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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 12 (June 15), 1998:
pp. 4668-4676
Characterization of t(2;5) Reciprocal Transcripts and Genomic
Breakpoints in CD30+ Cutaneous Lymphoproliferations
By
M. Beylot-Barry,
A. Groppi,
B. Vergier,
K. Pulford, and
J.P. Merlio for the French Study Group of Cutaneous Lymphoma
From CHU of Bordeaux and University of Bordeaux II, Bordeaux, France;
LRF Immunodiagnostics Unit, John Radcliffe Hospital; and the French
Study Group of Cutaneous Lymphoma, Créteil, France.
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ABSTRACT |
NPM-ALK chimeric transcripts, encoded by the t(2;5), lead to an
aberrant expression of ALK by CD30+ systemic lymphomas.
To determine if t(2;5) is involved in cutaneous lymphoproliferative
disorders, we studied 37 CD30+ cutaneous
lymphoproliferations, 27 mycosis fungoides (MF), and 16 benign
inflammatory disorders (BID). NPM-ALK transcripts were detected by
nested reverse transcription-polymerase chain reaction (RT-PCR) in 1 of 11 lymphomatoid papulosis (LyP), 7 of
15 CD30+ primary cutaneous T-cell lymphoma (CTCL), 3 of
11 CD30+ secondary cutaneous lymphoma, 6 of 27 MF, and 1 of 16 BID. However, the expression of NPM-ALK transcripts was not
associated with ALK1 immunoreactivity in MF, LyP, or BID cases. Only 1 CD30+ primary CTCL and 3 CD30+ secondary
cutaneous lymphoma were ALK1 immunoreactive. The ALK1+
cases were also characterized by amplification of tumor-specific genomic breakpoints on derivative chromosome 5. These cases, except for
1 secondary cutaneous lymphoma, were also characterized by reciprocal
breakpoints on derivative chromosome 2, leading to the expression of
reciprocal ALK-NPM transcripts. Amplification of chromosomal
breakpoints on both derivative chromosomes could represent an
alternative to conventional cytogenetics for the diagnosis of t(2;5)
and seems to be more reliable than the detection of cryptic NPM-ALK
transcripts by nested RT-PCR.
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INTRODUCTION |
UNLIKE SYSTEMIC CD30+
lymphomas with cutaneous involvement, CD30+ primary
cutaneous T-cell lymphomas (CTCL) are characterized by an indolent
course with spontaneous remission and good prognosis.1-4 Other CD30+ cutaneous disorders, such as lymphomatoid
papulosis (LyP), also belong to the spectrum of CD30+
lymphoproliferations and may be associated in some instances with
CD30+ CTCL or mycosis fungoides (MF).4-7 No
single morphological or biological feature can be used as a gold
standard to differentiate CD30+ primary cutaneous lymphoma
from CD30+ systemic lymphoma at the time of diagnosis and
therapeutic choice.3 Moreover, descriptions of a common
clonal cell origin between LyP and CD30+ cutaneous and
systemic lymphoma in the same patients8,9 have suggested
that these entities may be biologically related in some instances.
The t(2;5)(p23;q35) translocation fuses the NPM (nucleophosmin) gene at
5q35 with the newly identified ALK (anaplastic lymphoma kinase) gene at
2p23.10 This results in the expression of a chimeric fusion
protein NPM-ALK/p80 containing the entire intracellular portion of ALK,
including its tyrosine kinase domain but lacking its extracellular and
transmembrane domains.10,11 Whereas ALK protein is not
expressed by normal lymphocytes, the ubiquitously activated NPM
promoter drives the expression of a chimeric NPM-ALK protein with
oncogenic properties that may contribute to
lymphomagenesis.10,12-14 NPM-ALK transcripts have been
detected in a significant proportion (ranging from 16% to 66%) of
systemic CD30+ T-cell lymphoma.15-20 We and
others18 have shown the presence of NPM-ALK transcripts in
a subset of CD30+ cutaneous lymphoproliferations, including
LyP cases.21 However, several studies have suggested that
the absence of t(2;5) may be a common feature of CD30+
cutaneous lymphoproliferations, as opposed to its presence in CD30+ systemic lymphomas.17,20,22-24 Such
discrepancies have been observed for Hodgkin's
disease,25-31 suggesting either polymerase chain reaction
(PCR) artifacts or the presence of normal cells expressing NPM-ALK
transcripts within the above lymphoproliferations.32,33 This led us to further characterize NPM-ALK breakpoints and transcripts in CD30+ cutaneous lymphoproliferations. Therefore, we
designed DNA-PCR assays for the detection of the t(2;5) breakpoints on
both derivative chromosomes 5 and 2. We also developed a reverse
transcription-PCR (RT-PCR) assay for the detection of
the ALK-NPM reciprocal transcript. The findings were analyzed in view
of the results of immunodetection of the chimeric protein with either
the monoclonal ALK1 antibody or the polyclonal anti-p80 antibody.
Furthermore, we studied a larger series of CD30+ cutaneous
lymphomas, LyP, and a group of MF and benign inflammatory skin
disorders (BID) to determine whether NPM-ALK transcripts or protein
could be detected in epidermotropic T-cell lymphoma or in benign
cutaneous disorders.
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MATERIALS AND METHODS |
Patient samples.
This multicentric study included 37 patients with a CD30+
cutaneous lymphoproliferation. Twenty-three of them were included in a
previous study.21 The diagnosis was revised by the French Study Group of Cutaneous Lymphoma both for clinical and
histopathological data, especially for LyP cases. Expression of CD30
antigen by more than 75% of lymphomatous cells was required for the
diagnosis of CD30+ lymphoma. After a complete initial
staging procedure and a minimum 6 months of clinical follow-up, these
37 cases were divided into three anatomo-clinical groups: (1) LyP (n = 11), (2) CD30+ primary CTCL without extracutaneous
involvement for at least 6 months after diagnosis (n = 15, including 3 cases with a past record of LyP), and (3) CD30+ secondary
cutaneous lymphoma arising either in the course of a CD30+
systemic lymphoma (n = 4) or de novo with concomitant cutaneous and
systemic involvement (n = 7). In addition, 27 MF and 16 BID such as
eczemas (n =12) and psoriasis (n = 4) were studied by RT-PCR, DNA-PCR,
and immunohistochemistry.
Immunohistochemistry.
After a high-pressure cooking antigen-retrieval procedure, a
three-stage streptavidin-peroxidase assay (Dako, Les Ullis, France) was
used for the detection of the CD30, CD3, and L26 antigens (Dako). The
LSAB kit was used with the monoclonal ALK1 antibody34 and a
StreptABC HRPkit (Dako) was used with the polyclonal
anti-p8035 (Nichirei Co, Tokyo, Japan). These antibodies
were both applied at a 1:50 dilution for 16 hours at 4°C.
Primers and probes.
All primers were purchased from Eurogentec (Seraing, Belgium). Standard
procedures of RT-PCR for NPM-ALK transcript and genomic PCR (DNA-PCR)
on derivative chromosome 5 used the primers 5 NPM (5-TCCCTTGGGGGCTTTGAAATAACACC-3) and 3ALK
(5-CGAGGTGCGGAGCTTGCTCAGC-3), whereas nested procedures
used the internal primers 5NPMint
(5CCAGTGGTCTTAAGGTTGAAG-3) and 3ALKint
(5-TTGTACTCAGGGCTCTGCAGC-3), as previously described (Fig 1).21 To amplify the
reciprocal ALK-NPM breakpoints and cDNA, for the standard PCR we used
the primers 5ALK2 (5-ATCCTCTCTGTGGTGACCTC-3) and
3NPM2 (5-TGGAACCTTGCTACCACCTC-3).36
Internal primers 5ALK2int
(5-CCCTCGTGGCCGCCCTGGTC-3) and 3NPM2int
(5-GGGGCAGACCGCTTTCCAGA-3) were designed for nested
rounds. Amplifications were performed on a Hybaid OmniGene automated
thermal cycler (Hybaid, Teddington, UK). A junction-specific oligoprobe
NPM-ALK-J
(5GCTCCTGGTGCTTCCGGCGGTACACTACTAAGTGCTGTCCACT-3) was used
for Southern hybridization of NPM-ALK amplified cDNA, as previously
described.21 Southern analysis of the reciprocal ALK-NPM
amplified cDNA used the junction-specific oligoprobe ALK-NPM-J (5-TCCGGCATCATGATTGCTGTGGAGGAAGAT-3). Southern
hybridization of DNA-PCR products was performed with two exonic probes
flanking both sides of the breakpoint either on derivative chromosome 5 (NPM-5P, 5-GTGGTTCAGGGCCAGTGCATATT-3, and ALK-5P,
5-ATCTGCATGGCTTGCAGCTCCTG-3) or on derivative chromosome
2 (NPM-2P, 5-TATACTTAAGAGTTTCACATCC-3, and ALK-2P,
5-GTCCTGGCTTTCTCCGGCAT-3).
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| Fig 1.
Schematic diagram of NPM and ALK regions rearranged by
t(2;5) (according to Ladanyi and Cavalchire55) and mRNA.
NPM and ALK exons, whose normal sizes are unknown, are shown as open
boxes, and single lines represent introns. The relative approximate
positions of PCR primers and probes used in our study are indicated.
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RT-PCR detection of chimeric t(2;5)-encoded NPM-ALK transcript and
ALK-NPM reciprocal transcript.
RT-PCR was performed on total RNA extracted with Trizol reagent
(GIBCO-BRL, Gaithersburg, MD) from a 500-µm thick section of frozen
skin biopsy.21 The final reaction mix of reverse
transcription contained 1.5 µg of total RNA, 500 pmol of random
hexamers [pd(N)6; Boehringer Mannheim, Mannheim, Germany], 50 mmol/L
Tris-HCl, pH 8.3, 75 mmol/L KCl, 3 mmol/L MgCl2, 500 µmol/L of each deoxynucleotide-5-triphosphate, and 300 U of
Superscript II RT (GIBCO-BRL) in a final volume of 25 µL. Reverse
transcription was performed at 37°C for 60 minutes. After
heat-inactivation of the reverse transcriptase at 95°C for 2 minutes, half of the cDNA was amplified by PCR. The final reaction mix
contained 10 mmol/L Tris-HCl, pH 9, 50 mmol/L KCl, 1.5 mmol/L MgCl2, 200 µmol/L of each
deoxynucleotide-5-triphosphate, 0.1% Triton X-100, 25 pmol of
each primer (5NPM and 3ALK for NPM-ALK amplification and
5ALK2 and 3NPM2 for ALK-NPM amplification), and 1.5 U of
Taq polymerase (Promega, Madison, WI) in a final volume of 100 µL
overlaid with 75 µL of mineral oil. Tubes were heated at 94°C for
5 minutes and then were subjected to 40 cycles of PCR by denaturing at
94°C for 1 minute, annealing at 60°C for 1 minute, and
extending at 72°C for 2 minutes with a touch-down protocol that
decreased the annealing temperature by 1°C every 6 cycles from
60°C to 55°C. One microliter of the standard PCR product was
used as template for the nested PCR with the following nested primers:
5NPMint, 3ALKint for NPM-ALK amplification and 5ALK2int, 3ALK2int for ALK-NPM amplification. Each of the
36 amplification cycles was composed of a 1-minute
denaturation step at 94°C, a 1-minute annealing step at 60°C,
and a 1-minute elongation step at 72°C. The standard and nested
RT-PCR products (10 µL) were electrophoresed on a 2% NuSieve Agarose
gel (FMC, Rockland, MA), stained with ethidium bromide, and
photographed under UV light.
PCR amplification of t(2;5) breakpoints on derivative chromosomes 2 and 5.
DNA was extracted from frozen skin biopsies using a standard
phenol:chloroform protocol.37 The reaction mix contained
250 ng of genomic DNA, 10 mmol/L Tris-HCl, pH 8.3, 50 mmol/L KCl, 1.5 mmol/L MgCl2, 200 µmol/L of each
deoxynucleotide-5-triphosphate, 25 pmol of each primer
(5NPM and 3ALK for NPM-ALK or 5ALK2 and
3ALK2 for ALK-NPM), and 1.25 U of AmpliTaq polymerase
(Perkin-Elmer, Norwalk, CT) in a final volume of 50 µL overlaid with
75 µL of mineral oil. The tubes were heated at 94°C for 5 minutes
and then subjected to 36 cycles of PCR by denaturing at 94°C for 1 minute, annealing at 60°C for 1 minute, and extending at 72°C
for 3 minutes with a touch-down protocol that decreased the annealing
temperature by 1°C every 6 cycles from 60°C to 55°C. Three
microliters of a 1:8,000 dilution of the standard PCR products was used
as template for 36 cycles of nested PCR with the internal primers
(5NPMint and 3ALKint for NPM-ALK or 5ALK2int, and
3ALK2int for ALK-NPM). Standard or nested PCR products (10 µL)
were electrophoresed on a 1% NuSieve Agarose gel, stained with
ethidium bromide, visualized, and photographed under UV light.
Controls.
For the RT-PCR and DNA-PCR assays, the cDNA and genomic DNA of SU-DHL1
cell line (gift of Dr M. Cleary, Stanford University, Stanford,
CA) were used as positive controls. Titration studies were performed for the DNA-PCR on derivative chromosomes 2 and 5, using
SU-DHL1 DNA diluted in reactive lymph node DNA. The amplification of a
3,016-bp  -globin gene fragment was performed with primers 5Globin (5-GAAGAGCCAAGGACAGGTAC-3) and
3Globin (5-GTTTGATGTAGCCTCACTTC-3) for all DNA
samples to check the feasibility of long-range PCR. To avoid
cross-contamination, extraction of nucleic acids was performed in an
independent laboratory. Amplification, electrophoresis, and nested
procedure were performed in separate rooms. Nested RT-PCR without the
reverse transcription step and amplification of the reaction mix
without template were performed as negative controls. All results were
reproduced by two independent experiments for each sample.
Southern blot analysis.
PCR products (10 µL) electrophoresed on agarose gels were blotted
onto nylon membranes (Hybond-N+; Amersham International, Buckinghamshire, UK). Membranes were prehybridized and then hybridized at 42°C overnight in a solution of 5× standard saline citrate (SSC), 5× Denhardt's solution, 0.5% sodium dodecyl sulfate
(SDS), 0.2 g/L of salmon testes sonicated denatured DNA (Sigma, St
Louis, MO), and the appropriate  32P-dATP oligonucleotide
probe labeled at the 3end using terminal transferase
(GIBCO-BRL). Blots were washed twice in 2× SSC, 0.1% SDS for 10 minutes at room temperature and then twice in 1× SSC, 0.1% SDS
for 20 minutes at 5°C below the theoretical Tm. Blots were exposed
to x-ray film (Kodak X-Omat, Rochester, NY) with intensifying screens
at  80°C. Probes were removed from membranes by the
dehybridization procedure described by the manufacturer to be analyzed
with another probe after a radioautographic control.
Sequencing.
PCR products (35 µL) were purified through MicroSpin S-300 Columns
(Pharmacia Biotech, Uppsala, Sweden) and sequenced on both DNA strands
using the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit
(Perkin Elmer Applied Biosystems, Foster City, CA) on an automated
Applied ABI 377A DNA sequencer (Perkin Elmer Applied Biosystems).
Nucleotide sequence data were analyzed using the Sequence Navigator
software (Perkin Elmer Applied Biosystems). Sequence comparisons were
made with the Genbank database by using the Wisconsin Package (Genetics
Computer Group, Inc, Madison, WI), FASTA38
and BLAST39 programs.
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RESULTS |
The age of the patients with CD30+ cutaneous
lymphoproliferations (20 men and 17 women) ranged from 5 to 92 years
(median, 49 years; Table 1). Thirty-four
cases had a T-cell phenotype and 1 case of secondary cutaneous lymphoma
had a B-cell phenotype. Two cases of CD30+ primary
cutaneous lymphoma had a null phenotype, but the genomic study showed a
monoclonal rearrangement of the TCR chain gene in both cases (data
not shown).
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Table 1.
Correlation Between DNA-PCR for the Derivative
Chromosome 5 and Chromosome 2, RT-PCR for the Reciprocal
Translocation, and Immunohistochemistry Using ALK1 and Anti-p80
Antibodies in the Cases With Amplifiable NPM-ALK Transcripts by
RT-PCR
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RT-PCR for NPM-ALK transcripts.
Standard PCR of the RT-PCR assay allowed the detection of NPM-ALK
transcripts in 2 of the 15 CD30+ primary CTCL (cases no. 8 and 10) and in 3 of the 11 CD30+ secondary cutaneous
lymphoma (cases no. 1, 2, and 3; Fig 2 and data not shown). Furthermore, the nested PCR allowed the detection of
the NPM-ALK transcript in 1 of the 11 LyP (case no. 4); in 7 (cases no.
5 through 11) of the 15 CD30+ primary CTCL, including the
above-mentioned 2 cases; and in the 3 previously detected cases of the
11 CD30+ secondary cutaneous lymphomas. After ethidium
bromide staining showing the same sized amplicons, these results were
confirmed both by direct sequencing and by Southern blot hybridization
with the junction-specific oligoprobe NPM-ALK-J. The study of MF and BID samples did not show any NPM-ALK transcript after the standard PCR
of the RT-PCR assay. However, nested amplification showed NPM-ALK
specific amplicons in 6 of the 27 MF and in 1 eczema of the 16 BID
samples (data not shown).
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| Fig 2.
Detection of NPM-ALK and reciprocal ALK-NPM transcripts
by nested RT-PCR. Total RNA was extracted from frozen skin biopsies or
cultured cells and submitted to nested RT-PCR analysis, followed by
electrophoresis on 2% agarose gel. Lanes 1, 2 and 3, cases no. 1, 2, and 3, respectively, CD30+ secondary CLCL; lane 4, case
no. 4, LyP; lanes 5 through 11, cases no. 5 through 11, CD30+ primary CLCL; lane 12, t(2;5)+
SU-DHL-1 cell line; lane 0, no template; lane M, molecular weight marker 100-bp DNA ladder (GIBCO-BRL). Detection of NPM-ALK transcripts: (A) ethidium bromide staining and (B) radioautography. Detection of
ALK-NPM transcripts: (C) ethidium bromide staining and (D) radioautography. The gels were transferred to a nylon membrane, hybridized either with the NPM-ALK-J probe (B) or the ALK-NPM-J probe
(D), and radioautographed. Sizes are indicated in bases.
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Immunohistochemistry.
The polyclonal anti-p80 provided a staining of some large cells of 1 of
the 11 LyP (case no. 4). A cytoplasmic
staining of lymphomatous cells was also seen in 4 of the 15 CD30+ primary CTCL (cases no. 5, 6, 8, and 9) and in 3 of
the 11 CD30+ secondary cutaneous lymphomas (cases no. 1, 2, and 3). Keratinocytes or dendritic cells of the dermis were sometimes
stained by anti-p80. All p80+ cases were previously shown
to contain NPM-ALK transcripts by nested RT-PCR. However, not all cases
with NPM-ALK chimeric transcripts were stained by p80+. No
p80+ cells were detected in MF and BID sections. The
staining with the monoclonal ALK1 antibody was cytoplasmic and
nucleolar and restricted to tumoral cells of CD30+
lymphomas. Only 1 case of CD30+ primary CTCL was
ALK1+ (case no. 8; Fig 3). This case was 1 of the 2 cases
with a positive standard RT-PCR amplification, whereas the other 1 was
found to be negative for both p80 and ALK1 immunostaining (case no.
10). The 3 cases of CD30+ secondary CTCL with NPM-ALK
transcripts were stained for ALK1 (cases no. 1, 2, and 3; Fig 3). No
ALK1-immunoreactive cell was found in LyP sections and no
labeling of the epidermis was observed. None of the cases with a
negative NPM-ALK detection by RT-PCR and none of the MF and
BID was found to be stained for ALK1.
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| Fig 3.
Immunohistochemical detection of chimeric NPM-ALK protein
(×400). A case of CD30+ primary CLCL (case no. 8; A)
and a case of CD30+ secondary CLCL (case no. 1; B) both
with a positive NPM-ALK amplification by both RT-PCR and DNA-PCR were
stained with the monoclonal ALK1 antibody. A granular cytoplasmic and
strong nucleolar staining was observed on the large lymphomatous cells
in these 2 cases.
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Amplification of t(2;5) breakpoint on derivative chromosome 5.
After standard DNA-PCR, 4 of the 11 cases with a chimeric transcript
detected by nested RT-PCR showed a specific amplicon (Fig 4). The size of the chimeric amplicons
varied from case to case, ranging between 0.8 and 2.9 kb, according to
variable intronic breakpoints.23,40 These cases, which were
previously found to contain NPM-ALK transcripts by standard RT-PCR,
corresponded to 3 CD30+ secondary CTCL (cases no. 1, 2, and
3) and 1 CD30+ primary CTCL (case no. 8). The nested
amplification allowed the detection of size-specific amplicons in the
same cases (no. 1, 2, 3, and 8) and in 2 additional CD30+
primary CTCL (cases no. 7 and 9). Titration experiments showed that the
sensitivity of DNA-PCR was 10 3 for the standard PCR
and 104 for the nested PCR (data not shown).
Southern blot hybridization of PCR products confirmed the specificity
of these results, giving a positive signal with both NPM-5P and ALK-5P
in the 3 CD30+ secondary CTCL and in one CD30+
primary CTCL (case no. 8). However, the 2 other CD30+
primary CTCL with a visible amplicon after nested DNA-PCR (cases no. 7 and 9) hybridized only with ALK-5P but not with NPM-5P. Moreover, 1 case of LyP (case no. 4) was shown to give a positive hybridization
only with ALK-5P, although no signal was detectable on ethidium
bromide-stained gel. The nested DNA-PCR amplicon of case no. 7 was
sequenced and found to contain the 3 end of the ALK exon
targeted by the 3ALKint oligonucleotide and the flanking intron,
but lacked the 5 end of the NPM exon targeted by the 5NPMint oligonucleotide. In cases no. 4 and 9, the amount of amplicon was too low to perform sequencing. Size-specific amplicons of
cases no. 1, 2, 3, and 8 were sequenced and shown to contain chimeric
intronic sequences between the flanking 5NPM and 3ALK exons. Multiple alignment analysis of these sequences with the t(2;5)
genomic nucleotide sequence of SU-DHL-122 showed a perfect homology of the ranging from both exonic extremities over a variable intronic area from case to case depending on the location of the breakpoint (data not shown). In contrast, all MF and BID cases were
negative even after nested DNA-PCR.
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| Fig 4.
Amplification of genomic breakpoint on derivative
chromosome 5. Genomic DNA was subjected to standard and nested
amplification, followed by product separation on a 1% agarose gel.
Ethidium bromide staining of standard DNA-PCR products (A) and of
nested DNA-PCR products (D). Lanes 1 through 11, cases no. 1 through
11; lane 12, t(2;5)+ SU-DHL-1 cell line; lane M,
molecular weight marker 1-kb DNA ladder (GIBCO-BRL). The gels were
transferred to a nylon membrane and hybridized either with the ALK-5P
(radioautographies B and E) or the NPM-5P (radioautographies C and F).
The sizes are indicated in bases.
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Amplification of t(2;5) reciprocal breakpoint on derivative
chromosome 2.
Standard DNA-PCR allowed the detection of amplicons (0.6 to 2.2 kb) by
gel staining in 3 CD30+ secondary CTCL (cases no. 1, 2, and
3) and in 1 CD30+ primary CTCL (case no. 8;
Fig 5). These cases (no. 1, 2, 3, and 8)
were also positive at the DNA-PCR level on derivative chromosome 5 and
by both standard and nested RT-PCR analysis. Size-specific amplicons
were also obtained by nested DNA-PCR for cases no. 2, 3, and 8 but not
for case no. 1. Titration study showed that the sensitivity of this
DNA-PCR was 102 for the standard PCR and
103 for the nested PCR (results not shown). Southern
blot hybridization with both NPM-2P and ALK-2P confirmed these results
in 3 of the 4 cases. In case no. 1 (CD30+ secondary CTCL),
a positive hybridization of standard PCR products, was obtained only
with NPM-2P, but not with ALK-2P. Sequence analysis showed that the
standard DNA-PCR amplicon of case no. 1 contained the 3 end of
the NPM exon targeted by the 3NPM2int oligonucleotide and the
flanking intron, but lacked the 5 end of the ALK exon targeted
by the 5ALK2int primer. Amplicons of the nested DNA-PCR in cases
no. 2, 3, and 8 and the SU-DHL-1 cell line were purified and sequenced
(Fig 6). Multiple alignment analysis of
these sequences showed a perfect homology of chimeric intronic
sequences ranging from both exonic extremities over a variable area
from case to case depending on the position of the breakpoint (data not
shown). The complete sequence of the reciprocal breakpoint DNA fragment of the SU-DHL1 was characterized (Genbank accession no. AF032882). No
genomic breakpoint was amplified in the MF and BID cases.
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| Fig 5.
Amplification of genomic breakpoint on derivative
chromosome 2. Genomic DNA was subjected to standard and nested
amplification, followed by product separation on a 1% agarose gel.
Ethidium bromide staining of standard DNA-PCR products (A) and of the
nested DNA-PCR products (D). Lanes 1 through 11, cases 1 through 11;
lane 12, t(2;5)+ SU-DHL-1 cell line; lane M, molecular
weight marker 1-kb DNA ladder (GIBCO-BRL). The gels were transferred to
a nylon membrane and hybridized either with the ALK-2P
(radioautographies B and E) or the NPM-2P (radioautographies C and F).
The sizes are indicated in bases.
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| Fig 6.
Genomic nucleotide sequence of the reciprocal
translocation on the derivative chromosome 2 in SU-DHL1. The shaded
boxes at the 5 end and at the 3 end of the sequence
contain, respectively, the ALK exon sequence and the NPM exon sequence
flanking the breakpoint.
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Detection of ALK-NPM reciprocal transcript.
The standard RT-PCR assay showed no amplicon after electrophoresis
staining (Fig 2 and data not shown). Nested PCR allowed the
amplification of the same-sized products (127 bp) visible on gel
staining for the same 3 cases positive with the DNA-PCR assay on
derivative chromosome 2 (cases no. 2, 3, and 8; 2 CD30+
secondary CTCL and 1 CD30+ primary CTCL). The specificity
of the results was confirmed by Southern blotting with ALKNPM-J
probe and by DNA sequencing of the nested RT-PCR products. No
reciprocal transcript was detected in the other cases including MF and
BID cases even after nested RT-PCR and Southern blotting.
| |
DISCUSSION |
Our study demonstrates that lymphoproliferative or inflammatory
cutaneous diseases may contain NPM-ALK transcripts in some instances in
which no genomic breakpoints or expression of the chimeric NPM-ALK
protein can be detected. PCR contamination or artifacts were ruled out
by extensive controls such as negative amplification without the
reverse transcription step, hybridization, and sequencing of NPM-ALK
amplicons. Differences in the detection threshold of the different PCR
techniques may give a rationale for these discrepancies and the
differences between our previous results21,41 and those of
other groups that did not detect NPM-ALK transcripts or breakpoints in
CD30+ primary cutaneous
lymphoproliferations.17,20,22,24 These groups did not
perform a nested RT-PCR amplification that appeared in our hand as the
most sensitive assay, reaching a sensitivity threshold of
1:106. In addition, cells carrying t(2;5) theoretically
contain only one copy of the NPM-ALK chimeric gene on the derivative
chromosome 5, which is transcribed in several copies of mRNA.
Therefore, nested RT-PCR probably allows the detection of chimeric
transcripts in a higher number of cases than DNA-PCR. Such a nested
RT-PCR proved to be a reliable technique for the detection of NPM-ALK transcripts in nodal or systemic CD30+
lymphomas.15,31,42 In cutaneous samples, a small number of CD30+ cells may be intermingled within normal or
inflammatory cells, especially in LyP cases. However, ALK
immunoreactivity and t(2;5) breakpoints were detected in only 1 of 7 CD30+ cutaneous lymphoproliferations and in no BID or MF
cases containing NPM-ALK transcripts.
The existence of scarce cells expressing chimeric transcripts could
also explain our results. Whether these bystander cells are normal or
tumoral cells cannot be addressed but similar results have been
obtained by nested PCR assay for the detection of t(14;18) and t(9;22)
in normal tissues or blood samples.43-46 Therefore, the
detection of chimeric transcripts may overevaluate the implication of
t(2;5) in the genesis of CD30+ cutaneous
lymphoproliferations, because ALK immunoreactivity was not found in
most cases. Moreover, NPM-ALK transcripts were detected in some MF or
BID cases that did not contain CD30+ cells. Similarly,
NPM-ALK transcripts have frequently been detected in peripheral blood
cells of healthy donors by using nested RT-PCR assay.47
Such data are to be likened to the discordant results obtained by
several groups in Hodgkin's disease.26,27,33,48
Immunohistochemistry with monoclonal ALK1 and polyclonal anti-p80
antibodies provided a concordant staining of tumoral CD30+
cells in 1 CTCL and 3 secondary CTCL. These cases were further characterized by t(2;5) breakpoints. In addition, the p80 antibody also
stained large cells in 1 LyP case and in 3 CTCL. In the latter cases,
p80 staining was not correlated with the detection of the (2;5)
breakpoint. We previously observed a p80 staining of large dendritic
cells of the dermis.21 Moreover, several groups found p80
staining difficult to interpret in some instances.31,34 This may depend on the fixative or the fixation time, especially for
small specimens such as skin biopsies. Nonetheless, the ALK1 antibody
was restricted to CD30+ lymphoid cells.3,31
However, as for bcl2 immunoreactivity,49 ALK expression is
not specific for the 2;5 chromosomal translocation. ALK1 recognizes a
part of the tyrosine kinase domain of ALK, and other molecular events
such as t(1;2) or inversion on chromosome 2 may lead to an aberrant ALK
immunoreactivity of T-cell lymphomas.31,34,50 In addition,
a new subtype of large B-cell lymphomas was found to express the entire
ALK protein in the absence of t(2;5) translocation.51 Both
clinical and biological differences may exist between
NPM-ALK+ and ALK+ lymphoma. Moreover, NPM
sequences proved to be necessary for the oncogenicity of the chimeric
protein.12,13 Therefore, the identification of
t(2;5)+ lymphomas requires a combination of molecular and
immunohistochemical evidence in the lack of conventional cytogenetics.
In a few recent studies,19,22 DNA-PCR has been proposed as
an alternative procedure to conventional cytogenetics beside RT-PCR.
Indeed, genomic breakpoints are specific for each tumor and the
detection on both derivative chromosomes 2 and 5 could be a specific
PCR assay for the diagnosis of t(2;5). The size of the NPM and ALK
introns was confirmed to be short, approximately 1 and 2 kb,
respectively, suggesting that the largest possible size for the
chimeric NPM-ALK intron is about 3 kb.36 Indeed, the sum of
the sizes of the DNA-PCR products in our cases was about 3 kb. However,
our study points to the need to check size-specific amplicons obtained
by DNA-PCR, either by Southern hybridization with probes complementary
to both breakpoint sides or by sequencing. Size-specific amplicons may
be generated by PCR mispriming, as observed on derivative chromosome 5 for case 7 that contained only exonic and intronic ALK sequences, thus
hybridizing only with ALK-5P.
Amplification of the reciprocal ALK-NPM breakpoint on derivative
chromosome 2 was correlated with the ALK1+ immunophenotype
and with the amplification of the NPM-ALK breakpoint in all cases but
1. In this case (case no. 1), characterized by the presence of a
derivative chromosome 5 breakpoint, a small reciprocal chromosome
fragment, here 5q35, could have been lost on derivative chromosome 2, as described for reciprocal translocations involving
chromosome.15,52 A mispriming of 5ALK2 primer within intronic sequences of either NPM genes or NPM pseudogenes, both on a
nontranslocated chromosome 5, could explain this result. Therefore,
DNA-PCR for the amplification of derivative chromosome 2 breakpoint
provided an amplicon containing NPM exonic and intronic sequences but
no ALK sequences. Furthermore, the amplification of the derivative
chromosome 2 breakpoint allowed us to further characterize the genomic
nucleotide sequence of the SU-DHL1 cell line.
The reciprocal breakpoint was also shown to encode for chimeric
reciprocal ALK-NPM transcripts in all cases with positive DNA-PCR on
derivative chromosome 2. However, these transcripts were detected only
by nested RT-PCR, which may indicate a low level of expression. This
would be expected, because ALK promoter is normally silent in normal
lymphoid cells.10 Whether reciprocal ALK-NPM transcripts
have a biological function needs to be elucidated as for other
reciprocal translocations, such as t(9;22) or
t(12;21).53,54
Finally, our findings may be reconciled with those obtained by several
groups in cutaneous CD30+ disorders. Firstly,
t(2;5)-positive CD30+ primary CTCL may exist but appear to
be very rare as opposed to t(2;5)-positive CD30+ secondary
cutaneous lymphomas (1/26 v 3/11). The t(2;5) appeared not to
be implicated in LyP (no amplification of genomic breakpoints, no ALK1
immunoreactivity), as previously reported.22,23,34 Secondly, the presence of NPM-ALK transcripts, detected both in cutaneous lymphoproliferative and inflammatory disorders, does not
allow the distinction between CD30+ primary and secondary
cutaneous lymphomas by nested RT-PCR alone. Thirdly, ALK1
immunostaining was found to be specific for cutaneous lymphomas that
harbor t(2;5) by genomic PCR. In the absence of conventional
cytogenetics, the amplification of chromosomal breakpoints on either
derivative chromosomes 2 or 5 provided tumor-specific molecular
evidence for the presence of t(2;5) among lymphomas expressing NPM-ALK
transcripts. Our study clearly demonstrates the specificity of such
DNA-breakpoint amplification versus the detection of NPM-ALK
transcripts for the diagnosis and monitoring of patients with
t(2;5)-positive lymphomas.
| |
FOOTNOTES |
Submitted November 6, 1997;
accepted February 3, 1998.
Supported by grants from the Région Aquitaine and Comité de
Gironde et de Charente de la Ligue Contre le Cancer. K.P. was supported
by the Leukaemia Research Fund.
Address reprint requests to M. Beylot-Barry, MD, Equipe Histologie et
Pathologie du Système Immunitaire, Laboratoire
d'Histologie-Embryologie, UFR 3, Université de Bordeaux 2, 33076 Bordeaux Cedex, France; e-mail:
Marie.Beylot-Barry{at}histo.u-bordeaux2.fr.
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 following members of the French Study Group of Cutaneous Lymphoma
have contributed to this study: M.F. Avril and J. Bosq (Villejuif), M. Bagot and J. Wechsler (Creteil), L. Vaillant and A. de Muret (Tours),
C. Beylot (Pessac-Bordeaux), M. Delaunay (Bordeaux), S. Dalac and T. Petrella (Dijon), P. Joly and E. Thomine (Rouen), and C. Bodemer and S. Fraitag (Necker-Paris). We also thank J. Ferrer, C. Bartoli, J.C.
Garroste, and M. Turmo for their expert technical assistance and J.P.
Javerzat (Genetic Laboratory CNRS 9026) for his contribution. We thank
Dr M.L. Cleary for the gift of the SU-DHL1 cell line.
| |
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Abstract
Introduction
Abstract