Ecacy and Biomarkers of Advanced Non-small Cell Lung Carcinoma (NSCLC) Patients Receiving Different PD-1 Agents in Northern China: A Real-world Clinical Study

Aim and Methods: From May 2019 to January 2020, 116 advanced NSCLC patients with programmed death ligand-1 (PD-L1) expression > 1% were treated with nivolumab (44), pembrolizumab (21), and toripalimab (51) as a mono-therapy, respectively. The primary endpoints were dened as the objective response rate (ORR) after 3 and 6 months of therapy, and the progression-free survival (PFS). The observed ecacy of the different PD-1 antibodies was also compared. The gene mutation statuses of tumor protein p53 (TP53), epidermal growth factor receptor ( EGFR ), and anaplastic lymphoma kinase ( ALK ), the tumor mutational burden (TMB), along with the expression of CD47 were analyzed. Results: Toripalimab had a higher ORR and a longer PFS than the other two PD-1 agents after 3 months of evaluation (P = 0.0178). Thenon-classical mutations of EGFR (EGFRG719C and EGFRE709V) did not signicantly inuence the ecacy of the PD-1 inhibitors, while TP53 mutation, high TMB and elevated PD-L1 expression showed benets. EGFR co-mutated genes were enriched in the “Response to osmotic stress” , “response to oxidative stress” and “myeloid leukocyte activation” pathways. CD47 expression showed a negative correlation to the prognosis of anti-PD-1 therapy. Participants with ALK rearrangement exhibited poor clinical outcomes. Conclusions: Toripalimab seemed to have a more rapid effect and provide a longer PFS than pembrolizumab and nivolumab. The PD-1 blockade therapy was beneted by TP53 mutation, high TMB, and elevated PD-L1 expression. CD47 expression is a potential biomarker to predict the ecacy of PD-1 blockade treatment in patients with EGFR mutations.


Introduction
Global cancer statistics from 2018 revealed that lung cancer is still the most common malignant tumor (11.6% of all cancer patients) and the leading cause of death from cancer (18.4% of all cancer deaths) [1]. Due to inadequate tobacco control and a sharp increase in the number of young smokers, the incidence of lung cancer in China is increasing. Non-small cell lung cancer (NSCLC) accounts for 85% of the total lung cancer cases [2]. According to previous studies, the 5-year survival rate of NSCLC is 17%, and that of advanced NSCLC is only < 5%. Surgical resection is the most effective treatment for patients with stage I, II, or IIIA NSCLC. However, 70% of patients in the late stage cannot undergo surgery, and patients with metastatic or recurrent disease have a relatively limited response to conventional treatments. Therefore, it is necessary to explore novel therapies to meet the clinical needs of this patient group.
Traditional cancer immunotherapy relies mainly on direct activation of the immune system. Recently, however, the focus of immunotherapy has moved toward reducing immune tolerance of tumor cells [3].
The abnormal expression of immune checkpoint molecules is one of the mechanisms of tumor immune escape, which is caused by ligand-receptor interactions on the surface of immune and tumor cells [4].
Cytotoxic T lymphocyte-associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1), and their corresponding ligands B7-1/2 and PD-L1, have received the most clinical attention. Researchers have also developed monoclonal antibodies targeting these molecules, namely immune checkpoint inhibitors (ICIs). These ICIs deliver anticancer effects by suppressing and blocking the action of inhibitory receptors, such as PD-1 and CTLA-4, so that the immune system can effectively recognize and attack cancer cells [5,6].
From 2018 to 2020, 6 monoclonal antibody agents against PD-1, including nivolumab, pembrolizumab, toripalimab, sintilimab, camrelizumab, and tislelizumab, have been approved by the China Food and Drug Administration and marketed in China for a variety of cancer indications. Currently, only nivolumab monotherapy and the combination therapy of pembrolizumab plus chemotherapeutics have been approved as second-line and rst-line therapies, respectively, for NSCLC patients in China. Since different monoclonal PD-1 antibodies have different pharmacokinetic pro les related to the unique complementaritydetermining region sequences in the heavy and light chains, we conducted a clinical trial (Real-World Study of Four PD-1 Agents in China: NCT03966456) to compare the clinical e cacies of 3 anti-PD-1 monoclonal antibodies, including nivolumab, pembrolizumab, and toripalimab. Here, we report the therapeutic outcomes and potential predictive biomarkers for the different PD-1 antibodies analyzed in this study.

Study population
As part of an ongoing clinical trial (Real World Study of Four PD-1 Agents in China, NCT03966456), this clinical assessment was carried out using data from 116 NSCLC patients admitted to the A liated Hospital of Qingdao University between May 2019 and January 2020. The inclusion conditions were patients 18-85 years old who were diagnosed with advanced NSCLC with positive PD-L1 expression (tumor proportion score > 1%) by pathological examination. All patients were treated with a PD-1 monoclonal antibody as a mono-therapy. Patients with a classical epidermal growth factor receptor (EGFR) mutation were excluded, excepting EGFRG719C (18/29) and EGFRE709V (19/29). The Response Evaluation Criteria in Solid Tumors (RECIST version 1.1) were followed, and the patients must have had ≥ 1 measurable lesion per response. The Eastern Cooperative Oncology Group (ECOG) performance status was 0 or 1 for all participants. The study protocol and all amendments were approved by the institutional ethics committee of the A liated Hospital of Qingdao University (QYFYKYLL 471311920).

Biomarker studies
Pretreatment tumor biopsies from all participants were examined for PD-L1 expression by immunohistochemical (IHC) staining with the anti-human PD-L1 monoclonal antibody SP142 [7]. The PD-L1 positivity evaluated by certi ed pathologists was de ned as a strong PD-L1 staining on the cell membrane detected in ≥ 1% of tumor cells or any staining intensity observed in tumor-in ltrating immune cells. The number of deduced somatic mutations per megabase (Mb) was used to evaluate the TMB. Using 12 mutations/Mb as the cutoff value, TMB ≥ 12 mutations/Mb was de ned as a high TMB, and TMB < 12 mutations/Mb was de ned as a low TMB. Whole-exome sequencing of tumor tissue and matched peripheral blood mononuclear cells were pro led using a commercially available capture-based lung plasma sequencing panel (Burning Rock Biotech Ltd., Guangzhou, China) targeting 168 genes and spanning 160,000 human genomic regions. Brie y, fragmented DNA captured by probe baits was puri ed with magnetic beads, and the library was indexed and ampli ed by polymerase chain reaction (PCR). A bio-analyzer assay was then used to assess the quality and size range of the library. The pool of 30 normalized libraries was nally loaded onto a NextSeq 500 system (Illumina, Inc., San Diego, CA, USA) for paired-end sequencing.
Gene ontology functional analysis and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis of EGFR and co-mutation events Each group was further separated into 2 subgroups by the median percent spliced in (psi) value. Then, univariate Cox regression analysis was conducted to explore independent prognostic factors with the signi cance set as a P value < 0.05. Next, we selected the co-mutated genes with an EGFR mutation in our study as candidates for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses using Metascape (http://metascape.org/).A P-value of < 0.05 indicated statistical signi cance.

Western blot analysis
Proteins were extracted from the the Formalin-Fixed and Parr n-Embedded (FFPE) samples. The protein concentration was determined by a BCA kit (Pierce, Rockford, IL, USA). And then the proteins were separated by 10% SDS-PAGE, transferred to nitrocellulose membranes (Millipore, Bedford, MA, USA), blocked with 5% low fat dried milk for 2 h, and incubated overnight at 4 °C with corresponding primary antibodies directed against CD47 (Rabbit, 1:2000, ab175388, Abcam). Subsequently, the membranes were incubated with horseradish peroxidase (HRP)-conjugated donkey-anti-rabbit secondary antibody for Statistical analysis Data were analyzed by using the statistical software SPSS version 20.0 (IBM Corp, Armonk, NY, USA) and Graph Pad Prism 5.0 software (Graph Pad Software Inc., San Diego, CA, USA). Continuous variables with a normal distribution are presented as the mean ± standard deviation and were compared by the Student's t-test. Continuous variables with a non-normal distribution are expressed as the median (inter quartile range) and were compared with the Mann-Whitney test. Categorical variables are expressed as a number and proportion and were analyzed with the Fisher's exact or chi-squared (χ 2 ) test. The variables with a statistically signi cant difference were further explored using binary logistic regression analysis.
Irreversible organ injury and death were de ned as poor outcome events. The survival time was de ned as the period between the diagnosis of NSCLC and the occurrence of a poor outcome event. The Kaplan-Meier method and the Cox proportional hazards regression model were used to evaluate the prognostic factors. Differences were considered signi cant at a P-value of < 0.05.

Patient population
The baseline clinical characteristics of the 116 participants are summarized in Table 1. There were no signi cant differences in age among the 3 cohorts (P > 0.1), and the average age was 64 years old. More male than female patients were enrolled, which was consistent with a higher incidence of NSCLC in males than in females. All participants in this study received ≥ 1 prior line of treatment and anti-PD-1 mono-therapy as described above (nivolumab [  Subgroup analysis forclinical e cacy A total of 6 participants had died by January 23, 2020 including 4 who were treated with nivolumab, 1 who was treated with pembrolizumab, and 1 who was treated with toripalimab. The changes observed at 3 and 6 months from baseline were each analyzed ( Fig. 1A and 1B). According to the results shown in Fig. 1C and 1D, the toripalimab cohort exhibited a higher ORR while the pembrolizumab cohort showed a higher disease control rate, but these differences were not statistically signi cant (P > 0.05). The PFS curves of the 3 anti-PD-1agents were not signi cantly separated (Fig. 1E). The median PFS times for the nivolumab, pembrolizumab, and toripalimab groups were 9, 10, and 11 months, respectively (log rank P = 0.990). And there was no signi cant difference in e cacy between the 3 agents with respect to the histology (Fig. 2).
Functional enrichment analysis of co-mutated genes in patients with EGFR mutation and CD47 expression of the patients with EGFR mutation According to the next-generation sequencing analysis, peripheral-extracellular signal-regulated kinase (P-MAPK1), neuroblastoma RAS (NRAS), TP53, tumor necrosis factor (TNF), and caspase-3 have high comutation frequencies with EGFR non-classical mutations (Fig. 3I). These genes were further assessed by functional and pathway enrichment analyses. The most signi cant enrichment results are shown in Fig. 3H. The commonly enriched terms were "proteoglycans in cancer," "response to osmotic stress," "response to oxidative stress," and "myeloid leukocyte activation". Western blot analysis showed that the patient with lower CD47 expression exhibited better e cacy to PD-1 therapy. But higher CD47 expression maybe associated with disease progression under PD-1 therapy (Fig. 3I).

E cacy for the squamous cell carcinoma subtype
Analysis of the e cacy of the different PD-1 agents for treatment of the squamous cell carcinoma subtype is shown in Fig. 4A and 4B. In the squamous cell carcinoma subtype, the ORRs of nivolumab, pembrolizumab, and toripalimab after 3 months of therapy were 16.7%, 0.0%, and 50%, respectively (P = 0.2062); and after 6 months of therapy, they were 38.9%, 66.7%, and 50.0%, respectively (P = 0.5878). Survival analysis (Fig. 4C)  Correlation of TP53 mutation, ALK rearrangement, and TMB with clinical e cacy Among all 116 participants, just 78 had a TP53 mutation. The percentage of participants with a TP53 mutation was not signi cantly different between the 3 PD-1 antibody cohorts (Fig. 5A). According to our study, 80.77% (63/78) of the participants with a TP53 mutational so had a KRAS mutation. In addition, the participants with a TP53 mutation had a longer PFS (12 [95% CI: 10.887-13.113] and 8 [95% CI: 6.680-9.320] months for patients with a TP53 mutation and wild-type TP53, respectively P = 0.019) (Fig. 5B). Furthermore, the meta-analysis of our data showed that PD-1 therapy was more effective in patients with a TP53 mutation ( Fig. 5C and 5D). Among the 3 PD-1 antibodies studied herein, the participants with a TP53 mutation appeared to be more sensitive to nivolumab after 6 months of therapy (Fig. 5D).
In our study, there were only 4 patients with ALK rearrangement, all of whom showed disease progression before the evaluation after 3 months of treatment. The receiver operating characteristic (ROC) curve analysis showed that the TMB was not associated with the e cacy of PD-1 treatment at the 3-month evaluation (P > 0.1) (Fig. 5E). However, at the 6-month evaluation, a higher TMB was signi cantly bene cial to the PD-1 therapy (P < 0.05) (Fig. 5E). At the 3-month evaluation, a higher PD-L1 expression level correlated with a better ORR, indicating that a high PD-L1 expression level is conducive to early treatment e cacy. However, at the 6-month evaluation, the difference was not signi cant (Fig. 5F).

Discussion
One of the aims of this study was to analyze the e cacies of different monoclonal anti-PD-1 antibodies in northern China. In a previous study, a comparison of the amino acid sequences of nivolumab and pembrolizumab showed that they are basically the same except for the variable region [8]. In addition, Fesses et al. compared the molecular, preclinical, and early clinical characteristics of nivolumab and pembrolizumab. They found signi cant molecular similarities between these drugs, indicating that the differences observed in the clinical data were not likely to be drug-dependent and likely to be drugindependent. The observed differences may be due to variations between the participant populations in the clinical trials. In our study, toripalimab was found to have a higher ORR and longer PFS than the other 2 PD-1 agents, especially at the 3-month evaluation.
The PD-L1 of tumor cells is currently the most widely studied protein and the only Federal Drug Agencyapproved biomarker for clinical use. According to the KEYNOTE-042 study results published at the 2018 American Society of Clinical Oncology conference, for advanced NSCLC patients with PD-L1 ≥ 1% and negative for EGFR or ALK mutations, the overall survival(OS) of those receiving rst-line pembrolizumab therapy was higher than that of those receiving chemotherapy. This nding showed that pembrolizumab is more bene cial for NSCLC patients with PD-L1 ≥ 1% [9]. The TMB is the sum of non-synonymous mutations in the coding region of the somatic genome. Non-synonymous somatic mutations can alter the amino acid sequence of the protein encoded by the affected gene, thereby forming a neoantigen and helping to enhance the immunogenicity of tumor cells [10]. There are various methods to calculate the TMB, and they generally require whole-exome sequencing of ≥ 200 cancer-related genes [11]. The TMB was rst reported as a biomarker in the rst edition of the 2019 National Comprehensive Cancer Network guide for screening NSCLC patients for whom nivolumab combined with ipilimumab or nivolumab monotherapy may be effective. In the present study, higher TMB and PD-L1 expression levels were also demonstrated to bene t PD-1 blockade therapy. We found that the TMB in uenced the ORR signi cantly after 6 months of therapy and that the PD-L1 expression in uenced the ORR signi cantly after 3 months of therapy.
The correlation between mutations of cancer driver genes and the e cacy of PD-1 monoclonal antibody therapy has also attracted attention. An EGFR mutation in adenocarcinoma lung cancer is a good predictive factor for the e cacy of EGFR tyrosine kinase inhibitors [12][13][14]. In the KEYNOTE-001 study, the median OS of EGFR mutation-positive patients treated with pembrolizumab was signi cantly shorter than that in wild-type EGFR patients [15]. Gainor et al. also found that compared with wild-type EFGR NSCLC patients, EGFR mutation-positive NSCLC patients had a much lower ORR [16]. However, our study found that a non-classical EGFR mutation, such as G719C or E709V, did not signi cantly in uence the e cacy of PD-1 therapy for NSCLC patients in northern China. According to the results of the functional enrichment analysis of co-mutated genes in patients with an EGFR mutation, the commonly enriched terms were "proteoglycans in cancer," "response to osmotic stress," "response to oxidative stress," and "myeloid leukocyte activation" (Fig. 8B). And more the co-mutations, better the prognosis (Fig. 8A). These pathways associated activation of cell signaling pathways of proliferation, angiogenesis, and cell motility (Fig. 8C). The most enriched signal pathway of these mutations is "proteoglycans in cancer".
Proteoglycans (PGs) are heterogeneous glycoproteins, expressed in cells of the tumor microenvironment and on tumor cells, which could regulation immunosensitization [17]. "Response to osmotic stress", "response to oxidative stress" and "myeloid leukocyte activation" pathways were also signi cantly enrichment herein. Both osmotic stress and oxidative stress are associated with in ammation. And in ammation was associated with immune activation [18]. Myeloid leukocyte activation may activate B cells [19] and improve the tumor microenvironment to make it sensitive to PD-1 agents [20]. Take above all, these gene characteristic maybe in uence the patients response to PD-1 therapy. As we all known the expression of CD47 is involved in imparting resistance to programmed cell death (PD)-1/PD-ligand 1 inhibitors [21]. According to our study, the EGFR-mutated patients who were achieved PR after 6 mtherapy showed a lower CD47 expression. But the patients with disease progression exhibited a higher CD47 expression. Maybe CD47 expression is the potential biomarker to patients with EGFR mutation treated with PD-1 agents.
Another important driver gene of NSCLC is ALK. The ATLANTIC study showed that for patients with ALK rearrangement, if the PD-L1-positive cell expression rate is ≥ 25%, immunotherapy will still work [22]. Moreover, it has been reported that NSCLC patients with ALK gene fusion might bene t from PD-1 treatment [22][23][24]. However, according to our clinical observations, those with ALK gene fusion showed a poor response to PD-1 therapy. Previous studies have indicated that a high TMB and PD-L1 expression level were both independently correlated with survival bene ts from anti-PD-1 therapy. In a recent study, researchers also observed that the median OS was longer in the TP53-mutated group than in the wildtype TP53 group (18.1 months vs. 8.1 months). Furthermore, the median PFS was signi cantly longer and the ORR was higher in the TP53-mutated patients [24]. In accordance with these ndings, we also found that a TP53 mutation was bene cial for PD-1 therapy.
This study had several limitations. The sample size of the group of participants treated with PD-1 antibodies in our study was small, especially when classi ed according to the therapy. Additionally, the OS was not determined for most of the participants, and the e cacy of PD-1 therapy was evaluated using only the ORR and the PFS. It can take years before an effective improvement or death occurs to enable further assessment of e cacy. When available, the nal set of data, including the effectiveness and late toxicity endpoints, will provide valuable insight into the differences in survival and quality of life of the NSCLC patients.

Conclusions
Immune checkpoint blockade targeting PD-1 has dramatically changed the landscape for treatments in patients with NSCLC. Herein, we compared the e cacy of the 3 early accessible PD-1 agents for NSCLC patients in northern China, nivolumab, pembrolizumab, and toripalimab, and found that toripalimabconferred the most bene t. Mutation of TP53, high TMB, and elevated PD-L1 expression were bene cial to the PD-1 blockade therapy. The e cacy of PD-1 inhibitors was not signi cantly in uenced by EGFR G719C or EGFR E709V. It is possible that proteoglycans, osmotic stress and oxidative stress, and myeloid leukocyte activation, which are regulated by EGFR, P-MAPK1, NRAS, TP53, TNF, and caspase-3,