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 In several studies,1-3 as well as in our own unpublished data, a preferential usage of several VH genes is observed.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 (χ2 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 1; χ2 analysis,P = .004).
The most frequent VH genes used in unmutated and mutated B-CLL across 4 studies
| Homology to germline VH gene . | Unmutated/mutated cases, by study* . | |||
|---|---|---|---|---|
| No. 1 . | No. 2 . | No. 3 . | No. 4 . | |
| VH1-69 | ||||
| Below 98% | 21/0 | 8/2 | 5/1 | 9/1 |
| Below 96% | NM | 8/2 | 5/1 | 9/1 |
| VH3-07 | ||||
| Below 98% | 0/4 | 0/5 | 1/8 | 0/5 |
| Below 96% | NM | 1/4 | 1/8 | 0/5 |
| VH3-21 | ||||
| Below 98% | 2/13 | 0/1 | 0/0 | 1/1 |
| Below 96% | 12/3 | 0/1 | 0/0 | 1/1 |
| VH3-23 | ||||
| Below 98% | 0/3 | 0/7 | 2/1 | 3/8 |
| Below 96% | NM | 1/6 | 2/1 | 4/7 |
| VH3-30 | ||||
| Below 98% | 2/4 | 3/8 | 2/0 | 6/4 |
| Below 96% | NM | 4/4 | 2/0 | 6/4 |
| Total number of patients | 119 | 74 | 64 | 84 |
| Overall frequency of cases, unmutated/mutated cases, % | ||||
| Below 98% | 58/42 | 35/65 | 48/52 | 45/55 |
| Below 96% | NM | 47/53 | 55/45 | 57/43 |
| Template type on which analysis was performed | DNA | DNA | RNA | RNA or DNA |
| Forward primer(s) | VH1 to VH6 | FR1/2† | VHL1 to VHL6 | VH1 to VH6 |
| Reverse primers(s) | JH | JH cons† | CH | JH |
| Homology to germline VH gene . | Unmutated/mutated cases, by study* . | |||
|---|---|---|---|---|
| No. 1 . | No. 2 . | No. 3 . | No. 4 . | |
| VH1-69 | ||||
| Below 98% | 21/0 | 8/2 | 5/1 | 9/1 |
| Below 96% | NM | 8/2 | 5/1 | 9/1 |
| VH3-07 | ||||
| Below 98% | 0/4 | 0/5 | 1/8 | 0/5 |
| Below 96% | NM | 1/4 | 1/8 | 0/5 |
| VH3-21 | ||||
| Below 98% | 2/13 | 0/1 | 0/0 | 1/1 |
| Below 96% | 12/3 | 0/1 | 0/0 | 1/1 |
| VH3-23 | ||||
| Below 98% | 0/3 | 0/7 | 2/1 | 3/8 |
| Below 96% | NM | 1/6 | 2/1 | 4/7 |
| VH3-30 | ||||
| Below 98% | 2/4 | 3/8 | 2/0 | 6/4 |
| Below 96% | NM | 4/4 | 2/0 | 6/4 |
| Total number of patients | 119 | 74 | 64 | 84 |
| Overall frequency of cases, unmutated/mutated cases, % | ||||
| Below 98% | 58/42 | 35/65 | 48/52 | 45/55 |
| Below 96% | NM | 47/53 | 55/45 | 57/43 |
| Template type on which analysis was performed | DNA | DNA | RNA | RNA or DNA |
| Forward primer(s) | VH1 to VH6 | FR1/2† | VHL1 to VHL6 | VH1 to VH6 |
| Reverse primers(s) | JH | JH cons† | CH | JH |
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 IgVHmutation 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.
IgVH3-21 gene usage in chronic lymphocytic leukemia
We 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 λ light chains and shorter average length of the complementarity determining region (CDR) 3.1-1 Interestingly, this subset of mutated VH3-21 cases also showed an inferior survival compared with the remaining mutated CLL cases. We have therefore suggested that mutated VH3-21 cases might constitute a new entity of CLL.
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.1-1-1-4 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.1-1-1-5 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.1-3-1-5Kröber et al have recently shown that a cutoff level at 97% gave the best separation of the 2 subsets regarding overall survival.1-6 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 Vλ2-14 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.
References
We thank Jan Philippé and Elke Boone for their helpful comments.
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