Gene Expression Patterns Related to Vascular Invasion and Aggressive Features in Endometrial Cancer
2011; Elsevier BV; Volume: 178; Issue: 2 Linguagem: Inglês
10.1016/j.ajpath.2010.10.040
ISSN1525-2191
AutoresMonica Mannelqvist, Ingunn M. Stefansson, Geir Bredholt, Trond Hellem Bø, Anne M. Øyan, Inge Jonassen, Karl‐Henning Kalland, Helga B. Salvesen, Lars A. Akslen,
Tópico(s)RNA Research and Splicing
ResumoThe presence of tumor cells entering vascular channels is a prognostic marker for many cancers, including endometrial carcinoma. Vascular invasion is considered to be an early step in the metastatic process and important for the progress of malignant tumors. Here, we investigated the gene expression patterns related to vascular involvement in 57 primary endometrial cancers, using DNA microarray and quantitative PCR techniques. A vascular invasion signature of 18 genes was significantly associated with patient survival and clinicopathological phenotype. Vascular involvement was also related to gene sets for epithelial-mesenchymal transition, wound response, endothelial cells, and vascular endothelial growth factor (VEGF) activity. With immunohistochemical validation, both collagen 8 and matrix metalloproteinase 3 (MMP3) were associated with vascular invasion, whereas ANGPTL4 and IL-8 were associated with patient survival. Our findings indicate that vascular involvement within primary tumors is associated with gene expression profiles related to angiogenesis and epithelial-mesenchymal transition. These data could contribute to an improved understanding of potential targets for metastatic spread and may provide clinically important information for better management of endometrial cancer. The presence of tumor cells entering vascular channels is a prognostic marker for many cancers, including endometrial carcinoma. Vascular invasion is considered to be an early step in the metastatic process and important for the progress of malignant tumors. Here, we investigated the gene expression patterns related to vascular involvement in 57 primary endometrial cancers, using DNA microarray and quantitative PCR techniques. A vascular invasion signature of 18 genes was significantly associated with patient survival and clinicopathological phenotype. Vascular involvement was also related to gene sets for epithelial-mesenchymal transition, wound response, endothelial cells, and vascular endothelial growth factor (VEGF) activity. With immunohistochemical validation, both collagen 8 and matrix metalloproteinase 3 (MMP3) were associated with vascular invasion, whereas ANGPTL4 and IL-8 were associated with patient survival. Our findings indicate that vascular involvement within primary tumors is associated with gene expression profiles related to angiogenesis and epithelial-mesenchymal transition. These data could contribute to an improved understanding of potential targets for metastatic spread and may provide clinically important information for better management of endometrial cancer. Invasion and metastatic spread are important for the progress of malignant tumors,1Hanahan D. Weinberg R.A. The hallmarks of cancer.Cell. 2000; 100: 57-70Abstract Full Text Full Text PDF PubMed Scopus (23139) Google Scholar and the occurrence of metastases represents an advanced stage of most cancers. The presence of vascular invasion (ie, tumor cells entering vascular channels as demonstrated by microscopic examination of the primary tumor) has been a strong prognostic factor in studies of various malignant tumors, including breast cancer, prostate cancer, endometrial cancer, and cutaneous melanoma.2Ferrari M.K. McNeal J.E. Malhotra S.M. Brooks J.D. Vascular invasion predicts recurrence after radical prostatectomy: stratification of risk based on pathologic variables.Urology. 2004; 64: 749-753Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 3Mohammed R.A. Martin S.G. Gill M.S. Green A.R. Paish E.C. Ellis I.O. Improved methods of detection of lymphovascular invasion demonstrate that it is the predominant method of vascular invasion in breast cancer and has important clinical consequences.Am J Surg Pathol. 2007; 31: 1825-1833Crossref PubMed Scopus (155) Google Scholar, 4Stefansson I.M. Salvesen H.B. Immervoll H. Akslen L.A. Prognostic impact of histological grade and vascular invasion compared with tumour cell proliferation in endometrial carcinoma of endometrioid type.Histopathology. 2004; 44: 472-479Crossref PubMed Scopus (68) Google Scholar, 5Straume O. Akslen L.A. Independent prognostic importance of vascular invasion in nodular melanomas.Cancer. 1996; 78: 1211-1219Crossref PubMed Scopus (56) Google Scholar Vascular invasion is regarded as an indicator of metastatic spread evident in the primary tumor.6Woodhouse E.C. Chuaqui R.F. Liotta L.A. General mechanisms of metastasis.Cancer. 1997; 80: 1529-1537Crossref PubMed Google Scholar Less is known about the molecular pathogenesis and characteristics of these early steps of metastatic dissemination. We recently found that vascular invasion by tumor cells in endometrial cancer was associated with features of aggressive tumors and reduced survival, as shown by multivariate prognostic analysis.7Mannelqvist M. Stefansson I. Salvesen H.B. Akslen L.A. Importance of tumour cell invasion in blood and lymphatic vasculature among patients with endometrial carcinoma.Histopathology. 2009; 54: 174-183Crossref PubMed Scopus (40) Google Scholar We then showed that vascular invasion was more frequent in tumors with an activated angiogenesis and less mature tumor vessels.8Stefansson I.M. Salvesen H.B. Akslen L.A. Vascular proliferation is important for clinical progress of endometrial cancer.Cancer Res. 2006; 66: 3303-3309Crossref PubMed Scopus (113) Google Scholar Our findings support a relationship between active angiogenesis, immature vessels, and metastatic spread in these malignant tumors. In the present study, tissues from primary endometrial cancers were examined for differential gene expression according to their status of vascular involvement. Although studies of various tumors have reported gene signatures related to the presence of metastases,9van 't Veer L.J. Dai H. van de Vijver M.J. He Y.D. Hart A.A. Mao M. Peterse H.L. van der Kooy K. Marton M.J. Witteveen A.T. Schreiber G.J. Kerkhoven R.M. Roberts C. Linsley P.S. Bernards R. Friend S.H. Gene expression profiling predicts clinical outcome of breast cancer.Nature. 2002; 415: 530-536Crossref PubMed Scopus (7982) Google Scholar the idea of vascular invasion representing a morphological marker of metastatic spread has received little attention. We here present novel data indicating that vascular involvement within primary tumors is associated with certain gene expression profiles related to angiogenesis and epithelial-mesenchymal transition, and the findings indicate that individual markers are involved in this process, including ANGPTL4, collagen VIII (COL8), matrix metalloproteinase 3 (stromelysin 1, progelatinase) (MMP3), and IL-8. Our data might contribute to an improved understanding of targets for metastatic spread and may provide clinically important information for better management of endometrial cancer. Microarray and quantitative PCR (qPCR) experiments were performed on a prospectively collected test series of fresh-frozen specimens. Clinicopathological correlates and outcome were also studied in a retrospective validation series.10Salvesen H.B. Carter S.L. Mannelqvist M. Dutt A. Getz G. Stefansson I.M. Raeder M.B. Sos M.L. Engelsen I.B. Trovik J. Wik E. Greulich H. Bo T.H. Jonassen I. Thomas R.K. Zander T. Garraway L.A. Oyan A.M. Sellers W.R. Kalland K.H. Meyerson M. Akslen L.A. Beroukhim R. Integrated genomic profiling of endometrial carcinoma associates aggressive tumors with indicators of PI3 kinase activation.Proc Natl Acad Sci USA. 2009; 106: 4834-4839Crossref PubMed Scopus (251) Google Scholar During the years 2001 to 2003, 57 cases of endometrial cancer (median age 63.0 years) were prospectively collected at the Department of Gynecology and Obstetrics, Haukeland University Hospital, University of Bergen, Norway. Patients were treated with hysterectomy and salpingo-oophorectomy. Fresh tumor tissue was carefully dissected from the surgical specimens and divided into two: one part was immediately frozen in liquid nitrogen and stored at −80°C for later use; the other half was fixed in formalin for histology. The 57 samples studied were selected at random from a population-based tissue bank of gynecologic cancers with tumor purity ensured to be ≥50% (and >80% neoplastic tissue in the majority of cases). A population-based series including all 316 cases of endometrial carcinoma diagnosed in Hordaland County (∼450,000 inhabitants, or 10% of the total Norwegian population) during the years 1981–1990 was used for validation. Of the 316 cases, 12 were excluded because of changed diagnosis at reclassification and 5 because of a diagnosis based on cytology only. Of the remaining 299 cases, paraffin blocks from the primary tumor were available in 286 cases. There was no significant difference in the distribution of clinicopathological variables between series I and II. The following histological variables were included for both series: histological type of primary tumor, histological grade (FIGO), presence of vascular invasion, tumor necrosis, number of mitoses, progesterone and estrogen receptor status, and presence of metastasis. Lymphocytic invasion around vessels at the periphery of the tumor was also recorded.8Stefansson I.M. Salvesen H.B. Akslen L.A. Vascular proliferation is important for clinical progress of endometrial cancer.Cancer Res. 2006; 66: 3303-3309Crossref PubMed Scopus (113) Google Scholar Presence of tumor cells within vascular spaces was examined on standard H&E sections as previously described.4Stefansson I.M. Salvesen H.B. Immervoll H. Akslen L.A. Prognostic impact of histological grade and vascular invasion compared with tumour cell proliferation in endometrial carcinoma of endometrioid type.Histopathology. 2004; 44: 472-479Crossref PubMed Scopus (68) Google Scholar Vascular invasion was considered present when more than one space was occupied by tumor cells; no spaces or a single space occupied was recorded as no vascular invasion. For series I, patients were followed from the time of primary surgery until June 2007 or death. Median follow-up time for survivors was 3.6 years (range, 0.8–5.5 years). All events (14 recurrences, 7 deaths from cancer) were recorded; deaths due to other causes were censored. No patient was lost to follow-up. For series II, patients were followed from the time of primary surgery until death or last follow up as of August 2004. Median follow-up time for survivors was 17 years (range, 10–23 years); 74 patients died from endometrial cancer. Total RNA was isolated from tumor samples as previously described.10Salvesen H.B. Carter S.L. Mannelqvist M. Dutt A. Getz G. Stefansson I.M. Raeder M.B. Sos M.L. Engelsen I.B. Trovik J. Wik E. Greulich H. Bo T.H. Jonassen I. Thomas R.K. Zander T. Garraway L.A. Oyan A.M. Sellers W.R. Kalland K.H. Meyerson M. Akslen L.A. Beroukhim R. Integrated genomic profiling of endometrial carcinoma associates aggressive tumors with indicators of PI3 kinase activation.Proc Natl Acad Sci USA. 2009; 106: 4834-4839Crossref PubMed Scopus (251) Google Scholar T7 RNA polymerase promoter containing cDNAs and Cy3- and Cy5-labeled cRNAs were generated and hybridized to Agilent 21k and 22k microarrays (Agilent Technologies, Santa Clara, CA).10Salvesen H.B. Carter S.L. Mannelqvist M. Dutt A. Getz G. Stefansson I.M. Raeder M.B. Sos M.L. Engelsen I.B. Trovik J. Wik E. Greulich H. Bo T.H. Jonassen I. Thomas R.K. Zander T. Garraway L.A. Oyan A.M. Sellers W.R. Kalland K.H. Meyerson M. Akslen L.A. Beroukhim R. Integrated genomic profiling of endometrial carcinoma associates aggressive tumors with indicators of PI3 kinase activation.Proc Natl Acad Sci USA. 2009; 106: 4834-4839Crossref PubMed Scopus (251) Google Scholar, 11Halvorsen O.J. Oyan A.M. Bø T.H. Olsen S. Rostad K. Haukaas S.A. Bakke A.M. Marzolf B. Dimitrov K. Stordrange L. Lin B. Jonassen I. Hood L. Akslen L.A. Kalland K.H. Gene expression profiles in prostate cancer: association with patient subgroups and tumour differentiation.Int J Oncol. 2005; 26: 329-336PubMed Google Scholar, 12Rostad K. Mannelqvist M. Halvorsen O.J. Oyan A.M. Bo T.H. Stordrange L. Olsen S. Haukaas S.A. Lin B. Hood L. Jonassen I. Akslen L.A. Kalland K.H. ERG upregulation and related ETS transcription factors in prostate cancer.Int J Oncol. 2007; 30: 19-32PubMed Google Scholar Generation of Cy3 cRNA targets from human umbilical vein endothelial cell (HUVEC) and human dermal microvascular cell (HMVEC) total RNAs and Cy5 cRNA reference targets from Stratagene universal RNA (Stratagene, La Jolla, CA) and hybridization using human 44k whole-genome microarrays (G4112; Agilent Technologies) were done as previously described.13Øyan A.M. Anensen N. Bø T.H. Stordrange L. Jonassen I. Bruserud Ø. Kalland K.H. Gjertsen B.T. Genes of cell-cell interactions, chemotherapy detoxification and apoptosis are induced during chemotherapy of acute myeloid leukemia.BMC Cancer. 2009; 9: 77Crossref PubMed Scopus (19) Google Scholar Microarrays were scanned and features were extracted using the Agilent microarray scanner bundle. Data including foreground and background intensity for microarray spots were processed using the J-Express software.14Dysvik B. Jonassen I. J-Express: exploring gene expression data using Java.Bioinformatics. 2001; 17: 369-370Crossref PubMed Scopus (281) Google Scholar For each spot the background corrected intensity value for each channel (Cy5, Cy3) was calculated. The lowess method15Yang Y.H. Buckley M.J. Speed T.P. Analysis of cDNA microarray images.Brief Bioinform. 2001; 2: 341-349Crossref PubMed Scopus (165) Google Scholar was used to correct for dye-specific effects, under the assumption that most genes would have unchanged expression levels. Filtration was done as previously described,11Halvorsen O.J. Oyan A.M. Bø T.H. Olsen S. Rostad K. Haukaas S.A. Bakke A.M. Marzolf B. Dimitrov K. Stordrange L. Lin B. Jonassen I. Hood L. Akslen L.A. Kalland K.H. Gene expression profiles in prostate cancer: association with patient subgroups and tumour differentiation.Int J Oncol. 2005; 26: 329-336PubMed Google Scholar and missing values were estimated and inserted using the LSimpute_adaptive method.16Bø T.H. Dysvik B. Jonassen I. LSimpute: accurate estimation of missing values in microarray data with least squares methods.Nucleic Acids Res. 2004; 32: e34Crossref PubMed Scopus (277) Google Scholar Batch adjustment was also performed.17Engelsen I.B. Mannelqvist M. Stefansson I.M. Carter S.L. Beroukhim R. Øyan A.M. Otte A.P. Kalland K.H. Akslen L.A. Salvesen H.B. Low BMI-1 expression is associated with an activated BMI-1-driven signature, vascular invasion, and hormone receptor loss in endometrial carcinoma.Br J Cancer. 2008; 98: 1662-1669Crossref PubMed Scopus (41) Google Scholar The Leave One Out Cross-Validation (LOOCV) method was used to test the predictability of presence (VI+) or absence (VI−) of vascular invasion, using all except one sample for training data in each round. In this method, a subset of genes is selected to maximize the possibility of discriminating the two groups, based on the training samples. Thus, the gene set is not fixed, but changes each round, based on which samples are left out. The classifier is tested on the one remaining patient, and the result (VI+ or VI−) is compared with the true status. The selected genes were used in a diagonal linear discriminant model for prediction. To test prediction accuracy achieved against pure chance, a permutation test was performed, in which labels (VI+ or VI−) were randomly permuted 1000 times, and subsequently LOOCV was performed for each label permutation. The results were compared with those obtained with the original labeling to estimate the significance of the result. The significance analysis of microarrays (SAM) method18Tusher V.G. Tibshirani R. Chu G. Significance analysis of microarrays applied to the ionizing radiation response.Proc Natl Acad Sci USA. 2001; 98 ([Erratum appeared in Proc Natl Acad Sci U S A 2001, 98:10515]): 5116-5121Crossref PubMed Scopus (9873) Google Scholar was applied to look for differential expression between samples labeled vascular invasion positive (VI+) and negative (VI−), with an additional requirement of at least twofold change between the categories. The PANTHER gene expression tool (http://www.pantherdb.org/tools/)was applied to map and correlate gene sets with molecular functions, biological processes, and biological pathways.19Leblond C.P. Inoue S. Structure, composition, and assembly of basement membrane.Am J Anat. 1989; 185: 367-390Crossref PubMed Scopus (227) Google Scholar, 20Thomas P.D. Kejariwal A. Guo N. Mi H. Campbell M.J. Muruganujan A. Lazareva-Ulitsky B. Applications for protein sequence-function evolution data: mRNA/protein expression analysis and coding SNP scoring tools.Nucleic Acids Res. 2006; 34: W645-W650Crossref PubMed Scopus (406) Google Scholar A minimum fold change (FC) of 1.5 and a false discovery rate (FDR) of <25% regarding vascular invasion yielded 127 up-regulated and 92 down-regulated genes; these were used for PANTHER analysis. Groups with P values of 0.65) to 1 of the 28 signature genes, resulting in a final expanded signature of 468 genes.Table 1Clinicopathologic Characteristics According to Vascular Invasion in 57 Endometrial Carcinomas from which Fresh Tissues Were Prospectively CollectedVariablesVI negative, no. (%)VI positive, no. (%)P value⁎Pearson's chi-square test.Histologic type0.017 Endometrioid34 (97)17 (77) Non-endometrioid1 (3)5 (23)Histologic grade<0.001 Grades 1 and 233 (94)11 (50) Grade 32 (6)11 (50)Necrosis<0.001 Absent20 (57)2 (9) Present15 (43)20 (91)Mitoses†Median value used as cutpoint.NS‡No significant difference between VI+ and VI− patients. Low28 (80)14 (64) High7 (20)8 (36)ER0.269 Positive27 (77)14 (64) Negative8 (23)8 (36)PR0.053 Positive30 (86)14 (64) Negative5 (14)8 (36)FIGO stage0.009 I/II33 (94)15 (68) III/IV2 (6)7 (32)ER, estrogen receptor; PR, progesterone receptor; FIGO, International Federation of Gynecology and Obstetrics; NS, no significant difference. Pearson's chi-square test.† Median value used as cutpoint.‡ No significant difference between VI+ and VI− patients. Open table in a new tab ER, estrogen receptor; PR, progesterone receptor; FIGO, International Federation of Gynecology and Obstetrics; NS, no significant difference. Because vascular invasion is related to the epithelial-mesenchymal transition, known to be induced by tumor growth factor β (TGF-β), which is also related to ANGPTL4, two gene sets related to TGF-β signaling were included: a TGF-β response signature reported by Padua et al25Padua D. Zhang X.H. Wang Q. Nadal C. Gerald W.L. Gomis R.R. Massagué J. TGFbeta primes breast tumors for lung metastasis seeding through angiopoietin-like 4.Cell. 2008; 133: 66-77Abstract Full Text Full Text PDF PubMed Scopus (782) Google Scholar (153 genes) and a 217-gene TGF-β signature (Ambion GeneAssist pathway atlas; Applied Biosystems, Austin, TX). Mapping was done using gene symbols and gene ID, and 121 and 163 common genes, respectively, were found. We studied the 13 genes in a VEGF-related gene signature published by Hu et al26Hu Z. Fan C. Livasy C. He X. Oh D.S. Ewend M.G. Carey L.A. Subramanian S. West R. Ikpatt F. Olopade O.I. van de Rijn M. Perou C.M. A compact VEGF signature associated with distant metastases and poor outcomes.BMC Med. 2009; 7: 9Crossref PubMed Scopus (149) Google Scholar that are highly expressed in patients with distant breast cancer metastasis, compared with patients with primary tumors only; all 13 genes were identified, including ANGPTL4 in duplicate. An average expression value across the 13 genes [RRAGD, FABP5, UCHL1, GAL, PLOD1 (previously PLOD), DDIT4, VEGFA (previously VEGF), ADM, ANGPTL4, NDRG1, PNP (previously NP),SLC16A3, and FLVCR2 (previously C14orf58)]was calculated for each patient and divided into two groups with high and low expression by the median value. Samples were clustered (unsupervised hierarchical clustering, weighted average linkage WPGMA, Pearson's correlation) based on the expression levels of the signature genes. The resulting clusters of samples were tested for associations with vascular invasion status. For a TaqMan low-density array (Applied Biosystems, Foster City, CA), qPCR confirmation genes (87 genes in addition to controls) and candidate genes significantly up-regulated or down-regulated in relation to vascular invasion (from SAM; n = 30) were included and were combined with 57 hypothesis-based genes related to angiogenesis and invasion. From a base of 300 genes compiled from the literature, hypothesis-based genes were selected based on their overall associations in our dataset (series I) with five basic clinicopathological variables: vascular invasion, mitosis, tumor cell necrosis, FIGO stage, and metastatic phenotype (ranked by the combined P value, a product of individual P values). For TaqMan low-density array, a 384-well microfluidic card was constructed with two control genes [GAPDH (assay ID Hs99999905_m1) and ACTB (assay ID Hs99999903_m1)] and selected candidate genes, all in duplicate. cDNA synthesis and PCR protocols were performed as previously described.17Engelsen I.B. Mannelqvist M. Stefansson I.M. Carter S.L. Beroukhim R. Øyan A.M. Otte A.P. Kalland K.H. Akslen L.A. Salvesen H.B. Low BMI-1 expression is associated with an activated BMI-1-driven signature, vascular invasion, and hormone receptor loss in endometrial carcinoma.Br J Cancer. 2008; 98: 1662-1669Crossref PubMed Scopus (41) Google Scholar SDS2.2 software (Applied Biosystems) was used for analysis of the qPCR data. Data were imported to J-Express, and genes significantly related to vessel invasion (t-test, P < 0.05; fold change ≥ 2.0) were used for further studies. A summary score of the vascular invasion signature was obtained as the sum of normalized expression values for up-regulated genes with subtraction of corresponding values for down-regulated genes: signature summary score = Σ up-regulated genes − Σ down-regulated genes. Staining was performed on 5-μm sections of paraffin-embedded tissues, using either regular slides or tissue microarray slides.27Stefansson I.M. Salvesen H.B. Akslen L.A. Prognostic impact of alterations in P-cadherin expression and related cell adhesion markers in endometrial cancer.J Clin Oncol. 2004; 22: 1242-1252Crossref PubMed Scopus (135) Google Scholar Staining was performed with antibodies against ANGPTL4, collagen VIII, MMP3, IL-8, and N-cadherin. (For immunohistochemical protocols, see Supplemental Table S1 at http://ajp.amjpathol.org.) Tissue microarray slides were evaluated in a standard light microscope for immunohistochemical staining (by M.M., I.M.S., H.B.S.). Cytoplasmic staining intensity in tumor cells (grades 0–3) and staining area (0, no tumor cells positive; 1, 50%) were determined and multiplied, giving a staining index with scores 0–9.28Aas T. Børresen A.L. Geisler S. Smith-Sørensen B. Johnsen H. Varhaug J.E. Akslen L.A. Lønning P.E. Specific P53 mutations are associated with de novo resistance to doxorubicin in breast cancer patients.Nat Med. 1996; 2: 811-814Crossref PubMed Scopus (691) Google Scholar Cases were divided into two or four subgroups based on median or quartiles for staining index and also considering the size of these subgroups, number of events, and survival similarities. Regular slides were evaluated in a standard light microscope (by M.M., L.A.A.). The invasive tumor front was examined, and staining intensity in endothelial cells and tumor stroma was considered. Two categories were compared: cases with vascular invasion and lymphocytes around vessels (n = 20), and cases without such alterations (n = 20). Western blotting was used to examine the specificity of the antibodies as described earlier29Bachmann I.M. Puntervoll H.E. Otte A.P. Akslen L.A. Loss of BMI-1 expression is associated with clinical progress of malignant melanoma.Mod Pathol. 2008; 21: 583-590Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar Primary antibodies were diluted in non-fat 5% dry milk:ANGPTL4 1:500, collagen 8 1:500, IL-8 1:75, MMP3 1:50; incubation for 1 hour at room temperature. Secondary antibodies were diluted in non-fat dry milk: Envision rabbit 1:500 against ANGPTL4 and collagen 8 (Dako, Glostrup, Denmark), Envision mouse 1:500 against MMP3, and PowerVision poly-GRP anti-goat IgG (ImmunoVision, Springdale, AR) 1:100 against IL-8. Endometrial cancer cell lines KLE, RL95-2, AN3 CA, and HEC-1-A (American Type Culture Collection, ATCC, Manassas, VA) and MFE-280, MFE-296, and EFE-184 (German Collection of Microorganisms and Cell Cultures, DSMZ, Braunschweig, Germany) were grown in recommended media supplemented with 2 mmol/L L-glutamine and 1× penicillin-streptomycin-amphotericin B. Conditioned media were collected after 18 hours incubation (37°C, 5% CO2) in basal medium EBM-2 (Lonza, Allendale, NJ), and the medium was cleared by centrifugation. HUVEC and HMVEC cells (Lonza) were grown in EGM-2 and EGM-2MV media (Lonza), respectively, to subconfluence. Cells were then exposed to conditioned medium and nonconditioned medium (control) for 18 hours (37°C, 5% CO2). Cells were harvested using 350 μL TRK lysis buffer per well (E.Z.N.A. gel extraction kit;Omega Bio-Tek, Norcross, GA). Total RNA was purified in accordance with the manufacturer's manual (E.Z.N.A. total RNA kit I; Omega Bio-Tek). Statistical analyses were performed with the PASW statistical software package version 17 (SPSS, Chicago, IL). Associations between different categorical variables were assessed by Pearson's χ2 test or Fisher's exact test. Correlations between two continuous variables are given by Pearson's correlation coefficient and between a categorical and a continuous variable by Spearman's correlation coefficient. Kappa-statistics was used for interobserver reproducibility. Univariate survival analyses were performed using the Kaplan-Meier method (log-rank significance test). Vascular invasion and standard clinicopathological variables and biological markers were examined by log-log plot to determine how these variables could be incorporated in multivariate survival analysis using Cox' proportional hazards regression models (likelihood ratiosignificance test). Vascular invasion (ie, the presence of tumor cells in vascular channels) was observed in 22 of 57 cases in our prospective series (series I) (39%). This feature was associated with characteristics of aggressive tumors, such as nonendometrioid histological type (P = 0.017), high histological grade (P < 0.001), presence of tumor necrosis (P < 0.001), and advanced tumor stage by FIGO classification (P = 0.009) (Table 1). Vascular invasion was also associated with decreased patient survival (log-rank test, P = 0.028) (Figure 1A). These findings were confirmed in a separate and independent validation series of 286 cases (series II) (see Supplemental Table S2 at http://ajp.amjpathol.org); vascular invasion was present in 36% of cases and was strongly associated with reduced patient survival (Figure 1B). Furthermore, vascular invasion wa
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