Post-irradiation dysbiosis in patients with nasopharyngeal carcinoma having received radiotherapy - A pilot study.

OBJECTIVE: To compare the changes in the sinonasal mucosa microbiome in patients with nasopharyngeal carcinoma (NPC) before and after radiotherapy (RT), and to explore the pathogenesis of post-irradiation chronic rhinosinusitis (PI-CRS) and its association with dysbiosis. STUDY DESIGN: Prospective cohort study. SETTING: Unicenter, Tertiary referral hospital. METHODS: Included patients newly diagnosed with NPC. Samples of ostiomeatal complex mucosa were collected before and after RT. Microbiome analysis was conducted using 16S rRNA sequencing, and statistical analysis was performed. Subgroup analyses based on RT modality (proton therapy or photon therapy) RESULTS: Total of 18 patients were enrolled in the study, with 62.1% receiving intensity-modulated proton therapy (IMPT). Corynebacterium was the most dominant genus identified in both the pre- and post-RT groups, with a visible increase in Staphylococcus and a decrease in Fusobacterium genus in post-RT group. Alpha-diversity did not significantly differ between groups, although the beta-diversity analysis revealed a dispersed microbiota in the post-RT group. The functional prediction indicated a higher relative abundance of taxonomies associated with biofilm formation in the post-RT group. The subgroup analysis revealed the above changes to be more significant in patients who received photon therapy (Intensity modulated radiation therapy, IMRT). CONCLUSIONS: This is the first study to analyze the microbiome of patients with NPC after IMPT. We identified similarities between the post-RT microenvironment and that reported in patients with CRS, with a more apparent change noted in patients treated with IMRT. Further investigation is required to further elucidate the pathogenesis of PI-CRS and its relationship to post-RT dysbiosis, particularly IMPT.

Prodrug-conjugated tumor-seeking commensals for targeted cancer therapy.

Prodrugs have been explored as an alternative to conventional chemotherapy; however, their target specificity remains limited. The tumor microenvironment harbors a range of microorganisms that potentially serve as tumor-targeting vectors for delivering prodrugs. In this study, we harness bacteria-cancer interactions native to the tumor microbiome to achieve high target specificity for prodrug delivery. We identify an oral commensal strain of Lactobacillus plantarum with an intrinsic cancer-binding mechanism and engineer the strain to enable the surface loading of anticancer prodrugs, with nasopharyngeal carcinoma (NPC) as a model cancer. The engineered commensals show specific binding to NPC via OppA-mediated recognition of surface heparan sulfate, and the loaded prodrugs are activated by tumor-associated biosignals to release SN-38, a chemotherapy compound, near NPC. In vitro experiments demonstrate that the prodrug-loaded microbes significantly increase the potency of SN-38 against NPC cell lines, up to 10-fold. In a mouse xenograft model, intravenous injection of the engineered L. plantarum leads to bacterial colonization in NPC tumors and a 67% inhibition in tumor growth, enhancing the efficacy of SN-38 by 54%.

Influence of Pre-treatment Saliva Microbial Diversity and Composition on Nasopharyngeal Carcinoma Prognosis.

Background: The human microbiome has been reported to mediate the response to anticancer therapies. However, research about the influence of the oral microbiome on nasopharyngeal carcinoma (NPC) survival is lacking. We aimed to explore the effect of oral microbiota on NPC prognosis. Methods: Four hundred eighty-two population-based NPC cases in southern China between 2010 and 2013 were followed for survival, and their saliva samples were profiled using 16s rRNA sequencing. We analyzed associations of the oral microbiome diversity with mortality from all causes and NPC. Results: Within- and between-community diversities of saliva were associated with mortality with an average of 5.29 years follow-up. Lower Faith's phylogenetic diversity was related to higher all-cause mortality [adjusted hazard ratio (aHR), 1.52 (95% confidence interval (CI), 1.06-2.17)] and NPC-specific mortality [aHR, 1.57 (95% CI, 1.07-2.29)], compared with medium diversity, but higher phylogenetic diversity was not protective. The third principal coordinate (PC3) identified from principal coordinates analysis (PCoA) on Bray-Curtis distance was marginally associated with reduced all-cause mortality [aHR, 0.85 (95% CI, 0.73-1.00)], as was the first principal coordinate (PC1) from PCoA on weighted UniFrac [aHR, 0.86 (95% CI, 0.74-1.00)], but neither was associated with NPC-specific mortality. PC3 from robust principal components analysis was associated with lower all-cause and NPC-specific mortalities, with HRs of 0.72 (95% CI, 0.61-0.85) and 0.71 (95% CI, 0.60-0.85), respectively. Conclusions: Oral microbiome may be an explanatory factor for NPC prognosis. Lower within-community diversity was associated with higher mortality, and certain measures of between-community diversity were related to mortality. Specifically, candidate bacteria were not related to mortality, suggesting that observed associations may be due to global patterns rather than particular pathogens.

Characterization of the nasopharynx microbiota in patients with nasopharyngeal carcinoma vs. healthy controls.

PURPOSE: Nasopharyngeal carcinoma (NPC), an epithelial-originated malignant tumor, has a special geographic distribution. However, the etiology of NPC has not been examined in detail. Increasing pieces of evidence indicate that the microbiome may contribute to head and neck squamous cell carcinoma. Until now, there is limited information on the role of the microbiome in NPC, so we assessed variations in the nasopharynx microbiota of patients with NPC relative to the bacterial in health controls. METHODS: Nasopharynx lavage fluid (NLF) samples were collected from 11 NPC patients and 5 volunteer controls. 16S rRNA sequencing and comparative analyses of NLF bacterial microbiome between NPC patients and controls were performed. RESULTS: NLF microbial alpha-diversity by the Shannon index and Simpson index decreased significantly in the NPC patients when compared with the controls. Beta-diversity by principal component analysis exhibited separated patterns of the NPC patients and healthy controls. Thirty-one genera differed significantly between the NPC patient group and healthy control group. The abundance of 17 bacteria was correlated with primary tumor size and invaded lymph node size. Functional gene prediction analysis showed that 9 gene function pathways were significantly different between the two groups. CONCLUSION: Our results demonstrated that the nasopharynx microbiota in NPC patients was different from that of the healthy controls, suggesting that the nasopharynx microenvironment might be related to NPC.

Investigation of salivary function and oral microbiota of radiation caries-free people with nasopharyngeal carcinoma.

Radiation caries have been reported to be correlated with radiotherapy-induced destruction of salivary function and changes in oral microbiota. There have been no published reports detailing patients who have remained radiation caries-free following radiotherapy for nasopharyngeal carcinoma. The aim of this study was to investigate the relationship between salivary function, oral microbiota and the absence of radiation caries. Twelve radiation caries-free patients and nine patients exhibiting radiation caries following irradiated nasopharyngeal carcinoma were selected. V40, the dose at which the volume of the contralateral parotid gland receives more than 40 Gy, was recorded. Stimulated saliva flow rate, pH values and buffering capacity were examined to assess salivary function. Stimulated saliva was used for molecular profiling by Denaturing Gradient Gel Electrophoresis. Mutans streptococci and Lactobacilli in saliva were also cultivated. There were no significant differences in V40 between radiation caries-free individuals and those with radiation caries. Compared with normal values, the radiation caries-free group had significantly decreased simulated saliva flow rate, while there were no significant differences in the saliva pH value and buffering capacity. Similar results were observed in the radiation caries group. There was no statistical difference in microbial diversity, composition and log CFU counts in cultivation from the radiation caries-free group and the radiation caries group. Eleven genera were detected in these two groups, among which Streptococcus spp. and Neisseria spp. had the highest distribution. Our results suggest that changes in salivary function and in salivary microbiota do not explain the absence of radiation caries in radiation caries-free individuals.

[Analysis of the nutritional status and nosocomial infection during chemoradiotherapy in advanced nasopharyngeal carcinoma patients].

OBJECTIVE: To analyze the nutritional status and nosocomial infection of nasopharyngeal carcinoma patients before and after the chemoradiotherapy treatment. METHOD: An analysis was made for the nutritional and nosocomial infection status of 82 cases before and after chemoradiotherapy treatment. RESULT: Statistically significant differences were revealed between indexes related with nutritional status such as body mass, hemoglobin, serum albumin before and after the treatment. Sixty-three patients occurred nosocomial infection. The infection rate was 76.83%. The main risk factor was oropharynx mucosal lesion and the rate is 92.68%. Isolates of 39 bacteria were found, of which Gram-negative organisms were 58.97%, Fungi were 30.77%, Gram-positive ones were 7.69%, Herpes zoster were 2.56%. CONCLUSION: Chemoradiotherapy has negative influence on nutritional status of patients. Medical personnel should pay attention to patients' nutritional status and do a good job of nutritional status monitoring, nutrition support, dieting guidance, reducing side effects, in order to improve the patient's tolerability and quality of life. The nosocomial infection rate of Gram-negative bacteria of oropharyngeal mucosal is the highest in patients with advanced nasopharyngeal cancer during chemoradiotherapy. It is very important for us to prevent and control nosocomial infection.

Nasopharyngeal carcinoma mimicking Aspergillosis rhinosinusitis: an unusual case report and review of the literature.

Clinical symptoms and imaging features of fungal infection are confused with those of atypical nasopharyngeal carcinoma (NPC), and therefore development of a more effective diagnostic method is essential. It is a common knowledge that there is a significant association between Epstein-Barr virus (EBV) and nonkeratinizing NPC. However, fungal infection may be considered to be a vital etiologic agent contributing to the NPC and more evidence is needed to be approved this theory. We report on a rare case of a patient with atypical nasopharyngeal carcinoma (NPC) who suffered from chronic fungal infection and was diagnosed initially as Aspergillosis. Following anti-aspergillus infection therapy, the repeated deep biopsy of the maxillary sinus and MRI confirmed the diagnosis of nasopharyngeal carcinoma (NPC).

Changes of saliva microbiota in nasopharyngeal carcinoma patients under chemoradiation therapy.

OBJECTIVE: A growing body of evidence has implicated human oral microbiota in the aetiology of oral and systemic diseases. Nasopharyngeal carcinoma (NPC), an epithelial-originated malignancy, has a complex aetiology not yet fully understood. Chemoradiation therapy of NPC can affect oral microbiota and is usually accompanied by plaque accumulation. Thus, the study aimed to understand the diversity, divergence and development of the oral microbiota in NPC patients and their associated treatment, which might provide useful insights into disease aetiology and treatment side effects. DESIGN: A longitudinal study was designed that included three Chinese adults with NPC. Saliva samples were collected at three time points: prior to the chemoradiation treatment (carcinoma baseline, or CB), 7 months post-treatment (carcinoma-after-therapy phase 1 or CA1) and 12 months post-treatment (carcinoma-after-therapy phase 2 or CA2). Pyrosequencing of the bacterial 16S ribosomal DNA (rDNA) V1-V3 hypervariable region was employed to characterise the microbiota. Saliva samples of three healthy subjects from our former study were employed as healthy controls. Principal coordinates analysis (PCoA), Metastats and random forest prediction models were used to reveal the key microbial members associated with NPC and its treatment programme. RESULTS: (1) In total, 412 bacterial species from at least 107 genera and 13 phyla were found in the saliva samples of the NPC patients. (2) PCoA revealed that not only were the microbiota from NPC patients distinct from those of healthy controls (p<0.001) but also that separation was found on the saliva microbiota between pre- and post-therapy (p<0.001) in the NPC samples. (3) At the genus level and the operational taxonomic unit (OTU) level, Streptococcus was found with lower abundance in NPC samples. (4) Chemoradiation therapy did not incur similar changes in microbiota structure among the three NPC patients; the microbiota in one of them stayed largely steady, while those in the other two showed significant alteration. CONCLUSIONS: This is the first study employing culture-independent techniques to interrogate the phylogenetic diversity, divergence and temporal development of oral microbiota in NPC patients. Our results indicated that certain bacterial taxa might be associated with NPC and that oral microbiota of NPC patients might respond to the chemoradiation therapy in a host-specific manner. Further investigation with larger sample size should help to validate the links between oral microbiota and NPC.

Biofilm formations in nasopharyngeal tissues of patients with nasopharyngeal osteoradionecrosis.

OBJECTIVE: Determine the presence of nasopharynx biofilms in patients with nasopharyngeal cancer (NPC) and osteoradionecrosis (ORN) and patients with NPC but no ORN. STUDY DESIGN: Cross-sectional study. SETTING: Tertiary referral medical center. SUBJECTS AND METHODS: We enrolled 27 patients with NPC from our outpatient clinic during January 2010 to June 2012. These patients were diagnosed with NPC between 1980 and 2012, and all had received radiotherapy. Of these 27 patients, 15 presented with ORN, and 12 did not. The nasopharynx biopsied specimens were processed and analyzed within 2 hours of collection with the FilmTracer LIVE_DEAD Biofilm Viability Kit (Molecular Probes, Invitrogen, Carlsbad, California). A blinded investigator determined the formation of biofilms by fluorescence microscopy. Bacterial cultures were collected. RESULTS: Eleven of 15 (73%) ORN patients had biofilm formations in nasopharynx biopsy specimens. Five of these samples (45%) yielded positive cultures, and 4 of these cultures indicated the presence of methicillin-resistant Staphylococcus aureus (MRSA). Only 1 of 12 NPC patients without ORN had nasopharynx biofilm formation, and all culture results were negative. CONCLUSION: Biofilm formations were common in nasopharynx samples of NPC patients with ORN but rare in samples of NPC patients without ORN. The presence of biofilms, especially MRSA, may have a role in the disease progression of ORN or may contribute to the chronicity and resistance to antibiotic treatment.

Bacteriology of postradiotherapy chronic rhinosinusitis in nasopharyngeal carcinoma patients and chronic rhinosinusitis.

The purpose of this study was to investigate and compare the bacteriology of postradiotherapy chronic rhinosinusitis (postRT-CRS) and chronic rhinosinusitis (CRS) by evaluating the aspiration materials of the maxillary sinus of patients with postRT-CRS and patients with CRS. We collected the secretions of the maxillary sinus from 30 nasopharyngeal carcinoma patients with postRT-CRS and 30 patients with CRS for aerobe/facultative anaerobe bacteria culture. The most common isolates in the postRT-CRS group were Streptococcus viridans, Staphylococcus aureus and Haemophilus influenzae, while those in the CRS group were Haemophilus influenzae, Pseudomonas aeruginosa and Staphylococcus aureus. Isolated gram-positive coccus rate in postRT-CRS patients was significantly higher than in CRS patients (62.50% compared with 30.00%, respectively; P < 0.05), and isolated gram-negative bacilli rate in postRT-CRS patients was significantly lower than in CRS patients (31.25% compared with 70.00%, respectively; P < 0.05). However, the incidence of positive cultures was not significantly different between the postRT-CRS group and the CRS group (P > 0.05). This study found that there were some differences in bacteriology between postRT-CRS and CRS. Gram-positive coccus was the predominant aerobic/facultative anaerobe pathogenic bacterium in patients with postRT-CRS, and gram-negative bacilli was predominant in CRS patients.

Intracellular co-localization of SPLUNC1 protein with nanobacteria in nasopharyngeal carcinoma epithelia HNE1 cells depended on the bactericidal permeability increasing protein domain.

Epithelial surfaces constitute natural immunobarriers against environmental threats. These barriers are brimming with fluids that bind, transport, cleave or degrade bacterial cells and their endotoxic by-products. Saliva and the airway surface-lining fluid (ASL) comprise the important fluid constituents. Short palate, lung and nasal epithelium clone 1 (SPLUNC1) is a potential host defensive protein that is secreted from the submucosal gland to the saliva and nasal lavage fluid. However, its antimicrobial spectrum and antimicrobial mechanism is not clear. Through green fluorescence protein (GFP) mediated subcellular localization experiments in nasopharyngeal carcinoma (NPC) HNE1 cell line, we determined that the intracellular GFP-tagged SPLUNC1 protein binds to a miniscule microorganisms, approximately 50-400nm in size, after the bactericidal permeability increasing protein (BPI) domain was deleted, GFP-tagged truncated SPLUNC1 protein lost its function of binding to the miniscule microorganisms. We verified that these microorganisms are nanobacteria (NB) with a negative staining using transmitted electronic microscope (TEM) and immunofluorescent analysis using an NB-specific antibody. We isolated and cultured the NB from the cultured nasopharyngeal carcinoma epithelia HNE1 cell supernatant. We found that the NB did not absorb the Hoechst stain, even when we extended the staining time to 35min. However, with the time extension the larger sized NB (larger than 300nm) did stain positively. From the biopsy specimen of NPC, we also detected the NB, which can lead to the swelling of mitochondria in the infected host cells. We hypothesize that SPLUNC1 and NB co-localization is due to the GFP-tagged SPLUNC1 protein binding to the lipopolysaccharide (LPS) of the Gram-negative NB, which can play an important role in the host defense of nasopharyngeal epithelium. This research sheds new light on the mechanism of SPLUNC1 involvement in the host upper respiratory tract defense system.

The persistence of Streptococcus mutans in nasopharyngeal carcinoma patients after radiotherapy.

The aim of the study was to examine the persistence of oral Streptococcus mutans in nasopharyngeal carcinoma patients after radiotherapy. Ten subjects, ranging in age from 20 to 67 years, participated. DMFT/DMFS, salivary level of mutans streptococci and oral health status were recorded. Pooled plaque samples were obtained from the cervical margins and the interproximal regions of all the teeth and the occlusal surfaces of the molars prior to, immediately after, 3 and 6 months after the completion of radiotherapy. At least 10 colonies of S. mutans were isolated from each subject and totally 645 isolates were genotyped by restriction endonuclease analysis. The results showed that the salivary level of S. mutans increased significantly with the reduction of salivary flow rate after radiotherapy. Each subject had at least 1 genotype of S. mutans isolated throughout the follow-up period. In 3 subjects who initially carried 2 or more genotypes, 1 or 2 genotypes of S. mutans could not be detected 3 months after treatment. Moreover, the genotypes that became undetectable were predominant bacteria in the first sampling. The result indicated that most S. mutans genotypes were persistent after radiotherapy but some genotypes that might not adapt to the alteration of oral environment became undetectable.

Nasopharyngeal granulomatous inflammation and tuberculosis complicating undifferentiated carcinoma.

OBJECTIVE: Four cases of nasopharyngeal granulomatous inflammation after radiotherapy for undifferentiated carcinoma were analyzed for tuberculosis, and the histologic features were compared. STUDY DESIGN AND SETTING: We conducted a retrospective study with analysis of tuberculosis by Ziehl Neelsen staining and polymerase chain reaction analysis for Mycobacterium tuberculosis DNA on histologic materials. RESULTS: Three patients had previous nasopharyngeal undifferentiated carcinoma, one had previous metastatic undifferentiated carcinoma to cervical lymph nodes, and all patients received similar radiotherapy regimen. The light microscopic features were similar with epithelioid histiocytes and granulomas with Langhan's giant cells. In 3 cases, acid-fast bacilli were identified by Ziehl Neelsen stain, and 1 was negative. The results of 2 cases were confirmed by polymerase chain reaction analysis for Myocbacterium tuberculosis DNA. CONCLUSION: Granulomatous reaction after radiotherapy of nasopharyngeal undifferentiated carcinoma can be caused by tuberculosis. SIGNIFICANCE: Diligent search for organisms in postirradiation granulomatous inflammation is warranted to avoid missing an occult tuberculosis infection.

Expression of Epstein-Barr virus-encoded small nuclear RNA 1 in Japanese nasopharyngeal carcinomas.

An examination was made of the incidence of the Epstein-Barr virus (EBV) genome and its exact localization in 39 cases of nasopharyngeal carcinoma (NPC) in Japanese patients by means of in situ hybridization (ISH) with a digoxigenin-labeled Epstein-Barr virus-encoded small nuclear RNA 1 (EBER1) oligonucleotide probe. Hybridization signals were observed in the nucleus of tumor cells in all 39 NPCs, including keratinizing carcinomas. The signals varied greatly in intensity from case to case and even from cell to cell in the same tumor, but were recognized in most tumor cells in each case. Signals could occasionally be seen in limiting number of infiltrating small lymphocytes but were absent in all tumors of the tongue, midpharynx and hypopharynx. Combined immunohistochemistry-ISH studies indicated that EBER1 signals were restricted to tumor cells positive for cytokeratin. As a result of this study, it is now possible to perform large-scale retrospective analyses using routine formalin-fixed, paraffin-embedded tissue sections and to combine ISH for the EBV genome with immunohistochemistry for cytokeratin to determine the epithelial features of EBV genome-possessing cells. All NPCs were clearly shown to be EBV-infected, thus indicating that EBV is essential for the oncogenesis of NPCs.

Subgingival microbiota of shallow periodontal pockets in individuals after head and neck irradiation.

This study aimed at investigating the subgingival plaque microorganisms of shallow pockets (< or = 5 mm) in subjects who previously received irradiation in the head and neck region for treatment of nasopharyngeal carcinoma. Direct microscopy and anaerobic culture were used. Subgingival paper point samples were taken from 6 tooth-sites (one/sextant) per subject for direct microscopy (n = 108). Another set of paper points was taken from the deepest of the previously selected sites (one per subject) with: group A) no bleeding on probing to the sulcus depth (n = 9) and group B) bleeding on probing to the sulcus depth (n = 6) for microscopic and anaerobic culture study. Under the microscope, the microflora was found to be a complex mixture comprising gram-positive and gram-negative cocci, rods and filaments, fusiforms, curved rods and spirochetes. Low level of fungi were observed and mycelia were occasionally detected. There was no significant variation in the plaque bacterial morphotypes observable according to sites of isolation and no significant difference between group A and group B in morphotypes of the different microflora. The predominant cultivable microflora comprised several species of facultative and obligate anaerobic bacteria: Gemella, Peptostreptococcus, Staphylococcus, Stomatococcus, Streptococcus, Actinomyces, Eubacterium, Lactobacillus, Propionibacterium, Neisseria, Veillonella, Bacteroides, Campylobacter, Capnocytophaga, Fusobacterium, Kingella, Porphyromonas and Prevotella species. There was no difference between the two groups except the significantly higher proportion of Kingella dentrificans isolated from group B sites. However, colonization of the gingival sulcus in these individuals by microbes that are normal flora of: skin (Peptostreptococcus prevotii and Propionibacterium granulosum) and gut (Eubacterium aerofaciens, Fusobacterium mortiferum and Fusobacterium varium) was detected. These findings appear to suggest that the major components of the subgingival microflora of shallow sites in previously head- and neck-irradiated individuals are similar to that of gingivitis sites in the normal population although they may contain bacterial or fungal species uncommon in normal subjects.

Detection of EBV in tumor tissue and peripheral blood by polymerase chain reaction in patients with nasopharyngeal carcinoma.

Sixty-nine untreated patients with a pathologically verified nasopharyngeal carcinoma were selected for the study of detection of EBV in nasopharyngeal tumor and peripheral blood by polymerase chain reaction (PCR). Primers were directed to conserved regions of EBV genome encoding capsid protein gp 220 (Bam HI L region). A distinct 239 bp band of the PCR products indicated the presence of EBV. Results showed that EBV DNA was obtained in 91.3% of 69 NPC patients and 16.7% of 18 healthy individuals on nasopharyngeal tissue, and the difference was statistically significant between the above two groups. Nevertheless, no EBV DNA was verified from the mononuclear cells of the peripheral blood of the two groups. There was no relationship between the positive EBV DNA and the titer of serological markers. Meanwhile, the positive EBV DNA did not show any relationship with the histology type, tumor and nodal bulk, or even metastasis. Although a high positive rate of EBV DNA was detected in nasopharyngeal tumor of patients, additional environmental and genetic factors must still be considered.