Characterization of feline immunoglobulin heavy chain variable region genes for the molecular diagnosis of B-cell neoplasia

J. A. Werner, J. C. Woo, William Vernau, P. S. Graham, Robert A Grahn, Leslie A Lyons, Peter F Moore

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Abstract

To develop a molecular-based assay so that the diagnosis of feline B-cell neoplasia can be facilitated, we have characterized 24 feline immunoglobulin heavy chain variable region (IGH V) complementary DNA (cDNA) transcripts. Structural homology with rearranged human IGH V genes was found, and the sequence information was used to design a feline-specific polymerase chain reaction (PCR)-based assay to amplify the complementarity determining region 3 as a marker for B-cell clonality. Conserved primers derived from the second and third framework regions of V gene segments were used in conjunction with 2 sequence-specific primers and 1 degenerate primer derived from the J gene segments. Each PCR reaction was run in duplicate, and both native and denatured PCR products were evaluated using polyacrylamide gel electrophoresis. Formalin-fixed, paraffin-embedded (FFPE) tissue sections from cats with confirmed B-cell neoplasia (diffuse large B-cell lymphoma, plasmacytoma, and myeloma) were examined, and 15/22 (68.2%) cats produced results indicative of the presence of a monoclonal population of B cells. The evaluation of denatured PCR products (heteroduplex analysis) facilitated a more accurate interpretation in 3/15 (20%) cats. Pseudoclonality was a major reason for the failure to detect monoclonality. Poor DNA quality is a significant concern and was responsible for the removal of 2 cats from the study. Using this assay, FFPE normal feline lymphoid tissues and unfixed peripheral blood mononuclear cells were determined to be composed of polyclonal populations of B cells. This assay represents a useful adjunctive diagnostic tool for the diagnosis and investigation of feline B-cell lymphoproliferative disorders.

Original languageEnglish (US)
Pages (from-to)596-607
Number of pages12
JournalVeterinary Pathology
Volume42
Issue number5
DOIs
StatePublished - Sep 2005

Fingerprint

immunoglobulin heavy chains
Immunoglobulin Heavy Chains
Felidae
B-lymphocytes
B-Lymphocytes
cats
neoplasms
Cats
Genes
Neoplasms
Polymerase Chain Reaction
genes
polymerase chain reaction
Paraffin
Formaldehyde
Heteroduplex Analysis
assays
Complementarity Determining Regions
formalin
alkanes

Keywords

  • Cats
  • Clonality
  • Complementarity determining region 3
  • Heteroduplex analysis
  • Immunoglobulin heavy chain gene
  • Lymphoma
  • Myeloma
  • Plasmacytoma
  • Polymerase chain reaction

ASJC Scopus subject areas

  • veterinary(all)

Cite this

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title = "Characterization of feline immunoglobulin heavy chain variable region genes for the molecular diagnosis of B-cell neoplasia",
abstract = "To develop a molecular-based assay so that the diagnosis of feline B-cell neoplasia can be facilitated, we have characterized 24 feline immunoglobulin heavy chain variable region (IGH V) complementary DNA (cDNA) transcripts. Structural homology with rearranged human IGH V genes was found, and the sequence information was used to design a feline-specific polymerase chain reaction (PCR)-based assay to amplify the complementarity determining region 3 as a marker for B-cell clonality. Conserved primers derived from the second and third framework regions of V gene segments were used in conjunction with 2 sequence-specific primers and 1 degenerate primer derived from the J gene segments. Each PCR reaction was run in duplicate, and both native and denatured PCR products were evaluated using polyacrylamide gel electrophoresis. Formalin-fixed, paraffin-embedded (FFPE) tissue sections from cats with confirmed B-cell neoplasia (diffuse large B-cell lymphoma, plasmacytoma, and myeloma) were examined, and 15/22 (68.2{\%}) cats produced results indicative of the presence of a monoclonal population of B cells. The evaluation of denatured PCR products (heteroduplex analysis) facilitated a more accurate interpretation in 3/15 (20{\%}) cats. Pseudoclonality was a major reason for the failure to detect monoclonality. Poor DNA quality is a significant concern and was responsible for the removal of 2 cats from the study. Using this assay, FFPE normal feline lymphoid tissues and unfixed peripheral blood mononuclear cells were determined to be composed of polyclonal populations of B cells. This assay represents a useful adjunctive diagnostic tool for the diagnosis and investigation of feline B-cell lymphoproliferative disorders.",
keywords = "Cats, Clonality, Complementarity determining region 3, Heteroduplex analysis, Immunoglobulin heavy chain gene, Lymphoma, Myeloma, Plasmacytoma, Polymerase chain reaction",
author = "Werner, {J. A.} and Woo, {J. C.} and William Vernau and Graham, {P. S.} and Grahn, {Robert A} and Lyons, {Leslie A} and Moore, {Peter F}",
year = "2005",
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doi = "10.1354/vp.42-5-596",
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T1 - Characterization of feline immunoglobulin heavy chain variable region genes for the molecular diagnosis of B-cell neoplasia

AU - Werner, J. A.

AU - Woo, J. C.

AU - Vernau, William

AU - Graham, P. S.

AU - Grahn, Robert A

AU - Lyons, Leslie A

AU - Moore, Peter F

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N2 - To develop a molecular-based assay so that the diagnosis of feline B-cell neoplasia can be facilitated, we have characterized 24 feline immunoglobulin heavy chain variable region (IGH V) complementary DNA (cDNA) transcripts. Structural homology with rearranged human IGH V genes was found, and the sequence information was used to design a feline-specific polymerase chain reaction (PCR)-based assay to amplify the complementarity determining region 3 as a marker for B-cell clonality. Conserved primers derived from the second and third framework regions of V gene segments were used in conjunction with 2 sequence-specific primers and 1 degenerate primer derived from the J gene segments. Each PCR reaction was run in duplicate, and both native and denatured PCR products were evaluated using polyacrylamide gel electrophoresis. Formalin-fixed, paraffin-embedded (FFPE) tissue sections from cats with confirmed B-cell neoplasia (diffuse large B-cell lymphoma, plasmacytoma, and myeloma) were examined, and 15/22 (68.2%) cats produced results indicative of the presence of a monoclonal population of B cells. The evaluation of denatured PCR products (heteroduplex analysis) facilitated a more accurate interpretation in 3/15 (20%) cats. Pseudoclonality was a major reason for the failure to detect monoclonality. Poor DNA quality is a significant concern and was responsible for the removal of 2 cats from the study. Using this assay, FFPE normal feline lymphoid tissues and unfixed peripheral blood mononuclear cells were determined to be composed of polyclonal populations of B cells. This assay represents a useful adjunctive diagnostic tool for the diagnosis and investigation of feline B-cell lymphoproliferative disorders.

AB - To develop a molecular-based assay so that the diagnosis of feline B-cell neoplasia can be facilitated, we have characterized 24 feline immunoglobulin heavy chain variable region (IGH V) complementary DNA (cDNA) transcripts. Structural homology with rearranged human IGH V genes was found, and the sequence information was used to design a feline-specific polymerase chain reaction (PCR)-based assay to amplify the complementarity determining region 3 as a marker for B-cell clonality. Conserved primers derived from the second and third framework regions of V gene segments were used in conjunction with 2 sequence-specific primers and 1 degenerate primer derived from the J gene segments. Each PCR reaction was run in duplicate, and both native and denatured PCR products were evaluated using polyacrylamide gel electrophoresis. Formalin-fixed, paraffin-embedded (FFPE) tissue sections from cats with confirmed B-cell neoplasia (diffuse large B-cell lymphoma, plasmacytoma, and myeloma) were examined, and 15/22 (68.2%) cats produced results indicative of the presence of a monoclonal population of B cells. The evaluation of denatured PCR products (heteroduplex analysis) facilitated a more accurate interpretation in 3/15 (20%) cats. Pseudoclonality was a major reason for the failure to detect monoclonality. Poor DNA quality is a significant concern and was responsible for the removal of 2 cats from the study. Using this assay, FFPE normal feline lymphoid tissues and unfixed peripheral blood mononuclear cells were determined to be composed of polyclonal populations of B cells. This assay represents a useful adjunctive diagnostic tool for the diagnosis and investigation of feline B-cell lymphoproliferative disorders.

KW - Cats

KW - Clonality

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KW - Heteroduplex analysis

KW - Immunoglobulin heavy chain gene

KW - Lymphoma

KW - Myeloma

KW - Plasmacytoma

KW - Polymerase chain reaction

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U2 - 10.1354/vp.42-5-596

DO - 10.1354/vp.42-5-596

M3 - Article

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JO - Veterinary Pathology

JF - Veterinary Pathology

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