{"paper_id":"da293eea-ec8d-44ab-8965-af9c41db5dd7","body_text":"456\npISSN 2383-7837\neISSN 2383-7845\n© 2017 The Korean Society of Pathologists/The Korean Society for Cytopathology\nThis is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ \nby-nc/ 4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.\nThe Potential Roles of MELF-Pattern, Microvessel Density, and VEGF \nExpression in Survival of Patients with Endometrioid Endometrial \nCarcinoma: A Morphometrical and Immunohistochemical Analysis \nof 100 Cases\nDmitry Aleksandrovich Zinovkin \nMd Zahidul Islam Pranjol1 \nDaniil Rudolfovich Petrenyov2 \nEldar Arkadievich Nadyrov3 \nOleg Gennadievich Savchenko4\nUniversity Research Laboratory, Gomel State \nMedical University, Gomel, Belarus; 1University of \nExeter Medical School, Institute of Biomedical and \nClinical Science, Exeter, Devon, United Kingdom; \n2Laboratory of Endocrinology and Biochemistry, \nInstitute of Radiobiology National Academy \nof Sciences, Gomel; \n3Laboratory of Clinical \nResearch, Republican Research Center for \nRadiation Medicine and Human Ecology, Gomel; \n4Department of Oncology, Gomel State Medical \nUniversity, Gomel, Belarus\nBackground: In this study, we hypothesized that microcystic, elongated, fragmented (MELF)-pat-\ntern, vascular endothelial growth factor (VEGF) expression by cancer cells and microvessel density \nof cancer stroma may be associated with progression of endometrioid adenocarcinoma. Methods: \nThe study used data from the Belarus Cancer Registry and archival histological material of 100 \npatients with retrospectively known good (survival) and poor (disease progression and death) out-\ncomes. All cases were immunohistochemically stained for CD34 and VEGF . Two independent \nsamples were compared for the characteristics of signs, and obtained results were analyzed by \nreceiver operating characteristic analysis, Mann-Whitney U test, \nχ2 test (Yates correction), and \nMantel-Cox test. Multivariate Cox hazard analysis and Spearman correlation test were used. A p-\nvalue of less than .05 was considered statistically significant. \nResults: The observed survival rate \nof patients with endometrioid adenocarcinoma was significantly lower (p = .002) in MELF-pattern \npositive patients when compared with MELF-pattern negative patients. The overall survival rate of \npatients whose tumors had more than 114 vessels/mm2 of tissue was significantly low (p < .001). \nInterestingly, a similar observation was found in patients with increased vessel area, evidenced by \nVEGF expression in the glandular tumor component. Conclusions: Our study suggests, for the \nfirst time, that these criteria may be used as risk factors of endometrioid adenocarcinoma pro-\ngression during 5 years after radical surgical treatment. However, a large independent cohort of \nsamples should be considered in the future to validate our findings.\nKey Words: Carcinoma, endometrioid; Vascular endothelial growth factor; Prognosis; MELF; Vessel \ndensity\nReceived: May 14, 2017\nRevised: July 6, 2017\nAccepted: July 19, 2017\nCorresponding Author\nDmitry Aleksandrovich Zinovkin, MD\nUniversity Research Laboratory, Gomel State \nMedical University, Lange Str. 5, Gomel, Belarus\nTel: +375-29-182-7416\nFax: +375-232-77-36-72\nE-mail: zinovkin2012@gmail.com\nJournal of Pathology and Translational Medicine 2017; 51: 456-462\nhttps://doi.org/10.4132/jptm.2017.07.19\n▒ ORIGINAL ARTICLE ▒\nThe stromal microenvironment of tumors is gradually becoming \na main focus in the field of cancer research. It is believed that \nmalignancy is a result of complex molecular and cellular inter-\nactions between the elements of tumor microenvironment and \nsurrounding host tissues which induce selection and expansion \nof the neoplastic cells.\n1 In 2005, Zigrino et al.2 reported an inter-\naction of tumor cells with the stromal elements during tumor \nprogression and paid special attention to the ability of the neo-\nplastic cells to modify stroma by changing the adjacent connective \ntissue and modulating cellular metabolism of the host. In such \ncircumstances, a new stroma is formed in areas of tumor invasion, \nincluding the parts of distant metastases, creating favorable \nconditions for the aggressive potential of tumor cells. This event \nin the areas of active cancer cell invasion is common in tumor \nprogression.\n2 \nMurray et al.3 introduced the acronym “MELF” (microcystic, \nelongated, fragmented) which describes the unusual changes in-\ncurred by the endometrioid adenocarcinoma (EA) when invading \ninto the myometrium. These changes are characterized by the \nformation of microcysts lined with eosinophilic cytoplasm, elon-\n\n\nhttp://jpatholtm.org/https://doi.org/10.4132/jptm.2017.07.19\nMELF, Vessels in Endometrial Carcinoma  •     457\ngated glandular structures, and clusters of individual cells. 3 \nThough earlier it was believed that this fibromyxoid stromal \nreaction was initially a degenerative process, a number of studies \nhave shown that the MELF pattern can be a specific tumor stroma \nreaction, similar to the epithelial-mesenchymal interactions \nobserved in other tumors.\n4\nAngiogenesis is defined by formation of new blood vessels \nfrom preexisting ones, playing a key role in uncontrolled prolif-\neration of cells, survival of localized malignant cells and distant \ntumor invasion. Increased microvessel density, an indirect marker \nof intense tumor vascularization, is known to be associated with \nboth evolution of the disease and patient survival. The formation \nof new vessels depends on the interaction between different \nhormones/growth factors and microvascular endothelial cells \nlining the existing microvessels.\n5,6 The endometrium expresses \nseveral growth factors involved in angiogenesis, including epidermal \ngrowth factor, transforming growth factor, and vascular endo-\nthelial growth factor (VEGF). VEGF is one of the most common \npromoters of angiogenesis, expressed even by the normal endo-\nmetrium. As an angiogenetic factor, VEGF stimulates proliferation \nof endothelial cells and also increases vascular permeability and \nprotein extravasations.\n7-9\nIn this study, we hypothesized that MELF-pattern, VEGF \nexpression by cancer cells, and microvessel density may be asso-\nciated with the progression of EA and survival of patients. \nTherefore, we investigated the role of vessel and stroma of tumor \nmicroenvironment and reported for the first time that these criteria \nmay be used as prognostic factors for EA of the uterine corpus.\nMATERIALS AND METHODS\nEthical approval \nThe study was approved by the Committees for Medical and \nHealth Sciences of Research Ethics of Republican Research \nCenter for Radiation Medicine and Human Ecology and Gomel \nState Medical University. Dispensation from the requirement of \npatient consent was granted.\nPatient characteristics\nThis retrospective study involved women with endometrial \nEA who were treated between January 2010 and December \n2012 in the Grodno region, Republic of Belarus. The inclusion \ncriteria for the study were stage I–III (International Federation \nof Gynecology and Obstetrics [FIGO], 2009), the presence of \nEA as the main cause of death, progression of the tumor, age \nbetween 45 and 80, hysterectomy, and an absence of malignant \ntumors in other parts of the body. The control subjects were selected \ncases of EA stage I–III with 5-year survival, age between 45 and \n80, and no history of other malignant tumors. The exclusion \ncriteria for the study were as follows: stage IV (FIGO, 2009), \ndeath from postoperative complications during the first month \nafter hysterectomy, death from causes not related to EA progression, \nLynch syndrome, synchronous and metachronous malignancies, \nand age less than 45 or more than 80. \nA total of 100 out of 156 cases of EA during the study period \nwere determined to be eligible for inclusion in the study. The \npower analysis demonstrated that the number of patients was \nsufficient to conduct further research. The study used data from \nthe Belarus Cancer Registry and archival histological material of \n100 patients with histopathological diagnosis of EA. Histologic \ntyping was performed according to the histological classification \nof endometrial cancer by the World Health Organization. Patients \nwere divided into two groups. The first group included 48 subjects \nwho had recurrence or died of EA within 5 years after diagnosis \n(unfavorable outcome). The second group consisted of 52 subjects \nwho had no recurrence or death within 5 years after diagnosis \n(favorable outcome). The average age in the group with favorable \noutcome was 62.7\n ± 10.1 years and it was 65.2 ± 9.6 years in the \ngroup with an unfavorable outcome. Patient characteristics by \nFIGO stage and tumor grade are presented in Table 1.\nHematoxylin and eosin staining protocol\nFive-micrometer-thick sections were prepared from the archival \nhistological blocks. They were mounted on microscopic slides. \nNext, the sections were deparaffinized in two portions of xylene \nand rehydrated in descending concentrations of ethanol. Subse-\nquently, they were stained with hematoxylin and eosin by standard \nmethods. The sections were cleansed in carbolic xylene, dehy-\ndrated in ascending alcohol concentrations, dried and mounted \nunder coverslips using Biomount medium (DAKO, Glostrup, \nTable 1. Characteristics of the patient groups by FIGO stage (2009) \nand tumor grade\nCharacteristic Unfavorable outcome group \n(n = 48)\nFavorable outcome group \n(n = 52)\nFIGO\n   I 20 23\n   II 23 23\n   III 5 6\nGrade\n   G1 17 16\n   G2 25 31\n   G3 6 5\nFIGO, International Federation of Gynecology and Obstetrics.\n\nhttp://jpatholtm.org/ https://doi.org/10.4132/jptm.2017.07.19\n458     •  Zinovkin DA, et al.\nDenmark).\nPrimary antibodies and detection system \nPrimary antibodies used in this study include the following: \nready-to-use monoclonal rabbit anti-CD34 (clone EP88) and \nready-to-use polyclonal rabbit anti-VEGF (Diagnostic Biosystems, \nPleasanton, CA, USA). Mouse/Rabbit PolyVue Plus HRP/DAB \nDetection System (Diagnostic Biosystems) was used for primary \nantibodies visualization.\nImmunohistochemical staining protocol\nThe 4–5-μm-thick sections of tissue on l-polylysine coated glass \nslides were deparaffinized and washed with distilled water for 3 \nminutes. Antigen retrieval was performed using antigen unmasking \nsolutions Tris-EDTA buffer (1 mM, pH 9.0) and citrate buffer \n(1 mM, pH 6.0), with preheating in the microwave at 800 W for \n5 minutes and at 600 W for 10 minutes, respectively. The sections \nwere then allowed to cool in the same solution. Endogenous \nperoxidase blocking was performed in 5% hydrogen peroxide for \n20 minutes, and blocking of nonspecific antibody binding was \nensured by incubating the sections in 5% casein in Tris-buffered \nsolution for 1 hour. Following a brief wash in Tris-buffered solution, \nthe sections were incubated in moist chamber at room temperature \nfor 2 hours with corresponding primary antibodies. Tissue sections \nwere then incubated at room temperature for 30 minutes with anti-\nmouse horseradish peroxidase secondary antibodies. Between each \nstep the sections were washed twice with Tris-buffered solution \nfor 5 minutes each. The reaction product was visualized with \n3.3’-diaminobenzidine staining for 5 minutes, followed by Mayer’s \nhematoxylin counter-staining.\n10\nMorphometry\nDetermination of tumor vessels of microvasculature was carried \nout in the field with the largest number of capillaries (hot spots). \nThe number and the area of vessels per 1 mm2 were determined \nby counting the number in 5 fields under the magnification of \n× 400. These results were converted to 1 mm2 area of the tumor \ntissue. The microscope Nikon Eclipse 50i with digital camera DS-\nF1 and NIS-Elements software (Nikon, Tokyo, Japan) was used \nfor this morphometric work.\nStatistical analysis\nAll data were presented by the median, lower and upper quar-\ntiles. A two-tailed Fisher test was used to compare the groups \naccording to the presence or absence of MELF-pattern. Mann-\nWhitney test and receiver operating characteristic (ROC)-analysis \nwere used for comparing the study groups based on the evaluated \ncriteria. Determining the confidence interval (CI) and the area \nunder the ROC-curve were the compulsory component of the \nROC-analysis. The quality prediction model was labelled excel-\nlent at area under the curve 0.9–1.0, very good at 0.8–0.9, good \nat 0.7–0.8, medium at 0.6–0.7, and unsatisfactory at 0.5–0.6. \nAccording to the threshold indicator, the patients were divided \ninto two groups for 5-year survival analysis by Mantel-Cox test. A \nSpearman correlation test was used for groups. A multivariate \nCox proportional hazard analysis was developed using stepwise \nregression (forward selection, enter/remove limits p\n = .10) to identify \nindependent predictors of outcomes. A p-value of less than .05 \nwas considered statistically significant. R v.3.4.0 free soft was \nused for statistical analysis. \nRESULTS\nMELF-pattern\nDistinctive changes in the glands that characterized the MELF-\npattern were related with fibromyxoid stromal reaction. For instance, \ninvasion of the myometrium by tumor glands showed that there \nis an absence of fibroblastic reaction (Fig. 1A). Intriguingly, fibro-\nmyxoid reaction compressing cancer glands were observed in \nthe MELF pattern as expected (Fig. 1B).\nThe MELF-pattern was observed in eight cases (16.7%) in the \ngroup with favorable outcome. Fibromyxoid changes were typical \nfor the MELF-pattern which was observed in 17 cases (56.7%) \nof EA in the group with unfavorable outcome. Statistical differ-\nence (p\n = .014) was detected by comparing the number of the \nMELF-pattern present in the two groups. The observed survival \nrate of a patient with EA was significantly lower (p = .002) when \nMELF pattern was present compared with when MELF-pattern \nwas not present (Fig. 2A).\nNumber of vessels\nIn the group with favorable outcome of the disease, the vessels \nwere mostly detected in a small or moderate amount, with an oval-\nshaped lumen. Slight atypia was observed in the endothelium \nwhere the basement membrane was visualized throughout the \ncross-section of the vessels (Fig. 1C). On the contrary, the vessels \nof the microvasculature within the hot spot areas in cases with \nunfavorable outcome had mostly irregularly-shaped lumen. \nThey were closely located to each other, often forming a densely \nbranching network. It should be noticed that the endothelium \nhad an irregular shape and an irregular intermittent basement \nmembrane in the unfavorable outcome group (Fig. 1D).\n\nhttp://jpatholtm.org/https://doi.org/10.4132/jptm.2017.07.19\nMELF, Vessels in Endometrial Carcinoma  •     459\nFig. 1. (A) Stroma without fibroblastic reaction and tumor glands invading the myometrium. (B) MELF-pattern of the stroma with fibromyxoid \nreaction compressing the cancer glands. (C) Vessels in EA stroma with round lumen in group of patients with favorable outcome (arrows, \nCD34 immunostaining). (D) A large number of unusual vessels with dilated lumens in group of patients with unfavorable outcome (arrows, \nCD34 immunostaining). (E) Weak focal expression of VEGF in glands of EA, commonly detectable in patients with good outcome (VEGF immu-\nnostaining). (F) Diffuse strong expression of VEGF in glands of EA in cases of unfavorable outcome (VEGF immunostaining). MELF, microcystic, \nelongated, fragmented; EA, endometrioid adenocarcinoma; VEGF, vascular endothelial growth factor.\nA\nC\nE\nB\nD\nF\n\nhttp://jpatholtm.org/ https://doi.org/10.4132/jptm.2017.07.19\n460     •  Zinovkin DA, et al.\nIn the group of patients with unfavorable outcome, the median \nnumber of vessels in 1 mm2 of EA tissue was 139.1 (range, 74.1 \nto 174.6), and in the group with favorable outcome the median \nwas 95.5 (range, 57.0 to 171.0). A significantly increased number \nof vessels were detected in the group with unfavorable outcome \n(p\n < .001; z = 5.625), compared to favorable outcome group.\nThe ROC-analysis of this index showed that the area under \nthe ROC-curve was 92.3% (95% CI, 82.5 to 97.6; p < .001). \nThe sensitivity was 86.7% (95% CI, 69.3 to 96.2), the specificity \nwas 96.7% (95% CI, 82.8 to 99.9), and the threshold value of \nthe index was 114.0 vessels/mm\n2.\nAfter studying the overall survival rate of patients with EA \ndepending on the number of vessels in 1 mm2 of tumor, it was \nfound that the survival rate was statistically lower (p < .001) in \npatients whose tumors had more than 114.0 vessels/mm2 of tumor \ntissue (Fig. 2B). \nArea of the vessels\nThe median of the vessel area in 1 mm2 of tumor tissue in \ngroup 1 was 4,904.1 µm2/mm2 (range, 4,400.1 to 6,245.1 µm2/\nmm2). The median of this index in the second group was 2,818.9 \nµm2/mm2 (range 1,348.2 to 5,449.8 µm2/mm2). A significantly \nlarger area of vessels was detected in the unfavorable outcome \ngroup (p\n < .001, z = 6.247) compared with the favorable out-\ncome group. \nAfter performing ROC-analysis of the vessel area in 1 mm2 of \ntumor tissue, it was found that the area under the ROC-curve \nwas 97.0% (95% CI, 89.0 to 99.7; p\n < .001). The sensitivity, \nspecificity, and threshold value of the index were 100% (95% \nCI, 88.4 to 100.0), 96.7% (95% CI, 82.8 to 99.9), and 3,541.2 \nµm\n2/mm2, respectively.\nThe study of the overall survival rate of patients with EA \ndepending on the vessel area of 1 mm2 of tumors showed that \nthe survival rate was statistically lower (p < .001) in patients \nwhose vascular area in tumors was more than 3,541.2 µm2/mm2 \n(Fig. 2C). \nVEGF\nThe expression of VEGF, one of the main stimulators of angio-\ngenesis, was observed in all cases of EA. Diffuse expression of \nthis marker was detected in the stroma and glandular component \nof EA. However, in cases of favorable outcome, a weak staining \nwas observed in the cytoplasm, indicating a lower expression of \nVEGF (Fig. 1E). In the glandular component of the tumor, VEGF \nDays\n0               1,000           2,000            3,000          4,000\n100\n80\n60\n40\n20\n0\nSurvival (%)\n≤ 3,541.2 μm2/mm2\n> 3,541.2 μm2/mm2\nC\nDays\n0               1,000           2,000            3,000          4,000\n100\n80\n60\n40\n20\n0\nSurvival (%)\n≤ 114 mm2\n> 114 mm2\nB\nDays\n0               1,000           2,000            3,000          4,000\n100\n80\n60\n40\n20\n0\nSurvival (%)\n≤ 58.1%\n> 58.1%\nD\nDays\n0               1,000           2,000            3,000          4,000\n100\n80\n60\n40\n20\n0\nSurvival (%)\nMELF–\nMELF+\nA\nFig. 2. Cumulative proportion survival. (A) MELF-pattern. (B) Number of vessels per 1 mm2. (C) Area of vessels per 1 mm2. (D) VEGF expres-\nsion by tumor glands. MELF, microcystic, elongated, fragmented; VEGF, vascular endothelial growth factor. \n\nhttp://jpatholtm.org/https://doi.org/10.4132/jptm.2017.07.19\nMELF, Vessels in Endometrial Carcinoma  •     461\nexpression was, however, more evident and uniform. In the group \nwith unfavorable outcome, an opposite result of immunohisto-\nchemistry was observed: the VEGF expression was strong, detected \nas brown staining foci in the cytoplasm of the tumor cells (Fig. 1F). \nIn cases with unfavorable outcome, the median of VEGF expres-\nsion was 82.1% (range, 59.1% to 100.0%) and it was statistically \nhigher (p < .001; z = 6.616) in comparison with the median of cases \nwith favorable outcome, which was 49.0% (range, 20.8% to 62.1%). \nThe ROC-analysis of VEGF expression showed that the area \nunder the ROC-curve was 99.8% (95% CI, 93.6 to 100.0; p < \n.001). The sensitivity was 100.0% (95% CI, 88.4 to 100.0) and \nthe specificity was 96.7% (95% CI, 82.8 to 99.9). The threshold \nvalue of the index was 58.1%. The survival rate was statistically \nlower (p < .001) in patients whose VEGF expression of the glandular \ntumor component was more than 58.1% (Fig. 2D), as expected.\nCorrelation analysis\nOur study demonstrated a significant correlation between the \nMELF-pattern and VEGF expression in both groups. For instance, \nthe correlation between the two was r = .541 (p < .001). This was \nalso observed between VEGF expression and the area of vessels \n(r\n = .762, p < .001) and number of vessels (r = .648, p < .001). Corre-\nlation analysis describes the changes in cancer stroma caused by \nVEGF expression in cancer cells.\nMultivariate Cox’s proportional hazard model\nA multivariate Cox’s regression analysis revealed that MELF-\npattern and the area and number of vessels per 1 mm2 of tumor \ntissue are independent prognostic factors of 5-year survival of \npatients with EA (Table 2).\nDISCUSSION\nOur study shows that the MELF pattern is more frequently \nobserved in cases with unfavorable outcome than in those with \nfavorable outcome, suggesting that the presence of MELF pattern \nmay be a prognostic factor for patient survival. It can be assumed \nthat MELF is a “medium” which enhances the spread of the tumor \ncells. After aggressive radiation therapy, a similar change of fibro-\nmyxoid response in the stoma was observed in squamous cell \ncarcinoma of the vulva, just as MELF pattern in EA.\n11 Immu-\nnohistochemical and genetic study of MELF pattern in EA dis-\nclosed stromal cell separation and disappearance and downregu-\nlation of E-cadherin expression.\n12 As pointed out by several \nauthors, these changes are probably crucial in increasing the inva-\nsive capacity of EA and intensification of its metastatic potential. \nIn the univariate analysis, the presence of stromal fibromyxoid \nreactions by MELF pattern was associated with an unfavorable \nprognosis of EA.\n8,10,11\nThe number and area of microvessels in our study showed \nstatistically significant difference between the survival rates of \npatients with favorable outcome and unfavorable outcome. This \ncan be used as a strong potential prognostic factor in the survival \nof patients with EA. Microvessel density in tumor-invaded tissue \nis increased by local angiogenesis that results in enhanced cancer \ncell proliferation during tumor progression. In gynecological \ncancer, angiogenesis is one of the crucial factors of tumor progres-\nsion and plays a significant role in the maintenance of the growth \nof malignancies and their metastatic potential.\n13 Some authors \nsuggested that the density of vessels of microvasculature is an \nindirect marker of the intensity of tumor vascularization, which \nis known to be associated with the progression of endometrial \ncancer and 5-year survival rate.\n14,15 In such reports, immunohis-\ntochemical marker CD34 was proven useful in the detection of \nthese endothelial cells.16 The immunohistochemical expression \nof CD34 by endothelial cells allows counting the number and \narea of tumor vessels, which are prognostic signs that do not \ndepend on other tumor characteristics, such as expressions of \nproliferation markers and adhesion molecules.\n17\nOur study shows VEGF expression as a predictor of survival \nin patients with EA. For instance, the higher the expression of \nVEGF in cancer cells, the lower the survival of the patients, as \npredicted in our study. Nowadays, VEGF is the most frequently \nstudied angiogenic promoter; its expression is observed in the \nnormal endometrium as well as in other uterine malignancies, \nalthough it is higher in cancer tissue when compared with normal.\n18 \nVEGF stimulates endothelial cell proliferation, but it also increases \nvascular permeability, which helps the tumor cells to migrate to \nmetastatic sites.19-21 Saito et al.22 reported that based on the surgical \nmaterials of 85 cases of EA, there was a significant VEGF expres-\nsion in both highly differentiated and moderately differentiated \ntumors compared to poorly differentiated ones. In addition, it was \nreported that estrogen levels decrease the expression of VEGF, \nwhich may be an indication of increased survival of patients \nwith EA.\n23 \nTable 2. Multivariate Cox’s proportional hazard model analysis of \nprognostic factors in patients with endometrioid adenocarcinoma\nFactor p-value Hazard ratio 95% CI\nMELF-pattern .013 2.20 1.18–4.09\nNo. of vessels .009 3.31 1.33–8.16\nArea of vessels < .001 1.03 1.01–1.17\nCI, confidence interval; MELF, microcystic, elongated, fragmented.\n\nhttp://jpatholtm.org/ https://doi.org/10.4132/jptm.2017.07.19\n462     •  Zinovkin DA, et al.\nWe showed for the first time that there is a significant presence \nof MELF pattern and an increased number and area of vessels in \ncases of EA with unfavorable outcome. In our study, VEGF expres-\nsion correlated with the area and number of vessels, but it did \nnot have any predictive force according to multivariate Cox’s \nproportional hazard analysis. Although our data suggest that \nthese criteria may be used as prognostic factors of EA during \nthe 5 years after radical surgical treatment, a larger independent \ncohort of samples should be studied to verify these findings.\nConflicts of Interest\nNo potential conflict of interest relevant to this article was \nreported.\nREFERENCES\n1. Gacche RN, Meshram RJ. Targeting tumor micro-environment for \ndesign and development of novel anti-angiogenic agents arresting \ntumor growth. Prog Biophys Mol Biol 2013; 113: 333-54.\n2. Zigrino P , Löffek S, Mauch C. Tumor-stroma interactions: their role \nin the control of tumor cell invasion. Biochimie 2005; 87: 321-8.\n3. Murray SK, Young RH, Scully RE. Unusual epithelial and stromal \nchanges in myoinvasive endometrioid adenocarcinoma: a study of \ntheir frequency, associated diagnostic problems, and prognostic \nsignificance. Int J Gynecol Pathol 2003; 22: 324-33.\n4. Dogan Altunpulluk M, Kir G, Topal CS, Cetiner H, Gocmen A. The \nassociation of the microcystic, elongated and fragmented (MELF) \ninvasion pattern in endometrial carcinomas with deep myometrial \ninvasion, lymphovascular space invasion and lymph node metas-\ntasis. J Obstet Gynaecol 2015; 35: 397-402.\n5. Kukreja I, Kapoor P , Deshmukh R, Kulkarni V . VEGF and CD 34: a \ncorrelation between tumor angiogenesis and microvessel density-an \nimmunohistochemical study. J Oral Maxillofac Pathol 2013; 17: 367-73.\n6. Z\n˙yła MM, Kostrzewa M, Litwin´ska E, Szpakowski A, Wilczyn´ski \nJR, Stetkiewicz T. The role of angiogenic factors in endometrial \ncancer. Prz Menopauzalny 2014; 13: 122-6.\n7. Stefansson IM, Salvesen HB, Immervoll H, Akslen LA. Prognostic \nimpact of histological grade and vascular invasion compared with \ntumour cell proliferation in endometrial carcinoma of endometrioid \ntype. Histopathology 2004; 44: 472-9.\n8. Stefansson IM, Salvesen HB, Akslen LA. Vascular proliferation is \nimportant for clinical progress of endometrial cancer. Cancer Res \n2006; 66: 3303-9.\n9. Stewart CJ, Crook ML, Manso L. Fascin expression in low-grade \nuterine endometrioid adenocarcinoma: correlation with microcystic, \nelongated and fragmented (MELF)-type alteration at the deep inva-\nsive margin. Histopathology 2011; 59: 73-80.\n10. Bajracharya D, Shrestha B, Kamath A, Menon A, Radhakrishnan R. \nImmunohistochemical correlation of matrix metalloproteinase-2 \nand tissue inhibitors of metalloproteinase-2 in tobacco associated \nepithelial dysplasia. Dis Markers 2014; 2014: 197813.\n11. Zaino RJ. Unusual patterns of endometrial carcinoma including \nMELF and its relation to epithelial mesenchymal transition. Int J \nGynecol Pathol 2014; 33: 357-64.\n12. Stewart CJ, Crook ML. Galectin-3 expression in uterine endometrioid \nadenocarcinoma: comparison of staining in conventional tumor \nglands and in areas of MELF pattern myometrial invasion. Int J \nGynecol Pathol 2010; 29: 555-61.\n13. Erdem O, Erdem M, Erdem A, Memis L, Akyol G. Expression of \nvascular endothelial growth factor and assessment of microvascular \ndensity with CD 34 and endoglin in proliferative endometrium, \nendometrial hyperplasia, and endometrial carcinoma. Int J Gynecol \nCancer 2007; 17: 1327-32.\n14. Aybatli A, Sayin C, Kaplan PB, Varol F, Altaner S, Süt N. The inves-\ntigation of tumoral angiogenesis with HIF-1 alpha and microvessel \ndensity in women with endometrium cancer. J Turk Ger Gynecol \nAssoc 2012; 13: 37-44.\n15. Haldorsen IS, Stefansson I, Grüner R, et al. Increased microvascular \nproliferation is negatively correlated to tumour blood flow and is \nassociated with unfavourable outcome in endometrial carcinomas. \nBr J Cancer 2014; 110: 107-14.\n16. Ozdemir O. Mast cell density, angiogenesis, and their significance \nin tumor development. Gynecol Oncol 2006; 100: 628-9.\n17. Simionescu C, Ma\n˘rga˘ritescu C, Stepan A, Pirici D, Ciurea R, Cernea \nN. Tumor angiogenesis, macrophages and mast cell microdensities \nin endometrioid endometrial carcinoma. Oncol Lett 2013; 6: 415-20.\n18. Nunobiki O, Nakamura M, Taniguchi E, et al. Adrenomedullin, \nBcl-2 and microvessel density in normal, hyperplastic and neoplastic \nendometrium. Pathol Int 2009; 59: 530-6.\n19. Schmid BC, Oehler MK. Improvements in progression-free and \noverall survival due to the use of anti-angiogenic agents in gyneco-\nlogic cancers. Curr Treat Options Oncol 2015; 16: 318.\n20. Saarelainen SK, Staff S, Peltonen N, et al. Endoglin, VEGF, and its \nreceptors in predicting metastases in endometrial carcinoma. Tumour \nBiol 2014; 35: 4651-7.\n21. Wang J, Taylor A, Showeil R, et al. Expression profiling and signifi-\ncance of VEGF-A, VEGFR2, VEGFR3 and related proteins in endo-\nmetrial carcinoma. Cytokine 2014; 68: 94-100.\n22. Saito M, Sato Y, Watanabe J, Kuramoto H, Kaba S, Fukuda T. Angio-\ngenic factors in normal endometrium and endometrial adenocarci-\nnoma. Pathol Int 2007; 57: 140-7.\n23. Matias-Guiu X, Davidson B. Prognostic biomarkers in endometrial \nand ovarian carcinoma. Virchows Arch 2014; 464: 315-31.","source_license":"CC0","license_restricted":false}