Estimation of Immunohistochemical Expression of VEGF in Ductal Carcinomas of the Breast

Vascular endothelial growth factor A (VEGF-A) is a very important growth factor in angiogenesis and holds potential as both a predictive marker for anti-angiogenic cancer treatment and a prognostic variable. Consequently, reliable estimation of VEGF expression is crucial. Investigators immunostained whole tumor sections for VEGF-A, VEGF-B, and VEGFR-1 of invasive ductal carcinomas of the breast and scored the tumors manually with staining intensity as the only parameter and by a combination of qualitative and quantitative information. The investigators also introduce an automated method for analyzing VEGF expression (so-called AI score) using the same tumor sections. Analysis of 100% of the tumor area was performed and the results were compared with the reduced analysis of 25% of the tumor area. These analyses were performed at ×5 and ×10 magnification, and each analysis was repeated in a second run with a new delineation of the tumor area. The AI scores were correlated to the manual scoring of VEGF intensity, but reproducibility of manual IHC scores was rather poor. The AI scores were reproducible, and the restricted analysis of 25% of the tumor area at ×5 magnifications was the most efficient considering time consumption and data load.     

Serrated Pathway Adenocarcinomas: Molecular and Immunohistochemical Insights into Their Recognition

Introduction

Colorectal adenocarcinomas (CRC) developed through serrated pathway seem to present particular behavior compared with the non-serrated ones, but recognition of them is difficult to do. The aim of our paper was to establish some criteria to facilitate their identification.

Materials and Methods

In 170 consecutive CRCs, we performed immunohistochemical staining with Cytokeratin 7 (CK7) and Cytokeratin 20 (CK20) and also with p53 and MLH-1. At the same time, we analyzed BRAF and K-ras mutations and the microsatellite status of CRC.

Results

26.47% of cases expressed CK7, and 16.47% were CK20-negative. Diffuse positivity for CK7 was associated in the proximal colon with CK20 negativity or weak positivity, BRAF mutations, lack of K-ras mutations, and p53 and MLH-1 negativity. All these cases were microsatellite-unstable and were diagnosed in stage II. Those cases from the distal colon and rectum that expressed CK7 were K-ras-mutated and had low p53 index and MLH-1 positivity, independent of the CK20 expression.

Conclusions

CK7, associated with MLH-1 and p53 expression, and also with the microsatellite status, BRAF and K-ras pattern, might be used to identify the CRC potentially going through serrated pathway. The serrated pathway adenocarcinomas of the proximal colon that do not display the morphological features of this pattern are more frequent CK7+/p53−/MLH-1−/BRAF-mutated/K-ras-wt/MSI cases, but those located in the distal colorectal segments seem to be CK7+/CK20+/p53−/MLH-1+/BRAF wt/K-ras-mutated/MSS cases.     

The cDNA Microarray Profiling of Protein Kinases and Phosphatases: Molecular Portrait of Human Prostate Carcinomas

Hybridization with cDNA arrays was used to obtain expression profiles of 263 protein-tyrosine kinase (PTK), protein-tyrosine phosphatase (PTP), dual-specific phosphatase (DuSP), and other genes for the normal prostate tissue, primary prostate carcinomas (PC) of 84 patients, 7 xenografts, and 5 carcinoma cell lines. Analysis of 96 profiles revealed eight clusters of genes coexpressed in PC (coefficient of correlation r > 0.7). According to the known functions of their genes, the clusters were designated as proliferating-cell (CDC42, TOP2A, FGFR3, MYC, etc.), neoangiogenesis and blood-cell (LCK, VAV1, KDR, VEGF, MMP9, SYK, PTPRS, and FLT4), invasion-1 and invasion-2 (ADAM17, TRPM2, DUSP6, VIM, CAV1, CAV2, JAK1, PTPNS1, FYN, and PDGFB), HER2, and PSA/PSM/HER3. Basing on expression profiles of 66 genes, a molecular classification of PC was generated and allowed discrimination between PC and cell lines or xenografts at 98.9% probability. The results suggested that, along with PSA, PSM (FOLH1), callicreine-2, and -2-macroglobulin, cell signaling genes EGFR, HER2, HER3, TOP2, KRT8, KRT18, VEGF, CD44, VIM, CAV1, and CAV2 may serve as diagnostic and prognostic markers in PC. The HER2, VEGF, and CD44 genes and the MMP and ADAM families were assumed to be promising targets for inhibitors of PC cell proliferation and metastasis.     

Molecular Genetic Analyses of Plant Polyamines

Polyamines are small, positively charged aliphatic amines that play a variety of roles in plant physiology. Putrescine, spermidine, and spermine are usually what are collectively meant by the term polyamines, although plants also have a variety of other related compounds and secondary product conjugates to polyamines. Organisms synthesize putrescine, spermidine, and spermine by pathways leading from ornithine, arginine, and SAM, with three of the important enzymes being amino acid decarboxylases. There has been recent progress in understanding plant polyamines, both their function and the regulation of their synthesis, as a result of molecular genetic investigations. The cDNAs for many of the key enzymes have been cloned and se-quenced, and studies on regulation of the enzymes have begun. Mutational and transgenic approaches are being used to perturb the pathway. Some of the phenotypes observed suggest interactions between polyamines and either ethylene or cytokinin, consistent with some of the correlations observed many years previously by polyamine physiologists. These studies, while still in their early stages, should improve our understanding of polyamine synthesis, but difficult problems remain to be solved before we can answer the question: What are the biological functions and associated mechanisms of action of polyamines?     

Relationship between the Inflammatory Molecular Profile of Breast Carcinomas and Distant Metastasis Development

Inflammatory conditions may promote tumor progression and aggressiveness. In previous reports, we found a group of breast cancer tumors characterized by metalloprotease-11 (MMP-11) expression by intratumoral mononuclear inflammatory cells (MICs), which was associated with distant metastasis development. Thus, in the present study we evaluated the relationship between MMP-11 expression by MICs, distant metastasis development, and a wide panel of inflammatory factors in breast carcinoma. In an initial approach, we analyzed 65 factors associated with tumor progression and inflammation, in a tumor population classified in good or bad prognosis, based on MMP-11 expression by intratumoral MICs. The most differentially expressed factors were then analyzed in a wider tumor population classified according to MMP-11 expression by MICs and also according to metastasis development. These analyses were carried out by Real-time PCR. The results showed that of the 65 starting factors analyzed, those related with MMP-11 expression by MICs were: IL-1, −5, −6, −8, −17, −18, MMP-1, TIMP-1, ADAM-8, −10, −15, −23, ADAMTS-1, −2, −15, Annexin A2, IFNβ, Claudin-3, CCL-3, MyD88, IRAK-4 and NFκB. Of them, factors more differentially expressed between both groups of tumors were IL-1, IL-5, IL-6, IL-17, IFNβ and NFκB. Thereafter, we confirmed in the wider tumor population, that there is a higher expression of those factors in tumors infiltrated by MMP-11 positive MICs. Altogether these results indicate that tumors developing worse prognosis and identified by MMP-11 expression by intratumoral MICs, shows an up-regulation of inflammatory-related genes.     

Molecular Biomarker Analyses Using Circulating Tumor Cells

Background

Evaluation of cancer biomarkers from blood could significantly enable biomarker assessment by providing a relatively non-invasive source of representative tumor material. Circulating Tumor Cells (CTCs) isolated from blood of metastatic cancer patients hold significant promise in this regard.

Methodology/Principal Findings

Using spiked tumor-cells we evaluated CTC capture on different CTC technology platforms, including CellSearch® and two biochip platforms, and used the isolated CTCs to develop and optimize assays for molecular characterization of CTCs. We report similar performance for the various platforms tested in capturing CTCs, and find that capture efficiency is dependent on the level of EpCAM expression. We demonstrate that captured CTCs are amenable to biomarker analyses such as HER2 status, qRT-PCR for breast cancer subtype markers, KRAS mutation detection, and EGFR staining by immunofluorescence (IF). We quantify cell surface expression of EGFR in metastatic lung cancer patient samples. In addition, we determined HER2 status by IF and FISH in CTCs from metastatic breast cancer patients. In the majority of patients (89%) we found concordance with HER2 status from patient tumor tissue, though in a subset of patients (11%), HER2 status in CTCs differed from that observed in the primary tumor. Surprisingly, we found CTC counts to be higher in ER+ patients in comparison to HER2+ and triple negative patients, which could be explained by low EpCAM expression and a more mesenchymal phenotype of tumors belonging to the basal-like molecular subtype of breast cancer.

Conclusions/Significance

Our data suggests that molecular characterization from captured CTCs is possible and can potentially provide real-time information on biomarker status. In this regard, CTCs hold significant promise as a source of tumor material to facilitate clinical biomarker evaluation. However, limitations exist from a purely EpCAM based capture system and addition of antibodies to mesenchymal markers could further improve CTC capture efficiency to enable routine biomarker analysis from CTCs.     

我国巢鼠属分类和分子系统学分析

本研究对2018至2021年采集的9号巢鼠(Micromys minutus)标本、22号红耳巢鼠(M. erythrotis)标本和19号待厘定的巢鼠属标本,进行形态分类和分子系统学分析,进一步揭示我国巢鼠属的分类和系统分化问题。待厘定的巢鼠属标本形态特征为：标本体背毛黑棕,体腹毛基灰色,毛尖灰白,体侧毛色具明显区分,尾背部毛色黑棕,尾腹部毛色灰棕色;尾长长于头体长的120%;头骨背面观可见颧弓明显弯曲;颅全长[(18.59 ± 0.48)mm]和颅基长[(17.43 ± 0.48 mm)]较长,腭长[(9.35 ± 0.11)mm]较长,脑颅高[(7.43 ± 0.06)mm]较高。待厘定的巢鼠属标本形态特征与巢鼠和红耳巢鼠均存在差异。待厘定巢鼠属标本与巢鼠和红耳巢鼠之间的遗传距离分别为0.115和0.136,接近于巢鼠与红耳巢鼠之间的遗传距离(0.126)。利用Cyt b基因全序列和核基因IRBP1、RAG1和RAG2序列分别构建的巢鼠属系统发生树均以较高的置信度分化成3个进化支,即巢鼠、红耳巢鼠和待厘定的巢鼠属样本的进化支。形态学和分子系统学分析结果均支持待厘定的巢鼠属标本为独立物种分类单元,对应于文献记载的巢鼠川西亚种(M. m. pygmaeus)。根据产地、遗传距离和形态分化,建议将巢鼠川西亚种提升为种,命名为川西巢鼠(M. pygmaeus comb. nov.)。利用Cyt b基因全序列构建的巢鼠系统发生树分化成6个进化谱系：日韩谱系、欧洲谱系、俄罗斯新西伯利亚谱系、中国东北和俄罗斯远东谱系、中国安徽谱系和中国台湾谱系。     

Molecular Characteristics and Metastasis Predictor Genes of Triple-Negative Breast Cancer: A Clinical Study of Triple-Negative Breast Carcinomas

Background

Triple-negative breast cancer is a subtype of breast cancer with aggressive tumor behavior and distinct disease etiology. Due to the lack of an effective targeted medicine, treatment options for triple-negative breast cancer are few and recurrence rates are high. Although various multi-gene prognostic markers have been proposed for the prediction of breast cancer outcome, most of them were proven clinically useful only for estrogen receptor-positive breast cancers. Reliable identification of triple-negative patients with a favorable prognosis is not yet possible.

Methodology/Principal Findings

Clinicopathological information and microarray data from 157 invasive breast carcinomas were collected at National Taiwan University Hospital from 1995 to 2008. Gene expression data of 51 triple-negative and 106 luminal breast cancers were generated by oligonucleotide microarrays. Hierarchical clustering analysis revealed that the majority (94%) of triple-negative breast cancers were tightly clustered together carrying strong basal-like characteristics. A 45-gene prognostic signature giving 98% predictive accuracy in distant recurrence of our triple-negative patients was determined using the receiver operating characteristic analysis and leave-one-out cross validation. External validation of the prognostic signature in an independent microarray dataset of 59 early-stage triple-negative patients also obtained statistical significance (hazard ratio 2.29, 95% confidence interval (CI) 1.04–5.06, Cox P = 0.04), outperforming five other published breast cancer prognostic signatures. The 45-gene signature identified in this study revealed that TGF-β signaling of immune/inflammatory regulation may play an important role in distant metastatic invasion of triple-negative breast cancer.

Conclusions/Significance

Gene expression data and recurrence information of triple-negative breast cancer were collected and analyzed in this study. A novel set of 45-gene signature was found to be statistically predictive in disease recurrence of triple-negative breast cancer. The 45-gene signature, if further validated, may be a clinically useful tool in risk assessment of distant recurrence for early-stage triple-negative patients.     

Immunohistochemical and Biochemical Analyses of LGI3 in Monkey Brain: LGI3 Accumulates in Aged Monkey Brains

Leucine-rich glioma inactivated (LGI) 3 encodes a leucine-rich repeat protein. The precise function of LGI3, however, remains unknown. We have previously shown that amyloid-β peptide (Aβ) upregulates LGI3 and that Aβ and LGI3 colocalize on plasma membranes of cultured rat astrocytes. In the present study, we performed immunohistochemical and biochemical analyses of LGI3 using various aged monkey brains. Immunohistochemistry showed that LGI3 was present in almost all neural cells and mainly localized at plasma membranes and nuclei. In aged monkey brains, we found that LGI3 accumulated on or near the plasma membranes of neurons, and colocalized with endocytosis-associated proteins and lipid raft markers. Double immunohistochemistry also showed that LGI3 colocalized with Aβ in astrocytes of aged brains. Moreover, Western blot analyses revealed that LGI3 may be cleaved in brain. Additionally, in aged monkeys LGI3 accumulated in microsomal and nuclear brain fractions.     

六株成团肠杆菌 (Enterobacteragglomerans)接合子的分子生物学分析

对 6株成团肠杆菌 (Enterobacteragglomerans)接合子的分子生物学进行了分析 .6株菌与nifHDK基因有杂交 .菌株总DNA经BamHⅠ酶切后与pEA9 DNA进行Southern杂交 ,只有 2株菌具有完整的质粒DNA ,其余菌株质粒DNA发生了 15 3～ 137 7kb不同程度的缺失 .用切割位点较少的限制性内切酶XbaⅠ酶切 6株菌的总DNA ,经脉冲场凝胶电泳 (PFGE)后用pEA9 DNA为探针进行Southern杂交 ,每株菌的pEA9 DNA明显大于用BamHⅠ酶切后的杂交结果 ,表明质粒与染色体发生了整合 .转座子Tn5或插入序列IS 12 2 2和IS 12 71可能参与质粒与染色体的整合过程 .     

Aplysia Ganglia Preparation for Electrophysiological and Molecular Analyses of Single Neurons

A major challenge in neurobiology is to understand the molecular underpinnings of neural circuitry that govern a specific behavior. Once the specific molecular mechanisms are identified, new therapeutic strategies can be developed to treat abnormalities in specific behaviors caused by degenerative diseases or aging of the nervous system. The marine snail Aplysia californica is well suited for the investigations of cellular and molecular basis of behavior because neural circuitry underlying a specific behavior could be easily determined and the individual components of the circuitry could be easily manipulated. These advantages of Aplysia have led to several fundamental discoveries of neurobiology of learning and memory. Here we describe a preparation of the Aplysia nervous system for the electrophysiological and molecular analyses of individual neurons. Briefly, ganglion dissected from the nervous system is exposed to protease to remove the ganglion sheath such that neurons are exposed but retain neuronal activity as in the intact animal. This preparation is used to carry out electrophysiological measurements of single or multiple neurons. Importantly, following the recording using a simple methodology, the neurons could be isolated directly from the ganglia for gene expression analysis. These protocols were used to carry out simultaneous electrophysiological recordings from L7 and R15 neurons, study their response to acetylcholine and quantitating expression of CREB1 gene in isolated single L7, L11, R15, and R2 neurons of Aplysia.     

Genetical and Molecular Analyses of qa-2 Transformants in NEUROSPORA CRASSA

Neurospora crassa qa-2+ transformants from five different donor DNA clones were analyzed by genetical and molecular techniques. None of the 32 transformants have the qa-2+ DNA replacing the qa-2- gene in linkage group VII. In one transformant, the qa-2+ DNA was inserted adjacent to the qa-2- gene. Thirty-one transformants have the qa-2+ inserts at sites not linked, or not closely linked, to the qa-2 locus in LG VII. Plasmid sequences were integrated along with the qa-2+ gene in 28 transformants. In the unlinked duplication-type transformants, catabolic dehydroquinase (the qa-2+ gene product) was induced at 5-100% of the wild-type-induced enzyme activity, with 24 transformants in the 5-80% range. The reduced levels of enzyme activity may be due to "position effects" of sequences adjacent to the integration site either in the N. crassa genomic DNA or in the flanking plasmid (pBR322 or pBR325) sequences. Unexpected gene conversion-like events, in which a qa-2+ gene was changed to qa-2-, were observed in tetrads from intercrosses between unlinked duplication-type transformants and in selfings of such transformants.     

Canine Spontaneous Head and Neck Squamous Cell Carcinomas Represent Their Human Counterparts at the Molecular Level

Spontaneous canine head and neck squamous cell carcinoma (HNSCC) represents an excellent model of human HNSCC but is greatly understudied. To better understand and utilize this valuable resource, we performed a pilot study that represents its first genome-wide characterization by investigating 12 canine HNSCC cases, of which 9 are oral, via high density array comparative genomic hybridization and RNA-seq. The analyses reveal that these canine cancers recapitulate many molecular features of human HNSCC. These include analogous genomic copy number abnormality landscapes and sequence mutation patterns, recurrent alteration of known HNSCC genes and pathways (e.g., cell cycle, PI3K/AKT signaling), and comparably extensive heterogeneity. Amplification or overexpression of protein kinase genes, matrix metalloproteinase genes, and epithelial–mesenchymal transition genes TWIST1 and SNAI1 are also prominent in these canine tumors. This pilot study, along with a rapidly growing body of literature on canine cancer, reemphasizes the potential value of spontaneous canine cancers in HNSCC basic and translational research.