Leukemia (2004) 18, 826–831 & 2004 Nature Publishing Group All rights reserved 0887-6924/04 $25.00 www.nature.com/leu Activation-induced cytidine deaminase expression in follicular : association between AID expression and ongoing mutation in FL

MS Hardianti1, E Tatsumi1, M Syampurnawati1, K Furuta1, K Saigo2, Y Nakamachi3, S Kumagai3, H Ohno4,5, S Tanabe6,7, M Uchida6 and N Yasuda8

1International Center for Medical Research (ICMR), Graduate School of Medicine, Kobe University, Kobe, Japan; 2Blood Transfusion Division, Graduate School of Medicine, Kobe University, Japan; 3Department of Laboratory Medicine, Graduate School of Medicine, Kobe University, Japan; 4Department of Hematology and Oncology, Graduate School of Medicine, Kyoto University, Kyoto, Japan; 5Department of Hematology, Nara Social Health Insurance Hospital, Nara, Japan; 6Department of Hematology, Hyogo Prefectural Amagasaki Hospital, Amagasaki, Japan; 7Department of Internal Medicine, Torigata Hospital, Osaka, Japan; and 8Department of General Internal Medicine, Kyoto City Hospital, Kyoto, Japan

Activation-induced cytidine deaminase (AID) is required for role in the resistance of some FL cases to a certain therapy.8 The (SHM) and class switch recombination mutational analysis of the Ig gene in FLs has revealed some (CSR) of the immunoglobulin (Ig) gene. AID has been reported heterogeneity in SHM characteristic in terms of intraclonal to be specifically expressed in the (GC). 9,10 (FL) cells are known to be exposed to microheterogeneity and selection. GC reaction, as characterized by a high degree of SHM with Based on the reported heterogeneity of the SHM pattern in FL some heterogeneity in terms of intraclonal microheterogeneity and the possible role of AID involved, AID expression was and antigen selection. The heterogeneity of SHM pattern in FL investigated in the present study. AID mRNA was investigated in intrigued us to investigate the AID expression. AID expression the fresh cells of 15 FL cases and four FL cell lines. AID mRNA was investigated in 19 FL materials consisting of 15 cases of FL fresh cells and four cell lines. In all, 10 fresh cells and three cell was expressed in 10 cases and three cell lines. Furthermore, lines expressed AID, but the others did not. SHM was SHM in IgVH genes was analyzed in 12 FL cases and all cell investigated in 12 fresh cells and four cell lines. The ongoing lines. Independent of the expression or absence of AID mutation was significantly different between AID-positive and transcripts, the IgVH genes in all FL samples were somatically AID-negative FL fresh cells (unpaired Student’s t-test, mutated, indicating that AID can start to be switched off in FL. P ¼ 0.047). Ongoing mutation was not seen in any of the cell The switch off of AID expression in some FL cases may be lines. AID expression was associated with the ongoing muta- tion in FL fresh cells (two-tailed Pearson’s coefficient correla- related to the achievement of high-affinity , as indicated tion, r ¼ 0.899, P ¼ 0.01). The switch off of AID expression may by the cessation of the ongoing mutation in AID-negative FL start in the B-lineage differentiation stage counterpart of FL fresh cells. after optimizing SHM, indicated by the cessation of the ongoing mutation in AID-negative FL fresh cells. Leukemia (2004) 18, 826–831. doi:10.1038/sj.leu.2403323 Materials and methods Published online 26 February 2004 Keywords: FL; AID expression; SHM; ongoing mutation Patients and cell lines

Our series consisted of 19 FL materials comprising 15 fresh cells Introduction of FL cases derived from tissues and four FL cell lines, namely FL18,11 FL 218,12 FL31812 and FL518. The The immunoglobulin (Ig) gene undergoes V(D)J recombination, diagnosis of the 15 cases and the donor patient of FL-518 were class switch recombination (CSR) and somatic hypermutation based on the pathological diagnosis, cell-surface antigen (SHM) to encode a high-affinity antibody. The latter two analysis of lymph-node cell suspension and chromosomal processes mainly occur in the germinal center (GC) of secondary analysis of t(14;18)(q32;q21) (Table 1). lymphoid organ, which is formed by the activated B-cell.1 The isolation of the activation-induced cytidine deaminase (AID) gene, whose product is an enzyme required for SHM and CSR, Reverse transcription polymerase chain reaction was a major achievement for clarifying the molecular mechan- (RT-PCR) for AID ism of the SHM and CSR processes despite the fact that its precise actions remain elusive.2 An initial report showed that 3 cDNA was synthesized using random primer (TaKaRa, Kyoto, AID is specifically expressed in GC. Japan) and M-MLV reverse transcriptase (GibcoBRL, NY, USA) Follicular (FLs) are thought to originate from GC B m 4 from 2 g of sample derived RNA extracted by Trizol (Invitro- cells, based on the accumulation of SHM in their Ig genes. The m 5,6 gen, Carlsbad, CA, USA). cDNA (2 l) was then amplified using tumor growth of FLs may be driven by antigen selection, the PCR reaction mixture from Takara Ex-Taq (Takara, Shiga, which involves an active SHM process reflected by the ongoing Japan) in a total volume of 50 ml, following the supplier’s mutations that result in considerable intraclonal microheter- 7 instruction, using a specific AID primers covering the region ogeneity. Ongoing mutation has also been reported to play a from exons 1 to 5, yielding a product of 646 bp.13,14 Briefly, 5 pmol each of an upstream primer (50-GAG GCA AGA AGA Correspondence: E Tatsumi, International Center for Medical Research CAC TCT GG-30) and a downstream primer (50-GTG ACA TTC (ICMR), School of Medicine, Kobe University, Kusunoki-Cho, Chuo- CTG GAA GTT GC-30) were used. After 5 min of incubation at þ Ku, Kobe 650-0017, Japan; Fax: 78 382 5716; 1 E-mail: [email protected] 94 C, 30 cycles of PCR were performed under the following Received 9 June 2003; accepted 7 January 2004; Published online 26 conditions: a denaturation step at 941C for 1 min, an annealing February 2004 step at 561C for 1 min and an extension step at 721C for 2 min AID expression in FL MS Hardianti et al 827 Table 1 Characteristics of FL materials

FL material Sex Age (years) CD5 CD10 CD19 CD20 sIg t(14;18)(q32;q21)

Case 1 M 52 – + + + Ml + Case 2 M 61 – – + + Gk + Case 3 M 81 – + + + Mk ND Case 4 M 22 – + + + Mk + Case 5 F 38 – – + + Mk ND Case 6 M 53 – – + + Mk + Case 7a F 67 – + ND + Mk ND Case 8 F 48 – + + + Mk + Case 9 F 42 ND – + + Ml + Case 10a F 69 ND + ND + Mk ND Case 11 M 66 – – ND + Ml ND Case 12a M 59 – + ND + Mk + Case 13a M72–+ND+Ml ND Case 14a M57–+ND+Ml ND Case 15 M 61 – + + + Mk ND FL18b M69–+++Gk+ FL218b M73––++Gl + FL318b M67–+++Mk+ FL518b M61–+++Mk+ aExpression of bcl2 protein was detected by immunostaining of the pathological specimen. bAccompanying data represented the donor patient data and CSA of the cell line. M, male; F, female; ND, not determined.

(10 min in the last cycle). PCR products (10 ml) were electro- competent cells (TA cloning kit; Invitrogen, Carlsbad, CA, USA). phoresed in a 3% low melting NuSieve GTG Agarose gel (BMA, Following an overnight culture, 15 colonies were picked from a Rockland, ME, USA) in 1 Â TBE and visualized by staining with Luria Bertani (LB) agar plate based on X-Gal screening (Nacalai ethidium bromide. This primer pair can also detect splice Tesque.Inc, Kyoto, Japan). After the insert was checked by PCR, variants consisting of a longer (939 bp) and a shorter (530 bp) five to 10 positive colonies were subcultured overnight in 5 ml product, which were reported to perturb the functionality of of LB medium. Minipreps of plasmid were prepared from the wild-type AID by preventing SHM or interrupting ongoing SHM cultures by alkaline lysis and purified on DNA affinity column in CLL cases.14 by QIAprep Miniprep kit (QIAGENGmbH, Germany). Sequen- Densitometry of AID mRNA levels was carried out using cing was carried out on an ABI Prism 310 sequencer (Applied Scion Image-Release Beta 4.0.2. for Windows. The expression Biosystems, Fostercity, CA, USA) using the dye terminator cycle levels were normalized to the expression level of b-actin sequencing method (BigDye version 3, Applied Biosystems). mRNA15 of each sample.

Mutational analysis SHM and ongoing mutation study Final sequences were compared to those of the published A seminested PCR was performed as described previously,16 germline genes with the highest homology in the V-Base (http:// with 500 ng of genomic DNA extracted from lymph node tissues www.mrc-cpe.cam.ac.uk/). The number of somatic mutations for fresh cells and from cell pellets for cell lines, using a within the complementarity determining region (CDR)2 and Genomix kit (Talent SRL, Trieste, Italy). In brief, DNA samples framework (FR)3 in the consensus sequence of each sample were first amplified using the PCR reaction mixture from Ex-Taq were determined. As mutation patterns within CDRs are difficult Takara in a total volume of 50 ml by using 5 pmol of an upstream to interpret, only the R (replacement) to S (silent) mutation ratio primer (FR2A 50-TGG ATC CGC CAG GCT TCN GG-30) and in the FR3 was considered as an indication for antigen 5 pmol of a downstream primer (LJH 50-TGA GGA GAC GGT selection.17 A sequence was regarded as being antigen selected GAC C-30). After 10 min at 951C, 30 cycles of PCR were when the R to S ratio in FR was less than 1.6.18 performed under the following conditions: a denaturation step at 961C for 15 s, an annealing step at 601C for 45 s and extension step at 721C for 45 s (7 min for the last cycle). For the reamplification, the downstream primer was replaced by nested Ongoing mutation analysis consensus primer (VLJH 50-GGT GAC CAG GGT CCC TTG GCC CCA G 30), and 1% of the amplified product (0.5 ml) was used as Sequences from the same DNA insert were compared within template. PCR was performed according to the same protocol clones and aligned with the consensus one. The consensus described above for a total of 24 cycles. PCR products (10 ml) sequence was derived from the most dominant sequences were electrophoresed in a 3% low melting agarose gel in among clones.19 Ongoing mutation was determined by dividing 1 Â TBE buffer and visualized by staining with ethidium the cumulative number of partially shared mutations (mutations bromide. shared by some clones but not by all the IgVH gene clones) and PCR product (100ml) was electrophoresed in a 3% low unique mutations (mutations unique to a distinct IgVH gene melting agarose gel in 1 Â TAE buffer and recovered from the clone) with the expected number of mutations calculated based gel, purified using a MinElute Gel Extraction Kit (QIAGEN, MD, on the error rate of PCR19 (in our experimental conditions, it was USA) and ligated into the pCR 2.1 vector to transfect TOP10 4.5 Â 10À4 change/base/PCR cycle).

Leukemia AID expression in FL MS Hardianti et al 828 Statistical analysis The IgVH sequences of the 16 FL materials (12 fresh cells and four cell lines) analyzed in our study were deposited in the Statistical analysis of the difference of the ongoing mutation GenBank under accession numbers AY245254-480, AY450647- between AID-positive and AID-negative FL fresh cell categories 662 and AY450667-9. The VH genes were derived from the VH3 was performed by the unpaired Student’s t-test, and Po0.05 was in 10 materials, VH4 in five materials and VH1 in one material. considered as statistically significant. The correlation between Such usage without apparent bias from that in normal peripheral AID expression and ongoing mutation was analyzed by blood lymphocytes has also been reported (Table 2).21 determining the two-tailed Pearson’s correlation coefficient, The distribution of mutations in CDR2 and FR3, the mutation with r40.5 considered as statistically significant. Both tests were rate and the antigen selection calculation are also shown in performed using SPSS version 10 for Windows. Table 2. Irrespective of the AID expression, all materials displayed SHM in their IgVH genes, with mutation rates ranging from 4.16 to 15.6%, with an average of 10.07%. Similar frequencies have been 21,22 Results reported by others. The possible role of a specific antigen in the clonal growth of the tumor cells was indicated by the evidence AID mRNA expression of antigenic selection in eight of 16 materials (50%) as shown by an R to S ratio in FR3 of less than 1.6.18 The AID transcript by was detected RT-PCR in 13 out of 19 FL Ongoing mutation analysis materials, representing 10 out of 15 fresh cells of FL cases (66.6%) and in three out of four cell lines (75%) (Table 2). The As shown in Table 3, the ongoing mutation in seven AID- expression of the two splice variant products was always positive FL fresh cells was generally high, except in case 7, as detected in cases expressing wild-type AID, but not in the cases indicated by an average of 17.93-fold higher than the expected in which the wild-type AID was not expressed (data not shown). number of additional mutations due to PCR error. Besides expressing the wild-type AID, all AID-positive FL fresh cells also expressed splice variants of AID mRNA. The products of these IgVH gene usage and mutational analysis splice variants in the FL cases did not seem to inhibit the function of the wild-type AID mRNA products, in contrast to 14 The SHM analysis of IgVH gene was carried out for 16 materials, those in CLL cases. For these seven cases, the AID/b-actin ratio consisting of 12 out of 15 fresh cells of FL cases and four cell was plotted in Figure 1. lines. The SHM of IgVH gene was not analyzed in three FL cases In case 7, there were two additional partially shared due to the lack of clonality as revealed by the blurred band of mutations of the transition type (codon 78: C to T), which was IgH DNA PCR product visualized on the agarose gel (data not only 4.4-fold higher than the number of mutations expected due shown). This may have resulted from the limited amount of to PCR error (0.45). In clone 5 of case 10, there was one tumor cells in the tissue or the lack of primer site in the IgVH additional base substitution (codon 83: A to T) that caused a stop gene, which may have been caused by frequent mutations in the codon. Besides the additional partially shared and unique 20 IgVH gene. The AID/b-actin ratios determined by densitometry mutations, there was one base deletion in codon 91(A) of clone analysis in 12 fresh cells of FL cases analyzed for SHM are 5 of case 1, which might have resulted from the ongoing shown in Table 3. mutation activity.23

Table 2 AID expression, IgVH gene usage and mutational analysis of FL materials

a b Material AID mRNA VH usage No of bases analyzed CDR2 FR3 Total Mutation rate (%) R/S

R S R S R S CDR2 FR3

Case 1 + VH4/DP-63 144 3 1 5 7 8 8 11 3 0.7 Case 2 + VH3/DP-51 147 0 3 1 5 1 8 6.12 0 0.2 Case 3 + VH3/DP-53 147 4 2 3 4 7 6 8.84 2 0.8 Case 4 – VH4/DP-71 141 6 1 7 2 13 3 11.3 6 3.5 Case 5 – VH3/DP-47 147 4 2 5 6 9 8 11.6 2 0.8 Case 6 – VH3/DP-47 147 6 1 8 3 14 4 12.2 6 2.6 Case 7 + VH3/DP-47 144 4 0 10 5 14 5 13.19 inf 2 Case8 + VH1/DP-8 144 2 1 4 0 6 1 4.86 2 inf Case9 + VH3/DP-63 141 1 1 7 3 8 4 8.51 1 2.3 Case10 + VH3/DP-49 144 5 2 10 3 15 5 13.8 2.5 3.3 Case11 – VH3/YAC6 144 8 2 6 2 14 4 12.5 4 3 Case12 – VH4/DP-79 141 7 0 8 6 15 6 15.6 inf 1.3 Case13 + ND Case14 + ND Case15 + ND FL-18 + VH4/DP-79 144 1 1 3 1 4 2 4.16 1 3 FL-218 + VH4/DP-71 144 4 4 6 4 10 8 12.5 1 1.5 FL-318 + VH3/DP-54 147 2 3 7 1 9 4 8.84 0.6 7 FL-518 – VH3/DP-51 147 3 2 2 2 5 4 6.12 1.5 1 aNumber within parenthesis indicates the level of AID/b-actin ratio. bMutation rate was determined by dividing the total number of R and S mutations in CDR2 and FR3 by the number of bases analyzed. CDR, complementarity determining region; FR, framework region; R, replacement mutation; S, silent mutation; inf, infinity; ND, not determined.

Leukemia AID expression in FL MS Hardianti et al 829 Table 3 Distribution of somatic point mutations expressed among clones in FL materials

FL material AID mRNAa GenBank Number of Number of Point mutationsb Number of Ongoing accession clones analyzed bases sequenced mutation expected mutationd per clone by PCR errorc Total Sharede Partially Uniqueg sharedf

Case 1 + (14.4) AY245254-9 6 144 29 19 3 7 0.39 25.6 Case 2 + (1.5) AY245260-3 6 147 14 9 3 2 0.4 12.5 Case 3 + (2.2) AY245264-8 7 147 24 17 0 7 0.46 15.2 Case 4 – (0) AY245269-70 6 141 19 18 0 1 0.38 2.6 Case 5 – (0) AY245271-73 7 147 23 21 0 2 0.46 4.3 Case 6 – (0) AY245274 6 147 24 24 0 0 0.4 0 Case 7 + (7) AY450647-8 7 144 27 25 2 0 0.45 4.4 Case 8 + (3.6) AY450649-53 6 144 12 8 0 4 0.39 10.3 Case9 + (13) AY450654-8 6 141 15 12 0 4 0.38 10.6 Case 10 + (20) AY450659-62 5 144 41 26 2 13 0.32 46.9 Case 11 – (0) AY450666 7 144 18 18 0 0 0.45 0 Case 12 – (0) AY450667-9 6 141 31 28 2 1 0.38 7.9 FL18 + AY245275 5 144 9 9 0 0 0.32 0 FL218 + AY245276-7 5 144 23 22 0 1 0.32 3.12 FL318 + AY245278 10 147 17 17 0 0 0.66 0 FL518 – AY245279-80 10 147 12 11 0 1 0.66 1.5 aNumber within parenthesis indicates the level of AID/b-actin ratio. b Point mutations in the IgVH gene transcripts of FL materials. cBased on the Taq error rate of 4.5 Â 10À4 change/base/PCR cycle. dDetermined by dividing the cumulative number of partially shared and unique mutations by the expected number of mutations calculated based on the error rate of PCR. e Shared, mutations shared by all the IgVH gene clones analyzed. f Partially shared, mutations shared by some clones but not by all the IgVH gene clones analyzed. g Unique, mutations unique to a distinct IgVH gene transcript analyzed.

codon 94: A to G). Thus, one or two additional unique mutations in these cases is hardly an evidence for the ongoing mutation. In case 12, there were two additional partially shared mutations of transition type (codon 79: T to C) and one additional unique mutation of transversion type (codon 63: C to G), which was 7.9-fold higher than the number of expected additional mutations due to PCR error (0.38). The difference in the ongoing mutation between the AID- positive and AID-negative FL fresh cells was significant (P ¼ 0.047). AID expression and ongoing mutation were also well correlated in the FL fresh cells (r ¼ 0.899, P ¼ 0.01). None of the four cell lines showed considerable intraclonal microheterogeneity indicating the ongoing mutation. FL18 and FL318, both of which are AID positive, did not have any additional mutation. Each of FL218 (AID positive) and FL518 (AID negative) had only one additional unique nucleotide change, which is only 3.1-fold (FL218) and 1.5-fold (FL518) higher than the expected number of mutations due to PCR error (0.32 and 0.66, respectively). The one additional unique mutation in FL218 was of transition type (codon 93: G to A) Figure 1 AID/b-Actin ratio in AID-positive FL fresh cells. and so was the one in FL518 (codon 64: A to G).

Among the five AID-negative FL fresh cells, the ongoing mutation was generally low, yielding an average of 2.96-fold Discussion higher than the expected number of additional mutations due to PCR error. Two AID-negative FL cases did not have any Switch-off point of AID expression in B-lineage additional mutation (cases 6 and 11). In three cases (case 4, 5 differentiation and 12), some additional mutations were observed in low frequency. Cases 4 and 5 showed only one and two additional While an initial report showed that AID is specifically expressed unique nucleotide changes, respectively, which was 2.6-fold in the GC,3 the exact point in B-lineage differentiation where higher (case 4) and 4.3-fold higher (case 5) than the number of AID expression is switched off remains to be identified. It was expected mutations due to PCR error (0.38 and 0.46, suggested that the expression of AID is probably restricted only respectively). Moreover, the one unique nucleotide change in to a short period of time during normal B-cell maturation, while case 4 was of transition type (codon 80: T to C), and so were the the constitutive expression of AID could be observed in the two unique nucleotide changes in case 5 (codon 54: T to C and neoplastic condition.24 However, broad studies on the SHM

Leukemia AID expression in FL MS Hardianti et al 830 pattern, which is known to be useful for tracing the progenitors Our primary finding was that a significant difference in of human B-cell lymphomas,18 suggested the concordance of intraclonal microheterogeneity, indicating the ongoing muta- the expression of AID in both the normal and neoplastic tion, was found between AID-positive FL fresh cells and AID- condition. negative ones (P ¼ 0.047). In the fresh cells of FL cases, the AID In our previous study,25 the AID expression was found in 13 of expression was associated with ongoing mutation (r ¼ 0.899, 15 Burkitt lymphoma (BL) cell lines and five of five cases of fresh P ¼ 0.01). This could explain the reported heterogeneity found BL cells. SHM was found in four of four analyzed AID-positive in fresh FL cells.10,22 BL lines, whereas no SHM was found in the two rare AID- None of the FL cell lines (three AID-positive and one AID- negative BL lines. One of the two AID-negative BL lines (Tree92) negative) showed ongoing mutation. A given subclone might expressed terminal deoxynucleotidyl transferase and recombi- have prevailed due to some growth advantage during main- nation activation gene 1 and 2.26 Thus, the absence of AID in tenance, resulting in the disappearance of subclones that might the two rare BL lines was of primary-negative type (negative have exhibited the ongoing mutation. The absence of B-cell before the primary expression). This primary-negative AID receptor crosslinking and intimate T– contacts through expression in the two rare BL cell lines is in contrast to the various surface molecules in the in vitro culture leads to the absence of AID expression in our FL materials, in which AID cessation of the operation of the SHM machinery, as concluded was switched off after the completion of SHM. In this situation, based on one study using the BL2 cell line.28 Interaction through the absence of AID can be referred to as secondary negative. CD80-CD28 and CD40-CD40L was also reported to be It appeared from our data that the SHM rate was generally necessary but not sufficient for inducing SHM.29 A very rare FL lower in AID-positive FL fresh cells than in AID-negative ones, cell line exhibiting the ongoing mutation, HF-1.3.4, was derived except for two cases (cases 7 and 10) (Table 2). When the ratio from the parental HF-1 FL cell line after stimulation with B-cell- of AID/b-actin of AID-positive FL fresh cells were plotted, there specific growth factor.30 So far, Ramos is the only cell line that seemed to be a tendency for SHM rate to increase along with naturally displays the ongoing mutation in vitro due to the clonal AID expression level (Figure 1). Considerable intraclonal instability of its Ig variable region.31 AID expression was shown microheterogeneity, indicating the ongoing mutation, was a to be correlated with the mutation rate in Ramos subclones, and common feature in those AID-positive FL fresh cells, except in 1 a long-term culture period was shown to favor the outgrowth of case (case 7). In contrast, in the five AID-negative FL fresh cells, nonmutating cells expressing lower levels of AID. the SHM rate was generally high (Table 2), accompanied by a Concerning our previously reported finding that BL generally low degree or lack of detectable ongoing mutation (Table 3). expressed AID25 irrespective of the variety of the ongoing Case 7, which was AID positive but had a high SHM rate and mutation,32–34 AID is necessary to initiate but may not be low intraclonal microheterogeneity, is of importance, as it sufficient to maintain an active ongoing mutation process,35 as possibly represents a borderline stage between common AID- seen in some BL not displaying the ongoing mutation.32 An positive FL cases and AID-negative FL cases, before AID additional factor may be required to maintain the ongoing expression is switched off. Some mechanisms to switch off the mutation process in some cases of BL.36 Alternatively, some expression of AID in FL fresh cells might be affected by the level impairment in AID function may cause the arrest of the ongoing of SHM and ongoing mutation activity. mutation in some cases of BL, as suggested by an experiment The possible switch-off point of AID expression in the normal where a nonfunctional AID mutant arrested the process of B-cell counterpart of FL in our results accords with the result of ongoing mutation in a hypermutating BL cell line.37 Impairment Greeve et al,24 where AID was expressed in normal centroblasts of AID function by the products of splice variant AID mRNAs but not in , both of which are thought to be possible was also reported in an AID-positive mutated CLL case progenitors of FL. However, our results contradicted another (homology of 93%) that lacked intraclonal microhetero- part of their results, in which they found AID being expressed in geneity.14 These reports14,37 and our results suggest a possible five of five FL cases. This difference may be resolved by association between AID and ongoing mutation in FL. The analyzing more samples, since there have been several reports association between AID and CSR has been observed in stage of showing heterogeneity in the SHM pattern in FL, suggesting the CLL,38 and such point needs to be clarified as a next step in FL possible heterogeneous expression of AID in this entity.10,22 stage. Another report by Smit et al27 showed the expression of AID Since analyzing the ongoing mutation clarifies the properties mRNA above the limit of detection only in nine out of 36 cases of B-cell lymphoma in disease progression and the resistance of (25%). The quantitative RT-PCR method used in their study FL to certain therapy, such analyses should be beneficial for might have caused this lower incidence than ours. predicting the disease prognosis.8,39 Thus, the association between AID and ongoing mutation in FL deserves further investigation. Association between AID expression and ongoing mutation in FL cells Acknowledgements Previous studies of Ig gene SHM in FL reported abundant heterogeneity of the SHM pattern. Evidence of possible MSH is a Graduate Student of Foreign Scholarship of Japan antigenic selection was found in 30% and intraclonal variation (MONBUSHO) under the superiority of Professor Masafumi in 78.2% of FL cases,10 while another study observed antigenic Matsuo. selection in 84% and intraclonal variation in 71% of cases.22 10,22 Based on the reported heterogeneity in FL, the difference in References the AID expression found among the FL materials in our series and others,24,27 and based on the basic proposed function of 2 1 Janeway CA, Travers P, Walport M. The generation of diversity in AID in activating the SHM machinery, it can be presumed that the humoral immune response. In: Immunobiology: The Immune some difference in the SHM characteristic might exist between System in Health and Disease, 4th edn. New York: Garland, 1999, the AID-positive and AID-negative FL. pp 90–101.

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