ALBERT

Description: Immunoglobulin heavy chain variable gene (IGHV) replacement or "VH replacement" (VHR) modifies a rearranged IGHV-D-J sequence by replacing the original IGHV gene with another. This process leaves a detectible "footprint" at the IGHV-D junction of the existing sequence. Roughly 33% of chronic lymphocytic leukemia (CLL) cases exhibit stereotyped B cell receptors (BCRs) often characterized by signature VH CDR3 amino acids. Various mechanisms have been put forth to account for stereotypy in CLL. An overarching hypothesis is that the stereotyped BCRs are antigen driven. Within this concept, a variety of mechanisms could lead to the signatures including somatic mutations and addition/deletion of nucleotides at junctional regions. Here we explore the possibility that VHR provides another mechanism to account for some of the stereotyped rearrangements and some of their signature VH CDR3 amino acid residues in CLL. We examined IG sequences of 26,642 CLL cases and ~16 million healthy controls (HC) to find relic footprints as indicators of VHR. This was done using the VHRFA program developed by Lin Huang et al (PLoS ONE, 2013), as well as our own program which duplicates the VHRFA results but is better able to process large numbers of sequences. The frequency of VHR was similar in CLL and HC (11.6 and 11.9%, respectively). Focusing solely on CLL sequences to define a relationship between VHR and stereotypy, we found highly significant differences in VHR frequencies between stereotyped (n=8,568) and non-stereotyped cases (n=18,074), with stereotyped cases exhibiting VHR at a greatly reduced frequency (7.7% vs. 13.5%, respectively). When comparing VHR frequencies between stereotyped cases and non-stereotyped cases that used the same IGHV, we found that the number of subsets with low VHR exceeded those with elevated VHR ~2:1, accounting for the overall VHR in stereotyped cases being lower than non-stereotyped cases. Further restricting comparisons of stereotyped subsets to non-stereotyped cohorts by matching VH CDR3 length led to similar conclusions. Within stereotyped cases there was a wide distribution of VHR, ranging from 55.6% to 0.1%. Restricting VH CDR3 lengths to "short" (5 - ≤13), "medium" (13.1 - ≤20) and "long" (20.1 - ≤28), the corresponding VHR increased monotonically with length (1.1, 8.2, and 11.9% respectively). Notably, subsets showing elevated VH replacement included better prognosis subsets, #4, 77 and 201 (23.8, 22.1, and 28.6%, respectively). Among low VHR frequency subsets were those associated with worse prognosis, #1, 2, 5, 6, 8, 9 and 10 (VHR frequencies: 0.2, 0.1, 0.9, 2.3, 7.7, 9.0 %, respectively). This was most strikingly exhibited by subsets #1 and #2, both of which comprise patients with poor clinical courses. Each of these sets of sequences displayed virtually no examples of VHR (0.2 and 0.1%, respectively). This might be predicted because these two subsets have relatively short VH CDR3 lengths (subset #1: 13 aa; subset #2: 9 aa), based on the length association mentioned above. Detailed analyses of the presence of footprints and the position of these in the rearranged IGHV-D-J indicated that for some subsets, certain signature VH CDR3 amino acids could be the result of VHR. For example in subset #201, sequence analysis suggests that VHR is responsible for an arginine and for a glutamine in the 5' portion of the VH CDR3. Similarly, VHR may craft the characteristic glutamine on the 5' end of the subset #6 VH CDR3. Thus, our studies indicate that, as a whole, CLL IGHV-D-J sequences use VHR at a frequency comparable to that of normal B cells and significantly less than that of non-stereotyped rearrangements. However, certain stereotyped cases are dramatically enriched for evidence of VHR. Moreover among these cases, the footprints found in the VH CDR3s of stereotyped cases can be shown to directly code for signature amino acids in VH CDR3s. Finally, stereotyped cases with high levels of VHR tend to be those with better clinical courses, whereas those worse outcome stereotyped cases exhibit less evidence for this process. This latter finding is consistent with the concept that VHR is one of the molecular mechanisms used by developing B cells to edit BCRs having high affinity for autoantigens. Since many CLL BCRs are autoreactive, including those found to have high levels of VHR such as subset #4, this implies a fundamental defect in tolerance mechanisms in those normal B cells that eventually became leukemic. Disclosures Agathangelidis: Gilead: Research Funding. Hadzidimitriou:Janssen: Honoraria, Research Funding; Gilead: Research Funding; Abbvie: Research Funding. Ghia:AbbVie, Inc: Honoraria, Research Funding; Acerta: Honoraria, Research Funding; BeiGene: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Sunesis: Honoraria, Research Funding. Stamatopoulos:Gilead: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding; Janssen: Honoraria, Research Funding. Chiorazzi:AR Pharma: Equity Ownership; Janssen, Inc: Consultancy.

Description: Despite a wealth of information about the structure of surface membrane immunoglobulin (smIg) on chronic lymphocytic leukemia (CLL) cells, little is known about epitopes reacting with their binding sites. Probing phage-displayed peptide libraries, we identified and characterized mimetopes for Igs of 4 patients with IGHV mutated CLL (M-CLL) and 4 with IGHV unmutated CLL (U-CLL). Six of these mAbs were representatives of stereotyped B-cell receptors characteristic of CLL. We found that mimetic epitopes for U- and M-CLL Igs differed significantly. M-CLL–derived peptides exhibited better amino acid motifs, were more similar to each other, aligned more easily, and formed tighter clusters than U-CLL–derived peptides. Mono-, oligo-, and polyreactivity of peptides correlated with structural changes within antigen-binding sites of selecting M-CLL mAbs. Although M-CLL–isolated peptides and certain U-CLL mAbs bound more effectively to the selecting mAb, others were not as specific, reacting with M-CLL and U-CLL mAbs; these data suggest that in vivo structurally diverse epitopes could bind smIgs of distinct CLL clones, thereby altering survival and growth. Finally, an M-CLL–derived peptide inhibited, in a dose-dependent manner, binding of its homologous mAb to human B lymphocytes; therefore peptides that inhibit or alter the consequences of antigen-smIg interactions may represent therapeutic modalities in CLL.

Description: In chronic lymphocytic leukemia (CLL) monoclonal B cells expand and progressively accumulate in the bone marrow, eventually migrating to secondary lymphoid organs for even greater proliferation. At both sites, CLL cells engage in complex, incompletely defined cellular and molecular interactions involving multiple cell types such as T cells, myeloid cells, mesenchymal stromal cells, and matrix, collectively referred to as the "tumor microenvironment". This microenvironment is critical for the survival and proliferation of CLL cells, and data indicate that T cells and myeloid cells have an important role in these processes. In this study, we focus on two cells types: CD4+ T lymphocytes and myeloid-derived suppressor cells (MDSCs). In CLL patients, these populations are altered and impact on clinical outcome. CD4+ T cells comprise several subtypes, and CLL patients often have expanded Th2 and Tregs populations, consistent with the immunosuppressive status of these patients. Moreover, patients with higher numbers of another CD4+ subset, Th17 cells that produce IL-17 and other pro-inflammatory cytokines, can have longer survival times. Although studied minimally in CLL, MDSCs are known suppressors of T cell proliferation in vitro, and expand along with malignant cells in several cancers. However, no information is available about their effects on CD4+ T cell differentiation or on B-cell biology in CLL. In a cohort of 56 untreated CLL patients, we first explored correlation of the numbers of MDSCs and autologous T cells, using flow cytometry. CD3+ cell numbers significantly paralleled total MDSCs and monocyte-like MDSCs (mMDSCs) (P = 0.002, Spearman r = 0.44; P = 0.004, Spearman r = 0.41, respectively). Interestingly, MDSCs correlated with CD4+ and CD8+ T-cells (P 〈 0.001, Spearman r = 0.646; P 〈 0.001, Spearman r = 0.61, respectively). However, the correlation of MDSC subpopulations with CD4+ and CD8+ cells differed: mMDSCs associated significantly with CD4+ cells (P 〈 0.001, Spearman r = 0.73) and granulocyte-like MDSCs (gMDSCs) with CD8+ cells (P= 0.008; Spearman r = 0.45). Furthermore, although gMDSCs did not correlate with the numbers of CD4+ T-cells, we observed that they positively paralleled Tregs defined as CD3+/CD4+/CD25+/CD127-/FoxP3+ cells (P = 0.020, Spearman r = 0.44). Other subpopulations are currently under study. To address the effect of MDSCs on CD4+ cell differentiation, we FACS sorted CD3+/CD45RO- naïve CD4+ lymphocytes and stimulated them in vitro with anti-CD3/CD28 beads and IL2 in the presence or absence of mMDSCs (HLA-DRlo/CD11b+/CD33+/CD14+), gMDSCs (HLA-DRlo/CD11b+/CD33+/CD15+) or monocytes (HLA-DRhi/CD11b+/CD14+); these studies involved samples from 3 CLLs and 3 healthy controls (HCs). On day 7, cells were harvested and cytokine production was quantified by intracellular flow cytometry as the percentages of the following populations: Th1 (INFγ), Th2 (IL-4), Tregs (FoxP3), Th17 (IL-17A and IL-17F), Th9 (IL-9) and Th22 (IL-22). Culturing CLL or HC T cells in the absence of MDSCs revealed a lower percentage of cytokine-producing cells (24% vs. 55%; P = 0.017) in CLL, which was mainly due to a reduction in IL-4+ cells (P = 0.066). However, when analyzing the effects of MDSC subsets on the polarization of CLL or HC T cell, gMDSCs promoted significantly more FoxP3+ and less IL-22+ cells in CLL than in HC (P = 0.025 and P = 0.048, respectively). When analyzing only CLL T cells, supplementation with mMDSCs induced a reduction in IL-22+ cells (P = 0.027) and an insignificant increase of IL-4+ and IL-17+ cells. Conversely monocytes supported an expansion of INFγ+ T-cells (P=0.066), and gMDSCS promoted an increase of IL-9+ cells (P = 0.046) and a reduction of FoxP3+ cells (P = 0.019). In summary, in CLL the absolute numbers of total MDSCs and T cells are tightly linked. There is a significant correlation between CD4+ T cells and mMDSCs, and between CD8+ T cells and gMDSCs. Additionally, in CLL naïve CD4+ differentiation appears reduced compared to HC, in concordance with lower T-cell responses previously reported. Moreover, the preliminary aspects of the study suggest that CLL mMDSCs promote an expansion of Th2, Th17 cells and a reduction of Th22 cells, and monocytes enhance Th1s. Unexpectedly, since we observed a significant positive correlation in the PBMCs, gMDSCs may reduce Tregs and augment Th9. These findings depict differential consequences of CLL T cell - MDSC / mMDSC / gMDSC interactions. Disclosures Stamatopoulos: Abbvie: Honoraria, Other: Travel expenses; Gilead: Consultancy, Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Janssen: Honoraria, Other: Travel expenses, Research Funding.

Description: B cell receptor signaling is a key factor in chronic lymphocytic leukemia (CLL), evinced by inhibitory drug ibrutinib's efficacy. Studies of normal and CLL B cells indicate surface membrane IgM or IgD engagement has diverse signaling consequences. But little is known about relative amounts of sIg/co-receptor components within intraclonal fractions and how their compositions affect signaling. We studied relative densities of IgM and IgD and Ig-associated stimulatory and inhibitory co-receptors in two subsets based on: [1] relative densities of IgM and IgD and [2] reciprocal expression of CXCR4/CD5 indicating activation state. Samples from 5 U and 5 M-CLL patients, pre and during (4-6 weeks) ibrutinib treatment, were tested by conventional and by imaging flow cytometry using an ISX Mark II providing multiple spectral images of individual cells in a flow setting. After subfractionating clones for IgM (IgMDim, IgMInt, IgMBright) or IgD (IgDDim, IgDInt, IgDBright) densities, we quantified relative densities of stimulatory/inhibitory molecules on the subsets. A directly proportional change was observed for CD5, CD19, CD20, Siglec10, CD25, HLA-DR, and CD38 as IgM moved from Dim to Bright. Exceptions were CXCR4, which dramatically decreased as IgM density increased, and CD22, that had a constant density in all fractions. Similar changes were seen for IgD except for CD22 which increased in IgDBright density. Again CXCR4 showed the opposite pattern. CD25 and HLADR remained constant within IgD increments. Stimulatory markers CD25 and HLADR changed upward only in the transition to IgMBright. Together these imply signaling through IgM heightens as IgM density increases, but does not through IgD, likely due to increased Siglec 10 and CD22. The reduced amounts of smCXCR4 suggest impaired ability of cells with high smIgM or smIgD to traffic. We examined subpopulations based on CXCR4 and CD5. IgD and IgM densities increased from resting (RF) CXCR4Bright/CD5Dim to intermediate (INT) CXCR4Int/CD5Int to proliferative (PF) CXCR4Dim/CD5Bright fractions, although the degree of upregulation was more marked for IgD than IgM. All stimulatory/inhibitory coreceptors also increased in density from RF to PF. Interestingly, CD22 and IgD retained a constantdensity from RF to INT but increased considerably at the PF. Although stimulatory molecules CD25, CD38 and HLADR had upward trends, peaking occurred in the PF. Collectively, this implies signaling capacity through smIgM amplifies toward the PF due to higher smIgM density and IgD upregulation. Finally, we evaluated CLL clones as a whole as well as based on the subsets above for ibrutinib treatment induced changes. Clonally, a density decrement occurred for smIgD and an increment for smIgM. Data for the other molecules fell into 3 categories based on relative density changes: Decreasing: CD5, CD20, CD38, CD25, HLA-DR; Increasing: CXCR4: Invariant: CD22, CD19, Siglec10. For subfractionating based on smIgs density, ibrutinib drastically reduced density differences for co-receptors and activation markers among IgM or IgD density subpopulations with IgMBright and IgDBright fractions more affected than the Dim and Intermediate density counterparts. Similarly for the CXCR4/CD5 subpopulations, lower differences in relative expression of coreceptors from RF to PF were found. As for IgM and IgD levels, the PF was the most reduced. The composite effect was a reduced slope of change among the original sets of density change categories (IgMDim -〉 IgMBright; IgDDim -〉 IgDBright; RF to PF), leading to more phenotypically homogenous subpopulations. In summary, prior to ibrutinib therapy, both the Ig and most of the associated molecules increase density in subsets marked by increased IgM and IgD densities and marked by decreasing CXCR4/CD5 densities. However, there was a major difference in density correlations for CXCR4 and CD22. CXCR4, levels were lowest on IgMBright and IgDBright cells which differs from published data using anti-IgM beads. For CD22 the highest levels were in IgDBright but not in IgMBright and in CXCR4/CD5 subset with the highest IgD and IgM (CXCR4Dim/CD5Bright). In contrast, ibrutinib treatment led to an overall change in coreceptor molecules altering considerably the density relationships with IgM and IgD. Concomitantly, these culminate in a downregulatory membrane stimulatory environment. Disclosures No relevant conflicts of interest to declare.

Description: B-CLL cells frequently express antigen receptors with varying degrees of self-reactivity. In this context, it is likely that the precursors of leukemic clones have attempted to reconfigure their receptors to eliminate or reduce affinity for autoantigen. IGHV replacement is a form of receptor reconfiguration that retains the existing IGHD/J rearrangement and swaps the originally rearranged IGHV gene for another upstream IGHV gene, exploiting cryptic heptamer-like sequences in the coding regions of most human IGHV genes. In this study, we explored the extent of IGHV replacement in 583 IGHVDJ rearrangements from patients with B-CLL and compared these findings to normal B cells. B-CLL IGH sequences used IGHV1/2/3/4/5/6/7 subgroup genes in 144/33/255/122/14/9/6 cases, respectively. IGHV3 and IGHV4 genes were used predominantly in mutated (M) sequences (136/255 and 75/122 cases respectively); in contrast, 83% (120/144) of IGHV1 genes were used in unmutated (UM) sequences. IGHV replacement in B-CLL was examined using a search method, also applied in the normal repertoire, looking for the presence of pentameric footprints, which are short stretches of 3′ nucleotides from the original IGHV that remain in the IGHVD joint after each round of IGHV replacement. This method uses a set of 33 pentamers deriving from the 3′ends of human IGHV genes carrying cryptic heptamers to identify potential IGHV replacement products. After alignment with sequences in the IGHVD junctions in B-CLL, only exact matches were accepted. At least one pentameric footprint consistent with IGHV replacement was detected in the IGHVD junction of 34 (19 M/15 UM) of 583 B-CLL cases (5.8%). Twenty-seven pentamers were detected in this cohort of sequences. B-CLL rearrangements with evidence of IGHV replacement footprints had longer HCDR3 regions (median,19 amino acids) compared to the series as a whole (median,17 amino acids). The most frequent IGHV genes that probably replaced a previously rearranged IGHV gene were IGHV1-69 (6 cases/5UM), IGHV3-23 (6 cases/1UM), IGHV3-7 (4 cases/1UM), IGHV3-33 (3 cases/2UM). In 30/34 cases (88%), the amino acids encoded by these footprint sequences in the HCDR3 where positively or negatively charged, a finding associated with autoreactivity. These residues likely contribute to antigen specificity and affinity and can be important for B-CLL cells, which are antigen-experienced, activated cells. In conclusion, IGHV replacement in CLL appears to occur at a frequency (5.8%), which is not inherently different from that reported in the normal B-cell repertoire (5%). Although IGHV replacement is ostensibly initiated to modify autoreactivity, its products still often resemble autoantibodies (increased HCDR3 length, aminoacid composition and charge). However since some of these HCDR3 properties also exist in anti-microbial mAbs, their retention after secondary rearrangements may be a protective as well as a potentially harmful mechanism. Such a selection for microbial reactivity supports the notion that B-CLL derives from cells expressing mAb with shared reactivity between autoantigens and microbes. In support of this possibility is the finding that a UM-IGHV1-69 recombinant mAb with evidence of IGHV replacement reacted against 5 different bacterial strains.

Description: Immunoglobulin heavy variable (IGHV) gene replacement is a RAG-mediated recombination event that modifies a rearranged IGHV-IGHD-IGHJ sequence by replacing the original IGHV with another IGHV. As the excision of the original IGHV occurs at a cryptic heptamer near the end of FR3 of the original IGHV, a trace (“footprint”) remains embedded in the V-D (“N1”) junction of the heavy complementarity-determining region 3 (HCDR3) sequence of the new rearrangement. Since IGHV-replaced antibody sequences are over-represented in several autoantibody-associated syndromes, it is assumed that the process is designed to eliminate unwanted antibody specificities, particularly those which are autoreactive. Because CLL antibodies/B cell receptors (BCRs) are often autoreactive, we sought to determine if IGHV replacement plays a significant role in the formation of these antibodies. To this end, we analyzed IGHV-IGHD-IGHJ rearrangements in a CLL sample set of 6,121 sequences and in another set of 3,326 sequences from normal B lymphocytes culled from the GenBank Database. Sequences without positional information for their IGHV match were eliminated, resulting in 6023 CLL and 3202 normal sequences. From among these, we looked for the presence of IGHV footprints in the N1 and N2 junctional regions. Sequences were processed by IMGT to determine the particular IGHV used as well as junctional N1, N2 sequences. The resulting junctional regions were analyzed using the Java-based VH Replacement Footprint Analyzer (VHRFA) program [Huang, L et al (2013) PLOS ONE] to identify footprints (≥5-mer) in the N1 and N2 regions. Various CLL subgroups based on ZAP-70 expression, CD38 expression, IGHV mutation status, BCR stereotypy, and genomic aberrations were also examined for differences in the presence of footprints. Comparison of CLL with normal sequences in the N1 regions revealed a slight yet significant difference in footprint frequency (12.62% versus 11.06%, respectively; P = 0.03). Comparison of IGHV-mutated (M) versus unmutated (U) subgroups of CLL also disclosed a significant difference in frequencies (M: 13.58%, U: 11.51%, P = 0.02). Using footprints in the N2 regions as an internal control, we found the frequency of ≥5-mer replacement footprint motifs to be significantly higher in N1 over N2 (P = 1.1E-7), supporting the validity of this approach. Other comparisons led to a significant enrichment of footprint frequencies, such as 13.71% for the ZAP-70-negative group (P = 0.029), 13.36% for the CD38-negative group (P = 0.013), and 14.70% for the 13q-deletion group (P = 0.016) compared with the normal group. Note, an overall comparison of stereotyped versus non-stereotyped cases revealed highly statistically significant differences in N1 footprint frequencies (9.4% for the 2084 cases having defined subsets vs 14.32% for the 3,939 cases without defined subsets; P = 2.7E-8). Focusing on stereotyped cases that exhibited evidence for IGHV replacement, we found significantly different footprint frequencies in the N1 region among different stereotyped subsets, even when restricting the comparison to subsets utilizing the same IGHV and belonging to the same mutational category (U-CLL or M-CLL). Relevant examples include differential frequencies among U-CLL, IGHV1-69-expressing subsets #3, #5, #6, #7, and #59 (0 - 15.5%; P = 0.01) and M-CLL, IGHV4-34-expressing subsets #4, #16, #29, and #201 (0 - 40.9%; P = 0.005). In summary, CLL had an elevated IGHV replacement rate compared to normal controls. In addition, M-CLL, ZAP-70-neg, CD38-neg and 13q del CLL subgroups were also elevated above normal controls. The most dramatic differences in IGHV replacement frequency were found between CLL non-stereotyped and stereotyped CLL cases, with an intriguing difference within stereotyped subgroups exhibiting the same IGHV where IGHV replacement varied by as much as 40-fold. Because higher levels of IGHV replacement are found in ZAP-70-neg, CD38-neg, 13q del CLL, and non-stereotyped cases that in general are less aggressive, the presence of IGHV replacement may pinpoint CLL clones whose precursor B cells had edited their BCRs away from autoreactivity and therefore antigen-drive. This conclusion suggests a differential role for autoantigen drive in certain stereotyped subsets: CLL subsets not exhibiting IGHV replacement appear to be more susceptible to antigen drive than those that do. Disclosures: No relevant conflicts of interest to declare.

Description: Chronic lymphocytic leukemia (CLL) cells are thought to have diminished cell-cycling capacity, a view challenged by their phenotypic resemblance to activated human B lymphocytes. The present study addresses the cell-cycling status of CLL cells, focusing on those leukemic cells expressing CD38, a molecule involved in signaling and activation that also serves as a prognostic marker in this disease. CD38+ and CD38− members of individual CLL clones were analyzed for coexpression of molecules associated with cellular activation (CD27, CD62L, and CD69), cell-cycle entry (Ki-67), signaling (ZAP-70), and protection from apoptosis (telomerase and Bcl-2). Regardless of the size of the CD38+ fraction within a CLL clone, CD38+ subclones are markedly enriched for expression of Ki-67, ZAP-70, human telomerase reverse transcriptase, and telomerase activity. Although the percentage of cells (approximately 2%) entering the cell cycle as defined by Ki-67 expression is small, the absolute number within a clone can be sizeable and is contained primarily within the CD38+ fraction. Despite these activation/proliferation differences, both CD38+ and CD38− fractions have similar telomere lengths, suggesting that CD38 expression is dynamic and transient. These findings may help explain why high percentages of CD38+ cells within clones are associated with poor clinical outcome.

Description: CLL results from the accumulation of monoclonal B lymphocytes that derive from a small fraction of cells with proliferative activity. Because expression of the DNA mutator, activation-induced cytidine deaminase (AID) is restricted to these dividing cells, they can develop new DNA abnormalities leading to more lethal disease. Hence, such cells are important targets for therapy. Our previous study indicated that the B-cell subset with low levels of CXCR4 and high levels of CD5 (CXCR4DimCD5Bright) is enriched in these cells ("proliferative fraction", PF), whereas the less vital, resting cells exhibit a CXCR4BrightCD5Dim phenotype ("resting fraction", RF). In this study, we focused on analyzing the significantly differentially expressed genes (DEGs) between PF and RF. PF and RF were isolated from 26 CLL (13 U-CLL and 13 M-CLL) and microarrays (llumina HT12) were performed. Selected DEGs between PF and RF were confirmed by rtQ-PCR and/or by flow cytometry. Array data were interpreted using Gene Set Enrichment Analysis (GSEA) and Ingenuity Pathway Analysis (IPA). First, focusing on the Immunologic Signature of B lymphocytes with GSEA, we found the PF was enriched in gene sets shared with IgM memory cells and pre-germinal center B cells, whereas RF was enriched in gene sets in common with naïve, IgD+ B cells. Notably, the PF also shared gene sets with myeloid dendritic cells and monocytes. Protein expression of 10 myeloid markers (CD68, CD1c, CD11a, CD11b, CD11c, CD13, CD31, CD205, CXCR3 and CLECL1) was documented in PF B cells from 11 U-CLL and 11 M-CLL patients, and each was more highly expressed in the PF than RF. No difference in myeloid markers was observed between U-CLL and M-CLL, suggesting that this expression is independent of IGHV mutation status. DEGs were also determined based on expression ratios for PF and RF for each patient; t tests were performed using R. With a fold change cutoff of 〉 1.5 or 〈 -1.5 and a level of significance of P 〈 0.01, we identified 198 genes significantly upregulated in PF and 88 in RF. The top biological-function categories identified using IPA indicated that these genes related to cellular development (n=37), cellular growth and proliferation (n=49), cellular movement (n=42) and cell survival and death (n=50). In addition, these DEGs mapped to 8 canonical pathways with Z-scores ≥2, the highest being the integrin signaling pathway (Z-score = 2.887). Twelve of 13 genes were upregulated in the PF and correlated significantly with the integrin pathway: ACTG, ARPC5, ARPC1B, BCAR3, CAPNS1, ITGAX, ITGB1, ITGB2, ITGB7, PFN1, RAC2 and RHOC. Upregulation of integrin subunits was confirmed by Q-PCR and cell surface staining by flow cytometry. Preliminary cellular adhesion experiments suggest PF bind to fibronectin coated plates, and those cells that bind survive better. Next, comparing PF and RF expression ratios for U-CLL vs. M-CLL revealed 502 DEGs in U-CLL and 179 in M-CLL; 144 genes were shared by both U-CLL and M-CLL. IPA analysis of the latter genes correlated best with integrin signaling, and the potential upstream regulators of these were IFNg, IL1, F2 and IFNa. The Rho Family GTPases signaling pathway was significant (10 genes) for DEGs unique to U-CLL. No significant pathway or bio-function relationship was observed in DEGs only in M-CLL. Finally, IPA analysis showed IL4 as an upstream regulator of DEGs unique to the PF of U-CLL based on upregulation of 12 IL4 target genes (APRT, CCL5, CDKN1A, DECR1, IL17RB, ITGAL, ITGB1, LTA, MAPKAPK3, PIGR, SAMSN1, TIMP1). Three other IL-4 targeted genes were paradoxically under-represented in the U-CLL PF (BCL2, CCR7 and VIPR1). The upregulated findings are consistent with PF B cells inducing T cells to produce IL-4 via co-receptors on B/myeloid cells that foster a Th2 response (e.g., CLECL1). In conclusion, gene expression profiling indicates that cells of the PF display a dual activated B cell/myeloid cell phenotype suggesting enhanced antigen-presentation capacities. Integrin signaling appears to be a key pathway for these cells which could foster cell proliferation, survival, and migration, especially in U-CLL clones where integrin activation can lead to Rho GTPases activation. Finally, genes regulated by IL-4 in the PF could be induced by interactions of autologous T cells with CLL B cells. These findings suggest antigen-presentation and integrin and IL-4 signaling pathways as therapeutic targets in CLL, particularly for U-CLL. Disclosures Barrientos: AbbVie: Consultancy, Research Funding; Gilead: Consultancy, Research Funding; Janssen: Consultancy.

Description: B cells originate in the bone marrow where precursors pass through a series of highly regulated processes to generate a functional B-cell receptor (BCR). IGHV-D-J and IGLV-J rearrangements are achieved via a combinatorial process with associated amino acid additions/deletions at recombination sites leading to an immense variety of structurally diverse BCRs. Due to the large number of possibilities, potentially harmful BCRs recognizing self-antigens can be generated. In the early stages of B cell development a number of distinct selection mechanisms eliminate higher affinity auto-reactive B cells. Chronic lymphocytic leukemia (CLL) is a CD5+ B cell malignancy. Besides having a unique phenotype not always assignable to a defined B-cell subset, many CLL B cells express auto-reactive BCRs; those that do not can often be shown to have derived from self-reactive clones. In addition, certain CLL patients express virtually identical IGHV-D-J rearrangements that can be organized into categories called "stereotyped BCRs". To date, stereotyped BCRs have not been conclusively shown to exist in the normal B-cell repertoire. This might be due to strict autoreactive censoring that eliminates stereotyped BCRs during B-cell ontogeny and/or because the number of IGHV-D-J rearrangements sequenced from individuals has not been sufficient to detect infrequent Igs. Therefore, using ultradeep, next generation IGHV-D-J sequencing we searched for CLL-specific stereotyped BCR rearrangements in the early phases of normal B cell maturation, before all mechanisms of selection are complete. We sorted several B-cell populations from bone marrow samples obtained from healthy patients undergoing hip replacement without an autoimmunity or chronic infection history. The MiSeq platform was used to determine the IGHV-D-J repertoire of pro-, pre-, immature, transitional, naive, and memory B cells, and of plasmablasts. Sequences were analyzed using the pRESTO bioinformatic tool and R. Using VDJ rearrangement and CDR3 length criteria we focused on sequences potentially similar to those in CLL. Sequences were submitted to an online tool (ARReST/Assign subset) to be attributed to a particular CLL stereotyped subset based on a degree of certitude. We identified 156 sequences belonging to defined CLL stereotyped subsets with varying levels of similarity (extreme to low), most residing in early/antigen-independent compartments such as pre-B cells and immature, transitional, and naive B cells. When stereotyped rearrangements of all degrees of certainty were considered, the greatest number of CLL-like stereotypes were found among the most immature B-cell populations, with frequency decreasing with advanced maturation (e.g., 0.04% in pro-B cells vs. 0.028 in naive and 0.01% in memory B cells). This is consistent with the notion that auto-reactive stereotyped rearrangements are eliminated from the normal repertoire. However, when using only those IGHV-D-J rearrangements with extreme or very high certitude, CLL-like stereotyped sequences were found at the highest frequency in the naive B-cell subset (0.02% vs 0.0058% in immature and 0.007 in pre-B cells). In addition, we identified at least one sequence for a majority of the most frequent 18 stereotyped subsets, although the proportional representation of each subset in our data is different from that seen in CLL. Specifically, the highest scoring recurrent subsets in our analysis were subsets 3 (61% of total CLL sequences), 5 (9.5%) and 64B (7%), which are not the most common in the known CLL cohort. Rather, we found only a few sequences belonging to the most common subsets such as subsets 1 and 2 (4.5% and 2% of the total CLL sequences), and none belonging to subset 4, although this subset is always isotype class-switched and IGHV-mutated and hence would not be expected to be found in the developing or naive repertoire. Finding a significant presence of CLL-like stereotyped sequences when using ultra-deep DNA sequencing techniques in early phases of B-cell development and in the naive B-cell fraction suggests that CLL-like B cells are present among normal B cell populations albeit at very low frequencies. Moreover the presence of only certain of the most prevalent stereotypes such as subset 3 and 5, respectively the 5th and 6th biggest subsets, suggests these clones might not exhibit high auto-reactive affinity allowing them to more readily escape mechanisms of selection. Disclosures Stamatopoulos: Abbvie: Honoraria, Other: Travel expenses; Gilead: Consultancy, Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Janssen: Honoraria, Other: Travel expenses, Research Funding.

Description: The transforming events that lead to B-cell chronic lymphocytic leukemia (B-CLL) are unknown, and although genetic abnormalities appear to promote disease progression, none of these is seen in every patient. Therefore, we have applied a highly sensitive and specific method to probe, in greater depth, for genetic changes that can occur in B-CLL, and to explore if these correlate with disease development or progression. Utilizing ROMA to measure changes in gene copy number, we screened the entire genome of over two dozen B-CLL cases to identify characteristic and novel chromosomal changes. DNA from leukemic cells of patients was compared to the normal DNA from their unaffected neutrophils and to an unrelated, normal human control. By comparing normal DNA from each patient to an unrelated normal, we avoid inadvertently mistaking common copy number variation between people as possible candidates for lesions in B-CLL. ROMA was performed on both 85,000 and 390,000 probe microarrays enabling us to screen for far smaller lesions than are detectable by routine techniques, as well as resolve regions of interest, possibly pinpointing important genes in the etiology and progression of B-CLL. Data were also obtained by using the Illumina platform. These two methods (ROMA and Illumina), while not identical, are largely in agreement. We have observed virtually all the major cytogenetic imbalances previously reported, and in many cases to a higher resolution. Although there are fewer lesions present in B-CLL than in more advanced stages of solid cancers, many early stage cancers exhibit only a few common lesions. The most frequent deletion and often the sole abnormality found in B-CLL occurs at chromosome 13q13.4. Two micro-RNAs, miR-15a and miR-16-1, have been implicated in the smallest region of this deletion. ROMA analysis has further delineated the deletions on 13q, suggesting that there may be two epicenters, one encompassing these micro-RNA genes and one encompassing a neighboring region that does not span miR-15a or miR-16-1. A novel finding is the existence in certain patients (5/26) of numerous single probe deletions or amplifications. Since we compare B-CLL to the normal DNA from the same patient, and to a known control, even single probe events can be relevant. Many B-CLLs display single probe aberrations and these events are currently under investigation. In addition, we have observed two patients with major genomic instability, as evidenced by large and multiple regions that change copy number. The analysis of large numbers of B-CLL cases is essential to identifying and understanding the ongoing progression of genetic lesions and the role they play in clinical outcome. Initial results clearly indicate the need to obtain more data to identify critically mutated genes within the leukemic cells that cause them to become more aggressive. The identification and characterization of these genetic changes may be used as stratification tools for new diagnostic and therapeutic approaches to this currently incurable disease.