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Additional file 2. Study Protocol.

Additional file 4: Table S1. Baseline characteristics. Table S2a. 6 most common serious adverse event (grade 2 or more). Table S2b. 8 most common adverse event (any grade). Table S3. Response to neoadjuvant epirubicin and docetaxel in stage II-III breast cancers, and outcome at surgery. Table S4. Treatment response. Table S5a. Pretreatment mutations and prediction to epirubicin treatment. Table S5b. Pretreatment mutations and prediction to docetaxel treatment. Table S6. Mutations in pretreatment biopsies and prediction to epirubicin treatment. Table S7. Mutations in post-epirubicin biopsies and prediction to docetaxel treatment.

Additional file 3: Figure S1. CONSORT diagram for the Dose-Dense trial. Figure S2. Survival in the Dose-Dense trial after a minimum follow-up of five years or until death. Figure S3. Average sequencing coverage for each sample subject to whole exome sequencing (WES). Figure S4. Average sequencing coverage for each pretreatment tumor sample subject to amplicon-based sequencing. Figure S5. Oncoplot depicting the frequency of mutations in tumor samples undergoing whole exome sequencing (WES). Figure S6. Merged overview of copy number alterations (CNAs) in the sample set. Figure S7. Scatter plot showing Pearson correlation between copy number alterations (CNAs) and tumor mutation burden. Figure S8. Estimated contribution of mutational processes for each of the patients according to the classification by COSMIC. Figure S9. Graphical representation of clonal evolution from the pretreatment setting to post-epirubicin and post-docetaxel. Figure S10. Circos plots, coxcomb plots and heatmaps illustrating copy number logRs and mutation rVAFs pretreatment, post-epirubicin and post-docetaxel.

Additional file 1. The Dose Dense Trial (Methods and Results) and Sequencing approaches.

Additional file 5: Supplementary Table S3. Genes with >99 confidence level difference in methylation ratio between a minority (one third or less) of samples versus the majority.

Additional file 2: Supplementary information – workflow

Additional file 3: Supplementary Table S1. Pan-cancer panel of 283 tumor suppressor genes for which promoters are included in methylation analyses. The panel was generated based on CGPv2/3-panels [1], Roche’s Comprehensive Cancer Design along with manual literature search.

Additional file 5: Supplementary Table S3. Genes with >99 confidence level difference in methylation ratio between a minority (one third or less) of samples versus the majority.

Additional file 1: Supplementary Figure 1. Fraction of methylated alleles in promoter region of selected tumour suppressor genes. (A) Regions with high methylation levels across samples from all 34 healthy individuals. (B) Regions with low methylation levels across the same samples. Note the different scale on the Y-axis for panel A and B. Data for AIP were lacking for samples 32, 33, 34 due to low coverage (see details in Materials and methods). Supplementary Figure 2. Plot examplifying consistent high and low methylated CpGs in the same promoter, across patients. Fraction of methylated alleles across CpGs in the promoter region of RB1 in the two samples S7 and S24 are displayed. These two samples were selected because they were the one with highest and lowest overall methylation across the 283 investigated tumour suppressor genes, respectively (ref. Supplementary figure 3), and as such should represent the extremes. Still within the RB1 promoter, they reveal a very similar pattern of some CpGs being highly methylated, while others are hardly methylated at all. Supplementary Figure 3. Distribution of overall average methylation across 283 tumour suppressor gene promoters in 34 healthy individuals. (A) Bars indicate the average fraction of methylated alleles for all CpGs covered per patient. Dotted red lines indicate the upper and lower border of the 95% confidence interval for the average values per patient (CI for individual observations). Sample S24 falls below the lower border of the CI, indicating general hypo-methylation. Samples S4, S8 and S7 fall above the upper border of the CI, indicating general hyper-methylation. (B) Q-Q plot based on the same data as displayed in (A). S24 is encircled in green, while S4, S8 and S7 are encircled in red.

Background The tumor suppressor gene p53 (TP53) controls numerous signaling pathways and is frequently mutated in human cancers. Novel p53 isoforms suggest alternative splicing as a regulatory feature of p53 activity. Results In this study we have analyzed mRNA expression of both wild-type and mutated p53 and its respective Δp53 isoform in 88 tumor samples from breast cancer in relation to clinical parameters and molecular subgroups. Three-dimensional structure differences for the novel internally deleted p53 isoform Δp53 have been predicted. We confirmed the expression of Δp53 mRNA in tumors using quantitative real-time PCR technique. The mRNA expression levels of the two isoforms were strongly correlated in both wild-type and p53-mutated tumors, with the level of the Δp53 isoform being approximately 1/3 of that of the full-length p53 mRNA. Patients expressing mutated full-length p53 and non-mutated (wild-type) Δp53, "mutational hybrids", showed a slightly higher frequency of patients with distant metastasis at time of diagnosis compared to other patients with p53 mutations, but otherwise did not differ significantly in any other clinical parameter. Interestingly, the p53 wild-type tumors showed a wide range of mRNA expression of both p53 isoforms. Tumors with mRNA expression levels in the upper or lower quartile were significantly associated with grade and molecular subtypes. In tumors with missense or in frame mutations the mRNA expression levels of both isoforms were significantly elevated, and in tumors with nonsense, frame shift or splice mutations the mRNA levels were significantly reduced compared to those expressing wild-type p53. Conclusion Expression of p53 is accompanied by the functionally different isoform Δp53 at the mRNA level in cell lines and human breast tumors. Investigations of "mutational hybrid" patients highlighted that wild-type Δp53 does not compensates for mutated p53, but rather may be associated with a worse prognosis. In tumors, both isoforms show strong correlations in different mutation-dependent mRNA expression patterns.

BACKGROUND: Tamoxifen, taken for five years, is the standard adjuvant treatment for postmenopausal women with primary, estrogen-receptor-positive breast cancer. Despite this treatment, however, some patients have a relapse. METHODS: We conducted a double-blind, randomized trial to test whether, after two to three years of tamoxifen therapy, switching to exemestane was more effective than continuing tamoxifen therapy for the remainder of the five years of treatment. The primary end point was disease-free survival. RESULTS: Of the 4742 patients enrolled, 2362 were randomly assigned to switch to exemestane, and 2380 to continue to receive tamoxifen. After a median follow-up of 30.6 months, 449 first events (local or metastatic recurrence, contralateral breast cancer, or death) were reported--183 in the exemestane group and 266 in the tamoxifen group. The unadjusted hazard ratio in the exemestane group as compared with the tamoxifen group was 0.68 (95 percent confidence interval, 0.56 to 0.82; P