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Pitfalls in TCR gene clonality testing: teaching cases.

Groenen PJ, Langerak AW, van Dongen JJ, van Krieken JH - J Hematop (2008)

Bottom Line: Several immunobiological and technical pitfalls that should be taken into account to avoid misinterpretation of data are addressed in this report.Furthermore, we discuss the need to integrate the molecular data with those from immunohistology, and preferably also flow cytometric immunophenotyping, for appropriate interpretation.Such an interactive, multidisciplinary diagnostic model guarantees integration of available data to reach the most reliable diagnosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Radboud University Nijmegen Medical Centre, Geert Grooteplein 24, 6525 GA, Nijmegen, The Netherlands, P.Groenen@pathol.umcn.nl.

ABSTRACT
Clonality testing in T-lymphoproliferations has technically become relatively easy to perform in routine laboratories using standardized multiplex polymerase chain reaction protocols for T-cell receptor (TCR) gene analysis as developed by the BIOMED-2 Concerted Action BMH4-CT98-3936. Expertise with clonality diagnostics and knowledge about the biology of TCR gene recombination are essential for correct interpretation of TCR clonality data. Several immunobiological and technical pitfalls that should be taken into account to avoid misinterpretation of data are addressed in this report. Furthermore, we discuss the need to integrate the molecular data with those from immunohistology, and preferably also flow cytometric immunophenotyping, for appropriate interpretation. Such an interactive, multidisciplinary diagnostic model guarantees integration of available data to reach the most reliable diagnosis.

No MeSH data available.


PCR analysis of TCRB gene rearrangements. a Schematic diagram of the human TCRB locus on chromosome band 7q34. The figure is adapted from the international ImMunoGeneTics database [8, 9]. Only one of the rearrangeable nonpolymorphic functional Vβ gene segments is depicted (in blue). Both Cβ gene segments (Cβ1 and Cβ2, depicted in green) are preceded by a Dβ gene (Dβ1 and Dβ2, depicted in pink) and a Jβ cluster, depicted in light blue. b Schematic diagram of Vβ–Jβ and Dβ–Jβ rearrangements. The 23 Vβ primers, 13 Jβ primers, and two Dβ primers are combined in three tubes: tube A with 23 Vβ primers and nine Jβ primers, tube B with 23 Vβ primers and four Jβ primers, and tube C with two Dβ primers and 13 Jβ primers. The Vβ primers cover approximately 90% of all Vβ gene segments. Adapted from BIOMED-2 report [14]
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Fig1: PCR analysis of TCRB gene rearrangements. a Schematic diagram of the human TCRB locus on chromosome band 7q34. The figure is adapted from the international ImMunoGeneTics database [8, 9]. Only one of the rearrangeable nonpolymorphic functional Vβ gene segments is depicted (in blue). Both Cβ gene segments (Cβ1 and Cβ2, depicted in green) are preceded by a Dβ gene (Dβ1 and Dβ2, depicted in pink) and a Jβ cluster, depicted in light blue. b Schematic diagram of Vβ–Jβ and Dβ–Jβ rearrangements. The 23 Vβ primers, 13 Jβ primers, and two Dβ primers are combined in three tubes: tube A with 23 Vβ primers and nine Jβ primers, tube B with 23 Vβ primers and four Jβ primers, and tube C with two Dβ primers and 13 Jβ primers. The Vβ primers cover approximately 90% of all Vβ gene segments. Adapted from BIOMED-2 report [14]

Mentions: In the late 1990s, a European consortium of ~45 laboratories (BIOMED-2 Concerted Action BMH4-CT98-3936) was initiated with the aim to establish a highly reliable standard in polymerase chain reaction (PCR)-based clonality testing, for B-cell as well as for T-cell malignancies. Figure 1a illustrates a schematic diagram of the human TCRB locus, showing one of the Vβ gene elements from the Vβ gene segments, 39–47 are qualified as functional and belong to 23 families. Both Cβ gene segments (Cβ1 and Cβ2) are preceded by a Dβ gene (Dβ1 and Dβ2) and a Jβ cluster, which comprises six (Jβ1.1–Jβ1.6) and seven (Jβ2.1–Jβ2.7) functional Jβ segments. A schematic diagram of the human TCRG locus on chromosome band 7p14 is illustrated in Fig. 2a. The TCRG locus contains only nine Vγ gene segments that have been shown to undergo rearrangement. The BIOMED-2-approved set of multiplex TCRB and TCRG PCR tubes, with the position of the primers, are shown in Figs. 1b and 2b, respectively. In the BIOMED-2 approach, the issue of false-negativity was addressed at several levels: (1) design of complete sets of primers to cover all possible V–J rearrangements; (2) inclusion of incomplete rearrangements as additional targets (e.g., Dβ–Jβ); (3) evaluation of multiple targets per sample. This concept of complementarity of targets was only feasible for routine testing by designing multiplex PCR reaction mixtures consisting of multiple primers. The other challenge was to prevent false-positivity, which was achieved by introducing standardized, reliable methods for evaluation of PCR products: heteroduplex analysis [10, 11], and GeneScan fragment analysis [12, 13] (Fig. 3). Following its technical evaluation [14], the multiplex protocol was successfully applied to different well-defined WHO lymphoma entities with unprecedented high frequencies of malignant cases showing clonality [15–20]. The sensitivity of the BIOMED-2 multiplex PCR clonality assays has been evaluated during the primer design and testing phase. The detection of virtually all TCR gene rearrangements has a sensitivity of at least 10% [14]. Multiplex PCR-based clonality testing and assessment by GeneScan and/or heteroduplex analysis have become a worldwide standard [21–24]. Technically, TCR clonality testing and assessment by GeneScan and/or heteroduplex analysis has become relatively easy to perform. However, knowledge of and experience with TCR rearrangement analysis and inclusion of quality checks in the routine diagnostic setting are essential to avoid misinterpretation of the data.Fig. 1


Pitfalls in TCR gene clonality testing: teaching cases.

Groenen PJ, Langerak AW, van Dongen JJ, van Krieken JH - J Hematop (2008)

PCR analysis of TCRB gene rearrangements. a Schematic diagram of the human TCRB locus on chromosome band 7q34. The figure is adapted from the international ImMunoGeneTics database [8, 9]. Only one of the rearrangeable nonpolymorphic functional Vβ gene segments is depicted (in blue). Both Cβ gene segments (Cβ1 and Cβ2, depicted in green) are preceded by a Dβ gene (Dβ1 and Dβ2, depicted in pink) and a Jβ cluster, depicted in light blue. b Schematic diagram of Vβ–Jβ and Dβ–Jβ rearrangements. The 23 Vβ primers, 13 Jβ primers, and two Dβ primers are combined in three tubes: tube A with 23 Vβ primers and nine Jβ primers, tube B with 23 Vβ primers and four Jβ primers, and tube C with two Dβ primers and 13 Jβ primers. The Vβ primers cover approximately 90% of all Vβ gene segments. Adapted from BIOMED-2 report [14]
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2713482&req=5

Fig1: PCR analysis of TCRB gene rearrangements. a Schematic diagram of the human TCRB locus on chromosome band 7q34. The figure is adapted from the international ImMunoGeneTics database [8, 9]. Only one of the rearrangeable nonpolymorphic functional Vβ gene segments is depicted (in blue). Both Cβ gene segments (Cβ1 and Cβ2, depicted in green) are preceded by a Dβ gene (Dβ1 and Dβ2, depicted in pink) and a Jβ cluster, depicted in light blue. b Schematic diagram of Vβ–Jβ and Dβ–Jβ rearrangements. The 23 Vβ primers, 13 Jβ primers, and two Dβ primers are combined in three tubes: tube A with 23 Vβ primers and nine Jβ primers, tube B with 23 Vβ primers and four Jβ primers, and tube C with two Dβ primers and 13 Jβ primers. The Vβ primers cover approximately 90% of all Vβ gene segments. Adapted from BIOMED-2 report [14]
Mentions: In the late 1990s, a European consortium of ~45 laboratories (BIOMED-2 Concerted Action BMH4-CT98-3936) was initiated with the aim to establish a highly reliable standard in polymerase chain reaction (PCR)-based clonality testing, for B-cell as well as for T-cell malignancies. Figure 1a illustrates a schematic diagram of the human TCRB locus, showing one of the Vβ gene elements from the Vβ gene segments, 39–47 are qualified as functional and belong to 23 families. Both Cβ gene segments (Cβ1 and Cβ2) are preceded by a Dβ gene (Dβ1 and Dβ2) and a Jβ cluster, which comprises six (Jβ1.1–Jβ1.6) and seven (Jβ2.1–Jβ2.7) functional Jβ segments. A schematic diagram of the human TCRG locus on chromosome band 7p14 is illustrated in Fig. 2a. The TCRG locus contains only nine Vγ gene segments that have been shown to undergo rearrangement. The BIOMED-2-approved set of multiplex TCRB and TCRG PCR tubes, with the position of the primers, are shown in Figs. 1b and 2b, respectively. In the BIOMED-2 approach, the issue of false-negativity was addressed at several levels: (1) design of complete sets of primers to cover all possible V–J rearrangements; (2) inclusion of incomplete rearrangements as additional targets (e.g., Dβ–Jβ); (3) evaluation of multiple targets per sample. This concept of complementarity of targets was only feasible for routine testing by designing multiplex PCR reaction mixtures consisting of multiple primers. The other challenge was to prevent false-positivity, which was achieved by introducing standardized, reliable methods for evaluation of PCR products: heteroduplex analysis [10, 11], and GeneScan fragment analysis [12, 13] (Fig. 3). Following its technical evaluation [14], the multiplex protocol was successfully applied to different well-defined WHO lymphoma entities with unprecedented high frequencies of malignant cases showing clonality [15–20]. The sensitivity of the BIOMED-2 multiplex PCR clonality assays has been evaluated during the primer design and testing phase. The detection of virtually all TCR gene rearrangements has a sensitivity of at least 10% [14]. Multiplex PCR-based clonality testing and assessment by GeneScan and/or heteroduplex analysis have become a worldwide standard [21–24]. Technically, TCR clonality testing and assessment by GeneScan and/or heteroduplex analysis has become relatively easy to perform. However, knowledge of and experience with TCR rearrangement analysis and inclusion of quality checks in the routine diagnostic setting are essential to avoid misinterpretation of the data.Fig. 1

Bottom Line: Several immunobiological and technical pitfalls that should be taken into account to avoid misinterpretation of data are addressed in this report.Furthermore, we discuss the need to integrate the molecular data with those from immunohistology, and preferably also flow cytometric immunophenotyping, for appropriate interpretation.Such an interactive, multidisciplinary diagnostic model guarantees integration of available data to reach the most reliable diagnosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Radboud University Nijmegen Medical Centre, Geert Grooteplein 24, 6525 GA, Nijmegen, The Netherlands, P.Groenen@pathol.umcn.nl.

ABSTRACT
Clonality testing in T-lymphoproliferations has technically become relatively easy to perform in routine laboratories using standardized multiplex polymerase chain reaction protocols for T-cell receptor (TCR) gene analysis as developed by the BIOMED-2 Concerted Action BMH4-CT98-3936. Expertise with clonality diagnostics and knowledge about the biology of TCR gene recombination are essential for correct interpretation of TCR clonality data. Several immunobiological and technical pitfalls that should be taken into account to avoid misinterpretation of data are addressed in this report. Furthermore, we discuss the need to integrate the molecular data with those from immunohistology, and preferably also flow cytometric immunophenotyping, for appropriate interpretation. Such an interactive, multidisciplinary diagnostic model guarantees integration of available data to reach the most reliable diagnosis.

No MeSH data available.