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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.


Related in: MedlinePlus

TCR clonality profiles from a diagnostic tissue suspicious for cutaneous T-cell lymphoma. GeneScan results of the Vγ–Jγ TCRG gene rearrangements using the BIOMED-2 TCRG-tubes A and B (a); and of the Vβ–Jβ and Dβ–Jβ TCRB gene rearrangements using the BIOMED-2 TCRB-tubes A, B and C, respectively (b). Shown are the results of the extracted DNA from the patients’ tissue (case 1) in duplicate (100 ng input/PCR), a monoclonal control sample (from a T-cell lymphoma tissue), and a polyclonal control (from a reactive lesion). TCR clonality analysis of case 1 showed two clonally rearranged TCRG genes, which fits to biallelic TCRG clonal rearrangements (a). Also a clonal TCRB-DJ incomplete rearrangement was detected (b: TCRB tube C). The rearrangement pattern of the TCRB-tube A shows a peak pattern, which is interpreted as polyclonal (low signal) (b). The peak at 273 bp is a so-called false peak as described before [16, Table 25], since this peak is particularly seen in samples with low numbers of T cells or in samples in which the clonal TCRB product is seen in one of the other TCRB tubes, which fits with the current case
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Fig6: TCR clonality profiles from a diagnostic tissue suspicious for cutaneous T-cell lymphoma. GeneScan results of the Vγ–Jγ TCRG gene rearrangements using the BIOMED-2 TCRG-tubes A and B (a); and of the Vβ–Jβ and Dβ–Jβ TCRB gene rearrangements using the BIOMED-2 TCRB-tubes A, B and C, respectively (b). Shown are the results of the extracted DNA from the patients’ tissue (case 1) in duplicate (100 ng input/PCR), a monoclonal control sample (from a T-cell lymphoma tissue), and a polyclonal control (from a reactive lesion). TCR clonality analysis of case 1 showed two clonally rearranged TCRG genes, which fits to biallelic TCRG clonal rearrangements (a). Also a clonal TCRB-DJ incomplete rearrangement was detected (b: TCRB tube C). The rearrangement pattern of the TCRB-tube A shows a peak pattern, which is interpreted as polyclonal (low signal) (b). The peak at 273 bp is a so-called false peak as described before [16, Table 25], since this peak is particularly seen in samples with low numbers of T cells or in samples in which the clonal TCRB product is seen in one of the other TCRB tubes, which fits with the current case

Mentions: Case 1 concerns a skin lesion of a patient suspected for cutaneous T-cell lymphoma. Two skin biopsies, from the palm of the hand and from the arm were used for diagnosis. The histology of the lesion, showing a polymorphous infiltrate consisting of CD2-positive lymphocytes, is shown in Fig. 5. For molecular analysis, snap-frozen tissue biopsies from two sites were used, containing 80% T cells of which at least 10% were suspected to be malignant T cells. The quality of the extracted DNA from both biopsies was fine, showing an amplification up to 400 bp of the BIOMED-2 control gene PCR (using the 100-, 200-, 300-, 400- amplicons) [16]. The clonality profile of this patient is presented in Fig. 6. For this patient, we showed that both skin biopsies from the patient showed identically sized TCR gene rearrangements, which supports the clonal nature of the skin lesions.Fig. 5


Pitfalls in TCR gene clonality testing: teaching cases.

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

TCR clonality profiles from a diagnostic tissue suspicious for cutaneous T-cell lymphoma. GeneScan results of the Vγ–Jγ TCRG gene rearrangements using the BIOMED-2 TCRG-tubes A and B (a); and of the Vβ–Jβ and Dβ–Jβ TCRB gene rearrangements using the BIOMED-2 TCRB-tubes A, B and C, respectively (b). Shown are the results of the extracted DNA from the patients’ tissue (case 1) in duplicate (100 ng input/PCR), a monoclonal control sample (from a T-cell lymphoma tissue), and a polyclonal control (from a reactive lesion). TCR clonality analysis of case 1 showed two clonally rearranged TCRG genes, which fits to biallelic TCRG clonal rearrangements (a). Also a clonal TCRB-DJ incomplete rearrangement was detected (b: TCRB tube C). The rearrangement pattern of the TCRB-tube A shows a peak pattern, which is interpreted as polyclonal (low signal) (b). The peak at 273 bp is a so-called false peak as described before [16, Table 25], since this peak is particularly seen in samples with low numbers of T cells or in samples in which the clonal TCRB product is seen in one of the other TCRB tubes, which fits with the current case
© Copyright Policy
Related In: Results  -  Collection

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

Fig6: TCR clonality profiles from a diagnostic tissue suspicious for cutaneous T-cell lymphoma. GeneScan results of the Vγ–Jγ TCRG gene rearrangements using the BIOMED-2 TCRG-tubes A and B (a); and of the Vβ–Jβ and Dβ–Jβ TCRB gene rearrangements using the BIOMED-2 TCRB-tubes A, B and C, respectively (b). Shown are the results of the extracted DNA from the patients’ tissue (case 1) in duplicate (100 ng input/PCR), a monoclonal control sample (from a T-cell lymphoma tissue), and a polyclonal control (from a reactive lesion). TCR clonality analysis of case 1 showed two clonally rearranged TCRG genes, which fits to biallelic TCRG clonal rearrangements (a). Also a clonal TCRB-DJ incomplete rearrangement was detected (b: TCRB tube C). The rearrangement pattern of the TCRB-tube A shows a peak pattern, which is interpreted as polyclonal (low signal) (b). The peak at 273 bp is a so-called false peak as described before [16, Table 25], since this peak is particularly seen in samples with low numbers of T cells or in samples in which the clonal TCRB product is seen in one of the other TCRB tubes, which fits with the current case
Mentions: Case 1 concerns a skin lesion of a patient suspected for cutaneous T-cell lymphoma. Two skin biopsies, from the palm of the hand and from the arm were used for diagnosis. The histology of the lesion, showing a polymorphous infiltrate consisting of CD2-positive lymphocytes, is shown in Fig. 5. For molecular analysis, snap-frozen tissue biopsies from two sites were used, containing 80% T cells of which at least 10% were suspected to be malignant T cells. The quality of the extracted DNA from both biopsies was fine, showing an amplification up to 400 bp of the BIOMED-2 control gene PCR (using the 100-, 200-, 300-, 400- amplicons) [16]. The clonality profile of this patient is presented in Fig. 6. For this patient, we showed that both skin biopsies from the patient showed identically sized TCR gene rearrangements, which supports the clonal nature of the skin lesions.Fig. 5

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.


Related in: MedlinePlus