Nicholas P. Tobin

Down-Regulation of the Oncogene Cyclin D1 Increases Migratory Capacity in Breast Cancer and Is Linked to Unfavorable Prognostic Features.

The oncogene cyclin D1 is highly expressed in many breast cancers and, despite its proliferation-activating properties, it has been linked to a less malignant phenotype. To clarify this observation, we focused on two key components of malignant behavior, migration and proliferation, and observed that quiescent G(0)/G(1) cells display an increased migratory capacity compared to cycling cells. We also found that the down-regulation of cyclin D1 in actively cycling cells significantly increased migration while also decreasing proliferation. When analyzing a large set of premenopausal breast cancers, we observed an inverse proliferation-independent link between cyclin D1 and tumor size and recurrence, suggesting that this protein might abrogate infiltrative malignant behavior in vivo. Finally, gene expression analysis after cyclin D1 down-regulation by siRNA confirmed changes in processes associated with migration and enrichment of our gene set in a metastatic poor prognosis signature. This novel function of cyclin D1 illustrates the interplay between tumor proliferation and migration and may explain the attenuation of malignant behavior in breast cancers with high cyclin D1 levels.

Prognostic Significance in Breast Cancer of a Gene Signature Capturing Stromal PDGF Signaling

In this study, we describe a novel gene expression signature of platelet-derived growth factor (PDGF)-activated fibroblasts, which is able to identify breast cancers with a PDGF-stimulated fibroblast stroma and displays an independent and strong prognostic significance. Global gene expression was compared between PDGF-stimulated human fibroblasts and cultured resting fibroblasts. The most differentially expressed genes were reduced to a gene expression signature of 113 genes. The biological significance and prognostic capacity of this signature were investigated using four independent clinical breast cancer data sets. Concomitant high expression of PDGFβ receptor and its cognate ligands is associated with a high PDGF signature score. This supports the notion that the signature detects tumors with PDGF-activated stroma. Subsequent analyses indicated significant associations between high PDGF signature score and clinical characteristics, including human epidermal growth factor receptor 2 positivity, estrogen receptor negativity, high tumor grade, and large tumor size. A high PDGF signature score is associated with shorter survival in univariate analysis. Furthermore, the high PDGF signature score acts as a significant marker of poor prognosis in multivariate survival analyses, including classic prognostic markers, Ki-67 status, a proliferation gene signature, or other recently described stroma-derived gene expression signatures.

Digital image analysis outperforms manual biomarker assessment in breast cancer

In the spectrum of breast cancers, categorization according to the four gene expression-based subtypes ‘Luminal A,’ ‘Luminal B,’ ‘HER2-enriched,’ and ‘Basal-like’ is the method of choice for prognostic and predictive value. As gene expression assays are not yet universally available, routine immunohistochemical stains act as surrogate markers for these subtypes. Thus, congruence of surrogate markers and gene expression tests is of utmost importance. In this study, 3 cohorts of primary breast cancer specimens (total n=436) with up to 28 years of survival data were scored for Ki67, ER, PR, and HER2 status manually and by digital image analysis (DIA). The results were then compared for sensitivity and specificity for the Luminal B subtype, concordance to PAM50 assays in subtype classification and prognostic power. The DIA system used was the Visiopharm Integrator System. DIA outperformed manual scoring in terms of sensitivity and specificity for the Luminal B subtype, widely considered the most challenging distinction in surrogate subclassification, and produced slightly better concordance and Cohen’s κ agreement with PAM50 gene expression assays. Manual biomarker scores and DIA essentially matched each other for Cox regression hazard ratios for all-cause mortality. When the Nottingham combined histologic grade (Elston–Ellis) was used as a prognostic surrogate, stronger Spearman’s rank-order correlations were produced by DIA. Prognostic value of Ki67 scores in terms of likelihood ratio χ2 (LR χ2) was higher for DIA that also added significantly more prognostic information to the manual scores (LR−Δχ2). In conclusion, the system for DIA evaluated here was in most aspects a superior alternative to manual biomarker scoring. It also has the potential to reduce time consumption for pathologists, as many of the steps in the workflow are either automatic or feasible to manage without pathological expertise.

Molecular subtype and tumor characteristics of breast cancer metastases as assessed by gene expression significantly influence patient post-relapse survival

An enhanced understanding of the biology of breast cancer metastases is needed to individualize patient management. Here, we show that tumor characteristics of breast cancer metastases significantly influence post-relapse survival, emphasizing that molecular investigation at relapse offers clinically relevant information, with the potential to improve patient management and survival.BACKGROUND We and others have recently shown that tumor characteristics are altered throughout tumor progression. These findings emphasize the need for re-examination of tumor characteristics at relapse and have led to recommendations from ESMO and the Swedish Breast Cancer group. Here, we aim to determine whether tumor characteristics and molecular subtypes in breast cancer metastases confer clinically relevant prognostic information for patients. PATIENTS AND METHODS The translational aspect of the Swedish multicenter randomized trial called TEX included 111 patients with at least one biopsy from a morphologically confirmed locoregional or distant breast cancer metastasis diagnosed from December 2002 until June 2007. All patients had detailed clinical information, complete follow-up, and metastasis gene expression information (Affymetrix array GPL10379). We assessed the previously published gene expression modules describing biological processes [proliferation, apoptosis, human epidermal receptor 2 (HER2) and estrogen (ER) signaling, tumor invasion, immune response, and angiogenesis] and pathways (Ras, MAPK, PTEN, AKT-MTOR, PI3KCA, IGF1, Src, Myc, E2F3, and β-catenin) and the intrinsic subtypes (PAM50). Furthermore, by contrasting genes expressed in the metastases in relation to survival, we derived a poor metastasis survival signature. RESULTS A significant reduction in post-relapse breast cancer-specific survival was associated with low-ER receptor signaling and apoptosis gene module scores, and high AKT-MTOR, Ras, and β-catenin module scores. Similarly, intrinsic subtyping of the metastases provided statistically significant post-relapse survival information with the worst survival outcome in the basal-like [hazard ratio (HR) 3.7; 95% confidence interval (CI) 1.3-10.9] and HER2-enriched (HR 4.4; 95% CI 1.5-12.8) subtypes compared with the luminal A subtype. Overall, 25% of the metastases were basal-like, 32% HER2-enriched, 10% luminal A, 28% luminal B, and 5% normal-like. CONCLUSIONS We show that tumor characteristics and molecular subtypes of breast cancer metastases significantly influence post-relapse patient survival, emphasizing that molecular investigations at relapse provide prognostic and clinically relevant information. CLINICALTRIALS.GOV: This is the translational part of the Swedish multicenter and randomized trial TEX, clinicaltrials.gov identifier nct01433614 (http://www.clinicaltrials.gov/ct2/show/nct01433614).

Role of Tumor Pericytes in the Recruitment of Myeloid-Derived Suppressor Cells

BACKGROUND Pericytes are members of the tumor stroma; however, little is known about their origin, function, or interaction with other tumor components. Emerging evidence suggest that pericytes may regulate leukocyte transmigration. Myeloid-derived suppressor cells (MDSC) are immature myeloid cells with powerful inhibitory effects on T-cell-mediated antitumor reactivity. METHODS We generated subcutaneous tumors in a genetic mouse model of pericyte deficiency (the pdgfb (ret/ret) mouse) and littermate control mice (n = 6-25). Gene expression profiles from 253 breast cancer patients (stage I-III) were evaluated for clinic-pathological parameters and survival using Cox proportional hazard ratios (HRs) and 95% confidence intervals (CIs) based on a two-sided Wald test. RESULTS We report that pericyte deficiency leads to increased transmigration of Gr1(+)/CD11b(+) cells in experimentally induced tumors. Pericyte deficiency produced defective tumor vasculature, resulting in a more hypoxic microenvironment promoting IL-6 upregulation in the malignant cells. Silencing IL-6 expression in tumor cells attenuated the observed differences in MDSC transmigration. Restoring the pericyte coverage in tumors abrogated the increased MDSC trafficking to pericyte-deficient tumors. MDSC accumulation in tumors led to increases in tumor growth and in circulating malignant cells. Finally, gene expression analysis from human breast cancer patients revealed increased expression of the human MDSC markers CD33 and S100A9 with concomitant decreased expression of pericyte genes and was associated with poor prognosis (HR = 1.88, 95% CI = 1.08 to 3.25, P = .03). CONCLUSIONS Our data uncovers a novel paracrine interaction between tumor pericytes and inflammatory cells and delineates the cellular events resulting in the recruitment of MDSC to tumors. Furthermore, we propose for the first time a role for tumor pericytes in modulating the expression of immune mediators in malignant cells by promoting a hypoxic microenvironment.

Decreased expression of Yes-associated protein is associated with outcome in the luminal A breast cancer subgroup and with an impaired tamoxifen response.

BackgroundYes-associated protein (YAP1) is frequently reported to function as an oncogene in many types of cancer, but in breast cancer results remain controversial. We set out to clarify the role of YAP1 in breast cancer by examining gene and protein expression in subgroups of patient material and by downregulating YAP1 in vitro and studying its role in response to the widely used anti-estrogen tamoxifen.MethodsYAP1 protein intensity was scored as absent, weak, intermediate or strong in two primary breast cancer cohorts (n = 144 and n = 564) and mRNA expression of YAP1 was evaluated in a gene expression dataset (n = 1107). Recurrence-free survival was analysed using the log-rank test and Cox multivariate analysis was used to test for independence. WST-1 assay was employed to measure cell viability and a luciferase ERE (estrogen responsive element) construct was used to study the effect of tamoxifen, following downregulation of YAP1 using siRNAs.ResultsIn the ER+ (Estrogen Receptor α positive) subgroup of the randomised cohort, YAP1 expression was inversely correlated to histological grade and proliferation (p = 0.001 and p = 0.016, respectively) whereas in the ER- (Estrogen Receptor α negative) subgroup YAP1 expression correlated positively to proliferation (p = 0.005). Notably, low YAP1 mRNA was independently associated with decreased recurrence-free survival in the gene expression dataset, specifically for the luminal A subgroup (p < 0.001) which includes low proliferating tumours of lower grade, usually associated with a good prognosis. This subgroup specificity led us to hypothesize that YAP1 may be important for response to endocrine therapies, such as tamoxifen, extensively used for luminal A breast cancers. In a tamoxifen randomised patient material, absent YAP1 protein expression was associated with impaired tamoxifen response which was significant upon interaction analysis (p = 0.042). YAP1 downregulation resulted in increased progesterone receptor (PgR) expression and a delayed and weaker tamoxifen in support of the clinical data.ConclusionsDecreased YAP1 expression is an independent prognostic factor for recurrence in the less aggressive luminal A breast cancer subgroup, likely due to the decreased tamoxifen sensitivity conferred by YAP1 downregulation.

Use of Molecular Tools to Identify Patients With Indolent Breast Cancers With Ultralow Risk Over 2 Decades

Importance The frequency of cancers with indolent behavior has increased with screening. Better tools to identify indolent tumors are needed to avoid overtreatment. Objective To determine if a multigene classifier is associated with indolent behavior of invasive breast cancers in women followed for 2 decades. Design, Setting, and Participants This is a secondary analysis of a randomized clinical trial of tamoxifen vs no systemic therapy, with more than 20-year follow-up. An indolent threshold (ultralow risk) of the US Food and Drug Administration–cleared MammaPrint 70-gene expression score was established above which no breast cancer deaths occurred after 15 years in the absence of systemic therapy. Immunohistochemical markers (n = 727 women) and Agilent microarrays, for MammaPrint risk scoring (n = 652 women), were performed from formalin-fixed paraffin-embedded primary tumor blocks. Participants were postmenopausal women with clinically detected node-negative breast cancers treated with mastectomy or lumpectomy and radiation enrolled in the Stockholm tamoxifen (STO-3) trial, 1976 to 1990. Exposures After 2 years of tamoxifen vs no systemic therapy, regardless of hormone receptor status, patients without relapse who reconsented were further randomized to 3 additional years or none. Main Outcomes and Measures Breast cancer–specific survival assessed by Kaplan-Meier analyses and multivariate Cox proportional hazard modeling, adjusted for treatment, patient age, year of diagnosis, tumor size, grade, hormone receptors, and ERBB2/HER2 and Ki67 status. Results In this secondary analysis of node-negative postmenopausal women, conducted in the era before mammography screening, among the 652 women with MammaPrint scoring available (median age, 62.8 years of age), 377 (58%) and 275 (42%) were MammaPrint low and high risk, respectively, while 98 (15%) were ultralow risk. At 20 years, women with 70-gene high and low tumors but not ultralow tumors had a significantly higher risk of disease-specific death compared with ultralow-risk patients by Cox analysis (hazard ratios, 4.73 [95% CI, 1.38-16.22] and 4.54 [95% CI, 1.40-14.80], respectively). There were no deaths in the ultralow-risk tamoxifen-treated arm at 15 years, and these patients had a 20-year disease-specific survival rate of 97%, whereas for untreated patients the survival rate was 94%. Recursive partitioning identified ultralow risk as the most significant predictor of good outcome. In tumors “not ultralow risk,” tumor size greater than 2 cm was the most predictive of outcome. Conclusions and Relevance The ultralow-risk threshold of the 70-gene MammaPrint assay can identify patients whose long-term systemic risk of death from breast cancer after surgery alone is exceedingly low.

Multi-level gene expression signatures, but not binary, outperform Ki67 for the long term prognostication of breast cancer patients

Proliferation‐related gene signatures have been proposed to aid breast cancer management by providing reproducible prognostic and predictive information on a patient‐by‐patient basis. It is unclear however, whether a less demanding assessment of cell division rate (as determined in clinical setting by expression of Ki67) can function in place of gene profiling.

Contrasting breast cancer molecular subtypes across serial tumor progression stages: biological and prognostic implications

The relevance of the intrinsic subtypes for clinical management of metastatic breast cancer is not comprehensively established. We aimed to evaluate the prevalence and prognostic significance of drifts in tumor molecular subtypes during breast cancer progression. A well-annotated cohort of 304 women with advanced breast cancer was studied. Tissue microarrays of primary tumors and synchronous lymph node metastases were constructed. Conventional biomarkers were centrally assessed and molecular subtypes were assigned following the 2013 St Gallen guidelines. Fine-needle aspirates of asynchronous metastases were transcriptionally profiled and subtyped using PAM50. Discordant expression of individual biomarkers and molecular subtypes was observed during tumor progression. Primary luminal-like tumors were relatively unstable, frequently adopting a more aggressive subtype in the metastases. Notably, loss of ER expression and a luminal to non-luminal subtype conversion was associated with an inferior post-recurrence survival. In addition, ER and molecular subtype assessed at all tumor progression stages were independent prognostic factors for post-recurrence breast cancer mortality in multivariable analyses. Our results demonstrate that drifts in tumor molecular subtypes may occur during tumor progression, conferring adverse consequences on outcome following breast cancer relapse.

Guidance molecule SEMA3A restricts tumor growth by differentially regulating the proliferation of tumor-associated macrophages

Accumulation of tumor-associated macrophages (TAM) correlates with malignant progression, immune suppression, and poor prognosis. In this study, we defined a critical role for the cell-surface guidance molecule SEMA3A in differential proliferative control of TAMs. Tumor cell-derived SEMA3A restricted the proliferation of protumoral M2 macrophages but increased the proliferation of antitumoral M1, acting through the SEMA3A receptor neuropilin 1. Expansion of M1 macrophages in vivo enhanced the recruitment and activation of natural killer (NK) cells and cytotoxic CD8(+) T cells to tumors, inhibiting their growth. In human breast cancer specimens, we found that immunohistochemical levels of SEMA3A correlated with the expression of genes characteristic of M1 macrophages, CD8(+) T cells, and NK cells, while inversely correlating with established characters of malignancy. In summary, our results illuminate a mechanism whereby the TAM phenotype is controlled and identify the cell-surface molecule SEMA3A as a candidate for therapeutic targeting. Cancer Res; 76(11); 3166-78. ©2016 AACR.