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Comprehensive analysis of genomic alterations detected by next-generation sequencing-based tissue and circulating tumor DNA assays in Chinese patients with non-small cell lung cancer

While tumor genotyping is the standard treatment for patients with non-small cell lung cancer (NSCLC), spatial and temporal tumor heterogeneity and insufficient specimens can lead to limitations in the use of tissue-based sequencing. Circulating tumor DNA (ctDNA) fully encompasses tumor-specific sequence alterations and offers an alternative to tissue sample biopsies. However, few studies have evaluated whether the frequency of multiple genomic alterations observed following ctDNA sequencing is similar to that observed following tissue sequencing in NSCLC. Therefore, in the present study, targeted next-generation sequencing (NGS) was performed on tissue and plasma ctDNA samples in 99 patients with NSCLC. Overall, the frequencies of genetic alterations detected in ctDNA were positively correlated with those detected via tissue profiling (r=0.812; P=0.022). Genomic data revealed significant mutual exclusivity between alterations in epidermal growth factor receptor (EGFR) and tumor protein 53 (TP53; P=0.020), and between alterations in EGFR and KRAS (P=0.008), as well as potential mutual exclusivity between alterations in EGFR and Erb-B2 receptor tyrosine kinase 2 (P=0.059). Furthermore, the EGFR mutant allele frequency (MAF) was positively correlated with the TP53 MAF in individual tumors (r=0.773; P=0.005), and there was a marked difference in the EGFR MAF between patients with and without the TP53 mutation (P=0.001). Levels of the tumor serum marker CA242 in patients with ctDNA-detectable mutations were higher compared with those in patients without ctDNA-detectable mutations. The data from the present study highlight the importance of tissue and plasma ctDNA screening by NGS to guide personalized therapy and promote the clinical management of patients with NSCLC.


Introduction
Non-small cell lung cancer (NSCLC) is a leading cause of cancer-associated mortality worldwide, accounting for ~85% of lung cancer cases (1,2). An estimated 1.8 million new lung cancer cases and 1.6 million lung cancer-associated mortalities were reported worldwide in 2012 (3). Advances in genomic research and targeted therapy have greatly improved the development of therapeutic strategies and the clinical outcomes of patients with NSCLC (4,5). Activation of specific tyrosine kinases, including those derived from epidermal growth factor receptor (EGFR) mutations or the rearrangement of the anaplastic lymphoma kinase (ALK) gene, promotes development in the corresponding molecular inhibitors in EGFR mutation and ALK translocation positive subgroups (6)(7)(8). In addition to EGFR and ALK, a number of other oncogenic driver genes, such as ROS1, BRAF, MET, RET and Erb-B2 receptor tyrosine kinase 2 (ERBB2) have been identified as relevant targets in NSCLC (9).
Next-generation sequencing (NGS) has led to considerable advances in the comprehensive analysis of genomic alterations in cancer research and clinical application (10,11). At present, the detection of genomic alterations in tumors is primarily reliant on cancer tissue analysis. Despite the benefits and therapeutic insights offered by tissue sample testing, spatial and temporal tumor heterogeneity, and insufficient and unacquirable specimens can lead to limitations in the use of tissue-based sequencing (12,13). Circulating tumor DNA (ctDNA), released into the plasma from apoptotic and necrotic tumor cells derived from primary tumors and metastatic lesions, are comprised of tumor-specific sequence alterations and may be an alternative target to tissue sample biopsies (14)(15)(16). An increasing body of evidence has indicated that ctDNA testing could be utilized for patients without acquirable tissue samples to screen for genetic variations and thus guide treatment decisions in patients with NSCLC (17)(18)(19).
Despite the increasing utilization of plasma ctDNA mutations in guiding clinical decisions, few studies have evaluated whether the frequency of multiple genomic alterations observed following ctDNA sequencing is similar to that observed following tissue sequencing in NSCLC (17,20). Therefore, in order to investigate the associations in the frequency of comprehensive genomic alterations between ctDNA and tissue profiling in Chinese patients with NSCLC, the present study analyzed 59 plasma ctDNA samples and 40 tissue samples via targeted NGS. The results provide important insights into the potential benefits of ctDNA profiling in patients with NSCLC in clinical practice.

Materials and methods
Patients and sample collection. The present study enrolled 99 patients with NSCLC who were baseline or previously treated at the Hunan Cancer Hospital (Changsha, China) between September 2017 and April 2018. The specimens included 59 plasma and 40 tissue samples. The inclusion criteria for the patients were as follows: i) Patients were diagnosed with NSCLC by histopathology or cytology; ii) tissue specimens were confirmed by qualified pathologists, from Hunan Cancer Hospital who were blinded to the study and ensured >30% tumor content; and iii) at least 150 ng and 15 ng DNA from each tissue and ctDNA sample, respectively, were successfully extracted. Patients who had other malignant tumors and serious mental illness were excluded. Written informed consent was provided by all participants. All experiments were approved by and performed in accordance with the relevant guidelines and regulations of the Committee of Medical Ethics of Hunan Cancer Hospital.
Targeted deep sequencing. ctDNA was isolated from ≥2 ml plasma with a QIAamp Circulating Nucleic Acid kit (Qiagen GmbH) according to the manufacturer's protocol. Tissue DNA was extracted using the QIAamp Genomic DNA kit (Qiagen GmbH). The quality of the DNA and DNA quantification were assessed using the Agilent 2100 BioAnalyzer (Agilent Technologies, Inc.) and Qubit ds DNA HS assay kit (Thermo Fisher Scientific, Inc.).
Sequencing libraries were constructed according to the Illumina standard library construction instructions (Illumina, Inc.). Genomic DNA extracted from tissue samples was first sheared to 250 bp with an ultrasonoscope. The fragmented DNA was subjected to end-repairing, A-tailing and ligation to the adapters with barcode sequences. Subsequently, PCR was performed and the resulting products were purified with AMPure XP magnetic beads (Agencourt AMPure XP kit; Beckman Coulter, Inc.) according to the manufacturer's protocol. The various libraries were then hybridized with a nine-gene panel, which was enriched for the coding regions and selected introns of genes with known relevance to NSCLC. The target-enriched libraries were then pooled and sequenced on an Illumina HiSeq2500 NGS platform (Illumina, Inc.). The sequencing depth was >10,000x. Reads were aligned to the human genomic reference sequences (hg19) using the Burrows-Wheeler alignment (BWA) tool (21). Local realignment and base quality score recalibration were conducted using GATK software (version 2.3; software.broadinstitute. org/gatk) (22). MuTect2 (version 1.1.1; software.broadinstitute. org/cancer/cga/mutect) with the recommended parameters was used to identify single-nucleotide variants (SNVs) and small insertions or deletions (INDELs) (23). Copy number variants (CNV) calling was performed with CONTRA software (version 2.0.4; contra-cnv.sourceforge.net) (24).
The cancer genome Atlas (TCGA) datasets. Multiplatform genomics data was previously published in The Cancer Genome Atlas (TCGA; cancergenome.nih.gov). In total, 1,144 NSCLC cases comprising 660 patients with lung adenocarcinoma (AD) and 484 patients with lung squamous cell carcinoma (SC) were included in TCGA cohort with gene mutation data (25).

Detection of tumor serum markers.
Tumor serum markers were detected by a C12 multi-tumor marker protein chip system (Shanghai HealthDigit Co., Ltd.). Based on the manufacturer's instruction, 100 µl of the different calibrators, quantitative quality control products and serum specimen were transferred to the reaction well of the chip, which was subsequently incubated at 37˚C for 30 min with agitation. The mixture was discarded and the well was washed four times with the wash solution. Next, 100 µl reaction solution was added to the well and incubated at 37˚C for 30 min with agitation. The mixture was discarded and the well was washed four times with the wash solution. Subsequently, detection solution A and B were added to the surface of the chip and incubated at room temperature for 1.5 min. The chip was placed into the biochip scanner (HD-2001A; Shanghai HealthDigit Co., Ltd.) to analyze each tumor serum marker.
Statistical analysis. SPSS statistical software (version 21.0; IBM Corp.) was used to analyze the data. Differences in continuous variables were assessed using unpaired Student's t-test. Associations between different genotypes and clinical characteristics were analyzed using Fisher's exact test or χ 2 test. Correlations between variables were assessed using Spearman's rank correlation coefficient. Patterns of co-alteration or mutual exclusivity between EGFR and other driver genes were analyzed by Fisher's exact test. The odds ratio (OR) is a statistic that quantifies the strength of the association between two events. In the current study, EGFR mutations and other driver gene mutations were classified as events A and B, respectively. The odds ratio was calculated as the ratio of the odds of A in the presence of B and the odds of A in the absence of B. P<0.05 was considered to indicate a statistically significant result.

Results
NGS-based tissue and ctDNA assays to identify genomic alterations among 99 patients with NSCLC. In the present study, a total of 99 patients diagnosed with NSCLC were enrolled. NGS was performed for specimens, including 59 plasma ctDNA and 40 tissue samples. Among the patients, the median age at diagnosis was 56 years (range, 39-83 years), 73.7% of the patients were male, 64.6% of the patients were smokers, 58.6% of the patients were diagnosed with AD, and 67.7% of the patients had stage IV cancer. The clinical and pathological characteristics of the patients are listed in Table I. All 99 samples were profiled by targeted sequencing with a panel of nine of the most common driver genes [EGFR, ALK, ROS1, BRAF, MET, RET, ERBB2, KRAS and tumor protein 53 (TP53)] in NSCLC. A total of 38 out of 40 (95.0%) tissue samples and 38 out of 59 (64.4%) ctDNA samples exhibited at least one genetic alteration. TP53 was the most commonly mutated gene (56.6%), followed by EGFR (36.8%). Compared with TCGA data, significantly more somatic mutations were observed in EGFR and ERBB2 (P<0.001), and significantly fewer mutations in TP53 (P=0.045) were identified among the cohort of Chinese patients with NSCLC (Table II). Although the KRAS mutation rate according to TCGA was higher than that of the cohort in the present study, no significant difference was observed (P= 0.179; Table II). Further analysis demonstrated that the frequency of TP53 alterations was markedly higher in SC than in AD (P= 0.009; Table III), and the frequency of EGFR alterations was associated with being female, AD and non-smokers (P<0.001, P=0.001 and P<0.001, respectively; Table III). KRAS alterations were more likely to occur in male patients and smokers, although no statistically significant differences were observed (P=0.086 and P=0.105, respectively). ERBB2 alterations were more frequently observed in non-smokers (P<0.001). Furthermore, in the present study, no ERBB2 alterations were observed in patients with localized NSCLC, and statistical analysis revealed that ERBB2 alterations were potentially associated with metastatic stage in NSCLC (P=0.108; Table III).   Overall, 50.5% (50/99) of the patients with NSCLC harbored at least one targetable alteration (data not shown). Regarding pathological type, the frequency of the targetable alterations between patients with AD (63.8%) and SC (30.6%) were significantly different (P= 0.002; Table IV). The landscape of genomic mutations demonstrated that an EGFR mutation was seen alongside each other gene (MET, ERBB2, KRAS and ALK) mutation in only one patient each (1%). In addition, the present study demonstrated that EGFR amplification coexisted with EGFR mutations, including SNVs and INDELs, but was mutually exclusive with the EGFR T790M mutation. Furthermore, no patients with concurrent ERBB2 mutation and amplification were observed. All four patients that possessed the EGFR T790M mutation also harbored other EGFR mutations (Fig. 1). To investigate the patterns of concurrent alterations and mutual exclusivity, pairwise associations between somatic events were examined. Mutual exclusivity was observed between alterations in EGFR and TP53 [odds ratio (OR)= 0.324; P= 0.020; Table V] and between those in EGFR and KRAS (OR= 0.091; P= 0.008) in patients with NSCLC. Furthermore, potential mutual exclusivity between EGFR and ERBB2 alterations was also observed, but this was not statistically significant (OR=0.161; P=0.059; Table V).
Comparison of the frequency of genomic variants between tissue and ctDNA samples. In order to investigate the use of ctDNA sequencing, the present study compared the prevalence of genomic alterations, including non-synonymous SNVs, CNVs and rearrangements, between tissue and ctDNA samples. Overall, there were marked similarities in the frequency of variants when comparing the tissue and ctDNA samples ( Fig. 2A). Further analysis indicated that the prevalence of alterations among the most common NSCLC driver genes in ctDNA was positively correlated with that observed in tumor tissues (r=0.812; P=0.022; Fig. 2B).

Association between EGFR and TP53 mutant allele frequencies (MAFs).
Evolutionary studies of NSCLC have demonstrated that TP53 and EGFR mutations are the two most dominant clonal mutations (26); thus, the present study hypothesized that there may be a correlation between the MAFs of TP53 and EGFR in individual patients. As expected, a significantly linear relationship was observed (r=0.773; P=0.005; Fig. 3A). However, when the MAFs of TP53 and KRAS were compared, no linear correlation was identified (r=0.700; P=0.233; Fig. 3B). In addition, the present study observed significant differences in the MAF of EGFR between patients with and without TP53 mutations (P=0.001; Fig. 3C). By contrast, no clear difference in the MAF of TP53 was observed between patients with and without EGFR mutations (P=0.122; Fig. 3D).

Discussion
Through NGS, the present study comprehensively analyzed genomic alterations in tissue and plasma ctDNA samples derived from Chinese patients with NSCLC. In the present study, the associations between the frequencies of several driver gene alterations and clinical characteristics were observed. Of note, TP53 alterations were associated with SC, ERBB2 alterations were associated with smoking, and KRAS alterations seemed to primarily arise in male or smoking patients with NSCLC. In addition, ERBB2 alterations, including somatic  mutations and CNVs, were only encountered in patients exhibiting metastasis, implying that aberrations in ERBB2 may occur later during NSCLC progression. The differences in the prevalence of several driver gene mutations (such as EGFR, ERBB2 and TP53) between Chinese and TCGA cohorts was indicative of the specificity of the gene mutational spectrum in Chinese patients with NSCLC. Furthermore, although the frequency of targetable alterations in patients with AD was markedly higher than in patients with SC, the data from the present study demonstrated that a proportion of patients with SC could potentially benefit from targeted therapy and that the detection of genomic alterations in patients with SC is therefore required.
Multiple resistance mechanisms against first-generation EGFR-TKI treatment, including EGFR amplification, the EGFR T790M mutation and ERBB2 alterations, have been described previously (32). Based on the genomic landscape, the present study demonstrated that EGFR amplification coexisted with EGFR somatic mutations, but was mutually exclusive with EGFR T790M mutations; there were no cases of simultaneous ERBB2 somatic mutations and amplification. This implies that patients with NSCLC may not exhibit mutations and amplification of the same genes in resistance to EGFR-TKI. Further analysis revealed that an EGFR mutation was seen alongside each other gene (MET, ERBB2, KRAS and ALK) mutation in only one patient each, demonstrating the importance of multi-gene panel testing in NSCLC. However, no co-occurrence of EGFR alterations with RET or BRAF alterations was observed. The statistical analyses revealed that EGFR alterations were significantly mutually exclusive with TP53 or KRAS alterations and potentially mutually exclusive with ERBB2 alterations. These findings are indicative of mutual exclusivity between EGFR alterations and alterations in other driver genes in NSCLC.
A previous study reported that EGFR and TP53 are the two dominant clonal mutant genes in NSCLC (26). The significant linear correlation between the MAFs of TP53 and EGFR in individual patients further confirmed that TP53 and EGFR mutations regularly co-occur as clonal events in the evolution of NSCLC. Notably, marked differences in the EGFR MAFs of patients with and without TP53 mutations were observed. A recent study suggested that the abundance of EGFR-activating mutations was correlated with the efficacy of EGFR-TKIs in advanced NSCLC (33). According to the results of the present study, there may be differences in the therapeutic effect of EGFR-TKIs in patients with EGFR-activating mutations with and without TP53 mutations. Another finding was the difference in the number of mutations among baseline tumors with and without TP53 mutations. Several studies have reported an association between the number of mutations and prognosis, or between mutations in specific genes and prognosis (27)(28)(29)(30)(31). As the present study did not obtain complete information on the therapeutic effect and prognosis in a large proportion of patients, determination of the efficacy of EGFR-TKIs and prognosis in tumors with and without TP53 mutations could not be assessed.
At present, the detection of genomic alterations primarily relies on the analysis of cancer tissues obtained via surgical excision or biopsy. However, tumor tissues are not always available for all patients, and tissue biopsy of a single site may not fully reflect the tumor genomic landscape due to tumor genetic heterogeneity (34). Therefore, in the present study, the use of plasma ctDNA was assessed for patients without sufficient tissue samples to screen for genomic alterations to guide personalized therapy. In the analysis of the ctDNA and tissue profiles, the landscape and frequencies of genomic alterations in ctDNA samples demonstrated a strong similarity with those observed in tissue samples, supporting the feasibility and use of plasma ctDNA testing among patients with NSCLC. The association between the frequency of plasma ctDNA-detectable mutations and clinical characteristics was also assessed in the present study, which revealed that the frequency of ctDNA-detectable  mutations was significantly associated with clinical stage, consistent with previous results (15). In addition, it was observed that serum CA242 levels were significantly higher in patients with ctDNA-detectable mutations when compared with patients without ctDNA-detectable mutations. A prior study reported that serum CA242 levels were associated with clinical stage in NSCLC (35). Therefore, the present study provides a novel line of evidence confirming the prior result that ctDNA-detectable mutations are associated with clinical stage in NSCLC.
In conclusion, the present study identified targetable genetic alterations in 50.5% of patients with NSCLC using targeted NGS and discovered significant correlations between the frequency of genomic variants in tissue and plasma ctDNA samples. In addition, the results of the present study demonstrated that the MAF of EGFR was associated with that of TP53 in individual tumors and that the EGFR MAF in patients with the TP53 mutation was different to that observed in patients without the TP53 mutation. Although further studies are required to confirm these findings, these findings provide an improved understanding of the spectra of genomic alterations detected by tissue and plasma ctDNA assays in Chinese patients with NSCLC.