Next-generation sequencing of peripheral B-lineage cells pinpoints the circulating clonotypic cell pool in multiple myeloma

Thiele, Benjamin; Kloster, Marie; Alawi, Malik; Indenbirken, Daniela; Trepel, Martin; Grundhoff, Adam; Binder, Mascha

doi:10.1182/blood-2014-02-556746

Cited by 12 publications

(10 citation statements)

References 23 publications

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“…Based on previous studies, 21–23 we established optimal PCR and NGS conditions for comprehensive immune repertoire analysis and high-sensitivity detection of V(D)J rearrangements from leukocyte DNA. A sequencing depth of 80,000 reads per sample was sufficient to comprehensively analyze the B-lineage repertoire from 500 ng genomic or 250 ng cell-free DNA, which can typically be extracted from 1–5 mL of blood ( Online Supplementary Figure S1A ).…”

Section: Resultsmentioning

confidence: 99%

Monitoring multiple myeloma by next-generation sequencing of V(D)J rearrangements from circulating myeloma cells and cell-free myeloma DNA

et al. 2017

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Recent studies suggest that circulating tumor cells and cell-free DNA may represent powerful non-invasive tools for monitoring disease in patients with solid and hematologic malignancies. Here, we conducted a pilot study in 27 myeloma patients to explore the clonotypic V(D)J rearrangement for monitoring circulating myeloma cells and cell-free myeloma DNA. Next-generation sequencing was used to define the myeloma V(D)J rearrangement and for subsequent peripheral blood tracking after treatment initiation. Positivity for circulating myeloma cells/cell-free myeloma was associated with conventional remission status (P<0.001) and 91% of non-responders/progressors versus 41% of responders had evidence of persistent circulating myeloma cells/cell-free myeloma DNA (P<0.001). About half of the partial responders showed complete clearance of circulating myeloma cells/cell-free myeloma DNA despite persistent M-protein, suggesting that these markers are less inert than the M-protein, rely more on cell turnover and, therefore, decline more rapidly after initiation of effective treatment. Positivity for circulating myeloma cells and for cell-free myeloma DNA were associated with each other (P=0.042), but discordant in 30% of cases. This indicates that cell-free myeloma DNA may not be generated entirely by circulating myeloma cells and may reflect overall tumor burden. Prospective studies need to define the predictive potential of high-sensitivity determination of circulating myeloma cells and DNA in the monitoring of multiple myeloma.

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Section: Resultsmentioning

confidence: 99%

Monitoring multiple myeloma by next-generation sequencing of V(D)J rearrangements from circulating myeloma cells and cell-free myeloma DNA

et al. 2017

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“…The existence of B-lineage cells other than plasma cells with the major clonotype and their physiological relevance has been discussed (19,42–49). However, the phenotypic range of clonotypic cells could not be studied at the single-cell level in high-dimensional space.…”

Section: Discussionmentioning

confidence: 99%

“…Neither RNA- nor DNA-based bulk immunoglobulin gene sequencing can quantify clone frequencies or assigned immune phenotypes to the identified clones could be inaccurate as a result of contamination with other cells (19). Yet, the success of B cell-targeting cellular therapeutics in subsets of patients (20,21) suggests the involvement of cells phenotypically different from the majority of aberrant-phenotype plasma cells in multiple myeloma pathophysiology.…”

Section: Introductionmentioning

confidence: 99%

Clonal Expansion and Interrelatedness of Distinct B-Lineage Compartments in Multiple Myeloma Bone Marrow

Hansmann

Han

Penter

et al. 2017

Cancer Immunology Research

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Multiple myeloma is characterized by the clonal expansion of malignant plasma cells in the bone marrow. But the phenotypic diversity and the contribution of less predominant B-lineage clones to the biology of this disease have been controversial. Here we asked whether cells bearing the dominant multiple myeloma immunoglobulin rearrangement occupy phenotypic compartments other than that of plasma cells. To accomplish this, we combined 13-parameter FACS index sorting and t-Stochastic Neighbor Embedding (t-SNE) visualization with high-throughput single-cell immunoglobulin sequencing to track selected B-lineage clones across different stages of human B-cell development. As expected, the predominant clones preferentially mapped to aberrant plasma cell compartments, albeit phenotypically altered from wild type. Interestingly, up to 1.2 % of cells of the predominant clones co-localized with B-lineage cells of a normal phenotype. In addition, minor clones with distinct immunoglobulin sequences were detected in up to 9 % of sequenced cells but only 2 out of 12 of these clones showed aberrant immune phenotypes. The majority of these minor clones showed intraclonal silent nucleotide differences within the CDR3s and varying frequencies of somatic mutations in the immunoglobulin genes. Therefore the phenotypic range of multiple myeloma cells in the bone marrow is not confined to aberrant-phenotype plasma cells but extends to low frequencies of normal-phenotype B cells in line with the recently reported success of B cell-targeting cellular therapies in some patients. The majority of minor clones result from parallel non-malignant expansion.

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“…After isolation of RNA from 5 £ 10 6 -3 £ 10 7 PBMCs per sample using the RNeasy Mini Kit (QIAGEN, Hilden, Germany), cDNA synthesis with the Mint-2 cDNA synthesis kit (Evrogen, Moscow, Russia) allowed subsequent immunoglobulin isotype specific amplification as described previously. 29 Afterwards, preparation for NGS including amplicon extension with Illumina adapter sequences and unique barcodes was performed as described above.…”

Section: Next-generation Sequencing (Ngs) Of Immunoglobulin Heavy Chamentioning

confidence: 99%

Dynamic changes of the normal B lymphocyte repertoire in CLL in response to ibrutinib or FCR chemo-immunotherapy

et al. 2018

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Using next-generation immunoglobulin () sequencing and flow cytometry, we characterized the composition, diversity and dynamics of non-malignant B cells in patients undergoing treatment with the Bruton tyrosine kinase (BTK) inhibitor ibrutinib or chemo-immunotherapy with fludarabine, cyclophosphamide, and rituximab (FCR). During ibrutinib therapy, non-malignant B cell numbers declined, but patients maintained stable diversity and constant fractions of-mutated B cells. This indicates partial preservation of antigen-experienced B cells during ibrutinib therapy, but impaired replenishment of the normal B cell pool with naïve B cells. In contrast, after FCR we noted a recovery of normal B cells with a marked predominance of B cells with unmutated . This pattern is compatible with a deletion of pre-existing antigen-experienced B cells followed by repertoire renewal with antigen-naïve B cells. These opposite patterns in B cell dynamics may result in different responses towards neoantigens versus recall antigens, which need to be further defined.

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Next-generation sequencing of peripheral B-lineage cells pinpoints the circulating clonotypic cell pool in multiple myeloma

Abstract: Key Points Clonotypic B cells, long suspected to represent circulating stem-like cells, are consistently absent in the blood of myeloma patients. Malignant plasma cells frequently circulate in the peripheral blood, show evidence for clonal evolution, and may spread the disease.

Cited by 12 publications

References 23 publications

Monitoring multiple myeloma by next-generation sequencing of V(D)J rearrangements from circulating myeloma cells and cell-free myeloma DNA

Monitoring multiple myeloma by next-generation sequencing of V(D)J rearrangements from circulating myeloma cells and cell-free myeloma DNA

Clonal Expansion and Interrelatedness of Distinct B-Lineage Compartments in Multiple Myeloma Bone Marrow

Dynamic changes of the normal B lymphocyte repertoire in CLL in response to ibrutinib or FCR chemo-immunotherapy

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