Predictive and Prognostic Value of Hepatic Steatosis in Conversion Therapy for Colorectal Liver-limited Metastases

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1 Article Predictive and Prognostic Value of Hepatic Steatosis in Conversion Therapy for Colorectal Liver-limited Metastases Mi Jian 1,, Wenju Chang 1,, Li Ren 1,, Tianyu Liu 1, Yijiao Chen 1, Ye Wei 1, Qi Lin 1, Jianmin Xu 1, Xinyu Qin 1, * 1 Colorectal cancer center, Department of General Surgery, Zhongshan Hospital, Fudan University, Shanghai, China; jianmi1990@126.com (M.J.); chang_erich@hotmail.com (W.C.); renli1969@foxmail.com (L.R.); @fudan.edu.cn (T.L.); cyj @gmail.com (Y.C.); @126.com (Y.W.); @163.com (Q.L.); xujmin@aliyun.com (J.X.); * Correspondence: qin.xinyu@zs-hospital.sh.cn; Tel.: These authors contributed equally to this work Abstract: This study is aimed to assess the role of hepatic steatosis (HS) on outcome of conversion therapy for patients with initially unresectable synchronous colorectal liver-limited metastases (CLLMs). We identified 346 patients with initially unresectable CLLMs received conversion therapy under the guidance of multidisciplinary team (MDT) in Zhongshan hospital (2013 to 2016). HS status of all patients was evaluated before the first circle of conversion therapy. The objective response rate (ORR), hepatectomy rate, predictor of conversion hepatectomy, and overall survival (OS) were compared using propensity-score matching (PSM). Predictive value of HS in conversion hepatectomy was validated with a separate cohort of 60 patients initially unresectable CLLMs who received conversion therapy (2017 to 2018). Before conversion therapy start, 108 (31.2%) patients were detected with HS status. In study set after PSM, compared with non-hs group, HS group supplied improved ORR (36.7% vs 23.9%, P=0.020), hepatectomy rate from MDT (27.7% vs. 16.9%, P<0.001) following conversion therapy. Multivariate analysis confirmed that HS (OR, 5.234; 95%CI, , P<0.001), targeted therapy and transarterial chemoembolization treatment were independent predictors of hepatectomy rate. In the validation cohort, patients with HS had an improved conversion hepatectomy rate (43.7% vs. 15.9%, P=0.038) following conversion therapy. Hepatic steatosis status could be a predictor of conversion hepatectomy in patients with synchronous colorectal liver-limited metastases. This effect appears to be independent of use of targeted therapy. Keywords: Colorectal cancer; Liver metastases; Conversion therapy; Hepatic steatosis; Objective response rate Introduction Liver metastases are one of the leading cause of death in patients with colorectal cancer (CRC) [1]. Approximately 25% of patients present with liver metastases at the time of the first diagnosis, and up to 50% will further develop recurrence in the liver during their disease course [2, 3]. The latest developments of systemic chemotherapies with or without molecular targeted agents have 2018 by the author(s). Distributed under a Creative Commons CC BY license.

2 2 of dramatically improved the tumor response rates and prognostic outcomes in CRC patients with liverlimited metastases (CLLMs) [2, 4-6]. However, surgical resection remains the only potentially curative therapeutic option for CLLMs [7]. Highly effective chemotherapy regimens enable radical resection for initially unresectable liver metastasis from CRC, which is termed conversion therapy. Conversion therapy after oxaliplatin- or irinotecan-based systemic chemotherapy with molecular targets for initially unresectable liver metastasis has been achieved in approximately 12-54% of patients [8, 9]. The latest ESMO guidelines recommend all CRC patents with potential resectable liver metastases undergo conversion therapy, and secondary resection is a chance of cure for patients with effective conversion [10]. Although after conversion hepatectomy, patients can achieve survival rates similar to those of patients who underwent liver resection initially, the high cost with obvious toxicity events was mentioned by previous studied [2]. Therefore, how to identify which CRC patients are more likely to benefit from conversion therapy is important to improve the conversion hepatectomy rate followed intensive conversion therapy. As demonstrated by Folprecht and colleagues, there is a strong correlation between response rates and hepatectomy rate among patients who have isolated liver involvement [11]. In the metastatic setting, tumor microenvironment of the target organ is a key factor modulates tumor cell invasion and chemotherapeutic response [12, 13]. Hepatic steatosis (HS) alters the component diversity of liver microenvironment, and it may affect metastases foci formation and chemotherapeutic response in patients with CLLMs. HS is reported to be % of the general population in various countries [14-16], and very common in China. Studies have demonstrated that HS may be a negative prognostic factor for the onset and progression of CLLMs, and HS are associated with lower incidence of liver metastases and improved prognosis in patients with CLLMs [17, 18]. However, whether HS could influence the efficacy of conversion therapy for unresectable CLLMs is still unknow. The aim of this study is to analyze the effect of hepatic steatosis on initially unresectable CLLMs patients who undergone conversion therapy, and to determine whether HS can be a predictive factor to identify benefit patients from conversion therapy. 2. Results 2.1. Patient Characteristics in Study Set before Propensity-score Matching Between March 2013 and Dec 2016, 346 patients with initially unresectable synchronous liverlimited CLLMs were included in our study. Overall, 108 patients were classified as showing evidence of HS. Significant group-dependent difference were observed in terms of metastases size (maximum length of CLLMs > 5cm, 20.4% vs. 32.8%, P =0.038), numbers (number of CLLMs >3, 38.9% vs. 56.3%, P =0.003), extent of CLLMs (48.1% vs. 64.7%, P =0.004) (Table 1, Table S1). Conversion therapy regiments was similar in both groups (Table 1, Table S2). Overall, 280 patients (280/346, 80.9%) were assigned to receive first-line doublet chemotherapy regiments (FOLFOX/FOLFIRI) and chemotherapy treatment in 324 patients (324/346, 93.6%) were not exceeding 12 cycles. In addition, only 31.4% of patients in HS group and 34% in non-hs group accepted the targeted therapy. In study set, only 51 patients were treated with cetuximab in first-line regiments, although 225 patients were verified as RAS wild-type. Notably, for patients with progressive hepatic metastases, transarterial chemoembolization (TACE) were considered as an local treatment option. In this study, a total of 68 patients underwent TACE treatment during conversion therapy period. 2.2 Patient characteristics in Study Set after Propensity-score Matching Using propensity score matching according to age, maximum length of CLLMs, number of CLLMs, extent of CLLMs and cycles of first-line chemotherapy, 101 patients in HS group and 201 patients in non-hs group were selected. After matching, the characteristics and conversion regiments were conserved between the HS group and non-hs group (Table 1, Table S1).

3 3 of Table 1. Baseline Variables in HS and non-hs Groups before and after propensity-score matching. Before PSM After PSM Characteristic HS non-hs P HS non-hs P (n = 108) (n = 238) (n =101) (n = 201) Sex, M:F 81:27 177: :25 151: Age, y, mean ±standard deviation 57.6± ± ± ± Body mass index >25 kg/ m2 36 (33.3) 65 (22.7) (31.7) 52 (25.9) ECOG PS 0:1 89:19 202: :17 171: Pretreatment CEA value > 5ng/mL 93 (86.1) 213 (89.5) (87.1) 179 (89.1) Primary tumor T1/T2/T3/T4 1/5/57/45 8/18/116/ /3/58/39 7/12/99/ Lymph node involvement 50 (50.5) 106 (43.3) (42.6) 91 (45.3) Rectum: Colon 33:75 81: :60 82: Differentiation, G1/G2: G3/other 40:68 85: :61 74: Histological type, Adenocarcinoma: Mucinous 99:9 214: :9 184: Metastatic lesion characteristics Maximum length of CLLMs, 5 cm: >5 cm 86:22 160: :22 156: Number of CLLMs, 3: >3 66:42 104: :42 103: Extent of CLLMs, Unilobar: Bilobar distribution 56:52 84: :52 82: RAS status* Wild-type 69 (64.5) 156 (76.5) 69 (68.3) 137 (81.3) Mutant-type 38 (35.5) 48 (23.5) 32 (31.7) 32 (18.8) Conversion Therapy Regiments Treatment sequencing selection Systemic therapy first 30 (27.8) 80 (33.6) 31 (30.7) 64 (31.8) Primary tumor resection first 78 (72.2) 158 (66.4) 70 (69.3) 137 (68.2) First-line chemotherapy regiments CapeOX/FOLFOX/FOLFIRI 22/69/17 44/164/ /63/17 39/138/ Targeted therapy Non/Cetuximab/Bevacizumab 74/17/17 157/34/ /16/12 141/27/ Cycles of first-line chemotherapy <8 / 8-12 / >12 60/45/3 128/91/ /42/3 102/86/ Cycles of targeted therapy <8 / 8-12 / >12 17/15/2 48/26/ /11/2 35/19/ Use of TACE, 15 (13.9) 43 (18.1) (12.9) 31 (15.4) NOTE. Data are given as No. (%) unless otherwise noted. Abbreviations: PSM, propensity score matched; HS, hepatic steatosis; non-hs, none of hepatic steatosis; CLLMs, colorectal liver-limited metastases; CEA,carcinoembryonic antigen; TACE, transarterial chemoembolization.

4 4 of * There were only 309 patients performing gene test,among whom 309 patients detected KRAS status, 116 patients detected NRAS status and 192 patients detected BRAF status Ras mutations were detected in tumor samples harboring mutations in KRAS exon2 or/and NRAS(G12A,G12D,G12V, and G13D). 2.3 Outcomes of Conversion Therapy in Study Set After propensity-score matching (PSM), objective response rate (ORR) in HS group was improved than that in non-hs group (36.7% vs. 23.9%, P =0.020), with the disease control rate (DCR) were comparable in two groups (74.2% vs 69.7%, P =0.404). Following conversion therapy for every 6-8 circles, the possibility of hepatectomy were evaluated by multidisciplinary team (MDT). Total 28 patients (27.7%) in HS group and 35 patients (16.9%) in non-hs group were determined to be eligible for radical liver metastases resection. Actually, 3 patients (one in HS group, and two in non-hs group) refused further surgical intervention. Another one patient in HS group, R0 resection could not be obtained at exploration. Ultimately, 26 patients (25.7%) in HS group and 33 patients (16.4%) in non- HS group achieved a R0 resection. And the conversion hepatectomy rate was significantly higher in the HS group than non-hs group (27.7% vs. 16.6%; odds ratio (OR), 5.56; P <0.001). Similar, the R0 hepatectomy rate was higher in the HS group than the non-hs group (25.9% vs. 16.4%, P =0.005). Table 2. Outcomes of conversion therapy by hepatic steatosis in study set and validation set Study set Validation set Variable HS non-hs P HS non-hs P (n=101) (n=201) (n=16) (n=44) Efficacy results according to RECIST 1.1 CR 1 (1.0) 2 (1.0) 0 (0.0) 1 (2.3) PR 36 (35.6) 47 (22.9) 9 (56.3) 15 (34.1) SD 38 (37.6) 92 (45.8) 4 (25.0) 14 (31.8) PD 26 (25.8) 61 (30.3) 3 (18.7) 14 (31.8) ORR 37 (36.7) 48 (23.9) (56.3) 16 (36.4) DCR 75 (74.2) 140 (69.7) (65.9) 29 (81.3) Hepatectomy rate from MDT < YES 28 (27.7) * 35 (16.9) * 7 (43.7) 7 (15.9) NO 73 (72.3) 166 (83.1) 9 (56.3) 37 (84.1) Actual hepatectomy rate R0 26 (25.7) 33 (16.4) 7 (43.7) 7 (15.9) R1 1 (1.0) 0 (0.0) 9 (56.3) 37 (84.1) Surgery sequencing selection Simultaneous resection 4 (4.0) 5 (2.5) 3 (18.9) 3 (6.8) Staged resection 23 (22.7) 28 (13.9) 4 (25.0) 4 (9.1) Primary tumor resected only 40 (39.6) 84 (41.8) 2 (12.5) 6 (13.6) NOTE. Data are given as No. (%) unless otherwise noted. Abbreviations: HS, hepatic steatosis; non-hs, none of hepatic steatosis RECIST=Response Evaluation Criteria in Solid Tumors; CR, complete response; PR=partial response; SD, stable disease; PD, progressive disease; ORR, overall response rate; DCR, disease control rate *There were 3 patients performed successful conversion but did not undergo hepatectomy for poor tolerance to surgery or personal reason.

5 5 of Predictors of Conversion Hepatectomy in Study Set In univariate analysis, we indicated that HS (OR, 5.56; 95%CI, ; P <0.001), number of CLLMs >3 (OR, 0.49; 95%CI, ; P =0.014), TACE treatment (OR, 0.07; 95%CI, ; P =0.011), and targeted therapy (OR, 2.01; 95%CI, ; P =0.017) were predictors of conversion hepatectomy from MDT (Table S3). Those variables with P <0.05 were analyzed in multivariate regression model, confirmed that all of the following predictors: HS (OR, 5.23; 95%CI ; P < 0.001), use of TACE (OR, 0.08; 95%CI ; P =0.017) and targeted therapy (OR, 2.48; 95%CI ; P =0.026) were independent predictors of conversion hepatectomy from MDT in Table 3. Table 3. Univariate and multivariate logistic analyses of conversion hepatectomy rate in study set and validation set. Conversion hepatectomy rate n n (%) Univariable P Odds Ratio 95% CI P In study set Hepatic steatosis No Yes Targeted therapy No Yes Use of TACE No Yes In validation set (16.9) < reference < (27.7) (17.1) Reference (29.3) (24.0) Reference (2.3) Hepatic steatosis No Yes Targeted therapy No Yes Cycles of first-line 44 7 (15.9) Reference (43.7) (10.0) reference (36.7) chemotherapy < 8 cycle 44 3 (6.8) < reference cycle (68.8) Abbreviations: TACE, transarterial chemoembolization Patient Characteristics, Outcome of Conversion Therapy and Predictors of Conversion Hepatectomy 131 in Validation Set 132 In the validation set, among the 60 patients in the validation set, 16 (26.7%) were diagnosed with 133 HS and 44 (73.3%) were diagnosed without HS (Table S4). There were no significant differences 134 between the 2 groups in terms of basic demographics, characteristics of primary tumor and CLLMs 135 and conversion therapy regiments. 136 Following conversion therapy for every 6-8 circles, the possibility of hepatectomy were also 137 evaluated by MDT. hepatectomy rate in HS group was improved significantly than that in non-hs 138 group (43.7% vs.16.9%, P =0.038) and the actual hepatectomy rate was identical to hepatectomy rate

6 6 of from MDT. However, ORR were comparable in patients with HS or without HS (56.3% vs. 36.4%, P =0.167), which is not consistent with study set (Table 2). In validation set, by using multivariate regression model, we confirmed that all of the following predictors: HS (OR, 7.39; 95%CI ; P = 0.031), targeted therapy (OR, 7.52; 95%CI ; P =0.042) and cycles of first-line chemotherapy (OR, 0.05; 95%CI ; P =0.001) were independent predictors of conversion hepatectomy from MDT in Table 3 and Table S Overall Survival in Study Set and Validation Set For all 302 patients in study set after PSM, the median follow-up was 24 months. During followup, 208 patients (69%) died with a 1-, 3-year OS were 84% and 28%, respectively. Patients with HS status had benefit median overall survival (OS) than non-hs group (3-yr OS, 39% vs.23%, P =0.027, hazard ratio (HR) 0.50, 95%CI ) (Fig. 1a). However, such an improvement in OS in patients with HS were not observed in validation set. After a median follow-up time of 11 months (range, 5 to 23), OS were comparable in in both groups (1-yr OS, 88% vs.53%, P=0.889, HR 0.94, 95%CI ) (Fig. 1b) Figure 1. Survival of patients with synchronous CLLMs undergoing conversion therapy. (a)kaplan Meier curves for overall survival between HS group and non-hs group in study set. (b)kaplan Meier curves for overall survival between HS group and non-hs group in validation set. Abbreviations: HS, hepatic steatosis; non-hs, none of hepatic steatosis; HR, hazard ratio. 3. Discussion In this study, we found that possibility of hepatectomy after conversion therapy were predominantly predicted by two biologic features: hepatic steatosis status and use of targeted therapy. Compared with patients without HS, patients with HS had a higher conversion hepatectomy rate (27.7% vs. 16.9%, P <0.001), a higher actual hepatectomy rate (26.7% vs. 16.4%, P =0.005), and a better rate of objective response rate (36.7% vs. 23.9%, P = 0.020). In a separate validation cohort of patients who receive conversion therapy since Jan to obtain a resection criterion, HS status was also associated with a better conversion hepatectomy rate (43.7% vs. 15.9%, P=0.038). However, in separate validation we did not observe the association between HS and ORR (56.3% vs %, P =0.167). Previous reports have shown the prognostic effects of HS in patients with both primary CRC and resectable metastatic CRC, and to the best of our knowledge, this is the first report to show the impact of HS on hepatectomy rate after conversion therapy on unresectable CLLMs [18, 22, 23]. Our findings suggest that in the era of conversion therapy, hepatic steatosis status and use of targeted therapy have greater predictive importance than the clinicopathologic and other chemotherapy-related factors that have traditionally been reported to be associated with outcome (number, size or distribution of liver metastases, RAS mutation status, etc.) [2, 24, 25]. One possible

7 7 of explanation for improved hepatectomy rate is the inhibiting effect of pretreatment HS on growth of CLLMs. Clinical study and animal experiments reported that pretreatment HS can decrease malignant metastases growth in the liver and alleviate tumor load, which depend on lipid accumulation, reduced angiogenic activity and reduced number of cancer-associated fibroblasts in the liver parenchyma [17, 26, 27]. In general, hepatic steatosis provides an unsuitable environment for survival or development of metastatic cells. We also analyzed the impact of HS prior to treatment on survival in patients with unresectable synchronous CLLMs undergoing conversion therapy. Previous reports showed that the presence of pretreatment HS is associated with a lower incidence of CLLMs in patients and favorable prognosis in patients with CRC, while results reported by Ze and Kim et al showed pretreatment HS is positively associated the incidence of colorectal adenoma or adenocarcinoma [22, 23]. Those studies above suggest pretreatment HS may have different effects in different stages of CRC. Until now, there is no literature to report the cause effect relationship between pretreatment HS and conversion therapy for CLLMs. In our study set, for patients with unresectable synchronous CLLMs undergoing conversion therapy, pretreatment HS is also associated with improved prognosis. However, in validation set, we didn't observe the same impact of pretreatment HS on overall survival. Such discordance maybe attributed to a short follow-up period in validation set. In the present study, we used a validation set of patients who were diagnosed as unresectable CLLMs later. Even in this validation set, the conversion hepatectomy rate was also better in patients with HS than in patients without HS (43.7% vs 15.9%), suggesting that the effect of pretreatment HS on conversion hepatectomy rate was further confirmed. In previous reports on studies of unresectable colorectal liver metastases, multiple reasons have been proposed for the higher conversion hepatectomy rate of patients, including the lower rate of RAS mutations, smaller size or fewer number of CLLMs, use of targeted therapy, use of doublet or triplet chemotherapy, cycles of chemotherapy, all of which were regarded as predictors of higher R0 resection rate after conversion therapy to systemic or local treatment [3, 7, 8, 24]. However, because cycles of first-line chemotherapy were always prolonged or changed to second-line regiments when the shrinkage of tumors was not apparently, cycles of first-line chemotherapy were not likely to be the main reasons for conversion hepatectomy differences between patients with and without HS [7]. Furthermore, local treatment, like TACE, is also not likely to be a main reason for such differences because local treatment was also used when the shrinkage of tumors by systemic chemotherapy was not apparently. In addition, we did not observe the effect of RAS mutation on the outcome by conversion therapy both in study set or validation set, which may be resulted from a low rate of use of targeted therapy. To explore the underlying molecular mechanism, further investigations regarding reasons for the differences of oncologic outcomes between patients with are needed using the colorectal liver metastases specimens. The current study possesses some limitations. First, it followed a retrospective design, considering inevitably missed information about the history of alcohol consumption. Second, pretreatment HS and liver metastasis were mainly diagnosed via abdominal ultrasonography, computed tomography (CT), or magnetic resonance imaging and were not confirmed by biopsy, possibly leading to false-positive diagnoses, although the imaging results were conducted and evaluated by imaging specialists. Finally, we cannot study the effect of CLLMs under different grades of steatosis because of the limitations of the imaging results. Despite the limitations above, the predicating effect of HS on conversion to hepatectomy is convincible. 4. Materials and Methods 4.1 Patient Selection and Evaluations A total of 613 patients were diagnosed with resectable primary tumor and nonresectable synchronous liver-limited metastases, and followed regularly by MDT at Zhongshan Hospital, Fudan University between March 2013 and March Only 406 patients were arranged to receive conversion therapy. The possibility of resection for tumor or metastases was assessed by a local multidisciplinary team including more than three liver surgeons and one radiologist. Other criteria

8 8 of for eligibility were Eastern Cooperative Oncology Group performance status of 0 to 1, life expectancy > 3 months, and adequate hematologic, hepatic, and renal function. Patients with a past history of other cancers or splenectomy, or without pre-treatment non-enhanced CT scan were excluded. The final study set included 346 patients (Fig. 2). For our validation set, we selected 60 patients with synchronous colorectal liver-limited metastases who received conversion therapy from January 2017 to March 2018 (Fig. 2). Prior to conversion therapy, all patients were assessed by physical examination, routine hematology, carcinoembryonic antigen (CEA) levels and biochemistry analyses. CT scan or magnetic resonance image of the abdomen was done to define the extent of liver disease. Positron emission tomography CT (PET/CT) was done to rule out any extrahepatic metastasis Figure 2. Patient selection 4.2 Hepatic Steatosis Evaluation Non-enhanced CT was performed with a tube potential of 120 kvp. Tube current was adjusted by automatic exposure control with a noise index of 10 in a slice thickness of 5 mm. All CT images were reviewed by using a picture archiving and communication system workstation (Digital Imaging and Communications in Medicine, DICOM, USA). The mean CT attenuation values (in Hounsfield Units (HU)) of the liver and the spleen were obtained using standard region of interest method (Fig. 3). After measurement of the CT attenuation of the liver and spleen, liver-spleen ratio (liver attenuation (HU)/spleen attenuation (HU)) was calculated. Those patients with a liver-spleen ratio lower than 1.1 were diagnosed as HS, as previously reported [17, 19]. Since chemotherapy may have an effect on HS, we investigated the presence of HS in non-enhanced CT image prior to conversion therapy. 252

9 9 of Figure 3. Measurement of hepatic steatosis. Representative tomographic images showing the points for the measurement of attenuation and the obtained values. (a)normal liver and (b)hepatic steatosis 4.3 First-line chemotherapy Regimen and Decision of Conversion FOLFOX (oxaliplatin 85 mg/m 2 and levofolinate calcium 200 mg/m 2, followed by 5-FU, as a 400 mg/m 2 intravenous bolus and 2,400 mg/m 2 continuous infusion during 46 h) was administered every 2 weeks. CapeOX (oxaliplatin 130 mg/m 2 over 2 h on day 1 plus oral capecitabine 1,000 mg/m 2 twice daily on days 1 14) was administered every 3 weeks. FOLFIRI (150 mg/m 2 irinotecan, and 200 mg/m 2 levofolinate calcium, followed by 5-FU, as a 400 mg/m 2 intravenous bolus and 2,400 mg/m 2 continuous infusion during 46 h) was administered every 2 weeks. The choice of addition of cetuximab (400 mg/m 2 initial infusion every 2 week) or bevacizumab (5mg/kg initial infusion every 2 week) depended on the doctors discretion; however, cetuximab antibodies were exclusively used for CRC with wildtype RAS. Subjective symptoms, physical examination results, performance status and all adverse reactions were recorded before each treatment cycle. Treatment courses were repeated every 2 weeks for a total of four courses unless there was evidence of progressive disease. 4.4 Assessment of Response Tumor response was measured after every 4 cycles by CT scan and the Response Evaluation Criteria in Solid Tumors version 1.1 (RECIST v1.1) was applied to grade the best response in each case [20]. All cases were discussed for eligibility for surgery by a multidisciplinary team including medical oncologists, radiologists and surgical oncologists. Patients with resectable disease were offered liver surgery within 2 6 weeks of the last treatment cycle. Patients who showed progression or no response were started on secondary chemotherapy. 4.5 Long-Term Prognosis Overall survival (OS) was defined as the time interval between the starting date of conversion therapy to patient's death or to last follow-up. Patients who had not died at the time analysis were censored. 4.6 Statistical analysis The computer program Statistical Package for The Social Sciences version 19.0 for Windows (SPSS, Inc, Chicago, IL, USA) was used for statistical analysis. The propensity score model of presence of HS was constructed via stepwise variable selection into a multivariable logistic regression model. Candidate variables included all variables significantly associated with hepatectomy rate from MDT via univariable analysis, with a threshold of P<0.20 required for initial inclusion and P<0.10 required to remain in the model [21]. The categorical parameters were compared using two-sided Pearson s χ2 test, Fisher s exact test, as appropriate. Summary statistics on time-to-event variables were

10 10 of calculated according to the Kaplan-Meier method and were compared by means of the log-rank test. P value less than 0.05 was considered as statistically significant. 5. Conclusions In conclusion, beyond targeted therapy, pretreatment hepatic steatosis is associated with improved hepatectomy rate in conversion therapy for unresectable colorectal liver-limited metastases, and can serve as a valuable predictor of high possibility of conversion to hepatectomy in clinical practice. Supplementary Materials: The following are available online, Table S1. Baseline Variables in HS and non-hs Groups before and after PSM. Table S2. Conversion Therapy Regiments in non-hs and HS Groups. Table S3. Univariate and multivariate logistic regression of conversion hepatectomy rate in study set. Table S4 Baseline Variables in HS and non-hs Groups in validation set. Table S5. Univariate and multivariate logistic regression of conversion hepatectomy rate in validation set Author Contributions: Mi Jian, Wenju Chang, and Li Ren contributed equally to this article. Conceptualization, Ye Wei, Jianmin Xu and Xinyu Qin; Data curation, Jianmin Xu; Formal analysis, Mi Jian; Funding acquisition, Jianmin Xu; Investigation, Mi Jian and Li Ren; Methodology, Tianyu Liu; Project administration, Xinyu Qin; Resources, Jianmin Xu; Software, Yijiao Chen; Supervision, Xinyu Qin; Validation, Ye Wei and Qi Lin; Visualization, Mi Jian; Writing original draft, Mi Jian, Wenju Chang and Li Ren; Writing review & editing, Wenju Chang and Jianmin Xu. Funding: This study was supported by grants from the National Natural Science Foundation of China (No ) and the Shanghai Engineering Research Center of Colorectal Cancer Minimally Invasive (17DZ ). Acknowledgments: The authors wish to express their gratitude to Xiangdong Peng for statistical support and advice. Conflicts of Interest: The authors declare no conflict of interest. Ethical approval: All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. Informed consent: Informed consent was obtained from all individual participants included in the study. References 1. Engstrand, J.; Nilsson, H.; Stromberg, C.; Jonas, E.; Freedman, J. Colorectal cancer liver metastases - a population-based study on incidence, management and survival. BMC CANCER 2018, 18, Nozawa, H.; Ishihara, S.; Kawai, K.; Hata, K.; Kiyomatsu, T.; Tanaka, T.; Nishikawa, T.; Otani, K.; Yasuda, K.; Sasaki, K.; Kaneko, M.; Murono, K. Conversion to Resection in Patients Receiving Systemic Chemotherapy for Unresectable and/or Metastatic Colorectal Cancer Predictive Factors and Prognosis. CLIN COLORECTAL CANC 2018, 17, e91-e Ye, L.; Liu, T.; Ren, L.; Wei, Y.; Zhu, D.; Zai, S.; Ye, Q.; Yu, Y.; Xu, B.; Qin, X.; Xu, J. Randomized Controlled Trial of Cetuximab Plus Chemotherapy for Patients With KRAS Wild-Type Unresectable Colorectal Liver- Limited Metastases. J CLIN ONCOL 2013, 31, Huiskens, J.; van Gulik, T.M.; van Lienden, K.P.; Engelbrecht, M.R.; Meijer, G.A.; van Grieken, N.C.; Schriek, J.; Keijser, A.; Mol, L.; Molenaar, I.Q.; Verhoef, C.; de Jong, K.P.; Dejong, K.H.; Kazemier, G.; Ruers, T.M.; de Wilt, J.H.; van Tinteren, H.; Punt, C.J. Treatment strategies in colorectal cancer patients with initially unresectable liver-only metastases, a study protocol of the randomized phase 3 CAIRO5 study of the Dutch Colorectal Cancer Group (DCCG). BMC CANCER 2015, 15, Jawed, I.; Wilkerson, J.; Prasad, V.; Duffy, A.G.; Fojo, T. Colorectal Cancer Survival Gains and Novel Treatment Regimens: A Systematic Review and Analysis. JAMA ONCOL 2015, 1,

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