Tumor invasion loci in resected margins of perihilar cholangiocarcinoma and their impact on survival: a retrospective 13-year longitudinal cohort study

Tumor invasion loci in resected margins of perihilar cholangiocarcinoma and their impact on survival: a retrospective 13-year longitudinal cohort study | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Tumor invasion loci in resected margins of perihilar cholangiocarcinoma and their impact on survival: a retrospective 13-year longitudinal cohort study Yury Kovalenko, Anna Olifir, Andrei Glotov, Anatoly Popov, Sergey Trifonov, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6620965/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 21 Oct, 2025 Read the published version in World Journal of Surgical Oncology → Version 1 posted 9 You are reading this latest preprint version Abstract Background Perihilar cholangiocarcinoma (PHC) presents significant challenges in achieving curative resection, with current definitions of radicality (R0/R1/R2) based solely on tumor presence at margins lacking prognostic precision. This study investigates the impact of tumor invasion loci within resection margins on survival and proposes a novel prognostic classification. Methods A 13-year retrospective cohort study of 83 PHC patients undergoing liver/bile duct resection was conducted. Histopathological evaluation assessed seven invasion loci: bile duct margins, adipose tissue, hepatic parenchyma, vascular/lymphatic structures, perineural spaces, lymph nodes, and radial margins. Resection radicality was redefined as RN0-7, combining the number of tumor-positive loci (R + 1 to R + 6) with lymph node status (N). Survival analysis utilized Kaplan-Meier and Cox regression methods. Results Tumor invasion loci were identified in 91.6% of specimens. Multivariate analysis revealed perineural invasion (HR = 14.48, p = 0.03), lymph node involvement (HR = 2.16, p = 0.01), and higher TNM stage (p = 0.006) as key prognostic factors. Survival rates declined progressively with increasing invasion loci: 5-year survival exceeded 60 months for patients with ≤ 2 loci (RN0-2), but dropped significantly with ≥ 3 loci (p < 0.00001). Traditional UICC R0/R1 classification poorly correlated with outcomes, as 36.1% classified as R0 exhibited poor survival if multiple loci were involved. Conclusion The number of tumor invasion loci in resection margins, combined with lymph node status, provides superior prognostic stratification compared to conventional R0/R1 criteria. The proposed RN0-7 classification highlights the importance of quantifying anatomical sites of residual disease, offering a foundation for standardized histopathological evaluation and personalized adjuvant strategies. This approach may enhance prognostic accuracy and guide therapeutic decisions in PHC, where randomized trials remain impractical. perihilar cholangiocarcinoma resection margin tumor invasion loci radicality survival analysis prognostic classification Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 Figure 16 Figure 17 1. Background Perihilar cholangiocarcinoma accounts for no more than 3% of all gastrointestinal tumors 1 – 3 which greatly limits the ability to investigate this condition in depth, while ethical considerations make comparative randomized trials impractical at present. In addition, the anatomy and the type of the tumor expansion make radical resections less likely 4 – 5 . Literature sources indicate that the frequency of R0 resections varies greatly, from 15 to 95% 6–10 and on average is 73·7% 11 . In addition, the 5-year survival rate varies between 20–40% 11 . Currently, there is no common understanding on what radicality is and how to define the resection margin 2 , 7 . For example, a number of authors define an R0 resection as the absence of tumor cells at the margins of the hepatic duct and the radial margin 12 – 13 . Note that different surgical approaches cannot significantly affect the prognosis, whereas patients with an R0 resection typically have better survival rates 14 . According to the Union for International Cancer Control (UICC), there are three types of the resection margin: a negative resection margin (R0), microscopically present tumor cells in the resection margin (R1) and a macroscopically present residual tumor (R2) 15 . The resection margin usually refers to the site of separation and intersection of anatomical structures. First of all, it is the margin of the bile duct, the dissection plane in the hepatoduodenal ligament, where structures are surgically released or dissected from the surrounding tissues 16 – 19 . We would like to emphasize that the existing estimates of the ductal margin (DM) and even the radial margin (RM) of the resection cannot objectively reflect how radical the resection is 20 . We should note that in general the concept of an R0 resection offers great value, primarily in terms of long-term prognosis and the possibility of drug therapy. Therefore, a number of authors note the importance of developing a common universal definition of "pR0" and standardization 21 – 22 . Research in context Evidence before this study We searched PubMed for peer-reviewed, original studies (with no start date and up to June 2023), using the search terms cholangiocarcinoma, perihilar cholangiocarcinoma, resection margin, radial margin, radicality, radical resections, prognosis. Typically, when studies refer to the resection margin, only the DM of the bile duct is mentioned, and the RM is often overlooked. To our knowledge, several studies has addressed the RM status in detail in resected PHC. The UICC currently grades surgical margins as R0 or R1 based on whether tumor cells are present or absent. In addition, there is no consensus as to what an R0 resection actually is and how wide the tumor-free margin should be. The existing concept of the resection margin does not objectively reflect the surgical radicality since it does not take into account histopathological risk factors, and there is no focus on prognosis. Therefore, the lack of complete information on the resection status compromises the validity of survival studies. Added value of this study As far as we know, this is the first study presenting a new concept for evaluating resection margins and radicality of surgery in patients with perihilar cholangiocarcinoma in a homogeneous, clinically well-characterized cohort of patients. Furthermore, this is the first study to provide detailed morphological data on all tumor-affected microstructures within the resection margin. We provide a different understanding, and thus a different approach, for evaluating resected samples with a proven correlation of survival rates with risk factors (invasion loci) since we know that the mere presence of tumor cells in the resection margin does not correlate with survival rates in all patients, and vice versa, the absence of these cells does not guarantee that relapse will not occur. Our concept was to evaluate every potential tumor locus within the true resection margin and to count them all combined with the lymph node status (N). For example, we found that the long-term overall survival reliably depended on the number of tumor-affected loci and the presence of positive lymph nodes. In addition, we found a trend showing that the more we had such loci, the more progressively the survival rate declined during follow-up. Notably, the survival rate for all patients exceeded the 5-year mark with one or two affected loci out of seven possible, and the mere presence of tumor cells at the resection margin had no practical importance. Furthermore, while our study has grown over the past 13 years as the number of patients has increased, we have performed numerous recalculations, proving the above pattern. Implications of all the available evidence The results of our cohort study provide the best proof that the evaluation of the resection margin solely based on whether or not cells are present—even in the so-called true margin—does not objectively represent the surgical radicality and offers no potential advantage for the long-term prognosis. Our concept of "the influence of the number of tumor invasion loci in resected margins on the survival rate" should serve as the foundation for a best-practice consensus to develop clinical and morphological guidelines in patients with portal cholangiocarcinoma and can potentially be applied to other malignancies. Randomized clinical trials on the subject are likely to be neither feasible nor of practical benefit in the coming years since the disease is quite rare, the tumor heterogeneity is significant with respect to its mutational status, patients require long-term and cost-intensive treatment and no effective drug therapy options exist. Our data also provide new insights for creating prognostic models and finding new targets for targeted therapies. 1.1 Objective The first objective was to study and determine the significance of tumor invasion loci in the true resection margin in terms of long-term survival. The second objective was to formulate the concept of radical resections and develop a morphological classification based on the evaluation of all tumor invasion loci, taking into account the individual prognosis. 2. Patients and Methods 2.1 Study Population From 2011 to 2023, patients with perihilar cholangiocarcinoma were treated in the Department of Abdominal Oncology of the Vishnevsky National Medical Research Center of Surgery. The patients were taken from a prospective database and studied retrospectively. The study included 83 liver and bile duct resections. The sample was limited to only those patients who had a complete morphological examination after liver and bile duct resection to properly identify all tumor invasion loci and reveal the true margin status, both the ductal margin and the radial margin. The exclusion criteria have been identified for this study: resection of the liver and/or bile ducts with a macroscopic residual margin, the lack of the possibility of histological examination of biopsy material (that is the histological blocks, for one reason or another, did not allow conducting a full study (there was a nonobservance of the making blocks technology, loss of certain parameters (proteins) in the analyzed tissue specimen which were specific for this disease, etc.), including immunohistochemical study), combined hepato-cholangiocellular carcinoma, underwent only palliative and symptomatic treatment, the patient refusal of proposed surgical treatment. The study was approved by the Center's local ethics committee. 2.2 Preoperation Management and Surgery The patient examination procedure usually included ultrasonography, CT, MRI (with MRCP and 3-D reconstruction) with contrast, tumor markers (CA 19·9, CEA, AFP). Decompression of the bile ducts, mainly by the antegrade route, was the first stage in patients with obstructive jaundice. The type of surgical intervention was planned depending on the location of the tumor in the liver according to Bismuth-Corlette, the presence of vascular invasion and the future liver remnant, and in some cases the possibility of performing a reliable hepaticojejunal anastomosis. Intraoperatively, when the bile duct confluence was involved (Bismuth-Corlette type IV), the choice of the type of hepatectomy primarily depended on the results of intraoperative biopsy and intraoperative ultrasonography. All patients underwent lymph node dissection according to the JSBS 23 . The excised lymph nodes were represented by 12, 8 and 13 groups. Portal vein resection was performed when it was involved in a tumor invasion according to CT data and confirmed macroscopic evaluation of the vessel invasion during surgery. If possible, dissection was carried out by a single block of nerve trunks within the nerve plexus, connective tissue, adipose tissue and lymphatic vessels of the hepatoduodenal ligament to evaluate the loci of invasion and the RM status. 2.3 Pathological Evolution and Definition of the surgical margins Histological examination was performed according to the College of American Pathologists (CAP) guidelines and the current WHO classification of tumors of the digestive system (5th edition, 2019) 24 . The tumor was staged according to the current TNM classification for perihilar cholangiocarcinoma (extrahepatic bile duct cancer, AJCC 8th edition) 15 . All possible tumor invasion loci were evaluated histologically as microvessels (V), surrounding adipose tissue (SAT), lymph nodes (N) and vessels (L), proximal and distal bile duct margins (BD), perineural invasion (Pn), and hepatic parenchyma (Hep). If necessary, CD31 immunohistochemical testing (IHC) was performed to confirm lymphovascular invasion. The resulting tissue samples were fixed in 4% buffered formalin for 24–48 hours. The specimens were prepared in a standard manner for microscopy with hematoxylin and eosin staining. For IHC they were prepared on positively charged slides. The latter was performed on serial paraffin sections. All surgical margins were stained to match the color marker for subsequent identification under a microscope (Fig. 1 , 2 ). A positive ductal margin was defined by the presence of tumor cells in the proximal (hepatic side) and/or distal (duodenal side) parts of the bile duct. A positive resection margin was defined by the presence of tumor cells at a distance less than 1 mm from the dissection plane of the hepatic hilum, hepatic parenchyma and hepatoduodenal ligament structures (Fig. 3 – 6 ). All histology specimens were evaluated by two or three experienced histologists. 2.4 Follow-Up All patients were monitored for relapse after discharge from the hospital. CT or MRI scans were conducted every 3–4 months for a year and then every 6 months for the next two years. Thereafter, once a year or as needed. PET-CT was performed in doubtful cases. 2.5. Ethical considerations The study complied with the norms of the Declaration of Helsinki and was fully approved by the Local Ethics Committee of the A.V. Vishnevsky National Medical Research Center of Surgery under protocol No. 08–19 on May 6, 2019. All participants gave informed consent prior to the study. 2.6 Statistical Analysis Nonparametric statistical methods were used to process the results. The median (range) was defined as the average of two median values of a number of its ranked elements. The survival rate was analyzed using the Kaplan-Meyer method. To investigate the simultaneous influence of dependent variables on survival, both mono- and multivariate Cox regression analyses were used. The log-rank test was used to compare two survival curves. All tests were bilateral, and the results were considered significant at p < 0·05, with at least 95% accuracy. All statistical calculations were performed using STATISTICA® version 13 (StatSoft, USA). 3. Results 3.1 Patient and Operative Procedures The study included 83 patients with perihilar cholangiocarcinoma who underwent liver resection and/or extrahepatic bile duct resection. There were 12 (14·4%) patients who underwent extrahepatic bile duct resection only, 58 (69·9%) patients with hepatectomy combined with extrahepatic bile duct resection (among them 19 (32·8%) with right-sided hemihepatectomy and 39 (67·2%) with left-sided hemihepatectomy), 12 (14·4%) patients with extended hemihepatectomy and 1 (1·2%) patient with extrahepatic bile duct resection combined with pancreatoduodenectomy. The Bismuth-Corlette distribution of patients was as follows: I, II – 10 (12%), IIIA/B – 21 (25·3%), IV – 52 (62·6%). Liver resection with segment I – 32 (38·5%). Resection of the portal vein was performed in 12 (14·4%) patients; there was one case (1·2%) of combination of portal vein and artery resection. 3.2 Histopathological Findins The distribution of patients by disease stage (pTNM) was as follows: I (T1N0M0) – 7 (8·4%), II (T2a/bN0M0) – 33 (39·7%), III А/В (T3/4N0M0) – 16 (19·3%), III С (T2-4N1M0) – 22 (26·5%), IVА (T3/4N2M0) – 5 (6%). Lymph node involvement was observed in 26 (32·9%) cases. According to the UICC classification: R0–30 (36·1%), R1–50 (60·2%), R2–3 (3·6%). The analysis of the morphological characteristics of the tumor included all possible parameters reflecting the tumor expansion: the degree of differentiation of tumor cells, microvascular invasion, lymphovascular invasion, liver invasion, duct resection margin, perineural invasion, tumor cells in adipose tissue, tumor growth shape and the depth of tumor invasion (Table 1 ). Table 1 Morphological features perihilar cholangiocarcinoma patients Characteristics and sites of tumor invasion Number abs % Differentiation degree (n = 83) High 22 26·5 Moderate 39 47 Low 22 26·5 Microvascular invasion (n = 72) Yes 30 41·7 No 42 58·3 Lymphovascular invasion (n = 70) Yes 63 90 No 7 10 Tumor cells in the bile duct resection margin (n = 83) Yes 56 67·4 No 27 32·5 Liver invasion (n = 71) Yes 41 57·7 No 30 42·3 Perineural invasion (n = 74) Yes 66 89·2 No 8 10·8 Tumor cells in surrounding adipose tissue (n = 74) Yes 67 90·5 No 7 9·5 Table 1 : Morphological features perihilar cholangiocarcinoma patients. Characteristics and sites of tumor invasion Number abs % Depth of invasion of the bile duct wall (n = 71) Total invasion 63 88·7 Tumor growth shape (n = 71) exophyte 7 9·9 endophyte 64 90·1 We should note that, due to tumor expansion through hepatoduodenal ligament structures, we observe high incidence of invasion into the lymphatic ducts, surrounding adipose tissue and nerve trunks, significantly complicating the goal of radical surgery. 3.3 Tumor Invasion Places and Surgical Margins Tumor invasion loci at the true resection margin, i.e., DM and RM, in combination with lymph node evaluation, were used to evaluate resection radicality. A total of 7 sites of possible tumor invasion were evaluated. These are the resection margins of the bile duct, adipose tissue, hepatic parenchyma, venous/arterial and lymphatic vessels, nerve trunks and lymph nodes. Resection without all 6 positive loci was considered radical and was designated as R 0 . According to our proposed prognosis-targeted classification (detailed in Section 3.4), depending on the number of positive loci (R + ), we denoted the resection status, respectively, as R + 1 , R + 2 , R + 3 , R + 4 , R + 5 , R + 6 in combination with status N (RN). In total, there were 7 (8·43%) patients with RN 0 status, 3 (3·61%) with RN + 1 , 7 (8·43%) with RN + 2 , 3 (3·61%) with RN + 3 , 6 (7·23%) with RN + 4 , 15 (18·07%) with RN + 5 , 21 (25·30%) with RN + 6 , 21 (25·30%) with RN + 7 . Histological evaluation of all 7 loci revealed tumor cells in 76 (91·56%) of 83 resected samples (Table 2 ). There were 37 (44·5%) proximal positive margins and 19 (22·9%) distal positive margins. The RM was evaluated taking into account the different number of identified tumor invasion loci. We should emphasize that in certain situations, even with the most thorough evaluation of the RM resection status, it proved challenging for us to determine the "true" dissecting plane in a resected specimen. Table 2 Evaluation of invasion loci in resection margins. Invasive areas Аbs % Microvascular invasion (n = 72) 30 41·7 Lymphovascular invasion (n = 70) 63 90 Tumor cells in bile duct resection margins (n = 83) 41 49·4 Liver invasion (n = 71) 41 57·7 Perineural invasion (n = 74) 66 89·.2 Surrounding adipose tissue (n = 74) 67 90·5 Regional lymph nodes (n = 73) 26 35·6 We analyzed the RM in 80 histological specimens for the presence or absence of tumor cells; however, as the table illustrates, we were unable to evaluate some specimens for all six loci (structures) of invasion (Fig. 8 – 12 ). 3.4 Prognostic Factors, Classification and Survival In a multivariate analysis (11 pathomorphological factors included in the analysis), the disease stage according to pTNM (Disease Stage 8 th ed) (p = 0·006), the presence of cells in lymph nodes (p = 0·01), the degree of differentiation (p = 0·02) and perineural invasion (p = 0·03) were statistically significant prognostic factors (Table 3 ). Depending on the liver resection with or without the first segment, comparative analysis showed no difference (p = 0·14). 12-, 36-, 60-, 84-, 120-month survival rates were 70%, 30%, 18%, 16% and 16%, respectively (Fig. 13 ). The median follow-up was 27 months. In 9 patients, the survival rate exceeded 60 months. The maximum follow-up period was 156 months (one patient). Table 3 : The dependence of survival on the factors in the multivariable analyses. Pathomorphological factors Multivariable analyses*, р Risk Ratio (95% Confidence Interval) Tumor cells in the surrounding adipose tissue 0·50 Lymphovascular invasion 0·51 Microvascular invasion 0·08 Perineural invasion 0·03 14·48 (1·16–180·75) Depth of bile duct wall invasion 0·71 Regional lymph nodes 0·01 2·16 (1·18 − 3·96) Tumor differentiation stage 0·02 1·63 (1·05 − 2·51) Tumor cells in bile duct resection margins 0·82 Liver invasion 0·08 Form of tumor growth 0·18 pTNM (Disease Stage 8 th ed) 0·006 1·05 (1·01–1·09) * Statistical validity (р) of the analysis performed reached 0·00002. A comparative study by the number of loci (RN 0-7 ) revealed a pattern: the more affected loci a patient has, the worse the survival rate is for this patient. Due to the small number of patients in groups RN 0 , RN 1 and RN 2 and no difference in survival between them, all patients were combined to one group RN 0-2 . Statistical calculations for this group were carried out the statistics of a small sample. Thus, when comparing the groups RN 0-2 and RN 3-7 , we observed a statistically significant difference (p=0·00000) (Fig. 14), with survival exceeding 60 months in all patients in the RN 0-2 group. When comparing the groups RN 0-3 and RN 4-7 , RN 0-4 and RN 5-7 , RN 0-5 and RN 6-7 , we also obtained a statistically significant difference (p=0·00000, p=0·00030, p=0·03, respectively) (Fig. 15-18). Since we found a significant difference in survival depending on the number of tumor-affected places, we proposed a new prognosis-targeted classification (Table 4). Radicality degree (R) Number of tumor-affected places R 0 R 0 R + R +1 R +2 R +3 R +4 R +5 R +6 Table 4 : Classification of operations depending on radicality. Thus, taking into account the created prognosis-targeted classification, the radicality of resection in combination with the N status was designated as RN from 0 to 7 with a mandatory indication of the invasion locus (BD 0/1 , L 0/1 , V 0/1 , Pn 0/1 , N 0/1 , Hep 0/1 , SAT 0/1 ). 4 Discussion Currently, the only effective treatment for perihilar cholangiocarcinoma is surgery. Over the last two decades, its constant goal has been and still is to achieve the R0 status. Radical surgical intervention has become more aggressive, combining extensive hemihepatectomy with resection of extrahepatic bile ducts, lymphadenectomy, vascular resection and, in some cases, pancreatoduodenal resection 25 . Nonetheless, even this expanded surgical scope did not yield considerable success in reaching the radicality of surgery 11 , 25 . Studies have shown that the R0 resection has become a difficult and nearly impossible task 13 , 21 . One of the primary reasons as to why this problem remains unsolved is the lack of a unified concept of the radicality of resection, as well as no understanding concerning what negative margins of resection actually are. Furthermore, the criteria specialists use to determine R0 are vague, and there is some confusion when it comes to evaluating tumor expansion as criteria for radicality 17 , 26 – 29 . R0 resection is commonly thought to signify the absence of tumor cells in the margins of the common bile duct and hepatic duct, and, unfortunately, the radial margin is very rarely mentioned 16 – 19 . Thus, recent studies have shown that morphological reports provide poor evaluation of surgical margins, especially in Western medical centers, where their coverage rate ranges from 10–45% 27 . According to Kentaro Shinohara et al., literature sources describe only one study that discusses in detail the RM status in patients after resection of distal cholangiocarcinoma; however, the authors found no single report on perihilar cholangiocarcinoma 16 . To the best of our knowledge, this is the first study to explore the radicality of resection from a position of invasion places (bile ducts, arterial and venous vessels, the liver parenchyma, lymphatic vessels, nerves and surrounding adipose tissue) and their number in the resection margins, and in the absence of the possibility of assessing these places in the edge - additional assessment them in the tumor tissue. In addition, our study details the assessment of DM and RM, i.e. the so-called true resection margin. It should be noted that our hypothesis that radical R0 status should be considered only from the position of assessing all possible invasion places in the resection margin was confirmed by statistical analysis of the prospective database in 2019. Also, in connection with the addition of patients, all data were recalculated, and their repeated statistical processing was carried out in 2024. Currently, there are a lot of studies devoted to the significance of pathomorphological factors as markers of poor prognosis, such as low differentiation degree, infiltrative form of tumor growth, micro- and lymphovascular invasion, positive resection margin, perineural invasion, affected lymph nodes 20 , 22 , 29 . Significant pathomorphological factors in the multivariate analysis we completed for our study were the presence of tumor cells in regional lymph nodes and perineural invasion (p = 0·00002). A number of factors such as micro- and lymphovascular invasion, positive resection margin, liver invasion and tumor cells in the surrounding adipose tissue have not shown a reliable relationship with prognosis, but their significance has been demonstrated by a number of other studies 19 , 27 . The obtained result can be explained by the fact that very often it is impossible to determine which factor is independent and which is a confounder, i.e. in such cases, we take into account a “principle of confounders.” In our opinion, the radicality of resection should be considered not through a surgical prism, but from the point of view of the oncological position, i.e. position of long-term prognosis and search for possible points for solving the existing problem. It is very important not just to discuss the resection margins, but to discuss in which structures the tumor cells are located, as well as how many structures (places) are affected by the tumor. We should note that the pTNM disease stage (p = 0.0061 in the multivariate analysis) in our study was a significant factor reflecting how survival depended on a set of pathomorphological factors in the long-term period. These findings support our concept of "dependence of survival on the number of positive invasion loci" concerning long-term prognosis following resection. The TNM classification considers, in addition to clinical parameters, a combination of pathomorphological invasion loci, such as the bile duct, surrounding adipose tissue, vessels, liver parenchyma and lymph node tissue; however, it poorly reflects the resection status and is thus based primarily on the evaluation of tumor prevalence rather than prognosis 11 , 15 . According to our concept, we considered the number of tumor invasion sites in the true resection margin in combination with the N status. The evaluation showed that tumor cells were found in 91% of the resected samples. At the same time, according to the UICC classification, tumor cells were discovered in only 64% of the resected samples, implying that one-third of the patients would not have the radicality of surgery clearly defined, which previously prevented us from making an accurate prognosis. It is important to point out that a comparative analysis of the "number of positive loci" found several trends. We would like to focus on them. First, our statistical analysis revealed that survival worsened depending on the number of positive invasion loci, exceeding the 5-year limit in all patients with the RN 0 and RN + 1−2 status. Second, we observed that as the number of positive loci increased, survival declined progressively. As a result, we believe that the radicality of resection should be determined not only by the presence/absence of tumor cells in the resection margin, but primarily by the number of loci (anatomical structures) affected by the tumor in the resected margin, and that the prognosis should be based on a combination of the R and N statuses. Our classification of resection radicality compared to the UICC classification (R 0 , R 1 and R 2 ) has the designations R 0 , R + 1 , R + 2 , R + 3 , R + 4 , R + 5 , R + 6 in combination with the lymph node evaluation (RN 0 − 7 ) and the invasion locus in the true resection margin always indicated (BD 0/1 , L 0/1 , V 0/1 , Pn 0/1 , Hep 0/1 , SAT 0/1 ). It shows the number and location of the tumor invasion and thus indicates a long-term prognosis following surgery. The present study has some limitations, mainly related to the rarity of this disease. The main limitation of this study is the retrospective nature of the analysis; additionally, it was conducted in a single center. Therefore, unexpected biases cannot be completely excluded. In conclusion, we provide strong evidence for the value of the number of all possible invasion loci (R + ) in the true resection margin combined with the N status, enabling us to complete a comprehensive evaluation of resected tumor samples and make a long-term prognosis with good accuracy (RN 0−7 ). The presented morphologic classification has the potential to profoundly alter the tactics and treatment of patients because it has a high prognostic value. Finally, to the best of our knowledge, we provide a novel approach to evaluating resection margins, as well as the possibility of having the first practical basis for creating a long-term prognosis with high accuracy when selecting patients for drug therapy. Declarations Authors contributions: All authors – Conceptualization, Data curation, Formal Analysis, Investigation, Methodology, Validation, Visualization, Writing (original draft, review, and editing). Ethics approval: The study complied with the norms of the Declaration of Helsinki and was fully approved by the Local Ethics Committee of the A.V. Vishnevsky National Medical Research Center of Surgery under protocol No. 08-19 on May 6, 2019. Consent to participate: All participants gave informed consent prior to the study. Consent for publication: All authors have given their consent for publication. Availability of data and materials: A patient database available upon official request. Acknowledgments: N/A. Funding: N/A. References Landis SH, Murray T, Bolden S, Wingo PA. Cancer statistics. CA Cancer J Clin 1998; 48(1):6-29. Banales JM, Marin JJG, Lamarca A, et al. Cholangiocarcinoma 2020: the next horizon in mechanisms and management. 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Classification of biliary tract cancers established by the Japanese Society of Hepato-Biliary-Pancreatic Surgery: 3(rd) English edition. J Hepatobiliary Pancreat Sci 2015; 22(3):181-96. Nagtegaal ID, Odze RD, Klimstra D, et al. WHO Classification of Tumours Editorial Board. The 2019 WHO classification of tumours of the digestive system. Histopathology 2020; 76(2):182-188. Nagino M, Ebata T, Yokoyama Y, et al. Evolution of surgical treatment for perihilar cholangiocarcinoma: a single-center 34-year review of 574 consecutive resections. Ann Surg 2013; 258(1):129-40. Washington MK, Berlin J, Branton PA, et al. Protocol for the examination of specimens from patients with carcinoma of the perihilar bile ducts. Arch Pathol Lab Med 2010; 134(4):e19-24. Castellano-Megías VM, Ibarrola-de Andrés C, Colina-Ruizdelgado F. Pathological aspects of so called "hilar cholangiocarcinoma". World J Gastrointest Oncol 2013; 5(7):159-70. Nakanuma Y, Sasaki M, Sato Y, et al. Multistep carcinogenesis of perihilar cholangiocarcinoma arising in the intrahepatic large bile ducts. World J Hepatol 2009; 1(1):35-42. Nakanuma Y, Jang KT, Fukushima N, et al. A statement by the Japan-Korea expert pathologists for future clinicopathological and molecular analyses toward consensus building of intraductal papillary neoplasm of the bile duct through several opinions at the present stage. J Hepatobiliary Pancreat Sci 2018; 25(3):181-187. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 21 Oct, 2025 Read the published version in World Journal of Surgical Oncology → Version 1 posted Editorial decision: Revision requested 01 Jun, 2025 Reviews received at journal 31 May, 2025 Reviews received at journal 20 May, 2025 Reviewers agreed at journal 20 May, 2025 Reviewers agreed at journal 20 May, 2025 Reviewers invited by journal 20 May, 2025 Editor assigned by journal 15 May, 2025 Submission checks completed at journal 12 May, 2025 First submitted to journal 08 May, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6620965","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":455611264,"identity":"7ae92926-3006-4f2b-85c6-ebde9feb504f","order_by":0,"name":"Yury Kovalenko","email":"","orcid":"","institution":"A.V. Vishnevsky National Medical Research Center","correspondingAuthor":false,"prefix":"","firstName":"Yury","middleName":"","lastName":"Kovalenko","suffix":""},{"id":455611265,"identity":"ea806ba8-fdf2-4c1a-bf87-084c7fa3ae49","order_by":1,"name":"Anna Olifir","email":"","orcid":"","institution":"A.V. Vishnevsky National Medical Research Center","correspondingAuthor":false,"prefix":"","firstName":"Anna","middleName":"","lastName":"Olifir","suffix":""},{"id":455611266,"identity":"cfa803b1-bc99-4e07-929b-9a818b955050","order_by":2,"name":"Andrei Glotov","email":"","orcid":"","institution":"A.V. Vishnevsky National Medical Research Center","correspondingAuthor":false,"prefix":"","firstName":"Andrei","middleName":"","lastName":"Glotov","suffix":""},{"id":455611267,"identity":"ca680978-2b11-4b41-9116-5ee78d3a180e","order_by":3,"name":"Anatoly Popov","email":"","orcid":"","institution":"A.V. Vishnevsky National Medical Research Center","correspondingAuthor":false,"prefix":"","firstName":"Anatoly","middleName":"","lastName":"Popov","suffix":""},{"id":455611268,"identity":"83dd2c4a-8224-4392-a078-889a621de07e","order_by":4,"name":"Sergey Trifonov","email":"","orcid":"","institution":"A.V. Vishnevsky National Medical Research Center","correspondingAuthor":false,"prefix":"","firstName":"Sergey","middleName":"","lastName":"Trifonov","suffix":""},{"id":455611270,"identity":"8b56412e-88a9-45ed-a588-1916218709a7","order_by":5,"name":"Dmitry Blagovestnov","email":"","orcid":"","institution":"Russian Medical Academy of Continuous Professional Education","correspondingAuthor":false,"prefix":"","firstName":"Dmitry","middleName":"","lastName":"Blagovestnov","suffix":""},{"id":455611271,"identity":"ee962c9b-8498-4538-9c44-331fb143acf6","order_by":6,"name":"Tatyana Shevchenko","email":"","orcid":"","institution":"A.V. Vishnevsky National Medical Research Center","correspondingAuthor":false,"prefix":"","firstName":"Tatyana","middleName":"","lastName":"Shevchenko","suffix":""},{"id":455611273,"identity":"5e0de89c-e964-4d6e-804b-04dfe320c373","order_by":7,"name":"Beslan Gurmikov","email":"","orcid":"","institution":"A.V. Vishnevsky National Medical Research Center","correspondingAuthor":false,"prefix":"","firstName":"Beslan","middleName":"","lastName":"Gurmikov","suffix":""},{"id":455611274,"identity":"d6a9a6af-3958-4134-b87c-955c50e736f1","order_by":8,"name":"André Pontes-Silva","email":"","orcid":"","institution":"Federal University of São Carlos","correspondingAuthor":false,"prefix":"","firstName":"André","middleName":"","lastName":"Pontes-Silva","suffix":""},{"id":455611279,"identity":"e49d3092-7c02-479b-8f2d-c985114fa716","order_by":9,"name":"Yury Zharikov","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0UlEQVRIie3OMQuCQBTA8ScHNz11PTnxMwiBtPVVDgSnEwS3kECCWsS5j9FHEARbpDkQoo8QCI7SRVOL2tZw/+Xx4P3gAeh0/1gFQNRABmA8AJj7EyG+IriYgCKUve2ssLqK9El2d51jEWxv2RqBt/4kca4CVqcmRY5t0MlGPWbJaeK3ACFSgR6Lok5SRRDnSY3jh6RyXEaMvXkQyFnYkPiwgDgtEGKWAp2irnlcMqRIk0liKdLjIDbskue9HHaejeQ8SQDs5/dOZ+51Op1Ot6AX43o09YP0c/cAAAAASUVORK5CYII=","orcid":"","institution":"FSAEI HE I.M. Sechenov First MSMU of MOH of Russia (Sechenovskiy University)","correspondingAuthor":true,"prefix":"","firstName":"Yury","middleName":"","lastName":"Zharikov","suffix":""}],"badges":[],"createdAt":"2025-05-08 13:08:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6620965/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6620965/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12957-025-04006-7","type":"published","date":"2025-10-21T16:17:26+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":82891481,"identity":"1a8324ca-1ff5-4096-a201-5ad16ac8b615","added_by":"auto","created_at":"2025-05-16 12:13:18","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":373578,"visible":true,"origin":"","legend":"\u003cp\u003eMacroscopic assessment of surgical specimen for PHC. Formalin-fixed specimen after inking of the different resection margins: hilar and hepatoduodenal ligament - dark blue, hepatic parenchyma - red.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6620965/v1/391959670172dbca9403d971.png"},{"id":82892832,"identity":"5031ce77-40ad-48bd-967c-a1dd23ddb1d4","added_by":"auto","created_at":"2025-05-16 12:21:19","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":254733,"visible":true,"origin":"","legend":"\u003cp\u003eMacroscopic assessment of surgical specimen for PHC. A resected left lobe of the liver with extrahepatic bile ducts and the gallbladder. The circumferential margin of the resection is stained black\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6620965/v1/9214ed69da14e8357aee625f.png"},{"id":82892835,"identity":"a04822eb-1534-4d2c-801c-01d21c4dc43b","added_by":"auto","created_at":"2025-05-16 12:21:19","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":482265,"visible":true,"origin":"","legend":"\u003cp\u003eMicroscopic assessment. A wall of the bile duct. The circumferential margin of the resection is stained green. 50x magnification.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6620965/v1/3ac6ba489e780c1925db2081.png"},{"id":82891491,"identity":"83837c2c-57e9-447f-81ac-6314cd1c2544","added_by":"auto","created_at":"2025-05-16 12:13:19","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":319031,"visible":true,"origin":"","legend":"\u003cp\u003eMicroscopic assessment. The bile duct and surrounding tissues. The distance to the circumferential margin is less than 1 mm. 400x magnification.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6620965/v1/416c58049be28c20fb6fa4e4.png"},{"id":82894317,"identity":"29f6985b-9e56-42e4-830d-e8c2ee6959cd","added_by":"auto","created_at":"2025-05-16 12:37:19","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1570130,"visible":true,"origin":"","legend":"\u003cp\u003eMicroscopic assessment. The bile duct and surrounding tissues. The distance to the circumferential margin is less than 1 mm. 200x magnification.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-6620965/v1/5c888e461663bc795fcf45a7.png"},{"id":82891490,"identity":"6d67142c-46c5-4055-befa-c2056ff83260","added_by":"auto","created_at":"2025-05-16 12:13:19","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":146768,"visible":true,"origin":"","legend":"\u003cp\u003eMacroscopic assessment of surgical specimen for PHC. Consecutive sections of the wall of the bile ducts and surrounding tissues.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-6620965/v1/75eab01358e097c3e45348f5.png"},{"id":82891497,"identity":"47896a91-f111-4ae5-8c2a-8d69726eb040","added_by":"auto","created_at":"2025-05-16 12:13:19","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1168802,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eFigure 8\u003c/em\u003e: Microscopic assessment. Resection margins along adjacent invasion loci in fibrous tissue and adipose tissue. 30х magnification.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-6620965/v1/016abf7ab446db15a9eb993e.png"},{"id":82891494,"identity":"73b51491-dbbe-4d04-90e7-0e5619b1ff55","added_by":"auto","created_at":"2025-05-16 12:13:19","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":142831,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eFigure\u003c/em\u003e 9: Microscopic assessment. Resection margins along invasion loci in nerve trunks. 50х magnification.\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-6620965/v1/5436c1bd42cfa87a4e96f18d.png"},{"id":82891493,"identity":"ee921ac9-85cf-44e2-b3f1-3d0ac4aab94a","added_by":"auto","created_at":"2025-05-16 12:13:19","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":292076,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eFigure\u003c/em\u003e \u003cem\u003e10\u003c/em\u003e: Microscopic assessment. Resection margins along invasion loci in vascular spaces. 200x magnification.\u003c/p\u003e","description":"","filename":"floatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-6620965/v1/4e9faa24825f54635369c8ff.png"},{"id":82892841,"identity":"c659d7b7-75b1-4c0e-bdd3-c7f925fafd2c","added_by":"auto","created_at":"2025-05-16 12:21:19","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":267037,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eFigure\u003c/em\u003e \u003cem\u003e11:\u003c/em\u003eMicroscopic assessment. Morphological signs of lymphovascular invasion. 200x magnification\u003c/p\u003e","description":"","filename":"floatimage11.png","url":"https://assets-eu.researchsquare.com/files/rs-6620965/v1/3d8e0401647807fea1e391fd.png"},{"id":82892838,"identity":"1286f21a-3a7f-42de-87c2-48b70cb052a8","added_by":"auto","created_at":"2025-05-16 12:21:19","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":387099,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eFigure\u003c/em\u003e \u003cem\u003e12\u003c/em\u003e: Microscopic assessment. Tumor invasion into liver tissue. 100x magnification\u003c/p\u003e","description":"","filename":"floatimage12.png","url":"https://assets-eu.researchsquare.com/files/rs-6620965/v1/df057f93ca27d855384fc60d.png"},{"id":82891505,"identity":"f4f30e1f-1b05-4d9d-8979-5b92fc6a97d0","added_by":"auto","created_at":"2025-05-16 12:13:19","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":97536,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eFigure 13\u003c/em\u003e: Overall survival for all patients with PHC.\u003c/p\u003e","description":"","filename":"13.png","url":"https://assets-eu.researchsquare.com/files/rs-6620965/v1/0949c595d94e0e6869d8ac90.png"},{"id":82891510,"identity":"ec989683-77ce-44ba-9eec-93d0a2745ce2","added_by":"auto","created_at":"2025-05-16 12:13:19","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":96374,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eFigure\u003c/em\u003e \u003cem\u003e14:\u003c/em\u003e Overall survival for operated patients with PHC according to R status (UICC).\u003c/p\u003e","description":"","filename":"14.png","url":"https://assets-eu.researchsquare.com/files/rs-6620965/v1/c08c1b03e74a6c00fe4a8ed6.png"},{"id":82891513,"identity":"d5b3bea2-ef62-4717-a018-f0e9a013a601","added_by":"auto","created_at":"2025-05-16 12:13:19","extension":"png","order_by":14,"title":"Figure 14","display":"","copyAsset":false,"role":"figure","size":96512,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eFigure\u003c/em\u003e 15: Overall survival for operated patients with PHC according to the number of tumor-affected places (RN\u003csub\u003e0-2 \u003c/sub\u003eand RN\u003csub\u003e3-7\u003c/sub\u003e); p \u0026lt; 0·00000 (log-rank test).\u003c/p\u003e","description":"","filename":"15.png","url":"https://assets-eu.researchsquare.com/files/rs-6620965/v1/396246754f3fa2f4bdee610f.png"},{"id":82891521,"identity":"17802adc-1caf-4fa6-889b-44968eb5cf43","added_by":"auto","created_at":"2025-05-16 12:13:20","extension":"png","order_by":15,"title":"Figure 15","display":"","copyAsset":false,"role":"figure","size":99698,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eFigure\u003c/em\u003e \u003cem\u003e16:\u003c/em\u003e Overall survival for operated patients with PHC according to the number of tumor-affected places (RN\u003csub\u003e0-3\u003c/sub\u003e and RN\u003csub\u003e4-7\u003c/sub\u003e); p \u0026lt; 0·00000 (log-rank test).\u003c/p\u003e","description":"","filename":"16.png","url":"https://assets-eu.researchsquare.com/files/rs-6620965/v1/74a4c065bd85a513abcddd02.png"},{"id":82891506,"identity":"974d3a62-b6ba-41dc-9319-56e18dbdc421","added_by":"auto","created_at":"2025-05-16 12:13:19","extension":"png","order_by":16,"title":"Figure 16","display":"","copyAsset":false,"role":"figure","size":97462,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eFigure 17\u003c/em\u003e: Overall survival for operated patients with PHC according to the number of tumor-affected places (RN\u003csub\u003e0-4\u003c/sub\u003e and RN\u003csub\u003e5-7\u003c/sub\u003e); p \u0026lt; 0·00030 (log-rank test).\u003c/p\u003e","description":"","filename":"17.png","url":"https://assets-eu.researchsquare.com/files/rs-6620965/v1/fd564c6d20dbc3054b129432.png"},{"id":82891500,"identity":"735a4c1e-7d89-4b06-91ef-6d603e3dc57a","added_by":"auto","created_at":"2025-05-16 12:13:19","extension":"png","order_by":17,"title":"Figure 17","display":"","copyAsset":false,"role":"figure","size":98379,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eFigure\u003c/em\u003e \u003cem\u003e18\u003c/em\u003e: Overall survival for operated patients with PHC according to the number of tumor-affected places (RN\u003csub\u003e0-5\u003c/sub\u003e and RN\u003csub\u003e6-7\u003c/sub\u003e); p \u0026lt; 0·00 (log-rank test).\u003c/p\u003e","description":"","filename":"18.png","url":"https://assets-eu.researchsquare.com/files/rs-6620965/v1/fad0dd2e7c3e1d45d8ad6f85.png"},{"id":94490333,"identity":"a7cc19e4-de97-4394-b167-30d600ba5f4b","added_by":"auto","created_at":"2025-10-27 17:09:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7324836,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6620965/v1/f52009b6-8e73-412c-8e1f-113ea1f113be.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Tumor invasion loci in resected margins of perihilar cholangiocarcinoma and their impact on survival: a retrospective 13-year longitudinal cohort study","fulltext":[{"header":"1. Background","content":"\u003cp\u003ePerihilar cholangiocarcinoma accounts for no more than 3% of all gastrointestinal tumors \u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e which greatly limits the ability to investigate this condition in depth, while ethical considerations make comparative randomized trials impractical at present. In addition, the anatomy and the type of the tumor expansion make radical resections less likely\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eLiterature sources indicate that the frequency of R0 resections varies greatly, from 15 to 95% \u003csup\u003e6\u0026ndash;10\u003c/sup\u003e and on average is 73\u0026middot;7%\u003csup\u003e11\u003c/sup\u003e. In addition, the 5-year survival rate varies between 20\u0026ndash;40%\u003csup\u003e11\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCurrently, there is no common understanding on what radicality is and how to define the resection margin\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. For example, a number of authors define an R0 resection as the absence of tumor cells at the margins of the hepatic duct and the radial margin \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eNote that different surgical approaches cannot significantly affect the prognosis, whereas patients with an R0 resection typically have better survival rates\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. According to the Union for International Cancer Control (UICC), there are three types of the resection margin: a negative resection margin (R0), microscopically present tumor cells in the resection margin (R1) and a macroscopically present residual tumor (R2)\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. The resection margin usually refers to the site of separation and intersection of anatomical structures. First of all, it is the margin of the bile duct, the dissection plane in the hepatoduodenal ligament, where structures are surgically released or dissected from the surrounding tissues\u003csup\u003e\u003cspan additionalcitationids=\"CR17 CR18\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. We would like to emphasize that the existing estimates of the ductal margin (DM) and even the radial margin (RM) of the resection cannot objectively reflect how radical the resection is\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWe should note that in general the concept of an R0 resection offers great value, primarily in terms of long-term prognosis and the possibility of drug therapy. Therefore, a number of authors note the importance of developing a common universal definition of \"pR0\" and standardization\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eResearch in context\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEvidence before this study\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe searched PubMed for peer-reviewed, original studies (with no start date and up to June 2023), using the search terms cholangiocarcinoma, perihilar cholangiocarcinoma, resection margin, radial margin, radicality, radical resections, prognosis. Typically, when studies refer to the resection margin, only the DM of the bile duct is mentioned, and the RM is often overlooked. To our knowledge, several studies has addressed the RM status in detail in resected PHC. The UICC currently grades surgical margins as R0 or R1 based on whether tumor cells are present or absent. In addition, there is no consensus as to what an R0 resection actually is and how wide the tumor-free margin should be. The existing concept of the resection margin does not objectively reflect the surgical radicality since it does not take into account histopathological risk factors, and there is no focus on prognosis. Therefore, the lack of complete information on the resection status compromises the validity of survival studies.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAdded value of this study\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAs far as we know, this is the first study presenting a new concept for evaluating resection margins and radicality of surgery in patients with perihilar cholangiocarcinoma in a homogeneous, clinically well-characterized cohort of patients. Furthermore, this is the first study to provide detailed morphological data on all tumor-affected microstructures within the resection margin. We provide a different understanding, and thus a different approach, for evaluating resected samples with a proven correlation of survival rates with risk factors (invasion loci) since we know that the mere presence of tumor cells in the resection margin does not correlate with survival rates in all patients, and vice versa, the absence of these cells does not guarantee that relapse will not occur. Our concept was to evaluate every potential tumor locus within the true resection margin and to count them all combined with the lymph node status (N). For example, we found that the long-term overall survival reliably depended on the number of tumor-affected loci and the presence of positive lymph nodes. In addition, we found a trend showing that the more we had such loci, the more progressively the survival rate declined during follow-up. Notably, the survival rate for all patients exceeded the 5-year mark with one or two affected loci out of seven possible, and the mere presence of tumor cells at the resection margin had no practical importance. Furthermore, while our study has grown over the past 13 years as the number of patients has increased, we have performed numerous recalculations, proving the above pattern.\u003c/p\u003e \u003cp\u003e \u003cb\u003eImplications of all the available evidence\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe results of our cohort study provide the best proof that the evaluation of the resection margin solely based on whether or not cells are present\u0026mdash;even in the so-called true margin\u0026mdash;does not objectively represent the surgical radicality and offers no potential advantage for the long-term prognosis. Our concept of \"the influence of the number of tumor invasion loci in resected margins on the survival rate\" should serve as the foundation for a best-practice consensus to develop clinical and morphological guidelines in patients with portal cholangiocarcinoma and can potentially be applied to other malignancies. Randomized clinical trials on the subject are likely to be neither feasible nor of practical benefit in the coming years since the disease is quite rare, the tumor heterogeneity is significant with respect to its mutational status, patients require long-term and cost-intensive treatment and no effective drug therapy options exist. Our data also provide new insights for creating prognostic models and finding new targets for targeted therapies.\u003c/p\u003e \u003cdiv id=\"Sec2\" class=\"Section2\"\u003e \u003ch2\u003e\u003cem\u003e1.1 Objective\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eThe first objective was to study and determine the significance of tumor invasion loci in the true resection margin in terms of long-term survival. The second objective was to formulate the concept of radical resections and develop a morphological classification based on the evaluation of all tumor invasion loci, taking into account the individual prognosis.\u003c/p\u003e \u003c/div\u003e"},{"header":"2. Patients and Methods","content":"\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Study Population\u003c/h2\u003e \u003cp\u003eFrom 2011 to 2023, patients with perihilar cholangiocarcinoma were treated in the Department of Abdominal Oncology of the Vishnevsky National Medical Research Center of Surgery. The patients were taken from a prospective database and studied retrospectively. The study included 83 liver and bile duct resections. The sample was limited to only those patients who had a complete morphological examination after liver and bile duct resection to properly identify all tumor invasion loci and reveal the true margin status, both the ductal margin and the radial margin.\u003c/p\u003e \u003cp\u003eThe exclusion criteria have been identified for this study: resection of the liver and/or bile ducts with a macroscopic residual margin, the lack of the possibility of histological examination of biopsy material (that is the histological blocks, for one reason or another, did not allow conducting a full study (there was a nonobservance of the making blocks technology, loss of certain parameters (proteins) in the analyzed tissue specimen which were specific for this disease, etc.), including immunohistochemical study), combined hepato-cholangiocellular carcinoma, underwent only palliative and symptomatic treatment, the patient refusal of proposed surgical treatment.\u003c/p\u003e \u003cp\u003e The study was approved by the Center's local ethics committee.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Preoperation Management and Surgery\u003c/h2\u003e \u003cp\u003eThe patient examination procedure usually included ultrasonography, CT, MRI (with MRCP and 3-D reconstruction) with contrast, tumor markers (CA 19\u0026middot;9, CEA, AFP). Decompression of the bile ducts, mainly by the antegrade route, was the first stage in patients with obstructive jaundice. The type of surgical intervention was planned depending on the location of the tumor in the liver according to Bismuth-Corlette, the presence of vascular invasion and the future liver remnant, and in some cases the possibility of performing a reliable hepaticojejunal anastomosis. Intraoperatively, when the bile duct confluence was involved (Bismuth-Corlette type IV), the choice of the type of hepatectomy primarily depended on the results of intraoperative biopsy and intraoperative ultrasonography. All patients underwent lymph node dissection according to the JSBS\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. The excised lymph nodes were represented by 12, 8 and 13 groups.\u003c/p\u003e \u003cp\u003ePortal vein resection was performed when it was involved in a tumor invasion according to CT data and confirmed macroscopic evaluation of the vessel invasion during surgery. If possible, dissection was carried out by a single block of nerve trunks within the nerve plexus, connective tissue, adipose tissue and lymphatic vessels of the hepatoduodenal ligament to evaluate the loci of invasion and the RM status.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Pathological Evolution and Definition of the surgical margins\u003c/h2\u003e \u003cp\u003eHistological examination was performed according to the College of American Pathologists (CAP) guidelines and the current WHO classification of tumors of the digestive system (5th edition, 2019)\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. The tumor was staged according to the current TNM classification for perihilar cholangiocarcinoma (extrahepatic bile duct cancer, AJCC 8th edition)\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. All possible tumor invasion loci were evaluated histologically as microvessels (V), surrounding adipose tissue (SAT), lymph nodes (N) and vessels (L), proximal and distal bile duct margins (BD), perineural invasion (Pn), and hepatic parenchyma (Hep). If necessary, CD31 immunohistochemical testing (IHC) was performed to confirm lymphovascular invasion. The resulting tissue samples were fixed in 4% buffered formalin for 24\u0026ndash;48 hours. The specimens were prepared in a standard manner for microscopy with hematoxylin and eosin staining. For IHC they were prepared on positively charged slides. The latter was performed on serial paraffin sections. All surgical margins were stained to match the color marker for subsequent identification under a microscope (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). A positive ductal margin was defined by the presence of tumor cells in the proximal (hepatic side) and/or distal (duodenal side) parts of the bile duct. A positive resection margin was defined by the presence of tumor cells at a distance less than 1 mm from the dissection plane of the hepatic hilum, hepatic parenchyma and hepatoduodenal ligament structures (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAll histology specimens were evaluated by two or three experienced histologists.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Follow-Up\u003c/h2\u003e \u003cp\u003eAll patients were monitored for relapse after discharge from the hospital. CT or MRI scans were conducted every 3\u0026ndash;4 months for a year and then every 6 months for the next two years. Thereafter, once a year or as needed. PET-CT was performed in doubtful cases.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Ethical considerations\u003c/h2\u003e \u003cp\u003eThe study complied with the norms of the Declaration of Helsinki and was fully approved by the Local Ethics Committee of the A.V. Vishnevsky National Medical Research Center of Surgery under protocol No. 08\u0026ndash;19 on May 6, 2019. All participants gave informed consent prior to the study.\u003cem\u003e\u003c/p\u003e\u003cp\u003e2.6 Statistical Analysis\u003c/em\u003e\u003c/p\u003e \u003cp\u003eNonparametric statistical methods were used to process the results. The median (range) was defined as the average of two median values of a number of its ranked elements. The survival rate was analyzed using the Kaplan-Meyer method. To investigate the simultaneous influence of dependent variables on survival, both mono- and multivariate Cox regression analyses were used. The log-rank test was used to compare two survival curves. All tests were bilateral, and the results were considered significant at p\u0026thinsp;\u0026lt;\u0026thinsp;0\u0026middot;05, with at least 95% accuracy. All statistical calculations were performed using STATISTICA\u0026reg; version 13 (StatSoft, USA).\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Patient and Operative Procedures\u003c/h2\u003e \u003cp\u003eThe study included 83 patients with perihilar cholangiocarcinoma who underwent liver resection and/or extrahepatic bile duct resection. There were 12 (14\u0026middot;4%) patients who underwent extrahepatic bile duct resection only, 58 (69\u0026middot;9%) patients with hepatectomy combined with extrahepatic bile duct resection (among them 19 (32\u0026middot;8%) with right-sided hemihepatectomy and 39 (67\u0026middot;2%) with left-sided hemihepatectomy), 12 (14\u0026middot;4%) patients with extended hemihepatectomy and 1 (1\u0026middot;2%) patient with extrahepatic bile duct resection combined with pancreatoduodenectomy. The Bismuth-Corlette distribution of patients was as follows: I, II \u0026ndash; 10 (12%), IIIA/B \u0026ndash; 21 (25\u0026middot;3%), IV \u0026ndash; 52 (62\u0026middot;6%). Liver resection with segment I \u0026ndash; 32 (38\u0026middot;5%). Resection of the portal vein was performed in 12 (14\u0026middot;4%) patients; there was one case (1\u0026middot;2%) of combination of portal vein and artery resection.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Histopathological Findins\u003c/h2\u003e \u003cp\u003eThe distribution of patients by disease stage (pTNM) was as follows: I (T1N0M0) \u0026ndash; 7 (8\u0026middot;4%), II (T2a/bN0M0) \u0026ndash; 33 (39\u0026middot;7%), III А/В (T3/4N0M0) \u0026ndash; 16 (19\u0026middot;3%), III С (T2-4N1M0) \u0026ndash; 22 (26\u0026middot;5%), IVА (T3/4N2M0) \u0026ndash; 5 (6%). Lymph node involvement was observed in 26 (32\u0026middot;9%) cases. According to the UICC classification: R0\u0026ndash;30 (36\u0026middot;1%), R1\u0026ndash;50 (60\u0026middot;2%), R2\u0026ndash;3 (3\u0026middot;6%).\u003c/p\u003e \u003cp\u003eThe analysis of the morphological characteristics of the tumor included all possible parameters reflecting the tumor expansion: the degree of differentiation of tumor cells, microvascular invasion, lymphovascular invasion, liver invasion, duct resection margin, perineural invasion, tumor cells in adipose tissue, tumor growth shape and the depth of tumor invasion (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMorphological features perihilar cholangiocarcinoma patients\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" morerows=\"1\" nameend=\"c2\" namest=\"c1\" rowspan=\"2\"\u003e \u003cp\u003eCharacteristics and sites of tumor invasion\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eNumber\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eabs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eDifferentiation degree (n\u0026thinsp;=\u0026thinsp;83)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHigh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e26\u0026middot;5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eModerate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e47\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLow\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e26\u0026middot;5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eMicrovascular invasion (n\u0026thinsp;=\u0026thinsp;72)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e41\u0026middot;7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e58\u0026middot;3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eLymphovascular invasion (n\u0026thinsp;=\u0026thinsp;70)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e90\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eTumor cells in the bile duct resection margin\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;83)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e67\u0026middot;4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e32\u0026middot;5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eLiver invasion\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;71)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e57\u0026middot;7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e42\u0026middot;3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003ePerineural invasion (n\u0026thinsp;=\u0026thinsp;74)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e89\u0026middot;2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10\u0026middot;8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eTumor cells in surrounding adipose tissue\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;74)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e90\u0026middot;5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9\u0026middot;5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e: Morphological features perihilar cholangiocarcinoma patients.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" morerows=\"1\" nameend=\"c2\" namest=\"c1\" rowspan=\"2\"\u003e \u003cp\u003eCharacteristics and sites of tumor invasion\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eNumber\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eabs\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e%\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDepth of invasion of the bile duct wall\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;71)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTotal invasion\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e88\u0026middot;7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eTumor growth shape\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;71)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eexophyte\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9\u0026middot;9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eendophyte\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e90\u0026middot;1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eWe should note that, due to tumor expansion through hepatoduodenal ligament structures, we observe high incidence of invasion into the lymphatic ducts, surrounding adipose tissue and nerve trunks, significantly complicating the goal of radical surgery.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Tumor Invasion Places and Surgical Margins\u003c/h2\u003e \u003cp\u003eTumor invasion loci at the true resection margin, i.e., DM and RM, in combination with lymph node evaluation, were used to evaluate resection radicality. A total of 7 sites of possible tumor invasion were evaluated. These are the resection margins of the bile duct, adipose tissue, hepatic parenchyma, venous/arterial and lymphatic vessels, nerve trunks and lymph nodes. Resection without all 6 positive loci was considered radical and was designated as R\u003csub\u003e0\u003c/sub\u003e. According to our proposed prognosis-targeted classification (detailed in Section 3.4), depending on the number of positive loci (R\u003csub\u003e+\u003c/sub\u003e), we denoted the resection status, respectively, as R\u003csub\u003e+\u0026thinsp;1\u003c/sub\u003e, R\u003csub\u003e+\u0026thinsp;2\u003c/sub\u003e, R\u003csub\u003e+\u0026thinsp;3\u003c/sub\u003e, R\u003csub\u003e+\u0026thinsp;4\u003c/sub\u003e, R\u003csub\u003e+\u0026thinsp;5\u003c/sub\u003e, R\u003csub\u003e+\u0026thinsp;6\u003c/sub\u003e in combination with status N (RN). In total, there were 7 (8\u0026middot;43%) patients with RN\u003csub\u003e0\u003c/sub\u003e status, 3 (3\u0026middot;61%) with RN\u003csub\u003e+\u0026thinsp;1\u003c/sub\u003e, 7 (8\u0026middot;43%) with RN\u003csub\u003e+\u0026thinsp;2\u003c/sub\u003e, 3 (3\u0026middot;61%) with RN\u003csub\u003e+\u0026thinsp;3\u003c/sub\u003e, 6 (7\u0026middot;23%) with RN\u003csub\u003e+\u0026thinsp;4\u003c/sub\u003e, 15 (18\u0026middot;07%) with RN\u003csub\u003e+\u0026thinsp;5\u003c/sub\u003e, 21 (25\u0026middot;30%) with RN\u003csub\u003e+\u0026thinsp;6\u003c/sub\u003e, 21 (25\u0026middot;30%) with RN\u003csub\u003e+\u0026thinsp;7\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003eHistological evaluation of all 7 loci revealed tumor cells in 76 (91\u0026middot;56%) of 83 resected samples (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). There were 37 (44\u0026middot;5%) proximal positive margins and 19 (22\u0026middot;9%) distal positive margins. The RM was evaluated taking into account the different number of identified tumor invasion loci. We should emphasize that in certain situations, even with the most thorough evaluation of the RM resection status, it proved challenging for us to determine the \"true\" dissecting plane in a resected specimen.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEvaluation of invasion loci in resection margins.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eInvasive areas\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eАbs\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e%\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMicrovascular invasion (n\u0026thinsp;=\u0026thinsp;72)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e41\u0026middot;7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLymphovascular invasion (n\u0026thinsp;=\u0026thinsp;70)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e90\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTumor cells in bile duct resection margins (n\u0026thinsp;=\u0026thinsp;83)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e49\u0026middot;4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLiver invasion (n\u0026thinsp;=\u0026thinsp;71)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e57\u0026middot;7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePerineural invasion (n\u0026thinsp;=\u0026thinsp;74)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e89\u0026middot;.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSurrounding adipose tissue (n\u0026thinsp;=\u0026thinsp;74)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e90\u0026middot;5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRegional lymph nodes (n\u0026thinsp;=\u0026thinsp;73)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e35\u0026middot;6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eWe analyzed the RM in 80 histological specimens for the presence or absence of tumor cells; however, as the table illustrates, we were unable to evaluate some specimens for all six loci (structures) of invasion (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e12\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Prognostic Factors, Classification and Survival\u003c/h2\u003e \u003cp\u003eIn a multivariate analysis (11 pathomorphological factors included in the analysis), the disease stage according to pTNM (Disease Stage 8 th ed) (p\u0026thinsp;=\u0026thinsp;0\u0026middot;006), the presence of cells in lymph nodes (p\u0026thinsp;=\u0026thinsp;0\u0026middot;01), the degree of differentiation (p\u0026thinsp;=\u0026thinsp;0\u0026middot;02) and perineural invasion (p\u0026thinsp;=\u0026thinsp;0\u0026middot;03) were statistically significant prognostic factors (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Depending on the liver resection with or without the first segment, comparative analysis showed no difference (p\u0026thinsp;=\u0026thinsp;0\u0026middot;14). 12-, 36-, 60-, 84-, 120-month survival rates were 70%, 30%, 18%, 16% and 16%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e13\u003c/span\u003e). The median follow-up was 27 months. In 9 patients, the survival rate exceeded 60 months. The maximum follow-up period was 156 months (one patient).\u003c/p\u003e\u003cp\u003e\u003cem\u003eTable 3\u003c/em\u003e: The dependence of survival on the factors in the multivariable analyses.\u0026nbsp;\u003c/p\u003e\n\u003ctable id=\"Tabb\" border=\"1\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePathomorphological factors\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMultivariable analyses*, р\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eRisk Ratio (95% Confidence Interval)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTumor cells in the surrounding adipose tissue\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u0026middot;50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLymphovascular invasion\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u0026middot;51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMicrovascular invasion\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u0026middot;08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePerineural invasion\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u0026middot;03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14\u0026middot;48 (1\u0026middot;16\u0026ndash;180\u0026middot;75)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDepth of bile duct wall invasion\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u0026middot;71\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRegional lymph nodes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u0026middot;01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u0026middot;16 (1\u0026middot;18\u0026thinsp;\u0026minus;\u0026thinsp;3\u0026middot;96)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTumor differentiation stage\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u0026middot;02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u0026middot;63 (1\u0026middot;05\u0026thinsp;\u0026minus;\u0026thinsp;2\u0026middot;51)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTumor cells in bile duct resection margins\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u0026middot;82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLiver invasion\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u0026middot;08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eForm of tumor growth\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u0026middot;18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003epTNM (Disease Stage 8 th ed)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u0026middot;006\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u0026middot;05 (1\u0026middot;01\u0026ndash;1\u0026middot;09)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003c/p\u003e\n\u003cp\u003e* Statistical validity (р) of the analysis performed reached 0\u0026middot;00002.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab3\" border=\"1\"\u003e\u003c/table\u003e\n\u003c/div\u003e\u003cp\u003eA comparative study by the number of loci (RN\u003csub\u003e0-7\u003c/sub\u003e) revealed a pattern: the more affected loci a patient has, the worse the survival rate is for this patient. Due to the small number of patients in groups RN\u003csub\u003e0\u003c/sub\u003e, RN\u003csub\u003e1\u003c/sub\u003e and RN\u003csub\u003e2\u003c/sub\u003e and no difference in survival between them, all patients were combined to one group RN\u003csub\u003e0-2\u003c/sub\u003e. Statistical calculations for this group were carried out the statistics of a small sample. Thus, when comparing the groups RN\u003csub\u003e0-2\u003c/sub\u003e and RN\u003csub\u003e3-7\u003c/sub\u003e, we observed a statistically significant difference (p=0\u0026middot;00000) (Fig. 14), with survival exceeding 60 months in all patients in the RN\u003csub\u003e0-2\u0026nbsp;\u003c/sub\u003egroup. When comparing the groups RN\u003csub\u003e0-3\u003c/sub\u003e and RN\u003csub\u003e4-7\u003c/sub\u003e, RN\u003csub\u003e0-4\u003c/sub\u003e and RN\u003csub\u003e5-7\u003c/sub\u003e, RN\u003csub\u003e0-5\u003c/sub\u003e and RN\u003csub\u003e6-7\u003c/sub\u003e, we also obtained a statistically significant difference (p=0\u0026middot;00000, p=0\u0026middot;00030, p=0\u0026middot;03, respectively) (Fig. 15-18).\u003c/p\u003e\n\u003cp\u003eSince we found a significant difference in survival depending on the number of tumor-affected places, we proposed a new prognosis-targeted classification (Table 4).\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"489\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 243px;\"\u003e\n \u003cp\u003eRadicality degree (R)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 246px;\"\u003e\n \u003cp\u003eNumber of tumor-affected places\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 243px;\"\u003e\n \u003cp\u003eR\u003csub\u003e0\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 246px;\"\u003e\n \u003cp\u003eR\u003csub\u003e0\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"6\" style=\"width: 243px;\"\u003e\n \u003cp\u003eR\u003csub\u003e+\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 246px;\"\u003e\n \u003cp\u003eR\u003csub\u003e+1\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 246px;\"\u003e\n \u003cp\u003eR\u003csub\u003e+2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 246px;\"\u003e\n \u003cp\u003eR\u003csub\u003e+3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 246px;\"\u003e\n \u003cp\u003eR\u003csub\u003e+4\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 246px;\"\u003e\n \u003cp\u003eR\u003csub\u003e+5\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 246px;\"\u003e\n \u003cp\u003eR\u003csub\u003e+6\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cem\u003eTable 4\u003c/em\u003e: Classification of operations depending on radicality.\u003c/p\u003e\n\u003cp\u003eThus, taking into account the created prognosis-targeted classification, the radicality of resection in combination with the N status was designated as RN from 0 to 7 with a mandatory indication of the invasion locus (BD\u003csub\u003e0/1\u003c/sub\u003e, L\u003csub\u003e0/1\u003c/sub\u003e, V\u003csub\u003e0/1\u003c/sub\u003e, Pn\u003csub\u003e0/1\u003c/sub\u003e, N\u003csub\u003e0/1\u003c/sub\u003e, Hep\u003csub\u003e0/1\u003c/sub\u003e, SAT\u003csub\u003e0/1\u003c/sub\u003e).\u003c/p\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eCurrently, the only effective treatment for perihilar cholangiocarcinoma is surgery. Over the last two decades, its constant goal has been and still is to achieve the R0 status. Radical surgical intervention has become more aggressive, combining extensive hemihepatectomy with resection of extrahepatic bile ducts, lymphadenectomy, vascular resection and, in some cases, pancreatoduodenal resection\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Nonetheless, even this expanded surgical scope did not yield considerable success in reaching the radicality of surgery\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Studies have shown that the R0 resection has become a difficult and nearly impossible task \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOne of the primary reasons as to why this problem remains unsolved is the lack of a unified concept of the radicality of resection, as well as no understanding concerning what negative margins of resection actually are. Furthermore, the criteria specialists use to determine R0 are vague, and there is some confusion when it comes to evaluating tumor expansion as criteria for radicality \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan additionalcitationids=\"CR27 CR28\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eR0 resection is commonly thought to signify the absence of tumor cells in the margins of the common bile duct and hepatic duct, and, unfortunately, the radial margin is very rarely mentioned\u003csup\u003e\u003cspan additionalcitationids=\"CR17 CR18\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Thus, recent studies have shown that morphological reports provide poor evaluation of surgical margins, especially in Western medical centers, where their coverage rate ranges from 10\u0026ndash;45%\u003csup\u003e27\u003c/sup\u003e. According to Kentaro Shinohara et al., literature sources describe only one study that discusses in detail the RM status in patients after resection of distal cholangiocarcinoma; however, the authors found no single report on perihilar cholangiocarcinoma\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTo the best of our knowledge, this is the first study to explore the radicality of resection from a position of invasion places (bile ducts, arterial and venous vessels, the liver parenchyma, lymphatic vessels, nerves and surrounding adipose tissue) and their number in the resection margins, and in the absence of the possibility of assessing these places in the edge - additional assessment them in the tumor tissue. In addition, our study details the assessment of DM and RM, i.e. the so-called true resection margin.\u003c/p\u003e \u003cp\u003eIt should be noted that our hypothesis that radical R0 status should be considered only from the position of assessing all possible invasion places in the resection margin was confirmed by statistical analysis of the prospective database in 2019. Also, in connection with the addition of patients, all data were recalculated, and their repeated statistical processing was carried out in 2024.\u003c/p\u003e \u003cp\u003eCurrently, there are a lot of studies devoted to the significance of pathomorphological factors as markers of poor prognosis, such as low differentiation degree, infiltrative form of tumor growth, micro- and lymphovascular invasion, positive resection margin, perineural invasion, affected lymph nodes\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eSignificant pathomorphological factors in the multivariate analysis we completed for our study were the presence of tumor cells in regional lymph nodes and perineural invasion (p\u0026thinsp;=\u0026thinsp;0\u0026middot;00002). A number of factors such as micro- and lymphovascular invasion, positive resection margin, liver invasion and tumor cells in the surrounding adipose tissue have not shown a reliable relationship with prognosis, but their significance has been demonstrated by a number of other studies\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. The obtained result can be explained by the fact that very often it is impossible to determine which factor is independent and which is a confounder, i.e. in such cases, we take into account a \u0026ldquo;principle of confounders.\u0026rdquo;\u003c/p\u003e \u003cp\u003eIn our opinion, the radicality of resection should be considered not through a surgical prism, but from the point of view of the oncological position, i.e. position of long-term prognosis and search for possible points for solving the existing problem. It is very important not just to discuss the resection margins, but to discuss in which structures the tumor cells are located, as well as how many structures (places) are affected by the tumor.\u003c/p\u003e \u003cp\u003eWe should note that the pTNM disease stage (p\u0026thinsp;=\u0026thinsp;0.0061 in the multivariate analysis) in our study was a significant factor reflecting how survival depended on a set of pathomorphological factors in the long-term period. These findings support our concept of \"dependence of survival on the number of positive invasion loci\" concerning long-term prognosis following resection. The TNM classification considers, in addition to clinical parameters, a combination of pathomorphological invasion loci, such as the bile duct, surrounding adipose tissue, vessels, liver parenchyma and lymph node tissue; however, it poorly reflects the resection status and is thus based primarily on the evaluation of tumor prevalence rather than prognosis\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAccording to our concept, we considered the number of tumor invasion sites in the true resection margin in combination with the N status. The evaluation showed that tumor cells were found in 91% of the resected samples. At the same time, according to the UICC classification, tumor cells were discovered in only 64% of the resected samples, implying that one-third of the patients would not have the radicality of surgery clearly defined, which previously prevented us from making an accurate prognosis.\u003c/p\u003e \u003cp\u003eIt is important to point out that a comparative analysis of the \"number of positive loci\" found several trends. We would like to focus on them. First, our statistical analysis revealed that survival worsened depending on the number of positive invasion loci, exceeding the 5-year limit in all patients with the RN \u003csub\u003e0\u003c/sub\u003e and RN\u003csub\u003e+\u0026thinsp;1\u0026minus;2\u003c/sub\u003e status. Second, we observed that as the number of positive loci increased, survival declined progressively. As a result, we believe that the radicality of resection should be determined not only by the presence/absence of tumor cells in the resection margin, but primarily by the number of loci (anatomical structures) affected by the tumor in the resected margin, and that the prognosis should be based on a combination of the R and N statuses.\u003c/p\u003e \u003cp\u003eOur classification of resection radicality compared to the UICC classification (R\u003csub\u003e0\u003c/sub\u003e, R\u003csub\u003e1\u003c/sub\u003e and R\u003csub\u003e2\u003c/sub\u003e) has the designations R\u003csub\u003e0\u003c/sub\u003e, R\u003csub\u003e+\u0026thinsp;1\u003c/sub\u003e, R\u003csub\u003e+\u0026thinsp;2\u003c/sub\u003e, R\u003csub\u003e+\u0026thinsp;3\u003c/sub\u003e, R\u003csub\u003e+\u0026thinsp;4\u003c/sub\u003e, R\u003csub\u003e+\u0026thinsp;5\u003c/sub\u003e, R\u003csub\u003e+\u0026thinsp;6\u003c/sub\u003e in combination with the lymph node evaluation (RN\u003csub\u003e0\u0026thinsp;\u0026minus;\u0026thinsp;7\u003c/sub\u003e) and the invasion locus in the true resection margin always indicated (BD\u003csub\u003e0/1\u003c/sub\u003e, L\u003csub\u003e0/1\u003c/sub\u003e, V\u003csub\u003e0/1\u003c/sub\u003e, Pn\u003csub\u003e0/1\u003c/sub\u003e, Hep\u003csub\u003e0/1\u003c/sub\u003e, SAT\u003csub\u003e0/1\u003c/sub\u003e). It shows the number and location of the tumor invasion and thus indicates a long-term prognosis following surgery.\u003c/p\u003e \u003cp\u003eThe present study has some limitations, mainly related to the rarity of this disease. The main limitation of this study is the retrospective nature of the analysis; additionally, it was conducted in a single center. Therefore, unexpected biases cannot be completely excluded.\u003c/p\u003e \u003cp\u003eIn conclusion, we provide strong evidence for the value of the number of all possible invasion loci (R\u003csub\u003e+\u003c/sub\u003e) in the true resection margin combined with the N status, enabling us to complete a comprehensive evaluation of resected tumor samples and make a long-term prognosis with good accuracy (RN \u003csub\u003e0\u0026minus;7\u003c/sub\u003e). The presented morphologic classification has the potential to profoundly alter the tactics and treatment of patients because it has a high prognostic value. Finally, to the best of our knowledge, we provide a novel approach to evaluating resection margins, as well as the possibility of having the first practical basis for creating a long-term prognosis with high accuracy when selecting patients for drug therapy.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthors contributions:\u0026nbsp;\u003c/strong\u003eAll authors \u0026ndash; Conceptualization, Data curation, Formal Analysis, Investigation, Methodology, Validation, Visualization, Writing (original draft, review, and editing).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eEthics approval:\u0026nbsp;\u003c/strong\u003eThe study complied with the norms of the Declaration of Helsinki and was fully approved by the Local Ethics Committee of the A.V. Vishnevsky National Medical Research Center of Surgery under protocol No. 08-19 on May 6, 2019.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate:\u003c/strong\u003e All participants gave informed consent prior to the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u003c/strong\u003e All authors have given their consent for publication.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials:\u003c/strong\u003e A patient database available upon official request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u0026nbsp;\u003c/strong\u003eN/A.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eN/A.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLandis SH, Murray T, Bolden S, Wingo PA. 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A statement by the Japan-Korea expert pathologists for future clinicopathological and molecular analyses toward consensus building of intraductal papillary neoplasm of the bile duct through several opinions at the present stage. \u003cem\u003eJ Hepatobiliary Pancreat Sci\u003c/em\u003e 2018; 25(3):181-187. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"world-journal-of-surgical-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"wjso","sideBox":"Learn more about [World Journal of Surgical Oncology](http://wjso.biomedcentral.com)","snPcode":"12957","submissionUrl":"https://submission.nature.com/new-submission/12957/3","title":"World Journal of Surgical Oncology","twitterHandle":"@OncoBioMed","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"perihilar cholangiocarcinoma, resection margin, tumor invasion loci, radicality, survival analysis, prognostic classification","lastPublishedDoi":"10.21203/rs.3.rs-6620965/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6620965/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003ePerihilar cholangiocarcinoma (PHC) presents significant challenges in achieving curative resection, with current definitions of radicality (R0/R1/R2) based solely on tumor presence at margins lacking prognostic precision. This study investigates the impact of tumor invasion loci within resection margins on survival and proposes a novel prognostic classification.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eA 13-year retrospective cohort study of 83 PHC patients undergoing liver/bile duct resection was conducted. Histopathological evaluation assessed seven invasion loci: bile duct margins, adipose tissue, hepatic parenchyma, vascular/lymphatic structures, perineural spaces, lymph nodes, and radial margins. Resection radicality was redefined as RN0-7, combining the number of tumor-positive loci (R\u0026thinsp;+\u0026thinsp;1 to R\u0026thinsp;+\u0026thinsp;6) with lymph node status (N). Survival analysis utilized Kaplan-Meier and Cox regression methods.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eTumor invasion loci were identified in 91.6% of specimens. Multivariate analysis revealed perineural invasion (HR\u0026thinsp;=\u0026thinsp;14.48, p\u0026thinsp;=\u0026thinsp;0.03), lymph node involvement (HR\u0026thinsp;=\u0026thinsp;2.16, p\u0026thinsp;=\u0026thinsp;0.01), and higher TNM stage (p\u0026thinsp;=\u0026thinsp;0.006) as key prognostic factors. Survival rates declined progressively with increasing invasion loci: 5-year survival exceeded 60 months for patients with \u0026le;\u0026thinsp;2 loci (RN0-2), but dropped significantly with \u0026ge;\u0026thinsp;3 loci (p\u0026thinsp;\u0026lt;\u0026thinsp;0.00001). Traditional UICC R0/R1 classification poorly correlated with outcomes, as 36.1% classified as R0 exhibited poor survival if multiple loci were involved.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThe number of tumor invasion loci in resection margins, combined with lymph node status, provides superior prognostic stratification compared to conventional R0/R1 criteria. The proposed RN0-7 classification highlights the importance of quantifying anatomical sites of residual disease, offering a foundation for standardized histopathological evaluation and personalized adjuvant strategies. This approach may enhance prognostic accuracy and guide therapeutic decisions in PHC, where randomized trials remain impractical.\u003c/p\u003e","manuscriptTitle":"Tumor invasion loci in resected margins of perihilar cholangiocarcinoma and their impact on survival: a retrospective 13-year longitudinal cohort study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-16 12:13:14","doi":"10.21203/rs.3.rs-6620965/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-06-01T14:35:26+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-31T19:30:08+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-21T01:30:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"226454067487223952857593792827433214018","date":"2025-05-20T18:53:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"136520905591355237014599164464059236469","date":"2025-05-20T17:47:46+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-20T14:36:44+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-15T16:38:30+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-12T23:38:48+00:00","index":"","fulltext":""},{"type":"submitted","content":"World Journal of Surgical Oncology","date":"2025-05-08T12:56:54+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"world-journal-of-surgical-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"wjso","sideBox":"Learn more about [World Journal of Surgical Oncology](http://wjso.biomedcentral.com)","snPcode":"12957","submissionUrl":"https://submission.nature.com/new-submission/12957/3","title":"World Journal of Surgical Oncology","twitterHandle":"@OncoBioMed","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"11220e18-9da9-4012-9ee8-6ca2d59bd2ae","owner":[],"postedDate":"May 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-10-27T16:28:51+00:00","versionOfRecord":{"articleIdentity":"rs-6620965","link":"https://doi.org/10.1186/s12957-025-04006-7","journal":{"identity":"world-journal-of-surgical-oncology","isVorOnly":false,"title":"World Journal of Surgical Oncology"},"publishedOn":"2025-10-21 16:17:26","publishedOnDateReadable":"October 21st, 2025"},"versionCreatedAt":"2025-05-16 12:13:14","video":"","vorDoi":"10.1186/s12957-025-04006-7","vorDoiUrl":"https://doi.org/10.1186/s12957-025-04006-7","workflowStages":[]},"version":"v1","identity":"rs-6620965","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6620965","identity":"rs-6620965","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}