Discordant genetic changes in ovarian and endometrial endometrioid carcinomas: a potential pitfall in molecular diagnosis

Endometrioid carcinoma simultaneously involving ovaries as well as the uterine corpus may present a diagnostic dilemma because of the difficulty in determining whether the lesions are separate primary tumors or metastases. It has been reported that the detection of clonality using microsatellite markers may be useful in solving this dilemma. To determine the usefulness of this technique, we compared the genetic alterations in microsatellite markers present in matched pairs of ovarian tumors from 12 patients. The study includes four ovarian cancer FIGO stage I and eight stage III/IV patients, and four patients also with independent endometrial carcinoma of the uterus. DNA from paraffin-embedded tissue was extracted and amplified using a multiplex polymerase chain reaction, after which the status of microsatellite instability and loss of heterozygosity in four microsatellite loci (BAT25, BAT26, D17S250, and D5S346) were determined. In the four patients with stage I ovarian cancer, four microsatellite markers were identical in one patient and three were identical in the remaining three patients. In high-stage patients, three markers were identical in at least 4/8 cases. In three of four patients with uterine involvement, three of the four markers were identical in the uterine tumor and one of the corresponding ovarian tumors. These results suggest that genetic discordance does not indicate independent origin or metastasis of the tumor but instead a progression of genetic changes at separate sites probably due to the marked genetic instability existing in these tumors. Because of these discordant genetic changes, great caution should be taken when distinguishing between primary and metastatic tumors on the basis of microsatellite markers.     

Expression of inhibitory natural killer receptors on tumor-infiltrating CD8+ T lymphocyte lineage in human endometrial carcinoma

To investigate the expression of natural killer receptors (NKRs) within the human tumor milieu, we directly examined the in vivo expressions of various NKRs on tumor-infiltrating lymphocytes (TILs) derived from human endometrial carcinoma (EC). In total, 22 patients with stage IA-IIIA EC were enrolled. TILs were isolated from tissue specimens by means of a mechanical dispersal technique. The subpopulations of immunocytes were quantified, and expressions of NKRs on CD8+ T cells were analyzed by triple-color flow cytometry. CD8+ T cells express higher ratios of CD94 and NKG2A in TILs than in peripheral blood mononuclear cells (PBMCs) in human EC. Flow cytometry reveals that 15.90% of CD3+CD8+ TILs compared with 2.10% of CD3+CD8+ PBMCs express the NKG2A molecules (P < 0.001). The percentage expressions of CD94 are 8.40% in CD3+CD8+ TILs and 3.80% in CD3+CD8+ PBMCs (P= 0.013). The numbers of CD8+ T cells expressing CD158b and NKB1 are higher in CD3+CD8+ PBMCs in EC than in normal (CD158b: 10.70% vs 2.60%, P < 0.001; NKB1: 2.20% vs 0.40%, P= 0.018, respectively). Increased expression of CD94/NKG2A restricted to tumor-infiltrating CD8+ T cell subsets may shape the cytotoxic responses, which indicate a possible role of tumor escape from host immunity in human EC.     

The detection of microsatellite instability in blind endometrial samples—a potential novel screening tool for endometrial cancer in women from hereditary nonpolyposis colorectal cancer families?

Microsatellite instability (MSI) is the phenotypic molecular characteristic of the majority of tumors associated with the hereditary nonpolyposis colorectal cancer syndrome (HNPCC). Women in this group have an increased risk of endometrial cancer (EC). This study aimed to determine whether MSI could be demonstrated in blind endometrial samples from women with EC, HNPCC kindreds undergoing screening for EC, and women with normal endometrium. Twenty-four women with EC, 20 women from HNPCC kindreds, and 20 women undergoing gynecological surgery for benign indications underwent blind sampling. MSI analysis was performed by conventional polymerase chain reaction using fluorescent-labeled primers and automated analysis. Twelve microsatellites were studied with MSI defined as evident when novel alleles were seen in endometrial biopsy samples compared to genomic DNA. Of the 24 EC samples obtained, sufficient DNA for analysis was extracted in 17 cases. Three cases had evidence of MSI in at least 7/12 loci. None of the endometrium from the two other study groups revealed evidence of MSI. This is the first demonstration of MSI in blind endometrial biopsies. The ability to demonstrate MSI in heterogeneous endometrial samples suggests potential for the development of a novel EC screening tool for women in HNPCC kindreds.     

BRCA1 expression in a large series of sporadic ovarian carcinomas: a Gynecologic Oncology Group study

Abstract.   Thrall M, Gallion HH, Kryscio R, Kapali M, Armstrong DK, Deloia JA. BRCA1 expression in a large series of sporadic ovarian carcinomas: a Gynecologic Oncology Group study. Int J Gynecol Cancer 2006; 16(Suppl. 1): 166–171.
BRCA1 is a tumor suppressor gene that, when mutated, is associated with the development of hereditary ovarian cancer. A role for BRCA1 in the pathoetiology of sporadic ovarian epithelial cancer (OEC) development has been suggested, although spontaneous mutations of the BRCA1 gene in this disease are uncommon. Loss of gene function by epigenetic alteration is observed more commonly, while other means of gene inactivation have not been intensively investigated. We examined expression and localization of the BRCA1 gene product by immunohistochemistry and sought to clarify the relationship between protein expression and tumor stage, grade, histopathologic subtype, and outcome. Among 230 spontaneous OEC tumors, we found a statistically significant decrease in BRCA1 protein expression with advancing stages of OEC. There was no relationship between expression and tumor grade. There was a statistically significant relationship between the pathologic subtypes of OEC and BRCA1 expression. Minimal BRCA1 expression was protective for survival. These findings confirm a high rate of loss of BRCA1 protein expression in sporadic OEC and suggest a role of BRCA1 in the progression of sporadic ovarian carcinoma.     

The distribution of α inhibin and α glutathione S-transferase (δ4–5 isomerase) in the ovaries of patients with endometrial carcinoma

Abstract. Tiltman AJ, Allard U. The distribution of α inhibin and α glutathione S-transferase (δ^4–5 isomerase) in the ovaries of patients with endometrial carcinoma.
Estrogen is to thought to play a role in the pathogenesis of low grade but not high grade endometrial carcinomas. The dominant circulating estrogen in post menopausal women is estrone which is formed by aromatization of androstenedione. δ^4–5 isomerase, active in the conversion of dehydroepiandrosterone to androstenedione, may be demonstrated immunohistochemically by the antibody to α glutathione S-transferase (αGST). Inhibin, normally acting to suppress FSH secretion, also has an LH-dependent paracrine stimulatory effect on ovarian stromal cells to produce androstenedione. The purpose of this study was to compare the distributions of αGST and α inhibin in the ovaries of patients with low grade and high grade endometrial carcinomas. The results show a statistically significant increase in intracytoplasmic αGST staining in patients with low grade endometrioid adenocarcinomas compared to high grade carcinomas. There was also a statistically significant correlation between the distribution of αGST and α inhibin. These findings lend some support to the hypothesis that estrogen plays a role in the pathogenesis of low grade carcinomas; that the increase in estrone is partly due to increased production of androstenedione by the ovary and that this increased production could be the consequence of increased inhibin paracrine activity.     

Leucocyte intracellular pH and Na+/H+ exchanger isoform-1 activity in postpartum women with pre-eclampsia

Objective To investigate leucocyte  Na⁺/H⁺  exchanger isoform 1 activity in postpartum pre-eclamptics.
Design Exchanger isoform-1 activity and intracellular resting pH were established in leucocytes isolated from two study groups.
Sample Leucocytes isolated from 10 women who had had pre-eclamptic pregnancies more than five months postpartum, and from 10 age-matched normotensive women who were more than five months postpartum.
Setting Hypertension Clinic, Antenatal Assessment Area, Leicester Royal Infirmary.
Methods A well validated technique involving flurometry using a pH sensitive dye (BCECF-AM) was performed to determine exchanger isoform-1 activity and intracellular pH. Determination of exchanger isoform-1 protein abundance was performed by western blotting. Exchanger isoform-3 protein abundance was examined to rule out the possibility of activity due to this particular isoform.
Results Intracellular pH was significantly lower in the postpartum pre-eclamptic group (7.11 +/- 0.02), compared with the postpartum normotensive controls (7.33 +/- 0.04;   P <0.001  ). Exchanger isoform-1 efflux rate (in mmol/L/minute) was significantly higher in the postpartum pre-eclamptic group (35.91 +/- 3.1), compared with the postpartum normotensives (23.94 +/- 2.0;   P = 0.005  ). Exchanger isoform-1 protein density was established to be similar among the two subject groups. No exchanger isoform-3 protein was identified by western blotting.
Conclusion Our results suggest that elevated exchanger isoform-1 activity is an important finding in women who have suffered from pre-eclampsia. This increased activity is not due to an increase in exchanger isoform-1 protein abundance or the presence of exchanger isoform-3.     

Combined E-cadherin, α-catenin, and β-catenin expression is a favorable prognostic factor in endometrial carcinoma

Cell adhesion molecules, such as epithelial cadherin (E-cadherin), might be involved in the processes of tumor invasion and differentiation. The aim of this study was to investigate the expression of E-cadherin, alpha-catenin, and beta-catenin in endometrial carcinoma and to determine the prognostic value of these factors. We have investigated the expression of E-cadherin, alpha-catenin, and beta-catenin by immunohistochemistry in 225 endometrial carcinomas. The correlation between the E-cadherin and the catenins and their correlation with several histologic and clinical parameters were analyzed. Negative E-cadherin, alpha-catenin, and beta-catenin expression was observed in 44%, 47%, and 33% of endometrial carcinomas, respectively, and was correlated with histologic FIGO grade 3 (P < 0.001). Negative E-cadherin expression was more often observed in nonendometrioid endometrial carcinomas (NEECs) than in endometrioid carcinomas (75% versus 43%; P= 0.04). Combined positive E-cadherin, alpha-catenin, and beta-catenin expression was an independent positive prognostic factor for survival in patients with grade 1-2 carcinomas (P= 0.02). Negative E-cadherin expression was found to be associated with histologic grade 3 and with NEEC. Combined positive E-cadherin, alpha-catenin, and beta-catenin expression was a significant prognostic factor.     

The activity of letrozole in patients with advanced or recurrent endometrial cancer and correlation with biological markers – a study of the National Cancer Institute of Canada Clinical Trials Group

A multicenter phase II trial was conducted to define the activity of letrozole in postmenopausal women with recurrent or advanced endometrial carcinoma, who had no more than one prior line of progestins and never had chemotherapy (except adjuvant). Archival paraffin-embedded tumor samples were retrieved to determine the expression level of estrogen (ER) and progesterone receptor (PgR), p53, HER-2, bcl-2 and PTEN protein, and phosphorylation status of protein kinase B (PKB/Akt). Thirty-two eligible patients were treated with letrozole at 2.5 mg daily continuously, of whom 10 (31%) had prior progestins. Of the 28 patients evaluated for response, one complete and two partial responses were noted; overall response was 9.4% (95% confidence interval 2-25%). Eleven patients had stable disease for a median duration of 6.7 months (range 3.7-19.3 months). Amongst 22 patients who had tumor blocks available, the proportion showing positive expression of the following markers includes: PgR (86%), ER (86%), PTEN (82%), phosphorylated PKB/Akt (59%), bcl-2 (45%), p53 (32%), and HER-2 (0%). None of these markers correlated with response to letrozole or disease progression. In conclusion, letrozole is well tolerated but has little overall activity in this cohort of women with endometrial cancer.     

Multi-institutional phase 2 study of TLK286 (TELCYTA™, a glutathione S-transferase P1-1 activated glutathione analog prodrug) in patients with platinum and paclitaxel refractory or resistant ovarian cancer

The purpose of this study was to determine the safety and efficacy of TLK286 (TELCYTA(TM)), a glutathione analog prodrug, in patients with platinum and paclitaxel refractory or resistant ovarian carcinoma. Thirty-six patients with measurable disease were enrolled. TLK286 was administered at 1000 mg/m2 intravenously every 3 weeks. The endpoints were objective response rate assessed by Response Evaluation Criteria in Solid Tumors (RECIST) and survival. Adverse events were graded using the National Cancer Institute Common Toxicity Criteria. Thirty-four platinum refractory or resistant patients (94%) were evaluable for objective tumor response. Five patients (15%) had objective tumor responses, including one durable complete response (CR) of greater than 3 years and continuing. The disease stabilization rate was 50%, including one CR (3%), four partial responses (12%), and 12 durable disease stabilizations (35%). Responses were accompanied by improvement in clinical symptoms and Eastern Cooperative Oncology Group Performance Status (ECOG PS) and decline in CA125 levels. Median survival was 423 days with survival of 60% at 1 year and 40% at 18 months. TLK286 was well tolerated in this population. TLK286 is an active agent in chemotherapy-resistant ovarian cancer. Further studies of TLK286 in platinum and paclitaxel refractory or resistant ovarian cancer are in progress.     

The antiobesity drug Orlistat induces cytotoxic effects, suppresses Her-2/neu (erbB-2) oncogene overexpression, and synergistically interacts with trastuzumab (Herceptin™) in chemoresistant ovarian cancer cells

Abstract.   Menendez JA, Vellon L, Lupu R. The antiobesity drug Orlistat induces cytotoxic effects, suppresses Her-2/neu (erbB-2) oncogene overexpression, and synergistically interacts with trastuzumab (Herceptin^™) in chemoresistant ovarian cancer cells. Int J Gynecol Cancer 2006; 16: 219–221.     

Induction of endometrial cycles and ovulation in a woman with combined 17α-hydroxylase/17,20-lyase deficiency due to compound heterozygous mutations on the P45017α gene

Objective: To describe the case of a Japanese woman with combined 17α-hydroxylase/17,20-lyase deficiency (congenital adrenal hyperplasia type V) and to discuss possible therapeutic procedures in such patients.Design: Case report.Setting: University hospital.Patient(s): A 26-year-old woman with secondary amenorrhea and primary sterility.Intervention(s): Nucleotide sequencing of the P45017α gene (CYP17), induction of endometrial maturation with steroid hormone replacement, and ovulation induction with gonadotropin.Main Outcome Measure(s): Nucleotide sequence of CYP17, endometrial thickness and follicle diameter measured by transvaginal ultrasonography, and histologic evaluation of the endometrium.Result(s): Two different mutations were detected on CYP17: One was a deletion of the phenylalanine codon (TTC) at either amino acid 53 or 54 in exon 1, and the other was a missense mutation with the substitution of histidine (CAC) by leucine (CTC) at position 373 in exon 6. Repeated histologic evaluations performed during treatment with P consistently revealed an unripe endometrium with glands of the early secretory phase and markedly scanty stroma. Ultrasound examination revealed follicular growth and ovulation after gonadotropin administration, but insufficient thickness of the endometrium.Conclusion(s): Ovulation induction was possible in this patient with 17α-hydroxylase/17,20-lyase deficiency, but the endometrial response to steroid hormone replacement was extremely poor.     

Ancillary p16<Superscript>INK4a</Superscript> adds no meaningful value to the performance of ER/PR/Vim/CEA panel in distinguishing between primary endocervical and endometrial adenocarcinomas in a tissue microarray study

Purpose  Endocervical adenocarcinomas (ECA) and endometrial adenocarcinomas (EMA) are uterine malignancies that have differing biological behavior. The choice of appropriate therapeutic plan depends indeed on the tumor’s site of origin. In this study, we not only compare the individual expression status of five immunomarkers (ER, PR, Vim, CEA, and p16^INK4a), but also evaluate whether p16^INK4a adds value to the ER/PR/Vim/CEA panel characteristics in distinguishing between primary ECA and EMA. Methods  A tissue microarray (TMA) was constructed using paraffin-embedded, formalin-fixed tissues from 35 hysterectomy specimens, including 14 ECA and 21 EMA. TMA sections were immunostained with five anti-bodies, by avidin–biotin complex (ABC) method for antigen visualization. The staining intensity and area extent of the immunohistochemical (IHC) reactions were appraised by using the semi-quantitative scoring system. Results  The four respective markers (ER, PR, Vim, CEA) and their combined panel expressions showed significant (p < 0.05) frequency differences between ECA and EMA tumors. The p16^INK4a marker also revealed a significant frequency difference (p < 0.05) between the two sites of origin, but did not demonstrate to have any supplementary value to the 4-marker panel. Conclusion  According to our data, when there is histomorphological and clinical doubt as to the primary site of origin, we recommend that the conventional 4-marker (ER/PR/Vim/CEA) panel is appropriate. Ancillary p16^INK4a-marker testing does not add value to the 4-marker panel in distinguishing between primary ECA and EMA. C.-C. Yao and L.-F. Kok have equally contributed to this article.