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Blood, Vol. 92 No. 8 (October 15), 1998:
pp. 2688-2695
RAPID COMMUNICATION
The Cryptic inv(2)(p23q35) Defines a New Molecular Genetic Subtype
of ALK-Positive Anaplastic Large-Cell Lymphoma
By
Iwona Wlodarska,
Chris De Wolf-Peeters,
Brunangelo Falini,
Gregor Verhoef,
Stephan W. Morris,
Anne Hagemeijer, and
Herman Van denBerghe
From the Center for Human Genetics, Department of Pathology, and
Department of Hematology, K.U. Leuven, Leuven, Belgium; the Institute
of Hematology, University of Perugia, Perugia, Italy; and the
Department of Experimental Oncology, St. Jude Children's Research
Hospital, Memphis, TN.
 |
ABSTRACT |
Recently, a distinctive entity characterized by expression of the
anaplastic lymphoma kinase (ALK) protein [most frequently due to the
t(2;5)(p23;q35)-associated NPM-ALK fusion] has emerged within the
heterogenous group of non-Hodgkin's lymphomas (NHL) classified as
anaplastic large-cell lymphoma (ALCL). Sporadic variant
2p23/ALK abnormalities identified in ALK-positive ALCL indicate that genes other than NPM may also be involved in the deregulation of ALK and lymphomagenesis. We report here three cases with an inv(2)(p23q35) detected by fluorescence in situ hybridization (FISH) in young male patients with
ALK-positive ALCL. In contrast to ALCL cases with the
classical t(2;5)(p23;q35) that usually show both cytoplasmic and
nuclear or predominantly nuclear alone localization of the NPM-ALK
chimeric product, in all three cases with an inv(2)(p23q35) the ALK
protein accumulated in the cytoplasm only, supporting the previous
assumption that the oncogenic potential of ALK may not be dependent on
its nuclear localization. As the first step to identify the
ALK partner gene involved in the inv(2)(p23q35), we
performed extensive FISH studies and demonstrated that the 2q35
breakpoint occurred within the 1,750-kb region contained within the
914E7 YAC. Moreover, a striking association of the inv(2)(p23q35) with
a secondary chromosomal change, viz, ider(2)(q10)inv(2)(p23q35),
carrying two additional copies of the putative ALK-related
fusion gene, was found in all three patients, suggesting that, in
contrast to the standard t(2;5)/NPM-ALK fusion, multiple copies
of the putative 2q35-ALK chimeric gene may be required for
efficient tumor development. In summary, we demonstrate that the
inv(2)(p23q35), a variant of the t(2;5)(p23;q35), is a recurrent
chromosomal abnormality in ALK-positive ALCL, the further
characterization of which should provide new insight into the
pathogenesis of these lymphomas.
© 1998 by The American Society of Hematology.
| |
INTRODUCTION |
ANAPLASTIC LARGE-CELL lymphoma (ALCL),
recognized as a distinctive subtype of non-Hodgkin's lymphoma (NHL) in
the recent REAL classification,1 comprises lymphomas
characterized by large pleomorphic tumor cells, expression of CD30/Ki-1
antigen, a member of the TNF-receptor family,2 and
a T or null immunophenotype. This particular subgroup appears to have a
better overall survival than other large-cell lymphomas and occurs
predominantly in children and adolescent patients.3-5 Until
now, the only recurrent chromosomal abnormality identified in ALCL was
the t(2;5)(p23;q35). As demonstrated by Morris et al,6 this
translocation leads to the fusion of the nucleophosmin gene
(NPM) on chromosome 5q35 to the 2p23 gene encoding the
novel receptor tyrosine kinase ALK, which is highly related to
leukocyte tyrosine kinase (LTK) and normally expressed only
in the nervous system.7,8 The t(2;5)(p23;q35) has been observed in approximately 30% to 50% of ALCL cases documented by
cytogenetics9 and in between 12% and 80% of ALCL cases
analyzed by reverse transcription-polymerase chain reaction (RT-PCR)
(reviewed in Sarris et al10). Immunostaining using
polyclonal and monoclonal antibodies directed against the cytoplasmic
portion of the ALK protein have proved to be a very efficient approach
to identify ALCL with 2p23/ALK rearrangements.11-17
Anti-ALK immunostaining combined with cytogenetics, fluorescence in
situ hybridization (FISH), and/or molecular
studies showed that the ALK gene can also be deregulated by
mechanisms other than the t(2;5), such as
t(1;2)(q25;p23),13 cryptic insertion of the ALK
gene in the vicinity of NPM in a t(2;5)-negative
condition,14 and inv(2)(p23q35).14 The latter
abnormality, which is not recognizable by classical cytogenetics, was
detected by us in 1 of 13 ALK-positive ALCL cases analyzed at random by
two-color FISH with ALK and 5q35-specific probes. Our studies
reported here demonstrate that the inv(2)(p23q35) results in the
translocation of a portion of the ALK gene locus from 2p23 to
2q35. Moreover, we detected two additional copies of the rearranged
ALK gene on the i(2)(q10) chromosome in all analyzed cells from
this patient. By analogy with the t(2;5)/NPM-ALK, the 2q35
region presumably contains an as-yet-unidentified gene that, like
NPM, can deregulate ALK and be involved in the
pathogenesis of ALCL. We have collected now three such cases,
indicating that the inv(2)(p23q35) is a recurrent abnormality in
ALK-positive ALCL, and have studied them with FISH to better
characterize the 2q35 breakpoint.
| |
MATERIALS AND METHODS |
Patients.
Tumor samples from the patients were referred to the Centre for Human
Genetics and Department of Pathology for analysis from the Department
of Hematology, University of Leuven (Leuven, Belgium). The main
clinical and hematological findings of the reported cases are
summarized in Table 1.
Histopathology and phenotyping.
A portion of the biopsy material from each of the three cases was
snap-frozen and stored at  80°C until use. The remaining material
was fixed in buffered formalin and/or B5 and embedded in
paraffin. Immunophenotyping was performed on paraffin-embedded and/or frozen material with a panel of monoclonal antibodies to CD2 (OKT11), CD3 (Leu4), CD4 (Leu3a/OKT4), CD5 (Leu1), CD7 (Leu9), CD8
(OKT8), CD19 (Leu12), CD20 (L26), CD22 (Leu14), and CD30 (BerH2) using
a streptavidin-biotin-peroxidase three-stage technique. Immunohistological detection of ALK protein was performed on both frozen and paraffin sections. The latter were subjected to microwaving (750 W for 3 cycles of 5 each) using 1 mmol/L EDTA buffer, pH 8.0,18,19 as the antigen retrieval solution. After
microwave heating, the sections were allowed to cool at room
temperature for approximately 20 minutes and then washed in
Tris-buffered saline and stained by the immunoalkaline phosphatase
technique, as previously described.20 Briefly, sections
were incubated with ALK113 and ALKc monoclonal
antibodies17 (as undiluted supernatant), followed by rabbit
antimouse Ig (Dako, Glostrup, Denmark) and APAAP complexes. To maximize
the sensitivity of the method, the incubations with rabbit antimouse Ig
and APAAP complexes were repeated once. All antibody steps were for 30 minutes with intervening 5-minute washes in 0.05 mol/L Tris-buffered
saline, pH 7.6. Endogenous alkaline phosphatase was blocked with 1 mmol/L levamisole. Slides were then counterstained for 5 minutes in
Gill's hematoxylin and mounted in Kaiser's glycerol gelatin (Merck,
Darmstadt, Germany).
Cytogenetics.
One-day cultures of lymphoma cells were used for cytogenetic analysis
in all cases. Ten to 24 G-banded karyotypes were analyzed and
classified according to the International System for Human Cytogenetic
Nomenclature.21
FISH analysis.
FISH was performed as previously described.22 Probes
applied in FISH experiments are listed in Table
2. The ALK and NPM loci
were investigated using an ALK P1 clone (designated
ALK-DMPC-HFF#1-1111H1) and three cosmid clones (13, 15-2, and
47C12) from the 5q35 region located immediately centromeric to the
NPM locus.23 Rearrangement of ALK was
further analyzed with the Vysis LSI ALK probe assay (Vysis,
Inc, Downer's Grove, IL) that contains two differently labeled probes
located either 3 telomeric (spanning 250 kb and labeled with
SpectrumOrange) or 5 centromeric (spanning 300 kb and labeled with
SpectrumGreen) of the t(2;5) breakpoint of the ALK gene at
2p23. YACs assigned to 2q were selected from the STS-based map reported
by Chumakov et al.24 SHIP (SH2-containing inositol 5-phosphatase) gene probes (h4.2-A2/Hg4.1-A2) at 2q3725 and
subtelomeric probes for 2p (86J13/PAC) and 2q (210E14/P1)26 were gifts of L.R. Rohrschneider (Fred Hutchinson Cancer Research Center, Seattle, WA) and L. Kearney (Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, UK), respectively. In case no. 2, which contained polyploid cells, chromosome 2 was identified by
cohybridization with a digoxigenin-11-dUTP-labeled centromere-specific probe (D2Z; Oncor, Gaithersburg, MD) and G-banding using
DAPI counterstaining. Between 5 and 12 abnormal metaphases were studied in each experiment. The FISH data were collected on a Leitz DMRB fluorescence microscope equipped with a cooled black and white CCD
camera run by SmartCapture software (Vysis, Stuttgart, Germany).
| |
RESULTS |
Histopathology and phenotyping.
Hematoxylin-eosin-stained biopsy sections from all three cases showed
essentially identical findings. The normal lymph node parenchyma was
largely replaced by a dense, monotonous proliferation of atypical
cells. These cells were characterized by a large, occasionally slightly
indented nucleus with prominent nucleoli and an ample amount of
eosinophilic cytoplasm.
Immunophenotyping demonstrated that the neoplastic cells in all three
cases failed to express either pan-B-cell or pan-T-cell markers but
did express CD30 and ALK protein. The latter expression was mainly
found in the cytoplasm, with no clearcut nuclear or nucleolar staining
seen in frozen or in paraffin sections (Fig 1A), in contrast to ALCL characterized by a
t(2;5)(p23;q35) showing a cytoplasmic and nuclear anti-ALK
immunostaining (Fig 1B). The diagnosis of ALK-positive ALCL was made
based on these data.
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| Fig 1.
Examples of anti-ALK immunostaining. (A) Large anaplastic
cells from case no. 1 with the inv(2)(p23q35) showing strong diffuse
ALK positivity confined to the cytoplasm. (B) In comparison, a case of
CD30+ ALCL with the t(2;5)(p23;q35) showing expression of
the NPM-ALK protein both in the cytoplasm and the nucleus is shown (A
and B: lymph node formalin-fixed paraffin sections immunostained with
the ALKc monoclonal antibody; APAAP technique; original magnification
×800).
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Cytogenetics and FISH.
The results of cytogenetic analysis are summarized in Table
3. The common karyotypic feature of all
three cases was the presence of i(2)(q10). An example of the karyotype
from case no. 3, illustrating the i(2)(q10), is shown in Fig
2. The presence of inv(2)(p23q35) was
initially detected in studies performed on case no. 1 after two-color
FISH assay using an ALK P1 clone and 5q35 cocktail probes failed to identify the typical t(2;5)(p23;q35).17 Rather,
this cytogenetically cryptic abnormality resulted in the relocation of
this ALK P1 clone, which contains the 3 portions of the locus that are typically incorporated into the NPM-ALK fusion gene
from 2p23 to 2q35 instead of to the NPM locus at 5q35 (Fig
3A). The observed pattern of
ALK P1 clone hybridization also suggested that the associated
i(2)(q10) chromosome in this case is composed of two long arms produced
by the inv(2)(p23q35) [ider(2)(q10)inv(2)(p23q35), further referred to
as simply i(2)(q10)]. These suspected chromosome 2 abnormalities were
further confirmed by FISH using the 2p and 2q subtelomeric probes,
86J13 and 210E14, respectively. Clone 86J13 hybridized to the short (p)
arm of normal chromosome 2, to the long (q) arm of the inv(2)(p23q35),
and to both arms of the i(2)(q10) (Fig 3B), whereas clone 210E14 showed
a hybridization signal on the q arm of normal chromosome 2 and the p
arm of the inv(2)(p23q35) (data not shown).
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| Fig 3.
Examples of FISH experiments performed on case
no. 1 (A through D), case no. 2 (E), and case no. 3 (F). Applied probes
included ALK P1 (red) and 5q35 cosmids (green) (A), 86J13
(2psubtel) (B), LSI ALK (5 end of ALK [green]; 3
end of ALK [red]) (C), ALK P1 (red) and 914E7 (green)
(D through F), and D2Z (red) (E). Long arrows, short arrows, and
arrowheads indicate the normal chromosomes 2, inv(2)(p23q35), and
i(2)(q10), respectively.
|
|
Considering that a rearrangement of the ALK gene by
inv(2)(p23q35) could not be unequivocally demonstrated using the
ALK P1 probe containing only the 3 end of ALK, we
applied the recently developed Vysis LSI ALK FISH assay that
consists of two differently labeled probes for the centromeric (5) and
telomeric (3) sides of the t(2;5)(p23;q35) breakpoint of the
ALK gene. Using this assay, ALK rearrangement was
readily demonstrated by separation of the centromeric (green) and
telomeric (red) ALK probes on the inv(2)(p23q35) chromosome
(Fig 3C).
To determine the position of the 2q35 breakpoint of the inv(2)(p23q35),
FISH with a panel of YACs previously mapped to 2q35-q37 and with probes
from the SHIP gene (2q37) was performed using metaphases from
case no. 1. The results are summarized in Table 2. Briefly, two YAC
clones (947F4 and 884F10) showed signals on the long arms of the
inv(2)(p23q35) and i(2)(q10), six 2q probes (951B8, 743C9, 749F2,
770F5, 752E10, h4.2-A2/Hg4.1-A2[SHIP]) hybridized to the
short arm of the inv(2)(p23q35) and did not hybridize with the
i(2)(q10), and the 914E7 YAC showed a split signal on the p and q arms
of inv(2)(p23q35) and labeled both arms of the i(2)(q10), indicating
that it contained the 2q35 breakpoint region (Fig 3D). The FISH results
for case no. 1 are shown schematically in Fig 4.
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| Fig 4.
Schematic representation of the normal chromosome 2, inv(2)(p23q35), and ider(2)(q10) inv(2)(p23q35), together with the FISH
pattern of applied probes.
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Cytogenetic analysis of case no. 2 showed nine polyploid metaphases
with i(2)(q10), in addition to 15 normal cells. Despite the poor
quality of chromosomes in the polyploid cells that precluded further
karyotypic analysis, FISH experiments with the ALK P1 clone
clearly showed hybridization signals at 2p23, at 2q35, and on both arms
of the i(2)(q10) in all 7 analyzed polyploid cells (Fig 3E). Additional
application of the LSI ALK FISH assay demonstrated separation
of the 5 end and 3 end ALK probes on 1 or 2 copies of
chromosome 2 per cell (whereas in other copies of chromosome 2 both
probes hybridized together at 2p23), and the presence of the red signal
corresponding to the 3 end ALK probe on both arms of the
i(2)(q10) (not shown). These findings are consistent with the presence
of the inv(2)(p23q35) and ider(2)(q10)inv(2)(p23q35) in this case. The
number of aberrant chromosomes 2 in the cells analyzed was variable,
and either one inv(2)(p23q35) and two i(2)(q10), or two inv(2)(p23q35)
and one, or two i(2)(q10) per cell could be detected. The 914E7 YAC
clone previously found to span the 2q35 breakpoint in case no. 1 showed
the same hybridization pattern in case no. 2 (Fig 3E).
All 10 karyotyped metaphase cells from case no. 3 showed the presence
of the i(2)(q10) chromosome accompanied by additional karyotypic
abnormalities (Fig 2). FISH with the ALK P1 clone showed hybridization signals on both long arms of the i(2)(q10). However, in
contrast to the previous cases, both chromosomes 2, when present, seemed to be normal and yielded hybridization signals at 2p23 only.
Only one metaphase of a total of 41 cells tested with the ALK
P1, 86J13, and the 914E7 and 743C9 YAC probes showed the presence of
both aberrant chromosomes, inv(2)(p23q35) and i(2)(q10) (not shown).
Among the remaining analyzed metaphases, 21 had two normal chromosomes
2 and 19 cells showed the presence of only one normal chromosome 2, in
addition to the i(2)(q10). For this reason, we could not demonstrate a
split signal of the 914E7 YAC on the inv(2)(p23q35), because the
abnormality was not present in the cells analyzed in the particular
experiment from which images were captured for publication (Fig 3F).
However, considering that the 914E7 YAC clone hybridized to the
i(2)(q10), whereas the neighboring distal YAC 743C9 did not, we presume
that the 2q35 breakpoint in case no. 3 occurs in the same region as the
two previous cases.
| |
DISCUSSION |
Histopathological, cytogenetic, and FISH data presented here clearly
indicate that the inv(2)(p23q35) is a recurrent chromosomal abnormality
in ALK-positive ALCL. This variant rearrangement, in a manner similar
to the classical t(2;5)(p23;q35), targets the ALK gene on
chromosome 2 and leads to aberrant expression of its product, as
demonstrated by positive immunostaining with anti-ALK antibody. By
analogy to the t(2;5)/NPM-ALK rearrangement, it may be
predicted that the 2q35 region to which the 3 portion of the
ALK gene has been relocated by the inv(2)(p23q35) is the site
of a gene involved in a fusion with ALK and, in consequence, that this gene drives the expression and kinase activation of the
chimeric ALK protein. This partner gene has not yet been identified, but using FISH with a panel of DNA probes for 2q, we demonstrated that
the breakpoint is encompassed within the 1,750-kb insert of the 914E7
YAC clone. No known expressed sequence tags (ESTs) have been reported
in this region to date. Localization of the 2q35 breakpoint
within the same YAC clone in all three cases included in this study
indicates that the inv(2)(p23q35) is likely to result in the consistent
generation of a specific ALK fusion gene and chimeric product.
Identification of the inv(2)(p23q35) by classical cytogenetics is
difficult, because this rearrangement involves terminal bands on both
arms of chromosome 2 that are of the same size and similar banding
patterns. Given that the inv(2)(p23q35) was consistently associated
with i(2)(q10) in all cases reported here, the presence of i(2)(q10)
may serve as a useful cytogenetic indicator for this cryptic and
indiscernible aberration. Of course, PCR approaches (either RNA- or
DNA-based) designed to identify the classical NPM-ALK fusion
would be falsely negative for the involvement of the ALK gene
in the genesis of this molecular subtype of ALCL. Until the
characterization of the 2q35 gene locus altered by this abnormality,
the most efficient means to identify these lymphomas would seem to be
the use of anti-ALK immunostaining to verify expression of the protein,
followed by FISH analysis with ALK probes, as described here.
Our FISH analysis showed that the i(2)(q10) originates from the
inv(2)(p23q35) [ider(2)(q10)inv(2)(p23q35)] and thus represents a
secondary chromosomal abnormality resulting in tetrasomy of 2q and the
occurrence of two additional copies of the putative chimeric gene
involving ALK. However, in none of the cases studied, have we
observed the original subclone containing exclusively the
inv(2)(p23q35). These findings may be to some extent functionally analogous to the amplification of the Ph/BCR-ABL observed
during the development of some cases of blast crisis in chronic
myelogenous leukemia (CML)27 and to the gain
of an extra copy of the der(11)t(11;21)(q23;q11) during the
transformation of myelodysplastic syndrome (MDS) cases with the
t(11;21).28 However, in these two instances, the appearance of additional copies of an aberrant chromosome/gene is a phenomenon associated with transformation of an indolent condition to an aggressive one, whereas in the ALCL cases reported here, the i(2)(q10) occurred in all abnormal metaphases from the time of diagnosis. Whereas
a single copy of the classical t(2;5)/NPM-ALK rearrangement seems to be sufficient for malignant transformation, and a causative role for this chimera in the pathogenesis of ALCL has been recently supported by demonstration of its oncogenic properties by in vitro transformation assays and in a mouse model,29-31 the
above-described findings lead us to speculate that for unknown reasons
the oncogenic potential of the putative 2q35-ALK-encoded
fusion protein may be less pronounced or its expression level
insufficient for efficient tumor development; therefore, an extra
dosage of this chimeric gene could favor malignant transformation.
Immunohistopathology indicated that all three cases described here
represent examples of the common type of ALCL. As expected from the
monomorphic character of the neoplastic population,14 these
lymphomas were shown to express the ALK protein using both the
ALK1 (data not shown)13 and ALKc monoclonal
antibodies.17 With both antibodies, a clear cytoplasmic
staining was obtained, whereas the additional nuclear and/or
nucleolar staining that seems to characterize
t(2;5)/NPM-ALK-positive ALCL cases was not obviously present
(Fig 1). This finding is in agreement with the very recently reported
observations of Mason et al,32 who demonstrated that, in
ALCL with the (1;2)(q25;p23), an altered ALK protein accumulates only
within the cytoplasm. Moreover, these investigators observed a similar
pattern of immunostaining with an engineered TPR-ALK hybrid protein, in
which the NPM segment was replaced by the TPR homodimerization domain
that is present in the transforming TPR-MET chimeric protein. The
investigators concluded that the nuclear/nucleolar accumulation of
NPM-ALK, which occurs apparently because of heterodimerization of the
chimera with the normal NPM protein and resultant nucleo-cytoplasmic
shuttling mediated by NPM, is not a prerequisite for an oncogenic ALK
kinase activity. Rather, the NPM gene in
t(2;5)/NPM-ALK-positive ALCL functions to provide a promoter
that drives expression of the ALK gene in lymphoid cells (in
which the gene is normally silent) and also to activate the ALK kinase
domain by encoding a homodimerization motif to mimic ligand-induced
receptor cross-linking. Our observation that the putative ALK fusion
protein produced by the inv(2)(p23q35) is found within the cytoplasm
only suggests that the 2q35 partner gene altered by the abnormality
encodes a cytoplasmic protein with oligomerization capabilities. It is
noteworthy that a significant minority (about 20%) of ALK-positive
lymphomas reported in a recent study of 123 cases possessed anti-ALK
immunoreactivity that was restricted to the cytoplasm16
and, therefore, perhaps due to ALK rearrangements other than
NPM-ALK. Our identification of the inv(2)(p23q35) in 3 of 12 ALK-positive ALCL cases collected in our laboratory and documented by
cytogenetic and/or by FISH (10 cases in Pittaluga et
al14 and 2 additional cases reported here) suggests that
this genetic abnormality could be responsible for the development of a
considerable number of these lymphomas, an assumption that can now be
tested with the FISH assays used here.
In conclusion, we have identified the inv(2)(p23q35) as a second
recurrent chromosomal abnormality in ALK-positive ALCL. Like the
classical t(2;5)(p23;q35), this variant rearrangement was detected in
young adults and was shown to rearrange the ALK gene at
2p23, leading to aberrant ALK protein expression detectable by
immunostaining with ALK-specific antibodies. The consistent association
of the inv(2) with the secondary chromosomal aberration [ider(2)(q10)inv(2)(p23q35)], which results in extra copies of the
rearranged ALK gene and accumulation of the aberrant ALK
protein in malignant cells that is confined to the cytoplasm, further distinguishes this genetic subtype of ALCL from other ALK-positive lymphomas. These findings should be helpful in further understanding the mechanisms by which aberrant ALK activity contributes to the development of NHL.
| |
FOOTNOTES |
Submitted July 28, 1998;
accepted August 4, 1998.
Supported in part by the Italian Association for Cancer Research
(A.I.R.C.), National Cancer Institute (NCI) Grant No. CA 69129 (S.W.M.), NCI Cancer Center CORE Grant No. CA 21765, and the American
Lebanese Syrian Associated Charities (ALSAC), St. Jude Children's
Research Hospital. Also supported by a grant from the project
"Cryptic genomic changes in haematological malignancies" funded
by the Flemish government as a contribution to the action "Kom op
tegen Kanker/Vlaams Kanker Liga," and by the Biomed Concerted Action, CT 94-1703.
This text present research results of the Belgian program on
Interuniversity Poles of Attraction initiated by the Belgian State,
Prime Minister's Office, Science Policy Programming. The scientific
responsibility is assumed by the authors.
Address reprint requests to Herman Van den Berghe, Center for Human
Genetics, Herestraat 49, B-3000 Leuven, Belgium.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank Magda Dehaen, Griet Hasevoets, and Xiaoli Cui for
technical assistance and Rita Logist for help in preparation of the
manuscript. We also thank Dr Karen Pulford (The Leukaemia Research Fund
Immunodiagnostics Unit, University Department of Cellular Science, John
Radcliffe Hospital, Oxford, UK) for supplying the ALK1 monoclonal
antibody and Dr John Proffitt (Vysis, Inc) for supplying the VYSIS LSI
ALK assay probes for our use before their commercial release.
| |
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Abstract
Introduction
Abstract
