Chronic Lymphocytic Leukemia Subset Research Articles

Temporal Dynamics of Clonal Evolution in Chronic Lymphocytic Leukemia with Stereotyped IGHV4-34/IGKV2-30 Antigen Receptors: Longitudinal Immunogenetic Evidence

Chronic lymphocytic leukemia (CLL) patients assigned to stereotyped subset 4 possess distinctive patterns of intraclonal diversification (ID) within their immunoglobulin (IG) genes. Although highly indicative of an ongoing response to antigen(s), the critical question concerning the precise timing of antigen involvement is unresolved. Hence, we conducted a large-scale longitudinal study of eight subset 4 cases totaling 511 and 398 subcloned IG heavy and kappa sequences. Importantly, we could establish a hierarchical pattern of subclonal evolution, thus revealing which somatic hypermutations were negatively or positively selected. In addition, distinct clusters of subcloned sequences with cluster-specific mutational profiles were observed initially; however, at later time points, the minor cluster had often disappeared and hence not been selected. Despite the high intensity of ID, it was remarkable that certain residues remained essentially unaltered. These novel findings strongly support a role for persistent antigen stimulation in the clonal evolution of CLL subset 4.

Distinct transcriptional control in major immunogenetic subsets of chronic lymphocytic leukemia exhibiting subset-biased global DNA methylation profiles

Chronic lymphocytic leukemia (CLL) can be divided into prognostic subgroups based on the IGHV gene mutational status, and is further characterized by multiple subsets of cases with quasi-identical or stereotyped B cell receptors that also share clinical and biological features. We recently reported differential DNA methylation profiles in IGHV-mutated and IGHV-unmutated CLL subgroups. For the first time, we here explore the global methylation profiles of stereotyped subsets with different prognosis, by applying high-resolution methylation arrays on CLL samples from three major stereotyped subsets: the poor-prognostic subsets #1 (n = 15) and #2 (n = 9) and the favorable-prognostic subset #4 (n = 15). Overall, the three subsets exhibited significantly different methylation profiles, which only partially overlapped with those observed in our previous study according to IGHV gene mutational status. Specifically, gene ontology analysis of the differentially methylated genes revealed a clear enrichment of genes involved in immune response, such as B cell activation (e.g., CD80, CD86 and IL10), with higher methylation levels in subset #1 than subsets #2 and #4. Accordingly, higher expression of the co-stimulatory molecules CD80 and CD86 was demonstrated in subset #4 vs. subset #1, pointing to a key role for these molecules in the crosstalk of CLL subset #4 cells with the microenvironment. In summary, investigation of three prototypic, stereotyped CLL subsets revealed distinct DNA methylation profiles for each subset, which suggests subset-biased patterns of transcriptional control and highlights a key role for epigenetics during leukemogenesis.

PDE4 Inhibitors Block TLR7 and TLR9-Driven Signaling, Proliferation and Cytokine Secretion in CLL Cells As Well As Proliferation Driven by Exposure to Apoptotic Cells

AbstractAbstract 1774A growing body of literature supports the concept that at least a subset of chronic lymphocytic leukemia (CLL) B cell-receptors (BCRs) are reactive with antigens exposed on apoptotic cells. Work done in the autoimmunity field suggests that through their BCRs, autoreactive B cells can internalize RNA and DNA-containing autoantigens, thereby activating endosomal Toll-like receptor (TLR) 7 and TLR9, respectively, with subsequent TLR-mediated signaling, proliferation and cytokine secretion. cAMP signaling modulates inflammatory signaling in immune system cells and our prior studies have shown that PKA, EPAC and CREB-mediated cAMP signaling can be potently induced in CLL cells with inhibitors of the cyclic nucleotide phosphodiesterase (PDE) PDE4 family in the absence of additional exogenous stimulators of adenylate cyclase. We therefore examined the ability of PDE4 inhibitor-mediated cAMP signaling to regulate TLR7 and 9-mediated signaling in CLL cells. Treatment of CLL cells with the TLR7 and 9 agonists R848 and CpG-B oligonucleotide, respectively, induced IRAK1 degradation and activation of three signaling pathways known to be downstream of TLR7 and 9: the transcription factors NFkB and IRF5 as well as the MAP kinase JNK. The prototypic PDE4 inhibitor rolipram inhibited R848-induced TLR7 and CpG-B-induced TLR9 signaling in CLL cells as judged by Western analysis and immunofluorescent studies of NFkB and IRF5 nuclear translocation as well as JNK phosphorylation. Rolipram also blocked R848 and CpG-B-induced CLL proliferation and TNF secretion. In contrast, rolipram augmented R848 and CpG-B-induced CLL IL-10 synthesis, in keeping with prior studies documenting cAMP response elements in the IL-10 promoter. To test whether RNA and/or DNA antigens exposed in apoptotic cells might induce proliferation in CLL cells through BCR-mediated internalization and subsequent activation of TLR7 and TLR9, we irradiated CLL cells and added them in a graded fashion to live autologous CLL cells. Such exposure to irradiated cells undergoing apoptosis drove CLL proliferation in a manner that was sensitive to TLR7 and TLR9 antagonists, IRAK4 kinase inhibitors and the PDE4 inhibitor rolipram. Our work supports the concept that BCR and TLR-driven responses to apoptotic RNA and DNA-containing complexes may drive proliferation in CLL cells and that PDE4 inhibitors and other agents that target TLR signaling may prove to be effective therapeutic agents in this disease. Disclosures:No relevant conflicts of interest to declare.

CD49d Is Overexpressed in Trisomy 12 Chronic Lymphocytic Leukemia by an Epigenetic-Dependent Transcriptional Control

AbstractAbstract 929CD49d is an adhesion molecule with a variable expression in chronic lymphocytic leukemia (CLL). Only one third of CLL display CD49d expression in a significant fraction of cells, the remaining cases either expressing very low levels or completely lacking its expression. The negative prognostic impact of CD49d in CLL and its key role in microenvironmental interactions have been broadly described by several studies, whereas information on the mechanisms regulating CD49d expression in CLL is still lacking.Aiming to understand the genetic mechanisms underlying the different expression pattern of CD49d in CLL, CD49d expression was investigated by flow cytometry in the neoplastic component of 972 CLL patients, and correlated with cytogenetic profiles assessed by fluorescence in situ hybridization (FISH). Using the 30% positive cut-off value, 603 cases (62%) were CD49d-, whereas 369 cases (38%) were CD49d+. Correlation of CD49d expression with the major cytogenetic alterations (del13q14.3, trisomy 12, del11q22-q23-, del17p13.1), highlighted a very strong association between CD49d expression and the presence of trisomy 12 (p<0.0001). In particular, high CD49d expression was found in 93/113 cases (82.3%) with trisomy 12 alone, and 140/170 cases (82.3%) bearing trisomy 12 either alone or in the presence of other cytogenetic lesions. Notably, 50% (15/30) of CD49d- cases bearing trisomy 12 displayed a CD49d bimodal expression, with a CD49d positive population ranging from 10 to 28%. Even in the context of CD49d+ CLL, trisomy 12 CLL showed significantly higher CD49d mean fluorescence intensity (MFI) levels (MFI=2323±149, n=56) as compared to CLL cases with normal FISH (MFI=1530±118, n=56, p=0.0001). The association between trisomy 12 and CD49d expression was corroborated by results from FISH analysis in the flow cytometrically sorted (>99% purity) CD49d- and CD49d+ components from 3 CLL cases characterized by both CD49d bimodal expression (27%, 57%, and 75% of CD49d positive cells), and the presence of trisomy 12 in a fraction of the cells (25%, 42% and 63% respectively). In all cases, the CD49d- components almost completely lacked trisomy 12 (5%, 4%, and 0%), whereas the CD49d+ fractions displayed 70%, 62%, and 75% of trisomy 12, respectively (p<0.0001).A gene expression profiling was performed comparing CD49d+/trisomy12 (n=5) versus CD49d-/normal FISH CLL (n=5). According to bioinformatics tools for global analysis of gene function, the “Chromatin modification” (p<0.0001), “Methyltransferase activity” (p=0.017), and “Histone deacetylase (HDAC) activity” (p<0.0001) categories were found among the top-ranked Gene Ontology categories selected as containing differentially expressed genes. These categories included the genes of lysine demethylases KDM5A (fold change=1.8, p=0.01) and KDM2B (fold change=1.6, p=0.007), and lysine methyltransferase SETD8 (fold change=1.4, p<0.001), all up-regulated in the trisomy 12 group and mapping on chromosome 12, and the methyltransferase DNMT3A, down-regulated in trisomy 12 CLL (fold change=-2.4, p=0.01). Collectively, these results point to an epigenetic regulation of CD49d expression in CLL. To address this point, DNA methylation was studied within the CpG island (68 CpGs) of the CD49d gene 5’UTR region (725 pb before the translation start codon ATG) in CD49d+/trisomy12 (n=10) and CD49d-/normal FISH (n=9) purified CLL cells, by means of bisulfite genomic sequencing (at least 10 clones per sample). The CD49d-/normal FISH group showed a significantly higher degree of methylation of the CD49d 5’ UTR gene in comparison to the CD49d+/trisomy12 group (average amount of methylated CpGs=9.8% vs. 0.6%, p<0.0001). Focusing on a smaller region (214 bp before ATG) consisting in 24 CpGs, even higher methylation levels (average amount of methylated CpGs= 13.8%) were found in the CD49d-/normal FISH group.Altogether, our results demonstrate that the overall down-regulated CD49d expression characterizing CLL is subjected to epigenetic control. Re-expression of CD49d may occur through a fine regulation of methyltransferase and chromatin modification processes, particularly active in trisomy 12 CLL, where CD49d expression may have a role in determining the clinical and biological features of this particular CLL subset. Disclosures:No relevant conflicts of interest to declare.

Distinct Profiles of in Vivo Class Switch Recombination in Chronic Lymphocytic Leukemia Subsets with Stereotyped B Cell Receptors, Suggestive of Distinct Modes of Activation by Antigen

AbstractAbstract 1777Antigen stimulation through the B cell receptor (BcR) is critical in CLL ontogeny as evidenced by the distinct prognosis of cases with different mutational status of the clonotypic BcRs and the existence of subsets of patients with stereotyped BcRs. Of note, for several subsets analyzed thus far, stereotypy extends from shared primary BcR sequences to shared clinicobiological features and, even, outcome. Hence, it may be hypothesized that the clinical course of CLL perhaps reflects the outcome of ongoing BcR signaling in the context of other co-signals. Previous studies have documented high expression of activation-induced cytidine deaminase (AID) and in vivo class switch recombination (CSR) in a proportion of CLL cases, especially those with unmutated IGHV genes (U-CLL). Here we sought evidence of isotype switch events in 486 CLL cases, by far the largest series analyzed to-date, focusing on potential associations with the expression of particular BcRs. All 486 cases were selected due to expressing MD isotypes as determined by peripheral blood flow cytometry (FC) (298/486 caes; 61.3%) or for expressing tumor-specific Cμ and Cδ transcripts as determined by RT-PCR (188/486 cases; 38.7%). They were all subjected to RT-PCR for μ, δ, α and γ transcripts utilizing primers specific for the respective C genes along with leader primers corresponding to the IGHV subgroup of the tumor-specific VDJ rearrangement. All obtained RT-PCR products were sequenced in order to determine the correspondence with the clone-specific VDJ rearrangement. Using the 98% cut-off value for identity to the germline (GI), 340/468 cases (70%) expressed mutated IGHV genes (M-CLL), whereas the remaining (146/486 cases, 30%) cases concerned U-CLL. Stereotyped BcRs characteristic of major CLL subsets were expressed by 71 cases: subset #1, n=25; subset #2, n=23; subset #3, n=8; subset #6, n=7; subset #7, n=8. Except for subset #2 (IGHV3–21), all other subsets concerned U-CLL; subset #3, 6 and 7 BcRs utilize the IGHV1–69 gene, whereas BcRs of subset #1, the largest CLL subset overall, utilize IGHV genes of phylogenetic clan I (IGHV1/5/7). Overall, RT-PCR products for Cα and/or Cγ transcripts in addition to Cμ/Cδ were obtained in 78/486 cases (16%); in 55/78 cases, transcripts were tumor-specific VDJ sequences, for an overall frequency of 11.3% (55/486 cases). In particular, dominant cDNA bands of α isotypes were found in 27/486 cases (5.6%), dominant cDNA bands of γ isotypes were found in 21/486 cases (4.3%), while cDNA bands for both α and γ isotypes were found in 7/486 cases (1.4%). In keeping with the literature, the frequency of isotype switch events was significantly higher in U-CLL vs. M-CLL (33/146 vs. 22/340 cases, respectively; p<0.001). Of great interest, a significantly different frequency of such events was also observed in distinct stereotyped subsets. In particular, alternative tumor-derived Cα and/or Cγ transcripts were detected in 13/25 subset #1 cases vs. 0/23 subset #2 vs. 2/8 subset #3 vs. 0/7 subset #6 vs. 3/8 subset #8. In conclusion, we confirm and significantly extend previous reports that ongoing CSR occurs in a sizeable fraction of CLL, mostly U-CLL. Importantly, we provide for the first time evidence for subset-biased profiles of in vivo CSR, documenting a very high frequency of alternative tumor-derived transcripts in certain subsets, especially subset #1, reported to exhibit a particularly aggressive clinical behavior. Altogether, these findings link distinct CLL BcRs exhibiting particular biological features and behavior to specific modes of (micro)environmental interactions leading to clonal evolution and, perhaps, adverse outcome. Disclosures:No relevant conflicts of interest to declare.

Expression of ZAP-70 Does Not Accelerate Leukemia Development and Progression in the Eμ-TCL1 Transgenic Mouse Model of Chronic Lymphocytic Leukemia

AbstractAbstract 925One of the most relevant prognostic factors in chronic lymphocytic leukemia (CLL) is expression of the protein tyrosine kinase ZAP-70. Typically, patients whose leukemic cells express ZAP-70 at 30–100% of the levels in normal T cells have aggressive disease, whereas patients whose leukemic cells do not express ZAP-70 or express only low levels of this protein have indolent disease. Previously, we and others demonstrated that ZAP-70 modulates B-cell receptor signaling and thus affects the capacity of the leukemic cells to respond to antigen stimulation. However, a direct link between an altered antigen response and CLL pathogenesis has still not been established and, more importantly, the question whether ZAP-70 directly contributes to the aggressiveness of the disease or is just a marker of aggressive CLL still remains to be answered.We have now addressed these issues by analyzing in vivo the impact of forced expression of ZAP-70 on the development and behavior of leukemias that arise in the Eμ-TCL1 transgenic (tg) mouse model of CLL. This model is characterized by the development of antigen-driven leukemias that resemble human CLL in many aspects but are always ZAP-70-negative. To force the expression of ZAP-70 in TCL1 leukemias, we generated two tg lines with targeted expression of ZAP-70 in the B cell compartment (ZAP70high and ZAP70low) and crossed them with Eμ-TCL1 tg mice. B cells in ZAP70high tg mice express similar levels of ZAP-70 as normal mouse T cells, whereas the levels of ZAP-70 in B cells of ZAP70lowtg mice are approximately 10 times lower.Both cohorts of Eμ-TCL1/ZAP70 double tg mice developed characteristic TCL1 leukemias. Eμ-TCL1/ZAP70low tg mice developed leukemias with onset and rate of progression similar to their ZAP-70-negative littermates, indicating that low levels of ZAP-70 do not alter the development and behavior of the disease. Surprisingly, Eμ-TCL1/ZAP70high tg mice developed leukemias with an approximately 2 month delay compared to their ZAP-70-negative Eμ-TCL1 tg littermates, which was contrary to the expectation that high ZAP-70 expression will accelerate leukemia development. The delay in leukemia development was especially evident at 6 months of age, when leukemic cells could be detected in the PB of 77% (10/13) of Eμ-TCL1 tg mice and only 24% (4/17) of Eμ-TCL1/ZAP70hightg mice (P=0.011).Since ZAP-70 expression can affect the migratory and adhesion capacity of human CLL cells in vitro, we first investigated if the delayed appearance of leukemic cells in the PB of Eμ-TCL1/ZAP70high tg mice could be due to increased retention of the leukemic cells in the lymphoid tissues. Assessment of tumor burden in the spleen, peritoneal cavity (PC), bone marrow and PB of 7 months old mice showed that the number of tumor cells in each compartment was significantly lower in Eμ-TCL1/ZAP70hightg mice than their Eμ-TCL1 littermates, suggesting that the delay in leukemia appearance is not caused by increased tissue retention but rather by reduced tumor growth.To investigate if ZAP-70 impairs tumor growth by affecting proliferation, we performed in vivo BrdU incorporation analysis of leukemic cells from spleen and PC of Eμ-TCL1 and Eμ-TCL1/ZAP70high tg mice. Spleen and PC samples were analyzed because they are the major sites of leukemia proliferation in Eμ-TCL1 tg mice. Interestingly, while the percentage of proliferating leukemic cells in the spleens of Eμ-TCL1 and Eμ-TCL1/ZAP70high tg mice was similar (mean % of BrdU+ cells ±SD: 6.81 ±1.67 and 6.15 ±2.92, respectively; P=n.s.), the percentage of proliferating leukemic cells in the PC of Eμ-TCL1/ZAP70high tg mice was significantly lower (mean % of BrdU+cells ±SD: 1.74 ±1.05 and 0.56 ±0.39, respectively; P=0.024).In summary, this study shows that ZAP-70 expression, per se, is unable to accelerate leukemia development and progression in an established in vivo model of CLL and suggests that ZAP-70 is not directly responsible for the greater disease severity in the poor prognosis subset of CLL. In addition, this study reveals that ZAP-70 in certain tissue environments can function as a negative regulator of leukemic cell proliferation, contrary to the widespread perception of ZAP-70 as a positive regulator of leukemic cell responses. Disclosures:No relevant conflicts of interest to declare.

Gene Expression Profiles Document That Recently- and Previously-Divided CLL Fractions Represent a Continuum but Suggest Differing Modes of Activation for These Fractions in U-CLL and M-CLL

AbstractAbstract 317By using reciprocal densities of surface membrane CXCR4 and CD5, chronic lymphocytic leukemia (CLL) B cells can be divided into 3 fractions indicating time since last division (proliferative, intermediate, and resting). It has been suggested that cells in these fractions represent a continuum from resting to intermediate to proliferative. In this study, we made intraclonal gene expression profile (GEP) comparisons of these fractions from 17 CLL patients to try to confirm this notion and interclonal comparisons between U-CLL and M-CLL patients to determine if pathways involved in the actions of these fractions differed between patient subgroups.PBMCs from 8 U-CLL and 9 M-CLL patients were sorted into 3 fractions (CD19+CD3−CD5hiCXCR4lo, PROLIF), (CD19+CD3−CD5intCXCR4int, INTERM), and (CD19+CD3−CD5loCXCR4hi, REST); RNA was purified from each, and gene expression microarrays using Illumina HumanHT12 beadchips performed. To determine differentially expressed genes in intraclonal comparisons, expression value ratios for fractions from each patient were computed, log-transformed, and Student t-test performed using R (www.r-project.org); for interclonal comparisons, raw GEP data between subpopulations were compared: U-PROLIF and M-PROLIF, and U-REST and M-REST. Sets of significant genes (≥1.5 fold change and P<0.01) were analyzed using Ingenuity Pathway Analysis (IPA) and Gene Set Enrichment Analysis (GSEA).Upon plotting intraclonal average log ratios of PROLIF/INTERM vs INTERM/REST, it was clear that gene expression levels changed in the same direction, i.e. PROLIF>INTERM>REST, or PROLIF<INTERM<REST, consistent with a continuum between the 3 fractions. Within this pattern, 36 genes were significant for both plotted ratios. Of these, 29 were overexpressed, along with CD5; CD68, ITGAX, CCND2, CRIP1 and LGALS1 were the highest. Functional analysis using IPA showed these genes to be related to NFkB signaling and cell trafficking. Seven genes (ADARB1, BACH2, CNTNAP2, HRK, RHPN2, PRPML, and RXPA) were significantly downregulated, along with CXCR4.Next we characterized GEP differences between the PROLIF and REST fractions, identifying 390 genes up-regulated in PROLIF and 244 in REST. The top 5 upregulated PROLIF genes were CD68, LY96, ITGAX, CCND2 and CRIP1, and the top 5 REST genes were BACH2, CXCR4, ADARB1, RHPN2 and HRK. Functionally, the upregulated PROLIF genes were related to BCR signaling, cytokines (IFNa, IL12), NFkB, and Akt, whereas the upregulated REST genes related to BCL2, cell death and cell movement. By GSEA, 813/881 gene sets, defined by expression neighborhoods centered on cancer associated genes, were upregulated in the PROLIF with 436 gene sets significant at a false discovery rate (FDR) <10%; 206 sets were significantly enriched with p value <0.01. For the REST, 68/881 gene sets were upregulated, with none significant even at FDR <25%.Finally, we examined PROLIF and REST fractions from U-CLL vs M-CLL patients. In this interclonal analysis, 93 genes were significantly different between U-PROLIF and M-PROLIF. The top 5 in U-PROLIF were MSI2, TGFBR3, TP53I3, RGCC and IGSF3, and the top 5 in M-PROLIF were MTSS1, BACE2, BRI3BP, AP3B1 and UBE2G2. Similarly, there were 125 genes that were significantly different between U-REST and M-REST. The top 5 in U-REST were DUSP26, CLEC2B, MDK, and EGR2 and in M-REST were NAPSA, RAB24, TARDBP, KCNN4 and ADD3. Interestingly, U-PROLIF and M-PROLIF differed in pathway assignments, with upregulated genes in U-PROLIF contributing to cell signaling and activation, particularly implicating Akt, ERK and P38MAPK.The intraclonal gene GEP analysis on these 3 fractions confirms that CLL clones contain a spectrum of cells that transition in a sequential manner from PROLIF to INTERM to REST fractions. Functional analyses show that genes upregulated in PROLIF correlate with cell signaling and proliferation, while genes upregulated in REST relate to cell death. Thus the PROLIF fraction is enriched in recently divided cells that likely exit from lymphoid tissue and the REST in older, less vital cells that either traffic to lymphoid tissue or die. The interclonal analysis implies that the stimuli and/or the responses of cells in the PROLIF and REST fractions differ between U-CLL and M-CLL. This last novel finding suggests either distinct cells of origin or distinct activation pathways for the IGHV-defined CLL subsets. Disclosures:Barrientos:gilead and pharmacyclics research funding: Research Funding.

Active Crosstalk with the Microenvironment Leading to Clonal Evolution in Chronic Lymphocytic Leukemia with Stereotyped IGHV4–34/IGKV2–30 Antigen Receptors.

AbstractAbstract 2878We recently demonstrated that intraclonal diversification (ID) in the immunoglobulin (IG) genes of patients with chronic lymphocytic leukemia (CLL) was limited, with the outstanding exception of subset #4 cases (IGHV4–34/IGKV2–30). Subset #4 cases express IgG-switched antigen receptors carrying long VH CDR3s enriched in positively charged amino acid residues (especially arginine), with acidic residues introduced by somatic hypermutation (SHM) in critical positions of both the heavy and light chain variable domains. This group of patients, characterized clinically by an early age at diagnosis and an indolent disease course, exhibited distinctive patterns of intraclonal diversification (ID) within their IG genes. This may be considered as evidence for an ongoing response to active interaction with antigen (Ag), however, the critical question about the precise timing of Ag involvement and its role in clonal evolution remains unknown. To obtain insight into these issues, we conducted a large-scale subcloning study of the IG genes in a total of 514 and 398 subcloned IG heavy and kappa sequences, obtained from overtime samples from 8 subset #4 cases. All non-ubiquitous sequence changes from the germline among subcloned sequences of the same patient from the same timepoint were evaluated and recorded as follows: (i) unconfirmed mutation (UCM) - a mutation observed in only one subcloned sequence; (ii) confirmed mutation (CM) - a mutation observed in more than one but in less than all subcloned sequences. Overall, all cases carried intraclonally diversified IG genes. Detailed analysis of the topology and characteristics of mutations revealed: i) restricted ID patterns, in the sense of identical mutations at certain VH/VK positions amongst subclones of different cases, e.g. despite glycine at codon 28 (VH CDR1) being mutated in 413/514 (80%) sequences, the only observed substitution was to an acidic residue, ii) ID hotspots, i.e. mutations in certain codons were repeatedly observed during clonal evolution, and iii) predominance of conservative amino acid changes. Furthermore, the analysis of consecutive samples enabled us to trace the diversification of the CLL antibody over time, and describe the level of ID as increasing, decreasing, or complex when a mutation appears, disappears and then re-emerges at a subsequent time point, thereby also revealing which mutations were negatively or positively selected. Consequently, a stepwise accumulation of mutations could be observed with several CMs at an early timepoint becoming ubiquitous mutations, i.e. present in all subcloned sequences of subsequent timepoints. In addition, distinct clusters of subcloned sequences with cluster-specific mutational profiles were observed initially, however at later timepoints the minor cluster had often disappeared and hence been selected against. Despite the high intensity of ID, it was remarkable that certain residues such as in the VH FR1 motif responsible for recognizing the I/i NAL epitope remained essentially unaltered (only 6/514 sequences carried alterations at codon 7 VH FR1). In conclusion, this study defines a clear role for Ag selection in the clonal evolution of CLL subset #4. Whilst the critical eliciting Ag cannot be definitively determined, it is tempting to hypothesize that the distinctive modifications introduced by SHM in the stereotyped BcR likely represent a mechanism for negating auto-reactivity mediated by the arginine-rich VH CDR3s and inducing an anergic state which could potentially be re-activated by subsequent (auto)antigenic stimulation as evidenced by ongoing SHM. It remains to be established whether this stimulation is also accompanied by a change in functional status. Disclosures:No relevant conflicts of interest to declare.

Incidence and clinical implications of ATM aberrations in chronic lymphocytic leukemia

A subset of chronic lymphocytic leukemia (CLL) carries mutations in ataxia telangiectasia mutated (ATM). Such ATM mutations may be particularly relevant in the setting of del11q, which invariably results in the deletion of one ATM allele. To improve our understanding of the frequency and type of ATM mutations that exist in CLL, we resequenced all ATM coding exons in 24 CLL with del11q using direct sequencing. We detected two missense mutations, resulting in an ATM mutation frequency of 8%; nonsense and frameshift mutations were not identified. Given the low ATM mutation frequency detected in this cohort, we proceeded with measurements of nonmutational ATM aberrations in CLL through analysis of the activation state of ATM in response to external irradiation. The phosphorylation state of ATM at Ser-1981 was measured using quantitative immunoblotting in purified CLL cells isolated from 251 CLL patients; data were normalized to simultaneous measurements of total ATM protein and actin. Resulting p-ATM/ATM and p-ATM/actin ratios were subsequently analyzed for prognostic significance inclusive and exclusive of TP53 exons 2-10 mutations. From these analyses, conducted in a large prospectively enrolled CLL patient cohort, neither the p-ATM/ATM nor the p-ATM/actin ratios were found to be prognostic for short survival. These data in aggregate demonstrate a low frequency of ATM aberrations in an unselected CLL cohort and do not support a major prognostic role for ATM aberrations in CLL, thus motivating renewed research efforts aimed at understanding the pathobiology of 11q deletions in CLL. © 2012 Wiley Periodicals, Inc.

Alemtuzumab use in relapsed and refractory chronic lymphocytic leukemia: a history and discussion of future rational use

In this review, we outline the clinical experience with single-agent alemtuzumab as a treatment for relapsed and refractory chronic lymphocytic leukemia (CLL) in both prospective and retrospective trials and describe the multiagent use of the drug with the goal of updating clinicians on recent developments and possible future rational combinations. Alemtuzumab, an antibody targeting the lymphocyte-specific surface marker CD52, is an approved agent for the treatment of CLL. Despite its demonstrated efficacy, likely secondary to concerns regarding infectious complications, it is most commonly used in the relapsed and refractory setting. Given alemtuzumab's unique mechanism of action it has been demonstrated to have activity in disease that is refractory to both alkylating agents and purine analogs. Furthermore, it has activity in TP53-mutated disease, which has the worst prognosis of any subset of CLL. Alemtuzumab has greater efficacy on circulating disease relative to nodal disease. Rational combinations are attempting to use these attributes to increase response rates in patients with relapsed and refractory disease.

A novel chronic lymphocytic leukemia subset expressing mutated IGHV3-7-encoded rheumatoid factor B-cell receptors that are functionally proficient

A novel chronic lymphocytic leukemia subset expressing mutated IGHV3-7-encoded rheumatoid factor B-cell receptors that are functionally proficient

EVI-1 modulates leukemogenic potential and apoptosis sensitivity in human acute lymphoblastic leukemia

The transcriptional regulator ecotropic viral integration site-1 (EVI-1) has mainly been studied for its role in myeloid malignancies, in which high EVI-1 levels are associated with particularly aggressive disease. The role of EVI-1 in lymphoid cells, however, is largely unknown. Here we show that EVI-1 is indeed expressed in lymphoid malignancies such as acute lymphoblastic leukemia (ALL) and a subset of chronic lymphocytic leukemia. Expression data from pediatric ALL further suggest that high EVI-1 levels are associated with poor prognosis. Suppression of EVI-1 expression by RNA interference reduces cell growth and enhances apoptosis sensitivity in response to various stimuli in lymphoblastic leukemia cells. At the molecular level, EVI-1 modulates expression of several apoptosis-related genes (such as BCL2, BCL-x, XIAP, NOXA, PUMA, TRAIL-R1). Furthermore, EVI-1 knockdown strongly impairs in vivo engraftment of lymphoblastic leukemia cells upon transplantation in immune-permissive NOD/SCID/IL2Rγ(null) mice, conferring a survival benefit when compared with mice transplanted with control cells. Thus, our data show that EVI-1 is expressed not only in myeloid but also in lymphoid leukemias, and contributes to the leukemogenic potential and apoptosis resistance of ALL cells.

Next generation RNA‐sequencing in prognostic subsets of chronic lymphocytic leukemia

Advances in next-generation RNA-sequencing have revealed the complexity of transcriptomes by allowing both coding and noncoding(nc)RNAs to be analyzed. However, limited data exist regarding the whole transcriptional landscape of chronic lymphocytic leukemia(CLL). In this pilot-study, we evaluated RNA-sequencing in CLL by comparing two subsets which carry almost identical or "stereotyped" B-cell receptors with distinct clinical outcome, that is the poor-prognostic subset #1 (n 5 4) and the more favorable-prognostic subset #4(n 5 4). Our analysis revealed that 156 genes (e.g. LPL, WNT9A) and 76 ncRNAs, (e.g. SNORD48, SNORD115) were differentially expressed between the subsets. This technology also enabled us to identify numerous subset-specific splice variants (n 5 406), which were predominantly expressed in subset #1, including a splice-isoform of MSI2 with a novel start exon. A further important application of RNA-sequencing was for mutation detection and revealed 16–30 missense mutations per sample; notably many of these changes were found in genes with a strong potential for involvement in CLL pathogenesis, e.g., ATM and NOTCH2.This study not only demonstrates the effectiveness of RNA-sequencing for identifying mutations, quantifying gene expression and detecting splicing events, but also highlights the potential such global approaches have to significantly advance our understanding of the molecular mechanisms behind CLL development.

Mantle cell lymphoma displays a homogenous methylation profile: A comparative analysis with chronic lymphocytic leukemia

Mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL) are mature CD5(+) B-cell malignancies with different biological/clinical characteristics. We recently reported an association between different prognostic subgroups of CLL (i.e., IGHV mutated and unmutated) and genomic methylation pattern. However, the relationship between DNA methylation and prognostic markers, such as the proliferation gene expression signature, has not been investigated in MCL. We applied high-resolution methylation microarrays (27,578 CpG sites) to assess the global DNA methylation profiles in 20 MCL (10 each with high/low proliferation signature) and 30 CLL (15 poor-prognostic IGHV unmutated subset #1 and 15 good-prognostic IGHV mutated subset #4) samples. Notably, MCL and each CLL subset displayed distinct genomic methylation profiles. After unsupervised hierarchical clustering, 17/20 MCL cases formed a cluster separate from CLL, while CLL subsets #1 and #4 formed subclusters. Surprisingly, few differentially methylated genes (n = 6) were identified between high vs. low proliferation MCL. In contrast, distinct methylation profiles were demonstrated for MCL and CLL. Importantly, certain functional classes of genes were preferentially methylated in either disease. For instance, developmental genes, in particular homeobox transcription factor genes (e.g., HLXB9, HOXA13), were more highly methylated in MCL, whereas apoptosis-related genes were enriched among targets methylated in CLL (e.g., CYFIP2, NR4A1). Results were validated using pyrosequencing, RQ-PCR and reexpression of specific genes. In summary, the methylation profile of MCL was homogeneous and no correlation with the proliferation signature was observed. Compared to CLL, however, marked differences were discovered such as the preferential methylation of homeobox genes in MCL.

Disruption of BIRC3 associates with Fludarabine Chemorefractoriness in TP53 Wild Type Chronic Lymphocytic Leukemia

Abstract 466The genetic lesions identified to date do not fully recapitulate the molecular pathogenesis of chronic lymphocytic leukemia (CLL) and do not entirely explain the development of severe complications, such as chemorefractoriness. BIRC3, along with TRAF2 and TRAF3, cooperate in the same protein complex that negatively regulates MAP3K14, the central activator of non-canonical NF-kB signaling. Following the initial observation of recurrent BIRC3 mutations in splenic marginal zone lymphomas, we performed targeted re-sequencing of the BIRC3 coding sequence and splicing sites across the spectrum of B-cell neoplasia. By this screening, BIRC3 mutations were identified only in CLL (2/20), while were absent in diffuse large B-cell lymphoma (0/30), Burkitt lymphoma (0/38), follicular lymphoma (0/20), extranodal marginal zone lymphoma (0/65), hairy cell leukemia (0/19), and multiple myeloma (0/22). This observation prompted the investigation of prevalence and clinical impact of BIRC3 genetic lesion in CLL. The study population included 4 clinical CLL panels representative of different disease phases: i) a cohort of fludarabine-refractory CLL (n=49); ii) a cohort of fludarabine-sensitive CLL (n=62); iii) clonally related Richter syndrome (RS) (n=33; all diffuse large B cell lymphomas); and iv) a consecutive series of newly diagnosed and previously untreated CLL (n=308). BIRC3 was analyzed for mutations by Sanger sequencing (exons 2–9) and for copy number abnormalities by FISH (probe RP11-177O8). BIRC3 was affected in 12/49 (24%) fludarabine-refractory CLL by inactivating mutations (7 frameshift and 1 non-sense) and gene deletions (n=7) that distributed in a mutually exclusive fashion with TP53 disruption (p=.004) (Fig 1A and 1B). Two fludarabine-refractory CLL carried a biallelic inactivation of the BIRC3 gene by mutation of one allele and deletion of the second allele. All inactivating mutations were somatically acquired, were predicted to generate aberrant transcripts carrying premature stop codons, and caused elimination or truncation of the C-terminal RING domain, whose E3 ubiquitin ligase activity is essential for MAP3K14 proteosomal degradation by BIRC3. Western blot analysis confirmed the expression of aberrant bands corresponding in size to the predicted truncated BIRC3, and revealed constitutive non-canonical NF-kB activation in fludarabine-refractory CLL harboring BIRC3 disruption by mutations or deletion, as documented by NFkB2 processing from p100 to p52 (Fig. 1). BIRC3 was the sole gene of the TRAF3/MAP3K14-TRAF2/BIRC3 complex targeted by molecular lesions after extensive mutation and FISH analysis of TRAF2, TRAF3 and MAP3K14 in the fludarabine-refractory CLL cohort (n=49). To investigate whether BIRC3 genetic lesions were restricted to chemorefractory cases, we analyzed BIRC3 for mutations and deletions in other CLL subsets. Fludarabine-sensitive CLL were consistently devoid of BIRC3 disruption in all cases (n=62), suggesting that BIRC3 genetic lesions specifically associate with a chemorefractory phenotype among CLL requiring treatment (Fig. 1A). BIRC3 disruption was restricted to 1/33 (3.0%) clonally-related RS, corroborating the notion that RS is molecularly distinct from chemorefractory progression without transformation (Fig. 1A). In a consecutive series evaluated at CLL diagnosis, BIRC3 disruption was rare (13/308, 4%, all TP53 wild type), associated with primary chemorefractoriness among cases requiring treatment, and predicted poor outcome (Fig. 1A). By univariate analysis, the crude impact of BIRC3 disruption on survival was a ∼5.3 fold increase in the hazard of death (HR: 5.31; 95% CI: 2.62–10.73) and a significant overall survival (OS) shortening (median OS in BIRC3 disrupted cases: 3.1 years vs not reached in BIRC3 wild type cases; p<.001) (Fig. 1D). Multivariate analysis selected BIRC3 disruption as an independent risk factor of OS (HR: 4.25; 95% CI: 1.74–10.36; p=.001) after controlling for confounding clinical (age and Rai stage) and genetic (IGHV mutation status, 11q22-q23 deletion, TP53 disruption, NOTCH1 mutations) variables at diagnosis. These data document that disruption of BIRC3, a critical regulator of non-canonical NF-kB signaling, associates with fludarabine-refractoriness among TP53 wild type CLL and identifies a subgroup of patients showing poor outcome. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Clinical Monoclonal B Cell Lymphocytosis Is a Strong Favorable Prognosticator Independent From Biological and Clinical Parameters,

Abstract 3887Monoclonal B-cell lymphocytosis (MBL) is an asymptomatic monoclonal expansion of <5.0 × 109/L circulating CLL (chronic lymphocytic leukemia)-phenotype B-cells. CLL-like MBL, observed in the clinical setting and defined as clinical MBL (cMBL), demonstrated a substantial risk of leukemic progression to symptomatic disease requiring treatment that can be predicted by biological prognosticators (Rawstron, 2008; Rossi, 2009). Based on this background, the aims of our study were: (i) to define clinical features at diagnosis and a biological profile of cMBL and Rai 0 subsets; (ii) to determine treatment free survival (TFS) within cMBL in comparison with Rai 0 CLL; (iii) to identify clinical and biological variables at diagnosis that predict the risk of evolution from MBL to CLL requiring treatment; and finally (iiii) to validate cMBL as an independent prognostic factor. For this purpose, we investigated 516 patients, median age 66 years (range 33–89), 288 males and 228 females. According to the IWCLL 2008 guidelines, 109 cases were diagnosed as cMBL and 407 cases as CLL (Rai 0: 106; I-II: 290; III-IV: 11). Cases presented for initial evaluation at our Institution between June 1989 and June 2009. The database was updated for the analysis in January 2011. The median follow up was 6.5 years (range 1–20). Among cMBL, median absolute lymphocyte count was 7.3 × 109/L (range 3.2–12.6 × 109/L) and median CLL-phenotype lymphocyte count was 3.5 × 109/L (range 0.6–4.9 × 109/L). cMBL were characterized by both lower beta2-microglobulin (beta2-M) and soluble CD23 (sCD23) levels (P=0.008 and P=0.0005) than Rai 0 CLL. Moreover cMBL showed a more favorable genetic profile compared to Rai 0 CLL: more specifically, cMBL showed both a significant higher prevalence of normal karyotype (45/72 [63%]] vs 30/75 [40%]) and a lower incidence of del11q/del17p (5/72[7%] vs 11/75[15%] (P=0.020). IgVH mutational status as well as ZAP-70 and CD38 percentages did not differ significantly between these two groups. Lymphocyte doubling time was <12 months in 6/109 (5.5%) cMBL cases compared to 19/106 (18%) Rai 0 CLL (P=0.005). With regard to clinical outcome, only 12/109 (11%) cMBL patients required treatment, while 35/106 (33%) Rai 0 CLL underwent chemotherapy (P=0.00012). Noteworthy, also the time to treatment was significantly longer in cMBL patients (71.5 months vs 53.5 months, P=0.03). The slower disease kinetics was consistent with a longer TFS in cMBL compared to Rai 0 CLL (72% vs 42% at 14 years, P=0.00001, Figure). Interestingly, while, within Rai 0 CLL subset, biological prognosticators such as IgVH status (P=0.01), ZAP-70 (P=0.0002) and cytogenetics (P=0.02) as well as markers of tumor burden such as beta2-M (P=0.002) and sCD23 (P=0.0006) were confirmed to predict TFS, within cMBL none of these factors was able to predict progression free survival (PFS) and/or TFS, although some biological prognosticators (ZAP-70 and IgVH) were represented in similar percentages between these two groups. It might be hypothesized that some other factors related to the immune function (e.g., phenotype and function of T-lymphocyte subsets [G.D.P., unpublished observation]) could be able to explain the different time to progression of the cMBL patients. Finally, multivariate analysis, combining cMBL and Rai 0 CLL, documented that having a diagnosis of cMBL is an independent protective factor against the risk of treatment (P=0.011), together with a normal karyotype (P=0.020) and a lower ZAP-70 expression (P=0.0001). In conclusion, cMBL represents a biological and clinical distinct entity from Rai 0 CLL with a more favorable outcome, displaying some degree of heterogeneity in the risk of treatment requirement, probably due to both humoral and cellular immune dysfunctions. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

The Prognostic Significance of Various 13q14 Deletions in Chronic Lymphocytic Leukemia

To further our understanding of the biology and prognostic significance of various chromosomal 13q14 deletions in chronic lymphocytic leukemia (CLL). We analyzed data from SNP 6.0 arrays to define the anatomy of various 13q14 deletions in a cohort of 255 CLL patients and have correlated two subsets of 13q14 deletions (type I exclusive of RB1 and type II inclusive of RB1) with patient survival. Furthermore, we measured the expression of the 13q14-resident microRNAs by quantitative PCR (Q-PCR) in 242 CLL patients and subsequently assessed their prognostic significance. We sequenced all coding exons of RB1 in patients with monoallelic RB1 deletion and have sequenced the 13q14-resident miR locus in all patients. Large 13q14 (type II) deletions were detected in approximately 20% of all CLL patients and were associated with shortened survival. A strong association between 13q14 type II deletions and elevated genomic complexity, as measured through CLL-FISH or SNP 6.0 array profiling, was identified, suggesting that these lesions may contribute to CLL disease evolution through genomic destabilization. Sequence and copy number analysis of the RB1 gene identified a small CLL subset that is RB1 null. Finally, neither the expression levels of the 13q14-resident microRNAs nor the degree of 13q14 deletion, as measured through SNP 6.0 array-based copy number analysis, had significant prognostic importance. Our data suggest that the clinical course of CLL is accelerated in patients with large (type II) 13q14 deletions that span the RB1 gene, therefore justifying routine identification of 13q14 subtypes in CLL management.

1.12 Transcriptome Sequencing Reveals Novel Mutations and Differential Gene Expression in Stereotyped Subsets of Chronic Lymphocytic Leukemia

1.12 Transcriptome Sequencing Reveals Novel Mutations and Differential Gene Expression in Stereotyped Subsets of Chronic Lymphocytic Leukemia

The coexistence of chronic lymphocytic leukemia and myeloproliperative neoplasms: A retrospective multicentric GIMEMA experience

Although the coexistence of chronic lymphocytic leukemia (CLL) and myeloproliferative neoplasms (MPN) has been sporadically reported in the literature, no systematic studies on this disease association are available. We retrospectively analyzed 46 patients affected by CLL/MPN referred by 15 Italian GIMEMA centers. The aim of this retrospective multicenter study was to define the following: clinico-biological characteristics, possible familiarity, clinical course of both diseases, and influence of MPN chemotherapy on the course of CLL. Among 46 patients, 30 patients were males, 16 patients were females; median age was 71 years. Only one case had familiar CLL. Myeloproliferative disorders consisted of essential thrombocytemia in 18 cases, polycythemia vera in 10 cases, chronic myeloid leukemia in 9 cases, primary myelofibrosis in 6 cases, and MPN/myelodysplastic syndrome in 3 cases. The lymphoproliferative disorder was diagnosed as monoclonal B-cell lymphocytosis in 8 patients and as Binet Stage A CLL in 38 patients. After a median follow-up of 49 months, 9 patients experienced progressive CLL and only 6 patients required treatment after a median of 57.5 months. The biological profile confirmed a subset of low-risk CLL. Twenty patients received chemotherapy for MPN without influence on the course of CLL: lymphocyte counts remained unchanged after 3, 6, and 12 months of treatment. This series is the largest so far reported in literature. The diagnosis of concomitant CLL/MPN is a rare event and lymphoproliferative disorders present a clinical indolent course with a low-risk biological profile. MPN therapy does not interfere with the prognosis of patients with CLL.

Telomere length and telomerase levels delineate subgroups of B-cell chronic lymphocytic leukemia with different biological characteristics and clinical outcomes

B-cell chronic lymphocytic leukemia is a clinically heterogeneous disease; some patients rapidly progress and die within a few years of diagnosis, whereas others have a long life expectancy with minimal or no treatment. Telomere length and telomerase levels have been proposed as prognostic factors; however, very few cases have been characterized for both parameters and no study has analyzed the prognostic value of the telomere/telomerase profile. One hundred and seventy-three cases of chronic lymphocytic leukemia were characterized for telomere lengths and telomerase levels by real-time polymerase chain reaction. Data were correlated with established prognostic markers, IGVH mutational status and chromosomal aberrations, and clinical outcome. Telomere lengths were inversely correlated with telomerase levels (r(s) = -0.213; P = 0.012), and most of the cases of chronic lymphocytic leukemia with high levels (above median) of telomerase had short (below median) telomeres (P = 0.0001). Telomerase levels were higher and telomeres were shorter in unmutated IGVH cases than in mutated IGVH ones (P<0.0001). Chronic lymphocytic leukemias with 11q, 17p deletion or 12 trisomy had significantly higher levels of telomerase and shorter telomeres than those with no chromosomal aberration or the sole 13q deletion (P < 0.001). Telomere length/telomerase level profiles identified subgroups of patients with different clinical outcomes (P < 0.0001), even within the subsets of chronic lymphocytic leukemia defined by IGVH mutational status or chromosomal aberrations. Short telomere/high telomerase profile was independently associated with more rapid disease progression. Comprehensive analyses of telomeres, telomerase, chromosomal aberrations, and IGVH mutational status delineate groups of chronic lymphocytic leukemias with distinct biological characteristics and clinical outcomes. The telomere/telomerase profile may be particularly useful in refining the prognosis of chronic lymphocytic leukemia patients with mutated IGVH and no high-risk chromosomal aberrations.