External validation of a prognostic model based on total tumor load of sentinel lymph node for early breast cancer patients

Background: A prognostic model based on the results of molecular analysis of sentinel lymph nodes (SLN) is needed to replace the information that staging the entire axilla provided. The aim of the study is to conduct an external validation of a previously developed model for the prediction of 5-year DFS in a group of breast cancer patients that had undergone SLN biopsy assessed by the One Step Nucleic Acid Amplification (OSNA) method. Methods: We collected retrospective data of 889 patients with breast cancer, who had not received systemic treatment before surgery, and who underwent SLN biopsy and evaluation of all SLN by OSNA. The discrimination ability of the model was assessed by the area under the ROC curve (AUC ROC), and its calibration by comparing 5-years DFS Kaplan–Meier estimates in quartile groups of model predicted probabilities (MPP). Results: The AUC ROC ranged from 0.78 (at 2 years) to 0.73 (at 5 years) in the training set, and from 0.78 to 0.71, respectively, in the validation set. The MPP allowed to distinguish four groups of patients with heterogeneous DFS (log-rank test p < 0.0001). In the highest risk group, the HR were 6.04 [95% CI 2.70, 13.48] in the training set and 4.79 [2.310, 9.93] in the validation set. Conclusions: The model for the prediction of 5-year DFS was successfully validated using the most stringent form of validation, in centers different from those involved in the development of the model. The external validation of the model confirms its utility for the prediction of 5-year DFS and the usefulness of the TTL value as a prognostic variable.This study was supported by Sysmex España S.L

Surgical treatment of breast cancer in young women

International audienc

Utilidad de la gammagrafía con Tc99m-Sestamibi de doble fase para el diagnóstico de localización de glándulas paratiroides patológicas en el hiperparotiroidismo primario / Antonio Piñero Madrona ; directores Pascual Parrilla Paricio, José Manuel Rodríguez González.

Tesis-Universidad de Murcia.MEDICINA ESPINARDO. DEPOSITO. MU-Tesis 529.Consulte la tesis en: BCA. GENERAL. ARCHIVO UNIVERSITARIO. T.M.-1570

Reactivation of the Tumour Suppressor <em>RASSF1A</em> in Breast Cancer by Simultaneous Targeting of DNA and E2F1 Methylation

<div><h3>Background</h3><p>Tumour suppressor genes are often transcriptionally silenced by promoter hypermethylation, and recent research has implicated alterations in chromatin structure as the mechanistic basis for this repression. In addition to DNA methylation, other epigenetic post-translational modifications that modulate the stability and binding of specific transcription factors to gene promoters have emerged as important mechanisms for controlling gene expression. The aim of this study was to analyse the implications of these mechanisms and their molecular connections in the reactivation of <em>RASSF1A</em> in breast cancer.</p> <h3>Methods</h3><p>Compounds that modulate the intracellular concentration of adenosine, such as dipyridamole (DIPY), greatly increase the antiproliferative effects of 3-<em>O</em>-(3,4,5-trimethoxybenzoyl)-(−)-catechin (TMCG), a synthetic antifolate derived from the structure of tea catechins. Quantitative real-time PCR arrays and MALDI-TOF mass spectrometry indicated that this combination (TMCG/DIPY) induced apoptosis in breast cancer cells by modulating the methylation levels of DNA and proteins (such as E2F1), respectively. Chromatin immunoprecipitation (ChIP) assays were employed to confirm that this combination induced chromatin remodelling of the <em>RASSF1A</em> promoter and increased the occupancy of E2F1 at the promoter of this tumour suppressor gene.</p> <h3>Results</h3><p>The TMCG/DIPY combination acted as an epigenetic treatment that reactivated <em>RASSF1A</em> expression and induced apoptosis in breast cancer cells. In addition to modulating DNA methylation and chromatin remodelling, this combination also induced demethylation of the E2F1 transcription factor. The ChIP assay showed enhancement of E2F1 occupancy at the unmethylated <em>RASSF1A</em> promoter after TMCG/DIPY treatment. Interestingly, inhibition of E2F1 demethylation using an irreversible inhibitor of lysine-specific demethylase 1 reduced both TMCG/DIPY-mediated <em>RASSF1A</em> expression and apoptosis in MDA-MB-231 cells, suggesting that DNA and protein demethylation may act together to control these molecular and cellular processes.</p> <h3>Conclusions/Significance</h3><p>This study demonstrates that simultaneous targeting of DNA and E2F1 methylation is an effective epigenetic treatment that reactivates <em>RASSF1A</em> expression and induces apoptosis in breast cancer cells.</p> </div

TMCG/DIPY modulates the posttranslational state of E2F1 and promotes its binding to the <i>RASSF1A</i> promoter.

<p>(A) Schematic representation of the E2F1 protein. Residues susceptible to methylation (K185), acetylation (K117, K120, and K125), and phosphorylation (S31 and S364) are shown. (B) MALDI-TOF mass spectra of tryptic digests of immunoprecipitated E2F1. Peptides were analysed in untreated MDA-MB-231 cells (control) or in those treated for 3 days with TMCG/DIPY (10 μM/5 μM). (C) <i>In vivo</i> binding of E2F1 to the RASSF1A promoter. ChIP assays using untreated MDA-MB-231 cells (control) or those treated for 3 days with TMCG/DIPY (10 μM/5 μM). The enrichment of the E2F1 protein on the <i>RASSF1A</i> promoter is shown. Immunoprecipitation (IP) using the anti-E2F1 or an IgG was performed in triplicate. The results are presented as a percentage of the input DNA. (D) The proposed mechanism for the regulation of E2F1. E2F1 is regulated by several posttranslational modifications, including methylation (Me), acetylation (Ac) and phosphorylation (P) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052231#pone.0052231-Kontaki1" target="_blank">[6]</a>. The effect of TMCG/DIPY (red dashed line) on the E2F1 status is shown. E2F1 is reversibly methylated by the enzymatic actions of LSD1 and Set9.</p

The TMCG/DIPY combination modulates gene promoter methylation in breast cancer cells and induces the expression of RASSF1A in MDA-MB-231 cells.

<p>(A) The methylation status of 15 gene promoters that have been reported to be altered in a variety of breast cancers as investigated by qRT-PCR array. Cells were treated for 3 days with vehicle (control) or with a combination of 10 μM TMCG and 5 μM DIPY (TMCG/DIPY). The MDA abbreviation indicates MDA-MB-231 cells. (B) Semiquantitative reverse transcription-PCR was used to detect RASSF1A (249 bp) and β-actin (142 bp) mRNA expression. Lane 1, DNA markers; Lane 2, expression in MCF10; Lane 3, expression in MDA-MB-231 cells treated for 3 days with vehicle; Lane 4, expression in MDA-MB-231 cells treated for 3 days with a combination of 10 μM TMCG and 5 μM DIPY. The data shown here are from a representative experiment repeated five times with similar results. (C) Immunohistochemical analysis of the expression of the RASSF1A protein in untreated MDA-MB-231 cells (control) and in cells treated for 3 days with a combination of 10 μM TMCG and 5 μM DIPY (TMCG/DIPY). The cells were stained with an anti-RASSF1A antibody (red) and DAPI (blue). Merged images are shown. (D) RASSF1A shRNA inhibits TMCG/DIPY-induced apoptosis in MDA-MB-231 cells. The effective silencing of RASSF1A was tested by confocal microscopy. shRNAs (HSS117376, HSS117377, and HSS174151) transfection significantly (<i>P</i><0.05) increased MDA-MB-231 cell growth (the data, monitored 4 days after transfection, are expressed relative to 100% growth of the untransfected/untreated control cells). For TMCG/DIPY treatment, siCN- and siRASSF1A-transfected cells were treated for 3 days with a combination of 10 μM TMCG and 5 μM DIPY. Apoptosis was determined by fluorescence microscopy after DNA staining with Hoechst 33342. Differences in apoptosis between siCN- and siRASSF1A-TMCG/DIPY treated cells were statistically significant (<i>P</i><0.05). Morphological changes before and after TMCG/DIPY treatments were visualised by bright field microscopy.</p

Inhibition of DNA methylation by TMCG/DIPY treatment induces chromatin remodelling in MDA-MB-231 breast cancer cells.

<p>(A) Semiquantitative determination of DNMT1 mRNA in breast cancer cells. The estimated relative levels of mRNA relative to that of β-actin were calculated and then compared to the expression levels in untreated controls. The histograms represent the number of copies of mRNA for every 1×10³ copies of β-actin. MCF7 and MDA-MB-231 cells were treated with vehicle (control) or a combination of 10 μM TMCG and 5 μM DIPY (TMCG/DIPY) for 3 days. There were no significant differences (<i>ns</i>) in DNMT1 mRNA expression between TMCG/DIPY-treated cells and untreated controls. (B) Genomic map of the <i>RASSF1A</i> CpG island. The positions of CpG sites in the genomic sequence are indicated by the thin vertical lines. Bent arrow: the position of the transcription start site. Potential transcription factor binding sites (MZF1, GATA2, AML-1a, and Sp1) resulting from the TFSEARCH query <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052231#pone.0052231-Yan1" target="_blank">[14]</a> are marked in the promoter region. The functional E2F binding site (ERE2, identified at site −150/-143 bp within the <i>RASSF1A</i> CpG island) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052231#pone.0052231-Chang1" target="_blank">[28]</a> is also indicated. The bottom row of horizontal lines indicates the locations of <i>RASSF1A</i> promoter fragments used for the ChIP assay (P1, P2, and P3). (C) ChIP assay of the <i>RASSF1A</i> CpG island. Chromatin DNA was immunoprecipitated with antibodies specific for acetyl-H4, MeCP2, HDAC1, and HDAC3. DNA fragments corresponding to <i>RASSF1A</i> promoter regions P1, P2, and P3 were amplified by PCR. The histograms represent PCR analyses of ChIP in MDA-MB-231 in the three regions before and after 3 days of treatment with TMCG/DIPY (10 μM/5 μM). PCR was performed on immunoprecipitated DNA and total input DNA. The corresponding signal intensity enrichment of each ChIP-PCR analysis for the three regions is plotted. The enrichment levels are derived from fold changes between the normalised <i>RASSF1A</i> intensity in each immunoprecipitated DNA and the normalised <i>RASSF1A</i> intensity in the input DNA. In all cases, the changes after TMCG/DIPY treatment were statistically significant (<i>P</i><0.05) compared with protein occupancies in untreated controls.</p

TMCG and DIPY act synergistically to induce apoptosis in breast cancer cells.

<p>(A) The left and central panels represent the effects of various DIPY concentrations on MDA-MB-231 and MCF7 cell growth, respectively, after 3 days of treatment in the absence (DIPY series; •) or the presence of 10 µM TMCG (TMCG/DIPY series; □). The right panel represents 4T1 cells that were treated for 3 days with vehicle only (DMSO; control), DIPY (5 µM), TMCG (10 µM) or a combination of the same concentration of DIPY plus TMCG. In all cases, the data are expressed relative to 100% growth or viability of the untreated control cells. *<i>P</i><0.05 compared with single TMCG and DIPY treatments. (B) Morphology of untreated breast cancer cells (control) compared with those subjected to 3 days of treatment with 10 μM TMCG and 5 μM DIPY (TMCG/DIPY). (C) The histograms represent the effects of DIPY and TMCG/DIPY treatment on apoptosis in MCF7 cells (*<i>P</i><0.05 relative to DIPY-treated cells). The dose-dependent effects after 3 days of treatment with DIPY were analysed using increasing concentrations of DIPY in the absence (DIPY series) or the presence of 10 μM TMCG (TMCG/DIPY series). (D) Apoptosis induction in MDA-MB-231 and 4T1 breast cancer cells after 3 days of treatment with vehicle (control), DIPY (5 μM), TMCG (10 μM) or a combination of the same concentration of DIPY plus TMCG was visualised by fluorescence microscopy of DNA stained with Hoechst 33342. The arrows indicate apoptotic cells. (E) MDA-MB-231 cells treated for 3 days with vehicle (control) or a combination of 10 µM TMCG and 5 µM DIPY (TMCG/DIPY) were examined for γH2AX nuclear foci (red). The nuclei were counterstained with DAPI (blue), and merged images are shown.</p

MALDI-TOF mass spectroscopy properties of immunoprecipitated E2F1 tryptic digests.

a<p>The characteristics peptides involving posttranslational modifications of E2F1 (methylation  =  Me, acetylation  =  Ac, and phosphorylation  = P), as well as their measured and theoretical <i>m/z</i> are shown; ^bPeptides were analyzed in untreated MDA-MB-231 cells (control), treated for 3-days with 10 μM TMCG plus 5 μM DIPY (TMCG/DIPY) or treated for 3-days with 10 μM TMCG plus 5 μM DIPY in the presence of 50 μM 2PCPA (TMCG/DIPY/2PCPA); ^cRelative intensities of specific tryptic peptides were normalized with respect to an internal matrix control.</p