HCMV IE expression in breast cancer tissue and clinical prognostic markers.

<p>Expression of estrogen (A) and progesterone (B) receptor and Elston grading from breast cancer tissue of the SLN-negative and SLN-positive group are shown and are plotted in relation to HCMV IE grading (B, D and F) respectively.</p

Patient characteristics.

<p>Patient characteristics.</p

Additional file 2: of Exome sequencing of primary breast cancers with paired metastatic lesions reveals metastasis-enriched mutations in the A-kinase anchoring protein family (AKAPs)

Table S2. DNA concentrations. Complete list of DNA concentrations after extraction from tumor- or normal tissues and blood. For Cohort 1 total input amounts to WGA and recovered DNA amounts after is listed. (PDF 248 kb

Additional file 1: of Exome sequencing of primary breast cancers with paired metastatic lesions reveals metastasis-enriched mutations in the A-kinase anchoring protein family (AKAPs)

Table S1. Patient table cohort 2. The study material includes 20 patients and was collected at Karolinska University Hospital between the years 2000 and 2011. The following inclusion criteria were applied; metastatic adenocarcinoma; detailed clinical data available; axillary and distant metastasis available; required amount of paraffin embedded tissue. The study was approved by the Ethics committee at the Karolinska Institute. (PDF 637 kb

Additional file 3: of Exome sequencing of primary breast cancers with paired metastatic lesions reveals metastasis-enriched mutations in the A-kinase anchoring protein family (AKAPs)

Figure S2. Mean coverage of whole-exome sequencing in a) cohort 1 and b) cohort 2. b) Exome capture and sequencing was performed by SeqWright Genomic Services (GE Healthcare, Houston, USA). Briefly, exome capture was performed using Sure Select XT2 Human All ExonV5 (Agilent Technologies) according to manufacturers instructions. Paired end sequencing was performed on Illumina HiSeq 2500. Raw sequencing reads were quality and adapter trimmed using trim_galore, where the first 13 bases of Illumina adapter were used and stringency parameter was set to 2. Reads having lengths less than 70 after trimming were filtered out with their paired mates. Trimmed reads were aligned to the human reference genome build hg19 using bwa-mem with default parameters. The aligned reads were marked for duplicates by Picard, realigned around known indels and base-quality recalibrated by GATK. Somatic single nucleotide variants (SNVs) were detected by Mutect with the high-confidence mode. Somatic short indels were detected by Varscan2 with minimum variant allele frequency of 0.05. Copy number alterations were detected by ADTEx/Ascat. All pipelines and analyses were run using Anduril, a workflow framework for scientific data analysis. (PDF 915 kb

Immunohistochemical analysis of breast cancer and SLN samples.

<p>The positivity of HCMV was graded into four grades based on the estimated percentage of HCMV IE positive cells in tumors from breast and paired SLN: Grade I (<25% cells positive for HCMV IE), grade II (25–49%), grade III (50–75%) and grade IV (>75%), *n = number.</p

HCMV IE grade in cancer cells from breast and SLN, and in inflammatory cells from SLN samples.

<p>Patients were divided into 2 groups based on the presence or absence of SLN metastasis in both breast (A) and SLN (B and C) specimens. The positivity of HCMV was graded into four grades based on the percentage of HCMV IE positive cells in tumors from breast and paired SLN: Grade I (<25% cells positive for HCMV IE), grade II (25–49%), grade III (50–75%) and grade IV (>75%). The presence of HCMV IE-infected inflammatory cells in SLN specimen is shown in (C).</p

Additional file 8: of Exome sequencing of primary breast cancers with paired metastatic lesions reveals metastasis-enriched mutations in the A-kinase anchoring protein family (AKAPs)

Figure S5. AKAP gene expression. Boxplots showing summed expression of AKAP 8,7,3,1 and AKAPs 5,11,9,10,12 gene expression within each PAM50 molecular subgroup, a-b; TCGA data, c-d; risk cohort, d-e; cohort 1. P-values are indicative of ANOVA followed by post-hoc Tukey for Basal vs. other subtypes individually. ***; p ≤ 0.001, **; p ≤ 0.01. (PDF 586 kb

Immunohistochemistry of breast cancer and SLN samples.

<p>HCMV proteins were detected in the tissue sections from breast cancer patients (A–D) and tissue sections from sentinel lymph node (G–J) by immunohistochemistry. HCMV IE expression is confined to tumor cells both in breast and SLN specimens (A and G 20x; B and H 40x). (C, D, I, and J), same sections show immunoreactivity to HCMV LA protein (C, I 20x and D, J 40X). Cytokeratin was used as an epithelial marker and positive control (E, K) and omitting primary antibody was used as a negative control (F and L, 20x). Scale bars: (B, D, H, J, 100 <b>µ</b>m), (Others 80 <b>µ</b>m).</p

Additional file 5: of Exome sequencing of primary breast cancers with paired metastatic lesions reveals metastasis-enriched mutations in the A-kinase anchoring protein family (AKAPs)

Figures S3a and S3b. Estimation of LOH fraction and tumor content. Estimation of LOH fraction and tumor content using a Beta-Normal mixture model. Histograms show the distribution of major allele frequencies (range: 50–100%) and red curves show the mixture model estimated from the data. The set of SNPs called in the germline sample was considered in the tumor sample (primary and metastasis independently). When genomes contain regions of LOH, the distribution will be bimodal, as illustrated in the inset (top right). The first component (closer to 50%; red in inset) represents heterozygous SNPs in regions without LOH and was modeled as a normal distribution with a mean close to 50% in a perfect sample, but will increase towards 100% as allelic dropout increases. The second component (closer to 100%; blue in inset) represents homozygous SNPs in regions with LOH, was modeled as a beta distribution with two parameters (the mean of this component should be close to 100% and increase as LOH fraction increases and decrease as with non-cancer cell contamination. The mixture distribution thus had four free parameters plus the mixture proportion, the latter representing the estimated LOH fraction of the sample. Fitting this mixture to the observations yielded estimates for LOH fraction, tumor fraction and allelic dropout rate for each sample (Additional file 3: Figure S2). Model fitting was performed using the EstimatedDistribution function of Mathematica 9.0 (Wolfram Research Inc.). (ZIP 421 kb