Clinical Laboratory Method for Detection of IGHV Mutation Status in Patients with CLL Validated by Igblast and IMGT/V-QUEST
Chronic lymphocytic leukemia (CLL) is the most frequent type of adult leukemia in Western countries. Recently, new molecular prognostic markers like 17p deletion, 11q deletion, 13q deletion, trisomy 12, the mutational status of the immunoglobulin variable heavy chain genes (IGHV) genes, expression of ZAP-70 and CD38 were identified as prognostically significant. The CLL patients with mutated IGHV have a more favorable prognosis while non-mutated cases with the mutation's number less than 2% compared to the germline sequence suffer from more aggressive diseases. Here, we describe a clinical laboratory method for the detection of the mutation status of IGHV in patients with CLL using reverse transcription PCR and dideoxysequencing, and the evaluation using two immunoglobulin databases IMGT/V-QUEST and IgBLAST. We analyzed 37 different clonal rearrangements in 35 patients. Using two different databases, we identified 13 mutated and 24 non-mutated clones. The most preferred subfamilies were VH1, VH3, and VH4. The CLLs using the subfamily 1-69 were all non-mutated. Unlike previous reports, there were no significant differences between the used databases observed. The clinical trials are already incorporating new prognostic molecular markers such IGHV mutational status, so it is important to use standardized clinical laboratory methods and databases for a reliable identification of the mutation status in CLL.
Shanafelt TD. Predicting clinical outcome in CLL: how and why. Hematology. Am Soc Hematol Educ Program. 2009:421-9.
Hallek M, German CLL Study Group, 2008. Prognostic factors in chronic lymphocytic leukemia. Ann Oncol. 2008; Suppl.4: iv51-iv53.
Van Dongen, JJM, Langerak AW, Bruggemenn M, Evans PAS, Hummel M et al. Design and standardization of PCR primers and protocols for detection ov clonal immunoglobulin and T-cell receptor gene recombinations i suspect lymphorpoliferations: Report of the BIOMED-2 concerted action BMH4-CT98-3936. Leukemia 2003; 17: 2257-2317.
Szankasi P, Bahler DW. Clinical laboratory analysis of immunoglobulin heavy chain variable region genes for chronic lymphocytic leukemia prognosis. J Molec Diagn. 2010; 12: 244-249.
Tobin G, Thunberg U, Karlsson K, Murray F, Laurell A, et al. Subsets with restricted immunoglobulin gene rearrangements features indicate a role of antigen selection in the development of chronic lymphocytic leukemia. Blood 2004; 104: 2879-2885.
Hamblin TJ, Davis Z, Gardiner A, Oscier DG, Stevenson FK. Non-mutated Ig VH genes are associated with a more aggressive form of chronic lymphocytic leukemia. Blood 1999; 94: 1848-1854.
Giudicelli V, Chaume D, Lefranc MP. IMGT/V-QUEST, an integrated software program for immunoglobulin and T cell receptor V-J and V-D-J rearrangement analysis. Nucleic Acid Res. 2004; 32: W435-W440.
Ghia P, Stamatopoulos K, Belessi C, Moreno C, Stilgenbauer S, et al.: ERIC recommendations on IGHV gene mutational status analysis in chronic lymphocytic leukemia. Leukemia 2007; 21: 1-3
Pekova S, Baran-Marszak F, Schwarz J, Matoska V. Mutated or non-mutated? Which database to choose when determinig the IgVH mutational status in chronic lymphocytic leukemia? Haematologica 2006; 91: e11-e12.
Davi F, Rosenquist R, Ghia P, Belessi C, Stamatopouloss K. Determination of IGHV gene mutational status in chronic lymphocytic leukemia: bioinformatics advances meet clinical needs. Leukemia 2008; 22, 212-214; doi:10.1038/sj.leu.2404969;
Souto-Carneiro MM, Krenn V, Hermann R, Konig A, Muller-Hermelink HK. IgVH genes from different anatomical regions, with different histopathological patterns of rheumatoid arthritis patient suggest cyclic-reentry of mature synovial B-cells in the hypermutation process. Arthritis Res. 2000; 2: 303-314.
Pekova S, Markova J, Pajer P, Dvorak M, Cetkovsky, P, Schwarz J. Touch-down reverse transcriptase-PCR detection of IgVH rearrangement and Sybr-green-based real-time RT-PCR quantitation of minimal residual disease in patients with chronic lymphocytic leukemia. Mol Diagn. 2005; 9: 23-34.
Marasca R, Maffei R, Morselli M, Zucchini P, Castelli I, et al. Immunoglobulin mutational status detected through single-round amplification of partial VH region represents a good prognostic marker for clinical outcome in chronic lymphocytic leukemia. J Molec Diagnostics. 2005; 7: 566-574.
Valekova L, Fedorova J, Rumanova S, Stasko J, Flochova E, et al. Gastric MALT lymphoma: the role of flow cytometry. Acta Med Martiniana 2007; 3: 24-28.
Kjarfan-Dabaja MA, Chavez JC, Khorfan KA, Pinilla-Ibarz J. Clinical and therapeutic implications of the mutational status of IGHV in patients with chronic lymphocytic leukemia. Cancer 2008; 13: 898-906.
Shanafelt TD. Predicting clinical outcome in CLL: how and why? Hematology Am Soc Hematol Educ Program. 2009; 421-429.