In the past decades our knowledge concerning breast cancer progression from ductal carcinoma in situ (DCIS) to invasive cancer has grown rapidly. However, molecular factors driving the progression are still largely unknown. In the first study, we investigated tumor evolution in breast cancer by analyzing TP53 mutation status in tumors from various stages of the disease. Presence of the same TP53 mutations in both DCIS and invasive components from the same tumor indicates same cellular origin. The role of mutant TP53 in the progression of breast cancer is less clear and may vary between subtypes. In the second study, we studied the prognosis of basal-like DCIS in a large population-based cohort. Basal-like DCIS was associated with about doubled but not statistically significant risk for local recurrence compared with the other molecular subtypes. Molecular subtype was a better prognostic parameter than histopathological grade. In the third study, we studied markers in primary DCIS in relation to type of recurrence. Interestingly, recurrences after an ER-/HER2+, ER negative or EGFR positive primary DCIS were more often of the in situ type. The molecular subtype ER+/HER2+, FOXA1 positivity and FOXC1 positivity were risk factors for any recurrence. In the fourth study, we proposed a histological classification system for a new entity: neoductgenesis. We also evaluated histologic criteria for neoductgenesis. According to our criteria, good agreements among pathologists were achieved. Neoductgenesis was related to more aggressive tumor biology and to mammographic features. The result indicates potential benefits for women earlier considered having pure DCIS but later diagnosed as breast carcinoma with neoductgenesis, suggesting a need to develop appropriate treatment regiments. Our findings have to be repeated and the relation to prognosis warrants further studies.

WWOX is a tumor suppressor and the down-regulation of WWOX has been demonstrated in prostate, lung, breast, gastric cancers. However, the role of WWOX in renal cell carcinoma (RCC) remains unknown. It has been demonstrated that WWOX addressed in mitochondria, golgi apparatus, rough ER, lysosome, plasma membrane and nuclear. The Subcellular localization of WWOX has been controversial. There are two parts in this study: (I) The expression of WWOX in RCC and the probability of WWOX to be a diagnostic and a prognostic marker. (II) The regulation of WWOX by phosphorylation. For the study of WWOX expression in RCC, we prepared polyclonal WWOX antibody and characterized the specificity of the antibody. We applied this specific antibody to 33 NT paring RCC tissue specimen for immunoblotting study and 138 cases of paraffin-embedded specimens for IHC, respectively. Our results demonstrated that hWOX1 was specifically down-regulated in clear cell type RCC (p=0.018). The percentage of down-regulation in patient specimen is 60.7 % and 90.7 % in immunoblotting and IHC study, respectively. And in clear cell and clear-granular combined type RCC, down-regulation of WWOX was significantly correlated with the survival rate of patients (p=0.0482). Therefore, WWOX could be used as a diagnostic and a prognostic marker in clear cell type RCC. Besides, we performed bioinformatics to predict the phosphorylation site of WWOX and investigated the effect of phosphorylation on WWOX subcellular localization. Our results demonstrated that hWOX1 was phosphorylated by PKC at Thr49 and Thr102 and the phosphorylation regulated the subcellular localization of WWOX.

El carcinoma basocelular (CBC) es el tumor maligno más frecuente y en nuestro país su incidencia ha aumentado progresivamente en los últimos años. El tratamiento del CBC se basa en la cirugía, pero ante el envejecimiento de la población ha surgido la necesidad de poder tratar los pacientes con terapias menos agresivas. Imiquimod es un potente inductor de citocinas, principalmente el interferón, con propiedad inmunomoduladora sobre el sistema inmune innato y adquirido. La inmunoterapia con imiquimod crema al 5% permite tratar los pacientes de modo ambulatorio y ha demostrado ser un tratamiento eficaz en el CBC. El mecanismo de acción de imiquimod en el CBC no es bien conocido y el presente trabajo ha estudiado los efectos de la aplicación tópica de imiquimod al 5% en la apoptosis del CBC, en la expresión de las proteínas bcl2, p53 y ki67 del CBC, y en el infiltrado inflamatorio peritumoral del CBC.En el 2001 se diseñó un ensayo clínico abierto con 55 CBC tratados con imiquimod crema al 5%. En todos los CBC se obtuvieron 3 biopsias, una antes del tratamiento, otra durante y otra 6 semanas después del mismo. A partir de muestras congeladas se estudió el índice apoptótico (Tunel) del CBC, y a partir de muestras parafinadas se estudió la expresión de bcl2, p53 y ki67 del CBC. En 25 CBC también se estudió el infiltrado inflamatorio peritumoral: células totales, CD3 +, CD8 +, CD56 +, Grancima B +, CD20 +, CD68 + y S-100 +. Todos los pacientes fueron seguidos durante 18 meses para evaluar la tasa de recidiva de CBC después del tratamiento. Entre 2001 y 2002 se diseñó un ensayo clínico randomizado con imiquimod crema al 5% y su excipiente en 30 CBC. En todos los CBC se obtuvieron 3 biopsias, una antes del tratamiento y dos durante el mismo. A partir de muestras congeladas se estudió el índice apoptótico (Tunel) del CBC, y a partir de muestras parafinadas se estudió la expresión de bcl2, p53 y ki67 del CBC. En 12 CBC también se estudió la densidad del infiltrado inflamatorio peritumoral y se realizaron estudios ecográficos con Dermascan C. Los principales resultados del estudio abierto fueron que imiquimod aumentó de modo significativo el índice apoptótico del CBC, la densidad del infiltrado inflamatorio peritumoral, el número de células CD3 +, CD8 +, CD20 +, CD68 +, S-100 + y grancima B +. Por el contrario imiquimod diminuyó de modo significativo la expresión de bcl2 del CBC. Imiquimod no modificó de modo significativo la expresión de p53 y ki67 del CBC. Después de 18 meses de seguimiento 41 CBC (75%) no mostraron signos de recidiva y fueron consideraros curados. Los resultados del estudio randomizado confirmaron que imiquimod crema al 5%, a diferencia de su excipiente, aumentó de modo significativo el índice apoptótico del CBC y la densidad del infiltrado inflamatorio peritumoral, y diminuyó de modo significativo la expresión de bcl2 del CBC. La expresión de p53 y ki67 del CBC no se modificaron de modo significativo ni con imiquimod ni con su excipiente. Como conclusión se puede afirmar que imiquimod aumenta el índice apoptótico del CBC, disminuye la expresión de bcl2 del CBC e induce una respuesta inmune antitumoral frente al CBC mediada por células CD3 +, CD8+, CD20 +, CD68 +, S-100 + y grancima B +. / Basal cell carcinoma (BCC) is the most frequent type of malignant tumor and its incidence has gradually increased during the last few years. The treatment of BCC is based on surgery. However, the aging of the population has made it necessary to treat patients with less aggressive therapies. Imiquimod is a potent cytokine inducer (especially of interferon) with immunomodulating properties on the innate and acquired immune systems. Immunotherapy with imiquimod 5% cream enables outpatient treatment and has been proved to be an efficient treatment for BCC. The mechanism of action of imiquimod on BCC is quite unknown. The purpose of this study was to assess the effects of imiquimod 5% external application on BCC apoptosis, on the expression of bcl2, p53 and ki67 proteins in BCC and on the peritumoral inflammatory infiltrate of BCC.An open trial was designed in 2001 with 55 BCCs treated with imiquimod 5% cream. Three biopsies were performed on each BCC: one before the treatment, another one during the treatment and a final one 6 weeks after its completion. The BCC apoptotic indexes (Tunel) were studied using frozen samples. Moreover, bcl2, p53 and ki67 expression in the BCC was studied using paraffin embedded samples. The peritumoural inflammatory infiltrate was studied in 25 BCCs, as well: total number of cells, CD3+, CD8+, CD56+, granzyme B+, CD20+, CD68+ and S-100+. All patients were followed-up for 18 months to evaluate the index of BCC recurrence after the treatment. A randomized clinical trial was carried out between 2001 and 2002 to evaluate imiquimod 5% cream and its excipient in 30 BCC. Three biopsies were performed on each BCC, one before the treatment and two during the treatment. The BCC apoptotic indexes (Tunel) were studied using frozen samples. Furthermore, bcl2, p53 and ki67 expression in the BCC was studied using paraffin-embedded samples. The density of the peritumoural inflammatory infiltrate was assessed in 12 BCCs. In addition to this, ultrasound studies were performed with Dermascan C. The main results obtained in the open study were the following: imiquimod significantly increased the BCC apoptotic index, the density of the peritumoural inflammatory infiltrate, the number of CD3+, CD8+, CD20+, CD68+, S-100+ and granzyme B+ cells. On the contrary, imiquimod remarkably decreased the expression of bcl2 in the BCC. Imiquimod did not modify significantly the expression of p53 and ki67 in the BCC. After 18 months of follow-up, 41 BCCs (75%) did not show evidence of recurrence, and were therefore considered healed of BCC. The results of the randomized study proved that, unlike its excipient, imiquimod 5% cream significantly increased the apoptotic index of the BCC and the density of the peritumoural inflammatory infiltrate, as well as decreased the expression of bcl2 in the BCC. The expression of p53 and ki67 in the BCC was not modified significantly neither using imiquimod nor its excipient. Thus, it may be concluded that imiquimod increases the apoptotic index of BCC, decreases the bcl2 expression and induces an antitumoural immune response against the BCC thanks to the intervention of CD3+, CD8+, CD20+, CD68+, S-100+ and granzyme B + cells.

El cáncer de mama es una enfermedad heterogénea sobre la que se han realizados grandes esfuerzos para conocer su biología. El objetivo es poder identificar subtipos de tumores agresivos y pacientes con alto riesgo de recidivas y metástasis; los cuales se verían beneficiados de nuevos regímenes terapéuticos más apropiados.El aumento en la clasificación de variantes histológicas del cáncer de mama, ha hecho que disminuya la prevalencia del subgrupo clásico "carcinoma ductal infiltrante convencional".Muchos estudios han demostrado la correlación de determinadas estirpes histológicas con su desarrollo biológico; así, algunos fenotipos se han identificado como más agresivos y relacionados con mayor porcentaje de recidivas (116,117).La evaluación de los datos recogidos en nuestro estudio pretende demostrar los objetivos expuestos al principio:1. Determinar las características epidemiológicas de las mujeres que presentan una neoplasia mamaria con este patrón: micropapilar infiltrante.2. Estudiar el comportamiento del Carcinoma Micropapilar Infiltrante puro y mixto, respecto a una serie de variables pronósticas, con el fin de redefinir criterios diagnósticos del mismo. 3. Definir los aspectos clínicos, el patrón de diagnóstico por imagen más característico y la actitud terapéutica del carcinoma micropapilar infiltrante.4. Comparar el comportamiento de este grupo con los subgrupos de carcinoma ductal infiltrante (G-I y G-III).5. Analizar el intervalo de supervivencia libre de enfermedad y tasa de mortalidad, para valorar alguna modificación en la conducta terapéutica a seguir en estas pacientes.

Renal cell carcinoma (RCC) accounts for 85% of kidney cancer incidence in the US. Since 1950 there has been a 126% increase in kidney cancer incidence in the US. Thirty percent of new patients present with a localized easily treatable carcinoma while 30% of patients present with a high-grade metastatic carcinoma. Five-year survival rates for metastatic RCC is 6-12 months (Lipworth et al, 2006). Current disease treatment options for metastasis include chemotherapy and radiation (8% response rate), immunotherapy (15-30% response rate) and newly developed angiogenesis inhibitors which are in phase III trials (Staehler et el, 2005). In RCC cells, it has been shown that PPARγ agonists inhibit cell proliferation, induce apoptosis, and induce anti-angiogenic effects in vitro. Unlike most tumor types, PPARγ is downregulated in tissue samples from 47 RCC patients. However, in cell culture studies PPARγ expression does not correlate with growth inhibitory or apoptotic effects of PPARγ agonists in renal cell lines indicating that PPARγ independent responses may play a large role in actions associated with the PPARγ agonists (Yuan et al, 2006). 1,1-Bis(3'-indolyl)-1-(p-substitutedphenyl)methanes containing p-trifluoromethyl, p-t-butyl and p-phenyl substituents activate peroxisome proliferator-activated receptor (PPAR) and inhibit growth of ACHN and 786-0 renal cell carcinoma cell lines. PPAR is overexpressed in ACHN cells and barely detectable in 786-0 cells, and treatment with the t-butyl analog (DIM-C-pPhtBu) induces cell cyle inhibition. DIM-C-pPhtBu also induced several common PPAR-independent proapoptotic responses in ACHN and 786-0 cells, including increased expression of nonsteroidal antiimflammatory drug-activated gene-1 (NAG-1) and endoplasmic reticulum stress which activates death receptor 5 and the extrinsic pathway of apoptosis. In addition, DIM-C-pPhtbu (40 mg/kg/d) also inhibited tumor growth in an orthotopic mouse model for renal carcinogenesis, and this was accompanied by induction of apoptosis in renal tumors treated with DIM-C-pPhtBu but not in tumors treated with the corn oil vehicle (control). Thus, DIM-C-pPhtBu and related compounds represent a novel class of mechanism-based drugs that have potential for treatment of renal adenocarcinoma for which there are currently limited options for successful chemotherapy.

The F-box protein Skp2 (S-phase kinase associated protein 2) positively regulates the G1-S transition by controlling the cell cycle inhibitor p27Kip1. The p42/p44 mitogen-activated protein (MAP) kinase activation is also necessary for the cell cycle progression. p27Kip1 acts as a negative regulator of the cell cycle by inhibiting the activity of cyclin/cdk complexes during G0 and G1. RT-PCR, Western blotting and immunohistochemical staining were used to assay their relationship with cervical lesion development. In RT-PCR and western blotting, Skp2 mRNA and protein were expressed mostly in carcinoma tissues. At Fisher¡¦s exact test showed that Skp2, p27Kip1 and p42/p44 MAP Kinase are strongly associated with disease progession respectively (P < 0.0001, P < 0.0001, P = 0.0043). We also found a postive correlation between the expression of Skp2 and p42/p44 MAP Kinase (P = 0.0097).

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in the world, especially in Asia and Africa countries. The distribution pattern shows geographical variation and pathogesis in multifactors as environment, infection, nutrition, metabolism, and endocrine contribute to hepatocarcinogenesis. Alpha fetal protein (AFP), the major tumor marker used at present accounts only 50% HCC diagnostic efficiency. This study aims to identify potential tumor markers or suppressor genes for further application in early HCC diagnoses and treatments. Therefore we utilized available cDNA microarray databases in conjunction with other bioinformatic resources to identify our candidate genes related to HCCs. cDNA microarray technology and bioinformatic resources which enable investigators to obtain comprehensive data with respect to gene-expression profiles, is progressing rapidly. The Okabe¡¦s and Stanford¡¦s HCC database were our major data-mining material. A total of 85 potential tumor markers and 106 potential tumor suppressors were found via preliminary in silico datamining. We furthermore narrowed down to 14 candidate tumor markers and 7 candidate tumor suppressor genes by the way of quantitative RT-PCR technologies were applied in various HCC cancer cell lines and 21 patient¡¦s in pair, tumor/non-tumor tissues to confirm gene expression profile. The results revealed that 6 genes (PRO2000, PYGB, STMN1, AFM, C8FW, NNMT) conformed to our data-mining studies.

Hepatocellular carcinoma (HCC) is one of the most prevalent cancers worldwide. Hepatocarcinogenesis is considered a multifactorial and mulitstep process that involves the activation of oncogenes or the inactivation of tumor suppressor genes. Tumor suppressor gene PTEN (also known as MMAC or TEP1) is located on human chromosome 10q23. The 403¡Vamino acid PTEN protein encodes dual specificity protein phosphatases. Mutation of the PTEN is a common event in advanced stage of diverse human cancers. In our previous studies, immunohistochemical analysis indicated that reduced PTEN expression was found in nearly 40% of HCC specimens. Furthermore, restored PTEN expression by adenovirus gene delivery effectively inhibited the in vitro and in vivo tumorigenicity of Mahlavu cells, a human HCC cell line with PTEN inactivation. In the present study, we further characterize whether PTEN gene delivery still suppressed the oncogenic potential in HCC cell lines with functional PTEN. By expression and sequencing analysis, we identified human SK-Hep-1 cells as the hepatoma cell line with functional PTEN expression. The optimal condition for adenovirus vector to infect SK-Hep-1 cells was determined at the multiplicity of infection (MOI) of 50-100. Tough SK-Hep-1 cells were effectively transduced with exogenous PTEN gene, the enhanced PTEN expression by adenovirus gene delivery did not alter the phosphoryation extent of Akt in SK-Hep1 cells. Nevertheless, PTEN gene delivery reduced the proliferation of SK-Hep-1 cells by ~20%. In addition, the motility of PTEN-transduced SK-Hep-1 cells significantly decreased comparing to cells of control groups. Western blot analysis suggested the decreased cell motility might be attributed to the reduced phosphorylation of focal adhesion kinase (FAK) by PTEN gene delivery. Above all, PTEN gene delivery profoundly reduced the colony formation of SK-Hep-1 cells in soft-agar. However, PTEN gene delivery did not affect the secretion of matrix metallo-proteinases (MMPs) release. Animal studies will be carried out in the future to validate the present in vitro findings. In summary, PTEN gene delivery holds promise for treatment of HCC even when the hepatoma cells possess functional PTEN gene.

Head and neck cancer (HNC) makes up 6 ¢H of the cancer patients in the world every year. This disease usually occurs in males and the incidence is increasing year by year. According to statistical analysis, HNC has less than 50 ¢H five-year survival rate. Therefore, the research of HNC seems imminent so that may lead to the development of new approaches of diagnosis and therapy. Recent research had shown that expression of podoplanin caused cellular proliferation, and may be associated with tumor invasion, metastasis and malignant prognosis. Podoplanin, a mucin-type transmembrane sialoglycoprotein, is highly expressed in lymphatic endothelial cells but not expressed in vascular endothelial cells. The purpose of this study was to determine the clinical and pathological significance of podoplanin in oral squamous cell carcinoma (OSCC). Therefore, we collected clinical specimen and associated patient history of OSCC. Further, we used the human cell lines of HNSCC (Fadu, Hep2) to investigate the molecular regulation of podoplanin. Podoplanin expression was analyzed by RT-PCR and Western blot assay firstly. As shown, podoplanin was found to be overexpressed in tumors compared with normal adjacent tissues. Further, immunohistochemical analysis revealed the location of podoplanin expression in OSCC tissues. The results showed that podoplanin had higher expression in T4 stage tumor section than in normal adjacent tissues of OSCC samples, or in T1 stage. Here, podoplanin was highly expressed in the OSCC tumor cell and lymphatics of stage T4 OSCC tissue. Furthermore, we found that overexpression of podoplanin in OSCC patients was associated with decreased five-years survival rate. In the univariate analysis, several factors were statistically significantly associated with disease-specific survival rate, including Tumor stage, Nodal stage, and podoplanin expression. In the subsequent multivariate analysis, only Tumor stage and Nodal stage showed a trend toward worse disease-specific survival. To further investigate the regulatory mechanism of podoplanin and its position of expression within the cell, immunofluorescence and transfection were utilized to assay. The results showed that podoplanin was expressed in the nuclear membrane of the Fadu and Hep2 cell lines, and the PI3K/AKT signaling pathway was involved. We suggest that the role of podoplanin in OSCC should be further investigated for potential future treatment.

published_or_final_version / Medicine / Master / Master of Medical Sciences