Case Report
BIRC6-ALK, a Novel Fusion Gene in ALK Break-Apart FISH-Negative Lung Adenocarcinoma, Responds to Crizotinib Ling Shan, PhD,* Peidi Jiang, MD,† Feng Xu, MD,‡ Weilong Zhang,§ Lei Guo,* Jian Wu, MD, PhD,║ Yixin Zeng, MD, PhD,¶#** Yuchen Jiao, MD, PhD,§ and Jianming Ying, MD, PhD*
C
rizotinib was approved for the treatment of advanced non–small-cell lung cancer patients with ALK fusion as detected by the ALK fluorescence in situ hybridization (FISH) test. The assay clearly defined that isolated 5′ ALK signals are considered ALK negative because the 5′ probe is not located in the kinase region. However, here, we describe a case with a negative FISH result that was later identified as ALK+ by VENTANA ALK (D5F3) immunohistochemisty (IHC) and responded well to crizotinib. Targeted next generation sequencing revealed a new ALK partner gene, baculoviral inhibition of apoptosis protein repeat containing six (BIRC6), and the paracentric inversion for generating this fusion gene.
CASE PRESENTATION A 45-year-old Chinese woman with an 8 pack-year smoking history was referred to Cancer Hospital, Chinese Academy of Medical Sciences in February 2014 for bilateral pleural and pericardial effusion. Computed tomography of the chest revealed a left hilar mass, measured 3.1 × 3.4 cm2, and multiple enlarged lymph nodes in the right lymph node stations 1, 2R, 4R, and 6. An incision biopsy of lymph nodes revealed a metastatic adenocarcinoma. Molecular pathologic analysis showed
Departments of *Pathology, †Medical Oncology, ‡Diagnostic Imaging, §Laboratory of Cell and Molecular Biology and State Key Laboratory of Molecular Oncology, Cancer Institute and Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China; ║MyGenostics Inc., Baltimore, MD, USA; ¶Cancer Institute & Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College; #Department of Experimental Research, Sun Yat-sen University Cancer Center, Guangzhou, China; and **State Key Laboratory of Oncology in Southern China, Guangzhou, China. Ling Shan and Peidi Jiang contributed equally to this work. Yuchen Jiao and Jianming Ying contributed equally to this work. Disclosure: The authors declare no conflict of interest. Address for correspondence: Jianming Ying, MD, PhD, Department of Pathology, Cancer Institute and Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100021, China. E-mail: [email protected]; Yuchen Jiao, MD, PhD, Laboratory of Cell and Molecular Biology and State Key Laboratory of Molecular Oncology, Cancer Institute and Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China. E-mail: jiaoyuchen@ cicams.ac.cn DOI: 10.1097/JTO.0000000000000467 Copyright © 2015 by the International Association for the Study of Lung Cancer ISSN: 1556-0864/15/1006-0e37
no EGFR or KRAS mutations. FISH analysis revealed a negative result: over 50% of cancer cells demonstrated isolated 5′ ALK signals (green) and 2 copies of fused 5′ and 3′ ALK signals (Fig. 1B). However, IHC assay demonstrated strong ALK expression (Fig. 1A). Biotinylated oligoprobes were designed tiling along the nonrepeated regions of ALK gene containing all the exons and introns to capture fragmented ALK gene. The captured DNA was then sequenced on HiSeq 2000 sequencer (Illumina, San Diego, CA) for paired 100 bp read. The next generation sequence analysis revealed a new ALK partner gene, BIRC6, located at 2.4 Mb downstream of ALK. Direct Sanger sequencing demonstrated that intron 10 of BIRC6 is disrupted at a point, 7.2 kb downstream of exon 10, and is inverted to connect to a position 411 bp upstream of exon 20 of ALK (Fig. 2A). BIRC6 is a member of inhibitors of apoptosis proteins. In the predicted fusion protein, the N-terminal portion of BIRC6 is fused to the intracellular region of ALK containing the transmembrane domain and tyrosine kinase domain, which is the binding site for crizotinib (Fig. 2B). Given the evidence that strong ALK expression was detected by IHC assay, the patient received crizotinib orally at a dose of 250 mg twice daily in March 2014 with an impressive symptomatic improvement and computed tomography response after 1 month of therapy (Fig. 1C–G). The patient remains on treatment with crizotinib with no evidence of progression as of November 2014.
DISCUSSION This is the first report of a novel BIRC6-ALK fusion gene in non– small-cell lung cancer. According to FISH interpretation criteria, isolated 5′ ALK signals are considered ALK negative. In this case, given the short distance (~3 Mb) between 2 breakpoints in ALK and BIRC6, the inversion may not be revealed by breaking-apart signals. However, it is still difficult to explain the multiple single copies of 5′ ALK signals. It indicated that there is not a simple inversion but a possibility of a complex rearrangement.1 Isolated 5′ ALK signals have been previously reported regardless whether EML4 was still the fusion partner, and patients whose tumors harbored this type of signals responded well to crizotinib.2–4 Therefore ALK FISH negative criteria are challenged. If FISH is the initial diagnosis in clinical practice, isolated 5′ ALK signals should raise the pathologists’ notice to apply the alternative confirmatory assays, ideally IHC assay, to confirm the ALK fusion status.
Journal of Thoracic Oncology ® • Volume 10, Number 6, June 2015
Copyright © 2014 by the International Association for the Study of Lung Cancer
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Journal of Thoracic Oncology ® • Volume 10, Number 6, June 2015
Shan et al.
FIGURE 1. A. VENTANA ALK (D5F3) IHC assay revealed intense granular cytoplasmic staining in this case. Original magnification ×200. B, Specimen hybridized with the Vysis break-apart ALK FISH assay showing multiple single 5′ ALK signals (green) with two copies of fused 5′ and 3′ ALK signals (white arrows and a box). C, E, CT scan of mediastinal lymph nodes and thorax before initiation of crizotinib. D, F, CT scan of mediastinal lymph nodes and thorax after 1 month therapy of crizotinib. G, CT scan of thorax after 3-month therapy of crizotinib. IHC, immunohistochemistry; FISH, fluorescence in situ hybridization; CT, computed tomography.
ACKNOWLEDGMENTS
This study was supported by a grant from Youth Backbone Program (to Jianming Ying) of Cancer Hospital, CAMS, Beijing, China. REFERENCES 1. Peled N, Palmer G, Hirsch FR, et al. Next-generation sequencing identifies and immunohistochemistry confirms a novel crizotinib-sensitive ALK rearrangement in a patient with metastatic non-small-cell lung cancer. J Thorac Oncol 2012;7:e14–e16.
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2. Yoshida A, Tsuta K, Nitta H, et al. Bright-field dual-color chromogenic in situ hybridization for diagnosing echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase-positive lung adenocarcinomas. J Thorac Oncol 2011;6:1677–1686. 3. Camidge DR, Kono SA, Flacco A, et al. Optimizing the detection of lung cancer patients harboring anaplastic lymphoma kinase (ALK) gene rearrangements potentially suitable for ALK inhibitor treatment. Clin Cancer Res 2010;16:5581–5590. 4. Ren S, Hirsch FR, Varella-Garcia M, et al. Atypical negative ALK break-apart FISH harboring a crizotinib-responsive ALK rearrangement in non-small-cell lung cancer. J Thorac Oncol 2014;9: e21–e23.
Copyright © 2015 by the International Association for the Study of Lung Cancer
Copyright © 2014 by the International Association for the Study of Lung Cancer
Journal of Thoracic Oncology ® • Volume 10, Number 6, June 2015
BIRC6-ALK Responds to Crizotinib
FIGURE 2. A, Both the ALK gene and the BIRC6 gene map to chromosome 2p, but have opposite orientations. BIRC6 located at 2.4 Mb downstream of ALK and 9.5 Mb upstream of EML4. Direct Sanger sequencing in this case showed that BIRC6 is disrupted at a position, 7.2 kb downstream of exon 10 and is inverted to connect to a position 411 bp upstream of exon 20 of ALK. Given the short distance (~3 Mb) between 2 breakpoints in ALK and BIRC6, the inversion was not revealed by breakingapart signals in FISH analysis. B, Fusion of the N-terminal portion of BIRC6 (comprising BIR and UBCc domain) to the intracellular region of ALK (containing the TM and tyrosine kinase domain). BIR, a baculoviral inhibition of apoptosis protein repeat domain; UBCc, a ubiquitin-conjugating enzyme E2, catalytic domain; TM, transmembrane domain.
Copyright © 2015 by the International Association for the Study of Lung Cancer
Copyright © 2014 by the International Association for the Study of Lung Cancer
e39
BIRC6-ALK, a Novel Fusion Gene in ALK Break-Apart FISH-Negative Lung Adenocarcinoma, Responds to Crizotinib Ling Shan, PhD,* Peidi Jiang, MD,† Feng Xu, MD,‡ Weilong Zhang,§ Lei Guo,* Jian Wu, MD, PhD,║ Yixin Zeng, MD, PhD,¶#** Yuchen Jiao, MD, PhD,§ and Jianming Ying, MD, PhD*
C
rizotinib was approved for the treatment of advanced non–small-cell lung cancer patients with ALK fusion as detected by the ALK fluorescence in situ hybridization (FISH) test. The assay clearly defined that isolated 5′ ALK signals are considered ALK negative because the 5′ probe is not located in the kinase region. However, here, we describe a case with a negative FISH result that was later identified as ALK+ by VENTANA ALK (D5F3) immunohistochemisty (IHC) and responded well to crizotinib. Targeted next generation sequencing revealed a new ALK partner gene, baculoviral inhibition of apoptosis protein repeat containing six (BIRC6), and the paracentric inversion for generating this fusion gene.
CASE PRESENTATION A 45-year-old Chinese woman with an 8 pack-year smoking history was referred to Cancer Hospital, Chinese Academy of Medical Sciences in February 2014 for bilateral pleural and pericardial effusion. Computed tomography of the chest revealed a left hilar mass, measured 3.1 × 3.4 cm2, and multiple enlarged lymph nodes in the right lymph node stations 1, 2R, 4R, and 6. An incision biopsy of lymph nodes revealed a metastatic adenocarcinoma. Molecular pathologic analysis showed
Departments of *Pathology, †Medical Oncology, ‡Diagnostic Imaging, §Laboratory of Cell and Molecular Biology and State Key Laboratory of Molecular Oncology, Cancer Institute and Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China; ║MyGenostics Inc., Baltimore, MD, USA; ¶Cancer Institute & Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College; #Department of Experimental Research, Sun Yat-sen University Cancer Center, Guangzhou, China; and **State Key Laboratory of Oncology in Southern China, Guangzhou, China. Ling Shan and Peidi Jiang contributed equally to this work. Yuchen Jiao and Jianming Ying contributed equally to this work. Disclosure: The authors declare no conflict of interest. Address for correspondence: Jianming Ying, MD, PhD, Department of Pathology, Cancer Institute and Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100021, China. E-mail: [email protected]; Yuchen Jiao, MD, PhD, Laboratory of Cell and Molecular Biology and State Key Laboratory of Molecular Oncology, Cancer Institute and Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China. E-mail: jiaoyuchen@ cicams.ac.cn DOI: 10.1097/JTO.0000000000000467 Copyright © 2015 by the International Association for the Study of Lung Cancer ISSN: 1556-0864/15/1006-0e37
no EGFR or KRAS mutations. FISH analysis revealed a negative result: over 50% of cancer cells demonstrated isolated 5′ ALK signals (green) and 2 copies of fused 5′ and 3′ ALK signals (Fig. 1B). However, IHC assay demonstrated strong ALK expression (Fig. 1A). Biotinylated oligoprobes were designed tiling along the nonrepeated regions of ALK gene containing all the exons and introns to capture fragmented ALK gene. The captured DNA was then sequenced on HiSeq 2000 sequencer (Illumina, San Diego, CA) for paired 100 bp read. The next generation sequence analysis revealed a new ALK partner gene, BIRC6, located at 2.4 Mb downstream of ALK. Direct Sanger sequencing demonstrated that intron 10 of BIRC6 is disrupted at a point, 7.2 kb downstream of exon 10, and is inverted to connect to a position 411 bp upstream of exon 20 of ALK (Fig. 2A). BIRC6 is a member of inhibitors of apoptosis proteins. In the predicted fusion protein, the N-terminal portion of BIRC6 is fused to the intracellular region of ALK containing the transmembrane domain and tyrosine kinase domain, which is the binding site for crizotinib (Fig. 2B). Given the evidence that strong ALK expression was detected by IHC assay, the patient received crizotinib orally at a dose of 250 mg twice daily in March 2014 with an impressive symptomatic improvement and computed tomography response after 1 month of therapy (Fig. 1C–G). The patient remains on treatment with crizotinib with no evidence of progression as of November 2014.
DISCUSSION This is the first report of a novel BIRC6-ALK fusion gene in non– small-cell lung cancer. According to FISH interpretation criteria, isolated 5′ ALK signals are considered ALK negative. In this case, given the short distance (~3 Mb) between 2 breakpoints in ALK and BIRC6, the inversion may not be revealed by breaking-apart signals. However, it is still difficult to explain the multiple single copies of 5′ ALK signals. It indicated that there is not a simple inversion but a possibility of a complex rearrangement.1 Isolated 5′ ALK signals have been previously reported regardless whether EML4 was still the fusion partner, and patients whose tumors harbored this type of signals responded well to crizotinib.2–4 Therefore ALK FISH negative criteria are challenged. If FISH is the initial diagnosis in clinical practice, isolated 5′ ALK signals should raise the pathologists’ notice to apply the alternative confirmatory assays, ideally IHC assay, to confirm the ALK fusion status.
Journal of Thoracic Oncology ® • Volume 10, Number 6, June 2015
Copyright © 2014 by the International Association for the Study of Lung Cancer
e37
Journal of Thoracic Oncology ® • Volume 10, Number 6, June 2015
Shan et al.
FIGURE 1. A. VENTANA ALK (D5F3) IHC assay revealed intense granular cytoplasmic staining in this case. Original magnification ×200. B, Specimen hybridized with the Vysis break-apart ALK FISH assay showing multiple single 5′ ALK signals (green) with two copies of fused 5′ and 3′ ALK signals (white arrows and a box). C, E, CT scan of mediastinal lymph nodes and thorax before initiation of crizotinib. D, F, CT scan of mediastinal lymph nodes and thorax after 1 month therapy of crizotinib. G, CT scan of thorax after 3-month therapy of crizotinib. IHC, immunohistochemistry; FISH, fluorescence in situ hybridization; CT, computed tomography.
ACKNOWLEDGMENTS
This study was supported by a grant from Youth Backbone Program (to Jianming Ying) of Cancer Hospital, CAMS, Beijing, China. REFERENCES 1. Peled N, Palmer G, Hirsch FR, et al. Next-generation sequencing identifies and immunohistochemistry confirms a novel crizotinib-sensitive ALK rearrangement in a patient with metastatic non-small-cell lung cancer. J Thorac Oncol 2012;7:e14–e16.
e38
2. Yoshida A, Tsuta K, Nitta H, et al. Bright-field dual-color chromogenic in situ hybridization for diagnosing echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase-positive lung adenocarcinomas. J Thorac Oncol 2011;6:1677–1686. 3. Camidge DR, Kono SA, Flacco A, et al. Optimizing the detection of lung cancer patients harboring anaplastic lymphoma kinase (ALK) gene rearrangements potentially suitable for ALK inhibitor treatment. Clin Cancer Res 2010;16:5581–5590. 4. Ren S, Hirsch FR, Varella-Garcia M, et al. Atypical negative ALK break-apart FISH harboring a crizotinib-responsive ALK rearrangement in non-small-cell lung cancer. J Thorac Oncol 2014;9: e21–e23.
Copyright © 2015 by the International Association for the Study of Lung Cancer
Copyright © 2014 by the International Association for the Study of Lung Cancer
Journal of Thoracic Oncology ® • Volume 10, Number 6, June 2015
BIRC6-ALK Responds to Crizotinib
FIGURE 2. A, Both the ALK gene and the BIRC6 gene map to chromosome 2p, but have opposite orientations. BIRC6 located at 2.4 Mb downstream of ALK and 9.5 Mb upstream of EML4. Direct Sanger sequencing in this case showed that BIRC6 is disrupted at a position, 7.2 kb downstream of exon 10 and is inverted to connect to a position 411 bp upstream of exon 20 of ALK. Given the short distance (~3 Mb) between 2 breakpoints in ALK and BIRC6, the inversion was not revealed by breakingapart signals in FISH analysis. B, Fusion of the N-terminal portion of BIRC6 (comprising BIR and UBCc domain) to the intracellular region of ALK (containing the TM and tyrosine kinase domain). BIR, a baculoviral inhibition of apoptosis protein repeat domain; UBCc, a ubiquitin-conjugating enzyme E2, catalytic domain; TM, transmembrane domain.
Copyright © 2015 by the International Association for the Study of Lung Cancer
Copyright © 2014 by the International Association for the Study of Lung Cancer
e39
