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A Novel Linc00308/D21S2088E Intergenic Region ALK Fusion and Its Enduring Clinical Responses to Crizotinib

  • Jian Zhang
    Affiliations
    Department of Thoracic Surgery, The Third Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong, People’s Republic of China
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  • Chang Zou
    Affiliations
    Clinical Medical Research Center, The Second Clinical Medical College of Jinan University, The First Affiliated Hospital of Southern University, Shenzhen People’s Hospital, Shenzhen, Guangdong, People’s Republic of China
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  • Chenzhi Zhou
    Affiliations
    State Key Laboratory of Respiratory Disease, National Clinical Research Center for Respiratory Disease, Guangzhou Institute of Respiratory Health, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong, People’s Republic of China
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  • Yifeng Luo
    Affiliations
    Institute of Precision Medicine, The First Affiliated Hospital of Sun Yat-Sen University, Guangzhou, Guangdong, People’s Republic of China
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  • Qiong He
    Affiliations
    Molecular Diagnosis Center, The First Affiliated Hospital of Sun Yat-Sen University, Guangzhou, Guangdong, People’s Republic of China

    Institute of Precision Medicine, The First Affiliated Hospital of Sun Yat-Sen University, Guangzhou, Guangdong, People’s Republic of China
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  • Yu Sun
    Affiliations
    Molecular Diagnosis Center, The First Affiliated Hospital of Sun Yat-Sen University, Guangzhou, Guangdong, People’s Republic of China
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  • Jianwen Zhou
    Affiliations
    Institute of Precision Medicine, The First Affiliated Hospital of Sun Yat-Sen University, Guangzhou, Guangdong, People’s Republic of China
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  • Zunfu Ke
    Correspondence
    Corresponding author. Address for correspondence: Zunfu Ke, MD, PhD, Molecular Diagnosis Center, The First Affiliated Hospital, Sun Yat-sen University, No. 58, Zhongshan Second Road, Guangzhou, Guangdong, People’s Republic of China.
    Affiliations
    Institute of Precision Medicine, The First Affiliated Hospital of Sun Yat-Sen University, Guangzhou, Guangdong, People’s Republic of China

    Molecular Diagnosis Center, The First Affiliated Hospital of Sun Yat-Sen University, Guangzhou, Guangdong, People’s Republic of China
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Open AccessPublished:March 23, 2020DOI:https://doi.org/10.1016/j.jtho.2020.03.009
      Approximately 5% of patients with NSCLC present ALK gene rearrangements, which define a distinct molecular subgroup of NSCLC.
      • Barlesi F.
      • Mazieres J.
      • Merlio J.P.
      • et al.
      Routine molecular profiling of patients with advanced non-small-cell lung cancer: results of a 1-year nationwide programme of the French Cooperative Thoracic InterGroup (IFCT).
      Nevertheless, in addition to the classic ALK fusion partner EML4, other emerging ALK fusion partners bring great challenges to the targeted crizotinib therapy in clinics.
      First-line crizotinib versus Chemotherapy in ALK-Positive Lung Cancer.
      Herein we report a novel ALK fusion partner, the Linc00308/D21S2088E intergenic region, that conferred responsiveness to crizotinib in a patient with lung adenocarcinoma (LUAD).
      A 61-year-old Chinese man with smoking history was found to have an irregular nodule, predominantly in the hilum of the lung, under computed tomography. Tissue biopsy (Fig. 1A) and subsequent imaging were carried out, resulting in a diagnosis of IIIB adenocarcinoma. In the absence of a standard treatment in this setting, the patient’s formaldehyde-fixed paraffin-embedded (FFPE) tissue was submitted for genomic testing by targeted next-generation sequencing to uncover genetic alterations in the initial tumor. The mutation profile revealed a novel rearrangement variant generated by a fusion of the Linc00308/D21S2088E intergenic region on 21q21.1 to the intron 19 of ALK on 2p23 (Fig. 1B). According to the DNA sequencing structure, the fusion transcript consisted of the intergenic region (novel exon) and the exons 20 to 29 of the ALK gene, the latter encoding the complete ALK intracellular kinase domain (Fig. 2A and B). This fusion product was further validated at the mRNA level by reverse-transcriptase polymerase chain reaction and Sanger sequencing of the tumor FFPE RNA sample (Fig. 2C and D). Before treatment with crizotinib, the FFPE sample was reconfirmed by conventional fluorescence in situ hybridization assay and immunohistochemistry (ALK D5F3 antibody) (Fig. 2EG). Then, the patient was initiated on oral crizotinib therapy at a dosage of 250 mg twice daily. Computed tomography scans revealed considerable tumor shrinkage of the target lesions after 4 months, with the longest diameter of lung mass decreasing from 2.93 to 1.92 mm and sustained response after 6 months (Fig. 3A). Simultaneously, compared with the baseline circulating tumor cells level (13/mL) (Fig. 3B), the circulating tumor cell number significantly decreased at 4 months after therapy and remained at 1/mL for a period of 4 months (from April 2019 to August 2019). Repeated circulating tumor DNA analysis revealed that the mutant allele frequency of the intergenic region ALK fusion in the plasma was closely associated with the tumor burden, accurately reflecting the dynamic tumor response to crizotinib (Fig. 3C). Currently, crizotinib is being continued with good tolerance. In addition, as shown in Figure 4A and B, crizotinib treatment significantly inhibited ALK phosphorylation at position Y1507 and Y1604 in LUAD cells isolated from fresh tumor tissue. Moreover, the viability of intergenic region ALK-positive LUAD cells was highly suggestive of crizotinib treatment in vitro (Fig. 4C) and in vivo (Fig. 4D). To further assess the function of this novel ALK fusion protein in oncogenesis, plasmids expressing intergenic region ALK fusion protein were transfected into Ba/F3 cells whose growth is dependent on interleukin-3. In the absence of interleukin-3, Ba/F3 cells expressing intergenic region ALK grew at an exponential rate, confirming the kinase-dependent oncogenic activity of intergenic region ALK (Fig. 5A). Simultaneously, self-phosphorylation of ALK at position Y1507 and Y1604 could be observed in the intergenic region ALK-expressing 293T cells (Fig. 5B), suggesting the constitutive activation of the intergenic region ALK fusion protein. In 293T cells overexpressing the intergenic region ALK, we observed the significantly increased phosphorylation levels of MEK1/2 in the MAPK pathway, AKT in the PI3K-AKT pathway, and STAT3 in the JAK-STAT pathway (Fig. 5B and C). Meanwhile, the addition of crizotinib inhibited ALK phosphorylation and the activation of its downstream effector molecules, MEK1/2, AKT, and STAT3 in the intergenic region ALK-expressed 293T cells (Fig. 5B and C).
      Figure thumbnail gr1
      Figure 1A novel Linc00308/D21S2088E intergenic region ALK fusion was discovered in a patient with lung adenocarcinoma. (A) Immunohistochemistry results revealed that the tumor cells were positive for TTF-1, Napsin A, CK7, and negative for P40. (B) Schematic of intergenic region ALK fusion mRNA and protein expression. The intergenic region fusion breakpoint of chr21 is located in the region between Linc00308 and D21S2088E. In this region, an exon was confirmed through transcription expression by RNA-Seq. CK7, cytokeratin 7; RNA-Seq, RNA sequencing; TTF-1, thyroid transcription factor-1.
      Figure thumbnail gr2
      Figure 2The fusion transcript was identified using RNA-seq and validated using reverse-transcriptase polymerase chain reaction, fluorescence in situ hybridization, and immunohistochemistry. (A) Integrated Genomics Viewer from capture-based DNA-Seq revealed that the breakpoint was located in chr21:23877671 and chr2:29447271, respectively. (B) HISAT2 mapping to hg19 analysis using RNA-seq indicated many expressed RNA reads in the downstream area of 3′ end ALK breakpoint and the intergenic region, and none in the upstream area of 5′ end ALK breakpoint. (C) Agarose gel electrophoresis verified reverse-transcriptase polymerase chain reaction products spanning the fusion location point between Linc00308/D21S2088E and ALK. The patient’s white blood cells were used as a negative control. (D) Sanger sequencing of gene rearrangement revealed a clear fusion of ALK with the intergenic region between Linc00308 and D21S2088E. (E) Hematoxylin and eosin staining revealed the cellular structure and characteristics of lung adenocarcinoma. (F) In the fluorescence in situ hybridization image, a split signal of the 5′ and 3′ of the ALK gene was observed. (G) Immunohistochemistry staining indicated a strong expression of ALK (D5F3 antibody). DNA-Seq, DNA sequencing; FFPE, formaldehyde-fixed paraffin-embedded; RNA-Seq, RNA sequencing.
      Figure thumbnail gr3
      Figure 3Dynamic monitoring of the response of the patient with lung adenocarcinoma to crizotinib. (A) Chest CT scans on October 15, 2018 revealed the presence of an irregular nodule predominantly in the hilum of the lung. Significant (February 15, 2019) and (June 15, 2019) consistent reduction of the tumor volume was observed by the follow-up CT scans after the first-line therapy with crizotinib. (B) Noninvasive detection and monitoring of CTC number and CT scan during the patient’s clinical course. (C) Dynamic change of intergenic region ALK mutant allele frequency in ctDNA during the treatment course. CT, computed tomography; CTC, circulating tumor cells; ctDNA, circulating tumor DNA; NGS, next-generation sequencing; po bid, orally twice a day.
      Figure thumbnail gr4
      Figure 4Evaluation of drug sensitivity of Linc00308/D21S2088E intergenic region ALK fusion to crizotinib. (A) LUADCs were isolated from fresh tumor tissues of the patient with intergenic region ALK fusion using the Thermoresponsive NanoVelcro system. Cell passage of LUADCs in vitro did not affect ALK status. (B) ALK protein was detected in LUADCs and its phosphorylation level was decreased by crizotinib. (C) A line graph of MTT assay revealed that the proliferation of LUADCs was significantly inhibited by crizotinib compared with DMSO control. (D) Crizotinib suppressed tumor formation of LUADCs in mice. ∗p < 0.01. LUADCs, Lung adenocarcinoma cells.
      Figure thumbnail gr5
      Figure 5The oncogenic activity of Linc00308/D21S2088E intergenic region ALK fusion and its effect on ALK self-phosphorylation and the phosphorylation of other key proteins located in its downstream signaling pathways, including MEK1/2 in the MAPK pathway, AKT in PI3K-AKT pathway, and STAT3 in the JAK-STAT pathway. (A) Intergenic region ALK-overexpressed Ba/F3 cells grew in an interleukin-3–independent manner, which could be inhibited by crizotinib; whereas Ba/F3 cells with empty vector did not. (B, C) The phosphorylation levels of ALK, MEK1/2, AKT, and STAT3 in293 T cells could be inhibited by crizotinib. 293 T cells were transfected with pcDNA3.0-intergenic region ALK and empty vector, respectively. Representative results are from three independent experiments. ∗p < 0.01. GADPH, glyceraldehyde 3-phosphate dehydrogenase; po bid, orally twice a day.
      A vast array of oncogenic ALK variants are formed by the fusion of 3′-half of the ALK gene which retains its kinase catalytic domain, and the 5′-part of a partner gene that provides its promoter.
      • Heydt C.
      • Kostenko A.
      • Merkelbach-Bruse S.
      • Wolf J.
      • Buttner R.
      ALK evaluation in the world of multiplex testing: Network Genomic Medicine (NGM): the Cologne model for implementing personalised oncology.
      Structural studies have revealed that fusion with multiple 5′ partners facilitates multimerization and autophosphorylation of ALK kinase and increases the oncogenic potential of ALK, as evidenced by EML4-ALK
      • Wang D.
      • Li D.
      • Qin G.
      • et al.
      The structural characterization of tumor fusion genes and proteins.
      in NSCLC. In this study, we identified a new fusion partner of ALK, the Linc00308/D21S2088E (two noncoding genes
      • Jiang T.
      • Guo J.
      • Hu Z.
      • Zhao M.
      • Gu Z.
      • Miao S.
      Identification of potential prostate cancer-related pseudogenes based on competitive endogenous RNA network hypothesis.
      ,
      • Manichaikul A.
      • Wang X.Q.
      • Sun L.
      • et al.
      Genome-wide association study of subclinical interstitial lung disease in MESA.
      ) intergenic region, in LUAD. To our knowledge, an intergenic region between CENPA and DPYSL5-ALK fusion was reported previously.
      • Fei X.
      • Zhu L.
      • Zhou H.
      • Qi C.
      • Wang C.
      A novel intergenic region between CENPA and DPYSL5-ALK Exon 20 fusion variant responding to crizotinib treatment in a patient with lung adenocarcinoma.
      Nevertheless, they did not clarify whether the rearrangement affected the expression or function of the ALK gene. Here, we not only detected the presence of intergenic region ALK fusion transcripts, but also found that intergenic region ALK fusion could enhance ALK kinase activity and activate the phosphorylation of downstream AKT, MEK1/2, and STAT3 in 293T cells. Thus, we have provided proof that patients with advanced NSCLC harboring a Linc00308/D21S2088E intergenic region ALK may benefit from crizotinib, expanding the spectrum of ALK fusion variants to optimize treated strategy.

      Acknowledgments

      This work was supported by grants from National Key Research and Development Program of China ( 2017YFC1308800 ), National Natural Science Foundation of China to Zunfu Ke ( 30900650 , 81372501 , 81572260 , and 81773299 ), and Guangdong Natural Science Foundation ( 2018A050506036 ).

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