Bjørn Moe

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

BackgroundCancer-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.MethodsCAFs 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.ResultsExposing 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, β1 and α5 was consistently enhanced, and a dramatic augmentation and redistribution of focal contacts was observed.ConclusionsOur 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.

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.

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.

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

Summary 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.

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.

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‐α, ER‐β, 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.