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Summary The potential prognostic influence of genetic aberrations on chronic lymphocytic leukaemia (CLL) can vary based on various factors, such as the immunoglobulin heavy variable (IGHV) status. We conducted an integrative analysis on genetic abnormalities identified through cytogenetics and targeted next‐generation sequencing in 536 CLL patients receiving first‐line chemo(immuno)therapies (CIT) as part of two prospective trials. We evaluated the prognostic implications of the main abnormalities, with specific attention to their relative impact according to IGHV status. In the entire cohort, unmutated (UM)‐IGHV, complex karyotype, del(11q) and ATM mutations correlated significantly with shorter progression‐free survival (PFS). Focusing on the subset of mutated IGHV (M‐IGHV) patients, univariate analysis showed that complex karyotype, del(11q), SF3B1 and SAMHD1 mutations were associated with significant lower PFS. The prognostic influence varied based on the patient's IGHV status, as these abnormalities did not affect outcomes in the UM‐IGHV subgroup. TP53 mutations had no significant impact on outcomes in the M‐IGHV subgroup. Our findings highlight the diverse prognostic influence of genetic aberrations depending on the IGHV status in symptomatic CLL patients receiving first‐line CIT. The prognosis of gene mutations and cytogenetic abnormalities needs to be investigated with a compartmentalized methodology, taking into account the IGVH status of patients receiving first‐line BTK and/or BCL2 inhibitors. [ABSTRACT FROM AUTHOR]

Chromosomal abnormalities are frequent in chronic lymphocytic leukemia (CLL), and most have prognostic value. In addition to the four well-known abnormalities (13q, 11q and 17p deletions, and trisomy 12), other recurrent aberrations have been linked to the disease outcome and/or drug resistance. Moreover, the complex karyotype has recently emerged as a prognostic marker for patients undergoing immunochemotherapy or targeted therapies. Here, we describe the main chromosomal abnormalities identified in CLL and related disorders (small lymphocytic lymphoma and monoclonal B-cell lymphocytosis) by reviewing the most recent literature and discussing their detection and clinical impact. Lastly, we provide technical guidelines and a strategy for the cytogenetic assessment of CLL. [ABSTRACT FROM AUTHOR]

Massive genomic analyses have underscored the diversity of chronic lymphocytic leukaemia ( CLL) between patients. Genetic heterogeneity of tumour clones within a patient may fuel tumour evolution. Several recurrently deregulated intra-cellular pathways are candidates for targeted therapies that are very promising and are dramatically changing clinical patients' perspectives. In this review we present an overview of the genetic and epigenetic features of CLL and their clinical and biological implications. [ABSTRACT FROM AUTHOR]

Abstract: Burkitt-type acute leukemia cells were present in the bone marrow of a patient with B-prolymphocytic leukemia diagnosed from peripheral blood cell morphology. Immunophenotype analysis confirmed morphological patterns. Cytogenetic and fluorescence in situ hybridization (FISH) analysis showed an identical t(8;22)(q24;q21) with MYC locus rearrangement in blood and bone marrow cells, with additional chromosome abnormalities in the bone marrow. In addition, the loss of one copy of the TP53 gene and identical IGH DNA clonal rearrangements were shown with FISH and polymerase chain reaction analysis respectively in the two types of leukemic cells. These data indicated the common origin of the two coexisting leukemias and are the first example of such occurrence in a leukemic patient. [Copyright &y& Elsevier]

We describe a patient affected by a T-cell primary central nervous system lymphoma (PCNSL) with highly aberrant specific B-cell markers (CD79a and CD20). An unusual imaging presentation leads us to misdiagnose this lesion for a meningioma and perform surgical resection. We think that this infrequent anatomic presentation might be due to the aberrant specific B-cell markers (CD79a and CD20) genotype expression. We believe this case to be relevant in order to appreciate the diagnosis of cerebral lymphomas according to various presentations. We wonder whether it was not the aberrant genotype that contributed to this quirky presentation and ultimately if surgery in PCNSL should not be discussed? Furthermore, this case calls attention to the complexity of lineage assignment, imaging diagnosis, and treatment strategy in PCNSL. [ABSTRACT FROM AUTHOR]

Successive relapses from donor and host cells in a patient with DEAD-box helicase 41 (DDX41)-associated myelodysplastic syndrome: The lessons to be learned Keywords: DEAD-box helicase 41 (DDX41); microenvironment; myelodysplastic syndrome; primed donor leucocyte infusions EN DEAD-box helicase 41 (DDX41) microenvironment myelodysplastic syndrome primed donor leucocyte infusions 623 626 4 11/11/22 20221115 NES 221115 Germline pathogenic variants (PV) that predispose to hereditary haematological malignancies are being described in a growing number of genes. Chimerism monitoring using polymerase chain reaction (PCR) of small tandem repeats and quantitative PCR demonstrated a full donor chimerism for 8 years (Figure 2A) but at this time point, he relapsed into an IR-MDS. [Extracted from the article]

Background: Novel, less toxic, cost-effective and safe therapeutic strategies are needed to improve treatment of chronic lymphocytic leukemia (CLL). Ascorbic acid (AA, vitamin C) has shown a potential anti-cancer therapeutic activity in several cancers. However, the anti-cancer effects of ascorbic acid on CLL B-cells have not been extensively studied. We aimed in this study to evaluate the in vitro therapeutic activity using clinically relevant conditions. Methods: Primary CLL B-cells and two CLL cell lines were exposed to a dose that is clinically achievable by AA oral administration (250 μM), and cell death and potential mechanisms were assessed. The role of the protective CLL microenvironment was studied. Synergistic interaction between AA and CLL approved drugs (Ibrutinib, Idelalisib and Venetoclax) was also evaluated. Results: Ascorbic acid is cytotoxic for CLL B-cells at low dose (250 μM) but spares healthy B-cells. Ascorbic-acid-induced cytotoxicity involved pro-oxidant damage through the generation of reactive oxygen species in the extracellular media and in CLL cells, and induced caspase-dependent apoptosis. We also found that AA treatment overcame the supportive survival effect provided by microenvironment including bone marrow mesenchymal stem cells, T-cell cues (CD40L + IL-4), cytokines and hypoxia. Our data suggest that resistance to AA could be mediated by the expression of the enzyme catalase in some CLL samples and by the glucose metabolite pyruvate. We also demonstrated that AA synergistically potentiates the cytotoxicity of targeted therapies used in or being developed for CLL. Conclusion: These preclinical results point to AA as an adjuvant therapy with potential to further improve CLL treatments in combination with targeted therapies. [ABSTRACT FROM AUTHOR]

The article discusses automated blood cell counts and morphological and cytogenetic analyses of 11 cases of chronic B cell lymphoproliferative disorders at diagnosis with near‐tetraploidy. Topics include Near‐tetraploidy (NT) has a rare cytogenetic abnormality defined as a number of chromosomes comprised between 81 and 103; and leukemic chronic lymphoproliferative disorders with tetraploidy or NT managed at institution have analyzed from 2006 to 2017.

Mature T-cell and natural killer (NK)-cell neoplasms (MTNKNs) are a highly heterogeneous group of lymphomas that represent 10–15 % of lymphoid neoplasms and have usually an aggressive behavior. Diagnosis can be challenging due to their overlapping clinical, histological and immunophenotypic features. Genetic data are not a routine component of the diagnostic algorithm for most MTNKNs. Indeed, unlike B-cell lymphomas, the genomic landscape of MTNKNs is not fully understood. Only few characteristic rearrangements can be easily identified with conventional cytogenetic methods and are an integral part of the diagnostic criteria, for instance the t(14;14)/inv(14) or t(X;14) abnormality harbored by 95 % of patients with T-cell prolymphocytic leukemia, or the ALK gene translocation observed in some forms of anaplastic large cell lymphoma. However, advances in molecular and cytogenetic techniques have brought new insights into MTNKN pathogenesis. Several recurrent genetic alterations have been identified, such as chromosomal losses involving tumor suppressor genes (SETD2, CDKN2A, TP53) and gains involving oncogenes (MYC), activating mutations in signaling pathways (JAK-STAT, RAS), and epigenetic dysregulation, that have improved our understanding of these pathologies. This work provides an overview of the cytogenetics knowledge in MTNKNs in the context of the new World Health Organization classification and the International Consensus Classification of hematolymphoid tumors. It describes key genetic alterations and their clinical implications. It also proposes recommendations on cytogenetic methods for MTNKN diagnosis. [ABSTRACT FROM AUTHOR]

Elderly patients with chronic lymphocytic leukemia (CLL) are underrepresented in trials evaluating fludarabine, cyclophosphamide, and rituximab (FCR). We assessed four cycles of FCR with two additional rituximab doses on day 14 of cycles 1 and 2 in 194 untreated CLL patients > 65 years (median age 71.2) without del17p. Four FCR cycles were administered to 90.7% (176/194), with (n= 74) or without (n= 102) dose-delay and/or dose-reduction. A total of 50% grade 3/4 neutropenia occurred after each cycle. Only 6.2% cycles were associated with severe infection. Complete remission (CR) was achieved in 19.7%, and partial remission (PR) in 73.9% of patients. Minimal residual disease (MRD) was negative in 36.7%. Overall survival at 36 months was estimated at 87.4%. Oral FC and dose-dense rituximab is feasible and active in fit elderly CLL patients. However, myelosuppression is significant and frequent dose adaptations are required implying that these results cannot be generalized to unfit or frail elderly CLL. [ABSTRACT FROM PUBLISHER]

Although TP53, NOTCH1, and SF3B1 mutations may impair prognosis of patients with chronic lymphocytic leukemia (CLL) receiving frontline therapy, the impact of these mutations or any other, alone or in combination, remains unclear at relapse. The genome of 114 relapsed/refractory patients included in prospective trials was screened using targeted next-generation sequencing of the TP53, SF3B1, ATM, NOTCH1, XPO1, SAMHD1, MED12, BIRC3, and MYD88 genes. We performed clustering according to both number and combinations of recurrentgene mutations. The number of genes affected by mutation was ≥2, 1, and 0 in 43 (38%), 49 (43%), and 22 (19%) respectively. Recurrent combinations of ≥2 mutations of TP53, SF3B1, and ATM were found in 22 (19%) patients. This multiple-hit profile was associated with a median progression-free survival of 12 months compared with 22.5 months in the remaining patients (P 5 .003). Concurrent gene mutations are frequent in patients with relapsed/refractory CLL and are associated with worse outcome. [ABSTRACT FROM AUTHOR]

Background: Chronic lymphocytic leukemia (CLL), the most common adulthood leukemia, is characterized by the accumulation of abnormal CD5+ B lymphocytes, which results in a progressive failure of the immune system. Despite intense research efforts, drug resistance remains a major cause of treatment failure in CLL, particularly in patients with dysfunctional TP53. The objective of our work was to identify potential approaches that might overcome CLL drug refractoriness by examining the pro-apoptotic potential of targeting the cell surface receptor CD47 with serum-stable agonist peptides. Methods and Findings: In peripheral blood samples collected from 80 patients with CLL with positive and adverse prognostic features, we performed in vitro genetic and molecular analyses that demonstrate that the targeting of CD47 with peptides derived from the C-terminal domain of thrombospondin-1 efficiently kills the malignant CLL B cells, including those from high-risk individuals with a dysfunctional TP53 gene, while sparing the normal T and B lymphocytes from the CLL patients. Further studies reveal that the differential response of normal B lymphocytes, collected from 20 healthy donors, and leukemic B cells to CD47 peptide targeting results from the sustained activation in CLL B cells of phospholipase C gamma-1 (PLCγ1), a protein that is significantly over-expressed in CLL. Once phosphorylated at tyrosine 783, PLCγ1 enables a Ca2+-mediated, caspase-independent programmed cell death (PCD) pathway that is not down-modulated by the lymphocyte microenvironment. Accordingly, down-regulation of PLCγ1 or pharmacological inhibition of PLCγ1 phosphorylation abolishes CD47-mediated killing. Additionally, in a CLL-xenograft model developed in NOD/scid gamma mice, we demonstrate that the injection of CD47 agonist peptides reduces tumor burden without inducing anemia or toxicity in blood, liver, or kidney. The limitations of our study are mainly linked to the affinity of the peptides targeting CD47, which might be improved to reach the standard requirements in drug development, and the lack of a CLL animal model that fully mimics the human disease. Conclusions: Our work provides substantial progress in (i) the development of serum-stable CD47 agonist peptides that are highly effective at inducing PCD in CLL, (ii) the understanding of the molecular events regulating a novel PCD pathway that overcomes CLL apoptotic avoidance, (iii) the identification of PLCγ1 as an over-expressed protein in CLL B cells, and (iv) the description of a novel peptide-based strategy against CLL. [ABSTRACT FROM AUTHOR]

In systemic mastocytosis (SM), clinical problems arise from factor-independent proliferation of mast cells (MCs) and the increased release of mediators by MCs, but no human cell line model for studying NIC activation in the context of SM is available. We have created a stable stem cell factor (SCF) -dependent human MC line, ROSAKIT WT, expressing a fully functional immunoglobulin E (IgE) receptor. Transfection with KIT D816V converted ROSAKIT WT cells into an SCF-independent clone, ROSAKIT D816V, which produced a mastocytosis-like disease in NSG mice. Although several signaling pathways were activated, ROSAKIT D816V did not exhibit an increased, but did exhibit a decreased responsiveness to IgE-dependent stimuli. Moreover, NSG mice bearing ROSAKIT D816V-derived tumors did not show mediator-related symptoms, and KIT D816V-positive MCs obtained from patients with SM did not show increased IgE-dependent histamine release or CD63 upregulation. Our data show that KIT D816V is a disease-propagating oncoprotein, but it does not activate MCs to release proinflammatory mediators, which may explain why mediator-related symptoms in SM occur preferentially in the context of a coexisting allergy. ROSAKIT D816V may provide a valuable tool for studying the pathogenesis of mastocytosis and should facilitate the development of novel drugs for treating SM patients. [ABSTRACT FROM AUTHOR]

Translocations involving MYC are rare in chronic lymphocytic leukemia (CLL), and up to now, their prognostic significance remains unclear. We report the characteristics of 21 patients with CLL and nine patients with prolymphocytic leukemia (PLL), diagnosed in multiple centers ( n = 13), which showed an MYC translocation demonstrated by fluorescence in situ hybridization. The prevalence was estimated to be <1%. Advanced age and male predominance were observed. Morphological analysis frequently revealed the presence of prolymphocytes. A typical 'CLL-immunophenotype' was found in four of nine cases with PLL. Moreover, CD5 and CD23 were frequently expressed in PLL. The latter findings are atypical for PLL and may suggest transformation or progression of an underlying CLL. MYC translocations were frequently observed with concomitant adverse cytogenetic markers, such as del(11q) ( n = 8/30) and/or del(17p)/monosomy 17 ( n = 7/30). In addition, the presence of unbalanced translocations ( n = 24 in 13/30 cases) and complex karyotype ( n = 16/30) were frequent in cases with MYC translocations. Altogether, del(17p)/monosomy 17, del(11q), and/or complex karyotype were observed in 22 of 30 patients. Survival outcome was poor: the median time to treatment was only 5 months, and overall survival (OS) from clinical diagnosis and from genetic detection was 71 and 19 months, respectively. In conclusion, CLL/PLL with MYC translocations is a rare entity, which seems to be associated with adverse prognostic features and unfavorable outcome. [ABSTRACT FROM AUTHOR]

Summary: Loss-of-function mutations affecting one or both copies of the Ten-Eleven-translocation (TET)2 gene have been described in various human myeloid malignancies. We report that inactivation of Tet2 in mouse perturbs both early and late steps of hematopoiesis including myeloid and lymphoid differentiation in a cell-autonomous manner, endows the cells with competitive advantage, and eventually leads to the development of malignancies. We subsequently observed TET2 mutations in human lymphoid disorders. TET2 mutations could be detected in immature progenitors endowed with myeloid colony-forming potential. Our results show that the mutations present in lymphoid tumor cells may occur at both early and later steps of lymphoid development and indicate that impairment of TET2 function or/and expression predisposes to the development of hematological malignancies. [Copyright &y& Elsevier]

Background: The sensitivity of chronic lymphocytic leukemia (CLL) cells to current treatments, both in vitro and in vivo, relies on their ability to activate apoptotic death. CLL cells resistant to DNA damage-induced apoptosis display deregulation of a specific set of genes. Methods: Microarray hybridization (Human GeneChip, Affymetrix), immunofluorescent in situ labeling coupled with video-microscopy recording/analyses, chromatin-immunoprecipitation (ChIP), polymerase chain reactions (PCR), realtime quantitative PCR (RT-QPCR) and bisulfite genome sequencing were the main methods applied. Statistical analyses were performed by applying GCRMA and SAM analysis (microarray data) and Student's t-test or Mann & Whitney's U-test. Results: Herein we show that, remarkably, in a resistant male CLL cells the vast majority of genes were downregulated compared with sensitive cells, whereas this was not the case in cells derived from females. This gene down-regulation was found to be associated with an overall gain of heterochromatin as evidenced by immunofluorescent labeling of heterochromatin protein 1α (HP-1), trimethylated histone 3 lysine 9 (3metH3K9), and 5-methylcytidine (5metC). Notably, 17 genes were found to be commonly deregulated in resistant male and female cell samples. Among these, RELB was identified as a discriminatory candidate gene repressed in the male and upregulated in the female resistant cells. Conclusion: The molecular defects in the silencing of RELB involve an increase in H3K9- but not CpG-island methylation in the promoter regions. Increase in acetyl-H3 in resistant female but not male CLL samples as well as a decrease of total cellular level of RelB after an inhibition of histone deacetylase (HDAC) by trichostatin A (TSA), further emphasize the role of epigenetic modifications which could discriminate two CLL subsets. Together, these results highlighted the epigenetic RELB silencing as a new marker of the progressive disease in males. [ABSTRACT FROM AUTHOR]

Several prognostic factors can predict the rapid progression in chronic lymphocytic leukaemia (CLL), including IGHV mutational status, cytogenetic abnormalities and, more recently, LPL/ ADAM29 expression. In contrast, few studies have been devoted to the influence of these factors on clinical outcome in responding patients after therapy. We here propose to analyse the impact of IGHV gene status, LPL and ADAM29 gene expression on disease-free survival (DFS) and overall survival (OS) in 41 stage B or C CLL patients in remission after oral fludarabine plus cyclophosphamide. The median follow-up was of 64 (16–74) months. Sequencing of IGHV showed mutated (M) VH genes in 16 of 41 cases and unmutated (UM) in 25 cases. Analysis of LPL and ADAM29 expression in 35 of 41 cases showed overexpression of ADAM29 in 17 cases (14 M and three UM) and LPL in 18 cases (all UM). Patients expressing UM IGHV and LPL had shorter DFS and OS when compared to patients expressing M IGHV and/or ADAM29. Furthermore, blood minimal residual disease (MRD) evaluation using four-colour flow cytometry was performed in 33 out the 41 patients. We showed that patients who achieved phenotypic remission displayed longer DFS than those with MRD+. Our results support the use of LPL and ADAM29 gene expression associated to IGHV mutational status for predicting the clinical outcome of patients treated by oral fludarabine + cyclophosphamide and could be considered for treatment strategies. [ABSTRACT FROM AUTHOR]

Objectives: The t(12;14)(p13;q11) – a recurrent translocation in childhood T-cell acute lymphoblastic leukemia (T-ALL) – has very recently been molecularly characterized in one case, which displayed overexpression of the cyclin D2 gene ( CCND2). Patients and methods: We have characterized two pediatric t(12;14)-positive T-ALLs using fluorescence in situ hybridization (FISH), cDNA microarray, and real-time polymerase chain reaction (PCR). Results: FISH revealed breakpoints (BPs) in the T-cell receptor alpha/delta locus (14q11) and in the vicinity of the CCND2 gene at 12p13. To investigate the expression of genes in 12p13, cDNA microarray analysis was performed. Expression data for eight genes, including CCND2, surrounding the 12p BP were compared with those in other T-ALLs. The t(12;14)-positive T-ALL displayed an increased expression of CCND2 compared to the controls, whereas the expression of the other genes was similar in all T-ALLs. Expression of CCND2 and two additional genes ( PARP11 and FGF23), close to the 12p BP, was investigated with real-time PCR of the two t(12;14)-positive cases and four controls. Neither PARP11 nor FGF23 displayed expression differences among the T-ALLs, whereas CCND2 was clearly overexpressed in both t(12;14)-positive cases as compared to the mean expression level in the controls. Conclusion: We have confirmed, in two additional cases, that the recurrent T-ALL-associated t(12;14) results in overexpression of cyclin D2. The t(12;14) is the first neoplasia-associated translocation shown to result in overexpression of cyclin D2. Furthermore, it is the first example of a T-cell neoplasm with a targeted deregulation of a member of a cyclin-encoding gene family. [ABSTRACT FROM AUTHOR]

Abstract: A case of de novo acute myeloblastic leukemia (AML) M2, with trisomy 4 and double minute (dmin) chromosomes is reported. Amplification of the MYC gene ascertained by FISH was associated with dmin. A review of the literature of trisomy 4-dmin-associated AML shows that this entity preferentially occurs in elderly women and is not always associated with previously identified exposition to mutagens. [Copyright &y& Elsevier]

The resistance to apoptosis of chronic lymphocytic leukemia (CLL) cells partly results from the deregulated production of survival signals from leukemic cells. Despite the development of new therapies in CLL, drug resistance and disease relapse still occur. Recently, neutrophil gelatinase-associated lipocalin (NGAL), a secreted glycoprotein, has been suggested to have a critical role in the biology of tumors. Thus, we investigated the relevance of NGAL in CLL pathogenesis, analyzed the expression of its cellular receptor (NGAL-R) on malignant B cells and tested whether CLL cells are resistant to apoptosis through an autocrine process involving NGAL and NGAL-R. We observed that NGAL concentrations were elevated in the serum of CLL patients at diagnosis. After treatment (and regardless of the therapeutic regimen), serum NGAL levels normalized in CLL patients in remission but not in relapsed patients. In parallel, NGAL and NGAL-R were upregulated in leukemic cells from untreated CLL patients when compared to normal peripheral blood mononuclear cells (PBMCs), and returned to basal levels in PBMCs from patients in remission. Cultured CLL cells released endogenous NGAL. Anti-NGAL-R antibodies enhanced NGAL-R+ leukemia cell death. Conversely, recombinant NGAL protected NGAL-R+ CLL cells against apoptosis by activating a STAT3/Mcl-1 signaling pathway. Our results suggest that NGAL and NGAL-R, overexpressed in untreated CLL, participate in the deregulation of the apoptotic machinery in CLL cells, and may be potential therapeutic clues for CLL treatment. [ABSTRACT FROM AUTHOR]