Comprehensive assessment of potential multiple myeloma immunoglobulin heavy chain V-D-J intraclonal variation using massively parallel pyrosequencing

Renee C. Tschumper, Yan Asmann, Asif Hossain, Paul M. Huddleston, Xiaosheng Wu, Angela Dispenzieri, Bruce W. Eckloff, Diane F Jelinek

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

Multiple myeloma (MM) is characterized by the accumulation of malignant plasma cells (PCs) in the bone marrow (BM). MM is viewed as a clonal disorder due to lack of verified intraclonal sequence diversity in the immunoglobulin heavy chain variable region gene (IGHV). However, this conclusion is based on analysis of a very limited number of IGHV subclones and the methodology employed did not permit simultaneous analysis of the IGHV repertoire of non-malignant PCs in the same samples. Here we generated genomic DNA and cDNA libraries from purified MM BMPCs and performed massively parallel pyrosequencing to determine the frequency of cells expressing identical IGHV sequences. This method provided an unprecedented opportunity to interrogate the presence of clonally related MM cells and evaluate the IGHV repertoire of non-MM PCs. Within the MM sample, 37 IGHV genes were expressed, with 98.9% of all immunoglobulin sequences using the same IGHV gene as the MM clone and 83.0% exhibiting exact nucleotide sequence identity in the IGHV and heavy chain complementarity determining region 3 (HCDR3). Of interest, we observed in both genomic DNA and cDNA libraries 48 sets of identical sequences with single point mutations in the MM clonal IGHV or HCDR3 regions. These nucleotide changes were suggestive of putative subclones and therefore were subjected to detailed analysis to interpret: 1) their legitimacy as true subclones; and 2) their significance in the context of MM. Finally, we report for the first time the IGHV repertoire of normal human BMPCs and our data demonstrate the extent of IGHV repertoire diversity as well as the frequency of clonally-related normal BMPCs. This study demonstrates the power and potential weaknesses of in-depth sequencing as a tool to thoroughly investigate the phylogeny of malignant PCs in MM and the IGHV repertoire of normal BMPCs.

Original languageEnglish (US)
Pages (from-to)502-513
Number of pages12
JournalOncotarget
Volume3
Issue number4
StatePublished - Apr 2012

Fingerprint

Immunoglobulin Heavy Chains
Multiple Myeloma
Gene Library
Plasma Cells
Complementarity Determining Regions
Genomic Library
myeloma immunoglobulins
Genes
Illegitimacy
Phylogeny
Point Mutation
Immunoglobulins
Nucleotides
Clone Cells
Bone Marrow

Keywords

  • Heterogeneity
  • IGHV
  • Massively parallel sequencing
  • Multiple myeloma

ASJC Scopus subject areas

  • Oncology

Cite this

Comprehensive assessment of potential multiple myeloma immunoglobulin heavy chain V-D-J intraclonal variation using massively parallel pyrosequencing. / Tschumper, Renee C.; Asmann, Yan; Hossain, Asif; Huddleston, Paul M.; Wu, Xiaosheng; Dispenzieri, Angela; Eckloff, Bruce W.; Jelinek, Diane F.

In: Oncotarget, Vol. 3, No. 4, 04.2012, p. 502-513.

Research output: Contribution to journalArticle

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abstract = "Multiple myeloma (MM) is characterized by the accumulation of malignant plasma cells (PCs) in the bone marrow (BM). MM is viewed as a clonal disorder due to lack of verified intraclonal sequence diversity in the immunoglobulin heavy chain variable region gene (IGHV). However, this conclusion is based on analysis of a very limited number of IGHV subclones and the methodology employed did not permit simultaneous analysis of the IGHV repertoire of non-malignant PCs in the same samples. Here we generated genomic DNA and cDNA libraries from purified MM BMPCs and performed massively parallel pyrosequencing to determine the frequency of cells expressing identical IGHV sequences. This method provided an unprecedented opportunity to interrogate the presence of clonally related MM cells and evaluate the IGHV repertoire of non-MM PCs. Within the MM sample, 37 IGHV genes were expressed, with 98.9{\%} of all immunoglobulin sequences using the same IGHV gene as the MM clone and 83.0{\%} exhibiting exact nucleotide sequence identity in the IGHV and heavy chain complementarity determining region 3 (HCDR3). Of interest, we observed in both genomic DNA and cDNA libraries 48 sets of identical sequences with single point mutations in the MM clonal IGHV or HCDR3 regions. These nucleotide changes were suggestive of putative subclones and therefore were subjected to detailed analysis to interpret: 1) their legitimacy as true subclones; and 2) their significance in the context of MM. Finally, we report for the first time the IGHV repertoire of normal human BMPCs and our data demonstrate the extent of IGHV repertoire diversity as well as the frequency of clonally-related normal BMPCs. This study demonstrates the power and potential weaknesses of in-depth sequencing as a tool to thoroughly investigate the phylogeny of malignant PCs in MM and the IGHV repertoire of normal BMPCs.",
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