IGK Rearrangements Research Articles

Monitoring measurable residual disease in paediatric acute lymphoblastic leukaemia using immunoglobulin gene clonality based on next-generation sequencing

BackgroundAssessment of measurable residual disease (MRD) is an essential prognostic tool for B-lymphoblastic leukaemia (B-ALL). In this study, we evaluated the utility of next-generation sequencing (NGS)–based MRD assessment in real-world clinical practice.MethodThe study included 93 paediatric patients with B-ALL treated at our institution between January 2017 and June 2022. Clonality for IGH or IGK rearrangements was identified in most bone marrow samples (91/93, 97.8%) obtained at diagnosis.ResultsIn 421 monitoring samples, concordance was 74.8% between NGS and multiparameter flow cytometry and 70.7% between NGS and reverse transcription-PCR. Elevated quantities of clones of IGH alone (P < 0.001; hazard ratio [HR], 22.2; 95% confidence interval [CI], 7.1–69.1), IGK alone (P = 0.011; HR, 5.8; 95% CI, 1.5–22.5), and IGH or IGK (P < 0.001; HR, 7.2; 95% CI, 2.6–20.0) were associated with an increased risk of relapse. Detection of new clone(s) in NGS was also associated with inferior relapse-free survival (P < 0.001; HR, 18.1; 95% CI, 3.0–108.6). Multivariable analysis confirmed age at diagnosis, BCR::ABL1-like mutation, TCF3::PBX1 mutation, and increased quantity of IGH or IGK clones during monitoring as unfavourable factors.ConclusionIn conclusion, this study highlights the usefulness of NGS-based MRD as a routine assessment tool for prognostication of paediatric patients with B-ALL.

Studies on clinicopathological features of duodenal-type follicular lymphoma of 18 patients

To investigate the clinical and pathological characteristics of duodenal-type follicular lymphoma (D-FL), and to deepen the understanding of Duodenal-type follicular lymphoma. The clinical symptoms, endoscopic features, pathologic features, immunophenotype, molecular pathological features and treatment follow-up of 18 D-FL patients diagnosed in Department of Pathology, Beijing Tiantan Hospital affiliated to Capital Medical University between January 2020 and July 2023 were summarized. A total of 18 patients with D-FL were included, including 10 males and 8 females. The median age was 49 (32-69) years respectively. Most of the patients were found during gastroenteroscopy or presented with the common gastrointestinal symptoms of stomach pain, acid reflux, vomiting and diarrhea. Most endoscopic findings were multiple small gray and white polyposis. In the pathological morphology, the mucous layer and submucous layer showed lymphoid follicular structures with full and strained follicles. The immunophenotype showed that the tumor cells strongly expressed CD20 and BCL2 and had low proliferation activity. Immunoglobulin clonal analysis of 1 case showed IgK monoclonal rearrangement (1/1). FISH showed 1 case of BCL2 gene rearrangement (1/3). All patients did not receive targeted chemotherapy and adopted a wait-and-see strategy. Median follow-up was 12 (2-34) months. This study shows that D-FL is an indolent lymphoma, which tends to occur in the duodenum and has a good prognosis.

Optimal Rituximab Monotherapy in Splenic Marginal Zone Lymphoma (SMZL): A Case Report and Brief Review

Introduction:: Splenic marginal zone Lymphoma (SMZL) is a rare, chronic B lymphocyte proliferative disease. Generally, SMZL is accompanied by circulating atypical villous lymphocytes, known as SMZL with villous lymphocytes. Rituximab is a chimeric monoclonal antibody to CD20; recent but limited studies have confirmed its effectiveness in treating SMZL. Given the low incidence and selection of treatment, statistical comparisons of rituximab monotherapy with other available treatment options with the full range of data from previous clinical studies remain sparse. Here, we report a case of SMZL with villous lymphocytes treated by rituximab monotherapy, which is especially infrequently reported. Case Report:: A 63-year-old Chinese female was presented to the hospital with complaints of splenomegaly and pain in the spleen area. Immunohistochemistry analysis was positive for IGH, IGK, and IGL clonal rearrangement. Villous lymphocytes were found in peripheral blood and bone marrow, along with further immunotyping results. The case was considered as SMZL with villous lymphocytes. Based on the SMZLSG prognosis assessment, we applied rituximab monotherapy. After eight cycles of rituximab treatment, the patient’s condition improved markedly, with blood constituent and size of the spleen returning to normal levels, achieving complete response, with no significant side effect observed. Discussion:: The patient provides a typical SMZL with villous lymphocytes case treated with rituximab monotherapy. Currently, the main treatment options include splenectomy and rituximab. After synthesizing a series of current views, we put forward our opinion about the selection of therapy for SMZL patients in order to gain maximum benefits for patients in need of treatment. Conclusion:: Our analysis found no statistically significant difference between rituximab monotherapy and rituximab combined with chemotherapy, while rituximab treatments resulted in better therapeutic effects than chemotherapy. Rituximab monotherapy has favorable therapeutic effects and minor adverse effects (AEs) in treating SMZL.

Molecular pathology assists the diagnosis of lymphoepithelial sialadenitis, Sjögren's syndrome and extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue

Background/purposeLymphoepithelial sialadenitis (LESA), Sjögren's syndrome (SS), and salivary MALT lymphoma are diseases characterized by lymphoepithelial lesions, and the differential diagnosis between them in the salivary glands is challenging. This study aimed to explore clinicopathological and genetic characteristics of the three diseases. Materials and methodsWe retrospectively analyzed the clinical features, the histomorphology, immunohistochemistry, and genetic profiling by polymerase chain reaction (PCR) and next-generation sequencing (NGS). ResultsThere included 68 LESAs, 25 SSs, and 62 MALT lymphomas. Ten cases relapsed in total, and 3 of MALT lymphomas died due to high-level transformation. Immunohistochemical double staining showed FCRL4 cells co-expressed Pax-5 and Ki-67, suggesting FCRL4 cells were proliferative B-cells. The expression level of the FCRL4 was significantly higher in MALT lymphoma than LESA and SS. The detection rates of clonal IGH, IGK, and IGL gene rearrangements in MALT lymphoma with a sensitivity of 83.33%. Monoclonal immunoglobulin gene rearrangements were confirmed in five suspected patients by NGS (100%). ConclusionFCRL4 B cells might play an important role in the formation of lymphoepithelial lesions and might be as a diagnostic positive marker of salivary MALT lymphoma. The application of multiple detection methods could significantly improve the diagnostic accuracy for MALT lymphomas from LESA and SS.

Nemo-Dependent, ATM-Mediated Signals from RAG DNA Breaks at Igk Feedback Inhibit V κ Recombination to Enforce Igκ Allelic Exclusion.

Monoallelic AgR gene expression underlies specific adaptive immune responses. AgR allelic exclusion is achieved by sequential initiation of V(D)J recombination between alleles and resultant protein from one allele signaling to prevent recombination of the other. The ATM kinase, a regulator of the DNA double-strand break (DSB) response, helps enforce allelic exclusion through undetermined mechanisms. ATM promotes repair of RAG1/RAG2 (RAG) endonuclease-induced DSBs and transduces signals from RAG DSBs during Igk gene rearrangement on one allele to transiently inhibit RAG1 protein expression, Igk accessibility, and RAG cleavage of the other allele. Yet, the relative contributions of ATM functions in DSB repair versus signaling to enforce AgR allelic exclusion remain undetermined. In this study, we demonstrate that inactivation in mouse pre-B cells of the NF-κB essential modulator (Nemo) protein, an effector of ATM signaling, diminishes RAG DSB-triggered repression of Rag1/Rag2 transcription and Igk accessibility but does not result in aberrant repair of RAG DSBs like ATM inactivation. We show that Nemo deficiency increases simultaneous biallelic Igk cleavage in pre-B cells and raises the frequency of B cells expressing Igκ proteins from both alleles. In contrast, the incidence of biallelic Igκ expression is not elevated by inactivation of the SpiC transcriptional repressor, which is induced by RAG DSBs in an ATM-dependent manner and suppresses Igk accessibility. Thus, we conclude that Nemo-dependent, ATM-mediated DNA damage signals enforce Igκ allelic exclusion by orchestrating transient repression of RAG expression and feedback inhibition of additional Igk rearrangements in response to RAG cleavage on one Igk allele.

One-Step Next-Generation Sequencing of Immunoglobulin and T-Cell Receptor Gene Recombinations for MRD Marker Identification in Acute Lymphoblastic Leukemia.

Within the EuroClonality-NGS group, immune repertoire analysis for target identification in lymphoid malignancies was initially developed using two-stage amplicon approaches, essentially as a progressive modification of preceding methods developed for Sanger sequencing. This approach has, however, limitations with respect to sample handling, adaptation to automation, and risk of contamination by amplicon products. We therefore developed one-step PCR amplicon methods with individual barcoding for batched analysis for IGH, IGK, TRD, TRG, and TRB rearrangements, followed by Vidjil-based data analysis.

Validation of Next Generation Sequencing-Based Clonality Assays for Diagnosis and Minimal Residual Disease Tracking in Lymphoid Malignancies

Introduction:B or T cell receptor (BCR/TCR) clonal rearrangements have served as important diagnostic markers and minimal residual disease (MRD) tracking markers to guide treatment decisions in lymphoid malignancies. Next generation sequencing (NGS)-based BCR/TCR clonality assessment allows easier sample preparation, higher sensitivity and simpler standardization than flow cytometry and polymerase chain reaction (PCR)-based assays. Here, we developed NGS-based BCR/TCR clonality assays to identify and track disease-associated clonotypes of IGH, IGK, IGL, TCRB and TCRG rearrangements and BCL1/2-IGH translocations in lymphoid malignant cells. Our studies validated the analytical performance of the assays using genomic DNA (gDNA) from cell lines and patient samples diagnosed with acute lymphoblastic leukemia (ALL), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), lymphoma and lymphoid blast phase chronic myeloid leukemia (BP-CML), as well as peripheral blood gDNA and circulating tumor DNA (ctDNA) from lymphoma patients.Methods:Our BCR/TCR clonality assays were based on two rounds of multiplex PCR followed by NGS. In the first-round PCR, the sequences of complementary determining region 3 (CDR3) in rearranged immune receptor genes were amplified by multiplex primers, and sample-specific index and NGS adapters were then added in the second-round PCR. Sequencing was performed on NovaSeq 6000 System and processed with customized bioinformatics pipelines. BCR clonality assays could identify IGH (V H-D H-J H or D H-J H), IGK (V κ-J κ, V κ-Kde and intronRSS-Kde) and IGL (V λ-J λ) rearrangements, as well as BCL1-IGH and BCL2-IGH translocations. TCR clonality assays could detect TCRB (V β-D β-J β) and TCRG (V γ-J γ) rearrangements. To evaluate the performance of our assays, we detected BCR or TCR clonality in gDNA from 4 cell lines and 40 clinical samples with ALL, MM, CLL and lymphoma, as well as paired chronic- and blast-phase samples of CML. Limit-of-detection (LOD) was estimated by clinical samples and cell lines with a background of peripheral blood gDNA from healthy donors. Linearity of detection was established with gDNA of cell lines spiked into normal gDNA to generate across orders of magnitude of clonal frequencies. To ensure consistent performance of the assays, we tested separate reactions and a single mixed reaction for IGH VDJ, IGH DJ, IGK and IGL. In addition, peripheral blood gDNA matching aforementioned lymphoma cases and ctDNA samples were also tested by our assays.Results:Both BCR clonality of B-cell lymphoid malignancies (B-ALL, MM, CLL and lymphoma) and TCR clonality of T-cell lymphoid malignancies (T-ALL and lymphoma) showed above 90% positive detection rate and mostly positive in more than one receptor gene. Chronic and blast phase samples from the same CML patient showed an identical dominant clonotype. The assays had high sensitivity, with LoD defined between 1 to 2 malignant cells in both BCR and TCR clonality assessment. Linearity was observed with clonal frequencies from 1 to 10 -6, which indicated consistence between observed and expected frequencies. The sequencing results with a single adjusted mixed reaction for IGH VDJ, IGH DJ, IGK and IGL were comparable to that with separate reactions, suitable for both diagnosed samples and MRD samples, suggesting the robustness of the assays. Our testing results also showed that peripheral blood gDNA of lymphoma patients carried identical clonotypes found in malignant tissues and ctDNA.Conclusion:We characterized and validated the performance of our NGS -based BCR/TCR clonality assays. We also demonstrated its potential application as a highly sensitive tool for diagnosis and MRD tracking for lymphoid malignancies, including ALL, MM, CLL, lymphoma and even CML at risk of lymphoid blast transformation. DisclosuresNo relevant conflicts of interest to declare.

Immune Repertoire Analysis of Multiple Myeloma Research Samples Using NGS Characterization of Multiple B Cell Receptors in a Single Reaction

IntroductionB cell repertoire analysis by next-generation sequencing (NGS) is at the forefront of leukemia and lymphoma research. Some advantages provided by NGS-based techniques include a lower limit-of-detection and simpler paths to standardization compared to other methods. Importantly, in research of post-germinal B cell disorders, such as multiple myeloma (MM), NGS methods allow for the study of clonal lineage based on somatic hypermuation patterns. Current targeted NGS assays require multiple libraries to survey each B cell receptor chain (IGH, IgK, IgL), and this fact is highlighted when initial clonality detection fails due to mutations under primer binding sites. This issue can be especially true in MM which has a high rate of SHM. To address these issues, we have developed an assay for B cell analysis, based on Ion AmpliSeq™ technology, which enables efficient detection of IGH, IgK, and IgL chain rearrangements in a single reaction.MethodsThe B cell pan-clonality panel (Oncomine™ BCR Pan-Clonality Assay) targets the framework 3 (FR3) portion of the variable gene and the joining gene region of heavy- and light-chain loci (IGH, IgK, IgL) for all alleles found within the IMGT database, enabling readout of the complementary-determining region 3 (CDR3) sequence of each immunoglobulin chain. To maximize sensitivity, we included primers to amplify IgK loci rearrangements involving Kappa deletion element and the constant region intron. To evaluate assay performance, we conducted reproducibility studies and clonality assessment using gDNA from a total of 45 MM research samples. All MM cases examined in this work were confirmed clonal previously by light chain restriction via flow cytometry or IHC/ISH in tissue sections - 16 of the 45 MM samples were identified as lambda light chain restricted. For comparison, a small cohort of 12 B-ALL samples were also included in the study. Sequencing and repertoire analyses were performed using the Ion GeneStudio S5 System and Ion Reporter 5.16 analysis software.ResultsClonality assessment of MM clinical research samples show an 93% overall positive detection rate by an assay which combines the IGH, IgK, and IgL chains in a single reaction using published guidelines for clonality assignment. Thirty-four of 45 samples show positive detection of an IGH rearrangement, while 41 of 45 showed positive detection of at least one light chain receptor. In total, 42 of 45 samples were deemed clonal by the single tube assay based on detection for one or more receptor. Clonality results for this sample set are well correlated with orthogonal data from flow, IHC/ISH, or alternate NGS assays. A clonal lambda light chain was identified in 14 of 16 samples determined to be lambda restricted by flow cytometry. In two of the lambda restricted samples only a clonal lambda rearrangement was identified, showing the benefit of including primers targeting both the kappa and lambda light chains in a pan-clonality NGS assay. Both the MM and B-ALL cohorts were evaluated for biased IGHV gene usage. IGHV3-11 was observed in 5 of 45 MM and 5 of 12 B-ALL samples. IGHV4-34, typically linked to autoreactive antibodies and underrepresented in germinal center and memory B-cells, was nonetheless found in 5 of 45 MM samples surveyed. Estimates of somatic hypermutation rates were calculated using the BCR pan-clonality assay. Most MM samples, as expected, contained some somatic hypermutation with 6 of 45 samples showing greater than 10% mutation rates. Automated lineage analysis, based on somatic hypermuation signatures within each sample, identified 8 of 45 MM samples which contained 5 or more clones in the primary clonal lineage, with one case containing a lineage with 23 clones. Two MM samples showed no somatic hypermutation as measured using the FR3 primers contained in the BCR pan-clonality assay. These samples were also evaluated using an FR1-J targeted NGS assay, which confirmed relatively low mutation rates for these MM samples at 0.44% and 1.3%, respectively.ConclusionsThese results demonstrate the utility of a novel assay for combined repertoire analysis of B cell receptor heavy and light chains in a single library preparation reaction. We expect this assay to simplify laboratory workflows and including analysis tools such as automated somatic hypermutation rate calculation and clonal lineage identification may open new paths for research in lymphoid cell disorders.For research use only. DisclosuresLowman: Thermo Fisher Scientific: Current Employment. Toro: Thermo Fisher Scientific: Current Employment. Pickle: Thermo Fisher Scientific: Current Employment. Ostresh: Thermo Fisher Scientific: Current Employment. Sarda: Thermo Fisher Scientific: Current Employment. Yang: Thermo Fisher Scientific: Current Employment.

FISH-Guided Evaluation of Hyperdiploidy and Other Cytogenetic Abnormalities in Childhood Burkitt Lymphoma

Background: Burkitt lymphoma (BL) is the most common lymphoma in childhood. Apart from MYC rearrangement, considered the hallmark of the disease, BL often presents with additional chromosome aberrations, the biological and clinical significance of which is not fully understood. Materials and methods: The study included 72 children (55 boys, 17 girls), aged 2-16 years (median 9), with BL diagnosed on the basis of histopathology and a demonstrable MYC rearrangement. Touch imprints from the diagnostic biopsy samples were investigated with i-FISH for MYC, BCL2, BCL6, IGH, IGK and IGL rearrangements genes and copy number aberrations involving 1q21/1p32, 7cen/7q31, 9cen/9p21, 13q14/13q34 and 17cen/17p13. Deviations from the diploid status were further investigated for aneusomies of the carrier chromosome with the use of appropriate chromosome enumeration probes. Results: MYC gene was demonstrated in all cases with MYC/IGH fusion in 83.3% (60/72). 47 cases (65.3%) were found with least one additional aberration, most commonly with 1q gain (29.2%), 7q gain (19.4%), 13q deletion (19.4%), hemizygous 9p loss (8.3%) and hyperdiploidy (8.3%). Advanced clinical stage (IV), 7q gain (but not trisomy 7) and -9/9p- were significantly associated with shorter overall survival. There was no instance of relapse or death among the hyperdiploid cases. Conclusions: This extensive FISH investigation on imprints of affected tissue provides clinically meaningful information on the genetic profile of BL and may prove valuable in the management of patients with no karyotype available. In particular, the demonstration of hyperdiploidy through the use of chromosome enumeration probes could offer the means for the delineation of clonal evolution pathways and the recognition of a subgroup of children with excellent prognosis who could be cured with low-intensity chemotherapy regimens. DisclosuresKattamis: VIFOR: Consultancy; CRISPR/Vertex: Consultancy, Honoraria; Agios Pharmaceuticals: Consultancy; IONIS: Consultancy; BMS/Celgene: Consultancy, Honoraria, Research Funding; Chiesi: Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Amgen: Consultancy.

Fish evaluation of additional cytogenetic aberrations and hyperdiploidy in childhood Burkitt lymphoma

Beyond MYC rearrangement, Burkitt lymphoma (BL) often presents with additional aberrations. Biopsy touch imprints from 72 children with BL were tested with interphase fluorescence in-situ hybridization (i-FISH) for MYC, BCL2, BCL6, IGH, IGK and IGL rearrangements and copy-number aberrations involving 1q21/1p32, 7cen/7q31, 9cen/9p21, 13q14/13q34 and 17cen/17p13. Diploid status deviations were investigated with chromosome enumeration probes. MYC rearrangement was demonstrated in all cases. Additional aberrations included +1q (21/72:29.2%), +7q (14/72:19.4%), 13q- (14/72:19.4%), 9p-(6/72:8.3%) and hyperdiploidy (6/72:8.3%). Advanced clinical stage IV, +7q and 9p- were associated with shorter overall survival, with stage IV and +7q retaining prognostic significance on multivariate analysis. No relapse or death was reported among the hyperdiploid cases. This i-FISH investigation provides information on the genetic profile of BL and may prove valuable for patients with no karyotype analysis. Demonstration of hyperdiploidy could evolve research on clonal evolution pathways and probably identify a subgroup of children with favorable prognosis.

Assessing the Utility of Immunoglobulin and T Cell Receptor Gene Rearrangement Studies for Cerebrospinal Fluid Specimens

INTRODUCTION : Immunoglobulin (IGH and IGK) and T cell receptor (TCRG) rearrangements (-R) by polymerase chain reaction (PCR) have emerged as important ancillary tools for assessing clonality of B-cell and T-cell processes, respectively, in specimens such as lymph node, bone marrow and peripheral blood. However, the utility of these gene rearrangement studies in cerebrospinal fluid (CSF) samples has not yet been systematically examined. Cytomorphology and flow cytometry (FC) are currently the standard tools for the assessment of the CSF for leptomeningeal involvement by hematological malignancies. There are inherent limitations to both of these diagnostic methods; thus, it is important to establish the role of IGH/IGK- R and TCRG- R studies in the examination of CSF.METHODS : Pathology reports of 87 consecutive CSF specimens from 61 patients submitted for IGH, IGK and/or TCRG -R studies in the past three years (2015-2017) were retrospectively reviewed. PCR for IGH (using FR3 and JH primers), IGK and TCRG rearrangements was performed using the BIOMED2 assays. The PCR products for all three reactions were analyzed via polyacrylamide gel or capillary electrophoresis. The interpretation was rendered by an attending molecular pathologist. These results were assessed in the context of the corresponding flow cytometry report.RESULTS : Majority of the patients (82% of patients) had a prior diagnosis of a hematologic malignancy (Table 1), including diffuse large B-cell lymphoma (31% of specimens) and acute leukemias (17%), while the remaining patients presented with CNS symptoms with/without a brain mass. Nearly equal numbers of cases were requested by the clinicians concurrently with flow cytometry (46 cases; 53%) or by the hematopathologist evaluating the CSF FC results (41 cases; 47%). In the latter group, PCR was requested due to an atypical finding based on CSF flow cytometry, which most commonly was an abnormal population with an immunophenotype was different from the diagnostic specimen or a suspicious population that was too small to be diagnostic.Of the 61 IGH- R and 35 IGK -R tests requested, the majority of the specimens (74% and 63%, respectively) demonstrated a pseudoclonal pattern or poor amplification because of the paucity of B cells. Of the cases with sufficient cellular material, a monoclonal population was detected by IGH- R and IGK -R in more than half of the cases (56% and 54%, respectively). In contrast, in 75% (24/32) of the cases where TCRG-R was requested, there was an adequate number of T-cells for analysis; of these 24 informative cases, 9 (38%) were monoclonal and 15 (62%) were polyclonal.Overall, 37 of the 87 cases (43%) yielded an informative result for one or more of IGH, IGK, and/or TCRG -R. 38% (14/37) of these cases showed a result that clarified or supported an atypical/suspicious finding by FC, whether submitted by the pathologist (70%) or treating clinician (30%). Eight of the 37 cases (22%) were submitted by the pathologist to confirm the FC findings. In 7 of these 8 cases, the PCR findings corroborated the FC results; there was one discordant case in which a polyclonal T-cell population was seen in a positive FC case of T-lymphoblastic lymphoma/leukemia. Remainder of the cases (14/37, 38%) were submitted by the treating clinicians; if they were submitted together with a concurrent FC analysis (12 of 14 cases), the PCR results confirmed unambiguous FC results (whether negative or positive). In 6 of the 14 cases in which a IGH -R and/or IGK -R identified a monoclonal population, the atypical or neoplastic population constituted <5% of the analyzed cells by FC.CONCLUSIONS : The paucicellular nature of the CSF specimens provides a challenge not only for cytomorphology and FC but also for PCR for IGH, IGK, and TCRG rearrangements. Nonetheless, an informative clonality result was observed in 43% of the cases submitted for PCR analysis. The results of 60% of these cases (26% of the total submitted cases) were helpful for clarifying and/or supporting an FC finding. Thus, PCR for IGH, IGK and TCRG -R of CSF specimens is a useful ancillary diagnostic aid and can help in the interpretation and confirmation of flow cytometry results, particularly when the neoplastic lymphoid cells are of low abundance or exhibit an immunophenotype that is distinct from the diagnostic tumor. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Multiple Immunoglobulin κ Gene Rearrangements within a Single Clone Unraveled by Next-Generation Sequencing–Based Clonality Assessment

Clonality assessment of the Ig heavy- and light-chain genes (IGH and IGK) using GeneScan analysis is an important supplemental assay in diagnostic testing for lymphoma. Occasionally cases with an IGK rearrangement pattern that cannot readily be assigned to a monoclonal lymphoma are encountered, whereas the occurrence of biclonal lymphomas is rare, and the result of the IGH locus of these cases is in line with monoclonality. Three such ambiguous cases were assessed for clonality using next-generation sequencing. Information on the sequences of the rearrangements, combined with knowledge of the complex organization of the IGK locus, pointed to two explanations that can attribute seemingly biclonal IGK rearrangements to a single clone. In two cases, this explanation involved inversion rearrangements on the IGK locus, whereas in the third case, the cross-reactivity of primers generated an additional clonal product. In conclusion, next-generation sequencing-based clonality assessment allows for the detection of both inversion rearrangements and the cross-reactivity of primers, and can therefore facilitate the interpretation of cases of lymphoma with complex IGK rearrangement patterns.

In Vivo and Ex Vivo Patient-Derived Tumor Xenograft Models of Lymphoma for Drug Discovery.

In the hemato-oncology field, remarkable scientific progress has been achieved, primarily propelled by the discovery of new technologies, improvement in genomics, and novel in vitro and in vivo models. The establishment of multiple cell line collections and the development of instrumental mouse models enhanced our ability to discover effective therapeutics. However, cancer models that faithfully mimic individual cancers are still imperfect. Patient-derived tumor xenografts (PDTXs) have emerged as a powerful tool for identifying the mechanisms which drive tumorigenesis and for testing potential therapeutic interventions. The recognition that PDTXs can maintain many of the donor samples' properties enabled the development of new strategies for discovering and implementing therapies. Described in this article are protocols for the generation and characterization of lymphoma PDTXs that may be used as the basis of shared procedures. Universal protocols will foster the model utilization, enable the integration of public and private repositories, and aid in the development of shared platforms. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Tissue handling and cryopreservation of primary and PDTX samples Basic Protocol 2: Performing tumor implant in immunocompromised mice PDTX models Alternate Protocol 1: Intra-medullary femoral injection Alternate Protocol 2: Intravenous injection Alternate Protocol 3: Intraperitoneal injection Support Protocol 1: Phenotypical characterization of PDTXs by flow cytometry Support Protocol 2: Biological and molecular characterization of PDTX tumors by PCR detection of IGK, IGH, and TCR rearrangements Basic Protocol 3: Harvesting PDTX-derived tumor cells for ex vivo experiments Basic Protocol 4: In vivo testing of multiple compounds in a PDTX mouse model.

Characterization of the Immunoglobulin Heavy- and Light-Chain Repertoires in a Single Reaction

Background B cell repertoire analysis by next-generation sequencing (NGS) has shown particular utility in the field of hematological oncology research. Some advantages provided by NGS-based techniques include a lower limit-of-detection and simpler paths to standardization compared to flow-based methods, and the elimination of specifically designed primers often required for qPCR-based methods. Owing to primer-primer interactions and incompatibility of reaction conditions, current multiplex PCR assays require separate PCR reactions to survey each immunoglobulin chain (IGH, IGK, IGL), often leading to a longer time-to-answer for samples in which no marker is initially detected. We have developed an assay for receptor analysis based on Ion AmpliSeq technology to circumvent these issues, allowing the effective use of up to thousands of primers in a single reaction. The highly multiplexed, pan-clonality NGS assay provides for efficient detection of IGH, IGK, and IGL chain rearrangements in a single reaction. Methods We developed a single primer panel targeting the framework 3 (FR3) portion of the variable gene and the joining gene region of heavy- and light-chain loci (IGH, IGK, IGL) for all alleles found within the IMGT database, enabling readout of the complementary-determining region 3 (CDR3) sequence of each immunoglobulin chain. To maximize sensitivity, we included primers to amplify IGK loci rearrangements involving Kappa deletion and C intron elements. To evaluate performance, we conducted clonality assessment and limit-of-detection testing used gDNA from a total of 45 research samples representing common B cell malignancies. We included samples derived from peripheral blood, bone marrow, and FFPE-preserved tissues at input levels ranging from 100ng to 2µg. Finally, we further characterized the samples via a separate AmpliSeq-based multiplex PCR assay targeting rearranged TCRB and TCRG chains. Sequencing and clonality analysis was performed using the Ion GeneStudio S5 System and Ion Reporter 5.16. Results Clonality assessments carried out using gDNA collected from both cell line and clinical research samples (CLL, B-ALL, Multiple Myeloma, Burkitt's Lymphoma, NHL, and DLBCL) show a &amp;gt;90% overall positive detection rate. Assessment of linearity-of-response and limit-of-detection was carried out using cell lines diluted in PBL to between 10-3 and 10-6 by mass. The multi-receptor assay performs as expected, with linear response to the cell line frequency across the range tested, including the ability to detect clones of interest at 10-6. Conclusions These results demonstrate the robustness of our newly developed Ion AmpliSeq-assay for B cell receptor heavy and light chains. We expect this assay to simplify the workflow for clonality assessment and rare clone detection in B cell malignancy research. For research use only. Disclosures No relevant conflicts of interest to declare.

Improving the Definition of Response Assessment: Prognostic Value of Minimal Residual Disease Combined with PET/CT at Day 100 Post Autologous Stem Cell Transplantation in Multiple Myeloma

Introduction: Response assessment at day 100 post Autologous Stem Cell Transplant (ASCT) is associated with long-term relapsed free survival (RFS) and overall survival (OS) in multiple myeloma (MM). The International Myeloma Working Group (IMWG) are the preferred criteria to define best response to treatment and define relapse. In the last years, response assessment has incorporated minimal residual disease (MRD) status -associated with improved RFS and OS (Munshi et al); and PET/CT combined with clinical characteristics -also associated with favorable outcomes (Zamagni et al. NCT01910987; MMY3033). The 2016 IMWG MRD criteria, combined imaging (PET/CT) plus next-generation sequencing (NGS) MRD-negative to define complete response (CR). To our knowledge, there is limited data examining the correlation and prognostic value of MRD and FDG-PET/CT at day 100 post ASCT in MM. IN this study, we aimed to determine the prognostic valued of MRD by NGS combined with PET/CT in RFS and OS status after high dose chemotherapy and ASCT in MM. Methods: Patients who underwent ASCT for MM at Mayo Clinic Arizona and had MRD and PET/CT data were included in the study. Clinical data was obtained via retrospective chart review. Cytogenetic risk (CyR) was classified using the mSMART criteria . Disease and ASCT related characteristics were compared by MRD status. MRD was measured by NGS on bone marrow aspirates using the previosly validated clonoSEQ ® Assay (Adaptive Biotechnologies Corporation, Seattle, USA) tracking the IgH, IgK and IgL rearrangements at a minimum sensitivity level of 10-5. MRD was defined by residual clonal cells per million nucleated cells as: negative= 0, borderline= 1-5, positive &amp;gt;5. PET/CT scans were performed locally at baseline and at day 100. Comparisons were performed using the chi-square test for categorical variables, Wilcoxon rank-sum test for continuos variables, McNemar's test and Cohens's Kappa for agreement measures. Results: A total of 103 patients had matched MRD and PET/CT assessment around day 100 (+/-9 days) and were included in the analysis. Median age at diagnosis was 62 years (range, 54-66 years), 71 patients (68.9%) were men. CyR was standard risk in 49 (47.6%), high-risk in 39 (37.9%) and unknown in 15 (14.6%) patients. Most 75 (72.8%) patients were MRD positive, 16 (15.5%) were MRD negative, and 12 (11.7%) borderline. The median main MRD clone detected was 64 (range 0-91,874). 70 patients (68%) and 33 (32%) had a negative and positive PET/CT respectively. The median follow-up time was 18 months (range, 13-31 months). At the time of data analysis, 10 patients (9.7%) had relapsed and only 4 (3.9%) had died. There was a high-correlation between MRD status and PET/CT, 31 patients (93.9%) with positive PET/CT were also MRD positive (p=0.0027). There were no statistical differences between PET/CT and CyR (p=0.95). We analyzed the correlation using the FREQ procedure (McNemars's test); there was a strong association between positive PET/CT and positive MRD in 31/33 patients (93.9%, high sensitivity), and low association for negative PET/CT the negative/borderline MRD in 26/70 (37.1%, low specificity; p&amp;lt;0.001). The agreement measure between the PET/CT and MRD using negative/borderline combined had a kappa of 0.23 (95% CI 0.11, 0.35) indicating a fair agreement beyond chance (Figure 1). PET/CT-CT was a statistically significant predictor of worse RFS (HR 3.53, 95%CI: 1.02-12.24, p&amp;lt;0.0337) and OS (HR 11.38, 95%CI: 1.18-109.56, p&amp;lt;0.0078) (Figure 2-3, respectively). MRD was not predictive of neither RFS (HR 1.72, 95%CI: 0.36-8.14, p&amp;lt;0.49) or OS (p&amp;lt;0.16). Conclusions: In conclusion, we demonstrate that the combination of MRD by NGS (clonoSEQ ®) and PET/CT at day 100 are complementary and have a high sensitivity (true positive rate) and fair correlation of agreement but low specificity (true negative rate). PET/CT was the best most sensitive technique to prognosticate RFS and OS. We did not find prognostic correlation of MRD with RFS and OS. However, our findings might be confounded by the low risk of relapse and death, a longer follow-up may demonstrate clinically important differences. Our results add evidence that MRD plus PET/CT improve the definition of CR in MM patients post ASCT. Prospective studies are needed to elucidate the optimal timing and role of combined MRD, PET/CT with other prognostic markers of clinical outcomes. Disclosures Larsen: Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen Oncology: Honoraria, Membership on an entity's Board of Directors or advisory committees. Fonseca:Juno: Consultancy; Kite: Consultancy; Aduro: Consultancy; OncoTracker: Consultancy, Membership on an entity's Board of Directors or advisory committees; Merck: Consultancy; Bayer: Consultancy; Janssen: Consultancy; Takeda: Consultancy; Novartis: Consultancy; Pharmacyclics: Consultancy; Sanofi: Consultancy; Oncopeptides: Consultancy; GSK: Consultancy; AbbVie: Consultancy; Adaptive Biotechnologies: Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy; BMS: Consultancy; Celgene: Consultancy.

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.

Elucidating the Role of NEMO and SpiC in DNA Double Strand Break Induced Inhibition of V(D)J Recombination

Abstract V(D)J recombination is regulated such that most lymphocytes assemble and express an antigen receptor from only one allele (allelic exclusion). In pre-B cells, RAG-induced DNA double strand breaks (DSBs) at Igκ loci signal via ATM to rapidly repress Rag1/2 transcription, inhibit accessibility of Igk loci, and limit V recombination. We hypothesize that RAG DSB-induced repression of Rag1/2 is critical to transiently limit V recombination, ensure allelic exclusion, and inhibit oncogenic Ig translocations. Fittingly, Atm−/− mice have higher frequencies of developing B cells with RAG DSBs at both Igh and Igk loci and of mature B cells with bi-allelic expression of these genes. Yet, given the multifunctional roles of ATM in the DSB response, Atm−/− phenotypes cannot be directly attributed to Rag1/2 repression. To test our hypothesis, we study mouse models lacking downstream effectors of ATM: i) SpiC, which suppresses Igk accessibility in response to RAG DSBs, and ii) NFκB essential modulator (NEMO), which represses Rag1/2 in response to genotoxic DSBs. I show here that the frequency of surface bi-allelic Igκ expression is normal on SpiC−/− B cells and increased on Nemo−/− B cells. I also show that Nemo−/− pre-B cells exhibit impaired Rag1/2 repression in response to RAG DSBs and concomitant increased RAG cleavage at Igk. Our data support that RAG DSB-induced repression of Rag1/2 transiently inhibits additional Igk rearrangements to enforce Igκ allelic exclusion independent of ATM-mediated DSB repair. I am using Nemo−/− mouse models to determine if DSB-induced Rag1/2 repression requires Nemo in pro-B cells, and if this mechanism is critical to suppress oncogenic Ig translocations independent of defects in DSB repair/checkpoint activation.

IGH and IGK Rearrangement and IGH Somatic Hypermutation Analysis Using Next-generation Sequencing for the Detection of Clonality in High-risk Korean Multiple Myeloma Patients

Clonality assessment in patients with multiple myeloma (MM) is important both for initial diagnosis and detecting minimal residual disease. Next-generation sequencing (NGS)-based immunoglobulin gene rearrangement assays may be useful for assessing clonality, although their applicability has not been convincingly demonstrated. Hence, we evaluated the primer set most widely used with the only commercially available NGS-based clonality assay in high-risk MM patients for the first time.

PF175 MULTICENTRE STANDARDIZATION OF MINIMAL RESIDUAL DISEASE DETECTION AND QUANTITATION USING THE EUROCLONALITY‐NGS ASSAY

Background:Amplicon‐based next‐generation sequencing (NGS) is increasingly used for identification of clonal immunoglobulin (IG) and T‐cell receptor (TR) gene rearrangements as markers for minimal residual disease (MRD) monitoring in acute lymphoblastic leukaemia (ALL). A standardized assay integrating amplicon library preparation as well as bioinformatic data analysis for marker identification has been established and validated in a multicentre study by the EuroClonality‐NGS working group, including laboratories from the EuroMRD consortium (Brüggemann et al., Leukemia, in press). The use of NGS for MRD detection represents a universal assay which offers better specificity compared to the currently used real‐time quantitative (q)PCR as well as information about background repertoire, however a reliable quantitation of MRD levels by this approach still represents a challenge.Aims:14 laboratories from the EuroMRD consortium and EuroClonality‐NGS working group performed IG / TR marker identification and MRD quantitation in 3 follow‐up samples to assess the reproducibility of MRD detection and quantitation.Methods:All participating laboratories followed the standardized library preparation protocols developed by the EuroClonality‐NGS working group and performed 2 sequencing runs. The 1st run aimed at marker identification of potential target IGH, IGK, TRB, TRG and TRD rearrangements present in the diagnostic sample. Based on the screening results, at least one gene locus was selected by each laboratory for a 2nd run of all 3 follow‐up samples diluted into a central polyclonal buffy coat control with 500ng input DNA used (equivalent to 75,000 cells) for MRD detection and assessment of false positivity. MRD levels in these samples were assessed by qPCR independently. Libraries were prepared by 2 rounds of PCR in 12 laboratories, whereas 2 laboratories used a modified 1‐round approach. The EuroClonality‐NGS‐developed central In‐Tube Quality/Quantitation Control (cIT‐QC) ‐ spike‐ins of known DNA quantity, was used by 2 labs. Sequencing was performed on Illumina MiSeq (2x250 v2 kit) or IonTorrent S5 XL instruments. Results were analysed with ARResT/Interrogate.Results:The 6 rearrangements (3 IGH‐VJ, 1 IGH‐DJ, 1 TRD, 1 TRG) present in the diagnostic sample above the 5% threshold were identified as markers in 71/76 cases (93%). MRD detection was performed in the IGH‐VJ system by all 14 laboratories, IGH‐DJ by 4, TRD by 4 and, TRG by 2, with mean coverage of 992,129 reads per sample. In one case IGH‐VJ libraries did not meet the quality control standards for analysis and were therefore excluded. In 12 of the remaining 13 laboratories, all 3 IGH‐VJ targets were detected in all 3 follow‐up samples, as well as in all IGH‐DJ, TRD and TRG libraries. MRD levels (4.35E‐3; 1.8E‐3 and 1.72E‐4) correlated well with the expected dilution (8E‐3; 4E‐3 and 4E‐4) and qPCR results in TRG systems, but appeared over‐estimated in other systems in the absence of normalization to the cIT‐QC (selected IGH‐VJ target means vs. cIT‐QC normalized value: 6.34E‐2 vs 1.19E‐02; 3.18E‐2 vs. 8.40E‐03 and 3.98E‐3 vs. 2.31E‐04).Summary/Conclusion:We have shown that the EuroClonality‐NGS‐developed approach can be successfully used for MRD‐PCR marker identification. In addition, MRD detection using this assay showed very good inter‐laboratory concordance. For accurate quantitation of MRD the use of the cIT‐QC is essential and should be further validated in a large‐scale multicentre testing before the method can be used in a clinical setting.

Contribution of immunoglobulin lambda light chain gene rearrangement analysis in the diagnosis of B‐cell neoplasms

Identification of clonal IGH, IGK and IGL gene rearrangements offers diagnostic adjunct in suspected B-cell neoplasms. However, many centres omit IGL analysis as its value is uncertain. A review of 567 cases with IGH, IGK and IGL rearrangement assessed using BIOMED-2 assays showed clonal immunoglobulin gene rearrangement in 54% of cases, of which 24% had a clonal IGL rearrangement. In two cases, the clonal rearrangement was detected exclusively by IGL analysis. This finding demonstrates the added value of IGL analysis for clonality assessment, especially in suspected B-cell neoplasms in which a clonal IGH and/or IGK rearrangement is not detected or is equivocal.