Large Breast Cancer Cohort Research Articles

Postdiagnostic Calcium Channel Blocker Use and Breast Cancer Mortality: A Population-based Cohort Study.

There have long been concerns that calcium channel blockers (CCBs), widely used to treat hypertension, may contribute to malignant growth through the evasion of apoptosis and proliferation of cancer cells. Worryingly, a recent cohort study found breast cancer patients who used CCBs had higher death rates, but interpreting these results was difficult as they were based on all-cause mortality and medication use before cancer diagnosis. We used UK population-based data to more robustly investigate the association between CCB use and cancer-specific mortality. We selected a cohort of patients with breast cancer diagnosed between 1998 and 2012 from English cancer registries. We linked to prescription and clinical records from the Clinical Practice Research Datalink, and to death records from the Office for National Statistics. We used adjusted, time-dependent Cox regression models to calculate hazard ratios (HRs) comparing breast cancer-specific and all-cause mortality between postdiagnostic CCB users and nonusers. Our cohort included 23,669 breast cancer patients, of whom 5,141 used CCBs and 3,053 died due to their breast cancer during follow-up. After adjustment, CCB users had similar breast cancer-specific mortality to nonusers (HR = 0.98, 95% confidence interval [CI] = 0.88, 1.08). There was no evidence of a dose-response relationship. We found similar associations for specific CCBs, and for all-cause mortality. In this large population-based breast cancer cohort, we did not find any evidence that CCB use is associated with increased mortality.

Mediator complex (MED) 7: a biomarker associated with good prognosis in invasive breast cancer, especially ER+ luminal subtypes

BackgroundMediator complex (MED) proteins have a key role in transcriptional regulation, some interacting with the oestrogen receptor (ER). Interrogation of the METABRIC cohort suggested that MED7 may regulate lymphovascular invasion (LVI). Thus MED7 expression was assessed in large breast cancer (BC) cohorts to determine clinicopathological significance.MethodsMED7 gene expression was investigated in the METABRIC cohort (n = 1980) and externally validated using bc-GenExMiner v4.0. Immunohistochemical expression was assessed in the Nottingham primary BC series (n = 1280). Associations with clinicopathological variables and patient outcome were evaluated.ResultsHigh MED7 mRNA and protein expression was associated with good prognostic factors: low grade, smaller tumour size, good NPI, positive hormone receptor status (p < 0.001), and negative LVI (p = 0.04) status. Higher MED7 protein expression was associated with improved BC-specific survival within the whole cohort and ER+/luminal subgroup. Pooled MED7 gene expression data in the external validation cohort confirmed association with better survival, corroborating with the protein expression. On multivariate analysis, MED7 protein was independently predictive of longer BC-specific survival in the whole cohort and Luminal A subtype (p < 0.001).ConclusionsMED7 is an important prognostic marker in BC, particularly in ER+luminal subtypes, associated with improved survival and warrants future functional analysis.

Abstract P4-06-13: BRCA1/2 mutations identified by screening a large unselected breast cancer cohort in Sweden

Abstract Background Treatment options for BRCA1/2 breast cancer include new therapeutic agents, such as poly (ADP-ribose) polymerase (PARP) inhibitors, which selectively target BRCA defective cells. According to current Swedish screening guidelines, eligibility for clinical BRCA1/2 hereditary mutation testing is mainly based on family history of breast or ovarian cancer and early age onset. We aimed at examining the prevalence and characteristics of BRCA1/2 mutation carriers by screening a large unselected breast cancer cohort in Sweden, and comparing our results with BRCA mutation carriers already identified through the national BRCA testing program. Methods Germline DNA (blood) from 5122 women diagnosed with breast cancer between 2001-2008 (LIBRO1 study) were analysed for BRCA1/2 mutations by targeted sequencing (next generation sequencing, NGS), of which 5099 samples passed quality control. All patients provided informed consent. Information on patient and tumor characteristics was collected from the LIBRO1 database. Clinical BRCA testing information was obtained from the BRCA Lab (Lund University, Sweden), which carries out mutation screening for all oncogenetic clinics in Sweden. Multinomial logit models were used to compare tumor characteristics of BRCA1 and BRCA2 versus non-BRCA carriers. Multivariable logistic regression models were used to examine for differences between BRCA carriers identified through the national BRCA testing program and additional BRCA carriers found by sequencing the entire study population (not tested or not identified under current screening guidelines). Results In total, 92 (1.8%) BRCA1/2 mutation carriers were identified retrospectively by NGS. The prevalence of BRCA1/2 mutations was 1.6% (38/2363) between years 2001-2004; and 2.0% (54/2736) between years 2005-2008. After controlling for age and year of diagnosis, BRCA2 mutation carriers were in general similar to non-BRCA carriers regarding tumor characteristics (hormone receptor status, grade, tumor size and proliferation index), except for nodal involvement. BRCA1 mutation carriers, however, had more aggressive tumor characteristics than non-BRCA breast cancer patients. Overall, 55/92 BRCA1/2 mutation carriers (59.8%) found by NGS were not already identified through the national clinical BRCA testing program. The BRCA carriers identified by clinical testing were more likely high-risk individuals, i.e. younger, less likely to have experienced menopause, and more likely to be associated with a familiar ovarian cancer compared to those not identified through clinical testing, after adjusting for year of diagnosis. A larger proportion of BRCA2 (34/42, 80%) than BRCA1 mutations (25/50, 50%) were missed by selectively testing, mainly high-risk individuals. Conclusion BRCA1/2 mutations were found in approximately 2.0% of unselected BC patients. Six out of ten BRCA mutation carriers were not identified through the national testing program, which follows the screening guidelines. Revised guidelines might be needed for the effective identification of BRCA1/2 germline mutations. Citation Format: Li J, Wen SWX, Eriksson M, Kvist A, Christensen HN, Torstensson A, Easton DF, Teo S-H, Borg Å, Grönberg H, Czene K. BRCA1/2 mutations identified by screening a large unselected breast cancer cohort in Sweden [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P4-06-13.

Abstract P4-06-15: Targeted sequencing of BRCA1/2 across a large unselected breast cancer cohort in Sweden

Abstract Background Observed BRCA1/2 mutation frequencies can vary depending on the population screened, screening criteria, founder effects, and methods used for testing. We aimed at examining BRCA1/2 germline mutations, identified through targeted sequencing, in a large unselected breast cancer population in Sweden. Methods Sequencing libraries of germline DNA from 5122 breast cancer patients diagnosed between 2001-2008 (LIBRO1) were prepared (48.48 Fluidigm Access Array system, Fluidigm Corp, USA) and sequenced (Next-generation sequencing [NGS], Illumina HiSeq 2500, v2 chemistry, University of Cambridge, UK). A total of 5099 samples (97.8%) passed quality control and were analysed. BRCA1/2 carriers identified by NGS were compared with those who were already identified by clinical BRCA screening (n=418) performed by the BRCA Lab (Lund University). This unit carries out mutation screening for all oncogenetic clinics in Sweden using denaturing high performance liquid chromatography (DHPLC) and multiple ligation-dependent probe amplification (MLPA). Results A total of 50 BRCA1 mutation carriers were identified, of which 28 were unique pathogenic germline mutations. Frameshift insertions and deletions made up 34/50 (68%) of the BRCA1 mutations. Exon 11 harbored 33/50 (66%) of the BRCA1 mutations. The most common mutation was c.3048_3052dupTGAGA (n=8), which is a founder mutation originating from the West coast of Sweden. Three other Swedish founder mutations were also identified (c.1082_1092del [n=5], c.3626delT [n=3] and c.2475delC [n=2]). For BRCA2, 42 mutation carriers were identified; 33 unique deleterious BRCA2 mutations (27 frameshift deletions, 3 frameshift insertions, 9 truncating and 3 splice sites). More than half of the mutations (24/42, 57%) were found on exon 11. Of the 418 women who had attended clinical BRCA testing, 38 deleterious mutations were found. Our screening method confirmed 34 of these mutations, as two each of BRCA1 and BRCA2 mutations were missed, since NGS was proven unsuitable for the detection of large exon duplications. NGS did, however, identify three more carriers not previously identified by clinical testing (c.3048_3052dup, c.2577delA and c.7442delT). Overall, 55/92 BRCA1/2 mutation carriers (59.8%) identified in the present study by NGS were not clinically tested. Conclusion NGS is comparable with current BRCA testing tools for the identification of BRCA1/2 germline mutations, suggesting that the technology has the potential to be used in BRCA1/2 clinical testing in unselected breast cancer patients. Citation Format: Li J, Wen SWX, Eriksson M, Kvist A, Christensen HN, Torstensson A, Easton DF, Teo S-H, Borg Å, Grönberg H, Czene K. Targeted sequencing of BRCA1/2 across a large unselected breast cancer cohort in Sweden [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P4-06-15.

Abstract P3-04-03: Identification, clinical characteristics and treatment outcomes of somatic human epidermal growth factor receptor 2 (ERBB2) mutations in metastatic breast cancer patients

Abstract Background The human epidermal growth factor receptor 2 (ERBB2) can be altered by somatic mutations (with/without ERRB2 amplification) that likely drive tumorigenesis. Here, we present the first study that thoroughly investigates the behavior of patients with ERBB2 mutated tumors in a large unselected cohort of metastatic breast cancer (MBC) patients. Patients and Methods We included retrospectively all MBC patients with sufficient tumor material available, independent of hormone receptor or ERBB2 amplification status, diagnosed between 2000 and 2015 at the Multidisciplinary Breast Center of University Hospitals Leuven. Genomic DNA extracted from primary breast tumors was subjected to deep targeted re-sequencing using an assay covering 5 exons (exons 8, 17, 19, 20 and 21) known to accumulate ERBB2 hotspot mutations. Clinical characteristics, treatment response and outcomes (excluding bilateral BC) were compared between patients with and without ERBB2 mutations. Results We established and validated a research use only next-generation sequencing assay across five exons of ERBB2. Somatic ERBB2 mutations with an allelic frequency of ≥5% in at least one independent analysis, were observed in 1.8% (13/721) of MBC patients. MBC patients with ERBB2 mutant primary tumors appear to have similar patient and tumor characteristics (TNM stage, grade, and receptor and HER2 status) compared to non-mutant patients and ERBB2 mutations occur in all molecular subtypes (7 ER+/HER2-, 4 HER2+, 2 triple negative). However, we see a small trend towards enrichment in the invasive lobular carcinomas (ILC) in the ERBB2 mutant patients vs. the non-mutant patients (4/13 (31%) vs. 97/695 (14%), p=0.086). ER+ patients with an ERBB2 mutant tumor who received first line aromatase inhibitor (AI) (n=3) had a worse median time to progression (TTP) compared to non-mutant patients (n=156) (TTP 103 vs. 311 days, p=0.04), AI use in any line had also a worse TTP (n=8 vs. 376; TTP 74 vs. 213 days, p=0.006). TTP was not significantly different for other standard therapies, but numbers were small. Median distant disease free survival (DDFS) defined as time from primary diagnosis to first metastasis, was slightly shorter for ERBB2 mutant tumors (n=9 vs 514; DDFS 1.4 vs. 2.9 years, p=0.066). However, in ER+/HER2- ERBB2 mutant tumors, median DDFS was significantly shorter in non-mutant patients (n=5 vs. 328; DDFS 0.8 vs. 4.0 years, p=0.02). Median overall survival (OS) after diagnosis of MBC was numerically lower for mutant patients in all subtypes vs. non-mutant patients (n=11 vs. 669; OS 1.1 vs. 2.3 years, p=0.457), and in ER+/HER2- disease (n=6 vs. 431; OS 1.0 vs. 2.9 years, p=0.07). Conclusion In our large MBC cohort, patients with ERBB2 mutant tumors appear to have similar tumor and patient characteristics as ERBB2 non-mutant tumors. ERBB2 mutations do not seem to occur more or less often in specific breast cancer subtypes except for a slight increase for ILC. In ER+/HER2- MBC, patients with ERBB2 mutant tumors appear to be adversely prognostic; having a shorter DDFS after early breast cancer treatment, and in metastatic disease, they respond worse on AI and have a trend for worse OS compared to non-mutant cases. Citation Format: Jongen L, Floris G, Lambrechts D, Laenen A, Neven P, Mann G, Cutler Jr. RE, Lalani AS, Wildiers H. Identification, clinical characteristics and treatment outcomes of somatic human epidermal growth factor receptor 2 (ERBB2) mutations in metastatic breast cancer patients [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-04-03.

Abstract P1-06-01: Putting multigene signatures to the test: Prognostic assessment in population-based contemporary clinical breast cancer

Abstract Background Gene expression signatures hold promise for a molecularly driven division of primary breast cancer with clinical implications. A gap still remains in the application/validation of such signatures in actual clinical treatment groups from unselected, population-based, primary breast cancer receiving current standard of care therapy. We analyzed classification proportions and overall survival (OS) of 14 reported gene expression phenotypes (GEPs) and risk predictors (RPs) in seven clinical treatments groups from an 3273-sample breast cancer cohort representative of population-based disease in the South Swedish healthcare region. Patients and methods Between 2010-09-01 to 2015-03-31, 5101 (87%) of 5892 patients with invasive primary disease in the healthcare region were included in the SCAN-B study (ClinicalTrials.gov ID: NCT02306096). Inclusion criteria included no generalized/prior contralateral disease and known surgery/treatment status (neo- or adjuvant). 3273 tumors were profiled by RNA sequencing and matched to clinicopathological patient data from the National Breast Cancer Register, with distribution of clinicopathological characteristics reflecting proportions in the catchment region. RNA profiles were classified according to 14 reported gene signatures featuring both GEPs (PAM50, IC10, CIT, TNBCtype) and specific risk predictors (e.g. Oncotype Dx, 70-gene, 76-gene, ROR-variants, genomic grade index). Classifications were investigated for association with patient OS by univariate and multivariate analyses in seven adjuvant clinical treatment groups: TNBC-ACT (adjuvant chemotherapy, n=228), TNBC-untreated (n=83), HER2+/ER- with trastuzumab + ACT treatment (n=101), HER2+/ER+ with trastuzumab + ACT + endocrine treatment (n=210), ER+/HER2- with endocrine treatment (n=1477), ER+/HER2- with endocrine + ACT treatment (n=637), and ER+/HER2- untreated (n=216). Results For the majority of signatures, analysis of classification demonstrated prognostic value limited to ER+/HER2- tumors given follow-up time. Several signatures (including Oncotype Dx, 70-gene, ROR-variants) showed strong predictive value in identifying a subset of ER+/HER2- patients receiving a combination of endocrine and ACT therapy with excellent overall survival (&amp;gt;96%), indicating appropriate therapy selection. In addition, for both ER+/HER2- treatment groups signature analysis identified high-risk groups of patients in clear need of additional treatment beyond standard therapeutic regimes, even with less than 5-years of follow-up. Conclusions Our results support the prognostic association of gene expression signatures in large unselected population-based primary breast cancer cohorts even with a short follow-up of OS.Importantly, prognostic associations are limited to specific subgroups for different classifiers and in population-based breast cancer some clinically important subgroups constitute a small proportion of cases. In this context, continued population-based inclusion and broad transcriptional profiling of breast cancer patients provides an opportunity for application to broader patient groups (e.g. TNBC and HER2+), and for consensus classification of individual risk assessments that could potentially provide more stable predictions. Citation Format: Staaf J, Vallon-Christersson J, Häkkinen J, Saal LH, Hegardt C, Larsson C, Ehinger A, Ryden L, Loman N, Malmberg M, Borg Å. Putting multigene signatures to the test: Prognostic assessment in population-based contemporary clinical breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-06-01.

Selective serotonin reuptake inhibitor use and breast cancer survival: a population-based cohort study

BackgroundNearly 50% of breast cancer patients suffer from depression or anxiety. Selective serotonin reuptake inhibitors (SSRIs), the first-line pharmacological treatment for depression, have been implicated in breast cancer development through increased prolactin levels and tamoxifen metabolism inhibition. Previous studies of breast cancer progression have focused on tamoxifen users, or have been limited by their small sample size and methodology. Therefore, we used UK population-based data to more robustly investigate the association between SSRI use and cancer-specific mortality.MethodsA cohort of patients with newly-diagnosed breast cancer between 1998 and 2012 was selected from English cancer registries and linked to prescription records from the Clinical Practice Research Datalink, and to death records from the Office for National Statistics. We used Cox regression models to calculate hazard ratios (HRs) comparing mortality between post-diagnostic SSRI users and non-users (using time-dependant covariates), after adjusting for demographics, comorbidities and pre-diagnosis use of hormone replacement therapy or oral contraceptives. We conducted several additional analyses to assess causality.ResultsOur cohort included 23,669 breast cancer patients, of which 2672 used SSRIs and 3053 died due to their breast cancer during follow-up. After adjustment, SSRI users had higher breast cancer-specific mortality than non-users (HR = 1.27; 95% confidence interval (CI) 1.16, 1.40). However, this association was attenuated when restricting to patients with a prior history of depression (HR = 1.14; 95% CI 0.98, 1.33), and when comparing to users of other antidepressant medications (HR = 1.06; 95% CI 0.93, 1.20). There was some evidence of higher mortality among long-term SSRI users, even when restricting to patients with prior depression (HR = 1.54; 95% CI 1.03, 2.29).ConclusionsIn this large breast cancer cohort, SSRI use was associated with a 27% increase in breast cancer mortality. The cause of this is unknown; however, confounding by indication seems likely as it was largely attenuated when restricting to patients with prior depression, or when comparing SSRIs to other antidepressant medications. Clinicians should not be unduly concerned when prescribing SSRIs to breast cancer patients, but the increase in mortality among long-term SSRI users warrants further investigation.

Immunophenotypic profile of tumor buds in breast cancer

BackgroundTumor buds are associated with lympho-vascular invasion and lymph node metastases leading to the assumption that they are involved in the early metastatic process. Hence, it would be important to know if tumor buds can be targeted with the most widely used targeted therapies in breast cancer (BC) and if changes in hormone and Her2 status occur. The aim of this study was to answer these questions by determining whether hormone receptor (HR) and Her2 status are expressed in the tumor buds of a large cohort of BCs. DesignWe constructed a tumor bud next-generation tissue microarray (ngTMA) consisting of n = 199 BCs of non-special type. Generally, two 1 mm punches were taken from the tumor bud areas in the periphery (PTB) and within the tumor center (ITB). HR and Her2 status was assessed using immunohistochemistry and fluorescence in situ hybridization, respectively. HR status was positive if ≥1% of tumor bud cells were positive. Her2 status was considered positive if bud cells showed strong complete membranous Her2 over-expression or Her2 amplification. ResultsMost tumor buds were positive for estrogen (ER) (PTB: 86%; ITB: 88.3) and progesterone receptor (PgR) (PTB: 72%; ITB: 72.8%) and Her2 was positive in: PTB 11.5% and ITB 11%. A difference between the main tumor mass and tumor buds (PTB and ITB) was seen for PgR in 3.5% of cases (n = 7). No differences were seen for ER and Her2 between tumor buds and main tumor mass. ConclusionMost tumor buds (96.5%) share the same HR and Her2 expression profile of the main tumor mass, implying that tumor buds relay on the same pathways as the main tumor mass and might be equally responsive to targeted therapies.

Proteomic profiling of breast cancer metabolism identifies SHMT2 and ASCT2 as prognostic factors

BackgroundBreast cancer tumors are known to be highly heterogeneous and differences in their metabolic phenotypes, especially at protein level, are less well-understood. Profiling of metabolism-related proteins harbors the potential to establish new patient stratification regimes and biomarkers promoting individualized therapy. In our study, we aimed to examine the relationship between metabolism-associated protein expression profiles and clinicopathological characteristics in a large cohort of breast cancer patients.MethodsBreast cancer specimens from 801 consecutive patients, diagnosed between 2009 and 2011, were investigated using reverse phase protein arrays (RPPA). Patients were treated in accordance with national guidelines in five certified German breast centers. To obtain quantitative expression data, 37 antibodies detecting proteins relevant to cancer metabolism, were applied. Hierarchical cluster analysis and individual target characterization were performed. Clustering results and individual protein expression patterns were associated with clinical data. The Kaplan-Meier method was used to estimate survival functions. Univariate and multivariate Cox regression models were applied to assess the impact of protein expression and other clinicopathological features on survival.ResultsWe identified three metabolic clusters of breast cancer, which do not reflect the receptor-defined subtypes, but are significantly correlated with overall survival (OS, p ≤ 0.03) and recurrence-free survival (RFS, p ≤ 0.01). Furthermore, univariate and multivariate analysis of individual protein expression profiles demonstrated the central role of serine hydroxymethyltransferase 2 (SHMT2) and amino acid transporter ASCT2 (SLC1A5) as independent prognostic factors in breast cancer patients. High SHMT2 protein expression was significantly correlated with poor OS (hazard ratio (HR) = 1.53, 95% confidence interval (CI) = 1.10–2.12, p ≤ 0.01) and RFS (HR = 1.54, 95% CI = 1.16–2.04, p ≤ 0.01). High protein expression of ASCT2 was significantly correlated with poor RFS (HR = 1.31, 95% CI = 1.01–1.71, p ≤ 0.05).ConclusionsOur data confirm the heterogeneity of breast tumors at a functional proteomic level and dissects the relationship between metabolism-related proteins, pathological features and patient survival. These observations highlight the importance of SHMT2 and ASCT2 as valuable individual prognostic markers and potential targets for personalized breast cancer therapy.Trial registrationClinicalTrials.gov, NCT01592825. Registered on 3 May 2012.

Data-driven analysis of immune infiltrate in a large cohort of breast cancer and its association with disease progression, ER activity, and genomic complexity.

The tumor microenvironment is now widely recognized for its role in tumor progression, treatment response, and clinical outcome. The intratumoral immunological landscape, in particular, has been shown to exert both pro-tumorigenic and anti-tumorigenic effects. Identifying immunologically active or silent tumors may be an important indication for administration of therapy, and detecting early infiltration patterns may uncover factors that contribute to early risk. Thus far, direct detailed studies of the cell composition of tumor infiltration have been limited; with some studies giving approximate quantifications using immunohistochemistry and other small studies obtaining detailed measurements by isolating cells from excised tumors and sorting them using flow cytometry. Herein we utilize a machine learning based approach to identify lymphocyte markers with which we can quantify the presence of B cells, cytotoxic T-lymphocytes, T-helper 1, and T-helper 2 cells in any gene expression data set and apply it to studies of breast tissue. By leveraging over 2,100 samples from existing large scale studies, we are able to find an inherent cell heterogeneity in clinically characterized immune infiltrates, a strong link between estrogen receptor activity and infiltration in normal and tumor tissues, changes with genomic complexity, and identify characteristic differences in lymphocyte expression among molecular groupings. With our extendable methodology for capturing cell type specific signal we systematically studied immune infiltration in breast cancer, finding an inverse correlation between beneficial lymphocyte infiltration and estrogen receptor activity in normal breast tissue and reduced infiltration in estrogen receptor negative tumors with high genomic complexity.

Abstract 3286: Correlation between vitamin D levels and DNA repair capacity in breast cancer patients stratified by molecular subtypes

Abstract Vitamin D exists as vitamin D2 and D3, which are metabolized to 25-hydroxyvitamin D [25(OH)D], the major circulating vitamin D metabolite. Besides its physiological functions, vitamin D levels have also been studied as a risk factor for several hormonal cancers including breast cancer (BC). Worldwide, BC accounts for nearly a quarter of all cancers in women. Nutritional studies report that vitamin D intake is associated with a lower BC risk. Several discrepancies exist regarding the role of serum vitamin D in BC risk. While some studies report BC risk reduction by vitamin D only in premenopausal, others propose that it only occurs in postmenopausal women. BC tumors may (+) or may not (-) have three hormonal receptors: estrogen (ER), progesterone (PR), and HER2. Based on their status, four principal molecular BC subtypes have been identified: luminal A (ER+/−, PR+/−, HER2-), luminal B (ER+/−, PR+/−, HER2+), HER2+ (ER−, PR−, HER2+), and triple negative (TN) (ER−, PR−, HER2−). If BC is analyzed in terms of molecular subtypes, low vitamin D levels have been associated with aggressive phenotypes and worse prognosis. Vitamin D also influences estrogen synthesis. Since we have previously shown that a low DNA repair capacity (DRC), measured through the nucleotide excision repair pathway, is a risk factor for BC and vitamin D has also been found to affect DNA repair, the focus of this study is to examine the role of plasma vitamin D levels and DRC in BC. The main aim is to elucidate whether there is an association between vitamin D and DRC levels among the four molecular BC subtypes. We hypothesize that a negative correlation between 25(OH)D and DRC levels will be observed among these subtypes. As an initial effort, 47 BC cases and 20 controls without BC were selected from our large BC cohort. DRC was measured in lymphocytes of untreated women using the host cell reactivation assay. Pathology reports were examined to divide BC cases according to their molecular BC subtype: luminal A (n=13), luminal B (n=11), HER2+ (n=10), and TN (n=13). Plasma 25(OH)D levels were measured using the UniCel DxC System at a CLIA-certified lab. Our results show a negative correlation between 25(OH)D and DRC levels (p=0.04). Statistically significant differences were found for vitamin D levels among the different groups (p=0.0019, ANOVA). Moreover, higher 25(OH)D levels (47.97±2.4 ng/mL) were found in ER- BC cases (p=0.03, t-test). When comparing vitamin D levels in BC subtypes, a significant difference was found in HER2+ and TN groups when compared with the control group (p&amp;lt;0.05, Kruskal-Wallis). Based on these preliminary results we conclude that plasma vitamin D levels may be correlated with DRC levels in women with BC. Ongoing studies with a larger sample size are aimed at elucidating more precisely the potential association between vitamin D and DRC in these molecular BC subtypes. Citation Format: Carmen Ortiz, Jarline Encarnación, Ralphdy Vergne, Wanda Vargas, Jaime Matta. Correlation between vitamin D levels and DNA repair capacity in breast cancer patients stratified by molecular subtypes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3286. doi:10.1158/1538-7445.AM2017-3286

Abstract 2410: Toward mutation analysis of regulatory elements: Epigenetic profiling of primary breast tumors

Abstract Non-coding mutations found in regulatory elements can function as driver mutations in breast cancer by changing the binding affinity of transcription factors for DNA, thereby resulting in direct change of expression of genes that promote cancer development. Identifying such additional driver mutations can reveal the molecular mechanisms favorable to breast cancer development and progression, as well as reveal new biomarkers to better tailor personalized/precision cancer medicine. In this study we have collected 20 primary luminal breast tumors and optimized experimental workflow to dissociate solid tumors and map open chromatin using ATAC-seq. In our initial experiments using ATAC-seq profiling of bulk tumor tissues, we were able to call an average of 15x103 peaks. Subsequently, flow cytometry analysis showed the presence of 15-25% of immune cells in our primary tumors. Therefore, we have optimized a workflow to eliminate immune cells and focus mainly on epithelial tumor cells. Primary breast tumors were digested using collagenase and further dissociated with dispase. Cells were sorted into two populations (mammary epithelial and immune cells) using anti-CD45, anti-CD49f and anti-EpCAM antibodies. Sorted mammary epithelial cells were then used for ATAC- and RNA-seq library preparation as well as for generation of patient derived organoids. Our new workflow resulted in an increased number of called peaks (40x103 vs 15x103), as well as a significant increase in the percentage of unique peaks compared to bulk sequencing (45% vs 15%). By refining our workflow to enrich for tumour content, we will continue our ongoing effort to profile these open chromatin regions and contextualize the mutations within in a large cohort of luminal breast cancers using targeted sequencing. These data will be compared with large-scale whole genome data generated by our group and made publicly available by others. Citation Format: Samah El Ghamrasni, Paul Guilhamon, Rene Quevedo, Cindy Yang, Mathieu Lupien, Trevor Pugh. Toward mutation analysis of regulatory elements: Epigenetic profiling of primary breast tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2410. doi:10.1158/1538-7445.AM2017-2410

Expression and Clinical Significance of Herpes Virus Entry Mediator (HVEM) in Breast Cancer.

Immune checkpoint blockades are currently actively investigated in invasive breast cancers. Given the complexity of immune regulation, multiple inhibitory molecules within the immune checkpoint framework would be involved in tumor immune escape. Evaluation of the components within the framework is a prerequisite for not only identification of additional treatment targets and optimization of immunotherapeutic strategies but also understanding the prognostic value of these molecules. We examined a recently described component, herpes virus entry mediator (HVEM), in a large cohort of invasive breast cancers using immunohistochemistry, and evaluated its clinical relevance. HVEM expression was associated with aggressive tumor features, namely high grade (p<0.001), high pT (p=0.001) and pN stage (p=0.008), and was most prevalently found in human epidermal growth factor receptor 2-overexpressed subtype (67%). Interestingly, a negative association with programmed death-ligand 1 (p=0.021) has been observed. The prognostic impact of HVEM depended on the level of tumor-infiltrating lymphocytes (TILs), with the worst outcome occurring in patients with low TIL, HVEM-positive tumors. HVEM plays significant oncogenic roles in breast carcinogenesis, and may also be a tumor-specific target.

Highlights of This Issue

Resistance to anti-androgen therapy in prostate cancer (PCa) can occur through increased intratumoral/adrenal production of androgens, overexpression of the androgen receptor (AR), and/or AR mutants that exhibit altered ligand specificity or activate transcription in a ligand-independent manner. Given the diversity of mechanisms that can lead to endocrine therapy resistance, it has been difficult to develop drugs that directly and effectively inhibit AR in advanced disease. However, taking the alternative approach of targeting critical processes downstream of AR, Stice and colleagues demonstrate that inhibition of the AR-regulated cyclin D1-CDK 4/6 signaling axis results in substantial tumor inhibition in the most treatment recalcitrant animal models of PCa. These results suggest that CDK4/6 inhibitors (CDK4/6i) are a viable alternative to taxanes, the current standard of care in endocrine therapy-resistant prostate cancer.Circulating microvesicles (MVs) have been described as important players in cell-to-cell communication and tumor-derived MVs can include potentially oncogenic information in cancers. In plasma of leukemia patients, circulating MVs were shown to carry leukemia specific fusion transcripts. Here, using gene expression profiling it is demonstrated that MVs cargo of leukemic cells encloses oncogenic fusion transcripts and mRNA related to basic functions of leukemic cells. In co-culture with mesenchymal stem cells, MVs transferred to target cells enhanced their proliferation. As carriers of oncogenic protein coding messages, MVs potentially jeopardize cell-directed therapy and facilitate the spread to other compartments of the body.Hypoxia-induced changes in composition and stiffness of the extracellular matrix (ECM) in the tumor microenvironment (TME) are well-established. Ju and colleagues now define the role of hypoxia in the regulation of ECM-binding receptors by analyzing the expression of individual integrin subunits within a large clinical cohort of breast cancer (BCa) patients and a panel of 20 BCa cell lines. Furthermore, it was demonstrated that integrin α5 is required for BCa metastasis to lymph nodes and lungs. In clinical specimens, increased integrin α5 expression correlates with poor prognosis, suggesting that targeting integrin α5 has therapeutic benefit for patients with hypoxic tumors.Tyro3, Axl and Mertk (TAM receptors) have been implicated in human cancers, whereby aberrantly elevated expression and signaling is associated with cancer progression, metastasis, and resistance to targeted therapies. Here, Kasikara and colleagues explore the function of TAMs as phosphatidylserine (PS) sensing receptors and reveal that activation of TAMs by apoptotic cells and Gas6 promotes epithelial cell efferocytosis, AKT-mediated chemoresistance, and up-regulation of PD-L1. TAM-mediated up-regulation of PD-L1 could be partially blocked by PS-targeting antibodies or Annexin V; thus, providing a rationale that PS-targeting, anti-TAMs, and anti-PD1 based therapeutics have merit as combinatorial checkpoint inhibitors.

The Utrecht cohort for Multiple BREast cancer intervention studies and Long-term evaLuAtion (UMBRELLA): objectives, design, and baseline results

PurposeIn oncology, RCTs are often beset by slow recruitment, limited generalizability, and strong preferences for interventions by patients and physicians. The cohort multiple randomized controlled trial (cmRCT) is an innovative design with the potential to overcome those challenges. In cmRCT, a prospective cohort serves as an infrastructure for multiple RCTs. We implemented cmRCT in a clinical breast cancer setting by creating UMBRELLA—a large prospective cohort of breast cancer and DCIS patients/survivors.MethodsFor all participants, clinical data and patient-reported outcomes (PROs, i.e., quality of life, fatigue, anxiety and depression, physical activity, work ability, and cosmetic satisfaction) are being collected at regular time-intervals for a period of 10 years. These data are being used both for observational and randomized studies. For each intervention to be tested against standard care, a subcohort of eligible patients is identified within UMBRELLA. From this subcohort, a random sample of patients is offered the intervention. Their outcomes are compared to the outcomes of patients receiving standard care.ResultsSo far, between October 2013 and July 2016, we have recruited 1308 participants. In this period, 1308/1486 (88%) patients who were invited for participation in UMBRELLA consented to cohort participation. Of these patients, 1138 (87%) gave broad consent for randomization to future interventions. Return rate for PROs at baseline were 80%, and varied from 67 to 74% during follow-up. Several observational studies—and the first randomized intervention study—are currently ongoing.ConclusionsResults from UMBRELLA show that this novel study design is feasible and acceptable to patients in a clinical breast cancer setting. We invite researchers who are interested in conducting randomized or observational studies within the UMBRELLA cohort to contact the UMBRELLA scientific advisory board.

Abstract P1-06-07: Clinical and molecular relevance of intra-tumor genetic heterogeneity in breast cancer: Integrative analysis of data from The Cancer Genome Atlas

Abstract Background: Intra-tumor heterogeneity (ITH) plays a pivotal role in driving breast cancer progression and therapeutic resistance. Emerging evidence has indicated that the extent of genetic heterogeneity may serve as a clinically useful biomarker. While several studies have suggested the prognostic value of ITH in several cancer types, the clinical significance of genetic ITH and molecular portraits that correlated with different ITH levels were poorly understood in breast cancer. The establishment of algorithms estimating genetic ITH based on sequencing of bulk tumor DNA offered us an opportunity to explore the clinical implication of ITH in large breast cancer cohorts and, for the first time, to use integrative genomic analyses to reveal molecular portraits related to intra-tumor genetic heterogeneity. Methods: We assessed 916 female breast cancer patients from The Cancer Genome Atlas. Mutant-allele tumor heterogeneity (MATH) values were calculated from whole-exome sequencing data. We used integers nearest to the tertiles of the MATH values as cutoff points to divide the patients into three groups nearly equal in size. The association between MATH value and clinical characteristics was evaluated, followed by survival analyses in these different MATH groups. We then compared the rates of total non-silent somatic mutations among the different MATH groups, and further determined the mutations independently associated with high MATH by logistic regression adjusting for T classification and clinical subtypes. Similar methods, superadding somatic copy number alteration (SCNA) burden in logistic model, were used to evaluate SCNA events that were significantly associated with high MATH level. Gene enrichment between the high and rest MATH groups was analyzed using Gene Set Enrichment Analysis. Results: The patients were divided into low (MATH value lower than 33), intermediate (MATH between 33 and 46) and high (MATH higher than 46) MATH groups. High T stage, African American race, and triple-negative or basal-like subtype were associated with a higher MATH level (all P&amp;lt;0.001). In hormone receptor-positive and human epidermal growth factor receptor-negative patients, the high MATH group showed a tendency toward a worse overall survival (P=0.052); however, while in triple-negative breast cancer, both high and low MATH indicated a worse outcome (P=0.032). Furthermore, the TP53 mutation rate increased as MATH was elevated (P&amp;lt;0.001), whereas CDH1 mutations were correlated with a lower level of MATH (P=0.002). Several focal and arm-level SCNA events were more common in the high MATH group, including Chr8q24 with only the MYC gene in the “peak” region. Similarly, high MATH was associated with gene set enrichment related to the MYC pathway and proliferation. Conclusion: Our study extended the knowledge concerning the clinical role of ITH in breast cancer, especially the distinct pattern of prognostic values in different clinical subtypes, which may help promote the clinical utilization of genetic ITH. Our attempt at exploring the molecular features related to ITH might provide clues for the source and consequences of ITH, inspiring subsequent experiments investigating the laws underling tumor heterogeneity. Citation Format: Ma D, Jiang Y-Z, Liu X-Y, Liu Y-R, Yu K-D, Shao Z-M. Clinical and molecular relevance of intra-tumor genetic heterogeneity in breast cancer: Integrative analysis of data from The Cancer Genome Atlas [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-06-07.

Abstract P1-05-13: The metastatic breast cancer project: Translational genomics through direct patient engagement

Abstract Background: The Metastatic Breast Cancer Project is a nationwide research initiative that directly engages patients through social media and advocacy groups and seeks to empower them to share their samples and clinical information to accelerate research. Because the vast majority of patients are treated in the community setting, we sought to determine the feasibility of remotely obtaining tumor and saliva samples as well as medical records from a large cohort of metastatic breast cancer (MBC) patients who receive their care in diverse settings around the country. Methods: In collaboration with patients and advocacy groups, we developed a website to allow MBC patients across the U.S. to participate. Enrolled patients are sent a saliva kit and asked to mail back a saliva sample, which is used to extract germline DNA. We contact participants' medical providers and obtain medical records and part of their tumor biopsy. Whole exome and transcriptome sequencing is performed on tumor and germline samples. Clinically annotated genomic data are used to identify mechanisms of response and resistance to therapies. The database will be shared widely with researchers. Study updates and discoveries are shared with participants regularly. Results: In the first 8 months, 2285 MBC patients from all 50 states enrolled. 2163 (95%) completed the 16-question survey about their cancer, treatments, and demographic information. 1232 completed the online consent form permitting acquisition and analysis of medical records, tumor tissue, and saliva samples. 556 saliva samples have been received. Initial medical record and tumor sample requests have been made for patients who have provided saliva samples. To date, we have obtained medical records from 102 patients (93% success rate) and tumor samples from 32 patients (77% success rate). Whole exome and transcriptome sequencing has been successfully completed on initial samples received and is ongoing for additional samples. Conclusions: Partnering directly with patients through social media and advocacy groups enables rapid identification of thousands of patients willing to share tumors, saliva, and medical records to accelerate research. This approach allows for rapid identification of patients with rare phenotypes such as extraordinary responders, who have been challenging to identify with traditional approaches. Remote acquisition of medical records, saliva samples, and tumor tissue for patients located throughout the U.S. is feasible. Genomic analysis and medical record abstraction for these patients is underway. As data is generated, a clinically annotated database will be shared widely with the research community. Citation Format: Painter CA, Anastasio E, Krevalin M, Kim D, Larken K, Lennon N, Frank E, Winer EP, Lander ES, Golub T. The metastatic breast cancer project: Translational genomics through direct patient engagement [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-05-13.

Abstract PD1-05: Breast cancer brain metastases show limited intrinsic subtype switching, yet exhibit acquired ERBB2 amplifications and activating mutations

Abstract BACKGROUND: Metastasis is the major cause of mortality in breast cancer (BrCa) patients. Our understanding of brain metastasis (BrM) is limited, reflected by a lack of effective treatments. We aimed to (1) determine BrCa gene signature differences between primary tumors and matched BrM and (2) uncover BrM-specific alterations that may be clinically actionable. MATERIALS and METHODS: NanoString expression profiling of 127 genes from 5 major prognostic tests (MammaPrint, EndoPredict, PAM50, OncotypeDX, MGI) was performed on 20 patient-matched primary (10 ER-neg, 10 ER-pos) and metastatic brain tumors. Subtype classification was performed using genefu. Protein changes in ER and HER2 (ERBB2) were confirmed by IHC. BrM-specific ERBB2 gains were corroborated in a publicly available dataset of 18 additional patient-matched cases (dbGAP phs000730.v1.p1). To test whether ERBB2 amplification and base pair mutation is metastasis-site specific, we further analyzed an expanded cohort of 7,884 breast tumors enriched for metastatic samples (52%) including liver (16.7%), lung (4.3%), bone (3.6%), and brain (2.0%) using comprehensive hybrid-capture sequencing of ERBB2. RESULTS: 17/20 BrM retained the PAM50 subtype of the primary BrCa. Despite this concordance, 17/20 BrM harbored expression changes (&amp;lt; or &amp;gt; 2-fold) in clinically actionable genes including gains of FGFR4 (30%), FLT1 (20%), AURKA (10%) and loss of ESR1 expression (45%). The most recurrently upregulated gene was ERBB2, showing a &amp;gt;2-fold expression increase in 35% of BrM. 3 of 13 (23.3%) cases originally HER2-negative, and thus HER2-therapy naive, in the primary BrCa were IHC-positive (3+) in the paired BrM with an observed metastasis-specific amplification of the ERBB2 locus. In an independent dataset, 2 of 9 (22.2%) HER2-negative BrCa switched to HER2-positive with one BrM acquiring ERBB2 amplification and the other showing metastastic enrichment of the activating V777L ERBB2 mutation. Analysis of a large cohort of breast tumors (n=7,884) showed that across all organs ERBB2 amplification and/or base pair mutation was similar (p=0.18) between primary (13%) and metastatic disease (12%), however, a strong and significant enrichment was seen for BrM (primary 13% vs BrM 24%, p&amp;lt;0.0005). CONCLUSIONS: Taken together, these results demonstrate that the majority (85%) of patient-matched BrM retain the intrinsic subtype of the primary cancer. However, despite this transcriptional similarity, alterations in clinically actionable genes are common, with BrM acquiring ERBB2 amplifications and/or base pair mutations at a frequency of ∼20%, even in HER2-therapy naive tumors. In a large cohort of primary and metastatic breast cancers, there is also a unique enrichment for ERBB2 alterations in BrM. This study provides a strong rationale to molecularly profile metastatic lesions to both better understand biological mechanisms of metastases and to perhaps refine therapeutic decision-making in advanced cancers. Citation Format: Priedigkeit N, Hartmaier RJ, Chen Y, Vareslija D, Basudan A, Thomas R, Leone JP, Lucas PC, Bhargava R, Hamilton RL, Chmielecki J, Davidson NE, Oesterreich S, Brufsky AM, Young L, Lee AV. Breast cancer brain metastases show limited intrinsic subtype switching, yet exhibit acquired ERBB2 amplifications and activating mutations [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr PD1-05.

Abstract P2-05-25: Predictive value of ultra-high ESR1 mRNA expression in early breast cancer

Abstract Background Quantitative determination of estrogen receptor mRNA expression in luminal breast tumors is predictive for benefit from adjuvant tamoxifen compared to placebo treatment as has been shown in the large randomized NSABP B-14 trial, while protein determination by IHC or LBA is not (Kim et al JCO 2011). Interestingly, the ultrahigh expression of ESR1 mRNA (above ER score 10 by Oncotype test) has been indicative for tamoxifen benefit. This predictive cut-off value of mRNA expression is significantly higher than the diagnostic cut-off (at ER score 6.5). Here we tested wether the ultrahigh expression of ESR1 mRNA determined by commercial MammaTyper® testing is predictive for survival after neoadjuvant chemotherapy treatment of advanced breast cancer. Materials and Methods Pretreatment core cut biopsies from n=54 patients with PBC treated within a randomized phase II trial (2) of anthracyline/taxane based NAC with available clinical follow-up information were examined. RNA was extracted from the FFPE sections and ESR1 mRNA from each section was measured by commercial assays. For technical comparison of ESR1 mRNA values by Oncotype DX versus MammaTyper® from n=113 surgical samples were analyzed by both commercial assays in a blinded fashion. Statistical analysis was performed using the SAS JMP® 9.0.0 software. Results Quantification of ESR1 mRNA expression after RNA extraction from separate slices of 113 primary breast tumors and determination by different commercial RT-qPCR assays resulted in high correlation of continuous expression results (Spearman r=0,85; p&amp;lt;0,0001). The rate of ESR1 mRNA negative cases by both methods by predefined diagnostic cut-offs was low in this cohort (1/113 and 6/113, respectively) resulting in high concordance for positive ER status by both methods.The median expression of ER score and ESR1 40-DDCq was high (10,2 and 39,8, respectively) and almost exactly at the predictive ER score cut-off. Hence, the Tamoxifen benefit cut-off of ER score 10 by Oncotype is comparable with a 40-DDCqvalue of 39,6 for ESR1 mRNA determination by MammaTyper®, which resembles an ESR1 mRNA expression 3fold above the diagnostic cut-off. In the independent chemotherapy cohort theoptimal discrimination for overall survival could be achieved by an elevated ESR1 mRNA expression exactly at 39,6 resulting in 100% overall survival for ultra-high expressors and 75 % overall survival for lower ESR1 mRNA expression after 5 years (p=0,006). Conclusion Previous data suggest that ultrahigh expression of ESR1 mRNA is predictive for improved overall survival and tamoxifen benefit (1). Here we show that ultrahigh expression of ESR1 mRNA is also prognostic in more advanced breast tumors after neoadjuvant chemotherapy. These findings validate the importance of quantitative determination of estrogen receptor expression and substantiate the understanding of receptor expression being a continuous determinant with indication specific cut-off values. Ultrahigh expression of ESR1 seems to identify a distinct subset of luminal breast tumors with superior prognosis and benefit from tamoxifen treatment. These findings warrant further investigation, which are currently being done in independent large breast cancer cohorts. Citation Format: Wirtz RM, Scheffen I, Marme F, Laible M, Sclombs K, Schumacher C, Schneeweiss A, Eidt S, Sinn H-P. Predictive value of ultra-high ESR1 mRNA expression in early breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-05-25.

PD-L1 expression and tumor infiltrating PD-1+ lymphocytes associated with outcome in HER2+ breast cancer patients

Clinical trials showing programmed death (PD)-1-PD-ligand 1 (L1) axis as a promising therapeutic target in melanoma and non-small cell lung cancers have made the pathway a focus of recent attention. However, the data regarding PD-L1/PD-1 in breast cancer are inconsistent. Given the heterogeneity of breast cancers, the clinical relevance of PD-L1 and PD-1 tumor infiltrating lymphocytes (TIL) may vary according to subtypes of breast cancer. We aim to investigate PD-L1 expression in a large cohort of breast cancers and analyze its clinico-pathological as well as outcome relationship according to molecular subtypes. Also, we evaluate the relationship of PD-1 TIL and PD-L1 expression with patients' survival, particularly for breast cancers with high TIL. Immunohistochemical analysis of PD-L1 on tissue arrays for 1091 breast cancer patients and PD-1 TIL on 97 whole sections was performed. Associations of PD-L1 with luminal cancers (p<0.001) and features associated with that subtype [lower histologic grade, absence of necrosis, ER, PR, and AR expression (p<0.001)] were observed. However, in HER2+breast cancers, PD-L1 was an independent poor prognostic indicator (DFS: HR=1.866, p=0.001; OS: HR=1.517, p=0.036). Interestingly, HER2+cancers showed a lower PD-1 TIL level compared to the other high TIL cases (p=0.011). Cases with low PD-TIL but high PD-L1 expression showed the worst survival. This could be indicative of an active immune suppression by PD-L1 expression. Our data showed the relevance of PD-L1 expression in HER2+breast cancer. A combined evaluation of PD-L1 and PD-1 TIL in the prognosis of breast cancer might also be of value in treatment prediction.