Microbiota diversity in non-small cell lung cancer gut and mouth cavity microbiota diversity in non-small cell lung cancer patients

ORIGINAL PAPER Polish Journal of Microbiology 2023, Vol. 72, No 4, 467–475 https://doi.org/10.33073/pjm-2023-044 * Corresponding authors: J. Škarda, Department of Clinical and Molecular Pathology, Faculty of Medicine and Dentistry, Palacky University and University Hospital Olomouc, Olomouc, Czech Republic; Institute of Molecular and Clinical Pathology and Medical Genetics, University Hospital Ostrava, Ostrava, Czech Republic; Faculty of Medicine University of Ostrava, Ostrava, Czech Republic; e‑mail:[email protected]; +420597372305 V. Skopelidou, Institute of Molecular and Clinical Pathology and Medical Genetics, University Hospital Ostrava, Ostrava, Czech Republic; Faculty of Medicine University of Ostrava, Ostrava, Czech Republic; e‑mail:[email protected] © 2023 Aneta Brisudová et al. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 License (https://creativecommons.org/licenses/by-nc-nd/4.0/). Microbiota Diversity in Non‑Small Cell Lung Cancer Gut and Mouth Cavity Microbiota Diversity in Non‑Small Cell Lung Cancer Patients ANETA BRISUDOVÁ1, HANA BIELNIKOVÁ-KRYŠTOFOVÁ2, 3, OLDŘICH MOTYKA4, 5, DOMINIKA FRITZOVÁ1, VLADIMÍRA KAŤUCHOVÁ1, NATÁLIE PONIKELSKÁ2, 3, DANIELA SKANDEROVÁ1, VLADISLAV RACLAVSKÝ6, JAROSLAV MICHÁLEK1, MARCEL MITTÁK3, 7, PETRA ŠVECOVÁ8, PETR JAKUBEC8, DENISA ROZSIVALOVÁ8, MAREK SZKORUPA9, JIŘÍ KLEIN10, JOZEFŠKARDA1, 2, 3*, ZDENĚK KOLÁŘ1 and VALERIA SKOPELIDOU2, 3* 1 Department of Clinical and Molecular Pathology, Faculty of Medicine and Dentistry, Palacky University and University Hospital Olomouc, Olomouc, Czech Republic 2 Institute of Molecular and Clinical Pathology and Medical Genetics, University Hospital Ostrava, Ostrava, Czech Republic 3 Faculty of Medicine University of Ostrava, Ostrava, Czech Republic 4 Faculty of Mining and Geology, VŠB – Technical University of Ostrava, Ostrava, Czech Republic 5 Nanotechnology Centre, CEET, VŠB – Technical University of Ostrava, Ostrava, Czech Republic 6 Department of Microbiology, Faculty of Medicine and Dentistry, Palacky University and University Hospital Olomouc, Olomouc, Czech Republic 7 Department of Surgical Studies, University Hospital Ostrava, Ostrava, Czech Republic 8 Department of Respiratory Diseases, Faculty of Medicine and Dentistry, Palacky University and University Hospital Olomouc, Olomouc, Czech Republic 9 Department of Surgery I, Faculty of Medicine and Dentistry, Palacky University and University Hospital Olomouc, Olomouc, Czech Republic 10 Surgical Clinic, Thomas Baťa Regional Hospital, Czech Republic Submitted 10 August, accepted 27 October 2023, published online 22 December 2023 Abstract Keywords: lung cancer, oncogenic mutations, microbiota, non-small cell lung carcinoma Lung malignancies have a substantial impact on cancer incidence and mortality worldwide. Even though many factors involved in the development of the disease are known, many questions remain unanswered. Previous studies suggest that the intestinal micro - bi ota may have a role in developing malignant diseases. According to some findings, the microbiota has proven to be a key modulator of carcinogenic processes and the immune response against cancer cells, potentially influencing the effectiveness of immunotherapy. In our study, we characterized culturable microorganisms associated with non-small cell lung cancer (NSCLC) that can be recovered from rectal swabs and mouthwash. In addition, we also explored differences in the culturable microbiota with two main types of NSCLC – adenocarcinoma (ADC) and squamous cell carcinoma (SCC). With 141 patients included in the study (86ADC and 55SCC cases), a significant difference was observed between the two types in seven bacterial species (Collinsella, Corynebacterium, Klebsiella, Lactobacillus, Neisseria, Rothia, and Streptococcus), including the site of origin. The relationship between microbial dysbiosis and lung cancer is poorly understood; future research could shed light on the links between gut microbiota and lung cancer development. Brisudová A. et al. 4468 Introduction Lung carcinoma (LC) is a heterogeneous disease that is the most significant cause of death in both men and women worldwide. The World Health Organization (WHO) classifies lung cancer into two types: small cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC), which accounts for more than 85% of cases. NSCLCs are divided into adenocarcinomas (ADC, 40–50%), squamous cell carcinomas (SCC, 20–30%), large cell carcinomas (LCC, 3–10%) and other uncommon carcinomas (Ettinger et al. 2013). Histological features and marker expression remain the basis of tumor diagnosis; nevertheless, examination of genetic mutations within lung tumors becomes very important (Chen et al. 2017). It is generally known that the incidence of SCC is significantly linked to smoking; on the other hand, there is a high proportion of non-smokers in the ADC group (Song et al. 2012). Therefore, variables other than smoking should impact the development of ADC, and one of the candidates, in addition to hereditary predisposition, is the activity of the microbiota. As aresult, we sought to investigate whether there are significant differences in microbiota composition between ADC and SCC. Various diagnostic and therapeutic procedures have been developed with the expansion of modern genetic and molecular techniques. Essential mutations in specific genes, including NSCLC, can lead to cancer formation. These mutations occur in oncogenes or tumor suppressor genes. Epidermal growth factor receptor (EGFR), anaplastic lymphoma kinase (ALK), ROS Proto-Oncogene1, Receptor Tyrosine Kinase (ROS1) (Shaw et al. 2014; Mazières et al. 2015) and programmed cell death ligand 1 (PD-L1) are frequently monitored oncogenes that affect NSCLC development (Lindeman et al. 2013; Oxnard et al. 2013). The discovery of targeted therapies aiming at a subset of molecular markers has changed the management strategy of NSCLC. In patients with proven activating mutations in the EGFR gene, tyrosine kinase inhibitors (TKI) are frequently used as a first-line therapy. For instance, patients with ALK/ROS1 could benefit from crizotinib (Song et al. 2017). Previously, the lungs were assumed to be sterile; however, it is now known that they are constantly exposed to varied bacteria inhaled through the air and from the upper respiratory tract (Dickson and Huffnagle 2015). The microbiota represents more than 40 billion cells of the human body. The human microbiome positively and negatively influences systemic metabolic, endocrine, and immunological systems (Gopalakrishnan et al. 2018). Recent studies have revealed that the microbiota of healthy and tumoraffected lung tissue differ in numerous ways. Furthermore, NSCLC is linked to persistent inflammation mediated by particular immunological mediators. Many microorganism species commonly infect the lungs of cancer patients (Wong-Rolle et al. 2021). Many of our critical physiological processes, including the immune response, tumor growth, and responsiveness to anticancer therapy, are influenced by microbiota from our gut, skin, oral, respiratory, and genital tracts. The complex interactions between human cells and microbes may extend to cancer (Human Microbiome Project Consortium 2012). Microorganisms are involved in 20% of human malignancies (de Martel et al. 2012; Matson et al. 2018; Routy et al. 2018). Bacteria were discovered in human tumors more than a century ago, but the mechanism of their influence on the development and progression of cancer is still unexplained (Nejman et al. 2020). Tumor microbiota has a relatively low biomass, and it is problematic to detect any possible contamination in profiling the tumor microbiome. Mao et al. (2018) showed the Mycobacterium tuberculosis bacterium, which produces inflammatory markers, to cause significant changes in lung tissue, similar to the model established for Helicobacter pylori in gastric cancer. The microbiota was previously closely related to gastrointestinal malignancies; it affects carcinogenesis and immunotherapy of malignancies, including NSCLC (Yang et al. 2020; Liu et al. 2021). Among others, the microbiota is involved in the body’s response to targeted therapy with tyrosine kinase inhibitors and monoclonal antibodies. According to recent research, the microbiota in the gut corresponds with clinical response to targeted therapy (Liang et al. 2009; Davis et al. 2018). Unlike most research, which utilizes next-generation sequencing (NGS) to characterize the microbiome as the genetic correlate of microbiota, we investigated the ability of conventional culture to achieve the same at the level of easily cultured bacteria. Such a method can potentially overcome the fundamental limitation of microbiome investigations, which is the lack of species resolution, and shed insight on the microbiota of NSCLC patients from a previously unknown perspective. We utilized two easily accessible types of samples to facilitate performance: mouth wash and rectal swab. In this study, we describe the observed differences and explore their significance about ADC versus SCC and mouth versus rectum origin. Experimental Materials and Methods Patients. Altogether, 141 NSCLC patients seeking care at the Department of Respiratory Diseases, University Hospital Olomouc, were recruited for the study Microbiota NSCLC 4 469 (TableI). However, some of the patients diagnosed to have a lung lesion on X-ray and thus suspected to suffer lung cancer were also sampled at the time of yet unclear diagnosis to ensure the culture results not to be biased by any healthcare interventions assumed to follow. Most of them turned out to carry a metastasis of an extrapulmonary cancer or lymphoma. These patients were included as a quasi-control group to support the correct interpretation of the differences observed when comparing the microbiota of adenocarcinoma versus squamous cell carcinoma patients. Patients were recruited from April 2019 to October 2022, regardless of disease stage, age, smoking status, or gender. Microbial cultivation. Rectal swabs were collected using the Transystem™ 116C Traditional Bacteriology Transport Swabs (COPAN Diagnostics Inc., USA); 10 ml of sterile saline was moved into the mouth cavity and collected into a sterile tube. On the same day, both samples were delivered to the laboratory. Petri plates with Columbia Blood Agar (CBA), MacConkey Agar (MCA), Brain Heart Infusion (BHI) agar with 10% sheep blood, Schaedler Agar (SChA), and nalidixic acid and sulfamethazine (NAS) (Waite et al. 2012) agar plates were inoculated for bacterial cultivation. Concerning the purpose of different plates, CBA was inoculated to grow a broad spectrum of aerobic bacteria, and MCA to grow the subset of non-fastidious gram-negative bacteria to enable better differentiation of their colony morphology compared to CBA. BHI and Schaedler Agar were inoculated to grow anaerobic bacteria, and the semi-selective NAS agar was inoculated and cultured under microaerophilic conditions to facilitate the growth of streptococci, Streptococcus anginosus group (SAG), in particular. Sabouraud Glucose Agar with chloramphenicol (SGA) was inoculated for fungal cultivation (all from Oxoid, UK). Plates were incubated at 37°C in ambient air supplemented with 5% carbon dioxide (CBA, MCA), at 37°C in an anaerobic gas mixture (BHI, SChA, NAS; 80% nitrogen, 10% carbon dioxide, 10% hydrogen), and 30°C in ambient air (SGA). All aerobic plates were kept in a moist chamber during incubation to protect agar from drying and secure the best growth of microbes. Bacterial plates were incubated for 3 days; SGA was incubated for 5 days. All colonial morphotypes were subcultured for 24hours on appropriate media and conditions to verify pure culture and achieve the best results following identification; the cultivation period was appropriately prolonged in slowly growing isolates to collect sufficient cells for identification. Bacterial isolates were subcultured on Columbia Blood Agar, and toothpick sampling was used to prepare a direct smear on MALDI TOF target plate, which was then overlaid by 1 µl of 70% formic acid, air dried, and overlaid by 1 µl of matrix solution (α-cyano-4-hydroxycinnamic acid solution, HCCA; Sigma-Aldrich, USA). A MALDI Microflex® LT instrument (Bruker Corporation, USA) was used for identification in automated mode by the manufacturers’ instructions (MALDI Biotyper® 3.1 User manual revision1). In cases when the confidence score values dropped below 2.0, user-targeted laser shots were performed additionally to improve the score. Because of the limited resolution power in the case of the Streptococcus mitis group (S. mitis, Streptococcus oralis, Streptococcus pneumoniae, Streptococcus pseudopneumoniae), members of this group are referred to as one complex species. Statistical analysis. Multiple Correspondence Analysis was applied to the microbiome data to assess the relationships between all the observed variables. Both the TNM level and the site were – independently – used as supplementary variables in the multivariate model so that they would not affect the construction of the model and allow the determination of the overall dependence of microbiome composition on those parameters. The chi-squared test was applied for further bivariate assessments. Kaplan-Meier survival curves were plotted for all the categorical variables and Peto and Peto modification of the Gehan-Wilcoxon test was used to assess the differences between the curves. All the statistical analyses and visualizations were performed in the R environment (R Core Team 2021). The multivariate assessment was performed using the R package FactoMineR (Lê et al. 2008); the survival analysis was performed using survminer (Kassambara et al. 2021) and survival R packages (Therneau et al. 2023). The chi-square test or Fisher’s exact test was applied appropriately to evaluate the statistical significance of differences in the frequency of each identified species between ADC and SCC. Results Fig. 1 demonstrates the relative frequencies of 8 bacterial species that were either overrepresented (> 1.5 × more frequent) or underrepresented (< 0.5 × less frequent) in Age median 72 70 Male/Female 54/32 39/16 I 12 13 Stage II 9 10 III 23 17 IV 42 15 Smoker 26 22 Ex-smoker 45 26 Non-smoker 15 7 Table I Characterization of the patient cohort. ADC SCC Brisudová A. et al. 4470 mouth wash of ADC versus SCC patients. The highest significance was observed in Lactobacillus fermentum (10% in ADC versus 30% in SCC, p = 0,002), followed by S. mitis, S. oralis, S. pneumoniae, S. anginosus, Streptococcus mutans, Streptococcus sanguinis and Neisseria macacae (Table II). The overrepresentation of Streptococcus constellatus and Rothia aeria in ADC was not found to be significant. Compared to the quasi-control group of patients without confirmed lung cancer, the association between S. mutans and S. anginosus in mouth wash was further supported as significant. A similar analysis of rectal samples (Fig. 2, TableIII) showed less pronounced differences between ADC and SCC, namely, only six bacterial species met the criterium of overor underrepresentation (> 1,5 × or < 0,5 × times, respectively), and these differences were Rothia aeria 9 10.5 1 0.2 14 21.5 52.5 – – Streptococcus constellatus 13 15.1 3 5.5 16 24.6 2.7 0.080 0.152 Neisseria macacae 21 24.4 6 10.9 13 20.0 2.2 0.047 0.495 Streptococcus mutans 24 27.9 7 12.7 9 13.8 2.2 0.034 0.035 Streptococcus sanguinis 25 29.1 8 14.5 16 24.6 2.0 0.047 0.514 Streptococcus anginosus 46 53.5 19 34.5 23 35.4 1.5 0.028 0.023 Lactobacillus fermentum 9 10.5 17 30.9 14 20.9 0.3 0.002 0.065 Candida tropicalis 2 2.4 11 20.8 1 0.01 0.1 – – Table II Comparison of bacterial species cultured from mouth wash in adenocarcinoma (ADC; n = 85) and squamous cell carcinoma (SCC; n = 53) in NSCLC patients versus patients without confirmed lung cancer (n = 65). The species are ranked according to the measure of overrepresentation in ADC versus SCC patients (column 6, ADC/SCC ratio). Only those overrepresented ≥ 1,5 × or underrepresented ≤ 0,5 × are listed. NSCLC – non-small cell lung cancer Species ADC patients SCC patients No lung cancer ADC/SCC ratio p-value ADC/SCC p-value ADC/non n%n%n% Fig. 1. Relative abundance (%) of bacterial species overrepresented ≥ 1.5 ×, or underrepresented ≤ 0.5 ×, in ADC versus SCC patients’ mouth wash. Y axis – number of patients, blue columns – ADC patients, orange columns – SCC patients, * – significant difference (p ≤ 0.05), ADC – adenocarcinoma, SCC – squamous cell carcinoma Microbiota NSCLC 4 471 significant in four of the six species. The highest significance was observed in S. anginosus (50% in ADC versus 22% in SCC, p = 0.002), followed by Klebsiella oxytoca and Corynebacterium aurimucosum. The overrepresentation of S. mitis, S. oralis, S. pneumoniae and Collinsella aerofaciens in ADC was not found to be significant. Compared to the quasi-control group of patients without confirmed lung cancer, the association between S. anginosus in rectal swabs of ADC patients was not supported as significant, in contrast to the decreased occurrence of C. aurimucosum. As the composition of especially the mucosal microbiota can also be influenced by smoking, which is an important factor involved in the origin and development Fig. 2. Relative abundance (%) of bacterial species overrepresented ≥ 1.5 ×, or underrepresented ≤ 0.5 ×, in ADC versus SCC patients’ rectal swabs. Y axis – number of patients, blue columns – ADC patients, orange columns – SCC patients, *–significant difference (p ≤ 0.05), ADC – adenocarcinoma, SCC – squamous cell carcinoma Table III Comparison of bacterial species cultured from rectal swabs in adenocarcinoma (ADC; n = 85) and squamous cell carcinoma (SCC; n = 52) in NSCLC patients versus patients without confirmed lung cancer (n = 65). The species are ranked according to the measure of overrepresentation in ADC versus SCC patients (column 6, ADC/SCC ratio). Only those overrepresented ≥ 1,5 × or underrepresented ≤ 0,5 × are listed. Species ADC patients SCC patients No lung cancer ADC/SCC ratio p-value ADC/SCC p-value ADC/non n%n%n% Klebsiella oxytoca 11 12,9 1 1,9 8 12,3 6,8 – 0,688 Streptococcus anginosus 42 49,4 12 22,6 33 50,8 2,2 0,002 0,109 Streptococcus MOPP* 20 23,5 6 11,3 12 18,5 2,1 0,074 0,453 Collinsella aerofaciens 28 32,9 10 18,9 13 20,0 1,7 0,072 0,086 Corynebacterium aurimucosum 12 14,1 15 28,3 19 29,2 0,5 0,041 0,024 Alistipes shahii 2 2,4 5 9,6 2 30,8 0,2 – – * – MALDI-TOF MS technology fails to differentiate the species Streptococcus mitis, Streptococcus oralis, Streptococcuspneumoniae, and Streptococcus pseudopneumoniae, reliably. Therefore, the numbers are summed under a complex of species abbreviated as Streptococcus MOPP. Brisudová A. et al. 4472 of NSCLC, we also assessed for all species (associated with adenocarcinoma) and their occurrence across the entire set when comparing the status of patients classified as smokers (n = 46), ex-smokers (n = 71) and non-smokers (n = 20). No statistically significant dependence on smoking status was found for any of the evaluated species, including their combinations (e.g., smokers + ex-smokers versus non-smokers; nonsmokers + ex-smokers versus smokers, etc.). Furthermore, smoking status was not found to have a significant effect on overall survival, as is apparent from the Kaplan-Meier curves presented in Fig. 3. Peto and Peto modification of the Gehan-Wilcoxon test was used to assess the differences between the two curves and also found no significant difference (p = 0.5). Stage IA3 had higher occurrences of Alistipes shahii and L. fermentum. L. fermentum was also overrepresen ted in the IIA stage. IIB had higher occurrences of S. pneumoniaeandR. aeria. Lower occurrences of S. pneumoniae and S. mutans characterized stage IIIA. Ahigher occurrence of S. anginosus was also characteristic of stage IVB. Discussion Lung cancer is a heterogeneous disease with the leading cause of death worldwide. Although smoking is recognized to be the primary cause of lung cancer, specific microbiota can be related to or possibly contribute to lung cancer carcinogenesis (Chen, Domingue and Sears 2017). In most situations, microbiome analysis using 16S rRNA gene sequencing is utilized to identify microbiota composition, resulting in resolution limited to the genus level. In this study, we characterized ADC and SCC culturable microbiota, which allows for comprehensive and reliable species resolution in most cases. On the other hand, the ability to analyze culturable species only represents the main limitation of our study. Thus, our aim was not to compete with or replace microbiome-based studies but to complement them. To make recruiting participants less complicated, we focused on two easily accessible types of samples: mouth wash and rectal swab. As a result, we could not investigate any samples recovered directly from the lower respiratory tract, which is another limitation of our study. However, since oral microorganisms are the primary source of bacterial microbiota in the lungs, any significant changes in oral microbiota should be mirrored in the lungs (Dickson and Huffnagle 2015). The gut microbiota was included in our study because of its generally known ability to affect immunological responses not only locally but also systemically. The proposed existence of a gut-lung axis (Enaud et al. 2020) was another stimulus to study the gut and oral microbiota in parallel. Mouth wash culture results in ADC versus SCC and quasi‑control group. R. aeria was the species showing the highest overrepresentation in ADC versus SCC patients; however, it was found in very few cases, which limits the informativeness of the result. Similarly, the second-ranked S. constellatus, astreptococcal species causing pyogenic infections in various body sites, showed low numbers without statistical significance. Fig. 3. Correlation of smoking status and overall survival probability presented using Kaplan-Meier survival curves. N signifies non-smokers, while Y represents smokers. Microbiota NSCLC 4 473 However, it is worth mentioning its association (together with S. anginosus, see further in the text), with early gastric cancer (Zhou et al. 2022). Whereas little is known about the pathogenic or pro-oncogenic potential of N. maccacae ranked 3, the No. 4 S. mutants has been described to promote tumor progression in oral squamous cell carcinoma (Tsai et al. 2022). S. sanguinis has no apparent link to any tumor. However, it is recognized to be associated with infective endocarditis, and the elevation of pro-inflammatory genes has been observed in mouse models (Hashizume-Takizawa et al. 2019). The picture is brighter in the case of S. anginosus, which appears to be directly linked to the development of esophageal, gastric, pharyngeal, and oral cancer (Zhang et al. 2019). Although other members of oral microbiota (Porphyromonas gingivalis, Fusobacterium nucleatum, Treponema denticola) have also been linked to oral cancer, none of them is known to cause infection of the respiratory pathways, whereas S. anginosus was described to do so in at least two chronic respiratory diseases, namely cystic fibrosis, and chronic obstructive pulmonary disease (Parkins et al. 2008; Navratilova et al. 2016). S. anginosus, when present in the oral microbiota, may thus be capable of affectingrespiratory pathways and increase the risk of ADC much more than SCC, which is strongly linked to smoking. On the other hand, the difference is not as distinct and unequivocal (53.5% in ADC versus 34.6% in SCC, p = 0,028) as it might be in the case of a straightforward mechanism. Therefore, S. anginosus interstrain differences in pro-inflammatory action described earlier. Interestingly, the association between S. anginosus presence in microbiota and risk of ADC was also confirmed by rectal swab culture (49.4% in ADC versus 22.6% in SCC, p = 0,002). This suggests that this bacterium is perfectly suited to colonize both the mouth cavity and the gut, with no preference for one of these environments over the other. We can only speculate about possible other mechanisms of S. anginosus pro-oncogenic activity beyond the general pro-inflammatory action. To mention at least one of them, the production of hydrogen sulfide (H2S) can be found in a long list of S. anginosus virulence factors (Asam and Spellerberg, 2014) and H2S has also been described to create a favorable immune microenvironment for colon cancer (Yue et al. 2023). Regarding the species underrepresented in ADC versus SCC, the probiotic species L. fermentum, which is widely known for its anti-inflammatory properties, was significantly underrepresented in ADC. The commensal yeast species Candida tropicalis was found in low numbers and the difference was not significant. When the data from the ADC versus SCC mouth cavity microbiota relation were compared to the quasicontrol group, the significance was convincingly confirmed in the case of the overrepresentation of S. mutans and S. anginosus and underrepresentation of L. fermentum, but questioned in N. macacae and S. sanguinis, where any hints about a possible role in tumorigenesis are also lacking in the available literature. Rectal swab culture results in ADC versus SCC. Not surprisingly, less pronounced differences between ADC and SCC patients were found in the complex culturable gut microbiota sampled by rectal swabs. K. oxytoca was the species showing the highest overrepresentation in ADC versus SCC patients. However, it was found in very few cases, which limits the informativeness of the result. S. anginosus was the only one that met the criterion of statistical significance among the other species found to be overrepresented in ADC versus SCC; the same was true for C. aurimucosum in the species underrepresented in ADC versus SCC. Interestingly, C. aurimucosum was also found in individuals previously less responsive to NSCLC targeted therapy (Routy et al. 2018; Sędzikowska and Szablewski 2021). When the data from the ADC versus SCC rectal swab microbiota relation were compared to the quasicontrol group to guarantee a more reliable interpretation, the significance of S. anginosus overrepresentation (p = 0,109) was not convincingly confirmed, whereas C. aurimucosum underrepresentation (p = 0,024) was. Taken together, the considerable overrepresentation of S. anginosus in both the mouth wash and rectal swabs of ADC versus SCC patients is the most noticeable discovery of our study, which has yet to be reported in any microbiome-based study. Because ADC represents one of the most frequent forms of NSCLC, our finding of relative overrepresentation of streptococcal species in the mouth wash of ADC patients (S. anginosus, S. constellatus, S. mutans and S. sanguinis) is well in accordance with the results of (Tsay et al. 2018) who described the increased abundance of streptococcal species in lower respiratory pathways of lung cancer patients compared to controls. Conclusions To summarize, our findings show significant differences in the oral cavity microbiota between ADC and SCC patients. The list of bacterial species shown to be overrepresented in ADC versus SCC cases includes species with pro-inflammatory potential. In contrast, the only bacterium with anti-inflammatory potential, L. fermentum, was found to be underrepresented in ADC. As a result, our findings are consistent with the widely accepted role of chronic inflammation in carcinogenesis, in which inflammation serves as a bridge between intrinsic (oncogenes, tumor suppressors, and genome stability genes) and extrinsic (immune and stromal components) factors involved in tumor development. Brisudová A. et al. 4474 It also supports the notion that microbiota may one day serve as a potential risk marker and an appealing target for cancer-prevention intervention. ORCID Valeria Skopelidou https://orcid.org/0000-0001-7466-7535 Acknowledgments This work was supported by the project No. NV19-03-00069, Correlation of molecular biomarkers potentially important for immunotherapy with gut microbiome quality in NSCLC patients with the Agency for Medical Research of the Czech Republic. 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