Study of gene expression in thyrotropin-stimulated thyroid cells by cDNA expression array: ID3 transcription modulating factor as an early response protein and tumor marker in thyroid carcinomas.

Induction of cell proliferation by mitogen or growth factor stimulation leads to the specific induction or repression of a large number of genes. To identify genes differentially regulated by the cAMP-dependent transduction pathway, which is poorly characterized so far, we used the cDNA expression array technology. Hybridizations of Atlas human cDNA expression arrays with (32)P-labeled cDNA probes derived from control or thyrotropin (TSH)-stimulated dog thyrocytes in primary culture generated expression profiles of hundreds of genes simultaneously. Among the genes that displayed modified expression, we selected the transcription factor ID3, whose expression was increased by a cAMP-dependent stimulus. ID3 overexpression after TSH stimulation was first verified by Northern blotting analysis, and its mRNA regulation was then investigated in response to a variety of agents acting on thyrocyte proliferation and/or differentiation. We show that: (1) ID3 mRNA induction was stronger after stimulation of the cAMP cascade, but was not restricted to this signaling pathway, as phorbol myristate ester (TPA) and insulin also stimulated mRNA accumulation; (2) in contrast, powerful mitogens for thyroid cells, epidermal growth factor and hepatocyte growth factor, did not significantly modify ID3 mRNA levels; (3) ID3 protein levels closely parallelled mRNA levels, as revealed by immunofluorescence experiments showing a nuclear signal regulated by TSH; (4) in papillary thyroid carcinomas, ID3 mRNA was downregulated. Our results suggest that ID3 expression might be more related to the differentiating process induced by TSH than to the proliferative action of this hormone.

PIM-1 kinase expression in adipocytic neoplasms: diagnostic and biological implications

The differential diagnosis of soft tissue tumours poses a considerable challenge for pathologists, especially adipocytic tumours, as these may show considerable overlap in clinical presentation and morphological features with many other mesenchymal neoplasms. Hence, a specific and reliable marker that identifies adipocytic differentiation is much sought. We investigated the immunohistochemical expression of PIM-1 kinase in 35 samples of soft tissue tumours using tissue microarray technology and 49 full sections of adipocytic (n = 26) and non-adipocytic tumours (n = 23). Benign and malignant adipocytic tumours showed strong expression of PIM-1 while the non-adipocytic tumours were either negative or showed only weak staining for the protein. In myxoid liposarcomas, PIM-1 showed a distinct, unique vacuolar staining pattern, clearly outlining fine cytoplasmic lipid vacuoles. By contrast, non-adipocytic myxoid tumours (myxoma, chordoma and myxoid chondrosarcoma) did not show this vacuolar pattern of PIM-1 staining, although vacuolated cells were present on H&E. This differential expression was confirmed at a gene expression level in selected cases. Our results indicate that the expression of PIM-1 in adipose tissue may be a useful marker of adipocytic differentiation, in particular if the staining is both of high intensity and present in a unique, vacuolar pattern.

Discordant quantitative detection of putative biomarkers in nodal micrometastases of colorectal cancer: biological and clinical implications

Aims: Nodal expression of the carcinoembryonic antigen ( CEA), cytokeratin 20 ( CK20), and guanylyl cyclase C ( GCC) genes was measured in tandem in patients with colorectal cancer ( CRC) to assess whether there would be sufficient agreement between these markers in their ability to detect micrometastasis to qualify one of them as a universal marker, and whether frozen and paraffin wax embedded tissues would yield similar results.

JMJD6 is a driver of cellular proliferation and motility and a marker of poor prognosis in breast cancer

We developed an analytic strategy that correlates gene expression and clinical outcomes as a means to identify novel candidate oncogenes operative in breast cancer. This analysis, followed by functional characterization, resulted in the identification of Jumonji Domain Containing 6 (JMJD6) protein as a novel driver of oncogenic properties in breast cancer.

Proteomic analysis of colorectal cancer metastasis:stathmin-1 revealed as a player in cancer cell migration and prognostic marker

Metastasis accounts largely for the high mortality rate of colorectal cancer (CRC) patients. In this study, we performed comparative proteome analysis of primary CRC cell lines HCT-116 and its metastatic derivative E1 using 2-D DIGE. We identified 74 differentially expressed proteins, many of which function in transcription, translation, angiogenesis signal transduction, or cytoskeletal remodeling pathways, which are indispensable cellular processes involved in the metastatic cascade. Among these proteins, stathmin-1 (STMN1) was found to be highly up-regulated in E1 as compared to HCT-116 and was thus selected for further functional studies. Our results showed that perturbations in STMN1 levels resulted in significant changes in cell migration, invasion, adhesion, and colony formation. We further showed that the differential expression of STMN1 correlated with the cells' metastatic potential in other paradigms of CRC models. Using immunohistochemistry, we also showed that STMN1 was highly expressed in colorectal primary tumors and metastatic tissues as compared to the adjacent normal colorectal tissues. Furthermore, we also showed via tissue microarray analyses of 324 CRC tissues and Kaplan-Meier survival plot that CRC patients with higher expression of STMN1 have poorer prognosis.

CD44v8-10 Is a Cancer-Specific Marker for Gastric Cancer Stem Cells

The surface marker CD44 has been identified as one of several markers associated with cancer stem cells (CSC) in solid tumors, but its ubiquitous expression in many cell types, including hematopoietic cells, has hindered its use in targeting CSCs. In this study, 28 paired primary tumor and adjacent nontumor gastric tissue samples were analyzed for cell surface protein expression. Cells that expressed pan-CD44 were found to occur at significantly higher frequency in gastric tumor tissues. We identified CD44v8-10 as the predominant CD44 variant expressed in gastric cancer cells and verified its role as a gastric CSC marker by limiting dilution and serial transplantation assays. Parallel experiments using CD133 failed to enrich for gastric CSCs. Analyses of another 26 primary samples showed significant CD44v8-10 upregulation in gastric tumor sites. Exogenous expression of CD44v8-10 but not CD44 standard (CD44s) increased the frequency of tumor initiation in immunocompromised mice. Reciprocal silencing of total CD44 resulted in reduced tumor-initiating potential of gastric cancer cells that could be rescued by CD44v8-10 but not CD44s expression. Our findings provide important functional evidence that CD44v8-10 marks human gastric CSCs and contributes to tumor initiation, possibly through enhancing oxidative stress defense. In addition, we showed that CD44v8-10 expression is low in normal tissues. Because CD44 also marks CSCs of numerous human cancers, many of which may also overexpress CD44v8-10, CD44v8-10 may provide an avenue to target CSCs in other human cancers.

CD38 expression level and pattern of expression remains a reliable and robust marker of progressive disease in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) follows a variable clinical course which is difficult to predict at diagnosis. We assessed somatic mutation (SHM) status, CD38 and ZAP-70 expression in 87 patients (49 male, 38 female) with stage A CLL and known cytogenetic profile to compare their role in predicting disease progression, which was assessed by the treatment free interval (TFI) from diagnosis. Sixty (69%) patients were SHM+, 24 (28%) were CD38+ and ten (12%) were ZAP-70+. The median TFI for: (i) SHM + versus SHM- patients was 124 versus 26 months; hazard ratio (HR) = 3.6 [95% confidence interval (CI) = 1.8 - 7.3; P = 0.001]: (ii) CD38- versus CD38+ patients was 120 versus 34 months; HR = 2.4 (95% CI = 1.4 - 5.3; P = 0.02); and (iii) ZAP70- versus ZAP70+ was 120 versus 16 months; HR = 3.4 (95% CI = 1.4 - 8.7; P = 0.01). SHM status and CD38 retained prognostic significance on multivariate analysis whereas ZAP-70 did not. We conclude that ZAP-70 analysis does not provide additional prognostic information in this group of patients.

Flippase (FLP) recombinase-mediated marker recycling in the dermatophyte Arthroderma vanbreuseghemii.

Biological processes can be elucidated by investigating complex networks of relevant factors and genes. However, this is not possible in species for which dominant selectable markers for genetic studies are unavailable. To overcome the limitation in selectable markers for the dermatophyte Arthroderma vanbreuseghemii (anamorph: Trichophyton mentagrophytes), we adapted the flippase (FLP) recombinase-recombination target (FRT) site-specific recombination system from the yeast Saccharomyces cerevisiae as a selectable marker recycling system for this fungus. Taking into account practical applicability, we designed FLP/FRT modules carrying two FRT sequences as well as the flp gene adapted to the pathogenic yeast Candida albicans (caflp) or a synthetic codon-optimized flp (avflp) gene with neomycin resistance (nptII) cassette for one-step marker excision. Both flp genes were under control of the Trichophyton rubrum copper-repressible promoter (PCTR4). Molecular analyses of resultant transformants showed that only the avflp-harbouring module was functional in A. vanbreuseghemii. Applying this system, we successfully produced the Ku80 recessive mutant strain devoid of any selectable markers. This strain was subsequently used as the recipient for sequential multiple disruptions of secreted metalloprotease (fungalysin) (MEP) or serine protease (SUB) genes, producing mutant strains with double MEP or triple SUB gene deletions. These results confirmed the feasibility of this system for broad-scale genetic manipulation of dermatophytes, advancing our understanding of functions and networks of individual genes in these fungi.

Germin, a protein marker of early plant development, is an oxalate oxidase

Germin is a homopentameric glycoprotein, the synthesis of which coincides with the onset of growth in germinating wheat embryos. There have been detailed studies of germin structure, biosynthesis, homology with other proteins, and of its value as a marker of wheat development. Germin isoforms associated with the apoplast have been speculated to have a role in embryo hydration during maturation and germination. Antigenically related isoforms of germin are present during germination in all of the economically important cereals studied, and the amounts of germin-like proteins and coding elements have been found to undergo conspicuous change when salt-tolerant higher plants are subjected to salt stress. In this report, we describe how circumstantial evidence arising from unrelated studies of barley oxalate oxidase and its coding elements have led to definitive evidence that the germin isoform made during wheat germination is an oxalate oxidase. Establishment of links between oxalate degradation, cereal germination, and salt tolerance has significant implications for a broad range of studies related to development and adaptation in higher plants. Roles for germin in cell wall biochemistry and tissue remodeling are discussed, with special emphasis on the generation of hydrogen peroxide during germin-induced oxidation of oxalate.

Is television viewing time a marker of a broader pattern of sedentary behavior?

Background: Television (TV) viewing time is associated with abnormal glucose metabolism, the metabolic syndrome, and risk of type 2 diabetes; associations are stronger and more consistent in women. One explanation of this difference may be that TV viewing is a marker of an overall pattern of sedentary behavior in women.

Purpose: We sought to examine associations of TV viewing time with other sedentary behaviors and with leisure-time physical activity in a large sample of Australian adults.

Methods: Adults aged between 20 and 65 years (n=2,046) completed a  self-administered questionnaire on TV viewing, five other leisure-time sedentary behaviors, and leisure-time physical activity. Mean adjusted time spent in other sedentary behaviors and in physical activity was compared across TV-time  categories previously shown to be associated with abnormal glucose  metabolism.

Results: After adjustment for body mass index and sociodemographic variables, women’s time spent watching TV was associated positively with time in other sedentary behaviors and negatively with leisure-time physical activity, but no such associations were observed in men.

Conclusions: TV viewing time may be a robust marker of a sedentary lifestyle in women but not in men. Gender differences in the pattern of sedentary behaviors may explain at least in part the gender differences in the previously reported associations of TV viewing time with biological attributes related to type 2   diabetes.

Large-scale transcriptome analyses reveal new genetic marker candidates of head, neck, and thyroid cancer

A detailed genome mapping analysis of 213,636 expressed sequence tags (EST) derived from nontumor and tumor tissues of the oral cavity, larynx, pharynx, and thyroid was done. Transcripts matching known human genes were identified; potential new splice variants were flagged and subjected to manual curation, pointing to 788 putatively new alternative splicing isoforms, the majority (75%) being insertion events. A subset of 34 new splicing isoforms (5% of 788 events) was selected and 23 (68%) were confirmed by reverse transcription-PCR and DNA sequencing. Putative new genes were revealed, including six transcripts mapped to well-studied chromosomes such as 22, as well as transcripts that mapped to 253 intergenic regions. In addition, 2,251 noncoding intronic RNAs, eventually involved in transcriptional regulation, were found. A set of 250 candidate markers for loss of heterozygosis or gene amplification was selected by identifying transcripts that mapped to genomic regions previously known to be frequently amplified or deleted in head, neck, and thyroid tumors. Three of these markers were evaluated by quantitative reverse transcription-PCR in an independent set of individual samples. Along with detailed clinical data about tumor origin, the information reported here is now publicly available on a dedicated Web site as a resource for further biological investigation. This first in silico reconstruction of the head, neck, and thyroid transcriptomes points to a wealth of new candidate markers that can be used for future studies on the molecular basis of these tumors. Similar analysis is warranted for a number of other tumors for which large EST data sets are available.

Immunocytochemical study of Ki-67 as a prognostic marker in canine mammary neoplasia

Background: Canine mammary tumors are challenging for clinicians and pathologists because of complex histologic classification, low specificity of cytologic diagnosis, and unpredictable biological behavior. In histologic specimens, expression of tumor proliferation marker Ki-67, a nuclear nonhistone protein, has been shown to have prognostic value for canine mammary tumors and to correlate with malignancy and low survival rates. Objective: The objective of this study was to measure the proliferation index of canine mammary tumors by immunochemical detection of Ki-67 in cytologic specimens and to determine its relationship to clinical and pathologic variables and patient outcome. Methods: Spontaneous mammary tumors from 31 female dogs were surgically excised. Imprint specimens for cytologic evaluation were wet-fixed in ethanol; histologic specimens were prepared routinely. Immunostaining was performed with the PH 177 monoclonal antibody against Ki-67; proliferation index was graded from negative to +++. Dogs were followed for 18 months. Multivariate logistic regression analysis was used to determine correlations between immunocytochemical results, tumor and clinical variables, and patient outcome. Results: Ki-67 proliferation indices in cytologic specimens were significantly lower for nonmalignant tumors than for malignant tumors. High index values of Ki-67 were positively correlated with metastasis, death from neoplasia, low disease-free survival rates, and low overall survival rate. With the exception of 4 specimens for which cellularity was insufficient, positive expression of Ki-67 in cytologic specimens correlated with that of histologic specimens. Conclusions: The prognostic value of the Ki-67 index in canine mammary tumors by using wet-fixed cytology imprint specimens was similar to that observed previously for histologic specimens. Immunocytochemical detection of Ki-67 could improve the accuracy and value of cytology by providing safe and rapid information about malignancy and patient outcome. © 2004 American Society for Veterinary Clinical Pathology.

Homeobox gene expression profile indicates HOXA5 as a candidate prognostic marker in oral squamous cell carcinoma

The search for molecular markers to improve diagnosis, individualize treatment and predict behavior of tumors has been the focus of several studies. This study aimed to analyze homeobox gene expression profile in oral squamous cell carcinoma (OSCC) as well as to investigate whether some of these genes are relevant molecular markers of prognosis and/or tumor aggressiveness. Homeobox gene expression levels were assessed by microarrays and qRT-PCR in OSCC tissues and adjacent non-cancerous matched tissues (margin), as well as in OSCC cell lines. Analysis of microarray data revealed the expression of 147 homeobox genes, including one set of six at least 2-fold up-regulated, and another set of 34 at least 2-fold down-regulated homeobox genes in OSCC. After qRT-PCR assays, the three most up-regulated homeobox genes (HOXA5, HOXD10 and HOXD11) revealed higher and statistically significant expression levels in OSCC samples when compared to margins. Patients presenting lower expression of HOXA5 had poorer prognosis compared to those with higher expression (P=0.03). Additionally, the status of HOXA5, HOXD10 and HOXD11 expression levels in OSCC cell lines also showed a significant up-regulation when compared to normal oral keratinocytes. Results confirm the presence of three significantly upregulated (>4-fold) homeobox genes (HOXA5, HOXD10 and HOXD11) in OSCC that may play a significant role in the pathogenesis of these tumors. Moreover, since lower levels of HOXA5 predict poor prognosis, this gene may be a novel candidate for development of therapeutic strategies in OSCC.

STEAP1 protein overexpression is an independent marker for biochemical recurrence in prostate carcinoma

Aims: To investigate the prognostic value of expression levels of the genes STEAP1 and STEAP2, and of STEAP1 protein, in prostate carcinomas (PCa). Methods and results: STEAP1 and STEAP2 transcript levels were evaluated by RT-qPCR in samples from 35 PCa, 24 adjacent non-neoplastic prostate (AdjP) tissues, five cases of benign prostatic hyperplasia (BPH), and two histologically normal prostates (N). STEAP1 expression was assessed by immunohistochemistry in samples from 198 PCa, 76 AdjP, 22 BPH, and two N. The findings were compared with clinical and pathological parameters and patient outcome. STEAP1 and STEAP2 transcript analysis showed no differences between the groups tested. Although not significant, higher STEAP1 mRNA levels were detected in tumours with high Gleason scores and in patients who presented with biochemical recurrence (BCR). STEAP1 overexpression was detected in PCa, and was significantly associated with high-grade Gleason scores, seminal vesicle invasion, BCR, and worse outcome (metastasis or PCa-specific death). STEAP1 overexpression was significantly associated with shorter BCR-free survival. Multivariate analysis revealed that STEAP1 is an independent marker for BCR. Conclusions: These findings provide evidence that STEAP1 is a biomarker of worse prognosis in PCa patients. © 2013 John Wiley & Sons Ltd.