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Short Communications |
From the Departments of Pathology*
and Human
Genetics,
Memorial Sloan-Kettering Cancer
Center, New York, New York; and the Departments of Pathology and
Microbiology,
University of Nebraska Medical
Center, Omaha, Nebraska
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In 1994, the t(2;5) was found to involve a novel gene at 2p23 encoding a tyrosine kinase, ALK (anaplastic lymphoma kinase), and the NPM (nucleophosmin) gene at 5q35, which encodes a nucleolar phosphoprotein.10 The resulting fusion gene encodes a chimeric protein, NPM-ALK, with a molecular weight of 80 kd, consisting of the N-terminal portion of NPM fused to the catalytic domain of ALK.10 ALK is a tyrosine kinase receptor belonging to the insulin growth factor receptor superfamily, highly related to the leukocyte tyrosine kinase (LTK) gene but normally expressed only in the nervous system.11,12 The fusion with NPM contributes the NPM promoter and the NPM oligomerization domain to NPM-ALK, and removes the ALK extracellular and transmembrane domains. As a result, the ALK kinase domain within NPM-ALK is constitutively activated through autophosphorylation, and its expression is deregulated and ectopic, both in terms of cell type and cellular compartment (cytoplasm; reviewed in Ref. 13 ). Downstream targets of the ALK kinase domain that may be relevant in mediating the oncogenicity of NPM-ALK are being identified.14
Because of the highly restricted expression of native ALK in the nervous system and its absence in normal lymphoid tissues, immunohistochemical detection of aberrantly expressed ALK protein using monoclonal15,16 or polyclonal17,18 antibodies to the ALK kinase domain (retained in NPM-ALK) was found to be a sensitive and specific method for detecting NPM-ALK-positive ALCL. Interestingly, ALK immunostaining was observed in both cytoplasm and nucleus in most cases, but only in cytoplasm in some cases. The nuclear localization of NPM-ALK is due to the formation of heteromeric complexes with native NPM, which contains a nuclear localization signal.14 Initially, the occasional variability in subcellular localization of ALK immunostaining was thought to reflect unknown factors affecting either the heteromerization of NPM-ALK with NPM, or the entry of the resulting heteromeric complexes into the nucleus. However, it soon became apparent that ALCL with exclusively cytoplasmic ALK immunoreactivity usually lacked NPM-ALK by reverse transcriptase-polymerase chain reaction (RT-PCR).15,16,19 At the same time, using an artificial TPR-ALK construct, it was shown that only cytoplasmic localization is required for transformation by the ALK portion of NPM-ALK.20 Taken together, these results suggested that in some ALCL, ALK may become oncogenically activated through fusion with other translocation partners unassociated with nuclear transport.
Studies of large series of Ki-1 ALCL by ALK immunostaining now indicate that up to 20% of cases show cytoplasmic staining only.16,21,22 Furthermore, Western blot analysis has identified at least four different types of aberrant ALK-positive proteins in different cases of this subset of Ki-1 ALCL that show ALK immunostaining confined to the cytoplasm.23 Thus, up to 20% of ALK+ Ki-1 ALCL may contain variant ALK translocations, and these may be of at least four types. By cytogenetic analysis, several variant translocations involving 2p23 have been reported in Ki-1 ALCL. These include t(2;13)(p23;q34),24 t(1;2)(q25;p23),25 a cryptic inv(2)(p23q35),26 , t(1;2)(q21;p23), and t(2;3)p23;q21).27 Of these, only the t(1;2)(q25;p23) has so far been cloned. Using a PCR-based genomic walking technique, Lamant et al28 demonstrated that the gene involved at 1q25 is TPM3, which encodes nonmuscular tropomyosin and was previously known to similarly rearrange with and activate the NTRK1 receptor tyrosine kinase in some papillary thyroid carcinomas (reviewed in Ref. 29 ).
In the present report, we describe the cloning of a novel variant ALK gene fusion, ATIC-ALK, which is associated with the previously reported recurrent cryptic inversion, inv(2)(p23q35).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Among 26 cases of ALCL diagnosed at Memorial Sloan-Kettering
Cancer Center that had material available for molecular studies, we
identified 13 cases (50%) negative for NPM-ALK
by reverse transcriptase-polymerase chain reaction (RT-PCR), performed
as reported previously,9
using the primers
NPM-5' and ALK-3' listed in Table 1
. Molecular data on 10 of 13
NPM-ALK+ and 8 of 13
NPM-ALK- cases have been described in part in
previous studies.9,30,31
All cases were
immunohistochemically positive for Ki-1 antigen (CD30) using monoclonal
antibody Ber-H2 (Dako, Santa Barbara, CA). Lineage phenotype and
genotype were determined according to standard immunophenotypic and
molecular genetic methods, as described in more detail
elsewhere.4
Case histories of the two patients that were
studied in more detail (see Results) are summarized below.
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Case 1
This 52-year-old woman was diagnosed overseas with malignant
lymphoma in a left axillary mass and was treated with four cycles of
chemotherapy without response. She came to MSKCC 4 months later for a
second opinion. Clinical restaging showed left axillary adenopathy,
focal infiltration of fat and adjacent muscle, and retraction and
thickening of overlying skin. Bone marrow biopsy did not show
infiltration by lymphoma. Biopsies of axillary and skin tumors showed
large, polymorphic cells, with amphophilic cytoplasm, round and
lobulated nucleus with two or three nucleoli. Frequent mitotic figures,
necrosis, and phagocytosis were also seen (Figure 1)
. The tumor showed the following
staining features: CD30+, epithelial membrane antigen (EMA) +, CD43+,
CD3-, CD45RO-, CD20-. No clonal rearrangement involving the
immunoglobulin heavy chain gene (IGH) was detected by
Southern blotting, but the TCRß gene did show clonal rearrangement.
This pattern was consistent with a Ki-1-positive T cell ALCL.
Cytogenetic analysis of this biopsy showed the following clonal
karyotype: 46, XX, del(1)(p32),der(3)dic(1;3)(q13;q27),
der(6)t(1;?;6)(q25;?;p25), hsr(7)(p22), I(8)(q10),
der(12)t(9;12)(q13;p13), add (13)(p11),
der(15;15)(q10;q10), add(16)(q22), add(19) (q13),
add(21)(p11), +mar. The patient returned overseas and was lost to
follow-up.
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At the age of 12, this girl was first diagnosed with a nodal
diffuse large cell lymphoma. The clinical stage was IIIA (negative bone
marrow, no extranodal sites). She was treated with chemotherapy
and external radiation, and achieved complete remission. Three years
later, she developed a relapse and was treated with a similar
chemoradiotherapy combination, and achieved a prolonged second complete
remission. Twelve years later, she developed a second nodal relapse and
started a new chemotherapy protocol. She died 3 months later due to
sepsis and granulocytopenia. Biopsy of the second nodal recurrence
showed rounded, monomorphic tumor cells with round nuclei and one or
two nucleoli (Figure 1)
. Numerous mitotic figures were seen. The tumor
showed the following immunostaining: CD30+, EMA+, CD45+, CD43+, CD20-,
CD15-. No clonal rearrangement involving IGH was detected
by Southern blot analysis, but the TCRß gene was clonally rearranged.
This pattern was consistent with a Ki-1-positive T cell ALCL.
Cytogenetic analysis showed the following clonal abnormalities: 47, XX,
+2, del(6)(q21), t(8;13;20)(p11.2;p11.2;p11.2).
ALK Immunostaining
ALCL were subjected to immunostaining with a polyclonal antibody generated to amino acid residues 419–520 of NPM ALK, designated ALK-11 (gift of S. W. Morris),18 after heat-induced epitope retrieval in citrate buffer for 10 minutes. Equivalent results were obtained at dilutions of 1:1000 and 1:2000. This antibody produced strong nuclear and cytoplasmic staining in all 5 ALCL tested that were positive for NPM-ALK+ by RT-PCR (results not shown). Cases positive with ALK-11 were further tested with the ALK-1 monoclonal antibody, generated to the same amino acid residues of NPM-ALK as the ALK-11 antibody (Dako),15 at a dilution of 1:50, after heat-induced epitope retrieval in citrate buffer for 20 minutes. Immunoperoxidase staining was performed on paraffin sections, using a standard avidin-biotin peroxidase procedure.
Fluorescence in Situ Hybridization (FISH) with ALK and 2q35 Probes
Bicolor FISH studies were performed on cytologic touch preparations of Case 1 and on extracted nuclei from paraffin-embedded tissue blocks from Case 2 and the two ALK-11+ but ALK-1- cases (see Results) using the Vysis LSI ALK probe assay (Vysis, Inc., Downers Grove, IL) according to the manufacturer’s instructions. In addition, FISH studies with a 2p23 (ALK) breakpoint spanning probe (600 kb, courtesy of Dr. J. Proffitt, Vysis, Inc.) and yeast artificial chromosome (YAC) 914E7 (a 1750-kb CEPH YAC probe mapped to 2q35, Research Genetics, Huntsville, AL) were also performed on Case 1 and FISH studies with an ALK P1 clone (designated ALK-DMPC-HFF#1–1111H1, courtesy of Dr. S. W. Morris) and 914E7 were performed on Case 2. With respect to the latter hybridizations, probe mixtures containing 200 ng biotin-labeled YAC 914E7 (Nick Translation Kit, Vysis) and Spectrum Orange-labeled 2p23 (ALK) breakpoint spanning probe or digoxygenin-labeled ALKP1 was applied to a slide and sealed under a coverslip. The cells and probes were codenatured at 85°C for 5 minutes and incubated overnight at 37°C in a humidity chamber. Detection of signals was performed as described in detail elsewhere.32 As negative controls, metaphase cells obtained from a cytogenetically normal lymph node and cytologic touch preparations of normal skeletal muscle were simultaneously hybridized with these probes.
Images were prepared using the Cytovision Image Analysis System (Applied Imaging, Santa Clara, CA). One hundred interphase nuclei with strong and well-delineated signals were examined by two different individuals. A separation of the Spectrum Orange- and Spectrum Green-labeled 2p23 breakpoint flanking probes (3' telomeric and 5' centromeric respectively to the 2p23 breakpoint of the t(2;5)(p23;q35) associated with non-Hodgkin’s lymphoma; Vysis LSI ALK probe assay) was interpreted as a rearrangement of the ALK gene.
Inverse PCR
For both inverse PCR and conventional RT-PCR, total RNA was extracted using Trizol method (Tel-Test, Friendswood, TX), and the adequacy of the extracted RNA was confirmed by amplification of a 247-bp fragment of the ubiquitous phosphoglycerate kinase (PGK) transcript, using primers PGK-FWD and PGK-REV. For inverse PCR, double-stranded cDNA was synthesized as follows using a cDNA Synthesis Kit (Boehringer Mannheim, Indianapolis, IN). Reverse transcription was performed on 1 µg of RNA, and primed with 2 pMol of ALKREV primer using AMV reverse transcriptase. The ALKREV primer binds 98 bp from the ALK fusion point in NPM-ALK and TPM3-ALK. Second-strand cDNA synthesis was performed using Escherichia coli DNA polymerase I and RNase H. The resulting double-stranded cDNA was then blunt-ended with T4 DNA polymerase and subsequently purified using the QIAquick PCR Purification Kit (Qiagen, Valencia, CA). The cDNA was then circularized by overnight incubation at room temperature in the presence of 1 U/µl T4 DNA ligase (Boehringer Mannheim) in a final volume of 30 µl. The ligation reaction was stopped by 65°C incubation for 10 minutes. The circularized cDNA was then relinearized by digestion with PstI, which cuts the ALK cDNA between the ALKREV3 and ALKFWD4 primer binding sites (oriented 5' end to 5' end). After a manual hot start, the cDNA was then amplified by PCR (35 cycles: 95°C for 45 seconds, 58°C for 1 minute, 72°C for 3 minutes, final extension 72°C for 10 minutes) with primers ALKREV3 and ALKFWD4 (15 pMol of each) using 2U/µl rTth DNA Polymerase (Perkin Elmer, Branchburg, NJ). Nested PCR was performed on 1 µl of the first PCR product using primers ALKREV4 and ALKFWD5, Taq polymerase (DNA PCR Beads, Pharmacia Biotech, Piscataway, NJ), for 35 cycles (95°C for 1 minute, 62°C for 45 seconds, 72°C for 1 minute, final extension 72°C for 10 minutes).
RT-PCR for ATIC-ALK
RT-PCR was performed using ATIC-FWD and ALKREV primers. First, reverse transcription was performed for 30 minutes at 42°C on 1 µg of RNA using 10x buffer II, 25 mmol/L MgCl2, 50 mmol/L random hexamers, 10 mmol/L dNTP, 40U/µl RNase inhibitor, 200U/µl reverse transcriptase, and DEPC-treated H2O for a final volume of 20 µl. A negative control (10 µl of DEPC-water) was included at this stage. The reverse transcriptase was inactivated at 99°C for 5 minutes. Eighty µl of master mix (water, 10x buffer II, MgCl2 for a final concentration of 1.5 mmol/L, 15 pMol of each primer, 5U/µl of AmpliTaq DNA Polymerase, Perkin Elmer, Norwalk, CT) were added to the tube. PCR consisted of 35 cycles of 95°C for 1 minute, 60°C for 1 minute, 72°C for 1 minute, final extension of 72°C for 10 minutes. The PCR products were electrophoresed in 2% NuSieve agarose gel (FMC Bioproducts, Rockland, ME) and visualized by ethidium bromide staining.
YAC DNA Extraction
YACs 914E7 and 777D5 were obtained from Research Genetics. One colony was inoculated in 5 ml YPD medium (yeast extract, peptone, dextrose; Bio 101, Vista, CA) and incubated in an orbital shaker for 24 hours at 30°C. Eight hundred µl of this product were combined with 200 µl of glycerol and stored at -70°C. The remaining product was inoculated in 100 ml YPD medium and incubated in an orbital shaker for more 24 hours at 30°C. The YAC was centrifuged for 10 minutes at 3000 rpm and the pellet was resuspended in a lysis buffer (20% sodium dodecyl sulfate, 10% Triton X-100, 5 mol/L NaCl, 1 mol/L Tris, pH 8.0, 0.5 mol/L EDTA, pH 8.0). Seventy-five microliters of glass beads and 200 µl of 1:1 phenol:chloroform were added to the lysate and it was mixed in a vortex for 5 minutes. Two hundred microliters of TE buffer (10 mmol/L Tris, 1 mmol/L EDTA) was added to the lysate and it was mixed again. After 5 minutes centrifugation at room temperature, the clear supernatant was transferred to a new tube. Then, 750 µl of 100% isopropanol was added to it, mixed gently by inversion, and left for 5 minutes at room temperature. After centrifugation, a pink pellet was seen. The dried pellet was then resuspended in 300 µl TE buffer (pH 7.4). Fifteen microliters of 1 mg/ml RNase A was added and the product was incubated for 30 minutes at 37°C. The pellet was again precipitated with 100% isopropanol and 3 Mol/L NaAc. After centrifugation, it was washed in 70% ethanol and dissolved in TE buffer. The DNA was electrophoresed in a 1% agarose gel to evaluate its quality.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The study group consisted of 13 ALCL of non-B cell lineage that
lacked NPM-ALK by RT-PCR (see Materials and Methods). There
were 7 female and 6 male patients, with median age of 47.3 years
(range, 13–70 years). These 13 cases were subjected to immunostaining
with polyclonal ALK-11 antibody to the ALK kinase domain.
Four T cell ALCL cases were positive. These four cases were further
tested by immunostaining with the ALK-1 monoclonal antibody, and by
interphase FISH analysis for ALK rearrangement. Two cases,
Cases 1 and 2, were also positive with ALK-1 (Figure 1)
. Case 1 also
showed ALK rearrangement by FISH using 2p23
(ALK) breakpoint flanking probes. Specifically, a
separation of these breakpoint flanking probes was detected in 97% of
the interphase nuclei analyzed in Case 1 (Figure 2A)
using the two-color Vysis
ALK probe FISH assay, indicative of an ALK
rearrangement. Moreover, a third copy of the Spectrum Orange signal of
this probe set, which is located telomeric to the 2p23 breakpoint, was
observed in all abnormal cells of Case 1 (Figure 2A)
. FISH studies with
the two-color Vysis ALK probe FISH assay were unsuccessful
in Case 2, where only extracted nuclei from paraffin-embedded tissue
blocks were available. Brief case histories for these two patients are
presented above in Materials and Methods. The remaining two cases that
were negative by ALK-1 immunohistochemistry were also negative by
ALK FISH.
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As schematized in Figure 3
and
described in more detail in Materials and Methods, we performed inverse
PCR with nested amplification to isolate the ALK
translocation partner in this case. There were two inverse PCR product
bands: a broad 200- to 300-bp band (A), and a fainter band of
approximately 120 bp (B; Figure 4
). Both
products were analyzed by direct automated sequencing. Sequence
analysis of the 120-bp B band showed an in-frame fusion between
ATIC and ALK, occurring at codon 162 of the
former and codon 1058 of ALK, the same codon involved in the
NPM-ALK fusion. The broad 200- to 300-bp A band
was a nonspecific PCR product.
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Based on the ATIC-ALK chimeric transcript identified by
inverse PCR, we designed primer ATIC-FWD to produce a 169-bp RT-PCR
product in conjunction with the ALKREV primer. RT-PCR with these
primers yielded only a single strong 370-bp band in both cases (Figures 3 and 4)
, instead of the expected 169-bp product. Sequence analysis of
this 370-bp band also showed an in-frame fusion between ATIC
and ALK, occurring again at codon 1058 of ALK,
but at a different point in ATIC, codon 229 instead of 162
(Figure 3)
. In light of this result, we suspect that this major fusion
transcript may have been either obscured in the inverse PCR by the
nonspecific 200- to 300-bp product (see above) or that the shorter
fusion transcript may have been more efficiently isolated for technical
reasons. This shorter fusion transcript, which was detected only in
Case 1 by the nested amplification of the inverse PCR procedure, likely
arose by alternative splicing of the major fusion
product. The intervening portion of ATIC may
therefore correspond to one or more exons (the exon structure of
ATIC is largely unknown33
). This shorter minor
splice form is unlikely to be biologically significant because of its
low expression level and because it lacks the ATIC
dimerization domain (see Discussion). As an incidental observation, our
sequencing data confirmed that ATIC codon 164 reads GAC (for
aspartic acid), as in reference 34, instead of GGC (for glycine)
reported in reference 35. Furthermore, a search of the expressed
sequence tag database (dbEST) identified five perfect matches for GAC
(D83877, H09885, H06543, R20197, AA112359) and none for GGC at this
codon.
To assess Case 2 for the presence of the ATIC-ALK fusion, we
performed RT-PCR using the same primers as above, namely ATIC-FWD and
ALKREV. This yielded the same 370-bp RT-PCR product (Figure 5)
, confirmed by sequencing to be the
ATIC-ALK fusion transcript.
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ATIC was previously mapped to 2q34-q35 by FISH.33 YAC 914E7 at 2q35 was reported by Wlodarska et al to be rearranged by the cryptic inv(2)(p23q35).26 To confirm that this YAC contains the ATIC gene, we performed DNA PCR on purified YAC DNA using primers ATIC-FWD and ATIC-REV. The expected 71-bp product was amplified from YAC 914E7 DNA, but not from an unrelated YAC (777D5 from 1q21; result not shown), confirming that ATIC maps to YAC 914E7.
Confirmation of ATIC-ALK Fusion by FISH in Cases 1 and 2
FISH studies performed on Case 1 with the Spectrum Orange-labeled
2p23 (ALK) breakpoint spanning probe and the
biotin-labeled YAC 914E7 revealed a distinct or separate orange and
green signal consistent with the presence of a normal chromosome 2
homologue and three orange and green signals lying directly adjacent or
juxtaposed to each other indicative of 2p23 and 2q35 rearrangements in
96% of the interphase nuclei analyzed (Figure 2B)
. In Case 2, FISH
analysis of nuclei from paraffin-embedded tissue blocks with
ALK P1 and YAC 914E7 probes revealed a distinct or separate
red (rhodamine) and green (fluorescein isothiocyanate) signal
consistent with the presence of a normal chromosome 2 homologue and two
red and green signals lying directly adjacent or juxtaposed to each
other, indicative of two copies of ATIC-ALK fusion in 88%
of the interphase nuclei analyzed (not illustrated). The limited tissue
quantity available permitted analysis of 50 interphase nuclei in this
case. Thus, the findings in both cases were compatible with the
presence of an inv(2)(p23q35) and an additional copy of
ATIC-ALK.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Studies of ATIC deletion mutants have confirmed the existence of two non-overlapping functional domains, separated by a linker region.33 Based on these deletion studies and on crystallography data, a working model of ATIC has been proposed in which residues 1 to 169 encode the IMPCH function, residues 170 to 199 encode the linker region, and residues 200 to 592 encode the AICARFT activity.33,35 Furthermore, crystallography and equilibrium sedimentation studies indicate that ATIC exists mainly as a homodimer.33 Gel filtration and ultracentrifugation studies of additional ATIC deletion mutants suggest that the linker region contains a dimerization domain (G. P. Beardsley, personal communication). The first 229 amino acid residues of the predicted ATIC-ALK protein are identical to those of ATIC (the valine codon at the fusion point is conserved). Thus, in addition to an active promoter, ATIC appears to contribute a dimerization domain to ATIC-ALK, which should lead to constitutive autophosphorylation and activation of the ALK kinase domain. These properties are shared by NPM (see Introduction) and TPM3. In ALCL with the t(1;2)(q25;p23), TPM3 contributes to TPM3-ALK an active promoter, and activation of the ALK catalytic domain probably results from homodimerization through the TPM3 protein-protein interaction domain.28 Like ATIC-ALK, TPM3-ALK accumulates only within the cytoplasm.20,28
As discussed in the Introduction, various lines of evidence suggest that up to 20% of ALK+ ALCL contain variant ALK translocations. Furthermore, these may be of at least four types, according to the Western blot studies of Pulford et al.23 They identified four different types of putative variant ALK fusion proteins with molecular weights of 85, 97, 104, and 113 kd (compared to 80 kd for NPM-ALK and 200 kd for native ALK), in three of which tyrosine kinase activity was also confirmed.23 Whether any of these correspond to ATIC-ALK or TPM3-ALK is presently unclear. None of their cases had a documented inv(2)(p23q35), whereas one case with a aberrant ALK protein of 104 kd had a t(1;2), but with a 1q21 break instead of the 1q25 break associated with TPM3-ALK.28 NPM, ATIC, and TPM3 contribute 116, 229, and 255 amino acid residues to their respective ALK fusion proteins. Based on an average of 120 d per amino acid residue, the predicted molecular weight of the ATIC-ALK protein is about 94 kd, and that of TPM3-ALK is 97 kd. Aside from the approximate nature of these estimates, posttranslational modifications may alter protein mobility, making it difficult to directly assign bands on Western blots to specific predicted fusion proteins.
The inv(2)(p23q35) was first reported in 199719 and was then described in detail in three cases by Wlodarska et al.26 This inversion is cryptic, ie, it is not apparent on conventional Giemsa-banded cytogenetic preparations, because terminal bands of similar size and staining pattern are exchanged. This may explain why no 2p23 or 2q35 breaks were apparent in the conventional karyotypes of cases 1 and 2 (see Case Summaries). Furthermore, Wlodarska et al26 found a consistent association of the inv(2)(p23q35) with a secondary chromosomal aberration, ider(2)(q10)inv(2)(p23q35), which results in extra copies of the rearranged ALK gene. FISH analysis demonstrated at least one additional copy of the fusion gene in both of our cases. Consistent amplification of ATIC-ALK suggests that it may be less oncogenic than NPM-ALK, and therefore requires extra copies to exert an equivalent cellular effect. The phenomenon may be analogous to the consistent amplification of the PAX7-FKHR variant fusion gene in alveolar rhabdomyosarcoma.36,37
ATIC-ALK may make up a significant proportion of variant ALK fusions. In the present series, ATIC-ALK accounted for 2 of 15 (13%) cases positive for ALK by immunostaining or for NPM-ALK by RT-PCR. Considering the cytogenetic reports of the inv(2)(p23q35), three cases of ATIC-ALK have been previously reported.26 Aside from TPM3-ALK, reported in three cases,28 no other known ALK variant translocations have been recurrent.
As the third reported translocation partner of ALK, the
finding of ATIC highlights the promiscuous nature of many
genes involved in oncogenic translocations.38
NPM also rearranges with other genes, resulting in the
NPM-RAR
fusion in rare cases of acute
promyelocytic leukemia and the NPM-MLF1 fusion
seen in some cases of myelodysplastic syndrome and acute myeloid
leukemia.39,40
In ALCL, no variant fusions involving
NPM but not ALK have so far been identified,
although there are cytogenetic case reports of a
t(3;5)(q12;q35)7
and a
t(1;5)(q32;q35).41
In the absence of molecular
studies, however, the latter two cases may simply contain a cryptic
complex t(2;5). Interestingly, a recent report has also implicated
ALK activation by rearrangement as a recurrent alteration in
a mesenchymal tumor, the inflammatory myofibroblastic
tumor.42
The potential clinical significance of these variant ALK fusions is that ALK+ ALCL, defined either by immunohistochemistry, or by molecular or cytogenetic detection of NPM-ALK, is a prognostically favorable subset of ALCL. Before the development and application of ALK immunodetection, this important observation was statistically hampered by the limited numbers of cases with cytogenetic or molecular data.1,43,44 Recent retrospective analyses of large series of ALCL by ALK immunostaining have established that ALK+ ALCL occurs in significantly younger patients, is more often extranodal, and has a markedly better clinical outcome.22,45,46 Furthermore, a recent multivariate analysis indicates that the survival advantage of patients with ALK+ ALCL is not merely secondary to their younger age.46 Although this accounts for the bimodal age distribution of Ki-1 ALCL, the age ranges for ALK+ and ALK- cases still overlap considerably.
Pathologically, ALK+ ALCL are of non-B cell lineage and almost always coexpress CD30 and EMA.21,46 However, ALK+ ALCL may be morphologically indistinguishable from ALK- cases. ALK expression crosses all morphological forms of ALCL, including appearances which are neither anaplastic nor large cell.21,22,47 Hence, the simpler term ALK+ NHL has been proposed by some.21,22 In these clinicopathological studies of ALK+ ALCL, cases with variant ALK fusions have been lumped with the more common NPM-ALK cases. Although it is reasonable to expect that their clinical behavior may be closer to that of NPM-ALK+ ALCL than ALCL lacking any ALK alterations, it is only with the cloning of these variant ALK fusions that a systematic clinical comparison becomes possible.
Finally, it is tempting to speculate about possible therapeutic implications of the ATIC-ALK fusion. The AICARFT reaction mediated by ATIC is a folate-dependent reaction, and as such is thought to account in part for the anti-purine effects of antifolates such as methotrexate whose primary target is dihydrofolate reductase.34 This raises the intriguing possibility that, as the ATIC-ALK fusion represents a reciprocal rearrangement (leaving only one normal ATIC allele/cell), Ki-1 ALCL bearing this genetic alteration could be more sensitive to antifolates because of lower cellular ATIC activity, due to haploinsufficiency and/or potential dominant negative effects of heterodimerization of ATIC-ALK with residual native ATIC.
Note added in proof: Since our study was completed, Ma et al48 have reported in abstract form the independent cloning of the identical ATIC-ALK fusion and its detection in five cases with the inv(2)(p23q35).
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Acknowledgements |
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Footnotes |
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G. W. B. C. was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (São Paulo, Brazil). J. A. B. was supported in part by the Nebraska State Department of Health (grant LB595).
Accepted for publication November 14, 1999.
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B. Falini and D. Y. Mason Proteins encoded by genes involved in chromosomal alterations in lymphoma and leukemia: clinical value of their detection by immunocytochemistry Blood, January 15, 2002; 99(2): 409 - 426. [Abstract] [Full Text] [PDF]
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S. J. Meech, L. McGavran, L. F. Odom, X. Liang, L. Meltesen, J. Gump, Q. Wei, S. Carlsen, and S. P. Hunger Unusual childhood extramedullary hematologic malignancy with natural killer cell properties that contains tropomyosin 4-anaplastic lymphoma kinase gene fusion Blood, August 15, 2001; 98(4): 1209 - 1216. [Abstract] [Full Text] [PDF]
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J. A. Bridge, M. Kanamori, Z. Ma, D. Pickering, D. A. Hill, W. Lydiatt, M. Y. Lui, G. W. B. Colleoni, C. R. Antonescu, M. Ladanyi, et al. Fusion of the ALK Gene to the Clathrin Heavy Chain Gene, CLTC, in Inflammatory Myofibroblastic Tumor Am. J. Pathol., August 1, 2001; 159(2): 411 - 415. [Abstract] [Full Text]
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B. Maes, V. Vanhentenrijk, I. Wlodarska, J. Cools, B. Peeters, P. Marynen, and C. De Wolf-Peeters The NPM-ALK and the ATIC-ALK Fusion Genes Can Be Detected in Non-Neoplastic Cells Am. J. Pathol., June 1, 2001; 158(6): 2185 - 2193. [Abstract] [Full Text] [PDF]
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H. Stein, H.-D. Foss, H. Durkop, T. Marafioti, G. Delsol, K. Pulford, S. Pileri, and B. Falini CD30+ anaplastic large cell lymphoma: a review of its histopathologic, genetic, and clinical features Blood, December 1, 2000; 96(12): 3681 - 3695. [Abstract] [Full Text] [PDF]
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R. Suzuki, Y. Kagami, K. Takeuchi, M. Kami, M. Okamoto, R. Ichinohasama, N. Mori, M. Kojima, T. Yoshino, H. Yamabe, et al. Prognostic significance of CD56 expression for ALK-positive and ALK-negative anaplastic large-cell lymphoma of T/null cell phenotype Blood, November 1, 2000; 96(9): 2993 - 3000. [Abstract] [Full Text] [PDF]
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M. Ladanyi Aberrant ALK Tyrosine Kinase Signaling : Different Cellular Lineages, Common Oncogenic Mechanisms? Am. J. Pathol., August 1, 2000; 157(2): 341 - 345. [Full Text] [PDF]
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X174/HaeIII size marker bands
(in bp).
