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CORRESPONDENCE With interest we read the report of Tobin et
al1 describing a new subset of B-cell chronic
lymphocytic leukemia (B-CLL). After mutation analysis of rearranged
immunoglobulin variable heavy chain (IgVH) genes, they
observed that B-CLL patients with somatically mutated
VH3-21 genes have an unfavorable clinical course
similar to B-CLL patients with unmutated immunoglobulin genes. Their presented data and conclusions need some comments. In several
studies,1-3 as well as in our own unpublished data, a preferential usage of several VH genes is observed. Data of
a selection of frequently used VH genes are summarized in
Table 1. In all studies
VH1-69 is highly represented in B-CLL and is almost
exclusively unmutated. But the frequent occurrence of the VH3-21 gene found by Tobin et al, namely in 13% of all
CLL, could not be confirmed by the 3 other studies indicated (Table 1;
studies 2-4). This discrepancy of usage of the VH3-21 gene
is statistically significant ( Several explanations for these different findings can be proposed: there might be a wrong annotation due to closely related germline IgVH genes. VH3-48 is the immunoglobulin gene with the highest homology to the VH3-21, namely, 10 basepair differences between both genes at nucleotide level; but it seems unlikely that, due to mutations at most of these positions, the germline gene is not correctly assigned. Or there might be methodologic differences between the different studies. Each of the studies inevitably excluded samples that failed on the IgVH mutation analysis, which is interesting information since this might give some insight in the sensitivity of the assays to detect clonal rearrangements. Finally, there might be a different patient population treated at each center or a particular patient group selected for IgVH sequencing. Next to the difference in usage of the VH3-21 gene, the relatively low mutation load observed in the somatically mutated cases described by Tobin et al is striking. Thirteen patients showed a sequence homology to the germline VH3-21 gene between 92.8% and 98%. Ten of those show a homology between 96% and 98% (Table 1, study 1). The threshold value of 98% to distinguish unmutated from mutated IgVH genes was originally chosen because polymorphisms, which are quite common in VH genes, can account for 2% of disparity.4 But an alternative use of a 96% threshold is currently under debate. Kröber et al,5 analyzing 300 CLL patients, found that a cutoff value of 96% separates more significantly 2 groups with a different overall survival than the cutoff value of 98%. When a threshold of 96% sequence homology is used for the patients studied by Tobin et al,1 12 of 15 B-CLL cases using the VH3-21 gene are within the unmutated cases group, which is known to have an unfavorable prognosis. Therefore the definition of a new subset of CLL patients as suggested by Tobin et al seems premature. Multicenter studies of a large number of patients for their IgVH mutation status and analyses of clinical data are needed to explain the observed differences in VH-gene usage and define the threshold with the most significant prognostic value.
Friedel Nollet, Barbara Cauwelier, Johan Billiet, Dominik Selleslag, Achiel Van
Hoof, Andries Louwagie, and Arnold Criel We thank Jan Philippé and Elke Boone for their helpful comments. References
1.
Tobin G, Thunberg U, Johnson A, et al.
Somatically mutated Ig VH3-21 genes characterize a new subset of chronic lymphocytic leukemia.
Blood.
2002;99:2262-2264 2. Fais F, Ghiotto F, Hashimoto S, et al. Chronic lymphocytic leukemia B cells express restricted sets of mutated and unmutated antigen receptors. J Clin Invest. 1998;102:1515-1525[Medline] [Order article via Infotrieve].
3.
Hamblin TJ, Davis Z, Gardiner A, Oscier DG, Stevenson FK.
Unmutated Ig VH genes are associated with a more aggressive form of chronic lymphocytic leukemia [see comments].
Blood.
1999;94:1848-1854 4. Matsuda F, Shin EK, Nagaoka H, et al. Structure and physical map of 64 variable segments in the 3'0.8-megabase region of the human immunoglobulin heavy-chain locus. Nat Genet. 1993;3:88-94[CrossRef][Medline] [Order article via Infotrieve]. 5. Kröber A, Bühler A, Kienle D, et al. Analysis of VDJ rearrangement structure and VH mutation status in chronic lymphocytic leukemia [abstract]. Blood 2001;98:358a. 6. Aubin J, Davi F, Nguyen-Salomon F, et al. Description of a novel FR1 IgH PCR strategy and its comparison with three other strategies for the detection of clonality in B cell malignancies. Leukemia. 1995;9:471-479[Medline] [Order article via Infotrieve].
Response:IgVH3-21 gene usage in chronic lymphocytic leukemiaWe have recently reported a restricted usage of the
VH3-21 gene in mutated B-cell chronic lymphocytic leukemia
(CLL), where the VH3-21-utilizing cases displayed
distinctive characteristics such as clonal expression of In their letter Nollet et al have compared our findings with 2 prior studies on VH gene usage and their own unpublished results, and they found it remarkable that we have shown a significantly higher number of VH3-21-utilizing CLL cases compared with the other studies.1-4 Nollet et al have speculated on the basis of the divergent results. First, they have addressed the correctness of the VH gene alignment. We have used 3 different germline databases, and it is not likely that we have aligned them incorrectly, especially since all 3 databases indicate VH3-21 gene usage and the closest related gene, VH3-48, displays 7-bp differences to the VH3-21 gene. Second, Nollet et al have suggested that methodological differences could account for the disagreement between the studies. Certainly, different primer systems may have varied capability to amplify different VH genes, but we find it rather unlikely that this would explain the diverse results. Third, they have argued that the selection of patients could differ between the studies. We believe that the selection of patient material is the most probable factor explaining the different findings, since none of the referred studies are population-based. Prior studies have reported different frequencies of the mutated and unmutated subsets, and the median survival for these 2 groups has varied, thus indicating that different patient populations have been selected for VH gene analysis.1-5 In addition, we cannot rule out that the difference in frequency of VH3-21 usage compared with previous studies reflects a different genetic background or environmental factors in the Scandinavian population/countries. Nollet et al have also brought up the discussion about the appropriate cutoff level to distinguish mutated and unmutated CLL. We have chosen the mutation border of 98% to eliminate the possibility of polymorphic sites in accordance with previous CLL studies.3-5 Kröber et al have recently shown that a cutoff level at 97% gave the best separation of the 2 subsets regarding overall survival.6 Using this threshold, some of our mutated VH3-21 cases would indeed be interpreted as unmutated. But the true biologic level that distinguishes somatically mutated VH genes from unmutated ones is currently unknown and may also differ for different VH genes. Most of our mutated VH3-21 cases have a low frequency of mutations, but almost half of the mutations were found in hypermutation hotspot regions (RGYW/WRCY), indicating that they were introduced by the somatic hypermutation mechanism rather than representing VH3-21 gene polymorphisms. To solve this issue, future studies are warranted to analyze the germline VH genes in cases that display a low number of somatic hypermutations and compare the germline sequence with the sequence of the clonal rearrangement. Despite the VH gene mutation status, we still believe that
the VH3-21-utilizing cases represent an additional entity,
especially since they display certain features with short and in some
cases identical CDR3 structure and a highly preferential
V
Gerard Tobin, Ulf Thunberg, Anna Johnson, Ola Söderberg, Johan Botling, Gunilla Enblad, Jan Sällström, Christer Sundström, Göran Roos, and Richard Rosenquist
References
1.
Tobin G, Thunberg U, Johnson A, et al.
Somatically mutated Ig VH3-21 genes characterize a new subset of chronic lymphocytic leukemia.
Blood.
2002;99:2262-2264 2. Fais F, Ghiotto F, Hashimoto S, et al. Chronic lymphocytic leukemia B cells express restricted sets of mutated and unmutated antigen receptors. J Clin Invest. 1998;102:1515-1525[Medline] [Order article via Infotrieve].
3.
Hamblin TJ, Davis Z, Gardiner A, Oscier DG, Stevenson FK.
Unmutated Ig V(H) genes are associated with a more aggressive form of chronic lymphocytic leukemia.
Blood.
1999;94:1848-1854
4.
Damle RN, Wasil T, Fais F, et al.
Ig V gene mutation status and CD38 expression as novel prognostic indicators in chronic lymphocytic leukemia.
Blood.
1999;94:1840-1847
5.
Maloum K, Davi F, Merle-Beral H, et al.
Expression of unmutated VH genes is a detrimental prognostic factor in chronic lymphocytic leukemia.
Blood.
2000;96:377-379 6. Kröber A, Seiler T, Benner A, et al. VH mutation status, CD38 expression level, genomic aberrations, and survival in chronic lymphocytic leukemia. Blood. In press.   CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
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2 analysis for
VH3-21 usage: studies 1 versus 2, P = .013;
studies 1 versus 3, P = .008; studies 1 versus 4, P = .020). In addition, a statistically significant
difference is observed for the overall mutation frequency between
studies 1 and 2 (Table 
light
chains and shorter average length of the complementarity determining
region (CDR) 3.
2-14 usage. We have now identified 24 cases with
VH3-21 usage (out of 215 analyzed CLL cases), where 17 were
mutated and 7 unmutated according to the 98% cutoff level. The
VH3-21 CLL cases from our published study originated from
the central and northern counties in Sweden, but we have now also found
VH3-21-utilizing CLL cases from both Finland and southern
Sweden. In addition, a German research group has also identified
VH3-21-positive cases with similar genotypic findings that
showed an inferior outcome irrespective of their mutational status (S. Stilgenbauer, personal communication, April 3, 2002). But it
will be important to study larger numbers of VH3-21-positive cases to confirm our findings and to fully
evaluate the prognostic impact of VH3-21 gene usage in CLL.
Additionally, multicenter and population-based studies are needed to
investigate the clinical significance of VH gene usage in
general in CLL.
