Evaluation of gut leakage and bacterial translocation as efficacy biomarkers of immunotherapy in advanced non-small cell lung cancer (NSCLC)

Introduction

Background

Standard treatment of advanced non-small cell lung cancer (NSCLC) relies predominantly on immune checkpoint inhibitors (ICIs) as first-line therapy, targeting programmed death-1 (PD-1) or programmed death ligand-1 (PD-L1) (1-4). Alone or in association with platinum-based doublet chemotherapy, ICIs have transformed the prognosis of patients treated for advanced NSCLC (4-6). Several studies even suggest that the benefits of ICIs are likely to extend to earlier stages of NSCLC, with remarkable results when administered as perioperative therapy (7-9).

Rationale and knowledge gap

Despite the potential advancement in monitoring responses via blood tests, such as detecting circulating tumor DNA (10,11), current initial prognostic markers, such as PD-L1, remain unreliable due to variations in histological diagnoses (12-14). Furthermore, the biological mechanisms that lead to primary or secondary resistance to ICIs remain elusive.

As most patients treated with ICIs for advanced NSCLC will present with primary or secondary resistance, there is an urgent need to find new predictive markers of therapeutic response (to ICIs).

Among the mechanisms of resistance to ICIs that have been identified, such as mutational load (15,16) or low tumor infiltration by T cells (17), several studies have focused on the role of the gut microbiota and the microbiological environment. The composition of the intestinal microbiota, partly influenced by anti-biotherapy (18,19), seems to have a clear influence on the efficacy of ICIs (20-22).

Citrulline is a non-protein amino acid produced primarily by the enterocytes of the small bowel, and its production is closely linked to the quality of the intestinal barrier and the translocation of bacteria from the intestinal lumen into the bloodstream (23,24). Citrulline production is affected by all conditions impacting enterocyte mass and function, such as Crohn’s disease, short bowel syndrome or chemotherapy and radiotherapy treatments (24) whereas it does not depend on nutritional status or systemic inflammatory state. Validated as a biomarker of the quality of the intestinal barrier, citrulline also seems to have an impact on the regulation of the immune system, particularly on T-CD8 lymphocytes (25,26).

We conducted a pilot study on patients with advanced NSCLC treated with nivolumab in the second line setting or more for advanced NSCLC (27). We found that high baseline citrulline levels (>20 µM) was associated with tumor response, clinical benefit and longer overall survival (OS) (7.9 months with high citrulline level versus 1.6 months, P<0.001) and progression-free survival (PFS) (median not reached with high citrulline level versus 2.2 months, P<0.001) (27). We also showed that the early use of antibiotics (EUA) and the composition of the blood microbiome influenced the response to nivolumab in NSCLC, corroborating several results showing the impact of antibiotic therapy on the efficacy of ICIs (28-30).

Objective

We aimed to investigate the role of plasma citrulline in two cohorts of patients with advanced NSCLC treated with ICIs in the first line setting or more, as well as to assess the potential impact of the blood microbiome and EUA on therapeutic response. We present this article in accordance with the REMARK reporting checklist (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2024-1083/rc).

Methods

We included all consecutive patients with advanced NSCLC who received ICIs in first-line therapy at the Department of Respiratory Diseases and Thoracic Oncology of the Ambroise Paré Hospital-APHP (Boulogne-Billancourt, France), between November 2017 to April 2023 (cohort #1). We also used a validation cohort (cohort #2) of 85 consecutive patients with advanced NSCLC who received ICIs as second-line treatment between June 2015 and January 2018 in the Department of Medical Oncology of the Cochin Hospital-APHP (Paris, France).

The exclusion criteria included digestive or systemic conditions known to influence plasma citrulline levels, contraindications to ICIs, and an estimated life expectancy of less than 3 months.

Demographic and pathological characteristics were collected retrospectively, including systemic treatment (immunotherapy, immunotherapy and platinum-based chemotherapy), histology, staging, performance status (PS) and EUA.

We assessed tumor response using computed tomography (CT) scans performed every 2 or 3 cycles of treatment according to the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 criteria, evaluated by an expert thoracic radiologist and validated during a multidisciplinary meeting. The response criteria were defined as the overall response rate (ORR) for complete or partial response, refractory rate as progression at 1st follow-up (T1) according to RECIST 1.1 criteria and clinical benefit as treatment with ICIs administered for 6 months or longer.

Ethical approval and informed consent

The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. All patients included provided written informed consent for the use of their blood samples, and the study was approved by the local ethics committee, Comité de Protection des personnes – Ile de France (CPP IDF n°8) (No. 2014-A00187-40).

Plasma samples and citrulline assay

Patients’ blood samples were prospectively collected at baseline (T0), T1 and progression (T2), and plasma citrulline concentration was determined by ion exchange chromatography, as previously described (27). Citrulline levels were dichotomized using an optimal threshold, as described below. Plasma samples were prospectively collected at T0, T1 and T2, and stored in a freezer at a temperature of −81 ℃ until citrulline and blood microbiome analyses.

Blood microbiome

The microbiome of blood samples was analyzed by sequencing the V3–4 regions of the bacterial 16S rRNA gene at time point T0, using services provided by Vaiomer (Toulouse, France). In summary, polymerase chain reaction (PCR) amplification of the V3–4 regions of the 16S ribosomal gene was performed using universal primers. Quantitative PCR (qPCR) was carried out on a VIIA 7^® PCR system (Life Technologies), employing Sybr Green technology. The number of 16S rRNA gene copies was quantified by comparing with a standard curve derived from serial dilutions of plasmid standards (Vaiomer Universal plasmids). Sequencing libraries for each sample were prepared by incorporating sequencing adapters. Sequencing fragments were detected with MiSeq Illumina^® technology, and metagenomic sequences from the microbiota were processed using a bioinformatics pipeline designed by Vaiomer, adhering to FROGS guidelines. OTUs were generated via single-linkage clustering, with taxonomic assignments made to define the community profiles.

Statistical analyses

Given that this is an exploratory study, we did not calculate a minimum sample size for our analysis. To determine the optimal citrulline cutoff value as a predictive biomarker for the response to ICIs in NSCLC, we performed an analysis of the area under the receiver operating characteristic curve (AUC) based on citrulline levels and scannographic response (RECIST 1.1). This analysis was carried out in both the primary and validation cohorts. The results identified cutoff values of 27.6 and 28.0 µmol/L as providing the best sensitivity and specificity, respectively. Therefore, we selected a cutoff of 28 µmol/L for further analyses. To assess whether the missing data were missing completely at random (MCAR), we performed Little’s MCAR test, showing that the missing data followed a random distribution.

Continuous variables were compared using the Mann-Whitney test, while categorical variables were assessed using the Chi-squared test. Additionally, comparisons of refractory rates, objective response rate (ORR) and clinical benefit rates were conducted using the Chi-squared test according to citrulline levels. Concerning blood microbiome analyses, the reads acquired from the MiSeq sequencing platform underwent processing via the Vaiomer bioinformatics pipeline. This involved several steps, including quality filtering, clustering into operational taxonomic units (OTUs) utilizing the Swarm algorithm, and taxonomic classification. Additionally, the Linear Discriminant Analysis Effect Size (LEfSe) algorithm was employed to pinpoint statistically significant disparities in microbiome composition based on clinical parameters such as tumor response, clinical benefit and citrulline levels. PFS was assessed utilizing the Kaplan-Meier method, with P values calculated through the Gehan-Breslow-Wilcoxon test to emphasize early progression. We used the Log-rank test to analyze OS. Statistical analyses (except for blood microbiome analyses) were conducted using GraphPad Prism 2019 (California, USA) and missing data were handled using the case deletion method.

Results

Patients’ characteristics

Median citrulline level in cohort #1 at T0 was 30 [interquartile range (IQR), 23.0–37.0] µM, with a low citrulline level defined as less than 28 µM and a high citrulline level as greater than or equal to 28 µM.

In cohort #1, 89 patients were treated with ICIs in first line for an advanced NSCLC between November 2017 and April 2023. Patients were mostly male (53%), current or former smokers (95%), median age 65 (IQR, 60–72) years, PS 0–1 (86%), presenting an adenocarcinoma (75%) with stage IV disease (89%) and PD-L1 ≥50% (58%) (Table 1).

The treatment choice was decided in a multidisciplinary consultation meeting according to routine clinical practice.

Patients received either pembrolizumab alone (43%) or pembrolizumab combined with platinum-based doublet chemotherapy (57%), followed by maintenance treatment with pembrolizumab (+/− pemetrexed). Median number of pembrolizumab injections was 6 (IQR, 2–12.75), and median follow-up time was 349 (IQR, 177–705) days. At the end of follow-up, 25 patients were still treated with pembrolizumab. Patients’ refractory rate to treatment was 25% (n=22), ORR was 62% (n=56), and the clinical benefit rate was 39% (n=35).

In cohort #2 (n=85), patients were mostly male (60%), current or former smokers (92%), median age 65 (IQR, 59.5–70) years, PS 0–1 (52%), treated in second line for a stage IV (99%) adenocarcinoma (67%) with nivolumab (Table S1). Patient’s refractory rate to treatment was 53% (n=45), ORR was 25% (n=21) and clinical benefit rate was 34% (n=29).

Plasma citrulline

In cohort #1, citrulline levels were available for 75 patients at T0, 64 patients at T1 and 30 patients at T2 (Figure 1). We found 30 patients with low citrulline level (<28 µM) and 45 patients with high citrulline levels (≥28 µM). Patients with low citrulline levels were found to have a higher incidence of liver metastases (P=0.007) and lower hemoglobin level (P=0.003) (Table 1). Median citrulline level at T0 was 30 (IQR, 23.0–37.0) µmol/L, 29.9 (IQR, 21.3–40.0) µmol/L at T1 and 27.6 (IQR, 20.2–38.6) µmol/L at T2 in cohort #1.

Figure 1 Flow chart. Patients treated with ICIs in first line for a NSCLC between November 2017 to April 2023, plasma citrulline available at T0, T1, T2 and blood microbiome analyses available at T0. T0, baseline; T1, first follow-up; T2, progression. ICIs, immune checkpoint inhibitors; NSCLC, non-small cell lung cancer.

In cohort #2, the median citrulline level at T0 was 33 (IQR, 26.0–41.50) µmol/L, with 24 patients with low citrulline level (<28 µM) and 62 patients with high citrulline level (≥28 µM). Patients with high citrulline level were found to be older than patients with low citrulline levels (P=0.03, Table S1).

In cohort #1, high baseline citrulline levels (T0) were associated with a low refractory rate to ICIs (P<0.001), improved ORR (P=0.006), and a higher clinical benefit rate (P=0.01) compared to patients with lower baseline citrulline levels. Similarly, high citrulline levels at T1 were associated with a decreased refractory rate (P=0.02) and a better ORR (P=0.002) and clinical benefit rate (P=0.001) (Figure 2). Patients with clinical benefit had significantly higher citrulline levels at T0 (P=0.03) and T1 (P<0.001), with decreasing rates at T2 (Figure 3). Patients with high baseline citrulline level had significantly longer PFS and OS. Median PFS was 26.3 months with high citrulline at T0 and 7.2 months with low citrulline level (P=0.02), and median OS was 32.4 and 16.30 months (P=0.050) (Figure 4). In cohort #1, patients who received ICIs alone exhibited similar citrulline levels at T0 compared to those receiving ICIs in combination with platinum-based doublet chemotherapy (P=0.56) but displayed lower citrulline levels at T1 (P=0.01).

Figure 2 Citrulline level (high ≥28 µM, low <28 µM) and refractory rate, ORR and clinical benefit at T0 and T1 in cohort #1. *, P<0.05; **, P<0.01; ***, P<0.001. T0, baseline; T1, first follow-up. ORR, objective response rate.

Figure 3 Plasma citrulline levels evolution throughout ICIs treatment according to clinical benefit. *, P<0.05; ***, P<0.001. T0, baseline; T1, first follow-up; T2, progression. ICIs, immune checkpoint inhibitors.

Figure 4 PFS and OS according to low and high citrulline levels in cohort #1 (A and B respectively) and in cohort #2 (C and D respectively). OS, overall survival; PFS, progression-free survival.

In cohort #2, high baseline citrulline levels were associated with a lower refractory rate (P=0.03), a better ORR (P=0.002) and clinical benefit rate (P=0.001) (Figure 2).

Patients with higher baseline citrulline levels exhibited significantly longer PFS (median PFS: 3.85 vs. 1.8 months, P=0.002) and OS (median OS: 14.5 vs. 6.5 months, P=0.03) compared to those with lower citrulline levels (Figure 4).

EUA and blood microbiome

Thirty-one patients from cohort #1 underwent EUA, defined as the use of antibiotic therapy within two months before and one month after the start of ICIs treatment. We found no statistical difference with regard to refractory rate, ORR and clinical benefit between patients who had EUA and the others.

T0 blood microbiome analyses were available for 42 patients of cohort #1, with a median of 19,550 (IQR, 18,951–23,430) gene copies/µL (Figure 1). Twenty-three patients were considered as responders according to RECIST 1.1 criteria and 19 as non-responders. Two patients in the responder group were considered as outliers, with aberrant results compared to the control samples. Responder patients had significantly less 16S rRNA gene copy number in their blood samples than non-responder patients (P=0.01, median =15,462 vs. 23,234 gene copies/µL, respectively) (Figure 5).

Figure 5 16S rRNA gene copy number expression in blood sample in responder and non-responder group. **, P≤0.01.

We found no significant difference concerning the blood microbiome composition between responder and non-responder patients (Figure S1).

Discussion

In our study, elevated citrulline levels at T0 were correlated with a lower refractory rate, improved ORR, clinical benefit, PFS, and OS among patients receiving first-line ICIs for advanced NSCLC. Similarly, high citrulline levels at T1 were associated with a reduced refractory rate, enhanced ORR, and clinical benefit. We also found that responder patients had a lower quantity of 16S rRNA gene copies in their bloodstream.

To our knowledge, our study is the first to demonstrate the value of citrulline as a predictive marker of ICIs’ efficacy in the first-line setting for NSCLC. Our findings align with our previous pilot study (27) that highlighted the importance of citrulline as a predictive marker of Nivolumab efficacy in the second-line or more advanced NSCLC.

Predominantly synthesized by the enterocytes of the small bowel, circulating citrulline concentrations typically average 40±10 (IQR, 20–60) µmol/L in healthy individuals (24). Our citrulline level used as a cut-off was 28 µmol/L, which is higher than the 20 µmol/L used in our previous publication (23). This can be explained by the fact that our patients were in first-line treatment and therefore less exposed to prior treatments altering citrulline production, notably chemotherapy (31) and radiotherapy (32).

Interestingly, our results showed an increased level of citrulline between T0 and T1 for patients presenting a clinical benefit of ICIs. Activation of T-cells by ICIs can lead to increased arginine production, mediated by the activation of argininosuccinate synthase (ASS1), an enzyme that converts citrulline into arginine. This pathway is particularly important in arginine-limited environments, such as the tumor microenvironment. In this regard, Werner et al. demonstrated that activated T-cells can import citrulline and use it to regenerate arginine, which is crucial for T-cell proliferation that are the key for ICIs efficacy (33). Moreover, Mao et al. showed that citrulline conversion by the ASS1 enzyme is necessary for the activation of pro-inflammatory macrophages and the modulation of the local immune response (34).

Furthermore, protein citrullination, a post-translational process mediated by peptidylarginine deiminase (PAD) enzymes, may play a role in the antitumor immune response. Citrullinated peptides can serve as neoantigens, stimulating a specific immune response against tumor cells. Katayama et al. demonstrated that citrullinated peptides, via the class II major histocompatibility complex (MHC-II), can be presented on the cell surface, thereby promoting a targeted immune response against tumor cells (35).

Thus, we hypothesize that these mechanisms, which are central for the efficacy of ICIs depending on T-lymphocyte activation, may lead to an increased demand for citrulline in the tumor microenvironment. This could result in an upregulation of citrulline production through mechanisms that needs to be investigate.

We also observed that responder patients had significantly fewer copies of the 16S rRNA gene in their blood at baseline, indicating a potentially superior quality of the intestinal barrier in these patients and a more controlled and favorable immune environment. Among the factors that influence tumor microbiota composition, processes such as bacterial translocation through local mucosal barriers or adjacent tissue, as well as hematogenous spread, may play significant roles (36). Hematogenous spread, specifically, could be facilitated by the chemotactic gradient of necrotic cellular debris, potentially leading to intratumoral colonization (37). Thus, intratumoral bacterial load, and more specifically certain species such as F. nucleatum (38) and Methylobacterium (39) is associated with T-cell dysfunction and decreased tumor-infiltrating T-cell density (40), favoring tumor progression and resistance to ICIs.

Therefore, the potential of citrulline as a predictive biomarker for response to ICIs seems to be closely associated with the integrity of the intestinal barrier, serving as an indicator of bacterial translocation and in the most severe case systemic infection, as demonstrated in the study by de Mooij et al. for patients receiving chemotherapy (41). Hence, citrulline levels may reflect alterations in blood and tumor microbiota associated with bacterial translocation, thereby indirectly indicating changes in the tumor-specific immune response. Additionally, citrulline could function as an indirect marker of the quality of T-cell mediated immune response, of which it may serve as a substrate.

Additional studies investigating the correlation between citrulline and the blood and tumor microbiome will be necessary to provide further insights into this matter.

Our study suggests a strong link between therapeutic response to ICIs in advanced-stage NSCLC and the gut leakage and bacterial translocation, which could constitute a therapeutic target in patients with low citrulline levels. In this regard, Davar et al. have demonstrated the benefits of fecal transplantation from donors who had a complete or partial response to ICIs on the efficacy of anti-PD-1 therapies in melanoma patients initially refractory to ICIs (22). This approach enabled the reprogramming of the tumor immune environment by modulating both systemic and intratumoral immune responses. Patients who received fecal transplantation exhibited higher percentages of activated CD8⁺ T cells in their bloodstream and tumors, with enhanced cytolytic functions. Additionally, there was a downregulation of cytokines associated with tumor progression (MCP-1, IL-8, IL-18) and an upregulation of cytokines associated with tumor responses (IL-5, IL-10, IL-13, TNF). This resulted in a scanographic response in 20% of initially refractory patients and improved PFS and OS (22).

Nevertheless, we did not observe any disparity in the composition of the blood microbiome between responder and non-responder patients. This could be due to the limited number of samples available at T0 and the absence of analysis at T1.

Our study has several limitations. We had a limited number of samples for blood microbiome analyses at T0 and no samples at T1. Additionally, we did not collect stool or tumor samples for comparison with blood microbiome data.

However, our study also possesses several strengths. Citrulline levels at both T0 and T1 were accessible for a substantial number of patients undergoing first-line therapy with ICIs for NSCLC with all samples collected prospectively. We also used an independent external validation cohort, with consistent results on the impact of citrulline on response to immunotherapy.

Conclusions

Our study supports the potential use of citrulline as a predictive biomarker of response to first-line ICIs in patients with advanced NSCLC. Additionally, our findings suggest that the integrity of the intestinal barrier and the blood and tumor microenvironment may play pivotal roles in determining patients’ prognosis in this context. Further prospective studies are required to validate these findings.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the REMARK reporting checklist. Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2024-1083/rc

Data Sharing Statement: Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2024-1083/dss

Peer Review File: Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2024-1083/prf

Funding: None.

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2024-1083/coif). E.G.L. reports the consulting fees from AstraZeneca, Boehringer-Ingelheim, Bristol-Myers-Squib, Janssen, LillY, MSD, Pfizer, Roche, Sanofi, Takeda; and Support for attending meetings and/or travel from Roche, Pfizer, Takeda. The other authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the local ethics committee, CPP IDF n°8 (No. 2014-A00187-40). Written informed consent was obtained from all the patients included.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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