. Data are representative of two independent experiments. *p<0.05; **p<0.005; ***p<0.0005 unpaired, two-sided, Student’s t test. Mann–Whitney test. studies have that CXCR4 ALL homing and in the BM (Juarez et al., 2007; Sipkins et al., 2005). studies revealed that ALLs induce a but downregulation of CXCL12 expression in vivo (Figure 3D). However, when CXCR4 levels between ALLs and non-leukemic B cell progenitors developing in the we noted that ALLs express significantly higher amounts of CXCR4 in vivo (Figure and a CXCL12 in significantly more than non-leukemic developing B cell subsets (Figure These observations suggest that ALLs are more in the BM than non-leukemic B cell LTβR signaling AML growth and As AML also induces the downregulation of multiple hematopoietic cytokines expressed by MSCs, IL7 (Baryawno et al.,

I - Detection of Minimal Residual Disease in Acute Leukemia.- New possibilities for cytogenetic analysis of leukemic cells.- Breakpoint analysis in CML: potentials for detection of minimal residual disease.- The mammalian ETS genes: two unique chromosomal locations in cat, mice and man and novel translocated position in human leukemias.- Activated RAS oncogenes in acute leukemia.- DNA rearrangements as unique markers of clonal evolution, recurrence and translocation.- The application of monoclonal antibodies for the detection and classification of AML.- Towards detection of minimal disease: discrimination of AML precursors from normal myeloid precursors using a combination of surface makers.- An immunological approach to analyse the kinetics of minimal residual disease in acute leukemia.- Visualization of minor cell populations with simultaneous three-parameter flow cytometry: BN rat marrow and spleen model.- Growth kinetics of minimal residual disease in the brown Norway rat acute myelocytic leukemia.- Detection of minimal residual acute lymphoblastic leukemia by immunological marker analysis: possibilities and limitations.- Surveillance of terminal deoxynucleotidyl transferase-positive cells in peripheral blood of patients with acute lymphoblastic leukemia.- In vitro colony forming cells of acute lymphoblastic leukemia: analysis of 24 cases with recombinant interleukin 2 as growth stimulus.- II - Intensive Chemotherapy Regimens for `Minimal Residual Disease' in Acute Myeloblastic and Lymphoblastic Leukemia.- Biologic and treatment determinants of curability in acute myologenous leukemia.- High-dose cytosine-arabinoside plus AMSA for reinduction or consolidation-maintenance in acute myelogenous leukemia.- Treatment of residual disease in AML: interim analysis of a southeastern cancer study group prospective randomized clinical trial.- L-20 protocol for adult patients with acute lymphoblastic leukemia: a protocol utilizing prognostic factors, intensive chemotherapy and autologus "purged" marrow transplantation to eradicate minimal residual disease.- Treatment of minimal residual disease in adult ALL: the German national study.- Treatment of minimal residual disease in "poor risk" acute lymphoblastic leukaemia with high-dose cytosine arabinoside.- III Autologous Bone Marrow Transplantation for the Eradication of `Minimal Residual Leukemia'.- Autologous bone marrow transplantation in first remission AML using non-purged marrow - update.- Double autografting: a potential curative regimen for acute leukaemia?.- Autologous bone marrow transplantation in acute nonlymphocytic leukemia. A study of ex vivo marrow treatment with 4-hydroperoxycyclophosphamide.- Attempts to eliminate residual acute myeloid leukemia from autologous bone marrow grafts through in vitro chemotherapy - a review.- Monoclonal antibody purged autologous bone marrow transplantation for relapsed non T-cell acute lymphoblastic leukemia.- Allogeneic and autologous marrow transplantation: ex vivo purging with monoclonal antibody or immunotoxins to remove leukemic cells or to prevent graft versus host disease.- Detection and selective destruction of tumor cells by the lipophilic dye, merocyanine 540.- IV Allogeneic Bone Marrow Transplantation for the Eradication of `Minimal Residual Leukemia'.- Factors influencing long-term leukemia-free survival after allogeneic bone marrow transplantation for acute leukemia.- Bone marrow transplantation with HLA identical donors in the acute leukemias - Baltimore experience.- Results of allogeneic marrow transplantaton in patients transplanted for acute leukemia: a long-term follow-up.- Bone marrow transplantation in first CR of acute leukaemia using T-depleted marrow from HLA identical sibling donors.- Soybean lectin agglutination and E-rosette depletion for removal of T-cells from HLA-identical marrow grafts: results in 60 consecutive patients transplanted for hematologic malignancy.- Allogeneic bone marrow transplantation in adult leukemia: result of T-cell depletion by soybean lectin fractionation.- Rapid lymphocyte depletion by a new elutriator rotor for the prevention of acute graft versus host disease after allogeneic bone marrow transplantation.- Problems and prospect of histoincompatible bone marrow transplantation studied in rhesus monkeys.- Allogeneic marrow transplantation for the treatment of leukemia. Role of the major histocompatibility complex.- Summing up.- Minimal residual disease in leukemia: 1986.- Index of subjects.

The management of patients with leukaemia: the role of cytogenetics in this molecular era.

NCI First International Workshop on the Biology, Prevention and Treatment of Relapse after Allogeneic Hematopoietic Cell Transplantation: Report from the Committee on Prevention of Relapse Following Allogeneic Cell Transplantation for Hematologic Malignancies

Flow cytometry (FCM) is a powerful tool for classifying acute leukemia (AL) to acute lymphoblastic leukemia (ALL) or acute myeloid leukemia (AML) and further subtyping. Accurate diagnosis of AL improves the outcome. Mosul, a city in Iraq, had no FCM laboratory yet, mainly due to repeated wars in the area. The Japan Chernobyl Foundation (JCF), a Japanese non-profit humanitarian organization, helped establish the first FCM Laboratory in Mosul. We aimed to evaluate the project from a scholarly point of view with a focus on childhood ALL. An observational study was done after JCF provided the BD FACSCanto II system and all related materials. Anonymous data of the patients, including age, sex, address, initial complete blood count (CBC) with bone marrow aspirate (BMA) results, and coded FCM reports, were prospectively collected from February 2021 to January 2024. In addition to clinical notes on pediatric ALL cases, their treatment, and outcomes with a median follow-up of 26.2 (range, 0-44.3) months. Childhood ALL cases were 76, including (60, 78.9%) B-lineage ALL (B-ALL) and (16, 21.1%) T-lineage ALL (T-ALL) cases. B-ALL cases were classified as pro-B, common-B, pre-B, and mature-B, with frequencies of 11.7%, 76.7%, 10.0%, and 1.6%, respectively. T-ALL included early T-cell precursor (ETP), pro-T, pre-T, cortical-T, and medullary-T, with frequencies of 6.25%, 18.75%, 18.75%, 31.25%, and 25.00%, respectively. Furthermore, 20 pediatric AML cases were identified and sub-typed. Among ALL cases, the male-to-female ratio (M/F) was 1.5. There were 24 (31.6%) cases with white blood cell (WBC) counts of ≥50×109/L and 29 (38.2%) who were aged ≥10 years or ≤12 months. There was a substantial association between high WBC and male sex with the T-ALL subtype. Based on clinical criteria and immunophenotyping of ALL, 47 (61.8%) of patients were identified as a high-risk (HR) group, while 29 (38.2%) were of standard-risk (SR) group. Relapses were reported in 8 (11.6%) patients with ALL, principally in the HR group. The induction mortality rate was 4.2%. Septic death was the leading cause of death (8/17, 47.1%), especially in those younger than 2 years old. The overall survival (OS) in ALL cases was 73.7%. The OS and event-free survival (EFS) for the SR group of ALL were 86.2% and 82.8%, respectively. JCF's role was crucial in providing Mosul City, Iraq, with the first FCM Unit. The project made a breakthrough in AL diagnosis and established one of the important and supposed routine steps represented by ALL immunophenotyping. The HR group represented a significantly large portion of our ALL cases. Although the outcome was satisfactory for the SR group, the survival rate for the HR group was dismal. Further efforts are needed to scale up diagnostic and therapeutic capabilities to improve the outcome of ALL in Mosul City.

Historical review of lymphomas.

Optimizing antimetabolite-based chemotherapy for the treatment of childhood acute lymphoblastic leukaemia.