Association of Next Generation Sequencing and Routine Testing with Survival of Advanced Non-Small Cell Lung Cancer Patients in Clinical Practice

Background: The rapid development of detection technologies has resulted in an increased use of next generation sequencing (NGS) in advanced non-small cell lung cancer (NSCLC) patients. Here, we investigated the clinical benets of different detection technologies for patients with advanced lung cancer in a real world setting. Methods: This retrospective study enrolled advanced NSCLC patients diagnosed between January 1, 2013 and June 30, 2019 in Zhejiang Cancer Hospital who received genetic testing. Patients received either NGS or routine testing (EGFR and/or ALK and/or ROS1 only). Although different panels were used for NGS, they all included nine genes mentioned as molecular biomarkers in the NCCN guidelines (EGFR, ALK, ROS1, KRAS, HER2, BRAF, MET, RET, and NTRK). Clinical outcomes were compared between advanced NSCLC patients who received NGS and those undergoing routine testing. Overall survival (OS) and survival rates were analyzed. Propensity score analysis was used to prevent survival bias associated with patient characteristics. Results: Of 6,451 patients with advanced NSCLC included in the analysis, 5,666 (87.8%) patients underwent routine testing and 785 (12.2%) were tested by NGS. The median OS was 22.6 months and the 5-year survival rate was 22.89%. Comparison of OS showed an absolute difference between the two cohorts according to unadjusted survival curves (24.8 months vs. 22.1 months, P=0.0097), whereas no difference in OS was observed using propensity score analysis (P=0.533). The 1-year survival rate was signicantly higher in patients tested by NGS than in those undergoing routine testing (82.0% vs. 70.7%, P=0.0004). In patients with uncommon mutations and in those receiving targeted therapies, propensity score analysis showed that the 1- and 2-year mortalities were signicantly lower in the NGS group than in the routine testing group without mutations. This could be due to the higher rate of patients receiving targeted therapy (68.0% vs. 53.6%; P=0.042) and off-label drugs


Background
The incidence of lung cancer is increasing worldwide, and non-small cell lung cancer (NSCLC) is one of the main pathological types of lung cancer [1]. NSCLC is classi ed into two major histological subtypes, adenocarcinoma and squamous-cell carcinoma, and adenocarcinoma accounts for approximately 85% of lung cancer cases [2]. The emergence of targetable oncogenic driver alterations, most notably epidermal growth factor receptor (EGFR) activating mutations and anaplastic lymphoma kinase (ALK) and ROS1 gene rearrangements, has transformed NSCLC treatment models by incorporating tumor genotyping into therapeutic strategies [3][4][5][6]. The response rate of drugs targeting cancers harboring driver mutations is > 70%, and these drugs have consistently improved patient survival outcomes. As a result, molecular testing for EGFR, ALK, and ROS1 has been recommended for all adenocarcinoma NSCLC patients since the early days of individualized precision medicine.
Advances in gene detection technology and the rapid development of new drugs have led to the identi cation of a growing number of driver gene mutations such as ret proto-oncogene (RET) rearrangements, neurotrophic tyrosine receptor kinase 1 (NTRK1), ERBB2 (HER2), and BRAF mutation, MET gene alterations, and other examples of actionable alterations with proven therapeutic options [7][8][9][10][11][12]. Companion diagnostic tests for gene mutations that impact the success of anticancer drugs were conducted separately for each mutation, which detected EGFR, ALK, and ROS1 as the main genes. Although the incidence of rare driver gene mutations (mutation rate < 5%) such as those of MET, BRAF, NTRK, and HER2 is relatively low and may vary according to ethnic differences, agents targeting these driver mutations are being developed, and the number of emerging biomarkers and targets continues to grow. Targeted therapy is associated with a survival bene t in these patients. Consequently, and increasing number patients undergo multigene sequencing of tumors to identify genomic alterations that can be effectively targeted, and many clinicians are using next generation sequencing (NGS) methods to detect gene mutations associated with lung cancer treatment.
Studies have demonstrated the feasibility of routine NGS detection in patients with NSCLC, and showed that genetic testing using NGS is useful for the design of lung cancer treatment strategies [14,15]. However, routine testing may not be su cient because ethnicity is an important factor determining the response to therapy and outcomes [16]. Presley et al. [17] compared survival outcomes between advanced NSCLC patients who received broad-based genomic sequencing and those who received routine testing for EGFR mutations and/or ALK rearrangements alone in the Community Oncology Setting. They showed that broad-based genomic sequencing provided direct information on treatment in a minority of patients. Singal et al. [18] assessed whether a database with comprehensive genomic pro ling could identify and extend associations in NSCLC, and the results demonstrated the feasibility of creating a clinicogenomic database derived from routine clinical experience. Therefore, whether NGS can bring survival bene ts to patients compared with routine testing remains controversial. The paucity of appropriate studies and the incomplete understanding of genomic architecture across Chinese populations are additional challenges. In this study, routine testing (EGFR and/or ALK and/or ROS1 only) was compared with NGS panel testing to identify the optimal strategy. We analyzed the difference in the survival bene t between advanced NSCLC patients who used NGS and those undergoing routine testing as well as the potential effect of each test on therapeutic strategies.

Study design
This retrospective cohort study used the Database of Zhejiang Cancer Hospital to identify patients diagnosed with stage IIIB/IV advanced NSCLC who received antineoplastic treatment and either next generation sequencing (NGS) or routine testing (EGFR and/or ALK and/or ROS1 only). The histological classi cation of NSCLC was based on the World Health Organization criteria (2015 version) [19]. Various panels for NGS were used, but all of them included the nine genes used as molecular biomarkers in the National Comprehensive Cancer Network (NCCN) guidelines list of driver mutations (EGFR, ALK, ROS1,  KRAS, HER2, BRAF, MET, RET, and NTRK). Propensity score-matched survival analysis was used to evaluate differences in overall survival (OS) between the groups.

Study sample
Advanced NSCLC patients who were diagnosed between January 1, 2013 and June 30, 2019 and had genetic testing results were included in the study. The cohort included advanced NSCLC patients or earlystage NSCLC that subsequently developed recurrent or progressive disease during the period. Each patient had a diagnosis of lung cancer, at least two documented clinical visits on or after January 1, 2011, pathology consistent with non-squamous NSCLC, and con rmation of advanced NSCLC on or after January 1, 2011. Patients with evidence of other concurrent active cancers within 6 months prior to diagnosis of advanced NSCLC, other than non-melanoma skin cancer, were excluded. Patients with NGS testing were considered the treatment group, whereas patients with routine testing for EGFR/ALK/ROS1 were considered the control group as standard-of-care group in this study. Patients who met the following inclusion criteria were included in the study: 1) recorded clinicopathological information, including age and sex, and clinical data, such as pathological typing, genetic testing results and treatment type, 2) pathological examination of tumor specimens with proven records of at least one common driver gene test (EGFR or ALK or ROS1), 3) all patients received at least one line of systemic antineoplastic treatment for advanced NSCLC. Patients were excluded if: (1) clinical data including age, gender, and stage were missing, (2) pathologic examination showed NSCLC NOS or small cell lung cancer, (3) follow-up survival results were missing.

Outcomes
The last follow-up date of the study was June 30, 2020. OS was de ned as the time from the start of rstline treatment to the date of death. Analysis was performed using 12-month or 24-month mortality. OS was used as the outcome in the propensity score-matched survival analysis. Secondary outcomes included genetic alterations and treatments received.

Statistical analysis
The χ2 test was used for comparison of the distribution of cohort characteristics and treatment types between the NGS group and routine testing group. Propensity score matching was used to address potential confounding due to the signi cantly different cohort characteristics. Multivariable logistic regression analysis was used to estimate the propensity score based on age, sex, smoking status, stage, year of diagnosis, EGFR or ALK mutation, comorbidity, a single line of treatment, and immunotherapy in line 1 or line 2 vs. line 3 or line 4 of treatment. The quality of data for each variable was considered before their inclusion in propensity score-matched survival analysis. According to the study by Presley et al. [17] and to prevent survival bias caused by certain patient characteristics, six covariates were set as exact covariate matches as follows: EGFR or ALK mutation, receipt of only one line of treatment, year of diagnosis, receipt of immunotherapy in line 1 or line 2 of treatment, and receipt of immunotherapy in line 3 or line 4 of treatment. For the variables with missing data (smoking status, and EGFR or ALK mutation), a dummy variable was used because the missing variables were not missing at random. One-to-one matching was performed using a nearest neighbor algorithm with ratio of 1 and a caliper of 0.01 standard deviation. Survival outcomes were estimated and compared by the Kaplan-Meier method (time 0 = start of rst line treatment) and the log-rank test. Median survival and hazard ratio (HR) (log rank) and 95% con dence interval (CI) were reported. All statistical tests were two-sided, and a P value <0.05 was considered statistically signi cant. R version 3.6.0 (R-project, Institute for Statistics and Mathematics: packages MatchIt V3.0.2) was used for all analyses.

Baseline characteristics
Of 6,451 patients diagnosed with advanced NSCLC, 85 (12.2%) received NGS testing ( Table 1). The median age at diagnosis was 61 years (range, 19-90 years), 45.2% of patients had a history of smoking, and 41.6% of patients were female. In this cohort, 95.8% of patients received EGFR testing and 95.3% received ALK testing (in <5% of the remaining samples, the testing status of both genes was unknown). There was a signi cant association between immunotherapy in the rst four lines of treatment and NGS testing.

Survival analysis
All survival analyses were performed using the Kaplan-Meier method. For the entire cohort, the median OS was 22.6 months and the 5-year survival rate was 22.89%. There was a signi cant difference in survival between patients who received targeted therapy and those who did not in the unadjusted survival curves (HR, 0.52 [95% CI, 0.49-0.56], log-rank P < 0.0001, Figure 1A) or among the 2177 matched pairs with wellmatched characteristics and balance in the propensity score analysis (HR, 0.56 [95% CI, 0.52-0.60], logrank P < 0.0001, Fig. 1B).
Survival of patients with NGS testing and routine testing OS was compared between patients who underwent NGS testing and those who underwent routine testing. In the unadjusted survival curves, there was an absolute difference between the two cohorts (HR, 0.87 [95% CI, 0.78-0.96], log-rank P = 0.0097, Fig. 2A), especially in the 12-and 24-month mortality rates. In the propensity score analysis, there was no obvious OS difference between the two cohorts (760 matched patients, HR, 0.95 [95% CI, 0.82-1.11], log-rank P = 0.53, Fig. 2B). Patients who received NGS had a signi cantly higher 1-year survival rate than that of patients with routine testing (HR, 0.66 [95% CI, 0.53-0.83], log-rank P = 0.0004), as well as a higher rate of patients receiving targeted therapy (68.0% vs. 53.6%, P < 0.0001, Fig. 3A/B) and patients receiving off-label drugs (28.10% vs. 11.40%, P < 0.0001, Fig.  3C/D). The NGS testing cohort had a greater number of different types of off-label drugs.
Next, the patients were divided into four groups: patients with uncommon mutations who received NGS, those who received routine testing, those who received targeted therapies, and those who did not receive targeted therapies. The results of unadjusted survival curves showed that the median OS was longer in the two groups with targeted therapies, especially in the groups with NGS testing (Supplementary Fig. 2).
Among uncommon mutation patients with targeted therapies, propensity score analysis showed that the 12-and 24-month mortality was signi cantly higher in the NGS testing group than in the routine testing group (Supplementary Fig. 2). This suggested that NGS was a better detection method for patients with uncommon mutations. This could be associated with the fact that the rate of off-label treatments was signi cantly higher in the NGS group than in the routine testing group (45.4% vs. 9.6%, P < 0.0001, Supplementary Fig. 3), and indicated that NGS testing resulted in a more objective and precise clinical therapy for uncommon mutation patients.

Discussion
Two different survival analysis methods were used to show that NGS detection technology based on accessible targeted therapy could extend the overall survival of advanced NSCLC patients by nearly 3 months compared with routine detection technology including EGFR and/or ALK and/or ROS1 in the Chinese population. In particular, the 1-year survival rate of patients undergoing NGS testing was signi cantly higher than that of patients undergoing routine testing. Propensity score analysis con rmed the signi cant difference in the 1-year survival rate between the two groups. These differences may be attributed to the higher proportion of patients receiving targeted therapy and off-label treatment in the NGS group than in the routine testing group. To the best of our knowledge, this is the rst presentation of real world clinical data supporting that NGS is superior for clinical molecular pro ling of advanced NSCLC patients, which may improve the survival of patients with advanced NSCLC in the Chinese population. However, we also need to consider the availability of targeted drugs in China.
Genotyping is an essential step for the selection of targeted regimens in lung cancer, especially in lung adenocarcinoma. The survival bene ts of targeted therapy are higher than those of chemotherapy in patients with lung cancer. The present results showed that there was a signi cant difference in survival between patients with and without targeted therapy. The median OS of NSCLC patients treated with targeted therapy was 30 months, and the 5-year survival rate was 29.6% in this study. These ndings are consistent with the results of previous studies [14,18] and suggested that driver and genotype-directed therapy improved the survival of patients. Comprehensive survival analysis of patients receiving NGS and routine testing was performed to explore the difference between the two techniques in clinical practice. The results showed a difference in OS between patients detected by NGS and routine testing; the OS of NSCLC patients in the NGS group was 2.7 months longer than that of the routine detection group (P = 0.0097). In particular, the 1-year survival rate was signi cantly higher in the NGS group than in the routine testing group (82.03% vs. 70.76%, P<0.0001). This may be related to the obviously higher rate of patients who received targeted therapy. However, propensity score analysis showed no difference in OS between the two groups, and the 5-year survival did not differ signi cantly, which was consistent with previous data [14]. The use of NGS in advanced NSCLC patients is already fully endorsed in many international guidelines. According to previous reports, the value of routine testing and NGS testing is still controversial. Singal et al. [18] demonstrated the feasibility of creating a comprehensive genomic pro ling derived from routine clinical experience and the use of large-scale clinicogenomic data sets can augment various stages of drug development. Presley et al. [17] demonstrated that broad-based genomic sequencing was not associated with better survival compared with routine testing among advanced NSCLC patients receiving treatment. Similar results were obtained in the present study, which showed no obvious OS difference using propensity score analysis (P = 0.53). This may indicate that the overall survival bene t of NGS in lung adenocarcinoma patients is not large, and a speci c population is necessary to regulate the detection by NGS.
NGS may increase the opportunities for targeted therapies. Wu et al. [20] described the Pan-Asian guidelines established by ESMO and the Chinese Society of Clinical Oncology recommending testing of all advanced non-squamous NSCLC patients for EGFR mutation, ALK rearrangement, ROS1 rearrangement, and BRAF mutation. The IALSC Updated Molecular Testing Guidelines recommend testing for EGFR, ALK, and ROS1 along with the inclusion of additional genes (ERBB2, MET, BRAF, KRAS, and RET) for NGS panels [7]. These ndings suggest that NGS testing is becoming increasingly important for con rming the genetic features of patients, and effective targeted drugs may be needed to demonstrate the improved outcomes of NSCLC patients using NGS. However, only three categories of targeted drugs (EGFR, ALK, and ROS1) have been approved in China, and access to other drugs is limited. In this study, NGS testing was not superior for guiding targeted therapy in second-and third-line treatments, which may be due to the limited targeted drugs against EGFR mutations or ALK fusions that can be easily checked by routing testing. NGS is used in second-and third-line treatments to explore drug resistance mechanisms, as the corresponding targeted drugs are limited. Therefore, the proportion of off-label patients increased after NGS testing in China. In addition, the 36-month mortality and OS did not differ signi cantly between the NGS testing and routine testing groups, suggesting that not all targeted drugs offered an OS bene t. Because off-label medication is usually selected after the failure of standard treatment, the patient bene t from off-label drugs may be lower than that of rst line treatment.
NGS testing can identify speci c mutations that cannot be found by routine testing. In the present study, the OS of uncommon mutation patients and that of patients receiving targeted therapies with NGS testing was signi cantly longer than that of patients with routine testing. On the other hand, the rate of off-label treatments was signi cantly higher in the NGS than in the routine testing group (45.4% vs. 9.6%; P < 0.0001), suggesting that NGS testing resulted in a more objective and precise clinical therapy for patients with uncommon mutations. In addition, NGS may help patients with speci c mutations participate in clinical trials (9.6% vs. 3.9%). Zehir et al. [13] established a large-scale, prospective clinical sequencing initiative using a comprehensive assay (MSK-IMPACT trial), and found that only 11% of patients were enrolled in genomic-matched clinical trials among 10,000 patients. In the present study, the proportion of patients matched to clinical trials based on NGS was 9.6%, which was slightly lower than that of the previous study. This may be because clinical trials in China were somewhat similar to foreign community models. The study of Presley et al. [17] focused mainly on community-based practice, and demonstrated that the number of patients treated in the community setting who have access to clinical trials for advanced NSCLC remained low. A previous study [21] explored changes in clinical trials of cancer drugs in China over the decade of 2009-2018, and the results showed an increase in the number of clinical trials over time. The proportion of Phase 1 trials being initiated increased as well. However, the number of clinical trials focusing on epidemiological characteristics unique to the Chinese population remains low, and the geographical distribution of leading clinical trial units is unbalanced. Therefore, increased access to research clinical trials in China may improve the use of mutational data by NGS testing. The crossover of survival analysis curves of NGS and routine testing may be due to the fact that NGS panels derived from routine clinical practice may be mixed, and some patients cannot afford expensive targeted drugs, or the conditions were not met for inclusion in clinical studies. Overall, although the bene ts of NGS for NSCLC patients are limited, NGS remains essential in clinical practice.
The present study had several limitations, which need to be considered for data interpretation. First, this was a retrospective study, which may affect the results. For example, patient heterogeneity, inconsistency of treatment options selected by clinicians, and the fact that not all patients received NGS detection at diagnosis were factors that need to be considered. Second, this was not a randomly assigned study and this may induce analysis bias. Third, other outcome points including overall response rate and progression-free survival were not analyzed in this study. Fourth, despite the propensity analysis, confounding factors were not fully excluded, and unknown confounding factors may have been included. For patients undergoing NGS testing, the NGS panel contained the gene mutations mentioned in the NCCN guidelines; however, the panels were derived from different testing platforms and were not all the same. We considered that our study still had some clinical signi cance. Our article conducted out a large sample to con rm the NGS value compared by routine testing and it also suggest that NGS may need to be used more accurately. Future studies should assess the value of NGS in clinical trials of new drugs and the bene ts of NGS in clinical practice after the approval of new drugs.

Conclusions
NGS technology allowed the detection of uncommon driver mutations, which may lead to a greater bene t from targeted treatments and participation in clinical trials in NSCLC; however, it was not independently associated with a large survival bene t in advanced NSCLC patients. The patient populations need to be further categorized to explore the value of NGS in clinical practice.

Declarations
Ethics approval and consent to participate This study was conducted in accordance with the Declaration of Helsinki. The protocol was approved by the Institutional Ethics Committee at Zhejiang Cancer Hospital.

Consent for publication
All authors read this manuscript and approve for publication.     The comparison of the ration of the patients that received targeted therapy and off-label treatment according to NGS or Routine testing and used for a propensity score-matched survival analysis. The obviously higher ratio of the patients that received targeted therapy (68.0% vs. 53.6%; P<0.0001) and that received off-label drugs (28.10% vs. 11.40%; P <0.0001).

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download. NGSSFigure.docx