Clinical value of fluorescence in situ hybridization with MDM2 and DDIT3 probe in diagnosis of liposarcoma / 北京大学学报(医学版)

OBJECTIVE@#To investigate the value of using MDM2 amplification probe and DDIT3 dual-color, break-apart rearrangement probe fluorescence in situ hybridization (FISH) technique in the diagnosis of liposarcoma.@*METHODS@#In the study, 62 cases of liposarcoma diagnosed in Peking University First Hospital from January 2015 to December 2019 were analysed for clinicopathological information. Of these 62 cases of liposarcoma, all were analysed for MDM2 amplification and 48 cases were analysed for DDIT3 rearrangement using a FISH technique. Our study aimed to evaluate the status of MDM2 and DDIT3 by FISH in liposarcoma and correlate it with diagnosis of different subtypes of liposarcoma. The subtypes of liposarcoma were classified according to the FISH results, combined with the relevant clinicopathological features.@*RESULTS@#The patients aged 31-89 years (mean: 59 years) with a 1.75:1 male to female ratio. Histologically, there were 20 cases of atypical lipomatous tumour/well-differentiated liposarcoma (ALT/WDLPS), 26 cases of dedifferentiated liposarcoma (DDLPS), 13 myxoid liposarcoma (MLPS) and 3 pleomorphic liposarcoma (PLPS). Tumors with DDLPS (23/26) and WDLPS (8/20) were localized retroperitoneally, while both tumours of MLPS and PLPS were localized extra-retroperitoneally, and the difference of sites among the four subtypes of liposarcoma was statistically significant (P < 0.05). Histologically, varied mucoid matrix could be observed in the four subtypes of liposarcoma, and the difference was statistically significant (P < 0.05). MDM2 gene amplification was demonstrated in all cases of ALT/WDLPS and DDLPS (100%, 20/20 and 26/26 respectively); DDIT3 gene rearrangement was noted only in MLPS (100%, 13/13); most cases of DDLPS (96.2%, 25/26) and ALT/WDLPS (83.3%, 5/6, 6 cases selected for detection) demonstrated the picture of amplification of the DDIT3 telomeric tag. According to the instructions of DDIT3 break-apart rearrangement probe, the 5' telomere probe and 3' centromere probe spanned but did not cover the DDIT3 gene itself, on the contrary, the 5' telomere probe covered the CDK4 gene, while the DDIT3 and CDK4 gene were located adjacent to each other on chromosome, therefore, when the amplification signal appeared on the telomeric tag of the DDIT3 rearrangement probe, it indeed indicated the CDK4 gene amplification rather than the DDIT3 gene rearrangement. Then the 10 cases with DDIT3 telomeric tag amplification were selected for CDK4 and DDIT3 gene amplification probe FISH tests, and all the cases showed CDK4 gene amplification (100%, 10/10) and two of the 10 cases demonstrated co-amplification of CDK4 and DDIT3 (20%, 2/10); DDIT3 polysomy detected by DDIT3 gene rearrangement probe was found in 1 case of DDLPS and 2 cases of PLPS (66.7%, 2/3) with morphology of high-grade malignant tumour and poor prognosis.@*CONCLUSION@#Our results indicate that a diagnosis of different subtype liposarcoma could be confirmed based on the application of MDM2 and DDIT3 FISH, combined with clinicopathological findings. It is also noteworthy that atypical signals should be correctly interpreted to guide correct treatment of liposarcomas.

Diagnostic value of MDM2 RNA in situ hybridization in atypical lipomatous tumor/well-differentiated liposarcoma and dedifferentiated liposarcoma / 中华病理学杂志

Objective: To investigate the value of MDM2 RNA in situ hybridization (RNA-ISH) in diagnosing atypical lipomatous tumor/well-differentiated liposarcoma (ALT/WDL) and dedifferentiated liposarcoma (DDL). Methods: A total of 26 ALT/WDL/DDLs diagnosed from March 2017 to May 2019 in West China Hospital, Sichuan University, Chengdu, China and 18 control cases were included. MDM2 RNA-ISH was performed on all samples and compared with the fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) regarding their performance in detecting MDM2. Results: All samples were detected successfully using the three methods. Among 26 ALT/WDL/DDLs, all cases showed MDM2 amplification and positivity for MDM2 RNA-ISH (26/26, 100%). Twenty-four (24/26, 92.3%) of the 26 tested cases were positive for MDM2 IHC while two of them were negative. Eighteen control cases were all negative for MDM2 FISH and RNA-ISH, and 15 (15/18) cases were negative for MDM2 IHC. The sensitivity and specificity of RNA-ISH were both 100%, and those of MDM2 IHC were 92.3% and 83.3%, respectively. Diffuse staining was identified in all MDM2 RNA-ISH positive ALT/WDL/DDLs, but identified in only 8/24 (33.3%) of the MDM2 IHC positive cases. Among the 11 ALT/WDL/DDL samples evaluated on tissue microarray, the positive rate of MDM2 RNA-ISH was 100% with diffuse staining in all cases. The positive rate of MDM2 IHC was 9/11 while only 1 of the 9 cases showed diffuse staining. The result of MDM2 RNA-ISH was identical to that of MDM2 FISH and was overall consistent with that of MDM2 IHC (Kappa=0.763, P<0.001). Conclusions: In ALT/WDL/DDLs, results of MDM2 RNA-ISH are highly consistent with those of FISH. MDM2 RNA-ISH is more sensitive and more specific and has more diffuse positive signals than the IHC. The findings indicate that MDM2 RNA-ISH is highly valuable for the diagnosis and differential diagnosis of ALT/WDL/DDLs.

Diseminación clonal de KPC-2 en Klebsiella pneumoniae resistente a carbapenémicos / Clonal dissemination of KPC-2 in carbapenem resistant Klebsiella pneumoniae

Objetivo: Determinar los mecanismos de resistencia antibiótica y la epidemiología molecular de aislados clínicos de Klebsiella pneumoniae resistentes a carbapenémicos. Materiales y métodos: 30 aislados multirresistentes de K. pneumoniae fueron obtenidos a partir de: urocultivo, aspirado traqueal, secreción de herida, sonda vesical, hemocultivo, líquido peritoneal, punta de catéter, colección abdominal y secreción bronquial. Los aislados fueron colectados de noviembre de 2012 a abril de 2013. La identificación y susceptibilidad antibiótica fue determinada por el sistema automatizado VITEK 2. Para la amplificación de genes de resistencia se empleó PCR, la determinación de las Secuencias Tipo (ST) fue obtenida por tipificación multilocus de secuencias (MLST) y la relación clonal fue establecida por electroforesis en gel de campo pulsado (PFGE). Resultados: Todos los aislados mostraron fenotipos multirresistentes, excepto a colistina y tigeciclina. El 100% de los aislados fue productor de la carbapenemasa KPC-2. La determinación de la presencia de genes codificantes de β-lactamasas de Espectro Extendido mostró que el 67% de los aislados fue positivo para el gen blaCTX-M, el 100% fue positivo para el gen blaSHV y 93% fue positivo para el gen blaTEM. El análisis de la relación clonal de los 30 aislados agrupó a 20 en un mismo pulso tipo. El análisis por MLST demostró que la ST predominante fue ST258 presente en el 60% de la población, seguida de ST1199 presente en el 20% de la población analizada. Conclusiones: Los resultados obtenidos demuestran la importancia de implementar y combinar estudios epidemiológicos, clínicos y moleculares para comprender la distribución de la resistencia entre bacterias de interés clínico.

Objective: To determine the mechanism of antibiotic resistance and molecular epidemiology of carbapenem resistant isolates of Klebsiella pneumoniae. Materials and Methods: 30 multidrug resistant isolates of K. pneumoniae were obtained from urine culture, tracheal aspirate, wound secretion, bladder catheter, blood culture, peritoneal fluid, catheter tip, abdominal collection, and bronchial secretion. K. pneumoniae isolates were collected between November 2012 and April 2013. Identification and susceptibility were determined by the VITEK 2 system. Resistance genes were identified by PCR, sequence type (ST) was established by multilocus sequence typing (MLST), and clonal relationship was defined by pulsed field gel electrophoresis (PFGE). Results: All isolates were multidrug resistant and susceptible to colistin and tigecycline. 100% of isolates produced KPC-2 carbapenemase. This study detected Extended Spectrum β-Lactamases enconding genes. 67% of isolates were positive for blaCTX-M, 100% were positive for blaSHV, and 93% of isolates were positive for blaTEM. Analysis of the clonal relationship clustered 20 isolates in the same clonal complex. Multilocus sequence typing showed the predominant sequence type ST 258 in 60% of population. ST 1199 were present in 20% of bacterial population. Conclusion: Molecular epidemiology, clinical research and molecular biology studies improve understanding of mechanisms of resistance distribution among bacteria of clinical interest.

Probable Association Between Oral Lichen Planus and presence of Helicobacter Pylori: a Preliminary Study in a Chilean Population / Probable Asociación entre Liquen plano Oral y la presencia de Helicobacter pylori: estudio Preliminar en Chile

ABSTRACT: Oral Lichen planus (OLP) is one of the main inflammatory diseases of the oral mucosa that is considered as a potentially malignant disorder. The exact pathogenesis of OLP remains to be completely understood. However, presence of bacteria has been associated to the inflammatory response observed in OLP. Particularly, Helicobacter pylori a major etiological agent of gastrointestinal inflammatory diseases and risk factor for gastric cancer, has been associated to Lichen planus. Here we studied a group of Chilean patients if there is any association between the presence of Helicobacter pylori and the clinical manifestation of OLP. We found a significant difference between the patients positive for H. pylori and the age of OLP diagnosis, suggesting that oral H. pylori might induce the disease at an earlier age. However, we could not confirm a statistically significance between the presence of the bacteria and OLP.

RESUMEN: Liquen Plano Oral (LPO) es una enfermedad inflamatoria de la mucosa oral considerada como desorden potencialmente maligno. La patogénesis exacta de LPO es desconocida. Sin embargo, se ha asociado la presencia de bacterias como responsables de la inflamación observada en LPO. Particularmente, Helicobacter pylori (H. pylori), agente etiológico principal de enfermedades inflamatorias gastrointestinales y factor de riesgo de cáncer gástrico, ha sido asociado con LPO. Se estudió la posible asociación entre H. pylori y manifestaciones clínicas de LPO en un grupo de pacientes Chilenos. Se encontró diferencia significativa entre los pacientes positivos para H. pylori y la edad de diagnóstico de LPO, sugiriendo que H. pylori podría inducir la enfermedad a temprana edad. Sin embargo, no se pudo confirmar significancia estadística entre la presencia de esta bacteria y la presencia de displasia en LPO.

Antibacterial activity of phytopathogenic Streptomyces strains against bacteria associated to clinical diseases / Atividade antimicrobiana de Streptomyces fitopatogênicas contra bactérias associadas a doenças de importância clínica

The genus Streptomyces is associated with the ability to produce and excrete a variety of bioactive compounds, such as antibiotic, antifungal and antiviral. Biological active polyketide and peptide compounds with applications in medicine, agriculture and biochemical research are synthesized by PKS-I and NRPS genes. The evaluation of the presence of these genes associated with the biosynthesis of secondary metabolites in different phytopathogenic Streptomyces strains were performed using degenerated primers. The positive signal was observed in 58/63 Streptomyces strains for NRPS gene, 43/63 for PKS-I, and for PKS-II all the 63 strains showed positive signal of amplification. These strains also were tested with double layer agar-well technique against bacterial with clinical importance, and it was possible to observe the Streptomyces spp. strains were able to inhibit the growth of 14, 20, 13 and 3 isolates Gram-positive and Gram-negative bacteria, Staphylococcus aureus (ATCC 25923), Bacillus cereus (ATCC 14579), Pseudomonas aeruginosa (ATCC 27853) and Escherichia coli (ATCC 11775) respectively. The Streptomyces sp. strains IBSBF 2019 and IBSBF 2397 showed antibacterial activity against all four bacteria-target tested.(AU)

O gênero Streptomyces apresenta alta capacidade de produzir e excretar uma grande variedade de compostos biologicamente ativos, como antibióticos, antifúngicos e antivirais. Compostos biologicamente ativos de policetídeos e peptídeos com aplicações na medicina, agricultura e pesquisas bioquímicas são sintetizados pelos genes PKS-I e NRPS. A avaliação da presença desses genes associados à biossíntese de metabólitos secundários em diferentes linhagens de Streptomyces fitopatogênicas foi realizada através do uso de primers degenerados. O sinal positivo foi observado em 58/63 linhagens de Streptomyces para o gene NRPS, 43/63 para o gene PKS-I e, para o gene PKS-II, todas as 63 linhagens apesentaram o sinal positivo de amplificação. Essas linhagens também foram testadas através da técnica de dupla camada contra bactérias de importância clínica e foi possível observar que as linhagens de Streptomyces spp. foram capazes de inibir o crescimento de 14, 20, 13 e 3 isolados de bactérias Gram-positivas e Gram-negativas, Staphylococcus aureus (ATCC 25923), Bacillus cereus (ATCC 14579), Pseudomonas aeruginosa (ATCC 27853) e Escherichia coli (ATCC 11775), respectivamente. As linhagens de Streptomyces sp. ISBSF 2019 e 2397 apresentaram atividade antibacteriana contra todas as bactérias-alvo testadas.(AU)

Malignant transformation in a Breast Adenomyoepithelioma Caused by Amplification of c-MYC: A Common pathway to Cancer in a Rare Entity / 한국유방암학회지

Breast adenomyoepitheliomas are composed of a biphasic proliferation of myoepithelial cells around small epithelial-lined spaces. Due to the rarity of adenomyoepitheliomas, the molecular data describing them are limited. Adenomyoepitheliomas are considered to be benign or have low malignant potential, and be prone to local recurrence. Malignant transformation has been associated with homozygous deletion of CDKN2A or somatic mutations in TERT, but remains unexplained in many cases. Here, we describe a case of carcinomatous transformation of both epithelial and myoepithelial cells in an estrogen receptor-negative adenomyoepithelioma caused by amplification of MYC. Break-apart fluorescence in situ hybridization revealed an increase in the MYC gene copy number (3–4 copies/cell in 37%, > 4 copies/cell in 40%). Deregulation of MYC is responsible for uncontrolled proliferation and cellular immortalization in basal-like breast cancers. Our case demonstrates that genomic instability events associated with gene amplification may be involved in the carcinogenesis of malignant adenomyoepitheliomas.

An improved association analysis pipeline for tumor susceptibility variant in haplotype amplification area / 南方医科大学学报

OBJECTIVE@#Haplotype amplification on germline variants is suggested to imply potential selective advantages and clonal expansion susceptibility and has become an important signature for seeking cancer susceptibility gene.Here we propose an improved association method that fully considers the haplotype amplification status.@*METHODS@#The haplotype amplification status was estimated by the variant allelic frequencies.We adopted a permutation test on variant allelic frequencies to divide the candidate variants into multiple groups.A likelihood clustering method was then applied to establish the neighborhood system of the hidden Markov random field framework.A filtering pipeline was introduced into the proposed method to further refine the candidate variants, including a Wilson's interval filter and a false discovery rate controller.The final candidate set along with the haplotype amplification status was collapsed into the weighted virtual sites for association tests.@*RESULTS@#Through simulated tests on a series of datasets, we compared the type Ⅰ error rates of different minor allele frequencies, which stably fell within 2%, suggesting good robustness of the algorithm.In addition, we compared another 5 published association approaches for Type-Ⅰ and Type-Ⅱ error rates with the proposed method, which resulted in the error rates all within 2%, demonstrating significant advantages and a good statistical ability of the proposed method.@*CONCLUSIONS@#The proposed method can accurately identify tumor susceptibility variants in haplotype amplification area with good robustness and stability.

Fuente de alimentación de ejemplares de Triatoma sordida en un área con alto riesgo de domiciliación en el Chaco Paraguayo / Blood meal source of specimens of Triatoma sordida in an area with high risk of domiciliation in the Paraguayan Chaco

En el presente estudio evaluamos indicadores entomológicos (índices de infección, colonización e infección natural) de ejemplares de Triatoma sordida capturados en el intra y peridomicilio de viviendas del Chaco Paraguayo en el período 2014 al 2016. El objetivo fue comparar con los hallazgos previamente reportados en el periodo 2010 al 2013 en la misma región. Además se ha incorporado la detección de fuente de alimentación como complemento para definir el riesgo de domiciliación de este vector secundario. Se aplicaron técnicas moleculares asociados a indicadores entomológicos y epidemiológicos a 220 ejemplares de T. sordida capturados en 67 viviendas de 24 localidades del Chaco. Se detectó infestación y colonización por T. sordida en el intradomicilio 13/67 (19%) y 5/13 (38%) y en el peridomicilio 54/67 (81%) y 43/54 (80%), respectivamente. Se detectó infección con T. cruzi en un 17,3% de los ejemplares analizados. La fuente de alimentación pudo detectarse en tan solo 13 de 220 ejemplares (6%), todos resultaron positivos para sangre de gallina y correspondían a captura en el peridomicilio. El índice de infestación intradomiciliar fue del 19%, superior al de años anteriores y similar a las zonas históricamente endémicas de la Región Occidental (18%-20%). El riesgo de transmisión intradomiciliar sigue siendo elevado porque en 3 de 5 viviendas con ninfas de T. sordida, los ejemplares estaban infectados con T. cruzi. Los indicadores entomológicos obtenidos en el presente estudio concuerdan con resultados previos de nuestro equipo, y confirman la capacidad vectorial de este triatomino secundario en la transmisión de la enfermedad de Chagas(AU)

Clinical Implications According to Diagnostic Methods of Human Epidermal Growth Factor Receptor 2 Positivity in Breast Cancer: A Retrospective Study

PURPOSE: According to American Society of Clinical Oncology/College of American Pathologists guidelines, breast cancer is human epidermal growth factor receptor 2 (HER2) positive if there is HER2 protein overexpression at a 3+ level on immunohistochemistry (IHC 3+) or gene amplification (more than six copies per nucleus) on fluorescence in situ hybridization (FISH+). However, there have been few reports on whether outcomes differ based on diagnosis by these two techniques. In this study, we compared outcomes based on the two methods in patients with HER2-positive breast cancer. METHODS: This study was a retrospective analysis of HER2-positive breast cancer in 18,304 patients, including 14,652 IHC 3+ patients and 3,652 FISH+ patients from the Korean Breast Cancer Society Registry. We compared breast cancer-specific survival and overall survival based on IHC 3+ and FISH+ status with or without trastuzumab. RESULTS: Breast cancer-specific survival was significantly different between the IHC 3+ and FISH+ groups, with 5-year cumulative survival rates of 95.0% for IHC 3+ and 98.5% for FISH+ patients who did not receive trastuzumab (p=0.001) in Kaplan-Meier methods. However, there were no significant differences in breast cancer-specific survival and overall survival between IHC 3+ and FISH+ groups regardless of trastuzumab treatment in Cox proportional hazards models. CONCLUSION: The survival outcomes were not affected by the different two diagnostic methods of HER2-positive breast cancer. Further research to evaluate differences in prognosis and other characteristics according to the diagnostic methods of HER2 positivity is needed in the future.

Outcomes of long term treatments of type I hereditary angioedema in a Turkish family

Abstract: Background: Hereditary angioedema is a rare autosomal dominantly inherited immunodeficiency disorder characterized by potentially life-threatening angioedema attacks. Objective: We aimed to investigate the clinical and genetic features of a family with angioedema attacks. Methods: The medical history, clinical features and C1-INH gene mutation of a Turkish family were investigated and outcomes of long-term treatments were described. Results: Five members had experienced recurrent swellings on the face and extremities triggered by trauma. They were all misdiagnosed as familial Mediterranean fever (FMF) depending on frequent abdominal pain and were on colchicine therapy for a long time. They had low C4 and C1-INH protein concentrations and functions. A mutation (c.1247T>A) in C1-INH gene was detected. They were diagnosed as having hereditary angioedema with C1-INH deficiency (C1-INH hereditary angioedema) for the first time. Three of them benefited from danazol treatment without any significant adverse events and one received weekly C1 esterase replacement treatment instead of danazol since she had a medical history of thromboembolic stroke. Study limitations: Small sample size of participants. Conclusion: Patients with C1-INH hereditary angioedema may be misdiagnosed as having familial Mediterranean fever in regions where the disorder is endemic. Medical history, suspicion of hereditary angioedema and laboratory evaluations of patients and their family members lead the correct diagnoses of hereditary angioedema. Danazol and C1 replacement treatments provide significant reduction in hereditary angioedema attacks.

Selection of suitable reference genes for abiotic stress-responsive gene expression studies in peanut by real-time quantitative PCR

Background: Because of its strong specificity and high accuracy, real-time quantitative PCR (RT-qPCR) has been a widely used method to study the expression of genes responsive to stress. It is crucial to have a suitable set of reference genes to normalize target gene expression in peanut under different conditions using RT-qPCR. In this study, 11 candidate reference genes were selected and examined under abiotic stresses (drought, salt, heavy metal, and low temperature) and hormone (SA and ABA) conditions as well as across different organ types. Three statistical algorithms (geNorm, NormFinder and BestKeeper) were used to evaluate the expression stabilities of reference genes, and the comprehensive rankings of gene stability were generated. Results: The results indicated that ELF1B and YLS8 were the most stable reference genes under PEG-simulated drought treatment. For high-salt treatment using NaCl, YLS8 and GAPDH were the most stable genes. Under CdCl2 treatment, UBI1 and YLS8 were suitable as stable reference genes. UBI1, ADH3, and ACTIN11 were sufficient for gene expression normalization in low-temperature experiment. All the 11 candidate reference genes showed relatively high stability under hormone treatments. For organs subset, UBI1, GAPDH, and ELF1B showed the maximum stability. UBI1 and ADH3 were the top two genes that could be used reliably in all the stress conditions assessed. Furthermore, the necessity of the reference genes screened was further confirmed by the expression pattern of AnnAhs. Conclusions: The results perfect the selection of stable reference genes for future gene expression studies in peanut and provide a list of reference genes that may be used in the future.

Escherichia coli expressing endoglucanase gene from Thai higher termite bacteria for enzymatic and microbial hydrolysis of cellulosic materials

Background: Endoglucanase plays a major role in initiating cellulose hydrolysis. Various wild-type strains were searched to produce this enzyme, but mostly low extracellular enzyme activities were obtained. To improve extracellular enzyme production for potential industrial applications, the endoglucanase gene of Bacillus subtilis M015, isolated from Thai higher termite, was expressed in a periplasmic-leaky Escherichia coli. Then, the crude recombinant endoglucanase (EglS) along with a commercial cellulase (Cel) was used for hydrolyzing celluloses and microbial hydrolysis using whole bacterial cells. Results: E. coli Glu5 expressing endoglucanase at high levels was successfully constructed. It produced EglS (55 kDa) with extracellular activity of 18.56 U/mg total protein at optimal hydrolytic conditions (pH 4.8 and 50°C). EglS was highly stable (over 80% activity retained) at 40­50°C after 100 h. The addition of EglS significantly improved the initial sugar production rates of Cel on the hydrolysis of carboxymethyl cellulose (CMC), microcrystalline cellulose, and corncob about 5.2-, 1.7-, and 4.0-folds, respectively, compared to those with Cel alone. E. coli Glu5 could secrete EglS with high activity in the presence of glucose (1% w/v) and Tween 80 (5% w/v) with low glucose consumption. Microbial hydrolysis of CMC using E. coli Glu5 yielded 26 mg reducing sugar/g CMC at pH 7.0 and 37°C after 48 h. Conclusions: The recombinant endoglucanase activity improved by 17 times compared with that of the native strain and could greatly enhance the enzymatic hydrolysis of all studied celluloses when combined with a commercial cellulase.

HER2 Status and Its Heterogeneity in Gastric Carcinoma of Vietnamese Patient

BACKGROUND: Human epidermal growth factor receptor 2 (HER2) is related to the pathogenesis and poor outcome of numerous types of carcinomas, including gastric carcinoma. Gastric cancer patients with HER2 positivity have become potential candidates for targeted therapy with trastuzumab. METHODS: We investigated 208 gastric cancer specimens using immunohistochemistry (IHC), fluorescence in situ hybridization and dual in situ hybridization (ISH). We also investigated the concordance between IHC and ISH. The correlation between HER2 status and various clinicopathological findings was also investigated. RESULTS: In total, 15.9% (33/208) and 24.5% (51/208) of gastric cancers showed HER2 gene amplification and protein overexpression, respectively. A high level of concordance between ISH and IHC analyses (91.3%, κ = 0.76) was found. A significant correlation between HER2 status and intestinal-type (p < .05) and differentiated carcinomas (p < .05) was also noted. The HER2 heterogeneity was high in gastric cancers; we found 68.8% phenotypic heterogeneity and 57.6% genotypic heterogeneity. Heterogeneity in HER2 protein expression and gene amplification showed a close association with diffuse histologic type and IHC 2+. CONCLUSIONS: HER2 protein overexpression and gene amplification were detected in 24.5% and 15.9% of gastric cancer specimens, respectively. Intestinal-type showed a higher level of HER2 protein overexpression and gene amplification than diffuse type. HER2 status also showed a significant relationship with well- and moderately-differentiated carcinomas. The ratio of phenotypic and genotypic heterogeneity of HER2 was high in gastric carcinomas and was associated with HER2 IHC 2+ and diffuse histologic type.

Aurora Kinase A Is a Prognostic Marker in Colorectal Adenocarcinoma

BACKGROUND: Aurora kinase A (AURKA), or STK15/BTAK, is a member of the serine/threonine kinase family and plays important roles in mitosis and chromosome stability. This study investigated the clinical significance of AURKA expression in colorectal cancer patients in Korea. METHODS: AURKA protein expression was evaluated by immunohistochemistry in 151 patients with colorectal adenocarcinoma using tissue microarray blocks. We analyzed the relationship between clinicopathological characteristics and AURKA expression. In addition, the prognostic significance of various clinicopathological data for progression-free survival (PFS) was assessed. Also we evaluated copy number variations by array comparative genomic hybridization and AURKA gene amplification using fluorescence in situ hybridization in colorectal carcinoma tissues. RESULTS: AURKA gene amplification was found more frequently in the 20q13.2–13.33 gain-positive group than the group with no significant gain on the AURKA-containing locus. AURKA protein expression was detected in 45% of the cases (68/151). Positive staining for AURKA was observed more often in male patients (p = .035) and distally located tumors (p = .021). PFS was shorter in patients with AURKA expression compared to those with low-level AURKA expression (p < .001). Univariate analysis revealed that AURKA expression (p = .001), age (p = .034), lymphatic invasion (p = .001), perineural invasion (p = .002), and TNM stage (p = .013) significantly affected PFS. In a multivariate analysis of PFS, a Cox proportional hazard model confirmed that AURKA expression was an independent and significant prognostic factor in colorectal adenocarcinoma (hazard ratio, 3.944; p < .001). CONCLUSIONS: AURKA could serve as an independent factor to predict a poor prognosis in Korean colorectal adenocarcinoma patients.

NanoString nCounter® Approach in Breast Cancer: A Comparative Analysis with Quantitative Real-Time Polymerase Chain Reaction, In Situ Hybridization, and Immunohistochemistry / 한국유방암학회지

PURPOSE: Accurate testing for estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) is essential for breast cancer treatment. At present, immunohistochemistry (IHC)/florescence in situ hybridization (FISH) are widely accepted as the standard testing methods. To investigate the value of NanoString nCounter®, we performed its comparative analysis with IHC/FISH and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) for the assessment of ER, PR, and HER2. METHODS: Data on IHC/FISH results for ER, PR, and HER2 in 240 patients from a single tertiary hospital in Korea were collected and compared with NanoString nCounter® and qRT-PCR results at a single institution. RESULTS: Expression levels for each gene using NanoString nCounter® showed good correlation with the corresponding data for protein expression by IHC (p<0.001) and gene amplification status for HER2 (p<0.001). Comparisons between gene expression and IHC data showed good overall agreement with a high area under the curve (AUC) for ESR1/ER (AUC=0.939), PgR/PR (AUC=0.796), and HER2/HER2 (AUC=0.989) (p<0.001). CONCLUSION: The quantification of ER, PgR, and HER2 mRNA expression with NanoString nCounter® may be a viable alternative to conventional IHC/FISH methods.

Higher prevalence and gene amplification of HPV16 in oropharynx as compared to oral cavity

ABSTRACT Objective The objective of this study was to clarify differences regarding HPV16 infection and gene amplification between the oral cavity and oropharynx in healthy individuals. Material and Methods The subjects were 94 healthy asymptomatic individuals (41 males, 53 females; mean age 58.6 years, range 16-97 years) who visited the Department of Oral and Maxillofacial Reconstructive Surgery of the Hiroshima University Hospital from 2014 to 2015. Oral epithelial cells were collected from oral rinse and pharynx gargle samples and placed in saline. The human endogenous retrovirus gene ERV3-1 was used as a reference to estimate the number of human cells in each sample. DNA samples were extracted from approximately 10,000 human cells and tested for HPV16 DNA by PCR using a type-specific primer. Similarly, we analyzed the HPV16 viral copy number in HPV16-positive cases using real-time PCR to examine genomic amplification. Results The percentage of HPV16-positive cases was higher in the gargle (28.7%) as compared to the rinse (16.0%) samples. In the oral rinse samples, males (26.8%) showed a significantly higher rate of HPV16 than females (7.5%) (P=0.021). Importantly, in older subjects (aged 60-89 years), gargle samples showed a significantly higher rate of HPV16 (33.3%) than oral rinse samples (13.7%) (P=0.034). The average number of viral copies was approximately 8 times higher in the gargle than in the oral rinse samples (0.16±0.27 vs. 1.35±1.26 copy numbers per cell), a significant difference (P<0.001). Conclusion Our findings suggest that the oropharynx is more susceptible to HPV16 infection as compared to the oral cavity, while HPV16 gene amplification is also more commonly found in the oropharynx.

Utility of multiplex real-time PCR in the diagnosis of extrapulmonary tuberculosis

Abstract Objective The diagnosis of extrapulmonary tuberculosis is still a challenge because of its pauci-bacillary nature. The aim of the study was to evaluate the role of a multiplex PCR assay in the diagnosis of extrapulmonary tuberculosis and to compare the efficiency of two targets, IS6110 and MPB64 to detect Mycobacterium tuberculosis. Methods 150 extrapulmonary samples (61 pus/aspirate, 46 tissue, 32 body fluids, and 11 urine) from clinically suspected cases of tuberculosis were included in the study. All the samples were subjected to direct fluorescent microscopy, TB culture (BacT/ALERT 3D, biomerieux, Durham, North Carolina, USA) and a Multiplexed Tandem PCR targeting two mycobacterial DNA sequences, IS6110 and MPB64. Master-Mix reagents and primers were prepared by AusDiagnostics Pvt. Ltd (Alexandria, New South Wales, Australia). The performance of the assay was assessed using a composite gold standard, which included clinical characteristics, microbiology smear as well as culture, histopathology, cytology, radiology, and response to antitubercular therapy. Results 20.3%, 23.6%, and 45.3% of specimens were positive by smear, culture, and PCR, respectively. The sensitivity and specificity of the multiplex PCR was 91.9% and 88.4%, respectively, using the composite gold standard. Positive and negative predictive values of the PCR were estimated as 85.1% and 93.8%, respectively. Higher positivity was observed with target IS6110 (44.6%) as compared to target MPB64 (18.9%). The sensitivities of IS6110 and MPB64 individual targets were 90.3% and 64.5%, respectively, and specificities were 88.4% and 97.7%, respectively. Conclusion PCR can play an important role in rapid and accurate diagnosis of extrapulmonary tuberculosis. IS6110 alone is an effective target in our part of the country.

A molecular platform for the diagnosis of multidrug-resistant and pre-extensively drug-resistant tuberculosis based on single nucleotide polymorphism mutations present in Colombian isolates of Mycobacterium tuberculosis

Developing a fast, inexpensive, and specific test that reflects the mutations present in Mycobacterium tuberculosis isolates according to geographic region is the main challenge for drug-resistant tuberculosis (TB) control. The objective of this study was to develop a molecular platform to make a rapid diagnosis of multidrug-resistant (MDR) and extensively drug-resistant TB based on single nucleotide polymorphism (SNP) mutations present in therpoB, katG, inhA,ahpC, and gyrA genes from Colombian M. tuberculosis isolates. The amplification and sequencing of each target gene was performed. Capture oligonucleotides, which were tested before being used with isolates to assess the performance, were designed for wild type and mutated codons, and the platform was standardised based on the reverse hybridisation principle. This method was tested on DNA samples extracted from clinical isolates from 160 Colombian patients who were previously phenotypically and genotypically characterised as having susceptible or MDR M. tuberculosis. For our method, the kappa index of the sequencing results was 0,966, 0,825, 0,766, 0,740, and 0,625 forrpoB, katG, inhA,ahpC, and gyrA, respectively. Sensitivity and specificity were ranked between 90-100% compared with those of phenotypic drug susceptibility testing. Our assay helps to pave the way for implementation locally and for specifically adapted methods that can simultaneously detect drug resistance mutations to first and second-line drugs within a few hours.

Aldosterona e IL-17 en la génesis de la hipertensión arterial mineralocorticoídea, un estudio ex vivo / Aldosterone and il-17 in the genesis of mineralocorticoid arterial hypertension: an ex vivo study

Background: Treatment of dendritic cells (DC) with aldosterone induces the secretion of IL-6 and TGF-beta. The polarization of naïve T cells to helper 17 T lymphocytes with DCs pre-incubated with aldosterone, has been described in vivo, generating an IL-17 hyper-secreting phenotype, a cytokine associated with cardiac and renal fibrosis. There are mineralocorticoid receptors (MR) in immune cells and their activation may determine the inflammatory (M1) or adaptive (M2) macrophage phenotype. Aldosterone levels could regulate immunogenic gene expression in these cells, modulating the liberation of specific cytokines. Aim: To assess in humans the association of aldosterone levels and IL-17 with inflammatory markers in peripheral blood mononuclear cells (PBMC). Material and Methods: In blood samples of 176 participants aged 18 to 67 years (61 percent women) with a body mass index of 27.1 +/- 4.8 kg/m2, aldosterone, plasma renin activity (ARP), cortisol, C reactive protein, andIL-17 were measured. mRNA was isolated from PBMCs to measure the expression of MR RAC-1, HO-1, TLR-4, CD-14, NGAL and IL-17 by real time polymerase chain reaction. Results: Aldosterone correlated positively with ARP and the expression of CD-14 in PBMCs. Plasma levels of IL-17 were positively associated with the expression of MR, Rac1a and NGAL. Conclusions: Aldosterone and IL-17 levels were associated with inflammatory activation markers in PBMC, which could activate MRand promote a subclinical inflammatory status inducing hypertension.

Application of Van-Clear and xylene in the detection of cervical hTERC gene by fluorescence in situ hybridization / 中南大学学报(医学版)

OBJECTIVE@#To observe the effect of Van-Clear on vamplification of human telomerase RNA component (hTERC) gene in cervical tissues by fluorescence in situ hybridization, and to determine the potential for Van-Clear to replace xylene.
@*METHODS@#A total of 278 specimens of cervix uteri were collected from inpatients of Department of Gynaecology in Boai Hospital of Zhongshan from January to February, 2015, with 81 cases of normal specimens, 68 cases of cervical intraepithelial neoplasia (CIN) I, 57cases of CIN2, 42 cases of CIN3 and 30 cases of cervical invasive cancer. Double samples were collected from the same region. Fluorescence in situ hybridization was applied to detect the changes in the amplification of hTERC gene in 2 groups of specimens from the cervical biopsy.
@*RESULTS@#Differences in the positive expression rate of hTERC gene between the 2 groups of cervical lesions at all levels were not statistically significant (P>0.05).
@*CONCLUSION@#There is no significant difference in the positive rate of hTERC gene expression between the slices made by Van-clear and xylene. As an environmental-friend product, Van-Clear possesses certain value in detection of cervical hTERC gene by fluorescence in situ hybridization.