B-cell Repertoire Research Articles

Detection Of Classical Hodgkin Lymphoma In Peripheral Blood Using High-Throughput Sequencing Assay

BackgroundIt was only a little more than a decade ago that the origin of the pathognomonic Reed Sternberg cells of Classical Hodgkin lymphoma (CHL) was established as B-cell malignancy characterized by a clonal expansion (Marafioti et al. Blood 2000). A previous report suggested that clonotypic B cells may be present in the blood of newly diagnosed CHL patients by virtue of identifying some light chain restricted populations by flow cytometry (Jones et al. Blood 2009). However, definitive investigation to assess whether CHL clones are detectable in the blood has been lacking. We developed the LymphoSIGHT™ platform, a high-throughput sequencing-based method, to detect evidence of lymphoid malignancies in peripheral blood samples, as this could potentially be used for detection of minimal residual disease (MRD) after treatment (Faham et al. Blood 2012). This sequencing platform has a sensitivity to detect one lymphoma cell per million leukocytes in peripheral blood. We conducted a pilot study to assess the ability of this platform to detect the lymphoma clone in peripheral blood samples from 13 CHL patients at the time of diagnosis or disease recurrence. MethodsThis study was IRB-approved and consent was obtained from all patients. Using universal primer sets, we amplified immunoglobulin heavy chain (IGH) variable, diversity, and joining gene segments from genomic DNA in tumor biopsy and peripheral blood samples (plasma and peripheral blood mononuclear cell [PBMC] compartments) collected at initial diagnosis or at the time of recurrence. Amplified products were sequenced and analyzed using standardized algorithms for clonotype determination. Tumor-specific clonotypes were identified for each patient based on their high-frequency within the B-cell repertoire in the lymph node biopsy sample. The presence of the tumor-specific clonotype was then quantitated in plasma and PBMC compartments from a blood sample obtained around the time of primary tissue biopsy. A quantitative and standardized measure of clone level among all leukocytes in the sample was determined using internal reference DNA. ResultsClinical and histopathological characteristics are summarized in Table 1. We detected a high-frequency IGH clonal rearrangement in 8 of 13 (62%) lymph node biopsy samples. We observed a trend for a low rate of lymphoma clonotype identification in untreated patients with early stage disease (50%, 4 of 8 patients), and a higher rate of identification in patients with relapsed disease (80%, 4 of 5). In the 8 patients with an identified lymphoma specific clonotype in the biopsy sample, the clonotype was also detected in the plasma and/or PBMC compartment in 7 (88%) patients (Table 1). We detected a lymphoma clonotype more frequently in plasma (88%, 7 of 8) than in PBMC (33%, 2 of 6). ConclusionsOur data is the first to show that circulating clonal tumor cells can be detected in the blood of patients with CHL, providing new opportunities to explore novel methods to detect minimal residual disease. Additional cases are currently being analyzed to determine the sensitivity and specificity in early stage versus advanced stage disease, and to determine whether the detection of MRD post therapy would correlate with clinical relapse. Disclosures:Carlton:Sequenta, Inc: Employment, Equity Ownership. Kong:sequenta, Inc.: Employment, Equity Ownership. Faham:Sequenta, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

DNA Sequencing-Based Monitoring Of Serum Predicts Clinical Relapse Before CT Imaging in Diffuse Large B-Cell Lymphoma

BackgroundDiffuse large B-cell lymphoma (DLBCL) is the most common lymphoma with >40,000 cases annually in the US. While most patients are cured by frontline immunochemotherapy, up to 20% recur and salvage therapy is poor. Relapse detection relies on CT imaging, which is hampered by radiation exposure, high costs, high false positive rates, and infrequently detects relapse prior to symptoms. Novel methods to detect early relapse may improve outcomes. The LymphoSIGHT™ platform is a high-throughput DNA sequencing method that can detect minimal residual disease (MRD) of lymphoid malignancies in peripheral blood (PB) (Faham et al. Blood 2012) with a sensitivity of one lymphoma cell per million leukocytes. We showed it can also detect circulating tumor DNA in the PB of DLBCL pts (Armand Br J Haematol 2013). We assessed the sensitivity and specificity of LymphoSIGHT™ to detect circulating tumor DNA in serum after frontline therapy in DLBCL. MethodsAll pts were newly diagnosed DLBCL undergoing frontline EPOCH-based chemotherapy. Post-therapy surveillance included clinic visits, PB samples, and contrast CT scans at 3, 4, 6, 12 and 12 mos intervals for the first 5 years, respectively. Pre-treatment lymph node biopsies and PB samples collected at initial diagnosis and during surveillance visits were sent for VDJ amplification and MRD sequencing assessment. Using universal primer sets, we amplified immunoglobulin heavy and kappa chain (IGH and IGK) variable, diversity, and joining gene segments from genomic DNA in tumor biopsy and PB samples. Amplified products were sequenced, and tumor-specific clonotypes were identified for each pt based on their high frequency within the B-cell repertoire in the lymph node. The presence of the tumor-specific clonotype was then quantitated in PB samples. ResultsTwenty-seven pts, treated between July 1996 and July 2005, had adequate DNA from pre-treatment FFPE lymph node biopsies, and 25 (92.6%) had a high-frequency clonal rearrangement (MRD) detected. The median pt age was 49 years (20-75) and an IPI risk groups included 8 low, 3 low-intermediate, 11 high-intermediate and 3 high. The pts had a median of 12 surveillance CT scans (range 1-13) and 221 serum samples analyzed for MRD. Two pts progressed early after cycles 1 and 6. The first pt had progressive disease by CT scan after cycle 1 and remained MRD positive. The second pt remained MRD positive at the end of cycle 6, at which time a CT showed CR-unconfirmed, and progressed one month later. In the remaining 23 pts who achieved CR, seven relapsed at a median of 20.3 (range 5.6-151.6) months. The 16 pts in sustained CR had a median follow-up of 99.7 months (range 3.0-136.3) and none were MRD positive post-treatment (specificity 100%; 88.9-100%). There were 0 false positives in 200 post-treatment samples tested (specificity 100%, 99-100%). Of 7 relapsing pts, 6 had serum samples within 1 year of recurrence. Of these, 5 pts had a positive MRD (range 3.2 to 11.2 months) (sensitivity 83%; 44-98%) at a median of 9.2 months (range 3.2 to 11.2 months) prior to a positive CT scan (Figure). One relapsing pt only had a serum sample obtained 18 mos before CT recurrence and it was MRD negative. ConclusionsOur data suggest that a DNA sequencing-based MRD test of serum can predict clinical relapse with high sensitivity and specificity in DLBCL prior to CT progression. MRD assay detection may significantly reduce the cost, inconvenience and toxicity of surveillance. Importantly, early detection of recurrence may significantly improve pt outcomes. Additional pts are being analyzed and updated results will be available.This work was supported by the Intramural Research Program of NCI at NIH and Sequenta, Inc.▪ Disclosures:Kong:Sequenta: Employment, Equity Ownership. Zheng:Sequenta: Employment, Equity Ownership. Willis:Sequenta: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Faham:Sequenta: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

From B-Cell Tolerance to Autoimmunity

Despite numerous mechanisms that exist to purge the B-cell repertoire of potentially dangerous autoreactive cells, it is now clear that numerous cells with the capability of binding self determinants survive negative selection and persist peripherally as anergic or ignorant cells. Exactly how these B cells are normally constrained, and then aberrantly activated in the context of autoimmunity, are two major questions in B-cell biology. A better understanding of the mechanisms involved in these processes could provide important insights to the regulation of alloreactivity. One clue to our understanding of autoimmunity comes from the nature of the autoantigens commonly targeted in systemic autoimmune diseases. Autoantigens often consist of macromolecular complexes that incorporate self-nucleic acids, and numerous in vitro studies have now shown that many of these canonical autoantigens are essentially potent endogenous autoadjuvants. Mice expressing a low affinity BCR specific for autologous IgG2a can be potently activated by DNA or RNA-associated immune complexes through a mechanism dependent on both the BCR and either TLR9 or TLR7, and serve as a useful prototype for autoreactive B cells in general. A number of groups have now tested this BCR/TLR paradigm in vivo. As expected, Tlr9-/- autoimmune-prone mice fail to produce autoantibodies directed against chromatin, while Tlr7-/- mice fail to produce autoantibodies directed against numerous RNA-associated proteins. However, the Tlr9-/-mice develop accelerated clinical disease, while the Tlr7-/-mice exhibit remarkably prolonged survival. We have now shown that BCR/TLR9 and BCR/TLR7 induce inherently different functional outcomes in B cells. Quite remarkably, both in vitro and in vivo, BCR/TLR7-dependent activation of autoreactive B cells leads to a more prolonged response and increased numbers of antibody producing cells. This response can be defined by a unique gene-expression profile and associated with proteins known to promote plasmablast differentiation. By contrast, BCR/TLR9 activation appears to initially limit autoreactive B-cell expansion; although in the context of systemic autoimmunity TLR9 is required for the production of DNA-reactive autoantibodies. Together these data indicate that the outcome BCR/TLR9 engagement of autoreactive B cells is highly dependent on environmental cues, and suggest that BCR/TLR7 B-cell activation is a key factor in the initiation of systemic lupus erythematosus and other systemic disorders. Disclosures:Marshak-Rothstein:Idera Pharmaceuticals: Consultancy; Abbvie: Consultancy; Genentech: Honoraria.

Antiviral treatment improves disrupted peripheral B lymphocyte homeostasis in chronic hepatitis B virus-infected patients

Disruption of peripheral blood B-cell homeostasis and variation of surface receptors occur with certain infections and autoimmune diseases. However, the impact of antiviral therapy on B-cell alteration during chronic hepatitis B (CHB) infection remains unclear. Our study aims to document the effects of B-cell alteration in CHB patients treated with tenofovir or adefovir. A total of 21 CHB patients and 10 healthy donors were recruited into the study. We identified B-cell subsets by flow cytometry and observed changes in the B-cell repertoire of CHB patients upon tenofovir or adefovir antiviral treatment. The total and percent of B cells and CD5 + B-cell subsets were significantly increased in CHB patients compared to healthy donors. Total and percent of CD5 + B cells gradually decreased following the diminution of the HBV DNA load after tenofovir and adefovir treatment. Upon tenofovir treatment, the percent of memory CD27 + B cells was increased but the absolute number declined, whereas naïve CD27- B cells declined in both percent and absolute number. In the adefovir treatment group, neither naïve nor memory B cells were altered by the treatment. Furthermore, CHB patients displayed higher levels of activation markers (CD69 and CD24) and trended towards restored B-cell homeostasis after antiviral treatment. In conclusion, disrupted B-cell homeostasis is an important feature of CHB patients and is partially restored after control of viral replication by antiviral treatment. B-cell antiviral immunity is improved by restoring B-cell homeostasis and activation.

A Mouse Variable Gene Fragment Binds to DNA Independently of the BCR Context: A Possible Role for Immature B-Cell Repertoire Establishment

B-cell maturation occurs in several steps and requires constant stimulus for its continuing development. From the emergence of the pre-B-cell receptor, signal transduction stimulates and supports B-cell development. Current viewpoints indicate that both positive selection pressure for autoantigens and tonic signaling constitutively stimulate B-cell maturation. In this work, we tested for the presence of a putative DNA binding site in a variable gene segment in a germline configuration, independently of VDJ recombination. After a survey of the public antibody databases, we chose a single mouse heavy variable gene segment that is highly represented in anti-nucleic acid antibodies and tested it for ssDNA binding. A phage display approach was used to search for intrinsic binding to oligo deoxythymidine. The results revealed that binding to an antigen can be influenced by the use of a specific DNA binding V gene segment. Our data support the idea that some variable genes have intrinsic reactivity towards specific types of endogenous autoantigens, and this property may contribute to the establishment of the immature B-cell repertoire.

Genetic measurement of memory B-cell recall using antibody repertoire sequencing

Annual influenza vaccinations aim to protect against seasonal infections, and vaccine strain compositions are updated every year. This protection is based on antibodies that are produced by either newly activated or memory B cells recalled from previous encounters with influenza vaccination or infection. The extent to which the B-cell repertoire responds to vaccination and recalls antibodies has so far not been analyzed at a genetic level--which is to say, at the level of antibody sequences. Here, we developed a consensus read sequencing approach that incorporates unique barcode labels on each starting RNA molecule. These labels allow one to combine multiple sequencing reads covering the same RNA molecule to reduce the error rate to a desired level, and they also enable accurate quantification of RNA and isotype levels. We validated this approach and analyzed the differential response of the antibody repertoire to live-attenuated or trivalent-inactivated influenza vaccination. Additionally, we analyzed the antibody repertoire in response to repeated yearly vaccinations with trivalent-inactivated influenza vaccination. We found antibody sequences that were present in both years, providing a direct genetic measurement of B-cell recall.

Immunoglobulin G4-associated cholangitis: Dominating immunoglobulin G4-positive clones within the B-cell receptor repertoire indicate light at the end of a long tunnel

Immunoglobulin G4-associated cholangitis: Dominating immunoglobulin G4-positive clones within the B-cell receptor repertoire indicate light at the end of a long tunnel

An in vitro culture system for studying the human B cell repertoire (P1459)

Abstract Defining the human B-cell repertoire is important for understanding human B-cell development and autoimmunity. Currently, re-expression of immunoglobulins from single B cells is the standard method to study human B-cell repertoire; however, this method is labor-intensive and costly as it requires large numbers of recombinant antibodies to be generated by single-cell PCR and expression of recombinant heavy and light chain pairs. We have developed an alternative method to characterize the B-cell repertoire by culture that supports the proliferation and differentiation of human pre-B, immature/transitional, and mature B cells recovered from bone marrow, fetal liver, or peripheral blood. Briefly, single B cells are cultured in IL-2, IL-4, IL-21, and BAFF on CD154+ stromal cells. This culture supports extensive cell proliferation and differentiation into IgG-secreting plasmacytes. The monoclonal IgG antibodies in each cell culture can be readily screened by ELISA, Luminex, or immunofluorescence against endogenous and exogenous antigens. We find that autoantibodies from pre-B cell cultures have higher avidities for self-antigens than do those from immature/transitional and mature B cell cultures, suggesting the loss of high avidity cells at the first tolerance checkpoint. V(D)J rearrangements are easily recovered from selected clones for sequencing and expression. This culture method offers a pathway to determine B-cell repertoires with high throughput and reduced cost.

Memory to self-antigens in Systemic Lupus Erythematosus (P4020)

Abstract Systemic Lupus Erythematosus (SLE) is a systemic autoimmune disease associated with auto-IgG antibodies, which are produced due to defective clearance of apoptotic cells (AC) and loss of tolerance to self-antigens. SLE follows flare and remission courses; disease symptoms can come rapidly and unpredictably, suggesting the presence of immune memory to self-antigens in patients. However, experimental evidence and molecular mechanisms behind this are not known. We set up a mouse model by injecting normal mice with syngenic AC weekly for 4 weeks that breaks tolerance and induces auto-IgG production. One month later the serum auto-IgG drops to pre-immune level, these mice can rapidly respond to a single AC injection and produce increased auto-IgGs. This memory-like response is transferable into naïve mice. Serum level of auto-IgG subclass IgG2, but not IgG1, is dramatically increased in the memory response compared to that in the primary response, which contribute to pathogenic effects. The memory auto-IgGs are produced via both germinal center and plasmablast pathways. Spectratyping shows that B-cell repertoire is more skewed in memory response than that in pre-immune and primary response. These observations demonstrate an inducible immune memory to self-antigen in normal mice and establish a model for study of SLE flare-like response in mice. In addition, this study suggests that the formation of immune memory to self-antigens contributes to the disease relapse in SLE patients.

Clues to pathogenesis of Waldenström macroglobulinemia and immunoglobulin M monoclonal gammopathy of undetermined significance provided by analysis of immunoglobulin heavy chain gene rearrangement and clustering of B-cell receptors

We characterized immunoglobulin heavy chain (IGH) gene rearrangements and searched for clusters of stereotyped B-cell receptors in 123 patients with Waldenström macroglobulinemia (WM; n = 59) or immunoglobulin M monoclonal gammopathy of undetermined significance (IgM-MGUS) (n = 64). A productive monoclonal IGHV-D-J rearrangement was obtained in 99/123 patients (80%). Immunoglobulin heavy chain variable (IGHV) genes were mutated in 94/99 patients (95%) with a median somatic hypermutation rate of 6.7% (2.1–14.5). Compared with the normal B-cell repertoire, patients with WM/IgM-MGUS showed an over-representation of the IGHV3 subgroup (83% vs. 55%, p < 0.0001) and an under-representation of IGHV1 (7% vs. 14%, p = 0.04) and IGHV4 (7% vs. 23%, p = 0.0001) subgroups. At the gene level, in WM/IgM-MGUS there was an over-representation of IGHV3-23 (24% vs. 12%, p = 0.0003), IGHV3-64 (3% vs. < 1%, p = 0.003), IGHV3-7 (12% vs. 4%, p = 0.0001) and IGHV3-74 (9% vs. 2%, p < 0.0001), while IGHV4-39 was never used (0 vs. 5%, p = 0.03). Intra-WM/IgM-MGUS search for HCDR3 similarity showed no association fulfilling criteria for stereotyped receptors. WM/IgM-MGUS sequences were unrelated to known chronic lymphocytic leukemia (CLL), splenic marginal zone lymphoma (SMZL) or mantle cell lymphoma (MCL) subsets. In conclusion, the IGHV gene usage in WM and IgM-MGUS is remarkably biased as compared to the normal B-cell repertoire. WM and IgM-MGUS-specific HCDR3 clusters do not occur with a frequency detectable with currently available databases, not supporting a B-cell receptor-driven pathogenesis in WM and IgM-MGUS.

Antibody deficiency in patients with ataxia telangiectasia is caused by disturbed B- and T-cell homeostasis and reduced immune repertoire diversity

Ataxia telangiectasia (AT) is a multisystem DNA-repair disorder caused by mutations in the ataxia telangiectasia mutated (ATM) gene. Patients with AT have reduced B- and T-cell numbers and a highly variable immunodeficiency. ATM is important for V(D)J recombination and immunoglobulin class-switch recombination (CSR); however, little is known about the mechanisms resulting in antibody deficiency severity. We sought to examine the immunologic mechanisms responsible for antibody deficiency heterogeneity in patients with AT. In this study we included patients with classical AT plus early-onset hypogammaglobulinemia (n = 3), classical AT (n = 8), and variant AT (late onset, n = 4). We studied peripheral B- and T-cell subsets, B-cell subset replication history, somatic hypermutation frequencies, CSR patterns, B-cell repertoire, and ATM kinase activity. Patients with classical AT lacked ATM kinase activity, whereas patients with variant AT showed residual function. Most patients had disturbed naive B-cell and T-cell homeostasis, as evidenced by low cell numbers, increased proliferation, a large proportion CD21(low)CD38(low) anergic B cells, and decreased antigen receptor repertoire diversity. Impaired formation of T cell-dependent memory B cells was predominantly found in patients with AT plus hypogammaglobulinemia. These patients had extremely low naive CD4(+) T-cell counts, which were more severely reduced compared with those seen in patients with classical AT without hypogammaglobulinemia. Finally, AT deficiency resulted in defective CSR to distal constant regions that might reflect an impaired ability of B cells to undergo multiple germinal center reactions. The severity of the antibody deficiency in patients with AT correlates with disturbances in B- and T-cell homeostasis resulting in reduced immune repertoire diversity, which consequently affects the chance of successful antigen-dependent cognate B-T interaction.

High-throughput sequencing of the paired human immunoglobulin heavy and light chain repertoire

Each B-cell receptor consists of a pair of heavy and light chains. High-throughput sequencing can identify large numbers of heavy- and light-chain variable regions (V(H) and V(L)) in a given B-cell repertoire, but information about endogenous pairing of heavy and light chains is lost after bulk lysis of B-cell populations. Here we describe a way to retain this pairing information. In our approach, single B cells (>5 × 10(4) capacity per experiment) are deposited in a high-density microwell plate (125 pl/well) and lysed in situ. mRNA is then captured on magnetic beads, reverse transcribed and amplified by emulsion V(H):V(L) linkage PCR. The linked transcripts are analyzed by Illumina high-throughput sequencing. We validated the fidelity of V(H):V(L) pairs identified by this approach and used the method to sequence the repertoire of three human cell subsets-peripheral blood IgG(+) B cells, peripheral plasmablasts isolated after tetanus toxoid immunization and memory B cells isolated after seasonal influenza vaccination.

Frontiers in B-cell immunology

Frontiers in B-cell immunology

Comparison of High-Throughput Sequencing and Flow Cytometry for Measuring Minimal Residual Disease in Pediatric Acute Lymphoblastic Leukemia: A Children's Oncology Group Cohort

AbstractAbstract 1440 Background:Measurement of minimal residual disease (MRD) during and after induction therapy has emerged as the most important predictor of outcome in pediatric acute lymphoblastic leukemia (ALL). Despite this, over 1/3 of relapses occur in patients who are MRD negative. In addition, ∼50% of children that have detectable MRD do not relapse. The Children Oncology Group (COG) trials use flow cytometry (FC) with a sensitivity of 10−4 for MRD detection and subsequent intensification of therapy in MRD+ patients. A more sensitive tool for monitoring MRD could lead to the identification of more patients who are likely to relapse, while a more specific assay could prevent unwarranted therapy intensification. To this end, we are employing the LymphoSIGHT platform developed by Sequenta Inc., which utilizes high-throughput sequencing for identification of clonal gene rearrangements in the B-cell repertoire and subsequent MRD measurement. In this blinded pilot study (COG AALL12B1), we compared the ability of the sequencing assay to measure MRD to that of FC in diagnostic and post-induction samples from 6 ALL patients. Methods:Using universal primer sets, we amplified immunoglobulin heavy chain (IgH@) variable (V), diversity (D), and joining (J) gene segments from genomic DNA in diagnostic and follow-up bone marrow samples from 6 ALL patients. Amplified products were sequenced to obtain >1 million reads per sample and were analyzed using algorithms for clonotype determination. Tumor-specific clonotypes were identified for each patient based on their high-frequency within the B-cell repertoire in the diagnostic sample. The presence of the tumor-specific clonotype was then monitored in post-induction samples. Absolute quantification was performed by normalizing the patient’s reads to internal reference DNA. We then analyzed concordance between MRD results obtained by sequencing and FC. Results:We detected a high-frequency IgH clonal rearrangement in 5/6 diagnostic ALL samples. MRD was assessed in the 5 post-induction samples from these patients (Table 1). Deep coverage of all MRD samples was obtained, with each original IgH molecule generating ∼20 sequencing reads, ensuring the detection of a single leukemic cell if present in the sample. Leukemic clones were detected in 4/5 follow-up samples (Table 1). In the positive samples, the number of detected leukemic molecules ranged from 12 to over 6,000 and the MRD level ranged from 0.008% to 0.3%. MRD results were concordant with FC in 3 of 5 patients and were consistent with the patient’s clinical courses. In one patient we detected MRD at 0.008%, a level below the sensitivity of FC, which was negative. In another sample, FC detected MRD of 0.01–0.1%, but no leukemic clones were detected by the sequencing assay despite the fact that the sample contained sufficient cell input (almost 2 million cells). The patient remained in continuous remission. Evaluation of additional paired diagnostic and post-induction samples and their association with clinical outcomes is ongoing. Conclusions:We show the application of a high-throughput sequencing method for MRD detection in childhood ALL. IgH clonal rearrangements were detected in 5/6 (83%) of samples using the sequencing assay. The absence of a clonal rearrangement in 1/6 of patients was anticipated and is likely to be mitigated by the presence of a clonal rearrangement in another immunoglobulin or T cell receptor gene. Experiments are ongoing to assess the presence of clonal rearrangements in these receptors (i.e., IgH D-J, IgK, TRB@, TRD@ or TRG@) in the diagnostic samples. In 3/5 patients there was concordance between FC and sequencing-based MRD detection. In one patient, sequencing detected MRD at a level below the threshold of FC. The last patient was negative by sequencing but positive by FC and has not relapsed. Further analysis of the sensitivity and specificity of the sequencing platform compared to FC using additional paired diagnostic and post-induction samples is ongoing.Table 1Comparison of Day 29 (end of induction) MRD results using sequencing and FC methodsSampleNumber of input cellsNumber of input leukemic clone moleculesSequencing MRD (%)Flow cytometry MRD (%)Clinical outcome1162,945120.008<0.01CR25,789,0336,3560.1>0.1Relapse4291,352480.020.01-0.1CR51,901,076000.01-0.1CR61,968,5115,8550.3>0.1CR Disclosures:Faham:Sequenta, Inc.: Employment, Equity Ownership, Research Funding. Moorhead:Sequenta, Inc.: Employment, Equity Ownership, Research Funding. Zheng:Sequenta, Inc.: Employment, Equity Ownership, Research Funding.

Detection of Diffuse Large B-Cell Lymphoma in Peripheral Blood Using High-Throughput Sequencing Assay.

AbstractAbstract 2666 Background:In patients with diffuse large B-cell lymphoma (DLBCL), circulating lymphoma cells in the bloodstream are rarely detected by conventional morphology or flow cytometry evaluation. We developed a high-throughput sequencing based platform, LymphoSIGHT, to detect evidence of lymphoid malignancies in peripheral blood samples, as this could potentially be used for detection of minimal residual disease after treatment. This sequencing method has a sensitivity to detect one lymphoma cell per million leukocytes in peripheral blood. We herein report the results of our pilot study assessing the ability of this method to detect the lymphoma clone in peripheral blood samples from 5 DLBCL patients at the time of diagnosis. Methods:This study has been approved by IRB and consent has been obtained from patients. Using universal primer sets, we amplified immunoglobulin heavy chain (IgH@) variable, diversity, and joining gene segments from genomic DNA in tumor biopsy and peripheral blood samples (plasma and peripheral blood mononuclear cell (PBMC) compartments) collected at initial diagnosis. Amplified products were sequenced to obtain >1 million reads (>10× sequencing coverage per IgH molecule), and were analyzed using standardized algorithms for clonotype determination. Tumor-specific clonotypes were identified for each patient based on their high-frequency within the B-cell repertoire in the lymph node biopsy sample. The presence of the tumor-specific clonotype was then quantitated in cell-free and PBMC compartments from the diagnostic blood sample. A quantitative and standardized measure of clone level among all leukocytes in the diagnostic sample was determined using internal reference DNA. Results:We detected a high-frequency IgH clonal rearrangement in all 5 lymph node biopsy samples. The lymphoma clonotype that was identified in the tumor biopsy was also detected in the plasma and/or PBMC compartment in all 5 patients at diagnosis. Specifically, the lymphoma clonotype was detected in the plasma compartment in 4 patients, while 3 patients demonstrated the presence of the lymphoma clonotype in the PBMC compartment (Table 1). We hypothesize that the positive lymphoma clone in the plasma is due to rapid proliferation and necrosis of the primary tumor, releasing the degraded component of lymphoma into the blood stream. However, in this small sample size, we did not observe an obvious correlation between the level of detection (PBMC or plasma) and clinical parameters (LDH, stage, size of tumor, tumor Ki67, cell-of-origin). All patients achieved complete response after initial treatment and four are being followed. We plan to analyze blood specimens while they are in remission. Conclusions:IgH clonal rearrangements were detected by sequencing in all tumor biopsy samples. Importantly, all peripheral blood samples showed signs of circulating lymphoma material in either the plasma or PBMC compartment at diagnosis. Analysis of diagnostic and post-therapy samples from additional DLBCL patients is ongoing. These data will determine whether the sequencing assay is a strong indicator for response to therapy and relapse monitoring.Table 1Detection of circulating tumor material in DLBCL patients at diagnosis.PBMC compartmentPlasma compartmentPatientNumber of clone moleculesFrequency among IgH sequences (%)Number of clone moleculesFrequency among IgH sequences (%)12,31813.52970.520032.733,70150040014095.5570.052124.4 Disclosures:Faham:Sequenta, Inc.: Employment, Equity Ownership, Research Funding. Carlton:Sequenta, Inc.: Employment, Equity Ownership, 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.

A Generalized Mathematical Model To Estimate T- and B-Cell Receptor Diversities Using AmpliCot

The efficiency of the adaptive immune system is dependent on the diversity of T- and B-cell receptors, which is created by random rearrangement of receptor gene segments. AmpliCot is an experimental technique that allows the measurement of the diversity of the T- and B-cell repertoire. This procedure has the advantage over other cloning and sequencing techniques of being time- and expense-effective. In previous studies, receptor diversity, measured with AmpliCot, has been inferred assuming a second-order kinetics model. The latter implies that the relation between diversity and concentration × time (Cot) values is linear. We show that a more detailed model, involving heteroduplex and transient-duplex formation, leads to significantly better fits of experimental data and to nonlinear diversity-Cot relations. We propose an alternative fitting procedure, which is straightforward to apply and which gives an improved description of the relationship between Cot values and diversity.

Endogenous antigen tunes the responsiveness of naive B cells but not T cells

In humans up to 75% of newly generated B cells and about 30% of mature B cells exhibit some degree of autoreactivity1. Yet, how B cells establish and maintain tolerance in the face of autoantigen exposure during and after development is not certain. Studies of model BCR transgenic systems have highlighted the critical role played by functional unresponsiveness or ‘anergy’2,3. Unlike T cells, evidence suggests that receptor editing and anergy, rather than deletion, account for much of B cell tolerance4,5. However, it remains unclear whether the mature diverse B cell repertoire of mice contains anergic autoreactive B cells, and if so, whether antigen was encountered during or after their development. By taking advantage of a reporter mouse in which B cell antigen receptor (BCR) signaling rapidly and robustly induces GFP expression under the control of the Nur77 regulatory region, antigen-dependent and – independent BCR signaling events in vivo during B cell maturation were visualized. Here we show that B cells encounter antigen during development in the spleen, and that this antigen exposure in turn tunes the responsiveness of BCR signaling in B cells at least partly by down-modulating expression of surface IgM but not IgD BCRs, and by modifying basal calcium levels. By contrast, no analogous process occurs in naive mature T cells. Our data demonstrate not only that autoreactive B cells persist in the mature repertoire, but that functional unresponsiveness or ‘anergy’ exists in the mature B cell repertoire along a continuum, a fact that has long been suspected, but never yet shown. These results have important implications for understanding how tolerance in T and B cells is differently imposed, and how these processes might go awry in disease.

Secreted human Ro52 autoantibody proteomes express a restricted set of public clonotypes

Long-lived secreted autoantibody responses in systemic autoimmunity are generally regarded to be polyclonal and to express a diverse B-cell repertoire. Here, we have used a proteomic approach based on de novo sequencing to determine the clonality and V region structures of human autoantibodies directed against a prototypic systemic autoantigen, Ro52 (TRIM21). Remarkably, anti-Ro52 autoantibodies from patients with primary Sjögren’s syndrome, systemic lupus erythematosus, systemic sclerosis or polymyositis were restricted to two IgG1 kappa clonotypes that migrated as a single species on isoelectric focusing; shared a common light chain paired with one of two closely-related heavy chains; and were public in unrelated patients. Targeted mass spectrometry using these uniquely mutated V region peptides as surrogates detected anti-Ro52 autoantibodies in human sera with high sensitivity and specificity compared with traditional ELISA. Mass spectrometry-based detection of specific autoantibody motifs provides a powerful new tool for analysis of humoral autoimmunity.

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.