Análise do padrão de envolvimento de linfonodos no carcinoma de células escamosas da orofaringe submetido à cirurgia robótica transoral / Analysis of the pattern of lympho node involvement in oropharynx squamous cell carcinoma submitted to transoral robotic surgery

A inovação das ressecções robóticas transorais permitiram o acesso a regiões da orofaringe cujas incisões convencionais tinham maior morbidade, reservando o acesso cervical para os casos com indicação de esvaziamento cervical. O estudo da linfangiogênese dos tumores de orofaringe se faz essencial, com vistas a individualização do tratamento do pescoço destes pacientes. Para tanto, foram analisados 156 prontuários de pacientes submetidos à orofaringectomia por via transoral robótica para o tratamento de carcinoma escamoso no período de 2012 a 2020 no Instituto Nacional de Câncer. A incidência de acometimento do nível IIA neste estudo foi de 39% (n=61) e III de 19,2% (n=30). O nível IIB estava majoritariamente associado ao acometimento do nível IIA. O baixo acometimento dos níveis cervicais IA, IB e V demonstra que ressecção profilática destes não apresentou benefícios associado à elevada morbidade. O conhecimento do padrão de disseminação linfonodal do câncer de orofaringe pode determinar a melhor abordagem cirúrgica do pescoço

The innovation of transoral robotic resections allowed access to regions of the oropharynx where conventional incisions had greater morbidity, reserving cervical access for cases requiring neck dissection. The study of lymphangiogenesis of oropharyngeal tumors is essential, with a view to individualizing the treatment of the neck of these patients. To this end, 156 medical records of patients who underwent robotic transoral oropharyngectomy for the treatment of squamous cell carcinoma from 2012 to 2020 at the National Cancer Institute were analyzed. The incidence of level IIA involvement in this study was 39% (n=61) and III was 19.8% (n=31). Level IIB was mostly associated with level IIA involvement. The low involvement of cervical levels IA, IB and V demonstrates that prophylactic resection of these did not provide benefits associated with high morbidity. Knowledge of the lymph node spread pattern of oropharyngeal cancer can determine the best surgical approach to the neck

Wessely corneal ring phenomenon: An unsolved pathophysiological dilemma.

The cornea is a densely innervated avascular tissue showing exceptional inflammatory and immune responses. The cornea is a site of lymphangiogenic and angiogenic privilege devoid of blood and lymphatic vessels that limits the entry of inflammatory cells from the adjacent and highly immunoreactive conjunctiva. Immunological and anatomical differences between the central and peripheral cornea are also necessary to sustain passive immune privilege. The lower density of antigen-presenting cells in the central cornea and the 5:1 peripheral-to-central corneal ratio of C1 are two main features conferring passive immune privilege. C1 activates the complement system by antigen-antibody complexes more effectively in the peripheral cornea and, thus, protects the central corneas' transparency from immune-driven and inflammatory reactions. Wessely rings, also known as corneal immune rings, are noninfectious ring-shaped stromal infiltrates usually formed in the peripheral cornea. They result from a hypersensitivity reaction to foreign antigens, including those of microorganism origin. Thus, they are thought to be composed of inflammatory cells and antigen-antibody complexes. Corneal immune rings have been associated with various infectious and noninfectious causes, including foreign bodies, contact lens wear, refractive procedures, and drugs. We describe the anatomical and immunologic basis underlying Wessely ring formation, its causes, clinical presentation, and management.

Intratumoral lymphatic vascular density is an independent factor for disease-free and overall survival in advanced stage oral squamous cell carcinoma.

OBJECTIVE: The presence of lymphatic and blood vessels in oral squamous cell carcinoma (OSCC) should play a key role in progression and dissemination. This study aimed to evaluate the correlation between the lymphatic and blood vessel densities with prognostic outcomes in advanced stage OSCC. STUDY DESIGN: Immunohistochemical reactions for D-240, CD34, and CD105 were performed in 88 advanced stage OSCC cases located at the oral tongue and the floor of the mouth. The lymphatic vascular density (LVD), blood vascular density (BVD), and neoformed vascular density (NVD) were assessed by counting positive reactions in 4 hotspot areas, both intratumoral (IT) and peritumoral (PT), at high magnification (× 40). RESULTS: High IT LVD was associated with extracapsular spread of lymph node metastasis (P = .03). Recurrence rates were correlated with IT LVD (P < .0001), IT BVD (P = .036), and IT NVD (P = .047), and overall survival was associated with high IT LVD (P = .0016) and IT NVD (P = .009). Yet, IT LVD was an independent factor for disease-free survival and for overall survival based on the Cox proportional hazards model. CONCLUSIONS: Our results suggest that high IT LVD has a strong impact on survival outcomes in advanced stage OSCC.

Cancer treatment in the lymphatic system: A prospective targeting employing nanostructured systems.

Cancer related to lymphangiogenesis has gained a great deal of attention in recent decades ever since specific markers of this intriguing system were discovered. Unlike the blood system, the lymphatic system has unique features that can advance cancer in future metastasis, or, conversely, can provide an opportunity to prevent or treat this disease that affects people worldwide. The aim of this review is to show the recent research of cancer treatment associated with the lymphatic system, considered one of the main gateways for disseminating metastatic cells to distant organs. Nanostructured systems based on theranostics and immunotherapies can offer several options for this complex disease. Precision targeting and accumulation of nanomaterials into the tumor sites and their elimination, or targeting the specific immune defense cells to promote optimal regression of cancer cells are highlighted in this paper. Moreover, therapies based on nanostructured systems through lymphatic systems may reduce the side effects and toxicity, avoid first pass hepatic metabolism, and improve patient recovery. We emphasize the general understanding of the association between the immune and lymphatic systems, their interaction with tumor cells, the mechanisms involved and the recent developments in several nanotechnology treatments related to this disease.

A modified mouse-tail lymphedema model.

One of the main obstacles to studying the pathophysiology of lymphedema development is the lack of appropriate experimental models. Fol-lowing up on a mouse-tail method that has been described, we performed changes to the method which made it easier to perform in our hands and demonstrated similar results. Twenty C57Black mice were operated on using the previous tech-nique and euthanized after 3 or 6 weeks. Another twenty mice were submitted to the new technique developed in our laboratory and euthanized at the same time points. Tissue samples were collected from the proximal part of the tail (control) and from the distal part (lymphedema) for both mod-els. Animals in both operative groups developed marked edema in the distal part of the tail. This was characterized by lymph vessels dilation, edema, inflammatory cell infiltration, and adipose tissue deposition. Lymphedema was detected after 3 weeks in both models, reaching its maximum after 6 weeks. Adipocytes detected by histology (Oil red O staining) and molecular markers for adipogenesis, lymphangiogenesis and inflam-mation (lipin 1 and 2, SLP76, and F4-80) were demonstrated to be increased equally in both models. In conclusion, both models provide a reliable method to study lymphedema pathophys-iology. However, our modified technique is easier and faster to perform while still providing reliable and consistent results.

Donor-host Lymphatic Anastomosis After Murine Lung Transplantation.

BACKGROUND: Establishing lung lymphatic drainage is thought to be important for successful lung transplantation. To date, there has been a complete absence of knowledge of how lymphatic connections are reestablished after lung transplant, despite evidence suggesting that this does indeed occur. The present study aimed to elucidate whether and how lymphatic anastomosis occurs after lung transplant. METHODS: An orthotopic murine model of lung transplant using lymphatic reporter mice and whole mount immunohistochemistry was used to evaluate the lymphatic vasculature and donor-host connections after lung transplantation. RESULTS: Immunohistochemistry of transplanted lungs demonstrated robust lymphatic vessels, and functional assays demonstrated lymphatic drainage in the transplanted lung that was comparable with that in native lungs. Lymphatic vessels in the donor lung exhibited active sprouting toward the host at the anastomosis within the first 3 days after lung transplantation, with more numerous and complex lymphatic sprouting developing thereafter. Donor lymphatic vessels were numerous at the site of anastomosis by day 14 after lung transplantation and formed physical connections with host lymphatic vessels, demonstrating a mechanism by which lymphatic drainage is reestablished in the transplanted lung. CONCLUSIONS: Lymphatic drainage after lung transplantation is established by active sprouting of donor lymphatic vessels towards the host and the formation of donor-host lymphatic connections at the level of the transplant anastomosis.

Gene expression and promoter methylation of angiogenic and lymphangiogenic factors as prognostic markers in melanoma.

The high mortality rate of melanoma is broadly associated with its metastatic potential. Tumor cell dissemination is strictly dependent on vascularization; therefore, angiogenesis and lymphangiogenesis play an essential role in metastasis. Hence, a better understanding of the players of tumor vascularization and establishing them as new molecular biomarkers might help to overcome the poor prognosis of melanoma patients. Here, we further characterized a linear murine model of melanoma progression and showed that the aggressiveness of melanoma cells is closely associated with high expression of angiogenic factors, such as Vegfc, Angpt2, and Six1, and that blockade of the vascular endothelial growth factor pathway by the inhibitor axitinib abrogates their tumorigenic potential in vitro and in the in vivo chicken chorioallantoic membrane assay. Furthermore, analysis of The Cancer Genome Atlas data revealed that the expression of the angiogenic factor ANGPT2 (P-value = 0.044) and the lymphangiogenic receptor VEGFR-3 (P-value = 0.002) were independent prognostic factors of overall survival in melanoma patients. Enhanced reduced representation bisulfite sequencing-based methylome profiling revealed for the first time a link between abnormal VEGFC, ANGPT2, and SIX1 gene expression and promoter hypomethylation in melanoma cells. In patients, VEGFC (P-value = 0.031), ANGPT2 (P-value < 0.001), and SIX1 (P-value = 0.009) promoter hypomethylation were independent prognostic factors of shorter overall survival. Hence, our data suggest that these angio- and lymphangiogenesis factors are potential biomarkers of melanoma prognosis. Moreover, these findings strongly support the applicability of our melanoma progression model to unravel new biomarkers for this aggressive human disease.

Síndromes MonoMAC e Emberger em paciente com mutação no gene GATA2 / MonoMAC and Emberger syndromes in a patient with GATA2 mutation

As mutações que ocorrem no gene GATA2 podem ocasionar um amplo espectro de doenças genéticas. Os pacientes podem ter anormalidades na hematopoiese, na linfangiogenesis e na resposta imunológica. Os fenótipos incluem algumas síndromes caracterizadas por monocitopenia e infecção por micobactéria (síndrome MonoMac), síndrome mielodisplásica familiar, leucemia mieloide crônica ou aguda, síndrome de Emberger (linfedema primário), e mais raramente neutropenia, anemia aplástica e deficiência isolada de células NK. A idade da apresentação clínica pode variar desde a infância até a idade adulta. A deficiência autossômica dominante de GATA2 pode permanecer clinicamente silenciosa por décadas, ou mesmo durante toda a vida. Descrevemos o caso de uma jovem brasileira que apresentou a maioria dos problemas ligados à mutação no gene GATA2, observando-se as duas síndromes: MonoMAC e Emberger.

GATA2 mutations may cause a wide spectrum of genetic disorders. Patients may have several abnormalities in hematopoiesis, lymphangiogenesis and immune response. The phenotypes include monocytopenia and mycobacterial infection (MonoMAC) syndrome, familial myelodysplastic syndrome (MDS), chronic or acute myeloid leukemia (CML or AML), Emberger syndrome and, more rarely, neutropenia, aplastic anemia and isolated NKcell deficiency. Age at clinical onset ranges from early childhood to late adulthood. Autosomal dominant GATA2 deficiency may remain clinically silent for decades or even for life. We report a case of a Brazilian young patient who had most of the problems related to GATA2 mutation as well as MonoMAC and Emberger syndromes.

Vascular endothelial growth factor C treatment for mouse hind limb lymphatic revascularization.

Spontaneous lymphatic revascularization is a challenge and the establishment of new therapeutic strategies may improve life quality for patients suffering from lymphatic disorders. This study was designed to verify if VEGFC treatment improves lymphatic vascularization in a time-dependent manner in mouse hindlimb (HL) after resection of the inguinal lymph node. Lymphatic vascular density (Vv) and length (Lv) were evaluated by stereology after immunohistochemistry. The control Group (CG) was not manipulated but received saline instead of VEGFC treatment. The surgery Group (SG) had the left inguinal lymph node resected but did not received VEGFC treatment. VEGFC Treated Group (TG) had the node resected and received VEGFC treatment. VEGFC and VEGFR3 local expression were assessed by qPCR. There was an effect of time over Vv and Lv in the SG and significant difference between CG and SG in the regions studied (proximal, medium and distal regions) of the left HL (LHL). The Lv showed significant difference between CG and SG only in the medium region. The Vv and the Lv for TG were higher than the other groups. VEGFC and VEGFR3 gene expression presented time effect in all regions of the LHL for SG and TG. Both VEGFC and VEGFR3 gene expression presented significant difference between CG and SG, between SG and TG and between CG and TG. This study showed significant decrease in lymphatic vascularization in the left hindlimb of mice after surgical removal of the inguinal lymph node and adjacent lymphatic vessels. Exogenous VEGFC could recover lymphatic vascularization through stimulating neolymphangiogenesis.

Immunohistochemical study of CD34 and podoplanin in periodontal disease.

BACKGROUND AND OBJECTIVE: The aim of this study was to evaluate the angiogenesis and lymphangiogenesis in gingival tissue biopsy specimens of individuals with clinically healthy gingiva, chronic gingivitis, and chronic periodontitis (n = 30 per clinical condition). MATERIAL AND METHODS: Histological sections were stained using hematoxylin and eosin as well as immunohistochemically with hematopoietic progenitor cell antigen CD34 and podoplanin (PDPN) antibodies to evaluate the microvascular count, area, and perimeter of blood and lymphatic vessels, respectively. RESULTS: The results revealed a correlation between the microvascular count of blood and lymphatic vessels (P = 0.03; however, in individuals with chronic periodontitis, fewer lymphatic vessels were present than in the clinically healthy gingival tissue (P = 0.01), which was not observed in the case of microvascular area and perimeter. Podoplanin labeling was present in the epithelium, and the intensity of labeling was positively correlated to the intensity of the inflammatory infiltrate (P = 0.03). CONCLUSION: In this study, we concluded that an increase in the number of blood and lymphatic vessels was not observed in bouth gingivitis and periodontitis samples. Podoplanin expression is highly associated with an increased inflammatory infiltration suggesting that PDPN might play an additional role in periodontal disease, other than solely as a lymphangiogenesis marker.

Histomorphometric evaluation of the Ki-67 proliferation rate and CD34 microvascular and D2-40 lymphovascular densities drives the pulmonary typical carcinoid outcome.

Ki-67 has shown promise as a prognostic factor in pulmonary carcinoids. In this study, we sought to validate the importance of Ki-67 and study the relationships between Ki-67 and other stromal biomarkers of vascular density. We examined Ki-67, CD34, and D2-40 in tumor tissues from 128 patients with surgically excised typical carcinoid of the lung. We used immunohistochemistry and morphometry to evaluate the amount of tumor staining for cellular proliferation (Ki-67), microvascular density (CD34-MVD), and D2-40 lymphovascular density. The main outcome was overall survival, considered as life expectancy until death from metastasis. Specimens from patients with central tumors showed high CD34-MVD (P = .01), which was also significantly associated with a compromised surgical margin, lymph node metastasis, and clinical stage Ib. Equally significant was high D2-40 lymphovascular density in central specimens with a compromised surgical margin and lymph node metastasis. A high Ki-67 proliferation rate was significantly associated with tumors from patients with clinical stage IIb, IIIa, and IV disease. Multivariate Cox model analysis demonstrated that tumor location and stage, surgical margin, tumor size, and N stage were significantly related to survival time (P < .05). Quantitative staining of the tumor for Ki-67 and CD34-MVD served as prognostic factors (P < .05), which were more relevant than the surgical and pathological stage. Ki-67 greater than 5% and CD34-MVD greater than 7% staining comprise a subset of patients with higher death hazard; this outcome may harbor evidence for further prospective studies of target therapy after surgical resection.

Immunohistochemical expression of podoplanin (D2-40), lymphangiogenesis, and neoangiogenesis in tooth germ, ameloblastomas, and ameloblastic carcinomas.

BACKGROUND: Ameloblastoma is a benign but locally aggressive odontogenic tumor, while ameloblastic carcinoma is its malignant counterpart. Angiogenesis and lymphangiogenesis in malignancies have been correlated with higher aggressiveness and poor prognosis, as well as greater expression of podoplanin by tumoral cells. METHODS: Immunohistochemical expression of podoplanin, CD34, and CD105 (endoglin) was evaluated in 53 ameloblastomas and three ameloblastic carcinomas; additionally, immunohistochemistry for podoplanin was also performed in 10 tooth germs. Microvessel density of blood and lymphatic vessels was calculated and compared between ameloblastomas and ameloblastic carcinomas. Immunoexpression of podoplanin by ameloblastic cells was evaluated in tooth germs, ameloblastomas, and ameloblastic carcinomas. RESULTS: Podoplanin was similarly expressed by odontogenic epithelial cells of tooth germs and ameloblastomas, while its expression was lower in ameloblastic carcinomas. There was no difference in microvessel density assessed by CD34 between ameloblastomas and ameloblastic carcinomas; nevertheless, the latter presented higher amounts of lymphatic and new formed blood vessels. CONCLUSIONS: Results suggest that podoplanin does not seem to be involved in invasion mechanisms of ameloblastic carcinomas, as its expression was decreased in the malignant tumoral cells. On the other hand, the increased lymphatic microvessel density and neoangiogenesis found in ameloblastic carcinomas could be related to its aggressiveness and potential for metastasis.

Effects of fatty acid synthase inhibitors on lymphatic vessels: an in vitro and in vivo study in a melanoma model.

Fatty acid synthase (FASN) is responsible for the endogenous production of fatty acids from acetyl-CoA and malonyl-CoA. Its overexpression is associated with poor prognosis in human cancers including melanomas. Our group has previously shown that the inhibition of FASN with orlistat reduces spontaneous lymphatic metastasis in experimental B16-F10 melanomas, which is a consequence, at least in part, of the reduction of proliferation and induction of apoptosis. Here, we sought to investigate the effects of pharmacological FASN inhibition on lymphatic vessels by using cell culture and mouse models. The effects of FASN inhibitors cerulenin and orlistat on the proliferation, apoptosis, and migration of human lymphatic endothelial cells (HDLEC) were evaluated with in vitro models. The lymphatic outgrowth was evaluated by using a murine ex vivo assay. B16-F10 melanomas and surgical wounds were produced in the ears of C57Bl/6 and Balb-C mice, respectively, and their peripheral lymphatic vessels evaluated by fluorescent microlymphangiography. The secretion of vascular endothelial growth factor C and D (VEGF-C and -D) by melanoma cells was evaluated by ELISA and conditioned media used to study in vitro lymphangiogenesis. Here, we show that cerulenin and orlistat decrease the viability, proliferation, and migration of HDLEC cells. The volume of lymph node metastases from B16-F10 experimental melanomas was reduced by 39% in orlistat-treated animals as well as the expression of VEGF-C in these tissues. In addition, lymphatic vessels from orlistat-treated mice drained more efficiently the injected FITC-dextran. Orlistat and cerulenin reduced VEGF-C secretion and, increase production of VEGF-D by B16-F10 and SK-Mel-25 melanoma cells. Finally, reduced lymphatic cell extensions, were observed following the treatment with conditioned medium from cerulenin- and orlistat-treated B16-F10 cells. Altogether, our results show that FASN inhibitors have anti-metastatic effects by acting on lymphatic endothelium and melanoma cells regardless the increase of lymphatic permeability promoted by orlistat.

Estudo imunoistoquímico do CD34 e podoplanina na doença periodontal / Immunohistochemical study of CD34 and podoplanin in periodontal disease

A angiogênese e a linfangiogênese são alterações também decorrentes da inflamação gengival provocada por microrganismos presentes no biofilme dental, bem como pela a migração de células de defesa e secreção de mediadores inflamatórios no local da agressão. Este estudo teve por objetivo avaliar a angiogênese e linfangiogênese em 90 espécimes de biópsias de tecido gengival clinicamente saudável, com gengivite e com periodontite crônicas. Os cortes histológicos foram avaliados pela coloração de hematoxilina e eosina e pela técnica de imunoistoquímica através da imunomarcação de CD34 e podoplanina, para avaliar, respectivamente, o índice angiogênico e linfangiogênico, por meio da técnica de contagem microvascular. Os resultados mostraram que há correlação entre os índices (p=0,030), porém, mostrou que na periodontite há menos números de vasos linfáticos do que no tecido gengival clinicamente saudável (p=0,016). A podoplanina mostrou marcação no epitélio e que há relação da intensidade de marcação com a intensidade do infiltrado inflamatório, sendo mais intensa a marcação na presença de infiltrado inflamatório severo (p=0,033). Concluiu-se neste estudo que há menor número de vasos sanguíneos na periodontite em comparação com a gengiva clinicamente saudável. As sinalizações presentes no processo inflamatório, bem como o real papel da vasculatura sanguínea e linfática gengival ainda não estão totalmente elucidadas. (AU)

Angiogenesis and lymphangiogenesis are changes that occur due to gingival inflammation caused by microorganisms present in the biofilm, as well as the migration of immune cells and secretion of mediators in the aggressed site. This study aimed to research angiogenesis and lymphangiogenesis in 90 specimens of clinically healthy, with gingivitis and chronic periodontitis gingival tissue biopsies. The histological sections were evaluated by hematoxylin and eosin and the immunohistochemical technique through immunostaining for CD34 and podoplanin. To evaluate the angiogenic and lymphangiogenic indexes we performed a microvessel counting technique. The results showed that there is a correlation between the indexes (p = 0.030), however, we observed that periodontitis showed less lymphatic vessels than clinically healthy gingival tissue (p = 0.016). Podoplanin showed positive staining in the basal layers of the epithelium, and we observed a relationship between immunostaining intensity and the intensity of inflammatory infiltrate, with more intense staining in the presence of severe inflammatory infiltrate (p = 0.033). For this study, we concluded that there are fewer blood vessels in periodontitis compared with clinically healthy gingiva. The signaling present in the inflammatory process and the actual role of gingival blood and lymphatic vasculature are not fully understood, with further studies on angiogenesis and lymphangiogenesis being suggested. (AU)

Análise da imunoexpressão da podoplanina e da triptase em carcinoma epidermóide de língua e sua relação com parâmetros clinicopatológicos / Immunohistochemical analysis of podoplanin and tryptase in squamous cell carcinoma of the tongue and its relationship with clinicopathological parameters

O carcinoma epidermóide de língua oral (CELO) apresenta um comportamento biológico agressivo, com elevada propensão ao desenvolvimento de metástases nodais. Nesse contexto, a linfangiogênese é considerada um fenômeno importante para a disseminação das células tumorais e pode sofrer influência de estímulos do microambiente. Os mastócitos têm sido relacionados à progressão de neoplasias malignas, no entanto o seu papel na formação de vasos linfáticos ainda não está bem estabelecido. O propósito desta pesquisa foi avaliar possíveis correlações entre a densidade linfática, a contagem de mastócitos e o perfil clinicopatológico em casos de CELO, incluindo o estadiamento clínico TNM, a gradação histológica de malignidade (Bryne, 1998) e a presença/ausência de metástases nodais. A amostra foi constituída por 50 casos de CELO, dos quais 26 apresentavam metástase nodal, e os 24 restantes eram isentos de metástases. A densidade linfática foi estabelecida como a média de vasos linfáticos imunomarcados pelo anticorpo anti-podoplanina (D2-40), identificados em cinco campos microscópicos (200x). Para a análise dos mastócitos, foram quantificadas as células imunorreativas ao anticorpo anti-triptase, em cinco campos (400x). Destaca-se que ambas as imunomarcações foram analisadas no centro tumoral e no front de invasão. A densidade linfática intratumoral (DLI) foi superior nos casos em estágios clínicos avançados (III-IV), quando comparados àqueles em estágios iniciais (I-II), assim como nos casos metastáticos em relação aos não-metastáticos (p<0,05).

Não houve diferenças estatisticamente significativas entre os casos de baixo grau e alto grau de malignidade no tocante à DLI (p>0,05). De outro modo, a densidade linfática peritumoral (DLP) e as contagens de mastócitos não demonstraram relações significativas com nenhum dos parâmetros clinicopatológicos avaliados (p>0,05). Também não foram encontradas correlações significativas entre as densidades linfáticas e as contagens de mastócitos, seja na região intratumoral (r = -0,004; p=0,977) ou na peritumoral (r = -0,154; p=0,285). Os resultados do presente estudo sugerem que os vasos linfáticos intratumorais contribuem na progressão do CELO. Por sua vez, a DLP pode não ser suficiente para justificar diferenças no comportamento biológico do CELO, o que sustenta a hipótese de envolvimento de outros mecanismos na disseminação metastática das células malignas, que complementariam os efeitos da linfangiogênese. Os mastócitos, ainda que realizem diversas funções pró- e antitumorais, parecem não influenciar diretamente o potencial de agressividade do CELO. Adicionalmente, é possível que a quantidade destas células não seja um fator determinante para a formação de vasos linfáticos. (AU)

Oral tongue squamous cell carcinoma (OTSCC) has an aggressive biological behavior, with a high propensity for the development of lymph node metastases. In this context, lymphangiogenesis is considered an important phenomenon for the spread of tumor cells and may be influenced by microenvironmental stimuli. Mast cells have been implicated in tumor progression, although their influence in the formation of lymphatic vessels is not well established. The aim of this study was to analyze, in a case series of OTSCC (n=50), possible correlations between lymphatic vessel density (LVD), mast cell count and clinicopathological features, including tumor-node-metastasis (TNM) stage, histological grade of malignancy (Bryne, 1998), and nodal metastasis. LVD was established as the mean number of lymphatic vessels immunostained by anti-podoplanin (D2-40) antibody, identified in five microscopic fields (200x). For the analysis of mast cells, tryptase-immunoreactive cells were quantified in five fields (400x). Both immunostainings were analyzed in the tumor center and invasion front. Intratumoral lymphatic density (ILD) was higher in cases in advanced clinical stages (III-IV), compared to those in initial stages (I-II), as well as in metastatic cases in respect of non-metastatic (p<0,05).

There were no statistically significant differences between low-grade and high-grade malignancy cases with respect to ILD (p>0,05). Peritumoral lymphatic density (PLD) and mast cell counts showed no significant relations with any of the clinicopathological parameters evaluated (p>0,05). Also there were no significant correlations between LVD and mast cell counts, whether in intratumoral (r = -0,004; p=0,977) or peritumoral region (r = -0,154; p=0,285). The results of the present study suggest that intratumoral lymphatic vessels may contribute in part to the progression of OTSCC, although PLD may be insufficient to justify differences in biological behavior. This supports the hypothesis of involvement of other mechanisms in metastatic spread of malignant cells, which could complement the effects of lymphangiogenesis. Although mast cells perform several pro- and antitumoral functions, they do not appear to directly influence aggressiveness of OTSCC. In addition, the quantity of these cells may not be essential for lymphatic vessel formation. (AU)

Lymphangiogenesis and angiogenesis in oral cavity and lower lip squamous cell carcinoma / Linfangiogênese e angiogênese em carcinomas de células escamosas de lábio inferior e da cavidade oral

ABSTRACT INTRODUCTION: Tumors of the lip and oral cavity differ in various aspects; therefore a clarification of the distinctions among these sites may help to better understand the biologic behavior of neoplasms occurring in these locations. OBJECTIVE: Considering that angiogenesis and lymphangiogenesis are two major elements that can influence various aspects of tumor biology, we aimed to compare these factors between squamous cell carcinoma of the lower lip and oral cavity. METHODS: A total of 84 primary squamous cell carcinomas including 45 oral and 39 lower lip tumors were selected and immunohistochemically stained with monoclonal antibody against D2-40 and CD105. Mean microvessel density was assessed in tumoral tissue, while lymphatic vessel density was calculated in both neoplastic tissue and invasion front. Data were statistically analyzed using t-test and p-values of <0.05 were considered significant. RESULTS: We found a mean microvessel density ± standard deviation of 31.94 ± 18.9 in oral cavity and 27.54 ± 20.8 in lower lip squamous cell carcinomas, with no significant difference (p = 0.32). Mean lymphatic vessel density ± standard deviation was 13.05 ± 8.2 and 16.57 ± 10.79 in of oral cavity and lower lip neoplastic tissue, respectively. The corresponding values were 9.94 ± 5.59 and 12.50 ± 7.8 in the invasive front. Significant differences were not observed in either of the lymphatic vessel density variables between the two sites. CONCLUSION: According to our results, it seems that the search for additional factors other than those related to the vasculature should continue, to help clarify the differences in biologic behavior between lower lip and oral cavity squamous cell carcinomas.

Resumo Introdução: Os tumores de lábio e da cavidade oral diferem em vários aspectos; portanto, o conhecimento das diferenças entre eles pode ajudar na melhor compreensão do comportamento biológico das neoplasias que ocorrem nesses locais. Objetivo: Considerando que a angiogênese e a linfangiogênese são dois elementos importantes que podem influenciar diversos aspectos da biologia dos tumores, objetivamos comparar esses fatores entre o carcinoma de células escamosas (CCE) de lábio inferior e da cavidade oral. Método: No total, foram selecionados 84 CCEs primários (45 tumores da cavidade oral e 39 tumores de lábio). Esses tumores foram corados por processo imunohistoquímico com anticorpo monoclonal anti-D2-40 e CD105. Avaliamos a densidade média de microvasos (DMV) no tecido tumoral, enquanto que a densidade vascular linfática (DVL) foi calculada tanto no tecido neoplásico como no front de invasão. Os dados foram estatisticamente analisados com o uso do teste t e valores de p < 0,05 foram considerados significantes. Resultados: Chegamos a uma média para DMV ± DP de 31,94 ± 18,9 para CCEs na cavidade oral e de 27,54 ± 20,8 no lábio inferior, sem diferença significante (p = 0,32). As médias para DVL ± DP foram de 13,05 ± 8,2 e 16,57 ± 10,79 no tecido neoplásico da cavidade oral e lábio inferior, respectivamente. Os valores correspondentes foram 9,94 ± 5,59 e 12,50 ± 7,8 no front invasivo. Não foram observadas diferenças significantes nas duas variáveis DVL entre os dois locais. Conclusão: De acordo com os nossos resultados, a pesquisa por fatores adicionais, além daqueles relacionados à vasculatura, deve ter continuidade, para auxiliar no esclarecimento das diferenças do comportamento biológico entre CCEs no lábio inferior e na cavidade oral.

Evaluation of hypoxia, angiogenesis, and lymphangiogenesis in actinic cheilitis.

BACKGROUND: Actinic cheilitis is a potentially malignant condition caused mainly by chronic sun exposure. Here we aim to evaluate the role of hypoxia, angiogenesis, and lymphatic density in the clinical and morphological progression of a series of cases of actinic cheilitis. MATERIALS AND METHODS: Immunohistochemistry was used to evaluate positivity to hypoxia-inducible factor (HIF)-1α, vascular endothelial growth factor (VEGF)-C, and D2-40 in 40 cases of actinic cheilitis of the lower lip. RESULTS: The cases studied exhibited variable degrees of positivity to the markers. The median number of lymphatic vessels was 3.2, 2.4, and 3.0 in lesions showing no epithelial dysplasia (NED) and with mild (MED) and moderate (MOED) epithelial dysplasia, respectively. The median VEGF-C positivity index was 82.44% (NED), 92.74% (MED), and 82.83% (MOED), and the median HIF-1α positivity index was 11.57% (NED), 5.26% (MED), and 13.55% (MOED). No significant differences in lymphatic density or median VEGF-C and HIF-1α positivity indices were observed between histological grades or clinical presentations of actinic cheilitis (P > 0.05). CONCLUSIONS: Although representing early events in lip carcinogenesis, the present results suggest that hypoxia, angiogenesis, and lymphangiogenesis do not influence the morphological or clinical progression of actinic cheilitis.

Promoting inflammatory lymphangiogenesis by vascular endothelial growth factor-C (VEGF-C) aggravated intestinal inflammation in mice with experimental acute colitis.

Angiogenesis and lymphangiogenesis are thought to play a role in the pathogenesis of inflammatory bowel diseases (IBD). However, it is not understood if inflammatory lymphangiogenesis is a pathological consequence or a productive attempt to resolve the inflammation. This study investigated the effect of lymphangiogenesis on intestinal inflammation by overexpressing a lymphangiogenesis factor, vascular endothelial growth factor-C (VEGF-C), in a mouse model of acute colitis. Forty eight-week-old female C57BL/6 mice were treated with recombinant adenovirus overexpressing VEGF-C or with recombinant VEGF-C156S protein. Acute colitis was then established by exposing the mice to 5% dextran sodium sulfate (DSS) for 7 days. Mice were evaluated for disease activity index (DAI), colonic inflammatory changes, colon edema, microvessel density, lymphatic vessel density (LVD), and VEGFR-3mRNA expression in colon tissue. When acute colitis was induced in mice overexpressing VEGF-C, there was a significant increase in colonic epithelial damage, inflammatory edema, microvessel density, and neutrophil infiltration compared to control mice. These mice also exhibited increased lymphatic vessel density (73.0±3.9 vs 38.2±1.9, P<0.001) and lymphatic vessel size (1974.6±104.3 vs 1639.0±91.5, P<0.001) compared to control mice. Additionally, the expression of VEGFR-3 mRNA was significantly upregulated in VEGF-C156S mice compared to DSS-treated mice after induction of colitis (42.0±1.4 vs 3.5±0.4, P<0.001). Stimulation of lymphangiogenesis by VEGF-C during acute colitis promoted inflammatory lymphangiogenesis in the colon and aggravated intestinal inflammation. Inflammatory lymphangiogenesis may have pleiotropic effects at different stages of IBD.

Expressão de VEGF-C, VEGF-D, mensuração da densidade linfática e da proliferação endotelial linfática em neoplasias de glândulas salivar / Expression of VEGF-C, VEGF-D density measurement of lymphatic and lymphatic endothelial proliferation in salivary gland neoplasms

As neoplasias de glândulas salivares exibem uma grande diversidade morfológica e comportamentos biológicos variados o que suscita o interesse na pesquisa destas lesões. A disseminação das células tumorais é um passo inicial para a progressão de neoplasias malignas e, dentro deste contexto, os vasos linfáticos neoformados são considerados essenciais para que ocorra essa disseminação. O papel do VEGF (fator de crescimento endotelial vascular) na formação dos vasos é fato conhecido mas, pouco se sabe a respeito de sua participação em tumores de glândula salivar. Desta forma, o objetivo deste estudo foi avaliar a expressão do VEGF-C e VEGF-D, a densidade linfática tumoral (D2-40) e a proliferação endotelial linfática (dupla marcação D2-40/Ki-67) em uma série de neoplasias de glândulas salivares. A amostra foi composta por 20 adenomas pleomórficos, 20 carcinomas adenóides císticos, 20 carcinomas mucoepidermóides e 10 casos de tecido glandular salivar com características de normalidade para efeito comparativo. Todos os casos estudados exibiram expressão positiva para VEGF-C em região peritumoral e intratumoral, não sendo encontrada diferenças de imunoexpressão entre os grupos. No entanto, o grupo dos carcinomas adenóides císticos demonstrou diferença significativa da imunoexpressão do VEGF-C segundo o padrão cribriforme e sólido (p = 0,004). A maioria dos casos constantes do presente estudo, apresentou fraca marcação para VEGF-D em região peritumoral e intratumoral...

Salivary gland neoplasms exhibit a great morphological diversity and varied biological behavior which raises the interest in the study of these lesions. The spread of tumor cells is an early step in the progression of malignancies and the neoformed lymphatic vessels are considered essential in tumor dissemination. Vascular endotelial growth fator (VEGF) is a family of proteins involved in angiogenesis e lymphangiogenesis. However, in salivar tumors we have limited information on the expression. The aim of this study was to assess the expression of VEGF-C and VEGF-D, lymphatic vessel density (single-staining D2-40) and lymphatic endothelial proliferation (double labeling D2-40/Ki-67) in a series of salivary glands neoplasms. We selected 20 cases of pleomorphic adenoma, 20 of mucoepidermoide carcinoma, 20 of adenoid cystic carcinoma and 10 tissue sample of normal salivary gland. All cases studied showed positive expression of VEGF-C in intratumoral and peritumoral region, no differences in immunoreactivity was found between the groups. However, the group of adenoid cystic carcinoma showed a significant difference in immunoreactivity of VEGF-C by the cribriform and solid pattern (p = 0.004). Most of the cases included in this study showed weak immunoreactivity for VEGF-D in intratumoral and peritumoral region. In the assessment of lymphatic endotelial density peritumoral, intratumoral and total, the groups showed an increasing gradient, with lower values for the group of pleomorphic adenomas followed by mucoepidermoid carcinoma and adenoid cystic carcinoma. Lymphatic endothelial cell density was higher in malignant than benign tumors. No correlation was observed between the immunoreactivity of VEGF-C and VEGF-D in relation to tumor lymphatic density and lymphatic endothelial proliferation.

Interaction of stromal and microvascular components in keratocystic odontogenic tumors.

OBJECTIVE: Little is known about the interaction of stromal components in odontogenic tumors. Thus, the aim of this study was to investigate mast cells (MCs), myofibroblasts, macrophages, and their possible association with angiogenesis and lymphangiogenesis in keratocystic odontogenic tumors (KCOTs). MATERIAL AND METHODS: Thirty cases of KCOTs were included and analyzed by immunohistochemistry for mast cell tryptase, α-SMA, CD34, CD163, and D240. For comparative purpose, 15 radicular cysts (CRs) and 7 pericoronal follicles (PFs) were included. RESULTS: There was an increase in MCs for RCs and this difference was significant when they were compared to KCOTS and PFs. A significant increase in the density of MFs was observed for KCOTs when compared to RCs and PFs (P = 0.00). No significant difference in CD163-positive macrophages (P = 0.084) and CD34-positive vessels (P = 0.244) densities was observed between KCOTs, RCs, and PFs, although KCOTs showed a higher density of all proteins. Significant difference in lymphatic vessel density was observed for KCOTs when compared to RCs and PFs (P = 0.00). Positive correlation was observed between mast cell tryptase and CD34 in KCOTs (P = 0.025). CONCLUSIONS: A significant interaction between the MC population and CD34-positive vessels in KCOTs supported the hypothesis that MCs and blood vessels contribute to the stromal scaffold of KCOT.