IgH Sequences Research Articles

The common variable immunodeficiency IgM repertoire narrowly recognizes erythrocyte and platelet glycans

BackgroundAutoantibody-mediated cytopenias (AICs) regularly occur in profoundly IgG-deficient common variable immunodeficiency (CVID) patients. The isotypes, antigenic targets, and origin(s) of their disease-causing autoantibodies are unclear. ObjectiveTo determine reactivity, clonality and provenance of AIC-associated IgM autoantibodies in CVID patients. MethodsWe utilized glycan arrays, patient erythrocytes, and platelets to determine targets of CVID IgM autoantibodies. Glycan binding profiles were used to identify auto-reactive clones across B cell subsets, specifically circulating marginal zone-like (MZ) B cells, for sorting and IGH sequencing. The locations, transcriptomes and responses of tonsillar MZ B cells to different T helper cell subsets were determined by confocal microscopy, RNA-sequencing, and co-cultures, respectively. ResultsAutoreactive IgM coated erythrocytes and platelets from many CVID patients with AICs (CVID+AIC). On glycan arrays, CVID+AIC plasma IgM narrowly recognized erythrocytic i antigens and platelet i-related antigens and failed to bind hundreds of pathogen- and tumor-associated carbohydrates. Polyclonal i antigen-recognizing B-cell receptors were highly enriched among CVID+AIC circulating marginal zone (MZ) B cells. Within tonsillar tissues, MZ B cells secreted copious IgM when activated by the combination of IL-10 and IL-21 or when cultured with IL10/IL-21 secreting FOXP3-CD25hiTfh cells. In lymph nodes from immunocompetent controls, MZ B cells, plentiful FOXP3+ regulatory T cells, and rare FOXP3-CD25+ cells that represented likely CD25hiTfh cells, all localized outside of GCs. In CVID+AIC lymph nodes, cellular positions were similar but CD25hiTfh cells greatly outnumbered regulatory cells. ConclusionsOur findings indicate glycan-reactive IgM autoantibodies produced outside of GCs may contribute to the autoimmune pathogenesis of CVID.

APPLICABILITY OF IMMUNOGLOBULIN SEQUENCING TO ACCURATELY MONITOR DISEASE: AN ANALYSIS OF UNIQUENESS AMONG DIFFUSE LARGE B‐CELL LYMPHOMA PATIENTS TESTED BY CLONOSEQ

Introduction: Diffuse Large B-Cell Lymphoma (DLBCL), an aggressive form of non-Hodgkin lymphoma, most commonly affects older adults. ∼80% of patients achieve complete response to frontline therapy; roughly half are cured while half relapse and receive salvage treatment such as transplant or CAR-T therapy which may be curative. Due to the aggressiveness of DLBCL and therapies offering potential cure, there is growing interest in a monitoring tool that provides more accurate information than standard clinical imaging. clonoSEQ® is a next-generation sequencing assay, which monitors measurable residual disease (MRD) levels from plasma of DLBCL patients. Using data from 1268 patients we assessed Ig loci uniqueness. Methods: Following identification of a dominant sequence(s) in a high tumor burden sample, the clonoSEQ algorithm quantifies tumor-associated sequences in MRD samples. The algorithm assigns a uniqueness score to each dominant sequence based on analyses of Ig locus V, D, and J segments and non-templated nucleotides. This score reflects the probability a sequence could be independently recreated in a non-malignant cell, including in another person, and is incorporated into the limit of detection (LOD). Sequences with a lower uniqueness must be observed at a higher rate to be above the LOD and reflect confidence they are tumor related. To assess the uniqueness of sequences detected across DLBCL patients, we examined our database containing 3657 dominant sequences from 1268 patients. 969 of these patients are from 27 clinical studies, and 302 are from use of the commercial assay. We assessed the dominant sequences for their uniqueness distribution across IgH, IgK, and IgL loci. Results: Across the population of 1268 DLBCL patients and 3657 dominant sequences, there was a range of 1–9 dominant sequences with 1115 (87.9%) patients having 1–4 (avg 2.89; med 3). We found that 1187 (93.6%) patients had at least one dominant IgH sequence. The remaining 81 (6.4%) patients had only dominant light chain sequences with 66 (5.2%) patients having IgK only. We assessed the distribution of uniqueness across the Ig loci in these patients. Figure 1a shows that IgH sequences were most unique, followed by IgL, IgH (D-J segments), and IgK. Figure 1b indicates the uniqueness score for each patient with only one dominant sequence, with just 31 (2.4%) subjects with expected ‘backgrounds’ of >1/100,000 of all Ig sequences. Keyword: diagnostic and prognostic biomarkers Conflicts of interests pertinent to the abstract A. Jacob Employment or leadership position: Adaptive Biotechnologies Stock ownership: yes, as an employee L. W. Lee Employment or leadership position: Adaptive Biotechnologies Stock ownership: yes, as an employee K. Akers Employment or leadership position: Adaptive Biotechnologies Stock ownership: yes, as an employee I. Kirsch Employment or leadership position: Adaptive Biotechnologies Stock ownership: yes, as an employee H. Simmons Employment or leadership position: Adaptive Biotechnoloigies Stock ownership: yes, as an employee

Insights into IGH clonal evolution in BCP-ALL: frequency, mechanisms, associations, and diagnostic implications.

The malignant transformation leading to a maturation arrest in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) occurs early in B-cell development, in a pro-B or pre-B cell, when somatic recombination of variable (V), diversity (D), and joining (J) segment immunoglobulin (IG) genes and the B-cell rescue mechanism of VH replacement might be ongoing or fully active, driving clonal evolution. In this study of newly diagnosed BCP-ALL, we sought to understand the mechanistic details of oligoclonal composition of the leukemia at diagnosis, clonal evolution during follow-up, and clonal distribution in different hematopoietic compartments. Utilizing high-throughput sequencing assays and bespoke bioinformatics we identified BCP-ALL-derived clonally-related IGH sequences by their shared 'DNJ-stem'. We introduce the concept of 'marker DNJ-stem' to cover the entirety of, even lowly abundant, clonally-related family members. In a cohort of 280 adult patients with BCP-ALL, IGH clonal evolution at diagnosis was identified in one-third of patients. The phenomenon was linked to contemporaneous recombinant and editing activity driven by aberrant ongoing DH/VH-DJH recombination and VH replacement, and we share insights and examples for both. Furthermore, in a subset of 167 patients with molecular subtype allocation, high prevalence and high degree of clonal evolution driven by ongoing DH/VH-DJH recombination were associated with the presence of KMT2A gene rearrangements, while VH replacements occurred more frequently in Ph-like and DUX4 BCP-ALL. Analysis of 46 matched diagnostic bone marrow and peripheral blood samples showed a comparable clonal and clonotypic distribution in both hematopoietic compartments, but the clonotypic composition markedly changed in longitudinal follow-up analysis in select cases. Thus, finally, we present cases where the specific dynamics of clonal evolution have implications for both the initial marker identification and the MRD monitoring in follow-up samples. Consequently, we suggest to follow the marker DNJ-stem (capturing all family members) rather than specific clonotypes as the MRD target, as well as to follow both VDJH and DJH family members since their respective kinetics are not always parallel. Our study further highlights the intricacy, importance, and present and future challenges of IGH clonal evolution in BCP-ALL.

Lineage Reconstruction of In Vitro Identified Antigen-Specific Autoreactive B Cells from Adaptive Immune Receptor Repertoires.

The emergence, survival, growth and maintenance of autoreactive (AR) B-cell clones, the hallmark of humoral autoimmunity, leave their footprints in B-cell receptor repertoires. Collecting IgH sequences related to polyreactive (PR) ones from adaptive immune receptor repertoire (AIRR) datasets make the reconstruction and analysis of PR/AR B-cell lineages possible. We developed a computational approach, named ImmChainTracer, to extract members and to visualize clonal relationships of such B-cell lineages. Our approach was successfully applied on the IgH repertoires of patients suffering from monogenic hypomorphic RAG1 and 2 deficiency (pRD) or polygenic systemic lupus erythematosus (SLE) autoimmune diseases to identify relatives of AR IgH sequences and to track their fate in AIRRs. Signs of clonal expansion, affinity maturation and class-switching events in PR/AR and non-PR/AR B-cell lineages were revealed. An extension of our method towards B-cell expansion caused by any trigger (e.g., infection, vaccination or antibody development) may provide deeper insight into antigen specific B-lymphogenesis.

Needle in a Haystack: A Pilot Study Combining Single-Cell Multiomics with Clinical NGS-MRD Sequencing to Search for Circulating Clonotypic Dedifferentiated Myeloma Cells

Multiple myeloma (MM) is a heterogenous disease in which clonal evolution or preexisting reservoirs of phenotypically divergent cells may be harbingers of therapeutic resistance. Prior research has suggested the presence of myeloma cells with immunophenotypic and expression profiles dedifferentiated from that of terminally differentiated plasma cells that may be found in circulation, marrow, or potentially other lymphoid compartments; however, this has not been conclusively demonstrated in patients. The identification of such clonotypic myeloma cells could lead to a better understanding of myeloma biology and resistance, and inform therapies targeted at these populations, and single-cell technologies provide the granularity to characterize them. Next-generation sequencing (NGS)-based measurable residual disease (MRD) clinical-grade assays (eg. clonoSEQ®, Adaptive Biosciences) identify individuals’ unique heavy and light chain sequences. Theoretically this sequencing data could be overlaid with research-grade single-cell multiomics to identify clusters of clonotypic cells with variable differentiation. In this pilot study using the Enhanced Single Cell Analysis with Protein Expression (ESCAPE) platform (Singleron Biotechnologies) we aimed to identify peripherally circulating clonotypic MM cells by their NGS-MRD sequence and simultaneously determine whether their multiomic profile reflected possible dedifferentiated clusters found in paired marrow. Using scRNA-Seq (10x Genomics) data previously generated at our site from bone marrow mononuclear cells we identified MM cell clusters enriched for differentially expressed genes known to be expressed in B-cells and plasmablasts. These genes included CD19, PAX5, BCL6, EZH2, STMN1, HMGB2, MKI67 and CD27. We next sought to assay the peripheral blood mononuclear cells (PBMC) of patients (n=2) with an overrepresentation of cells in these clusters. To evaluate both the transcriptomic and immunophenotypic profiles of the PBMCs at single-cell resolution we utilized Singleron's Immune Profiling panel on the ESCAPE platform. Using Singleron's MapSuite™ analysis pipeline, we annotated cell-type classifications, and highlighted cells within the mature B-cell/plasma cell differentiation spectrum (Figure A). Within this subset, we attempted to identify gene expression reflective of the previously identified "dedifferentiated” marrow clusters with specific genes of interest, but found that there was not significant overlap. Heatmaps comparing the 5 peripheral subclusters with marrow plasma cells demonstrate some concordance with the marrow subclusters, with examination of these overlaps ongoing, but these were not the "dedifferentiated” marrow clusters. The included patients also had clonoSEQ® identification sequences of heavy chain and/or light chain available. By cross-referencing the NGS-MRD sequence data (clonoSEQ®, Adaptive Biosciences), we were able to use ESCAPE-Seq to identify definitive clonotypic circulating MM cells within the PBMC samples at single cell granularity. Among PBMCs from sample 2 we were able to identify circulating clonotypic myeloma cells with 100% match to the IgH and IgK MRD sequences, which also represented the dominant V(D)J clonotype in peripheral circulating. Concomitant single-cell surface protein analysis of this clonotypic cell population demonstrated significant expression of CD38, without significant differential expression of CD138 nor surface markers of B-cells (Figure B). While these data are preliminary and studies are ongoing, we successfully leveraged clinical-level MRD sequencing data to inform single-cell multiomics, a potentially powerful translational research pathway for any hematologic malignancy in which NGS-MRD is clinically available. We definitively identified a small subset of circulating MM cells and used the ESCAPE platform to efficiently generate a multiomics profile. This profile suggested elevated CD38 expression without elevated CD138 expression, which previously has been associated with dedifferentiated plasma cells and plasmablasts, although further analyses are ongoing. In future studies, we will enrich for B-lymphoid spectrum cells prior to single-cell analytics. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Specificity & Precision of Minimal Residual Disease Monitoring in DLBCL Using Ig-HTS

Background: Dynamic minimal residual disease (MRD) monitoring in the diffuse large B-cell lymphoma (DLBCL) setting has been shown to predict treatment failure at early timepoints and relapse at later timepoints. Precise and reliable approaches are critically important for accurate detection of residual disease. We sought to evaluate the precision of heavy-chain (IGH) and light-chain (IGK/L) MRD measurement within a standard clinical workflow. Methods: We developed a database of clinical clonoSEQ (Adaptive Biotechnologies) and clonoSIGHT reports (Sequenta, Inc.) identifying the dominant malignant IGH or IGK/L clonotypes from the tumor biopsy samples of 285 patients collected as a part of routine clinical care for MRD monitoring. Included patients had been diagnosed with DLBCL at either Stanford University or the National Cancer Institute, 130 of whom had samples sequenced with the clonoSEQ assay and 155 with the clonoSIGHT assay. A control database was generated from all publicly available immunoSEQ (Adaptive Biotechnologies) B-cell repertoires with IGH or IGK/L sequencing, spanning both malignant and healthy patient contexts. The patients within the clinical DLBCL and control databases were distinct without any overlap. We assessed whether each malignant IGH or IGK/L clonotype from the clinical DLBCL database shared overlap with clonotypes present in the control IGH or IGK/L repertoires. A match was considered positive only if the sequence context of the malignant clonotype was an exact match with the sequence of a control clonotype; exact substrings were allowed given the differences in potential primer sites between assays. Results: The clinical database included 341 dominant IGK/L sequences from 187 patients and 271 dominant IGH sequences from 195 patients, where dominance was determined by the clinical assay. We generated a control IGK/L database of 618,775 sequences from 71 subjects and a control IGH database of 65,017,932 sequences from 1698 subjects. IGH demonstrated high precision (99.3%) with only two clinically dominant sequences demonstrating overlap with the control repertoire. Despite the smaller control database, IGK/L demonstrated significantly reduced precision of 92% (Fisher's exact p-value < 0.001) with 29 sequences demonstrating overlap with the control repertoire, all of which were limited to the subset of patients who had been sequenced by clonoSEQ. At the patient level, 19 of the 105 patients with clonoSEQ IGK/L sequencing had clinically trackable (Fig. 1A), putatively malignant sequences that were shared with the control database (precision 84.7%). The frequency of these clinically dominant sequences in the control repertoires ranged from 8.27x10-8 to 0.575 with a mean frequency of 0.000598. Most non-unique clonotypes were shared across multiple control subjects (range 1-37, mean 13, Fig. 1B). Conclusions: The high rate of overlap seen between putatively malignant, dominant IGK/L clonotypes and a publicly available control repertoire suggests substantially reduced validity of approaches relying on IGK/L tracking for minimal residual disease monitoring. The overlapping light chain clonotypes were not only present at relatively high frequencies in both the clinical samples and control repertoire, but were also seen across multiple control subjects, suggesting MRD assays that track only IGK/L may be unable to reliably differentiate malignant clonotypes from background light chain repertoires. Caution should be used when MRD is detected using IGK/IGL given reduced specificity and precision of these sequences. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

EXTH-92. TARGETING PRIMARY CENTRAL NERVOUS SYSTEM B CELL LYMPHOMA IGH CLONOTYPES USING NOVEL RNA-NPS

Abstract BACKGROUND Recurrent PCNSBLs represent a therapeutic challenge. Up to 60% of PCNSBL patients relapse to later face survival rates as low as 22%. Unfortunately, tumor heterogeneity and off-target effects have limited the success of immunotherapy against PCNSBL. METHODS We propose a novel immunotherapy to overcome PCNSBL heterogeneity and off-target effects in an exquisitely tumor specific manner using nanoparticle vaccination, capable of delivering personalized tumor derived mRNAs, that induces systemic orchestration of innate and adaptive immunity. We target tumor antigens derived from the B cell receptor (i.e., heavy chain immunoglobulin - IgH) clonotypes. IgH clonotypes are hypervariable gene rearrangements clonally generated by B cells. Tumor IgH clonotypes are unique for each malignant B cell clone and hence attractive immune targets, not shared by normal B cell clones avoiding undesirable off-target effects. RESULTS RNA-NPs can reprogram tumor microenvironment while activating the innate immunity via IFN type I (i.e., IFNα) and priming of hypervariable region clonotype specific T cell responses in naïve mice. We determined the rearranged IgH sequences (predominant clone 99% and nine additional clones with frequencies &amp;lt; 1%) of clinically relevant inbred murine PCNSBL models (BAL17 and A20) by PCR. The number of identified clonotypes confirmed the IgH variability observed in human B cell hematological malignancies. In preliminary experiments targeting lymphoma derived single clonotypes with RNA-NPs, we showed the feasibility of priming in-vivo T cells specific against hypervariable regions after 3 weekly i.v. RNA-NPs (median IFNγ: 58 pg/ml; range: 50-70 pg/ml vs controls &amp;lt; 30 pg/ml; p=0.008). Targeting of clonotype RNA-NPs was associated with decreased tumor growth (p=0.04). Interestingly, we have observed tumor reactive lymphangiogenesis that communicates with regional skull bone marrow observed in 3D microscopy that might direct future routes of RNA-NP administration. CONCLUSION Our RNA-NP systemic vaccination platform can induce PCNSBL clonotype specific T cell responses, sparing normal tissues.

Development and application of a next-generation sequencing protocol and bioinformatics pipeline for the comprehensive analysis of the canine immunoglobulin repertoire.

Profiling the adaptive immune repertoire using next generation sequencing (NGS) has become common in human medicine, showing promise in characterizing clonal expansion of B cell clones through analysis of B cell receptors (BCRs) in patients with lymphoid malignancies. In contrast, most work evaluating BCR repertoires in dogs has employed traditional PCR-based approaches analyzing the IGH locus only. The objectives of this study were to: (1) describe a novel NGS protocol to evaluate canine BCRs; (2) develop a bioinformatics pipeline for processing canine BCR sequencing data; and (3) apply these methods to derive insights into BCR repertoires of healthy dogs and dogs undergoing treatment for B-cell lymphoma. RNA from peripheral blood mononuclear cells of healthy dogs (n = 25) and dogs newly diagnosed with intermediate-to-large B-cell lymphoma (n = 18) with intent to pursue chemotherapy was isolated, converted into cDNA and sequenced by NGS. The BCR repertoires were identified and quantified using a novel analysis pipeline. The IGK repertoires of the healthy dogs were far less diverse compared to IGL which, as with IGH, was highly diverse. Strong biases at key positions within the CDR3 sequence were identified within the healthy dog BCR repertoire. For a subset of the dogs with B-cell lymphoma, clonal expansion of specific IGH sequences pre-treatment and reduction post-treatment was observed. The degree of expansion and reduction correlated with the clinical outcome in this subset. Future studies employing these techniques may improve disease monitoring, provide earlier recognition of disease progression, and ultimately lead to more targeted therapeutics.

Long-Term Follow-up and Single-Cell Multiomics Characteristics of Infusion Products in Patients with Chronic Lymphocytic Leukemia Treated with CD19 CAR-T Cells

Long-Term Follow-up and Single-Cell Multiomics Characteristics of Infusion Products in Patients with Chronic Lymphocytic Leukemia Treated with CD19 CAR-T Cells

Retrospective Analysis of Minimal Residual Disease Testing By High Throughput Immunosequencing Versus High Sensitivity Flow Cytometry in Multiple Myeloma

Introduction:Detection of minimal residual disease (MRD) is one of the strongest predictors of outcome in multiple myeloma (MM). Until recently, the most commonly available method to detect MRD in clinical practice has been high sensitivity flow cytometry (FC) which can detect MRD with at 10 -5 sensitivity. In recent years, next-generation sequencing (NGS) has become a viable method to assess the MRD in MM patients with a 10 -6 sensitivity. NGS appears to have some advantages over HC-FC by circumventing subjectivity of analysis. However, real-world comparison between these two methodologies in the literature is limited and is important to inform daily hematopathology and oncology ordering practices.Methods:We retrospectively identified all cases of MM with NGS MRD data from bone marrow specimens at the Moffitt Cancer Center and collated corresponding flow MRD data and clinical data (OS, patient demographics) electronically and via chart review.10-color flow cytometry was performed on a Gallios System and analyzed on Kaluza (Beckman Coulter, IN). Two million events were collected on all cells. Validated lower limit of detection was at least 0.01%. Antibodies included CD28, CD81, CD56, CD138, CD319, CD20, CD19, CD117, CD38, CD45, CD27, CD200 (BD, Biolegend, Beckman Coulter).clonoSEQ ® (Adaptive Biotechnologies, Seattle, WA) testing was performed which uses multiplex polymerase chain reaction (PCR) and NGS to identify, characterize, and monitor clonotypes of immunoglobulin (Ig) IgH (V-J), IgH (D-J), IgK, and IgL receptor gene sequences, and translocated BCL1/IgH (J) and BCL2/IgH (J) sequencesStatistical analysis was performed by Spearman correlation coefficient and Kaplan-Meier analysis.Results:192 samples from 122 unique patients were identified that had both NGS and FC data performed on the same sample. FC+ values ranged from 1x10 -7 to 0.39. NGS+ values ranged from 2.3 x 10 -7 to 0.15. Spearman correlation coefficient showed moderate concordance between NGS and FC at r=0.67 (p<0.001). Six samples were positive by FC (mean tumor burden (MTB)= 0.0007) but missed by NGS; whereas 59 samples were positive by NGS (MTB= 0.002) but missed by flow cytometry. Two cases by FC were equivocal and these were both definitively designated as MRD+ by NGS. Overall survival was worse for MRD+ (by NGS or FC) vs MRD(-) (Figure 1).Conclusion:Our study confirms the importance of MRD detection in MM and shows the robust utility of NGS for MRD detection in routine hematopathology practice. While both FC and NGS are complementary given that each can potentially detect MRD missed by another method, the data supports the increased sensitivity of NGS over FC. [Display omitted] DisclosuresNishihori: Novartis: Research Funding; Karyopharm: Research Funding. Hussaini: Stemeline Therapeutics: Honoraria.

Concurrent Monitoring of Peripheral Blood Circulating Tumor DNA and Circulating Tumor Cells in Relapsed/Refractory Follicular Lymphoma Patients Post Axicabtagene Ciloleucel, a Single Center Experience

Concurrent Monitoring of Peripheral Blood Circulating Tumor DNA and Circulating Tumor Cells in Relapsed/Refractory Follicular Lymphoma Patients Post Axicabtagene Ciloleucel, a Single Center Experience

Retrospective Analysis of Minimal Residual Disease Testing By High Throughput Immunosequencing Versus High Sensitivity Flow Cytometry and qPCR in B-Lymphoblastic Leukemia

Introduction: Detection of minimal residual disease (MRD) is one of the most powerful predictors of prognosis in patients with B-Lymphoblastic Leukemia (ALL). Standard methods to detect MRD in ALL include high-resolution flow cytometry (FC) (sensitivity 10 -4 to 10 -5) and qPCR for Ph+ ALL (sensitivity 10 -5 to -6). More recently, high-throughput sequencing (NGS) has been leveraged to detect MRD (sensitivity 10 -6). Retrospective, non-clinical trial based real-world data comparing between these methodologies in the literature is limited to absent. We report our experience at a high-volume cancer center comparing NGS, qPCR, and FC.Methods: All cases of ALL with NGS MRD data at the Moffitt Cancer Center were identified. Corresponding FC, qPCR (p190 and p210) and clinical data were collected electronically and via chart review.10-color flow cytometry was performed on a Gallios System and analyzed on Kaluza (Beckman Coulter, IN). 750,000 events were collected on all cells. Validated lower limit of detection was at least 0.01%. Antibodies included CD15, CD130, CD10, CD58, CD22, CD33, CD13, CD123, CD19, CD20, CD45, CD38, CD34, CD81, CD200 (BD, Biolegend, Beckman Coulter).Quantidex™ BCR-ABL IS Kit (RT qPCR) was used to quantify BCR-ABL (p190 and p210) and ABL transcripts in total RNA in patients with ALL on Applied Biosystems 7500 Fast Dx Real-Time PCR instrument. The results were expressed by normalized BCR-ABL to ABL (control) ratio.clonoSEQ ® (Adaptive Biotechnologies, Seattle, WA) testing was performed which uses multiplex polymerase chain reaction (PCR) and NGS to identify, characterize, and monitor clonotypes of immunoglobulin (Ig) IgH (V-J), IgH (D-J), IgK, and IgL receptor gene sequences, and translocated BCL1/IgH (J) and BCL2/IgH (J) sequences.Statistical analysis was performed by Spearman correlation coefficient and Kaplan-Meier analysis.Results: 95 samples from 54 unique patients were identified that had both NGS and FC data. A subset of patients had qPCR data (n=31 samples). FC+ values ranged from 0.00004 to 0.75. NGS+ values ranged from 8.25E-08 to 0.49. Spearman correlation coefficient showed moderate concordance between NGS and FC at r=0.62 (p<0.001). Spearman correlation coefficient for qPCR vs Flow was r= 0.08 (p=0.68). Three samples were positive by FC (mean tumor burden (MTB= 0.002) but missed by NGS; whereas 31 samples were detected by NGS (MTB= 0.0016) that were missed by flow cytometry. By FC, 9 samples were equivocal of which 7 were definitively designated as MRD+ by NGS. There were 7 qPCR+/NGS- samples (MTB= 2.5 x 10 -5) and 6 qPCR-/NGS+ cases (MTB= 0.012). Overall survival was worse for MRD+ (by NGS, qPCR, or FC) vs MRD(-) (Figure 1).Conclusion:Our study confirms the importance of MRD detection in ALL and shows the robust utility of NGS for MRD detection in routine hematopathology practice. While qPCR, FC and NGS are complementary given that each can potentially detect MRD missed by another method, the data supports the increased sensitivity of NGS over FC. [Display omitted] DisclosuresShah: Servier Genetics: Other; Jazz Pharmaceuticals: Research Funding; Incyte: Research Funding; BeiGene: Consultancy, Honoraria; Acrotech/Spectrum: Honoraria; Pharmacyclics/Janssen: Honoraria, Other: Expenses; Kite, a Gilead Company: Consultancy, Honoraria, Other: Expenses, Research Funding; Precision Biosciences: Consultancy; Amgen: Consultancy; Pfizer: Consultancy, Other: Expenses; Novartis: Consultancy, Other: Expenses; Bristol-Myers Squibb/Celgene: Consultancy, Other: Expenses; Adaptive Biotechnologies: Consultancy. Hussaini: Stemeline Therapeutics: Honoraria.

Utility Of Non-Invasive Monitoring Of Circulating Tumor DNA At Diagnosis, Interim Therapy, and Relapse Of DLBCL Using High-Throughput Sequencing Of Immunoglobulin Genes

BackgroundRecent studies have shown limited utility of routine surveillance imaging for patients with diffuse large B-cell lymphoma achieving remission after R-CHOP (Thompson et al, ASCO 2013 #8503; Ghesquieres et al, ICML 2013 #226). Separately, response assessment using early interim PET-CT during R-CHOP has suboptimal accuracy. We hypothesized that non-invasive tumor burden monitoring using peripheral blood could have utility for diagnosis and monitoring of DLBCL. MethodsWe prospectively profiled two cohorts of patients with DLBCL at various stages in their disease, including at diagnosis, during various therapies, after achieving remission, and upon relapse or progression. Cohort1 patients were treated with curative intent at Stanford University with blood collected at multiple time points during follow-up. Cohort2 patients participated in a multi-center Austrialian Phase 2 study (ALLG NHL21), where patients with poor risk DLBCL as identified by residual disease on interim PET/CT after 4 R-CHOP cycles received early treatment intensification using R-ICE/ASCT; blood samples were collected before the 1st and after the 4thR-CHOP cycles. Tumor-derived clonotypic IgH and IgK sequences were defined in diagnostic tissues grossly involved with DLBCL, and then tracked using high-throughput sequencing of immunoglobulin genes (Faham et al 2012 Blood). ResultsWe successfully defined for 77% (30/39) of patients the tumor-specific immunoglobulin sequences required for non-invasive surveillance, with calibration success correlating with input tissue mass. Using 194 assays on 79 blood specimens from these patients, we detected disease in 75% of patients at diagnosis of DLBCL, in 18% at interim time-points during therapy, 0% during radiographic/clinical remissions, and 80% at relapse or disease progression. When considering radiographic/clinical follow-up, noninvasive monitoring had 0% false positive rate but 26% false negative rate, with the latter likely due to low tumor burden at time of phlebotomy. Disease burden detectable in the blood was significantly correlated between IgH and IgK assays, and between the cellular (leukocyte) and acellular (plasma) fractions (r=0.77, p=0.004). Strikingly, clonotypic IgH/IgK sequences were significantly more abundant in plasma than in circulating leukocytes, and identified exclusively in plasma in 5% of blood samples with available paired cellular/acellular fractions. This allowed more accurate disease detection and monitoring using plasma circulating tumor DNA than blood leukocytes, including in cases of radiographically defined isolated CNS involvement, and in histological transformation of FL. Within the limitations of this small study employing frequent surveillance imaging, we observed no significant lead time between detection of disease in the plasma and radiographic/clinical evidence of progression. ConclusionsNon-invasive surveillance of circulating tumor DNA has utility for tumor burden at diagnosis and relapse of DLBCL using high-throughput sequencing of immunoglobulin genes, with superior detection in blood plasma. The current assay has limited sensitivity at early interim time points during therapy. Disclosures:Kong:Sequenta Inc.: Employment. Faham:Sequenta, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Germinal Center Program De-Synchronization and Intra-Patient Heterogeneity in Follicular Lymphoma B-Cells Revealed By Integrative Single-Cell Analysis

IntroductionFollicular lymphoma (FL) is an indolent B-cell neoplasm with histological, phenotypical and transcriptional features of germinal center (GC) B-cells, suggesting that FL B-cells are “frozen” in a GC B-cell state. In normal GC B-cells, affinity maturation relies on iterative cycles of somatic hypermutation and proliferation followed by affinity-based selection. Thus GC B-cells are functionally heterogeneous. Although FL sub-clonal genetic diversity is well documented, tumor cell functional heterogeneity in FL has not been studied in detail. Bulk-level transcriptomic analyses fail to capture cellular heterogeneity and are thus insufficient to compare FL B-cells to the heterogeneous GC B-cell subset. Here, we used integrative single-cell analysis of phenotype, gene expression and IGH sequence to assess and compare functional heterogeneity in FL B-cells and GC B-cells.MethodsNormal B-cells were sorted from spleens or tonsil of healthy human donors (n=4), and FL B-cells from diagnostic lymph node biopsies (n=5), all available as frozen viable cell suspensions. Single normal or malignant B-cells were sorted by flow cytometry for parallel analysis of phenotype (9-color panel), gene expression (91-gene panel by microfluidic RT-qPCR), and IGH sequence (RT-PCR followed by Sanger sequencing; FL B-cells only). Custom and existing bioinformatics analysis pipelines were combined for quality control and cell filtering, dimensionality reduction (PCA, t-SNE), clustering, pseudo-time analysis, IGH sequence analysis and integrative data analysis.ResultsIn normal B-cells, single-cell gene expression profiling segregated major cell types (antibody-producing cells, memory B-cells and GC B-cells) and enabled the reconstruction of differentiation trajectories within cell types. Specifically, our data revealed a cyclic continuum of transitional GC B-cell states where synchronized expression of gene modules characterized human GC B-cell functional heterogeneity. Single-cell gene expression profiles defined FL B-cells as an independent cell type, distinct from GC B-cells. In particular, the expression of most characteristic GC-associated genes was de-synchronized in FL B-cells from all patients. Clustering of single FL B-cell 91-gene expression profiles revealed inter-patient and intra-patient heterogeneity, and defined common FL B-cell subsets found in most patients. Intra-patient heterogeneity was not linked to sub-clonal divergence as defined by IGH sequencing.ConclusionsUsing integrative single-cell analyses, we have shown that synchronized expression of characteristic gene modules drives GC B-cells through a cyclic continuum of transitional states. De-synchronization of that program in FL B-cells, and substantial intra-patient gene expression heterogeneity, suggest that FL B-cells are functionally plastic and not blocked to a GC B-cell state. DisclosuresNo relevant conflicts of interest to declare.

Role of clonoSEQ®, a Next-Generation Sequencing (NGS) Assay and PET/CT As a Measure of Minimal Residual Disease Negativity Among Patients with Multiple Myeloma

Role of clonoSEQ®, a Next-Generation Sequencing (NGS) Assay and PET/CT As a Measure of Minimal Residual Disease Negativity Among Patients with Multiple Myeloma

Real World Observational Study Using Clonoseq® Next Generation Sequencing in Hematologic Malignancies: The ’Watch’ Registry

Real World Observational Study Using Clonoseq® Next Generation Sequencing in Hematologic Malignancies: The ’Watch’ Registry

Validation and interpretation of IGH and TCR clonality testing by Ion Torrent S5 NGS for diagnosis and disease monitoring in B and T cell cancers

Cancers of B and T lymphocytes are the most common hematologic malignancies in the US. Molecular assays for assessing clonal rearrangements of the immunoglobulin receptor (IGH) and T-cell receptor (TCR), commonly referred to as B- and T-cell clonality, as well as determination of IGH somatic mutation status, enables improved diagnostic accuracy and disease monitoring. Here we describe validation of NGS LymphoTrack (IGH, TCRG, Invivoscribe, Inc) with Ion Torrent S5 sequencing, which employs a different sequencing chemistry and has not been previously reported for NGS clonality to our knowledge. We also demonstrate the concordance of clonality by LymphoTrack with S5 sequencing with other molecular methodologies and with clinical measurements of disease. We show that LymphoTrack with S5 sequencing identifies previously detected IGH and TCRG clonal sequences across matched biopsy specimens and clinical timepoints, enabling more precise and sensitive disease monitoring for B- and T-cell cancers compared to PCR fragment or capillary sequencing. In sum, our study demonstrates that the LymphoTrack assays with Ion Torrent S5 sequencing 1) can be used successfully for IGH and TCR clonality with reproducible results; 2) generates and quantifies clonal sequences to enable highly precise comparison of samples; 3) are substantially more sensitive than PCR fragment and return clonality results in specimens that failed PCR fragment assays; and 4) the TCRG assays are highly concordant with clinical and histopathologic diagnoses. Taken together, the LymphoTrack with Ion S5 NGS clonality assays offer a sensitive and precise method for diagnostic testing and disease monitoring in B- and T-cell cancers.

Abstract IA35: Follicular lymphoma dynamics through single-cell analysis

Abstract Follicular lymphoma (FL) is the second most common non-Hodgkin lymphoma in the Western world, generally characterized by a disseminated disease at diagnosis, an indolent clinical course, and recurrent, increasingly chemoresistant relapses. Overt FL is preceded by an insidious phase of asymptomatic growth and might emerge from common precursor clones, evolving over decades, and which might participate in subsequent relapses. Consequently, the cell of origin remains ambiguous. Although FL results from the malignant transformation of germinal center B cells, FL precursors emerge much earlier in B-cell ontogeny with the hallmark t(14;18) translocation and ensuing constitutive expression of BCL2 in bone marrow pre-B cells. While t(14;18) is considered the necessary early first hit to transformation, BCL2 as such is a very weak driver, as evidenced by the detection of t(14;18) in peripheral blood from a large fraction of the adult healthy population and the long latency and low penetrance observed in various BCL2 mouse models. Recent data of clonal dynamics in human and mouse models strongly suggest that additional illegitimate events likely accumulate over iterative GC passages, generating a highly mutagenic dynamics linked with slow oncogenic progression. Combined with the power of next-generation sequencing, molecular interrogations of t(14;18)+ clones in healthy individuals and FL patients recently provided the extent of FL genetic diversity in space (distinct tissues) and time (diagnosis vs. relapse). Thus, the path to transformation appears as a complex multi-hit process occurring along B-cell ontogeny, escalating along successive derailments of B-cell receptor diversification mechanisms, and subversion of specific immunologic properties of B cells. This protracted clonal evolution generates a massive accumulation of subclones and an associated complex overall genetic heterogeneity. This raises several questions. While FL originate from germinal center B cells, it is unclear to what extent overt lymphoma B cells retain GC B-cell functional dynamics or are blocked in a particular stage of the GC reaction. Furthermore, although one might intuitively assume that distinct subclones likely have a different gene expression profile associated to specific functions (and potentially driving distinct capacity to respond to therapy), it is still unclear to which extent subclonal genetic heterogeneity imprints functional heterogeneity. Because subclonal heterogeneity is hidden in bulk, where all distinct subclones are mixed and averaged, we recently used integrative single-cell analysis of phenotype, gene expression, and IGH sequence to track the characteristic human GC B-cell program in FL B cells. We used pseudo-time approaches to model the cyclic continuum of GC B-cell transitional states and identified characteristic patterns of synchronously expressed gene clusters in GC B cells. Strikingly this GC-specific gene expression synchrony was lost in single lymphoma B cells. Yet, distinct and conserved FL-specific cell states co-existed within single patient biopsies. Our data show that lymphoma B cells are not blocked in a GC B-cell state but may adopt new dynamic modes of functional diversity, opening novel definitions of lymphoma identity. Citation Format: Bertrand Nadel. Follicular lymphoma dynamics through single-cell analysis [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr IA35.

Abstract 3305: Comparative analysis of RNA versus DNA as input material for IGH repertoire sequencing based detection of rare clonal B cells at a frequency of 10E-6

Abstract Background: Next generation sequencing of rearranged IGH chains has emerged as a reproducible and highly sensitive approach for detecting rare B cell clones, e.g. malignant B cell, in peripheral blood. Historically, efforts to evaluate the frequency of rare B cells by IGH chain sequencing have utilized DNA input given potential challenges in accurately quantifying template copy number from RNA data owing to B cell subtype specific variation in the expression of the B cell receptor. Hypothetically, however, RNA input based monitoring could be advantageous both owing to reduced input requirements and superior ability to detect B cell malignancies of plasmablast and plasma cell origin, where the BCR is robustly expressed. Here we compared the ability of RNA and DNA based IGH chain sequencing to detect two Burkitt's Lymphoma B cell lines (CA46 and GA10) at a frequency of 10E-6 from peripheral blood. We discuss advantages of either approach for detection of rare B cell clones. Methods: IGH chain libraries were prepared using the Oncomine IGH-SR assay (framework 3 and joining gene multiplex primers) from gDNA or total RNA extracted from peripheral blood and spiked with Burkitt's lymphoma or CLL cell line gDNA or total RNA to a frequency of 10E-6 by mass ratio. Libraries were sequenced via the Gene Studio S5 followed by Ion Reporter analysis to identify clonotypes and evaluate B cell clone frequencies across samples. Automated downsampling analysis was used to confirm libraries were sequenced to saturation. Library preparation and analysis was performed in replicate to quantify sensitivity of detection. Results: For each cell line, we prepared and sequenced (1) 30 libraries derived from amplification of 2ug gDNA spiked with 2pg cell line gDNA and (2) 10 libraries derived from amplification of 100ng RNA spiked with .1pg cell line total RNA. The Burkitt's lymphoma cell lines were detected in 10/30 and 8/30 gDNA libraries respectively, for CA46 and GA-10) consistent with the historical performance of orthologous DNA-based sequencing approaches. For RNA libraries, the Burkitt's lymphoma cell lines were detected in each library (10/10 and 10/10, respectively). Conclusions: Here we demonstrate detection of B cell lines at a frequency down to 10E-6 from DNA and RNA input. Importantly, we find that RNA based IGH sequencing may significantly reduce input requirements for rare clone detection, potentially enabling routine detection of clones down to 10E-6 frequency from a single library. Citation Format: Michelle Toro, Loni Pickle, Jayde Chang, Timothy Looney, Geoffrey Lowman, Mark Andersen, Fiona Hyland. Comparative analysis of RNA versus DNA as input material for IGH repertoire sequencing based detection of rare clonal B cells at a frequency of 10E-6 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3305.

Analytical evaluation of the clonoSEQ Assay for establishing measurable (minimal) residual disease in acute lymphoblastic leukemia, chronic lymphocytic leukemia, and multiple myeloma

BackgroundThe clonoSEQ® Assay (Adaptive Biotechnologies Corporation, Seattle, USA) identifies and tracks unique disease-associated immunoglobulin (Ig) sequences by next-generation sequencing of IgH, IgK, and IgL rearrangements and IgH-BCL1/2 translocations in malignant B cells. Here, we describe studies to validate the analytical performance of the assay using patient samples and cell lines.MethodsSensitivity and specificity were established by defining the limit of detection (LoD), limit of quantitation (LoQ) and limit of blank (LoB) in genomic DNA (gDNA) from 66 patients with multiple myeloma (MM), acute lymphoblastic leukemia (ALL), or chronic lymphocytic leukemia (CLL), and three cell lines. Healthy donor gDNA was used as a diluent to contrive samples with specific DNA masses and malignant-cell frequencies. Precision was validated using a range of samples contrived from patient gDNA, healthy donor gDNA, and 9 cell lines to generate measurable residual disease (MRD) frequencies spanning clinically relevant thresholds. Linearity was determined using samples contrived from cell line gDNA spiked into healthy gDNA to generate 11 MRD frequencies for each DNA input, then confirmed using clinical samples. Quantitation accuracy was assessed by (1) comparing clonoSEQ and multiparametric flow cytometry (mpFC) measurements of ALL and MM cell lines diluted in healthy mononuclear cells, and (2) analyzing precision study data for bias between clonoSEQ MRD results in diluted gDNA and those expected from mpFC based on original, undiluted samples. Repeatability of nucleotide base calls was assessed via the assay’s ability to recover malignant clonotype sequences across several replicates, process features, and MRD levels.ResultsLoD and LoQ were estimated at 1.903 cells and 2.390 malignant cells, respectively. LoB was zero in healthy donor gDNA. Precision ranged from 18% CV (coefficient of variation) at higher DNA inputs to 68% CV near the LoD. Variance component analysis showed MRD results were robust, with expected laboratory process variations contributing ≤3% CV. Linearity and accuracy were demonstrated for each disease across orders of magnitude of clonal frequencies. Nucleotide sequence error rates were extremely low.ConclusionsThese studies validate the analytical performance of the clonoSEQ Assay and demonstrate its potential as a highly sensitive diagnostic tool for selected lymphoid malignancies.