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Consultations in Molecular Diagnostics |
From the Institute for Clinical and Experimental Pathology,
*
Associated Regional and University Pathologists (ARUP) Laboratories, Salt Lake City, Utah; the Department of Pathology,
University of Utah Health Sciences Center, Salt Lake City, Utah; and the Department of Laboratory Medicine,
Deaconess Medical Center, Billings, Montana
Anaplastic large-cell lymphoma (ALCL) is a subtype of aggressive non-Hodgkin’s lymphoma first described in 1985,1 and characterized by the expression of CD30/Ki-1 antigen. By current definition, ALCLs exhibit a T-cell or null phenotype and the majority of cases demonstrate expression of the anaplastic lymphoma kinase (ALK) protein.2 Most ALK-expressing ALCLs harbor the t(2;5)(p23;q35) chromosomal aberration3, 4, 5 that involves the 5'-oligomerization motif region of nucleophosmin (NPM) gene on chromosome 5, and the 3'-cytoplasmic tyrosine kinase catalytic domain of anaplastic lymphoma kinase (ALK) gene of chromosome 2 to form the NPM-ALK fusion gene.6 This rearrangement places the ALK gene under the control of the NPM promoter and results in deregulated expression of the ALK protein. Normal expression of ALK appears to be stringently controlled and limited to the cytoplasm of the testis, ganglion cells of the intestine, and neural tissues.6 The NPM-ALK fusion protein has been shown by immunohistochemistry to localize in the cytoplasm and the nucleus of the neoplastic cells, thereby providing a distinctive marker for t(2;5)-positive ALCL. The t(2;5)-positive ALCLs account for 80 to 85% of the ALK-positive lymphomas.7
The remaining 15 to 20% of ALK-positive ALCLs harbor variant fusion partners and exhibit immunohistological patterns different from that observed for t(2;5).8 In these cases, ALK expression is predominantly cytoplasmic and is not present in a nuclear or nucleolar localization.8 This is because of ALK forming fusion partners with genes other than NPM. Interestingly, a number of non-t(2;5) translocations have also been identified in a nonhematopoietic soft-tissue neoplasm known as inflammatory myofibroblastic tumor (IMT).9, 10, 11 Remarkably, IMT may bear a histological resemblance to morphological variants of ALCL such as the lymphohistiocytic, small cell, and sarcomatoid variants.8, 12 The propensity of ALK for partnering with a diverse variety of genes in both ALCLs and IMTs raises the possibility that other yet unidentified genes that partner with ALK may be involved in the pathogenesis of these tumors. In a quest to identify the ALK fusion partner in a non-t(2,5) ALCL with cytoplasmic expression of the ALK protein, we used the 5'-rapid amplification of cDNA ends (RACE) technique that revealed a fusion between tropomyosin 3 (TPM3) and the ALK genes. Molecular analysis for T-cell clonality also complemented immunohistochemical studies in the definitive assignment of T-cell lineage to the tumor.
Patient History
A 32-year-old Caucasian male presented with a recent history of painful swellings in the right axillary and inguinal regions. The patient denied having fevers, sweats, or weight loss. A computerized tomography scan showed right axillary and inguinal adenopathy but no intra-abdominal or pelvic node enlargement. The masses continued to enlarge despite antibiotic therapy. An excisional biopsy of a 3.0 x 2.5 x 3.0 cm right axillary lymph node was performed. Routine and histopathological examination revealed diffuse effacement of nodal architecture by a proliferation of intermediate-to-large pleomorphic cells with vesicular folded nuclei containing conspicuous nucleoli and with abundant eosinophilic cytoplasm. Many of the cells exhibited nuclei arranged in a wreath-like or horseshoe configuration (Figure 1A)
. Occasional mitotic figures and focal areas of confluent necrosis were noted. Extranodal extension into perinodal adipose tissue was also seen. Immunohistochemical studies showed that the neoplastic cells were negative for CD45 and CD3, but showed strong reactivity for CD30 (Figure 1B)
and epithelial membrane antigen. The tumor cells were also negative for CD20, CD79a, CD15, and CD45RO. Importantly, the tumor cells showed strong reactivity for ALK1 in a cytoplasmic and membranous distribution (Figure 1C)
, without nuclear localization. This pattern contrasted with the characteristic distribution described for t(2;5)-positive ALCLs that typically demonstrate ALK expression in a nuclear and cytoplasmic distribution. The histopathological findings were considered as being compatible with the lymphohistiocytic variant of ALCL. Molecular studies were requested to provide an explanation for the cytoplasmic and membrane distribution of ALK identified by immunohistochemistry.
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This case presented here exhibited an unusual expression of ALK protein in the cytoplasm and cytoplasmic membrane suggesting an ALK-positive neoplasm with a gene fusion partner other than NPM. We sought to determine the identity of the partner gene in the variant ALK fusion using a 5'-RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE) protocol (Ambion, Austin, TX). RNA ligase-mediated rapid amplification of cDNA ends represents a major improvement to the classic RACE technique in that it is designed to amplify cDNA only from full-length, capped mRNA.13, 14, 15
This strategy was chosen because the ALK-positive ALCLs carry a 5'-translocation of a short (<1000 bp) section of a fusion gene partner with a larger (>1500 bp) 3'-portion of the ALK gene. In brief, total RNA was isolated from the snap-frozen tumor sample using the TRIzol method (Invitrogen, Carlsbad, CA), and subjected to calf intestinal phosphatase treatment to remove free 5'-phosphates from molecules such as ribosomal RNA, fragmented mRNA, tRNA, and contaminating genomic DNA. This was followed by phenol-chloroform purification and alcohol precipitation of the RNA. The calf intestinal phosphatase-treated RNA was treated with tobacco acid phosphatase to remove the cap structure from full-length mRNA, leaving a 5'-monophosphate. This prepares the full-length mRNA population within the sample to receive the 5'-RACE RNA adapter oligomer by ligation using T7 RNA ligase. Other RNA species are not ligated with this oligomer because they lack the necessary 5'-phosphate group. After ligation of the adapter oligomer to the 5'-end of the decapped mRNA, cDNA was formed by a random decamer-primed reverse transcriptase reaction. The ALK fusion transcript was preferentially amplified from the cDNA by nested polymerase chain reaction (PCR) using the 5'-RACE adapter and ALK-specific primers. The products of RLM-RACE and PCR were visualized after agarose gel electrophoresis and ethidium bromide staining (Figure 2)
. The RLM-RACE procedure produced two major and a number of minor PCR products. The major DNA fragments were 
500 and 150 bp and the minor products ranged from 200 to 1000 bp as estimated by linear size standards in a 2% agarose gel (Figure 2A)
. The 500- and 1000-bp bands were purified from the gel and cloned into the pCR-BluntII-TOPO vector (Invitrogen). To ascertain that the cloned RLM-RACE product contained the ALK gene, DNA from selected clones were evaluated by PCR using ALK-specific primers that interrogate a 75-bp region downstream of the known fusion point in the ALK gene (Figure 2B)
. Plasmid DNA from a positive clone was purified and submitted for automated DNA sequencing on the Applied Biosystems 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA). The sequencing data confirmed the presence of a 1015-bp partial transcript of an ALK fusion gene containing the complete coding sequence of the 5'-fusion partner gene and a short sequence of the ALK gene (Figure 3)
. A BLAST homology search (www.ncbi.nlm.nih.gov/BLAST/) revealed that the fusion partner gene sequence (758 bp) contained 94 bases of 5'-noncoding sequence and 664 bases homologous with the coding sequence of tropomyosin 3 (TPM3). The remaining 257-bp sequence was homologous with ALK gene sequence beginning with codon 1058 through to codon 1144. The fusion point in the mRNA transcript was between the first nucleotide (G) in codon 222 in exon 7 of TPM3 and the second nucleotide in codon 1058 in exon 20 of ALK. The fusion completes the appropriate ALK codon 1058 (GTG) and permits the in-frame translation of the C-terminal ALK kinase domain (Figure 3)
. This represents an example of a previously described t(1;2)(q25;p23) translocation, involving TPM3 and ALK.16
To assess the clonality status and thereby determine the lineage of the tumor cells in our patient sample, PCR for clonal rearrangements of the immunoglobulin heavy chain17
and T-cell receptor 
-chain18
genes was performed as previously described. The T-cell receptor PCR yielded a monoclonal band (Figure 4)
and IgH PCR yielded a polyclonal pattern.
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The vast majority of ALK-positive ALCLs carry the t(2;5)(p23;q35) genetic abnormality, a translocation that has been extensively characterized because of its utility as a disease marker for ALCL.12 The 680-amino acid fusion protein is comprised of the N-terminal 116 amino acids from NPM and the C-terminal 563 amino acids from ALK. The localization of the fusion protein in the nucleus and cytoplasm results from the hetero-oligomerization of NPM-ALK with normal NPM, which carries nuclear localization signal domains. The oligomerization of the fusion protein also causes the constitutive activation of the tyrosine kinase catalytic domain of ALK, resulting in multiple signaling cascades that lead to increased mitogenesis and cell survival. ALK protein-positive ALCLs exhibit a better prognosis than ALK protein-negative ALCLs.19 A small percentage of ALCLs express ALK in the cytoplasm only, suggesting that other genes can deregulate the expression of the ALK gene. To date there are nine non-t(2;5) genetic rearrangements identified that involve the ALK gene, of which six are associated with ALCL. These are t(X;2)(q11;p23), t(1;2)(q25;p23), inv(2)(p23;q35), t(2;3)(p23;q21), t(2;17)(p23;q23), and t(2;17)(p23;q35). In each of these translocations the partner gene appears to promote the dimerization of the chimeric ALK protein, which leads to autophosphorylation and constitutive tyrosine kinase activity. The non-t(2;5)-positive ALCL-associated ALK-fusion partners that have been identified so far include moesin (MSN), tropomyosin 3 (TPM3),16 5-aminoimidazole-4-carboximide-1-b-D-ribonucleotide transformylase/inosine monophosphate cyclohydrolase (ATIC),20, 21, 22 TRK-fused gene (TFG),23, 24 clathrin heavy chain (CLTC),12 and most recently ALK lymphoma oligomerization partner on chromosome 17 (ALO17).25 At the time of preparation of this manuscript NPM-ALK, ATIC-ALK, TFG-ALKL, TFG-ALKS, and ALO17-ALK have only been identified in ALCLs, TPM3-ALK, and CLTC-ALK have been described in both ALCLs and IMTs, whereas RanBP2-ALK, TPM4-ALK, and a fusion involving cysteinyl tRNA synthetase (CARS-ALK) have only been described in IMTs.12, 25
As is evident from the facile identification of the fusion partners listed above, 5'-RACE PCR affords the identification of unknown flanking cDNA sequences, when a portion of the 3'-cDNA sequence is already known. In the case of the ALK gene in which several translocation partners have been associated, 5'-RACE PCR provides an opportunity to identify novel translocation partners that could further elucidate the pathogenesis of neoplasia with ALK deregulation. In addition, in circumstances in which the abnormal fusion partner has already been described such as in the case described in this report, 5'-RACE could be a more convenient option than conventional PCR, because it would be applicable to all possible translocation partners. On the other hand, conventional PCR is limited to the identification of known translocation partners, and as is the case with ALK fusions, could require as many as 10 or more different primer pairs before identification of the specific translocation partner is achieved. Thus, RACE PCR is advantageous in that it is informative whether or not the fusion partner gene is already described.
In summary, RLM-RACE analysis of RNA isolated from our ALCL specimen that showed cytoplasmic and membrane expression of ALK resulted in the identification of a variant ALK fusion gene. The fusion gene occurred as a result of a t(1;2)(q25;p23) aberration that juxtaposed the 5'-dimerization domains of TPM3 on chromosome 1, and the 3'-cytoplasmic tyrosine kinase catalytic domain of ALK on chromosome 2. PCR for clonal rearrangements of the TCR chain gene was monoclonal, thus indicating that the tumor cells were unequivocally of T-cell derivation, and ruling out a diagnosis of IMT. As with the t(2;5) ALK-positive ALCL there appears to be a favorable prognosis associated with the expression of the variant ALK fusion gene.26
In this regard, the patient described herein remains disease-free 1 year after a multiagent chemotherapy regimen.
Footnotes
Address reprints requests to Kojo S. J. Elenitoba-Johnson, M.D., Division of Anatomic Pathology, University of Utah Health Sciences Center, 50 North Medical Dr., Salt Lake City, UT 84132. E-mail: kojo.elenitobaj{at}path.utah.edu
N. S. R. and S. D. J. contributed equally to this study.
Accepted for publication January 9, 2003.
References
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