Association between Fusobacterium nucleatum and patient prognosis in metastatic colon cancer

Recent evidence suggests that Fusobacterium nucleatum (Fn) is associated with the development and progression of colorectal cancer. We aimed to delineate the clinical implications of Fn in metastatic colon cancer. We performed quantitative polymerase chain reaction (qPCR) using DNA samples from synchronous metastatic colon cancer patients with either formalin-fixed paraffin-embedded (FFPE) archival primary site tumor samples or fresh colon tissues. Progression-free survival (PFS)1 and PFS2 were defined as PFS of first- and second-line palliative settings. qPCR for Fn was successfully performed using 112 samples (FFPE, n = 61; fresh tissue, n = 51). Forty-one and 68 patients had right-sided and left-sided colon cancer, respectively. Patients with Fn enriched right-sided colon cancers had shorter PFS1 (9.7 vs. 11.2 months) than the other subgroups (HR 3.54, 95% confidence interval [CI] 1.05–11.99; P = 0.04). Fn positive right-sided colon was also associated with shorter PFS2 (3.7 vs. 6.7 months; HR 2.34, 95% CI 0.69–7.91; P = 0.04). In the univariate analysis, PFS1 was affected by differentiation and Fn positive right-sided colon cancer. The multivariate analysis showed that differentiation (HR 2.68, 95% CI 1.40–5.14, P = 0.01) and Fn positive right-sided colon (HR 0.40, 95% CI 0.18–0.88, P = 0.02) were associated with PFS1. Fn enrichment in right sided colon was not associated with overall survival (OS). Fn enrichment has significantly worse prognosis in terms of PFS1 and PFS2 in patients with right-sided metastatic colon cancers.

www.nature.com/scientificreports/ amount of Fn also correlates with poor overall survival (OS) in advanced stages and is more prevalent in the right-sided colon 17,18 . Several mechanisms of Fn's role in chemoresistance in CRC have been suggested in pre-clinical models 19 . Fn promotes carcinogenesis through surface adhesion virulence factors such as FadA and Fap2, which inhibit the activity of immune cells 20,21 . Furthermore, Fn harbors chemoresistance by modulating the autophagy pathway by targeting innate immune signaling 11 . Despite these findings, the role of Fn on chemoresistance remains elusive in the clinical setting.
In this study, we aimed to delineate the clinical implications of Fn in patients with synchronous metastatic colon cancer. To this end, we used surgical specimens to perform quantitative polymerase chain reaction (qPCR), detect Fn, and analyze the prognostic role of Fn. We determined whether Fn enrichment by primary tumor location (left-sided vs. right-sided) was correlated with patient prognosis.

Results
Baseline characteristics according to Fn enrichment. Among the patients with synchronous metastatic colon cancer, qPCR was successfully performed using 112 patient colon tumor samples, including 61 FFPE and 51 fresh tissue samples. We also analyzed fresh tumor specimens with adjacent normal tissue (n = 34), as shown in Fig. 1. Compared to adjacent normal tissues, tumor tissues had four times higher level of Fn (P = 0.01).
We then compared mPFS1, mPFS2, mPFS3, and mOS among the four groups ( Supplementary Fig. S1A). Fn-enriched right-sided colon cancers had the shortest mPFS1 of 9.7 months, whereas Fn-enriched left-sided colon had the longest mPFS1 of 14.5 months (P = 0.02). Although the difference in mPFS1 was not statistically significant (P = 0.20), the mPFS1 in patients with Fn negative right-sided colon cancer (10.4 months) was comparable to that in patients with left-sided colon cancer without Fn enrichment (10.2 months). Similar trends were  Table 2). The multivariate analysis further showed that both differentiation (HR 2.68, 95% CI 1.40-5.14, P = 0.01) and Fn positive right-sided colon cancer (HR 0.40, 95% CI 0.18-0.88, P = 0.02) were significant determinants of PFS1. In both univariate and multivariate analyses, Fn enriched right-sided colon cancer was not an independent risk factor of OS (Supplementary Table 1). Tumor differentiation was an independent factor in both univariate (HR 1.31, 95% CI 1.57-6.73) and multivariate analyses (HR 4.00, 95% CI 0.18-0.88, P = 0.02).

Validation in The Cancer
Genome Atlas (TCGA) metastatic colon cancer. Next, we applied our findings to a large cancer genome cohort as independent validation 22 . In the TCGA colon cancer cohort, a total of 53 patients was stage IV metastatic colon cancer. Consistent with our finding, patients with Fn enriched rightsided colon cancers (n = 7) had shorter median PFS1 (mPFS1) than other subgroups did (8.5 vs. 20.4 months; HR 6.3, 95% CI 1.59-25.08; log-rank test, P = 0.008) (Fig. 3A and Supplementary Fig. S3A). Furthermore, patients who harbor Fn enriched right-sided colon cancers (n = 7) were significantly associated with shorter    3B and Supplementary  Fig. S3B).

Discussion
The gut microbiome affects carcinogenesis, progression, and response to treatment in CRC 19 . Among diverse microbiomes, Fn is one of the most commonly found in tissue samples of colon cancers and contributes to chemoresistance 11,23 . Similarly, our study showed that Fn was enriched four-fold higher in colon tissues than in normal adjacent tissues. Fn enriched right-sided colon cancer was associated with a poor prognosis in terms of mPFS. In contrast, Fn negative right-sided colon cancer had comparable mPFS and mOS with left-sided colon cancer. In addition to the tumor right-sidedness, Fn positivity contributed to the lack of response to systemic chemotherapy in palliative settings. Collectively, these findings suggest that Fn contributes to chemoresistance and may be a potential predictive or prognostic biomarker in metastatic and recurrent rectal cancer. Although the previous report suggests that the amount of tissue Fn was associated with poor cancer-specific mortality 17,24 , the data on cancer treatment were limited, and the role of Fn on chemoresistance was not addressed in clinical samples. Therefore, our study tried to add a missing link between chemoresistance of Fn in preclinical studies 11,23 and the clinical validation. Considering that the development of chemoresistance is a critical issue in metastatic colon cancer, we focused on synchronous metastatic colon cancer patients. We showed shorter PFS1 and PFS2 in metastatic patients than other reports with heterogeneous colorectal cancer patients with all tumor stages 17,24 .
Several studies have shown that primary tumor location affected prognosis in patients with right-sided colon cancer having worse prognosis in recurrent and metastatic settings 25,26 . Fn has been found in abundance in proximal locations such as cecum, ascending, and transverse colon compared to distal locations, including sigmoid colon and rectum 18 . One potential mechanism underlying chemoresistance to 5-FU and oxaliplatin is the ability of Fn to target innate immune signaling pathways such as Toll-like receptor 4 (TLR4) and activating Myeloid differentiation primary response 88 (MyD88) in cancer cells to activate autophagy pathway 11 . The abundance and negative prognostic implications of Fn in right-sided colon cancer warrant treatment strategies specifically targeting Fn. Treatment with metronidazole for Fn enriched patient-derived xenograft showed a decrease in both the amount of Fn and tumor growth 12 . However, metronidazole does not target only Fn but also normal anaerobes in the intestines, which may disrupt the composition of normal flora 27 . Thus, Fn-specific treatment with antimicrobial agents is limited in practicality.
The presence of Fn is associated with microsatellite instability (MSI-high) and shorter survival 13,14,17,28 . Two virulence factors, Fap2 and FadA, have been identified as potential immune modulators 20,21 . Fap2 protein binds to T cell immunoreceptor with Ig and ITIM domain and suppresses natural killer cell cytotoxicity 21 . FadA binds to vascular endothelial cadherin (CDH5) and E-cadherin 20 . The binding to CDH5 receptors activates the inflammatory genes of NF-κB and cytokines, and the binding to E-cadherin expressed on colon cells activates Wnt genes and oncogenes, thereby promoting an immune-suppressive environment.
Until recently, the current standard of palliative treatment in colon cancer was limited to cytotoxic agents with targeted agents such as cetuximab and bevacizumab 5 . Recently, an anti-PD-1 agent pembrolizumab showed superior efficacy in MSI-H/mismatch repair deficient metastatic CRC based on the pivotal Keynote-177 trial 29 . The study demonstrated the doubling of PFS in response to pembrolizumab compared to standard chemotherapy with or without bevacizumab or cetuximab 29 . Although MSI-H incidence in metastatic CRC setting is less than 5%, pembrolizumab is a promising new standard of treatment option for this subset of patients 30 . Whether treatment with immune checkpoint inhibitors such as pembrolizumab is more effective in Fn enriched colon cancer harboring MSI-H and whether changes in enrichment status of Fn are evident after immunotherapy are interesting questions to be studied in the future. In our study, 55% of patients did not have MSI status because we retrospectively reviewed and included surgical specimens that date back to the time when MSI was not performed routinely. Only one patient with MSI-H colon cancer was included and treated with cytotoxic chemotherapy.
Limitations to this study include small sample size and its retrospective nature. Although we collected 112 tissue samples, further analysis of primary tumor location and Fn positivity resulted in fewer patients and uneven distribution in subgroups. Considering shorter PFS in response to palliative chemotherapy and poor prognosis of Fn positivity in both our cohort and independent TCGA metastatic colon cancer data, a larger number of patients in prospective settings warrants further validation and expansion of our findings.
In conclusion, Fn enriched right-sided metastatic, and recurrent colon cancer was significantly associated with worse PFS, indicating that Fn enriched right-sided colon responded less to palliative cytotoxic chemotherapy. Further analysis, including more extensive patient sampling and prospective cohort, are needed to validate the proposed role of Fn in chemoresistance.

Methods
Study population. From January 2009 to July 2019, data of patients with metastatic and recurrent colon cancer in Yonsei Cancer Center, Korea, were collected retrospectively. A total of 112 patients had surgical colon specimens available either as Formalin-Fixed Paraffin-Embedded (FFPE) or fresh tissue for analysis of Fn using qPCR.
Clinicopathological variables including age, sex, primary tumor location, histology, sites of metastasis, mutations such as KRAS, NRAS, BRAF, and microsatellite status were collected. The staging was determined using the 8th edition of the American Joint Committee on Cancer guideline of the primary tumor, node, and metastasis classification 31 . Right-sided colon cancers were defined as tumors occurring from cecum to transverse colon, and left-sided colon cancers as tumors arising from splenic flexure to sigmoid colon 26  www.nature.com/scientificreports/ Patients were treated with first-line palliative chemotherapeutic agents such as FOLFOX (5-fluorouracil, leucovorin, and oxaliplatin), FOLFIRI (5-fluorouracil, leucovorin, and irinotecan) with or without targeted agents such as bevacizumab or cetuximab, CAPEOX (capecitabine and oxaliplatin), and capecitabine. These regimens have been described in detail in another study 32 . Response evaluation was performed using computed tomography scanning every two months, every four cycles for FOLFOX and FOLFIRI, and every three cycles for capecitabine.
All authors followed Good Clinical Practice, and the study was conducted according to the principles of the Declaration of Helsinki. All enrolled patients provided written informed consent. The protocol was approved by the Institutional Review of Severance Hospital (IRB 4-2014-0239).
DNA isolation and quantitative PCR analysis of Fn. Genomic DNA was isolated from clinical samples using the QIAamp DNA Mini Kit (Qiagen, Crawley, UK). Briefly, the samples were suspended with protease K in ATL buffer and incubated at 55 °C for 2 h. Both AL buffer and absolute ethanol were added to the samples before applying the QIAamp spin column. Each sample was centrifuged and washed according to the manufacturer's protocol. DNA was eluted from the column with 50 μL of the supplied AE buffer. The quality and quantity of the isolated DNA were determined using a NanoDrop spectrophotometer (ND-1000; Thermo Scientific, MA, USA).
To detect the Fn sequence, specific primers and probes were designed to recognize the 16S ribosomal RNA gene. Amounts of Fn DNA were determined using quantitative real-time PCR using the TaqMan assay system. Statistical analysis. For all statistical analysis, differences were considered to be statistically significant at P < 0.05. Baseline characteristics were compared using the Kruskal-Wallis test or Mann-Whitney U test. Progression-free survival (PFS) was defined as the time from the start of palliative chemotherapy to the date of progression, last follow-up, or death. The time points for PFS1, PFS2, and PFS3 were defined as the start of first-, second-, and third-line palliative chemotherapy, respectively. OS was calculated from the date of initial diagnosis to the date of last follow-up or death. The Kaplan-Meier method was used to estimate median PFS and OS, and the Cox proportional hazard model was used for multivariate analysis. All analyses were conducted using SPSS statistical software ver. 25 (IBM, Chicago, IL, USA) and GraphPad Prism 8 (GraphPad Software, Inc., San Diego, CA).

Data availability
The datasets used or analyzed from this trial may be available upon reasonable request to the corresponding author.