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NEOPLASIA
From Molecular Immunology Group, Tenovus Laboratory,
Southampton University Hospitals, Southampton, United Kingdom; and
Cattedra e U.O. di Ematologia, Ospedale A. Sclavo, Universita'
degli Studi di Siena, Siena, Italy.
Hairy cell leukemia (HCL) derives from a mature B cell and
expresses markers associated with activation. Analysis of
immunoglobulin variable region genes has revealed somatic
mutation in most cases, consistent with an origin from a cell that has
encountered the germinal center. One unusual feature of hairy cells
(HCs) is the frequent expression of multiple immunoglobulin
heavy chain isotypes, with dominance of immunoglobulin (Ig)-G3, but
only a single light chain type. The origin and clonal relationship of
these isotype variants have been unclear. In order to probe the isotype
switching status of HCL, RNA transcripts of
VHDJH- constant region sequences from 5 cases
of typical HCL, all expressing multiple surface immunoglobulin isotypes, were analyzed. Tumor
VHDJH-Cµ sequences were
identified and found to be somatically mutated (range, 1.4% to 6.5%),
with a low level of intraclonal heterogeneity. Additional
immunoglobulin isotypes of identical VHDJH
sequence were also identified, including IgD (5 of 5), IgG3 (5 of 5),
IgG1 (3 of 5), IgG2 (2 of 5), IgA1 (4 of 5), and IgA2 (1 of 5).
Derivation of multiple isotypes from individual cells was demonstrated
by analyzing transcripts in single sorted cells from one patient, with
evidence for coexistence of isotype variants in 10 of 10 cells. These
findings indicate that clonally related multiple isotypes coexist in
single HCs, with individual isotypes presumably generated via RNA
splicing. Production of IgG3 appears common, but IgG1, IgG2, IgA1, and
IgA2 also arise, indicating a continuing influence of a directed
process on the tumor clone. These HCs appear to be arrested at the
point of isotype switch, where RNA processing may precede deletional recombination.
(Blood. 2001;98:1174-1181) Hairy cell leukemia (HCL) is a relatively rare
leukemia of B-cell origin, which derives its name from the
characteristic cytoplasmic projections of the pathognomonic cell.
Clinically, HCL is characterized by pancytopenia with monocytopenia and
splenomegaly. Hairy cells (HCs) circulate in the blood and infiltrate
the bone marrow. Characteristically, HCs populate the splenic red pulp,
and eventually the white pulp atrophies and is replaced, but lymph
nodes are generally not involved.1 HCs express FMC7, a
normal B-cell activation marker, and commonly express the The status of the immunoglobulin genes provides an indicator of the
maturation stage reached by B-cell tumors.5 Naive B cells
prior to antigen encounter have unmutated V genes, while B cells that
have entered the site of somatic mutation, generally considered to be
in the germinal center of the lymph node, are likely to have
accumulated somatic mutations.6 For B-cell tumors, the
mutational status can have clinical relevance, as found in chronic
lymphocytic leukemia (CLL) where the approximately 50% of cases with
unmutated VH genes have a significantly worse
prognosis.7,8 The majority of B-cell tumors show evidence
of somatic mutation, and those located in germinal center sites
commonly continue to accumulate mutations after
transformation.9-11 However, tumors of more mature B
cells, such as multiple myeloma, have VH genes with mutated
but homogeneous sequences, indicative of silencing of mutational
activity at the postfollicular stage.12,13
Isotype switching events can clearly take place in the germinal
center environment,14 but normal IgM+
memory cells can also exit from this site, circulate in the
blood,15 and move to the marrow.16 In
general, B-cell tumors have reflected the known sequence of isotype
switching events in normal cells and express IgM(IgD) or, following
deletional recombination, a downstream isotype.17 However,
immunophenotypic studies have indicated dual isotype expression in some
B-cell tumors, and switching events leading to additional isotypes have
been confirmed by analysis of RNA transcripts in CLL18-20
and in diffuse large B-cell lymphoma (DLBCL).21 In
myeloma, where the tumor clone has usually undergone isotype switching,
and where there is no phenotypic evidence for IgM+
precursor cells, transcripts of CDR3-Cµ have also been
detected.22,23 Clearly B-cell tumor clones can include
cells at different stages of both somatic mutation and isotype switching.
Analysis of functionally rearranged VH gene segments in HCL
has indicated that most sequences contain somatic mutations consistent with a cell of origin that has encountered the germinal
center.24 The level of mutation in the functional
V The most unusual feature of HCs is expression of multiple
immunoglobulin isotypes on the tumor cell surface in about 40% of cases.28 Although preferential expression of IgG3 has been
documented,29 multiple isotype expression on HCs has been
reported previously.30-33 It was initially suggested that
this may be the result of passively adsorbed polyclonal immunoglobulin,
since HCs have avid Fc receptors,34-36 but
rosetting techniques and the use of F(ab')2
reagents have confirmed expression of multiple immunoglobulin isotypes
in HCL.37
An analysis of the clonal relatedness of the multiple isotypes
expressed was made by generating heterohybridomas from HCs by fusion to
a mouse myeloma cell line.38 It was found by Southern blotting that hybridomas contained the tumor VH
rearrangement, and that individual hybridomas secreted IgM, IgG, or
IgA, indicating that the tumor clone was undergoing isotype
switching.38 However, this approach and the possible
perturbing effect of heterohybridization did not allow further insight
into the production of the multiple isotypes.38 Since
conventional isotype switching involves intrachromosomal recombination,
with looping out and deletion of intervening constant-region genes,
expression of multiple isotypes within a tumor clone is exceptional.
Either the clone is heterogeneous, with subsets undergoing a switch to
different isotypes, or processing of RNA transcripts to generate
different isotypes within the same cell is occurring.39 Our analysis of transcripts of 5 cases of HCL in which all cells express multiple immunoglobulin isotypes appears consistent with the latter.
Patients
Phenotypic analysis
Identification and analysis of the tumor-related
VHDJH-Cµ and
VHDJH-C For identification of tumor VH-Cµ
transcripts, a mixture of 5'-primers specific for each of the
VH leader sequences of VH1 through
VH7 families (VH leader mix)11 was
used together with a 3'-primer specific for Cµ
(Cµ16) (Table 1). In all
cases, polymerase chain reaction (PCR) was carried out in a final
volume of 50 µL with 20 pmol each primer, 50 µmol deoxynucleotide triphosphates (dNTPs), and 2.5 U Taq DNA polymerase with
reaction buffer (Qiagen, Crawley, United Kingdom). Amplification
consisted of an initial denaturation step of 5 minutes at 94°C,
followed by 30 cycles of 94°C for 1 minute, 56°C for 1 minute, and
72°C for 1 minute, with a final extension step of 5 minutes at
72°C. In order to prevent contamination, the previously described
general procedure were followed, including a control for each PCR with no added template to check for any contaminant.40 For the
identification of VH-C
Amplified products were run on 1.5% to 2% agarose gel and purified with the Gene Clean III kit (Bio 101, Vista, CA). DNA was ligated into pGEM-T vector (Promega, Southampton, United Kingdom). Cloning was performed with Escherichia coli strain JM109 (Promega), and sequencing was performed with the Big Dye sequencing reaction kit, a GeneAmp 9600 PCR system, and an ABI Prism 377 DNA sequencer (PerkinElmer, Warrington, United Kingdom). M13 forward and reverse primers or Sp6 primer was used to sequence in both directions. The data were analyzed by means of MacVector 4.5.3 software and aligned to Entrez and V-BASE databases.41,62 Analysis of VH gene usage and mutation pattern was performed as previously described.11 Alternative transcripts were considered tumor derived when they shared the same CDR3 sequence of the tumor VHDJH-Cµ transcript. Intraclonal heterogeneity was assessed in the cloned products and was distinguished from Taq infidelity by an increased frequency as compared with Taq error rate and by the finding of the same mutations in more than one clone and/or in different PCRs. Qiagen Taq polymerase error rate was assessed by means of the
following strategy. A pair of primers, 5'H- Identification of
VHDJH-C  - or C -specific 3'-primers.
After the CDR3-constant region products were identified by PCR,
cloning, and sequencing, then clonal CDR1-specific 5'-primers were
designed and used with the same C (CFF1)
or C (CFF1) primers to confirm the presence of the alternative transcripts and to obtain extended VHDJH-constant sequence. This strategy also
allowed comparison of the pattern of mutation among different isotypes
in the tumor. The positioning of the constant-region primers
facilitated discrimination among different subclasses in the PCR
product and could provide an analysis of C and
C subclasses present within the tumor clone. Table 1
describes primer sequences and location. In all cases, conventional
PCRs, without the need of nested or seminested PCRs, were performed as
described above, by means of 30 cycles with 65°C annealing
temperature. Amplified products were then cloned, sequenced,
and analyzed.
Analysis of tumor-derived transcripts in HCL single cells Material from patient 103 was available for further analysis. Leukemic cells were preincubated, and labeled with CD11c. HCs were identified as high SSC, high FSC PBMCs, and CD11c+ and sorted on a Becton Dickinson FACS Vantage instrument equipped with an automated cell-deposition unit (Becton Dickinson). Sorting of single events had previously been confirmed by separation of 2.49 µm Nile Red beads (Becton Dickinson). In addition, sorting and V gene analysis of normal CD19+ B cells were found to give single, unrelated sequences (C.I.M., unpublished data, September 2000). Single leukemic cells were sorted into 0.2-mL PCR tubes containing 10 µL of 1 × Superscript II First-Strand Buffer (Gibco BRL, Life Technologies, Paisley, United Kingdom). Sorted cells were snap-frozen on dry ice and stored at 80°C. Reverse
transcription to cDNA was carried out in a final volume of 25 µL with
random hexamers and Superscript II RNase H Reverse
Transcriptase according to the manufacturer's instructions (Gibco BRL).
Individual IgM, IgD, and IgG transcripts were identified from single
cells by means of a seminested PCR approach, while IgA transcripts were
identified by means of a nested PCR (Figure
2). In the first round, 3 µL cDNA was
amplified by means of VH4 leader primer and a respective
downstream Cµ16, C
For secondary PCR, 3 µL of the first PCR DNA product was amplified by
means of the internal 103-CDR1 primer with individual 3'-CH
primers. For the identification of IgA transcripts, the internal
C
Immunophenotype and immunoglobulin isotype surface expression Table 2 summarizes the clinical and immunophenotypic characteristics of the 5 HCL patients who entered this study. Phenotypic analysis of PBMCs confirmed diagnosis of HCL since all cases were CD19+, CD20+, CD25+, FMC7+, CD103+, CD11c+, CD79b+, with strong expression of SmIg and negative expression of CD23. The leukemic population, identified as CD19+/CD103+ cells, represented 20% to 77% of PBMCs. It is noteworthy that no cases expressed CD38 or the memory B-cell marker CD27.42
To determine the surface expression of immunoglobulin isotypes on the
neoplastic cells of our patients, we used FITC-conjugated F(ab')2 isotype-specific monoclonal antibodies
(Figure 1 illustrates patient 163 as an example). As described in Table
3, all patients expressed IgM, IgD, and
IgG at varying levels. IgA was expressed in 2 cases (patients 103 and
163) in 94% and 85% of the tumor population, respectively. In
patients 83 and 93, we could not detect the clear presence of the IgA
protein although a small shift of the sample peak as compared with the
isotype control peak was observed. Only patient 42 was phenotypically
completely negative for IgA. Although the level of expression of each
isotype appeared different within some of the cases, there was no
indication of separate populations expressing different
immunoglobulin classes.
Analysis of tumor-derived VH genes Preparations of cDNA from all 5 patients were amplified, cloned, and sequenced initially by means of a VH leader mix and Cµ primers. The identification of tumor-derived VH genes was based on a common CDR3 signature sequence among multiple clones from each patient's amplified cDNA. Table 4 shows the number of tumor-derived clones identified and the total number of clones sequenced for the IgM transcript. Sequences not related to the tumor clone differed individually and were most likely to derive from normal B cells. The VH gene used by each tumor, together with deviation in homology from the germline counterpart, is also shown in Table 4. The deduced amino acid sequences are shown in Figure 3. Nucleotide sequences have been deposited in the GenBank database (accession numbers: AF302819, AF302820, AF302821, AF302822, AF302823).
VH gene segments were derived from V3-33 in 2 cases, and V3-23, V4-31, and V1-02 in the other 3 cases. CDR3 length varied from 12 to 19 codons (mean, 16 codons). JH4b was used in 3 cases, while JH6b and JH2 were used in the remaining 2 cases. According to the rule proposed by Corbett et al,43 D gene segment could be assigned with confidence in 3 cases (patients 163, 42, and 93), In patient 83, CDR3 alignment did not reach the interval of confidence, while in patient 103 the D segment was not identified. All tumor-related VH genes revealed nucleotide substitutions with the percentage of homology ranging from 93.2% to 98.6%. Intraclonal heterogeneity In all our 5 cases, intraclonal heterogeneity exceeding Taq error (0.8 × 103 bp1) was observed. The
general level of intraclonal variation was low, ranging from
1.2 × 103 (patient 163) to 3.6 × 103
bp1 (patient 103) with a median of
2 × 103 bp1 in the IgM
transcripts analyzed, and it is difficult to assess the significance of
this level. However, the reality of mutations was supported by the
presence of repeats among different PCRs. In fact, there was clear
evidence of repeated identical intraclonal changes common to at least 2 different isotype transcripts in all cases: in particular, identical
nucleotide variations were seen in tumor-derived IgM and IgD clones in
patient 163; in IgM and IgG in patient 103; in IgD, IgG, and IgA in
patient 42; and in all isotypes in patients 83 and 92. Figure
4 illustrates intraclonal heterogeneity
in patient 83 as an example.
Transcripts for alternative isotypes After identification of the VHDJH-Cµ transcripts, we sought VHDJH-C ,
VHDJH-C, and
VHDJH-C tumor-derived transcripts, with results shown in Table
5. For all patients, we found transcripts
for IgD, and full
VHDJH-C-derived sequences had a
mutational pattern identical to the clonal IgM transcript. Tumor-derived VHDJH-C clonal
sequences were also identified in 5 of 5 patients, with IgG3
transcripts in all patients, IgG1 in 3 of 5, and IgG2 in 2 of 5. In
terms of frequency within the IgG class, IgG3 appeared predominant
(Table 5) in all patients except for patient 93, where IgG1 and IgG2
were most evident. IgA-derived clonal sequences were detected in 4 of 5 patients, with predominance of IgA1 (Table 5).
Full-length VHDJH sequences linked to
C Expression of immunoglobulin protein generally correlated with
detection of transcripts. All cases expressed both the message and the
protein for IgM, IgD, and IgG isotypes. However, for IgA there was some
disparity. This was most evident in patients 42 and 163. In patient 42, Multiple tumor-derived transcripts in single HCs Table 6 summarizes the results obtained from 10 individual HCs. PCR controls without template were negative in all cases, excluding possibility of contamination. All individual cells were tumor- derived and, apart from 2 nonidentical base changes in 2 separate cells, probably due to Taq infidelity, shared an identical pattern of VH mutation to the tumor sequence, previously identified by whole population analysis, irrespective of isotype. All cells expressed the IgG3 transcript. In one cell, IgM, D, G3, and A transcripts could be identified. Seven out of 10 cells expressed at least 3 different isotypes (3 IgM/IgD/IgG3; 4 IgD/IgG3/IgA), and the remaining 2 cells expressed 2 different isotypes (1 IgD/IgG3; 1 IgG3/IgA).
HCL appears to be derived from a mature B cell, and the presence
of somatic mutations in VH genes confirms that the cell of origin has encountered a site where mutation is activated. The clear
evidence for intraclonal variation also indicates that mutation continued at a low level after transformation, consistent with a
possible influence of activated T cells and antigen.44 The most distinctive feature of approximately 40% of cases of HCL, and of
all our randomly selected cases, is the expression of multiple immunoglobulin isotypes, apparently by the majority of cells in the
clone. In normal B cells, isotype switching generally occurs by
recombination between donor and acceptor switch (S) sites located 5' of
each constant-region gene,45-48 with consequent looping
out and deletion of the intervening CH genes. The outcome
of this deletional mechanism is that a B cell will usually express only a single isotype. The exceptions are IgM and IgD, which are commonly coexpressed owing to the lack of a conventional S The deletional model, however, has to accommodate observations that
normal B cells in the mouse can express IgM together with downstream
isotypes, and that mouse B cells expressing IgM and IgG1 contain
nuclear RNAs that have both Cµ and C Most studies of human B cells have centered on Burkitt lymphoma cell lines, where isotype switching appears to occur mainly by deletional recombination, with 75% of cases deleting Cµ on both allelic chromosomes.54 Normal Epstein-Barr virus-transformed B cells show a similar tendency to undergo switching on both alleles, often to the same constant region.55,56 However, the switching process on the nonproductive allele can also be surprisingly variable, with complex deletions and duplications observed.57 In the cases of HCLs that express a single downstream isotype, most appear to have undergone this process, often on both alleles.58 Analysis of the switching process in spontaneous human B-cell tumors that synthesize both IgM and a downstream isotype has been more limited. One difficulty is that such tumors, although clonally related, may be arrested at different points of differentiation. This was evident in our study of a subset of lymphoplasmacytoid leukemia, where the tumor clone can express and secrete IgM and IgG.59 Analysis of VH gene sequences in these cases revealed clonal relatedness between the isotypes. However somatic mutational patterns differed in 2 of 5 patients, clearly showing that the tumor population was heterogeneous, with a divergent mutational activity at the IgM and IgG stages.59 Cases with identical mutational patterns were not assessed, since material for single-cell analysis would be required. In our cases of DLBCL, clonally related isotype-switch variants again showed distinct mutational patterns, with clustering into an IgMD/IgG3 set or an IgG1 set.21 Evidence for existence of subpopulations in at least some tumors was therefore clear. The cases of HCL we have studied show no evidence for the existence of
subpopulations, either phenotypically or in somatic mutational
pattern; this suggests that most cells were synthesizing multiple
isotypes. However, because suitable material was available for one
case, we were able to take the investigation further by analyzing
events in single cells. The finding that 9 of 10 cells contained
coexisting transcripts of preswitched (IgM/IgD) and postswitched
(IgG/IgA) immunoglobulins allowed us to conclude that
individual cells were able to synthesize multiple isotypes. It excluded
the alternative possibility that subpopulations were present, each
having undergone deletional switch events to different isotypes. It is
also in complete accord with the phenotypic data. Strikingly, in one
cell, IgM, IgD, IgG, and IgA isotypes were detected. In all cases,
transcripts were derived from the same VHDJH
recombination and are therefore from the functionally re-arranged allele. The most likely explanation is that, as for the mouse B cells
studied previously, the cells are processing long nuclear RNA
transcripts.39,52 At present, it is not known whether this involves the cis or the trans allele. Previous
analysis of constant-region genes in cases of HCL expressing multiple
isotypes had shown that 2 of 3 cases had abnormalities in the
JH-Cµ intronic region28 similar
to those identified previously by Laffan and Luzzatto.60 The abnormalities arose from the presence of an inverted
Cµ-containing sequence 5' to a normal C It has been suggested that the stage of RNA processing may be passed through by normal B cells, and that trans-spliced chimeric germ line transcripts may serve as "bridging templates" for normal immunoglobulin class-switching recombination.39 It is possible that this subset of HCL is arrested at a stage in which isotype switching is being attempted. The potentially functional transcripts that we have identified from VHDJH-constant region sequences appear in the main to produce immunoglobulin protein. Events in the tumor cells of HCL appear to support the concept that multiple isotype-switch variants are first generated by RNA processing. If so, HCL may provide a useful model to probe these events and to investigate the reason for arrest.
Submitted September 22, 2000; accepted April 12, 2001.
Supported by Associazione Italiana contro le Leucemie (Italy); Multiple Myeloma Research Foundation (United States); and Leukaemia Research Fund (United Kingdom).
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: Francesco Forconi, Molecular Immunology Group, Tenovus Laboratory, Southampton University Hospitals, Tremona Rd, Southampton S016 6YD, United Kingdom; e-mail: ff1{at}soton.ac.uk.
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  F. Forconi, E. Sozzi, D. Rossi, S. S. Sahota, T. Amato, D. Raspadori, L. Trentin, L. Leoncini, G. Gaidano, and F. Lauria Selective influences in the expressed immunoglobulin heavy and light chain gene repertoire in hairy cell leukemia Haematologica, May 1, 2008; 93(5): 697 - 705. [Abstract] [Full Text] [PDF]
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N. Zojer, H. Ludwig, M. Fiegl, F. K. Stevenson, and S. S. Sahota Patterns of somatic mutations in VH genes reveal pathways of clonal transformation from MGUS to multiple myeloma Blood, May 15, 2003; 101(10): 4137 - 4139. [Abstract] [Full Text] [PDF]
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S. S. Sahota, F. Forconi, C. H. Ottensmeier, D. Provan, D. G. Oscier, T. J. Hamblin, and F. K. Stevenson Typical Waldenstrom macroglobulinemia is derived from a B-cell arrested after cessation of somatic mutation but prior to isotype switch events Blood, July 30, 2002; 100(4): 1505 - 1507. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
chain of
the interleukin-2 receptor CD25. Other important diagnostic
markers are CD11c, HC2, B-Ly7, and HML-1 (CD103), the latter 2 recognizing the 
7 subunit of the same integrin and being the most
specific for the diagnosis of HCL.
genes of 3 cases of HCL appeared low, probably
reflecting the generally lower rate of mutation in VL as
compared with VH.
/
light chains (Dako, Glostrup, Denmark); and anti-SmIg [surface membrane Ig], anti-IgA,
anti-IgD, anti-IgG, and anti-IgM (Caltag Laboratories, Burlingame,
CA). In particular, expression of immunoglobulin isotypes on
HCs was performed by 3-color staining by means of fluorescein
isothiocyanate (FITC)-conjugated F(ab')2 anti-IgG,
anti-IgM, anti-IgD, anti-IgA; peridinin chlorophyll protein
(PerCP)-conjugated anti-CD19; and phycoerythrin-conjugated
anti-CD11c. HCs were identified as high side scatter (SSC) and
high forward scatter (FSC) PBMCs,
CD19+/CD11c+ (Figure
, 
, and
AA substitution (amino acid G