Mapping antigenic epitopes of potato virus Y with antibodies affinity-purified by using overlapping synthetic peptides

Synthetic, overlapping peptides representing the entire amino acid sequence of potato virus Y (PVY) coat protein were used to affinity-purify antibodies from polyclonal antisera to PVY. In testing the binding of the purified antibodies to PVY particles, antigenic epitopes were identified. The N-terminal and C-terminal regions of the PVY coat protein were found to contain most of the antigenic epitopes. The results will facilitate the development of detection methods for PVY based on synthetic peptides

Hyaluronan synthases (HAS1-3) and hyaluronidases (HYAL1-2) in the accumulation of hyaluronan in endometrioid endometrial carcinoma

<p>Abstract</p> <p>Background</p> <p>Hyaluronan accumulation correlates with the degree of malignancy in many solid tumor types, including malignant endometrial carcinomas. To elucidate the mechanism of hyaluronan accumulation, we examined the expression levels of the hyaluronan synthases (<it>HAS1</it>, <it>HAS2 </it>and <it>HAS3</it>) and hyaluronidases (<it>HYAL1 </it>and <it>HYAL2</it>), and correlated them with hyaluronan content and HAS1-3 immunoreactivity.</p> <p>Methods</p> <p>A total of 35 endometrial tissue biopsies from 35 patients, including proliferative and secretory endometrium (n = 10), post-menopausal proliferative endometrium (n = 5), complex atypical hyperplasia (n = 4), grade 1 (n = 8) and grade 2 + 3 (n = 8) endometrioid adenocarcinomas were divided for gene expression by real-time RT-PCR, and paraffin embedded blocks for hyaluronan and HAS1-3 cytochemistry.</p> <p>Results</p> <p>The mRNA levels of <it>HAS1-3 </it>were not consistently changed, while the immunoreactivity of all HAS proteins was increased in the cancer epithelium. Interestingly, <it>HAS3 </it>mRNA, but not HAS3 immunoreactivity, was increased in post-menopausal endometrium compared to normal endometrium (p = 0.003). The median of <it>HYAL1 </it>mRNA was 10-fold and 15-fold lower in both grade 1 and grade 2+3 endometrioid endometrial cancers, as compared to normal endometrium (p = 0.004-0.006), and post-menopausal endometrium (p = 0.002), respectively. <it>HYAL2 </it>mRNA was also reduced in cancer (p = 0.02) and correlated with <it>HYAL1</it> (r = 0.8, p = 0.0001). There was an inverse correlation between <it>HYAL1 </it>mRNA and the epithelial hyaluronan staining intensity (r = -0.6; P = 0.001).</p> <p>Conclusion</p> <p>The results indicated that <it>HYAL1 </it>and <it>HYAL2 </it>were coexpressed and significantly downregulated in endometrioid endometrial cancer and correlated with the accumulation of hyaluronan. While immunoreactivity for HASs increased in the cancer cells, tumor mRNA levels for <it>HAS</it>s were not changed, suggesting that reduced turnover of HAS protein may also have contributed to the accumulation of hyaluronan.</p

Expression of Hyaluronan Synthases (HAS1–3) and Hyaluronidases (HYAL1–2) in Serous Ovarian Carcinomas: Inverse Correlation between HYAL1 and Hyaluronan Content

<p>Abstract</p> <p>Background</p> <p>Hyaluronan, a tumor promoting extracellular matrix polysaccharide, is elevated in malignant epithelial ovarian tumors, and associates with an unfavorable prognosis. To explore possible contributors to the accumulation of hyaluronan, we examined the expression of hyaluronan synthases (<it>HAS1</it>, <it>HAS2 </it>and <it>HAS3</it>) and hyaluronidases (<it>HYAL1 </it>and <it>HYAL2</it>), correlated with hyaluronidase enzyme activity hyaluronan content and HAS1–3 immunoreactivity.</p> <p>Methods</p> <p>Normal ovaries (n = 5) and 34 serous epithelial ovarian tumors, divided into 4 groups: malignant grades 1+2 (n = 10); malignant grade 3 (n = 10); borderline (n = 4) and benign epithelial tumors (n = 10), were analyzed for mRNA by real-time RT-PCR and compared to hyaluronidase activity, hyaluronan staining, and HAS1–3 immunoreactivity in tissue sections of the same specimens.</p> <p>Results</p> <p>The levels of <it>HAS2 </it>and <it>HAS3 </it>mRNA (<it>HAS1 </it>was low or absent), were not consistently increased in the carcinomas, and were not significantly correlated with HAS protein or hyaluronan accumulation in individual samples. Instead, the median of <it>HYAL1 </it>mRNA level was 69% lower in grade 3 serous ovarian cancers compared to normal ovaries (P = 0.01). The expression of <it>HYAL1</it>, but not <it>HYAL2</it>, significantly correlated with the enzymatic activity of tissue hyaluronidases (r = 0.5; P = 0.006). An inverse correlation was noted between <it>HYAL1 </it>mRNA and the intensity of hyaluronan staining of the corresponding tissue sections (r = -0.4; P = 0.025).</p> <p>Conclusion</p> <p>The results indicate that in serous epithelial ovarian malignancies <it>HAS </it>expression is not consistently elevated but <it>HYAL1 </it>expression is significantly reduced and correlates with the accumulation of hyaluronan. (233 words)</p

Twist and snai1 expression in pharyngeal squamous cell carcinoma stroma is related to cancer progression

<p>Abstract</p> <p>Background</p> <p>Epithelial-mesenchymal transition (EMT) is a crucial process in tumorigenesis since tumor cells attain fibroblast-like features enabling them to invade to surrounding tissue. Two transcription factors, <it>TWIST </it>and <it>SNAI1</it>, are fundamental in regulating EMT.</p> <p>Methods</p> <p>Immunohistochemistry was used to study the expression of TWIST and SNAI1 in 109 pharyngeal squamous cell carcinomas.</p> <p>Results</p> <p>Tumors with intense stromal staining of TWIST relapsed more frequently (p = 0.04). Tumors with both positive TWIST and SNAI1 immunoreactivity in the stroma were at least Stage II (p = 0.05) and located more often in hypopharynx (p = 0.035). Tumors with negative immunostaining of TWIST and SNAI1 in the stromal compartment were smaller (T1-2) (p = 0.008), less advanced (SI-II) (p = 0.031) and located more often in the oropharynx (p = 0.007). Patients with negative SNAI1 and TWIST immunostaining in tumor stroma had a better 5-year disease-specific and overall survival (p = 0.037 and p = 0.014 respectively).</p> <p>Conclusion</p> <p>TWIST and SNAI1 expression in stromal cells is associated with clinical and histopathological characteristics that indicate progressive disease. Negative expression of these EMT-promoting transcription factors predicts a better outcome.</p

Performance of automated scoring of ER, PR, HER2, CK5/6 and EGFR in breast cancer tissue microarrays in the Breast Cancer Association Consortium

Breast cancer risk factors and clinical outcomes vary by tumour marker expression. However, individual studies often lack the power required to assess these relationships, and large-scale analyses are limited by the need for high throughput, standardized scoring methods. To address these limitations, we assessed whether automated image analysis of immunohistochemically stained tissue microarrays can permit rapid, standardized scoring of tumour markers from multiple studies. Tissue microarray sections prepared in nine studies containing 20 263 cores from 8267 breast cancers stained for two nuclear (oestrogen receptor, progesterone receptor), two membranous (human epidermal growth factor receptor 2 and epidermal growth factor receptor) and one cytoplasmic (cytokeratin 5/6) marker were scanned as digital images. Automated algorithms were used to score markers in tumour cells using the Ariol system. We compared automated scores against visual reads, and their associations with breast cancer survival. Approximately 65–70% of tissue microarray cores were satisfactory for scoring. Among satisfactory cores, agreement between dichotomous automated and visual scores was highest for oestrogen receptor (Kappa = 0.76), followed by human epidermal growth factor receptor 2 (Kappa = 0.69) and progesterone receptor (Kappa = 0.67). Automated quantitative scores for these markers were associated with hazard ratios for breast cancer mortality in a dose-response manner. Considering visual scores of epidermal growth factor receptor or cytokeratin 5/6 as the reference, automated scoring achieved excellent negative predictive value (96–98%), but yielded many false positives (positive predictive value = 30–32%). For all markers, we observed substantial heterogeneity in automated scoring performance across tissue microarrays. Automated analysis is a potentially useful tool for large-scale, quantitative scoring of immunohistochemically stained tissue microarrays available in consortia. However, continued optimization, rigorous marker-specific quality control measures and standardization of tissue microarray designs, staining and scoring protocols is needed to enhance results.Peer reviewe

E-cadherin breast tumor expression, risk factors and survival : Pooled analysis of 5,933 cases from 12 studies in the Breast Cancer Association Consortium

E-cadherin (CDH1) is a putative tumor suppressor gene implicated in breast carcinogenesis. Yet, whether risk factors or survival differ by E-cadherin tumor expression is unclear. We evaluated E-cadherin tumor immunohistochemistry expression using tissue microarrays of 5,933 female invasive breast cancers from 12 studies from the Breast Cancer Consortium. H-scores were calculated and case-case odds ratios (OR) and 95% confidence intervals (CIs) were estimated using logistic regression. Survival analyses were performed using Cox regression models. All analyses were stratified by estrogen receptor (ER) status and histologic subtype. E-cadherin low cases (N = 1191, 20%) were more frequently of lobular histology, low grade, > 2 cm, and HER2-negative. Loss of E-cadherin expression (score <100) was associated with menopausal hormone use among ER-positive tumors (ever compared to never users, OR = 1.24, 95% CI = 0.97-1.59), which was stronger when we evaluated complete loss of E-cadherin (i.e. H-score = 0), OR = 1.57, 95% CI = 1.06-2.33. Breast cancer specific mortality was unrelated to E-cadherin expression in multivariable models. E-cadherin low expression is associated with lobular histology, tumor characteristics and menopausal hormone use, with no evidence of an association with breast cancer specific survival. These data support loss of E-cadherin expression as an important marker of tumor subtypes.Peer reviewe

Transcription factors zeb1, twist and snai1 in breast carcinoma

Peer reviewe

Mapping antigenic epitopes of potato virus Y with antibodies affinity-purified by using overlapping synthetic peptides

Synthetic, overlapping peptides representing the entire amino acid sequence of potato virus Y (PVY) coat protein were used to affinity-purify antibodies from polyclonal antisera to PVY. In testing the binding of the purified antibodies to PVY particles, antigenic epitopes were identified. The N-terminal and C-terminal regions of the PVY coat protein were found to contain most of the antigenic epitopes. The results will facilitate the development of detection methods for PVY based on synthetic peptides