Prediction of Relapse After Therapy Withdrawal in Women with Endometrial Hyperplasia: A Long-term Follow-up Study.

AIM: To investigate whether risk of relapse of endometrial hyperplasia persists many years after successful primary therapy and whether clinical or biological markers observed at primary diagnosis may predict relapse. MATERIALS AND METHODS: A series of 57 women with endometrial hyperplasia received levonorgestrel-impregnated intrauterine system or oral progestin for three months during 1998-2000. Index biopsies were classified according to WHO1994 and D-score systems, and immunohistochemical staining for estrogen receptor α (ERα), estrogen receptor ß (ERß), progesterone receptor A (PRA), progesterone receptor B (PRB), B-cell lymphoma 2/apoptosis regulator (BCL2), BCL2-associated X protein/apoptosis regulator (BAX), paired box 2 (PAX2), and phosphatase and tensin homolog (PTEN) reported as H-scores. RESULTS: Over a follow-up of 157.8 months, 23% (10/43) of patients experienced relapse. No correlation with age, body mass index, parity, WHO94 classification, or D-score was found. Only PRA (p=0.004) and PRB (p=0.038) showed certain correlation with relapse. CONCLUSION: Endometrial hyperplasia recurs many years after successful progestin therapy. Increased expression of PRB and reduced expression of PRA significantly correlated with relapse. Our results support the importance of continuous endometrial protection and the need for new clinical surveillance guidelines.

Increased gene expression of the ABCC5 transporter without distinct changes in the expression of PDE5 in human cervical cancer cells during growth.

Carcinoma of the uterine cervix represents the second most frequent female malignancy worldwide, but few biochemical tumour markers have been implemented into clinical practice. Elevated extracellular guanosine 3', 5'-cyclic monophosphate (cGMP) levels have been reported to be a sensitive, early and reliable marker for screening relapse in carcinoma of the uterine cervix. The mechanism behind this observation remains unknown. The possibility exists that the cancer cells develop resistance to the antiproliferative effect of high intracellular cGMP levels. The enhanced cGMP expression may originate from either an increase in cellular export capacity by increased expression of member 5 in subfamily C of ATP-Binding-Cassette transporters (ABCC5), or increased substrate (cGMP) levels for this pump. The latter situation occurs with increased expression of inducible nitric oxide synthase (iNOS) and/or soluble guanylyl cyclase (sGC) and/or reduced expression of member 5 of the cyclic nucleotide phosphodiesterases (PDE5). Four transformed human cell lines derived from carcinomas of the uterine cervix (C-4 I, C-33 A, SiHa and ME-180 cells) and one non-transformed human cell line (WI-38) were included in the study in order to unveil which biokinetic components are involved. The expressions of iNOS, sGC, PDE5 and ABCC5 in the initial and final phase of the exponential growth curve were compared. Assuming that the WI-38 control cells mimic the situation in a normal tissue, iNOS remains un-expressed during proliferation, and the expression of sGC is low but shows a clear increase during exponential growth. PDE5 is highly expressed and increases (≈130%) during growth whereas ABCC5 exhibited low to moderate expression, with a moderate increase (≈40%) during growth. The malignant cells exhibited moderate ABCC5 expression with a distinct increase during exponential growth, whereas PDE5 expression remained virtually unchanged. Dysregulation of the cGMP biokinetics in growing malignant cells may account for the elevation of extracellular cGMP observed in patients with carcinoma of the uterine cervix.

Cancer-associated fibroblasts from human NSCLC survive ablative doses of radiation but their invasive capacity is reduced.

BACKGROUND: Cancer-Associated Fibroblasts (CAFs) are significant components of solid malignancies and play central roles in cancer sustainability, invasion and metastasis. In this study we have investigated the invasive capacity and matrix remodelling properties of human lung CAFs after exposure to ablative doses of ionizing radiation (AIR), equivalent to single fractions delivered by stereotactic ablative radiotherapy (SART) for medically inoperable stage-I/II non-small-cell lung cancers. METHODS: CAFs were isolated from lung tumour specimens from 16 donors. Initially, intrinsic radiosensitivity was evaluated by checking viability and extent of DNA-damage response (DDR) at different radiation doses. The migrative and invasive capacities of CAFs were thereafter determined after a sub-lethal single radiation dose of 18 Gy. To ascertain the mechanisms behind the altered invasive capacity of cells, expression of matrix metalloproteinases (MMPs) and their endogenous inhibitors (TIMPs) were measured in the conditioned media several days post-irradiation, along with expression of cell surface integrins and dynamics of focal contacts by vinculin-staining. RESULTS: Exposing CAFs to 1 × 18 Gy resulted in a potent induction of multiple nuclear DDR foci (> 9/cell) with little resolution after 120 h, induced premature cellular senescence and inhibition of the proliferative, migrative and invasive capacity. AIR promoted MMP-3 and inhibited MMP-1 appearance to some extent, but did not affect expression of other major MMPs. Furthermore, surface expression of integrins α2, ß1 and α5 was consistently enhanced, and a dramatic augmentation and redistribution of focal contacts was observed. CONCLUSIONS: Our data indicate that ablative doses of radiation exert advantageous inhibitory effects on the proliferative, migratory and invasive capacity of lung CAFs. The reduced motility of irradiated CAFs might be a consequence of stabilized focal contacts via integrins.

MCF-7 cell apoptosis and cell cycle arrest: non-genomic effects of progesterone and mifepristone (RU-486).

UNLABELLED: The pharmacology of progestins includes actions initiated by various cellular targets, including classic receptors characterized as nuclear transcription factors (nPR), G-protein-coupled membrane receptors (mPR), enzymes, membrane channels and transporters. The effects initiated by targets other than nPR are termed non-genomic and there is an increasing recognition that these effects also play an important role in the regulation of cell growth. MATERIALS AND METHODS: The nPR-positive breast cancer (MCF-7) and the nPR-negative uterine cervix cancer (C4-I) cell lines were exposed to progesterone (PG) and mifepristone (MF) during a culture period of 96 h. Daily cell count, cell cycle analysis and apoptosis assay were performed. RESULTS: It was possible to separate the nPR initiated effects (growth stimulation) from the non-genomic effects (growth inhibition) in the MCF-7 cells. Below 1 µM PG treatment gave a small, but distinct increase in cell density which was effectively blocked by MF. Such an effect was absent from the nPR-negative C4-I cells. For a range of concentrations between 1 µM and 100 µM, the effect of both PG and MF developed over time and showed concentration dependency. The PG concentrations needed to reduce cell density by 50% (IC(50)) were 12.8 ± 1.1 µM and 6.5 ± 0.2 µM for the MCF-7 and C4-I cells, respectively. MF appeared to be equally or slightly more potent, with respective IC(50) values of 6.9 ± 0.5 µM and 5.3 ± 0.3 µM. The cell density reduction was both a result of cell cycle arrest and apoptosis. The combination of PG and MF had a potentiated effect on cell density reduction, cell cycle arrest and apoptosis. CONCLUSION: The antiproliferative/cytotoxic effect of PG and MF in concentrations between 1 and 100 µM is of a non-genomic nature.

Down-regulated progesterone receptor A and B coinciding with successful treatment of endometrial hyperplasia by the levonorgestrel impregnated intrauterine system.

OBJECTIVE: To investigate whether regression of endometrial hyperplasia observed after 3 months of treatment with levonorgestrel impregnated intrauterine system device (LNG-IUS) was sustained after 6 months and whether these effects were still occurring synchronously with extinguished expression of progesterone receptors and increased apoptosis. DESIGN: Retrospective population-based observational study. SETTING: Six local hospitals and one university hospital in northern Norway. POPULATION: Patients (n = 41) with low and medium risk endometrial hyperplasia. METHODS: Histopathological treatment response comparing LNG-IUS (n = 25) and standard per oral medroxyprogesterone (n = 16). Expression of progesterone receptor A (PR-A), progesterone receptor B (PR-B), ER-alpha, ER-beta, Bcl-2, BAX, Caspase-3 and metallothionein (MT) were investigated by immunohistochemistry; results were evaluated by a semi-quantitative H-score. MAIN OUTCOME MEASURES: Response to progestin treatment. RESULTS: All the LNG-IUS treated patients had therapy response after 6 months. PR-A and PR-B in glands were almost extinguished for IUD users compared to the oral group. Estrogen receptors were also reduced. Co-existent changes in apoptosis were differently modulated in glands and stroma in the two treatment groups. Bcl-2 was different in glands and stroma in responders and non-responders to oral therapy. CONCLUSION: The study confirms that LNG-IUS can be safely used for 6 months as treatment for endometrial hyperplasia. The clinical effect is accompanied by almost extinguished PR-receptors in glands coinciding with modulation of apoptosis. The results strongly indicate that progestins activate non-classical initiated signaling pathways.

Prognostic markers for coexistent carcinoma in high-risk endometrial hyperplasia with negative D-score: significance of morphometry, hormone receptors and apoptosis for outcome prediction.

OBJECTIVES: Hysterectomy represents the current routine therapy for high-risk endometrial precancers. More sophisticated methods are needed for treatment decision among women who want to preserve fertility and seriously ill patients. Among women diagnosed with high-risk hyperplasia, approximately 40% show signs of endometrial cancer in the hysterectomy specimen. Thus, more sophisticated methods are needed to select the women at risk. SETTING: University Hospital of Tromsø, Regional Center for Gynecological Oncology in northern Norway. POPULATION: From 1999 to 2004, 258 consecutive patients had endometrial hyperplasia diagnosed by D-score; 57 among these were high-risk cases (D-score < 0) and 10 had coexisting endometrial carcinoma. No further cancers were detected after long-term follow-up (4-10 years). DESIGN: From the initial histological specimens, material from the 10 patients with cancer and from the 13 cases without cancer (high-risk D-score < 0) was analyzed with selected histomorphometric (architectural and nuclear) and immunohistochemical (hormone receptors and apoptotic) features blinded to the investigator. METHOD: Original slides were used for computerized histomorphometry (4-class rule and related procedures). Serial sections from the paraffin embedded material were used for immunohistochemical investigations. Immunohistochemical expression in glands and stroma was evaluated by the semi-quantitative H-score (ER-alpha, ER-beta, PR-A, PR-B, RCAS-1, Bcl-2, BAX, and Caspase-3). RESULTS: The histomorphometric 4-class rule differentiates between presence and absence of cancers with a sensitivity of 80% and specificity of 77%. Several morphometric and immunohistochemical features were significantly different in cases with cancer and hyperplasia. CONCLUSIONS: Histomorphometry seems superior in predicting coexistent carcinoma in high-risk endometrial hyperplasia and should be considered for clinical use.

Development of a new method to harvest chondroprogenitor cells from underneath cartilage defects in the knees.

BACKGROUND: Mesenchymal progenitor cells from bone marrow hold great potential as a cell source for cartilage repair. Aspiration from the iliac crest is the most widely used method to harvest bone marrow cells for cartilage repair. The objective of our study was to establish a new method to isolate mesenchymal progenitor cells by direct aspiration of bone marrow from the subchondral spongious bone underneath cartilage defects during microfracture treatment and to confirm the chondrogenic potential of the resulting cell cultures. METHODS: Bone marrow was aspirated arthroscopically from patients treated for isolated cartilage defects. Adherent stromal cells were isolated, expanded in monolayer cultures, and characterized by flow cytometry. Chondrogenic induction of cells was achieved by combination of spheroid cultures in hanging drops and the concomitant use of transforming growth factor-beta (TGFbeta). Articular chondrocytes established in three-dimensional (3D) cultures were used as positive cartilage-forming units, and skin fibroblasts were used as negative controls. Three-dimensional constructs were stained for immunohistochemical and histological examination, and a real-time polymerase chain reaction (PCR) was performed to quantify the expression of aggrecan, collagen types 1 and 2, and Sox9. RESULTS: Mesenchymal stem cell-like progenitor cells (MSCs) displaying chondrogenic differentiation capacity were harvested arthroscopically from underneath cartilage lesions on distal femurs using the one-hole technique. Stem cell-related surface antigens analyzed by flow cytometry confirmed the nature of the isolated adherent cells. MSC spheroids stained positive for glycosaminoglycans and collagen type 2. Realtime PCR showed that MSCs in 3D spheroids significantly increased gene expression of collagen type 2, aggrecan, and Sox 9 and down-regulated expression of collagen type 1 when compared to the mRNA levels measured in MSCs monolayers. CONCLUSIONS: We describe a new technique that may be applied for harvesting bone marrow cells from cartilage defects during arthroscopic intervention of the knee. Cells harvested in this way hold full chondrogenic differentiation potential. Our data imply that MSC storage may be established by using marrow from this approach, bypassing the need for cell aspiration from the iliac crest.

High concentrations of progesterone and mifepristone mutually reinforce cell cycle retardation and induction of apoptosis.

High concentrations of progesterone (PG) and mifepristone (MF) reinforce the effect of each other, reducing the endometrial cancer (Ishikawa) cell densities in vitro. Whether this effect is caused by cell cycle retardation, induction of apoptosis, or a combination of both was questioned. During a 5 days period in the absence of PG, the G(1)/G(0) fraction increased from 60% to 82% . After the first day of exposure with 32 and 95 muM PG the respective G(1)/G(0) fractions increased to 79% and 82% . A similar pattern was evident after exposure to MF. After the first day, MF caused a clear increase in the G(1)/G(0) fraction from 53% (control) to 72% (70 microM MF). In a third series of experiments, PG and MF were combined. After the first day, the G(1)/G(0) fractions were 50% in absence of active agents, 67% in presence of 32 microM PG, 66% in presence of 23 microM MF and 76% when PG (32 microM) and MF (23 microM) were combined. Both PG and MF induced apoptosis, which showed a time- and concentration-dependent pattern. In the control series the % apoptotic Ishikawa cells increased 2 to 3-fold during the experimental period and the effect of PG on apoptosis developed to a maximum after 4-5 days. On the fifth day, the trend was identical in three different assays commonly used to quantify different stages of apoptosis. The fractions of apoptotic cells, in the presence of 70 microM PG, increased from 2.5% to 28.2% (JC-1), from 1.4% to 13.2% (Annexin-FITC) and from 2.8% to 27.7% (DNA fragmentation assay), respectively. After five days, in a separate experiment, the fraction of apoptotic cells, as determined by JC-1 assay was 3.2% in the absence of active agents, 5.0% in the presence of 32 microM PG, 7.3% in the presence of 23 muM MF, and 12.4% in the presence of both PG (32 microM) and MF (23 microM). The present study shows that supraphysiological PG concentrations and high pharmacological concentrations of MF cause cell cycle retardation and induce apoptosis. In combination, PG and MF mutually reinforce these effects.

Levonorgestrel, medroxyprogesterone and progesterone cause a concentration-dependent reduction in endometrial cancer (Ishikawa) cell density, and high concentrations of progesterone and mifepristone act in synergy.

Endometrial hyperplasia is a precursor lesion of endometrial carcinoma. Clinical studies of endometrial hyperplasia have shown that levonorgestrel (LNG) is more therapeutically effective than medroxyprogesterone acetate (MPA). The present pharmacological in vitro study was performed to compare progestin effects on human endometrial cancer (Ishikawa) cells. Supraphysiological concentrations of progesterone (PG) and high concentrations of LNG and MPA were employed to determine the order of potency in reducing cell density. The order of potency was LNG>MPA>PG with respective 50% inhibitory concentrations (IC(50)) of 3.9+/-0.4, 30.4+/-3.4 and 45.3+/-2.7 microM. Mifepristone (MF) is a potent antiprogestin, but was unable to antagonize the PG-induced cell density reduction. For MF concentrations from 0.2 to 70 microM alone, a PG-mimetic effect was observed with an IC(50) value of 19.0+/-1.7 muM. When PG and MF were combined, a marked reinforcement of the effect was seen. These observations indicate that extranuclear initiated signaling pathways are involved in the reduction of endometrial cancer cells exposed to high concentrations of PG and MF.

Early effects of high concentrations of progesterone and mifepristone A gene expression study of endometrial cancer cells (Ishikawa).

Patients with endometrial hyperplasia representing preliminary stages of endometrial cancer have shown to respond to therapy in 100% of the cases when treated with levonorgestrel-impregnated intrauterine device. Anti-proliferative effect has also been reported after application of an anti-progestin impregnated intrauterine device which showed to induce endometrial atrophy. The intention of the present study was to obtain more information of novel therapeutic targets for hormonal treatment in endometrial hyperplasia and endometrial cancers. Gene expression of signaling pathways after stimulation of Ishikawa cells with high doses of progesterone (32 microM) or Mifepristone (32 microM) was performed. After using an oligo microarrays representing 24,650 human genes and 37,580 gene transcripts, 6154 genes remained after pre-processing and filtering. This resulted in a total of 993 up-regulated genes with 189 genes for progesterone and 255 genes for Mifepristone. The 550 down-regulated genes were distributed with 256 genes for progesterone, 127 genes for RU 486. The results showed that genes presenting the epidermal growth factor (EGF)/MAP-kinase pathway were significantly over-represented by progesterone treatment, whereas, by Mifepristone treatment genes involved in the p53 pathway were also up-regulated (data not shown). These genes may be interesting as potential new therapeutic targets in endometrial hyperplasia and endometrial cancer, as candidate genes for therapy response or as candidate markers for tumor progression.

Gene expression in endometrial cancer cells (Ishikawa) after short time high dose exposure to progesterone.

The potent antiproliferative effect of progestins has been utilized in clinical regimens for treatment of endometrial proliferative disorders. The progestin infiltrated intrauterine device used as therapy for endometrial carcinoma as well as endometrial hyperplasia yields a hundred-fold increase of local progestin concentration in the endometrium compared to that of oral treatment. The genetic basis for the complex effects of high dose progestins and the different signalling pathways regulated by these genes have never been accurately surveyed. The aim of the present study was to determine the gene expression pattern in highly differentiated endometrial cancer cells (Ishikawa) after short time exposure to high progesterone doses. In eight independent experiments, cells were treated with progesterone (30microg/ml) for 4h and gene expression was compared to that of untreated cells, which served as controls. Microarray analysis revealed 247 differentially expressed genes of which 126 were up-regulated and 121 were down-regulated. Of these, 135 genes are known to be involved in biological processes like cell cycle, cell proliferation and differentiation, developmental processes, immune responses, intracellular protein traffic and transport. Our study shows that microarray analysis can detect relevant gene expression changes in endometrial cells treated with progestin, including those involved in several alternative transcriptional factors and signalling pathways. Many of the differentially expressed genes were not previously known to be affected by progesterone or have unknown biological functions. Characterization of these genes may give new insights into molecular responses to treatment with high progesterone doses. Alternative signalling pathways for progesterone, rather than the classical steroid receptors pathways are also suggested.

Prognosis of early cervical cancer (FIGO Stages IA2, IB, and IIA) in northern Norway predicted by malignancy grading score and objective morphometric image analysis.

Recurrence of early-stage cervical cancer after primary surgery represents a considerable clinical problem, and, so far, few reliable markers for prediction of recurrence exist. Thus, the prognostic value of the malignancy grading score (MGS) classification system was evaluated in 82 patients with early-stage cervical cancer (International Federation of Gynecology and Obstetrics stages IA2, IB, and IIA) and long-time follow-up (5-16 years). Recurrence or not, the likelihood of lymph node metastases and reproducibility of the MGS semiquantitative system were tested. The prognostic power of the MGS to identify high-risk cases prone to recurrence in patients lacking lymph node metastases at primary surgery was a main purpose of the present study. The semiquantitative MGS classification system was performed independently by 2 pathologists unaware of prognosis and clinical data using light microscopy. Routine hematoxylin and eosin sections from surgical specimens were used, and investigation area was defined in the deep part of the tumor. Data-based image analysis was also used to investigate if objective morphometric parameters could add any prognostic power to MGS. The 5-year survival for the whole patient group was 92%. Malignancy grading score of greater than 17 risk points was statistically highly significant in predicting relapse and lymph node metastases (n = 82). High-risk cases lacking lymph node metastases (n = 70) were also statistically associated with high MGS. Depth of invasion and vascular invasion were statistically related to recurrence. Objective image analysis of nuclear parameters was of no additional statistical value for the prediction of outcome. The MGS classification system proved to be a useful tool in predicting recurrence and lymph node metastases and, most importantly, was a predictor of high-risk patients without metastases at primary surgery.