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NEOPLASIA
From the Molecular Immunology Group, Tenovus
Laboratory, Southampton University Hospitals, Southampton,
England.
Primary diffuse large B-cell lymphomas (DLBCLs) are aggressive
tumors accounting for approximately 40% of B-cell malignancies. The
immunoglobulin (Ig) variable region genes have undergone rearrangement and are commonly somatically mutated. The majority show intraclonal variation which indicates that somatic mutation has continued after
transformation. Typically, cells of DLBCLs express Ig of a
single isotype, but there may be accompanying cells that express alternative isotypes. To probe the status of the isotype switch process
in DLBCL, 4 cases of tumor-derived constant region transcripts of all isotypes were investigated. Following the identification of the
VDJ sequences, the presence of the major isotype expected from
immunohistochemical analysis was confirmed at the RNA level. Another
3-4 alternative isotypes were revealed in all cases, some of which
could also be detected by immunohistochemistry. All cases were
somatically mutated with an intraclonal variation. In 2 cases there
were clearly distinct patterns of somatic mutation between isotypes,
which was consistent with independent evolution of the tumor
subpopulations. There was apparent clustering of mutational patterns
into either an IgMD/IgG3/IgA set or an IgG1/IgA set, indicating that
the switch to IgA can occur by different routes. Alternative isotype
expression is evident in DLBCL at both the RNA and protein levels. The
pattern of mutation indicates that switching is occurring in
subpopulations of the tumor after malignant transformation. The
findings support the concept that isotype switch events may be a
feature of DLBCL.
(Blood. 2000;96:2550-2556) Primary diffuse large B-cell lymphomas (DLBCLs) are
clinically aggressive lymphomas that account for approximately 40% of all B-cell malignancies. They were previously thought to represent malignant counterparts of germinal center B cells.1
However, recent analysis using microarrays suggests that there are at
least 2 subsets, with gene expression patterns similar either to
germinal center B cells or to activated B cells.2 DLBCLs
are composed of sheets of blasts, which typically express the B-cell
markers CD19, CD20, and CD22 and the surface immunoglobulin (Ig). The Ig variable region genes in DLBCL are commonly somatically
mutated,3-7 and this applies also to morphologically
distinct subsets of DLBCL that are sited in the central nervous system
(CNS) or skin and to T-cell-rich B-cell lymphoma.6,8-10
Somatic mutation can continue in B-cell lymphomas located in a germinal
center environment, thereby leading to intraclonal sequence
variation.3,11-14 In DLBCL, this feature has been observed
in some studies3,15,16 but not in all
studies.5 These differences may reflect the recently
observed heterogeneity of DLBCLs2 or the narrowing of
intraclonal heterogeneity, which can occur after
treatment.3,12
Isotype switch events in IgM+/IgD+ (IgM/D) B
cells generally occur by deletional recombination,17 with
sequences between the µ-switch region (S-µ) and the switch regions
upstream of the C- This point was investigated in a study of a subset of
lymphoplasmacytoid lymphoma, where tumor cells expressing IgM and IgG protein were evident.23 Transcript analysis of 5 cases
showed that both isotypes were clonally related. Interestingly, in 2 of
5 cases, where cells were apparently expressing both IgM and IgG
protein, somatic mutational patterns were divergent, which is
consistent with 2 separate clonally related tumor populations. This
demonstrated the danger of drawing conclusions from Ig phenotypic profiles. It also indicated that some tumor cells can undergo isotype switch and can continue to coexist with the nonswitched population.
In GC-derived lymphomas, such as follicular lymphomas (FLs) or DLBCLs,
there are cases in which the tumor cells express isotype proteins other
than IgM.24-26 We previously investigated 1 FL case and 2 cases of DLBCL at the transcript level3 and could detect clonally related alternative isotype transcripts. The evidence points
to the use of deletional switch mechanisms for the production of these
transcripts. Using fiber-FISH (fluorescence in situ hybridization) on a
group of FL patients, Vaandrager et al27 observed that there was no mechanism for class switching other than deletion, in
spite of detecting complex constant region rearrangements downstream of
the functional constant region on the productive allele. Tumors of GC
origin, such as Burkitt's lymphoma, can be induced to switch in vitro
by a deletional mechanism.28 However, there are also models in which one cell can produce more than one isotype
simultaneously, perhaps indicating RNA processing or
trans-splicing.29
In this study we investigated the nature of isotype switch variants in
primary DLBCL. We found evidence for class switching to a range of
isotypes with distinct somatic mutational patterns, which is consistent
with the existence of isotype switch variant subpopulations within
the tumor.
Clinical information and immunochemistry
The patients were staged according to the Ann Arbor
criteria31 (Table 1). Routine
flow cytometric analyses were available for 3 cases. Additionally,
sections were cut from paraffin-embedded material and stained using an
established indirect immunoperoxidase method with appropriate
controls.30,32 Primary monoclonal antibodies (mAbs) were
specific for IgD (Dako, High Wycombe, England) and IgM, IgG,
and IgA (M. Glennie, Tenovus, Southampton, England).
Identification of the tumor-related variable heavy chain region genes Double sets of 5 µm frozen tissue were cut in a cryostat. RNA was extracted with RNAzol (Cinna Biotecx Laboratories, Houston, TX). Reverse transcription was carried out with oligo-d(T)18 primer and Superscript reverse transcriptase (RT) (Gibco Life Technologies, Oxford, England) in a final volume of 20 µL. Both procedures followed the manufacturers' protocols. For VH gene analysis, 50 µL polymerase chain reactions (PCRs) were performed using 2 µL complementary DNA (cDNA), Taq, and buffer solutions from Qiagen (Qiagen, Crawley, England). Mixed 5'-oligonucleotide primers (5'-primers) specific for the VH leader or VH framework region 1 (FWR1) sequences33,34 were used together with a consensus 3'-JH primer (3'-primer).3,35 At least 2 separate PCRs were performed for each sample.The products were analyzed on agarose gels and purified with the Gene
Clean kit (Bio 101, Vista, CA). DNA was ligated into a pGEM-T vector
(Promega, Southampton, England), cloned into JM109 (Promega), and
sequenced with Big Dye, a GeneAmp 9600 PCR system, and an ABI Prism 377 DNA sequencer (PerkinElmer Biosystems, Warrington, England). M13
forward and reverse primers were used to sequence in both directions.
The Taq error rate was determined using a known plasmid
template from the leader to C-µ in a nested PCR. We sequenced 20 clones with an error rate of 0.2 of 1000 nucelotides. Sahota et al36 additionally assessed the Taq
error rate starting from a DNA template ( Identification of isotype transcripts For the identification of the tumor-derived isotype profiles, nested PCRs were performed. A touchdown protocol was used, starting with 65°C annealing and then reducing the annealing temperature by 1 degree per cycle to a final temperature of 59°C. This annealing temperature was maintained for a total of 20 cycles (first round) or 25 cycles (second round). Initial amplification from cDNA was carried out with the family-specific VH leader or FRW1 5'-primers together with an outer constant region 3'-primer.38,39 In case 5049, where the germline was identified as V4-34, analysis of isotype variants was carried out with a V4-34-specific FRW1 primer. Sequences for the external IgM primer, the primer pairs for IgG and IgA, and the external IgE primer are shown in Table 2. The IgG primers allow distinction between the IgG subclasses. For the secondary reaction, internal FRW1, FRW2, or FRW3 primers were used with an internal constant region 3'-primer. Where the secondary reactions failed with the consensus primers, patient-specific primers, based on the 3'-end of the VH gene segment and the first half of CDR3 (Table 2), were used together with 20 pmol/50 µL inner constant region 3'-primer. At least 2 separate PCR amplifications were performed for each constant region and biopsy. Denaturation, annealing, and extension were for 5, 5, and 15 seconds, respectively. Cloning and sequencing were performed as for the VH sequences. Products with a length of less than 150 nucleotides were ligated directly without purification to improve the efficiency of the ligation.
Histology and VH gene analysis All 4 cases were classified as primary DLBCL and had extensive replacement of the primary site by tumor cells. Predominant repeated VHDJH sequences with a CDR3 "clonal signature" were identified in all cases (Table 1), which indicated derivation from the tumor cells.40 Other sequences detected were individually distinct and likely to be derived from normal B cells. All tumor-related VH genes revealed a significant level of somatic mutation, with 89.2%-94.6% homology to the closest germline VH segment. The deduced amino acid sequences and the replacement/silent mutations of the dominant subclone of each case are summarized in Figure 1. No unequivocal D segment alignments could be made.41
Patterns of somatic mutation To investigate intraclonal variation in the tumor-derived sequences, multiple clones were analyzed. Detailed results for the alignment to VH and JH germlines for case 5334 are shown in Figure 2. The majority of somatic mutations were present in all clones. Additionally there were mutations found in only some subclones. When the same mutation was detected in more than one subclone (eg, in codon 8 or 28), it was unlikely to be related to Taq errors and was consistent with ongoing mutational activity. Some mutations were found in only one sequence. While for each individual mutation the possibility remains that the mutation might be due to Taq error, the overall level of mutations was 5.6, 14.7, and 11.8 mutations of 1000 nucleotides sequenced for cases 5126, 5184, and 5334, respectively, which was a 28-fold to 73-fold excess of the Taq error rate (see "Materials and methods"). Therefore, the large majority of the observed nucleotide changes are likely to represent intraclonal sequence variation. Case 5049 showed no evidence for ongoing mutation in 10 of 10 identical sequences from leader to JH, thereby initially implying that this case was clonally homogeneous. However, further amplification of 49 clonally related sequences from leader and FWR into the constant regions using nonnested and seminested PCR revealed the presence of intraclonal heterogeneity at a low level of 2.4 of 1000 nucleotides for all 59 sequences, approximately a 10-fold excess of the Taq error (Figure 3).
Transcripts for alternative isotypes Immunophenotypically, all cases were reported as IgM+. To assess the presence of isotype transcript tumor-derived VHDJH, clonal sequences linked to C-µ, C- , C- , C- , or C- were sought using frozen tissue.
For all 4 cases we were able to demonstrate the presence of transcripts
for IgM, IgD, IgG, and IgA (Table 3). Tumor-related IgE sequences were not detected, even though unrelated IgE sequences could be amplified. In case 5049, transcript analysis was
facilitated because the V4-34 gene allows the use of a
specific FWR1 primer. This was used in single-round PCRs together with outer constant region primers. To further probe the presence of transcripts for the IgG subclasses and to expand on the number of IgA
transcripts detected by the initial reactions, nested PCRs were
obtained with a CDR3-specific 5'-primer and inner constant region
primers. A total of 59 clonally related sequences were found. Figure 3
shows all subclones, with their observed frequency indicated to the
left of a particular sequence. We detected the µ-, -, -1-,
-3-, and -transcripts. Intraclonal heterogeneity was clearly
documented in these sequences, both within and between isotypes.
Assessment of the mutational patterns of different isotypes revealed
that the µ- and The
Analysis of Ig isotype protein expression Table 4 shows the immunophenotypic analyses performed. Flow cytometry at diagnosis identified only the dominant isotype, and minor populations of other isotypes were not sought at this stage. For the immunohistochemical analyses, the difficulties of using anti-Ig mAbs in paraffin-embedded material are well known, and the staining for alternative isotypes was only taken as positive when accompanied by a characteristic perinuclear pattern.30 Case 5049 expressed IgM/D/ by flow
cytometry, and immunohistochemistry also revealed a subpopulation of
IgG+ tumor cells, but no clear evidence for IgA. However,
transcripts for IgM, IgD, IgG, and IgA were detectable (Table 3).
Morphologically, about one-third of the cells expressed IgG protein,
implying that a subgroup of cells were double-positives for IgM/G. For
case 5126, tumor cells were  +, and it was possible to
use anti- as a background control. In addition to the major
IgM+ population, a few IgG+ and
IgA+ cells with characteristic staining patterns were
observed. Transcripts for IgM, IgD, IgG, and IgA, the latter only by
nested PCR, were detected. There were no flow cytometric data available
for case 5184. Immunohistochemistry showed that all cells were
-light chain restricted and expressed IgM. Additionally 30%
coexpressed IgD. Although protein expression was not detectable, we
were able to additionally detect IgG and IgA transcripts. In case 5334, initial phenotyping by flow cytometry had shown staining for IgM/D/. In the histochemical profile, only plasma cells showed convincing staining for IgM, IgG, and IgA, which is probably due to poor conservation of the specimen. At an RNA level we could detect transcripts for IgM, D, G, and A, only with a nested PCR
approach, which possibly reflected this reduced quality.
A consensus is beginning to emerge on the nature of
VH genes used by tumor cells in DLBCL. While
there has been some controversy concerning a possible bias in
VH gene usage and in the level of ongoing
somatic mutation,5 there now seems to be agreement that
there is no obvious bias and that most cases show evidence for
intraclonal variation.3,4,6,10,16 However, it is clear
from clinical course, morphology, and more recently from microarray
analysis that DLBCL is a heterogeneous disease.2 Expression of Ig is almost always retained by neoplastic B cells, but
some cases of DLBCL appear Ig In a previous investigation of 2 cases of IgM+ DLBCL, we
found the expected CDR3 C-µ transcripts and also detected CDR3
C- IgD transcripts were present in all cases, presumably arising from RNA
processing.44 More surprisingly, IgG and IgA transcripts were also detected in all cases. For IgG, it was possible from the
sequences to assign the subclass: 2 cases were IgG1 only; 1 case, IgG3
only; and 1 case, IgG1 and IgG3. There was no other IgG subclass
detected, and IgE was not found in these cases. Dominance of a switch
to IgG1 and IgG3 indicates a possible influence of IL-10, a cytokine
known to stimulate switching of CD40-activated naive B cells to these
isotypes in vitro.45,46 A similar parallel switching to
IgG1 and IgG3 has been observed in vivo in Lyme borreliosis, where
interferon (IFN)- The finding of clonally related IgMD, IgG1, IgG3, and IgA transcripts indicates that the tumor cells are heterogeneous in their response to switch stimuli and that only some cells have undergone the presumed deletional recombination events. In all cases the level of somatic mutation was quite high in all
isotypes, including IgMD, and although ongoing somatic mutational activity tends to blur mutational differences, there were clearly distinct patterns evident in the switch variants. The close similarity between IgMD and IgG3, especially present in case 5126 and confirmed in
case 5049, suggests direct switching, with little or no further accumulation of mutations. In contrast, there were markedly different mutational patterns between IgMD and IgG1, evident in case 5049 and
confirmed in case 5184, which suggests that the switch to IgG1 had
occurred prior to the acquisition of the majority of the mutations seen
in the major IgMD population. On the route to IgG1, mutational activity
continued, either in an unidentified IgMD sister cell or in the
IgG1-switched cells. The history of IgA transcripts is more complex
because it is most similar to the IgMD and IgG3 sequences (cases 5049 and 5126). However, there are common mutations between IgA and IgG1 in
cases 5334 and 5184 and in one set of sequences in case 5049. Although
the data are limited, there is a suggestion that IgA might be generated
by 2 routes, one via IgG3 and the other via IgG1. By either route, few
further mutations appear to accumulate. Control of the switch to IgA is
not completely understood and may vary with the location of the B cell.
Knockout mouse models have shown that switching in the mucosal site can
occur in the absence of T cells.48 Experiments in vitro
have indicated that the CD40 engagement of naive B cells is sufficient
to induce switching to IgA by the release of endogenous transforming growth factor (TGF)- In summary, events in DLBCL indicate that the tumor cell population is arrested at a stage where both somatic mutation and isotype switch events can continue after transformation. Most of the mutational activity appears to be over by the IgMD+ stage, although there may be a further low level following the isotype switch. The IgG isotype variants probably reflect an influence of TH1 cells. The variants appear functional at the RNA level, and protein expression is seen at least for some of the transcripts. Mutational patterns are consistent with the existence of subpopulations within the tumor and with parallel switch events to either IgG3 or IgG1, which can then switch to IgA. The findings do not necessarily reflect dysregulation, but rather that the tumor cells are responding to normal signals without being able to achieve a fully differentiated state.
The authors are grateful to Emeritus Professor Dennis Wright and Dr Bridget Wilkins, the Department of Histopathology, Southampton University, Southampton, England, for expert help in evaluating the tumor morphology and immunocytochemistry.
Submitted March 20, 2000; accepted June 6, 2000.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Christian H. Ottensmeier, Molecular Immunology Group, Tenovus Laboratory, Cancer Sciences Division, University of Southampton, Tremona Road, Southampton SO16 6YD, England; e-mail: cho{at}soton.ac.uk.
1.
Harris NL, Jaffe ES, Stein H, et al.
A revised European-American classification of lymphoid neoplasms: a proposal from the International Lymphoma Study Group.
Blood.
1994;84:1361-1392 2. Alizadeh AA, Eisen MB, Davis RE, et al. Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling. Nature. 2000;403:503-511[Medline] [Order article via Infotrieve].
3.
Ottensmeier CH, Thompsett AR, Zhu D, et al.
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.
1998;91:4292-4299 4. Kuppers 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. 1997;27:1398-1405[Medline] [Order article via Infotrieve].
5.
Hsu FJ, Levy R.
Preferential use of the VH4 Ig gene family by diffuse large-cell lymphoma.
Blood.
1995;86:3072-3082
6.
Aarts WM, Willemze R, Bende RJ, et al.
VH gene analysis of primary cutaneous B-cell lymphomas: evidence for ongoing somatic hypermutation and isotype switching.
Blood.
1998;92:3857-3864 7. Driessen A, Tierens A, Ectors N, et al. Primary diffuse large B cell lymphoma of the stomach: analysis of somatic mutations in the rearranged immunoglobulin heavy chain variable genes indicates antigen selection. Leukemia. 1999;13:1085-1092[Medline] [Order article via Infotrieve].
8.
Thompsett AR, Ellison DW, Stevenson FK, Zhu D.
V(H) gene sequences from primary central nervous system lymphomas indicate derivation from highly mutated germinal center B cells with ongoing mutational activity.
Blood.
1999;94:1738-1746
9.
Montesinos-Rongen M, Kuppers R, Schluter D, et al.
Primary central nervous system lymphomas are derived from germinal-center B cells and show a preferential usage of the V4-34 gene segment.
Am J Pathol.
1999;155:2077-2086
10.
Brauninger A, Kuppers R, Spieker T, et al.
Molecular analysis of single B cells from T-cell-rich B-cell lymphoma shows the derivation of the tumor cells from mutating germinal center B cells and exemplifies means by which immunoglobulin genes are modified in germinal center B cells.
Blood.
1999;93:2679-2687 11. Levy R, Levy S, Cleary ML, et al. Somatic mutation in human B-cell tumors. Immunol Rev. 1987;43-58. 12. 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. 1994;86:505-512[Medline] [Order article via Infotrieve].
13.
Zelenetz AD, Chen TT, Levy R.
Clonal expansion in follicular lymphoma occurs subsequent to antigenic selection.
J Exp Med.
1992;176:1137-1148 14. Stamatopoulos K, Kosmas C, Papadaki T, et al. Follicular lymphoma immunoglobulin kappa light chains are affected by the antigen selection process, but to a lesser degree than their partner heavy chains. Br J Haematol. 1997;96:132-146[Medline] [Order article via Infotrieve]. 15. Kume M, Suzuki R, Yatabe Y, et al. Somatic hypermutations in the VH segment of immunoglobulin genes of CD5-positive diffuse large B-cell lymphomas. Jpn J Cancer Res. 1997;88:1087-1093[Medline] [Order article via Infotrieve].
16.
Lossos IS, Okada CY, Tibshirani R, et al.
Molecular analysis of immunoglobulin genes in diffuse large B-cell lymphomas.
Blood.
2000;95:1797-1803 17. Lorenz M, Radbruch A. Developmental and molecular regulation of immunoglobulin class switch recombination. Curr Top Microbiol Immunol. 1996;217:151-169[Medline] [Order article via Infotrieve]. 18. Fais F, Sellars B, Ghiotto F, et al. Examples of in vivo isotype class switching in IgM+ chronic lymphocytic leukemia B cells. J Clin Invest. 1996;98:1659-1666[Medline] [Order article via Infotrieve].
19.
Sahota SS, Garand R, Mahroof R, et al.
V(H) gene analysis of IgM-secreting myeloma indicates an origin from a memory cell undergoing isotype switch events.
Blood.
1999;94:1070-1076
20.
Corradini P, Boccadoro M, Voena C, Pileri A.
Evidence for a bone marrow B cell transcribing malignant plasma cell VDJ joined to Cmu sequence in immunoglobulin (IgG)- and IgA-secreting multiple myelomas.
J Exp Med.
1993;178:1091-1096
21.
Billadeau D, Ahmann G, Greipp P, Van Ness B.
The bone marrow of multiple myeloma patients contains B cell populations at different stages of differentiation that are clonally related to the malignant plasma cell.
J Exp Med.
1993;178:1023-1031 22. Billadeau D, Greipp P, Ahmann G, Witzig T, Van Ness B. Detection of B-cells clonally related to the tumor population in multiple myeloma and MGUS. Curr Top Microbiol Immunol. 1995;194:9-16[Medline] [Order article via Infotrieve].
23.
Sahota SS, Garand R, Bataille R, Smith JA, Stevenson FK.
VH gene analysis of clonally-related IgM and IgG from human lymphoplasmacytoid B-cell tumors with CLL features and high serum monoclonal IgG.
Blood.
1998;91:238-243 24. Cossman J, Neckers LM, Hsu S, Longo D, Jaffe ES. Low-grade lymphomas: expression of developmentally regulated B-cell antigens. Am J Pathol. 1984;115:117-124[Abstract]. 25. Kadowaki N, Hayashi T, Amakawa R, et al. Class switch recombination of the immunoglobulin heavy chain gene frequently occurs in B-cell lymphomas associated with rearrangement of the BCL2 gene. Int J Hematol. 1995;61:69-75[Medline] [Order article via Infotrieve]. 26. Warnke R, Miller R, Levy R. Immunologic phenotype in large-cell lymphoma [letter]. N Engl J Med. 1980;303:1303[Medline] [Order article via Infotrieve].
27.
Vaandrager JW, Schuuring E, Kluin-Nelemans HC, et al.
DNA fiber fluorescence in situ hybridization analysis of immunoglobulin class switching in B-cell neoplasia: aberrant CH gene rearrangements in follicle center-cell lymphoma.
Blood.
1998;92:2871-2878
28.
Cerutti A, Zan H, Schaffer A, et al.
CD40 ligand and appropriate cytokines induce switching to IgG, IgA, and IgE and coordinated germinal center and plasmacytoid phenotypic differentiation in a human monoclonal IgM+IgD+ B cell line.
J Immunol.
1998;160:2145-2157 29. Fujieda S, Lin YQ, Saxon A, Zhang K. Multiple types of chimeric germ-line Ig heavy chain transcripts in human B cells: evidence for trans-splicing of human Ig RNA. J Immunol. 1996;157:3450-3459[Abstract].
30.
Cuevas EC, Bateman AC, Wilkins BS, et al.
Microwave antigen retrieval in immunocytochemistry: a study of 80 antibodies.
J Clin Pathol.
1994;47:448-452 31. Rosenberg SA. Validity of the Ann Arbor staging classification for the non-Hodgkin's lymphomas. Cancer Treat Rep. 1977;61:1023-1027[Medline] [Order article via Infotrieve]. 32. Hsu SM, Raine L, Fanger H. The use of antiavidin antibody and avidin-biotin-peroxidase complex in immunoperoxidase technics. Am J Clin Pathol. 1981;75:816-821[Medline] [Order article via Infotrieve]. 33. Segal GH, Jorgensen T, Scott M, Braylan RC. Optimal primer selection for clonality assessment by polymerase chain reaction analysis, II: Follicular lymphomas. Hum Pathol. 1994;25:1276-1282[Medline] [Order article via Infotrieve]. 34. Segal GH, Jorgensen T, Masih AS, Braylan RC. Optimal primer selection for clonality assessment by polymerase chain reaction analysis, I: Low grade B-cell lymphoproliferative disorders of nonfollicular center cell type. Hum Pathol. 1994;25:1269-1275[Medline] [Order article via Infotrieve].
35.
Diss TC, Pan L, Peng H, Wotherspoon AC, Isaacson PG.
Sources of DNA for detecting B cell monoclonality using PCR.
J Clin Pathol.
1994;47:493-496
36.
Sahota SS, Davis Z, Hamblin TJ, Stevenson FK.
Somatic mutation of bcl-6 genes can occur in the absence of VH mutations in chronic lymphocytic leukemia.
Blood.
2000;95:3534-3540 37. Cook GP, Tomlinson IM. The human immunoglobulin V(H) repertoire. Immunol Today. 1995;16:237-242[Medline] [Order article via Infotrieve].
38.
Sahota SS, Leo R, Hamblin TJ, Stevenson FK.
Ig V(H) gene mutational patterns indicate different tumor cell status in human myeloma and monoclonal gammopathy of undetermined significance.
Blood.
1996;87:746-755 39. Van der Stoep N, Korver W, Logtenberg T. In vivo and in vitro IgE isotype switching in human B lymphocytes: evidence for a predominantly direct IgM to IgE class switch program. Eur J Immunol. 1994;24:1307-1311[Medline] [Order article via Infotrieve].
40.
Hawkins RE, Zhu D, Ovecka M, et al.
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.
1994;83:3279-3288 41. 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. 1997;270:587-597[Medline] [Order article via Infotrieve]. 42. Chothia C, Lesk AM, Gherardi E, et al. Structural repertoire of the human V(H) segments. J Mol Biol. 1992;227:799-817[Medline] [Order article via Infotrieve]. 43. Warnke R, Miller R, Grogan T, et al. Immunologic phenotype in 30 patients with diffuse large-cell lymphoma. N Engl J Med. 1980;303:293-300[Abstract]. 44. Rothman P, Coffman RL. Immunoglobulin heavy chain class-switching. In: Herzenberg LA,Weir DM,Herzenberg L,Blackwell C, eds. Weir's Handbook of Experimental Immunology. Vol I. 5th ed. Cambridge, MA: Blackwell Science; 1997:19.11-19.14.
45.
Briere F, Servet-Delprat C, Bridon JM, Saint-Remy JM, Banchereau J.
Human interleukin 10 induces naive surface immunoglobulin D+ (sIgD+) B cells to secrete IgG1 and IgG3.
J Exp Med.
1994;179:757-762
46.
Malisan F, Briere F, Bridon JM, et al.
Interleukin-10 induces immunoglobulin G isotype switch recombination in human CD40-activated naive B lymphocytes.
J Exp Med.
1996;183:937-947 47. Widhe M, Ekerfelt C, Forsberg P, Bergstrom S, Ernerudh J. IgG subclasses in Lyme borreliosis: a study of specific IgG subclass distribution in an interferon-gamma-predominated disease. Scand J Immunol. 1998;47:575-581[Medline] [Order article via Infotrieve]. 48. Salvi S, Holgate ST. Could the airway epithelium play an important role in mucosal immunoglobulin A production? Clin Exp Allergy. 1999;29:1597-1605[Medline] [Order article via Infotrieve].
49.
Zan H, Cerutti A, Dramitinos P, Schaffer A, Casali P.
CD40 engagement triggers switching to IgA1 and IgA2 in human B cells through induction of endogenous TGF-beta: evidence for TGF-beta but not IL-10-dependent direct S mu-S alpha and sequential S mu-S gamma, S gamma-S alpha DNA recombination.
J Immunol.
1998;161:5217-5225
© 2000 by The American Society of Hematology.
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  F. Forconi, S. S. Sahota, D. Raspadori, M. Ippoliti, G. Babbage, F. Lauria, and F. K. Stevenson Hairy cell leukemia: at the crossroad of somatic mutation and isotype switch Blood, November 15, 2004; 104(10): 3312 - 3317. [Abstract] [Full Text] [PDF]
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F. Forconi, S. S. Sahota, D. Raspadori, C. I. Mockridge, F. Lauria, and F. K. Stevenson Tumor cells of hairy cell leukemia express multiple clonally related immunoglobulin isotypes via RNA splicing Blood, August 15, 2001; 98(4): 1174 - 1181. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
, and CD45RO
-globin) in tumor
samples with a similar rate of error at 0.26 of 1000 nucleotides.
The sequences were analyzed with Mac Vector 4.5.3 software (Oxford
Molecular, England), and aligned to Entrez and
V-BASE.
