Novel dual-function CellDetect® staining technology: wedding morphology and tinctorial discrimination to detect cervical neoplasia

<p>Abstract</p> <p>Background</p> <p>A persistent goal of oncologic histochemistry is to microscopically identify neoplasia tinctorially. Consequently, the newly developed CellDetect^®staining technology, that appears to exhibit this property, warrants clinical evaluation. The objective of this study was to compare the diagnostic results using CellDetect^®to the outcomes of standard microscopic examination based on hematoxylin and eosin (H&E) staining for the recognition of different squamous epithelial phenotypes of the uterine cervix.</p> <p>Methods</p> <p>Pairs of adjacent sections were made from 60 cervical biopsy cases that were diagnosed originally as either normal or neoplastic (CIN, SCC). One section of the pair was stained for H&E; the second section, with CellDetect^®. Based on the examination of these pairs by two experienced pathologists, we investigated the following issues:(1) diagnostic agreement between the pathologists on each pair; (2) agreement between H&E and CellDetect^®for each pair (3) tinctorial characteristics in micro-regions (n = 130) evaluated as either normal, reactive or neoplastic.</p> <p>Results</p> <p>Qualitatively, CellDetect^®-stained preparations displayed cyto-morphological detail comparable to H&E images. Tinctorially, <it>non-neoplastic </it>cells appeared green/blue when stained withCellDetect^®, contrasting with cytologically <it>neoplastic </it>foci, where cells of every grade were red/magenta in color. Due to these tinctorial characteristics, even small foci of neoplasia could be readily distinguished that were inconspicuous on H&E at low magnification. In some instances, this prompted re-examination of the H&E and revision of the diagnosis. Quantitatively, we found that despite diagnostic variation between pathologists, in about 3% of the cases, each pathologist made the same diagnosis regardless of whether CellDetect^®or H&E was used, i.e. there was 100% self-agreement for each pathologist between stains. Particularly noteworthy was the finding of a 0% false negative rate, coupled with a 10-15% false positive rate. Regarding specificity, the performance in <it>reactive </it>squamous processes was similar to that observed for morphologically normal squamous epithelium.</p> <p>Conclusions</p> <p>In this first order assessment of clinical applicability, CellDetect^®staining technology was at least comparable to results using H&E, and perhaps surperior. CellDetect^®provided a uniquely useful tinctorial clue for the detection of neoplasia, which exhibited an impressive 0% false negative rate. A more extensive, blinded study is needed to confirm these promising findings.</p

Noninvasive test for the diagnosis of ovarian hormone-secreting-neoplasm in postmenopausal women

Context: The diagnosis of ovarian hormone-secreting neoplasm in postmenopausal women is currently based on imaging modalities and selective venography. However, these diagnostic tests are not always accurate. In order to improve and simplify the diagnosis, we propose a noninvasive hormonal test. Objective: To report our experience using noninvasive hormonal test for the diagnosis of ovarian hormone producing tumor in two postmenopausal women. Design and intervention: Evaluation of androgen and estradiol serum levels following 1. Adrenal hormonal depression, 2. ovarian hormonal depression and 3. ovarian hormonal stimulation. Setting: Tertiary care medical center. Main outcome measures: Changes in androgen and estradiol levels. Results: In the first case, total testosterone, free androgen index and estradiol serum levels decreased following ovarian depression by GnRH-antagonist (6.9 nmol/L, 67 nmol/L and <70 pmol/L, respectively) and subsequently increased after ovarian stimulation with LH (11.5 nmol/L, 117 nmol/L and 176 pmol/L, respectively). Histological evaluation revealed steroid cell tumor in one ovary. In the second case, estradiol serum levels decreased following ovarian depression by GnRH-antagonist (73 pmol/L) and subsequently increased following ovarian stimulation with FSH (118 pmol/L). Histological evaluation revealed granulosa cell tumor in one ovary. Conclusions: To our knowledge, these are the first cases of ovarian hormone-producing tumors in postmenopausal women diagnosed by noninvasive hormonal test. The proposed test can be considered in postmenopausal women suspected of having androgen and/or estrogen producing tumors

The mechanism of action of the histone deacetylase inhibitor vorinostat involves interaction with the insulin-like growth factor signaling pathway.

A correlation between components of the insulin-like growth factor (IGF) system and endometrial cancer risk has been shown in recent studies. The antitumor action of vorinostat, a histone deacetylase inhibitor, involves changes in the expression of specific genes via acetylation of histones and transcription factors. The aim of this study was to establish whether vorinostat can modify the expression of specific genes related to the IGF-I receptor (IGF-IR) signaling pathway and revert the transformed phenotype. Human endometrioid (Type I, Ishikawa) and uterine serous papillary (Type II, USPC-2) endometrial cancer cell lines were treated with vorinostat in the presence or absence of IGF-I. Vorinostat increased IGF-IR phosphorylation, produced acetylation of histone H3, up-regulated pTEN and p21 expression, and reduced p53 and cyclin D1 levels in Ishikawa cells. Vorinostat up-regulated IGF-IR and p21 expression, produced acetylation of histone H3, and down-regulated the expression of total AKT, pTEN and cyclin D1 in USPC-2 cells. Of interest, IGF-IR activation was associated with a major elevation in IGF-IR promoter activity. In addition, vorinostat treatment induced apoptosis in both cell lines and abolished the anti-apoptotic activity of IGF-I both in the absence or presence of a humanized monoclonal IGF-IR antibody, MK-0646. Finally, vorinostat treatment led to a significant decrease in proliferation and colony forming capability in both cell lines. In summary, our studies demonstrate that vorinostat exhibits a potent apoptotic and anti-proliferative effect in both Type I and II endometrial cancer cells, thus suggesting that endometrial cancer may be therapeutically targeted by vorinostat

Insulin-like growth factor-I receptor inhibition by specific tyrosine kinase inhibitor NVP-AEW541 in endometroid and serous papillary endometrial cancer cell lines

Endometrial cancer is the most widespread gynecological cancer in Western countries and constitutes a major public health issue. The role of the insulin-like growth factor (IGF) system in endometrial biology as well as in endometrial cancer has been well established. The IGF-I receptor (IGF-IR) emerged in recent years as a promising therapeutic target in a number of cancers. NVP-AEW541 (Novartis Pharma) is a pyrrolo(2,3-d)pyrimidine derivative with specific IGF-IR tyrosine kinase inhibitory activity. NVP-AEW541 has been shown to specifically abrogate IGF-I-mediated IGF-IR autophosphorylation and to reduce activation of the IGF-IR downstream signaling pathways. The aim of the present study was to investigate the potential anti-proliferative activities of NVP-AEW541 in Type I (endometrioid) and Type II (uterine serous papillary endometrial carcinoma, USPC) endometrial cancer cell lines. Results obtained showed that NVP-AEW541 abolished the IGF-I stimulated IGF-IR phosphorylation in all of the cell lines investigated (ECC-1, Ishikawa, USPC-1, USPC-2), whereas it abolished AKT and ERK phosphorylation in ECC-1 and USPC-1 cells. Furthermore, the inhibitor prevented from IGF-I from exerting its antiapoptotic effect in ECC-1, USPC-1 and USPC-2 cells. In addition, proliferation assays showed that NVP-AEW541 caused a significant decrease in proliferation rate in all of the cell lines. NVP-AEW541 had no major effect on the insulin receptor. In summary, our results suggest that specific IGF-IR inhibition by NVP-AEW541 is a promising therapeutic tool in endometrial cancer

Effect of vorinostat on IGF-I-mediated signal transduction.

<p>Ishikawa and USPC-2 cells were treated for 24 h with vorinostat (or left untreated) and/or IGF-I during the last 10 min of the incubation period. Whole-cell lysates (100 µg) were resolved on SDS-PAGE and immunoblotted with antibodies against pIGF-IR, TIGF-IR, pAKT, TAKT, pERK1/2, TERK1/2, p21, and actin. The figure shows the result of a typical experiment, repeated three times with similar results. IGF-IR phosphorylation in USPC-2 cells was significantly lower than in Ishikawa cells and was detected by Western blot analysis only after longer exposure times of blots to X-ray film. Therefore, this experiment does not allow comparison between both cell lines in terms of levels of expression or phosphorylation.</p

Regulation of IGF-IR promoter activity by vorinostat in endometrial cancer cells.

<p>Ishikawa and USPC-2 cells were transiently transfected with an IGF-IR promoter-luciferase reporter plasmid, p(−476/+640)LUC, which contains most of the proximal region of the IGF-IR promoter, and a ß-galactosidase vector. Promoter activity is expressed as luciferase values normalized for ß-galactosidase. A value of 100% was given to the promoter activity in the absence of vorinostat. Results are mean ± S.E.M. (three independent experiments); p*<0.01 <i>versus</i> untreated cells.</p

Effect of vorinostat on endometrial cancer cells proliferation.

<p>Cells were plated in 24-well plates at a density of 2×10⁴ cells/well for Ishikawa (A) and 3×10⁴ cells/well for USPC-2 (B). The bars represent the mean ± S.E.M. of three independent experiments, performed each in triplicate; * p<0.05 <i>versus</i> untreated cells.</p

Effect of vorinostat on the mTor and Ampk signaling pathway in endometrial cancer cells.

<p>Ishikawa and USPC-2 cells were treated with vorinostat and/or IGF-I, and cell lysates (100 µg) were prepared after 24 h. Western blot analysis was performed with antibodies against pmTor, T-mTor, p-Ampk, T-Ampk, p21, cyclin D1, PI3K and actin. The figure shows the results of a typical experiment, repeated three times with similar results.</p

Effect of vorinostat on apoptosis and acetyl-histone H3.

<p>Western blot analysis of PARP (A) on Ishikawa and USPC-2 cells and caspase 3 (B), Bcl2 (C) and caspase 8 (D) on USPC-2 cells. Cells were treated with vorinostat for 24 h in the absence or presence of IGF-I. After incubation, cell lysates were prepared and electrophoresed (100 µg for Bcl2 or 70 µg for caspases 3 and 8). Caspase 3 expression in USPC-2 cells was detected after a long exposure time of blots to X-ray film. Results are representative of three independent experiments.</p