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Stela Iurciuc,1,2 Anca Maria Cimpean,3 Florin Mitu,4 Rodica Heredea,5 Mircea Iurciuc1,2 1Department of Preventive Medicine, Angiogenesis Research Center, “Victor Babes” University of Medicine and Pharmacy, 2Clinical Emergency County Hospital “Pius Brînzeu,” 3Department of Microscopic Morphology/Histology, Angiogenesis Research Center, “Victor Babes” University of Medicine and Pharmacy, Timisoara, 4Department of Cardiology, “Grigore T Popa” University of Medicine and Pharmacy, Iasi, 5Department of Pathology, Louis Turcanu Children Hospital, Timisoara, Romania Abstract: The true onset of atherosclerosis remains one of the biggest challenges for cardiologists. Is atheroma plaque development considered the earliest step of vascular aging? If so, when it starts? Before or after birth? If it starts before birth or early during childhood, it seems that Thomas Sydenham was right: “A man is as old as his arteries.” Except disorganization of elastic fibers, less is known about the morphology of vascular aging and also about the molecular events influencing the age of arteries, arterial stiffness, and their role in the appearance of future complications. Cellular and molecular events responsible for the switch from physiologic to pathologic aging of human arteries are less known. Epigenetic, genetic, and environmental influences at the onset of early vascular aging (EVA) should specifically influence the process. This paper briefly reviews the controversial data regarding vascular aging with an emphasis on the less known facts about the morphology of EVA. Keywords: arterial stiffness, blood pressure variability, arterial aging, telomeres

Backround: Depression is a significant concern in clinical and preclinical psychoneurobiological sciences due to its high prevalence and its individual and collective consequences. Identifying efficient biomarkers for accurate diagnosis is crucial, with ideal biomarkers having detectable serum levels and conformational and thermal stability. This study aims to identify stable plasma biomarkers for the diagnosis and prognosis of major depressive disorder, as the pathogenesis of the disorder remains incompletely understood, affecting diagnosis accuracy., Methods: Thus, this study included ten MDD patients and eight healthy controls. The present work analyzed miRNAs in patients with major depressive disorder compared to healthy controls., Results: Eleven specific miRNAs, particularly hsa-miR-874-3p; hsa-let-7d-5p; and hsa-miR-93-3p showed upregulation-type plasma variations in the group of patients with major depressive disorder. miRNA functionality is linked to depressive pathophysiology., Conclusions: This study identifies a "bouquet" of miRNAs with significant upregulation variations in patients with major depressive disorder, suggesting further research to determine their suitability for personalization and evaluation, ultimately becoming integral components of major depression serological evaluations.

Background and Objectives : Our aim was to perform a retrospective analysis of the volume of cervical screening tests, the number of patients treated with an excision method, and the incidence of invasive and non-invasive cervical during a pandemic and pre-pandemic period of 24 months. Materials and Methods : The study compared 404 patients who underwent cervical cone biopsy for cervical cancer. The study examined patients' specimens based on histopathological characteristics and categorized cervical lesions based on pap smear. Results : There was a statistically significant age difference between the two study periods. The mean difference was 32 years before the pandemic and 35 years during the pandemic ( p -value > 0.05). The biggest patient loss ratio identified by age group was in the 50-59-year group, with a 14.53% loss in the pre-pandemic period and a 9.1% loss in the pandemic period. In the pandemic period, patients from rural areas presented in the clinical trial with a lower rate of 39.52% (83 patients) vs. 60.47% (127 patients) in urban areas. A higher percentage of patients experiencing cervicorrhagia as a clinical manifestation in the pandemic period vs. the pre-pandemic period, with an increase in more severe lesions in the pandemic period, had a statistical significance of 8% more newly diagnosed compared to the pre-pandemic period. Conclusions : The addressability of the patients during the COVID period was not affected in a drastic way in our study. We encountered a decrease in appointments in the age group of 50-59 years and a decrease in patients with rural residence. In our study, we found an increase in cervical bleeding as a reason for consultation in the pandemic period with a higher lesion degree, both on a pap smear and on a cervical biopsy.

The expression and function of podoplanin (PDPN) in the normal human placenta has been debated in placental evaluation. This study emphasizes the importance of a multimodal approach of PDPN expression in normal human placentas. A complete examination is performed using immunohistochemistry, RNAscope and automated Digital Image examination (DIA) interpretation. QuPath DIA-based analysis automatically generated the stromal and histological scores of PDPN expression for immunohistochemistry and RNAscope stains. The umbilical cord's isolated fibroblasts and luminal structures expressed PDPN protein and PDPN_mRNA. RNAscope detected PDPN_mRNA upregulation in syncytial placental knots trophoblastic cells, but immunohistochemistry did not certify this at the protein level. The study found a significant correlation between the IHC and RNAscope H-Score ( p = 0.033) and Allred Score ( p = 0.05). A successful multimodal strategy for PDPN assessment in human placentas confirmed PDPN expression heterogeneity in the full-term human normal placenta and umbilical cord at the protein and mRNA level. In placental syncytial knots trophoblastic cells, PDPN showed mRNA overexpression, suggesting a potential role in placenta maturation.

This systematic review evaluates the clinical outcomes and molecular predictors of response to pembrolizumab in patients with advanced and metastatic cervical cancer. We adhered to the PRISMA guidelines for systematic reviews, conducting a database search in PubMed, Scopus, and Embase. The eligibility criteria centered on clinical outcomes, including the overall survival (OS), progression-free survival (PFS), and immune-related biomarkers post-pembrolizumab therapy. We included both prospective and retrospective studies that detailed clinical outcomes and molecular characteristics predictive of therapeutic response. Our search yielded six studies involving 846 patients treated with pembrolizumab from 2017 to 2022. The meta-analysis of these studies showed that pembrolizumab, used as monotherapy or in combination with chemotherapy, extended the OS by a weighted median of 10.35 months and the PFS by 8.50 months. The treatment demonstrated a pooled objective response rate (ORR) of 22.39%, although the I 2 test result of 67.49% showed a high heterogeneity among the studies. Notably, patients with high PD-L1 expression (CPS ≥ 10) experienced improved outcomes in terms of the PFS and OS. The most common complications were fatigue, diarrhea, and immune-related adverse events. Pembrolizumab significantly enhances clinical outcomes in metastatic cervical cancer, particularly among patients with high PD-L1 expression. The drug maintains a good safety profile, reinforcing its treatment potential for patients with advanced and metastatic cervical cancer. Future studies should explore long-term effects and strategies to integrate pembrolizumab optimally into current treatment regimens, aiming to maximize patient benefits and effectively manage side effects.

Background Human embryo vasculogenesis (blood vessel development starting from endothelial precursors) includes the ability of mesenchymal cells and pluripotent stem cells to differentiate into endothelial cells. Quantification of endothelial progenitor cells is difficult to assess during the early steps of human embryo development due to several factors, especially due to the paucity of human embryo tissue which is usually discarded after early-stage pregnancy abortive methods. CD133 (Promimin-1) is a general marker of progenitor cells, but combined with other endothelial markers such as CD34, it may identify endothelial progenitor cells during embryonic development. CD34 immunohistochemistry was previously performed by our team to identify human embryo capillaries and comparatively assess microvessel density between different human embryonic tissues. TIE2 is an angiopoietin receptor strongly involved in the newly formed blood vessel maturation due to its expression in some mesenchymal precursors for future pericytes. CD34 assesses the presence of endothelial cells but its single use does not evaluate the endothelial progenitor state as CD133 may do nor vessel maturation as TIE2 may do. Data about the dynamics of CD133/TIE2 expression in the early stages of human embryo development are scarce. Hence, in this study, we aimed to comparatively assess the dynamic of CD133+ endothelial precursors and TIE2 expression on five and seven-week-old human embryonic tissues with a special emphasis on their expression on embryonic vascular beds. Methodology CD133 and TIE2 immunohistochemistry was performed on five and seven-week-old human embryonic tissues followed by their quantification using the Qu Path digital image analysis (DIA) automated method. Results CD133 and TIE2 showed divergent patterns of expression during the initial phases of human embryonic development, specifically in the vascular endothelium of tiny capillaries. The expression of CD133 in endothelial cells lining the perfused lumen gradually decreased from five to seven-week-old embryos. It remained expressed with greater intensity in cells located at the tip of the vascular bud that emerged into pre-existing capillaries. TIE2 was much more specific than CD133, being restricted to the level of the vascular endothelium; therefore, it was easier to quantify using digital image analysis. The endothelium of the embryonic aorta was an exception to the divergent expression, as CD133 and TIE2 were consistently co-expressed in the seven-week-old embryo. The Qu Path DIA assessment increased the accuracy of CD133 and TIE2 evaluation, being the first time they were quantified by using automated software and not manually. Conclusions High heterogeneity of CD133 and TIE2 was observed between five and seven-week-old embryonic tissues as well as between different embryonic regions from the same gestational age. The unique finding of CD133/TIE2 co-expression persistence inside aortic endothelium needs further studies to elucidate the role of this co-expression., Competing Interests: The authors have declared that no competing interests exist., (Copyright © 2024, Tomescu et al.)

Vasculogenesis, which refers to the development of blood vessels from precursor cells, is a process that occurs predominantly during early embryonic life. It plays a crucial role in the establishment of the primitive vascular network. Vasculogenesis diminishes throughout the fetal vascular remodeling process, giving way to angiogenesis, which becomes the predominant mechanism after birth. At first, the development of the kidney's blood vessels depends on vasculogenesis, and then both vasculogenesis and angiogenesis happen simultaneously. Both processes are necessary for the normal development of the renal vasculature. Although the kidneys are highly vascularized, our understanding of normal kidney vasculogenesis is still incomplete. This lack of knowledge may explain the limited data available on the role of vasculogenesis in the progression and spread of renal cancers. In other types of cancer, researchers have well documented the phenomenon of tumor vasculogenesis. However, there is currently limited and fragmented information about the occurrence of clear-cell renal cell carcinomas (cc-RCC). In this article, we provide a comprehensive review of the current understanding of normal kidney vasculogenesis and vasculogenic pathways in clear cell renal cell carcinoma (cc-RCC). We specifically focus on cellular precursors, growth factors, and the influence of the normal and tumor environments on these processes. It will carefully look at how tumor vasculogenesis might affect the growth and metastasis of clear cell renal cell carcinoma (cc-RCC), as well as how it might affect the effectiveness of drugs and the development of therapy resistance., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Cosma, Fenesan, Nesiu, Melnic, Ferician, Ferician, Ceban, Sarb and Cimpean.)

Objective: The study investigates morphological variants of tertiary lymphoid structures (TLSs) in relation to cervical cancer development, from intraepithelial neoplastic lesions to invasive carcinomas with locoregional lymph node metastases., Materials and Methods: This retrospective analysis comprised 100 cervical cancer cases who had had total hysterectomy with lymphadenectomy in the Obstetrics and Gynecology Clinic of the Municipal Emergency Clinical Hospital of Timisoara, Romania, from 2020 to 2023. Bilateral ilio obturator lymphadenectomy and total hysterectomy were used to acquire biopsy samples. The presence of germinal centers, other stromal structures, TLS density, topography relative to the tumor lesion, and malignant cell islets are used to evaluate and classify TLS., Results: We first globally evaluated the total number of TLSs (TLS.T). We observed topographically two places in the cervical stroma: TLS immediately peritumorally positioned and TLS away from tumor lesions. Invasive carcinomas have bigger superficial TLSs with a well-defined germinal center. As they approached the tumor, TLSs increased in size and density. We also detected a special type of TLS associated with nerve fibers, which we named tertiary lymphoid structures associated with nerves (TLS.N). The total number of TLSs did not correlate with age, but 85.71% of patients presenting TLS.N were aged between 59 and 72 years old. Our findings showed a strong correlation between age (postmenopausal, p = 0.005) and TLS-N presence. Similarly, TLS parameters evolved with tumor differentiation. Only in the TLS.N group did the tumoral grading (G) 3 correlate with TLS (p = 0.041), while TLS.T did not correlate with G. All TLS.N. patients, except one, had lymphovascular invasion and massive histiocytosis. On the first point, TLS.N correlated with lymphovascular invasion (p = 0.032)., Conclusion: Tertiary lymphoid structures associated with nerves have not been previously reported in cervical cancer, and their effects on prognosis and aggression are unknown. There was a substantial association between TLSs.N presence and age over 60, suggesting it is exclusive to menopausal women. They were also substantially connected with lymphovascular invasion and G3, suggesting they may be a poor cervical cancer prognostic factor., Competing Interests: The authors have declared that no competing interests exist., (Copyright © 2024, Bălan et al.)

Type I collagen, prevalent in the extracellular matrix, is biocompatible and crucial for tissue engineering and wound healing, including angiogenesis and vascular maturation/stabilization as required processes of newly formed tissue constructs or regeneration. Sometimes, improper vascularization causes unexpected outcomes. Vascularization failure may be caused by extracellular matrix collagen and non-collagen components heterogeneously. This study compares the angiogenic potential of collagen type I-based scaffolds and collagen type I/glycosaminoglycans scaffolds by using the chick embryo chorioallantoic membrane (CAM) model and IKOSA digital image analysis. Two clinically used biomaterials, Xenoderm (containing type I collagen derived from decellularized porcine extracellular matrix) and a dual-layer collagen sponge (DLC, with a biphasic composition of type I collagen combined with glycosaminoglycans) were tested for their ability to induce new vascular network formation. The AI-based IKOSA app enhanced the research by calculating from stereomicroscopic images angiogenic parameters such as total vascular area, branching sites, vessel length, and vascular thickness. The study confirmed that Xenoderm caused a fast angiogenic response and substantial vascular growth, but was unable to mature the vascular network. DLC scaffold, in turn, produced a slower angiogenic response, but a more steady and organic vascular maturation and stabilization. This research can improve collagen-based knowledge by better assessing angiogenesis processes. DLC may be preferable to Xenoderm or other materials for functional neovascularization, according to the findings.

Background/aim: Microfluidic experimental models allow to study the mutual interrelation between tumor development and the microvasculature avoiding animal use and lacking interspecies differences. This study aimed to develop and characterize a 3D tissue culture model employing a two-compartment microfluidic chip-perfused platform to visualize and quantify human bone marrow-derived mesenchymal stem cells (hBM-MSCs) and MCF-7 breast cancer cell-cell interactions in real time., Materials and Methods: MCF-7 cells were implanted in the tumor chamber and hBM-MSCs were injected into microvascular channels. hBM-MSCs culture media was perfused into microvascular compartments. The microfluidic device was microscopically examined weekly for four weeks., Results: VE- and E-cadherin immunofluorescence validated hBM-MSCs differentiation into endothelial cells and MCF-7 cell tumor formation. hBM-MSCs differentiation was highly heterogeneous along the microvascular channels, due to different perfusion flow. hBM-MSCs lining microvascular channels acquired VE-cadherin positive endothelial phenotype and continuously covered microchannels as an endothelium like layer. MCF-7 cells were constantly grown as spheroidal aggregates and later formed a compact area of E-cadherin-positive tumor cells inside tumor compartment., Conclusion: Our study provides valuable knowledge on the properties of hBM-MSCs as vasculogenesis-supporting cells when co-cultured with MCF-7 cells on a 3D perfused biomimetic microfluidic device. This newly established model may serve as an experimental platform for testing anti-tumor/anti-angiogenic drugs., (Copyright © 2024 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: Biomaterials are essential in modern medicine, both for patients and research. Their ability to acquire and maintain functional vascularization is currently debated. The aim of this study was to evaluate the vascularization induced by two collagen-based scaffolds (with 2D and 3D structures) and one non-collagen scaffold implanted on the chick embryo chorioallantoic membrane (CAM)., Materials and Methods: Classical stereomicroscopic image vascular assessment was enhanced with the IKOSA software by using two applications: the CAM assay and the Network Formation Assay, evaluating the vessel branching potential, vascular area, as well as tube length and thickness., Results: Both collagen-based scaffolds induced non-inflammatory angiogenesis, but the non-collagen scaffold induced a massive inflammation followed by inflammatory-related angiogenesis. Vessels branching points/Region of Interest (Px^2) and Vessel branching points/Vessel total area (Px^2), increased exponentially until day 5 of the experiment certifying a sustained and continuous angiogenic process induced by 3D collagen scaffolds., Conclusion: Collagen-based scaffolds may be more suitable for neovascularization compared to non-collagen scaffolds. The present study demonstrates the potential of the CAM model in combination with AI-based software for the evaluation of vascularization in biomaterials. This approach could help to reduce and replace animal experimentation in the pre-screening of biomaterials., (Copyright © 2024, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: A real challenge for patients with rheumatoid arthritis (RA) and rheumatologists is primary nonresponse status (PNRS) or secondary nonresponse status (SNRS) to various therapies. Despite their detrimental influence on patient life quality, PNRS and SNRS have no accurate definition and no early predictive criteria for their development exist. Patients with RA under 40 years of age are rare, hence PNRS and SNRS data for this age group are scarce. This study examined the PNRS and SNRS according to sex, age, BMI, therapy type, and duration., Patients and Methods: Retrospectively, 115 patients with RA having PNRS and/or SNRS were stratified by age (22-39, 40-59, and 60-81). The association between body mass index (BMI), proinflammatory cytokines inhibitors, JAK inhibitors, and TNF-alpha inhibitors, sex, age, and PNRS and SNRS was examined., Results: All three proinflammatory cytokine inhibitors (rituximab, tocilizumab, and abatacept) were associated with PNRS and SNRS in women with a high BMI aged 40-59 years. Abatacept-related PNRS and SNRS was significant in women with normal BMI aged 60-81 years. Adalimumab, infliximab, and golimumab affected SNRS differently in women with normal BMI aged 22-39 years and women with high BMI aged 60-81 years. Etanercept and infliximab were associated with SNRS status in men with high-BMI aged 40-59 years., Conclusion: PNRS and SNRS development in patients with RA is significantly influenced by age, sex, and BMI, but most importantly is closely and differentially related to therapy type and duration., (Copyright © 2023, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background: Breast cancer (BC) stroma has CD34- and αSMA-positive cancer-associated fibroblasts (CAFs) differently distributed. During malignant transformation, CD34-positive fibroblasts decrease while αSMA-positive CAFs increase. The prevalence of αSMA-positive CAFs in BC stroma makes microscopic examination difficult without digital image analysis processing (DIA). DIA was used to compare CD34- and αSMA-positive CAFs among breast cancer molecular subgroups. DIA-derived data were linked to age, survival, tumor stroma vessels, tertiary lymphoid structures (TLS), invasion, and recurrence., Methods: Double immunostaining for CD34 and αSMA showed different CAF distribution patterns in normal and BC tissues. Single CD34 immunohistochemistry on supplemental slides quantified tumor stroma CD34_CAFs. Digital image analysis (DIA) data on CAF density, intensity, stromal score, and H-score were correlated with clinico-pathologic factors., Results: CD34/αSMA CAF proportion was significantly related to age in Luminal A (LA), Luminal B (LB), and HER2 subtypes. CD34_CAF influence on survival, invasion, and recurrence of LA, LB-HER2, and TNBC subtypes was found to be significant. The CD34/αSMA-expressing CAFs exhibited a heterogeneous impact on stromal vasculature and TLS., Conclusion: BC stromal CD34_CAFs/αSMA_CAFs have an impact on survival, invasion, and recurrence differently between BC molecular subtypes. The tumor stroma DIA assessment may have predictive potential to prognosis and long-term follow-up of patients with breast cancer., Competing Interests: The authors have no conflict of interest.

Background: Tertiary lymphoid structures (TLSs) mediate local antitumor immunity, and interest in them significantly increased since cancer immunotherapy was implemented. We examined TLS- tumor stromal blood vessel interplay for each breast cancer (BC) molecular subtype related to recurrence, lymphovascular invasion (LVI), and perineural invasion (PnI)., Methods: TLSs were quantified on hematoxylin and eosin stain specimens followed by CD34/smooth muscle actin (SMA) double immunostaining for stromal blood vessel maturation assessment. Statistical analysis linked microscopy to recurrence, LVI, and PnI., Results: TLS negative (TLS-) subgroups in each BC molecular subtype (except to Luminal A) have higher LVI, PnI, and recurrence. A significant rise in LVI and PnI were observed for the HER2+/TLS- subgroup ( p < 0.001). The triple negative breast cancer (TNBC)/TLS- subgroup had the highest recurrence and invasion risk which was also significantly related to tumor grade. PnI but not LVI significantly influenced recurrence in the TNBC/TLS+ subgroup ( p < 0.001). TLS-stromal blood vessel interrelation was different amongst BC molecular subtypes., Conclusion: BC invasion and recurrence are strongly influenced by TLS presence and stromal blood vessels, especially for HER2 and TNBC BC molecular subtypes.

Background: Overexpression of chloride intracellular channel protein 1 (CLIC1) in tumor cells has been confirmed, but it has received less attention in the tumor blood vessel endothelium. Aim: The assessment of CLIC1 expression in ccRCC tumor blood vessels and its relationship with TNM parameters and tumor cell CLIC1 expression. Methods: CLIC1 immunostaining in ccRCC was evaluated in 50 cases in both malignant cells and tumor blood vessels (CLIC1 microvessel density-CLIC1-MVD) and was correlated with TNM staging parameters. Results: CLIC1-MVD was observed in approximately 65% of cases, and CLIC1 co-localization in both tumor and endothelial cells was observed in 59% of cases. ccRCC was classified into four groups (Classes 0−3) based on the percentage of positive tumor cells, with each group including sub-groups defined by CLIC1 expression in the endothelium. Class 3 (60−100% positive tumor cells) had the highest CLIC1-MVD, with an impact on T and M parameters (p value = 0.007 for T, and p value = 0.006 for M). For cases with CLIC1 intracellular translocation, there was a strong correlation between CLIC1-MVD and M (p value < 0.001). Conclusions: Co-expression of ccRCC tumor and endothelial cells promotes tumor progression and metastasis and should be investigated further as a potential therapeutic target for ccRCC and other human malignancies.

Background/aim: We previously described four different vascular patterns (reticular, diffuse, fasciculate, and trabecular) in renal cell carcinoma (RCC) suggesting an early and heterogeneous acquisition of perivascular cells most probably due to a particular PDGF pathway gene expression profile. The aim of the study was to study PDGF pathway gene expression profiles, separately for each vascular pattern., Materials and Methods: TaqMan assay for the PDGF pathway was performed on twelve cases of ccRCC previously evaluated by histopathology, immunohistochemistry, and RNAscope. Gene expression profile was correlated with grade, invasion, vascular patterns, and VEGF., Results: PIK3C3 and SLC9A3 genes were overexpressed in all vascular patterns, but they were significantly correlated with high VEGF mRNA in the reticular and diffuse pattern. STAT1, JAK2, SHC2, SRF and CHUK (IKK) were exclusively overexpressed in cases with diffuse vascular pattern. SLC9A3, CHUK and STAT3 were overexpressed in G2 tumors., Conclusion: Three ccRCC subgroups were defined: 1) PIK3C3 (VSP34)/SLC9A3 which may be proper for anti PIK3C3 inhibitors; 2) VEGF high subgroup where association of anti VEGF may be a benefit and 3) JAK2/STAT1 subgroup, potentially being eligible for anti JAK/STAT therapy associated with IKK inhibitors., (Copyright © 2022, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background . Infantile hemangiomas may have unexpected behavior. Initial regression (spontaneously or drug-induced) may be followed by unexplained recurrences. At this moment, there are no well-established criteria to predict infantile hemangioma reccurrences. Methods . We compared the VEGF pathway gene expression profile for one case of involuting infantile hemangioma versus one case of recurrent proliferative infantile hemangioma using TaqMan Array. Results . We found ten genes upregulated for both involuting and recurrent proliferative hemangiomas: ACTB, KRAS, MAP2K1, HRAS, NOS3, BAD, HSPB1, HPRT1, GUSB, and CASP9. Thirteen genes were downregulated for both involuting and proliferative hemangiomas: FIGF, ACTG1, GRB2, MAPKAPK2, ACTG2, MAP2K2, MAPK3, HSP90AA1, MAP2K6, NRAS, ACTA1, KDR, and MAPK1. Three genes showed divergent expression between proliferating and involuting hemangiomas. Proliferating hemangioma had MAPK14 and AKT1 gene upregulation and ACTA2 downregulation. Involuting infantile hemangioma was characterized by ACTA2 upregulation and AKT1 and MAPK14 downregulation. Conclusions . Three genes, AKT1, p38/MAPK14, and ACTA2, were found to have divergent expression in proliferating and involuting infantile hemangiomas. Excepting AKT1, which was mentioned in the last ISSVA classification (strictly related to Proteus Syndrome), none of the other genes were reported. An accurate gene expression profile mapping of infantile hemangiomas together with a gene expression-based hemangioma classification is stringently needed.

Background/aim: Chloride intracellular channel protein 1 (CLIC1) is known as a promoter of cancer progression, metastasis, and angiogenesis. Thus, CLIC1 could be a future therapeutic target. This study aimed to evaluate the effect of anti-CLIC1 antibodies on tumour cells and vessels of human renal cell carcinoma (RCC) in rabbit cornea and chick embryo chorioallantoic membrane (CAM) models., Materials and Methods: Human cc-RCC xenografts on rabbit cornea and CAM surface were performed. Anti-CLIC1 antibodies were applied for 5 consecutive days on both tumor models. We comparatively evaluated treated and untreated tumors by combining ultrasonography with microscopic techniques., Results: RCC implants rapidly recruited blood vessels and had an exponential growth rate on both tumor models. Anti-CLIC1 antibodies suppressed tumor growth by inducing tumor cell necrosis. Tumor vessels regressed rapidly but not completely during anti-CLIC1 antibodies based therapy., Conclusion: Anti-CLIC1 antibodies induced tumor necrosis and tumor vasculature regression in human cc-RCC xenografts in both in vivo experimental models., (Copyright © 2022 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: Understanding tumor vasculogenesis is a cornerstone for the inhibition of tumor progression. This study aimed to generate an in vivo breast cancer environment to analyze the patterns of tumor vasculogenesis., Materials and Methods: Human mesenchymal stem cells (hMSC) and breast cancer MCF-7 cells (MCF-7) were seeded onto a chorioallantoic membrane (CAM) and, after a 7-day incubation, we performed a morphological and immunohistochemical analysis of CAM., Results: hMSC and MCF-7 activated vasculogenesis and hematopoiesis on CAM. They stimulated the development of cord/capillary-like structures (CLS), formed by endothelial-like cells and hematopoietic cells. CLS presented a polygonal pattern, evolving towards a clearly visible plexus. Immunohistochemically, CLS were CD105+/AC133+/Oct3/4+, and the intensity was weak-moderate in the endothelial-like cells (inconstant) and weak in the hematopoietic cells., Conclusion: Tumor and embryonic vasculogenesis share a common paradigm, while CD105, AC133, and Oct3/4 were found to play a role in establishing the vasculogenic and hematopoietic stage., (Copyright © 2022 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: Colon cancer liver metastases with desmoplastic growth pattern (dGP) have a highly heterogeneous therapy response. The aim of the study was to evaluate the dGP liver metastasis molecular profile from a chemo-naive patient by mimicking metastatic process on an experimental chick embryo chorioallantoic membrane (CAM) model., Materials and Methods: Three successive CAM passages of dGP human colorectal liver metastases were immunophenotyped for keratin (K) 8, and 20, CLIC1, VEGF, EGFR, CD34, podoplanin, Ki67, E-cadherin and vimentin., Results: Metastatic cells gradually lost K20 while K8, E-cadherin and vimentin heterogeneously increased during passages. VEGF, CLIC 1, EGFR expression increased in metastatic cells especially at the tumor graft periphery. Scattered proliferating and non-proliferating podoplanin-positive tumor cells, lymphatic and blood vessels were heterogeneously detected in tumor xenografts depending on passage stage., Conclusion: By mimicking repetitive metastatic processes we proved that metastatic cells change their phenotype. This may explain why not all metastases have a similar response to therapy., (Copyright © 2021 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: Chloride intracellular channel protein 1 (CLIC1) activates inflammasomes in rheumatoid (RA) and psoriatic (PsA) arthritis. We studied CLIC1 expression in RA and PsA patients' skin with vasculitis and its variability depending on the therapy used., Materials and Methods: CLIC1 immunoexpression was evaluated in the vascular (CLIC1-V) and stromal (CLIC1-S) compartments of the RA and PsA skin biopsies of patients treated with methotrexate (MTX), leflunomid (LFN), corticotherapy (CT), or biological therapies., Results: MTX significantly reduced CLIC1-S expression (p=0.016), whereas LFN decreased CLIC1-V (p<0.001). LFN therapy duration also correlated with CLIC1-V (p<0.001). CT decreased CLIC1-S expression (p=0.006). CLIC1-S expression persisted in skin biopsies despite of erythrocyte sedimentation rate (ESR, p=0.018) and C reactive protein (CRP, p=0.0026) normalisation. For PsA, CLIC1-S expression significantly related to MTX (p<0.022). Both CLIC1-S (p<0.001) and CLIC1-V (p=0.007) decreased by biological therapies in RA., Conclusion: CLIC1 expression is strongly influenced by the therapy used. Our data strongly support the extensive evaluation of CLIC1 in RA as a potential marker of inflammation and tool to predict therapy response., (Copyright © 2021 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: Renal cell carcinoma is strongly vascularized, and formation of new blood vessels is a complex and multi-step process. In this study, we analysed the subtypes of intermediate blood vessels, as shown by double immunohistochemistry., Materials and Methods: Tumour-associated blood vessels were identified by double immunostaining based on CD34 and smooth muscle cell actin. Blood vessels were classified both quantitatively and qualitatively based on the expression of the aforementioned two markers. The main criteria to sub-classify intermediate blood vessels was the presence, distribution, and arrangement of perivascular cells., Results: We described three subtypes of intermediate blood vessels found particularly in the tumour area: Subtype 1 lacked perivascular cells, subtype 2 showed scattered pericytes attached to the vascular wall, and subtype 3 showed a continuous layer of perivascular cells on one side., Conclusion: We describe for the first time three subtypes of renal cell carcinoma-associated intermediate blood vessels, which could be important in prognosis and as potential targets for anti-vascular therapy., Competing Interests: The Authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest, or non-financial interest in the subject matter or materials discussed in this manuscript., (Copyright 2021, International Institute of Anticancer Research.)

Background/aim: Angiogenic growth factors expression is not known in the normal cornea. The aim was to study corneal gene expression profile of VEGF and PDGF pathways influencing the avascular state of cornea., Materials and Methods: cDNA synthesis was performed from mRNA extracted from five fresh pig corneas followed by cDNA synthesis and analysis of VEGF and PDGF pathways by TaqMan Array gene expression profile., Results: Normal pig cornea lacks VEGFR2 and VEGFR3 gene expression. MK2 and AKT1 genes were significantly overexpressed (p=0.000684, p=0.050995, respectively). Six PDGF pathway genes were overexpressed: TIAM1 (p=0.047), PIK3CA (p=0.00005), IKBKG (p=0.000006), PAK4 (p=0.034), RAC1 (p=0.000006 and PTGS2, p=0.00375). PDGF A was up-regulated, but not with a statistical significance (p=0.79911), while PDGFRα was down-regulated and PDGFRβ was not expressed., Conclusion: Normal cornea avascularity is given by growth factor receptors down-regulation. Rapid corneal neovascularisation is induced by activation of the main angiogenic growth factors that induce angiogenic cascade and vessel recruitment., (Copyright© 2021, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: Chloride intracellular channel protein (CLIC1), E- and P-cadherin (Ecad, Pcad) are certified factors of aggressivity, but they have not been studied in breast cancer to date. The aim was to study CLIC1, Ecad and Pcad impact on breast cancer in terms of defining new high-risk subgroups., Materials and Methods: Ninety-seven breast cancer biopsies were immunohistochemically evaluated for CLIC1, Ecad and Pcad expression related to molecular subtypes. CLIC1 expression was assessed in both tumor cells (CLIC1T) and blood vessels (CLIC1V)., Results: For 23% of Luminal A cases, both cadherins and CLIC1V were positive. Luminal B/HER2 subtype, had two specific phenotypes: Ecad-/Pcad-/CLIC1T-/CLIC1V+ and Ecad+/Pcad-/CLIC1T-/CLIC1V+. All TNBC cases were clustered into two subgroups: 60% were Ecad+/Pcad+/CLIC1T+/CLIC1V+) while 40% were Ecad+/Pcad+/CLIC1T+/CLIC1V-)., Conclusion: CLIC1, Ecad and Pcad association stratifies molecular types of breast cancer in subgroups that may explain different response to therapy and different aggressiveness previously observed by other authors within the same molecular subtype., (Copyright © 2021 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Most of angiogenesis assays were designed and developed during Folkman's era. But the growth of new blood vessels in several pathologic conditions as tumor development or inflammation were observed long time ago.The development of new blood vessels was early observed by ancient Egyptians who tried to destroy them by applying empirical methods. From the first observations regarding angiogenesis to a personalized therapy targeting newly formed blood vessels a lot of experimental in vitro and in vivo angiogenesis assays have been developed. The present work will overview the oldest and less known part of angiogenesis assays development, and in addition, it will present the newest data in the experimental field of angiogenesis which is rapidly improved by the needs of new antiangiogenic and antivascular therapy development.

Background/aim: Invasive bladder cancer mortality remains high despite progresses made in early diagnosis and surgical procedures. Thus, there is a need to define new markers for bladder cancer. CLIC1 has not been previously studied in bladder cancer and thus, we aimed to assess its immunohistochemical expression in relation to different stages of bladder cancer development., Materials and Methods: Immunohistochemistry for CLIC1 was applied in 50 cases of muscle invasive bladder cancer., Results: CLIC1 was not expressed in the normal urothelium, but a strong reaction was observed in dysplastic urothelium, carcinoma in situ and in 94% of the cases with invasive urothelial carcinoma; however, it was not expressed in squamous cell carcinoma cases. No correlation was found between the immunohistochemical expression of CLIC1 and the stage and grade of the tumour., Conclusion: CLIC1 was overexpressed in urinary bladder dysplastic epithelium, carcinoma in situ and invasive carcinoma. CLIC1 constitutes a new potential marker of invasive bladder cancer., (Copyright © 2020 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: E- and P-cadherin (E-cadh, P-cadh) control tumor cell invasion, metastatic or stemness potential and chemotherapy resistance. The study aimed to assess E- and P-cadherin expression in breast cancer molecular subtypes., Materials and Methods: Immunohistochemistry for E-cadh and P-cadh was performed for 97 breast cancer cases. Membrane (M), cytoplasmic (C) or mixed (MC) patterns of E-cadh and P-cadh were considered in our evaluation., Results: E-cadh and P-cadh C pattern was significantly correlated in the HER2 subtype (p=0.031). P-cadh M pattern was highly specific for the HER2 subtype (p=0.002). Only P-cadh C characterized the triple negative breast cancer subtype (p=0.015). For Luminal B/HER2 cases, P-cadh M pattern was strongly coexpressed with the E-cadh MC pattern (p=0.012). Progesterone receptor (PR) expression influenced E-cadh M pattern in the Luminal B/HER2 subtype (p=0.042)., Conclusion: E- and P-cadherins define distinct subgroups within breast cancer molecular subtypes. Our findings support the inclusion of E- and P-cadherin into breast cancer molecular classification., (Copyright© 2020, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Chronic stress is regarded as a significant factor in the etiology of the many diseases. Numerous methods have been developed through which the effect of chronic stress is examined. The aim of this study is to demonstrate the new experimental model for analysis of immuno-suppression induced by chronic restraint stress, through challenge with conditionally tumorigenic cell line BHK-21/C13. 20 male NMRI mice were randomly divided into 2 groups-control and experimental. Each mouse was subcutaneously inoculated with BHK-21/C13 cells. Stress in the experimental group was induced for 20 days. After the experiment, tumor masses were removed, and analyzed using histology and immunohistochemistry techniques. We found a statistically significant difference (p = 0.034) in tumor expression and tumor volumes (p = 0.0061) between groups, as well as in immunopositivity on Ki67, cytochrome C and matrix metalloproteinase 9. Absence of immune infiltrate was noticed in experimental, and the presence of inflammatory infiltrate at tumor invasion front in control group.

Purpose . Clinically apparent anastomotic leakage (AL) after low anterior rectal resection (LAR; <7 cm from anal verge) using circular double-stapled anastomosis without defunctioning stoma is up to 37.5%. However, it is unclear whether there is reduction of LAR after 21 postoperative days without defunctioning stoma but with extraluminal anastomotic application of fibrin sealant. Methods . Forty-eight-week-old pigs underwent LAR and circular double-stapled anastomosis in end-to-end technique (descendo-rectostomy). Animals were randomized into therapy and control group (cg). Therapy group (n = 20) received additional extraluminal circular anastomotic application of fibrin sealant. Objective was to assess incidence of clinically apparent and nonclinically apparent leakage through the 21st postoperative day. Remaining animals were sacrificed on the 21st day, and anastomotic region was analyzed. In case of earlier diagnosed AL, animals were sacrificed. Results . In cg, we observed clinically and nonclinically AL in 20% (n = 4). No animal was identified with a nonclinical-apparent leakage in this group, and all 4 animals with leakages presented clinical signs. In the therapy group, no animal (0/20) developed clinically apparent leakage signs. There were no leakages in this group, but 3 animals had ulcerative lesions without leak and without clinical signs. These lesions were observed intraluminally at crossing of staple lines after 21 days. In one of these animals, incomplete leakage was observed, blocked by fibrin sealant. Conclusion . In circular stapled colorectal anastomosis, circular fibrin glue sealant successfully protected anastomotic intraluminal wall defects at crossing of staple lines, reducing leakage rate from 20% to 0% (cg vs therapy group) after 21 postoperative days.

Background: The role of podoplanin (PDPN) and homebox prospero gene 1 (PROX1) in early stages of pancreatic islet changes induced by hypercaloric diet is unclear. The aim of this study was to study PDPN and PROX1 variability in pancreatic islets after a hypercaloric diet in a rat experimental model., Materials and Methods: Pancreatic biopsies harvested from Sprague-Dawley rats at 3, 6, and 9 weeks following hypercaloric diet intake were evaluated for morphological and molecular changes of Langerhans islets based on PDPN and PROX1 expression Results: Six weeks of hypercaloric diet induced hypertrophy of pancreatic islets with focal expression of Pdpn and Prox1 mRNA. At 9 weeks of hypercaloric diet, strong peri-insular inflammation was found around hypertrophic islets highly expressing PDPN, and lacking Prox1 mRNA and protein expression., Conclusion: This is the first report of Pdpn and Prox1 mRNA expression variability and involvement in early steps of pancreatic islet changes following hypercaloric food intake., (Copyright© 2019, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: Chloride intracellular channel 1 (CLIC1) represents a promising target for personalized therapy. Our aim was to assess CLIC1 expression in clear cell renal cell carcinoma (cc RCC) and identify its possible prognostic role., Materials and Methods: Fifty cases of cc RCC were evaluated and selected for immunohistochemistry. CLIC1 expression was correlated with tumor grade, invasion and heterogeneity., Results: A total of 87.5% of the cases were CLIC1 positive, with either a homogeneous (31.42%) or a heterogeneous (68.57%) pattern. Low, mild and strong CLIC1 expressing tumors were defined based on nuclear (N), cytoplasmic (C), membrane (M) or combinations of them (NC, NM, CM, NCM) in terms of CLIC1 distribution. A significant correlation was found between tumor grade and percent of positive tumor cells (p=0.017). For G3 tumors, CLIC1 cytoplasmic expression was strongly correlated with high expression status (p=0.025) and tumor heterogeneity (p=0.004). CLIC1 expression was also correlated with metastasis (p=0.046)., Conclusion: We defined four cc RCC groups depending on G, CLIC1 expression and pattern: i) G3/NM/low CLIC1 + , ii) G2/CM/mild CLIC1 + iii) G1 or G2/NM or CM /high CLIC1 + , and iv) G2/M /high CLIC1 ., (Copyright© 2019, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: Chemotherapy-induced cardiotoxicity may be observed during treatment or may cause severe cardiac failure as the main cause of death, even several years after therapy implementation. Herein, the aim was to establish the early diagnosis of cardiotoxicity through the periodic evaluation of the left ventricular (LV) and vascular remodeling parameters, in patients with acute lymphoblastic leukemia (ALL)., Materials and Methods: The study population included 35 patients diagnosed with ALL, evaluated before and 3 months after starting chemotherapy, measuring systolic and diastolic parameters of the LV and intima-media thickness (IMT), arterial stiffness aortic pulse wave velocity (PWVAo) and ankle-brachial index (ABI)., Results: After the first 2 cycles of chemotherapy, all patients experienced a drop in LV ejection fraction (LVEF) (p<0.001), and 12 patients suffered a decrease of LVEF<50%. The ABI (p<0.05) and the global longitudinal strain (GLS) (p<0.001) decreased, while IMT and PWVAo (p<0.001) increased, proving a subclinical deterioration of the LV function and vascular remodeling., Conclusion: Assessment of cardiovascular risk factors before chemotherapy initiation in ALL patients may be helpful for an early diagnosis of chemotherapy-induced cardiotoxicity, thus contributing to early treatment and a subsequent decrease of death caused by such cardiovascular complications., (Copyright© 2019, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: Mast cells (MCs) represent the most controversial non-malignant element of the tumor microenvironment. Our aim was to study how MCs density and distribution (intratumoral-MCit versus peritumoral-MCpt) relate to tumor grade and molecular subtypes., Materials and Methods: MCs tryptase immunohistochemistry was performed on 80 cases of breast carcinomas., Results: For Luminal A tumors, a partial correlation was detected between MCit and progesterone receptor (PR) (p=0.005). Luminal B tumors showed a significant correlation between MCpt and age (p=0.009), estrogen receptor (ER) (p=0.017) and PR (p=0.035). MCit and MCpt were strongly interrelated in this subtype (p=0.002) and in triple-negative breast cancers (p=0.002). In HER2 subtype, MCpt tumors were significantly correlated with HER2 (p=0.044). In G2 tumors, MCpt correlated with ER (p=0.015) and PR (p=0.038) while in G3 tumors ER correlated with both MCit (p=0.009) and MCpt (p=0.000487) tumors., Conclusion: MCs dynamics are strongly influenced by hormone receptors and HER2 status. MCit increased in aggressive tumor types and is a worse prognostic factor., (Copyright© 2019, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

CD99 (MIC2; single-chain type-1 glycoprotein) is a heavily O-glycosylated transmembrane protein (32 kDa) present on leukocytes and activated endothelium. Expression of CD99 on endothelium is important in lymphocyte diapedesis. CD99 is a diagnostic marker for Ewing's Sarcoma (EWS), as it is highly expressed by these tumors. It has been reported that CD99 can affect the migration, invasion and metastasis of tumor cells. Our results show that CD99 is also highly expressed in the tumor vasculature of most solid tumors. Furthermore, we found that in vitro CD99 expression in cultured endothelial cells is induced by starvation. Targeting of murine CD99 by a conjugate vaccine, which induced antibodies against CD99 in mice, resulted in inhibition of tumor growth in both a tumor model with high CD99 (Os-P0109 osteosarcoma) and low CD99 (CT26 colon carcinoma) expression. We demonstrated that vaccination against CD99 is safe, since no toxicity was observed in mice with high antibody titers against CD99 in their sera during a period of almost 11 months. Targeting of CD99 in humans is more complicated due to the fact that the human and mouse CD99 protein are not identical. We are the first to show that growth factor activated endothelial cells express a distinct human CD99 isoform. We conclude that our observations provide an opportunity for specific targeting of CD99 isoforms in human tumor vasculature.

Background/aim: The aim of this study was to evaluate SOX2 expression in pituitary adenomas and its correlation to their secretory state and clinicopathological parameters., Patients and Methods: Thirty-four patients were clinically evaluated and surgery was recommended for tumor removal. Histopathological diagnosis by hematoxylin eosin staining was followed by immunohistochemistry for pituitary hormones and SOX2 co-expression., Results: Fourteen of the 34 cases were GH-secreting adenomas, 10 were prolactinomas and 10 non-functioning pituitary adenomas. SOX2-positive expression was detected in 47.05% of total cases: 8 GH-secreting adenomas (57.14%), 6 prolactinomas (60%) and 2 non-functioning adenomas (20%). SOX2 positivity was significantly higher amongst secreting adenomas (p=0.041). SOX2-negative tumors were significantly associated with corticotrophin deficiency (p=0.047) and gonadotrophin deficiency (p=0.041). No correlation with tumor size or extrasellar extension was detected., Conclusion: SOX2 is differentially expressed in pituitary adenomas and influences the secretory state or clinical behavior of pituitary adenomas., (Copyright© 2019, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Microscopic and molecular events related to alveolar ridge augmentation are less known because of the lack of experimental models and limited molecular markers used to evaluate this process. We propose here the chick embryo chorioallantoic membrane (CAM) as an in vivo model to study the interaction between CAM and bone substitutes (B) combined with hyaluronic acid (BH), saline solution (BHS and BS, respectively), or both, aiming to point out the microscopic and molecular events assessed by Runt-related transcription factor 2 (RUNX 2), osteonectin (SPARC), and Bone Morphogenic Protein 4 (BMP4). The BH complex induced osteoprogenitor and osteoblastic differentiation of CAM mesenchymal cells, certified by the RUNX2 +, BMP4 +, and SPARC + phenotypes capable of bone matrix synthesis and mineralization. A strong angiogenic response without inflammation was detected on microscopic specimens of the BH combination compared with an inflammatory induced angiogenesis for the BS and BHS combinations. A multilayered organization of the BH complex grafted on CAM was detected with a differential expression of RUNX2, BMP4, and SPARC. The BH complex induced CAM mesenchymal cells differentiation through osteoblastic lineage with a sustained angiogenic response not related with inflammation. Thus, bone granules resuspended in hyaluronic acid seem to be the best combination for a proper non-inflammatory response in alveolar ridge augmentation. The CAM model allows us to assess the early events of the bone substitutes⁻mesenchymal cells interaction related to osteoblastic differentiation, an important step in alveolar ridge augmentation.

Aggressive tumor cells can adopt an endothelial cell-like phenotype and contribute to the formation of a tumor vasculature, independent of tumor angiogenesis. This adoptive mechanism is referred to as vascular mimicry and it is associated with poor survival in cancer patients. To what extent tumor cells capable of vascular mimicry phenocopy the angiogenic cascade is still poorly explored. Here, we identify pericytes as important players in vascular mimicry. We found that pericytes are recruited by vascular mimicry-positive tumor cells in order to facilitate sprouting and to provide structural support of the vascular-like networks. The pericyte recruitment is mediated through platelet-derived growth factor (PDGF)-B. Consequently, preventing PDGF-B signaling by blocking the PDGF receptors with either the small tyrosine kinase inhibitor imatinib or blocking antibodies inhibits vascular mimicry and tumor growth. Collectively, the current study identifies an important role for pericytes in the formation of vascular-like structures by tumor cells. Moreover, the mechanism that controls the pericyte recruitment provides therapeutic opportunities for patients with aggressive vascular mimicry-positive cancer types. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland., (© 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.)

Aim To assess the onset of early left ventricular (LV) systolic and diastolic function impairment and the subclinical atherosclerosis following chemotherapy in patients diagnosed with acute myeloid leukemia (AML)., Materials and Methods: Thirty patients diagnosed with AML with no cardiac history, having LV ejection fraction (LVEF) >50%, were evaluated at baseline and 6 months after starting four cycles of chemotherapy. We measured LV function, global longitudinal strain and subclinical atherosclerosis markers: intima-media thickness (IMT), arterial stiffness aortic pulse wave velocity (PWVAo) and ankle-brachial index (ABI)., Results: LVEF had decreased at 6 months after treatment initialization (p<0.001), the same changes being observed for LV fraction shortening (p<0.001), mitral annular plane systolic excursion and S' wave (p<0.001 and p<0.05). Bilateral IMT and PWVAo significantly increased, 12 out of 30 patients (40%) had LVEF ≤50% after 6 months of chemotherapy, five of them receiving daunorubicin at more than 500 mg/m 2 /injection., Conclusion: LV function is impaired after 6 months of chemotherapy, with early changes of subclinical atherosclerosis becoming evident., (Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

The formation of new blood vessels, or angiogenesis, is a complex process that plays important roles in growth and development, tissue and organ regeneration, as well as numerous pathological conditions. Angiogenesis undergoes multiple discrete steps that can be individually evaluated and quantified by a large number of bioassays. These independent assessments hold advantages but also have limitations. This article describes in vivo, ex vivo, and in vitro bioassays that are available for the evaluation of angiogenesis and highlights critical aspects that are relevant for their execution and proper interpretation. As such, this collaborative work is the first edition of consensus guidelines on angiogenesis bioassays to serve for current and future reference.

Aim: To characterize baby hamster kidney fibroblast (BHK 21/C13) cells and test the effects of antibodies against podoplanin and disodium cromolyn on BHK 21/C13 cell line-derived tumors grown on chick embryo chorioallantoic membrane (CAM)., Material and Methods: BHK 21/C13 cell-derived fibrosarcomas developed in hamsters were implanted on CAM and treated with anti-podoplanin antibodies and disodium cromolyn. BHK 21/C13 cell immunophenotype was assessed., Results: Fibrosarcoma cells were positive for vimentin, CD117, smooth muscle actin, vascular endothelial growth factor epidermal growth factor receptor, homebox prospero gene 1 and negative for platelet-derived growth factor B, neuron-specific enolase, S100, CD34, Ewing sarcoma and podoplanin. CAM-grown fibrosarcomas were highly sensitive to disodium cromolyn and anti-podoplanin antibodies., Conclusion: Immunophenotyping BHK 21/C13 cells and their response to drugs represent the first step in revealing cell line utility and a reliable tool for experimental cancer research., (Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background: Few data are available regarding the epithelial to mesenchymal transition (EMT) /mesenchymal to epitheilal transition (MET) in the liver metastasis of digestive cancers. The aim of this study was to establish EMT/MET metastatic tumor cell plasticity according to the histological growth pattern of liver metastases., Materials and Methods: Biopsies from 25 patients with liver metastasis (desmoplastic, replacement and pushing type) were evaluated. Double immunostaining of E-cadherin/vimentin, keratin 8,18/vimentin and E-cadherin/ keratin 8,18 were performed., Results: The following cell types were noted: epithelial, mesenchymal, non-differentiated and differentiated hybrid mesenchymal/ epithelial and non-hybrid phenotype. All cases had mesenchymal/ epithelial phenotype cells. A significant correlation was found between the non-differentiated hybrid mesenchymal/ epithelial phenotype metastatic cells and histological growth pattern for gastric and colorectal cancer., Conclusion: A MET-targeting strategy, in conjunction with conventional chemotherapy, may be useful for the treatment of liver metastases., (Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: Probably due to their low occurrence, chromophobe and papillary renal cell carcinomas are less well characterized and, currently, there are no reliable prognostic markers for this group of patients. Moreover, the optimal therapy for patients with non-clear renal cell carcinoma (RCC) is unknown yet. Although elevated levels of Galectin-3 (Gal-3) were associated with poor prognosis in conventional RCC, the impact of this protein on carcinogenesis of chromophobe and papillary entities has not been previously described., Materials and Methods: Gal-3 expression was investigated in 34 consecutive cases of RCCs, including 19 papillary carcinomas and 15 chromophobe carcinomas., Results: Immunohistochemical analysis of Gal-3 in tumor cells showed 3 patterns of expression: membranous, cytoplasmic and nuclear staining. Most tumors included in our study showed a cytoplasmic expression and it was almost equally distributed between the histologic subtypes. However, only nuclear staining of Gal-3 was associated with both Fuhrman grade and tumor stage (p=0.016 and p=0.032, respectively) in chromophobe subtype., Conclusion: Our results indicate that the nuclear expression of Gal-3 has an essential role in the development of chromophobe carcinoma. The association with advanced tumor stage and nuclear grade suggests that this protein is an indicator of aggressiveness in the chromophobe subtype, thus targeting anti-nuclear transport may prove an effective therapy for this particular group of patients., (Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: To assess the concordance between IHC and FISH and its influence on tumor type, grade and lympho-vascular invasion (LVI)., Materials and Methods: HER2 immunohistochemistry (IHC) to 45 cases of bladder carcinoma, followed by fluorescent in situ hybridization (FISH) were applied., Results: 31.12% cases were IHC positive. Less than 35% of HER2-negative cases presented LVI and this percent increased to 54.54% for +1 HER2 cases. LVI increases up to 57.14% for +2 HER2 cases and slightly decreased for +3 HER2 cases to 42.85%. IHC/FISH concordance was found for 73.34% cases but 31.57% were previously negative and 36.36% scored as +1 by IHC had gene amplification as shown by FISH analysis. T3 was correlated with HER2-IHC (p=0.05) and HER2-FISH (p=0.01)., Conclusion: Improved HER2 assessement is needed for urothelial carcinomas. HER2-IHC scored as 0-2 should be validated by and reclassified according to FISH analysis., (Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)