Analysis of IgVH gene mutations in B cell chronic lymphocyticleukaemia according to antigen-driven selection identifiessubgroups with different prognosis and usage of the canonicalsomatic hypermutation machinery

B-cell chronic lymphocytic leukaemia (B-CLL) cells were

originally considered to be the neoplastic counterpart of B

lymphocytes unable to undergo somatic hypermutation (SHM;

Hamblin, 2002; Chiorazzi & Ferrarini, 2003). The demonstra-

tion that approximately 50% of B-CLL cases express a mutated

configuration of immunoglobulin (Ig)VH genes (Schroeder &

Dighiero, 1994; Oscier et al, 1997; Fais et al, 1998) changed

this view suggesting the existence of at least two distinct B-CLL

subsets that probably originated from pre- or post-germinal

centre (GC) B cells (Naylor & Capra, 1999). This different

histogenesis has important prognostic implications, as B-CLL

cases with a mutated (M) configuration of IgVH genes have a

better prognosis than those with unmutated (UM) B-CLL

(Damle et al, 1999; Hamblin et al, 1999; Maloum et al, 2000;

Krober et al, 2002; Lin et al, 2002). More recently, the

expression of a homogeneous phenotype and gene profile

related to experienced B cells virtually in all B-CLL cases (Klein

et al, 2001; Rosenwald et al, 2001; Damle et al, 2002) have

strongly questioned such a pathogenetic model, rather favour-

ing a common transformation mechanism for both M and UM

B-CLLs.

The SHM process, as it occurs during a T-cell-dependent

GC differentiation of normal B cells, introduces mutations in a

non-random manner generating several biased nucleotide

substitutions. Among them, the skewing of replacement (R)

mutations from framework (FR) to complementarity-deter-

mining regions (CDR) of IgVH genes is the result of a positive

selection operated by the antigen, aimed at preserving the

Massimo Degan,1 Riccardo Bomben,1

Michele Dal Bo,1 Antonella Zucchetto,1

Paola Nanni,1 Maurizio Rupolo,2

Agostino Steffan,3 Vincenza Attadia,1

Pier Ferruccio Ballerini,4 Daniela

Damiani,5 Carlo Pucillo,6 Giovanni Del

Poeta,7 Alfonso Colombatti8 and Valter

Gattei1

1Clinical and Experimental Haematology

Research Unit, 2Medical Oncology, 3Blood Bank,

IRCCS, Aviano (PN), 4Division of Internal

Medicine, De Gironcoli Hospital, Conegliano

(TV), 5Division of Hematology, and 6Laboratory

of Immunology, University of Udine, Udine,7S. Eugenio Hospital, University of Tor Vergata,

Rome, and 8Experimental Oncology 2 and Centro

di Riferimento Oncologico, IRCCS, Aviano (PN),

Italy

Received 23 January 2004; accepted for

publication 22 March 2004

Correspondence: Valter Gattei MD, Clinical and

Experimental Hematology Research Unit,

Centro di Riferimento Oncologico, IRCCS, Via

Pedemontana Occidentale, 12, Aviano (PN),

Italy. E-mail: vgattei@cro.it

Summary

Cases of B-cell chronic lymphocytic leukaemia (B-CLL) with mutated (M)

IgVH genes have a better prognosis than unmutated (UM) cases. We analysed

the IgVH mutational status of B-CLL according to the features of a canonical

somatic hypermutation (SHM) process, correlating this data with survival. In

a series of 141 B-CLLs, 124 cases were examined for IgVH gene per cent

mutations and skewing of replacement/silent mutations in the framework/

complementarity-determining regions as evidence of antigen-driven

selection; this identified three B-CLL subsets: significantly mutated (sM),

with evidence of antigen-driven selection, not significantly mutated (nsM)

and UM, without such evidence and IgVH gene per cent mutations above or

below the 2% cut-off. sM B-CLL patients had longer survival within the good

prognosis subgroup that had more than 2% mutations of IgVH genes. sM,

nsM and UM B-CLL were also characterized for the biased usage of IgVH

families, intraclonal IgVH gene diversification, preference of mutations to

target-specific nucleotides or hotspots, and for the expression of enzymes

involved in SHM (translesion DNA polymerase f and g and activation-

induced cytidine deaminase). These findings indicate the activation of a

canonical SHM process in nsM and sM B-CLLs and underscore the role of

the antigen in defining the specific clinical and biological features of B-CLL.

Keywords: chronic lymphocytic leukaemia, somatic hypermutation, antigen

selection, translesion DNA polymerases, prognosis.

research paper

ª 2004 Blackwell Publishing Ltd, British Journal of Haematology, 126, 29–42 doi:10.1111/j.1365-2141.2004.04985.x

structural integrity of Igs by concomitantly providing a high

mutational rate in antigen-binding regions (Chang & Casali,

1994; Dorner et al, 1998a; Lossos et al, 2000). Moreover, the

excess of transitions over transversions, as well as of mutations

targeting specific nucleotides or nucleotide motifs, may be

explained by the activity of a specific mutational machinery

(Smith et al, 1996; Dorner et al, 1998b; Neuberger et al, 1998).

This includes the DNA polymerases (pol) f and g, which are

efficient translesion and error-prone DNA polymerases, i.e.

capable of carrying out lesion bypassing DNA synthesis by

copying DNA with a high error rate (Zan et al, 2001; Zeng

et al, 2001; Storb & Stavnezer, 2002). Moreover, the activation-

induced cytidine deaminase (AID), a member of the cytidine

deaminase family exclusively expressed by GC B cells, is

another essential enzyme for both SHM and class switch

recombination of normal B cells (Muramatsu et al, 1999, 2000;

Cerutti et al, 2002; Storb & Stavnezer, 2002).

While some reports indicate a broad expression of AID in

B-CLL (Albesiano et al, 2003; Oppezzo et al, 2003), with the

highest levels being confined to UM CD38high subsets

(McCarthy et al, 2003; Degan et al, 2004), expression of

translesion DNA polymerases has not been yet investigated in

this disease. However, the finding of an intraclonal VHDJH

gene diversification in most B-CLL cases (Gurrieri et al, 2002)

suggests the presence of mutational machinery that is still

functionally active following neoplastic transformation. Finally,

evidence of antigen-driven selection in B-CLL, as evaluated by

computing the unbalanced distribution of R/silent (S) muta-

tions in CDR/FR, although reported by some studies (Hamblin

et al, 1999; Gurrieri et al, 2002; Messmer et al, 2004), has

never been correlated with survival.

In the present study, the IgVH mutational status was

investigated in a large cohort of B-CLLs by using a strategy of

cloning and sequencing of at least 5–10 transcripts for each

patient. By extending some of our preliminary observations

(Gattei et al, 2003), we demonstrated that the application of

statistical models aimed at evaluating the impact of positive

selection operated by the antigen (Chang & Casali, 1994;

Lossos et al, 2000) enabled the identification of three B-CLL

subsets, each with a different prognosis and usage of the

canonical SHM machinery.

Patients, materials and methods

B-CLL patients and cell samples and immunophenotypicalanalysis

The study was performed on peripheral blood (PB) samples,

collected after informed consent for routine diagnostic and

follow-up procedures, from 141 patients affected by typical B-

CLL. Diagnosis was made according to standard criteria, inclu-

ding expression of CD19, CD5, CD23 and low levels of clonally

restricted surface Igs (Matutes et al, 1994). The median age at

diagnosis was 64 years (range 32–97 years) and the male to female

ratio was 1Æ1 (75 males, 66 females). The clinical stage at diagnosis

according to Rai et al (1975) was available for 127 of 141 patients

as follows: stage 0, 33% (n ¼ 42); stage I, 39Æ4% (n ¼ 50);

stage II, 23Æ6% (n ¼ 30); stage III, 1Æ6% (n ¼ 2); stage IV, 2Æ4%

(n ¼ 3). Mononuclear cells were isolated by centrifugation on

Ficoll–Hypaque (Pharmacia, Uppsala, Sweden) gradient and

either used directly or cryopreserved in liquid nitrogen until use.

Expression of phenotypic markers, including CD38, was inves-

tigated by flow cytometry, as previously described (Gattei et al,

1997; Damle et al, 1999; Del Poeta et al, 2001). Cases in which

CD38 was detected in <30% of cells were identified as CD38low,

while cases with ‡30% CD38 B-CLL cells were considered

CD38high (Damle et al, 1999; Del Poeta et al, 2001).

Amplification, cloning and sequencing of VHDJH

transcripts

Total RNA was extracted and reverse-transcribed as described

(Gattei et al, 1997). The resulting cDNAs, checked for first-

strand synthesis (Degan et al, 2000), were amplified using a

mixture of sense primers annealing either to the VH1 through

VH6 leader sequences or to the 5¢-end of VH1–VH6 FR1

utilized in conjunction with a mixture of antisense primers

complementary to the germ line JH regions, as reported (Fais

et al, 1998; Damle et al, 1999; Hamblin et al, 1999; Gurrieri

et al, 2002; Krober et al, 2002). The purified amplified

products, inserted into the PCR2.1-TOPO vector (Invitrogen

S.R.L., San Giuliano Milanese, Milan, Italy), were expanded in

TOP10 One ShotTM competent cells (Invitrogen) and cloned.

Plasmid DNAs were isolated from overnight cultures of

randomly selected colonies and sequenced by using an

automatic DNA sequencer (ABI PRISM 3100, Applied

Biosystem, Foster City, CA, USA; Beckman CEQ2000XL,

Fullerton, CA, USA). When tested for the DNA polymerase

error rate according to Pasqualucci et al (1998), this strategy

yielded an incorporating error rate of 2Æ8 · 10)4/bp. In

additional experiments, plasmid DNA from two distinct

colonies from two different B-CLL patients (B176 and B211)

were amplified and cloned by exactly reproducing the proce-

dures described above. Among 20 sequenced colonies

(6732 bp), a single mutation was detected (DNA polymerase

error rate ¼ 1Æ5 · 10)4/bp). Comparisons between the ob-

tained sequences and those of the various germ line IgVHDJH

genes were performed with the Ig basic local alignment search

tool (IgBLAST) directory (http://www.ncbi.nlm.nih.gov/ig-

blast) utilizing the MacVector 7.1 sequence analysis software

(Accelrys; Symantec Co., Accelrys European Headquarters,

Cambridge, UK). According to this algorithm, a stretch of

seven or more nucleotides was required for D and JH

assignment. Only when the same rearrangement was identified

in at least 5–10 clones (three or four clones in 11 cases), a given

IgVH sequence was further analysed. As described (Gurrieri

et al, 2002), alignment of the IgVH sequences available for each

patient revealed, along with mutations shared by all the

transcripts analysed, a number of partially shared or unique

mutations. For this reason, all mutational analyses (see below)

M. Degan et al

30 ª 2004 Blackwell Publishing Ltd, British Journal of Haematology, 126, 29–42

were carried out in each IgVH transcript separately, and the per

cent mutation assigned to a given B-CLL was the mean value of

the per cent mutations found in each individual transcript.

IgVH mutation analysis

A census of somatic point mutations was performed by solely

considering the IgVH sequence, i.e. from FR1 to FR3. As

evidence of antigen-driven selection (Chang & Casali, 1994;

Dorner et al, 1998a; Lossos et al, 2000; Gurrieri et al, 2002),

the probability that an excess or scarcity of R mutations

occurred by chance in IgVH CDR or FR, respectively, was

computed by applying the available binomial and multinomial

distribution models (Chang & Casali, 1994; Lossos et al, 2000).

Both these models calculate the expected frequency of

mutations by taking into account the base composition of a

given germ line IgVH sequence (Chang & Casali, 1994; Lossos

et al, 2000). In the absence of a positive selective pressure

operated by the antigen, nucleotide changes yielding R or

S mutation will be distributed throughout the coding sequence

with a pattern superimposible to that of randomly occurring

mutations (P > 0Æ05). Conversely, a skewing of R mutation

from FR to CDR and/or of S mutations from CDR to FR will

indicate a positive selection operated by the antigen; in this

case, the probability that these mutations are due by chance

will be <0Æ05 (Chang & Casali, 1994; Lossos et al, 2000). In the

present study, P-values were computed by applying the

multinomial distribution model, taking advantage of the

ready-to-use computer program at the http://www-stat.stan-

ford.edu/immunoglobulin website (Lossos et al, 2000). Due to

some flaws of the available JAVA applet, the multinomial

model was not appropriate when analysing specific IgVH (3–53

and 4–39, overall accounting for nine cases of 77 M B-CLL in

our series). In these cases, calculations of biased R/S mutations

were made according to the binomial distribution model

utilizing the reported formula (Chang & Casali, 1994). The

expected values of transitions or transversions, of mutations

targeting specific nucleotides as well as the RGYW (i.e. purine/

G/pyrimidine/A or T) or its inverse (WRCY) motifs were

computed as reported (Smith et al, 1996; Dorner et al, 1998b;

Neuberger et al, 1998).

Expression of translesion DNA polymerases g and f andAID

Expression of DNA pol g and f was evaluated by quantitative

real-time polymerase chain reaction (QRT-PCR), while AID

expression was investigated by conventional reverse transcrip-

tion-PCR (RT-PCR) on a selected cohort of B-CLL cases (52

cases). To avoid interference with normal B and T cells that

allegedly express these enzymes, only cases with more than 95%

of neoplastic cells in PB samples were analysed. Briefly, cDNAs

from B-CLL cells were amplified with primer pairs specific for

pol g, pol f and the housekeeping gene b2-microglobulin

(b2M), as described (Zan et al, 2001; Spessotto et al, 2002). The

incorporation of the SYBR� Green dye (Applied Biosystems)

into the PCR products was monitored in real-time with a

sequence detection system (ABI PRISM 7700), resulting in a

calculation of threshold cycles (Ct value), i.e. the PCR cycle

number corresponding to the beginning of the exponential

growth of PCR products. Ct values were converted into

attomoles by means of QRT-PCR experiments carried out

with serial dilution of known concentrations of pol g, pol f and

b2M-specific amplicons. Results were reported as the ratio of

the relative expression levels of pol g/b2M or pol f/b2M.

Activation-induced cytidine deaminase expression was

checked in the same cohort of cases essentially as described

(Albesiano et al, 2003; McCarthy et al, 2003; Oppezzo et al,

2003). Briefly, cDNAs were amplified with primers chosen to

anneal to sequences corresponding to the first (sense, position

45–64) and the fifth (antisense, position 675–653) exons of

AID-specific cDNA (Albesiano et al, 2003; McCarthy et al,

2003; Oppezzo et al, 2003). Amplified products (10 ll) were

run on 1Æ5% ethidium bromide-stained agarose gels and

analysed under ultra-violet light.

Statistical analysis

Complete survival data were available for 141 patients. The

mean follow-up was 67 months (range 8–220 months). Patient

survivals were analysed using the Kaplan–Meier survival curves

and log-rank test (Armitage & Berry, 1987). Correlation

between IgVH mutational status and CD38 expression was

performed by the chi-square test (Armitage & Berry, 1987).

Differences between expected and observed values of transi-

tions and transversions, mutations targeting specific nucleo-

tides, as well as RGYW/WRCY motifs in IgVH sequences were

assessed by the Student’s t-test for paired samples, by

separately evaluating each transcript (Armitage & Berry,

1987). Differences between the relative expression levels of

pol f/g ratios were assessed by the Student’s t-test for unpaired

samples. Statistical significance was always accepted when

P < 0Æ05.

Results

IgVH per cent mutations, biased IgVH usage, intraclonalIgVH diversification, CD38 expression and survivals

The mutational status of IgVH genes was investigated in 124 of

141 B-CLLs by sequencing at least 5–10 transcripts (three to

four in 11 cases) for each patient. Values of IgVH gene

mutations (Fig 1), represent the mean values of per cent

mutations due to a certain degree of intraclonal IgVH

diversification (see below). According to this analysis, 47

B-CLL cases (38%) had neoplastic cells bearing less than 2%

average mutations when compared with the nearest germ line

sequence (UM B-CLL), while 77 cases (62%) displayed ‡2%

average mutations and were classified as M B-CLLs (Damle

et al, 1999; Hamblin et al, 1999; Maloum et al, 2000; Krober

IgVH Gene Mutations and Prognosis in B-CLL

ª 2004 Blackwell Publishing Ltd, British Journal of Haematology, 126, 29–42 31

et al, 2002). Tables I and II summarize VHDJH assignments

and the means of per cent mutations for UM and M B-CLLs

respectively.

In agreement with previous reports (Keating et al, 2003;

Messmer et al, 2004), a higher number of B-CLLs expressing

the VH1 family was found in the UM, when compared with the

M, B-CLL subgroups (37Æ5% vs. 10Æ1%; Table III). Conversely,

M B-CLLs preferentially expressed VH genes belonging to the

VH3 family (58Æ2% vs. 31Æ3%; Table III). No differences were

observed between UM and M B-CLLs regarding the expression

of other IgVH families, including VH4 (about 20% in both

subgroups) and other rarely expressed IgVH genes, such as

VH2, VH5 and VH6 (Table III).

Table II also reports a detailed analysis of IgVH point

mutations for the M B-CLL group. According to Gurrieri et al

(2002), they were divided into ‘shared’ (i.e. mutations found in

all the IgVH transcripts analysed), ‘partially shared’ (i.e.

mutations present in a part of the analysed IgVH transcripts)

and ‘unique’ (i.e. mutations found only in distinct IgVH

transcripts) mutations (Tables II and IV). Among 79 IgVH

(449 IgVH transcripts), 63 (about 80%) exhibited either no

intraclonal IgVH diversification (i.e. shared mutations only, 13

IgVH, 16Æ5%) or a low number of unique and/or partially

shared mutations, always in the presence of a great excess of

shared mutations (Tables II and IV). Conversely, in 16 of 79

IgVH, the sum of unique and partially shared mutations

exceeded the number of shared mutations (Tables II and IV),

thus revealing a higher degree of intraclonal IgVH diversifica-

tion.

Data on CD38 expression was available for 140 of 141

patients, of which 51 cases (36Æ4%) were CD38high B-CLL, and

the remaining 89 cases (63Æ6%) were classified as CD38low

B-CLL (Damle et al, 1999; Del Poeta et al, 2001). In agreement

with some of the current literature (Damle et al, 1999), we

found a statistically significant correlation between CD38high

and low mutations of IgVH genes by applying a chi-square test

(P < 0Æ01; Fig 1).

A comparison of patient survivals with CD38 expression

(140 cases) or IgVH mutational status (124 cases) also

demonstrated the positive impact of CD38low and IgVH

mutations above the standard cut-off of 2% on prognosis

(Fig 2) (Damle et al, 1999; Del Poeta et al, 2001; Krober et al,

2002; Lin et al, 2002).

Altogether, IgVH per cent mutations and usage, intraclonal

IgVH diversification, CD38 expression and survivals, as found

on our cohort of B-CLL patients, were in keeping with the

above reports.

Evidence of antigen-driven selection identified three B-CLLsubsets

The hypothesis that M B-CLL cases may derive as a transfor-

mation of a competent B cell (Naylor & Capra, 1999), led us to

investigate whether the IgVH gene mutational status found in

Fig 1. CD38 expression and IgVH mutational status in B-cell chronic lymphocytic leukaemia (B-CLL). B-CLL cases were arranged according to the

IgVH mutational status, expressed as per cent mutation (upper panel); the lower panel indicates, for each B-CLL case, the corresponding expression

level for CD38, reported as percentage of positive B cells. Cases for which IgVH mutational status and CD38 expression data were not simultaneously

available are reported as open histograms. Dotted lines refer to the established cut-offs of 2% mutation (upper panel) and 30% CD38+ cells (lower

panel). Asterisks indicate three cases (B241, B235, B260) with biclonal disease, whose per cent mutations were separately reported. A chi-square

correlation between IgVH mutational status (cut-off: 2% mutations) and CD38 expression (cut-off: 30% of positive cells) is also reported (P < 0Æ01).

n.a., not available.

M. Degan et al

32 ª 2004 Blackwell Publishing Ltd, British Journal of Haematology, 126, 29–42

B-CLL cells was consistent with that of B cells positively

selected by the antigen (Chang & Casali, 1994; Dorner et al,

1998a; Lossos et al, 2000). Within IgVH genes, sequences

encoding for the antigen-contacting CDRs are inherently more

prone to R mutations than those encoding for the respective

FRs (Chang & Casali, 1994), this susceptibility being further

stressed by antigen selection (Chang & Casali, 1994; Dorner

et al, 1998a; Lossos et al, 2000). In our study, among 77 M

B-CLLs, 449 IgVH transcripts were analysed by applying the

multinomial distribution model to assess the probability that

biased R/S mutations in IgVH gene CDR/FR arise randomly

(Lossos et al, 2000). When the multinomial model gave a

result that was not appropriate (52/449 IgVH transcripts,

11Æ5%), we took advantage of the binomial distribution model

according to Chang and Casali (1994). Overall, a given IgVH

transcript was defined as having evidence of antigen-driven

selection if the applied statistical model yielded a significant

result (P < 0Æ05) either in CDRs or FRs (Dorner et al, 1998a;

Sakai et al, 2000).

By using such an approach, we identified 52 of 77 M B-CLLs

for which evidence of antigen-driven selection was found in all

or in part of the IgVH transcripts analysed (Table II). These

B-CLL cases were classified as ‘significantly mutated’ (sM

B-CLL). Table II summarizes the characteristics of the sM B-CLL

cases, including VHDJH gene assignment, per cent mutations,

number of IgVH transcripts analysed and number of IgVH

transcripts showing evidence of antigen-driven selection in

CDR and/or FR. Among 289 analysed IgVH transcripts

(average 5Æ7, range 3–10), 247 showed a significant excess of

R mutations in CDR (25 IgVH transcripts) or a scarcity of

R mutations in FR (114 IgVH transcripts) or both (108 IgVH

transcripts), while the remaining 42 IgVH transcripts did not

display evidence of antigen-driven selection. Taken together,

36 of 52 sM B-CLLs (69Æ2%) showed evidence of antigen-

driven selection in all of the transcripts analysed, while in 16

cases this evidence was documented only in some IgVH

transcripts (Table II). The remaining B-CLL cases were

classified as ‘not significantly mutated’ (nsM, 25 cases) or

‘unmutated’ (UM, 47 cases) when the per cent mismatch was

above or below the 2% cut-off respectively. For all these cases,

the average number of IgVH transcripts analysed for each

B-CLL patients was 6Æ1 (range 5–10) and none showed

evidence of antigen-driven selection using the two available

statistical models.

Characterization of sM and nsM B-CLLs: biased IgVH

usage and intraclonal IgVH diversification

The sM subgroup of B-CLL cases displayed a pattern of IgVH

usage similar to that reported for M B-CLLs (Table III).

However, when compared with M B-CLL, a more evident

skewing towards the preferential usage of IgVH3 family was

documented in sM B-CLL. In fact, almost 2/3 of sM B-CLLs

rearranged IgVH genes of the VH3 family, whereas nsM B-CLLs

Table I. Mutation characteristics of IgVHDJH genes in the unmutated

B-cell chronic lymphocytic leukaemia (B-CLL) subgroup.

CLL case

VHDJH gene

% mutation*VH D JH

B 48 1-18 3-10 4 0Æ0B 201 3-15 3-3 4 0Æ0B 206 3-11 6-6 6 0Æ0B 265 3-73 3-22 3 0Æ0B 210 3-53 3-3 4 0Æ1B 16 3-13 3-3 4 0Æ2B 209 1-2 6-19 4 0Æ2B 232 4-39 6-13 5 0Æ2B 169 1-2 1-26 4 0Æ3B 205 3-21 5-24 6 0Æ3B 47 1-18 3-10 4 0Æ4B 149 4-59 3-22 6 0Æ4B 42 4-39 6-13 5 0Æ5B 255 5-51 3-22 4 0Æ5B 249 5-51 3-22 4 0Æ5B 202 1-69 3-16 3 0Æ6B 179 4-39 6-13 4 0Æ7B 191 3-11 3-9 6 0Æ7B 241 2-26 6-13 4 0Æ7

2-5 1-7 4 1Æ9B 116 1-69 2-2 2 0Æ8B 173 3-30 7-21 1 0Æ8B 220 1-69 3-22 6 0Æ8B 128 3-33 3-3 3 0Æ9B 159 6-1 2-2 4 0Æ9B 21 3-23 5-10 4 0Æ9B 196 4-34 6-13 6 1Æ0B 223 1-69 n.a. 3 1Æ0B 118 1-2 2-21 6 1Æ1B 18 1-2 1-26 6 1Æ1B 176 1-69 6-13 3 1Æ1B 83 3-23 2-8 6 1Æ1B 119 4-31 3-3 5 1Æ1B 166 4-39 4-17 5 1Æ1B 158 1-69 7-27 1 1Æ1B 244 3-30 n.a. 5 1Æ1B 161 4-59 3-22 4 1Æ2B 183 1-69 3-16 4 1Æ3B 245 3-33 7-27 4 1Æ3B 174 1-69 3-3 6 1Æ3B 17 1-46 3-10 6 1Æ4B 14 4-34 3-3 6 1Æ6B 110 1-18 6-19 4 1Æ6B 15 3-21 5-18 6 1Æ6B 184 3-48 2-8 4 1Æ7B 165 1-2 3-10 5 1Æ7B 39 1-2 6-13 4 1Æ7B 75 4-39 2-8 4 1Æ7

n.a., not assigned.

*Values represent the mean value of 5–10 transcripts analysed for each

B-CLL case.

IgVH Gene Mutations and Prognosis in B-CLL

ª 2004 Blackwell Publishing Ltd, British Journal of Haematology, 126, 29–42 33

Table II. Mutation characteristics of IgVHDJH genes in the mutated B-cell chronic lymphocytic leukaemia (B-CLL) subgroup.

CLL case

VHDJH genesTranscripts

analysed

IgVH point mutations*

% mutation�

IgVH transcripts with P < 0Æ05�

Ag selection§VH D JH Shared Partially shared Unique CDR only FR only CDR + FR

B 186 1-69 3-16 4 5 30 0 0 2Æ0 0 0 0 )B 49 3-30 3-21 6 5 10 16 4 2Æ1 0 0 0 )B 180 3-23 2-15 5 7 42 0 0 2Æ2 n.a.– n.a. n.a. +

B 153 3-30 5-12 4 4 20 3 2 2Æ3 0 1 1 +

B 216 3-53 7-27 4 7 49 0 0 2Æ4 0 0 0 )B 217 4-39 3-22 4 7 49 0 1 2Æ4 7 0 0 +

B 224 3-33 3-9 3 5 35 2 1 2Æ6 0 0 0 )B 67 1-46 3-10 6 6 6 21 25 2Æ9 0 0 2 +

B 156 1-58 3-10 3 8 64 7 0 3Æ0 0 0 0 )B 182 3-53 4-11 4 5 45 4 3 3Æ4 5 0 0 +

B 227 3-48 6-19 5 4 40 0 0 3Æ4 0 4 0 +

B 218 3-64 2-2 6 10 90 11 2 3Æ5 0 0 0 )B 188 3-7 4-23 3 5 55 0 0 3Æ7 0 0 0 )B 194 4-39 6-25 4 5 55 0 2 3Æ7 0 0 0 )B 195 4-39 6-25 4 5 55 0 4 3Æ9 0 0 0 )B 219 3-48 n.a. 5 5 13 33 13 4Æ0 0 3 0 +

B 68 4-34 6-19 4 5 60 0 1 4Æ1 0 0 0 )B 189 3-21 n.a. 6 5 50 6 5 4Æ2 0 0 0 )B 114 3-48 6-19 5 6 66 4 2 4Æ2 0 3 2 +

B 98 4-28 3-22 3 7 91 0 0 4Æ4 0 0 7 +

B 125 4-34 6-19 4 6 78 0 1 4Æ6 0 0 0 )B 236 3-48 5-24 4 6 36 35 10 4Æ6 0 6 0 +

B 154 4-39 6-13 4 5 65 0 3 4Æ7 0 0 0 )B 261 3-21 6-13 4 5 60 4 5 4Æ7 0 0 5 +

B 229 3-23 5-12 5 4 56 0 0 4Æ8 0 0 4 +

B 231 3-23 5-12 5 6 84 0 1 4Æ8 0 0 6 +

B 198 3-48 3-22 4 6 84 2 0 4Æ9 0 0 6 +

B 221 3-7 3-3 4 6 84 0 6 5Æ1 0 0 0 )B 164 3-21 5-24 6 3 42 0 3 5Æ1 0 0 3 +

B 177 3-23 2-15 5 4 56 0 3 5Æ1 4 0 0 +

B 215 4-4 7-27 4 10 150 0 3 5Æ2 0 3 0 +

B 240 3-11 3-21 4 7 105 0 5 5Æ4 0 0 7 +

B 207 4-59 2-15 3 9 144 0 2 5Æ5 0 0 0 )B 242 2-5 n.a. 5 5 10 50 22 5Æ5 0 1 1 +

B 80 4-28 3-3 4 5 85 0 0 5Æ7 0 0 5 +

B 13 4-28 3-10 5 6 96 5 2 5Æ8 3 2 2 +

B 235 4-59 2-15 2 5 85 0 1 5Æ9 0 5 0 +

3-9 3-3 6 4 132 0 1 11Æ4 4 0 0 +

B 222 3-72 2-21 4 5 85 2 3 6Æ0 0 4 0 +

B 84 5-51 3-3 4 5 90 0 0 6Æ1 0 0 0 )B 181 4-31 7-27 4 5 35 53 0 6Æ1 0 5 0 +

B 264 3-33 n.a. 6 5 5 64 22 6Æ2 0 0 1 +

B 263 4-39 n.a. 4 6 12 81 19 6Æ3 1 0 3 +

B 259 4-39 2-8 3 7 125 0 2 6Æ4 0 0 0 )B 239 1-3 6-13 4 9 171 0 4 6Æ7 0 0 0 )B 230 2-5 n.a. 4 5 95 0 6 6Æ7 0 1 0 +

B 260 3-33 n.a. 3 6 24 48 46 6Æ7 0 2 3 +

1-69 n.a. 4 5 15 48 55 8Æ0 0 0 4 +

B 237 3-7 3-4 4 5 95 4 2 6Æ9 0 3 2 +

B 107 3-23 5-24 4 4 64 9 11 7Æ1 0 4 0 +

B 271 3-23 7-27 4 7 140 6 4 7Æ3 0 0 0 )B 170 3-15 3-3 3 3 66 0 0 7Æ3 0 3 0 +

B 155 3-11 2-21 6 5 100 4 1 7Æ3 1 0 2 +

M. Degan et al

34 ª 2004 Blackwell Publishing Ltd, British Journal of Haematology, 126, 29–42

preferentially used the VH4 family (44Æ0%), IgVH3 genes being

employed only in 40% of cases.

As summarized in Tables II and IV, a certain degree of

intraclonal IgVH diversification (Gurrieri et al, 2002) was

found by analysing the 54 (296 IgVH transcripts) and the 25

(153 IgVH transcripts) IgVH from the sM and nsM B-CLL

subsets. In both subgroups, the vast majority of IgVH

transcripts showed a large excess of shared mutations associ-

ated or not with a low number of unique and/or partially

shared mutations (Tables II and IV). However, within the

group of B-CLL displaying the highest degree of intraclonal

IgVH diversification (i.e. that with the sum of unique and

partially shared mutations exceeding the number of shared

mutations), almost all cases (14 of 16) belonged to the sM

B-CLL subset, suggesting a role of the antigen in determining

the complexity of intraclonal IgVH diversification.

Characterization of sM, nsM and UM B-CLLs: analysis ofbiased IgVH mutations

The mutational machinery responsible for SHM in normal

B cells has been shown to introduce mutations in a non-random

manner (Smith et al, 1996; Dorner et al, 1998a,b; Neuberger

et al, 1998). Therefore, we investigated the occurrence of these

mutations in IgVH sequences from sM, nsM and UM B-CLLs in

the present series. In total, we analysed 351 IgVH transcripts from

67 cases belonging to the sM (27 cases), nsM (14 cases) and UM

(26 cases) B-CLL subgroups. As summarized in Fig 3, a

Table II. continued

B 243 4-59 n.a. 3 4 12 16 59 7Æ5 0 4 0 +

B 126 4-31 4-17 5 5 5 47 61 7Æ7 0 0 0 )B 274 3-7 3-16 4 6 132 0 3 7Æ7 0 0 6 +

B 254 4-59 2-15 2 8 168 13 5 7Æ8 0 0 0 )B 257 3-33 n.a. 3 5 110 3 2 7Æ8 0 5 0 +

B 167 3-7 2-2 5 8 175 10 2 7Æ9 0 3 2 +

B 253 3-23 n.a. 4 6 18 108 21 8Æ4 0 0 5 +

B 197 3-23 7-27 4 7 21 132 22 8Æ5 0 0 7 +

B 273 3-23 3-22 5 6 138 12 3 8Æ8 0 6 0 +

B 163 3-23 7-27 4 3 81 0 0 9Æ2 0 0 3 +

B 168 3-33 3-9 2 4 108 0 3 9Æ4 0 0 4 +

B 234 2-5 n.a. 3 5 10 96 33 9Æ4 0 0 2 +

B 99 3-33 7-27 4 5 135 0 1 9Æ5 0 5 0 +

B 238 4-59 n.a. 4 5 140 0 2 9Æ7 0 0 0 )B 262 4-39 n.a. 5 5 15 128 0 9Æ7 0 0 5 +

B 211 3-7 3-22 3 10 290 0 5 10Æ1 0 6 2 +

B 225 3-72 3-16 4 9 270 2 5 10Æ3 0 9 0 +

B 157 3-11 4-11 6 6 180 4 2 10Æ4 0 6 0 +

B 152 3-23 4-23 5 5 150 0 1 10Æ5 0 0 0 )B 258 3-30 n.a. 3 6 186 0 0 10Æ6 0 0 0 )B 226 4-61 2-2 6 5 155 4 1 10Æ9 0 0 0 )B 250 3-33 2-21 3 6 192 0 1 11Æ0 0 6 0 +

B 127 1-3 3-3 5 5 170 0 3 11Æ6 0 2 3 +

B 185 1-69 4-23 5 3 114 0 0 13Æ0 0 0 3 +

B 204 1-2 n.a. 5 5 195 0 4 13Æ6 0 5 0 +

B 208 2-5 n.a. 4 7 294 2 1 14Æ3 0 7 0 +

n.a., not assigned.

*IgVH point mutations have been divided into ‘shared’ (i.e. present in all the IgVH transcripts), ‘partially shared’ (i.e. present in some but not all IgVH

transcripts) and ‘unique’ (i.e. present only in distinct IgVH transcript) mutations, according to Gurrieri et al (2002).

�Mean value among the various IgVH transcripts analysed for each B-CLL case.

�P-values were obtained by computing the probability that an excess or scarcity of replacement mutations occurred by chance in IgVH comple-

mentarity-determining region (CDR) or framework region (FR), respectively, by applying the available multinomial (Lossos et al, 2000) and binomial

(Chang & Casali, 1994) distribution models, as described in Patients, materials and methods.

§Mutated B-CLL cases with evidence of antigen-driven selection (P < 0Æ05 in CDR only and/or FR only and/or CDR + FR) in all or in part of the

IgVH transcripts analysed were re-classified as ‘significantly mutated’ and indicated as ‘+’; mutated B-CLL cases without evidence of antigen-driven

selection in all the IgVH transcripts analysed were re-classified as ‘not significantly mutated’ and indicated as ‘)’.

–An insertion of 21 nucleotides in the CDR2 was documented in all the transcripts analysed; the per cent mutations reported does not take into

account this insertion; although neither the binomial nor the multinomial distribution models were applicable, this case has also been included in this

subgroup.

IgVH Gene Mutations and Prognosis in B-CLL

ª 2004 Blackwell Publishing Ltd, British Journal of Haematology, 126, 29–42 35

significant excess of transitional over transversional mutations

was documented in IgVH transcripts belonging to the sM and

nsM B-CLL subgroups (Fig 3A). This excess was mainly due to

higher mutations targeting the nucleotide A, although relative

C- (nsM B-CLLs) or C- and G-preferences (sM B-CLL) were also

found (Fig 3B). Similarly, both nsM and sM B-CLLs displayed a

higher number of mutations targeting the RGYW and its inverse

(WRCY) motifs (Fig 3C). Conversely, no evidence of biased

nucleotide substitutions was observed by analysing the low

number of mutations found in UM B-CLLs. Taken together,

mutational analysis of IgVH sequences are compatible with the

activity of a canonical hypermutation machinery in the nsM and

sM B-CLL subgroups.

Characterization of sM, nsM and UM B-CLLs: expressionof DNA pol f, DNA pol g and AID

The notion that both nsM and sM B-CLLs displayed

intraclonal IgVH diversification and had a pattern of IgVH

mutations consistent with the activation of a canonical SHM

machinery, prompted us to investigate the expression of the

enzymes involved in SHM of normal GC-derived B cells in

these subgroups, such as translesion DNA polymerases and

AID (Muramatsu et al, 1999, 2000; Zan et al, 2001; Zeng et al,

2001; Cerutti et al, 2002; Storb & Stavnezer, 2002).

Figure 4 reports the mean values of the relative expression

levels of DNA pol g, DNA pol f along with the mean values of

the DNA pol f/g expression ratio found in 52 B-CLL samples

Table III. Biased IgVH usage in unmutated (UM), mutated (M),

significantly mutated (sM) and not significantly mutated (nsM) B-cell

chronic lymphocytic leukaemia (B-CLL) subgroup.

IgVH family

B-CLL subgroup

UM (48)* M (79) sM (54) nsM (25)

VH1 37Æ5 (18)� 10Æ1 (8) 9Æ3 (5) 12Æ0 (3)

VH2 4Æ2 (2) 3Æ8 (3) 7Æ4 (4) 0Æ0VH3 31Æ3 (15) 58Æ2 (46) 64Æ8 (35) 40Æ0 (10)

VH4 20Æ8 (10) 26Æ6 (21) 18Æ5 (10) 44Æ0 (11)

VH5 4Æ2 (2) 1Æ3 (1) 0Æ0 4Æ0 (1)

VH6 2Æ1 (1) 0Æ0 0Æ0 0Æ0VH7 0Æ0 0Æ0 0Æ0 0Æ0

*Values in parentheses represent the number of IgVH found for each

B-CLL subgroup, exactly as reported in Table I (UM) and Table II

(M).

�Values represented the percentage of cases utilizing a given IgVH

family; the corresponding total number of cases are given in paren-

theses.

Table IV. Intraclonal IgVH diversification in the mutated B-cell

chronic lymphocytic leukaemia (B-CLL) subgroup.

Intraclonal IgVH diversification M (79)* sM (54) nsM (25)

No i.d.� 13 (16Æ5)� 8 (14Æ8) 5 (20)

s. > u./p.s.§ 29 (36Æ8) 17 (31Æ5) 12 (48)

s. > u. + p.s.– 21 (26Æ5) 15 (27Æ8) 6 (24)

u. + p.s. > s.** 16 (20Æ2) 14 (25Æ9) 2 (8)

*Values in parentheses represent the number of IgVH found for each

B-CLL subgroup, exactly as reported in Table II, including the two

biclonal B-CLLs (B235 and B260).

�IgVH showing no intraclonal diversification (i.d.).

�Values refer to the number of IgVH displaying a given level of

intraclonal diversification; percentages are given in parentheses.

§IgVH displaying either unique (u., 27 IgVH) or partially shared (p.s., 2

IgVH) mutations; in this subgroup, shared (s.) mutations always

exceeded u./p.s. mutations.

–IgVH displaying both unique and partially shared (u. + p.s.) muta-

tions; in this subgroup, shared (s.) mutations always exceeded u. + p.s.

mutations.

**IgVH displaying both unique and partially shared (u. + p.s.) muta-

tions; in this subgroup, u. + p.s. mutations always exceeded shared (s.)

mutations.

Fig 2. Survival of B-cell chronic lymphocytic leukaemia (B-CLL)

patients based on CD38 expression and IgVH mutational status.

(A) Kaplan–Meier curves comparing survival based on the detection of

CD38 in <30% (CD38low) or in ‡30% (CD38high) of B-CLL cells

(CD38low: 50 cases; CD38high: 90 cases); log-rank test, P ¼ 0Æ003.

(B) Kaplan–Meier curves comparing survival in B-CLL cases with a

mutated (‡2% mutations) or unmutated (<2% mutations) configuration

of IgVH genes (‡2%: 77 cases; <2%: 47 cases); log-rank test, P ¼ 0Æ003.

M. Degan et al

36 ª 2004 Blackwell Publishing Ltd, British Journal of Haematology, 126, 29–42

(UM ¼ 21 cases; nsM ¼ 10 cases; sM ¼ 21 cases), all selected

as having a very low contamination by normal B and T cells.

Notwithstanding a certain heterogeneity also within the same

B-CLL subgroup, significantly higher expression levels of DNA

pol g could be observed both in the nsM and sM B-CLL

subgroups, when compared with UM B-CLLs (Fig 4). Con-

versely, the mean value of DNA pol f expression did not change

in the various subgroups (Fig 4), hence the DNA pol f/gexpression ratio, which was high in UM B-CLLs, was signifi-

cantly down-regulated both in sM and nsM B-CLLs (Fig 4).

Expression of AID was investigated by a conventional

RT-PCR strategy, utilizing primers located in exons 1 and 5 of

the AID gene. As shown in Fig 5 this strategy identified, in all

AID-positive cases, four spliced variants: (i) a 924-bp fragment

corresponding to the described high-molecular weight variant

and due to the retention of intron 4; (ii) a 631-bp fragment

corresponding to the wild-type form of AID; (iii) a fragment of

601 bp due to a deletion of 30 nucleotides of the initial portion

of exon 4; and (iv) a 515-bp fragment due to the omission of

exon 4 (Albesiano et al, 2003; McCarthy et al, 2003; Oppezzo

et al, 2003; Degan et al, 2004). AID expression was found in 21

(40%) of 52 B-CLL cases; when stratified by IgVH mutational

status, AID-positive cases accounted for 57% (12/21) of UM B-

CLLs and 29% (9/31) of the sM and nsM B-CLL cases together,

Fig 3. Mutational analysis of IgVH sequences in unmutated (UM), not

significantly mutated (nsM) and significantly mutated (sM) B-cell

chronic lymphocytic leukaemia (B-CLL). (A) Comparison between

expected and observed number of transitions (trs) or transversions

(trv). The expected number (open bars) of trs or trv was computed by

considering that randomly occurring point mutations should be 1/3

transitions and 2/3 transversions. Closed bars refer to observed

mutations found in the three B-CLL subgroups. (B) Comparison be-

tween expected and observed number of mutations targeting specific

nucleotides in transitional mutations. The expected number of muta-

tions targeting specific nucleotides (open bars) was computed as fol-

lows: frequency of occurrence of the nucleotide of interest in a given

germ line IgVH sequence multiplied by the total number of observed

mutations and the result divided by three, as, theoretically, each

nucleotide has the same probability to be mutated into one of the other

three nucleotides. Closed bars refer to observed mutations found in the

three B-CLL subgroups. (C) The expected frequency of mutations

targeting the RGYW (i.e. purine/G/pyrimidine/A or T) or its inverse

(WRCY) motifs (open bars) is the percentage of RGYW/WRCY motifs

in a given IgVH sequence. The actual frequency of mutations targeting

the motifs of interest (closed bars) was computed by taking into ac-

count the total number of mutations in a given IgVH sequence and the

number of mutations targeting the motifs. Differences between

expected and observed values of transitions and transversions, muta-

tions targeting specific nucleotides, as well as RGYW/WRCY motifs in

IgVH sequences were assessed by means of the Student’s t-test for

paired samples by separately evaluating each transcript. Asterisks

indicate conditions in which were statistically significant (P < 0Æ05).

Fig 4. Expression of translesion DNA pol g and f by QRT-PCR.

cDNAs from unmutated (UM), not significantly mutated (nsM) and

significantly mutated (sM) B-cell chronic lymphocytic leukaemia

(B-CLL) (52 cases) were amplified with primer pairs specific for pol g,

pol f and the housekeeping gene b2M. The incorporation of the

SYBR� Green dye into the PCR products was monitored in real-time

and the resulting threshold cycles (Ct; i.e. the PCR cycle number

corresponding to the beginning of the exponential growth of PCR

products) were computed. Ct values were converted into attomoles by

means of QRT-PCR experiments carried out with serial dilution of

known concentrations of pol g, pol f and b2M-specific amplicons.

Results for each B-CLL subgroup represent the mean values of the

relative expression levels of pol g/b2M (open bars), pol f/b2M (striped

bars) and pol f/g ratio (closed bars). Reported P-values refer to dif-

ferences between the relative expression levels of pol f/g ratios, as

assessed by the Student’s t-test for unpaired samples.

IgVH Gene Mutations and Prognosis in B-CLL

ª 2004 Blackwell Publishing Ltd, British Journal of Haematology, 126, 29–42 37

with no significant differences between these latter subgroups

(Fig 5).

Characterization of sM, nsM and UM B-CLLs: correlationwith survival

By comparing the three newly identified B-CLL subsets, i.e.

sM, nsM and UM B-CLLs, for survival, we found that the

group of patients with evidence of antigen-driven selection

(sM B-CLL) had significantly longer survival when compared

with all the other cases, i.e. UM + nsM B-CLLs (Fig 6A), and

with UM B-CLL patients alone (Fig 6B). More importantly,

the comparison between the survival of sM and nsM B-CLL

patients revealed that evidence of antigen-driven selection

was able to identify a subset of patients with longer survival

also within the good prognosis group with mutated IgVH

Fig 6. Survival of B-cell chronic lymphocytic leukaemia (B-CLL) patients based on antigen-driven selection evidence. Antigen-driven selection was

assessed by applying the available distribution models. Accordingly, B-CLL were divided into three groups: sM B-CLL, with evidence of antigen-

driven selection; nsM B-CLL, without evidence of antigen-driven selection and per cent mutations ‡2%; UM B-CLL, without evidence of antigen-

driven selection and per cent mutations <2%. Kaplan–Meier curves comparing survival in B-CLL cases with (A) sM vs. (nsM + UM) configurations

of IgVH genes (sM: 52 cases; nsM + UM: 72 cases); log-rank test, P ¼ 0Æ0017; (B) sM vs. UM configurations of IgVH genes (sM: 52 cases; nsM: 47

cases); log-rank test, P ¼ 0Æ0005; (C) sM vs. nsM configurations of IgVH genes (sM: 52 cases; nsM: 25 cases); log-rank test, P ¼ 0Æ043; (D) nsM vs.

UM configurations of IgVH genes (nsM: 25 cases; UM: 47 cases); log-rank test, P ¼ 0Æ227.

Fig 5. Expression of activation-induced cytidine

deaminase (AID) by RT-PCR. cDNAs from

unmutated (UM), not significantly mutated

(nsM) and significantly mutated (sM) B-cell

chronic lymphocytic leukaemia (B-CLL)

(52 cases) were amplified with primers anneal-

ing to sequences corresponding to the first

(sense, position 45–64) and the fifth (antisense,

position 675–653) exons of AID-specific cDNA.

Amplified products (10 ll) were run on 1Æ5%

ethidium bromide-stained agarose gels and

analysed under ultra-violet light. Enlargement of

the product lane of an AID+ B-CLL case (B128)

enabled at least four spliced variants to be

determined. Asterisks refer to molecular weight

markers. Positive (+) and negative ()) controls

were the mature B-cell line Ci-1 and the omis-

sion of cDNA respectively.

M. Degan et al

38 ª 2004 Blackwell Publishing Ltd, British Journal of Haematology, 126, 29–42

gene configuration (Fig 6C). Consistently, no differences in

terms of overall survival were detected between those patients

affected by a disease characterized by more than 2% IgVH

gene mutations but lacking evidence of antigen-driven

selection (nsM B-CLL) and the bad prognosis B-CLL patient

subset displaying less than 2% mutations of IgVH genes

(Fig 6D).

Discussion

By combining the analysis of IgVH gene per cent mutations

with the application of algorithms evaluating skewing of R/S

mutations in FR/CDR as evidence of antigen-driven selection

(Chang & Casali, 1994; Smith et al, 1996; Dorner et al,

1998a,b; Neuberger et al, 1998; Lossos et al, 2000) in a large

series of B-CLL cases, we were able to identify at least three

B-CLL subsets with different prognoses and usage of the

canonical SHM machinery.

The strategy chosen for determining the IgVH mutational

status included the use of RNA as source material and the

sequencing of RT-PCR products upon their cloning into

appropriate plasmids. This approach, although still not widely

applied (Damle et al, 1999; Hamblin et al, 1999; Maloum et al,

2000; Jelinek et al, 2001; Krober et al, 2002; Lin et al, 2002;

McCarthy et al, 2003; Oppezzo et al, 2003), has the advantages

of reducing the possibility of amplifying aberrantly rearranged

VH genes (Hamblin et al, 1999) and of revealing those cases in

which an intraclonal IgVH diversification has occurred (Gurrieri

et al, 2002). In this regard, we found that a certain degree of

intraclonal IgVH diversification was a common event in M B-

CLL, occurring in about 80% of cases in our series. These results

are consistent with those reported by Gurrieri et al (2002),

although obtained by using a different approach. The possibility

that our finding might be biased by errors introduced by our

PCR and cloning strategy could be ruled out by the observation

that this approach yielded a DNA polymerase error rate of less

than one base out of 3000–6000 bp. In keeping with previous

studies (Damle et al, 1999; Hamblin et al, 1999; Maloum et al,

2000; Krober et al, 2002; Lin et al, 2002), our results included a

significant correlation between low mutations and CD38high

expression (Damle et al, 1999; Del Poeta et al, 2001; Lin et al,

2002). CD38, however, could not be considered a reliable

surrogate of IgVH mutational status (Hamblin et al, 2000;

Matrai et al, 2001; Thunberg et al, 2001) also in our study. In

addition, both CD38 and IgVH mutations had prognostic

relevance as, at the respective standard cut-off values of 30%

positive cells and 2% mutations, we were able to split patients

into subgroups with different survivals, as previously reported

by several authors (Damle et al, 1999; Hamblin et al, 1999; Del

Poeta et al, 2001; Jelinek et al, 2001; Matrai et al, 2001; Krober

et al, 2002; Lin et al, 2002).

The usage of a certain value of IgVH per cent mutations to

separate M from UM B-CLL cases (Damle et al, 1999; Hamblin

et al, 1999), surely of undiscussed clinical relevance, represents

an arbitrary method that only partially takes into account the

physiology of the SHM process, as it occurs during

T-dependent GC differentiation of normal B cells (Chang &

Casali, 1994; Smith et al, 1996; Dorner et al, 1998a,b; Neu-

berger et al, 1998; Lossos et al, 2000). Since IgVH mutations,

introduced as the result of a physiological SHM process, fulfil

precise criteria due to the antigenic stimulation acting in

concert with the hypermutation machinery (Chang & Casali,

1994; Smith et al, 1996; Dorner et al, 1998a,b; Neuberger et al,

1998; Lossos et al, 2000), we speculated that these factors

might be important in defining specific clinical and biological

features of B-CLL. Thus, we investigated whether our B-CLL

cases displayed evidence of positive selection operated by the

antigen by evaluating the excess and/or scarcity of R mutations

in CDR or FR of a given IgVH sequence respectively. This was

accomplished by utilizing a multinomial statistical algorithm

(Dorner et al, 1998a; Lossos et al, 2000; Sakai et al, 2000) or

the binomial distribution model described by of Chang and

Casali (1994). The use of this latter method was restricted to

those cases (about 11% of IgVH transcripts from the M B-CLL

subgroup) in which analyses were not amenable with the JAVA

applet available for the application of the multinomial

algorithm. According to this strategy, we identified three B-

CLL subsets: sM, with evidence of antigen-driven selection,

nsM and UM, without this evidence and IgVH gene per cent

mutations above or below the 2% cut-off.

These three newly identified B-CLL subsets were also

characterized for the biased usage of IgVH families, the

complexity of intraclonal IgVH diversification, the preference

of mutations to target-specific nucleotides or hotspots, as well

as for the expression of enzymes involved in SHM process of

normal B cells (Muramatsu et al, 1999, 2000; Zan et al, 2001;

Zeng et al, 2001; Cerutti et al, 2002; Storb & Stavnezer, 2002;

Albesiano et al, 2003; McCarthy et al, 2003; Oppezzo et al,

2003). Data accumulated in the present study favours the idea

of a mechanism similar to the canonical IgVH mutation

machinery of normal B cells operating in both sM and nsM

B-CLLs (Messmer et al, 2004). This conclusion is supported by

the demonstration of a similar degree of intraclonal IgVH

diversification, along with an excess of transitions over

transversions or of mutations clustering within RGWY/WRCY

motifs in both sM and nsM B-CLL subsets (Gurrieri et al,

2002; Messmer et al, 2004). Consistently, high levels of both

DNA pol f and g were found in nsM and sM B-CLL but not in

UM B-CLL. This data, if associated with the finding of a

relative A-preference in nsM and sM B-CLLs, is in agreement

with the notion that DNA pol g is an A-T mutator during the

SHM process (Zeng et al, 2001). The additional expression, in

all B-CLL subgroups, of high levels of the error-prone DNA

pol i, a known G-C mutator (Faili et al, 2002), could explain

the finding of a certain degree of G- and/or C-preference in

nsM and sM B-CLLs (R. Bomben, unpublished data).

Specific sets of IgVH genes were preferentially expressed by

sM, nsM and UM B-CLLs. In agreement with previous studies

(Albesiano et al, 2003; Keating et al, 2003; Messmer et al,

2004), UM and M B-CLLs preferentially utilized the IgVH1

IgVH Gene Mutations and Prognosis in B-CLL

ª 2004 Blackwell Publishing Ltd, British Journal of Haematology, 126, 29–42 39

and IgVH3 families respectively. However, when considering

the sM and nsM B-CLL subsets, a more striking skewing

towards the usage of IgVH3 family was documented in sM

B-CLLs, while nsM cases more frequently employed genes

from the IgVH4 family. A different behaviour between sM and

nsM B-CLLs was also observed when analysing the intraclonal

IgVH diversification. In this regard, although the vast majority

of IgVH transcripts displayed an excess of shared mutations

associated or not with few unique and/or partially shared

mutations, the small subset of IgVH transcripts characterized

by the highest degree of intraclonal IgVH diversification almost

exclusively belonged to the sM B-CLL subset. Intraclonal IgVH

diversification has been so far described by a single study

reporting only B-CLL cases lacking evidence of antigen-driven

selection (Gurrieri et al, 2002). In addition to confirming in a

wider cohort of cases that a certain degree of intraclonal IgVH

diversification is not an unfrequent event in B-CLL, we

provided evidence that a positive selection operated by the

antigen can play a role in determining the intrinsic complexity

of intraclonal IgVH diversification.

Strikingly, by examining the prognostic value of antigen-

driven selection, we were able to demonstrate a significantly

longer survival of sM B-CLL patients even within the good

prognosis subgroup with more than 2% mutations of IgVH

genes. Consistently, those B-CLL patients affected by a disease

with more than 2% mutations but lacking evidence of antigen-

driven selection had survivals comparable with bad prognosis

patients, i.e. displaying less than 2% mutations of IgVH genes.

The reasons for this different clinical behaviour remain to be

established. A putative hypothesis could be that the neoplastic

clone from sM B-CLLs continues to be under the positive

pressure exerted by the antigen also following the neoplastic

transformation, while, in nsM B-CLL, the leukaemic growth

has mostly become independent from any external factor,

including the antigen. The higher complexity level of intra-

clonal IgVH diversification found in sM B-CLLs, when

compared with nsM cases, is in keeping with such a view.

Alternatively, the notion that nsM and sM B-CLL cells

preferentially use different IgVH genes may support the

hypothesis that these B-CLL subsets are the result of the

neoplastic transformation of normal B-cell clones selected by

different antigens (Keating et al, 2003; Messmer et al, 2004).

Finally, it cannot be excluded that leukaemic cells from nsM B-

CLLs might represent the neoplastic counterpart of B cells

discarded during a T-dependent SHM process that, by

dysfunction of specific apoptotic pathways (Caligaris-Cappio

& Hamblin, 1999; Lin et al, 2002), are not eliminated by

apoptosis and are more resistant to therapy.

Unmutated B-CLLs, frequently expressing CD38 and AID

(Albesiano et al, 2003; McCarthy et al, 2003; Oppezzo et al,

2003; Degan et al, 2004) also in our series, might be viewed as

the neoplastic counterpart of an ‘early-GC’ B cell. The demon-

stration that somatic point mutation of BCL6 gene, a marker of

GC passage (Migliazza et al, 1995), may also occur in UM B-

CLLs (Sahota et al, 2000), are in agreement with this hypothe-

sis. Moreover, the expression of low levels of translesion DNA

pol g are in keeping with a still incomplete activation of the

SHM machinery (Zeng et al, 2001) and may explain the low

mutation rate of this B-CLL subset (Klein et al, 1998). However,

the finding that UM B-CLLs preferentially expressed IgVH genes

from the IgVH1 family again underscore the role of a putative

selection by specific antigens also for this B-CLL subset. In this

regard, it cannot be excluded that UM B-CLLs may derive from

memory B cells selected by specific antigen via a T-independent

pathway, thus expressing none or few mutations not fulfilling

the features of a canonical T-dependent SHM process (Klein

et al, 2001; Rosenwald et al, 2001).

From a clinical standpoint, this study underscores the role of

the antigen in defining the clinical behaviour of B-CLLs and

suggests a certain caution in the application of the per cent

mutations as a marker of prognostic value in the absence of

evidence of antigen-driven selection. The availability of ready-

to-use computer programs aimed at evaluating the biased R/S

mutations within the various IgVH segments (Chang & Casali,

1994; Lossos et al, 2000) could be of help for the rapid

detection of good prognosis B-CLL subsets during routine

diagnostic/prognostic procedures.

Acknowledgments

The authors thank M. Maio (Cancer Bioimmunotherapy,

CRO, Aviano) and A. Cerutti (Weill Medical College of

Cornell University, New York) for helpful discussion. This

work was supported in part by the Associazione Italiana per la

Ricerca sul Cancro (AIRC), Milan, Italy; and the Ministero

della Salute (Ricerca Finalizzata IRCCS and ‘Alleanza Contro il

Cancro’), Rome, Italy.

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