A rad50 germline mutation induces tumorigenesis and ataxia-telangiectasia phenotype in a transparent medaka model.

The MRE11A-RAD50-NBS1 complex activates the ataxia-telangiectasia mutated (ATM) pathway and plays a central role in genome homeostasis. The association of RAD50 mutations with disease remains unclear; hence, we adopted a medaka rad50 mutant to demonstrate the significance of RAD50 mutation in pathogenesis using the medaka as an experimental animal. A 2-base pair deletion in the rad50 gene was introduced into transparent STIII medaka using the CRISPR/Cas9 system. The mutant was analyzed histologically for tumorigenicity and hindbrain quality, as well as for swimming behavior, to compare with existing ATM-, MRE11A-, and NBS1-mutation-related pathology. Our results revealed that the medaka rad50 mutation concurrently reproduced tumorigenesis (8 out of 10 rad50Δ2/+ medaka), had a decrease in median survival time (65.7 ± 1.1 weeks in control vs. 54.2 ± 2.6 weeks in rad50Δ2/+ medaka, p = 0.001, Welch's t-test), exhibited semi-lethality in rad50Δ2/Δ2 medaka and most of the major ataxia-telangiectasia phenotypes, including ataxia (rheotaxis ability was lower in rad50Δ2/+ medaka than in the control, Mann-Whitney U test, p < 0.05), and telangiectasia (6 out of 10 rad50Δ2/+ medaka). The fish model may aid in further understanding the tumorigenesis and phenotype of ataxia-telangiectasia-related RAD50 germline mutations and in developing novel therapeutic strategies against RAD50 molecular disorders.

Microsatellite instability in the high-grade dysplasia component of duodenal adenoma is associated with progression to adenocarcinoma.

PURPOSE: We examined the microsatellite instability of duodenal tumors to evaluate their molecular features associated with the adenoma-carcinoma sequence. METHODS: Fifty-two non-ampullary duodenal epithelial tumors collected by endoscopic mucosal resection or surgical resection were studied. When a tumor had two or more dysplasia grades, the highest grade was considered. Representative areas were macro-dissected and subjected to a microsatellite instability analysis and immunohistochemical staining. RESULTS: The 52 tumors were classified as either adenoma with low-grade dysplasia (n = 18), adenoma with high-grade dysplasia (n = 20), or adenocarcinomas (n = 14). Among these, 3 adenocarcinoma cases showed microsatellite instability and the remaining 49 tumors showed microsatellite stability. Of the 14 adenocarcinoma cases, 3 contained both high-grade dysplasia and adenocarcinoma components, and 11 contained only the adenocarcinoma component. Interestingly, all three adenocarcinoma + high-grade dysplasia cases were microsatellite instability-high in both the adenocarcinoma and high-grade dysplasia components. Immunohistochemical staining of mismatch repair proteins showed mismatch repair deficiency in three microsatellite instability-high adenocarcinoma + high-grade dysplasia cases. CONCLUSIONS: Only adenocarcinoma cases with high-grade dysplasia components were microsatellite instability-high (in both the adenocarcinoma and high-grade dysplasia components). This suggests that microsatellite instability in the high-grade dysplasia component of duodenal adenoma is associated with progression to adenocarcinoma.

Can Individuals with Suboptimal Antibody Responses to Conventional Antiviral Vaccines Acquire Adequate Antibodies from SARS-CoV-2 mRNA Vaccination?

In Japan, healthcare workers (HCWs) are vaccinated against measles, rubella, chickenpox, mumps, and hepatitis B to prevent nosocomial infection; however, some do not produce sufficient antibodies ("suboptimal responders"). This study compared immune responses to a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 mRNA) vaccine among HCWs with normal and suboptimal responses to conventional vaccines. In this prospective cohort study, 50 HCWs received two doses of BNT162b2 mRNA vaccine 3 weeks apart. SARS-CoV-2 anti-spike antibodies were measured 11 times, starting before the first vaccination and ending 5 months after the second vaccination. Antibody titers of four suboptimal and 46 normal responders were compared. SARS-CoV-2 neutralizing antibody activity was measured twice in suboptimal responders, 1 week/1 month and 5 months after the second vaccination. The SARS-CoV-2 anti-spike antibody was detectable in the samples from suboptimal and normal responders at each timepoint after vaccination. Suboptimal responders exhibited SARS-CoV-2 neutralizing antibody activity 1 week/1 month as well as 5 months after the second vaccination; however, activity was slightly reduced at 5 months. Our findings show that suboptimal responders do acquire adequate SARS-CoV-2 anti-spike and SARS-CoV-2 neutralizing antibodies from vaccination to prevent SARS-CoV-2. SARS-CoV-2 mRNA vaccines should thus be recommended for both normal and suboptimal responders to conventional vaccines.

Cytomorphometric and flow cytometric analyses using liquid-based cytology materials in subtypes of lung adenocarcinoma.

BACKGROUND: The histological classifications of invasive lung adenocarcinoma subtypes are considered to predict patient prognosis after surgical treatment. The objectives of this study were to evaluate cytomorphological characteristics and proliferative activities among the histological predominant patterns by performing cytomorphometric and flow cytometric analyses using liquid-based cytology materials. METHODS: Cytological samples fixed by liquid-based cytology preservatives from 53 surgically-resected lung adenocarcinoma specimens were obtained between August 2018 and November 2019. The Papanicolaou-stained and paired Ki-67-stained slides were analyzed for calculating nuclear morphology (nuclear area, nuclear perimeter and nuclear circularity) and Ki-67 labeling index using software. The cell proliferation index (CPIx) was calculated and cellular information including cell cycle stage of tumor cells was obtained by flow cytometry. RESULTS: The 53 cases included papillary (n = 29), acinar (n = 8), lepidic (n = 5), and solid (n = 4) subtypes, and invasive mucinous adenocarcinoma (n = 7) were also included. In the lepidic pattern, nuclear area (79.6 ± 28.8 µm2 ) and perimeter (34.1 ± 6.1 µm) were relatively larger and longer than those of the other predominant patterns. The Ki-67 labeling index of the solid pattern (27.9 ± 12.5%) was highest compared with those of other predominant patterns. There were statistically significant differences in the lepidic versus solid patterns and the papillary versus solid patterns (p = .013 and p = .039, respectively). The calculated mean CPIx of the lepidic and the acinar patterns were approximately two-fold higher than those of the other predominant patterns. CONCLUSION: By revealing the differences of cytomorphological characteristics, these methodologies might be used for diagnosing cytopathological materials using digital cytopathology.

Sonographically Measured Adipose Tissue Thickness Correlates with Laboratory Test Abnormalities Reflecting Metabolic State in Elderly Women.

Background: Accumulation of adipose tissue progresses to metabolic diseases. Sonography is a convenient modality for measuring the thickness of adipose tissue. The present study aimed to clarify the site of adipose tissue thickness that correlated best with laboratory test values reflecting metabolic abnormalities. Methods: Subjects comprised 37 elderly women with metabolic diseases or an almost healthy state (median age, 71 years; interquartile range, 62-78 years). Abdominal visceral adipose tissue (VAT), subcutaneous adipose tissue, peritoneal adipose tissue, perirenal adipose tissue, and epicardial adipose tissue (EAT) thicknesses were measured. Correlations were evaluated between laboratory test values and these adipose tissue thicknesses. Results: VAT thickness measured at the level of the umbilicus correlated positively with values of triglycerides (TGs) (r = 0.593, P = 0.0009) and hemoglobin A1c (r = 0.490, P = 0.0081) and negatively with the value of high-density lipoprotein cholesterol (r = -0.521, P = 0.0045), even after adjusting for body mass index. Significant positive correlations were also found between EAT thickness and TGs (r = 0.542, P = 0.0029). Conclusions: Among the adipose tissue thicknesses measured at several sites by sonography, VAT thickness correlated most closely with laboratory test values representing metabolic abnormalities in elderly women.

Establishing an internal quality control method for the stable extraction of nucleic acids of severe acute respiratory syndrome coronavirus 2 and RT-PCR-based detection.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the coronavirus disease 2019 (COVID-19), is detected using real-time RT-PCR. However, there are limitations pertaining to quality control, particularly with respect to establishing quality control measures for extraction of viral nucleic acids. Here, we investigated the quality control measures for the various processes using an extrinsic quality control substance and quality control charts. METHODS: An extrinsic quality control substance was added to the sample, and then, real-time RT-PCR was performed. Samples with negative test results and the corresponding data were analyzed; a quality control chart was created and examined. RESULTS: Data analysis and the quality control charts indicated that SARS-CoV-2 could be reliably detected using real-time RT-PCR, even when different nucleic acid extraction methods were used or when different technicians were employed. CONCLUSION: With the use of quality control substances, it is possible to achieve quality control throughout the process-from nucleic acid extraction to nucleic acid detection-even upon using varying extraction methods. Further, generating quality control charts would guarantee the stable detection of SARS-CoV-2.

Next-Generation Sequencing for Non-Ampullary Duodenal Carcinoma Suggesting the Existence of an Adenoma-Carcinoma Sequence.

Duodenal tumors with a sporadic adenoma-carcinoma sequence are extremely rare. For such clinically suspected cases without a specific family history, performing a comprehensive gene search is important to understand the germline mutation background. We present a 68-year-old woman without a genetic or familial history of familial adenomatous polyposis (FAP), Peutz-Jeghers syndrome, or Lynch syndrome who presented to Kosei Hospital, Japan, with exertional dyspnea induced by abdominal pain lasting 3 weeks. A duodenal tumor was suspected by contrast-enhanced computed tomography. Esophagogastroduodenoscopy showed a lesion accompanied by a white microprotuberance on the descending part of the duodenum opposite the papilla, with a giant ulcerative lesion at the center of the white lesion. Biopsy revealed a low-grade adenoma, high-grade adenoma, and adenocarcinoma. Immunohistochemical analysis of the adenoma and adenocarcinoma showed Ki-67, p53, cytokeratin 20, caudal-type homeobox 2, and carcinoembryonic antigen positivity and cytokeratin 7 negativity. The findings suggested the presence of an adenoma-adenocarcinoma sequence in duodenal carcinoma. However, in the mutational analysis using next-generation sequencing, c.4348C>T (p.Arg1450Ter) mutation in APC was detected in all normal mucosal, adenoma, and carcinoma tissues. This mutation is common in FAP patients. Even if the presence of an adenoma-adenocarcinoma sequence in duodenal carcinoma is suggested in cases without a familial FAP history, as in this case, genetic analysis may reveal FAP. Thus, performing a comprehensive genetic analysis of duodenal carcinoma patients with a possible adenoma-carcinoma sequence is necessary to explore their genetic background.

STK11 p.F354L Germline Mutation in a Case of Multiple Gastrointestinal Tumors.

Serine/threonine kinase 11 (STK11) is known as a critical tumor-suppressor gene that is frequently mutated in a broad spectrum of human cancers. Among these, the p.F354L mutation of STK11 has been identified in sporadic colon or lung cancer cases. Here, we report the case of a 75-year-old male patient who underwent surgical treatment for multiple tumors of the gastrointestinal system. Genetic mutations were screened in all resected samples, including duodenal high-grade adenoma, gastric high-grade adenoma, rectal adenocarcinoma, and liver metastasis of rectal adenocarcinoma, by next-generation sequencing for mutational hotspots involving 50 oncogenes and tumor suppressor genes. The characteristic hamartomatous polyp of Peutz-Jeghers syndrome was not detected in any tumor specimen. However, all samples as well as the normal rectal mucosa harbored the genetic mutation p.F354L in STK11. In addition, somatic mutations coexisted in the tumor samples, including KRAS p.A146T, TP53 p.G238X, and APC p.T1556fs in the duodenal adenoma; TP53 p.G238Y and APC p.T1556fs in the gastric adenoma; and TP53 p.R282W in the rectal adenocarcinoma and metastatic liver cancer. No somatic mutation was detected in the normal rectal mucosa as a control sample. To our knowledge, this is the first report of an STK11 germline mutation in a patient with multiple tumors of the gastrointestinal tract.

Discrimination of well-differentiated liposarcoma from benign lipoma on sonography: an uncontrolled retrospective study.

PURPOSE: Well-differentiated liposarcoma, the most common subtype of liposarcoma, should be discriminated from benign lipoma. However, features on sonography for discriminating these two types of tumor have not been fully investigated. The present study was therefore aimed at clarifying differences in sonographic findings between well-differentiated liposarcoma and lipoma. METHODS: The study population comprised 23 cases of well-differentiated liposarcoma and 181 cases of lipoma. We investigated differences in sonographic appearance and pathological findings between the two types of tumor. RESULTS: Well-differentiated liposarcoma tended to develop more frequently in older patients and in the lower extremities including the gluteal region, compared with lipoma. Concerning sonographic findings, both tumors exhibited well-defined margins and heterogeneous internal echogenicity, including typical tiny striated hyperechoic lines. Well-differentiated liposarcoma was characterized by a higher frequency of the following findings compared with lipoma: (1) deep location, (2) irregular shape, (3) large diameter, (4) hyperechogenicity compared to surrounding tissue, and (5) presence of vascularity on Doppler sonography (p < 0.01 each). Notably, hyperechogenicity corresponded to the intermingled sclerosing component within the adipocytic component when sonographic findings were compared with those of pathology. CONCLUSION: The present study suggests that several sonographic findings including hyperechogenicity and presence of vascularity might be key features for discriminating well-differentiated liposarcoma from lipoma.

C-X-C motif receptor 3A enhances proliferation and invasiveness of colorectal cancer cells, and is mediated by C-X-C motif ligand 10.

The chemokine CXCL10 and its receptor CXCR3 have been demonstrated to be implicated in cancer cell proliferation and metastasis. CXCR3 has three splice variants: CXCR3A, CXCR3B and CXCR3-alt. CXCR3A and B serve multiple roles in the growth and invasiveness of a number of cancer types. However, the roles of CXCR3 isoforms in colorectal cancer (CRC) cells remain unclear. In the current study, the effects of CXCL10 and CXCR3 isoforms on proliferation and invasion of CRC cells was examined. Proliferation and invasiveness of the CRC cell line HCT116, which were transfected with CXCR3A or CXCR3B in the presence of CXCL10, were evaluated in vitro using MTT, scratch wound healing and transwell assays. MTT assay indicated that regardless of the presence or absence of CXCL10, the proliferative ability of CXCR3A-transfected HCT116 cells was enhanced compared with blank and mock cells. Scratch wound healing and transwell assays indicated that invasiveness of CXCR3A-transfected cells was greater compared with blank and mock cells. However, HCT116 cells transfected with CXCR3B did not exhibit changes in their proliferative or invasive ability. mRNA expression of MMP9, which is associated with signaling downstream of the CXCL10/CXCR3A pathway, was increased 4-fold in CXCR3A-transfected HCT116 cells compared with control cells. The results of the present study indicated that CXCL10-enhanced proliferation and invasiveness of the CRC cell line HCT116 was likely mediated by CXCR3A, but not by CXCR3B.

Subtyping and EGFR mutation testing from blocks of cytological materials, based on liquid-based cytology for lung cancer at bronchoscopic examinations.

BACKGROUND: Liquid-based cytology (LBC) allows immunohistochemistry (IHC), fluorescence in situ hybridization, and molecular testing to be performed in fixed cell materials. We examined the feasibility of subtyping and EGFR mutation testing of bronchoscopic samples from patients with lung cancer using cell blocks (CB) based on LBC fixation (LBC-CB). METHODS: We included 35 consecutive patients with peripheral lung nodules who underwent endobronchial ultrasonography with a guide sheath in our hospital. Thirty of these patients were diagnosed with lung cancer by obtaining cytological samples. Cytological subtyping was performed with IHC using LBC-CB, and the Cobas EGFR Mutation Test ver. 2 was performed using extracted genomic DNA from the LBC-CB, formalin-fixed paraffin-embedded (FFPE) tissue, and matched plasma. RESULTS: Of the 30 cases, 25 were classified cytomorphologically as adenocarcinoma (ADC, n = 17) and squamous-cell carcinoma (SQCC, n = 8). The remaining five cases were classified by IHC as favor ADC (n = 3) and favor SQCC (n = 2) according to the WHO criteria. In the final ADC group (n = 20), EGFR mutations on the LBC-CB were identified in eight cases (40%; 1 exon 19 deletion, 6 L858R, and 1 L861Q). Mutations in FFPE samples were identified in seven cases (35%) at the same site in each case. Plasma EGFR mutations were identified in four cases (20%) at the same site. The CB detection rate was higher than for FFPE and plasma. CONCLUSION: LBC-CB is suitable for subtyping and EGFR mutation testing in lung cancers.

Use of a plasma test for verifying epidermal growth factor receptor gene (EGFR) mutations in fluid samples from non-small cell lung cancer patients.

Because epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) are effective in the treatment of non-small cell lung cancer (NSCLC) patients with EGFR mutations, it is critical to obtain accurate EGFR mutation test results. For NSCLC patients, EGFR mutation testing is performed using the commercial Cobas EGFR Mutation test v2.0, which can be used to analyze both formalin-fixed, paraffin-embedded (FFPE) tissue (FFPE test, or FT) and plasma samples (plasma test, or PT). Since primary tumor tissues are often unavailable from relapsed patients, fluid samples are often used for EGFR mutation testing, but they are often tested using the FT. Here, we report three cases in which EGFR mutations were detected using the FT with FFPE primary tumor tissue samples, but were not detected using fluid samples (two pleural effusion and one cerebrospinal fluid sample). Because the FT may not be capable of detecting EGFR mutations in fluid samples, we used the PT, which is more sensitive, to verify the presence of EGFR mutations using the same fluid samples. As expected, the PT detected the same EGFR mutations in fluid samples as the FT did in FFPE primary tumor tissue samples.

Sonographic Appearances of Liposarcoma: Correlations with Pathologic Subtypes.

Liposarcoma is the second most common malignant soft-tissue tumor. This entity is pathologically categorized into 4 subtypes: well-differentiated, myxoid, dedifferentiated and pleomorphic. Although features on magnetic resonance imaging and computed tomography for these 4 subtypes have been reported quite precisely, those on sonography have not been fully investigated. The present study was therefore aimed at clarifying the sonographic appearances of each liposarcoma subtype and assessing correlations with histopathology. The study population was made up of 35 cases, including 21 cases of well-differentiated liposarcoma, 6 cases of myxoid liposarcoma, 6 cases of dedifferentiated liposarcoma and 2 cases of pleomorphic liposarcoma. Compared with the other subtypes, well-differentiated liposarcoma was characterized by the high frequency of the following findings: isoechogenicity, tiny hyperechoic lines and hypovascularity (p < 0.01, in each). Myxoid liposarcomas were characterized by low echogenicity, intermingled with anechoic areas and moderate vascularity (p < 0.01, in each). Dedifferentiated liposarcomas showed a specific biphasic pattern of hyperechoic and hypoechoic areas and hypervascularity (p < 0.01, in each). Pleomorphic liposarcomas showed a specific gyrus-like mixture of hyperechoic and hypoechoic areas (p < 0.01). In conclusion, the present study revealed different characteristics of sonographic appearance among the 4 histopathologic subtypes of liposarcoma.

Genotyping and cytomorphological subtyping of lung adenocarcinoma based on liquid-based cytology.

BACKGROUND: Liquid-based cytology (LBC) samples allow immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), and molecular testing of nucleic acids to be performed in the remaining fixed cells. The current study aimed to examine the relationship between gene mutational status and cytomorphological features in primary lung adenocarcinoma (ADC) using LBC materials. METHODS: Forty consecutive patients with primary lung ADC underwent surgical resection in our hospital. Cytological material was obtained by scraping the cut-surface of the lesion, and samples were fixed and stored as LBC materials using CytoRich Red. Epidermal growth factor receptor (EGFR) and Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations, anaplastic lymphoma kinase (ALK), and c-ros oncogene 1 (ROS1) gene rearrangements were detected, and cytomorphological studies were performed. RESULTS: Twenty cases (50%) were positive for EGFR mutation and four (10%) were positive for KRAS mutation. ALK gene rearrangement was identified in one case (2.5%) by IHC and FISH, and ROS1 gene rearrangement was identified in one case (2.5%) by IHC and real-time polymerase chain reaction. The KRAS-positive group included higher proportions of cases with an inflammatory background (100%), predominantly papillary architecture (75%), and papillary-type ADC pattern (75%) compared with the EGFR-positive group and the other group, which included ALK and ROS1 gene rearrangements. CONCLUSIONS: LBC material is suitable for use in molecular testing. Differences in major gene aberrations detected by this method might predict specific cytomorphological features.

Serum levels of candidate microRNA diagnostic markers differ among the stages of non-small-cell lung cancer.

Circulating microRNAs (miRNAs) are promising markers for cancer diagnosis and prognosis. Numerous studies evaluating miRNAs as markers for non-small cell lung cancer (NSCLC) have been conducted in recent years; however, the majority of candidate markers proposed via individual studies were inconsistent and no marker miRNAs for the diagnosis of early stage NSCLC have been established. In the present study, miR-145, miR-20a, miR-21 and miR-223, which were previously reported as candidate diagnostic markers of NSCLC, were re-evaluated. The serum levels of these miRNAs were quantified in 56 patients with stage I-IV NSCLC using the TaqMan microRNA assays and separately compared the levels at each stage with those in 26 control patients. The level of miR-145 was significantly reduced in patients with NSCLC, regardless of clinical stage, and its level increased following tumor resection in patients with stage I-II disease. These results indicate that miR-145 is relevant as a diagnostic marker for stages I-IV NSCLC. Additionally, the levels of miR-20a and miR-21 demonstrated notable differences among patients at different clinical stages. These miRNAs distinguished patients in a number of, but not all, stages of NSCLC from cancer-free control patients. These results indicated that it is essential to analyze miRNA levels at each stage separately in order to evaluate marker miRNAs for NSCLC diagnosis.

APC:T1556fs and STK11 mutations in duodenal adenomas and adenocarcinomas.

PURPOSE: Duodenal adenoma and adenocarcinoma (AC) are rare tumors, and few studies have examined their genetic features. We aimed to determine the key genetic changes in duodenal adenoma and AC, and to clarify the possible involvement of the adenoma-carcinoma sequence in duodenal tumor carcinogenesis. METHODS: Nineteen duodenal tumors collected by endoscopic mucosal resection or surgical resection were classified as AC, adenoma with high-grade dysplasia (HGD), or adenoma with low-grade dysplasia (LGD) per the World Health Organization tumor classification. When a tumor contained two or more components with different dysplasia grades, the highest grade was assigned as the tumor grade. Representative areas of these components with different grades were microdissected and evaluated by a genomic analysis. Mutational hotspots involving 50 oncogenes and tumor suppressor genes were analyzed by next-generation sequencing, and their association with the dysplasia grade was investigated. RESULTS: We analyzed 27 tumor components of AC or adenoma, with 11 normal mucosal samples obtained from 19 patients with duodenal tumors. The most prevalent abnormality among 50 genes tested was the KRAS mutation, which was detected in 12/19 (63.2%) patients, followed by APC and TP53 mutations (47.4 and 36.8%, respectively). According to the tumor dysplasia grading of each component, KRAS mutations were found in 5/8 (62.5%) tumors with AC components, 6/9 (66.7%) tumors with HGD components, and 3/10 (30.0%) tumors with LGD components. TP53 mutations were found in 4/8 (50.0%) tumors with AC components, 3/9 (33.3%) tumors with HGD components, and 1/10 (10.0%) tumors with LGD components. APC mutations were found in 2/8 (25.0%) tumors with AC components, 6/9 (66.7%) tumors with HGD components, and 5/10 (50.0%) tumors with LGD components. Notably, an APC:T1556fs mutation was detected in six cases (31.6%), five of which were adenoma cases. Furthermore, STK11 mutations were confirmed in 2/8 (25.0%) AC cases and in 1/11 (9.1%) adenoma cases. CONCLUSION: APC:T1556fs and STK11 mutations found in duodenal adenomas/ACs highlight the importance of proteins encoded by these genes in tumor development. APC mutations were identified in duodenal adenomas more frequently than in duodenal ACs, which differed from the observations of typical adenoma-carcinoma sequences seen in colorectal cancer, suggesting the limited involvement of this mechanism in duodenal cancer development.

KRAS Mutation as a Potential Prognostic Biomarker of Biliary Tract Cancers.

BACKGROUND: The aim of this study was to identify the unique molecular characteristics of biliary tract cancer (BTC) for the development of novel molecular-targeted therapies. MATERIALS AND METHODS: We performed mutational analysis of KRAS, BRAF, PIK3CA, and FBXW7 and immunohistochemical analysis of EGFR and TP53 in 63 Japanese patients with BTC and retrospectively evaluated the association between the molecular characteristics and clinicopathological features of BTC. RESULTS: KRAS mutations were identified in 9 (14%) of the 63 BTC patients; no mutations were detected within the analyzed regions of BRAF, PIK3CA, and FBXW7. EGFR overexpression was observed in 5 (8%) of the 63 tumors, while TP53 overexpression was observed in 48% (30/63) of the patients. Overall survival of patients with KRAS mutation was significantly shorter than that of patients with the wild-type KRAS gene (P = 0.005). By multivariate analysis incorporating molecular and clinicopathological features, KRAS mutations and lymph node metastasis were identified to be independently associated with shorter overall survival (KRAS, P = 0.004; lymph node metastasis, P = 0.015). CONCLUSIONS: Our data suggest that KRAS mutation is a poor prognosis predictive biomarker for the survival in BTC patients.

Development of Stemness in Cancer Cell Lines Resistant to the Anticancer Effects of Zoledronic Acid.

BACKGROUND: Drug resistance is closely related to cancer cell stemness, that is acquired along with resistance to various anticancer agents. However, this has not been investigated as a potential mechanism underlying cancer cell resistance to zoledronate, that is used to suppress bone metastasis. MATERIALS AND METHODS: Zoledronate-resistant A549 lung cancer and MG63 osteosarcoma cell lines were established by repeated treatment with sub-lethal concentrations of zoledronate. Expression levels of the stem cell marker NANOG, cMYC, octamer-binding transcription factor 4, and sex-determining region Y-box 2 were evaluated and sphere formation was compared between parental and resistant cell lines. Tumourigenicity was assessed in vivo. RESULTS: Stem cell marker expression was up-regulated and sphere formation was enhanced in resistant compared to parental cells and showed greater tumour formation capacity in mice. CONCLUSION: Repeated treatment of malignant tumour cell lines with zoledronate, induces the development of drug resistance and stemness.

Relationship between epicardial adipose tissue thickness and early impairment of left ventricular systolic function in patients with preserved ejection fraction.

Epicardial adipose tissue (EAT) is metabolically bioactive fat. The present study aimed to clarify the relationship between EAT amount and early impairment of left ventricular (LV) systolic function in patients with preserved ejection fraction (EF), all evaluated echocardiographically. Participants comprised 62 elderly women (mean age ± standard deviation, 68 ± 11 years) with lifestyle-related diseases and EF ≥ 60 %. EAT amount was evaluated as thickness. Parameters suggesting early impairment of systolic function such as decreases in systolic mitral annular velocity (S') and tissue mitral annular displacement percentage (TMAD %) were evaluated along with EF. Correlations between EAT thickness and these LV systolic functions were assessed. Influences of various factors on the resultant significant relationships were also assessed. EAT thickness correlated inversely with S' and TMAD % (r = -0.402, p = 0.001 and r = -0.585, p < 0.001, respectively), but did not correlate with EF (r = 0.054, not significant). These significant relationships were maintained after considering factors such as body mass index, age, presence of lifestyle-related diseases and blood test results. A significant relationship existed between EAT amount and early impairment of LV systolic function in patients with preserved EF. Accumulation of EAT might contribute to the initial development of LV systolic dysfunction.