Discordance of PIK3CA and TP53 mutations between breast cancer brain metastases and matched primary tumors.

There is limited knowledge of the biology of breast cancer (BC) brain metastasis (BM). We primarily aimed to determine the mutations in BCBM and to compare the mutational pattern with the matched primary breast cancer (BC). Secondary aims were to determine mutations in each subgroup (Luminal A-/B-like, HER2+ and TNBC) of BCBM, and to determine survival according to specific mutations. We investigated 57 BCBMs, including 46 cases with matched primary tumors (PT) by targeted Next Generation Sequencing (NGS) using the Cancer Hotspot Panel v2 (ThermoFisher Scientific) covering 207 targeted regions in 50 cancer related genes. Subtype according to immunohistochemistry was re-evaluated. NGS results fulfilling sequencing quality criteria were obtained from 52 BM and 41 PT, out of which 37 were matched pairs. Pathogenic mutations were detected in 66% of PTs (27/41), and 62% of BMs (32/52). TP53 mutations were most frequent; 49% (20/41) of PTs and 48% (25/52) in BMs, followed by PIK3CA mutations; 22% (9/42) in PTs and 25% (13/52) in BMs. Mutations in CDH1, EGFR, HRAS, RB1 CDKN2A and PTEN were detected in single pairs or single samples. Mutational pattern was discordant in 24% of matched pairs. We show a discordance of PIK3CA and TP53 mutations of roughly 25% indicating the need to develop methods to assess mutational status in brain metastasis where analysis of cell-free DNA from cerebrospinal fluid (CSF) has shown promising results.

Efficacy and tolerability of metronomic chemotherapy in patients with metastatic breast cancer - an international experience in West Sweden and in the South of Ireland.

INTRODUCTION: Metronomic chemotherapy (MCT) is the continuous administration of low dose chemotherapy. It has significant clinical efficacy with minimal toxicity as compared to conventional chemotherapy regimens. Thus represents an attractive treatment modality in selected patients with advanced breast cancer. METHODS: Patients who received MCT in the form of Capecitabine/Cyclophosphamide for the treatment of advanced breast cancer between May 2014 and October 2018 in Sahlgrenska University Hospital in Sweden and in Cork University Hospital, University Hospital Kerry and the South Infirmary-Victoria University Hospital in Ireland were identified. Medical records were retrospectively reviewed to collect data. All survival analyses were described by Kaplan-Meier curves and analysed with log-rank tests. The primary end-point was time on treatment, used as a surrogate marker for efficacy. RESULTS: 148 patients were identified (84 - Sweden, 64 - Ireland), with a median age of 64.2 (range 31-89). The overall mean time on treatment for all patients in both countries is 9.05 months (range 0.36 - 67.21). In patients with bone only disease the mean time on treatment was 10.1 months (range 0.7 - 67.2), compared to patients with visceral disease of 8.91 months (range 0.36 - 39.77). Treatment was ended in the majority of patients because of progression of disease, representing 108 patients (72.9%). CONCLUSION: This is an observational, retrospective study demonstrating the real world effectiveness of MCT in the treatment of advanced breast cancer. In this cohort of unselected pre-treated patients, the efficacy of MCT was comparable with the survival outcomes of landmark clinical trials.

A comparison between young and old patients with triple-negative breast cancer: biology, survival and metastatic patterns.

PURPOSE: To determine the biology, recurrence rate, metastatic patterns and survival times in primary triple-negative breast cancer (TNBC) with focus on the comparison between younger and elderly patients. METHODS: Patients with primary TNBC stage I-IV diagnosed from 2007 to 2015 were identified and information on tumor biology, stage, treatment, recurrences and death recorded. RESULTS: A total of 524 patients, median age 60 years (range 24-94) with a median follow-up of 55 months (range 0-129) were identified. Stage was similar in younger (< 40 years) (n = 58) and older (> 74 years) (n = 96) patients (p = 0.37). A statistically significant difference was found concerning histopathologic grade (p = 0.006) and Ki67 (median 80% versus 70%; p = 0.002) but not for LVI (p = 0.9) with more aggressive tumors among younger patients. Adjuvant/neoadjuvant chemotherapy was more frequently given to younger compared with older patients (96% versus 12%; p = 0.0005). Only brain (p = 0.016) and liver (p = 0.047) metastases were more often registered among younger patients while other locations were similar. Shorter survival times, recurrence-free survival (RFS), distant disease-free survival (DDFS) and breast cancer-specific survival (BCSS) were found in the older group, although not after adjusting for adjuvant/neoadjuvant chemotherapy. Most deaths (68%) in the older group were caused by TNBC. When comparing patients > 75 years (n = 92) with ≤ 75 years (n = 432), a worse outcome among older was also observed: RFS (p = 0.00012), DDFS (p = 0.00041), BCSS (p < 0.0001) and survival following distant metastasis (p = 0.0064) CONCLUSIONS: Primary TNBC in younger patients is more often of poor differentiation grade and highly proliferative compared with older patients. The majority of older patients still have grade III tumors with a Ki67 > 60% and outcome is poor. Few older patients in our study were treated with chemotherapy both in adjuvant and palliative setting, underlining the need for more prospective trials and treatment options suitable for this patient population.

PAM50 Provides Prognostic Information When Applied to the Lymph Node Metastases of Advanced Breast Cancer Patients.

Purpose: Transcriptional pathway activity and the molecular subtypes of breast cancer metastases have been shown to significantly influence patient postrelapse survival. Here, we further determine the relevance of clinically employed gene signatures in the advanced breast cancer (ABC) setting.Experimental Design: Sufficient RNA for expression profiling was obtained from distant metastatic or inoperable loco-regional relapse tissue by fine-needle aspiration from 109 patients of the Swedish TEX clinical trial. Gene signatures (GGI, 70 gene, recurrence score, cell-cycle score, risk of recurrence score, and PAM50) were applied to all metastases, and their relationship to long- (5-year) and short-term (1.5-year) postrelapse survival at all and locoregional lymph nodes (n = 40) versus other metastatic sites (n = 69) combined was assessed using Kaplan-Meier and/or multivariate Cox regression analyses.Results: The majority of metastases were classified into intermediate or high-risk groups by all signatures, and a significant association was found between metastatic signature subgroups and primary tumor estrogen receptor status and histologic grade (P < 0.05). When considering all sites of metastasis, only PAM50 was statistically significant in Kaplan-Meier analysis (Log-rank P = 0.008 and 0.008 for long- and short-term postrelapse breast cancer-specific survival, respectively). This significance remained in both uni- and multivariate models when restricting analyses to lymph node metastases only, and a similar trend was observed in other metastatic sites combined, but did not reach formal significance.Conclusions: Our findings are the first to demonstrate that the PAM50 signature can provide prognostic information from the lymph node metastases of ABC patients. Clin Cancer Res; 23(23); 7225-31. ©2017 AACR.

Low-Dose Oral Cyclophosphamide and Methotrexate Maintenance for Hormone Receptor-Negative Early Breast Cancer: International Breast Cancer Study Group Trial 22-00.

PURPOSE: To evaluate the benefit of low-dose cyclophosphamide and methotrexate (CM) maintenance, which previously demonstrated antitumor activity and few adverse effects in advanced breast cancer, in early breast cancer. PATIENTS AND METHODS: International Breast Cancer Study Group (IBCSG) Trial 22-00, a randomized phase III clinical trial, enrolled 1,086 women (1,081 intent-to-treat) from November 2000 to December 2012. Women with estrogen receptor- and progesterone receptor-negative (< 10% positive cells by immunohistochemistry) early breast cancer any nodal and human epidermal growth factor receptor 2 status, were randomly assigned anytime between primary surgery and 56 days after the first day of last course of adjuvant chemotherapy to CM maintenance (cyclophosphamide 50 mg/day orally continuously and methotrexate 2.5 mg twice/day orally on days 1 and 2 of every week for 1 year) or to no CM. The primary end point was disease-free survival (DFS), which included invasive recurrences, second (breast and nonbreast) malignancies, and deaths. RESULTS: After a median of 6.9 years of follow-up, DFS was not significantly better for patients assigned to CM maintenance compared with patients assigned to no CM, both overall (hazard ratio [HR], 0.84; 95% CI, 0.66 to 1.06;P = .14) and in triple-negative (TN) disease (n = 814; HR, 0.80; 95% CI, 0.60 to 1.06). Patients with TN, node-positive disease had a nonstatistically significant reduced HR (n = 340; HR, 0.72; 95% CI, 0.49 to 1.05). Seventy-one (13%) of 542 patients assigned to CM maintenance did not start CM. Of 473 patients who received at least one CM maintenance dose (including two patients assigned to no CM), 64 (14%) experienced a grade 3 or 4 treatment-related adverse event; elevated serum transaminases was the most frequently reported (7%), followed by leukopenia (2%). CONCLUSION: CM maintenance did not produce a significant reduction in DFS events in hormone receptor-negative early breast cancer. The trend toward benefit observed in the TN, node-positive subgroup supports additional exploration of this strategy in the TN, higher-risk population.

Proteomics profiling identify CAPS as a potential predictive marker of tamoxifen resistance in estrogen receptor positive breast cancer.

BACKGROUND: Despite the success of tamoxifen since its introduction, about one-third of patients with estrogen (ER) and/or progesterone receptor (PgR) - positive breast cancer (BC) do not benefit from therapy. Here, we aim to identify molecular mechanisms and protein biomarkers involved in tamoxifen resistance. RESULTS: Using iTRAQ and Immobilized pH gradient-isoelectric focusing (IPG-IEF) mass spectrometry based proteomics we compared tumors from 12 patients with early relapses (<2 years) and 12 responsive to therapy (relapse-free > 7 years). A panel of 13 proteins (TCEAL4, AZGP1, S100A10, ALDH6A1, AHNAK, FBP1, S100A4, HSP90AB1, PDXK, GFPT1, RAB21, MX1, CAPS) from the 3101 identified proteins, potentially separate relapse from non-relapse BC patients. The proteins in the panel are involved in processes such as calcium (Ca(2+)) signaling, metabolism, epithelial mesenchymal transition (EMT), metastasis and invasion. Validation of the highest expressed proteins in the relapse group identify high tumor levels of CAPS as predictive of tamoxifen response in a patient cohort receiving tamoxifen as only adjuvant therapy. CONCLUSIONS: This data implicate CAPS in tamoxifen resistance and as a potential predictive marker.

Comparative proteome analysis revealing an 11-protein signature for aggressive triple-negative breast cancer.

BACKGROUND: Clinical outcome of patients with triple-negative breast cancer (TNBC) is highly variable. This study aims to identify and validate a prognostic protein signature for TNBC patients to reduce unnecessary adjuvant systemic therapy. METHODS: Frozen primary tumors were collected from 126 lymph node-negative and adjuvant therapy-naive TNBC patients. These samples were used for global proteome profiling in two series: an in-house training (n = 63) and a multicenter test (n = 63) set. Patients who remained free of distant metastasis for a minimum of 5 years after surgery were defined as having good prognosis. Cox regression analysis was performed to develop a prognostic signature, which was independently validated. All statistical tests were two-sided. RESULTS: An 11-protein signature was developed in the training set (median follow-up for good-prognosis patients = 117 months) and subsequently validated in the test set (median follow-up for good-prognosis patients = 108 months) showing 89.5% sensitivity (95% confidence interval [CI] = 69.2% to 98.1%), 70.5% specificity (95% CI = 61.7% to 74.2%), 56.7% positive predictive value (95% CI = 43.8% to 62.1%), and 93.9% negative predictive value (95% CI = 82.3% to 98.9%) for poor-prognosis patients. The predicted poor-prognosis patients had higher risk to develop distant metastasis than the predicted good-prognosis patients in univariate (hazard ratio [HR] = 13.15; 95% CI = 3.03 to 57.07; P = .001) and multivariable (HR = 12.45; 95% CI = 2.67 to 58.11; P = .001) analysis. Furthermore, the predicted poor-prognosis group had statistically significantly more breast cancer-specific mortality. Using our signature as guidance, more than 60% of patients would have been exempted from unnecessary adjuvant chemotherapy compared with conventional prognostic guidelines. CONCLUSIONS: We report the first validated proteomic signature to assess the natural course of clinical TNBC.

Retinoic acid receptor alpha is associated with tamoxifen resistance in breast cancer.

About one-third of oestrogen receptor alpha-positive breast cancer patients treated with tamoxifen relapse. Here we identify the nuclear receptor retinoic acid receptor alpha as a marker of tamoxifen resistance. Using quantitative mass spectrometry-based proteomics, we show that retinoic acid receptor alpha protein networks and levels differ in a tamoxifen-sensitive (MCF7) and a tamoxifen-resistant (LCC2) cell line. High intratumoural retinoic acid receptor alpha protein levels also correlate with reduced relapse-free survival in oestrogen receptor alpha-positive breast cancer patients treated with adjuvant tamoxifen solely. A similar retinoic acid receptor alpha expression pattern is seen in a comparable independent patient cohort. An oestrogen receptor alpha and retinoic acid receptor alpha ligand screening reveals that tamoxifen-resistant LCC2 cells have increased sensitivity to retinoic acid receptor alpha ligands and are less sensitive to oestrogen receptor alpha ligands compared with MCF7 cells. Our data indicate that retinoic acid receptor alpha may be a novel therapeutic target and a predictive factor for oestrogen receptor alpha-positive breast cancer patients treated with adjuvant tamoxifen.

Vascular endothelial growth factor receptor 2 and downstream p38 mitogen-activated protein kinase are possible candidate markers of intrinsic resistance to adjuvant endocrine treatment in steroid receptor positive breast cancer.

A cross talk between tyrosine kinase receptors and mitogen-activated protein kinases (MAPKs) is proposed as involved in endocrine resistance. We wanted to investigate intratumoral levels of vascular endothelial growth factor receptor 2 (VEGFR2) and p38 MAPK in relation to relapse-free (RFS) and breast cancer corrected survival (BCCS) after adjuvant endocrine treatment, mainly tamoxifen for 2 or 5 years. We also wanted to investigate these markers in relation to early and late recurrences. VEGFR2 (n = 381) and p38 (n = 174) were determined by enzyme-linked immuno-sorbent assays in tumor homogenates from primary BC diagnosed 1993-1996. Wide ranges of VEGFR2 and p38 proteins were found; median 0.72 pg/µg DNA (range 0.0-11.66), and 0.04 pg/µg DNA (range 0.0-6.79), respectively. Detectable levels of p38 were registered in 65% and classified positive. Higher VEGFR2 were correlated to higher VEGF (P = 0.005), p38 MAPK (P = 0.018), negative ER (P = 0.008), larger tumors (P = 0.001), histopathological grade III (P = 0.018), distant metastasis (P = 0.044), shorter RFS (P = 0.013), and shorter BCCS (P = 0.017). Expression of p38 was significantly correlated with negative PgR (P = 0.044) and with early relapses (P = 0.021), while no difference was seen during the later follow-up period (P = 0.73). Higher VEGFR2 had a significant negative impact on both early (P = 0.029) and later recurrences (P = 0.018), while VEGF only predicted later relapses (P = 0.037). Our preliminary results suggest higher intratumoral levels of VEGFR2 and p38 MAPK as candidate markers of intrinsic resistance for adjuvant endocrine therapy.

Vascular endothelial growth factor is a strong predictor of early distant recurrences in a prospective study of premenopausal women with lymph-node negative breast cancer.

We investigate the prognostic significance of the pro-angiogenic cytokine vascular endothelial growth factor (VEGF), urokinase-type plasminogen activator (uPA), plasminogen activator inhibitor 1 (PAI-1), and S-phase fraction (SPF) for distant disease free survival (DDFS) in 219 premenopausal patients with node-negative breast cancer (NNBC). In univariate analysis significantly shorter DDFS was observed for patients with high VEGF (p=0.006), high uPA (p=0.001), and high SPF (p<0.001). The prognostic significance of VEGF varied over time being very strong for early relapses (0-2.25 years follow-up) (HR=7.9; p=0.006) while no difference was seen in the subsequent follow-up period (HR=1.3; p=0.62). In a series of bivariate analyses VEGF provided prognostic information during the whole observation period (0-72 months) in addition to age, tumour size, oestrogen receptor (ER), progesterone receptor (PgR), and uPA. Also this effect was more pronounced during the first follow-up period suggesting VEGF as a marker of early recurrences.

Prognostic correlation of basic fibroblast growth factor and vascular endothelial growth factor in 1307 primary breast cancers.

This study was designed to investigate the possible relationship between the protein expression of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) with p53 status, breast cancer prognostic factors, metastatic site, and survival after adjuvant therapy. Basic fibroblast growth factor and VEGF expression were determined by enzyme-linked immunosorbent assays in cytosol specimens obtained from 1307 patients with T1-3 primary breast cancer (789 node-negative, 518 node-positive) diagnosed between 1990 and 1997. The median follow-up time was 70 months. Increased bFGF expression was more frequently found in tumors with low VEGF expression (r = -0.286; P = 0.095). Increased bFGF was associated with smaller tumors (P < 0.001), absence of axillary metastasis (P = 0.003), low S-phase fraction (P < 0.001), and longer recurrence-free survival (RFS; P = 0.0038) and overall survival (OS; P = 0.0316). Vascular endothelial growth factor was a prognostic factor for RFS (P < 0.0001) and OS (P < 0.0001) in univariate and multivariate analyses (RFS: 95% CI, 1.1-1.7; P = 0.036; OS: 95% CI, 1.2-2.2; P = 0.002), whereas bFGF expression was not correlated with RFS or OS. Increased VEGF content was correlated with shorter survival after adjuvant endocrine therapy (RFS, P = 0.0004; OS, P = 0.0009). Patients with estrogen receptor-negative disease were excluded from the analysis. Basic fibroblast growth factor was not a prognostic factor after adjuvant systemic therapy, nor was it related to metastatic site. Expression of VEGF is an independent prognostic factor for patients with primary breast cancer. High bFGF expression was related to good prognostic features and longer survival times, but did not add prognostic information in multivariate analysis. The results might implicate that different angiogenic pathways exist in human breast cancer.