Somatic tumor mutations detected by targeted next generation sequencing in minute amounts of serum-derived cell-free DNA

The use of blood-circulating cell-free DNA (cfDNA) as 'liquid-biopsy' is explored worldwide, with hopes for its potential in providing prognostic or predictive information in cancer treatment. In exploring cfDNA, valuable repositories are biobanks containing material collected over time, however these retrospective cohorts have restrictive resources. In this study, we aimed to detect tumor-specific mutations in only minute amounts of serum-derived cfDNA by using a targeted next generation sequencing (NGS) approach. In a retrospective cohort of ten metastatic breast cancer patients, we profiled DNA from primary tumor tissue (frozen), tumor-adjacent normal tissue (formalin-fixed paraffin embedded), and three consecutive serum samples (frozen). Our presented workflow includes comparisons with matched normal DNA or in silico reference DNA to discriminate germline from somatic variants, validation of variants through the detection in at least two DNA samples of an individual, and the use of public databases on variants. By our workflow, we were able to detect a total of four variants traceable as circulating tumor DNA (ctDNA) in the sera of three of the ten patients

Cell-free DNA mutations as biomarkers in breast cancer patients receiving tamoxifen

The aim was to identify mutations in serum cell-free DNA (cfDNA) associated with disease progression on tamoxifen treatment in metastatic breast cancer (MBC). Sera available at start of therapy, during therapy and at disease progression were selected from 10 estrogen receptor (ER)-positive breast cancer patients. DNA from primary tumor and normal tissue and cfDNA from minute amounts of sera were analyzed by targeted next generation sequencing (NGS) of 45 genes (1,242 exons). At disease progression, stop-gain single nucleotide variants (SNVs) for CREBBP (1 patient) and SMAD4 (1 patient) and non-synonymous SNVs for AKAP9 (1 patient), PIK3CA (2 patients) and TP53 (2 patients) were found. Mutations in CREBBP and SMAD4 have only been occasionally reported in breast cancer. All mutations, except for AKAP9, were also present in the primary tumor but not detected in all blood specimens preceding progression. More sensitive detection by deeper re-sequencing and digital PCR confirmed the occurrence of circulating tumor DNA (ctDNA) and these biomarkers in blood specimens

Loss of Y-Chromosome during Male Breast Carcinogenesis

Loss of Y-chromosome (LOY) is associated with increased cancer mortality in males. The prevalence of LOY in male breast cancer (BC) is unknown. The aim of this study is to assess the presence and prognostic effect of LOY during male BC progression. We included male BC patients diagnosed between 1989 and 2009 (n = 796). A tissue microarray (TMA) was constructed to perform immunohistochemistry and fluorescent in situ hybridization (FISH), using an X and Y probe. We also performed this FISH on a selected number of patients using whole tissue slides to study LOY during progression from ductal carcinoma in situ (DCIS) to invasive BC. In total, LOY was present in 12.7% (n = 92) of cases, whereby LOY was associated with ER and PR negative tumors (p = 0.017 and p = 0.01). LOY was not associated with the outcome. Using whole slides including invasive BC and adjacent DCIS (n = 22), we detected a concordant LOY status between both components in 17 patients. In conclusion, LOY is an early event in male breast carcinogenesis, which generally starts at the DCIS stage and is associated with ER and PR negative tumors

Cell-free DNA mutations as biomarkers in breast cancer patients receiving tamoxifen

The aim was to identify mutations in serum cell-free DNA (cfDNA) associated with disease progression on tamoxifen treatment in metastatic breast cancer (MBC). Sera available at start of therapy, during therapy and at disease progression were selected from 10 estrogen receptor (ER)-positive breast cancer patients. DNA from primary tumor and normal tissue and cfDNA from minute amounts of sera were analyzed by targeted next generation sequencing (NGS) of 45 genes (1,242 exons). At disease progression, stop-gain single nucleotide variants (SNVs) for CREBBP (1 patient) and SMAD4 (1 patient) and non-synonymous SNVs for AKAP9 (1 patient), PIK3CA (2 patients) and TP53 (2 patients) were found. Mutations in CREBBP and SMAD4 have only been occasionally reported in breast cancer. All mutations, except for AKAP9, were also present in the primary tumor but not detected in all blood specimens preceding progression. More sensitive detection by deeper re-sequencing and digital PCR confirmed the occurrence of circulating tumor DNA (ctDNA) and these biomarkers in blood specimens

Expression and Localization of Ferritin-Heavy Chain Predicts Recurrence for Breast Cancer Patients with a <i>BRCA1/2</i> Mutation

The ferritin-heavy chain (FTH1) is the catalytic subunit of the ferroxidase ferritin, which prevents oxidative DNA damage via intracellular iron storage. FTH1 was shown to be a prognostic marker for triple-negative breast cancer (BC) patients and associated with an enrichment of CD8+ effector T cells. However, whether the expression and localization of FTH1 are also associated with clinical outcome in other BC subtypes is unknown. Here, we investigated the association of FTH1 with time to survival in BCs from 222 BRCA1/2 mutation carriers by immunohistochemistry on tissue microarrays. In addition, for 51 of these patients, the association between FTH1 and specific subsets of T cells was evaluated on whole slides using automatic scoring algorithms. We revealed that nuclear FTH1 (nFTH1) expression, in multivariable analyses, was associated with a shorter disease-free (HR = 2.71, 95% CI = 1.49–4.92, p = 0.001) and metastasis-free survival (HR = 3.54, 95% CI = 1.45–8.66, p = 0.006) in patients carrying a BRCA1/2 mutation. However, we found no relation between cytoplasmic FTH1 expression and survival of BRCA1/2 mutation carriers. Moreover, we did not detect an association between FTH1 expression and the amount of CD45+ (p = 0.13), CD8+ (p = 0.18), CD4+ (p = 0.20) or FOXP3+ cells (p = 0.17). Consequently, the mechanism underlying the worse recurrence-free survival of nFTH1 expression in BRCA1/2 mutation carriers needs further investigation.</p

Expression and Localization of Ferritin-Heavy Chain Predicts Recurrence for Breast Cancer Patients with a <i>BRCA1/2</i> Mutation

The ferritin-heavy chain (FTH1) is the catalytic subunit of the ferroxidase ferritin, which prevents oxidative DNA damage via intracellular iron storage. FTH1 was shown to be a prognostic marker for triple-negative breast cancer (BC) patients and associated with an enrichment of CD8+ effector T cells. However, whether the expression and localization of FTH1 are also associated with clinical outcome in other BC subtypes is unknown. Here, we investigated the association of FTH1 with time to survival in BCs from 222 BRCA1/2 mutation carriers by immunohistochemistry on tissue microarrays. In addition, for 51 of these patients, the association between FTH1 and specific subsets of T cells was evaluated on whole slides using automatic scoring algorithms. We revealed that nuclear FTH1 (nFTH1) expression, in multivariable analyses, was associated with a shorter disease-free (HR = 2.71, 95% CI = 1.49–4.92, p = 0.001) and metastasis-free survival (HR = 3.54, 95% CI = 1.45–8.66, p = 0.006) in patients carrying a BRCA1/2 mutation. However, we found no relation between cytoplasmic FTH1 expression and survival of BRCA1/2 mutation carriers. Moreover, we did not detect an association between FTH1 expression and the amount of CD45+ (p = 0.13), CD8+ (p = 0.18), CD4+ (p = 0.20) or FOXP3+ cells (p = 0.17). Consequently, the mechanism underlying the worse recurrence-free survival of nFTH1 expression in BRCA1/2 mutation carriers needs further investigation.</p

Prognostic significance of nuclear expression of UMP-CMP kinase in triple negative breast cancer patients

We have previously identified UMP-CMP kinase (CMPK1) as a prognostic marker for triple negative breast cancer (TNBC) by mass spectrometry (MS). In this study we evaluated CMPK1 association to prognosis in an independent set of samples by immunohistochemistry (IHC) and assessed biological pathways associated to its expression through gene set enrichment analysis (GSEA). A total of 461 TNBC paraffin-embedded tissues were collected from different academic hospitals in Europe, incorporated into tissue micro-arrays (TMA), and stained for CMPK1 expression. We also collected gene expression data of 60 samples, which were also present in the TMA, for GSEA correlation analysis. CMPK1 IHC staining showed both cytoplasmic and nuclear components. While cytoplasmic CMPK1 did not show any association to metastasis free survival (MFS), nuclear CMPK1 was associated to poor prognosis independently from other prognostic factors in stratified Cox regression analyses. GSEA correlation analysis of the nuclear CMPK1-stratified gene expression dataset showed a significant enrichment of extracellular matrix (ECM; positive correlation) and cell cycle (negative correlation) associated genes. We have shown here that nuclear CMPK1 is indicative of poor prognosis in TNBCs and that its expression may be related to dysregulation of ECM and cell cycle molecules

Metabotropic glutamate receptor 1 is associated with unfavorable prognosis in ER-negative and triple-negative breast cancer

New therapies are an urgent medical need in all breast cancer subgroups. Metabotropic glutamate receptor 1 (mGluR1) is suggested as a potential new molecular target. We

The Prevalence of CD146 Expression in Breast Cancer Subtypes and Its Relation to Outcome

CD146, involved in epithelial-to-mesenchymal transition (EMT), might affect cancer aggressiveness. We here investigated the prevalence of CD146 expression in breast cancer subtypes, its relation to prognosis, the relation between CD146 and EMT and the outcome to tamoxifen. Primary breast cancer tissues from 1342 patients were available for this retrospective study and immunohistochemically stained for CD146. For survival analyses, pure prognosis was studied by only including lymph-node negative patients who did not receive (neo)adjuvant systemic treatment (n = 551). 11% of the tumors showed CD146 expression. CD146 expression was most prevalent in triple-negative cases (64%, p < 0.001). In univariable analysis, CD146 expression was a prognostic factor for both metastasis-free survival (MFS) (p = 0.020) and overall survival (OS) (p = 0.037), but not in multivariable analysis (including age, tumor size, grade, estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2) and Ki-67). No correlation between CD146 and EMT nor difference in outcome to first-line tamoxifen was seen. In this large series, our data showed that CD146 is present in primary breast cancer and is a pure prognostic factor for MFS and OS in breast cancer patients. We did not see an association between CD146 expression and EMT nor on outcome to tamoxifen

SIAH2 protein expression in breast cancer is inversely related with ER status and outcome to tamoxifen therapy

Our previous study demonstrated that high mRNA levels for Seven in Absentia Homolog 2 (SIAH2) correlated with high Estrogen Receptor (ER) mRNA levels and with longer progression-free survival (PFS) after first-line tamoxifen. Others showed high SIAH2 protein levels in ER-negative breast cancer associated with an unfavorable relapse-free survival. In the current study, we investigated SIAH2 protein expression to clarify the discrepancy between protein and mRNA findings and to determine its diagnostic value in breast cancer patients. Tissue microarrays (TMAs) containing core specimens of primary breast tumors were immunohistochemically stained for SIAH2 protein. The TMAs analyzed a cohort of 746 patients with primary breast cancer (PBC) and a cohort of 245 patients with ER-positive metastatic breast cancer (MBC) treated with first-line tamoxifen. SIAH2 staining was scored for intensity and proportion of positive tumor cells and evaluated for its relationship with metastasis-free survival (MFS) and PFS. Multivariate survival analyses included traditional prognostic or predictive factors, respectively. The PBC-cohort had 263 patients with high SIAH2 protein expression and decreased expression of ER protein and mRNA levels (P = 0.005 and P = 0.003, respectively). High SIAH2 levels correlated with significant unfavorable MFS in lymph node negative, ER-positive breast cancer patients. The MBC-cohort had 86 patients with increased SIAH2 protein expression. High SIAH2 expression was associated with an unfavorable PFS after first-line tamoxifen in multivariate analyses (HR = 1.45; 95% CI, 1.07-1.96; P = 0.015). In conclusion, SIAH2 protein expression is especially observed in ER-negative tumors. Its prognostic value in breast cancer does not add to current prognostic markers. The proportion of SIAH2-positive cells can be used as biomarker to predict tamoxifen treatment failure in MBC patients