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S12967-021-02982-4.Pdf Xia et al. J Transl Med (2021) 19:308 https://doi.org/10.1186/s12967-021-02982-4 Journal of Translational Medicine RESEARCH Open Access Molecular characteristics and clinical outcomes of complex ALK rearrangements identifed by next-generation sequencing in non-small cell lung cancers Peiyi Xia1†, Lan Zhang1†, Pan Li1†, Enjie Liu1, Wencai Li1, Jianying Zhang2, Hui Li3, Xiaoxing Su3 and Guozhong Jiang1* Abstract Background: Complex kinase rearrangement, a mutational process involving one or two chromosomes with clustered rearrangement breakpoints, interferes with the accurate detection of kinase fusions by DNA-based next- generation sequencing (NGS). We investigated the characteristics of complex ALK rearrangements in non-small cell lung cancers using multiple molecular tests. Methods: Samples of non-small cell lung cancer patients were analyzed by targeted-capture DNA-based NGS with probes tilling the selected intronic regions of fusion partner genes, RNA-based NGS, RT-PCR, immunohistochemistry (IHC) and fuorescence in situ hybridization (FISH). Results: In a large cohort of 6576 non-small cell lung cancer patients, 343 (5.2%) cases harboring ALK rearrange- ments were identifed. Fourteen cases with complex ALK rearrangements were identifed by DNA-based NGS and classifed into three types by integrating various genomic features, including intergenic (n 3), intragenic (n 5) and “bridge joint” rearrangements (n 6). All thirteen cases with sufcient samples actually expressed= canonical EML4-ALK= fusion transcripts confrmed by RNA-based= NGS. Besides, positive ALK IHC was detected in 13 of 13 cases, and 9 of 11 cases were positive in FISH testing. Patients with complex ALK rearrangements who received ALK inhibitors treatment (n 6), showed no diference in progression-free survival (PFS) compared with patients with canonical ALK fusions n = 36, P 0.9291). = = Conclusions: This study frstly reveals the molecular characteristics and clinical outcomes of complex ALK rearrange- ments in NSCLC, sensitive to ALK inhibitors treatment, and highlights the importance of utilizing probes tilling the selected intronic regions of fusion partner genes in DNA-based NGS for accurate fusion detection. RNA and protein level assay may be critical in validating the function of complex ALK rearrangements in clinical practice for optimal treatment decision. *Correspondence: [email protected] †Peiyi Xia, Lan Zhang and Pan Li contributed equally to this work 1 Department of Pathology, The First Afliated Hospital of Zhengzhou University, Zhengzhou University, Jian She Dong Road 1, Zhengzhou 450052, Henan, China Full list of author information is available at the end of the article © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Xia et al. J Transl Med (2021) 19:308 Page 2 of 12 Keywords: ALK fusion, Complex rearrangements, Non-small cell lung cancer, Next-generation sequencing, Targeted therapy Background treatments. A combination methodology of DNA- Rearrangements of the anaplastic lymphoma kinase based NGS technique followed by RNA-based NGS (ALK) gene have been identifed in approximately 3–7% provides a unique opportunity to explore the muta- of non-small cell lung cancer (NSCLC) patients, with tional processes in cancer genomes. Although there echinoderm microtubule-associated protein like-4 has been landmark study characterizing the complex (EML4) representing the most common fusion partner intergenic-breakpoint fusions [14], it was largely based [1, 2]. ALK-rearranged NSCLC defne a distinct molec- on exome and selected introns in ALK gene. It is lack of ular subset with high sensitivity to ALK tyrosine kinase the study using DNA-based NGS designed for intronic inhibitors (TKIs). Crizotinib, a well-tolerated frst regions from fusion partner genes known to likely har- generation ALK inhibitor [3, 4], has been approved by bor the genomic breakpoint. Food and Drug Administration in US for the treatment Herein, we utilized DNA-based NGS panel specifcally of ALK-rearranged NSCLC in 2011. Second genera- designed with multiple probes tilling selected intronic tion ALK inhibitors, such as alectinib and ceritinib, are regions of fusion partner genes, and identifed three efective not only in crizotinib-naive patients [5], but types of complex ALK rearrangements in 14 cases from a also in patients with acquired resistance to crizotinib large cohort of NSCLC patients. Further functional vali- [6–9]. Te identifcation of ALK rearrangements and dation performed by RNA or protein assay elucidated the the approval of a number of ALK TKIs have revolution- importance of DNA and RNA-based NGS for the com- ized the treatment of patients harboring ALK fusions. prehensive detection of kinase fusions and guiding opti- Terefore, accurate detection for ALK rearrangements mal treatment decision. is crucial. A challenge for precision oncology is identifying novel or complex translocations. Traditional methods, Methods including fuorescence in situ hybridization (FISH) and Patients and samples immunohistochemistry (IHC), have limitations, such Samples from a cohort of 6576 patients with NSCLC as FISH does not permit identifcation of ALK partner from January 2018 to July 2020 were collected for molec- genes or non-canonical breakpoints, and ALK IHC ular testing. Pathological and clinical information was could be confounded in principle by overexpression of obtained from clinical records. Te study was approved ALK driver rather than a true fusion protein [10]. While by the Institutional Review Board of the First Afliated next-generation sequencing (NGS) techniques pro- Hospital of Zhengzhou University. All patients provided vide an efective and accurate detection for known and informed written consent for these genomic analyses. novel oncogenic fusions, and have been widely applied in clinical diagnostics. Complex kinase rearrangements, herein referred to a DNA/RNA extraction mutational process involving one or two chromosomes Te pathological diagnosis of each case was confrmed with clustered rearrangement breakpoints. Recent on routine hematoxylin and eosin stained slides, and studies have revealed that complex genomic rearrange- the corresponding optimal blocks containing a mini- ments generated 74% of known fusion oncogenes in mum of 20% tumor cells were forwarded for DNA/RNA human lung adenocarcinoma of non-smokers, includ- extraction. Genomic DNA (gDNA) and total RNA were ing EML4-ALK, CD74-ROS1, and KIF5B-RET [11]. extracted from the formalin-fxed parafn-embedded However, complex genomic rearrangements frequently (FFPE) tumor tissue samples using AllPrep DNA/RNA hindered proper capture in DNA-based NGS assay [12]. FFPE Kit (Qiagen, USA) according to the manufac- Accumulating evidences have suggested that genomic turer’s instructions. As a control, gDNA from the white breakpoints identifed by DNA sequencing are an unre- blood cell samples was extracted using MagPure Blood liable predictor of breakpoint at the transcript level DNA DA Kit (Magen, China) according to the manufac- owing to genomic complexities [13, 14]. Te identif- turer’s instructions. Te quality of purifed DNA/RNA cation and clinically functional validation of complex were assayed by gel electrophoresis and quantifed by ® kinase rearrangements remain elusive, which makes Qubit 4.0 Fluorometer (Life Technologies, USA). Te the oncologists confused to choose the appropriate amounts of extracted DNA more than 30 ng were con- sidered sufcient for analysis. In the extracted FFPE RNA Xia et al. J Transl Med (2021) 19:308 Page 3 of 12 samples, the 28S and 18S rRNA bands were degraded, FISH and ≥ 200 ng RNA were optimal for high analytical In brief, FFPE tumor tissue samples was analyzed by sensitivity. FISH using the Vysis LSI ALK Dual color, Break Apart Rearrangement Probe (Abbott/Vysis, Abbott Park, IL, DNA‑based NGS USA). In 50 scored tumour cells of every sample, if more Te purifed gDNA was frst fragmented into DNA pieces than 15% of the scored tumour cells had split one or both ′ ′ ′ about 300-bp using enzymatic method (5X WGS Frag- ALK 5 and 3 probe signals or had isolated 3 signals, the mentation Mix, Qiagen, USA), followed by end repairing, sample was considered to be FISH positive. Every FISH T-adaptors ligation, and PCR amplifcation, resulting in slide was evaluated by two pathologists independently. pre-library. An in-house designed panel targeting most exons and selected introns in 86 cancer-related genes was IHC used to capture DNA fragments to detect SNV/Indel,
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