A Case of Lung Adenocarcinoma Response to Alectinib Harboring a Rare EML4-ALK Variant, Exon 6 of EML4 Fused to Exon 18 of ALK

Authors: Lirong Liu MM1,2, Fangfang Hou MM1,2,3, Yufeng Liu MM3, Wenzhu Li MM1,2, and Haibo Zhang MD1,2,4,5
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  • 1 Department of Oncology, The Second Affiliated Hospital of Guangzhou University of Chinese Medicine;
  • | 2 Department of Oncology, Guangdong Provincial Hospital of Traditional Chinese Medicine;
  • | 3 The Second Clinical Medical College of Guangzhou University of Chinese Medicine;
  • | 4 Guangdong-Hong Kong-Macau Joint Laboratory on Chinese Medicine and Immune Disease Research; and
  • | 5 Guangdong Provincial Key Laboratory of Clinical Research on Traditional Chinese Medicine Syndrome, Guangzhou, Guangdong, China.

More than 20 types of ALK fusion variant subtypes have been identified, including different fusion partner genes or EML4-ALK fusions with different breakpoints. However, different ALK fusions show different sensitivities to ALK-tyrosine kinase inhibitors (ALK-TKIs) and the emergence of rare fusions brings great challenges to the target therapy in clinic. We report a rare EML4-ALK (E6;A18) fusion in a patient with lung adenocarcinoma that responded well to alectinib. This is the second case of this rare variant reported but the first report of response to an ALK-TKI. This evidence is the first to show that alectinib may be effective for this rare fusion type of non–small cell lung cancer, and these findings have important implications for drug selection in patients with this subtype. Further studies are needed to understand the function of this variant.

ALK rearrangement is found in approximately 5% of patients with non–small cell lung cancer (NSCLC).1 The overall response rate and progression-free survival of patients with ALK-positive NSCLC are significantly improved in the development and clinical application of ALK inhibitors, including the first-generation (crizotinib), second-generation (eg, alectinib, ceritinib, brigatinib), and even third-generation ALK inhibitors (lorlatinib).26 More than 20 types of ALK fusion variant subtypes have been identified, including different fusion partner genes or EML4-ALK fusion with different breakpoints. However, different ALK fusions show different sensitivities to ALK-tyrosine kinase inhibitors (ALK-TKIs).7,8 There is only one publication of EML4-ALK (E6;A18) fusion, a rare fusion type, that showed resistance to crizotinib in a 48-year-old male patient with lung adenocarcinoma.9 Here, we report a rare EML4-ALK (E6;A18) fusion with stable protein expression in a patient with lung adenocarcinoma who responded well to alectinib.

This study was approved by the ethics committee of The Second Affiliated Hospital of Guangzhou University of Chinese Medicine. Written informed consent was obtained from the patient for the publication of case details and images.

Case Presentation

In March 2020, a 64-year-old woman who had never smoked and had no significant medical history was initially admitted to our hospital because she had been experiencing shoulder and neck pain for 2 months. Cervical MRI indicated bone destruction of the C5 vertebral body, which was considered to be a metastatic tumor. 18F-FDG PET/CT revealed left lower lobe atelectasis. There were hypermetabolic nodules in the left hilar suggestive of lung cancer with obstructive pneumonia (Figure 1A). The left hilar nodules were diagnosed as adenocarcinoma by endobronchial ultrasound transbronchial needle aspiration under bronchofiberscopy. The final diagnosis was clinical stage IV (T2bN2bM1c) lung adenocarcinoma according to the lung cancer TNM classification.10 We identified a rare fusion of EML4 with ALK through next-generation sequencing (NGS; BGISEQ-500 platform [MGI Tech Co. Ltd], commercial panel, only DNA-based) technology in which exon 6 of EML4 was fused to exon 18 of ALK (E6;A18) (Figure 2). Immunohistochemistry (Ventana ALK [D5F3] Assay, Roche Diagnostics) showed that ALK was positive (Figure 3B). To our knowledge, only one case of this rare variant has been reported,9 and that patient died 2 weeks after starting treatment with crizotinib, suggesting that patient was resistant to crizotinib and had a poor prognosis.

Figure 1.
Figure 1.

Dynamic monitoring of the patient’s response to alectinib. (A1–A3) PET/CT shows hypermetabolic nodules in left hilar suggestive of lung cancer with obstructive pneumonia and bone and right adrenal metastases. (B1–B3) CT after 2 cycles of carboplatin/pemetrexed with bevacizumab shows left lower lobe atelectasis aggravated with a large amount of pleural effusion. (C1–C3) CT shows partial response after 8 weeks of alectinib. (D1–D3) CT shows continuous response when taking alectinib for 6 months.

Citation: Journal of the National Comprehensive Cancer Network 2021; 10.6004/jnccn.2021.7077

Figure 2.
Figure 2.

A fusion variant of EML4 intron 6 with ALK intron 17 was identified by NGS analysis of genomic DNA and supposed to cause a rare EML4-ALK fusion transcript in which exon 6 of EML4 was fused to exon 18 of ALK (E6:A18). (A) Paired-end sequencing data from tumor tissue samples indicated somatic intrachromosomal EML4-ALK fusion as demonstrated by Integrative Genomics Viewer program (Broad Institute; https://igv.org/app).16 (B) Alignment of NGS reads to supposed-reference sequence. (C) Schematic diagram of the predicted domains of the EML4-ALK fusion protein compared with the 2 most common EML4-ALK variants, V1 and V3. (D) Secondary structure prediction for the acquired region in Phyre2 software.

Abbreviations: ALK, anaplastic lymphoma kinase; CC, coiled coil domain; Fxa inhibition, coagulation Factor Xa inhibitory site; NGS, next-generation sequencing; SS, secondary structure; TMD, transmembrane domain; WD, tryptophan and asparticacid.

Citation: Journal of the National Comprehensive Cancer Network 2021; 10.6004/jnccn.2021.7077

Figure 3.
Figure 3.

(A) Ultrasound-guided fine-needle aspiration cytology of the left hilar nodules showed adenocarcinoma cells (hematoxylin-eosin, original magnification ×100). (B) Immunohistochemistry showed that ALK(D5F3) was positive.

Citation: Journal of the National Comprehensive Cancer Network 2021; 10.6004/jnccn.2021.7077

We first gave our patient 2 cycles of pemetrexed (500 mg/m2 intravenously on day 1 every 3 weeks) and carboplatin (area under the plasma concentration–time curve, 5 mg/mL per min intravenously on day 1 every 3 weeks) in combination with bevacizumab (7.5 mg/kg intravenously on day 1 every 3 weeks). However, chest CT showed left lower lobe atelectasis aggravated with a large amount of pleural effusion (Figure 1B). The clinical condition of the patient worsened. Her ostalgia worsened and she experienced an obvious cough and shortness of breath. Alectinib (600 mg orally twice daily) was started in May 2020 with a rapid clinical response. One week after taking alectinib, her cough and shortness of breath were significantly relieved. The ostalgia was gradually alleviated and she was able to stop the analgesics after 3 weeks. As of this writing, the patient has taken alectinib for more than 9 months, with a continuous partial response noted in September 2020 by CT (Figure 1C, D).

Discussion

This is the first report that describes the rare variant EML4-ALK fusion (E6;A18) response to alectinib. The unique previous publication of a case involving an EML4-ALK (E6;A18) fusion revealed that a 48-year-old male patient with lung adenocarcinoma with EML4-ALK (E6;A18) was resistant to crizotinib. His disease progressed rapidly and he died 2 weeks after taking crizotinib.9 Based on this case report, we recommended chemotherapy as the first-line treatment for our patient; unfortunately, chemotherapy was ineffective.

Without more options, we focused on the rare ALK fusion again. In the previous EML4-ALK (E6;A18) case report, the investigators proposed that the transmembrane domain in exon 18–containing variants may be related to the differences in EML4-ALK protein stability and resistance to crizotinib.9 However, there was no fluorescence in situ hybridization or immunohistochemical analysis to verify the fusion gene or protein expression after the investigators revealed a novel EML4-ALK fusion (E6;A18) by Sanger sequencing of reverse transcriptase-PCR product. With the same breakpoint of ALK exon 18, different fusion partner, Chen et al11 reported a noncoding partner (Chr2 30297612) with exon 18 of ALK in a patient with lung adenocarcinoma who had stable ALK fusion protein expression and experienced a response to crizotinib. Based on the stable overexpression of the ALK protein in the tumor cells of our patient (Figure 3), and the better efficacy of alectinib reported in the ALEX, J-ALEX, and ALESIA studies,1214 we administered alectinib to our patient. The symptoms of cough, shortness of breath, and pain were relieved within 1 week, and the imaging evaluation at the eighth week showed ongoing partial response and revealed good therapeutic effect over 9 months to the cutoff date.

This is the second reported case of a patient carrying a rare variant EML4-ALK (E6;A18) fusion. Unlike the first patient with EML4-ALK (E6;A18) resistant to crizotinib, our case showed for the first time that alectinib is effective for this rare variant EML4-ALK (E6;A18) fusion. Compared with the canonical EML4-ALK fusion, this new fusion (E6;A18) acquires 85 amino acids, from positions 229 to 314. The added region contains 12 amino acids derived from introns and 73 amino acids derived from exons. Similar to the Chr2 30297612-ALK (Chr2:30297612; A18) fusion case, there was stable ALK fusion protein expression in our EML4-ALK (E6;A18) fusion case, which indicates that the transmembrane domain in exon 18 may not destabilize EML4-ALK (E6;A18) protein structure. Indeed, EML4-ALK (E6;A18) had complete EML4 trimerization domain. The trimerization domain of EML4 is essential for ALK autophosphorylation and activation.7 Therefore, we speculate that EML4-ALK (E6;A18) fusion with immunohistochemistry-verified protein overexpression could benefit from ALK-TKI therapy. A secondary structure prediction in Phyre2 software (Structural Bioinformatics Group) for the new sequence found that the acquired region 229–314 was displayed as several beta sheets followed by an alpha helix (Figure 2D).15 In addition, ligand binding site prediction for the acquired peptide region 229–314 showed no binding potential. Therefore, the newly added 85 amino acids may not have a particularly significant impact on the function of fusion protein. However, further studies are needed to understand the function of this variant. In addition to this method, an NGS platform that incorporates RNA sequencing could also be useful in querying expression of rare fusion proteins. Combination detection is strongly recommended for this rare fusion to optimize the treatment strategy.

Conclusions

This is the second case of a patient with lung adenocarcinoma harboring a rare EML4-ALK (E6;A18) fusion reported, but the first report of response to an ALK-TKI. Our data suggested that alectinib may be effective for this rare fusion type of NSCLC and provided evidence for drug selection in patients with this subtype for the first time.

Acknowledgments

The authors thank the patient and her family.

References

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    Yoshida T, Oya Y, Tanaka K, et al. Differential crizotinib response duration among ALK fusion variants in ALK-positive non-small-cell lung cancer. J Clin Oncol 2016;34:33833389.

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    Anai S, Takeshita M, Ando N, et al. A case of lung adenocarcinoma resistant to crizotinib harboring a novel EML4-ALK variant, exon 6 of EML4 fused to exon 18 of ALK. J Thorac Oncol 2016;11:e126128.

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    Amin MB, Edge S, Greene F, et al. eds. AJCC Cancer Staging Manual, 7th edition. New York: Springer International Publishing; 2017.

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    Chen X, Zhao G, Zhong P, et al. Chr2 30297612-ALK, a novel intergenic fusion with exon18 of ALK, responds to crizotinib. Clin Lung Cancer 2020;21:e564566.

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    Peters S, Camidge DR, Shaw AT, et al. Alectinib versus crizotinib in untreated ALK-positive non-small-cell lung cancer. N Engl J Med 2017;377:829838.

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    Zhou C, Kim SW, Reungwetwattana T, et al. Alectinib versus crizotinib in untreated Asian patients with anaplastic lymphoma kinase-positive non-small-cell lung cancer (ALESIA): a randomised phase 3 study. Lancet Respir Med 2019;7:437446.

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    Hida T, Nokihara H, Kondo M, et al. Alectinib versus crizotinib in patients with ALK-positive non-small-cell lung cancer (J-ALEX): an open-label, randomised phase 3 trial. Lancet 2017;390:2939.

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Submitted February 23, 2021; final revision received June 17, 2021; accepted for publication June 21, 2021. Published online November 11, 2021.

Disclosures: The authors have disclosed that they have not received any financial consideration from any person or organization to support the preparation, analysis, results, or discussion of this article.

Funding: This work was supported by funding from Science and Technology Planning Project of Guangdong Province (No. 2020B1212030006).

Correspondence: Haibo Zhang, MD, Department of Oncology, The Second Affiliated Hospital of Guangzhou University of Chinese Medicine, No. 111 Dade Road, Yuexiu District, Guangzhou 510120, China. Email: haibozh@gzucm.edu.cn
  • View in gallery

    Dynamic monitoring of the patient’s response to alectinib. (A1–A3) PET/CT shows hypermetabolic nodules in left hilar suggestive of lung cancer with obstructive pneumonia and bone and right adrenal metastases. (B1–B3) CT after 2 cycles of carboplatin/pemetrexed with bevacizumab shows left lower lobe atelectasis aggravated with a large amount of pleural effusion. (C1–C3) CT shows partial response after 8 weeks of alectinib. (D1–D3) CT shows continuous response when taking alectinib for 6 months.

  • View in gallery

    A fusion variant of EML4 intron 6 with ALK intron 17 was identified by NGS analysis of genomic DNA and supposed to cause a rare EML4-ALK fusion transcript in which exon 6 of EML4 was fused to exon 18 of ALK (E6:A18). (A) Paired-end sequencing data from tumor tissue samples indicated somatic intrachromosomal EML4-ALK fusion as demonstrated by Integrative Genomics Viewer program (Broad Institute; https://igv.org/app).16 (B) Alignment of NGS reads to supposed-reference sequence. (C) Schematic diagram of the predicted domains of the EML4-ALK fusion protein compared with the 2 most common EML4-ALK variants, V1 and V3. (D) Secondary structure prediction for the acquired region in Phyre2 software.

    Abbreviations: ALK, anaplastic lymphoma kinase; CC, coiled coil domain; Fxa inhibition, coagulation Factor Xa inhibitory site; NGS, next-generation sequencing; SS, secondary structure; TMD, transmembrane domain; WD, tryptophan and asparticacid.

  • View in gallery

    (A) Ultrasound-guided fine-needle aspiration cytology of the left hilar nodules showed adenocarcinoma cells (hematoxylin-eosin, original magnification ×100). (B) Immunohistochemistry showed that ALK(D5F3) was positive.

  • 1.

    Kwak EL, Bang Y, Camidge DR, et al. Anaplastic lymphoma kinase inhibition in non-small-cell lung cancer. N Engl J Med 2010;363:16931703.

  • 2.

    Solomon BJ, Mok T, Kim DW, et al. First-line crizotinib versus chemotherapy in ALK-positive lung cancer. N Engl J Med 2014;371:21672177.

  • 3.

    Hida T, Nokihara H, Kondo M, et al. Alectinib versus crizotinib in patients with ALK-positive non-small-cell lung cancer (J-ALEX): an open-label, randomised phase 3 trial. Lancet 2017;390:2939.

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 4.

    Soria JC, Tan DSW, Chiari R, et al. First-line ceritinib versus platinum-based chemotherapy in advanced ALK-rearranged non-small-cell lung cancer (ASCEND-4): a randomised, open-label, phase 3 study. Lancet 2017;389:917929.

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 5.

    Gettinger SN, Bazhenova LA, Langer CJ, et al. Activity and safety of brigatinib in ALK-rearranged non-small-cell lung cancer and other malignancies: a single-arm, open-label, phase 1/2 trial. Lancet Oncol 2016;17:16831696.

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 6.

    Shaw AT, Felip E, Bauer TM, et al. Lorlatinib in non-small-cell lung cancer with ALK or ROS1 rearrangement: an international, multicentre, open-label, single-arm first-in-man phase 1 trial. Lancet Oncol 2017;18:15901599.

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 7.

    Bayliss R, Choi J, Fennell DA, et al. Molecular mechanisms that underpin EML4-ALK driven cancers and their response to targeted drugs. Cell Mol Life Sci 2016;73:12091224.

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 8.

    Yoshida T, Oya Y, Tanaka K, et al. Differential crizotinib response duration among ALK fusion variants in ALK-positive non-small-cell lung cancer. J Clin Oncol 2016;34:33833389.

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 9.

    Anai S, Takeshita M, Ando N, et al. A case of lung adenocarcinoma resistant to crizotinib harboring a novel EML4-ALK variant, exon 6 of EML4 fused to exon 18 of ALK. J Thorac Oncol 2016;11:e126128.

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 10.

    Amin MB, Edge S, Greene F, et al. eds. AJCC Cancer Staging Manual, 7th edition. New York: Springer International Publishing; 2017.

  • 11.

    Chen X, Zhao G, Zhong P, et al. Chr2 30297612-ALK, a novel intergenic fusion with exon18 of ALK, responds to crizotinib. Clin Lung Cancer 2020;21:e564566.

  • 12.

    Peters S, Camidge DR, Shaw AT, et al. Alectinib versus crizotinib in untreated ALK-positive non-small-cell lung cancer. N Engl J Med 2017;377:829838.

  • 13.

    Zhou C, Kim SW, Reungwetwattana T, et al. Alectinib versus crizotinib in untreated Asian patients with anaplastic lymphoma kinase-positive non-small-cell lung cancer (ALESIA): a randomised phase 3 study. Lancet Respir Med 2019;7:437446.

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 14.

    Hida T, Nokihara H, Kondo M, et al. Alectinib versus crizotinib in patients with ALK-positive non-small-cell lung cancer (J-ALEX): an open-label, randomised phase 3 trial. Lancet 2017;390:2939.

    • Crossref
    • PubMed
    • Search Google Scholar
    • Export Citation
  • 15.

    Kelley LA, Mezulis S, Yates CM, et al. The Phyre2 web portal for protein modeling, prediction and analysis. Nat Protoc 2015;10:845858.

  • 16.

    Robinson JT, Thorvaldsdóttir H, Winckler W, et al. Integrative genomics viewer. Nat Biotechnol 2011;29:2426.

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