A quantitative evaluation method utilizing the homology concept to assess the state of chromatin within the nucleus of lung cancer.

Homology is a mathematical tool to quantify "the contact degree", which can be expressed in terms of Betti numbers. The Betti numbers used in this study consisted of two numbers, b0 (a zero-dimensional Betti number) and b1 (a one-dimensional Betti number). We developed a chromatin homology profile (CHP) method to quantify the chromatin contact degree based on this mathematical tool. Using the CHP method we analyzed the number of holes (surrounded areas = b1 value) formed by the chromatin contact and calculated the maximum value of b1 (b1MAX), the value of b1 exceeding 5 for the first time or Homology Value (HV), and the chromatin density (b1MAX/ns2). We attempted to detect differences in chromatin patterns and differentiate histological types of lung cancer from respiratory cytology using these three features. The HV of cancer cells was significantly lower than that of non-cancerous cells. Furthermore, b1MAX and b1MAX/ns2 showed significant differences between small cell and non-small cell carcinomas and between adenocarcinomas and squamous cell carcinomas, respectively. We quantitatively analyzed the chromatin patterns using homology and showed that the CHP method may be a useful tool for differentiating histological types of lung cancer in respiratory cytology.

Impact of LBC Fixative Type and Fixation Time on Molecular Analysis of Pancreatic Cancer Cells: A Comparative Study of Cell Morphology, Antigenicity and Nucleic Acids.

Introduction: Liquid-based cytology (LBC) has been widely used since 2000. Next-Generation Sequencing (NGS) analysis of residual specimens in LBC fixative may also be performed for pancreatic cancer in the near future. We examined cell morphology, antigenicity and nucleic acids in pancreatic cancer cells at different fixation times using two types of LBC fixatives. Methods: PANC-1 cells were fixed in 1 ml CytoRich Red (CR), CytoRich Blue (CB), 95% ethanol (95% AL) or 10% neutral buffered formalin (10% NBF) and evaluated for cell area, antigenicity and nucleic acids with fixation times of 1 hour and 1, 3, 9, and 14 days. Antigenicity was evaluated by immunocytochemical staining for p53 and CK20, and nucleic acid fragmentation was assessed by real-time PCR. Results: There was no difference in total cell area between 1 hour and 14 day fixation times for the CR group, but the CB group showed cell contraction with 9 days fixation. In immunocytochemical staining, the CR group showed high p53 and CK20 positivity even after 14 days fixation. The CB group had a lower p53 positive rate than the CR group from 1 hour fixation. For nucleic acid fragmentation, Ct values for the CR group increased with fixation time. The CB group had consistently low Ct values. Conclusion: Different LBC fixatives and fixation time can have varying effects on cell morphology, antigenicity and nucleic acids in pancreatic cancer cells. Therefore, fixative type and fixation time should be considered for molecular testing on residual samples in LBC fixatives.

Cancer-related gene mutations and intratumoral genetic heterogeneity in human epidermal growth factor receptor 2 heterogeneous gastric cancer.

Human epidermal growth factor receptor 2 (HER2) protein overexpression is associated with HER2 gene amplification, a critical driver oncogenetic change in gastric cancer. HER2 heterogeneity in advanced gastric cancer is associated with a poor prognosis and affects the clinical efficacy of trastuzumab. However, the mechanisms of HER2 heterogeneity are not fully understood. Here, we examined whether HER2 heterogeneous gastric cancer exhibited intratumoral genetic heterogeneity in other cancer-related genes. Two cases of advanced gastric cancer with HER2 heterogeneity were selected, and samples of HER2-positive and HER2-negative areas in each case were analyzed using a cancer-associated multiple gene panel. In both cases, TP53 mutations were observed in both HER2-positive and HER2-negative areas, whereas many of the potential driver and passenger mutations differed between HER2-positive and HER2-negative areas. Overall, our findings demonstrated that HER2 heterogeneous gastric cancer exhibited intratumoral genetic heterogeneity in other cancer-related genes and that the molecular mechanisms could differ between HER2-positive and -negative areas.

SOX11-induced decrease in vimentin and an increase in prostate cancer cell migration attributed to cofilin activity.

SOX11 is a transcription factor in the SOX family of genes that regulate multiple cellular events by influencing the expression of key genes in developmental, physiological, and tumorigenic cells. To elucidate the role of SOX11 in prostate cancer cells, PC-3 prostate cancer cells were cloned (S6 and S9 cells) to highly express SOX11. We demonstrated that both S6 and S9 lose vimentin expression, acquiring epithelial marker proteins, which indicates the Epithelial state phenotype. S6 and S9 cells have cancer-promoting characteristics that include higher migratory properties compared with control cells. The mechanisms that are responsible for the enhanced migration are cofilin activity and keratin 18 expression. TCGA (The Cancer Genome Atlas) dataset analysis revealed that metastatic prostate cancer tumors tend to have more SOX11 gene amplification compared with primary tumors. These results suggest the tumor promotive role and epithelial protein induction of SOX11 in prostate cancer cell.

Antifibrotic Agent Pirfenidone Suppresses Proliferation of Human Pancreatic Cancer Cells by Inducing G0/G1 Cell Cycle Arrest.

BACKGROUND: Pirfenidone (PFD), which is an antifibrotic agent used for treatment of idiopathic pulmonary fibrosis, induces G0/G1 cell cycle arrest in fibroblasts. We hypothesized that PFD-induced G0/G1 cell cycle arrest might be achieved in other types of cells, including cancer cells. Here we investigated the effects of PFD on the proliferation of pancreatic cancer cells (PCCs) in vitro. METHOD: Human skin fibroblasts ASF-4-1 cells and human prostate stromal cells (PrSC) were used as fibroblasts. PANC-1, MIA PaCa-2, and BxPC-3 cells were used as human PCCs. Cell cycle and apoptosis were analyzed using flow cytometer. RESULTS: First, we confirmed that PFD suppressed cell proliferation of ASF-4-1 cells and PrSC and induced G0/G1 cell cycle arrest. Under these experimental conditions, PFD also suppressed cell proliferation and induced G0/G1 cell cycle arrest in all PCCs. In PFD-treated PCCs, expression of p21 was increased but that of CDK2 was not clearly decreased. Of note, PFD did not induce significant apoptosis among PCCs. CONCLUSIONS: These results demonstrated that the antifibrotic agent PFD might have antiproliferative effects on PCCs by inducing G0/G1 cell cycle arrest. This suggests that PFD may target not only fibroblasts but also PCCs in the tumor microenvironment of pancreatic cancer.

Association of HER2 gene amplification and tumor progression in early gastric cancer.

Overexpression of human epidermal growth factor receptor 2 (HER2) protein in association with HER2 gene amplification is found in 7-34% of gastric cancers. In breast cancer, HER2 overexpression is a prognostic factor in advanced cases and is associated with tumor progression in ductal carcinoma in situ. However, the biological and clinical significance of HER2 status in early gastric cancer is unknown. Here, we aimed to examine the correlation between HER2 gene amplification and tumor progression in early gastric cancer. The HER2 status was evaluated in 149 lesions from 141 consecutive patients with early gastric cancer who underwent endoscopic resection by immunohistochemistry and dual color in situ hybridization. HER2 gene amplification was detected in 35 (23.5%) of 149 lesions, and of those, 26 cases (74.3%) showed intratumoral heterogeneity. HER2 gene amplification was found in noninvasive carcinoma, and there was a significant correlation between HER2 status and T factor (P = 0.0290). Our study demonstrated that HER2 gene amplification occurred during the early stages of gastric cancer and showed heterogeneity in several cases. HER2 gene amplification may be involved in tumor progression in early gastric cancer.

Elimination of p19ARF -expressing cells protects against pulmonary emphysema in mice.

Senescent cells accumulate in tissues during aging and are considered to underlie several aging-associated phenotypes and diseases. We recently reported that the elimination of p19ARF -expressing senescent cells from lung tissue restored tissue function and gene expression in middle-aged (12-month-old) mice. The aging of lung tissue increases the risk of pulmonary diseases such as emphysema, and cellular senescence is accelerated in emphysema patients. However, there is currently no direct evidence to show that cellular senescence promotes the pathology of emphysema, and the involvement of senescence in the development of this disease has yet to be clarified. We herein demonstrated that p19ARF facilitated the development of pulmonary emphysema in mice. The elimination of p19ARF -expressing cells prevented lung tissue from elastase-induced lung dysfunction. These effects appeared to depend on reduced pulmonary inflammation, which is enhanced after elastase stimulation. Furthermore, the administration of a senolytic drug that selectively kills senescent cells attenuated emphysema-associated pathologies. These results strongly suggest the potential of senescent cells as therapeutic/preventive targets for pulmonary emphysema.

Cytological features of adenocarcinoma admixed with small cell neuroendocrine carcinoma of the uterine cervix.

Adenocarcinoma admixed with neuroendocrine carcinoma of the uterine cervix is a rare malignancy with a poor prognosis, and few reports have described the cytological features of this carcinoma. To characterize the cytological features of this malignancy in cervical smears, we report a case of a 52-year-old Japanese woman with cervical adenocarcinoma admixed with small cell neuroendocrine carcinoma (SCNEC). Cytologically, there were two types of cells with different sizes. The smaller cells formed clusters, which showed a partially Indian file pattern, a high nuclear/cytoplasmic ratio, and hyperchromatic nuclei. In contrast, the larger cells showed cytological features of adenocarcinoma, indicating a glandular-like pattern. Histological examination of biopsy specimens revealed that the tumors were composed of almost equal areas of SCNEC and adenocarcinoma. Neuroendocrine differentiation was confirmed by immunohistochemistry for synaptophysin and CD56. Thus, when adenocarcinoma cells are detected in smears, attempts to search for SCNEC cells should be made by combined cytological and histological analyses in order to reach an accurate diagnosis of the carcinoma in the uterine cervix.

Elimination of p19ARF-expressing cells enhances pulmonary function in mice.

Senescent cells accumulate in many tissues as animals age and are considered to underlie several aging-associated pathologies. The tumor suppressors p19ARF and p16INK4a, both of which are encoded in the CDKN2A locus, play critical roles in inducing and maintaining permanent cell cycle arrest during cellular senescence. Although the elimination of p16INK4a-expressing cells extends the life span of the mouse, it is unclear whether tissue function is restored by the elimination of senescent cells in aged animals and whether and how p19ARF contributes to tissue aging. The aging-associated decline in lung function is characterized by an increase in compliance as well as pathogenic susceptibility to pulmonary diseases. We herein demonstrated that pulmonary function in 12-month-old mice was reversibly restored by the elimination of p19ARF-expressing cells. The ablation of p19ARF-expressing cells using a toxin receptor-mediated cell knockout system ameliorated aging-associated lung hypofunction. Furthermore, the aging-associated gene expression profile was reversed after the elimination of p19ARF. Our results indicate that the aging-associated decline in lung function was, at least partly, attributed to p19ARF and was recovered by eliminating p19ARF-expressing cells.

ASF-4-1 fibroblast-rich culture increases chemoresistance and mTOR expression of pancreatic cancer BxPC-3 cells at the invasive front in vitro, and promotes tumor growth and invasion in vivo.

Pancreatic cancer develops dense stromal tissue through the desmoplastic reaction. The aim of the present study was to assess the effects of a fibroblast-rich environment on the malignant potential of pancreatic cancer. Cells from the human pancreatic cancer cell line BxPC-3 were mixed at a ratio of 1:3 (fibroblast-rich) or 1:1 (fibroblast-poor) with cells from the human skin fibroblast line ASF-4-1. In the fibroblast-rich co-culture, tumor budding was observed and BxPC-3 cells were found to be more resistant to gemcitabine than those in the fibroblast-poor co-culture. Immunohistochemistry revealed that the expression of mammalian target of rapamycin was increased at the invasive front of fibroblast-rich co-cultures. In addition, in mouse xenografts of fibroblast-rich co-cultures, tumors were larger and had a higher Ki-67 index compared with that of the fibroblast-poor co-culture xenografts. These results indicate that fibroblast-rich co-cultures may promote the malignant potential of the pancreatic cancer cell line BxPC-3, both in vitro and in vivo.

Human Equilibrative Nucleoside Transporter 1 Expression in Endoscopic Ultrasonography-Guided Fine-Needle Aspiration Biopsy Samples Is a Strong Predictor of Clinical Response and Survival in the Patients With Pancreatic Ductal Adenocarcinoma Undergoing Gemcitabine-Based Chemoradiotherapy.

OBJECTIVES: This study aimed to clarify whether pretreatment human equilibrative nucleoside transporter (hENT1) expressions in endoscopic ultrasonography-guided fine-needle aspiration biopsy (EUS-FNAB) specimens obtained from resectable, borderline resectable, and locally advanced unresectable pancreatic ductal adenocarcinoma (PDAC) are concordant with those in the resected specimen after gemcitabine-based chemoradiotherapy (Gem-CRT) and to validate the utility of hENT1 expression using EUS-FNAB samples as a prognostic marker. METHODS: We evaluated the relationship between hENT1 expressions assessed by immunohistochemical staining and clinical outcomes in 51 of 76 patients with PDAC who were diagnosed by EUS-FNAB and received preoperative Gem-CRT. RESULTS: The concordance rate of hENT1 expressions was 89.2% (K = 0.681). Median survival time (month) in the 51 whole patients and 37 patients with resection was significantly longer in hENT1 positive than in hENT1 negative: 25.0 and 30.0 versus 9.0 and 9.0, respectively. A multivariate analysis confirmed that hENT1 expression was an independent prognostic factor in both whole patients and those with resection. Regardless of T3 and T4, hENT1-positive patients with resection had significantly better prognosis than hENT1-negative patients, whose prognosis was similar to those without resection. CONCLUSIONS: The assessment of hENT1 expression using EUS-FNAB samples before Gem-CRT provides important information on patients with PDAC who can benefit from curative-intent resection.

Significant intratumoral heterogeneity of human epidermal growth factor receptor 2 status in gastric cancer: A comparative study of immunohistochemistry, FISH, and dual-color in situ hybridization.

The assessment of human epidermal growth factor receptor 2 (HER2) status is crucial for selecting patients with gastric cancer who may benefit from HER2-targeted therapy. Accurate assessment using biopsy specimens is important for patients with advanced-stage cancer. Intratumoral heterogeneity of HER2, however, is a major challenge in HER2 testing. Here, we aimed to examine whether assessment of HER2 status could be accurately carried out with currently used methods, namely, immunohistochemistry (IHC), FISH, and dual-color in situ hybridization (DISH). Human epidermal growth factor receptor 2 status was evaluated in 108 biopsy tissues from patients with gastric adenocarcinoma and 70 matched surgical specimens by IHC, FISH, and DISH; HER2 amplification was detected in 11 (10.2%) out of 108 biopsy specimens. The IHC and FISH results were well correlated, and FISH and DISH results were consistent for all cases. The overall concordance rate of HER2 status between biopsy tissues and surgical specimens was 91.4%. All six discordant cases were false negative on biopsy; of these cases, five showed HER2 heterogeneity on surgical resection. Assessment of the HER2 status of biopsy tissues could predict the status of the whole tumor; however, a proportion of these cases may be discordant because of intratumoral heterogeneity.

Tenascin-C may accelerate cardiac fibrosis by activating macrophages via the integrin αVß3/nuclear factor-κB/interleukin-6 axis.

Tenascin-C (TN-C) is an extracellular matrix protein not detected in normal adult heart, but expressed in several heart diseases closely associated with inflammation. Accumulating data suggest that TN-C may play a significant role in progression of ventricular remodeling. In this study, we aimed to elucidate the role of TN-C in hypertensive cardiac fibrosis and underlying molecular mechanisms. Angiotensin II was administered to wild-type and TN-C knockout mice for 4 weeks. In wild-type mice, the treatment induced increase of collagen fibers and accumulation of macrophages in perivascular areas associated with deposition of TN-C and upregulated the expression levels of interleukin-6 and monocyte chemoattractant protein-1 as compared with wild-type/control mice. These changes were significantly reduced in TN-C knockout/angiotensin II mice. In vitro, TN-C accelerated macrophage migration and induced accumulation of integrin αVß3 in focal adhesions, with phosphorylation of focal adhesion kinase (FAK) and Src. TN-C treatment also induced nuclear translocation of phospho-NF-κB and upregulated interleukin-6 expression of macrophages in an NF-κB-dependent manner; this being suppressed by inhibitors for integrin αVß3 and Src. Furthermore, interleukin-6 upregulated expression of collagen I by cardiac fibroblasts. TN-C may enhance inflammatory responses by accelerating macrophage migration and synthesis of proinflammatory/profibrotic cytokines via integrin αVß3/FAK-Src/NF-κB, resulting in increased fibrosis.

Report of a case of acinar cell carcinoma with its differential diagnosis on endoscopic ultrasound-guided fine-needle aspiration cytology.

Acinar cell carcinomas (ACCs) of the pancreas are a rare tumor accounting for only about 2% of all pancreas tumors. We report herein on this case and discuss how to distinguish ACCs from neuroendocrine tumors (NETs) and solid pseudo-papillary tumor (SPTs) morphologically and immunohistochemically. In cytological findings, the nuclear-cytoplasmic ratio was high, and the cytoplasm was granular, but zymogen granules were not evident. The nucleus was biased in location, assuming small circular and irregular forms. Chromatin was fine granular in shape and distributed nonuniformly, accompanied by evident nucleoli. Immunohistochemically was positive for ß-catenin (cell membrane and part of nuclei), synaptophysin (focal), chromogranin A (focal) and chymotrypsin were all positive. Although the cytological distinction of ACCs from NETs and SPTs is difficult, the nuclear chromatin pattern and nuclear inclusion bodies, pseudopapillary arrangement and hyaline globules seem to play an important role in the cytological differential diagnosis. Furthermore, not only enzymatic and neuroendocrine markers, but also antibodies to ß-catenin, vimentin and so on seem to be useful in the differential diagnosis.

Inflammatory suppressive effect of prostate cancer cells with prolonged exposure to transforming growth factor ß on macrophage-differentiated cells via downregulation of prostaglandin E2.

Transforming growth factor ß1 (TGFß1) regulates a variety of cellular functions, including cell growth, apoptosis and differentiation. The aim of the current study was to investigate the alterations of phenotypic events in the long-term exposure of prostate cancer (PCa) cells to TGFß1 and its effect on macrophage-differentiated cells. The PCa cell line, PC-3, and the subclone, M1, were exposed to TGFß1 for short- or long-term periods. TGFß1 signaling was assessed by Smad3 phosphorylation, and non-canonical signaling was analyzed by quantitative polymerase chain reaction-based regulatory gene expression profiles. TGFß1-exposed PCa cells were also co-cultured with phorbol 12-myristate 13-acetate (PMA)-treated THP-1 macrophages as a model of the tumor microenvironment. The phosphorylation of Smad3 in the PCa cells with long-term exposure was lower than that in the PCa cells with short-term exposure. Interleukin-6 mRNA expression in the PMA-treated THP-1 macrophages was significantly downregulated by co-culture with the PCa cells with long-term exposure. Cyclooxygenase-2 expression in the long-term TGFß1-exposed PCa cells was lower than that in the control PCa cells, and the production of prostaglandin E2 (PGE2) in the long-term TGFß1-exposed PCa cells was also significantly lower. The results of the current study demonstrated that the long-term TGFß1 exposure of PCa cells induces phenotypic changes, including the downregulation of PGE2 production. This indicates that prolonged TGFß-exposed PCa cells may change the cytokine production of macrophages in the tumor microenvironment.

Splenectomy increases the number of circulating hematopoietic stem/progenitor cells in patients with hepatitis C virus-associated liver cirrhosis.

AIM: The spleen is not believed to contribute to hematopoiesis in healthy adults. However, several reports have demonstrated that the spleen in adults contains a large number of hematopoietic stem/progenitor cells (HSC). Although splenectomy increases platelet and leukocyte counts, the effects of splenectomy on circulating HSC have not been elucidated. In this study, we evaluated the association between the number of circulating HSC and splenectomy in patients with hepatitis C virus (HCV)-associated liver cirrhosis (LC). METHODS: In 48 patients with various stages of HCV-associated chronic liver disease and seven patients with LC who underwent splenectomy, and 10 healthy volunteers, we determined the numbers of circulating CD34+ cells and colony-forming unit culture by flow cytometry and methylcellulose culture, respectively. Plasma stromal cell-derived factor-1α (SDF-1α) concentrations were measured using an enzyme-linked immunosorbent assay. RESULTS: The numbers of circulating CD34+ cells and colony-forming unit culture decreased but the plasma SDF-1α concentration increased with the progression of liver disease. There was an inverse correlation between the number of circulating HSC and the plasma SDF-1α concentration. The numbers of circulating HSC and platelets were determined before and after splenectomy in seven patients with LC. In these patients, the numbers of circulating HSC and platelets increased significantly after splenectomy and the enhancing effect persisted for a long time. CONCLUSION: Our data suggest that the spleen plays an important role in modulating HSC dynamics in patients with HCV-associated chronic liver disease. Our results also imply that splenectomy may improve liver function in patients with LC.

[Endoscopic ultrasound-guided fine needle aspiration cytology with on-site diagnosis by pathologist].

Endoscopic ultrasound-guided fine needle aspiration cytology/biopsy is now widely used to diagnose pancreatic tumors. The procedure was introduced to Mie University Hospital in 2006. A pathologist and cytotechnologist collaborate in the endoscopy room, and immediate on-site diagnosis is routinely performed in our hospital. If the pathologist is not able to come to the bed-side, telediagnosis is done using a digital camera attached to a smart phone. The authors emphasize that communication between physicians, cytotechnologists, and pathologists is very important. Therefore, the presence of pathologists in the endoscopy room promotes physicians' skills as well as the accuracy of this procedure.