RESUMEN
AIMS: To determine the role of Na+/H+ exchanger regulatory factor (NHERF1) in breast cancerogenesis and progression. METHODS AND RESULTS: NHERF1 expression was examined in normal tissue, ductal carcinoma in situ (DCIS), invasive carcinoma (IBC), synchronous metastatic lymph node and metachronous distant metastases of a retrospective series of breast cancers. Fifty-one IBC, 42 DCIS and normal tissues were examined immunohistochemically, and the colocalization between NHERF1 and HER2/neu was studied by immunofluorescence. NHERF1 showed a different localization and pattern of expression in the different compartments of the breast. The mean value of cytoplasmic NHERF1 expression in paired samples was significantly higher in DCIS, IBC, distant metastases and metastatic lymph nodes with respect to normal tissues. Moreover, in metastatic lymph nodes NHERF1 was exclusively cytoplasmic. In the membrane NHERF1 was colocalized with overexpressed HER2/neu in DCIS, IBC and distant metastases. CONCLUSIONS: Breast cancerogenesis is characterized by increased cytoplasmic expression of NHERF1 as the tumour progresses, suggesting a role in this process. The switch from apical membranous to cytoplasmic expression is compatible with a dual role for NHERF1 as a tumour suppressor or tumour promoter dependent on its subcellular localization.
Asunto(s)
Biomarcadores de Tumor/análisis , Neoplasias de la Mama/metabolismo , Carcinoma in Situ/metabolismo , Carcinoma Ductal de Mama/metabolismo , Fosfoproteínas/biosíntesis , Intercambiadores de Sodio-Hidrógeno/biosíntesis , Neoplasias de la Mama/patología , Carcinoma in Situ/patología , Carcinoma Ductal de Mama/patología , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Inmunohistoquímica , Hibridación Fluorescente in Situ , Estadificación de Neoplasias , Receptor ErbB-2/biosíntesisRESUMEN
CONTEXT: Learning the characteristics of frozen tissue samples stored in tumor banks for biological studies remains a problem. OBJECTIVE: To assess the use of touch imprint cytology on fresh tissue samples as a rapid and reliable method of determining the presence and quantity of neoplastic cells before freezing. DESIGN: Touch imprint cytology was performed on 259 specimens of operable breast cancer. Touch imprints were prepared from fresh tissue specimens before freezing samples for storage. Each tumor sample was imprinted on a glass slide and stained with hematoxylin-eosin. Tumor cellularity was quantified as negative, poor, moderate, or rich. RESULTS: A significant correlation was found between samples with a tumor size greater than 2 cm and high tumor cellularity (P = .03; chi(2) test). Furthermore, 35% of ductal tumors showed higher tumor cellularity compared with lobular tumors (P < .001; chi(2) test). No association was found between lymph node status and tumor grade. When samples for which more than 2 imprints were available were examined, tumor cellularity among imprints of the same sample showed an overall agreement of 0.67 (P < .001; kappa statistic). It was also determined that the higher the cellularity, the higher the agreement. Our data also showed concordance of 0.87 (P < .001; kappa statistic) between touch imprint cytology imprints and histologic sections from contiguous tumor. Moreover, 11 randomly selected samples underwent DNA extraction, polymerase chain reaction, and sequencing to verify the feasibility of DNA analyses. We found that DNA from touch imprint cytology was amplifiable and suitable for direct sequencing. CONCLUSIONS: Touch imprint cytology may represent an important step in the quality control of tumor cellularity of breast cancer specimens designed to be stored in tumor biobanks and a valid method for assessing the suitability of such tissue for further biomorphologic and biomolecular applications.
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Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Técnicas Citológicas , ADN de Neoplasias/aislamiento & purificación , Bancos de Tejidos , Femenino , Humanos , Reacción en Cadena de la Polimerasa , Control de Calidad , Bancos de Tejidos/normasRESUMEN
RhoA protein is over-expressed in breast cancer and other solid tumors and has been used in tumor biopsies as a quantitative tumor marker for progression, stage and prognosis in molecular detection strategies. Measuring protein markers in plasma or blood cells is preferred to tumor biopsies as it represents a minimally invasive, repeatable measurement that can be followed over time. In this study we evaluated the hypothesis that quantitative RhoA protein expression in circulatory lymphocytes is identically associated with the same tumor clinico-pathological features found in biopsies. RhoA protein levels were analyzed by Western blotting in circulating lymphocytes isolated from 52 consecutive patients with breast cancer and in 34 paired breast tumor biopsies from the same case study, and compared with the following clinico-pathological features of the patients: histological grade, tumor size, steroid receptor status, lymphonode status, proliferative activity and prognosis [Nottingham Prognostic Index (NPI)]. We observed that the level of circulatory, peripheral lymphocyte RhoA expression reflected that found in the matched biopsy of the same patient. Furthermore, similarly to previous reports regarding breast cancer tissue biopsies, the level of RhoA protein expression in both biopsies and in circulatory lymphocytes was positively associated with tumor size, grade, proliferative activity of the tumor biopsy and NPI, while there was no significant association of RhoA protein expression with either estrogen- or progesterone-receptor expression. Our study demonstrated that the association of lymphocyte RhoA protein expression with classical clinico-pathological parameters closely corresponded with that observed for RhoA protein expression in the tumor biopsies. We propose that measurement of RhoA expression in the circulatory lymphocytes of breast cancer patients can be used to predict breast cancer occurrence, progression and prognosis and may prove valuable in the management of cancer patients.
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Neoplasias de la Mama/enzimología , Neoplasias de la Mama/patología , Linfocitos/enzimología , Proteína de Unión al GTP rhoA/metabolismo , Anciano , Progresión de la Enfermedad , Femenino , Humanos , Persona de Mediana Edad , Estadísticas no ParamétricasRESUMEN
The chromosomal changes in eight familial BRCAx breast cancers (i.e., negative for BRCA1 or BRCA2) were analyzed by comparative genomic hybridization (CGH) to investigate intratumor heterogeneity. This was the first step in a study of most frequent chromosomal aberrations in BRCAx familial breast cancers. Laser microdissection analysis of paraffin tissue samples was followed by whole-genome amplification. CGH was performed on DNA isolated from two to three different cell groups per case to detect any cytogenetic aberrations in important clones that might have been missed when analyzing DNA extracted from large numbers of cells. The results were compared, to evaluate the influence of tumor heterogeneity on CGH, and the heterogeneity was confirmed comparing CGH with fluorescence in situ hybridization results. Different chromosomal aberrations were detected between adjacent clones within the same section, which highlights the utility of microdissection in addressing the problem of heterogeneity in whole-genome studies. Some chromosomal regions were more frequently altered in the eight BRCAx tumors; loss of 2q, 3p, 3q, 8p, 9p, and 15q and gains of 1p, 4p, 4q, 5p, 6q, 12q, and 19p were the most common. Further studies focusing on specific genes and sequences with more sensitive approaches, such as array-CGH, are warranted to confirm these findings.
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Proteína BRCA1/genética , Proteína BRCA2/genética , Neoplasias de la Mama/genética , Aberraciones Cromosómicas , ADN de Neoplasias/genética , Heterogeneidad Genética , Genoma Humano , Adulto , Anciano , Proteínas Reguladoras de la Apoptosis , Cromosomas Humanos/genética , Femenino , Humanos , Hibridación Fluorescente in Situ , Masculino , Persona de Mediana Edad , Mutación/genética , Hibridación de Ácido Nucleico , Reacción en Cadena de la PolimerasaRESUMEN
The antineoplastic effect of paclitaxel is mainly related to its ability to bind the beta subunit of tubulin, thus preventing tubulin chain depolarization and inducing apoptosis. The relevance of the Class I beta-tubulin characteristics have also been confirmed in the clinical setting where mutations of paclitaxel-binding site of beta-tubulin Class I have been related to paclitaxel resistance in non small cell lung and ovarian cancers. In the present study, we verified the hypothesis of a relationship between molecular alterations of beta-tubulin Class I and paclitaxel sensitivity in a panel of breast cell lines with different drug IC(50). The Class I beta-tubulin gene cDNA has been sequenced detecting heterozygous missense mutations (exon 1 and 4) only in MCF-7 and SK-BR-3 lines. Furthermore, the expression (at both mRNA and protein level) of the different isotypes have been analyzed demonstrating an association between low cell sensitivity to paclitaxel and Class III beta-tubulin expression increasing. Antisense oligonucleotide (ODN) experiments confirmed that the inhibition of Class III beta-tubulin could at least partially increase paclitaxel-chemosensitivity. The hypothesis of a relationship between beta-tubulin tumor expression and paclitaxel clinical response has been finally verified in a series of 92 advanced breast cancer patients treated with a first line paclitaxel-based chemotherapy. Thirty-five percent (95% CI: 45-31) of patients with high Class III beta-tubulin expression showed a disease progression vs. only 7% of patients with low expression (35% vs. 7%, p < 0.002). Our study suggests that Class III beta-tubulin tumor expression could be considered a predictive biomarker of paclitaxel-clinical resistance for breast cancer patients.
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Antineoplásicos Fitogénicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Citoesqueleto/fisiología , Paclitaxel/farmacología , Tubulina (Proteína)/fisiología , Secuencia de Aminoácidos , Secuencia de Bases , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Procesos de Crecimiento Celular/efectos de los fármacos , Procesos de Crecimiento Celular/fisiología , Línea Celular Tumoral , Citoesqueleto/efectos de los fármacos , Citoesqueleto/metabolismo , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Humanos , Persona de Mediana Edad , Datos de Secuencia Molecular , Mutación , Isoformas de Proteínas , Estudios Retrospectivos , Tubulina (Proteína)/biosíntesis , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismoRESUMEN
AIM: To investigate H pylori expression in gastric cancer patients in relation to primary tumor angiogenic markers, such as microvessel density (MVD), thymidine phosphorylase (TP), vascular endothelial growth factor receptor-1 (VEGF-R1), p53 and circulating VEGF levels. METHODS: Angiogenic markers were analyzed immunohistochemically in 56 primary gastric cancers. H pylori cytotoxin (vacA) and the cytotoxin-associated gene (cagA) amplification were evaluated using PCR assay. Serum H pylori IgG antibodies and serum/plasma circulating VEGF levels were detected in 39 and 38 patients by ELISA, respectively. RESULTS: A total of 69% of patients were positive for circulating IgG antibodies against H pylori. cagA-positive H pylori strains were found in 41% of gastric patients. vacA was found in 50% of patients; s1 strains were more highly expressed among vacA-positive patients. The presence of the s1 strain was significantly associated with cagA (P = 0.0001). MVD was significantly correlated with both tumor VEGF expression (r = 0.361, P = 0.009) and serum VEGF levels (r = -0.347, P = 0.041). Conversely, neither VEGF-R1 expression nor MVD was related to p53 expression. However, H pylori was not related to any angiogenic markers except for the plasma VEGF level (P = 0.026). CONCLUSION: H pylori antigen is related to higher plasma VEGF levels, but not to angiogenic characteristics. It can be hypothesized that the toxic effects of H pylori on angiogenesis occurs in early preclinical disease phase or in long-lasting aggressive infections, but only when high H pylori IgG levels are persistent.
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Infecciones por Helicobacter/complicaciones , Helicobacter pylori/patogenicidad , Neovascularización Patológica/fisiopatología , Neoplasias Gástricas/irrigación sanguínea , Neoplasias Gástricas/microbiología , Adulto , Anciano , Anciano de 80 o más Años , Antígenos Bacterianos/genética , Antígenos Bacterianos/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Femenino , Regulación Bacteriana de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Infecciones por Helicobacter/genética , Infecciones por Helicobacter/metabolismo , Helicobacter pylori/genética , Helicobacter pylori/inmunología , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina G/genética , Masculino , Microcirculación , Persona de Mediana Edad , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , Receptores de Factores de Crecimiento Endotelial Vascular/genética , Receptores de Factores de Crecimiento Endotelial Vascular/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Timidina Fosforilasa/genética , Timidina Fosforilasa/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Factor A de Crecimiento Endotelial Vascular/sangre , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
While several studies have reported that thymidylate synthase (TS) tumour expression can be a reliable predictive marker of clinical response to 5-Fluorouracil (5-FU) for advanced colorectal cancer patients, only a few studies that searched for predictive factors of irinotecan (CPT-11) clinical response are available. The aim of the present study has been to verify the predictive value of immunohistochemical topoisomerase-I (Topo-I) and TS primary tumour expression in a consecutive series of 62 advanced colorectal cancer patients that received a first line 5-FU/CPT-11 chemotherapy. TS and Topo-I immunostaining was observed in 76% and 43% of tumours, respectively, resulting in a significant relationship within each tumour (r=0.365, p<0.004). Patients with different TS tumour expression showed a similar percentage of Objective Clinical Response, OR (40% vs. 28% of OR in low and high TS-expressing tumours, respectively, p=ns); also, patients with different Topo-I tumour expression did not show a different probability of OR (39% vs. 29% of OR in high and low Topo-I expressing tumours, respectively; p=ns). The tumour expression of these 2 biomarkers also did not impact on time to progression and overall survival of patients. Furthermore, the combined analysis of TS and Topo-I tumour status did not permit to individualize subgroups of patients with different probability of OR. With multivariate analysis, only patient Performance Status significantly impacted on OS (Hazard ratio 4.87; p=0.02) of these patients. We can conclude that high TS tumour expression seems not to preclude a clinical activity for 5-FU/CPT-11 polichemotherapy in advanced colorectal cancer patients; furthermore, clinical response and prognosis of colorectal cancer patients treated with 5-FU/CPT-11 regimen do not differ in tumours with different TS or Topo-I expression.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Camptotecina/análogos & derivados , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , ADN-Topoisomerasas de Tipo I/biosíntesis , Perfilación de la Expresión Génica , Timidilato Sintasa/biosíntesis , Camptotecina/administración & dosificación , Neoplasias Colorrectales/patología , Femenino , Fluorouracilo/administración & dosificación , Humanos , Infusiones Intravenosas , Inyecciones Intravenosas , Irinotecán , Leucovorina/administración & dosificación , Masculino , Análisis Multivariante , Pronóstico , Resultado del TratamientoRESUMEN
Vascular endothelial growth factor (VEGF) is known to play a central role in tumour angiogenesis. Up to now inconclusive data have been published on the clinical-biological significance of circulating VEGF and on the most suitable blood fraction for measuring it. The aims of this pilot study were to assess VEGF in blood compartments of 16 healthy control volunteers and 56 gastrointestinal cancer patients, prospectively collected, to identify the most suitable blood fraction for the determination of VEGF and to evaluate its possible clinical-biological significance. Samples of serum (S) and plasma (P) in both sodium citrate (SC) and sodium citrate-theophylline-adenosine-dipyridamole (CTAD) were collected from venous blood. After the centrifugation and separation methods VEGF levels were detected by ELISA in: S, plasma-platelets poor (P-PP), plasma-activated platelets rich (P-APR) and blood-lysed whole (B-LW). The best differentiation between healthy control volunteers and cancer patients in VEGF level was seen for P-APRCTAD (mean value: 278 pg/ml vs 77 pg/ml; p=0.0036 by t-test). No significant correlation among the blood fractions of VEGF analysed and clinical-pathological features was found. Our data suggest that P-APRCTAD blood fraction, obtained according to well standardised conditions, could represent the most suitable compartment for the assessment of VEGF. We suggest that VEGF levels in P-APRCTAD could play a role as an angiogenic marker of malignant gastrointestinal transformation. Further studies on a larger series of patients and healthy controls with the same experimental methodological conditions are required to confirm our preliminary conclusions.