*Article RAD51Bme* **Levels as a Potential Predictive Biomarker for PD-1 Blockade Response in Non-Small Cell Lung Cancer**

**Inês Maria Guerreiro 1,\*,**†**, Daniela Barros-Silva 2,**†**, Paula Lopes 2,3, Mariana Cantante 2,3, Ana Luísa Cunha 2,3, João Lobo 2,3,4, Luís Antunes 5, Ana Rodrigues 1, Marta Soares 1, Rui Henrique 2,3,4 and Carmen Jerónimo 2,4,\***


Received: 21 February 2020; Accepted: 1 April 2020; Published: 2 April 2020

**Abstract:** Lung cancer (LC) cells frequently express high levels of programmed death-ligand 1 (PD-L1). Although these levels grossly correlate with the likelihood of response to specific checkpoint inhibitors, the response prediction is rather imperfect, and more accurate predictive biomarkers are mandatory. We examined the methylation profile of *RAD51B* (*RAD51Bme*) as a candidate predictive biomarker for anti-PD-1 therapy efficacy in non-small cell lung cancer (NSCLC), correlating with patients' outcome. PD-L1 immunoexpression and *RAD51Bme* levels were analysed in NSCLC samples obtained from patients not treated with anti-PD-1 (Untreated Cohort (#1)) and patients treated with PD-1 blockade (Treated Cohort (#2)). Of a total of 127 patients assessed, 58.3% depicted PD-L1 positivity (PD-L1+). *RAD51Bme* levels were significantly associated with PD-L1 immunoexpression. Patients with PD-1 blockade clinical benefit disclosed higher *RAD51Bme* levels (*p* = 0.0390) and significantly lower risk of disease progression (HR 0.37; 95% CI: 0.15–0.88; *p* = 0.025). Combining *RAD51Bme*<sup>+</sup> with PD-L1<sup>+</sup> improved the sensitivity of the test to predict immunotherapy response. PD-L1<sup>+</sup> was also associated with lower risk of death (HR 0.35; 95% CI: 0.15–0.81; *p* = 0.014). Thus, *RAD51Bme* levels might be combined with validated predictive biomarker PD-L1 immunostaining to select patients who will most likely experience clinical benefit from PD-1 blockade. The predictive value of *RAD51Bme* should be confirmed in prospective studies.

**Keywords:** *RAD51B* methylation; PD-L1 expression; predictive biomarker; PD-1 blockade

### **1. Introduction**

Lung cancer is the leading cause of cancer death in Europe, with an estimated 470,000 new cases (311,000 in men and 158,200 in women) in 2018 [1]. The estimated mortality in 2018 was 20.1% in both genders, being the most common cause of death from cancer in men (267,000 deaths, 24.8%) and the second most frequent in women (121,000 deaths, 14.2%) [1]. Most patients are diagnosed at advanced stages, with an overall 5-year survival rate of 4–17% depending on the stage and regional differences [2]. The incidence of lung cancer is directly related to tobacco smoking, which is the primary cause of lung cancer, accounting for about 80% to 90% of cases [3]. The risk of lung cancer increases with the extent of smoking measured by the number of packs of cigarettes smoked per day and with the number of years of smoking (pack-years of smoking history) [4].

Since the emergence of personalised targeted therapies, pathology plays a critical role because histologic and genetic features of lung cancer are important determinants of molecular testing and treatment decisions [5–7]. Lung cancer can be classified in non-small cell lung cancer (NSCLC) and small-cell lung cancer [5]. NSCLC is the most frequent class of lung cancer, representing 80% of all cases [4] and includes non-squamous carcinoma and squamous cell carcinoma as major types [5]. Non-squamous carcinoma includes adenocarcinoma, which is the most common subtype of lung cancer [4]. When clear adenocarcinoma, squamous or neuroendocrine morphology or staining pattern is not present, NSCLC is generally classified as not otherwise specified (NOS) [5].

Several predictive biomarkers indicative of therapeutic efficacy have emerged in lung cancer [6]. Immunotherapy, mainly immune checkpoint inhibitors, has changed the treatment paradigm of NSCLC. Immune checkpoints are important to control the immune responses in order to protect tissues from damage when the immune system is activated [8]. The expression of immune checkpoint proteins can be dysregulated by cancer cells, enabling immune evasion, a cancer hallmark [8,9]. Programmed cell death protein 1 (PD-1) is an immune checkpoint receptor expressed on the surface of activated T cells, including a large proportion of tumour-infiltrating lymphocytes from many tumours [8,10]. The binding to its ligands, PD-L1 and PD-L2, inhibits the response of cytotoxic T cells, hence the activation of the pathway PD-1/PD-L1 is a mechanism of immune-escape [11]. PD-L1 is commonly upregulated at the tumour cell surface [8] and is generally expressed in 20% to 40% of NSCLC [12]. There is evidence that infiltrating lymphocytes, mutational burden, and the expression of PD-L1 [13,14] are predictive biomarkers for treatment with checkpoint inhibitors. However, prediction of response is rather imperfect and, thus, more accurate predictive biomarkers are mandatory.

Genome instability leading to the accumulation of genomic aberrations is another characteristic of cancer cells [9]. Double-strand DNA breaks (DSB) may lead to mutations, chromosomal translocations, cell senescence and apoptosis [15,16]; hence, repair mechanisms are essential to maintain genome stability. Homologous recombination repair (HRR) is the leading DNA repair mechanism of double-strand DNA breaks (DSB) that uses the homologous region of the sister chromatid as the replicative template in order to reliably repair DSB [16]. *RAD51* protein has an important activity in HRR, promoting the insertion of the broken ends of the DSB into the sister chromatid [17,18]. Its action is dependent on *RAD51*-like proteins: *RAD51B*, *RAD51C*, *RAD51D*, *XRCC2* and *XRCC3* [17–19]. Defects in the HRR pathway entail cell proliferation despite DNA damage, promoting cancer development [20]. HRR pathway deficiencies seem to be associated with higher expression of PD-L1 and linked to an immune-evasive tumour phenotype [16]. Rieke et al. found that HRR genes hypermethylation is inversely correlated with mRNA transcription and associated with PD-L1 expression in head and neck, lung, and cervix squamous cell carcinomas [18]. As such, the methylation status of these genes could represent new predictive biomarkers for immune checkpoint inhibition.

The aim of this study is to investigate the association of immune checkpoint PD-L1 expression and the status of DNA repair gene *RAD51B* promoter methylation (*RAD51Bme*) in advanced NSCLC, correlating with patients' outcome. Additionally, the potential of *RAD51Bme* levels as a candidate predictive biomarker for PD-1 blockade response in NSCLC was also assessed.
