Nuclear CSPP1 expression defined subtypes of basal-like breast cancer.

BACKGROUND: The multi-exon CSPP1 gene, encoding for centrosome and microtubule-associated proteins involved in ciliogenesis and cell division, is a candidate oncogene in luminal breast cancer but expression of CSPP1 proteins remained unexplored. METHODS: CSPP1 gene and protein expression was examined in normal mammary tissue, human breast cancer cell lines, and primary breast cancer biopsies from two patient cohorts. Cell type and epitope-dependent subcellular-specific CSPP1 staining pattern in normal mammary gland epithelium and cancer biopsies were correlated to molecular and clinical parameters. RESULTS: A novel, nuclear localised CSPP1 isoform was exclusively detected in luminal epithelial cells, whereas cytoplasmic CSPP-L was generally expressed in normal mammary epithelium. Luminal cell-related nuclear CSPP1 expression was preserved in type-matched cell lines and carcinomas, and correlated to gene copy number and mRNA expression. In contrast, basal-like carcinomas displayed generally lower CSPP1 mRNA expression. Yet, a subgroup of basal-like breast carcinomas depicted nuclear CSPP1 expression, displayed luminal traits, and differed from nuclear CSPP1 devoid counterparts in expression of eight genes. Eight-gene signature defined groups of basal-like tumours from an independent cohort showed significant differences in survival. CONCLUSIONS: Differential expression of a nuclear CSPP1 isoform identified biologically and clinically distinct subgroups of basal-like breast carcinoma.

Flotillins as regulators of ErbB2 levels in breast cancer.

Amplification and overexpression of the receptor tyrosine kinase ErbB2 occur in up to 30% of human breast cancers, and high ErbB2 levels are correlated with poor prognosis for breast cancer patients. In contrast to the epithelial growth factor receptor (ErbB1), ErbB2 is not downregulated by ligand-induced mechanisms. Here we show that flotillins are involved in the stabilization of ErbB2 at the plasma membrane. In SKBR3 breast cancer cells and breast cancer tissue, a positive correlation between flotillin and ErbB2 expression levels could be demonstrated. Moreover, the tissue microarray analyses of biopsies from 194 patients diagnosed with carcinomas of the breast showed that flotillin-2 emerged as a potential predictor of prognosis in breast cancer. Depletion of flotillin-1 and flotillin-2 leads to internalization and degradation of ErbB2. Furthermore, flotillin-1 and -2 were found to be in a molecular complex with ErbB2 and Hsp90. The depletion of one of these proteins results in disruption of this complex, followed by destabilization of ErbB2 at the membrane, and its internalization and degradation. As a consequence, ErbB2-triggered downstream signalling is inhibited. Our data demonstrate a novel mechanism for interfering with ErbB2 signalling, which potentially can have clinical impact.

Prognostic importance of DNA ploidy and DNA index in stage I and II endometrioid adenocarcinoma of the endometrium.

BACKGROUND: We evaluated the prognostic importance of DNA ploidy in stage I and II endometrioid adenocarcinoma (EAC) of the endometrium with a focus on DNA index. PATIENTS AND METHODS: High-resolution DNA ploidy analysis was carried out in tumor material from 937 consecutive patients with International Federation of Gynecology and Obstetrics (FIGO) stage I and II EAC of the endometrium. RESULTS: Patients with diploid (N = 728), aneuploid tumor with DNA index ≤ 1.20 (N = 118), aneuploid tumors with DNA index >1.20 (N = 39) and tetraploid tumor (N = 52) had 5-year recurrence rates 8%, 14%, 20% and 12%, respectively. Patients with aneuploid tumor with DNA index >1.20 had a poorer 5-year progression-free survival (67%) and overall survival (72%) compared with the patients with aneuploid tumor with DNA index ≤ 1.20 (81% and 89%, respectively). Aneuploid tumors with DNA index ≤ 1.20 relapsed mainly in the vagina and pelvis, whereas aneuploid tumors with DNA index >1.20 relapsed predominantly outside pelvis. CONCLUSIONS: The recurrence risk for the patients with aneuploid tumor is higher than the patients with diploid tumor in EAC of the endometrium. Based on DNA index with cut-off 1.20, aneuploid tumors can be separated into two subgroups with different recurrence pattern and survival.

Flow cytometric measurement of cellular FLICE-inhibitory protein (c-FLIP) in ovarian carcinoma effusions.

OBJECTIVE: The objective of this study was to establish a flow cytometry assay for measuring c-FLIP in serous effusions. In addition, we studied the clinical relevance in ovarian carcinoma effusions of this inhibitor protein in the death receptor signalling pathway of apoptosis. METHODS: Two c-FLIP antibodies were tested using Western blotting and the best performing one was used for titration of c-FLIP expression in a panel of five cell lines, consisting of ovarian carcinoma, breast carcinoma and malignant mesothelioma. The concentration that provided the best signal-to-noise ratio was used for comparison of the performance of three fixation and permeabilization protocols. The best performing protocol was chosen for analysis of 69 ovarian carcinoma effusions. c-FLIP expression was analysed for association with clinicopathological parameters and survival. RESULTS: Rabbit polyclonal c-FLIP by Abcam and the IntraStain kit by Dako performed best. c-FLIP expression was detected in tumour cells in all 69 effusions (expression range 21-100%, median = 80%). No association was found between c-FLIP expression and clinicopathological parameters, including chemoresponse and survival. However, an inverse correlation was found between c-FLIP levels and expression of the previously studied apoptosis marker cleaved caspase-3 (P = 0.029). CONCLUSIONS: An assay for measuring c-FLIP in cytology specimens is presented. c-FLIP is frequently expressed in ovarian carcinoma effusions, but its expression appears to be unrelated to disease aggressiveness.

Measurement of apoptosis in cytological specimens by flow cytometry: comparison of Annexin V, caspase cleavage and dUTP incorporation assays.

OBJECTIVE: To compare the performance of different assays for measuring apoptosis in cytological specimens. METHODS: Apoptosis was assessed in 27 specimens (22 effusions, five fine needle aspirates; 20 malignant, seven reactive) using flow cytometry, applying assays for the measurement of annexin V expression, caspase-3 and -8 cleavage and deoxynucleotidyl transferase deoxyuridine triphosphates (dUTP) incorporation. Results were studied for differences between reactive and malignant specimens, as well as performance across assays. RESULTS: Wide variation in the degree of apoptosis was observed in both benign and malignant specimens using all assays. However, the percentage of annexin V-positive cells was higher compared with those showing caspase cleavage or dUTP incorporation in the majority of cases, irrespective of specimen type. Comparative analysis of benign and malignant specimens showed no significant differences in expression of any of the studied parameters. However, tumour cells and reactive mesothelial cells in pleural effusions had a significantly lower level of dUTP incorporation compared with their counterparts in peritoneal specimens (P = 0.001). CONCLUSIONS: The present data are in agreement with our previous observation in ovarian carcinoma effusions, that measurement of apoptosis by the annexin V assay provides higher expression values than those obtained by other assays, suggesting that this assay does not accurately reflect the degree of apoptosis in benign or malignant cells in effusions.

Effect of varicose vein surgery on venous reflux scoring and plethysmographic assessment of venous function.

BACKGROUND: Colour duplex ultrasonography (CDU) is widely recommended before varicose vein surgery, combined with quantification of venous reflux by plethysmography where required. This study assessed venous haemodynamics before and after varicose vein surgery by venous outflow plethysmography (VOP), venous reflux plethysmography (VRP) and by adoption of a modified segmental venous reflux score (VRS). The effect of wearing one or two class I medical compression stockings was also assessed. The aim of the study was to identify parameters which reflect the outcome of treatment using medical compression stockings or surgical intervention. METHODS: 24 legs of 21 patients with superficial vein incompetence of clinical grade C(2-4a) (CEAP) were assessed before and a mean of 8 S.D. 4 months after superficial vein surgery. Investigations were CDU, as well as VOP and VRP using mercury in rubber gauges fitted either around the calf or the forefoot. Venous reflux was semi-quantitatively graded by CDU in relation to the actual vein diameter and transformed into a VRS with respect to the number of involved serial vein segments. The venous reflux rates were measured in standing patients after knee bending before and after application of one or two superimposed compression stockings (class I). RESULTS: According to VRP, one compression stocking reduced the maximum venous reflux rates (VR(max)) by about 30% which was comparable with the effect of surgery on VR(max). Two superimposed compression stockings were almost twice as effective and diminished VR(max) pre- and post operatively by around 60%. Varicose surgery reduced the maximum venous outflow rates significantly (pre-op: 166 S.D. 77 ml/min x 100 ml tissue, post op: 120 S.D. 34) and improved VRS (pre-op median 5.0 IQR: 4.5-5.5, post-op median 0.5 IQR: 0-1.0). Surgery had no effect on venous refilling time or venous reflux rates when measured without compression stockings. CONCLUSION: Venous reflux assessed by plethysmography was moderated by the use of compression stockings pre-operatively but did not reflect the outcome of surgical treatment of superficial venous reflux. Increased venous volume and venous outflow were restored to the levels of normal contralateral limbs by surgery. The VRS decreased considerably following surgery, reflecting the effect of surgical treatment on the number of incompetent venous segments. Changes in this parameter did not correlate with any of the plethysmographic measurements.

Effect of shear stress on the expression of coagulation and fibrinolytic factors in both smooth muscle and endothelial cells in a co-culture model.

UNLABELLED: Blood vessels are subjected to forces due to the flow. Endothelial cells (EC) are recipients, cross-talk with smooth muscle cells (SMC), and regulate physiology. It was hypothesized that both EC and SMC respond to shear stress, which alters the expression of factors in coagulation and fibrinolysis. METHODS: A co-culture of human saphenous vein EC (HSVEC) and human saphenous vein SMC (HSVSMC) was exposed to shear, following which the cells were separated. Gene expression of tissue factor, thrombomodulin (TM), plasminogen activator inhibitor-1 (PAI-1), tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA) were analyzed with real-time RT-PCR. Protein expression was studied with ELISA. In HSVEC, the expression of PAI-1 (x2.1), tPA (x1.8), uPA (x1.6), tissue factor (x2.5) and TM (x1.9) was upregulated after 4 h of shear compared to controls. After 24 h of shear, expression was still upregulated in tPA (x2.3) and TM (x1.6). In HSVSMC, change in expression of PAI-1 (x2.1) was present after 4 h and in uPA (x2.1), and TM (x0.4) after 24 h. Both HSVEC and HSVSMC responded to shear, which led to altered expression of coagulation and fibrinolytic factors. This indicates that SMC, and interactions between EC and SMC, are more important in the regulation of vascular wall hemostasis than earlier studies have reported.

DNA- versus RNA-based methods for human papillomavirus detection in cervical neoplasia.

OBJECTIVE: To compare DNA-based and mRNA-based methods for detection of high-grade cervical neoplasia in Norway. METHODS: HPV prevalence was analyzed in 383 women with positive index cytology, selected from gynecology clinics. All patients were investigated by a new PAP smear, histology, and two commercially available HPV tests: Hybrid Capture II (Digene, Gaithersburg, MD) and the Pre Tect HPV-Proofer (NorChip AS). Cases with positive DNA test and negative mRNA test and cases with high-grade histology and negative HPV tests were retested with PCR and sequencing. We regarded the infection as latent or transient if sequencing revealed an HPV type included in both assays. RESULTS: High-risk HPV was detected in 99.7% of the histological confirmed high-grade lesions (CIN2+) (290/291). The DNA test was positive in 95% (275/291), and the mRNA test was positive in 77% (225/291) of the histological confirmed high-grade lesions. All invasive carcinomas were mRNA positive. The DNA test was significantly more often positive in benign and low-grade lesions, some of which were found to be false positive due to cross-contamination with unrelated types. High-grade histology was detected in 83% of women with normal cytology and positive mRNA test. Latent or transient infections were detected in 11 low-grade and 12 high-grade preinvasive lesions. Sequencing revealed high-risk HPV types included only in the DNA test in 35 high-grade preinvasive lesions, HPV 52 and 58 were the most prevalent HPV types. CONCLUSIONS: These HPV tests have the potential to improve the detection rate of high-grade cervical neoplasia, with some limitations. The mRNA test seems to be more appropriate for risk-evaluation. Larger scale, population based studies are necessary to evaluate the predictive values of HPV testing in Norway.

The prognostic significance of thymidine phosphorylase, thymidylate synthase and dihydropyrimidine dehydrogenase mRNA expressions in breast carcinomas.

Thymidine phosphorylase (TP), thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) have been indicated as possible predictive markers for epithelial malignancies. All these three enzymes are actively involved in 5-FU metabolism. In this report, we investigated mRNA expression of these factors with real-time quantitative PCR in a series of 86 micro-selected breast carcinomas and 8 micro-selected tumour-adjacent normal breast epithelial specimens. Highly variable mRNA expressions of these factors were observed in both normal and cancerous samples. TP and TS mRNA expressions in breast carcinomas were elevated, but only TS mRNA expression showed a trend for statistical difference, compared with the expression in normal breast epithelial samples. Although the DPD mRNA expression range in tumours was also elevated, the average mean was reduced in tumours compared to that in normal samples. No association between mRNA expressions of TP, TS and DPD and clinicopathological features such as histological grade, tumour size, node status, S-phase fraction, ploidy, and clinical stage was found. A negative association between DPD mRNA expression and age was, however, revealed. Ten-year follow-up analysis showed no association between TP and DPD mRNA expression and clinical outcome. An high level of TS mRNA expression, however, was associated with a shorter clinical survival, indicating its potential role as a clinical marker in breast carcinoma.

Treatment of descending thoracic aneurysms by endovascular stent grafting.

PURPOSE: Endovascular stent-graft treatment for true aneurysms of the descending thoracic aorta is a valid and effective alternative to conventional surgery. A review of our experience with 21 consecutive patients is reported and technical considerations are discussed. METHODS: Twenty-one patients (mean age 73 years) with true aneurysms of the descending thoracic aorta (n = 14) or contained rupture (n = 7) were treated between October 1999 and July 2001. Seven patients (33%) underwent emergency endovascular procedure. Postoperatively, the patients were followed with CT scans at 1, 3, 6, and 12 months. Follow-up, which averaged 17 months, was 100% complete. THIRTY-DAY RESULTS: No conversions to open repair were necessary. Two patients died (10%), one of acute intestinal ischemia and the other because of multiorgan failure. Four patients showed endoleaks immediately after stenting. Two patients required new endovascular stentgrafts, while the remaining two were treated conservatively. Besides endoleaks, eight major complications occurred in six patients (two stroke, two paraplegia, two respiratory insufficiency, and one renal failure). MID-TERM RESULTS: Three more patients died during the follow-up period. One patient died of heart failure after a complicated postoperative course, 91 days after stenting. The second patient died because of aortic rupture, 139 days after stenting. The third patient died of heart failure, 15 months after the endovascular procedure. The remaining 16 patients are alive and have been regularly controlled by CT scans. No late migration or endoleaks have been detected. In all the survivors, the size of the aneurysm was unchanged or diminished. CONCLUSIONS: Treatment of descending thoracic aortic aneurysms by endovascular stentgraft devices has good early and mid-term results. More accurate selection of patients may further reduce mortality and morbidity.

Clinicopathological associations of CD44 mRNA and protein expression in primary breast carcinomas.

AIMS: The purpose of this study was to examine the occurrence of CD44 isoforms in breast carcinomas and their role in predicting clinical outcome. METHODS AND RESULTS: Shock-frozen tumour tissues from 110 patients with breast carcinoma were examined by immunohistochemistry using antibodies directed against CD44s, v5, v6, v7 and v3-10. In addition, 80 of these tumours were available for quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of CD44s and CD44v6. Immunohistochemically, the positive tumours showed cytoplasmic and/or membranous staining with all antibodies. Staining results did not correlate with histological subtype, lymph node status, status of steroid receptors, tumour size or age. Neither was any correlation found for overall and disease-free survival. Quantitative real-time RT-PCR of CD44s and CD44v6, however, revealed that expression of CD44v6 mRNA was significantly associated with lower pathological grade (Pearson chi(2) test P = 0.009; linear-by-linear association P = 0.003). Linear-by-linear association between CD44s mRNA expression and lower pathological grade was also seen (P = 0.02). Survival analysis with the Kaplan-Meier method demonstrated that increased CD44s mRNA expression was significantly associated with both disease-free survival and overall survival (P = 0.0185 and P = 0.0344, respectively). A similar trend for CD44v6 mRNA expression was seen in these cases, but the difference was not significant. CONCLUSIONS: Quantitative real-time RT-PCR revealed clinical correlations of CD44s and CD44v6 mRNA expression in breast carcinomas while immunohistochemistry for the protein expression of CD44s and other CD44 variants did not. This contradictory result merits further studies concerning the clinical impact of CD44 molecules in breast carcinomas.

Treatment of femoral pseudoaneurysms. Percutaneous US-guided thrombin injection versus US-guided compression.

PURPOSE: Thrombin injection in femoral pseudoaneurysms has been suggested to be superior to traditional US-guided compression. Our aim was to evaluate results with compression therapy with special reference to use of thrombin in case of failure. We also studied 7 patients who underwent primary thrombin injection. MATERIAL AND METHODS: We retrospectively reviewed all (n=44) femoral artery pseudoaneurysms diagnosed at our department during October 1998-May 1999. US-guided compression with the Femostop device or US-guided thrombin injection (100-1000 IU) was the first choice according to the physicians' preference, followed by the other regime if the first choice was non-successful. RESULTS: Thirty-nine (89%) of the patients received anticoagulation treatment and/or concomitant antiplatelet drugs. Out of the 44 patients, 37 were treated with compression as the first choice. This regime was successful in 22 (59%). This group included 2 lesions that resolved spontaneously after initially failed compression and 1 deep venous thrombosis after treatment. The persistent 15 pseudoaneurysms after failed compression received thrombin injection, and it was also the primary therapy in 7 patients. Complete thrombosis within the pseudoaneurysm was immediately induced after treatment. One early recurrence required a second injection. No complication of thrombin was noted and no surgery was required. CONCLUSION: US-guided thrombin injection is an effective treatment for embolisation of pseudoaneurysms. The technique is superior to compression therapy.

Nottingham Health Profile and Short-Form 36 Health Survey questionnaires in patients with chronic lower limb ischemia: before and after revascularization.

OBJECTIVE: The purpose of this study was to compare the usefulness of the Nottingham Health Profile (NHP) and the Short-Form 36 Health Survey (SF-36) as general outcome measures after vascular intervention for lower limb ischemia with respect to patients' quality of life, on the basis of validity, reliability, and responsiveness analyses. PATIENTS AD METHODS: Eighty patients, 40 with claudication and 40 with critical ischemia, were assessed before and one month after revascularization by using comparable domains of the NHP and the SF-36 questionnaires. RESULTS: The SF-36 scores were less skewed and were distributed more homogeneously than the NHP scores. Discriminate validity results showed that NHP was better than SF-36 in discriminating among levels of ischemia with respect to pain and physical mobility. For both questionnaires, the reliability standards were satisfactory in most respects. The NHP was more responsive than the SF-36 in detecting within-patient changes. All of the NHP domains not zero at baseline were improved significantly one month after hemodynamically successful revascularization for patients with claudication, whereas patients with critical ischemia showed significant abatement of pain and improvements in physical mobility and social isolation. The SF-36 scores indicated a significant decrease in bodily pain and improvements in physical functioning and vitality for patients with claudication, and decrease in bodily pain and improvement in physical functioning for patients with critical ischemia. CONCLUSIONS: The findings indicated that both NHP and SF-36 were reliable. The SF-36 scores were less skewed than the NHP scores, whereas NHP discriminated better among levels of ischemia and was more responsive in detecting quality-of-life changes over time than SF-36 in these particular patients.

The kinase insert domain-containing receptor (KDR) is regulated by shear stress.

OBJECTIVE: Intimal hyperplasia develops in areas with low shear stress. The aim of the present study was to investigate if the mRNA expression of vascular endothelial growth factor (VEGF), Fms-like tyrosine kinase-1 receptor (Flt-1) and kinase insert domain-containing receptor (KDR) is regulated by shear stress. DESIGN: Endothelial cells from human umbilical veins were in an in vitro system subjected to different levels of shear stress during 1 and 12 h. The mRNA expression of VEGF, Flt-1 and KDR was measured with RT-PCR. eNOS served as positive control and actin as housekeeping gene. RESULTS: The KDR expression was isolated upregulated 3-4 times after 12 h exposure to high shear stress. CONCLUSION: The genetic expression of KDR is upregulated by shear stress and this response is time dependent.

Angiogenesis in invasive breast carcinoma--a prospective study of tumour heterogeneity.

Assessment of angiogenesis has been reported to be an independent prognostic factor in breast cancer, while other studies have been negative. This study prospectively investigates the degree of intratumoral microvessel heterogeneity and the possible influence on the results. From 21 invasive breast cancers six 4 micro sections were cut. Sections (n=126) were stained immunohistochemically with a CD31 monoclonal antibody (JC70). In each section, three areas with the most intense neovascularisation (hot spots) were identified and the microvessel density (MVD) was obtained by counting vessels at 200x magnification. The variation between sections contributed more to the total variance than variation between different tumours: 45.0 and 37.3%, respectively, according to a nested ANOVA analysis. Paired comparisons of two sections at a time from the same tumour showed a concordance in 59.0% (95% Confidence Interval (CI): (55.3-62.8)) with reference to a tentative cut-off level. Our study demonstrates that assessment of MVD in hot spots is questionable to measure angiogenesis due to the considerable intratumoral heterogeneity.

Expression levels of the nerve growth factor receptors TrkA and p75 in effusions and solid tumors of serous ovarian carcinoma patients.

PURPOSE: The purpose of this study was to analyze the expression of the high- and low-affinity nerve growth factor (NGF) receptors TrkA and p75 in effusions and in primary and metastatic tumors of serous ovarian carcinoma patients, as well as to evaluate their association with clinicopathological parameters and disease outcome. EXPERIMENTAL DESIGN: Sections from 77 malignant effusions and 78 primary and metastatic lesions were evaluated for protein expression of TrkA and p75 using immunohistochemistry (IHC). Expression of the phosphorylated form of TrkA (p-TrkA) was evaluated in 75 effusions using IHC. TrkA and p75 mRNA expression was studied in 44 effusions using reverse transcription-PCR (RT-PCR). RESULTS: TrkA protein membrane expression was detected in carcinoma cells in 30 of 77 (39%) effusions and 64 of 78 (82%) solid tumors. The decrease in TrkA expression in effusions approached, but did not reach, statistical significance when only corresponding lesions were analyzed (P = 0.06 in the comparison of effusions and primary tumors, P = 0.09 for effusions and metastases). Conversely, p75 protein membrane expression was more common in effusions, which was detected in 16 of 77 (21%) effusions as compared with 6 of 78 (8%) solid tumors (P > 0.05 in analysis of corresponding lesions). Expression of p-TrkA in carcinoma cells was limited to 5 of 75 effusions. Interestingly, 11 of 16 p75-positive effusions were also immunoreactive for the antibody against TrkA (P = 0.001), suggesting NGF activation using two signaling pathways. TrkA and p75 protein expression in tumor cells was similar in pleural and peritoneal effusions (P > 0.05). Using reverse transcription-PCR, TrkA mRNA was detected in 2 of 45 effusions, whereas p75 mRNA was present in 3 of 45 specimens. TrkA and p75 showed no association with tumor grade, Fédération Internationale des Gynaecologistes et Obstetristes stage, chemotherapy status, the extent of residual disease, or survival (P > 0.05). CONCLUSIONS: TrkA and p75 are both expressed in advanced-stage ovarian carcinoma, but whereas p75 expression is elevated in effusions, TrkA shows an opposite trend. The different expression of NGF receptors in effusions may relate to the different microenvironment and growth factor availability in body cavities, as also supported by the infrequent activation of TrkA in effusions. The similar expression of TrkA and p75 in carcinoma cells in pleural and peritoneal effusions provides further evidence for our hypothesis that there are few, if any, phenotypic differences between ovarian carcinoma cells at these two sites. TrkA and p75 expression in effusions does not appear to be a predictor of disease outcome in advanced-stage serous ovarian carcinoma.

Ets-1 mRNA expression in effusions of serous ovarian carcinoma patients is a marker of poor outcome.

Ets-1 proto-oncogene is a transcription factor with a role in the activation of metastasis-associated molecules. We recently found that Ets-1 mRNA expression in solid tumors is a marker of poor prognosis in ovarian carcinoma. The objective of this study was to compare the expression of Ets-1 mRNA in effusions and primary and metastatic tumors of serous ovarian carcinoma patients and to evaluate its prognostic role in effusions. Sections from 67 malignant effusions and 90 primary and metastatic lesions were evaluated for expression of Ets-1 using mRNA in situ hybridization. Expression of Ets-1 mRNA was detected in carcinoma cells in 24 of 67 (36%) effusions. Expression in cancer cells was similar in peritoneal and pleural effusions. In solid lesions Ets-1 expression was detected in both tumor cells and stromal cells in 34 of 90 (38%) lesions. Ets-1 expression in tumor cells showed a strong association with that of stromal cells (p <0.001). Ets-1 expression in effusions showed an association with mRNA expression of basic fibroblast growth factor, previously studied in this patient cohort (p = 0.019). Ets-1 expression in solid lesions showed an association with mRNA expression of vascular endothelial growth factor (p <0.001 for both carcinoma and stromal cells), basic fibroblast growth factor (p = 0.007 for carcinoma cells, p = 0.006 for stromal cells), and interleukin-8 (IL-8) (p = 0.001 for tumor cells). Ets-1 mRNA showed upregulation in metastases when compared with effusion specimens (p = 0.028). In univariate survival analysis Ets-1 expression in carcinoma cells in effusions correlated with poor survival (p = 0.003). Our findings confirm the role of Ets-1 as a novel prognostic marker in advanced-stage ovarian carcinoma and extend it to effusion specimens. The elevated expression in solid metastases supports a central role in tumor progression as well. The association between Ets-1 mRNA expression and the expression of angiogenic genes, documented also in our previous study, points to the close link between these molecules, in agreement with the role of angiogenic genes in the transcriptional activation of Ets-1. The identical phenotype of carcinoma cells in pleural and peritoneal effusions provides further evidence for our theory that cells at these sites share similar genotypic and phenotypic profiles.

Aneurysm sac hygroma: a cause of endotension.

PURPOSE: To describe a new pathophysiological mechanism for endotension. CASE REPORTS: Four patients developed aneurysm sac expansion after repair of abdominal aortic aneurysms, one with a conventional polytetrafluoroethylene (PTFE) graft and the others with a variety of commercially made endografts (2 PTFE, 1 Dacron). Pressures within the sacs were nonpulsatile and approximately half the systemic blood pressure. Attenuation on computed tomography (CT) was significantly less in the sac than in the graft in 3 of the patients. A clear, highly viscous fluid was aspirated from all 4 sacs, supporting the diagnosis of aneurysm sac hygroma. Prominent local hyperfibrinolysis in the sac was combined with signs of local coagulation activation. CONCLUSIONS: A new mechanism for continued sac expansion based on aneurysm sac hygroma is proposed. Measurement of attenuation may be of diagnostic value. It is further proposed that local hyperfibrinolysis/coagulation may promote rebleeding, liquefaction, and continued expansion analogous to the chronic subdural hematoma.

Detection of monocyte/macrophage cell populations in effusions: a comparative study using flow cytometric immunophenotyping and immunocytochemistry.

The objective of the present study was to compare the efficiency of immunophenotyping using flow cytometry (FCM) and immunocytochemistry (ICC) in the detection of macrophages in serous effusions. Cytoblock sections from 90 effusions were stained for the monocyte/macrophage marker CD14, using ICC. Fresh-frozen samples of all cases were analyzed for CD14 expression, using FCM. Epithelial, lymphoid, and mesothelial cell populations were identified using antibodies against Ber-EP4, CD45, and N-cadherin, respectively. Results were compared with clinical parameters and morphological diagnosis. Thirty-nine specimens were cytologically diagnosed as malignant, containing tumor cells of nonhematologic origin, whereas 46 were interpreted as benign. Two additional specimens were diagnosed as indeterminate or suspicious for malignancy, and 3 specimens contained lymphoma cells. CD14-positive cells were detected in 85/90 (94%) of effusions using FCM, and in all 90 specimens using ICC. The percentage of CD14-positive cells was highly variable, but in some specimens was as high as 76% using FCM and 85% using ICC. A good association was observed between the two methods in the detection of CD14-positive cells (P < 0.001). The presence of macrophages in effusions showed an association with female gender, using both FCM (P = 0.002) and ICC (P = 0.011), but none with effusion site, patient age, clinical and cytological diagnosis, or presence of Ber-EP4-positive cells (P > 0.05). The presence of Ber-EP4-positive cells showed a strong association with the cytological diagnosis of malignancy (P < 0.001). In conclusion, macrophages are a significant cell population in effusions, of both benign and malignant etiology, due to both their size and their possible confusion with cancer cells. Both FCM and ICC aid in the recognition of these cells, and thus provide an effective tool for the identification of different cell populations in effusions.

The role of desmin and N-cadherin in effusion cytology: a comparative study using established markers of mesothelial and epithelial cells.

The objective of the present study was to analyze the role of the mesothelial markers desmin and N-cadherin in the diagnostic panel of serous effusions. A total of 181 pleural and peritoneal effusions consisted of 101 cases cytologically diagnosed as malignant (89 carcinomas, 12 mesotheliomas), 78 benign, and 2 inconclusive specimens. All specimens were immunostained using 11 antibodies, against epithelial membrane antigen, Ber-EP4, carcinoembryonic antigen, E-cadherin, CA 125, N-cadherin, desmin, calretinin, p53, vimentin, and CD45. After evaluation of immunocytochemistry results, 110 specimens were diagnosed as malignant (98 carcinomas, 12 mesotheliomas) and 71 as benign (56 cellular, 15 paucicellular). The presence of desmin was detected in benign mesothelial cells in 47 of 56 (84%) reactive cellular specimens compared with 1 of 12 (8%) malignant mesotheliomas and 2 of 98 (2%) carcinomas. N-cadherin was expressed in 48 of 56 (86%) reactive cases, 12 of 12 (100%) mesotheliomas, and 47 of 98 (48%) carcinomas. In carcinomas, N-cadherin expression was most often seen in ovarian carcinoma but was also found in other carcinomas. Calretinin, an established marker of mesothelial cells, was detected in 52 of 56 (93%) reactive specimens, 11 of 12 (93%) mesotheliomas, and 3 of 98 (3%) carcinomas. Evaluation of staining results led to reclassification of six malignant specimens as benign, whereas 17 cases diagnosed as benign and the two diagnosed as inconclusive were classified as malignant. In conclusion, desmin appears to be a promising marker for the distinction between reactive mesothelium and malignant epithelial cells in terms of both specificity and sensitivity, and its complementary use with calretinin is recommended. Unlike calretinin, it may also prove valuable for the distinction between benign and malignant mesothelial cells. N-cadherin does not have a role in the distinction between mesothelial and epithelial cells. However, it may prove useful in the characterization of carcinomas of unknown origin. As has previously been shown, a significant number of diagnoses that are based on morphologic examination alone are modified after the use of a broad antibody panel.