Original Article

Analysis of the immunoglobulin heavy chain gene variable region of 101 cases with peripheral B cell neoplasms and B cellchronic lymphocytic leukemia in the Japanese population

Naoya Nakamura, Tetsuo Kuze, Yuko Hashimoto, Kazuhiro Tasaki, Hiroshi Hojo, Yoshikazu Sasaki,Michiko Sato and Masafumi Abe

Department of Pathology, Fukushima Medical University School of Medicine, Fukushima, Japan

Recent advances in molecular biologic techniques haveprovided new insights into the biologic characterization of Bcells and B cell neoplasms.1–5 The availability of a completemap of the immunoglobulin heavy chain variable region gene(VH gene) repertoire allows us to analyze the degree ofsomatic mutations of B cells at their differentiation stageduring the immune response.5 Polymerase chain reaction(PCR) analysis of the VH gene of B cells gives germlinesequence data for pregerminal center (GC) B cells (naive orvirgin B) or somatic hypermutation data for GC and post-GCB cells.6,7 The GC B cells particularly show intraclonal diver-sity by ongoing somatic hypermutation.6 On this basis, itcould be possible to define the clonal origin of B cell neo-plasms by the analysis of the rearranged VH gene. Bothchronic lymphocytic leukemia (CLL) and mantle cell lym-phoma (MCL) lack significant mutations in their rearrangedVH genes, suggesting their naive B cell origin,8–11 whereasfollicular lymphoma (FL),12–14 mucosa-associated lymphoidtissue (MALT) lymphoma,15 diffuse large B cell lymphoma(DLBCL)16–18 and plasma cell myeloma (PCM)19 exhibited ahypermutated VH gene, suggesting GC and post-GC cellorigin. Among the latter types of B cell neoplasms, FLparticularly shows ongoing somatic mutation.13,14 However,some cases of CLL with hypermutated VH gene20–25 and acase of MCL with a low degree of somatic mutation26 havebeen reported, so that rearranged VH genes of CLL and MCLdo not seem to always be uniform. These observations,therefore, prompted us to investigate the VH gene sequenceof B cell neoplasms in the Japanese population.

In this article, we studied the complementarity determiningregion 2 (CDR2) and the framework 3 (FW3) regions of VHgenes in 101 Japanese cases of peripheral B cell neoplasmby PCR. We demonstrated MALT lymphoma, FL, DLBCL andPCM with somatic hypermutation, which were very similar topreviously reported cases. More than half of the CLL/smalllymphocytic lymphoma (SLL) cases (eight of 13) showedhypermutated VH genes and more cases with VH4 family.

Pathology International 1999; 49: 595–600

We have analyzed the immunoglobulin heavy chain (VH)gene variable regions (CDR2 and FW3) of 101 Japanesecases with peripheral B cell neoplasms. When all except onecase with a deletion were graphed by frequency of replace-ment mutation, the 100 cases could be separated into twogroups: 24 cases with zero, one and two mutations(germline or low frequency of somatic mutation); and 76cases with three or more mutations (medium to high fre-quency of somatic mutation). While most mantle cell lym-phoma cases (11/13) showed germline or low frequency ofsomatic mutation, all cases of mucosa-associated lymphoidtissue (MALT) lymphoma (11/11), follicular lymphoma (threeof three cases), plasma cell myeloma (seven of seven cases)and most cases of diffuse large B cell lymphoma (DLBCL;42/47) belonged to the latter group. These 76 cases, there-fore, may be considered to show somatic hypermutation.More than half of chronic lymphocytic leukemia/small lym-phocytic lymphoma cases (CLL/SLL; eight of 13) showed a hypermutated VH gene and the ratio of replacement mu-tation : silent mutation in CDR2 of CLL/SLL was consid-erably higher compared with DLBCL and MALT lymphoma,showing somatic hypermutation. When comparing VH genetype of B cell-CLL (B-CLL) among our series and those in theliterature, more cases of CD51 B-CLL in the Westernliterature have the VH5 and VH6 family types, while morecases in Japan are reported to have VH4 family. Theoccurrence of VH families in B-CLL between Japanese andWestern people seems to be comparable.

Key words: B cell chronic lymphoid leukemia, B cell neoplasm,immunoglobulin heavy chain gene, immunoglobulin heavy chaingene variable region family type, polymerase chain reaction,somatic mutation

Correspondence: Naoya Nakamura, MD, Department of Pathology,Fukushima Medical University School of Medicine, 1-Hikarigaoka,Fukushima-shi 960-1295, Japan. Email: nao@cc.fmu.ac.jp

Received 21September 1998. Accepted for publication 2 February1999.

When comparing VH family type of B cell CLL (B-CLL) amongour series and those in the literature, VH family type of B-CLLbetween the Japanese cases and the Western cases seemsto be comparable.

MATERIALS AND METHODS

Data from 190 cases of non-Hodgkin’s lymphoma with a Bcell phenotype were retrieved from the files of malignant lym-phoma obtained at the Department of Pathology, FukushimaMedical University School of Medicine from 1980 to 1998.Among them, 101 cases with peripheral B cell neoplasms, inwhich complete oligonucleotide sequences (OS) of CDR2and FW3 were identified by PCR techniques, were used inthis study. The histologic diagnosis was established accord-ing to the REAL classification.27 There were 13 identified of 16 examined cases of CLL/SLL, three of three cases ofimmunocytoma/lymphoplasmacytoma (IC/LP), 13 of 18cases of MCL, three of three cases of FL, 11 of 20 cases ofmarginal zone B cell MALT lymphoma, one of two cases of hairy cell leukemia (HCL), 47/120 cases of DLBCL, three ofthree cases of Burkitt’s lymphoma (BL) and seven of 15cases of plasma cell myeloma/plasmacytoma (PCM).

DNA was extracted from peripheral blood cells, paraffin-embedded tissues and frozen tissues. The variable regions(CDR2 and FW3) and VDJ region (CDR3) of theimmunoglobulin heavy chain (IgH) gene were amplified bysemi-nested PCR using primers of FR2A, LJH and VLJH,according to the previously described method.14,18 Poly-merase chain reaction products were estimated to beapproximately 220–300 b.p. in length. Oligonucleotide se-quences of CDR2 and FW3 were analyzed by the directsequence method (Thermo Sequenase core sequencing kit

with 7-deaza-deoxyguanosine triphosphate; Amersham,Cleveland, OH, USA) and the HITACHI SQ-5500 (Tokyo,Japan) and then compared with germline sequencesrecorded in the GenBank database.

RESULTS

Oligonucleotide sequences of CDR2 and FW3 obtained byPCR and the direct sequence method were compared to theclosest germline sequence in the database. Replacementmutations are single-base substitutions resulting in aminoacid replacement while silent mutations are single-basesubstitutions without amino acid replacement. The frequencyof replacement mutations of all cases are summarized inTable 1 and Fig. 1. There were 76 cases with three or morereplacement mutations (medium to high frequency of somaticmutation) of the VH gene. These 76 cases included eight of13 cases of CLL/SLL (61.5%), two of three cases of IC/LP(66.7%), two of 13 cases of MCL (15.5%), three of threecases of FL (100%), 11 of 11 cases of MALT lymphoma(100%), one of one case of HCL (100%), 42 of 47 cases ofDLBCL (89.4%), none of three cases of BL (0%) and seven ofseven cases of PCM (100%). The average total mutation,replacement mutation and replacement/silent ratio of eachhistologic subtype are shown in Table 2. All cases except fourwere found with an in-frame sequence. Four cases with anout-of-frame sequence consisted of one case of CLL with a55 b.p. deletion and a new stop codon and three cases ofDLBCL: an anaplastic variant with 1 b.p. deletion, ananaplastic variant with a 9 b.p. insertion, and a centroblasticvariant with a 15 b.p. deletion. In Table 3, immunophenotype,somatic mutation frequency and VH family type of B-CLL/SLLdata are shown. Table 4 shows the VH family type of CD51

596 N. Nakamura et al.

Table 1 Distribution of cases of each histologic subtype according to frequency of replacement mutations in the CDR2 and FW3 variableregions of the immunoglobulin heavy chain gene

Frequency of replacement mutations

Histologic No.subtype cases 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 >20

CLL/SLL 12* 2 3 2 1 1 1 1 1IC/LP 3 1 1 1MCL 13 3 4 4 1 1FL 3 1 1 1MALT 11 1 2 3 1 3 1HCL 1 1DLBCL 45 1 2 2 3 4 2 7 5 4 1 1 5 1 2 1 1 5BL 3 1 2PCM 7 1 1 1 1 2 1

Total 100 7 7 11 0 3 6 5 5 8 10 6 6 3 7 2 3 0 0 2 3 6

* A case of CLL with a large deletion was not included in this figure.CLL/SLL, chronic lymphocytic leukemia/small lymphocytic lymphoma; IC/LP, immunocytoma/lymphoplasmacytoma; MCL, mantle cell lymphoma; FL,

follicular lymphoma; MALT, mucosa-associated lymphoid tissue lymphoma; HCL, hairy cell leukemia; DLBCL, diffuse large B cell lymphoma; BL, Burkitt’slymphoma; PCM, plasma cell myeloma/plasmacytoma; CDR2, complementarity determining region 2; FW3, framework 3.

and CD5– B-CLL among our series and those reported in theliterature.

DISCUSSION

We have analyzed the rearranged VH genes of Japanesepatients with peripheral B cell neoplasms. All but one casewas shown in the results. The frequency of replacementmutations showed that the 100 cases seemed to constitutetwo groups; one consisted of 24 cases with zero to two

replacement mutations (germline or low frequency of muta-tion). The other group consisted of 76 cases with three ormore replacement mutations (moderate to high frequency ofmutation). Separation by frequency of replacement mutationwas clearer than that by frequency of both replacement andsilent mutation (detail not shown). The latter group, consistingof cases with moderate to high frequency of mutation,included all cases of MALT lymphoma (11/11), FL (three ofthree) and PCM (seven of seven) and most cases of DLBCL(42/47, 89.4%), while 11 of 13 cases of MCL exhibitedgermline or low frequency of mutation. Therefore, the caseswith three or more replacement mutations may be consideredto be somatic hypermutation cases in the present series.

Although B-CLL has a characteristic phenotype of CD5,CD23, and low levels of surface IgM and IgD, no typicalchromosomal aberrations or oncogenic abnormalities asso-ciated with B-CLL have been reported.28–30 The normal coun-terpart cells of B-CLL have been suggested to be naive Bcells because some B-CLL cases show a germ line ofrearranged VH gene, but the true situation remains unclear.B-CLL is the commonest form of leukemia in Westerncountries, accounting for nearly 30% of all leukemias,31 but isless common in Japan where it accounts for only 2% of allleukemias.32 A difference in cell morphologic appearancebetween Western and Japanese cases has been reported. InWestern countries, approximately 90% of B-CLL cases showa proliferation of well-differentiated small lymphocytes and B-CLL with atypical morphology are uncommon,33,34 while B-CLL cases with a predominant population of large lym-phocytes or atypical lymphocytes are not rare in Japan.35

However, immunophenotypic and karyotypic examinations

VH gene analysis of B cell lymphoma and B-CLL 597

Figure 1 Frequency of replacementmutation of B cell neoplasms in thecomplementarity determining region 2(CDR2) and the framework 3 (FW3)variable regions of immunoglobulinheavy chain gene. One hundred of the101 cases are arranged by frequencyof replacement mutation. See Table 1for details. The case not shown is acase of B cell chronic lymphocyticleukemia with a deletion.

Table 2 Average mutation frequency and replacement mutation(R): silent mutation (S) ratio in each histologic subtype

Average frequency R : SR 1 S R CDR2 FW3

CLL/SLL 10.5 (7.8) 7.3 (5.4) 8.43 0.9IC/LP 11.3 (7.7) 8.3 (5.7) 4.67 1.83MCL 2.9 (2.0) 1.8 (1.2) 1.67 1.44FL 18.0 (12.4) 10.6 (7.6) 1.86 1.27MALT 13.6 (9.3) 9.4 (6.4) 2.67 2.02HCL 12.0 (8.2) 11.0 (7.5) z 7DLBCL 16.2 (11.1) 10.4 (7.2) 2.96 1.38BL 3.3 (2.3) 1.7 (1.1) 3 0.5PCM 16.6 (11.1) 10.7 (7.2) 2.33 1.54

CLL/SLL, chronic lymphocytic leukemia/small lymphocytic lymphoma;IC/LP, immunocytoma/lymphoplasmacytoma; MCL, mantle cell lym-phoma; FL, follicular lymphoma; MALT, mucosa-associated lymphoidtissue lymphoma; HCL, hairy cell leukemia; DLBCL, diffuse large B cell lymphoma; BL, Burkitt’s lymphoma; PCM, plasma cell myeloma/plasmacytoma; CDR2, complementarity determining region 2; FW3,framework 3.

Figures in parentheses are percentages.

have shown that the Japanese CLL cases show little dif-ference from the reported Western cases.36–38 We havecompared somatic mutation frequency and VH gene type ofB-CLL in the present series to those of B-CLL reported in theliterature. Many reports of somatic mutation analysis of B-CLL have been published so far: B-CLL was consideredinitially to lack significant hypermutation of the rearranged VHgene compared with MALT and DLBCL which have hyper-mutated VH genes, but CD51 B-CLL with somaticallymutated VH genes have been described.8–11,20–25 In addition, B-CLL is known to lack intraclonal diversity. Oscier et al.recently reported that B-CLL with trisomy12 showed germlinesequence, while B-CLL with 13q14 showed somatic hyper-mutation.25 They demonstrated that B-CLL cases withexpression of IgM and IgD included cases with germlinesequence as well as cases with somatic hypermutation. Theexistence of isotype-switched B-CLL with germline or littlesomatic mutation of the VH gene are also known.39 Thepattern of somatic mutation of B-CLL is heterogeneous. The

proportion of B-CLL with a somatically mutated VH gene isestimated to be 40% of all cases of B-CLL.40 Our present datashowed that incidence of CLL/SLL cases with somatichypermutation (eight of 13, 61.5%) seemed to be higher thanthat of cases with germline sequence in Japan.

Functional VH genes are classified into seven families andthe germline complexity of these families is reported to be25–30% for the VH1 family, 5–10% for the VH2 family,50–55% for the VH3 family, 5–18% for the VH4 family, 1–2%for the VH5 family and 1% for the VH6 family.41,42 While theEpstein–Barr virus-transformed peripheral blood lymphocytecell lines show a VH gene utilization roughly proportional tothe estimated family size, more cases of CD51 B-CLL in theWest have VH5 and VH6 families.43–45 The VH gene type ofthe Western cases is biased to the respective gene com-plexity for each VH family. The biased VH gene type is sug-gested to be related to antigen selection by auto-antigens.30

CD51 B-CLL in the Japanese is found in a high proportion ofVH4 type compared with other VH families. When comparing

598 N. Nakamura et al.

Table 4 Comparison of VH family type of B-CLL between our present series and reported cases

Examined VH family type (%)Author cases VH1 VH2 VH3 VH4 VH5 VH6

CD51

Deane and Norton43 34* 14 2 55 16 9 5Mayer et al.44 40* 21 7 32 16 12 14Ikematsu et al.45 21* 14 10 24 48 5 0Present series 10† 0 0 60 40

CD52

Maloum et al.47 11† 18 18 27 36Ikematsu et al.45 5 0 0 80 20 0 0Present series 2† 0 0 100 0

* VH gene type was detected by Southern blotting. Cases with cross-hybridization are included.† VH gene type is detected by the oligonucleotide sequencing method.VH, variable region of the immunoglobulin heavy gene; B-CLL, B cell chronic lymphocytic leukemia.

Table 3 Immunophenotype, somatic mutation frequency and VH family usage of B-CLL/SLL

Immunophenotype (CD) R : S R : S No. mutations : no. germline VHCase Age : sex 5 21 22 23 (CDR2) (FW3) (closest germline) family

CLL(03) 65 : M 1 – 1 1 8 : 3 5 : 6 22 : 147 (4 : GL49) 4CLL(04) 56 : M 1 1 NT 1 2 : 0 3 : 2 7 : 144 (4 : DP67) 4CLL(08) 59 : M 1 – 1 1 Out of frame(*) (3 : GL16) 3CLL(09) 69 : M 1 1 – 1 2 : 0 0 : 1 3 : 153 (3.15 : DP38) 3CLL(10) 73 : M 1 1 NT 1 15 : 0 3 : 2 20 : 147 (3 : VZM) 3CLL(12) 72 : M 1 NT NT 1 1 : 0 1 : 1 3 : 147 (3.30 : DP49) 3CLL(15) 66 : M 1 NT NT 1 1 : 0 1 : 3 5 : 147 (3.23 : DP47) 3CLL(16) 77 : M 1 NT NT 1 0 : 0 0 : 0 0 : 147 (4 : VH5) 4CLL(17) 67 : M 1 NT NT 1 2 : 0 3 : 7 12 : 147 (4 : VH5) 4CLL(18) 49 : M 1 – – 1 7 : 1 1 : 0 9 : 147 (3.48 : G26G) 3CLL(02) 70 : F – 1 1 1 10 : 1 7 : 5 23 : 147 (3.11 : DP35) 3CLL(05) 71 : F – – – – 9 : 2 5 : 4 20 : 147 (3.9 : DP31) 3SLL(02) 78 : F 1 – – 1 0 : 0 0 : 0 0 : 147 (3.21 : DP77) 3

* A 55 b.p. deletion and a stop codon of (TGA) appear in the VH gene of CLL(08).R : S, replacement mutation number : silent mutation number. B-CLL, B cell chronic lymphocytic leukemia; SLL, small lymphocytic lymphoma. CDR2,

complementarity determining region 2; FW3, framework 3.

the VH family type of the Japanese cases with the Westerncases, the high proportion of VH4 type of the Japanese casesis considered to be a distinguishing characteristic.

CD5– B-CLL is known to represent less than 5% of B-CLL.CD5– B-CLL exhibits a certain clinical course and is cyto-logically and immunophenotypically different from CD51

B-CLL.46 The proportion of cases with advanced disease,splenomegaly, mixed cytology type, high amounts of surfaceimmunoglobulin and less expression of CD23 in CD5– B-CLLis high compared with that of CD51 B-CLL. VH familyexpression of CD5– B-CLL seems to be different from that ofCD51 B-CLL. While, in the West, CD5– B-CLL is frequentlyfound in the VH4 family, CD5– B-CLL in the Japanese isfrequently found in the VH3 family.45,47 CD5– B-CLL in theJapanese may appear close to the respective gene com-plexity for each VH family. Thus, VH family type of B-CLL inJapanese and Western patients is comparable. A furtherstudy of somatic mutation in B-CLL among various races willbe required.

In conclusion, our somatic mutation analysis of therearranged VH gene in B cell neoplasms in the Japanesepopulation revealed the following: (i) while many MCL casesshowed germline or low frequency of somatic mutation,MALT lymphoma, FL, PCM and most cases of DLBCL showsomatic hypermutation; (ii) many B-CLL/SLL showed somatichypermutation with a considerably high ratio of replacementmutation:silent mutation (R:S); and (iii) the VH family type ofB-CLL in the Japanese cases seems to be different from thatof the Western cases.

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