Distinctive immunoglobulin VH gene usage in Japanese patients with chronic lymphocytic leukemia

Leukemia Research 30 (2006) 272–276

Distinctive immunoglobulin VH gene usage in Japanese patients with chronic lymphocytic leukemia Hiromi Koiso a, Arito Yamane b, Takeki Mitsui a, Takafumi Matsushima a, Norifumi Tsukamoto a, Hirokazu Murakami c , Syuichi Miyawaki d , Yoshihisa Nojima a , Masamitsu Karasawa b,∗ a

Department of Medicine and Clinical Science, Graduate School of Medicine, Gunma University, Gunma, Japan Blood Transfusion Service, University Hospital, Faculty of Medicine, Gunma University School of Medicine, 3-39-22, Showa-machi, Maebashi, Gunma 371 8511, Japan c School of Health Science, Faculty of Medicine, Gunma University, Gunma, Japan d Internal Medicine, Saiseikai Maebashi Hospital, Gunma, Japan

b

Received 28 June 2005 Available online 12 September 2005

Abstract The incidence of chronic lymphocytic leukemia (CLL) is low in Asian countries including Japan, while CLL is the most common type of leukemia in the west. To elucidate which factors contribute to the low frequency of CLL in Japan, we investigated immunoglobulin (Ig) heavy chain gene usage and somatic hypermutation status in 44 Japanese patients with CLL. Only one of these patients used VH 1–69. The finding is in striking contrast to data from western countries, where the frequency of CLL expressing VH 1–69 ranges from 12 to 21%. © 2005 Elsevier Ltd. All rights reserved. Keywords: Chronic lymphocytic leukemia; Immunoglobulin heavy chain gene; Somatic hypermutation; VH 1–69; Japanese population; CD38

1. Introduction B-cell chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of morphologically mature lymphocytes expressing CD5 and CD23 together with low surface expression of B-cell receptors and related molecules such as CD79. Immunoglobulin (Ig) gene segments undergo antigen-independent rearrangement in B-lymphocytes before they exit the bone marrow. At this stage, somatic mutations in the Ig heavy chain variable region (VH ) genes of lymphocytes are not evident. After reaching the germinal center, somatic mutations accumulate in B-cells to increase antigen affinity. Although B-CLL has long been viewed as a leukemia of naive B-cells, this notion has recently been challenged [1–3]. Damle et al. and Hamblin et al. have shown that B-CLL cells apparently arise from B-lymphocytes with

∗

Corresponding author. Tel.: +81 27 220 8673; fax: +81 27 220 8671. E-mail address: [email protected] (M. Karasawa).

0145-2126/$ – see front matter © 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.leukres.2005.07.007

non-mutated and with mutated Ig VH genes. Moreover, mutation status correlates with disease prognosis, since patients with non-mutated genes have an unfavorable clinical outcome compared with those whose Ig VH genes have mutated [4,5]. Many subsequent studies have consistently confirmed that Ig VH mutation status is a distinct and reliable prognostic marker of B-CLL [6–11]. The major Ig VH gene complex on chromosome 14 comprises approximately 50 functional Ig VH segments within seven families. Studies of Ig VH gene usage in CLL cells have revealed non-random differential use of the genes, representing a disease-specific restriction that does not reflect the variation in the normal B-cell repertoire. Of these VH genes, VH 1–69 that associates with the non-mutated subset in CLL patients is over-represented [1–3,5,12,13]. Furthermore, the subset expressing VH 1–69 preferentially uses D3 together with JH6 [3,12] and has long third complementary determining regions (CDR3s). Other frequently used VH genes are VH 4–34 and VH 3–23 that usually belong to the mutated subset [3,5].

H. Koiso et al. / Leukemia Research 30 (2006) 272–276

The incidence of CLL geographically varies. In contrast to western countries, where CLL is the most frequent type of leukemia, CLL is rare in Asia [14–17]. The estimated incidence of CLL in Japan is from 4–5-fold [17] to 39–92-fold [14] lower than that in the west. We considered that elucidation of the factors that reduce the risk of CLL would be important for understanding the pathogenesis of this disease. We therefore analyzed VH gene usage, mutation status and CD38 expression, which is also related to the clinical outcome of B-CLL, among Japanese patients [4,6–10,18–21].

2. Materials and methods 2.1. Patients We enrolled 44 patients with B-CLL (29 males and 15 females) who were referred to our institutions between March 1999 and March 2004. The median age at the time of diagnosis was 68 years (37–92 years) and all patients had at least 10% peripheral leukemic cells. The diagnosis was based on immunophenotypic analysis and cell morphology determined from Wright-stained peripheral blood and bone marrow smears. To exclude mantle cell lymphoma (MCL) in the leukemic phase, cyclin D1 (CyD1) expression was analyzed using quantitative real-time RT-PCR [22], which confirmed lower expression of CyD1 in these patients as compared with representative MCL patients. The median follow-up period of these patients was 4.0 years (0.5–34.2 years). G-banding cytogenetic analysis was performed in 32 of the B-CLL patients and metaphases were absent in 2 of them. Karyotypic abnormalities were detected in 11 patients (10 with 45, X–Y and 1 with complex abnormalities including trisomy 12). Fluorescent in situ hybridization (FISH) of trisomy 12 in interphase nuclei was performed on specimens from 22 B-CLL patients. Of these, five were positive for trisomy 12 (22.7%). This study was approved by the Institutional Review Board and written informed consent was provided by all patients before sampling. 2.2. Immunophenotype Cell surface antigens were analyzed by flow cytometry as described [22]. In brief, for three-color analysis of surface antigens, a peripheral nucleated cell suspension was incubated with combinations of fluorescein isothionate (FITC), phycoerythrin (PE)- and peridinin chlorophyll (PerCP)conjugated antibodies in PBS for 30 min on ice. After washing, stained cells were sorted using a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA) and the data were analyzed using CELLQuest software (Becton Dickinson). FITC-conjugated anti-mouse IgG1 (control), anti-CD5 and anti-CD19 antibodies were purchased from BD Pharmingen (San Diego, CA), and anti-kappa antibody was purchased from Becton Dickinson. PE-conjugated anti-mouse IgG1, anti-CD19, anti-CD23, anti-CD27, anti-

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CD79b, anti-IgM, anti-IgD, and anti-IgG antibodies were purchased from BD Pharmingen and anti-lambda antibody was purchased from Becton Dickinson. PerCP-conjugated anti-CD45 antibody (for gating) was purchased from Becton Dickinson. The cut-off point for CD38 expression was set at 30% [4]. 2.3. RNA isolation and first strand cDNA synthesis Mononuclear cells collected from peripheral blood (42 patients), bone marrow (1 patient), or lymph nodes (1 patient) using lymphocyte separation medium (Organon Teknika, Durham, NC, USA) were cryopreserved at −70 ◦ C in aliquots of 2 × 107 cells per tube. Total RNA was extracted from these cells with RNAzol B (TEL-TEST, Friendswood, TX, USA) and suspended in 20 ␮l RNase-free dH2 O. The RNA solution (2 ␮l) was reverse transcribed in a total volume of 20 ␮l using SUPERSCRIPT II RNase H (−) reverse transcriptase (GIBCO BRL, Gaithersburg, MD, USA) and 100 ng of random hexamer (TAKARA, Kyoto, Japan) according to the manufacturer’s instructions. Reverse transcription proceeded at 42 ◦ C for 50 min. 2.4. Sequence analysis VH gene To determine VH gene family usage in B-CLL cells, the cDNAs were amplified using forward VH family-specific framework region (FR) primers (FR1 to FR7) together with the appropriate reverse CH isotype specific primers (IgM or IgG) [3] using a 9600 Gene Amp System (Perkin-Elmer Cetus, Emeryville, CA, USA). When these primers failed to determine VH gene family usage due to non-specific amplified bands or contamination with non-specific amplified bands, a different set of primers was applied [23]. VH gene sequences were determined by re-amplifying original cDNAs using the corresponding VH leader and CH primers [3]. Most of the amplified samples were directly sequenced using the BigDye Terminator Cycle Sequencing FS Ready Reaction kit (Applied Biosystems, Foster City, CA, USA) on an ABI Prism 310 Genetic Analyzer (Applied Biosystems). Some samples were sequenced by TA-cloning. Nucleotide sequences were compared with those in the VQUEST or IgBLAST databases. Non-mutated and mutated VH sequences were defined as those with <2% sequence deviation from the most similar germ line Ig VH genes and with
2% sequence difference, respectively [4,5]. 2.5. Statistical evaluation Survival curves from diagnosis were constructed using the Kaplan–Meier method and significance was examined by the log-rank test (Mantle-Cox.) using Stat View software (Version 5.0). Correlation between CD38 expression level and Ig VH mutation status was evaluated by the Mann–Whitney U-test. Statistical significance was established at P < 0.05.

H. Koiso et al. / Leukemia Research 30 (2006) 272–276

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Table 1 VH gene family usage and mutation status; values in the table represent the number of cases VH family

n

Mutated VH gene

Unmutated VH gene

VH1 VH2 VH3 VH4 VH5 VH6 VH7

4 2 23 12 1 1 0

2 0 12 10 0 1 0

2 2 11 2 1 0 0

Total

43

25

18

3. Results 3.1. VH gene usage We sequenced IgH variable region genes from 43 of the 44 patients, since no bands were amplified in samples from 1 patient. The usage frequency of the seven VH gene families in the 43 patients was as follows (Table 1): VH 1, 4/43 (9.3%); VH 2, 2/43 (4.6%); VH 3, 23/43 (53.5%); VH 4, 12/43 (27.9%); VH 5, 1/43 (2.3%); VH 6, 1/43 (2.3%); and VH 7, 0/43 (0%). Subfamily usage was identified by sequencing the VH genes. Of these, the most remarkable finding was that VH 1–69 was used in only one patient (2.3%). This result is in striking contrast to those in western countries, where VH 1–69 is one of the most frequently used genes. Subfamily analysis revealed that only four genes accounted for 49% of the total usage; VH 4–34 (9/43, 20.9%), VH 3–23 (6/43, 14.0%), VH 3–21 (3/43, 7%), and VH 3–30 (3/43, 7.0%). These findings are analogous to those of other reports. 3.2. VH gene somatic mutation status and prognosis Eighteen (41.9%) and 25 (58.1%) patients displayed nonmutated and mutated VH gene usage, respectively. The ratio (%) of non-mutated patients in this study was comparable to that of previous reports citing ranges between 40 and 50% (3–5, 8, 9). Other studies have shown that the prognosis obviously differs between the two groups. The present study found that the survival was significantly shorter for patients with non-mutated, than mutated genes as estimated by the Kaplan–Meier method (Fig. 1A). The predicted 50% survival rate for the patients with non-mutated and mutated genes was 109 months, and above 50%, respectively, and the difference was significant (P = 0.029, log-rank test). 3.3. CD38 expression analysis in B-CLL At a 30% cut-off level for CD38 reactivity in CD19+ CD5+ cells [4], 29.5% (n = 13) and 70.5% (n = 31) of the patients were CD38 positive and negative, respectively. Survival did not significantly differ between the two groups (P = 0.519, log-rank test; Fig. 1B). Furthermore, CD38 expression and

Fig. 1. Overall survival of patients with CLL. Survival significantly differs based on VH mutation status (A) but not on CD38 expression (B).

VH gene status did not significantly correlate (P = 0.121, Mann–Whitney U-test).

4. Discussion Several studies have indicated that Ig VH usage in B-CLL is not random. Of these, the usage of VH 1–69 is disproportionately high [1–3,5,12,13]. The VH 1–69 locus is highly polymorphic, consisting of two sets of 51p1-related and hv1263-related gene families. Each haplotype contains 0 to 2 51p1 related genes that probably result from gene duplication [24,25] and almost all CLL leukemic cells that express VH 1–69 also express 51p1-related allelic variants and not hv1263-related genes. The CLL subset expressing VH 1–69 has several unique features such as being almost exclusively non-mutated, preferentially using specific diversity and JH6 gene segments, and having long CDR3s [12]. Moreover, Widhopf et al. recently differentiated using the VH 1–69 allele, 163 CLL patients among an unrelated cohort of 1220 and found that 15 of the 163 had virtually identical CDR3s and the same A27 kappa light-chain [13]. Other investigators have also reported subgroups of CLL patients with identical or highly similar VH , DH , JH segments and shared restricted CDR3 configurations [11,26–28]. Taken together, these data suggest that leukemic cells in subsets of CLL are selected by antigens regardless of VH gene mutation status. In obvious contrast to western countries, where the incidence of CLL expressing VH 1–69 ranges from 12 to 21% ([12,13,26] and Table 2), our study revealed VH 1–69 usage in only 1 among 44 patients.

H. Koiso et al. / Leukemia Research 30 (2006) 272–276 Table 2 VH1 gene family and VH1-69 usage in literatures; NA: not available References

n

VH1/total (%)

VH1-69/total (%)

Western countries Tobin G et al. (2004) Johnson TA et al. (1997) Hamblin et al. (1999) Widhopf GF II et al. (2004)

407 117 84 1188

28 35 26 27

14 22.2 11.9 13.8

21 13 43

14 0 9

ND ND 2.3

Japan Ikematsu et al. (1994) Nakamura N (1999) Present study

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factors among the population or differences in antigens that stimulate B-cells based on distinctive environments.

Acknowledgements

To our knowledge, only two publications have described VH family usage in the Japanese population [29,30]. The proportion of CLL expressing the VH 1 family in Japan is not so prominent as in western countries (Table 2), although the VH 1–69 frequency in these studies is unknown because subfamilies were not analyzed. Another example of regional differences in the incidence of VH genes usage might be VH 3–21, which appears at a higher frequency in Sweden compared with other countries [11,26]. However, the incidence of CLL expressing VH 3–21 (7%) in the present study was similar to that of the Swedish population (9%) [11,26]. This VH 3–21 subset also uniquely displays highly restricted usage of the Vlambda 2–14 gene and homologous CDR3s sequences [11,26]. In our study, 41.9% of the CLL patients harbored non-mutated VH genes, the incidence of which was similar to that of other reports. The absence of a VH mutation and CD38 positivity are associated with an unfavorable clinical outcome [4–10,18–21]. Some reports have suggested that the expression of surface CD38 on CLL cells could be a surrogate marker of VH mutation status [4], but others have reported that VH status poorly correlates with CD38 expression [6,7,9]. Therefore, the correlation between VH mutations and CD38 expression is controversial and each might be an independent prognostic factor [7]. Although VH gene mutation status is not reversible, the CD38 expression level does not remain constant throughout the course of the disease in a considerable number of patients [7]. We confirmed the prognostic significance of VH gene mutation status in Japanese CLL patients. Survival rates were lower and the clinical course was more aggressive among patients with a non-mutated, than a mutated VH gene. However, the present study did not confirm a prognostic impact of CD38 expression. Whether this is due to the influence of genetic factors, a methodological difference or bias in the small patient cohort remains unclear. Moreover, VH gene status did not significantly correlate with CD38 expression in our CLL patients. Our results suggest that the low incidence of CLL patients expressing VH1–69 partly explains the low susceptibility to CLL in the Japanese population. The absence of overrepresented VH1–69 gene usage in the Japanese population might have resulted from the influence of different genetic

Contributions. H. Koiso contributed to collection, analysis and interpretation of data and provided drafting of the article. A. Yamane, T. Mitsui, contributed to data collection and assembly. T. Matsushima and N. Tsukamoto contributed to data collection and logistic support. H. Murakami and S. Miyawaki contributed to the conception and design of the study. Y. Nojima contributed to logistical support and provided critical revisions of the article, and gave final approval. M. Karasawa contributed to the concept and design, provided drafting of the article and gave final approval.

References [1] Schroeder Jr HW, Dighiero G. The pathogenesis of chronic lymphocytic leukemia: analysis of the antibody repertoire. Immunol Today 1994;15:288–94. [2] Oscier DG, Thompsett A, Zhu D, Stevenson FK. Differential rates of somatic hypermutation in V(H) genes among subsets of chronic lymphocytic leukemia defined by chromosomal abnormalities. Blood 1997;89:4153–60. [3] Fais F, Ghiotto F, Hashimoto S, Sellars B, Valetto A, Allen SL, et al. Chronic lymphocytic leukemia B-cells express restricted sets of mutated and unmutated antigen receptors. J Clin Invest 1998;102:1515–25. [4] Damle RN, Wasil T, Fais F, Ghiotto F, Valetto A, Allen SL, et al. Ig V gene mutation status and CD38 expression as novel prognostic indicators in chronic lymphocytic leukemia. Blood 1999;94:1840–7. [5] 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;94:1848–54. [6] Jelinek DF, Tschumper RC, Geyer SM, Bone ND, Dewald GW, Hanson CA, et al. Analysis of clonal B-cell CD38 and immunoglobulin variable region sequence status in relation to clinical outcome for B-chronic lymphocytic leukaemia. Br J Haematol 2001;115:854– 61. [7] Hamblin TJ, Orchard JA, Ibbotson RE, Davis Z, Thomas PW, Stevenson FK, et al. CD38 expression and immunoglobulin variable region mutations are independent prognostic variables in chronic lymphocytic leukemia, but CD38 expression may vary during the course of the disease. Blood 2002;99:1023–9. [8] Lin K, Sherrington PD, Dennis M, Matrai Z, Cawley JC, Pettitt AR. Relationship between p53 dysfunction, CD38 expression, and IgV(H) mutation in chronic lymphocytic leukemia. Blood 2002;100:1404–9. [9] Krober A, Seiler T, Benner A, Bullinger L, Bruckle E, Lichter P, et al. V(H) mutation status, CD38 expression level, genomic aberrations, and survival in chronic lymphocytic leukemia. Blood 2002;100:1410–6. [10] Ghia P, Guida G, Stella S, Gottardi D, Geuna M, Strola G, et al. The pattern of CD38 expression defines a distinct subset of chronic lymphocytic leukemia (CLL) patients at risk of disease progression. Blood 2003;101:1262–9. [11] Tobin G, Thunberg U, Johnson A, Eriksson I, Soderberg O, Karlsson K, et al. Chronic lymphocytic leukemias utilizing the VH3-21 gene display highly restricted Vlambda2-14 gene use and homologous CDR3s: implicating recognition of a common antigen epitope. Blood 2003;101:4952–7.

276

H. Koiso et al. / Leukemia Research 30 (2006) 272–276

[12] 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. [13] Widhopf II GF, Rassenti LZ, Toy TL, Gribben JG, Wierda WG, Kipps TJ. Chronic lymphocytic leukemia B-cells of more than 1% of patients express virtually identical immunoglobulins. Blood 2004;104:2499–504. [14] Nishiyama H, Mokuno J, Inoue T. Relative frequency and mortality rate of various types of leukemia in Japan. Gann 1969;60:71–81. [15] Weiss NS. Geographical variation in the incidence of the leukemias and lymphomas. Natl Cancer Inst Monogr 1979;53:139–42. [16] Gale RP, Cozen W, Goodman MT, Wang FF, Bernstein L. Decreased chronic lymphocytic leukemia incidence in Asians in Los Angeles County. Leuk Res 2000;24:665–9. [17] Tamura K, Sawada H, Izumi Y, Fukuda T, Utsunomiya A, Ikeda S, et al. Chronic lymphocytic leukemia (CLL) is rare, but the proportion of T-CLL is high in Japan. Eur J Haematol 2001;67:152–7. [18] Ibrahim S, Keating M, Do KA, O’Brien S, Huh YO, Jilani I, et al. CD38 expression as an important prognostic factor in B-cell chronic lymphocytic leukemia. Blood 2001;98:181–6. [19] Del Poeta G, Maurillo L, Venditti A, Buccisano F, Epiceno AM, Capelli G, et al. Clinical significance of CD38 expression in chronic lymphocytic leukemia. Blood 2001;98:2633–9. [20] Morabito F, Mangiola M, Oliva B, Stelitano C, Callea V, Deaglio S, et al. Peripheral blood CD38 expression predicts survival in B-cell chronic lymphocytic leukemia. Leuk Res 2001;25:927–32. [21] Durig J, Naschar M, Schmucker U, Renzing-Kohler K, Holter T, Huttmann A, et al. CD38 expression is an important prognostic marker in chronic lymphocytic leukaemia. Leukemia 2002;16:30–5. [22] Koiso H, Tsukamoto N, Miyawaki S, Shinonome S, Nojima Y, Karasawa M. Quantitative analysis of Cyclin D1 and CD23 expression in

[23]

[24]

[25]

[26]

[27]

[28]

[29]

[30]

mantle cell lymphoma and B-chronic lymphocytic leukemia. Leuk Res 2002;26:809–15. Kuppers R, Zhao M, Rajewsky K, Hansmann ML. Detection of clonal B-cell populations in paraffin-embedded tissues by polymerase chain reaction. Am J Pathol 1993;143:230–9. Sasso EH, Willems van Dijk K, Bull AP, Milner EC. A fetally expressed immunoglobulin VH1 gene belongs to a complex set of alleles. J Clin Invest 1993;91:2358–67. Sasso EH, Johnson T, Kipps TJ. Expression of the immunoglobulin VH gene 51p1 is proportional to its germline gene copy number. J Clin Invest 1997;97:2074–80. Tobin G, Thunberg U, Karlsson K, Murray F, Laurell A, Willander K, et al. Subsets with restricted immunoglobulin gene rearrangement features indicate a role for antigen selection in the development of chronic lymphocytic leukemia. Blood 2004;104:2879–85. Ghiotto F, Fais F, Valetto A, Albesiano E, Hashimoto S, Dono M, et al. Remarkably similar antigen receptors among a subset of patients with chronic lymphocytic leukemia. J Clin Invest 2004;113:1008–16. Messmer BT, Albesiano E, Efremov DG, Ghiotto F, Allen SL, Kolitz J, 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. Ikematsu W, Ikematsu H, Okamura S, Otsuka T, Harada M, Niho Y. Surface phenotype and Ig heavy-chain gene usage in chronic B-cell leukemias: expression of myelomonocytic surface markers in CD5chronic B-cell leukemia. Blood 1994;83:2602–10. Nakamura N, Kuze T, Hashimoto Y, Tasaki K, Hojo H, Sasaki Y, et al. Analysis of the immunoglobulin heavy chain gene variable region of 101 cases with peripheral B-cell neoplasms and B-cell chronic lymphocytic leukemia in the Japanese population. Pathol Int 1999;49:595–600.