Brief Report
A Novel Fusion of TPR and ALK in Lung Adenocarcinoma Yoon-La Choi, MD, PhD,*† Maruja E. Lira, BS,‡ Mineui Hong, MD,* Ryong Nam Kim, PhD,§ So-Jung Choi PhD,** Ji-Young Song, MS,† Kinnari Pandy, MS,‡ Derrick L. Mann, PhD,║ Joshua A. Stahl, PhD,║ Heather E. Peckham, PhD,║ Zongli Zheng, MD, PhD,¶# Joungho Han, PhD,† Mao Mao, MD, PhD,‡ and Jhingook Kim, MD, PhD**
Introduction: Anaplastic lymphoma kinase (ALK) fusion is the most common mechanism for overexpression and activation in non– small-cell lung carcinoma. Several fusion partners of ALK have been reported, including echinoderm microtubule-associated protein-like 4, TRK-fused gene, kinesin family member 5B, kinesin light chain 1 (KLC1), protein tyrosine phosphatase and nonreceptor type 3, and huntingtin interacting protein 1 (HIP1). Methods and Results: A 60-year-old Korean man had a lung mass which was a poorly differentiated adenocarcinoma with ALK overexpression. By using an Anchored Multiplex polymerase chain reaction assay and sequencing, we found that tumor had a novel translocated promoter region (TPR)-ALK fusion. The fusion transcript was generated from an intact, in-frame fusion of TPR exon 15 and ALK exon 20 (t(1;2)(q31.1;p23)). The TPR-ALK fusion encodes a predicted protein of 1192 amino acids with a coiled-coil domain encoded by the 5’-2nd of the TPR and juxtamembrane and kinase domains encoded by the 3’-end of the ALK. Conclusions: The novel fusion gene and its protein TRP-ALK, harboring coiled-coil and kinase domains, could possess transforming potential and responses to treatment with ALK inhibitors. This case is the first report of TPR-ALK fusion transcript in clinical tumor samples and could provide a novel diagnostic and therapeutic candidate target for patients with cancer, including non–small-cell lung carcinoma. Key Words: Lung cancer, ALK, TPR, Translocation. (J Thorac Oncol. 2014;9: 563–566)
Departments of *Pathology and **Thoracic Surgery, Samsung Medical Center, Sungkyunkwan University College of Medicine, Seoul, Korea; †Laboratory of Cancer Genomics and Molecular Pathology, Samsung Biomedical Research Institute, Samsung Medical Center, Seoul, Korea; ‡Oncology Research Unit, Pfizer Inc., San Diego, California; §Department of Pharmacy, College of Pharmacy, Seoul National University, Seoul, Korea; ║ArcherDx Inc., Boulder, Colorado; ¶Department of Pathology, Massachusetts General Hospital, Boston, Massachusetts; and #Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, Stockholm, Sweden. Disclosure: M.E. Lira, K. Pandya, and M. Mao are employed by Pfizer Inc., and have their own stock in Pfizer Inc. The other authors declare no conflict of interest. Address for correspondence: Jhingook Kim, Department of Thoracic Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Ilwon-dong, Gangnam-gu, Seoul, 135–710, Korea. E-mail: [email protected] Copyright © 2014 by the International Association for the Study of Lung Cancer ISSN: 1556-0864/14/0904-0563
Journal of Thoracic Oncology ® • Volume 9, Number 4, April 2014
CASE DESCRIPTION A 60-year-old Korean man was referred to a hospital because of a cough that persisted for 3 months. The patient had a history of tuberculosis 20 years ago. He was an exsmoker with a 30-pack-year history of regular smoking. On computed tomography scan of the chest, a 51-mm irregular mass and stable tuberculosis lesions were seen in the right upper lobe (Fig. 1A). The patient underwent lobectomy, which removed a lobulating grayish and yellow-tan solid tumor (Fig. 1B) that invaded the parietal pleural of the chest wall. Microscopic examination revealed that the tumor was a poorly differentiated adenocarcinoma with solid type (Fig. 1C). No mucinous components or signet-ring cells, which are usual features of ALK-positive adenocarcinoma, were identified. The tumor showed lymphatic invasion and metastasis into a single regional lymph node. The tumor did not have mutations in either epidermal growth factor receptor (EGFR) or Kirsten rat sarcoma viral oncogene homolog (KRAS). ALK antibody staining (dilution 1:50; clone 5A4; Novocastra, Newcastle, United Kingdom) was diffusively cytoplasmic and slightly granular (Fig. 1D). Break-apart fluorescence in situ hybridization (FISH) analysis for ALK (Abbott Molecular, Abbott Park, IL) showed split 5’- and 3’-probe signals with occasional multiple signals (Fig. 1E and F). During screening for ALK fusions in 533 lung adenocarcinoma tumors using an assay that we developed,1 we discovered that the tumor in this case had elevated ALK 3’ expression but low scores for known ALK fusion variants. These results indicated that the tumor might harbor a novel ALK fusion. Using an Anchored Multiplex polymerase chain reaction (PCR) assay for low amounts of low-quality formalin-fixed samples (Dr. Zheng, personal communication) and sequencing (Ion Personal Genome Machine [PGM]; Life Technologies, Carlsbad, CA), we determined that tumor had a novel TPR-ALK fusion. Reverse-transcriptase PCR followed by Sanger sequencing confirmed that the fusion transcript was generated from an intact, in-frame fusion of TPR exon 15 and ALK exon 20 (Fig. 2A). To assess this fusion event at the genomic level, we performed genomic PCR. The results indicated that the chromosomal translocation t(1;2)(q31.1;p23) had occurred by recombination between nucleotide 186,325,172 (nucleotide 246 downstream of the TPR exon 15) on chromosome 1 and nucleotide 29,446,944 (nucleotide 550 upstream of the ALK exon 20) on chromosome 2. This translocation resulted in a novel TPR-ALK
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Journal of Thoracic Oncology ® • Volume 9, Number 4, April 2014
Choi et al.
FIGURE 1. A, Chest computed tomography with a 51-mm irregular mass in the right upper lobe (arrow). B, Cut surface of the lobectomy specimen showing a lobulating grayish and yellow-tan solid mass. C, Hematoxylin and eosin staining shows adenocarcinoma with a solid pattern (×200). D, ALK immunohistochemical staining shows diffuse cytoplasmic pattern (×200). E and F, FISH assay of the ALK genomic rearrangement with split 5’- and 3’-probe signals (red and green arrows) with occasionally increased ALK copy number.
fusion gene. The genomic sequence and cDNA sequence harboring and surrounding the translocation fusion points and the TPR-ALK fusion transcript were deposited in the National Center for Biotechnology Information databank. The TPRALK fusion encodes a predicted protein of 1192 amino acids with a coiled-coil domain encoded by the 5’-end of the TPR and JM and kinase domains encoded by the 3’-end of the ALK (Fig. 2B). The patient was treated with first-line adjuvant chemotherapy using vinorelbine and cisplain for four cycles. After 18 months of follow-up, no evidence of recurrent tumor or distant metastasis was noted.
DISCUSSION Lung cancer is a leading cause of global cancer mortality. In addition to the two oncogenes EGFR and KRAS, ALK is emerging as a molecular screening target for therapeutic strategies for pulmonary adenocarcinoma. The most common mechanism of ALK expression in tumors results from fusion of the ALK gene with another gene through chromosomal translocation.2 To date, five genes have been reported as fusion partners for ALK in lung adenocarcinomas: echinoderm microtubule-associated protein-like 4 (EML4), TRKfused gene, kinesin family member 5B (KIF5B), kinesin light
564
chain 1 (KLC1), and protein tyrosine phosphatase and nonreceptor type 3. Recently, we reported that HIP1 was a fusion partner for ALK (in press). TPR encodes a large coiled-coil protein that forms intranuclear filaments attached to the inner surface of nuclear pore complexes (NPCs),3 which joins the inner and outer nuclear membranes and allows passive diffusion of ions and small molecules. The coiled-coil structure includes the first 1600 amino acids of TPR and facilitates the assembly of homopolymer or heteropolymer filaments.4 The TPR protein directly interacts with several NPC components. TPR is essential for the nuclear export of mRNAs and some proteins.5 Its position on the cytoplasmic side of NPCs suggests that it might extend fibrils from the nuclear pore into the cytoplasm.6 Oncogenic fusions of TPR with partner genes occur in some neoplasms. Gonzatti-Haces et al.7 first described a fusion of TPR with the met proto-oncogene (MET) oncogene. TPR-MET is created by a fusion between a 5’-region of TPR on chromosome 1 and a 3’-region of MET on chromosome 7. Mutations in MET frequently deregulate the enzymatic activity of the tyrosine kinase receptor, activating its oncogenic potential. The fusion of TPR to MET facilitates autophosphorylation of the fusion protein, which shows oncogenic activity. The TPR-MET gene is overexpressed in gastric carcinoma and precursor lesions.8
Copyright © 2014 by the International Association for the Study of Lung Cancer
Journal of Thoracic Oncology ® • Volume 9, Number 4, April 2014
A
TPR
TPR and ALK in Lung Adenocarcinoma
ALK
exon 15
exon 20
….ACATG …TTTGGTTA GCAGGCC.……..…….. TGTAC…….
1q31.1
Chr1
TPR intron 246 bp
ALK intron 550 bp
Chr2
TPR intron
ALK intron
TPR exon 15
Genomic rearrangement for TPR-ALK
B
1
ALK exon 20
TPR-ALK fusion transcript cDNA
629
Coiled coil
TPR 1
1
2363
Coiled coil
FIGURE 2. A, Schematic structure of the genomic DNA sequence (upper) and DNA sequence chromatograms (lower) showing fusion points for genomic rearrangement and TPR-ALK fusion transcript cDNA. B, Functional domain structures of TPR, ALK, and TPR-ALK proteins. TPR, translocated promoter region; ALK, anaplastic lymphoma kinase; JM, juxtamembrane.
1192
629
Coiled coil
TPR-ALK
ALK
2p23
J M
Kinase
1058
1620
J M
Kinase
TRK-T1 is formed by an intrachromosomal rearrangement that recombines the 5’-end of the TPR gene to a region encoding the tyrosine kinase domain of TRK.9 This fusion gene is found in papillary thyroid carcinoma and promotes neoplastic transformation of the thyroid epithelium.10 A TPRfibroblast growth factor receptor 1 (FGFR1) fusion was identified in myeloproliferative syndrome.11 TPR-FGFR1 might promote the malignant transformation of cells, as observed with other FGFR1 translocations. Different FGFR1 fusions are related to subtly distinct disease phenotypes. Clinical presentations of patients vary with fusion subtypes. For example, patients with breakpoint cluster region (BCR)-FGFR1 t(8;22) are clinically and hematologically similar to patients with t(9;22), which causes chronic myelogenous leukemia. However, patients with FGFR1 oncogene partner (FOP)FGFR1 t(6;8) or CEP110-FGFR1 t(8;9) show pathological phenotypes similar to chronic myelomonocytic leukemia.12 Among the known five fusion proteins that have ALK as a partner, KIF5B-ALK and KLC1-ALK show different staining patterns from the conventional EML4-ALK by anti-ALK immunohistochemistry. EML4-ALK tends to have a diffuse cytoplasmic staining pattern, but fusion proteins of ALK with KIF5B or KLC1 exhibit cytoplasmic granular staining with highlighted peripheral areas. This staining pattern suggests that the immunostaining phenotypes of tumor cells might be determined by the proteins that fuse with ALK. The novel fusion protein TPR-ALK presented in this case report showed a diffuse cytoplasmic staining pattern similar to the pattern of EML4-ALK.
We report a naturally occurring in vivo TPR-ALK fusion. A previous report showed that an artificial, in vitro–engineered TPR-ALK fusion protein could transform rat fibroblasts, and that retrovirus-mediated gene transfer of TPR-ALK into murine bone marrow developed immunoblastic B-cell lymphomas in the transplanted mice.13,14 A putative dimerization motif in the nucleophosmin portion of the fusion protein is essential for activating anaplastic large cell lymphoma cells and causing malignant transformation through phosphorylation of downstream molecules in tumor cells.15 Thus, the coiled-coil domain of TPR, which is similar to the dimerization motif in NPM, might contribute to the oncogenic potential of TPR-ALK. The previous results with artificially engineered ALK fusion proteins strongly suggest that the novel naturally occurring in vivo TPR-ALK fusion generated by a translocation might have been oncogenic and caused the lung cancer and lymph node metastasis observed in this case report. Although the response of TPR-ALK to ALK inhibitors in lung cancers is unknown, ALK inhibitors might be an efficient treatment for patients with TPR-ALK–positive cancer. The fusion TPR-ALK gene reported here and its fusion protein might be useful as biomarkers or therapeutic targets for future cancer diagnostics and treatment.
CONCLUSION This case is the first report of TPR-ALK fusion transcript in non–small-cell lung carcinomas. Fusions of TPR with several partner genes occur and show oncogenic effects in some neoplasms. TPR protein contains coiled-coil domains
Copyright © 2014 by the International Association for the Study of Lung Cancer
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Journal of Thoracic Oncology ® • Volume 9, Number 4, April 2014
Choi et al.
and the fusion protein possibly dimerizes constitutively, possessing transforming potential and responses to treatment with ALK inhibitors.
ACKNOWLEDGMENTS
This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (NRF-2013R1A2A2A01068922). REFERENCES 1. Lira ME, Kim TM, Huang D, et al. Multiplexed gene expression and fusion transcript analysis to detect ALK fusions in lung cancer. J Mol Diagn 2013;15:51–61. 2. Kwak EL, Bang YJ, Camidge DR, et al. Anaplastic lymphoma kinase inhibition in non-small-cell lung cancer. N Engl J Med 2010;363:1693–1703. 3. Bangs P, Burke B, Powers C, et al. Functional analysis of Tpr: identification of nuclear pore complex association and nuclear localization domains and a role in mRNA export. J Cell Biol 1998;143:1801–1812. 4. Albers K, Fuchs E. The molecular biology of intermediate filament proteins. Int Rev Cytol 1992;134:243–279. 5. Coyle JH, Bor YC, Rekosh D, et al. The Tpr protein regulates export of mRNAs with retained introns that traffic through the Nxf1 pathway. RNA 2011;17:1344–1356. 6. Bangs PL, Sparks CA, Odgren PR, Fey EG. Product of the oncogene-activating gene Tpr is a phosphorylated protein of the nuclear pore complex. J Cell Biochem 1996;61:48–60.
566
7. Gonzatti-Haces M, Seth A, Park M, et al. Characterization of the TPRMET oncogene p65 and the MET protooncogene p140 protein-tyrosine kinases. Proc Natl Acad Sci U S A 1988;85:21–25. 8. Soman NR, Correa P, Ruiz BA, Wogan GN. The TPR-MET oncogenic rearrangement is present and expressed in human gastric carcinoma and precursor lesions. Proc Natl Acad Sci U S A 1991;88:4892–4896. 9. Greco A, Pierotti MA, Bongarzone I, et al. TRK-T1 is a novel oncogene formed by the fusion of TPR and TRK genes in human papillary thyroid carcinomas. Oncogene 1992;7:237–242. 10. Russell JP, Powell DJ, Cunnane M, et al. The TRK-T1 fusion protein induces neoplastic transformation of thyroid epithelium. Oncogene 2000;19:5729–5735. 11. Li F, Zhai YP, Tang YM, et al. Identification of a novel partner gene, TPR, fused to FGFR1 in 8p11 myeloproliferative syndrome. Genes Chromosomes Cancer 2012;51:890–897. 12. Macdonald D, Reiter A, Cross NC. The 8p11 myeloproliferative syndrome: a distinct clinical entity caused by constitutive activation of FGFR1. Acta Haematol 2002;107:101–107. 13. Mason DY, Pulford KA, Bischof D, et al. Nucleolar localization of the nucleophosmin-anaplastic lymphoma kinase is not required for malignant transformation. Cancer Res 1998;58:1057–1062. 14. Kutok JL, Aster JC. Molecular biology of anaplastic lymphoma kinase-positive anaplastic large-cell lymphoma. J Clin Oncol 2002;20:3691–3702. 15. Bonvini P, Gastaldi T, Falini B, Rosolen A. Nucleophosmin-anaplastic lymphoma kinase (NPM-ALK), a novel Hsp90-client tyrosine kinase: down-regulation of NPM-ALK expression and tyrosine phosphorylation in ALK(+) CD30(+) lymphoma cells by the Hsp90 antagonist 17-allylamino,17-demethoxygeldanamycin. Cancer Res 2002;62:1559–1566.
Copyright © 2014 by the International Association for the Study of Lung Cancer
A Novel Fusion of TPR and ALK in Lung Adenocarcinoma Yoon-La Choi, MD, PhD,*† Maruja E. Lira, BS,‡ Mineui Hong, MD,* Ryong Nam Kim, PhD,§ So-Jung Choi PhD,** Ji-Young Song, MS,† Kinnari Pandy, MS,‡ Derrick L. Mann, PhD,║ Joshua A. Stahl, PhD,║ Heather E. Peckham, PhD,║ Zongli Zheng, MD, PhD,¶# Joungho Han, PhD,† Mao Mao, MD, PhD,‡ and Jhingook Kim, MD, PhD**
Introduction: Anaplastic lymphoma kinase (ALK) fusion is the most common mechanism for overexpression and activation in non– small-cell lung carcinoma. Several fusion partners of ALK have been reported, including echinoderm microtubule-associated protein-like 4, TRK-fused gene, kinesin family member 5B, kinesin light chain 1 (KLC1), protein tyrosine phosphatase and nonreceptor type 3, and huntingtin interacting protein 1 (HIP1). Methods and Results: A 60-year-old Korean man had a lung mass which was a poorly differentiated adenocarcinoma with ALK overexpression. By using an Anchored Multiplex polymerase chain reaction assay and sequencing, we found that tumor had a novel translocated promoter region (TPR)-ALK fusion. The fusion transcript was generated from an intact, in-frame fusion of TPR exon 15 and ALK exon 20 (t(1;2)(q31.1;p23)). The TPR-ALK fusion encodes a predicted protein of 1192 amino acids with a coiled-coil domain encoded by the 5’-2nd of the TPR and juxtamembrane and kinase domains encoded by the 3’-end of the ALK. Conclusions: The novel fusion gene and its protein TRP-ALK, harboring coiled-coil and kinase domains, could possess transforming potential and responses to treatment with ALK inhibitors. This case is the first report of TPR-ALK fusion transcript in clinical tumor samples and could provide a novel diagnostic and therapeutic candidate target for patients with cancer, including non–small-cell lung carcinoma. Key Words: Lung cancer, ALK, TPR, Translocation. (J Thorac Oncol. 2014;9: 563–566)
Departments of *Pathology and **Thoracic Surgery, Samsung Medical Center, Sungkyunkwan University College of Medicine, Seoul, Korea; †Laboratory of Cancer Genomics and Molecular Pathology, Samsung Biomedical Research Institute, Samsung Medical Center, Seoul, Korea; ‡Oncology Research Unit, Pfizer Inc., San Diego, California; §Department of Pharmacy, College of Pharmacy, Seoul National University, Seoul, Korea; ║ArcherDx Inc., Boulder, Colorado; ¶Department of Pathology, Massachusetts General Hospital, Boston, Massachusetts; and #Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, Stockholm, Sweden. Disclosure: M.E. Lira, K. Pandya, and M. Mao are employed by Pfizer Inc., and have their own stock in Pfizer Inc. The other authors declare no conflict of interest. Address for correspondence: Jhingook Kim, Department of Thoracic Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Ilwon-dong, Gangnam-gu, Seoul, 135–710, Korea. E-mail: [email protected] Copyright © 2014 by the International Association for the Study of Lung Cancer ISSN: 1556-0864/14/0904-0563
Journal of Thoracic Oncology ® • Volume 9, Number 4, April 2014
CASE DESCRIPTION A 60-year-old Korean man was referred to a hospital because of a cough that persisted for 3 months. The patient had a history of tuberculosis 20 years ago. He was an exsmoker with a 30-pack-year history of regular smoking. On computed tomography scan of the chest, a 51-mm irregular mass and stable tuberculosis lesions were seen in the right upper lobe (Fig. 1A). The patient underwent lobectomy, which removed a lobulating grayish and yellow-tan solid tumor (Fig. 1B) that invaded the parietal pleural of the chest wall. Microscopic examination revealed that the tumor was a poorly differentiated adenocarcinoma with solid type (Fig. 1C). No mucinous components or signet-ring cells, which are usual features of ALK-positive adenocarcinoma, were identified. The tumor showed lymphatic invasion and metastasis into a single regional lymph node. The tumor did not have mutations in either epidermal growth factor receptor (EGFR) or Kirsten rat sarcoma viral oncogene homolog (KRAS). ALK antibody staining (dilution 1:50; clone 5A4; Novocastra, Newcastle, United Kingdom) was diffusively cytoplasmic and slightly granular (Fig. 1D). Break-apart fluorescence in situ hybridization (FISH) analysis for ALK (Abbott Molecular, Abbott Park, IL) showed split 5’- and 3’-probe signals with occasional multiple signals (Fig. 1E and F). During screening for ALK fusions in 533 lung adenocarcinoma tumors using an assay that we developed,1 we discovered that the tumor in this case had elevated ALK 3’ expression but low scores for known ALK fusion variants. These results indicated that the tumor might harbor a novel ALK fusion. Using an Anchored Multiplex polymerase chain reaction (PCR) assay for low amounts of low-quality formalin-fixed samples (Dr. Zheng, personal communication) and sequencing (Ion Personal Genome Machine [PGM]; Life Technologies, Carlsbad, CA), we determined that tumor had a novel TPR-ALK fusion. Reverse-transcriptase PCR followed by Sanger sequencing confirmed that the fusion transcript was generated from an intact, in-frame fusion of TPR exon 15 and ALK exon 20 (Fig. 2A). To assess this fusion event at the genomic level, we performed genomic PCR. The results indicated that the chromosomal translocation t(1;2)(q31.1;p23) had occurred by recombination between nucleotide 186,325,172 (nucleotide 246 downstream of the TPR exon 15) on chromosome 1 and nucleotide 29,446,944 (nucleotide 550 upstream of the ALK exon 20) on chromosome 2. This translocation resulted in a novel TPR-ALK
563
Journal of Thoracic Oncology ® • Volume 9, Number 4, April 2014
Choi et al.
FIGURE 1. A, Chest computed tomography with a 51-mm irregular mass in the right upper lobe (arrow). B, Cut surface of the lobectomy specimen showing a lobulating grayish and yellow-tan solid mass. C, Hematoxylin and eosin staining shows adenocarcinoma with a solid pattern (×200). D, ALK immunohistochemical staining shows diffuse cytoplasmic pattern (×200). E and F, FISH assay of the ALK genomic rearrangement with split 5’- and 3’-probe signals (red and green arrows) with occasionally increased ALK copy number.
fusion gene. The genomic sequence and cDNA sequence harboring and surrounding the translocation fusion points and the TPR-ALK fusion transcript were deposited in the National Center for Biotechnology Information databank. The TPRALK fusion encodes a predicted protein of 1192 amino acids with a coiled-coil domain encoded by the 5’-end of the TPR and JM and kinase domains encoded by the 3’-end of the ALK (Fig. 2B). The patient was treated with first-line adjuvant chemotherapy using vinorelbine and cisplain for four cycles. After 18 months of follow-up, no evidence of recurrent tumor or distant metastasis was noted.
DISCUSSION Lung cancer is a leading cause of global cancer mortality. In addition to the two oncogenes EGFR and KRAS, ALK is emerging as a molecular screening target for therapeutic strategies for pulmonary adenocarcinoma. The most common mechanism of ALK expression in tumors results from fusion of the ALK gene with another gene through chromosomal translocation.2 To date, five genes have been reported as fusion partners for ALK in lung adenocarcinomas: echinoderm microtubule-associated protein-like 4 (EML4), TRKfused gene, kinesin family member 5B (KIF5B), kinesin light
564
chain 1 (KLC1), and protein tyrosine phosphatase and nonreceptor type 3. Recently, we reported that HIP1 was a fusion partner for ALK (in press). TPR encodes a large coiled-coil protein that forms intranuclear filaments attached to the inner surface of nuclear pore complexes (NPCs),3 which joins the inner and outer nuclear membranes and allows passive diffusion of ions and small molecules. The coiled-coil structure includes the first 1600 amino acids of TPR and facilitates the assembly of homopolymer or heteropolymer filaments.4 The TPR protein directly interacts with several NPC components. TPR is essential for the nuclear export of mRNAs and some proteins.5 Its position on the cytoplasmic side of NPCs suggests that it might extend fibrils from the nuclear pore into the cytoplasm.6 Oncogenic fusions of TPR with partner genes occur in some neoplasms. Gonzatti-Haces et al.7 first described a fusion of TPR with the met proto-oncogene (MET) oncogene. TPR-MET is created by a fusion between a 5’-region of TPR on chromosome 1 and a 3’-region of MET on chromosome 7. Mutations in MET frequently deregulate the enzymatic activity of the tyrosine kinase receptor, activating its oncogenic potential. The fusion of TPR to MET facilitates autophosphorylation of the fusion protein, which shows oncogenic activity. The TPR-MET gene is overexpressed in gastric carcinoma and precursor lesions.8
Copyright © 2014 by the International Association for the Study of Lung Cancer
Journal of Thoracic Oncology ® • Volume 9, Number 4, April 2014
A
TPR
TPR and ALK in Lung Adenocarcinoma
ALK
exon 15
exon 20
….ACATG …TTTGGTTA GCAGGCC.……..…….. TGTAC…….
1q31.1
Chr1
TPR intron 246 bp
ALK intron 550 bp
Chr2
TPR intron
ALK intron
TPR exon 15
Genomic rearrangement for TPR-ALK
B
1
ALK exon 20
TPR-ALK fusion transcript cDNA
629
Coiled coil
TPR 1
1
2363
Coiled coil
FIGURE 2. A, Schematic structure of the genomic DNA sequence (upper) and DNA sequence chromatograms (lower) showing fusion points for genomic rearrangement and TPR-ALK fusion transcript cDNA. B, Functional domain structures of TPR, ALK, and TPR-ALK proteins. TPR, translocated promoter region; ALK, anaplastic lymphoma kinase; JM, juxtamembrane.
1192
629
Coiled coil
TPR-ALK
ALK
2p23
J M
Kinase
1058
1620
J M
Kinase
TRK-T1 is formed by an intrachromosomal rearrangement that recombines the 5’-end of the TPR gene to a region encoding the tyrosine kinase domain of TRK.9 This fusion gene is found in papillary thyroid carcinoma and promotes neoplastic transformation of the thyroid epithelium.10 A TPRfibroblast growth factor receptor 1 (FGFR1) fusion was identified in myeloproliferative syndrome.11 TPR-FGFR1 might promote the malignant transformation of cells, as observed with other FGFR1 translocations. Different FGFR1 fusions are related to subtly distinct disease phenotypes. Clinical presentations of patients vary with fusion subtypes. For example, patients with breakpoint cluster region (BCR)-FGFR1 t(8;22) are clinically and hematologically similar to patients with t(9;22), which causes chronic myelogenous leukemia. However, patients with FGFR1 oncogene partner (FOP)FGFR1 t(6;8) or CEP110-FGFR1 t(8;9) show pathological phenotypes similar to chronic myelomonocytic leukemia.12 Among the known five fusion proteins that have ALK as a partner, KIF5B-ALK and KLC1-ALK show different staining patterns from the conventional EML4-ALK by anti-ALK immunohistochemistry. EML4-ALK tends to have a diffuse cytoplasmic staining pattern, but fusion proteins of ALK with KIF5B or KLC1 exhibit cytoplasmic granular staining with highlighted peripheral areas. This staining pattern suggests that the immunostaining phenotypes of tumor cells might be determined by the proteins that fuse with ALK. The novel fusion protein TPR-ALK presented in this case report showed a diffuse cytoplasmic staining pattern similar to the pattern of EML4-ALK.
We report a naturally occurring in vivo TPR-ALK fusion. A previous report showed that an artificial, in vitro–engineered TPR-ALK fusion protein could transform rat fibroblasts, and that retrovirus-mediated gene transfer of TPR-ALK into murine bone marrow developed immunoblastic B-cell lymphomas in the transplanted mice.13,14 A putative dimerization motif in the nucleophosmin portion of the fusion protein is essential for activating anaplastic large cell lymphoma cells and causing malignant transformation through phosphorylation of downstream molecules in tumor cells.15 Thus, the coiled-coil domain of TPR, which is similar to the dimerization motif in NPM, might contribute to the oncogenic potential of TPR-ALK. The previous results with artificially engineered ALK fusion proteins strongly suggest that the novel naturally occurring in vivo TPR-ALK fusion generated by a translocation might have been oncogenic and caused the lung cancer and lymph node metastasis observed in this case report. Although the response of TPR-ALK to ALK inhibitors in lung cancers is unknown, ALK inhibitors might be an efficient treatment for patients with TPR-ALK–positive cancer. The fusion TPR-ALK gene reported here and its fusion protein might be useful as biomarkers or therapeutic targets for future cancer diagnostics and treatment.
CONCLUSION This case is the first report of TPR-ALK fusion transcript in non–small-cell lung carcinomas. Fusions of TPR with several partner genes occur and show oncogenic effects in some neoplasms. TPR protein contains coiled-coil domains
Copyright © 2014 by the International Association for the Study of Lung Cancer
565
Journal of Thoracic Oncology ® • Volume 9, Number 4, April 2014
Choi et al.
and the fusion protein possibly dimerizes constitutively, possessing transforming potential and responses to treatment with ALK inhibitors.
ACKNOWLEDGMENTS
This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (NRF-2013R1A2A2A01068922). REFERENCES 1. Lira ME, Kim TM, Huang D, et al. Multiplexed gene expression and fusion transcript analysis to detect ALK fusions in lung cancer. J Mol Diagn 2013;15:51–61. 2. Kwak EL, Bang YJ, Camidge DR, et al. Anaplastic lymphoma kinase inhibition in non-small-cell lung cancer. N Engl J Med 2010;363:1693–1703. 3. Bangs P, Burke B, Powers C, et al. Functional analysis of Tpr: identification of nuclear pore complex association and nuclear localization domains and a role in mRNA export. J Cell Biol 1998;143:1801–1812. 4. Albers K, Fuchs E. The molecular biology of intermediate filament proteins. Int Rev Cytol 1992;134:243–279. 5. Coyle JH, Bor YC, Rekosh D, et al. The Tpr protein regulates export of mRNAs with retained introns that traffic through the Nxf1 pathway. RNA 2011;17:1344–1356. 6. Bangs PL, Sparks CA, Odgren PR, Fey EG. Product of the oncogene-activating gene Tpr is a phosphorylated protein of the nuclear pore complex. J Cell Biochem 1996;61:48–60.
566
7. Gonzatti-Haces M, Seth A, Park M, et al. Characterization of the TPRMET oncogene p65 and the MET protooncogene p140 protein-tyrosine kinases. Proc Natl Acad Sci U S A 1988;85:21–25. 8. Soman NR, Correa P, Ruiz BA, Wogan GN. The TPR-MET oncogenic rearrangement is present and expressed in human gastric carcinoma and precursor lesions. Proc Natl Acad Sci U S A 1991;88:4892–4896. 9. Greco A, Pierotti MA, Bongarzone I, et al. TRK-T1 is a novel oncogene formed by the fusion of TPR and TRK genes in human papillary thyroid carcinomas. Oncogene 1992;7:237–242. 10. Russell JP, Powell DJ, Cunnane M, et al. The TRK-T1 fusion protein induces neoplastic transformation of thyroid epithelium. Oncogene 2000;19:5729–5735. 11. Li F, Zhai YP, Tang YM, et al. Identification of a novel partner gene, TPR, fused to FGFR1 in 8p11 myeloproliferative syndrome. Genes Chromosomes Cancer 2012;51:890–897. 12. Macdonald D, Reiter A, Cross NC. The 8p11 myeloproliferative syndrome: a distinct clinical entity caused by constitutive activation of FGFR1. Acta Haematol 2002;107:101–107. 13. Mason DY, Pulford KA, Bischof D, et al. Nucleolar localization of the nucleophosmin-anaplastic lymphoma kinase is not required for malignant transformation. Cancer Res 1998;58:1057–1062. 14. Kutok JL, Aster JC. Molecular biology of anaplastic lymphoma kinase-positive anaplastic large-cell lymphoma. J Clin Oncol 2002;20:3691–3702. 15. Bonvini P, Gastaldi T, Falini B, Rosolen A. Nucleophosmin-anaplastic lymphoma kinase (NPM-ALK), a novel Hsp90-client tyrosine kinase: down-regulation of NPM-ALK expression and tyrosine phosphorylation in ALK(+) CD30(+) lymphoma cells by the Hsp90 antagonist 17-allylamino,17-demethoxygeldanamycin. Cancer Res 2002;62:1559–1566.
Copyright © 2014 by the International Association for the Study of Lung Cancer
