Radiological review of prior screening mammograms of screen-detected breast cancer.

OBJECTIVE: To perform a radiological review of mammograms from prior screening and diagnosis of screen-detected breast cancer in BreastScreen Norway, a population-based screening program. METHODS: We performed a consensus-based informed review of mammograms from prior screening and diagnosis for screen-detected breast cancers. Mammographic density and findings on screening and diagnostic mammograms were classified according to the Breast Imaging-Reporting and Data System®. Cases were classified based on visible findings on prior screening mammograms as true (no findings), missed (obvious findings), minimal signs (minor/non-specific findings), or occult (no findings at diagnosis). Histopathologic tumor characteristics were extracted from the Cancer Registry of Norway. The Bonferroni correction was used to adjust for multiple testing; p < 0.001 was considered statistically significant. RESULTS: The study included mammograms for 1225 women with screen-detected breast cancer. Mean age was 62 years ± 5 (SD); 46% (567/1225) were classified as true, 22% (266/1225) as missed, and 32% (392/1225) as minimal signs. No difference in mammographic density was observed between the classification categories. At diagnosis, 59% (336/567) of true and 70% (185/266) of missed cancers were classified as masses (p = 0.004). The percentage of histological grade 3 cancers was higher for true (30% (138/469)) than for missed (14% (33/234)) cancers (p < 0.001). Estrogen receptor positivity was observed in 86% (387/469) of true and 95% (215/234) of missed (p < 0.001) cancers. CONCLUSIONS: We classified 22% of the screen-detected cancers as missed based on a review of prior screening mammograms with diagnostic images available. One main goal of the study was quality improvement of radiologists' performance and the program. Visible findings on prior screening mammograms were not necessarily indicative of screening failure. KEY POINTS: • After a consensus-based informed review, 46% of screen-detected breast cancers were classified as true, 22% as missed, and 32% as minimal signs. • Less favorable prognostic and predictive tumor characteristics were observed in true screen-detected breast cancer compared with missed. • The most frequent mammographic finding for all classification categories at the time of diagnosis was mass, while the most frequent mammographic finding on prior screening mammograms was a mass for missed cancers and asymmetry for minimal signs.

Interval and Consecutive Round Breast Cancer after Digital Breast Tomosynthesis and Synthetic 2D Mammography versus Standard 2D Digital Mammography in BreastScreen Norway.

Background Screening that includes digital breast tomosynthesis (DBT) with two-dimensional (2D) synthetic mammography (SM) or standard 2D digital mammography (DM) results in detection of more breast cancers than does screening with DM alone. A decrease in interval breast cancer rates is anticipated but is not reported. Purpose To compare rates and characteristics of (a) interval breast cancer in women screened with DBT and SM versus those screened with DM alone and (b) screen-detected breast cancer at consecutive screenings with DM. Materials and Methods This prospective cohort study from BreastScreen Norway included women screened with DBT and SM (study group) or DM alone (control group) between February 2014 and December 2015 (baseline). All women, except nonattendees, women with breast cancer, and those who exceeded the upper age limit, were consecutively screened with DM after 2 years. Interval breast cancer, sensitivity, and specificity were estimated for women screened at baseline. Recall, screen-detected breast cancer, and positive predictive value were analyzed for consecutively screened women. A χ2 test, t test (P < .001 after Bonferroni correction indicated a significant difference), and binomial regression model were used to analyze differences across groups. Results A total of 92 404 women who underwent baseline screening (mean age, 59 years ± 6 [standard deviation]) were evaluated; 34 641 women in the study group (mean age, 59 years ± 6) were screened with DBT and SM and 57 763 women in the control group (mean age, 59 years ± 6) were screened with DM. A total of 26 474 women in the study group (mean age, 60 years ± 5) and 45 543 women in the control group (mean age, 60 years ± 5) were consecutively screened with DM. Rates of interval breast cancer were 2.0 per 1000 screened women in the study group and 1.5 per 1000 screened women in the control group (P = .12). No differences in histopathologic characteristics of interval breast cancer were observed. In the consecutive screening round, rates of screen-detected breast cancer were 3.9 per 1000 screened women (study group) and 5.6 per 1000 screened women (control group) (P = .001). Rates of histologic grade 1 invasive cancer were 0.5 per 1000 screened women (study group) and 1.3 per 1000 screened women (control group) (P = .001). Conclusion No differences in interval breast cancer rates or tumor characteristics were observed in women screened with DBT and SM compared with women screened with DM. Higher rates of low-grade screen-detected tumors were observed in the control group at consecutive screening. © RSNA, 2019 Online supplemental material is available for this article.

Surgical treatment of breast cancer in Norway 2003-2018. / Kirurgisk behandling av brystkreft i Norge 2003­18.

BACKGROUND: Breast-conserving surgery is recommended in Norway and internationally in cases of early-stage breast cancer. We analysed the surgical methods used for breast-cancer patients by hospital providing treatment, age at the time of diagnosis, detection method and histopathological characteristics of the tumours in the period 2003 to 2018. MATERIAL AND METHOD: Data on women of all ages diagnosed with invasive breast cancer (n = 47 004) were retrieved from the Cancer Registry of Norway's databases. We excluded women with distant metastases at the time of diagnosis (n = 1 773) and those for whom no surgical method was recorded (n = 2 638). The detection method was defined as breast cancer detected by screening, in inter-screening intervals, or outside BreastScreen Norway. The surgical methods chosen were compared by means of descriptive analyses. RESULTS: Slightly over half (23 661 of 42 593, i.e. 55.6 %) of the women in whom breast cancer was detected in the study period underwent breast-conserving surgery. The percentage increased from 1 189/2 423 (49.1 %) in 2003 to 2 070/2 958 (70.0 %) in 2018. There were large differences across hospitals. In the period 2015-2018 we found the highest proportion of breast-conserving surgery, 175/187 (93.6 %) for breast cancer detected by screening to be performed at Ålesund Hospital, and the lowest proportion, 121/351 (34.5 %) among women with breast cancer detected outside BreastScreen Norway, to be performed at Radiumhospitalet. Breast-conserving surgery was used most frequently on women with small tumours without spreading to axillary lymph nodes. INTERPRETATION: We found considerable differences in the surgical methods used across hospitals and for different detection methods.

Tumor expression, plasma levels and genetic polymorphisms of the coagulation inhibitor TFPI are associated with clinicopathological parameters and survival in breast cancer, in contrast to the coagulation initiator TF.

INTRODUCTION: Hypercoagulability in malignancy increases the risk of thrombosis, but is also involved in cancer progression. Experimental studies suggest that tissue factor (TF) and tissue factor pathway inhibitor (TFPI) are involved in cancer biology as a tumor- promoter and suppressor, respectively, but the clinical significance is less clear. Here, we aimed to investigate the clinical relevance of TF and TFPI genetic and phenotypic diversity in breast cancer. METHODS: The relationship between tumor messenger RNA (mRNA) expression and plasma levels of TF and TFPI (α and ß), tagging single nucleotide polymorphisms (tagSNPs) in F3 (TF) (n=6) and TFPI (n=18), and clinicopathological characteristics and molecular tumor subtypes were explored in 152 treatment naive breast cancer patients. The effect of tumor expressed TF and TFPIα and TFPIß on survival was investigated in a merged breast cancer dataset of 1881 patients. RESULTS: Progesterone receptor negative patients had higher mRNA expression of total TFPI (α+ß) (P=0.021) and TFPIß (P=0.014) in tumors. TF mRNA expression was decreased in grade 3 tumors (P=0.003). In plasma, total TFPI levels were decreased in patients with larger tumors (P=0.013). SNP haplotypes of TFPI, but not TF, were associated with specific clinicopathological characteristics like tumor size (odds ratio (OR) 3.14, P=0.004), triple negativity (OR 2.4, P=0.004), lymph node spread (OR 3.34, P=0.006), and basal-like (OR 2.3, P=0.011) and luminal B (OR 3.5, P=0.005) molecular tumor subtypes. Increased expression levels of TFPIα and TFPIß in breast tumors were associated with better outcome in all tumor subtypes combined (P=0.007 and P=0.005) and in multiple subgroups, including lymph node positive subjects (P=0.006 and P=0.034). CONCLUSIONS: This study indicates that genetic and phenotypic variation of both TFPIα and TFPIß, more than TF, are markers of cancer progression. Together with the previously demonstrated tumor suppressor effects of TFPI, the beneficial effect of tumor expressed TFPI on survival, renders TFPI as a potential anticancer agent, and the clinical significance of TFPI in cancer deserves further investigation.

Increased coagulation activity and genetic polymorphisms in the F5, F10 and EPCR genes are associated with breast cancer: a case-control study.

BACKGROUND: The procoagulant state in cancer increases the thrombotic risk, but also supports tumor progression. To investigate the molecular mechanisms controlling cancer and hemostasis, we conducted a case-control study of genotypic and phenotypic variables of the tissue factor (TF) pathway of coagulation in breast cancer. METHODS: 366 breast cancer patients and 307 controls were genotyped for SNPs (n = 41) in the F2, F3 (TF), F5, F7, F10, TFPI and EPCR genes, and assayed for plasma coagulation markers (thrombin generation, activated protein C (APC) resistance, D-dimer, antithrombin, protein C, protein S, and TF pathway inhibitor (TFPI)). Associations with breast cancer were evaluated using logistic regression to obtain odds ratios (ORs) and 95% confidence intervals (CIs), or the chi-square test. RESULTS: Four SNPs in F5 (rs12120605, rs6427202, rs9332542 and rs6427199), one in F10 (rs3093261), and one in EPCR (rs2069948) were associated with breast cancer. EPCR rs2069948 was associated with estrogen receptor (ER) and progesterone receptor (PR) positivity, while the SNPs in F5 appeared to follow hormone receptor negative and triple negative patients. The prothrombotic polymorphisms factor V Leiden (rs6025) and prothrombin G20210A (rs1799963) were not associated with breast cancer. High APC resistance was associated with breast cancer in both factor V Leiden non-carriers (OR 6.5, 95% CI 4.1-10.4) and carriers (OR 38.3, 95% CI 6.2-236.6). The thrombin parameters short lag times (OR 5.8, 95% CI 3.7-9.2), short times to peak thrombin (OR 7.1, 95% CI 4.4-11.3), and high thrombin peak (OR 6.1, 95% CI 3.9-9.5) predicted presence of breast cancer, and high D-dimer also associated with breast cancer (OR 2.0, 95% CI 1.3-3.3). Among the coagulation inhibitors, low levels of antithrombin associated with breast cancer (OR 5.7, 95% CI 3.6-9.0). The increased coagulability was not explained by the breast cancer associated SNPs, and was unaffected by ER, PR and triple negative status. CONCLUSIONS: A procoagulant phenotype was found in the breast cancer patients. Novel associations with SNPs in F5, F10 and EPCR to breast cancer susceptibility were demonstrated, and the SNPs in F5 were confined to hormone receptor negative and triple negative patients. The study supports the importance of developing new therapeutic strategies targeting coagulation processes in cancer.

Long-term follow-up of complex oncoplastic breast-conserving surgery, standard breast conservation and skin-sparing mastectomy in DCIS - a register-based study.

BACKGROUND: Few studies evaluate oncological safety in complex oncoplastic breast-conserving surgery(C-OBCS) for DCIS. It still needs to be defined whether it is equivalent to standard breast conservation(S-BCS) or an alternative to skin-sparing mastectomy(SSM). This study compares local recurrence rates(LR), disease-free survival(DFS) and overall survival (OS) between the three surgical techniques. METHODS: We conducted a retrospective register-based study on LR, DFS and OS of patients operated with S-BCS(n=1388), C-OBCS (n=106) or skin-sparing mastectomy (n=218) for DCIS diagnosed 2007-2020. Data was extracted from the Norwegian Breast Cancer Registry. RESULTS: In the S-BCS, C-OBCS and SSM groups, median age was 60, 58 and 51 years (p<0.001), median size 15, 25, and 40 mm (p<0.001) and median follow-up 55, 48 and 76 months. At ten years, the overall LR was 12.7%, 14.3% for S-BCS, 11.2% for C-OBCS and 6.8% for SSM. Overall DFS at ten years was 82.3%, 80.5% for S-BCS, 82.4% for C-OBCS and 90.4% for SSM. At ten years, the OS was 93.8%, 93.0% in S-BCS, 93.3% in C-OBCS and 96.6% in the SSM group. Weighted Kaplan Meier plots showed that SSM had a significantly higher DFS than S-BCS (p=0.003) and C-OBCS (p=0.029). DFS in C-OBCS versus S-BCS and the difference in OS was not significant (p=0.264). CONCLUSION: SSM had a significantly higher DFS than S-BCS and C-OBCS. The difference in DFS between S-BCS and C-OBCS, and OS between the three groups was not statistically significant. Our study suggests that C-OBCS is a safe alternative to S-BCS and SSM.

Breast cancer missed at screening; hindsight or mistakes?

PURPOSE: To investigate radiologists' interpretation scores of screening mammograms prior to diagnosis of screen-detected and interval breast cancers retrospectively classified as missed or true negative. METHODS: We included data on radiologists' interpretation scores at screening prior to diagnosis for 1223 screen-detected and 1007 interval cancer cases classified as missed or true negative in an informed consensus-based review. All prior screening examinations were independently scored 1-5 by two radiologists; score 1 by both was considered concordant negative, score ≥ 2 by one radiologist discordant, and score ≥ 2 by both concordant positive. We analyzed associations between interpretation, review categories, mammographic features and histopathological findings using descriptive statistics and logistic regression. RESULTS: Among screen-detected cancers, 31% of missed and 10% of true negative cancers had discordant or concordant positive interpretation at prior screening. The corresponding percentages for interval cancer were 21% and 8%. Age-adjusted odds ratio (OR) and 95% confidence interval (CI) for missed screen-detected cancer was 3.8 (95% CI: 2.6-5.4) after discordant and 5.5 (95% CI: 3.2-9.5) after concordant positive interpretation, using concordant negative as reference. Corresponding ORs for missed interval cancer were 3.0 (95% CI: 2.0-4.5) for discordant and 6.3 (95% CI: 2.3-17.5) for concordant positive interpretation. Asymmetry was the dominating mammographic feature at prior screening for all, except concordant positive screen-detected cancers where a mass dominated. Histopathological characteristics did not vary statistically with interpretation. CONCLUSIONS: Most cancers were interpreted negatively at screening prior to diagnosis. Increased risk for missed screen-detected or interval cancer was observed after positive interpretation at prior screening.

Oncological outcomes after simple and skin-sparing mastectomy of ductal carcinoma in situ: A register-based cohort study of 576 Norwegian women.

BACKGROUND: For Ductal Carcinoma in Situ (DCIS), recurrence is shown to be higher after skin-sparing (SSM) versus simple (SM) mastectomy. This study aimed to compare the two groups recurrence rates, disease-free survival (DFS), and overall (OS) survival. METHODS: We conducted a retrospective register-based cohort study of women operated with SSM (n = 338) or SM (n = 238) for DCIS between 2007 and 2017. Data from the Norwegian Breast Cancer Registry was used to estimate recurrences rates, DFS and OS. RESULTS: Mean age was 51 and 61 years in the SSM and SM groups, respectively. Median follow-up time was 77 months for SSM (range: 21-152 months) vs 84 months for SM (range: 7-171 months). After five years of follow-up, the overall recurrence rate (OR) was 2.1%; 3.9% for SSM and 0.9% for SM. After ten years, the rates were 3.0%, 6.2% for SSM and still 0.9% for SM. DFS was after ten years 92.2%; 91.8% for SSM, and 92.4% for SM. OS was 95.0%; 97.5% for SSM and 93.3% for SM at ten years. For SSM, involved margins represented a significant risk for recurrence. CONCLUSION: The recurrence rate was higher in the SSM versus the SM group. Whether the difference is due to the operating procedures or underlying risk factors remains unknown. When stratifying for the difference in age, there was no statistical difference in DFS or OS. Involved margins in the SSM group were associated with an increased risk of recurrence.

High-throughput screen in vitro identifies dasatinib as a candidate for combinatorial treatment with HER2-targeting drugs in breast cancer.

Human epidermal growth factor receptor 2-positive (HER2+) breast cancer is an aggressive subtype of this disease. Targeted treatment has improved outcome, but there is still a need for new therapeutic strategies as some patients respond poorly to treatment. Our aim was to identify compounds that substantially affect viability in HER2+ breast cancer cells in response to combinatorial treatment. We performed a high-throughput drug screen of 278 compounds in combination with trastuzumab and lapatinib using two HER2+ breast cancer cell lines (KPL4 and SUM190PT). The most promising drugs were validated in vitro and in vivo, and downstream molecular changes of the treatments were analyzed. The screen revealed multiple drugs that could be used in combination with lapatinib and/or trastuzumab. The Src-inhibitor dasatinib showed the largest combinatorial effect together with lapatinib in the KPL4 cell line compared to treatment with dasatinib alone (p < 0.01). In vivo, only lapatinib significantly reduced tumor growth (p < 0.05), whereas dasatinib alone, or in combination with lapatinib, did not show significant effects. Protein analyses of the treated xenografts showed significant alterations in protein levels compared to untreated controls, suggesting that all drugs reached the tumor and exerted a measurable effect. In silico analyses suggested activation of apoptosis and reduced activity of survival pathways by all treatments, but the opposite pattern was observed for the combinatorial treatment compared to lapatinib alone.

True and Missed Interval Cancer in Organized Mammographic Screening: A Retrospective Review Study of Diagnostic and Prior Screening Mammograms.

RATIONALE AND OBJECTIVES: To explore radiological aspects of interval breast cancer in a population-based screening program. MATERIALS AND METHODS: We performed a consensus-based informed review of mammograms from diagnosis and prior screening from women diagnosed with interval cancer 2004-2016 in BreastScreen Norway. Cases were classified as true (no findings on prior screening mammograms), occult (no findings at screening or diagnosis), minimal signs (minor/non-specific findings) and missed (obvious findings). We analyzed mammographic findings, density, time since prior screening, and histopathological characteristics between the classification groups. RESULTS: The study included 1010 interval cancer cases. Mean age at diagnosis was 61 years (SD = 6), mean time between screening and diagnosis 14 months (SD = 7). A total of 48% (479/1010) were classified as true or occult, 28% (285/1010) as minimal signs and 24% (246/1010) as missed. We observed no differences in mammographic density between the groups, except from a higher percentage of dense breasts in women with occult cancer. Among cancers classified as missed, about 1/3 were masses and 1/3 asymmetries at prior screening. True interval cancers were diagnosed later in the screening interval than the other classification categories. No differences in histopathological characteristics were observed between true, minimal signs and missed cases. CONCLUSION: In an informed review, 24% of the interval cancers were classified as missed based on visibility and mammographic findings on prior screening mammograms. Three out of four true interval cancers were diagnosed in the second year of the screening interval. We observed no statistical differences in histopathological characteristics between true and missed interval cancers.

Treatment of Ductal Carcinoma in situ: A Register-Based Study of Norwegian Women Diagnosed between 1995 and 2018.

Introduction: The incidence of ductal carcinoma in situ (DCIS) has increased after implementation of mammographic screening. The lesion represents management challenges due to its undetermined growth pattern. We aimed to explore treatment of women aged 48-71 years diagnosed with DCIS between 1995 and 2018, by detection mode and histopathological characteristics. Material and Methods: Data on surgical treatment and radiation therapy (RT) of 4,995 women diagnosed with DCIS were retrieved from the Cancer Registry of Norway. We described the percentage and frequency of breast-conserving treatment (BCT) for participants in BreastScreen Norway (screen-detected) and nonparticipants. We estimated the relative risk (RR) of BCT, using log-binomial regression models. Results: Use of BCT increased from about 40% in 1995 to 85% in 2018. Use of BCT was more common among older than younger women and more commonly used for screen-detected versus tumors detected outside the screening program. Nine out of ten women with tumors ≤10 mm were treated with BCT and two out of ten with tumors >50 mm. RT was given to 89.3% of the women with tumors ≤10 mm, 34.1% of those with tumors classified as van Nuys' grade 1 and <10 mm and 96.0% of the tumors >50 mm. Use of BCT was less common for tumors >50 mm compared to <10 mm (RR adjusted for age, detection mode, van Nuys' grade, and localization: 0.26, 95% CI: 0.19-0.36). Conclusion: BCT was increasingly used among women diagnosed with DCIS in Norway during the period from 1995 to 2018, particularly for screen-detected, small lesions with low van Nuys' grade.

miR-101-5p Acts as a Tumor Suppressor in HER2-Positive Breast Cancer Cells and Improves Targeted Therapy.

Purpose: Human epidermal growth factor receptor 2 positive (HER2+) breast cancers responding poorly to targeted therapy need improved treatment options. miR-101-5p has shown tumor-suppressive properties in multiple cancer forms, and we assessed the effect and mechanism of action of this miRNA in HER2+ breast cancer. Methods: Expression levels of miR-101-5p in two clinical datasets, TCGA and METABRIC, were compared between tumor and normal adjacent samples, and across molecular subtypes and HER2 status. The ability of miR-101-5p to sensitize for treatment with lapatinib, tucatinib and trastuzumab was explored in HER2+ breast cancer cells responding poorly to such targeted drugs. Proliferation and apoptosis assays and downstream protein analysis were performed. Results: Expression levels of miR-101-5p were significantly lower in tumor compared to normal adjacent tissue (p < 0.001), and particularly low in HER2+ tumors, both the HER2-enriched subtype (p ≤ 0.037) and clinical HER2-status (p < 0.001). In a HER2+ cell line (KPL4) responding poorly to targeted drugs, miR-101-5p overexpression inhibited proliferation (p < 0.001), and combinatorial treatment with lapatinib and trastuzumab significantly further decreased this inhibition (p = 0.004). Proteomic data and in silico analyses revealed PI3K/Akt- and HER2-pathways among the predicted regulated pathways. miR-101-5p alone (p = 0.018) and in combination with lapatinib and trastuzumab (p < 0.001) induced apoptosis, while the targeted drugs alone did not exert any significant effect neither on proliferation nor apoptosis. Conclusion: miR-101-5p acts as a tumor suppressor by inducing apoptosis in HER2+ breast cancer and sensitizes cells with initially poor response to lapatinib and trastuzumab.

NRF2 drives an oxidative stress response predictive of breast cancer.

Many breast cancer patients are diagnosed with small, well-differentiated, hormone receptor-positive tumors. Risk of relapse is not easily identified in these patients, resulting in overtreatment. To identify metastasis-related gene expression patterns, we compared the transcriptomes of the non-metastatic 67NR and metastatic 66cl4 cell lines from the murine 4T1 mammary tumor model. The transcription factor nuclear factor, erythroid 2-like 2 (NRF2, encoded by NFE2L2) was constitutively activated in the metastatic cells and tumors, and correspondingly a subset of established NRF2-regulated genes was also upregulated. Depletion of NRF2 increased basal levels of reactive oxygen species (ROS) and severely reduced ability to form primary tumors and lung metastases. Consistently, a set of NRF2-controlled genes was elevated in breast cancer biopsies. Sixteen of these were combined into a gene expression signature that significantly improves the PAM50 ROR score, and is an independent, strong predictor of prognosis, even in hormone receptor-positive tumors.

MicroRNA in combination with HER2-targeting drugs reduces breast cancer cell viability in vitro.

HER2-positive (HER2 +) breast cancer patients that do not respond to targeted treatment have a poor prognosis. The effects of targeted treatment on endogenous microRNA (miRNA) expression levels are unclear. We report that responsive HER2 + breast cancer cell lines had a higher number of miRNAs with altered expression after treatment with trastuzumab and lapatinib compared to poorly responsive cell lines. To evaluate whether miRNAs can sensitize HER2 + cells to treatment, we performed a high-throughput screen of 1626 miRNA mimics and inhibitors in combination with trastuzumab and lapatinib in HER2 + breast cancer cells. We identified eight miRNA mimics sensitizing cells to targeted treatment, miR-101-5p, mir-518a-5p, miR-19b-2-5p, miR-1237-3p, miR-29a-3p, miR-29c-3p, miR-106a-5p, and miR-744-3p. A higher expression of miR-101-5p predicted better prognosis in patients with HER2 + breast cancer (OS: p = 0.039; BCSS: p = 0.012), supporting the tumor-suppressing role of this miRNA. In conclusion, we have identified miRNAs that sensitize HER2 + breast cancer cells to targeted therapy. This indicates the potential of combining targeted drugs with miRNAs to improve current treatments for HER2 + breast cancers.

Coagulation factor V is a marker of tumor-infiltrating immune cells in breast cancer.

Background: Factor (F) V is an essential cofactor in blood coagulation, however, F5 expression in breast tumors has also been linked to tumor aggressiveness and overall survival. The specific role of FV in breast cancer is yet unknown. We therefore aimed at dissecting the biological relevance of FV in breast cancer. Methods: Gene expression data from a Scandinavian breast cancer cohort (n = 363) and the cancer genome atlas (TCGA) (n = 981) and 12 replication cohorts were used to search for F5 co-expressed genes, followed by gene ontology analysis. Pathological and bioinformatic tools were used to evaluate immune cell infiltration and tumor purity. T cell activation, proliferation and migration were studied in FV treated Jurkat T cells. Results: F5 co-expressed genes were mainly associated with immune system processes and cell activation. Tumors with high expression of F5 were more infiltrated with both lymphoid (T cells, NK cells, and B cells) and myeloid cells (macrophages and dendritic cells), and F5 expression was negatively correlated with tumor purity (ρ = -0.32). Confirming a prognostic role, data from the Kaplan-Meier plotter showed that high F5 expression was associated with improved relapse-free survival. The strongest association was observed in basal-like breast cancer (HR = 0.55; 95% CI, 0.42-0.71). Exogenous FV did not substantially affect activation, proliferation or migration of human T cells. Conclusions: F5 was identified as a novel marker of immune cell infiltration in breast cancer, and the prognostic role of F5 was verified. FV emerge as an interesting immunological biomarker with potential therapeutic relevance for the cancer-inflammation-thrombosis circuit.

An independent poor-prognosis subtype of breast cancer defined by a distinct tumor immune microenvironment.

How mixtures of immune cells associate with cancer cell phenotype and affect pathogenesis is still unclear. In 15 breast cancer gene expression datasets, we invariably identify three clusters of patients with gradual levels of immune infiltration. The intermediate immune infiltration cluster (Cluster B) is associated with a worse prognosis independently of known clinicopathological features. Furthermore, immune clusters are associated with response to neoadjuvant chemotherapy. In silico dissection of the immune contexture of the clusters identified Cluster A as immune cold, Cluster C as immune hot while Cluster B has a pro-tumorigenic immune infiltration. Through phenotypical analysis, we find epithelial mesenchymal transition and proliferation associated with the immune clusters and mutually exclusive in breast cancers. Here, we describe immune clusters which improve the prognostic accuracy of immune contexture in breast cancer. Our discovery of a novel independent prognostic factor in breast cancer highlights a correlation between tumor phenotype and immune contexture.

A novel hypoxia response element regulates oxygen-related repression of tissue factor pathway inhibitor in the breast cancer cell line MCF-7.

BACKGROUND: Hypoxia is one of the most pervasive physiological stresses in solid tumors. We have previously demonstrated that tissue factor (TF) pathway inhibitor (TFPI) expression was transcriptionally repressed by the activation of hypoxia inducible factor (HIF)-1α under hypoxic conditions. However, the role of HIF-2α, also known as endothelial PAS domain-containing protein 1 (EPAS1), on TFPI expression remains unclear. AIM: To explore the role of HIF-2α/EPAS1 in the regulation of TFPI expression under hypoxia in breast cancer cells. METHODS AND RESULTS: Quantitative RT-PCR showed that total TFPI mRNA and protein levels were decreased by the overexpression of HIF-2α/EPAS1 in MCF7 cells. Luciferase reporter assay and chromatin immunoprecipitation (ChIP) assay indicated a HIF-2α/EPAS1 responsive region located in the TFPI promoter region at -170 to +21 relative to the transcriptional start site. Subsequent mutagenesis demonstrated a functional hypoxia response element (HRE) 5'-AAACAGGA-3' for HIF-2α/EPAS1 within the TFPI promoter located at -45 to -38. In breast cancer patients, a positive correlation between HIF-2α/EPAS1 and total TFPI mRNA expression was observed by using gene expression analysis. CONCLUSIONS: This study provides evidence that HIF-2α/EPAS1 is involved in the regulation of TFPI gene expression in breast cancer cells, suggesting that the activation of coagulation and the increased risk of thrombosis observed in breast cancer patients may correlate with local hypoxic regulation of coagulation factors and their inhibitors.

Drug-screening and genomic analyses of HER2-positive breast cancer cell lines reveal predictors for treatment response.

BACKGROUND: Approximately 15%-20% of all diagnosed breast cancers are characterized by amplified and overexpressed HER2 (= ErbB2). These breast cancers are aggressive and have a poor prognosis. Although improvements in treatment have been achieved after the introduction of trastuzumab and lapatinib, many patients do not benefit from these drugs. Therefore, in-depth understanding of the mechanisms behind the treatment responses is essential to find alternative therapeutic strategies. MATERIALS AND METHODS: Thirteen HER2 positive breast cancer cell lines were screened with 22 commercially available compounds, mainly targeting proteins in the ErbB2-signaling pathway, and molecular mechanisms related to treatment sensitivity were sought. Cell viability was measured, and treatment responses between the cell lines were compared. To search for response predictors and genomic and transcriptomic profiling, PIK3CA mutations and PTEN status were explored and molecular features associated with drug sensitivity sought. RESULTS: The cell lines were divided into three groups according to the growth-retarding effect induced by trastuzumab and lapatinib. Interestingly, two cell lines insensitive to trastuzumab (KPL4 and SUM190PT) showed sensitivity to an Akt1/2 kinase inhibitor. These cell lines had mutation in PIK3CA and loss of PTEN, suggesting an activated and druggable Akt-signaling pathway. Expression levels of five genes (CDC42, MAPK8, PLCG1, PTK6, and PAK6) were suggested as predictors for the Akt1/2 kinase-inhibitor response. CONCLUSION: Targeting the Akt-signaling pathway shows promise in cell lines that do not respond to trastuzumab. In addition, our results indicate that several molecular features determine the growth-retarding effects induced by the drugs, suggesting that parameters other than HER2 amplification/expression should be included as markers for therapy decisions.

DNA methylation at enhancers identifies distinct breast cancer lineages.

Breast cancers exhibit genome-wide aberrant DNA methylation patterns. To investigate how these affect the transcriptome and which changes are linked to transformation or progression, we apply genome-wide expression-methylation quantitative trait loci (emQTL) analysis between DNA methylation and gene expression. On a whole genome scale, in cis and in trans, DNA methylation and gene expression have remarkably and reproducibly conserved patterns of association in three breast cancer cohorts (n = 104, n = 253 and n = 277). The expression-methylation quantitative trait loci associations form two main clusters; one relates to tumor infiltrating immune cell signatures and the other to estrogen receptor signaling. In the estrogen related cluster, using ChromHMM segmentation and transcription factor chromatin immunoprecipitation sequencing data, we identify transcriptional networks regulated in a cell lineage-specific manner by DNA methylation at enhancers. These networks are strongly dominated by ERα, FOXA1 or GATA3 and their targets were functionally validated using knockdown by small interfering RNA or GRO-seq analysis after transcriptional stimulation with estrogen.

Decreased expression of B7-H3 reduces the glycolytic capacity and sensitizes breast cancer cells to AKT/mTOR inhibitors.

B7 family proteins are important immune response regulators, and can mediate oncogenic signaling and cancer development. We have used human triple-negative breast cancer cell lines with different expression levels of B7-H3 to evaluate its effects on the sensitivity to 22 different anticancer compounds in a drug screen. API-2 (triciribidine) and everolimus (RAD-001), two inhibitors that target the PI3K/AKT/mTOR pathway, showed enhanced inhibition of cell viability and proliferation in B7-H3 knockdown tumor cells compared to their B7-H3 expressing counterparts. Similar inhibition was seen in control cells treated with an anti-B7-H3 monoclonal antibody. In B7-H3 overexpressing cells, the effects of the two drugs were reduced, supported also by in vivo experiments in which B7-H3 overexpressing xenografts were less sensitive to everolimus than control tumors. In API-2 and everolimus-treated B7-H3 overexpressing cells, phospho-mTOR levels were decreased. However, phosphorylation of p70S6K was differentially regulated in B7-H3 cells treated with API-2 or everolimus, suggesting a different B7-H3-mediated mechanism downstream of mTOR. Both API-2 and everolimus decreased the glycolysis of the cells, whereas knockdown of B7-H3 decreased and B7-H3 overexpression increased the glycolytic capacity. In conclusion, we have unveiled a previously unknown relationship between B7-H3 expression and glycolytic capacity in tumor cells, and found that B7-H3 confers resistance to API-2 and everolimus. The results provide novel insights into the function of B7-H3 in cancer, and suggest that targeting of B7-H3 may be a novel alternative to improve current anticancer therapies.