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[Introduction] Proliferative lesions in the breast have been implicated in the development of breast cancer. Previous studies showed that some proliferative lesions and adjacent breast cancers shared common genetic alterations, suggesting that these originated from the same ancestral cell. However, the clonal structure of normal epithelia and their clonal history during evolution to cancer are poorly understood. In this study, we analyzed genetic profiles of normal epithelia and proliferative lesions in the cancer-borne breast to illustrate the clonal evolution of cancer from a normal epithelial cell. [Methods] Single cell-derived organoids (n=47) were established from breast milk of 4 healthy women aged 22–36 and normal breast tissue of 15 breast cancer patients aged 29–83 to evaluate somatic mutation rate in normal epithelial cells. Multiple normal lobules and proliferative lesions together with cancer lesions were collected using laser-capture micro-dissection (LCM) from fresh frozen (n=3) or formalin-fixed paraffin-embedded (n=5) surgical specimens in 9 premenopausal breast cancer patients. Somatic mutations and copy number alterations were evaluated using whole-genome sequencing. [Results] The mutation profile of single cell-derived organoids suggests that somatic mutations accumulate in normal mammary epithelial cells at a constant rate of 19.4/genome/year before menopause, and the mutation rate decreases to 6.9/genome/year after menopause. Parity was negatively associated with mutation number (-49.3 per life birth). In total, we analyzed 143 LCM samples, including those from 72 normal lobules, 43 proliferative lesions, and 19 non-invasive and 9 invasive cancer samples. Five cases showed a large expansion of proliferative lesions sharing a substantial number of somatic mutations with cancer. These lesions expanded over a distance of 35-90 mm, sharing tens to hundreds of mutations including those in breast cancer-related driver genes, such as PIK3CA, AKT1, GATA3, CBFB and PTEN, while harboring private mutations or copy number alterations of their own. Of interest, the cancers in 4 out of these 5 cases was luminal-A type invasive ducal carcinoma or ER-positive HER2-negative ductal carcinoma in situ, and characterized in common by the presence of der(1;16), concurrent whole-arm 1q gain and 16q loss, in both cancer and proliferative lesions. Phylogenetic analysis adapted with the mutation rate in normal cells predicted that der(1;16) had been acquired between puberty and early 20’s, and the common ancestors of non-cancerous and cancerous lesions emerged by early 30’s, >10 years earlier than at the time of cancer diagnosis. By contrast, analysis of non-cancerous lobules unrelated to cancer showed that der(1;16)-negative non-cancer clones that had emerged after puberty stayed within a single lobule or spatially confined to adjacent lobules and rarely expanded to a large area as observed for those carrying der(1;16), even if the clones had acquired mutations in driver genes such as PIK3CA and PIK3R1, which highlighted the role of der(1;16) in wide clonal expansion. [Conclusions] Our results suggest that in some breast cancer cases, particularly in those with der(1;16), a highly recurrent translocation accounting for the major subset of Luminal A breast cancer, the clones with the funder driver alterations expanded macroscopically long before the onset of cancer, in which further clonal evolutions recursively occur multi-focally, giving rise to multiple proliferative lesions and ultimately, invasive cancers. Our findings provide new insight into the early development of breast cancer. Citation Format: Tomomi Nishimura, Nobuyuki Kakiuchi, Kenichi Yoshida, Takaki Sakurai, Tatsuki R. Kataoka, Eiji Kondoh, Yoshitsugu Chigusa, Masahiko Kawai, Morio Sawada, Takuya Inoue, Yasuhide Takeuchi, Hirona Maeda, Satoko Baba, Yusuke Shiozawa, Ryunosuke Saiki, Masahiro M. Nakagawa, Yasuhito Nannya, Yotaro Ochi, Tomonori Hirano, Yukiko Inagaki-Kawata, Kosuke Aoki, Masahiro Hirata, Eiji Suzuki, Masahiro Takada, Masahiro Kawashima, Kosuke Kawaguchi, Kenichi Chiba, Yuichi Shiraishi, Junko Takita, Satoru Miyano, Masaki Mandai, Kengo Takeuchi, Hironori Haga, Masakazu Toi, Seishi Ogawa. Clonal evolution of mammary epithelial cells into breast cancers [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-13-04.

Histological analysis of ICC and the incidence of HCC developed in AlbcreKP; RosaNotchOE and AlbcreKP; RosaNotchOE; Hes1flox/flox mice at 8 weeks of age.

Leading edge genes in gene set enrichment analysys using HALLMARK gene set of KRAS_SIGNALING_UP.

Intestinal metaplasia (IM) is a risk factor for gastric cancer following infection with Helicobacter pylori. To explore the susceptibility of pure gastric IM to cancer development, we investigated genetic alterations in single IM gastric glands. We isolated 50 single IM or non-IM glands from the inflamed gastric mucosa of 11 patients with intramucosal gastric carcinoma (IGC) and 4 patients without IGC; 19 single glands in the noninflamed gastric mucosa of 11 individuals from our cohort and previous dataset were also included as controls. Whole-exome sequencing of single glands revealed significantly higher accumulation of somatic mutations in various genes within IM glands compared with non-IM glands. Clonal ordering analysis showed that IM glands expanded to form clusters with shared mutations. In addition, targeted-capture deep sequencing and copy number (CN) analyses were performed in 96 clustered IM or non-IM gastric glands from 26 patients with IGC. CN analyses were also performed on 41 IGC samples and The Cancer Genome Atlas-Stomach Adenocarcinoma datasets. These analyses revealed that polyclonally expanded IM commonly acquired CN aberrations (CNA), including amplification of chromosomes 8, 20, and 2. A large portion of clustered IM glands typically consisted of common CNAs rather than other cancer-related mutations. Moreover, the CNA patterns of clustered IM glands were similar to those of IGC, indicative of precancerous conditions. Taken together, these findings suggest that, in the gastric mucosa inflamed with H. pylori infection, IM glands expand via acquisition of CNAs comparable with those of IGC, contributing to field cancerization.Significance:This study contributes to our understanding of gastric intestinal metaplasia as a risk factor for gastric adenocarcinoma via their multifocal expansion and acquisition of CNAs and somatic mutations.

Intrahepatic cholangiocarcinoma (ICC) is frequently driven by aberrant KRAS activation and develops in the liver with chronic inflammation. Although the Notch signaling pathway is critically involved in ICC development, detailed mechanisms of Notch-driven ICC development are still unknown. Here, we use mice whose Notch signaling is genetically engineered to show that the Notch signaling pathway, specifically the Notch/Hes1 axis, plays an essential role in expanding ductular cells in the liver with chronic inflammation or oncogenic Kras activation. Activation of Notch1 enhanced the development of proliferating ductal cells (PDC) in injured livers, while depletion of Hes1 led to suppression. In correlation with PDC expansion, ICC development was also regulated by the Notch/Hes1 axis and suppressed by Hes1 depletion. Lineage-tracing experiments using EpcamcreERT2 mice further confirmed that Hes1 plays a critical role in the induction of PDC and that ICC could originate from PDC. Analysis of human ICC specimens showed PDC in nonneoplastic background tissues, confirming HES1 expression in both PDC and ICC tumor cells. Our findings provide novel direct experimental evidence that Hes1 plays an essential role in the development of ICC via PDC.Significance:This study contributes to the identification of the cells of origin that initiate ICC and suggests that HES1 may represent a therapeutic target in ICC.

Supplementary Data from Expansion of Gastric Intestinal Metaplasia with Copy Number Aberrations Contributes to Field Cancerization

Supplemental Data

Intestinal metaplasia (IM) is a risk factor for gastric cancer following infection with Helicobacter pylori. To explore the susceptibility of pure gastric IM to cancer development, we investigated genetic alterations in single IM gastric glands. We isolated 50 single IM or non-IM glands from the inflamed gastric mucosa of 11 patients with intramucosal gastric carcinoma (IGC) and 4 patients without IGC; 19 single glands in the noninflamed gastric mucosa of 11 individuals from our cohort and previous dataset were also included as controls. Whole-exome sequencing of single glands revealed significantly higher accumulation of somatic mutations in various genes within IM glands compared with non-IM glands. Clonal ordering analysis showed that IM glands expanded to form clusters with shared mutations. In addition, targeted-capture deep sequencing and copy number (CN) analyses were performed in 96 clustered IM or non-IM gastric glands from 26 patients with IGC. CN analyses were also performed on 41 IGC samples and The Cancer Genome Atlas-Stomach Adenocarcinoma datasets. These analyses revealed that polyclonally expanded IM commonly acquired CN aberrations (CNA), including amplification of chromosomes 8, 20, and 2. A large portion of clustered IM glands typically consisted of common CNAs rather than other cancer-related mutations. Moreover, the CNA patterns of clustered IM glands were similar to those of IGC, indicative of precancerous conditions. Taken together, these findings suggest that, in the gastric mucosa inflamed with H. pylori infection, IM glands expand via acquisition of CNAs comparable with those of IGC, contributing to field cancerization. Significance: This study contributes to our understanding of gastric intestinal metaplasia as a risk factor for gastric adenocarcinoma via their multifocal expansion and acquisition of CNAs and somatic mutations.

In Helicobacter pylori (Hp)-uninfected individuals, diffuse-type gastric cancer (DGC) was reported as the most common type of cancer. However, the carcinogenic mechanism of Hp-uninfected sporadic DGC is largely unknown. We performed whole-exome sequencing of Hp-uninfected DGCs and Hp-uninfected normal gastric mucosa. For advanced DGCs, external datasets were also analyzed. Eighteen patients (aged 29–78 years) with DGCs and nine normal subjects (28–77 years) were examined. The mutation burden in intramucosal DGCs (10–66 mutations per exome) from individuals aged 29–73 years was not very different from that in the normal gastric glands, which showed a constant mutation accumulation rate (0.33 mutations/exome/year). Unbiased dN/dS analysis showed that CDH1 somatic mutation was a driver mutation for intramucosal DGC. CDH1 mutation was more frequent in intramucosal DGCs (67%) than in advanced DGCs (27%). In contrast, TP53 mutation was more frequent in advanced DGCs (52%) than in intramucosal DGCs (0%). This discrepancy in mutations suggests that CDH1-mutated intramucosal DGCs make a relatively small contribution to advanced DGC formation. Among the 16 intramucosal DGCs (median size, 6.5 mm), 15 DGCs were pure signet ring cell carcinoma (SRCC) with reduced E-cadherin expression and a low proliferative capacity (median Ki-67 index, 2.4%). Five SRCCs reviewed endoscopically over 2–5 years showed no progression. Impaired E-cadherin function due to CDH1 mutation was considered as an early carcinogenic event of Hp-uninfected intramucosal SRCC. Genetic and clinical analyses suggest that Hp-uninfected intramucosal SRCCs may be less likely to develop into advanced DGCs.

Background: Chronic kidney disease is frequently associated with persistent inflammation, which results in fibrosis in the stroma, a reduced number of renal tubules, the formation of multiple cystic lesions, ultimately terminating in renal failure and hemodialysis. Given the high incidence of renal cell carcinoma (RCC) in hemodialysis patients, suggesting a relationship between tissue remodeling by chronic inflammation and carcinogenesis. However, little is known about the genetic background of cancer development from remaining tubules and cystic lesions in hemodialysis patients. Method: We enrolled 5 patients under hemodialysis who were accompanied by acquired cystic kidney disease and underwent radical nephrectomy for RCC. Surgical specimens were fixed with alcohol-based solution and paraffin-embedded, and after H&E staining, subjected to laser capture microdissection (LCM) to collect remaining tubules and cysts containing approximately 200 cells. DNA was extracted and analyzed for somatic mutations and copy number alteration by whole-exome sequencing. Result: In total, we collected 161 LCM samples, including 118 from proximal tubules, 17 from collecting ducts, and 26 from cysts. Median variant allele frequencies of detected mutations were 0.237 in proximal tubules, 0.133 in collecting duct, and 0.381 in cysts, indicating larger clonal expansion in proximal tubules and cysts than in collecting ducts. Proximal tubules and cysts contained recurrent mutations in FAT1 (11% and 8%, respectively), STAG2 (5% and 8%), and PTEN (3% and 4%), whereas no recurrent driver mutations were identified in collecting ducts. In copy number analysis, 26% of proximal tubules, 35% of collecting ducts, and 80% of cysts had copy number alterations. Of note, samples from cysts had more copy number alterations compared to remaining tubules (on average, 2.3 vs. 1.1). Proximal tubules had recurrent copy number gains of chromosomes 3, 7, 10, 18, and 20, and loss of chromosome 22 and samples from collecting ducts showed gains of chromosome 7 and loss of chromosome 18. In cysts, gains of chromosomes 2, 3, 7, 10, 12, and 16 and loss of chromosomes 15, 16, 21, and 22 were frequently observed. Copy number profile in cysts was similar to that of papillary RCC and acquired cystic disease-associated RCC. Conclusion: In the end-stage cystic kidney, proximal tubules and cysts showed an enrichment of driver mutations commonly found in papillary RCC and RCC with acquired cystic disease, reflecting the fact that the incidence of these two types of RCC increases as the duration of dialysis becomes longer. In addition, clear cell RCC drivers, such as VHL mutation and loss of chromosome 3, were not observed, possibly explaining the rare occurrence of clear cell RCC in hemodialysis patients. Since cysts had more frequent copy number alterations than remaining tubules, cysts in the end-stage kidney might be precursor lesions of RCC. Citation Format: Kosuke Ieiri, Nobuyuki Kakiuchi, Tomonori Hirano, Tomomi Nishimura, Koichi Watanabe, Hiroko Tanaka, Satoru Miyano, Dai Takamatsu, Keisuke Monji, Eiji Kashiwagi, Masaki Shiota, Junichi Inokuchi, Masatoshi Eto, Seishi Ogawa. Genomic analysis of end-stage renal disease [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6066.

Introduction: The majority of patients with pancreatic cancer are inoperable at the time of diagnosis and even those cases with successful tumor resection for early-stage tumors frequently had second tumors within the remaining pancreas shortly after the surgery, contributing to a poor clinical outcome. Moreover, synchronous multiple pancreatic cancers were often reported. One of the long-standing issues about such synchronous and/or metachronous multiple pancreatic cancers is their clonal origins. In this study, we aimed to reveal the origin of multiple pancreatic cancers using an unbiased detection of somatic mutations in primary and metachronous cancers as well as adjacent precursor lesions. Methods: Serially obtained formalin-fixed paraffin-embedded surgical specimens from 18 patients who had undergone curative surgery for an early-stage pancreatic cancer were subjected to laser microdissection for the enrichment of tumor and precancerous lesions, from which DNA was extracted and analyzed for somatic mutations using whole-exome sequencing (WES) with matched normal DNA. Based on shared and private mutations across different samples, we interrogated history of clonal evolution of these lesions. Results: We analyzed 18 patients with multiple pancreatic cancers of 2 metachronous and synchronous, 14 metachronous, and 2 synchronous tumors. In metachronous cases, pathology for primary cancer were margin-negative in all patients, and the median interval between the initial and second surgery was 38.9 months (7 - 85 months). In addition, a total of 20 pancreatic intraepithelial neoplasia (PanIN) from 5 of these cases were analyzed. We identified a median of 66 (range: 31-232) and 20 (14-42) somatic mutations in cancer and PanIN lesions, respectively. None of the patients have known pathogenic germline variants. All samples had one or more driver mutations. In most cases (N=17), sharing many somatic mutations, multiple tumors had a common clonal origin. In another synchronous case, the synchronous tumors shared only one mutation, suggesting that they were clonally independent tumors. By contrast, none of the mutations other than hotspot KRAS mutations were shared between precursor and cancer lesions. While multiple independent clones were observed in precancerous lesions, most of multiple cancers originated from a common ancestor. Moreover, negative pathology of the margins at the initial surgery suggested that those multiple lesions arose from distant dissemination or metastasis, rather than contiguous, intraductal invasion. Conclusions: In conclusion, our study suggests that even early pancreatic cancer might be disseminated within the pancreas and contribute to synchronous and metachronous cancers. Citation Format: Tomonori Hirano, Nobuyuki Kakiuchi, Yasuhide Takeuchi, Tomomi Nishimura, Toshihiko Masui, Kazuyuki Nagai, Takayuki Anazawa, Sachiko Minamigushi, Hironori Haga, Norimitsu Uza, Hiroshi Seno, Yuzo Kodama, Atsuhiro Masuda, Takeshi Tanaka, Seishi Ogawa, Hiroko Tanaka, Satoru Miyano. Origin of synchronous or metachronous multiple pancreatic cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1511.

Intrahepatic cholangiocarcinoma (ICC) is frequently driven by aberrant KRAS activation and develops in the liver with chronic inflammation. Although the Notch signaling pathway is critically involved in ICC development, detailed mechanisms of Notch-driven ICC development are still unknown. Here, we use mice whose Notch signaling is genetically engineered to show that the Notch signaling pathway, specifically the Notch/Hes1 axis, plays an essential role in expanding ductular cells in the liver with chronic inflammation or oncogenic Kras activation. Activation of Notch1 enhanced the development of proliferating ductal cells (PDC) in injured livers, while depletion of Hes1 led to suppression. In correlation with PDC expansion, ICC development was also regulated by the Notch/Hes1 axis and suppressed by Hes1 depletion. Lineage-tracing experiments using EpcamcreERT2 mice further confirmed that Hes1 plays a critical role in the induction of PDC and that ICC could originate from PDC. Analysis of human ICC specimens showed PDC in nonneoplastic background tissues, confirming HES1 expression in both PDC and ICC tumor cells. Our findings provide novel direct experimental evidence that Hes1 plays an essential role in the development of ICC via PDC. Significance: This study contributes to the identification of the cells of origin that initiate ICC and suggests that HES1 may represent a therapeutic target in ICC.

Background Accurate preoperative assessment of the longitudinal extension of perihilar cholangiocarcinoma (PHCC) is essential for treatment planning. Mapping biopsies for PHCC remain challenging owing to technical difficulties and insufficient sample amounts. The aim of this study was to investigate the usefulness of a novel technique for mapping biopsies of PHCC. Methods Our novel method focused on a biliary stent delivery system for mapping biopsies. Fifty patients with PHCC undergoing endoscopic transpapillary mapping biopsy using the novel method were reviewed from August 2015 to June 2019. Results The median number of biopsy samples was six (range 1 – 17), and the rate of adequate sampling was 91.4 % (266 /291). Biopsy from the intrahepatic bile duct was possible in 82.0 % of patients (41 /50), and negative margins were confirmed in the resected specimens from 34 /39 patients who underwent surgery (87.2 %). None of the patients had post-endoscopic retrograde cholangiopancreatography pancreatitis. Conclusions With our novel method, accurate assessment of the longitudinal extension of PHCC might be expected with minimal trauma to the duodenal papilla.

Hard, hydrophilic ions that hardly transport over the water-oil interface by imposing external electric potential could undergo facile transport with a trace of ligand. Such phenomena, called "shuttling", are elucidated by microscopic investigation with molecular dynamics simulations. The catalytic role manifests itself in a 2-D free-energy surface within the nanometer range of the interface. The free-energy landscape clearly distinguishes the condition that the catalytic shuttling plays a vital role in the ion transport. The mechanism associated with transient complex formation at the interface is shown to be widely relevant to the ion kinetics and extends the conventional concept of facilitated ion transport.

Tobacco smoking causes lung cancer1–3, a process that is driven by more than 60 carcinogens in cigarette smoke that directly damage and mutate DNA4,5. The profound effects of tobacco on the genome of lung cancer cells are well-documented6–10, but equivalent data for normal bronchial cells are lacking. Here we sequenced whole genomes of 632 colonies derived from single bronchial epithelial cells across 16 subjects. Tobacco smoking was the major influence on mutational burden, typically adding from 1,000 to 10,000 mutations per cell; massively increasing the variance both within and between subjects; and generating several distinct mutational signatures of substitutions and of insertions and deletions. A population of cells in individuals with a history of smoking had mutational burdens that were equivalent to those expected for people who had never smoked: these cells had less damage from tobacco-specific mutational processes, were fourfold more frequent in ex-smokers than current smokers and had considerably longer telomeres than their more-mutated counterparts. Driver mutations increased in frequency with age, affecting 4–14% of cells in middle-aged subjects who had never smoked. In current smokers, at least 25% of cells carried driver mutations and 0–6% of cells had two or even three drivers. Thus, tobacco smoking increases mutational burden, cell-to-cell heterogeneity and driver mutations, but quitting promotes replenishment of the bronchial epithelium from mitotically quiescent cells that have avoided tobacco mutagenesis. Whole-genome sequencing of normal bronchial epithelium from 16 individuals shows that tobacco smoking increases genomic heterogeneity, mutational burden and driver mutations, whereas stopping smoking promotes replenishment of the epithelium with near-normal cells.

Chronic inflammation is accompanied by recurring cycles of tissue destruction and repair and is associated with an increased risk of cancer1–3. However, how such cycles affect the clonal composition of tissues, particularly in terms of cancer development, remains unknown. Here we show that in patients with ulcerative colitis, the inflamed intestine undergoes widespread remodelling by pervasive clones, many of which are positively selected by acquiring mutations that commonly involve the NFKBIZ, TRAF3IP2, ZC3H12A, PIGR and HNRNPF genes and are implicated in the downregulation of IL-17 and other pro-inflammatory signals. Mutational profiles vary substantially between colitis-associated cancer and non-dysplastic tissues in ulcerative colitis, which indicates that there are distinct mechanisms of positive selection in both tissues. In particular, mutations in NFKBIZ are highly prevalent in the epithelium of patients with ulcerative colitis but rarely found in both sporadic and colitis-associated cancer, indicating that NFKBIZ-mutant cells are selected against during colorectal carcinogenesis. In further support of this negative selection, we found that tumour formation was significantly attenuated in Nfkbiz-mutant mice and cell competition was compromised by disruption of NFKBIZ in human colorectal cancer cells. Our results highlight common and discrete mechanisms of clonal selection in inflammatory tissues, which reveal unexpected cancer vulnerabilities that could potentially be exploited for therapeutics in colorectal cancer. In patients with ulcerative colitis, chronic inflammation can lead to remodelling of the colorectal epithelium through positive selection of clones with mutations in genes related to IL-17 signalling, which, however, might be negatively selected during colitis-associated carcinogenesis.

Computational analysis of difference spectra between two analogous systems is a challenging issue, as reliable estimation of a tiny difference spectrum requires an extraordinary precision of the two original spectra. We have developed an alternative new method to calculate the difference spectra under background-free conditions, which greatly improved the efficiency of computation. In this paper, we report further improvement by using efficient parallel implementation and the time correlation formula based on time derivative quantities. As a consequence, the present work achieved further remarkable acceleration in the calculations of difference infrared and Raman spectra in the order of magnitude and thereby allowed us by analyzing these difference spectra at a practical cost of computation.

[Introduction] The majority of patients with pancreatic cancer are inoperable at the time of diagnosis and even those cases with successful tumor resection for early-stage tumors frequently had second tumors within the remaining pancreas shortly after the surgery, contributing to a poor clinical outcome. Moreover, synchronous multiple pancreatic cancers were often reported. One of the long-standing issues about such synchronous and/or metachronous multiple pancreatic cancers is their clonal origins. In this study, we aimed to reveal the origin of multiple pancreatic cancers using an unbiased detection of somatic mutations in primary and metachronous cancers as well as adjacent precursor lesions. [Methods] Serially obtained formalin-fixed paraffin-embedded surgical specimens from 17 patients who had undergone curative surgery for an early-stage pancreatic cancer were subjected to laser microdissection for the enrichment of tumor and precancerous lesions, from which DNA was extracted and analyzed for somatic mutations using whole-exome sequencing (WES) with matched normal DNA. Based on shared and private mutations across different samples, we interrogated history of clonal evolution of these lesions. [Results] We analyzed 17 patients with multiple pancreatic cancers of 2 metachronous and synchronous, 10 metachronous, and 2 synchronous tumors. In metachronous cases, pathology for primary cancer were margin-negative in all patients, and the median interval between the initial and second surgery was 37.7 months (12 - 85 months). In addition, we analyzed 5 adjacent pancreatic intraepithelial neoplasia (PanIN) in one of these cases. We identified a median of 66 (range: 31-232) and 20 (14-42) somatic mutations in cancer and PanIN lesions, respectively. None of the patients have known pathogenic germline variants. All samples had one or more driver mutations. In most cases (N=16), sharing many somatic mutations, multiple tumors had a common clonal origin. In another synchronous case, the synchronous tumors shared only one mutation, suggesting that they were clonally independent tumors. By contrast, none of the mutations other than hotspot KRAS mutations were shared between precursor and cancer lesions. While multiple independent clones were observed in precancerous lesions, most of multiple cancers originated from a common ancestor. Moreover, negative pathology of the margins at the initial surgery suggested that those multiple lesions arose from distant dissemination or metastasis, rather than contiguous, intraductal invasion. [Conclusions] In conclusion, our study suggests that even early pancreatic cancer might be disseminated within the pancreas and contribute to synchronous and metachronous cancers. Citation Format: Tomonori Hirano, Nobuyuki Kakiuchi, Takeuchi Yasuhide, Tomomi Nishimura, Toshihiko Masui, Kazuyuki Nagai, Takayuki Anazawa, Sachiko Minamiguchi, Hironori Haga, Norimitsu Uza, Hiroshi Seno, Yuzo Kodama, Atsuhiro Masuda, Takeshi Tanaka, Yuichi Shiraishi, Satoru Miyano, Seishi Ogawa. Genetic analysis of synchronous or metachronous multiple pancreatic cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6198.

Transport of ions through liquid-liquid interfaces is of fundamental importance to a wide variety of applications. However, since it is quite challenging for experimentalists to directly and selectively observe molecules at the interfaces, microscopic mechanisms of ion transport have been largely presumed from kinetic information. This Perspective illustrates recent examples that molecular dynamics simulations with proper free energy surfaces clarified mechanistic pictures of ion transport. The key is a proper choice of coordinates and defining/calculating free energy surfaces in multidimensional space. Once the free energy surfaces for realistic systems are available, they naturally provide new insight into the ion transport in unprecedented details, including water finger, transient ion pairing, and electron transfer.

In Helicobacter pylori (Hp)-uninfected individuals, diffuse-type gastric cancer (DGC) was reported as the most common type of cancer. However, the carcinogenic mechanism of Hp-uninfected sporadic DGC is largely unknown.We performed whole-exome sequencing of Hp-uninfected DGCs and Hp-uninfected normal gastric mucosa. For advanced DGCs, external datasets were also analyzed.Eighteen patients (aged 29-78 years) with DGCs and nine normal subjects (28-77 years) were examined. The mutation burden in intramucosal DGCs (10-66 mutations per exome) from individuals aged 29-73 years was not very different from that in the normal gastric glands, which showed a constant mutation accumulation rate (0.33 mutations/exome/year). Unbiased dN/dS analysis showed that CDH1 somatic mutation was a driver mutation for intramucosal DGC. CDH1 mutation was more frequent in intramucosal DGCs (67%) than in advanced DGCs (27%). In contrast, TP53 mutation was more frequent in advanced DGCs (52%) than in intramucosal DGCs (0%). This discrepancy in mutations suggests that CDH1-mutated intramucosal DGCs make a relatively small contribution to advanced DGC formation. Among the 16 intramucosal DGCs (median size, 6.5 mm), 15 DGCs were pure signet ring cell carcinoma (SRCC) with reduced E-cadherin expression and a low proliferative capacity (median Ki-67 index, 2.4%). Five SRCCs reviewed endoscopically over 2-5 years showed no progression.Impaired E-cadherin function due to CDH1 mutation was considered as an early carcinogenic event of Hp-uninfected intramucosal SRCC. Genetic and clinical analyses suggest that Hp-uninfected intramucosal SRCCs may be less likely to develop into advanced DGCs.

Calculation of time correlation functions is a primary task in the computational analysis of sum frequency generation spectroscopy. This paper resolved basic issues to extract interface signals from the calculation. These issues stem from the boundary to restrict the bulk region, which renders the practical computation feasible at a finite and affordable cost. The boundary is found to have significant influences on the time correlation functions, which is closely related to the quadrupole contribution in the nonlinear susceptibility. Thus, we thoroughly examined these influences to establish a proper treatment in performing reliable spectroscopic analysis. We elucidated the distinction of the present boundary effects from the quadrupole contribution and also established a proper center of molecule to minimize the quadrupole effect in the time correlation functions. In the case of liquid water, the proper center was found to be close to the center of mass of a water molecule.

Summary Tobacco smoking causes lung cancer1–3, driven by the 60+ carcinogens in cigarette smoke that directly damage and mutate DNA4,5. The profound effects of tobacco on the lung cancer genome have been well documented6–10, but we lack equivalent data for normal bronchial cells. We sequenced whole genomes of 632 colonies derived from single bronchial epithelial cells across 16 subjects. Tobacco smoking was the major influence on mutation burden, adding 1000-10,000+ mutations/cell, massively increasing both within-subject and between-subject variance, and generating several distinct signatures of substitutions and indels. A population of cells in subjects with smoking history had mutation burdens equivalent to that expected for never-smokers: these cells had less damage from tobacco-specific mutational processes, were four-fold more frequent in ex-smokers than current smokers, and had significantly longer telomeres than their more mutated counterparts. Driver mutations increased in frequency with age, affecting 4-14% of cells in middle-aged never-smokers. In current smokers, ≥25% of cells carried driver mutations and 0-6% cells had 2 or even 3 drivers. Thus, tobacco smoking increases mutation burden, cell-to-cell heterogeneity and driver mutations, but quitting promotes replenishment of bronchial epithelium from mitotically quiescent cells that have avoided tobacco mutagenesis.

Background Few reports have evaluated the effectiveness of laser-cut, covered, self-expandable metal stents (LC-CSEMS) for unresectable malignant distal biliary obstruction (MDBO) and whether reintervention is feasible after placement. We describe our experience with LC-CSEMS placement for unresectable MDBO. Methods Patients undergoing LC-CSEMS placement for unresectable MDBO from November 2014 to December 2018 were reviewed. Recurrent biliary obstruction (RBO), median time to RBO (TRBO), and reintervention were analyzed. Results 52 patients who underwent LC-CSEMS placement for unresectable MDBO were included in the analysis. The RBO rate was 15 % and the median TRBO was 445 days. Reintervention was attempted in nine patients and stent removal was successful in eight patients. Conclusions Our experience suggests the effectiveness of LC-CSEMS in patients with unresectable MDBO in terms of stent patency and feasibility of reintervention.

The electron transfer (ET) reaction of ferrocene and ferricyanide at the water-dichloromethane interface, a typical liquid-liquid ET in electrochemistry, was intensively investigated with a three-dimensional free energy surface that fully describes the transport, association, and solvent fluctuation in the ET processes. The calculated free energy surface provides the comprehensive picture of the ET mechanism at the liquid-liquid interface. The present calculation revealed the heterogeneous route of ET that takes place over the interface, rather than the homogeneous one. The present conclusion is found to be consistent with previous results of electrochemical experiments by careful re-examination of the analysis.

Background and Aims: Ulcerative colitis is the most frequent type of inflammatory bowel disease and is characterized by colonic epithelial cell damage. Although involvement of autoimmunity has been suggested in ulcerative colitis, specific autoantigens/antibodies have yet to be elucidated. Methods: Using 23 recombinant integrin proteins, we performed enzyme-linked immunosorbent assays on sera from patients with ulcerative colitis and controls. Integrin expression and IgG binding in the colon tissues of patients with ulcerative colitis and controls were examined using immunofluorescence and coimmunoprecipitation, respectively. The blocking activity of autoantibodies was examined using solid-phase binding and cell adhesion assays. Results: Screening revealed that patients with ulcerative colitis had IgG antibodies against integrin αvβ6. In the training and validation groups, 103 of 112 (92.0%) patients with ulcerative colitis and only 8 of 155 (5.2%) controls had anti–integrin αvβ6 antibodies (P < .001), resulting in a sensitivity of 92.0% and a specificity of 94.8% for diagnosing ulcerative colitis. Anti–integrin αvβ6 antibody titers coincided with ulcerative colitis disease activity, and IgG1 was the major subclass. Patient IgG bound to the integrin αvβ6 expressed on colonic epithelial cells. Moreover, IgG of patients with ulcerative colitis blocked integrin αvβ6–fibronectin binding through an RGD (Arg-Gly-Asp) tripeptide motif and inhibited cell adhesion. Conclusions: A significant majority of patients with ulcerative colitis had autoantibodies against integrin αvβ6, which may serve as a potential diagnostic biomarker with high sensitivity and specificity., 指定難病「潰瘍性大腸炎」の自己抗体発見 --新たな診断や治療開発へ--. 京都大学プレスリリース. 2021-03-09.

We propose a constraint algorithm of molecular dynamics (MD) in the case that the Lagrange multipliers would bring about singularity, such as the constraint of linear molecules. Present extension t...

[Introduction] Early detection of pancreatic cancer is a key to curable surgery. However, many patients are diagnosed with an advanced disease and even in those who have an early stage tumor successfully resected, the development of metachronous cancer in the residual pancreas prevents a long-term survival. Thus, it is important to control metachronous tumors to improve clinical outcomes, whose pathogenesis, however, is poorly understood. In this study, we aimed to reveal the origin of metachronous tumors using an unbiased detection of somatic mutations in primary and metachronous tumors as well as adjacent precursor lesions. [Methods] Serially obtained formalin-fixed paraffin-embedded surgical specimens from 12 patients who had undergone curative surgery for an early-stage pancreatic cancer were subjected to laser microdissection for enrichment of tumor and precancerous components, from which DNA was extracted and analyzed for somatic mutations using whole-exome sequencing (WES) with matched normal DNA. Based on shared and private mutations across different samples, we interrogated history of clonal evolution of these lesions. [Results] Pathology for resected primary cancer was margin-negative in all patients. The median interval between the initial and second surgery was 34.6 months (12 - 65.1 months). Paired primary and metachronous cancers were analyzed in all 12 patients. An additional 5 pancreatic intraepithelial neoplasia (PanIN) samples were also analyzed in one case. We identified a median of 86 (range: 40-145) and 20 (14-42) somatic mutations using WES in cancers and PanIN lesions, respectively. None of the patients have known pathogenic germline variants. All samples had one or more driver mutations. In each patient, all driver mutations and many passenger mutations were shared between primary and metachronous cancer samples, suggesting that those metachronous tumors are evolutionally closely related to the primary cancer, despite long years before recurrence. Negative pathology of the margins at the initial surgery suggested that those metachronous lesions originated from distant dissemination or metastasis, rather than contiguous, intraductal invasion. By contrast, none of the mutations other than hotspot KRAS mutations were shared between precursor lesions and cancer samples. Despite multiple independent clones in the precancerous lesion, all metachronous tumors originated from the primary lesions in this study. [Conclusions] Our study suggests that even early pancreatic cancer might be disseminated within the pancreas and give rise to metachronous cancers, suggesting the importance of close monitoring of recurrence in the residual pancreas. Citation Format: Tomonori Hirano, Nobuyuki Kakiuchi, Yasuhide Takeuchi, Tomomi Nishimura, Toshihiko Masui, Sachiko Minamiguhi, Hironori Haga, Kenichi Chiba, Hiroko Tanaka, Yuichi Shiraishi, Satoru Miyano, Uza Norimitsu, Yuzo Kodama, Hiroshi Seno, Seishi Ogawa. Genetic analysis of metachronous pancreatic cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2185.

Skin is the largest organ system and is exposed to ultraviolet (UV) or sun light, a well-known mutagenic insult in various skin cancers, depending on the body site. A previous study showed that keratinocytes in sun-exposed eyelids harbor a high number of somatic mutations affecting a number of common driver mutations found in skin cancers, including NOTCH1, NOTCH2, NOTCH3, FAT1, TP53, and RBM10, suggesting pervasive expansion of clones before the development of skin cancer. However, clonal expansion in other skin sites in association not only with UV but also other insults, such as chronic inflammation is yet to be evaluated. Thus, to fully characterize the clonal expansion in the skin, we performed sequencing analysis of apparently normal skin, as well as that affected by chronic inflammatory conditions such as psoriasis and atopic dermatitis. We analyzed small fragments (0.2mm2) of epidermal samples accrued from surplus biopsy materials using exome sequencing. Analysis of bulk epidermal samples revealed a median of ~30 mutations/exome. Sun-exposed skin was notable for a high mutation burden (>200 mutations/exome). On the basis of the analysis of their variant allele frequencies, the median clone size was estimated to be ~0.1mm2. Several cases harbored large of clones that spanned multiple samples, which were invariably found in sun-exposed skin. In one of such cases, a clone spanned a region having more than 2mm in diameter. Skin affected by chronic inflammation showed a higher number of mutations. dN/dS analysis revealed positively selected driver genes that were similar to the previous report. Diseased skin showed a similar mutation profile that is seen in apparently normal sun-exposed skin. Analysis of mutational signature disclosed signatures that are related ageing (SBS1) and UV light exposure (SBS7). The contribution of SBS7 was prominent in the sun-exposed area but also detected in areas not exposed to sun light, such as the back, buttock, or thighs. By contrast, inflammation-affected skin samples are characterized by a large contribution of the SBS1 signature, likely reflecting increased cell cycles. In conclusion, our analysis shows UV light is a relevant driver of clonal expansion of skin keratinocytes. Chronic inflammation accelerates an accumulation of somatic mutations in and/or clonal expansion of keratinocytes. Citation Format: Yoshihiro Ishida, Nobuyuki Kakiuchi, Yoichi Fujii, Tomonori Hirano, Yoshikage Inoue, Tomomi Nishimura, Tatsuki Ogasawara, Hirona Maeda, Yuichi Shiraishi, Kenichi Chiba, Hiroko Tanaka, Satoru Miyano, Atsushi Otsuka, Kenji Kabashima, Seishi Ogawa. Clonal expansion of skin keratinocyte [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2675.

Chronic inflammation is a major cause of morbidity and mortality in the human population and is associated with a substantial cancer risk. It has recently been demonstrated that extensive tissue remodeling can take place even in apparently normal tissues in an age-dependent manner and is mediated by expansion of numerous clones carrying common cancer-related mutations. Given the inflammation-associated cancer risks, clonal expansion may also play a role in inflammation-associated tissue remodeling. However, it is largely unknown whether this actually happens, with what mutations it occurs if ever, and how it modifies cancer development. To address these issues, we studied ulcerative colitis (UC) which is a chronic inflammatory bowel disease. Long-standing UC is associated with an increased risk (15-20%) of colitis-associated colorectal cancer (CAC). We performed whole exome sequencing (WES) of single crypts (n=441) and bulk crypts (n=569) from patients with UC and control individuals, as well as CAC samples (n=99). WES of single crypts from control individuals revealed UC crypts showed a more than 3 times higher mutation rate than non-UC crypts. We analyzed history of crypt expansion in clusters of single crypts. From mutations detected in each crypt, we reconstructed phylogenetic trees of clusters of crypts. Non-UC trees showed many early branches before 20 years of age, whereas, UC trees were characterized by a prominently long trunk with many short branches, suggesting crypts rapidly expanded from a common ancestral crypt during the past few years. Bulk-crypt analysis from surgically resected UC rectums showed many expanded clones, among which the maximum clone was as large as 19 cm2 in size. While no driver gene was detected in control individuals, the dN/dS analysis on 399 non-dysplasia UC samples revealed 14 driver genes, within which NFKBIZ were most frequently mutated, followed by PIGR and ZC3H12A. These drivers displayed a surprising overlap to signaling pathways converging on NFKBIZ. Among these pathways most prominent was IL-17 signaling, in which IL-17 receptors (IL17RA/IL17RC) and its adaptor, TRAF3IP2 were significantly mutated in UC epithelium. A driver role of NFKBIZ mutations was confirmed by growth advantage of Nfkbiz-KO cells compared with control cells under IL-17A stimulation in vitro or in a DSS-induced colitis model. Despite a substantial overlap of driver genes between CAC and UC epithelium, their frequencies significantly differed between both tissues. NFKBIZ mutations, which is most prevalent in UC non-dysplasia, were never found in 99 CAC samples. We confirmed a negative role of NFKBIZ mutations by compromised cell-competition of NFKBIZ-disrupted colorectal cancer cells and significantly attenuated colitis-induced tumor formation in Nfkbiz-deficient mice. Our results highlight common and discrete mechanisms of clonal selection in inflammatory tissues, which unexpectedly reveal cancer vulnerability potentially utilized for therapeutics of colorectal cancer. Citation Format: Nobuyuki Kakiuchi, Kenichi Yoshida, Motoi Uchino, Takako Kihara, Akaki Kotaro, Yoshikage Inoue, Kenji Kawada, Satoshi Nagayama, Akira Yokoyama, Tomonori Hirano, Yasuhide Takeuchi, Hiroyuki Miyoshi, Yoshiharu Sakai, Hironori Haga, Seiichi Hirota, Hiroki Ikeuchi, Osamu Takeuchi, Satoru Miyano, Hiroshi Seno, Seishi Ogawa. Analysis of clonal expansion in epithelium affected by ulcerative colitis reveals frequent mutations affecting IL-17 signaling pathway and novel cancer vulnerability [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5875.

Background Lung cancer is the leading cause of cancer death, of which 80-90% are attributed to tobacco smoking. Our understanding of how tobacco exposure affects mutational burden, mutational signatures, driver mutations and clonal dynamics in normal lung tissue is limited. Also, genetic differences between normal cells and cancer cells in lung has not been fully elucidated. Methods To access the landscape of somatic mutations in normal bronchial epithelia, we sequenced whole genomes of 632 single-cell-derived colonies of lung basal cells from 16 patients, including 3 children, 4 never-smokers, 6 ex-smokers and 3 current smokers. Five patients had squamous cell carcinomas or carcinoma in situ, which we also sequenced to compare the genetic alteration between normal and cancer cells. Results A positive correlation between the number of base substitutions and age was observed, and 22 somatic mutations accumulated per year (95% confidence interval:20-25; P=10−8). Previous or current smoking significantly increased mutational burden: 2330 substitutions in ex-smokers and 5300 in current smokers. In addition, tobacco smoking is massively increasing both between-subject and within-subject variance of mutation burden. A population of cells in subjects with smoking history had mutation burdens equivalent to that expected for never-smokers: these cells had less damage from tobacco-specific mutational processes, and were four-fold more frequent in ex-smokers than current smokers. Signature analysis revealed that the same mutational signatures seen in lung cancers operate both in patients with and without smoking history: endogeneous signatures, including COSMIC signatures 1 and 5, and APOBEC-related signatures. Three mutational signatures were largely restricted to current or ex-smokers, including known smoking-related COSMIC signature 4 characterized by C>A transversions, signature 16 characterized by T>C mutations with extremely strong transcription strand bias and a new signature characterized by T>A and T>C mutations. dN/dS method identified driver genes in normal bronchial epithelium, including NOTCH1, TP53 and FAT1, which were overlapped with those seen in squamous cell lung cancers and other normal squamous tissues such as esophagus and skin. Driver mutations increased in frequency with age, affecting 4-14% of cells in middle-aged never-smokers. In current smokers, ≥25% of cells carried driver mutations and 0-6% cells had 2 or even 3 drivers. Layering driver mutations onto phylogenetic trees revealed that driver mutations occurred in early life. Compared to the normal bronchial epithelial cells, lung cancers and precancerous lesions were characterized by extensive copy number changes or structural variants and distinct set of driver mutations. Conclusions Tobacco smoking increases mutation burden, cell-to-cell heterogeneity and driver mutations. Our data of genetic lesions in normal bronchial cells provides insights into genetic alterations that drive carcinogenesis in lung. Citation Format: Kenichi Yoshida, Kate HC Gowers, Henry Lee-Six, Deepak P. Chandrasekharan, Tim Coorens, Elizabeth F. Maughan, Kathryn Beal, Andrew Menzies, Fraser R. Millar, Elizabeth Anderson, Sarah E. Clarke, Adam Pennycuick, Ricky M. Thackeray, Colin R. Butler, Nobuyuki Kakiuchi, Tomonori Hirano, Robert E. Hynds, Michael R. Stratton, Inigo Martincorena, Sam M. Janes, Peter J. Campbell. Tobacco exposure and somatic mutations in normal bronchial epithelia [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2342.

Clonal expansion in aged normal tissues has been implicated in cancer development. However, its chronology and risk-dependence are poorly understood. Esophageal squamous cell carcinoma (ESCC) is a predominant esophageal cancer among Asian populations and substantially affected by heavy smoking and drinking, likely through a ‘field effect’. To elucidate the role of these lifestyle risks on ESCC development, we investigated clonal expansion in physiologically normal esophageal epithelia (PNE) using multiple microscale sampling, as small as 0.2 mm2 in size, followed by an unbiased detection of somatic mutations with whole exome sequencing. Mutations were detected in most of PNE samples (151/157), where none of the mutations were shared between samples collected >10 mm apart. The number of mutations and their allele frequency increased with age, suggesting age-related clonal expansion in PNE (ARCE), which was significantly promoted by heavy smoking and drinking. Mutations were dominated by age-related patterns and a still poorly-defined, ‘esophagus-specific signature, as well as a COSMIC 16-like signature. The latter has recently been related to alcohol drinking and was enriched in high-risk samples, which was confirmed by whole genome sequencing of single cell-derived colonies. As many as 10 genes were significantly mutated or positively selected in ARCE. Among most commonly affected genes were NOTCH1, TP53, FAT1, PPM1D, NOTCH2, and NOTCH3, which substantially differed from those in ESCC, showing prominent over-representation of NOTCH1, PPM1D, FAT1 and NOTCH2, and significant underrepresentation of TP53, NFE2L2, and CDKN2A were significantly underrepresented, suggesting different mechanisms of positive selection between ARCE and ESCC. Driver mutations were detected more frequently and in higher numbers in high-risk PNE samples than low-risk ones, with accentuated NOTCH1, TP53 and PPM1D mutations. Analyses of densely collected micro-scale samples (0.2 mm2) disclosed fine structure of ARCE with its chronological history. Driver-mutated clones emerge multifocally from early childhood as early as Citation Format: Akira Yokoyama, Nobuyuki Kakiuchi, Tetsuichi Yoshizato, Yasuhito Nannya, Hiromichi Suzuki, Yasuhide Takeuchi, Yusuke Shiozawa, Yusuke Sato, Kosuke Aoki, Soo kim, Yoichi Fujii, Kenichi Yoshida, Keisuke Kataoka, Masahiro M. Nakagawa, Yoshikage Inoue, Tomonori Hirano, Yuichi Shiraishi, Kenichi Chiba, Hiroko Tanaka, Masashi Sanada, Shinya Ohashi, Shin’ichi Miyamoto, Shigeru Tsunoda, Koshi Mimori, Sachiko Minamiguchi, Satoru Miyano, Hideki Makishima, Manabu Muto, Seishi Ogawa. Chronology and risk-dependence of age-related remodelling of oesophageal epithelia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3322.

Pancreatic neuroendocrine neoplasms (PanNENs) are pancreatic tumors with neuroendocrine differentiation. PanNENs are classified into three grades according to the proliferating cell fraction as measured by Ki-67 or mitotic index. Among them, PanNEN grade 3 (PanNEN G3) shows the highest Ki-67 index > 20%, which is morphologically divided into two categories, well-differentiated (PanNET G3) and poorly differentiated (PanNEC) tumors. Although sharing some overlapping histological features, both tumors also differ from each other with respect of clinical characteristics. To date, the molecular basis of these difference and similarity are poorly understood. To understand their molecular pathogenesis, we performed comprehensive genetic analysis on PanNEN G3. In total, we enrolled 25 PanNEN G3 cases, including 13 PanNET G3, 10 PanNEC, and 2 PanNEN G3 NOS, which were analyzed for somatic mutations. All tumors showed neuroendocrine markers such as synaptophysin or chromogranin A. The median Ki67 index of PanNET G3 was 30.5% (15-78%), which was lower than that of PanNEC (median 70.9%, range 44-100 %). Genomic DNA was extracted from microdissected tumors and paired normal tissues from formalin-fixed paraffin-embedded PanNEN G3 specimens guided by H&E stained sections and subjected to whole-exome sequencing (WES). Copy number alterations were also analyzed on the basis of sequencing data. All tumor specimens were centrally reviewed by the two expert pathologists and assigned to PanNET G3, PanNEC, or PanNEN G3 not otherwise specified (PanNEN G3 NOS). WES analysis detected a total of 1,688 somatic mutations with a median of 45 (range 21-450)/sample. There was no clear difference in mutational burden between PanNET G3 and PanNEC. Combined with copy number alterations, mutations most frequently affected TP53 (40%), followed by MEN1(28%), SMAD4 (28%), KRAS(24%), and CDKN2A (20%). Frequently mutated in PanNET G1/2, MEN1, DAXX, and ATRX mutations were associated with well-differentiated morphology (n=7/7), while tumors with KRAS or RB1 mutations exhibited poorly differentiated histology (n=5/5). MEN1, DAXX, and ATRX mutations frequently co-occurred with mutations in mTOR pathway genes, such as NF1, TSC2, or DEPDC5 (q Our results help understand a molecular basis of PanNEN G3, contributing to a better understanding and classification of PanNET G3 and PanNEC. Citation Format: Nobuyuki Kakiuchi, Kenichi Yoshida, Yusuke Shiozawa, Akira Yokoyama, Keisuke Kataoka, Yoshikage Inoue, Yasuhide Takeuchi, Tomonori Hirano, Yoichi Fujii, Hiroo Ueno, Susumu Hijioka, Nobumasa Mizuno, Waki Hosoda, Yasushi Yatabe, Kenichi Chiba, Hiroko Tanaka, Yuichi Shiraishi, Satoru Miyano, Toshihiko Masui, Shinji Uemoto, Akihiko Yoshizawa, Hironori Haga, Norimitsu Uza, Hiroshi Seno, Yuzo Kodama, Seishi Ogawa. Genetic analysis of pancreatic neuroendocrine neoplasms grade 3 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3429.

To examine factors affecting Hortonian overland flow generation in forested hillslopes, we measured overland flow at plots in Japanese cypress (Chamaecyparis obtusa), Japanese ceder (Cryptomeria japonica),and broadleaf forests of three study sites (Kochi, Mie, and Tokyo),which had different geology and climate. Overland flow occurred at all plots. Runoff ratios of overland flow were different among the three study sites rather than among forest species. By introducing overland flow index for each plot, we investigated relationships between the overland flow index and potential factors affecting overland flow generation. The index was not significantly associated with slope gradient and saturated hydraulic conductivity, whereas the index tended to be smaller as ground cover ratio increasing. This suggests that ground cover reduced rainfall impact to soil surface and thus contributed to preventing formation of structure crust, which inhibited infiltration and enhanced overland flow. The overland flow index increased as increases in silt and clay content in surface soils, implying that silt and clay content was an implicit factor influencing crust formation as a potential component of structure crust.; Language: Japanese

森林植生の樹種および管理状態と流域の短期流出特性との関連性を明らかにするために成木川流域源頭部に位置する間伐遅れの針葉樹林流域(1.29 ha)，落葉広葉樹林流域(1.28 ha)とヒノキ育成林流域(0.62 ha)の3 つの小流域において水文観測を行った。植生樹種及び間伐などの森林管理と林床被覆状態や土壌表層部の樹木の根系分布は密接に関連していると考えられ，間伐遅れのスギ・ヒノキ人工林では林床面が裸地化し，明瞭な根系層(樹木の細根が密集した表層土壌層位)が形成されていたのに対して，ヒノキ育成林及び落葉広葉樹林では林床面が下層植生や堆積リター層に覆われ，根系層は形成されていなかった。ヒノキ林・スギ林プロットの降雨に対する地表流の流出応答は育成林・広葉樹林プロットよりも大きかった。また，各プロットでは土壌の最終浸透能(6.4～26.8 mm／5min)よりも低いと考えられる降雨強度(4.0 mm／5min 未満)においても地表流が発生していたことに加えて，ヒノキ人工林斜面では根系流(根系層内を選択的に流下する表層流)が発生していたために，各プロットで観測される地表流の流出形態はヒノキ林・スギ林プロットでは根系流であり，育成林・広葉樹林プロットではリターフロー(落葉層などの堆積リター層内を流下する水流)ではないかと推察された。小規模秋雨イベントと中規模台風イベントともに各小流域の‘新しい水’の流出率は0.2～2.0％ と非常に小さいことから，‘新しい水’の構成成分は主に河道への直接降雨と河道近傍で発生する根系流やリターフローである可能性が高いと考えられた。成木川流域における間伐遅れの針葉樹林流域では根系流が存在するために他の2 流域にくらべて短期流出に対する‘新しい水’の寄与量が相対的に大きくなると考えられた。

There is a growing opinion that poorly managed plantation forests in Japan are contributing to increased storm runoff and erosion. Here we present evidence to the contrary from runoff plots at two scales (hillslope and 0·5 x 2 m plots) for several forest conditions in the Mie and Nariki catchments. Runoff coefficients from small plots in untended hinoki forests were variable but typically higher than from better managed or deciduous forests during small storms at Nariki; at Mie, runoff during small events was highly variable from all small plots but runoff coefficients were similar for hinoki plots with and without understory vegetation, while the deciduous plot had lower runoff coefficients. Storm runoff was less at the hillslope scale than the plot scale in Mie; these results were more evident at sites with better ground cover. During the largest storms at both sites, differences in runoff due to forest condition were not evident regardless of scale. Dynamic soil moisture tension measurements at Nariki indicated that during a large storm, flow in the upper organic-rich and root-permeated soil horizons was 3·2 times higher than measured overland runoff from a small hinoki plot with poor ground cover and 8·3 times higher than runoff from a deciduous forest plot. On the basis of field observations during storms, at least a portion of the monitored 'Hortonian overland flow' was actually occurring in this near-surface 'biomat'. Therefore our field measurements in both small and large plots potentially included biomat flow in addition to short-lived Hortonian runoff. Because overland flow decreased with increasing scale, rill erosion did not occur on hillslopes. Additionally, runoff coefficients were not significantly different among cover conditions during large storms; thus, the 'degraded' forest conditions appear not to greatly enhance peak flows or erosion potential at larger scales, especially when biomat flow is significant.