Immunohistochemical expression of OCT4 and CD44 in major and minor salivary gland neoplasms

Abstract The aim of this study was to identify tumor parenchyma cells exhibiting immunohistochemical profile of stem cells by evaluating the immunoreactivity of OCT4 and CD44 in a number of cases of salivary gland neoplasms. The sample consisted of 20 pleomorphic adenomas, 20 mucoepidermoid carcinomas, and 20 adenoid cystic carcinomas located in major and minor salivary glands. The expression of OCT4 and CD44 was evaluated by the percentage of positive cells and the intensity of expression. All studied cases showed positive expression of OCT4 and CD44 and higher values than the control groups. For OCT4, luminal and non-luminal cells were immunostained in the case of pleomorphic adenomas and adenoid cystic carcinomas. Moreover, the immunoreactivity of CD44 was particularly evident in the non-luminal cells of these lesions. In mucoepidermoid carcinomas, there was immunoreactivity for both markers in squamous and intermediate cells and absence of staining in mucous cells. For both markers, a significantly higher immunostaining was verified in neoplasms located in the major salivary glands compared with lesions in minor salivary glands (p<0.001). In the total sample and in minor salivary glands, malignant neoplasms exhibited higher immunoreactivity for OCT4 than pleomorphic adenoma. A significant moderate positive correlation (r = 0.444 and p ≤ 0.001) was found between OCT4 and CD44 immunoexpression in the total sample. The high expression of OCT4 and CD44 may indicate that these proteins play an important role in identifying tumor stem cells.

Expression of CD44 in previously grafted alveolar bone / Expresión del marcador óseo CD44 en hueso alveolar previamente injertado

Objetive: To determine the expressions of the bone surface marker CD44 in samples of alveolar bone previously regenerated with allograft, xenograft, and mixed, using the technique of guided bone regeneration. Material and Methods: This exploratory study was approved by the institutional research and ethics committee. By means of intentional sampling and after obtaining informed consent for tissue donation, 20 samples of alveolar bone previously regenerated with guided bone regeneration therapy with particulate bone graft and membrane were taken during implant placement. The samples were stained with hematoxylin-eosin for histological analysis, and by immunohistochemistry for the detection of CD44. Results: Sections with hematoxylin-eosin showed bone tissue with the presence of osteoid matrix and mature bone matrix of usual appearance. Of the CD44+ samples, 80% were allograft and 20% xenograft. The samples with allograft-xenograft were negative. There were no differences in the intensity of CD44 expression between the positive samples. The marker was expressed in osteocytes, stromal cells, mononuclear infiltrate, and some histiocytes. Eighty percent of the CD44+ samples and 100% of the samples in which 60 or more cells were labelled corresponded to allografts (p=0.000). A total of 67% of the samples from the anterior sector, and 40% from the posterior sector were CD44+ (p=0.689). Conclusion: This study shows for the first time that guided bone regeneration using allografts is more efficient for the generation of mature bone determined by the expression of CD44, compared to the use of xenografts and mixed allograft-xenograft, regardless of the regenerated anatomical area.

Objetivo: Determinar la expresión del marcador de membrana óseo CD44 en muestras de hueso alveolar previamente regenerado con aloinjerto, xenoinjerto y mezcla mediante la técnica de regeneración ósea guiada. Material y Métodos: Con aval del Comité de Investigación y Ética, se realizó un estudio exploratorio. Por muestreo intencional y firma de consentimiento informado de donación, se tomaron durante la colocación del implante, 20 muestras de hueso alveolar previamente regenerado con terapia de regeneración ósea guiada con injerto óseo particulado y membrana. Las muestras fueron teñidas con hematoxilina-eosina para el análisis histológico y por inmunohistoquímica para la detección del CD44. Resultados: : Los cortes con hematoxilina-eosina mostraron tejido óseo con presencia de matriz osteoide y matriz ósea madura de aspecto usual. De las muestras CD44+, 80% fueron de aloinjerto y 20% de xenoinjerto. Las muestras con aloinjerto-xeoninjerto fueron negativas. No hubo diferencias en la intensidad de la expresión del CD44 entre las muestras positivas. El marcador se expresó en osteocitos, células estromales, infiltrado mononuclear y algunos histiocitos. El 80% de las muestras CD44+ y el 100% de las muestras con marcación de 60 o más células correspondían a aloinjertos (p=0,000). El 67% de las muestras del sector anterior y el 40% del sector posterior fueron CD44+ (p=0,689). Conclusión: Este estudio muestra por primera vez que la regeneración ósea guiada usando aloinjertos, es más eficiente para la generación de hueso maduro determinado por la expresión de CD44, comparado con el uso de xenoinjertos y mezcla de aloinjerto-xenoinjerto, independientemente del sector anatómico regenerado.

Avaliação da expressão de CD44, SOX2 e STAT3 e no câncer de mama localmente avançado e metastático / Evaluation of the expression of CD44, SOX2 and STAT3 in locally advanced and metastatic breast cancer

Introdução: O câncer de mama (CM) está associado ao aumento contínuo na incidência, com altas taxas de mortalidade em vários países. Existem quatro imunofenotipos de CM, luminal A, Luminal B, triplo negativo e HER2+. Algumas proteínas que estão presentes nas células tumorais e no microambiente tumoral fazem parte do processo da tumorigênese e metástases. A molécula de CD44 é considerada um importante marcador de resposta inflamatória e está presente em todos os leucócitos e na superfície de células tronco tumorais, estando envolvido com invasão tumoral e metástase. Alguns autores demonstraram que o signal transduce randactivator of transcription 3 (STAT3) e sex determining region Y related group box 2 (SOX2) estão relacionados com a regulação da divisão celular de células somáticas e com a tumorigênese e a metástase no CM. Objetivo: Avaliar a expressão de CD44+, SOX2 e STAT3 no sangue de pacientes com câncer de mama localmente avançado e metastático. Métodos: É um estudo de coorte transversal realizado no Hospital de Câncer de Pernambuco (HCP) e no Laboratório de Pesquisa Translacional do Instituto de Medicina Integral Prof. Fernando Figueira (IMIP) no período de março de 2017 a abril de 2018. Participaram deste estudo, 65 pacientes do sexo feminino com diagnóstico de câncer de mama e idade entre 28 a 64 anos. Destas, 51 mulheres tinham tumor de mama em estádio III (localmente avançado) e 14 com doença metastática (estágio IV). Como grupo controle, foram 24 mulheres clinicamente saudáveis com idade entre 18 anos a 65 anos. As análises da expressão de CD44+, SOX2 e STAT3 foi realizada por citometria de fluxo. Resultados: foram observados níveis elevados de células CD44+, CD24neg e CD44+/CD24neg CD44+/CD24negSOX2+ no sangue das pacientes com tumores de mama LB, HER2+ e Triplo negativos comparados aos controles (p<0,0001). Níveis baixos CD44+/CD24negSTAT3+ no sangue das pacientes comparado aos controles. Foram observados níveis elevados de células CD44+/CD24neg e CD44+/CD24negSTAT3+ no com CM localmente avançado comparado a CM metastático (p<0,05). As pacientes com CM triplo negativo apresentaram níveis elevados de linfócitos TCD3+SOX2+ comparado aos grupos com subtipos LB e HER2+ (p<0,05). O grupo de pacientes CM HER2+ apresentou baixos níveis de TCD3+SOX2+ comparado aos controles e LA (p<0,05). Níveis elevados de linfócitos TCD3 totais p=0,0007 e baixos de TCD3+SOX2+ p=0,02 no CM localmente avançado comparado a doença metastática. Níveis elevados de linfócitos TCD3+SOX2+ em pacientes linfonodos negativos quando comparado aos grupos N1 (p=0,004) e N2 (p<0,0001). Foram observados níveis elevados de células CD24neg e CD44+/CD24negSTAT3+ e de linfócitos TCD3+, e baixos níveis de TCD3+STAT3+ no CMTM localmente avançado comparado ao metastático. Conclusão: Os níveis das moléculas CD44, CD24, SOX2+ e STAT3+ no sangue mostraram ser alterados nas pacientes com CM em estágios mais avançados independente do imunofenótipo

Introduction: Breast cancer (BC) is associated with a continuous increase in incidence, with high mortality rates in several countries. There are four immunophenotypes of BC, luminal A, Luminal B, triple negative (TNBC), and HER2 +. Some proteins that are present in tumor cells, and the tumor microenvironment are part of the process of tumorigenesis and metastases. The CD44 molecule is considered an important marker of the inflammatory response. It is present in all leukocytes and on the surface of tumor stem cells, being involved with tumor invasion and metastasis. Some authors have demonstrated that the signal transducer and activator of transcription 3 (STAT3) and sex determining region Y related group box 2 (SOX2) are related to the regulation of somatic cell division and tumorigenesis and metastasis in BC. Objective: To evaluate the expression of CD44 +, SOX2, and STAT3 in the blood of patients with locally advanced and metastatic breast cancer. Methods: A cross-sectional study was carried out at the Pernambuco Cancer Hospital (HCP) and at the Translational Research Laboratory of the Instituto de Medicina Integral Prof. Fernando Figueira (IMIP) from March 2017 to April 2018. 65 female patients diagnosed with breast cancer participated in this study, aged between 28 and 64 years. Of these, 51 women had stage III (locally advanced) breast tumor, and 14 with metastatic disease (stage IV). As a control group, there were 24 clinically healthy women aged 18 to 65 years. The analysis of CD44 +, SOX2, and STAT3 expression was performed by flow cytometry. Results: elevated levels of CD44 +, CD24neg and CD44+/CD24neg, CD44+/CD24neg SOX2+ cells were observed in the blood of patients with LB, HER2+ and TNBC compared to controls (p<0.0001). Low CD44+/CD24negSTAT3+ levels in patients' blood compared to controls. Elevated levels of CD44+/CD24neg and CD44+/CD24negSTAT3 + cells were observed in locally advanced compared to metastatic BC (p <0.05). Patients with TNBC had elevated TCD3+SOX2+ lymphocytes compared to groups with subtypes LB and HER2+ (p <0.05). HER2+ patient had low levels of TCD3+ SOX2+ compared to controls and LA (p <0.05). High levels of total TCD3 (p= 0.0007) and low levels of TCD3 + SOX2 + (p = 0.02) in locally advanced BC compared to metastatic disease. Elevated levels of TCD3+SOX2+ in negative lymph node patients compared to groups N1 (p = 0.004) and N2 (p <0.0001). High levels of CD44+/CD24negSTAT3+ cells and TCD3+ were observed, and low levels of TCD3+ STAT3+ in locally advanced TNBC compared to metastatic. Conclusion: the levels of CD44, CD24, SOX2 +, and STAT3 + molecules in the blood were altered in breast cancer more advanced independent immunophenotype

Avaliação dos níveis de células CD44high/CD24low E TCD3+, e da expressão dos fatores de transcrição SOX2 e STAT3 no carcinoma escamoso de pênis / Evaluation of CD44highCD24low and TCD3 + cell levels, and expression of SOX2 and STAT3 transcription factors in penile squamous cell carcinoma

Introdução: O câncer de pênis (CaPe) tem uma alta incidência em alguns países subdesenvolvidos. O tratamento padrão é a remoção do tumor primário e a linfadenectomia inguinal, em alguns pacientes. O fator prognóstico mais importante é a doença linfonodal, porém os métodos de estadiamento, disponíveis na atualidade são pouco precisos, e a alta taxa de morbidade da linfadenectomia tem estimulado o estudo de biomarcadores preditivos de metástases em linfonodos, selecionando os pacientes que necessitam da linfadenectomia. Os marcadores STAT3 e CD44, CD24, e SOX2+ são conhecidos por serem considerados marcadores de diagnóstico e prognóstico em outros cânceres, mas sem estudos no câncer de pênis. Objetivo: Avaliar os níveis das células CD44high/CD24low E TCD3+, e a expressão dos fatores de transcrição SOX2 e STAT3 no carcinoma escamoso de pênis. Métodos: Estudo transversal realizado no Hospital de Câncer de Pernambuco (HCP) e Laboratório de Pesquisa Translacional do Instituto de Medicina Integral Prof. Fernando Figueira (IMIP), no período de março de 2015 a dezembro de 2017 em uma população de 38 pacientes com CaPe e controles. Foram realizadas análises de SOX2, STAT3, CD24 e CD44 no sangue e tecido tumoral por citometria de fluxo. Resultados: A mediana da idade foi de 61 anos (37 - 80 anos). A maioria dos pacientes apresentou tumor localizado na glande (76,3%), tipo histológico usual (71,0%), de grau 1 (86,8%), sem invasão vascular e perineural (86,8% e 68,4%), sem presença de metástases (86,8%). Com relação ao estadiamento, 55,3% dos pacientes apresentavam estágio pT2 e 34,2% pT1. Foram encontrados níveis elevados de CD44highCD24low, CD44highCD24lowpSTAT3+ e CD44highCD24lowSOX2+nos pacientes, quando comparados aos controles (p<0,05). Foram observados níveis percentuais reduzidos de leucócitos SOX2+ e TCD3+/SOX2+, e elevados leucócitos pSTAT3 nos pacientes, quando comparados aos dos controles saudáveis. Foram constatados níveis elevados de CD44highCD24low e de leucócitos SOX2+ no grupo de pacientes com invasão perineural, tamanho tumoral > 3 cm e em estádio pT2 (p<0,05). Foram encontradas: associação dos níveis de célulasCD44high/CD24low e no sangue periférico com o tamanho do tumor (p=0,04) e status de sobrevida (p=0,01) e de CD44high/CD24lowSOX2+ no sangue e tumor com status de sobrevida nos pacientes;expressão reduzida de pSTAT3 no sangue de pacientes com invasão perineural e elevados no tecido tumoral no grupo em estádio pT1;alta expressão de TCD3+ no sangue e tecido tumoral de pacientes com tumor ≤3cm;aumento de TCD3+SOX2+ no sangue de pacientes sem invasão perineural e em estádio pT1.Observou-se correlação de CD44highCD24lowpSTAT3+ (r=0,669; p=0,024), de pSTAT3 (r=0,487; p=0,018) e de TCD3+SOX2+ (r=0,404; p=0,029) entre o sangue e o tecido tumoral. Conclusão: As moléculas CD44, CD24 e SOX2 foram marcadores de doença avançada, sendo associados ao pior prognóstico no câncer de pênis, porém, p-STAT3 e TCD3+ foram associados a um prognóstico mais favorável neste estudo

Introduction: Penile cancer (CaPe) has a high incidence in some underdeveloped countries. The standard treatment is removal of the primary tumor and inguinal lymphadenectomy in some patients. At the moment, the most important prognostic factor is lymph node disease, but the staging methods currently available are not very accurate and the high morbidity rate of lymphadenectomy has prompted the study of biomarkers that may predict lymph node metastasis, and aid in the selection of patients who need lymphadenectomy. The markers STAT3 and CD44, CD24, SOX2 + are known to be markers of diagnosis and prognosis in other cancers, but there are no such studies on penile cancer. Aim: To evaluate the levels of CD44high/ CD24low and TCD3+ cells, and the expression of SOX2 and STAT3 transcription factors in squamous cell carcinoma of the penis. Methods: A cross-sectional study was conducted at the Hospital de Cancer de Pernambuco (HCP) and Translational Research Laboratory at the Prof. Fernando Figueira Institute of Integral Medicine (IMIP), from March 2015 to December 2017 of a population of 38 patients with CaPe and controls. Analyses of SOX2, STAT3, CD24 and CD44 were performed on blood and tumor tissue by flow cytometry. Results: The median age was 61 years (37-80 years). Most of the patients presented tumor localized in the glans (76.3%), histological type (71.0%), grade 1 (86.8%), without vascular and perineural invasion (86.8% and 68.4% %), without metastases (86.8%). Regarding staging, 55.3% of the patients had pT2 and 34.2% pT1. High levels of CD44highCD24low, CD44highCD24lowpSTAT3+and CD44highCD24low,SOX2+ were found in the patients compared to the controls (p <0.05). Reduced percentage levels of SOX2+ and TCD3+/ SOX2+ leukocytes and high pSTAT3 leukocytes were observed in the patients when compared to healthy controls. There were elevated levels of CD44highCD24low and SOX2+ leukocytes in the group of patients with perineural invasion, tumor size> 3 cm and at pT2 stage (p <0.05). Peripheral blood CD44highCD24low levels were found to be associated with the tumor size(p = 0.04) and survival status (p = 0.01) and CD44highCD24lowSOX2+ levels in the blood and tumor with survival status in patients. Reduced levels of pSTAT3 in the blood of patients with perineural invasion and elevated tumor tissue in the pT1 stage. There were elevated levels of TCD3 + in the blood and tumor tissue of patients with tumors of ≤3cm. Increased TCD3 + SOX2 + was found in the blood of patients without perineural and pT1 stage invasion. CD44highCD24lowpSTAT3 + (r = 0.669; p = 0.024), pSTAT3 (r = 0.487, p = 0.018) and TCD3 + SOX2 + (r = 0.404, p = 0.029) correlations were observed between blood and tumor tissue. Conclusion: CD44, CD24 and SOX2 molecules were markers of advanced disease, and were associated with the worst prognosis in penile cancer. However, p-STAT3 and TCD3 + were associated with a more favorable prognosis in this study

Prognostic significance of receptor for hyaluronan acid-mediated motility (CD168) in acute pediatric leukemias - assessment of clinical outcome, post induction, end of treatment and minimal residual disease

ABSTRACT Introduction: The extracellular matrix protein hyaluronan acid plays an active in role in tumor cell proliferation and invasion. Hyaluronan acid receptors, namely CD168 or the receptor for hyaluronan acid-mediated motility (RHAMM) and CD44 have been implicated in promoting malignancy. There is a lacuna in data on the expression of the receptor in pediatric leukemias. Methods: Pediatric patients with acute leukemia who were diagnosed, treated and followed up in our center were enrolled. The bone marrow biopsies performed prior to treatment were subjected to immunohistochemical staining (54 biopsies: acute lymphoblastic leukemia - 45, acute myeloid leukemia - 9). Blast counts were carried out at diagnosis, end of the induction phase and end of chemotherapy, the minimal residual disease was assessed and follow up details were collected. Positivity was correlated with initial blast count, post-induction blast count, minimal residual disease and patient survival. Results: There was no correlation between the initial blast count and the percentage of blasts with RHAMM expression. The positive correlation between percentage of blasts expressing RHAMM and the post-induction blast count was moderate in acute myeloid leukemia (0.74) and mild in acute lymphoblastic leukemia (0.48). There was a statistically significant difference in RHAMM expression between the two minimal residual disease risk groups (p-value = 0.012) with a negative prognostic effect of RHAMM expression. Moreover, a negative prognostic effect of RHAMM expression was noted when patient survival was considered. Conclusion: This study shows that blasts in acute myeloid leukemia show more RHAMM positivity than those of acute lymphoblastic leukemia indicating the aggressive nature of this type of leukemia. In acute leukemias, patients with high percentages of RHAMM-positive blasts had more post-induction blasts, blasts in minimal residual disease and poorer prognosis.

Effect of 0, 15% sodium hialuronate and 0, 5% carboxymethylcellulose on tear film breakup time in healthy dogs and in dogs with keratoconjunctivitis sicca / Efeito do hialuronato de sódio a 0, 15% e da carboximetilcelulose sódica a 0, 5% no tempo de ruptura do filme lacrimal de cães saudáveis e com ceratoconjuntivite seca

This study aimed to evaluate the effect of the 0.15% sodium hyaluronate (SH) and of 0.5% carboxymethylcellulose (CMC) on tear film breakup time (TFBUT) in 10 healthy dogs and in 32 eyes of dogs with keratoconjunctivis sicca (KCS). In addition, the goblet cell density (GCD) of this population was quantified. TFBUT was assessed at baseline and at different time points following the instillation of SH and CMC. KCS was graded as mild, moderate, and severe. GCD were quantified from conjunctival biopsies. The number of GCD differed significantly between patients with mild and moderate KCS (P<0.01). TFBUT of healthy dogs increased only for 1 minute after treatment with SH (P<0.01). Regarding baseline and treatments, SH significantly increased TFBUT for up to 30 minutes on the ocular surface, in comparison to CMC, in all categories of KCS (P<0.01). TFBUT and GCD correlated positively when the healthy and diseased eyes were grouped (r=0.41, P=0.006). It can be concluded that in dogs with KCS, SH lasts longer periods on the ocular surface than CMC, but such agents does not increase TFBUT in healthy dogs. Additionally, tear film stability tends to reduce in a linear fashion from the mild to severe form of KCS.(AU)

Objetivou-se avaliar os efeitos do hialuronato de sódio a 0,15% (HS) e da carboximetilcelulose a 0,5% (CMC) no teste de ruptura do filme lacrimal (TRFL) em 10 cães saudáveis e em 32 olhos de cães com ceratoconjuntivite seca (CCS). Ademais, quantificou-se a densidade de células caliciformes (DCC) deles. Mensurou-se o TRFL em momentos distintos antes e após a instilação do HS e da CMC. Graduou-se a CCS em leve, moderada e severa. Quantificou-se a DCC a partir de biópsias conjuntivais. A DCC diferiu apenas entre pacientes com CCS leve e severa (P<0,01). Em cães saudáveis, o TRFL se elevou apenas após um minuto do tratamento com HS (P<0,01). Relativamente ao período basal e entre os tratamentos, o HS elevou o TRFL de forma mais eficaz e permaneceu por até 30 minutos na superfície ocular, comparativamente à CMC, em todas as categorias de CCS (P<0,01). Ao se agruparem os olhos saudáveis e os com CCS, o TRFL se correlacionou com a DCC (r=0.41, P=0.006). Conclui-se que o HS permanece por maior tempo na superfície ocular que a CMC em cães com CCS, mas que tais substâncias não elevam o TRFL em cães saudáveis. Ademais, a estabilidade do filme lacrimal tende a se reduzir de modo linear da forma leve até à severa da CCS.(AU)

Impact of extracellular alkalinization on the survival of human CD24-/CD44+ breast cancer stem cells associated with cellular metabolic shifts

Cancer stem cells reside in a distinct region within the tumor microenvironment that it is believed to play a fundamental role in regulating stemness, proliferation, survival, and metabolism of cancer cells. This study aimed to analyze the effect of extracellular alkalinization on metabolism and survival of human CD24-/CD44+ breast cancer stem cells (BCSCs). BCSCs were cultured in alkalinized DMEM-F12 and incubated at 37°C, 5% CO2, and 20% O2 for 30 min, 6, 24, and 48 h. After each incubation period, we analyzed the modulation of various mRNA expressions related to pH and cellular metabolic regulation using the qRT-PCR. Metabolic state was measured using colorimetric and fluorometric assays. To examine cell proliferation and apoptosis, we used trypan blue and annexin V/propidium iodide assay, respectively. This study demonstrated that alkalinization could stimulate extracellular carbonic anhydrase (CAe) activity, as well as CA9 and HIF1α expression. Under alkaline pH and HIF1α regulation, glucose consumption, extracellular lactate production, and LDH activity of BCSCs were upregulated while O2 consumption was downregulated. These metabolic shifts seemed to promote apoptosis and suppress the proliferation of BCSCs. To conclude, modulation of the extracellular environment through alkalinization could change the metabolic states of BCSCs, which in turn affect the cell survival.

Valor prognóstico da imunoexpressão de podoplanina e de CD44v6 na recidiva locorregional dos pacientes com câncer de lábio / Prognostic value of podoplanin and CD44v6 immunoexpressions on locoregional recurrence of the patients with lip cancer

O objetivo deste estudo consistiu em avaliar a expressão imuno-histoquímica da podoplanina e do CD44v6 pelas células malignas, verificando a associação destas proteínas com as variáveis clínicas, microscópicas, com o índice histopatológico de malignidade e com a sobrevivência livre de doença de 91 pacientes portadores de carcinomas espinocelulares (CEC) de lábio inferior, tratados no Centro de Tratamento e Pesquisa do Hospital do Câncer A.C.Camargo, São Paulo. Os tumores foram corados, separadamente, com os anticorpos anti-podoplanina e anti-CD44v6, sendo avaliada a imunoexpressão destas proteínas pelas células neoplásicas, no front de invasão tumoral, por meio de um método semi-quantitativo de escores. A associação da expressão da podoplanina e do CD44v6 com as variáveis demográficas, clínicas e microscópicas foi feita pelo teste do qui-quadrado ou exato de Fisher. As taxas de sobrevivência livre de doença, acumuladas em cinco e dez anos, foram calculadas pelo teste de Kaplan-Meier e a influência das variáveis clínicas e microscópicas no prognóstico avaliadas pelo modelo de regressão de Cox. A correlação entre a podoplanina e o CD44v6 foi analisada pelo teste de Spearman. Em todos os testes estatísticos utilizou-se um nível de significância de 5%. Os resultados mostraram uma predominância da forte expressão membranosa e citoplasmática da podoplanina pelas células malignas. Verificou-se uma associação significativa da podoplanina citoplasmática com a recidiva locorregional (p=0,028) e da podoplanina membranosa com o índice histopatológico de malignidade tumoral (p=0,026). O CD44v6 foi fortemente expresso pelas células neoplásicas de 95,4% dos CECs e significativamente, associado com o estadiamento clínico T (p=0,034). Não houve correlação entre a podoplanina e o CD44v6 nos CECs de lábio inferior. A forte expressão de podoplanina membranosa (p=0,016) e citoplasmática (p=0,030) pelas células malignas foi fator de prognóstico favorável independente na sobrevivência livre de doença. Concluímos que a podoplanina e o CD44v6 são fortemente expressos pelas células neoplásicas e que a forte imunoexpressão membranosa e citoplasmática da podoplanina pode auxiliar na identificação do risco de recidiva locorregional nos pacientes portadores de carcinoma espinocelular de lábio inferior.(AU)

The aim of this study was evalute the podoplanin and CD44v6 immunohistochemical expression by malignant cells and its association with the clinical and microscopic variables, tumor histopathological grading and disease-free survival of 91 patients with lip squamous cell carcinomas (SCC), submitted to surgical treatment at Research and Treatment Center of the Cancer Hospital A.C. Camargo, São Paulo. The tumors were stained separately, with the antibodies anti-podoplanin and anti-CD44v6, and the immunoexpression of these proteins, by the neoplastic cells in the invasion front, was evaluated by a semi-quantitative scores method. Chi-square test or Fishers exact test was used to analyze the association of podoplanin and CD44v6 expression with demographic, clinical, and microscopic variables. Disease-free survival in five and ten years, were calculated by the Kaplan-Meier method and the influence of clinical and microscopic variables on prognosis were evaluated by the Cox regression model. The correlation between podoplanin and CD44v6 expression was analyzed by Spearman's test and a significance level of 5% was used in all statistical tests. The results showed a predominance of strong membranous and cytoplasmic podoplanin expression by malignant cells. An association between cytoplasmic podoplanin and locorregional recurrence (p=0,028) and membranous podoplanin with tumor histopathological grading (p=0,026). CD44v6 was strongly expressed in 95.4% of the SCCs neoplastic cells and significantly associated with the clinical staging T (p=0,034). There was no correlation between podoplanin and CD44v6 expression in the lower lip SCC. The strong expression of membranous (p=0.016) and cytoplasmic (p=0.030) podoplanin by malignant cells was a favorable independent prognostic factor in disease-free survival. Concluding, the podoplanin and CD44v6 are strongly expressed by neoplastic cells and the strong membranous and cytoplasmic immunoexpression of podoplanin can help the identification of locoregional recurrence risk in patients with squamous cell carcinoma of the lower lip.(AU)

Differential Expression of Stem Cell Markers in Human Adamantinomatous Craniopharyngioma and Pituitary Adenoma

Background/Aims: Although craniopharyngioma (CP) is histologically benign, it is a pituitary tumour that grows rapidly and often recurs. Adamantinomatous CP (ACP) was associated with an activating mutation in ß-catenin, and it has been postulated that pituitary stem cells might play a role in oncogenesis in human ACP. Stem cells have also been identified in pituitary adenoma. Our aim was to characterize the expression pattern of ABCG2, CD44, DLL4, NANOG, NOTCH2, POU5F1/OCT4, SOX2, and SOX9 stem cell markers in human ACP and pituitary adenoma. Methods and Results: We studied 33 patients (9 ACP and 24 adenoma) using real-time quantitative PCR (RT-qPCR) and immunohistochemistry. SOX9 was up-regulated in ACP, exhibiting positive immunostaining in the epithelium and stroma, with the highest expression in patients with recurrence. CD44 was overexpressed in ACP as confirmed by immunohistochemistry. SOX2 did not significantly differ among the tumour types. The RT-qPCR array showed an increased expression of MKI67,OCT4/POU5F1, and DLL4 in all tumours. NANOG was decreased in ACP. ABCG2 was down-regulated in most of the tumours. NOTCH2 was significantly decreased in the adenomas. Conclusion: Our results confirm the presence of stem cell markers in human pituitary tumours as well as the different expression patterns of ACP and adenoma. These findings suggest that ACP may originate from a more undifferentiated cell cluster. Additionally, SOX9 immunodetection in the stroma and the highest expression levels related to the relapse of patients suggest a contribution to the aggressive behaviour and high recurrence of this tumour type.

Isolation of breast cancer stem cell from MDA-MB231 cell line using vincristine / Aislamiento de células madre de cáncer de mama de la línea celular MDA-MB231 utilizando vincristina

Cancer has been considered as a stem cell disease. Suspension culture combined with anti-cancer drugs has recently been proposed for isolation of cancer stem cells (CSCs). In the current study, Vincristine as an anti-cancer drug combined with suspension culture was used for isolation and purification of CSCs from human breast cancer cell line (MDA-MB231). The cells were treated with different concentrations of vincristine (0, 2, 4, 6 and 8 ng/ml). Stem cells were identified with the expression of OCT4, nanog, SOX2 and nucleostemin genes by RT-PCR. Mammosphere forming unit was measured upon suspension culture containing EGF, bFGF, LIF, B27, insulin and BSA. The isolated mammospheres were investigated for CD44 expression. Results showed that 4 ng/ml of vincristine for 72 hours could be utilized as the best and most reliable dose which eliminates around 80 % of non-cancer stem cells with no destructive effect on CSCs' viability (P> 0.05). RT-PCR demonstrated that drug treated cells expressed OCT4, nanog, SOX2 and nucleostemin. Mammosphere formation unit of cells pretreated with vincristine was significantly higher than unpretreated ones (P>0.05). Immunofluorescence staining for CD44 depicted high expression of CSC marker among the isolated mammospheres. Vincristine combined with suspension culture can be considered as an appropriate method to isolate CSC.

El cáncer ha sido considerado como una enfermedad de células madre. Recientemente se ha propuesto cultivo en suspensión en combinación con medicamentos contra el cáncer para aislamiento de las células madre del cáncer (CMC). En este estudio se utilizó la vincristina como fármaco anticanceroso combinado con cultivo en suspensión para el aislamiento y purificación de las células madre cancerosas, de la línea celular de cáncer de mama humano (MDA-MB231). Las células se trataron con diferentes concentraciones de vincristina (0, 2, 4, 6 y 8 ng/ml). Las células madre se identificaron mediante la expresión de los genes OCT4, Nanog, SOX2 y nucleostemin por RT-PCR. La unidad de formación mammosphere se midió a través de cultivo en suspensión que contenía EGF, bFGF, LIF, B27, insulina y BSA. Los mammospheres aislados fueron estudiados para la expresión de CD44. Los resultados mostraron que 4 ng/ml de vincristina durante 72 horas podrían ser utilizados como la mejor y más fiable dosis que permite eliminar alrededor del 80 % de las células madre no cancerosas, sin causar un efecto destructivo sobre la viabilidad de las CMC (P> 0,05). La RT-PCR mostró que en las células tratadas con él fármaco hubo expresión de los genes OCT4, Nanog, SOX2 y nucleostemin. La unidad de formación de las células pretratadas con vincristina fue significativamente más alta que las unidades sin tratamiento previo (P>0,05). La inmunofluorescencia para CD44 muestró una alta expresión del marcador de CMC entre mammospheres aisladas. La vincristina en combinación con el cultivo en suspensión puede ser considerado como un método apropiado para aislar CMC.

Imunoexpressão de marcadores de células-tronco, CD44 e CD133, no câncer gástrico primário emetástases linfonodais

O câncer gástrico é a quarta neoplasia mais comum em todo o mundo, representando a segunda causa de mortalidade por câncer. Apesar do tratamento com cirurgia e quimioterapia, a sobrevida global em cinco anos de pacientes com câncer gástrico permanece baixa. Uma possível explicação para ineficácia da terapia é a presença de células-tronco cancerosas, uma subpopulação de células tumorais que apresentam características de células-tronco. Estas células, assim como as células tronco embrionárias, são consideradas imortais, podem se auto-renovar e se transformar em qualquer célula do corpo. Vários marcadores, incluindo CD44 e CD133, têm sido relatados como marcadores de células-tronco, normais e cancerosas, e utilizados para isolar células cancerosas de tumores sólidos. O objetivo desse trabalho foi avaliar a expressão de CD44 e de CD133, no câncer gástrico primário e metástases linfonodais, através de imunohistoquímica, e relacioná-la com as variáveis clínico-patológicas de tipo histológico, sexo, idade, localização anatômica, dimensão do tumor, invasão angiolinfática, infiltração perineural, classificação TNM (TN) e acometimento linfonodal. Este estudo foi desenvolvido a partir de um conjunto de 72 casos de adenocarcinoma gástrico, dos Arquivos do Serviço de Patologia e Medicina Legal da Universidade Federal do Ceará (DPML-UFC). Utilizou-se a técnica de tissue microarray asociada à imunohistoquímica comanticorpo monoclonal anti-CD44 e policlonal anti-CD133...

Gastric cancer is the fourth most common cancer worldwide, accounting for the second leading cause of cancer mortality. Despite treatment with surgery and chemotherapy, the overall five-year survival of patients with gastric cancer remains low. One possible explanation for the ineffectiveness of therapy is the presence of cancer stem cells, a subpopulation of tumor cells that have stem cell characteristics. It has been reported that these, as well as embryonic stem cells, are immortal, can self-renew and to differentiate to be transformed in any cell type in the body. Several markers, including CD44 and CD133, have been reported as stem cell markers in both normal and cancerous cells and have been used to isolate cancer cells from solid tumors. The aim of this study was to evaluate the expression of CD44 and CD133 in primary gastric cancer and lymph node metastases by immunohistochemical and to relate it to clinicopathologic variables as histological type, gender, age, anatomical site, tumor size, angiolymphatic invasion, infiltration perineural, TNM classification (TN) and lymph node involvement. This study was developed from a set of 72 cases of gastric adenocarcinoma, from the Archives of Pathology and Forensic Medicine of the Federal University of Ceará Service (DPML-UFC). Tissue microarray andimmunohistochemistry were utilized, with anti-CD44 monoclonal antibody and polyclonal anti-CD133...

Estudio de la captación de ácido hialurónico por Ascaris lumbricoides / Study of hyaluronic acid capture by Ascaris lumbricoides

El ácido hialurónico (AH) tiene importantes funciones en la inmunidad. En experiencias previas se demostró que extractos de adultos de A. lumbricoides y concentrados larvarios, tienen capacidad de unión a AH. El objetivo de este trabajo fue estudiar la captación de AH por este helminto. Se trabajó con tres extractos del parásito adulto ([EA]B,C,D) y 3 concentrados de larvas ([CLAL1]: 1100 a 1200 larvas/ mL; [CLAL2]: 400 a 600 larvas/ mL y [CLAL3]: 100 a 200 larvas/ mL). Se empleó la técnica modificada de Inhibición de la Adhesión para detección del Receptor CD44 soluble de hialuronato en suero humano. Se definió CexpAdhE AH como el cociente entre los eritrocitos adheridos por el AH en presencia y ausencia del parásito y se definió IexpCP AH, como la cantidad de eritrocitos que se dejaron de adherir debido a la captación de AH por el parásito, referido al número total de eritrocitos. Los resultados mostraron diferencias significativas en CexpAdhE AH y en IexpCP AH, por efecto de la concentración larvaria y del [EA]. Las medias aritméticas de CexpAdhE AH y de IexpCP AH para los concentrados larvarios fueron 0,636 y 0,21 ([CLAL1]); 0,819 y 0,068 ([CLAL2]); 0,97 y 0,013 ([CLAL3]). Las medianas de CexpAdhE AH y de IexpCP AH para los extractos fueron [EA]C 0,275 y 0,4; [EA]B: 0,20 y 0,43; [EA]D: 0,075 y 0,495. La experiencia permitiría suponer que el parásito puede captar AH para interferir en la respuesta inmune del hospedador.

Hyaluronan Acid (HA) has important functions in immunity. Previous experiences have shown that A. lumbricoides's extracts of adult specimens and larval concentrates have hyaluronan binding capacity. The aim of this study was to analyse the HA capture by this helminth. Three extracts of adult specimens ([AE]B,C,D) and 3 larval concentrates ([ALLC1]: 1100 to 1200 larvae/ mL; [ALLC2]: 400 to 600 larvae/ mL and [ALLC3]: 100 to 200 larvae/ mL) were studied. The modified test of serum soluble CD44 Detection by Aggregation Inhibition was used. CexpAdhE AH was defined as the quotient between erythrocytes adhered by the HA in presence and in absence of the parasite, and IexpCP AH was defined as the amount of erythrocytes that stopped adhering due to the HA capture by the parasite in relation to the total number of erythrocytes. The results showed significant differences in CexpAdhE AH and IexpCP AH as the result of larvae concentration and of the extracts. The arithmetic means of CexpAdhE AH and IexpCP AH for the larvae were 0.636 and 0.21 ([ALLC1]); 0.819 and 0.068 ([ALLC2]); 0.97 and 0.013 ([ALLC3]). Medians of CexpAdhE AH and IexpCP AH for the extracts were [EA]C:0.275 and 0.4; [EA]B: 0.20 and 0.43; [EA]D: 0.075 and 0.495. From the experience, it could be assumed that the parasite can capture the HA to interfere in the host's immune response.

Ascaris lumbricoides: Capacidad de unión a hialuronato / Ascaris lumbricoides: Hyaluronan binding capacity

El ácido hialurónico tiene importantes funciones en los procesos inflamatorios y de reparación tisular. Sus principales receptores son CD44, RHAMM e ICAM-I. Debido a la variedad de estrategias utilizadas por los parásitos para evadir la respuesta inmune del hospedador y considerando las múltiples funciones e importancia fisiológica del ácido hialurónico, el objetivo de este trabajo fue determinar si Ascaris lumbricoides tiene capacidad de unión a hialuronato. Se trabajó con 36 extractos de A. lumbricoides obtenidos por remoción quirúrgica de la cutícula y ruptura mecánica refrigerada. Se modificó la técnica de detección de CD44 soluble en suero por Inhibición de la Agregación por adhesión. Los resultados demostraron que 23 de los 36 extractos estudiados tenían capacidad de unión a hialuronato. Este hecho podría deberse a la existencia de algún receptor en el parásito que une hialuronato y que eventualmente competiría con los receptores habituales del hospedador. A. lumbricoides podría utilizar este mecanismo para evadir la respuesta inmune.

Hyaluronan acid has important functions in inflammatory and tissue reparation processes. Its main receptors are CD44, RHAMM and ICAM-I. Owing to the varied strategies of the parasites to evade the host´s immune response, and also considering the multiple functions and physiological importance of hyaluronan acid, the aim was to study if Ascaris lumbricoides has hyaluronan binding capacity. Extracts of A. lumbricoides were prepared by surgical remotion of the cuticle and refrigerated mechanical rupture. The study was done on 36 parasite extracts. The test of serum soluble CD44 Detection by Aggregation Inhibition was modified. Of the 36 extracts studied, 23 presented hyaluronan binding capacity. This fact can possibly be due to the existence of a receptor with hyaluronan acid binding capacity in the parasite, which eventually might compete with the usual receptors of the host. A. lumbricoides might use this mechanism to evade the immune response.

Importance of hyaluronan biosynthesis and degradation in cell differentiation and tumor formation

Hyaluronan is an important connective tissue glycosaminoglycan. Elevated hyaluronan biosynthesis is a common feature during tissue remodeling under both physiological and pathological conditions. Through its interactions with hyaladherins, hyaluronan affects several cellular functions such as cell migration and differentiation. The activities of hyaluronan-synthesizing and -degrading enzymes have been shown to be regulated in response to growth factors. During tumor progression hyaluronan stimulates tumor cell growth and invasiveness. Thus, elucidation of the molecular mechanisms which regulate the activities of hyaluronan-synthesizing and -degrading enzymes during tumor progression is highly desired

Unión diferencial de ácido hialurónico en dos sublíneas celulares CD44+. Posible implicancia en la infiltración tumoral / Differential binding of hyaluronic acid in 2 CD44+ sublines. Relationship with tumor infiltration

We have established and characterized a cell line (LBL) from a spontaneous murine T lymphoma LB. Histopathological analysis has demonstrated LB primary tumor infiltration in spleen, lymph nodes, liver, thymus, bone marrow and lung. However LBL cells infiltrated all these organs except lung. Two sublines with different growth behavior were derived from LBL cell line. One of them grew in suspension as clusters (LBLc) while the other one grew as adherent monolayers (LBLa). Growth rate, response to mitogenic stimuli and apoptosis induction were different among the parental cell line and the derived sublines. CD44 was expressed constitutively in LBL and LBLa cells. In contrast LBLc cells only expressed similar levels of this molecule when stimulated with PMA. LBLa cells showed hyaluronic acid (HA) binding properties, while LBL and LBLc cells were not able to bind HA even when activated with PMA. We postulate that differences in HA binding could be related with different infiltration behaviors.

Modulación de isoformas de la molécula de adhesión CD44 en un modelo murino de isquemia y reperfusión intestinal / Isoforms modulation of CD44 adhesion molecule in a murine model of ischemia and intestinal reperfusion

Gut ischemia-reperfusion (G-IR) induces a systemic inflammatory response, in which leukocyte contribution to this injury in distant organs is important. ICAM-1 as well as CD11/CD18 have been involved in leukocyte infiltration in liver and lungs. CD44 adhesion molecule plays an essential role in other inflammatory processes such as rheumatoid arthritis and allergic contact dermatitis, however its implication in G-IR has not been described. In order to establish a possible role of CD44 in the development of systemic inflammation by G-IR, we have studied CD44 mRNA expression by RT-PCR in a murine model of gut ischemia reperfusion. Animals subjected to G-IR showed an increased number of CD44 variable isoforms expressed in liver and spleen compared to non-treated animals or animals subjected to laparotomy. This finding indicates that G-IR specifically induces the expression of different CD44 variable isoforms. Liver CD44 upregulation in animals subjected to G-IR suggests a contribution of this molecule to lymphocyte activation and migration to this injured organ. Moreover, increased isoform expression in spleen may be induced by the proinflammatory environment resulting from a systemic depuration activity.