Bax expression in untreated breast cancer: an immunocytometric study of 255 cases.

BACKGROUND: Bax is one of the main effectors of apoptosis in breast cancer. However, in contrast with the antiapoptotic protein Bcl-2, which has been extensively studied in this tumor, there are relatively few clinical studies on the biological role of Bax in breast cancer. MATERIALS AND METHODS: The expression of the apoptosis-related Bax gene was studied in a series of 255 previously untreated breast cancers by means of immuno-flow cytometry. Additionally, and by the same method, the expression of the Bcl-2, VEGF and Nup88 genes were also studied. As variables of the study for the final statistical analysis, the histological variety of the tumors, histological and nuclear grade, the expression of hormone receptors, p53, Ki-67 or c-erb-B2, axillary node invasion, tumor size and DNA-ploidy were also included. RESULTS: The expression of the proapoptotic Bax protein was significantly associated with the expression of Nup88 (p<0.0001), VEGF (p=0.0014) and Bcl-2 (p=0.0063), all measured by the same method. An inverse correlation with c-erb-B2 expression, which almost attained statistical significance (p=0.058) was also registered. CONCLUSION: This study adds evidence to the little explored link between apoptosis and angiogenesis. Furthermore, it discloses a previously unreported relationship between Bax and Nup88 expression.

The small variant of the apoptosis-associated X-chromosome RBM10 gene is co-expressed with caspase-3 in breast cancer.

BACKGROUND: There are very few studies on the final triggers of apoptosis, the caspases, in breast cancer. MATERIALS AND METHODS: Caspase-3 expression was studied by means of reverse transcript polymerase chain reaction (RT-PCR) in a series of 108 previously untreated patients with breast cancer. Expression levels were correlated with those obtained using the same technique of the apoptosis-associated X-chromosome genes RBMX, RBM3, RBM10 small and RBM10 large variant; Bcl-2 and Bax; the angiogenesis-associated genes VEGF and CD105 (endoglin); hMAM and Nup88. The correlation with the expression of hormone receptors, c-erb-B2, mutant p53 and Ki-67, all measured by means of immunohistochemistry, was also studied, as well as that with standard clinical parameters such as histological type, tumor size, axillary metastasis and DNA-ploidy. RESULTS: The only statistically significant correlations observed between caspase-3 mRNA expression and the parameters tested were a direct one with both the Bax (p = 0.007) and the small variant of the X-chromosome RBM10 gene (p = 0.018), and an inverse one with the angiogenesis-associated CD105 (endoglin) gene (p = 0.044). CONCLUSION: These results indicate that very few genes are involved in the last steps of the apoptotic cascade in breast cancer, among them one of the X-chromosome RBM family. They also support the relatively unexplored link between apoptosis and angiogenesis.

The S-phase fraction of the aneuploid cell subpopulation is the biologically relevant one in aneuploid breast cancers.

BACKGROUND: In the case of DNA-aneuploid tumors there are no clear guidelines as to which S-phase fraction is the more relevant one: that corresponding to either the diploid or the aneuploid population, or rather an average of both. MATERIALS AND METHODS: We studied 280 breast cancer specimens from previously untreated patients. Histologically, 231 were ductal infiltrating carcinomas, 30 lobular infiltrating carcinomas and 19 corresponded to other, less frequent varieties. Postsurgically, 164 cases (58.6%) were classified as T1, 87 (31.1%) as T2 and 7 as T3. The remaining 22 cases were multifocal, diffuse tumors. Flow cytometry was performed on fresh tumor tissue, and immunohistochemistry for hormone receptors, Ki67, c-erb-B2 and p53 on paraffin-embedded material. RESULTS: In diploid tumors, a high S-phase (above the 75th percentile) correlated significantly with Ki67 expression > or =20% (p<0.0001). In aneuploid tumors, however, this was only the case for the aneuploid fraction of tumor cells (p< 0.0001). A high S-phase of diploid tumors correlated directly and significantly with a high histologic grade (p=0.04), a high nuclear grade (p=0.01), tumor size (p=0.0008), and inversely with estrogen (p<0.0001) and progesterone (p<0.0001) receptor expression. In aneuploid tumors, the aneuploid tumor fraction showed a direct and significant correlation with a high histologic grade (p=0.005), a high nuclear grade (p=0.001), mutant p53 expression (p=0.0009), and inversely with estrogen (p<0.0001) and progesterone (p=0.0001) receptor expression. A high S-phase of the diploid cell fraction of aneuploid tumors, on the other hand, just showed an inverse correlation with high nuclear grade of the tumors (p=0.02), and none whatsoever with all other tested parameters.

Down-regulation of BRMS1 mRNA Expression in Breast Cancer is not Related to the Presence of Axillary Node Metastasis.

BACKGROUND: BRMS1, recently identified in MDA-MB-435 breast cancer cells, seems to act as a potent antimetastatic gene in experimental tumor models. MATERIALS AND METHODS: To verify if BRMS1 exerts its action in a similar way in clinical tumors, BRMS1-mRNA expression was investigated in a series of 107 human breast carcinomas and correlated with the presence or not of axillary node metastases. Additionally, BRMS1 expression was correlated with all available clinical (histologic variety, histologic and nuclear grade) and biological parameters (ploidy, expression of Ki67, hormone receptors, c-erb-B2 and p53), as well as with the expression of hMAM- and Nup88-mRNA, previously shown by us to correlate with very low and very high aggressiveness of breast cancer, respectively. RESULTS: Down-regulation of BRMS1 expression in the tumors did not correlate with the presence of axillary node metastases. Furthermore, BRMS1 expression did not correlate with any other of the studied clinical or biological tumor parameters. CONCLUSION: In clinical breast cancers, down-regulation of BRMS1 expression seems not to mimic the very clear-cut antimetatstatic properties displayed in experimental tumor models.

The X-chromosome RBMX Gene is Expressed in Mammary Carcinoma.

BACKGROUND: A gene located on the q11.23 region of the male chromosome, RBMY, which plays a role in spermatogenesis, is down-regulated in testicular cancer. RBMY is a diverged X-Y shared gene. The corresponding X chromosome gene, RBMX, is located on Xq26. MATERIALS AND METHODS: We studied fresh tissues from 122 infiltrating breast cancers (99 ductal infiltrating, 19 lobular infiltrating and 4 tubular carcinomas) for the expression of RBMX by means of differential RT-PCR (reverse transcription-polymerase chain reaction), using beta-actin as an internal control and normalization standard. The obtained results were compared with all available clinical and molecular data of the studied tumors (estrogen and progesterone receptors (ER & PR), c-erb-B2, p53, Ki67, DNA-ploidy, Bcl-2, VEGF, CD105 (endoglin), histologic variety, histologic and nuclear grade and axillary node invasion). RESULTS: RBMX RT-PCR was successful in 120/122 instances. All 120 cases expressed RBMX. The only significant correlation found between RBMX expression and all the variables tested was an inverse one with CD105 (endoglin) mRNA-expression (r=-3063; p=0.0007). CONCLUSION: The X-chromosome RBMX gene is expressed in all breast cancers. The expression is inversely correlated with the expression of the angiogenesis-associated CD105 (endoglin) gene. The precise meaning of this association has still to be elucidated.

Elevated mammaglobin (h-MAM) expression in breast cancer is associated with clinical and biological features defining a less aggressive tumour phenotype.

BACKGROUND: Mammaglobin (h-MAM) is expressed mainly by breast epithelial cells, and this feature has been used to detect circulating breast cancer cells and occult metastases in sentinel axillary lymph nodes of breast cancer patients. However, the biological role of mammaglobin is completely unknown. METHODS: We studied 128 fresh-frozen breast cancer specimens by means of reverse transcriptase-polymerase chain reaction and quantified their h-MAM mRNA expression. This was then correlated with histological and nuclear grade, oestrogen and progesterone receptor expression, c-erb-B2 and mutant p53 expression, as well as with cellular proliferation measured by means of the Ki67 labelling index, DNA ploidy and S-phase, and finally with the presence or not of invaded axillary nodes in the mastectomy specimen. RESULTS: In the univariate analysis, high h-MAM expression (above the median for the whole group) correlated significantly (P < 0.05) with oestrogen and progesterone receptor expression, diploid DNA content, low Ki67 labelling index, low nuclear grade and almost significantly (P = 0.058) with the absence of axillary nodal invasion in the mastectomy specimen. In a final, multivariate model, only progesterone receptor expression, diploid DNA content and absence of nodal invasion were found to be independently associated with high h-MAM expression. CONCLUSION: All of the features associated with mammaglobin expression reflect, without exception, a less aggressive tumour phenotype. Further studies are needed to clarify whether this is attributable to h-MAM expression itself, or to another mechanism of which mammaglobin expression forms part.

Immunofluorometric study of Bcl-2 and Bax expression in clinical fresh tumor samples from breast cancer patients.

BACKGROUND: The balance between the expression of the antiapoptotic gene Bcl-2 and the proapoptotic gene Bax is considered a good indicator of the apoptotic activity of tumor cells. Bcl-2 and Bax expression seem also to individually play a prognostic role in breast cancer. Our aim was to study the expression of both genes in fresh breast cancer samples, and to correlate the obtained results with other available clinical and biological parameters of the tumors. MATERIALS AND METHODS: Fresh tumor specimens from 86 breast cancer patients were studied by means of immunofluorocytometry for the expression of the apoptosis-associated Bcl-2 and Bax genes. Additionally, DNA-ploidy was also measured. Paraffin blocks corresponding to the same tumors were used for immunohistochemistry, to study the expression of hormone receptors (ER and PR), p53, c-erb-B2 and the Ki67 labelling index. Fourteen patients had been treated with four cycles of induction chemotherapy (cyclo-phosphamide, adriamycin and 5-fluorouracil), and separate statistical analyses were carried out both for the whole group, and for the 62 patients not having received any treatment whatsoever, in order to exclude any potential influence of the chemotherapeutic treatment on the expression of the studied antigens. RESULTS: Bcl-2 expression correlated significantly with estrogen (p = 0.002) and progesterone (p = 0.012) receptor expression, as well as with c-erb-B2 (p = 0.045) expression in chemotherapy-naïve tumors, the correlation being completely lost if treated tumors were added to the study group. A high apoptotic index (Bcl2/Bax < 0.5) correlated significantly with progesterone receptor expression (p = 0.037) and c-erb-B2 expression (p = 0.018), and this correlation was maintained, whether previously treated tumors were included into the study or not (p = 0.038 and p = 0.027, respectively). Bax expression did not correlate with any other clinical or biological parameters of the tumors, including Bcl-2 expression. CONCLUSION: Bcl-2 and Bax-expression can be easily determined in clinical breast cancer specimens by means of immunofluorocytometry. Bcl-2-expression significantly correlates with hormone-receptor- and c-erb-B2-expression exclusively in previously untreated tumors. This, however, seems only to be the case when considering Bcl-2 expression in isolation, since a high apoptotic index, which considers the ratio of Bcl-2 versus Bax expression in the same tumor, seems not to be affected by the previous use of chemotherapy.

Flow cytometry vs. Ki67 labelling index in breast cancer: a prospective evaluation of 181 cases.

BACKGROUND: Excessive proliferation is one of the first steps in oncogenic activation and one of the most important biological features defining the aggressiveness of tumors. Quantifying the proportion of tumor cells in S-phase by means of flow cytometry has shown, in the past, to be useful for defining high-risk subgroups in breast cancer. Several antigens closely associated with proliferation are also detectable by means of immunohistochemistry, offering in theory an easy to perform and cheap alternative to flow cytometry for measuring proliferation. To test this hypothesis, we compared both methods prospectively in a series of breast cancers. MATERIALS AND METHODS: We studied the proliferation rate of 181 breast cancers (152 ductal infiltrating, 17 lobular infiltrating, 12 other histological varieties), operated upon at our institution, by means of flow cytometry and the Ki67 labelling index, using the MIBI antibody. Ploidy (expressed as DNA content or DNA-index), S-phase fraction and the Ki67 labelling index were the variables of the study. The S-phase fraction was considered separately for diploid and aneuploid tumors, following the 1992 Maine Consensus guidelines and was judged abnormally elevated if higher than the 75th percentile for each group. The Ki67 labelling index was expressed as percent positive tumor cells, positive cells being those showing specific nuclear staining. RESULTS: DNA-ploidy and the Ki67 labelling index could be evaluated in all tumors. Of the total, 96 (53%) were diploid and 85 (47%) aneuploid. S-phase fraction could be measured in 172 out of the 181 tumors (95%). The 75th percentile cut-offs for diploid and aneuploid tumors were 9.9% and 15.8%, respectively. We found a significant correlation beween rising DNA content and increasing Ki67 index (r = 0.18; p = 0.022), as well as between the percentage of cells in S-phase of the whole tumor population and Ki67 (r = 0.22; p = 0.0055). A Ki67 cut-off of 50% or higher identified most aneuploid tumors, or a small group of diploid ones with a high S-phase fraction (specificity = 96.7%; positive predictive value 92.5%), however at the price of a very low sensitivity (62.6%). This was due to the presence of many aneuploid tumors with a low S-phase fraction. CONCLUSION: The Ki67 labelling index and S-phase fraction are significantly correlated. However, flow cytometry provides additional indirect information on tumor aggressiveness associated with DNA-ploidy. Further studies are needed to determine whether Ki67 alone is sufficient as a routinely applicable method.

Diferencias bioquímicas entre los quistes de mama benignos tipo II únicos y asociados a tipo I / Biochemical differences between single benign type ii breast cysts and those associated with type ii breast cysts

Introducción. Los quistes mamarios tipo II se caracterizan por presentar en el líquido altas concentraciones de Na+, albúmina, pH y cloruros (Cl-) y parecen corresponder al estadio más avanzado de la enfermedad macroquística. En el presente estudio hemos querido analizar la posible influencia de la coexistencia de quistes tipo I, reflejo de la actividad de la enfermedad, sobre las características bioquímicas de los quistes tipo II. Pacientes y métodos. El grupo estudio incluyó 124 líquidos de quistes tipo II (Na+/K+ > 1,5), de los cuales 72 fueron únicos y 52 asociados a quistes tipo I. En ellos determinamos las concentraciones de Na+, K+, Cl-, glucosa, albúmina, pH y volumen. Resultados. Los quistes tipo II únicos presentaron mayores valores de pH (p = 0,0306) e índice Na+K+ (p = 0,0205), así como menores de K+ (p = 0,0313) y volumen (p = 0,0014). No se constataron diferencias entre ambos grupos en las mujeres en fase folicular, pero sí en las de fase luteínica y menopáusicas. Cuando el dintel clasificador de los quistes fue establecido en una relación Na+/K+ > 3, observamos que los quistes tipo II únicos presentaban valores mayores de pH y menor volumen. Conclusiones. Los resultados anteriores indican que los quistes tipo II únicos presentan unas características bioquímicas distintas de cuando se asocian con quistes tipo I, de tal modo que en esta situación adquieren ciertas propiedades de estos últimos, lo cual puede ser el exponente de una fase más activa de la enfermedad (AU)

Estudio de las concentraciones del antígeno prostático específico en secreciones memerias benignas / Prostate-specific antigen in nipple discharge due to non-malignant disease

Introducción. El antígeno prostático específico (PSA) es una serinproteasa que puede ser detectada en las secreciones mamarias. Su comportamiento parece ser opuesto al de otros marcadores tumorales y puede ayudar a establecer el diagnóstico de carcinomas mamarios. Hemos realizado el presente trabajo con objeto de estudiar su comportamiento en secreciones mamarias no malignas. Pacientes y métodos. Hemos determinado, mediante un fluoroinmunoanálisis (EG&G Wallac, Finlandia) las concentraciones de PSA en 79 secreciones mamarias, correspondiendo a 67 ectasias ductales, 5 mastitis y 7 papilomas intraductales. Resultados. En las ectasias ductales los valores de PSA estuvieron comprendidos entre 0,1 y 89,9 ng/ml (mediana 6,0) y fueron mayores (p = 0,085) en la fase luteínica (intervalo: 0,189,9; mediana 9,2) que en la folicular (intervalo: 0,1-81 ng/ml; mediana 5,8). En las mastitis las cifras de PSA estuvieron comprendidas entre 0,1 y 375 ng/ml (mediana 33), mientras que en los papilomas intraductales oscilaron entre 0,1 y 23 ng/ml (mediana 0,1). Observamos sólo diferencias significativas (p = 0,010) entre las concentraciones del PSA de las ectasias y papilomas. Cifras de PSA superiores a 1 ng/ml se constataron en 48 de las 67 ectasias, 4 de las 5 mastitis y 2 de los 7 papilomas. Conclusiones. Los resultados anteriores nos sugieren que es posible detectar altas concentraciones de PSA en las secreciones mamarias por procesos no malignos mamarios, principalmente ectasias y mastitis, mientras que en los papilomas intraductales las concentraciones antigénicas fueron mucho menores. Asimismo, nuestros resultados apoyan la posible utilidad del PSA en las secreciones mamarias como indicador de riesgo de cáncer de mama tal como han sugerido ciertos grupos (AU)

[Carcinoembryonic antigen levels (CEA) in 67 breast secretions caused by mammary duct ectasia]. / Estudio de las concentraciones del antígeno carcinoembrionario (CEA) en 67 secreciones mamarias por ectasias ductales mamarias.

In order to study the behavior of the carcinoembryonic antigen (CAE), we assayed the concentrations of CEA in 67 nipple discharges of women with duct ectasia of the breast by means of a fluoroimmunoassay (EG & G. Wallac. Finland). The CEA concentrations ranged from 0.1 to 854 ng/ml (median 38.7), these being higher (p: 0.065) in the luteal phase (34 cases; range: 5.0-854, median 61.7) than in follicular phase of the menstrual cycle (29 cases; range: 0.1-452; median 30.5). Concentrations greater than 400 ng/ml were observed in two cases (2.98%) and none of the cases showed CEA values higher than 1000 ng/ml. Our results suggest that nipple discharges by ductal ectasia of the breast can be associated with increases in CEA, which can be explained by its own physiopathology, although it rarely (3%) exceeded 400 ng/ml.

Estudio de las concentraciones del antígeno carcinoembrionario (CEA) en 67 secreciones mamarias por ectasias ductales mamarias / Study of the carcinoembryonic antigen (CEA) in 67 mammary secretions due to mammary ductal ectasias

Con objeto de estudiar su comportamiento, hemos determinado, mediante un fluoroinmunoanálisis (EG & G. Wallac. Finlandia), las concentraciones del antígeno carcinoembrionario (CEA) en 67 secreciones mamarias por ectasia ductal. Aquellas oscilaron entre 0,1 y 854 ng/ml (mediana 38,7) y fueron superiores (p. 0,065) en la fase luteínica (34 casos; i: 5,0-854, mediana 61,7) que en la folicular (29 casos; i: 0,1-451, mediana 30,5 ng/ml). Concentraciones > 400 ng/ml fueron constatadas en 2 casos (2,98 por ciento) y en ninguna ocasión se superaron los 1.000 ng/ml. Nuestros resultados nos sugieren que las secreciones mamarias por ectasia ductal pueden cursar con incrementos de CEA, hecho que se puede explicar por su propia fisiopatología, si bien raras veces (3 por ciento) se superaron los 400 ng/ml. (AU)