Marion Ferreira
email: marion-ferreira0404@gmail.com

Thomas Secher

Nathalie Heuzé-Vourc'h

Karen L Reckamp

Nathalie Heuze-Vourc'h

Immune Checkpoint and Anti-Angiogenic Antibodies

Keywords: lung cancer, immune checkpoint inhibitors, anti-angiogenic, antibodies, cancer treat-23 ment 24 25

published or not. The documents may come

Introduction

Lung cancer is responsible for significant morbidity and mortality worldwide; it is with advanced or metastatic cancers [START_REF] Guo | Nobel Prize in Medicine Goes to Cancer Immunotherapy (Editorial for BMC Cancer)[END_REF]. The choice of standard therapeutic options for patients with locally advanced, and metastatic NSCLC has been expanded by monoclonal antibodies (Abs) like ICIs, but also anti-angiogenic Abs and other targeted Abs [START_REF] Pabani | Current Landscape of Immunotherapy for the Treatment of Metastatic Non-Small-Cell Lung Cancer[END_REF].

Several types of Abs are currently used for the treatment of NSCLC, but this review will discuss ICI and anti-angiogenic Abs. In fact, some Abs are directed to the epidermal growth factor receptor (EGFR) like necitumumab, a monoclonal antibody (Ab) and blocks the interaction between EGFR and its ligands. It was approved by the FDA in 2015 for first-line treatment of metastatic squamous NSCLC in combination with chemotherapy [START_REF] Thatcher | Necitumumab plus Gemcitabine and Cisplatin versus Gemcitabine and Cisplatin Alone as First-Line Therapy in Patients with Stage IV Squamous Non-Small-Cell Lung Cancer (SQUIRE): An Open-Label, Randomised, Controlled Phase 3 Trial[END_REF] but the National Comprehensive Cancer Network (NCCN) guidelines recom-mends against the use of this Ab, based on its toxicity, cost and limited improvement in efficacy when compared to chemotherapy.

We will discuss commonly used and marketed ICIs for patients with NSCLC including: nivolumab [START_REF] Brahmer | Nivolumab versus Docetaxel in Advanced Squamous-Cell Non-Small-Cell Lung Cancer[END_REF][START_REF] Carbone | First-Line Nivolumab in Stage IV or Recurrent Non-Small-Cell Lung Cancer[END_REF][START_REF] Hellmann | Nivolumab plus Ipilimumab in Lung Cancer with a High Tumor Mutational Burden[END_REF][START_REF] Borghaei | Nivolumab versus Docetaxel in Advanced Nonsquamous Non-Small-Cell Lung Cancer[END_REF], pembrolizumab [START_REF] Herbst | Pembrolizumab versus Docetaxel for Previously Treated, PD-L1-Positive, Advanced Non-Small-Cell Lung Cancer (KEY-NOTE-010): A Randomised Controlled Trial[END_REF][START_REF] Reck | Pembrolizumab versus Chemotherapy for PD-L1-Positive Non-Small-Cell Lung Cancer[END_REF][START_REF] Langer | Carboplatin and Pemetrexed with or without Pembrolizumab for Advanced, Non-Squamous Non-Small-Cell Lung Cancer: A Randomised, Phase 2 Cohort of the Open-Label KEYNOTE-021 Study[END_REF][START_REF] Garon | Pembrolizumab for the Treatment of Non-Small-Cell Lung Cancer[END_REF], cemiplimab [START_REF] Sezer | Cemiplimab Monotherapy for First-Line Treatment of Advanced Non-Small-Cell Lung Cancer with PD-L1 of at Least 50%: A Multicentre, Open-Label, Global, Phase 3, Randomised, Controlled Trial[END_REF], durvalumab [START_REF] Antonia | Durvalumab after Chemoradiotherapy in Stage III Non-Small-Cell Lung Cancer[END_REF], atezolizumab [START_REF] Rittmeyer | Atezolizumab versus Docetaxel in Patients with Previously Treated Non-Small-Cell Lung Cancer (OAK): A Phase 3, Open-Label, Multicentre Randomised Controlled Trial[END_REF][START_REF] Fehrenbacher | Atezolizumab versus Docetaxel for Patients with Previously Treated Non-Small-Cell Lung Cancer (POPLAR): A Multicentre, Open-Label, Phase 2 Randomised Controlled Trial[END_REF][START_REF] Gandara | Blood-Based Tumor Mutational Burden as a Predictor of Clinical Benefit in Non-Small-Cell Lung Cancer Patients Treated with Atezolizumab[END_REF], and ipilimumab [START_REF] Hellmann | Nivolumab plus Ipilimumab in Lung Cancer with a High Tumor Mutational Burden[END_REF][START_REF] Hodi | Nivolumab plus Ipilimumab or Nivolumab Alone versus Ipilimumab Alone in Advanced Melanoma (CheckMate 067): 4-Year Outcomes of a Multicentre, Randomised, Phase 3 Trial[END_REF]. They act by targeting immune checkpoints expressed by tumor infiltrating lymphocytes (TILs) -programmed-death 1 (PD-1) or cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) -or expressed by cancer and tumor infiltrating immune cells -programmed-death ligand 1 (PD-L1) [START_REF] Steven | Immunotherapy for Lung Cancer[END_REF]. Anti-angiogenic Abs used in NSCLC are bevacizumab [START_REF] Sandler | Paclitaxel-Carboplatin Alone or with Bevacizumab for Non-Small-Cell Lung Cancer[END_REF][START_REF] Reck | Phase III Trial of Cisplatin plus Gemcitabine with Either Placebo or Bevacizumab as First-Line Therapy for Nonsquamous Non-Small-Cell Lung Cancer: AVAil[END_REF][START_REF] Ferrara | Single or Combined Immune Checkpoint Inhibitors Compared to First-Line Platinum-Based Chemotherapy with or without Bevacizumab for People with Advanced Non-Small Cell Lung Cancer[END_REF] and ramucirumab [START_REF] Garon | Ramucirumab plus Docetaxel versus Placebo plus Docetaxel for Second-Line Treatment of Stage IV Non-Small-Cell Lung Cancer after Disease Progression on Platinum-Based Therapy (REVEL): A Multicentre, Double-Blind, Randomised Phase 3 Trial[END_REF][START_REF] Nakagawa | Ramucirumab plus Erlotinib in Patients with Untreated, EGFR-Mutated, Advanced Non-Small-Cell Lung Cancer (RELAY): A Randomised, Double-Blind, Placebo-Controlled, Phase 3 Trial[END_REF].

The choice of ICI molecule depends on the expression of tumor PD-L1 for some molecules or associations and the choice of anti-angiogenic therapy is based on patients' contraindications and tumor histology. ICIs have proven to be better tolerated than chemotherapy [START_REF] Carbone | First-Line Nivolumab in Stage IV or Recurrent Non-Small-Cell Lung Cancer[END_REF][START_REF] Reck | Pembrolizumab versus Chemotherapy for PD-L1-Positive Non-Small-Cell Lung Cancer[END_REF] but, the response to single-agent ICI is not durable in most patients and only a minority have a durable benefit [START_REF] Rittmeyer | Atezolizumab versus Docetaxel in Patients with Previously Treated Non-Small-Cell Lung Cancer (OAK): A Phase 3, Open-Label, Multicentre Randomised Controlled Trial[END_REF][START_REF] Herbst | Long-Term Follow-up in the KEYNOTE-010 Study of Pembrolizumab (Pembro) for Advanced NSCLC, Including in Patients (Pts) Who Completed 2 Years of Pembro and Pts Who Received a Second Course of Pembro[END_REF]. Even if ICI represent one of the most promising therapeutic approaches in metastatic NSCLC, combination treatments are frequently used including chemotherapy, multiple ICIs or ICI and other Abs, in order to improve their efficacy.

Considering the various therapeutic options for locally advanced and metastatic lung cancer and differences in use according to geographic area, we present a comprehensive review of the marketed ICI and antiangiogenic Abs approved in European Union and the US to treat NSCLC patients. We will focus our discussion on NSCLC without oncogenic drivers except for anti-angiogenic Abs which can be used in combination with tyrosine kinase inhibitors (TKI) in case of EGFR-mutated tumors. The European Medicines Agency (EMA) is a decentralized agency of the European Union (EU) responsible for the scientific evaluation, supervision and safety monitoring of medicines. It serves the EU and other countries: Iceland, Norway, and Liechtenstein. United Kingdom (UK) and Switzerland are therefore not affected by this review. Even before their withdrawal from the EU, UK did not use EMA recommendations for their drug approvals; Switzerland has its own marketing authorization system (Swissmedic's). Herein, we briefly describe the different molecules and their development in thoracic oncology and compare pharmacokinetic data and marketing authorizations by the EMA and US Food and Drug Administration (FDA).

Immune checkpoint inhibitors

Cancer growth and spread are not only dependent on tumor cell characteristics but are also affected by the interaction with the immune system [START_REF] Hanahan | Hallmarks of Cancer: The next Generation[END_REF]. To prevent any unwanted damage caused by activated T cells to surrounding tissues, the immune system has evolved a variety of mechanisms, or 'checkpoints' that are used to modulate the duration and amplitude of the immune responses [START_REF] Pardoll | The Blockade of Immune Checkpoints in Cancer Immunotherapy[END_REF]. This dampens the immune response and protects against damage caused by unwanted inflammation or autoimmunity. This is achieved primarily by upregulation of 'co-inhibitory' receptors (TIGIT, TIM-3…) that act to 'turn off' activated T cells. Ultimately, it is the balance between co-stimulatory and co-inhibitory signals that dictates the fate of activated T cells [START_REF] Steven | Immunotherapy for Lung Cancer[END_REF]. In cancer, T-cells primed to respond to tumor cells are exposed continuously to tumor antigens during active malignancy, which may result in upregulation of multiple inhibitory receptors, culminating decreased activity against tumor cells, phenomenon known as T-cell exhaustion [START_REF] Wherry | Molecular and Cellular Insights into T Cell Exhaustion[END_REF]. These multifaceted interactions can be manipulated to drive effective anticancer immunity. For example, several agonist antibodies targeting immune co-stimulatory receptors (CD27, CD40, OX40…) have been evaluated clinically in cancer for now more than years; but none have been approved to date, nor have begun any phase III randomized trials [START_REF] Mayes | The Promise and Challenges of Immune Agonist Antibody Development in Cancer[END_REF]. On the other hand, T-cell exhaustion could be overcome by modulating the inhibitory pathways with antagonist Abs [START_REF] Wherry | Molecular and Cellular Insights into T Cell Exhaustion[END_REF].

CTLA-4 protein is expressed on the surface of T cells and competes with CD28 for B7 binding in an inhibitory fashion, therefore acting as suppressor of T-cell activation [START_REF] Phan | Cancer Regression and Autoimmunity Induced by Cytotoxic T Lymphocyte-Associated Antigen 4 Blockade in Patients with Metastatic Melanoma[END_REF].

Abs to CTLA-4 block the inhibition of CD28/B7-dependent T-cell activation and in turn prolong anti-tumor activity (Figure 1).

In lung cancer, only one anti-CTLA-4 antibody is currently used: ipilimumab; a fully human IgG1 kappa monoclonal (Table 1). The efficiency of ICIs targeting to CTLA-4 were initially demonstrated for the treatment of metastatic melanoma [30] before being expanded in metastatic lung cancer.

Anti-PD-1 and PD-L1 antibodies

One mechanism of immune suppression in NSCLC is the expression of inhibitory molecules in the tumor microenvironment. PD-L1 (B7-H1) is expressed on tumor cells in approximately half of NSCLC; its expression can contribute to poor prognosis by suppressing T-cell function and promoting tumor cell immune escape [START_REF] Mu | High Expression of PD-L1 in Lung Cancer May Contribute to Poor Prognosis and Tumor Cells Immune Escape through Suppressing Tumor Infiltrating Dendritic Cells Maturation[END_REF]. Both PD-L1 and PD-L2 (B7-DC) bind to PD-1 but PD-L1 expression is predominantly confined to the tumor microenvironment on the contrary of PD-L2 which is mainly expressed on dendritic cells and macrophages [START_REF] Brahmer | Immune Checkpoint Inhibitors: Making Immunotherapy a Reality for the Treatment of Lung Cancer[END_REF] (Figure 1). PD-1 checkpoint blockade uses Abs directed against either the receptor (PD-1) or its ligand (PD-L1). Early in the development of these therapies, two anti-PD-1 Abs were investigated and are currently used in metastatic lung cancer. Nivolumab is a fully human IgG4 monoclonal antibody targeting PD-1 (Table 1) and pembrolizumab is a humanized IgG4 antibody targeting PD-1 (Table 1). Recently, another antibody has been approved in the US: cemiplimab is a highly potent, fully human, IgG4, directed against PD-1 [START_REF] Sezer | Cemiplimab Monotherapy for First-Line Treatment of Advanced Non-Small-Cell Lung Cancer with PD-L1 of at Least 50%: A Multicentre, Open-Label, Global, Phase 3, Randomised, Controlled Trial[END_REF].

There are currently 2 anti-PD-L1 antibodies that are used in locally advanced or metastatic lung cancer: atezolizumab, a humanized IgG1 antibody targeting PD-L1 (Table 1) and durvalumab, a fully human IgG1 kappa antibody targeting also PD-L1 (Table 1).

Anti-angiogenic antibodies

Angiogenesis is frequently upregulated in malignant solid tumors and is critical for tumor growth, proliferation and metastases [START_REF] Reck | Antiangiogenic Therapy for Patients with Aggressive or Refractory Advanced Non-Small Cell Lung Cancer in the Second-Line Setting[END_REF]. The vascular endothelial growth factors (VEGF-A, -B, -C and -D) and their transmembrane tyrosine kinase receptors (VEGFR-1, -2 and -3) are critical proangiogenic factors in malignant tissues [START_REF] Bergers | Tumorigenesis and the Angiogenic Switch[END_REF] (Figure 1). In NSCLC, increased VEGF expression is common and associated with adverse clinical outcomes.

Two Abs are used in lung cancer: bevacizumab, a recombinant humanized monoclonal IgG1 antibody that neutralizes VEGF's biologic activity through a steric inhibition of its binding to VEGF receptor. Ramucirumab is a recombinant human monoclonal IgG1 antibody with high affinity for the extracellular domain of VEGFR-2, inhibiting ligand binding (VEGF-A, -C and -D) and activation of downstream proangiogenic pathways [START_REF] Reck | Antiangiogenic Therapy for Patients with Aggressive or Refractory Advanced Non-Small Cell Lung Cancer in the Second-Line Setting[END_REF] (Table 1). benefit in the treatment of several cancers, including metastatic NSCLC. In general, monoclonal Abs, including immunomodulatory Abs, have proven to be well tolerated [START_REF] Suntharalingam | Cytokine Storm in a Phase 1 Trial of the Anti-CD28 Monoclonal Antibody TGN1412[END_REF]. The pharmacokinetic (PK) of the approved ICI is similar to that of endogenous immunoglobulin G (IgG). The typical volume of distribution of monoclonal Abs is comparable to the plasma volume (2 to 4 L) [START_REF] Zhao | Clinical Pharmacology Considerations in Biologics Development[END_REF]. Drug-receptor binding affinity and association-dissociation kinetics have an important role in distribution [START_REF] Deng | Monoclonal Antibodies: What Are the Pharmacokinetic and Pharmacodynamic Considerations for Drug Development?[END_REF]. Elimination occurs by both specific (target-mediated) and nonspecific (Fc-mediated-routes, accounting for the nonlinear and linear elimination PK [START_REF] Zhao | Clinical Pharmacology Considerations in Biologics Development[END_REF]. Following target saturation, the linearnonspecific -route of elimination is predominant; so, the half-life of ICI varies from 3 to 4 weeks approximately [START_REF] Zhao | Clinical Pharmacology Considerations in Biologics Development[END_REF][START_REF] Mould | Drug Development of Therapeutic Monoclonal Antibodies[END_REF].

Ipilimumab was the first ICI to be made commercially available for the treatment of metastatic melanoma in 2011 in the US. It is administered intravenously, with a mean half-life of 15.4 days [START_REF] Centanni | Clinical Pharmacokinetics and Pharmacodynamics of Immune Checkpoint Inhibitors[END_REF] (Table 1). The following factors do not represent contraindications to the use because they had no clinically important effect on the clearance of ipilimumab: age, sex, performance status (PS), renal impairment (glomerular filtration rate ≥15 mL/min/1.73 m2), or mild hepatic impairment (total bilirubin >1 to 1.5 times the upper limit of normal or aspartate transaminase levels > upper limit of normal) (data available on Bristol Myers Squibb. YERVOY®(Ipilimumab)).

Nivolumab is given intravenously, with a mean half-life of 25 days [START_REF] Centanni | Clinical Pharmacokinetics and Pharmacodynamics of Immune Checkpoint Inhibitors[END_REF] (Table 1). No dose adjustment is recommended in patients with renal failure, and mild or moderate hepatic impairment because no side effects are expected under these conditions (data available on Bristol Myers Squibb. OPDIVO™ (Nivolumab)).

Pembrolizumab is administered intravenously, with a mean half-life of 22 days [START_REF] Centanni | Clinical Pharmacokinetics and Pharmacodynamics of Immune Checkpoint Inhibitors[END_REF] (Table 1). No dose adjustment is recommended in patients with renal failure, and mild or moderate hepatic impairment (data available on Merck Sharp & Dohme Corp.

KEYTRUDA®(Pembrolizumab)).

Cemiplimab is given intravenously, with a half-life of 20.3 days (Table 1). Mild or moderate renal failure (estimated glomerular filtration rate (eGFR) ≥ 30 mL/min/1.73 m2) and mild to moderate liver failure (bilirubin < 3× upper limit of normal and any aspartate transaminase levels) had no clinically significant effect on the systemic exposure of cemiplimab (data available on accessdata.fda.gov with reference ID: 4750303) Atezolizumab is given intravenously, with a half-life of 27 days [START_REF] Centanni | Clinical Pharmacokinetics and Pharmacodynamics of Immune Checkpoint Inhibitors[END_REF] (Table 1). Mild or moderate renal failure (estimated glomerular filtration rate (eGFR) ≥ 30 mL/min/1.73 m2) and mild to moderate liver failure (bilirubin < 3× upper limit of normal and any aspartate transaminase levels) had no clinically significant effect on the systemic exposure of atezolizumab (data available on Genentech Inc. TECENTRIQ®(Atezolizumab)).

Durvalumab is administered intravenously, with a half-life of 18 days [START_REF] Centanni | Clinical Pharmacokinetics and Pharmacodynamics of Immune Checkpoint Inhibitors[END_REF] (Table 1).

Mild (creatinine clearance 60 to 89 mL/min) or moderate renal impairment (creatinine clearance 30 to 59 mL/min), and mild hepatic impairment (bilirubin ≤ upper limit of normal and aspartate transaminase levels > upper limit of normal or bilirubin > 1 to 1.5 x upper limit of normal and any aspartate transaminase levels) had no clinically significant effect on the pharmacokinetics of durvalumab (data available on AstraZeneca Pharmaceuticals L.P. IMFINZI®(Durvalumab)).

ICIs commonly display modest interpatient variability in PK. Some covariates could explain part of the observed variability. They varied between the approved ICIs, but sex, Eastern Cooperative Oncology Group (ECOG) PS, body weight, tumor type, tumor burden, baseline lactate dehydrogenase (LDH), estimated glomerular filtration rate, albumin, can be considered in the differences [START_REF] Feng | Model-Based Clinical Pharmacology Profiling of Ipilimumab in Patients with Advanced Melanoma[END_REF][START_REF] Bajaj | Model-Based Population Pharmacokinetic Analysis of Nivolumab in Patients With Solid Tumors[END_REF][START_REF] Ahamadi | Model-Based Characterization of the Pharmacokinetics of Pembrolizumab: A Humanized Anti-PD-1 Monoclonal Antibody in Advanced Solid Tumors[END_REF].

PK and precaution for use of anti-angiogenic Abs

Bevacizumab is given intravenously, with a half-life of 19.9 days (Table 1). No dose adjustment is recommended in patients with renal failure, and mild or moderate hepatic impairment [START_REF] Lu | Clinical Pharmacokinetics of Bevacizumab in Patients with Solid Tumors[END_REF].

Ramucirumab is administered intravenously, with a half-life of 14 days and with no dose adjustment in case of renal failure or mild/moderate hepatic impairment [44] (Table 1).

The use of anti-angiogenic molecules is indicated in selected patients only, because of its toxicity. Generally, in subjects with NSCLC, they are safe and well tolerated but some clinical or biological adverse events can interfere with the use of these molecules.

The most common adverse events are hypertension, proteinuria, and epistaxis. Infrequent serious adverse events include neutropenia complications, thromboembolic events, and pulmonary hemorrhage. In order to reduce the incidence of severe hemorrhage, the first randomized clinical trials of bevacizumab in NSCLC excluded: 1) subjects with squamous histology; 2) subjects with significant hemoptysis (≥ 1 teaspoon); 3) subjects with tumors invading or abutting major blood vessels or with central tumor localization or with tumor cavitation, based on a radiological assessment; 4) subjects with hemorrhagic disorders or in treatment with anticoagulant therapy; 5) subjects with brain metastases; 6) subjects with ECOG>1; and 7) elderly patients (age ≥75 years) [START_REF] Reck | Predicting and Managing the Risk of Pulmonary Haemorrhage in Patients with NSCLC Treated with Bevacizumab: A Consensus Report from a Panel of Experts[END_REF]. But after several years and real-life observations, safety and efficacy of bevacizumab is not affected by patient age, PS, anticoagulation therapy, and brain metastases. The only absolute contraindications to its use are squamous histology and a history of clinically significant hemoptysis [START_REF] Russo | Bevacizumab in the Treatment of NSCLC: Patient Selection and Perspectives[END_REF].

Treatment paradigm for NSCLC in 2021

The treatment for locally advanced or metastatic NSCLC has changed dramatically in the last 15 years. Since 2007, anti-angiogenic Abs have been used in advanced/metastatic recurrent non-squamous NSCLC [START_REF] Cohen | FDA Drug Approval Summary: Bevacizumab (Avastin) plus Carboplatin and Paclitaxel as First-Line Treatment of Advanced/Metastatic Recurrent Nonsquamous Non-Small Cell Lung Cancer[END_REF]. In March 2015, the EMA and FDA approved the first anti-PD-1 antibody as second line option for treatment of patients with advanced squamous NSCLC [START_REF] Brahmer | Nivolumab versus Docetaxel in Advanced Squamous-Cell Non-Small-Cell Lung Cancer[END_REF]. Additionally, a series of inhibitors related to CTLA-4 and PD-1/PD-L1 immune checkpoints have led to the use of immunotherapy for most NSCLC patients, and significantly revised the chemotherapy treatment model. The use of therapeutic Abs (anti-angiogenic or ICI) has become the cornerstone of first line treatment, either as a single modality or with chemotherapy. These advances in lung cancer management, along with targeted agents like tyrosine kinase inhibitors for lung cancer with oncogenic drivers or lifestyle alterations such as decreasing smoking among men, resulted in a decrease in population-level mortality from NSCLC in the US [START_REF] Howlader | The Effect of Advances in Lung-Cancer Treatment on Population Mortality[END_REF] similar to European Union. We will review the phase II/III clinical trials that led to the marketing authorizations of anti-angiogenic and ICI Abs currently used in NSCLC is the US and in European Union and in the US only (Table 2). We will present the indications according to the expression levels of PD-L1, using the same strata as in the clinical trials (> 50%, 1-49% and <1%).

Front line treatment in the US and in European Union

PD-L1 > 50%

Patients with metastatic NSCLC and with PD-L1 > 50% have multiple immunotherapy treatment options. In both the US and European Union, monotherapy with pembrolizumab is an approach that may be used, and ICI and chemotherapy could also be given as front-line therapy to these patients.

Pembrolizumab was first used as monotherapy for second-line treatment and beyond (see section below). In the front-line setting, a randomized phase III trial, KEY-NOTE-024, recruited 305 previously-untreated patients with NSCLC having PD-L1 expression in more than 50% tumor cells and no EGFR/ALK mutations to either receive pembrolizumab or platinum-based chemotherapy [START_REF] Reck | Pembrolizumab versus Chemotherapy for PD-L1-Positive Non-Small-Cell Lung Cancer[END_REF]. Patients treated with pembrolizumab showed better progression-free survival (PFS), overall survival (OS), and overall response rate (ORR) than those treated with chemotherapy [START_REF] Reck | Pembrolizumab versus Chemotherapy for PD-L1-Positive Non-Small-Cell Lung Cancer[END_REF][START_REF] Reck | Updated Analysis of KEYNOTE-024: Pembrolizumab Versus Platinum-Based Chemotherapy for Advanced Non-Small-Cell Lung Cancer With PD-L1 Tumor Proportion Score of 50% or Greater[END_REF]. Based on these results, the FDA and EMA approved pembrolizumab as a single-agent first-line immunotherapy in patients with advanced NSCLC harboring high PDL1 expression (PD-L1 > 50%). The 3-year survival results of KEYNOTE-024 indicated that the pembrolizumab monotherapy significantly prolonged the median OS length (26.3 versus 14.2 months) and the 3-year OS rate (43.7 versus 24.9%) than is those treated with chemotherapy [START_REF] Reck | Updated Analysis of KEYNOTE-024: Pembrolizumab Versus Platinum-Based Chemotherapy for Advanced Non-Small-Cell Lung Cancer With PD-L1 Tumor Proportion Score of 50% or Greater[END_REF].

Moreover, the KEYNOTE-042 phase III trial, with the same patient groups, divided each treatment arm into three subgroups based on the level of PD-L1 expression (PD-L1 > 50%, > 20%, > 1%) [START_REF] Mok | Pembrolizumab versus Chemotherapy for Previously Untreated, PD-L1-Expressing, Locally Advanced or Metastatic Non-Small-Cell Lung Cancer (KEYNOTE-042): A Randomised, Open-Label, Controlled, Phase 3 Trial[END_REF]. All groups showed significantly better OS with pembrolizumab rather than chemotherapy but the OS of patients who received pembrolizumab in the high PD-L1 expression arm (>50%) showed maximum benefit. These results supported pembrolizumab administration as a single-drug in patients with advanced NSCLC harboring PD-L1>50%. in the absence of contraindication to bevacizumab. Bevacizumab is the only anti-angiogenic agent approved for the first-line treatment of NSCLC in selected patients [START_REF] Jenab-Wolcott | Bevacizumab: Current Indications and Future Development for Management of Solid Tumors[END_REF].

Recently, another combination of two ICIs and chemotherapy has been approved.

The CheckMate-9LA study randomized patients to receive nivolumab plus ipilimumab combined with histology-based, platinum doublet chemotherapy (for two cycles), or chemotherapy alone (for four cycles) [START_REF] Paz-Ares | First-Line Nivolumab plus Ipilimumab Combined with Two Cycles of Chemotherapy in Patients with Non-Small-Cell Lung Cancer (CheckMate 9LA): An International, Randomised, Open-Label, Phase 3 Trial[END_REF]. The OS, as well as secondary endpoints of PFS and ORR, were superior for the combination nivolumab plus ipilimumab, given concomitantly with two cycles of chemotherapy, versus chemotherapy alone. This immune-chemotherapy combination is approved regardless the PD-L1 expression and the tumor histology (Table 2).

PD-L1 1-49 %

For patients with PD-L1 between 1-49%, treatment options shared by the US and the European Union are pembrolizumab and chemotherapy association regardless the histology type [START_REF] Gandhi | Pembrolizumab plus Chemotherapy in Metastatic Non-Small-Cell Lung Cancer[END_REF][START_REF] Paz-Ares | Pembrolizumab plus Chemotherapy for Squamous Non-Small-Cell Lung Cancer[END_REF], atezolizumab-chemotherapy-bevacizumab only for non-squamous NSCLC [START_REF] Socinski | Atezolizumab for First-Line Treatment of Metastatic Nonsquamous NSCLC[END_REF] and combination of nivolumab, ipilimumab and two cycles of chemotherapy for all PD-L1 expressions [START_REF] Paz-Ares | First-Line Nivolumab plus Ipilimumab Combined with Two Cycles of Chemotherapy in Patients with Non-Small-Cell Lung Cancer (CheckMate 9LA): An International, Randomised, Open-Label, Phase 3 Trial[END_REF] (Table 2).

PD-L1 < 1%

For patients with PD-L1 < 1%, treatment options shared by the US and the European Union are pembrolizumab and chemotherapy association regardless the histology type [START_REF] Gandhi | Pembrolizumab plus Chemotherapy in Metastatic Non-Small-Cell Lung Cancer[END_REF][START_REF] Paz-Ares | Pembrolizumab plus Chemotherapy for Squamous Non-Small-Cell Lung Cancer[END_REF], or atezolizumab-chemotherapy-bevacizumab only for non-squamous NSCLC [START_REF] Socinski | Atezolizumab for First-Line Treatment of Metastatic Nonsquamous NSCLC[END_REF] (Table 2). In European Union only, the combination of nivolumab, ipilimumab with two cycles of chemotherapy for all PD-L1 expressions can be used [START_REF] Paz-Ares | First-Line Nivolumab plus Ipilimumab Combined with Two Cycles of Chemotherapy in Patients with Non-Small-Cell Lung Cancer (CheckMate 9LA): An International, Randomised, Open-Label, Phase 3 Trial[END_REF].

Front line treatment in the US only

Most treatment options in the US and in European Union are similar but there are some indications currently specific for the US.

PD-L1 > 50%

The FDA has granted approval to atezolizumab monotherapy as treatment for patients with metastatic NSCLC whose tumors have PD-L1 expression greater than 50%, without EGFR/ALK mutations. In fact, the Impower110 study compared atezolizumab with chemotherapy among patients with high PD-L1 expression [START_REF] Herbst | Atezolizumab for First-Line Treatment of PD-L1-Selected Patients with NSCLC[END_REF]. The median OS was 20.1 months with atezolizumab versus 13.1 months with chemotherapy, at a median follow-up of 15.7 months.

Recently, another ICI monotherapy has been approved in the US. The EMPOW-ER-Lung 1 phase III open-label study randomized patients to receive cemiplimab of platinum-based chemotherapy. Primary endpoints were assessed in the intention-to-treat population and in a prespecified PD-L1 of at least 50% population [START_REF] Sezer | Cemiplimab Monotherapy for First-Line Treatment of Advanced Non-Small-Cell Lung Cancer with PD-L1 of at Least 50%: A Multicentre, Open-Label, Global, Phase 3, Randomised, Controlled Trial[END_REF]. Cemiplimab reduced the risk of death by 43% compared to chemotherapy. This was achieved with crossover rate to cemiplimab greater than 70% following disease progression on chemotherapy, as well as the largest population of patients with pre-treated and clinically stable brain metastases among advanced NSCLC pivotal trials to date. Therefore, in the US, for these patients with PD-L1 expression ≥ 50%, the choice between pembrolizumab, cemiplimab and atezolizumab can be made as ICI monotherapy for front line treatment.

An ICI combination without chemotherapy is also possible in the US: ipilimumab and nivolumab (see section below) (Table 2).

PD-L1 1-49 %

For patients with PD-L1 expression 1-49%, 2 additional options are available in the USA compared to European Union. In 2019, FDA expanded pembrolizumab indication for first-line treatment of NSCLC with PD-L1 expression greater than 1%. This approval was based on the KEYNOTE-042 trial [START_REF] Mok | Pembrolizumab versus Chemotherapy for Previously Untreated, PD-L1-Expressing, Locally Advanced or Metastatic Non-Small-Cell Lung Cancer (KEYNOTE-042): A Randomised, Open-Label, Controlled, Phase 3 Trial[END_REF]: in the PD-L1 ≥ 1% population (overall population), the median OS was 16.7 and 12.1 months for the pembrolizumab and chemotherapy arms, respectively. In the ≥ 50% subgroup, the estimated median OS was 20 months and 12.2 months for those receiving pembrolizumab and chemotherapy, respectively. There were no significant differences in progression-free survival or overall response rate between arms in any population.

The second additional option compared to European Union is the ICI combination nivolumab and ipilimumab, based on the CheckMate227 study [START_REF] Hellmann | Nivolumab plus Ipilimumab in Lung Cancer with a High Tumor Mutational Burden[END_REF][START_REF] Hellmann | Nivolumab plus Ipilimumab in Advanced Non-Small-Cell Lung Cancer[END_REF]. This trial recruited treatment-naïve patients with advanced NSCLC. They were randomized to nivolumab plus ipilimumab, nivolumab with or without chemotherapy, and histology-based chemotherapy arms [START_REF] Hellmann | Nivolumab plus Ipilimumab in Lung Cancer with a High Tumor Mutational Burden[END_REF][START_REF] Hellmann | Nivolumab plus Ipilimumab in Advanced Non-Small-Cell Lung Cancer[END_REF]. According to PD-L1 expression, patients were enrolled into PD-L1 > 1% and <1% cohorts, and further randomized 1:1:1 to nivolumab plus ipilimumab, platinum-based chemotherapy, or nivolumab monotherapy (PD-L1 > 1% group) or nivolumab plus platinum-based chemotherapy (PD-L1 < 1% group). The study protocol was later modified to include a co-primary endpoint of PFS in patients with high tumor-mutational burden (TMB), as defined by ≥10 mutations per megabase. Among patients with high TMB, nivolumab plus ipilimumab arm showed a significantly prolonged PFS than chemotherapy arm (7.2 versus 5.5 months) but median OS was not significantly different among high TMB. The final analysis for Checkmate-227 was based on the PD-L1 ≥ 1% cohort and demonstrated a significantly higher median OS in patients who received ipilimumab plus nivolumab arm than in patients treated with chemotherapy alone (median OS: 17.1 months vs 14.9 months) [START_REF] Hellmann | Nivolumab plus Ipilimumab in Advanced Non-Small-Cell Lung Cancer[END_REF]. Median PFS was also improved with nivolumab plus ipilimumab over chemotherapy for all patients regardless of PD-L1 expression, but was numerically higher for patients in the PD-L1 expression < 1% cohort.

The final analysis did not demonstrate improved OS based on TMB. In CheckMate568, the association of efficacy with PD-L1 expression and TMB was assessed in patients who received first-line nivolumab plus ipilimumab [START_REF] Ready | First-Line Nivolumab Plus Ipilimumab in Advanced Non-Small-Cell Lung Cancer (CheckMate 568): Outcomes by Programmed Death Ligand 1 and Tumor Mutational Burden as Biomarkers[END_REF]. Higher response rates and improved PFS were observed in patients with TMB of 10 or more mutations per megabase versus TMB of fewer than 10 mutations per megabase, irrespective of PD-L1 expression. Currently, nivolumab and ipilimumab combination therapy is approved in the US based on PD-L1 expression alone, for patients with PD-L1 expression > 1% (Table 2).

PD-L1 < 1%

There is no difference in therapeutic strategy for patients with PD-L1 expression less than 1% (Table 2). Nivolumab + ipilimumab + chemotherapy 6 Awaiting a recommendation at the end of January 2021

Table 2. Algorithm for front line immune checkpoint inhibitors treatement of patients with NSCLC based on PD-L1 expression

1 Chemotherapy is cisplatin or carboplatin + pemetrexed for non-squamous NSCLC. Chemotherapy is carboplatin + paclitaxel for squamous NSCLC 2 Continuation maintenance until progression, unacceptable toxicity, with pemetrexed in non-squamous NSCLC 3 Chemotherapy is carboplatin + paclitaxel 4 Continuation maintenance until progression, unacceptable toxicity 5 Double continuation maintenance with bevacizumab-atezolizumab until progression or unacceptable toxicity 6 Chemotherapy is cisplatin or carboplatin + pemetrexed for non-squamous NSCLC. Chemotherapy is carboplatin + paclitaxel for squamous NSCLC. 2 cycles of chemotherapy.

Patients who are not eligible to receive ICI

If first-line ICI cannot be used due to contraindications or active autoimmune disease, standard platinum-based doublet therapy is often given. The addition of bevacizumab to chemotherapy can also be discussed in eligible patients. Bevacizumab was evaluated in 2006 in a phase III clinical with stage IIIB-IV non-squamous NSCLC patients [START_REF] Sandler | Paclitaxel-Carboplatin Alone or with Bevacizumab for Non-Small-Cell Lung Cancer[END_REF]. Patients were treated with carboplatin-paclitaxel every three weeks for six cycles with or without bevacizumab at 15 mg/kg. Bevacizumab was then administered as maintenance therapy until evidence of disease progression or unacceptable toxicity occurred. OS was significantly higher in bevacizumab group (12.3 versus 10.3 months) and PFS was increased too. The same results were observed in the AVAiL trial with chemotherapy by cisplatin and gemcitabine, with or without bevacizumab, for the two doses tested (7.5 or 15 mg/kg) [START_REF] Reck | Phase III Trial of Cisplatin plus Gemcitabine with Either Placebo or Bevacizumab as First-Line Therapy for Nonsquamous Non-Small-Cell Lung Cancer: AVAil[END_REF]. After that, the phase III POINTBREAK trial randomized patients in two arms: pemetrexed-carboplatin-bevacizumab followed by pemetrexed plus bevacizumab in maintenance or paclitaxel-carboplatin-bevacizumab followed by maintenance therapy with bevacizumab alone [START_REF] Patel | PointBreak: A Randomized Phase III Study of Pemetrexed plus Carboplatin and Bevacizumab Followed by Maintenance Pemetrexed and Bevacizumab versus Paclitaxel plus Carboplatin and Bevacizumab Followed by Maintenance Bevacizumab in Patients with Stage IIIB or IV Nonsquamous Non-Small-Cell Lung Cancer[END_REF]. The primary end-point of OS did not reach statistical significance, although in the group treat with pemetrexed and bevacizumab in maintenance therapy, an increased PFS was reported. Another important phase III trial was AVAPERL study: patients were randomized, after four cycles of chemotherapy with cisplatin-pemetrexed-bevacizumab, to receive maintenance therapy with bevacizumab alone or with pemetrexed and bevacizumab [START_REF] Barlesi | Maintenance Bevacizumab-Pemetrexed after First-Line Cisplatin-Pemetrexed-Bevacizumab for Advanced Nonsquamous Nonsmall-Cell Lung Cancer: Updated Survival Analysis of the AVAPERL (MO22089) Randomized Phase III Trial[END_REF]. The PFS was improved in the double maintenance group (10.2 versus 6.6 months) and the result for OS was the same, but it was not statistically significant.

VEGF/VEGFR antibody therapy in patients with EGFR mutations

TKI are standard treatment options for NSCLC with EGFR mutations. Some preclinical studies had shown that the EGFR signaling pathway can upregulate VEGF expression [START_REF] Yen | Heregulin Selectively Upregulates Vascular Endothelial Growth Factor Secretion in Cancer Cells and Stimulates Angiogenesis[END_REF] and that VEGF/VEGFR can play a role in EGFR-TKI resistance [START_REF] Naumov | Combined Vascular Endothelial Growth Factor Receptor and Epidermal Growth Factor Receptor (EGFR) Blockade Inhibits Tumor Growth in Xenograft Models of EGFR Inhibitor Resistance[END_REF]. This led to the investigation of combination treatments with EGFR-TKIs and anti-angiogenic Ab.

In European Union, bevacizumab in combination with erlotinib, has been indicated for first-line treatment of adult patients with unresectable advanced, metastatic or recurrent NSCLC with EGFR mutations since 2016. It is recommended that the treatment with bevacizumab in addition to erlotinib is continued until disease progression. The pivotal JO25567 study was a randomized phase II study conducted to assess the safety and efficacy of first-line bevacizumab in combination with erlotinib compared to erlotinib alone [START_REF] Seto | Erlotinib Alone or with Bevacizumab as First-Line Therapy in Patients with Advanced Non-Squamous Non-Small-Cell Lung Cancer Harbouring EGFR Mutations (JO25567): An Open-Label, Randomised, Multicentre, Phase 2 Study[END_REF] in Japanese patients with advanced NSCLC with EGFR mutations.

Seventy-seven patients were randomly assigned to receive erlotinib and bevacizumab and 77 to erlotinib alone. Median PFS was 16.0 months with erlotinib plus bevacizumab and 9.7 months with erlotinib alone. OS was not improved in patients receiving the combination. The safety analysis population comprised 75 patients in erlotinib plus bevacizumab group and 77 in erlotinib group, who received at least one dose of study drug. There was no difference in incidence of serious adverse events between the 2 groups. Nevertheless, more grade > 3 adverse events were reported with erlotinib plus bevacizumab (90.7%) than with erlotinib (53.2%). It was primarily due to grade 3 hypertension, but it was controllable with antihypertensive medications in most cases. Proteinuria and bleeding were also more frequently reported with the association treatment but were manageable and did not lead to early discontinuations [START_REF] Kato | Erlotinib Plus Bevacizumab Phase Ll Study in Patients with Advanced Non-Small-Cell Lung Cancer (JO25567): Updated Safety Results[END_REF].

More recently, in 2020, the FDA approved ramucirumab in combination with erlotinib for first-line treatment of metastatic NSCLC with EGFR mutations. Efficacy was evaluated in the RELAY trial, a multinational, randomized, double-blind, placebo-controlled, world-wild, multicenter study in patients with previously untreated metastatic NSCLC whose tumors have EGFR exon 19 deletion or exon 21 (L858R) substitution mutations [START_REF] Nakagawa | Ramucirumab plus Erlotinib in Patients with Untreated, EGFR-Mutated, Advanced Non-Small-Cell Lung Cancer (RELAY): A Randomised, Double-Blind, Placebo-Controlled, Phase 3 Trial[END_REF]. A total of 449 patients were randomized (1:1) to receive either ramucirumab 10 mg/kg or placebo every 2weeks, in combination with erlotinib once daily, until disease progression or unacceptable toxicity. Median PFS was 19.4 months in the ramucirumab plus erlotinib arm compared with 12.4 months in the placebo plus erlotinib arm. ORR was 76% in the ramucirumab plus erlotinib arm and 75% in the pla-cebo plus erlotinib arm, with median duration of response of 18.0 months and 11.1 months, respectively. The follow up was not sufficient to determine whether the combination led to an OS benefit. The most common adverse reactions observed in patients treated with ramucirumab with erlotinib at a rate of ≥20% and ≥2% higher than placebo with erlotinib were infections, hypertension, stomatitis, proteinuria, alopecia, epistaxis, and peripheral edema. Most patients who can receive ICI therapy are treated with first-line immunotherapy with or without chemotherapy, and ICI re-challenge is not a standard second line treatment. Ramucirumab is an anti-angiogenic Ab that can be used in the 2nd line setting NSCLC. The REVEL study was a phase III, placebo-controlled trial which included patients with metastatic NSCLC who had progressed during or after platinum-based chemotherapy, with or without bevacizumab [START_REF] Garon | Ramucirumab plus Docetaxel versus Placebo plus Docetaxel for Second-Line Treatment of Stage IV Non-Small-Cell Lung Cancer after Disease Progression on Platinum-Based Therapy (REVEL): A Multicentre, Double-Blind, Randomised Phase 3 Trial[END_REF]. Patients were randomly allocated to receive docetaxel and either ramucirumab or placebo. Median OS was 9.1 months versus 10.5 months in the placebo and ramucirumab group respectively. The addition of ramucirumab to docetaxel can be considered in younger patients with good PS as a second line treatment.

Using a therapeutic strategy containing bevacizumab or ramucirumab is feasible in selected patients because of the efficacy and safety related to the administration of these Abs [START_REF] Lima | Addition of Bevacizumab to Chemotherapy in Advanced Non-Small Cell Lung Cancer: A Systematic Review and Meta-Analysis[END_REF]. Ramucirumab was safe across all NSCLC histologies in the second line setting.

The anti-angiogenic Ab, bevacizumab, is only used in non-squamous NSCLC because of more frequent and severe pulmonary hemorrhage in patients with squamous NSCLC, as shown in a phase II trial in 2004 [START_REF] Johnson | Randomized Phase II Trial Comparing Bevacizumab plus Carboplatin and Paclitaxel with Carboplatin and Paclitaxel Alone in Previously Untreated Locally Advanced or Metastatic Non-Small-Cell Lung Cancer[END_REF]. In fact, squamous cell tumors are more likely centrally located and cavitated compared to adenocarcinoma; but it is not clear whether the histology, as is, is an independent risk factor or a marker of increased risk. Another problem was the presence of brain metastasis, at risk of bleeding: a factor long considered as an exclusion criterion for treatment with bevacizumab. A retrospective analysis of 17 studies on the use of bevacizumab in patients with untreated metastases for lung, breast, kidney or colorectal cancer showed that the use of bevacizumab did not increase risk of bleeding [START_REF] Besse | Bevacizumab Safety in Patients with Central Nervous System Metastases[END_REF]. The BRAIN phase II trial enrolled patients with untreated brain metastases and the rate of central nervous system hemorrhage was comparable to that of previous bevacizumab study, in which the presence of untreated brain metastases was an exclusion criteria [START_REF] Besse | Bevacizumab in Patients with Nonsquamous Non-Small Cell Lung Cancer and Asymptomatic, Untreated Brain Metastases (BRAIN): A Nonrandomized, Phase II Study[END_REF]. Without detailing all the studies supporting these results, we can say that many of the factors previously considered as exclusion criteria, such as anticoagulant treatment, central tumor location, the presence of cavitation or brain metastases, and an advanced age, are not currently valid [START_REF] Crinò | Safety and Efficacy of First-Line Bevacizumab-Based Therapy in Advanced Non-Squamous Non-Small-Cell Lung Cancer (SAiL, MO19390): A Phase 4 Study[END_REF][START_REF] Reck | Predicting and Managing the Risk of Pulmonary Haemorrhage in Patients with NSCLC Treated with Bevacizumab: A Consensus Report from a Panel of Experts[END_REF][START_REF] Marom | Tumor Cavitation during Therapy with Antiangiogenesis Agents in Patients with Lung Cancer[END_REF][START_REF] Leighl | Efficacy and Safety of Bevacizumab-Based Therapy in Elderly Patients with Advanced or Recurrent Nonsquamous Non-Small Cell Lung Cancer in the Phase III BO17704 Study[END_REF]. In fact, the only exclusion criteria that are absolute contraindications to the use of bevacizumab are squamous histology and the presence of hemoptysis [START_REF] Reck | Predicting and Managing the Risk of Pulmonary Haemorrhage in Patients with NSCLC Treated with Bevacizumab: A Consensus Report from a Panel of Experts[END_REF].

If chemotherapy alone has been prescribed in the first line, monotherapy with ICI may be considered. As a second-line therapy for stage IIIB or IV squamous NSCLC with disease recurrence after one prior platinum-containing regimen, in a phase III study, CheckMate 017, nivolumab was compared to docetaxel [START_REF] Brahmer | Nivolumab versus Docetaxel in Advanced Squamous-Cell Non-Small-Cell Lung Cancer[END_REF]. Nivolumab was shown to be more efficacious than chemotherapy: OS was improved in the ICI group ( The third monotherapy ICI available is atezolizumab, approved in 2016 by the FDA and EMA for the treatment of patients with NSCLC whose cancer had progressed during or after standard chemotherapy. This approval was based on two trials, phase II POPLAR [START_REF] Fehrenbacher | Atezolizumab versus Docetaxel for Patients with Previously Treated Non-Small-Cell Lung Cancer (POPLAR): A Multicentre, Open-Label, Phase 2 Randomised Controlled Trial[END_REF] and phase III OAK study [START_REF] Rittmeyer | Atezolizumab versus Docetaxel in Patients with Previously Treated Non-Small-Cell Lung Cancer (OAK): A Phase 3, Open-Label, Multicentre Randomised Controlled Trial[END_REF]. The randomized OAK trial compared atezolizumab to docetaxel and found a median OS of 13,8 months versus 9,6 months in the chemotherapy group.

locally advanced inoperable NSCLC (IIIA/B/C stages)

In patients with stage III NSCLC that is not resectable, ICI has been considered.

According to the PACIFIC study [START_REF] Antonia | Durvalumab after Chemoradiotherapy in Stage III Non-Small-Cell Lung Cancer[END_REF], durvalumab is given for 12 months if no progression after concomitant radio-chemotherapy is seen, but only if PD-L1 > 1% in some countries. In the US and France, durvalumab is allowed in patients with a PD-L1 < 1%.

Treatment decision algorithm in European Union and in the US

For the use of ICI, contraindications to those molecules should always be kept in mind (uncontrolled autoimmune pathologies, uncontrolled HIV-HBV-HCV infections, for example) but this is to be discussed on a case-by-case basis, during multidisciplinary meeting [73].

In the front-line setting, the first selection is then made based on the expression of PD-L1 (Figure 2 and 3). In chemotherapy-immunotherapy trials, patients had a PS 0 or 1 and a median age of 65 years [START_REF] Gandhi | Pembrolizumab plus Chemotherapy in Metastatic Non-Small-Cell Lung Cancer[END_REF][START_REF] Paz-Ares | Pembrolizumab plus Chemotherapy for Squamous Non-Small-Cell Lung Cancer[END_REF]. Therefore, the pembrolizumab + chemotherapy regimen is mostly reserved for patients who are not too old and in good general condition. However, we observe the same data in the 1st line immunotherapy alone studies (PS 0-1 and median age 65 [START_REF] Mok | Pembrolizumab versus Chemotherapy for Previously Untreated, PD-L1-Expressing, Locally Advanced or Metastatic Non-Small-Cell Lung Cancer (KEYNOTE-042): A Randomised, Open-Label, Controlled, Phase 3 Trial[END_REF]); in practice, immunotherapy as monotherapy seems to have better tolerance and is therefore to be favored in older patients and in less good general condition. In any case, there is no restriction of approvals and marketing authorizations in European Union and in the US based on age or PS. Finally, in case of non-squamous NSCLC, the use of the quadruple combination ABCP must include verification that contraindications to antiangiogenic agents do not exist [START_REF] Russo | Bevacizumab in the Treatment of NSCLC: Patient Selection and Perspectives[END_REF]. Regarding nivolumab-ipilimumab-chemotherapy combination, the study group included 70% non-squamous NSCLC, 40% PD-L1 negative and 20% hepatic metastases [START_REF] Paz-Ares | First-Line Nivolumab plus Ipilimumab Combined with Two Cycles of Chemotherapy in Patients with Non-Small-Cell Lung Cancer (CheckMate 9LA): An International, Randomised, Open-Label, Phase 3 Trial[END_REF]; factors should be considered to reserve this combination for certain types of patients (Figure 2 and 3).

In the second line, the treatment is directly dictated in part by the treatment received in the front line. If chemotherapy as monotherapy has been used, regardless of PD-L1 expression, atezolizumab or nivolumab as monotherapy may be considered, in the absence of contraindications to ICI (Figure 2 and 3). Again, the trials included 0-1 PS patients with a median age of 65 years. The OAK study using atezolizumab [START_REF] Rittmeyer | Atezolizumab versus Docetaxel in Patients with Previously Treated Non-Small-Cell Lung Cancer (OAK): A Phase 3, Open-Label, Multicentre Randomised Controlled Trial[END_REF] included nearly 50% of patients over 65 years of age and 10% of them had stable brain metastases.

The Checkmate studies with nivolumab [START_REF] Brahmer | Nivolumab versus Docetaxel in Advanced Squamous-Cell Non-Small-Cell Lung Cancer[END_REF][START_REF] Borghaei | Nivolumab versus Docetaxel in Advanced Nonsquamous Non-Small-Cell Lung Cancer[END_REF] included 41% of patients over 65 in the nivolumab arm but 93% had no brain metastases. The center-specific experience can then also be taken into account to choose between these 2 ICI. The expression of PD-L1 is not restrictive for the prescription of these 2 molecules, unlike pembrolizumab, where the tumor must express more than 1% of PD-L1 (Figure 2 and3). The Keynote studies [START_REF] Herbst | Pembrolizumab versus Docetaxel for Previously Treated, PD-L1-Positive, Advanced Non-Small-Cell Lung Cancer (KEY-NOTE-010): A Randomised Controlled Trial[END_REF] included 15% of patients with stable brain metastases. The clinician is therefore free to choose ICI as monotherapy in 2nd line if the patient did not receive in the front line setting and does not have contraindications.

If an ICI has been used as a first-line monotherapy, it is possible to treat the patient with a chemotherapy and anti-angiogenic combination. This in the absence of contraindications to bevacizumab [START_REF] Russo | Bevacizumab in the Treatment of NSCLC: Patient Selection and Perspectives[END_REF] and for non-squamous NSCLC only [START_REF] Lima | Addition of Bevacizumab to Chemotherapy in Advanced Non-Small Cell Lung Cancer: A Systematic Review and Meta-Analysis[END_REF]: the regimen is then platinum-based chemotherapy, pemetrexed or paclitaxel, and bevacizumab. If platinum-based chemotherapy has been used in first line with an ICI, the second line chemotherapy association could be paclitaxel-bevacizumab. Finally, a last possible chemotherapy anti-angiogenic Ab combination is docetaxel with ramucirumab [START_REF] Garon | Ramucirumab plus Docetaxel versus Placebo plus Docetaxel for Second-Line Treatment of Stage IV Non-Small-Cell Lung Cancer after Disease Progression on Platinum-Based Therapy (REVEL): A Multicentre, Double-Blind, Randomised Phase 3 Trial[END_REF] for non-squamous NSCLC only (Figure 2 and3). There are therefore more options for non-squamous than for NSCLC squamous in second line of treatment.

27 the

 27 leading cause of cancer death. It comprises non-small cell lung cancer (NSCLC), the 28 most frequent subtype, and small cell lung cancer (SCLC). Despite recent therapeutic 29 progresses, it still constitutes a major public health problem, due to a diagnosis fre-30 quently made at advanced stage with a high cost to society. A better understanding of 31 tumor and immune signaling pathways has enabled the recent discovery of new molec-32 ular targets. Immunotherapy with immune checkpoint inhibitors (ICIs) has changed the 33 treatment paradigm in many tumor types and improved survival in a subset of patients

Figure 1 .

 1 Figure 1. Characteristics of immune checkpoint inhibitors and anti-angiogenic antibodies used in NSCLC. Created with BioRender.com

The addition of PD- 1 /

 1 PD-L1 inhibition to standard chemotherapy in treatment-naïve NSCLC patients was investigated, regardless of PD-L1 expression. KEYNOTE-189 was a phase III trial that assessed first-line platinum-based chemotherapy with or without pembrolizumab in EGFR/ALK-wild type, non-squamous NSCLC patients[START_REF] Gandhi | Pembrolizumab plus Chemotherapy in Metastatic Non-Small-Cell Lung Cancer[END_REF]. The median OS was not reached at the time of analysis for the pembrolizumab-chemotherapy arm versus 11.3 months for placebo-chemotherapy arm, and the OS advantage was achieved in all PD-L1 subgroups. On the basis of these results, pembrolizumab in combination with pemetrexed and carboplatin as front-line treatment in metastatic non-squamous NSCLC became a new standard, regardless of PD-L1 expression. Similar treatment was then evaluated in squamous NSCLC: the KEYNOTE-407 trial randomized patients to receive 4 cycles of carboplatin and paclitaxel or nab-paclitaxel with or without pembrolizumab[START_REF] Paz-Ares | Pembrolizumab plus Chemotherapy for Squamous Non-Small-Cell Lung Cancer[END_REF]. In the same way, patients in the pembrolizumab-containing group showed a significantly improved OS compared with those in the chemotherapy group (15.9 versus 11.3 months). The combination with pembrolizumab and platinum-based chemotherapy is therefore used in the front line setting regardless of the level of PD-L1 expression or the tumor histology.Another possible treatment option in the US and in European Union for patients with a PD-L1 greater than 50% is a combination of chemotherapy, atezolizumab and bevacizumab for non-squamous NSCLC. The Impower150 trial randomized patients to 3 groups: atezolizumab-bevacizumab-carboplatin-paclitaxel (ABCP), atezolizumab-carboplatin-paclitaxel (ACP) and bevacizumab-carboplatin-paclitaxel (BCP). Both the median PFS and OS were improved in the atezolizumab-containing arm (PFS: 8.3 versus 6.8 months; OS: 19.2 versus 14.7 months) compared with the patients treated with BCP [54]. Therefore, this combination is authorized in the front line setting in the US and in European Union for advanced NSCLC regardless of PD-L1 expression,

5. 3

 3 Second-line treatment and beyond in European Union and the USSecond line treatment for metastatic lung cancer is determined by the type of agents received in the first line setting. There are some challenges in second line-treatment: patients are pre-treated, often with reduced PS, thus tolerability becomes more important; responses rates are lower; thus, tumor control becomes important; and patients can be more symptomatic, thus symptom control and symptom improvement become important.

  pembrolizumab 200 mg every three weeks were similar to the pembrolizumab exposure data observed in patients with other solid tumors (mainly melanoma and NSCLC) receiving pembrolizumab 2 mg/kg every three weeks, supporting a fixed 200 mg every three dose for the head and neck cancer indication[72]. Based on these data, FDA and EMA recommended a fixed dose of pembrolizumab (200 mg) for the treatment of patients with metastatic NSCLC, irrespective of prior line of therapy.

Figure 3 .

 3 Figure 3. Potential treatment paradigm in European Union for NSCLC without actionable mutations in front line. Adapted from [74,75].

Table 1 .

 1 Characteristics of marketed antibodies in NSCLC. According to[START_REF] Centanni | Clinical Pharmacokinetics and Pharmacodynamics of Immune Checkpoint Inhibitors[END_REF]

	3 weeks (200 mg)
	6 weeks (400 mg)

  a lower median PFS (2,3 versus 4,2 months). Following these results, nivolumab was approved by the FDA and EMA as second-line therapy for metastatic NSCLC with progression on or after standard chemotherapy. PD-L1 testing was not required for nivolumab administration. This molecule was therefore the first ICI used in

	thoracic oncology.
	Unlike nivolumab, which received non-restricted FDA or EMA approval,
	pembrolizumab received accelerated approval with companion diagnostic PD-L1 assay
	of 1% or more for advanced NSCLC that progressed during or after front line chemo-
	therapy. The KEYNOTE-010 study recruited patients with NSCLC in second line setting
	and evaluated two dosing regimens for pembrolizumab (2 and 10 mg/kg) compared to
	docetaxel [8]. Pembrolizumab demonstrated an improved OS benefit at both tested doses
	in the total population compared with chemotherapy: OS was 10,4 and 12,7 months for
	the 2 and 10 mg/kg pembrolizumab doses respectively, compared with 8,5 months for the
	docetaxel group. Differences in PFS were not statistically significant between either of the
	pembrolizumab doses and docetaxel. Thus, on 2016, EMA and FDA approved
	pembrolizumab for treatment of patients with metastatic NSCLC whose tumors express
	PD-L1 greater than ≥1%) with disease progression on or after platinum-containing
	chemotherapy. Previous analyses indicated that the observed pembrolizumab exposures
	in patients with squamous cell carcinoma of the head and neck treated with

9,2 versus 6 months), as well as PFS (3,5 versus 2,8 months). Subsequently, the CheckMate 057 phase III study randomized patients with a metastatic non-squamous NSCLC in two groups: nivolumab versus docetaxel

[START_REF] Borghaei | Nivolumab versus Docetaxel in Advanced Nonsquamous Non-Small-Cell Lung Cancer[END_REF]

. Nivolumab demonstrated superior OS (12,2 versus 9,4 months) despite

Funding: This research received no external funding.

Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.

Data Availability Statement: Data sharing not applicable

Acknowledgments: Thanks to BioRender.com for figure creation


Adélaïde Chesnay

Éric Bailly

Guillaume Desoubeaux
email: guillaume.desoubeaux@univ-tours.fr

Demonstration of the

Keywords: Candida auris, povidone-iodine, EN 1275

HAL is

Dear Editor, First isolated from the external ear canal of a Japanese patient in 2009 [START_REF] Satoh | Candida auris sp. nov., a novel ascomycetous yeast isolated from the external ear canal of an inpatient in a Japanese hospital[END_REF] , Candida auris is an emerging yeast pathogen that has been put in the spotlight over the last decade as a global health threat; this species has been associated with multiple nosocomial outbreaks in several healthcare facilities worldwide, both in colonization and invasive infection cases [START_REF] Schelenz | First hospital outbreak of the globally emerging Candida auris in a European hospital[END_REF] , and is sometimes challenging for its identification [START_REF] Desoubeaux | Candida auris in contemporary mycology labs: A few practical tricks to identify it reliably according to one recent French experience[END_REF] and for choosing the adequate antifungal therapy because of elevated minimal inhibition concentrations (MICs) [START_REF] Lockhart | Simultaneous Emergence of Multidrug-Resistant Candida auris on 3 Continents Confirmed by Whole-Genome Sequencing and Epidemiological Analyses[END_REF] . However, accurate data are still lacking about C. auris, especially about its sensitivity to antiseptics that are usually supposed to play a critical role in the fight against nosocomial transmission.

Chlorine-based surface disinfectants and improved hydrogen peroxide disinfectants were already demonstrated to be highly active against Candida species, including C. auris [START_REF] Cadnum | Effectiveness of Disinfectants Against Candida auris and Other Candida Species[END_REF][START_REF] Moore | Yeasticidal activity of chemical disinfectants and antiseptics against Candida auris[END_REF] (at the opposite of quaternary ammonium disinfectants [START_REF] Cadnum | Effectiveness of Disinfectants Against Candida auris and Other Candida Species[END_REF] ), according to the European and International Standards EN 13624:2013 and ASTM E2197-11.

Nevertheless, no commercial disinfectant product has been officially registered so far by the health authorities for specific use against C. auris. Data appear even more limited about the yeasticidal activity of antiseptics: only a chlorhexidine-based product [chlorhexidine 2% (w/v) chlorhexidine gluconate in 70% (v/v)] and an iodinebased antiseptic [10% (v/v) povidone-iodine] were shown effective so far, against C. auris, according to the European Standard EN 13624:2013 [START_REF] Moore | Yeasticidal activity of chemical disinfectants and antiseptics against Candida auris[END_REF] . Thus, the aim of this study was to assess the in vitro yeasticidal activity of three different iodine-based antiseptic products against C. auris.

The isolate of C. auris, number 171 10744, was obtained from a 58 year-old-Lebanese man who had been hospitalized for liver transplantation [START_REF] Desoubeaux | Candida auris in contemporary mycology labs: A few practical tricks to identify it reliably according to one recent French experience[END_REF] (clinical collection of the Parasitology-Mycology laboratory of Tours University Hospital, France). Species identification was confirmed by MALDI-TOF mass spectrometry and DNA sequencing [START_REF] Desoubeaux | Candida auris in contemporary mycology labs: A few practical tricks to identify it reliably according to one recent French experience[END_REF] . C. auris suspensions were obtained on malt extract media (PO 5055 A, OXOID, Hampshire, UK) [START_REF]Chemical Disinfectants and Antiseptics -Quantitative suspension Test for the evaluation of basic fungicidal or basic yeasticidal activity of chemical disinfectants and antiseptics -Test method and requirements (Phase 1)[END_REF] , and prepared to yield a final organism density comprised between 1.5 x 10 7 and 5.0 x 10 7 colony-forming units per milliliter (CFU/mL). The products to be tested included three commercial 'ready-to-use' povidoneiodine-based antiseptics: one foaming solution for cutaneous application [4% (w/v) povidone-iodine; MEDA PHARMA, Paris -France] named Betadine Scrub 4% ® for preoperative hand washing or antiseptic washing, and two solutions for cutaneous application [10% (w/v) povidone-iodine; MEDA PHARMA, Paris -France] referred to as Betadine dermique 10% ® used for the antisepsis of healthy or damaged skin and mucous membranes, and [5% (w/v) povidone-iodine, in 70% (v/v) ethanol 96%; MEDA PHARMA, Paris -France] so called Betadine alcoolique 5% ® used for surgical or skin antisepsis before minor surgery. All the aforementioned products were diluted with water for injection for the testing procedure: at 80% for all three, then at 0.25% for Betadine Scrub 4% ® , at 0.10% for Betadine dermique 10% ® , and at 2% for Betadine alcoolique 5% ® . The method for evaluating the yeasticidal efficacy was based on a quantitative dilution-neutralization testing according to the EN 1275:2005 standard [START_REF]Chemical Disinfectants and Antiseptics -Quantitative suspension Test for the evaluation of basic fungicidal or basic yeasticidal activity of chemical disinfectants and antiseptics -Test method and requirements (Phase 1)[END_REF] .

All the iodine-based antiseptics, at a concentration of 80% (as recommended by EN 1275:2005 standard [START_REF]Chemical Disinfectants and Antiseptics -Quantitative suspension Test for the evaluation of basic fungicidal or basic yeasticidal activity of chemical disinfectants and antiseptics -Test method and requirements (Phase 1)[END_REF] ), decreased C. auris viability to below the limit of efficiency defined for antiseptics [START_REF]Chemical Disinfectants and Antiseptics -Quantitative suspension Test for the evaluation of basic fungicidal or basic yeasticidal activity of chemical disinfectants and antiseptics -Test method and requirements (Phase 1)[END_REF] , achieving >4 Log 10 reduction (Table 1). Yeasticidal concentrations were even largely lower for Betadine scrub ® and alcoholic Betadine alcoolique ® , at 0.25% and 2% respectively. Same kind of tests were carried out on the Candida albicans ATCC 10231 reference strain and exhibited similar efficiency of the iodine-based antiseptics (Table 1).

As for C. auris, Betadine dermique ® showed lower yeasticidal concentrations for C. albicans at 0.1%. In addition, C. glabrata (i.e. a yeast species of clinical interest and which has proved problematic in surgical intensive care departments) was also tested and actually found less susceptible to povidone-iodine antiseptics than C. auris: for instance following 15 min contact with Betadine Scrub® diluted at 0.25%, the yeast survival rate was only 0.07% for C. auris vs. 1.5% for C. glabrata ATCC MYA2950 reference strain, and 0.25% for the clinical strains; for Betadine alcoolique®, only 0.02% for C. auris vs. 1.8% for C. glabrata reference strain and 0.36% for the clinical strains; and eventually for Betadine dermique®, only 0.01% for C. auris vs. 0.1% for C. glabrata reference strain (by the way, these data underline an efficacy of povidone-iodine antiseptics also against

C. glabrata).

Regarding the literature, there are only three studies that evaluated povidone-iodine against C. auris in vitro. Abdolrasouli et al. reported a growth inhibition for 12 clinical C. auris isolates by povidone-iodine concentrations between 0.07% and 1.25%, with a 3-min minimum contact time [START_REF] Abdolrasouli | In vitro efficacy of disinfectants utilised for skin decolonisation and environmental decontamination during a hospital outbreak with Candida auris[END_REF] . However, this study used a microdilution method that was not referred to any standard. In another study using a quantitative suspension test referring to the phase 2 application standards EN 13624:2013 7 , Moore et al. reported that povidone-iodine suspension was effective against four isolates of C. auris after a 2-min contact time, but with a product concentration at 10%, largely higher than ours. Finally in the third study, Kean et al. investigated the activity of povidone-iodine on fungal biofilms of four C. auris isolates 10 , using a three-dimensional complex model.

Povidone-iodine concentrations of 1.25 to 2.5% were required to inhibit the biofilms after a 5-min contact time, while reduced sensitivity to clinically-relevant chlorhexidine concentrations was concomitantly reported [START_REF] Kean | The comparative efficacy of antiseptics against Candida auris biofilms[END_REF] . Our study is the first one to evaluate the in vitro yeasticidal activity of three different iodine-based antiseptics against C. auris, according to the European Standard EN 1275:2005 [START_REF]Chemical Disinfectants and Antiseptics -Quantitative suspension Test for the evaluation of basic fungicidal or basic yeasticidal activity of chemical disinfectants and antiseptics -Test method and requirements (Phase 1)[END_REF] . Our findings are in agreement with the few literature data available, suggesting that povidone-iodine based antiseptics express an excellent yeasticidal activity against C. auris, herein at much lower dilutions than the 80% recommended by the standard. Some limitations can be underscored in our study. The in vitro behavior of the two fungal strains that have been tested appeared quite variable: in suspension and in culture, C. albicans and C. auris showed different characteristics from each other requiring minimal technical adaptations and some C. auris strains are known to exhibit differential phenotypes for aggregating and non-aggregating on which antiseptics could have different actions.

In conclusion, nosocomial transmission of C. auris appears to be multifactorial, involving a rapid and persistent skin colonization in affected patients that readily contaminate their immediate environment and the caregivers [START_REF] Schelenz | First hospital outbreak of the globally emerging Candida auris in a European hospital[END_REF][START_REF] Vallabhaneni | Investigation of the First Seven Reported Cases of Candida auris, a Globally Emerging Invasive, Multidrug-Resistant Fungus-United States, May 2013-August 2016[END_REF] .

Controlling and preventing the spread of C. auris requires the isolation of any colonized/infected individual, the detection of contact cases, the detection of environmental contamination, but also the reinforcement of standard hygiene measures. Thus, disinfectants and antiseptics should have to play a critical role in such a context. The results of this study, carried out according to the technical recommendations of the EN 1275:2005 standard, support the use of commercial povidone-iodine products as antiseptics for the healthcare fighting against C. auris.

Table 1: logarithmic decimal reduction in Candida auris and Candida albicans viability after exposure to povidone-iodine-based antiseptics, according to the EN 1275:2005 standard 1 mL of Candida test suspension was added to 1 mL of water for injection, and then incubated for 2 minutes (min) at 20°C. Afterwards, 8 mL of each povidone-iodine-based solution were added at the desired concentration; this mixture was incubated for 15 min at 20°C.

Thereafter, 1 mL of the resulting suspension was transferred to a tube containing 8 mL of sodium thiosulfate neutralizing solution and 1 mL of water for injection. After 5 min of neutralization at 20°C, aliquots of 1 mL of the aforementioned solution were plated (in duplicate) on malt extract media and incubated at 30°C for 48 h to subsequently enumerate the living yeasts by counting the CFU. Yeasticidal activity was expressed as the logarithmic decimal reduction in viability in comparison with the control situation without antiseptic.

ACKNOWLEDGEMENTS

The authors thank the direction of the research of Tours hospital for its precious help.

ETHICS

No approval was necessary.