The Genomic Characteristics of ALK Fusion Positive Tumors in Chinese NSCLC Patients

Anaplastic lymphoma kinase (ALK) fusion events account for ~3–7% genetic alterations in patients with non-small cell lung cancer (NSCLC). In this study, we identified the ALK fusion patterns and a novel ALK fusion partner in 44 ALK positive NSCLC patients using a customized HapOncoCDx panel, and identified ALK fusion partners. The most common partner is EML4, forming the variant 1 (v1, E13:A20, 18/44), variant 2 (v2, E20:A20, 5/44), and variant 3 (v3, E6:A20, 13/44). Moreover, we detected a new ALK fusion partner HMBOX1. At the mutation level, TP53 is the most frequently mutated gene (24%), followed by ALK (12%) and STED2 (12%). The median tumor mutation burden (TMB) of these samples is 2.29 mutations/Mb, ranging from 0.76 mut/Mb to 16.79 muts/Mb. We further elaborately portrayed the TP53 mutation sites on the peptide sequence of the encoded protein by lollipop. The mutational signature and copy number alterations (CNAs) of the samples were also analyzed. The CNA events were found in 13 (13/44) patients, and the most commonly amplified genes were MDM2 (n = 4/13) and TERT (n = 4/13). Together, these results may guide personalized clinical management of patients with ALK fusion in the era of precision medicine.


INTRODUCTION
Anaplastic lymphoma kinase (ALK) fusion events, which are the result of ALK rearrangements, account for ∼3-7% genetic alterations in non-small cell lung cancer (NSCLC) patients (1, 2). These oncogenic mutations could lead to the constitutive activation of the ALK tyrosine kinase domain, and further cause tumorigenesis (3). Hitherto, multiple ALK fusion partners have been identified, and the most normal one is echinoderm microtubule-associated protein-like 4 (EML4), which were observed in nearly 80% of all the ALK fusion cases (2). It is worth noting that more than a dozen of different EML4-ALK variants have been identified in NSCLC patients. The most common variants are variant 1 (v1, E13:A20), variant 2 (v2, E20:A20), and variant 3 (v3, E6:A20) (4).
Currently, ALK tyrosine kinase inhibitors (TKIs) are recommended for the treatment of NSCLC patients harboring ALK fusion (5). Prior to ALK TKIs treatment, the median overall survival (OS) of ALK fusion-positive metastatic NSCLC patients receiving chemotherapy was around 12 months (6). However, under the sequential treatment with ALK fusion TKIs, the OS of the patients were extended to 5 years (7). The first approved is Crizotinib which is the first generation TKI. Compared to traditional chemotherapy, it improves the PFS and OS of ALK fusion NSCLC patients significantly (8). Nevertheless, nearly all of the patients would develop drug resistance within 2 years after the initial treatment. The drug resistance was possibly caused by a secondary mutation in the kinase domain of ALK, the activation of alternative pathways, ALK amplification, or epithelial-mesenchymal transition (9). To overcome the resistance, the second-generation ALK TKIs were developed including Ceritinib, Alectinib, and Brigatinib. They were approved for the treatment of metastatic NSCLC patients with ALK fusion and had progressed on or intolerant to Crizotinib. Notably, Ceritinib and Alectinib were approved for the firstline treatment of the ALK fusion positive metastatic NSCLC patients (10)(11)(12)(13)(14)(15)(16). In addition, as the third generation ALK inhibitor, Lorlatinib has also been approved for the treatment of metastatic NSCLC patients with ALK fusion, on condition that the disease has progressed on Crizotinib or at least one other ALK inhibitor such as Alectinib or Ceritinib for metastatic disease (17). It is worth noting that different ALK inhibitors have different potencies and spectrums against acquired resistance mutations (18).
In the era of precision medicine, the genomic profiles of the patients may guide the planning of treatment strategy. For the ALK fusion positive NSCLC patients, detailed genomic profiles can elucidate the fusion partner and the rearranged breakpoint. Moreover, the proposed resistant mutations are critical for clinical treatment guidelines. Furthermore, several studies reported that molecular profiling is also associated with the prognosis of patients. Christopoulos et al. reported that the concomitant TP53 mutation is a strong indicator of poor prognosis in ALK fusion positive NSCLC patients (19). Their study also reported that EML4-ALK fusion variant V3 was associated with a more aggressive phenotype and worse overall survival due to earlier failure of several therapeutic modalities. In addition, they found that V3 and TP53 double positive patients had a very high risk of death with a median OS of around 2 years.
With the development of next-generation sequencing technologies, it is becoming more affordable to obtain the genomic landscape of cancer patients. In this study, we aim to demonstrate the genomic landscape of ALK fusion-positive tumors in 44 Chinese NSCLC patients sequenced with our customized HapOncoCDx panel which involves hybridization capture and deep sequencing of all protein-coding exons of 464 cancer-associated genes and other selected introns of other oncogenes and tumor suppressor genes, and illustrate their genomic mutation patterns and characteristics, which potentially helps to develop treatment strategy.

Patients and Samples
Forty-four patients were enrolled from 1349 NSCLC patients in this study. Tumor tissues were collected during surgery, and were formalin fixed, paraffin-embedded (FFPE) and archived. Peripheral blood (PBL) samples were also collected from each patient as control.

DNA Extraction
DNA samples were extracted from Formalin-fixed paraffinembedded (FFPE) samples with QIAamp DNA FFPE tissue kit (Qiagen). Extraction of PBL DNA was conducted using the RelaxGene Blood DNA system (Tiangen Biotech Co., Ltd., Beijing, China) according to the manufacturer's protocol. All the DNA samples were quantified both using the Qubit 2.0 fluorometer and the Qubit dsDNA HS Assay kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's protocol.

Library Construction and Sequencing
One hundred nanogram of DNA from each sample was sheared by the dsDNA Fragmentase (New England BioLabs, Inc., Ipswich, MA, USA), and then performed size selection (150-250 bp) using Ampure XP beads (Beckman Coulter, Inc., Brea, CA, USA). Library construction was performed using the KAPA Library Preparation kit (Kapa Biosystems, Inc., Wilmington, MA, USA) according to the manufacturer's protocol. The concentration of the library were assessed using the e Qubit dsDNA HS Assay kit, and fragment length was determined on a 4200 Bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA, USA). Target enrichment was carried out using the Agilent SureSelect XT HS kit (Agilent Technologies) according to the manufacturer's Protocol. DNA sequencing was then performed on the Illumina Novaseq 6000 system at an average depth of 2000X.

Sample Collection and Patient Characteristics
Of the 1349 NSCLC cases, ALK rearrangements were detected in 44 cases (3.26 %). Those 44 Chinese patients with locally advanced or metastatic NSCLC were enrolled in this study, of which 20 (45.5%) were female. All patients carry ALK fusion events. Their mean age was 52.5 with ranging from 29 to 73. NGS was performed on 44 pairs of tumor and white blood cell samples. All the samples that passed the histology quality control (HQC) yielded sufficient amounts of DNA for NGS.

Identification of ALK Rearrangements Using Targeted Sequencing
In order to identify ALK rearrangement from the DNA of patients' FFPE samples, we designed probes to cover the intron 18 and intron 19 of ALK, as well as introns of some well-known ALK fusion partners. We identified ALK rearrangements and corresponding breakpoints in the sequencing data of these patients. The statistical summary and breakpoints of the rearrangement events are listed in Table 1 and shown in Figures 1, 2, respectively. We found that 43 out of 44 patients had an EML4-ALK fusion, with variant 1 (v1, E13:A20), variant 2 (v2, E20:A20), and variant 3 (v3, E6:A20) detected in 18, 5, and 13 patients, respectively. We also identified one novel ALK fusion partner HMBOX1.
We further studied their mutational signatures. We observed that C>T transition occurred most frequently, followed by C>A transition (Figure 4). This pattern is consistent with COSMIC signature 1 that had been found in most cancer samples.
Different driver gene mutations revealed inter-tumor heterogeneity. TP53 mutations in exon 5-8 were observed, and we further elaborately portrayed the TP53 mutation sites on the peptide sequence in a lollipop plot (Figure 5).

DISCUSSION
In this study, we identified ALK rearrangement events in 44 Chinese NSCLC patients using NGS technologies. Consistent with other studies, the most common ALK fusion partner is EML4, and the fusion occurs in the forms of the three most common variants. We also report a novel ALK fusion partner HMBOX1. It implied that NGS-based assessment for ALK fusions was accurate and comprehensive, having the unique advantages in detecting unknown ALK fusion partners, and identifying the exact breakpoints compared to the traditional methods, like FISH and IHC.
At the same time, we characterized the mutational profiles of the patients. The results were consistent with other studies, in terms of the relatively lower frequency of TP53 mutations, lower TMB, and fewer co-mutations compared to ALKnegative NSCLC patients (29). Besides, we identified the copy number alterations in their genome. Apart from the genes with a high frequency of copy number amplification, such as MET, MDM2, and TERT, we also identified some genes with copy number loss, such as CD274, CDKN2A, and JAK2. This information is important for guiding optimal clinical treatment. For instance, the copy number loss of CD274 probably indicates a low expression of PD-L1. MDM2 amplification had been reported to associate with a poor clinical outcome and significantly increased tumor growth rate with anti-PD-1/PD-L1 immunotherapies (30).
In conclusion, using our customized HapOncoCDx panel, we not only successfully detected the ALK fusion events in 44 Chinese NSCLC patients, but also explored their genomic mutational landscapes. To the best of our knowledge, this is the first study that exhibited the mutational landscape of Chinese NSCLC patients with ALK rearrangement. This result can provide genomic information for personalized clinical FIGURE 6 | Copy number aberrations in 13 ALK fusion positive NSCLC patients. The names of the aberrant genes are shown in the y-axis, while each column represents a patient. The type of copy number aberrations, including gain, normal, and loss are indicated by red, yellow, and blue, respectively. management for patients with ALK fusion in the era of precision medicine.

DATA AVAILABILITY STATEMENT
The raw sequence data reported in this paper have been deposited in the Genome Sequence Archive in BIG Data Center, Beijing Institute of Genomics (BIG), Chinese Academy of Sciences, under accession numbers HRA000138, HRA000138 that can be accessed at http://bigd.big.ac.cn/gsa-human.

ETHICS STATEMENT
This study was approved by the ethics committee of the Second XIANGYA Hospital of Central South University and complied with Good Clinical Practices, the principles of the Declaration of Helsinki and all applicable regulatory requirements. All patients provided written informed consent prior to any studyspecific procedures.

AUTHOR CONTRIBUTIONS
SL, TH, SG, and SC designed the study. WH, YZ, LY, YLo, DZ, HZ, and YLi carried out the sequencing experiment and collected data. TH, YZ, and WL performed the bioinformatics analysis. TH, SL, and ML wrote this manuscript. JD and SC revised this manuscript. SG and SC supervised the study.