XCR1 Expression and Biased VH Gene Usage Are Distinct Features of Diffuse Large B-Cell Lymphoma...

556 Am J Clin Pathol 2011;135:556-564556 DOI: 10.1309/AJCPCTDC5PY3LXBP

© American Society for Clinical Pathology

Hematopathology / XCR1 and VH Gene in BM DLBCL

XCR1 Expression and Biased VH Gene Usage Are Distinct Features of Diffuse Large B-Cell Lymphoma Initially Manifesting in the Bone Marrow

Yoriko Yamashita, MD, PhD,1 Dai Kajiura, MD, PhD,1 Lee Tang, MD,1 Yuichi Hasegawa, MD, PhD,2 Tomohiro Kinoshita, MD, PhD,3 Shigeo Nakamura, MD, PhD,4 Shinya Akatsuka, PhD,1 Shinya Toyokuni, MD, PhD,1 and Naoyoshi Mori, MD, PhD1

Key Words: Diffuse large B-cell lymphoma; Bone marrow; Intravascular large B-cell lymphoma, Asian variant; Chemokine receptor; XCR1; VH gene usage

DOI: 10.1309/AJCPCTDC5PY3LXBP

A b s t r a c t

A total of 29 cases of diffuse large B-cell lymphoma initially manifesting in the bone marrow (BM-DLBCL) were analyzed for VH gene sequence, and expression microarray of chemokines and chemokine receptors and immunohistochemical analysis were done. Seminested polymerase chain reaction (PCR) and sequencing analyses of 18 cases revealed that the VH gene usage in 6 cases was restricted to VH3-7, in 3 cases to VH4-34, and in 2 cases to VH4-39, which were all previously reported to be autoreactive. In total, 14 of 18 VH genes were those associated with autoimmune diseases, including VH3-21, VH3-23, and VH3-48. Furthermore, cDNA microarray analysis specific for chemokine and chemokine receptors revealed that chemokine receptor XCR1 expression was significantly elevated in the BM-DLBCL cases (P < .05), which was confirmed by quantitative reverse transcriptase–PCR and immunohistochemical analysis. Expression of the chemokine receptor XCR1 and frequent usage of autoreactive VH genes seem to be distinct characteristics of BM-DLBCL.

We previously reported 37 cases of diffuse large B-cell lymphoma demonstrating bone marrow involvement at ini-tial examination. We termed these cases as diffuse large B-cell lymphoma initially manifesting in the bone marrow (BM-DLBCL).1 According to previous reports, BM-DLBCL is often associated with histologic hemophagocytosis in the BM, intravascular packing of tumor cells, and involvement of extranodal organs such as the spleen and liver.1,2 In 1997, Murase et al3 reported autopsy findings of a newly recognized B-cell lymphoma in which they defined the disease entity by including cases that fulfilled at least 3 of the following 4 con-ditions: (1) hemophagocytosis in the hematopoietic system, (2) hepatomegaly and/or splenomegaly, (3) BM invasion of the lymphoma cells, and (4) a lack of overt lymphadenopathy and tumor formation. Then they termed the disease “Asian variant of intravascular large B-cell lymphoma” (AIVL) and further characterized the entity by clinical, pathologic, and cytogenetic approaches.4 Similar cases have been reported by other groups of Western pathologists and clinicians.5,6 Most recently, an international consensus meeting was held to dis-cuss the definition, diagnosis, and management of this newly defined intravascular B-cell lymphoma,7 which was thereafter included in the new World Health Organization classifica-tion.8 Thus, we consider that these entities, BM-DLBCL and AIVL, should be included in the same comprehensive clinicopathologic category, setting the subtle differences1 in pathologic findings aside.

There are only a few studies that analyzed the origin or the differentiation state of the tumor cells in AIVL or BM-DLBCL, and there are no markers to distinguish these entities from other B-cell lymphomas. VH family usage and the somatic hypermutation rate can provide some information

Am J Clin Pathol 2011;135:556-564 557557 DOI: 10.1309/AJCPCTDC5PY3LXBP 557


Hematopathology / Original Article

about the character and the differentiation state of neoplastic B cells.9

Human B lymphocytes first arise and develop in the BM, and then most of them mature in the spleen to become follicular or marginal zone B cells.10 Murine B1 cells were first reported as functionally distinct B cells that are CD5+ and secrete IgM autoantibodies.11 B1 cells and splenic marginal zone B cells participate in the natural immune memory.12 Much less is known about human B-cell subpopulations; however, human IgM+/IgD+/CD27+ memory B cells in the blood have the same origin as circulating splenic marginal zone B cells. These cells resemble murine B1a cells,13 but whether these cells are human B1 cells remains mostly unknown.10 CD5 is a marker for murine B1a cells or human mantle cell lymphoma. However, hetero-geneity of CD5 expression has been observed in BM-DLBCL and AIVL.1,14 Furthermore, serum IgM autoantibodies have been detected in some AIVL cases.15,16

The aim of this study was to elucidate the differentiation state and VH gene usage of BM-DLBCL. Thus, sequence analysis of the immunoglobulin VH gene was performed. Furthermore, an expression microarray analysis specific for chemokines and chemokine receptors was also performed to establish biomarkers distinguishing BM-DLBCL from other B-cell lymphomas.

Materials and Methods

CasesBM-DLBCL cases were selected as previously reported.1

Clinical data are summarized in ❚Table 1❚. The experimental designs of genomic and expression studies were reviewed and approved by the Committee for Bioethics (No. 670) of Nagoya University Graduate School of Medicine, Nagoya, Japan. Formalin-fixed, paraffin-embedded tumor samples of 29 cases of BM-DLBCL, 23 cases of control DLBCL, 6 fol-licular lymphoma cases, 3 mantle cell lymphoma cases, and 9 mucosa-associated lymphoid tissue (MALT) lymphoma cases underwent immunohistochemical analysis. All cases were specifically reviewed by more than 2 registered pathologists (Y.Y., S.N., and N.M.).

VH Gene Sequence AnalysisDNA for polymerase chain reaction (PCR) analysis was

extracted from 14 periodate-lysine-paraformaldehyde–fixed frozen tissue samples using the QIAamp DNA Mini Kit (Qiagen, Valencia, CA) or from 4 formalin-fixed, paraffin-embedded samples (cases 6, 11, 14, and 18; Table 1) using the DNeasy Blood & Tissue Kit (Qiagen). Seminested PCR was performed using the FR2A consensus primer for con-served framework 2, and consensus primers for the J region

external LJH and internal VLJH, as previously described.17 The PCR products were separated by electrophoresis on Nusieve 3:1 Agarose gels using Nusieve GTG Agarose (Cambrex BioScience, Rockland, ME), and single bands were subsequently cloned into a pGEM T-Easy TA cloning vector (Promega, Madison, WI).

Sequencing was performed bidirectionally using the Big Dye Terminator Kit, version 3.1 (Applied Biosystems [ABI], Foster City, CA) with M13 primers, M13F and M13R, using an ABI Prism 310 Genetic Analyzer (ABI). At least 3 clones were sequenced in each case. When the sequences of the 3 clones were exactly the same, intraclonal diversity was con-sidered absent. If the sequence of 1 or more of the 3 was dif-ferent, 7 additional clones were sequenced to evaluate a total of 10 clones. DNA sequences were analyzed by IgBLAST (http://www.ncbi.nlm.nih.gov/igblast/).

Histologic and Immunohistochemical AnalysesFor histologic and immunohistochemical examination,

tissue samples were fixed with 10% formaldehyde and embed-ded in paraffin. Then, 2- to 4-μm-thick sections were prepared

❚Table 1❚Summary of Clinical Data on BM-Derived Diffuse Large B-Cell Lymphoma Cases

Case No./ Sex/Age (y) Involved Sites (Organs) Outcome

1/F/84 BM, liver, spleen, iliopsoas muscle Died, 13 mo2/F/56 BM, spleen Died, 26 mo3/F/49 BM Died, 14 mo4/F/59 BM, liver, kidney, adrenal gland, Died, 5 mo brain, retina 5/F/54 BM Died, 37 mo6/F/76 BM, liver, spleen Died, 1 d7/F/70 BM, liver, spleen NA8/F/61 BM, liver, spleen AWOD, 107 mo9/M/74 BM Died, 2 mo10/M/66 BM, liver, spleen Died, 2 mo11/M/55 BM Died, 36 mo12/M/81 BM, spleen Died, 5 mo13/F/73 BM, liver, spleen, lung, adrenal Died, 2 mo gland, kidney 14/F/72 BM, liver Died, 35 mo15/F/70 BM Died, 6 mo16/M/54 BM, liver, spleen, brain, lung, kidney Died, 16 mo17/M/51 BM, liver, spleen Died, 7 mo18/F/68 BM, lung, spleen Died, 26 mo19/M/54 BM Died, 19 mo20/F/64 BM, lung, liver, spleen Died, 12 d21/F/70 BM, spleen Died, 14 d22/F/77 BM, kidney, stomach Died, 9 mo23/M/69 BM, liver, spleen Died, 10 mo24/M/66 BM, liver Died, 7 mo25/M/67 BM, liver, spleen Died, 15 mo26/F/82 BM Died, 10 d27/F/63 BM, liver, spleen Died, 2 mo28/M/58 BM, spleen AWOD, 7 mo29/M/67 BM, liver, spleen AWOD, 13 mo

AWOD, alive without disease; BM, bone marrow; NA, data not available.



Yamashita et al / XCR1 and VH Gene in BM DLBCL

and stained with H&E. The labeled streptavidin-biotin-perox-idase method with the Ultra Tech kit (Immunotech, Marseille, France) was used for immunohistochemical analyses. Primary antibodies used were CD5 and CD10 (Novocastra, Wetzlar, Germany); CD20 (L26), CD79a (mb-1), BCL-6, and IRF-4/MUM-1 (DAKO, Glostrup, Denmark); and XCR1 (CCXCR1) (GeneTex, San Antonio, TX). For XCR1, intense staining observed in the membrane of the majority of tumor cells was defined as 2+. Perinuclear staining or weak membrane stain-ing that was observed in more than half of the tumor cells was defined as 1+. We defined +/– as when fewer than 50% of the tumor cells were positive but not totally negative.

Chemokine and Chemokine Receptor Microarray and Quantitative Reverse Transcriptase–PCR

For microarray and real-time quantitative reverse tran-scriptase (RT)-PCR, total RNA was extracted from periodate-lysine-paraformaldehyde–fixed frozen samples using the RecoverAll Nucleic Acid Isolation Kit (Ambion, Austin, TX). High-quality RNA suitable for microarray and RT-PCR was obtained from 4 and 9 BM-DLBCL samples, respectively. For cDNA microarray, efficiently amplified and labeled RNA was then hybridized to the Chemokines & Receptors Oligo GEArray, which has 128 probes on each membrane, accord-ing to the manufacturer’s protocol (SABiosciences, Frederick, MD). Data were analyzed using the GEArray Expression Analysis Suite software (SABiosciences). For quantitative RT-PCR, 200 ng of isolated total RNA was reverse transcribed using the Superscript III First-Strand Synthesis System for RT-PCR (Invitrogen, Carlsbad, CA), and quantitative PCR was performed using a 7300 real-time PCR system (ABI) with the PlatinumSYBR Green qPCR Supermix-UDG kit (Invitrogen), following the manufacturer’s instructions.

The sequences of primers were as follows: XCR1, 5'-TCCTGTTCTGCTACGTG-3' (sense) and 5'-AGGGCGTATTCTAGCTG-3' (antisense) and control β-actin, 5'-CGGGACCTGACTGACTA-3' (sense) and 5'- GAAGGAAGGCTGGAAGAGT-3' (antisense) (annealing at 55°C). The product sizes of XCR1 and β-actin were 205 and 235 base pairs, respectively. Transcripts were quantified in duplicate. To control for variation in the amount of RNA, XCR1 expression in each sample was normalized to human β-actin using the comparative Ct method. The microarray and quantitative PCR data were analyzed by using the Mann-Whitney U test.

Results

Results of the VH gene sequence analysis are summarized in ❚Table 2❚. VH gene usage in 6 cases was restricted to VH3-7, in 3 cases to VH4-34, and in 2 cases to VH4-39. VH genes

in the other cases were VH1-18, VH3-11, VH3-21, VH3-23, VH3-48, VH3-74, and VH4-61. In summary, 14 of 18 VH genes expressed autoreactive VH domains. The mutation fre-quency of the 14 VH genes ranged from 2.1% to 23.8% (mean, 14.3%). Thus, somatic hypermutation was detected in all 18 cases, suggesting the post–germinal center status of the neo-plastic B cells. Ongoing mutation was detected in 4 cases.

Immunohistochemical results of other known diagnos-tic markers of B-cell lymphomas are summarized in ❚Table 3❚. CD5 was positive in 8 (28%) of 29 cases. Because the majority of cases were IgM+ (23/29 [79%]), there may have been a genomic deletion of the switch μ region, resulting in a failure of class switch recombination in these tumor cells, a finding similar to that in a previous report describing cases of central nervous system lymphoma.18 PCR analysis revealed a partial deletion of the region in 4 of 11 examined BM-derived DLBCL cases (data not shown), and no deletion was observed in the 10 control DLBCL cases expressing IgG. However, a deletion of the switch μ region was also present in 4 of 10 IgM-expressing DLBCL cases without BM involvement; this finding identifies a deleted switch μ region as a common feature of DLBCLs expressing IgM. In the 29 cases, IRF-4/MUM-1 was positive in 26 (90%), CD10 in 2 (7%), and BCL-6 in 3 (10%). Thus, the majority of these cases were considered to be of the non–germinal center phenotype.

Microarray analysis of the BM-DLBCL samples was performed, and samples from non–germinal center type

❚Table 2❚Results of Sequence Analysis of the Immunoglobulin VH Gene

Case Homology (vs Mutation IntraclonalNo. VH Family Germline; %) Frequency (%) Diversity

1 VH3-7* 80.7 19.3 –2 VH3-7* 85.7 14.3 +3 VH3-7* 85.7 14.3 +4 VH3-7* 81.4 18.6 –5 VH3-7* 91 9 +6 VH3-7* 88.9 11.1 –7 VH4-34* 79.9 20.1 –8 VH4-34* 80.4 19.6 –9 VH4-34* 86.1 13.9 –10 VH4-39* 76.2 23.8 –11 VH4-39* 85.9 14.1 –12 VH3-48* 86.6 13.4 –13 VH3-23* 82.5 17.5 –14 VH3-21* 97.9 2.1 –15 VH4-61 88.9 11.1 –16 VH1-18 90.2 9.8 +17 VH3-74 87.8 12.2 –18 VH3-11 86.9 13.1 –

+, present; –, absent.* Encodes autoantibodies as follows: VH3-7 and VH3-48, rheumatoid arthritis;

VH3-23, DNA-binding autoantibody; VH4-34, autoimmune hemolytic anemia; VH4-34 and VH4-39, multiple sclerosis; and VH3-21 and VH3-23, hepatitis C virus–related mixed cryoglobulinemia.




DLBCLs without BM involvement were used as control sam-ples. Clustering analysis of the chemokine- and chemokine receptor–specific expression microarray analysis revealed that, in contrast with the control DLBCLs, which all clustered together, the BM-DLBCL samples formed 2 distinct clusters ❚Figure 1A❚. Up-regulated genes, sorted by the mean ratio of the 2 groups, are shown in ❚Table 4❚. Of the 8 genes that were largely down-regulated in the BM-DLBCL samples compared with the control DLBCL samples (<5.3-fold), 7 were shown to be significant by the Mann-Whitney U test ❚Table 5❚. In contrast, 4 genes that were largely up-regulated (>3.3-fold) were not significant (Table 4). However, 6 genes, including chemokine receptor XCR1, were significantly overexpressed (P < .05) in the BM-derived DLBCL cases (Table 4). Four of the six genes, namely, toll-like receptor 4, endothelial cell growth factor 1 (platelet-derived), interleukin 8 receptor-β, and platelet factor 4, were likely to be products of background reactive cells such as neutrophils, endothelial cells, or plate-lets. Because XCR1 clustered together with tumor necrosis factor α (Figure 1A) and both seemed to be specific products of the lymphoma cells, XCR1 was chosen for further analysis. Quantitative RT-PCR revealed that XCR1 was significantly overexpressed in the BM-DLBCL samples compared with the control DLBCL samples (P < .01) ❚Figure 1B❚.

Then, we performed immunohistochemical analysis to determine whether the lymphoma cells really expressed XCR1 protein. Two-plus staining of XCR1, in which intense XCR1 immunostaining was observed in the membrane of the majority of tumor cells, was detected in 22 (76%) of the 29 cases ❚Image 1❚. None of the control DLBCL cases were 2+; 1+ staining was observed in 6 (21%) of the 29 BM-DLBCL cases and 4 (17%) of the 23 control cases (Image 1). In 19 (83%) of the control DLBCL cases, staining was graded as +/–, with only weak staining seen (Image 1), or as nega-tive (Image 1). Other subtypes of B-cell lymphoma, such as follicular lymphoma, mantle cell lymphoma, and MALT lymphoma, showed variable positivity, and 2+ staining was seen in only 1 of 9 cases of MALT lymphoma. The results of immunohistochemical analyses are summarized in ❚Table 6❚.

Discussion

BM-DLBCL,1,2 AIVL,16,19,20 and Western cases of intravascular lymphomatosis (IVL)6 have been described in the literature by several groups. Whether these entities describe a single disease or several different, overlapping disease entities remains elusive. However, the clinical and

❚Table 3❚Immunohistochemical and Histologic Findings in Bone Marrow Samples in Diffuse Large B-Cell Lymphoma

Case No. CD20 CD79a IgM CD5 CD10 BCL6 IRF-4/MUM1 XCR1*

1 + + + – – – + 2+2 + + – – – – + 2+3 + + + – – – + +4 + + + + – – + 2+5 + + + + – – + 2+6 + + + – – – + 2+7 + + + – – – + 2+8 + + + – – – + 2+9 + + + – – – + 2+10 + + + – – – + 2+11 + + + – + + – +12 + + + – – – + +13 + + + – – – + 2+14 + + – – + + – 2+15 + + – – – – + +16 + + + + – – + 2+17 + + + + – – + +18 + + + – – – + 2+19 + + + + – – + 2+20 + + + + – – + 2+21 + + + + – + + +22 + + + – – – + 2+23 + + + – – – + –24 + + – – – – + 2+25 + + + – – – – 2+26 + + – + – – + 2+27 + + + – – – + 2+28 + + – – – – + 2+29 + + + – – – + 2+

+, positive; –, negative.* For XCR1, 2+ indicates intense staining in the membrane of the majority of tumor cells; +, perinuclear staining or weak membrane staining in more than half of the tumor cells.




pathologic findings of the Asian cases seem to be differ-ent from the previous definition of “Western” IVL,7,14,21 and we suggest that AIVL and BM-DLBCL be recognized in the same disease group, different from Western IVL. It

is interesting that 7 of 29 BM-DLBCL cases in this study had lymphoma involvement restricted to the BM (Table 1), confirmed by clinical examination and BM biopsy. Thus, we prefer using the term BM-DLBCL, which allows this

BM-DLBCLs

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20

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5 10 14 15 16 C1 C2 C3 C4 C5 C6 C7 C8 C9 C10

Control DLBCLs

CD20orf175CMTM2CMTM5CCL1CMTM6GPR109BMMP2CCBP2C5MMP7CCL7CCR2CCL8CCR8CCR1CCL4CXCL5CCR6CCL18CMTM7TREM2NFKB1CCL24CCL5CCL4L1HIF1AECGF1CCL2CXCR4CCL13CCL27CXCL12CXCR7CCL15CCL16C5AR1SLIT2PF4TCP10GPR81TREM1RGS3VHLXCR1TNF

BM-DLBCLs

16 10 14 4

Control DLBCLs

C4 C1 C3 C6 C7 C5 C2

A

B

❚Figure 1❚ Up-regulation of XCR1 messenger RNA in bone marrow–derived (BM) diffuse large B-cell lymphoma (DLBCL) samples. A, Clustering analysis of the chemokine- and chemokine receptor–specific expression microarray. Red genes are up-regulated and green genes are down-regulated in BM-DLBCL samples. BM-DLBCLs form 2 distinct clusters. XCR1 clustered together with tumor necrosis factor (TNF) at the bottom (arrow). B, Real-time quantitative reverse transcriptase–polymerase chain reaction. XCR1 is significantly up-regulated in BM-DLBCL samples (P < .01; Mann-Whitney U test).




category to be more easily distinguished from the Western type of IVL than the term AIVL.

Consistent with the findings by Murase et al,14 most cases in this study were negative for BCL-6 and positive for IRF-4/MUM-1, thus indicating a non–germinal center phenotype.22 However, 4 of 18 cases also showed intraclonal diversity. VH3-23–rearranged IgM+/IgD+/CD27+ B cells in the blood of patients with human X-linked hyper-IgM syndrome, which lacks CD40 and CD40 ligand interaction, undergo somatic hypermutation,23 and autoantibody-producing B cells of auto-immune mice also undergo somatic hypermutation outside the germinal center.24 Murine B1 cells have restricted autoreac-tive B cell receptors, such as VH11 and VH12.12,25 Recently, human B1 progenitor cells were identified in the BM,18 and human autoreactive B cells, which have a restricted VH reper-toire, may still have an important role in human innate immu-nity.26 However, human immature B2-B cells are also mostly autoreactive; thus, the normal counterpart of BM-DLBCL remains obscure.

An analysis of immunoglobulin variable gene usage has revealed that restricted VH family genes are frequently detect-ed in some autoimmune diseases.27 VH4-34, formerly known as VH4-21, was first detected in patients with cold agglutinin disease28 and thereafter in systemic lupus erythematosus29 and multiple sclerosis.30 VH3-7 and VH3-48 have been spe-cifically detected in B-cell clones established with nurse-like

❚Table 4❚Top 12 Up-Regulated Genes

Position UniGene RefSeq No. Symbol Description Ratio* P†

1 80 Hs.524811 NM_032554 GPR81 G protein–coupled receptor 81 7.68 .452 81 Hs.509554 NM_001530 HIF1A Hypoxia-inducible factor 1, α subunit (basic helix-loop-helix 4.54 .131 transcription factor) 3 103 Hs.351 NM_004610 TCP10 T-complex 10 (mouse) 3.57 .0894 59 Hs.522891 NM_000609 CXCL12 Chemokine (C-X-C motif) ligand 12 (stromal cell–derived factor 1) 3.31 .0895 105 Hs.174312 NM_138554 TLR4 Toll-like receptor 4 3.17 <.056 72 Hs.592212 NM_001953 ECGF1 Endothelial cell growth factor 1 (platelet-derived) 3.1 <.057 89 Hs.846 NM_001557 IL8RB Interleukin-8 receptor, β 3.03 <.058 20 Hs.247838 NM_002991 CCL24 Chemokine (C-C motif) ligand 24 3.02 .7059 52 Hs.471751 NM_020311 CXCR7 Chemokine (C-X-C motif) receptor 7 2.87 .50810 95 Hs.81564 NM_002619 PF4 Platelet factor 4 (chemokine [C-X-C motif] ligand 4) 2.78 <.0511 97 Hs.1905 NM_000948 PRL Prolactin 2.63 <.0512 113 Hs.248116 NM_005283 XCR1 Chemokine (C motif) receptor 1 2.57 <.05

RefSeq, National Center for Biotechnology Information Reference Sequence.* Average of control samples/average of bone marrow samples of diffuse large B-cell lymphoma.† Mann-Whitney U test. Of the 12 genes, 6 were significantly up-regulated in the bone marrow–derived diffuse large B-cell lymphoma cases. Four largely up-regulated

(>3.3-fold) genes were not significant mostly because of the variability in the samples.

❚Table 5❚Top 8 Down-Regulated Genes

Position UniGene RefSeq No. Symbol Description Ratio* P†

1 31 Hs.301921 NM_001295 CCR1 Chemokine (C-C motif) receptor 1 9.88 <.012 29 Hs.251526 NM_006273 CCL7 Chemokine (C-C motif) ligand 7 9.02 <.013 38 Hs.113222 NM_005201 CCR8 Chemokine (C-C motif) receptor 8 7.47 <.014 30 Hs.652137 NM_005623 CCL8 Chemokine (C-C motif) ligand 8 6.60 <.015 47 Hs.99272 NM_138460 CMTM5 CKLF-like MARVEL transmembrane domain containing 5 6.24 <.056 5 Hs.473029 NM_080829 C20orf175 Chromosome 20 open reading frame 175 5.88 <.017 32 Hs.644637 NM_000648 CCR2 Chemokine (C-C motif) receptor 2 5.84 <.058 9 Hs.54460 NM_002986 CCL11 Chemokine (C-C motif) ligand 11 5.35 .13057

RefSeq, National Center for Biotechnology Information Reference Sequence.* Average of control samples/average of bone marrow samples of diffuse large B-cell lymphoma.† Mann-Whitney U test. Of the 8 genes largely down-regulated in the bone marrow diffuse large B-cell lymphoma (DLBCL) samples compared with control DLBCL samples

(< 5.3-fold), 7 were significant.

❚Table 6❚XCR1 Immunohistochemical Results

Bone Marrow– Control Derived DLBCLs DLBCLs FL MCL MALT

2+ 22 0 0 0 11+ 6 4 2 0 5+/– 0 4 2 1 1– 1 15 2 2 2Total 29 23 6 3 9

DLBCL, diffuse large B-cell lymphoma; FL, follicular lymphoma; MALT, mucosa-associated lymphoid tissue lymphoma; MCL, mantle cell lymphoma; 2+, intense membrane staining in >50% of tumor cells; 1+, weak staining in >50% of tumor cells; +/–, staining <50% of tumor cells; –, no staining.

* MALT includes 6 gastric and 3 thyroid cases, and 4 cases had strongly staining large tumor cells.




expression system, VH3-7, VH3-23, and VH4-39 have also been reported to be autoreactive VH domains.34 It is important to note that VH genes of conventional DLBCL are not biased, even in the CD5+ cases.35 Thus, our finding that VH usage is biased toward an autoimmune repertoire seems to be another distinct feature of BM-DLBCLs.

cells from patients with rheumatoid arthritis,31 and VH3-7 was also selectively expressed by patients with Sjögren syn-drome.32 VH3-21 and VH3-23 are autoreactive VH genes that were frequently detected in natural IgM antibodies of patients with hepatitis C virus–related mixed cryoglobulinemia.33 Furthermore, based on an analysis using an in vitro bacterial

❚Image 1❚ Immunohistochemical analyses of XCR1 expression. CD20+ neoplastic B cells show intense cytoplasmic and membranous staining of XCR1 in a bone marrow–derived diffuse large B-cell lymphoma case.




4. Murase T, Nakamura S, Kawauchi K, et al. An Asian variant of intravascular large B-cell lymphoma: clinical, pathological and cytogenetic approaches to diffuse large B-cell lymphoma associated with haemophagocytic syndrome. Br J Haematol. 2000;111:826-834.

5. Morice WG, Rodriguez FJ, Hoyer JD, et al. Diffuse large B-cell lymphoma with distinctive patterns of splenic and bone marrow involvement: clinicopathologic features of two cases. Mod Pathol. 2005;18:495-502.

6. Ponzoni M, Ferreri AJM. Intravascular lymphoma: a neoplasm of “homeless” lymphocytes? Hematol Oncol. 2006;24:105-112.

7. Ponzoni M, Ferreri AJM, Campo E, et al. Definition, diagnosis, and management of intravascular large B-Cell lymphoma: proposals and perspectives from an international consensus meeting. J Clin Oncol. 2007;25:3168-3173.

8. Nakamura S, Ponzoni M, Campo E. Intravascular large B-cell lymphoma. In: Swerdlow S, Campo E, Harris NL, et al, eds. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. 4th ed. Lyon, France: IARC Press; 2008:252-253.

9. Stevenson F, Sahota S, Zhu DL, et al. Insight into the origin and clonal history of B-cell tumors as revealed by analysis of immunoglobulin variable region genes. Immunol Rev. 1998;162:247-259.

10. Pillai S, Cariappa A. The follicular versus marginal zone B lymphocyte cell fate decision. Nat Rev Immunol. 2009;9:767-777.

11. Hayakawa K, Hardy RR, Honda M, et al. Ly-1 B-cells: functionally distinct lymphocytes that secrete IgM autoantibodies. Proc Natl Acad Sci U S A. 1984;81:2494-2498.

12. Martin F, Kearney JF. B-cell subsets and the mature preimmune repertoire: marginal zone and B1B cells as part of a “natural immune memory.” Immunol Rev. 2000;175:70-79.

13. Weller S, Braun MC, Tan BK, et al. Human blood IgM “memory” B cells are circulating splenic marginal zone B cells harboring a prediversified immunoglobulin repertoire. Blood. 2004;104:3647-3654.

14. Murase T, Yamaguchi M, Suzuki R, et al. Intravascular large B-cell lymphoma (IVLBCL): a clinicopathologic study of 96 cases with special reference to the immunophenotypic heterogeneity of CD5. Blood. 2007;109:478-485.

15. Murase T, Tashiro K, Suzuki T, et al. Detection of antibodies to Fasciola and Anisakis in Japanese patients with intravascular lymphomatosis [letter]. Blood. 1998;92:2182-2183.

16. Nakamura S, Murase T, Kinoshita T. Intravascular large B-cell lymphoma: the heterogeneous clinical manifestations of its classical and hemophagocytosis-related forms. Haematologica. 2007;92:434-436.

17. Ramasamy I, Brisco M, Morley A. Improved PCR method for detecting monoclonal immunoglobulin heavy-chain rearrangement in B-cell neoplasms. J Clin Pathol. 1992;45:770-775.

18. Montecino-Rodriguez E, Leathers H, Dorshkind K. Identification of a B-1B cell–specified progenitor. Nat Immunol. 2006;7:293-301.

19. Shimazaki C, Inaba T, Nakagawa M. B-cell lymphoma–associated hemophagocytic syndrome. Leuk Lymphoma. 2000;38:121-130.

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XCR1, chemokine (C motif) receptor 1, also known as GPR5 and CCXCR1,36 is a chemokine receptor belonging to the G protein–coupled receptor superfamily. Members of this family are characterized by the presence of 7 transmembrane domains and numerous conserved amino acids. This receptor is most closely related to RBS11 and the MIP1-α/RANTES receptor.37 It transduces a signal by increasing the level of intracellular calcium ions,36 and its antagonist, the viral mac-rophage inflammatory protein-II, blocks signaling.38 XCR1 is expressed in human T cells, B cells, and neutrophils.39 Most recently, XCR1 was shown to be exclusively expressed in CD8+ dendritic cells in mice40; however, another recently published report showed functional expression of XCR1 in human oral epithelial cells.41 Only a few reports to date have discussed the role of XCR1 expression in B cells. A recent study showed high expression of XCR1 in extragas-tric MALT lymphomas and infrequent expression of XCR1 in extranodal DLBCLs.42 Whether XCR1 expression in BM-DLBCLs reflects some role of XCR1 in normal human B-cell function or is only aberrantly expressed in neoplastic cells remains to be clarified.

Expression of the chemokine receptor XCR1 and biased immunoglobulin gene recombination to autoreactive VH families are distinct features of the neoplastic B cells in BM-DLBCL. Our findings will potentially be helpful for the differential diagnosis and understanding of the biologic char-acteristics of the neoplastic cells of BM-DLBCL.

From the Departments of 1Pathology and Biological Responses, Nagoya University Graduate School of Medicine, Nagoya, Japan; 2Clinical and Experimental Hematology, Tsukuba University Hospital, Tsukuba, Japan; and 3Hematology and Oncology and 4Pathology and Clinical Laboratories, Nagoya University Hospital, Nagoya, Japan.

Supported by a Grant-in-Aid from the Japan Society of the Promotion of Science, Tokyo, Japan.

Address reprint requests to Dr Yamashita: Dept of Pathology and Biological Responses, Nagoya University Graduate School of Medicine, 65 Tsuruma-cho, Showa-ku, Nagoya, 466-8550, Japan.

Acknowledgment: We thank Y. Tanaka and N. Misawa for excellent technical assistance.

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