Combined analysis of albumin in situ hybridisation and C reactive protein immunohistochemistry for the diagnosis of intrahepatic cholangiocarcinoma: towards a molecular classification paradigm.

AIMS: Intrahepatic cholangiocarcinoma (iCCA) is a diagnosis of exclusion that can pose a challenge to the pathologist despite thorough clinical workup. Although several immunohistochemical markers have been proposed for iCCA, none of them reached clinical practice. We here assessed the combined usage of two promising diagnostic approaches, albumin in situ hybridisation (Alb-ISH) and C reactive protein (CRP) immunohistochemistry, for distinguishing iCCA from other adenocarcinoma primaries. METHODS: We conducted Alb-ISH and CRP immunohistochemistry in a large European iCCA cohort (n=153) and compared the results with a spectrum of other glandular adenocarcinomas of different origin (n=885). In addition, we correlated expression patterns with clinicopathological information and mutation data. RESULTS: Alb-ISH was highly specific for iCCA (specificity 98.8%) with almost complete negativity in perihilar CCA and only rare positives among other adenocarcinomas (sensitivity 69.5%). CRP identified the vast majority of iCCA cases (sensitivity 84.1%) at a lower specificity of 86.4%. Strikingly, the combination of CRP and Alb-ISH boosted the diagnostic sensitivity to 88.0% while retaining a considerable specificity of 86.1%. Alb-ISH significantly correlated with CRP expression, specific tumour morphologies and small or large duct iCCA subtypes. Neither Alb-ISH nor CRP was associated with iCCA patient survival. 16 of 17 recurrent mutations in either IDH1, IDH2 and FGFR2 affected Alb-ISH positive cases, while the only KRAS mutation corresponded to an Alb-ISH negative case. CONCLUSIONS: In conclusion, we propose a sequential diagnostic approach for iCCA, integrating CRP immunohistochemistry and Alb-ISH. This may improve the accuracy of CCA classification and pave the way towards a molecular-guided CCA classification.

Deep Learning-Enabled Diagnosis of Liver Adenocarcinoma.

BACKGROUND & AIMS: Diagnosis of adenocarcinoma in the liver is a frequent scenario in routine pathology and has a critical impact on clinical decision making. However, rendering a correct diagnosis can be challenging, and often requires the integration of clinical, radiologic, and immunohistochemical information. We present a deep learning model (HEPNET) to distinguish intrahepatic cholangiocarcinoma from colorectal liver metastasis, as the most frequent primary and secondary forms of liver adenocarcinoma, with clinical grade accuracy using H&E-stained whole-slide images. METHODS: HEPNET was trained on 714,589 image tiles from 456 patients who were randomly selected in a stratified manner from a pool of 571 patients who underwent surgical resection or biopsy at Heidelberg University Hospital. Model performance was evaluated on a hold-out internal test set comprising 115 patients and externally validated on 159 patients recruited at Mainz University Hospital. RESULTS: On the hold-out internal test set, HEPNET achieved an area under the receiver operating characteristic curve of 0.994 (95% CI, 0.989-1.000) and an accuracy of 96.522% (95% CI, 94.521%-98.694%) at the patient level. Validation on the external test set yielded an area under the receiver operating characteristic curve of 0.997 (95% CI, 0.995-1.000), corresponding to an accuracy of 98.113% (95% CI, 96.907%-100.000%). HEPNET surpassed the performance of 6 pathology experts with different levels of experience in a reader study of 50 patients (P = .0005), boosted the performance of resident pathologists to the level of senior pathologists, and reduced potential downstream analyses. CONCLUSIONS: We provided a ready-to-use tool with clinical grade performance that may facilitate routine pathology by rendering a definitive diagnosis and guiding ancillary testing. The incorporation of HEPNET into pathology laboratories may optimize the diagnostic workflow, complemented by test-related labor and cost savings.

Spatiotemporal analysis of tumour-infiltrating immune cells in biliary carcinogenesis.

BACKGROUND: Intraductal papillary neoplasms (IPN) and biliary epithelial neoplasia (BilIN) are well-defined precursor lesions of biliary tract carcinoma (BTC). The aim of this study was to provide a comprehensive characterisation of the inflammatory microenvironment in BTC precursor lesions. METHODS: Immunohistochemistry was employed to assess tumour-infiltrating immune cells in tissue samples from patients, for whom precursor lesions were identified alongside invasive BTC. The spatiotemporal evolution of the immune microenvironment during IPN-associated carcinogenesis was comprehensively analysed using triplet sample sets of non-neoplastic epithelium, precursor lesion and invasive BTC. Immune-cell dynamics during IPN- and BilIN-associated carcinogenesis were subsequently compared. RESULTS: Stromal CD3+ (P = 0.002), CD4+ (P = 0.007) and CD8+ (P < 0.001) T cells, CD20+ B cells (P = 0.008), MUM1+ plasma cells (P = 0.012) and CD163+ M2-like macrophages (P = 0.008) significantly decreased in IPN compared to non-tumorous biliary epithelium. Upon transition from IPN to invasive BTC, stromal CD68+ (P = 0.001) and CD163+ (P < 0.001) macrophages significantly increased. In contrast, BilIN-driven carcinogenesis was characterised by significant reduction of intraepithelial CD8+ T-lymphocytic infiltration from non-tumorous epithelium via BilIN (P = 0.008) to BTC (P = 0.004). CONCLUSION: IPN and BilIN are immunologically distinct entities that undergo different immune-cell variations during biliary carcinogenesis. Intraepithelial CD8+ T-lymphocytic infiltration of biliary tissue decreased already at the IPN-precursor stage, whereas BilIN-associated carcinogenesis showed a slowly progressing reduction towards invasive carcinoma.

Integrative analysis reveals early and distinct genetic and epigenetic changes in intraductal papillary and tubulopapillary cholangiocarcinogenesis.

OBJECTIVE: A detailed understanding of the molecular alterations in different forms of cholangiocarcinogenesis is crucial for a better understanding of cholangiocarcinoma (CCA) and may pave the way to early diagnosis and better treatment options. DESIGN: We analysed a clinicopathologically well-characterised patient cohort (n=54) with high-grade intraductal papillary (IPNB) or tubulopapillary (ITPN) neoplastic precursor lesions of the biliary tract and correlated the results with an independent non-IPNB/ITPN associated CCA cohort (n=294). The triplet sample set of non-neoplastic biliary epithelium, precursor and invasive CCA was analysed by next generation sequencing, DNA copy number and genome-wide methylation profiling. RESULTS: Patients with invasive CCA arising from IPNB/ITPN had better prognosis than patients with CCA not associated with IPNB/ITPN. ITPN was localised mostly intrahepatic, whereas IPNB was mostly of extrahepatic origin. IPNB/ITPN were equally associated with small-duct and large-duct type intrahepatic CCA. IPNB exhibited mutational profiles of extrahepatic CCA, while ITPN had significantly fewer mutations. Most mutations were shared between precursor lesions and corresponding invasive CCA but ROBO2 mutations occurred exclusively in invasive CCA and CTNNB1 mutations were mainly present in precursor lesions. In addition, IPNB and ITPN differed in their DNA methylation profiles and analyses of latent methylation components suggested that IPNB and ITPN may have different cells-of-origin. CONCLUSION: Integrative analysis revealed that IPNB and ITPN harbour distinct early genetic alterations, IPNB are enriched in mutations typical for extrahepatic CCA, whereas ITPN exhibited few genetic alterations and showed distinct epigenetic profiles. In conclusion, IPNB/ITPN may represent a distinctive, intermediate form of intrahepatic and extrahepatic cholangiocarcinogenesis.

COVID-19 pneumonia and its lookalikes: How radiologists perform in differentiating atypical pneumonias.

PURPOSE: To examine the performance of radiologists in differentiating COVID-19 from non-COVID-19 atypical pneumonia and to perform an analysis of CT patterns in a study cohort including viral, fungal and atypical bacterial pathogens. METHODS: Patients with positive RT-PCR tests for COVID-19 pneumonia (n = 90) and non-COVID-19 atypical pneumonia (n = 294) were retrospectively included. Five radiologists, blinded to the pathogen test results, assessed the CT scans and classified them as COVID-19 or non-COVID-19 pneumonia. For both groups specific CT features were recorded and a multivariate logistic regression model was used to calculate their ability to predict COVID-19 pneumonia. RESULTS: The radiologists differentiated between COVID-19 and non-COVID-19 pneumonia with an overall accuracy, sensitivity, and specificity of 88% ± 4 (SD), 79% ± 6 (SD), and 90% ± 6 (SD), respectively. The percentage of correct ratings was lower in the early and late stage of COVID-19 pneumonia compared to the progressive and peak stage (68 and 71% vs 85 and 89%). The variables associated with the most increased risk of COVID-19 pneumonia were band like subpleural opacities (OR 5.55, p < 0.001), vascular enlargement (OR 2.63, p = 0.071), and subpleural curvilinear lines (OR 2.52, p = 0.021). Bronchial wall thickening and centrilobular nodules were associated with decreased risk of COVID-19 pneumonia with OR of 0.30 (p = 0.013) and 0.10 (p < 0.001), respectively. CONCLUSIONS: Radiologists can differentiate between COVID-19 and non-COVID-19 atypical pneumonias at chest CT with high overall accuracy, although a lower performance was observed in the early and late stage of COVID 19 pneumonia. Specific CT features might help to make the correct diagnosis.

HER2 gene (ERBB2) amplification is a low-frequency driver with potential predictive value in gallbladder carcinoma.

Gallbladder carcinoma (GBC) is an aggressive type of cancer with a dismal prognosis. Recent case reports have highlighted the human epidermal growth factor receptor 2 (HER2) as a promising target for individualized therapy in biliary tract cancer; however, current data on HER2 positivity in GBC is contradictory. This study aimed to assess the proportion of HER2 positivity and its clinical implications in a large and well-characterized European GBC cohort. HER2 status was determined in 186 cases of surgically resected gallbladder adenocarcinoma and a subset of coexistent high-grade biliary intraepithelial neoplasia (BilIN, n = 74) in accordance with the up-to-date consensus for HER2 testing in gastric cancer by immunohistochemistry and dual-color chromogenic in situ hybridization. Positivity for HER2 was observed in 5.4% of all cases (n = 10). In those patients with concomitant high-grade BilIN, two of four positive samples also showed amplification in the precursor lesion, while in the two remaining cases, positivity was either confined to invasive tumor or high-grade BilIN, exclusively. Equivocal staining found in eleven cases was not accompanied by gene amplification. Staging of the HER2-positive group was significantly different from the HER2-negative group with most cases presenting at stage IV, paralleled by a trend towards decreased survival. One patient who received dual HER2 inhibition almost went into full clinical remission despite treatment initiation in a metastasized state. Our results reveal a low prevalence of HER2 positivity and highlight HER2 gene amplification as an early, potentially driving event in gallbladder carcinogenesis. Prospective standardized HER2 testing and randomized control studies are needed to prove clinical efficacy of targeted HER2 inhibition in GBC.

HER2 gene (ERBB2) amplification is a rare event in non-liver-fluke associated cholangiocarcinogenesis.

BACKGROUND: Cholangiocarcinoma is a rapidly fatal cancer entity with a median survival of less than one year. In contrast to many other malignancies, no substantial therapeutic breakthrough has been made in the past few decades, thereby limiting the treatment to cytotoxic chemotherapy with little beneficial effect for most patients. Targeted therapy tailored to the individual has shown substantial success in the recent past as a promising avenue for cancer therapy. METHODS: In this study, we determined the frequency of amplification of the HER2 gene in a comprehensive and well-characterized European cholangiocarcinoma cohort encompassing 436 patients including intrahepatic (n = 155), proximal (n = 155) and distal (n = 126) cholangiocarcinoma by strict application of a combined immunohistochemical and in situ hybridization algorithm following the current guidelines for HER2 assessment in gastric cancer. RESULTS: We identified a proportion of 1.4% (n = 6) patients that demonstrated HER2 gene amplification, with the highest rate among the distal cholangiocarcinoma patients (2.4%). None of the patients with equivocal (2+) immunohistochemical staining results exhibited gene amplification molecularly. In four of the five patients with HER2 positivity, gene amplification was already present in concomitantly tested high-grade biliary intraepithelial neoplasia (80%). HER2 gene amplification was not significantly associated with other clinical parameters, including survival. CONCLUSIONS: This study identifies HER2 gene amplification as a rare event in cholangiocarcinoma of the Western population, occurring already in high-grade BilIN in a subset of patients. Furthermore, we provide a robust testing algorithm that may be used prior to therapy administration in future clinical trials evaluating the role of HER2 as a predictive marker in cholangiocarcinoma.

Low frequency of mismatch repair deficiency in gallbladder cancer.

BACKGROUND: DNA mismatch repair (MMR) deficiency is a major pathway of genomic instability in cancer. It leads to the accumulation of numerous mutations predominantly at microsatellite sequences, a phenotype known as microsatellite instability (MSI). MSI tumors have a distinct clinical behavior and commonly respond well to immune checkpoint blockade, irrespective of their origin. Data about the prevalence of MSI among gallbladder cancer (GBC) have been conflicting. We here analyzed a well-characterized cohort of 69 Western-world GBCs. METHODS: We analyzed the mononucleotide MSI marker panel consisting of BAT25, BAT26, and CAT25 to determine the prevalence of MMR deficiency-induced MSI. RESULTS: MSI was detected in 1/69 (1.4%) of analyzed GBCs. The detected MSI GBC had a classical histomorphology, i.e. of acinar/tubular/glandular pancreatobiliary phenotype, and showed nuclear expression of all four MMR proteins MLH1, MSH2, MSH6, and PMS2. The MSI GBC patient showed a prolonged overall survival, despite having a high tumor stage at diagnosis. The patient had no known background or family history indicative of Lynch syndrome. CONCLUSIONS: Even though the overall number of MSI tumors is low in GBC, the potentially therapeutic benefit of checkpoint blockade in the respective patients may justify MSI analysis of GBC.

Programmed cell death ligand 1 (PD-L1, CD274) in cholangiocarcinoma - correlation with clinicopathological data and comparison of antibodies.

BACKGROUND: Cholangiocarcinoma (CCA) may arise in the intra- or extrahepatic biliary tract and is associated with a poor prognosis. Despite recent advances, to date there is still no established targeted therapeutic approach available. Non-surgical therapeutic agents are urgently needed, as most patients are non-eligible to surgical resection. Anti-PD-L1 therapy prevents cancer cells from evading the immune system and has emerged as a new treatment option in several cancer entities. Recently, PD-L1 expression has been analyzed in comparably small CCA patient cohorts. However, a systematic validation of different PD-L1 antibodies has not been performed in CCA so far. METHODS: We stained a tissue microarray consisting of 170 patients, including 72 intrahepatic cholangiocarcinomas (iCCAs), 57 perihilar cholangiocarcinomas (pCCAs) and 41 distal cholangiocarcinomas (dCCAs) by immunohistochemistry and evaluated PD-L1 positivity in tumor and stromal cells. We analyzed three different PD-L1 antibodies (clones 28-8, SP142, and SP263) that are frequently used and recommended for predictive diagnostic testing in other cancer types. RESULTS: For PD-L1 antibody clone SP263, 5% of iCCAs, 4% of pCCAs and 3% of dCCAs exhibited PD-L1 expression on tumor cells, thereby showing the highest frequencies of PD-L1 positivity. Accordingly, highest PD-L1 positivity rates of stromal cells with 31% in iCCA, 40% in pCCA and 61% in dCCA were detected for clone SP263. Agreement of PD-L1 positivity in tumor cells was moderate for clone 28-8 and SP263 (κ = 0.44) and poor between 28-8 and SP142 (κ = 0.13), as well as  SP142 and SP263 (κ = 0.11), respectively. Statistical analyses of PD-L1 expression (clone SP263) on tumor cells with clinicopathological data revealed a positive correlation with shortened overall survival in CCA patients. CONCLUSIONS: Selection of appropriate PD-L1 antibodies and careful evaluation of immunohistochemical staining patterns have a significant impact on PD-L1 testing in CCA. Clinical trials are necessary to investigate the putative beneficial effects of PD-L1 targeted immunotherapy in CCA patients.

[Spindle and giant cell type undifferentiated carcinoma of the distal bile duct: a case report]. / Undifferenziertes Karzinom des distalen Gallengangs vom Spindel- und Riesenzelltyp: Ein Fallbericht.

Undifferentiated carcinoma of the biliary tract is rare and more frequently occurs in the gall bladder than in the extrahepatic bile ducts. We report on an extremely rare case of a spindle and giant cell type undifferentiated carcinoma of the distal bile duct. In the presented tumor, atypical glands and single-cell clusters arising from the bile duct epithelium gradually transitioned into a predominantly sarcomatoid architecture, which constituted more than 98 % of the whole tumor volume. Focally, osteoclast-like giant cells were intermixed with the spindle cells. The tumor showed a high proliferation activity (Ki-67) and was demonstrated to harbor mutations in the genes for cyclin D3 (CCND3), fibroblast growth factor receptor 4 (FGFR4), neurofibromin 1 (NF1) and NOTCH3, as assessed by next-generation sequencing analysis. The presented case underscores the relevance of this tumor entity beyond the pancreas and gall bladder and emphasizes the indispensable combination of morphology and immunohistochemistry regarding the diagnostic process.

Mismatch repair deficiency is a rare but putative therapeutically relevant finding in non-liver fluke associated cholangiocarcinoma.

BACKGROUND: A major molecular pathway of genetic instability in cancer is DNA mismatch repair deficiency. High-level microsatellite instability (MSI-H) is currently the best predictor of responsiveness towards immune checkpoint blockade. Data about the prevalence of high-level microsatellite instability in cholangiocarcinoma (CCA) has been conflicting. METHODS: We employed a cohort comprising 308 Western-world, non-liver fluke-associated CCAs (159 intrahepatic, 106 perihilar, and 43 distal). We analysed the mononucleotide microsatellite instability marker panel consisting of BAT25, BAT26, and CAT25 and detected MSI-H in 4/308 CCAs (1.3%). RESULTS: Patients affected by MSI-H CCA had mostly an atypical histomorphology (p = 0.004), showed a longer overall survival, although having a high tumour stage, and were of younger age. Correlation analysis of microsatellite instability status with tumour-infiltrating immune cells, MHC I, and PD-L1 expression in the same cholangiocarcinoma cohort showed higher numbers of CD8 + T cells, FOXP3 + regulatory T cells, CD20 + B cells and high or at least moderate MHC I expression levels in MSI-H CCAs. CONCLUSIONS: Even though the overall number of MSI-H CCAs is low, the dismal prognosis of the disease and the therapeutic option of immune checkpoint blockade in the respective patients justify MSI testing of cholangiocarcinoma, particularly in younger patients showing an atypical histomorphology.

BRAF V600E-specific immunohistochemistry reveals low mutation rates in biliary tract cancer and restriction to intrahepatic cholangiocarcinoma.

BRAF mutations have emerged as an important predictive biomarker for metastasized melanoma. Other types of cancer may also benefit from BRAF mutation-targeted therapies. In biliary tract cancer, reported BRAF mutation rates are highly controversial, ranging from 0 to 33% in adenocarcinoma of the gallbladder and 0 to 22% in cholangiocarcinoma. We here analyzed tissue microarrays of a large cohort of biliary tract cancer (n=377) including 159 intrahepatic cholangiocarcinomas, 149 extrahepatic cholangiocarcinomas, and 69 adenocarcinomas of the gallbladder for BRAF V600E mutation using a highly sensitive immunohistochemical screening approach implementing the BRAF V600E protein-specific antibody VE1. All VE1-positive cases as well as 42 VE1-negative cases were additionally analyzed by Sanger sequencing. In total, only 5 VE1-positive cases were detected (5/377; 1%). BRAF V600E mutation was confirmed by direct sequencing in all cases. All 5 mutated cases were intrahepatic cholangiocarcinomas (5/159; 3%). None of the extrahepatic cholangiocarcinomas and adenocarcinomas of the gallbladder were VE1 positive. Apart from the subtype restriction of BRAF V600E mutation to intrahepatic cholangiocarcinoma and a female predominance (4 female, 1 male), no significant correlation with clinicopathological data and patient outcome was detected. In conclusion, we demonstrate that BRAF V600E mutation is a rare event in biliary tract cancer, accounting for only 1% of all subtypes, and is restricted to intrahepatic cholangiocarcinoma. In addition, we demonstrate that VE1 immunohistochemistry is a feasible approach to routinely screen for BRAF V600E mutation in biliary tract cancer patients, thereby facilitating the detection of rare patients who may benefit from BRAF mutation-targeted therapies.

Differential expression of the tumor suppressor A-kinase anchor protein 12 in human diffuse and pilocytic astrocytomas is regulated by promoter methylation.

The scaffold protein A-kinase anchor protein 12 (AKAP12) exerts tumor suppressor activity and is downregulated in several tumor entities. We characterized AKAP12 expression and regulation in astrocytomas, including pilocytic and diffusely infiltrating astrocytomas. We examined 194 human gliomas and 23 normal brain white matter samples by immunohistochemistry or immunoblotting for AKAP12 expression. We further performed quantitative methylation analysis of the AKAP12 promoter by MassARRAY® of normal brain, World Health Organization (WHO) grade I to IV astrocytomas, and glioma cell lines. Our results show that AKAP12 is expressed in a perivascular distribution in normal CNS, strongly upregulated in tumor cells in pilocytic astrocytomas, and weakly expressed in diffuse astrocytomas of WHO grade II to IV. Methylation analyses revealed specific hypermethylation of AKAP12α promoter in WHO grade II to IV astrocytomas. Restoration experiments using 5-aza-2'-deoxycytidine in primary glioblastoma cells decreased AKAP12α promoter methylation and markedly increased AKAP12α mRNA levels. In summary, we demonstrate that AKAP12 is differentially expressed in human astrocytomas showing high expression in pilocytic but low expression in diffuse astrocytomas of all WHO-grades. Our results further indicate that epigenetic mechanisms are involved in silencing AKAP12 in diffuse astrocytomas; however, a tumor suppressive role of AKAP12 in distinct astrocytoma subtypes remains to be determined.

Follicle-stimulating hormone receptor expression in soft tissue sarcomas.

AIMS: In adult humans, the follicle-stimulating hormone receptor (FSHR) is expressed only in the granulosa cells of the ovary and the Sertoli cells of the testis. Recently, it has been shown that FSHR is expressed selectively on the surface of blood vessels in a wide range of tumours. So far, the expression of FSHR in mesenchymal tumours has not been studied. METHODS AND RESULTS: We performed a semiquantitative evaluation of FSHR protein expression in a large cohort of soft tissue sarcomas (STS; n = 335), including 11 subtypes. FSHR-positive vessels were detected in all sarcoma subtypes analysed. Among liposarcomas, significantly more cases of dedifferentiated liposarcomas (28 of 44) showed FSHR expression compared to well-differentiated liposarcomas (WDLS; four of 21; P < 0.001). Vessels in lipomas (n = 9) and non-neoplastic fat were FSHR-negative. FSHR expression was also detected in tumour cells of all sarcoma subtypes examined, with the lowest incidence in WDLS (three of 21; 14.3%) and the highest frequency in undifferentiated high-grade pleomorphic sarcomas (41 of 60; 68.3%). CONCLUSIONS: These data supplement the previously reported results of FSHR expression in endothelial cells of various cancer types and form a solid basis for further studies of FSHR in mesenchymal neoplasms.

Noncompaction myocardium in association with type Ib glycogen storage disease.

Noncompaction myocardium is a rare disorder assumed to occur as an arrest of the compaction process during the normal development of the heart. Left ventricular noncompaction has been reported to be associated with a variety of cardiac and extracardiac, especially neuromuscular abnormalities. Moreover, it has been suggested that metabolic alterations could be responsible for the noncompaction. However, no association of noncompaction myocardium with type Ib glycogen storage disease (GSD) has been reported so far. Type Ib GSD is due to a defect of a transmembrane protein which results, similar to type Ia GSD, in hypoglycemia, a markedly enlarged liver and, additionally, in neutropenia, recurrent infections, and inflammatory bowel disease. Until now, no muscular or cardiac involvement has been described in type Ib GSD patients. The present case represents the first report of a noncompaction myocardium in a child with type Ib GSD who died of sudden clinical deterioration at the age of four.

Down-regulation of tumor suppressor A kinase anchor protein 12 in human hepatocarcinogenesis by epigenetic mechanisms.

UNLABELLED: The A kinase anchor protein 12 (AKAP12) is a central mediator of protein kinase A and protein kinase C signaling. Although AKAP12 has been described to act as a tumor suppressor and its expression is frequently down-regulated in several human malignancies, the underlying molecular mechanisms responsible for the AKAP12 reduction are poorly understood. We therefore analyzed the expression of AKAP12 and its genetic and epigenetic regulatory mechanisms in human hepatocarcinogenesis. Based on tissue microarray analyses (n = 388) and western immunoblotting, we observed a significant reduction of AKAP12 in cirrhotic liver (CL), premalignant lesions (DN), and hepatocellular carcinomas (HCCs) compared to histologically normal liver specimens (NL). Analyses of array comparative genomic hybridization data (aCGH) from human HCCs revealed chromosomal losses of AKAP12 in 36% of cases but suggested additional mechanisms underlying the observed reduction of AKAP12 expression in hepatocarcinogenesis. Quantitative methylation analysis by MassARRAY of NL, CL, DN, and HCC tissues, as well as of various tumorigenic and nontumorigenic liver cell lines revealed specific hypermethylation of the AKAP12α promoter but not of the AKAP12ß promoter in HCC specimens and in HCC cell lines. Consequently, restoration experiments performed with 5-aza-2'deoxycytidine drastically increased AKAP12α mRNA levels in a HCC cell line (AKN1) paralleled by AKAP12α promoter demethylation. As hypermethylation is not observed in CL and DN, we investigated microRNA-mediated posttranscriptional regulation as an additional mechanism to explain reduced AKAP12 expression. We found that miR-183 and miR-186 are up-regulated in CL and DN and are able to target AKAP12. CONCLUSION: In addition to genetic alterations, epigenetic mechanisms are responsible for the reduction of the tumor suppressor gene AKAP12 in human hepatocarcinogenesis.