Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT98-3936.
In a European BIOMED-2 collaborative study, multiplex PCR assays have successfully been developed and standardized for the detection of clonally rearranged immunoglobulin (Ig) and T-cell receptor (TCR) genes and the chromosome aberrations t(11;14) and t(14;18). This has resulted in 107 different primers in only 18 multiplex PCR tubes: three VH-JH, two DH-JH, two Ig kappa (IGK), one Ig lambda (IGL), three TCR beta (TCRB), two TCR gamma (TCRG), one TCR delta (TCRD), three BCL1-Ig heavy chain (IGH), and one BCL2-IGH. The PCR products of Ig/TCR genes can be analyzed for clonality assessment by heteroduplex analysis or GeneScanning. The detection rate of clonal rearrangements using the BIOMED-2 primer sets is unprecedentedly high. This is mainly based on the complementarity of the various BIOMED-2 tubes. In particular, combined application of IGH (VH-JH and DH-JH) and IGK tubes can detect virtually all clonal B-cell proliferations, even in B-cell malignancies with high levels of somatic mutations. The contribution of IGL gene rearrangements seems limited. Combined usage of the TCRB and TCRG tubes detects virtually all clonal T-cell populations, whereas the TCRD tube has added value in case of TCRgammadelta(+) T-cell proliferations. The BIOMED-2 multiplex tubes can now be used for diagnostic clonality studies as well as for the identification of PCR targets suitable for the detection of minimal residual disease.
van Dongen
J J M
JJ
Department of Immunology, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands. j.j.m.vandongen@erasmusmc.nl
Langerak
A W
AW
Brüggemann
M
M
Evans
P A S
PA
Hummel
M
M
Lavender
F L
FL
Delabesse
E
E
Davi
F
F
Schuuring
E
E
García-Sanz
R
R
van Krieken
J H J M
JH
Droese
J
J
González
D
D
Bastard
C
C
White
H E
HE
Spaargaren
M
M
González
M
M
Parreira
A
A
Smith
J L
JL
Morgan
G J
GJ
Kneba
M
M
Macintyre
E A
EA
D002869:Chromosome Aberrations; D002999:Clone Cells; D017931:DNA Primers; D005062:European Union; D015329:Gene Rearrangement, T-Lymphocyte; D006801:Humans; D007136:Immunoglobulins / Q000235:genetics*; D008232:Lymphoproliferative Disorders / Q000175:diagnosis* / Q000235:genetics*; D018365:Neoplasm, Residual / Q000175:diagnosis / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards*; D011948:Receptors, Antigen, T-Cell / Q000235:genetics*; D012015:Reference Standards; D015203:Reproducibility of Results
D017931:DNA Primers; D007136:Immunoglobulins; D011948:Receptors, Antigen, T-Cell