Similar studies regarding ICI therapy have focused on the microbiome in patients who have not yet received ICI therapy. Those studies revealed that the microbiome diversity and composition before ICI therapy was a predictive biomarker for ICI response. Although variations in gut microbiome composition were observed in the previous studies, there has been minimal information regarding gut microbiome status during ICI therapy. This information is important for efforts to enhance ICI therapy through biotics therapy (e.g., pre-, pro-, and synbiotics) and/or FMT. This analysis of oral and gut microbiome profiles in Japanese NSCLC patients during ICI therapy produced several novel findings.
The gut microbiome might have an important role in the ICI response in NSCLC patients, although the oral microbiome conveyed information distinct from the gut microbiome. Greater numbers of both oral and gut microbiome species were observed in ICI responders than in ICI non-responders. Additionally, those microbiomes mainly consisted of minor species (< 1%) at the genus level. Moreover, the 4th and 5th majority of gut microbiome species in ICI responder were differed from that in ICI non-responder, while 1st to 5th majority of oral microbiome were same between two groups.
An important tool for objective evaluation of the above data involves analysis of microbiome diversity: the numbers or abundances of microorganisms colonizing the gut. Greater alpha diversity indicates larger numbers of species in the gut, which implies a distinct gut microbiome composition. Several studies have reported that the ICI response was influenced by the alpha diversity of the gut microbiome before ICI therapy [8, 27]. In the present study, high alpha diversity was observed in the ICI responder gut microbiome. Thus, our results indicated that ICI responders had more abundant gut microbes, compared with ICI non-responders, during ICI therapy.
Our study also revealed a significant association between beta diversity and ICI response. Higher beta diversity indicates a significant difference in gut microbiome composition between two samples. In this study, the intersample distance was significantly shorter in ICI responders than in ICI non-responders. Therefore, the ICI responder gut microbiome had significant similarity, compared with the ICI non-responder gut microbiome, which implies a simple approach to control the gut microbiome by adding biotics therapy to ICI therapy.
Additionally, we identified specific gut microbiome species in Japanese NSCLC patients receiving ICI therapy by using LEfSe. The results indicated that Blautia was enriched in ICI responders, whereas RF32 unclassified was enriched in ICI non-responders. Several studies have shown significant associations between various gut microbiome components (e.g., Bifidobacterium longum , Collinsella aerofaciens , Enterococcus faecium , Akkermansia muciniphila , Ruminococcaceae , Faecalibacterium genii  and Firmicutes ) and clinical response to ICI therapy. These results differed among individual studies, including the present study, presumably due to factors such as patient disease (e.g., melanoma, renal cell carcinoma, and NSCLC) and ICI regimen. Indeed, Frankel et al  indicated that the gut microbiome composition depended on the ICI regimen. Moreover, these differences might be caused by ethnicities. Nishijima et al  compared the gut microbiome between the Japanese population and individuals from 11 other nations. Notably, the Japanese gut microbiome was considerably different from the microbiomes of other populations; it was characterized by the highest abundances of Blautia, Bifidobacterium, Collinsella, Streptococcus, and an unclassified Clostridiales genus, compared with the microbiomes from the remaining 11 countries.
In addition to its status as a species characteristic of the gut microbiome in Japanese individuals, Blautia coccoides is regarded as an effective probiotic species. B. coccoides is an anaerobe and Gram-positive species found in human fecal samples. Reduced numbers of B. coccoides are associated with several benign diseases (e.g., hepatic cirrhosis and encephalopathy, irritable bowel syndrome, acute diarrhea, idiopathic inflammatory bowel disease, intestinal inflammation, and diabetes mellitus) and some malignancies (e.g., colorectal and breast cancers) [30, 31]. Furthermore, some reports have revealed that increased numbers of B. coccoides might be beneficial for human health. B. coccoides is increased among individuals with diets high in resistant starch and arabinoxylan ; moreover, it can reduce NF-κB activity in human colon cancer cells . Additionally, Blautia obeum is a gut microbiome component involved in the transformation of carcinogenic heterocyclic amines, and reduced abundance of this species may increase heterocyclic amine-induced colorectal cancer risk . Myles et al  reported that high omega-3 intake altered colonic inflammation and increased Blautia abundance in a murine model. Therefore, Blautia may be a key gut microbiome component involved in the ICI response in Japanese NSCLC patients. Conversely, the relative abundance of RF32 unclassified has been positively correlated with colonic damage and inflammation , which are presumably negative influences on immunity in NSCLC patients.
The findings in this study indicate that controlling both gut microbiome diversity and the abundances of specific gut microbiome species during ICI therapy might lead to ICI response enhancement in Japanese NSCLC patients. Future therapies targeting the gut microbiome by means of pre-, pro-, or synbiotics to enhance the ICI response might be considered from our findings.
This study had the following limitations. First, only a single sample collection was performed. Thus, it was unclear how the microbiome diversity and composition might have changed before and after ICI therapy. Second, the sample size was small, which may have interfered with meaningful conclusions. Third, this study allowed various ICI regimens with or without combination chemotherapy. In the future, we plan to perform a large, multicenter, prospective observational study to evaluate the association between ICI response and changes in gut microbiome by collecting samples at multiple points (before and during ICI therapy) for NSCLC patients receiving a specific ICI regimen.
In conclusion, this study revealed a strong association between gut microbiome diversity and ICI response in Japanese NSCLC patients. Moreover, specific gut microbiome compositions may influence the ICI response. These findings might be useful in identifying biomarkers to predict ICI response, as well as in developing biotic therapies to enhance the ICI response.