Stromal cells engineered to express T cell factors induce robust CLL cell proliferation in vitro and in PDX co-transplantations allowing the identification of RAF inhibitors as anti-proliferative drugs.

Several in vitro models have been developed to mimic chronic lymphocytic leukemia (CLL) proliferation in immune niches; however, they typically do not induce robust proliferation. We prepared a novel model based on mimicking T-cell signals in vitro and in patient-derived xenografts (PDXs). Six supportive cell lines were prepared by engineering HS5 stromal cells with stable expression of human CD40L, IL4, IL21, and their combinations. Co-culture with HS5 expressing CD40L and IL4 in combination led to mild CLL cell proliferation (median 7% at day 7), while the HS5 expressing CD40L, IL4, and IL21 led to unprecedented proliferation rate (median 44%). The co-cultures mimicked the gene expression fingerprint of lymph node CLL cells (MYC, NFκB, and E2F signatures) and revealed novel vulnerabilities in CLL-T-cell-induced proliferation. Drug testing in co-cultures revealed for the first time that pan-RAF inhibitors fully block CLL proliferation. The co-culture model can be downscaled to five microliter volume for large drug screening purposes or upscaled to CLL PDXs by HS5-CD40L-IL4 ± IL21 co-transplantation. Co-transplanting NSG mice with purified CLL cells and HS5-CD40L-IL4 or HS5-CD40L-IL4-IL21 cells on collagen-based scaffold led to 47% or 82% engraftment efficacy, respectively, with ~20% of PDXs being clonally related to CLL, potentially overcoming the need to co-transplant autologous T-cells in PDXs.

Higher-order connections between stereotyped subsets: implications for improved patient classification in CLL.

Chronic lymphocytic leukemia (CLL) is characterized by the existence of subsets of patients with (quasi)identical, stereotyped B-cell receptor (BcR) immunoglobulins. Patients in certain major stereotyped subsets often display remarkably consistent clinicobiological profiles, suggesting that the study of BcR immunoglobulin stereotypy in CLL has important implications for understanding disease pathophysiology and refining clinical decision-making. Nevertheless, several issues remain open, especially pertaining to the actual frequency of BcR immunoglobulin stereotypy and major subsets, as well as the existence of higher-order connections between individual subsets. To address these issues, we investigated clonotypic IGHV-IGHD-IGHJ gene rearrangements in a series of 29 856 patients with CLL, by far the largest series worldwide. We report that the stereotyped fraction of CLL peaks at 41% of the entire cohort and that all 19 previously identified major subsets retained their relative size and ranking, while 10 new ones emerged; overall, major stereotyped subsets had a cumulative frequency of 13.5%. Higher-level relationships were evident between subsets, particularly for major stereotyped subsets with unmutated IGHV genes (U-CLL), for which close relations with other subsets, termed "satellites," were identified. Satellite subsets accounted for 3% of the entire cohort. These results confirm our previous notion that major subsets can be robustly identified and are consistent in relative size, hence representing distinct disease variants amenable to compartmentalized research with the potential of overcoming the pronounced heterogeneity of CLL. Furthermore, the existence of satellite subsets reveals a novel aspect of repertoire restriction with implications for refined molecular classification of CLL.

Profiling of biological and environmental risk factors in immunogenetic subgroups of chronic lymphocytic leukemia - Czech national study.

AIMS: This is a nation-wide survey of chronic lymphocytic leukemia (CLL) patients at six large hematology centers in the Czech Republic. The aim was to identify specific populations, social, and health characteristics of CLL subgroups divided according to the immunogenetic features of their B cell receptors (BCRs) and clonality. PATIENTS AND METHODS: Questionnaires directed to specific health, social, and environmental conditions were collected in a cohort of 573 CLL patients. For these patients, immunoglobulin heavy chain gene rearrangements were also analyzed in order to gain information about their clonality, IGHV mutational status, and the presence of stereotyped BCRs. Data extracted from the questionnaires were analyzed statistically in the context of immunogenetic features of the cohort. RESULTS: There were no statistically significant differences in the data collected in the survey between patients with mutated and patients with unmutated IGHV. However, patients with oligoclonal CLL reported health conditions such as hypercholesterolemia, hypertension, herpes simplex, tumors, and also, separately, CLL in 1st degree relatives, more often than their monoclonal counterparts. In patients with stereotyped BCRs, we found more frequent alcohol consumption and gastric infections in subset #1 cases and frequent cholecystectomies and familial CLL in subset #2 cases. CONCLUSION: To the best of our knowledge, this study is the first to investigate CLL immunogenetic features and clonality in the context of epidemiological data. We reported statistically significant associations suggesting the influence of certain health and social conditions on a number of clonal populations expanding in CLL and also on characteristic BCR features, especially stereotypy.

Impact of gene mutations and chromosomal aberrations on progression-free survival in chronic lymphocytic leukemia patients treated with front-line chemoimmunotherapy: Clinical practice experience.

The impact of genetic aberrations on rituximab-based therapeutic regimens has been intensely studied in chronic lymphocytic leukemia (CLL). According to the current consensus chemoimmunotherapy consisting of rituximab and DNA-damaging drugs is not suitable for patients with TP53 defects. In our study, we focused on CLL patients with an intact TP53 gene and investigated four recurrently mutated genes in CLL, genomic aberrations by FISH, and IGHV status with the aim of analyzing their impact on progression-free survival (PFS) after front-line therapy with FCR (fludarabine, cyclophosphamide, rituximab) or BR (bendamustine, rituximab) regimens. Using next-generation sequencing, we analyzed 120 patients treated with FCR and 57 patients treated with BR at a university hospital. We used a 10% cut-off for variant allele frequency and recorded the following mutation frequencies in the pre-therapy samples: ATM 23%, SF3B1 20%, NOTCH1 19% and BIRC3 11%. The data on cytogenetic aberrations (11q22, 13q14, trisomy 12) and IGHV mutation status were also considered in PFS analyses. In univariate analyses, we observed a negative impact of BIRC3 mutations and 11q22 deletion in both regimens, while the unmutated IGHV status was associated with a significantly shorter PFS only in the FCR-treated cohort. In a multivariate analysis, only deletion 11q22 in both regimens, and the unmutated IGHV in the FCR cohort maintained an independent association with the reduced PFS. Notably, sole 11q22 deletion, without an ATM mutation on the other allele, manifested the shortest PFS of all analyzed markers. Deletion 11q22 and IGHV status predict PFS in previously untreated CLL patients.

Fragment analysis represents a suitable approach for the detection of hotspot c.7541_7542delCT NOTCH1 mutation in chronic lymphocytic leukemia.

The hotspot c.7541_7542delCT NOTCH1 mutation has been proven to have a negative clinical impact in chronic lymphocytic leukemia (CLL). However, an optimal method for its detection has not yet been specified. The aim of our study was to examine the presence of the NOTCH1 mutation in CLL using three commonly used molecular methods. Sanger sequencing, fragment analysis and allele-specific PCR were compared in the detection of the c.7541_7542delCT NOTCH1 mutation in 201 CLL patients. In 7 patients with inconclusive mutational analysis results, the presence of the NOTCH1 mutation was also confirmed using ultra-deep next generation sequencing. The NOTCH1 mutation was detected in 15% (30/201) of examined patients. Only fragment analysis was able to identify all 30 NOTCH1-mutated patients. Sanger sequencing and allele-specific PCR showed a lower detection efficiency, determining 93% (28/30) and 80% (24/30) of the present NOTCH1 mutations, respectively. Considering these three most commonly used methodologies for c.7541_7542delCT NOTCH1 mutation screening in CLL, we defined fragment analysis as the most suitable approach for detecting the hotspot NOTCH1 mutation.

COBLL1, LPL and ZAP70 expression defines prognostic subgroups of chronic lymphocytic leukemia patients with high accuracy and correlates with IGHV mutational status.

The clinical course of chronic lymphocytic leukemia (CLL) is highly variable. Patients with unmutated IGHV (U-CLL) usually progress rapidly, whereas patients with mutated IGHV (M-CLL) have a more indolent disease. The expression of several genes correlates closely with the IGHV mutational status and could be used to assess prognosis in CLL. We analyzed the prognostic relevance of COBLL1, LPL, and ZAP70 gene expression, which correlated with IGHV mutational status (p < 0.0001), in 117 CLL patients and established a prognostic parameter dividing the tested cohort according to the disease aggressiveness. Our prognostic parameter was validated on an independent cohort of 161 CLL patients and achieved a high accuracy (94%). Patients divided according to the prognostic parameter differ in overall survival and time to first treatment (p < 0.0001, HR = 2.300/5.970, 95% CI: 1.587-3.450/4.621-15.86). Our approach provides a reliable alternative method to prognosis assessment via IGHV mutational status analysis.

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.

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.

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).

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.

Monoallelic and biallelic inactivation of TP53 gene in chronic lymphocytic leukemia: selection, impact on survival, and response to DNA damage.

Deletion of TP53 gene, under routine assessment by fluorescence in situ hybridization analysis, connects with the worst prognosis in chronic lymphocytic leukemia (CLL). The presence of isolated TP53 mutation (without deletion) is associated with reduced survival in CLL patients. It is unclear how these abnormalities are selected and what their mutual proportion is. We used methodologies with similar sensitivity for the detection of deletions (interphase fluorescence in situ hybridization) and mutations (yeast functional analysis) and analyzed a large consecutive series of 400 CLL patients; a subset of p53-wild-type cases (n = 132) was screened repeatedly during disease course. The most common type of TP53 inactivation, ie, mutation accompanied by deletion of the remaining allele, occurred in 42 patients (10.5%). Among additional defects, the frequency of the isolated TP53 mutation (n = 20; 5%) and the combination of 2 or more mutations on separate alleles (n = 5; 1.3%) greatly exceeded the sole deletion (n = 3; 0.8%). Twelve patients manifested defects during repeated investigation; in all circumstances the defects involved mutation and occurred after therapy. Monoallelic defects had a negative impact on survival and impaired in vitro response to fludarabine. Mutation analysis of the TP53 should be performed before each treatment initiation because novel defects may be selected by previous therapies.

Inactivation of p53 and amplification of MYCN gene in a terminal lymphoblastic relapse in a chronic lymphocytic leukemia patient.

B-cell chronic lymphocytic leukemia (CLL) is an incurable disease with a highly variable clinical course. A proportion of patients eventually progress to a higher stage of malignancy. A recent association has been observed between the presence of aberrant somatic hypermutations in leukemic cells (hypermutations occurring outside of the immunoglobulin locus) and the transformation to a diffuse large B-cell lymphoma or prolymphocytic leukemia. In this study, we report on the rarely observed blastic transformation in a CLL patient who had previously been shown to harbor aberrant somatic hypermutations in the TP53 tumor-suppressor gene (Mol Immunol 2008;45:1525-29). The enzyme responsible, the activation-induced cytidine deaminase, was still active within the transformation, as evidenced by the ongoing class-switch recombination of cytoplasmic immunoglobulins. The transformation was accompanied by a complete p53 inactivation, as well as complex karyotype changes including prominent amplification of MYCN oncogene. Our case-study supports the view that the aberrant somatic hypermutation is associated with transformation of CLL to a more aggressive malignancy.

Spectrum of low density lipoprotein receptor mutations in Czech hypercholesterolemic patients.

The aim of our study was to define mutations causing familial hypercholesterolemia (FH) phenotype in Czech hypercholesterolemic individuals. A combination of heteroduplex analysis, SSCP, DGGE, DNA sequencing and PCR/restriction analysis was used for this purpose. Molecular searching in the promoter region and coding sequence of the low density lipoprotein receptor (LDLR) gene in 130 patients from 68 unrelated families resulted in the identification of 37 sequence variations. Thirty of them are most likely disease causing mutations. Nineteen mutations were novel (two nonsense, five missense, six nucleotide(s) insertions and six nucleotide(s) deletions). Their pathological effect can be predicted on the basis of their position with respect to previously reported mutations with an estimated reduction of the receptor activity and/or premature termination of translation. These results expand our knowledge of mutations responsible for FH. Seven nucleotide variations were characterized as silent polymorphisms.