Serum elastase-1 predicts malignancy in intraductal papillary mucinous neoplasm of the pancreas.

BACKGROUND: The indication for surgical resection of intraductal papillary mucinous neoplasms (IPMNs) is defined by imaging features, such as mural nodules. Although carbohydrate antigen (CA) 19-9 was selected as a parameter for worrisome features, no serum biomarkers were considered when deciding on surgical indications in the latest international consensus guideline. In this study, we assessed whether clinical factors, imaging findings, and serum biomarkers are useful in predicting malignant IPMNs. METHODS: A total of 234 resected IPMN cases in Chiba University Hospital from July 2005 to December 2021 were retrospectively analyzed. RESULTS: Among the 234 patients with resected IPMNs diagnosed by preoperative imaging, 117 were diagnosed with malignant pathologies (high-grade dysplasia and invasive IPMNs) according to the histological classification. In the multivariate analysis, cyst diameter ≥30 mm; p = 0.035), enhancing mural nodules on multidetector computed tomography (≥5 mm; p = 0.018), and high serum elastase-1 (≥230 ng/dl; p = 0.0007) were identified as independent malignant predictors, while CA19-9 was not. Furthermore, based on the receiver operator characteristic curve analyses, elastase-1 was superior to CA19-9 for predicting malignant IPMNs. Additionally, high serum elastase-1 levels (≥230 ng/dl; p = 0.0093) were identified as independent predictors of malignant IPMNs in patients without mural nodules on multidetector computed tomography (MDCT) in multivariate analysis. CONCLUSION: The serum elastase-1 level was found to be a potentially useful biomarker for predicting malignant IPMNs.

Clinical benefits of pulmonary resection for lung metastases from pancreatic cancer.

PURPOSE: Systemic chemotherapy is generally used for metastatic pancreatic cancer; however, pulmonary resection may be a treatment option for lung oligometastases from pancreatic cancer. The current study aimed to clarify the oncological outcomes and clinical benefits of pulmonary resection for lung metastases. METHODS: Of 510 patients who underwent pancreatic resection for pancreatic cancer, 44 patients with recurrence of isolated lung metastases and one patient with simultaneous lung metastases were evaluated. RESULTS: Of the 45 patients, 20 patients were selected as candidates for pulmonary resection based on clinical factors such as recurrence-free interval (RFI) from pancreatectomy to lung metastases, number of lung metastases, and serum CA19-9 level. The post-recurrent survival of patients with pulmonary resection was significantly better than that of patients without pulmonary resection. Fourteen of the 20 patients with pulmonary resection developed tumor recurrence with a median disease-free survival (DFS) of 15 months. Univariate analyses revealed that an RFI from pancreatectomy to lung metastases of ≥28 months was associated with better DFS after pulmonary resection. Of the 14 patients with an RFI of ≥28 months, pulmonary resection resulted in prolonged chemotherapy-free interval in 12 patients. Furthermore, repeat pulmonary resection for recurrent tumors after pulmonary resection led to further cancer-free interval in some cases. CONCLUSIONS: Although many patients had tumor recurrence after pulmonary resection, pulmonary resection for lung metastases from pancreatic cancer may provide prolonged cancer-free interval without the need for chemotherapy. Pulmonary resection should be performed for the patients with a long RFI from pancreatectomy to lung metastases.

Inducing multiple nicks promotes interhomolog homologous recombination to correct heterozygous mutations in somatic cells.

CRISPR/Cas9-mediated gene editing has great potential utility for treating genetic diseases. However, its therapeutic applications are limited by unintended genomic alterations arising from DNA double-strand breaks and random integration of exogenous DNA. In this study, we propose NICER, a method for correcting heterozygous mutations that employs multiple nicks (MNs) induced by Cas9 nickase and a homologous chromosome as an endogenous repair template. Although a single nick near the mutation site rarely leads to successful gene correction, additional nicks on homologous chromosomes strongly enhance gene correction efficiency via interhomolog homologous recombination (IH-HR). This process partially depends on BRCA1 and BRCA2, suggesting the existence of several distinct pathways for MN-induced IH-HR. According to a genomic analysis, NICER rarely induces unintended genomic alterations. Furthermore, NICER restores the expression of disease-causing genes in cells derived from genetic diseases with compound heterozygous mutations. Overall, NICER provides a precise strategy for gene correction.

The Outcome of Conversion to Hand-Assisted Laparoscopic Surgery in Laparoscopic Liver Resection.

BACKGROUND: Hand-assisted laparoscopic surgery (HALS) is known as a useful option. However, the outcome and predictor of conversion to HALS in laparoscopic liver resection (LLR) are unclear. METHODS: Data from consecutive patients who planned pure LLR between 2011 and 2020 were retrospectively reviewed. Univariate and multivariate analyses were performed and compared pure LLR, HALS, and converted open liver resection (OLR). RESULTS: Among the 169 LLRs, conversion to HALS was performed in 19 (11.2%) and conversion to OLR in 16 (9.5%). The most frequent reasons for conversion to HALS were failure to progress (11 cases). Subsequently, bleeding (3 cases), severe adhesion (2 cases), and oncological factors (2 cases) were the reasons. In the multivariable analysis, the tumor located in segments 7 or 8 (p = 0.002) was evaluated as a predictor of conversion to HALS. Pure LLR and HALS were associated with less blood loss than conversion to OLR (p = 0.005 and p = 0.014, respectively). However, there was no significant difference in operation time, hospital stay, or severe complications. CONCLUSIONS: The predictor of conversion to HALS was a tumor located in segments 7 or 8. The outcome of conversion to HALS was not inferior to pure LLR in terms of bleeding, operation time, hospital stay, or severe complication.

[Intraoperative Blood Flow Assessment of Gastric Tube Using Indocyanine Green fluorography during Pancreaticoduodenectomy Following Esophagectomy-Case Reports].

Case 1: A 73-year-old male, who had an intraductal papillary mucinous adenocarcinoma or resectable pancreatic cancer at the uncinate process of the pancreas five years after subtotal esophagectomy for esophageal cancer, underwent pylorus preserving pancreaticoduodenectomy(PPPD). Case 2: A 68-year-old male, who also had a resectable pancreatic cancer at the uncinate process of the pancreas 3 years after subtotal esophagectomy for esophageal cancer, underwent PPPD following neoadjuvant chemotherapy. In both cases, right gastroepiploic artery and vein were preserved to maintain the perfusion of the gastric tube during surgery. Indocyanine Green(ICG)fluorography was performed just before duodenal-jejunal anastomosis, which visually showed the well-perfused gastric tube. Both patients had no necrosis of the gastric tube, nor gastrointestinal obstruction after surgery. Intraoperative ICG fluorography was useful to evaluate the blood flow of the remaining gastric tube visually during PPPD for post-esophagectomy patients.

[Conversion Surgery Following Systemic Chemotherapy for Advanced Gallbladder Cancer with Peritoneal Dissemination-A Case Report].

A 73-year-old female was diagnosed with gallbladder cancer, but the future liver remnant volume was deemed insufficient for curative resection. Consequently, transileocolic portal vein embolization was performed. During laparotomy, multiple nodules were palpable on the peritoneal surface of the pelvic floor. Subsequently, staging laparoscopy confirmed the pathological diagnosis of adenocarcinoma in the resected nodules, indicating peritoneal dissemination of gall bladder cancer. Due to this peritoneal dissemination, surgical resection was deemed inappropriate, and the patient was initiated on systemic chemotherapy consisting of gemcitabine and cisplatin. Following 22 courses of chemotherapy, contrast-enhanced computed tomography demonstrated no significant changes in the size of the primary tumor or its location relative to the main vessels, although a small metastatic lesion was identified in the gallbladder bed. At the second staging laparoscopy, any nodules suggesting peritoneal dissemination were observed. Based on these findings, we decided to perform curative resection. The surgical procedure involved right hepatectomy plus segment 4a resection, extrahepatic bile duct resection, and hepaticojejunostomy. Pathological examination revealed ypT3bN0M1(HEP), ypStage ⅣB, with the achievement of R0 resection. The patient survived with no recurrences for 40 months after surgery. These results suggest that aggressive therapeutic strategies, including conversion surgery following systemic chemotherapy, may be beneficial for patients initially deemed unresectable due to gallbladder cancer.

[Retrospective Study of Preoperative Serial Pancreatic Juice Aspiration Cytological Examination(SPACE)for Early Preoperative Detection of Pancreatic Cancer].

Serial pancreatic juice aspiration cytological examination(SPACE)has been reported as a reliable preoperative diagnostic method for early pancreatic cancer, when combined with imaging findings suspecting early pancreatic cancer. Among 259 patients with suspected pancreatic cancer who underwent pancreatic resection at our hospital, SPACE was preoperatively performed in 14 cases(5.4%). Of these 14 cases, final pathological diagnosis was pancreatic cancer in 12 patients (86%), including 5 patients with Stage ⅠA pancreatic cancer(35.7%), all of whom had a mass on preoperative CT or EUS. On the other hand, in the other 2 cases(14.3%), CT/EUS detected no mass but focal pancreatic parenchymal atrophy and main pancreatic duct stenosis which were the imaging findings suspecting very early pancreatic cancer such as cancer in situ. Although preoperative SPACE results of these 2 cases were class Ⅳ, final pathological results of resected specimen were low-grade PanIN in both cases. SPACE was considered useful for preoperative diagnosis of pancreatic cancer in our study, however further study is needed to examine its diagnostic accuracy for early pancreatic cancer which does not appear as a mass in any imaging modality.

Predictors of a difficult Pringle maneuver in laparoscopic liver resection and evaluation of alternative procedures to assist bleeding control.

PURPOSE: To evaluate the predictors of a difficult Pringle maneuver (PM) in laparoscopic liver resection (LLR) and to assess alternative procedures to PM. METHODS: Data from patients undergoing LLR between 2013 and 2020 were reviewed retrospectively. Univariate and multivariate analyses were performed and the outcomes of patients who underwent PM or alternative procedures were compared. RESULTS: Among 106 patients who underwent LLR, PM could not be performed in 18 (17.0%) because of abdominal adhesions in 14 (77.8%) and/or collateral flow around the hepatoduodenal ligament in 5 (27.8%). Multivariate analysis revealed that Child-Pugh classification B (p = 0.034) and previous liver resection (p < 0.001) were independently associated with difficulty in performing PM in LLR. We evaluated pre-coagulation of liver tissue using microwave tissue coagulators, saline irrigation monopolar, clamping of the hepatoduodenal ligament using an intestinal clip, and hand-assisted laparoscopic surgery as alternatives procedures to PM. There were no significant differences in blood loss (p = 0.391) or transfusion (p = 0.518) between the PM and alternative procedures. CONCLUSIONS: Child-Pugh classification B and previous liver resection were identified as predictors of a difficult PM in LLR. The alternative procedures were found to be effective.

RNF168 E3 ligase participates in ubiquitin signaling and recruitment of SLX4 during DNA crosslink repair.

SLX4/FANCP is a key Fanconi anemia (FA) protein and a DNA repair scaffold for incision around a DNA interstrand crosslink (ICL) by its partner XPF nuclease. The tandem UBZ4 ubiquitin-binding domains of SLX4 are critical for the recruitment of SLX4 to damage sites, likely by binding to K63-linked polyubiquitin chains. However, the identity of the ubiquitin E3 ligase that mediates SLX4 recruitment remains unknown. Using small interfering RNA (siRNA) screening with a GFP-tagged N-terminal half of SLX4 (termed SLX4-N), we identify the RNF168 E3 ligase as a critical factor for mitomycin C (MMC)-induced SLX4 foci formation. RNF168 and GFP-SLX4-N colocalize in MMC-induced ubiquitin foci. Accumulation of SLX4-N at psoralen-laser ICL tracks or of endogenous SLX4 at Digoxigenin-psoralen/UVA ICL is dependent on RNF168. Finally, we find that RNF168 is epistatic with SLX4 in promoting MMC tolerance. We conclude that RNF168 is a critical component of the signal transduction that recruits SLX4 to ICL damage.

Early- and long-term outcomes of liver transplantation with rescue allocation grafts.

In France, liver grafts which have been refused by at least five centers are proposed as rescue allocation (RA). The aim of this study is to clarify the feasibility and safety of RA grafts in liver transplantation (LT). Short- and long-term outcomes of patients who received RA grafts (RA group) were compared with those of patients who received standard allocation (SA) grafts (SA group). From a total of 1635 patients, 102 patients received RA grafts. Before matching, the RA group was characterized primarily by less severe liver disease, but the quality of graft was worse. After matching recipients' characteristics of 102 patients who used RA grafts with 306 patients who used SA grafts, recipients' characteristics were well balanced (1:3 matching). Although the rate of primary dysfunction was significantly higher in the RA group, there is no significant difference in the occurrence of major complications, length of hospitalization, and mortality between two groups. Graft survival (GS) and overall survival (OS) in the RA group were not significantly different from the SA group (GS; HR = 1.03 P = .89, OS; HR = 1.03 P = .90). In the French allocation system, the feasibility and safety of RA grafts might be comparable to SA grafts for carefully selected patients.

Digenic mutations in ALDH2 and ADH5 impair formaldehyde clearance and cause a multisystem disorder, AMeD syndrome.

Rs671 in the aldehyde dehydrogenase 2 gene (ALDH2) is the cause of Asian alcohol flushing response after drinking. ALDH2 detoxifies endogenous aldehydes, which are the major source of DNA damage repaired by the Fanconi anemia pathway. Here, we show that the rs671 defective allele in combination with mutations in the alcohol dehydrogenase 5 gene, which encodes formaldehyde dehydrogenase (ADH5FDH ), causes a previously unidentified disorder, AMeD (aplastic anemia, mental retardation, and dwarfism) syndrome. Cellular studies revealed that a decrease in the formaldehyde tolerance underlies a loss of differentiation and proliferation capacity of hematopoietic stem cells. Moreover, Adh5-/-Aldh2 E506K/E506K double-deficient mice recapitulated key clinical features of AMeDS, showing short life span, dwarfism, and hematopoietic failure. Collectively, our results suggest that the combined deficiency of formaldehyde clearance mechanisms leads to the complex clinical features due to overload of formaldehyde-induced DNA damage, thereby saturation of DNA repair processes.

UBC13-Mediated Ubiquitin Signaling Promotes Removal of Blocking Adducts from DNA Double-Strand Breaks.

Chemical modifications and adducts at DNA double-strand break (DSB) ends must be cleaned before re-joining by non-homologous end-joining (NHEJ). MRE11 nuclease is essential for efficient removal of Topoisomerase II (TOP2)-DNA adducts from TOP2 poison-induced DSBs. However, mechanisms in MRE11 recruitment to DSB sites in G1 phase remain poorly understood. Here, we report that TOP2-DNA adducts are expeditiously removed through UBC13-mediated polyubiquitination, which promotes DSB resection in G2 phase. We found that this ubiquitin signaling is required for efficient recruitment of MRE11 onto DSB sites in G1 by facilitating localization of RAP80 and BRCA1 to DSB sites and complex formation between BRCA1 and MRE11 at DSB sites. UBC13 and MRE11 are dispensable for restriction-enzyme-induced "clean" DSBs repair but responsible for over 50% and 70% of NHEJ-dependent repair of γ-ray-induced "dirty" DSBs, respectively. In conclusion, ubiquitin signaling promotes nucleolytic removal of DSB blocking adducts by MRE11 before NHEJ.

Ubiquitination of DNA Damage-Stalled RNAPII Promotes Transcription-Coupled Repair.

Transcription-coupled nucleotide excision repair (TC-NER) is initiated by the stalling of elongating RNA polymerase II (RNAPIIo) at DNA lesions. The ubiquitination of RNAPIIo in response to DNA damage is an evolutionarily conserved event, but its function in mammals is unknown. Here, we identified a single DNA damage-induced ubiquitination site in RNAPII at RPB1-K1268, which regulates transcription recovery and DNA damage resistance. Mechanistically, RPB1-K1268 ubiquitination stimulates the association of the core-TFIIH complex with stalled RNAPIIo through a transfer mechanism that also involves UVSSA-K414 ubiquitination. We developed a strand-specific ChIP-seq method, which revealed RPB1-K1268 ubiquitination is important for repair and the resolution of transcriptional bottlenecks at DNA lesions. Finally, RPB1-K1268R knockin mice displayed a short life-span, premature aging, and neurodegeneration. Our results reveal RNAPII ubiquitination provides a two-tier protection mechanism by activating TC-NER and, in parallel, the processing of DNA damage-stalled RNAPIIo, which together prevent prolonged transcription arrest and protect against neurodegeneration.

[Laparoscopic Local Gastrectomy for Gastric Schwannoma and Extramurally Developed Gastric GIST].

Among gastric submucosal tumors, neurogenic tumors are considered to be rare diseases. We experienced a case of laparoscopic local gastrectomy of gastric schwannoma coexisting with extramurally developed gastric GIST found accidentally during surgery. A 61-year-old man was pointed out a gastric submucosal tumor with a diameter of 15 mm in a medical checkup. Endoscopic ultrasound-guided fine needle aspiration(EUS-FNA)was performed, and immunostaining showed that c-kit(-), CD34(-), S-100(+), SMA(-), MIB-1<2%. Diagnosis was gastric schwannoma. We performed laparoscopic local gastrectomy. During the surgery another extramural nodule was accidentally found with a diameter of 8 mm at the anterior wall of the gastric body near lesser curvature. Immunostaining showed c-kit(+), CD34(+)and was diagnosed GIST. Because a gastric schwannoma coexisting with GIST is a rare case, we decided to report it by adding discussion with some literatures.

Ischemic Cholangiopathy Following Transcatheter Arterial Chemoembolization for Recurrent Hepatocellular Carcinoma After Hepatectomy: an Underestimated and Devastating Complication.

BACKGROUND: Ischemic cholangiopathy (IC) has a known poor prognosis. However, the risks and outcomes of this complication after transcatheter arterial chemoembolization (TACE) in hepatectomized patients are poorly documented. This study aimed to evaluate the incidence of and to identify the predictive factors for IC following TACE for recurrent hepatocellular carcinoma (HCC) after hepatectomy. METHOD: From a cohort with a total of 486 patients who underwent resection for HCC, we included all consecutive patients who were treated with TACE for recurrent HCC after hepatectomy between 2000 and 2017. IC was defined by the coexistence of biological cholestasis and morphological lesions. RESULTS: A total of 156 patients underwent TACE for the treatment of HCC recurrence after hepatectomy. Of them, eight (5.1%) developed IC. Their prognosis was poor compared with patients without IC (3-year survival 23.4% vs 76.2%; P = 0.008). Two factors, namely, time between hepatectomy and TACE (4.8 months vs. 16.0 months, P = 0.001) and TACE for a remnant liver mobilized during hepatectomy (P = 0.001), were associated with IC. Receiver operating characteristic (ROC) curve analysis showed that 7 months was the more discriminant cutoff for the time period. IC occurred in 33.3% of the patients with the two factors, in 5.0% of those with one factor, and 0% in the absence of any factors. CONCLUSION: TACE for treating HCC recurrence carries a high risk of IC when performed early after hepatectomy in a previously mobilized liver. Our results might aid in identifying candidates for TACE for recurrent HCC, considering the major effect on patient outcomes.

CRISPR-Cas3 induces broad and unidirectional genome editing in human cells.

Although single-component Class 2 CRISPR systems, such as type II Cas9 or type V Cas12a (Cpf1), are widely used for genome editing in eukaryotic cells, the application of multi-component Class 1 CRISPR has been less developed. Here we demonstrate that type I-E CRISPR mediates distinct DNA cleavage activity in human cells. Notably, Cas3, which possesses helicase and nuclease activity, predominantly triggered several thousand base pair deletions upstream of the 5'-ARG protospacer adjacent motif (PAM), without prominent off-target activity. This Cas3-mediated directional and broad DNA degradation can be used to introduce functional gene knockouts and knock-ins. As an example of potential therapeutic applications, we show Cas3-mediated exon-skipping of the Duchenne muscular dystrophy (DMD) gene in patient-induced pluripotent stem cells (iPSCs). These findings broaden our understanding of the Class 1 CRISPR system, which may serve as a unique genome editing tool in eukaryotic cells distinct from the Class 2 CRISPR system.

Roles of Pin1 as a Key Molecule for EMT Induction by Activation of STAT3 and NF-κB in Human Gallbladder Cancer.

BACKGROUND: Despite developments in multidisciplinary treatment, the prognosis for advanced gallbladder cancer (GBC) still is poor because of its rapid progression. Epithelial-mesenchymal transition (EMT) plays a central role in promoting tumor invasion and metastasis in malignancies thorough signal transducer and activator of transcription-3 (STAT3) and nuclear factor κB (NF-κB) activation. Whereas Pin1 mediates STAT3 and NF-κB activation, the involvement of Pin1 in GBC progression is unclear. METHODS: Factors regulating Pin1-related STAT3 and NF-κB activation were evaluated using surgical specimens collected from 76 GBC patients, GBC cells, and orthotopic GBC xenograft mice. RESULTS: In the patients with GBC, high Pin1 expression in GBC was associated with aggressive tumor invasion and increased tumor metastasis, and was an independent factor for a poor prognosis. Pin1 expression was correlated with phosphorylation of STAT3(Ser727) and NF-κB-p65(Ser276), thereby activating STAT3 and NF-κB in GBC. Pin1-mediated STAT3 and NF-κB activation induced EMT in GBC. When Pin1 knockdown was performed in GBC cells, the phosphorylation of STAT3(Ser727) and NF-κB-p65(Ser276) was inhibited, and STAT3 and NF-κB activation was suppressed. Inactivation of STAT3 and NF-κB in Pin1-depleted cells decreased snail and zeb-2 expression, thereby reducing the rate of mesenchymal-like cells, suggesting that EMT was inhibited in GBC cells. PiB, a Pin1-specific inhibitor, inhibited EMT and reduced tumor cell invasion by inactivating STAT3 and NF-κB in vitro. Moreover, PiB treatment inhibited lymph node metastasis and intrahepatic metastasis in orthotopic GBC xenograft tumor in vivo. CONCLUSIONS: Pin1 accelerates GBC invasion and metastasis by activating STAT3 and NF-κB. Therefore, Pin1 inhibition by PiB is an excellent therapy for GBC by safely inhibiting its metastasis.