The Diversity of Gut Microbiome is Associated With Favorable Responses to Anti–Programmed Death 1 Immunotherapy in Chinese Patients With NSCLC

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§ The Diversity of Gut Microbiome is Associated With Favorable Responses to Anti-Programmed Death 1 Immunotherapy in Chinese Patients With NSCLC > Discussion In the present study, we have investigated gut microbiota profiles in Chinese patients with advanced NSCLC receiving nivolumab treatment.Our results indicate that patients with better clinical responses exhibit higher diversity as well as a distinct gut microbiome composition.In addition, the association between gut microbiome diversity and systematic immune signatures highlights memory CD8 þ T cells and NK cells as potential mediators of antitumor immunity upon ICI treatment.The diversity of gut microbiome is defined as the number and abundance distribution of distinct types of micro-organisms colonizing within the gut.Abnormal diversity (dysbiosis) in the gut has been proven to be associated with human diseases, such as obesity, inflammatory bowel disease, cancer, etc. 20,21,33,34 Recently, Gopalakrishnan et al. 35 reported that patients with a high diversity of gut microbiome favored the clinical outcomes of anti-PD-1 immunotherapy in melanoma patients in the Western population.In our study, both alpha diversity and beta diversity at baseline are dramatically different between R and NR as well.A higher alpha diversity index represented by both the Shannon index and inverse Simpson indexes in R group suggests more species harboring in the gut, which is largely consistent with a differential composition of gut microbiome between R and NR.The higher the diversity of gut microbiota is, the longer PFS the patients undergo.Visualized beta diversity through PCoA analysis of 16S sequencing also displays apparently two clusters based on clinical responses to nivolumab treatment.Our results thus show that regardless of genetic background and geographic location with different lifestyles and diet styles, the diversity of gut microbiota might be a common benefit factor to ICI treatment.In our opinion, this is rational.It is widely accepted that gut microbiota modulates immune responses in a global way including antitumor immunity.36,37 Both innate and adaptive immune responses can be reshaped by gut microbiome.Although how the diversity of gut microbiota reshapes systemic immune responses is still not clearly addressed, microbiota-derived short chain fat acids and other metabolites should be good candidates to exert strong immune regulation.19,38 Further study will focus on the relevant metabolites analysis. Several clinical factors are reported to be involved in affecting the efficacy of ICI therapy, including smoking, previous treatment, and ATB usage.15,32,39 Among them, ATB is supposed to be the most relevant to gut microbiota.It has been reported previously that recent ATB usage in NSCLC patients was associated with shorter OS, although there was no difference in terms of objective response or PFS.32 In another study, ATB treatment is identified as the key factor significantly associated with both PFS and OS. 15 In our study, we retrospectively investigated ATB usage among 37 patients.The composition of gut microbiota at baseline between participants with or without ATB before first nivolumab infusion was similar (Supplementary Fig. 2), which excludes the possible effects of ATB on gut microbiota in our study.Median PFS was similar between ATB-treated and -naïve patients (median PFS: 126 days versus 117.9 days, p > 0.05) (Supplementary Fig. 3).One possibility lies in the number limitation in our study.Another possibility is that gut microbiome possess the ability of resilience.40 The majority of gut microbiota can be normalized after 1 week once ATB usage stops.41 We also observed that gut microbiota remained stable in longitudinal samples within one individual in our study.Such a trait might ensure patient long-term benefit from ICI treatment. Among all the clinical manifestations, smoking is the only factor that affects the responses to nivolumab treatment in our study.The roles of smoking on carcinogenesis refer to both cancer cell itself and immune responses of the host.It has been hypothesized that higher mutational burden could be induced by smoking, which has shown an increased response to ICI. 42,43 Median PFS was longer in current and former smokers compared with nonsmokers in nivolumab monotherapy for first-line treatment of advanced NSCLC as well.44 Smoking is reported to affect both innate and adaptive immunity, including memory T cells in the periphery.45 In our study, memory T cell profiles at baseline are similar between smoking and nonsmoking patients (Supplementary Fig. 4).This result confers that memory T cells are more likely affected by gut microbiota rather than other factors such as smoking.It has also been reported that chemotherapy could influence the gut microbiota of cancer patients largely due to the disruption of the intestinal barrier and subsequent bacteria translocation.In our study all the participants have received nivolumab therapy after failure of traditional chemotherapy.There was no difference in the interval from the date of last chemotherapy to that of first nivolumab infusion between R and NR groups (Supplementary Fig. 5, left).Correlation analysis showed that the diversity of gut microbiota was irrelevant to the time-off of chemotherapy in our cohort study as well (Supplementary Fig. 5, right). In addition to diversity, gut microbiome composition is also related to favorable responses to ICI treatment.In a pre-clinical study, gut commensal Bifidobacterium was shown to facilitate anti-PD-L1 efficacy.46 Clinical investigations reported that Faecalibacterium and some species belonging to Bacteroides genus (such as B. fragilis or B. thetaiotaomicron) are positively associated with the efficacy of PD-1 and CTLA-4 blockade, respectively.14,23 In our study, seven genera were identified different between R and NR.Ruminococcus_unclassified was enriched in NR whereas Bifidobacterium longum, Prevotella copri, and Alistipes putredinis were enriched in R. It has been shown in another study that Bifidobacterium longum could increase the production of pro-inflammatory cytokine tumor necrosis factor-alpha and significantly improve tumor control upon anti-PD-L1 therapy in a B16.SIY tumor mouse model.46 More interestingly, most bacteria enriched in R in our study belong to pro-inflammatory gram-negative bacteria.There are several previous studies reporting the association of gram-negative bacteria, such as Akkermansia muciniphila, Alistipes, and Porphyromonas with better clinical response to immunotherapy.14,15,36 It is already evident that the increase of pro-inflammatory gramnegative bacteria in the gut of older patients is associated with systematic and chronic inflammation.It is largely mediated by more release of lipopolysaccharide. In another study, mice colonized with Prevotella copri displayed increased inflammation in dextran sulfate sodium-induced colitis.Prevotella copri was strongly correlated with the disease severity in rheumatoid arthritis, which is a systemic autoimmune disease with the proliferation of pro-inflammatory T cells.47 In the antitumor setting, the exacerbation of immune responses might assist the ability of immune surveillance in the patients.This is also consistent with the report that patients who develop immune-related adverse events are more sensitive to ICI treatment.48 Although the direct evidence on the linkage between the inflammation and ICI benefit in the population still needs to be investigated, it is not out of expectation that systemic antitumor immune responses are diverse in patients with different gut microbiome composition.Gut microbiota thus becomes an intrinsic biosensor to sensitize anti-PD-1 treatment, which in turn leads to different clinical outcomes.This can be partially supported by the results from immune signature exploration in our study.Previous studies already highlighted the roles of PD-1 in reshaping immune responses in the mice.49 ICI therapy, to some extent, facilitates the investigation on PD-1 in the regulation of human immunity.By using multi-color flow cytometry, immune signatures including immune cell subsets and their function-related signatures have been detected.Furthermore, correlation analyses were performed between individual immune signatures and microbiota diversity.Among multiple signatures, the frequency and function of memory CD8 þ T cells are mostly dedicated.Memory CD8 þ T cells are a bunch of long-lived cells which are capable of rapid antigenspecific responses and play critical roles in immune recall responses. 50In addition to the percentage of CD8 þ Tm subsets such as effector memory T (Tem) cells and central memory T (Tcm) cells, the functional subset with Granzyme B production is also positively correlated with the diversity index, which can partially explain why patients with high microbiota diversity respond better to ICI therapy.The increase in Ki67 þ CD8 þ Tm in the patients with high microbiota diversity implies that gut microbiota also modulate the proliferation of certain immune cell populations.Recent studies already reported the effects of PD-1 blockade on the proliferation of CD8 þ T cells, which is beneficial for ICI treatment.51 ICOS and TIM3 expressions on specific T cells subsets are also related to antitumor immunity.52,53 Whereas CD8 þ T cells infiltrating in tumor microenvironment are thought to be the executors of tumor killing, circulating CD8 þ T cell subsets reported here are probably inclined to reflect the roles of gut microbiota on systematic immune responses.How peripheral memory CD8 þ T cell signatures affect the efficacy of ICI treatment will be investigated in the future.Once verified, they might become antitumor immune mediators against NSCLC. Our study shows that the diversity of gut microbiota is associated with favorable response to nivolumab therapy against NSCLC in Chinese cohorts.Considering the correlation between microbiota diversity and peripheral memory T cells and NK cells being reshaped upon ICI treatment, we propose that patients with more diverse gut microbiota at baseline benefit from ICI immunotherapy to a greater extent.This might be mediated by reprogramming systemic antitumor immune responses.The significance of our study lies in that the modulation of gut microbiota before the treatment might provide an alternative way to enhance the efficacy of ICI immunotherapy once validated in large cohort studies.A latest publication has introduced 11 bacterial strains from healthy donors' feces to induce interferon-g producing CD8 þ T cells in the intestine, which mediates effective antitumor immunity in a mouse model.54 Once clearly addressed, customized microbiome will exhibit great prospect as biotherapeutics in assistance with ICI treatment against advanced NSCLC.Project (17431906103), Shanghai Economic and Information Commission for oncology data platforms (201602010) and Shanghai Scientific Research Projects (14140902800).The authors thank Prof. Guo-ping Zhao for scientific suggestions on the project.Drs.Hong Li and Guoqing Zhang from Bigdata Center of Shanghai Insistute of Biological Science CAS helped to construct an integrative database with both clinical and multi-omics source data. Figure: 1 Figure 1 . Figure 1.Study scheme.

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§ The Diversity of Gut Microbiome is Associated With Favorable Responses to Anti-Programmed Death 1 Immunotherapy in Chinese Patients With NSCLC > Introduction Advanced NSCLC is an incurable disease with a poor prognosis and is the leading cause of cancer-related death. 1 With impressive advancement in immune checkpoint inhibitor (ICI) therapies targeting programmed death 1 (PD-1) and programmed death ligand 1 (PD-L1) axis against multiple malignancies, these agents exhibit clinical efficacy against patients with advanced NSCLC both in second-line and first-line treatment when compared to conventional chemotherapy.At present, five therapeutic monoclonal antibodies targeting PD-1 or PD-L1 have been approved by the U.S. Food and Drug Administration (FDA) for the treatment of 11 cancer indications including NSCLC. 2 Nivolumab (Opdivo) is a fully human immunoglobulin G4 monoclonal antibody against PD-1.It blocks the binding of PD-1 to its ligand PD-L1 and restores the function of "exhausted" T cells in tumor sites.The unleashed lymphocytes exert killing ability, resulting in tumor shrinkage.3 It was firstly approved by the FDA in 2015 for the treatment of patients with advanced NSCLC after the failure of platinum-based chemotherapy, and by the National Medical Products Administration in 2018 after the phase III clinical trial CheckMate-078 for previously treated patients with NSCLC. 4 According to results from CheckMate-078, nivolumab yields superior overall survival (OS) compared with the docetaxel arm (median OS: 12 mo versus 9.6 mo). 5 However, only 20% of patients with advanced NSCLC benefit from the treatment, whereas up to 50% experience treatmentrelated adverse events in which grade 3-4 toxicities occur in 10% patients receiving nivolumab.6 Considering high cost of the agents, limited benefit population, and potential severe side effects, biomarkers for selecting patients with likely clinical efficacy from ICI treatment in advanced NSCLC would be of paramount importance. So far, PD-L1 expression on tumor cells is regarded as the most available biomarker to predict the efficacy of ICIs in NSCLC and other malignancies.A linear relationship has been reported between the proportion of ICI benefit and PD-L1 expression in NSCLC tumor sections.7,8 Nevertheless, the responses to ICI treatment have still been observed in those with low or undetectable levels of PD-L1 expression.9 To date, other biomarkers such as high tumor mutation burden, nonsynonymous mutations and neoantigens, CD8 þ T cell counts in tumor regions, defects of mismatch repair genes or high microsatellite instability, microbiota, and metabolic profiles are also supposed to be predictive for favorable responses to ICI treatment, among which gut microbiome property is of great value.[10][11][12][13][14][15][16][17] Human gut harbors more than 10 13 microbes, which outnumbers host cells 10-fold.More and more evidence supports the critical roles of gut microbiota in influencing human health in part through shaping systematic immune responses.18 Certain metabolites such as shortchain fatty acids and bile acid are considered to be key mediators.19 Dysbiosis of intestinal flora has been shown to be associated with multiple diseases.[20][21][22] Recent studies have also revealed that gut microbiota could mediate diverse responses to immunotherapy and might serve as a potential predictive biomarker.Gut commensals such as Bacteroides thetaiotaomicron or B. fragilis are predicable for anti-cytotoxic T-lymphocyte associated protein 4 (CTLA-4) treatment in a mouse melanoma model.23 In patients with melanoma receiving anti-PD-1 therapy, significant difference in the diversity and composition of gut microbiome was observed between the responders and nonresponders to the therapy.14 Germ-free mice receiving fecal transplantation from the responders tended to be more sensitive to anti-PD-1 treatment in contrast to those receiving feces from the nonresponders.14,15 In one cohort study enrolling 60 patients with advanced NSCLC and 40 with renal cell carcinoma undergoing PD-1 blockade therapy, Akkermansia muciniphila was reported to be overrepresented in the responders.15 Nevertheless, the previous studies were performed predominantly in the patients from Western countries.Gut microbiome structures have been elucidated remarkably variable among different races and ethnicities.24 Extrinsic factors, such as geographical location or diet and life styles, and intrinsic factors such as genetics or early-life exposures are main contributors to such divergences.[25][26][27] Therefore, it is still significant to reveal the characteristics of gut microbiota in regional patients with advanced NSCLC and the potential roles of gut microbiota in the prediction for the responses to ICI treatment.

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Every source the paper cites for this claim was checked and supports it.

§ The Diversity of Gut Microbiome is Associated With Favorable Responses to Anti-Programmed Death 1 Immunotherapy in Chinese Patients With NSCLC > Discussion In the present study, we have investigated gut microbiota profiles in Chinese patients with advanced NSCLC receiving nivolumab treatment.Our results indicate that patients with better clinical responses exhibit higher diversity as well as a distinct gut microbiome composition.In addition, the association between gut microbiome diversity and systematic immune signatures highlights memory CD8 þ T cells and NK cells as potential mediators of antitumor immunity upon ICI treatment.The diversity of gut microbiome is defined as the number and abundance distribution of distinct types of micro-organisms colonizing within the gut.Abnormal diversity (dysbiosis) in the gut has been proven to be associated with human diseases, such as obesity, inflammatory bowel disease, cancer, etc. 20,21,33,34 Recently, Gopalakrishnan et al. 35 reported that patients with a high diversity of gut microbiome favored the clinical outcomes of anti-PD-1 immunotherapy in melanoma patients in the Western population.In our study, both alpha diversity and beta diversity at baseline are dramatically different between R and NR as well.A higher alpha diversity index represented by both the Shannon index and inverse Simpson indexes in R group suggests more species harboring in the gut, which is largely consistent with a differential composition of gut microbiome between R and NR.The higher the diversity of gut microbiota is, the longer PFS the patients undergo.Visualized beta diversity through PCoA analysis of 16S sequencing also displays apparently two clusters based on clinical responses to nivolumab treatment.Our results thus show that regardless of genetic background and geographic location with different lifestyles and diet styles, the diversity of gut microbiota might be a common benefit factor to ICI treatment.In our opinion, this is rational.It is widely accepted that gut microbiota modulates immune responses in a global way including antitumor immunity.36,37 Both innate and adaptive immune responses can be reshaped by gut microbiome.Although how the diversity of gut microbiota reshapes systemic immune responses is still not clearly addressed, microbiota-derived short chain fat acids and other metabolites should be good candidates to exert strong immune regulation.19,38 Further study will focus on the relevant metabolites analysis. Several clinical factors are reported to be involved in affecting the efficacy of ICI therapy, including smoking, previous treatment, and ATB usage.15,32,39 Among them, ATB is supposed to be the most relevant to gut microbiota.It has been reported previously that recent ATB usage in NSCLC patients was associated with shorter OS, although there was no difference in terms of objective response or PFS.32 In another study, ATB treatment is identified as the key factor significantly associated with both PFS and OS. 15 In our study, we retrospectively investigated ATB usage among 37 patients.The composition of gut microbiota at baseline between participants with or without ATB before first nivolumab infusion was similar (Supplementary Fig. 2), which excludes the possible effects of ATB on gut microbiota in our study.Median PFS was similar between ATB-treated and -naïve patients (median PFS: 126 days versus 117.9 days, p > 0.05) (Supplementary Fig. 3).One possibility lies in the number limitation in our study.Another possibility is that gut microbiome possess the ability of resilience.40 The majority of gut microbiota can be normalized after 1 week once ATB usage stops.41 We also observed that gut microbiota remained stable in longitudinal samples within one individual in our study.Such a trait might ensure patient long-term benefit from ICI treatment. Among all the clinical manifestations, smoking is the only factor that affects the responses to nivolumab treatment in our study.The roles of smoking on carcinogenesis refer to both cancer cell itself and immune responses of the host.It has been hypothesized that higher mutational burden could be induced by smoking, which has shown an increased response to ICI. 42,43 Median PFS was longer in current and former smokers compared with nonsmokers in nivolumab monotherapy for first-line treatment of advanced NSCLC as well.44 Smoking is reported to affect both innate and adaptive immunity, including memory T cells in the periphery.45 In our study, memory T cell profiles at baseline are similar between smoking and nonsmoking patients (Supplementary Fig. 4).This result confers that memory T cells are more likely affected by gut microbiota rather than other factors such as smoking.It has also been reported that chemotherapy could influence the gut microbiota of cancer patients largely due to the disruption of the intestinal barrier and subsequent bacteria translocation.In our study all the participants have received nivolumab therapy after failure of traditional chemotherapy.There was no difference in the interval from the date of last chemotherapy to that of first nivolumab infusion between R and NR groups (Supplementary Fig. 5, left).Correlation analysis showed that the diversity of gut microbiota was irrelevant to the time-off of chemotherapy in our cohort study as well (Supplementary Fig. 5, right). In addition to diversity, gut microbiome composition is also related to favorable responses to ICI treatment.In a pre-clinical study, gut commensal Bifidobacterium was shown to facilitate anti-PD-L1 efficacy.46 Clinical investigations reported that Faecalibacterium and some species belonging to Bacteroides genus (such as B. fragilis or B. thetaiotaomicron) are positively associated with the efficacy of PD-1 and CTLA-4 blockade, respectively.14,23 In our study, seven genera were identified different between R and NR.Ruminococcus_unclassified was enriched in NR whereas Bifidobacterium longum, Prevotella copri, and Alistipes putredinis were enriched in R. It has been shown in another study that Bifidobacterium longum could increase the production of pro-inflammatory cytokine tumor necrosis factor-alpha and significantly improve tumor control upon anti-PD-L1 therapy in a B16.SIY tumor mouse model.46 More interestingly, most bacteria enriched in R in our study belong to pro-inflammatory gram-negative bacteria.There are several previous studies reporting the association of gram-negative bacteria, such as Akkermansia muciniphila, Alistipes, and Porphyromonas with better clinical response to immunotherapy.14,15,36 It is already evident that the increase of pro-inflammatory gramnegative bacteria in the gut of older patients is associated with systematic and chronic inflammation.It is largely mediated by more release of lipopolysaccharide. In another study, mice colonized with Prevotella copri displayed increased inflammation in dextran sulfate sodium-induced colitis.Prevotella copri was strongly correlated with the disease severity in rheumatoid arthritis, which is a systemic autoimmune disease with the proliferation of pro-inflammatory T cells.47 In the antitumor setting, the exacerbation of immune responses might assist the ability of immune surveillance in the patients.This is also consistent with the report that patients who develop immune-related adverse events are more sensitive to ICI treatment.48 Although the direct evidence on the linkage between the inflammation and ICI benefit in the population still needs to be investigated, it is not out of expectation that systemic antitumor immune responses are diverse in patients with different gut microbiome composition.Gut microbiota thus becomes an intrinsic biosensor to sensitize anti-PD-1 treatment, which in turn leads to different clinical outcomes.This can be partially supported by the results from immune signature exploration in our study.Previous studies already highlighted the roles of PD-1 in reshaping immune responses in the mice.49 ICI therapy, to some extent, facilitates the investigation on PD-1 in the regulation of human immunity.By using multi-color flow cytometry, immune signatures including immune cell subsets and their function-related signatures have been detected.Furthermore, correlation analyses were performed between individual immune signatures and microbiota diversity.Among multiple signatures, the frequency and function of memory CD8 þ T cells are mostly dedicated.Memory CD8 þ T cells are a bunch of long-lived cells which are capable of rapid antigenspecific responses and play critical roles in immune recall responses. 50In addition to the percentage of CD8 þ Tm subsets such as effector memory T (Tem) cells and central memory T (Tcm) cells, the functional subset with Granzyme B production is also positively correlated with the diversity index, which can partially explain why patients with high microbiota diversity respond better to ICI therapy.The increase in Ki67 þ CD8 þ Tm in the patients with high microbiota diversity implies that gut microbiota also modulate the proliferation of certain immune cell populations.Recent studies already reported the effects of PD-1 blockade on the proliferation of CD8 þ T cells, which is beneficial for ICI treatment.51 ICOS and TIM3 expressions on specific T cells subsets are also related to antitumor immunity.52,53 Whereas CD8 þ T cells infiltrating in tumor microenvironment are thought to be the executors of tumor killing, circulating CD8 þ T cell subsets reported here are probably inclined to reflect the roles of gut microbiota on systematic immune responses.How peripheral memory CD8 þ T cell signatures affect the efficacy of ICI treatment will be investigated in the future.Once verified, they might become antitumor immune mediators against NSCLC. Our study shows that the diversity of gut microbiota is associated with favorable response to nivolumab therapy against NSCLC in Chinese cohorts.Considering the correlation between microbiota diversity and peripheral memory T cells and NK cells being reshaped upon ICI treatment, we propose that patients with more diverse gut microbiota at baseline benefit from ICI immunotherapy to a greater extent.This might be mediated by reprogramming systemic antitumor immune responses.The significance of our study lies in that the modulation of gut microbiota before the treatment might provide an alternative way to enhance the efficacy of ICI immunotherapy once validated in large cohort studies.A latest publication has introduced 11 bacterial strains from healthy donors' feces to induce interferon-g producing CD8 þ T cells in the intestine, which mediates effective antitumor immunity in a mouse model.54 Once clearly addressed, customized microbiome will exhibit great prospect as biotherapeutics in assistance with ICI treatment against advanced NSCLC.Project (17431906103), Shanghai Economic and Information Commission for oncology data platforms (201602010) and Shanghai Scientific Research Projects (14140902800).The authors thank Prof. Guo-ping Zhao for scientific suggestions on the project.Drs.Hong Li and Guoqing Zhang from Bigdata Center of Shanghai Insistute of Biological Science CAS helped to construct an integrative database with both clinical and multi-omics source data. Figure: 1 Figure 1 . Figure 1.Study scheme.

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§ The Diversity of Gut Microbiome is Associated With Favorable Responses to Anti-Programmed Death 1 Immunotherapy in Chinese Patients With NSCLC > Discussion In the present study, we have investigated gut microbiota profiles in Chinese patients with advanced NSCLC receiving nivolumab treatment.Our results indicate that patients with better clinical responses exhibit higher diversity as well as a distinct gut microbiome composition.In addition, the association between gut microbiome diversity and systematic immune signatures highlights memory CD8 þ T cells and NK cells as potential mediators of antitumor immunity upon ICI treatment.The diversity of gut microbiome is defined as the number and abundance distribution of distinct types of micro-organisms colonizing within the gut.Abnormal diversity (dysbiosis) in the gut has been proven to be associated with human diseases, such as obesity, inflammatory bowel disease, cancer, etc. 20,21,33,34 Recently, Gopalakrishnan et al. 35 reported that patients with a high diversity of gut microbiome favored the clinical outcomes of anti-PD-1 immunotherapy in melanoma patients in the Western population.In our study, both alpha diversity and beta diversity at baseline are dramatically different between R and NR as well.A higher alpha diversity index represented by both the Shannon index and inverse Simpson indexes in R group suggests more species harboring in the gut, which is largely consistent with a differential composition of gut microbiome between R and NR.The higher the diversity of gut microbiota is, the longer PFS the patients undergo.Visualized beta diversity through PCoA analysis of 16S sequencing also displays apparently two clusters based on clinical responses to nivolumab treatment.Our results thus show that regardless of genetic background and geographic location with different lifestyles and diet styles, the diversity of gut microbiota might be a common benefit factor to ICI treatment.In our opinion, this is rational.It is widely accepted that gut microbiota modulates immune responses in a global way including antitumor immunity.36,37 Both innate and adaptive immune responses can be reshaped by gut microbiome.Although how the diversity of gut microbiota reshapes systemic immune responses is still not clearly addressed, microbiota-derived short chain fat acids and other metabolites should be good candidates to exert strong immune regulation.19,38 Further study will focus on the relevant metabolites analysis. Several clinical factors are reported to be involved in affecting the efficacy of ICI therapy, including smoking, previous treatment, and ATB usage.15,32,39 Among them, ATB is supposed to be the most relevant to gut microbiota.It has been reported previously that recent ATB usage in NSCLC patients was associated with shorter OS, although there was no difference in terms of objective response or PFS.32 In another study, ATB treatment is identified as the key factor significantly associated with both PFS and OS. 15 In our study, we retrospectively investigated ATB usage among 37 patients.The composition of gut microbiota at baseline between participants with or without ATB before first nivolumab infusion was similar (Supplementary Fig. 2), which excludes the possible effects of ATB on gut microbiota in our study.Median PFS was similar between ATB-treated and -naïve patients (median PFS: 126 days versus 117.9 days, p > 0.05) (Supplementary Fig. 3).One possibility lies in the number limitation in our study.Another possibility is that gut microbiome possess the ability of resilience.40 The majority of gut microbiota can be normalized after 1 week once ATB usage stops.41 We also observed that gut microbiota remained stable in longitudinal samples within one individual in our study.Such a trait might ensure patient long-term benefit from ICI treatment. Among all the clinical manifestations, smoking is the only factor that affects the responses to nivolumab treatment in our study.The roles of smoking on carcinogenesis refer to both cancer cell itself and immune responses of the host.It has been hypothesized that higher mutational burden could be induced by smoking, which has shown an increased response to ICI. 42,43 Median PFS was longer in current and former smokers compared with nonsmokers in nivolumab monotherapy for first-line treatment of advanced NSCLC as well.44 Smoking is reported to affect both innate and adaptive immunity, including memory T cells in the periphery.45 In our study, memory T cell profiles at baseline are similar between smoking and nonsmoking patients (Supplementary Fig. 4).This result confers that memory T cells are more likely affected by gut microbiota rather than other factors such as smoking.It has also been reported that chemotherapy could influence the gut microbiota of cancer patients largely due to the disruption of the intestinal barrier and subsequent bacteria translocation.In our study all the participants have received nivolumab therapy after failure of traditional chemotherapy.There was no difference in the interval from the date of last chemotherapy to that of first nivolumab infusion between R and NR groups (Supplementary Fig. 5, left).Correlation analysis showed that the diversity of gut microbiota was irrelevant to the time-off of chemotherapy in our cohort study as well (Supplementary Fig. 5, right). In addition to diversity, gut microbiome composition is also related to favorable responses to ICI treatment.In a pre-clinical study, gut commensal Bifidobacterium was shown to facilitate anti-PD-L1 efficacy.46 Clinical investigations reported that Faecalibacterium and some species belonging to Bacteroides genus (such as B. fragilis or B. thetaiotaomicron) are positively associated with the efficacy of PD-1 and CTLA-4 blockade, respectively.14,23 In our study, seven genera were identified different between R and NR.Ruminococcus_unclassified was enriched in NR whereas Bifidobacterium longum, Prevotella copri, and Alistipes putredinis were enriched in R. It has been shown in another study that Bifidobacterium longum could increase the production of pro-inflammatory cytokine tumor necrosis factor-alpha and significantly improve tumor control upon anti-PD-L1 therapy in a B16.SIY tumor mouse model.46 More interestingly, most bacteria enriched in R in our study belong to pro-inflammatory gram-negative bacteria.There are several previous studies reporting the association of gram-negative bacteria, such as Akkermansia muciniphila, Alistipes, and Porphyromonas with better clinical response to immunotherapy.14,15,36 It is already evident that the increase of pro-inflammatory gramnegative bacteria in the gut of older patients is associated with systematic and chronic inflammation.It is largely mediated by more release of lipopolysaccharide. In another study, mice colonized with Prevotella copri displayed increased inflammation in dextran sulfate sodium-induced colitis.Prevotella copri was strongly correlated with the disease severity in rheumatoid arthritis, which is a systemic autoimmune disease with the proliferation of pro-inflammatory T cells.47 In the antitumor setting, the exacerbation of immune responses might assist the ability of immune surveillance in the patients.This is also consistent with the report that patients who develop immune-related adverse events are more sensitive to ICI treatment.48 Although the direct evidence on the linkage between the inflammation and ICI benefit in the population still needs to be investigated, it is not out of expectation that systemic antitumor immune responses are diverse in patients with different gut microbiome composition.Gut microbiota thus becomes an intrinsic biosensor to sensitize anti-PD-1 treatment, which in turn leads to different clinical outcomes.This can be partially supported by the results from immune signature exploration in our study.Previous studies already highlighted the roles of PD-1 in reshaping immune responses in the mice.49 ICI therapy, to some extent, facilitates the investigation on PD-1 in the regulation of human immunity.By using multi-color flow cytometry, immune signatures including immune cell subsets and their function-related signatures have been detected.Furthermore, correlation analyses were performed between individual immune signatures and microbiota diversity.Among multiple signatures, the frequency and function of memory CD8 þ T cells are mostly dedicated.Memory CD8 þ T cells are a bunch of long-lived cells which are capable of rapid antigenspecific responses and play critical roles in immune recall responses. 50In addition to the percentage of CD8 þ Tm subsets such as effector memory T (Tem) cells and central memory T (Tcm) cells, the functional subset with Granzyme B production is also positively correlated with the diversity index, which can partially explain why patients with high microbiota diversity respond better to ICI therapy.The increase in Ki67 þ CD8 þ Tm in the patients with high microbiota diversity implies that gut microbiota also modulate the proliferation of certain immune cell populations.Recent studies already reported the effects of PD-1 blockade on the proliferation of CD8 þ T cells, which is beneficial for ICI treatment.51 ICOS and TIM3 expressions on specific T cells subsets are also related to antitumor immunity.52,53 Whereas CD8 þ T cells infiltrating in tumor microenvironment are thought to be the executors of tumor killing, circulating CD8 þ T cell subsets reported here are probably inclined to reflect the roles of gut microbiota on systematic immune responses.How peripheral memory CD8 þ T cell signatures affect the efficacy of ICI treatment will be investigated in the future.Once verified, they might become antitumor immune mediators against NSCLC. Our study shows that the diversity of gut microbiota is associated with favorable response to nivolumab therapy against NSCLC in Chinese cohorts.Considering the correlation between microbiota diversity and peripheral memory T cells and NK cells being reshaped upon ICI treatment, we propose that patients with more diverse gut microbiota at baseline benefit from ICI immunotherapy to a greater extent.This might be mediated by reprogramming systemic antitumor immune responses.The significance of our study lies in that the modulation of gut microbiota before the treatment might provide an alternative way to enhance the efficacy of ICI immunotherapy once validated in large cohort studies.A latest publication has introduced 11 bacterial strains from healthy donors' feces to induce interferon-g producing CD8 þ T cells in the intestine, which mediates effective antitumor immunity in a mouse model.54 Once clearly addressed, customized microbiome will exhibit great prospect as biotherapeutics in assistance with ICI treatment against advanced NSCLC.Project (17431906103), Shanghai Economic and Information Commission for oncology data platforms (201602010) and Shanghai Scientific Research Projects (14140902800).The authors thank Prof. Guo-ping Zhao for scientific suggestions on the project.Drs.Hong Li and Guoqing Zhang from Bigdata Center of Shanghai Insistute of Biological Science CAS helped to construct an integrative database with both clinical and multi-omics source data. Figure: 1 Figure 1 . Figure 1.Study scheme.

Probably not a substantial claim. The paper’s conclusions may not depend on it.

The cited sources support only part of this claim — read each source’s verdict below.

§ The Diversity of Gut Microbiome is Associated With Favorable Responses to Anti-Programmed Death 1 Immunotherapy in Chinese Patients With NSCLC > Discussion In the present study, we have investigated gut microbiota profiles in Chinese patients with advanced NSCLC receiving nivolumab treatment.Our results indicate that patients with better clinical responses exhibit higher diversity as well as a distinct gut microbiome composition.In addition, the association between gut microbiome diversity and systematic immune signatures highlights memory CD8 þ T cells and NK cells as potential mediators of antitumor immunity upon ICI treatment.The diversity of gut microbiome is defined as the number and abundance distribution of distinct types of micro-organisms colonizing within the gut.Abnormal diversity (dysbiosis) in the gut has been proven to be associated with human diseases, such as obesity, inflammatory bowel disease, cancer, etc. 20,21,33,34 Recently, Gopalakrishnan et al. 35 reported that patients with a high diversity of gut microbiome favored the clinical outcomes of anti-PD-1 immunotherapy in melanoma patients in the Western population.In our study, both alpha diversity and beta diversity at baseline are dramatically different between R and NR as well.A higher alpha diversity index represented by both the Shannon index and inverse Simpson indexes in R group suggests more species harboring in the gut, which is largely consistent with a differential composition of gut microbiome between R and NR.The higher the diversity of gut microbiota is, the longer PFS the patients undergo.Visualized beta diversity through PCoA analysis of 16S sequencing also displays apparently two clusters based on clinical responses to nivolumab treatment.Our results thus show that regardless of genetic background and geographic location with different lifestyles and diet styles, the diversity of gut microbiota might be a common benefit factor to ICI treatment.In our opinion, this is rational.It is widely accepted that gut microbiota modulates immune responses in a global way including antitumor immunity.36,37 Both innate and adaptive immune responses can be reshaped by gut microbiome.Although how the diversity of gut microbiota reshapes systemic immune responses is still not clearly addressed, microbiota-derived short chain fat acids and other metabolites should be good candidates to exert strong immune regulation.19,38 Further study will focus on the relevant metabolites analysis. Several clinical factors are reported to be involved in affecting the efficacy of ICI therapy, including smoking, previous treatment, and ATB usage.15,32,39 Among them, ATB is supposed to be the most relevant to gut microbiota.It has been reported previously that recent ATB usage in NSCLC patients was associated with shorter OS, although there was no difference in terms of objective response or PFS.32 In another study, ATB treatment is identified as the key factor significantly associated with both PFS and OS. 15 In our study, we retrospectively investigated ATB usage among 37 patients.The composition of gut microbiota at baseline between participants with or without ATB before first nivolumab infusion was similar (Supplementary Fig. 2), which excludes the possible effects of ATB on gut microbiota in our study.Median PFS was similar between ATB-treated and -naïve patients (median PFS: 126 days versus 117.9 days, p > 0.05) (Supplementary Fig. 3).One possibility lies in the number limitation in our study.Another possibility is that gut microbiome possess the ability of resilience.40 The majority of gut microbiota can be normalized after 1 week once ATB usage stops.41 We also observed that gut microbiota remained stable in longitudinal samples within one individual in our study.Such a trait might ensure patient long-term benefit from ICI treatment. Among all the clinical manifestations, smoking is the only factor that affects the responses to nivolumab treatment in our study.The roles of smoking on carcinogenesis refer to both cancer cell itself and immune responses of the host.It has been hypothesized that higher mutational burden could be induced by smoking, which has shown an increased response to ICI. 42,43 Median PFS was longer in current and former smokers compared with nonsmokers in nivolumab monotherapy for first-line treatment of advanced NSCLC as well.44 Smoking is reported to affect both innate and adaptive immunity, including memory T cells in the periphery.45 In our study, memory T cell profiles at baseline are similar between smoking and nonsmoking patients (Supplementary Fig. 4).This result confers that memory T cells are more likely affected by gut microbiota rather than other factors such as smoking.It has also been reported that chemotherapy could influence the gut microbiota of cancer patients largely due to the disruption of the intestinal barrier and subsequent bacteria translocation.In our study all the participants have received nivolumab therapy after failure of traditional chemotherapy.There was no difference in the interval from the date of last chemotherapy to that of first nivolumab infusion between R and NR groups (Supplementary Fig. 5, left).Correlation analysis showed that the diversity of gut microbiota was irrelevant to the time-off of chemotherapy in our cohort study as well (Supplementary Fig. 5, right). In addition to diversity, gut microbiome composition is also related to favorable responses to ICI treatment.In a pre-clinical study, gut commensal Bifidobacterium was shown to facilitate anti-PD-L1 efficacy.46 Clinical investigations reported that Faecalibacterium and some species belonging to Bacteroides genus (such as B. fragilis or B. thetaiotaomicron) are positively associated with the efficacy of PD-1 and CTLA-4 blockade, respectively.14,23 In our study, seven genera were identified different between R and NR.Ruminococcus_unclassified was enriched in NR whereas Bifidobacterium longum, Prevotella copri, and Alistipes putredinis were enriched in R. It has been shown in another study that Bifidobacterium longum could increase the production of pro-inflammatory cytokine tumor necrosis factor-alpha and significantly improve tumor control upon anti-PD-L1 therapy in a B16.SIY tumor mouse model.46 More interestingly, most bacteria enriched in R in our study belong to pro-inflammatory gram-negative bacteria.There are several previous studies reporting the association of gram-negative bacteria, such as Akkermansia muciniphila, Alistipes, and Porphyromonas with better clinical response to immunotherapy.14,15,36 It is already evident that the increase of pro-inflammatory gramnegative bacteria in the gut of older patients is associated with systematic and chronic inflammation.It is largely mediated by more release of lipopolysaccharide. In another study, mice colonized with Prevotella copri displayed increased inflammation in dextran sulfate sodium-induced colitis.Prevotella copri was strongly correlated with the disease severity in rheumatoid arthritis, which is a systemic autoimmune disease with the proliferation of pro-inflammatory T cells.47 In the antitumor setting, the exacerbation of immune responses might assist the ability of immune surveillance in the patients.This is also consistent with the report that patients who develop immune-related adverse events are more sensitive to ICI treatment.48 Although the direct evidence on the linkage between the inflammation and ICI benefit in the population still needs to be investigated, it is not out of expectation that systemic antitumor immune responses are diverse in patients with different gut microbiome composition.Gut microbiota thus becomes an intrinsic biosensor to sensitize anti-PD-1 treatment, which in turn leads to different clinical outcomes.This can be partially supported by the results from immune signature exploration in our study.Previous studies already highlighted the roles of PD-1 in reshaping immune responses in the mice.49 ICI therapy, to some extent, facilitates the investigation on PD-1 in the regulation of human immunity.By using multi-color flow cytometry, immune signatures including immune cell subsets and their function-related signatures have been detected.Furthermore, correlation analyses were performed between individual immune signatures and microbiota diversity.Among multiple signatures, the frequency and function of memory CD8 þ T cells are mostly dedicated.Memory CD8 þ T cells are a bunch of long-lived cells which are capable of rapid antigenspecific responses and play critical roles in immune recall responses. 50In addition to the percentage of CD8 þ Tm subsets such as effector memory T (Tem) cells and central memory T (Tcm) cells, the functional subset with Granzyme B production is also positively correlated with the diversity index, which can partially explain why patients with high microbiota diversity respond better to ICI therapy.The increase in Ki67 þ CD8 þ Tm in the patients with high microbiota diversity implies that gut microbiota also modulate the proliferation of certain immune cell populations.Recent studies already reported the effects of PD-1 blockade on the proliferation of CD8 þ T cells, which is beneficial for ICI treatment.51 ICOS and TIM3 expressions on specific T cells subsets are also related to antitumor immunity.52,53 Whereas CD8 þ T cells infiltrating in tumor microenvironment are thought to be the executors of tumor killing, circulating CD8 þ T cell subsets reported here are probably inclined to reflect the roles of gut microbiota on systematic immune responses.How peripheral memory CD8 þ T cell signatures affect the efficacy of ICI treatment will be investigated in the future.Once verified, they might become antitumor immune mediators against NSCLC. Our study shows that the diversity of gut microbiota is associated with favorable response to nivolumab therapy against NSCLC in Chinese cohorts.Considering the correlation between microbiota diversity and peripheral memory T cells and NK cells being reshaped upon ICI treatment, we propose that patients with more diverse gut microbiota at baseline benefit from ICI immunotherapy to a greater extent.This might be mediated by reprogramming systemic antitumor immune responses.The significance of our study lies in that the modulation of gut microbiota before the treatment might provide an alternative way to enhance the efficacy of ICI immunotherapy once validated in large cohort studies.A latest publication has introduced 11 bacterial strains from healthy donors' feces to induce interferon-g producing CD8 þ T cells in the intestine, which mediates effective antitumor immunity in a mouse model.54 Once clearly addressed, customized microbiome will exhibit great prospect as biotherapeutics in assistance with ICI treatment against advanced NSCLC.Project (17431906103), Shanghai Economic and Information Commission for oncology data platforms (201602010) and Shanghai Scientific Research Projects (14140902800).The authors thank Prof. Guo-ping Zhao for scientific suggestions on the project.Drs.Hong Li and Guoqing Zhang from Bigdata Center of Shanghai Insistute of Biological Science CAS helped to construct an integrative database with both clinical and multi-omics source data. Figure: 1 Figure 1 . Figure 1.Study scheme.

Probably not a substantial claim.

The sources with a verdict support this claim; the rest weren’t checked yet, have no full text we could read, or their verdicts aren’t rated yet.

§ The Diversity of Gut Microbiome is Associated With Favorable Responses to Anti-Programmed Death 1 Immunotherapy in Chinese Patients With NSCLC > Discussion In the present study, we have investigated gut microbiota profiles in Chinese patients with advanced NSCLC receiving nivolumab treatment.Our results indicate that patients with better clinical responses exhibit higher diversity as well as a distinct gut microbiome composition.In addition, the association between gut microbiome diversity and systematic immune signatures highlights memory CD8 þ T cells and NK cells as potential mediators of antitumor immunity upon ICI treatment.The diversity of gut microbiome is defined as the number and abundance distribution of distinct types of micro-organisms colonizing within the gut.Abnormal diversity (dysbiosis) in the gut has been proven to be associated with human diseases, such as obesity, inflammatory bowel disease, cancer, etc. 20,21,33,34 Recently, Gopalakrishnan et al. 35 reported that patients with a high diversity of gut microbiome favored the clinical outcomes of anti-PD-1 immunotherapy in melanoma patients in the Western population.In our study, both alpha diversity and beta diversity at baseline are dramatically different between R and NR as well.A higher alpha diversity index represented by both the Shannon index and inverse Simpson indexes in R group suggests more species harboring in the gut, which is largely consistent with a differential composition of gut microbiome between R and NR.The higher the diversity of gut microbiota is, the longer PFS the patients undergo.Visualized beta diversity through PCoA analysis of 16S sequencing also displays apparently two clusters based on clinical responses to nivolumab treatment.Our results thus show that regardless of genetic background and geographic location with different lifestyles and diet styles, the diversity of gut microbiota might be a common benefit factor to ICI treatment.In our opinion, this is rational.It is widely accepted that gut microbiota modulates immune responses in a global way including antitumor immunity.36,37 Both innate and adaptive immune responses can be reshaped by gut microbiome.Although how the diversity of gut microbiota reshapes systemic immune responses is still not clearly addressed, microbiota-derived short chain fat acids and other metabolites should be good candidates to exert strong immune regulation.19,38 Further study will focus on the relevant metabolites analysis. Several clinical factors are reported to be involved in affecting the efficacy of ICI therapy, including smoking, previous treatment, and ATB usage.15,32,39 Among them, ATB is supposed to be the most relevant to gut microbiota.It has been reported previously that recent ATB usage in NSCLC patients was associated with shorter OS, although there was no difference in terms of objective response or PFS.32 In another study, ATB treatment is identified as the key factor significantly associated with both PFS and OS. 15 In our study, we retrospectively investigated ATB usage among 37 patients.The composition of gut microbiota at baseline between participants with or without ATB before first nivolumab infusion was similar (Supplementary Fig. 2), which excludes the possible effects of ATB on gut microbiota in our study.Median PFS was similar between ATB-treated and -naïve patients (median PFS: 126 days versus 117.9 days, p > 0.05) (Supplementary Fig. 3).One possibility lies in the number limitation in our study.Another possibility is that gut microbiome possess the ability of resilience.40 The majority of gut microbiota can be normalized after 1 week once ATB usage stops.41 We also observed that gut microbiota remained stable in longitudinal samples within one individual in our study.Such a trait might ensure patient long-term benefit from ICI treatment. Among all the clinical manifestations, smoking is the only factor that affects the responses to nivolumab treatment in our study.The roles of smoking on carcinogenesis refer to both cancer cell itself and immune responses of the host.It has been hypothesized that higher mutational burden could be induced by smoking, which has shown an increased response to ICI. 42,43 Median PFS was longer in current and former smokers compared with nonsmokers in nivolumab monotherapy for first-line treatment of advanced NSCLC as well.44 Smoking is reported to affect both innate and adaptive immunity, including memory T cells in the periphery.45 In our study, memory T cell profiles at baseline are similar between smoking and nonsmoking patients (Supplementary Fig. 4).This result confers that memory T cells are more likely affected by gut microbiota rather than other factors such as smoking.It has also been reported that chemotherapy could influence the gut microbiota of cancer patients largely due to the disruption of the intestinal barrier and subsequent bacteria translocation.In our study all the participants have received nivolumab therapy after failure of traditional chemotherapy.There was no difference in the interval from the date of last chemotherapy to that of first nivolumab infusion between R and NR groups (Supplementary Fig. 5, left).Correlation analysis showed that the diversity of gut microbiota was irrelevant to the time-off of chemotherapy in our cohort study as well (Supplementary Fig. 5, right). In addition to diversity, gut microbiome composition is also related to favorable responses to ICI treatment.In a pre-clinical study, gut commensal Bifidobacterium was shown to facilitate anti-PD-L1 efficacy.46 Clinical investigations reported that Faecalibacterium and some species belonging to Bacteroides genus (such as B. fragilis or B. thetaiotaomicron) are positively associated with the efficacy of PD-1 and CTLA-4 blockade, respectively.14,23 In our study, seven genera were identified different between R and NR.Ruminococcus_unclassified was enriched in NR whereas Bifidobacterium longum, Prevotella copri, and Alistipes putredinis were enriched in R. It has been shown in another study that Bifidobacterium longum could increase the production of pro-inflammatory cytokine tumor necrosis factor-alpha and significantly improve tumor control upon anti-PD-L1 therapy in a B16.SIY tumor mouse model.46 More interestingly, most bacteria enriched in R in our study belong to pro-inflammatory gram-negative bacteria.There are several previous studies reporting the association of gram-negative bacteria, such as Akkermansia muciniphila, Alistipes, and Porphyromonas with better clinical response to immunotherapy.14,15,36 It is already evident that the increase of pro-inflammatory gramnegative bacteria in the gut of older patients is associated with systematic and chronic inflammation.It is largely mediated by more release of lipopolysaccharide. In another study, mice colonized with Prevotella copri displayed increased inflammation in dextran sulfate sodium-induced colitis.Prevotella copri was strongly correlated with the disease severity in rheumatoid arthritis, which is a systemic autoimmune disease with the proliferation of pro-inflammatory T cells.47 In the antitumor setting, the exacerbation of immune responses might assist the ability of immune surveillance in the patients.This is also consistent with the report that patients who develop immune-related adverse events are more sensitive to ICI treatment.48 Although the direct evidence on the linkage between the inflammation and ICI benefit in the population still needs to be investigated, it is not out of expectation that systemic antitumor immune responses are diverse in patients with different gut microbiome composition.Gut microbiota thus becomes an intrinsic biosensor to sensitize anti-PD-1 treatment, which in turn leads to different clinical outcomes.This can be partially supported by the results from immune signature exploration in our study.Previous studies already highlighted the roles of PD-1 in reshaping immune responses in the mice.49 ICI therapy, to some extent, facilitates the investigation on PD-1 in the regulation of human immunity.By using multi-color flow cytometry, immune signatures including immune cell subsets and their function-related signatures have been detected.Furthermore, correlation analyses were performed between individual immune signatures and microbiota diversity.Among multiple signatures, the frequency and function of memory CD8 þ T cells are mostly dedicated.Memory CD8 þ T cells are a bunch of long-lived cells which are capable of rapid antigenspecific responses and play critical roles in immune recall responses. 50In addition to the percentage of CD8 þ Tm subsets such as effector memory T (Tem) cells and central memory T (Tcm) cells, the functional subset with Granzyme B production is also positively correlated with the diversity index, which can partially explain why patients with high microbiota diversity respond better to ICI therapy.The increase in Ki67 þ CD8 þ Tm in the patients with high microbiota diversity implies that gut microbiota also modulate the proliferation of certain immune cell populations.Recent studies already reported the effects of PD-1 blockade on the proliferation of CD8 þ T cells, which is beneficial for ICI treatment.51 ICOS and TIM3 expressions on specific T cells subsets are also related to antitumor immunity.52,53 Whereas CD8 þ T cells infiltrating in tumor microenvironment are thought to be the executors of tumor killing, circulating CD8 þ T cell subsets reported here are probably inclined to reflect the roles of gut microbiota on systematic immune responses.How peripheral memory CD8 þ T cell signatures affect the efficacy of ICI treatment will be investigated in the future.Once verified, they might become antitumor immune mediators against NSCLC. Our study shows that the diversity of gut microbiota is associated with favorable response to nivolumab therapy against NSCLC in Chinese cohorts.Considering the correlation between microbiota diversity and peripheral memory T cells and NK cells being reshaped upon ICI treatment, we propose that patients with more diverse gut microbiota at baseline benefit from ICI immunotherapy to a greater extent.This might be mediated by reprogramming systemic antitumor immune responses.The significance of our study lies in that the modulation of gut microbiota before the treatment might provide an alternative way to enhance the efficacy of ICI immunotherapy once validated in large cohort studies.A latest publication has introduced 11 bacterial strains from healthy donors' feces to induce interferon-g producing CD8 þ T cells in the intestine, which mediates effective antitumor immunity in a mouse model.54 Once clearly addressed, customized microbiome will exhibit great prospect as biotherapeutics in assistance with ICI treatment against advanced NSCLC.Project (17431906103), Shanghai Economic and Information Commission for oncology data platforms (201602010) and Shanghai Scientific Research Projects (14140902800).The authors thank Prof. Guo-ping Zhao for scientific suggestions on the project.Drs.Hong Li and Guoqing Zhang from Bigdata Center of Shanghai Insistute of Biological Science CAS helped to construct an integrative database with both clinical and multi-omics source data. Figure: 1 Figure 1 . Figure 1.Study scheme.

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The cited sources support only part of this claim — read each source’s verdict below.

§ The Diversity of Gut Microbiome is Associated With Favorable Responses to Anti-Programmed Death 1 Immunotherapy in Chinese Patients With NSCLC > Introduction Advanced NSCLC is an incurable disease with a poor prognosis and is the leading cause of cancer-related death. 1 With impressive advancement in immune checkpoint inhibitor (ICI) therapies targeting programmed death 1 (PD-1) and programmed death ligand 1 (PD-L1) axis against multiple malignancies, these agents exhibit clinical efficacy against patients with advanced NSCLC both in second-line and first-line treatment when compared to conventional chemotherapy.At present, five therapeutic monoclonal antibodies targeting PD-1 or PD-L1 have been approved by the U.S. Food and Drug Administration (FDA) for the treatment of 11 cancer indications including NSCLC. 2 Nivolumab (Opdivo) is a fully human immunoglobulin G4 monoclonal antibody against PD-1.It blocks the binding of PD-1 to its ligand PD-L1 and restores the function of "exhausted" T cells in tumor sites.The unleashed lymphocytes exert killing ability, resulting in tumor shrinkage.3 It was firstly approved by the FDA in 2015 for the treatment of patients with advanced NSCLC after the failure of platinum-based chemotherapy, and by the National Medical Products Administration in 2018 after the phase III clinical trial CheckMate-078 for previously treated patients with NSCLC. 4 According to results from CheckMate-078, nivolumab yields superior overall survival (OS) compared with the docetaxel arm (median OS: 12 mo versus 9.6 mo). 5 However, only 20% of patients with advanced NSCLC benefit from the treatment, whereas up to 50% experience treatmentrelated adverse events in which grade 3-4 toxicities occur in 10% patients receiving nivolumab.6 Considering high cost of the agents, limited benefit population, and potential severe side effects, biomarkers for selecting patients with likely clinical efficacy from ICI treatment in advanced NSCLC would be of paramount importance. So far, PD-L1 expression on tumor cells is regarded as the most available biomarker to predict the efficacy of ICIs in NSCLC and other malignancies.A linear relationship has been reported between the proportion of ICI benefit and PD-L1 expression in NSCLC tumor sections.7,8 Nevertheless, the responses to ICI treatment have still been observed in those with low or undetectable levels of PD-L1 expression.9 To date, other biomarkers such as high tumor mutation burden, nonsynonymous mutations and neoantigens, CD8 þ T cell counts in tumor regions, defects of mismatch repair genes or high microsatellite instability, microbiota, and metabolic profiles are also supposed to be predictive for favorable responses to ICI treatment, among which gut microbiome property is of great value.[10][11][12][13][14][15][16][17] Human gut harbors more than 10 13 microbes, which outnumbers host cells 10-fold.More and more evidence supports the critical roles of gut microbiota in influencing human health in part through shaping systematic immune responses.18 Certain metabolites such as shortchain fatty acids and bile acid are considered to be key mediators.19 Dysbiosis of intestinal flora has been shown to be associated with multiple diseases.[20][21][22] Recent studies have also revealed that gut microbiota could mediate diverse responses to immunotherapy and might serve as a potential predictive biomarker.Gut commensals such as Bacteroides thetaiotaomicron or B. fragilis are predicable for anti-cytotoxic T-lymphocyte associated protein 4 (CTLA-4) treatment in a mouse melanoma model.23 In patients with melanoma receiving anti-PD-1 therapy, significant difference in the diversity and composition of gut microbiome was observed between the responders and nonresponders to the therapy.14 Germ-free mice receiving fecal transplantation from the responders tended to be more sensitive to anti-PD-1 treatment in contrast to those receiving feces from the nonresponders.14,15 In one cohort study enrolling 60 patients with advanced NSCLC and 40 with renal cell carcinoma undergoing PD-1 blockade therapy, Akkermansia muciniphila was reported to be overrepresented in the responders.15 Nevertheless, the previous studies were performed predominantly in the patients from Western countries.Gut microbiome structures have been elucidated remarkably variable among different races and ethnicities.24 Extrinsic factors, such as geographical location or diet and life styles, and intrinsic factors such as genetics or early-life exposures are main contributors to such divergences.[25][26][27] Therefore, it is still significant to reveal the characteristics of gut microbiota in regional patients with advanced NSCLC and the potential roles of gut microbiota in the prediction for the responses to ICI treatment.

Probably not a substantial claim. The paper’s conclusions may not depend on it.

The cited sources support only part of this claim — read each source’s verdict below.

§ The Diversity of Gut Microbiome is Associated With Favorable Responses to Anti-Programmed Death 1 Immunotherapy in Chinese Patients With NSCLC > Discussion In the present study, we have investigated gut microbiota profiles in Chinese patients with advanced NSCLC receiving nivolumab treatment.Our results indicate that patients with better clinical responses exhibit higher diversity as well as a distinct gut microbiome composition.In addition, the association between gut microbiome diversity and systematic immune signatures highlights memory CD8 þ T cells and NK cells as potential mediators of antitumor immunity upon ICI treatment.The diversity of gut microbiome is defined as the number and abundance distribution of distinct types of micro-organisms colonizing within the gut.Abnormal diversity (dysbiosis) in the gut has been proven to be associated with human diseases, such as obesity, inflammatory bowel disease, cancer, etc. 20,21,33,34 Recently, Gopalakrishnan et al. 35 reported that patients with a high diversity of gut microbiome favored the clinical outcomes of anti-PD-1 immunotherapy in melanoma patients in the Western population.In our study, both alpha diversity and beta diversity at baseline are dramatically different between R and NR as well.A higher alpha diversity index represented by both the Shannon index and inverse Simpson indexes in R group suggests more species harboring in the gut, which is largely consistent with a differential composition of gut microbiome between R and NR.The higher the diversity of gut microbiota is, the longer PFS the patients undergo.Visualized beta diversity through PCoA analysis of 16S sequencing also displays apparently two clusters based on clinical responses to nivolumab treatment.Our results thus show that regardless of genetic background and geographic location with different lifestyles and diet styles, the diversity of gut microbiota might be a common benefit factor to ICI treatment.In our opinion, this is rational.It is widely accepted that gut microbiota modulates immune responses in a global way including antitumor immunity.36,37 Both innate and adaptive immune responses can be reshaped by gut microbiome.Although how the diversity of gut microbiota reshapes systemic immune responses is still not clearly addressed, microbiota-derived short chain fat acids and other metabolites should be good candidates to exert strong immune regulation.19,38 Further study will focus on the relevant metabolites analysis. Several clinical factors are reported to be involved in affecting the efficacy of ICI therapy, including smoking, previous treatment, and ATB usage.15,32,39 Among them, ATB is supposed to be the most relevant to gut microbiota.It has been reported previously that recent ATB usage in NSCLC patients was associated with shorter OS, although there was no difference in terms of objective response or PFS.32 In another study, ATB treatment is identified as the key factor significantly associated with both PFS and OS. 15 In our study, we retrospectively investigated ATB usage among 37 patients.The composition of gut microbiota at baseline between participants with or without ATB before first nivolumab infusion was similar (Supplementary Fig. 2), which excludes the possible effects of ATB on gut microbiota in our study.Median PFS was similar between ATB-treated and -naïve patients (median PFS: 126 days versus 117.9 days, p > 0.05) (Supplementary Fig. 3).One possibility lies in the number limitation in our study.Another possibility is that gut microbiome possess the ability of resilience.40 The majority of gut microbiota can be normalized after 1 week once ATB usage stops.41 We also observed that gut microbiota remained stable in longitudinal samples within one individual in our study.Such a trait might ensure patient long-term benefit from ICI treatment. Among all the clinical manifestations, smoking is the only factor that affects the responses to nivolumab treatment in our study.The roles of smoking on carcinogenesis refer to both cancer cell itself and immune responses of the host.It has been hypothesized that higher mutational burden could be induced by smoking, which has shown an increased response to ICI. 42,43 Median PFS was longer in current and former smokers compared with nonsmokers in nivolumab monotherapy for first-line treatment of advanced NSCLC as well.44 Smoking is reported to affect both innate and adaptive immunity, including memory T cells in the periphery.45 In our study, memory T cell profiles at baseline are similar between smoking and nonsmoking patients (Supplementary Fig. 4).This result confers that memory T cells are more likely affected by gut microbiota rather than other factors such as smoking.It has also been reported that chemotherapy could influence the gut microbiota of cancer patients largely due to the disruption of the intestinal barrier and subsequent bacteria translocation.In our study all the participants have received nivolumab therapy after failure of traditional chemotherapy.There was no difference in the interval from the date of last chemotherapy to that of first nivolumab infusion between R and NR groups (Supplementary Fig. 5, left).Correlation analysis showed that the diversity of gut microbiota was irrelevant to the time-off of chemotherapy in our cohort study as well (Supplementary Fig. 5, right). In addition to diversity, gut microbiome composition is also related to favorable responses to ICI treatment.In a pre-clinical study, gut commensal Bifidobacterium was shown to facilitate anti-PD-L1 efficacy.46 Clinical investigations reported that Faecalibacterium and some species belonging to Bacteroides genus (such as B. fragilis or B. thetaiotaomicron) are positively associated with the efficacy of PD-1 and CTLA-4 blockade, respectively.14,23 In our study, seven genera were identified different between R and NR.Ruminococcus_unclassified was enriched in NR whereas Bifidobacterium longum, Prevotella copri, and Alistipes putredinis were enriched in R. It has been shown in another study that Bifidobacterium longum could increase the production of pro-inflammatory cytokine tumor necrosis factor-alpha and significantly improve tumor control upon anti-PD-L1 therapy in a B16.SIY tumor mouse model.46 More interestingly, most bacteria enriched in R in our study belong to pro-inflammatory gram-negative bacteria.There are several previous studies reporting the association of gram-negative bacteria, such as Akkermansia muciniphila, Alistipes, and Porphyromonas with better clinical response to immunotherapy.14,15,36 It is already evident that the increase of pro-inflammatory gramnegative bacteria in the gut of older patients is associated with systematic and chronic inflammation.It is largely mediated by more release of lipopolysaccharide. In another study, mice colonized with Prevotella copri displayed increased inflammation in dextran sulfate sodium-induced colitis.Prevotella copri was strongly correlated with the disease severity in rheumatoid arthritis, which is a systemic autoimmune disease with the proliferation of pro-inflammatory T cells.47 In the antitumor setting, the exacerbation of immune responses might assist the ability of immune surveillance in the patients.This is also consistent with the report that patients who develop immune-related adverse events are more sensitive to ICI treatment.48 Although the direct evidence on the linkage between the inflammation and ICI benefit in the population still needs to be investigated, it is not out of expectation that systemic antitumor immune responses are diverse in patients with different gut microbiome composition.Gut microbiota thus becomes an intrinsic biosensor to sensitize anti-PD-1 treatment, which in turn leads to different clinical outcomes.This can be partially supported by the results from immune signature exploration in our study.Previous studies already highlighted the roles of PD-1 in reshaping immune responses in the mice.49 ICI therapy, to some extent, facilitates the investigation on PD-1 in the regulation of human immunity.By using multi-color flow cytometry, immune signatures including immune cell subsets and their function-related signatures have been detected.Furthermore, correlation analyses were performed between individual immune signatures and microbiota diversity.Among multiple signatures, the frequency and function of memory CD8 þ T cells are mostly dedicated.Memory CD8 þ T cells are a bunch of long-lived cells which are capable of rapid antigenspecific responses and play critical roles in immune recall responses. 50In addition to the percentage of CD8 þ Tm subsets such as effector memory T (Tem) cells and central memory T (Tcm) cells, the functional subset with Granzyme B production is also positively correlated with the diversity index, which can partially explain why patients with high microbiota diversity respond better to ICI therapy.The increase in Ki67 þ CD8 þ Tm in the patients with high microbiota diversity implies that gut microbiota also modulate the proliferation of certain immune cell populations.Recent studies already reported the effects of PD-1 blockade on the proliferation of CD8 þ T cells, which is beneficial for ICI treatment.51 ICOS and TIM3 expressions on specific T cells subsets are also related to antitumor immunity.52,53 Whereas CD8 þ T cells infiltrating in tumor microenvironment are thought to be the executors of tumor killing, circulating CD8 þ T cell subsets reported here are probably inclined to reflect the roles of gut microbiota on systematic immune responses.How peripheral memory CD8 þ T cell signatures affect the efficacy of ICI treatment will be investigated in the future.Once verified, they might become antitumor immune mediators against NSCLC. Our study shows that the diversity of gut microbiota is associated with favorable response to nivolumab therapy against NSCLC in Chinese cohorts.Considering the correlation between microbiota diversity and peripheral memory T cells and NK cells being reshaped upon ICI treatment, we propose that patients with more diverse gut microbiota at baseline benefit from ICI immunotherapy to a greater extent.This might be mediated by reprogramming systemic antitumor immune responses.The significance of our study lies in that the modulation of gut microbiota before the treatment might provide an alternative way to enhance the efficacy of ICI immunotherapy once validated in large cohort studies.A latest publication has introduced 11 bacterial strains from healthy donors' feces to induce interferon-g producing CD8 þ T cells in the intestine, which mediates effective antitumor immunity in a mouse model.54 Once clearly addressed, customized microbiome will exhibit great prospect as biotherapeutics in assistance with ICI treatment against advanced NSCLC.Project (17431906103), Shanghai Economic and Information Commission for oncology data platforms (201602010) and Shanghai Scientific Research Projects (14140902800).The authors thank Prof. Guo-ping Zhao for scientific suggestions on the project.Drs.Hong Li and Guoqing Zhang from Bigdata Center of Shanghai Insistute of Biological Science CAS helped to construct an integrative database with both clinical and multi-omics source data. Figure: 1 Figure 1 . Figure 1.Study scheme.

Probably not a substantial claim. The paper’s conclusions may not depend on it.

Every source the paper cites for this claim was checked and supports it.

§ The Diversity of Gut Microbiome is Associated With Favorable Responses to Anti-Programmed Death 1 Immunotherapy in Chinese Patients With NSCLC > Introduction Advanced NSCLC is an incurable disease with a poor prognosis and is the leading cause of cancer-related death. 1 With impressive advancement in immune checkpoint inhibitor (ICI) therapies targeting programmed death 1 (PD-1) and programmed death ligand 1 (PD-L1) axis against multiple malignancies, these agents exhibit clinical efficacy against patients with advanced NSCLC both in second-line and first-line treatment when compared to conventional chemotherapy.At present, five therapeutic monoclonal antibodies targeting PD-1 or PD-L1 have been approved by the U.S. Food and Drug Administration (FDA) for the treatment of 11 cancer indications including NSCLC. 2 Nivolumab (Opdivo) is a fully human immunoglobulin G4 monoclonal antibody against PD-1.It blocks the binding of PD-1 to its ligand PD-L1 and restores the function of "exhausted" T cells in tumor sites.The unleashed lymphocytes exert killing ability, resulting in tumor shrinkage.3 It was firstly approved by the FDA in 2015 for the treatment of patients with advanced NSCLC after the failure of platinum-based chemotherapy, and by the National Medical Products Administration in 2018 after the phase III clinical trial CheckMate-078 for previously treated patients with NSCLC. 4 According to results from CheckMate-078, nivolumab yields superior overall survival (OS) compared with the docetaxel arm (median OS: 12 mo versus 9.6 mo). 5 However, only 20% of patients with advanced NSCLC benefit from the treatment, whereas up to 50% experience treatmentrelated adverse events in which grade 3-4 toxicities occur in 10% patients receiving nivolumab.6 Considering high cost of the agents, limited benefit population, and potential severe side effects, biomarkers for selecting patients with likely clinical efficacy from ICI treatment in advanced NSCLC would be of paramount importance. So far, PD-L1 expression on tumor cells is regarded as the most available biomarker to predict the efficacy of ICIs in NSCLC and other malignancies.A linear relationship has been reported between the proportion of ICI benefit and PD-L1 expression in NSCLC tumor sections.7,8 Nevertheless, the responses to ICI treatment have still been observed in those with low or undetectable levels of PD-L1 expression.9 To date, other biomarkers such as high tumor mutation burden, nonsynonymous mutations and neoantigens, CD8 þ T cell counts in tumor regions, defects of mismatch repair genes or high microsatellite instability, microbiota, and metabolic profiles are also supposed to be predictive for favorable responses to ICI treatment, among which gut microbiome property is of great value.[10][11][12][13][14][15][16][17] Human gut harbors more than 10 13 microbes, which outnumbers host cells 10-fold.More and more evidence supports the critical roles of gut microbiota in influencing human health in part through shaping systematic immune responses.18 Certain metabolites such as shortchain fatty acids and bile acid are considered to be key mediators.19 Dysbiosis of intestinal flora has been shown to be associated with multiple diseases.[20][21][22] Recent studies have also revealed that gut microbiota could mediate diverse responses to immunotherapy and might serve as a potential predictive biomarker.Gut commensals such as Bacteroides thetaiotaomicron or B. fragilis are predicable for anti-cytotoxic T-lymphocyte associated protein 4 (CTLA-4) treatment in a mouse melanoma model.23 In patients with melanoma receiving anti-PD-1 therapy, significant difference in the diversity and composition of gut microbiome was observed between the responders and nonresponders to the therapy.14 Germ-free mice receiving fecal transplantation from the responders tended to be more sensitive to anti-PD-1 treatment in contrast to those receiving feces from the nonresponders.14,15 In one cohort study enrolling 60 patients with advanced NSCLC and 40 with renal cell carcinoma undergoing PD-1 blockade therapy, Akkermansia muciniphila was reported to be overrepresented in the responders.15 Nevertheless, the previous studies were performed predominantly in the patients from Western countries.Gut microbiome structures have been elucidated remarkably variable among different races and ethnicities.24 Extrinsic factors, such as geographical location or diet and life styles, and intrinsic factors such as genetics or early-life exposures are main contributors to such divergences.[25][26][27] Therefore, it is still significant to reveal the characteristics of gut microbiota in regional patients with advanced NSCLC and the potential roles of gut microbiota in the prediction for the responses to ICI treatment.

Probably not a substantial claim.

The cited sources support only part of this claim — read each source’s verdict below.

§ The Diversity of Gut Microbiome is Associated With Favorable Responses to Anti-Programmed Death 1 Immunotherapy in Chinese Patients With NSCLC > Introduction Advanced NSCLC is an incurable disease with a poor prognosis and is the leading cause of cancer-related death. 1 With impressive advancement in immune checkpoint inhibitor (ICI) therapies targeting programmed death 1 (PD-1) and programmed death ligand 1 (PD-L1) axis against multiple malignancies, these agents exhibit clinical efficacy against patients with advanced NSCLC both in second-line and first-line treatment when compared to conventional chemotherapy.At present, five therapeutic monoclonal antibodies targeting PD-1 or PD-L1 have been approved by the U.S. Food and Drug Administration (FDA) for the treatment of 11 cancer indications including NSCLC. 2 Nivolumab (Opdivo) is a fully human immunoglobulin G4 monoclonal antibody against PD-1.It blocks the binding of PD-1 to its ligand PD-L1 and restores the function of "exhausted" T cells in tumor sites.The unleashed lymphocytes exert killing ability, resulting in tumor shrinkage.3 It was firstly approved by the FDA in 2015 for the treatment of patients with advanced NSCLC after the failure of platinum-based chemotherapy, and by the National Medical Products Administration in 2018 after the phase III clinical trial CheckMate-078 for previously treated patients with NSCLC. 4 According to results from CheckMate-078, nivolumab yields superior overall survival (OS) compared with the docetaxel arm (median OS: 12 mo versus 9.6 mo). 5 However, only 20% of patients with advanced NSCLC benefit from the treatment, whereas up to 50% experience treatmentrelated adverse events in which grade 3-4 toxicities occur in 10% patients receiving nivolumab.6 Considering high cost of the agents, limited benefit population, and potential severe side effects, biomarkers for selecting patients with likely clinical efficacy from ICI treatment in advanced NSCLC would be of paramount importance. So far, PD-L1 expression on tumor cells is regarded as the most available biomarker to predict the efficacy of ICIs in NSCLC and other malignancies.A linear relationship has been reported between the proportion of ICI benefit and PD-L1 expression in NSCLC tumor sections.7,8 Nevertheless, the responses to ICI treatment have still been observed in those with low or undetectable levels of PD-L1 expression.9 To date, other biomarkers such as high tumor mutation burden, nonsynonymous mutations and neoantigens, CD8 þ T cell counts in tumor regions, defects of mismatch repair genes or high microsatellite instability, microbiota, and metabolic profiles are also supposed to be predictive for favorable responses to ICI treatment, among which gut microbiome property is of great value.[10][11][12][13][14][15][16][17] Human gut harbors more than 10 13 microbes, which outnumbers host cells 10-fold.More and more evidence supports the critical roles of gut microbiota in influencing human health in part through shaping systematic immune responses.18 Certain metabolites such as shortchain fatty acids and bile acid are considered to be key mediators.19 Dysbiosis of intestinal flora has been shown to be associated with multiple diseases.[20][21][22] Recent studies have also revealed that gut microbiota could mediate diverse responses to immunotherapy and might serve as a potential predictive biomarker.Gut commensals such as Bacteroides thetaiotaomicron or B. fragilis are predicable for anti-cytotoxic T-lymphocyte associated protein 4 (CTLA-4) treatment in a mouse melanoma model.23 In patients with melanoma receiving anti-PD-1 therapy, significant difference in the diversity and composition of gut microbiome was observed between the responders and nonresponders to the therapy.14 Germ-free mice receiving fecal transplantation from the responders tended to be more sensitive to anti-PD-1 treatment in contrast to those receiving feces from the nonresponders.14,15 In one cohort study enrolling 60 patients with advanced NSCLC and 40 with renal cell carcinoma undergoing PD-1 blockade therapy, Akkermansia muciniphila was reported to be overrepresented in the responders.15 Nevertheless, the previous studies were performed predominantly in the patients from Western countries.Gut microbiome structures have been elucidated remarkably variable among different races and ethnicities.24 Extrinsic factors, such as geographical location or diet and life styles, and intrinsic factors such as genetics or early-life exposures are main contributors to such divergences.[25][26][27] Therefore, it is still significant to reveal the characteristics of gut microbiota in regional patients with advanced NSCLC and the potential roles of gut microbiota in the prediction for the responses to ICI treatment.

Probably not a substantial claim. The paper’s conclusions may not depend on it.

The sources with a verdict support this claim; the rest weren’t checked yet, have no full text we could read, or their verdicts aren’t rated yet.

§ The Diversity of Gut Microbiome is Associated With Favorable Responses to Anti-Programmed Death 1 Immunotherapy in Chinese Patients With NSCLC > Discussion In the present study, we have investigated gut microbiota profiles in Chinese patients with advanced NSCLC receiving nivolumab treatment.Our results indicate that patients with better clinical responses exhibit higher diversity as well as a distinct gut microbiome composition.In addition, the association between gut microbiome diversity and systematic immune signatures highlights memory CD8 þ T cells and NK cells as potential mediators of antitumor immunity upon ICI treatment.The diversity of gut microbiome is defined as the number and abundance distribution of distinct types of micro-organisms colonizing within the gut.Abnormal diversity (dysbiosis) in the gut has been proven to be associated with human diseases, such as obesity, inflammatory bowel disease, cancer, etc. 20,21,33,34 Recently, Gopalakrishnan et al. 35 reported that patients with a high diversity of gut microbiome favored the clinical outcomes of anti-PD-1 immunotherapy in melanoma patients in the Western population.In our study, both alpha diversity and beta diversity at baseline are dramatically different between R and NR as well.A higher alpha diversity index represented by both the Shannon index and inverse Simpson indexes in R group suggests more species harboring in the gut, which is largely consistent with a differential composition of gut microbiome between R and NR.The higher the diversity of gut microbiota is, the longer PFS the patients undergo.Visualized beta diversity through PCoA analysis of 16S sequencing also displays apparently two clusters based on clinical responses to nivolumab treatment.Our results thus show that regardless of genetic background and geographic location with different lifestyles and diet styles, the diversity of gut microbiota might be a common benefit factor to ICI treatment.In our opinion, this is rational.It is widely accepted that gut microbiota modulates immune responses in a global way including antitumor immunity.36,37 Both innate and adaptive immune responses can be reshaped by gut microbiome.Although how the diversity of gut microbiota reshapes systemic immune responses is still not clearly addressed, microbiota-derived short chain fat acids and other metabolites should be good candidates to exert strong immune regulation.19,38 Further study will focus on the relevant metabolites analysis. Several clinical factors are reported to be involved in affecting the efficacy of ICI therapy, including smoking, previous treatment, and ATB usage.15,32,39 Among them, ATB is supposed to be the most relevant to gut microbiota.It has been reported previously that recent ATB usage in NSCLC patients was associated with shorter OS, although there was no difference in terms of objective response or PFS.32 In another study, ATB treatment is identified as the key factor significantly associated with both PFS and OS. 15 In our study, we retrospectively investigated ATB usage among 37 patients.The composition of gut microbiota at baseline between participants with or without ATB before first nivolumab infusion was similar (Supplementary Fig. 2), which excludes the possible effects of ATB on gut microbiota in our study.Median PFS was similar between ATB-treated and -naïve patients (median PFS: 126 days versus 117.9 days, p > 0.05) (Supplementary Fig. 3).One possibility lies in the number limitation in our study.Another possibility is that gut microbiome possess the ability of resilience.40 The majority of gut microbiota can be normalized after 1 week once ATB usage stops.41 We also observed that gut microbiota remained stable in longitudinal samples within one individual in our study.Such a trait might ensure patient long-term benefit from ICI treatment. Among all the clinical manifestations, smoking is the only factor that affects the responses to nivolumab treatment in our study.The roles of smoking on carcinogenesis refer to both cancer cell itself and immune responses of the host.It has been hypothesized that higher mutational burden could be induced by smoking, which has shown an increased response to ICI. 42,43 Median PFS was longer in current and former smokers compared with nonsmokers in nivolumab monotherapy for first-line treatment of advanced NSCLC as well.44 Smoking is reported to affect both innate and adaptive immunity, including memory T cells in the periphery.45 In our study, memory T cell profiles at baseline are similar between smoking and nonsmoking patients (Supplementary Fig. 4).This result confers that memory T cells are more likely affected by gut microbiota rather than other factors such as smoking.It has also been reported that chemotherapy could influence the gut microbiota of cancer patients largely due to the disruption of the intestinal barrier and subsequent bacteria translocation.In our study all the participants have received nivolumab therapy after failure of traditional chemotherapy.There was no difference in the interval from the date of last chemotherapy to that of first nivolumab infusion between R and NR groups (Supplementary Fig. 5, left).Correlation analysis showed that the diversity of gut microbiota was irrelevant to the time-off of chemotherapy in our cohort study as well (Supplementary Fig. 5, right). In addition to diversity, gut microbiome composition is also related to favorable responses to ICI treatment.In a pre-clinical study, gut commensal Bifidobacterium was shown to facilitate anti-PD-L1 efficacy.46 Clinical investigations reported that Faecalibacterium and some species belonging to Bacteroides genus (such as B. fragilis or B. thetaiotaomicron) are positively associated with the efficacy of PD-1 and CTLA-4 blockade, respectively.14,23 In our study, seven genera were identified different between R and NR.Ruminococcus_unclassified was enriched in NR whereas Bifidobacterium longum, Prevotella copri, and Alistipes putredinis were enriched in R. It has been shown in another study that Bifidobacterium longum could increase the production of pro-inflammatory cytokine tumor necrosis factor-alpha and significantly improve tumor control upon anti-PD-L1 therapy in a B16.SIY tumor mouse model.46 More interestingly, most bacteria enriched in R in our study belong to pro-inflammatory gram-negative bacteria.There are several previous studies reporting the association of gram-negative bacteria, such as Akkermansia muciniphila, Alistipes, and Porphyromonas with better clinical response to immunotherapy.14,15,36 It is already evident that the increase of pro-inflammatory gramnegative bacteria in the gut of older patients is associated with systematic and chronic inflammation.It is largely mediated by more release of lipopolysaccharide. In another study, mice colonized with Prevotella copri displayed increased inflammation in dextran sulfate sodium-induced colitis.Prevotella copri was strongly correlated with the disease severity in rheumatoid arthritis, which is a systemic autoimmune disease with the proliferation of pro-inflammatory T cells.47 In the antitumor setting, the exacerbation of immune responses might assist the ability of immune surveillance in the patients.This is also consistent with the report that patients who develop immune-related adverse events are more sensitive to ICI treatment.48 Although the direct evidence on the linkage between the inflammation and ICI benefit in the population still needs to be investigated, it is not out of expectation that systemic antitumor immune responses are diverse in patients with different gut microbiome composition.Gut microbiota thus becomes an intrinsic biosensor to sensitize anti-PD-1 treatment, which in turn leads to different clinical outcomes.This can be partially supported by the results from immune signature exploration in our study.Previous studies already highlighted the roles of PD-1 in reshaping immune responses in the mice.49 ICI therapy, to some extent, facilitates the investigation on PD-1 in the regulation of human immunity.By using multi-color flow cytometry, immune signatures including immune cell subsets and their function-related signatures have been detected.Furthermore, correlation analyses were performed between individual immune signatures and microbiota diversity.Among multiple signatures, the frequency and function of memory CD8 þ T cells are mostly dedicated.Memory CD8 þ T cells are a bunch of long-lived cells which are capable of rapid antigenspecific responses and play critical roles in immune recall responses. 50In addition to the percentage of CD8 þ Tm subsets such as effector memory T (Tem) cells and central memory T (Tcm) cells, the functional subset with Granzyme B production is also positively correlated with the diversity index, which can partially explain why patients with high microbiota diversity respond better to ICI therapy.The increase in Ki67 þ CD8 þ Tm in the patients with high microbiota diversity implies that gut microbiota also modulate the proliferation of certain immune cell populations.Recent studies already reported the effects of PD-1 blockade on the proliferation of CD8 þ T cells, which is beneficial for ICI treatment.51 ICOS and TIM3 expressions on specific T cells subsets are also related to antitumor immunity.52,53 Whereas CD8 þ T cells infiltrating in tumor microenvironment are thought to be the executors of tumor killing, circulating CD8 þ T cell subsets reported here are probably inclined to reflect the roles of gut microbiota on systematic immune responses.How peripheral memory CD8 þ T cell signatures affect the efficacy of ICI treatment will be investigated in the future.Once verified, they might become antitumor immune mediators against NSCLC. Our study shows that the diversity of gut microbiota is associated with favorable response to nivolumab therapy against NSCLC in Chinese cohorts.Considering the correlation between microbiota diversity and peripheral memory T cells and NK cells being reshaped upon ICI treatment, we propose that patients with more diverse gut microbiota at baseline benefit from ICI immunotherapy to a greater extent.This might be mediated by reprogramming systemic antitumor immune responses.The significance of our study lies in that the modulation of gut microbiota before the treatment might provide an alternative way to enhance the efficacy of ICI immunotherapy once validated in large cohort studies.A latest publication has introduced 11 bacterial strains from healthy donors' feces to induce interferon-g producing CD8 þ T cells in the intestine, which mediates effective antitumor immunity in a mouse model.54 Once clearly addressed, customized microbiome will exhibit great prospect as biotherapeutics in assistance with ICI treatment against advanced NSCLC.Project (17431906103), Shanghai Economic and Information Commission for oncology data platforms (201602010) and Shanghai Scientific Research Projects (14140902800).The authors thank Prof. Guo-ping Zhao for scientific suggestions on the project.Drs.Hong Li and Guoqing Zhang from Bigdata Center of Shanghai Insistute of Biological Science CAS helped to construct an integrative database with both clinical and multi-omics source data. Figure: 1 Figure 1 . Figure 1.Study scheme.

Probably not a substantial claim. The paper’s conclusions may not depend on it.

The cited sources support only part of this claim — read each source’s verdict below.

§ The Diversity of Gut Microbiome is Associated With Favorable Responses to Anti-Programmed Death 1 Immunotherapy in Chinese Patients With NSCLC > Discussion In the present study, we have investigated gut microbiota profiles in Chinese patients with advanced NSCLC receiving nivolumab treatment.Our results indicate that patients with better clinical responses exhibit higher diversity as well as a distinct gut microbiome composition.In addition, the association between gut microbiome diversity and systematic immune signatures highlights memory CD8 þ T cells and NK cells as potential mediators of antitumor immunity upon ICI treatment.The diversity of gut microbiome is defined as the number and abundance distribution of distinct types of micro-organisms colonizing within the gut.Abnormal diversity (dysbiosis) in the gut has been proven to be associated with human diseases, such as obesity, inflammatory bowel disease, cancer, etc. 20,21,33,34 Recently, Gopalakrishnan et al. 35 reported that patients with a high diversity of gut microbiome favored the clinical outcomes of anti-PD-1 immunotherapy in melanoma patients in the Western population.In our study, both alpha diversity and beta diversity at baseline are dramatically different between R and NR as well.A higher alpha diversity index represented by both the Shannon index and inverse Simpson indexes in R group suggests more species harboring in the gut, which is largely consistent with a differential composition of gut microbiome between R and NR.The higher the diversity of gut microbiota is, the longer PFS the patients undergo.Visualized beta diversity through PCoA analysis of 16S sequencing also displays apparently two clusters based on clinical responses to nivolumab treatment.Our results thus show that regardless of genetic background and geographic location with different lifestyles and diet styles, the diversity of gut microbiota might be a common benefit factor to ICI treatment.In our opinion, this is rational.It is widely accepted that gut microbiota modulates immune responses in a global way including antitumor immunity.36,37 Both innate and adaptive immune responses can be reshaped by gut microbiome.Although how the diversity of gut microbiota reshapes systemic immune responses is still not clearly addressed, microbiota-derived short chain fat acids and other metabolites should be good candidates to exert strong immune regulation.19,38 Further study will focus on the relevant metabolites analysis. Several clinical factors are reported to be involved in affecting the efficacy of ICI therapy, including smoking, previous treatment, and ATB usage.15,32,39 Among them, ATB is supposed to be the most relevant to gut microbiota.It has been reported previously that recent ATB usage in NSCLC patients was associated with shorter OS, although there was no difference in terms of objective response or PFS.32 In another study, ATB treatment is identified as the key factor significantly associated with both PFS and OS. 15 In our study, we retrospectively investigated ATB usage among 37 patients.The composition of gut microbiota at baseline between participants with or without ATB before first nivolumab infusion was similar (Supplementary Fig. 2), which excludes the possible effects of ATB on gut microbiota in our study.Median PFS was similar between ATB-treated and -naïve patients (median PFS: 126 days versus 117.9 days, p > 0.05) (Supplementary Fig. 3).One possibility lies in the number limitation in our study.Another possibility is that gut microbiome possess the ability of resilience.40 The majority of gut microbiota can be normalized after 1 week once ATB usage stops.41 We also observed that gut microbiota remained stable in longitudinal samples within one individual in our study.Such a trait might ensure patient long-term benefit from ICI treatment. Among all the clinical manifestations, smoking is the only factor that affects the responses to nivolumab treatment in our study.The roles of smoking on carcinogenesis refer to both cancer cell itself and immune responses of the host.It has been hypothesized that higher mutational burden could be induced by smoking, which has shown an increased response to ICI. 42,43 Median PFS was longer in current and former smokers compared with nonsmokers in nivolumab monotherapy for first-line treatment of advanced NSCLC as well.44 Smoking is reported to affect both innate and adaptive immunity, including memory T cells in the periphery.45 In our study, memory T cell profiles at baseline are similar between smoking and nonsmoking patients (Supplementary Fig. 4).This result confers that memory T cells are more likely affected by gut microbiota rather than other factors such as smoking.It has also been reported that chemotherapy could influence the gut microbiota of cancer patients largely due to the disruption of the intestinal barrier and subsequent bacteria translocation.In our study all the participants have received nivolumab therapy after failure of traditional chemotherapy.There was no difference in the interval from the date of last chemotherapy to that of first nivolumab infusion between R and NR groups (Supplementary Fig. 5, left).Correlation analysis showed that the diversity of gut microbiota was irrelevant to the time-off of chemotherapy in our cohort study as well (Supplementary Fig. 5, right). In addition to diversity, gut microbiome composition is also related to favorable responses to ICI treatment.In a pre-clinical study, gut commensal Bifidobacterium was shown to facilitate anti-PD-L1 efficacy.46 Clinical investigations reported that Faecalibacterium and some species belonging to Bacteroides genus (such as B. fragilis or B. thetaiotaomicron) are positively associated with the efficacy of PD-1 and CTLA-4 blockade, respectively.14,23 In our study, seven genera were identified different between R and NR.Ruminococcus_unclassified was enriched in NR whereas Bifidobacterium longum, Prevotella copri, and Alistipes putredinis were enriched in R. It has been shown in another study that Bifidobacterium longum could increase the production of pro-inflammatory cytokine tumor necrosis factor-alpha and significantly improve tumor control upon anti-PD-L1 therapy in a B16.SIY tumor mouse model.46 More interestingly, most bacteria enriched in R in our study belong to pro-inflammatory gram-negative bacteria.There are several previous studies reporting the association of gram-negative bacteria, such as Akkermansia muciniphila, Alistipes, and Porphyromonas with better clinical response to immunotherapy.14,15,36 It is already evident that the increase of pro-inflammatory gramnegative bacteria in the gut of older patients is associated with systematic and chronic inflammation.It is largely mediated by more release of lipopolysaccharide. In another study, mice colonized with Prevotella copri displayed increased inflammation in dextran sulfate sodium-induced colitis.Prevotella copri was strongly correlated with the disease severity in rheumatoid arthritis, which is a systemic autoimmune disease with the proliferation of pro-inflammatory T cells.47 In the antitumor setting, the exacerbation of immune responses might assist the ability of immune surveillance in the patients.This is also consistent with the report that patients who develop immune-related adverse events are more sensitive to ICI treatment.48 Although the direct evidence on the linkage between the inflammation and ICI benefit in the population still needs to be investigated, it is not out of expectation that systemic antitumor immune responses are diverse in patients with different gut microbiome composition.Gut microbiota thus becomes an intrinsic biosensor to sensitize anti-PD-1 treatment, which in turn leads to different clinical outcomes.This can be partially supported by the results from immune signature exploration in our study.Previous studies already highlighted the roles of PD-1 in reshaping immune responses in the mice.49 ICI therapy, to some extent, facilitates the investigation on PD-1 in the regulation of human immunity.By using multi-color flow cytometry, immune signatures including immune cell subsets and their function-related signatures have been detected.Furthermore, correlation analyses were performed between individual immune signatures and microbiota diversity.Among multiple signatures, the frequency and function of memory CD8 þ T cells are mostly dedicated.Memory CD8 þ T cells are a bunch of long-lived cells which are capable of rapid antigenspecific responses and play critical roles in immune recall responses. 50In addition to the percentage of CD8 þ Tm subsets such as effector memory T (Tem) cells and central memory T (Tcm) cells, the functional subset with Granzyme B production is also positively correlated with the diversity index, which can partially explain why patients with high microbiota diversity respond better to ICI therapy.The increase in Ki67 þ CD8 þ Tm in the patients with high microbiota diversity implies that gut microbiota also modulate the proliferation of certain immune cell populations.Recent studies already reported the effects of PD-1 blockade on the proliferation of CD8 þ T cells, which is beneficial for ICI treatment.51 ICOS and TIM3 expressions on specific T cells subsets are also related to antitumor immunity.52,53 Whereas CD8 þ T cells infiltrating in tumor microenvironment are thought to be the executors of tumor killing, circulating CD8 þ T cell subsets reported here are probably inclined to reflect the roles of gut microbiota on systematic immune responses.How peripheral memory CD8 þ T cell signatures affect the efficacy of ICI treatment will be investigated in the future.Once verified, they might become antitumor immune mediators against NSCLC. Our study shows that the diversity of gut microbiota is associated with favorable response to nivolumab therapy against NSCLC in Chinese cohorts.Considering the correlation between microbiota diversity and peripheral memory T cells and NK cells being reshaped upon ICI treatment, we propose that patients with more diverse gut microbiota at baseline benefit from ICI immunotherapy to a greater extent.This might be mediated by reprogramming systemic antitumor immune responses.The significance of our study lies in that the modulation of gut microbiota before the treatment might provide an alternative way to enhance the efficacy of ICI immunotherapy once validated in large cohort studies.A latest publication has introduced 11 bacterial strains from healthy donors' feces to induce interferon-g producing CD8 þ T cells in the intestine, which mediates effective antitumor immunity in a mouse model.54 Once clearly addressed, customized microbiome will exhibit great prospect as biotherapeutics in assistance with ICI treatment against advanced NSCLC.Project (17431906103), Shanghai Economic and Information Commission for oncology data platforms (201602010) and Shanghai Scientific Research Projects (14140902800).The authors thank Prof. Guo-ping Zhao for scientific suggestions on the project.Drs.Hong Li and Guoqing Zhang from Bigdata Center of Shanghai Insistute of Biological Science CAS helped to construct an integrative database with both clinical and multi-omics source data. Figure: 1 Figure 1 . Figure 1.Study scheme.

Probably not a substantial claim. The paper’s conclusions may not depend on it.

The cited sources support only part of this claim — read each source’s verdict below.

§ The Diversity of Gut Microbiome is Associated With Favorable Responses to Anti-Programmed Death 1 Immunotherapy in Chinese Patients With NSCLC > Discussion In the present study, we have investigated gut microbiota profiles in Chinese patients with advanced NSCLC receiving nivolumab treatment.Our results indicate that patients with better clinical responses exhibit higher diversity as well as a distinct gut microbiome composition.In addition, the association between gut microbiome diversity and systematic immune signatures highlights memory CD8 þ T cells and NK cells as potential mediators of antitumor immunity upon ICI treatment.The diversity of gut microbiome is defined as the number and abundance distribution of distinct types of micro-organisms colonizing within the gut.Abnormal diversity (dysbiosis) in the gut has been proven to be associated with human diseases, such as obesity, inflammatory bowel disease, cancer, etc. 20,21,33,34 Recently, Gopalakrishnan et al. 35 reported that patients with a high diversity of gut microbiome favored the clinical outcomes of anti-PD-1 immunotherapy in melanoma patients in the Western population.In our study, both alpha diversity and beta diversity at baseline are dramatically different between R and NR as well.A higher alpha diversity index represented by both the Shannon index and inverse Simpson indexes in R group suggests more species harboring in the gut, which is largely consistent with a differential composition of gut microbiome between R and NR.The higher the diversity of gut microbiota is, the longer PFS the patients undergo.Visualized beta diversity through PCoA analysis of 16S sequencing also displays apparently two clusters based on clinical responses to nivolumab treatment.Our results thus show that regardless of genetic background and geographic location with different lifestyles and diet styles, the diversity of gut microbiota might be a common benefit factor to ICI treatment.In our opinion, this is rational.It is widely accepted that gut microbiota modulates immune responses in a global way including antitumor immunity.36,37 Both innate and adaptive immune responses can be reshaped by gut microbiome.Although how the diversity of gut microbiota reshapes systemic immune responses is still not clearly addressed, microbiota-derived short chain fat acids and other metabolites should be good candidates to exert strong immune regulation.19,38 Further study will focus on the relevant metabolites analysis. Several clinical factors are reported to be involved in affecting the efficacy of ICI therapy, including smoking, previous treatment, and ATB usage.15,32,39 Among them, ATB is supposed to be the most relevant to gut microbiota.It has been reported previously that recent ATB usage in NSCLC patients was associated with shorter OS, although there was no difference in terms of objective response or PFS.32 In another study, ATB treatment is identified as the key factor significantly associated with both PFS and OS. 15 In our study, we retrospectively investigated ATB usage among 37 patients.The composition of gut microbiota at baseline between participants with or without ATB before first nivolumab infusion was similar (Supplementary Fig. 2), which excludes the possible effects of ATB on gut microbiota in our study.Median PFS was similar between ATB-treated and -naïve patients (median PFS: 126 days versus 117.9 days, p > 0.05) (Supplementary Fig. 3).One possibility lies in the number limitation in our study.Another possibility is that gut microbiome possess the ability of resilience.40 The majority of gut microbiota can be normalized after 1 week once ATB usage stops.41 We also observed that gut microbiota remained stable in longitudinal samples within one individual in our study.Such a trait might ensure patient long-term benefit from ICI treatment. Among all the clinical manifestations, smoking is the only factor that affects the responses to nivolumab treatment in our study.The roles of smoking on carcinogenesis refer to both cancer cell itself and immune responses of the host.It has been hypothesized that higher mutational burden could be induced by smoking, which has shown an increased response to ICI. 42,43 Median PFS was longer in current and former smokers compared with nonsmokers in nivolumab monotherapy for first-line treatment of advanced NSCLC as well.44 Smoking is reported to affect both innate and adaptive immunity, including memory T cells in the periphery.45 In our study, memory T cell profiles at baseline are similar between smoking and nonsmoking patients (Supplementary Fig. 4).This result confers that memory T cells are more likely affected by gut microbiota rather than other factors such as smoking.It has also been reported that chemotherapy could influence the gut microbiota of cancer patients largely due to the disruption of the intestinal barrier and subsequent bacteria translocation.In our study all the participants have received nivolumab therapy after failure of traditional chemotherapy.There was no difference in the interval from the date of last chemotherapy to that of first nivolumab infusion between R and NR groups (Supplementary Fig. 5, left).Correlation analysis showed that the diversity of gut microbiota was irrelevant to the time-off of chemotherapy in our cohort study as well (Supplementary Fig. 5, right). In addition to diversity, gut microbiome composition is also related to favorable responses to ICI treatment.In a pre-clinical study, gut commensal Bifidobacterium was shown to facilitate anti-PD-L1 efficacy.46 Clinical investigations reported that Faecalibacterium and some species belonging to Bacteroides genus (such as B. fragilis or B. thetaiotaomicron) are positively associated with the efficacy of PD-1 and CTLA-4 blockade, respectively.14,23 In our study, seven genera were identified different between R and NR.Ruminococcus_unclassified was enriched in NR whereas Bifidobacterium longum, Prevotella copri, and Alistipes putredinis were enriched in R. It has been shown in another study that Bifidobacterium longum could increase the production of pro-inflammatory cytokine tumor necrosis factor-alpha and significantly improve tumor control upon anti-PD-L1 therapy in a B16.SIY tumor mouse model.46 More interestingly, most bacteria enriched in R in our study belong to pro-inflammatory gram-negative bacteria.There are several previous studies reporting the association of gram-negative bacteria, such as Akkermansia muciniphila, Alistipes, and Porphyromonas with better clinical response to immunotherapy.14,15,36 It is already evident that the increase of pro-inflammatory gramnegative bacteria in the gut of older patients is associated with systematic and chronic inflammation.It is largely mediated by more release of lipopolysaccharide. In another study, mice colonized with Prevotella copri displayed increased inflammation in dextran sulfate sodium-induced colitis.Prevotella copri was strongly correlated with the disease severity in rheumatoid arthritis, which is a systemic autoimmune disease with the proliferation of pro-inflammatory T cells.47 In the antitumor setting, the exacerbation of immune responses might assist the ability of immune surveillance in the patients.This is also consistent with the report that patients who develop immune-related adverse events are more sensitive to ICI treatment.48 Although the direct evidence on the linkage between the inflammation and ICI benefit in the population still needs to be investigated, it is not out of expectation that systemic antitumor immune responses are diverse in patients with different gut microbiome composition.Gut microbiota thus becomes an intrinsic biosensor to sensitize anti-PD-1 treatment, which in turn leads to different clinical outcomes.This can be partially supported by the results from immune signature exploration in our study.Previous studies already highlighted the roles of PD-1 in reshaping immune responses in the mice.49 ICI therapy, to some extent, facilitates the investigation on PD-1 in the regulation of human immunity.By using multi-color flow cytometry, immune signatures including immune cell subsets and their function-related signatures have been detected.Furthermore, correlation analyses were performed between individual immune signatures and microbiota diversity.Among multiple signatures, the frequency and function of memory CD8 þ T cells are mostly dedicated.Memory CD8 þ T cells are a bunch of long-lived cells which are capable of rapid antigenspecific responses and play critical roles in immune recall responses. 50In addition to the percentage of CD8 þ Tm subsets such as effector memory T (Tem) cells and central memory T (Tcm) cells, the functional subset with Granzyme B production is also positively correlated with the diversity index, which can partially explain why patients with high microbiota diversity respond better to ICI therapy.The increase in Ki67 þ CD8 þ Tm in the patients with high microbiota diversity implies that gut microbiota also modulate the proliferation of certain immune cell populations.Recent studies already reported the effects of PD-1 blockade on the proliferation of CD8 þ T cells, which is beneficial for ICI treatment.51 ICOS and TIM3 expressions on specific T cells subsets are also related to antitumor immunity.52,53 Whereas CD8 þ T cells infiltrating in tumor microenvironment are thought to be the executors of tumor killing, circulating CD8 þ T cell subsets reported here are probably inclined to reflect the roles of gut microbiota on systematic immune responses.How peripheral memory CD8 þ T cell signatures affect the efficacy of ICI treatment will be investigated in the future.Once verified, they might become antitumor immune mediators against NSCLC. Our study shows that the diversity of gut microbiota is associated with favorable response to nivolumab therapy against NSCLC in Chinese cohorts.Considering the correlation between microbiota diversity and peripheral memory T cells and NK cells being reshaped upon ICI treatment, we propose that patients with more diverse gut microbiota at baseline benefit from ICI immunotherapy to a greater extent.This might be mediated by reprogramming systemic antitumor immune responses.The significance of our study lies in that the modulation of gut microbiota before the treatment might provide an alternative way to enhance the efficacy of ICI immunotherapy once validated in large cohort studies.A latest publication has introduced 11 bacterial strains from healthy donors' feces to induce interferon-g producing CD8 þ T cells in the intestine, which mediates effective antitumor immunity in a mouse model.54 Once clearly addressed, customized microbiome will exhibit great prospect as biotherapeutics in assistance with ICI treatment against advanced NSCLC.Project (17431906103), Shanghai Economic and Information Commission for oncology data platforms (201602010) and Shanghai Scientific Research Projects (14140902800).The authors thank Prof. Guo-ping Zhao for scientific suggestions on the project.Drs.Hong Li and Guoqing Zhang from Bigdata Center of Shanghai Insistute of Biological Science CAS helped to construct an integrative database with both clinical and multi-omics source data. Figure: 1 Figure 1 . Figure 1.Study scheme.

Probably not a substantial claim. The paper’s conclusions may not depend on it.

A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

§ The Diversity of Gut Microbiome is Associated With Favorable Responses to Anti-Programmed Death 1 Immunotherapy in Chinese Patients With NSCLC > Baseline Gut Microbiota Diversity is Associated With Favorable Responses to Nivolumab

It may state two separate claims as one.

• AssumptionRests on an assumption: The baseline fecal samples available from 25 of the 37 enrolled patients are representative of the full enrolled cohort.

A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

§ The Diversity of Gut Microbiome is Associated With Favorable Responses to Anti-Programmed Death 1 Immunotherapy in Chinese Patients With NSCLC > Gut Microbiota Composition is Stable During Nivolumab Treatment Whether gut microbiota profile is influenced during ICI treatment is very interesting due to the different outcomes of individual patients.In our study using timeserial fecal sampling during the treatment, dynamic analysis of gut microbiota was feasible.The Shannon diversity index remained stable based on Kruskal-Wallis test (p > 0.999) from baseline to different assessment timepoints (T1 to T8) as well as at progression timepoints (Fig. 4A, left).Paired samples at baseline and at the T1 (Fig. 4A, middle) or baseline and progression timepoint (Fig. 4A, right) displayed no differences in Shannon index based on Wilcoxon matched-pairs signed rank test (p > 0.05).We then compared gut microbiota composition at the genera level using 10 paired fecal samples at baseline and timepoint with disease progression.As shown in Figure 4B, the compositions of fecal microbiota were apparently similar.We further conducted PCoA analysis of gut microbiome in longitudinal samples of 10 patients (7 from R and 3 from NR).While gut microbiota from one patient at different sampling timepoints (represented by one shape) were clustered very well, R and NR individuals were separated clearly as well (pink and purple circles) (Fig. 4C).Analyzing precisely, the relative abundance of main bacterial phylum remained stable over time (Fig. 4D).Seven genera which were differentially abundant in R and NR at baseline showed mild turbulence during the therapy.But no statistical difference was noticed (Fig. 4E).Altogether, these data indicate that nivolumab treatment does not modify gut microbiome community in patients with advanced NSCLC.

It may state two separate claims as one.

A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

§ The Diversity of Gut Microbiome is Associated With Favorable Responses to Anti-Programmed Death 1 Immunotherapy in Chinese Patients With NSCLC > Compositional Difference in Gut Microbiome in Response to Nivolumab

It may state two separate claims as one.

• AssumptionRests on an assumption: The baseline fecal samples available from 25 of the 37 enrolled patients are representative of the full enrolled cohort.

A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

§ The Diversity of Gut Microbiome is Associated With Favorable Responses to Anti-Programmed Death 1 Immunotherapy in Chinese Patients With NSCLC > Compositional Difference in Gut Microbiome in Response to Nivolumab

It may state two separate claims as one.

• AssumptionRests on an assumption: The baseline fecal samples available from 25 of the 37 enrolled patients are representative of the full enrolled cohort.

A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

§ The Diversity of Gut Microbiome is Associated With Favorable Responses to Anti-Programmed Death 1 Immunotherapy in Chinese Patients With NSCLC > Gut Microbiota Composition is Stable During Nivolumab Treatment Whether gut microbiota profile is influenced during ICI treatment is very interesting due to the different outcomes of individual patients.In our study using timeserial fecal sampling during the treatment, dynamic analysis of gut microbiota was feasible.The Shannon diversity index remained stable based on Kruskal-Wallis test (p > 0.999) from baseline to different assessment timepoints (T1 to T8) as well as at progression timepoints (Fig. 4A, left).Paired samples at baseline and at the T1 (Fig. 4A, middle) or baseline and progression timepoint (Fig. 4A, right) displayed no differences in Shannon index based on Wilcoxon matched-pairs signed rank test (p > 0.05).We then compared gut microbiota composition at the genera level using 10 paired fecal samples at baseline and timepoint with disease progression.As shown in Figure 4B, the compositions of fecal microbiota were apparently similar.We further conducted PCoA analysis of gut microbiome in longitudinal samples of 10 patients (7 from R and 3 from NR).While gut microbiota from one patient at different sampling timepoints (represented by one shape) were clustered very well, R and NR individuals were separated clearly as well (pink and purple circles) (Fig. 4C).Analyzing precisely, the relative abundance of main bacterial phylum remained stable over time (Fig. 4D).Seven genera which were differentially abundant in R and NR at baseline showed mild turbulence during the therapy.But no statistical difference was noticed (Fig. 4E).Altogether, these data indicate that nivolumab treatment does not modify gut microbiome community in patients with advanced NSCLC.

It may state two separate claims as one.

A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

§ The Diversity of Gut Microbiome is Associated With Favorable Responses to Anti-Programmed Death 1 Immunotherapy in Chinese Patients With NSCLC > Baseline Gut Microbiota Diversity is Associated With Favorable Responses to Nivolumab

The quote only partly states this claim.

• AssumptionRests on an assumption: The baseline fecal samples available from 25 of the 37 enrolled patients are representative of the full enrolled cohort.

A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

§ The Diversity of Gut Microbiome is Associated With Favorable Responses to Anti-Programmed Death 1 Immunotherapy in Chinese Patients With NSCLC > Baseline Gut Microbiota Diversity is Associated With Favorable Responses to Nivolumab

Probably not a substantial claim. The paper’s conclusions may not depend on it.

• AssumptionRests on an assumption: The baseline fecal samples available from 25 of the 37 enrolled patients are representative of the full enrolled cohort.

A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

§ The Diversity of Gut Microbiome is Associated With Favorable Responses to Anti-Programmed Death 1 Immunotherapy in Chinese Patients With NSCLC > Patient Characteristics Forty-two Chinese patients with advanced NSCLC were consented and enrolled.All patients were followedup until disease progression or death.Among them, 1 patient died after a single infusion of nivolumab.Two patients were lost during the follow-up study.Two patients discontinued therapy because of grade 3-4 adverse events.The remaining 37 patients were grouped according to clinical assessment.Twenty-three patients being evaluated as either partial response (n ¼ 5) or stable disease (n ¼ 18) were classified as R whereas the others (n ¼ 14) with disease progression after the first clinical assessment were classified as NR.Fecal samples at baseline were obtained from 25 patients, among whom 13 patients were R and 12 patients were NR (Fig. 1).Clinical information revealed that the majority of patients enrolled in the study were male (78.3%).All of the patients had received at least one or two-rounds of chemotherapy before enrollment.No difference was observed in sex, disease history, disease stage, and the treatment between R and NR patients except smoking history (p ¼ 0.008).Recent studies reported negative association between ATB prescription and clinical outcome of PD-1 inhibitors.15,32 When we examined ATB usage in our cohort, 11 of 37 patients (29.7%) were prescribed ATB within 2 month.However, the R/NR ratio with or without ATB usage was similar (Table 1).Therefore, smoking is the only clinical factor associated with the responses to nivolumab in our study.

Probably not a substantial claim. The paper’s conclusions may not depend on it.

A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

§ The Diversity of Gut Microbiome is Associated With Favorable Responses to Anti-Programmed Death 1 Immunotherapy in Chinese Patients With NSCLC > Patient Characteristics Forty-two Chinese patients with advanced NSCLC were consented and enrolled.All patients were followedup until disease progression or death.Among them, 1 patient died after a single infusion of nivolumab.Two patients were lost during the follow-up study.Two patients discontinued therapy because of grade 3-4 adverse events.The remaining 37 patients were grouped according to clinical assessment.Twenty-three patients being evaluated as either partial response (n ¼ 5) or stable disease (n ¼ 18) were classified as R whereas the others (n ¼ 14) with disease progression after the first clinical assessment were classified as NR.Fecal samples at baseline were obtained from 25 patients, among whom 13 patients were R and 12 patients were NR (Fig. 1).Clinical information revealed that the majority of patients enrolled in the study were male (78.3%).All of the patients had received at least one or two-rounds of chemotherapy before enrollment.No difference was observed in sex, disease history, disease stage, and the treatment between R and NR patients except smoking history (p ¼ 0.008).Recent studies reported negative association between ATB prescription and clinical outcome of PD-1 inhibitors.15,32 When we examined ATB usage in our cohort, 11 of 37 patients (29.7%) were prescribed ATB within 2 month.However, the R/NR ratio with or without ATB usage was similar (Table 1).Therefore, smoking is the only clinical factor associated with the responses to nivolumab in our study.

It may state two separate claims as one.

A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

§ The Diversity of Gut Microbiome is Associated With Favorable Responses to Anti-Programmed Death 1 Immunotherapy in Chinese Patients With NSCLC > Patient Characteristics Forty-two Chinese patients with advanced NSCLC were consented and enrolled.All patients were followedup until disease progression or death.Among them, 1 patient died after a single infusion of nivolumab.Two patients were lost during the follow-up study.Two patients discontinued therapy because of grade 3-4 adverse events.The remaining 37 patients were grouped according to clinical assessment.Twenty-three patients being evaluated as either partial response (n ¼ 5) or stable disease (n ¼ 18) were classified as R whereas the others (n ¼ 14) with disease progression after the first clinical assessment were classified as NR.Fecal samples at baseline were obtained from 25 patients, among whom 13 patients were R and 12 patients were NR (Fig. 1).Clinical information revealed that the majority of patients enrolled in the study were male (78.3%).All of the patients had received at least one or two-rounds of chemotherapy before enrollment.No difference was observed in sex, disease history, disease stage, and the treatment between R and NR patients except smoking history (p ¼ 0.008).Recent studies reported negative association between ATB prescription and clinical outcome of PD-1 inhibitors.15,32 When we examined ATB usage in our cohort, 11 of 37 patients (29.7%) were prescribed ATB within 2 month.However, the R/NR ratio with or without ATB usage was similar (Table 1).Therefore, smoking is the only clinical factor associated with the responses to nivolumab in our study.

Probably not a substantial claim.

A result the paper reports from its own data — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

§ The Diversity of Gut Microbiome is Associated With Favorable Responses to Anti-Programmed Death 1 Immunotherapy in Chinese Patients With NSCLC > Patient Characteristics Forty-two Chinese patients with advanced NSCLC were consented and enrolled.All patients were followedup until disease progression or death.Among them, 1 patient died after a single infusion of nivolumab.Two patients were lost during the follow-up study.Two patients discontinued therapy because of grade 3-4 adverse events.The remaining 37 patients were grouped according to clinical assessment.Twenty-three patients being evaluated as either partial response (n ¼ 5) or stable disease (n ¼ 18) were classified as R whereas the others (n ¼ 14) with disease progression after the first clinical assessment were classified as NR.Fecal samples at baseline were obtained from 25 patients, among whom 13 patients were R and 12 patients were NR (Fig. 1).Clinical information revealed that the majority of patients enrolled in the study were male (78.3%).All of the patients had received at least one or two-rounds of chemotherapy before enrollment.No difference was observed in sex, disease history, disease stage, and the treatment between R and NR patients except smoking history (p ¼ 0.008).Recent studies reported negative association between ATB prescription and clinical outcome of PD-1 inhibitors.15,32 When we examined ATB usage in our cohort, 11 of 37 patients (29.7%) were prescribed ATB within 2 month.However, the R/NR ratio with or without ATB usage was similar (Table 1).Therefore, smoking is the only clinical factor associated with the responses to nivolumab in our study.

Probably not a substantial claim.

A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

§ The Diversity of Gut Microbiome is Associated With Favorable Responses to Anti-Programmed Death 1 Immunotherapy in Chinese Patients With NSCLC > Discussion In the present study, we have investigated gut microbiota profiles in Chinese patients with advanced NSCLC receiving nivolumab treatment.Our results indicate that patients with better clinical responses exhibit higher diversity as well as a distinct gut microbiome composition.In addition, the association between gut microbiome diversity and systematic immune signatures highlights memory CD8 þ T cells and NK cells as potential mediators of antitumor immunity upon ICI treatment.The diversity of gut microbiome is defined as the number and abundance distribution of distinct types of micro-organisms colonizing within the gut.Abnormal diversity (dysbiosis) in the gut has been proven to be associated with human diseases, such as obesity, inflammatory bowel disease, cancer, etc. 20,21,33,34 Recently, Gopalakrishnan et al. 35 reported that patients with a high diversity of gut microbiome favored the clinical outcomes of anti-PD-1 immunotherapy in melanoma patients in the Western population.In our study, both alpha diversity and beta diversity at baseline are dramatically different between R and NR as well.A higher alpha diversity index represented by both the Shannon index and inverse Simpson indexes in R group suggests more species harboring in the gut, which is largely consistent with a differential composition of gut microbiome between R and NR.The higher the diversity of gut microbiota is, the longer PFS the patients undergo.Visualized beta diversity through PCoA analysis of 16S sequencing also displays apparently two clusters based on clinical responses to nivolumab treatment.Our results thus show that regardless of genetic background and geographic location with different lifestyles and diet styles, the diversity of gut microbiota might be a common benefit factor to ICI treatment.In our opinion, this is rational.It is widely accepted that gut microbiota modulates immune responses in a global way including antitumor immunity.36,37 Both innate and adaptive immune responses can be reshaped by gut microbiome.Although how the diversity of gut microbiota reshapes systemic immune responses is still not clearly addressed, microbiota-derived short chain fat acids and other metabolites should be good candidates to exert strong immune regulation.19,38 Further study will focus on the relevant metabolites analysis. Several clinical factors are reported to be involved in affecting the efficacy of ICI therapy, including smoking, previous treatment, and ATB usage.15,32,39 Among them, ATB is supposed to be the most relevant to gut microbiota.It has been reported previously that recent ATB usage in NSCLC patients was associated with shorter OS, although there was no difference in terms of objective response or PFS.32 In another study, ATB treatment is identified as the key factor significantly associated with both PFS and OS. 15 In our study, we retrospectively investigated ATB usage among 37 patients.The composition of gut microbiota at baseline between participants with or without ATB before first nivolumab infusion was similar (Supplementary Fig. 2), which excludes the possible effects of ATB on gut microbiota in our study.Median PFS was similar between ATB-treated and -naïve patients (median PFS: 126 days versus 117.9 days, p > 0.05) (Supplementary Fig. 3).One possibility lies in the number limitation in our study.Another possibility is that gut microbiome possess the ability of resilience.40 The majority of gut microbiota can be normalized after 1 week once ATB usage stops.41 We also observed that gut microbiota remained stable in longitudinal samples within one individual in our study.Such a trait might ensure patient long-term benefit from ICI treatment. Among all the clinical manifestations, smoking is the only factor that affects the responses to nivolumab treatment in our study.The roles of smoking on carcinogenesis refer to both cancer cell itself and immune responses of the host.It has been hypothesized that higher mutational burden could be induced by smoking, which has shown an increased response to ICI. 42,43 Median PFS was longer in current and former smokers compared with nonsmokers in nivolumab monotherapy for first-line treatment of advanced NSCLC as well.44 Smoking is reported to affect both innate and adaptive immunity, including memory T cells in the periphery.45 In our study, memory T cell profiles at baseline are similar between smoking and nonsmoking patients (Supplementary Fig. 4).This result confers that memory T cells are more likely affected by gut microbiota rather than other factors such as smoking.It has also been reported that chemotherapy could influence the gut microbiota of cancer patients largely due to the disruption of the intestinal barrier and subsequent bacteria translocation.In our study all the participants have received nivolumab therapy after failure of traditional chemotherapy.There was no difference in the interval from the date of last chemotherapy to that of first nivolumab infusion between R and NR groups (Supplementary Fig. 5, left).Correlation analysis showed that the diversity of gut microbiota was irrelevant to the time-off of chemotherapy in our cohort study as well (Supplementary Fig. 5, right). In addition to diversity, gut microbiome composition is also related to favorable responses to ICI treatment.In a pre-clinical study, gut commensal Bifidobacterium was shown to facilitate anti-PD-L1 efficacy.46 Clinical investigations reported that Faecalibacterium and some species belonging to Bacteroides genus (such as B. fragilis or B. thetaiotaomicron) are positively associated with the efficacy of PD-1 and CTLA-4 blockade, respectively.14,23 In our study, seven genera were identified different between R and NR.Ruminococcus_unclassified was enriched in NR whereas Bifidobacterium longum, Prevotella copri, and Alistipes putredinis were enriched in R. It has been shown in another study that Bifidobacterium longum could increase the production of pro-inflammatory cytokine tumor necrosis factor-alpha and significantly improve tumor control upon anti-PD-L1 therapy in a B16.SIY tumor mouse model.46 More interestingly, most bacteria enriched in R in our study belong to pro-inflammatory gram-negative bacteria.There are several previous studies reporting the association of gram-negative bacteria, such as Akkermansia muciniphila, Alistipes, and Porphyromonas with better clinical response to immunotherapy.14,15,36 It is already evident that the increase of pro-inflammatory gramnegative bacteria in the gut of older patients is associated with systematic and chronic inflammation.It is largely mediated by more release of lipopolysaccharide. In another study, mice colonized with Prevotella copri displayed increased inflammation in dextran sulfate sodium-induced colitis.Prevotella copri was strongly correlated with the disease severity in rheumatoid arthritis, which is a systemic autoimmune disease with the proliferation of pro-inflammatory T cells.47 In the antitumor setting, the exacerbation of immune responses might assist the ability of immune surveillance in the patients.This is also consistent with the report that patients who develop immune-related adverse events are more sensitive to ICI treatment.48 Although the direct evidence on the linkage between the inflammation and ICI benefit in the population still needs to be investigated, it is not out of expectation that systemic antitumor immune responses are diverse in patients with different gut microbiome composition.Gut microbiota thus becomes an intrinsic biosensor to sensitize anti-PD-1 treatment, which in turn leads to different clinical outcomes.This can be partially supported by the results from immune signature exploration in our study.Previous studies already highlighted the roles of PD-1 in reshaping immune responses in the mice.49 ICI therapy, to some extent, facilitates the investigation on PD-1 in the regulation of human immunity.By using multi-color flow cytometry, immune signatures including immune cell subsets and their function-related signatures have been detected.Furthermore, correlation analyses were performed between individual immune signatures and microbiota diversity.Among multiple signatures, the frequency and function of memory CD8 þ T cells are mostly dedicated.Memory CD8 þ T cells are a bunch of long-lived cells which are capable of rapid antigenspecific responses and play critical roles in immune recall responses. 50In addition to the percentage of CD8 þ Tm subsets such as effector memory T (Tem) cells and central memory T (Tcm) cells, the functional subset with Granzyme B production is also positively correlated with the diversity index, which can partially explain why patients with high microbiota diversity respond better to ICI therapy.The increase in Ki67 þ CD8 þ Tm in the patients with high microbiota diversity implies that gut microbiota also modulate the proliferation of certain immune cell populations.Recent studies already reported the effects of PD-1 blockade on the proliferation of CD8 þ T cells, which is beneficial for ICI treatment.51 ICOS and TIM3 expressions on specific T cells subsets are also related to antitumor immunity.52,53 Whereas CD8 þ T cells infiltrating in tumor microenvironment are thought to be the executors of tumor killing, circulating CD8 þ T cell subsets reported here are probably inclined to reflect the roles of gut microbiota on systematic immune responses.How peripheral memory CD8 þ T cell signatures affect the efficacy of ICI treatment will be investigated in the future.Once verified, they might become antitumor immune mediators against NSCLC. Our study shows that the diversity of gut microbiota is associated with favorable response to nivolumab therapy against NSCLC in Chinese cohorts.Considering the correlation between microbiota diversity and peripheral memory T cells and NK cells being reshaped upon ICI treatment, we propose that patients with more diverse gut microbiota at baseline benefit from ICI immunotherapy to a greater extent.This might be mediated by reprogramming systemic antitumor immune responses.The significance of our study lies in that the modulation of gut microbiota before the treatment might provide an alternative way to enhance the efficacy of ICI immunotherapy once validated in large cohort studies.A latest publication has introduced 11 bacterial strains from healthy donors' feces to induce interferon-g producing CD8 þ T cells in the intestine, which mediates effective antitumor immunity in a mouse model.54 Once clearly addressed, customized microbiome will exhibit great prospect as biotherapeutics in assistance with ICI treatment against advanced NSCLC.Project (17431906103), Shanghai Economic and Information Commission for oncology data platforms (201602010) and Shanghai Scientific Research Projects (14140902800).The authors thank Prof. Guo-ping Zhao for scientific suggestions on the project.Drs.Hong Li and Guoqing Zhang from Bigdata Center of Shanghai Insistute of Biological Science CAS helped to construct an integrative database with both clinical and multi-omics source data. Figure: 1 Figure 1 . Figure 1.Study scheme.

It may state two separate claims as one.

• AssumptionRests on an assumption: The baseline fecal samples available from 25 of the 37 enrolled patients are representative of the full enrolled cohort.

A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

§ The Diversity of Gut Microbiome is Associated With Favorable Responses to Anti-Programmed Death 1 Immunotherapy in Chinese Patients With NSCLC > Discussion In the present study, we have investigated gut microbiota profiles in Chinese patients with advanced NSCLC receiving nivolumab treatment.Our results indicate that patients with better clinical responses exhibit higher diversity as well as a distinct gut microbiome composition.In addition, the association between gut microbiome diversity and systematic immune signatures highlights memory CD8 þ T cells and NK cells as potential mediators of antitumor immunity upon ICI treatment.The diversity of gut microbiome is defined as the number and abundance distribution of distinct types of micro-organisms colonizing within the gut.Abnormal diversity (dysbiosis) in the gut has been proven to be associated with human diseases, such as obesity, inflammatory bowel disease, cancer, etc. 20,21,33,34 Recently, Gopalakrishnan et al. 35 reported that patients with a high diversity of gut microbiome favored the clinical outcomes of anti-PD-1 immunotherapy in melanoma patients in the Western population.In our study, both alpha diversity and beta diversity at baseline are dramatically different between R and NR as well.A higher alpha diversity index represented by both the Shannon index and inverse Simpson indexes in R group suggests more species harboring in the gut, which is largely consistent with a differential composition of gut microbiome between R and NR.The higher the diversity of gut microbiota is, the longer PFS the patients undergo.Visualized beta diversity through PCoA analysis of 16S sequencing also displays apparently two clusters based on clinical responses to nivolumab treatment.Our results thus show that regardless of genetic background and geographic location with different lifestyles and diet styles, the diversity of gut microbiota might be a common benefit factor to ICI treatment.In our opinion, this is rational.It is widely accepted that gut microbiota modulates immune responses in a global way including antitumor immunity.36,37 Both innate and adaptive immune responses can be reshaped by gut microbiome.Although how the diversity of gut microbiota reshapes systemic immune responses is still not clearly addressed, microbiota-derived short chain fat acids and other metabolites should be good candidates to exert strong immune regulation.19,38 Further study will focus on the relevant metabolites analysis. Several clinical factors are reported to be involved in affecting the efficacy of ICI therapy, including smoking, previous treatment, and ATB usage.15,32,39 Among them, ATB is supposed to be the most relevant to gut microbiota.It has been reported previously that recent ATB usage in NSCLC patients was associated with shorter OS, although there was no difference in terms of objective response or PFS.32 In another study, ATB treatment is identified as the key factor significantly associated with both PFS and OS. 15 In our study, we retrospectively investigated ATB usage among 37 patients.The composition of gut microbiota at baseline between participants with or without ATB before first nivolumab infusion was similar (Supplementary Fig. 2), which excludes the possible effects of ATB on gut microbiota in our study.Median PFS was similar between ATB-treated and -naïve patients (median PFS: 126 days versus 117.9 days, p > 0.05) (Supplementary Fig. 3).One possibility lies in the number limitation in our study.Another possibility is that gut microbiome possess the ability of resilience.40 The majority of gut microbiota can be normalized after 1 week once ATB usage stops.41 We also observed that gut microbiota remained stable in longitudinal samples within one individual in our study.Such a trait might ensure patient long-term benefit from ICI treatment. Among all the clinical manifestations, smoking is the only factor that affects the responses to nivolumab treatment in our study.The roles of smoking on carcinogenesis refer to both cancer cell itself and immune responses of the host.It has been hypothesized that higher mutational burden could be induced by smoking, which has shown an increased response to ICI. 42,43 Median PFS was longer in current and former smokers compared with nonsmokers in nivolumab monotherapy for first-line treatment of advanced NSCLC as well.44 Smoking is reported to affect both innate and adaptive immunity, including memory T cells in the periphery.45 In our study, memory T cell profiles at baseline are similar between smoking and nonsmoking patients (Supplementary Fig. 4).This result confers that memory T cells are more likely affected by gut microbiota rather than other factors such as smoking.It has also been reported that chemotherapy could influence the gut microbiota of cancer patients largely due to the disruption of the intestinal barrier and subsequent bacteria translocation.In our study all the participants have received nivolumab therapy after failure of traditional chemotherapy.There was no difference in the interval from the date of last chemotherapy to that of first nivolumab infusion between R and NR groups (Supplementary Fig. 5, left).Correlation analysis showed that the diversity of gut microbiota was irrelevant to the time-off of chemotherapy in our cohort study as well (Supplementary Fig. 5, right). In addition to diversity, gut microbiome composition is also related to favorable responses to ICI treatment.In a pre-clinical study, gut commensal Bifidobacterium was shown to facilitate anti-PD-L1 efficacy.46 Clinical investigations reported that Faecalibacterium and some species belonging to Bacteroides genus (such as B. fragilis or B. thetaiotaomicron) are positively associated with the efficacy of PD-1 and CTLA-4 blockade, respectively.14,23 In our study, seven genera were identified different between R and NR.Ruminococcus_unclassified was enriched in NR whereas Bifidobacterium longum, Prevotella copri, and Alistipes putredinis were enriched in R. It has been shown in another study that Bifidobacterium longum could increase the production of pro-inflammatory cytokine tumor necrosis factor-alpha and significantly improve tumor control upon anti-PD-L1 therapy in a B16.SIY tumor mouse model.46 More interestingly, most bacteria enriched in R in our study belong to pro-inflammatory gram-negative bacteria.There are several previous studies reporting the association of gram-negative bacteria, such as Akkermansia muciniphila, Alistipes, and Porphyromonas with better clinical response to immunotherapy.14,15,36 It is already evident that the increase of pro-inflammatory gramnegative bacteria in the gut of older patients is associated with systematic and chronic inflammation.It is largely mediated by more release of lipopolysaccharide. In another study, mice colonized with Prevotella copri displayed increased inflammation in dextran sulfate sodium-induced colitis.Prevotella copri was strongly correlated with the disease severity in rheumatoid arthritis, which is a systemic autoimmune disease with the proliferation of pro-inflammatory T cells.47 In the antitumor setting, the exacerbation of immune responses might assist the ability of immune surveillance in the patients.This is also consistent with the report that patients who develop immune-related adverse events are more sensitive to ICI treatment.48 Although the direct evidence on the linkage between the inflammation and ICI benefit in the population still needs to be investigated, it is not out of expectation that systemic antitumor immune responses are diverse in patients with different gut microbiome composition.Gut microbiota thus becomes an intrinsic biosensor to sensitize anti-PD-1 treatment, which in turn leads to different clinical outcomes.This can be partially supported by the results from immune signature exploration in our study.Previous studies already highlighted the roles of PD-1 in reshaping immune responses in the mice.49 ICI therapy, to some extent, facilitates the investigation on PD-1 in the regulation of human immunity.By using multi-color flow cytometry, immune signatures including immune cell subsets and their function-related signatures have been detected.Furthermore, correlation analyses were performed between individual immune signatures and microbiota diversity.Among multiple signatures, the frequency and function of memory CD8 þ T cells are mostly dedicated.Memory CD8 þ T cells are a bunch of long-lived cells which are capable of rapid antigenspecific responses and play critical roles in immune recall responses. 50In addition to the percentage of CD8 þ Tm subsets such as effector memory T (Tem) cells and central memory T (Tcm) cells, the functional subset with Granzyme B production is also positively correlated with the diversity index, which can partially explain why patients with high microbiota diversity respond better to ICI therapy.The increase in Ki67 þ CD8 þ Tm in the patients with high microbiota diversity implies that gut microbiota also modulate the proliferation of certain immune cell populations.Recent studies already reported the effects of PD-1 blockade on the proliferation of CD8 þ T cells, which is beneficial for ICI treatment.51 ICOS and TIM3 expressions on specific T cells subsets are also related to antitumor immunity.52,53 Whereas CD8 þ T cells infiltrating in tumor microenvironment are thought to be the executors of tumor killing, circulating CD8 þ T cell subsets reported here are probably inclined to reflect the roles of gut microbiota on systematic immune responses.How peripheral memory CD8 þ T cell signatures affect the efficacy of ICI treatment will be investigated in the future.Once verified, they might become antitumor immune mediators against NSCLC. Our study shows that the diversity of gut microbiota is associated with favorable response to nivolumab therapy against NSCLC in Chinese cohorts.Considering the correlation between microbiota diversity and peripheral memory T cells and NK cells being reshaped upon ICI treatment, we propose that patients with more diverse gut microbiota at baseline benefit from ICI immunotherapy to a greater extent.This might be mediated by reprogramming systemic antitumor immune responses.The significance of our study lies in that the modulation of gut microbiota before the treatment might provide an alternative way to enhance the efficacy of ICI immunotherapy once validated in large cohort studies.A latest publication has introduced 11 bacterial strains from healthy donors' feces to induce interferon-g producing CD8 þ T cells in the intestine, which mediates effective antitumor immunity in a mouse model.54 Once clearly addressed, customized microbiome will exhibit great prospect as biotherapeutics in assistance with ICI treatment against advanced NSCLC.Project (17431906103), Shanghai Economic and Information Commission for oncology data platforms (201602010) and Shanghai Scientific Research Projects (14140902800).The authors thank Prof. Guo-ping Zhao for scientific suggestions on the project.Drs.Hong Li and Guoqing Zhang from Bigdata Center of Shanghai Insistute of Biological Science CAS helped to construct an integrative database with both clinical and multi-omics source data. Figure: 1 Figure 1 . Figure 1.Study scheme.

Probably not a substantial claim. It may be too vague to check against evidence.

What the paper did — it cites no source for this, so there is none to check. The quote shows it is in the paper.

§ The Diversity of Gut Microbiome is Associated With Favorable Responses to Anti-Programmed Death 1 Immunotherapy in Chinese Patients With NSCLC > Patient Characteristics Forty-two Chinese patients with advanced NSCLC were consented and enrolled.All patients were followedup until disease progression or death.Among them, 1 patient died after a single infusion of nivolumab.Two patients were lost during the follow-up study.Two patients discontinued therapy because of grade 3-4 adverse events.The remaining 37 patients were grouped according to clinical assessment.Twenty-three patients being evaluated as either partial response (n ¼ 5) or stable disease (n ¼ 18) were classified as R whereas the others (n ¼ 14) with disease progression after the first clinical assessment were classified as NR.Fecal samples at baseline were obtained from 25 patients, among whom 13 patients were R and 12 patients were NR (Fig. 1).Clinical information revealed that the majority of patients enrolled in the study were male (78.3%).All of the patients had received at least one or two-rounds of chemotherapy before enrollment.No difference was observed in sex, disease history, disease stage, and the treatment between R and NR patients except smoking history (p ¼ 0.008).Recent studies reported negative association between ATB prescription and clinical outcome of PD-1 inhibitors.15,32 When we examined ATB usage in our cohort, 11 of 37 patients (29.7%) were prescribed ATB within 2 month.However, the R/NR ratio with or without ATB usage was similar (Table 1).Therefore, smoking is the only clinical factor associated with the responses to nivolumab in our study.

Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

What the paper did — it cites no source for this, so there is none to check. The quote shows it is in the paper.

§ The Diversity of Gut Microbiome is Associated With Favorable Responses to Anti-Programmed Death 1 Immunotherapy in Chinese Patients With NSCLC > Patients

Probably not a substantial claim. The paper’s conclusions may not depend on it.

§ The Diversity of Gut Microbiome is Associated With Favorable Responses to Anti-Programmed Death 1 Immunotherapy in Chinese Patients With NSCLC > Discussion In the present study, we have investigated gut microbiota profiles in Chinese patients with advanced NSCLC receiving nivolumab treatment.Our results indicate that patients with better clinical responses exhibit higher diversity as well as a distinct gut microbiome composition.In addition, the association between gut microbiome diversity and systematic immune signatures highlights memory CD8 þ T cells and NK cells as potential mediators of antitumor immunity upon ICI treatment.The diversity of gut microbiome is defined as the number and abundance distribution of distinct types of micro-organisms colonizing within the gut.Abnormal diversity (dysbiosis) in the gut has been proven to be associated with human diseases, such as obesity, inflammatory bowel disease, cancer, etc. 20,21,33,34 Recently, Gopalakrishnan et al. 35 reported that patients with a high diversity of gut microbiome favored the clinical outcomes of anti-PD-1 immunotherapy in melanoma patients in the Western population.In our study, both alpha diversity and beta diversity at baseline are dramatically different between R and NR as well.A higher alpha diversity index represented by both the Shannon index and inverse Simpson indexes in R group suggests more species harboring in the gut, which is largely consistent with a differential composition of gut microbiome between R and NR.The higher the diversity of gut microbiota is, the longer PFS the patients undergo.Visualized beta diversity through PCoA analysis of 16S sequencing also displays apparently two clusters based on clinical responses to nivolumab treatment.Our results thus show that regardless of genetic background and geographic location with different lifestyles and diet styles, the diversity of gut microbiota might be a common benefit factor to ICI treatment.In our opinion, this is rational.It is widely accepted that gut microbiota modulates immune responses in a global way including antitumor immunity.36,37 Both innate and adaptive immune responses can be reshaped by gut microbiome.Although how the diversity of gut microbiota reshapes systemic immune responses is still not clearly addressed, microbiota-derived short chain fat acids and other metabolites should be good candidates to exert strong immune regulation.19,38 Further study will focus on the relevant metabolites analysis. Several clinical factors are reported to be involved in affecting the efficacy of ICI therapy, including smoking, previous treatment, and ATB usage.15,32,39 Among them, ATB is supposed to be the most relevant to gut microbiota.It has been reported previously that recent ATB usage in NSCLC patients was associated with shorter OS, although there was no difference in terms of objective response or PFS.32 In another study, ATB treatment is identified as the key factor significantly associated with both PFS and OS. 15 In our study, we retrospectively investigated ATB usage among 37 patients.The composition of gut microbiota at baseline between participants with or without ATB before first nivolumab infusion was similar (Supplementary Fig. 2), which excludes the possible effects of ATB on gut microbiota in our study.Median PFS was similar between ATB-treated and -naïve patients (median PFS: 126 days versus 117.9 days, p > 0.05) (Supplementary Fig. 3).One possibility lies in the number limitation in our study.Another possibility is that gut microbiome possess the ability of resilience.40 The majority of gut microbiota can be normalized after 1 week once ATB usage stops.41 We also observed that gut microbiota remained stable in longitudinal samples within one individual in our study.Such a trait might ensure patient long-term benefit from ICI treatment. Among all the clinical manifestations, smoking is the only factor that affects the responses to nivolumab treatment in our study.The roles of smoking on carcinogenesis refer to both cancer cell itself and immune responses of the host.It has been hypothesized that higher mutational burden could be induced by smoking, which has shown an increased response to ICI. 42,43 Median PFS was longer in current and former smokers compared with nonsmokers in nivolumab monotherapy for first-line treatment of advanced NSCLC as well.44 Smoking is reported to affect both innate and adaptive immunity, including memory T cells in the periphery.45 In our study, memory T cell profiles at baseline are similar between smoking and nonsmoking patients (Supplementary Fig. 4).This result confers that memory T cells are more likely affected by gut microbiota rather than other factors such as smoking.It has also been reported that chemotherapy could influence the gut microbiota of cancer patients largely due to the disruption of the intestinal barrier and subsequent bacteria translocation.In our study all the participants have received nivolumab therapy after failure of traditional chemotherapy.There was no difference in the interval from the date of last chemotherapy to that of first nivolumab infusion between R and NR groups (Supplementary Fig. 5, left).Correlation analysis showed that the diversity of gut microbiota was irrelevant to the time-off of chemotherapy in our cohort study as well (Supplementary Fig. 5, right). In addition to diversity, gut microbiome composition is also related to favorable responses to ICI treatment.In a pre-clinical study, gut commensal Bifidobacterium was shown to facilitate anti-PD-L1 efficacy.46 Clinical investigations reported that Faecalibacterium and some species belonging to Bacteroides genus (such as B. fragilis or B. thetaiotaomicron) are positively associated with the efficacy of PD-1 and CTLA-4 blockade, respectively.14,23 In our study, seven genera were identified different between R and NR.Ruminococcus_unclassified was enriched in NR whereas Bifidobacterium longum, Prevotella copri, and Alistipes putredinis were enriched in R. It has been shown in another study that Bifidobacterium longum could increase the production of pro-inflammatory cytokine tumor necrosis factor-alpha and significantly improve tumor control upon anti-PD-L1 therapy in a B16.SIY tumor mouse model.46 More interestingly, most bacteria enriched in R in our study belong to pro-inflammatory gram-negative bacteria.There are several previous studies reporting the association of gram-negative bacteria, such as Akkermansia muciniphila, Alistipes, and Porphyromonas with better clinical response to immunotherapy.14,15,36 It is already evident that the increase of pro-inflammatory gramnegative bacteria in the gut of older patients is associated with systematic and chronic inflammation.It is largely mediated by more release of lipopolysaccharide. In another study, mice colonized with Prevotella copri displayed increased inflammation in dextran sulfate sodium-induced colitis.Prevotella copri was strongly correlated with the disease severity in rheumatoid arthritis, which is a systemic autoimmune disease with the proliferation of pro-inflammatory T cells.47 In the antitumor setting, the exacerbation of immune responses might assist the ability of immune surveillance in the patients.This is also consistent with the report that patients who develop immune-related adverse events are more sensitive to ICI treatment.48 Although the direct evidence on the linkage between the inflammation and ICI benefit in the population still needs to be investigated, it is not out of expectation that systemic antitumor immune responses are diverse in patients with different gut microbiome composition.Gut microbiota thus becomes an intrinsic biosensor to sensitize anti-PD-1 treatment, which in turn leads to different clinical outcomes.This can be partially supported by the results from immune signature exploration in our study.Previous studies already highlighted the roles of PD-1 in reshaping immune responses in the mice.49 ICI therapy, to some extent, facilitates the investigation on PD-1 in the regulation of human immunity.By using multi-color flow cytometry, immune signatures including immune cell subsets and their function-related signatures have been detected.Furthermore, correlation analyses were performed between individual immune signatures and microbiota diversity.Among multiple signatures, the frequency and function of memory CD8 þ T cells are mostly dedicated.Memory CD8 þ T cells are a bunch of long-lived cells which are capable of rapid antigenspecific responses and play critical roles in immune recall responses. 50In addition to the percentage of CD8 þ Tm subsets such as effector memory T (Tem) cells and central memory T (Tcm) cells, the functional subset with Granzyme B production is also positively correlated with the diversity index, which can partially explain why patients with high microbiota diversity respond better to ICI therapy.The increase in Ki67 þ CD8 þ Tm in the patients with high microbiota diversity implies that gut microbiota also modulate the proliferation of certain immune cell populations.Recent studies already reported the effects of PD-1 blockade on the proliferation of CD8 þ T cells, which is beneficial for ICI treatment.51 ICOS and TIM3 expressions on specific T cells subsets are also related to antitumor immunity.52,53 Whereas CD8 þ T cells infiltrating in tumor microenvironment are thought to be the executors of tumor killing, circulating CD8 þ T cell subsets reported here are probably inclined to reflect the roles of gut microbiota on systematic immune responses.How peripheral memory CD8 þ T cell signatures affect the efficacy of ICI treatment will be investigated in the future.Once verified, they might become antitumor immune mediators against NSCLC. Our study shows that the diversity of gut microbiota is associated with favorable response to nivolumab therapy against NSCLC in Chinese cohorts.Considering the correlation between microbiota diversity and peripheral memory T cells and NK cells being reshaped upon ICI treatment, we propose that patients with more diverse gut microbiota at baseline benefit from ICI immunotherapy to a greater extent.This might be mediated by reprogramming systemic antitumor immune responses.The significance of our study lies in that the modulation of gut microbiota before the treatment might provide an alternative way to enhance the efficacy of ICI immunotherapy once validated in large cohort studies.A latest publication has introduced 11 bacterial strains from healthy donors' feces to induce interferon-g producing CD8 þ T cells in the intestine, which mediates effective antitumor immunity in a mouse model.54 Once clearly addressed, customized microbiome will exhibit great prospect as biotherapeutics in assistance with ICI treatment against advanced NSCLC.Project (17431906103), Shanghai Economic and Information Commission for oncology data platforms (201602010) and Shanghai Scientific Research Projects (14140902800).The authors thank Prof. Guo-ping Zhao for scientific suggestions on the project.Drs.Hong Li and Guoqing Zhang from Bigdata Center of Shanghai Insistute of Biological Science CAS helped to construct an integrative database with both clinical and multi-omics source data. Figure: 1 Figure 1 . Figure 1.Study scheme.

Probably not a substantial claim. The paper’s conclusions may not depend on it.

A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

§ The Diversity of Gut Microbiome is Associated With Favorable Responses to Anti-Programmed Death 1 Immunotherapy in Chinese Patients With NSCLC > Discussion In the present study, we have investigated gut microbiota profiles in Chinese patients with advanced NSCLC receiving nivolumab treatment.Our results indicate that patients with better clinical responses exhibit higher diversity as well as a distinct gut microbiome composition.In addition, the association between gut microbiome diversity and systematic immune signatures highlights memory CD8 þ T cells and NK cells as potential mediators of antitumor immunity upon ICI treatment.The diversity of gut microbiome is defined as the number and abundance distribution of distinct types of micro-organisms colonizing within the gut.Abnormal diversity (dysbiosis) in the gut has been proven to be associated with human diseases, such as obesity, inflammatory bowel disease, cancer, etc. 20,21,33,34 Recently, Gopalakrishnan et al. 35 reported that patients with a high diversity of gut microbiome favored the clinical outcomes of anti-PD-1 immunotherapy in melanoma patients in the Western population.In our study, both alpha diversity and beta diversity at baseline are dramatically different between R and NR as well.A higher alpha diversity index represented by both the Shannon index and inverse Simpson indexes in R group suggests more species harboring in the gut, which is largely consistent with a differential composition of gut microbiome between R and NR.The higher the diversity of gut microbiota is, the longer PFS the patients undergo.Visualized beta diversity through PCoA analysis of 16S sequencing also displays apparently two clusters based on clinical responses to nivolumab treatment.Our results thus show that regardless of genetic background and geographic location with different lifestyles and diet styles, the diversity of gut microbiota might be a common benefit factor to ICI treatment.In our opinion, this is rational.It is widely accepted that gut microbiota modulates immune responses in a global way including antitumor immunity.36,37 Both innate and adaptive immune responses can be reshaped by gut microbiome.Although how the diversity of gut microbiota reshapes systemic immune responses is still not clearly addressed, microbiota-derived short chain fat acids and other metabolites should be good candidates to exert strong immune regulation.19,38 Further study will focus on the relevant metabolites analysis. Several clinical factors are reported to be involved in affecting the efficacy of ICI therapy, including smoking, previous treatment, and ATB usage.15,32,39 Among them, ATB is supposed to be the most relevant to gut microbiota.It has been reported previously that recent ATB usage in NSCLC patients was associated with shorter OS, although there was no difference in terms of objective response or PFS.32 In another study, ATB treatment is identified as the key factor significantly associated with both PFS and OS. 15 In our study, we retrospectively investigated ATB usage among 37 patients.The composition of gut microbiota at baseline between participants with or without ATB before first nivolumab infusion was similar (Supplementary Fig. 2), which excludes the possible effects of ATB on gut microbiota in our study.Median PFS was similar between ATB-treated and -naïve patients (median PFS: 126 days versus 117.9 days, p > 0.05) (Supplementary Fig. 3).One possibility lies in the number limitation in our study.Another possibility is that gut microbiome possess the ability of resilience.40 The majority of gut microbiota can be normalized after 1 week once ATB usage stops.41 We also observed that gut microbiota remained stable in longitudinal samples within one individual in our study.Such a trait might ensure patient long-term benefit from ICI treatment. Among all the clinical manifestations, smoking is the only factor that affects the responses to nivolumab treatment in our study.The roles of smoking on carcinogenesis refer to both cancer cell itself and immune responses of the host.It has been hypothesized that higher mutational burden could be induced by smoking, which has shown an increased response to ICI. 42,43 Median PFS was longer in current and former smokers compared with nonsmokers in nivolumab monotherapy for first-line treatment of advanced NSCLC as well.44 Smoking is reported to affect both innate and adaptive immunity, including memory T cells in the periphery.45 In our study, memory T cell profiles at baseline are similar between smoking and nonsmoking patients (Supplementary Fig. 4).This result confers that memory T cells are more likely affected by gut microbiota rather than other factors such as smoking.It has also been reported that chemotherapy could influence the gut microbiota of cancer patients largely due to the disruption of the intestinal barrier and subsequent bacteria translocation.In our study all the participants have received nivolumab therapy after failure of traditional chemotherapy.There was no difference in the interval from the date of last chemotherapy to that of first nivolumab infusion between R and NR groups (Supplementary Fig. 5, left).Correlation analysis showed that the diversity of gut microbiota was irrelevant to the time-off of chemotherapy in our cohort study as well (Supplementary Fig. 5, right). In addition to diversity, gut microbiome composition is also related to favorable responses to ICI treatment.In a pre-clinical study, gut commensal Bifidobacterium was shown to facilitate anti-PD-L1 efficacy.46 Clinical investigations reported that Faecalibacterium and some species belonging to Bacteroides genus (such as B. fragilis or B. thetaiotaomicron) are positively associated with the efficacy of PD-1 and CTLA-4 blockade, respectively.14,23 In our study, seven genera were identified different between R and NR.Ruminococcus_unclassified was enriched in NR whereas Bifidobacterium longum, Prevotella copri, and Alistipes putredinis were enriched in R. It has been shown in another study that Bifidobacterium longum could increase the production of pro-inflammatory cytokine tumor necrosis factor-alpha and significantly improve tumor control upon anti-PD-L1 therapy in a B16.SIY tumor mouse model.46 More interestingly, most bacteria enriched in R in our study belong to pro-inflammatory gram-negative bacteria.There are several previous studies reporting the association of gram-negative bacteria, such as Akkermansia muciniphila, Alistipes, and Porphyromonas with better clinical response to immunotherapy.14,15,36 It is already evident that the increase of pro-inflammatory gramnegative bacteria in the gut of older patients is associated with systematic and chronic inflammation.It is largely mediated by more release of lipopolysaccharide. In another study, mice colonized with Prevotella copri displayed increased inflammation in dextran sulfate sodium-induced colitis.Prevotella copri was strongly correlated with the disease severity in rheumatoid arthritis, which is a systemic autoimmune disease with the proliferation of pro-inflammatory T cells.47 In the antitumor setting, the exacerbation of immune responses might assist the ability of immune surveillance in the patients.This is also consistent with the report that patients who develop immune-related adverse events are more sensitive to ICI treatment.48 Although the direct evidence on the linkage between the inflammation and ICI benefit in the population still needs to be investigated, it is not out of expectation that systemic antitumor immune responses are diverse in patients with different gut microbiome composition.Gut microbiota thus becomes an intrinsic biosensor to sensitize anti-PD-1 treatment, which in turn leads to different clinical outcomes.This can be partially supported by the results from immune signature exploration in our study.Previous studies already highlighted the roles of PD-1 in reshaping immune responses in the mice.49 ICI therapy, to some extent, facilitates the investigation on PD-1 in the regulation of human immunity.By using multi-color flow cytometry, immune signatures including immune cell subsets and their function-related signatures have been detected.Furthermore, correlation analyses were performed between individual immune signatures and microbiota diversity.Among multiple signatures, the frequency and function of memory CD8 þ T cells are mostly dedicated.Memory CD8 þ T cells are a bunch of long-lived cells which are capable of rapid antigenspecific responses and play critical roles in immune recall responses. 50In addition to the percentage of CD8 þ Tm subsets such as effector memory T (Tem) cells and central memory T (Tcm) cells, the functional subset with Granzyme B production is also positively correlated with the diversity index, which can partially explain why patients with high microbiota diversity respond better to ICI therapy.The increase in Ki67 þ CD8 þ Tm in the patients with high microbiota diversity implies that gut microbiota also modulate the proliferation of certain immune cell populations.Recent studies already reported the effects of PD-1 blockade on the proliferation of CD8 þ T cells, which is beneficial for ICI treatment.51 ICOS and TIM3 expressions on specific T cells subsets are also related to antitumor immunity.52,53 Whereas CD8 þ T cells infiltrating in tumor microenvironment are thought to be the executors of tumor killing, circulating CD8 þ T cell subsets reported here are probably inclined to reflect the roles of gut microbiota on systematic immune responses.How peripheral memory CD8 þ T cell signatures affect the efficacy of ICI treatment will be investigated in the future.Once verified, they might become antitumor immune mediators against NSCLC. Our study shows that the diversity of gut microbiota is associated with favorable response to nivolumab therapy against NSCLC in Chinese cohorts.Considering the correlation between microbiota diversity and peripheral memory T cells and NK cells being reshaped upon ICI treatment, we propose that patients with more diverse gut microbiota at baseline benefit from ICI immunotherapy to a greater extent.This might be mediated by reprogramming systemic antitumor immune responses.The significance of our study lies in that the modulation of gut microbiota before the treatment might provide an alternative way to enhance the efficacy of ICI immunotherapy once validated in large cohort studies.A latest publication has introduced 11 bacterial strains from healthy donors' feces to induce interferon-g producing CD8 þ T cells in the intestine, which mediates effective antitumor immunity in a mouse model.54 Once clearly addressed, customized microbiome will exhibit great prospect as biotherapeutics in assistance with ICI treatment against advanced NSCLC.Project (17431906103), Shanghai Economic and Information Commission for oncology data platforms (201602010) and Shanghai Scientific Research Projects (14140902800).The authors thank Prof. Guo-ping Zhao for scientific suggestions on the project.Drs.Hong Li and Guoqing Zhang from Bigdata Center of Shanghai Insistute of Biological Science CAS helped to construct an integrative database with both clinical and multi-omics source data. Figure: 1 Figure 1 . Figure 1.Study scheme.

Probably not a substantial claim. It may be too vague to check against evidence.

§ The Diversity of Gut Microbiome is Associated With Favorable Responses to Anti-Programmed Death 1 Immunotherapy in Chinese Patients With NSCLC > Discussion In the present study, we have investigated gut microbiota profiles in Chinese patients with advanced NSCLC receiving nivolumab treatment.Our results indicate that patients with better clinical responses exhibit higher diversity as well as a distinct gut microbiome composition.In addition, the association between gut microbiome diversity and systematic immune signatures highlights memory CD8 þ T cells and NK cells as potential mediators of antitumor immunity upon ICI treatment.The diversity of gut microbiome is defined as the number and abundance distribution of distinct types of micro-organisms colonizing within the gut.Abnormal diversity (dysbiosis) in the gut has been proven to be associated with human diseases, such as obesity, inflammatory bowel disease, cancer, etc. 20,21,33,34 Recently, Gopalakrishnan et al. 35 reported that patients with a high diversity of gut microbiome favored the clinical outcomes of anti-PD-1 immunotherapy in melanoma patients in the Western population.In our study, both alpha diversity and beta diversity at baseline are dramatically different between R and NR as well.A higher alpha diversity index represented by both the Shannon index and inverse Simpson indexes in R group suggests more species harboring in the gut, which is largely consistent with a differential composition of gut microbiome between R and NR.The higher the diversity of gut microbiota is, the longer PFS the patients undergo.Visualized beta diversity through PCoA analysis of 16S sequencing also displays apparently two clusters based on clinical responses to nivolumab treatment.Our results thus show that regardless of genetic background and geographic location with different lifestyles and diet styles, the diversity of gut microbiota might be a common benefit factor to ICI treatment.In our opinion, this is rational.It is widely accepted that gut microbiota modulates immune responses in a global way including antitumor immunity.36,37 Both innate and adaptive immune responses can be reshaped by gut microbiome.Although how the diversity of gut microbiota reshapes systemic immune responses is still not clearly addressed, microbiota-derived short chain fat acids and other metabolites should be good candidates to exert strong immune regulation.19,38 Further study will focus on the relevant metabolites analysis. Several clinical factors are reported to be involved in affecting the efficacy of ICI therapy, including smoking, previous treatment, and ATB usage.15,32,39 Among them, ATB is supposed to be the most relevant to gut microbiota.It has been reported previously that recent ATB usage in NSCLC patients was associated with shorter OS, although there was no difference in terms of objective response or PFS.32 In another study, ATB treatment is identified as the key factor significantly associated with both PFS and OS. 15 In our study, we retrospectively investigated ATB usage among 37 patients.The composition of gut microbiota at baseline between participants with or without ATB before first nivolumab infusion was similar (Supplementary Fig. 2), which excludes the possible effects of ATB on gut microbiota in our study.Median PFS was similar between ATB-treated and -naïve patients (median PFS: 126 days versus 117.9 days, p > 0.05) (Supplementary Fig. 3).One possibility lies in the number limitation in our study.Another possibility is that gut microbiome possess the ability of resilience.40 The majority of gut microbiota can be normalized after 1 week once ATB usage stops.41 We also observed that gut microbiota remained stable in longitudinal samples within one individual in our study.Such a trait might ensure patient long-term benefit from ICI treatment. Among all the clinical manifestations, smoking is the only factor that affects the responses to nivolumab treatment in our study.The roles of smoking on carcinogenesis refer to both cancer cell itself and immune responses of the host.It has been hypothesized that higher mutational burden could be induced by smoking, which has shown an increased response to ICI. 42,43 Median PFS was longer in current and former smokers compared with nonsmokers in nivolumab monotherapy for first-line treatment of advanced NSCLC as well.44 Smoking is reported to affect both innate and adaptive immunity, including memory T cells in the periphery.45 In our study, memory T cell profiles at baseline are similar between smoking and nonsmoking patients (Supplementary Fig. 4).This result confers that memory T cells are more likely affected by gut microbiota rather than other factors such as smoking.It has also been reported that chemotherapy could influence the gut microbiota of cancer patients largely due to the disruption of the intestinal barrier and subsequent bacteria translocation.In our study all the participants have received nivolumab therapy after failure of traditional chemotherapy.There was no difference in the interval from the date of last chemotherapy to that of first nivolumab infusion between R and NR groups (Supplementary Fig. 5, left).Correlation analysis showed that the diversity of gut microbiota was irrelevant to the time-off of chemotherapy in our cohort study as well (Supplementary Fig. 5, right). In addition to diversity, gut microbiome composition is also related to favorable responses to ICI treatment.In a pre-clinical study, gut commensal Bifidobacterium was shown to facilitate anti-PD-L1 efficacy.46 Clinical investigations reported that Faecalibacterium and some species belonging to Bacteroides genus (such as B. fragilis or B. thetaiotaomicron) are positively associated with the efficacy of PD-1 and CTLA-4 blockade, respectively.14,23 In our study, seven genera were identified different between R and NR.Ruminococcus_unclassified was enriched in NR whereas Bifidobacterium longum, Prevotella copri, and Alistipes putredinis were enriched in R. It has been shown in another study that Bifidobacterium longum could increase the production of pro-inflammatory cytokine tumor necrosis factor-alpha and significantly improve tumor control upon anti-PD-L1 therapy in a B16.SIY tumor mouse model.46 More interestingly, most bacteria enriched in R in our study belong to pro-inflammatory gram-negative bacteria.There are several previous studies reporting the association of gram-negative bacteria, such as Akkermansia muciniphila, Alistipes, and Porphyromonas with better clinical response to immunotherapy.14,15,36 It is already evident that the increase of pro-inflammatory gramnegative bacteria in the gut of older patients is associated with systematic and chronic inflammation.It is largely mediated by more release of lipopolysaccharide. In another study, mice colonized with Prevotella copri displayed increased inflammation in dextran sulfate sodium-induced colitis.Prevotella copri was strongly correlated with the disease severity in rheumatoid arthritis, which is a systemic autoimmune disease with the proliferation of pro-inflammatory T cells.47 In the antitumor setting, the exacerbation of immune responses might assist the ability of immune surveillance in the patients.This is also consistent with the report that patients who develop immune-related adverse events are more sensitive to ICI treatment.48 Although the direct evidence on the linkage between the inflammation and ICI benefit in the population still needs to be investigated, it is not out of expectation that systemic antitumor immune responses are diverse in patients with different gut microbiome composition.Gut microbiota thus becomes an intrinsic biosensor to sensitize anti-PD-1 treatment, which in turn leads to different clinical outcomes.This can be partially supported by the results from immune signature exploration in our study.Previous studies already highlighted the roles of PD-1 in reshaping immune responses in the mice.49 ICI therapy, to some extent, facilitates the investigation on PD-1 in the regulation of human immunity.By using multi-color flow cytometry, immune signatures including immune cell subsets and their function-related signatures have been detected.Furthermore, correlation analyses were performed between individual immune signatures and microbiota diversity.Among multiple signatures, the frequency and function of memory CD8 þ T cells are mostly dedicated.Memory CD8 þ T cells are a bunch of long-lived cells which are capable of rapid antigenspecific responses and play critical roles in immune recall responses. 50In addition to the percentage of CD8 þ Tm subsets such as effector memory T (Tem) cells and central memory T (Tcm) cells, the functional subset with Granzyme B production is also positively correlated with the diversity index, which can partially explain why patients with high microbiota diversity respond better to ICI therapy.The increase in Ki67 þ CD8 þ Tm in the patients with high microbiota diversity implies that gut microbiota also modulate the proliferation of certain immune cell populations.Recent studies already reported the effects of PD-1 blockade on the proliferation of CD8 þ T cells, which is beneficial for ICI treatment.51 ICOS and TIM3 expressions on specific T cells subsets are also related to antitumor immunity.52,53 Whereas CD8 þ T cells infiltrating in tumor microenvironment are thought to be the executors of tumor killing, circulating CD8 þ T cell subsets reported here are probably inclined to reflect the roles of gut microbiota on systematic immune responses.How peripheral memory CD8 þ T cell signatures affect the efficacy of ICI treatment will be investigated in the future.Once verified, they might become antitumor immune mediators against NSCLC. Our study shows that the diversity of gut microbiota is associated with favorable response to nivolumab therapy against NSCLC in Chinese cohorts.Considering the correlation between microbiota diversity and peripheral memory T cells and NK cells being reshaped upon ICI treatment, we propose that patients with more diverse gut microbiota at baseline benefit from ICI immunotherapy to a greater extent.This might be mediated by reprogramming systemic antitumor immune responses.The significance of our study lies in that the modulation of gut microbiota before the treatment might provide an alternative way to enhance the efficacy of ICI immunotherapy once validated in large cohort studies.A latest publication has introduced 11 bacterial strains from healthy donors' feces to induce interferon-g producing CD8 þ T cells in the intestine, which mediates effective antitumor immunity in a mouse model.54 Once clearly addressed, customized microbiome will exhibit great prospect as biotherapeutics in assistance with ICI treatment against advanced NSCLC.Project (17431906103), Shanghai Economic and Information Commission for oncology data platforms (201602010) and Shanghai Scientific Research Projects (14140902800).The authors thank Prof. Guo-ping Zhao for scientific suggestions on the project.Drs.Hong Li and Guoqing Zhang from Bigdata Center of Shanghai Insistute of Biological Science CAS helped to construct an integrative database with both clinical and multi-omics source data. Figure: 1 Figure 1 . Figure 1.Study scheme.

Probably not a substantial claim. The paper’s conclusions may not depend on it.

§ The Diversity of Gut Microbiome is Associated With Favorable Responses to Anti-Programmed Death 1 Immunotherapy in Chinese Patients With NSCLC > Discussion In the present study, we have investigated gut microbiota profiles in Chinese patients with advanced NSCLC receiving nivolumab treatment.Our results indicate that patients with better clinical responses exhibit higher diversity as well as a distinct gut microbiome composition.In addition, the association between gut microbiome diversity and systematic immune signatures highlights memory CD8 þ T cells and NK cells as potential mediators of antitumor immunity upon ICI treatment.The diversity of gut microbiome is defined as the number and abundance distribution of distinct types of micro-organisms colonizing within the gut.Abnormal diversity (dysbiosis) in the gut has been proven to be associated with human diseases, such as obesity, inflammatory bowel disease, cancer, etc. 20,21,33,34 Recently, Gopalakrishnan et al. 35 reported that patients with a high diversity of gut microbiome favored the clinical outcomes of anti-PD-1 immunotherapy in melanoma patients in the Western population.In our study, both alpha diversity and beta diversity at baseline are dramatically different between R and NR as well.A higher alpha diversity index represented by both the Shannon index and inverse Simpson indexes in R group suggests more species harboring in the gut, which is largely consistent with a differential composition of gut microbiome between R and NR.The higher the diversity of gut microbiota is, the longer PFS the patients undergo.Visualized beta diversity through PCoA analysis of 16S sequencing also displays apparently two clusters based on clinical responses to nivolumab treatment.Our results thus show that regardless of genetic background and geographic location with different lifestyles and diet styles, the diversity of gut microbiota might be a common benefit factor to ICI treatment.In our opinion, this is rational.It is widely accepted that gut microbiota modulates immune responses in a global way including antitumor immunity.36,37 Both innate and adaptive immune responses can be reshaped by gut microbiome.Although how the diversity of gut microbiota reshapes systemic immune responses is still not clearly addressed, microbiota-derived short chain fat acids and other metabolites should be good candidates to exert strong immune regulation.19,38 Further study will focus on the relevant metabolites analysis. Several clinical factors are reported to be involved in affecting the efficacy of ICI therapy, including smoking, previous treatment, and ATB usage.15,32,39 Among them, ATB is supposed to be the most relevant to gut microbiota.It has been reported previously that recent ATB usage in NSCLC patients was associated with shorter OS, although there was no difference in terms of objective response or PFS.32 In another study, ATB treatment is identified as the key factor significantly associated with both PFS and OS. 15 In our study, we retrospectively investigated ATB usage among 37 patients.The composition of gut microbiota at baseline between participants with or without ATB before first nivolumab infusion was similar (Supplementary Fig. 2), which excludes the possible effects of ATB on gut microbiota in our study.Median PFS was similar between ATB-treated and -naïve patients (median PFS: 126 days versus 117.9 days, p > 0.05) (Supplementary Fig. 3).One possibility lies in the number limitation in our study.Another possibility is that gut microbiome possess the ability of resilience.40 The majority of gut microbiota can be normalized after 1 week once ATB usage stops.41 We also observed that gut microbiota remained stable in longitudinal samples within one individual in our study.Such a trait might ensure patient long-term benefit from ICI treatment. Among all the clinical manifestations, smoking is the only factor that affects the responses to nivolumab treatment in our study.The roles of smoking on carcinogenesis refer to both cancer cell itself and immune responses of the host.It has been hypothesized that higher mutational burden could be induced by smoking, which has shown an increased response to ICI. 42,43 Median PFS was longer in current and former smokers compared with nonsmokers in nivolumab monotherapy for first-line treatment of advanced NSCLC as well.44 Smoking is reported to affect both innate and adaptive immunity, including memory T cells in the periphery.45 In our study, memory T cell profiles at baseline are similar between smoking and nonsmoking patients (Supplementary Fig. 4).This result confers that memory T cells are more likely affected by gut microbiota rather than other factors such as smoking.It has also been reported that chemotherapy could influence the gut microbiota of cancer patients largely due to the disruption of the intestinal barrier and subsequent bacteria translocation.In our study all the participants have received nivolumab therapy after failure of traditional chemotherapy.There was no difference in the interval from the date of last chemotherapy to that of first nivolumab infusion between R and NR groups (Supplementary Fig. 5, left).Correlation analysis showed that the diversity of gut microbiota was irrelevant to the time-off of chemotherapy in our cohort study as well (Supplementary Fig. 5, right). In addition to diversity, gut microbiome composition is also related to favorable responses to ICI treatment.In a pre-clinical study, gut commensal Bifidobacterium was shown to facilitate anti-PD-L1 efficacy.46 Clinical investigations reported that Faecalibacterium and some species belonging to Bacteroides genus (such as B. fragilis or B. thetaiotaomicron) are positively associated with the efficacy of PD-1 and CTLA-4 blockade, respectively.14,23 In our study, seven genera were identified different between R and NR.Ruminococcus_unclassified was enriched in NR whereas Bifidobacterium longum, Prevotella copri, and Alistipes putredinis were enriched in R. It has been shown in another study that Bifidobacterium longum could increase the production of pro-inflammatory cytokine tumor necrosis factor-alpha and significantly improve tumor control upon anti-PD-L1 therapy in a B16.SIY tumor mouse model.46 More interestingly, most bacteria enriched in R in our study belong to pro-inflammatory gram-negative bacteria.There are several previous studies reporting the association of gram-negative bacteria, such as Akkermansia muciniphila, Alistipes, and Porphyromonas with better clinical response to immunotherapy.14,15,36 It is already evident that the increase of pro-inflammatory gramnegative bacteria in the gut of older patients is associated with systematic and chronic inflammation.It is largely mediated by more release of lipopolysaccharide. In another study, mice colonized with Prevotella copri displayed increased inflammation in dextran sulfate sodium-induced colitis.Prevotella copri was strongly correlated with the disease severity in rheumatoid arthritis, which is a systemic autoimmune disease with the proliferation of pro-inflammatory T cells.47 In the antitumor setting, the exacerbation of immune responses might assist the ability of immune surveillance in the patients.This is also consistent with the report that patients who develop immune-related adverse events are more sensitive to ICI treatment.48 Although the direct evidence on the linkage between the inflammation and ICI benefit in the population still needs to be investigated, it is not out of expectation that systemic antitumor immune responses are diverse in patients with different gut microbiome composition.Gut microbiota thus becomes an intrinsic biosensor to sensitize anti-PD-1 treatment, which in turn leads to different clinical outcomes.This can be partially supported by the results from immune signature exploration in our study.Previous studies already highlighted the roles of PD-1 in reshaping immune responses in the mice.49 ICI therapy, to some extent, facilitates the investigation on PD-1 in the regulation of human immunity.By using multi-color flow cytometry, immune signatures including immune cell subsets and their function-related signatures have been detected.Furthermore, correlation analyses were performed between individual immune signatures and microbiota diversity.Among multiple signatures, the frequency and function of memory CD8 þ T cells are mostly dedicated.Memory CD8 þ T cells are a bunch of long-lived cells which are capable of rapid antigenspecific responses and play critical roles in immune recall responses. 50In addition to the percentage of CD8 þ Tm subsets such as effector memory T (Tem) cells and central memory T (Tcm) cells, the functional subset with Granzyme B production is also positively correlated with the diversity index, which can partially explain why patients with high microbiota diversity respond better to ICI therapy.The increase in Ki67 þ CD8 þ Tm in the patients with high microbiota diversity implies that gut microbiota also modulate the proliferation of certain immune cell populations.Recent studies already reported the effects of PD-1 blockade on the proliferation of CD8 þ T cells, which is beneficial for ICI treatment.51 ICOS and TIM3 expressions on specific T cells subsets are also related to antitumor immunity.52,53 Whereas CD8 þ T cells infiltrating in tumor microenvironment are thought to be the executors of tumor killing, circulating CD8 þ T cell subsets reported here are probably inclined to reflect the roles of gut microbiota on systematic immune responses.How peripheral memory CD8 þ T cell signatures affect the efficacy of ICI treatment will be investigated in the future.Once verified, they might become antitumor immune mediators against NSCLC. Our study shows that the diversity of gut microbiota is associated with favorable response to nivolumab therapy against NSCLC in Chinese cohorts.Considering the correlation between microbiota diversity and peripheral memory T cells and NK cells being reshaped upon ICI treatment, we propose that patients with more diverse gut microbiota at baseline benefit from ICI immunotherapy to a greater extent.This might be mediated by reprogramming systemic antitumor immune responses.The significance of our study lies in that the modulation of gut microbiota before the treatment might provide an alternative way to enhance the efficacy of ICI immunotherapy once validated in large cohort studies.A latest publication has introduced 11 bacterial strains from healthy donors' feces to induce interferon-g producing CD8 þ T cells in the intestine, which mediates effective antitumor immunity in a mouse model.54 Once clearly addressed, customized microbiome will exhibit great prospect as biotherapeutics in assistance with ICI treatment against advanced NSCLC.Project (17431906103), Shanghai Economic and Information Commission for oncology data platforms (201602010) and Shanghai Scientific Research Projects (14140902800).The authors thank Prof. Guo-ping Zhao for scientific suggestions on the project.Drs.Hong Li and Guoqing Zhang from Bigdata Center of Shanghai Insistute of Biological Science CAS helped to construct an integrative database with both clinical and multi-omics source data. Figure: 1 Figure 1 . Figure 1.Study scheme.

Probably not a substantial claim. The paper’s conclusions may not depend on it.

§ The Diversity of Gut Microbiome is Associated With Favorable Responses to Anti-Programmed Death 1 Immunotherapy in Chinese Patients With NSCLC > Introduction Advanced NSCLC is an incurable disease with a poor prognosis and is the leading cause of cancer-related death. 1 With impressive advancement in immune checkpoint inhibitor (ICI) therapies targeting programmed death 1 (PD-1) and programmed death ligand 1 (PD-L1) axis against multiple malignancies, these agents exhibit clinical efficacy against patients with advanced NSCLC both in second-line and first-line treatment when compared to conventional chemotherapy.At present, five therapeutic monoclonal antibodies targeting PD-1 or PD-L1 have been approved by the U.S. Food and Drug Administration (FDA) for the treatment of 11 cancer indications including NSCLC. 2 Nivolumab (Opdivo) is a fully human immunoglobulin G4 monoclonal antibody against PD-1.It blocks the binding of PD-1 to its ligand PD-L1 and restores the function of "exhausted" T cells in tumor sites.The unleashed lymphocytes exert killing ability, resulting in tumor shrinkage.3 It was firstly approved by the FDA in 2015 for the treatment of patients with advanced NSCLC after the failure of platinum-based chemotherapy, and by the National Medical Products Administration in 2018 after the phase III clinical trial CheckMate-078 for previously treated patients with NSCLC. 4 According to results from CheckMate-078, nivolumab yields superior overall survival (OS) compared with the docetaxel arm (median OS: 12 mo versus 9.6 mo). 5 However, only 20% of patients with advanced NSCLC benefit from the treatment, whereas up to 50% experience treatmentrelated adverse events in which grade 3-4 toxicities occur in 10% patients receiving nivolumab.6 Considering high cost of the agents, limited benefit population, and potential severe side effects, biomarkers for selecting patients with likely clinical efficacy from ICI treatment in advanced NSCLC would be of paramount importance. So far, PD-L1 expression on tumor cells is regarded as the most available biomarker to predict the efficacy of ICIs in NSCLC and other malignancies.A linear relationship has been reported between the proportion of ICI benefit and PD-L1 expression in NSCLC tumor sections.7,8 Nevertheless, the responses to ICI treatment have still been observed in those with low or undetectable levels of PD-L1 expression.9 To date, other biomarkers such as high tumor mutation burden, nonsynonymous mutations and neoantigens, CD8 þ T cell counts in tumor regions, defects of mismatch repair genes or high microsatellite instability, microbiota, and metabolic profiles are also supposed to be predictive for favorable responses to ICI treatment, among which gut microbiome property is of great value.[10][11][12][13][14][15][16][17] Human gut harbors more than 10 13 microbes, which outnumbers host cells 10-fold.More and more evidence supports the critical roles of gut microbiota in influencing human health in part through shaping systematic immune responses.18 Certain metabolites such as shortchain fatty acids and bile acid are considered to be key mediators.19 Dysbiosis of intestinal flora has been shown to be associated with multiple diseases.[20][21][22] Recent studies have also revealed that gut microbiota could mediate diverse responses to immunotherapy and might serve as a potential predictive biomarker.Gut commensals such as Bacteroides thetaiotaomicron or B. fragilis are predicable for anti-cytotoxic T-lymphocyte associated protein 4 (CTLA-4) treatment in a mouse melanoma model.23 In patients with melanoma receiving anti-PD-1 therapy, significant difference in the diversity and composition of gut microbiome was observed between the responders and nonresponders to the therapy.14 Germ-free mice receiving fecal transplantation from the responders tended to be more sensitive to anti-PD-1 treatment in contrast to those receiving feces from the nonresponders.14,15 In one cohort study enrolling 60 patients with advanced NSCLC and 40 with renal cell carcinoma undergoing PD-1 blockade therapy, Akkermansia muciniphila was reported to be overrepresented in the responders.15 Nevertheless, the previous studies were performed predominantly in the patients from Western countries.Gut microbiome structures have been elucidated remarkably variable among different races and ethnicities.24 Extrinsic factors, such as geographical location or diet and life styles, and intrinsic factors such as genetics or early-life exposures are main contributors to such divergences.[25][26][27] Therefore, it is still significant to reveal the characteristics of gut microbiota in regional patients with advanced NSCLC and the potential roles of gut microbiota in the prediction for the responses to ICI treatment.

Probably not a substantial claim. It may state two separate claims as one.

What the paper did — it cites no source for this, so there is none to check. The quote shows it is in the paper.

§ The Diversity of Gut Microbiome is Associated With Favorable Responses to Anti-Programmed Death 1 Immunotherapy in Chinese Patients With NSCLC > Patients

Probably not a substantial claim. The paper’s conclusions may not depend on it.

A conclusion the paper draws from its own results — it cites no other source for it, so there is none to check. The quote shows it is in the paper.

§ The Diversity of Gut Microbiome is Associated With Favorable Responses to Anti-Programmed Death 1 Immunotherapy in Chinese Patients With NSCLC > Discussion In the present study, we have investigated gut microbiota profiles in Chinese patients with advanced NSCLC receiving nivolumab treatment.Our results indicate that patients with better clinical responses exhibit higher diversity as well as a distinct gut microbiome composition.In addition, the association between gut microbiome diversity and systematic immune signatures highlights memory CD8 þ T cells and NK cells as potential mediators of antitumor immunity upon ICI treatment.The diversity of gut microbiome is defined as the number and abundance distribution of distinct types of micro-organisms colonizing within the gut.Abnormal diversity (dysbiosis) in the gut has been proven to be associated with human diseases, such as obesity, inflammatory bowel disease, cancer, etc. 20,21,33,34 Recently, Gopalakrishnan et al. 35 reported that patients with a high diversity of gut microbiome favored the clinical outcomes of anti-PD-1 immunotherapy in melanoma patients in the Western population.In our study, both alpha diversity and beta diversity at baseline are dramatically different between R and NR as well.A higher alpha diversity index represented by both the Shannon index and inverse Simpson indexes in R group suggests more species harboring in the gut, which is largely consistent with a differential composition of gut microbiome between R and NR.The higher the diversity of gut microbiota is, the longer PFS the patients undergo.Visualized beta diversity through PCoA analysis of 16S sequencing also displays apparently two clusters based on clinical responses to nivolumab treatment.Our results thus show that regardless of genetic background and geographic location with different lifestyles and diet styles, the diversity of gut microbiota might be a common benefit factor to ICI treatment.In our opinion, this is rational.It is widely accepted that gut microbiota modulates immune responses in a global way including antitumor immunity.36,37 Both innate and adaptive immune responses can be reshaped by gut microbiome.Although how the diversity of gut microbiota reshapes systemic immune responses is still not clearly addressed, microbiota-derived short chain fat acids and other metabolites should be good candidates to exert strong immune regulation.19,38 Further study will focus on the relevant metabolites analysis. Several clinical factors are reported to be involved in affecting the efficacy of ICI therapy, including smoking, previous treatment, and ATB usage.15,32,39 Among them, ATB is supposed to be the most relevant to gut microbiota.It has been reported previously that recent ATB usage in NSCLC patients was associated with shorter OS, although there was no difference in terms of objective response or PFS.32 In another study, ATB treatment is identified as the key factor significantly associated with both PFS and OS. 15 In our study, we retrospectively investigated ATB usage among 37 patients.The composition of gut microbiota at baseline between participants with or without ATB before first nivolumab infusion was similar (Supplementary Fig. 2), which excludes the possible effects of ATB on gut microbiota in our study.Median PFS was similar between ATB-treated and -naïve patients (median PFS: 126 days versus 117.9 days, p > 0.05) (Supplementary Fig. 3).One possibility lies in the number limitation in our study.Another possibility is that gut microbiome possess the ability of resilience.40 The majority of gut microbiota can be normalized after 1 week once ATB usage stops.41 We also observed that gut microbiota remained stable in longitudinal samples within one individual in our study.Such a trait might ensure patient long-term benefit from ICI treatment. Among all the clinical manifestations, smoking is the only factor that affects the responses to nivolumab treatment in our study.The roles of smoking on carcinogenesis refer to both cancer cell itself and immune responses of the host.It has been hypothesized that higher mutational burden could be induced by smoking, which has shown an increased response to ICI. 42,43 Median PFS was longer in current and former smokers compared with nonsmokers in nivolumab monotherapy for first-line treatment of advanced NSCLC as well.44 Smoking is reported to affect both innate and adaptive immunity, including memory T cells in the periphery.45 In our study, memory T cell profiles at baseline are similar between smoking and nonsmoking patients (Supplementary Fig. 4).This result confers that memory T cells are more likely affected by gut microbiota rather than other factors such as smoking.It has also been reported that chemotherapy could influence the gut microbiota of cancer patients largely due to the disruption of the intestinal barrier and subsequent bacteria translocation.In our study all the participants have received nivolumab therapy after failure of traditional chemotherapy.There was no difference in the interval from the date of last chemotherapy to that of first nivolumab infusion between R and NR groups (Supplementary Fig. 5, left).Correlation analysis showed that the diversity of gut microbiota was irrelevant to the time-off of chemotherapy in our cohort study as well (Supplementary Fig. 5, right). In addition to diversity, gut microbiome composition is also related to favorable responses to ICI treatment.In a pre-clinical study, gut commensal Bifidobacterium was shown to facilitate anti-PD-L1 efficacy.46 Clinical investigations reported that Faecalibacterium and some species belonging to Bacteroides genus (such as B. fragilis or B. thetaiotaomicron) are positively associated with the efficacy of PD-1 and CTLA-4 blockade, respectively.14,23 In our study, seven genera were identified different between R and NR.Ruminococcus_unclassified was enriched in NR whereas Bifidobacterium longum, Prevotella copri, and Alistipes putredinis were enriched in R. It has been shown in another study that Bifidobacterium longum could increase the production of pro-inflammatory cytokine tumor necrosis factor-alpha and significantly improve tumor control upon anti-PD-L1 therapy in a B16.SIY tumor mouse model.46 More interestingly, most bacteria enriched in R in our study belong to pro-inflammatory gram-negative bacteria.There are several previous studies reporting the association of gram-negative bacteria, such as Akkermansia muciniphila, Alistipes, and Porphyromonas with better clinical response to immunotherapy.14,15,36 It is already evident that the increase of pro-inflammatory gramnegative bacteria in the gut of older patients is associated with systematic and chronic inflammation.It is largely mediated by more release of lipopolysaccharide. In another study, mice colonized with Prevotella copri displayed increased inflammation in dextran sulfate sodium-induced colitis.Prevotella copri was strongly correlated with the disease severity in rheumatoid arthritis, which is a systemic autoimmune disease with the proliferation of pro-inflammatory T cells.47 In the antitumor setting, the exacerbation of immune responses might assist the ability of immune surveillance in the patients.This is also consistent with the report that patients who develop immune-related adverse events are more sensitive to ICI treatment.48 Although the direct evidence on the linkage between the inflammation and ICI benefit in the population still needs to be investigated, it is not out of expectation that systemic antitumor immune responses are diverse in patients with different gut microbiome composition.Gut microbiota thus becomes an intrinsic biosensor to sensitize anti-PD-1 treatment, which in turn leads to different clinical outcomes.This can be partially supported by the results from immune signature exploration in our study.Previous studies already highlighted the roles of PD-1 in reshaping immune responses in the mice.49 ICI therapy, to some extent, facilitates the investigation on PD-1 in the regulation of human immunity.By using multi-color flow cytometry, immune signatures including immune cell subsets and their function-related signatures have been detected.Furthermore, correlation analyses were performed between individual immune signatures and microbiota diversity.Among multiple signatures, the frequency and function of memory CD8 þ T cells are mostly dedicated.Memory CD8 þ T cells are a bunch of long-lived cells which are capable of rapid antigenspecific responses and play critical roles in immune recall responses. 50In addition to the percentage of CD8 þ Tm subsets such as effector memory T (Tem) cells and central memory T (Tcm) cells, the functional subset with Granzyme B production is also positively correlated with the diversity index, which can partially explain why patients with high microbiota diversity respond better to ICI therapy.The increase in Ki67 þ CD8 þ Tm in the patients with high microbiota diversity implies that gut microbiota also modulate the proliferation of certain immune cell populations.Recent studies already reported the effects of PD-1 blockade on the proliferation of CD8 þ T cells, which is beneficial for ICI treatment.51 ICOS and TIM3 expressions on specific T cells subsets are also related to antitumor immunity.52,53 Whereas CD8 þ T cells infiltrating in tumor microenvironment are thought to be the executors of tumor killing, circulating CD8 þ T cell subsets reported here are probably inclined to reflect the roles of gut microbiota on systematic immune responses.How peripheral memory CD8 þ T cell signatures affect the efficacy of ICI treatment will be investigated in the future.Once verified, they might become antitumor immune mediators against NSCLC. Our study shows that the diversity of gut microbiota is associated with favorable response to nivolumab therapy against NSCLC in Chinese cohorts.Considering the correlation between microbiota diversity and peripheral memory T cells and NK cells being reshaped upon ICI treatment, we propose that patients with more diverse gut microbiota at baseline benefit from ICI immunotherapy to a greater extent.This might be mediated by reprogramming systemic antitumor immune responses.The significance of our study lies in that the modulation of gut microbiota before the treatment might provide an alternative way to enhance the efficacy of ICI immunotherapy once validated in large cohort studies.A latest publication has introduced 11 bacterial strains from healthy donors' feces to induce interferon-g producing CD8 þ T cells in the intestine, which mediates effective antitumor immunity in a mouse model.54 Once clearly addressed, customized microbiome will exhibit great prospect as biotherapeutics in assistance with ICI treatment against advanced NSCLC.Project (17431906103), Shanghai Economic and Information Commission for oncology data platforms (201602010) and Shanghai Scientific Research Projects (14140902800).The authors thank Prof. Guo-ping Zhao for scientific suggestions on the project.Drs.Hong Li and Guoqing Zhang from Bigdata Center of Shanghai Insistute of Biological Science CAS helped to construct an integrative database with both clinical and multi-omics source data. Figure: 1 Figure 1 . Figure 1.Study scheme.

It may state two separate claims as one.

§ The Diversity of Gut Microbiome is Associated With Favorable Responses to Anti-Programmed Death 1 Immunotherapy in Chinese Patients With NSCLC > Introduction Advanced NSCLC is an incurable disease with a poor prognosis and is the leading cause of cancer-related death. 1 With impressive advancement in immune checkpoint inhibitor (ICI) therapies targeting programmed death 1 (PD-1) and programmed death ligand 1 (PD-L1) axis against multiple malignancies, these agents exhibit clinical efficacy against patients with advanced NSCLC both in second-line and first-line treatment when compared to conventional chemotherapy.At present, five therapeutic monoclonal antibodies targeting PD-1 or PD-L1 have been approved by the U.S. Food and Drug Administration (FDA) for the treatment of 11 cancer indications including NSCLC. 2 Nivolumab (Opdivo) is a fully human immunoglobulin G4 monoclonal antibody against PD-1.It blocks the binding of PD-1 to its ligand PD-L1 and restores the function of "exhausted" T cells in tumor sites.The unleashed lymphocytes exert killing ability, resulting in tumor shrinkage.3 It was firstly approved by the FDA in 2015 for the treatment of patients with advanced NSCLC after the failure of platinum-based chemotherapy, and by the National Medical Products Administration in 2018 after the phase III clinical trial CheckMate-078 for previously treated patients with NSCLC. 4 According to results from CheckMate-078, nivolumab yields superior overall survival (OS) compared with the docetaxel arm (median OS: 12 mo versus 9.6 mo). 5 However, only 20% of patients with advanced NSCLC benefit from the treatment, whereas up to 50% experience treatmentrelated adverse events in which grade 3-4 toxicities occur in 10% patients receiving nivolumab.6 Considering high cost of the agents, limited benefit population, and potential severe side effects, biomarkers for selecting patients with likely clinical efficacy from ICI treatment in advanced NSCLC would be of paramount importance. So far, PD-L1 expression on tumor cells is regarded as the most available biomarker to predict the efficacy of ICIs in NSCLC and other malignancies.A linear relationship has been reported between the proportion of ICI benefit and PD-L1 expression in NSCLC tumor sections.7,8 Nevertheless, the responses to ICI treatment have still been observed in those with low or undetectable levels of PD-L1 expression.9 To date, other biomarkers such as high tumor mutation burden, nonsynonymous mutations and neoantigens, CD8 þ T cell counts in tumor regions, defects of mismatch repair genes or high microsatellite instability, microbiota, and metabolic profiles are also supposed to be predictive for favorable responses to ICI treatment, among which gut microbiome property is of great value.[10][11][12][13][14][15][16][17] Human gut harbors more than 10 13 microbes, which outnumbers host cells 10-fold.More and more evidence supports the critical roles of gut microbiota in influencing human health in part through shaping systematic immune responses.18 Certain metabolites such as shortchain fatty acids and bile acid are considered to be key mediators.19 Dysbiosis of intestinal flora has been shown to be associated with multiple diseases.[20][21][22] Recent studies have also revealed that gut microbiota could mediate diverse responses to immunotherapy and might serve as a potential predictive biomarker.Gut commensals such as Bacteroides thetaiotaomicron or B. fragilis are predicable for anti-cytotoxic T-lymphocyte associated protein 4 (CTLA-4) treatment in a mouse melanoma model.23 In patients with melanoma receiving anti-PD-1 therapy, significant difference in the diversity and composition of gut microbiome was observed between the responders and nonresponders to the therapy.14 Germ-free mice receiving fecal transplantation from the responders tended to be more sensitive to anti-PD-1 treatment in contrast to those receiving feces from the nonresponders.14,15 In one cohort study enrolling 60 patients with advanced NSCLC and 40 with renal cell carcinoma undergoing PD-1 blockade therapy, Akkermansia muciniphila was reported to be overrepresented in the responders.15 Nevertheless, the previous studies were performed predominantly in the patients from Western countries.Gut microbiome structures have been elucidated remarkably variable among different races and ethnicities.24 Extrinsic factors, such as geographical location or diet and life styles, and intrinsic factors such as genetics or early-life exposures are main contributors to such divergences.[25][26][27] Therefore, it is still significant to reveal the characteristics of gut microbiota in regional patients with advanced NSCLC and the potential roles of gut microbiota in the prediction for the responses to ICI treatment.

Probably not a substantial claim. The paper’s conclusions may not depend on it.

What the paper did — it cites no source for this, so there is none to check. The quote shows it is in the paper.

§ The Diversity of Gut Microbiome is Associated With Favorable Responses to Anti-Programmed Death 1 Immunotherapy in Chinese Patients With NSCLC > Patients

Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

§ The Diversity of Gut Microbiome is Associated With Favorable Responses to Anti-Programmed Death 1 Immunotherapy in Chinese Patients With NSCLC > Discussion In the present study, we have investigated gut microbiota profiles in Chinese patients with advanced NSCLC receiving nivolumab treatment.Our results indicate that patients with better clinical responses exhibit higher diversity as well as a distinct gut microbiome composition.In addition, the association between gut microbiome diversity and systematic immune signatures highlights memory CD8 þ T cells and NK cells as potential mediators of antitumor immunity upon ICI treatment.The diversity of gut microbiome is defined as the number and abundance distribution of distinct types of micro-organisms colonizing within the gut.Abnormal diversity (dysbiosis) in the gut has been proven to be associated with human diseases, such as obesity, inflammatory bowel disease, cancer, etc. 20,21,33,34 Recently, Gopalakrishnan et al. 35 reported that patients with a high diversity of gut microbiome favored the clinical outcomes of anti-PD-1 immunotherapy in melanoma patients in the Western population.In our study, both alpha diversity and beta diversity at baseline are dramatically different between R and NR as well.A higher alpha diversity index represented by both the Shannon index and inverse Simpson indexes in R group suggests more species harboring in the gut, which is largely consistent with a differential composition of gut microbiome between R and NR.The higher the diversity of gut microbiota is, the longer PFS the patients undergo.Visualized beta diversity through PCoA analysis of 16S sequencing also displays apparently two clusters based on clinical responses to nivolumab treatment.Our results thus show that regardless of genetic background and geographic location with different lifestyles and diet styles, the diversity of gut microbiota might be a common benefit factor to ICI treatment.In our opinion, this is rational.It is widely accepted that gut microbiota modulates immune responses in a global way including antitumor immunity.36,37 Both innate and adaptive immune responses can be reshaped by gut microbiome.Although how the diversity of gut microbiota reshapes systemic immune responses is still not clearly addressed, microbiota-derived short chain fat acids and other metabolites should be good candidates to exert strong immune regulation.19,38 Further study will focus on the relevant metabolites analysis. Several clinical factors are reported to be involved in affecting the efficacy of ICI therapy, including smoking, previous treatment, and ATB usage.15,32,39 Among them, ATB is supposed to be the most relevant to gut microbiota.It has been reported previously that recent ATB usage in NSCLC patients was associated with shorter OS, although there was no difference in terms of objective response or PFS.32 In another study, ATB treatment is identified as the key factor significantly associated with both PFS and OS. 15 In our study, we retrospectively investigated ATB usage among 37 patients.The composition of gut microbiota at baseline between participants with or without ATB before first nivolumab infusion was similar (Supplementary Fig. 2), which excludes the possible effects of ATB on gut microbiota in our study.Median PFS was similar between ATB-treated and -naïve patients (median PFS: 126 days versus 117.9 days, p > 0.05) (Supplementary Fig. 3).One possibility lies in the number limitation in our study.Another possibility is that gut microbiome possess the ability of resilience.40 The majority of gut microbiota can be normalized after 1 week once ATB usage stops.41 We also observed that gut microbiota remained stable in longitudinal samples within one individual in our study.Such a trait might ensure patient long-term benefit from ICI treatment. Among all the clinical manifestations, smoking is the only factor that affects the responses to nivolumab treatment in our study.The roles of smoking on carcinogenesis refer to both cancer cell itself and immune responses of the host.It has been hypothesized that higher mutational burden could be induced by smoking, which has shown an increased response to ICI. 42,43 Median PFS was longer in current and former smokers compared with nonsmokers in nivolumab monotherapy for first-line treatment of advanced NSCLC as well.44 Smoking is reported to affect both innate and adaptive immunity, including memory T cells in the periphery.45 In our study, memory T cell profiles at baseline are similar between smoking and nonsmoking patients (Supplementary Fig. 4).This result confers that memory T cells are more likely affected by gut microbiota rather than other factors such as smoking.It has also been reported that chemotherapy could influence the gut microbiota of cancer patients largely due to the disruption of the intestinal barrier and subsequent bacteria translocation.In our study all the participants have received nivolumab therapy after failure of traditional chemotherapy.There was no difference in the interval from the date of last chemotherapy to that of first nivolumab infusion between R and NR groups (Supplementary Fig. 5, left).Correlation analysis showed that the diversity of gut microbiota was irrelevant to the time-off of chemotherapy in our cohort study as well (Supplementary Fig. 5, right). In addition to diversity, gut microbiome composition is also related to favorable responses to ICI treatment.In a pre-clinical study, gut commensal Bifidobacterium was shown to facilitate anti-PD-L1 efficacy.46 Clinical investigations reported that Faecalibacterium and some species belonging to Bacteroides genus (such as B. fragilis or B. thetaiotaomicron) are positively associated with the efficacy of PD-1 and CTLA-4 blockade, respectively.14,23 In our study, seven genera were identified different between R and NR.Ruminococcus_unclassified was enriched in NR whereas Bifidobacterium longum, Prevotella copri, and Alistipes putredinis were enriched in R. It has been shown in another study that Bifidobacterium longum could increase the production of pro-inflammatory cytokine tumor necrosis factor-alpha and significantly improve tumor control upon anti-PD-L1 therapy in a B16.SIY tumor mouse model.46 More interestingly, most bacteria enriched in R in our study belong to pro-inflammatory gram-negative bacteria.There are several previous studies reporting the association of gram-negative bacteria, such as Akkermansia muciniphila, Alistipes, and Porphyromonas with better clinical response to immunotherapy.14,15,36 It is already evident that the increase of pro-inflammatory gramnegative bacteria in the gut of older patients is associated with systematic and chronic inflammation.It is largely mediated by more release of lipopolysaccharide. In another study, mice colonized with Prevotella copri displayed increased inflammation in dextran sulfate sodium-induced colitis.Prevotella copri was strongly correlated with the disease severity in rheumatoid arthritis, which is a systemic autoimmune disease with the proliferation of pro-inflammatory T cells.47 In the antitumor setting, the exacerbation of immune responses might assist the ability of immune surveillance in the patients.This is also consistent with the report that patients who develop immune-related adverse events are more sensitive to ICI treatment.48 Although the direct evidence on the linkage between the inflammation and ICI benefit in the population still needs to be investigated, it is not out of expectation that systemic antitumor immune responses are diverse in patients with different gut microbiome composition.Gut microbiota thus becomes an intrinsic biosensor to sensitize anti-PD-1 treatment, which in turn leads to different clinical outcomes.This can be partially supported by the results from immune signature exploration in our study.Previous studies already highlighted the roles of PD-1 in reshaping immune responses in the mice.49 ICI therapy, to some extent, facilitates the investigation on PD-1 in the regulation of human immunity.By using multi-color flow cytometry, immune signatures including immune cell subsets and their function-related signatures have been detected.Furthermore, correlation analyses were performed between individual immune signatures and microbiota diversity.Among multiple signatures, the frequency and function of memory CD8 þ T cells are mostly dedicated.Memory CD8 þ T cells are a bunch of long-lived cells which are capable of rapid antigenspecific responses and play critical roles in immune recall responses. 50In addition to the percentage of CD8 þ Tm subsets such as effector memory T (Tem) cells and central memory T (Tcm) cells, the functional subset with Granzyme B production is also positively correlated with the diversity index, which can partially explain why patients with high microbiota diversity respond better to ICI therapy.The increase in Ki67 þ CD8 þ Tm in the patients with high microbiota diversity implies that gut microbiota also modulate the proliferation of certain immune cell populations.Recent studies already reported the effects of PD-1 blockade on the proliferation of CD8 þ T cells, which is beneficial for ICI treatment.51 ICOS and TIM3 expressions on specific T cells subsets are also related to antitumor immunity.52,53 Whereas CD8 þ T cells infiltrating in tumor microenvironment are thought to be the executors of tumor killing, circulating CD8 þ T cell subsets reported here are probably inclined to reflect the roles of gut microbiota on systematic immune responses.How peripheral memory CD8 þ T cell signatures affect the efficacy of ICI treatment will be investigated in the future.Once verified, they might become antitumor immune mediators against NSCLC. Our study shows that the diversity of gut microbiota is associated with favorable response to nivolumab therapy against NSCLC in Chinese cohorts.Considering the correlation between microbiota diversity and peripheral memory T cells and NK cells being reshaped upon ICI treatment, we propose that patients with more diverse gut microbiota at baseline benefit from ICI immunotherapy to a greater extent.This might be mediated by reprogramming systemic antitumor immune responses.The significance of our study lies in that the modulation of gut microbiota before the treatment might provide an alternative way to enhance the efficacy of ICI immunotherapy once validated in large cohort studies.A latest publication has introduced 11 bacterial strains from healthy donors' feces to induce interferon-g producing CD8 þ T cells in the intestine, which mediates effective antitumor immunity in a mouse model.54 Once clearly addressed, customized microbiome will exhibit great prospect as biotherapeutics in assistance with ICI treatment against advanced NSCLC.Project (17431906103), Shanghai Economic and Information Commission for oncology data platforms (201602010) and Shanghai Scientific Research Projects (14140902800).The authors thank Prof. Guo-ping Zhao for scientific suggestions on the project.Drs.Hong Li and Guoqing Zhang from Bigdata Center of Shanghai Insistute of Biological Science CAS helped to construct an integrative database with both clinical and multi-omics source data. Figure: 1 Figure 1 . Figure 1.Study scheme.

Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.