Kenneth R. Shroyer

The Transcription Factor ZEB1 Is Aberrantly Expressed in Aggressive Uterine Cancers

The transcription factor ZEB1 (deltaEF1 in mice) has been implicated in cellular processes during development and tumor progression including epithelial to mesenchymal transition. deltaEF1 null mice die at birth, but heterozygotes expressing a LacZ reporter inserted into the deltaEF1 gene live and reproduce. Using these mice, we observed ZEB1 promoter activity in the virgin myometrium, and stroma and myometrium of the pregnant uterus. ZEB1 protein is up-regulated in the myometrium and endometrial stroma after progesterone or estrogen treatment of ovariectomized mice. In the normal human uterus, ZEB1 protein is increased in the myometrium and stroma during the secretory stage of the menstrual cycle. ZEB1 is not expressed in the normal endometrial epithelium. In malignancies of the uterus, we find that ZEB1 (a) is overexpressed in malignant tumors derived from the myometrium (leiomyosarcomas), (b) is overexpressed in tumor-associated stroma of low-grade endometrioid adenocarcinomas, and (c) is aberrantly expressed in the tumor epithelial cells of aggressive endometrial cancers. Specifically, in grade 3 endometrioid adenocarcinomas and uterine papillary serous carcinomas, ZEB1 could be expressed in the epithelial-derived carcinoma cells as well as in the stroma. In malignant mixed Müllerian tumors, the sarcomatous component always expresses ZEB1, and the carcinomatous component can also be positive. In summary, ZEB1 is normally regulated by both estrogen and progesterone receptors, but in uterine cancers, it is likely no longer under control of steroid hormone receptors and becomes aberrantly expressed in epithelial-derived tumor cells, supporting a role for ZEB1 in epithelial to mesenchymal transitions associated with aggressive tumors.

Quantitative and qualitative differences in protein expression between papillary thyroid carcinoma and normal thyroid tissue

In order to better understand basic mechanisms of tumor development and identify potential new biomarkers, we have performed difference gel electrophoresis (DIGE) and peptide mass fingerprinting on pooled protein extracts from patients with papillary thyroid carcinoma (PTC) compared with matched normal thyroid tissue. Image analysis of DIGE gels comparing PTC and matched normal thyroid tissue protein indicated that 25% of the protein spots were differentially expressed at a 2.5‐fold cutoff and 35% at two‐fold. Comparison between two different pools of protein from normal thyroid tissues revealed differential protein expression of only 4% at 2.5‐fold and 6% at two‐fold cutoff. One hundred ninety‐two protein spots were identified by MALDI‐TOFMS, representing 90 distinct proteins. Excluding albumin, globins and thyroglobulin, imaging software determined 31 proteins to be differentially expressed at the two‐fold (or greater) level. Individual gel comparisons (PTC vs. matched normal) from five patients established that 15/31 (48%) of these proteins exhibited statistically significant differential expression. Previously identified molecular markers in this group of proteins include cathepsin B, cytokeratin 19, and galectin‐3. Novel differentially expressed proteins include S100A6, moesin, HSP70 (BiP), peroxiredoxin 2, protein phosphatase 2, selenium binding protein 1, vitamin D binding protein, and proteins involved in mitochondrial function. The use of two‐dimensional gel electrophoresis (2DGE) revealed a significantly altered protein mass and/or pI in 10%–15% of proteins, suggesting alternatively spliced forms and other posttranslational modification of proteins revealed by this approach. We confirmed S100A6 as a potentially useful biomarker using immunohistochemical analysis (85% sensitivity and 69% specificity for distinguishing benign from malignant thyroid neoplasms). In summary, proteomic analysis of PTC using DIGE and mass spectrometry has confirmed several known biomarkers, uncovered novel potential biomarkers, and provided insights into global pathophysiologic changes in PTC. Many of the differences observed would not have been detected by genomic or other proteomic approaches.

Human papillomavirus testing and molecular markers of cervical dysplasia and carcinoma.

Cervical cancer is the second most common cancer in women worldwide. Human papillomavirus (HPV) is the etiologic agent for the vast majority of premalignant and malignant lesions, and high‐risk HPV types can be detected in almost all cases of cervical dysplasia and carcinoma. HPV testing has been widely adopted for the triage of patients after a cervical cytology screening test (Papanicolaou smear or liquid‐based cervical cytology such as ThinPrep or SurePath) interpretation of atypical squamous cells of undetermined significance (ASCUS), and HPV testing is increasingly used for screening in conjunction with cervical cytology. Although cervical cytology is a highly effective screening test for cancer, it has limited specificity for clinically significant lesions in cases with low‐grade cytologic abnormalities. Up to a quarter of all patients may have a false‐negative result on the basis of cervical cytology testing alone. This review focuses on HPV testing methods and molecular markers and their clinical relevance. HPV testing and surrogate molecular markers of HPV infection (p16INK4a) may help identify cases that are associated with underlying high‐grade premalignant or malignant lesions and may also reduce aggressive treatment of patients with low‐grade lesions. Cancer (Cancer Cytopathol) 2007;.

ZEB1 expression in type I vs type II endometrial cancers: a marker of aggressive disease

Zinc-finger E-box-binding homeobox 1 (ZEB1) is a transcription factor containing two clusters of Kruppel-type zinc-fingers, by which it binds E-box-like sequences on target DNAs. A role for ZEB1 in tumor progression, specifically, epithelial to mesenchymal transitions, has recently been revealed. ZEB1 acts as a master repressor of E-cadherin and other epithelial markers. We previously demonstrated that ZEB1 is confined to the stromal compartment in normal endometrium and low-grade endometrial cancers. Here, we quantify ZEB1 protein expression in endometrial samples from 88 patients and confirm that it is expressed at significantly higher levels in the tumor-associated stroma of low-grade endometrioid adenocarcinomas (type I endometrial cancers) compared to hyperplastic or normal endometrium. In addition, as we previously reported, ZEB1 is aberrantly expressed in the epithelial-derived tumor cells of highly aggressive endometrial cancers, such as FIGO grade 3 endometrioid adenocarcinomas, uterine serous carcinomas, and malignant mixed Müllerian tumors (classified as type II endometrial cancers). We now demonstrate, in both human endometrial cancer specimens and cell lines, that when ZEB1 is inappropriately expressed in epithelial-derived tumor cells, E-cadherin expression is repressed, and that this inverse relationship correlates with increased migratory and invasive potential. Forced expression of ZEB1 in the nonmigratory, low-grade, relatively differentiated Ishikawa cell line renders them migratory. Conversely, reduction of ZEB1 in a highly migratory and aggressive type II cell line, Hec50co, results in reduced migratory capacity. Thus, ZEB1 may be a biomarker of aggressive endometrial cancers at high risk of recurrence. It may help identify women who would most benefit from chemotherapy. Furthermore, if expression of ZEB1 in type II endometrial cancers could be reversed, it might be exploited as therapy for these highly aggressive tumors.

Validation of a novel immunocytochemical assay for topoisomerase II-α and minichromosome maintenance protein 2 expression in cervical cytology

Cervical cytopathology has limited specificity for the detection of underlying clinically significant lesions in cases with low‐grade cytologic abnormalities. The current study evaluated the performance of a novel immunocytochemical test (ProEx C) for topoisomerase II alpha (TOP2A) and minichromosome maintenance protein 2 (MCM2) in normal versus high‐grade squamous intraepithelial lesion (HSIL) and positive control (SiHa) pooled cytology preparations and in a pilot series of prospectively collected patient specimens.

Detection of human papillomavirus DNA by in situ DNA hybridization and polymerase chain reaction in premalignant and malignant oral lesions

The sensitivity of detection of human papillomavirus (HPV) DNA in premalignant and malignant oral lesions by in situ hybridization (ISH) and polymerase chain reaction (PCR) were compared. With both methods HPV DNA was found in 4 of 24 cases of epithelial dysplasia, 4 of 14 cases of verrucous hyperplasia, and 1 of 10 cases of squamous cell carcinoma. The 10 cases of smokeless tobacco keratoses and 3 cases of verrucous carcinoma that we examined were all negative for HPV DNA. The PCR for the E6 open reading frame of HPV-16 correctly identified all cases that were positive by ISH. Only a single case that was positive by PCR was negative by ISH for HPV DNA. However, the PCR demonstrated the presence of HPV-16 infection in one case, which had hybridized most intensely with the probe for types 31/33/35 in the ISH. This discrepancy probably is due to the high degree of cross-hybridization in the ISH assay. PCR appears to be an effective technique for identifying HPV-16 DNA sequences in biopsy material from premalignant and malignant oral lesions.

Estrogen and Progesterone Receptor and p53 Gene Expression in Adenoid Cystic Cancer

Objectives The current study examined the role of estrogen receptors (ER), progesterone receptors (PR) and p53 expression in adenoid cystic carcinoma (ACC) to determine if simple expression or possible overexpression of these products might influence the development and natural course of this cancer. Study Design ER and PR status and p53 overexpression were retrospectively evaluated utilizing immunohistochemical evaluation of 47 ACC specimens. Methods Formalin-fixed paraffin-embedded tissues from 47 ACC specimens and 47 samples of normal salivary gland tissue were evaluated histochemically for the presence of ER, PR and p53. Immunoreactivity was scored using a 0 to +3 scale in which staining was either (0) negative, (+1) spotty, (+2) weakly positive, or (+3) strongly positive. Results ER was expressed in 8 of 47 tumors while PR was expressed in 4 of 47 tumors. p53 aberrations were demonstrated in 26 of 47 tumors. Tumors showed varying degrees of immunopositivity ranging from 0 to +3. Conclusions These studies suggest that p53 aberrations may be involved in ACC tumor progression and that ER and PR may play a role in ACC development.