Immune Repertoire Analysis of Multiple Myeloma Research Samples Using NGS Characterization of Multiple B Cell Receptors in a Single Reaction
Bibliographic record
Abstract
Abstract Introduction B 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. Methods The 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. Results Clonality 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. Conclusions These 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. Disclosures Lowman: 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.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.001 | 0.001 |
| Meta-epidemiology (narrow) | 0.001 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.002 | 0.001 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.001 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.001 | 0.000 |
| Insufficient payload (model declined to judge) | 0.003 | 0.001 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".