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From www.bloodjournal.org by guest on October 28, 2014. For personal use only.
1998 92: 2987-2989
FGFR3 Gene Mutations Associated With Human Skeletal Disorders Occur
Rarely in Multiple Myeloma
Nicola Stefano Fracchiolla, Stefano Luminari, Luca Baldini, Luigia Lombardi, Anna Teresa Maiolo and
Antonino Neri
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2987
2. Peto R, Pike MC, Armitage P, Breslow NE, Cox DR, Howard SV,
Mantel N, McPherson K, Peto J, Smith PG: Design and analysis of
randomized clinical trials requiring prolonged observation of each
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3. Peto R, Pike MC, Armitage P, Breslow NE, Cox DR, Howard SV,
Mantel N, McPherson K, Peto J, Smith PG: Design and analysis of
randomized clinical trials requiring prolonged observation of each
patient. II. Analysis and examples. Br J Cancer 35:1, 1977
4. The Italian Cooperative Study Group on Chronic Myeloid Leukemia: Interferon alfa-2a as compared with conventional chemotherapy
for the treatment of chronic myeloid leukemia. N Engl J Med 330:820,
1994
5. Hasford J, Pfirrmann M, Hehlmann R, Allan NC, Kluin-Nelemans
JC, Alimena G, Steegmann JL, Ansari H: A new prognostic score for
survival of patients with chronic myeloid leukemia treated with
interferon alfa. J Nat Cancer Inst 90:850, 1998
6. Mahon F, Montastruc M, Faberes C, Reiffers J: Predicting
complete cytogenetic response in chronic myelogenous leukemia
patients treated in chronic myelogenous leukemia patients treated with
recombinant interferon-a. Blood 84:3592, 1994
7. Guilhot F, Chastang C, Michallet M, Guerci A, Harousseau JL,
Maloisel F, Bouabdallah R, Guyotat D, Cheron N, Nicolini F, Abgrall
JF, Tanzer J: Interferon alpha2b (IFN) and cytarabine (Ara-C) increase
survival and cytogenetic response in chronic myeloid leukemia (CML).
Results of a randomized trial. N Engl J Med 337:223, 1997
8. Allan NC, Richards SM, Shepherd PCA: Interferon-a therapy
with busulphan or hydroxyurea compared with either BU or HU alone
in treatment of chronic phase CML. Results from the MRC III trial. Int J
Hematol 64:S68, 1996 (abstr 258, suppl 1)
9. Shepherd PCA, Richards SM, Allan NC: Progress with interferon
in CML—Results of the MRC UK CML III study. Bone Marrow
Transplant 17:S15, 1996 (suppl 3)
10. CML Trialists’ Collaborative Group: Interferon alfa versus
chemotherapy for chronic myeloid leukemia: A meta-analysis of seven
randomized trials. J Natl Cancer Inst 89:1616, 1997
11. Hehlmann R, Heimpel H, Hasford J, Kolb HJ, Pralle H, Hossfeld
DK, Queisser W, Lo¨ffler H, Hochhaus A, Heinze B, Georgii A, Bartram
CR, Griesshammer M, Bergmann L, Essers U, Falge C, Queisser U,
Meyer P, Schmitz N, Eimermacher H, Walther F, Fett W, Kleeberg UR,
Ka¨bisch A: Randomized comparison of interferon-a with busulfan and
hydroxyurea in chronic myelogenous leukemia. Blood 84:4064, 1994
FGFR3 Gene Mutations Associated With Human Skeletal Disorders
Occur Rarely in Multiple Myeloma
To the Editor:
Fibroblast growth factor receptor 3 (FGFR3) is one of four distinct
tyrosine-kinase receptors (FGFR1-4) that are capable of binding a
repertoire of at least nine related mitogenic fibroblast growth factors
(FGFs). FGFRs encode proteins that all contain three glycosylated
extracellular Ig-like domains, a transmembrane domain (TM), and a
split cytoplasmic tyrosine-kinase domain. Point mutations in distinct
domains of the FGFR3 gene are associated with autosomal dominant
human skeletal disorders, such as achondroplasia, thanatophoric dysplasia types I and II, and hypochondroplasia.1,2 Recent reports indicate that
the point mutations associated with these disorders produce constitutively activated FGFR3, which shows autophosphorylation in the
absence of ligand and is no longer regulated by FGF binding.3-6
We and others have recently provided the first evidence of FGFR3
gene involvement in human cancer.7,8 In particular, the FGFR3 gene
located at 4p16.3 is translocated to chromosome 14q32 as a result of a
novel and karyotypically undetectable t(4;14)(p16.3;q32) chromosomal
translocation in multiple myeloma (MM), a malignant proliferation of
plasma cells. Molecular studies have shown this lesion in five MMderived cell lines and in four primary tumors. Although the breakpoints
on 4p16.3 are located approximately 50 to 120 kb centromeric to
FGFR3, the gene is overexpressed in these cases, but absent or barely
detectable in cell lines without the translocation. Interestingly, FGFR3
gene mutations associated with distinct human skeletal disorders2 have
also been identified in some MM tumors carrying the t(4;14)(p16.3;
q32): in particular, the Y373C mutation in the KMS-11 cell line,7,8 the
K650E mutation in the OPM2 cell line,7 and the K650M mutation in a
primary MM tumor.7
These findings prompted us to look for FGFR3 mutations known to
be associated with skeletal disorders in a representative panel of MM,
including 80 primary cases (60 patients at first diagnosis, 12 at relapse,
and 8 affected by plasma cell leukemia) and 10 MM-derived cell lines
(including the KMS-11 and OPM2 cell lines). The analysis was
performed by means of the polymerase chain reaction–single-strand
conformation polymorphism (PCR-SSCP) direct sequencing of genomic
DNA. We amplified five distinct genomic FGFR3 fragments containing
codons affected by mutations: codon 248, the entire TM domain
Fig 1. Schematic representation of the primers from the human FGFR3 gene used in the study. The FGFR3 exons are indicated by white boxes,
and the introns are indicated by lines. The 38 untranslated region is indicated by the dashed box. The approximate locations of the primers, the
length of the amplified fragments, and the approximate positions of codons 248, 540, 650, and 807 are indicated. The nucleotide sequence of
FGFR3 cDNA and the intron-exon organization of the gene have been previously reported.12,13 The sequences of the primers are as follows: 248F
(intron 6), 58-CCTGAGCGTCATCTGCC-38, and 248R (exon 7), 58-CCATTGCATCCCACACGG-38; TD5 (exon 10), 58-AGGAGCTGGTGGAGGCTGA-38,
and TD3 (exon 10), 58-GGAGATCTTGTGCACGGTGG-3814; 540F (exon 13), 58-ACTGACAAGGACCTGTCGGAC-38, and 540R (exon 13), 58GCCCTGCGTGCAGGCGCC-38; 650F (exon 15), 58-GCATCCACAGGGACCTGG-38, and 650R (intron 15), 58-AGGCGGTGTTGGCGCCAG-38; 14S (exon
15), 58-GTGCACAACCTCGACTAC-38 (this primer was used with 650R to obtain a DNA fragment suitable for the restriction enzyme analysis of
codon 650); 807F (exon 19), 58-CCTGTCGGCGCCTTTCGAGCAGTAC-38, and 807R (exon 19), 58-CACCAGCAGCAGGGTGGGCTGCTAG-38.15
From www.bloodjournal.org by guest on October 28, 2014. For personal use only.
2988
CORRESPONDENCE
constitutive oncogenic signal for the growth and/or survival of malignant plasma cells. This possibility is supported by the evidence that the
bone marrow environment and, in particular, the stromal cells with
which the plasma cells interact10 are able to produce FGFs.11 The FGFR3
mutations reported in MM probably represent somatic events, suggesting that
FGFR3 gene may be deregulated by different mechanisms. However, we
were unable to detect FGFR3 mutations associated with skeletal disorders in
our series of samples, except in the cell lines previously reported.7,8 This
finding suggests that such mutations represent rare events in MM and support
the hypothesis that they may occur after the translocation and deregulation of
the FGFR3 gene, thus contributing to tumor progression by means of
ligand-independent activation.
ACKNOWLEDGMENT
We are grateful to Dr T. Otsuki, Dr F. Malavasi, and Dr A. Solomon
for providing us with the some of the MM-derived cell lines (KMM1,
KMS-11, KMS-12, LP-1, and UTMC-2) used in this study and to G
Ciceri for technical assistance. The cell lines U266, Sultan, ARH-77,
and RPMI 8226 were obtained from ATCC and the OPM2 cell line was
obtained from DSMZ. This work was supported by a grant from the
Associazione Italiana Ricerca sul Cancro (AIRC) to A.N. and a grant
‘‘Ricerca Corrente 1994’’ from the Ministero Italiano della Sanita` to
Ospedale Maggiore IRCCS.
Nicola Stefano Fracchiolla
Stefano Luminari
Luca Baldini
Luigia Lombardi
Anna Teresa Maiolo
Antonino Neri
Servizio di Ematologia
Istituto di Scienze Mediche
Universita` di Milano
Ospedale Maggiore
IRCCS
Milan, Italy
Fig 2. PCR-SSCP analysis of the FGFR3 gene. N, normal control;
migrating fragments different from the normal control are indicated
by arrows.
(codons 371, 373, 375, and 380), codon 540, codon 650, and codon 807
(Fig 1). The mutations at codon 650 were also investigated by means of
a restriction enzyme analysis of the PCR-amplified fragment using Mbo
II and Bbs I enzymes, as previously described.9 We detected allelic
variations of the FGFR3 gene only in the fragment specific for the TM
domain. An abnormal fragment with the same pattern of migration was
observed in 2 cases (the LP-1 cell line and a primary tumor; Fig 2); in
both cases, a novel single basepair mutation involving codon 384 in the
form of a T to C transition (TTC-CTC) led to a conservative Phe = Leu
amino acid substitution (data not shown). Interestingly, this mutation
abrogates a Mbo II restriction site and creates a new Mnl I site that
allows restriction enzyme analysis of the PCR-amplified fragment. The
apparently similar intensity of the normal and mutated bands in both
cases, as well as the detection of the mutation in 2 of 100 normal
individuals by means of restriction enzyme analysis, suggest that it may
represent a rare genetic polymorphism. Finally, the FGFR3 gene was
apparently not expressed in the LP-1 cell line; it remains to be seen
whether this particular variant may affect FGFR3 biological activity.
Although no specific genetic lesions have been found to be associated
with MM (unlike other types of lymphoid neoplasms), cytogenetic and
more recent molecular analyses suggest that chromosomal translocations involving the Ig locus on chromosome 14q32 may play an
important role in gene deregulation.7,8 In this context, the recent
identification of the t(4;14)(p16.3;q32) in MM, associated with an
apparent deregulation of the FGFR3 gene, may provide some insights
into the pathogenesis of this neoplasia. Although more work is needed
to assess the role and frequency of the t(4;14) in MM, it can be
suggested that deregulation of FGFR3 gene expression may lead to a
REFERENCES
1. Muenke M, Schell U: Fibroblast-growth-factor receptor mutations
in human skeletal disorders. Trends Genet 11:308, 1995
2. Webster MK, Donoghue DJ: FGFR activation in skeletal disorders: Too much of a good thing. Trends Genet 13:178, 1997
3. Naski MC, Wang Q, Xu J, Ornitz DM: Graded activation of
fibroblast growth factor receptor 3 by mutations causing achondroplasia
and thanatophoric dysplasia. Nat Genet 13:233, 1996
4. Webster MK, Donoghue DJ: Constitutive activation of fibroblast
growth factor receptor 3 by the transmembrane domain point mutation
found in achondroplasia. EMBO J 15:520, 1996
5. Webster MK, d’Avis PY, Robertson SC, Donoghue DJ: Profound
ligand-independent kinase activation of fibroblast growth factor receptor 3 by the activation loop mutation responsible for a lethal skeletal
dysplasia, thanatophoric dysplasia type II. Mol Cell Biol 16:4081, 1996
6. d’Avis PY, Robertson SC, Meyer AP, Bardwell WM, Webster MK,
Donoghue DJ: Constitutive activation of fibroblast growth factor
receptor 3 by mutations responsible for the lethal skeletal dysplasia
thanatophoric dysplasia type I. Cell Growth Differ 9:71, 1998
7. Chesi M, Nardini E, Brents LA, Schroch E, Ried T, Kuehl WM,
Bergsagel PL: Frequent translocation t(4;14)(p16.3;q32.3) in multiple
myeloma is associated with increased expression and activating mutations of fibroblast growth factor receptor 3. Nat Genet 16:260, 1997
8. Richelda R, Ronchetti D, Baldini L, Cro L, Viggiano L, Marzella
R, Rocchi M, Otsuki T, Lombardi L, Maiolo AT, Neri A: A novel
From www.bloodjournal.org by guest on October 28, 2014. For personal use only.
CORRESPONDENCE
chromosomal translocation t(4;14)(p16.3;q32) in multiple myeloma
involves the fibroblast growth-factor receptor 3 gene. Blood 10:4062, 1997
9. Tavormina PL, Shiang R, Thompson LM, Zhu YZ, Wilkin DJ,
Lachman RS, Wilcox WR, Rimoin DL, Cohn DH, Wasmuth JJ:
Thanatophoric dysplasia (types I and II) caused by distinct mutations in
fibroblast growth factor receptor 3. Nat Genet 9:321, 1995
10. Caligaris-Cappio F, Bergui L, Gregoretti MG, Gaidano G,
Gaboli M, Schena M, Zallone AZ, Marchisio PC: Role of bone marrow
stromal cells in the growth of human multiple myeloma. Blood 77:2688,
1991
11. Allouche M: Basic fibroblast growth factor and hematopoiesis.
Leukemia 9:937, 1995
12. Keegan K, Johnson DE, Williams LT, Hayman MJ: Isolation of
2989
an additional member of the fibroblast growth factor receptor family,
FGFR-3. Proc Natl Acad Sci USA 88:1095, 1991
13. Perez-Castro AV, Wilson J, Altherr MR: Genomic organization
of the human fibroblast growth factor receptor 3 (FGFR3) gene and
comparative sequence analysis with the mouse fgfr3 gene. Genomics
41:10, 1997
14. Shiang R, Thompson LM, Zhu YZ, Church DM, Fielder TJ,
Bocian M, Winckur ST, Wasmuth JJ: Mutations in the transmembrane
domain of FGFR3 cause the most common genetic form of dwarfism,
achondroplasia. Cell 78:335, 1994
15. Rousseau F, Saugier P, Le Merrer M, Munnich A, Delezoide A-L,
Maroteaux P, Bonaventure J, Narcy F, Sanak M: Stop codon FGFR3
mutations in thanatophoric dwarfism type 1. Nat Genet 10:11, 1995
Interleukin-2 Receptor Subunit Expression and Function on Human Peripheral T
Cells Is Not Dependent on the Anticoagulant
To the Editor:
In a recent report, David et al1 addressed the ongoing controversy on
expression levels of interleukin-2 receptor (IL-2R) a, b, and g chains on
various mononuclear cells from the peripheral blood. Using freshly
isolated peripheral blood mononuclear cells (PBMCs) from (sodium)
heparinized blood and fluorescein isothiocyanate (FITC)-labeled commercial monoclonal antibodies, they showed that all three IL-2R chains
usually are hardly detectable on either CD4 or CD8 T cells from healthy
donors and from hemochromatosis patients. These results are in contrast
with the much higher levels of IL-2R subunits on T cells observed by
several investigators, including ourselves.2-9 David et al1 tentatively
explain the discrepancy by invoking effects of anticoagulant and of
storage. Indeed, if Ca21 chelators were used instead of heparin, the
levels of all three IL-2R chains on T cells apparently increased, and
overnight storage of heparinized blood also seemed to upregulate IL-2R
subunit expression.
These observations are very important, because they not only seem to
settle a long-standing controversy on IL-2R expression, but they also
imply that the use of Ca21 chelators as anticoagulant instead of heparin
could dramatically influence the sensitivity of the T cells to IL-2.
Because IL-2 and other common g-chain triggering cytokines are
central to almost any T-cell function, EDTA or citrate anticoagulants
should be avoided if subsequent functional testing is envisioned. A lot of
immunological research is based on buffy coats, which routinely are
anticoagulated with citrate. In our own studies of T-cell function during
human immunodeficiency virus (HIV) infection, we have systematically used EDTA blood as starting material, because it is readily
available and because we did not find a functional difference between
lymphocytes derived from blood anticoagulated with heparin, citrate, or
EDTA in preliminary experiments. In view of the findings of David et
al,1 we felt obliged to carefully control the effect of Ca21 chelators on
IL-2R expression and function and we did not observe any significant
influence of the anticoagulant.
In three separate experiments, blood from five healthy control
subjects (all lab personnel) was drawn at 10 AM in three different tubes
from Sarstedt containing either sodium heparin (final concentration, 0.3
mg/mL), potassium-EDTA (final concentration, 1.6 mg/mL), or sodiumcitrate (final concentration, 10.6 mmol/L). The largest part of each tube
was immediately processed for mononuclear cell (PBMC) separation,
using Histopaque 1077 (Sigma, Bornem, Belgium), whereas the rest
was kept at room temperature. At 2 PM, 50 µL of whole blood and 50 µL
of PBMCs (containing 200,000 cells), derived from each of the three
anticoagulant tubes, were incubated for 20 minutes at 4°C with 0.1 µg of
the nonconjugated reference monoclonals anti-Tac (IL-2Ra–specific;
obtained from Dr Thomas Waldman, National Institutes of Health,
Bethesda, MD) and with 2R-B (IL-2Rb–specific; from Dr Takashi
Uchiyama, Institute for Virus Research, Kyoto University, Kyoto,
Japan). As an isotypic (IgG1) control, we used purified 56D3 directed
against an irrelevant parasitic antigen (provided by Dr J. Brandt,
Institute of Tropical Medicine, Antwerpen, Belgium). After washing
with phosphate-buffered saline (PBS), containing 0.5% bovine serum
albumin, 1 µL of FITC-labeled F(ab8) 2 goat antimouse IgG (Tago,
Burlingame, CA) was added for another 20 minutes. After washing
again, the remaining binding sites on the FITC-conjugate were blocked
with 5 µL of mouse serum. Next, 5 µL of phycoerythrin (PE)-labeled
anti-CD4 and 5 µL of peridinin-chlorophyll A protein (PercP)-labeled
anti-CD3 (both from Becton Dickinson, Erembodegem, Belgium) were
added for the last 20 minutes. The tubes with whole blood were then
subjected to the Becton Dickinson lysing solution. All preparations
were washed once and fixed with 1% paraformaldehyde. The samples
were analyzed on a FACScan (Becton Dickinson) using the LYSYS I
software.
Based on the scatter and the CD3/CD4 expression, the CD41 and
CD42 T lymphocytes were gated separately and the distribution of the
first fluorescence was represented in a histogram for each subset. An
example of this analysis is shown in Fig 1. It is evident that, within both
the CD41 and CD42 T-cell populations, the expression profile of
IL-2Ra is rather broad and tends to be bimodal (a negative and a
positive subpopulation), whereas the curve of IL-2Rb is unimodal and
shows a shift to the right, which is most evident in the CD42 subset. We
chose to express the results for both chains as percentage of positive
cells, after establishing a narrow threshold at a relative fluorescence
intensity of 10, based on the background of the control monoclonal. A
summary of the results is shown in Table 1. No significant difference
was observed in the level of IL-2R a and b chains on CD41 or CD42 T
cells, according to the anticoagulant used and regardless of whether the
cells were stained in the context of whole blood or PBMCs. Comparing
the mean fluorescence intensity of all gated cells (instead of the
percentage of positive cells) showed similar results and confirmed that
the low level of IL-2Rb expression on CD41 T cells significantly
differed from background (data not shown).
We next wanted to know whether the anticoagulant influences the
sensitivity to IL-2. To this end, we cultured the three preparations of
PBMCs at a final concentration of 106/mL in RPMI, supplemented with
antibiotics (GIBCO, Paisley, UK) and 10% bovine calf serum (Hyclone,