池田 純一郎

Cerebellar liponeurocytoma (cLNC), categorized as a World Health Organization grade II tumor, is a rare neoplasm characterized by advanced neuronal/neurocytic differentiation and focal lipid accumulation in neuroepithelial tumor cells. However, the expression and genetic profiling of cLNC have been poorly studied. A 44-year-old woman with a three-year history of cerebellar ataxia and numbness in lower extremities underwent radiological examination revealing multiple contrast-enhancing tumors at the floor of the fourth ventricle and in the lower vermis, and spinal dissemination. The high uptake of 11 C-methionine in positron emission tomography (Met-PET) supported the preoperative cLNC diagnosis. Subtotal removal of the tumor around the obex and inferior vermis was performed. Histologically, the tumor was composed of small, uniform cells with round nuclei in a sheet-like fashion. Tumor cells were diffusely reactive for the neuronal markers synaptophysin and neurofilament. Vacuolate cells with a displacement of nuclei suggested the accumulation of lipid, which was further supported by immunohistochemical staining of S-100. These findings confirmed the diagnosis of cLNC. Next-generation sequencing of tumoral DNA detected a splice site mutation in the ATRX gene. Further reports of cLNC cases with detailed expression and genetic profiles are essential for precise diagnosis and clarifying the oncogenic pathway in cLNC.

The 4F2 cell-surface antigen heavy chain (4F2hc) forms a heterodimeric complex with L-type amino acid transporter 1 (LAT1) and transports large neutral essential amino acids. However, in contrast to the traditional role of LAT1 in various cancers, the role of 4F2hc has largely remained unknown. The role of 4F2hc in prostate cancer was studied. Treatment of C4-2 cells with si4F2hc was found to suppress cellular growth, migratory and invasive abilities, with this effect occurring through the cell cycle, with a significant decrease in S phase and a significant increase in G0/G1 phase, suggesting cell cycle arrest. In addition, it was proven by RNA seq that the key to 4F2hc's impact on cancer is SKP2. si4F2hc upregulates the protein expression of cyclin-dependent kinase inhibitors (P21cip1, P27kip1) through the downstream target SKP2. Furthermore, the expression of 4F2hc and LAT1 in prostate cancer cells suggests the importance of 4F2hc. Multivariate analysis showed that high 4F2hc expression was an independent prognostic factor for progression-free survival (HR 11.54, p = 0.0357). High 4F2hc was related to the clinical tumour stage (p = 0.0255) and Gleason score (p = 0.0035). Collectively, 4F2hc contributed significantly to prostate cancer (PC) progression. 4F2hc may be a novel marker and therapeutic target in PC.

INTRODUCTION: Although 18F-FDG PET was originally developed to evaluate benign and malignant tumors, the frequency of detection of benign adrenocortical adenomas showing FDG-PET accumulation has increased. However, the details of FDG-PET-accumulated benign adrenocortical adenomas have not been elucidated. METHODS: To elucidate the pathophysiology of FDG-PET-positive cortisol-producing adrenal tumors, we performed clinicopathological and genetic analyses of adrenocortical adenomas examing FDG-PET in 30 operated patients with unilateral cortisol-producing adrenal tumors (26 adrenal adenomas and 4 adrenal cancers). RESULTS: All adrenocortical carcinomas and 17/26 (65%) benign adrenocortical adenomas showed high FDG accumulation (SUVmax ≥ 3). In adrenocortical adenomas with high FDG accumulation (SUVmax ≥ 3), SUVmax showed a positive correlation with the CT Hounsfield units. A higher SUVmax showed a clear black adenoma appearance with predominantly compact cells, which exhibited high T1 and T2 signals, a lack of signal drop on out-of-phase imaging on MRI, and less accumulation on 131-I adsterol scintigraphy. Furthermore, RNA-sequencing analysis revealed significant increases in the lysosomal and autophagy pathways and metabolic pathways, including glycolysis through glucose transporter (GLUT) 1 and 3, in black adenomas with high-level FDG accumulation. DISCUSSION: A black adenoma is blackish due to lipofuscin, which accumulates as a result of damaged mitochondria or proteins that escape lysosomal degradation or autophagy. Since FDG in PET is taken up via GLUTs, alteration of the intracellular metabolic dynamics associated with mitochondrial damage in black adenomas may increase PET accumulation. CONCLUSION: Black adrenal adenomas should be considered with adrenal tumors showing PET accumulation and low lipid contents.

Bronchiolitis obliterans syndrome (BOS) occurring after hematopoietic stem cell transplantation (HSCT) for hematologic malignancies is a progressive and refractory disease, and lung transplantation (LTx) seems to be the only promising treatment. We report two cases of BOS after HSCT, which showed distinct clinical courses and were successfully treated with LTx. The respiratory symptoms and function of the two patients progressively deteriorated to a critical level during the waiting period. In one patient, recurrent and intractable pneumothoraxes consistent with thoracic air-leak syndrome (TALS) occurred, which were associated with pleuroparenchymal fibroelastosis. TALS could accelerate clinical deterioration, thus permitting a shorter waiting period for LTx.

Sialadenoma papilliferum (SP) is a rare benign tumor of the salivary glands, and only 3 unequivocal cases of SP arising in the bronchus have been reported. We herein describe the histomorphologic and molecular features of 4 bronchial SP cases and discuss the differential diagnosis of this entity and the relationship with its clinicopathologic mimics, in particular, glandular papilloma and mixed squamous cell and glandular papilloma (GP/MP). We encountered 2 male and 2 female patients with bronchial SP (mean: 66.8 y old). All 4 tumors arose in the central bronchus and were characterized by a combination of surface exophytic endobronchial papillary proliferation and a submucosal multicystic component with complex architecture. The neoplastic epithelium consisted predominantly of nonciliated stratified columnar cells with ciliated, squamous, and mucinous cells present focally. While 2 tumors (50%) harbored a BRAF V600E mutation by molecular and immunohistochemical analysis, similar to GP/MP, no KRAS, HRAS, AKT1, or PIK3CA mutations were detected in any of the cases. Two patients were treated with limited resection, while 2 patients underwent lobectomy based on the diagnosis of adenocarcinoma or possible squamous cell carcinoma in situ in the preoperative biopsy. All survived without recurrence or metastasis for 23 to 122 months after treatment. SP can develop in the central bronchus as the bronchial counterpart of the salivary gland tumor and should be considered in the differential diagnosis of endobronchial tumors. In addition, some histologic resemblance and frequent BRAF V600E mutation raise the possibility of SP and GP/MP being on the same disease spectrum.

von Hippel-Lindau (VHL) disease is characterized by neoplastic and cystic lesions, such as central nervous system (CNS) hemangioblastoma and clear cell renal cell carcinoma (RCC), arising in multiple organs. Here, we report a case of an RCC that metastasized to a spinal hemangioblastoma in a patient diagnosed with VHL disease. This is a unique case study because visceral neoplasms rarely metastasize to the CNS. The patient had undergone posterior fossa surgery for the removal of hemangioblastomas in the right cerebellar hemisphere as a child. He was diagnosed with RCC at the age of 20 years, and he underwent partial nephrectomy at the age of 35 years. The patient underwent surgical removal of a spinal tumor from Th8, which was also diagnosed as a hemangioblastoma at the age of 40. However, the residual spinal tumor rapidly regrew within 1.5 years. A second surgery was performed due to progressive leg motor weakness. The resected tumor from the second surgery had two distinct components between the tumor center and the margin. Immunohistochemistry of CD10, PAX 8, and inhibin A demonstrated the predominant region of the tumor was RCC. Pathological findings confirmed tumor-to-tumor metastasis of the RCC migrating into residual spinal hemangioblastoma. It can be challenging to distinguish hemangioblastoma from RCC in neuroimaging. We suggest that tumor-to-tumor metastasis should be considered as a differential diagnosis if benign tumors grow rapidly, even if the pathological diagnosis does not initially confirm malignancy. The biological mechanisms of RCC migrating into residual hemangioblastoma are discussed.

A 45-year-old woman was diagnosed with myelodysplastic syndrome (MDS) with trisomy 8 and Behçet-like disease (BLD) with multiple colorectal ulcers. Nonspecific inflammatory cells were infiltrated in the intestinal mucosa, whereas fluorescence in situ hybridization (FISH) analysis revealed only sporadic trisomy 8-positive cells. She presented massive lower gastrointestinal bleeding early after bone marrow transplantation but achieved long-term remission of both MDS and BLD. This is the first report of massive gastrointestinal bleeding after transplantation for MDS with BLD. Based on FISH analysis, dysregulation of systemic inflammation may be involved in BLD rather than direct invasion by trisomy 8-positive MDS clones.

To our knowledge, the in situ phase of small cell lung carcinoma (SCLC) has not been reported previously although in situ/pre-invasive lesions are commonly observed in murine models. We report a patient diagnosed with SCLC presenting mostly as carcinoma in situ after photodynamic therapy (PDT) under the diagnosis of bronchial squamous cell carcinoma (SqCC) in situ.

BACKGROUND: Approximately 60% of adrenocortical carcinomas (ACC) are functional, and Cushing's syndrome is the most frequent diagnosis that has been revealed to have a particularly poor prognosis. Since 30% of ACC present steroid hormone-producing disorganization, measurement of steroid metabolites in suspected ACC is recommended. Previous reports demonstrated that steroid hormone precursors or their urine metabolites, which can be assessed using liquid chromatography tandem mass spectrometry (LC-MS/MS) or gas chromatography mass spectrometry (GC-MS) respectively, are useful for distinguishing ACC from cortisol-producing adenomas (CPA); however, despite high precision, LC-MS/MS and GC-MS require a highly trained team, are expensive and have limited capacity. METHODS: Here, we examined 12 serum steroid metabolites using an immunoassay, which is a more rapid and less costly method than LC-MS/MS, in cortisol-producing ACC and CPA. Further, the correlation of each steroid metabolite to the classification stage and pathological status in ACC was analyzed. RESULTS: Reflecting disorganized steroidogenesis, the immunoassay revealed that all basal levels of steroid precursors were significantly increased in cortisol-producing ACC compared to CPA; in particular, 17-hydroxypregnenolone (glucocorticoid and androgen precursor) and 11-deoxycorticosterone (mineralocorticoid precursor) showed a large area under the ROC curve with high sensitivity and specificity when setting the cut-off at 1.78 ng/ml and 0.4 mg/ml, respectively. Additionally, a combination of androstenedione and DHEAS also showed high specificity with high accuracy. In cortisol-producing ACC, 11-deoxycortisol (glucocorticoid precursor) showed significant positive correlations with predictive prognostic factors used in ENSAT classification, while testosterone showed significant positive correlations to the Ki67-index in both men and women. CONCLUSION: Less expensive and more widely available RIA and ECLIA may also biochemically distinguish ACC from CPA and may predict the clinicopathological features of ACC.

We treated a man with co-incident Marfan-like connective tissue disease with morphologic left ventricular non-compaction (LVNC). He underwent valve-sparing aortic root replacement because of aortic root dilation at 43 years old. Pathological findings of the aorta revealed cystic medio-necrosis, consistent with Marfan syndrome. He developed congestive heart failure caused by LVNC at 47 years old. His daughter had scoliosis, and he had several physical characteristics suggestive of Marfan syndrome. We herein report a rare case of a patient who had Marfan-like connective disease with an LVNC appearance.

OBJECTIVES: Complete hydatidiform moles (CHMs) are androgenetic and have a high rate of progression to gestational trophoblastic neoplasia (GTN). CHMs are negative when immunostained for p57KIP2 protein, the product of the maternally expressed gene on chromosome 11p15.5, whereas biparental partial hydatidiform moles and hydropic abortion are positive for p57KIP2. This study presents two cases of p57KIP2-positive androgenetic CHMs and explores the cause of this inconsistency. METHODS: Androgenetic CHMs were diagnosed using multiplex short tandem repeat polymorphism analysis. Single-nucleotide polymorphism arrays were performed for molecular karyotyping. RESULTS: Among the consecutive 188 androgenetic CHMs, two cases were positive for p57KIP2. The first case remitted spontaneously, whereas the second case developed into low-risk GTN. The first case was positive for p57KIP2 in all villi. The karyotype was 48,XX,+7,+11, with the additional chromosome 11 confirmed to be of maternal origin. The second case presented a mosaic of both positively and negatively stained villi. The karyotype was 46,XX. CONCLUSIONS: The cause of one of the CHMs was trisomy with an additional maternal chromosome 11. Although rare, the confirmation of p57KIP2-positive androgenetic CHM status is necessary to manage GTN risk.

Irradiation, or chemoradiotherapy, is a curative treatment for oropharyngeal squamous cell carcinoma (OPSCC). Its invasiveness, however, can often negate its efficacy. Therefore, developing methods to predict which patients would benefit from irradiation is urgent. Promoter DNA hypermethylation was recently reported to correlate with favorable OPSCC prognosis. It is still unclear, however, whether there is an association between promoter DNA methylation and response to irradiation. In this study, we analyzed DNA methylation in the specimens from 40 OPSCC patients who had undergone irradiation, using the Infinium assay. Our results showed significant correlation between high levels of promoter DNA methylation and better response to treatment (P < 0.01). We used the 10 most differentially-methylated genes between responders and non-responders to develop a panel of predictive markers for efficacy. Our panel had high sensitivity, specificity and accuracy (92%, 93% and 93%, respectively). We conducted pyrosequencing to quantitatively validate the methylation levels of 8 of the 10 marker genes (ROBO1, ULK4P3, MYOD1, LBX1, CACNA1A, IRX4, DPYSL3 and ELAVL2) obtained by Infinium. The validation by pyrosequencing showed that these 8 genes had a high prediction performance for the training set of 40 specimens and for a validation set of 35 OPSCC specimens, showing 96% sensitivity, 89% specificity and 94% accuracy. Methylation of these markers correlated significantly with better progression-free and overall survival rates, regardless of human papillomavirus status. These results indicate that increased DNA methylation is associated with better responses to irradiation therapy and that DNA methylation can help establish efficacy prediction markers in OPSCC.

Aim: Esophageal squamous cell carcinoma (ESCC) is a refractory digestive organ cancer that requires better treatment strategies. We have recently reported that the antidiabetic drug metformin exerts antitumor effects on ESCC by inhibition of nuclear factor kappa B (NF-κB) nuclear translocation. In the present study, we focused on caspase recruitment domain family member 9 (CARD9), an essential signal adapter in NF-κB activation to examine whether it can be used as a prognostic factor in ESCC. Methods: We investigated CARD9 expression immunohistochemically in clinical samples obtained from 93 patients with ESCC who underwent curative esophagectomy. CARD9 expression was analyzed for correlation with clinicopathological characteristics and ESCC prognosis. The molecular effects were investigated by knocking down ESCC cells. Comprehensive RNA expression changes in these ESCC cells were detected by next-generation sequencing (NGS). Results: High CARD9 expression is significantly correlated with advanced tumor depth (P < .001), positive lymph node metastasis (P = .005) and advanced stage (P = .001). Kaplan-Meier method and the log-rank test showed that overall survival (OS) and disease-free survival (DFS) were significantly poor in the high CARD9 expression group (OS: P = .027, DFS: P = .005). Univariate and multivariate analysis showed that high CARD9 expression is a significant poor prognostic factor for DFS. Cell proliferation and migration were suppressed by CARD9 knockdown. NGS detected altered the expression of some RNAs including maternally expressed 3 (MEG3). Conclusion: High CARD9 expression is significantly associated with poor prognosis. Therefore, CARD9 expression may be a prospective prognostic biomarker in ESCC.

•A cul-de-sac endometrioid carcinoma adjacent to extraovarian endometriosis was identified during remission of endometrial cancer.•The origin of the cul-de-sac tumor was malignant transformation of deep infiltrating endometriosis.•Endometriosis-related cancer was identified in a woman with endometrial cancer during remission.•Hyperestrogenism due to infertility treatment may contribute to malignant transformation of deep infiltrating endometriosis.

© 2020, The Author(s), under exclusive licence to Springer Nature Limited. Serine racemase (SRR) catalyses not only the racemization but also the dehydration of l-serine and d-serine, resulting in the formation of pyruvate and ammonia. Although SRR activity is important in the central nervous system, SRR has not been linked to cancer metabolism before. Here we show that SRR supports proliferation of colorectal-cancer cells. We find that SRR expression is upregulated in colorectal adenoma and adenocarcinoma lesions compared with non-neoplastic mucosa in human colorectal-cancer specimens. SRR-mediated dehydration of serine contributes to the pyruvate pool in colon-cancer cells, enhances proliferation, maintains mitochondrial mass and increases basal reactive oxygen species production, which has anti-apoptotic effects. Moreover, SRR promotes acetylation of histone H3 by maintaining intracellular acetyl-CoA levels. Inhibition of SRR suppresses growth of colorectal tumours in mice and augments the efficacy of 5-fluorouracil treatment. Our findings highlight a previously unknown mechanism through which a racemase supports cancer-cell growth and suggest that SRR might be a molecular target for colorectal-cancer therapy.

We present evidence for the decomposition and oxidation of amino acids in aqueous solution following irradiation with a nonequilibrium plasma jet. Of 15 amino acids tested in cell culture medium, plasma irradiation induced a marked chemical change in methionine and tryptophan due to the effective production of reactive oxygen species by plasma-water interaction. We also report that plasma-treated methionine and tryptophan aqueous solutions can kill cancer cells, greatly decreasing the viability of human endometrial carcinoma (HEC-1) cancer cells due to the presence of decomposition or oxidation products generated from the amino acid. Plasma-treated methionine and tryptophan aqueous solutions also induced an anti-cancer effect on cancer-initiating cells.

? 2019, The Author(s). In Figure 5 there is a missing axis label. The y-axis of the bottom graph should be labelled “Intensity (arb. units)” The correct Figure 5 appears below as Figure 1. Additionally, this Article contains errors in Reference 44 which is incorrectly given as: Ito, T., Uchida, G., Nakajima, A., Takenaka, K. &amp; Setsuhara, Y. Selective production of reactive oxygen and nitrogen species in the plasma-treated water by using a nonthermal high-frequency plasma jet. Jpn. J. Appl. Phys. 56, 01AC06 (2016) The correct reference is listed below as ref. 1:.

Angiosarcoma is a rare malignant tumour, which accounts for 1-2% of all malignant soft-tissue tumours. Most cases of angiosarcoma arise spontaneously, and malignant transformation of vascular malformation to angiosarcoma is extremely rare. We describe the case of a 70-year-old woman with a massive arteriovenous malformation in her shoulder, which gradually enlarged, despite repeated surgeries and radiation therapy over 53 years. She also presented with rapidly growing haemorrhagic masses in her oral cavity. Excision biopsy was performed, and the pathohistological diagnosis was angiosarcoma. Positron emission tomography-computed tomography revealed high fluorodeoxyglucose accumulation in the oral cavity and right shoulder, the latter of which was consistent with the location of the arteriovenous malformation. The masses in the oral cavity were diagnosed as metastatic angiosarcoma from the right shoulder, where the massive arteriovenous malformation was suspected to have malignantly transformed. This report describes a possible case of malignant transformation of arteriovenous malformation to angiosarcoma.

BACKGROUND: Microsatellite instability (MSI) and programmed death-ligand 1 (PD-L1) are candidate predictors for the response to immune checkpoint inhibitors, and may predict chemotherapy sensitivity. We investigated the simultaneous expression of mutL homolog 1 (MLH1), a mismatch repair gene, and PD-L1 in gastric cancers. METHODS: We examined MLH1 and PD-L1 expression in surgical specimens from 285 gastric cancer patients treated with or without preoperative chemotherapy, and assessed the relation between expression results and both histological response and recurrence-free survival (RFS). RESULTS: Of 285 patients, 28 (9.8%) and 70 (24.6%) exhibited negative MLH1 and high PD-L1 expression, respectively. Most MLH1-negative tumors (85.7%) showed high MSI, and these tumors exhibited high PD-L1 expression more frequently than MLH1-positive tumors (57.1% vs. 21.0%, P < 0.001). MLH1-negative patients were significantly less likely to respond to preoperative chemotherapy than MLH1-positive patients (16.7% vs. 61.2%, P = 0.005), whereas there was no significant difference between high- and low-PD-L1 expression patients (55.9% vs. 56.6%, P = 0.95). RFS in patients without preoperative chemotherapy was significantly longer in the MLH1-negative group than in the MLH1-positive group (HR 0.30; 95% CI 0.09-0.95; P = 0.030), whereas in patients with preoperative chemotherapy there was no significant difference in RFS between the two groups (HR 0.70; 95% CI 0.30-1.63; P = 0.41). PD-L1 expression was not associated with RFS in patients with or without chemotherapy. CONCLUSIONS: Loss of MLH1 was associated with chemoresistance and did not prolong survival following neoadjuvant chemotherapy. The strong association between MLH1 and MSI status suggests that immune checkpoint inhibitors may be preferable to conventional chemotherapy for MLH1-negative gastric cancer.

© 2019 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association. Endometrioid carcinoma (EC) is one of the most common malignancies of the female genital system. We reported previously that aldehyde dehydrogenase 1 (ALDH1), a predominant isoform of the ALDH family in mammals and a potential marker of normal and malignant stem cells, is related to the tumorigenic potential of EC. We compared the levels of various proteins in human EC cells with high and low ALDH1 expression using shotgun proteomics and found that serum deprivation-response protein (SDPR) was preferentially expressed in cells with high ALDH1 expression. Also known as cavin-2, SDPR is a member of the cavin protein family, which is required for the formation of caveolae. Using SDPR-knockout EC cells generated using the CRISPR/Cas9 system, we revealed that SDPR was correlated with invasion, migration, epithelial-mesenchymal transition, and colony formation, as well as the expression of ALDH1. RNA sequencing showed that integrin-linked kinase (ILK) signaling is involved in the effect of SDPR on ALDH1. Immunohistochemical analysis revealed that the localization of ILK at the cell cortex was disrupted by SDPR knockout, potentially interfering with ILK signaling. Moreover, immunohistochemical analysis of clinical samples showed that SDPR is related to histological characteristics associated with invasiveness, such as poor differentiation, lymphatic invasion, and the microcystic, elongated, and fragmented histopathological pattern. This is, to our knowledge, the first report that SDPR is related to tumor progression.

Clean operating margins in breast cancer surgery are important for preventing recurrence. However, the current methods for determining margins such as intraoperative frozen section analysis or imprint cytology are not satisfactory since they are time-consuming and cause a burden on the patient and on hospitals with a limited accuracy. A "click-to-sense" probe is developed based on the detection of acrolein, which is a substance released by oxidatively stressed cancer cells and can be visualized under fluorescence microscopy. Using live breast tissues resected from breast cancer patients, it is demonstrated that this method can quickly, selectively, and sensitively differentiate cancer lesion from normal breast gland or benign proliferative lesions. Since acrolein is accumulated in all types of cancers, this method could be used to quickly assess the surgical margins in other types of cancer.

© 2019 IOP Publishing Ltd. The performance characteristics of two plasma sources, based on different types of discharge, were assessed. Three primary aspects of these sources were examined. These were electrical parameters (voltage and current flowing through a target), plasma parameters (gas temperature and electron density, determined using optical emission spectroscopy), and levels of gas-phase oxygen atoms (measured by vacuum ultraviolet absorption spectroscopy) and liquid-phase ▪OH radicals (generated by plasma treatment of water and detected using an electron spin resonance spin-trapping technique). As a result, there were few significant differences such as electron density, oxygen atom density, and gas temperature between the two plasma sources. However, the time-averaged electrical currents flowing to the target and the amount of liquid-phase ▪OH radicals showed a large difference, especially when the plume made contact with the target. Thus, many points of similarity but some differences in the two devices were found by the benchmarking study. These shall contribute to our understanding of the mechanisms for each consequence in medical applications.

Glutamine (Gln) is important not only for cell proliferation but also for differentiation. Although Gln is essential for plasmacytic differentiation of lymphocytes, no study has been done on the effect of Gln on differentiation of tumor cells, such as lymphoma. Here we examined the effect of Gln on plasmacytic differentiation of lymphoplasmacytic lymphoma (LPL) with its cell lines, MWCL-1 and RPCI-WM1. Gln promoted plasmacytic differentiation of LPL, and p38 MAPK signaling pathway mediated such differentiation. We previously reported that the subpopulation with plasmacytic differentiation was vulnerable to apoptosis in LPL. Although it is difficult to lead these findings to the radical therapy, they might help the treatment of LPL, in which stimulation of p38 MAPK by Gln induced differentiation of LPL into vulnerable subpopulation.

© 2018 The Japan Society of Applied Physics. We describe the effects of the plasma-activation of a medium on the killing of cancer cells, in which the plasma-activated medium is produced by the irradiation of the cell culture medium using a low-frequency (LF) or very-high-frequency (VHF) plasma jet. The plasma-activated medium produced by the direct contact of the LF plasma jet with the liquid surface induces much lower cancer cell viability, and large amounts of oxidation products, such as gluconic acid and methionine sulfoxide, can be detected in the plasma-activated medium. Our experiments show that the process of using plasma created by the direct contact of a LF plasma jet leads to a plasma-activated medium with stronger cancer-cell killing ability compared with a no-contact process. Also, the oxidation products, such as methionine sulfoxide in the plasma-activated medium become useful proxies for lethality.

The pathologic feature of intraductal papillomas is defined as a papillary structure composed of a fibrovascular stromal core lined by luminal epithelial cells and myoepithelial cells. We used droplet digital PCR for the mutational analysis of AKT1 (E17K) and PIK3CA (H1047R, E542K, and E545K) in 60 papillomas. AKT1 and PIK3CA mutations were detected in 12 (20%) and 17 (28%) of the papillomas, respectively. In five tumors harboring mutations, mutational analysis of AKT1 or PIK3CA was performed separately using luminal epithelial cells and myoepithelial cells sorted using anti-cytokeratin 19 antibody and anti-α smooth muscle actin antibody. The two types of cells from a given papilloma had the identical mutation. Three patients with the PIK3CA mutation-positive papilloma developed breast cancers at the resection site of the papilloma, but none of these subsequent breast cancers had the PIK3CA mutation. These results indicate that a papilloma stems from a bipotent progenitor cell that contains the AKT1 or PIK3CA mutation and proliferates and differentiates to form the papilloma. Papilloma can be a risk factor for developing breast cancer but is unlikely to be its obligate precursor.

© 2018 United States & Canadian Academy of Pathology. Adenylosuccinate lyase (ADSL) is an enzyme that plays important roles in de novo purine synthesis. Although ADSL was reported to be upregulated in various malignancies, such as colorectal, breast, and prostate cancer, as well as gliomas, the mechanism by which elevated ADSL expression contributes to cancer has not been elucidated. We previously performed a shotgun proteomics analysis to characterize specific proteins associated with the properties of the aldehyde dehydrogenase (ALDH)-high cell population, which was reported to be involved in tumorigenic potential, and showed that ADSL expression is upregulated in the ALDH-high population of endometrial cancer. Here, we showed that ADSL is involved in endometrial cancer aggressiveness by regulating expression of killer cell lectin-like receptor C3 (KLRC3), which is a receptor expressed on natural killer cells. Immunohistochemical analysis indicated that ADSL expression increased as endometrioid carcinoma specimens became more poorly differentiated and higher degree of primary tumor progression. Knockdown of ADSL in endometrial cancer cells decreased cell proliferation, migration, and invasive capability, and caused the cells to adopt a more rounded shape. DNA microarray analysis and quantitative real-time PCR showed that KLRC3 expression was decreased in ADSL knockdown cells. Knockdown of KLRC3 in endometrial cancer cells resulted in the same phenotype as knockdown of ADSL. Moreover, fumarate, which could be produced by ADSL and was recently shown to be an oncometabolite, recovered KLRC3 expression in ADSL knockdown cells, suggesting that fumarate produced by ADSL could regulate KLRC3 expression. Our findings indicate that ADSL enhances cell proliferation, migration, and invasive capability through regulation of KLRC3 expression by fumarate.

Enchondromatosis is a rare skeletal disorder characterized by the development of multiple enchondromas, which can also manifest non-cartilage tumors including gliomas. Here, we describe a genetic analysis of a low-grade glioma that developed in an enchondromatosis case. A 32-year-old man with a long history of enchondromatosis developed a left frontal tumor. The histopathological findings of his surgical specimen revealed characteristics of a low-grade glioma with an IDH1 c.395G>A (R132H) mutation and 1p/19q codeletion, which led to a definitive diagnosis of oligodendroglioma. A common point mutation in IDH1 (R132H) was detected in the patient's enchondroma and glioma-matched pair specimens. To the best of our knowledge, this is the first case of molecularly confirmed oligodendroglioma associated with enchondromatosis. Furthermore, identification of a common IDH1 mutation in enchondroma and oligodendroglioma-matched pair specimens supports the hypothesis that IDH1/2 mosaicism initiates tumorigenesis.

© 2017 Japanese Society of Pathology and John Wiley & Sons Australia, Ltd Medical non-thermal plasma (NTP) treatments for various types of cancers have been reported. Cells with tumorigenic potential (cancer-initiating cells; CICs) are few in number in many types of tumors. CICs efficiently eliminate anti-cancer chemicals and exhibit high-level aldehyde dehydrogenase (ALDH) activity. We previously examined the effects of direct irradiation via NTP on cancer cells; even though we targeted CICs expressing high levels of ALDH, such treatment affected both non-CICs and CICs. Recent studies have shown that plasma-activated medium (PAM) (culture medium irradiated by NTP) selectively induces apoptotic death of cancer but not normal cells. Therefore, we explored the anti-cancer effects of PAM on CICs among endometrioid carcinoma and gastric cancer cells. PAM reduced the viability of cells expressing both low and high levels of ALDH. Combined PAM/cisplatin appeared to kill cancer cells more efficiently than did PAM or cisplatin alone. In a mouse tumor xenograft model, PAM exerted an anti-cancer effect on CICs. Thus, our results suggest that PAM effectively kills both non-CICs and CICs, as does NTP. Therefore, PAM may be a useful new anti-cancer therapy, targeting various cancer cells including CICs.

Cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs) in tumor stroma play a key role in disease progression. Recent studies using mice models suggest that CAFs are partly derived from bone marrow and TAMs primarily originate from bone marrow-derived inflammatory monocytes. However, the origin of these cells in humans remains unclear. Hence, we investigated their human origin, using specimens from human secondary tumors that developed after sex-mismatched bone marrow transplantation, by modified immunofluorescent in situ hybridization analysis and triple immunostaining. We observed that most of the α-smooth muscle actin (αSMA)-positive CAFs in the mammary gland, liver, and oral mucosa specimens obtained 3-19 years after bone marrow transplantation are recipient-derived cells. In contrast, the majority of the peritumoral αSMA-negative fibroblast-like cells are actually bone marrow-derived HLA-DR-positive myeloid cells, such as macrophages and dendritic cells. Furthermore, almost all CD163-positive TAMs and macrophages present in the non-tumor areas are derived from bone marrow.

Resistance against anti-HER2 drugs in HER2-positive breast cancer is a major obstacle to the improving prognosis. Transforming growth factor beta (TGF beta) is a cytokine involved in the acquisition of more malignant phenotypes through epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC) properties. The aim of this study was to investigate the effects of TGF beta and its downstream SMAD pathway on resistance to anti-HER2 drugs. HER2-positive breast cancer cell lines were stimulated with TGF beta for 14 days. Then, the sensitivity to trastuzumab and lapatinib and the expression levels of various EMT and CSC markers were examined. The correlation of nuclear SMAD3 expression in untreated breast tumor tissues with trastuzumab efficacy in neoadjuvant settings was examined. The effect of a small-molecule inhibitor of SMAD3 (SIS3) on resistance to anti-HER2 drugs was explored. We found that continuous activation of the TGF beta-SMAD3 pathway induced resistance to anti-HER2 drugs and CSC traits in HER2-positive breast cancer cells. The induction of drug resistance by TGF beta required strong activation of SMAD3. In fact, activated SMAD3 regulated multiple genes that harbor SMAD-binding elements and are involved in trastuzumab resistance. Nuclear SMAD3 expression in tumor tissue was inversely correlated with sensitivity to neoadjuvant treatment with trastuzumab. SIS3 not only prevented the acquisition of resistance to anti-HER2 drugs but also restored trastuzumab sensitivity in trastuzumab-resistant cells. This study indicates that the TGF beta-SMAD3 pathway plays an important role in the induction and maintenance of resistance to anti-HER2 drugs. Thus, SMAD3 is a potential therapeutic target that can inhibit resistance and restore sensitivity to anti-HER2 drugs.

The examination of hematoxylin and eosin (H&E)-stained tissues on glass slides by conventional light microscopy is the foundation for histopathological diagnosis. However, this conventional method has some limitations in x-y axes due to its relatively narrow range of observation area and in z-axis due to its two-dimensionality. In this study, we applied a CUBIC pipeline, which is the most powerful tissue-clearing and three-dimensional (3D)-imaging technique, to clinical pathology. CUBIC was applicable to 3D imaging of both normal and abnormal patient-derived, human lung and lymph node tissues. Notably, the combination of deparaffinization and CUBIC enabled 3D imaging of specimens derived from paraffin-embedded tissue blocks, allowing quantitative evaluation of nuclear and structural atypia of an archival malignant lymphoma tissue. Furthermore, to examine whether CUBIC can be applied to practical use in pathological diagnosis, we performed a histopathological screening of a lymph node metastasis based on CUBIC, which successfully improved the sensitivity in detecting minor metastatic carcinoma nodules in lymph nodes. Collectively, our results indicate that CUBIC significantly contributes to retrospective and prospective clinicopathological diagnosis, which might lead to the establishment of a novel field of medical science based on 3D histopathology.

Multiphoton excitation microscopy (MPM) is regarded as an effective tool that enables the visualization of deep regions within living tissues and organs, with little damage. Here, we report novel non-labeling MPM (NL-MPM) imaging of fresh human colorectal mucosa, which is useful for discriminating cancer lesions from normal tissues quantitatively without any need for resection, fixation, or staining. Using NL-MPM, we visualized three components in human colorectal mucosa, epithelial cells, immune cells, and basement membranes, based on their characteristic patterns of fluorescence. These patterns are characterized by the different auto-fluorescence properties of nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate, and flavin adenine dinucleotide and from second harmonic generation (SHG). NL-MPM images were at least as informative to pathologists as were 'conventional' images of fixed tissue sections stained with hematoxylin and eosin. Additionally, two quantitative parameters extracted from NL-MPM images - the nucleus diameter (index N) and the intensity of SHG in the basement membrane (index S) - rendered it possible to diagnose cancer regions effectively. In conclusion, NL-MPM is a novel, promising method for real-time clinical diagnosis of colorectal cancers, and is associated with minimal invasiveness.

Argininosuccinate synthetase 1 (ASS1) is a rate-limiting enzyme in arginine biosynthesis. Although ASS1 expression levels are often reduced in several tumors and low ASS1 expression can be a poor prognostic factor, the underlying mechanism has not been elucidated. In this study, we reveal a novel association between ASS1 and migration/invasion of endometrial tumors via regulation of mechanistic target of rapamycin complex (mTORC) 1 signaling. ASS1-knockout cells showed enhanced migration and invasion in response to arginine following arginine starvation. In ASS1-knockout cells, DEPTOR, an inhibitor of mTORC1 signal, was downregulated and mTORC1 signaling was more activated in response to arginine. ASS1 epigenetically enhanced DEPTOR expression by altering the histone methylation. Consistent with these findings, tumor cells at the invasive front of endometrioid carcinoma cases showed lower ASS1 and DEPTOR expression. Our findings suggest that ASS1 levels in each tumor cell are associated with invasion capability in response to arginine within the tumor microenvironment through mTORC1 signal regulation.

Marginal zone lymphoma (MZL) is a low-grade B-cell lymphoma derived from marginal zone B cells. Because of a lack of specific immunohistochemical markers, MZL is mainly diagnosed based on the cytological appearance and growth pattern of the tumor. Marginal zone B cells were recently shown to selectively express immunoglobulin superfamily receptor translocation-associated 1 (IRTA1), but the antibody used in that study is not commercially available. We therefore investigated the IRTA1 expression in nonneoplastic lymphoid tissues and 261 malignant lymphomas, examining the ability of a commercially available antibody to accurately diagnose MZL. Among 37 MZLs, 23 of 25 extranodal MZLs of mucosa-associated lymphoid tissue (MALT lymphomas), 3 of 6 splenic MZLs and 3 of 6 nodal MZLs were positive for IRTA1. Among the 98 diffuse large B-cell lymphomas, 33 were positive for IRTA1, including 1 of 38 follicular lymphomas, and all precursor B-lymphoblastic (2/2) and T-lymphoblastic (7/7) leukemia/lymphomas. Other mature B-cell and T-cell lymphomas, and Hodgkin lymphoma were negative for IRTA1. In MALT lymphoma, positive cells were detected mainly in intraepithelial and subepithelial marginal zone B cells. In 1 case of grade 3 follicular lymphoma, IRTA1 was also expressed in the area of large cell transformation. When tumors were classified as germinal center B cell like (GCB) or non-GCB using the algorithm of Hans, positive expression of IRTA1 was correlated significantly with non-GCB diffuse large B-cell lymphomas (P &lt; .05). These results demonstrated the ability of the commercially available IRTA1 antibody to distinguish MALT lymphoma from other low-grade B-cell lymphomas. (C) 2016 Elsevier Inc. All rights reserved.

Nonequilibrium atmospheric pressure plasma (NEAPP) is a novel approach for blood coagulation, wound healing, and tumor elimination. NEAPP not only directly but also indirectly affects living cells via the medium exposed to NEAPP-yielding devises, called plasma-activated medium (PAM). The conservable and portable PAM serves as an alternative and advantageous approach over direct NEAPP. Here we examined the effect of PAM on lymphoplasmacytic lymphoma (LPL) cell lines. We found that PAM induced plasma cell differentiation and reduced tumorigenic population. PAM increased the expression level of PRDM1 alpha, which is a transcription factor promoting plasma cell differentiation, suggesting that plasma cell differentiation of LPL might be mediated by PRDM1 alpha. We previously reported that plasma cell component of LPL is vulnerable to apoptosis and less tumorigenic. These findings suggested that PAM treatment might become a novel therapy against LPL by inducing the transition from tumorigenic to non-tumorigenic population. (c) 2017 Elsevier GmbH. All rights reserved.

Cyclin G1 (CycG1) and Cyclin G2 (CycG2) play similar roles during the DNA damage response (DDR), but their detailed roles remain elusive. To investigate their distinct roles, we generated knockout mice deficient in CycG1 (G1KO) or CycG2 (G2KO), as well as double knockout mice (DKO) deficient in both proteins. All knockouts developed normally and were fertile. Generation of mouse embryonic fibroblasts (MEFs) from these mice revealed that G2KO MEFs, but not G1KO or DKO MEFs, were resistant to DNA damage insults caused by camptothecin and ionizing radiation (IR) and underwent cell cycle arrest. CycG2, but not CycG1, co-localized with.H2AX foci in the nucleus after gamma-IR, and gamma H2AXmediated DNA repair and dephosphorylation of CHK2 were delayed in G2KO MEFs. H2AX associated with CycG1, CycG2, and protein phosphatase 2A (PP2A), suggesting that.H2AX affects the function of PP2A via direct interaction with its B'. subunit. Furthermore, expression of CycG2, but not CycG1, was abnormal in various cancer cell lines. Kaplan-Meier curves based on TCGA data disclosed that head and neck cancer patients with reduced CycG2 expression have poorer clinical prognoses. Taken together, our data suggest that reduced CycG2 expression could be useful as a novel prognostic marker of cancer.

Cancer-initiating cells (CICs) are specialized cells that have the ability to self-renew and are multipotent. We recently demonstrated that Forkhead box O3a (FoxO3a)-expressing cells exhibited a CIC-like potential in Hodgkin's lymphoma (HL). A proportion of HL patients are infected with Epstein-Barr virus (EBV). EBV-encoded latent membrane protein (LMP) 1 downregulates FoxO3a, suggesting that FoxO3a expression may be abolished in EBV-positive HL. Inhibitors of DNA-binding (ID) proteins are highly conserved transcription factors mediating stem cell functions. To the best of our knowledge, no study has investigated possible associations among ID1, FoxO3a and LMP1 expression in HL to date. We immunohistochemically evaluated the expression of the three abovementioned factors in HL patients. The ID1 expression level was inversely correlated with that of FoxO3a (P=0.00035). LMP1-positive HL cells abundantly expressed ID1 (P=0.029), but not FoxO3a (P=0.00085). Thus, our previous observation that FoxO3a may serve as a marker of CICs may not be applicable in EBV-positive HL patients, but rather ID1 may be a candidate CIC marker in this type of HL.

Low-temperature plasma (LTP) treatment promotes blood clot formation by stimulation of the both platelet aggregation and coagulation factors. However, the appearance of a membrane-like structure in clots after the treatment is controversial. Based on our previous report that demonstrated characteristics of the form of coagulation of serum proteins induced by LTP treatment, we sought to determine whether treatment with two plasma instruments, namely BPC-HP1 and PN-110/120TPG, formed clots only from red blood cells (RBCs). LTP treatment with each device formed clots from whole blood, whereas LTP treatment with BPC-HP1 formed clots in phosphate-buffered saline (PBS) containing 2 x 10(9)/mL RBCs. Light microscopic analysis results showed that hemolysis formed clots consisting of materials with membrane-like structures from both whole blood and PBS-suspended RBCs. Moreover, electron microscopic analysis results showed a monotonous material with high electron density in the formed clots, presenting a membrane-like structure. Hemolysis disappeared with the decrease in the current through the targets contacting with the plasma flare and clot formation ceased. Taken together, our results and those of earlier studies present two types of blood clot formation, namely presence or absence of hemolysis capability depending on the current through the targets. (C) 2016 Elsevier Inc. All rights reserved.

carcinoma (EC) is one of the most common malignancies of the female genital system. Although the behavior of EC ranges from an excellent prognosis to aggressive disease with a poor outcome, the factors that determine its diversity have not been determined. Here, we show that S100A4, a calcium-binding protein of the EF-hand type, is correlated with the proliferation and invasion ability of EC. We demonstrated previously that EC cells with high aldehyde dehydrogenase (ALDH) activity were more tumorigenic than ALDH-lo cells. Screening by shotgun proteomics demonstrated that the expression level of S100A4 in ALDH-hi EC cells was significantly higher than that in ALDH-lo cells. S100A4-knockout cells generated by the CRISPR/Cas9 system showed reduced proliferation and invasion. These cells showed impaired AKT phosphorylation and matrix metalloproteinase-2 activation, accounting for their impaired proliferation and invasion, respectively. Furthermore, in clinical EC samples, elevated expression of S100A4 was highly related to myometrial and lymphatic invasion in well to moderately differentiated EC. Notably, strong and diffuse expression of S100A4 was observed in tumor tissues with a microcystic, elongated and fragmented ("MELF") pattern, which is associated with a highly invasive EC phenotype. Collectively, our results demonstrate not only that high expression of S100A4 contributes to an aggressive phenotype of EC, but also that its elevated expression is closely related to the MELF histopathological pattern.

Transcriptional coactivator with PDZ-binding motif (TAZ) is a crucial component of the Hippo tumor suppressor pathway, interacting with transcriptional factors to regulate cell proliferation, apoptosis and tumorigenesis. TAZ and its paralog, Yes-associated protein (YAP), are activated at high frequencies during the progression towards malignancy in various tumors. Recently, YAP has been identified to modulate oncogenic features in endometrial adenocarcinoma, and it has also been reported that the nuclear expression of YAP is correlated with the poorly-differentiated form of endometrioid adenocarcinoma. In contrast to YAP, no studies have investigated TAZ expression in endometrioid adenocarcinoma. In the present study, TAZ expression was immunohistochemically examined in 55 clinical samples of endometrioid adenocarcinoma, and the clinical implications were evaluated. The results demonstrated that TAZ was located primarily in the cell nuclei, and that high TAZ expression was significantly correlated with high tumor-factor (P=0.024), stage (P=0.041) and histological grade (P=0.001), lymph node metastasis (P=0.046), recurrence (P=0.002) and a poor prognosis (P=0.007). Furthermore, univariate analysis identified that high TAZ expression was a poor prognostic factor for overall and disease-free survival. To the best of our knowledge, the present case is the first to report the clinical implications of TAZ in endometrioid adenocarcinoma of the uterus. TAZ may become a marker of a poor prognosis in endometrioid adenocarcinoma.

Cancer cells with tumorigenic potential are limited to a small subpopulation known as cancer-initiating cells (CICs). Recently we investigated a candidate of CICs of lymphoplasmacytic lymphoma (LPL), which is positive for both B-cell marker CD20 and plasma-cell marker CD138. We reported that the subpopulation of CD20(-) CD138(-) phenotype, in which both markers were negative was a candidate of CICs in LPL using LPL cell line, MWCL-1. CICs are known to be plastic under stressed condition, in which non-CICs are changed to CICs. In the present study, we investigated the plasticity of CICs of LPL, and found that hypoxia induced the conversion of CD20(+) CD138(-) to CD20(-) CD138(-) phenotype. We then searched for markers preferentially expressed in CD20(-) CD138(-) subpopulation, and the chemokine receptor CXCR7 was isolated. When cultured with CXCL12, a ligand of CXCR7, the number of CD20(-) CD138(-) cells increased in a time- and dose-dependent manner. In addition, hypoxia enhanced the expression level of CXCL12 in MWCL-1. In clinical samples of LPL, a few tumor cells expressed CXCR7, in which CD20 expression was not detected. These results indicated that hypoxia and CXCL12-CXCR7 axis appeared to be advantageous microenvironments to CD20(-) CD138(-) cells.

Angiogenic factor with G-patch and FHA domain 1 (AGGF1) is a novel angiogenic factor that was first described in Klippel-Trenaunay syndrome, a congenital vascular disease associated with capillary and venous malformations. AGGF1, similar to vascular endothelial growth factor (VEGF), has been shown to promote strong angiogenesis in chick embryos in vivo. Blocking AGGF1 expression prevented vessel formation, which suggests AGGF1 is a potent angiogenic factor linked to vascular malformations. So far, AGGF1 expression studies in human vascular lesions have not been performed. Here, we immunohistochemically investigated AGGF1 expression in venous, arteriovenous or capillary malformations, and infantile or congenital hemangioma. We found that AGGF1 was mostly expressed in endothelial cells with plump morphology. Moreover, the majority of mast cells strongly expressed AGGF1. Notwithstanding our incomplete knowledge of the molecular mechanism of AGGF1 in angiogenesis, our results show for the first time that AGGF1 is expressed in plump endothelial cells and mast cells.

The objective of this study is to determine whether there is a pathway different from the natural blood coagulation process involved in the plasma-induced activation of platelets and coagulation factors. Plasma treatment of bleeding wounds generated glomerular structures, which could be detected in high-magnification scanning electron microscopy images, and plasma flares produced aggregation on the surface of solutions containing bovine serum albumin, ovalbumin (OVA), or similar protein material. Ultramicroscopic analysis of the plasma-generated aggregation of OVA indicated a possible mechanism to explain the particle formation and fusion as a monotonous solid appearance. This study suggests that plasma coagulation involves aggregations of materials in blood in addition to the activation of platelets and coagulation factors by plasma treatment.

To examine the effect of NEAPP on cancer-initiating cells (CICs) that are believed to cause recurrence or metastasis due to their resistance to anti-cancer drugs, the treatment of nonequilibrium atmospheric pressure plasma (NEAPP) combined with anti-cancer drug cisplatin and NEAPP irradiation of tumor xenograft mice are performed in human uterine endometrioid adenocarcinoma cells. NEAPP treatment affects both ALDH-low (non-CICs) and ALDH-high cells (CICs) more efficiently than cisplatin. In tumor xenograft mice, ALDH expression reduces in tumor with NEAPP irradiation. These results suggest that NEAPP treatment is effective not only on non-CICs but also on CICs.

The anti-Hu antibody is one of the most famous onco-neural antibodies related to paraneoplastic neurological syndrome, and is associated with small cell lung carcinoma in most cases. Here, we report a case of poorly differentiated hepatocellular carcinoma accompanied by paraneoplastic peripheral neuropathy positive for the anti-Hu antibody. Image inspection before operation revealed that no tumors were found in organs other than the liver, including lung, and that the liver tumor had no metastatic lesion. The liver tumor showed histological appearance of poorly differentiated carcinoma with cartilaginous metaplasia and partial blastoid cell appearance. Most tumor cells presented trabecular-like structure lined by sinusoidal vessels. Immunohistochemically, the tumor cells were positive for low molecular weight cytokeratin and vimentin, partially positive for cytokeratin 19 and CD56, but negative for synaptophysin, chromogranin A and alpha-fetoprotein. Based on the trabecular-like morphology and the results of immunohistochemical staining, we concluded that the tumor was diagnosed as poorly differentiated hepatocellular carcinoma. Anti-Hu antibody-positive paraneoplastic peripheral neuropathy accompanied with liver tumor is extremely rare as far as is known. The presented case indicates that poorly differentiated carcinoma has the potential to be the responsible lesion of anti-Hu antibody-positive paraneoplastic neurological syndrome and systemic work-up is important for the management of this neurological disorder.