Tracking of clonal immunoglobulin V(D)J rearrangement sequences by next generation sequencing is highly sensitive for minimal residual disease in multiple myeloma. However, previous studies have found variable rates of V(D)J sequence identification at baseline, which could limit tracking. Here, we aimed to define the factors influencing the identification of clonal V(D)J sequences. Bone marrow mononuclear cells from 177 myeloma patients underwent V(D)J sequencing by the LymphoTrack assays (Invivoscribe). As a molecular control for tumor cell content, we sequenced the samples using our in-house myeloma panel myTYPE. V(D)J sequence clonality was identified in 81% of samples overall, as compared with 95% in samples where tumor-derived DNA was detectable by myTYPE. Clonality was detected more frequently in patients with lambda-restricted disease, mainly because of increased detection of kappa gene rearrangements. Finally, we describe how the tumor cell content of bone marrow aspirates decrease gradually in sequential pulls because of hemodilution: From the initial pull used for aspirate smear, to the final pull that is commonly used for research. In conclusion, baseline clonality detection rates of 95% or higher are feasible in multiple myeloma. Optimal performance depends on the use of good quality aspirates and/or subsequent tumor cell enrichment.

Minimal residual disease (MRD) tracking, by next generation sequencing of immunoglobulin sequences, is moving towards clinical implementation in multiple myeloma.However, there is only sparse information available to address whether clonal sequences remain stable for tracking over time, and to what extent light chain sequences are sufficiently unique for tracking. Here, we analyzed immunoglobulin repertoires from 905 plasma cell myeloma and healthy control samples, focusing on the third complementarity determining region (CDR3). Clonal heavy and/or light chain expression was identified in all patients at baseline, with one or more subclones related to the main clone in 3.2%. In 45 patients with 101 sequential samples, the dominant clonal CDR3 sequences remained identical over time, despite differential clonal evolution by whole exome sequencing in 49% of patients. The low frequency of subclonal CDR3 variants, and absence of evolution over time in active multiple myeloma, indicates that tumor cells at this stage are not under selective pressure to undergo antibody affinity maturation. Next, we establish somatic hypermutation and

Context.— Minimal residual disease (MRD) is a major prognostic factor in multiple myeloma, although validated technologies are limited. Objective.— To standardize the performance of the LymphoTrack next-generation sequencing (NGS) assays (Invivoscribe), targeting clonal immunoglobulin rearrangements, in order to reproduce the detection of tumor clonotypes and MRD quantitation in myeloma. Design.— The quantification ability of the assay was evaluated through serial dilution experiments. Paired samples from 101 patients were tested by LymphoTrack, using Sanger sequencing and EuroFlow's next-generation flow (NGF) assay as validated references for diagnostic and follow-up evaluation, respectively. MRD studies using LymphoTrack were performed in parallel at 2 laboratories to evaluate reproducibility. Results.— Sensitivity was set as 1.3 tumor cells per total number of input cells. Clonality was confirmed in 99% and 100% of cases with Sanger and NGS, respectively, showing great concordance (97.9%), although several samples had minor discordances in the nucleotide sequence of rearrangements. Parallel NGS was performed in 82 follow-up cases, achieving a median sensitivity of 0.001%, while for NGF, median sensitivity was 0.0002%. Reproducibility of LymphoTrack-based MRD studies (85.4%) and correlation with NGF (R2 > 0.800) were high. Bland-Altman tests showed highly significant levels of agreement between flow and sequencing. Conclusions.— Taken together, we have shown that LymphoTrack is a suitable strategy for clonality detection and MRD evaluation, with results comparable to gold standard procedures.

Introduction Minimal residual disease (MRD) negativity after initial therapy is a strong predictor of survival in multiple myeloma. Tracking of clonal immunoglobulin V(D)J rearrangements by next generation sequencing is highly sensitive for MRD and does not require immediate analysis of fresh samples. However, previous studies have found variable rates of baseline V(D)J sequence capture, which could limit tracking. In this study, we aimed to define the sample-related and disease-related factors that influence V(D)J capture. Methods We included 177 patients with plasma cell myeloma who had available stored mononuclear cells from a baseline bone marrow aspirate. Each sample was sequenced by two assays: The LymphoTrack® VDJ assays from Invivoscribe, and our in-house myeloma panel myTYPE, as a molecular control for detectable tumor derived DNA in the samples. MyTYPE positivity was defined by one or more single nucleotide variant, insertion, deletion, translocation or copy number variation that is known to occur in myeloma. Results and discussion The V(D)J capture rate in our whole cohort was 81 %, as compared with 95 % in the myTYPE positive samples, demonstrating the importance of tumor cell content for V(D)J capture. This was confirmed in multivariate logistic regression (Figure 1), where myTYPE positivity was a strong independent predictor of V(D)J capture success, with an odds ratio (OR) of 6.61 (95 % CI 2.22-24.81, p = 0.002). Plasma cell content estimated from bone marrow aspirate smears also contributed to the multivariate model, with an OR of 1.3 for each 10 % increase in plasma cell content (95 % CI 0.96-1.84, p=0.109), but this did not reach statistical significance after accounting for the strong effect of myTYPE. Finally, having lambda light chain restricted plasma cells was a strong predictor of V(D)J capture success (OR 6.91, 95 % CI 2.4-25.32, p = 0.001). Higher V(D)J capture rate in lambda-restricted myeloma as compared with kappa-restricted was mostly driven by a difference in immunoglobulin kappa gene (IGK) rearrangement capture (73 vs. 44 %, p < 0.001). As a potential explanation, we found up to 4 unique IGK rearrangements that are amenable to capture in lambda-restricted cases, as well as dramatically lower somatic hypermutation (SHM) of the IGK variable region in clonal rearrangements, as compared with kappa-restricted cases. SHM has previously been shown to cause V(D)J capture failure by interfering with PCR primer annealing. Both of these factors can be attributed to IGK inactivation by rearrangements involving the "kappa deleting element" region, affecting both IGK alleles in lambda-restricted plasma cells (Perfetti et al, Immunology, 2004). As an explanation for low plasma cell content in the samples used in this study, we describe how the tumor cell content of bone marrow aspirates decrease gradually in sequential pulls because of hemodilution: from the initial pull used for aspirate smear, to the final pull that is commonly used for research. Supporting the important role of hemodilution, we found V(D)J capture rates of 97 % in clinical samples (early pull aspirates) from our institution that were analyzed with the same NGS assays, as long as the bone marrow plasma cell infiltration was above 5 %. V(D)J capture probability appears to be determined by two factors: The abundance of clonal cells (i.e. tumor cell content), and the degree to which clonal sequences can be amplified by the assay (which is negatively affected by SHM). Thus, increasing the tumor cell content in samples as much as practically possible (i.e., optimal bone marrow aspirates and enrichment of CD138+ plasma cells) may compensate for SHM and improve V(D)J capture rates beyond 95 %. Conclusion V(D)J capture rates of at least 95 % are feasible in multiple myeloma using LymphoTrack® NGS assays, when the sample quality is good. The most important reason for V(D)J capture failure is low tumor cell content due to bone marrow aspirate hemodilution. Optimal performance depends on the use of early pull aspirates and/or subsequent tumor cell enrichment. Figure 1: Predicting V(D)J capture. Regression lines and individual data points are colored according to myTYPE status (red = positive; blue = negative); V(D)J capture probability on the y-axis (capture yes/no on the second y-axis) and bone marrow plasma cell percentage by aspirate smear on the x-axis; split into panels according to light chain restriction (right = lambda; left = kappa). Disclosures Ho: Invivoscribe, Inc.: Honoraria. Arcila:Invivoscribe, Inc.: Consultancy, Honoraria. Jacobsen:Invivoscribe, Inc.: Employment. Huang:Invivoscribe, Inc.: Employment. Miller:Invivoscribe, Inc.: Employment, Equity Ownership. Landgren:Pfizer: Consultancy; Karyopharm: Consultancy; Amgen: Consultancy, Research Funding; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.