Next-Generation Sequencing Identified a Novel Crizotinib-Sensitive PLB1-ALK Rearrangement in Lung Large-Cell Neuroendocrine Carcinoma

Abstract

Clinical Practice Points•Lung large-cell neuroendocrine carcinoma (LCNEC) is diagnosed in approximately 3% of all patients with lung cancer, and is generally associated with high rate of metastases and poor survival. Anaplastic lymphoma kinase (ALK) rearrangements are rare in patients with LCNEC, who could benefit greatly from ALK tyrosine kinase inhibitors.•We report a case of LCNEC harboring a novel PLB1-ALK rearrangement sensitive to crizotinib by next-generation sequencing (NGS). The patient’s disease responded to crizotinib for > 5 months.•NGS demonstrates an additional advantage of being able to concurrently detect different mutations in genetic testing and plays an important role in detecting ALK-rearranged non–small-cell lung cancer. Screening for ALK rearrangement by NGS in patients with LCNEC may help in selecting potential candidates for targeted therapy. •Lung large-cell neuroendocrine carcinoma (LCNEC) is diagnosed in approximately 3% of all patients with lung cancer, and is generally associated with high rate of metastases and poor survival. Anaplastic lymphoma kinase (ALK) rearrangements are rare in patients with LCNEC, who could benefit greatly from ALK tyrosine kinase inhibitors.•We report a case of LCNEC harboring a novel PLB1-ALK rearrangement sensitive to crizotinib by next-generation sequencing (NGS). The patient’s disease responded to crizotinib for > 5 months.•NGS demonstrates an additional advantage of being able to concurrently detect different mutations in genetic testing and plays an important role in detecting ALK-rearranged non–small-cell lung cancer. Screening for ALK rearrangement by NGS in patients with LCNEC may help in selecting potential candidates for targeted therapy. Lung high-grade neuroendocrine carcinoma includes the following two clinicopathologic entities, classified according to their morphologic and biological features: lung large-cell neuroendocrine carcinoma (LCNEC) and small-cell lung cancer (SCLC).1Travis W.D. Advances in neuroendocrine lung tumors.Ann Oncol. 2010; 21: vii65-vii71Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar Anaplastic lymphoma kinase (ALK) rearrangements have been identified in 3% to 7% of patients with non-SCLC (NSCLC), who could benefit greatly from ALK tyrosine kinase inhibitors (TKIs). However, ALK rearrangements are rare in LCNEC. We report a case of advanced LCNEC with a novel crizotinib-sensitive phospholipase B1 (PLB1)-ALK rearrangement. The patient experienced a partial response and a 5-month-long progression-free survival. A 41-year-old man, a current smoker, presented to Henan Cancer Hospital of Zhengzhou University with coughing for 1 month. Computed tomographic (CT) scans revealed a 42 × 40 × 40 mm mass in the superior lobe of the right lung with hilum of the right lung and mediastinal lymph node metastasis. Brain magnetic resonance imaging (MRI) revealed a 16 × 9 mm nodular lesion with surrounding edema in the temporal lobe of the brain (Figure 1). Lung biopsy established the pathologic diagnosis of LCNEC (Figure 2A). Immunohistochemistry (IHC) was positive for TTF-1 (thyroid transcription factor 1), CK, CK7, LCA, SyN, NapsinA, and 40% for Ki-67, but was negative for CK5/6 and P40 (Figure 2B-E). The patient was diagnosed with advanced LCNEC with mediastinal lymph node, right hilar, and brain involvement. At the request of the patient, we conducted the ALK Ventana-D5F3 IHC and fluorescence in-situ hybridization (FISH) on biopsy samples. IHC revealed abnormal ALK protein positivity, and FISH for ALK gene revealed split signals in 56% of tumor cells (Figure 2F and G). Subsequent capture-based next-generation sequencing (NGS) (Gene Detection Panel for Target Drug of Lung Cancer; Burning Rock, Guangzhou, China) was performed on biopsy samples using a gene panel comprising 8 lung cancer–related genes. A novel PLB1-ALK (P56:A20) rearrangement (with an abundance of 28.33%) was identified whereby the 56th intron of PLB1 and the 19th intron of ALK were broken and rearranged (Figure 3).Figure 2Micrographs of Large-Cell Neuroendocrine Lung Carcinoma. Low-Power Image of HE-Stained Section From Large-Cell Neuroendocrine Lung Carcinoma (A). Immunohistochemical Analysis Revealed Immunoreactivity to CK (B), Ki-67 (C), Synaptophysin (D), and TTF-1 (E). ALK Rearrangement was Also Confirmed by ALK (D5F3) Immunostaining (F). FISH Analysis of ALK Locus Using Break-Apart Probe Strategy. Approximately 56% of Tumor Cells had Rearrangement at ALK Locus, as Demonstrated by Split Red/Green Signals (Arrows) (G). Original Magnification ×100 for HE Staining and IHC; and ×1000 for FISH imagesShow full captionAbbreviations: ALK = anaplastic lymphoma kinase; FISH = fluorescence in-situ hybridization; HE = hematoxylin and eosin; IHC = immunohistochemistry; PLB1 = phospholipase B1; TTF-1 = thyroid transcription factor 1.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Sequencing Reads of ALK and PLB1 Shown Using Integrative Genomics ViewerView Large Image Figure ViewerDownload Hi-res image Download (PPT) Abbreviations: ALK = anaplastic lymphoma kinase; FISH = fluorescence in-situ hybridization; HE = hematoxylin and eosin; IHC = immunohistochemistry; PLB1 = phospholipase B1; TTF-1 = thyroid transcription factor 1. This study was approved by the medical ethics committee of Henan Cancer Hospital of Zhengzhou University. Written informed consent was received from the patient at the time of diagnosis. The patient received 2 cycles of etoposide (100 mg/m2 day 1–3) plus carboplatin (AUC = 5) but experienced disease progression. After a general discussion, on the basis of the ALK rearrangement, the patient was recommended to receive crizotinib (250 mg twice daily). After 1 month’s treatment with crizotinib, the symptoms improved, and the chest CT and brain MRI revealed partial response of the disease, which suggested that the patient’s disease had responded to crizotinib, both in the central nervous system and extracranially. However, after 5 months, a repeat chest CT and enhanced brain MRI revealed increase in the size of the right lung lesion and progression of the brain metastases, respectively, indicating progressive disease. The patient then received 1 month of anlotinib (a novel multitarget TKI, inhibiting tumor angiogenesis and proliferative signaling), 12 mg po qd (d1-14, 21days as a cycle), followed by 2 cycles of irinotecan (60 mg/m2 on days 1 and 8) plus nedaplatin (85 mg/m2) every 3 weeks, which failed to control the tumor. Considering the possibility of drug resistance mutations, the patient began ceritinib (750 mg/day) treatment. After 2 months of ceritinib treatment, repeat CT and MRI scans showed stable lung lesions and intracranial lesions. However, 4.5 months later, an increase in the size of the right lung lesion and the enlarged brain metastasis indicated progressive disease. As a high-grade neuroendocrine carcinoma, LCNEC, diagnosed in approximately 3% of all patients with lung cancer, is generally associated with high rate of metastases and poor survival. The optimal systemic treatment for LCNEC is still under debate. Chemotherapy remains the standard treatment for advanced high-grade neuroendocrine carcinoma, despite the unsatisfying survival rates. Cisplatin and etoposide chemotherapy has an overall response rate of 20% to 25%, and it remains the primary treatment option for inoperable patients.2Frizziero M. Spada F. Lamarca A. et al.Carboplatin in combination with oral or intravenous etoposide for extra-pulmonary, poorly-differentiated neuroendocrine carcinomas.Neuroendocrinology. 2019; 109: 100-112Crossref PubMed Scopus (9) Google Scholar ALK rearrangements have been identified in NSCLC patients who could greatly benefit from ALK TKIs. In addition to echinoderm microtubule associated protein-like 4 (EML4)-ALK, increasingly novel rearrangement partners for ALK, such as cut-like homeobox 1 gene (CUX1)-ALK and S1 RNA binding domain 1 (SRBD1)-ALK, have been detected by NGS, and most of them appear to be sensitive to ALK TKIs.3Zhang M. Wang Q. Ding Y. et al.CUX1-ALK, a novel ALK rearrangement that responds to crizotinib in non–small cell lung cancer.J Thorac Oncol. 2018; 13: 1792-1797Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar,4Hou X. Xu H. Chen L. SRBD1-ALK, a novel ALK fusion gene identified in an adenocarcinoma patient by next-generation sequencing.J Thorac Oncol. 2019; 14: e72-e73Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar However, ALK rearrangements are extremely rare in primary LCNEC. With the widespread use of NGS with an additional advantage of being able to concurrently detect different mutations in genetic testing, several cases of ALK rearrangements sensitive to ALK TKIs have been published in primary LCNEC.5Zheng Q. Zheng M. Jin Y. et al.ALK-rearrangement neuroendocrine carcinoma of the lung: a comprehensive study of a rare case series and review of literature.Onco Targets Ther. 2018; 11: 4991-4998Crossref PubMed Scopus (14) Google Scholar, 6Shimizu N. Akashi Y. Fujii T. et al.Use of ALK immunohistochemistry for optimal therapeutic strategy of pulmonary large-cell neuroendocrine carcinoma and identification of a novel KIF5B-ALK fusion oncokinase.Anticancer Res. 2019; 39: 413-420Crossref PubMed Scopus (5) Google Scholar, 7Hayashi N. Fujita A. Saikai T. et al.Large cell neuroendocrine carcinoma harboring an anaplastic lymphoma kinase (ALK) rearrangement with response to alectinib.Intern Med. 2018; 57: 713-716Crossref PubMed Scopus (14) Google Scholar We systematically reviewed the previous case reports of LCNEC patients with ALK fusion mutation; we summarize the detailed clinical characteristics and outcome of anti-ALK–targeted therapy in Table 1.5Zheng Q. Zheng M. Jin Y. et al.ALK-rearrangement neuroendocrine carcinoma of the lung: a comprehensive study of a rare case series and review of literature.Onco Targets Ther. 2018; 11: 4991-4998Crossref PubMed Scopus (14) Google Scholar, 6Shimizu N. Akashi Y. Fujii T. et al.Use of ALK immunohistochemistry for optimal therapeutic strategy of pulmonary large-cell neuroendocrine carcinoma and identification of a novel KIF5B-ALK fusion oncokinase.Anticancer Res. 2019; 39: 413-420Crossref PubMed Scopus (5) Google Scholar, 7Hayashi N. Fujita A. Saikai T. et al.Large cell neuroendocrine carcinoma harboring an anaplastic lymphoma kinase (ALK) rearrangement with response to alectinib.Intern Med. 2018; 57: 713-716Crossref PubMed Scopus (14) Google Scholar, 8Omachi N. Shimizu S. Kawaguchi T. et al.A case of large-cell neuroendocrine carcinoma harboring an EML4-ALK rearrangement with resistance to the ALK inhibitor crizotinib.J Thorac Oncol. 2014; 9: e40-e42Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 9Hoton D. Humblet Y. Libbrecht L. Phenotypic variation of an ALK-positive large-cell neuroendocrine lung carcinoma with carcinoid morphology during treatment with ALK inhibitors.Histopathology. 2018; 72: 707-710Crossref PubMed Scopus (10) Google Scholar Here we report a case of LCNEC in a patient with a novel PLB1-ALK rearrangement who received crizotinib and experienced a 5-month progression-free survival—longer than that of any other treatment option. To our knowledge, this is the first report of a case with LCNEC harboring the crizotinib-sensitive PLB1-ALK rearrangement, demonstrating that ALK rearrangements, while rare, can be detected in LCNEC, and, consistent with our current knowledge, they serve as the oncogenic driver. The phospholipase B1 gene, which is located on human chromosome 2, is known to play pivotal roles in encoding a membrane-associated phospholipase. In addition, PLB1 exerts lipase and retinyl ester hydrolase activities.Table 1Clinical Characteristics and Therapeutic Effect of ALK Rearrangement in Large-Cell Neuroendocrine CarcinomaStudyYearGender/Age (Years)Stage at DiagnosisSmoking StatusFusion PartnerALK Inhibitor TherapyClinical OutcomePFS (Months)Omachi8Omachi N. Shimizu S. Kawaguchi T. et al.A case of large-cell neuroendocrine carcinoma harboring an EML4-ALK rearrangement with resistance to the ALK inhibitor crizotinib.J Thorac Oncol. 2014; 9: e40-e42Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar2014F/43IVNEML4CrizotinibProgression1.4Hayashi7Hayashi N. Fujita A. Saikai T. et al.Large cell neuroendocrine carcinoma harboring an anaplastic lymphoma kinase (ALK) rearrangement with response to alectinib.Intern Med. 2018; 57: 713-716Crossref PubMed Scopus (14) Google Scholar2017F/75IVbNNAAlectinibPR6+Hoton9Hoton D. Humblet Y. Libbrecht L. Phenotypic variation of an ALK-positive large-cell neuroendocrine lung carcinoma with carcinoid morphology during treatment with ALK inhibitors.Histopathology. 2018; 72: 707-710Crossref PubMed Scopus (10) Google Scholar2017F/69IVNNACrizotinibProgression6CeritinibProgression9Zheng5Zheng Q. Zheng M. Jin Y. et al.ALK-rearrangement neuroendocrine carcinoma of the lung: a comprehensive study of a rare case series and review of literature.Onco Targets Ther. 2018; 11: 4991-4998Crossref PubMed Scopus (14) Google Scholar2018M/44IVbYNANNANAZheng5Zheng Q. Zheng M. Jin Y. et al.ALK-rearrangement neuroendocrine carcinoma of the lung: a comprehensive study of a rare case series and review of literature.Onco Targets Ther. 2018; 11: 4991-4998Crossref PubMed Scopus (14) Google Scholar2018F/47IVaNEML4CrizotinibPR10Shimizu6Shimizu N. Akashi Y. Fujii T. et al.Use of ALK immunohistochemistry for optimal therapeutic strategy of pulmonary large-cell neuroendocrine carcinoma and identification of a novel KIF5B-ALK fusion oncokinase.Anticancer Res. 2019; 39: 413-420Crossref PubMed Scopus (5) Google Scholar2018M/73IVbYKIF5BCrizotinibProgression8AlectinibSD24+Abbreviations: ALK = anaplastic lymphoma kinase; NA = not available; PFS = progression-free survival; PR = partial response; SD = stable disease; EML4 = echinoderm microtubule associated protein-like 4; KIF5B = the kinesin family 5B gene. Open table in a new tab Abbreviations: ALK = anaplastic lymphoma kinase; NA = not available; PFS = progression-free survival; PR = partial response; SD = stable disease; EML4 = echinoderm microtubule associated protein-like 4; KIF5B = the kinesin family 5B gene. NGS has displayed impressive capability to detect ALK-rearranged NSCLC and has evolved into a clinically practical alternative for diagnosis of ALK rearrangements besides techniques based on FISH, IHC, and reverse transcriptase PCR.10Chang W.C. Kim H.K. Shin B.K. Clinicopathological features and diagnostic methods of ALK fusion–positive non–small cell lung cancer in Korea.Oncol Rep. 2020; 43: 218-228PubMed Google Scholar Although NGS played a pivotal role in directing the patient’s targeted therapy, which can prolong survival and improve quality of life, patients with LCNEC do not routinely receive a recommendation for molecular testing because of the extremely low probability of detecting a targetable genetic alteration. But genomic subtyping has shown potential in prognostication and therapeutic decision making for patients with LCNEC. We propose that screening for ALK rearrangement by NGS in patients with LCNEC may help in selecting potential candidates for targeted therapy. The authors have stated that they have no conflict of interest. We thank the study patient and his family. This work was supported by a project cosponsored by the Henan Province and Ministry of Health of Medical Science and Technology Program ( 201601026 ), and in part by the National Natural Science Foundation of China ( 81272600 ), Zhongyuan1000 Talents Plan, Natural Science Foundation of Henan Province ( 162300410300 ), the 51282 project Leading Talent of Henan Provincial Health Science and Technology Innovation Talents ( [2016]32 ). It was also supported by the Program for Science and Technology Innovation Talents in Universities of Henan Province ( 18HASTIT044 ). The funders had no role in study design, data collection, and analysis, decision to publish, or preparation of the report.