Tumor mutation burden as a biomarker for lung cancer patients treated with pemetrexed and cisplatin (the JIPANG‐TR)

Abstract The JIPANG study is a randomized phase III study of pemetrexed/cisplatin (Pem/Cis) versus vinorelbine/cisplatin (Vnr/Cis) for completely resected stage II‐IIIA non‐squamous non‐small cell lung cancer (Ns‐NSCLC). This study did not meet the primary endpoint (recurrence‐free survival, RFS) but Pem/Cis had a similar efficacy to Vnr/Cis with a better tolerability. Tumor mutation burden (TMB) is thought to have a predictive value of immune checkpoint inhibitors. However, the relevance of TMB to cytotoxic chemotherapy remains unknown. This exploratory study investigates the relationship between tumor mutation profiles and clinical outcome of Pem/Cis. Formalin‐fixed, paraffin‐embedded tumor tissues (n = 389) were obtained from the patients. Mutation status of tissue DNA was analyzed by targeted deep sequencing. Epidermal growth factor receptor (EGFR) mutations were detected frequently in Ns‐NSCLC (139/374). Patients without any EGFR mutations experienced longer RFS in the Pem/Cis arm versus Vnr/Cis arms. Pem/Cis in patients with high TMB (≥12‐16 mut/Mb) tended to have improved survival. In patients with wild‐type EGFR, TMB ≥ 12 mut/Mb was significantly associated with improved RFS with Pem/Cis versus Vnr/Cis (not reached vs 52.5 months; hazard ratio (HR) 0.477). It could be proposed that TMB was predictive of RFS benefit with Pem/Cis versus Vnr/Cis in Ns‐NSCLC. Further investigation is required to determine whether TMB combined with EGFR mutation status could be used as a predictive biomarker.


| INTRODUC TI ON
Patients with early stage non-small cell lung cancer (NSCLC) are operable but cure of a significant proportion is disrupted by recurrence.
Adjuvant chemotherapy in early stage NSCLC patients is currently considered the standard treatment and associated with an approximately 5% survival benefit at 5 years. 1 Cisplatin plus vinorelbine is recommended as a standard adjuvant treatment for stage II-III resected NSCLC patients. Therefore, randomized controlled studies have been conducted to investigate treatments with higher therapeutic effects.
Clinical application of biomarkers is warranted for operable cancer patients to identify patients at increased risk for recurrence such as Oncotype DX and MammaPrint for breast cancer patients.
Clinically relevant predictive biomarkers can identify patients most likely to benefit from adjuvant chemotherapy supporting the decision-making of postoperative adjuvant therapy and to predict its efficacy.
The JIPANG study is a randomized phase III study of pemetrexed/cisplatin (Pem/Cis) versus vinorelbine/cisplatin (Vnr/Cis) for completely resected stage II-IIIA non-squamous non-small cell lung cancer (Ns-NSCLC). 2 This phase III study did not meet the primary endpoint (recurrence-free survival, RFS) but Pem/Cis had a similar efficacy to Vnr/Cis with better tolerability. Exploratory analysis demonstrated hazard ratios in patients with and without epidermal growth factor receptor (EGFR) mutations were 1.38 (95% CI, 0.95-1.99) for Pem/Cis and Vnr/Cis 0.87 (95% CI, 0.69-1.09), respectively. Pemetrexed and cisplatin combination therapy is considered as one of the options for postoperative chemotherapy for stage II-IIIA Ns-NSCLC, especially EGFR wild type. 2 We have planned an exploratory biomarker study (JIPANG-TR) to identify the predictive biomarkers, the arm of which is beneficial for each patient through next-generation sequencing (NGS)-based mutation profiling of tumor tissues.
Amplicon deep sequencing is a powerful technology to analyze formalin-fixed, paraffin-embedded (FFPE) tumor samples. Tumor mutation burden (TMB) is reported as the total number of non-synonymous variants or single nucleotide variants per tumor genomic region. 3 Relatively large targeted deep sequencing provides TMB as well as mutation status of hundreds of genes. [4][5][6] Lung cancer compared with other solid cancers is characterized by a relatively high level of non-synonymous mutations resulting in the production of neo-epitopes of which TMB in lung cancer is a predictor of response to immunotherapies. [7][8][9] However, the relevance of TMB to cytotoxic chemotherapy is not yet fully understood. This study investigates the association of TMB and other mutation profiles with clinical outcomes of two platinum-containing regimens to determine which patients benefited by each adjuvant chemotherapy.

| Clinical specimens and outcome
A total of 389 (48.4%) of the 803 patients in the JIPANG study enrolled to the JIPANG-TR study between March 2012 and August 2016 at each institute. All patients provided written informed consent to participate in the study, including the collection of tumor tissue for analysis. Clinical outcome of overall survival (as a primary endpoint) and recurrence-free survival (as a secondary endpoint) was elucidated in the JIPANG study. 2 Overall survival was defined as the time from randomization to death from any cause. Recurrencefree survival was defined as time from randomization to disease recurrence, or death, whichever occurred first.
This study was designed as a prospective and exploratory study aimed at characterizing somatic mutations in tumor tissues and comparing tumor mutation status, including TMB and recurrence-free survival, to Pem/Cis or Vnr/Cis adjuvant therapy. This study was conducted in compliance with the Helsinki Declaration and the

Ethical Guidelines for Medical and Health Research Involving Human
Subjects by the Japanese government and has been approved by the ethics committee in each institute.

| Tissue processing
Tumor tissues were obtained at resectable operation and pathologically confirmed as non-squamous, non-small cell lung cancer. The

| Next-generation sequencing
The targeted DNA library comprising approximately 1.

| Statistical analysis
Patients were classified based on EGFR mutation status of tumor tissues. For biomarker analyses of each somatic mutation, the predictive and prognostic values were assessed by comparing the RFS of each arm (Pem/Cis and Vnr/Cis) in the JIPANG-TR study. The JMP (ver.
14.0, SAS Institute) and GraphPad Prism software (ver. 8, GraphPad Software Inc) were used for statistical analysis. Cox proportional hazards regression model was applied to perform univariate analyses. Relations between mutation status and patient characteristics were evaluated using the chi-squared (χ 2 ) test. Kaplan-Meier curves were used to estimate survival, and the log-rank test was used to compare times to events between groups. P-values of <.05 were considered statistically significant.  Table 1. Baseline patient's characteristics in the subgroup were generally comparable to the primary study. Two-year RFS was similar in the two treatment arms. The hazard ratios of the two treatment arms were not substantially different between the primary and subgroup studies. Thus, one may establish similarity of patient characteristics and efficacy between the primary and subgroup analysis cohorts.

| EGFR mutations and mutation burden
Nonsynonymous EGFR mutations were frequently identified in 139 samples (37.2%) by targeted sequencing (

| Association of mutation profile with clinical outcome
In this JIPANG-TR study, a subgroup (n = 374) of the patients enrolled in the JIPANG study was analyzed. There was no significant difference of RFS between the Pem/Cis and Vnr/Cis arms ( Figure S1A).
JIPANG study demonstrated that patients without common EGFR mutations detected by IVD kits showed favorable outcomes by Pem/ Cis compared with Vnr/Cis. 2 In the JIPANG-TR study, EGFR mutations were detected in 139 patients by targeted deep sequencing (  Figure S2B).

| Mutation burden and clinical outcome
To determine the preferable TMB cutoff level for predictive analyses, we conducted forest plot analyses for all cutoff levels (4-20 mutations/Mb) to assess the RFS of Pem/Cis or Vnr/Cis arms for 374 patients. Minimum hazard ratio (HR) of the high TMB group was 0.541 at 16 mutations/Mb for favorable outcomes in Pem/Cis with longer RFS (Figure 2A), whereas that of the low TMB group was 1.33 at 6 mutations/Mb for favorable outcomes in Vnr/Cis ( Figure 2B).
However, these were not significant.
When focusing on the subgroup with EGFR wild type, forest and Kaplan-Meier plots demonstrated longer RFS periods in EGFR wild-type patients treated with Pem/Cis than Vnr/Cis groups with high TMB but not low TMB values (Figures 3 and 4).