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Splenic Marginal Zone Lymphoma with Villous Lymphocytes Shows On-Going Immunoglobulin Gene Mutations

2003; Elsevier BV; Volume: 162; Issue: 2 Linguagem: Inglês

10.1016/s0002-9440(10)63862-x

1525-2191

Anne Tierens, Jan Delabie, Agnieszka Małecka, Junbai Wang, Alicja Gruszka, Daniel Catovsky, Estella Matutes,

Monoclonal and Polyclonal Antibodies Research

Splenic marginal zone lymphoma (also splenic lymphoma with villous lymphocytes) is a B-cell non-Hodgkin's lymphoma with a characteristic morphology and phenotype. We studied the pattern of somatic hypermutation of the rearranged immunoglobulin heavy chain genes on 23 cases and have correlated these data with survival as well as immunophenotypic and genetic characteristics of the cases. Two-thirds of the cases show immunoglobulin gene mutations, half of which show evidence of antigen selection, whereas one-third of the cases show no significant mutations. On-going mutation, a feature characteristic of follicular lymphoma, was demonstrated in all six cases randomly selected for this analysis, including one case with a low number of mutations (<2%). No statistical significant correlation was found between immunoglobulin mutation status and clinical, immunophenotypic, or genetic characteristics. Our results demonstrate that on-going somatic hypermutation is a prominent feature of splenic marginal zone lymphoma with circulating villous lymphocytes. On-going somatic hypermutation has previously been demonstrated in extra-nodal and nodal marginal zone lymphoma. Our results indicate that marginal zone lymphomas at different anatomical localizations may derive from a similar B-cell subset. Splenic marginal zone lymphoma (also splenic lymphoma with villous lymphocytes) is a B-cell non-Hodgkin's lymphoma with a characteristic morphology and phenotype. We studied the pattern of somatic hypermutation of the rearranged immunoglobulin heavy chain genes on 23 cases and have correlated these data with survival as well as immunophenotypic and genetic characteristics of the cases. Two-thirds of the cases show immunoglobulin gene mutations, half of which show evidence of antigen selection, whereas one-third of the cases show no significant mutations. On-going mutation, a feature characteristic of follicular lymphoma, was demonstrated in all six cases randomly selected for this analysis, including one case with a low number of mutations ( 30% of B cells stained with the mAb. Fluorescent in situ hybridization analyses were performed using standard methods. Mononuclear cells were fixed in methanol:glacial acetic acid (3:1) and stored at −20°C until needed. The slides were incubated with RNase A (0.1 mg/ml, Sigma, St. Louis, MO) at 37°C for 1 hour, and washed in 2× standard saline citrate (SSC) at room temperature for 5 minutes. The cells were then digested with pepsin (0.1 mg/ml, Sigma) at 37°C for 10 minutes, rinsed in phosphate-buffered saline at room temperature for 5 minutes, and in 2× SSC at 37°C for 30 minutes. Afterward, the slides were dehydrated in 70%, 90%, and 100% ethanol and dried. Subsequently, denaturation was performed on a dry-heat block at 72°C using a denaturation solution (70% formamide, 2× SSC, 0.05 mol/L sodium phosphate buffer, pH 7.0). The slides were then quickly rinsed in 2× SCC, dehydrated, and dried before hybridization. A biotinylated centromeric probe for chromosome 3 was obtained from Dr. Janet Shipley (ICR, Sutton, Surrey, UK). A p53 locus-specific probe (LSI p53 Spectrum Orange; Vysis, Richmond, UK) was used in combination with a probe specific for the chromosome 17 centromere (CEP17 Spectrum Green, Vysis). The probes were prepared and denatured according to the manufacturer's instructions. The hybridization was performed overnight at 37°C. The posthybridization procedure consisted of three washes in 1× SCC at 45°C, followed by three washes in 0.1× SSC at 60°C and one wash in 4× SSC/Triton X-100 at room temperature. The slides were then dehydrated in 70%, 90%, and 100% ethanol; dried; and mounted with Vectashield mounting medium with 4,6-diamidino-2-phenylindole (Vector Labs, Peterborough, UK). To detect the biotinylated probes, the slides were additionally incubated with avidin-fluorescein isothiocyanate (Vector Labs), washed in 4× SSC/Triton X-100, dehydrated, and mounted as above. To be able to study antigen selection, only expressed rearranged immunoglobulin genes have been studied. Poly A+ mRNA was isolated from either mononuclear cell suspensions of spleen tissues, peripheral blood, or bone marrow using Dynabeads Oligo (dT)25 following the manufacturer's recommendations (Dynal, Oslo, Norway). Five μL of the eluted mRNA were reverse-transcribed using Thermoscript reverse transcriptase and Oligo (dT) (Life Technologies, Inc., Grand Island, NY). Ten μL of a mastermix [0.01 mol/L dithiothreitol, 40 U of RNase out, 2 mmol/L dNTP mix, 15 U of Thermoscript reverse transcriptase in reverse transcriptase-polymerase chain reaction (PCR buffer)] was added to 10 μl of diethyl pyrocarbonate H2O containing 2 μmol/L of Oligo (dT)25 and 5 μl of mRNA. The samples were incubated at 60°C for 60 minutes and subsequently at 85°C for 5 minutes to stop the reaction. Finally, 1 μl of RNase H was added to the samples that were incubated for 20 minutes at 37°C. Five μL of cDNA was used to amplify the rearranged Ig heavy (H) chain genes using a mixture of six framework (FR) 1 IgH variable gene segment (VH) family-specific and three joining (JH) primers.21Tierens A Delabie J Pittaluga S Driessen A De Wolf-Peeters C Mutation analysis of the rearranged immunoglobulin heavy chain genes of marginal zone cell lymphomas indicates an origin from different marginal zone B lymphocyte subsets.Blood. 1998; 91: 2381-2386PubMed Google Scholar, 24Küppers R Zhao M Hansmann ML Rajewsky K Tracing B cell development in human germinal centres by molecular analysis of single cells picked from histological sections.EMBO J. 1993; 12: 4955-4967Crossref PubMed Scopus (590) Google Scholar Forty μL of a mastermix (200 μmol/L dNTPs, 2.5 mmol/L MgCL2, 100 nmol/L of each primer in PCR buffer, 5 μl of cDNA) was heated at 94°C for 10 minutes before adding 1.5 U Taq polymerase diluted in 10 μl of dH2O. The PCR conditions were as follows: 1 cycle at 95°C for 2 minutes, 59°C for 4 minutes, 72°C for 80 seconds, followed by 34 cycles at 95°C for 90 seconds, 59°C for 30 seconds, 72°C for 80 seconds, and a final cycle at 72°C for 5 minutes. An aliquot of 10 μl of PCR product was size fractionated on a agarose gel 1.5% in 1× TBE buffer. The gel bands containing the monoclonal IgH products were excised and purified using the Concert PCR purification kit (Life Technologies, Inc.). The purified PCR products were cloned using pGEM-T easy vector system (Promega, Madison, WI). Purified DNA from at least five clones per case were sequenced on both strands using the DNA sequencing kit (PE Applied Biosystems, Foster City, CA) and Universal M13 primers (Life Technologies). Only those cases in which identical sequences were obtained from the majority of clones, were included in the study and further analyzed. For the study of on-going somatic hypermutation, at least 20 clones were sequenced per selected case. Mac Vector 5.0 sequence analysis software (Oxford Molecular group Inc., Campbell, CA) was used for sequence analysis. Sequences were aligned with germline sequences derived from V Base database (V Base Sequence Directory, Tomlinson and colleagues, MRC Center for Protein Engineering, Cambridge, UK). The binomial distribution model was used to calculate the probability that the number of R and S mutations in the FR and complementarity determining region (CDR) sequences occurred by chance only.25Chang WC Casali P The CDR1 sequences of a major proportion of germline Ig VH genes are inherently susceptible to amino-acid replacement.Immunol Today. 1994; 15: 367-373Abstract Full Text PDF PubMed Scopus (374) Google Scholar This method gives a good estimation of antigen selection for the purpose of this study, although mutational hot-spots such as the targeting of RGYW sequences, characteristic for somatic hypermutation, are not taken into account.26Lossos IS Tibshirani R Narasimhan B Levy R The inference of antigen selection on Ig genes.J Immunol. 2000; 165: 5122-5126PubMed Google Scholar, 27Dunn-Walters DK Spencer J Strong intrinsic biases towards mutation and conservation of bases in human IgVH genes during somatic hypermutation prevent statistical analysis of antigen selection.Immunology. 1998; 95: 339-345Crossref PubMed Scopus (50) Google Scholar A P value of ≤0.05 was considered as statistically significant. The inherent susceptibility of R mutations of the CDRs and FRs have been calculated for each of the identified germline genes and is based on the chances of the occurrence in each codon of an amino acid replacement given any single nucleotide change not resulting in a termination codon. The likelihood of the occurrence of on-going somatic mutations versus Taq polymerase-induced errors in clonally-related sequences was calculated using the chi-square test. For the calculation, only unique on-going mutations in the clonally related sequences were counted. The rate of Taq polymerase errors was calculated by repeatedly amplifying and sequencing a cloned known germline Ig sequence using the same PCR conditions as described above. The error rate was determined as 0.3 per 1000 bp. Kaplan-Meier life tables were used to analyze the survival of the mutated and nonmutated SMZL cases. Two cutoff values, 98% and 99%, respectively, for the percentage of sequence difference with respect to the germline VH gene sequence were tested for their discriminative value. Indeed, sequence differences can be because of as yet uncharacterized polymorphisms and may not necessarily indicate somatic hypermutation. Differences between survival curves were determined according to the log-rank test. The chi-square test was used to analyze differences in clinical, immunophenotypic, and genetic features between mutated and unmutated cases. A summary of the clinical, immunophenotypic, and genotypic features of the cases is given in Table 1. The median age of the 23 patients that proved informative for this study was 67 years (range, 42 to 84 years) and male:female ratio 1.1. The median lymphocyte count was 19 × 109/L (range, 2.3 to 410) and all had a circulating clonal B-cell population as assessed by immunophenotyping. All patients presented with splenomegaly, but only a minority presented with lymphadenopathy and/or hepatomegaly.Table 1Summary of the Clinical Features, Immunophenotype, and Genotype of the SMZL CasesImmunophenotypeCaseSex/age (year)κ/λCD5CD23Cytogenetics and FISH for chromosome 3, p53, t(11;14)WBC (109/L)SM/LASPxDead/alive1M/68λ−−FISH: diploid 3, diploid p53ndSM+/LA−−No follow up2M/70κ+NDFISH: NDndnd−Dead, 12 months3F/65λ−−FISH: diploid 3, diploid p5314SM+/LA−−Alive, 72 months4M/84κ+/−−Complex karyotype/FISH: diploid 3, diploid p53410SM+/LA−−Dead, 10 months5M/75κ+−t(2;7)(p12; q21, +12; FISH: diploid 3, del p5346SM+/LA−+Alive, 24 months6M/NKλ+NDFISH: ND5SM+/LA−+Alive, 14 months7F/66κ−+FISH: ND13.9SM+/LA−+Alive, 5 months8F/49κ−+FISH: diploid 3, diploid p5392SM+/LA−NDNo follow up9M/72κ−−FISH: diploid 3, diploid p53199SM+/LA+NDNo follow up10M/56κ−−FISH: diploid 3, diploid p5311.8SM+/LA−+Alive, 72 months11M/NKκ−−FISH: ND18SM+/LA−+Dead, 60 months12F/74κ−−FISH: diploid 3, diploid p539.6SM+/LA−NDNo follow up13M/48κ−−FISH: diploid 3, del p532.3SM+/LA−+Alive, 48 months14M/42λ−−FISH: diploid 3, diploid p5320SM+/LA−−Alive, 84 months15F/73κ−−FISH: diploid 3, diploid p5342SM+/LA−NDNo follow up16F/48κ−−FISH: +3, diploid p535.5SM+/LA−+Alive, 84 months17M/50κ−−FISH: ND30SM+/LA−+Alive, 144 months18F/70κ+/−−FISH: diploid 3, diploid p5322.7SM+/LA++Alive, 48 months19F/67κ−−FISH: diploid 3, diploid p5320SM+/LA−+Alive, 12 months20F/74λ+−FISH: ND30SM+/LA−+Dead21F/76κ+−FISH: diploid 3, del p5338SM+/LA−+Dead, 32 months22F/66κ−−FISH: diploid 3, diploid p534.9SM+/LA−NDNo follow up23M/66λ−−FISH: NDndSM+/LA−+No follow upND, Not determined; NK, not known; SM, splenomegaly; LA, lymphadenopathy; SPx, splenectomy. Open table in a new tab ND, Not determined; NK, not known; SM, splenomegaly; LA, lymphadenopathy; SPx, splenectomy. All patients had circulating villous lymphocytes as assessed on May-Grünwald-Giemsa-stained peripheral blood smears. Light chain restriction of the B cells was demonstrated in all cases. The majority of the cases expressed FMC7 and were strongly positive for surface CD22 and/or CD79b. A minority of the cases expressed CD23 and less than one-third expressed CD5. According to the scoring system for the diagnosis of CLL6Matutes E Owusu-Ankomah K Morilla R Garcia-Marco J Houlihan A Que TH Catovsky D The immunological profile of B-cell disorders and proposal of a scoring system for the diagnosis of CLL.Leukemia. 1994; 8: 1640-1645PubMed Google Scholar all cases had scores ranging from 0 to 2. Trisomy 3 was demonstrated in one of the cases. Fluorescent in situ hybridization analysis showed a p53 gene deletion in three cases, one of which was associated with overexpression of the protein and a mutation in the p53 gene. Conventional karyotyping demonstrated a t(2;7)(p12;q21) in one patient and a complex karyotype in another. In 23 of 29 cases a definitive monoclonal IgH gene rearrangement could be identified. The lack of sufficient numbers of tumor cells in the samples or the lack of amplification of the neoplastic rearranged IgH sequences because of mutation, is likely the cause of negative results in six cases. The sequence analysis of the expressed rearranged IgH genes is summarized in Table 2. All sequences are available from GenBank under accession numbers AJ487485 to AJ487510. SMZL preferentially rearranged genes of the VH1 and VH3 families (21 of 23 cases). There was a random use of VH3 family genes, but one member of the VH1 family gene, VH1-2, was used in five of the cases. The IgH diversity gene segment (DH) gene could be identified in 18 of 23 cases. In most of the cases, homology of at least 10 consecutive nucleotides with the closest germline was retained for the DH gene assignment.28Corbett 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. 1997; 270: 587-597Crossref PubMed Scopus (249) Google Scholar In the remaining cases, a potential DH gene was identified when using a less strict criterium of a minimum of six successive matches or seven successive matches interrupted by one mismatch. No preferential rearrangement involving any of the DH genes was seen. In 50% of the cases JH4 family genes were used, whereas the other 50% rearranged either the JH6 gene and, to a lesser extent, JH3 and JH5 family genes.Table 2Sequence and Mutation Analysis of Ig VH Genes in SMZLCaseDH germlineJH germlineClosest VH germline% IdentityR/S observedP(R)P(S)Conclusion1D2-2JH63-30-3100%FR0/0NANANACDR0/0NANA2D3-10JH61–8100%FR0/0NANANACDR0/0NANA3NIJH51–46100%FR0/0NANANACDR0/0NANA4D2-15JH43–33100%FR0/0NANANACDR0/0NANA5D2-2/D6-13JH43–21100%FR0/0NANANACDR0/0NANA6D3-10JH41–299.1%FR0/1NANANACDR1/0NANA7D3-9JH51–299.1%FR2/0NANANACDR2/0NANA8D6-6JH43–798.6%FR2/00.4030.624RandomCDR0/10.4270.1369NIJH45–5198.3%FR1/00.1380.616RandomCDR1/20.1190.59810D2-21JH32–598.0%FR2/10.2590.407RandomCDR1/00.4090.16611D3-22JH61–296.9%FR3/10.2400.394RandomCDR3/00.1600.65412D6-13JH51–294.1%FR4/30.0500.204RandomCDR5/10.1130.36413D6-13JH63–1195.6%FR2/60.0230.002Ag selectionCDR0/20.0640.07614NIJH33–2392.4%FR4/40.0070.160Ag selectionCDR7/20.0450.21015D1-14JH43–5394.4%FR2/30.0080.193Ag selectionCDR7/00.0080.47316D2JH43–796.4%FR2/40.0660.019Ag selectionCDR1/10.2710.28117D1-7JH41–297.3%FR2/20.1870.296Ag selectionCDR2/00.2870.04118D3-9JH43–2191.2%FR5/50.0090.09Ag selectionCDR6/30.1540.41319NIJH61–1893.2%FR4/10.0230.216Ag selectionCDR6/40.0850.00820NIJH31–6994.6%FR3/40.0210.083Ag selectionCDR4/10.1740.37321D3-9JH43–1190.1%FR10/10.1160.095Ag selectionCDR10/10.0140.36122D3-3JH43–4888.0%FR5/15<0.001<0.001Ag selectionCDR7/00.1690.21523D3-3JH33–3090.5%FR5/50.0030.137Ag selectionCDR9/20.0080.254VH, IgH variable gene segment; DH, IgH diversity gene segment; JH, IgH joining gene segment; % identity, % homology of the rearranged VH gene with the closest germline; NI, not identified; R, replacement mutations; S, silent mutations; FR, framework regions; CDR, complementarity determining regions; p (R), probability that excess or scarcity of the R mutations in the VH gene CDRs or FRs resulted from chance only; p (S), probability that excess or scarcity of the S mutations in the VH gene CDRs or FRs resulted from chance only; NA, not applicable/not analyzed. Open table in a new tab VH, IgH variable gene segment; DH, IgH diversity gene segment; JH, IgH joining gene segment; % identity, % homology of the rearranged VH gene with the closest germline; NI, not identified; R, replacement mutations; S, silent mutations; FR, framework regions; CDR, complementarity determining regions; p (R), probability that excess or scarcity of the R mutations in the VH gene CDRs or FRs resulted from chance only; p (S), probability that excess or scarcity of the S mutations in the VH gene CDRs or FRs resulted from chance only; NA, not applicable/not analyzed. Nucleotide sequence analysis of the VH genes, illustrated in Table 2, allowed to distinguish two groups of SMZL as follows: those with 99% or 100% homology to germline genes in the database. We used the 99% cutoff value because polymorphisms and PCR errors cannot be excluded above this value. As case 7 illustrates (see further), this cutoff level is not too high. In 7 of 23 cases, IgVH genes were in germline or near-germline configuration, whereas in 16 SMZL cases, IgVH genes were somatically mutated. The homology with the closest identified germline VH gene ranged from 88 to 99%. In addition, we studied the mutation pattern of somatic hypermutations to investigate whether the lymphoma cells showed features of antigen selection. This analysis is based on the fact that as a result of antigen-selective pressure, the ratio of amino acid replacement (R) to silent mutations (S) is higher than expected in the CDR regions, which is consistent with the need to provide the best fit for the antigen. In contrast, the R/S ratio is lower in the FR regions to conserve the antibody structure. The distribution of R and S mutations in CDR and FR regions of the tumor-derived VH genes is summarized in Table 2. P values of ≤0.05 according to the binomial distribution model were considered statistically significant of antigen-selective pressure. A random distribution of somatic hypermutation was observed in 5 of 16 cases suggesting that the tumor cells derived from nonselected memory B cells. In 11 of 16 cases the pattern of R or S mutations was indicative of antigen-selective pressure. T