Patient characteristics
A total of 20 patients were recruited in this study. Patient characteristics are summarised in Table S1. Briefly, 80% of the patients were males with a median age of 65.5 years. Among patients, 75% were either smokers or ex-smokers and 85% reported drinking less than 10 drinks per week. Tumours were located either in the oral cavity (20%), oropharynx (25%), nasal cavity (10%), salivary glands (30%), or HN skin (15%). Half of the patients had early-stage disease (I/II) and the remaining had late-stage disease (III/IV). All patients completed the planned radiotherapy course except for one who discontinued treatment after completing two fractions and hence they were excluded from treatment-related factors analysis. Patients were treated with either radiotherapy alone (31.6%), postoperative radiotherapy (47.4%), chemoradiotherapy (15.8%), or postoperative chemoradiotherapy (5.3%). Overall, patients received an average of 58.62 ± 8.78 Gy cumulative dose in 2.53 ± 1.21 fraction over 5.53 ± 1.46 weeks with 79% treated for curative intent.
Among 19 patients who completed treatment, two received palliative treatment (36 Gy; 6 Gy/F) over 2 weeks. Due to the low exposure, they were excluded from treatment outcomes analyses. Among 17 patients included, 17.7%, 35.3%, 29.4%, and 17.7% experienced G1, G2, G3, and G4 OM respectively. Further, three patients (17.6%) developed recurrence within 12 months post-treatment completion.
Characterisation of HNC patients’ gut microbiota
First, we characterised the gut microbiota of all 20 patients. At the genus level, patients’ gut microbiota was predominantly composed of Bacteroides (39.9%), unclassified Ruminococcaceae (7.4%), Faecalibacterium (6.8%), Parabacteroides (5.6%), and unclassified Lachnospiraceae (4.8%) (Fig. 1A). The average number of positive OTUs was 603.9 [229 - 864 range] and the average Shannon index value was 3.2 [1.3 - 4.1 range] (Fig. S1A & S2A).
Sex was the only factor associated with a significant difference in the microbial diversity and richness between patients. Female patients had significantly lower OTUs richness (p= 0.0007) and alpha diversity (p= 0.0289). Moreover, the gut microbiota of male and female patients clustered in distinctive patterns as shown by PCoA (p= 0.0052) (Fig. 1B-D). Further, five genera, mainly Prevotella and Phascolarctobacterium, were enriched in males while unclassified Lactobacillales and P-75-a5 were increased in females (Fig. 1E).
Although there was no significant difference in the microbial richness and diversity based on other factors (Fig. S1B-M & S2C-N), specific genera were found to be enriched in specific subgroups. For instance, Faecalibacterium, Paraprevotella, and Ruminococcus-2 were enriched in <50, 55-65, and >65 age groups respectively (Fig. 1F). Further, patients with cutaneous tumours had an increased abundance of unclassified RF32 while SMB35 was increased among patients with salivary gland tumours (Fig. 1G). Phascolarctobacterium was increased in early-stage disease while Enterococcus was enriched in the advanced disease group (Fig. 1H). Moreover, Phascolarctobacterium was enriched in patients with HPV+ tumours (Fig. 1I). The unclassified Enterobacteriaceae was enriched in patients treated with radiotherapy alone while Faecalibacterium and Phascolarctobacterium were increased in those treated with postoperative radiotherapy and chemoradiotherapy respectively (Fig. 1J). Differential compositional changes based on other patient-related factors were also observed (Fig. S3A-H).
Risk factors associated with OM
Three patients were excluded from OM severity analysis (discontinued treatment/received low radiation doses). Patients were divided into either mild/moderate OM (G1-2) or severe OM (G3-4). In this cohort, there was no significant impact of all factors, except treatment type, on OM severity. Expectedly, 75% of patients with tumours in the oral cavity or oropharynx developed severe OM compared to only 22% of patients with tumours in other sites, but the difference was not statistically significant. However, those treated with chemoradiotherapy had significantly more severe OM (100%) compared to those who received radiotherapy without chemotherapy (30.8%) (p= 0.029) (Table 1).
Table 1: Risk factors associated with OM severity
|
G1-2 (n=9)
|
G3-4 (n=8)
|
P value
|
Age (Year; mean ± SD)
|
67.89 ± 10.83
|
62.13 ± 9.73
|
0.269
|
BMI (Mean ± SD)
|
28.62 ± 5.95
|
25.54 ± 3.28
|
0.241
|
Sex, n (%)
|
|
|
|
Male
|
6 (46.1)
|
7 (53.9)
|
0.577
|
Female
|
3 (75.0)
|
1 (25.0)
|
|
Tobacco smoking, n (%)
|
|
|
|
Non-smoker
|
2 (50.0)
|
2 (50.0)
|
>0.999
|
Ex-smoker/ Smoker
|
7 (53.9)
|
6 (46.1)
|
|
Alcohol (# drinks/week), n (%)
|
|
|
|
≤10
|
8 (57.1)
|
6 (42.9)
|
0.577
|
>10
|
1 (33.3)
|
2 (66.7)
|
|
Antibiotics (B/D radiotherapy), n (%)
|
|
|
|
Yes
|
4 (50.0)
|
4 (50.0)
|
>0.999
|
No
|
5 (55.6)
|
4 (44.4)
|
|
Tumour site, n (%)
|
|
|
|
Within the oral cavity (Oral cavity/ Oropharynx)
|
2 (25.0)
|
6 (75.0)
|
0.057
|
Outside the oral cavity (parotid gland/ nasal cavity/ HN skin)
|
7 (77.8)
|
2 (22.2)
|
|
Treatment type, n (%)
|
|
|
|
Radiotherapy
|
9 (69.2)
|
4 (30.8)
|
0.029*
|
Chemoradiotherapy
|
0
|
4 (100)
|
|
Cumulative dose (Gy; mean ± SD)
|
59.89 ± 4.26
|
62.84 ± 4.47
|
0.184
|
Treatment period (Week; mean ± SD)
|
5.78 ± 0.67
|
6.13 ± 1.13
|
0.445
|
B/D, Before or during radiotherapy; Unpaired T-test; Fisher's exact test; * p< 0.05
|
Gut microbiota traits associated with OM
Characterising the gut microbiota based on OM severity, the most abundant genera in the G1-2 OM group were Bacteroides (40%), Parabacteroides (7.8%), Faecalibacterium (6.9%), unclassified Ruminococcaceae (6.8%), and unclassified Clostridiales (4.7%) compared to Bacteroides (41.9%), Faecalibacterium (7.9%), unclassified Ruminococcaceae (7.2%), Prevotella (5.5%) and unclassified Lachnospiraceae (4.2%) in G3-4 OM group (Fig. 2A) (Table S2). Although there was no significant difference in the OTUs richness, alpha, and beta diversity between groups (Fig. S4A-C), Eubacterium, Victivallis, Ruminococcus, Oxalobacter, unclassified Victivallaceae, and unclassified desulfovibrionaceae were significantly increased in patients with G3-4 OM while unclassified RF32, Alistipes, and unclassified ML615J-28 were increased in those with G1-2 OM (Fig. 2B) (Table S4-S5).
Among the six genera enriched in the G3-4 OM group, the relative abundance of Eubacterium (p= 0.019), Victivallis (p= 0.016), and Ruminococcus (p= 0.027) was significantly higher in G3-4 compared to G1-2 OM group (Fig. 2C-E). Eubacterium and Ruminococcus genera were most abundant in patients with G3 OM while Victivallis was most abundant among patients with G4 OM (Fig. 2F-H). In contrast, the relative abundance of unclassified RF32 genus (p= 0.032) was significantly higher among patients with G1-2 OM and was most abundant among patients with G2 OM (Fig. 2I-J). Correlation analysis showed a significant positive correlation between the relative abundance of Victivallis and OM severity grade (r= 0.67, p= 0.003) (Fig. 3).
Risk factors associated with tumour recurrence
Among 17 patients included in tumour recurrence analysis, 14 patients did not develop tumour recurrence while 3 patients had recurrence within 12 months of treatment completion. Overall, there was no significant association between any of the patients and treatment-related factors and tumour recurrence (Table 2). Those who developed recurrence had tumours in the oropharynx, nasal cavity, or salivary gland. One of them had early-stage disease and two had advanced-stage disease. All of these patients received similar treatment; however, 2 out of these three patients had treatment breaks or delays.
Table 2: Patient and treatment-related factors associated with tumour recurrence
|
No REC
(n= 14)
|
REC
(n= 3)
|
P
value
|
Age (Year; mean ± SD)
|
63.57 ± 10.65
|
72.67 ± 5.69
|
0.178
|
Sex, n (%)
|
|
|
|
Male
|
12 (92.3)
|
1 (7.7)
|
0.121
|
Female
|
2 (50)
|
2 (50)
|
|
BMI (Mean ± SD)
|
28.09 ± 4.88
|
21.56 ± 1.12
|
0.088
|
Smoking, n (%)
|
|
|
|
Non-smoker
|
3 (75.0)
|
1 (25.0)
|
>0.999
|
Ex-smoker/Smoker
|
11 (84.6)
|
2 (15.4)
|
|
Alcohol (# drinks/week), n (%)
|
|
|
|
≤10
|
11 (78.6)
|
3 (21.4)
|
>0.999
|
>10
|
3 (100)
|
0
|
|
Antibiotics (B/D radiotherapy), n (%)
|
|
|
|
Yes
|
8 (100)
|
0
|
0.206
|
No
|
6 (66.7)
|
3 (33.3)
|
|
Tumour site, n (%)
|
|
|
|
Oral cavity
|
3 (100)
|
0
|
-
|
Oropharynx
|
4 (80)
|
1 (20)
|
|
Nasal cavity
|
1 (50)
|
1 (50)
|
|
Salivary gland
|
4 (80)
|
1 (20)
|
|
HN skin
|
2 (100)
|
0
|
|
Tumour stage, n (%)
|
|
|
|
Early stage (I/ II)
|
8 (88.9)
|
1 (11.1)
|
0.577
|
Advanced disease (III/ IV)
|
6 (75)
|
2 (25)
|
|
HPV+, n (%)
|
4 (100)
|
0
|
-
|
Treatment type, n (%)
|
|
|
|
Radiotherapy
|
10 (76.9)
|
3 (23.1)
|
0.541
|
Chemoradiotherapy
|
4 (100)
|
0
|
|
Cumulative dose (Gy; mean ± SD)
|
61.41 ± 3.39
|
60.67 ± 9.24
|
0.665
|
Dose/Fraction (Gy/F)
|
2.11 ± 0.23
|
2.13 ± 0.12
|
0.337
|
Treatment period (Week; mean ± SD)
|
6.00 ± 0.78
|
5.67 ± 1.51
|
0.941
|
Treatment intent, n (%)
|
|
|
|
Curative
|
13 (86.7)
|
2 (13.3)
|
0.331
|
Palliative
|
1 (50)
|
1 (50)
|
|
Treatment gaps/breaks, n (%)
|
|
|
|
Yes
|
2 (50)
|
2 (50)
|
0.121
|
No
|
12 (92.3)
|
1 (7.7)
|
|
REC: recurrence; B/D, Before or during; Unpaired T-test; Fisher's exact test
|
Gut microbiota traits associated with tumour recurrence
Characterising the gut microbiota based on tumour recurrence, the most abundant genera among patients with no recurrence (no REC) were Bacteroides (39%), Faecalibacterium (8.9%), unclassified Ruminococcaceae (7.2%), Parabacteroides (5.9%), and Prevotella (4.9%) compared to Bacteroides (50%), unclassified Clostridiales (6.4%), unclassified Ruminococcaceae (6.0%), Parabacteroides (5.7%), and Blautia (4.5%) in recurrence (REC) group (Fig. 4A) (Table S3). Generally, there was no significant difference in the number of OTUs, alpha, and beta diversity between groups (Fig. S5A-B & Fig. 4B). However, Faecalibacterium, Prevotella, and Phascolarctobacterium were enriched in patients with no recurrence, and Adlercreutzia, Pseudoramibacter_Eubacterium, Desulfitobacter, Eggerthella, Megasphaera, and p-75-a5 were increased in patients with recurrence (Fig. 4C) (Table S4-S5). The relative abundance of Faecalibacterium (p= 0.029), Prevotella (p= 0.031), and Phascolarctobacterium (p= 0.019) was significantly higher in patients with no recurrence (Fig. 4D-F). Further, patients who did not develop recurrence also had a significantly higher Prevotella to Bacteroides (P/B) ratio (p= 0.047) (Fig. 4G). Conversely, the relative abundance of Adlercreutzia (p= 0.006) and Eggerthella (p= 0.006) genera was significantly higher in patients with recurrence (Fig. 4H-I). There was no significant difference between recurrence and no recurrence groups in the relative abundance of other genera (Fig. S5C-F).