Aberrant Glycosylation in Pancreatic Ductal Adenocarcinoma 3D Organoids Is Mediated by KRAS Mutations.

Aberrant glycosylation in tumor cells is a hallmark during carcinogenesis. KRAS gene mutations are the most well-known oncogenic abnormalities but their association with glycan alterations in pancreatic ductal adenocarcinoma (PDAC) is largely unknown. We employed patient-derived 3D organoids to culture pure live PDAC cells, excluding contamination by fibroblasts and immune cells, to gasp the comprehensive cancer cell surface glycan expression profile using lectin microarray and transcriptomic analyses. Surgical specimens from 24 PDAC patients were digested and embedded into a 3D culture system. Surface-bound glycans of 3D organoids were analyzed by high-density, 96-lectin microarrays. KRAS mutation status and expression of various glycosyltransferases were analyzed by RNA-seq. We successfully established 16 3D organoids: 14 PDAC, 1 intraductal papillary mucinous neoplasm (IPMN), and 1 normal pancreatic duct. KRAS was mutated in 13 (7 G12V, 5 G12D, 1 Q61L) and wild in 3 organoids (1 normal duct, 1 IPMN, 1 PDAC). Lectin reactivity of AAL (Aleuria aurantia) and AOL (Aspergillus oryzae) with binding activity to α1-3 fucose was higher in organoids with KRAS mutants than those with KRAS wild-type. FUT6 (α1-3fucosyltransferase 6) and FUT3 (α1-3/4 fucosyltransferase 3) expression was also higher in KRAS mutants than wild-type. Meanwhile, mannose-binding lectin (rRSL [Ralstonia solanacearum] and rBC2LA [Burkholderia cenocepacia]) signals were higher while those of galactose-binding lectins (rGal3C and rCGL2) were lower in the KRAS mutants. We demonstrated here that PDAC 3D-cultured organoids with KRAS mutations were dominantly covered in increased fucosylated glycans, pointing towards novel treatment targets and/or tumor markers.

Triple modal treatment comprising with proton beam radiation, hyperthermia, and gemcitabine/nab-paclitaxel for locally advanced pancreatic cancer: a phase I/II study protocol (TT-LAP trial).

BACKGROUND: Locally advanced pancreatic ductal adenocarcinoma (PDAC), accounting for about 30% of PDAC patients, is difficult to cure by radical resection or systemic chemotherapy alone. A multidisciplinary strategy is required and our TT-LAP trial aims to evaluate whether triple-modal treatment with proton beam therapy (PBT), hyperthermia, and gemcitabine plus nab-paclitaxel is a safe and synergistically effective treatment for patients with locally advanced PDAC. METHODS: This trial is an interventional, open-label, non-randomized, single-center, single-arm phase I/II clinical trial organized and sponsored by the University of Tsukuba. Eligible patients who are diagnosed with locally advanced pancreatic cancer, including both borderline resectable (BR) and unresectable locally advanced (UR-LA) patients, and selected according to the inclusion and exclusion criteria will receive triple-modal treatment consisting of chemotherapy, hyperthermia, and proton beam radiation. Treatment induction will include 2 cycles of chemotherapy (gemcitabine plus nab-paclitaxel), proton beam therapy, and 6 total sessions of hyperthermia therapy. The initial 5 patients will move to phase II after adverse events are verified by a monitoring committee and safety is ensured. The primary endpoint is 2-year survival rate while secondary endpoints include adverse event rate, treatment completion rate, response rate, progression-free survival, overall survival, resection rate, pathologic response rate, and R0 (no pathologic cancer remnants) rate. The target sample size is set at 30 cases. DISCUSSION: The TT-LAP trial is the first to evaluate the safety and effectiveness (phases1/2) of triple-modal treatment comprised of proton beam therapy, hyperthermia, and gemcitabine/nab-paclitaxel for locally advanced pancreatic cancer. ETHICS AND DISSEMINATION: This protocol was approved by the Tsukuba University Clinical Research Review Board (reference number TCRB22-007). Results will be analyzed after study recruitment and follow-up are completed. Results will be presented at international meetings of interest in pancreatic cancer plus gastrointestinal, hepatobiliary, and pancreatic surgeries and published in peer-reviewed journals. TRIAL REGISTRATION: Japan Registry of Clinical Trials, jRCTs031220160. Registered 24 th June 2022, https://jrct.niph.go.jp/en-latest-detail/jRCTs031220160 .

Survival impact on triple-modal strategy comprising hyperthermia, external radiation, and chemotherapy for unresectable locally advanced (UR-LA) pancreatic ductal adenocarcinoma.

BACKGROUND: Present treatment strategy for unresectable locally advanced (UR-LA) pancreatic ductal adenocarcinoma (PDAC) patients is controversial. Hence, a triple-modal therapy, which is a multidisciplinary strategy, was designed for patients with UR-LA PDAC by adding hyperthermia to conventional chemoradiotherapy at our institution. In this study we aimed to evaluate the effectiveness of this strategy. METHODS: Data of 21 UR-LA PDAC patients who underwent the triple-modal treatment were retrospectively analyzed for evaluating the safety and oncological effect of the treatment. The treatment schedule included, five concurrent infusions of gemcitabine (800 mg/m2) followed by hyperthermia (1 h) and X-ray (2 Gy) or proton beam radiation (2.7 Gy) on days 1, 8, 15, 29, and 36. Additional radiotherapies applied a total dose of 50 Gy/25 fr for X-ray radiation or 67.5 Gy/25 fr for proton beam radiation. RESULTS: Median overall survival (OS) was 23.6 months. Conversion surgery was performed in 5 patients (23.8%), and a R0 margin could be achieved in 4 of them; however, their median OS (16.3 months) tended to be shorter than that of the patients who did not undergo resection (23.6 months, p = 0.562). Further, the median OS of patients who underwent proton beam radiation (28.0 months) was significantly longer than that of patients who underwent X-ray radiation (13.9 months, p = 0.045). Most adverse events were manageable, except for one grade 3 gastric ulcer. The median tumor size and marker reduction rates were -17% and -91%, respectively. The tumor responses were partial response, stable disease, and progressive disease in 3, 15, and 3 patients, respectively. CONCLUSION: Triple-modal strategy, especially when combined with proton beam radiation, is feasible and results in favorable survival outcomes in patients with UR-LA PDAC.

Exploring optimal examination to detect occult anastomotic leakage after rectal resection in patients with diverting stoma.

BACKGROUND: When considering "early stoma closure", both standardized inclusion/exclusion criteria and standardized methods to assess anastomosis are necessary to reduce the risk of occult anastomotic leakage (AL). However, in the immediate postoperative period, neither have the incidence and risk factors of occult AL in patients with diverting stoma (DS) been clarified nor have methods to assess anastomosis been standardized. The aim of this study was to elucidate the incidence and risk factors of occult AL in patients who had undergone rectal resection with DS and to evaluate the significance of computed tomography (CT) following water-soluble contrast enema (CE) to detect occult anastomotic leakage. METHODS: This was a single institutional prospective observational study of patients who had undergone rectal resection with the selective use of DS between May and October 2019. Fifteen patients had undergone CE and CT to assess for AL on postoperative day (POD) 7, and CT was performed just after CE. Univariate analysis was performed to assess the relationship between preoperative variables and the incidence of occult AL on POD 7. RESULTS: The incidence of occult AL on postoperative day 7 was 6 of 15 (40%). Hand-sewn anastomosis, compared with stapled anastomosis, was a significant risk factor. Five more cases with occult AL that could not be detected with CE could be detected on CT following CE; CE alone had a 33% false-negative radiological result rate. CONCLUSIONS: Hand-sewn anastomosis appeared to be a risk factor for occult AL, and CE alone had a high false-negative radiological result rate. When considering the introduction of early stoma closure, stapled anastomosis and CT following CE could be an appropriate inclusion criterion and preoperative examination, respectively.

Preclinical evaluation of heat-denatured [18F]FDG-labeled red blood cells for detecting splenic tissues with PET in rats.

INTRODUCTION: Heat-denatured 99mTc-labeled red blood cells (RBCs) are used for detecting splenic tissues with scintigraphy. The present study aimed to evaluate the feasibility of using heat-denatured [18F]fluorodeoxyglucose ([18F]FDG)-labeled RBCs in detecting splenic tissues using positron emission tomography (PET) in rats. METHODS: RBCs were washed with phosphate buffered saline, labeled with [18F]FDG at 38°C, and heat-denatured at 50°C for 15 min. In vitro stability was assessed by measuring extracellular radioactivity during the 0-180 min incubation at 37°C. Thin layer chromatography (TLC) of the extracellular fluid was performed. The autologous RBCs were intravenously injected in four rats and PET scanning was simultaneously performed for 30 min. Time-activity curves of several organs, including the spleen, were analyzed on the PET images. RESULTS: Labeling efficiency was 92%. Low levels of radioactivity were released from the labeled RBCs for 180 min. TLC revealed that 80% of the released radioactivity was due to [18F]FDG-6-phosphate. Whole body images showed strong uptake of heat-denatured [18F]FDG-labeled RBCs in the spleen soon after injection in all four rats. Time-activity curves revealed that the splenic uptake continued to increase for 30 min and the amount of radioactivity in the other organs, except the urinary bladder, decreased after the initial surge. CONCLUSIONS: Heat-denatured [18F]FDG-labeled RBCs are suitable spleen-specific agents for PET. This method is clinically relevant as an alternative for heat-denatured 99mTc-labeled RBC scintigraphy.

Phrenic nerve protection via packing of gauze into the pericardial space during ablation of cristal atrial tachycardia in a child.

The success of catheter ablation of focal atrial tachycardia is limited by possible collateral damage to the phrenic nerve. Protection of the phrenic nerve is required. Here we present a case of a 9-year-old girl having a history of an unsuccessful catheter ablation of a focal atrial tachycardia near the crista terminalis (because of proximity of the phrenic nerve) who underwent a successful ablation by means of a novel technique for phrenic nerve protection: packing of gauze into the pericardial space. This method is a viable approach for patients with a failed endocardial ablation due to the proximity of the phrenic nerve.

Treatment of facial telangiectasia with a small spot of intense pulsed light: a case series of three patients.

Intense pulsed light (IPL) technology has long been used in the treatment of facial telangiectasia. While the large spot size of traditional IPL devices offers rapid coverage, it has limitations in terms of visibility and uniform contact with the skin in contoured areas of the face. The novel IPL used in this study had a small spot size (6.35 mm) and shorter wavelength (500-635 nm), allowing the use of high fluence without burning the normal epidermal tissue surrounding the lesion, thus providing better efficacy. Treatment of facial telangiectasia using small-spot IPL is effective with a low risk of dermatological damage, and its uses for medical care are expected to diversify.

[A case of portal hypertension after 5-fluorouracil, leucovorin, and oxaliplatin (mFOLFOX6) chemotherapy].

A 46-year-old man with cancer of the sigmoid colon with hepatic metastasis underwent sigmoidectomy, partial hepatectomy, and cholecystectomy in May 2008. He subsequently received 10 cycles of a modified 5-fluorouracil, leucovorin, and oxaliplatin (mFOLFOX6) regimen as adjuvant chemotherapy from June 2008 to December 2008, following which he developed thrombocytopenia and splenomegaly. In May 2011, upper gastrointestinal endoscopy was performed, which revealed esophageal and gastric varices. The varices were treated endoscopically with ligation and balloon-occluded retrograde transvenous obliteration. A liver biopsy was performed to determine the cause of the portal hypertension in the absence of severe hepatic dysfunction or liver cirrhosis. The biopsy revealed obliteration of the peripheral portal veins with sinusoidal dilatation without fibrosis or inflammatory cell infiltration in the hepatic lobules. Oxaliplatin-based chemotherapy has been associated with hepatovascular injury, such as sinusoidal dilatation and fibrosis, resulting in non-cirrhotic portal hypertension as seen in this case.

Hypercobalaminemia induced by an energy drink after total gastrectomy: a case report.

We encountered a case of hypercobalaminemia induced by oral intake of an energy drink after total gastrectomy. The patient was referred to our hospital due to findings suspicious for gastric cancer on screening. A 20 mm type 0-IIc lesion was detected in the gastric subcardia on esophagogastroduodenoscopy. Total gastrectomy followed by Roux-en-Y reconstruction was performed. He was discharged without complications. His basal serum vitamin B12 level was initially maintained with monthly intramuscular injections of vitamin B12. After 9 months, his serum vitamin B12 level suddenly increased up to 36-fold higher than the normal range and persisted there for one year without vitamin B12 injections. The patient ultimately reported consuming half a bottle of an energy drink each day during this time period. This case demonstrates the risk of unexpected hypervitaminemia resulting from self-administration of nutritional supplements.

High-dose vitamins E and C supplementation prevents ribavirin-induced hemolytic anemia in patients with chronic hepatitis C.

AIM: In combination therapy using interferon (IFN) and ribavirin for chronic hepatitis C, reduced doses should be used due to ribavirin-induced hemolytic anemia. The present study aimed to elucidate whether high-dose vitamins E and C supplementation attenuated ribavirin-induced hemolytic anemia. METHODS: Twenty-one consecutive patients with chronic hepatitis C were enrolled in this study between July 2003 and December 2004, and received high-dose vitamins E (2000 mg) and C (2000 mg) supplementation, daily, in addition to IFN alfa-2b and ribavirin combination therapy (vitamins E/C group). Twenty-one sex- and age-matched patients who received a standard regimen of IFN alfa-2b and ribavirin for chronic hepatitis C between January 2001 and June 2003 were evaluated as the control group. RESULTS: Decrease in hemoglobin level was significantly prevented in the vitamins E and C group compared to that in the control group (P = 0.029). Three (14.3%) patients in the control group discontinued treatment because of anemia, while no treated patient dropped out of the study due to anemia. Sustained virological response was not significantly different between the two groups. CONCLUSION: High-dose vitamins E and C supplementation prevented ribavirin-induced hemolytic anemia during combination therapy with ribavirin and IFN alfa-2b in patients with chronic hepatitis C.

Expression of adrenomedullin2/intermedin in human brain, heart, and kidney.

Adrenomedullin2/intermedin (AM2/IMD) is a novel member of the calcitonin/calcitonin gene-related peptide (CGRP) family. In the present study, we developed a specific radioimmunoassay of human AM2/IMD. Expression of AM2/IMD was studied in the human brain, pituitary, heart and kidney obtained at autopsy by radioimmunoassay and immunocytochemistry. Immunoreactive-AM2/IMD was detected by radioimmunoassay in human brains (range; 0.163-1.495 pmol/g wet weight), pituitaries (4.46+/-0.689 pmol/g wet weight, mean+/-S.E.M, n=3), left ventricles of hearts (0.251+/-0.0321 pmol/g wet weight, n=4), kidneys (3.49+/-1.18 pmol/g wet weight, n=5), and plasma obtained at healthy subjects (24.7+/-1.78 pmol/l, n=3). Reverse-phase high performance liquid chromatography showed that immunoreactive-AM2/IMD in human brain, kidney and plasma extracts were eluted in the position of authentic AM2/IMD. Additional peaks eluted earlier were found in the brain tissue and plasma. Immunocytochemistry showed that immunoreactive-AM2/IMD was localized in paraventricular and supraoptic nuclei of hypothalamus, anterior and posterior lobes of pituitary, cardiomyocytes, pericardial adipocytes, vascular endothelial cells of pericardial veins, and vascular smooth muscle cells of coronary arteries and renal arterioles as well as in renal tubular cells. The present study has shown expression of AM2/IMD in various types of cells in the central nervous system and the cardiovascular system, and suggested possible (patho)physiological roles of AM2/IMD in these systems.

Enhanced antinociceptive effects of morphine in histamine H2 receptor gene knockout mice.

We have previously shown that antinociceptive effects of morphine are enhanced in histamine H1 receptor gene knockout mice. In the present study, involvement of supraspinal histamine H2 receptor in antinociception by morphine was examined using histamine H2 receptor gene knockout (H2KO) mice and histamine H2 receptor antagonists. Antinociception was evaluated by assays for thermal (hot-plate, tail-flick and paw-withdrawal tests), mechanical (tail-pressure test) and chemical (formalin and capsaicin tests) stimuli. Thresholds for pain perception in H2KO mice were higher than wild-type mice. Antinociceptive effects of intracerebroventricularly administered morphine were enhanced in the H2KO mice compared to wild-type mice. Intracerebroventricular co-administration of morphine and cimetidine produced significant antinociceptive effects in the wild-type mice when compared to morphine or cimetidine alone. Furthermore, zolantidine, a selective and hydrophobic H2 receptor antagonist, enhanced the effects of morphine in all nociceptive assays examined. These results suggest that histamine exerts inhibitory effects on morphine-induced antinociception through H2 receptors at the supraspinal level. Our present and previous studies suggest that H1 and H2 receptors cooperatively function to modulate pain perception in the central nervous system.

Immunocytochemical localization of adrenomedullin 2/intermedin-like immunoreactivity in human hypothalamus, heart and kidney.

Adrenomedullin 2/intermedin (AM2/IMD) is a novel member of the calcitonin/calcitonin gene-related peptide (CGRP) peptide family. AM2/IMD has a vasodilator action, and antidiuretic and antinatriuretic effects in mice. The aim of the present study is to clarify immunolocalization of AM2/IMD in human hypothalamus, heart and kidney obtained at autopsy. Immunocytochemistry showed AM2/IMD-immunoreactive cell bodies in the paraventricular and supraoptic nuclei of human hypothalamus. Both parvocellular and magnocellular cells in the paravetricular nucleus are immunostained with AM2/IMD. Immunostaining of serial sections showed co-localization of AM2/IMD-like immunoreactivity and vasopressin in the paraventricular nucleus. Myocardial cells of the heart and renal tubular cells were positively immunostained with AM2/IMD, whereas neither renal glomeruli nor vasculature in the heart and kidney were immunostained. Reverse-transcriptase polymerase chain reaction confirmed expression of AM2/IMD mRNA in the brain, pituitary, heart and kidney. The present study has shown the wide expression of AM2/IMD in human hypothalamus, heart and kidney, raising the possibility that this novel peptide may be related to the central and peripheral regulation of the circulation and water-electrolyte metabolism.

Expression of urocortin III/stresscopin in human heart and kidney.

Urocortin III (Ucn III)/stresscopin (SCP) is a novel peptide of the corticotropin-releasing factor (CRF) family and is a specific ligand for the CRF type 2 receptor. We wished to clarify whether Ucn III/SCP is expressed in the human heart and kidney. Immunoreactive Ucn III was detected by RIA in the human heart (0.74-1.15 pmol/g wet weight, mean +/- SEM; n = 4) and kidney (1.21 +/- 0.30 pmol/g wet weight), which were obtained at autopsy. These levels were comparable to the levels in pituitary (2.72 +/- 0.13 pmol/g wet weight; n = 3) and brain tissues ( approximately 1-2 pmol/g wet weight). Furthermore, immunoreactive Ucn III was present in human plasma (51.8 +/- 16.0 pmol/liter; n = 5) and urine (266 +/- 20 pmol/liter; n = 5) obtained from healthy subjects. Reverse-phase HPLC showed a broad peak of immunoreactive Ucn III eluting in the position of synthetic Ucn III in the heart, kidney, and hypothalamus. Material eluting in the position of SCP was also found in the HPLC analysis of these tissue extracts. Immunocytochemistry showed positive staining of Ucn III in the myocardium and the renal tubules, particularly distal tubules. RT-PCR showed expression of Ucn III mRNA in the brain, pituitary, heart, and kidney. The present study has shown expression of Ucn III/SCP in the human heart and kidney as well as brain and pituitary tissues and its presence in plasma and urine. Ucn III/SCP may therefore regulate the cardiac and renal function as a local factor and a circulating hormone.