Molecular evidence for antigen drive in the natural history of mantle cell lymphoma.

To further our understanding about antigen involvement in mantle cell lymphoma (MCL), we analyzed the expression levels of activation-induced cytidine deaminase (AID), a key player in B-cell responses to antigen triggering, in 133 MCL cases; assessed the functionality of AID by evaluating in vivo class switch recombination in 52 MCL cases; and sought for indications of ongoing antigen interactions by exploring intraclonal diversification within 14 MCL cases. The AID full-length transcript and the most frequent splice variants (AID-ΔE4a, AID-ΔE) were detected in 128 (96.2%), 96 (72.2%), and 130 cases (97.7%), respectively. Higher AID full-length transcript levels were significantly associated (P < 0.001) with lack of somatic hypermutation within the clonotypic immunoglobulin heavy variable (IGHV) genes. Median AID transcript levels were higher in lymph node material compared to cases in which peripheral blood was analyzed, implying that clonal behavior is influenced by the microenvironment. Switched tumor-derived IGHV-IGHD-IGHJ transcripts were identified in 5 of 52 cases (9.6%), all of which displayed somatic hypermutation and AID-mRNA expression. Finally, although most cases exhibited low levels of intraclonal diversification, analysis of the mutational activity revealed a precise targeting of somatic hypermutation indicative of an active, ongoing interaction with antigen(s). Collectively, these findings strongly allude to antigen involvement in the natural history of MCL, further challenging the notion of antigen naivety.

Not all IGHV3-21 chronic lymphocytic leukemias are equal: prognostic considerations.

An unresolved issue in chronic lymphocytic leukemia (CLL) is whether IGHV3-21 gene usage, in general, or the expression of stereotyped B-cell receptor immunoglobulin defining subset #2 (IGHV3-21/IGLV3-21), in particular, determines outcome for IGHV3-21-utilizing cases. We reappraised this issue in 8593 CLL patients of whom 437 (5%) used the IGHV3-21 gene with 254/437 (58%) classified as subset #2. Within subset #2, immunoglobulin heavy variable (IGHV)-mutated cases predominated, whereas non-subset #2/IGHV3-21 was enriched for IGHV-unmutated cases (P = .002). Subset #2 exhibited significantly shorter time-to-first-treatment (TTFT) compared with non-subset #2/IGHV3-21 (22 vs 60 months, P = .001). No such difference was observed between non-subset #2/IGHV3-21 vs the remaining CLL with similar IGHV mutational status. In conclusion, IGHV3-21 CLL should not be axiomatically considered a homogeneous entity with adverse prognosis, given that only subset #2 emerges as uniformly aggressive, contrasting non-subset #2/IGVH3-21 patients whose prognosis depends on IGHV mutational status as the remaining CLL.

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).

Multiple productive immunoglobulin heavy chain gene rearrangements in chronic lymphocytic leukemia are mostly derived from independent clones.

In chronic lymphocytic leukemia, usually a monoclonal disease, multiple productive immunoglobulin heavy chain gene rearrangements are identified sporadically. Prognostication of such cases based on immunoglobulin heavy variable gene mutational status can be problematic, especially if the different rearrangements have discordant mutational status. To gain insight into the possible biological mechanisms underlying the origin of the multiple rearrangements, we performed a comprehensive immunogenetic and immunophenotypic characterization of 31 cases with the multiple rearrangements identified in a cohort of 1147 patients with chronic lymphocytic leukemia. For the majority of cases (25/31), we provide evidence of the co-existence of at least two B lymphocyte clones with a chronic lymphocytic leukemia phenotype. We also identified clonal drifts in serial samples, likely driven by selection forces. More specifically, higher immunoglobulin variable gene identity to germline and longer complementarity determining region 3 were preferred in persistent or newly appearing clones, a phenomenon more pronounced in patients with stereotyped B-cell receptors. Finally, we report that other factors, such as TP53 gene defects and therapy administration, influence clonal selection. Our findings are relevant to clonal evolution in the context of antigen stimulation and transition of monoclonal B-cell lymphocytosis to chronic lymphocytic leukemia.

Fludarabine with cytarabine followed by reduced-intensity conditioning and allogeneic hematopoietic stem cell transplantation in patients with poor-risk chronic lymphocytic leukemia.

Allogeneic stem cell transplantation (SCT) is a treatment option for patients with poor-risk chronic lymphocytic leukemia (CLL). Sequential use of chemotherapy and reduced-intensity conditioning has been proposed to improve the treatment outcomes. Fludarabine (30 mg/m(2)/day) and cytarabine (2 g/m(2)/day) for 4 days (combination of fludarabine with cytarabine; FAraC) were used for cytoreduction. After 3 days of rest, reduced intensity conditioning (RIC) was carried out consisting of 4 Gy total body irradiation, 10-20 mg/kg/day antithymocyte globulin for 3 days, and 40-60 mg/kg/day cyclophosphamide for 2 days. The median time of neutrophil engraftment was 16 days. The most frequent toxicities were grades III/IV infections in 12 of 15 cases and gastrointestinal toxicities in 8 of 15 cases. Remission (complete remission + partial remission) was achieved in 14 of 15 patients (93 %), minimal residual disease negativity according to flowcytometric analysis was observed in 10 patients. Nonrelapse mortality after 1 and 2 years was 7 and 13 %, respectively. After the median follow-up from SCT of 30 months, 80 % of patients were alive (12/15), three patients have died, and three relapses occurred. The FAraC-RIC protocol seems to be a promising approach to the treatment of poor-risk CLL with a high response rate of 93 % and favorable progression-free survival and overall survival of 70 and 85 % at 2 years after SCT, respectively. Other prospective clinical trials are needed to confirm the results of this novel therapeutic strategy.

Missense mutations located in structural p53 DNA-binding motifs are associated with extremely poor survival in chronic lymphocytic leukemia.

PURPOSE: There is a distinct connection between TP53 defects and poor prognosis in chronic lymphocytic leukemia (CLL). It remains unclear whether patients harboring TP53 mutations represent a homogenous prognostic group. PATIENTS AND METHODS: We evaluated the survival of patients with CLL and p53 defects identified at our institution by p53 yeast functional assay and complementary interphase fluorescence in situ hybridization analysis detecting del(17p) from 2003 to 2010. RESULTS: A defect of the TP53 gene was identified in 100 of 550 patients. p53 mutations were strongly associated with the deletion of 17p and the unmutated IgVH locus (both P < .001). Survival assessed from the time of abnormality detection was significantly reduced in patients with both missense (P < .001) and nonmissense p53 mutations (P = .004). In addition, patients harboring missense mutation located in p53 DNA-binding motifs (DBMs), structurally well-defined parts of the DNA-binding domain, manifested a clearly shorter median survival (12 months) compared with patients having missense mutations outside DBMs (41 months; P = .002) or nonmissense alterations (36 months; P = .005). The difference in survival was similar in the analysis limited to patients harboring mutation accompanied by del(17p) and was also confirmed in a subgroup harboring TP53 defect at diagnosis. The patients with p53 DBMs mutation (at diagnosis) also manifested a short median time to first therapy (TTFT; 1 month). CONCLUSION: The substantially worse survival and the short TTFT suggest a strong mutated p53 gain-of-function phenotype in patients with CLL with DBMs mutations. The impact of p53 DBMs mutations on prognosis and response to therapy should be analyzed in investigative clinical trials.

High expression of lymphocyte-activation gene 3 (LAG3) in chronic lymphocytic leukemia cells is associated with unmutated immunoglobulin variable heavy chain region (IGHV) gene and reduced treatment-free survival.

Chronic lymphocytic leukemia (CLL) is characterized by a monoclonal expansion of mature B-lymphocytes. Mutational status of the immunoglobulin variable heavy chain region (IGHV) gene stratifies CLL patients into two prognostic groups. We performed microarray analysis of CLL cells using the Agilent platform to detect the most important gene expression differences regarding IGHV status in CLL cells. We analyzed a cohort of 118 CLL patients with different IGHV mutational status and completely characterized all described prognostic markers using expression microarrays and quantitative real-time RT-PCR (reverse transcription PCR). We detected lymphocyte-activation gene 3 (LAG3) as a novel prognostic marker: LAG3 high expression in CLL cells correlates with unmutated IGHV (P < 0.0001) and reduced treatment-free survival (P = 0.0087). Furthermore, quantitative real-time RT-PCR analysis identified a gene-set (LAG3, LPL, ZAP70) whose overexpression is assigned to unmutated IGHV with 90% specificity (P < 0.0001). Moreover, high expression of tested gene-set and unmutated IGHV equally correlated with reduced treatment-free survival (P = 7.7 * 10(-11) vs. P = 1.8 * 10(-11)). Our results suggest that IGHV status can be precisely assessed using the expression analysis of LAG3, LPL, and ZAP70 genes. Expression data of tested markers provides a similar statistical concordance with treatment-free survival as that of the IGHV status itself. Our findings contribute to the elucidation of CLL pathogenesis and provide novel prognostic markers for possible application in routine diagnostics.