GENES, CHROMOSOMES & CANCER 53:865–874 (2014)
Identification of Novel ALK Rearrangement A2M–ALK in a Neonate with Fetal Lung Interstitial Tumor Tadashi Onoda,1,2* Miyako Kanno,1 Hiroko Sato,1 Noriyuki Takahashi,1 Hiroko Izumino,1 Hiroshi Ohta,3 Takaki Emura,3 Hirohisa Katoh,3 Hiroyuki Ohizumi,3 Hiroya Ohtake,4 Hironobu Asao,2 Louis P. Dehner,5 Ashley D. Hill,6 Kiyoshi Hayasaka,1 and Tetsuo Mitsui1 1
Department of Pediatrics,Yamagata University Faculty of Medicine,Yamagata,Japan Department of Immunology,Yamagata University Faculty of Medicine,Yamagata,Japan 3 Second Department of Surgery,Yamagata University Faculty of Medicine,Yamagata,Japan 4 Department of Pathology,Yamagata University Faculty of Medicine,Yamagata,Japan 5 Department of Pathology and Immunology, and Pathology in Pediatrics,Washington University in St.Louis, St.Louis,MO 6 Division of Pathology,Children’s National Medical Center,Washington,DC 2
Fetal lung interstitial tumor (FLIT) is a recently reported type of congenital lung lesion comprising solid and cystic components. The pathological features include unique interstitial mesenchyme-based cell proliferation, and differ from other neoplasms represented by pleuropulmonary blastoma or congenital peribronchial myofibroblastic tumor. FLIT is extremely rare and its gene expression profile has not yet been reported. We provide the first report of a novel chromosomal rearrangement resulting in a-2-macroglobulin (A2M) and anaplastic lymphoma kinase (ALK) gene fusion in a patient with FLIT. The tumor cells contained a t(2;12)(p23;p13) and were mesenchymal in origin (e.g., inflammatory myofibroblastic tumors), suggesting the involvement of ALK in this case of FLIT. Break apart fluorescence in situ hybridization demonstrated chromosomal rearrangement at ALK 2p23. Using 50 -rapid amplification of cDNA ends, we further identified a novel transcript fusing exon 22 of A2M to exon 19 of ALK, which was confirmed by reverse-transcription polymerase chain reaction. The corresponding chimeric gene was subsequently confirmed by sequencing, including the genomic break point between intron 22 and 18 of A2M and ALK, respectively. Discovery of A2M as a novel ALK fusion partner, together with the involvement of ALK, provides new insights into the pathogenesis of FLIT, and suggests the potential for new therapeutic strategies C 2014 Wiley Periodicals, Inc. V based on ALK inhibitors.
INTRODUCTION
Fetal lung interstitial tumor (FLIT) has recently been proposed as a type of intrathoracic tumor in neonates (Dishop et al., 2010). It was reported as an extremely rare mesenchymal neoplasm comprising uniform immature interstitial cells with irregular air spaces, resembling fetal lung tissue at 20–24 weeks of gestation (Dishop et al., 2010; Lazar et al., 2011). FLIT was previously considered as a cystic neoplasm (e.g., pleuropulmonary blastoma [PPB]) among congenital pulmonary lesions (Priest et al., 1997, 2006; Langston, 2003; Dishop and coworkers, 2008, 2010; Hill et al., 2008). The molecular basis of FLIT remains unclear, and few publications have considered the appropriate therapeutic strategy in conjunction with long-term observation (Dishop et al., 2010). PPB is the most common primitive embryonal mesenchymal tumor of childhood (Dishop and Kuruvilla, 2008). It has an undifferentiated sarcomatous appearance and epithelial-lined structures combined with rhabdomyosarcomatous, fibrosarcomaC 2014 Wiley Periodicals, Inc. V
like, and cartilaginous foci (Priest et al., 1997, 2006; Dishop and Kuruvilla, 2008). PPB can be classified into three subtypes: type I (low-grade cystic), type II (intermixed), and type III (high-grade solid masses) (Priest et al., 1997, 2006, 2009; Dishop and Kuruvilla, 2008). Cytogenetically, trisomies 8 and 2, and the translocation t(12;17)(q24.1;q21) have been reported in PPB (Novak et al., 1997; Yang et al., 1997; Dishop and Kuruvilla, 2008; Behery et al., 2012) and a heterozygous DICER1 mutation has also been identified in familial PPB (Hill et al., 2009). Additional Supporting Information may be found in the online version of this article. Supported by: Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research, Grant number: 23791150. *Correspondence to: Tadashi Onoda, MD, PhD, Department of Pediatrics, Yamagata University Faculty of Medicine, 2-2-2 Iida-Nishi, Yamagata 990–9585, Japan. E-mail: [email protected] Received 27 March 2014; Accepted 12 June 2014 DOI 10.1002/gcc.22199 Published online 25 June 2014 in Wiley Online Library (wileyonlinelibrary.com).
866
ONODA ET AL.
In contrast, congenital peribronchial myofibroblastic tumor is a benign peribronchial mesenchymal tumor of infancy (McGinnis et al., 1993; Alobeid et al., 1997). It is a solid tumor, characterized by myofibroblastic differentiation with entrapped bronchial cartilage (Dishop and Kuruvilla, 2008). Inflammatory myofibroblastic tumors (IMTs) are another type of benign mesenchymal lesion composed of myofibroblasts and inflammatory cell infiltrations with myxoid to collagenous stroma (Gleason and Hornick, 2008). Aberrant expression of anaplastic lymphoma kinase (ALK) as an oncokinase has been a particular focus of study in IMTs (Barreca et al., 2011; Takeuchi et al., 2011; Marino-Enriquez and Dal Cin, 2013). ALK is a 1,620-amino acid, single-chain transmembrane receptor tyrosine kinase, first discovered in anaplastic large-cell lymphoma (Morris et al., 1994; Barreca et al., 2011; Marino-Enriquez and Dal Cin, 2013; Roskoski, 2013). Rearrangements involving the ALK locus on 2p23 have been reported in some childhood tumors including IMTs, rhabdomyosarcoma, neuroblastoma, and nonsmall-cell lung cancer (Kwak et al., 2010; Barreca et al., 2011; Marino-Enriquez and Dal Cin, 2013; Mosse et al., 2013). The mechanisms are usually thought to involve ALK overexpression or ALK domain activation induced by the fusion partner as a result of chromosomal translocation with the consequent constitutive activation of ALK playing a role in tumorigenesis. The selective ALK tyrosine kinase inhibitor crizotinib has recently been used for recurrent or refractory childhood tumors harboring ALK rearrangements (Mosse et al., 2013). The detection of ALK and its fusion partners thus provides potential new therapeutic approaches for undefined neonatal tumors carrying ALK rearrangements. In this report, we describe the first case of FLIT expressing a novel a-2-macroglobulin (A2M)–ALK fusion gene in a patient who remained disease-free for over 3 years without adjuvant chemotherapy. MATERIALS AND METHODS
Written informed consent was obtained from the patient’s parents in accordance with the Declaration of Helsinki. All experiments were approved by the institutional review board of Yamagata University Faculty of Medicine. Patient Materials
Parts of the surgically resected tumor specimen were fixed in 10% neutralized formalin and embedded in paraffin for conventional histopathologic Genes, Chromosomes & Cancer DOI 10.1002/gcc
examination, or frozen and embedded in OCT compound (Miles Inc., Diagnostic Division, Elkhart, IN) to obtain frozen sections. Total RNA and genomic DNA were extracted from a snapfrozen tumor specimen or peripheral blood from a healthy donor (control) in liquid nitrogen using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA) and a Wizard genomic DNA purification kit (Promega, Madison, WI), respectively. Pathology and Immunohistochemistry
Sections from formalin-fixed, paraffin-embedded (FFPE) tissue were stained with hematoxylin and eosin and reviewed. Immunohistochemical staining was performed on 3-mm-thick paraffin sections using standard staining procedures. Antibodies were obtained as follows: CAM5.2 (BD Biosciences, San Jose, CA), AE1/AE3 (AE1/AE3, Nichirei, Tokyo, Japan), epithelial membrane antigen (EMA; E29, Nichirei), vimentin (SP-20, Nichirei), smooth muscle actin (SMA; 1A4, Nichirei), desmin (D33, Nichirei), thyroid transcription factor 1 (TTF-1; SPT24, Nichirei), surfactant protein-A (SP-A; 32E12, Leica Biosystems, Newcastle, UK), carcinoembryonic antigen (CEA; ZC23, Nichirei), Ki-67 (SP6, Nichirei), and ALK (5A4, Abcam, Cambridge, UK). The intercalated antibody-enhanced polymer method was used for the sensitive detection of ALK, as described previously (Takeuchi et al., 2009). Cytogenetics and Fluorescence In Situ Hybridization
G-banded metaphase analysis was performed following short-term culture of the tumor specimens in RPMI 1640 supplemented with 10% fetal calf serum for 4 days. Twenty metaphases were evaluated, and the karyotype was written in accordance with the ISCN 2013 guidelines. ALK rearrangements were detected using Vysis LSI ALK Break apart FISH probes (Abbott Molecular Inc., Abbott Park, IL). FFPE unstained sections were subjected to hybridization with break apart ALK probes following the manufacturer’s directions. The green and orange probes hybridized with the 50 and 30 regions of ALK, respectively. The separation of probe signals indicated a chromosomal rearrangement involving ALK. 50 -Rapid Amplification of cDNA Ends and Polymerase Chain Reaction
The SMARTer RACE cDNA Amplification Kit (Clontech Laboratories Inc., Mountain View, CA)
A2M–ALK IN FETAL LUNG INTERSTITIAL TUMOR
was used according to the manufacturer’s instructions to perform 50 -rapid amplification of cDNA ends (RACE) to identify the ALK fusion partner. In brief, total RNA was extracted from a snapfrozen tumor specimen and first-strand cDNA corresponding to a novel ALK fusion gene was synthesized. cDNA containing the fusion point was further amplified by polymerase chain reaction (PCR) using a universal primer and ALK-SP1 (50 -TCGTCCTGTTCAGAGCACACTTCAGG-30 ) in exon 22 (Wang et al., 2012). The sequence for identifying the fusion partner was constructed using a nested universal primer and ALK-SP2 (50 -GACA CCTGGCCTTCATACACCTC-30 ) in exon 21 (Wang et al., 2012) using an ABI 3500xL Genetic Analyzer (Applied Biosystems, Foster City, CA). The fusion transcripts were confirmed by reversetranscription (RT)-PCR using the forward primer (50 -GTCCCAGTGGAGAAGGAACA-30 ) in exon 20 of A2M and the reverse primer (50 -AATCATGA TGCCGGAGAAAG-30 ) in exon 19 of ALK. The genomic break point of tumor genomic DNA was also confirmed by partial direct sequencing using the same primer sets.
RESULTS Case Presentation
The patient was a male infant born at term and admitted to a local hospital because of mild apnea. He had a normal prenatal course and no family history of malignancy. He was administered lowdose supplemental oxygen to stabilize his respiratory status. A chest X-ray revealed an unexpected intrathoracic mass in the left lower lung field (Supporting Information Fig. 1A). He was then transferred to our institution at 4 days of age. On admission, his respiration remained stable, even in room air. His lungs were clear to auscultation, and no cardiac murmur was noted. No surface lymph nodes were palpable. Laboratory investigation revealed white blood cell count 7.2 3 109/l, hemoglobin 14.0 g/dl, platelet count 30.8 3 109/l, aspartate transaminase 51 IU/l, alanine transaminase 15 IU/l, lactate dehydrogenase 408 IU/l, creatine kinase 419 IU/l, C-reactive protein 0.17 mg/dl, soluble interleukin (IL)-2 receptor 1,120 U/ml, afetoprotein 59,369 U/ml, serum human chorionic gonadotropin
Identification of Novel ALK Rearrangement A2M–ALK in a Neonate with Fetal Lung Interstitial Tumor Tadashi Onoda,1,2* Miyako Kanno,1 Hiroko Sato,1 Noriyuki Takahashi,1 Hiroko Izumino,1 Hiroshi Ohta,3 Takaki Emura,3 Hirohisa Katoh,3 Hiroyuki Ohizumi,3 Hiroya Ohtake,4 Hironobu Asao,2 Louis P. Dehner,5 Ashley D. Hill,6 Kiyoshi Hayasaka,1 and Tetsuo Mitsui1 1
Department of Pediatrics,Yamagata University Faculty of Medicine,Yamagata,Japan Department of Immunology,Yamagata University Faculty of Medicine,Yamagata,Japan 3 Second Department of Surgery,Yamagata University Faculty of Medicine,Yamagata,Japan 4 Department of Pathology,Yamagata University Faculty of Medicine,Yamagata,Japan 5 Department of Pathology and Immunology, and Pathology in Pediatrics,Washington University in St.Louis, St.Louis,MO 6 Division of Pathology,Children’s National Medical Center,Washington,DC 2
Fetal lung interstitial tumor (FLIT) is a recently reported type of congenital lung lesion comprising solid and cystic components. The pathological features include unique interstitial mesenchyme-based cell proliferation, and differ from other neoplasms represented by pleuropulmonary blastoma or congenital peribronchial myofibroblastic tumor. FLIT is extremely rare and its gene expression profile has not yet been reported. We provide the first report of a novel chromosomal rearrangement resulting in a-2-macroglobulin (A2M) and anaplastic lymphoma kinase (ALK) gene fusion in a patient with FLIT. The tumor cells contained a t(2;12)(p23;p13) and were mesenchymal in origin (e.g., inflammatory myofibroblastic tumors), suggesting the involvement of ALK in this case of FLIT. Break apart fluorescence in situ hybridization demonstrated chromosomal rearrangement at ALK 2p23. Using 50 -rapid amplification of cDNA ends, we further identified a novel transcript fusing exon 22 of A2M to exon 19 of ALK, which was confirmed by reverse-transcription polymerase chain reaction. The corresponding chimeric gene was subsequently confirmed by sequencing, including the genomic break point between intron 22 and 18 of A2M and ALK, respectively. Discovery of A2M as a novel ALK fusion partner, together with the involvement of ALK, provides new insights into the pathogenesis of FLIT, and suggests the potential for new therapeutic strategies C 2014 Wiley Periodicals, Inc. V based on ALK inhibitors.
INTRODUCTION
Fetal lung interstitial tumor (FLIT) has recently been proposed as a type of intrathoracic tumor in neonates (Dishop et al., 2010). It was reported as an extremely rare mesenchymal neoplasm comprising uniform immature interstitial cells with irregular air spaces, resembling fetal lung tissue at 20–24 weeks of gestation (Dishop et al., 2010; Lazar et al., 2011). FLIT was previously considered as a cystic neoplasm (e.g., pleuropulmonary blastoma [PPB]) among congenital pulmonary lesions (Priest et al., 1997, 2006; Langston, 2003; Dishop and coworkers, 2008, 2010; Hill et al., 2008). The molecular basis of FLIT remains unclear, and few publications have considered the appropriate therapeutic strategy in conjunction with long-term observation (Dishop et al., 2010). PPB is the most common primitive embryonal mesenchymal tumor of childhood (Dishop and Kuruvilla, 2008). It has an undifferentiated sarcomatous appearance and epithelial-lined structures combined with rhabdomyosarcomatous, fibrosarcomaC 2014 Wiley Periodicals, Inc. V
like, and cartilaginous foci (Priest et al., 1997, 2006; Dishop and Kuruvilla, 2008). PPB can be classified into three subtypes: type I (low-grade cystic), type II (intermixed), and type III (high-grade solid masses) (Priest et al., 1997, 2006, 2009; Dishop and Kuruvilla, 2008). Cytogenetically, trisomies 8 and 2, and the translocation t(12;17)(q24.1;q21) have been reported in PPB (Novak et al., 1997; Yang et al., 1997; Dishop and Kuruvilla, 2008; Behery et al., 2012) and a heterozygous DICER1 mutation has also been identified in familial PPB (Hill et al., 2009). Additional Supporting Information may be found in the online version of this article. Supported by: Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research, Grant number: 23791150. *Correspondence to: Tadashi Onoda, MD, PhD, Department of Pediatrics, Yamagata University Faculty of Medicine, 2-2-2 Iida-Nishi, Yamagata 990–9585, Japan. E-mail: [email protected] Received 27 March 2014; Accepted 12 June 2014 DOI 10.1002/gcc.22199 Published online 25 June 2014 in Wiley Online Library (wileyonlinelibrary.com).
866
ONODA ET AL.
In contrast, congenital peribronchial myofibroblastic tumor is a benign peribronchial mesenchymal tumor of infancy (McGinnis et al., 1993; Alobeid et al., 1997). It is a solid tumor, characterized by myofibroblastic differentiation with entrapped bronchial cartilage (Dishop and Kuruvilla, 2008). Inflammatory myofibroblastic tumors (IMTs) are another type of benign mesenchymal lesion composed of myofibroblasts and inflammatory cell infiltrations with myxoid to collagenous stroma (Gleason and Hornick, 2008). Aberrant expression of anaplastic lymphoma kinase (ALK) as an oncokinase has been a particular focus of study in IMTs (Barreca et al., 2011; Takeuchi et al., 2011; Marino-Enriquez and Dal Cin, 2013). ALK is a 1,620-amino acid, single-chain transmembrane receptor tyrosine kinase, first discovered in anaplastic large-cell lymphoma (Morris et al., 1994; Barreca et al., 2011; Marino-Enriquez and Dal Cin, 2013; Roskoski, 2013). Rearrangements involving the ALK locus on 2p23 have been reported in some childhood tumors including IMTs, rhabdomyosarcoma, neuroblastoma, and nonsmall-cell lung cancer (Kwak et al., 2010; Barreca et al., 2011; Marino-Enriquez and Dal Cin, 2013; Mosse et al., 2013). The mechanisms are usually thought to involve ALK overexpression or ALK domain activation induced by the fusion partner as a result of chromosomal translocation with the consequent constitutive activation of ALK playing a role in tumorigenesis. The selective ALK tyrosine kinase inhibitor crizotinib has recently been used for recurrent or refractory childhood tumors harboring ALK rearrangements (Mosse et al., 2013). The detection of ALK and its fusion partners thus provides potential new therapeutic approaches for undefined neonatal tumors carrying ALK rearrangements. In this report, we describe the first case of FLIT expressing a novel a-2-macroglobulin (A2M)–ALK fusion gene in a patient who remained disease-free for over 3 years without adjuvant chemotherapy. MATERIALS AND METHODS
Written informed consent was obtained from the patient’s parents in accordance with the Declaration of Helsinki. All experiments were approved by the institutional review board of Yamagata University Faculty of Medicine. Patient Materials
Parts of the surgically resected tumor specimen were fixed in 10% neutralized formalin and embedded in paraffin for conventional histopathologic Genes, Chromosomes & Cancer DOI 10.1002/gcc
examination, or frozen and embedded in OCT compound (Miles Inc., Diagnostic Division, Elkhart, IN) to obtain frozen sections. Total RNA and genomic DNA were extracted from a snapfrozen tumor specimen or peripheral blood from a healthy donor (control) in liquid nitrogen using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA) and a Wizard genomic DNA purification kit (Promega, Madison, WI), respectively. Pathology and Immunohistochemistry
Sections from formalin-fixed, paraffin-embedded (FFPE) tissue were stained with hematoxylin and eosin and reviewed. Immunohistochemical staining was performed on 3-mm-thick paraffin sections using standard staining procedures. Antibodies were obtained as follows: CAM5.2 (BD Biosciences, San Jose, CA), AE1/AE3 (AE1/AE3, Nichirei, Tokyo, Japan), epithelial membrane antigen (EMA; E29, Nichirei), vimentin (SP-20, Nichirei), smooth muscle actin (SMA; 1A4, Nichirei), desmin (D33, Nichirei), thyroid transcription factor 1 (TTF-1; SPT24, Nichirei), surfactant protein-A (SP-A; 32E12, Leica Biosystems, Newcastle, UK), carcinoembryonic antigen (CEA; ZC23, Nichirei), Ki-67 (SP6, Nichirei), and ALK (5A4, Abcam, Cambridge, UK). The intercalated antibody-enhanced polymer method was used for the sensitive detection of ALK, as described previously (Takeuchi et al., 2009). Cytogenetics and Fluorescence In Situ Hybridization
G-banded metaphase analysis was performed following short-term culture of the tumor specimens in RPMI 1640 supplemented with 10% fetal calf serum for 4 days. Twenty metaphases were evaluated, and the karyotype was written in accordance with the ISCN 2013 guidelines. ALK rearrangements were detected using Vysis LSI ALK Break apart FISH probes (Abbott Molecular Inc., Abbott Park, IL). FFPE unstained sections were subjected to hybridization with break apart ALK probes following the manufacturer’s directions. The green and orange probes hybridized with the 50 and 30 regions of ALK, respectively. The separation of probe signals indicated a chromosomal rearrangement involving ALK. 50 -Rapid Amplification of cDNA Ends and Polymerase Chain Reaction
The SMARTer RACE cDNA Amplification Kit (Clontech Laboratories Inc., Mountain View, CA)
A2M–ALK IN FETAL LUNG INTERSTITIAL TUMOR
was used according to the manufacturer’s instructions to perform 50 -rapid amplification of cDNA ends (RACE) to identify the ALK fusion partner. In brief, total RNA was extracted from a snapfrozen tumor specimen and first-strand cDNA corresponding to a novel ALK fusion gene was synthesized. cDNA containing the fusion point was further amplified by polymerase chain reaction (PCR) using a universal primer and ALK-SP1 (50 -TCGTCCTGTTCAGAGCACACTTCAGG-30 ) in exon 22 (Wang et al., 2012). The sequence for identifying the fusion partner was constructed using a nested universal primer and ALK-SP2 (50 -GACA CCTGGCCTTCATACACCTC-30 ) in exon 21 (Wang et al., 2012) using an ABI 3500xL Genetic Analyzer (Applied Biosystems, Foster City, CA). The fusion transcripts were confirmed by reversetranscription (RT)-PCR using the forward primer (50 -GTCCCAGTGGAGAAGGAACA-30 ) in exon 20 of A2M and the reverse primer (50 -AATCATGA TGCCGGAGAAAG-30 ) in exon 19 of ALK. The genomic break point of tumor genomic DNA was also confirmed by partial direct sequencing using the same primer sets.
RESULTS Case Presentation
The patient was a male infant born at term and admitted to a local hospital because of mild apnea. He had a normal prenatal course and no family history of malignancy. He was administered lowdose supplemental oxygen to stabilize his respiratory status. A chest X-ray revealed an unexpected intrathoracic mass in the left lower lung field (Supporting Information Fig. 1A). He was then transferred to our institution at 4 days of age. On admission, his respiration remained stable, even in room air. His lungs were clear to auscultation, and no cardiac murmur was noted. No surface lymph nodes were palpable. Laboratory investigation revealed white blood cell count 7.2 3 109/l, hemoglobin 14.0 g/dl, platelet count 30.8 3 109/l, aspartate transaminase 51 IU/l, alanine transaminase 15 IU/l, lactate dehydrogenase 408 IU/l, creatine kinase 419 IU/l, C-reactive protein 0.17 mg/dl, soluble interleukin (IL)-2 receptor 1,120 U/ml, afetoprotein 59,369 U/ml, serum human chorionic gonadotropin
