# Required Evidence for Clinical Applications of Liquid Biopsy Using Especially CTCs in Lung Cancer
## 1 Lung Cancer  
Despite novel treatment options, lung cancer still has a high mortality rate and short survival time, making it a devastating diagnosis for patients. Less than 20% will survive longer than 5 years, making it the leading cause of cancer-related death [1]. Non-small cell lung cancer (NSCLC) makes up the majority of cases, with most patients being (former) smokers. However, the number of NSCLC patients that have never smoked is increasing. These patients often only have less genomic aberrations than smokers, mostly single nucleotide mutation, rearrangements or deletions, in a tumor driver or tumor suppressor gene. These genes, such as an epidermal growth factor receptor (EGFR), are by themselves capable of driving a cell to survive and proliferate (Figure 1).  
Figure 1: Targetable genes and main pathways that lead to cellular proliferation.  
For those patients who present with advanced disease, prognosis is not only dependent on the patient but also on tumor characteristics, as these determine which treatments are effective. Chemotherapy can be given to all patients but is infamous for its side effects. In NSCLC it has limited efficacy, but is often the only option to suppress symptoms and improve the quality of life. Other therapies, i.e., targeted therapy and immunotherapy, which can have long lasting effects, have become available for NSCLC, but are only effective in a small proportion of patients. Patients with specific genomic aberrations (Figure 1), more often present in non-smoking patients, are eligible for targeted therapy [2; 3; 4; 5]. Specific tyrosine kinase inhibitors (TKIs) provide impressive tumor responses, but not when their associated mutation is absent [4; 6]. TKIs are capable of disturbing the kinase activity necessary for signal transmissions, inhibiting specific pathways and thereby tumor growth. While many tumors develop resistance within 1-2 years, many of these tumors contain secondary, resistent mutations for which also new specific TKIs are (becoming) available [4; 6; 7; 8]. They are divided into on-target at the specific receptor and off-target resistance mechanisms, that are often unknown. Therefore, obtaining tumor biopsy material to monitor the presence of resistant mutations has become a vital part of quality care.  
Immunotherapy with checkpoint inhibitors act by inhibiting an immune escape mechanism of tumor cells. The most common used therapeutics inhibit the programmed death receptor 1 and its ligand (PD-1 and PD-L1). PD-L1 restrains the immune system as a negative immune regulator and inhibits the lytic activity of effector immune cells. The inhibition of this receptor increases the recognition of tumor cells as foreign. This therapy has been recently introduced and although only 20%-25% of NSCLC patients respond to single agent immune checkpoint inhibitors, responses can last for years [9]. The presence of PD-L1 (the inhibited mechanism for immune evasion) is a major factor in determining the chance that a patient will respond, but it is not a robust predictor. Even when PD-L1 is present on the majority of tumor cells, response rates only reach 40%, while up to 10% of patients will respond to therapy when PD-L1 is not detected. Markers that can improve the prediction are therefore desperately required.