Toll-Like Receptor 7-Expressed Macrophages Are Involved in the Pathogenesis of Esophageal Achalasia and Esophagogastric Junction Outflow Obstruction.

INTRODUCTION: Esophageal achalasia is a typical esophageal motility disorder (EMD). Although viral infections have been hypothesized to play a role in the pathogenesis of esophageal achalasia, its etiology remains unclear. This study used esophageal muscle layer specimens collected during per-oral endoscopic myotomy (POEM) procedures to investigate the association between esophageal achalasia and esophagogastric junction outflow obstruction (EGJOO) and pattern recognition receptors. METHODS: Patients with esophageal achalasia and EGJOO who underwent POEM were allocated to the EMD group. Biopsies of the inner circular muscle were conducted during the POEM procedure. The control group comprised individuals diagnosed with esophageal squamous cell carcinoma who underwent surgical resection. Expression of pattern recognition receptors, including Toll-like receptor (TLR) 7, was examined by polymerase chain reaction. Immunohistochemical staining was performed to determine TLR7 expression sites in the esophageal muscle layer, and the relationship between TLR7 mRNA expression and clinical score was investigated. RESULTS: Our analysis revealed a notable upregulation of TLR7 mRNA levels within the muscle layer of esophageal achalasia and EGJOO, in contrast to those of control specimens. In contrast, the correlation between TLR7 and clinical score was not significant. Immunohistochemical staining revealed increased numbers of TLR7-expressing macrophages between the muscle layers. CONCLUSIONS: TLR7-expressing macrophages are involved in the innate immune response underlying esophageal achalasia and EGJOO. This result will lead to the elucidation of new pathogenetic mechanisms and the development of novel therapeutic targets.

Follicular dendritic cell sarcoma arising from the lymph node of the pancreatic head: a case report with literature review.

A 72-year-old man was referred to our hospital for the examination of a pancreatic head mass. Abdominal computed tomography revealed a contrasted 8-cm-diameter tumor extending from the dorsal pancreatic head to the porta hepatis. The preoperative diagnosis was challenging due to the absence of specific imaging findings and the inability to perform a biopsy. Positron emission tomography/computed tomography and diffusion-weighted imaging suggested a malignant tumor originating from the organs surrounding the pancreatic head. Subtotal stomach-preserving pancreaticoduodenectomy with regional lymph node dissection was performed, as dissection from the pancreatic head proved unfeasible. Pathological examination identified the tumor as an enlarged lymph node consisting of pleomorphic large cells forming clusters, positive for follicular dendritic cell markers cluster of differentiation (CD) 21 and CD23. No evidence of tumor capsule infiltration, other organ infiltration, or metastasis to other lymph nodes was observed. The final diagnosis was nodal follicular dendritic cell sarcoma (FDCS) originating from the pancreatic head lymph nodes. No recurrence occurred at 3 years postoperatively with no postoperative treatment. Intraperitoneal nodal FDCS is extremely rare, and occasionally, it can lead to postoperative recurrence and progression. It is crucial to differentiate neoplastic lymph node enlargement around the pancreatic head from nodal FDCS.

Three-dimensional analysis of ductular reactions and their correlation with liver regeneration and fibrosis.

The liver has multiple regeneration modes, including hepatocellular hypertrophy and self-renewal of hepatocytes. When hepatocyte proliferation is impaired, hepatic progenitor cells may proliferate through ductular reaction (DR), differentiate into hepatocytes, and contribute to fibrosis. However, the three-dimensional spatial relationship between DR and regenerating hepatocytes and dynamic changes in DR associated with fibrosis remain poorly understood. Here, we performed three-dimensional (3D) imaging of cleared 42 liver explants with chronic and acute liver diseases and 4 normal livers to visualize DR. In chronic hepatic liver diseases, such as viral hepatitis, steatohepatitis, autoimmune hepatitis, and cryptogenic cirrhosis, the total length and number of branches of DR showed a significant positive correlation. We studied the spatial relationship between DR and GS-expressing cells using glutamine synthetase (GS) and cytokeratin 19 (CK19) as markers of liver regeneration and DR, respectively. The percentage of CK19-positive cells that co-expressed GS was less than 10% in chronic liver diseases. In contrast, nearly one-third of CK19-positive cells co-expressed GS in acute liver diseases, and chronic cholestatic liver diseases, including primary biliary cholangitis and primary sclerosing cholangitis, showed no co-expression. We also found that DR was longer and had more branching in livers with progressive fibrosis compared to those with regressive fibrosis. Our results suggest that DR displays varying degrees of spatial complexity and contribution to liver regeneration. DR may serve as hepatobiliary junctions that maintain continuity between hepatocytes and bile ducts rather than hepatocyte regeneration in chronic liver diseases.

Exploration of Malignant Characteristics in Neoadjuvant Chemotherapy-Resistant Rectal Cancer, Focusing on Extramural Lesions.

BACKGROUND: Extramural vascular invasion (EMVI) and tumor deposits (TD) are poor prognostic factors in rectal cancer (RC), especially when resistant to neoadjuvant chemotherapy (NAC). We aimed to define differential expression in NAC responders and non-responders with concomitant EMVI and TD. METHODS: From 52 RC surgical patients, post-NAC resected specimens were extracted, comprising two groups: cases with residual EMVI and TD (NAC-resistant) and cases without (NAC-effective). Proteomic analysis was conducted to define differential protein expression in the two groups. To validate the findings, immunohistochemistry was performed in another cohort that included 58 RC surgical patients. Based on the findings, chemosensitivity and prognosis were compared. RESULTS: The NAC-resistant group was associated with a lower 3-year disease-free survival rate than the NAC-effective group (p = 0.041). Discriminative proteins in the NAC-resistant group were highly associated with the sulfur metabolism pathway. Among these pathway constituents, selenium-binding protein 1 (SELENBP1) expression in the NAC-resistant group decreased to less than one-third of that of the NAC-effective group. Immunohistochemistry in another RC cohort consistently validated the relationship between decreased SELENBP1 and poorer NAC sensitivity, in both pre-NAC biopsy and post-NAC surgery specimens. Furthermore, decrease in SELENBP1 was associated with a lower 3-year disease-free survival rate (p = 0.047). CONCLUSIONS: We defined one of the differentially expressed proteins in NAC responders and non-responders, concomitant with EMVI and TD. SELENBP1 was suspected to contribute to NAC resistance and poor prognosis in RC.

Three-dimensional analysis of perineural invasion in extrahepatic cholangiocarcinoma using tissue clearing.

Perineural invasion (PNI) is a characteristic invasion pattern of distal cholangiocarcinoma (DCC). Conventional histopathologic examination is a challenging approach to analyze the spatial relationship between cancer and neural tissue in full-thickness bile duct specimens. Therefore, we used a tissue clearing method to examine PNI in DCC with three-dimensional (3D) structural analysis. The immunolabeling-enabled 3D imaging of solvent-cleared organs method was performed to examine 20 DCC specimens from five patients and 8 non-neoplastic bile duct specimens from two controls. The bile duct epithelium and neural tissue were labeled with CK19 and S100 antibodies, respectively. Two-dimensional hematoxylin/eosin staining revealed only PNI around thick nerve fibers in the deep layer of the bile duct, whereas PNI was not identified in the superficial layer. 3D analysis revealed that the parts of DCC closer to the mucosa exhibited more nerves than the normal bile duct. The nerve fibers were continuously branched and connected with thick nerve fibers in the deep layer of the bile duct. DCC formed a tubular structure invading from the epithelium and extending around thin nerve fibers in the superficial layer. DCC exhibited continuous infiltration around the thick nerve fibers in the deep layer. This is the first study using a tissue clearing method to examine the PNI of DCC, providing new insights into the underlying mechanisms.

Use of time­density curves of dynamic contrast­enhanced computed tomography for determination of the histological therapeutic effects of neoadjuvant chemotherapy for pancreatic ductal adenocarcinoma.

The present study aimed to investigate the histological changes caused by neoadjuvant chemotherapy (NAC) for pancreatic ductal adenocarcinoma (PDAC), and to demonstrate the use of time­density curves (TDCs) of dynamic contrast­enhanced computed tomography (CECT) for determination of the histological therapeutic effects of NAC for PDAC. A total of 96 patients with PDAC were examined; 46 underwent NAC (NAC group) and 50 did not undergo NAC (non­NAC group). Based on histological therapeutic effect and using the area of residual tumor (ART) grading system, the NAC group was divided into low­responders and high­responders. Histological analysis was used to evaluate the densities of cancer cells, cancer­associated fibroblasts (CAFs), microvessels and stromal collagen fibers in the NAC and non­NAC groups. Radiological analysis was used to evaluate the TDCs of three slopes of the NAC group, namely slopes between the non­contrast and arterial phases (δ1 and δ1'), between the arterial and portal phases (δ2 and δ2'), and between the portal and equilibrium phases (δ3 and δ3'). δ1­Î´3 were before NAC, whereas δ1'­Î´3' were after NAC. Changes in δ1, δ2 and δ3 before and after NAC were denoted as δδ1 (=δ1'­Î´1), δδ2 (=δ2'­Î´2) and δδ3 (=δ3'­Î´3). ART grading system, histological examination and radiological examination data were also statistically analyzed. Histological examination revealed a significant decrease in cancer cells and CAFs, and a significant increase in stromal collagen fibers due to NAC (P<0.01). Radiological examination revealed that δ1' was significantly higher than δ1 in low­responders (P<0.05), whereas δ2' was significantly lower than δ2 in high­responders (P<0.01). δδ2 was significantly lower and δδ3 was significantly higher in high­responders than in low­responders (P<0.01 and P<0.05, respectively). Receiver operating characteristic curve showed that δδ2 and δδ3 were effective indicators of the histological therapeutic effect of NAC. In conclusion, the TDC of dynamic CECT may be useful for determining the histological therapeutic effect of NAC for PDAC.

18F­fluorodeoxyglucose positron emission tomography predicts recurrence and histological grade of extrahepatic bile duct cancer.

Malignant tumors in cholangiocarcinoma are diagnosed and staged using 18F-fluorodeoxyglucose positron emission tomography (FDG-PET) and clinical analysis. However, comprehensive analysis, including pathological analysis, has not yet been sufficiently performed. In the present study, the maximum standardized uptake value (SUVmax) was calculated using FDG-PET and its relationship with clinicopathological factors was analyzed. The present study included 86 patients who underwent preoperative FDG-PET/computed tomography (CT) and did not receive chemotherapy among 331 patients with hilar and distal cholangiocarcinoma. Receiver operating characteristic analysis with recurrence events was used to determine the SUVmax cutoff of 4.9. Immunohistochemical staining of glucose transporter 1 (Glut1), hypoxia-inducible factor-1α and Ki-67 was performed for pathological analysis. The standardized uptake value (SUV)-high group (SUVmax ≥4.9) had a higher postoperative recurrence rate (P<0.046) and higher Glut1 and Ki-67 expression rates (P<0.05 and P<0.0001, respectively). Furthermore, SUVmax and Glut1 expression (r=0.298; P<0.01) and SUVmax and Ki-67 expression rates (r=0.527; P<0.0001) were positively correlated. The preoperative measurement of SUVmax by PET-CT is useful in predicting recurrence as well as cancer malignancy.

A case of serous cystic neoplasm with tumor growth acceleration leading to extrapancreatic invasion.

Serous cystic neoplasm (SCN) is a potentially malignant and invasive disease. However, there are no established guidelines regarding the surgical management of SCN. Here, we report a case of SCN with jejunal invasion that ultimately required a distal pancreatectomy with partial resection of the jejunum. The patient was a 65-year-old female who was referred to our department after a diagnosis of SCN in the pancreatic tail. CT and MRI showed a 75-mm multifocal cystic mass with calcifications; the splenic vein and left adrenal vein were entrapped within the tumor. Furthermore, the tumor was in contact with the beginning of the jejunum. Finally, she underwent a posterior radical antegrade modular pancreatosplenectomy with a partial wedge-shaped resection of the jejunum. Histological findings indicated serous cystadenoma. In addition, the tumor cells were found to have infiltrated the jejunal muscularis propria in some areas, suggesting that the tumor had malignant potential. Currently, 14 months have passed since surgery and there is no evidence of metastasis or recurrence. Surveillance and the decision to perform surgical resection should be made based on tumor size and growth rate to avoid malignant transformation as well as to provide SCN patients with organ-sparing, less invasive surgery.

Case Report: A Giant Epignathus with a Well-Developed Skeleton of Head and Neck.

Epignathus is an extremely rare teratoma found in the oral cavity or oropharyngeal region of newborns, whose pathogenesis is poorly understood. We describe a giant epignathus arising from the oropharynx in a newborn. The giant tumor completely obstructed the airway of the newborn resulting in death. Histological and radiological examination of the tumor reveals the presence of a remarkably well-developed skeleton of the head and neck. A row of teeth, the axis and atlas, thyroid and salivary glands, trachea, and cerebral tissue are all detected within the tumor. These findings suggest that the epignathus is fetus-in-fetu which is considered a type 0 germ cell tumor in accordance with current literature.

Testicular teratomagenesis from primordial germ cells with overexpression of germinal center-associated nuclear protein.

Testicular teratomas are the major histologic type of testicular germ cell tumors and their incidence continues to grow. Moreover, teratomas can develop from undifferentiated cells in induced pluripotent stem (iPS) cell transplantation therapy, seriously hampering the progress of regenerative medicine. Germinal center-associated nuclear protein (GANP) is thought to be important to the biogenetic control of primordial germ cells and is among the genes susceptible to testicular germ cell tumors. Thus, we analyzed the expression of GANP in human testicular postpubertal-type teratomas and established a novel mouse model to reveal the association between GANP and teratomagenesis. We analyzed 31 cases of human testicular postpubertal-type teratomas and, in all cases, GANP was overexpressed. The aberrant expression was also detected in germ cell neoplasia in situ accompanied by the teratoma. GANP expression was particularly high in the epithelia of the epidermis, cutaneous appendages, and trachea-like ciliated epithelium. To further clarify the association between GANP and teratomagenesis, we established a novel teratomagenesis mouse model (CAG-ganpTg mice). In the GANP-teratoma mice, GANP-overexpressing teratomas were more frequent at the testes and the middle portion of the uterus than has been seen in the previously established mouse models. In conclusion, GANP is overexpressed in testicular postpubertal-type teratomas and is an essential teratomagenic factor. We also found that CAG-ganpTg mice are useful mouse models of teratomagenesis that mimics human midline teratomas and that teratomas may originate from the overexpression of GANP in primordial germ cells.

Heterogeneity of metabolic adaptive capacity affects the prognosis among pancreatic ductal adenocarcinomas.

BACKGROUND: Evolutionary cancer has a supply mechanism to satisfy higher energy demands even in poor-nutrient conditions. Metabolic reprogramming is essential to supply sufficient energy. The relationship between metabolic reprogramming and the clinical course of pancreatic ductal adenocarcinoma (PDAC) remains unclear. We aimed to clarify the differences in metabolic status among PDAC patients. METHODS: We collected clinical data from 128 cases of resectable PDAC patients undergoing surgery. Sixty-three resected tissues, 15 tissues from the low carbohydrate antigen 19-9 (CA19-9), 38-100 U/mL, and high CA19-9, > 500 U/mL groups, and 33 non-tumor control parts, were subjected to tandem mass spectrometry workflow to systematically explore metabolic status. Clinical and proteomic data were compared on the most used PDAC biomarker, preoperative CA19-9 value. RESULTS: Higher CA19-9 levels were clearly associated with higher early recurrence (p < 0.001), decreased RFS (p < 0.001), and decreased DSS (p = 0.025). From proteomic analysis, we discovered that cancer evolution-related as well as various metabolism-related pathways were more notable in the high group. Using resected tissue immunohistochemical staining, we learned that high CA19-9 PDAC demonstrated aerobic glycolysis enhancement, yet no decrease in protein synthesis. We found a heterogeneity of various metabolic processes, including carbohydrates, proteins, amino acids, lipids, and nucleic acids, between the low and the high groups, suggesting differences in metabolic adaptive capacity. CONCLUSIONS: Our study found metabolic adaptation differences among PDAC cases, pertaining to both cancer evolution and the prognosis. CA19-9 can help estimate the metabolic adaptive capacity of energy supply for PDAC evolution.

Cancerization of ducts in hilar cholangiocarcinoma.

Invasive cancers that arise from ductal structures can infiltrate and colonize pre-existing ducts in a process referred to as cancerization of ducts (COD). COD in cholangiocarcinoma is an under-studied process whose clinical significance remains poorly understood. Even though both cancerized ducts and biliary intraepithelial neoplasias (BilINs) show dysplastic changes, hallmarks of COD are (i) an abrupt transition from the normal/reactive epithelium to severe dysplasia and (ii) close proximity to invasive carcinoma with similar cytologic features. We investigated 113 cases of surgically resected hilar cholangiocarcinoma and identified COD in 37 cases (33%). Using immunohistochemistry, we found that COD and adjacent invasive carcinoma had a concordant pattern of p53 and SMAD4 staining in 95% (21/22) and 100% (21/21) of cases, respectively. In contrast, BilINs and cancerized ducts showed significantly lower levels of concordance in p53 and SMAD4 staining at 44% (8/18) and 47% (8/17) of cases, respectively (P = 0.0007 and 0.0001, respectively). By univariate analysis, positive lymph node metastasis (P = 0.027), positive final bile duct margin (P = 0.021), and the presence of COD (P = 0.020) were associated with decreased overall survival. We further performed multivariate analysis to demonstrate that positive lymph node metastasis (P = 0.031), positive final bile duct margin (P = 0.035), and COD (P = 0.0051) were correlated with decreased overall survival. Together, our study highlights that COD is a clinically significant process in hilar cholangiocarcinoma that can be identified using morphological criteria in conjunction with p53 and SMAD4 immunolabeling.

Eukaryotic initiation factor 2 signaling behind neural invasion linked with lymphatic and vascular invasion in pancreatic cancer.

Perineural invasion (PNI) is a typical poor prognostic factor in pancreatic ductal adenocarcinoma (PDAC). The mechanisms linking PNI to poor prognosis remain unclear. This study aimed to clarify what changes occurred alongside PNI in PDAC. A 128-patient cohort undergoing surgery for early-stage PDAC was evaluated. Subdivided into two groups, according to pathological state, a pancreatic nerve invasion (ne) score of less than three (from none to moderate invasion) was designated as the low-grade ne group. The high-grade (marked invasion) ne group (74 cases, 57.8%) showed a higher incidence of lymphatic metastasis (P = 0.002), a higher incidence of early recurrence (P = 0.004), decreased RFS (P < 0.001), and decreased DSS (P < 0.001). The severity of lymphatic (r = 0.440, P = 0.042) and venous (r = 0.610, P = 0.002) invasions was positively correlated with the ne score. Tumors having abundant stroma often displayed severe ne. Proteomics identified eukaryotic initiation factor 2 (EIF2) signaling as the most significantly enriched pathway in high-grade ne PDAC. Additionally, EIF2 signaling-related ribosome proteins decreased according to severity. Results showed that PNI is linked with lymphatic and vascular invasion in early-stage PDAC. Furthermore, the dysregulation of proteostasis and ribosome biogenesis can yield a difference in PNI severity.

Roles of the nervous system in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC), with its extremely poor prognosis, presents a substantial health problem worldwide. Outcomes have improved thanks to progress in surgical technique, chemotherapy, pre-/postoperative management, and centralization of patient care to high-volume centers. However, our goals are yet to be met. Recently, exome sequencing using PDAC surgical specimens has demonstrated that the most frequently altered genes were the axon guidance genes, indicating involvement of the nervous system in PDAC carcinogenesis. Moreover, perineural invasion has been widely identified as one poor prognostic factor. The combination of innovative technologies and extensive clinician experience with the nervous system come together here to create a new treatment option. However, evidence has emerged that suggests that the relationship between cancer and nerves in PDAC, the underlying mechanism, is not fully understood. In an attempt to tackle this lethal cancer, this review summarizes the anatomy and physiology of the pancreas and discusses the role of the nervous system in the pathophysiology of PDAC.

Time density curve of dynamic contrast-enhanced computed tomography correlates with histological characteristics of pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by an infiltrative growth pattern with intense desmoplastic stroma comprised of cancer-associated fibroblasts (CAFs). Additionally, the histological characteristics are considered to play a vital role in the poor prognosis of PDAC. However, the density of cancer cells, degree of desmoplasia and vascular proliferation varies in individual cases. We hypothesized that preoperative radiological images would reflect histological characteristics, such as cancer cell density, CAF density and microvessel density. To clarify the association between the histological characteristics and radiological images of PDAC, the cancer cell density, CAF density and microvessel density from surgical specimens were measured with immunostaining, and the time density curve of dynamic contrast-enhanced computed tomography (CECT) was analyzed. Overall, the initial slope between non-enhanced and arterial phases was correlated with microvessel density, and the second slope between arterial and portal phases was correlated with CAF and cancer cell densities. In conclusion, the present study suggested the possibility of estimating cancer cell, CAF and microvessel densities using the TDC of dynamic CECT.

A case report of mucinous adenocarcinoma derived from intra-ampullary papillary-tubular neoplasm with a malignant course.

BACKGROUND: Intra-ampullary papillary-tubular neoplasm (IAPN) has been classified as a Vater papillary tumor. The prognosis of IAPN is generally relatively good. Here, we describe a patient with a mucinous adenocarcinoma cluster in the Vater papilla of IAPN origin. CLINICAL PRESENTATION: The patient was a 66-year-old man who was admitted to our hospital after a diagnosis of pancreatic head carcinoma based on a pancreatic duct dilatation found on abdominal ultrasound. CT showed a 40 mm lesion in the pancreatic head and expansion of the main pancreatic duct to a maximum diameter of 9 mm on the caudal side of the lesion. The extrahepatic bile duct had also expanded to a maximum diameter of 8 mm. PET/CT showed fluorodeoxyglucose (FDG) accumulation of SUVmax 6.02 that corresponded to the tumor in the pancreatic head, though it did not suggest distant metastasis. The patient was diagnosed with pancreatic head carcinoma T3 N0 M0 Stage IIA and underwent a pancreaticoduodenectomy. Pathology indicated that the tumor in the pancreatic head was a benign inflammatory lesion. On the other hand, the papillotubular tumor pervading the lumen in the duodenal papillary common channel met the criteria for IAPN, and a mucinous adenocarcinoma cluster found in the surrounding stroma suggested malignant transformation of IAPN. No metastasis to lymph nodes was demonstrated. With regard to the mucus phenotype of each lesion, the IAPN was MUC2 and MUC5AC positive, while the mucinous adenocarcinoma was MUC2-positive and MUC5AC-negative. In addition, CD10 was negative in both lesions, suggesting that mucus transformation from the gastric type to the intestinal type was a key element. A blood test 10 months after surgery showed increased CA19-9 (105 U/mL) and CEA (7.1 ng/mL). Abdominal CT showed multiple cystoid nodes in the liver, which were diagnosed as multiple liver metastases of mucinous adenocarcinoma transformed from the IAPN. CONCLUSIONS: We reported a case with IAPN that developed in the Vater papilla, which took an extremely malignant course. IAPN generally has a good prognosis, but it is important to understand that a malignant course may occur.

Prognostic significance of the Ki67 index and programmed death-ligand 1 expression after radical cystectomy in patients with muscle-invasive bladder cancer.

OBJECTIVES: To investigate the association between Ki67 index and programmed death-ligand 1 (PD-L1) expression in muscle-invasive bladder cancer (MIBC) patients after RC. MATERIALS AND METHODS: We retrospectively evaluated 262 MIBC patients treated with RC between April 2004 and April 2020. The impact of Ki67 index and PD-L1 expression on prognosis was evaluated by univariate Cox regression analysis. In addition, a pathomolecular risk score, including Ki67 and PD-L1, was developed to predict prognosis and pathological factors. We also evaluated the link between the Ki67 index and PD-L1 under the IL-6 stimulation in the bladder cancer cell lines of T24 and 5637 cells. RESULTS: The median age and follow-up period was 69 years and 52 months, respectively. Ki67 index and PD-L1 expression were significantly associated with tumor recurrence. Univariate Cox regression analysis showed that pT3-4, mixed histology, lymphovascular invasion positive (LVI+), pN+, Ki67-high (>17%), and PD-L1+ were significantly associated with recurrence-free survival (RFS). The pathomolecular risk score was developed using resection margin+ (1 point), mixed histology (1 point), LVI+ (1 point), pN+ (1 point), and Ki67-high (1 point). RFS and overall survival were significantly shorter in patients with higher pathomolecular risk scores (>1) than in those with lower risk scores (≤1). Cell proliferation was significantly increased in the T24 and 5637 cells under the IL-6 stimulation, while PD-L1 expression was not. CONCLUSIONS: A significant effect of Ki67-high and PD-L1 expression on poor prognosis was observed in patients with MIBC. Further studies are necessary to elucidate the precise mechanisms of cell proliferation and PD-L1 expression in patients with MIBC.

Three-dimensional analysis of extrahepatic cholangiocarcinoma and tumor budding.

Advances in tissue clearing and microscopy make it possible to study human diseases in three dimensions (3D). High-grade tumor budding is known to be associated with poor prognosis in various cancers; however, little is known about the 3D architecture of tumor budding. Using tissue clearing, we analyzed the 3D structure of tumor budding and E-cadherin expression in 31 extrahepatic cholangiocarcinomas. A total of 31 thick slabs (up to 5 mm) were harvested from surgically resected tumor tissue, including 27 hilar and 4 distal cholangiocarcinomas. Twenty-eight cases were adenocarcinoma, and three were undifferentiated carcinoma. After clearing, the tissues were immunolabeled with antibodies to cytokeratin 19 and to E-cadherin, and then visualized using light-sheet and confocal laser scanning microscopy. Tumor budding was evaluated in hematoxylin and eosin-stained sections (2D) using standard pathological criteria. Of the 31 cancers, 13 showed low-grade tumor budding and 18 showed high-grade tumor budding. First, 3D analysis revealed that the neoplastic cells in tumor buds of adenocarcinoma were typically not individual islands of cells, but rather tips of attenuated protrusions connected to the main tumor. Second, adenocarcinomas with low-grade tumor budding were composed predominantly of tubules that only focally form cords at the periphery. By contrast, adenocarcinomas with high-grade tumor budding predominantly formed cords in both centers and peripheries of the tumors. Third, adenocarcinoma with low-grade tumor budding was characterized by a few short protrusions with few branches, whereas adenocarcinoma with high-grade tumor budding was characterized by longer protrusions with more branching. Finally, immunolabeling of E-cadherin was stronger in the center of the adenocarcinoma but decreased at the tips of protrusions. E-cadherin loss was more extensive in the protrusions of high-grade tumor budding than in the protrusions of low-grade tumor budding. Our findings suggest that tumor buds as seen in 2D are, in fact, cross-sections of attenuated but contiguous protrusions extending from the main tumor. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.