Monoclonal B‐cell lymphocytosis is closely related to chronic lymphocytic leukaemia and may be better classified as early‐stage CLL

The World Health Organization classification uses a cut‐off point of 5·0 × 10⁹/l cells with a chronic lymphocytic leukaemia (CLL)‐phenotype in peripheral blood to discriminate between monoclonal B‐lymphocytosis (MBL) and B‐CLL. This study analysed 298 MBL patients by multi‐parameter flow cytometry, chromosome banding analysis (CBA)/fluorescence in situ hybridization (FISH), and IGHV mutation status and compared them with 356 CLL patients. In MBL, CBA more frequently revealed a normal karyotype and FISH identified less frequently del(6q), del(13q) (as sole alterations), and del(17)(p13). Within the MBL cohort, a shorter time to treatment (TTT) was found for ZAP‐70‐positivity, 14q32/IGH‐translocations (CBA), del(11)(q22·3) (FISH) and unmutated IGHV status. Higher CD38 and ZAP‐70 expression, del(11)(q22·3) (FISH), trisomy 12 (FISH), and 14q32/IGH‐translocations (CBA) were correlated with a shorter TTT in the combined cohort (MBL + CLL); a sole del(13)(q14) (FISH) correlated with longer TTT. Regarding overall survival, unmutated IGHV status and ‘other’ alterations (CBA) had an adverse impact. There was no correlation between the concentration of CLL‐cells and TTT or overall survival. Multivariate analysis confirmed a negative impact on TTT for del(11)(q22·3)/ATM, trisomy 12 (both by FISH), and 14q32/IGH‐translocations by CBA. These data emphasize a close relationship between MBL and CLL regarding clinically relevant parameters and provide no evidence to strictly separate these entities by a distinct threshold of clonal B‐cells.     

Chronic lymphocytic leukaemia,monoclonal B‐lymphocytosis and pregnancy: five cases,a literature review and discussion of management

Chronic lymphocytic leukaemia (CLL) occurs rarely with pregnancy and monoclonal B‐Lymphocytosis (MBL) has not previously been described in this setting. CLL is predominantly a disease of the elderly and affects men twice as often as women and hence only an estimated 2% of patients are females of childbearing age. We identified only five reported cases of CLL in pregnancy in the literature. We describe two additional cases, plus three other women with CLL dealing with pregnancy‐related decisions. We review the literature and discuss proposals for management and issues that arise in this relatively uncommon occurrence. In contrast to many other haematological malignancies where longer remissions are typically associated with a lower risk of relapse, most patients with CLL who require treatment will ultimately relapse with current therapy. This complex setting requires careful consideration and well informed patients to assist with decisions related to pregnancy.     

Pro‐apoptotic TP53 homolog TAp63 is repressed via epigenetic silencing and B‐cell receptor signalling in chronic lymphocytic leukaemia

Chronic lymphocytic leukaemia (CLL) is an accumulative disorder marked by deficient apoptosis. The TP53 homolog TAp63 promotes apoptosis and chemosensitivity in solid tumours and its deregulation may contribute to CLL cell survival. We found that TAp63α was the most prevalent TP63 isoform in CLL. Compared to healthy B cells, TAp63 mRNA was repressed in 55·7% of CLL samples. TP63 promoter methylation was high in CLL and inversely correlated with TP63 protein expression in B‐cell lymphoma cell lines. siRNA‐mediated knockdown of TP63 resulted in partial protection from spontaneous apoptosis accompanied by reductions in PMAIP1 (NOXA), BBC3 (PUMA), and BAX mRNA in CLL cells and increased proliferation of Raji lymphoma cells. TAp63 mRNA levels were higher in CLL with unmutated IGHV. B‐cell receptor (BCR) engagement led to repression of TP63 mRNA expression in malignant B cells, while pharmacological inhibition of BCR signalling prevented TP63 downregulation. MIR21, known to target TAp63, correlated inversely with TAp63 expression in CLL, and BCR‐mediated downregulation of TP63 was accompanied by MIR21 upregulation in most CLL samples. Our data illustrate the pro‐apoptotic function of TP63, provide insights into the mechanisms of BCR‐targeting agents, and establish a rationale for designing novel approaches to induce TP63 in CLL and B‐cell lymphoma.     

Molecular and clinical features of chronic lymphocytic leukaemia with stereotyped B cell receptors: results from an Italian multicentre study

A fraction of chronic lymphocytic leukaemia (CLL) cases carry highly homologous B-cell receptors (BCR), i.e. characterized by non-random combinations of immunoglobulin heavy-chain variable ( IGHV ) genes and heavy-chain complementarity determining region-3 (HCDR3), often associated with a restricted selection of IGVK/L light chains. Such 'stereotyped' BCR occur more frequently in CLL with unmutated (UM) than mutated (M) IGHV genes. We analysed 1426 IG rearrangements (from 1398 CLL cases) by a clustering driven by HCDR3 similarities. Molecular findings were correlated to time-to-treatment (TTT) and presence of known prognosticators. Sixty-nine clusters (319 IG-rearrangements, 22·4%) with stereotyped BCR were identified. Among 30 confirmed clusters (≥3 IG-rearrangements/cluster), we found 14 novel clusters, of which 11 had M IG rearrangements (M clusters) and predominantly (8/11) used IGHV3 subgroup genes. Recurrent cluster-biased amino acid changes were found throughout IGHV sequences of these 'M clusters'. Regarding clinical outcome: (i) UM CLL from the IGHV1-2/1-3/1-18/1-46/7-4-1/IGKV1-39 cluster had poorer prognosis than UM/M cases, or UM cases using the same IGHV genes but not in clusters; (ii) M CLL from the IGHV3-21/IGLV3-21 cluster had TTT similar to UM CLL, and shorter than M CLL expressing IGHV3-21 but not in cluster. Altogether, our analysis identified additional molecular and clinical features for CLL expressing stereotyped BCR.     

B cells CD19+ in patients with B‐cell chronic lymphocytic leukaemia and autoimmune haemolytic anaemia

The study presents results of B and T lymphocytes population analysis in patients with chronic lymphocytic leukaemia B cells and autoimmune haemolytic anaemia (CLL‐B + AIHA). We evaluated the following groups of patients: (1) with newly recognized CLL‐B and co‐existent AIHA (untreated), (2) after short‐term treatment with corticosteroids, (3) after treatment with chemotherapy and corticosteroids. The control groups were made of patients with CLL‐B without AIHA. The populations of lymphocytes and determination of cells immunophenotype were performed by means of flow cytometry. The analysed data were obtained from 25 patients. The untreated patients with CLL‐B + AIHA presented significantly more numerous population of neoplastic cells CD19+ CD5+ in comparison with patients without AIHA. The patients with AIHA showed a reduced percentage of B CD19+ CD22+ cells in comparison with those without AIHA. Untreated patients with AIHA or after a short‐term corticosteroid treatment showed a higher ratio of the number of CD19+ CD5+ cells to the number of T CD4+ and T CD8+ lymphocytes than CLL‐B patients without AIHA. It can be presumed that the differences found may be related to the pathogenesis of the autoimmune haemolysis syndrome in patients with CLL‐B.     

Second malignancies in B‐cell chronic lymphocytic leukaemia: possible association with human papilloma virus

Second primary malignancies have long been associated with chronic lymphocytic leukaemia (CLL). We assessed secondary tumour samples from CLL and control patients for the presence of human papilloma virus (HPV). 132 CLL patients with 44 second malignancies were compared to a matched randomly‐identified control population of 264 non‐CLL patients with 54 solid malignancies. Polymerase chain reaction was performed with the highly conserved MY09/MY11 HPV primer. None of control samples were HPV‐positive, while 53% of samples from the CLL group were positive. This report describes preliminary evidence for the presence of HPV in secondary malignancies, in patients with CLL.     

High‐throughput sequencing for the identification of NOTCH1 mutations in early stage chronic lymphocytic leukaemia: biological and clinical implications

NOTCH1 mutations have recently emerged as new genetic lesions significantly correlated with survival in chronic lymphocytic leukaemia (CLL). We performed deep next generation sequencing of the NOTCH1 mutation hotspot in 384 cases at diagnosis, including 100 monoclonal B cell lymphocytosis (MBL) and 284 Binet stage A CLL cases, enrolled in the Gruppo Italiano Studio Linfomi O‐CLL1 multicentre trial. The NOTCH1 c.7541_7542delCT dinucleotide deletion was detected and confirmed by an extremely sensitive polymerase chain reaction‐based approach in 11% of MBL and 13·4% of CLL patients. Remarkably, the NOTCH1 mutation was often observed at low clonal level, mainly in MBL patients. Sequential analyses in a fraction of cases showed that the NOTCH1 mutation generally does not occur during the disease course and that the mutational load in positive cases tends to be stable over time. NOTCH1‐mutated cases, even at low clonal level, displayed a significant reduction in median progression‐free survival, although NOTCH1 mutation lost its prognostic impact in a multivariate analysis including 11q and/or 17p deletion, IGHV mutational status, and MBL or CLL status. Our data highlight the importance of using highly sensitive methods to measure NOTCH1 mutations, in order to improve prognostic stratification and obtain useful information for potential therapeutic approaches.     

Nature and nurture: a case of transcending haematological pre‐malignancies in a pair of monozygotic twins adding possible clues on the pathogenesis of B‐cell proliferations

We describe a comprehensive molecular analysis of a pair of monozygotic twins, who came to our attention when one experienced amaurosis fugax and was diagnosed with JAK2+ polycythaemia vera. He (Twin A) was also found to have an asymptomatic B‐cell chronic lymphocytic leukaemia (B‐CLL). Although JAK2?, Twin B was subsequently shown to have a benign monoclonal B‐cell lymphocytosis (MBL). Flow cytometric and molecular analyses of the B‐cell compartments revealed different immunoglobulin light and heavy chain usage in each twin. We hypothesized that whole exome sequencing could help delineating the pattern of germline B‐cell disorder susceptibility and reveal somatic mutations potentially contributing to the differential patterns of pre‐malignancy. Comparing bone marrow cells and T cells and employing in‐house engineered integrative analysis, we found aberrations in Twin A consistent with a myeloid neoplasm, i.e. in TET2, RUNX1, PLCB1 and ELF4. Employing the method for detecting high‐ranking variants by extensive annotation and relevance scoring, we also identified shared germline variants in genes of proteins interacting with B‐cell receptor signalling mediators and the WNT‐pathway, including IRF8, PTPRO, BCL9L, SIT1 and SIRPB1, all with possible implications in B‐cell proliferation. Similar patterns of IGHV‐gene usage to those demonstrated here have been observed in inherited acute lymphoblastic leukaemia. Collectively, these findings may help in facilitating identification of putative master gene(s) involved in B‐cell proliferations in general and MBL and B‐CLL in particular.     

B cell activation through CD40 and IL4R ligation modulates the response of chronic lymphocytic leukaemia cells to BAFF and APRIL

The two tumour necrosis factor family proteins BAFF (TNFSF13B) and APRIL (TNFSF13) and their receptors [BAFF‐R (TNFRSF13C), TACI (TNFRSF13B), BCMA (TNFRSF17)] play a critical role in the survival of normal B cells. The sensitivity of normal B cells to BAFF and APRIL can be modulated by signals regulated by their receptors. This modulation, however, has not been extensively investigated in chronic lymphocytic leukaemia (CLL) cells. We evaluated the expression, regulation and signalling of BAFF and APRIL receptors in normal and in CLL cells upon stimulation through CD40+IL4R and BCR. We further analysed the prognostic value of BAFF and APRIL receptors expression in patients with CLL. BCMA expression was significantly higher on CLL cells than on normal B cells. BCR and CD40+IL4R stimulation promoted an increase in TACI and BCMA expression, cell viability and activation in normal B cells. A similar effect was observed in CLL cells after CD40+IL4R but not BCR stimulation. BCMA expression correlated with unmutated IGHV genes, poor‐risk cytogenetics, and short progression‐free survival. These findings further characterize the link between CD40+IL4R regulatory signals, BAFF, APRIL and their receptors and the survival of leukaemic cells and clinical features of CLL.     

A phase 1/2 trial of ublituximab,a novel anti‐CD20 monoclonal antibody,in patients with B‐cell non‐Hodgkin lymphoma or chronic lymphocytic leukaemia previously exposed to rituximab

This phase 1/2 study evaluated the safety, pharmacokinetic behavior and anti‐tumour activity of ublituximab, a unique type I, chimeric, glycoengineered anti‐CD 20 monoclonal antibody, in rituximab‐relapsed or ‐refractory patients with B‐cell non‐Hodgkin lymphoma (B‐NHL ) or chronic lymphocytic leukaemia (CLL ). Induction therapy (doses of 450–1200 mg) consisted of 4 weekly infusions in cycle 1 for NHL and 3 weekly infusions in cycles 1 and 2 for CLL . Patients received ublituximab maintenance monthly during cycles 3–5, then once every 3 months for up to 2 years. Enrolled patients with B‐NHL (n  = 27) and CLL (n  = 8) had a median of 3 prior therapies. No dose‐limiting toxicities or unexpected adverse events (AE s) occurred. The most common AE s were infusion‐related reactions (40%; grade 3/4, 0%); fatigue (37%; grade 3/4, 3%); pyrexia (29%; grade 3/4, 0%); and diarrhoea (26%; grade 3/4, 0%). Common haematological AE s were neutropenia (14%; grade 3/4, 14%) and anaemia (11%; grade 3/4, 6%). The overall response rate for evaluable patients (n  = 31) was 45% (13% complete responses, 32% partial responses). Median duration of response and progression‐free survival were 9·2 months and 7·7 months, respectively. Ublituximab was well‐tolerated and efficacious in a heterogeneous and highly rituximab‐pre‐treated patient population.