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.

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.

Two conferences were sponsored by the International Society for Cutaneous Lymphomas (ISCL) to gain consensus on definitions and terminology for clinical use in erythrodermic cutaneous T-cell lymphoma (E-CTCL). Three subsets of E-CTCL were defined: Sezary syndrome ("leukemic phase" E-CTCL), erythrodermic mycosis fungoides (secondary E-CTCL that develops in patients with mycosis fungoides), and E-CTCL, not otherwise defined. The hematologic criteria recommended for Sezary syndrome are intended to identify patients with a worse prognosis compared with the other E-CTCL subsets and consist of one or more of the following: (1) an absolute Sezary cell count of 1000 cells/mm3 or more; (2) a CD4/CD8 ratio of 10 or higher caused by an increase in circulating T cells and/or an aberrant loss or expression of pan-T cell markers by flow cytometry; (3) increased lymphocyte counts with evidence of a T-cell clone in the blood by the Southern blot or polymerase chain reaction technique; or (4) a chromosomally abnormal T-cell clone. For staging purposes, it is proposed that these criteria define the B2 blood rating and that the B2 rating be considered equivalent to nodal involvement.

Update on erythrodermic cutaneous T-cell lymphoma: report of the International Society for Cutaneous Lymphomas.

PCR-based immunoglobulin (Ig)/T-cell receptor (TCR) clonality testing in suspected lymphoproliferations has largely been standardized and has consequently become technically feasible in a routine diagnostic setting. Standardization of the pre-analytical and post-analytical phases is now essential to prevent misinterpretation and incorrect conclusions derived from clonality data. As clonality testing is not a quantitative assay, but rather concerns recognition of molecular patterns, guidelines for reliable interpretation and reporting are mandatory. Here, the EuroClonality (BIOMED-2) consortium summarizes important pre- and post-analytical aspects of clonality testing, provides guidelines for interpretation of clonality testing results, and presents a uniform way to report the results of the Ig/TCR assays. Starting from an immunobiological concept, two levels to report Ig/TCR profiles are discerned: the technical description of individual (multiplex) PCR reactions and the overall molecular conclusion for B and T cells. Collectively, the EuroClonality (BIOMED-2) guidelines and consensus reporting system should help to improve the general performance level of clonality assessment and interpretation, which will directly impact on routine clinical management (standardized best-practice) in patients with suspected lymphoproliferations.

/pdf/euroclonality-biomed-2-guidelines-for-interpretation-and-mkge8cq2ra.pdf

EuroClonality/BIOMED-2 guidelines for interpretation and reporting of Ig/TCR clonality testing in suspected lymphoproliferations

Recent advances in flow cytometry: application to the diagnosis of hematologic malignancy

Hepatosplenic γδ T-cell lymphoma is a rare clinicopathologic entity with poor outcome: report on a series of 21 patients

Cutaneous T-cell infiltrates: analysis of T-cell receptor gamma gene rearrangement by polymerase chain reaction and denaturing gradient gel electrophoresis.

The recombination events of the gamma and beta T-cell receptor (TCR) loci were analysed in a series of 39 peripheral T-cell lymphomas (PTCLs) in association with the expression of TCR chains. In TCR alpha beta PTCLs, 22/23 cases showed a gamma-gene rearrangement while only 18/23 showed a concomitant beta-gene rearrangement. The germline configuration of the beta locus was found in angioimmunoblastic lymphadenopathy and lymphoepithelioid lymphomas. Three gamma delta PTCLs rearranged both gamma and beta genes. TCR silent PTCLs showed three different patterns of gamma- and beta-gene rearrangements. Three cases were in germline configuration for both loci; five cases had a rearranged gamma and a germline beta locus; and five cases had the two loci rearranged. Regarding the variable genes in the gamma-rearranged alleles, members of the V gamma I subgroup were the most frequently presented (39/50), followed by V gamma II, V gamma III, and V gamma IV (9/50, 1/50, and 1/50, respectively). Joining segment usage was as follows: J1 or J2 (32/50), JP1 or JP2 (17/50), and JP (1/50). Taken together, these data demonstrate that the gamma locus is more frequently rearranged whatever the TCR expression. The gamma-locus analysis provides a better diagnostic yield than the beta locus in the study of PTCL clonality.

Recombination pattern of the TCR gamma locus in human peripheral T-cell lymphomas.

Using Southern blotting for the diagnosis of clonality in peripheral T-cell lymphomas (PTCLs), analysis of the T-cell receptor (TCR) gamma gene rearrangement was shown to be more informative than that of the TCR beta gene rearrangement. In order to amplify every VJ gamma rearrangement, a polymerase chain reaction (PCR) procedure using newly designed GC-clamp primers has been developed. All primers can be mixed in a single multiplex PCR. PCR products are analysed by denaturing gradient gel electrophoresis (DGGE), providing tumour-specific imprints inasmuch as the procedure characterizes N sequence polymorphism at the VJ junctions. In a series of 30 PTCL cases, the PCR procedure demonstrated 27 cases to be clonally rearranged and failed in three cases. PCR was more accurate than Southern blotting, showing 47 rearranged gamma alleles, four of which were undetectable on the Southern blot. When lymphomas were studied at different sites and at relapse, the DGGE pattern remained unchanged. In PTCL, the proposed PCR is helpful for the diagnosis and staging of the disease and should improve the follow-up monitoring. The undetectability of clonal rearrangements in a few cases is discussed in the light of concepts of lymphomagenesis and T-cell differentiation.

VJ REARRANGEMENTS OF THE TCRγ LOCUS IN PERIPHERAL T‐CELL LYMPHOMAS: ANALYSIS BY POLYMERASE CHAIN REACTION AND DENATURING GRADIENT GEL ELECTROPHORESIS

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Cutaneous T-cell infiltrates: analysis of T-cell receptor gamma gene rearrangement by polymerase chain reaction and denaturing gradient gel electrophoresis.

Recombination pattern of the TCR gamma locus in human peripheral T-cell lymphomas.

VJ REARRANGEMENTS OF THE TCRγ LOCUS IN PERIPHERAL T‐CELL LYMPHOMAS: ANALYSIS BY POLYMERASE CHAIN REACTION AND DENATURING GRADIENT GEL ELECTROPHORESIS