Relevance of iNOS expression in tumor growth and maintenance of cancer stem cells in a bladder cancer model.

The expression of inducible nitric oxide (NO) synthase (iNOS) in human bladder cancer (BC) is a poor prognostic factor associated with invasion and tumor recurrence. Here, we evaluated the relevance of iNOS expression in BC progression and in cancer stem cell (CSC) maintenance in a murine BC model. Also, iNOS expression and CSC markers were analyzed in human BC samples. iNOS inhibitors (L-NAME or 1400W) or shRNA were used on murine BC model with different iNOS expressions and invasiveness grades: MB49 (iNOS+, non-muscle invasive (NMI)) and MB49-I (iNOS++, muscle invasive (MI)), in order to analyzed cell proliferation, tumor growth, angiogenesis, number of CSC, and pluripotential marker expression. iNOS, SOX2, Oct4, and Nanog expressions were also analyzed in human BC samples by qPCR and immunohistochemistry. iNOS inhibtion reduced parameters associated with tumor progression and reduced the number of CSC, wich resulted higher in MB49-I than in MB49, in concordance with the higher expression of SOX2, Oct4, and Nanog. The expression of SOX2 was notoriously diminished, when iNOS was inhibited only in the MI cell line. Similar results were observed in human samples, where MI tumors expressed higher levels of iNOS and pluripotential genes, in comparison to NMI tumors with a positive correlation between those and iNOS, suggesting that iNOS expression is associated with CSC. iNOS plays an important role in BC progression and CSC maintenance. Its inhibition could be a potential therapeutic target to eradicate CSC, responsible for tumor recurrences. KEY MESSAGES: • iNOS expression is involved in bladder tumor development, growth, and angiogenesis. • iNOS expression is involved in bladder cancer stem cell generation and maintenance, playing an important role regulating their self-renewal capacity, especially in muscle invasive murine bladder cancer cells. • iNOS expression is higher in human muscle invasive tumors, in association with a high expression of pluripotential genes, especially of SOX2.

Effect of nitric oxide inhibition in Bacillus Calmette-Guerin bladder cancer treatment.

BACKGROUND: Bacillus Calmette-Guerin (BCG) is the standard treatment for patients with high-risk non-muscle invasive bladder cancer (BC). Despite its success, about 30-50% of patients are refractory. It was reported that inducible nitric oxide synthase (iNOS) tumor expression is presented in 50% of human BC, associated with bad prognosis and BCG failure. OBJECTIVE: to evaluate in human bladder tumors the association between iNOS expression and the tumor microenvironment focusing on the immunosuppressive protein S100A9. Also, investigate in a preclinical murine MB49-BC model the tumor immunoresponse induced by BCG in combination with the nitric oxide production inhibitor l-NAME. RESULTS: In human bladder tumors, we detected a positive association between iNOS and S100A9 tumor expression, suggesting a relationship between both immunomodulatory proteins. We also found a positive correlation between iNOS tumor expression and the presence of S100A9+ tumor-infiltrating cells, suggesting an immunosuppressive tumor microenvironment induced by the nitric oxide production. Using the subcutaneous murine BC model, we show that similarly to the human pathology, MB49 tumors constitutively expressed iNOS and S100A9 protein. MB49 tumor-bearing mice presented an immunosuppressive systemic profile characterized by fewer cytotoxic cells (CD8+ and NK) and higher suppressor cells (Treg and myeloid-derived suppressor cells -MDSC-) compared to normal mice. BCG treatment reduced tumor growth, increasing local CD8+-infiltrating cells and induced a systemic increase in CD8+ and a reduction in Treg. BCG combined with l-NAME, significantly reduced tumor growth compared to BCG alone, diminishing iNOS and S100A9 tumor expression and increasing CD8+-infiltrating cells in tumor microenvironment. This local response was accompanied by the systemic increase in CD8+ and NK cells, and the reduction in Treg and MDSC, even more than BCG alone. Similar results were obtained using the orthotopic BC model, where an increase in specific cytotoxicity against MB49 tumor cells was detected. CONCLUSION: The present study provides preclinical information where NO inhibition in iNOS-expressing bladder tumors could contribute to improve BCG antitumor immune response. The association between iNOS and S100A9 in human BC supports the hypothesis that iNOS expression is a negative prognostic factor and a promising therapeutic target.

Receptor de Andrógenos y Cáncer de Mama Triple Negativo: implicaciones clínicas y correlación con factores pronósticos y predictivos / Androgen Receptor and triple negative breast cancer: clinical implications and correlation with prognostic and predictive factors

Introducción El Cáncer de Mama Triple Negativo (cmtn) es un grupo heterogéneo, de dispar evolución y sin terapia blanco específica. En la actualidad, se postula al Receptor de Andrógenos (ra) como un prometedor biomarcador en cmtn. Objetivos En el presente estudio analizamos un grupo de pacientes con cmtn con el fin de definir la prevalencia del Receptor de Andrógenos en nuestra población y correlacionarla con factores pronósticos y predictivos. Material y método Se realizó un estudio retrospectivo de determinación de Receptor de Andrógenos por inmunohistoquímica sobre los tacos histológicos de pacientes con diagnóstico de cmtn. Se correlacionó su expresión con las características clinicopatológicas y el impacto en la sobrevida. Resultados Se analizaron 179 pacientes con cmtn, diagnosticadas desde el 1º de enero de 2008 al 31 de diciembre de 2014. El 34,02% de los tacos fueron positivos para ra por ihq. El ra se correlacionó inversamente con Ki 67 y citoqueratinas basales. No se encontró relación con la sobrevida global ni con la sobrevida libre de enfermedad. Conclusiones El rol biológico del Receptor de Andrógenos esta aún en discusión. El cmtn ra positivo se relaciona con menor proliferación celular y menor presencia de marcadores basales. El presente trabajo no demostró impacto en la sobrevida global y en la sobrevida libre de enfermedad. Se deberá seguir estudiando al ra por su gran potencial como blanco terapéutico

Introduction Triple Negative Breast Cancer (tnbc) is a heterogeneous group, with aggressive evolution and without a specific therapeutic target. The Androgen Receptor (ar) is being postulated as a promising biomarker in tnbc. Objectives In this study, we analyze a group of patients with tnbc aiming to define the prevalence of the ar in our population, and the correlation of ar expression with prognostic and predictive factors and its impact on prognosis. Materials and method A retrospective study of determination of ar by immunohistochemistry was done in histological samples of the patients with an invasive Triple Negative Breast Cancer diagnosis. We correlated its expression with clinicopathologic features and clinical outcome. Results 179 patients with tnbc diagnosed from January 2008 to December 2014, where analyzed. An 34.02% of the samples were positive for ar. The ar inversely correlated with Ki 67 and basal cytokeratin. There wasn't found a correlation with overall survival or disease free survival. Conclusions The biological role of the Androgen Receptor is still on discussion. The positive ar tnbc is linked with less cellular proliferation and less presence of basal markers. Although its role as a prognostic factor couldn't be revealed by this job, ar receptor must be investigated to determinated their potential role as treatment target.

Estudio fenotípico de inestabilidad microsatelital en cáncer colorrectal. Correlación con parámetros histológicos y clínicos / Immunohistochemical study of microsatellite instability in colorectal cancer. Correlation with histological and clinical parameters

La detección de proteínas de reparación del ADN por inmunohistoquímica (IHQ) ha demostrado ser útil para determinar inestabilidad de microsatélites (IM). Las guías de Bethesda revisadas y el MsPath Score permiten detectar pacientes de alto riesgo. Se analizaron criterios histológicos sugestivos de IM en 115 pacientes con cáncer colorrectal (CCR). Se efectúo el MsPath Score e IHQ para la demostración de proteínas de reparación del ADN (MMR): MLH1 MSH2, MSH6 y PMS2. La distribución por sexos fue de 66 varones y 49 mujeres, con un rango etario de 16 a 78 años. Cincuenta y tres casos tuvieron rasgos morfológicos de IM: linfocitos intraepiteliales (n = 31), infiltrados tipo Crohn (n = 17), morfología mucinosa/células en anillo de sello (n = 32) y/o pobre diferenciación (n = 12) en forma simultánea o aisladamente. Se observó patrón aberrante para MMR en 16 casos: ausencia de expresión de MLH1/PMS2 (n = 11), MSH2/MSH6 (n = 3), MLH1 (n = 1) y PMS2 (n = 1). Los adenocarcinomas con rasgos morfológicos convencionales no mostraron alteraciones en las MMR (n = 62) (AU)

The detection of mismatch repair proteins (MMR) by immunohistochemistry (IHC) is a useful tool in the identification of microsatellital inestability (MI). The revised Bethesda guidelines and MsPath score allow the detection of patients at high-risk for MI. We studied 115 patients with colorectal cancer (CRC) and evaluated the histological criteria of MI. Detection of MMR: MLH1, MSH2, MSH6 and PMS2 were analyzed by IHC. Sixty-six male and 49 female patients (age range 16-78). Histological picture of MI was observed in 53 cases: intraepithelial lymphocytosis (n = 31), Crohn like infiltrates (n = 17), mucinous or ringed cell histology (n = 32) and/or poorly differentiated histology (n = 12). Aberrant immunostaining pattern of MMR was observed in 16 cases: negative MLH1/PMS2 (n = 11), negative MSH2/MSH6 (n = 3), negative MLH1 (n = 1) and negative PMS2 (n = 1). The 62 cases with conventional histology had normal immunostaining. Conventional histology (n = 62) did not show any alterations in MMR (AU)

Inducible nitric oxide synthase and PPARγ are involved in bladder cancer progression.

PURPOSE: We evaluated the role of inducible nitric oxide synthase and PPARγ as prognostic factors for bladder cancer. MATERIALS AND METHODS: Inducible nitric oxide synthase and PPARγ were evaluated by Western blot and immunohistochemistry in a mouse bladder cancer model of nonmuscle invasive and invasive MB49-I tumor cells, and in human bladder cancer samples. RESULTS: Inducible nitric oxide synthase expression was negative in mouse normal urothelium and higher in invasive than in noninvasive MB49 tumors. In vitro inducible nitric oxide synthase activity, determined as nitrite, was higher in MB49-I than in MB49 cells (p <0.001). In human samples expression was also associated with tumor invasion. Nuclear PPARγ expression was negative in normal mouse urothelium but higher in nonmuscle invasive MB49 than in MB49-I tumors. Similarly in human tumors low PPARγ was associated with poor prognosis factors, such as high histological grade (p = 0.0160) and invasion status (p = 0.0352). A positive correlation was noted between inducible nitric oxide synthase and PPARγ in low histological grade and nonmuscle invasive tumors (Pearson correlation index 0.6368, p = 0.0351, 0.4438 and 0.0168, respectively). As determined by gene reporter assay, PPARγ activity was induced by nitric oxide only in nonmuscle invasive MB49 cells (p <0.001). CONCLUSIONS: Results suggest that increased PPARγ controls inducible nitric oxide synthase expression at early tumor stages. However, regulation is lost at advanced tumor stages, when the increase in inducible nitric oxide synthase and the decrease in PPARγ seem to be associated with bladder cancer progression.

Linfoma gástrico primario anaplásico de células grandes CD30 (Ki-1) positivo / Primary gastric CD30 (Ki-1)-positive anaplastic large cell lymphoma

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[Hematodermic CD4+ CD56+ neoplasm in childhood]. / Neoplasia hematodérmica CD4+ CD56+ en la infancia.

Hematodermic CD4+ CD56+ neoplasm with plasmacytoid dendritic cell phenotype is a rare and aggressive neoplasm recently recognized by the WHO-EORTC classification. It generally appears in elderly adults, exceptionally in childhood. We present a 12-year-old girl with severe mental retardation, genetic clinical features and multiple nodular cutaneous lesions on legs and arms. Histologically the nodules showed diffuse dermal infiltrate of medium and small cells and expression of CD4, CD56, CD43, S100 and plasmacytoid dendritic markers: CD123, BDCA-2 under flow cytometry study. Peripheral blood and bone marrow were not involved. Clinical remission of cutaneous lesions was observed after two weeks of acute lymphoblastic leukemia therapy.

Neoplasia hematodérmica CD4+ CD56+ en la infancia / Hematodermic CD4+ CD56+ neoplasm in childhood

La neoplasia hematodérmica CD4+ CD56+ con fenotipo de célula dendrítica plasmocitoide es una rara y agresiva neoplasia recientemente reconocida por la WHO-EORTC classification. Afecta adultos de edad media y ancianos, siendo muy pocos los casos descriptos en niños. Presentamos el caso de una niña de 12 años con grave retraso mental, estigmas genéticos y múltiples lesiones cutáneas localizadas en miembros inferiores y superiores. Histológicamente se observó un infiltrado dérmico difuso de células pequeñas y medianas con expresión de CD4, CD56, CD43 y S100 así como de marcadores dendríticos plasmocitoides: CD 123 y BDCA-2 confirmados por citometría de flujo, sin compromiso de sangre periférica ni médula ósea. Cumpliendo dos semanas de tratamiento para leucemia linfoblástica aguda evolucionó con remisión clínica de las lesiones cutaneas.

Hematodermic CD4+ CD56+ neoplasm with plasmacytoid dendritic cell phenotype is a rare and aggressive neoplasm recently recognized by the WHO-EORTC classification. It generally appears in elderly adults, exceptionally in childhood. We present a 12-year-old girl with severe mental retardation, genetic clinical features and multiple nodular cutaneous lesions on legs and arms. Histologically the nodules showed diffuse dermal infiltrate of medium and small cells and expression of CD4, CD56, CD43, S100 and plasmacytoid dendritic markers: CD123, BDCA-2 under flow cytometry study. Peripheral blood and bone marrow were not involved. Clinical remission of cutaneous lesions was observed after two weeks of acute lymphoblastic leukemia therapy.

[Cutaneous B cell processes with nodular pattern]. / Procesos linfoides B cutáneos con patrón nodular.

Cutaneous lymphomas are low grade malignant neoplasms with favourable prognosis. Those related to the germinal centre with nodular pattern may be: follicular lymphomas (LFC) or extranodal marginal zone B-cell lymphomas (LMC). They are difficult to tell apart, and from reactive processes like cutaneous follicular hyperplasia and cutis immunocytomas. The objective of this study was to check the incidence and the value of both histology and immunohistochemistry in differential diagnosis. Fifty six patients with cutaneous lymphomas were selected within the period 1995-2004. The biopsies were studied with hematoxilin eosin and immunohistochemistry. Thirty two out of the fifty six cutaneous lymphoid infiltrates were of T origin (57.1%) and twenty four of B origin (42.8%), ten out of this last figure (17.7%) were lymphoid processes with nodular pattern Four LFC, three LMC and three HLC were diagnosed. Convergent follicles with scarce mantle and germinal centres with monomorph celullarity were observed in the LFC. Among the LMC, follicles with prominent mantle and nests of monocitoid cells in the mantle, interfollicular zone and in the germinal centers observed. In the HLC macrophages with detritus were found in the germinal centers. LFC showed: CD20 (+), CD 10 (+), bcl-2 (+) or (-), and bcl-6 (+) in the follicle and in the interfollicular area. LMC showed: CD 20 (+), bcl-2 (-), CD 10 (+/-), and bcl-6 (+) in the follicle, and bcl-2 (+), CD10 (-/+) and bcl-6 (-) in the interfollicular area. The HLC results were: bcl-2 (-), bcl-6 (+) and CD 10 (-) in the follicle and bcl-2 (+), bcl-6 (-) and CD 10 (-) in the interfollicular zone. We conclude that lymphoid B cell processes with nodular pattern are unusual. Histology and immunohistochemistry proved to be useful in the differential diagnosis of these lymphomas, and for differentiating these from lymphoid hyperplasias or non tumoral hyperplasias.

[Signet ring cell lymphoma mimicking mucin-producing carcinoma]. / Linfoma de células en anillo de sello que simula carcinoma mucosecretante.

Signet ring cell lymphoma is a rare neoplasm characterized by large, vacuolated and clear cells mimicking mucin-producing adenocarcinoma. It is localized in nodal and extranodal sites. A case of a 59 years old male, with a diffuse lymphoma signet ring cell type localized on oropharyngeal mucosa is reported. The histopathology study showed signet ring cells and the immunophenotype was: vimentine(+), CD45(+), CD20(+), Ig M(+), Kappa chain(+) and high index proliferative activity of neoplastic cells (Ki 67:70%). After a review of the literature and previous reports, we could not find a similar case in this anatomic site. The patient had a unfavourable clinical course and died two months after the diagnosis without receiving any treatment.

Linfoma de células en anillo de sello que simula carcinoma mucosecretante. / [Signet ring cell lymphoma mimicking mucin-producing carcinoma]

Signet ring cell lymphoma is a rare neoplasm characterized by large, vacuolated and clear cells mimicking mucin-producing adenocarcinoma. It is localized in nodal and extranodal sites. A case of a 59 years old male, with a diffuse lymphoma signet ring cell type localized on oropharyngeal mucosa is reported. The histopathology study showed signet ring cells and the immunophenotype was: vimentine(+), CD45(+), CD20(+), Ig M(+), Kappa chain(+) and high index proliferative activity of neoplastic cells (Ki 67:70

). After a review of the literature and previous reports, we could not find a similar case in this anatomic site. The patient had a unfavourable clinical course and died two months after the diagnosis without receiving any treatment.

Fracción de crecimiento tumoral en los linfomas no-Hodgkin: revisión del grado intermedio de la clasificación de la fórmula de trabajo para uso clínico / Fraction of tumoral growth in non-Hodgkin lymphomas: revision of the intermediate degree of the classification of the work formula for clinical use

La clasificación morfológica de los linfomas no-Hodgkin [LNH] ha sido por años controvertida. A pesar de los esfuerzos de los investigadores, los estudios histológicos de estos tumores no han permitido la predicción de la evolución clínica de los pacientes, especialmente en aquellos clasificados dentro del grupo intermedio de acuerdo con la Clasificación de la Fórmula de Trabajo para Uso Clínico [WF]. La determinación de la Fracción de Crecimiento [FC] tumoral ha demostrado su utilidad en la medición del grado de proliferación celular, que a su vez es una indicación del grado de malignidad de la lesión. Los objetivos de este trabajo fueron: 1- estudiar la correlación entre el grado de malignidad de la lesión [clasificación WF] y el valor de la FC y 2- proponer un método adicional para definir mejor el grado intermedio de la clasificación histológica. Se estudiaron 132 biopsias de pacientes con LNH por medio de Hematoxilina y Eosina y Peroxidasa-anti-Peroxidasa con anticuerpo Ki-67. Este anticuerpo identifica un antígeno nuclear presente en las fases G1, S, G2 y mitosis del ciclo celular. La FC es el porcentaje de células con Ki-67 positivo, obtenido por 2 observadores independientes contando un total de 200 células en un fotomicroscopio con un campo de 40 x. El diagnóstico histológico se realizó de acuerdo a la WF en grados Bajo [B], intermedio [I] y alto [A] y misceláneo [M]. En 19 biopsias del grado B, hubo 9 con FC40 por ciento. En 70 biopsias del grado I, hubo 24 con FC40 por ciento. En 31 casos del grado A, no hubo ninguna biopsia con FC40 por ciento. En el Grupo M [12 casos] hubo 9 biopsias con FC<20 por ciento y 3 entre 20 y 39 por ciento. Los resultados indican que: 1- Los altos grados de malignidad histológica se correlacionan con valores altos de la FC [Chi cuadrado, P<0.001]. 2- Bajos grados de malignidad histológica se asocian con bajos valores de la FC [Chi cuadrado, P<0.001]. 3- El grado intermedio histológico no se asocia con valores intermedios de la FC, sino que está compuesto por pacientes con valores bajos, medianos y altos de la FC. Concluímos que la FC se correlaciona bien con los grados histológicos A y B, pero no con el grado intermedio, que requiere una re-distribución teniendo en cuenta los valores de la FC.

Fracción de crecimiento tumoral en los linfomas no-Hodgkin: revisión del grado intermedio de la clasificación de la fórmula de trabajo para uso clínico / Fraction of tumoral growth in non-Hodgkin lymphomas: revision of the intermediate degree of the classification of the work formula for clinical use

La clasificación morfológica de los linfomas no-Hodgkin [LNH] ha sido por años controvertida. A pesar de los esfuerzos de los investigadores, los estudios histológicos de estos tumores no han permitido la predicción de la evolución clínica de los pacientes, especialmente en aquellos clasificados dentro del grupo intermedio de acuerdo con la Clasificación de la Fórmula de Trabajo para Uso Clínico [WF]. La determinación de la Fracción de Crecimiento [FC] tumoral ha demostrado su utilidad en la medición del grado de proliferación celular, que a su vez es una indicación del grado de malignidad de la lesión. Los objetivos de este trabajo fueron: 1- estudiar la correlación entre el grado de malignidad de la lesión [clasificación WF] y el valor de la FC y 2- proponer un método adicional para definir mejor el grado intermedio de la clasificación histológica. Se estudiaron 132 biopsias de pacientes con LNH por medio de Hematoxilina y Eosina y Peroxidasa-anti-Peroxidasa con anticuerpo Ki-67. Este anticuerpo identifica un antígeno nuclear presente en las fases G1, S, G2 y mitosis del ciclo celular. La FC es el porcentaje de células con Ki-67 positivo, obtenido por 2 observadores independientes contando un total de 200 células en un fotomicroscopio con un campo de 40 x. El diagnóstico histológico se realizó de acuerdo a la WF en grados Bajo [B], intermedio [I] y alto [A] y misceláneo [M]. En 19 biopsias del grado B, hubo 9 con FC<20 por ciento, 9 entre 20 y 39 por ciento y sólo una biopsia con FC>40 por ciento. En 70 biopsias del grado I, hubo 24 con FC<20 por ciento, 14 con FC entre 20 y 39 por ciento y 32 con FC>40 por ciento. En 31 casos del grado A, no hubo ninguna biopsia con FC<20 por ciento, hubo 7 con valores de FC entre 20 y 39 por ciento y 24 con valores >40 por ciento. En el Grupo M [12 casos] hubo 9 biopsias con FC<20 por ciento y 3 entre 20 y 39 por ciento. Los resultados indican que: 1- Los altos grados de malignidad histológica se correlacionan con valores altos de la FC [Chi cuadrado, P<0.001]. 2- Bajos grados de malignidad histológica se asocian con bajos valores de la FC [Chi cuadrado, P<0.001]. 3- El grado intermedio histológico no se asocia con valores intermedios de la FC, sino que está compuesto por pacientes con valores bajos, medianos y altos de la FC. Concluímos que la FC se correlaciona bien con los grados histológicos A y B, pero no con el grado intermedio, que requiere una re-distribución teniendo en cuenta los valores de la FC. (AU)