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Mucosal cancer-associated microbes and anastomotic leakage after resection of colorectal carcinoma
Surgical Oncology 32 (2020) 63–68
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Surgical Oncology journal homepage: http://www.elsevier.com/locate/suronc
Mucosal cancer-associated microbes and anastomotic leakage after resection of colorectal carcinoma Kosuke Mima a, b, Yuki Sakamoto a, Keisuke Kosumi c, Yoko Ogata a, Keisuke Miyake a, Yukiharu Hiyoshi a, Takatsugu Ishimoto a, Masaaki Iwatsuki a, Yoshifumi Baba a, Shiro Iwagami a, Yuji Miyamoto a, Naoya Yoshida a, Shuji Ogino c, d, e, f, Hideo Baba a, * a
Department of Gastroenterological Surgery, Graduate School of Medical Science, Kumamoto University, Kumamoto, Japan Department of Surgery, National Hospital Organization Kumamoto Medical Center, Kumamoto, Japan Department of Oncologic Pathology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA, USA d Broad Institute of MIT and Harvard, Cambridge, MA, USA e Program in MPE Molecular Pathological Epidemiology, Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA f Department of Epidemiology, Harvard T.H. Chan School of Public Health, Boston, MA, USA b c
A R T I C L E I N F O Presentation: This work was presented at the SSO 2019 Annual Cancer Symposium; March 27th – 30th, 2019. Keywords: Bacteria Complication Surgery
A B S T R A C T
Background: Clinical and experimental evidence suggests that colorectal mucosal microbiota changes during colorectal carcinogenesis and may impair colorectal anastomotic wound healing. Thus, we hypothesized that amounts of colorectal cancer-associated microbes in colorectal tissue might be associated with anastomotic leakage after resection for colorectal carcinoma. Methods: We analyzed 256 fresh frozen tissues of colorectal cancer from patients who underwent elective colorectal resection and anastomosis. Amounts of colorectal cancer-associated microbes, including Fusobacterium nucleatum, Escherichia coli possessing the polyketide synthase (pks) gene cluster, Enterococcus faecalis, and Bifi dobacterium genus, in colorectal cancer tissues were measured by quantitative polymerase chain reaction assay; we equally dichotomized positive cases (high versus low). Multivariable logistic regression analysis was con ducted to assess associations of these microbes with anastomotic leakage, adjusting for patient and tumor characteristics, and surgery-related factors. Results: Fusobacterium nucleatum, pks-positive Escherichia coli, Enterococcus faecalis, and Bifidobacterium genus were detected in colorectal carcinoma tissue in 140 (54%), 94 (36%), 193 (75%), and 89 (35%) of 256 cases, respectively. Compared with Bifidobacterium genus-negative cases, Bifidobacterium genus-high cases were asso ciated with an increased risk of anastomotic leakage (multivariable odds ratio, 3.96; 95% confidence interval, 1.50 to 10.51; Ptrend ¼ 0.004). The association of Fusobacterium nucleatum, pks-positive Escherichia coli, or Enterococcus faecalis with anastomotic leakage was not statistically significant. Conclusions: The amount of Bifidobacterium genus in colorectal tissue is associated with an increased risk of anastomotic leakage after resection for colorectal cancer. These findings need to be validated to target gastro intestinal microflora for the prevention of anastomotic leakage after colorectal resection.
1. Introduction More than 100 trillion microorganisms inhabit the human gastroin testinal tract and play important roles in health conditions, immunity, and diseases including colorectal cancer [1–3]. Human colorectal mucosal microbiota has been shown to change during the development
and progression of colorectal neoplasia [4–6]. Experimental evidence from animal models demonstrates that intestinal microbes, such as Fusobacterium nucleatum, Escherichia coli possessing the polyketide syn thase (pks) gene cluster, Enterococcus faecalis, and Bifidobacterium genus, can influence the development and progression of colorectal neoplasms by damaging DNA, producing metabolites, and modulating host immune
Abbreviations: CI, confidence interval; Ct, cycle threshold; OR, odds ratio; PCR, polymerase chain reaction. * Corresponding author. Department of Gastroenterological Surgery, Graduate School of Medical Science Kumamoto University, 1-1-1 Honjo, Chuo-ku, Kumamoto, 860-8556, Japan. E-mail address: [email protected] (H. Baba). https://doi.org/10.1016/j.suronc.2019.11.005 Received 27 January 2019; Received in revised form 10 November 2019; Accepted 17 November 2019 Available online 18 November 2019 0960-7404/© 2019 Elsevier Ltd. All rights reserved.
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response and intestinal inflammation [3,7]. Consistent with these lines of experimental evidence, human studies have shown associations of these microbes with antitumor immune response, intestinal inflamma tion, and colorectal neoplasms [8–13]. In 1313 colorectal cancers from U.S. nationwide prospective cohort studies, the amount of intratumor Bifidobacterium genus has been associated with the extent of signet ring cells in colorectal cancer tissue [14]. Although surgery with complete resection represents a potentially curative treatment for colorectal carcinoma, anastomotic leakage after colorectal resection remains the major cause for morbidity and mortality [15,16]. Emerging evidence from animal models indicates that colo rectal mucosal microbes may impair colorectal anastomotic wound healing after colorectal resection [17–23]. However, the relationship between the complex intestinal microbiota and wound healing of colo rectal anastomoses in humans cannot be completely recapitulated in animal models, analysis using human tissue is required for clinical application. We hypothesized that higher amounts of these colorectal cancer-associated microbes in human colorectal tissue might be associ ated with an increased risk of anastomotic leakage after resection of colorectal carcinoma. A better understanding of roles of the intestinal microbes in the incidence of anastomotic leakage after resection of colorectal cancer may provide new opportunities to target the intestinal microbes for postoperative recovery in patients with colorectal cancers. To test our hypothesis, we examined associations of the amounts of colorectal cancer-associated microbes, including Fusobacterium nuclea tum, pks-positive Escherichia coli, Enterococcus faecalis, and Bifidobacte rium genus, in human colorectal carcinoma tissue with the incidence of anastomotic leakage after resection of colorectal carcinoma.
Enterococcus faecalis and Bifidobacterium genus, and the reference human gene, SLCO2A1 were used as previously described [8,24–26]. Each re action contained 12.5 ng of genomic DNA and was assayed in 10 μL reactions containing 1 � final concentration LightCycler 480 Probe Master (Roche) and each TaqMan Gene Expression Assay (Applied Biosystems), in a 384-well optical PCR plate. Amplification and detec tion of DNA was performed with a LightCycler 480 Instrument II (Roche) using the following reaction conditions: 10 min at 95 � C and 45 cycles of 15 s at 95 � C and 1 min at 60 � C. The primer and probe sequences for each TaqMan Gene Expression Assay were as follows: Fusobacterium nucleatum forward primer, 50 TGGTGTCATTCTTCCAAAAATATCA-3’; Fusobacterium nucleatum reverse primer, 50 -AGATCAAGAAGGACAAGTTGCTGAA-3’; Fusobacte rium nucleatum FAM probe, 50 -ACTTTAACTCTACCATGTTCA -3’; pkspositive Escherichia coli forward primer, 50 - TGCTATGTATTCACG CAAACG-3’; pks-positive Escherichia coli reverse primer, 50 CGTTGCTCTCCATGACCTG-3’; pks-positive Escherichia coli probe, 50 CTGGCTGG-3’; Enterococcus faecalis forward primer, 50 AAACCTTTCCCTGGTGTTCA-3’; Enterococcus faecalis reverse primer, 50 CGTTAATCCCTGTTGAAGCAA-3’; Enterococcus faecalis probe, 50 TTCTGGCT-3’; Bifidobacterium genus forward primer, 50 -CGGGTGAG TAATGCGTGACC-3’; Bifidobacterium genus reverse primer, 50 - TGA TAGGACGCGACCCCA-3’; Bifidobacterium genus FAM probe, 50 CTCCTGGAAACGGGTG-3’; SLCO2A1 forward primer, 50 -ATCCC CAAAGCACCTGGTTT-3’; SLCO2A1 reverse primer, 50 -AGAGGCCAA GATAGTCCTGGTAA-3’; SLCO2A1 VIC probe, 50 CCATCCATGTCCTCATCTC-3’. Each specimen was analyzed in duplicate for each target in a single batch, and we used the average of the two cycle threshold (Ct) values for each target. The amount of Fusobacterium nucleatum, pks-positive Escherichia coli, Enterococcus faecalis, or Bifidobacterium genus in each specimen was calculated as a relative unitless value normalized with SLCO2A1 using the 2-ΔCt method (where ΔCt ¼ “the average Ct value of intestinal microbes” - “the average Ct value of SLCO2A1”) as previously described [8,27].
2. Methods 2.1. Subjects and specimens We analyzed 256 fresh frozen colorectal cancer tissue specimens from patients who underwent elective colorectal resection and anasto mosis for colorectal cancer at Kumamoto University Hospital in Japan. All patients had histologically confirmed colorectal carcinoma. The pathologic diagnoses and the clinicopathological factors were estab lished based on the American Joint Committee on Cancer/International Union Against Cancer staging system. Written informed consent was obtained from each patient. This study was approved by the Human Ethics Review Committee of the Graduate School of Medicine, Kuma moto University (Kumamoto, Japan). All patients received mechanical bowel preparation with 2L of polyethylene glycol 1 day before surgery. None of the patients received preoperative oral antibiotics. All of the patients received 1 g of cefme tazole intravenously after the induction of anesthesia and within 60 min before skin incision. Subsequently, 1 g of cefmetazole was additionally given every 3 h during surgery. The antibiotic was discontinued within 24 h after surgery. Colorectal carcinoma tissue specimens and normal colorectal mu cosa tissue specimens that were at least 5–10 cm from the edge of the tumor tissues were cut using a sterile blade and placed in a cryotube and frozen immediately in liquid nitrogen within 30 min after colorectal resection. The frozen tissue was stored at 80 � C until DNA extraction.
2.3. Definition of anastomotic leakage The definition of anastomotic leakage was used as previously re ported clinical trials [28,29]; peritonitis from any staple line, and pelvic abscess without radiologically proven leakage mechanism were included. Leakage was verified by clinical (inspection of drain contents), endoscopic (flexible sigmoidoscopy), or radiologic (rectal contrast study, computed tomography scan) interventions. 2.4. Statistical analysis All statistical analyses were conducted using JMP (version 12.2, SAS Institute, Cary, NC) and all P values were two-sided. Neither the amounts of intestinal microbes, including Fusobacterium nucleatum, pkspositive Escherichia coli, Enterococcus faecalis, and Bifidobacterium genus, nor the log-transformed value of the amounts of these intestinal mi crobes fit a normal distribution with the use of the Shapiro-Wilk test for normality (P < 0.0001). Thus, our primary hypothesis testing was the linear trend test in a logistic regression model to assess associations of the amounts of these intestinal microbes in colorectal carcinoma tissue (an ordinal predictor variable) with the incidence of anastomotic leakage after resection of colorectal carcinoma (a dichotomous outcome variable), as previously described [8,14,30]. Cases with detectable these microbes were categorized as low or high based on the median cutpoint amount of the microbes, while cases without detectable the microbes were categorized as “negative”. The linear trend test was performed using the ordinal predictor variable of the microbes (negative, low, and high) as a continuous variable in a logistic regression model. All statis tical tests were two-sided at α level of 0.005, considering the multiple comparisons and the consequent false positives [31].
2.2. Quantitative polymerase chain reaction (PCR) for intestinal microbes Genomic DNA was extracted from colorectal carcinoma tissue spec imens and normal colorectal mucosa tissue specimens, using QIAamp DNA Mini Kit (Qiagen). We performed quantitative PCR assays to measure the amount of tissue DNA of Fusobacterium nucleatum, pkspositive Escherichia coli, Enterococcus faecalis, or Bifidobacterium genus. Custom TaqMan primer/probe sets (Applied Biosystems) for the nusG gene of Fusobacterium nucleatum, the pks colibactin gene cluster of Escherichia coli, the 16S ribosomal RNA gene DNA sequences of 64
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We performed multivariable logistic regression analysis to adjust for potential confounders. The multivariable model initially included age (�64 vs. 65–74 vs. � 75), sex, body mass index (<30 kg/m2 vs. � 30 kg/ m2), diabetes (present vs. absent), serum albumin levels (<3 g/dl vs. � 3 g/dl), tumor location (colon vs. rectum), pT stage (pT1-3 vs. pT4), lymph node metastasis (negative vs. positive), distant metastasis (negative vs. positive), surgical approach (open or laparoscopy con verted vs. laparoscopy), operating time (<240 min vs. � 240 min), intraoperative bleeding (<300 mL vs. � 300 mL), and diverting ileos tomy (present vs. absent). A backward stepwise elimination with a threshold of P < 0.05 was used to select variables in the final models. To assess associations between the dichotomous category (absence and present) of the incidence of anastomotic leak and categorical data, the chi-square test was performed.
Table 1 Patient and tumor characteristics, and surgery-related factors according to the incidence of anastomotic leakage after resection of colorectal carcinoma. Characteristica
3. Results 3.1. Patient and tumor characteristics, and surgery-related factors in relation to anastomotic leakage after resection of colorectal carcinoma Anastomotic leakage was diagnosed in 29 (11%) of 256 cases. Pa tient and tumor characteristics, and surgery-related factors are sum marized according to the incidence of anastomotic leakage in Table 1. Male sex and rectal tumor location were associated with anastomotic leakage (P < 0.001; with α level of 0.005).
All patients (n ¼ 256)
Patients without anastomotic leak (n ¼ 227)
Patients with anastomotic leak (n ¼ 29)
Age (year) �64 65-74 �75
90 (35%) 84 (33%) 82 (32%)
83 (36%) 72 (32%) 72 (32%)
7 (24%) 12 (41%) 10 (35%)
Sex Men Women
152 (59%) 104 (41%)
127 (56%) 100 (44%)
25 (86%) 4 (14%)
Body mass index (kg/m2) <30 247 (96%) �30 9 (3.5%)
218 (96%) 9 (4%)
29 (100%) 0
Diabetes No Yes
212 (83%) 44 (17%)
188 (83%) 39 (17%)
24 (83%) 5 (17%)
Serum albumin levels (g/dl) <3 21 (8.2%) �3 235 (92%)
18 (7.9%) 209 (92%)
3 (10%) 26 (90%)
83 (32%)
78 (34%)
5 (17%)
128 (50%)
117 (52%)
11 (38%)
45 (18%)
32 (14%)
13 (45%)
pT stage (depth of tumor invasion) pT1-3 209 (82%) pT4 47 (18%)
184 (81%) 43 (19%)
25 (86%) 4 (14%)
Lymph node metastasis Negative 160 (62%) Positive 96 (38%)
141 (62%) 86 (38%)
19 (66%) 10 (34%)
210 (82%) 46 (18%)
184 (81%) 43 (19%)
26 (90%) 3 (10%)
67 (26%)
55 (24%)
12 (41%)
189 (74%)
172 (76%)
17 (59%)
Operating time (minutes) <240 62 (24%) �240 194 (76%)
60 (26%) 167 (74%)
2 (6.9%) 27 (93%)
Intraoperative bleeding (mL) <300 198 (77%) �300 58 (23%)
179 (79%) 48 (21%)
19 (66%) 10 (34%)
Diverting ileostomy No Yes
209 (92%) 18 (7.9%)
25 (86%) 4 (14%)
Tumor location Caecum, ascending colon, transverse colon Descending colon, sigmoid colon, rectosigmoid junction Rectum
3.2. Associations of the amounts of colorectal cancer-associated microbes in tumor tissue with anastomotic leakage after resection of colorectal carcinoma We measured the relative amounts of Fusobacterium nucleatum, pkspositive Escherichia coli, Enterococcus faecalis, and Bifidobacterium genus in fresh frozen tumor tissue of 256 colorectal carcinoma cases, using the quantitative PCR assay. Fusobacterium nucleatum, pks-positive Escherichia coli, Enterococcus faecalis, and Bifidobacterium genus were detected in colorectal carcinoma tissue in 140 (54%), 94 (36%), 193 (75%), and 89 (35%) of 256 cases, respectively. We categorized colo rectal carcinoma cases with detectable these four microbes as low or high based on the median cutpoint amount of the microbes. Table 2 shows the distribution of colorectal carcinoma cases ac cording to the amounts of the microbes and the incidence of anastomotic leakage. In our primary hypothesis testing, we conducted univariable and multivariable logistic regression analyses to assess associations of the amounts of the microbes in colorectal carcinoma tissue (as ordinal predictor variables) with the incidence of anastomotic leakage (as a dichotomous outcome variable) (Table 3). The amount of Bifidobacte rium genus in colorectal carcinoma tissue was associated with an increased risk of anastomotic leakage in univariable (Ptrend ¼ 0.002) and multivariable logistic regression analysis (Ptrend ¼ 0.004). Compared with Bifidobacterium genus-negative cases, Bifidobacterium genus-high cases were associated with an increased risk of anastomotic leakage (multivariable odds ratio, 3.96; 95% confidence interval, 1.50 to 10.51; Table 3 and Supplementary Table S1). The association of the amount of Fusobacterium nucleatum, pks-positive Escherichia coli, or Enterococcus faecalis with the incidence of anastomotic leakage was not statistically significant (Ptrend > 0.039; with α level of 0.005; Table 3). Associations of the amount of Bifidobacterium genus in colorectal carcinoma tissue with the patient and tumor characteristics, including tumor location, were not statistically significant (with α level of 0.005; Supplementary Table S2). In our exploratory analysis, we measured the relative amount of Bifidobacterium genus in normal colorectal mucosa tissues that were at least 5–10 cm distant from the edge of the tumor in 209 colorectal carcinoma cases, using the quantitative PCR assay. The amount of Bifi dobacterium genus in normal colorectal mucosa tissue appeared to be associated with an increased risk of anastomotic leakage in univariable
Distant metastasis Negative Positive Surgical approach Open or laparoscopy converted Laparoscopy
234 (91%) 22 (8.6%)
P valueb
0.37
<0.001
0.14
0.99
0.67
<0.001
0.49
0.72
0.23 0.06
0.010
0.12
0.32
a Percentage (%) indicates the proportion of cases with patient and tumor characteristics or surgery-related factors according to the incidence of anasto motic leakage. b To assess associations between the dichotomous category of the incidence of anastomotic leakage and categorical variables, the chi-square test was per formed. All statistical tests were two-sided at α level of 0.005, considering the multiple comparisons and the consequent false positives.
65
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Table 2 Distribution of cases according to the amounts of colorectal cancer-associated microbes in colorectal carcinoma tissue and the incidence of anastomotic leakage after resection of colorectal carcinoma. The amount of microbes in colorectal carcinoma tissue
All patients (n ¼ 256)
Fusobacterium nucleatum Negative 116 (46%) Low 70 (27%) High 70 (27%) pks-positive Escherichia coli Negative 162 (64%) Low 47 (18%) High 47 (18%) Enterococcus faecalis Negative 63 (25%) Low 95 (37%) High 98 (38%) Bifidobacterium genus Negative 167 (65%) Low 44 (17%) High 45 (18%)
Patients without anastomotic leak (n ¼ 227)
Patients with anastomotic leak (n ¼ 29)
104 (46%) 63 (28%) 60 (26%)
12 (41%) 7 (24%) 10 (35%)
148 (65%) 39 (17%) 40 (18%)
14 (48%) 8 (28%) 7 (24%)
55 (24%) 86 (38%) 86 (38%)
8 (28%) 9 (31%) 12 (41%)
155 (68%) 38 (17%) 34 (15%)
12 (41%) 6 (21%) 11 (38%)
Table 3 The association of the amounts of colorectal cancer-associated microbes in colorectal carcinoma tissue with the incidence of anastomotic leakage after resection of colorectal carcinoma.
Ptrenda
The amount of microbes in colorectal carcinoma tissue
Univariable OR (95% CI)
Multivariable OR (95% CI)a
Model for anastomotic leakage after resection of colorectal carcinoma (n ¼ 256, as an outcome variable) Fusobacterium nucleatum Negative 1 (reference) 1 (reference) Low 0.96 (0.34–2.52) 0.83 (0.27–2.34) High 1.44 (0.58–3.55) 1.23 (0.44–3.29) Ptrendb 0.45 0.73
0.45
0.13
pks-positive Escherichia coli
Negative Low High Ptrendb
1 (reference) 2.24 (0.77–6.15) 1.92 (0.62–5.44) 0.13
1 (reference) 2.17 (0.66–6.76) 2.71 (0.81–8.73) 0.039
Enterococcus faecalis
Negative Low High Ptrendb
1 (reference) 0.72 (0.26–2.02) 0.96 (0.37–2.59) 0.99
1 (reference) 0.89 (0.30–2.74) 0.88 (0.32–2.57) 0.83
Bifidobacterium genus
Negative Low High
1 (reference) 2.04 (0.67–5.62) 4.18 (1.68–10.34) 0.002
1 (reference) 2.33 (0.72–6.95) 3.96 (1.50–10.51)
0.99
0.002
a
Ptrend value was calculated by the linear trend test across the ordinal (negative, low, and high) categories of the amount of the intestinal microbes as a continuous variable in univariable logistic regression model for the incidence of anastomotic leakage (a dichotomous outcome variable). All statistical tests were two-sided at α level of 0.005, considering the multiple comparisons and the consequent false positives.
Ptrendb
0.004
Abbreviations: CI, confidence interval; OR, odds ratio. a The multivariable logistic regression analysis model initially included age, sex, body mass index, diabetes, serum albumin levels, tumor location, pT stage, lymph node metastasis, distant metastasis, surgical approach, operating time, intraoperative bleeding, and diverting ileostomy. A backward stepwise elimi nation with a threshold of P < 0.05 was used to select variables in the final models. b Ptrend value was calculated by the linear trend test across the ordinal (negative, low, and high) categories of the amount of the intestinal microbes as a continuous variable in logistic regression model for the incidence of anastomotic leakage (a dichotomous outcome variable). All statistical tests were two-sided at α level of 0.005, considering the multiple comparisons and the consequent false positives.
(Ptrend ¼ 0.005) and multivariable logistic regression analysis (Ptrend ¼ 0.040; Supplementary Table S3). 4. Discussion We conducted this study to test the hypothesis that higher amounts of colorectal cancer-associated microbes in colorectal tissue might be associated with an increased risk of anastomotic leakage after resection of colorectal carcinoma. We found that higher amounts of colorectal cancer-associated microbes, such as Bifidobacterium genus, in human colorectal carcinoma tissue were associated with an increased risk of anastomotic leakage. Although clinical studies have demonstrated associations of anasto motic leakage with patient and tumor characteristics (male sex, obesity, diabetes, and rectal tumor location), and surgery-related factors (oper ative time, intraoperative bleeding, and diverting ileostomy) [28,29], exact mechanisms of colorectal anastomotic leakage remain uncertain. Our human data suggest a possible link between colorectal mucosal microbes and anastomotic leakage, generating some mechanistic hy potheses for further investigation. Experimental evidence from animal models demonstrates that Bifi dobacterium genus in the gut appear to inhibit colorectal carcinogenesis through prevention of enteropathogenic infection or acidification, which can reduce secondary bile acid production [32]. However, in 1313 human colorectal cancer tissue specimens from U.S. nationwide prospective cohort studies, the amount of intratumor Bifidobacterium genus is correlated with the extent of signet ring cells, which has been associated with worse patient survival [14,33]. Experimental evidence demonstrates that prostaglandin-endoperoxide synthase 2 (PTGS2, cyclooxygenase-2) is essential for neovascularization of the colonic anastomosis and thereby promote colorectal anastomotic wound healing [34]. Bifidobacterium genus has been shown to decrease PTGS2 expres sion level in colonic cells [35]. Studies have shown the enrichment of Bifidobacterium genus in the hypoxic tumor microenvironment [36–38]. Although exact mechanisms underlying the association of Bifidobacte rium genus with colorectal anastomotic leakage need to be elucidated by further investigations, Bifidobacterium genus might impair anastomotic
wound healing through poor vascularization and tissue hypoxia in colorectal anastomotic tissues. It is also possible that Bifidobacterium genus may contribute to colorectal anastomotic leakage through microbe-microbe interactions as a member of human microbial ecosys tems [39]. An experimental study of a rat model has shown that Enterococcus faecalis may impair colorectal anastomotic would healing by producing collagenases and activating host metalloproteinase MMP9 in anasto motic tissues [18]. In the current study, the association of the amount of Enterococcus faecalis with the incidence of anastomotic leakage was not statistically significant. Our findings need to be validated by further studies. We recognize limitations of this study. First, we did not examine the microbiota and its metabolites in colorectal tissue, data on stool microbiota, or microbe-microbe interactions. Future comprehensive metagenomic analyses on colorectal tissue and stool may provide further insights on roles of intestinal microbiota in the development of colo rectal anastomotic leakage. Nonetheless, given complex roles of in teractions between microbial and host factors in human colorectal anastomotic wound healing, we believe that our human data on colo rectal cancer-associated microbes in relation to the incidence of anas tomotic leakage after colorectal resection represent valuable information. Second, we did not examine potential influences of me chanical bowel preparation with or without oral antibiotics before sur gery on the amount of the colorectal cancer-associated microbes in colorectal tissue because all patients routinely received mechanical bowel preparation without oral antibiotics before elective resection of 66
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colorectal cancer in the current study. Mechanical bowel preparation has been shown to influence both the luminal and mucosal microbiota in the human colon [40,41]. Clinical studies have shown that preoperative mechanical bowel preparation may decrease the amount of Bifido bacterium genus in the human colon, and significantly reduce anasto motic leakage after colorectal resection [42–46]. Future investigations are needed to examine potential influences of mechanical bowel prep aration with or without oral antibiotics before surgery on the intestinal and mucosal microflora in patients with colorectal cancer, and examine associations of the colorectal cancer-associated microbes in colorectal tissue with the incidence of anastomotic leakage after resection of colorectal cancer in patients who do not receive bowel preparation. Third, data on colorectal mucosal microbes from patients with benign colorectal disease were not available in the current study. Because colorectal mucosal microbiota in patients with colorectal cancer has been shown to differ significantly from that in patients with benign colorectal disease [4–6], future investigations are needed to examine associations of colorectal mucosal microbes with the incidence of anastomotic leakage after colorectal resection in patients with benign colorectal disease. A major strength of this study includes the use of our database which integrates patient and tumor characteristics, surgery-related factors, and the amounts of colorectal carcinoma-associated microbes in colorectal cancer and normal colorectal tissues. The comprehensiveness of this colorectal cancer database enabled us to assess the association between the amounts of colorectal mucosal microbes and anastomotic leakage, controlling for potential confounders.
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5. Conclusions The amounts of colorectal cancer-associated microbes, such as Bifi dobacterium genus, in colorectal tissue are associated with an increased risk of anastomotic leakage after resection for colorectal cancer. These findings need to be validated by further studies to target gastrointestinal microflora for the prevention of anastomotic leakage after colorectal resection. Author contributions Kosuke Mima and Yuki Sakamoto contributed equally as co-first authors. All authors contributed to review and revision. Kosuke Mima, Shuji Ogino, and Hideo Baba developed the main concept and designed the study. Kosuke Mima, Shuji Ogino, and Hideo Baba wrote grant ap plications. Kosuke Mima, Yuki Sakamoto, Yoko Ogata, Keisuke Miyake, Yukiharu Hiyoshi, Takatsugu Ishimoto, Masaaki Iwatsuki, Yoshifumi Baba, Shiro Iwagami, Yuji Miyamoto, Naoya Yoshida, and Hideo Baba were responsible for collection of tumor and normal colorectal tissue, and acquisition of clinical and tumor and normal colorectal tissue data, including histopathological characteristics. Kosuke Mima, Keisuke Kosumi, Shuji Ogino, and Hideo Baba performed data analysis and interpretation. Kosuke Mima, Shuji Ogino, and Hideo Baba drafted the manuscript. Yukiharu Hiyoshi, Yuji Miyamoto, Takatsugu Ishimoto, Masaaki Iwatsuki, Yoshifumi Baba, Shiro Iwagami, and Naoya Yoshida contributed to editing and critical revision for important intellectual contents. Funding This work was supported by grants from the U.S. National Institutes of Health (NIH) [R35 CA197735 to S.O.] and the Nodal Award (to S.O.) of the Dana-Farber Harvard Cancer Center. K.M. is supported by grants from Takeda Science Foundation, KANAE Foundation for the Promotion of Medical Science, YOKOYAMA Foundation for Clinical Pharmacology, the Uehara Memorial Foundation, and JSPS KAKENHI Grant Number 17H05094. K.K. is supported by grants from JSPS Fujita Memorial Fund for Medical Research and Overseas Research Fellowship from JSPS 67
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[25] K.S. Viljoen, A. Dakshinamurthy, P. Goldberg, et al., Quantitative profiling of colorectal cancer-associated bacteria reveals associations between fusobacterium spp., enterotoxigenic Bacteroides fragilis (ETBF) and clinicopathological features of colorectal cancer, PLoS One 10 (2015), e0119462. [26] A. Sivan, L. Corrales, N. Hubert, et al., Commensal Bifidobacterium promotes antitumor immunity and facilitates anti-PD-L1 efficacy, Science 350 (2015) 1084–1089. [27] K. Mima, R. Nishihara, Z.R. Qian, et al., Fusobacterium nucleatum in colorectal carcinoma tissue and patient prognosis, Gut 65 (2016) 1973–1980. [28] P. Matthiessen, O. Hallbook, J. Rutegard, et al., Defunctioning stoma reduces symptomatic anastomotic leakage after low anterior resection of the rectum for cancer: a randomized multicenter trial, Ann. Surg. 246 (2007) 207–214. [29] M. Frasson, B. Flor-Lorente, J.L. Rodriguez, et al., Risk factors for anastomotic leak after colon resection for cancer: multivariate analysis and nomogram from a multicentric, prospective, national study with 3193 patients, Ann. Surg. 262 (2015) 321–330. [30] K. Mima, R. Nishihara, J.A. Nowak, et al., MicroRNA MIR21 and T cells in colorectal cancer, Cancer Immunol. Res. 4 (2016) 33–40. [31] D.J. Benjamin, J.O. Berger, M. Johannesson, et al., Redefine statistical significance, Nature Human Behav. 2 (2018) 6–10. [32] A. O’Callaghan, D. van Sinderen, Bifidobacteria and their role as members of the human gut microbiota, Front. Microbiol. 7 (2016) 925. [33] K. Inamura, M. Yamauchi, R. Nishihara, et al., Prognostic significance and molecular features of signet-ring cell and mucinous components in colorectal carcinoma, Ann. Surg. Oncol. 22 (2015) 1226–1235. [34] K.W. Reisinger, D.H. Schellekens, J.W. Bosmans, et al., Cyclooxygenase-2 is essential for colorectal anastomotic healing, Ann. Surg. 265 (2017) 547–554. [35] J.T. Nurmi, P.A. Puolakkainen, N.E. Rautonen, Bifidobacterium Lactis sp. 420 upregulates cyclooxygenase (Cox)-1 and down-regulates Cox-2 gene expression in a Caco-2 cell culture model, Nutr. Cancer 51 (2005) 83–92. [36] S. Taniguchi, Y. Shimatani, M. Fujimori, Tumor-targeting therapy using geneengineered anaerobic-nonpathogenic Bifidobacterium longum, Methods Mol. Biol. 1409 (2016) 49–60.
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Contents lists available at ScienceDirect
Surgical Oncology journal homepage: http://www.elsevier.com/locate/suronc
Mucosal cancer-associated microbes and anastomotic leakage after resection of colorectal carcinoma Kosuke Mima a, b, Yuki Sakamoto a, Keisuke Kosumi c, Yoko Ogata a, Keisuke Miyake a, Yukiharu Hiyoshi a, Takatsugu Ishimoto a, Masaaki Iwatsuki a, Yoshifumi Baba a, Shiro Iwagami a, Yuji Miyamoto a, Naoya Yoshida a, Shuji Ogino c, d, e, f, Hideo Baba a, * a
Department of Gastroenterological Surgery, Graduate School of Medical Science, Kumamoto University, Kumamoto, Japan Department of Surgery, National Hospital Organization Kumamoto Medical Center, Kumamoto, Japan Department of Oncologic Pathology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA, USA d Broad Institute of MIT and Harvard, Cambridge, MA, USA e Program in MPE Molecular Pathological Epidemiology, Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA f Department of Epidemiology, Harvard T.H. Chan School of Public Health, Boston, MA, USA b c
A R T I C L E I N F O Presentation: This work was presented at the SSO 2019 Annual Cancer Symposium; March 27th – 30th, 2019. Keywords: Bacteria Complication Surgery
A B S T R A C T
Background: Clinical and experimental evidence suggests that colorectal mucosal microbiota changes during colorectal carcinogenesis and may impair colorectal anastomotic wound healing. Thus, we hypothesized that amounts of colorectal cancer-associated microbes in colorectal tissue might be associated with anastomotic leakage after resection for colorectal carcinoma. Methods: We analyzed 256 fresh frozen tissues of colorectal cancer from patients who underwent elective colorectal resection and anastomosis. Amounts of colorectal cancer-associated microbes, including Fusobacterium nucleatum, Escherichia coli possessing the polyketide synthase (pks) gene cluster, Enterococcus faecalis, and Bifi dobacterium genus, in colorectal cancer tissues were measured by quantitative polymerase chain reaction assay; we equally dichotomized positive cases (high versus low). Multivariable logistic regression analysis was con ducted to assess associations of these microbes with anastomotic leakage, adjusting for patient and tumor characteristics, and surgery-related factors. Results: Fusobacterium nucleatum, pks-positive Escherichia coli, Enterococcus faecalis, and Bifidobacterium genus were detected in colorectal carcinoma tissue in 140 (54%), 94 (36%), 193 (75%), and 89 (35%) of 256 cases, respectively. Compared with Bifidobacterium genus-negative cases, Bifidobacterium genus-high cases were asso ciated with an increased risk of anastomotic leakage (multivariable odds ratio, 3.96; 95% confidence interval, 1.50 to 10.51; Ptrend ¼ 0.004). The association of Fusobacterium nucleatum, pks-positive Escherichia coli, or Enterococcus faecalis with anastomotic leakage was not statistically significant. Conclusions: The amount of Bifidobacterium genus in colorectal tissue is associated with an increased risk of anastomotic leakage after resection for colorectal cancer. These findings need to be validated to target gastro intestinal microflora for the prevention of anastomotic leakage after colorectal resection.
1. Introduction More than 100 trillion microorganisms inhabit the human gastroin testinal tract and play important roles in health conditions, immunity, and diseases including colorectal cancer [1–3]. Human colorectal mucosal microbiota has been shown to change during the development
and progression of colorectal neoplasia [4–6]. Experimental evidence from animal models demonstrates that intestinal microbes, such as Fusobacterium nucleatum, Escherichia coli possessing the polyketide syn thase (pks) gene cluster, Enterococcus faecalis, and Bifidobacterium genus, can influence the development and progression of colorectal neoplasms by damaging DNA, producing metabolites, and modulating host immune
Abbreviations: CI, confidence interval; Ct, cycle threshold; OR, odds ratio; PCR, polymerase chain reaction. * Corresponding author. Department of Gastroenterological Surgery, Graduate School of Medical Science Kumamoto University, 1-1-1 Honjo, Chuo-ku, Kumamoto, 860-8556, Japan. E-mail address: [email protected] (H. Baba). https://doi.org/10.1016/j.suronc.2019.11.005 Received 27 January 2019; Received in revised form 10 November 2019; Accepted 17 November 2019 Available online 18 November 2019 0960-7404/© 2019 Elsevier Ltd. All rights reserved.
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response and intestinal inflammation [3,7]. Consistent with these lines of experimental evidence, human studies have shown associations of these microbes with antitumor immune response, intestinal inflamma tion, and colorectal neoplasms [8–13]. In 1313 colorectal cancers from U.S. nationwide prospective cohort studies, the amount of intratumor Bifidobacterium genus has been associated with the extent of signet ring cells in colorectal cancer tissue [14]. Although surgery with complete resection represents a potentially curative treatment for colorectal carcinoma, anastomotic leakage after colorectal resection remains the major cause for morbidity and mortality [15,16]. Emerging evidence from animal models indicates that colo rectal mucosal microbes may impair colorectal anastomotic wound healing after colorectal resection [17–23]. However, the relationship between the complex intestinal microbiota and wound healing of colo rectal anastomoses in humans cannot be completely recapitulated in animal models, analysis using human tissue is required for clinical application. We hypothesized that higher amounts of these colorectal cancer-associated microbes in human colorectal tissue might be associ ated with an increased risk of anastomotic leakage after resection of colorectal carcinoma. A better understanding of roles of the intestinal microbes in the incidence of anastomotic leakage after resection of colorectal cancer may provide new opportunities to target the intestinal microbes for postoperative recovery in patients with colorectal cancers. To test our hypothesis, we examined associations of the amounts of colorectal cancer-associated microbes, including Fusobacterium nuclea tum, pks-positive Escherichia coli, Enterococcus faecalis, and Bifidobacte rium genus, in human colorectal carcinoma tissue with the incidence of anastomotic leakage after resection of colorectal carcinoma.
Enterococcus faecalis and Bifidobacterium genus, and the reference human gene, SLCO2A1 were used as previously described [8,24–26]. Each re action contained 12.5 ng of genomic DNA and was assayed in 10 μL reactions containing 1 � final concentration LightCycler 480 Probe Master (Roche) and each TaqMan Gene Expression Assay (Applied Biosystems), in a 384-well optical PCR plate. Amplification and detec tion of DNA was performed with a LightCycler 480 Instrument II (Roche) using the following reaction conditions: 10 min at 95 � C and 45 cycles of 15 s at 95 � C and 1 min at 60 � C. The primer and probe sequences for each TaqMan Gene Expression Assay were as follows: Fusobacterium nucleatum forward primer, 50 TGGTGTCATTCTTCCAAAAATATCA-3’; Fusobacterium nucleatum reverse primer, 50 -AGATCAAGAAGGACAAGTTGCTGAA-3’; Fusobacte rium nucleatum FAM probe, 50 -ACTTTAACTCTACCATGTTCA -3’; pkspositive Escherichia coli forward primer, 50 - TGCTATGTATTCACG CAAACG-3’; pks-positive Escherichia coli reverse primer, 50 CGTTGCTCTCCATGACCTG-3’; pks-positive Escherichia coli probe, 50 CTGGCTGG-3’; Enterococcus faecalis forward primer, 50 AAACCTTTCCCTGGTGTTCA-3’; Enterococcus faecalis reverse primer, 50 CGTTAATCCCTGTTGAAGCAA-3’; Enterococcus faecalis probe, 50 TTCTGGCT-3’; Bifidobacterium genus forward primer, 50 -CGGGTGAG TAATGCGTGACC-3’; Bifidobacterium genus reverse primer, 50 - TGA TAGGACGCGACCCCA-3’; Bifidobacterium genus FAM probe, 50 CTCCTGGAAACGGGTG-3’; SLCO2A1 forward primer, 50 -ATCCC CAAAGCACCTGGTTT-3’; SLCO2A1 reverse primer, 50 -AGAGGCCAA GATAGTCCTGGTAA-3’; SLCO2A1 VIC probe, 50 CCATCCATGTCCTCATCTC-3’. Each specimen was analyzed in duplicate for each target in a single batch, and we used the average of the two cycle threshold (Ct) values for each target. The amount of Fusobacterium nucleatum, pks-positive Escherichia coli, Enterococcus faecalis, or Bifidobacterium genus in each specimen was calculated as a relative unitless value normalized with SLCO2A1 using the 2-ΔCt method (where ΔCt ¼ “the average Ct value of intestinal microbes” - “the average Ct value of SLCO2A1”) as previously described [8,27].
2. Methods 2.1. Subjects and specimens We analyzed 256 fresh frozen colorectal cancer tissue specimens from patients who underwent elective colorectal resection and anasto mosis for colorectal cancer at Kumamoto University Hospital in Japan. All patients had histologically confirmed colorectal carcinoma. The pathologic diagnoses and the clinicopathological factors were estab lished based on the American Joint Committee on Cancer/International Union Against Cancer staging system. Written informed consent was obtained from each patient. This study was approved by the Human Ethics Review Committee of the Graduate School of Medicine, Kuma moto University (Kumamoto, Japan). All patients received mechanical bowel preparation with 2L of polyethylene glycol 1 day before surgery. None of the patients received preoperative oral antibiotics. All of the patients received 1 g of cefme tazole intravenously after the induction of anesthesia and within 60 min before skin incision. Subsequently, 1 g of cefmetazole was additionally given every 3 h during surgery. The antibiotic was discontinued within 24 h after surgery. Colorectal carcinoma tissue specimens and normal colorectal mu cosa tissue specimens that were at least 5–10 cm from the edge of the tumor tissues were cut using a sterile blade and placed in a cryotube and frozen immediately in liquid nitrogen within 30 min after colorectal resection. The frozen tissue was stored at 80 � C until DNA extraction.
2.3. Definition of anastomotic leakage The definition of anastomotic leakage was used as previously re ported clinical trials [28,29]; peritonitis from any staple line, and pelvic abscess without radiologically proven leakage mechanism were included. Leakage was verified by clinical (inspection of drain contents), endoscopic (flexible sigmoidoscopy), or radiologic (rectal contrast study, computed tomography scan) interventions. 2.4. Statistical analysis All statistical analyses were conducted using JMP (version 12.2, SAS Institute, Cary, NC) and all P values were two-sided. Neither the amounts of intestinal microbes, including Fusobacterium nucleatum, pkspositive Escherichia coli, Enterococcus faecalis, and Bifidobacterium genus, nor the log-transformed value of the amounts of these intestinal mi crobes fit a normal distribution with the use of the Shapiro-Wilk test for normality (P < 0.0001). Thus, our primary hypothesis testing was the linear trend test in a logistic regression model to assess associations of the amounts of these intestinal microbes in colorectal carcinoma tissue (an ordinal predictor variable) with the incidence of anastomotic leakage after resection of colorectal carcinoma (a dichotomous outcome variable), as previously described [8,14,30]. Cases with detectable these microbes were categorized as low or high based on the median cutpoint amount of the microbes, while cases without detectable the microbes were categorized as “negative”. The linear trend test was performed using the ordinal predictor variable of the microbes (negative, low, and high) as a continuous variable in a logistic regression model. All statis tical tests were two-sided at α level of 0.005, considering the multiple comparisons and the consequent false positives [31].
2.2. Quantitative polymerase chain reaction (PCR) for intestinal microbes Genomic DNA was extracted from colorectal carcinoma tissue spec imens and normal colorectal mucosa tissue specimens, using QIAamp DNA Mini Kit (Qiagen). We performed quantitative PCR assays to measure the amount of tissue DNA of Fusobacterium nucleatum, pkspositive Escherichia coli, Enterococcus faecalis, or Bifidobacterium genus. Custom TaqMan primer/probe sets (Applied Biosystems) for the nusG gene of Fusobacterium nucleatum, the pks colibactin gene cluster of Escherichia coli, the 16S ribosomal RNA gene DNA sequences of 64
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We performed multivariable logistic regression analysis to adjust for potential confounders. The multivariable model initially included age (�64 vs. 65–74 vs. � 75), sex, body mass index (<30 kg/m2 vs. � 30 kg/ m2), diabetes (present vs. absent), serum albumin levels (<3 g/dl vs. � 3 g/dl), tumor location (colon vs. rectum), pT stage (pT1-3 vs. pT4), lymph node metastasis (negative vs. positive), distant metastasis (negative vs. positive), surgical approach (open or laparoscopy con verted vs. laparoscopy), operating time (<240 min vs. � 240 min), intraoperative bleeding (<300 mL vs. � 300 mL), and diverting ileos tomy (present vs. absent). A backward stepwise elimination with a threshold of P < 0.05 was used to select variables in the final models. To assess associations between the dichotomous category (absence and present) of the incidence of anastomotic leak and categorical data, the chi-square test was performed.
Table 1 Patient and tumor characteristics, and surgery-related factors according to the incidence of anastomotic leakage after resection of colorectal carcinoma. Characteristica
3. Results 3.1. Patient and tumor characteristics, and surgery-related factors in relation to anastomotic leakage after resection of colorectal carcinoma Anastomotic leakage was diagnosed in 29 (11%) of 256 cases. Pa tient and tumor characteristics, and surgery-related factors are sum marized according to the incidence of anastomotic leakage in Table 1. Male sex and rectal tumor location were associated with anastomotic leakage (P < 0.001; with α level of 0.005).
All patients (n ¼ 256)
Patients without anastomotic leak (n ¼ 227)
Patients with anastomotic leak (n ¼ 29)
Age (year) �64 65-74 �75
90 (35%) 84 (33%) 82 (32%)
83 (36%) 72 (32%) 72 (32%)
7 (24%) 12 (41%) 10 (35%)
Sex Men Women
152 (59%) 104 (41%)
127 (56%) 100 (44%)
25 (86%) 4 (14%)
Body mass index (kg/m2) <30 247 (96%) �30 9 (3.5%)
218 (96%) 9 (4%)
29 (100%) 0
Diabetes No Yes
212 (83%) 44 (17%)
188 (83%) 39 (17%)
24 (83%) 5 (17%)
Serum albumin levels (g/dl) <3 21 (8.2%) �3 235 (92%)
18 (7.9%) 209 (92%)
3 (10%) 26 (90%)
83 (32%)
78 (34%)
5 (17%)
128 (50%)
117 (52%)
11 (38%)
45 (18%)
32 (14%)
13 (45%)
pT stage (depth of tumor invasion) pT1-3 209 (82%) pT4 47 (18%)
184 (81%) 43 (19%)
25 (86%) 4 (14%)
Lymph node metastasis Negative 160 (62%) Positive 96 (38%)
141 (62%) 86 (38%)
19 (66%) 10 (34%)
210 (82%) 46 (18%)
184 (81%) 43 (19%)
26 (90%) 3 (10%)
67 (26%)
55 (24%)
12 (41%)
189 (74%)
172 (76%)
17 (59%)
Operating time (minutes) <240 62 (24%) �240 194 (76%)
60 (26%) 167 (74%)
2 (6.9%) 27 (93%)
Intraoperative bleeding (mL) <300 198 (77%) �300 58 (23%)
179 (79%) 48 (21%)
19 (66%) 10 (34%)
Diverting ileostomy No Yes
209 (92%) 18 (7.9%)
25 (86%) 4 (14%)
Tumor location Caecum, ascending colon, transverse colon Descending colon, sigmoid colon, rectosigmoid junction Rectum
3.2. Associations of the amounts of colorectal cancer-associated microbes in tumor tissue with anastomotic leakage after resection of colorectal carcinoma We measured the relative amounts of Fusobacterium nucleatum, pkspositive Escherichia coli, Enterococcus faecalis, and Bifidobacterium genus in fresh frozen tumor tissue of 256 colorectal carcinoma cases, using the quantitative PCR assay. Fusobacterium nucleatum, pks-positive Escherichia coli, Enterococcus faecalis, and Bifidobacterium genus were detected in colorectal carcinoma tissue in 140 (54%), 94 (36%), 193 (75%), and 89 (35%) of 256 cases, respectively. We categorized colo rectal carcinoma cases with detectable these four microbes as low or high based on the median cutpoint amount of the microbes. Table 2 shows the distribution of colorectal carcinoma cases ac cording to the amounts of the microbes and the incidence of anastomotic leakage. In our primary hypothesis testing, we conducted univariable and multivariable logistic regression analyses to assess associations of the amounts of the microbes in colorectal carcinoma tissue (as ordinal predictor variables) with the incidence of anastomotic leakage (as a dichotomous outcome variable) (Table 3). The amount of Bifidobacte rium genus in colorectal carcinoma tissue was associated with an increased risk of anastomotic leakage in univariable (Ptrend ¼ 0.002) and multivariable logistic regression analysis (Ptrend ¼ 0.004). Compared with Bifidobacterium genus-negative cases, Bifidobacterium genus-high cases were associated with an increased risk of anastomotic leakage (multivariable odds ratio, 3.96; 95% confidence interval, 1.50 to 10.51; Table 3 and Supplementary Table S1). The association of the amount of Fusobacterium nucleatum, pks-positive Escherichia coli, or Enterococcus faecalis with the incidence of anastomotic leakage was not statistically significant (Ptrend > 0.039; with α level of 0.005; Table 3). Associations of the amount of Bifidobacterium genus in colorectal carcinoma tissue with the patient and tumor characteristics, including tumor location, were not statistically significant (with α level of 0.005; Supplementary Table S2). In our exploratory analysis, we measured the relative amount of Bifidobacterium genus in normal colorectal mucosa tissues that were at least 5–10 cm distant from the edge of the tumor in 209 colorectal carcinoma cases, using the quantitative PCR assay. The amount of Bifi dobacterium genus in normal colorectal mucosa tissue appeared to be associated with an increased risk of anastomotic leakage in univariable
Distant metastasis Negative Positive Surgical approach Open or laparoscopy converted Laparoscopy
234 (91%) 22 (8.6%)
P valueb
0.37
<0.001
0.14
0.99
0.67
<0.001
0.49
0.72
0.23 0.06
0.010
0.12
0.32
a Percentage (%) indicates the proportion of cases with patient and tumor characteristics or surgery-related factors according to the incidence of anasto motic leakage. b To assess associations between the dichotomous category of the incidence of anastomotic leakage and categorical variables, the chi-square test was per formed. All statistical tests were two-sided at α level of 0.005, considering the multiple comparisons and the consequent false positives.
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Table 2 Distribution of cases according to the amounts of colorectal cancer-associated microbes in colorectal carcinoma tissue and the incidence of anastomotic leakage after resection of colorectal carcinoma. The amount of microbes in colorectal carcinoma tissue
All patients (n ¼ 256)
Fusobacterium nucleatum Negative 116 (46%) Low 70 (27%) High 70 (27%) pks-positive Escherichia coli Negative 162 (64%) Low 47 (18%) High 47 (18%) Enterococcus faecalis Negative 63 (25%) Low 95 (37%) High 98 (38%) Bifidobacterium genus Negative 167 (65%) Low 44 (17%) High 45 (18%)
Patients without anastomotic leak (n ¼ 227)
Patients with anastomotic leak (n ¼ 29)
104 (46%) 63 (28%) 60 (26%)
12 (41%) 7 (24%) 10 (35%)
148 (65%) 39 (17%) 40 (18%)
14 (48%) 8 (28%) 7 (24%)
55 (24%) 86 (38%) 86 (38%)
8 (28%) 9 (31%) 12 (41%)
155 (68%) 38 (17%) 34 (15%)
12 (41%) 6 (21%) 11 (38%)
Table 3 The association of the amounts of colorectal cancer-associated microbes in colorectal carcinoma tissue with the incidence of anastomotic leakage after resection of colorectal carcinoma.
Ptrenda
The amount of microbes in colorectal carcinoma tissue
Univariable OR (95% CI)
Multivariable OR (95% CI)a
Model for anastomotic leakage after resection of colorectal carcinoma (n ¼ 256, as an outcome variable) Fusobacterium nucleatum Negative 1 (reference) 1 (reference) Low 0.96 (0.34–2.52) 0.83 (0.27–2.34) High 1.44 (0.58–3.55) 1.23 (0.44–3.29) Ptrendb 0.45 0.73
0.45
0.13
pks-positive Escherichia coli
Negative Low High Ptrendb
1 (reference) 2.24 (0.77–6.15) 1.92 (0.62–5.44) 0.13
1 (reference) 2.17 (0.66–6.76) 2.71 (0.81–8.73) 0.039
Enterococcus faecalis
Negative Low High Ptrendb
1 (reference) 0.72 (0.26–2.02) 0.96 (0.37–2.59) 0.99
1 (reference) 0.89 (0.30–2.74) 0.88 (0.32–2.57) 0.83
Bifidobacterium genus
Negative Low High
1 (reference) 2.04 (0.67–5.62) 4.18 (1.68–10.34) 0.002
1 (reference) 2.33 (0.72–6.95) 3.96 (1.50–10.51)
0.99
0.002
a
Ptrend value was calculated by the linear trend test across the ordinal (negative, low, and high) categories of the amount of the intestinal microbes as a continuous variable in univariable logistic regression model for the incidence of anastomotic leakage (a dichotomous outcome variable). All statistical tests were two-sided at α level of 0.005, considering the multiple comparisons and the consequent false positives.
Ptrendb
0.004
Abbreviations: CI, confidence interval; OR, odds ratio. a The multivariable logistic regression analysis model initially included age, sex, body mass index, diabetes, serum albumin levels, tumor location, pT stage, lymph node metastasis, distant metastasis, surgical approach, operating time, intraoperative bleeding, and diverting ileostomy. A backward stepwise elimi nation with a threshold of P < 0.05 was used to select variables in the final models. b Ptrend value was calculated by the linear trend test across the ordinal (negative, low, and high) categories of the amount of the intestinal microbes as a continuous variable in logistic regression model for the incidence of anastomotic leakage (a dichotomous outcome variable). All statistical tests were two-sided at α level of 0.005, considering the multiple comparisons and the consequent false positives.
(Ptrend ¼ 0.005) and multivariable logistic regression analysis (Ptrend ¼ 0.040; Supplementary Table S3). 4. Discussion We conducted this study to test the hypothesis that higher amounts of colorectal cancer-associated microbes in colorectal tissue might be associated with an increased risk of anastomotic leakage after resection of colorectal carcinoma. We found that higher amounts of colorectal cancer-associated microbes, such as Bifidobacterium genus, in human colorectal carcinoma tissue were associated with an increased risk of anastomotic leakage. Although clinical studies have demonstrated associations of anasto motic leakage with patient and tumor characteristics (male sex, obesity, diabetes, and rectal tumor location), and surgery-related factors (oper ative time, intraoperative bleeding, and diverting ileostomy) [28,29], exact mechanisms of colorectal anastomotic leakage remain uncertain. Our human data suggest a possible link between colorectal mucosal microbes and anastomotic leakage, generating some mechanistic hy potheses for further investigation. Experimental evidence from animal models demonstrates that Bifi dobacterium genus in the gut appear to inhibit colorectal carcinogenesis through prevention of enteropathogenic infection or acidification, which can reduce secondary bile acid production [32]. However, in 1313 human colorectal cancer tissue specimens from U.S. nationwide prospective cohort studies, the amount of intratumor Bifidobacterium genus is correlated with the extent of signet ring cells, which has been associated with worse patient survival [14,33]. Experimental evidence demonstrates that prostaglandin-endoperoxide synthase 2 (PTGS2, cyclooxygenase-2) is essential for neovascularization of the colonic anastomosis and thereby promote colorectal anastomotic wound healing [34]. Bifidobacterium genus has been shown to decrease PTGS2 expres sion level in colonic cells [35]. Studies have shown the enrichment of Bifidobacterium genus in the hypoxic tumor microenvironment [36–38]. Although exact mechanisms underlying the association of Bifidobacte rium genus with colorectal anastomotic leakage need to be elucidated by further investigations, Bifidobacterium genus might impair anastomotic
wound healing through poor vascularization and tissue hypoxia in colorectal anastomotic tissues. It is also possible that Bifidobacterium genus may contribute to colorectal anastomotic leakage through microbe-microbe interactions as a member of human microbial ecosys tems [39]. An experimental study of a rat model has shown that Enterococcus faecalis may impair colorectal anastomotic would healing by producing collagenases and activating host metalloproteinase MMP9 in anasto motic tissues [18]. In the current study, the association of the amount of Enterococcus faecalis with the incidence of anastomotic leakage was not statistically significant. Our findings need to be validated by further studies. We recognize limitations of this study. First, we did not examine the microbiota and its metabolites in colorectal tissue, data on stool microbiota, or microbe-microbe interactions. Future comprehensive metagenomic analyses on colorectal tissue and stool may provide further insights on roles of intestinal microbiota in the development of colo rectal anastomotic leakage. Nonetheless, given complex roles of in teractions between microbial and host factors in human colorectal anastomotic wound healing, we believe that our human data on colo rectal cancer-associated microbes in relation to the incidence of anas tomotic leakage after colorectal resection represent valuable information. Second, we did not examine potential influences of me chanical bowel preparation with or without oral antibiotics before sur gery on the amount of the colorectal cancer-associated microbes in colorectal tissue because all patients routinely received mechanical bowel preparation without oral antibiotics before elective resection of 66
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colorectal cancer in the current study. Mechanical bowel preparation has been shown to influence both the luminal and mucosal microbiota in the human colon [40,41]. Clinical studies have shown that preoperative mechanical bowel preparation may decrease the amount of Bifido bacterium genus in the human colon, and significantly reduce anasto motic leakage after colorectal resection [42–46]. Future investigations are needed to examine potential influences of mechanical bowel prep aration with or without oral antibiotics before surgery on the intestinal and mucosal microflora in patients with colorectal cancer, and examine associations of the colorectal cancer-associated microbes in colorectal tissue with the incidence of anastomotic leakage after resection of colorectal cancer in patients who do not receive bowel preparation. Third, data on colorectal mucosal microbes from patients with benign colorectal disease were not available in the current study. Because colorectal mucosal microbiota in patients with colorectal cancer has been shown to differ significantly from that in patients with benign colorectal disease [4–6], future investigations are needed to examine associations of colorectal mucosal microbes with the incidence of anastomotic leakage after colorectal resection in patients with benign colorectal disease. A major strength of this study includes the use of our database which integrates patient and tumor characteristics, surgery-related factors, and the amounts of colorectal carcinoma-associated microbes in colorectal cancer and normal colorectal tissues. The comprehensiveness of this colorectal cancer database enabled us to assess the association between the amounts of colorectal mucosal microbes and anastomotic leakage, controlling for potential confounders.
(JP2017-775). The content is solely the responsibility of the authors and does not necessarily represent the official views of NIH. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Declaration of competing interest All authors declare that they have no competing financial interest. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.suronc.2019.11.005. References [1] L. Zitvogel, Y. Ma, D. Raoult, et al., The microbiome in cancer immunotherapy: diagnostic tools and therapeutic strategies, Science 359 (2018) 1366–1370. [2] J.A. Gilbert, M.J. Blaser, J.G. Caporaso, et al., Current understanding of the human microbiome, Nat. Med. 24 (2018) 392–400. [3] H. Tilg, T.E. Adolph, R.R. Gerner, et al., The intestinal microbiota in colorectal cancer, Cancer Cell 33 (2018) 954–964. [4] G. Nakatsu, X. Li, H. 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5. Conclusions The amounts of colorectal cancer-associated microbes, such as Bifi dobacterium genus, in colorectal tissue are associated with an increased risk of anastomotic leakage after resection for colorectal cancer. These findings need to be validated by further studies to target gastrointestinal microflora for the prevention of anastomotic leakage after colorectal resection. Author contributions Kosuke Mima and Yuki Sakamoto contributed equally as co-first authors. All authors contributed to review and revision. Kosuke Mima, Shuji Ogino, and Hideo Baba developed the main concept and designed the study. Kosuke Mima, Shuji Ogino, and Hideo Baba wrote grant ap plications. Kosuke Mima, Yuki Sakamoto, Yoko Ogata, Keisuke Miyake, Yukiharu Hiyoshi, Takatsugu Ishimoto, Masaaki Iwatsuki, Yoshifumi Baba, Shiro Iwagami, Yuji Miyamoto, Naoya Yoshida, and Hideo Baba were responsible for collection of tumor and normal colorectal tissue, and acquisition of clinical and tumor and normal colorectal tissue data, including histopathological characteristics. Kosuke Mima, Keisuke Kosumi, Shuji Ogino, and Hideo Baba performed data analysis and interpretation. Kosuke Mima, Shuji Ogino, and Hideo Baba drafted the manuscript. Yukiharu Hiyoshi, Yuji Miyamoto, Takatsugu Ishimoto, Masaaki Iwatsuki, Yoshifumi Baba, Shiro Iwagami, and Naoya Yoshida contributed to editing and critical revision for important intellectual contents. Funding This work was supported by grants from the U.S. National Institutes of Health (NIH) [R35 CA197735 to S.O.] and the Nodal Award (to S.O.) of the Dana-Farber Harvard Cancer Center. K.M. is supported by grants from Takeda Science Foundation, KANAE Foundation for the Promotion of Medical Science, YOKOYAMA Foundation for Clinical Pharmacology, the Uehara Memorial Foundation, and JSPS KAKENHI Grant Number 17H05094. K.K. is supported by grants from JSPS Fujita Memorial Fund for Medical Research and Overseas Research Fellowship from JSPS 67
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