Primary central nervous system lymphoma (PCNSL) represents 1% to 3% intracranial tumors. Most PCNSL are located in the brain, and 75% are large B-cell lymphomas. The largest subgroup of these tumors contains cells that resemble centroblasts and has been labelled diffuse centroblastic (polymorphous) lymphoma. To investigate the cell of origin and the clonal history of these tumors, we have analyzed VH gene of 5 cases of PCNSL, all confirmed by histological studies to be Epstein-Barr virus (EBV)-negative, high-grade diffuse B-cell lymphomas. The V4-34 gene of the VH4 family was used in 4 of 5 cases. All VHgenes were found to have accumulated very high levels of somatic mutation (14% to 25%). In 3 of 5 cases, intraclonal nucleotide heterogeneity, including codon deletion in some clones in 1 case, was observed, indicating that the VH genes were still under the influence of the somatic hypermutation mechanism. Analysis of the distribution of silent and replacement mutations showed evidence for preservation of immunoglobulin structure in all cases. These results suggest that, although there is no evidence for germinal center formation in the brain tissue, PCNSL is derived from a B cell with features associated with location in a germinal center environment.

PRIMARY CENTRAL NERVOUS system lymphoma (PCNSL) is an uncommon neoplasm, representing 1% to 2% of lymphomas and 1% to 3% of intracranial tumors.1,2 While most examples of PCNSL are sporadic, some are associated with congenital or acquired immunodeficiency. An increasing incidence of PCNSL can partly be attributed to a growing population of immunocompromised patients in which PCNSL is mainly associated with the acquired immunodeficiency syndrome, organ transplantation, and chemotherapy. PCNSL has been associated with a poor prognosis, which is worse than that for similar non-Hodgkin’s lymphomas outside the CNS.3However, recent trials of radiotherapy with various chemotherapeutic regimens have shown an improvement in survival.4,5 Most PCNSLs are located in the brain, and about 75% are large B-cell lymphomas (REAL classification). The largest subgroup of these large cell lymphomas contains cells that resemble centroblasts6 and has been labeled diffuse centroblastic (polymorphous) lymphoma (Kiel classification). Other B-cell PCNSLs are low-grade lymphocytic, lymphoplasmacytic, and plasmacytic lymphomas. T-cell PCNSLs are very rare, but large and small cell examples have been reported.1 

Generation of functional antigen receptors by somatic recombination of variable, diversity, and joining segments is a unique feature of B cells and T cells. In a normal immune response, B lymphocytes stimulated by antigen can undergo somatic hypermutation in their immunoglobulin (Ig) variable region genes in a germinal center microenvironment, thus generating a more diverse repertoire of antibodies. B cells with high affinity for antigen are selectively rescued from apoptosis to differentiate into either circulating memory cells or antibody-producing plasma cells.7,8 From the developmental point of view, B-cell tumors can be regarded as B cells arrested at a certain differentiation stage when neoplastic transformation took place. Analysis of Ig V region genes has provided invaluable information on the status of tumors in B-cell differentiation and has added another dimension to classification of B-cell tumors at the molecular level.9,10 In the case of follicular center lymphoma (FCL), V genes are often somatically mutated and in some cases significant accumulation of replacement mutations is found in the complementarity-determining regions (CDRs), suggesting a selective role for antigenic stimulation. Furthermore, intraclonal nucleic acid sequence variation appears to be a common feature of FCL, an indication that tumor cells are still under the influence of a mutation mechanism.11-14 Recent studies have also shown that VH genes in primary diffuse large cell lymphomas (DLCL) are somatically mutated,15-18 with a significant level of intraclonal heterogeneity, again indicating ongoing somatic mutation.16 Interestingly, the heterogeneity is reduced after successful chemotherapy, possibly due to selective pressure on the clone.16 There have been few studies of Ig V genes on PCNSL. Southern blotting and polymerase chain reaction (PCR) amplification of the VH CDR3 sequences have demonstrated Ig V-gene rearrangement in most cases, confirming the B-cell nature of PCNSL.19-21 To gain insight into the nature of the Ig V genes in PCNSL, we cloned and sequenced the complete VHgenes of 5 cases of PCNSL. We have found frequent usage of the V4-34 gene. We have also found that the VH genes are extensively mutated and show intraclonal heterogeneity in some cases, reminiscent of alterations influenced by the germinal center environment.

Immunohistochemistry and in situ hybridization.

Tumor biopsies were examined by standard histological techniques, including immunohistochemistry as previously described.22 A range of antibodies (DAKO, Glostrup, Denmark) to immunoglobulins, and B-cell and T-cell–specific antigens was used at the following concentrations: IgM 1:500; IgG 1:1,000; IgA 1:500; Kappa 1:500; lambda 1:500; CD20 1:400; CD79a 1:250; CD3 1:50; CD45RO 1:1,000. Sections (5 μm) were also probed for Epstein-Barr virus (EBV)-encoded RNA (EBER) transcripts using an in situ hybridization kit (Novocastra, Newcastle, UK) with appropriate positive and negative controls.

Preparation of cDNA.

Frozen tumour tissue (≈0.1 g) was cut into small pieces and ground into fine powder in liquid nitrogen in a mortar. The powder was transferred to a microcentrifuge tube, and total RNA was isolated using RNAzol according to the supplier’s instructions (Cinna Biotecxlabs Inc, Houston, TX). First-strand cDNA was synthesized from 5 μg of total RNA in a final volume of 33 μL using a cDNA synthesis kit and oligo(dT) primer (Pharmacia, Uppsala, Sweden).

Amplification, cloning, and sequencing of VH genes.

For amplification of VH genes from cDNA, 1 to 5 μL of cDNA was used as template for PCR using a mixture of oligonucleotide primers specific for each of the leader sequences of VH 1-6 families (Table 1), together with a mixture of primers complementary to the sequences of the germ line JH genes.23 In some cases, a Cμ constant region primer was also used. PCR was performed in a final volume of 50 μL containing 1 to 5 μL of cDNA, 20 pmol of each primer, 250 mmol/L of dNTPs, 2.5 U of Taq DNA polymerase with the reaction buffer supplied by the manufacturer (QIAgen, Hilden, Germany). PCR consisted of an initial denaturation step of 5 minutes at 94°C, followed by 35 cycles of 94°C for 60 seconds, 65°C for 60 seconds, and 72°C for 60 seconds, with a final extension step of 10 minutes at 72°C. PCR products (≈400 bp) were separated by agarose gel electrophoresis, purified using the GeneClean kit (Bio 101 Inc, Vista, CA), and cloned into the pGEM-T vector (Promega, Madison, WI). Randomly picked bacterial clones were sequenced on an ABI 377 automatic sequencer (Perkin-Elmer, Foster City, CA).

Table 1.

VH Leader and Cμ Primers for VH Gene Amplification

VH1/7Lead  5′-CTC ACC ATG GAC TGG ACC TGG AG-3′ 
VH2Lead  5′-ATG GAC ACA CTT TGC T(A/C)C AC(G/A) CTC-3′  
VH3Lead  5′-CCA TGG AGT TTG GGC TGA GCT GG-3′  
VH4Lead  5′-ACA TGA AAC A(C/T)C TGT GGT TCT TCC-3′  
VH5Lead  5′-ATG GGG TCA ACC GCC ATC CT(C/T) G-3′  
VH6Lead  5′-ATG TCT GTC TCC TTC CTC ATC TTC-3′  
Cμ  5′-GGA ATT CTC ACA GGA GAC GAG G-3′ 
VH1/7Lead  5′-CTC ACC ATG GAC TGG ACC TGG AG-3′ 
VH2Lead  5′-ATG GAC ACA CTT TGC T(A/C)C AC(G/A) CTC-3′  
VH3Lead  5′-CCA TGG AGT TTG GGC TGA GCT GG-3′  
VH4Lead  5′-ACA TGA AAC A(C/T)C TGT GGT TCT TCC-3′  
VH5Lead  5′-ATG GGG TCA ACC GCC ATC CT(C/T) G-3′  
VH6Lead  5′-ATG TCT GTC TCC TTC CTC ATC TTC-3′  
Cμ  5′-GGA ATT CTC ACA GGA GAC GAG G-3′ 
Analysis of VH gene usage and mutation pattern.

Tumor-related VH genes were identified as predominant repeated or similar sequences with clonally related CDR3.23Sequence alignment analysis was to the Entrez database (National Center for Biotechnology Information, Bethesda, MD) and to the VBase, which contains all known human germ line immunoglobulin V region genes24 using MacVector sequence analysis software (Oxford Molecular, Oxford, UK). The germ line genes with highest homology to the tumor-derived sequences are considered as the germ line gene donors. Statistical analysis of mutation distribution was performed according to Chang and Casali.25 In this method, each V gene is analyzed codon by codon for significance of deviation from the germ line sequence. A modified binomial distribution model is then applied to calculate whether the probability (p) of excess (in CDR) or scarcity (in framework region [FR]) of R mutations resulted from chance alone.

Clinical and histological data.

All 5 patients, of whom 4 were female, presented with CNS symptoms and signs. Their ages ranged from 50 to 71 years (median, 60 years). None had detectable lymphadenopathy, and none was immunocompromised. Neuroimaging showed mass lesions in the cerebrum (3 patients) or cerebellum (2 patients), which were thought to be neoplastic in nature. Biopsy of all tumors was undertaken at the Wessex Neurological Centre where concurrent radiological investigations failed to show tumor outside the CNS. In all cases, histological examination showed the typical architectural and cytological features of a high-grade (polymorphous) B-cell PCNSL (Fig 1), including the characteristic perivascular arrangement of neoplastic cells and invasion of adjacent brain tissue to form intraparenchymal masses.

Fig. 1.

PCNSL histology. (A) Brain at the periphery of a PCNSL characteristically shows accumulation of neoplastic cells in the perivascular spaces (periodic acid Schiff; original magnification [OM] × 250). (B) Invasion of surrounding brain by neoplastic cells occurs from the perivascular space (periodic acid Schiff; OM × 500). (C) A sheet of neoplastic lymphoid cells characterizes this intraparenchymal large cell PCNSL. Moderate nuclear pleomorphism, a variable number of nucleoli per cell, and mitotic figures are evident (hematoxylin and eosin; OM × 500). (D) All tumors in this study were positive for CD20 (L-26 antibody, immunoperoxidase; OM × 500).

Fig. 1.

PCNSL histology. (A) Brain at the periphery of a PCNSL characteristically shows accumulation of neoplastic cells in the perivascular spaces (periodic acid Schiff; original magnification [OM] × 250). (B) Invasion of surrounding brain by neoplastic cells occurs from the perivascular space (periodic acid Schiff; OM × 500). (C) A sheet of neoplastic lymphoid cells characterizes this intraparenchymal large cell PCNSL. Moderate nuclear pleomorphism, a variable number of nucleoli per cell, and mitotic figures are evident (hematoxylin and eosin; OM × 500). (D) All tumors in this study were positive for CD20 (L-26 antibody, immunoperoxidase; OM × 500).

Close modal

Immunohistochemistry confirmed a B-cell immunophenotype for the neoplastic cells in each case (Table 2). All tumors expressed CD20 (Fig 1) and CD79a. Antibodies to CD3 and CD45RO labeled moderate numbers of reactive T lymphocytes among the neoplastic cells. Immunohistochemistry with antibodies to IgM, IgG, IgA, and kappa and lambda light chains was feasible in 3 of 5 cases. IgM and kappa light chains were detected in neoplastic cells in all 3 cases. In 1 tumor, sparse IgG-positive cells were detected in addition to more widespread IgM. No IgA was detected. All 5 tumors were EBER-negative by in situ hybridization.

Table 2.

Clinical, Immunophenotypic, and EBV Data of PCNSL Cases

Patient Age/Sex Tumor Location CD20 CD79a CD3CD45RO IgM IgG IgA κ λ EBER
61/F  Cerebral  +  +  −  −  NA  NA  NA NA  NA  −  
2  71/M  Cerebella  +  +  − −  +  ±  −  +  −  −  
3  59/F Cerebral  +  +  −  −  +  −  −  −  −  
4  60/F  Cerebral  +  +  −  − NA  NA  NA  NA  NA  −  
5  50/F  Cerebella +  +  −  −  ±  −  −  +  − − 
Patient Age/Sex Tumor Location CD20 CD79a CD3CD45RO IgM IgG IgA κ λ EBER
61/F  Cerebral  +  +  −  −  NA  NA  NA NA  NA  −  
2  71/M  Cerebella  +  +  − −  +  ±  −  +  −  −  
3  59/F Cerebral  +  +  −  −  +  −  −  −  −  
4  60/F  Cerebral  +  +  −  − NA  NA  NA  NA  NA  −  
5  50/F  Cerebella +  +  −  −  ±  −  −  +  − − 

Abbreviations: +, positive; −, negative; ±, a few immunopositive cells only; NA, not available.

Usage of VH, D, and JH genes in the tumors.

Preparations of cDNA from all 5 patients were amplified, cloned, and sequenced. In all cases, predominant repeated or similar clones with identical CDR3 sequences were obtained, consistent with tumor derivation (Table 3). The usage of VH, D, and JH genes and the homology to the corresponding germ line genes are summarized in Table 3. Sequence analysis indicates that the V4-34 gene of the VH4 family was used in 4 of 5 patients, and in the remaining case, the VH gene was derived from V3-23, a member of the VH3 family. Assignment of D gene segments was based on the homology between CDR3 sequences and the germ line D genes. According to the rule proposed by Corbett et al26 that at least 10 consecutive nucleotides of identity are required to assign a D segment, D gene can only be identified with confidence in patient 4. However, shorter D segments or D segments with nucleotide substitutions could have been used in the other cases and are also shown in Table3. There was no apparent bias in JH gene usage. JH6 gene was used in cases 1 and 5, JH3 in case 2, and JH4 in case 4. The JH gene in case 3 was extensively truncated at the 5′ end, which could be derived from either JH1 or JH5.

Table 3.

Analysis of Tumor-Derived VH Gene Sequences

Patient VH Family GL DonorHomology (%) D Gene JH Gene Related Clones3-150/ Clones Sequenced
1  VH4  4-34  75 D3-10  JH6b  13/14  
2  VH3-23  81  D5-12  JH3  8/8  
VH4  4-34  86  D3-3   JH1/5b 6/8  
4  VH4  4-34  82  D3-10 JH4b  13/13  
5  VH4  4-34 84  D4-11  JH6c  8/9 
Patient VH Family GL DonorHomology (%) D Gene JH Gene Related Clones3-150/ Clones Sequenced
1  VH4  4-34  75 D3-10  JH6b  13/14  
2  VH3-23  81  D5-12  JH3  8/8  
VH4  4-34  86  D3-3   JH1/5b 6/8  
4  VH4  4-34  82  D3-10 JH4b  13/13  
5  VH4  4-34 84  D4-11  JH6c  8/9 
F3-150

Clones sharing identical or near identical CDR3 sequences.

Somatic hypermutations in tumor-derived VH genes.

The nucleotide sequences of the tumor VH genes and the deduced amino acid sequences are shown in Figs2 and 3. A large number of nucleotide substitutions was found in all tumor VH genes, ranging from 41 to 73 nucleotides (14% to 25% of the VH genes). In addition to single nucleotide change, double and triple substitution also frequently occurred, mostly within the same codon. No nucleotide insertion was observed, but triple in-frame as well as single nucleotide deletion was seen in a number of clones in case 1 (see below). Among the V4-34–derived sequences, common nucleotide substitutions leading to both silent and replacement mutations were seen in several codons, such as Gln5, Val71, and Ser35 (Fig 3). To see if there was significant clustering of replacement (R) mutations in the CDRs and silent mutations in the FRs, the distribution of somatic mutations was analyzed by the method of Chang and Casali25(Table 4). The analysis shows that the number of R mutations in FRs was significantly lower in all cases (P < .05), probably as a result of selection against R mutations within the FRs to preserve the structures of expressed immunoglobulins. In patient 2, the number of R mutations in CDRs was significantly higher than would be expected to arise solely by chance (P < .05), whereas in the other cases, the number of replacement mutations in the CDRs was not significantly higher than expected (P > .05).

Fig. 2.

Nucleotide and deduced amino acid sequences of VH genes derived from PCNSL. Comparisons were made with the most homologous germ line VH genes. Dashes represent identity with the representative germ line sequence. Replacement amino acids are starred. Blank space indicates nucleotide deletion. The binding sites for JHprimers are shown in italics. For patients 1 and 4, full JHsequences are shown for the clones obtained by PCR using the VH leader and the Cμ primers. Clones with nucleotide deletions in case 1 are underlined. The VH gene sequences have been deposited to the EMBL database with following accession numbers: patient 1, AJ235649-AJ235661; patient 2,AJ235662; patient 3, AJ235663; patient 4, AJ235664-AJ23566; patient 5,AJ235671-AJ235678.

Fig. 2.

Nucleotide and deduced amino acid sequences of VH genes derived from PCNSL. Comparisons were made with the most homologous germ line VH genes. Dashes represent identity with the representative germ line sequence. Replacement amino acids are starred. Blank space indicates nucleotide deletion. The binding sites for JHprimers are shown in italics. For patients 1 and 4, full JHsequences are shown for the clones obtained by PCR using the VH leader and the Cμ primers. Clones with nucleotide deletions in case 1 are underlined. The VH gene sequences have been deposited to the EMBL database with following accession numbers: patient 1, AJ235649-AJ235661; patient 2,AJ235662; patient 3, AJ235663; patient 4, AJ235664-AJ23566; patient 5,AJ235671-AJ235678.

Close modal
Fig. 3.

Deduced amino acid sequences of VH regions of PCNSL. Comparisons were made with the most homologous germ line VH genes. Amino acid numbering is according to Kabat et al.46 Dashes represent identity with the representative germ line sequence. Uppercase, replacement mutation; lowercase, silent mutation.

Fig. 3.

Deduced amino acid sequences of VH regions of PCNSL. Comparisons were made with the most homologous germ line VH genes. Amino acid numbering is according to Kabat et al.46 Dashes represent identity with the representative germ line sequence. Uppercase, replacement mutation; lowercase, silent mutation.

Close modal
Table 4.

Distributions of Mutations in Tumor-Derived VH Sequences

Patient Region ExpectedObserved P4-150
R S R S
1  FR  42  15  29  23  <.01  
 CDR  13 3  16  5  .07  
2  FR  33  11  19  15 <.01  
 CDR  9  2  15  6  .02  
3  FR 35  13  16  16  <.01  
 CDR  11  2  4  .12  
4  FR  31  11  23  14  .02 
 CDR  9  2  10  5  .14  
5  FR  26  14  16  <.01  
 CDR  8  2  10  .11 
Patient Region ExpectedObserved P4-150
R S R S
1  FR  42  15  29  23  <.01  
 CDR  13 3  16  5  .07  
2  FR  33  11  19  15 <.01  
 CDR  9  2  15  6  .02  
3  FR 35  13  16  16  <.01  
 CDR  11  2  4  .12  
4  FR  31  11  23  14  .02 
 CDR  9  2  10  5  .14  
5  FR  26  14  16  <.01  
 CDR  8  2  10  .11 
F4-150

P is the probability of obtaining the number of R mutations by chance.

Intraclonal nucleotide sequence variation.

In 3 cases (1, 4, and 5), intraclonal nucleotide variation was observed in VH genes (Fig 3). This sequence heterogeneity is considered mostly as the result of ongoing somatic mutation rather than being introduced in PCR amplification, as the levels of variation (>1 in 700 bp) were much higher than the PCR error frequency (≈1 in 5,000 bp in our laboratory) and several identical base changes were seen in different clones. This is supported also by the fact that no heterogeneity was observed in cases 2 and 3 with similar numbers of clones sequenced. The level of nucleotide variation in the 3 cases differs and the variations appeared to be distributed randomly. Most nucleotide variations were single base substitutions, resulting in both silent and replacement mutations. However, deletion of in-frame triple nucleotides, ie, an entire codon, in 3 different positions in the CDR2 was found in a number of clones in patient 1. Single nucleotide deletion was also found in 2 clones in this case, 1 in the FR2 and 1 in the FR3, leading to aberrant sequences. No nucleotide insertions were observed.

PCNSL is a rare tumor with increasing incidence during the past decade, both in immunocompromised and in general populations.1PCNSL shows morphological and immunohistochemical features similar to those of malignant lymphomas arising in sites outside the CNS. The B-cell nature of most of these tumors has not only been demonstrated by immunohistochemistry, but by Southern blotting and PCR amplification of the short VH CDR3 sequences.19-21 In this small study of full-length VH gene sequences, VH gene usage appeared to be restricted as the V4-34 gene was used in 4 of 5 cases. The V4-34 gene is used in ≈5% of healthy adults27,28 and appears to be overexpressed in some autoimmune diseases and B-cell tumors. The V4-34 gene has been shown to be mandatory for encoding the IgM proteins of cold agglutinin disease.29,30 In this case, the red blood cell I/i antigens appear to bind to the FR1 of the Ig. This binding outside the CDRs is indication of a B-cell superantigen. Recently, Hsu and Levy15 reported that in DLCL there was a preferential usage of genes from the VH4 family, especially the V4-34 gene (11 of 17 cases, 65%), although a recent study31using the V4-34 gene product-specific antibody, 9G4, showed that only 30% of DLCL expressed the V4-34 gene; and the sequence analysis by Küppers et al17 and a study of 11 cases of DLCL in this laboratory did not show bias toward V4-34 gene (C. Ottensmeier, personal communication, September 1998). VH4 family genes, but not V4-34, also appear to be preferentially used in the B cells from cerebrospinal fluid in multiple sclerosis.32 33 If such bias were to be confirmed in PCNSL, it might suggest a role for superantigen drive in lymphoma development. It is interesting that certain infections, including EBV, cytomegalovirus, and mycoplasma pneumoniae, cause a selective increase in serum levels of V4-34 encoded Ig. Although the cerebral tumors described are EBV-genome negative, this intriguing association may indicate a potential influence of pathogens on B cells expressing V4-34 encoded Ig.

The VH genes of all 5 cases were very heavily mutated. In fact, the somatic mutation frequency in these cases (mean ± standard deviation [SD]% = 18.4 ± 3.7%) is higher than in all of the other B-cell malignancies studied so far (summarized by Klein et al10). In DLCL, which involves cells morphologically and phenotypically similar to PCNSL, a mean mutation frequency of 11% (range, 4% to 22%) was reported.17 Despite high levels of somatic mutation in PCNSL, there is good evidence for selection against R mutations in the FRs, suggesting that functional expression of the Ig receptor may be advantageous for tumor growth. Some shared nucleotide substitutions leading to silent as well as replacement mutations were seen among the V4-34 encoded sequences. The intrinsic mutational “hot spots” are likely to be responsible for these common mutations, as the same changes in the V4-34 gene have been observed in different studies,15,34 and biased nucleotide substitution is a general feature of both human35 and murine IgV genes.36 

Intraclonal heterogeneity was observed in 3 cases, suggesting that the tumor cells retained the capacity to undergo further somatic hypermutation. In case 1, which was the most mutated, deletion of in-frame triple nucleotides occurred in 2 different positions in the CDR2; therefore the gene could still be expressed. However, in 2 clones, the reading-frame was disrupted by single nucleotide deletion and therefore no Ig could be produced. Possibly, these sequences were derived from the cells that contained deleterious mutations and were due to die, but had not yet been eliminated from the tumor population. Nucleotide deletion and insertion have also been reported in other B-cell tumors.37-39 However, insertional or deletional events are not unique to tumor cells because there is strong evidence that nucleotide insertion and deletion are frequently introduced in the somatic hypermutation process, and in-frame insertion and deletion have been found in normal B cells.40 41 

The pattern of somatic mutation and intraclonal variation indicates that the cell of origin of PCNSL has been exposed to the mutational mechanism, both before and after neoplastic transformation. This pattern is characteristic of tumors of germinal center origin, such as FCL, and is also seen in Burkitt’s lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma and DLCL.9,10 Stimulation of somatic mutation is generally considered to require a GC environment where activated T cells and cytokines are present.7,8 Cross-linking of surface Ig also may increase the rate of somatic mutation, possibly reflecting a role for antigen.42 

For PCNSL, there is no evidence for GC structure in the brain. However, PCNSL cells do express BCL-6 protein.43 PCNSLs are mainly located in the cerebral parenchyma and can infiltrate the pial or ependymal surfaces to spread along the edge of CSF pathways, with no particular anatomical location. One possibility is that the somatic mutation is occurring independently of GC structure. Support for some GC-independent mutational activity comes from mice deficient in lymphotoxin-α44 and Lyn kinase.45 Although these mice cannot form GCs, they are able to accumulate somatic mutations in V genes. However, levels are low and would not be expected to reach the very high levels found in the B cells that give rise to PCNSL.

An alternative possibility is that the B cells of PCNSL have undergone somatic mutation and neoplastic transformation in a draining cervical lymph node. These events could have occurred after antigenic stimulation, perhaps due to infection. However, the neoplastic cells have then to leave the lymph node and enter the cerebral parenchyma where they apparently remain. The puzzle is that cervical lymph nodes are not obviously involved at presentation. Availability of VH CDR3 sequence tags should now allow an approach to these questions by probing of blood and bone marrow of patients to track possible migration of tumor cells from the brain.

Supported by Tenovus and the Cancer Research Campaign, UK.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. section 1734 solely to indicate this fact.

1
Grant
 
JW
Isaacson
 
PG
Primary central nervous system lymphoma.
Brain Pathol
2
1992
97
2
van der Valk
 
P
Central nervous system lymphomas.
Curr Diagn Pathol
3
1996
45
3
Pollack
 
IF
Lunsford
 
LD
Flickinger
 
JC
Dameshek
 
HL
Prognostic factors in the diagnosis and treatment of primary central nervous system lymphoma.
Cancer
63
1989
939
4
Tomlinson
 
FH
Kurtin
 
PJ
Suman
 
VJ
Scheithauer
 
BW
O’Fallon
 
JR
Kelly
 
PJ
Jack
 
CR
O’Neill
 
BP
Primary intracerebral malignant lymphoma: A clinicopathological study of 89 patients.
J Neurosurg
82
1995
558
5
Abrey
 
L
DeAngelis
 
L
Yahalom
 
J
Long-term survival in primary CNS lymphoma.
J Clin Oncol
16
1998
859
6
Schwechheimer
 
K
Braus
 
DF
Schwarzkopf
 
G
Feller
 
AC
Volk
 
B
Muller-Hermelink
 
HK
Polymorphous high-grade B cell lymphoma is the predominant type of spontaneous primary cerebral malignant lymphomas. Histological and immunomorphological evaluation of computed tomography-guided stereotactic brain biopsies.
Am J Surg Pathol
18
1994
931
7
Berek
 
C
The development of B cells and the B-cell repertoire in the microenvironment of the germinal center.
Immunol Rev
126
1992
5
8
MacLennan
 
IC
Germinal centers.
Annu Rev Immunol
12
1994
117
9
Stevenson
 
F
Sahota
 
S
Zhu
 
D
Ottensmeier
 
C
Chapman
 
C
Oscier
 
D
Hamblin
 
T
Insight into the origin and clonal history of B-cell tumors as revealed by analysis of immunoglobulin variable region genes.
Immunol Rev
162
1998
247
10
Klein
 
U
Goossens
 
T
Fischer
 
M
Kanzler
 
H
Braeuninger
 
A
Rajewsky
 
K
Kuppers
 
R
Somatic hypermutation in normal and transformed human B cells.
Immunol Rev
162
1998
261
11
Levy
 
R
Levy
 
S
Cleary
 
ML
Carroll
 
W
Kon
 
S
Bird
 
J
Sklar
 
J
Somatic mutation in human B-cell tumors.
Immunol Rev
96
1987
43
12
Bahler
 
DW
Levy
 
R
Clonal evolution of a follicular lymphoma: Evidence for antigen selection.
Proc Natl Acad Sci USA
89
1992
6770
13
Zelenetz
 
AD
Chen
 
TT
Levy
 
R
Clonal expansion in follicular lymphoma occurs subsequent to antigenic selection.
J Exp Med
176
1992
1137
14
Zhu
 
D
Hawkins
 
RE
Hamblin
 
TJ
Stevenson
 
FK
Clonal history of a human follicular lymphoma as revealed in the immunoglobulin variable region genes.
Br J Haematol
86
1994
505
15
Hsu
 
FJ
Levy
 
R
Preferential use of the VH4 Ig gene family by diffuse large-cell lymphoma.
Blood
86
1995
3072
16
Ottensmeier
 
CH
Thompsett
 
AR
Zhu
 
D
Wilkins
 
BS
Sweetenham
 
JW
Stevenson
 
FK
Analysis of VH genes in follicular and diffuse lymphoma shows ongoing somatic mutation and multiple isotype transcripts in early disease with changes during disease progression.
Blood
91
1998
4292
17
Küppers
 
R
Rajewsky
 
K
Hansmann
 
ML
Diffuse large cell lymphomas are derived from mature B cells carrying V region genes with a high load of somatic mutation and evidence of selection for antibody expression.
Eur J Immunol
27
1997
1398
18
Taniguchi
 
M
Oka
 
K
Hiasa
 
A
Yamaguchi
 
M
Ohno
 
T
Kita
 
K
Shiku
 
H
De novo CD5+ diffuse large B-cell lymphomas express VH genes with somatic mutation.
Blood
91
1998
1145
19
Smith
 
WJ
Garson
 
JA
Bourne
 
SP
Kemshead
 
JT
Coakham
 
HB
Immunoglobulin gene rearrangement and antigenic profile confirm B cell origin of primary cerebral lymphoma and indicate a mature phenotype.
J Clin Pathol
41
1988
128
20
Kumanishi
 
T
Washiyama
 
K
Nishiyama
 
A
Abe
 
S
Saito
 
T
Ichikawa
 
T
Primary malignant lymphoma of the brain: Demonstration of immunoglobulin gene rearrangements in four cases by the Southern blot hybridization technique.
Acta Neuropathol (Berl)
79
1989
23
21
Albrecht
 
S
Bruner
 
JM
Segall
 
GK
Immunoglobulin heavy chain rearrangements in primary brain lymphomas. A study using PCR to amplify CDR-III.
J Pathol
169
1993
297
22
Ellison
 
DW
Steart
 
PV
Gatter
 
KC
Weller
 
RO
Apoptosis in cerebral astrocytic tumours and its relationship to expression of the bcl-2 and p53 proteins.
Neuropathol Appl Neurobiol
21
1995
352
23
Hawkins
 
RE
Zhu
 
D
Ovecka
 
M
Winter
 
G
Hamblin
 
TJ
Long
 
A
Stevenson
 
FK
Idiotypic vaccination against human B-cell lymphoma. Rescue of variable region gene sequences from biopsy material for assembly as single-chain Fv personal vaccines.
Blood
83
1994
3279
24
Cook
 
GP
Tomlinson
 
IM
The human immunoglobulin VH repertoire.
Immunol Today
16
1995
237
25
Chang
 
B
Casali
 
P
The CDR1 sequences of a major proportion of human germline Ig VH genes are inherently susceptible to amino acid replacement.
Immunol Today
15
1994
367
26
Corbett
 
SJ
Tomlinson
 
IM
Sonnhammer
 
ELL
Buck
 
D
Winter
 
G
Sequence of the human immunoglobulin diversity (D) segment locus: A systematic analysis provides no evidence for the use of DIR segments, inverted D segments, “minor” D segments or D-D recombination.
J Mol Biol
270
1997
587
27
Stevenson
 
FK
Smith
 
GJ
North
 
J
Hamblin
 
TJ
Glennie
 
MJ
Identification of normal B-cell counterparts of neoplastic cells which secrete cold agglutinins of anti-I and anti-i specificity.
Br J Haematol
72
1989
9
28
Schutte
 
ME
van Es
 
JH
Silberstein
 
LE
Logtenberg
 
T
VH4.21-encoded natural autoantibodies with anti-i specificity mirror those associated with cold hemagglutinin disease.
J Immunol
151
1993
6569
29
Pascual
 
V
Victor
 
K
Spellerberg
 
M
Hamblin
 
TJ
Stevenson
 
FK
Capra
 
JD
VH restriction among human cold agglutinins. The VH4-21 gene segment is required to encode anti-I and anti-i specificities.
J Immunol
149
1992
2337
30
Silberstein
 
LE
Jefferies
 
LC
Goldman
 
J
Friedman
 
D
Moore
 
JS
Nowell
 
PC
Roelcke
 
D
Pruzanski
 
W
Roudier
 
J
Silverman
 
GJ
Variable region gene analysis of pathologic human autoantibodies to the related i and I red blood cell antigens.
Blood
78
1991
2372
31
Funkhouser
 
WK
Warnke
 
RA
Preferential IgH V4-34 gene segment usage in particular subtypes of B-cell lymphoma detected by antibody 9G4.
Hum Pathol
29
1998
1317
32
Owens
 
GP
Kraus
 
H
Burgoon
 
MP
Smith-Jensen
 
T
Devlin
 
ME
Gilden
 
DH
Restricted use of VH4 germline segments in an acute multiple sclerosis brain.
Ann Neurol
43
1998
236
33
Qin
 
Y
Duquette
 
P
Zhang
 
Y
Talbot
 
P
Poole
 
R
Antel
 
J
Clonal expansion and somatic hypermutation of V(H) genes of B cells from cerebrospinal fluid in multiple sclerosis.
J Clin Invest
102
1998
1045
34
Chapman
 
CJ
Mockridge
 
CI
Hamblin
 
TJ
Stevenson
 
FK
Tracking of the V4-34 (VH4-21) gene in human tonsil reveals clonal isotype switch events and a highly variable degree of somatic hypermutation.
Clin Exp Immunol
105
1996
360
35
Dorner
 
T
Brezinschek
 
HP
Foster
 
SJ
Brezinschek
 
RI
Farner
 
NL
Lipsky
 
PE
Delineation of selective influences shaping the mutated expressed human Ig heavy chain repertoire.
J Immunol
160
1998
2831
36
Betz
 
AG
Neuberger
 
MS
Milstein
 
C
Discriminating intrinsic and antigen-selected mutational hotspots in immunoglobulin V genes.
Immunol Today
14
1993
405
37
Hashimoto
 
S
Dono
 
M
Wakai
 
M
Allen
 
SL
Lichtman
 
SM
Schulman
 
P
Vinciguerra
 
VP
Ferrarini
 
M
Silver
 
J
Chiorazzi
 
N
Somatic diversification and selection of immunoglobulin heavy and light chain variable region genes in IgG+ CD5+ chronic lymphocytic leukemia B cells.
J Exp Med
181
1995
1507
38
Kuppers
 
R
Rajewsky
 
K
Zhao
 
M
Simons
 
G
Laumann
 
R
Fischer
 
R
Hansmann
 
ML
Hodgkin disease: Hodgkin and Reed-Sternberg cells picked from histological sections show clonal immunoglobulin gene rearrangements and appear to be derived from B cells at various stages of development.
Proc Natl Acad Sci USA
91
1994
10962
39
Braeuninger
 
A
Kuppers
 
R
Strickler
 
JG
Wacker
 
HH
Rajewsky
 
K
Hansmann
 
ML
Hodgkin and Reed-Sternberg cells in lymphocyte predominant Hodgkin disease represent clonal populations of germinal center-derived tumor B cells [published erratum appears in Proc Natl Acad Sci USA 94:14211, 1997].
Proc Natl Acad Sci USA
94
1997
9337
40
Wilson
 
PC
de Bouteiller
 
O
Liu
 
YJ
Potter
 
K
Banchereau
 
J
Capra
 
JD
Pascual
 
V
Somatic hypermutation introduces insertions and deletions into immunoglobulin V genes.
J Exp Med
187
1998
59
41
Goossens
 
T
Klein
 
U
Kuppers
 
R
Frequent occurrence of deletions and duplications during somatic hypermutation: Implications for oncogene translocations and heavy chain disease.
Proc Natl Acad Sci USA
95
1998
2463
42
Denepoux
 
S
Razanajaona
 
D
Blanchard
 
D
Meffre
 
G
Capra
 
JD
Banchereau
 
J
Lebecque
 
S
Induction of somatic mutation in a human B cell line in vitro.
Immunity
6
1997
35
43
Larocca
 
LM
Capello
 
D
Rinelli
 
A
Nori
 
S
Antinori
 
A
Gloghini
 
A
Cingolani
 
A
Migliazza
 
A
Saglio
 
G
Cammilleri-Broet
 
S
Raphael
 
M
Carbone
 
A
Gaidano
 
G
The molecular and phenotypic profile of primary central nervous system lymphoma identifies distinct categories of the disease and is consistent with histogenetic derivation from germinal center-related B cells.
Blood
92
1998
1011
44
Matsumoto
 
M
Lo
 
SF
Carruthers
 
CJ
Min
 
J
Mariathasan
 
S
Huang
 
G
Plas
 
DR
Martin
 
SM
Geha
 
RS
Nahm
 
MH
Chaplin
 
DD
Affinity maturation without germinal centres in lymphotoxin-alpha-deficient mice.
Nature
382
1996
462
45
Kato
 
J
Motoyama
 
N
Taniuchi
 
I
Takeshita
 
H
Toyoda
 
M
Masuda
 
K
Watanabe
 
T
Affinity maturation in Lyn kinase-deficient mice with defective germinal center formation.
J Immunol
160
1998
4788
46
Kabat
 
EA
Wu
 
TT
Perry
 
HM
Gottesman
 
KS
Foeller
 
C
Sequences of Proteins of Immunological Interest
ed 5
1991
US Department of Health and Human Services, Public Health Service, National Institutes of Health, NIH Publication No. 91-3242
Bethesda, MD

Author notes

Address reprint requests to Delin Zhu, PhD, Molecular Immunology Group, Tenovus Research Laboratory, Southampton University Hospitals Trust, Tremona Rd, Southampton SO16 6YD, UK; e-mail: dz1@soton.ac.uk.

Sign in via your Institution