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Blood, 15 February 2003, Vol. 101, No. 4, pp. 1653-1653
CORRESPONDENCE
To the editor:
Translocation of the IgH locus is nearly ubiquitous in multiple
myeloma as detected by immuno-FISH
Fonseca et al1 have recently reported a high
frequency (46%) of IgH translocations to occur in monoclonal
gammopathy of undetermined significance. As this frequency seems to
approach the reported frequencies in multiple myeloma (MM;
60%-70%),2,3 it was suggested that IgH translocations
represent early cytogenetic events in plasma cell discrasias and are
likely of pathogenetic importance. They have plasma cells positively
identified by staining interphase cells with AMCA-conjugated
(blue fluorescent) anti-light chain
antibodies,
followed by denaturation and probing with green- and red-labeled
probes, thus obviating the need to culture or purify plasma cells in
vitro. Here we have furthered this approach by modifying the procedure
to a double-color immunofluorescence in situ hybridization
(immuno-FISH), where fluorescein isothiocyanate (FITC) is used
both for Ig staining and probing, and we found near ubiquity of
t(14q32) in MM, confirming the notion of IgH translocations being early
pathogenic events. Bone marrow (BM) samples (57) were obtained from patients with MM at
the time of diagnosis (47) or during follow-up, and cytospin slides
were prepared. t(14q32) was detected using a set of probes spanning the
IgH locus, labeled with either biotin or digoxigenin, as we described
earlier.4 For light chain-specific detection of the
plasma cells, FITC-conjugated antibodies directed against human  or
 were used (SBA, Birmingham, AL). For each sample, we analyzed 100 plasma cells using CytoVision Applied Imaging software (DSS Imagetech,
New Delhi, India). Of 47 patients studied at diagnosis, 45 were evaluable. Of these
samples, 43 (96%) carried a t(14q32). In the majority of cases, the
translocation was found in all plasma cells, whereas in one-third of
the patients, the percentage of plasma cells having an identifiable
t(14q32) varied from 14% to 82% (mean, 62%). In 10 patients at
follow-up, t(14q32) was essentially as frequent (data not shown); in
this situation, the presence of as low as 0.1% plasma cells in BM
still did allow positive identification of the t(14q32). This modified FISH technique to detect IgH translocations in
well-defined cells is restricted to the 3 usual fluorescent markers (FITC, cyanine 3.18 [CY3], and diamidinophenolindole
[DAPI]) and is easily applicable on normal fluorescent
microscopes. Cytoplasmic staining with an FITC-conjugated antibody
allows for positive identification of plasma cells, whereas
DAPI-mediated morphologic discrimination allows for probing with the
same FITC signal in nuclear localization (Figure 1). Because
of the sensitivity of the technique, it may be applied for
follow-up of patients during treatment regimens. Furthermore, the
technique may be applied for detection of partner chromosomes of
t(14q32) that, as well as other molecular characteristics, have
been suggested to have clinical2,5,6 and
pathophysiologic7 relevance for a molecular subdivision of
MM.
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| Figure 1.
Detection of t(14q32) by double-color FISH and
Ig-light-chain staining in interphase plasma cells.
Cytospin slides from BM samples were fixed in 10% acetic acid solution
in ethanol, followed by dehydration, denaturation (70% formamide), and
dehydration. After hybridization of the probes overnight at 37°C,
immunoglobulins were stained using FITC-conjugated goat anti-human
light-chain antibodies to positively identify plasma cells. The IgH2
and C- probes were detected following standard avidine-FITC and CY3
staining. The slides were mounted with antifade medium containing DAPI.
Fusion signals of the probes indicate nontranslocated IgH loci, whereas
split signals reflect a translocation of the IgH
locus.
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Earlier, we reported t(14q32) to occur in up to 95% of human myeloma
cell lines.4 Now, the frequency of t(14q32) in MM patients
at diagnosis seems to be as high, indicating that findings in cell
lines do not point to an in vitro artifact but rather indicate that IgH
translocations are an early pathogenic event in MM.
Gienke R. Boersma-Vreugdenhil, Ton Peeters, and Bert J. E. G. Bast
Correspondence: Gienke Boersma-Vreugdenhil,
Department of Immunology University Medical Center Utrecht, P O Box
85500, 3508 GA Utrecht, The Netherlands; e-mail:
g.r.boersma{at}lab.azu.nl
Acknowledgments
Supported by grant UU 2000-2278 of the Dutch Cancer Society KWF
References
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Fonseca R, Bailey RJ, Ahman GJ, et al.
Genomic abnormalities in monoclonal gammopathy of undetermined significance.
Blood.
2002;100:1417-1424[Abstract/Free Full Text].
2.
Avet-Loiseau H, Facon T, Grosbois B, et al.
Oncogenesis of multiple myeloma: 14q32 and 13q chromosomal abnormalities are not randomly distributed, but correlate with natural history, immunological features, and clinical presentation.
Blood.
2002;99:2185-2191[Abstract/Free Full Text].
3.
Nishida K, Tamura A, Nakazawa N, et al.
The Ig heavy chain is frequently involved in chromosomal translocations in multiple myeloma and plasma cell leukemia as detected by in situ hybridization.
Blood.
1997;90:526-534[Abstract/Free Full Text].
4.
Kuipers J, Vaandrager JW, Oldeweghuis D, et al.
FISH analysis shows the frequent occurrence of 14q32.3 rearrangements with involvement of the immunoglobulin switch regions in myeloma cell lines.
Cancer Genet Cytogenet.
1999;109:99-107[CrossRef][Medline]
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Fonseca R, Blood EA, Oken MM, et al.
Myeloma and the t(11;14)(q13;q32); evidence for a biologically defined unique subset of patients.
Blood.
2002;99:3735-3741[Abstract/Free Full Text].
6.
Moreau P, Facon T, Leleu X, et al.
Recurrent 14q32 translocations determine the prognosis of multiple myeloma especially in patients receiving intense chemotherapy.
Blood.
2002;100:1579-1583[Abstract/Free Full Text].
7.
Bergsagel PL, Kuehl WM.
Chromosome translocations in multiple myeloma.
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[Order article via Infotrieve].
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