Deletion of the inhibitory co-receptor CTLA-4 enhances and invigorates chimeric antigen receptor T cells.

Chimeric antigen receptor (CAR) T cell therapy targeting CD19 has achieved tremendous success treating B cell malignancies; however, some patients fail to respond due to poor autologous T cell fitness. To improve response rates, we investigated whether disruption of the co-inhibitory receptors CTLA4 or PD-1 could restore CART function. CRISPR-Cas9-mediated deletion of CTLA4 in preclinical models of leukemia and myeloma improved CAR T cell proliferation and anti-tumor efficacy. Importantly, this effect was specific to CTLA4 and not seen upon deletion of CTLA4 and/or PDCD1 in CAR T cells. Mechanistically, CTLA4 deficiency permitted unopposed CD28 signaling and maintenance of CAR expression on the T cell surface under conditions of high antigen load. In clinical studies, deletion of CTLA4 rescued the function of T cells from patients with leukemia that previously failed CAR T cell treatment. Thus, selective deletion of CTLA4 reinvigorates dysfunctional chronic lymphocytic leukemia (CLL) patient T cells, providing a strategy for increasing patient responses to CAR T cell therapy.

Critical care management of chimeric antigen receptor T-cell therapy recipients.

Chimeric antigen receptor (CAR) T-cell therapy is a promising immunotherapeutic treatment concept that is changing the treatment approach to hematologic malignancies. The development of CAR T-cell therapy represents a prime example for the successful bench-to-bedside translation of advances in immunology and cellular therapy into clinical practice. The currently available CAR T-cell products have shown high response rates and long-term remissions in patients with relapsed/refractory acute lymphoblastic leukemia and relapsed/refractory lymphoma. However, CAR T-cell therapy can induce severe life-threatening toxicities such as cytokine release syndrome, neurotoxicity, or infection, which require rapid and aggressive medical treatment in the intensive care unit setting. In this review, the authors provide an overview of the state-of-the-art in the clinical management of severe life-threatening events in CAR T-cell recipients. Furthermore, key challenges that have to be overcome to maximize the safety of CAR T cells are discussed.

Cut-and-Run: A Distinct Mechanism by which V(D)J Recombination Causes Genome Instability.

V(D)J recombination is essential to generate antigen receptor diversity but is also a potent cause of genome instability. Many chromosome alterations that result from aberrant V(D)J recombination involve breaks at single recombination signal sequences (RSSs). A long-standing question, however, is how such breaks occur. Here, we show that the genomic DNA that is excised during recombination, the excised signal circle (ESC), forms a complex with the recombinase proteins to efficiently catalyze breaks at single RSSs both in vitro and in vivo. Following cutting, the RSS is released while the ESC-recombinase complex remains intact to potentially trigger breaks at further RSSs. Consistent with this, chromosome breaks at RSSs increase markedly in the presence of the ESC. Notably, these breaks co-localize with those found in acute lymphoblastic leukemia patients and occur at key cancer driver genes. We have named this reaction "cut-and-run" and suggest that it could be a significant cause of lymphocyte genome instability.

The PAX5-JAK2 translocation acts as dual-hit mutation that promotes aggressive B-cell leukemia via nuclear STAT5 activation.

While PAX5 is an important tumor suppressor gene in B-cell acute lymphoblastic leukemia (B-ALL), it is also involved in oncogenic translocations coding for diverse PAX5 fusion proteins. PAX5-JAK2 encodes a protein consisting of the PAX5 DNA-binding region fused to the constitutively active JAK2 kinase domain. Here, we studied the oncogenic function of the PAX5-JAK2 fusion protein in a mouse model expressing it from the endogenous Pax5 locus, resulting in inactivation of one of the two Pax5 alleles. Pax5Jak2/+ mice rapidly developed an aggressive B-ALL in the absence of another cooperating exogenous gene mutation. The DNA-binding function and kinase activity of Pax5-Jak2 as well as IL-7 signaling contributed to leukemia development. Interestingly, all Pax5Jak2/+ tumors lost the remaining wild-type Pax5 allele, allowing efficient DNA-binding of Pax5-Jak2. While we could not find evidence for a nuclear role of Pax5-Jak2 as an epigenetic regulator, high levels of active phosphorylated STAT5 and increased expression of STAT5 target genes were seen in Pax5Jak2/+ B-ALL tumors, implying that nuclear Pax5-Jak2 phosphorylates STAT5. Together, these data reveal Pax5-Jak2 as an important nuclear driver of leukemogenesis by maintaining phosphorylated STAT5 levels in the nucleus.

JAK2 is dispensable for maintenance of JAK2 mutant B-cell acute lymphoblastic leukemias.

Activating JAK2 point mutations are implicated in the pathogenesis of myeloid and lymphoid malignancies, including high-risk B-cell acute lymphoblastic leukemia (B-ALL). In preclinical studies, treatment of JAK2 mutant leukemias with type I JAK2 inhibitors (e.g., Food and Drug Administration [FDA]-approved ruxolitinib) provided limited single-agent responses, possibly due to paradoxical JAK2Y1007/1008 hyperphosphorylation induced by these agents. To determine the importance of mutant JAK2 in B-ALL initiation and maintenance, we developed unique genetically engineered mouse models of B-ALL driven by overexpressed Crlf2 and mutant Jak2, recapitulating the genetic aberrations found in human B-ALL. While expression of mutant Jak2 was necessary for leukemia induction, neither its continued expression nor enzymatic activity was required to maintain leukemia survival and rapid proliferation. CRLF2/JAK2 mutant B-ALLs with sustained depletion or pharmacological inhibition of JAK2 exhibited enhanced expression of c-Myc and prominent up-regulation of c-Myc target genes. Combined indirect targeting of c-Myc using the BET bromodomain inhibitor JQ1 and direct targeting of JAK2 with ruxolitinib potently killed JAK2 mutant B-ALLs.

Molecular and cellular mechanisms of chemoresistance in paediatric pre-B cell acute lymphoblastic leukaemia.

Paediatric patients with relapsed B cell acute lymphoblastic leukaemia (B-ALL) have poor prognosis, as relapse-causing clones are often refractory to common chemotherapeutics. While the molecular mechanisms leading to chemoresistance are varied, significant evidence suggests interactions between B-ALL blasts and cells within the bone marrow microenvironment modulate chemotherapy sensitivity. Importantly, bone marrow mesenchymal stem cells (BM-MSCs) and BM adipocytes are known to support B-ALL cells through multiple distinct molecular mechanisms. This review discusses the contribution of integrin-mediated B-ALL/BM-MSC signalling and asparagine supplementation in B-ALL chemoresistance. In addition, the role of adipocytes in sequestering anthracyclines and generating a BM niche favourable for B-ALL survival is explored. Furthermore, this review discusses the role of BM-MSCs and adipocytes in promoting a quiescent and chemoresistant B-ALL phenotype. Novel treatments which target these mechanisms are discussed herein, and are needed to improve dismal outcomes in patients with relapsed/refractory disease.

Engineering of potent CAR NK cells using non-viral Sleeping Beauty transposition from minimalistic DNA vectors.

Natural killer (NK) cells have high intrinsic cytotoxic capacity, and clinical trials have demonstrated their safety and efficacy for adoptive cancer therapy. Expression of chimeric antigen receptors (CARs) enhances NK cell target specificity, with these cells applicable as off-the-shelf products generated from allogeneic donors. Here, we present for the first time an innovative approach for CAR NK cell engineering employing a non-viral Sleeping Beauty (SB) transposon/transposase-based system and minimized DNA vectors termed minicircles. SB-modified peripheral blood-derived primary NK cells displayed high and stable CAR expression and more frequent vector integration into genomic safe harbors than lentiviral vectors. Importantly, SB-generated CAR NK cells demonstrated enhanced cytotoxicity compared with non-transfected NK cells. A strong antileukemic potential was confirmed using established acute lymphocytic leukemia cells and patient-derived primary acute B cell leukemia and lymphoma samples as targets in vitro and in vivo in a xenograft leukemia mouse model. Our data suggest that the SB-transposon system is an efficient, safe, and cost-effective approach to non-viral engineering of highly functional CAR NK cells, which may be suitable for cancer immunotherapy of leukemia as well as many other malignancies.

Autologous CD7 CAR-T cells generated without T cell pre-selection in pediatric patients with relapsed/refractory T-ALL: A phase I trial.

Chimeric antigen receptor (CAR)-T cell therapy showed preliminary activity in patients with refractory or relapsed T cell acute lymphoblastic leukemia (r/r T-ALL). However, many obstacles remain, including manufacturing difficulties and risk of infections. This phase I study (NCT04840875) evaluated autologous CD7 CAR-T cells manufactured without pre-selection of healthy T cells in r/r T-ALL. Thirty patients (29 children and one adult) with a median of two lines of prior therapy but without detectable peripheral leukemia were enrolled. Excluding three cases of manufacturing failures, a total of 27 (90%) patients received infusions after products were confirmed free of leukemia contamination, including 16 (59%) meeting planned target doses. Common adverse events within 30 days included grade 3-4 cytopenias (100%), grade 1-2 (70%) and 3-4 (7%, including one dose-limiting toxicity) cytokine release syndrome, grade 1 neurotoxicity (7%), grade 2 infection (4%), and grade 2 graft-versus-host disease (4%). Two patients developed grade 2 infections after day 30. At day 30, 96% responded and 85% achieved complete remission (CR) or CR with incomplete hematologic recovery (CRi). Seventy-four percent underwent transplantation. Twelve-month progression-free survival with and without censoring transplantation was 22% (95% confidence interval 4%-100%) and 57% (41%-81%), respectively. These results support that autologous CD7 CAR-T therapy without T cell pre-selection is feasible in patients with r/r T-ALL.

Bispecific antibodies and autologous chimeric antigen receptor T cell therapies for treatment of hematological malignancies.

In recent years, the therapeutic landscape for hematological malignancies has markedly advanced, particularly since the inaugural approval of autologous chimeric antigen receptor T cell (CAR-T) therapy in 2017 for relapsed/refractory acute lymphoblastic leukemia (ALL). Autologous CAR-T therapy involves the genetic modification of a patient's T cells to specifically identify and attack cancer cells, while bispecific antibodies (BsAbs) function by binding to both cancer cells and immune cells simultaneously, thereby triggering an immune response against the tumor. The subsequent approval of various CAR-T therapies and BsAbs have revolutionized the treatment of multiple hematological malignancies, highlighting high response rates and a subset of patients achieving prolonged disease control. This review explores the mechanisms underlying autologous CAR-T therapies and BsAbs, focusing on their clinical application in multiple myeloma, ALL, and non-Hodgkin lymphoma. We provide comprehensive insights into their individual efficacy, limitations concerning broad application, and the potential of combination therapies. These upcoming strategies aim to propel the field forward, paving the way for safer and more effective therapeutic interventions in hematological malignancies.

Folylpolyglutamate synthetase inactivation in relapsed ALL induces a druggable folate metabolic vulnerability.

AIMS: The antifolate methotrexate (MTX) is an anchor drug used in acute lymphoblastic leukemia (ALL) with poorly understood chemoresistance mechanisms in relapse. Herein we find decreased folate polyglutamylation network activities and inactivating FPGS mutations, both of which could induce MTX resistance and folate metabolic vulnerability in relapsed ALL. METHODS: We utilized integrated systems biology analysis of transcriptomic and genomic data from relapse ALL cohorts to infer hidden ALL relapse drivers and related genetic alternations during clonal evolution. The drug sensitivity assay was used to determine the impact of relapse-specific FPGS mutations on sensitivity to different antifolates and chemotherapeutics in ALL cells. We used liquid chromatography-mass spectrometry (LC-MS) to quantify MTX and folate polyglutamate levels in folylpoly-γ-glutamate synthetase (FPGS) mutant ALL cells. Enzymatic activity and protein degradation assays were also conducted to characterize the catalytic properties and protein stabilities of FPGS mutants. An ALL cell line-derived mouse leukemia xenograft model was used to evaluate the in vivo impact of FPGS inactivation on leukemogenesis and sensitivity to the polyglutamatable antifolate MTX as well as non-polyglutamatble lipophilic antifolate trimetrexate (TMQ). RESULTS: We found a significant decrease in folate polyglutamylation network activities during ALL relapse using RNA-seq data. Supported by functional evidence, we identified multifactorial mechanisms of FPGS inactivation in relapsed ALL, including its decreased network activity and gene expression, focal gene deletion, impaired catalytic activity, and increased protein degradation. These deleterious FPGS alterations induce MTX resistance and inevitably cause marked intracellular folate shrinkage, which could be efficiently targeted by a polyglutamylation-independent lipophilic antifolate TMQ in vitro and in vivo. CONCLUSIONS: MTX resistance in relapsed ALL relies on FPGS inactivation, which inevitably induces a folate metabolic vulnerability, allowing for an efficacious antifolate ALL treatment strategy that is based upon TMQ, thereby surmounting chemoresistance in relapsed ALL.

ARID5B as a critical downstream target of the TAL1 complex that activates the oncogenic transcriptional program and promotes T-cell leukemogenesis.

The oncogenic transcription factor TAL1/SCL induces an aberrant transcriptional program in T-cell acute lymphoblastic leukemia (T-ALL) cells. However, the critical factors that are directly activated by TAL1 and contribute to T-ALL pathogenesis are largely unknown. Here, we identified AT-rich interactive domain 5B (ARID5B) as a collaborating oncogenic factor involved in the transcriptional program in T-ALL. ARID5B expression is down-regulated at the double-negative 2-4 stages in normal thymocytes, while it is induced by the TAL1 complex in human T-ALL cells. The enhancer located 135 kb upstream of the ARID5B gene locus is activated under a superenhancer in T-ALL cells but not in normal T cells. Notably, ARID5B-bound regions are associated predominantly with active transcription. ARID5B and TAL1 frequently co-occupy target genes and coordinately control their expression. ARID5B positively regulates the expression of TAL1 and its regulatory partners. ARID5B also activates the expression of the oncogene MYC Importantly, ARID5B is required for the survival and growth of T-ALL cells, and forced expression of ARID5B in immature thymocytes results in thymus retention, differentiation arrest, radioresistance, and tumor formation in zebrafish. Our results indicate that ARID5B reinforces the oncogenic transcriptional program by positively regulating the TAL1-induced regulatory circuit and MYC in T-ALL, thereby contributing to T-cell leukemogenesis.

Low-Level BCR::ABL1 Transcript at Diagnosis in Childhood Leukemia: A 10-Year Single Institution Study.

INTRODUCTION: Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL) is a high risk form of ALL associated with dismal outcomes in the pre-tyrosine kinase inhibitor (TKI) era. Addition of a TKI to chemotherapy improves outcomes. Therefore, testing for the presence of the Philadelphia chromosome by at least two methods at the time of diagnosis is critical. Diagnostic testing may include karyotype, fluorescent in situ hybridisation (FISH), and RT-PCR for the BCR::ABL1 transcript. The significance of low-level BCR::ABL1 transcript by RT-PCR in the absence of the Philadelphia chromosome on karyotype or by FISH is unknown. METHODS: This is a retrospective review of children diagnosed with acute leukemia at our institution from 2010 to 2020. Those positive for the BCR::ABL1 transcript by qualitative RT-PCR, and negative for t(9;22) by karyotype or FISH were analyzed for demographics, cytogenetic and molecular features at diagnosis and relapse, treatment and outcomes. The Kaplan-Meier method was used to estimate event-free and overall survival. RESULTS: Forty-seven of 306 (15%) patients with Ph- ALL had low-level BCR::ABL1 detected by RT-PCR. Most (77%) had B-cell ALL. The e1a2 transcript was detected most frequently, in 43 (91%) patients. BCR::ABL1 was quantifiable in 12/43 (28%) patients, with a median of 0.0008% (range 0.0003-0.095%). Seven patients (15%) relapsed. No patient with low-level BCR::ABL1 at diagnosis developed Ph + ALL at relapse. There was no difference in 5-year event-free (77% versus 81%, p = 0.407) or overall survival (86% versus 91%, p = 0.3) between children with low-level BCR::ABL1 (n = 47) and those without (n = 259). CONCLUSION: BCR::ABL1 low-level positivity in children with newly diagnosed Ph- ALL is a relatively common finding and did not adversely affect outcome for patients treated using a contemporary risk-adapted approach.

The impact of an additional copy of chromosome 21 in B-cell precursor acute lymphoblastic leukemia.

A common finding in pediatric B-cell precursor acute lymphoblastic leukemia (BCPALL) is that chromosome 21 is never lost and an extra chromosome 21 is often gained. This implies an important role for chromosome 21 in the pathobiology of BCPALL, emphasized by the increased risk of BCPALL in children with Down syndrome. However, model systems of chromosome 21 gain are lacking. We therefore developed a BCPALL cell line (Nalm-6, DUX4-rearranged) with an additional chromosome 21 by means of microcell-mediated chromosome transfer. FISH, PCR, multiplex ligation-dependent probe amplification, and whole exome sequencing showed that an additional chromosome 21 was successfully transferred to the recipient cells. Transcription of some but not all genes on chromosome 21 was increased, indicating tight transcriptional regulation. Nalm-6 cells with an additional chromosome 21 proliferated slightly slower compared with parental Nalm-6 and sensitivity to induction chemotherapeutics was mildly increased. The extra copy of chromosome 21 did not confer sensitivity to targeted signaling inhibitors. In conclusion, a BCPALL cell line with an additional human chromosome 21 was developed, validated, and subjected to functional studies, which showed a minor but potentially relevant effect in vitro. This cell line offers the possibility to study further the role of chromosome 21 in ALL.

Constitutional and acquired genetic variants in ARID5B in pediatric B-cell precursor acute lymphoblastic leukemia.

Constitutional polymorphisms in ARID5B are associated with an increased risk of developing high hyperdiploid (HeH; 51-67 chromosomes) pediatric B-cell precursor acute lymphoblastic leukemia (BCP ALL). Here, we investigated constitutional and somatic ARID5B variants in 1335 BCP ALL cases from five different cohorts, with a particular focus on HeH cases. In 353 HeH ALL that were heterozygous for risk alleles and trisomic for chromosome 10, where ARID5B is located, a significantly higher proportion of risk allele duplication was seen for the SNPs rs7090445 (p = 0.009), rs7089424 (p = 0.005), rs7073837 (p = 0.03), and rs10740055 (p = 0.04). Somatic ARID5B deletions were seen in 16/1335 cases (1.2%), being more common in HeH than in other genetic subtypes (2.2% vs. 0.4%; p = 0.002). The expression of ARID5B in HeH cases with genomic deletions was reduced, consistent with a functional role in leukemogenesis. Whole-genome sequencing and RNA-sequencing in HeH revealed additional somatic events involving ARID5B, resulting in a total frequency of 3.6% of HeH cases displaying a somatic ARID5B aberration. Overall, our results show that both constitutional and somatic events in ARID5B are involved in the leukemogenesis of pediatric BCP ALL, particularly in the HeH subtype.

Anti-tumor activity of niclosamide-mediated oxidative stress against acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) is a heterogeneous clonal disease originated from B- or T-cell lymphoid precursor cells. ALL is often refractory or relapses after treatment. Novel treatments are anxiously needed in order to achieve a better response and prolonged overall survival in ALL patients. In the present study, we aimed at examining the anti-tumor effect of niclosamide on ALL. We investigated the effects of niclosamide on the proliferation and apoptosis in vitro, the growth of ALL cells in xenografted NCG mice. The results showed that niclosamide treatment potently inhibited the growth of ALL cells and induced apoptosis via elevating the levels of reactive oxygen species (ROS) and activating TP53. These findings suggest that niclosamide may be a promisingly potential agent for ALL therapy.

Molecular characterization and biomarker identification in paediatric B-cell acute lymphoblastic leukaemia.

B-cell acute lymphoblastic leukaemia (B-ALL) is the most prevalent hematologic malignancy in children and a leading cause of mortality. Managing B-ALL remains challenging due to its heterogeneity and relapse risk. This study aimed to delineate the molecular features of paediatric B-ALL and explore the clinical utility of circulating tumour DNA (ctDNA). We analysed 146 patients with paediatric B-ALL who received systemic chemotherapy. The mutational landscape was profiled in bone marrow (BM) and plasma samples using next-generation sequencing. Minimal residual disease (MRD) testing on day 19 of induction therapy evaluated treatment efficacy. RNA sequencing identified gene fusions in 61% of patients, including 37 novel fusions. Specifically, the KMT2A-TRIM29 novel fusion was validated in a boy who responded well to initial therapy but relapsed after 1 year. Elevated mutation counts and maximum variant allele frequency in baseline BM were associated with significantly poorer chemotherapy response (p = 0.0012 and 0.028, respectively). MRD-negative patients exhibited upregulation of immune-related pathways (p < 0.01) and increased CD8+ T cell infiltration (p = 0.047). Baseline plasma ctDNA exhibited high mutational concordance with the paired BM samples and was significantly associated with chemotherapy efficacy. These findings suggest that ctDNA and BM profiling offer promising prognostic insights for paediatric B-ALL management.

The novel TERF2::PDGFRB fusion gene enhances tumorigenesis via PDGFRB/STAT5 signalling pathways and sensitivity to TKI in ph-like ALL.

Patients with Philadelphia chromosome-like acute lymphoblastic leukaemia (Ph-like ALL) often face a grim prognosis, with PDGFRB gene fusions being commonly detected in this subgroup. Our study has unveiled a newfound fusion gene, TERF2::PDGFRB, and we have found that patients carrying this fusion gene exhibit sensitivity to dasatinib. Ba/F3 cells harbouring the TERF2::PDGFRB fusion display IL-3-independent cell proliferation through activation of the p-PDGFRB and p-STAT5 signalling pathways. These cells exhibit reduced apoptosis and demonstrate sensitivity to imatinib in vitro. When transfused into mice, Ba/F3 cells with the TERF2::PDGFRB fusion gene induce tumorigenesis and a shortened lifespan in cell-derived graft models, but this outcome can be improved with imatinib treatment. In summary, we have identified the novel TERF2::PDGFRB fusion gene, which exhibits oncogenic potential both in vitro and in vivo, making it a potential therapeutic target for tyrosine kinase inhibitors (TKIs).

Oculocerebrorenal syndrome of Lowe (OCRL) controls leukemic T-cell survival by preventing excessive PI(4,5)P2 hydrolysis in the plasma membrane.

T-cell acute lymphoblastic leukemia (T-ALL) is one of the deadliest and most aggressive hematological malignancies, but its pathological mechanism in controlling cell survival is not fully understood. Oculocerebrorenal syndrome of Lowe is a rare X-linked recessive disorder characterized by cataracts, intellectual disability, and proteinuria. This disease has been shown to be caused by mutation of oculocerebrorenal syndrome of Lowe 1 (OCRL1; OCRL), encoding a phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] 5-phosphatase involved in regulating membrane trafficking; however, its function in cancer cells is unclear. Here, we uncovered that OCRL1 is overexpressed in T-ALL cells, and knockdown of OCRL1 results in cell death, indicating the essential role of OCRL in controlling T-ALL cell survival. We show OCRL is primarily localized in the Golgi and can translocate to plasma membrane (PM) upon ligand stimulation. We found OCRL interacts with oxysterol-binding protein-related protein 4L, which facilitates OCRL translocation from the Golgi to the PM upon cluster of differentiation 3 stimulation. Thus, OCRL represses the activity of oxysterol-binding protein-related protein 4L to prevent excessive PI(4,5)P2 hydrolysis by phosphoinositide phospholipase C ß3 and uncontrolled Ca2+ release from the endoplasmic reticulum. We propose OCRL1 deletion leads to accumulation of PI(4,5)P2 in the PM, disrupting the normal Ca2+ oscillation pattern in the cytosol and leading to mitochondrial Ca2+ overloading, ultimately causing T-ALL cell mitochondrial dysfunction and cell death. These results highlight a critical role for OCRL in maintaining moderate PI(4,5)P2 availability in T-ALL cells. Our findings also raise the possibility of targeting OCRL1 to treat T-ALL disease.

Distinct pattern of genomic breakpoints in CML and BCR::ABL1-positive ALL: analysis of 971 patients.

BACKGROUND: The BCR::ABL1 is a hallmark of chronic myeloid leukemia (CML) and is also found in acute lymphoblastic leukemia (ALL). Most genomic breaks on the BCR side occur in two regions - Major and minor - leading to p210 and p190 fusion proteins, respectively. METHODS: By multiplex long-distance PCR or next-generation sequencing technology we characterized the BCR::ABL1 genomic fusion in 971 patients (adults and children, with CML and ALL: pediatric ALL: n = 353; pediatric CML: n = 197; adult ALL: n = 166; adult CML: n = 255 patients) and designed "Break-App" web tool to allow visualization and various analyses of the breakpoints. Pearson's Chi-Squared test, Kolmogorov-Smirnov test and logistic regression were used for statistical analyses. RESULTS: Detailed analysis showed a non-random distribution of breaks in both BCR regions, whereas ABL1 breaks were distributed more evenly. However, we found a significant difference in the distribution of breaks between CML and ALL. We found no association of breakpoints with any type of interspersed repeats or DNA motifs. With a few exceptions, the primary structure of the fusions suggests non-homologous end joining being responsible for the BCR and ABL1 gene fusions. Analysis of reciprocal ABL1::BCR fusions in 453 patients showed mostly balanced translocations without major deletions or duplications. CONCLUSIONS: Taken together, our data suggest that physical colocalization and chromatin accessibility, which change with the developmental stage of the cell (hence the difference between ALL and CML), are more critical factors influencing breakpoint localization than presence of specific DNA motifs.

Choanephora infundibulifera Rhinosinusitis in Man with Acute Lymphoblastic Leukemia, Tennessee, USA.

Choanephora infundibulifera is a member of the Mucorales order of fungi. The species is associated with plants as a saprophyte or parasite and may be responsible for spoilage or disease but is an uncommon cause of human infection. We describe C. infundibulifera rhinosinusitis in a young man with leukemia in Tennessee, USA.