Leukemia (1997) 11, 1055–1062 1997 Stockton Press All rights reserved 0887-6924/97 $12.00 TECHNICAL REPORT BTS Leukemia Automated high resolution PCR fragment analysis for identification of clonally rearranged immunoglobulin heavy chain genes B Linke1, I Bolz1, A Fayyazi2, M von Hofen1, Ch Pott1, J Bertram, W Hiddemann1 and M Kneba1 1Department of Internal Medicine, Division of Hematology/Oncology and 2Institute of Pathology, Georg-August University, Robert-Koch-Str 40, D 37075 Goettingen, Germany The development of rapid polymerase chain reaction (PCR) region (CDR3).2,6–10 Clonal expansions of B cells carry ident- protocols for amplification of rearranged heavy chain immuno- ical copies of unique V -N-D -N-J junctional region globulin (IgH) gene sequences has facilitated the identification H H H of clonal IgH rearrangements in non-Hodgkin’s lymphomas sequences. Therefore, rearranged IgH genes are used as mark- (NHL) and leukemias of B lineage. In the present report we have ers for lineage and clonality in human lymphoid neoplasms explored the recently described improved strategy for assess- and DNA sequences at the junction of VH,DH and JH segments ment of clonality of rearranged immunoglobulin heavy chain can be used as clone-specific markers in individual (IgH) genes in more detail in a series of 101 B cell malignancies patients.6,9–20 and 50 polyclonal controls. The assay is based on an IgH-PCR In the past Southern blotting techniques with radiolabeled with an automated fluorescence-based strategy for PCR detec- probes for specific immune gene segments have been exten- tion of IgH gene rearrangements. Third complementarity 19,21–23 determining region (IgH-CDR3) sequences were amplified sively applied to the study of ALL and NHL patients. using fluorescent dye labeled consensus primers homologous However, this technique is time-consuming, labour-intensive, to the corresponding variable (VH) and joining (JH) gene seg- relatively insensitive and impractical for clinical studies. The ments in combination with a thermostable proofreading DNA development of PCR has markedly facilitated the characteriz- polymerase. PCR products were size separated on a high resol- ation of IgH junctions by direct DNA-sequencing and ution polyacrylamide gel and analyzed for clonality by exact size determination and fluorescence quantification in an auto- enhanced the sensitivity for the detection of these clonal mar- mated DNA sequencer. PCR findings obtained with the optim- kers. We recently showed that the amplification efficacy of ized IgH-CDR3-PCR assay showed an overall monoclonality IgH–CDR3 junctions with consensus PCR primers and the detection rate of 97% (97 of 101 cases with B cell neoplasms). accuracy of PCR results is significantly improved by appli- The specificity was 100% as determined by analysis of 50 con- cation of the thermostable UITma DNA polymerase with an trols, all of which gave polyclonal PCR results. We found a high inherent 3′ to 5′ exonuclease activity.24 In the present report rate of monoclonal IgH-CDR3-PCR results not only in the leuke- mias and diffuse lymphoma but also in the group of follicular we have explored this new strategy for detection of clonally lymphoma, where a high rate of false negative results is fre- rearranged IgH-CDR3 junctions in detail in a large series of B quently reported in the literature. In summary, we identified cell malignancies. Clone-specific IgH CDR3 sequences were monoclonal IgH-CDR3 junctions in 55 out of 59 cases (93%) amplified by the polymerase chain reaction (PCR) using flu- with B cell lymphoma and in 42 of 42 (100%) cases with leuke- orescent dye labeled consensus primers. PCR products were mia, immunocytoma and multiple myeloma. The results demon- size separated on a high resolution polyacrylamide gel and strate that automated fluorescence detection of IgH-CDR3-PCR products is an ideal tool for detection of clonal and polyclonal analyzed by fluorescence quantification and size determi- lymphoid B cells. In combination with allele-specific primers nation in an automated DNA sequencer. The gene scanning the procedure may improve current experimental approaches results were verified by sequencing randomly selected PCR to detect occult malginant B cells during initial staging and products. The results demonstrate that automated fluor- follow-up of NHL and ALL patients. escence detection of IgH-CDR3-PCR products is a powerful Keywords: lymphoma; PCR; IgH-CDR3; sequencing; gene tool for the detection of clonal and polyclonal B cells. scanning Materials and methods Introduction Clinical samples and cell lines During B cell development, IgH genes undergo a complex rearrangement process of their variable (VH), diversity (DH) A total of 101 newly diagnosed patients with B cell non-Hodg- 1–5 and joining (JH) gene segments. The recombination sites of kin’s lymphomas and B lineage acute or chronic leukemias the IgH genes form the structure of the highly diverse VH– were selected for this study (Table 1). All patients were admit- DH–JH junctions known as third complementarity determining ted to the Department of Internal Medicine of the University of Go¨ttingen between October 1985 and June 1996 (19 cases of chronic B cell leukemia (B-CLL), three hairy cell leukemia Correspondence: M Kneba (HCL), 12 acute B cell leukemias (ALL), two cases with mul- Received 8 October 1996; accepted 10 April 1997 tiple myeloma, six stage IV immunocytomas (IC), 19 cases of Technical report B Linke et al 1056 Table 1 IgH-CDR3, t(14;18) mbr and t(11;14) results Diagnosis n t(14;18) mbr- t(11;14) PCR IgH-CDR3-PCR/Gene-scanning results PCRa positive cases + positive cases n /nt(%) + p 1R 2R Total number of n /nt(%) positive cases B-NHL CB-CC 19 10/19 (53) 0/19 (0) 1/19 (5) 16/19 (84) 2/19 (11) 18/19 (95) CC 11 0/11 (0) 3/11 (27) 0/11 (0) 9/11 (82) 2/11 (18) 11/11 (100) DLC (IB, CB) 24 2/24 (8) 0/24 (0) 2/24 (8) 15/24 (63) 7/24 (29) 22/24 (92) Burkitt 5 0/5 (0) 0/5 (0) 1/5 (20) 4/5 (80) 0/5 (0) 4/5 (80) Total 59 13/59 (22) 3/59 (5) 4/59 (7) 44/59 (74) 11/59 (19) 55/59 (93) B cell leukemia and multiple myeloma CLL, ALL, IC, 40 ND ND 0/40 (0) 26/40 (65) 14/40 (35) 40/40 (100) HCL Myeloma 2 ND ND 0/2 (0) 2/2 (100) 0/2 (0) 2/2 (100) Total 101 4/101 (4) 72/101 (72) 25/101 (25) 97/101 (97) Controls 50 ND ND 50/50 (100) 0/50 (0) 0/50 (0) 0/50 (0) + + n, the number of tested cases in this diagnosis group; n 2/nt, denotes the number of cases with a monoclonal gene scanning result (n ) per number of tested cases (nt); DLC, diffuse large cells lymphoma, for the other diagnoses see Materials and methods; p, polyclonal; 1R and 2R, indicate the number of monoclonally rearranged alleles in individual cases. ND, not determined. aOnly the major breakpoint region of the bcl-2 gene was analyzed. low-grade centroblastic-centrocytic (CB-CC), 11 mantle cell PCR conditions (centrocytic, CC) lymphomas, 24 cases of high-grade centrobl- astic (CB) and immunoblastic NHL (IB), and five cases with The PCR primers and cycling conditions used in this study Burkitt’s lymphoma). The lymphomas were categorized followed the recently described protocol.24 Briefly, amplifi- 25 according to the updated Kiel classification, using conven- cation reactions with consensus VH and JH primers were per- tional morphological and immunohistological techniques. formed in an automated thermocycler (model 480; Perkin The B cell leukemia cases and the polyclonal blood and bone Elmer) with 0.5–1.0 mg of sample DNA in a final volume of marrow samples were classified by flow cytometry. As poly- 30 ml reaction buffer containing 40 mM of each deoxynucleo- clonal controls DNA samples from normal peripheral blood tide, 5 pmol of each primer and 2 units of U1Tma DNA poly- 24 lymphocytes (18 cases), patients with nonmalignant disorders merase (Perkin Elmer) for 35 cycles with primers VH and JH. (10 bone marrow samples) and diagnostic lymph node biopsy Cycle conditions were: denaturation at 96°C for 1 min. with specimens of patients with unspecific lymphadenitis (seven annealing/extension at 55°C for 45 s). Hot-start-initiated PCR cases), three cases with toxoplasmosis, Hodgkin’s disease was performed by addition of BioWax (Biozym, Hameln, (HD; five cases) and peripheral T cell NHL (seven cases) were Germany) to the reaction tubes. The VH primer was HPLC- studied. Fifteen of these negative controls were tested by purified and labeled at its 5′ end with 6-FAM (6- Southern analysis and all showed a germline JH-configuration. carboxyfluorescein) (ABI) generating blue fluorescence signals All patients were admitted to the Department of Internal Medi- after excitation with an argon ion laser in the automated cine of the University of Goettingen for diagnosis and treat- sequencer (model 373A; ABI). ment. The B cell line SKW 6.4 (American Type Culture Collec- tion TIB 215) served as monoclonal control. IgH Southern and IgH PCR results of 44 patients with lymphoma or leukemia of Fluorescent fragment analysis (gene scanning) B lineage have been reported recently in a short communication.24 For automated fluorescent fragment analysis, aliquots of the PCR products (1–2 ml) were mixed with equal amounts of DNA preparation loading buffer (5 volumes formamide:1 volume EDTA; 10% dextraneblue) and 0.5 ml of the internal size standard Genes- High molecular weight DNA was extracted using an auto- can-350-ROX (ABI). After denaturation for 3 min at 95°C pro- mated DNA extractor (model 341; Applied Biosystems (ABI), ducts were analyzed by automatic fluorescence quantification Weiterstadt, Germany) from snap-frozen peripheral blood, and size determination using the computer program GENES- bone marrow, or lymph node biopsy specimens that were col- CAN 672 (ABI).