Lydia Scarfò

Recurrent mutations refine prognosis in chronic lymphocytic leukemia

Through the European Research Initiative on chronic lymphocytic leukemia (CLL) (ERIC), we screened 3490 patients with CLL for mutations within the NOTCH1 (n=3334), SF3B1 (n=2322), TP53 (n=2309), MYD88 (n=1080) and BIRC3 (n=919) genes, mainly at diagnosis (75%) and before treatment (>90%). BIRC3 mutations (2.5%) were associated with unmutated IGHV genes (U-CLL), del(11q) and trisomy 12, whereas MYD88 mutations (2.2%) were exclusively found among M-CLL. NOTCH1, SF3B1 and TP53 exhibited variable frequencies and were mostly enriched within clinically aggressive cases. Interestingly, as the timespan between diagnosis and mutational screening increased, so too did the incidence of SF3B1 mutations; no such increase was observed for NOTCH1 mutations. Regarding the clinical impact, NOTCH1 mutations, SF3B1 mutations and TP53 aberrations (deletion/mutation, TP53ab) correlated with shorter time-to-first-treatment (P<0.0001) in 889 treatment-naive Binet stage A cases. In multivariate analysis (n=774), SF3B1 mutations and TP53ab along with del(11q) and U-CLL, but not NOTCH1 mutations, retained independent significance. Importantly, TP53ab and SF3B1 mutations had an adverse impact even in U-CLL. In conclusion, we support the clinical relevance of novel recurrent mutations in CLL, highlighting the adverse impact of SF3B1 and TP53 mutations, even independent of IGHV mutational status, thus underscoring the need for urgent standardization/harmonization of the detection methods.

General population low-count CLL-like MBL persists over time without clinical progression, although carrying the same cytogenetic abnormalities of CLL

Monoclonal B-cell lymphocytosis (MBL) is classified as chronic lymphocytic leukemia (CLL)-like, atypical CLL, and CD5(-) MBL. The number of B cells per microliter divides CLL-like MBL into MBL associated with lymphocytosis (usually detected in a clinical setting) and low-count MBL detected in the general population (usually identified during population screening). After a median follow-up of 34 months we reevaluated 76 low-count MBLs with 5-color flow cytometry: 90% of CLL-like MBL but only 44.4% atypical CLL and 66.7% CD5(-) MBL persisted over time. Population-screening CLL-like MBL had no relevant cell count change, and none developed an overt leukemia. In 50% of the cases FISH showed CLL-related chromosomal abnormalities, including monoallelic or biallelic 13q deletions (43.8%), trisomy 12 (1 case), and 17p deletions (2 cases). The analysis of the T-cell receptor β (TRBV) chains repertoire showed the presence of monoclonal T-cell clones, especially among CD4(high)CD8(low), CD8(high)CD4(low) T cells. TRBV2 and TRBV8 were the most frequently expressed genes. This study indicates that (1) the risk of progression into CLL for low-count population-screening CLL-like MBL is exceedingly rare and definitely lower than that of clinical MBL and (2) chromosomal abnormalities occur early in the natural history and are possibly associated with the appearance of the typical phenotype.

MicroRNA and proliferation control in chronic lymphocytic leukemia: functional relationship between miR-221/222 cluster and p27

We investigated functional relationships between microRNA 221/222 (miR-221/222) cluster and p27, a key regulator of cell cycle, in chronic lymphocytic leukemia (CLL). The enforced expression of miR-221/222 in the CLL cell line MEC1 induced a significant down-regulation of p27 protein and conferred a proliferative advantage to the transduced cells that exhibited faster progression into the S phase of the cell cycle. Accordingly, expression of miR-221/miR-222 and p27 was found to be inversely related in leukemic cells obtained from peripheral blood (PB) of 38 patients with CLL. Interestingly, when miR-221/222 and p27 protein were evaluated in different anatomic compartments (lymph nodes or bone marrow) of the same patients, increased expression of the 2 miRNAs became apparent compared with PB. This finding was paralleled by a low expression of p27. In addition, when CLL cells were induced in vitro to enter cell cycle (eg, with cytosine phosphate guanine oligodeoxynucleotide), a significant increase of miR-221/222 expression and a marked down-regulation of p27 protein were evident. These data indicate that the miR-221/222 cluster modulates the expression of p27 protein in CLL cells and lead to suggest that miR-221/222 and p27 may represent a regulatory loop that helps maintaining CLL cells in a resting condition.

Reprogramming cell death: BCL2 family inhibition in hematological malignancies

The BCL2 family members play a central role in regulating programmed cell death (apoptosis) and arbitrating the cellular fate through an accurate balance between pro-apoptotic (BAX, BAK, and BH3-only proteins) and pro-survival (BCL2 and its closest homologues, BCLXL, BCLW and MCL-1) factors. Deregulation of BCL2 family proteins contributes to programmed cell death evasion, that is a hallmark of human cancers and it is often related to (chemo)therapy resistance. High BCL2 levels have been detected in most human lymphoid malignancies, not limited to follicular lymphoma (where the role of BCL2 overexpression is driven by the t[14;18] translocation) but also B-cell chronic lymphocytic leukemia (CLL) and multiple myeloma. For all these reasons, the opportunity to induce apoptosis by targeting BCL2 proteins is considered a potentially promising therapeutic approach in hematological malignancies. BCL2 family inhibition strategies currently explored in phase 1, 2 and 3 clinical trials are essentially two: (1) the use of antisense-based strategies to knockdown BCL2 or BCLXL expression (e.g. oblimersen) or (2) the use of synthetic BH3 mimetics i.e. small molecules binding to anti-apoptotic inhibitors thereby allowing the pro-apoptotic activity of BH3-only molecules (e.g. obatoclax, AT-101, ABT-737 and its derivatives ABT-263 and ABT-199). Several of these drugs demonstrated relevant clinical activity as single-agent or in combination therapy, with the most significant drawbacks in clinical use being represented by challenging pharmacokinetic profile (e.g. iv administration, high-levels of plasma proteins binding) and on-target side effects (e.g. gastrointestinal toxicity and thrombocytopenia). Further clinical development of the current compounds (e.g. ABT-199), showing high efficacy but devoid of the most threatening drug-related toxicities, is eagerly awaited. Hopefully, in the next future, BCL2 inhibitors (alone or in combination with immuno- and/or chemo-therapeutic agents) will represent target-specific drugs expanding our therapeutic armamentarium in the fight against hematologic malignancies.

Distinct patterns of novel gene mutations in poor-prognostic stereotyped subsets of chronic lymphocytic leukemia : the case of SF3B1 and subset #2

Recent studies have revealed recurrent mutations of the NOTCH1, SF3B1 and BIRC3 genes in chronic lymphocytic leukemia (CLL), especially among aggressive, chemorefractory cases. Nevertheless, it is currently unknown whether their presence may differ in subsets of patients carrying stereotyped B-cell receptors and also exhibiting distinct prognoses. Here, we analyzed the mutation status of NOTCH1, SF3B1 and BIRC3 in three subsets with particularly poor prognosis, that is, subset #1, #2 and #8, aiming to explore links between genetic aberrations and immune signaling. A remarkably higher frequency of SF3B1 mutations was revealed in subset #2 (44%) versus subset #1 and #8 (4.6% and 0%, respectively; P<0.001). In contrast, the frequency of NOTCH1 mutations in subset #2 was only 8%, lower than the frequency observed in either subset #1 or #8 (19% and 14%, respectively; P=0.04 for subset #1 versus #2). No associations were found for BIRC3 mutations that overall were rare. The apparent non-random association of certain mutations with stereotyped CLL subsets alludes to subset-biased acquisition of genomic aberrations, perhaps consistent with particular antigen/antibody interactions. These novel findings assist in unraveling specific mechanisms underlying clinical aggressiveness in poor-prognostic stereotyped subsets, with far-reaching implications for understanding their clonal evolution and implementing biologically oriented therapy.

Immunogenetics shows that not all MBL are equal: the larger the clone, the more similar to CLL.

Chronic lymphocytic leukemia (CLL) -like monoclonal B-cell lymphocytosis (MBL) shares common immunophenotype and cytogenetic abnormalities with CLL, from which it is discriminated by a cutoff value of 5 × 10(9)/L circulating clonal B cells. However, the clonal size in MBL is extremely variable and allows discrimination of two distinct entities (high-count [HC] and low-count [LC]-MBL) based on a cutoff value of 0.5 × 10(9)/L clonal B cells. HC-MBL is associated with lymphocytosis and progresses to CLL requiring treatment at a rate of 1.1% per year, whereas LC-MBL is found in the general population only through high-sensitivity techniques and carries limited, if any, risk of progression. We performed an immunogenetic profiling of 333 cases with CLL-like MBL supplemented by detailed comparisons with CLL, focusing especially on CLL Rai stage 0 (CLL-0). LC- and HC-MBL had similar somatic hypermutation status, yet different IGHV gene repertoires and frequencies of B-cell receptor (BcR) stereotypy. In particular, stereotyped BcRs were infrequent in LC-MBL and were often not CLL specific. In contrast, HC-MBL exhibited clear immunogenetic similarities to CLL-0. These findings indicate that LC-MBL may not represent a true preleukemic condition, thus differing from HC-MBL/CLL-0 in which the identification of factors endowing malignant potential is strongly warranted.

Targeting B cell anergy in chronic lymphocytic leukemia

B-cell receptor (BCR) triggering and responsiveness have a crucial role in the survival and expansion of chronic lymphocytic leukemia (CLL) clones. Analysis of in vitro response of CLL cells to BCR triggering allowed the definition of 2 main subsets of patients and lack of signaling capacity was associated with constitutive activation of extracellular-regulated kinases 1/2 (ERK1/2) and nuclear factor of activated T cells c1 (NF-ATc1), consistent with the idea that at least one group of CLL patients derives from the abnormal expansion of anergic B cells. In the present work, we further investigated the anergic subset of CLL (defined as the one with constitutive ERK1/2 phosphorylation) and found that it is characterized by low levels of surface immunoglobulin M and impairment of calcium mobilization after BCR engagement in vitro. Chronic BCR triggering promoted CLL cell survival selectively in phosphorylated ERK1/2 samples and the use of mitogen-activated protein kinase and NF-AT signaling inhibitors specifically induced apoptosis in this group of patients. Apoptosis induction was preceded by an initial phase of anergy reversal consisting in the loss of ERK phosphorylation and NF-AT nuclear translocation and by the restoration of BCR responsiveness, reinforcing the idea that the anergic program favors the survival of leukemic lymphocytes.

Monoclonal B Cell Lymphocytosis in Hepatitis C Virus Infected Individuals

Monoclonal B cell lymphocytosis (MBL) is a preclinical condition characterized by an expansion of clonal B cells in the absence of B lymphocytosis (BALC < 5 × 109/L) in the peripheral blood, without clinical signs, suggestive of a lymphoproliferative disorder. B cell clonal expansions are also associated with hepatitis C virus (HCV) infection and they can evolve into lymphoproliferative disorders such as mixed cryoglobulinemia and non‐Hodgkin lymphomas (NHL). The relationship between MBL and HCV infection has not been established yet.

A novel Rag2−/−γc−/−-xenograft model of human CLL

Easily reproducible animal models that allow for study of the biology of chronic lymphocytic leukemia (CLL) and to test new therapeutic agents have been very difficult to establish. We have developed a novel transplantable xenograft murine model of CLL by engrafting the CLL cell line MEC1 into Rag2(-/-)gamma(c)(-/-) mice. These mice lack B, T, and natural killer (NK) cells, and, in contrast to nude mice that retain NK cells, appear to be optimal recipient for MEC1 cells, which were successfully transplanted through either subcutaneous or intravenous routes. The result is a novel in vivo model that has systemic involvement, develops very rapidly, allows the measurement of tumor burden, and has 100% engraftment efficiency. This model closely resembles aggressive human CLL and could be very useful for evaluating both the biologic basis of CLL growth and dissemination as well as the efficacy of new therapeutic agents.

Clinical effect of stereotyped B-cell receptor immunoglobulins in chronic lymphocytic leukaemia: a retrospective multicentre study

BACKGROUND About 30% of cases of chronic lymphocytic leukaemia (CLL) carry quasi-identical B-cell receptor immunoglobulins and can be assigned to distinct stereotyped subsets. Although preliminary evidence suggests that B-cell receptor immunoglobulin stereotypy is relevant from a clinical viewpoint, this aspect has never been explored in a systematic manner or in a cohort of adequate size that would enable clinical conclusions to be drawn. METHODS For this retrospective, multicentre study, we analysed 8593 patients with CLL for whom immunogenetic data were available. These patients were followed up in 15 academic institutions throughout Europe (in Czech Republic, Denmark, France, Greece, Italy, Netherlands, Sweden, and the UK) and the USA, and data were collected between June 1, 2012, and June 7, 2013. We retrospectively assessed the clinical implications of CLL B-cell receptor immunoglobulin stereotypy, with a particular focus on 14 major stereotyped subsets comprising cases expressing unmutated (U-CLL) or mutated (M-CLL) immunoglobulin heavy chain variable genes. The primary outcome of our analysis was time to first treatment, defined as the time between diagnosis and date of first treatment. FINDINGS 2878 patients were assigned to a stereotyped subset, of which 1122 patients belonged to one of 14 major subsets. Stereotyped subsets showed significant differences in terms of age, sex, disease burden at diagnosis, CD38 expression, and cytogenetic aberrations of prognostic significance. Patients within a specific subset generally followed the same clinical course, whereas patients in different stereotyped subsets-despite having the same immunoglobulin heavy variable gene and displaying similar immunoglobulin mutational status-showed substantially different times to first treatment. By integrating B-cell receptor immunoglobulin stereotypy (for subsets 1, 2, and 4) into the well established Döhner cytogenetic prognostic model, we showed these, which collectively account for around 7% of all cases of CLL and represent both U-CLL and M-CLL, constituted separate clinical entities, ranging from very indolent (subset 4) to aggressive disease (subsets 1 and 2). INTERPRETATION The molecular classification of chronic lymphocytic leukaemia based on B-cell receptor immunoglobulin stereotypy improves the Döhner hierarchical model and refines prognostication beyond immunoglobulin mutational status, with potential implications for clinical decision making, especially within prospective clinical trials. FUNDING European Union; General Secretariat for Research and Technology of Greece; AIRC; Italian Ministry of Health; AIRC Regional Project with Fondazione CARIPARO and CARIVERONA; Regione Veneto on Chronic Lymphocytic Leukemia; Nordic Cancer Union; Swedish Cancer Society; Swedish Research Council; and National Cancer Institute (NIH).