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Blood, Vol. 94 No. 10 (November 15), 1999:
pp. 3614-3617
CORRESPONDENCE
Complex Variant Translocation t(1;2) With
TPM3-ALK Fusion Due to Cryptic ALK Gene Rearrangement
in Anaplastic Large-Cell Lymphoma
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LETTER |
To the Editor:
Anaplastic large-cell lymphoma (ALCL) is frequently associated with the
recurrent translocation t(2;5)(p23;q35) that results in activation of
the anaplastic lymphoma kinase (ALK ) gene at 2p23 by fusion
to the ubiquitously expressed gene encoding the nucleolar
phosphoprotein nucleophosmin (NPM ) at 5q35. This
translocation leads to aberrant nuclear (and cytoplasmic) expression of
ALK, which is normally silent in hematopoietic
tissues.1,2
Approximately 20% of ALK-positive ALCLs do not express
ALK in the nucleus but show aberrant expression of ALK
restricted to the cytoplasm.3,4 These "cytoplasmic
ALK only" ALCL do not contain the t(2;5), suggesting that
other genetic abnormalities can result in aberrant ALK
expression. So far, only very limited data on cytogenetic alterations
of this subtype of ALCL are available. Nevertheless, in very recent
issues of BLOOD, "cytoplasmic ALK only" ALCL
cases have been described to contain variant rearrangements of the
chromosomal region 2p23. Wlodarska et al5 reported 3 cases
with cryptic inv(2)(p23q35). Rosenwald et al6 described 1 case each with t(1;2)(q21;p23) and t(2;3)(p23;q21). Finally, Lamant et
al7 reported a t(1;2)(q25;p23) in 1 patient. Cloning of the
translocation breakpoint showed a fusion of ALK to the TPM3 gene in 1q25 encoding a nonmuscle tropomyosin. A
TPM3-ALK fusion transcript was detectable by reverse
transcriptase-polymerase chain reaction (RT-PCR) in 2 of 3 additional
ALCL, with ALK staining restricted to the
cytoplasm.7
We recently investigated 3 cases of "cytoplasmic ALK
only" ALCL by means of fluorescence in situ hybridization (FISH).
Whereas 2 cases turned out to contain the previously described
inv(2)(p23q35), we report here a cryptic insertion into the ALK
gene observed in the third patient. The lymph node biopsy specimen was
obtained from a 5-year-old male patient with stage IVB disease.
Histological evaluation showed a CD30(Kil)-positive ALCL of null cell
phenotype. Using the ALK1 antibody (Dako, Glostrup, Denmark), only 20%
of the tumor cells stained positive, with strong cytoplasmic
ALK protein expression but lack of any nuclear ALK expression.
Chromosome analysis of R-banded metaphases derived from unstimulated
short-term lymph node cultures showed clonal aberrations in 6 of 16 metaphases. The karyotype was 50 ~ 51, XY, +X, del(1)(q21), +2,
der(2)?dup(2)(p25p21)dup(2)(p11p25) × 2, +6, +7, +17 [cp6]/46, XY
[10]. To investigate the case for a cryptic ALK
rearrangement, we performed FISH with the LSI ALK assay (Vysis, Downers
Grove, IL) according to the manufacturer's instructions. The LSI ALK assay consists of 2 differentially labeled probes for the centromeric (5') and telomeric (3') regions of the ALK gene that showed to be separated in cases with t(2;5) and inv(2). Using this assay, 38 of
100 interphase cells each displayed 2 isolated signals for the
centromeric (green) and telomeric (red) ALK probes, indicating 2 rearranged copies of the ALK gene in addition to 1 colocalization of each 1 signal derived from an intact ALK
locus. FISH on metaphases followed by subsequent R-banding analysis
showed the short arms of the intact chromosome 2 and both der(2)
chromosomes each to contain 1 signal for the centromeric and the
telomeric ALK probe. Nevertheless, the spatial separation of the 2 ALK probes on the der(2) chromosome suggests an insertion of
genomic material into the ALK gene on the der(2) (Fig 1A
through D).
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| Fig 1.
Metaphase FISH with LSI ALK probe (A) and subsequent
R-banding analysis (B) shows insertion of genomic material into the
ALK locus indicated by spatial separation of the red signal for
3'ALK (telomeric) and the green signal for 5'ALK
(centromeric) on the short arms of both der(2) chromosomes (arrows) as
compared with the intact chromosome 2 (arrowhead). (C) In interphase
nuclei of tumor cells, insertion into the ALK locus on both
derivative chromosome 2 leads to separation of 3' and 5'ALK
signals in addition to each 1 colocalized, indicating the intact
ALK locus. (D) In contrast to the larger nucleus of the tumor
cells, the smaller nuclei of nonneoplastic cells show 2 colocalized
signals for 3' and 5'ALK, which is the regular signal
constellation for 2 intact chromosomes 2. (B, small picture) FISH with
a chromosome 1 painting probe on R-banded chromosomes shows a major
part of the short arm of the der(2) chromosomes to derive from
chromosome 1. (E) RT-PCR amplifies a PCR product of approximately 300 bp specific for TPM3-ALK fusion (Lane 2). Lane 1, 100-bp
ladder; lane 3: H2O control.
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As for R-banding analysis, there was no evidence for material from
chromosomes other than chromosome 2 to be contained in the der(2)
chromosomes, suggesting that a complex inv(2) might lead to insertion
of 2q35 material into the ALK locus. To test this hypothesis,
FISH with YAC probes 884F10, 770F5, and 914E7 flanking and spanning the
2q35 breakpoint of the inv(2), respectively, was applied.5
These hybridizations did not provide evidence for the genomic material
inserted into the ALK locus to be derived from a complex inv(2).
Considering the loss of chromosome 1 material due to the del(1)(q21) in
the tumor cells, it was also tempting to speculate that the material
inserted into the ALK locus might be derived from the long arm
of chromosome 1. Consequently, FISH on archived R-banded slides was
performed with a whole chromosome 1 painting probe (AGS, Heidelberg,
Germany). Surprisingly, this analysis showed a major part of the short
arms of the der(2) chromosomes to be derived from chromosome 1 (Fig 1B,
small picture). Thus, the present case contains a complex t(1;2)
translocation that is hardly detectable by banding analyses due to the
underlying multiple break events.
Because this complex aberration might be a variant of the recently
cloned translocation t(1;2)(q25;p23) leading to a TPM3-ALK fusion, we aimed to investigate the case for this hybrid
transcript.7 No fresh material for molecular analyses was
available from initial diagnosis and the patient is in continuous
complete remission for 2.5 years now after initial treatment according
to the German NHL-BFM95 trial (group III-K3).8 Thus, RNA
was extracted from cells in Carnoy's fixative by means of RNAzol (WAK
Chemie, Bad Homburg, Germany), and first-strand synthesis was performed
with pooled ALK-specific primers (5'-AGC ACA CTT CAG GCA GCG
TCT TCA CAG CCA-3' and 5'-CAT TCC GGA CAC CTG GCC TTC ATA CAC CTC-3') using the Reverse Transcription System (Promega, Madison, WI). Nested
PCR according to Lamant et al7 indeed amplified the characteristic PCR product of approximately 300 bp (Fig 1E). Sequencing of this PCR product confirmed the presence of the TPM3-ALK
fusion transcript.
In summary, we identified a cryptic ALK gene rearrangement due
to an insertion that, by molecular cytogenetics and RT-PCR analysis,
turned out to be a complex variant translocation t(1;2) leading to
TPM3-ALK fusion. On the one hand, the present case confirms
TPM3-ALK fusion to be a recurrent mode of ALK
activation in ALCL with ALK expression restricted to the
cytoplasm.7 On the other hand, this case again indicates
that ALK gene rearrangements might be cytogenetically hidden,
eg, due to complex breakage events. Indeed, in the present case as well
as in the cases with inv(2), the ALK rearrangements would not
have been cytogenetically detected without applying molecular
cytogenetics. Thus, in our opinion, FISH with an ALK-specific
probe should be generally integrated into the cytogenetic analysis of
"cytoplasmic ALK only" ALCL so that cryptic ALK
rearrangements are not missed.
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ACKNOWLEDGMENT |
The authors are grateful to C. Becher, D. Schuster, and F. Jäger
for their excellent technical assistance. This work was supported by
the Deutsche Krebshilfe, the Wilhelm-Sander-Stiftung, and the IZKF
Kiel. S.G. is a scholar of the Hensel-Stiftung (Kiel, Germany).
Reiner Siebert
Stefan Gesk
Lana Harder
Doris Steinemann
Werner Grote
Brigitte Schlegelberger
Department
of Human Genetics
Markus Tiemann
Department of
Hematopathology
University of Kiel
Kiel, Germany
Iwona Wlodarska
Center for Human Genetics
K.U. Leuven
Leuven,
Belgium
Verena Schemmel
Study Center NHL-BFM
Department of Pediatric Hematology/Oncology
Medical School
Hannover
Hannover, Germany
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