Immunotherapy and PD-L1 Tumor Expression in Moroccan Non-Small Cell Lung Cancer Patients with Various Metastasis

approximately 85%


Introduction
Lung cancer is one of the most prevalent malignant diseases [1] and the leading cause of cancer-related deaths worldwide [2].In Morocco, lung cancer is the second most common cancer, with a prevalence of 13.9 % for both sexes and up to 25.6 % in males [3].Consequently, non-small cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancer cases [4].Most patients are diagnosed at an advanced stage of the disease

Immunotherapy and PD-L1 Tumor Expression in Moroccan
Non-Small Cell Lung Cancer Patients with Various Metastasis [5].In NSCLC, the spread of metastasis is a major concern [2] as they can affect various organs such as the bones, brain, and liver.Indeed, bone metastasis account for approximately 34% of cases, while brain and liver metastasis affect nearly 39% and 20% of NSCLC patients, respectively [6].Furthermore, studies have shown that different types of metastasis have distinct prognostic value in NSCLC patients, especially liver and multiorgan metastasis, which are associated with an increased risk of mortality [7].Emerging therapeutic approaches  Pathology Department, Ibn Rochd University Hospital, Casablanca, Morocco. 3 Immunology Laboratory, Faculty of Medicine and Pharmacy, Hassan II University of Casablanca, Morocco. 4 Mohammed VI Cancer Treatment Center, Ibn Rochd University Hospital, Casablanca, Morocco. 5 Ryad Oncology Clinic, Casablanca, Morocco.*For Correspondence: oussamaaazzane@gmail.com have improved the prognosis of NSCLC patients [8] and among them, immunotherapy using immune checkpoint inhibitors (ICIs), specifically anti-PD-1 (Pembrolizumab) or anti-PD-L1 (Atezolizumab), has transformed the treatment landscape for advanced or metastatic NSCLC patients [9] and has become a powerful therapeutic strategy [10,11].Despite the survival benefits offered by ICIs, it remains challenging to identify patients who will fully benefit from them.Currently, PD-L1 expression is the only clinically validated biomarker to identify patients most likely to respond to immunotherapy [12].However, certain clinical factors, such as performance status, the number of affected metastatic organs (metastatic burden), and the site of metastasis, have also emerged as potential predictors of immunotherapy efficacy [13,14].The results of the Keynote-189 study suggest that patients with brain metastasis should benefit from immunotherapy, contrary to other studies such as Keynote 024, which reached the opposite conclusion [6].The use of immunotherapy as a treatment, especially in NSCLC patients with different PD-L1 statuses and distant metastasis localized in specific organs such as the liver, brain, and bones, remains a complex issue to address.Therefore, the aim of this study is to evaluate the predictive significance of tumor PD-L1 expression as well as the number and site of metastasis in Moroccan NSCLC patients treated with immunotherapy.

Ethical Consideration
This study was approved by the local ethics committee of Ibn Rochd University Hospital (CHU) in Casablanca (Approval number: 03/2022).All patients provided informed consent before participating in the study.Thus, the protocol of our study adheres to the principles outlined in the Helsinki Declaration.

Patients
Between January 2019 and February 2023, 40 Moroccan patients with metastatic NSCLC were recruited from two different institutions: the Mohammed VI Center for Cancer Treatment at Ibn Rochd University Hospital in Casablanca and the Ryad Oncology Clinic in Casablanca.Eligible participants for our study must meet the following predefined criteria: age ≥ 18 years, presenting histologically confirmed NSCLC, having been treated with immunotherapy alone or in combination with chemotherapy, and having clinical and pathological data available.Exclusion criteria concern patients with other types of lung cancer or those under chemotherapy alone.The patients' characteristics are cited in Table 1.

Outcome Assessment
The assessment of response to immunotherapy was based on iRECIST criteria (Immunotherapy Response Evaluation Criteria in Solid Tumors, Version 1.1).Specific organ responses included complete responses (CR), partial responses (PR), stable disease (SD), and progressive disease (PD).The objective response rate (ORR) corresponds to the ratio of patients who achieved CR, PR, and SD to the total.Progression-free survival (PFS) was defined from the first day of ICI treatment until the day of disease progression assessment by the physician or death, regardless of the cause.Overall survival (OS) corresponds to the duration of immunotherapy treatment (deceased or alive).

Expression of PD-L1
Tumor expression of PD-L1 was assessed from formalin-fixed paraffin-embedded (FFPE) tumor samples using the 22C3 pharmDX test on the Dako Link 48 platform.Tumor cells showing partial or total membranous staining were considered positive.Thus, the tumor expression of PD-L1 was evaluated using the tumor proportion score (TPS), defined as the percentage of positive PD-L1 tumor cells (TC+) relative to the total number of TC.Based on PD-L1 expression, tumor cells were classified into three groups: negative expression (TPS < 1%), low expression (TPS from 1 to 49%), and high expression (TPS ≥ 50%) [15].

EGFR Test
Molecular alterations of EGFR were detected using real-time polymerase chain reaction (qPCR) with the cobas® mutation test.This test identifies various mutations within EGFR exons from FFPE tissues.Specific mutations targeted include those in exon 18 (G719A, G719C, and G719S), exon 19, exon 20 (S768I, T790M), and exon 21 (L858R and L861Q).The results revealed the presence either absence of specific EGFR gene mutations in the tested samples.

ALK Status
ALK translocation testing was performed using immunohistochemistry (IHC) with a rabbit monoclonal anti-ALK antibody (Clone D5F3, Ventana, Roche).A positive result is characterized by intense granular cytoplasmic staining observed within tumor cells.

Statistical Analysis
Statistical analysis was conducted using SPSS version 21 statistical software.The chi-square test was used to analyze the difference in the objective response rate (ORR) between subgroups.The Kaplan-Meier method was used to assess overall survival (OS) and progression-free survival (PFS), and the log-rank test was used to determine the significance of differences.We also performed univariate and multivariate Cox regression analysis to explore the impact of clinical variables on patients' PFS and OS.A P-value <0.05 was considered statistically significant.

Patient Characteristics
Table 1 presents the characteristics of the 40 patients included in this study.Among these patients, 90% (N=36) were men with a sex ratio of 9.The median age of the patients was 67 years (range 39 to 92 years), with 55% (N=22) aged ≥ 67 years.95% (N=38) of the patients had adenocarcinoma, 76.5% (N=27) had a performance status (PS) of 0, and 85% (N=34) were smokers.Additionally, 22.5% (N=9) of the patients had more than 3 affected

Results of PD-L1 tumor expression and response to immunotherapy
Figure 1 depicts the results of PD-L1 expression and response to immunotherapy based on ORR (Figure 1, A), PFS (Figure 1, B), and OS (Figure 1, C).The results reveal a significant difference in overall survival duration

The results concerning the metastatic burden, metastatic site, and response to immunotherapy
We classified patients into two groups based on the number of affected metastatic organs (MB <3 and

Study of PD-L1 Tumor Expression Based on Metastatic Burden and Sites
Our study reveals significant differences in ORR, PFS, and OS among patients based on their PD-L1 tumor expression, metastatic burden, and site.Consequently, certain patients exhibited significantly shorter ORR, lower PFS, and reduced OS (Figure 4, A, B, and C).

Univariate and multivariate overall survival analysis
In our study, we thoroughly examined factors significantly associated with unfavorable progression of PFS and OS.Univariate analysis revealed several significant variables related to unfavorable PFS and OS.Among these, metastatic burden (≥3) (p=0.001) and the presence of hepatic and osseous metastasis (p=0.046,p=0.000 respectively) were particularly notable for PFS (Table 2).Regarding OS, performance status (PS 1-2) (p=0.000),metastatic burden (≥3) (p=0.003),osseous metastasis (p=0.000), and tumor expression of PD-L1 (TPS ≥ 1%) (p=0.010) were identified as significant factors (Table 3).However, in multivariate analysis, some of these factors retained their importance as independent predictors of unfavorable PFS and OS.Specifically, the presence of osseous metastasis (p=0.001)remained a significant predictor for PFS (Table 2), while performance status (PS 1-2) (p=0.047) and the presence of osseous metastasis (p=0.002)remained important predictive factors for OS (Table 3).

Discussion
The objective of our study was to assess the predictive value of PD-L1 tumor expression and the burden and sites of metastasis in patients with non-small cell lung cancer (NSCLC) treated with immunotherapy.A significant difference in overall survival (OS) duration was observed between patients with negative PD-L1 expression (28.85 months) and patients with positive expression (17.19 months) (p=0.01).These results are in line with those reported by Kaiyan Chen et al (China, 2020) in NSCLC patients (OS: PD-L1 <1% = 29.3months, PD-L1 ≥1% = 15.20 months, p=0.0006) [16].Additionally, another study conducted by Si-yan Liu et al (China, 2018) found a significant difference in OS between PD-L1 (+) patients (OS=78.6 months) and PD-L1 (-) patients (OS=93.4months) (p=0.0005)[17].Our results should be interpreted considering several factors that may influence the final interpretation, including the type of analyzed specimen.In our study, we worked with tissue biopsies since the patients were inoperable at the time of diagnosis.It is worth noting that PD-L1 IHC tests can yield false negatives due to the intratumoral heterogeneity of PD-L1 expression, as emphasized in the recommendations from the PATTERN group of thoracic pathologists [18].Furthermore, treatment resistance, the presence of other oncogenic drivers, the burden, and the metastatic site must be taken into consideration.Moreover, concerning NSCLC treatments, immunotherapy represents a promising strategy aimed at mobilizing the immune system to recognize and potentially eliminate tumor cells, thus prolonging patient survival.
However, some patients develop resistance to treatment, whether it's primary resistance (lack of initial response or clinical benefit) or acquired resistance    (alterations in the INF-γ signaling pathway, loss of effector function of T lymphocytes, and upregulation of alternative immune checkpoint receptors).This occurs when tumor progression occurs after a median progression-free survival of 4 to 10 months [19,[20][21]22].Thus, among the 19 deceased patients, 9 may have acquired resistance to immunotherapy (PFS > 4 months, 3 with PD-L1 (+) and 6 with PD-L1 (-)).As for primary resistance to immunotherapy, 4 out of 19 deceased patients had the following profile (PFS < 4 months, adenocarcinoma NSCLC, EGFR-, ALK-, and smokers).This suggests that this resistance may be due to rare EGFR mutations, as observed in the study by How-wen Ko (Taoyuan, 2022), which demonstrated a positive relationship between smoking and the frequency of these rare EGFR mutations, reinforcing this hypothesis [23].Mutations in the HER-2 gene can also lead to tumor progression and patient death, which could be the case for one of the 19 deceased patients in our study who presented (PFS < 4 months, adenocarcinoma NSCLC, non-smoker, EGFR-, activity in brain metastasis of NSCLC with PD-L1 expression ≥1% [29]. In conclusion, our study highlights the importance of immunotherapy in the management of NSCLC while emphasizing the complexity of treatment responses.Our results suggest that tumor expression of PD-L1 and MB play a significant role in predicting the response to immunotherapy.A MB ≥ 3 appears to be a major risk factor, as well as the presence of bone and hepatic metastasis.Finally, immunotherapy remains a promising strategy, provided it is combined with the customization of care for NSCLC patients based on biomarkers and clinical characteristics to enhance treatment efficacy.Mehdi Karkouri).
ALK-, and PD-L1+).According to Mathieu Chevalier et al (Switzerland, 2021), 1 to 3% of NSCLC patients had HER-2 mutations, mainly observed in cases of adenocarcinoma, especially in women and non-smokers [24].Additionally, among the 19 studied patients, 2 of them had activating EGFR mutations, which may have contributed to their death.Finally, it is important to highlight that among the 19 deceased patients, 11 had a metastatic burden ≥ 2, and 15 had bone metastasis.These results confirm the significant association we found in our study between metastatic burden and immunotherapy effectiveness (Figure 2, A; Figure 3, A; Figue 4, A).These observations align with the results of a study conducted by Jiayi Deng et al (China, 2022) in NSCLC patients, which showed a significant decrease in PFS with increasing metastatic burden (metastatic burden ≥3: PFS=4.4 months, metastatic burden=2: PFS=14.4months, metastatic burden=1: PFS=25.2months, p=0.0052) [25].
Our results also revealed significantly unfavorable objective response rate (ORR), progression-free survival (PFS), and overall survival (OS) compared to patients without bone metastasis (ORR=17.7% vs. 78.3%,p=0.000;PFS=10.72 vs. 31.33months, p=0.000;OS=11.39 vs. 36.17months, p=0.000) (Figure 2, D; Figure 3, D; Figure 4, D).These results can be explained by the fact that the bone marrow exhibits notable immune vulnerability due to various factors, including the presence of immature and inhibitory immune cells, a high proportion of regulatory T cells (Tregs), and myeloid-derived suppressor cells (MDSCs).This immune vulnerability in the bone poses a major challenge in the treatment of bone metastasis and requires specific therapeutic approaches to target the immune microenvironment in this area [26].

The negative impact of liver metastasis on immunotherapy effectiveness can be attributed to several factors
Induction of hepatic immune tolerance (elimination of tumor-specific CD8+ T lymphocytes through Fas-FasL pathway-induced apoptosis by macrophages) [28].
Finally, our study shows that the effectiveness of immunotherapy depends on the tumor PD-L1 status, metastatic burden, and site (Table 4).Indeed, PD-L1 expression differs between the primary site and metastatic sites, which have distinct genetic and immunological profiles.It is worth noting that a study conducted by Sarah B. Goldberg et al (USA, 2020) in the context of a phase 2 clinical trial showed the efficacy of pembrolizumab in treating NSCLC patients with brain metastasis and PD-L1 expression (PD-L1 ≥1%), highlighting pembrolizumab's

Table 1 .
Characteristics of the Patients Recruited in This Study

Table 2 .
Predictive Factors of Progression-Free Survival (PFS) in Univariate and Multivariate Analysis between patients with negative PD-L1 expression (28.85 months) and those with positive expression (17.19 months) (p=0.01).

Table 3 .
Predictive Factors of Overall Survival (OS) in Univariate and Multivariate Analysis.