Section 3 -

Standardization of PCR Techniques for Assessing Lymphoid Clonality; the Biomed-2 Experience Thierry Jo Molina Université Paris-Descartes, Faculté de Médecine AP-HP Hôtel-Dieu Service d'Anatomie et de Cytologie Pathologiques 1 place du Parvis Notre Dame 75181 Paris Cedex O4 France

PCR techniques have been developed for now more than 15 years in laboratories involved in hematopathology mainly focusing in the detection of clonality of lymphoid cell populations targeting Immunoglobulin (Ig) and T-cell receptor (TCR) genes. These techniques are very useful in cases where histopathology and immunophenotypic analysis is equivocal. The overall result should nevertheless always be interpreted in relation to the clinical presentation. Most of the published methods use consensus primers for completely rearranged antigen receptor genes, mainly the Ig heavy chain (IgH) and TCR gamma (TCRG) genes. However, worldwide, many different protocols are used, each with different sensitivity and specificity. A multi-center study performed by a US Association of Molecular Pathology group showed a remarkable heterogeneity in the performance of and results from IgH PCR with diagnostic sensitivity ranging from 90% to as low as 20%, when evaluating the same specimen [1]. The significant drop-off in detection of formalin-fixed paraffin-embedded tissue was also clearly demonstrated in this study supporting the need of freezing part of a biopsy or of a tumor, especially when lymphoma is suspected. Such studies underline the need for standardization of PCR techniques and further validation, optimization of standardized protocols. This has been one of the major aim of the Biomed-2 concerted action BMH4 CT98-3936 directed by JJM Van Dongen, (Rotterdam, Netherlands) initiated to develop standardized reagents and methods for PCR-based clonality diagnostics. A total of 47 institutes from seven European countries collaborated in this project [2]. This action resulted in highly efficient multiplex PCR protocols using multiple primers for virtually all different functional gene segments of the Ig and TCR genes. After an initial testing of the multiplex PCR primer tubes, a large series of almost 600 WHO-classified lymphomas as well as more than 100 histomorphologically reactive lesions were evaluated to cover the spectrum of diagnostic situations in hematopathology. All cases were reviewed by national pathology panels and approximately 10% of the samples as well as unexpected results in the molecular assays were reviewed by the Biomed-2 Pathology Review Panel. In this concerted action, it was decided to include IgH, IgK (kappa), IgL (lambda) as well as TCRB (beta), TCRG (gamma), TCRD (delta) as targets. A total of 97 new primers were designed, representing 418 single PCR tests, then gathered in 14 Ig-TCR multiplex PCR tubes: 3 for complete IgH (V-J), 2 for IgH incomplete (D-J), 2 for IGK, 1 for IGL, 2 for complete TCRB, 1 for incomplete TCRB, 2 for TCRG, 1 for all types of TCRD. After the designing of the primers was approved by the group between October 1998 (design) and April 2001 (final primer approval), a standardized biomed-2 PCR protocol was developed based on pre-existing experience from earlier European collaborative studies. Genomic DNA was extracted from frozen biopsies and DNA samples were distributed to two different laboratories. A standardized PCR protocol was defined using a determined volume of reaction, a defined concentration of dNTPs, PCR primers (10pmol of each primer), DNA (100 ng), Taq polymerase as well as buffer; The MgCl2 concentrations could be a little optimized and cycling conditions were defined depending on the PCR equipment (classical vs newer). In each laboratory, the techniques for the analysis of PCR products were defined and standardized using either heteroduplex analysis of PCR products on a 6% polyacrylamide gel or capillary electrophoresis (CE) scanning using fluorochrome labelled primers. Some meetings were devoted to the consensus analysis of PCR products resulting from heteroduplex analysis or CE, because not all the teams were familiar enough with the interpretation and analysis, a round table discussion significantly iproved the reliability and reproduciblility of the results among the laboratories. A general testing on 90 southern-blot defined DNA samples was initially performed. Based on the experience of the 30 PCR laboratories all relevant background information per multiplex tubes was defined as well as the size ranges of the monoclonal and polyclonal PCR products. A control gene primer set for quality assessment of DNA from paraffin embedded tissue was also defined. The ability to amplify a 300 bp control led to 50 to 100% of clonality detection according to the target gene compared to frozen tissue. The ability to amplify 100 to 200bp led from 0 to 40% of clonality detection compared to frozen tissue. Therefore, although it was possible to use formalin fixed tissue, better results were obtained using frozen tissue. When evaluating large series of lymphomas and reactive tissues (overall 700),with techniques performed in duplicate in two different labs for the same sample, we found a limited intralaboratory variation (34 out of 3000 PCR in the group involved in the analysis of reactive lesions), concerning mainly weak clonal products being detected mainly in CE but not in Heteroduplex [3]. Interlaboratory discrepancies in the same group were observed in 70 out of the 3000 duplicate PCR tests. In such cases, a third laboratory was involved in order to come to final conclusions. Cases were often classified as clear polyclonal samples (polyclonal in all PCR reactions), probably polyclonal samples (weak clonal band in a limited number of multiplex PCRs and mostly detected with a single technique), probably clonal samples (clear clonality in a single Ig/TCR locus), clear clonal samples (clear clonality pattern in multiple multiplex PCRs). Interestingly, our study showed that IgKappa PCR in addition with IgH (complete and incomplete) were able to detect B cell clonality in 99% of all B-cell lymphomas tested including 109 follicular lymphomas, 41 Marginal zone lymphomas and 109 diffuse large B cell lymphomas, known to be heavily somatically mutated [4]. Analogously, TCRB (complete and incomplete) together with TCRG were able to detect T cell clonality in 99% of peripheral T cell lymphomas (PTCL), 100% in PTCL NOS and 95% even in Angioimmunoblastic (AIL) T cell lymphoma [5]. Studied separately, we found clonality in only 80% of anaplastic T/NK which is not surprising because some of these cases are known as null ALCL. Analyzing 106 histologically defined reactive lesions, 90% showed a polyclonal (75%) or probably polyclonal (15%) pattern. In 10% of the cases, a clear clonal pattern was found including two missed lymphomas in national review and nine difficult cases for histopathology such as early involvement of lymph node by AIL T cell lymphoma, mycosis fungoides or clonal monotypic plasmacytosis in a ruptured spleen of unknown significance. Based on our study [6], we suggest that this standardized protocol could be used in clinical management of suspected lymphoproliferations with a rational use of the different targets, taking into account the results of the more sensitive tests and the pathological pattern. However, we cannot exclude the interesting applications of using the three IgH (VJ) and the two IgK tubes for B cell clonality, and the three TCRB and the two TCRG tubes for T cell clonality. This approach has the advantage of confirmation of clonality based on multiple positive results. Our protocol needs to be evaluated and validated by other groups. A recent study by Mc Clure et al, [7] focusing on Ig genes target, found an overall good performance of our Biomed-2 protocol, and therefore constitutes a very interesting validation study. However, as any classification in pathology, our standardized Biomed-2 protocol will without any doubt,undergo further innovation and evolution in the future. References Bagg A, Braziel RM, Arber DA? Et al. Immunoglobulin heavy chain gene analysis in lymphomas. A multi-center study demonstrating the heterogeneity of performance of polymerase chain reaction assays. J Mol Diagn, 2002, 4, 81-89. Van Dongen JJM, Langerak AW, Brüggemann M, et al 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. Leukemia 2003;17:2257-2317. Langerak AW, Molina TJ, Lavender FL, et al .PCR-based clonality testing in tissue samples with reactive lymphoproliferations: usefulness and pitfalls: A study from the BIOMED-2 Concerted Action BMH4-CT98-3936. Leukemia, 2006 in press. Evans PAS, Pott Ch, Groenen PJTA et al . Significantly improved PCR-based clonality testing in B-cell malignancies by use of multiple immunoglobulin gene targets: Report of the BIOMED-2 Concerted Action BHM4-CT98-3936. Leukemia, 2006, submitted Brüggemann M, White H, Gaulard P, et al.. Powerful strategy for PCR-based clonality assessment in T-cell malignancies: Report of the BIOMED-2 Concerted Action BHM4-CT98-3936. Leukemia, 2006, in press Van Krieken JHJM, Langerak AW, MacIntyre EA, et al. Improved reliability of lymphoma diagnostics via PCR based clonality testing. Report of Biomed -2 concerted action BHM4-CT98-3936. Leukemia, 2006, submitted. McClure RF, Kaur P, Pagel E et al. Validation of immunoglobulin gene rearrangement detection by PCR using commercially available biomed-2 primers. Leukemia, 2006, 20, 176-9.