Diverse Microbiome Characteristics in Treatment Naïve Extranodal NK/T-Cell Lymphoma Patients

Background: It is still unclear why extranodal NK/T cell lymphoma (ENKTL) emerged in certain regions, such as Asia and South America, and initiated ENKTL lymphomagenesis in restricted upper airway or aerodigestive tract regions. Recently, reports have highlighted the profound relationship between the gut microbiome and immune system modulation. These associations can potentially influence cancer development and impact the effectiveness of chemotherapy. Therefore, we conducted a stool analysis to determine the newly diagnosed ENKTL (ND-ENKTL) microbiome characteristics. Additionally, a comparison analysis of microbiome composition among healthy individuals, newly diagnosed diffuse large B-cell lymphoma (ND-DLBCL), and ND-ENKTL was performed to identify potential clues that could influence ENKTL development and outcomes.

Methods: We have gathered and analyzed microbial data from 40 feces samples obtained from 40 ND-ENKTL patients from 2020 to 2022. Fecal samples from ENKTL patients were collected using a stool sampling kit (CJ Bioscience. Inc, Seoul, Korea) designed explicitly for microbial community analysis using shotgun sequencing. We used pre-analyzed gut microbiome data from the healthy control (HC) or newly diagnosed DLBCL (ND-DLBCL) group to compare the gut microbiome specificities.

Results : 32 (80.0%) presented stage I/II, and 7 (17.5%) or 6 (15.0%) had a high PINK (≥2) or high PINK-E score (≥3). In the alpha diversity analysis, ENKTL exhibited significantly lower diversity than HC. Additionally, we observed significant differences in microbial composition between these two groups (PERMANOVA, P-value=0.001). Species such as Escherichia coli, Citrobacter portucalensis and Citrobacter freundii were identified as significant biomarkers associated with dysbiosis in ENKTL. All these species belong to the Enterobacteriaceae family, a facultative aerobic opportunistic bacteria group. In the functional analysis, we observed an increase in genes related to aerobic bacterial metabolism in the ENKTL group. Conversely, a trend of decreased short-chain fatty acid (SCFA) metabolism and folate biosynthesis was identified. The noteworthy observation in ENKTL is the dominant Enterobacteriaceae family presence, which bears similarity to the microbiome associated with gut microbial dysbiosis in DLBCL, previously reported. While no significant differences were observed at the family level, a notable distinction was found between ENKTL and DLBCL at the genera. Specifically, a significantly higher proportion of Escherichia was dominant in ENKTL compared to DLBCL, which was dominant in Enterobacter and Citrobacter. In the chi-square analysis, a high abundance of Enterobacteriaceae presented statistically correlated with advanced stage (p=0.001) and PINK-high (≥2, p=0.003). Moreover, statistically significant associations were observed between the high proportion of the Escherichia genus and advanced stage (p=0.037), PINK-High (≥2, p=0.020), and a high PD-L1 CPS (≥10, p=0.029). During the median 21.1 months (95% CI 16.2-25.9) follow-up duration, the patients with high Enterobacteriaceae abundance exhibited significantly poorer progression-free survival (PFS) compared to those without such abundance (5.6 months vs. NR, p=0.001). Furthermore, patients with high Escherichia abundance demonstrated inferior PFS compared to those without (5.2 months vs. NR, p=0.001).

Conclusion: By comparing the intestinal microbes of ND-ENKTL with those of healthy individuals and ND-DLBCL, we could determine the distinct distribution of intestinal microbiome specific to ENKTL. Although the finding of the Enterobacteriaceae family was similar result with previously reported microbiome composition in DLBCL, we determined a high abundance of Escherichia genea in ENKTL but high Enterobacter and Citrobacter in DLBCL through subgroup analysis. Interestingly, Escherichia genea in ENKTL showed a statistical correlation with a high disease burden, PD-L1 expression, and inferior PFS. Based on the results, we expected that it would be possible to predict the disease characteristics and treatment susceptibility in ENKTL by intestinal microbes' composition and diversity in the future.