Impact of preoperative antibiotics and other variables on integrated microbiome-host transcriptomic data generated from colorectal cancer resections

Table 1 Specific Taqman polymerase chain reaction primer and probe sequences for the Fusobacterium nucleatum nusG gene and broad range Taqman primer and probe sequences for the bacterial 16S rRNA gene

Target gene	Sequence
F. nucleatum nusG gene forward primer[29]	5’-CAACCATTACTTTAACTCTACCATGTTCA-3’
F. nucleatum nusG gene reverse primer[29]	5’-GTTGACTTTACAGAAGGAGATTATGTAAAAATC-3’
F. nucleatum nusG gene probe[30]	5’-TCAGCAACTTGTCCTTCTTGATCTTTAAATGAACC-3’
Broad range 16S rRNA gene forward primer[31]	5’-TCCTACGGGAGGCAGCAGT-3’
Broad range 16S rRNA gene reverse primer[31]	5’- GGACTACCAGGGTATCTAATCCTGTT-3’
Broad range 16S rRNA gene probe[31]	5’-CGTATTACCGCGGCTGCTGGCAC-3’

F. nucleatum: Fusobacterium nucleatum.

Table 2 Comparison of patient characteristics between the subset of sporadic colorectal cancer samples analyzed with that of the total collection of archived samples in the Stony Brook Cancer Center Tissue Analytics Shared Resource (2010-2020)

	Subset analyzed, n = 51	Total collection, n = 190	P value
Age (yr) ± IQR	62 ± 20	65 ± 20	0.4329
Male sex (%)	30 (58.8)	115 (60.5)	0.8254
Race (%)			0.0295
White	38 (74.5)	163 (85.8)
Black	11 (21.6)	14 (7.4)
Asian	1 (2.0)	6 (3.2)
Other	1 (1.9)	7 (3.7)
Hispanic ethnicity (%)	6 (11.8)	22 (11.6)	0.9707
Family history 1st degree relative (%)	7 (13.7)	34 (17.9)	0.4817
BMI (kg/m2)	27.8 ± 8	28.2 ± 8	0.5683
Diabetes mellitus (%)	18 (35.3)	55 (28.9)	0.4853
Smoking (%)			0.1103
Current	7 (13.7)	10 (5.3)
Past	21 (41.2)	88 (46.3)
Never	23 (45.1)	92 (48.4)
Anatomic location (%)			0.0210
Left	31 (60.8)	81 (42.6)
Right	20 (39.2)	109 (57.4)
Cancer stage (%)			0.4578
Stage 0	1 (2.0)	6 (3.2)
Stage 1	9 (17.6)	27 (14.2)
Stage 2	14 (27.4)	63 (33.2)
Stage 3	25 (49.0)	75 (39.5)
Stage 4	2 (3.9)	19 (15.8)
Preoperative antibiotics (%)	16 (31.4)	91 (47.9)	0.0350

IQR: Inter-quartile range; BMI: Body mass index.

Table 3 Estimated differences in alpha diversity indices between the following groups

Variable	Sobs		ShannonH		ShannonE
	Estimate (95%CI)	P value	Estimate (95%CI)	P value	Estimate (95%CI)	P value
Tumor vs nontumor	-1.34 (-4.04, 1.35)	0.32	-0.05 (-0.25, 0.15)	0.61	-0.004 (-0.04, 0.03)	0.77
Pre-op antibiotics vs none	2.93 (-3.51, 9.37)	0.36	0.27 (-0.12, 0.66)	0.17	0.04 (-0.02, 0.09)	0.20
Left vs right	2.83 (-2.44, 8.10)	0.29	-0.10 (-0.43, 0.23)	0.53	-0.03 (-0.08, 0.02)	0.26
Diabetic vs nondiabetic	3.73 (-2.30, 9.75)	0.22	0.26 (-0.11, 0.62)	0.16	0.03 (-0.02, 0.09)	0.22
Black/AA race vs not Black/AA race	2.70 (-4.43, 9.83)	0.45	0.09 (-0.34, 0.52)	0.69	0.01 (-0.06, 0.07)	0.80

Linear mixed models were used to estimate differences. The P values are based on t-tests from the linear mixed models. CI: Confidence interval; AA: African Ancestry.

Table 4 PERMANOVA results for the following groups

Variable	P value
Tumor vs nontumor	0.002
Pre-operative antibiotics vs no preoperative antibiotics	0.001
Left vs right anatomic location	0.367
Diabetes mellitus vs not diabetic	0.061
Black/AA race vs not Black/AA race	0.258

AA: African Ancestry.

Table 5 Operational taxonomic units (genus level) with significant tumor histology*pre-operative antibiotics interaction terms

OTU (genus level)
Actinobacteria/Gardnerella
Actinobacteria/Eggerthella
Firmicutes/Bacilli/unspecified
Firmicutes/Staphylococcus
Firmicutes/Parvimonas
Firmicutes/Anaerostipes
Firmicutes/Howardella
Firmicutes/Solobacterium
Fusobacteria/Fusobacteriales/unspecified
Fusobacteria/Leptotrichia
Proteobacteria/Sutterella
Proteobacteria/Ralstonia
Proteobacteria/Enterobacter
Proteobacteria/Haemophilus

OTU: Operational taxonomic unit.

Table 6 The operational taxonomic units (genus level) without significant tumor histology*preoperative antibiotics interaction terms, with significant (P < 0.05 after Bonferroni correction) estimated tumor/nontumor ratios of relative abundance and 95% confidence intervals

OTU	Ratio (95%CI)	P value
Increased in tumor
Firmicutes/Carnobacteriaceae/unspecified	22.21 (7.57, 65.21)	< 0.0001
Firmicutes/ Peptoniphilus	2.13 (1.45, 3.13)	0.0042
Firmicutes/Catenibacterium	3.89 (1.79, 8.46)	0.0206
Firmicutes/Dialister	2.05 (1.48, 2.83)	0.0005
Fusobacteria/Fusobacterium	5.30 (2.82, 9.97)	< 0.0001
Proteobacteria/Campylobacter	7.40 (3.30, 16.62)	< 0.0001
Proteobacteria/Citrobacter	3.51 (2.12, 5.81)	< 0.0001
Decreased in tumor
Actinobacteria/Coriobacteriaceae/unspecified	0.45 (0.32, 0.64)	0.0003
Bacteroidetes/unspecified	0.49 (0.32, 0.74)	0.0240
Bacteroidetes/S24-7	0.50 (0.36, 0.70)	0.0016
Cyanobacteria/Chloroplast	0.44 (0.31, 0.64)	0.0021
Firmicutes/Lachnospiraceae/unspecified	0.76 (0.68, 0.84)	< 0.0001
Firmicutes/Marvinbryantia	0.39 (0.24, 0.65)	0.0113
Firmicutes/vadinBB60	0.50 (0.36, 0.70)	0.0052
Firmicutes/Turicibacter	0.44 (0.30, 0.66)	0.0074
Firmicutes/Allobaculum	0.43 (0.29, 0.62)	0.0002
Proteobacteria/Rickettsiales/mitochondria	0.32 (0.19, 0.53)	0.0003
Tenericutes/Anaeroplasma	0.42 (0.28, 0.62)	0.0004
Verrucomicrobia/Akkermansia	0.58 (0.42, 0.80)	0.0350

OTU: Operational taxonomic unit; CI: Confidence interval.

Table 7 Comparison of the mean quantitative polymerase chain reaction mean threshold cycles ± SD between genomic DNA extracted from paired frozen and formalin-fixed paraffin embedded samples

	F. nucleatum nusG		16S rRNA		Human SLCO2A1
	Frozen	FFPE	Frozen	FFPE	Frozen	FFPE
Tumor A	25.9 ± 1.2	35.4 ± 0.1	21.8 ± 0.1	31.4 ± 0.1	32.6 ± 2.5	39.3 ± 0.6
Nontumor A	37.2 ± 0.6	39.9/ND	22.2 ± 0.1	31.7 ± 0.3	34.8 ± 0.5	ND
Tumor B	26.0 ± 1.0	27.5 ± 1.0	21.6 ± 0.2	28.3 ± 0.1	33.2 ± 1.0	32.5 ± 1.3
Nontumor B	31.5 ± 1.4	35.1 ± 0.7	25.2 ± 0.1	31.2 ± 0.6	34.6 ± 0.9	38.2 ± 0.1

F. nucleatum: Fusobacterium nucleatum; FFPE: Formalin-fixed paraffin embedded; ND: Not detected.

Table 8 Differentially expressed genes, (+)antibiotics vs (-)antibiotics, common to both tumor and nontumor samples

Gene (increased)	Symbol	Tumor log2 fold change	Tumor adjusted P value	Nontumor log2 fold change	Nontumor adjusted P value
ENSG00000023902	PLEKHO1	1.127	0.0461	1.694	0.0130
ENSG00000253304	TMEM200B	1.326	0.0445	1.719	0.0481
ENSG00000187608	ISG15	1.534	0.0301	2.202	0.0102
ENSG00000185745	IFIT1	1.708	0.0243	2.245	0.0176
ENSG00000109705	NKX3-2	2.082	0.0476	2.859	0.0264
ENSG00000145936	KCNMB1	2.011	0.0480	2.874	0.0176

Table 9 Differentially expressed genes, Black/African Ancestry vs not Black/African Ancestry, common to both tumor and nontumor samples

Gene	Symbol	Tumor log2 fold change	Tumor adjusted P value	Nontumor log2 fold change	Nontumor adjusted P value
Decreased
ENSG00000155959	VBP1	-8.217	5.26 × 10-10	-8.318	5.26 × 10-10
ENSG00000102109	PCSK1N	-5.965	0.0050	-7.175	0.0004
Increased
ENSG00000225972	MTND1P23	4.0913	5.67 × 10-7	3.801	0.0001