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Chronic lymphocytic leukemia patients with a V1-69 gene rearrangement do not have inferior survival with respect to patients that express other unmutated VH genes
Leukemia Research 31 (2007) 245–248
Brief communication
Chronic lymphocytic leukemia patients with a V1-69 gene rearrangement do not have inferior survival with respect to patients that express other unmutated VH genes Irina Panovska-Stavridis ∗ , Martin Ivanovski, Nikola Siljanovski, Lidija Cevreska, Dimitar G. Efremov Department of Hematology, Faculty of Medicine, Vodnjanska 17, 1000 Skopje, Republic of Macedonia Received 9 May 2006; received in revised form 9 May 2006; accepted 13 May 2006 Available online 23 June 2006
Abstract Recent studies indicate that VH gene usage in B-CLL may have prognostic impact independently of VH gene mutation status. The V1-69 gene is the most frequently rearranged VH gene in B-CLL and is almost always unmutated. We therefore investigated whether patients with a V1-69 gene rearrangement differ in clinical course and outcome with respect to patients expressing other unmutated VH genes. We show that V1-69 B-CLLs constitute a uniform group of patients that more often present at advanced clinical stages and require early treatment, but their survival does not differ significantly from patients with other unmutated VH genes. © 2006 Elsevier Ltd. All rights reserved. Keywords: Chronic lymphocytic leukemia; Prognosis; VH gene mutation status; V1-69
1. Introduction B-cell chronic lymphocytic leukemia (CLL) is a heterogeneous disease with a highly variable clinical course, which can at least in part be predicted by the mutational status of the immunoglobulin variable heavy-chain (VH ) genes. Patients with CLL B-cells that express unmutated VH genes typically have a more aggressive disease and shorter survival than CLL patients with mutated VH genes [1,2]. Recent studies indicate that VH gene usage may have a prognostic impact that is independent of the VH gene mutation status. This is especially pertinent to the V3-21 gene which is associated with an unfavourable clinical course even in mutated cases [3]. A second example is the V3-72 gene which has been observed in a series of highly sta-
∗
Corresponding author. Tel.: +389 2 31 47 782; fax: +389 2 30 93 610. E-mail address: dr [email protected] (I. Panovska-Stavridis).
0145-2126/$ – see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.leukres.2006.05.008
ble and indolent CLL patients [4]. Finally, the V1-69 gene, which is the most frequently rearranged VH gene in CLL, is almost always unmutated and as such is invariably associated with unfavourable prognosis. The leukemic immunoglobulins encoded by this gene display characteristic features, such as biased usage of certain D and JH genes, long third complementarity determining regions (CDR3s) and frequent pairing with a few variable light-chain (VL ) genes [5,6]. Moreover, recent analysis of the expression of several genes that regulate B-cell receptor signaling, cell cycle progression and apoptosis have shown that V1-69 cases have a distinct expression pattern as compared to CLL B-cells that use other unmutated VH genes [7]. The striking molecular similarities between the V1-69 cases indicate that they represent a rather homogeneous group with certain biological features that are distinct from other VH -unmutated cases. Therefore, we investigated in a series of CLL patients with a long follow-up whether cases with a V1-69 gene rearrangement have a different clinical course
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I. Panovska-Stavridis et al. / Leukemia Research 31 (2007) 245–248
and outcome from patients that express other unmutated VH genes.
3. Results and discussion The leukemic immunoglobulin was encoded by the V169 gene in 25 of the 106 investigated CLL samples (23.6%). The VH gene sequence was always unmutated (>98% homology) and was completely identical to the germline V1-69 sequence in 22 cases. The V1-69 gene was joined to the D33 gene segment in 9 cases (Table 1). Six of these cases had a highly conserved heavy chain CDR3 containing the amino acid motif FWSGY, and in two of the three investigated cases the V1-69-encoded heavy chain was paired with a V1gencoded variable light-chain region. A second conserved CDR3 motif (DIVVVPAAI) was identified in three cases. In two of these cases the V1-69-encoded heavy chain was paired with the V02 or the highly homologous V018 variable light chain. V1-69 encoded immunoglobulins belonging to other previously described sets of stereotyped CLL antigen receptors were also sporadically identified in the remaining cases (cases MF9 and MJ21)[6]. The high frequency of V1-69 gene cases in CLL patients originating from a small geographic region and the significant structural similarities between the leukemic immunoglobulins further point to an important role for antigen stimulation in the pathogenesis of this CLL subset. Among the 40 randomly selected non-V1-69 CLLs, the VH gene sequence was unmutated in 19, mutated in 14, and could not be determined in seven cases. Comparison of the V1-69 cases with those expressing other unmutated VH genes showed that the two subsets do not differ with respect to age, gender, median Ly counts and total tumor mass (TTM)
2. Materials and methods Blood samples were collected from 106 consecutive CLL patients that had been diagnosed at our institution between 1977 and 2000. Diagnosis was based on standard morphologic and immunophenotypic criteria for CLL. Following RNA isolation from peripheral blood mononuclear cells by standard techniques, all samples were analyzed by allele-specific immunoglobulin VH gene fingerprinting to identify cases with a V1-69 gene rearrangement [8]. Nucleotide sequencing of the rearranged VH genes was subsequently performed in all V1-69-expressing cases and in 40 of the remaining cases using standard RT/PCR and nucleotide sequencing procedures. In selected cases the VL gene sequence was also determined. Candidate germline genes were assigned by searching the VBASE directory. Sequences with less than 2% differences from germline VH genes were considered unmutated. Correlations between the different CLL subsets were made with the use of the t-test, Wilcoxon rank-sum test and Fisher exact test. The median time to treatment and survival were estimated by the method of Kaplan and Meier and assessed by the log-rank test. Data for patients that had not received treatment were regarded as censored. Statistical analyses were performed using the SigmaStat 3.1 program (Systat Software Inc., Richmond, CA). Table 1 Structural characteristics of the V1-69 encoded immunoglobulin variable regions Patient
D gene
JH gene
CDR3 sequence
MO6 HA-IDR CLL#86 HA1 MJ2 MF15 MO10 MO13 HA2 MF12 MJ26 NI MO12 MF9 MD2 HA-SC MJ21 MF7 MJ5 MJ29 HA3 MM6 MJ19 MF5 CLL#132
D2-2 D2-2 D2-15 D3-3 D3-3 D3-3 D3-3 D3-3 D3-3 D3-3 D3-3 D3-3 D3-22 D3-16 D3-10 D3-10 D3-10 D1-26 D2-21 D2-2 D5-5 D5-5 D6-19 D6-19 D6-6
JH6 JH6 JH6 JH6 JH6 JH5 JH5 JH4 JH4 JH4 JH6 JH6 JH4 JH3 JH6 JH4 JH6 JH4 JH6 JH2 JH6 JH5 JH4 JH4 JH6
ERCGKL—DIVVVPAAIRYYGMDV MDP——DIVVVPAAIAYYYGMDV RRGGPSPVVDIVVVVAATRFIYYYYYYMDV AGRIVT-IFWSGYLPNYYYYGMDV GGWSTNYDFWSGYSPYYYYYYMDV DGLER–DFWSGYMRVTPYNWFDP ELVPPQ–FWSGYFYSWFDP DDT–YYDFWSGYYY DN–YYYDFWSGYYPGVD PTQFWSWGEFGYYFDY FRIEGVVSYYYYYYGMDV DLSPIWSGYYMRDYYYGMDV ASRGYYYD-SSGYYSGVY GGPYDYVWGSYRPNDAFDI GMVRGVITYYYYYYYMDV GTILWFGELLYPLDY AMVRGVITYYYYYYYMDV DNYLFKRIVGPLDY DDIVKIGPCGGDCYFPVGYYYYGMDV VRGWKYQLLYQEDWYFDL VSMGQLWLQRNYYYYMDV ESIGGYSYGYIYNWFDP PSTVTTYSSGWSTIFDY EEGVSSDSSGWNY DSAGIAARFISMTDYYYYYGMDV
VL /JL gene V02/J1 V018/J3 V1g/J3b
V1g/J2
V1c/J2 VA27/J5 V1c/J3b
V018/J1
I. Panovska-Stavridis et al. / Leukemia Research 31 (2007) 245–248
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Table 2 Clinical and laboratory features of CLL patients stratified according to VH gene usage and mutational status Patient characteristics
V1-69
Other VH unmutated
VH mutated
V1-69 vs. other VH unmutated
Male:female Median age (years) Median follow-up (range) Median Ly count at dg (×106 /ml) Total tumor mass scorea at dg
21:4 60 56 months (6–109) 53.0 11.92
13:6 60 59 months (20–97) 48.0 10.92
8:6 62 77 months (53–300) 29.8 6.03
P = n.s., Fisher exact test P = n.s., t-test P = n.s., t-test P = n.s., t-test P = n.s., t-test
Rai stage at dg Low risk Intermediate risk High risk
1 15 9
2 16 1
6 6 2
Low + intermediate vs. high risk, P = 0.027, Fisher exact test
a Total tumor mass score (TTM) was calculated as the sum of the square root of the number of peripheral blood lymphocytes per ml, the diameter of the largest palpable lymph node in centimeters (cm) and the enlargement of the spleen below the left costal margin in cm. High TTM was defined as a sum equal to or greater than 9.0.
Fig. 1. Kaplan–Meier survival curves of CLL patients stratified according to VH gene usage and mutation status. (A) Time from diagnosis to initial therapy. (B) Overall survival.
score at diagnosis (Table 2). However, the percentage of cases presenting at an advanced stage was significantly higher in the V1-69 subgroup (36% of cases in Rai high risk category) than in the subgroup with other unmutated VH genes (5% of cases in Rai high risk category, P = 0.027). Eighty percent of the V1-69 cases and 42% of the cases with other unmutated VH genes had received treatment immediately following diagnosis (Fig. 1(A)). However, median time to treatment was not significantly different between the two subgroups (1 month and 9 months for V1-69 and other VH unmutated cases, respectively, P = 0.085). To date, 24 of the V1-69 patients (96%) and 15 of the patients with other unmutated VH genes (79%) have died, with median survival times of 56 and 60 months, respectively (P = 0.9) (Fig. 1(B)). For comparison, median survival of the 14 VH mutated cases from this series was 125 months (P < 0.0001 with respect to both subgroups with unmutated VH genes).
Altogether, these data show that CLL patients with a V169 gene rearrangement constitute a uniform group characterized by a progressive clinical course and frequent presentation at an advanced disease stage. Although in our series CLL patients with a V1-69 gene rearrangement more frequently received treatment immediately following diagnosis, median time to treatment and median survival were not significantly different from patients expressing other unmutated VH genes. Thus, in this single-institution study that included a series of patients with a relatively long follow-up, V1-69 gene usage does not appear to have prognostic impact that is independent of its mutation status.
Acknowledgements This work was supported by grants from the International Centre for Genetic Engineering and Biotechnology and from
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I. Panovska-Stavridis et al. / Leukemia Research 31 (2007) 245–248
the Ministry of Education and Science of the Republic of Macedonia. The authors have no financial interests to disclose. Contributions: Irina Panovska contributed to the concept and design of the study, data generation and evaluation, statistical analysis and interpretation, and provided drafting of the article. Martin Ivanovski contributed to the collection and analysis of data. Lidija Cevreska and Nikola Siljanovski provided study materials and patient data. Dimitar Efremov contributed to the concept and design of the study, obtained funding, provided patient data and study materials, revised the article and gave final approval.
References [1] Hamblin JT, Davis Z, Gardiner A, et al. Unmutated Ig VH genes are associated with a more aggressive form of chronic lymphocytic leukemia. Blood 1999;94:1848–54.
[2] 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;94:1840–7. [3] Tobin G, Thunberg U, Johnson A, et al. Somatically mutated Ig V(H)321 genes characterize a new subset of chronic lymphocytic leukemia. Blood 2002;99:2262–4. [4] Guarini A, Gaidano G, Mauro FR, et al. Chronic lymphocytic leukemia patients with highly stable and indolent disease show distinctive phenotypic and genotypic features. Blood 2003;102:1035–41. [5] Johnson TA, Rassenti LZ, Kipps TJ. Ig VH1 genes expressed in B cell chronic lymphocytic leukemia exhibit distinctive molecular features. J Immunol 1997;158:235–46. [6] Messmer BT, Albesiano E, Efremov DG, et al. Multiple distinct sets of stereotyped antigen receptors indicate a role for antigen in promoting chronic lymphocytic leukemia. J Exp Med 2004;200:519–25. [7] Kienle D, Benner A, Krober A, et al. Distinct gene expression patterns in chronic lymphocytic leukemia defined by usage of specific VH genes. Blood 2006;107:2090–3. [8] Ivanovski M, Silvestri F, Pozzato G, et al. Somatic hypermutation, clonal diversity, and preferential expression of the VH 51p1/VL kv325 immunoglobulin gene combination in hepatitis C virus-associated immunocytomas. Blood 1998;91:2433–42.
Brief communication
Chronic lymphocytic leukemia patients with a V1-69 gene rearrangement do not have inferior survival with respect to patients that express other unmutated VH genes Irina Panovska-Stavridis ∗ , Martin Ivanovski, Nikola Siljanovski, Lidija Cevreska, Dimitar G. Efremov Department of Hematology, Faculty of Medicine, Vodnjanska 17, 1000 Skopje, Republic of Macedonia Received 9 May 2006; received in revised form 9 May 2006; accepted 13 May 2006 Available online 23 June 2006
Abstract Recent studies indicate that VH gene usage in B-CLL may have prognostic impact independently of VH gene mutation status. The V1-69 gene is the most frequently rearranged VH gene in B-CLL and is almost always unmutated. We therefore investigated whether patients with a V1-69 gene rearrangement differ in clinical course and outcome with respect to patients expressing other unmutated VH genes. We show that V1-69 B-CLLs constitute a uniform group of patients that more often present at advanced clinical stages and require early treatment, but their survival does not differ significantly from patients with other unmutated VH genes. © 2006 Elsevier Ltd. All rights reserved. Keywords: Chronic lymphocytic leukemia; Prognosis; VH gene mutation status; V1-69
1. Introduction B-cell chronic lymphocytic leukemia (CLL) is a heterogeneous disease with a highly variable clinical course, which can at least in part be predicted by the mutational status of the immunoglobulin variable heavy-chain (VH ) genes. Patients with CLL B-cells that express unmutated VH genes typically have a more aggressive disease and shorter survival than CLL patients with mutated VH genes [1,2]. Recent studies indicate that VH gene usage may have a prognostic impact that is independent of the VH gene mutation status. This is especially pertinent to the V3-21 gene which is associated with an unfavourable clinical course even in mutated cases [3]. A second example is the V3-72 gene which has been observed in a series of highly sta-
∗
Corresponding author. Tel.: +389 2 31 47 782; fax: +389 2 30 93 610. E-mail address: dr [email protected] (I. Panovska-Stavridis).
0145-2126/$ – see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.leukres.2006.05.008
ble and indolent CLL patients [4]. Finally, the V1-69 gene, which is the most frequently rearranged VH gene in CLL, is almost always unmutated and as such is invariably associated with unfavourable prognosis. The leukemic immunoglobulins encoded by this gene display characteristic features, such as biased usage of certain D and JH genes, long third complementarity determining regions (CDR3s) and frequent pairing with a few variable light-chain (VL ) genes [5,6]. Moreover, recent analysis of the expression of several genes that regulate B-cell receptor signaling, cell cycle progression and apoptosis have shown that V1-69 cases have a distinct expression pattern as compared to CLL B-cells that use other unmutated VH genes [7]. The striking molecular similarities between the V1-69 cases indicate that they represent a rather homogeneous group with certain biological features that are distinct from other VH -unmutated cases. Therefore, we investigated in a series of CLL patients with a long follow-up whether cases with a V1-69 gene rearrangement have a different clinical course
246
I. Panovska-Stavridis et al. / Leukemia Research 31 (2007) 245–248
and outcome from patients that express other unmutated VH genes.
3. Results and discussion The leukemic immunoglobulin was encoded by the V169 gene in 25 of the 106 investigated CLL samples (23.6%). The VH gene sequence was always unmutated (>98% homology) and was completely identical to the germline V1-69 sequence in 22 cases. The V1-69 gene was joined to the D33 gene segment in 9 cases (Table 1). Six of these cases had a highly conserved heavy chain CDR3 containing the amino acid motif FWSGY, and in two of the three investigated cases the V1-69-encoded heavy chain was paired with a V1gencoded variable light-chain region. A second conserved CDR3 motif (DIVVVPAAI) was identified in three cases. In two of these cases the V1-69-encoded heavy chain was paired with the V02 or the highly homologous V018 variable light chain. V1-69 encoded immunoglobulins belonging to other previously described sets of stereotyped CLL antigen receptors were also sporadically identified in the remaining cases (cases MF9 and MJ21)[6]. The high frequency of V1-69 gene cases in CLL patients originating from a small geographic region and the significant structural similarities between the leukemic immunoglobulins further point to an important role for antigen stimulation in the pathogenesis of this CLL subset. Among the 40 randomly selected non-V1-69 CLLs, the VH gene sequence was unmutated in 19, mutated in 14, and could not be determined in seven cases. Comparison of the V1-69 cases with those expressing other unmutated VH genes showed that the two subsets do not differ with respect to age, gender, median Ly counts and total tumor mass (TTM)
2. Materials and methods Blood samples were collected from 106 consecutive CLL patients that had been diagnosed at our institution between 1977 and 2000. Diagnosis was based on standard morphologic and immunophenotypic criteria for CLL. Following RNA isolation from peripheral blood mononuclear cells by standard techniques, all samples were analyzed by allele-specific immunoglobulin VH gene fingerprinting to identify cases with a V1-69 gene rearrangement [8]. Nucleotide sequencing of the rearranged VH genes was subsequently performed in all V1-69-expressing cases and in 40 of the remaining cases using standard RT/PCR and nucleotide sequencing procedures. In selected cases the VL gene sequence was also determined. Candidate germline genes were assigned by searching the VBASE directory. Sequences with less than 2% differences from germline VH genes were considered unmutated. Correlations between the different CLL subsets were made with the use of the t-test, Wilcoxon rank-sum test and Fisher exact test. The median time to treatment and survival were estimated by the method of Kaplan and Meier and assessed by the log-rank test. Data for patients that had not received treatment were regarded as censored. Statistical analyses were performed using the SigmaStat 3.1 program (Systat Software Inc., Richmond, CA). Table 1 Structural characteristics of the V1-69 encoded immunoglobulin variable regions Patient
D gene
JH gene
CDR3 sequence
MO6 HA-IDR CLL#86 HA1 MJ2 MF15 MO10 MO13 HA2 MF12 MJ26 NI MO12 MF9 MD2 HA-SC MJ21 MF7 MJ5 MJ29 HA3 MM6 MJ19 MF5 CLL#132
D2-2 D2-2 D2-15 D3-3 D3-3 D3-3 D3-3 D3-3 D3-3 D3-3 D3-3 D3-3 D3-22 D3-16 D3-10 D3-10 D3-10 D1-26 D2-21 D2-2 D5-5 D5-5 D6-19 D6-19 D6-6
JH6 JH6 JH6 JH6 JH6 JH5 JH5 JH4 JH4 JH4 JH6 JH6 JH4 JH3 JH6 JH4 JH6 JH4 JH6 JH2 JH6 JH5 JH4 JH4 JH6
ERCGKL—DIVVVPAAIRYYGMDV MDP——DIVVVPAAIAYYYGMDV RRGGPSPVVDIVVVVAATRFIYYYYYYMDV AGRIVT-IFWSGYLPNYYYYGMDV GGWSTNYDFWSGYSPYYYYYYMDV DGLER–DFWSGYMRVTPYNWFDP ELVPPQ–FWSGYFYSWFDP DDT–YYDFWSGYYY DN–YYYDFWSGYYPGVD PTQFWSWGEFGYYFDY FRIEGVVSYYYYYYGMDV DLSPIWSGYYMRDYYYGMDV ASRGYYYD-SSGYYSGVY GGPYDYVWGSYRPNDAFDI GMVRGVITYYYYYYYMDV GTILWFGELLYPLDY AMVRGVITYYYYYYYMDV DNYLFKRIVGPLDY DDIVKIGPCGGDCYFPVGYYYYGMDV VRGWKYQLLYQEDWYFDL VSMGQLWLQRNYYYYMDV ESIGGYSYGYIYNWFDP PSTVTTYSSGWSTIFDY EEGVSSDSSGWNY DSAGIAARFISMTDYYYYYGMDV
VL /JL gene V02/J1 V018/J3 V1g/J3b
V1g/J2
V1c/J2 VA27/J5 V1c/J3b
V018/J1
I. Panovska-Stavridis et al. / Leukemia Research 31 (2007) 245–248
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Table 2 Clinical and laboratory features of CLL patients stratified according to VH gene usage and mutational status Patient characteristics
V1-69
Other VH unmutated
VH mutated
V1-69 vs. other VH unmutated
Male:female Median age (years) Median follow-up (range) Median Ly count at dg (×106 /ml) Total tumor mass scorea at dg
21:4 60 56 months (6–109) 53.0 11.92
13:6 60 59 months (20–97) 48.0 10.92
8:6 62 77 months (53–300) 29.8 6.03
P = n.s., Fisher exact test P = n.s., t-test P = n.s., t-test P = n.s., t-test P = n.s., t-test
Rai stage at dg Low risk Intermediate risk High risk
1 15 9
2 16 1
6 6 2
Low + intermediate vs. high risk, P = 0.027, Fisher exact test
a Total tumor mass score (TTM) was calculated as the sum of the square root of the number of peripheral blood lymphocytes per ml, the diameter of the largest palpable lymph node in centimeters (cm) and the enlargement of the spleen below the left costal margin in cm. High TTM was defined as a sum equal to or greater than 9.0.
Fig. 1. Kaplan–Meier survival curves of CLL patients stratified according to VH gene usage and mutation status. (A) Time from diagnosis to initial therapy. (B) Overall survival.
score at diagnosis (Table 2). However, the percentage of cases presenting at an advanced stage was significantly higher in the V1-69 subgroup (36% of cases in Rai high risk category) than in the subgroup with other unmutated VH genes (5% of cases in Rai high risk category, P = 0.027). Eighty percent of the V1-69 cases and 42% of the cases with other unmutated VH genes had received treatment immediately following diagnosis (Fig. 1(A)). However, median time to treatment was not significantly different between the two subgroups (1 month and 9 months for V1-69 and other VH unmutated cases, respectively, P = 0.085). To date, 24 of the V1-69 patients (96%) and 15 of the patients with other unmutated VH genes (79%) have died, with median survival times of 56 and 60 months, respectively (P = 0.9) (Fig. 1(B)). For comparison, median survival of the 14 VH mutated cases from this series was 125 months (P < 0.0001 with respect to both subgroups with unmutated VH genes).
Altogether, these data show that CLL patients with a V169 gene rearrangement constitute a uniform group characterized by a progressive clinical course and frequent presentation at an advanced disease stage. Although in our series CLL patients with a V1-69 gene rearrangement more frequently received treatment immediately following diagnosis, median time to treatment and median survival were not significantly different from patients expressing other unmutated VH genes. Thus, in this single-institution study that included a series of patients with a relatively long follow-up, V1-69 gene usage does not appear to have prognostic impact that is independent of its mutation status.
Acknowledgements This work was supported by grants from the International Centre for Genetic Engineering and Biotechnology and from
248
I. Panovska-Stavridis et al. / Leukemia Research 31 (2007) 245–248
the Ministry of Education and Science of the Republic of Macedonia. The authors have no financial interests to disclose. Contributions: Irina Panovska contributed to the concept and design of the study, data generation and evaluation, statistical analysis and interpretation, and provided drafting of the article. Martin Ivanovski contributed to the collection and analysis of data. Lidija Cevreska and Nikola Siljanovski provided study materials and patient data. Dimitar Efremov contributed to the concept and design of the study, obtained funding, provided patient data and study materials, revised the article and gave final approval.
References [1] Hamblin JT, Davis Z, Gardiner A, et al. Unmutated Ig VH genes are associated with a more aggressive form of chronic lymphocytic leukemia. Blood 1999;94:1848–54.
[2] 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;94:1840–7. [3] Tobin G, Thunberg U, Johnson A, et al. Somatically mutated Ig V(H)321 genes characterize a new subset of chronic lymphocytic leukemia. Blood 2002;99:2262–4. [4] Guarini A, Gaidano G, Mauro FR, et al. Chronic lymphocytic leukemia patients with highly stable and indolent disease show distinctive phenotypic and genotypic features. Blood 2003;102:1035–41. [5] Johnson TA, Rassenti LZ, Kipps TJ. Ig VH1 genes expressed in B cell chronic lymphocytic leukemia exhibit distinctive molecular features. J Immunol 1997;158:235–46. [6] Messmer BT, Albesiano E, Efremov DG, et al. Multiple distinct sets of stereotyped antigen receptors indicate a role for antigen in promoting chronic lymphocytic leukemia. J Exp Med 2004;200:519–25. [7] Kienle D, Benner A, Krober A, et al. Distinct gene expression patterns in chronic lymphocytic leukemia defined by usage of specific VH genes. Blood 2006;107:2090–3. [8] Ivanovski M, Silvestri F, Pozzato G, et al. Somatic hypermutation, clonal diversity, and preferential expression of the VH 51p1/VL kv325 immunoglobulin gene combination in hepatitis C virus-associated immunocytomas. Blood 1998;91:2433–42.