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Abstract In cancer genome analysis, identifying pathogenic alterations and assessing their effects on oncogenic processes is important. Although whole exome sequencing (WES) can effectively detect such changes, driver alterations could not be identified in 27.8% of the cases, according to a previous study. The objectives of the present study were to evaluate the utility of whole genome sequencing (WGS) and clarify its differences with WES in terms of driver alteration detection. For this purpose, WGS analysis was conducted on 177 driverless WES samples, selected from 5,480 fresh frozen samples derived from 5,140 Japanese patients with cancer. These samples were selected as primary tumor, both WES and transcriptome profiling were performed, estimated tumor content of ≥ 30%, and no driver alterations were identified by WES. WGS identified driver and likely driver alterations in 68.4 and 22.6% of the samples, respectively. The most frequent alteration type was oncogene amplification, followed by tumor suppressor gene deletion and small variants located outside the coding region. In the remaining 9.0% of samples, no such signals were identified; therefore, further investigations are required. The current study clearly demonstrated the role and utility of WGS in identifying genomic alterations that contribute to tumorigenesis.

Abstract The detection of copy number variations (CNVs) and somatic mutations in cancer is important for the selection of specific drugs for patients with cancer. In cancers with sporadic tumor cells, low tumor content prevents the accurate detection of somatic alterations using targeted sequencing. To efficiently identify CNVs, we performed tumor cell enrichment using tissue suspensions of formalin-fixed paraffin-embedded (FFPE) tissue sections with low tumor cell content. Tumor-enriched and residual fractions were separated from FFPE tissue suspensions of intestinal and diffuse-type gastric cancers containing sporadic tumor cells, and targeted sequencing was performed on 225 cancer-related genes. Sequencing of a targeted panel of cancer-related genes using tumor-enriched fractions increased the number of detectable CNVs and the copy number of amplified genes. Furthermore, CNV analysis using the normal cell-enriched residual fraction as a reference for CNV scoring allowed targeted sequencing to detect CNV characteristics of diffuse-type gastric cancer with low tumor content. Our approach improves the CNV detection rate in targeted sequencing with tumor enrichment and the accuracy of CNV detection in archival samples without paired blood.

Abstract This study employed a digital image correlation method (DICM) to experimentally quantify horizontal strain distribution in silicone rubber bulk during horizontal displacement against a stainless-steel sphere with/without glycerol. The strain distribution at different depth levels was measured by capturing the position of white powders in transparent rubber bulk. The experimental results indicated that each point in the rubber bulk moved while describing a horizontal loop during horizontal displacement depending on the position and lubrication conditions. This caused changes in the horizontal strain during horizontal displacement. These results suggest that the hysteresis term could be caused by changes in the vertical and horizontal strains.

The transverse vaginal septum, a rare Müllerian duct anomaly, presents diagnostic and therapeutic challenges owing to its variable location, thickness, and potential association with uterine malformations. Therefore, an accurate diagnosis and selection of an appropriate treatment are important. Herein, the case of a 28-year-old nonpregnant woman with sexual dysfunction attributable to a transverse vaginal septum is presented. The septum, approximately 5 mm thick, was situated low on the vaginal wall near the urethral opening, with a small central aperture. Employing YV plasty, full extension of the posterior and lateral vaginal walls was achieved while minimizing the manipulation of the anterior wall to avoid urethral injury. Postoperatively, the patient achieved sexual function without vaginal stenosis. YV plasty is a minimally invasive and effective approach for preventing postoperative stenosis in the treatment of a thin transverse vaginal septum located low on the vaginal wall.

Introduction The risk of postoperative bleeding complications should be concerned to perform percutaneous nephrolithotripsy. Most of the vascular injuries occurred at the peripheral renal artery in the previous reports. We experienced a case of bleeding shock induced by the injury of the intercostal artery in the abdominal wall following percutaneous nephrolithotripsy. Case presentation A 56‐year‐old woman had been in the bleeding shock status on the 2nd day after percutaneous nephrolithotoripsy. Emergently, contrast‐enhanced computed tomography was performed and extravasation of contrast agents was seen in the abdominal wall. Injuries of the intercostal artery were identified in the angiography and controlled by transcatheter arterial embolization. Conclusion The intercostal arteries could be injured in the anterolateral zone of the abdominal wall over the end of the ribs. Contrast‐enhanced computed tomography was useful to detect the bleeding point. Transcatheter arterial embolization was an effective and safe method to control bleedings from them.

Ureaplasma parvum is one of the most common endemic mycoplasmas in the genitourinary tract and can cause amniotic fluid infection leading to preterm labor. We report a rare case of Ureaplasma parvum infection ascending from the vagina to the abdominal cavity after hysterectomy, causing vaginal cuff infection, postoperative peritonitis, and small bowel obstruction. A 29-year-old nulliparous woman presented with infected uterine cervical cancer. After radical hysterectomy for uterine cervical cancer, the patient had paralytic ileus with ascites and fever. Peritonitis was suspected; however, all cultures were negative, making it difficult to identify the causative organism. Polymerase chain reaction (PCR) of the ascites revealed Ureaplasma parvum, which could be treated with levofloxacin (LVFX). Open drainage to control the infection revealed a necrotic tissue around vaginal cuff and the small intestine encased in cocoon-like fibers like sclerosing encapsulating peritonitis. After the infection was improved, the bowel obstruction was also improved. Ureaplasma spp. can be difficult to culture. PCR testing for Ureaplasma infection should be considered when urogenital infection is suspected in patients prone to opportunistic infections, such as those with malignant tumors.

Abstract Background Small intestine carcinoma (SIC) cases in Japan have recently been treated with chemotherapy according to colorectal carcinoma classification, while papilla of Vater carcinoma (PVC) cases according to cholangiocarcinoma (CHC) classification. However, few research reports support the molecular genetic validity of these therapeutic choices. Patients and methods Here, we investigated the clinicopathological and molecular genetic factors of SIC and PVC. We used the data from the Japanese version of The Cancer Genome Atlas. Additionally, molecular genetic data on gastric adenocarcinoma (GAD), colorectal adenocarcinoma (CRAD), pancreatic ductal adenocarcinoma (PDAC), and CHC were also referred to. Results This study consisted of tumor samples from 12 patients of SIC and three patients of PVC treated from January 2014 to March 2019. Among them, six patients had pancreatic invasion. t‐Distributed stochastic neighbor embedding analysis showed that the gene expression pattern of SIC was similar not only to those of GAD and CRAD, but also to that of PDAC in the pancreatic invasion patients. In addition, PVC resembled the GAD, CRAD, and PDAC, rather than the CHC. The molecular genetic characteristics of the six patients with pancreatic invasion were: one had high microsatellite instability, two had a TP53 driver mutation, and three had tumor mutation burden values

Abstract Background Pancreatic cancer (PC) is one of the most aggressive cancers worldwide. Although many studies have investigated genomic alterations, the genomic landscape of Japanese PC patients has not been fully elucidated. Methods We used whole‐exome sequencing, cancer gene panel deep‐sequencing, and microarray gene expression profiling data derived from the Japanese version of the Cancer Genome Atlas (JCGA) in 93 PC cases. Results Somatic driver mutations were identified in 65.6% of samples in 19 genes. The median tumor mutation burden (TMB) value was 0.24 Muts/Mb (interquartile range, 0.15–0.64 Muts/Mb). The commonly mutated genes were KRAS (58%), TP53 (40%), CDKN2A (10%), SMAD4 (10%), FGFR2 (9%), and PKHD1 (9%). Frequent germline variation genes were BRCA1 (8%), CDH1 (5%), MET (5%), MSH6 (5%), and TEK (5%). Frequent chromosomal arm alterations included copy number gains in 2q (42%), 7q (24%), and 3q (24%), and copy number losses in 19p (62%), 19q (47%), 12q (34%), and 7q (30%). A prognostic analysis according to the presence of driver mutations showed that overall survival (OS) in the driver mutation‐positive group was significantly worse in comparison to that of the driver mutation‐negative group (median, 23.1 vs 46.7 mo; P = .010). A Cox proportional hazards analysis for OS identified driver mutation (hazard ratio [HR], 1.89; P = .025) and lymph node metastasis (HR, 3.27; P = .002) as independent prognostic factors. Conclusion The present results from the JCGA dataset constitute a fundamental resource for genomic medicine for PC patients, especially in Japan.

Abstract In pancreatic cancer (PC), Tumor mutation burden (TMB) has been reported to be lower than in other cancers, with its clinical significance remaining unclear. We analyzed the dataset of whole-exome sequencing and gene expression profiling of 93 resected PC cases. The median TMB was 0.24. The TMB was classified as High (≥ 5.0), Low (

Objectives: Port placements at the mid-abdomen (mainstay of robotic surgery [Rob]) appear to be difficult compared to that at lower abdomen (mainstay of conventional laparoscopy [Con-Lap]). We hypothesized that the reason for this may be the difference in port puncture places. Materials and Methods: We examined how the differences between the place and puncture order of ports affected Con-Lap cases with ports mainly placed in the lower abdomen and Rob cases with ports mainly placed in the middle abdomen. The trocar time was measured from the time when the puncture position and skin incision were determined and initiated, respectively, to the time when the port was punctured and fixed and used as the indicator of difficulty. Results: In the Con-Lap group analysis, the trocar time of the left lower port was longer (right lower: 77 s, middle lower: 117.5 s, and left lower: 138 s, P < 0.0001). In the Rob group analysis, the trocar time of the left most port was significantly longer (right-most: 89.0 s, right-middle: 92.5 s, left-middle: 121.0 s, and left-most: 197.0 s; P < 0.0001). In addition, the total trocar time was significantly longer in the first puncture at the right-middle port in the Rob group (right-most first: 8.4 min, right-middle first: 12.4 min, and left-middle first: 8.5 min, P = 0.0063). Conclusion: In the mid-abdomen port placement, mainstay of Rob cases, the puncture order, and port site have a significant impact on the difficulty of the procedure. It is preferable to avoid initially puncturing the right-middle port in case of the Rob.

Abstract Objective Pan‐cancer analysis across The Cancer Genome Atlas has revealed the molecular profiles of major types of carcinomas. High‐grade serous carcinomas (HGSCs) have been characterized; however, in ovarian cancer, the profile of carcinoma with minor histopathological changes remains unclear. This study aimed to perform the molecular profiling of rare malignant ovarian tumors, including non‐epithelial tumors (NETs; germ cell tumors and sex cord tumors) and clear cell carcinoma (CCC), to determine how they differ from the major HGSCs. Methods Sixty‐nine malignant ovarian tumors surgically resected at the Shizuoka Cancer Center between January 2014 and March 2019 were classified based on their histopathological types. The germline and somatic mutations in these carcinomas, including NETs, were determined using next‐generation sequencing. Gene expression analysis was performed to investigate the major pathways of drug resistance, which is a characteristic of CCC. Results NETs harbored copy‐neutral loss of heterozygosity, accompanied by a high homologous recombination deficiency score; germline mutations of PALB2 and BARD1 were identified in two patients with NET. In chemoresistant CCC, the epithelial‐mesenchymal transition pathway was activated regardless of ABC transporter expression. Conclusion This study revealed some genomic characteristics of rare malignant ovarian tumors, including NETs and CCC.

Abstract Background When a patient has multiple tumors in different organs, it is very important to identify whether the tumors are multiple cancers or metastasis from one tumor in order to establish an optimal treatment strategy. However, it is difficult to obtain an accurate diagnosis from conventional diagnostic strategies, including immunohistochemistry. We report two patients with multiple tumors in which a somatic mutation comparison using next-generation sequencing (NGS) was useful for the diagnosis of a metastatic tumor. Case presentations Patient 1: A 64-year-old man was diagnosed with gastric and lung cancer. After radical chemoradiotherapy for lung cancer, gastrectomy was planned for gastric cancer. At gastrectomy, the patient underwent a multiple omics analysis for “Project HOPE”. The gene mutational signature of the gastric tumor showed signature 4 of COSMIC mutational signature version 2, which was associated with smoking and has not been found in gastric cancer. To confirm that the gastric tumor was metastasis from lung cancer, we conducted a somatic mutation comparison of the two tumors with 409-gene panel sequencing, which revealed that 28 of 97 mutations in the lung tumor completely matched those of the gastric tumor. Based on these findings, the gastric tumor was diagnosed as metastasis from lung cancer. Patient 2: A 47-year-old woman underwent distal gastrectomy for gastric cancer. A colon tumor was detected 6 years after gastrectomy. The colon lesion was a submucosal tumor-like elevated tumor, and was suspected to be metastasis from gastric cancer. The patient underwent sigmoidectomy, and participated in “Project HOPE”. The possibility of primary colon cancer could not be ruled out, and we conducted a somatic mutation comparison of the two tumors as we did with Patient 1. Panel sequencing revealed 11 mutations in the gastric tumors, 4 of which completely matched those of the colon tumor. The colon tumor was diagnosed as metastasis from gastric cancer. Conclusion We reported two patients with multiple tumors in which a somatic mutation comparison using NGS was useful for the diagnosis of a metastatic tumor.

Abstract Background Multiple mutation (MM) within a single gene has recently been reported as a mechanism involved in carcinogenesis. The present study investigated the clinical significance of MMs in hepatocellular carcinoma (HCC). Methods Two hundred twenty-three surgically resected HCCs were subjected to gene expression profiling and whole-exome sequencing. Results MMs in individual genes were detected in 178 samples (MM tumors: 79.8%). The remaining samples all carried a single mutation (SM tumors: 20.2%). Recurrence-free survival in the MM group was significantly worse in comparison to the SM group (P = 0.012). A Cox proportional hazard analysis revealed that MM tumor was an independent predictor for worse a prognosis (hazard ratio, 1.72; 95% confidence interval, 1.01–3.17; P = 0.045). MMs were frequently observed across in various genes, especially MUC16 (15% of samples had at least one mutation in the gene) and CTNNB1 (14%). Although the MUC16 mRNA expression of MUC16 wild-type and MUC16 SM tumors did not differ to a statistically significant extent, the expression in MUC16 MM tumors was significantly enhanced in comparison to MUC16 SM tumors (P

Abstract The differences in genetic susceptibility to lung adenocarcinoma and squamous cell carcinoma remain unclear. We developed a customized, targeted gene sequencing panel for efficient and sensitive identification of germline variants, including whole-gene deletion types for cancer-related drug-metabolizing enzyme genes in lung adenocarcinoma and squamous cell carcinoma. The minor allele frequencies of the variants, confirmed as clinically significant in the Japanese population, did not differ significantly from those of normal participants listed in the public database. Genotype analysis comparing lung adenocarcinoma (n = 559) and squamous cell carcinoma (n = 151) indicated that the variants of DPYD (rs190771411, Fisher’s exact test, P = 0.045; rs200562975, P = 0.045) and ALDH2 (rs568781254, P = 0.032) were associated with an increased risk of squamous cell carcinoma compared to adenocarcinoma. Conversely, whole-gene deletion of CYP2A6 was associated with adenocarcinoma but not squamous cell carcinoma. Notably, whole-gene deletion of CYP2A6 was confirmed in 22 patients with lung adenocarcinoma but not in any patients with squamous cell carcinoma. Most patients with whole-gene deletion of CYP2A6 were female non-smokers. The discovery of a whole-gene deletion of CYP2A6 in patients with lung adenocarcinoma may have an important role in clinical practice and advance our understanding of CYP2A6 germline variants and their association with carcinogenesis or their susceptibility to lung adenocarcinoma.

Abstract Introduction The genomic characteristics of hepatocellular carcinoma (HCC) after a sustained virological response (SVR) and its differences according to whether an SVR was achieved by treatment with direct‐acting antivirals (DAA) or interferon (IFN) are still not fully understood. Methods Sixty‐nine surgically resected HCCs from patients with hepatitis C virus infection were analyzed by gene expression profiling and whole‐exome sequencing. Results Among the 69 HCC patients, 34 HCCs in which an SVR was not achieved at the time of surgery were classified as HCV‐positive, and 35 HCCs in which an SVR was achieved at the time of surgery were classified as HCV‐SVR. According to the HCV treatment, 35 HCV‐SVR HCCs were classified into two groups: eight tumors with DAA (HCV‐SVR‐DAA) and 24 tumors with interferon (HCV‐SVR‐IFN). The frequency of samples with ARID2 mutations was significantly lower in HCV‐SVR than in HCV‐positive tumors (p = 0.048). In contrast, the frequency of samples with PREX2 mutations was significantly higher in HCV‐SVR samples than in HCV‐positive samples (p = 0.048). Among the patients with HCV‐SVR, the frequency of samples with TP53 mutations was significantly higher in HCV‐SVR‐DAA tumors than in HCV‐SVR‐IFN tumors (p = 0.030). TP53 inactivation scores in HCV‐SVR‐DAA tumors were found to be significantly enhanced in comparison to HCV‐SVR‐IFN tumors (p = 0.022). In addition, chromosomal instability and PI3K/AKT/mTOR pathway signatures were enhanced in HCV‐SVR‐DAA tumors. HCV‐SVR‐DAA was significantly associated with portal vein invasion (p = 0.003) in comparison to HCV‐SVR‐IFN. Conclusion Our dataset potentially serves as a fundamental resource for the genomic characteristics of HCV‐SVR‐DAA tumors. Our comprehensive genetic profiling by WES revealed significant differences in the mutation rate of several driver genes between HCV‐positive tumors and HCV‐SVR tumors. Furthermore, it was revealed that the frequency of samples with mutations in TP53 was significantly higher in HCV‐SVR‐DAA tumors than in HCV‐SVR‐IFN tumors.

Abstract Targeted sequencing offers an opportunity to select specific drugs for cancer patients based on alterations in their genome. However, accurate sequencing cannot be performed in cancers harboring diffuse tumor cells because of low tumor content. We performed tumor cell enrichment using tissue suspension of formalin-fixed, paraffin-embedded (FFPE) tissue sections with low tumor cell content. The enriched fractions were used to efficiently identify mutations by sequencing a target panel of cancer-related genes. Tumor-enriched and residual fractions were isolated from FFPE tissue sections of intestinal and diffuse gastric cancers harboring diffuse tumor cells and DNA of suitable quality was isolated for next-generation sequencing. Sequencing of a target panel of cancer-related genes using the tumor-enriched fraction increased the number of detectable mutations and variant allele frequency. Furthermore, mutation analysis of DNA isolated from tumor-enriched and residual fractions allowed us to estimate germline mutations without a blood reference. This approach of tumor cell enrichment will not only enhance the success rate of target panel sequencing, but can also improve the accuracy of detection of somatic mutations in archived specimens.

Mature cystic teratoma of the ovary (MCT) occasionally undergoes malignant transformation (MT) that is resistant to chemotherapy and has a poor prognosis. We experienced a case of clinically aggressive MCT-MT that invades surrounding organs and tissues. Although tumor was resected entirely, a rapid tumor recurrence occurred during postoperative chemotherapy (paclitaxel + ifosfamide + cisplatin). The results of comprehensive genomic profiling test performed early in the postoperative period showed a high tumor mutational burden of 23 mutations/Mb. Treatment with nivolumab monotherapy has promptly been initiated and has been very successful for more than one year.

Beta-tricalcium phosphate (β-TCP) serves as a bone substitute in clinical practice because it is resorbable, biocompatible, osteointegrative, and osteoconductive. Particles of β-TCP are also inflammatory mediators although the mechanism of this function has not been fully elucidated. Regardless, the ability of β-TCP to stimulate the immune system might be useful for immunomodulation. The present study aimed to determine the effects of β-TCP particles on NLR family pyrin domain containing 3 (NLRP3) inflammasome complexes. We found that β-TCP activates NLRP3 inflammasomes, and increases interleukin (IL)-1β production in primary cultured mouse dendritic cells (DCs) and macrophages, and human THP-1 cells in caspase-1 dependent manner. In THP-1 cells, β-TCP increased also IL-18 production, and NLRP3 inflammasome activation by β-TCP depended on phagocytosis, potassium efflux, and reactive oxygen species (ROS) generation. We also investigated the effects of β-TCP in wild-type and NLRP3-deficient mice in vivo. Immune cell migration around subcutaneously injected β-TCP particles was reduced in NLRP3-deficient mice. These findings suggest that the effects of β-TCP particles in vivo are at least partly mediated by NLRP3 inflammasome complexes.

Abstract With the emergence of next-generation sequencing (NGS)-based cancer gene panel tests in routine oncological practice in Japan, an easily interpretable cancer genome database of Japanese patients in which mutational profiles are unaffected by racial differences is needed to improve the interpretation of the detected gene alterations. Considering this, we constructed the first Japanese cancer genome database, called the Japanese version of the Cancer Genome Atlas (JCGA), which includes multiple tumor types. The database includes whole-exome sequencing data from 4907 surgically resected primary tumor samples obtained from 4753 Japanese patients with cancer and graphically provides genome information on 460 cancer-associated genes, including the 336 genes that are included in two NGS-based cancer gene panel tests approved by the Pharmaceuticals and Medical Devices Agency. Moreover, most of the contents of this database are written in Japanese; this not only helps physicians explain the results of NGS-based cancer gene panel tests but also enables patients and their families to obtain further information regarding the detected gene alterations.

Abstract EMI Domain Containing 1 (EMID1) was identified as a potential candidate metastasis-promoting gene. We sought to clarify the molecular function of EMID1 and the protein expression. Overexpression and knockdown studies using mouse tumor cell lines identified two novel functions of EMID1: intracellular signaling involving enhancement of cell growth via cell cycle promotion and suppression of cell motility, and inhibition of cell–matrix adhesion by extracellularly secreted EMID1. EMID1 deposited on the culture dish induced self-detachment of cells that overexpressed the protein and inhibited adhesion of additionally seeded cells. This multifunctional property involving both intracellular signaling and the extracellular matrix suggests that EMID1 may be a matricellular proteins. Expression analysis using immunohistochemical staining revealed expression of EMID1 that was limited to chief cells of the gastric fundic gland and β cells of the pancreatic islets in normal adult human tissues, implying cell-specific functions of this molecule. In addition, increased expression of EMID1 protein detected in some cases of human cancers implies that EMID1 might be a new therapeutic target for cancer treatment.

Abstract Based on our previous phase II clinical trial of anti-programmed death-1 (PD-1) antibody nivolumab for platinum-resistant ovarian cancer (n = 19, UMIN000005714), we aimed to identify the biomarkers predictive of response. Tumor gene expression was evaluated by proliferative, mesenchymal, differentiated, and immunoreactive gene signatures derived from high-grade serous carcinomas and a signature established prior for ovarian clear cell carcinoma. Resulting signature scores were statistically assessed with both univariate and multivariate approaches for correlation to clinical response. Analyses were performed to identify pathways differentially expressed by either the complete response (CR) or progressive disease (PD) patient groups. The clear cell gene signature was scored significantly higher in the CR group, and the proliferative gene signature had significantly higher scores in the PD group where nivolumab was not effective (respective p values 0.005 and 0.026). Combinations of gene signatures improved correlation with response, where a visual projection of immunoreactive, proliferative, and clear cell signatures differentiated clinical response. An applicable clinical response prediction formula was derived. Ovarian cancer-specific gene signatures and related pathway scores provide a robust preliminary indicator for ovarian cancer patients prior to anti-PD-1 therapy decisions.

Background In cancer therapy, higher-resolution tumor-agnostic biomarkers that predict response to immune checkpoint inhibitor (ICI) therapy are needed. Mutation signatures reflect underlying oncogenic processes that can affect tumor immunogenicity, and thus potentially delineate ICI treatment response among tumor types.Methods Based on mutational signature analysis, we developed a stratification for all solid tumors in The Cancer Genome Atlas (TCGA). Subsequently, we developed a new software (Genomic Subtyping and Predictive Response Analysis for Cancer Tumor ICi Efficacy, GS-PRACTICE) to classify new tumors submitted to whole-exome sequencing. Using existing data from 973 pan-cancer ICI-treated cases with outcomes, we evaluated the subtype-response predictive performance.Results Systematic analysis on TCGA samples identified eight tumor genomic subtypes, which were characterized by features represented by smoking exposure, ultraviolet light exposure, APOBEC enzyme activity, POLE mutation, mismatch repair deficiency, homologous recombination deficiency, genomic stability, and aging. The former five subtypes were presumed to form an immune-responsive group acting as candidates for ICI therapy because of their high expression of immune-related genes and enrichment in cancer types with FDA approval for ICI monotherapy. In the validation cohort, the samples assigned by GS-PRACTICE to the immune-reactive subtypes were significantly associated with ICI response independent of cancer type and TMB high or low status.Conclusions The new tumor subtyping method can serve as a tumor-agnostic biomarker for ICI response prediction and will improve decision making in cancer treatment.

Abstract Background There has been a paradigm shift in cancer treatment from curing disease to both curing disease and caring for patients. In terms of care for patients, the opinions of patients and their families are important for improving medical services. Aim The opinions of patients and families were collected at Shizuoka Cancer Center (SCC) and examined from the standpoint of characteristics, response status, and temporal changes. Methods Patients' and families' opinions submitted to suggestion boxes at SCC over an 11‐year period (2005–2015) were analyzed. Opinions were categorized as complaints or compliments, with sub‐categories including “facilities, goods, and medical care,” “people,” “time,” and “other.” The status of facilities' response to complaints was categorized as “responded,” “did not respond”, and “difficult to respond”. Results Changes in the number of opinions and content over time were examined. In total, 3419 opinions were collected; 69.1% were complaints, and 30.5% were compliments. Of the complaints, 53.4% were related to “facilities, goods, and medical care” (mainly focusing on “poor product quality” and “shortage of goods”), 38.7% were related to “people,” and 7.7% to “time.” Of the compliments, 82.4% were about “people,” all of which concerned facility staff. Facilities' responses to complaints were as follows: “responded” (42.4%), “did not respond” (14.3%), and “difficult to respond” (43.3%). Conclusion Understanding patients' and families' opinions is effective for strengthening trust between patients and healthcare professionals, promoting holistic care.

Tertiary lymphoid structures (TLS) are transient ectopic lymphoid aggregates whose formation might be caused by chronic inflammation states, such as cancer. However, how TLS are induced in the tumor microenvironment (TME) and how they affect patient survival are not well understood. We investigated TLS distribution in relation to tumor infiltrating lymphocytes (TILs) and related gene expression in high-grade serous ovarian cancer (HGSC) specimens. CXCL13 gene expression correlated with TLS presence and the infiltration of T cells and B cells, and it was a favorable prognostic factor for patients with HGSC. Coexistence of CD8+ T cells and B cell lineages in the TME significantly improved the prognosis of HGSC and was correlated with the presence of TLS. CXCL13 expression was predominantly coincident with CD4+ T cells in TLS and CD8+ T cells in TILs, and it shifted from CD4+ T cells to CD21+ follicular DCs as TLS matured. In a mouse ovarian cancer model, recombinant CXCL13 induced TLS and enhanced survival by the infiltration of CD8+ T cells. These results suggest that TLS formation was associated with CXCL13-producing CD4+ T cells and that TLS facilitated the coordinated antitumor response of cellular and humoral immunity in ovarian cancer.

Abstract Background Mutation analysis using next-generation sequencing highlights the features of tumors with somatic alterations. However, the mutation profile of double cancer remains unclear. Here, we analyzed tumors derived from the same patient using whole exome sequencing (WES) to investigate the coherence of somatic mutations in double cancer. Methods First, the tumor mutational burden (TMB) was investigated using WES of 5521 tumor specimens from a Japanese pan-cancer cohort. The frequencies of mutation concordance were then compared in these cancers. Finally, we calculated the expected value of mutational concordance fitting a Poisson distribution to determine the relationship between double and metastatic cancers. Results In all, 44, 58, and 121 paired samples were diagnosed as double cancer, multifocal lesions (derived from identical tissues), and metastasis, respectively. Our analysis revealed that common somatic mutations were almost entirely absent in double cancer, whereas primary tumors and metastatic foci harbored several identical alterations. Concordance of the mutation profile in the same patient reflects the tumor origin and development, suggesting the potential for identifying double cancer based on common somatic mutations. Furthermore, according to a Poisson distribution, double cancer could be discriminated based on paired samples from the same patient. The probability of double cancer with more than 10 mutations was ≤1 part-per-billion (ppb, 10− 9). In multifocal lesions, 74% of tumor pairs accumulated ≤10 common mutations, implying a difference in tumor origin within identical tissues. Conclusions These findings indicate that counting common somatic mutations can indicate the differences in origin between tumors derived from the same patient. Our mutation coherence analysis can thus provide beneficial information for diagnosing double cancer.

Objective The need for tailoring ovarian cancer treatments to individual patients is increasing. This study aimed to evaluate the prognostic value of pretreatment laboratory test data for predicting the response and survival outcomes of platinum-based chemotherapy in ovarian cancer. Methods We enrolled 270 patients with ovarian cancer diagnosed at the Kyoto Medical Center (n=120; group A) and Kyoto University (n=150; group B). Data on 9 blood parameters (neutrophil to lymphocyte ratio [NLR], platelet to lymphocyte rate [PLR], C-reactive protein, lactate dehydrogenase [LDH], glucose, total cholesterol, high-density lipoprotein [HDL], low-density lipoprotein, and triglyceride levels), cancer pathology, cancer stage, cytoreduction outcomes, serum cancer antigen 125 levels, platinum-free interval (PFI), disease-free survival (DFS), and overall survival were assessed retrospectively. Results NLR, PLR, LDH, and HDL were significantly different in advanced stage patients (P

Abstract Background Uridine diphosphate glucuronosyltransferase 1 family polypeptide A1 (UGT1A1) is a predictive biomarker for the side-effects of irinotecan chemotherapy, which reduces the volume of tumors harboring UGT1A1 polymorphisms. We aimed to determine whether UGT1A1 polymorphisms can predict progression-free survival in patients with local cervical cancer treated with irinotecan chemotherapy. Methods We retrospectively analyzed the data of 51 patients with cervical cancer treated at a single institution between 2010 and 2015. All patients were diagnosed with 2009 International Federation of Gynecology and Obstetrics (FIGO) stage IB1, IB2, IIA, or IIB squamous cell carcinoma, underwent radical hysterectomy, and received irinotecan chemotherapy as neoadjuvant and/or adjuvant treatment. All patients were examined for irinotecan side effects using UGT1A1 tests. Conditional inference tree and survival analyses were performed considering the FIGO stage, age, the UGT1A1 status, and the number of metastatic lymph nodes to determine primary factors associated with progression-free survival. Results The tree-structured survival model determined high recurrence-risk factors related to progression-free survival. The most relevant factor was ≥2 metastatic lymph nodes (p = 0.004). The second most relevant factor was UGT1A1 genotype (p = 0.024). Among patients with ≤1 metastatic lymph node, those with UGT1A1 polymorphisms benefited from irinotecan chemotherapy and demonstrated significantly longer progression-free survival (p = 0.020) than those with wild-type UGT1A1. Conclusions Irinotecan chemotherapy might be beneficial in patients with cervical cancer, UGT1A1 polymorphisms, and ≤ 1 metastatic lymph nodes.

Abstract Background It is unclear whether clinical factors and immune microenvironment (IME) factors are associated with tumor mutation burden (TMB) in patients with nonsmall cell lung cancer (NSCLC). Materials and methods We assessed TMB in surgical tumor specimens by performing whole exome sequencing. IME profiles, including PD‐L1 tumor proportion score (TPS), stromal CD8 tumor‐infiltrating lymphocyte (TIL) density, and stromal Foxp3 TIL density, were quantified by digital pathology using a machine learning algorithm. To detect factors associated with TMB, clinical data, and IME factors were assessed by means of a multiple regression model. Results We analyzed tumors from 200 of the 246 surgically resected NSCLC patients between September 2014 and September 2015. Patient background: median age (range) 70 years (39‐87); male 37.5%; smoker 27.5%; pathological stage (p‐stage) I/II/III, 63.5/22.5/14.0%; histological type Ad/Sq, 77.0/23.0%; primary tumor location upper/lower, 58.5/41.5%; median PET SUV 7.5 (0.86‐29.8); median serum CEA (sCEA) level 3.4 ng/mL (0.5‐144.3); median serum CYFRA 21‐1 (sCYFRA) level 1.2 ng/mL (1.0‐38.0); median TMB 2.19/ Mb (0.12‐64.38); median PD‐L1 TPS 15.1% (0.09‐77.4); median stromal CD8 TIL density 582.1/mm2 (120.0‐4967.6);, and median stromal Foxp3 TIL density 183.7/mm2 (6.3‐544.0). The multiple regression analysis identified three factors associated with higher TMB: smoking status: smoker, increase PET SUV, and sCEA level: >5 ng/mL (P

Abstract Objective Low birth weight (LBW) is a major public health issue as it increases the risk of noncommunicable diseases throughout life. However, the genome-wide DNA methylation patterns of full-term LBW infants (FT-LBWs) are still unclear. This exploratory study aimed to analyze the DNA methylation differences in FT-LBWs compared with those in full-term normal birth weight infants (FT-NBWs) whose mothers were nonsmokers and had no complications. Initially, 702 Japanese women with singleton pregnancies were recruited. Of these, four FT-LBWs and five FT-NBWs were selected as references for DNA methylation analysis, and 862,260 CpGs were assessed using Illumina Infinium MethylationEPIC BeadChip. Gene ontology enrichment analysis was performed using DAVID v6.8 software to identify the biological functions of hyper- and hypomethylated DNA in FT-LBWs. Results 483 hyper-differentially methylated genes (DMGs) and 35 hypo-DMGs were identified in FT-LBW promoter regions. Hyper-DMGs were annotated to 11 biological processes; “macrophage differentiation” (e.g., CASP8), “apoptotic mitochondrial changes” (e.g., BH3), “nucleotide-excision repair” (e.g., HUS1), and “negative regulation of inflammatory response” (e.g., NLRP12 and SHARPIN). EREG was classified into “ovarian cumulus expansion” within the “organism growth and organization” category. Our data imply that LBW might be associated with epigenetic modifications, which regulate the immune system and cell maturation.

Radical hysterectomy is a standard operation for invasive cervical cancers. However, if the invasion to the parametrium is more advanced than estimation in the operation, it is difficult to perform usual radical hysterectomy. Superradical hysterectomy was developed by Prof. Ryukichi Mibayashi of Kyoto University and was published in 1941, and has been performed for the limited cases by a part of Japanese gynecologic surgeons. Superradical hysterectomy is a procedure in which the soft tissues in the pelvis are removed en bloc by sequential processing of the internal iliac vessels, which leads to a complete dissection of the lymphatic tissue in the pelvis to the pelvic wall.

Abstract Side population (SP) cells harbor malignant phenotypes in cancer. The aim of this study was to identify genes that modulate the proportion of ovarian cancer SP cells. Using a shRNA library targeting 15,000 genes, a functional genomics screen was performed to identify genes whose suppression increased the SP percentage. The biological effects caused by alteration of those identified genes were investigated in vitro and in vivo. We found that suppression of MSL3, ZNF691, VPS45, ITGB3BP, TLE2, and ZNF498 increased the proportion of SP cells. Newly generated SP cells exhibit greater capacity for sphere formation, single cell clonogenicity, and in vivo tumorigenicity. On the contrary, overexpression of MSL3, VPS45, ITGB3BP, TLE2, and ZNF498 decreased the proportion of SP cells, sphere formation capacity and single cell clonogenicity. In ovarian cancer cases, low expression of MSL3, ZNF691 and VPS45 was related to poor prognosis. Suppression of these six genes enhanced activity of the hedgehog pathway. Cyclopamine, a hedgehog pathway inhibitor, significantly decreased the number of SP cells and their sphere forming ability. Our results provide new information regarding molecular mechanisms favoring SP cells and suggest that Hedgehog signaling may provide a viable target for ovarian cancer.

Abstract Background Somatic PTEN mutation occurs in a proportion of ovarian endometrioid carcinomas. However, these cancers have seldom been reported in diseases associated with germline PTEN variants, such as Cowden syndrome (CS). Case presentation The present case was a 39-year-old woman with a left ovarian carcinoma who demonstrated a germline splice variant of PTEN (c.1026 + 1G > T) following genome-wide whole exome sequencing of her germline DNA. Histology of her resected tumor revealed endometrioid carcinoma of the same type as a right ovarian cancer resected eight years previously. These tumors showed null immunostaining for PTEN. She was genetically diagnosed with CS. Despite her clinical examinations had demonstrated several characteristic findings of CS, including mammary fibroma, esophageal and skin papilloma, colonic hamartoma, uterine myoma, and lipoma, the clinicians could not approach this diagnosis. Conclusion Ovarian endometrioid carcinoma is generally thought to develop from endometrial tissue menstruated from the uterus and implanted on the ovary. To date, ovarian cancers have not been listed as CS-related cancers; however, ovarian endometrioid cancer can have a potential association with CS in endometriosis cases.

Abstract Background Lynch syndrome (LS) is the commonest inherited cancer syndrome caused by pathogenic variants of germline DNA mismatch repair (g.MMR) genes. Genome‐wide sequencing is now increasingly applied for tumor characterization, but not for g.MMR. The aim of this study was to evaluate the incidence and pathogenicity of g.MMR variants in Japanese cancer patients. Methods Four g.MMR genes (MLH1, MSH2, MSH6, and PMS2) were analyzed by next generation sequencing in 1058 cancer patients (614 male, 444 female; mean age 65.6 years) without past diagnosis of LS. The g.MMR variant pathogenicity was classified based on the ClinVar 2015 database. Tumor MMR immunohistochemistry, microsatellite instability (MSI), and BRAF sequencing were also investigated in specific cases. Results Overall, 46 g.MMR variants were detected in 167 (15.8%) patients, 17 likely benign variants in 119 patients, 24 variants of uncertain significance (VUSs) in 68 patients, two likely pathogenic variants in two patients, and three pathogenic variants in three (0.3%) patients. The three pathogenic variants included two colorectal cancers with MLH1 loss and high MSI and one endometrial cancer with MSH6 loss and microsatellite stability. Two likely pathogenic variants were shifted to VUSs by ClinVar (2018). One colon cancer with a likely benign variant demonstrated MLH1 loss and BRAF mutation, but other nonpathogenic variants showed sustained MMR and microsatellite stability. Conclusions Universal sequencing of g.MMR genes demonstrated sundry benign variants, but only a small proportion of cancer patients had pathogenic variants. Pathogenicity evaluation using the ClinVar database agreed with MSI, MMR immunohistochemistry, and BRAF sequencing.

Abstract Here, we investigated the clinicopathological and mutation profiles of colorectal cancer (CRC) with POLE mutations. Whole‐exome sequencing was performed in 910 surgically resected primary CRCs. Tumors exceeding 500 counts of nonsynonymous single nucleotide variants (SNVs) were classified as hypermutators, whereas the remaining were classified as nonhypermutators. The hypermutators were subdivided into 2 groups. CRCs harboring more than 20% C‐to‐A and less than 3% C‐to‐G transversions were classified as POLE category tumors, whereas the remaining were classified as common‐hypermutators. Gene expression profiling (GEP) analysis was performed in 892 (98.0%) tumors. Fifty‐seven (6.3%) and 10 (1.1%) tumors were classified common‐hypermutators and POLE category tumors, respectively. POLE category tumors harbored a significantly higher SNV count than common‐hypermutators, and all POLE category tumors were associated with exonuclease domain mutations, such as P286R, F367C, V411L, and S297Y, in the POLE gene. Patients with POLE category tumors were significantly younger than those with nonhypermutators and common‐hypermutators. All POLE mutations in the early‐onset (age of onset ≤50 years old) POLE category (7 tumors) were P286R mutations. GEP analysis revealed that PD‐L1 and PD‐1 gene expression levels were significantly increased in both common‐hypermutators and POLE category tumors compared with those in nonhypermutators. CD8A expression was significantly upregulated in POLE category tumors compared with that in nonhypermutators. Thus, we concluded that CRCs with POLE proofreading deficiency had characteristics distinct from those of other CRCs. Analysis of POLE proofreading deficiency may be clinically significant for personalized management of CRCs.

Ovarian tumors can get extremely giant to occupy the whole abdominal cavity. We report a case of 36 kg solid ovarian tumor, which was the largest ovarian solid tumor that have been ever reported. A 54-year-old woman presented to our hospital with a chief complaint of markedly distended abdominal wall. Preoperative imaging examinations revealed that most of the tumor was uniform and its density was like that of subcutaneous fat. Pleural effusion was detected in the right thoracic region. We organized a multidisciplinary team and successfully resected the right adnexa. The patient had an uneventful postoperative course, and she was discharged on the 7th postoperative day and diagnosed with a fibroma of the ovary with Meigs syndrome. A comprehensive literature search revealed 48 cases of extremely giant ovarian tumor in these 20 years. Six out of 48 cases are solid. Twelve out of 48 cases are malignant or borderline malignant, and patients’ age and tumor size/weight were not related to the frequency of malignancy/borderline malignancy. As many as 4 out of 48 patients died before their first hospital visit or early after surgery. Clinicians should consider a considerable high mortality and frequency of severe surgical complications when planning the treatment strategy for extremely giant ovarian tumors.

We report a case of torsion in an otherwise-normal ovary with a giant hematosalpinx. A 23-year-old woman presented with complaints of abdominal pain and nausea. At initial visit, there was few abnormal findings of imaging tests, and we made a diagnosis of ovarian hemorrhage. Three days later, she came back with increased symptoms, and we detected the mass of a complex solid cystic structure with a unilocular cyst much larger than solid component. A diagnostic laparoscopy was performed immediately, and we could make a diagnosis of torsion in an otherwise-normal ovary with a giant hematosalpinx. We performed a salpingectomy and could preserve her ovary. This is the first case of torsion in an otherwise-normal ovary with a giant hematosalpinx which enlarged to a greater extent than the ovary.

Purpose: The adhesion molecule cluster of differentiation 44v9 interacts with and stabilizes the cystine/glutamate exchanger protein, which functions as a transporter of cystine. Stabilized cystine/glutamate exchanger increases extracellular cystine uptake and enhances glutathione production. Augmented levels of reduced glutathione mitigate reactive oxygen species and protect cancer cells from apoptosis. Salazosulfapyridine blocks cystine/glutamate exchanger activity and mitigates the supply of cystine to increase intracellular ROS production, thereby increasing cell susceptibility to apoptosis. This enhances the effect of anticancer drugs such as cisplatin. Currently, salazosulfapyridine is being developed as a promising anticancer agent. In the present study, we elucidated the molecular mechanism associated with salazosulfapyridine by investigating the salazosulfapyridine-induced changes in glutathione metabolism in cultured gastric cancer cell lines. Methods: The effect of salazosulfapyridine treatment on glutathione metabolism was investigated in 4 gastric cancer (AGS, MKN1, MKN45, and MKN74) and 2 colorectal cancer (HCT15 and HCT116) cell lines using metabolomic analyses. In addition, the effect of inhibition of the reduced form of nicotinamide adenine dinucleotide phosphate by 2-deoxyglucose on glutathione metabolism was evaluated. Results: Under hypoxia, enhanced glycolysis resulted in lactate accumulation with an associated reduction in nicotinamide adenine dinucleotide phosphate. Salazosulfapyridine treatment decreased the cysteine content and inhibited the formation of glutathione. Combined treatment with salazosulfapyridine and 2-deoxyglucose significantly inhibited cell proliferation. 2-Deoxyglucose, an inhibitor of glycolysis, depleted nicotinamide adenine dinucleotide phosphate required for the formation of glutathione. Conclusions: Our results indicate that in cancer cells having a predominant glycolytic pathway, metabolomic analyses under hypoxic conditions enable the profiling of global metabolism. In addition, inhibiting the supply of nicotinamide adenine dinucleotide phosphate by blocking glycolysis is a potential treatment strategy for cancer, in addition to cystine blockade by salazosulfapyridine.

Objective: To characterize the prevalence of PPFE (pleuroparenchymal fibroelastosis)-like finding on CT in daily practice and to identify the risk factors for its progression. Matelials & methods: 2416 consecutive daily CT examinations were screened for PPFE-like finding. CT images with PPFE-like finding were retrospectively reviewed for the extent, maximum height, presence or absence of intraalveolar fibroelastosis (IAFE), emphysema, interstitial lung disease (ILD), suprasternal depression at the level of clavicle end, and bronchiectasis in upper lobe, and anterior-posterior/transverse diameter ratio (AT ratio) of thoracic cage. Serial CT scans more than 3 years before the baseline scan were also reviewed and compared when available. Results: 380 patients (median age of 65, M:F = 153:227) were recognized as having PPFE-like finding. The lowest level of PPFE-like finding was T5 in median, horizontal extent was 0–25 % in 229/380, 25–50 % in 66 and >50 % in 24. Median height was 8.0 mm. IAFE was seen in 75, emphysema in 71, ILD in 59, chronic bronchial disorder in 71, suprasternal depression in 148, upper-lobe bronchial dilatation in 124. Mean AT ratio was 0.63. Serial images were available in 131 patients. 11 showed significant progression, 57 showed mild progression. Existing ILD lesion and >25 % horizontal extent were significantly related to significant progression. Conclusions: PPFE-like finding was seen relatively in high proportion. Extensive lesion in axial plane and co-existing ILD might be risk factors of progression.

Low-grade endometrial stromal sarcoma (LG-ESS) is a rare malignant disease and demonstrates various patterns in preoperative imaging. Therefore, accurate diagnosis is important. Given its unique form, we report a case of LG-ESS with a nodule-in-nodule appearance on preoperative imaging. A 41-year-old woman was referred to our department for further examination of a 45 mm diameter uterine corpus mass. Preoperative magnetic resonance imaging (MRI) revealed several small nodules within a larger nodule. T2-weighted images showed moderate-to-high signal intensity with focal bands of low signal intensity in the small nodules. The patient underwent total abdominal hysterectomy and bilateral salpingo-oophorectomy. Histopathological findings of the small nodules showed densely concentrated endometrial stromal cells reminiscent of a proliferative phase endometrium with a concentric arrangement of small spiral arteriole-like vessels. The small nodules exhibited an expansile growth pattern and were surrounded by less densely concentrated endometrial stromal cells intermingled with the normal uterine myometrium. LG-ESS with smooth muscle differentiation and sex cord-like elements was partially observed. In summary, LG-ESS demonstrating a unique nodule-in-nodule appearance on preoperative imaging histopathologically comprised tumor cells of varying densities. Our current case suggests that preoperative diagnostic imaging with MRI may be useful.

Snail is a transcription factor that induces epithelial-mesenchymal transition. Here the authors show that, in the mesenchymal subtype of ovarian cancer, Snail expression promotes tumorigenesis by inducing immune evasion through CXCR2-ligands-mediated recruitment of myeloid-derived suppressor cells.

Abstract Background The Cancer Genome Atlas Research Network reported that high-grade serous carcinoma (HGSC) can be classified based on gene expression profiles into four subtypes, termed “immunoreactive,” “differentiated,” “proliferative,” and “mesenchymal.” We previously established a novel histopathological classification of HGSC, corresponding to the gene expression subtypes: immune reactive (IR), papillo-glandular (PG), solid and proliferative (SP), and mesenchymal transition (MT). The purpose of this study is to identify distinct clinical findings among the four pathological subtypes of HGSC, as well as to predict pathological subtype based on preoperative images. Methods We retrospectively assessed 65 HGSC cases (IR: 17, PG: 7, SP: 14, MT: 27) and analyzed preoperative images. Results All IR cases originated from either the ovary or fallopian tube (P = 0.0269). Significantly more IR cases were diagnosed at earlier stages (P = 0.0013), and IR cases displayed lower levels of ascites (P = 0.0014), fewer peritoneal lesions (P = 0.0080), a sporadic pattern of peritoneal lesions (P = 0.0016), a lower incidence of omental cake (P = 0.0416), and fewer distant metastases (P = 0.0146) compared with the other subtypes. MT cases were more likely to be of peritoneal origin (P = 0.0202), presented at advanced stages with higher levels of ascites (P = 0.0008, 0.0052, respectively), and more frequently had a diffuse pattern of peritoneal lesions (P = 0.0059), omental cake (P = 0.0179), and distant metastasis (P = 0.0053). A decision tree analysis estimated the histopathological subtypes based on preoperative images, with a sensitivity of 67.3%. Conclusions Pathological subtypes of HGSC have distinct clinical behaviors, and preoperative images enable better prediction of pathological subtype. These findings may lead to individualized treatment plans if the effect of treatment based on the HGSC subtype is elucidated.

Abstract Identification of driver genes contributes to the understanding of cancer etiology and is imperative for the development of individualized therapies. Gene amplification is a major event in oncogenesis. Driver genes with tumor-specific amplification-dependent overexpression can be therapeutic targets. In this study, we aimed to identify amplification-dependent driver genes in 1,454 solid tumors, across more than 15 cancer types, by integrative analysis of gene expression and copy number. Amplification-dependent overexpression of 64 known driver oncogenes were found in 587 tumors (40%); genes frequently observed were MYC (25%) and MET (18%) in colorectal cancer; SKP2 (21%) in lung squamous cell carcinoma; HIST1H3B (19%) and MYCN (13%) in liver cancer; KIT (57%) in gastrointestinal stromal tumors; and FOXL2 (12%) in squamous cell carcinoma across tissues. Genomic aberrations in 138 known cancer driver genes and 491 established fusion genes were found in 1,127 tumors (78%). Further analyses of 820 cancer-related genes revealed 16 as potential driver genes, with amplification-dependent overexpression restricted to the remaining 22% of samples (327 tumors) initially undetermined genetic drivers. Among them, AXL, which encodes a receptor tyrosine kinase, was recurrently overexpressed and amplified in sarcomas. Our studies of amplification-dependent overexpression identified potential drug targets in individual tumors.

Cherubism is caused by mutations in SH3BP2. Studies of cherubism mice showed that tumor necrosis factor α (TNF-α)-dependent autoinflammation is a major cause of the disorder but failed to explain why human cherubism lesions are restricted to jaws and regress after puberty. We demonstrate that the inflammation in cherubism mice is MYD88 dependent and is rescued in the absence of TLR2 and TLR4. However, germ-free cherubism mice also develop inflammation. Mutant macrophages are hyperresponsive to PAMPs (pathogen-associated molecular patterns) and DAMPs (damage-associated molecular patterns) that activate Toll-like receptors (TLRs), resulting in TNF-α overproduction. Phosphorylation of SH3BP2 at Y183 is critical for the TNF-α production. Finally, SYK depletion in macrophages prevents the inflammation. These data suggest that the presence of a large amount of TLR ligands, presumably oral bacteria and DAMPs during jawbone remodeling, may cause the jaw-specific development of human cherubism lesions. Reduced levels of DAMPs after stabilization of jaw remodeling may contribute to the age-dependent regression.

Epithelial cell adhesion molecule (EpCAM)-based enumeration of circulating tumor cells (CTC) has prognostic value in patients with solid tumors, such as advanced breast, colon, and prostate cancer. However, poor sensitivity has been reported for non-small cell lung cancer (NSCLC). To address this problem, we developed a microcavity array (MCA) system integrated with a miniaturized device for CTC isolation without relying on EpCAM expression. Here, we report the results of a clinical study on CTCs of advanced lung cancer patients in which we compared the MCA system with the CellSearch system, which employs the conventional EpCAM-based method.Paired peripheral blood samples were collected from 43 metastatic lung cancer patients to enumerate CTCs using the CellSearch system according to the manufacturer's protocol and the MCA system by immunolabeling and cytomorphological analysis. The presence of CTCs was assessed blindly and independently by both systems.CTCs were detected in 17 of 22 NSCLC patients using the MCA system versus 7 of 22 patients using the CellSearch system. On the other hand, CTCs were detected in 20 of 21 small cell lung cancer (SCLC) patients using the MCA system versus 12 of 21 patients using the CellSearch system. Significantly more CTCs in NSCLC patients were detected by the MCA system (median 13, range 0-291 cells/7.5 mL) than by the CellSearch system (median 0, range 0-37 cells/7.5 ml) demonstrating statistical superiority (p = 0.0015). Statistical significance was not reached in SCLC though the trend favoring the MCA system over the CellSearch system was observed (p = 0.2888). The MCA system also isolated CTC clusters from patients who had been identified as CTC negative using the CellSearch system.The MCA system has a potential to isolate significantly more CTCs and CTC clusters in advanced lung cancer patients compared to the CellSearch system.

BackgroundExosomes play a major role in cell-to-cell communication, targeting cells to transfer exosomal molecules including proteins, mRNAs, and microRNAs (miRNAs) by an endocytosis-like pathway. miRNAs are small noncoding RNA molecules on average 22 nucleotides in length that regulate numerous biological processes including cancer pathogenesis and mediate gene down-regulation by targeting mRNAs to induce RNA degradation and/or interfering with translation. Recent reports imply that miRNAs can be stably detected in circulating plasma and serum since miRNAs are packaged by exosomes to be protected from RNA degradation. Thus, profiling exosomal miRNAs are in need to clarify intercellular signaling and discover a novel disease marker as well.Methodology/principal findingsExosomes were isolated from cultured cancer cell lines and their quality was validated by analyses of transmission electron microscopy and western blotting. One of the cell lines tested, a metastatic gastric cancer cell line, AZ-P7a, showed the highest RNA yield in the released exosomes and distinctive shape in morphology. In addition, RNAs were isolated from cells and culture media, and profiles of these three miRNA fractions were obtained using microarray analysis. By comparing signal intensities of microarray data and the following validation using RT-PCR analysis, we found that let-7 miRNA family was abundant in both the intracellular and extracellular fractions from AZ-P7a cells, while low metastatic AZ-521, the parental cell line of AZ-P7a, as well as other cancer cell lines showed no such propensity.Conclusions/significanceThe enrichment of let-7 miRNA family in the extracellular fractions, particularly, in the exosomes from AZ-P7a cells may reflect their oncogenic characteristics including tumorigenesis and metastasis. Since let-7 miRNAs generally play a tumor-suppressive role as targeting oncogenes such as RAS and HMGA2, our results suggest that AZ-P7a cells release let-7 miRNAs via exosomes into the extracellular environment to maintain their oncogenesis.

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