A Systematic Search Into The Role Of IGHV Gene Replacement In Shaping The Immunoglobulin Repertoire Of Chronic Lymphocytic Leukemia

Abstract 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.

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Research on the Characteristics of IGHV Gene In Chronic Lymphocytic Leukemia of China.

Blood ◽

10.1182/blood.v116.21.4609.4609 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4609-4609

Author(s):

Chun Qiao ◽

Kourong Miao ◽

Jianyong Li

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Heavy Chain ◽

Conflicts Of Interest ◽

Gene Families ◽

Lymphocytic Leukemia ◽

Chinese Patients ◽

Prognostic Impact ◽

Mutation Status ◽

Ighv Gene ◽

Significant Difference

Abstract Abstract 4609 Objective The usage, mutation status and prognostic impact of immunoglobulin heavy chain variable (IGHV) gene in Chinese patients with chronic lymphocytic leukemia (CLL) is unclear. We set out to define the characteristics of IGHV gene and its relevance to clinical and biological parameter in our patients. Methods IGHV gene mutations were detected by multiplex PCR in 202 Chinese CLL patients and the purified PCR amplification products were sequenced. IGHV somatic hypermutation status and gene usage were analyzed by IMGT/V-QUEST software. The association analysis between IGHV somatic mutation status and the clinical and biological features, including Binet staging, immunophenotype, cytogenetic aberrant, were also emphasized in this study. Results The results showed that 129 patients had mutated (M) IGHV, and the remaining 73 patients had unmutated (UM) IGHV according to the cutoff value of accordance rate 98%. The most frequent VH gene family was found to be VH3 (47.5%), followed by VH4 (34.7%), VH1 (11.4%), VH2 (2.5%), VH5(1.5%), VH7(1.5%) and VH6(0.9%) gene families, which was similar to other Asian populations. The overall survival (OS) time of UM IGHV group was significant shorter than M IGHV group (P=0.025). Significance was found in the expression of CD38 and ZAP-70 between patients with and without IGHV mutations (P<0.0001 and P=0.015, respectively). Binet staging was significantly different with IGHV mutation status (P<0.001). “Unmutated” sequences had significantly longer heavy chain complementarity-determining region 3 (HCDR3). Seven of these patients used VH1-69, which was similar to other Asia countries, but in striking contrast to those in Western countries, where VH1-69 was one of the most frequently used genes. FISH was performed in 117 cases, del(11q22) was considered as high risk factors, and 10(10/42, 23.8%) cases with UM IGHV gene. On the other hand, there were 7(7/75, 9.3%) cases with M IGHV gene (P=0.033). No significance was found in del(17p),del(13q),del(6q),add(12),IGH translocation between IGHV mutation and unmuatioan patients. A total of five stereotyped BCR were identified, IGHV3-21/IGHD3-9/IGHJ6, IGHV4-34/IGHD2-15/IGHJ6, IGHV1-3/IGHD6-19/IGHJ4, IGHV4-59/IGHD3-22/IGHJ6 and IGHV4-39/IGHD6-13/IGHJ5. Conclusions The usage of IGHV gene families indicates significant difference in Chinese CLL patients compared with Western patients, suggesting involvement of ethnic and/or environmental factors in CLL disease initiation. In the development course of CLL, BCR play an important role in the immunological recognition and selection. There are intimate relationships between mutation status of IGHV gene and prognosis. The usage of IGHV provides enlightment for the occurrence mechanism of CLL. Disclosures: No relevant conflicts of interest to declare.

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TLR, RAGE, and HMGB1 in the Inflammatory Response in Ph(-) Myeloproliferative Neoplasm

Blood ◽

10.1182/blood-2020-134988 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 31-32

Author(s):

Guanfang Shi ◽

Kiron Nair ◽

Preethi Ramachandran ◽

Chi Chen ◽

Ching Wong ◽

...

Keyword(s):

Inflammatory Response ◽

Real Time ◽

Inflammatory Cytokines ◽

Internal Control ◽

Real Time Pcr ◽

Mononuclear Cells ◽

Conflicts Of Interest ◽

Philadelphia Chromosome ◽

Myeloproliferative Neoplasm ◽

Significant Difference

Recent evidence of increased constitutional symptoms and inflammatory cytokines in Philadelphia chromosome negative (Ph (-)) MPN suggests that an inflammatory response is important in the pathogenesis of Ph (-) MPN. Toll-like receptors (TLR), Receptor for Advanced Glycation End products (RAGE) and High mobility group protein B1 (HMGB1) are the important pathways for the inflammatory response. All these three important pathway proteins were studied in MPN diseases in the current studies. Materials and Methods: TLR assay. TLR 2,3, 4, 7, 9 quantification was performed by immuno-staining of 1×106 mononuclear cells (peripheral blood) which were incubated with fluorescence-conjugated anti-TLR-2,3, 4, 7, 9 antibodies and assayed by flow cytometry. HMGB1assay:HMGB1 ELISA kit from Immuno-Biological Laboratories, Inc. (IBL-America) were used. The plasma samples were diluted four times with the provided sample dilution buffer, and assayed in duplicate according to the manufacturer's suggestion. RAGE (RT-PCR) Assay: Total RNA was extracted from normal control or patient mononuclear cells. Predesigned primers for RAGE, and internal control genes were ordered from Qiagen (Germantown, MD). Real-time PCR was performed using SsoAdvanced™ Universal SYBR® Green Supermix (Bio-Rad, Hercules, CA) on Bio-Rad iQ5 Multicolor Real-Time PCR Detection System. At least three house-keeping genes (ribosomal protein L4, TATA box binding protein, and tubulin-α 1b) were used as normalization controls. The expression of RAGE were compared with each internal control. Average of three was used to calculate the ratio of final patient to normal Results: Total of 97 patients with MPN were studied 1) TLR: TLR 3,7,9 was not significantly different from controls. But TLR 2 was significantly increased in both PV, as well as in the MPN group when PV, ET and MF were grouped together as MPN (Fig A). TLR 4 was not significantly increased in PV, ET, MF individually but was found to be significantly increased than the controls, when they are grouped together as MPN (Fig B). 2) RAGE: No significant difference was found between ET, PV, MF individually or when they were grouped together as MPN than the controls (Fig C). 3) HMGB1: No significant difference was seen between ET, PV, MF or when they were grouped as MPN (Fig D). Conclusion: Current study suggests that TLR pathway especially TLR2, and to a lesser extent TLR4 are the important pathways for inflammatory response with increased inflammatory cytokines in MPN, while HMGB1 and RAGE pathways were not different from controls. Figure Disclosures No relevant conflicts of interest to declare.

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Comparative Efficacy of First-Line Chemotherapy-Free Combinations in Chronic Lymphocytic Leukemia (CLL): A Network Meta-Analysis

Blood ◽

10.1182/blood-2020-136411 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 25-26

Author(s):

Philip A Haddad ◽

Nowell Ganey ◽

Kevin M. Gallagher

Keyword(s):

Chronic Lymphocytic Leukemia ◽

English Language ◽

Conflicts Of Interest ◽

Residual Disease ◽

Meta Analysis ◽

The Elderly ◽

Lymphocytic Leukemia ◽

First Line ◽

Significant Difference ◽

Anti Cd20

Introduction: Chronic Lymphocytic Leukemia (CLL) is an incurable B-cell malignancy which disproportionately affects the elderly. Although first-line chemoimmunotherapy (CIT) improved CLL clinical outcomes, recent randomized trials revealed superior outcomes with novel chemotherapy-free combinations (CFC) incorporating anti-CD20 monoclonal antibodies and inhibitors of BTK or Bcl-2. So far, these CFC have not been compared head-to-head. We conducted this network meta-analysis to evaluate their relative efficacy to each other. Methods: A review of the medical literature was conducted using online databases. Inclusion criteria consisted of English language; diagnosis of CLL; trials that explored the efficacy of first-line CFC with Obinutuzumab (O), Rituximab (R), Ibrutinib (IB), Acalabrutinib (ACAL), Venetoclax (VEN) compared to standard CIT that included Chlorambucil (CHLOR) with either R or O, Bendamustine+Rituximab, or Fludarabine+ Cyclophosphamide+R; and phase 3 randomized studies reporting responses, progression, death, and adverse (AE) events. A frequentists network meta-analysis was conducted using netmeta package and random-effects model. Results: Five studies comprising a total of 2,272 participants were included. When O-based CFC data was analyzed, only ACAL-O had a significant lower relative risk (RR) of progression and death (P&D). There were no significant differences with respect to overall response rates (ORR), complete remission (CR), minimal residual disease (MRD), or grade >3 adverse events (Grd3+) among O-based CFC. When R-based CFC data was analyzed, IB and IB-R were not different with respect to RR of P&D, ORR, CR, MRD, or Grd3+. When the data was analyzed as CFC versus combined CIT, only ACAL-O was found to be significantly superior to other O- and R-based CFC with respect to RR of P&D. ORR and Grad3+ rates of O- and R-based CFC were not significantly different. While ACAL-O, IB-O, and VEN-O had superior CR and MRD rates compared to other CFC, there were no significant differences among each other. Conclusions: This network meta-analysis is the first to compare and rank first-line CFC therapies in CLL. It indicates that ACAL-O has a superior profile having the lowest RR of P&D without significant difference in Grd3+ among CFC. Disclosures No relevant conflicts of interest to declare.

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Monocytes/Macrophages Are the Major Targets of the CCL3 Chemokine Produced by CD38+CD49d+ Chronic Lymphocytic Leukemia Cells.

Blood ◽

10.1182/blood.v114.22.2350.2350 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2350-2350

Author(s):

Antonella Zucchetto ◽

Dania Benedetti ◽

Claudio Tripodo ◽

Riccardo Bomben ◽

Fleur Bossi ◽

...

Keyword(s):

Endothelial Cells ◽

Chronic Lymphocytic Leukemia ◽

T Lymphocytes ◽

Conflicts Of Interest ◽

Cell Types ◽

Lymphocytic Leukemia ◽

Bone Marrow Biopsies ◽

Tnf Α ◽

Significant Difference

Abstract Abstract 2350 Poster Board II-327 Introduction: CD38 and CD49d are associated negative prognosticators in chronic lymphocytic leukemia (CLL). Recent gene expression profiling studies comparing CLL cases expressing low versus high levels of CD38 and CD49d, identified CCL3 as a gene upregulated by CD38+CD49d+ CLL. The release of CCL3 by cultured CLL cells was also demonstrated upon CD38 triggering, and CCL3 protein was found in CLL cells from bone marrow biopsies (BMB) of CD38+ cases (Zucchetto et al., Cancer Res, 2009; 69:4001-9). Given the role of CCL3 as potent chemoattractant for different cell types, we aimed at identifying the major targets of CCL3, as produced by CD38+CD49d+ CLL cells. Methods: CLL infiltrates of BMB were characterized by immunohistochemistry (IHC). Expression of the CCL3 receptors CCR1 and CCR5 by PB CLL subpopulations was evaluated by flow cytometry. T lymphocyte and monocyte migrations were performed by in-vitro transwell chemotaxis assays. Results: IHC analysis of BMB from 16 CLL cases revealed a higher number of infiltrating CD68+ cells in the context of CLL-involved areas of BMB from CD38+CD49d+CCL3+, compared to CD38−CD49d−CCL3− cases (p=0.01). CD3+ lymphocytes were interspersed in the CLL aggregates, but with no significant difference between the two subgroups. Evaluation of CCR1 and CCR5 in PB cell subpopulations from 40 CLL cases expressing or not surface CD38 and CD49d, showed the highest mean fluorescence intensity (MFI) levels for both CCR1 (624±60) and CCR5 (64±9) in the monocytic component, irrespective of CD38 and CD49d expression by CLL cells. Conversely, both CLL cells and residual T lymphocytes showed low MFI levels for CCR1 (19±4 and 14±3) and CCR5 (21±2 and 20±2). High CCR1 and CCR5 expression levels were detected in in-vitro differentiated monocytes from purified PB cells of four CD38+CD49d+ CLL. Accordingly, CCR1 expression was documented in macrophage-like cells in BMB from CD38+CD49d+ CLL. Next, we evaluated the capability of purified monocytes and T lymphocytes from 10 CLL cases to migrate in response to CCL3. In keeping with the strong expression of CCR1, monocytes migrated toward CCL3 at a concentration of 3 ng/mL (migration index, MI= 8.8±0.9, p=0.03), whereas T lymphocytes required a higher CCL3 concentration (100 ng/mL) to display slight migration capability (MI= 1.6±0.2, p=ns). The increased infiltration of macrophages in BMB from CCL3-producing CD38+CD49d+ CLL, prompted us to verify the capability of CCL3-stimulated macrophages to induce the expression by endothelial cells (EC) of the CD49d specific ligand VCAM-1. By using two different EC models (HUVEC and ADMEC), we documented a significant up-regulation of VCAM-1 by EC exposed to conditioned media (CM) collected from cultures of macrophages challenged in-vitro with CCL3 (p=0.002). Notably, increased levels of the pro-inflammatory cytokine TNF-α were detected in CCL3-CM (p=0.006), and neutralization of TNF-α by specific antibodies reverted the capability of CCL3-CM to induce VCAM-1 by EC models. In agreement with these in-vitro data, we found a more prominent meshwork of VCAM-1+ stromal/endothelial cells in lymphoid infiltrates from CD38+CD49d+ CLL compared to CD38−CD49d− cases (p=0.002), and engagement of CD49d by VCAM-1 was able to significantly delay the spontaneous apoptosis observed in cultured CLL cells. Conclusions: CD68+ monocytes/macrophages are likely the main targets for the CLL3 chemokine produced by CD38+CD49d+ CLL cells, and are active in determining, through the release of TNF-α and other yet unidentified cytokines, the overexpression of VCAM-1 by endothelial cells. Experiments aimed at investigating further roles of CD68+ monocytes/macrophage in CLL are currently matter of study. Disclosures: No relevant conflicts of interest to declare.

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The Outcome of CLL Patients with 97% Identity to Germline Is Inferior to Other ‘Mutated’ Cases Defined by a 98% Cut off

Blood ◽

10.1182/blood.v118.21.2842.2842 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2842-2842

Author(s):

Zadie Davis ◽

Anton Parker ◽

Daniel Catovsky ◽

David Oscier

Keyword(s):

Cox Regression ◽

Conflicts Of Interest ◽

Early Stage ◽

Prognostic Significance ◽

Published Data ◽

Early Stage Disease ◽

Ighv Gene ◽

Significant Difference ◽

Gene Usage ◽

The Uk

Abstract Abstract 2842 IGHV gene mutational load and use of specific IGHV genes and stereotypes have all been reported to have prognostic significance in CLL. In the UK CLL4 trial there were significant differences in response rate and progression free survival regardless of whether a 97% or 98% cut off was used, and the percentage of mutations which correlates best with clinical outcome remains controversial. We performed IGHV gene sequencing on 1071 patients with CLL or ‘clinical’ MBL (n= 153) in whom biomarkers, time to first treatment (TTFT) and overall survival (OS) were available. Four hundred and ninety six cases were entered into the UK CLL4 trial and 575 presented or were referred to the Royal Bournemouth Hospital. TTFT and OS were determined for cases with <96% identity and for each mutational point from 96% – 100% identity separately, excluding cases utilising IGHV3-21 assigned to stereotype subset 2. There was a significant difference in median TTFT and median OS between those with 97% identity (TTFT-20.9 and OS-99.3 months) and <97% identity (TTFT-118 and OS-191 months; p<0.001 and p<0.001), but not between cases with 97% and >97% identity (TTFT -13.1 months p=0.052 and OS-84.5 months p=0.177). When TTFT was determined for patients with early stage disease only (stage A CLL or CLL-like MBL, n=571), those with 97% identity, determined using either leader sequence or BIOMED 2 primers, had a significantly longer median TTFT than those with >97% identity (92.0 and 36.4 months respectively; p=0.012) and significantly shorter than those cases with <97% identity (273.1 months; p=0.012). If only stage A cases were analysed, those with 97% identity had a significantly longer median TTFT than those with >97% identity (TTFT; 68 vs. 26 months p=0.034). However, when compared to cases with <97% identity, there was a trend towards a shorter TTFT but significance was not reached (68 vs. 128 months p=0.060). A series of Cox Regression analyses were conducted to see if the prognostic value of the 98% cut off in MBL/stage A cases could be improved. In univariate analyses a model which incorporated <97%, 97%, >97% identity and stereotype subset 2 was the best discriminator of TTFT (p=0.0011) (Figure 1).Figure 1.Four subgroups of MBL/stage A CLL with differing TTFT based on stereotype subset 2 and relationship to 97% germline identityFigure 1. Four subgroups of MBL/stage A CLL with differing TTFT based on stereotype subset 2 and relationship to 97% germline identity Multivariate analysis, selected 97% and >97% identity as independent predictors of shorter TTFT (HR 2.5; 95% CI 1.3–4.9; p=0.007 and HR 4.2; 95% CI 2.9–6.1; p<0.001 respectively) in a model including <97%, 97%, >97% identity to germline, age at diagnosis, gender, expression of ZAP70, expression of CD38, del11q, del17p, stereotypy and stereotype subset 2 (Table 1). Further analyses were performed to investigate whether the differences in TTFT between cases with <97%, 97% or >97% identity could be explained by differences in IGHV gene usage. Sixty-one percent of cases with 97% identity to germline utilised only five genes; IGHV3-21, IGHV3-23, IGHV3-48, IGHV3-53 and IGHV1-18 and these genes were significantly over-represented in cases with 97% compared to either, cases with <97% (p>0.001) or >97% (p>0.001). When subset 2 cases were excluded, there was no difference in TTFT between cases using the above 5 genes and all other IGHV genes at this identity (p=0.288). In contrast to previously published data we found no difference in TTFT between mutated IGHV3-23 cases and other mutated cases (using a 98% cut-off), but IGHV3-23 cases with 97% identity had a shorter TTFT than cases with <97% identity (p<0.001). In conclusion the clinical course of cases with 97% identity, especially if diagnosed early in their disease, appears distinct from other cases defined as having mutated IGHV genes using the conventional 98% cut off. This is not accounted for by differences in IGHV gene usage, the incidence of stereotypy or other biomarkers and may reflect differences in response to BCR stimulation between cases with 97% and <97% identity.Table.Multivariate analysis for TTFT in MBL/stage A CLLOutcomeCovariateHazard Ratio (HR)95% CI for HRSignificance (p)TTFT97% identity2.51.3–4.90.007>97% identity4.22.9–6.1<0.001Subset 23.71.8–7.4<0.001Del11q1.71.1–2.70.014CD381.51.0–2.00.028Age at diagnosis0.980.96–0.99<0.001 Only covariates selected as significant are listed above. Disclosures: No relevant conflicts of interest to declare.

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CD1d Expression Is Higher In Chronic Lymphocytic Leukemia Patients With Unfavorable Prognosis

Blood ◽

10.1182/blood.v122.21.5278.5278 ◽

2013 ◽

Vol 122 (21) ◽

pp. 5278-5278

Author(s):

Agnieszka Bojarska-Junak ◽

Iwona Hus ◽

Anna Dmoszynska ◽

Jacek Rolinski

Keyword(s):

B Cells ◽

Chronic Lymphocytic Leukemia ◽

Disease Progression ◽

Conflicts Of Interest ◽

Staining Intensity ◽

Lymphocytic Leukemia ◽

Disease Stage ◽

Inkt Cells ◽

Cd38 Expression

Abstract Chronic lymphocytic leukemia (CLL) is characterized by a very heterogeneous clinical course, which is slow and indolent in most of the patients, however some patient experience rapid disease progression and anticancer therapy is required shortly after the diagnosis. Many issues in CLL development and progression are still unclear. The functional consequences of CD1d expression on tumour cells are not well understood. However, increasing evidence suggests that they may affect iNKT cells.The role of CD1d expression in CLL immunopathogenesis remains undefined. In this study, we investigated the potential role of CD1d in CLL by analyzing the level of CD1d expression on leukemic B cells in peripheral blood of120 patients and assessed its correlation with prognostic markers such as ZAP-70 and CD38 expression, Rai stages and unfavourable cytogenetic changes.Measuring CD1d expression by flow cytometry and qRT-PCR, we showed lower CD1d molecule and CD1d mRNA expression in B cells of CLL patients than of healthy controls. The frequency of CD1d+/CD19+ cells, CD1d staining intensity and CD1d transcript levels increased with the disease stage. CD1d expression was positively associated with ZAP-70 and CD38 expressions as well as with unfavourable cytogenetic changes (17p deletion, 11q deletio),. We established the relationship between high CD1d expression and shorter time to treatment and overall survival. The percentage of CD1d+/CD19+cells inversely correlated with the percentage of iNKT cells. iNKT cells ζ-chain expression was downregulated in the high-CD1d group.These results suggest that high CD1d expression is associated with poor prognosis of CLL and might be involved in disease progression. Disclosures: No relevant conflicts of interest to declare.

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Vegf and bFGF Single-Nucleotide Polymorphisms In Chronic Lymphocytic Leukemia: Impact On Susceptibility

Blood ◽

10.1182/blood.v122.21.5287.5287 ◽

2013 ◽

Vol 122 (21) ◽

pp. 5287-5287

Author(s):

Sandra Ballester ◽

Begoña Pineda ◽

Eduardo Tormo ◽

Blanca Navarro ◽

Ariadna Perez ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Conflicts Of Interest ◽

Expression Patterns ◽

Lymphocytic Leukemia ◽

P Value ◽

Nucleotide Polymorphisms ◽

Exact Test ◽

Cd38 Expression ◽

Mutational Status ◽

The Individual

Abstract Background B-cell chronic lymphocytic leukemia (B-CLL) is a heterogeneous disease with a highly variable clinical outcome. Recent studies have identified a number of different molecular prognostic markers (including mutational status of the IgVH gene, ZAP70 and CD38 expression) that allow to discriminate patients in prognostic subgroups. However, different expression patterns of angiogenic factors as VEGF, VEGFR1 and bFGF have been related with B-CLL susceptibility and treatment requirements. We have analyzed the polymorphisms: -710 C/T in VEGFR1, rs1109324, rs1547651, rs3025039 (936C/T) and rs833052 in VEGF and rs1449683 (223 C/T) in bFGF in order to determine the possible association with susceptibility in B-CLL. Methods Peripheral blood samples from 230 B-CLL patients and 476 healthy controls were genotyped using probes TaqMan SNP Genotyping Assays. Samples were providing from the Hospital Clinic of Valencia. Four SNPs in the VEGF gene, one SNP in the bFGF gene and one SNP in the VEGFR1 gene were evaluated. Statistical analysis was performed using SNPStats program (Catalan Institute of Oncology) and Fisher's exact test was applied to evaluate the significance. Results We have observed an increased frequency of the T allele in the rs1449683 SNP [OR 1.62 (95% CI: 0.98-2.66) p-value =0.063] and in the rs1547651 SNP [OR 0.72 (95% CI: 0.51-1.03), p-value=0.072] in our B-LLC patients when compared to control subjects. Moreover we observed that T allele carriers of rs3025039 (VEGF) have a significant protective effect concerning this disease [OR 0.59 (95% CI: 0.39-0.89) p-value=0.009]. Conclusion Our data indicate an increased frequency of the T allele in polymorphisms rs1449683 (bFGF) and rs1547651 (VEGF) in the group of patients, which possibly account for the individual susceptibility to develop B-CLL. On the other hand the data provided suggest that the T allele of VEGF rs3025039 is likely important genetic marker of susceptibility to B-CLL. Further studies regarding the role of pro-angiogenic markers in B-CLL would be beneficial to help elucidate pathogenic pathways in this disease. Disclosures: No relevant conflicts of interest to declare.

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Revisiting Hypogammaglobulinemia in Chronic Lymphocytic Leukemia: A Combined Clinicobiological Approach

Blood ◽

10.1182/blood.v124.21.5633.5633 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5633-5633 ◽

Cited By ~ 3

Author(s):

Panagiotis Baliakas ◽

Aliki Xochelli ◽

Eva Minga ◽

Anastasia Hadzidimitriou ◽

Vassiliki Douka ◽

...

Keyword(s):

Clinical Stage ◽

Normal Serum ◽

Lymphocytic Leukemia ◽

Cd38 Expression ◽

Serum Immunoglobulins ◽

Cytogenetic Aberrations ◽

Mutational Status ◽

Ighv Gene ◽

Immunoglobulin Subclasses ◽

Notch1 Mutations

Abstract Chronic lymphocytic leukemia (CLL) is characterized by progressive hypogammaglobulinemia that can affect one or more immunoglobulin subclasses. Although many underlying mechanisms have been suggested, the pathogenesis of this phenomenon remains to be elucidated. In the present study, we revisit hypogammaglobulinemia in CLL through a combined clinicobiological approach aiming at identifying associations with particular disease profiles that would offer pathogenetic insight and guidance for further research. The study group included 412 CLL patients with available information about serum immunoglobulins either at diagnosis (n=380) or before treatment initiation (n=32). Patient characteristics were as follows: median age: 65 years; males/females: 266/146; Binet stage A: 272/335, unmutated IGHV genes (U-CLL): 140/412 cases (34%); CD38 expression: 59/330 cases (18%); clonotypic IG of the MD or G isotype: 250 and 43 cases, respectively; isolated del(13q): 64/136 (47%); trisomy 12: 18/183 (10%); del(11q): 18/186 (10%); del(17p): 11/189 (6%); NOTCH1 del7544-45/p.P2514Rfs*4: 8/219 (4%). With a median follow up of 5 years, 152/329 cases (46%) received treatment. Decreased immunoglobulin serum levels in at least one subclass were identified in 220/412 patients (53%), as follows: (i) decreased IgM, 172/412 cases (41%); (ii) decreased IgG, 78/412 cases (19%); (iii) decreased IgA, 100/412 cases (24%). In 36/412 cases (9%), a decrease in all serum immunoglobulin subclasses was noted. No statistically significant differences were identified between patients with normal serum immunoglobulin levels versus those with hypogammaglobulinemia regarding age, gender, disease burden at diagnosis, IGHV gene mutational status, CD38 expression, cytogenetic aberrations, NOTCH1 mutations and the incidence of a second malignancy. However patients with hypogammaglobulinemia exhibited increased need for treatment compared to patients with normal serum immunoglobulins (91/175 vs 61/154 respectively, p=0.025). Among cases with hypogammaglobulinemia, 90 (41%) and 26 (12%) exhibited isolated IgM and IgA subclass deficiency, respectively; isolated IgG decrease, was relatively rare (10/220 cases, 4%). Interestingly, when comparing isolated IgA versus other subclass deficiencies, statistically significant associations were identified with (i) advanced clinical stage (Binet B/C, Rai III/IV) (p=0.002); (ii) female gender (p=0.041); and, (iii) NOTCH1 mutations (p=0.004). A propos of the latter, it is noteworthy that in 5/8 (63%) mutant NOTCH1 cases with hypogammaglobulinemia, the affected subclass was IgA. Within our cohort, we identified cases belonging to one of three different, well characterized subsets with stereotyped B-cell receptor immunoglobulin (BcR IG), namely: (1) subset #1 (clan I IGHV genes/IGKV1(D)-39): U-CLL, clinically aggressive, n=12; (2) subset #2 (IGHV3-21/IGLV3-21), mixed IGHV mutational status, noted clinical aggressiveness, n=5; and, (3) subset #4, mutated IGHV4-34/IGKV2-30 BcR IG, clinically indolent, n=12. Notably, all subset #2 cases showed low levels of at least one serum subclass, while in 4/5 and 3/5 cases, two or all three immunoglobulin subclasses were affected. Although numbers are small, the incidence of hypogammaglobulinemia in subset #2 was significantly (p<0.05) higher compared to either subset #1 or subset #4). Univariate analysis revealed clinical stage, CD38 expression and IGHV mutational status as statistically important parameters (p<0.05) for both time-to-first–treatment (TTFT) and overall survival (OS); in contrast, hypogammaglobulinemia had no impact either on on TTFT or OS. In multivariate analysis, clinical stage and IGHV gene mutational status retained independent significance. In conclusion, abnormalities of serum immunoglobulins are detected in CLL patients with heterogeneous clinicobiological profiles, including different disease burden (clinical stage), cytogenetic aberrations and IGHV gene mutational status. However, certain observations reported herein, in particular the high incidence of hypogammaglobulinemia in subset #2 and the association of NOTCH1 mutations with IgA subclass deficiency, are noteworthy and indicate the need for research towards unraveling causal mechanisms among the observed interwined events. Disclosures No relevant conflicts of interest to declare.

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Investigating the Role of the Gut Microbiome in the Inflammatory State of Myeloproliferative Neoplasms

Blood ◽

10.1182/blood-2018-99-109761 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3051-3051

Author(s):

Kenza Elalaoui ◽

Claudia Weihe ◽

Andrew Oliver ◽

Brianna Craver ◽

Hew Yeng Lai ◽

...

Keyword(s):

16S Rrna ◽

Gut Microbiota ◽

Inflammatory Cytokines ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Symptom Burden ◽

Rrna Gene ◽

Significant Difference ◽

Microbiome Data ◽

Normal Controls

Abstract The human microbiome is composed of trillions of micro-organisms living in symbiosis with the human body. The interest in the microbiome and the clinical impact of its disruption (dysbiosis) has grown exponentially in the past years, especially in the oncology field. Microbiota play an instructive role in chronic inflammatory disorders and determine response to checkpoint inhibitors cancers, such as melanoma. Increased inflammation in myeloproliferative neoplasms (MPN) drive many of the symptoms associated with this disease and inflammation also likely plays an important role as a driver of the disease. We hypothesized that the microbiome may be dysregulated in MPN which could contribute to the increased inflammatory cytokines seen in this disease and that the microbiome could also potentially impact symptom burden. We compared the gut microbiota of 25 patients with classical MPN, essential thrombocythemia (ET), polycythemia vera (PV) and primary and secondary myelofibrosis (MF) to that of 25 normal controls. Whenever possible, co-inhabiting adults such as a spouse were used as normal controls. Exclusion criteria were acute illness and antibiotics treatment for three months preceding the study. Each participant collected three stool specimens in the course of one week and a subset of the group (n=20) had peripheral blood drawn. The stool microbiome was analyzed using 16S rRNA sequencing and plasma inflammatory cytokines (TNFα, IL-6, IL-8, IL-17a, IL-10, IFNγ, IFNa2, IL-22, IL1-β, GRO, IP-10) were measured using Luminex multiplex analysis. Participants also completed an intake survey which included symptom burden (MPN-SAF TSS) and dietary intake. Characteristics of our MPN patient cohort and normal controls are shown in Table 1. Plasma levels of TNFα and IP-10 were significantly higher in the MPN group versus normal controls (respectively p=0.013 and p=0.0050). We divided the group into those with high (MPN-SAF > 20) and low (MPN-SAF ≤ 20) symptom burden, 7 (28%) patients had high symptom burden affecting considerably their quality of life. Fatigue and early satiety were the most common complaints. GRO (CXCL1) was significantly lower in MPN patients MPN patients with a lower symptom burden (p=0.0287) as compared to those with a high symptom burden. The 16S rRNA gene sequencing did not reveal a significant difference between MPN patients and their healthy counterparts (PERMANOVA, p = 0.83), a large amount of variance in the microbiome data was explained by the individual (PERMANOVA, p= 0.0001), in concordance with other studies suggesting that the microbiome is highly individualistic. To determine whether certain taxa are differentially represented in MPN patients as compared to normal controls, a SIMPER test was used. Prevotellaceae, a family of bacteria implicated in chronic inflammatory conditions, was 20% higher in MPN patients than in normal controls. To test which cytokines explained the most variance in the microbiome data, a distance-based linear model was used (DistLM). TNFα and IL-17a, taken alone, explain 18.7% and 14.5% respectively. The microbiome of patients with a high symptom burden was not significantly different from MPN patients with low symptom burden. We did find a significant difference between the microbiomes of patients treated with Ruxolitinib versus Hydroxyurea. Taken together, this pilot study suggests that inflammation associated with MPN may be driving changes in the microbiome at a finer level, undetected when comparing broad microbial diversity metrics between health and disease. It is still not clear whether the inflammation first modulates the microbiome or whether the gut microbiota triggers the inflammatory signals driving the disease. A larger number of subjects may help answer these questions. Disclosures No relevant conflicts of interest to declare.

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98% IGHV Gene Identity Is Still the Optimal Cutoff to Predict the Prognosis of Chronic Lymphocytic Leukemia Patients in China

Blood ◽

10.1182/blood-2018-99-118141 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 5545-5545

Author(s):

Yi Xia ◽

Ke Shi ◽

Qian Sun ◽

Chun Qiao ◽

Huayuan Zhu ◽

...

Keyword(s):

Prognostic Factor ◽

Chronic Lymphocytic Leukemia ◽

Conflicts Of Interest ◽

Somatic Hypermutation ◽

Lymphocytic Leukemia ◽

Study Cohort ◽

Optimal Cutoff ◽

Entire Cohort ◽

Mutational Status ◽

Significant Difference

Abstract Background: Immunoglobulin heavy chain variable region (IGHV) has been an important prognostic factor for chronic lymphocytic leukemia (CLL) for decades. 98% being a cut-off value for IGHV is a mathematical choice and researches on the best cut-off value have never been stopped. Chinese CLL patients are known to differ from Caucasian CLL patients on both clinical and genetical features. However, the optimal cutoff for IGHV mutational status has not yet been studied in this particular ethnic group. Method: We carried out a study on 595 Chinese CLL patients in order to find out whether 98% is the best cut-off value for IGHV in Chinese CLL patients. Genomic DNA from peripheral blood or bone marrow was subjected to PCR amplification following the IGH Somatic Hypermutation Assay v2.0 protocol (InVivoScribe). Sequences were aligned to ImMunoGeneTics/V-QUEry and Standardization (IMGT/-VQUEST) database. Result: 600 sequences were received after IGHV rearrangement sequencing. IGHV3-23, IGHV4-34, IGHV3-7, IGHV4-39 and IGHV1-69 were the most frequently used IGHV genes. 352 (58.7%) cases were IGHV-mutated while 248 (41.3%) cases were IGHV-unmutated if the classical 98% classification by ERIC was used. In order to determine the optimal cut-off value, we used 1% as the interval to divide the entire cohort into 7 groups according to the mutational rate, which were <95%, 95%-95.99%, 96%-96.99%, 97%-97.99%, 98%-98.99%, 99%-99.99% and 100% respectively. Binet A patients had a relatively indolent course of disease and cases with different IGHV mutational rates had no significant differences in time to first treatment (TTFT) apart from truly unmutated (100%) cases. For the whole study cohort, significant difference appeared at 98% interval (P<0.001 and P=0.005 for TTFT and OS respectively) while intervals less than 98% had no significant difference compared with the <95% group. Similarly, there was no clear dissimilarities among 98%, 99% and 100% intervals (Table 1a and b). All the other prognostic factors including del(17p), del(11q), TP53 mutation, MYD88 mutation, NOTCH1 mutation, SF3B1 mutation, CD38, ZAP-70, Binet staging, gender, β2-microglobulin and EBV-DNA were differently distributed between group <98% and group ³98%, but not among subgroups in ³98%. In multivariate analysis, the 98% IGHV was also an independent prognostic factor for TTFT and OS. Conclusion: 98% is the optimal cutoff value for IGHV mutational status to predict the prognosis of CLL patients in China. Table 1. Table 1. Disclosures No relevant conflicts of interest to declare.

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