Effect of antimicrobial therapy on progression-free survival of immunotherapy and chemo-/immunotherapy in patients with non-small cell lung cancer

The choice between immunotherapy with a checkpoint inhibitor (CPI) and chemo-/immunotherapy (CIT) in patients with NSCLC stage IV is often discussed. There are some data that the effect of CPI therapy is impaired by antimicrobial therapy (AMT). Little is known about the influence of AMT on CIT. We retrospectively analysed 114 patients (age 68 ± 8.5 years) with NSCLC stage IV. Patients were treated according to the guidelines with either CPI alone (pembrolizumab, nivolumab, atezolizumab, cemiplimab) or CIT (Carboplatin/Pemetrexed/Pembrolizumab, Carboplatin/Paclitaxel/Pembrolizumab). We registered patients’ characteristics including presence and timing of AMT. Group 1 consisted of 42 patients with AMT in the month before CPI or CIT, group 2 were 49 patients with AMT during CPI or CIT, and group 3 were 64 patients without AMT and CPI or CIT. Group 1–3 showed comparable patients characteristics. Using cox-regression analysis, we found that AMT in the month before CPI resulted in a decreased progression-free survival (PFS) compared to patients with CPI and no AMT (14 ± 1.02 vs. 4 ± 1.02 months, p = 0.002, 95% CI 1.88–9). In patients, who were treated with CIT, there was no difference in PFS in those with or without AMT in the month before therapy (10 ± 2.5 vs. 6 ± 1.2 months, p = 0.7). Interestingly, AMT during CIT or CPI therapy showed no effect on PFS. In a real-life setting, we found that AMT reduces PFS when given in the month before CIT therapy. AMT before or during CIT does not seem to influence PFS. As a consequence, AMT before start of therapy might be a factor that could lead to a preference of CIT instead of CPI in NSCLC stage IV patients.


Background
Immunotherapy with a checkpoint inhibitor (CPI) and chemo-/immunotherapy (CIT) have significantly changed therapy in non-small cell lung cancer (NSCLC) in recent years. There are some data that the effect of CPI therapy is influenced by the microbiome (Lee et al. 2021). A gut dysbiosis due to antimicrobial therapy (AMT) is associated with a dysfunctional immune system (Lee et al. 2021;Pflug et al. 2016). In a retrospective study, it was shown that AMT, given within a month before CPI, leads to a shorter progression-free survival (PFS) in patients with NSCLC (Uhlenbruch and Kruger 2021). Some studies showed negative effects of AMT on outcome in patients with chemotherapy, too. This was more pronounced in patients with CPI therapy, but also present under chemotherapy (Hekmatshoar et al. 2019).
In one study with 302 patients with NSCLC stage IV, Cortinelli et al. saw a significantly shorter PFS and overall survival (OS) in patients, who were treated with AMT. AMT had an influence on CPI but not on CIT (Cortellini et al. 2021). In this study, there was no discrimination with respect to comorbidities.
Alltogether knowledge about the influence of AMT on CIT is limited. Moreover, predictive parameters for the use of CPI alone versus CIT with respect to AMT are missing.
Therefore, the aim of our study was to analyse, if the effect of AMT is different on CPI and CIT with respect to comorbidities in patients with NSCLC stage IV disease.

Study design and participants
In a retrospective study design, we analysed 114 patients (age 68 ± 8.5 years) with NSCLC stage IV. All patients were treated in our lung cancer centre in the Florence-Nightingale Hospital in Düsseldorf/Germany. We collected data from 06/2017 to 01/22.
We registered patients' characteristics including presence and timing of AMT. We accepted prior therapy lines of NSCLC according to guidelines.
We chose variables, based on their prognostic impact in NSCLC and risk factors for infections. This included age, sex, BMI, ECOG performance status, lung cancer pathology (squamous and non-squamous NSCLC), comorbidities (COPD, diabetes mellitus, hypertension), PD-L1 status, AMT use, and timing of AMT.
Group 1 consisted of 42 patients with AMT in the month before CPI or CIT, group 2 were 49 patients with AMT during CPI or CIT, and group 3 were 64 patients without AMT and CPI or CIT. Patient characteristics are shown in Table 1.
Information about timing of AMT and PFS was taken from our electronic patient database. Only patients with all available data about AMT and PFS were included in the analysis.
AMT was defined as at least 5 day treatment with antimicrobial drugs. PFS was determined according to classical RECIST criteria in repeated restaging with computed tomography during antineoplastic therapy.

Statistics
Continuous variables are expressed as mean ± SD or median and compared using t test unless stated otherwise. Statistical analysis was performed using SPSS (Version 28, IBM, Armonk, NY). Cox regression was used to analyse the effect of several factors on progression-free survival in uni-and multivariable analyses.
Cox regression survival curves were generated to visualize the distribution of times from baseline to disease progression. All statistical tests were two-tailed and a p value < 0.05 was considered statistically significant.

Results
We included 114 patients in our analysis. 61 patients were treated with CPI, while 53 received CIT. 25 patients with CIT got AMT in the month before start of CIT and 16 patients during CIT. 17 patients with CPI were treated with AMT 1 month before start of CPI therapy and 23 patients during CPI therapy. At the time of final analysis, 80 patients had died and 34 patients were still treated with CPI.
Median PFS in group 1 (patients with AMT in the month before CPI or CIT) was comparable in those with CPI and CIT (4 ± 1.02 vs. 6 ± 1.2 months, p = 0.97).
Median PFS in group 2 (patients with AMT during CPI or CIT) was comparable in those with CPI and CTI (8 ± 2.1 vs. 3 ± 2.4 months, p = 0.135).
However, CIT patients without AMT versus CIT patients with AMT in the month before start of therapy showed comparable PFS (10 ± 2.5 vs. 6 ± 1.2 months, p = 0.7).
The Cox regression survival curve for PFS of patients with CPI and CTI is shown in Fig. 1.
The number of comorbidities in patients with or without AMT before start of CPI or CIT showed no significant differences.
Cox regression demonstrated no significant effect of any comorbidity on PFS, except for diabetes mellitus.
In addition to AMT in the month before CPI therapy, PFS was significantly negatively influenced by the presence of type II diabetes mellitus in CPI patients (p = 0.03).
The effect of CIT was not influenced by the presence of diabetes mellitus (p = 0.6).

Discussion
The main finding of our study is that AMT before start of CPI decreases PFS, whereas CIT is not influenced by AMT before start of therapy.
Previous studies have shown the possible negative effect of AMT in patients with NSCLC and CPI. However, in these previous studies, there was no focus on comorbidities. To the best of our knowledge, we are the first to demonstrate that especially AMT in the month before CPI results in a lower PFS, whereas CIT is not influenced by AMT in this setting. The reason might be that CIT is associated with resilience on the effect of the microbiome on CPI. This is reasonable, because chemotherapy is not influenced by AMT (Pflug et al. 2016;Galli et al. 2019).
Neither AMT, nor diabetes mellitus, which might lead to dysbiosis, was shown to influence PFS in patients with CIT.
The hypothesis for the explanation of our findings might be: AMT leads to relevant short-term changes in the microbiome, which might negatively influence the immunological answer to CPI. Once the immune system under the influence of CPI started to stimulate specific T-lymphocytes to attack tumor cells, the immunological answer to CPI might not be so vulnerable anymore as a consequence of AMT. This could explain why AMT given during CPI or CIT therapy, which also causes dysbiosis, showed no significant effect in our study. The negative effect of diabetes, that we found, might also be linked to dysbiosis (Moreno-Indias et al. 2014) and could be associated with reduced PFS under CPI therapy.
Patients treated with CIT could be more resistant to dysbiosis, because chemotherapy seems not to be influenced by the microbiome in the same way as it is in CPI.
Thus, AMT before start of therapy might be a factor that could lead to a preference of CIT instead of CPI in NSCLC stage IV patients.
Our study has several limitations. First, it is a retrospective study with a moderate number of patients. Second, we did not analyse the microbiome and its possible changes under AMT during the study. Third, patients with an infection often show more signs of systemic inflammation and are more often in a reduced clinical status as sequelae of the infection.
On the other hand, patients were only allowed to receive CPI or CIT, if they recovered from severe infection.
In conclusion, our data are hypothesis generating and need validation in a larger study collectively.