Immunoglobulin Heavy Chain Variable Gene Research Articles

CLL Cell Viability Promoted by Myosin Heavy Chain IIA Exposed Apoptotic Cells is BTK-dependent

AbstractAbstract 1767B-cell chronic lymphocytic leukemia (CLL) is a clonal CD5+CD19+ B-lymphocyte cancer with a B cell receptor (BCR) immunoglobulin heavy chain variable (IGHV) gene sequence that may be classified as unmutated (U-CLL) or mutated (M-CLL) depending on the level of IGHV mutation. Because U-CLL patients have a worse clinical prognosis than M-CLL patients, the structure of the BCR and the signaling consequences of BCR occupancy by antigen are considered crucial to disease development and possibly progression. Moreover, nearly one third of CLL patients express a CLL BCR that is virtually identical in sequence (stereotyped) to other CLL patient subgroups, an incidence that is extraordinarily unlikely by random chance, suggesting that common antigens could bind to these CLL BCRs and initiate signaling. Apoptotic autoantigens may be a source of such antigens. Indeed, CLL BCRs expressed as native or recombinant monoclonal antibodies (mAbs) can recognize autoantigens, such as nonmuscle myosin heavy chain IIA (myosin), actin, vimentin, cofilin-1, filamin B, insulin, DNA, IgG, myoglobin, thyroglobulin, cardiolipin, and oxidized low-density lipoprotein. These molecules are generally intracellular and may be exposed on the cell surface during apoptosis, permitting binding to CLL BCRs. Indeed, intracellular myosin is exposed on the surface of a subset of apoptotic cells, termed MEACs (myosin exposed apoptotic cells). Furthermore, MEACs bind to over 60% of CLL mAbs tested, supporting the idea that multiple autoantigens exposed on MEACs may stimulate CLL cells. This binding may furnish signals beneficial to the leukemic cell, because MEAC binding correlates with shorter patient survival.To test this hypothesis, the effect of MEAC binding on spontaneous CLL cell apoptosis was tested after co-culture of CLL peripheral blood mononuclear cells from a large patient cohort with MEACs (3:1–5:1 ratio) for 2–4 days using flow cytometry and AnnexinV and 7-actinomycin D staining to measure apoptosis in CD19+CD3− cells. A typical wide range of spontaneous apoptosis was observed (median = 26.98% (5.93%–91.35%) AnnexinV+). In this large patient cohort (n=64, with some patient samples tested 2–8 times), co-culture of CLL cells with MEACs resulted in a decrease in apoptosis in all patient samples as compared to cultures without MEACs (median = 18.58% (2.49%–76.35%) AnnexinV+), a highly significant result (P=0.0001, two-tailed paired t-test). In this cohort, U-CLL patients (n=23) had a slightly higher median decrease in apoptosis (30.39%) than M-CLL patients (n=33, 22.69%), but this was not significant (P=0.3940, two-tailed unpaired t-test with Welch’s correction). Furthermore, no apparent correlation between MEAC co-culture responsiveness and in vitro MEAC binding was observed in the 9 CLL patient samples exhibiting stereotyped CLL BCR. Finding that MEAC co-culture affects all CLL cells regardless of IGHV mutation status or stereotypy contrasts with MEAC binding to CLL mAbs in vitro, for which there is a marked preference for U-CLL and stereotyped mAbs. One possible explanation for the conflicting result is that CLL cells with BCRs that do not bind MEACs well receive an additional non-Ig receptor signal that boosts the BCR signal to make it near equivalent to the signal from BCRs on cells that bind MEACs well. In this model, inhibition of BCR signaling would abrogate the decrease in apoptosis produced by MEAC co-culture in all CLL cases. To test this, Bruton tyrosine kinase (BTK) inhibitors, ibrutinib or LFM-A13, were added to these co-cultures (n=12 and 18, respectively). Both inhibitors significantly abrogated the MEAC co-culture increase in viability (ibrutinib, P=0.0005; LFM-A13, P=0.0007; two-tailed paired t-test). Ibrutinib and LFM-A13 inhibited all samples regardless of IGHV mutation status (stereotyped BCR samples were not tested).In conclusion, these results are consistent with the theory that antigenic stimulation of CLL cells via the BCR promotes CLL cell viability and may influence the level of disease activity. MEACs may be a source of such antigenic stimulation. Interference with BCR signaling via BTK inhibition prevents this stimulation and may be one of the explanations for the clinical success of BCR signaling inhibitors in treating CLL. Disclosures:Barrientos:Gilead and Pharmacyclics: Research Funding.

Comparison of Minimal Residual Disease Detection Using High-Throughput Sequencing and Allele-Specific Oligonucleotide PCR Methods in Pediatric B-Lineage Acute Lymphoblastic Leukemia.

AbstractAbstract 2532▪▪This icon denotes a clinically relevant abstract Background:The clinical management of patients with pediatric B-lineage acute lymphoblastic leukemia (B-ALL) relies on combinations of multiagent anticancer drugs and risk-stratified treatment. The prognostic significance of minimal residual detection (MRD) in pediatric B-ALL has been demonstrated in multiple cohorts. Allele-specific oligonucleotide PCR (ASO-PCR) amplification of immunoglobulin or T-cell receptor rearrangements, a method for MRD detection, requires the development of patient-specific reagents and cannot detect clonal evolution. ASO-PCR also has limited coverage, with clonal rearrangements being detected in only 90% of patients. We developed the sequencing-based LymphoSIGHT platform to address these limitations. Here we report the results of a pilot study of MRD detection using both the sequencing assay and ASO-PCR in paired diagnostic and end-of-induction samples from 7 B-ALL patients. Analysis of 82 additional patients is ongoing. Methods:Using universal primer sets, we amplified immunoglobulin heavy chain (IgH@) variable (V), diversity, and joining gene segments from genomic DNA in diagnostic and follow-up bone marrow samples. Amplified products were sequenced to obtain >1 million reads (20× coverage per B-cell), and were analyzed using standardized algorithms for clonotype determination. Tumor-specific clonotypes were identified in the diagnostic sample of each patient based on high-frequency within the B-cell repertoire. The presence of the tumor-specific clonotype was then assessed in the end-of-induction sample. A quantitative and standardized measure of MRD level among all leukocytes in the sample was determined using internal reference DNA. Following identification of IgH clonal rearrangements and MRD assessment using the sequencing assay, we examined the MRD results obtained at Boston Children’s Hospital using ASO-PCR. Among the 7 patients analyzed to date, 6 patients were in complete remission at the time of the second sample; 1 patient had persistent evidence of disease. Sequencing was performed blinded to all clinical and ASO-PCR information on these patients. Results:With the sequencing platform, we detected a high-frequency IgH clonal rearrangement in all 7 diagnostic ALL samples. The leukemic clonotype that was identified at diagnosis was detected in the end-of-induction sample in each of the 7 patients. The quantitative range of the leukemic sequence in MRD samples ranged over 5 orders of magnitude. MRD results were concordant between sequencing and ASO-PCR in 5 of 7 patients. The detected MRD level differed by > 10 fold in 2 patients. In patient 1, sequencing detected high MRD, while low MRD was detected by ASO-PCR; this patient relapsed 1 year later while still on therapy. In patient 6, sequencing detected low MRD, while ASO-PCR detected high MRD. This patient remains in complete remission after 7 years. In patient 7, sequencing and ASO-PCR concordantly detected low MRD; this patient relapsed after completion of therapy. Conclusions:Results from the application of a high-throughput sequencing method for MRD detection in childhood B-ALL are shown. The sequencing assay does not require development of patient-specific reagents, which will reduce cost and laboratory turnaround time. This data, along with the laboratory workflow improvements, support the use of the sequencing assay as a next-generation MRD test for B-ALL. Analysis of samples from 82 patients is ongoing.Table 1Comparison of MRD results using sequencing and ASO-PCR methodsPatientNumber of input cellsNumber of input leukemic clone moleculesSequencing MRD (%)ASO-PCR MRD (%) (High MRD ≥ 0.1%)Clinical outcome1163,33317,1705.30.008Relapse22,043,8609,958,326∼10018.2Induction failure32,052,6512,4640.066.5Alive and well41,298,291670.0010.001Alive and well5984,91810,0360.50.2Alive and well6863,757474,93827.528.1Relapse731,536730.10.03Relapse Disclosures:Faham:Sequenta, Inc.: Employment, Equity Ownership, Research Funding. Fang:Sequenta, Inc.: Employment, Equity Ownership, Research Funding. Moorhead:Sequenta, Inc.: Employment, Equity Ownership, Research Funding. Zheng:Sequenta, Inc.: Employment, Equity Ownership, Research Funding.

Evaluation of BRAFV600E, NRAS and KRAS Mutations As Well As IGHV Usage for Diagnostic Use in Hairy Cell Leukemia, Hairy Cell Leukemia-Variant and Splenic Marginal Zone Lymphoma.

Abstract 2679 Background:The BRAF V600E mutation has recently been discovered in nearly all cases of hairy cell leukemia (HCL), but not in cases of HCL-variant (HCL-v). However, this perfect correlation has been challenged by studies reporting HCL cases without BRAF V600E. Interestingly, the immunoglobulin heavy chain variable region gene IGHV4–34, which has been associated with poor prognosis in HCL, appeared exclusively and to a high percentage in these BRAF V600E-negative cases of classic HCL and also in HCL-v (Xi et al., Blood, 2011). Further, splenic marginal zone lymphoma (SMZL) is a disease closely related to HCL and HCL-v and BRAF has been shown to be unmutated in this entity. Aims:1. To characterize our cohorts of HCL, HCL-v and SMZL for the presence of BRAF V600E and to correlate the results with IGHV gene usage. 2. We hypothesized that other genes of the RAF/RAS pathway might be affected. Thus we analysed NRAS, and KRAS in addition to BRAF for mutations in all three entities. Methods:We analyzed the bone marrow or peripheral blood of 314 cases (182 cases with HCL, 49 cases with HCL-v, and 83 cases with SMZL) at diagnosis as confirmed by multiparameter flow cytometry and cytomorphology. The BRAF V600E mutation was analyzed by an mRNA-based reverse transcription allele-specific real-time quantification (RQ-PCR) assay. The BRAF V600E expression was calculated as %BRAF V600E/BRAF wt. NRAS and KRAS were analyzed by melting curve analysis and subsequent Sanger sequencing. IGHV genes and mutation status were analyzed by the use of Biomed-2 primers. An identity of ≥98% of the analyzed IGHV sequence compared to published germline sequences was considered an unmutated IGHV status. Results:In our cohort the median percent leukemic cells was 16% (range 0.2–74%) for HCL, 33% (range 5–59%) for HCL-v and 29% (range: 1–84%) for SMZL as determined by multiparameter flow cytometry. The BRAF V600E mutation was detected in 178/182 (97.8%) of HCL cases, whereas 0/49 of HCL-v and 0/83 SMZL were positive. Thus, the BRAF V600E mutation is 100% specific for HCL regarding these three entities. The median BRAF V600E expression ratio of positive cases was 14.2 (range 0.22 – 280.3). After normalization to % pathological cells as assessed by multiparameter flow cytometry the median ratio was 173 (range:22–1,788). However, in 4 cases with 4%, 8%, 28% and 66% percent leukemic cells by multiparameter flow cytometry, which is within the clone size that can be clearly detected by the BRAF V600E-specific RQ-PCR assay, no mutation was detected. Thus, BRAF V600E detection used for the identification of HCL has a sensitivity of 97.8%. Further, NRAS and KRAS mutation screening in all cases with HCL, HCL-v, and SMZL did not detect any mutation except for one case with SMZL that harboured an NRAS Gly12Asp mutation. This case was found to have an MDS in parallel and thus the mutation more likely belongs to the MDS clone. Thus, analysis of NRAS and KRAS mutations does not further improve diagnostics in these diseases. Further, we analyzed the IGHV usage in all 4 BRAF unmutated HCL and in additional 60 cases (total n=64) with HCL and 41 cases with HCL-v. IGHV4–34 usage was very frequent in HCL-v with 14/41 (34.1%). In contrast, it was never detected in HCL including the BRAF wildtype cases. Thus, we were not able to confirm the usage of the IGHV4–34 gene, which was previously suggested for BRAF V600E negative HCL. On the other hand IGHV5–51 was most frequently found in HCL (9/64, 14.1%) but never detected in HCL-v. We detected an unmutated IGHV status in 12/62 (19.4%) of HCL, which was less frequent compared to 14/40 (35.0%) in HCL-v (p = 0.095). The IGHV mutation status was unmutated in 9/11 (81.8%) IGHV4–34 cases (100% identity to germline each). The four cases of HCL, which lacked BRAF V600E mutation, expressed the IGHV genes IGHV1–3*01 (96.5% identity), IGHV1–69*02 (94.0% identity), IGHV3–9*01 (96.9% identity) and IGHV6-1*01 (99.0% identity), which were also expressed by various BRAF V600E positive HCL cases in our cohort. Conclusions:1) In our cohort of 314 cases with HCL, HCL-v, and SMZL we confirm a high specificity (100%) and sensitivity (97.8%) for BRAF V600E mutations to detect HCL. 2) Other RAS pathway mutations (NRAS, KRAS) were not detected in any of the three analysed entities. 3) In the 4 rare cases of HCL with BRAF wt we were not able to confirm the previously postulated IGHV4–34 usage. 4) IGHV4–34 further delineates classic HCL from HCL-v. Disclosures:Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Dicker:MLL Munich Leukemia Laboratory: Employment. Eder:MLL Munich Leukemia Laboratory: Employment. Jeromin:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Equity Ownership.

Ultra-Deep Sequencing of De Novo IGHV Mutations in Activated CLL Cells: Evidence for Activation-Induced Deaminase Function.

AbstractAbstract 2545B-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/γcnull (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 Cμ (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 activity, 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. Disclosures:No relevant conflicts of interest to declare.

B‐cell receptor configuration and adverse cytogenetics are associated with autoimmune hemolytic anemia in chronic lymphocytic leukemia

The development of autoimmune hemolytic anemia (AIHA) in patients with chronic lymphocytic leukemia (CLL) is associated with specific biological features. The occurrence of AIHA was hereby investigated in a retrospective series of 585 CLL patients with available immunoglobulin heavy chain variable (IGHV) gene status. AIHA occurred in 73 patients and was significantly associated with an IGHV unmutated (UM) status (P < 0.0001) and unfavorable [del(17)(p13) and del(11)(q23)] cytogenetic lesions (P < 0.0001). Stereotyped HCDR3 sequences were identified in 29.6% of cases and were similarly represented among patients developing or not AIHA; notably, subset #3 was associated with a significantly higher risk of AIHA than the other patients (P = 0.004). Multivariate analysis showed that UM IGHV, del(17)(p13) and del(11)(q23), but not stereotyped subset #3, were the strongest independent variables associated with AIHA. Based on these findings, we generated a biological risk score for AIHA development according to the presence of none (low risk), one (intermediated risk), or two (high risk) of the independent risk factors. Overall, our data indicate that UM IGHV status and/or unfavorable cytogenetic lesions are associated with the risk of developing secondary AIHA in CLL patients and suggest a possible role of specific stereotyped B-cell receptor subsets in a proportion of cases.

Biallelic ATM Inactivation Significantly Reduces Survival in Patients Treated on the United Kingdom Leukemia Research Fund Chronic Lymphocytic Leukemia 4 Trial

The prognostic significance of ATM mutations in chronic lymphocytic leukemia (CLL) is unclear. We assessed their impact in the context of a prospective randomized trial. We analyzed the ATM gene in 224 patients treated on the Leukemia Research Fund Chronic Lymphocytic Leukemia 4 (LRF-CLL4) trial with chlorambucil or fludarabine with and without cyclophosphamide. ATM status was analyzed by denaturing high-performance liquid chromatography and was related to treatment response, survival, and the impact of TP53 alterations for the same patient cohort. We identified 36 ATM mutations in 33 tumors, 16 with and 17 without 11q deletion. Mutations were associated with advanced disease stage and involvement of multiple lymphoid sites. Patients with both ATM mutation and 11q deletion showed significantly reduced progression-free survival (median, 7.4 months) compared with those with ATM wild type (28.6 months), 11q deletion alone (17.1 months), or ATM mutation alone (30.8 months), but survival was similar to that in patients with monoallelic (6.7 months) or biallelic (3.4 months) TP53 alterations. This effect was independent of treatment, immunoglobulin heavy chain variable gene (IGHV) status, age, sex, or disease stage. Overall survival for patients with biallelic ATM alterations was also significantly reduced compared with those with ATM wild type or ATM mutation alone (median, 42.2 v 85.5 v 77.6 months, respectively). The combination of 11q deletion and ATM mutation in CLL is associated with significantly shorter progression-free and overall survival following first-line treatment with alkylating agents and purine analogs. Assessment of ATM mutation status in patients with 11q deletion may influence the choice of subsequent therapy.

Chronic lymphocytic leukemia with t(14;18)(q32;q21)

The t(14;18)(q32;q21) is a cytogenetic hallmark of follicular lymphoma and also occurs in approximately 20% of diffuse large B-cell lymphomas of follicle center cell origin. Relatively few cases of chronic lymphocytic leukemia/small lymphocytic lymphoma with t(14;18) have been reported previously. We report the clinicopathologic, cytogenetic, and molecular genetic features of 12 patients with chronic lymphocytic leukemia associated with t(14;18). There were 9 men and 3 women, with a median age of 51 years at diagnosis. To date, 11 patients have required chemotherapy, 6 before coming to our institution. At last follow-up, 5 patients have died of disease. Karyotypic analysis showed that 10 cases had t(14;18) in the stemline and 2 cases in the sideline; t(14;18) was the sole abnormality in the stemline in 2 cases. In 11 cases, other abnormalities were identified in the stemline or sidelines, most commonly trisomy 12 in 6 cases. Trisomy 12 was associated with atypical morphology and immunophenotype. Of 8 cases tested, 7 showed somatically mutated immunoglobulin heavy chain variable region genes. We conclude that the t(14;18) in chronic lymphocytic leukemia is associated with relatively young age at diagnosis, mutated immunoglobulin heavy chain variable region genes, and a clinical course that usually requires chemotherapy. The cytogenetic findings, in particular, t(14;18) in the stemline in 10 cases and as the sole karyotypic abnormality in 2 cases, suggest that t(14;18) is an early pathogenetic event in this small subset of chronic lymphocytic leukemia cases.

Altered BCR and CD40 signalling are associated with clinical outcome in small lymphocytic lymphoma/chronic lymphocytic leukaemia and marginal zone lymphoma patients

Altered <scp>BCR</scp> and <scp>CD</scp>40 signalling are associated with clinical outcome in small lymphocytic lymphoma/chronic lymphocytic leukaemia and marginal zone lymphoma patients

Molecular pathogenesis of chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults. Here, we highlight important genetic alterations that contribute to tumorigenesis, clinical progression, and chemorefractoriness of CLL. All CLLs share a common gene expression profile that suggests derivation from antigen-experienced B cells, a model supported by frequent B cell receptor repertoire skewing and stereotypy. Many CLL patients carry mutated immuno-globulin heavy-chain variable genes, while approximately 35% harbor unmutated IgV genes, which are associated with an inferior outcome. Deletion of chromosome 13q14, which is the most common genetic mutation at diagnosis, is considered an initiating lesion that frequently results in disruption of the tumor suppressor locus DLEU2/MIR15A/MIR16A. Next-generation sequencing has revealed additional recurrent genetic lesions that are implicated in CLL pathogenesis. These advancements in the molecular genetics of CLL have important implications for stratifying treatment based on molecular prognosticators and for targeted therapy.

IGHV gene features and MYD88 L265P mutation separate the three marginal zone lymphoma entities and Waldenström macroglobulinemia/lymphoplasmacytic lymphomas

To clarify the relationships between marginal zone lymphomas (MZLs) and Waldenström macroglobulinemia/lymphoplasmacytic lymphomas (WM/LPLs), immunoglobulin heavy chain variable gene (IGHV) features were analyzed and the occurrence of MYD88 L265P mutations was identified in a series of 123 patients: 53 MZLs from the spleen (SMZLs), 11 from lymph nodes (NMZLs), 28 mucosa-associated lymphatic tissue (MALT) lymphomas and 31 WM/LPLs. SMZLs were characterized by overrepresentation of IGHV1-2 gene rearrangements with a canonical motif, without selection pressure and with long CDR3 segments. NMZLs had increased frequencies of IGHV3 genes. The IGHV gene was unmutated in most cases, often with long CDR3 segments. MALT lymphomas were usually associated with a mutated IGHV gene, but with the absence of selection pressure. WM/LPLs were associated with an IGHV3-23 overrepresentation and high IGHV mutation rate, with features of selection pressure and short CDR3 segments. MYD88 L265P mutations were almost restricted exclusively to WM/LPL patients. Taken all diagnoses together, all patients with MYD88 L265P mutations had an immunoglobulin M peak and almost all patients except one had bone marrow infiltration. These results demonstrate that the history of antigen exposure of the four entities studied was different and MYD88 L265P was specifically associated with WM/LPLs. WM/LPL may thus be functionally associated with constitutive nuclear factor-κB activation.

The role of B cell receptor stimulation in CLL pathogenesis.

Chronic lymphocytic leukemia (CLL) is the most frequent leukemia in adults in the Western world and is characterized by the accumulation of monoclonal CD5(+) mature B cells in the blood. The disease has a highly variable clinical course. CLL is subdivided into two disease subtypes, whereby leukemias with hypermutated immunoglobulin heavy chain variable (IGHV) genes have a more favorable prognosis than those with unmutated IGHV genes, which tend to show advanced, progressive disease, adverse cytogenetic features and resistance to therapy. The current view is that both CLL types derive from antigen-experienced cells. Based on the finding that the IGHV repertoire is highly restricted and biased, as compared to the normal adult B-cell repertoire, it has been hypothesized that CLL cells are selected by some sort of antigenic pressure. Hereby, either autoantigens or antigens derived from apoptotic cells or pathogens are essential to trigger CLL pathogenesis. Although different cytogenetic aberrations were shown to contribute to CLL leukemogenesis, it remains unclear which abnormalities are primary events. Very recently, whole-genome sequencing identified genes that are recurrently mutated and provided novel insights into the mechanisms of oncogenic transformation. Because of the impact on prognosis, it is important to unravel the role of antigenic selection in CLL. Interestingly, B cell receptor (BCR) signaling is aberrantly increased in CLL and expression of tyrosine kinase ZAP70, which is able to signal downstream of the BCR, is a prognostic indicator. In this context we discuss the functional significance of antigenic selection in CLL and describe emerging agents to target BCR signaling that are currently being tested as a novel therapeutic strategy for CLL.

CD38 gene polymorphism and risk of chronic lymphocytic leukemia

rs6449182 CD38 gene polymorphism was determined by polymerase chain reaction with restriction of products in 328 chronic lymphocytic leukemia (CLL) patients and 271 age- and sex-matched controls. An association between GG genotype and CLL risk was found in the whole group of patients (OR=2.12; p=0.009) and in patients with unmutated immunoglobulin heavy chain variable genes (OR=2.17; p=0.011) comparing to the controls. In the subgroup of 174 controls with evaluated lipids the genotype distributions in CLL patients and dyslipidemic controls were similar. An association between GG genotype and CLL risk was significant compared to controls without lipids’ abnormalities (OR=3.92; p=0.006).

Reduced frequency of NKT-like cells in patients with progressive chronic lymphocytic leukemia

Natural killer T (NKT) cells are a subset of innate immune cells displaying a limited repertoire of antigen specificities and CD1d restriction. Little is known about contribution of NKT cells in cancer initiation and progression. In this study, the frequencies of NKT-like cells, B cells expressing CD1d molecule and CD4(+) regulatory (Treg) cells were analyzed in 40 patients with chronic lymphocytic leukemia (CLL) and 15 healthy subjects by flow cytometry. Our results showed that the frequency of CD3(+)CD56(+) NKT-like cells is significantly decreased in progressive (4.9 ± 0.8 % of total CD3(+) T cells) compared with indolent (8.1 ± 1.2 %, p = 0.036) patients and healthy subjects (10.6 ± 1.7 %, p = 0.003). However, no association was found between NKT-like cell frequency and immunoglobulin heavy chain variable region gene (IGHV) mutation or CD38 and ZAP70 expression. On the other hand, expression of CD1d molecule was significantly higher in leukemic B cells of patients with CLL (75 ± 1.5 % of total CD19(+) B cells) compared to B cells from healthy subjects (59.6 ± 2.2 %, p < 0.001), with no significant difference between progressive and indolent patients. Interestingly, the frequency of Treg cells was inversely correlated with that of NKT-like cells in patients with CLL (r = -0.4, p = 0.002). Our results suggest a protective role for NKT-like cells in patients with CLL, which seems to be downregulated presumably by Treg cells.

Variations of the ataxia telangiectasia mutated gene in patients with chronic lymphocytic leukemia lack substantial impact on progression-free survival and overall survival: a Cancer and Leukemia Group B study

The impact of mutation of the ATM (ataxia telangiectasia mutated) gene in chronic lymphocytic leukemia (CLL) treatment outcome has not been examined. We studied ATM mutations in 73 patients treated with fludarabine and rituximab. ATM gene mutation analysis was performed using temperature gradient capillary electrophoresis. The impact of detected variants on overall survival (OS) and progression-free survival (PFS) was tested with proportional hazards models. None of the 73 patients demonstrated truncating ATM mutations; 17 (23%, 95% confidence interval 14–35%) had non-silent variants (ATM-NSVs), including 13 known ATM polymorphisms and four missense variants. ATM-NSVs were not significantly associated with any baseline characteristics including immunoglobulin heavy chain variable gene (IGVH) status. In multivariable models, no significant differences in complete response (p =0.70), PFS (p =0.59) or OS (p =0.13) were observed. Our data indicate that truncating ATM mutations are rare in patients with CLL. Furthermore, in this dataset, these non-silent variants had limited impact on PFS and OS.

Signaling pathways activated by the B-cell receptor in chronic lymphocytic leukemia

Over the past decade, several features of the B-cell receptor (BCR) complex have emerged as major markers for prognostic classification of B-cell chronic lymphocytic leukemia (B-CLL). In particular, the absence of somatic mutations within the immunoglobulin variable heavy chain genes (IGHV), the presence of ZAP-70 and a higher ability of the BCR to translate signals within the cell have been associated with an aggressive clinical course. Indeed, the stratification of patients with B-CLL based on BCR features suggests that heterogeneity of B-CLL clinical courses may reflect BCR signaling differences that have arisen during the evolution of leukemia. Therefore, characterizing BCR signaling profiles may help to identify signaling markers useful for patient stratification, disease monitoring and therapeutic targeting in B-CLL.

Chronic Hepatitis C Virus Infection Breaks Tolerance and Drives Polyclonal Expansion of Autoreactive B Cells

Chronic Hepatitis C virus (HCV) infection has been linked with B cell lymphoproliferative disorders and several autoimmune-related diseases. The mechanisms of how chronic viral infection affects B cell development and predisposes the patients to autoimmune manifestations are poorly understood. In this study, we established an experimental system to probe the B cell responses and characterize the antibodies from chronic-HCV-infected individuals. We identified an unusual polyclonal expansion of the IgM memory B cell subset in some patients. This B cell subset is known to be tightly regulated, and autoreactive cells are eliminated by tolerance mechanisms. Genetic analysis of the immunoglobulin (Ig) heavy chain variable gene (V(H)) sequences of the expanded cell population showed that the levels of somatic hypermutation (SHM) correlate with the extent of cell expansion in the patients and that the V(H) genes exhibit signs of antigen-mediated selection. Functional analysis of the cloned B cell receptors demonstrated autoreactivity in some of the expanded IgM memory B cells in the patients which is not found in healthy donors. In summary, this study demonstrated that, in some patients, chronic HCV infection disrupts the tolerance mechanism that normally deletes autoreactive B cells, therefore increasing the risk of developing autoimmune antibodies. Long-term follow-up of this expanded B cell subset within the infected individuals will help determine whether these cells are predictors of more-serious clinical manifestations.

The immunoglobulin heavy chain locus: genetic variation, missing data, and implications for human disease

The immunoglobulin (IG) loci consist of repeated and highly homologous sets of genes of different types, variable (V), diversity (D) and junction (J), that rearrange in developing B cells to produce an individual's highly variable repertoire of expressed antibodies, designed to bind to a vast array of pathogens. This repeated structure makes these loci susceptible to a high frequency of insertion and deletion events through evolutionary time, and also makes them difficult to characterize at the genomic level or assay with high-throughput techniques. Given the central role of antibodies in the adaptive immune system, it is not surprising that early candidate gene approaches showed that germline polymorphisms in these regions correlated with susceptibility to both infectious and autoimmune diseases. However, more recent studies, particularly those using high-throughput genome-wide arrays, have failed to implicate these loci in disease. In this review of the IG heavy chain variable gene cluster (IGHV), we examine how poorly we understand the distribution of haplotype variation in this genomic region, and we argue that this lack of information may mask candidate loci in the IGHV gene cluster as causative factors for infectious and autoimmune diseases.

Comprehensive Assessment of Potential Multiple Myeloma Immunoglobulin Heavy Chain V-D-J Intraclonal Variation Using Massively Parallel Pyrosequencing

Multiple myeloma (MM) is characterized by the accumulation of malignant plasma cells (PCs) in the bone marrow (BM). MM is viewed as a clonal disorder due to lack of verified intraclonal sequence diversity in the immunoglobulin heavy chain variable region gene (IGHV). However, this conclusion is based on analysis of a very limited number of IGHV subclones and the methodology employed did not permit simultaneous analysis of the IGHV repertoire of non-malignant PCs in the same samples. Here we generated genomic DNA and cDNA libraries from purified MM BMPCs and performed massively parallel pyrosequencing to determine the frequency of cells expressing identical IGHV sequences. This method provided an unprecedented opportunity to interrogate the presence of clonally related MM cells and evaluate the IGHV repertoire of non-MM PCs. Within the MM sample, 37 IGHV genes were expressed, with 98.9% of all immunoglobulin sequences using the same IGHV gene as the MM clone and 83.0% exhibiting exact nucleotide sequence identity in the IGHV and heavy chain complementarity determining region 3 (HCDR3). Of interest, we observed in both genomic DNA and cDNA libraries 48 sets of identical sequences with single point mutations in the MM clonal IGHV or HCDR3 regions. These nucleotide changes were suggestive of putative subclones and therefore were subjected to detailed analysis to interpret: 1) their legitimacy as true subclones; and 2) their significance in the context of MM. Finally, we report for the first time the IGHV repertoire of normal human BMPCs and our data demonstrate the extent of IGHV repertoire diversity as well as the frequency of clonally-related normal BMPCs. This study demonstrates the power and potential weaknesses of in-depth sequencing as a tool to thoroughly investigate the phylogeny of malignant PCs in MM and the IGHV repertoire of normal BMPCs.

Association of interleukin-6 and interleukin-8 with poor prognosis in elderly patients with chronic lymphocytic leukemia

In population studies, the relative survival in chronic lymphocytic leukemia (CLL) decreases with age. In this study, we demonstrated in a cohort of 189 patients from a CLL clinic that overall survival was lower in the sub-cohort of patients aged ≥ 70 years, but causes of death were similar for all age groups, being progressive CLL, secondary malignancies and infections. As normal individuals age, the plasma levels of inflammatory cytokines, such as interleukin-6 (IL-6) and IL-8, can increase. In our patients with CLL, IL-6, IL-8 and tumor necrosis factor-α (TNF-α) levels increased with age to a greater degree than in normal individuals, and the levels correlated closely with plasma β2-microglobulin and with one another. In addition, in patients ≥ 70 years, IL-6 was found to be a better prognostic marker than immunoglobulin variable heavy chain gene (IgVH) status. In vitro studies demonstrated that IL-6 and IL-8 could enhance the binding of CLL cells to stromal cells, suggesting that their clinical activity may be mediated through their effects on the microenvironment. Thus, plasma IL-6 is an important prognostic marker for the elderly with CLL, and this study highlights that the utility of prognostic markers may depend on patient age.

Epigenetic Silencing of the Circadian Clock Gene CRY1 is Associated with an Indolent Clinical Course in Chronic Lymphocytic Leukemia

Disruption of circadian rhythm is believed to play a critical role in cancer development. Cryptochrome 1 (CRY1) is a core component of the mammalian circadian clock and we have previously shown its deregulated expression in a subgroup of patients with chronic lymphocytic leukemia (CLL). Using real-time RT-PCR in a cohort of 76 CLL patients and 35 normal blood donors we now demonstrate that differential CRY1 mRNA expression in high-risk (HR) CD38+/immunoglobulin variable heavy chain gene (IgVH) unmutated patients as compared to low-risk (LR) CD38−/IgVH mutated patients can be attributed to down-modulation of CRY1 in LR CLL cases. Analysis of the DNA methylation profile of the CRY1 promoter in a subgroup of 57 patients revealed that CRY1 expression in LR CLL cells is silenced by aberrant promoter CpG island hypermethylation. The methylation pattern of the CRY1 promoter proved to have high prognostic impact in CLL where aberrant promoter methylation predicted a favourable outcome. CRY1 mRNA transcript levels did not change over time in the majority of patients where sequential samples were available for analysis. We also compared the CRY1 expression in CLL with other lymphoid malignancies and observed epigenetic silencing of CRY1 in a patient with B cell acute lymphoblastic leukemia (B-ALL).