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Blood, Vol. 93 No. 2 (January 15), 1999:
pp. 632-642
Detection of Normal and Chimeric Nucleophosmin in Human Cells
By
Jacqueline L. Cordell,
Karen A.F. Pulford,
Barbara Bigerna,
Giovanna Roncador,
Alison Banham,
Emanuela Colombo,
Pier-Giuseppe Pelicci,
David Y. Mason, and
Brunangelo Falini
From the Leukaemia Research Fund Immunodiagnostics Unit, Nuffield
Department of Clinical Biochemistry and Cellular Science, John
Radcliffe Hospital, Oxford, UK; and the Institute of Hematology,
Policlinico Monteluce, Perugia, Italy.
 |
ABSTRACT |
In anaplastic large-cell lymphoma (ALCL), the (2;5) chromosomal
translocation creates a fusion gene encoding the 80-kD NPM-ALK hybrid
protein. This report describes three new monoclonal antibodies, two of
which recognize, by Western blotting, the N-terminal portion of NPM
present in the NPM-ALK fusion protein and also in two other NPM fusion
proteins (NPM-RAR and NPM-MLF1). The third antibody recognizes the
C-terminal portion (deleted in NPM-ALK) and reacts only with wild-type
NPM. The three antibodies immunostain wild-type NPM (in
paraffin-embedded normal tissue samples) in cell nuclei and in the
cytoplasm of mitotic cells. Cerebral neurones, exceptionally, show
diffuse cytoplasmic labeling. In contrast to normal tissues, the two
antibodies against the N-terminal portion of NPM labeled the cytoplasm
of neoplastic cells, in four ALK-positive ALCL, reflecting their
reactivity with NPM-ALK fusion protein, whereas the antibody to the
C-terminal NPM epitope labeled only cell nuclei. Immunocytochemical
labeling with these antibodies can therefore confirm that an
ALK-positive lymphoma expresses NPM-ALK (rather than a variant
ALK-fusion protein) and may also provide evidence for chromosomal
anomalies involving the NPM gene other than the classical (2;5)
translocation.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
IN 1984, A LARGE-CELL lymphoma,
encountered most frequently in children and young adults, was defined
on the basis of its distinctive morphology and expression of CD30
(Ki-1).1 This tumor, which came to be generally known as
anaplastic large-cell lymphoma (ALCL) or Ki-1 lymphoma, was
subsequently found to be associated with a (2;5) chromosomal
translocation.2,3
In 1994, Morris et al4 showed that this genetic anomaly
juxtaposes part of the nucleophosmin (NPM) gene on chromosome 5 to a portion of the ALK receptor tyrosine kinase gene on
chromosome 2, encoding its entire intracytoplasmic region.
Cross-linking of the resultant 80-kD NPM-ALK hybrid protein (via motifs
in the NPM moiety) activates its kinase activity,5,6 and
this appears to contribute directly to neoplastic transformation,
because transfection of murine hematopoietic cells with the NPM-ALK
gene induces a transplantable lymphoid tumor.7
The NPM gene is also involved in other neoplasia-associated
genetic abnormalities.8 In cases of acute promyelocytic
leukemia carrying the (5;17)(q33;q12) translocation, the same
N-terminal portion as that present in the NPM-ALK kinase fuses to part
of the RAR gene product.9,10 The
NPM gene can also fuse to the MLF1 gene in rare cases
of acute leukemia and myelodysplasia that carry a (3;5)(q25.1;q34-35)
chromosomal translocation.11,12 The resultant fusion
protein contains a longer sequence of the N-terminal portion of NPM
than is present in NPM-ALK (175 v 117 amino acids), and this
extra region contains a nuclear localization signal and acid residue
clusters capable of interacting with other proteins.13
The NPM-ALK fusion gene can be detected in tissue samples by
the reverse transcriptase-polymerase chain reaction (RT-PCR) technique.
However, this technique is potentially prone to artefact, a problem
that may account for discrepancies in the literature over the frequency
of the NPM-ALK gene in different categories of lymphoid
neoplasia14 and also for the controversial but unconfirmed claim that it is frequently present in Hodgkin's
disease.15-18 Furthermore, RT-PCR detection of the
NPM-ALK gene is best performed on fresh tissue and in the past
this has restricted the number of cases that could be studied.
The production of antibodies specific for the cytoplasmic portion of
the ALK protein therefore represented a significant advance in the
detection of NPM-ALK protein.19-22 Normal lymphoid tissue does not contain detectable ALK protein and a positive
immunocytochemical staining reaction usually indicates that a lymphoma
expresses the NPM-ALK fusion protein as a result of the t(2;5) anomaly.
Extensive immunohistochemical studies have now been performed using
anti-ALK antibodies,21-26 and a clearer perception has emerged of the pathologic and clinical features of ALK-positive lymphomas and of their relation to other lymphoma types. In recent publications, the term "ALK-positive lymphoma" was
proposed22,26 to distinguish these tumors from the
heterogeneous group of neoplasms covered by the term ALCL.
However, there have been a few reports of cases in which ALK-positivity
is caused by the presence of a protein other than the NPM-ALK
gene product and it is of obvious interest to recognize them. One
example is a rare large B-cell lymphoma expressing full-length ALK
protein,27 which can be recognized on the basis of its
unusual morphology and phenotype. It also shows a characteristic
granular intracytoplasmic labeling pattern for ALK that differs from
the diffuse cytoplasmic and nuclear labeling seen in classical
ALK-positive ALCL.
A case of ALK-positive ALCL carrying a (1;2) translocation has been
reported,6,21,28 and in this case, the ALK gene (at 2p23) was presumably linked to a gene on chromosome 1q25 rather than to
NPM. Two other ALK-positive lymphomas with variants of the
classical (2;5) translocation have been reported.25 In one case, fluorescence in situ hybridization (FISH) analysis
showed a cryptic NPM-ALK fusion gene, but in the other it
appeared that a gene located at 2q35 may have fused to ALK.
Other cases of ALCL with possible novel chromosomal translocations have
been reported,29-32 but they were not analyzed for ALK
expression.
One possible way to confirm that an ALK-positive case carries the (2;5)
translocation and to exclude variant ALK rearrangements would be to
immunostain for the N-terminal and the C-terminal portions of NPM,
because the former is present in NPM-ALK and the latter absent. In this
report, we describe monoclonal anti-NPM antibodies with the required
specificities that are suitable for Western blotting and that can
distinguish, by this technique, between wild-type NPM and NPM-ALK (and
also the NPM-RAR and NPM-MLF1 fusion proteins). They also label
routinely processed paraffin-embedded tissue and give differential
staining patterns that allow NPM-ALK-positive lymphomas to be
specifically detected. They are therefore of potential value for
exploring variants of the (2;5) translocation and for detecting tumors
that contain novel NPM fusion proteins.
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MATERIALS AND METHODS |
Cells and Tissues
Cell lines were cultured at 37°C in RPMI 1640 medium containing
10% fetal calf serum (FCS; Life Technologies Ltd,
Paisley, Scotland). Cytocentrifuge preparations of cell lines were made as previously described.33 Samples of human tonsil,
obtained from one of the authors' (D.Y.M.) laboratories, had been snap frozen and stored at  70°C.33 Other tissues
obtained from the same source had been fixed in formalin before
embedding in paraffin and comprised one sample each of tonsil, lymph
node, thymus, spleen, kidney, liver, pancreas, lung, brain, prostate,
and gut, and biopsies of 9 carcinomas (3 squamous cell and 2 basal cell
carcinomas of skin and 4 ductal carcinomas of breast) and of 4 cases of
ALCL. Additional routinely processed normal human tissue specimens (1 sample each of lymph node, spleen, muscle, and liver) were obtained from another of the authors' (B.F.) laboratories, as were bone marrow
biopsies that had been fixed in B5 fixative for 3 hours and decalcified
in EDTA before paraffin-embedding. All routinely processed
paraffin-embedded samples had been diagnosed by conventional histologic
criteria and immunohistologic labeling for cell lineage markers. No
differences in immunostaining were noted between material from
different sources.
Normal mouse tissues (1 sample each of lymph node, spleen, bone marrow,
muscle, and liver) were obtained in one of the authors' (B.F.)
laboratories and fixed in formalin for 24 hours or in B5 fixative for 2 hours before paraffin-embedding.
Recombinant Proteins
Proteins for immunization.
Three different recombinant proteins containing NPM were used to raise
antibodies. The monoclonal antibody NA24 (anti-N-terminus) was raised
to protein prepared in one of the authors' (D.Y.M.) laboratories as
follows: NPM-ALK cDNA plasmid in pBluescript was cut with
Pst I and the resulting 450-bp fragment, including the fusion
point with ALK, was gel-purified and cloned into the Pst I site of pBluescript SK (Stratagene, La Jolla,
CA) creating plasmid pAB192. This was then digested with
EcoRI, and the 450-bp fragment was gel-purified and cloned into
the EcoRI site of the vector pGEX1 (Pharmacia, Uppsala,
Sweden). A glutathione S-transferase (GST) fusion protein, containing the N-terminus of NPM fused to 14 amino acids from ALK, was
then expressed in Escherchia coli strain BL21 (DE3). Inclusion
bodies were prepared for immunization as previously described,34 and soluble protein for the enzyme-linked
immunosorbent assay (ELISA) was affinity purified using
glutathione-Sepharose (Pharmacia) according to the manufacturer's
instructions.
The monoclonal antibody NPMa (anti-N-terminus) was raised against
recombinant protein produced in another of the authors' (P.-G.P.)
laboratories by digesting NPM-ALK cDNA in pcDNA3 with EcoRI and Taq I. The resulting fragment was blunt ended
with Klenow enzyme and then cloned in the Sma I site of
pGEX-4T-3 (Pharmacia). The resulting GST-NPM-ALK fusion protein
included the entire NPM-ALK coding sequence except for the first nine
nucleotides of the NPM cloning sequence (which were lost during the
subcloning procedure). This plasmid was transformed into E coli
strain BL21 (DE3) and the recombinant GST-NPM-ALK fusion protein was
prepared as described.34
The third recombinant antigen used to produce the antibody NPMc was
also prepared in one of the authors' (P.-G.P.) laboratories. The
NPM cDNA was isolated from a library made from the KG1 cell line and its identity was confirmed by DNA sequence analysis. The NPM
coding sequence was then cloned into the Sma I site of the
pGEX-4T-3 vector (Pharmacia). The resulting GST-NPM fusion protein
included the entire NPM coding sequence, except for the first nine AA
(which were lost during the subcloning procedure). This plasmid was
transformed into E coli strain BL21 and the recombinant GST-NPM
fusion protein was prepared as described previously.34
Eukaryotic expression of NPM proteins.
A cDNA fragment corresponding to the whole open reading frame of
NPM-ALK protein was generated by PCR of total cDNA from the Karpas cell
line, using oligonucleotide primers spanning from the NPM ATG
to the ALK TGA triplets. The PCR product was cloned into the
pCR2.1 TA cloning system (Invitrogen, San Diego, CA), checked by
sequencing analysis, and subcloned into the eukaryotic expression
plasmid pCDNA3 (Invitrogen). The NPM and MLF1
full-length cDNAs were isolated by the PCR technique using total cDNA
from the KG1 cell line as a template. RAR cDNA was already
available in the laboratory.35
NPM-MLF1 and NPM-RAR chimeric cDNAs identical to the
natural fusion genes were then constructed by recombinant PCR using the
appropriate full-length cDNAs described above. The PCR-generated cDNAs
were checked by sequencing and then cloned into the pCDNA3 expression
plasmid. Aliquots of the CV1 cell line were transfected with the three
plasmids.35 After 36 hours, the transfected cells were
lysed in Laemmli buffer,36 and 30-µg aliquots of total lysate were analyzed by Western blotting.
Production of Monoclonal Antibodies
Monoclonal antibodies were raised by conventional techniques against
recombinant proteins.37,38 Antibody NA24 was obtained by
screening (by the ELISA technique) for supernatants that reacted with
NPM-ALK-GST recombinant protein but not with GST. Supernatants were
then screened by immunocytochemical labeling against the t(2;5)-positive SU-DHL-1 cell line.21
The fusions producing antibodies NPMa and NPMc were screened by an
immuno-alkaline phosphatase technique (APAAP)39 on paraffin sections of human tonsil and of a biopsy from a t(2;5)-positive ALCL
known to express the NPM-ALK protein.
Anti-ALK Antibodies
The ALK1 and ALKc antibodies were produced in the authors'
laboratories, as described previously.21,22
Immunocytochemical Labeling
Acetone-fixed cryostat sections were stained by immunoperoxidase or
APAAP techniques, as described previously.39 Sections of
paraffin-embedded tissue on Superfrost Plus slides (Speci-Microsystems Ltd, Carshalton, Surrey, UK) were dewaxed and then subjected to microwave treatment as described previously.40 Antibodies
were used as undiluted tissue culture supernatants.
Biochemical Characterization
Western blotting of cell extracts and of the three NPM fusion proteins
was performed by conventional techniques.21,36
| |
RESULTS |
Western Blotting With Anti-NPM Antibodies
When the monoclonal antibody NPMc was tested by Western blotting, it
detected a single band in extracts both of reactive lymphoid tissue
(tonsil) and of the t(2,5)-positive cell line SU-DHL-1 (Fig 1A). In contrast, antibodies NA24 and
NPMa both detected an additional band in the SU-DHL-1 extract, but not
in the extract of normal tonsil. This band corresponded in size (80 kD)
to the NPM-ALK fusion protein, and it was concluded that these two
antibodies detect epitopes in the N-terminal portion of NPM conserved
in the fusion protein, whereas NPMc detects an epitope encoded on the
C-terminal side of the NPM breakpoint. The antibodies also showed
differential reactivity by Western blotting against two recombinant
fusion proteins, NPM-MLF1 and NPM-RAR (Fig 1B).
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| Fig 1.
Western blotting reactivity of monoclonal anti-NPM
antibodies. (A) Wild-type NPM (38 kD) is detected in lysates of both
normal cells (tonsil) and of the t(2;5)-positive cell line SU-DHL-1
with each of the three antibodies. However, only the two antibodies to
the N-terminal region (NA24 and NPMa) react with the hybrid NPM-ALK
protein (80 kD). (B) The two antibodies to the N-terminal region (NA24
and NPMa) detect recombinant NPM-ALK and also recombinant NPM-MLF1 and
NPM-RAR proteins. Antibody NPMc (against the C-terminal region) is
unreactive with any of these fusion proteins.
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Immunocytochemical Staining
Normal tissues.
The anti-NPM antibody recognizing the C-terminus (antibody NPMc)
stained nucleoli in cytospin preparations of cell lines
(Fig 2) and in cryostat tissue sections. In
contrast, the two antibodies against the N-terminus gave only weak or
negative reactions on cytospin preparations and cryostat sections.
However, all three antibodies gave strong nuclear labeling in both
human and mouse tissues when paraffin-embedded tissue samples were
tested (Fig 2), and their reactivity was essentially indistinguishable.
The reactivity within nuclei was diffuse, although at higher antibody dilutions nucleolar labeling became evident. The intensity of nuclear
staining varied from cell to cell within individual sections, regardless of the antibody used, and epithelial cells tended to give
the most uniform labeling.
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| Fig 2.
Immunostaining of cytocentrifuged cell lines and
paraffin-embedded normal tissues for NPM (APAAP technique). Cell lines:
The t(2;5)-positive cell line Karpas 299 (a) and a murine mastocytoma
cell line (b) both show labeling of nucleoli with antibody to the
C-terminal portion of NPM (antibody NPMc). Note the cytoplasmic
labeling of a mitotic cell in (b). Kidney: (a) Many cells show nuclear
labeling for nucleophosmin. (b) and (c) show high power views of,
respectively, a glomerulus and tubular epithelium (antibody NA24).
Thymus: Two different anti-NPM antibodies give essentially identical
labeling patterns of cell nuclei in a Hassall's corpuscle and in the
surrounding medullary tissue. Tonsil: Nuclear labeling of lymphoid
cells is seen. The arrows in the low power view (left) indicate
scattered mitotic cells in which there is cytoplasmic labeling, seen at
high power on the right (antibody NA24). Cerebral cortex: Neural cells
show strong cytoplasmic staining, seen at low power (above) and high
power (below) (antibody NA24).
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Diffuse cytoplasmic staining was noted with all three antibodies in
human cerebral neuronal cells (Fig 2) and in 5% to 30% of
megakaryocytes in both normal and reactive bone marrow samples. The
only other normal cells that showed cytoplasmic labeling were mitotic
cells, for example, in the germinal centers of reactive lymphoid tissue
(Fig 2). Rare scattered nonmitotic cells also showed cytoplasmic
staining, and these often lay adjacent to each other. The nuclei of
erythroid precursors were frequently unstained with the anti-NPM
antibodies.
Neoplastic tissues.
A number of human neoplasms were investigated with the anti-NPM
antibodies (see Materials and Methods). The immunostaining pattern of
carcinomas with each of the antibodies was essentially identical to
that seen in normal tissues, ie, ubiquitous labeling of nuclei, and
diffuse cytoplasmic labeling of scattered mitotic and nonmitotic cells
(Fig 3).
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| Fig 3.
In a squamous cell carcinoma, the nuclei of all cells are
labeled (antibody NA24 APAAP technique). Occasional mitotic cells
(arrows) show diffuse cytoplasmic labeling.
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Four cases that fulfilled the conventional criteria for ALK-positive
lymphoma22,26 (and that showed nuclear and cytoplasmic immunostaining with each of the 2 anti-ALK antibodies) were tested with
the anti-NPM antibodies. The two anti-N-terminal antibodies NA24 and
NPMa (which react with both wild-type NPM and NPM-ALK) stained, as
expected, the nuclei of neoplastic cells, but also gave diffuse
cytoplasmic labeling (Figs 4 and
5). This was in contrast
to the immunostaining pattern of antibody NPMc, recognizing the
C-terminal region, which (apart from the expected cytoplasmic staining
of scattered mitotic and nonmitotic cells) was restricted to cell
nuclei (Figs 4 and 5).
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| Fig 4.
Staining of paraffin sections of an ALK-positive ALCL
(APAAP technique). (Upper) The reactivity of the same area of lymph
node with antibodies to the C-terminal epitope (antibody NPMc) and to
the N-terminal region (antibody NA24) is seen at low power (above) and
high power (below). The tumor cells surround a residual area of normal
B cells (asterisks). A clear difference between the labeling patterns
of the two antibodies is seen even at low magnification, accounted for
by the reactivity of the anti-N-terminal antibody with tumor cell
cytoplasm. Scattered mitotic cells showing diffuse cytoplasmic labeling
are seen in both sections (arrowed) and can be picked out in the
low-power view of the immunostaining for the C-terminus. (Lower) The
appearance of the tumor in sections stained by hematoxylin and eosin
and for ALK protein is shown at low power (left) and high power
(right).
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| Fig 5.
Staining of paraffin sections of a second
ALK-positive ALCL seen at low (left) and high (right) power (APAAP
technique). As in the case shown in Fig 4, a clear difference is seen
between the immunostaining for the C-terminal epitope (antibody NPMc)
and an N-terminal epitope (antibody NPMa). Immunostaining for ALK is
also shown. In the lower power view, the corresponding areas in three
sections are shown. The asterisks indicate residual normal lymphoid
tissue surrounded by tumor cells.
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DISCUSSION |
Nucleophosmin was first identified more than 15 years ago as B23, an
acidic 37-kD phosphoprotein associated with cell
nucleoli.41 Subsequently, the nuclear matrix protein
numatrin, which had been independently identified, was shown to be
identical to B23/nucleophosmin.42 It was evident from the
earliest studies, and confirmed by immunoelectron microscopy,43 that nucleophosmin is associated with the
nucleolus,44 although it was also recognized that, under
certain conditions (eg, treatment with cytotoxic drugs), it may enter
the nucleoplasm.45-47 However, it was reported in 1989 by
Borer et al48 that nucleophosmin shuttles continuously
between the cytoplasm and the nucleolus and that this reflects its role
as a carrier of newly synthesized ribosomal proteins into the
nucleolus.
Both polyclonal and monoclonal antibodies to nucleophosmin have
been reported, but their main use has been in biochemical or
immunocytochemical analysis of cell lines.41,42,44-47,49 Only a few studies of the immunocytochemical distribution of
nucleophosmin in human tissues have been reported, and these have
confirmed its ubiquitous expression in both normal and neoplastic
cells.50,51
In the present report, we describe three monoclonal antibodies that
detect NPM in paraffin-embedded tissue samples and confirm its
ubiquitous distribution within cell nuclei. Most previous immunocytochemical studies have emphasized its nucleolar localization, and it has been claimed that in colorectal tumors a change from a
nucleolar to a diffuse nuclear pattern is associated with
adenoma/carcinoma progression.51 However, in our
experience, nucleolar labeling was not obvious, but this probably
reflects NPM diffusion during tissue fixation, because a comparable
difference between cryostat and paraffin-embedded sections has been
noted previously.50 We also found that nucleophosmin is
present diffusely within the cytoplasm of mitotic cells, as has been
observed in cultured cells,52,53 and in a previous
immunohistologic study.51 We interpret the rare scattered
cells in all normal tissues that show cytoplasmic staining as cells
that have recently undergone mitosis (because such cells were often
seen lying in pairs adjacent to each other).
One novel observation was the diffuse cytoplasmic labeling of cerebral
neurones (Fig 2). There is evidence that phosphorylation of NPM is
implicated in the initiation of mitosis,54,55 so that its
unique distribution in neural cells, which are known to be nondividing,
is of interest and merits further study.
Two of the antibodies (NA24 and NPMa) recognize the N-terminal portion
of NPM, which is preserved in the fusion protein encoded by the
NPM-ALK fusion gene, whereas the third (NPMc) is specific for
the C-terminal region (absent from this hybrid gene product). Western
blotting with polyclonal anti-NPM antisera has been used previously to
demonstrate the differing molecular sizes of wild-type NPM and NPM-ALK
fusion proteins,5,6 but this is the first study in which
monoclonal antibodies against different regions of the NPM molecule
have been used to distinguish between these molecules.
The ALCLs tested in this study showed diffuse cytoplasmic staining with
antibody against the N-terminal NPM sequence present in NPM-ALK,
whereas the cytoplasm of the same cells was unstained with antibody to
the C-terminal portion of NPM. The cytoplasmic staining for the
N-terminal portion of NPM (preserved in NPM-ALK) mirrored the labeling
pattern obtained with anti-ALK (Figs 4 and 5), and the immunostaining
with antibodies to the N-terminal portion of NPM reactivity presumably
represents reactivity with cytoplasmic NPM-ALK, as shown schematically
in Fig 6.
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| Fig 6.
Schematic diagram of NPM and ALK proteins in
normal cells and in t(2;5)-positive lymphoma cells and the resulting
immunostaining patterns. This scheme assumes that NPM and NPM-ALK are
present in both monomeric and dimeric forms in approximately equal
numbers, and does not show larger oligomers that might
form.5 It may thus represent an oversimplification of what
occurs in vivo. (a) In a normal cell, NPM is confined to the nucleus.
In a cell carrying the (2;5) translocation, NPM-ALK is found in the
cytoplasm and other NPM-ALK that has heterodimerized with wild-type NPM
has acquired nuclear localization. (b) Immunostaining for N- and
C-terminal epitopes of NPM and for ALK show distinctive patterns,
reflecting the localization of the proteins in the scheme shown
above.
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The N-terminal antibodies also detected the chimeric proteins encoded
by the NPM-RAR and NPM-MLF1 fusion genes, which were created, respectively, by the (5;17) and the (3;5) chromosomal translocations.9,11,12 The first of these anomalies is
occasionally found in acute promyelocytic leukemia and the other has
been reported in rare cases of myelodysplasia/acute myeloid leukemia.
Because of their rarity, we have not studied any fresh clinical samples carrying these genetic lesions, but it is possible that differential staining with anti-N-terminal and anti-C-terminal anti-NPM
antibodies, of the type seen in ALCL (Figs 4 and 5), would be obtained.
More importantly, these antibodies could be used in the
immunocytochemical screening of human neoplasms for novel genetic
rearrangements that involve the NPM gene. Even if such
anomalies occur only rarely, they might identify novel fusion partners
for NPM that are involved more widely in neoplasia.
Immunostaining with these anti-NPM antibodies may also offer, in the
context of ALCL, a means of detecting genetic anomalies that fuse the
ALK gene to a gene other than NPM. In classical ALCLs
carrying the (2;5) translocation, NPM-ALK protein is found not only in
the cytoplasm, but also within the nucleus,6,21 reflecting
heterodimer formation between NPM-ALK and wild-type NPM. Nuclear
localization motifs are absent from NPM-ALK, but their presence in the
wild-type protein causes the NPM/NPM-ALK heterodimers/oligomers to
relocate to the nucleus.5,6 In contrast, if ALK
fuses to a gene other than NPM the resultant hybrid protein is
likely to be oncogenic if its kinase function is activated (eg, by
cross-linking), but it would not show any tendency to move to the
nucleus (unless, by chance, the partner protein was also
nuclear-associated). This has been directly demonstrated using
artificial constructs in which a cross-linking sequence (TPR) with no
affinity for the cell nucleus was fused to ALK.5,6 Expression of TPR-ALK in vivo and in vitro caused cell transformation, but the protein did not localize to the nucleus.
A (1;2) translocation, which is presumed to fuse an unknown gene on
chromosome 1 to the ALK gene on chromosome 2, has been reported
in a single case of ALCL.6,21,28 As predicted, the neoplastic cells in this lymphoma expressed an ALK protein that was
restricted to the cytoplasm.6 In two recent studies it has
been noted that approximately 15% of ALK-positive large-cell lymphomas
show this pattern,22,26 but little cytogenetic or RT-PCR
data were available for these cases. It would be of interest to
investigate biopsy samples from such cases to see if the predicted nuclear-restricted staining pattern is obtained with the
anti-N-terminal NPM antibodies reported in this study. Cases could
then be studied further by biochemical and molecular biological
techniques, offering the possibility of identifying novel oncogenic
sequences that may be implicated in other tumor types.
| |
ACKNOWLEDGMENT |
The SU-DHL-1 cell line was kindly provided by Dr M.L. Cleary (Stanford,
CA) and the NPM-ALK plasmid was kindly provided by Dr Stephan
Morris (Memphis, TN).
| |
FOOTNOTES |
Submitted June 8, 1998;
accepted September 17, 1998.
Supported by the Leukaemia Research Fund of Great Britain and A.I.R.C
(Associazione Italiana per la Ricerca sul Cancro).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests or requests for antibodies to David Y. Mason,
MD, Hematology Department, John Radcliffe Hospital,
Oxford, OX3 9DU, UK (antibody NA24) or to Brunangelo Falini,
MD, Institute of Hematology, Policlinico Monteluce, 06100 Perugia, Italy (antibodies NPMa and NPMc).
| |
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Top
Abstract
Introduction