CPT2 downregulation adapts HCC to lipid-rich environment and promotes carcinogenesis via acylcarnitine accumulation in obesity.

OBJECTIVE: Metabolic reprogramming of tumour cells that allows for adaptation to their local environment is a hallmark of cancer. Interestingly, obesity-driven and non-alcoholic steatohepatitis (NASH)-driven hepatocellular carcinoma (HCC) mouse models commonly exhibit strong steatosis in tumour cells as seen in human steatohepatitic HCC (SH-HCC), which may reflect a characteristic metabolic alteration. DESIGN: Non-tumour and HCC tissues obtained from diethylnitrosamine-injected mice fed either a normal or a high-fat diet (HFD) were subjected to comprehensive metabolome analysis, and the significance of obesity-mediated metabolic alteration in hepatocarcinogenesis was evaluated. RESULTS: The extensive accumulation of acylcarnitine species was seen in HCC tissues and in the serum of HFD-fed mice. A similar increase was found in the serum of patients with NASH-HCC. The accumulation of acylcarnitine could be attributed to the downregulation of carnitine palmitoyltransferase 2 (CPT2), which was also seen in human SH-HCC. CPT2 downregulation induced the suppression of fatty acid ß-oxidation, which would account for the steatotic changes in HCC. CPT2 knockdown in HCC cells resulted in their resistance to lipotoxicity by inhibiting the Src-mediated JNK activation. Additionally, oleoylcarnitine enhanced sphere formation by HCC cells via STAT3 activation, suggesting that acylcarnitine accumulation was a surrogate marker of CPT2 downregulation and directly contributed to hepatocarcinogenesis. HFD feeding and carnitine supplementation synergistically enhanced HCC development accompanied by acylcarnitine accumulation in vivo. CONCLUSION: In obesity-driven and NASH-driven HCC, metabolic reprogramming mediated by the downregulation of CPT2 enables HCC cells to escape lipotoxicity and promotes hepatocarcinogenesis.

Intrahepatic cholangiocarcinoma frequently shows loss of BAP1 and PBRM1 expression, and demonstrates specific clinicopathological and genetic characteristics with BAP1 loss.

AIMS: BAP1 and PBRM1 expression loss has been observed in multiple cancers, including intrahepatic cholangiocarcinoma (ICC). We investigated BAP1 and PBRM1 expression in ICC using immunohistochemistry, and analysed its association with clinicopathological and genetic features, including two histological subtypes. METHODS AND RESULTS: Whole-section slides of 108 consecutive primary ICC cases were immunostained against BAP1 and PBRM1. Complete loss of BAP1 and PBRM1 was observed in 21 (19.4%) and 25 (23.1%) cases, respectively, and partial loss was identified in four (3.7%) and nine (8.4%) cases. In all cases, normal bile ducts were strongly and diffusely positive for both BAP1 and PBRM1. ICC with BAP1 loss showed lower serum CA19-9 levels, less perineural invasion, rare mucin production, weaker immunoreactivity against S-100P and stronger immunoreactivity against N-cadherin and NCAM. IDH mutations were identified more frequently in ICCs with BAP1 loss. All ICC with BAP1 loss corresponded to small-duct type ICC. Multivariate Cox regression analysis showed that BAP1 loss was an independent prognostic factor for both overall and recurrence-free survival (P < 0.05). Conversely, PBRM1 loss was found in both small-duct type and large-duct type ICC, and was not associated significantly with any specific characteristics, including prognosis. CONCLUSION: BAP1 and PBRM1 loss is seen frequently in ICC. ICC with BAP1 loss shares features of small-duct type ICC.

Immunohistochemistry using monoclonal antibody MsMab-2 is useful to detect IDH1 R132L in intrahepatic cholangiocarcinoma.

Immunohistochemical analysis using specific antibodies is a useful and convenient method to detect proteins altered by somatic mutations. We previously generated the rat monoclonal antibody MsMab-2, which recognizes isocitrate dehydrogenase (IDH)1 R132L and IDH2 R172M. In the present study, we used an immunohistochemical method to examine MsMab-2 immunoreactivity in 95 cases of intrahepatic cholangiocarcinoma, including five IDH1 R132L and one IDH2 R172M mutant cases confirmed by direct sequencing. Tissue microarray section slides of all IDH1/2-mutant and wild-type cases, as well as whole section slides of IDH1 R132L and IDH2 R172M cases were immunostained using an autostainer. All IDH1 R132L cases showed positive staining for MsMab-2, while other IDH1/2 mutant and IDH1/2 wild-type cases were negative. Tumor cells of the immunopositive cases invariably showed strong reactivity using whole-section slides. We consider immunohistochemical analysis using MsMab-2 to be a useful means of detecting IDH1 R132L. Further analysis of its effectiveness against IDH2 R172M is necessary because of the small sample size in this study.

Multilocular thymic cyst detected during COVID­19 treatment in an HIV­positive adult man: A case report and literature review.

A multilocular thymic cyst (MTC) is a rare mediastinal tumor with multiloculated cyst-like structures in the anterior mediastinum. This tumfor is associated with inflammatory diseases, including human immunodeficiency virus (HIV) infection. The present study reports a case of MTC detected during coronavirus disease 2019 (COVID-19) treatment in an adult who was tested HIV positive. An anterior mediastinal tumor was incidentally detected on computed tomography in a 52-year-old man with a 20-year history of HIV infection on the 9th day of COVID-19. The patient was asymptomatic with no notable physical findings. Magnetic resonance imaging revealed a 28-mm bilocular cyst. Robot-assisted thoracoscopic tumor resection was performed. Pathological examination showed that the cyst was lined with squamous or cuboidal epithelium, and the cystic lesion wall was mainly composed of thymic tissue with follicular hyperplasia. Based on these findings, the patient was diagnosed with MTC. To date, only 15 MTC cases have been reported in patients with HIV, and the majority of cases showed HIV infection-related symptoms such as lymphoid interstitial pneumonia and parotid gland enlargement. The present case was atypical for an HIV-related MTC because it did not involve HIV infection-related symptoms, suggesting the possibility for an alternative etiology such as COVID-19. Further reports on MTC development in patients with COVID-19 are required to elucidate the relationship between MTC and COVID-19.

Gastric cancer with a giant lymph node metastasis: a case report and review of the literature.

A 78-year-old man presented with a large abdominal mass detected by ultrasonography during a regular checkup. Even if the mass was > 10 cm in diameter, he was asymptomatic. Computed tomography detected an oval-shaped mass, with a maximum diameter of 12 cm, adjacent to the greater curvature of the stomach. Esophagogastroduodenoscopy revealed a 20 mm slightly depressed (type 0-IIc) lesion on the posterior wall of the gastric antrum, which was confirmed to be adenocarcinoma. Three cycles of combination chemotherapy with S-1 and oxaliplatin were administered as neoadjuvant chemotherapy. After neoadjuvant chemotherapy, the patient underwent distal gastrectomy, and a histopathological study identified the 12 cm giant mass as a lymph node metastasis. The postoperative course was uneventful, and thus far, the patient has completed adjuvant chemotherapy without relapse. Cases of gastric cancer with a giant lymph node metastasis are extremely rare. In this study, we report the present case and review the previous literature.

A Kidney Transplant Patient Who Died of COVID-19-associated Severe Acute Respiratory Distress Syndrome.

We herein report a 67-year-old kidney transplant patient who died of COVID-19. He was treated with hydroxychloroquine and azithromycin and received mechanical ventilation that temporarily improved his respiratory status. Despite our efforts, however, he later developed respiratory failure and died 43 days after the disease onset. The autopsy revealed prominent organization of alveoli and alveolar ducts, with a massive accumulation of macrophages in the lungs. A few severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen-positive cells were detected in the lung, suggesting delayed virus clearance owing to his long-term immunosuppressed state, leading to constant lung damage and ultimately respiratory failure.

Tumor Budding in Intrahepatic Cholangiocarcinoma: A Predictor of Postsurgery Outcomes.

Intrahepatic cholangiocarcinoma (ICC) is an extremely aggressive carcinoma. Useful predictors for the patients' prognosis after surgery have not been fully established. From the University of Tokyo Hospital pathology archives, we reviewed 107 cases of ICC, 54 cases of perihilar cholangiocarcinoma, and 40 cases of extrahepatic cholangiocarcinoma (ECC); we also investigated the significance of tumor budding in ICC, in comparison with perihilar cholangiocarcinoma and ECC. The tumor-budding frequencies were different by tumor location: 40.2% (43/107) in ICC, 70.4% (38/54) in perihilar cholangiocarcinoma, and 60.0% (24/40) in ECC. Tumor budding in ICC was associated with many pathologic indicators associated with invasion, such as major vascular invasion (P=0.012) and Union for International Cancer Control stage (P=0.007). Univariate and multivariate Cox regression analyses revealed tumor budding as a powerful prognostic factor for both recurrence-free survival (RFS) and overall survival (OS) in ICC by univariate (RFS: hazard ratio [HR]: 2.666; 95% confidence interval [CI]: 1.517-4.683, OS: HR: 4.206; 95% CI: 2.447-7.230) and by multivariate analyses (RFS: HR: 3.038; 95% CI: 1.591-5.973, OS: HR: 4.547, 95% CI: 2.348-8.805). Tumor budding was also a significant prognostic factor of perihilar cholangiocarcinoma, but not of ECC. When ICC was divided into 2 subtypes, type 1 (hilar) and type 2 (peripheral), tumor budding was the strong prognostic factor in type 2 ICC, but not in type 1 ICC, suggesting that some differences in biological behavior exist between type 1 ICC and perihilar cholangiocarcinoma. Tumor budding is prognostically important in ICC, and its pathogenetic role in biliary tract carcinomas might be different by anatomic location.

EVI1 expression is associated with aggressive behavior in intrahepatic cholangiocarcinoma.

Ecotropic virus integration site 1 protein homolog (EVI1), a well-known oncogenic transcriptional factor of hematopoietic cells, contributes to pancreatic cancer oncogenicity through increased expression of KRAS. Because EVI1 was upregulated in cholangiocarcinoma by referring The Cancer Genome Atlas, we investigated the importance of EVI1 in intrahepatic cholangiocarcinoma (ICC) which has been regarded as a heterogeneous group of cancers. Immunohistochemical analysis results demonstrated that EVI1 was overexpressed in about half of ICC (53/101, 52.5%). Moreover, all intraductal papillary neoplasms of the bile duct cases expressed EVI1 regardless of histological grading and subtypes such as gastric, intestinal, pancreatobiliary, or oncocytic (20/20, 100%). EVI1-positive ICC showed higher frequencies of aggressive pathological indicators such as periductal infiltrative growth (p = 0.022), hilar invasion (p = 0.041), advanced UICC stage (p = 0.026), major vascular invasion (p = 0.026), and perineural invasion (p = 0.007) than EVI1-negative ICC. Patients with EVI1-positive ICC showed worse overall survival and recurrence-free survival in all resected cases and in curative resected cases. Recently, we proposed type 1/2 (large/small duct types) classification of ICC based on mucin productivity and immunophenotypes (S100P, N-cadherin, and NCAM). Type 1 predominantly consisted of EVI1-positive ICC (33/42 cases, 79%), and the frequency was significantly higher than type 2 (18/55 cases, 32.7%) (p < 0.0001). EVI1-positive ICC was likely to express stomach-specific claudin CLDN18 (correlation coefficient r = 0.55373) and mucin MUC5AC (r = 0.42718). EVI1-positive ICC is an aggressive ICC showing both large-duct and/or gastric phenotypes. Consequently, a transcriptional factor EVI1 is associated with aggressive behavior in ICC and can be a therapeutic target molecule, while EVI1 might be a key molecule for the development of intraductal papillary neoplasms of the bile duct.

Mutant IDH1 confers resistance to energy stress in normal biliary cells through PFKP-induced aerobic glycolysis and AMPK activation.

Metabolism is a critical regulator of cell fate determination. Recently, the significance of metabolic reprogramming in environmental adaptation during tumorigenesis has attracted much attention in cancer research. Recurrent mutations in the isocitrate dehydrogenase (IDH) 1 or 2 genes have been identified in several cancers, including intrahepatic cholangiocarcinoma (ICC). Mutant IDHs convert α-ketoglutarate (α-KG) to 2-hydroxyglutarate (2-HG), which affects the activity of multiple α-KG-dependent dioxygenases including histone lysine demethylases. Although mutant IDH can be detected even in the early stages of neoplasia, how IDH mutations function as oncogenic drivers remains unclear. In this study, we aimed to address the biological effects of IDH1 mutation using intrahepatic biliary organoids (IBOs). We demonstrated that mutant IDH1 increased the formation of IBOs as well as accelerated glucose metabolism. Gene expression analysis and ChIP results revealed the upregulation of platelet isoform of phosphofructokinase-1 (PFKP), which is a rate-limiting glycolytic enzyme, through the alteration of histone modification. Knockdown of the Pfkp gene alleviated the mutant IDH1-induced increase in IBO formation. Notably, the high expression of PFKP was observed more frequently in patients with IDH-mutant ICC compared to in those with wild-type IDH (p < 0.01, 80.9% vs. 42.5%, respectively). Furthermore, IBOs expressing mutant IDH1 survived the suppression of ATP production caused by growth factor depletion and matrix detachment by retaining high ATP levels through 5' adenosine monophosphate-activated protein kinase (AMPK) activation. Our findings provide a systematic understanding as to how mutant IDH induces tumorigenic preconditioning by metabolic rewiring in intrahepatic cholangiocytes.

Distinct Clinicopathologic and Genetic Features of 2 Histologic Subtypes of Intrahepatic Cholangiocarcinoma.

Previous studies have identified 2 clinically significant morphologic subtypes of intrahepatic cholangiocarcinoma (ICC) on the basis of anatomic location and/or histologic appearances. Recognizing that these classification schemes are not always applicable practically, this study aimed to establish a novel classification system based on mucin productivity and immunophenotype and to determine the rationale of this classification by examining the clinicopathologic and genetic characteristics of the 2 subtypes defined by this method. We retrospectively investigated 102 consecutive ICC cases and classified them on the basis of mucin productivity and immunophenotype (S100P, N-cadherin, and NCAM). We found that 42 and 56 cases were classified as type 1 and type 2 ICCs, respectively, and only 4 cases were of indeterminate type. Type 1 ICC, generally characterized by mucin production and diffuse immunoreactivity to S100P, arose less frequently in chronic liver diseases and showed higher levels of serum CEA and CA 19-9 than did type 2 ICC, which generally showed little mucin production and exhibited immunoreactivity to N-cadherin and/or NCAM. Type 1 ICC was characterized by several pathologic features, including higher frequencies of perineural invasion and lymph node metastasis. Although the log-rank test demonstrated that type 1 ICC had significantly worse survival, the multivariate Cox regression analysis showed no prognostic significance of this histologic subtype. Genetic analyses revealed that KRAS mutation was significantly more frequent in type 1 ICC, whereas IDH mutation and FGFR2 translocation were restricted to type 2 ICC. In conclusion, the present classification of ICC based on mucin productivity and immunophenotype identified 2 subtypes with clinicopathologic significance.

Histologic Assessment of Intratumoral Lymphoplasmacytic Infiltration Is Useful in Predicting Prognosis of Patients with Hepatocellular Carcinoma.

In the present study, we investigated the clinicopathologic significance of intratumoral lymphoplasmacytic infiltration in a large cohort of patients with solitary hepatocellular carcinoma (HCC). Based on examination of hematoxylin and eosin-stained sections, significant infiltration was defined as dense lymphoplasmacytic infiltration, either multifocal or diffuse, in 2 or more fields under low-power magnification. Of 544 cases, 216 (39.7%) were positive for significant infiltration (HCC-LI group), while 328 (60.3%) were negative (HCC-NLI group). There were no significant between-group differences in patient age, sex, or background etiology. The lower incidence of Child-Pugh stage B (P = 0.001) and lower level of indocyanine green retention rate at 15 minutes (P < 0.001) in the HCC-LI group indicated better liver function in this group. Histologically, tumors were significantly smaller in size in the HCC-LI group than in the HCC-NLI group (P < 0.001). In addition, prominent neutrophilic infiltration, interstitial fibrosis and tumor steatosis were significantly more frequent (P < 0.001) in the HCC-LI group, while tumor necrosis was significantly less frequent (P = 0.008). Kaplan-Meier analyses revealed that overall and recurrence-free survival were significantly better in the HCC-LI group (P < 0.001). Multivariate Cox regression analysis showed that intratumoral lymphoplasmacytic infiltration was independently prognostic of both overall and recurrence-free survival (P < 0.001), with absence of infiltration showing high Cox-hazard ratios for poor prognosis. In conclusion, intratumoral lymphoplasmacytic infiltration, as determined by assessment of hematoxylin and eosin-stained slides, was significantly associated with the clinical and pathologic features of HCC and has profound prognostic importance.

Clinicopathologic Characteristics of Hepatocellular Carcinoma With Reactive Ductule-like Components, a Subset of Liver Cancer Currently Classified as Combined Hepatocellular-Cholangiocarcinoma With Stem-Cell Features, Typical Subtype.

The aim of this study was to elucidate the clinicopathologic characteristics of hepatocellular carcinoma with reactive ductule-like components (HCC-RD), corresponding to combined hepatocellular-cholangiocarcinoma (CHC) with stem cell features, typical subtype. Retrospective clinicopathologic analysis was performed on HCCs surgically treated at the University of Tokyo Hospital between 1995 and 2013. RD components were defined as neoplastic ductular structures composed of small "stem/progenitor-like" cells. There were 46 HCC-RDs, comprising about 3% of all HCCs. Thirty-eight cases of CHC, classical type (classical CHC), were identified during the study period. When compared with conventional HCC, HCC-RD was characterized by younger patient age (P=0.016), higher frequency of female patients (P<0.001), and higher serum α-fetoprotein levels (P=0.005). Serum carbohydrate antigen 19-9 elevation was also more frequently observed in HCC-RD than in conventional HCC (P=0.002). Histologically, clear cell constituents and interstitial fibrosis were more frequent in HCC-RD than in conventional HCC (P=0.003 and <0.001, respectively). When compared with HCC-RD and conventional HCC, classical CHC was characterized by a poorly differentiated HCC component, frequent vascular invasion, and lymph node metastasis (P<0.05). There was little prognostic difference between HCC-RD and conventional HCC, whereas overall and disease-free survival in classical CHC was significantly worse than in conventional HCC. In conclusion, although HCC-RDs do have some unique clinicopathologic characteristics, they have no prognostic significance, and it is not reasonable to include these tumors in the CHC category.