An Organoid Biobank of Neuroendocrine Neoplasms Enables Genotype-Phenotype Mapping.

Gastroenteropancreatic (GEP) neuroendocrine neoplasm (NEN) that consists of neuroendocrine tumor and neuroendocrine carcinoma (NEC) is a lethal but under-investigated disease owing to its rarity. To fill the scarcity of clinically relevant models of GEP-NEN, we here established 25 lines of NEN organoids and performed their comprehensive molecular characterization. GEP-NEN organoids recapitulated pathohistological and functional phenotypes of the original tumors. Whole-genome sequencing revealed frequent genetic alterations in TP53 and RB1 in GEP-NECs, and characteristic chromosome-wide loss of heterozygosity in GEP-NENs. Transcriptome analysis identified molecular subtypes that are distinguished by the expression of distinct transcription factors. GEP-NEN organoids gained independence from the stem cell niche irrespective of genetic mutations. Compound knockout of TP53 and RB1, together with overexpression of key transcription factors, conferred on the normal colonic epithelium phenotypes that are compatible with GEP-NEN biology. Altogether, our study not only provides genetic understanding of GEP-NEN, but also connects its genetics and biological phenotypes.

Technical Strategy for Pancreatic Body Cancers: A Raison d'etre of Distal Pancreatectomy with Portal Resection.

BACKGROUND: Advancements in multiagent chemotherapy have expanded the surgical indications for pancreatic cancer. Although pancreaticoduodenectomy (PD) with portal vein resection (PVR) has become widely adopted, distal pancreatectomy (DP) with PVR remains rarely performed because of its technical complexity. This study was designed to assess the feasibility of DP-PVR compared with PD-PVR for pancreatic body cancers, with a focus on PV complications and providing optimal reconstruction techniques when DP-PVR is necessary. METHODS: A retrospective review was conducted on consecutive pancreatic body cancer patients who underwent pancreatectomy with PVR between 2005 and 2020. An algorithm based on the anatomical relationship between the arteries and PV was used for optimal surgical selection. RESULTS: Among 119 patients, 32 underwent DP-PVR and 87 underwent PD-PVR. Various reconstruction techniques were employed in DP-PVR cases, including patch reconstruction, graft interposition, and wedge resection. The majority of PD-PVR cases involved end-to-end anastomosis. The length of PVR was shorter in DP-PVR (25 vs. 40 mm; p < 0.001). Although Clavien-Dindo ≥3a was higher in DP-PVR (p = 0.002), inpatient mortality and R0 status were similar. Complete PV occlusion occurred more frequently in DP-PVR than in PD-PVR (21.9% vs. 1.1%; p < 0.001). A cutoff value of 30 mm for PVR length was determined to be predictive of nonrecurrence-related PV occlusion after DP-PVR. The two groups did not differ significantly in recurrence or overall survival. CONCLUSIONS: DP-PVR had higher occlusion and postoperative complication rates than PD-PVR. These findings support the proposed algorithm and emphasize the importance of meticulous surgical manipulation when DP-PVR is deemed necessary.

Robotic Splenic Vessels Preserving Distal Pancreatectomy in a Post-Distal Gastrectomy Patient.

BACKGROUND: The optimal procedure during distal pancreatectomy (DP) for patients who have undergone distal gastrectomy (DG) remains unclear. Several papers on remnant gastric ischemia have reported that the preserved splenic vessels are essential for the proximal remnant stomach.1-4 We evaluated the outcomes of DP for post-DG patients in our hospital and introduced robotic splenic vessels preserving DP (R-SPDP). METHODS: Postoperative short-term outcomes of DP for post-DG patients during 2014 and 2021 were evaluated. Next, R-SPDP was performed for a post-DG patient with the intention of preserving the remnant stomach safely. The double bipolar method was used to dissect the adhesions around the splenic vessels.5,6 The splenic artery was clamped at the root side to prevent bleeding.7 All short gastric arteries and veins, which were the main feeders of the remnant stomach, were preserved and resection was completed. After resection, the indocyanine green (ICG) fluorescence angiography confirmed blood flow in the short gastric arteries and veins and good return blood flow to the splenic vein.8 RESULTS: Of four patients (50.0%, of 8 DP patients) in whom the remnant stomach was preserved, one conventional DP case had poor ICG perfusion and presented with remnant stomach ischemia postoperatively. The R-SPDP case with good ICG perfusion had a total operation time of 371 minutes and intraoperative blood loss of 10 mL. The oral diet was started on postoperative Day 3, and the postoperative course was uneventful. CONCLUSIONS: R-SPDP can be a good option for post-DG patients to preserve the remnant stomach safely.

"Conversion surgery" for locally advanced pancreatic cancer: A position paper by the study group at the joint meeting of the International Association of Pancreatology (IAP) & Japan Pancreas Society (JPS) 2022.

Locally advanced pancreatic cancer (LAPC), which progresses locally and surrounds major vessels, has historically been deemed unresectable. Surgery alone failed to provide curative resection and improve overall survival. With the advancements in treatment, reports have shown favorable results in LAPC after undergoing successful chemotherapy therapy or chemoradiation therapy followed by surgical resection, so-called "conversion surgery", at experienced high-volume centers. However, recognizing significant regional and institutional disparities in the management of LAPC, an international consensus meeting on conversion surgery for LAPC was held during the Joint Congress of the 26th Meeting of the International Association of Pancreatology (IAP) and the 53rd Annual Meeting of Japan Pancreas Society (JPS) in Kyoto in July 2022. During the meeting, presenters reported the current best multidisciplinary practices for LAPC, including preoperative modalities, best systemic treatment regimens and durations, procedures of conversion surgery with or without vascular resections, biomarkers, and genetic studies. It was unanimously agreed among the experts in this meeting that "cancer biology is surpassing locoregional anatomical resectability" in the era of effective multiagent treatment. The biology of pancreatic cancer has yet to be further elucidated, and we believe it is essential to improve the treatment outcomes of LAPC patients through continued efforts from each institution and more international collaboration. This article summarizes the agreement during the discussion amongst the experts in the meeting. We hope that this will serve as a foundation for future international collaboration and recommendations for future guidelines.

Curative Surgery and Ki-67 Value Rather Than Tumor Differentiation Predict the Survival of Patients With High-grade Neuroendocrine Neoplasms.

OBJECTIVE: To elucidate the role of surgery in patients with high-grade neuroendocrine neoplasms (hg-NENs) and Ki-67 more than 20%. BACKGROUND: Although surgery is the first treatment choice in patients with low-grade NENs, whether it increases the survival of patients with hg-NENs is debatable. METHODS: Between 2005 and 2018, 63 patients pathologically diagnosed with hg-NENs treated at our institution were retrospectively analyzed. The risk factors for overall survival (OS) and recurrence-free survival were analyzed, and OS was compared between each treatment group. RESULTS: The median observation time was 21.2 months, and the median Ki-67 value was 52%. Patients with hg-NENs were classified into low Ki-67 (Ki-67 <52%) and high Ki-67 (Ki-67 ≥52%) groups. Multivariate analysis for OS identified surgery (P = 0.013) and low Ki-67 value (P = 0.007) as independent risk factors, whereas morphological differentiation defined by the WHO 2017 criteria showed no association with OS. Patients with low Ki-67 value subjected to R0/1, R2, and chemotherapy had a median survival time of 83.8, 16.6, and 28.1 months, respectively. The median survival time for R0/1 group was significantly longer than that for chemotherapy group ( P = 0.001). However, no difference in survival was reported between patients from R0/1 and chemotherapy groups with high Ki-67. Ki-67 value could determine recurrence-free survival ( P = 0.006) in patients who underwent R0/1 surgery for pancreatic hg-NENs. CONCLUSIONS: R0/1 surgery predicted prognoses in the low Ki-67 group. The indication of surgery for patients with hg-NENs did not depend on tumor differentiation.

Cell-specific overexpression of COMT in dopaminergic neurons of Parkinson's disease.

The mechanism by which dopaminergic neurons are selectively affected in Parkinson's disease is not fully understood. In this study, we found a dramatic increase in the expression of catechol-O-methyltransferase (COMT), along with a lower level of DNA methylation, in induced pluripotent stem cell-derived dopaminergic neurons from patients with parkin (PARK2) gene mutations compared to those from healthy controls. In addition, a significant increase in the expression of COMT was found in dopaminergic neurons of isogenic PARK2 induced pluripotent stem cell lines that mimicked loss of function of PARK2 by CRISPR Cas9 technology. In dopamine transporter (DAT)-Cre mice, overexpression of COMT, specifically in dopaminergic neurons of the substantia nigra, produced cataleptic behaviours associated with impaired motor coordination. These findings suggest that upregulation of COMT, likely resulting from DNA hypomethylation, in dopaminergic neurons may contribute to the initial stage of neuronal dysfunction in Parkinson's disease.

Highly multiplex guide RNA expression units of CRISPR/Cas9 were completely stable using cosmid amplification in a novel polygonal structure.

BACKGROUND: Genome editing using the CRISPR/Cas9 system is now well documented in basic studies and is expected to be applied to gene therapy. Simultaneous expression of multiplex guide RNA (gRNA) and Cas9/Cas9 derivative is attractive for the efficient knockout of genes and a safe double-nicking strategy. However, such use is limited because highly multiplex gRNA-expressing units are difficult to maintain stably in plasmids as a result of deletion via homologous recombination. METHODS: Lambda in vitro packaging was used instead of transformation for the construction and preparation of large, cos-containing plasmid (cosmid). Polymerase chain reaction fragments containing multiplex gRNA units were obtained using the Four-guide Tandem method. Transfection was performed by lipofection. RESULTS: We constructed novel cosmids consisting of linearized plasmid-DNA fragments containing up to 16 copies of multiplex gRNA-expressing units as trimer or tetramer (polygonal cosmids). These cosmids behaved as if they were monomer plasmids, and multiplex units could stably be maintained and amplified with a lack of deletion. Surprisingly, the deleted cosmid was removed out simply by amplifying the cosmid stock using lambda packaging. The DNA fragments containing multiplex gRNA-units and Cas9 were transfected to 293 cells and were found to disrupt the X gene of hepatitis B virus by deleting a large region between the predicted sites. CONCLUSIONS: We present a simple method for overcoming the problem of constructing plasmids stably containing multiplex gRNA-expressing units. The method may enable the production of very large amounts of DNA fragments expressing intact, highly-multiplex gRNAs and Cas9/Cas9 derivatives for safe and efficient genome-editing therapy using non-viral vectors.

High-level expression by tissue/cancer-specific promoter with strict specificity using a single-adenoviral vector.

Tissue-/cancer-specific promoters for use in adenovirus vectors (AdVs) are valuable for elucidating specific gene functions and for use in gene therapy. However, low activity, non-specific expression and size limitations in the vector are always problems. Here, we developed a 'double-unit' AdV containing the Cre gene under the control of an α-fetoprotein promoter near the right end of its genome and bearing a compact 'excisional-expression' unit consisting of a target cDNA 'upstream' of a potent promoter between two loxPs near the left end of its genome. When Cre was expressed, the expression unit was excised as a circular molecule and strongly expressed. Undesired leak expression of Cre during virus preparation was completely suppressed by a dominant-negative Cre and a short-hairpin RNA against Cre. Using this novel construct, a very strict specificity was maintained while achieving a 40- to 90-fold higher expression level, compared with that attainable using a direct specific promoter. Therefore, the 'double-unit' AdV enabled us to produce a tissue-/cancer-specific promoter in an AdV with a high expression level and strict specificity.

[Recent progress in adenovirus vectors: focusing on VA-deleted AdV].

First-generation adenovirus vectors (FG-AdVs) are widely used because transduction efficiency of the vectors is very high. However, severe immune responses especially to the liver have been a serious problem of this vector. We succeeded to identify a viral protein that cause the immune responses and reported ''low-inflammatory AdVs'' that mostly solve this problem. However, to develop the ultimate form of this vector, it is necessary to remove virus-associated RNA (VA RNA) genes from the AdV vector genome. VA RNAs are transcribed by polymerase III; they are not essential for viral growth but have important roles to make appropriate circumstances for this virus. Large amount of VA RNAs are required in the late phase to support viral growth. Hence it is difficult to establish 293 cell lines that can support replication of AdVs lacking VA RNA genes (VA-deleted AdVs) supplying sufficient amount of VA RNA in trans. Recently we have developed a method for efficient production of VA-deleted AdVs and succeeded to obtain a high titer of VA-deleted AdVs. Then we construct VA-deleted AdVs expressing shRNA that knockdown the replication of hepatitis C virus (HCV). In fact, VA-deleted AdVs expressing these shRNAs suppressed HCV replication more effectively than conventional FG-AdV. Therefore, we showed that VA RNAs expressed from FG-AdVs probably compete with shRNA in the maturation pathway and reduce the effect of shRNAs. We think that VA-deleted AdV may substitute for current FG-AdVs and become a standard AdV.

Age distribution and prevalence in different age groups of four myositis-specific autoantibodies, including anti-ARS, anti-MDA5, anti-Mi-2, and anti-TIF1γ antibodies.

We accumulated the demographic information and analyzed the prevalence of myositis-specific antibodies (MSAs) in a large cohort across Japan as standard testing for MSAs becomes more widely available. This retrospective, observational, cohort study analyzed the records of individuals aged 0-99 years who are tested for serum MSAs at SRL Incorporation from January 2014 to April 2020 across Japan. An enzyme-linked immunosorbent assay testing was applied to determine the presence of anti-aminoacyl tRNA synthetase (anti-ARS), anti-Mi-2, anti-melanoma differentiation-associated gene 5 (anti-MDA5), or anti-transcriptional intermediary factor 1-γ (anti-TIF1γ) (Medical and Biological Laboratories). Anti-TIF1γ antibody was detected more in male patients than female patients. In contrast, women were predominant in patients with other MSAs. More than half of the anti-ARS or anti-TIF1γ antibody-positive patients were over 60 years old, although anti-MDA5 or anti-Mi-2-positive patients were mostly under <60 years old. Anti-MDA5 antibody-positive patients were mostly aged 40-59 years, while other MSA groups were mostly 60-79 years. Anti-MDA5 antibody was detected most frequently in the age range of 0-29 years. Anti-TIF1γ antibody was the second most commonly detected autoantibody in the age range of 0-19 years. Anti-ARS antibody was the most frequently detected autoantibody after the age of 30 years, and the frequency of anti-ARS gradually increased at more advanced ages. The second and third most detected autoantibodies were anti-MDA5 and anti-TIF1γ, respectively, in ages 30-79 years. We performed a nationwide >3-year evaluation of MSA detection in a routine diagnostic setting. This paper provides clinical images concerning the relationship between four MSA types and the distribution of sex and age in a large population.

Surgical approaches for minimally invasive distal pancreatectomy: A systematic review.

BACKGROUND: Minimally invasive distal pancreatectomy (MIDP) was initially performed for benign tumors, but recently its indications have steadily broadened to encompass other conditions including pancreatic malignancies. Thorough anatomical knowledge is mandatory for precise surgery in the era of minimally invasive surgery. However, expert consensus regarding anatomical landmarks to facilitate the safe performance of MIDP is still lacking. METHODS: A systematic literature search was performed using keywords to identify articles regarding the vascular anatomy and surgical approaches/techniques for MIDP. RESULTS: All of the systematic reviews revealed that MIDP was not associated with an increase in postoperative complications. Moreover, most showed that MIDP resulted in less blood loss than open surgery. Regarding surgical approaches for MIDP, a standardized stepwise procedure improved surgical outcomes, including blood loss, operative time, and major complications. There are two approaches to the splenic vessels, superior and inferior; however, no study has ever directly compared them with respect to clinical outcomes. The morphology of the splenic artery affects the difficulty of approaching the artery's root. To select an appropriate dissecting layer when performing posterior resection, thorough knowledge of the anatomy of the fascia, left renal vein/artery, and left adrenal gland is needed. CONCLUSIONS: In MIDP, a standardized approach and precise knowledge of anatomy facilitates safe surgery and has the advantage of a shorter learning curve. Anatomical features and landmarks are particularly important in cases of radical MIDP and splenic vessel preserving MIDP.

Hormonal tumor mapping for liver metastases of gastroenteropancreatic neuroendocrine neoplasms: a novel therapeutic strategy.

PURPOSE: In patients with metastatic functional gastroenteropancreatic neuroendocrine neoplasms (GEP-NENs), it is unknown what degree of tumor reduction is required to eliminate hormonal symptoms. We aimed to reduce hormonal symptoms derived from advanced GEP-NENs by efficient minimal intervention, constructing a hormonal tumor map of liver metastases. METHODS: Between 2013 and 2019, we treated 12 insulinoma or gastrinoma patients with liver metastases. Liver segments containing hormone-producing tumors were identified by injecting calcium gluconate via the hepatic arteries and monitoring the change in serum hormone concentration in the three hepatic veins. A greater-than-twofold increase in hormone concentration indicated a tumor-feeding vessel. RESULTS: Cases included eight insulinomas and four gastrinomas. Primary lesions were functional in three patients and nonfunctional in 9. Nine patients showed hormonal step-up indicating the presence of functional lesions; eight showed step-up in tumor-bearing liver segments, while one with synchronous liver metastases showed step-up only in the pancreatic region. Five patients underwent surgery. Serum hormone concentration decreased markedly after removing the culprit lesions in 3; immediate improvement in hormonal symptoms was achieved in all patients. Three patients with previous surgical treatment who showed step-up underwent transcatheter arterial embolization, achieving temporary improvement of hormonal symptoms. Four patients showed unclear localization of the hormone-producing tumors; treatment options were limited, resulting in poor outcomes. CONCLUSION: Hormonal tumor mapping demonstrated heterogeneity in hormone production among primary and metastatic tumors of GEP-NENs. Minimally invasive treatment based on hormonal mapping may be a viable alternative to conventional cytoreduction.

International Expert Consensus on Precision Anatomy for minimally invasive distal pancreatectomy: PAM-HBP Surgery Project.

BACKGROUND: Surgical views with high resolution and magnification have enabled us to recognize the precise anatomical structures that can be used as landmarks during minimally invasive distal pancreatectomy (MIDP). This study aimed to validate the usefulness of anatomy-based approaches for MIDP before and during the Expert Consensus Meeting: Precision Anatomy for Minimally Invasive HBP Surgery (February 24, 2021). METHODS: Twenty-five international MIDP experts developed clinical questions regarding surgical anatomy and approaches for MIDP. Studies identified via a comprehensive literature search were classified using Scottish Intercollegiate Guidelines Network methodology. Online Delphi voting was conducted after experts had drafted the recommendations, with the goal of obtaining >75% consensus. Experts discussed the revised recommendations in front of the validation committee and an international audience of 384 attendees. Finalized recommendations were made after a second round of online Delphi voting. RESULTS: Four clinical questions were addressed, resulting in 10 recommendations. All recommendations reached at least a 75% consensus among experts. CONCLUSIONS: The expert consensus on precision anatomy for MIDP has been presented as a set of recommendations based on available evidence and expert opinions. These recommendations should guide experts and trainees in performing safe MIDP and foster its appropriate dissemination worldwide.

International expert consensus on precision anatomy for minimally invasive pancreatoduodenectomy: PAM-HBP surgery project.

BACKGROUND: The anatomical structure around the pancreatic head is very complex and it is important to understand its precise anatomy and corresponding anatomical approach to safely perform minimally invasive pancreatoduodenectomy (MIPD). This consensus statement aimed to develop recommendations for elucidating the anatomy and surgical approaches to MIPD. METHODS: Studies identified via a comprehensive literature search were classified using the Scottish Intercollegiate Guidelines Network method. Delphi voting was conducted after experts had drafted recommendations, with a goal of obtaining >75% consensus. Experts discussed the revised recommendations with the validation committee and an international audience of 384 attendees. Finalized recommendations were made after a second round of online Delphi voting. RESULTS: Three clinical questions were addressed, providing six recommendations. All recommendations reached at least a consensus of 75%. Preoperatively evaluating the presence of anatomical variations and superior mesenteric artery (SMA) and superior mesenteric vein (SMV) branching patterns was recommended. Moreover, it was recommended to fully understand the anatomical approach to SMA and intraoperatively confirm the SMA course based on each anatomical landmark before initiating dissection. CONCLUSIONS: MIPD experts suggest that surgical trainees perform resection based on precise anatomical landmarks for safe and reliable MIPD.

Construction of adenovirus vectors simultaneously expressing four multiplex, double-nicking guide RNAs of CRISPR/Cas9 and in vivo genome editing.

Simultaneous expression of multiplex guide RNAs (gRNAs) is valuable for knockout of multiple genes and also for effective disruption of a gene by introducing multiple deletions. We developed a method of Tetraplex-guide Tandem for construction of cosmids containing four and eight multiplex gRNA-expressing units in one step utilizing lambda in vitro packaging. Using this method, we produced an adenovirus vector (AdV) containing four multiplex-gRNA units for two double-nicking sets. Unexpectedly, the AdV could stably be amplified to the scale sufficient for animal experiments with no detectable lack of the multiplex units. When the AdV containing gRNAs targeting the H2-Aa gene and an AdV expressing Cas9 nickase were mixed and doubly infected to mouse embryonic fibroblast cells, deletions were observed in more than 80% of the target gene even using double-nicking strategy. Indels were also detected in about 20% of the target gene at two sites in newborn mouse liver cells by intravenous injection. Interestingly, when one double-nicking site was disrupted, the other was simultaneously disrupted, implying that two genes in the same cell may simultaneously be disrupted in the AdV system. The AdVs expressing four multiplex gRNAs could offer simultaneous knockout of four genes or two genes by double-nicking cleavages with low off-target effect.