==== Front PLoS One PLoS One plos plosone PLoS ONE 1932-6203 Public Library of Science San Francisco, CA USA 10.1371/journal.pone.0240791 PONE-D-20-07323 Research Article Medicine and Health Sciences Oncology Cancers and Neoplasms Carcinoma Hepatocellular Carcinoma Medicine and Health Sciences Oncology Cancers and Neoplasms Gastrointestinal Tumors Hepatocellular Carcinoma Medicine and Health Sciences Gastroenterology and Hepatology Liver Diseases Hepatocellular Carcinoma Medicine and Health Sciences Oncology Cancers and Neoplasms Carcinoma Adenocarcinoma Cholangiocarcinoma Medicine and Health Sciences Oncology Cancers and Neoplasms Gastrointestinal Tumors Cholangiocarcinoma Medicine and Health Sciences Oncology Cancers and Neoplasms Medicine and Health Sciences Surgical and Invasive Medical Procedures Digestive System Procedures Hepatectomy Medicine and Health Sciences Gastroenterology and Hepatology Liver Diseases Cirrhosis Medicine and Health Sciences Diagnostic Medicine Prognosis Medicine and Health Sciences Oncology Cancer Treatment Surgical Oncology Tumor Resection Medicine and Health Sciences Clinical Medicine Clinical Oncology Surgical Oncology Tumor Resection Medicine and Health Sciences Oncology Clinical Oncology Surgical Oncology Tumor Resection Medicine and Health Sciences Surgical and Invasive Medical Procedures Surgical Resection Tumor Resection Medicine and Health Sciences Oncology Cancer Treatment Elevated neutrophil-to-lymphocyte ratio and predominance of intrahepatic cholangiocarcinoma prediction of poor hepatectomy outcomes in patients with combined hepatocellular–cholangiocarcinoma NLR and ICC predicts poor prognosis of patients with resectable combined hepatocellular cholangiocarcinomaChiu Tai-Jan ConceptualizationData curationFormal analysisFunding acquisitionInvestigationMethodologyResourcesValidationWriting – original draftWriting – review & editing123 Chen Yi-Ju Data curationFormal analysisMethodologyResourcesValidation4 Kuo Fang-Ying Data curationFormal analysisMethodologyValidation5 https://orcid.org/0000-0001-5450-5045Chen Yen-Yang ConceptualizationData curationFunding acquisitionInvestigationProject administrationSupervisionVisualizationWriting – original draftWriting – review & editing12* 1 Division of Hematology-Oncology, Department of Internal Medicine, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan and Chang Gung University College of Medicine, Kaohsiung, Taiwan 2 Kaohsiung Chang Gung Cholangiocarcinoma and Pancreatic Cancer Group, Cancer Center, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan 3 Institute of Clinical Medical Sciences, Chang Gung University, Kaohsiung, Taiwan 4 Department of Anatomic Pathology, E-Da Hospital, I-Shou University, Kaohsiung, Taiwan 5 Department of Pathology, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Kaohsiung, Taiwan Scarpa Aldo Editor Universita degli Studi di Verona, ITALY Competing Interests: The authors have declared that no competing interests exist. * E-mail: chen.y9964@gmail.com 11 12 2020 2020 15 12 e024079113 3 2020 3 10 2020 © 2020 Chiu et al2020Chiu et alThis is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Objectives Although elevated neutrophil-to-lymphocyte ratio (NLR) has been associated with survival in some liver cancers, its prognostic relevance has not been studied in the context of combined hepatocellular cholangiocarcinoma CHCC-CC, a rare primary liver cancer. We investigated whether elevated NLR and a predominance of cholangiocarcinoma might predict poor prognosis in patients with resectable CHCC-CC. Methods We retrospectively reviewed the clinicopathologic data of forty-two patients with CHCC-CC receiving hepatectomies at our hospital. We used Kaplan-Meier and Cox regression to analyze survival. Results Two-year disease-free survival and five-year overall survival rates were 43.2% and 32.9%, respectively. Univariate analyses showed that patients with NLR ≥3 had significantly worse 2-year DFS and 5-year OS rates. Univariant Kaplan-Meier survival analysis also associated these rates with a predominance in intrahepatic cholangiocarcinoma, AJCC tumor stage, pathological T stage and lymph-vascular invasion. However, our multivariate analysis found NLR ≥3 to be the only independent predictor of disease recurrence and poorer survival. Conclusions Neutrophil-to-lymphocyte ratio was the most important independent predictor of poorer survival in patients with resectable CHCC-CC. Predominance of intrahepatic cholangiocarcinoma, advanced AJCC tumor stage and pathological T stage, and lymph-vascular invasion also may affect poor prognosis in patients receiving complete tumor resections. http://dx.doi.org/10.13039/501100011892Kaohsiung Chang Gung Memorial HospitalCMRPG8E0881, CMRPG8F1771/1772https://orcid.org/0000-0001-5450-5045Chen Yen-Yang http://dx.doi.org/10.13039/501100011892Kaohsiung Chang Gung Memorial HospitalCMRPG8E0811/0812/0813, CMRPG8C0531 and CMRPG8D0801Chiu Tai-Jan This study was funded by grant obtained by Dr. Yen-Yang Chen from Kaohsiung Chang Gung Memorial Hospital Taiwan (Grant nos. CMRPG8E0881, CMRPG8F1771/1772). This study was also funded by grants obtained by Dr. Tai-Jan Chiu from Kaohsiung Chang Gung Memorial Hospital Taiwan (Grant CMRPG8E0811/0812/0813, CMRPG8C0531 and CMRPG8D0801). Data AvailabilityAll relevant data are within the manuscript and its Supporting information files.Data Availability All relevant data are within the manuscript and its Supporting information files. ==== Body Introduction Combined hepatocellular cholangiocarcinoma (CHCC-CC) is an uncommon primary liver malignancy. It is unique in that it shares pathological characteristics of hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (IHC) coexisting within the same tumor. It makes up between 1% to 14.3% of all primary liver tumors depending on the study [1–9]. It is difficult to use image studies to accurately diagnose this disease prior to non-invasive preoperative examinations [10]. Currently, complete tumor resection is the only possible curative treatment. Although some studies have attempted to identify factors that can predict post-hepatectomy disease free survival and overall survival, their results have not been in agreement. While some of them have reported that CHCC-CC has a worse median survival than hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (IHC) [3, 9, 11], others report its survival rate to fall between the survival rates of HCC and IHC [12–14] or be equal to IHC but worse than HCC [15]. One study found IHC-dominant patients with recurrent tumors to have significantly shorter mean survival rates than those with HCC-dominance [16]. Thus, it remains unclear how a predominance of HCC cells vs. IHC cells might affect surgical outcomes in patients with CHCC-CC. Tumors induced systemic inflammatory cytokines and mediators could promote angiogenesis, DNA damage, and inhibition of apoptosis of cancer cells and associated with poor prognoses in different cancers [17–19]. While tumor was diagnosed and before treatments, inflammatory response could be easily detected by peripheral blood tests. Several peripheral blood parameters, including neutrophil-to-lymphocyte ratio (NLR), derived NLR (d-NLR), platelet-to-lymphocyte ratio, and monocyte-to-lymphocyte ratio, have been found to be indicators of systemic inflammation and have been found predict prognosis in several malignancies [20, 21]. Although some studies have associated elevated NLR with poorer prognosis in patients with intrahepatic cholangiocarcinoma and hepatocellular carcinoma [22, 23], very few have discussed have its relevance to prognosis of CHCC-CC patients after hepatectomy. Therefore, this study was conducted to evaluate the possible association of systemic inflammation (NLR) and other pathological factors with disease free and overall survival in CHCC-CC patients after tumor resection. Materials and methods Patients This study was approved by the Chang Gung Medical Foundation Institutional Review Board, the case number is 103-7412B. All methods were performed in accordance with the principles expressed in the Declaration of Helsinki. Written informed consent was obtained from all patients before surgery. All data were fully anonymized before we accessed them. Patients’ medical records were accessed between January 2000 and December 2013. We retrospectively reviewed the records of forty-two patients diagnosed as having CHCC-CC post hepatectomy between January 2000 and December 2013 at Kaohsiung Chang Gung Memorial Hospital in Kaohsiung a medical center located in southern Taiwan. All of these patients had intrahepatic CHCC-CC post complete tumor resections (M0) and no synchronous primary tumors. CHCC-CC was diagnosed pathologically based on microscope studies of thin-section specimens stained with hematoxylin and eosin. The immunoreactivity of each tumor was confirmed [24]. They only had type B combined disease (contiguous but independent masses of HCC and IHC) and type C combined disease (intimate intermingling of hepatocellular and glandular elements), based on Allen and Lisa classification. None of the patients had type A (separate masses constituting either HCC or IHC). None of the patients had thrombus extending to the level of the superior mesenteric vein or any antitumor treatments before operation. In addition, all patients had to be ≥18 years old and have a performance status (PS) of ≤2 on the Eastern Cooperative Oncology Group (ECOG) scale, adequate bone marrow, hepatic and renal function (creatinine clearance >60 mL/min), and a computed tomography or magnetic resonance image scan of the abdominal region within three weeks prior to the initiation of treatment. Before hepatectomy, each patient received ultrasonography, contrast-enhanced magnetic resonance (MR), and/or tri-phase contrast enhanced helical computed tomography (CT) for the diagnosis of CHCC-CC and evaluation of the tumor. Also prior to operation, the liver function of each patient was assessed using the Child-Pugh classification system and/or the indocyanine green (ICG) clearance test. Three days before hepataectomy, neutrophil and lymphocyte counts were measured. NLR was calculated by dividing the neutrophil measurement by the lymphocyte measurement. Clinicopathological information including age, gender, tumor (T) stage, nodal (N) status, TNM stage, and survival was obtained from the patients’ clinical records. This study was approved by the Chang Gung Medical Foundation Institutional Review Board. No written informed consent was required due to the retrospective design of the study. Pathologic examination As our literature review, there was no study to evaluate whether the predominance of intra-hepatic cholaginocarcinoma affected the outcome in resectable CHCC-CC patients. After discussion with our pathologist with expertise in hepatic tumors, we made the definition of predominance of intrahepatic cholangiocarcinoma while those whose IHC cells made up more than 55%. Patients with CHCC-CC were subdivided into those whose IHC cells made up 55% or less (n = 23) of all cells within the largest tumor and those whose IHC cells made up more than 55% (n = 19). Two pathologists reviewed all pathological findings and concluded that both HCC cells and IHC cells coexisted in liver tumors of our patients and that IHC cells made up at least more than 10% of the cells within the tumor in all patients. Pathological diagnosis of CHCC-CC was made microscopically using thin-section specimens stained with Hematoxylin and Eosin (H&E). Immunohistochemical staining of each tumor was also performed. HCC cells were confirmed based on immunohistological stains of hepatocyte paraffin 1 (Hep1) and ICC cells confirmed based on cytokeratin 7 (CK7). Definition of NLR NLR was calculated by dividing the absolute neutrophil count by the absolute lymphocyte count. NLR was determined within seven days before hepatectomy. Based on the time-dependent receiver operating characteristic (ROC) curve of our study, we set the NLR cutoff value to be 3.0(NLR < 3, NLR ≥ 3) (S1 Fig). Previous studies in intrahepatic cholangiocarcinoma have used a similar threshold [25, 26]. Follow-up Patients were followed up every two months the first three years post surgery and every six months thereafter. Disease free survival (DFS) was defined as the duration between hepatectomy and recurrence or between hepatectomy and the last observation for patients without recurrence. Overall survival was defined as the time between hepatectomy and death or between hepatectomy and last follow-up. Statistical analyses The differences in numerical data between the two groups were compared by Student t test. Categorical variables were compared using χ2-tests. The estimated OS was calculated using Kaplan-Meier, and the differences between groups were assessed using the log rank test. Univariate and multivariate analyses of the prognostic factors for survival were performed using the Cox proportional hazards model. All statistical operations were performed using SPSS 18 statistical software (IBM Corp., Armonk, NY, USA). All tests were two-sided. A p-value < 0.05 was considered significant. Result Clinicopathological characteristics The median age of 42 patients whose cases we reviewed was 58 years (range, 32–80 years). Twenty-nine (69%) were male and 13 (31%) female. The demographic and case characteristics of the patients are shown in Table 1. Classified using the TMN cancer staging system (American Joint Committee on Cancer), 15 patients (35.7%) had stage I tumors, 19 (42.5%) stage II, 4 (9.5%) stage III and 4 (9.5%) stage IVA. No patient had stage IVB (distant metastasis (M1)) disease. Only three patients had regional lymph nodes metastases (N1) and only one patient had tumor with portal vein invasion (T4). Thirty patients (71.4%) tested positive for HbsAg (surface antigen of the hepatitis B virus), 12 (28.6%) positive for HCV antibody (hepatitis C virus), and 31 (72.8%) had liver cirrhosis. Preoperatively, 23 patients (54.8%) had AFP (Alpha-Fetoprotein) levels > 15 ng/ml, three patients (7.1%) had CEA (carcinoembryonic antigen) > 5 ng/ml, and twelve patients (28.6%) CA 19–9 > 37 U/ml in. Nineteen patients (45.2%) had a predominance of IHC cells within the tumor. 10.1371/journal.pone.0240791.t001Table 1 Clinicopathologic features of 42 patients with resectable combined hepatocellular cholangiocarcinoma post operation. Parameters No. of cases (percentage) Age (years)(mean: 58.2, median: 58.5, range 32–80) Age≦60 22 (52.3%) Age > 60 20 (47.6%) Sex Male 29 (69%) Female 13 (31%) Clinical 7th AJCC stage I-II 34 (81%) III-IV 8 (19%) pathological T classification T1 15 (35.7%) T2 21 (50%) T3 5 (11.9%) T4 1 (2.4%) Pathological N classification negative 39 (92.9%) positive 3 (7.1%) Surgical margin <10 mm 18 (42.8%) ≥ 10 mm 24 (57.2%) Lymph-vascular invasion negative 26 (61.9%) positive 16 (38.1%) Perineural invasion negative 39 (92.9%) positive 3 (7.1%) Histology grade Well or Moderate 35(83.3%) Poorly 7 (16.7%) Hepatitis B Negative 12(28.6%) positive 30(71.4%) Hepatitis C Negative 30(71.4%) positive 12(28.6%) Liver cirrhosis no 11(26.2%) yes 31(72.8%) CEA ≤5 39(92.9%) >5 3(7.1%) CA-199 ≤ 35 30(71.4%) >35 12(28.6%) AFP ≤ 15 19(45.2%) >15 23(54.8%) NLR <3 21(50%) ≧3 21(50%) Intrahepatic cholangiocarcinoma cell ≤55% 23 (54.8%) >55% 19 (45.2%) Abbreviations: AFP, Alpha-Fetoprotein; CA19-9, cancer antigen 19–9; LMR, lymphocyte to monocyte ratio; NLR, neutrophil-to-lymphocyte ratio. Correlation between clinicopathologic parameters and NLR Patients were subdivided into those with NLR<3 (n = 21, 50%) and those with NLR≧3 (21, 50%). The two NLR groups had no significant differences with regard to age, gender, AFP, CA19-9, HbsAg, HCV Ab, liver cirrhosis, lymph node metastasis, perineural invasion, lymph-vascular invasion, histology grade, AJCC tumor stage or IHC cell predominance (Table 2). 10.1371/journal.pone.0240791.t002Table 2 Relationships between elevated neutrophil-to-lymphocyte ratio and clinicopathological factors. NLR <3 ≧3 P Age ≤ 60 11 (50%) 11(50%) 1.000 > 60 10 (50%) 10 (50%) Gender male 14 (48.3%) 15 (51.7%) 1.000 female 7 (53.8%) 6 (46.2%) AJCC staging I-II 19(55.9%) 15 (44.1%) 0.238 III-IV 2 (25%) 6 (75%) T stage I-II 20 (55.6%) 16 (44.4%) 0.184 III-IV 1 (16.7%) 5 (83.3%) N stage Negative 20 (51.3%) 19 (48.7%) 1.000 positive 1 (33.3%) 2 (66.7%) Lymph-vascular invasion Negative 14 (53.8%) 12 (46.2%) 0.751 Positive 7 (43.8%) 9 (56.2%) Perineural invasion Negative 19 (48.7%) 20 (51.3%) 1.000 Positive 2 (66.7%) 1 (33.3%) Histology grade Well or Moderate 17 (48.6%) 18 (51.4%) 1.000 Poorly 4 (57.1%) 3 (42.9%) Hepatitis B Negative 5 (41.7%) 7 (58.3%) 0.734 positive 16 (53.3%) 14 (46.7%) Hepatitis C Negative 17 (56.7%) 13 (43.3%) 0.306 Positive 4 (33.3%) 8 (66.7%) Liver cirrhosis No cirrhosis 7 (63.6%) 4 (36.4%) 0.484 Cirrhosis 14 (45.2%) 17 (54.8%) CEA ≤5 21 (53.8%) 18 (46.2%) 0.232 >5 0 (0%) 3 (100%) CA-199 ≤ 35 16 (53.3%) 14 (46.7%) 0.734 >35 5 (41.7%) 7 (58.3%) AFP ≤ 15 9 (47.4%) 10 (52.6%) 0.757 >15 12 (52.2%) 11 (47.8%) IHC percentage ≤55% 14 (60.9%) 9 (39.1%) 0.121 >55% 7 (36.8%) 12 (63.2%) Surgical outcomes Mean survival follow-up was 677 days (87–2371 days). Nineteen of the patients died during the course of this study. Mean disease free survival was 499 days, two-year recurrence free survival was 43.2%, and five-year overall survival 34.9% (Table 3). As can be seen in Fig 1, the following were found by univariate analyses to be associated with poor disease free survival: lymph-vascular invasion (P = 0.035, Fig 1A), IHC cell > 55% (P = 0.031, Fig 1B), AJCC stage III/IV (P < 0.001, Fig 1C), T3/4 disease (P < 0.001, Fig 1D), NLR ≧3 (P<0.001, Fig 1E). Overall survival was also significantly lower in patients with lymph-vascular invasion (p = 0.001, Fig 2A), IHC cell > 55% (P = 0.034, Fig 2B), AJCC tumor staging III/V (p < 0.001, Fig 2C), T3/4 disease (p < 0.001, Fig 2D), NLR ≧3 (p < 0.001, Fig 2E). 10.1371/journal.pone.0240791.g001Fig 1 Kaplan-Meier estimates of the probability of disease free survival (DFS). Positive Lymph-vascular invasion (LVI)(1a), intratumor cholagnocarcinoma ≥ 55% (1b), T-stage III/IV(1c), AJCC tumor stage III/IV(1d) and neutrophil-to-lymphocyte ratio (NLR) ≥3 (1e) were associated with poor DFS. 10.1371/journal.pone.0240791.g002Fig 2 Kaplan-Meier estimates of the probability of overall survival (OS). Positive lymph-vascular invasion (LVI) (2a), intratumor cholagnocarcinoma ≥ 55% (2b), T stage III/IV (2c), AJCC tumor staging III/VI (2d) and neutrophil-to-lymphocyte ratio (NLR) ≥3 (2e) had poorer OS. 10.1371/journal.pone.0240791.t003Table 3 Correlation between the clinicopathological features and 2-year recurrence-free survival and overall survival in combined hepatocellular cholangiocarcinoma. Variables No. of patients Cumulative P Cumulative P 2- year recurrence-free survival rate 5-year overall survival rate Age ≦60 22 46.5% 0.935 37.8% 0.493 >60 20 52.5% 23.2% Sex male 29 44.8% 0.232 29.2% 0.627 female 13 60.6% 57.7% AJCC staging I-II 34 58.8% <0.001* 44.2% <0.001* III-IV 8 12.5% 0% T stage T1-2 36 57.7% <0.001* 41.2% <0.001* T3-4 6 0% 0% N stage negative 39 51.2% 0.198 37.9% <0.001* positive 3 33.3% 0% Lymph-vascular invasion Negative 26 62.8% 0.035* 49.1% 0.001* Positive 16 29.2% 12.9% Perineural invasion Negative 39 50.5% 0.498 36.7% 0.162 Positive 3 33.3% 0% Histology grade Well or Moderate 35 48.1% 0.853 38.5% 0.867 Poorly 7 57.1% 0% Hepatitis B Negative 12 48.6% 0.882 41.9% 0.947 Positive 30 49.3% 34.6% Hepatitis C Negative 30 51.7% 0.865 35.3% 0.805 Positive 12 42.8% 34.6% Liver cirrhosis No cirrhosis 11 54.5% 0.833 51.9% 0.465 Cirrhosis 31 47.3% 34.2% CEA ≤5 39 51.7% 0.112 36.8% 0.012* >5 3 0% 0% CA-199 ≤ 35 30 50.4% 0.776 45.1% 0.205 >35 12 45.7% 0% AFP ≤ 15 19 41.5% 0.788 23.3% 0.303 >15 23 55.2% 41.9% NLR <3 21 84.0% 0.001* 60.0% 0.001* ≧3 21 9.2% 10.8% IHC cell percentage ≤55% 23 61.4% 0.031* 45.9% 0.034* >55% 19 35.1% 17.2% Prognostic significance of NLR The clinicopathological characteristics were included in our univariate and multivariate analyses. As shown in Table 3, in our univariate analysis, lymph-vascular invasion, IHC cell percentage, AJCC tumor stage, T stage and NLR were all significantly associated with DFS and OS. Multivariate cox proportional hazard regression analysis, however, revealed that only NLR ≧3 to independently predict poor DFS (HR, 41.679; 95% CI, 7.812–222.378; P = 0.001) and OS (HR, 4.148; 95% CI, 1.196–14.388; P = 0.025) in our CHCC-CC after hepatectomy (Table 4). 10.1371/journal.pone.0240791.t004Table 4 Risk factors affecting 2-year recurrence free survival and 5-year overall survival rate determined by Cox regression analysis. Factors HR 95%CI P value Disease free survival Gender female 0.328 0.112–0.967 0.043 CA-199 >35 7.719 1.789–33.316 0.006 NLR ≧3 41.679 7.812–222.378 0.001 T stage III-IV 219.682 16.966–2844.518 0.001 Overall survival NLR ≧3 4.148 1.196–14.388 0.025 AJCC staging III-IV 10.847 3.085–38.140 0.001 CI, confidence interval; HR, hazard ratios a. Age, N stage, AJCC tumor stage, lymph-vascular invasion, perineural invasion, histology grade, hepatits B or C, liver cirrhosis, CEA > 5, AFP>15 and intrahepatic cholangiocarcinoma ratio were not associated the recurrence b. Age, N stage, pathological T stage, lymph-vascular invasion, perineural invasion, histology grade, hepatits B or C, liver cirrhosis, CEA > 5, CA199, AFP>15 and intrahepatic cholangiocarcinoma ratio were not associatedmortality. Discussion Combined hepatocellular cholangiocarcinoma is a rare mixed primary liver malignancy in which HCC and IHC coexist. It is important to identify prognostic factors at the time of diagnosis because this information may help operative decision making and guide adjuvant or neoadjuvant treatment choices. This study found that an elevated NLR (≧3.0) independently predicted worse DFS and OS in patients with CHCC-CC treated with curative resection. NLR can be calculated from a simple blood test and can be assessed prior to the initiation of therapy. Previous studies have demonstrated an association between NLR and survival of IHC [22, 26], HCC [27, 28] and CHCC-CC [29]. Although the prognostic significance of inflammation-based NLR has been previously reported in various types of cancer, it remained unclear whether these scores might predict which patients might be at high risk of recurrence after surgery for CHCC-CC. Our current study found that NLR can also be used to predict this recurrence and overall survival. In the microenvironment of tumors, the mediators and cellular effectors of inflammation are important constituents [30]. Inflammation may promote the development, progression, angiogenesis, invasion and metastasis of some types of cancer [31, 32]. Inflammation activates proangiogenic factors including vascular endothelial growth factor or inflammatory cytokines, such as IL-1β by increasing neutrophils [33, 34]. In the inflammatory process, lymphocytes play a very important role in innate immunity and adaptive immune response and can eradicate tumor cells by inhibiting cell proliferation or migration [17, 30]. Thus, NLR is a very practical biomarker marker of inflammation. The other interesting finding in our study is that IHC cell predominance (>55%) plays an important prognostic factor of DFS and OS of CHCC-CC patients post tumor resection in Kaplan-Meier univariate analyses. There were just a few studies about predominance of HCC or IHC cells within the tumor in CHCC-CC. Ariizumi et al. reported that CHCC-CC patients had poor outcomes after hepatectomy regardless of the predominance of IHC cells within the tumor. In their study, twenty of forty-four patients (45%) had portal vein invasion and ten patients received transcatheter arterial chemoembolization or radiofrequency ablation before surgery. Portal vein invasion has been found to be a significant poor prognostic factor in HCC [35, 36] and IHC [37] patients receiving operation. Nearly fifty-percent patients with portal vein invasion before surgery may affect their DFS and OS significantly in Ariizumi’ study. Although their study showed portal invasion was more frequent in patients with >50% IHC cells within the tumor than in patients with ≤50% IHC cells, but there was no statistically significant difference. Our study just included one patient with portal vein invasion because most were diagnosed as having HCC prior to hepatectomy. At our hospital, HCC with portal vein invasion is a contra-indication for surgery. It is difficult to diagnose CHCC-CC accurately before surgery, because CT or MRI scans often do not show typical patterns of contrast uptake or washout. CHCC-CC may have features of both HCC and IHC when a hepatic tumor contains an area of hyper-enhancement in the early phase and an area of delayed enhancement in the late phase on dynamics computed tomography [38]. Survival and prognosis of CHCC-CC patients after hepatectomy varies. Depending on study, 5-year survival rates range from 0% to 62% [39]. The difference in medial survival of those studies may be due to the small number of patients and may not accurately represent the actual prognosis of patients with CHCC-CC. In the current study, median DFS and OS were 16.6 months and 22.6 months, respectively, indicating patients with CHCC-CC had poor OS and DFS despite the curative resection. Most studies have found survival rates to be poorer in CHCC-CC than in HCC and undecided with regard to IHC [9, 40]. We found that two-year disease free survival in patients with less than 55% and more than 55% IHC cells within the tumor to be 61.4% and 35.1%, respectively, and 5-year overall survival rates to be 45.9% and 17.2%, respectively, both significantly different in univariate Kaplan-Meier survival analyses. A predominance of IHC cells within CHCC-CC was an indicator of a more aggressive tumor, though in our multivariate cox regression analysis, did not find the predominance to significantly affect DFS and OS. We found no clinicopathological factors to be associated with IHC predominance in this study (Table 5). Our univariant Kaplan-Meier survival analyses also found AJCC tumor stage, pathological T stage and lymph-vascular invasion to predict DFS and OS, but our multivariant cox regression analyses did not. These pathological factors have been related to poor prognoses in previous studies [41, 42]. 10.1371/journal.pone.0240791.t005Table 5 Relationships between predominance of intrahepatic cholangiocarcinoma and clinicopathological factors. Intrahepatic cholangiocarcinoma cell ≤55% >55% P Age  ≤ 60 13 (59.1%) 9 (40.9%) 0.551  > 60 10 (50%) 10 (50%) Gender  male 18 (62.1%) 11 (37.9%) 0.278  female 5 (38.5%) 8 (61.5%) AJCC staging  I-II 21(61.8%) 13 (38.2%) 0.112  III-IV 2 (25%) 6 (75%) T stage  I-II 22 (61.1%) 14 (38.9%) 0.075  III-IV 1 (16.7%) 5 (83.3%) N stage  Negative 21 (53.8%) 18 (46.2%) 1.000  positive 2 (66.7%) 1 (33.3%) Lymph-vascular invasion  Negative 17 (65.4%) 9 (34.6%) 0.149  Positive 6 (37.5%) 10 (62.5%) Perineural invasion  Negative 22 (56.4%) 17 (43.6%) 0.581  Positive 1 (33.3%) 2 (66.7%) Histology grade  Well or Moderate 18 (51.4%) 17 (48.6%) 0.428  Poorly 5 (71.4%) 2 (28.6%) Hepatitis B  Negative 5 (41.7%) 7 (58.3%) 0.462  positive 18 (60%) 12 (40%) Hepatitis C  Negative 16 (53.3%) 14 (46.7%) 1.000  Positive 7 (58.3%) 5 (41.7%) Liver cirrhosis  No cirrhosis 4 (36.4%) 7 (63.6%) 0.283  Cirrhosis 19 (61.3%) 12 (38.7%) CEA  ≤5 21 (53.8%) 18 (46.2%) 1.000  >5 2 (66.7%) 1 (33.3%) CA-199  ≤ 35 17 (56.7%) 13 (43.3%) 0.961  >35 6 (50%) 6 (50%) AFP  ≤ 15 12 (63.2%) 7 (36.8%) 0.320  >15 11 (47.8%) 12 (52.2%) NLR  <3 14 (66.7%) 7 (33.3%) 0.121  ≧3 9 (42.9%) 12 (57.1%) This study has some limitations. First, it was a single center retrospective study. Second, the patient sample number was relatively small and selection bias may affect outcome. Diagnosis of CHCC-CC before surgery was difficult. The preoperative diagnoses of patients in this study were either HCC or IHC. Third, our hospital had no standardized treatment of recurrent CHCC-CC. Different treatment modalities might have affected overall survival. In conclusion, preoperative NLR ≧3 was significantly predicted poor DFS and OS in resectable CHCC-CC. This readily available pre-operative test might potentially be used in conjunction with such post-operative pathological findings as T tumor stage, AJCC tumor stage, lymph-vascular invasion and IHC percentage to improve assessment of tumor biology and treatment decision-making. Supporting information S1 Fig ROC curve of Neutrophil/lymphocyte ratio. Threshold values were determined using the ROC curves, and the value with the highest sensitivity and specificity was calculated. (TIF) Click here for additional data file. S1 Data Study dataset. Analytical dataset used in the study. (XLSX) Click here for additional data file. The authors thank Kaohsiung Chang Gung Memorial Hospital Cancer Center for patients’ clinical data collection. Abbreviations AFPAlpha-Fetoprotein CEACarcinoembryonic antigen CHCC-CCcombined hepatocellular cholangiocarcinoma CK7cytokeratin 7 CTcomputed tomography DFSdisease free survival ECOGEastern Cooperative Oncology Group H&EHematoxylin and Eosin HbsAgsurface antigen of the hepatitis B virus HCCHepatocellular carcinoma HCVhepatitis C virus Hep1hepatocyte paraffin 1 HRhazard ratios ICGindocyanine green IHCintrahepatic cholangiocarcinoma MRmagnetic resonance NLRneutrophil-to-lymphocyte ratio OSoverall survival PSperformance status 10.1371/journal.pone.0240791.r001 Decision Letter 0 Scarpa Aldo Academic Editor © 2020 Aldo Scarpa2020Aldo ScarpaThis is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Submission Version0 13 Jul 2020 PONE-D-20-07323 Elevated neutrophil-to-lymphocyte ratio and predominance of intrahepatic cholangiocarcinoma prediction of poor hepatectomy outcomes in  patients with combined hepatocellular–cholangiocarcinoma PLOS ONE Dear Dr. Chen, Thank you for submitting your manuscript to PLOS ONE. 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This study was also funded by grants obtained by Dr. Tai-Jan Chiu from Kaohsiung Chang Gung Memorial Hospital Taiwan (Grant CMRPG8E0811/0812/0813, CMRPG8C0531 and CMRPG8D0801). We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. a. Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: 'The author(s) received no specific funding for this work.' b. Please include your amended statements within your cover letter; we will change the online submission form on your behalf. 4. Please upload a copy of Figure 4f and 4g, to which you refer in your text. 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You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: Thank you for the privilege of reviewing the paper by Chiu, Chen et al examining N-L ratio (NLR) in patients with resection of combined HCC-Cholangioca (intrahepatic) with a predominance of CCA. Reference 28 signifies that the finding is not terribly novel, but it was a small study (n=59( The study adds incrementally to the body of literature that supports the utility of NLR as a prognostic marker for recurrence post hepatectomy for IHC, HCC here examining combined HCC-IHC. This is still worthwhile in my opinion since there isn’t a lot of data or large numbers in published studies to date. The angle of IHC predominance is of some interest, but there may be confounding, see below. Some clarifications would be useful Did any cases have branch portal vein invasion? If so it has to be included in the descriptive analysis and model Is there an approved definition of HCC-CCA or predominance of IHC vs HCC in mixed lesions? Consider the paper by Brunt et al PMID: 29360137. I believe the authors have been careful in their case selection but important to add some references here where possible Where did the cut-offs for Age CEA CA199 AFP NLR and IHC% come from? This would be important to describe, especially for NLR, some descriptive analysis on this in particular is warranted Did NLR correlate with CA19-9 or IHC percentage? Nice to describe and does not negative the validity of data if there was a relationship, but we have to overtly recognize the risk of confounding then In Table 4, please provide by footnote the factors not associated with disease recurrence or mortality For sake of sensitivity analysis how would dose NLR as a continuous or categorical variable predict endpoints when age AFP CA199 CEA and IHC% are included as continuous variables Figures, formatting needed, change days to years, text too small on axes, image quality poor and add nothing to data shown in tables Consider a graph for NLR above and below threshold that is adjusted for all other factors, this can be done in Cox-regression in SPSS, that includes all factors, making NLR a categorical variable and clicking plot, follow the prompts to show smoothed survival curves by selected cat variable (NLR). This way you have two graphs with risk adjusted outcomes for recurrence and OS Reviewer #2: THis is a descriptive, paper describing NLR in intrahepatic cholangiocarcinoma looking at a series of cases collected over 13 years at a single institution Suggestions: it is stated that cutoff of 3 determined from ROC curve, but curve not shown The authors suggest that this is a predictive model but is actually a descriptive case series. Reviewer #3: The manuscript titled “Elevated neutrophil-to-lymphocyte ratio and predominance of intrahepatic cholangiocarcinoma prediction of poor hepatectomy outcomes in patients with combined hepatocellular–cholangiocarcinoma” is a study about prognostic value of elevated neutrophil-to-lymphocyte ratio in surgical treated combined hepatocellular – cholangiocarcinoma. The Authors should be congratulated for collecting a clinical series of 42 patients resected with this rare disease The manuscripts have the following limitations: • The Authors should discuss the criteria for dividing patients according to the proportion of IHC cell (more or less than 55%); • The Authors should clarify the reason for reporting univariate analysis at 2-year for recurrence-free survival and at 5 years for overall survival; • The Authors should report the rate of positive and negative margins among patients submitted to resection; • The small sample size and the distribution of different characteristics may have influenced the power of statistical analysis, the authors should comment the HR rate for T stage for recurrent disease (HR= 219.682). I suggest to review the statistical analysis by a statistician. ********** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No Reviewer #3: Yes: Andrea Ruzzenente [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. 10.1371/journal.pone.0240791.r002 Author response to Decision Letter 0 Submission Version1 16 Sep 2020 Response to Editor comments: 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf. Response: We agree with this valuable comment. We will make sure our manuscript to meet PLOS ONE style 2. In the ethics statement in the manuscript and in the online submission form, please provide additional information about the patient records used in your retrospective study, specifically whether all data were fully anonymized before you accessed them. Response: We agree with this valuable comment. In the “Ethics approval and consent to participate”, we provide Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request 3. 'Founding This study was funded by grant obtained by Dr. Yen-Yang Chen from Kaohsiung Chang Gung Memorial Hospital Taiwan (Grant nos. CMRPG8E0881, CMRPG8F1771/1772). This study was also funded by grants obtained by Dr. Tai-Jan Chiu from Kaohsiung Chang Gung Memorial Hospital Taiwan (Grant CMRPG8E0811/0812/0813, CMRPG8C0531 and CMRPG8D0801). We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. a. Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: 'The author(s) received no specific funding for this work.' Response: we will remove funding information from text and update our Funding statement. b. Please include your amended statements within your cover letter; we will change the online submission form on your behalf. Response: Our amended statements will be within our cover letter 4. Please upload a copy of Figure 4f and 4g, to which you refer in your text. If the figure is no longer to be included as part of the submission please remove all reference to it within the text. Response: We correct our mistakes of Figure 4f and 4g to Figure 2f and 2g 5. Your ethics statement must appear in the Methods section of your manuscript. If your ethics statement is written in any section besides the Methods, please move it to the Methods section and delete it from any other section. Please also ensure that your ethics statement is included in your manuscript, as the ethics section of your online submission will not be published alongside your manuscript. Response: We rewrite our ethics statement in the Methods section of our manuscript Response to Reviewer 1’s Comments: 1. Did any cases have branch portal vein invasion? If so it has to be included in the descriptive analysis and model. Response: We agreed with these comments. In this study, we just found only one patient had tumor with portal vein invasion (T4). In the section of result, we showed that only one patient had tumor with portal vein invasion. At our hospital, HCC with portal vein invasion is a contra-indication for surgery. It is difficult to diagnose CHCC-CC accurately before surgery, because CT or MRI scans often do not show typical patterns of contrast uptake or washout. The patient number was too few, and we did not include this item in our analysis. However, in our study, there were sixteen patients with microscopic lymph-vascular invasion. We found cHCC-CC patients with lymph-vascular invasion predicted poor outcome after resection. 2. Is there an approved definition of HCC-CCA or predominance of IHC vs HCC in mixed lesions? Consider the paper by Brunt et al PMID: 29360137. I believe the authors have been careful in their case selection but important to add some references here where possible Response: I think this is a very important comment. We citated this paper in our reference [24]. In this retrospective study, we collected medical records of 42 patients with primary CHCC-CC treated with surgical resection between January 2000 and December 2013 at our hospital. Two pathologists performed immunohistochemical studies of tissue samples collected during surgery. Following Allen and Lisa classification, we included only patients with CHCC-CC classified as type C (intimate intermingling of hepatocellular and glandular elements) and type B (contiguous but independent masses of HCC and CC). We excluded patients with type A (separate masses constituting either HCC or CC). CHCC-CC was diagnosed pathologically based on microscope studies of thin-section specimens stained with hematoxylin and eosin. The immunoreactivity of each tumor was confirmed: hepatocyte paraffin 1 (Hep1) antibody and CK-7 (cytokeratin-7) in CHCC-CC. 3. Where did the cut-offs for Age CEA CA199 AFP NLR and IHC% come from? This would be important to describe, especially for NLR, some descriptive analysis on this in particular is warranted. Response: We agreed with these comments. a. The cut-offs values of CEA, CA199 and AFP were the upper limit of normal range in our hospital. b. The cut-off value for Age is the median age of these 42 patients. c. Definition of NLR: NLR was calculated by dividing the absolute neutrophil count by the absolute lymphocyte count. NLR was determined within seven days before hepatectomy. Based on the time-dependent receiver operating characteristic (ROC) curve of our study, we set the NLR cutoff value to be 3.0(NLR < 3, NLR ≥ 3). Previous studies in intrahepatic cholangiocarcinoma have used a similar threshold[25, 26]. d. IHC cell predominance (>55%): The definition was discussed with pathologists with expertise in hepatic tumors. However, there was no related reference. Ariizumi et al. reported that CHCC-CC patients had poor outcomes after hepatectomy regardless of the predominance of IHC cells within the tumor. Their definition of IHC predominance was >50% IHC cells within the tumor. 4. Did NLR correlate with CA19-9 or IHC percentage? Nice to describe and does not negative the validity of data if there was a relationship, but we have to overtly recognize the risk of confounding then Response: We agreed with these comments. Table 2 showed relationships between elevated neutrophil-to-lymphocyte ratio and clinicopathological factors. We did not find the relationship between NLR and CA199 or IHC predominance. 5. In Table 4, please provide by footnote the factors not associated with disease recurrence or mortality. Response: We agreed with these comments. We provided by footnote the factors not associated with disease recurrence or mortality. 6. For sake of sensitivity analysis how would dose NLR as a continuous or categorical variable predict endpoints when age AFP CA199 CEA and IHC% are included as continuous variables. Response: We agreed with these comments. In Table 2, we found that there was no relationship between NLR and age, AFP, CA199, CEA and IHC%. NLR could predict tumor recurrence and survival time although cox-regression analyses including age, AFP, CA199, CAE, AFP and IHC% Recurrence B 標準誤差 Wald 自由度 顯著性 Exp(B) 95.0% Exp(B) 的 CI 下限 上限 Age60 -.227 .446 .258 1 .611 .797 .333 1.911 NLR2 2.893 .736 15.431 1 .000 18.039 4.260 76.383 CEA1 .575 .820 .492 1 .483 1.778 .356 8.872 CA199a -.559 .567 .970 1 .325 .572 .188 1.738 AFP1 -.187 .467 .160 1 .689 .829 .332 2.073 Cho55 .901 .502 3.217 1 .073 2.461 .920 6.587 Overall survival B 標準誤差 Wald 自由度 顯著性 Exp(B) 95.0% Exp(B) 的 CI 下限 上限 Age60 -.754 .505 2.225 1 .136 .471 .175 1.267 NLR2 1.972 .652 9.149 1 .002 7.187 2.002 25.795 CEA1 1.440 .851 2.865 1 .090 4.223 .797 22.383 CA199a .738 .519 2.019 1 .155 2.091 .756 5.784 AFP1 -1.013 .531 3.647 1 .056 .363 .128 1.027 Cho55 1.338 .555 5.819 1 .016 3.812 1.285 11.305 7. Figures, formatting needed, change days to years, text too small on axes, image quality poor and add nothing to data shown in tables Response: We agree with this valuable comment and we re-format the figures 2). How many pathologist was read IHC data? What criteria to accept on grading score from different reader? Response: In the “immunohistochemical study”, MK immunostaining was evaluated independently by two pathologists blinded to the subjects’ clinical information. Each specimen was assigned a score of 1 to 4 based on the percentage of positive cells within a field of cells (100 x magnification): 1 for <5% of the cells, 2 for 6–35% of the cells, 3 for 36–70% of the cells, and 4 for >71% of the cells. Each specimen also received another score of 1 to 4 based on intensity of staining: 1 for negative staining, 2 for weak staining, 3 for moderate staining and 4 for strong staining. MK expression score was then calculated by multiplying the percentile and intensity scores. A score of ≥4 for MK protein expression levels indicated the tumor was positive Response to Reviewer 2’s Comments: it is stated that cutoff of 3 determined from ROC curve, but curve not shown Response: We agree with this valuable comment and we provide our ROC curve at supplementary Figure 1 Response to Reviewer 3’s Comments: 1. The Authors should discuss the criteria for dividing patients according to the proportion of IHC cell (more or less than 55%) Response: We agreed with these comments. However, from the literature review, there was no study to evaluate whether the percentage of intra-hepatic cholaginocarcinoma affect the outcome in CHCC-CC patients. After discussion with our pathologist with expertise in hepatic tumors, we made the definition of predominance of intrahepatic cholangiocarcinoma while those whose IHC cells made up more than 55% 2. The Authors should clarify the reason for reporting univariate analysis at 2-year for recurrence-free survival and at 5 years for overall survival; Response: We agreed with these comments. Survival and prognosis of CHCC-CC patients after hepatectomy varies. Depending on study, 5-year survival rates range from 0% to 62%[39]. In the current study, Mean survival follow-up was 677 days (87-2371 days). Nineteen of the patients died during the course of this study. Mean disease free survival was 499 days, two-year recurrence free survival was 43.2%, and five-year overall survival 34.9%. Therefore, we report the univariate analysis at 2- year for recurrence free survival and 5 years for overall survival. 3. The Authors should report the rate of positive and negative margins among patients submitted to resection. Response: We agree with this valuable comment. We reported our surgical margin (< 1cm or � 1cm) on Table 1. Eighteen patients (42.8%) had surgical margin < 1 cm. 4. The small sample size and the distribution of different characteristics may have influenced the power of statistical analysis, the authors should comment the HR rate for T stage for recurrent disease (HR= 219.682). I suggest to review the statistical analysis by a statistician. Response: We agree with this valuable comment. We discussed with the statistician. They thought that HR rate for pathological T stage for recurrence was affected significantly because the patient number of pathological T3-4 was only 6 patients. The sample size was too small and all these patients got recurrence within 4 months (40 days to 106 days). Attachment Submitted filename: Response to Reviewers.docx Click here for additional data file. 10.1371/journal.pone.0240791.r003 Decision Letter 1 Scarpa Aldo Academic Editor © 2020 Aldo Scarpa2020Aldo ScarpaThis is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Submission Version1 5 Oct 2020 Elevated neutrophil-to-lymphocyte ratio and predominance of intrahepatic cholangiocarcinoma prediction of poor hepatectomy outcomes in  patients with combined hepatocellular–cholangiocarcinoma PONE-D-20-07323R1 Dear Dr. Chen, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. 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For more information, please contact onepress@plos.org. Kind regards, Aldo Scarpa Academic Editor PLOS ONE Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: All comments have been addressed Reviewer #3: All comments have been addressed ********** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #3: Yes ********** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #3: Yes ********** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #3: Yes ********** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #3: Yes ********** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The authors have addressed my comments, other reviewer comments seem less comprehensively approached and overall there are minimal changes to the paper, nevertheless the data is what it is and for a purely descriptive study it is reasonable to publish Reviewer #3: The Authors addressed the comments of the reviewers, and they imporved the manuscript accordignto the suggestions of the reviewers ********** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #3: Yes: Andrea Ruzzenente 10.1371/journal.pone.0240791.r004 Acceptance letter Scarpa Aldo Academic Editor © 2020 Aldo Scarpa2020Aldo ScarpaThis is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 2 Dec 2020 PONE-D-20-07323R1 Elevated neutrophil-to-lymphocyte ratio and predominance of intrahepatic cholangiocarcinoma prediction of poor hepatectomy outcomes in patients with combined hepatocellular–cholangiocarcinoma Dear Dr. Chen: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Aldo Scarpa Academic Editor PLOS ONE ==== Refs References 1 Goodman ZD , Ishak KG , Langloss JM , Sesterhenn IA , Rabin L . Combined hepatocellular-cholangiocarcinoma. A histologic and immunohistochemical study . Cancer . 1985 ;55 (1 ):124 –35 . .2578078 2 Allen RA , Lisa JR . Combined liver cell and bile duct carcinoma . 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