<p dir="ltr">Subcutaneous injection for self-administration of biotherapeutics, such as monoclonal antibodies (mAbs), is becoming increasingly prominent within the pharmaceutical sector due to its benefits in patient compliance and cost-effectiveness. The success of this drug delivery process depends on the coupled mechanical and transport phenomena within the subcutaneous tissue, both during and after the injection. Yet, the details of these processes are not well-elucidated, sparking a surge in computational efforts to fill this knowledge gap. Remarkably, there are very few computational studies on subcutaneous injection into three-dimensional porous media that account for large tissue deformations, drug transport and absorption, the use medical devices, and human factors. Here, we develop a high-fidelity computational framework to study large-volume subcutaneous injection of mAbs. Our investigation begins with a linear poroelastic model without drug transport, which we employ to study the effect of tissue deformation on injection dynamics. We progressively enhance this model, advancing to a nonlinear porohyperelastic framework that include drug transport and absorption. To capture the anisotropy of subcutaneous tissue, we employ a fibril-reinforced porohyperelastic model. Furthermore, we integrate the multi-layered structure of skin tissue by creating data-driven geometrical models of the tissue layers derived from histological data. Our analysis explores the impact of different handheld autoinjectors on the injection dynamics for various patient-applied forces. We investigate the effect of different pre-injection techniques, such as the pinch and stretch methods, on the drug transport and absorption. Additionally, we evaluate the impact of several physiological variables, including flow rate, injection depth, and body mass index. Our simulations yield crucial insights essential for comprehending and improving subcutaneous drug administration of mAbs. Additionally, they offer a deeper understanding of the human aspect of the injection procedure, thereby paving the way for advancements in the development of patient-centered injection devices and techniques.</p>

L'athérosclérose est la principale cause de maladies coronariennes, affectant les artères de grand et moyen calibre. C'est une maladie inflammatoire chronique caractérisée par des plaques situées dans la couche de l’intima, composées de cellules inflammatoires, de cellules musculaires lisses, de composants fibreux et de lipides. Qu'il provienne de source alimentaire ou hépatique, le cholestérol qui s'accumule dans les macrophages des tissus périphériques, comme la paroi artérielle, engendre une réaction inflammatoire et doit être conséquemment mobilisé à l'aide d’accepteurs de cholestérol comme les lipoprotéines de haute densité (HDL). Ce processus spécifique est appelé transport inverse du cholestérol (mRCT). Des études ont démontré que l'apolipoprotéine A-I (apoA-I) pourrait être un acteur clé dans la régulation du mRCT, exerçant des effets différents de ceux du HDL. Plus important encore, le système lymphatique a récemment été identifié comme un nouvel acteur essentiel dans l'élimination du cholestérol de la lésion athérosclérotique (Martel et al., JCI 2013). Il a été démontré que sans vaisseaux lymphatiques fonctionnels, la mobilisation du cholestérol hors de la plaque ne peut pas être réalisée correctement et aggrave la maladie. Le réseau lymphatique est parallèle au système sanguin et il est présent dans presque tous les tissus du corps. C'est un acteur essentiel dans le maintien de l'homéostase des fluides, dans le transport des cellules immunitaires de la périphérie vers les ganglions lymphatiques correspondants, ainsi que dans l’absorption des lipides alimentaires de l'intestin vers la circulation sanguine. Le système lymphatique comprend les vaisseaux lymphatiques (LVs) initiaux et collecteurs, ainsi que les ganglions lymphatiques, qui ont une anatomie spécifique et des rôles distincts. La lymphe, le liquide clair qui circule dans les LVs, se jette dans la circulation sanguine au niveau de la veine sous-clavière. Les plaquettes sont responsables de la régulation de cette séparation des vaisseaux sanguins et lymphatiques via la formation d’un thrombus formé lors de l’interaction de leur récepteur CLEC-2 avec la podoplanine présente sur les cellules endothéliales lymphatiques. Il a également été démontré que l’activité plaquettaire était nécessaire tout au long de la vie pour maintenir l’intégrité des jonctions des LVs. L'athérosclérose est également caractérisée par une activation cellulaire et une apoptose accrue. Par conséquent, ces activités cellulaires peuvent entraîner la formation de particules submicroniques appelées vésicules extracellulaires qui ont des effets variables, mais souvent néfastes, sur l'endothélium sanguin et l'évolution de la plaque. La maladie cardiovasculaire a été associée à une augmentation du nombre des vésicules extracellulaires (EVs) en circulation, et nous croyons que ces véhicules pourraient être impliqués dans le dysfonctionnement lymphatique lié à l'athérosclérose. D'après des données récentes publiées au cours de ma maîtrise, l'amélioration du transport lymphatique pourrait limiter la progression de l'athérosclérose et favoriser la régression de la plaque. Nous avons montré que le transport lymphatique est altéré chez les jeunes souris prédisposés à développer l'athérosclérose, même avant l'apparition de la plaque. Nous avons prouvé que cet effet est d’abord associé à un défaut au niveau des vaisseaux collecteurs et nous suggérons que l'amélioration de la liaison du VEGF-C/ VEGFR3 puisse supprimer ce défaut spécifique. L'objectif global de cette thèse était de poursuivre dans cette voie et de mieux définir le rôle de l’important facteur de croissance lymphatique, VEGF-C, et de la lipoprotéine apoA-I dans la maintenance de l’intégrité et la fonction des vaisseaux lymphatiques. En outre, une meilleure description des composants de la lymphe, en particulier des agents libérés par les cellules, a été jugée nécessaire. La première publication nous a permis de montrer que, lorsqu'elles étaient injectées avec un mutant du facteur de croissance VEGF-C ciblant spécifiquement le récepteur VEGFR-3 (VEGF-C 152s), avant l'administration d'une diète pro-athérogène, les souris Ldlr-/- étaient protégées contre l’accumulation excessive dans la plaque et celle-ci était plus stable à long terme. La capacité de contraction soutenue des vaisseaux lymphatiques collecteurs et l'expression accrue de VEGFR-3 et de FOXC2 observée chez ces souris traitées avec VEGF-C-152s ont contribué à la clairance des composants nocifs contenus dans les tissus périphériques tels que les macrophages et le cholestérol. La deuxième publication a montré que des souris Ldlr-/- athérosclérotiques traitées à faible dose avec de l’apoA-I, présentaient un transport lymphatique accru et une hyperperméabilité des vaisseaux lymphatiques collecteurs abrogée, possiblement par une modulation de l’activité plaquettaire. La troisième publication est la première à démontrer la présence de vésicules extracellulaires d'origines hétérogènes dans la lymphe des souris et que le nombre de différents sous-types augmente chez les souris athérosclérotiques. Collectivement, ces études confirment la présence d'un dysfonctionnement lymphatique chez la souris avant même l'apparition de la plaque, et il est intéressant de noter que ce dysfonctionnement est principalement associé à un défaut des vaisseaux lymphatiques collecteurs, limitant ainsi le transport de la lymphe des tissus périphériques vers le sang. Différents traitements avec des facteurs de croissance et des lipoprotéines peuvent potentiellement moduler l’apparition et la progression de la lésion en améliorant la fonction lymphatique à différents stades de la maladie athérosclérotique. Nos découvertes concernant la présence de EVs dans la lymphe représentent leur potentiel en tant que biomarqueurs, mais également une nouvelle cible pour mieux comprendre la dysfonction lymphatique. / Atherosclerosis is the principal cause of coronary artery disease (CAD), affecting large- and medium-sized arteries. It is a chronic inflammatory disease characterized by intimal plaques composed of inflammatory cells, smooth muscle cells, fibrous components and lipids. Cholesterol that accumulates within macrophages in peripheral tissues, like the arterial wall, whether from dietary or synthetic sources, promotes inflammatory responses and needs to be excreted with the help of the cholesterol acceptor high density lipoprotein (HDL). This specific process is termed macrophage reverse cholesterol transport (mRCT) and studies have demonstrated that lipid free apolipoprotein A-I (apoA-I) could be a key player in mRCT regulation, exuding different effects than HDL. More importantly, recently, the lymphatic system has been identified as a novel prerequisite player in the removal of cholesterol out of the atherosclerotic lesion (Martel et al., JCI 2013). It has been demonstrated that without functioning lymphatic vessels cholesterol mobilization from the plaque cannot be properly achieved and aggravates the disease. The lymphatic network runs in parallel to the blood vasculature and is present in almost all the tissues of the body. It is a crucial player in maintaining fluid homeostasis, trafficking immune cells from the periphery to corresponding lymph nodes, as well as transporting lipids from the intestine to the circulation. The lymphatic system comprises the initial and collecting lymphatic vessels (LVs), as well as lymph nodes, all with a specific anatomy and distinctive roles. Lymph, the clear fluid that circulates within LVs drains towards the bloodstream at the level of the subclavian vein. Platelets are responsible to regulate this blood/lymphatic vessel separation by forming a clog, upon the interaction of their C-type lectin-like receptor 2 (CLEC-2) with podoplanin, present on lymphatic endothelial cells. Platelet activity has also been shown to be required throughout life in order to maintain LV junction integrity. Atherosclerosis is also characterized by increased cellular activation and apoptosis. Consequently, these cellular activities may result in the formation of submicron particles called extracellular vesicles (EVs) that have variable effects on the blood endothelium and subsequent plaque evolution. CAD has been associated with increased circulating EVs, and we suspect that these EVs might be involved in atherosclerosis-related lymphatic dysfunction. Based on recent data collected during my master’s degree, there is evidence that enhancing lymphatic transport could limit atherosclerosis progression and favour plaque regression. We showed that lymphatic transport is impaired in young, atherosclerosis-prone mice, even before atherosclerosis onset. We believe it to be potentially associated with a defect in the lymphatic pumping capacity, and we suggest that enhancing VEGF-C/VEGFR-3 binding can abolish this specific defect. The global objective of this thesis was to pursue along this path and better delineate the role of the important lymphatic-specific growth factor, VEGF-C and the lipoprotein apoA-I, on collecting LVs function. Furthermore, a better understanding of lymph components, especially cellular releasants was deemed necessary. The first publication allowed us to show that when injected with VEGF-C 152s, before the administration of a pro-atherogenic regimen, Ldlr-/- mice were protected from excessive plaque formation and long-term, had a more stable plaque. The sustained contraction capacity of the collecting lymphatic vessels and the enhanced expression of VEGFR-3 and FOXC2 observed in these VEGF-C-152s treated mice contributed to the clearance of harmful components contained in peripheral tissues such as the macrophages and cholesterol. The second publication showed that atherosclerotic Ldlr-/- mice treated with low-dose lipid-free apoA-I had enhanced lymphatic transport and abrogated collecting LV permeability possibly through modulation of platelet activity. The third publication is the first ever to demonstrate the presence of extracellular vesicles of heterogeneous origins in the lymph of mice, and that their levels differ in atherosclerosis. Collectively, these studies confirm that lymphatic dysfunction is present before the onset of atherosclerosis, and particularly of interest, that this dysfunction is primarily associated with a defect in the collecting vessels, thereby limiting the lymph transport from peripheral tissues to the blood. Different treatments with growth factors and lipoproteins have the potential to modulate the lesion onset and progression through the enhancement of lymphatic function, while our findings regarding the presence of EVs in lymph represents their potential as biomarkers, but also a new venue to better understand lymphatic dysfunction.

<p>Subcutaneous administration has become a common approach for drug delivery of biotherapeutics, such as monoclonal antibodies, which is achieved mainly by absorption through the lymphatic system. This dissertation focuses on the computational modeling of the fluid flow and solute transport in the skin tissue and the quantitative investigation of lymphatic uptake. First, the various mechanisms governing drug transport and lymphatic uptake of biotherapeutics through subcutaneous injection are investigated quantitatively through high-fidelity numerical simulations, including lymphatic drainage, blood perfusion, binding, and metabolism. The tissue is modeled as a homogeneous porous medium using both a single-layered domain and a multi-layered domain, which includes the epidermis, dermis, hypodermis (subcutaneous tissue), and muscle layers. A systematic parameter study is conducted to understand the roles of different properties of the tissue in terms of permeability, porosity, and vascular permeability. The role of binding and metabolism on drug absorption is studied by varying the binding parameters for different macromolecules after coupling the transport equation with a pharmacokinetic equation. The interstitial pressure plays an essential role in regulating the absorption of unbound drug proteins during the injection, while the binding and metabolism of drug molecules reduce the total free drugs. </p> <p>  </p> <p>The lymphatic vessel network is essential to achieve the functions of the lymphatic system. Thus, the drug transport and lymphatic uptake through a three-dimensional hybrid discrete-continuum vessel network in the skin tissue are investigated through high-fidelity numerical simulations. The explicit heterogeneous vessel network is embedded into the continuum model to investigate the role of explicit heterogeneous vessel network in drug transport and absorption. The solute transport across the vessel wall is investigated under various transport conditions. The diffusion of the drug solutes through the explicit vessel wall affects the drug absorption after the injection, while the convection under large interstitial pressure dominates the drug absorption during the injection. The effect of diffusion cannot be captured by the previously developed continuum model. Furthermore, the effects of injection volume and depth on the lymphatic uptake are investigated in a multi-layered domain. The injection volume significantly affects lymphatic uptake through the heterogeneous vessel network, while the injection depth has little influence. At last, the binding and metabolism of drug molecules are studied to bridge the simulation to the experimentally measured drug clearance. </p> <p><br></p> <p>Convective transport of drug solutes in biological tissues is regulated by the interstitial fluid pressure, which plays a crucial role in drug absorption into the lymphatic system through the subcutaneous (SC) injection.  An approximate continuum poroelasticity model is developed to simulate the pressure evolution in the soft porous tissue during an SC injection. This poroelastic model mimics the deformation of the tissue by introducing the time variation of the interstitial fluid pressure. The advantage of this method lies in its computational time efficiency and simplicity, and it can accurately model the relaxation of pressure. The interstitial fluid pressure obtained using the proposed model is validated against both the analytical and the numerical solution of the poroelastic tissue model. The decreasing elasticity elongates the relaxation time of pressure, and the sensitivity of pressure relaxation to elasticity decreases with the hydraulic permeability, while the increasing porosity and permeability due to deformation alleviate the high pressure. </p> <p><br></p> <p>At last, an improved Kedem-Katchalsky model is developed to study solute transport across the lymphatic vessel network, including convection and diffusion in the multi-layered poroelastic tissue with a hybrid discrete-continuum vessel network embedded inside. The effect of different drug solutes with different Stokes radii and different structures of the lymphatic vessel network, such as fractal trees and Voronoi structure, on the lymphatic uptake is investigated. The drug solute with a small size has a larger partition coefficient and diffusivity across the openings of the lymphatic vessel wall, which favors drug absorption. The Voronoi structure is found to be more efficient in lymphatic uptake. </p> <p><br></p> <p>The systematic and quantitative investigation of subcutaneous absorption based on high-fidelity numerical simulations can provide guidance on the optimization of drug delivery systems and is valuable for the translation of bioavailability from the pre-clinical species to humans. We provide a novel approach to studying the diffusion and convection of drug molecules into the lymphatic system by developing the hybrid discrete-continuum vessel network. The study of the solute transport across the discrete lymphatic vessel walls further improves our understanding of lymphatic uptake. The novel and time-efficient computational model for solute transport across the lymphatic vasculature connects the microscopic properties of the lymphatic vessel membrane to macroscopic drug absorption. The comprehensive hybrid vessel network model developed here can be further used to improve our understanding of the diseases caused by the disturbed lymphatic system, such as lymphedema, and provide insights into the treatment of diseases caused by the malfunction of lymphatics.</p>

<p>Увод: Туморска лимфангиогенеза је подручје активног истраживања у циљу проналаска параметара који би омогућили прецизније предвиђање понашања меланома него што то омогућавају традиционални хистолошки и клинички параметри. Иако су импресивна открића о улози лимфангиогенезе у промовисању метастазирања меланома коже, ипак су остала отворена бројна питања и још увек нису усаглашене препоруке које би биле примењиване у дијагностици и терапији. Циљ истраживања је био испитати да ли параметри лимфангиогенезе и Шилдсов индекс имају значај за прогнозу код пацијената са меланомом коже. Материјал и методе: У истраживање је укључено 100 пацијената са меланомом коже (52 без метастаза и 48 са метастазама у лимфним чворовима). За приказ ендотела лимфних судова исечци тумора су бојени имунохистохемијском методом са D2-40 антителима. Квантитативни параметри лимфангиогенезе &ndash; густина и површина лимфних судова добијени су компјутерском морфометријском анализом. За детекцију инвазије лимфних судова туморским ћелијама меланома урађено је дупло бојење D2-40 и S-100 антигена. Имунохистохемијски је испитана експресија васкуларног ендотелног фактора раста (VEGF)-C и VEGF-D у туморским ћелијама, макрофагима и фибробластима меланома. Шилдсов индекс је рачунат као логаритам производа дебљине меланома, квадриране перитуморске густине лимфних судова (ГЛС) и броја 2 за присутну лимфатичну инвазију. Резултати: Параметри који су статистички значајно доприносили појави метастаза меланома у лимфним чворовима, на униваријантној анализи, били су интратуморска ГЛС, перитуморска ГЛС, експресија VEGF-C и VEGF-D у туморским ћелијама, дебљина тумора, улцерација, дубља инвазија по Кларку, нодуларни тип меланома и мушки пол. Мултиваријантна анализа је показала да су значајни независни прогностички фактори за настанак метастаза позитивна експресија VEGF-C у туморским ћелијама, повећана дебљина тумора, повећана интратуморска ГЛС и перитуморска ГЛС. Као најјачи предиктор појаве метастаза показала се позитивна експресија VEGF-C у туморским ћелијама. Пацијенти са &lt;20% VEGF-C позитивних туморских ћелија имали су 20 пута већи ризик за развој метастаза него пацијенти са негативном експресијом. Параметри са утицајем на меланом-специфично преживљавање на униваријантној анализи су били интратуморска ГЛС, перитуморска ГЛС, експресија VEGF-C и VEGF-D у туморским ћелијама, док је мултиваријантна анализа издвојила повећану интратуморску и перитуморску ГЛС као независне факторе ризика за смртни исход. Интратуморска и перитуморска површина лимфних судова, лимфатична инвазија и експресија VEGF-C и VEGF-D у макрофагима и фибробластима нису биле значајно повезане са појавом метастаза нити са краћим меланом-специфичним преживљавањем. Ниједан параметар лимфангиогенезе није био значајно повезан са краћим преживљавањем без рецидива болести. Анализа оперативне карактеристичне криве (ROC) је показала да је Шилдсов индекс веома добар параметар за процену настанка метастаза, са сензитивношћу 81,3% и специфичношћу 75%. Већа површина испод ROC криве код Шилдсовог индекса (0,857) у поређењу са површином код других параметара, указује да је индекс најтачнији прогностички параметар за настанак метастаза, затим следи интратуморска ГЛС (површина 0,795), перитуморска ГЛС (површина 0,772), дебљина меланома (површина 0,762) и American Joint Committee on Cancer систем класификације (површина 0,752). Закључак: Истраживање туморске лимфангиогенезе идентификује нове параметре као значајније предикторе исхода болести од дебљине тумора, улцерације и осталих клиничко-патолошких параметара. Помоћу интратуморске и перитуморске ГЛС, експресије VEGF-C и VEGF-D у туморским ћелијама, као и прорачуна Шилдс индекса, може се предвидети метастатски капацитет меланома. Рутинска патохистолошка анализа меланома требало би да укључи квантитативну анализу мреже лимфних судова и семиквантитативну евалуацију експресије VEGF-C и VEGF-D у туморским ћелијама.</p> / <p>Uvod: Tumorska limfangiogeneza je područje aktivnog istraživanja u cilju pronalaska parametara koji bi omogućili preciznije predviđanje ponašanja melanoma nego što to omogućavaju tradicionalni histološki i klinički parametri. Iako su impresivna otkrića o ulozi limfangiogeneze u promovisanju metastaziranja melanoma kože, ipak su ostala otvorena brojna pitanja i još uvek nisu usaglašene preporuke koje bi bile primenjivane u dijagnostici i terapiji. Cilj istraživanja je bio ispitati da li parametri limfangiogeneze i Šildsov indeks imaju značaj za prognozu kod pacijenata sa melanomom kože. Materijal i metode: U istraživanje je uključeno 100 pacijenata sa melanomom kože (52 bez metastaza i 48 sa metastazama u limfnim čvorovima). Za prikaz endotela limfnih sudova isečci tumora su bojeni imunohistohemijskom metodom sa D2-40 antitelima. Kvantitativni parametri limfangiogeneze &ndash; gustina i površina limfnih sudova dobijeni su kompjuterskom morfometrijskom analizom. Za detekciju invazije limfnih sudova tumorskim ćelijama melanoma urađeno je duplo bojenje D2-40 i S-100 antigena. Imunohistohemijski je ispitana ekspresija vaskularnog endotelnog faktora rasta (VEGF)-C i VEGF-D u tumorskim ćelijama, makrofagima i fibroblastima melanoma. Šildsov indeks je računat kao logaritam proizvoda debljine melanoma, kvadrirane peritumorske gustine limfnih sudova (GLS) i broja 2 za prisutnu limfatičnu invaziju. Rezultati: Parametri koji su statistički značajno doprinosili pojavi metastaza melanoma u limfnim čvorovima, na univarijantnoj analizi, bili su intratumorska GLS, peritumorska GLS, ekspresija VEGF-C i VEGF-D u tumorskim ćelijama, debljina tumora, ulceracija, dublja invazija po Klarku, nodularni tip melanoma i muški pol. Multivarijantna analiza je pokazala da su značajni nezavisni prognostički faktori za nastanak metastaza pozitivna ekspresija VEGF-C u tumorskim ćelijama, povećana debljina tumora, povećana intratumorska GLS i peritumorska GLS. Kao najjači prediktor pojave metastaza pokazala se pozitivna ekspresija VEGF-C u tumorskim ćelijama. Pacijenti sa &lt;20% VEGF-C pozitivnih tumorskih ćelija imali su 20 puta veći rizik za razvoj metastaza nego pacijenti sa negativnom ekspresijom. Parametri sa uticajem na melanom-specifično preživljavanje na univarijantnoj analizi su bili intratumorska GLS, peritumorska GLS, ekspresija VEGF-C i VEGF-D u tumorskim ćelijama, dok je multivarijantna analiza izdvojila povećanu intratumorsku i peritumorsku GLS kao nezavisne faktore rizika za smrtni ishod. Intratumorska i peritumorska površina limfnih sudova, limfatična invazija i ekspresija VEGF-C i VEGF-D u makrofagima i fibroblastima nisu bile značajno povezane sa pojavom metastaza niti sa kraćim melanom-specifičnim preživljavanjem. Nijedan parametar limfangiogeneze nije bio značajno povezan sa kraćim preživljavanjem bez recidiva bolesti. Analiza operativne karakteristične krive (ROC) je pokazala da je Šildsov indeks veoma dobar parametar za procenu nastanka metastaza, sa senzitivnošću 81,3% i specifičnošću 75%. Veća površina ispod ROC krive kod Šildsovog indeksa (0,857) u poređenju sa površinom kod drugih parametara, ukazuje da je indeks najtačniji prognostički parametar za nastanak metastaza, zatim sledi intratumorska GLS (površina 0,795), peritumorska GLS (površina 0,772), debljina melanoma (površina 0,762) i American Joint Committee on Cancer sistem klasifikacije (površina 0,752). Zaključak: Istraživanje tumorske limfangiogeneze identifikuje nove parametre kao značajnije prediktore ishoda bolesti od debljine tumora, ulceracije i ostalih kliničko-patoloških parametara. Pomoću intratumorske i peritumorske GLS, ekspresije VEGF-C i VEGF-D u tumorskim ćelijama, kao i proračuna Šilds indeksa, može se predvideti metastatski kapacitet melanoma. Rutinska patohistološka analiza melanoma trebalo bi da uključi kvantitativnu analizu mreže limfnih sudova i semikvantitativnu evaluaciju ekspresije VEGF-C i VEGF-D u tumorskim ćelijama.</p> / <p>Introduction: Tumor lymphangiogenesis is a field of active research with the aim of finding parameters which would enable the more precise prediction of melanoma behavior in comparison to the prediction of traditional histological and clinical parameters. Although scientific findings about the role of lymphangiogenesis in developing cutaneous melanoma are impressive, there are unresolved questions and recommendations to be applied in diagnostics and therapy, which are still not in compliance. The aim of this research is to examine if parameters of lymphangiogenesis and the Shields Index are important for the prognosis in patients with cutaneous melanoma. Material and methods: The research included a hundred patients with cutaneous melanoma (52 without metastasis and 48 with metastasis of lymphatic nodes). To present endothelial lymphatic vessels, specimens were stained by an immunohistochemical method with D2-40 antibodies. Quantitative parameters of lymphangiogenesis &ndash; lymphatic vessel density (LVD) and area (LVA) were calculated by computer-assisted morphometric analysis. In order to detect the invasion of lymphatic vessels by melanoma tumor cells, a double staining of D2-40 and S-100 antigens was performed. The expression of vascular endothelial growth factor (VEGF)-C and VEGF-D was tested by an immunohistochemical method in tumor cells, macrophages and fibroblasts melanoma. The Shields Index was calculated as a logarithm by multiplying the melanoma thickness, square of peritumoral LVD and the number 2 for present lymphatic invasion. Results:Parameters that statistically had a significant contribution to the emergence of melanoma metastases in lymph nodes, during the univariate analysis, were intratumoral LVD, peritumoral LVD, VEGF-C and VEGF-D expression in tumor cells, tumor thickness, ulceration, deeper Clarkꞌs level of invasion, nodular type of melanoma and a male sex. The multivariate analysis showed that positive expression of VEGF-C in tumor cells, tumor thickness, increased intratumoral LVD and peritumoral LVD are significant independent prognostic factors. The most important predictor of metastases is a positive expression of VEGF-C in tumor cells. Patients with &lt;20% VEGF-C positive tumor cells were 20 times more at risk of developing metastases than patients with a negative expression. Parameters that influence melanoma-specific survival at the univariate analysis were intratumoral LVD, peritumoral LVD, VEGF-C and VEGF-D expression in tumor cells, while the multivariate analysis singled out an increased intratumoral and peritumoral LVD as independent factors of the death risk. Intratumoral and peritumoral LVA, lymphatic invasion and VEGF-C and VEGF-D expression in macrophages and fibroblasts were not significantly correlated either with the emergence of metastases or with melanoma-specific survival. Non-parameter of lymphangiogenesis was significantly related with shorter disease-free survival. The analysis of receiver operator characteristic (ROC) curve showed that the Shields Index is a very good parameter for the estimation of the appearance of metastases, with the sensitivity of 81.3% and specificity of 75%. A bigger area under the ROC curve with the Shields Index (0.857) in comparison to the area in other parameters shows that the index is the most accurate prognostic parameter for an emergence of metastases, followed by intratumoral LVD (area 0.795), peritumoral LVD (area 0.772), the thickness of melanoma (area 0.762), and the American Joint Committee on Cancer classification system (area 0.752). Conclusion: The research of tumor lymphangiogenesis identifies new parameters as significant predictors of the disease outcome more so than the parameters like tumor thickness, ulceration, and other clinical-pathological parameters. By intratumoral and peritumoral LVD, VEGF-C and VEGF-D expression in tumor cells, as well as by the calculation of Shields Index, metastatic capacity of melanoma can be predicted. A routine pathohistological analysis of melanoma should include quantitative analysis of the lymphatic vessels network and semiquantitative evaluation of VEGF-C and VEGF-D expression in tumor cells.</p>

<p>UVOD: Maligni tumori reproduktivnih organa nalaze se među vodećim uzrocima obolevanja i umiranja od malignih bolesti žena, kako u svetu, tako i u Srbiji. Jedan od najvažnijih puteva &scaron;irenja ovih bolesti je limfogeni, a konvencionalna radiolo&scaron;ka dijagnostika limfnih čvorova kod ovih pacijentkinja je neprecizna. Funkcionalna radiolo&scaron;ka dijagnostika, uključujući i magnentno rezonantnu sekvencu difuzionog kretanja (DWI) i iz nje izvedenu ADC mapu koja omogućava kvantitativnu analizu difuzionih osobina unutar limfnog čvora, daju obećavajuće rezultate u mogućnosti razlikovanja benignih od maligno izmenjenih limfnih čvorova male karlice i ingvinuma kod pacijentkinja obolelih od malignih tumora ženskih polnih organa. CILJ: Cilj studije je 1. utvrđivanje dijagnostičkih mogućnosti magnetnorezonantne sekvence difuzionog kretanja (DWI) u razlikovanju benignih od maligno izmenjenih limfnih čvorova male karlice i ingvinuma kod pacijentkinja obolelih od malignih tumora ženskih polnih organa, poređenjem preoperativno načinjenog magnetnorezonantnog pregleda i postoperativnog patohistolo&scaron;kog nalaza; 2. analiza povezanosti osobina metastatski izmenjenih limfnih čvorova na sekvenci difuzionog kretanja (DWI) i gradusa primarnog tumora, i 3. utvrđivanje uticaja tehničkih karakteristika sekvenci difuzinonog kretanja (DWI) na magnetnorezonantu procenu metastatske infiltracije karličnih i ingvinalnih limfnih čvorova i postoperativnog patohistolo&scaron;kog nalaza. MATERIJAL I METODE: Istraživanje je sprovedeno u periodu od 2013. do 2016.godine, kao prospektivna klinička studija u Centru za radiologiju, na Operativnom odeljenju Zavoda za ginekologiju, Klinike za ginekologiju i aku&scaron;erstvo i u Zavodu za patologiju Kliničkog Centra Vojvodine u Novom Sadu. Studija je obuhvatila 80 pacijentkinja obolelih od malignih tumora vulve, vagine, grlića materice, tela materice i jajnika. Na osnovu lokalizacije malignog tumora sve ispitanice su razvrstane u 5 grupa: grupa A- 3 žene obolele od carcinoma vulve, grupa B- 1 žena obolela od karcinoma vagine, grupa C-32 pacijentkinje obolele od karcinoma grlića materice, grupa D- 30 pacijentkinja obolelih od malignih tumora tela materice i grupa E- 14 žena obolelih od malignih tumora jajnika. Procena stadijuma bolesti definitivno je izvr&scaron;ena posle operacije na osnovu histopatolo&scaron;kog pregleda kompletnog hirur&scaron;kog materijala uključujući i pregled uklonjenih limfnih čvorova na osnovu aktuelne FIGO klasifikacije stadijuma bolesti zasebno za svaku pojedinačnu lokalizaciju malignog tumora. Svim pacijentkinjama je preoperativno načinjen magnetnorezonantni pregled male karlice na uređaju za magnetnu rezonancu 1.5 T General Electric Signa HDx u Centru za radiologiju, Kliničkog centra Vojvodine. Kod istih pacijentkinja naknadno je sprovedeno standardno hirur&scaron;ko lečenje po protokolu hirur&scaron;kog lečenja za dato maligno ginekolo&scaron;ko oboljenje sa karličnom i/ili ingvinalnom limfadenektomijom. Postoperativno je izvr&scaron;ena patohistolo&scaron;ka analiza hirur&scaron;ki uklonjenog materijala i limfnih čvorova razdvojenih po anatomskim grupama u karlici i ingvinalnoj regiji. REZULTATI: Ukupno 2320 limfnih čvorova je mapirano i patohistolo&scaron;ki pregledano kod 80 pacijenata. Metastaze u limfnim čvorovima patohistolo&scaron;ki su verifikovane kod 28 pacijenata (35%). Kod ovih 28 (35%) pacijentkinja, 152 (27,28%) od ukupno 557 limfnih čvorova bilo je metastatski izmenjeno na patohistolo&scaron;kom pregledu. Metastaze u limfnim čvorovima utvrđene su kod 2 pacijentkinje (7,14%) sa karcinomom vulve, 11 (39,28%) sa karcinomom cerviksa, 9 (32,14%) sa tumorima tela materice, te 6 (21,42%) sa tumorima jajnika. Od 28 pacijentkinja kod kojih su utvrđeni pozitivni limfni čvorovi, 14 pacijentkinja (50%) imalo je dobro diferentovan primarni tumor, 8 (28,57%) srednje diferentovan, dok je 6 (21,42%) imalo lo&scaron;e diferentovan primarni tumor. Od ukupno 152 metastatski izmenjena limfna čvora u na&scaron;oj studiji, 8 limfnih čvorova (5,26%) pripadalo je ingvinalnoj grupi od čega 5 (3,289%) povr&scaron;noj ingvinalnoj, a 3 ( 1,97%) dubokoj ingvinalnoj grupi, 8 (5,26%) parametrijalnoj grupi, 48 (31,58%) opturatornoj grupi, 40 (26,31%) spolja&scaron;njoj ilijačnoj grupi, 36 (23,684%) unutra&scaron;njoj ilijačnoj grupi, dok je 12 (7,89%) pripadalo zajedničkoj ilijačnoj grupi karličnih limfnih čvorova. Kraći prečnik limfnog čvora nije pokazao značajnu razliku između metastatskih ( mean &plusmn; SD, 8,3 &plusmn; 5.4 mm, raspon , 4.5-30 mm ) i limfnih čvorova koji nisu bili metastatski izmenjeni ( 6,3 mm &plusmn; 1,5 , 4,5-9,6 mm ; P= 0,191 ). Izmerena ADC vrednost bila je značajno niža kod metastatski izmenjenih limfnih čvorova (mean &plusmn; SD , ADC: 0,8725 x 10-3 mm2/s &plusmn; 0,0125) nego kod limfnih čvorova koji nisu bili metastatski izmenjeni (mean &plusmn; SD, ADC: 1,116 x 10- 3 mm2/s &plusmn; 0,1848; P=0,001). Prosečne vrednosti ADC kod b =800 s/mm2 i b =1200 s/mm2 nisu se značajno razlikovale između metastaski izmenjenih limfnih čvorova (mean &plusmn; SD, ADC: 0,8575 &plusmn; 0,0125 x 10-3 mm2/s, ADC:0,8859 &plusmn; 0,0125 x 10-3 mm2/s) i limfnih čvorova koji nisu metastatski izmenjeni (mean &plusmn; SD, ADC:1,0345 &plusmn; 0,1222 x 10-3 mm2/s, ADC:1,1125 &plusmn; 1638 x 10-3 mm2/s; P =0,657 i P = 0,877). Ako se koristi vrednost ADC od 0,860 x 10- 3 mm2 / s kao kritična vrednost za razlikovanje metastatskih od limfnih čvorova koji nisu metastatski izmenjeni, senzitivnost DWI MR iznosila je 89%, specifičnost 85% i ukupna tačnost 86%. Pozitivna prediktivna vrednost (PPV) DWI MR u detekciji limfnih metastaza u karličnoj i ingvinalnoj regiji iznosila je 30%. Negativna prediktivna vrednost (NPV) testa iznosila je 99%. Pozitivna prediktivna vrednost (PPV) MR zasnovana na kriterijumu ADC vrednosti značajno je veća u odnosu na sve kriterijuma veličine (P &lt; 0,001). Negativna prediktivna vrednost MR zasnovanoj na kriterijumima veličine limfnog čvora i na ACD vrednosti nisu se međusobno statistički značajno razlikovali (P&lt;0,05). Performanse dijagnostičke metode (MR) bile su značajno bolje za minimalnu ADC vrednost od svih kriterijuma baziranih na veličini limfnih čvorova ( P=0.001 za minimalnu ADC vrednost u odnosu na sve druge kriterijume). MRI na osnovu definisanog modela koji kombinuje kriterijum ADC vrednosti sa kriterijumom veličine ima sledeće dijagnostičke performanse za diferencijaciju malignih od benignih limfnih čvorova: senzitivnost od 95%, specifičnost 92%, sveukupna tačnost od 92,5%, pozitivnu prediktivnu vrednost od 46% i negativnu prediktivnu vrednost od 99.6%. ZAKLJUČAK: Kriterijum veličine limfnog čvora nije dovoljno precizan pokazatelj metastatske invazije limfnih čvorova. Sekvenca difuzionog kretanja (DWI) uvek se mora analizirati zajedno sa ADC mapom i visoko rezolutivnim T1 i T2 otežanim magnetnorezonantnim sekvencama. Studijom je dokazan visok stepen povezanosti između preoperativnog određivanja metastaske infiltracije karličnih i ingvinalnih limfnih čvorova malignih tumora ženskih polnih organa primenom sekvence difuzionog kretanja (DWI) i postoperativnog patohistolo&scaron;kog nalaza. Uz graničnu ADC vrednost od 0,860 x 10-3 mm2/ s, senzitivnost MRI DWI u otkrivanju metastatskih limfnih čvorova iznosi 89%, a specifičnost 85%. Kombinacija ADC vrednosti i morfolo&scaron;kih karakteristika limfnih čvorova konvencionalnim magnentno rezonantnim pregledom je najprecizniji prediktor postojanja metastatske infiltracije karličnih i ingvinalnih limfnih čvorova kod pacijentkinja sa malignim tumorima ženskih polnih organa. Tehničke karakteristike sekvenci difuzionog kretanja (DWI) u smislu razlike u visokim b vrednostima ne utiču na magnentno rezonantnu procenu metastatske infiltracije karličnih i ingvinalnih limfnih čvorova kod pacijentkinja sa malignim tumorima ženskih polnih organa. Studijom nije utvrđena statistički značajna razlika između preoperativno utvrđenih ADC vrednosti metastatski izmenjenih limfnih čvorova i stepena histolo&scaron;ke diferencijacije ovih tumora. Sekvenca difuzionog kretanja (DWI) je brza, jednostavna, neinvazivna metoda koja značajno doprinosi dijagnostičkim mogućnostima magnetne rezonance u razlikovanju benignih od malignih limfnih čvorova male karlice i ingvinuma.</p> / <p>INTRODUCTION: Malignant tumors of reproductive organs are among the leading causes of morbidity and mortality in women, both in Serbia and worldwide. Lymphatic spread is one of the most important pathways of tumor dissemination. However, conventional lymph node imaging in these patients is imprecise. Functional imaging, including diffusion-weighted magnetic resonance imaging (DWI MRI) and derived ADC map which allows quantitative analysis of diffusion parameters within a lymph node, provide promising results in discrimination benign from malignant pelvic and inguinal lymph nodes in patients with gynecological malignancies. AIM: Aim of the study was: 1. To assess diagnostic performances of DWI MRI in differentiation between benign and malignant pelvic and inguinal lymph nodes in patients with gynecological malignancies, by comparison of preoperative magnetic resonance and postoperative histopathological findings. 2. To analyze correlation between DWI characteristics of metastatic lymph nodes and grade of the primary tumor, and 3. To evaluate the influence of technical characteristics of DWI sequences on MR assessment of metastatic pelvic and inguinal lymph node and postoperative histopathological findings. MATERIAL and METHODS: The prospective clinical study was conducted in Center for Radiology, Surgery Department of Clinic for Gynecology and Obstetrics and Pathology Department of Clinical Center of Vojvodina from 2013 to 2016. It comprised 80 patients with malignant tumors of vulva, vagina, uterine cervix and body and ovaries. Based on the localization of the tumor, all patients were divided into 5 groups: group A-3 patients with vulvar cancer, group B- 1 patient with vaginal cancer, group C- 32 patients with cervical cancer, group D- 30 patients with uterine body tumors and group E- 14 patients with malignant ovarian tumors. Staging of the disease was performed after surgery based on histopathological examination of complete surgical specimen, including examination of removed lymph nodes, based on current FIGO classification separately for each primary tumor location. Preoperatively, all patients underwent MRI examination (1.5 T General Electric Signa HDx) at Center for Radiology, Clinical Center of Vojvodina. The same patients underwent standard surgical treatment according to the treatment protocol regarding the tumor type and stage, with complete pelvic and/or inguinal lymphadenectomy. Histopathological examination of surgically removed material and lymph nodes separated in pelvic and inguinal anatomic groups was performed after the surgery. RESULTS: The total of 2320 of lymph nodes were mapped and histopathologically examined in 80 patients included in the study. Metastases in lymph nodes were histopathologically confirmed in 28 patients (35%). In these 28(35%) patients, in 152 (27,28%) out of 557 lymph nodes histopathological examination confirmed metastases. Lymph node metastases were confirmed in 2 patients (7.14%) with vulvar cancer, 11 (39.28%) with cervical cancer, 9 (32.14%) with uterine body tumors and 6 (21.42%)patients with ovarian tumors. In 28 patients with positive lymph nodes, 14 patients (50%) had well differentiated primary tumor, 8 (28.57%) moderately differentiated, while 6 (21.42%) patients had poorly differentiated primary tumor. Out of 152 metastatic lymph nodes in our study, 8 lymph nodes (5.26%) were inguinal ( 5 (3.289%) superficial inguinal and 3 ( 1.97%) deep inguinal group), 8 (5.26%) were parametrial, 48 (31. 58%) obturatory, 40 (26.31%) external iliac, 36 (23.684%) internal iliac, while 12 (7. 89%) belonged to common iliac pelvic lymph nodes group. Shorter lymph node axis did not show significant difference between metastatic ( mean &plusmn; SD, 8.3 &plusmn; 5.4 mm, range , 4.5-30 mm ) and benign lymph nodes ( 6.3 mm &plusmn; 1.5 , 4.5-9.6 mm ; P= 0.191 ). Measured ADC values were significantly lower in metastatic (mean &plusmn; SD , ADC: 0.8725 x 10-3 mm2/s &plusmn; 0.0125) than benign lymph nodes (mean &plusmn; SD, ADC: 1.116 x 10-3 mm2/s &plusmn; 0.1848; P=0.001). Mean ADC values at b =800 s/mm2 and b =1200 s/mm2 did not differ significantly between metastatic (mean &plusmn; SD, ADC: 0.8575 &plusmn; 0.0125 x 10-3 mm2/s, ADC:0.8859 &plusmn; 0,0125 x 10-3 mm2/s) and benign lymph nodes (mean &plusmn; SD, ADC:1.0345 &plusmn; 0.1222 x 10-3 mm2/s, ADC:1.1125 &plusmn; 1638 x 10-3 mm2/s; P =0.657 i P = 0.877). If ADC value of 0.860 x 10- 3 mm2 / s is determined as a cut off value for discrimination of benign and malignant lymph nodes, DWI MRI sensitivity was 89%, specificity 85% and overall accuracy was 86%. Positive predictive value (PPV) of DWI MR in detection of pelvic and inguinal lymph node metastases was 30%. Negative predictive value (NPV) of the test was 99%. MRI PPV based on ADC value criteria was significantly higher compared to all size-based criteria (P &lt; 0,001). MRI NPV based on size based and ADC values criteria did not differ significantly (P&lt;0,05). Performances of diagnostic method (MRI) were significantly better for minimal ADC value compared to all lymph node size-based criteria ( P=0.001 for minimal ADC value compared to all other criteria). Combination of ADC value criteria and size-based criteria yields MRI the following diagnostic performances in discrimination between benign and malignant lymph nodes: sensitivity 95%, specificity 92%, overall accuracy 92.5%, positive predictive value 46% and negative predictive value 99.6%. CONCLUSION: Lymph node size is not sufficiently precise criteria for determination of metastatic lymph node involvement. DWI sequence always needs to be evaluated together with ADC map and high resolution T1W and T2W magnetic resonance sequences. The study shows high correlation between preoperative assessment of pelvic and inguinal lymph node metastases from gynecological malignancies using MRI DWI and postoperative histopathological findings. With a cut off ADC value of 0.860 x 10-3 mm2/ s, sensitivity of MRI DWI in metastatic lymph node detection is 89%, while specificity is 85%. Combination of ADC values and morphological lymph nodes characteristics assessed by conventional MRI is the most precise predictor of metastatic pelvic and inguinal lymph node invasion in patients with gynecological malignancies. Technical characteristics of DWI i.e. different high b-values do not influence MR assessment of metastatic pelvic and inguinal lymph node involvement in patients with gynecological malignancies. The study did not confirm statistically significant difference between preoperatively measured ADC valued of metastatic lymph nodes and histological grade of primary tumors. DWI MRI sequence is fast, simple, noninvasive method which aids significantly to MRI diagnostic performances in discrimination between benign and malignant pelvic and inguinal lymph nodes.</p>

OBJETIVOS: Avaliar características clínico-patológicas com ênfase na presença de variantes histológicas (VH) nos carcinomas uroteliais da bexiga e nas metástases linfonodais (ML). Caracterizar perfis imuno-histoquímicos relacionados aos subtipos moleculares e verificar suas associações clínico-patológicas. MÉTODOS: 183 carcinomas uroteliais de alto grau músculo-invasivos submetidos a cistectomia radical no Instituto do Câncer do Estado de São Paulo, HCFMUSP ou Hospital Alemão Oswaldo Cruz foram caracterizados quanto ao gênero, idade, tamanho, focalidade, configuração neoplásica, necrose, invasões angiolinfática e perineural, VH, carcinoma in situ, infiltrado inflamatório peri e intratumoral, estádio, margem cirúrgica, ML e VH na metástase. Resultados semi-quantitativos da pesquisa imuno-histoquímica (IH) de CK20 e GATA3 (associados ao subtipo luminal) e CK5, SALL4 e Vimentina (associados ao subtipo basal), que foi realizada em micromatrizes teciduais com abundante representação do tumor primário e das ML, tiveram valor de corte de 20%, definindo os casos em positivos e negativos. Casos negativos para CK20 ou GATA3 e positivos para CK5 foram agrupados no subtipo basal; casos positivos para CK20 ou GATA3 e negativos para CK5 agrupados no subtipo luminal e casos duplo positivos ou duplo negativos agrupados no subtipo indeterminado. As associações entre estes subtipos e as características clínico-patológicas foram realizadas através de testes qui-quadrado ou exato de Fisher para as variáveis categóricas. A comparação dos subtipos obtidos no centro do tumor e na ML foi realizada com testes de McNemar-Bowker e concordância pelo coeficiente Kappa. A análise de sobrevida foi realizada pelo método Kaplan-Meier e testes de Breslow. RESULTADOS: VH foram observadas em 116 (63,4%) casos e associadas a invasão linfovascular (p=0,043), VH na metástase (p < 0,001), pior sobrevida global (p=0,043). Havia 14 diferentes VH, a escamosa, mais comum, foi associada a tumores maiores (p=0,003); necrose (p=0,004); VH sarcomatoide (p=0,047), VH na metástase (p=0,003), sem impacto nas sobrevidas. VH micropapilífera, segunda mais observada, foi associada a invasão linfovascular (p=0,002); margem cirúrgica positiva (p=0,005); metástase linfonodal (p < 0,001), VH na metástase (p=0,007) e piores sobrevidas doença específica (p=0,016) e global (p=0,005). VHs foram observadas em 40,7% das metástases linfonodais, com apenas um caso discordante das VH observadas no tumor vesical. Nos dois perfis IH estabelecidos não houve impacto nas sobrevidas e o subtipo basal foi associado a tumores maiores (CK5/CK20 p=0,025; CK5/GATA3 p=0,006) e a variante escamosa (CK5/CK20 e CK5/GATA3 p < 0,001) e o subtipo luminal foi associado a variantes micropapilífera (CK5/CK20 e CK5/GATA3 p < 0,001) e plasmocitoide (CK5/CK20 p=0,003; CK5/GATA3 p=0,011). Foi moderado o grau de concordância do subtipo observado no centro comparado ao observado na ML (CK5/CK20 Kappa=0,57; CK5/GATA3 Kappa=0,48). CONCLUSÕES: A presença de VH nos carcinomas uroteliais da bexiga, além de acarretar heterogeneidade intratumoral, foi associada a características clínico-patológicas de agressividade, com impacto na sobrevida, sobretudo na variante micropapilífera. O perfil IH basal foi associado a tumores maiores com variante escamosa e o perfil IH luminal foi associado às variantes micropapilífera e plasmocitoide. Os perfis IHs não diferiram quanto às sobrevidas e houve moderada concordância com os perfis observados nas ML / OBJETIVES: Evaluate clinicopathological features emphasizing the presence of histological variants (HV) in bladder urothelial carcinomas and their lymph node metastasis (LNM). Characterize immunohistochemical profiles related to molecular subtypes and verify their clinicopathological associations. METHODS: 183 high-grade muscle-invasive urothelial carcinomas undergoing radical cystectomy at Instituto do Câncer do Estado de São Paulo, HCFMUSP or Hospital Alemão Oswaldo Cruz were classified according to gender, age, size, focality, neoplastic configuration, necrosis, lymphovascular and perineural invasion, HV, carcinoma in situ, peritumoral and intratumoral inflammatory infiltrate, stage, surgical margin, LNM and HV in metastasis. Semi-quantitative results of the immuhistochemical (IHC) studies for CK20 and GATA3 (associated with the luminal subtype) and CK5, SALL4 and Vimentin (associated with basal subtype), which were performed in tissue microarrays with abundant representation of the primary tumor and LNM, had a cutoff value of 20%, dividing cases into positive and negative. Negative cases for CK20 or GATA3 and positive for CK5 were grouped into the basal subtype; positive cases for CK20 or GATA3 and negative for CK5 were grouped into luminal subtype and double positive or double negative cases were grouped into the undetermined subtype. The associations between these subtypes and clinicopathological features were performed using chi-square or Fisher´s exact tests for the categorical variables. Comparison of the subtype obtained at the center of the tumor and LNM was performed using McNemar-Bowker test and agreement with Kappa coefficient. Survival analysis was performed using Kaplan-Meier method and Breslow tests. RESULTS: HV were observed in 116 (63.4%) cases and associated with lymphovascular invasion (p=0.043); HV in metastasis (p < 0.001), worse overall survival (p=0.043). There were 14 different HV, and squamous variant, the most common, was associated with larger tumors (p=0.003); necrosis (p=0.004), sarcomatoid variant (p=0,047), HV in metastasis (p=0.003), with no impact on survival. Micropapillary variant, the second most observed, was associated with lymphovascular invasion (p=0.002); positive surgical margin (p=0.005); LNM (p < 0.001); HV in metastasis (p=0.007) and worse disease-specific (p=0.016) an overall survival (p=0.005). HV were observed in 40.7% of the LNM, and only one case was discordant with HV observed in the bladder tumor. In both IHC profiles there was no impact on survival, and the basal subtype was associated with larger tumors (CK5/CK20 p=0.025; CK5/GATA3 p=0.006) and squamous variant (CK5/CK20 and CK5/GATA3 p < 0.001). Luminal subtype was associated with micropapillary variant (CK5/CK20 and CK5/GATA3 p < 0.001) and plasmacytoid variant (CK5/CK20 p=0.003; CK5/GATA3 p=0.011). There was moderate agreement between the subtype observed in the tumor center compared to that observed in LNM (CK5/CK20 Kappa=0.57; CK5/GATA3 Kappa=0.48). CONCLUSIONS: The presence of HV in bladder urothelial carcinomas, in addition to cause intratumoral heterogeneity, was associated with clinical and pathological characteristics of aggressiveness, with impact on survival, especially in the micropapillary variant. The basal IHC profile was associated with larger tumors and squamous variant, and the luminal IHC profile was associated with micropapillary and plasmacytoid variants. The IHC profiles did not differ on the survival rates and there was moderate agreement compared to the subtypes observed in LNM

Metástases em ovário geralmente se apresentam como o primeiro sinal de doença, com o tumor primário não sendo imediatamente reconhecido. A diferenciação mucinosa é o fenótipo mais comum entre essas metástases. Em algumas situações, quando essa apresentação está associada a carcinomas metastáticos capazes de simular tumores ovarianos primários, essa configuração pode levar até mesmo patologistas experientes a diagnosticar incorretamente um depósito secundário como uma neoplasia primária. A maioria dos casos problemáticos pode ser resolvida correlacionando-se os dados clínicos, as características macroscópicas, os critérios de histologia e o perfil imuno-histoquímico, mas sempre haverá alguns casos com características sobrepostas. Levando-se em conta que a invasão linfovascular conspícua é uma das características que favorecem metástase, nós hipotetizamos que diferentes padrões de microdensidade vascular intratumoral poderiam nos ajudar na definição da origem primária ou secundária do tumor. Um total de 124 casos de tumores mucinosos de ovário foram selecionados, apresentando histologia \"borderline\" e maligna. Eles foram separados em dois grupos (primários ou metastáticos), classificados de acordo com as informações clínicas disponíveis, características macroscópicas e microscópicas, e perfil imuno-histoquímico realizado em amostras de tumores em microarranjo de tecido (TMA). A densidade vascular linfática (DVL) foi analisada quantificando-se espaços vasculares intratumorais identificados pela podoplanina, um marcador endotelial linfático. De acordo com os nossos resultados a DVL foi maior nas neoplasias primárias do que nas secundárias, mas, após análise multivariada, os melhores preditores de um tumor ser secundário foram tamanho de 10,0 cm ou menos (OR 9,4; IC 95% 1,2-69,2), bilateralidade (OR 51,5; IC 95% 7,1-370,2) e negatividade para CK7 (OR 64,8; IC 95% 9,4- 447). De acordo com o conhecimento atual, a disseminação metastática não é um evento aleatório, mas um processo de várias etapas complexas e sequenciais, altamente organizado e tecido específico. É importante aprofundar a relação entre as células tumorais e seu microambiente porque as características moleculares envolvidas podem ser uma possível fonte de novos marcadores que podem permitir uma categorização mais precisa dos tumores mucinosos nos ovários / Ovarian metastases commonly present as the first sign of the disease, with the primary tumor not being immediately recognized. Mucinous differentiation is the most common phenotype among those metastases. In particular situations, when compounded by the known metastatic carcinomas capable of simulate primary tumors, this setting can lead even experienced pathologists to diagnose incorrectly a secondary deposit as a primary neoplasm. Correlating clinical data, macroscopic features, histology criteria and immunohistochemistry profile, can solve the majority of those problematic cases, but there will always be some cases in a gray zone with superimposed characteristics. Since conspicuous lymphovascular invasion is one of the characteristics favoring metastases, we hypothesized if different patterns of intratumoral vascular microdensity can help us in defining the tumor origin. A total of 124 cases of mucinous tumors in ovary were selected, presenting borderline and malignant histology. They were separated in two groups (primary and metastatic) classified according to the available clinical data, gross and microscopic features, and immunohistochemistry profile performed in tumor samples in tissue microarrays (TMA). The lymphatic vascular density (LVD) was analyzed using podoplanin, a lymphatic endothelial marker. According to our results LVD was greater in primary than in secondary neoplasms, but after multivariate analysis, the best predictors of a secondary deposit tumor were size 10,0 cm or less (OR 9.4; CI 95% 1.2- 69.2), bilaterality (OR 51.5; CI 95% 7.1-370.2) and CK7 negativity (OR 64.8; CI 95% 9.4-447). According to actual knowledge metastatic dissemination is not a random event, but a complex and sequential multistep process, highly organized and tissue-specific. It is important to go deeper into the relation between tumor cells and their microenvironment because its molecular features may be a possible source of new markers that could allow a more precise categorization of mucinous tumors in the ovaries

O carcinoma espinocelular oral (CECO) corresponde a aproximadamente 95% das neoplasias malignas que acometem a cavidade oral. Os fatores de risco clássicos incluem o tabagismo e etilismo; no entanto, as disfunções do sistema imune em decorrência do envelhecimento (imunossenescência) na patogênese do CECO são muito pouco estudados. Análises comparativas vêm sendo feitas para melhor caracterização do perfil de pacientes jovens e idosos acometidos pelo CECO, o qual permanece ainda controverso. Vários estudos têm mostrado que os macrófagos associados ao tumor (MATs) no CECO de pacientes idosos exibem um fenótipo M2 (pró-tumoral), com propriedades moduladoras do estroma vascular, promovendo a angiogênese. No entanto, considerando a imunossenescência, não se sabe o perfil de MATs e seus efeitos na angiogenese no CECO afetando pacientes jovens. Assim, este estudo analisou por meio da técnica imunoistoquímica, a frequência e localização de MATs em correlação com a angiogênese, no CECO, afetando pacientes jovens e idosos. Cinquenta e sete biópsias de CECO divididos em 3 grupos (I: <40 anos [n=17]; II: 40-65 anos [n=20]; III: >65 anos [n=20]) foram selecionados para compor o estudo, sendo classificados morfologicamente seguindo às recomendações da OMS (2017). Os grupos I, II e III foram comparados quanto à imunoexpressão de CD68 e CD163 para análise de MATs e de CD34 (vasos sanguíneos) e D2-40 (vasos linfáticos) para avaliação da densidade microvascular (DMV), área microvascular (AMV) e área vascular total (AVT). A análise imunoistoquímica evidenciou similar expressão de CD68 e CD163 nos três grupos (p>0,05). A avaliação da DMV, AMV e AVT sanguínea e linfática também exibiu padrões similares, com predominância significativa (p<0.05) de vasos sanguíneos, nos três grupos analisados. Não houve correlação significativa quando avaliados marcadores macrofágicos e angiogênicos. Nossos resultados mostram um similar perfil de MATs e angiogênese quando comparados os três grupos estudados, sugerindo participação de mecanismos moleculares do microambiente tumoral na manutenção da predominância de macrófagos M2 e vasos sanguíneos no CECO afetando pacientes jovens e idosos / Oral squamous cell carcinoma (OSCC) corresponds to approximately 95% of all cases of oral cavity malignancies. The classic risk factors include smoking and alcoholism; however, immune system dysfunctions due to aging (immunoscencence) in OSCC pathogenesis are poorly studied. Comparative analyzes have been made to better characterize the profile of young and old patients affected by the OSCC, which remains controversial. Several studies have shown that tumor-associated macrophages (TAMs) in OSCC of elderly patients exhibit a pro-tumoral M2 phenotype, with vascular stroma modulating properties, promoting angiogenesis. However, considering immunoscencence, the profile of TAMs and their effects on angiogenesis in OSCC affecting young patients is unknown. Thus, this study analyzed by immunohistochemical technique, the frequency and location of TAMs in correlation with angiogenesis in OSCC affecting young and elderly patients. Fiftyseven biopsies were divided into three groups (I: <40 years [n=17]; II: 40-65 years [n=20]; III: > 65 years [n=20]) and selected to compose this study, being classified morphologically following WHO (2017) recommendations. Groups I, II and III were compared for immunoexpression of CD68 and CD163 for analysis of TAMs, and CD34 (blood vessels) and D2-40 (lymphatic vessels) for evaluation of microvessel density (MVD), microvascular area (MVA) and total vascular area (TVA). Immunohistochemical analysis showed similar expression of CD68 and CD163 in the three groups (p>0.05). The evaluation of blood and lymphatic MVD, MVA and TVA also showed similar patterns, with a significant predominance (p<0.05) of blood vessels in the three groups analyzed. There was no significant correlation when evaluating macrophage and angiogenic markers. Our results show a similar profile of TAMs and angiogenesis when compared to the three groups studied, suggesting participation of molecular mechanisms of the tumor microenvironment in the maintenance of M2-polarized macrophages and blood vessels in OSCC affecting young and elderly patients

In einer retrospektiven Studie über den Zeitraum von 1990-2000 wurden in der Klinik für Kinderchirurgie des Universitätsklinikums Charité, Berlin die Kinder untersucht, bei denen eine lymphatische Malformation aufgetreten war. Es ergaben sich insgesamt 18 Patienten. Parallel wurden aus der Klinik für Gynäkologie und Geburtshilfe, Abteilung für Pränatale Diagnostik und Therapie des Universitätsklinikums Charité, Berlin retrospektiv für den oben genannten Zeitraum 31 Patientinnen untersucht, bei deren Feten ein zystisches Nackenhygrom im Verlauf der Schwangerschaft diagnostiziert worden war. Ziel der Arbeit war es, die geeigneteste Therapieform in der Behandlung von lymphatischen Malformationen im Kindesalter in besonderen Hinsicht auf die Prognose, Rezidivfreiheit und Spätergebnisse zu untersuchen. Parallel galt es zu evaluieren, ob in Anbetracht der pränatalen Diagnostizierbarkeit lympahtischer Malformationen und dem postnatal sich daraus ergebenden Krankheitsverlauf eine Schwangerschaftsunterbrechung in Erwägung gezogen werden sollte. / A ten year retrospective study of 18 children with lymphatic malformation and 31 pregnant women with fetal cystic hygromas was carried out at the Department of Pediatric Surgery and the Department of Obstetrics and Gynaecology at the University Hospital Charité, Berlin. The objective of this study was to analyze the most effective therapy for lymphatic malformation in children with regard to the children prognosis, the recurrences after treatment and the long-time-follow-up. Another object to analyze was the point of prenatal diagnosis of fetal cystic hygromas and the adverse fetal outcome.

Made available in DSpace on 2016-01-26T12:51:37Z (GMT). No. of bitstreams: 1 newtonbordin_tese.pdf: 2102625 bytes, checksum: c010c20492698be8a7290a7a377973b4 (MD5) Previous issue date: 2012-05-25 / Although lymphedema resulting from the treatment of breast cancer is secondary lymphedema, it follows the same diagnostic and treatment criteria as other types of lymphedema. Its prevalence varies from 7% to more than 50% of patients submitted to surgery depending on the surgical approach and associated treatment employed. Main: objectives of this study were to evaluate the efficacy of mechanical lymphatic drainage in reducing the volume of lymphedematous limbs after breast cancer treatment and identify the optimal time of treatment. Patients and Method: From July 2007 to July 2008, twenty-five patients with lymphedema resulting from breast cancer treatment were enrolled in this study by order of arrival at the Godoy Clinic. The ages of the patients ranged from 42 to 86 years old (mean: 55.6 years). Diagnosis of lymphoedema was made clinically and confirmed by volumetry. Lymphedema was confirmed with a difference in volume between the patient s arms of more than 200 ml. Exclusion criteria were the presence of active infection, joint problems including immobility due to neurological involvement or any clinical condition in which physical exercises are contraindicated, such as heart failure and advanced active cancer. This study was divided into two stages with all patients being evaluated by water-displacement volumetry before and after activities. In phase I (n = 25) the patients were submitted to one hour of activities and phase II (n = 13) patients were submitted to three hours of activities with volumetry being carried out every hour. The RAGodoy® electromechanical device, which makes 15 to 25 passive flexion and extension movements of the elbow joint per minute, was used for mechanical lymphatic drainage. The t-test with a Bonferroni alpha correction of 0.008 was used for statistical analysis and an alpha error of 5% (p-value &#8804; 0.05) was considered significant. The study was approved by the Research Ethics Committee of the Medical School in Sao Jose do Rio Preto. Results: The reduction in volume was significant in Phase I (one hour treatment: p < 0.001), with the initial mean weight being 2026.4 and final weight being 1967.2 (mean loss of 59.2 grams). In Phase II (three hours activities), the reductions were significant in the first and second hours (p <0.0005 and 0.002, respectively). However, no statically significant difference was seen using a Bonferroni alpha correction of 0.008 (p-value = 0.01) between the second and third hour. Conclusion: In conclusion the use of the device reduces the volume of lymphedematous limbs; it is most effective in the first hour, but provides significant reductions for a maximum of two hours. / O lindedema pós-câncer de mama enquadra-se no tipo secundário de linfedema e segue os mesmos padrões diagnósticos e terapêuticos dos demais tipos de linfedema. Sua prevalência varia entre 7% a mais de 50% das pacientes submetidas ao tratamento cirúrgico, dependendo da abordagem cirúrgica realizada e dos tratamentos complementares empregados. Objetivo: O objetivo deste estudo foi avaliar a eficácia da drenagem linfática mecânica na redução de volume dos membros linfedematosos pós-tratamento câncer de mama e a identificação do tempo ideal de tratamento. Pacientes e Método: De julho 2007 a julho de 2008, 25 pacientes com linfedema pós-tratamento de câncer de mama foram avaliadas neste estudo, selecionadas por ordem de chegada, na Clínica Godoy. A idade das pacientes variou entre 42 a 86 anos e a idade média foi de 55,6 anos. O diagnóstico do linfedema foi clínico e confirmado por volumetria. O linfedema foi confirmado quando a diferença de volume era maior que 200 ml entre os membros. Considerou-se como critério de exclusão a presença de infecção ativa, problemas articulares envolvendo a imobilidade devido a comprometimento neurológico ou qualquer condição clínica com contraindicação para atividade física como a insuficiência cardíaca e o câncer em atividade. O estudo foi dividido em duas fases sendo que todas pacientes realizaram a volumetria por técnica de deslocamento de água antes a após a atividade. Na fase I (n = 25) as pacientes realizaram uma hora de atividade e na fase II (n = 13) as pacientes realizaram três horas de atividade, sendo feito a volumetria ao final de cada hora. O dispositivo eletromecânico RAGodoy®, que faz de 15 a 25 movimentos de flexão e extensão passivas da articulação do cotovelo, foi usado para a drenagem mecânica do membro. Para análise estatística foi usado o teste-t com correção alfa de Bonferroni = 0,008, considerando erro alfa de 5% (valor p&#8804; 0,05) como significativo. O estudo foi aprovado pelo Comitê de Ética em Pesquisa da Faculdade de Medicina de São Jose do Rio Preto-SP. Resultados: A redução do volume foi significativa na Fase I (uma hora de tratamento: p < 0,001), sendo a média do peso inicial de 2026,4g e final de 1967,2g (perda média de 59,2 g). Na Fase II (três horas de atividade) as reduções foram significativas na primeira e segunda horas (p < 0,0005 e p < 0,002, respectivamente). Entretanto, entre a segunda e terceira hora, a diferença não foi estatisticamente significativa. Conclusões: Conclui-se que o dispositivo mecânico reduz o volume de membro linfedematoso durante sua aplicação, sendo mais eficaz na primeira hora mas proporciona redução significativa quando usado por até duas horas.