Impact of Neutrophil Extracellular Traps Identified by Citrullinated Histone H3 Immunohistochemistry for Postoperative Prognosis in Patients with Extrahepatic Cholangiocarcinomas.

BACKGROUND: Neutrophil extracellular traps (NETs) are extracellular chromatin structures composed of cytoplasmic, granular, and nuclear components of neutrophils. Recently, NETs have received much attention for their role in tumor biology; however, their impact on the postoperative prognosis of patients with extrahepatic cholangiocarcinomas (EHCCs) remains unclear. The purpose of this study was to clarify the impact of NETs identified by immunohistochemical citrullinated histone H3 (Cit-H3) staining on postoperative overall survival (OS) in patients with perihilar cholangiocarcinoma (PHCC) and distal cholangiocarcinoma (DCC). METHODS: This study included 318 patients with EHCC (PHCC, n = 192; DCC, n = 126) who underwent surgical resection with curative intent. Neutrophils and NETs were identified by immunohistochemistry using antibodies against CD15 and Cit-H3, respectively. Based on the distribution of CD15 and Cit-H3 expression in the tumor bed, the patients were classified into four groups: one negative group and three subgroups of the positive group (diffuse, intermediate, and focal subgroups). RESULTS: No significant difference was found in the postoperative OS rate depending on the distribution of CD15 expression in patients with PHCC or DCC. However, the three subgroups with positive Cit-H3 expression had significantly poorer OS than the negative group for both PHCC and DCC. Moreover, positive Cit-H3 was an independent OS factor in the multivariable analyses of PHCC (hazard ratio [HR] 1.69, 95% confidence interval [CI] 1.11-2.59, P = 0.0115) and DCC (HR 2.03; 95% CI 1.21-3.42, P = 0.0057). CONCLUSIONS: The presence of NETs in the tumor microenvironment may have adverse prognostic effects in patients with EHCCs.

The Short- and Long-Term Surgical Results of Consecutive Hepatopancreaticoduodenectomy for Wide-Spread Biliary Malignancy.

BACKGROUND: Cancer-free resection (R0) is one of the most important factors for the long-term survival of biliary carcinoma. For some patients with widespread invasive cancer located between the hilar and intrapancreatic bile duct, hepatopancreaticoduodenectomy (HPD) is considered a radical surgery for R0 resection. However, HPD is associated with high morbidity and mortality rates. Furthermore, previous reports have not shown lymph node metastasis (LNM) status, such as the location or number, which could influence the prognosis after HPD. In this study, first, we explored the prognostic factors for survival, and second, we evaluated whether the LNM status (number and location of LNM) would influence the decision on surgical indications in patients with widely spread biliary malignancy. METHODS: We retrospectively reviewed the medical records of 54 patients who underwent HPD with hepatectomy in ≥2 liver sectors from January 2003 to December 2021 (HPD-G). We also evaluated 54 unresectable perihilar cholangiocarcinoma patients who underwent chemotherapy from January 2010 to December 2021 (CTx-G). RESULTS: R0 resection was performed in 48 patients (89%). The median survival time (MST) and 5-year overall survival rate of the HPD-G and CTx-G groups were 36.9 months and 31.1%, and 19.6 months and 0%, respectively. Univariate and multivariate analyses showed that pathological portal vein involvement was an independent prognostic factor for survival (MST: 18.9 months). Additionally, patients with peripancreatic LNM had worse prognoses (MST: 13.3 months) than CTx-G. CONCLUSIONS: Patients with peripancreatic LNM or PV invasion might be advised to be excluded from surgery-first indications for HPD.

Validation study for prognostic scoring system for perihilar cholangiocarcinoma surgery using preoperative factors.

PURPOSE: Recently, systemic inflammatory responses (SIR) have been shown to play a pivotal role in the development and progression of cancer. We previously reported that four factors, serum carcinoembryonic antigen (> 7 ng/dL), serum albumin (< 3.5 g/dL), C-reactive protein (> 0.5 mg/dL), and platelet-lymphocyte ratio (PLR; > 150), were independent prognostic factors after perihilar cholangiocarcinoma (PHCC) surgery. We also advocated a prognosis predictive preoperative prognostic score (PPS) using these four factors and showed that PPS could predict patients' prognosis on survival. This retrospective study sought to validate preoperatively available prognostic factors for survival after major hepatectomy as reported previously, including PPS for PHCC. METHODS: We retrospectively validated our PPS score and reported SIR scoring systems using the data of 125 consecutive patients who underwent PHCC surgery from January 2010 to November 2020. RESULTS: PPS was an independent preoperative prognostic factors for survival. The T and N categories were independent prognostic factors. Other SIR scores were not independent preoperative factors in the univariate analysis. Among SIR scores, only the PPS was found to be associated with OS and disease-free survival. The PPS was also associated with histopathological factors (T and N categories). CONCLUSION: PPS could be useful in predicting long-term survival after PHCC and may be a more useful scoring system than other SIR systems.

Postoperative Prognosis According to Pathologic Categorization of Desmoplastic Reaction in Patients with Extrahepatic Cholangiocarcinoma.

BACKGROUND: Recent studies have demonstrated the importance of desmoplastic reaction (DR) in predicting postoperative prognosis for patients with colorectal carcinoma. However, the impact of DR on the prognosis of extrahepatic cholangiocarcinomas (EHCCs) is not established. This study aimed to clarify the associations of pathologic DR categories with clinicopathologic factors and postoperative prognosis of perihilar cholangiocarcinoma (PHCC) and distal cholangiocarcinoma (DCC). METHODS: A pathologic review of 174 patients with PHCC and 109 patients with DCC who underwent surgical resection was performed. The patients were classified into three DR categories (immature, intermediate, and mature) based on the histologic features within the fibrotic stroma in the invasive front. The association between DR categories and the distribution of fibroblasts with anti-α-smooth muscle actin (SMA) expression, seeming to be tumor-promoting cancer-associated fibroblasts (CAFs), was evaluated in 191 tissue microarray specimens of EHCCs. RESULTS: Intermediate/immature DR categories were significantly associated with a more invasive nature, including higher pT and pN stages and more tumor buds than the mature category in both PHCC and DCC. The DR categories could stratify overall survival (OS) and relapse-free survival (RFS) in both PHCC and DCC patients. In the multivariate analysis, the DR category was an independent prognostic factor for OS and RFS in both PHCC and DCC (p < 0.001). The mature and immature DR categories were significantly associated respectively with the confined and pervasive distribution of fibroblasts with α-SMA expression. CONCLUSION: In patients with EHCCs, DR categorization was an independent prognostic factor reflecting the distribution of tumor-promoting CAFs in the invasive front.

CCR7 Mediates Cell Invasion and Migration in Extrahepatic Cholangiocarcinoma by Inducing Epithelial-Mesenchymal Transition.

The epithelial-mesenchymal transition (EMT) contributes to the metastatic cascade in various tumors. C-C chemokine receptor 7 (CCR7) interacts with its ligand, chemokine (C-C motif) ligand 19 (CCL19), to promote EMT. However, the association between EMT and CCR7 in extrahepatic cholangiocarcinoma (EHCC) remains unknown. This study aimed to elucidate the prognostic impact of CCR7 expression and its association with clinicopathological features and EMT in EHCC. The association between CCR7 expression and clinicopathological features and EMT status was examined via the immunohistochemical staining of tumor sections from 181 patients with perihilar cholangiocarcinoma. This association was then investigated in TFK-1 and EGI-1 EHCC cell lines. High-grade CCR7 expression was significantly associated with a large number of tumor buds, low E-cadherin expression, and poor overall survival. TFK-1 showed CCR7 expression, and Western blotting revealed E-cadherin downregulation and vimentin upregulation in response to CCL19 treatment. The wound healing and Transwell invasion assays revealed that the activation of CCR7 by CCL19 enhanced the migration and invasion of TFK-1 cells, which were abrogated by a CCR7 antagonist. These results suggest that a high CCR7 expression is associated with an adverse postoperative prognosis via EMT induction and that CCR7 may be a potential target for adjuvant therapy in EHCC.

Identification of antibody against wingless­type MMTV integration site family member 7B as a biliary cancer tumor marker.

Biliary cancer has a poor prognosis due to a lack of specific biomarkers and difficulty in diagnosis. The present study aimed to identify serum tumor markers for the diagnosis of biliary cancer via serological identification of antigens by recombinant cDNA expression cloning. Wingless­type MMTV integration site family, member 7 (WNT7B) was identified as a target antigen, suggesting the presence of serum antibodies against this antigen. Deletion mutants were then prepared to evaluate the response to serum antibodies. When serum antibody levels against WNT7B deletion mutants (WNT7B-92­2, -92­260, 2-260 and 184-260) were examined using amplified luminescence proximity homogeneous assay­linked immunosorbent assay, the levels of the antibody against WNT7B with amino acids 184­260 were higher in patients with biliary cancer than in healthy donors. Therefore, the region covering residues 184­260 of WNT7B was decomposed to generate seven peptides, and the levels of antibodies against these peptides were measured. Among them, the levels of antibodies against WNT7B234­253 and WNT7B244­260 were higher in patients with biliary cancers than in healthy donors (WNT7B234­253, P=0.0009; WNT7B244­260, P=0.0005). The levels of the antibody against the former were specifically high in patients with biliary cancer but not in those with esophageal, gastric, colorectal, pancreatic, or breast cancer. Furthermore, analysis by the cutoff value of WNT7B234­253 defined by ROC showed a high sensitivity of 70% in patients with biliary cancer. Therefore, the serum levels of the antibody against WNT7B234­253 may be useful as a marker for biliary cancer diagnosis.

Validation study of postoperative liver failure and mortality risk scores after liver resection for perihilar cholangiocarcinoma.

Background: Surgery for perihilar cholangiocarcinoma (PHCC) remains a challenging procedure with high morbidity and mortality. The Academic Medical Center (Amsterdam UMC) and Memorial Sloan Kettering Cancer Center proposed a postoperative mortality risk score (POMRS) and post-hepatectomy liver failure score (PHLFS) to predict patient outcomes. This study aimed to validate the POMRS and PHLFS for PHCC patients at Hokkaido University. Methods: Medical records of 260 consecutive PHCC patients who had undergone major hepatectomy with extrahepatic bile duct resection without pancreaticoduodenectomy at Hokkaido University between March 2001 and November 2018 were evaluated to validate the PHLFS and POMRS. Results: The observed risks for PHLF were 13.7%, 24.5%, and 39.8% for the low-risk, intermediate-risk, and high-risk groups, respectively, in the study cohort. A receiver-operator characteristic (ROC) analysis revealed that the PHLFS had moderate predictive value, with an analysis under the curve (AUC) value of 0.62. Mortality rates based on the POMRS were 1.7%, 5%, and 5.1% for the low-risk, intermediate-risk, and high-risk groups, respectively. The ROC analysis demonstrated an AUC value of 0.58. Conclusions: This external validation study showed that for PHLFS the threshold for discrimination in an Eastern cohort was reached (AUC >0.6), but it would require optimization of the model before use in clinical practice is acceptable. The POMRS were not applicable in the eastern cohort. Further external validation is recommended.

Concomitant Hepatic Artery Resection for Advanced Perihilar Cholangiocarcinoma: A Narrative Review.

Perihilar cholangiocarcinoma (PHCC) is one of the most intractable gastrointestinal malignancies. These tumours lie in the core section of the biliary tract. Patients who undergo curative surgery have a 40-50-month median survival time, and a five-year overall survival rate of 35-45%. Therefore, curative intent surgery can lead to long-term survival. PHCC sometimes invades the surrounding tissues, such as the portal vein, hepatic artery, perineural tissues around the hepatic artery, and hepatic parenchyma. Contralateral hepatic artery invasion is classed as T4, which is considered unresectable due to its "locally advanced" nature. Recently, several reports have been published on concomitant hepatic artery resection (HAR) for PHCC. The morbidity and mortality rates in these reports were similar to those non-HAR cases. The five-year survival rate after HAR was 16-38.5%. Alternative procedures for arterial portal shunting and non-vascular reconstruction (HAR) have also been reported. In this paper, we review HAR for PHCC, focusing on its history, diagnosis, procedures, and alternatives. HAR, undertaken by established biliary surgeons in selected patients with PHCC, can be feasible.

Clinical Features of Pancreatic Neuroendocrine Microadenoma: A Single-Center Experience and Literature Review.

OBJECTIVES: Pancreatic neuroendocrine microadenoma (NEMA) is a nonfunctioning neuroendocrine tumor of less than 5 mm. Most studies of NEMA were based on autopsies, and few reports have revealed the clinical frequency of NEMA. We investigated the clinicopathological features of NEMA. METHODS: The pathological results of the pancreatic resection specimens of patients, older than 18 years, who underwent pancreatic resection at Hokkaido University Hospital between April 2008 and December 2020 were retrospectively reviewed. The NEMAs were re-examined in detail and examined by immunohistochemical staining. RESULTS: Among 850 patients enrolled in this study, 24 NEMAs were identified in 12 patients (1.4%). Of the 12 patients, 2 patients had multiple endocrine neoplasia type 1, and the others had no hereditary disease, including 2 patients with multiple NEMAs. A difference in the number of NEMA was observed between patients with multiple endocrine neoplasia type 1 and sporadic NEMA. Intratumoral Ki-67 heterogeneity was correlated with the Ki-67 index. One grade 2 NEMA (Ki-67 index, 4.6%) was detected, but ATRX and DAXX labeling showed intact nuclear protein expression. CONCLUSIONS: Multiple sporadic NEMAs and grade 2 NEMAs were observed, suggesting that NEMA may have malignant potential. Thus, NEMAs should be carefully monitored for lymph node metastasis and postoperative recurrence.

Predictors of Long-Term Survival in Pancreatic Ductal Adenocarcinoma after Pancreatectomy: TP53 and SMAD4 Mutation Scoring in Combination with CA19-9.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDA) is a fatal cancer for which even unfavorable clinicopathological factors occasionally fail to preclude long-term survival. We sought to establish a scoring system that utilizes measurable pre-intervention factors for predicting survival following surgical resection. METHODS: We retrospectively analyzed 34 patients who died from short-term recurrences and 32 long-term survivors among 310 consecutively resected patients with PDA. A logistic regression model was used to define factors related to clinical parameters, molecular profiles of 18 pancreatic cancer-associated genes, and aberrant expression of major tumor suppressors. RESULTS: Carbohydrate antigen 19-9 (CA19-9) had the best ability to classify patients with short-term recurrence and long-term survivors [odds ratio 21.04, 95% confidence interval (CI) 4.612-96.019], followed by SMAD4 and TP53 mutation scoring (odds ratio 41.322, 95% CI 3.156-541.035). Missense TP53 mutations were strongly associated with the nuclear expression of p53, whereas truncating mutations were associated with the absence of nuclear p53. The former subset was associated with a worse prognosis. The combination of aberrant SMAD4 and mutation types of TP53 exhibited a better resolution for distinguishing patients with short-term recurrences from long-term survivors (compared with the assessment of the number of mutated KRAS, CDKN2A, TP53, and SMAD4 genes). Calibration of mutation scores combined with CA19-9 in a logistic regression model setting demonstrated a practical effect in classifying long survivors and patients with early recurrence (c-statistic = 0.876). CONCLUSIONS: Genetic information, i.e., TP53 mutation types and SMAD4 abnormalities, combined with CA19-9, will be a valuable tool for improving surgical strategies for pancreatic cancer.

Bacteremia after hepatectomy and biliary reconstruction for biliary cancer: the characteristics of bacteremia according to occurrence time and associated complications.

PURPOSE: Bacteremia occurring after extensive hepatic resection and biliary reconstruction (Hx + Bx) for biliary cancer is a critical infectious complication. This study evaluated postoperative bacteremia and examined the potential usefulness of surveillance cultures. METHODS: We retrospectively reviewed 179 patients who underwent Hx + Bx for biliary cancer from January 2008 to December 2018 in our department. RESULTS: Bacteremia occurred in 41 (23.0%) patients. Patients with bacteremia had a longer operation time and more frequent intraoperative transfusion and more frequently developed organ/space surgical site infection (SSI) than those without bacteremia. The most frequently isolated bacterial species from blood cultures were Enterococcus faecium (29.3%), Enterobacter cloacae (24.4%), and Enterococcus faecalis (22.0%). The SIRS duration of bacteremia associated with organ/space SSI was significantly longer than that of other infectious complications (median 96 h vs. 48 h; p = 0.043). Bacteremia associated with organ/space SSI occurred most often by postoperative day (POD) 30. The concordance rate of bacterial species between blood and surveillance cultures within POD 30 was 67-82%. CONCLUSIONS: Bacteremia associated with organ/space SSI required treatment for a long time and typically occurred by POD 30. Postoperative surveillance cultures obtained during this period may be useful for selecting initial antibiotic therapy because of their high concordance rate with blood cultures.

Outcomes of laparoscopic total gastrectomy in elderly patients: a propensity score matching analysis.

PURPOSE: This study evaluated the short-term outcomes and prognosis after laparoscopic total gastrectomy (LTG) in elderly patients aged ≥ 80 years in a multicenter retrospective cohort study using propensity score matching. METHODS: We retrospectively enrolled 440 patients who underwent curative LTG for gastric cancer at six institutions between January 2004 and December 2018. Patients were categorized into an elderly patient group (EG; age ≥ 80 years) and non-elderly patient group (non-EG; age < 80 years). Patients were matched using the following propensity score covariates: sex, body mass index, American Society of Anesthesiologists physical status, extent of lymph node dissection, and Japanese Classification of Gastric Carcinoma stage. Short-term outcomes and prognoses were compared. RESULTS: We identified 37 propensity score-matched pairs. The median operative time was significantly shorter, and postoperative stay was longer in the EG. In terms of postoperative outcomes, the rates of all complications were comparable. The median follow-up period of the EG and non-EG was 11.5 (1-106.4) months and 35.7 (1-110.0) months, respectively; there were significant differences in 5-year overall survival between the two groups (EG, 58.5% vs. non-EG, 91.5%; P = 0.031). However, there were no significant differences in 5-year disease-specific survival (EG, 62.1% vs. non-EG, 91.5%; P = 0.068) or 5-year disease-free survival (EG, 52.9% vs. non-EG, 60.8%; P = 0.132). CONCLUSIONS: LTG seems to be safe and feasible in elderly patients. LTG had a limited effect on morbidity, disease recurrence, and survival in elderly patients. Therefore, age should not prevent elderly patients from benefitting from LTG.

Clinical and oncological benefits of left hepatectomy for Bismuth type I/II perihilar cholangiocarcinoma.

PURPOSE: This retrospective study aimed to clarify whether the postoperative prognosis differs between right and left hepatectomy for Bismuth type I/II perihilar cholangiocarcinoma. METHODS: Preoperative images of 195 patients with perihilar cholangiocarcinoma were reexamined. Patients with Bismuth type I/II perihilar cholangiocarcinoma without a difference in extraductal tumor invasion between the right and left sides of the hepatic portal region were classified into those undergoing left (L group) or right (R group) hepatectomy. RESULTS: Twenty-three patients (11.8%) were classified into the L group and 33 (16.9%) into the R group. All eight patients with pTis/1 belonged to the L group. The L group had significantly less liver failure than the R group (p = 0.001). One patient (4.3%) in the L group and four patients (12.1%) in the R group died from postoperative complications. Among 48 patients with pT2, the L group tended to have better overall survival (median, 12.2 vs. 5.6 years; p = 0.072), but not recurrence-free survival (median, 9.1 vs. 3.6 years; p = 0.477), in comparison to the R group. CONCLUSIONS: Postoperative survival after left hepatectomy for Bismuth type I/II perihilar cholangiocarcinoma is expected to be as long as that after right hepatectomy.

Targeted amplicon sequencing for primary tumors and matched lymph node metastases in patients with extrahepatic cholangiocarcinoma.

BACKGROUND: Lymph node metastasis (LNM) is one of the most adverse prognostic factors in extrahepatic cholangiocarcinoma (EHCC) cases. As next-generation sequencing technology has become more widely available, the genomic profile of biliary tract carcinoma has been clarified. However, whether LNMs have additional genomic alterations in patients with EHCC has not been investigated. Here, we aimed to compare the genomic alterations between primary tumors and matched LNMs in patients with EHCC. METHODS: Sixteen patients with node-positive EHCCs were included. Genomic DNA was extracted from tissue samples of primary tumors and matched LNMs. Targeted amplicon sequencing of 160 cancer-related genes was performed. RESULTS: Among the 32 tumor samples from 16 patients, 91 genomic mutations were identified. Genomic mutations were noted in 31 genes, including TP53, MAP3K1, SMAD4, APC, and ARID1A. TP53 mutations were most frequently observed (12/32; 37.5%). Genomic mutation profiles were highly concordant between primary tumors and matched LNMs (13/16; 81.3%), and an additional genomic mutation of CDK12 was observed in only one patient. CONCLUSION: Genomic mutations were highly concordant between primary tumors and matched LNMs, suggesting that genotyping of archived primary tumor samples may help predict genomic mutations of metastatic tumors in patients with EHCC.

Long-term survival after repetitive lymphadenectomy for nodal recurrence of pancreatic neuroendocrine neoplasms: a report of two cases.

Pancreatic neuroendocrine neoplasms (PNENs) are rare, but their incidence has increased in recent years. Curative surgery is recommended in several global guidelines for resectable PNENs. Lymph node recurrence after R0 resection for PNENs is infrequent, and global guidelines recommend surgical resection for recurrence, if resectable. However, data on the prognosis after surgical resection for nodal recurrence of PNENs are limited. We herein report two cases in which long-term survival was achieved after repetitive lymphadenectomy for nodal recurrence of PNENs. In both cases, the pathological findings for primary PNEN showed well-differentiated neuroendocrine neoplasms and R0 resection was successfully performed. The Ki-67 index increased with each resection in both cases. Both patients showed long-term survival (10 and 14 years, respectively). Repetitive lymphadenectomy for nodal recurrence of PNENs may improve patient prognosis.

The assessment of risk factors for postoperative delirium using cubic spline curves in gastroenterological surgery.

PURPOSE: Delirium is associated with longer hospital stays and increased medical costs and mortality. This study explored the risk factors for postoperative delirium in gastroenterological surgery and investigated the association between qualitative changes in risk factors and the incidence of postoperative delirium. METHODS: A total of 418 patients > 18 years old who underwent gastroenterological surgery at our department between April 2018 and September 2019 were included. Risk factors were identified by comparing patients with and without postoperative delirium. Continuous variables were evaluated graphically using cubic spline curves. A logistic regression analysis was performed to assess independent risk factors. RESULTS: The incidence of postoperative delirium was 6.9%. The cubic spline curve showed that the incidence of postoperative delirium began to increase at 50 years old and increased sharply at 70 years old. A multiple logistic regression analysis of patients > 50 years old identified 5 risk factors: age ≥ 70 years, preoperative serum albumin ≤ 3.8 g/dL, psychosis, sedative-hypnotics, and intensive-care unit admission. CONCLUSION: The risk of postoperative delirium increases progressively at 50 years old and sharply at 70 years old. Advanced age, preoperative hypoalbuminemia, psychosis, sedative-hypnotics, and intensive-care unit admission are risk factors for postoperative delirium in patients > 50 years old undergoing gastroenterological surgery.