The Association Between Gut Microbiome Affecting Concomitant Medication and The Related Effectiveness of Immunotherapy in Patients With Stage IV NSCLC

This historically matched cohort study investigated the inuence of microbiome-affecting-medication on the effectiveness of immunotherapy in patients with stage IV non-small-cell lung cancer (NSCLC). We postulated that if the effectiveness of immunotherapy is mediated by drug-related changes of the microbiome, a stronger association between the use of co-medication and overall survival (OS) will be observed in patients treated with immunotherapy as compared to patients treated with chemotherapy.


Introduction
Lung cancer is the leading cause of cancer-related deaths worldwide. In 2012, the World Health Organisation (WHO) estimated that of the 1.6 million lung cancer related deaths, non-small-cell lung cancer (NSCLC) is the most frequent histological type, representing 85% of cases [1].
Since the beginning of systemic treatment of stage III and IV NSCLC patients, platinum-doublet chemotherapy has been the rst-choice treatment. With the development of new therapies, more effective treatment options have become available in addition to the platinum-doublet therapy [2] [3]. For patients without driver mutations, immunotherapies targeting immune checkpoints, such as the PD-1/PD-L1 pathway, have become an important new treatment option. Currently, four PD-1/PD-L1 inhibitors (pembrolizumab, nivolumab, durvalumab and atezolizumab) have been approved for treatment of patients with advanced NSCLC.
The approval of immunotherapy, with or without chemotherapy, is based on several phase III studies demonstrating superior e cacy in a population meeting the strict in-and exclusion criteria [4] [5] [6]. For example, patients with present active infections, autoimmune conditions, and other comorbidities often requiring concomitant medication are not included in these trials. Therefore, the impact of these factors, including the use of concomitant medication on the e cacy of immunotherapy, cannot be studied from clinical trial data.
Recent studies demonstrate that the microbial ora plays an important role in modulation of immunotherapy effectiveness by affecting the tumor immuno-microenvironment [7]. It is well known that the microbiome can vary signi cantly from one individual to another, which has been proposed as an explanation of the variability of response to immunotherapy [8]. Routy et al. showed that changes in composition of the gut microbiome negatively in uence the outcome of PD-1 inhibition by immunotherapy in mice and patients. Additionally, the study found that the replacement of deleterious microbial ora by a favorable one, can restore the e cacy of the immunotherapy response in mice [9]. The composition of the human microbiome is in uenced by several factors such as host genetics, lifestyle factors and the use of medication, such as antibiotics. Furthermore, recent publications showed that other drugs, such as proton pump inhibitors, opioids, metformin and other antidiabetics can disturb the microbiome as well [10] [11].
Accumulating observational studies show that antibiotic treatment associates signi cantly with attenuated clinical outcomes in NSCLC patients treated with immunotherapy [9] [12] [13]. However, the majority of these studies analyzed the effects of antibiotics on survival outcomes in single-arm cohort studies including only immunotherapy patients. This makes the results susceptible for bias due to confounding by indication, a situation in which patient characteristics -rather than the presence of concomitant medication-are independent predictors of clinical outcomes. Therefore, it is still an active area of debate whether or not there is a causal link between antibiotic related changes of the microbiome and the decreased effectiveness of immunotherapy. Assuming that a prospective clinical trial randomizing based on concomitant medication will not conducted, additional observational studies with alternative designs are needed to resolving this matter, for example a study design including a control group not treated with immunotherapy. Therefore, we aimed to study the association between the use of concomitant medication affecting the microbiome and the effectiveness of immunotherapy by using a historically matched cohort study design. To control for confounding by indication we used patients treated with chemotherapy in the pre-immunotherapy era as comparator group. We presume that there is no relation between the microbiome and the effectiveness of chemotherapy, so with this design we can assess whether the strength of the association between microbiome-affecting co-medication is equal among patients treated with immuno-and chemotherapy. We postulated that if the effectiveness of immunotherapy is indeed mediated by drug-related changes of the microbiome, a stronger association between the use of co-medication and the overall survival (OS) could be observed in patients treated with immunotherapy as compared to chemotherapy.

Study design
This is a retrospective observational historical-matched cohort study using clinical data from six hospitals of the Santeon hospital network.

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The Santeon hospital network consists of seven large teaching hospitals and covers more than 11% of all the hospitalizations in the Netherlands. The Netherlands Cancer Registry (NCR) was used for identifying all patients diagnosed with NSCLC and for obtaining information on the date of diagnosis and the vital status. Individual patients are assigned a unique anonymous identi er, which enables them to be tracked in the Santeon Farmadatabase (SFD). The information of the SFD was used for collection of detailed information about the systemic treatments. [14] Finally, the patients' medical records were used to complement the database with detailed information about the clinical and demographic characteristics, the use of concomitant medication and the treatment response. All data were gathered and stored at a Research Electronic Data Capture database (REDcap). [15] Study population Patients diagnosed with stage IV NSCLC between 1st January 2015 to 1st January 2019 and who started rst-, second-or third-line immunotherapy before the 1st of January 2020 were assigned to the immunotherapy group.
We matched every patient in the immunotherapy group to a patient with stage IV NSCLC who received conventional chemotherapy in the pre-immunotherapy era, and has been diagnosed before 1st January 2015 (see

Clinical characteristics
The patient characteristics and demographics were collected manually from the patients' medical records, including age, gender, body mass index (BMI), Eastern Cooperative Oncology Group-Performance status (ECOG-PS), the histology subtype, brain metastases and lines of systemic treatments. First-line treatment (1L) was de ned as the initial systemic therapy used in the treatment of NSCLC. Second-line and third-line treatment (2L and 3L) were de ned as the therapy given after discontinuation due to disease progression or completion of rst or second-line treatment, respectively. Additionally, oncogenic driver mutation status (e.g. EGFR, ALK, ROS-1) and PD-L1 expression were collected if available.

Concomitant medication
Patient records were reviewed to collect information on the use of concomitant medication potentially affecting the microbiome [10] [11]. The medication classes collected were antibiotics, proton-pump inhibitors, metformin, antidiabetics and opioids. Exposure was de ned if any information on the use of these drugs was reported by the physician in the medical health record within a timeframe of one month before until one month after the start of the systemic therapy.
Clinical outcomes OS was de ned as the time from the start of systemic therapy to death. Patients still alive at the end of follow-up on 1 January 2020 were censored at this date.

Statistical analysis
Statistical Software (SPSS version 26 for Windows: IBM) was used for statistical analysis. Categorical and continuous variables were summarized using descriptive statistics. To compare the immunotherapy and chemotherapy group, we used chi-squared tests (categorical variables) and independent t-test (continuous variables). The potential impact of concomitant medication on OS was analyzed through multivariable cox regression analyses. Possible factors associated with OS were rst identi ed using a univariable analysis. All univariate predictors with a p-value ≤ 0.15 and three other relevant variables -type of treatment, the ECOG-PS and the use of antibiotics -were used to construct the multivariable model. In the nal models, backward selection was applied to eliminate non-signi cant variables (p-value ≤ 0.10). Finally, the models were examined for the existence of effect modi cation by statistical testing of an interaction term between concomitant drugs of interest and the type of treatment (chemotherapy or immunotherapy). In order to investigate the difference between the lines of treatment, an exploratory analysis was performed for 1L patients and for 2L + 3L patients using the same approach described above. Survival curves using the Kaplan-Meier method were constructed to visualize -where considered of relevance-contrast between the use of concomitant medication and the type of treatment (chemotherapy or immunotherapy).

Ethical statement
All methods were carried out in accordance with relevant guidelines and regulations. Our ethics committee -the Santeon institutional review board -approved the study (SDB 2019-013) and waived informed consent. The need for informed consent was waved because of anonymous data handling and the retrospective nature of the study.
The study was performed in accordance with the ethical standards of the institutional and national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Results
Baseline characteristics A total of 221 immunotherapy patients could be matched to patients treated with chemotherapy in the preimmunotherapy era resulting in a total of 442 patients available for our primary analysis. The baseline characteristics of the immunotherapy and chemotherapy group are summarized in Table 1. Both immuno-and chemotherapy groups had an average age of 64 years. The majority of patients was male (59.2%) and received immunotherapy as second or higher line of treatment (62%). The demographic and baseline characteristics of the patients were well balanced between the two treatment groups, with the exception of the ECOG-PS. In the immunotherapy group, there was a signi cantly higher proportion of patients with an ECOG -PS1 (62.5% versus 39.8%) as compared to the chemotherapy group. In both groups, the most common histology subtype was adenocarcinoma, although there were fewer patients in the immunotherapy group with squamous tumor histology than in the chemotherapy group (10.9% versus 18.9% respectively). Table 2 provides a list of concomitant medication in use at the time of start systemic treatment. The most commonly used drugs in both groups were PPIs (43.4% vs 45.7% for the immuno-and chemotherapy group). Patients who received immunotherapy used signi cantly less opioids (26.7% vs 37.6% p = 0.02) and used more antibiotics as compared to the patients who received chemotherapy (15.8% vs 19.5%, p = 0.32). Only a small percentage of patients in the immunotherapy and chemotherapy group used metformin (3.6% and 9.5% respectively) or other antidiabetics (2.3% and 3.2% respectively).

Overall survival outcomes in the total population
The results of the univariable and multivariable analysis for OS are summarized in Table 3. The multivariable model showed that the use of antibiotics and opioids was signi cantly associated with shorter OS, with a corresponding HR of 1.39 (95%CI 1.06-1.81) and a HR 1.33 (95%CI 1.07-1.66) respectively. None of the other tested drugs were signi cantly associated with OS. Overall, the strongest factor associated with OS was the type of systemic treatment (immunotherapy vs chemotherapy). Other factors identi ed as independently associated with OS in the multivariable model were type of histology (squamous vs. non-squamous) and line of treatment (≥ 2L vs. 1L).
For both antibiotics and opioids, the tests for interaction with treatment type were insigni cant, both with a p value of 0.50. The distinctive effects of antibiotics and opioids on OS for the immuno-and chemotherapy cohort were summarized in table 6a. The use of antibiotics has a negative effect on OS outcomes for both patients treated with immunotherapy (HR 1.20 95% CI 0.79-1.85) and for patients treated with chemotherapy (HR 1.46 (95%CI 1.04-2.05). The use of opioids was associated with worse OS outcomes, with a HR 1.44 (95%CI 1.01-2.06) in immunotherapy group and a HR of 1.24 (95%CI 0.94-1.63) in chemotherapy group. Although not all these distinctive effects were signi cant, all HRs were > 1, indicating a negative effect of antibiotics and opioids for both immunotherapy and chemotherapy treated patients.

Survival outcomes per treatment line
Restriction of the cohort to patients with 1L treatment yielded that the use of antibiotics and opioids was also signi cantly associated with reduced OS in that setting. The HRs were 1.49 (95% CI 0.97-2.29) for antibiotic use and 1.58 (95% CI 1.08-2.32) for opioid use (Table 4).Other factors signi cantly associated with OS in the multivariable model were the type of treatment (immunotherapy vs chemotherapy) and the presence of brain metastases. The tests for interaction between treatment type and the use of antibiotics and opioids were both not statistically signi cant, with corresponding p values of 0.89 and 0.15 respectively. The negative effects of antibiotics and opioids on OS were observed in patients treated with chemotherapy as well as in patients treated with immunotherapy (table 6b). With HRs of similar magnitude between the immuno-and chemotherapy group, although not all were signi cant. In a second line setting, the use of antibiotics and the use opioids were not statistically signi cantly associated with OS. The multivariable model for OS showed that only the type of treatment (immunotherapy vs chemotherapy) was signi cantly associated with a greater risk of death (Table 5) Figures 1a and 1b demonstrate the OS-curves for patients treated with immuno-versus chemotherapy, showing the differences in result when using/not using antibiotics in the 1L (1a) and in the 2L/3L (1b). Association per type of treatment was not different.

Discussion
In this historically matched cohort study of 442 stage IV NSCLC patients, the use of antibiotics and opioids were shown to be independently associated with worse survival outcomes after adjusting for other prognostic factors associated with a poorer prognosis. Moreover, the negative in uence of antibiotics and opioids on OS was observed both for treatment with chemotherapy as well as with immunotherapy. This nding suggests that the association between worse survival outcomes and the use of antibiotics and opioids more likely originates from its use being linked with confounding by indication rather than the disturbing effect these drugs have on the microbiome.
We believe that our historically matched cohort study design adds to what has been published about the topic so far because all previously published reports on the association of antibiotics with immunotherapy effectiveness are retrospective single arm cohort studies. Lurienne et al (2020) summarized these cohort studies (n=21) in a meta-analysis and reported a pooled HR for OS of 1.69 (95% CI 1.25-2.29) for NSCLC patients exposed to antibiotics when starting immunotherapy [17]. Our observed HR of 1.39 is in line with this pooled number, but appears of similar magnitude in patients treated with chemotherapy in the years before the introduction of immunotherapy as an option. This nding argues against causality because an effect of antibiotics on chemotherapy effectiveness as a result of microbiome disturbance is unlikely. Thus, we consider that residual confounding by indication could have biased previous reports. An exception is the recent study of Chalabi et al. In a post hoc analysis of a clinical trial of atezolizumab versus docetaxel in a second line setting, there was a larger association between antibiotics and worse OS in the atezolizumab study arm compared to chemotherapy [18].
Considering the randomized nature of the data, residual confounding by indication is limited. On the other hand, also from this study causality cannot be concluded. It is also conceivable that rapid progressive disease requiring antibiotics, prevented patients randomized to immunotherapy from early disease control resulting in earlier death eventually [19].
For opioid use our ndings align with what has been studied by Zheng et al 2020 [20]. The authors conducted a cohort analysis (n =203 patients) and a meta-analysis (n = 26 articles) to investigate the impact of opioids use on survival of cancer patients. The results of their analyses showed a negative association between cancer-speci c survival and the use of opioids. This is in line with their meta-analysis indicating that opioid use for cancer-related pain is associated with poor OS of cancer patients. Furthermore, other studies have reported that opioids may negatively affect cancer patients' survival through respiratory depression, delirium, addiction or directly by acting on tumor cells [21] [22]. Even though these authors did not investigate whether the negative effects of opioids on survival outcomes differ between patients treated with immunotherapy or chemotherapy, it supports our observed association between opioids and survival irrespective of the systemic treatment applied.
Noteworthy is that the use of PPIs, metformin and other antidiabetics were not associated with poor survival outcomes in our cohort. This might indicate that both the microbiome-hypothesis and the hypothesis of confounding factors should be rejected for these drugs. Interesting, however, is that some other studies observed associations between PPI use and worse outcomes [16]. That we were unable to replicate this might be explained by the very high prevalence of PPI use in our cohort that could have resulted in overshadowing an assumed proxy of PPI use for worse clinical condition. In the Netherlands, PPIs are recommended for every adult >65 years and using anti-platelet drugs, NSAIDS or corticosteroids. Nevertheless, our data suggest no effect of PPIs on effectiveness of immunotherapy in NSCLC patients. The frequency of patients using metformin (6.5%) and antidiabetics (2.7%) in our cohort we consider too small to conclude on any association between these drugs and survival outcomes.

Strengths and weaknesses
Our study has a number of notable strengths. The main strength is its historically-matched study design. This design enabled us to investigate the possibility of concomitant medication-use having a prognostic impact on patients' clinical survival, regardless of the treatment given, under the assumption that unmeasured confounding factors will be balanced between the two groups. Because from the moment immunotherapy became available in 2015, less patients were quali ed for chemotherapy, and thereby creating imbalance of prognostic variables of patients receiving chemotherapy in the immunotherapy era. Another positive aspect is that we were able to evaluate not only the effects of antibiotics on the therapeutic outcomes of NSCLC patients but also the effects of other drugs, known to be associated with changes of the microbiome composition. There are numerous observational studies investigating the impact of antibiotics, however, studies regarding the effects of other drugs are scarce. Lastly, compared to previous reports, we used a relatively large cohort with advanced NSCLC patients treated in six different hospital across the Netherlands.
A limitation of our study is the small sample size of patients relative to the exposure of concomitant medication.
Another limitation, in hindsight, is that we did not collect usable data about the use of corticosteroids in our patients. Corticosteroids have been linked to altered immunotherapy effectiveness as well and inclusion of this variable might have resulted in other HRs with our variables of interest [23] [24]. Some co-linearity of corticosteroids and antibiotics or PPIs cannot be ruled out. Finally, our study shares all limitations linked to retrospective studies, at least information bias.

Conclusion
In conclusion, we observed that the use of antibiotics and opioids is associated with shorter survival similarly in chemotherapy and immunotherapy treated NSCLC patients. This suggests that the association is likely to be a consequence of confounding by indication rather than disturbing the composition of the microbiome. advanced non-small cell lung cancer, melanoma, or urothel. Oncoimmunology, 9 (1), 1-9 (2020).

Declarations
24. Mountzios, G. et al. Steroid use independently predicts for poor outcomes in patients with advanced NSCLC and high PD-L1 expression receiving rst-line pembrolizumab Mmonotherapy.Clin Lung Cancer. 2020.      a. OS total population opioids (use vs no use). 3 Interaction between antibiotic use and type of treatment 4 Interaction between opioid use and type of treatment Figures Figure 1 Kaplan-Meiercurves showing overall survival within the chemo-and immunotherapy group with and without the use of antibiotics, strati ed for 1st line treatment (a) and 2nd/3rd line treatment (b).