Rapid, multifluorescent TCRG Vγ and Jγ typing: application to T cell acute lymphoblastic leukemia and to the detection of minor clonal populations

Abstract

Detection of clonal T cell receptor ␥ (TCRG) gene rearrangements by PCR is widely used in both the diagnostic assessment of lymphoproliferative disorders and the follow-up of acute lymphoblastic leukaemia (ALL), when residual positivity in excess of 10 −3 at morphological complete remission is increasingly recognised to be an independent marker of poor prognosis. This is largely based on specific detection of V-J rearrangements from childhood cases. We describe rapid, multifluorescent V␥ and J␥ PCR typing of multiplex amplified diagnostic samples, as applied to 46 T-ALL. These strategies allow selected analysis of appropriate cases, immediate identification of V␥ and J␥ segments in over 95% of alleles, improved resolution and precision sizing and a sensitivity of detection at the 10 −2 -10 −3 level. We demonstrate preferential V-J combinations but no difference in V-J usage between children and adults, nor between SIL-TALI-negative and -positive cases. A combination of fluorescent multiplex and V␥-J␥-specific monoplex follow-up, as described here, will allow detection of both significant clonal evolution and of the diagnostic clone at a level of prognostic significance, by techniques which can readily be applied to large-scale prospective studies for which real-time analysis is required.

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

“…Approximate relative use of the different Vg and Jg segments in precursor B-ALL, T-ALL, and T-NHL is shown in Table 18. 1,30,111,112 The identification of Vg and Jg usage is not of purely academic interest, since specific amplification is required for MRD analysis. 113 We undertook to develop a minimal number of multiplex TCRG strategies that would maintain optimal sensitivity and informativity, minimize the risk of false-positive results, and allow simple Vg and Jg identification, including by heteroduplex analysis or monofluorescent GeneScanning strategies.…”

“…Sensitivity of detection varied between 1 and 10%, as a function of the complexity of the polyclonal repertoire and the position of the clonal rearrangement relative to the polyclonal Gaussian peak. 112 Interpretation of cVg11 rearrangements can be difficult, since the normal repertoire is extremely restricted and since these primitive rearrangements are often present in subclones.…”

Response to antiviral treatment in hepatitis C virus-associated marginal zone lymphomas

“…PCR for bcl2-IgH, IgH and TCR gamma rearrangement was performed, when mentioned, as previously described. 37,38 HCV serology was determined by two ELISA tests of third generation. HCV RNA was quantified by RT-PCR (Amplicor HCV, Roche).…”

Clinicobiological, Immunophenotypic, and Molecular Characteristics of Monoclonal CD56−/+dim Chronic Natural Killer Cell Large Granular Lymphocytosis

“…PCR amplification of the TCR-␥ gene was done using the BIOMED-2 primer sequences and protocols, 25 with a limit of detection varying from 1 to 10%, depending on the polyclonal TCR repertoire complexity, and the type of TCR-␥ gene rearrangements. 26 …”