PTEN Regulates PI(3,4)P2 Signaling Downstream of Class I PI3K.

The PI3K signaling pathway regulates cell growth and movement and is heavily mutated in cancer. Class I PI3Ks synthesize the lipid messenger PI(3,4,5)P3. PI(3,4,5)P3 can be dephosphorylated by 3- or 5-phosphatases, the latter producing PI(3,4)P2. The PTEN tumor suppressor is thought to function primarily as a PI(3,4,5)P3 3-phosphatase, limiting activation of this pathway. Here we show that PTEN also functions as a PI(3,4)P2 3-phosphatase, both in vitro and in vivo. PTEN is a major PI(3,4)P2 phosphatase in Mcf10a cytosol, and loss of PTEN and INPP4B, a known PI(3,4)P2 4-phosphatase, leads to synergistic accumulation of PI(3,4)P2, which correlated with increased invadopodia in epidermal growth factor (EGF)-stimulated cells. PTEN deletion increased PI(3,4)P2 levels in a mouse model of prostate cancer, and it inversely correlated with PI(3,4)P2 levels across several EGF-stimulated prostate and breast cancer lines. These results point to a role for PI(3,4)P2 in the phenotype caused by loss-of-function mutations or deletions in PTEN.

Automated Analysis for the Prevalence of Cancer-Associated Fibroblasts in Resected Specimens of Intrahepatic Cholangiocarcinoma is a Simple and Reliable Evaluation System.

BACKGROUND: Intrahepatic cholangiocarcinoma (iCCA) has a high recurrence rate and poor prognosis, and chemotherapy options are limited. The prevalence of cancer-associated fibroblasts (CAFs) in iCCA has recently emerged as a prognostic marker and therapeutic target. A method to quantify the expression of CAFs is needed; however, a simple and reliable quantification method has not yet been established. OBJECTIVE: The aim of this study was to establish a simple and reliable method of quantifying CAFs. METHODS: A total of 71 patients with iCCA who underwent curative resection from November 2006 to October 2020 in our hospital were investigated. Immunohistochemistry for alpha-smooth muscle actin (α-SMA) was performed and α-SMA-positive cells were quantified by an automated analysis system (new method) and visually counted (conventional method). The times required for measurement and the prognosis were compared. RESULTS: The results of the quantification of CAFs by the new method were significantly correlated with the results by the conventional method, and the time required for measurement was significantly shorter with the new method. Patients with high-intensity CAFs showed a significantly poorer prognosis in terms of overall survival (OS) and the cumulative hepatic recurrence rate. In addition, high α-SMA levels were a significant risk factor for OS in multivariate analysis. CONCLUSIONS: This new method may contribute to the management of patients with iCCA, not only for the prediction of prognosis of patients with iCCA, but also for the indication of targeted therapy against CAFs.

[A Case of Long-Term Progress Control of Unresectable Progressive Gastric Cancer with Nivolumab Introduced during Effective Duration of Secondary Treatment].

A 65-year-old man with 1 month of general malaise was admitted to our hospital. Thoracoabdominal CT showed that the supra-clavicular, sub-carina, and para-aortic lymph nodes were swelling. Upper gastrointestinal endoscopy revealed 2 type 1 tumors at the esophagogastric junction, and the biopsy showed Group 5, well to moderately differentiated adenocarcinoma. The clinical diagnosis was cardiac gastric cancer and cStage Ⅳ(cT3N3M1[LYM]). We started capecitabine plus oxaliplatin as the first-line chemotherapy, and weekly paclitaxel plus ramucirumab was administered as the second-line treatment. The second-line treatment was successful, and the effect of PR was obtained. However, considering the period of TTF, while the therapeutic effect continued, we switched to third-line treatment with nivolumab after 7 courses of the second treatment. With the third-line treatment, PR was maintained for 1 year and 3 months, and good quality of life and performance status were obtained for a long period without irAE. However, after 32 courses, because the tumor marker was elevated and lymph nodes were enlarged, we judged PD and switched to the fourth-line treatment with nab-paclitaxel plus ramucirumab. The tumor marker levels decreased, the lymph nodes shrank, and PR was achieved again with the fourth-line treatment. The treatment is still ongoing 2 year and 8 months after the diagnosis.

[Two Cases of Large Gastrointestinal Stromal Tumors Safely and Completely Resected by Laparoscopic Surgery Followed by Neoadjuvant Imatinib Therapy].

Case 1, the patient was a 51-year-old man. Upper gastrointestinal endoscopy revealed a submucosal tumor with delle at the posterior wall of the gastric body, and the biopsy demonstrated a diagnosis of GIST. Abdominal CT scan showed a tumor at the size of 130×110×90 mm. Six months after administration of 400 mg/day of imatinib, the maximum diameter was reduced to 55 mm, then partial gastrectomy was performed by laparoscopic surgery. He continued to take imatinib after the surgery for 3 years, and he is alive without recurrence 4 years postoperatively. Case 2, the patient was a 68-year-old man. An abdominal CT scan showed a tumor at the size of 160×120×85 mm on the posterior outside of the stomach, but no submucosal tumor could be identified by upper gastrointestinal endoscopy. Gastric GIST was suspected and he started taking imatinib 400 mg/day. Because the Grade 3 generalized eruption was appeared, imatinib was discontinued, and then the dose was reduced. Nine months after the initiation of the treatment, the maximum diameter was reduced to 90 mm, and laparoscopic partial gastrectomy was performed. The patient is followed up without administration of imatinib after the surgery, and is alive without recurrence for 1 year and 6 months postoperatively. We report 2 cases that the large gastric GIST was able to be resected safely and completely due to tumor shrinkage by neoadjuvant imatinib therapy.

[A Case of Hepatocellular Carcinoma with Extrahepatic Growth-A Difficult Preoperative Diagnosis].

A 75-year-old man was admitted to our hospital for breathing difficulty. CT showed a 20 cm mass with clear boundaries and internal non-uniformity, which we suspected to be a gastrointestinal stromal tumor(GIST). Surgical resection was been considered to be risky because the mass was close to surrounding organs, such as the stomach, liver and diaphragm. Thus, we chose imatinib therapy. After 2 months, he was admitted to our hospital for anemia. CT showed the size of mass to be smaller, but the area of low density with internal non-uniformity had increased. We diagnosed intratumoral bleeding, and chose surgical resection. The mass was under the omentum, and had infiltrated the extrahepatic area and lesser curvature of the stomach. We diagnosed the mass derived from the stomach, and performed partial gastrectomy with partial liver resection. Pathological diagnosis was extrahepatically growing hepatocellular carcinoma(HCC, pT3N0M0, pStage Ⅲ).

[Two Cases of Resectable Gastric Cancer with Extensive Nodal Metastasis That Received Preoperative DOS Therapy].

Case 1: A 67-year-old male had a type 1 tumor in the stomach with a lymph node metastasis 50 mm in size. He was diagnosed with cT4aN(+)M0, cStage Ⅲ and received preoperative docetaxel plus oxaliplatin plus S-1(DOS)therapy. After 3 courses of the regimen, the patient underwent laparoscopic total gastrectomy. The final stage was ypT3N1(1/38) M0, ypStage ⅡB, R0, and the pathological response was Grade 2b. Case 2: A 64-year-old male had a type 3 tumor in the abdominal esophagus and a lymph node metastasis 15 mm in size. He was diagnosed with cT3N(+)M0, cStage Ⅲ and received preoperative DOS therapy. After 3 courses, he underwent laparoscopic esophagectomy. The final stage was ypT0N0M0, ypStage 0, R0, and the pathological response was Grade 3. DOS therapy may be effective as a neoadjuvant chemotherapy.

[A Case of Pancreatic Pseudocyst Recurrence after Surgery for Remnant Pancreatic Cancer].

The patient was a 72-year-old man with a history of pancreatic cancer and IPMA treated with distal pancreatectomy. He had recurrence-free period after adjuvant chemotherapy with S-1. But 6 years after the surgery, a diameter of 1 cm mass was noted in the remnant pancreas on MRI examination after hepatocellular carcinoma treatment. The mass was diagnosed as remnant pancreatic cancer, and he had undergone partial pancreatectomy of remnant pancreas. The pathological diagnosis was pancreatic ductal carcinoma with negative margin. However, 6 months after the reoperation, epigastric pain appeared, and CT scan showed a pseudocyst of 10 cm in size. The diagnosis was local recurrence with positive cytology, and then puncture drainage was performed. After repeated drainages, adhesion of the cystic lesion, and chemotherapy, the cytology became negative and the cystic lesion disappeared, but peritoneal dissemination metastasis also appeared. The patient died of the primary disease 7 years and 8 months after the first surgery and 1 year and 11 months after the second surgery. There has been no report of local recurrence in the form of pancreatic pseudocyst after pancreatic cancer surgery, and we report this case with literature discussion.

[A Case of Pancreaticoduodenectomy for Vater Papillary Cancer with Celiac Axis Stricture].

During the follow‒up of Vater papillary adenoma, a 74‒year‒old man was admitted to our hospital with a chief complaint of upper abdominal pain and diagnosed as cholangitis with obstructive jaundice. Cholestasis had been considered to be caused by papillary adenoma, however, EUS exam showed continuous bile duct wall irregularity from papilla of Vater. So we diagnosed as papillary carcinoma with extension to the distal bile duct. Preoperative CT showed the stenosis at the root of celiac artery, and hepatic blood flow was considered to be supplied via the pancreatic head arcade from superior mesenteric artery, so an anastomosis of gastroduodenal artery and inferior pancreaticoduodenal artery was performed during pancreaticoduodenectomy( PD). Like this case, when performing PD with celiac artery stricture, it is important to evaluate hepatic blood flow before and during surgery and prepare for the arterial reconstruction.

[Analysis of 16 Cases of Primary Duodenal Carcinoma in Our Hospital].

Primary duodenal carcinoma is a rare disease among gastrointestinal malignancies and has little evidence. We evaluated retrospectively the treatment status of 16 cases of primary duodenal carcinoma in our hospital between 2010 and 2019. The median age was 72(58-88)years and 63% of patients were male, and Each stage were Stage 0 in 4 cases, Stage Ⅰ in 1 case, Stage ⅢA in 2 cases, Stage ⅢB in 3 cases, and Stage Ⅳ in 6 cases(UICC 8th edition). Initial treatment was endoscopic therapy in 3 cases, surgery in 10 cases, chemotherapy in 1 case, and best supportive care in 2 case. The 2-year survival rate was 51.3% and the MST was 25.4 months in all cases. The Stage 0, Stage Ⅰ cases had all recurrence-free survival, while the Stage ⅢA or higher cases, 2-year survival rate was 33.8% and the MST was 20.0 months. Also, XELOX was often selected as the first-line treatment for chemotherapy regimens including recurrence treatment.

Lysophosphatidylinositol-acyltransferase-1 is involved in cytosolic Ca2+ oscillations in macrophages.

Lysophosphatidylinositol-acyltransferase-1 (LPIAT1) specifically catalyzes the transfer of arachidonoyl-CoA to lysophosphoinositides. LPIAT-/- mice have been shown to have severe defects in the brain and liver; however, the exact molecular mechanisms behind these conditions are not well understood. As immune cells have been implicated in liver inflammation based on disfunction of LPIAT1, we generated Raw264.7 macrophages deficient in LPIAT1, using shRNA and CRISPR/Cas9. The amount of C38:4 species in phosphoinositides, especially in PtdInsP2 , was remarkably decreased in these cells. Unlike in wild-type cells, LPIAT1-deficient cells showed prolonged oscillations of intracellular Ca2+ upon UDP stimulation, which is known to activate phospholipase Cß through the Gq-coupled P2Y6 receptor, even in the absence of extracellular Ca2+ . It is speculated that the prolonged Ca2+ response may be relevant to the increased risk of liver inflammation induced by LPIAT1 disfunction.

[A Long-Survived Case of Gastric Cancer with Multiple Liver Metastases Who Underwent Gastrectomy and Received Chemotherapy].

A 70-year-old man visited our hospital because of a body weight loss. Upper gastrointestinal fiberscope revealed a type 3 tumor and an enhanced MRI showed 30 or more liver metastases. He received docetaxel plus cisplatin plus S-1(DCS)therapy. Although main tumor had shrinked only partially, multiple liver metastases could not be detected. Thus, he was performed distal gastrectomy. After gastrectomy, he received S-1 plus oxaliplatin(SOX)therapy followed by S-1 therapy. Two years and 2 months after surgery, chemotherapy was finished because of no signs of tumor progression. He is alive without recurrence for 2 years and 11 months after gastrectomy.

[Two Cases of Liver Metastasis from Gastric Cancer Achieving Long-Term Survival Successfully Treated with Stereotactic Body Radiotherapy(SBRT)].

We report 2 cases with long-term survival following stereotactic body radiotherapy(SBRT)for liver metastasis from gastric cancer. Case 1 was a 65-year-old man. We performed distal gastrectomy for gastric cancer with liver metastasis prior to chemotherapy due to pyloric stenosis. Postoperative S-1 chemotherapy was administered. Two liver metastases observed before the operation were temporarily reduced in size but subsequently enlarged; therefore, SBRT was performed 13 months postoperatively. The liver metastases showed a complete response(CR)and the patient is alive 4 years and 11 months after SBRT(6 years postoperatively). Case 2 was a 71-year-old woman. After performing distal gastrectomy, liver metastasis emerged during postoperative S-1 adjuvant chemotherapy; therefore, SBRT was performed 11 months postoperatively. She was then administered weekly paclitaxel. However, she underwent a right hepatic lobectomy 13 months after SBRT for suspected remnant or marginal recurrence by abdominal enhanced CT. Histopathological examination showed that the tumors contained fibrotic connective tissue with no viable cancer cell remnants; therefore, the therapeutic effect was determined to be of Grade 3. The patient is alive without recurrence in the remnant liver 4 years and 7 months after SBRT(5 years and 6 months after the operation of gastric cancer). Thus, SBRT for liver metastasis from gastric cancer may be considered an effective local treatment.

[A Case Report of Long Survival in Pancreatic Cancer with Superior Mesenteric Arterial Invasion Following Multimodal Therapy].

The prognosis of pancreatic cancer with superior mesenteric arterial invasion is very poor and judgment of surgical indication is very difficult. We report a case that received multimodal therapy for pancreatic cancer with superior mesenteric arterial invasion. A 43-year-old woman consulted a local doctor because of upper abdominal pain. Ultrasonography revealed a nodule in the pancreatic body and elevated CA19-9 values. She was referred to our hospital for evaluation and therapy. Computed tomography showed a low-density area in the pancreatic body and around the superior mesenteric artery. The clinical diagnosis was pancreatic cancer with superior mesenteric arterial invasion. She received chemoradiation therapy(RT, 50.4 Gy, gemcitabine[GEM]plus TS-1)followed by distal pancreatectomy. She received adjuvant chemotherapy(TS-1)for 6 months. However, follow-up CTperformed 10 months after surgery revealed local recurrence. The patient received chemotherapy( GEM)for 8 months and GEM plus nab-PTX for 22 months. She died from the cancer 50 months after the primary operation.

[A Case of Chemotherapy with Abscess Drainage for Pancreatic Cancer Detected Due to Peritonitis].

A 69-year-old woman was admitted to our hospital because of abdominal pain. Abdominal CT revealed free air, so we performed an emergency operation. Although the perforation site could not be confirmed, pancreatic cancer invading the stomach, spleen, and transverse colon was found. As a splenic abscess and peritoneal dissemination were also found, we created a colostomy and placed drains. Although the postoperative course was good, the splenic abscess continuous with the tumor remained. We thought that early removal of the drain would be difficult, so chemotherapy was introduced while continuing drainage. Fortunately, the pancreatic cancer was controlled, and the abscess tended to shrink, so we removed the tube 137 days after the surgery. After that, we continued chemotherapy, but in the second year after the surgery, the liver metastasis acutely exacerbated and DIC also developed, resulting in cancer death. If surgical intervention is difficult, as in this case, chemotherapy may be considered as an option, keeping in mind the possibility of exacerbation of infection.

PKN2 is essential for mouse embryonic development and proliferation of mouse fibroblasts.

PKN2, a member of the protein kinase N (PKN) family, has been suggested by in vitro culture cell experiments to bind to Rho/Rac GTPases and contributes to cell-cell contact and cell migration. To unravel the in vivo physiological function of PKN2, we targeted the PKN2 gene. Constitutive disruption of the mouse PKN2 gene resulted in growth retardation and lethality before embryonic day (E) 10.5. PKN2-/- embryo did not undergo axial turning and showed insufficient closure of the neural tube. Mouse embryonic fibroblasts (MEFs) derived from PKN2-/- embryos at E9.5 failed to grow. Cre-mediated ablation of PKN2 in PKN2flox/flox MEFs obtained from E14.5 embryos showed impaired cell proliferation, and cell cycle analysis of these MEFs showed a decrease in S-phase population. Our results show that PKN2 is essential for mouse embryonic development and cell-autonomous proliferation of primary MEFs in culture. Comparison of the PKN2-/- phenotype with the phenotypes of PKN1 and PKN3 knockout strains suggests that PKN2 has distinct nonredundant functions in vivo, despite the structural similarity and evolutionary relationship among the three isoforms.

The effect and possible clinical efficacy of in vivo inhibition of neutrophil extracellular traps by blockade of PI3K-gamma on the pathogenesis of microscopic polyangiitis.

OBJECTIVE: Neutrophil extracellular traps (NETs) are peculiar structures composed of the externalized chromatin with intracellular proteins and formed by activated neutrophils in a reactive oxygen species (ROS)-dependent manner. Aberrant NETs are considered to be autoantigens for anti-neutrophil cytoplasmic antibodies (ANCAs) underling the development of microscopic polyangiitis (MPA). However, little is known regarding the therapeutic efficacy of in vivo inhibition of NET formation (NETosis) on MPA pathogenesis. This study determines whether reducing NETosis prevents ANCA production and improves characteristic involvement. METHODS: A mouse model of MPA induced by administering a novel extract from Candida albicans was devised. By applying this method to mice lacking phosphoinositide 3-kinase gamma (PI3K-gamma), which is indispensable for ROS production in neutrophils, we investigated the levels of in vivo NETs, ANCA titers and histological damage. RESULTS: Our model exhibited accumulation of NETs in vivo, elevation of ANCA titers and characteristic pathologies mimicking human MPA, including small-vessel vasculitis and crescentic glomerulonephritis. Strikingly, these abnormalities were reduced by genetically and/or pharmacologically blocking PI3K-gamma. Moreover, a pharmacological PI3K-gamma blockade decreased the levels of human NETs. CONCLUSION: Our results suggest that in vivo inhibition of NETosis by blocking PI3K-gamma could be a promising therapeutic strategy for the pathogenesis of MPA.

Identifying neurocognitive markers for outcome prediction of global functioning in individuals with first-episode and ultra-high-risk for psychosis.

AIM: There is an increasing need for identifying neurocognitive predictors of global functional outcome in early psychosis toward optimizing an early intervention strategy. METHODS: We conducted a longitudinal observational study to investigate an association between neurocognitive assessments at baseline and global functional outcome at an average of 1-year follow up. Participants included ultra-high-risk for psychosis (UHR) individuals who had not converted to psychosis during the follow-up period (UHR-NP) and those with first-episode psychosis (FEP). We evaluated neurocognition at baseline using the Brief Assessment of Cognition in Schizophrenia Japanese version, including Verbal Memory, Working Memory, Motor Speed, Verbal Fluency, Attention/Processing Speed, and Executive Function. We also assessed global functional outcome using the modified Global Assessment of Functioning (mGAF) scale both at baseline and after the follow-up period. RESULTS: Thirty-four UHR-NP individuals (34/47, 72%) and 29 FEP individuals (29/36, 81%) completed assessment of neurocognitive function at baseline and functional outcome at follow up. In the UHR-NP group, Attention/Processing Speed was significantly associated with the mGAF score at follow up. In the FEP group, Executive Function was significantly associated with the average mGAF score during follow up. CONCLUSION: Attention/Processing Speed and Executive Function at baseline may predict global functional outcome of early psychosis. These neurocognitive tests are easy to incorporate in clinical settings and, if replicated in independent samples, may be included in routine clinical assessments for prediction of functional outcome in early psychosis.

The PtdIns(3,4)P(2) phosphatase INPP4A is a suppressor of excitotoxic neuronal death.

Phosphorylated derivatives of phosphatidylinositol, collectively referred to as phosphoinositides, occur in the cytoplasmic leaflet of cellular membranes and regulate activities such as vesicle transport, cytoskeletal reorganization and signal transduction. Recent studies have indicated an important role for phosphoinositide metabolism in the aetiology of diseases such as cancer, diabetes, myopathy and inflammation. Although the biological functions of the phosphatases that regulate phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) have been well characterized, little is known about the functions of the phosphatases regulating the closely related molecule phosphatidylinositol-3,4-bisphosphate (PtdIns(3,4)P(2)). Here we show that inositol polyphosphate phosphatase 4A (INPP4A), a PtdIns(3,4)P(2) phosphatase, is a suppressor of glutamate excitotoxicity in the central nervous system. Targeted disruption of the Inpp4a gene in mice leads to neurodegeneration in the striatum, the input nucleus of the basal ganglia that has a central role in motor and cognitive behaviours. Notably, Inpp4a(-/-) mice show severe involuntary movement disorders. In vitro, Inpp4a gene silencing via short hairpin RNA renders cultured primary striatal neurons vulnerable to cell death mediated by N-methyl-d-aspartate-type glutamate receptors (NMDARs). Mechanistically, INPP4A is found at the postsynaptic density and regulates synaptic NMDAR localization and NMDAR-mediated excitatory postsynaptic current. Thus, INPP4A protects neurons from excitotoxic cell death and thereby maintains the functional integrity of the brain. Our study demonstrates that PtdIns(3,4)P(2), PtdIns(3,4,5)P(3) and the phosphatases acting on them can have distinct regulatory roles, and provides insight into the unique aspects and physiological significance of PtdIns(3,4)P(2) metabolism. INPP4A represents, to our knowledge, the first signalling protein with a function in neurons to suppress excitotoxic cell death. The discovery of a direct link between PtdIns(3,4)P(2) metabolism and the regulation of neurodegeneration and involuntary movements may aid the development of new approaches for the treatment of neurodegenerative disorders.