Intratumoral Leptotrichia is a novel microbial marker for favorable clinical outcomes in head and neck cancer patients

Dear editor: Head and neck squamous cell carcinoma (HNSCC) accounts for more than 70 million cancer cases annually around the world.1 The pathogenesis of HNSCC is not fully understood, but several risk factors for HNSCC, such as smoking, alcohol consumption, and human papillomavirus infection, have been reported. Recently, studies of the microbiomes of patients with head and neck cancer suggest that microbial variation correlates with the development of HNSCC, and some potentially oncogenic bacteria have been identified.2 However, there are limited reports exploring the microbiomes of patients with different stages of HNSCC. Data from other tumors demonstrate that microbial-host factors, independent of the genomic composition of the tumor, may determine tumor behavior and patient outcomes; therefore, we hypothesized that patients with different stages of HNSCC would have diverse microbial compositions. In this study, we sought to identify microbial differences between early-stage (T1–T2) and advanced-stage (T3–T4) HNSCC patients. 16S ribosomal RNA sequencing data and clinical profiles of HNSCC patients were obtained from the Cancer Genome Atlas (TCGA) and the Cancer Microbiome Atlas (TCMA) database as described in the Supporting Information. There were total of 153 patients (T1–T2, N = 61; T3–T4, N = 92) included in this study from the TCGA database (Table S1). To measure whether the microbial composition was different between normal tissues, earlystage tumor tissues, and advanced-stage tumor tissues, we used principal coordinate analysis (PCoA) and permutational multivariate analysis of variance (PERMANOVA) based on the Bray‒Curtis distance from the genus profile. The results presented a significantly different distribution between normal tissues and tumor tissues (normal vs. T1– T2, p-value = 0.035; normal vs. T3–T4, p-value = 0.004) (Figure 1A). Then, differences in alpha-diversity indices, including Chao1 index and Shannon index, were mea-

sured (Figure 1B).The results showed that the microbial alpha-diversity was significantly decreased in T3-T4 samples compared with normal samples, and in T3-T4 samples compared with T1-T2 samples.Mucosal sites in the head and neck region harbor a site-specific microbiome, which has an essential role in maintaining health and homeostasis.As high microbial diversity is considered to be a sign of health, and the microbiome is characterized by an overgrowth of diverse bacteria, such as Fusobacterium, Prevotella, Veillonella, and so forth, in head and neck malignancies, the decrease in alpha-diversity of T3-T4 samples was consistent with previous studies.We next sought to examine tissue microbiome composition in three groups.At the phylum level, the tissue microbiota was dominated by members of Bacteroidetes and Firmicutes, followed by Fusobacteria (Figure 1C), which were similar to those reported in previous studies. 2We then compared the relative abundance of the top 20 genera in the three groups, which revealed the enrichment of specific genera, such as Fusobacterium, Treponema, and Capnocytophaga, in T1-T2 and T3-T4 samples compared with normal samples (Figure 1D).
To extend our understanding of the role of the microbiome and its association with tumor stage and patient outcomes, we compared the relative abundances of bacterial taxa between T1-T2 tumor tissues and T3-T4 tumor tissues, finding significant differences in one phylum, one class, two orders, two families, and five genera (Figure 1E).Compared with samples from advanced-stage (T3-T4) patients, samples from early-stage (T1-T2) patients had significantly increased mean abundances of the genera Campylobacter, Neisseria, Rothia, Leptotrichia, and Tannerella.The parent taxa of the genus Rothia were also significantly increased in early-stage patients compared with advanced-stage patients, up to the phylum level.Campylobacter spp.were identified as strongly associated and enriched in colorectal cancer tissues, but their role in HNSCC remains controversial.Neisseria spp.and Rothia spp.were associated with a decreased risk of developing HNSCC 3 ; in addition, depletions of Neisseria and Rothia in HNSCC cases were associated with worse cancer-specific survival.Tannerella spp. was reported to be less abundant in HNSCC patients than in normal controls, despite their positive correlation with grade 2+ radiation-induced oral mucositis. 3,4o further investigate these findings, we tested the relationship between tumor microbial composition and overall survival (OS) in the HNSCC cohort by stratifying the patients into two groups based on whether the above five genera could be detected in tumor samples.Kaplan-Meier curves were plotted for survival distributions (Figure 1F).The median OS times of the Leptotrichia-positive and Leptotrichia-negative groups were 37.5 and 20.5 months, respectively.Patients with Leptotrichia-positive samples had significantly prolonged OS compared with those with Leptotrichia-negative samples (p-value = 0.0005) using univariate Cox proportional hazard models (Figure 1F).To decrease the influence of confounding factors, we performed a multivariate analysis based on basic characteristics and survival data of the TCGA cohort.Leptotrichia was found to be an independent factor of OS and was correlated with better OS (Table S2).In addition, the proportion of Leptotrichia-positive samples in early-stage (T1-T2) patients was approximately 1.5-fold higher than that in advanced-stage (T3-T4) patients, which means Leptotrichia was more prevalent in samples from earlystage HNSCC patients than in samples from advancedstage patients (Figure 1G).These results suggest that Leptotrichia may have a protective effect in the HNSCC tumor microenvironment; therefore, we conducted fluorescence in situ hybridization (FISH) and quantitative PCR (qPCR) in HNSCC samples from patients in Peking Union Medical College Hospital to detect the genomic DNA of Leptotrichia.Using Leptotrichia genomic DNA as probes, we detected fluorescence signals in HNSCC tumor slides (Figure 1H).By calculating signals per highpower field (HPF) in each sample, more FISH signals were identified in samples from stage T1-T2 patients than in samples from stage T3-T4 patients (Figure 1I).As fluorescence signals were relatively sparse in these samples, qPCR experiments based on a total of 15 HNSCC tumor samples and six matched normal samples were applied to better quantify the relative expression level of Leptotrichia.The results showed that the gene expression level of samples from stage T1-T2 patients was significantly higher (p-value = 0.0059) than those in samples from stage T3-T4 patients (Figure 1J).Additional analysis of the composition difference of microbiome in different locations of head and neck cancers is shown in Table S3.
Several studies have reported that Leptotrichia along with Fusobacterium might promote the development of colorectal cancer, and Leptotrichia is significantly enriched in early-stage colorectal cancer compared with late-stage colorectal cancer. 5However, as a common bacterial genus in a healthy oral cavity, the role of Leptotrichia in HNSCC has rarely been reported in the literature.Leptotrichia may exert a protective role against malignancy in the upper respiratory tract by inducing the host cellular immune response, which needs further exploration.
In summary, our results demonstrated that the microbial composition of early-stage HNSCC tumor tissues was distinct from that of advanced-stage HNSCC tumor tissues, indicating a probable microbial effect on tumor behavior.In addition, we discovered that Leptotrichia was significantly increased in early-stage patients compared to advanced-stage patients and was correlated with better OS, suggesting a protective value of Leptotrichia in the HNSCC tumor microenvironment.Our study provided a strong rationale for further investigations to validate these discoveries and to extend these initial findings for the potential clinical development of microbiota-based cancer therapies.

A U T H O R C O N T R I B U T I O N S
P.Z., Y.Z., and X.C. provided the overall study design and co-wrote the paper.S.Y. designed the study, collected samples, and drafted the paper.J.C. was responsible for the data analysis and co-writing of the manuscript with the help of F.Y.All authors approved the final version of the manuscript.

C O N F L I C T O F I N T E R E S T S TAT E M E N T
The authors declare they have no conflicts of interest.

D ATA AVA I L A B I L I T Y S TAT E M E N T
The patient cohort materials used for the current study are publicly available and can be accessed from the TCGA database (https://portal.gdc.cancer.gov/,https:// www.cbioportal.org/).The processed data and analysis codes are available upon reasonable request from the corresponding author.

F I G U R E 1
The association of Leptotrichia with head and neck squamous cell carcinoma (HNSCC) stages and patient outcomes.(A) Beta-diversity calculated by principal coordinate analysis (PCoA) plots.The p-value was derived from permutational multivariate analysis of variance (PERMANOVA).(B) Comparison of microbial alpha-diversity among groups.p-Values were calculated from the Wilcoxon rank-sum test.p < 0.01 was labeled with "+" and "*" represented p < 0.05.(C) The relative abundance of dominant phyla in different groups.(D) The relative abundance of the top 20 dominant genera in different groups.(E) Significant differences in tumor microbial communities between T1-T2 tissues and T3-T4 tissues.(F) Overall survival of HNSCC patients based on whether Leptotrichia was detected in tumor samples based on the TCGA dataset.(G) Detection rate of Leptotrichia in different stages of HNSCC based on the TCGA dataset.(H) Fluorescence in situ hybridization (FISH) of tumor tissues from stage T1-T2 HNSCC or stage T3-T4 HNSCC.Leptotrichia genomic DNA was used as a probe (red) for FISH.The arrows indicate FISH signals.Scale bars: 20 µm.(I) Detected FISH signal per high-power field (HPF) of samples.(J) The relative expression level of Leptotrichia genomic DNA in tumor tissues and paired normal tissues from stage T1-T2 HNSCC or stage T3-T4 HNSCC patients.

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U N D I N G I N F O R M AT I O N This study is supported by National High Level Hospital Clinical Research Funding (2022-PUMCH-B-094), CAMS Innovation Fund for Medical Sciences (CIFMS) (2021-I2M-1-023/2020-I2M-2-009), and Science & Technology Fundamental Resources Investigation Program (Grant No. 2022FY100800).