Nicholas Chiorazzi

The Feinstein Institute for Medical Research, New York City, New York, United States

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Publications (207)1507.7 Total impact

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    ABSTRACT: An unresolved issue in chronic lymphocytic leukemia (CLL) is whether IGHV3-21 gene usage, in general, or the expression of stereotyped B-cell receptor immunoglobulin defining subset #2 (IGHV3-21/IGLV3-21), in particular, determines outcome for IGHV3-21-utilizing cases. We reappraised this issue in 8593 CLL patients of whom 437 (5%) used the IGHV3-21 gene with 254/437 (58%) classified as subset #2. Within subset #2, immunoglobulin heavy variable (IGHV)-mutated cases predominated, whereas non-subset #2/IGHV3-21 was enriched for IGHV-unmutated cases (P = .002). Subset #2 exhibited significantly shorter time-to-first-treatment (TTFT) compared with non-subset #2/IGHV3-21 (22 vs 60 months, P = .001). No such difference was observed between non-subset #2/IGHV3-21 vs the remaining CLL with similar IGHV mutational status. In conclusion, IGHV3-21 CLL should not be axiomatically considered a homogeneous entity with adverse prognosis, given that only subset #2 emerges as uniformly aggressive, contrasting non-subset #2/IGVH3-21 patients whose prognosis depends on IGHV mutational status as the remaining CLL. © 2015 by The American Society of Hematology.
    Blood 01/2015; 125(5):856-9. · 9.78 Impact Factor
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    ABSTRACT: Chronic Lymphocytic Leukaemia (CLL) development and progression is thought to be driven by unknown antigens/autoantigens through the B cell receptor (BCR), and environmental signals for survival and expansion including Toll-like receptor (TLR) ligands. CD180/RP105, a membrane-associated orphan receptor of the TLR family, induces normal B cell activation and proliferation and is expressed by approximately 60% of CLL samples. Half of these respond to ligation with anti-CD180 antibody by increased activation/phosphorylation of protein kinases associated with BCR signaling. Hence CLL cells expressing both CD180 and the BCR could receive signals via both receptors. Here we investigated cross-talk between BCR and CD180-mediated signaling on CLL cell survival and apoptosis. Our data indicate that ligation of CD180 on responsive CLL cells leads to activation of either pro-survival BTK/PI3K/AKT-mediated, or pro-apoptotic p38MAPK-mediated signaling pathways, whilst sIgM ligation predominantly engages the BTK/PI3K/AKT pathway. Furthermore, pre-treatment of CLL cells with anti-CD180 redirects IgM-mediated signaling from the pro-survival BTK/PI3K/AKT towards the pro-apoptotic p38MAPK pathway. Thus pre-engaging CD180 could prevent further pro-survival signaling mediated via the BCR and, instead, induce CLL cell apoptosis, opening the door to therapeutic profiling and new strategies for the treatment of a substantial cohort of CLL patients.
    Molecular medicine (Cambridge, Mass.). 01/2015;
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    ABSTRACT: Human B-cell chronic lymphocytic leukemia (CLL) is a clonal CD5+CD19+ B-lymphocyte whose B-cell receptor may be classified as unmutated (U-CLL) or mutated (M-CLL) depending on the level of IGHV mutations. Aggressive CLL associates with acquisition of new gene mutations and cytogenetic aberrations, not necessarily in the IGHV or IGLV loci and perhaps caused by activation-induced deaminase (AID). To test if CLL cells can produce functional AID, CLL cells were activated in vitro with CD32-transfected murine L cells, anti-CD40 and interleukin-4 (7 and 14 days U-CLL1278, 0.0% mutated IGHV3-30 ; 14 days M-CLL1299, 4.9% mutated IGHV3-23 ), plus irradiated T lymphocytes (10 or 14 days M-CLL1299). CLL cells in these cultures produced detectable AID protein. To evaluate mutational activity, CLL IGHV cDNA was ultra-deep sequenced using the 454 FLX system (Roche) prior to (day 0) or after activation. The resulting 458,124 sequence reads were processed to generate fixed sequence length datasets. Individual subclone sequences occurring at least twice were extracted and unique de novo subclones not shared between day 0 and activation were analyzed for new mutations. All culture conditions showed increases in IGHV mutation frequencies relative to the IGHM constant region. U-CLL1278 showed increased mutation at AID hotspots and a lower transition mutation frequency. M-CLL1299 showed an overall high frequency of transitions and an increase in mutation at AID hotspots in T cell cultures. Thus, de novo mutations consistent with AID activity were found, with some differences between U-CLL and M-CLL. Mutationally-active AID in CLL could lead to adverse consequences.
    15th International Congress of Immunology, 12/2014: pages 13-16; Medimond S.r.l., Monduzzi Editore., ISBN: 978-88-7587-711-8
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    ABSTRACT: Νext generation sequencing studies in Homo sapiens have identified novel immunoglobulin heavy variable (IGHV) genes and alleles necessitating changes in the international ImMunoGeneTics information system (IMGT) GENE-DB and reference directories of IMGT/V-QUEST. In chronic lymphocytic leukaemia (CLL), the somatic hypermutation (SHM) status of the clonotypic rearranged IGHV gene is strongly associated with patient outcome. Correct determination of this parameter strictly depends on the comparison of the nucleotide sequence of the clonotypic rearranged IGHV gene with that of the closest germline counterpart. Consequently, changes in the reference directories could, in principle, affect the correct interpretation of the IGHV mutational status in CLL. To this end, we analyzed 8066 productive IG heavy chain (IGH) rearrangement sequences from our consortium both before and after the latest update of the IMGT/V-QUEST reference directory. Differences were identified in 405 cases (5 % of the cohort). In 291/405 sequences (71.9 %), changes concerned only the IGHV gene or allele name, whereas a change in the percent germline identity (%GI) was noted in 114/405 (28.1 %) sequences; in 50/114 (43.8 %) sequences, changes in the %GI led to a change in the mutational set. In conclusion, recent changes in the IMGT reference directories affected the interpretation of SHM in a sizeable number of IGH rearrangement sequences from CLL patients. This indicates that both physicians and researchers should consider a re-evaluation of IG sequence data, especially for those IGH rearrangement sequences that, up to date, have a GI close to 98 %, where caution is warranted.
    Immunogenetics 11/2014; · 2.49 Impact Factor
  • Shih-Shih Chen, Nicholas Chiorazzi
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    ABSTRACT: Chronic lymphocytic leukemia (CLL) is a genetically complex disease, with multiple factors impacting on onset, progression, and response to therapy. Genetic differences/abnormalities have been found in hematopoietic stem cells from patients, as well as in B lymphocytes of individuals with monoclonal B-lymphocytosis that may develop the disease. Furthermore, after the onset of CLL, additional genetic alterations occur over time, often causing disease worsening and altering patient outcomes. Therefore, being able to genetically-engineer mouse models that mimic CLL or at least certain aspects of the disease help understand disease mechanisms and improve treatments. This notwithstanding, since neither the genetic aberrations responsible for leukemogenesis and progression nor the promoting factors that support these are likely identical in character or influences for all patients, genetically-engineered mouse models will only completely mimic CLL when all of these factors are precisely defined. In addition, multiple genetically-engineered models may be required because of the heterogeneity in susceptibility genes among patients that impact on genetic and environmental characteristics influencing disease development and outcome. For these reasons, in this chapter we review the major murine genetically-engineered and human xenograft models in use at the present time, aiming to report the advantages and disadvantages of each.
    Seminars in Hematology 07/2014; · 2.46 Impact Factor
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    ABSTRACT: B-cell chronic lymphocytic leukemia (B-CLL) patients expressing unmutated immunoglobulin heavy variable regions (IGHVs) use the IGHV1-69 B cell receptor (BCR) in 25% of cases. Since HIV-1 envelope gp41 antibodies also frequently use IGHV1-69 gene segments, we hypothesized that IGHV1-69 B-CLL precursors may contribute to the gp41 B cell response during HIV-1 infection. To test this hypothesis, we rescued 5 IGHV1-69 unmutated antibodies as heterohybridoma IgM paraproteins and as recombinant IgG1 antibodies from B-CLL patients, determined their antigenic specificities and analyzed BCR sequences. IGHV1-69 B-CLL antibodies were enriched for reactivity with HIV-1 envelope gp41, influenza, hepatitis C virus E2 protein and intestinal commensal bacteria. These IGHV1-69 B-CLL antibodies preferentially used IGHD3 and IGHJ6 gene segments and had long heavy chain complementary determining region 3s (HCDR3s) (≥21 aa). IGHV1-69 B-CLL BCRs exhibited a phenylalanine at position 54 (F54) of the HCDR2 as do rare HIV-1 gp41 and influenza hemagglutinin stem neutralizing antibodies, while IGHV1-69 gp41 antibodies induced by HIV-1 infection predominantly used leucine (L54) allelic variants. These results demonstrate that the B-CLL cell population is an expansion of members of the innate polyreactive B cell repertoire with reactivity to a number of infectious agent antigens including intestinal commensal bacteria. The B-CLL IGHV1-69 B cell usage of F54 allelic variants strongly suggests that IGHV1-69 B-CLL gp41 antibodies derive from a restricted B cell pool that also produces rare HIV-1 gp41 and influenza hemagglutinin stem antibodies.
    PLoS ONE 03/2014; 9(3):e90725. · 3.53 Impact Factor
  • Jan A. Burger, Nicholas Chiorazzi
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    ABSTRACT: B cell receptor (BCR) signaling plays an important pathogenic role in chronic lymphocytic leukemia (CLL) and B cell lymphomas, based on structural restrictions of the BCR, and BCR-dependent survival and growth of the malignant B cells. In CLL and lymphoma subtypes, ligand-independent (‘tonic’) and ligand-dependent BCR signaling have been characterized, which can involve mutations of BCR pathway components or be triggered by (auto)antigens present in the tissue microenvironment. In CLL, based on high response rates and durable remissions in early-stage clinical trials, there is rapid clinical development of inhibitors targeting BCR-associated kinases [Bruton's tyrosine kinase (BTK), phosphoinositide 3-kinase (PI3K)δ], which will change treatment paradigms in CLL and other B cell malignancies. Here, we discuss the evolution of this field, from BCR-related prognostic markers, to mechanisms of BCR activation, and targeting of BCR-associated kinases, the emerging Achilles’ heel in CLL pathogenesis.
    Trends in Immunology 12/2013; 34(12). · 12.03 Impact Factor
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    ABSTRACT: Within T-cell-dependent germinal centers, p53 gene transcription is repressed by Bcl-6 and is thus less vulnerable to mutation. Malignant lymphomas within inflamed extranodal sites exhibit a relatively high incidence of p53 mutations. The latter might originate from normal B-cell clones manifesting activation-induced cytosine deaminase (AID) and up-regulated p53 following T-cell-independent (TI) stimulation. We here examine p53 gene transcription in such TI clones, with a focus on modulatory effects of prostaglandin E2 (PGE2), and evaluate progeny for p53 mutations. Resting IgM(+)IgD(+)CD27(-) B cells from human tonsils were labeled with CFSE and stimulated in vitro with complement-coated antigen surrogate, IL-4, and BAFF ± exogenous PGE2 (50 nM) or an analog specific for the EP2 PGE2 receptor. We use flow cytometry to measure p53 and AID protein within variably divided blasts, qRT-PCR of p53 mRNA from cultures with or without actinomycin D to monitor mRNA transcription/stability, and single-cell p53 RT-PCR/sequencing to assess progeny for p53 mutations. We report that EP2 signaling triggers increased p53 gene transcriptional activity in AID(+) cycling blasts (P<0.01). Progeny exhibit p53 mutations at a frequency (8.5×10(-4)) greater than the baseline error rate (<0.8×10(-4)). We conclude that, devoid of the repressive influences of Bcl-6, dividing B lymphoblasts in inflamed tissues should display heightened p53 transcription and increased risk of p53 mutagenesis.-Haque, S., Yan, X. J., Rosen, L., McCormick, S., Chiorazzi, N., Mongini, P. K. A. Effects of prostaglandin E2 on p53 mRNA transcription and p53 mutagenesis during T-cell-independent human B-cell clonal expansion.
    The FASEB Journal 10/2013; · 5.48 Impact Factor
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    ABSTRACT: For antigen recognition, lampreys use leucine-rich repeats (LRR) instead of immunoglobulin V-(D)-J domains to generate variable lymphocyte receptors (VLR) of three types, VLRA, VLRB, and VLRC. VLRB-bearing lymphocytes respond to immunization with proliferation and differentiation into plasmacytes that secrete multivalent VLRB antibodies. Here we immunized lampreys with B cells from patients with chronic lymphocytic leukemia (CLL) to generate recombinant monoclonal VLRB antibodies, one of which, VLR39, was specific for the donor CLL cells. The target epitope of VLR39 was shown to be the complementarity determining region 3 (CDR3) of the heavy chain variable region (VH) of the B cell receptor. Using this antibody to monitor the CLL donor after chemo-immunotherapy-induced remission, we detected VLR39(+) B cells in the patient 51 months later, before significant increase in lymphocyte count or CD5(+) B cells. This indication of reemergence of the leukemic clone was verified by VH sequencing. Lamprey antibodies can exhibit exquisite specificity for a protein epitope, a CLL signature VH CDR3 sequence in this case, and offer a rapid strategy for generating anti-idiotype antibodies for early detection of leukemia recurrence.
    Cancer immunology research. 10/2013; 1(4):223-228.
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    ABSTRACT: ABSTRACT Array comparative genomic hybridization (aCGH) has yet to be fully leveraged in a prognostic setting in chronic lymphocytic leukemia (CLL). Genomic imbalance was assessed in 288 CLL specimens using a targeted array. Based on 20 aberrations in a hierarchical manner, all 228 treatment-naive specimens were classified into a group with poor outcome (20.6%) exhibiting at least one aberration that univariately associated with adverse outcome (gain: 2p, 3q, 8q, 17q, loss: 7q, 8p, 11q, 17p, 18p), good outcome (32.5%) showing 13q14 loss without any of the other ten aberrations (gain: 1p, 7p, 12, 18p, 18q, 19, loss: 4p, 5p, 6q, 7p), or intermediate outcome (remainder). The three groups significantly separated with respect to time to first treatment and overall survival (P<0.001) and validation of the stratification scheme was performed in two independent datasets. Gain of 3q and 8q, and 17p loss were determined to be independent unfavorable prognostic biomarkers. TP53, NOTCH1, and SF3B1 mutations correlated with the presence of one poor outcome aCGH marker, at a considerably higher frequency than when only considering poor risk aberrations routinely detected by FISH. These data support genomic imbalance evaluation in CLL by aCGH to assist in risk stratification.
    Leukemia & lymphoma 09/2013; · 2.61 Impact Factor
  • Cancer Research 08/2013; 73(8 Supplement):3531-3531. · 9.28 Impact Factor
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    ABSTRACT: Chronic lymphocytic leukemia (CLL) is a clonal disease of a subset of human B lymphocytes. Although the cause of the disease is unknown, its development and evolution appear to be promoted by signals delivered when B-cell receptors (BCRs) engage (auto)antigens. Here, using a peptide phage display library of enhanced size and diverse composition, we examined the binding specificity of a recombinant monoclonal antibody (mAb) constructed with the heavy chain and light chain variable domains of a CLL BCR that does not exhibit somatic mutations. As determined by testing the peptides identified in the selected peptide phage pool, this CLL-associated unmutated mAb bound a diverse set of sequences, some of which clustered in families based on amino acid sequence. Synthesis of these peptides and characterization of binding with the CLL-associated mAb revealed that mAb-peptide interactions were generally specific. Moreover, the mAb-peptide interactions were of lower affinities (micromolar KD), as measured by surface plasmon resonance, than those observed with a CLL mAb containing somatic mutations (nanomolar KD) and with IGHV-mutated antibodies selected by environmental antigens. This information may be of value in identifying and targeting B lymphocytes expressing specific BCRs in CLL patients and healthy subjects with monoclonal B lymphocytosis.
    Molecular Medicine 08/2013; · 4.82 Impact Factor
  • Piers E. M. Patten, Charles C. Chu, Nicholas Chiorazzi
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    ABSTRACT: Chronic lymphocytic leukemia (CLL) comprises 90% of chronic lymphoid leukemias in the USA and Europe, and contributes 6.7% of total cases of non-Hodgkin lymphoma [1]. It is a neoplasm of relatively monomorphic, small, round B lymphocytes with a characteristic immunophenotype. These cells reside in the peripheral blood, bone marrow and lymph nodes. Small lymphocytic lymphoma has a very similar immunophenotype to that of CLL, presenting with lymphadenopathy as opposed to lymphocytosis. CLL has an extremely variable natural history with survival from diagnosis ranging from months to decades. Some individuals require little or therapeutic intervention and enjoy a normal life expectancy, whereas others require multiple courses of treatment and ultimately die from the disease. The disease is incurable.
    Non-Hodgkin Lymphomas: Advanced Diagnostics and Personalized Therapies, Edited by Antonino Carbone, Ana Younes, 06/2013: pages 196-214; Future Medicine Ltd., ISBN: eBook ISBN: 978-1-78084-171-7
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    ABSTRACT: Ag selection has been suggested to play a role in chronic lymphocytic leukemia (CLL) pathogenesis, but no large-scale analysis has been performed so far on the structure of the Ag-binding sites (ABSs) of leukemic cell Igs. We sequenced both H and L chain V(D)J rearrangements from 366 CLL patients and modeled their three-dimensional structures. The resulting ABS structures were clustered into a small number of discrete sets, each containing ABSs with similar shapes and physicochemical properties. This structural classification correlates well with other known prognostic factors such as Ig mutation status and recurrent (stereotyped) receptors, but it shows a better prognostic value, at least in the case of one structural cluster for which clinical data were available. These findings suggest, for the first time, to our knowledge, on the basis of a structural analysis of the Ab-binding sites, that selection by a finite quota of antigenic structures operates on most CLL cases, whether mutated or unmutated.
    The Journal of Immunology 05/2013; · 5.36 Impact Factor
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    ABSTRACT: Recent studies have revealed recurrent mutations of the NOTCH1, SF3B1 and BIRC3 genes in chronic lymphocytic leukemia (CLL), especially among aggressive, chemorefractory cases. Nevertheless, it is currently unknown whether their presence may differ in subsets of patients carrying stereotyped B-cell receptors (BcR) and also exhibiting distinct prognoses. Here, we analyzed the mutation status of NOTCH1, SF3B1 and BIRC3 in three subsets with particularly poor prognosis, i.e. subsets #1, #2 and #8, aiming to explore links between genetic aberrations and immune signaling. A remarkably higher frequency of SF3B1 mutations was revealed in subset #2 (44%) versus subsets #1 and #8 (4.6% and 0%, respectively; P<0.001). In contrast, the frequency of NOTCH1 mutations in subset #2 was only 8%; lower than the frequency observed in either subset #1 or #8 (19% and 14%, respectively; P=0.04 for subset #1 versus #2). No associations were found for BIRC3 mutations that overall were rare. The apparent non-random association of certain mutations with stereotyped CLL subsets alludes to subset-biased acquisition of genomic aberrations, perhaps consistent with particular antigen/antibody interactions. These novel findings assist in unraveling specific mechanisms underlying clinical aggressiveness in poor-prognostic stereotyped subsets, with far-reaching implications for understanding their clonal evolution and implementing biologically-oriented therapy.Leukemia accepted article preview online, 5 April 2013; doi:10.1038/leu.2013.98.
    Leukemia: official journal of the Leukemia Society of America, Leukemia Research Fund, U.K 04/2013; · 10.16 Impact Factor
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    ABSTRACT: (Auto)antigen engagement by the B-cell receptor (BCR) and possibly the sites where this occurs influence the outcome of chronic lymphocytic leukemia (CLL). To test if selection for autoreactivity leads to increased aggressiveness and if this selection plays out equally in primary and secondary tissues, we used T-cell leukemia (TCL)1 cells reactive with the autoantigen phosphatidylcholine (PtC). After repeated transfers of splenic lymphocytes from a single mouse with oligoclonal PtC-reactive cells, outgrowth of cells expressing a single IGHV-D-J rearrangement and superior PtC-binding and disease virulence occurred. In secondary tissues, increased PtC-binding correlated with enhanced BCR signaling and cell proliferation, whereas reduced signaling and division of cells from the same clone was documented in cells residing in the bone marrow, blood, and peritoneum, even though cells from the last site had highest surface membrane IgM density. Gene-expression analyses revealed reciprocal changes of genes involved in BCR-, CD40-, and PI3K-signaling between splenic and peritoneal cells. Our results suggest autoantigen-stimulated BCR signaling in secondary tissues promotes selection, expansion, and disease progression by activating pro-oncogenic signaling pathways, and that-outside secondary lymphoid tissues-clonal evolution is retarded by diminished BCR-signaling. This transferrable, antigenic-specific murine B-cell clone (TCL1-192) provides a platform to study the types and sites of antigen-BCR interactions and genetic alterations that result and may have relevance to patients.
    Proceedings of the National Academy of Sciences 04/2013; · 9.81 Impact Factor
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    ABSTRACT: Background. While many prognostic markers in B-cell chronic lymphocytic leukemia provide insight into the biology of the disease, few have been demonstrated to be useful in the daily patient management. B-cell receptor signaling is a driving event in B-cell chronic lymphocytic leukemia progression and markers of B-cell receptor responsiveness have been shown to be of prognostic value. Single cell network profiling, a multiparametric flow cytometry-based assay, allows functional signaling analysis at the level of the single cell. Design and Methods. B-cell receptor signaling proteins (i.e. p-SYK, p-NF-κB p65, p-ERK, p-p38, p-JNK) were functionally characterized by single cell network profiling in samples from patients with B-cell chronic lymphocytic leukemia in an exploratory study (n=27) after stimulation with anti-IgM. Significant association of single cell network profiling data with clinical outcome (i.e. time to first treatment) as assessed by Cox regression models were then confirmed in patient samples in other two sequential independent studies, i.e. test study 1 (n=30), and test study 2 (n=37). Results. In the exploratory study, higher responsiveness of the B-cell receptor signaling proteins to anti-IgM was associated with poor clinical outcomes. Patient clustering based on signaling response was at least as powerful in discriminating different disease courses as traditional prognostic markers. In an unselected subgroup of Binet Stage A patients (n=21), increased anti-IgM-modulated p-ERK signaling was shown to be a significant, independent predictor of shorter time to first treatment. This result was independently confirmed in 2 test cohorts from distinct patient populations. Conclusions. These findings support the utility of the single cell network profiling assay in elucidating signaling perturbations with the potential for the development of a clinically useful prognostic test in patients with early stage B-cell chronic lymphocytic leukemia. These data support the clinical relevance of B-cell receptor signaling in B-cell chronic lymphocytic leukemia, and suggest a key role of ERK activation in the physiopathology of this leukemia.
    Haematologica 04/2013; 98(4):626-34. · 5.94 Impact Factor
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    ABSTRACT: Abstract 315 Adoptive transfer of primary patient CLL cells into NOD/SCID/{gamma}cnull(NSG) mice results in engraftment and proliferation of CLL cells if autologous T cells are present. Formation of splenic follicles consisting of B cells interspersed and surrounded by T cells indicates engraftment. However, ultimately these CD20+ cells are lost several weeks later. We describe one of the mechanisms for this apparent loss: differentiation to plasma cells. Peripheral blood cells from 9 IgM+ CLL patients (6 U-CLL and 3 M-CLL) were adoptively transferred into NSG mice with enriched autologous CD3+ cells pre-activated with anti-CD3/28 beads. B and T cell engraftment and subset distributions were analyzed for 47 mice by immunohistochemistry (IHC) and flow cytometry (FC) at the time of sacrifice. The earliest and latest times of assessment were 12 and 124 days, respectively, after CLL cell injection. In some cases, CLL cells were labeled with CFSE to track cell division. At sacrifice, 3 engraftment patterns were observed. Pattern 1 (observed up to day 56) showed small follicles of CD20+ cells with low-moderate numbers of surrounding T cells. Intensely positive CD38 cells were inconspicuous. FC showed CD19+CD5+ cells with no increase in CD38 and variable CFSE dilution indicating lower levels of proliferation. Pattern 2 (observed throughout the study period) showed much higher T and B cell numbers. CD20+ cells were interspersed with and surrounded by principally CD4+ cells which were activated and functional as indicated by expression of Ki-67, PD-1, CD57, and T cell derived cytokines IFN{gamma} and IL5 in plasma. Follicles contained CD20 and cytoplasmic Ig+ (cIg+) cells that double stained for IRF-4 and Blimp-1, transcription factors required for B cell differentiation. While Bcl-6 staining in these cells was minimal or absent, follicles from all 9 patients contained activation-induced deaminase (AID)+ cells. Cells with dim IgM expression localized to follicles; however, cells with intense IgM, IgA, or IgG were present both within, surrounding, and outside follicles matched by similar CD38 staining. Smaller populations of CD138+ cells surrounded follicles and were interspersed throughout non-follicular splenic areas. FC showed a novel CD19+CD5-CFSE-CD38++ population containing a CD138+ subset. Pattern 3 (observed in a limited subset of cases not before day 63) had minimal CD20+ cells by IHC, but noticeable populations of cIg+CD38+ and CD138+ cells interspersed amongst plentiful T cells. Such cells corresponded with cells with plasma cell morphology. Confirmation that differentiated cells were from the patient clone was achieved in 3 ways. First, in FACS sorted CD19+CD5+ and CD19+CD5-38++ cells from a subset of pattern 2 cases, RT-PCR revealed that all fractions contained both IGHC unswitched and switched clones identical to those found in the patients. Second, cases with pattern 3 engraftment generated CLL clonal switched and unswitched cDNA sequences. Finally, adoptive transfer of highly purified CD5+CD19+ patient cells generated IRF-4+Blimp-1+CD138+ cells. The generation of switched cells from all 9 patients indicated functional AID. In one U- CLL case, ultra-deep sequencing on pre-transfer and post-transfer human cells taken from mouse spleen revealed a significant number of new IGHVDJ mutations in spleen-derived cells. Such mutations targeted nucleotides typical for AID's action. In conclusion, CLL cells can diversify, switch, and differentiate in NSG mice in response to autologous T cell signals. The extent of this maturation is a function of T cell numbers and activity and the duration of the experiment. Differentiation without significant Bcl-6 expression suggests that follicles in NSG mice are not recapitulating classic germinal center reactions, possibly giving clues to the origin of CLL. Several features of poor prognosis disease were demonstrated (e.g., increased CD38 and AID expression with the development of clonally related switched transcripts) that might mirror clinical disease features. AID expressed by CLL ells is fully functional as indicated by de novo somatic hypermutation and class switch recombination. Both U-CLL and M-CLL clones respond in a similar manner in this model, suggesting the importance of T- B cell interactions in all types of CLL. Finally, the demonstration that cells can differentiate when appropriately induced may lead to novel therapeutic options for CLL. DisclosuresNo relevant conflicts of interest to declare.
    ASH Annual Meeting Abstracts; 12/2012
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    ABSTRACT: Abstract 2545 B-cell chronic lymphocytic leukemia (CLL) clones often acquire new mutations and cytogenetic aberrations over time. In other human cancers, including lymphomas and solid tumors, activation-induced deaminase (AID), which normally causes immunoglobulin (Ig) somatic hypermutation (SHM) and isotype class switch recombination (CSR) in germinal center B cells, is expressed and functions abnormally to cause mutations promoting aggressiveness. In CLL, AID mRNA expression in the leukemic cells correlates with increased adverse cytogenetic aberrations and worse clinical outcomes. Furthermore, CLL cells activated by culture with CD32-transfected murine L cells, anti-CD40 and interleukin-4, produce AID protein with associated functions: DNA breaks, Ig CSR, and Ig heavy chain (IGH) variable (IGHV) gene SHM. To evaluate AID-mediated SHM in CLL more accurately, ultra-deep sequencing was performed on CLL clone's IGH cDNA prior to and after in vitro activation in one unmutated CLL (U-CLL) case (CLL1278, 0.0% mutated IGHV3-30) and one mutated CLL (M-CLL) case (CLL1299, 4.9% mutated IGHV3-23). Additionally, to examine activation of CLL IGH mutation in vivo, ultra-deep sequencing was performed on cells from one U-CLL case (CLL1083, 0.0% mutated IGHV4-b) prior to and after adoptive transfer into the NOD/SCID/{gamma}cnull (NSG) mouse, a xenograft model of CLL, where upregulation of AID protein occurs in CD5+CD19+ human CLL cells. Libraries were created for ultra-deep sequencing using the 454 FLX system (Roche) by PCR amplification with IGHV family-specific framework1 (Lprimer) and IGH constant region C (IGHM) (Rprimer) primers on cDNA obtained from CLL cells prior to (day 0) or after in vitro culture for 7 (CLL1278) or 14 days (CLL1278; CLL1299) or from NSG spleen CLL cells collected 35 days after transfer (CLL1083). The resulting 461,153 sequence reads were processed to generate separate datasets with fixed sequence block lengths for each primer. The Lprimer sequence blocks included only 5'IGHV sequence, while the Rprimer sequence blocks encompassed 3'IGHV, IGH diversity, and IGH joining genes (IGHVDJ) as well as 5'IGHM sequence. Individual subclone sequences that occurred at least twice were extracted from each of the datasets and the unique de novo subclones not shared between day 0 and activation were analyzed for mutations. All three CLL cases showed increases in 5'IGHV and 3'IGHVDJ subclones with activation. After in vitro activation, for CLL1278, 123,518 total sequence reads produced 68 unique subclones as compared to 33 at day 0; and for CLL1299, 163,358 total sequence reads produced 78 unique subclones as compared to 61 at day 0. Likewise, after in vivo activation in the NSG mouse, for CLL1083, 174,472 total sequence reads produced 91 unique subclones as compared to 56 at day 0. In contrast, all three CLL cases showed decreases in 5'IGHM subclones after activation. After in vitro activation, CLL1278 and CLL1299 decreased from 22 and 20 unique day 0 subclones to 13 and 16 unique subclones. Similarly, CLL1083 showed a decrease from 20 unique day 0 subclones to 11 unique subclones after transfer into the NSG mouse. After normalization for read number and block sequence length, all three CLL cases showed an increase in 5'IGHV mutation with CLL cell activation (fold change relative to 5'IGHM = 3.4, 2.2, and 4.6 for CLL1278, CLL1299, and CLL1083, respectively). This increase in IGHV mutation relative to IGHM following activation is consistent with AID activity. Furthermore, examination of mutation sites in these subclones revealed an increase in mutations in AID hotspot motifs (GYW or WRC) in the 5'IGHV of all three CLL cases with activation (fold change = 2.0, 1.9, and 2.5 for CLL1278, CLL1299, and CLL1083, respectively), which was not observed further downstream in 3'IGHVDJ and 5'IGHM. Thus, by analyzing a very large number of IGH sequences in CLL cells after activation in vitro or in vivo, a pattern of de novo mutations consistent with AID activity is found. Furthermore, since both U-CLL and M-CLL clones exhibited AID a tivity, these findings indicate that AID-mediated SHM is not limited by CLL IGHV mutation status. Finally, these data support a model of AID-promoted mistargeted mutations, which may lead to adverse cytogenetic aberrations and unfavorable outcomes in CLL. DisclosuresNo relevant conflicts of interest to declare.
    ASH Annual Meeting Abstracts; 12/2012

Publication Stats

10k Citations
1,507.70 Total Impact Points

Institutions

  • 2006–2014
    • The Feinstein Institute for Medical Research
      • Laboratory of Experimental Rheumatology
      New York City, New York, United States
    • Azienda Ospedaliera, Cosenza
      Cosenza, Calabria, Italy
  • 2001–2014
    • North Shore-Long Island Jewish Health System
      New York, New York, United States
  • 2002–2013
    • Hofstra North Shore-LIJ School of Medicine
      New York City, New York, United States
  • 2011
    • Duke University Medical Center
      • Duke Human Vaccine Institute
      Durham, NC, United States
    • Università degli Studi di Torino
      • Dipartimento di Scienze Cliniche e Biologiche
      Torino, Piedmont, Italy
  • 2010–2011
    • Albert Einstein College of Medicine
      New York City, New York, United States
  • 1996–2011
    • Università degli Studi di Genova
      • Dipartimento di Medicina sperimentale (DIMES)
      Genova, Liguria, Italy
  • 2003–2006
    • North Shore-LIJ Health System
      Manhasset, New York, United States
  • 2004
    • University of California, San Diego
      • Department of Medicine
      San Diego, California, United States
  • 2000
    • Amedeo Avogadro University of Eastern Piedmont
      Novara, Piedmont, Italy
  • 1998
    • CRO Centro di Riferimento Oncologico di Aviano
      Aviano, Friuli Venezia Giulia, Italy
  • 1978–1998
    • The Rockefeller University
      • • Laboratory of Molecular Genetics
      • • Laboratory of Investigative Dermatology
      New York City, NY, United States
  • 1990–1996
    • Cornell University
      • Department of Medicine
      Ithaca, NY, United States
  • 1995
    • New York University
      • Department of Medicine
      New York City, NY, United States
  • 1994
    • Lawrence Livermore National Laboratory
      Livermore, California, United States
  • 1988
    • Philadelphia University
      Philadelphia, Pennsylvania, United States
    • Hospital for Special Surgery
      New York City, New York, United States