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PURPOSE: Minimal residual disease (MRD) negativity is a strong predictor for outcome in multiple myeloma. To assess V(D)J clonotype capture using the updated Adaptive next-generation sequencing (NGS) MRD assay in a clinical setting, we analyzed baseline and follow-up samples from patients with multiple myeloma who achieved deep clinical responses. EXPERIMENTAL DESIGN: A total of 159 baseline and 31 follow-up samples from patients with multiple myeloma were sequenced using the NGS MRD assay. Baseline samples were also sequenced using a targeted multiple myeloma panel (myTYPE). We estimated ORs with 95% confidence intervals (CI) for clonotypes detection using logistic regression. RESULTS: The V(D)J clonotype capture rate was 93% in baseline samples with detectable genomic aberrations, indicating presence of tumor DNA, assessed through myTYPE. myTYPE-positive samples had significantly higher V(D)J clonotype detection rates in univariate (OR, 7.3; 95% CI, 2.8-22.6) and multivariate analysis (OR, 4.4; 95% CI, 1.4-16.9; P = 0.016). Higher disease burden was associated with higher probability of V(D)J clonotype capture, meanwhile no such association was found for age, gender, or type of heavy or light immunoglobulin chain. All V(D)J clonotypes detected at baseline were detected in MRD-positive samples indicating that the V(D)J clonotypes remained stable and did not undergo further rearrangements during follow-up. Of the 31 posttreatment samples, 12 were MRD-negative using the NGS MRD assay. CONCLUSIONS: NGS for V(D)J rearrangements in multiple myeloma offers a reliable and sensitive method for MRD tracking with high detection rates in the clinical setting.

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A correction to this paper has been published and can be accessed via a link at the top of the paper.

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Recent genomic research efforts in multiple myeloma have revealed clinically relevant molecular subgroups beyond conventional cytogenetic classifications. Implementing these advances in clinical trial design and in routine patient care requires a new generation of molecular diagnostic tools. Here, we present a custom capture next-generation sequencing (NGS) panel designed to identify rearrangements involving the IGH locus, arm level, and focal copy number aberrations, as well as frequently mutated genes in multiple myeloma in a single assay. We sequenced 154 patients with plasma cell disorders and performed a head-to-head comparison with the results from conventional clinical assays, i.e., fluorescent in situ hybridization (FISH) and single-nucleotide polymorphism (SNP) microarray. Our custom capture NGS panel had high sensitivity (>99%) and specificity (>99%) for detection of IGH translocations and relevant chromosomal gains and losses in multiple myeloma. In addition, the assay was able to capture novel genomic markers associated with poor outcome such as bi-allelic events involving TP53. In summary, we show that a multiple myeloma designed custom capture NGS panel can detect IGH translocations and CNAs with very high concordance in relation to FISH and SNP microarrays and importantly captures the most relevant and recurrent somatic mutations in multiple myeloma rendering this approach highly suitable for clinical application in the modern era.

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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 non-templated insertions as the most important determinants of light chain clonal uniqueness, identifying a potentially trackable sequence in the majority of patients. Taken together, we show that dominant clonal sequences identified at baseline are reliable biomarkers for long-term tracking of the malignant clone, including both IGH and the majority of light chain clones.

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IMPORTANCE: Multiple myeloma is consistently preceded by monoclonal gammopathy of undetermined significance (MGUS). Risk models that estimate the risk of progression from MGUS to multiple myeloma use data from a single time point, usually the initial workup. OBJECTIVE: To longitudinally investigate the alterations of serum immune markers with stable vs progressive MGUS. DESIGN, SETTING, AND PARTICIPANTS: This prospective cross-sectional cohort study included 77 469 adult participants aged 55 to 74 years in the screening arm of the National Cancer Institute Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial who had a diagnosis of progressing MGUS (n = 187) or stable MGUS (n = 498), including light-chain subtype, from November 1993, through December 2011. For each participant, all available serially stored prediagnostic serum samples (N = 3266) were obtained. Data analysis was performed from April 2018, to December 2018. MAIN OUTCOMES AND MEASURES: Serum protein and monoclonal immunoglobulin levels, serum free light chains, and serum light chains within each immunoglobulin class were measured. RESULTS: Of 685 individuals included in the study, 461 (67.3%) were men; the mean (SD) age was 69.1 (5.6) years. In cross-sectional modeling, risk factors associated with progressive MGUS were IgA isotype (adjusted odds ratio [OR], 1.80; 95% CI, 1.03-3.13; P = .04), 15 g/L or more monoclonal spike (adjusted OR, 23.5; 95% CI, 8.9-61.9; P < .001), skewed (<0.1 or >10) serum free light chains ratio (adjusted OR, 46.4; 95% CI, 18.4-117.0; P < .001), and severe immunoparesis (≥2 suppressed uninvolved immunoglobulins) (adjusted OR, 19.1; 95% Cl, 7.5-48.3; P < .001). Risk factors associated with progressive light-chain MGUS were skewed serum free light chains ratio (adjusted OR, 44.0; 95% CI, 14.2-136.3; P < .001) and severe immunoparesis (adjusted OR, 48.6; 95% CI, 9.5-248.2; P < .001). In longitudinal analysis of participants with serial samples prior to progression, 23 of 43 participants (53%) had high-risk MGUS before progression; 16 of these 23 (70%) experienced conversion from low-risk or intermediate-risk MGUS within 5 years. Similar results were found for light-chain MGUS. CONCLUSIONS AND RELEVANCE: The findings of evolving risk patterns support annual blood testing and risk assessment for patients with MGUS or light-chain MGUS.

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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.

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Caucasian Americans (CA) compared with African Americans (AA) have a twofold increased incidence of multiple myeloma (MM) and have an earlier age of diagnosis. However, there is sparse information regarding underlying biological differences across racial/ethnic groups. We characterized genetic alterations using a targeted next-generation sequencing assay called myTYPE, developed at MSKCC, allowing capture of somatic mutations, IgH translocations, gains/losses, and hyperdiploidy. Samples were obtained from the NIH Plasma Cell Dyscrasia Racial Disparity Cohort. In total, 68 patient samples were successfully sequenced and manually curated based on well-established databases. Of the 68 patient samples (47 CA, 21 AA), 84% had at least one type of genomic alteration. Importantly, the IgH translocation, t(11;14), was observed more frequently in the AA group (0 vs. 29%, p = 0.001). Known oncogenic somatic non-synonymous mutations were found in 18 genes and indels in 2 genes. KRAS mutations were the most common mutation found in 16% of patients followed by NRAS and BRAF mutations. TP53 somatic mutations appeared to be more common in CA but lacked significance. This proof-of-principle study indicates the presence of varying underlying tumor biology between racial groups and supports the need of future prospective trials to capture these molecular characteristics.

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