Huang, Bing Q

Abstract Purpose: This phase Ib/2 trial investigated pembrolizumab-containing trimodality therapy in patients with gastroesophageal junction (GEJ) adenocarcinoma. Patients and Methods: Patients with GEJ adenocarcinoma (cT1–3NanyM0) received neoadjuvant pembrolizumab-containing chemoradiation (CROSS regimen) followed by surgical resection and adjuvant pembrolizumab. The primary endpoints were tolerability in the first 16 patients and pathologic complete response [pCR (ypT0N0)]. Secondary endpoints included progression-free survival (PFS) and overall survival (OS). An independent propensity-score-matched cohort (treated with CROSS without immunotherapy) was used for comparison. Exploratory analyses included immune biomarkers in the tumor microenvironment (TME) and plasma. Results: We enrolled 31 eligible patients, of whom 29 received all expected doses of neoadjuvant pembrolizumab and 28 underwent R0 resection. Safety endpoints were met. The primary efficacy endpoint was not met [7/31 (22.6%) achieved pCR]. Patients with high [i.e., combined positive score (CPS) ≥ 10] baseline expression of programmed death (PD)-L1 in the TME had a significantly higher pCR rate than those with low expression [50.0% (4/8) vs. 13.6% (3/22); P = 0.046]. Patients with high PD-L1 expression also experienced longer PFS and OS than propensity-score-matched patients. Among trial patients with PD-L1 CPS < 10, unprespecified analysis explored whether extracellular vesicles (EV) could identify further responders: an elevated plasma level of PD-L1–expressing EVs was significantly associated with higher pCR. Conclusions: Adding pembrolizumab to trimodality therapy showed acceptable tolerability but did not meet the pre-specified pCR endpoint. Exploratory analyses suggested that high PD-L1 expression in the TME and/or on EVs may identify patients most likely to achieve tumor response.

AbstractExtracellular vesicles (EVs) are of growing interest due to their potential diagnostic, disease surveillance, and therapeutic applications. While several studies have evaluated EV isolation methods in various biofluids, there are few if any data on these techniques when applied to stool. The latter is an ideal biospecimen for studying EVs and colorectal cancer (CRC) because the release of tumour markers by luminal exfoliation into stool occurs earlier than vascular invasion. Since EV release is a conserved mechanism, bacteria in stool contribute to the overall EV population. In this study, we assessed five EV separation methods (ultracentrifugation [UC], precipitation [EQ‐O, EQ‐TC], size exclusion chromatography [SEC], and ultrafiltration [UF]) for total recovery, reproducibility, purity, RNA composition, and protein expression in stool supernatant. CD63, TSG101, and ompA proteins were present in EV fractions from all methods except UC. Human (18s) and bacterial (16s) rRNA was detected in stool EV preparations. Enzymatic treatment prior to extraction is necessary to avoid non‐vesicular RNA contamination. Ultrafiltration had the highest recovery, RNA, and protein yield. After assessing purity further, SEC was the isolation method of choice. These findings serve as the groundwork for future studies that use high throughput omics technologies to investigate the potential of stool‐derived EVs as a source for novel biomarkers for early CRC detection.

Abstract Background & Aims Polycystic liver diseases (PLDs) are genetic disorders characterized by progressive development of multiple biliary cysts. Recently, novel PLD‐causative genes, encoding for endoplasmic reticulum (ER)‐resident proteins involved in protein biogenesis and transport, were identified. We hypothesized that aberrant proteostasis contributes to PLD pathogenesis, representing a potential therapeutic target. Methods ER stress was analysed at transcriptional (qPCR), proteomic (mass spectrometry), morphological (transmission electron microscopy, TEM) and functional (proteasome activity) levels in different PLD models. The effect of ER stress inhibitors [4‐phenylbutyric acid (4‐PBA)] and/or activators [tunicamycin (TM)] was tested in polycystic (PCK) rats and cystic cholangiocytes in vitro. Results The expression levels of unfolded protein response (UPR) components were upregulated in liver tissue from PLD patients and PCK rats, as well as in primary cultures of human and rat cystic cholangiocytes, compared to normal controls. Cystic cholangiocytes showed altered proteomic profiles, mainly related to proteostasis (ie synthesis, folding, trafficking and degradation of proteins), marked enlargement of the ER lumen (by TEM) and hyperactivation of the proteasome. Notably, chronic treatment of PCK rats with 4‐PBA decreased liver weight, as well as both liver and cystic volumes, of animals under baseline conditions or after TM administration compared to controls. In vitro, 4‐PBA downregulated the expression (mRNA) of UPR effectors, normalized proteomic profiles related to protein synthesis, folding, trafficking and degradation and reduced the proteasome hyperactivity in cystic cholangiocytes, reducing their hyperproliferation and apoptosis. Conclusions Restoration of proteostasis in cystic cholangiocytes with 4‐PBA halts hepatic cystogenesis, emerging as a novel therapeutic strategy.

Polycystic liver disease (PLD) is a group of genetic disorders with limited treatment options and significant morbidity. Hepatic cysts arise from cholangiocytes exhibiting a hyperproliferative phenotype. Considering that hyperproliferation of many cell types is associated with alterations in autophagy, we hypothesized that autophagy is altered in PLD cholangiocytes, contributes to hepatic cystogenesis, and might represent a potential therapeutic target. We employed functional pathway cluster analysis and next‐generation sequencing, transmission electron microscopy, immunofluorescence confocal microscopy, and western blotting to assess autophagy in human and rodent PLD cholangiocytes. A three‐dimensional culture model was used to study the effects of molecular and pharmacologic inhibition of autophagy on hepatic cystogenesis in vitro , and the polycystic kidney disease–specific rat, an animal model of PLD, to study the effects of hydroxychloroquine, a drug that interferes with the autophagy pathway, on disease progression in vivo . Assessment of the transcriptome of PLD cholangiocytes followed by functional pathway cluster analysis revealed that the autophagy–lysosomal pathway is one of the most altered pathways in PLD. Direct evaluation of autophagy in PLD cholangiocytes both in vitro and in vivo showed increased number and size of autophagosomes, lysosomes, and autolysosomes; overexpression of autophagy‐related proteins (Atg5, Beclin1, Atg7, and LC3); and enhanced autophagic flux associated with activation of the cAMP–protein kinase A–cAMP response element–binding protein signaling pathway. Molecular and pharmacologic intervention in autophagy with ATG7 small interfering RNA, bafilomycin A 1 , and hydroxychloroquine reduced proliferation of PLD cholangiocytes in vitro and growth of hepatic cysts in three‐dimensional cultures. Hydroxychloroquine also efficiently inhibited hepatic cystogenesis in the polycystic kidney disease–specific rat. Conclusion: Autophagy is increased in PLD cholangiocytes, contributes to hepatic cystogenesis, and represents a potential therapeutic target for disease treatment. (H epatology 2018;67:1088–1108)

Hepatic cystogenesis in polycystic liver disease is associated with increased levels of cyclic adenosine monophosphate (cAMP) in cholangiocytes lining liver cysts. Takeda G protein receptor 5 (TGR5), a G protein–coupled bile acid receptor, is linked to cAMP and expressed in cholangiocytes. Therefore, we hypothesized that TGR5 might contribute to disease progression. We examined expression of TGR5 and Gα proteins in cultured cholangiocytes and in livers of animal models and humans with polycystic liver disease. In vitro, we assessed cholangiocyte proliferation, cAMP levels, and cyst growth in response to (1) TGR5 agonists (taurolithocholic acid, oleanolic acid [OA], and two synthetic compounds), (2) a novel TGR5 antagonist (m‐tolyl 5‐chloro‐2‐[ethylsulfonyl] pyrimidine‐4‐carboxylate [SBI‐115]), and (3) a combination of SBI‐115 and pasireotide, a somatostatin receptor analogue. In vivo, we examined hepatic cystogenesis in OA‐treated polycystic kidney rats and after genetic elimination of TGR5 in double mutant TGR5−/− ;Pkhd1del2/del2 mice. Compared to control, expression of TGR5 and Gαs (but not Gαi and Gαq) proteins was increased 2‐fold to 3‐fold in cystic cholangiocytes in vitro and in vivo. In vitro, TGR5 stimulation enhanced cAMP production, cell proliferation, and cyst growth by ∼40%; these effects were abolished after TGR5 reduction by short hairpin RNA. OA increased cystogenesis in polycystic kidney rats by 35%; in contrast, hepatic cystic areas were decreased by 45% in TGR5‐deficient TGR5−/−;Pkhd1del2/del2 mice. TGR5 expression and its colocalization with Gαs were increased ∼2‐fold upon OA treatment. Levels of cAMP, cell proliferation, and cyst growth in vitro were decreased by ∼30% in cystic cholangiocytes after treatment with SBI‐115 alone and by ∼50% when SBI‐115 was combined with pasireotide. Conclusion: TGR5 contributes to hepatic cystogenesis by increasing cAMP and enhancing cholangiocyte proliferation; our data suggest that a TGR5 antagonist alone or concurrently with somatostatin receptor agonists represents a potential therapeutic approach in polycystic liver disease. (Hepatology 2017;66:1197‐1218).