Heterogeneous immunogenomic features and distinct escape mechanisms in multifocal hepatocellular carcinoma.

BACKGROUND & AIMS: The presence of multifocal tumors, developed either from intrahepatic metastasis (IM) or multicentric occurrence (MO), is a distinct feature of hepatocellular carcinoma (HCC). Immunogenomic characterization of multifocal HCC is important for understanding immune escape in different lesions and developing immunotherapy. METHODS: We combined whole-exome/transcriptome sequencing, multiplex immunostaining, immunopeptidomes, T cell receptor (TCR) sequencing and bioinformatic analyses of 47 tumors from 15 patients with HCC and multifocal lesions. RESULTS: IM and MO demonstrated distinct clonal architecture, mutational spectrum and genetic susceptibility. The immune microenvironment also displayed spatiotemporal heterogeneity, such as less T cell and more M2 macrophage infiltration in IM and higher expression of inhibitory immune checkpoints in MO. Similar to mutational profiles, shared neoantigens and TCR repertoires among tumors from the same patients were abundant in IM but scarce in MO. Combining neoantigen prediction and immunopeptidomes identified T cell-specific neoepitopes and achieved a high verification rate in vitro. Immunoediting mainly occurred in MO but not IM, due to the relatively low immune infiltration. Loss of heterozygosity of human leukocyte antigen (HLA) alleles, identified in 17% of multifocal HCC, hampered the ability of major histocompatibility complex to present neoantigens, especially in IM. An integrated analysis of Immunoscore, immunoediting, TCR clonality and HLA loss of heterozygosity in each tumor could stratify patients into 2 groups based on whether they have a high or low risk of recurrence (p = 0.038). CONCLUSION: Our study comprehensively characterized the genetic structure, neoepitope landscape, T cell profile and immunoediting status that collectively shape tumor evolution and could be used to optimize personalized immunotherapies for multifocal HCC. LAY SUMMARY: Immunogenomic features of multifocal hepatocellular carcinoma (HCC) are important for understanding immune-escape mechanisms and developing more effective immunotherapy. Herein, comprehensive immunogenomic characterization showed that diverse genomic structures within multifocal HCC would leave footprints on the immune landscape. Only a few tumors were under the control of immunosurveillance, while others evaded the immune system through multiple mechanisms that led to poor prognosis. Our study revealed heterogeneous immunogenomic landscapes and immune-constrained tumor evolution, the understanding of which could be used to optimize personalized immunotherapies for multifocal HCC.

Spatial and temporal clonal evolution of intrahepatic cholangiocarcinoma.

BACKGROUND & AIMS: Intrahepatic cholangiocarcinoma (ICC) is the second-most lethal primary liver cancer. Little is known about intratumoral heterogeneity (ITH) and its impact on ICC progression. We aimed to investigate the ITH of ICC in the hope of helping to develop new therapeutic strategies. METHODS: We obtained 69 spatially distinct regions from six operable ICCs. Patient-derived primary cancer cells (PDPCs) were established for each region, followed by whole-exome sequencing (WES) and multi-level validation. RESULTS: We observed widespread ITH for both somatic mutations and clonal architecture, shaped by multiple mechanisms, like clonal "illusion", parallel evolution and chromosome instability. A median of 60.3% of mutations were heterogeneous, among which 85% of the driver mutations were located on the branches of tumor phylogenetic trees. Many truncal and clonal driver mutations occurred in tumor suppressor genes, such as TP53, SMARCB1 and PBRM1 that are involved in DNA repair and chromatin-remodeling. Genome doubling occurred in most cases (5/6) after the accumulation of truncal mutations and was shared by all intratumoral sub-regions. In all cases, ongoing chromosomal instability is evident throughout the evolutionary trajectory of ICC. The recurrence of ICC1239 provided evidence to support the polyclonal metastatic seeding in ICC. The change of mutation landscape and internal diversity among subclones during metastasis, such as the loss of chemoresistance mediator, can be used for new treatment strategies. Targeted therapy against truncal alterations, such as IDH1, JAK1, and KRAS mutations and EGFR amplification, was developed in 5/6 patients. CONCLUSIONS: Integrated investigations of spatial ITH and clonal evolution may provide an important molecular foundation for enhanced understanding of tumorigenesis and progression in ICC. LAY SUMMARY: We applied multiregional whole-exome sequencing to investigate the evolution of intrahepatic cholangiocarcinoma (ICC). The results revealed that many factors, such as parallel evolution and chromosome instability, may participate and promote the branch diversity of ICC. Interestingly, in one patient with primary and recurrent metastatic tumors, we found evidence of polyclonal metastatic seeding, indicating that symbiotic communities of multiple clones existed and were maintained during metastasis. More realistically, some truncal alterations, such as IDH1, JAK1, and KRAS mutations and EGFR amplification, could be promising treatment targets in patients with ICC.

Kinetic analysis for the isomerization of cellobiose to cellobiulose in subcritical aqueous ethanol.

The isomerization of cellobiose to cellobiulose, and other degradation reactions of cellobiose were investigated in subcritical aqueous ethanol with concentrations of ethanol ranging from 0 to 60% (w/w) and at temperatures ranging from 170 to 200 °C. The maximum yield of cellobiulose (ca. 40%) was obtained from the treatment of cellobiose in 60% (w/w) aqueous ethanol at 190 °C. Glucose and fructose were also detected as byproducts. The concentration-time integral method was employed to analyze the rate constants for the isomerization and degradation processes. The rate constant of cellobiose isomerization to cellobiulose was greater than those of the degradation reactions under all experimental conditions, and it increased significantly with treatment temperature and ethanol concentration. However, the use of higher temperatures and ethanol concentrations was restricted due to decomposition of the saccharides and the low solubility of cellobiose, respectively. The effect of initial feed concentration (0.5-5.5% w/w) was also studied. The maximum productivity of cellobiulose, 54.1 kg/(h m(3)-reactor), was accomplished at a feed concentration of 5.5% (w/w) in 20% (w/w) subcritical aqueous ethanol.

Production of keto-disaccharides from aldo-disaccharides in subcritical aqueous ethanol.

Isomerization of disaccharides (maltose, isomaltose, cellobiose, lactose, melibiose, palatinose, sucrose, and trehalose) was investigated in subcritical aqueous ethanol. A marked increase in the isomerization of aldo-disaccharides to keto-disaccharides was noted and their hydrolytic reactions were suppressed with increasing ethanol concentration. Under any study condition, the maximum yield of keto-disaccharides produced from aldo-disaccharides linked by ß-glycosidic bond was higher than that produced from aldo-disaccharides linked by α-glycosidic bond. Palatinose, a keto-disaccharide, mainly underwent decomposition rather than isomerization in subcritical water and subcritical aqueous ethanol. No isomerization was noted for the non-reducing disaccharides trehalose and sucrose. The rate constant of maltose to maltulose isomerization almost doubled by changing solvent from subcritical water to 80 wt% aqueous ethanol at 220 °C. Increased maltose monohydrate concentration in feed decreased the conversion of maltose and the maximum yield of maltulose, but increased the productivity of maltulose. The maximum productivity of maltulose was ca. 41 g/(h kg-solution).

Production of rare sugars from common sugars in subcritical aqueous ethanol.

A new isomerization reaction was developed to synthesize rare ketoses. D-tagatose, D-xylulose, and D-ribulose were obtained in the maximum yields of 24%, 38%, and 40%, respectively, from the corresponding aldoses, D-galactose, D-xylose, and D-ribose, by treating the aldoses with 80% (v/v) subcritical aqueous ethanol at 180°C. The maximum productivity of D-tagatose was ca. 80 g/(Lh). Increasing the concentration of ethanol significantly increased the isomerization of D-galactose. Variation in the reaction temperature did not significantly affect the production of D-tagatose from D-galactose. Subcritical aqueous ethanol converted both 2,3-threo and 2,3-erythro aldoses to the corresponding C-2 ketoses in high yields. Thus, the treatment of common aldoses in subcritical aqueous ethanol can be regarded as a new method to synthesize the corresponding rare sugars.

Kinetic analysis for the isomerization of glucose, fructose, and mannose in subcritical aqueous ethanol.

Fructose, glucose, and mannose were treated with subcritical aqueous ethanol for ethanol concentrations ranging from 0 to 80% (v/v) at 180-200 °C. The aldose-ketose isomerization was more favorable than ketose-aldose isomerization and glucose-mannose epimerization. The isomerization of the monosaccharides was promoted by the addition of ethanol. In particular, mannose was isomerized most easily to fructose in subcritical aqueous ethanol. The apparent equilibrium constants for the isomerizations of mannose to fructose, Keq,M→F, and glucose to fructose, Keq,G→F, were independent of ethanol concentration and increased with increasing temperature. Moreover, the Keq,M→F value was much larger than the Keq,G→F value. The enthalpies for the isomerization of mannose to fructose, ΔHM→F, and glucose to fructose, ΔHG→F, were estimated to be 18 and 24 kJ/mol, respectively, according to van't Hoff equation. Subcritical aqueous ethanol can be used to produce fructose from glucose and mannose efficiently.

Promotion or suppression of glucose isomerization in subcritical aqueous straight- and branched-chain alcohols.

The influence of water-miscible alcohols (methanol, 1-propanol, 2-propanol, and t-butyl alcohol) on the isomerization of glucose to fructose and mannose was investigated under subcritical aqueous conditions (180-200 °C). Primary and secondary alcohols promoted the conversion and isomerization of glucose to afford fructose and mannose with high and low selectivity, respectively. On the other hand, the decomposition (side-reaction) of glucose was suppressed in the presence of the primary and secondary alcohols compared with that in subcritical water. The yield of fructose increased with increasing concentration of the primary and secondary alcohols, and the species of the primary and secondary alcohols tested had little effect on the isomerization behavior of glucose. In contrast, the isomerization of glucose was suppressed in subcritical aqueous t-butyl alcohol. Both the conversion of glucose and the yield of fructose decreased with increasing concentration of t-butyl alcohol. In addition, mannose was not detected in reactions using subcritical aqueous t-butyl alcohol.

[Prophylaxsis against recurrance of hepatitis B virus after liver transplantation].

OBJECTIVE: To summarize the clinical data in preventing HBV recurrence after liver transplantation and explore a optimal individual protocol in prophylaxis of HBV recurrence. METHODS: We retrospected outcomes in 195 recipients who underwent a liver transplantation for HBV-related liver disease between June 2004 and July 2008. According to the anti-virus protocol these recipients are divided into two groups as following: group A received a protocol of combination treatment of lamivudine with HBIG, and group B with combination treatment of adefovir with HBIG. With mean follow-up of 23.7 months, HBV recurrent rate was observed in overall and each group separately. RESULTS: A total of 195 liver transplant recipients were identified that met the study criteria. At the sixth and eleventh month after operation, HBV recurrence appeared in 2 recipients, each in two groups, which were due to LAM cessation and HBV mutation respectively. Recurrent rate was 0.6% in group A, 3.7% in group B and 1% in total. There was no significant difference in HBV recurrent rate between group A and B. CONCLUSION: Lamivudine combined with HBIg should be considered as a reliable method in preventing HBV recurrence after liver transplantation. Better outcomes can be achieved by individual anti-virus protocol and HBIg administration according to HBV status in recipient.

Expression of mouse liver receptor homologue 1 in embryonic stem cells is directed by a novel promoter.

Liver receptor homologue 1 (LRH-1) plays important roles in many physiological processes and embryogenesis. However, little is known about the developmental regulation of lrh-1 expression. We identified a novel transcript of mouse lrh-1 (mlrh-1v2) from embryonic stem (ES) cells. mlrh-1v2 is expressed throughout embryogenesis and in several adult tissues, while the known transcript (mlrh-1v1) appears later during embryogenesis. mlrh-1v2 expression is directed by a new promoter which displays a strong activity in ES cells. The generation of the new transcript is conserved in rats. The identification of novel mlrh-1 variant and promoter is critical for elucidating LRH-1 functions in development and adult tissues.

Prospero-related homeobox (Prox1) is a corepressor of human liver receptor homolog-1 and suppresses the transcription of the cholesterol 7-alpha-hydroxylase gene.

Cholesterol 7-alpha-hydroxylase (CYP7A1) catalyzes a rate-limiting step in bile acid synthesis in liver, and its gene transcription is under complex regulation by multiple nuclear receptors in response to bile acids, cholesterol derivatives, and hormones. The liver receptor homolog-1 (LRH-1), a member of the fushi tarazu factor 1 subfamily of nuclear receptors, has emerged as an essential regulator for the expression of cyp7a1. In this report, we demonstrate Prox1, a prospero-related homeobox transcription factor, identified through a yeast two-hybrid screening, can directly interact with human LRH-1 (hLRH-1) and suppresses hLRH-1-mediated transcriptional activation of human cyp7a1 gene. Biochemical analysis demonstrates that Prox1 interacts with both the ligand binding domain (LBD) and the DNA binding domain (DBD) of hLRH-1. An LRKLL motif in Prox1 is important for the interaction with the LBD but not the DBD of hLRH-1. In hLRH-1 LBD, helices 2 and 10 are essential for Prox1 recruitment. The suppression by Prox1 on the transcriptional activity of hLRH-1 can be mediated through its interaction with the LBD or the DBD of hLRH-1. Gel shift assays reveal that Prox1 impairs the binding of hLRH-1 to the promoter of human cyp7a1 gene.