Integrative genomic analyses of European intrahepatic cholangiocarcinoma: Novel ROS1 fusion gene and PBX1 as prognostic marker.

BACKGROUND: Cholangiocarcinoma (CCA) is a fatal cancer of the bile duct with a poor prognosis owing to limited therapeutic options. The incidence of intrahepatic CCA (iCCA) is increasing worldwide, and its molecular basis is emerging. Environmental factors may contribute to regional differences in the mutation spectrum of European patients with iCCA, which are underrepresented in systematic genomic and transcriptomic studies of the disease. METHODS: We describe an integrated whole-exome sequencing and transcriptomic study of 37 iCCAs patients in Germany. RESULTS: We observed as most frequently mutated genes ARID1A (14%), IDH1, BAP1, TP53, KRAS, and ATM in 8% of patients. We identified FGFR2::BICC1 fusions in two tumours, and FGFR2::KCTD1 and TMEM106B::ROS1 as novel fusions with potential therapeutic implications in iCCA and confirmed oncogenic properties of TMEM106B::ROS1 in vitro. Using a data integration framework, we identified PBX1 as a novel central regulatory gene in iCCA. We performed extended screening by targeted sequencing of an additional 40 CCAs. In the joint analysis, IDH1 (13%), BAP1 (10%), TP53 (9%), KRAS (7%), ARID1A (7%), NF1 (5%), and ATM (5%) were the most frequently mutated genes, and we found PBX1 to show copy gain in 20% of the tumours. According to other studies, amplifications of PBX1 tend to occur in European iCCAs in contrast to liver fluke-associated Asian iCCAs. CONCLUSIONS: By analyzing an additional European cohort of iCCA patients, we found that PBX1 protein expression was a marker of poor prognosis. Overall, our findings provide insight into key molecular alterations in iCCA, reveal new targetable fusion genes, and suggest that PBX1 is a novel modulator of this disease.

The Cellular and Humoral Immune Response to SARS-CoV-2 Messenger RNA Vaccines Is Significantly Better in Liver Transplant Patients Compared with Kidney Transplant Patients.

Patients after organ transplantation have impaired immune response after vaccination against the SARS-CoV-2 virus. So far, published studies have reported quite different response rates to SARS-CoV-2 vaccination, ranging from 15-79% in liver and kidney transplant recipients. Up to one year after the first vaccine dose, we analyzed the humoral and cellular immune response of 21 liver transplant (LTX) patients after vaccination with mRNA vaccines compared with 28 kidney transplant (KTX) patients. We evaluated IgG against the SARS-CoV-2 spike protein as well as SARS-CoV-2 specific T cells using an ELISpot assay that detected IFN-γ- and/or IL-2-expressing T cells. We found a cellular and/or humoral immune response in 100% of the LTX patients compared with 68% of the KTX patients. Antibody titers against the spike protein of SARS-CoV-2 were significantly higher in the LTX group, and significantly more LTX patients had detectable specific IL-2-producing T cells. The immunosuppression applied in our LTX cohort was lower compared with the KTX cohort (14% triple therapy in LTX patients vs. 79% in KTX patients). One year after the first vaccination, breakthrough infections could be detected in 41% of all organ transplant patients. None of those patients suffered from a severe course of COVID-19 disease, indicating that a partial vaccination response seemed to offer protection to immunosuppressed patients. The better immune response of LTX patients after SARS-CoV-2 vaccination might be due to less intense immunosuppressive therapy compared with KTX patients.

Six-Month Follow-Up after Vaccination with BNT162b2: SARS-CoV-2 Antigen-Specific Cellular and Humoral Immune Responses in Hemodialysis Patients and Kidney Transplant Recipients.

Hemodialysis patients (HDP) and kidney transplant recipients (KTR) have a high risk of infection with SARS-CoV-2 with poor clinical outcomes. Because of this, vaccination of these groups of patients against SARS-CoV-2 is particularly important. However, immune responses may be impaired in immunosuppressed and chronically ill patients. Here, our aim was to compare the efficacy of an mRNA-based vaccine in HDP, KTR, and healthy subjects. DESIGN: In this prospective observational cohort study, the humoral and cellular response of prevalent 192 HDP, 50 KTR, and 28 healthy controls (HC) was assessed 1, 2, and 6 months after the first immunization with the BNT162b2 mRNA vaccine. RESULTS: After 6 months, 97.5% of HDP, 37.9% of KTR, and 100% of HC had an antibody response. Median antibody levels were 1539.7 (±3355.8), 178.5 (±369.5), and 2657.8 (±2965.8) AU/mL in HDP, KTR, and HC, respectively (p ≤ 0.05). A SARS-CoV-2 antigen-specific cell response to vaccination was found in 68.8% of HDP, 64.5% of KTR, and 90% of HC. CONCLUSION: The humoral response rates to mRNA-based vaccination of HDPs are comparable to HCs, but antibody titers are lower. Furthermore, HDPs have weaker T-cell response to vaccination than HCs. KTRs have very low humoral and antigen-specific cellular response rates and antibody titers, which requires other vaccination strategies in addition to booster vaccination.

Cirrhosis risk score of the donor organ predicts early fibrosis progression after liver transplantation.

BACKGROUND AND AIMS: Fibrosis progression (FP) after liver transplantation (LT) increases morbidity and mortality. Biomarkers are needed for early prediction of FP. A recipient's seven-gene cirrhosis risk score (CRS) has been associated with FP, especially in non-transplant cohorts. A broader validation of CRS, including the genotype of the donor-organ and HCV-negative patients is lacking. We therefore analyzed the impact of donor- and recipient-specific genotypes on FP after LT in a large cohort of HCV-positive and -negative patients. METHOD: Genotyping from liver biopsies (n=201 donors) and peripheral blood (n=442 recipients) was performed. Cirrhosis risk score was correlated with FP at 1 and 5 years after LT. RESULTS: Fibrosis >/=F2 was documented in 26.5% of the recipients' CRS group (R-CRS) (defined by recipient's genotype) and in 23.4% of the donors' CRS- group (D-CRS) (defined by donor's genotype). Cumulative incidence for fibrosis >/=F2 was higher in patients with D-CRS >0.7 (p=0.03). While the R-CRS showed no prognostic relevance, D-CRS >0.7 was associated with higher hazard ratios (HRs) for fibrosis >/=F2 (HR=2.04; p=0.01), especially in HCV-negative patients (HR=2.59, p=0.03). Donors' CRS >0.7 was associated with higher risk for >/=F2 in 1-year protocol biopsies (p<0.001). Among the patients in whom both the recipient's and donor's CRS were available, fibrosis >/=F2 was encountered more frequently in patients with a D-CRS >0.7, in combination with any R-CRS, compared to patients with D-CRS scores /=F2 in subgroups. CONCLUSION: High D-CRS >0.7 predicted early FP after LT, especially in HCV negative patients.

Expression and prognostic significance of insulin­like growth factor-2 receptor in human hepatocellular carcinoma and the influence of transarterial chemoembolization.

Hepatocellular carcinoma (HCC) is one of the most common human malignancies, the incidence of which is growing worldwide. The prognosis of HCC is very poor and it is often accompanied by a high rate of recurrence. Conventional chemotherapeutic approaches are largely inefficient. In order to develop novel effective methods for the early detection and prognosis of HCC, novel markers and therapeutic targets are urgently required. The present study focused on the effects of the expression of the tumor suppressor gene insulin­like growth factor­2 receptor (IGF2R) on patient survival and tumor recurrence in patients with HCC; this study paid specific attention to the influence of transarterial chemoembolization (TACE) prior to surgery. The mRNA expression levels of IGF2R were measured in primary human HCC and corresponding non­neoplastic tumor­surrounding tissue (TST) by reverse transcription­polymerase chain reaction (RT­PCR) (n=92). Subsequently, the associations between IGF2R expression and clinicopathological parameters, outcomes of HCC and TACE pretreatment prior to surgery were determined. Furthermore, the effects of the IGF2R gene polymorphisms rs629849 and rs642588 on susceptibility and on clinicopathological features of HCC were investigated. RT­PCR demonstrated that the mRNA expression levels of IGF2R were downregulated in HCC compared with in TST samples (P=0.004), which was associated with a worse recurrence­free survival of patients with HCC (P=0.002) and a lower occurrence of cirrhosis (P=0.05). TACE­pretreated patients with HCC (n=26) exhibited significantly higher IGF2R mRNA expression in tumor tissues (P=0.019). In addition, significantly more patients with HCC in the TACE­pretreated group exhibited upregulated IGF2R mRNA expression compared with in the non­treated patients (P=0.032). The IGF2R SNPs rs629849 and rs642588 were not significantly associated with HCC risk, whereas a homozygous IGF2R rs629849 GG genotype was associated with a significantly elevated risk of non­viral liver cirrhosis (P=0.05). In conclusion, these data suggested an important role for IGF2R expression in HCC, particularly with regards to TACE treatment prior to surgery.

Genomic perturbations reveal distinct regulatory networks in intrahepatic cholangiocarcinoma.

Intrahepatic cholangiocarcinoma remains a highly heterogeneous malignancy that has eluded effective patient stratification to date. The extent to which such heterogeneity can be influenced by individual driver mutations remains to be evaluated. Here, we analyzed genomic (whole-exome sequencing, targeted exome sequencing) and epigenomic data from 496 patients and used the three most recurrently mutated genes to stratify patients (IDH, KRAS, TP53, "undetermined"). Using this molecular dissection approach, each subgroup was determined to possess unique mutational signature preferences, comutation profiles, and enriched pathways. High-throughput drug repositioning in seven patient-matched cell lines, chosen to reflect the genetic alterations specific for each patient group, confirmed in silico predictions of subgroup-specific vulnerabilities linked to enriched pathways. Intriguingly, patients lacking all three mutations ("undetermined") harbored the most extensive structural alterations, while isocitrate dehydrogenase mutant tumors displayed the most extensive DNA methylome dysregulation, consistent with previous findings. CONCLUSION: Stratification of intrahepatic cholangiocarcinoma patients based on occurrence of mutations in three classifier genes (IDH, KRAS, TP53) revealed unique oncogenic programs (mutational, structural, epimutational) that influence pharmacologic response in drug repositioning protocols; this genome dissection approach highlights the potential of individual mutations to induce extensive molecular heterogeneity and could facilitate advancement of therapeutic response in this dismal disease. (Hepatology 2018).

Loss of organic cation transporter 3 (Oct3) leads to enhanced proliferation and hepatocarcinogenesis.

BACKGROUND: Organic cation transporters (OCT) are responsible for the uptake of a broad spectrum of endogenous and exogenous substrates. Downregulation of OCT is frequently observed in human hepatocellular carcinoma (HCC) and is associated with a poor outcome. The aim of our current study was to elucidate the impact of OCT3 on hepatocarcinogenesis. METHODS: Transcriptional and functional loss of OCT was investigated in primary murine hepatocytes, derived from Oct3-knockout (Oct3-/-; FVB.Slc22a3tm1Dpb ) and wildtype (WT) mice. Liver tumors were induced in Oct3-/- and WT mice with Diethylnitrosamine and Phenobarbital over 10 months and characterized macroscopically and microscopically. Key survival pathways were investigated by Western Blot analysis. RESULTS: Loss of Oct3-/- in primary hepatocytes resulted in significantly reduced OCT activity determined by [3H]MPP+ uptake in vivo. Furthermore, tumor size and quantity were markedly enhanced in Oct3-/- mice (p<0.0001). Oct3-/- tumors showed significant higher proliferation (p<0.0001). Ki-67 and Cyclin D expression were significantly increased in primary Oct3-/- hepatocytes after treatment with the OCT inhibitors quinine or verapamil (p<0.05). Functional inhibition of OCT by quinine resulted in an activation of c-Jun N-terminal kinase (Jnk), especially in Oct3-/- hepatocytes. CONCLUSION: Loss of Oct3 leads to enhanced proliferation and hepatocarcinogenesis in vivo.

Organic Cation Transporter 1 (OCT1) mRNA expression in hepatocellular carcinoma as a biomarker for sorafenib treatment.

BACKGROUND: The polyspecific organ cation transporter 1 (OCT1) is one of the most important active influx pumps for drugs like the kinase inhibitor sorafenib. The aim of this retrospective study was the definition of the role of intratumoral OCT1 mRNA expression in hepatocellular carcinoma (HCC) as a biomarker in systemic treatment with sorafenib. METHODS: OCT1 mRNA expression levels were determined in biopsies from 60 primary human HCC by real time PCR. The data was retrospectively correlated with clinical parameters. RESULTS: Intratumoral OCT1 mRNA expression is a significant positive prognostic factor for patients treated with sorafenib according to Cox regression analysis (HR 0.653, 95%-CI 0.430-0.992; p = 0.046). Under treatment with sorafenib, a survival benefit could be shown using the lower quartile of intratumoral OCT1 expression as a cut-off. Macrovascular invasion (MVI) was slightly more frequent in patients with low OCT1 mRNA expression (p = 0.037). Treatment-induced AFP response was not associated with intratumoral OCT1 mRNA expression levels (p = 0.633). CONCLUSIONS: This study indicates a promising role for intratumoral OCT1 mRNA expression as a prognostic biomarker in therapeutic algorithms in HCC. Further prospective studies are warranted on this topic.

Downregulation of organic cation transporter 1 (SLC22A1) is associated with tumor progression and reduced patient survival in human cholangiocellular carcinoma.

Cholangiocellular carcinoma (CCA) is a primary hepatic malignancy derived from cholangiocytes. The prognosis for CCA patients is very poor and conventional chemotherapy has been proven ineffective in improving long­term patient survival rates. Organic cation transporters (OCTs) mediate the transport of a broad spectrum of endogenous substrates and the detoxification of xenobiotics. Moreover, OCTs are considered responsible for the responsiveness towards platinum­based chemotherapies. Currently, there are no data available regarding the role of OCTs in CCA. Therefore, the aim of this study was to investigate the expression of OCT1 and OCT3 in CCA and the corresponding non-neoplastic tumor­surrounding tissue (TST). OCT1 (SLC22A1) and OCT3 (SLC22A3) mRNA expression was measured in primary human CCA by real-time PCR (n=27). Protein expression was determined by western blot analysis and immunohistochemistry. Data were correlated with the clinicopathological parameters of CCA. Real-time PCR demonstrated a downregulation of the expression of SLC22A1 and SLC22A3 in CCA, compared to that in TST (p<0.001). A low SLC22A1 expression was associated with a worse patient survival (p<0.05). The downregulation of SLC22A1 was significantly associated with advanced CCA stages, since tumors with a low SLC22A1 mRNA expression presented with larger tumor diameters (p=0.02). There were no significant differences in tumor characteristics or patient survival in relation to the level of SLC22A3 expression. Western blot analysis and immunohistochemistry confirmed the downregulation of OCT1 and OCT3 protein levels in cancerous tissue compared to those in TST. In conclusion, the downregulation of OCT1 is associated with tumor progression and worse overall patient survival rates.

Downregulation of organic cation transporters OCT1 (SLC22A1) and OCT3 (SLC22A3) in human hepatocellular carcinoma and their prognostic significance.

BACKGROUND: Organic cation transporters (OCT) are responsible for the uptake and intracellular inactivation of a broad spectrum of endogenous substrates and detoxification of xenobiotics and chemotherapeutics. The transporters became pharmaceutically interesting, because OCTs are determinants of the cytotoxicity of platin derivates and the transport activity has been shown to correlate with the sensitivity of tumors towards tyrosine kinase inhibitors. No data exist about the relevance of OCTs in hepatocellular carcinoma (HCC). METHODS: OCT1 (SLC22A1) and OCT3 (SLC22A3) mRNA expression was measured in primary human HCC and corresponding non neoplastic tumor surrounding tissue (TST) by real time PCR (n = 53). Protein expression was determined by western blot analysis and immunofluorescence. Data were correlated with the clinicopathological parameters of HCCs. RESULTS: Real time PCR showed a downregulation of SLC22A1 and SLC22A3 in HCC compared to TST (p ≤ 0.001). A low SLC22A1 expression was associated with a worse patient survival (p < 0.05). Downregulation was significantly associated with advanced HCC stages, indicated by a higher number of T3 tumors (p = 0.025) with a larger tumor diameter (p = 0.035), a worse differentiation (p = 0.001) and higher AFP-levels (p = 0.019). In accordance, SLC22A1 was less frequently downregulated in tumors with lower stages who underwent transarterial chemoembolization (p < 0.001) and liver transplantation (p = 0.001). Tumors with a low SLC22A1 expression (< median) showed a higher SLC22A3 expression compared to HCC with high SLC22A1 expression (p < 0.001). However, there was no significant difference in tumor characteristics according to the level of the SLC22A3 expression.In the western blot analysis we found a different protein expression pattern in tumor samples with a more diffuse staining in the immunofluorescence suggesting that especially OCT1 is not functional in advanced HCC. CONCLUSION: The downregulation of OCT1 is associated with tumor progression and a worse patient survival.

The recipient CYP2D6 allele 4-associated poor metabolizer status correlates with an early fibrosis development after liver transplantation.

CYP2D6 is part of the cytochrome P450 system, which catalyzes biotransformation of endogenous substrates and xenobiotics. Approximately 10% of the Caucasian population has two null alleles, resulting in a poor metabolizer (PM) status. Mostly, allele four (CYP2D6*4) is responsible for the PM status, which is suspected to be associated with an accelerated fibrosis progression (FP). The aim of the present study was to analyze the role of the CYP2D6*4 genotype for FP after liver transplantation (LT). Genotypes were determined in liver biopsies (donor) and peripheral blood (recipient) by fluorescence resonance energy transfer. Data were correlated with clinical variables and risk factors for fibrosis. We analyzed 517 LTs performed between 1997 and 2009. Overall donor and recipient allele frequencies were comparable (18.0%, 20.5%; P = 0.43). The donor genotype did not correlate with FP. In contrast, recipients carrying CYP2D6*4, showed a significant higher risk for an accelerated FP (P = 0.011) in HCV positive (P = 0.038) and HCV negative patients (P = 0.033). Results were confirmed by multivariate analysis (Hazard ratio 1.65; P = 0.001). The CYP2D6*4-associated PM status of the donor liver seems to have no influence on FP after LT. Recipients, carrying the allele, have an elevated risk for an accelerated FP.

Chemokine receptor 5Delta32 mutation reduces the risk of acute rejection in liver transplantation.

BACKGROUND: Chemokine receptor 5 (CCR-5) plays a central role in allograft rejection. CCR-5Delta32 mutation results in a non-functioning receptor. Homozygous CCR-5Delta32 patients show a significantly improved kidney graft survival rate compared to CCR-5 wild-type patients. Similar correlations between the CCR-5Delta32 genotype and acute rejection or graft survival rate were shown for heart, lung and islet cell transplantation. MATERIAL/METHODS: The aim of this study was to examine CCR-5Delta32 and acute rejection after liver transplantation (OLT). 158 OLT patients were genotyped. Data of grafts and patients were collected prospectively into a transplant database. RESULTS: There were no significant differences between groups regarding patient, donor or graft variables. CCR-5 wild-type patients had explicitly more acute rejection episodes (p=0.086) than patients with the heterozygous or homozygous Delta32-mutation. Homozygous Delta32 patients had no acute rejection episodes. 12.5% of heterozygous patients had one acute rejection episode as opposed to 30.6% of wild-type patients. Only wild-type patients experienced more than one rejection episode. CONCLUSIONS: Patients with the Delta32-mutation might be candidates for a minimized immunosuppressive therapy. CCR-5 could be relevant as a target for a new therapeutic approach.

Chemokine Receptor-5Delta32 Mutation is No Risk Factor for Ischemic-Type Biliary Lesion in Liver Transplantation.

It has been shown that certain chemokine receptor polymorphisms may correspond to certain complications after organ transplantation. Ischemic-type biliary lesion (ITBL) encounters for major morbidity and mortality in liver transplant recipients. So far, the exact cause for ITBL remains unclear. Certain risk factors for the development of ITBL like donor age and cold ischemic time are well described. In a previous study, a 32-nucleotide deletion of the chemokine receptor-5Delta32 (CCR-5Delta32) was strongly associated with the incidence of ITBL in adult liver transplantation. This study re-evaluates the association of CCR-5Delta32 gene polymorphism and the incidence of ITBL. 169 patients were included into this retrospective analysis. 134 patients were homozygous for wild-type CCR-5, 33 patients heterozygous, and 2 patients were homozygous for CCR-5Delta32 mutation. There were no major differences in donor or recipients demographics. No association was found between CCR-5Delta32 mutation and the development of ITBL. We conclude that CCR-5Delta32 is no risk factor for the development of ITBL in our patient cohort.