Induced pluripotent stem cell (iPSC) modeling validates reduced GBE1 enzyme activity due to a novel variant, p.Ile694Asn, found in a patient with suspected glycogen storage disease IV.

Background: Glycogen Storage disease type 4 (GSD4), a rare disease caused by glycogen branching enzyme 1 (GBE1) deficiency, affects multiple organ systems including the muscles, liver, heart, and central nervous system. Here we report a GSD4 patient, who presented with severe hepatosplenomegaly and cardiac ventricular hypertrophy. GBE1 sequencing identified two variants: a known pathogenic missense variant, c.1544G>A (p.Arg515His), and a missense variant of unknown significance (VUS), c.2081T>A (p. Ile694Asn). As a liver transplant alone can exacerbate heart dysfunction in GSD4 patients, a precise diagnosis is essential for liver transplant indication. To characterize the disease-causing variant, we modeled patient-specific GBE1 deficiency using CRISPR/Cas9 genome-edited induced pluripotent stem cells (iPSCs). Methods: iPSCs from a healthy donor (iPSC-WT) were genome-edited by CRISPR/Cas9 to induce homozygous p.Ile694Asn in GBE1 (iPSC-GBE1-I694N) and differentiated into hepatocytes (iHep) or cardiomyocytes (iCM). GBE1 enzyme activity was measured, and PAS-D staining was performed to analyze polyglucosan deposition in these cells. Results: iPSCGBE1-I694N differentiated into iHep and iCM exhibited reduced GBE1 protein level and enzyme activity in both cell types compared to iPSCwt. Both iHepGBE1-I694N and iCMGBE1-I694N showed polyglucosan deposits correlating to the histologic patterns of the patient's biopsies. Conclusions: iPSC-based disease modeling supported a loss of function effect of p.Ile694Asn in GBE1. The modeling of GBE1 enzyme deficiency in iHep and iCM cell lines had multi-organ findings, demonstrating iPSC-based modeling usefulness in elucidating the effects of novel VUS in ultra-rare diseases.

Neoadjuvant therapy alters the collagen architecture of pancreatic cancer tissue via Ephrin-A5.

BACKGROUND: The treatment of pancreatic cancer (PDAC) remains clinically challenging, and neoadjuvant therapy (NAT) offers down staging and improved surgical resectability. Abundant fibrous stroma is involved in malignant characteristic of PDAC. We aimed to investigate tissue remodelling, particularly the alteration of the collagen architecture of the PDAC microenvironment by NAT. METHODS: We analysed the alteration of collagen and gene expression profiles in PDAC tissues after NAT. Additionally, we examined the biological role of Ephrin-A5 using primary cultured cancer-associated fibroblasts (CAFs). RESULTS: The expression of type I, III, IV, and V collagen was reduced in PDAC tissues after effective NAT. The bioinformatics approach provided comprehensive insights into NAT-induced matrix remodelling, which showed Ephrin-A signalling as a likely pathway and Ephrin-A5 (encoded by EFNA5) as a crucial ligand. Effective NAT reduced the number of Ephrin-A5+ cells, which were mainly CAFs; this inversely correlated with the clinical tumour shrinkage rate. Experimental exposure to radiation and chemotherapeutic agents suppressed proliferation, EFNA5 expression, and collagen synthesis in CAFs. Forced EFNA5 expression altered CAF collagen gene profiles similar to those found in PDAC tissues after NAT. CONCLUSION: These results suggest that effective NAT changes the extracellular matrix with collagen profiles through CAFs and their Ephrin-A5 expression.

Novel insights into immunohistochemical analysis for diagnosing serous neoplasm of the pancreas: aquaporin 1, stereocilin, and transmembrane protein 255B.

AIMS: Serous (cystic) neoplasm (SCN) of the pancreas is generally benign, and surgical treatment is recommended in only a limited number of cases. To avoid unnecessary surgery, an accurate diagnosis of SCN is essential. In the present study, we aimed to identify new immunohistochemical markers with which to distinguish SCN from other tumours. METHODS AND RESULTS: We compared the comprehensive gene expression profiles of SCN with those of normal pancreas and pancreatic ductal adenocarcinoma (PDAC). We selected the candidate molecules that were up-regulated in SCN, were minimally expressed or unexpressed in PDAC, and had specific and available antibodies suitable for immunohistochemistry, and then analysed their immunohistochemical expression in various tumours. We selected aquaporin 1 (AQP1), stereocilin (STRC), fibroblast growth factor receptor 3 (FGFR3), and transmembrane protein 255B (TMEM255B), which were diffusely expressed in SCN cells in 79%, 100%, 100% and 100% of SCN cases. AQP1 was not expressed in other tumours, except in 20% of mucinous cystic neoplasms (MCNs) and 19% of PDACs. STRC was rarely expressed in MCNs, neuroendocrine neoplasms (NENs), and PDACs. FGFR3 was expressed in 31% of intraductal papillary mucinous neoplasms (IPMNs), 50% of intraductal oncocytic papillary neoplasms, 40% of NENs, 30% of acinar cell carcinomas, 40% of solid pseudopapillary neoplasms, and 52% of PDACs. TMEM255B was not expressed in the other tumours, except in 50% of MCNs, 80% of gastric-subtype IPMNs, and 29% of PDACs. All antigens were usually expressed in a small proportion of cells when they were positive in tumours other than SCN. CONCLUSIONS: These findings indicate that AQP1 and STRC, and potentially TMEM255B, may act as SCN markers.

IAP inhibitor, Embelin increases VCAM-1 levels on the endothelium, producing lymphocytic infiltration and antitumor immunity.

There is an increasing unmet need for successful immunotherapeutic interventions. Lymphocyte extravasation via tumor tissue endothelial cells (TECs) is required for lymphocyte infiltration into tumor sites. This study aimed to investigate the clinical significance of dysfunctional TECs in pancreatic ductal adenocarcinoma (PDAC) and identify chemical compounds that boost tumor-infiltrating lymphocyte (TIL) numbers. We performed immunohistochemical detection and clinicopathological analysis of VCAM-1 on TECs, which is essential for lymphocyte trafficking. We characterized the gene expression profiles of TECs from fresh PDAC tissues. We isolated compounds that upregulated VCAM-1 and E-selectin expression in TECs and examined their biological activities. Compared to endothelial cells from chronic pancreatitis tissue, TECs showed significantly lower VCAM-1 and E-selectin expression and significant weaknesses in lymphocyte adhesion and transmigration, resulting in decreased T cell infiltration around vessels. Patients with a relatively high percentage of VCAM-1+ vessels among all vessels in PDAC tissue had an improved prognosis. A bioinformatics survey demonstrated that TNFR1 signaling was involved in abnormal VCAM-1 and E-selectin expression in TECs. We screened compounds affecting TNFR1 signaling, and the IAP inhibitor, Embelin, induced these molecules on TECs and enhanced T cell adhesion to TECs and transmigration. Furthermore, in vivo, Embelin enhanced tumor-infiltrating T cell numbers, leading to an antitumor immune response. Embelin accelerates TIL infiltration and the antitumor immune response by recovering VCAM-1 expression in TECs. Our strategy may be a therapeutic approach for accelerating the immunotherapeutic response in immune-quiescent tumors, leading to clinical trials' success.

Expression of classical human leukocyte antigen class I antigens, HLA-E and HLA-G, is adversely prognostic in pancreatic cancer patients.

The expression of classical human leukocyte antigen class I antigens (HLA-I) on the surfaces of cancer cells allows cytotoxic T cells to recognize and eliminate these cells. Reduction or loss of HLA-I is a mechanism of escape from antitumor immunity. The present study aimed to investigate the clinicopathological impacts of HLA-I and non-classical HLA-I antigens expressed on pancreatic ductal adenocarcinoma (PDAC) cells. We performed immunohistochemistry to detect expression of HLA-I antigens in PDAC using 243 PDAC cases and examined their clinicopathological influences. We also investigated the expression of immune-related genes to characterize PDAC tumor microenvironments. Lower expression of HLA-I, found in 33% of PDAC cases, was significantly associated with longer overall survival. Higher expression of both HLA-E and HLA-G was significantly associated with shorter survival. Multivariate analyses revealed that higher expression of these three HLA-I antigens was significantly correlated with shorter survival. Higher HLA-I expression on PDAC cells was significantly correlated with higher expression of IFNG, which also correlated with PD1, PD-L1 and PD-L2 expression. In vitro assay revealed that interferon gamma (IFNγ) stimulation increased surface expression of HLA-I in three PDAC cell lines. It also upregulated surface expression of HLA-E, HLA-G and immune checkpoint molecules, including PD-L1 and PD-L2. These results suggest that the higher expression of HLA-I, HLA-E and HLA-G on PDAC cells is an unfavorable prognosticator. It is possible that IFNγ promotes a tolerant microenvironment by inducing immune checkpoint molecules in PDAC tissues with higher HLA-I expression on PDAC cells.

Neutrophilia and hyperamylasemia in patients with immediate food allergy.

BACKGROUND: Presently, little is known about the laboratory data several hours after oral food challenge (OFC) in patients with immunoglobulin (Ig)E-mediated immediate food allergy (FA). METHODS: One hundred and twelve subjects who underwent OFC at the present institute between 1 June 2016 and 31 March 2018, were enrolled in this study. Changes in laboratory data several hours after OFC were examined. RESULTS: OFC was positive in 76 patients and negative in 36. Increase in absolute neutrophil count (ANC) was significantly higher in OFC-positive than in OFC-negative subjects (median, 2,306/µL vs 637/µL; P < 0.00001). On multivariate regression analysis, a significant correlation was seen between neutrophilia and the development of gastrointestinal symptoms (t = 3.63; P < 0.001). Serum interleukin-6 increased in 43.8% of the patients with marked neutrophilia and had a significant positive correlation with ANC (r = 0.64; P < 0.001). Serum amylase increased in 33.3% of the OFC-positive patients and was >100 U/L (median, 642 U/L) in five patients in whom serum lipase also increased markedly (1,197 U/L). There was a significant negative correlation between increase in serum amylase and decrease in absolute eosinophil count (r = -0.36, P < 0.01). CONCLUSIONS: Marked neutrophilia was seen after OFC in patients with immediate FA presenting gastrointestinal symptoms, which may provide an insight into the relationship between symptoms and laboratory data. A considerable increase in serum amylase after OFC was also seen in patients with immediate FA, suggesting that the pancreas is a target organ for immediate FA.