Histopathological growth pattern and vessel co-option in intrahepatic cholangiocarcinoma

Surgical resection can lead to prolonged survival in patients with isolated liver metastases (LM) from various primary cancers. However, there are currently no validated predictive markers to discriminate between these oligo/argometastatic patients, who will benefit from surgery, and those with diffuse metastatic behavior in whom surgery will be futile. To evaluate whether the tumor microenvironment, or histopathological growth pattern (HGP), of LM reflects the type of metastatic progression independently of the origin of the primary cancer, we analyzed a combined series of patients who underwent surgery for colorectal LM (N=263) or non-colorectal LM (N=66). HGPs of LM were scored in each patient to distinguish between desmoplastic HGP (all LM showing a complete encapsulated pattern) and non-desmoplastic HGP (at least one LM with some infiltrating-replacement component). In the entire series, 5-year overall and progression-free survival were, 44.5% and 15.5%, respectively, with no significant differences between colorectal and non-colorectal LM. In patients with desmoplastic HGP, 5-year overall and progression-free survival were 57% and 32%, respectively, as compared to 41% and 12%, respectively, in patients with non-desmoplastic-HGP (p=0.03 and 0.005). Irrespective of cancer origin and compared to traditional risk factors, desmoplastic HGP was the most significant predictor for better post-operative overall survival (adjusted HR: 0.62; 95% CI: [0.49-0.97]; p=0.035) and progression-free survival (adjusted HR: 0.61; 95% CI: [0.42-0.87], p=0.006). This suggests that the HGP of LM may represent an accurate marker that reflects the mode of metastatic behavior, independently of primary cancer type.

Up to 50% of uveal melanomas (UM) metastasise to the liver within 10 years of diagnosis, and these almost always prove rapidly fatal. As histopathological growth patterns (HGPs) of liver metastases of the replacement and desmoplastic type, particularly from colon and breast carcinoma, may import valuable biological and prognostic information, we have studied HGP in a series of 41 UM liver metastases originating from 41 patients from the period 2006–2017. Twenty patients underwent enucleation while 21 had radiation therapy. Analysis of UM by array comparative genomic hybridisation revealed: 25 (64%) patients with high risk (monosomy3/8q gain); 13 (33%) intermediate risk (M3/8normal or disomy3/8q gain); and 1 low risk (disomy3/8normal). The principal HGP was replacement in 30 (73%) cases and desmoplastic in 11 (27%) cases. Cases with replacement demonstrated striking vascular co‐option/angiotropism. With the development of liver metastasis, only the replacement pattern, largest primary tumour diameter, and R2 (incomplete resection) status predicted diminished overall survival (OS; p < 0.041, p < 0.017, p < 0.047, respectively). On multivariate analysis, only HGP (hazard ratio; HR = 6.51, p = 0.008) and resection status remained significant. The genomic high‐risk variable had no prognostic value at this stage of liver metastasis. Chi‐square test showed no association of HGP with monosomy 3 or 8q gain. Eighteen of 41 (44%) patients are alive with disease and 23 (56%) patients died with follow‐up ranging from 12 to 318 months (mean: 70 months, median: 47 months). In conclusion, we report for the first time the frequency of the replacement and desmoplastic HGPs in liver UM metastases resected from living patients, and their potential important prognostic value for UM patients, as in other solid cancers. These results may potentially be utilised to develop radiological correlates and therapeutic targets for following and treating patients with UM metastases.

Histopathological growth patterns (HGPs) have shown important prognostic values for patients with colorectal cancer liver metastases, but the potential molecular mechanisms remain largely unknown. We performed an exploratory analysis by conducting the RNA sequencing of primary colorectal lesions, colorectal liver metastatic lesions and normal liver tissues. We found that desmoplastic HGPs of the metastatic lesions were significantly enriched in EMT, angiogenesis, stroma, and immune signaling pathways, while replacement HGPs were enriched in metabolism, cell cycle, and DNA damage repair pathways. With the exception of immune-related genes, the differentially expressed genes of the two HGPs from colorectal liver metastases were mostly inherited from the primary tumor. Moreover, normal liver tissue in the desmoplastic HGP subgroup was markedly enriched in the fibrinous inflammation pathway. We surmised that HGPs are observable morphological changes resulting from the regulation of molecular expressions, which is the combined effect of the heterogeneity and remodeling of primary tumors seeds and liver soils.

Primary Colorectal Cancer Histopathology Predicts the Histopathological Growth Pattern of Corresponding Liver Metastasis

Cholangiocarcinomas (CCs) are primary hepatobiliary carcinomas of increasing importance and with major biological and therapeutic challenges. CCs may occur in any segment along the biliary tract and are presently also classified into two major categories according to their anatomic location. CCs arising from small bile duct and ductules to segmental large bile ducts are designated as intrahepatic or peripheral CC (ICC). Tumors originating from large bile ducts at the hilum (right or left hepatic bile duct or at their junction) or along the extrahepatic biliary tree are designated as extrahepatic bile duct carcinomas (BDC) or extrahepatic CC. Features of cholangiocyte differentiation characterize them; traditionally, they are thought to derive from the malignant transformation of bile duct epithelial cells and histologically classified as adenocarcinoma and rare variants. Recent data emphasized the significant degree of CCs’ heterogeneity in terms of their epidemiology and risk factors, pathological and molecular features, pathogenesis, and clinical behaviors and treatment and underlined the role of hepatic stem/progenitor cells as cell origin of a proportion of CC and their possible overlap with the major primary malignant tumor of the liver, namely hepatocellular carcinoma (HCC); precursor lesions and early lesions have been characterized underlining the existence of multistep carcinogenesis process. Overall, these data result in proposal of new histological or molecular classifications that could soon replace current anatomic-based classification and have major impact on establishment of prognosis and on development of novel target treatment approaches.

Colorectal cancer liver metastases (CRCLMs) have two major histopathological growth patterns (HGPs): desmoplastic (DHGP) and replacement (RHGP). The DHGP tumours derive their vasculature by angiogenesis, while the RHGP tumours use vessel co-option. Various studies have associated RHGP tumours with an unfavourable prognosis, as well as high levels of resistance to anti-angiogenic agents and chemotherapy. Recently, we reported higher numbers of neutrophils in the tumour microenvironment (TME) of vessel co-opting tumours compared to their angiogenic counterparts. However, the molecular mechanisms underlying this phenotype are unclear. Herein, we suggested a positive correlation between the expression of angiopoietin-1 (Ang1) in the hepatocytes and the presence of neutrophils in vessel co-opting tumours. Importantly, upregulation of Ang1 in the hepatocytes is associated with the presence of runt-related transcription factor-1 (RUNX1) in the neighboring cancer cells in vitro and in vivo. Altogether, our data suggest the molecular mechanisms by which neutrophils are infiltrated in vessel co-opting CRCLM lesions. This finding may yield novel therapeutic strategies for CRCLM patients in future.

Objective To investigate the CT diagnosis of patients with the carcinoma of bile duct. Methods The data of 46 patients who were treated with surgery, percutaneous puncture biopsy and pathologically confirmed to have the carcinoma of bile duct were collected, and the manifestations of CT plain scanning and dynamic enhancement scanning were retrospectively analyzed. Results The liver CT plain scanning of the carcinoma of intrahepatic bile duct was manifested as similar round and irregular low-density tumor, intrahepatic cholangiectasis was observed in 66.7%, and atrophy of liver lobe was observed in 41.7%; the enhancement scanning was manifested as mild enhancement of peripheral tumor at the arterial phase. The delayed scanning showed that as time elongated, the focus became enhanced gradually, especially that after 9 minutes, the intensities of all the loci were higher than those of normal liver parenchyma. The carcinoma of bile duct at hilus hepatis all displayed as intrahepatic cholangiectasis, and the forms appeared as vine. 21 patients were displayed as soft tissue tumors of hepatic hilar region. Another 3 patients were only displayed as the thickening, stenosis or discontinuation of bile duct wall, and could be accompanied by portal lymph node, the lymph node of posterior abdominal membrane, epoploon metastasis and abdominal dropsy. The carcinoma of extrahepatic biliary duct was manifested as intrahepatic and extrahepatic cholangiectasis, the augmentation of cholecyst, the sudden desection of common bile duct or irregular stenosis of common bile duct. Conclusion CT can clearly display the lesion of the carcinoma of bile duct, the degree of cholangiectasis, and the range of surrounding infiltration, and CT is very significant in detecting the carcinoma of bile duct at early period. Key words: Bile duct neoplasms; Tomography; X-ray computer; Diagnostic imaging

We have shown that large and small cholangiocytes, which reside primarily in large and small intrahepatic bile ducts, respectively, have different functions and responses to injuries. However, there are no systematic studies of the molecular differences between small and large cholangiocytes, which would explain cholangiocyte heterogeneity. To evaluate the differential gene expression between small and large cholangiocytes, microarray analysis was performed. Primary cultures of small and large cholangiocytes were isolated from normal mice (BALB/c), and immortalized by the introduction of the SV40 large T antigen gene. After cloning, small and large cholangiocyte cell lines were established. Their characteristic features were confirmed by electron microscopy (EM) and measurement of transepithelial electrical resistance (TER), and secretin-stimulated cAMP levels. Isolated total RNAs were hybridized with microarrays (Atlas Glass Array Mouse 1.0 and 3.8), which detects 4850 cDNA expressions. After hybridization, the fluorescent signals were scanned by a GenePix fluorescent scanner and analyzed using ArrayGauge software. EM, TER and secretin-stimulated cAMP synthesis are consistent with the concept that small and large immortalized cholangiocytes originate from small and large ducts, respectively. When a cut-off value at the expression signal difference of 3.0 times was employed, 230 cDNAs among 4850 cDNAs (4.74%) were differentially expressed between small and large cholangiocytes. Of these 230 cDNAs, aquaporin 8, IL-2 receptor beta chain and caspase 9 were more strongly expressed by large cholangiocytes. Microarray successfully displayed characteristic differential cDNA expression between small and large cholangiocytes. This technique provides molecular information, which further supports our hypothesis that small and large bile ducts have different functions.

Intrahepatic cholangiocarcinoma (ICC) arises from the lining epithelium and peribiliary glands of the intrahepatic biliary tree and shows variable cholangiocytic differentiation. To date, ICC was largely classified into adenocarcinoma and rare variants. Herein, we propose to subclassify the former, based on recent progress in the study of ICC including the gross classification and hepatic progenitor/stem cells and on the pathological similarities between biliary and pancreatic neoplasms. That is, ICC is classifiable into the conventional (bile duct) type, the bile ductular type, the intraductal neoplasm type and rare variants. The conventional type is further divided into the small duct type (peripheral type) and large bile duct type (perihilar type). The former is a tubular or micropapillary adenocarcinoma while the latter involves the intrahepatic large bile duct. Bile ductular type resembles proliferated bile ductules and shows a replacing growth of the hepatic parenchyma. Hepatic progenitor cell or stem cell phenotypes such as neural cell adhesion molecule expression are frequently expressed in the bile ductular type. Intraductal type includes papillary and tubular neoplasms of the bile duct (IPNBs and ITNBs) and a superficial spreading type. IPNB and ITNB show a spectrum from a preneoplastic borderline lesion to carcinoma and may have pancreatic counterparts. At invasive sites, IPNB is associated with the conventional bile duct ICC and mucinous carcinoma. Biliary mucinous cystic neoplasm with ovarian-like stroma in its wall is different from IPNB, particularly IPNB showing cystic dilatation of the affected ducts. Rare variants of ICC include squamous/adenosquamous cell carcinoma, mucinous/signet ring cell carcinoma, clear cell type, undifferentiated type, neuroendocrine carcinoma and so on. This classification of ICC may open up a new field of research of ICC and contribute to the clinical approach to ICC.

The liver is the most frequent and often the only site of distant disease in colorectal cancer and, of all treatment protocols currently in use, resection is the most likely to result in long-term cure. Within the liver, tumour proximity to major vasculature and biliary structures poses a resection challenge, requiring a balance of achieving negative margins while preserving adequate vascular circulation and biliary drainage. The focus on parenchymal sparing resections are important but just as important may be the 'biological' behaviour of the tumour. In colorectal liver metastases (CRLM), biomarkers such as the Kirsten rat sarcoma oncogene homologue (KRAS) gene and histological growth patterns (HGPs) further improve the prognostication after resection. However, to date, the association between the KRAS status and HGPs in CRLM and their impact on resection margins around major vasculature or biliary structures in terms of overall survival and recurrence rates are unclear. The aim of this review was to explore the available evidence for the association between KRAS and HGPs in CRLM and attempt to define their impact on resection margins near major structures.

Preclinical data indicate the improvement of the antitumor activity of capecitabine by mitomycin C and docetaxel through upregulation of thymidine phosphorylase activity. Therefore, we have established a combination regimen of these drugs (DocMitoCape), which demonstrated preliminary activity especially in bile duct and pancreatic carcinoma. Here we report the safety and efficacy of the DocMitoCape regimen in pre-treated patients with gallbladder, bile duct, or pancreatic carcinoma. Treatment consisted of capecitabine (2,000 mg/m(2) days 1-14) in combination with docetaxel (40 mg/m(2) day 1) and mitomycin C (4 mg/m(2) day 1). Cycles were repeated on day 22. Toxicity was graded according to NCI-CTC criteria, and the antitumor activity was assessed by RECIST criteria. Twenty-eight pre-treated patients with a median age of 59 suffering from pancreatic, gallbladder, intra- (IHCCC) or extrahepatic (EHCCC) bile duct carcinoma were included. Eleven patients had received ≥2 lines of prior chemotherapy. A total of 183 and a median of six cycles were administered (range 1-21). The mean dose intensity was as follows (cycles 1-2/3-4; %): capecitabine 97/92, docetaxel 100/100, mitomycin C 99/100. Main adverse events grades 2/3/4 were (n): leukocytopenia 3/2/2, anemia 13/4/0, thrombocytopenia 3/1/0, nausea/vomiting 2/1/0, diarrhea 5/1/0, hand-foot-skin reaction 7/0/0. Six patients achieved partial and seven patients minor remissions, while six patients had stable disease adding to a tumor control rate of 68%. Median progression-free and overall survival was 4.5 (range 1.0-44.9) and 6.8 months (range 1.5-44.9), respectively, calculated from the start of treatment. In all, the DocMitoCape regimen exhibited a favorable safety profile and a high rate of tumor stabilizations in patients with pre-treated gallbladder, bile duct and pancreatic carcinoma. It might be considered after failure of standard regimens in these types of cancer.

Pancreatic and duodenal homeobox 1 (Pdx1) is a transcription factor that is crucial in embryogenic development and differentiation of pancreas, and its overexpression is reportedly involved in the progression of many malignancies, including pancreatic carcinoma. In this study, the role of Pdx1 was examined in cholangiocarcinogenesis. Forty-three cases of human cholangiocarcinoma (CC) and 66 cases of hepatolithiasis or primary sclerosing cholangitis (PSC) with biliary intraepithelial neoplasia (BilIN) lesions and also eight fetal and 20 adult normal livers were examined immunohistochemically. Pdx1 was constantly expressed in the nuclei of fetal bile ducts, but was virtually absent in the large bile ducts of adults. By contrast, Hairy and enhancer of split 1 (Hes1), which represses pancreatic exocrine and endocrine differentiation, was expressed frequently in the adult bile ducts. Pdx1 was expressed in 67% of invasive CCs. In large bile ducts, expression of Pdx1 increased while that of Hes1 decreased during the progression of BilIN lesions to CC. Expression of Pdx1 correlated with proliferative activities in CCs. In an in vitro study, all three CC cell lines expressed Pdx1 mRNA and protein. Up-regulation of Pdx1 is a feature of cholangiocarcinogenesis associated with chronic cholangitis. Furthermore, expression of Pdx1 in CC is related to increased proliferative activity in CCs.

Parasitic infection of the biliary tree is caused by liver flukes, namely Clonorchis sinensis and Opisthorchis viverrini. These flukes reside in the peripheral small bile ducts of the liver and produce chronic inflammation of the bile duct, bile duct dilatation, mechanical obstruction, and bile duct wall thickening. On imaging, peripheral small intrahepatic bile ducts are dilated, but the large bile ducts and extrahepatic bile ducts are not dilated or slightly dilated. There is no visible caused of obstruction. Sometimes, in heavy infection, adult flukes are demonstrated on sonography, CT or MR cholangiography as small intraluminal lesions. The flukes in the gallbladder may appear as floating, small objects on sonography. Chronic infection may result in cholangiocarcinoma of the liver parenchyma or along the bile ducts. Human infection of Fasciola hepatica, a cattle flukes, may occur inadvertently, and the flukes migrate in the liver (hepatic phase) and reside the bile ducts (biliary phase). Image findings in the hepatic phase present with multiple, small, clustered, necrotic cavities or abscesses in the peripheral parts of the liver, showing "tunnels and caves" sign, reflecting parasite migration in the liver parenchyma. In the biliary phase, the flukes are demonstrated in the intra- and extrahepatic bile ducts and the gallbladder as small intraluminal flat objects, sometimes moving spontaneously. Bile ducts are dilated.

[Background and Aims]Enhancer of Zeste Homolog 2 (EZH2) is one of the polycomb proteins and involved in the progress of several solid tumors by methylating histone3 Lysin27 (H3K27) and silencing various genes. However, the role of EZH2 in cholangiocarcinoma has not been clarified. In this study, we investigated the significance of EZH2 expression in intrahepatic and extrahepatic cholangiocarcinoma progression.[Patients and Methods]Fourty-seven intrahepatic cholangiocarcinoma (ICC) patients and 45 extrahepatic cholangiocarcinoma (ECC) patients treated with curative resection from 1993 to 2010 were enrolled. We examined the EZH2 expression by immunohistochemistry, and relationship between EZH2 expression and clinicopathological factors, such as gender, age, CA19-9, tumor diameter, tumor number, histological vascular invasion, differentiation, lymph node metastasis and prognosis. To assess whether EZH2 relates to angiogenesis and tumor growth, Ki67 expression and microvessel density (MVD) using CD34 were also examined by immunohistochemistry. EZH2 expression was investigated eight cholangiocarcinoma cell lines. Using small interfering RNA against EZH2, the influence of EZH2 knock-down on cholangiocarcinoma cell proliferation and invasion was studied in vitro. [Results]In ICC, 24 patients (51.0%) shows a high expression of EZH2 when the cutoff value was determined to be 10%. The EZH2 high expression is significantly correlated with tumor diameter &amp;gt; 3.5cm (P=0.0113) and MVD score (P=0.039). The high EZH2 expression group showed significantly worse overall survival (p=0.045). In ECC, 19 patients(42.2%) showed a high EZH2 expression. The EZH2 high expression is significantly correlated with positive lymph node metastasis (p=0.0469), high Ki67 expression (P=0.0017). The high EZH2 expression group showed significantly worse overall survival (P=0.0261). All of the eight cholangiocarcinoma cell lines expressed EZH2 in a various level. When we knock down the expression of EZH2 in RBE cells, the expression of H3K27 was reduced and cell growth decreased. However, EZH2 down-regulation didn't affect cell invasion.[Conclusion]EZH2 may relate to the progression of intra and extrahepatic cholangiocarcinoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1055. doi:1538-7445.AM2012-1055

Von Meyenburg complexes (VMCs), or bile duct microhamartomas, are among the constellation of defects of ductal plate malformation. These present as multiple small intrahepatic cysts and are diagnosed incidentally. Association...