Use of V H, D and J H immunoglobulin gene segments in Brazilian patients with chronic lymphocytic leukaemia (CLL)

HUMAN AND MEDICAL GENETICS

SHORT COMMUNICATION

Use of V_H, D and J_H immunoglobulin gene segments in Brazilian patients with chronic lymphocytic leukaemia (CLL)

Beatriz Jatobá Pimentel^{I, II}; Cláudio Gustavo Stefanoff^III; Aline Santos Moreira^II; Héctor N. Seuánez^{I, II}; Ilana Renault Zalcberg^III

^IDepartamento de Genética, Instituto de Biologia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil

^IIDivisão de Genética, Centro de Pesquisa, Instituto Nacional de Câncer, Rio de Janeiro, RJ, Brazil

^IIILaboratório de Biologia Molecular, Centro de Transplante de Medula Óssea, Instituto Nacional de Câncer, Rio de Janeiro, RJ, Brazil

^{Send correspondence to}

ABSTRACT

Chronic lymphocytic leukaemia (CLL) is a haematological malignancy for which reliable prognostic markers are needed in view of its clinical heterogeneity. In approximately 50% of CLL patients, immunoglobulin (Ig) rearrangements are modified by somatic hypermutation (SHM), a process that represents a reliable prognostic indicator of favourable progression. In this study, we investigated SHM in 37 Brazilian CLL patients and identified the preferential involvement of specific immunoglobulin gene families and segments through PCR-amplified fragments or subcloned fragments. Forty-one rearrangements were observed and 37 of them were functional. A 98% homology cut-off with germinal sequences showed 18 patients (48.7%) with SHM. Unmutated cases showed a poorer clinical outcome. V_H3 was the most frequent V_H family, followed by V_H4. The V_H4-39 segment was the most frequently used, mainly in unmutated cases, while the V_H3 family was predominant in mutated cases. The D3 and J_H4/J_H6 families were the most frequently observed.

Key-words: chronic lymphocytic leukaemia, immunoglobulin rearrangements, somatic hypermutation.

Chronic lymphocytic leukaemia (CLL) is the most frequent lymphoproliferative malignancy affecting adults in the western world (Calligaris-Cappio and Hamblin, 1999), characterized by accumulation of CD5⁺ B cells in peripheral blood (Rozman and Montserrat, 1995). Diagnosis relies on the finding, in peripheral blood, of more than 5x10⁹ B-lymphocytes per litre with a CD5⁺, CD19⁺ and CD23⁺ immunophenotypic profile. CLL is a remarkably heterogeneous disease with respect to prognosis and clinical course, once some patients may survive for prolonged periods without requiring therapy, while in others a prompt fatal outcome occurs, even after aggressive treatment (Damle et al., 1999; Hallek, 2000). Although two different criteria - Rai (Rai et al., 1975) and Binet (Binet et al., 1981) - have been proposed for staging this clinically heterogeneous malignancy, at early stages none of these criteria can accurately distinguish patients with an ensuing indolent course from those with a subsequent poor outcome (Maloum et al., 2000). Other prognostic factors are therefore needed for determining, once the diagnosis is made, the therapy of choice for CLL patients (Dighiero and Hamblin, 2002).

One of the most accurate CLL prognostic factors is the occurrence of somatic hypermutation in Ig rearrangements, because patients with mutated Ig genes show an indolent clinical course compared to those with unmutated genes (Damle et al., 1999; Hamblin et al., 1999). As some Ig rearrangements have been more frequently found in unmutated cases while others are more frequent in mutated ones (Fais et al., 1998; Duke et al., 2003; Guarini et al., 2003), it has been postulated that these differences might be associated to geographic or genetic factors (Stevenson and Calligaris-Cappio, 2004) or to differences of antigen types to which cells might be reactive (Chiorazzi and Ferrarini, 2003; Ghiotto et al., 2004; Kolar and Capra, 2004).

In this paper, we report the first study of Ig rearrangements in Brazilian CLL patients and the results concerning the frequency with which V_H, D and J_H segments are involved in Ig rearrangements in mutated and unmutated cases.

Samples of peripheral blood (n = 25) or bone marrow (n = 12) were obtained from 37 patients (19 male and 18 female) with CLL, previously diagnosed by immunophenotyping. Their ages ranged from 38 to 76 years (median = 67 years). Their platelet count ranged from 100,000 to 360,000/mm³ (median = 215,000/mm³), haemoglobin levels from 9.0 to 16.1 g/dL (median 13.5 g/dL), and lymphocytes from 5,000 to 58,200/mm³ (median = 19,663/mm³). Binet stage (in 24 patients) was: A (n = 16), B (n = 06) and C (n = 02). The median follow-up time for 20 patients with complete clinical data was 23 months, ranging from 6 to 144 months (data from one patient with follow-up shorter than six months - LLC 32 - were excluded from analysis, for a better evaluation of disease progression). At the time of this study, treatment had been established for nine patients, three of which were already deceased.

Mononuclear cells were isolated from peripheral blood or bone marrow in Ficoll® (Sigma) density gradients. DNA was extracted using DNAzol® (Life Technologies) following the manufacturer's instructions.

Heavy chain Ig genes were amplified for clonality analysis as previously reported (Ramasamy et al., 1992; Yamada et al., 1989). Monoclonality was confirmed by the presence of a single DNA band resulting from co-migration of similar-sized fragments in polyacrylamide gels, while the presence of two DNA bands was interpreted as indicative of two different rearrangements within a same clone.

For sequence analysis, six separate PCR reactions were carried out per patient. Each reaction used one of six, family-specific (V_H1-V_H6), sense, leader primer with one antisense J_H-primer, as previously reported (Campbell et al., 1992), and with a V_H1 primer especially designed for co-amplification of V_H7 sequences. Whenever amplifications were unsuccessful, subsequent PCR assays were carried out with FR1 primers (specific for families V_H1, V_H2, V_H3, V_H4a, V_H4b, V_H5 and V_H6) instead of leader primers, with a V_H1 primer especially designed for co-amplification of V_H7, as described by Pritsch et al. (1993). Reaction products were run in 1.5% agarose gels, for determining whether the amplifications had been successful and for V_H-family identification. Amplified products were purified in GFX columns (Amersham Biosciences) for direct DNA sequencing or cloning, following the manufacturer's recommendations.

PCR products were cloned in order to (i) label and sequence amplified 5' Ig regions with FR1 primers; (ii) to separate V_H families with two rearrangements; and (iii) to allow sequencing those PCR products whose sequencing was unclear. Cloning was carried out using the pMOSBlue (Amersham Biosciences) plasmid vector, as specified by the manufacturer. Plasmids from recombinant colonies were extracted by Miniprep and used for sequencing.

DNA fragments were labelled with the DYEnamic ET Dye Terminator Cycle Sequencing Kit for MegaBACE DNA Analysis Systems® (Amersham Biosciences) according to the manufacturers instructions, and samples were run in a MegaBACE1000 automated DNA sequencer. Two different, non-cloned, PCR products were sequenced in both directions for each patient, and labelling was carried out with the same primers used in the PCR reactions. Whenever sequence differences were detected, one additional sequencing protocol was performed. Sequencing of cloned fragments, in both directions, was carried out with primers M13F e M13R, from at least three colonies per case in cases with a single rearrangement, and from at least six colonies per case in cases with two rearrangements. Sequences were analysed and edited with SeqMan 4.0 software (DNASTAR Inc.).

V_H, D and J_H sequences were submitted to the IMGT, IgBlast and V-base databases, for analysing and identifying rearranged segments with the highest homology to the germinal V_H, D and J_H regions. Conflicting results regarding D segments were analysed according to the criteria of Corbett et al. (1997) or of Fais et al. (1998). The CDR3 region was identified according to Fais et al. (1998).

Mutations were identified by comparisons of rearranged and germinal Ig sequences at the DNA and protein levels, although assignment of patients to the unmutated (UM) or mutated (M) group was based only on DNA data. Only the FR1, CDR1, FR2, CDR2 and FR3 regions were analysed, since changes in the CDR3 and FR4 regions could have been originated during the process of junction. A 98% homology cut-off was chosen for recognising somatic hypermutation, thus excluding a component of variance due to polymorphisms.

To analyse our data we used simple standard statistical tests performed on spreadsheets (Microsoft Excel) or commercially available at IFA Services Statistics.

Forty-one rearrangements, from 37 patients, were sequenced. Comparisons with germline V_H, D and J_H segments in databases allowed identification of sequence homologies and mutations. This analysis showed mutated rearrangements in 18 out of the 37 patients (48.7%), indicated by <98% homology with germline sequences, and unmutated rearrangements in 19 patients (51.3%), with >98% homology with germline sequences. These results are similar to those previously reported (Damle et al., 1999; Hamblin et al., 1999).

Four of the 37 patients carried two Ig rearrangements, but in all of them only one was functional. In three non-functional rearrangements, a stop codon was found at their CDR3 junction, while in another rearrangement the lack of function remained unclear, due to our inability to fully amplify this region. It contained three stop codons in its V_H region, probably generated by somatic hypermutation (SHM) without selection (data not shown). Rearrangements are described in Tables 1 and 2.

Comparisons of clinical data (excluding patient LLC32) showed no significant differences in lymphocyte and platelet counts, haemoglobin level, age, sex, and stage status between the UM and M cases (data not shown). When comparing treatment inclusion and outcome, non-significant but relatively small probability test values (p values = 0.070 and 0.058, respectively, in Fisher's exact test) were found, indicating that a similar investigation should be performed on a larger number of subjects to assess the true nature of the observed differences.

Although complete clinical data were not available for every patient, the M group showed a better outcome than the UM group, in agreement with previous reports (Damle et al., 1999; Hamblin et al., 1999; Maloum et al., 2000). This provided further evidence of the importance of determining the mutational status of CLL patients in order to make a prognosis, although the small number of patients analysed in this study should be taken into account.

In this study, V_H3 was the most frequently used V_H family (37.8%), followed by V_H4 (35.2%), V_H1 (10.8%), V_H5 (8.1%), V_H2 (5.4%) and V_H6 (2.7%). When comparing M and UM cases with regard to families, 61.1% of M cases were represented by the V_H3 family and 47.3% of UM cases by the V_H4 family.

V_H4-39 was the most frequently used segment (in seven rearrangements), followed by V_H3-23, V_H3-33, V_H4-34, and V_H5-51 (three rearrangements each), V_H1-69, V_H3-7, V_H3-11, V_H3-53, and V_H4-59 (two rearrangements each), and V_H1-18, V_H1-8, V_H2-26, V_H2-70, V_H3-30, V_H3-72, V_H4-31, and V_H6-1 (one rearrangement each).

Comparing the use of V_H segments in UM and M cases, VH4-39 was the most frequently used in UM cases (26.3%), followed by VH4-59, VH1-69 and VH5-51 (10.5% each); in M cases, VH3 segments were more frequently used, except for VH3-23 that was more frequent in UM cases.

Although we found that the V_H3 family was the most frequently involved in functional rearrangements, followed by V_H4 and V_H1, in agreement with Pasqualucci et al. (2000), several reports have shown V_H3 followed by V_H1 and V_H4 (Fais et al., 1998; Pritsch et al., 1999; Sakai et al., 2000; Duke et al., 2003; Vilpo et al., 2003) or V_H4 > V_H3 > V_H1 profiles (Hashimoto et al., 1995). The occurrence of V_H1 as the third most frequent family coincided with the profile of normal cells (Rassenti et al., 1995; Matsuda et al., 1998), although, in most CLL studies, it was found only when the V_H1-69 segment was excluded from the analyses (Oscier et al., 1997; Duke et al., 2003). The findings of only two V_H1-69 segments in our patients represented a relevant difference with respect to other reports. This, however, might be due to the small number of patients analysed or, alternatively, to individual variations in number of the germline V_H1-69 segment in our population (Stewart et al., 1993; Cook and Tomlinson, 1995).

We did not find any rearrangement involving the V_H3-21 segment, a very frequent occurrence in CLL (Duke et al., 2003) and usually related to a very poor prognosis in mutated cases (Tobin et al., 2002; Dighiero, 2004), although the relation of mutational status and the molecular profile of other V_H segments were coincident with other reports (Duke et al., 2003; Guarini et al., 2003; Stevenson and Calligaris-Cappio, 2004).

D segments were not completely resolved by sequencing in six rearrangements, due to loss of the initial J_H region (the annealing region) when sequencing. D5-5 was the most frequently used D segment (five rearrangements), followed by D3-3 (four rearrangements), D3-10 and D3-16 (three rearrangements each), D3-22 and D6-19 (two rearrangements each), and D1-1, D1-26, D2-2, D2-8, D2-21, D5-12, D5-18, D5-24, D6-6 and D6-13 (one rearrangement each). Two rearrangements did not contain a D segment. The use of D segments differed between UM and M patients; in UM cases, the most frequent segments were D3-3, D3-16, D3-22 and D5-5, while in M cases an equal distribution of D segments was observed.

D5-5 was the most frequent D segment in productive rearrangements, unlike previous reports pointing to D3-3 as the most frequent segment involved in CLL rearrangements (Vilpo et al., 2003), mainly in UM cases (Hamblin et al., 1999; Duke et al., 2003). In M cases, we found D5-5 and D3-10 to be the most frequent segments, unlike another report in which D3-22 was the most frequent (Duke et al., 2003). Interestingly, in our patients, both rearrangements involving D3-22 segments were found in UM cases.

The J_H family was not completely resolved by sequencing in six rearrangements, due to loss of the initial J_H region (the annealing region) when sequencing. J_H4 was the most frequently used J_H family (12 rearrangements), followed by J_H6 (10 rearrangements), J_H5 (five rearrangements), and J_H1 (one rearrangement). The J_H6 family was more frequently used in UM cases, while J_H4 was more frequently used in M cases. Our findings regarding J_H families were coincident with previous studies, with J_H4 being more frequently represented in M rearrangements and J_H6 in UM rearrangements (Fais et al., 1998; Pritsch et al., 1999; Duke et al., 2003; Vilpo et al., 2003).

The length of the CDR3 protein region, determined in 31 functional rearrangements, ranged from 12 to 28 amino acids. UM cases showed a longer CDR3 region (from 13 to 28 amino acids; median = 21) than M cases (from 12 to 20 amino acids; median = 15). This difference was statistically significant (p < 0.001, by both Student's t-test and the Wilcoxon test), in agreement with previous data in the literature (Opezzo et al., 2002; Duke et al., 2003). Here again, we do not know if these findings reflect genetic and/or environmental factors or result from the small number of patients analysed by us. It is noteworthy that the genetic background of the Brazilian population is markedly different from the European and North American populations in ancestry and miscegenation. Moreover, environmental factors might be responsible for the presence of different types of antigens, accounting for specific antigenic stimulations (Ghiotto et al., 2004; Kolar and Capra, 2004) and leading to the occurrence of different rearrangements in different geographic regions (Stevenson and Calligaris-Cappio, 2004). To elucidate this point, a much larger Brazilian population study should be conducted. Moreover, the lack of data on the incidence or prevalence of CLL in Brazil makes it impossible to know whether these epidemiological parameters are similar to those in other western countries, a fact that might explain the difficulties in finding larger cohorts for confirming our results.

Acknowledgments

Work supported by: Instituto Nacional do Câncer, Fundação Ary Frauzino, CNPq and CAPES (Brazil), and Swissbridge Foundation (Switzerland).

Internet Resources

Received: August 29, 2007; Accepted: January 16, 2008.

Associate Editor: Emmanuel Dias Neto

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Publication Dates

• Publication in this collection
  18 Aug 2008
• Date of issue
  2008

History

• Received
  29 Aug 2007
• Accepted
  16 Jan 2008