RESUMO
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.
Assuntos
Regiões Determinantes de Complementaridade/genética , Rearranjo Gênico de Cadeia Pesada de Linfócito B , Rearranjo Gênico de Cadeia Leve de Linfócito B , Sequenciamento de Nucleotídeos em Larga Escala , Mieloma Múltiplo/patologia , Biomarcadores Tumorais , Medula Óssea/patologia , Células da Medula Óssea/metabolismo , Ensaios Clínicos como Assunto/estatística & dados numéricos , Evolução Clonal , Células Clonais/patologia , Genes de Imunoglobulinas , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/genética , Mieloma Múltiplo/genética , Neoplasia Residual/diagnóstico , Neoplasia Residual/genética , RNA Mensageiro/genética , RNA Neoplásico/genética , Hipermutação Somática de Imunoglobulina , Éxons VDJRESUMO
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.
Assuntos
Mieloma Múltiplo , Humanos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Mieloma Múltiplo/diagnóstico , Mieloma Múltiplo/genética , Neoplasia Residual/diagnóstico , Neoplasia Residual/genética , Reprodutibilidade dos TestesRESUMO
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.