Immune stress suppresses innate immune signaling in preleukemic precursor B-cells to provoke leukemia in predisposed mice.

The initial steps of B-cell acute lymphoblastic leukemia (B-ALL) development usually pass unnoticed in children. Several preclinical studies have shown that exposure to immune stressors triggers the transformation of preleukemic B cells to full-blown B-ALL, but how this takes place is still a longstanding and unsolved challenge. Here we show that dysregulation of innate immunity plays a driving role in the clonal evolution of pre-malignant Pax5+/- B-cell precursors toward leukemia. Transcriptional profiling reveals that Myd88 is downregulated in immune-stressed pre-malignant B-cell precursors and in leukemic cells. Genetic reduction of Myd88 expression leads to a significant increase in leukemia incidence in Pax5+/-Myd88+/- mice through an inflammation-dependent mechanism. Early induction of Myd88-independent Toll-like receptor 3 signaling results in a significant delay of leukemia development in Pax5+/- mice. Altogether, these findings identify a role for innate immunity dysregulation in leukemia, with important implications for understanding and therapeutic targeting of the preleukemic state in children.

Extranodal natural killer/T-cell lymphoma nasal type in a western population: Molecular profiling identifies new therapeutic targets.

(A) Correlation matrix of unsupervised co-regulated genes, based on the 208 genes included in the NanoString platform. Some of the clusters of co-regulated genes corresponded to the following: Inflammatory cells; Epstein-Barr virus; B-cells; Cytotoxic T-cells; T-cells; and Proliferation. (B) Analysis of genomic alterations by targeted sequencing. Distribution of mutations in the 62 analyzed genes. Rows correspond to sequenced genes, columns represent individual patients. Color coding: green, missense; blue, synonymous; pink, frameshift; violet, Indel; red, stop gained; yellow, UTR.

The Terminal Extensions of Dbp7 Influence Growth and 60S Ribosomal Subunit Biogenesis in Saccharomyces cerevisiae.

Ribosome synthesis is a complex process that involves a large set of protein trans-acting factors, among them DEx(D/H)-box helicases. These are enzymes that carry out remodelling activities onto RNAs by hydrolysing ATP. The nucleolar DEGD-box protein Dbp7 is required for the biogenesis of large 60S ribosomal subunits. Recently, we have shown that Dbp7 is an RNA helicase that regulates the dynamic base-pairing between the snR190 small nucleolar RNA and the precursors of the ribosomal RNA within early pre-60S ribosomal particles. As the rest of DEx(D/H)-box proteins, Dbp7 has a modular organization formed by a helicase core region, which contains conserved motifs, and variable, non-conserved N- and C-terminal extensions. The role of these extensions remains unknown. Herein, we show that the N-terminal domain of Dbp7 is necessary for efficient nuclear import of the protein. Indeed, a basic bipartite nuclear localization signal (NLS) could be identified in its N-terminal domain. Removal of this putative NLS impairs, but does not abolish, Dbp7 nuclear import. Both N- and C-terminal domains are required for normal growth and 60S ribosomal subunit synthesis. Furthermore, we have studied the role of these domains in the association of Dbp7 with pre-ribosomal particles. Altogether, our results show that the N- and C-terminal domains of Dbp7 are important for the optimal function of this protein during ribosome biogenesis.

Venetoclax improves CD20 immunotherapy in a mouse model of MYC/BCL2 double-expressor diffuse large B-cell lymphoma.

BACKGROUND: Approximately one-third of diffuse large B cell lymphoma (DLBCL) patients exhibit co-expression of MYC and BCL2 (double-expressor lymphoma, DEL) and have a dismal prognosis. Targeted inhibition of the anti-apoptotic protein BCL2 with venetoclax (ABT-199) has been approved in multiple B-cell malignancies and is currently being investigated in clinical trials for DLBCL. Whether BCL2 anti-apoptotic function represents a multifaceted vulnerability for DEL-DLBCL, affecting both lymphoma B cells and T cells within the tumor microenvironment, remains to be elucidated. METHODS: Here, we present novel genetically engineered mice that preclinically recapitulate DEL-DLBCL lymphomagenesis, and evaluate their sensitivity ex vivo and in vivo to the promising combination of venetoclax with anti-CD20-based standard immunotherapy. RESULTS: Venetoclax treatment demonstrated specific killing of MYC+/BCL2+ lymphoma cells by licensing their intrinsically primed apoptosis, and showed previously unrecognized immunomodulatory activity by specifically enriching antigen-activated effector CD8 T cells infiltrating the tumors. Whereas DEL-DLBCL mice were refractory to venetoclax alone, inhibition of BCL2 significantly extended overall survival of mice that were simultaneously treated with a murine surrogate for anti-CD20 rituximab. CONCLUSIONS: These results suggest that the combination of anti-CD20-based immunotherapy and BCL2 inhibition leads to cooperative immunomodulatory effects and improved preclinical responses, which may offer promising therapeutic opportunities for DEL-DLBCL patients.

Ectonucleoside triphosphate diphosphohydrolase-1 (CD39) impacts TGF-ß1 responses: insights into cardiac fibrosis and function following myocardial infarction.

Extracellular purine nucleotides and nucleosides released from activated or injured cells influence multiple aspects of cardiac physiology and pathophysiology. Ectonucleoside triphosphate diphosphohydrolase-1 (ENTPD1; CD39) hydrolyzes released nucleotides and thereby regulates the magnitude and duration of purinergic signaling. However, the impact of CD39 activity on post-myocardial infarction (MI) remodeling is incompletely understood. We measured the levels and activity of ectonucleotidases in human left ventricular samples from control and ischemic cardiomyopathy (ICM) hearts and examined the impact of ablation of Cd39 expression on post-myocardial infarction remodeling in mice. We found that human CD39 levels and activity are significantly decreased in ICM hearts (n = 5) compared with control hearts (n = 5). In mice null for Cd39, cardiac function and remodeling are significantly compromised in Cd39-/- mice following myocardial infarction. Fibrotic markers including plasminogen activator inhibitor-1 (PAI-1) expression, fibrin deposition, α-smooth muscle actin (αSMA), and collagen expression are increased in Cd39-/- hearts. Importantly, we found that transforming growth factor ß1 (TGF-ß1) stimulates ATP release and induces Cd39 expression and activity on cardiac fibroblasts, constituting an autocrine regulatory pathway not previously appreciated. Absence of CD39 activity on cardiac fibroblasts exacerbates TGF-ß1 profibrotic responses. Treatment with exogenous ectonucleotidase rescues this profibrotic response in Cd39-/- fibroblasts. Together, these data demonstrate that CD39 has important interactions with TGF-ß1-stimulated autocrine purinergic signaling in cardiac fibroblasts and dictates outcomes of cardiac remodeling following myocardial infarction. Our results reveal that ENTPD1 (CD39) regulates TGF-ß1-mediated fibroblast activation and limits adverse cardiac remodeling following myocardial infarction.NEW & NOTEWORTHY We show that CD39 is a critical modulator of TGF-ß1-mediated fibroblast activation and cardiac remodeling following myocardial infarction via modulation of nucleotide signaling. TGF-ß1-induced CD39 expression generates a negative feedback loop that attenuates cardiac fibroblast activation. In the absence of CD39 activity, collagen deposition is increased, elastin expression is decreased, and diastolic dysfunction is worsened. Treatment with ecto-apyrase attenuates the TGF-ß1-induced profibrotic cardiac fibroblast phenotype, revealing a novel approach to combat post-myocardial infarction cardiac fibrosis.

Robotic Aortic Valve Replacement Using the Intuity Bioprosthesis Through a Right Lateral Approach.

Robotic technology offers excellent visualization and surgical precision but has not been established for aortic valve surgery. We present the first 2 cases of patients with severe aortic stenosis who underwent successful robotic aortic valve replacement using a lateral approach and the Edwards Intuity® rapid-deployment bioprosthesis. Postoperative recovery was excellent, and both patients were discharged home on the fourth postoperative day. We believe this approach is valuable for robotic aortic valve replacement and may help to finally bridge the gap into routine clinical use, becoming a valuable option for the surgical treatment of the aortic valve in selected patients.

Recurrent NOMO1 Gene Deletion Is a Potential Clinical Marker in Early-Onset Colorectal Cancer and Is Involved in the Regulation of Cell Migration.

The incidence of early-onset colorectal cancer (EOCRC; age younger than 50 years) has been progressively increasing over the last decades globally, with causes unexplained. A distinct molecular feature of EOCRC is that compared with cases of late-onset colorectal cancer, in EOCRC cases, there is a higher incidence of Nodal Modulator 1 (NOMO1) somatic deletions. However, the mechanisms of NOMO1 in early-onset colorectal carcinogenesis are currently unknown. In this study, we show that in 30% of EOCRCs with heterozygous deletion of NOMO1, there were pathogenic mutations in this gene, suggesting that NOMO1 can be inactivated by deletion or mutation in EOCRC. To study the role of NOMO1 in EOCRC, CRISPR/cas9 technology was employed to generate NOMO1 knockout HCT-116 (EOCRC) and HS-5 (bone marrow) cell lines. NOMO1 loss in these cell lines did not perturb Nodal pathway signaling nor cell proliferation. Expression microarrays, RNA sequencing, and protein expression analysis by LC-IMS/MS showed that NOMO1 inactivation deregulates other signaling pathways independent of the Nodal pathway, such as epithelial-mesenchymal transition and cell migration. Significantly, NOMO1 loss increased the migration capacity of CRC cells. Additionally, a gut-specific conditional NOMO1 KO mouse model revealed no subsequent tumor development in mice. Overall, these findings suggest that NOMO1 could play a secondary role in early-onset colorectal carcinogenesis because its loss increases the migration capacity of CRC cells. Therefore, further study is warranted to explore other signalling pathways deregulated by NOMO1 loss that may play a significant role in the pathogenesis of the disease.

Estrogen Receptor-ß Agonists Modulate T-Lymphocyte Activation and Ameliorate Left Ventricular Remodeling During Chronic Heart Failure.

BACKGROUND: CD4+ T cells temporally transition from protective to pathological during ischemic heart failure (HF; 8 weeks postmyocardial infarction). Cellular mechanisms mediating this shift are unknown. METHODS: RNA-sequencing of cardiac CD4+ T cells and flow cytometric analysis of immune cells was conducted. RESULTS: RNA-sequencing of CD4+ T cells from the failing hearts of male mice indicated activation of ER (estrogen receptor)-α signaling. Flow cytometric analysis showed that ERα in CD4+ T cells decreases significantly at 3-day postmyocardial infarction but increases during HF. To antagonize ERα, we tested a novel ERß agonist (OSU-ERb-012) to inhibit T cells and blunt left ventricular remodeling. Proliferation assays showed that OSU-ERb-012 dose-dependently inhibited proliferation and proinflammatory cytokine expression in anti-CD3/CD28 stimulated splenic T cells isolated from both the sexes. For in vivo efficacy, 10- to 12-week-old male and ovariectomized female mice were randomized at 4 weeks postmyocardial infarction and treated with either vehicle or drug (60 mg/kg per day; oral). While vehicle-treated HF mice displayed progressive left ventricular dilatation with significantly increased end-systolic and end-diastolic volumes from 4 to 8 weeks postmyocardial infarction, treatment with OSU-ERb-012 significantly blunted these changes and stopped left ventricular remodeling in both the sexes. Reduction in tibia-normalized heart and left ventricular weights, cardiomyocyte hypertrophy and interstitial fibrosis further supported these results. Additionally, OSU-ERb-012 treatment selectively inhibited cardiac, splenic, and circulating CD4+ T cells without affecting other myeloid and lymphoid cells in the HF mice. CONCLUSIONS: Our studies indicate that ERß agonists and OSU-ERb-012, in particular, could be used as selective immunomodulatory drugs to inhibit CD4+ T cells during chronic HF.

Transient Inhibition of the JAK/STAT Pathway Prevents B-ALL Development in Genetically Predisposed Mice.

Preventing development of childhood B-cell acute lymphoblastic leukemia (B-ALL), a disease with devastating effects, is a longstanding and unsolved challenge. Heterozygous germline alterations in the PAX5 gene can lead to B-ALL upon accumulation of secondary mutations affecting the JAK/STAT signaling pathway. Preclinical studies have shown that this malignant transformation occurs only under immune stress such as exposure to infectious pathogens. Here we show in Pax5+/- mice that transient, early-life administration of clinically relevant doses of ruxolitinib, a JAK1/2 inhibitor, significantly mitigates the risk of B-ALL following exposure to infection; 1 of 29 animals treated with ruxolitinib developed B-ALL versus 8 of 34 untreated mice. Ruxolitinib treatment preferentially targeted Pax5+/- versus wild-type B-cell progenitors and exerted unique effects on the Pax5+/- B-cell progenitor transcriptional program. These findings provide the first in vivo evidence for a potential strategy to prevent B-ALL development. SIGNIFICANCE: JAK/STAT inhibition suppresses tumorigenesis in a B-ALL-susceptible mouse model, presenting a novel approach to prevent B-ALL onset.

BCL2 translocation in high grade B cell lymphoma (NOS, DH/TH) is associated with reduced progression free survival.

High Grade B Cell Lymphoma, NOS, and High Grade B Cell Lymphoma with Dual Hit or Triple Hit have been recently recategorized in the 2016 revision of the WHO classification of lymphoid neoplasms. In this study we have characterized the genetic, histopathological, and clinical features of a series of this type of lymphoid neoplasia (17 HGBCL NOS and 53 HGBCL DH/TH).HGBCL NOS showed better response to first line treatment than HGBCL with DH/TH but no significant differences in PFS or OS were found between the two categories. Survival analysis in the whole cohort of cases found that only the presence of BCL2 translocation was significantly associated with PFS. Other clinical features such as IPI, LDH or stage were equivalent in both categories. Furthermore, both high grade and DLBCL morphological patterns showed equivalent PFS and OS in this set of High grade BCL NOS/DH/TH.Key pointsBCL2 translocation in High Grade B Cell Lymphoma NOS and High Grade B Cell Lymphoma with DH/TH is associated with reduced progression free survival.Both high grade and DLBCL morphological patterns showed equivalent outcome regarding PFS and OS in HGBCL.

The Second Oncogenic Hit Determines the Cell Fate of ETV6-RUNX1 Positive Leukemia.

ETV6-RUNX1 is almost exclusively associated with childhood B-cell acute lymphoblastic leukemia (B-ALL), but the consequences of ETV6-RUNX1 expression on cell lineage decisions during B-cell leukemogenesis are completely unknown. Clinically silent ETV6-RUNX1 preleukemic clones are frequently found in neonatal cord blood, but few carriers develop B-ALL as a result of secondary genetic alterations. The understanding of the mechanisms underlying the first transforming steps could greatly advance the development of non-toxic prophylactic interventions. Using genetic lineage tracing, we examined the capacity of ETV6-RUNX1 to instruct a malignant phenotype in the hematopoietic lineage by cell-specific Cre-mediated activation of ETV6-RUNX1 from the endogenous Etv6 gene locus. Here we show that, while ETV6-RUNX1 has the propensity to trigger both T- and B-lymphoid malignancies, it is the second hit that determines tumor cell identity. To instigate leukemia, both oncogenic hits must place early in the development of hematopoietic/precursor cells, not in already committed B-cells. Depending on the nature of the second hit, the resulting B-ALLs presented distinct entities that were clearly separable based on their gene expression profiles. Our findings give a novel mechanistic insight into the early steps of ETV6-RUNX1+ B-ALL development and might have major implications for the potential development of ETV6-RUNX1+ B-ALL prevention strategies.

Liquid biopsy: a non-invasive approach for Hodgkin lymphoma genotyping.

The Hodgkin lymphoma (HL) genomic landscape is hardly known due to the scarcity of tumour cells in the tissue. Liquid biopsy employing circulating tumour DNA (ctDNA) can emerge as an alternative tool for non-invasive genotyping. By using a custom next generation sequencing (NGS) panel in combination with unique molecule identifiers, we aimed to identify somatic variants in the ctDNA of 60 HL at diagnosis. A total of 277 variants were detected in 36 of the 49 samples (73·5%) with a good quality ctDNA sample. The median number of variants detected per patient was five (range 1-23) with a median variant allele frequency of 4·2% (0·84-28%). Genotyping revealed somatic variants in the following genes: SOCS1 (28%), IGLL5 (26%), TNFAIP3 (23%), GNA13 (23%), STAT6 (21%) and B2M (19%). Moreover, several poor prognosis features (high LDH, low serum albumin, B-symptoms, IPI ≥ 3 or at an advanced stage) were related to significantly higher amounts of ctDNA. Variant detection in ctDNA by NGS is a feasible approach to depict the genetic features of HL patients at diagnosis. Our data favour the implementation of liquid biopsy genotyping for the routine evaluation of HL patients.

Tracking the Antibody Immunome in Sporadic Colorectal Cancer by Using Antigen Self-Assembled Protein Arrays.

Sporadic Colorectal Cancer (sCRC) is the third leading cause of cancer death in the Western world, and the sCRC patients presenting with synchronic metastasis have the poorest prognosis. Genetic alterations accumulated in sCRC tumor cells translate into mutated proteins and/or abnormal protein expression levels, which contribute to the development of sCRC. Then, the tumor-associated proteins (TAAs) might induce the production of auto-antibodies (aAb) via humoral immune response. Here, Nucleic Acid Programmable Protein Arrays (NAPPArray) are employed to identify aAb in plasma samples from a set of 50 sCRC patients compared to seven healthy donors. Our goal was to establish a systematic workflow based on NAPPArray to define differential aAb profiles between healthy individuals and sCRC patients as well as between non-metastatic (n = 38) and metastatic (n = 12) sCRC, in order to gain insight into the role of the humoral immune system in controlling the development and progression of sCRC. Our results showed aAb profile based on 141 TAA including TAAs associated with biological cellular processes altered in genesis and progress of sCRC (e.g., FSCN1, VTI2 and RPS28) that discriminated healthy donors vs. sCRC patients. In addition, the potential capacity of discrimination (between non-metastatic vs. metastatic sCRC) of 7 TAAs (USP5, ML4, MARCKSL1, CKMT1B, HMOX2, VTI2, TP53) have been analyzed individually in an independent cohort of sCRC patients, where two of them (VTI2 and TP53) were validated (AUC ~75%). In turn, these findings provided novel insights into the immunome of sCRC, in combination with transcriptomics profiles and protein antigenicity characterizations, wich might lead to the identification of novel sCRC biomarkers that might be of clinical utility for early diagnosis of the tumor. These results explore the immunomic analysis as potent source for biomarkers with diagnostic and prognostic value in CRC. Additional prospective studies in larger series of patients are required to confirm the clinical utility of these novel sCRC immunomic biomarkers.

Knowledge-Based Volumetric Modulated Arc Therapy Treatment Planning for Breast Cancer.

Purpose: To create and to validate knowledge-based volumetric modulated arc therapy (VMAT) models for breast cancer treatments without lymph node irradiation. Materials and Methods: One hundred VMAT-based breast plans (manual plans [MP]) were selected to create two knowledge-based VMAT models (breast left and breast right) using RapidPlan™. The plans were generated on Eclipse v15.5 (Varian Medical Systems, Palo Alto, CA) with 6 MV of a Novalis Tx equipped with a high-resolution multileaf collimator. The models were verified based on goodness-of-fit statistics using the coefficients of determination (R 2) and Chi-square (χ2), and the goodness-of-estimation statistics through the mean square error (MSE). Geometrical and dosimetrical constraints were identified and removed from the RP models using statistical evaluation metrics and plots. For validation, 20 plans that integrate the models and 20 plans that do not were reoptimized with RP (closed and opened validation). Dosimetrical parameters of interest were used to compare MP versus RP plans for the Heart, Homolateral_Lung, Contralateral_Lung, and Contralateral_Breast. Optimization planning time and user independency were also analyzed. Results: The most unfavorable results of R2 in both models for the organs at risk were as follows: for Contralateral_Lung 0.51 in RP right breast (RP_RB) and for Heart 0.60 in RP left breast (RP_LB). The most unfavorable results of χ2 test were: for Contralateral_Breast 1.02 in RP_RB and for Heart 1.03 in RP_LB. These goodness-of-fit results show that no overfitting occurred in either of the models. There were no unfavorable results of mean square error (MSE, all < 0.05) in any of the two models. These goodness-of-estimation results show that the models have good estimation power. For closed validation, significant differences were found in RP_RB for Homolateral_Lung (all P ≤ 0.001), and in the RP_LB differences were found for the heart (all P ≤ 0.04) and for Homolateral_Lung (all P ≤ 0.022). For open validation, no statistically significant differences were obtained in either of the models. RP models had little impact on reducing optimization planning times for expert planners; nevertheless, the result showed a 30% reduction time for beginner planners. The use of RP models generates high-quality plans, without differences from the planner experience. Conclusion: Two RP models for breast cancer treatment using VMAT were successfully implemented. The use of RP models for breast cancer reduces the optimization planning time and improves the efficiency of the treatment planning process while ensuring high-quality plans.

Conditional expression of HGAL leads to the development of diffuse large B-cell lymphoma in mice.

Diffuse large B-cell lymphomas (DLBCLs) are clinically and genetically heterogeneous tumors. Deregulation of diverse biological processes specific to B cells, such as B-cell receptor (BCR) signaling and motility regulation, contribute to lymphomagenesis. Human germinal center associated lymphoma (HGAL) is a B-cell-specific adaptor protein controlling BCR signaling and B lymphocyte motility. In normal B cells, it is expressed in germinal center (GC) B lymphocytes and promptly downregulated upon further differentiation. The majority of DLBCL tumors, primarily GC B-cell types, but also activated types, express HGAL. To investigate the consequences of constitutive expression of HGAL in vivo, we generated mice that conditionally express human HGAL at different stages of hematopoietic development using 3 restricted Cre-mediated approaches to initiate expression of HGAL in hematopoietic stem cells, pro-B cells, or GC B cells. Following immune stimulation, we observed larger GCs in mice in which HGAL expression was initiated in GC B cells. All 3 mouse strains developed DLBCL at a frequency of 12% to 30% starting at age 13 months, leading to shorter survival. Immunohistochemical studies showed that all analyzed tumors were of the GC B-cell type. Exon sequencing revealed mutations reported in human DLBCL. Our data demonstrate that constitutive enforced expression of HGAL leads to DLBCL development.

The L-α-Lysophosphatidylinositol/G Protein-Coupled Receptor 55 System Induces the Development of Nonalcoholic Steatosis and Steatohepatitis.

BACKGROUND AND AIMS: G protein-coupled receptor (GPR) 55 is a putative cannabinoid receptor, and l-α-lysophosphatidylinositol (LPI) is its only known endogenous ligand. Although GPR55 has been linked to energy homeostasis in different organs, its specific role in lipid metabolism in the liver and its contribution to the pathophysiology of nonalcoholic fatty liver disease (NAFLD) remains unknown. APPROACH AND RESULTS: We measured (1) GPR55 expression in the liver of patients with NAFLD compared with individuals without obesity and without liver disease, as well as animal models with steatosis and nonalcoholic steatohepatitis (NASH), and (2) the effects of LPI and genetic disruption of GPR55 in mice, human hepatocytes, and human hepatic stellate cells. Notably, we found that circulating LPI and liver expression of GPR55 were up-regulated in patients with NASH. LPI induced adenosine monophosphate-activated protein kinase activation of acetyl-coenzyme A carboxylase (ACC) and increased lipid content in human hepatocytes and in the liver of treated mice by inducing de novo lipogenesis and decreasing ß-oxidation. The inhibition of GPR55 and ACCα blocked the effects of LPI, and the in vivo knockdown of GPR55 was sufficient to improve liver damage in mice fed a high-fat diet and in mice fed a methionine-choline-deficient diet. Finally, LPI promoted the initiation of hepatic stellate cell activation by stimulating GPR55 and activation of ACC. CONCLUSIONS: The LPI/GPR55 system plays a role in the development of NAFLD and NASH by activating ACC.

Inhibition of inflammatory signaling in Pax5 mutant cells mitigates B-cell leukemogenesis.

PAX5 is one of the most frequently mutated genes in B-cell acute lymphoblastic leukemia (B-ALL), and children with inherited preleukemic PAX5 mutations are at a higher risk of developing the disease. Abnormal profiles of inflammatory markers have been detected in neonatal blood spot samples of children who later developed B-ALL. However, how inflammatory signals contribute to B-ALL development is unclear. Here, we demonstrate that Pax5 heterozygosis, in the presence of infections, results in the enhanced production of the inflammatory cytokine interleukin-6 (IL-6), which appears to act in an autocrine fashion to promote leukemia growth. Furthermore, in vivo genetic downregulation of IL-6 in these Pax5 heterozygous mice retards B-cell leukemogenesis, and in vivo pharmacologic inhibition of IL-6 with a neutralizing antibody in Pax5 mutant mice with B-ALL clears leukemic cells. Additionally, this novel IL-6 signaling paradigm identified in mice was also substantiated in humans. Altogether, our studies establish aberrant IL6 expression caused by Pax5 loss as a hallmark of Pax5-dependent B-ALL and the IL6 as a therapeutic vulnerability for B-ALL characterized by PAX5 loss.

Biological Features and Prognostic Impact of Bone Marrow Infiltration in Patients with Diffuse Large B-Cell Lymphoma.

The biology and clinical impact of bone marrow (BM) infiltration in patients with diffuse large B-cell lymphoma (DLBCL) remains unclear in the rituximab era. We retrospectively analyzed 232 patients diagnosed with DLBCL at our center between 1999 and 2014. Concordant-presence of large cells similar to those of the lymph node biopsy- and discordant-infiltration by small cells forming lymphoid aggregates, lacking cytological atypia-BM infiltration was defined by histological criteria and further characterized by flow cytometry (FCM). Cell of origin (COO) was determined using Hans' algorithm. For the clonal relationship between tumor and discordant BM, the VDJH rearrangement was analyzed. Survival analyses were restricted to 189 patients treated with rituximab and chemotherapy. Thirty-six (16%) had concordant, and 37 (16%) discordant BM infiltration. FCM described different indolent lymphomas among discordant cases, clonally related with DLBCL in 10/13 available samples. Median follow-up was 58 months. 5-year-progression-free survival (PFS) for non-infiltrated, discordant and concordant groups was 68%, 65% and 30%, respectively (p < 0.001). Combining COO and BM infiltration, patients with discordant BM and non-germinal center B-cell COO also had decreased 5-year-PFS (41.9%). In multivariate analysis, concordant BM had an independent effect on PFS (HR 2.5, p = 0.01). Five-year cumulative incidence of central nervous system (CNS) relapse was 21%, 4% and 1% in concordant, discordant and non-infiltrated groups, respectively (p < 0.001). In conclusion, concordant BM infiltration represents a subset with poor prognosis, whereas the prognostic impact of discordant BM infiltration could be limited to non-CGB cases.

Infectious stimuli promote malignant B-cell acute lymphoblastic leukemia in the absence of AID.

The prerequisite to prevent childhood B-cell acute lymphoblastic leukemia (B-ALL) is to decipher its etiology. The current model suggests that infection triggers B-ALL development through induction of activation-induced cytidine deaminase (AID; also known as AICDA) in precursor B-cells. This evidence has been largely acquired through the use of ex vivo functional studies. However, whether this mechanism governs native non-transplant B-ALL development is unknown. Here we show that, surprisingly, AID genetic deletion does not affect B-ALL development in Pax5-haploinsufficient mice prone to B-ALL upon natural infection exposure. We next test the effect of premature AID expression from earliest pro-B-cell stages in B-cell transformation. The generation of AID off-target mutagenic activity in precursor B-cells does not promote B-ALL. Likewise, known drivers of human B-ALL are not preferentially targeted by AID. Overall these results suggest that infections promote B-ALL through AID-independent mechanisms, providing evidence for a new model of childhood B-ALL development.