Clonality assessment and detection of clonal diversity in classic Hodgkin lymphoma by next-generation sequencing of immunoglobulin gene rearrangements

Clonality analysis in classic Hodgkin lymphoma (cHL) is of added value for correctly diagnosing patients with atypical presentation or histology reminiscent of T cell lymphoma, and for establishing the clonal relationship in patients with recurrent disease. However, such analysis has been hampered by the sparsity of malignant Hodgkin and Reed-Sternberg (HRS) cells in a background of reactive immune cells. Recently, the EuroClonality-NGS Working Group developed a novel next-generation sequencing (NGS)-based assay and bioinformatics platform (ARResT/Interrogate) to detect immunoglobulin (IG) gene rearrangements for clonality testing in B-cell lymphoproliferations. Here, we demonstrate the improved performance of IG-NGS compared to conventional BIOMED-2/EuroClonality analysis to detect clonal gene rearrangements in 16 well-characterized primary cHL cases within the IG heavy chain (IGH) and kappa light chain (IGK) loci. This was most obvious in formalin-fixed paraffin-embedded (FFPE) tissue specimens, where three times more clonal cases were detected with IG-NGS (9 cases) compared to BIOMED-2 (3 cases). In total, almost four times more clonal rearrangements were detected in FFPE with IG-NGS (N = 23) as compared to BIOMED-2/EuroClonality (N = 6) as judged on identical IGH and IGK targets. The same clonal rearrangements were also identified in paired fresh frozen cHL samples. To validate the neoplastic origin of the detected clonotypes, IG-NGS clonality analysis was performed on isolated HRS cells, demonstrating identical clonotypes as detected in cHL whole-tissue specimens. Interestingly, IG-NGS and HRS single-cell analysis after DEPArray™ digital sorting revealed rearrangement patterns and copy number variation profiles indicating clonal diversity and intratumoral heterogeneity in cHL. Our data demonstrate improved performance of NGS-based detection of IG gene rearrangements in cHL whole-tissue specimens, providing a sensitive molecular diagnostic assay for clonality assessment in Hodgkin lymphoma.

Previously Unknown Stage of TCR Beta VDJ Recombination As a Novel Rich Source of Markers for Clonality Assessment and MRD Monitoring in T-ALL

Background. Genomic rearrangements of T and B cell receptor genes (BCR and TCR) have become the gold standard marker for clonality assessment and minimal residual disease monitoring (MRD) in acute lymphoblastic leukemia (ALL). B-ALL can bear both TCR and BCR rearrangements, whereas T-ALL contains mainly TCR rearrangements. The limitation on the number of potential markers decreases T-ALL cases suitable for MRD monitoring and its reliability. We discovered a missed stage of TRB locus VDJ-recombination during which rearrangement between D1 and D2 segments occurs. Here we examined this new type of rearrangement in pediatric T-ALL as a potential clonal marker for MRD diagnostics for the first time. Methods. Identification of TRB D1-D2 rearrangements was performed using targeted high-throughput sequencing on the Illumina MiSeq instrument [1]. Genomic libraries for sequencing were obtained using PCR with intronic primers flanking the TRBD1 and TRBD2 genes. Genomic DNA for the libraries was extracted from T-ALL patients' bone marrow and peripheral blood of healthy volunteers. DNA from normal CD4+ and CD8+ T-cells was used as a positive control. DNA from normal B-cells and the RMS cell line was used as a negative control. Extraction of TRB D1-D2 rearrangements from sequencing data was performed using modified MiXCR software. Post-analysis was performed using VDJtools software. Results. This study analyzed a cohort of 144 children aged 2-16 years with T-ALL and 12 healthy individuals aged 25-40 years. We identified TRB D1-D2 rearrangements in all analyzed samples except negative controls (B-cells and RMS cell line). The dominant TRB D1-D2 clone in all normal samples occupied 25-30% and had zero length of the D1-D2 junction. The clonal size of the second clone in the top was 3-8%. Based on this observation, we set a 30% cut-off for zero-length D1-D2 junction rearrangements and a 10% cut-off for others to identify abnormally proliferated T-ALL specific clones. We identified 221 different leukemia-related D1-D2 rearrangements in 124 T-ALL samples in total. We intersected them with those in normal samples to test the uniqueness of leukemia-related rearrangements as potential markers. 89% of analyzed rearrangements were specific for leukemia samples, so they can be used for tracking leukemic cells. 11% of rearrangements were present in at least one normal sample and can be used for clonality analysis but are useless for MRD monitoring due to its relatively high generation probability. Zero-length D-D junction was detected as a major clonal marker (25-100%) in 42 T-ALL samples. Nevertheless, 23 out of these 42 samples contained additional unique rearrangements useful for MRD. 29 out of the other 82 samples included one major rearrangement (clonal rate 80-90%), six samples had two major rearrangements with equal clonal rates (30-50%), and the rest of the samples contained 3-5 rearrangements representing oligoclonality. The length of the D-D junction among detected rearrangements in both leukemic and normal samples has a bimodal distribution which is probably linked to two different levels of TdT or exonuclease activity during D1-D2 recombination. Additionally, we observed a strong correlation (Mann-Whitney-U-test, p-value < 0.001) between the presence of TRB D1-D2 rearrangements in T-ALL samples and the CD117 negative immunophenotype of leukemic cells. Conclusions. Obtained results show that TRB D1-D2 rearrangements are a common feature of normal and malignant T-cells. Unique leukemia-related TRB D1-D2 rearrangements can be detected in 73% of pediatric T-ALL cases. It can be routinely detected by PCR with subsequent NGS and can be easily integrated into an existing multiplex system for T-ALL clonality and MRD analysis. References. Komkov et al. High-throughput sequencing of T-cell receptor alpha chain clonal rearrangements at the DNA level in lymphoid malignancies. British Journal of Haematology. 2020 Mar. 188(5): 723-731. Acknowledgment. This work was supported by Russian Science Foundation grant № 20-75-10091 to AK. Disclosures No relevant conflicts of interest to declare.

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. Disclosures No relevant conflicts of interest to declare.

Molecular Diagnosis of T-cell Lymphoma: A correlative study of PCR-based T-cell clonality assessment and targeted NGS

Immunomorphological diagnosis of T-cell lymphomas (TCL) may be challenging, especially on needle biopsies. Multiplex polymerase chain reaction (PCR) assays to assess T-cell receptor (TCR) gene rearrangements are now widely used to detect T-cell clones and provide diagnostic support. However, PCR assays only detect 80% of TCL, and clonal lymphocyte populations may also appear in non-neoplastic conditions. More recently, targeted next-generation sequencing (t-NGS) technologies have been deployed to improve lymphoma classification. To the best of our knowledge, the comparison of these techniques' performance in TCL diagnosis has not been reported yet. In this study, 82 TCL samples and 25 non-neoplastic T-cell infiltrates were divided into two cohorts (test and validation) and analyzed with both multiplex PCR and t-NGS to investigate TCR gene rearrangements and somatic mutations, respectively. The detection of mutations appeared to be more specific (100.0%) than T-cell clonality assessment (41.7% to 45.5%), whereas no differences were observed in terms of sensitivity (95.1% to 97.4%). Furthermore, t-NGS provided a reliable basis for TCL diagnosis in samples with partially degraded DNA that was impossible to assess with PCR. Finally, although multiplex PCR assays appeared to be less specific than t-NGS, both techniques remain complementary, as PCR recovered some t-NGS negative cases.

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 >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.

CD4 and CD8 double-negative immunophenotype of thymoma-associated lymphocytes in a dog

Persistent small-cell lymphocytosis in dogs with a concurrent mediastinal mass has been associated with both thymoma and small-cell lymphoma. In thymomas, neoplastic thymic epithelial cells induce overproduction and release of polyclonal lymphocytes, whereas thymic lymphoma results in thymic effacement by a clonal expansion of neoplastic lymphocytes and subsequent leukemic phase of lymphoma. Flow cytometry has been used to differentiate these 2 entities by immunophenotyping mediastinal mass aspirates. It has been reported that cases with mediastinal masses in which ≥ 10% of the associated small-cell lymphocytes were double positive for CD4 and CD8 were thymomas, whereas masses associated with < 10% were suggestive of lymphoma. We report a unique case of thymoma-associated lymphocytosis lacking the classic CD4+CD8+ immunophenotype. Our findings suggest that there may be more diversity in the thymoma-associated lymphocyte immunophenotype than has been identified previously; immunophenotyping alone might not be sufficient to differentiate thymic small-cell lymphoma from thymoma-associated lymphocytosis. In dogs with mediastinal masses and peripheral lymphocytosis, employing a variety of testing modalities to avoid misdiagnosis is prudent. These modalities include cytologic and/or histologic evaluation, immunophenotyping, and clonality assessment.

CXCR4 S338X clonality is an important determinant of ibrutinib outcomes in patients with Waldenström macroglobulinemia

Key Points CXCR4 S338X clonality ≥25% is associated with lower very good partial response and shorter progression-free survival to ibrutinib. CXCR4 S338X clonality assessment represents a novel biomarker to predict outcomes to ibrutinib in Waldenström macroglobulinemia patients.