Digital Pathology May Help Explain the Clinical Significance of Intratumoral Bacteria in Patients with Nasopharyngeal Carcinoma

The microbiota, especially gut bacteria, play a key role in shaping the immune system and modulating immunological responses against invading pathogens and cancer cells. Recently, the extensive interactions between microbiota and cancer cells and the role of the microbiota-tumor interplay in tumor progression have been discovered. Although the gut microbiota has emerged as an attractive biomarker for predicting survival and treatment response in patients with various cancer types, little is known about the prognostic role of intratumoral microbiota in patients with nasopharyngeal carcinoma.

In a recent multicenter study, researchers at the Sun Yat-sen University Cancer Center investigated the ability of intratumoral microbiota to predict survival in patients with nasopharyngeal carcinoma. The study showed that high intratumoral bacterial load was associated with poor patient survival and low levels of immune cell infiltration into the tumor.1

“In this multicenter cohort study, we confirmed the existence of microbiota within nasopharyngeal carcinoma tissues. Intratumoral bacterial load was associated with poor survival of nasopharyngeal carcinoma patients and negatively correlated with T lymphocyte infiltration,” said Na Liu, MD, PhD, Professor in the Department of Experimental Research, Sun Yat-sen University Cancer Center and corresponding author of the study.

Commenting on the implications of their findings, Liu noted: “Our study demonstrated that intratumoral bacterial load is a robust and reliable prognostic indicator in nasopharyngeal carcinoma patients and has the potential to guide treatment decisions for patients at different risk of malignant progression.”

Their findings were published in JAMA Oncology earlier this month.


Study Rationale: Understating the Prognostic Role of Intratumoral Bacteria in Nasopharyngeal Carcinoma

“The microbiota has gained increasing attention as a crucial player in the tumor microenvironment that modulates tumor progression and influences cancer prognosis. Although the primary habitat of the human commensal microbiota is the gut, thriving microbial populations exist throughout much of the body, including the respiratory, skin, oral, and genital tracts,” Dr Liu said.

“Previous profiling studies of over 1500 tumors across seven cancer types have shown distinct intratumoral and intracellular microbial compositions in different tumor types. Further analysis uncovered associations between microbial composition and clinical features,” she added.

Dr Liu also noted that the nasopharyngeal epithelium, where nasopharyngeal carcinoma develops, is a crucial habitat for bacteria in the upper respiratory tract. Nevertheless, the presence and clinical significance of the intratumoral microbiota in patients with nasopharyngeal carcinoma remain largely unknown.


Approach: Combining Digital Pathology with Clinical Oncology and Advanced Molecular Profiling

To understand the clinical significance of intratumoral microbiota in patients with nasopharyngeal carcinoma, the team combined clinical oncology, digital pathology, and microbiome research strategies in a large retrospective cohort of 802 patients from Sun Yat-sen University Cancer Center (Guangzhou, China) and Zhejiang Cancer Hospital (Hangzhou, China).

Nasopharyngeal carcinoma specimens were obtained between January 2004 and November 2016, and patient follow-up was carried out through November 2020. “This was the first and largest cohort study of this kind,” Dr Liu noted.

The study cohort included patients aged between 18 and 70 years with histologically proven nasopharyngeal carcinoma without distant metastasis at diagnosis. None of the patients had received treatment before the collection of biopsy specimens. Clinical measures included disease-free, metastasis-free, and overall survival.

To assess the composition and load of intratumoral bacteria, the team performed bacterial analysis using 16S rRNA gene sequencing and quantitative polymerase chain reaction, along with a series of contaminant control processes. Digital pathology analyses of biopsy tissues were conducted to evaluate the association between intratumoral bacteria and immune cell infiltration in tumors. Digital pathology analyses involved registration of sequential tissue sections, alignment of tissue specimens, and manual annotation of the tumor areas by two pathologists using hematoxylin and eosin slides and immunohistochemistry slides. Segmentation of nuclei and identification and counting of cells with positive staining were performed in total tissues and tumor areas using digital pathology software.


Bacteria Are Present in Nasopharyngeal Carcinoma Tissues

In situ hybridization and immunohistochemical analyses showed that intratumoral bacteria were present in nasopharyngeal carcinoma tissues. Phylogenetic analysis based on 16S rRNA sequencing demonstrated that Proteobacteria were the predominant bacterial phylum in nasopharyngeal carcinoma tissues. At the genus level, Corynebacterium and Staphylococcus were present in 81.0% and 76.0% of the tissues (relative abundance: 12.9% and 7.4%, respectively).

To understand the origins of intratumoral bacteria, researchers cultivated bacteria from nasopharyngeal carcinoma tissues. Metagenomic sequencing of cultured bacteria and comparison of sequences between intratumoral bacteria and nasopharyngeal bacteria from the same patient revealed that most intratumoral bacteria may have originated from microbiota populations of the nasopharynx.


High Load of Intratumoral Bacteria Is Associated with Tumor Relapse

Correlation analysis of clinical outcomes and intratumoral bacterial load was conducted using 241 fresh-frozen nasopharyngeal carcinoma specimens (training cohort). Compared with patients with low intratumoral microbiota load, those with high bacterial load had worse rates of overall survival (hazard ratio [HR]: 3.41, 95% confidence interval [CI]: 1.90–6.11, P < 0.001), disease-free survival (HR: 2.90; 95% CI: 1.72–4.90, P < 0.001), and distant metastasis-free survival (HR: 3.18, 95% CI, 1.58–6.39, P < 0.001).

Similarly, high load of intratumoral bacteria was significantly associated with disease-free survival in the internal validation cohort (n = 233; HR: 3.32, 95% CI: 2.11–5.21), P < 0.001) and the external validation cohort (n = 234; HR: 2.24, 95% CI: 1.44–3.47), P < 0.001).


High Load of Intratumoral Bacteria Is Associated with Poor T Cell Infiltration

To analyze the relationship between intratumoral bacteria and T cell infiltration in nasopharyngeal carcinoma tissues, the team conducted transcriptomic and digital pathology analyses in 12 paired samples. Pathway enrichment analysis showed that tumors with a high load of intratumoral bacteria were enriched in cell cycle and metastasis-associated pathways.

Interestingly, compared with nasopharyngeal carcinoma tissues with low levels of intratumoral bacteria, those with high levels of bacteria contained significantly lower levels of various immune cell types, including T cells (P = 0.04), cytotoxic T lymphocytes (P = 0.01), and natural killer cells (P = 0.01). “These immune cells are crucial for the elimination of tumor cells and may represent a link between a high load of intratumoral microbiota and poor clinical outcomes in patients with nasopharyngeal carcinoma,” Dr Liu explained.


Unanswered Questions

Dr Liu noted that there are various questions that remain to be addressed. “For our work, although a negative correlation between high bacterial load and T lymphocyte infiltration was observed, the causal relationship and underlying mechanisms are not yet understood,” she said. She added that the interaction between intratumoral bacteria and another microbial risk factor for nasopharyngeal carcinoma, Epstein-Barr virus, warrants further exploration.


  1. Qiao H, Tan X-R, Li H, et al. Association of Intratumoral Microbiota With Prognosis in Patients With Nasopharyngeal Carcinoma From 2 Hospitals in China. JAMA Oncol. July 2022. doi:10.1001/jamaoncol.2022.2810


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