Fina Climent

RANK Induces Epithelial–Mesenchymal Transition and Stemness in Human Mammary Epithelial Cells and Promotes Tumorigenesis and Metastasis

Paracrine signaling through receptor activator of NF-κB (RANK) pathway mediates the expansion of mammary epithelia that occurs during pregnancy, and activation of RANK pathway promotes mammary tumorigenesis in mice. In this study we extend these previous data to human cells and show that the RANK pathway promotes the development of mammary stem cells and breast cancer. Overexpression of RANK (FL-RANK) in a panel of tumoral and normal human mammary cells induces the expression of breast cancer stem and basal/stem cell markers. High levels of RANK in untransformed MCF10A cells induce changes associated with both stemness and transformation, including mammary gland reconstitution, epithelial-mesenchymal transition (EMT), increased migration, and anchorage-independent growth. In addition, spheroids of RANK overexpressing MCF10A cells display disrupted acinar formation, impair growth arrest and polarization, and luminal filling. RANK overexpression in tumor cells with nonfunctional BRCA1 enhances invasiveness in acinar cultures and increases tumorigenesis and metastasis in immunodeficient mice. High levels of RANK were found in human primary breast adenocarcinomas that lack expression of the hormone receptors, estrogen and progesterone, and in tumors with high pathologic grade and proliferation index; high RANK/RANKL expression was significantly associated with metastatic tumors. Together, our findings show that RANK promotes tumor initiation, progression, and metastasis in human mammary epithelial cells by increasing the population of CD44(+)CD24(-) cells, inducing stemness and EMT. These results suggest that RANK expression in primary breast cancer associates with poor prognosis.

VAV3 mediates resistance to breast cancer endocrine therapy

IntroductionEndocrine therapies targeting cell proliferation and survival mediated by estrogen receptor α (ERα) are among the most effective systemic treatments for ERα-positive breast cancer. However, most tumors initially responsive to these therapies acquire resistance through mechanisms that involve ERα transcriptional regulatory plasticity. Herein we identify VAV3 as a critical component in this process.MethodsA cell-based chemical compound screen was carried out to identify therapeutic strategies against resistance to endocrine therapy. Binding to ERα was evaluated by molecular docking analyses, an agonist fluoligand assay and short hairpin (sh)RNA–mediated protein depletion. Microarray analyses were performed to identify altered gene expression. Western blot analysis of signaling and proliferation markers, and shRNA-mediated protein depletion in viability and clonogenic assays, were performed to delineate the role of VAV3. Genetic variation in VAV3 was assessed for association with the response to tamoxifen. Immunohistochemical analyses of VAV3 were carried out to determine its association with therapeutic response and different tumor markers. An analysis of gene expression association with drug sensitivity was carried out to identify a potential therapeutic approach based on differential VAV3 expression.ResultsThe compound YC-1 was found to comparatively reduce the viability of cell models of acquired resistance. This effect was probably not due to activation of its canonical target (soluble guanylyl cyclase), but instead was likely a result of binding to ERα. VAV3 was selectively reduced upon exposure to YC-1 or ERα depletion, and, accordingly, VAV3 depletion comparatively reduced the viability of cell models of acquired resistance. In the clinical scenario, germline variation in VAV3 was associated with the response to tamoxifen in Japanese breast cancer patients (rs10494071 combined P value = 8.4 × 10−4). The allele association combined with gene expression analyses indicated that low VAV3 expression predicts better clinical outcome. Conversely, high nuclear VAV3 expression in tumor cells was associated with poorer endocrine therapy response. Based on VAV3 expression levels and the response to erlotinib in cancer cell lines, targeting EGFR signaling may be a promising therapeutic strategy.ConclusionsThis study proposes VAV3 as a biomarker and a rationale for its use as a signaling target to prevent and/or overcome resistance to endocrine therapy in breast cancer.

Resistance to Taxanes in Triple-Negative Breast Cancer Associates with the Dynamics of a CD49f+ Tumor-Initiating Population.

Summary Taxanes are a mainstay of treatment for breast cancer, but resistance often develops followed by metastatic disease and mortality. Aiming to reveal the mechanisms underlying taxane resistance, we used breast cancer patient-derived orthoxenografts (PDX). Mimicking clinical behavior, triple-negative breast tumors (TNBCs) from PDX models were more sensitive to docetaxel than luminal tumors, but they progressively acquired resistance upon continuous drug administration. Mechanistically, we found that a CD49f+ chemoresistant population with tumor-initiating ability is present in sensitive tumors and expands during the acquisition of drug resistance. In the absence of the drug, the resistant CD49f+ population shrinks and taxane sensitivity is restored. We describe a transcriptional signature of resistance, predictive of recurrent disease after chemotherapy in TNBC. Together, these findings identify a CD49f+ population enriched in tumor-initiating ability and chemoresistance properties and evidence a drug holiday effect on the acquired resistance to docetaxel in triple-negative breast cancer.

Lymphangioleiomyomatosis Biomarkers Linked to Lung Metastatic Potential and Cell Stemness.

Lymphangioleiomyomatosis (LAM) is a rare lung-metastasizing neoplasm caused by the proliferation of smooth muscle-like cells that commonly carry loss-of-function mutations in either the tuberous sclerosis complex 1 or 2 (TSC1 or TSC2) genes. While allosteric inhibition of the mechanistic target of rapamycin (mTOR) has shown substantial clinical benefit, complementary therapies are required to improve response and/or to treat specific patients. However, there is a lack of LAM biomarkers that could potentially be used to monitor the disease and to develop other targeted therapies. We hypothesized that the mediators of cancer metastasis to lung, particularly in breast cancer, also play a relevant role in LAM. Analyses across independent breast cancer datasets revealed associations between low TSC1/2 expression, altered mTOR complex 1 (mTORC1) pathway signaling, and metastasis to lung. Subsequently, immunohistochemical analyses of 23 LAM lesions revealed positivity in all cases for the lung metastasis mediators fascin 1 (FSCN1) and inhibitor of DNA binding 1 (ID1). Moreover, assessment of breast cancer stem or luminal progenitor cell biomarkers showed positivity in most LAM tissue for the aldehyde dehydrogenase 1 (ALDH1), integrin-ß3 (ITGB3/CD61), and/or the sex-determining region Y-box 9 (SOX9) proteins. The immunohistochemical analyses also provided evidence of heterogeneity between and within LAM cases. The analysis of Tsc2-deficient cells revealed relative over-expression of FSCN1 and ID1; however, Tsc2-deficient cells did not show higher sensitivity to ID1-based cancer inhibitors. Collectively, the results of this study reveal novel LAM biomarkers linked to breast cancer metastasis to lung and to cell stemness, which in turn might guide the assessment of additional or complementary therapeutic opportunities for LAM.

ALLOGENEIC HEMATOPOIETIC STEM CELL TRANSPLANTATION (ALLO-HSCT) FOR PATIENTS WITH RELAPSED/REFRACTORY SYSTEMIC ANAPLASTIC LARGE CELL LYMPHOMA (R/R SALCL). A RETROSPECTIVE ANALYSIS OF THE LYMPHOMA WORKING PARTY-EBMT.

Introduction: Long‐term survival of advanced‐stage and relapsed extranodal NK/T cell lymphoma (ENKTL) is 33%–45%. Our previous study of GLIDE (gemcitabine, L‐asparaginase, ifosfamide, dexamethasone and etoposide) chemotherapy reported complete response (CR) rate, and 3‐year overall survival (OS) of these patients were 57.1% and 56%, respectively. The role of ASCT as consolidation in these patients is unclear. To address this issue, we analyzed the efficacy and safety of our treatment strategy, GLIDE induction followed by ASCT, in newly diagnosed stage IV and relapsed ENKTL. Patients and methods: We treated 60 patients with newly diagnosed stage IV(n = 49) and relapsed (n = 11) ENKTL from 2010 to 2016. The median age at recruitment was 38 years and the median follow‐ up period was 13.4 months. Patients were treated with GLIDE every 4 weeks, and responses were evaluated with PET/CT every 2 cycles. Patients achieving CR underwent ASCT or continued with GLIDE up to 6 cycles. Others finished 6 cycles of GLIDE. Overall response rate (ORR), CR, OS and progression free survival (PFS) were calculated using standard methods. Statistical analysis was done using Fishers exact test or Chi‐square test/Kruskal–Wallis test. Kaplan–Meier method was used for time‐to‐event analysis including overall survival and progression free survival. The Log‐rank test was used to evaluate the difference in time‐to‐event endpoints between patient groups. Results: Fifty‐seven patients had finished planed treatment with 1 withdraw of informed consent after cycle 1, and 2 death of sepsis during cycle 1 and cycle 2 respectively. Twenty‐one patients underwent ASCT. The ORR was 81.4%, and the CR was 69.5% with early CR (CR after 2 cycles) of 57.6%. Estimated 5‐year OS and PFS rates of the whole cohort and patients underwent ASCT were 68.7%, 54.0%, 79.6% and 85.2%, respectively. Univariate analysis revealed that ECOG ≤1, IPI ≤2, early CR and ASCT were associated with less relapse and death. Multivariate analysis showed ECOG ≥2 was an independent risk factor for disease progression (HR = 4.321, 95% CI 1.127 ~ 16.572, P = 0.033) and death (HR = 46.254, 2.150 ~ 993.190, P = 0.014), and ASCT is associated with better PFS (HR = 0.058, 95%CI 0.007 ~ 0.495, P = 0.009) and OS (HR = 0.019,95% CI 0.001 ~ 0.596, P = 0.024). Figure 1 highlights the OS and PFS of whole cohort (A) and ASCT patients (B). Myelosuppression was the most common adverse reaction(AE). The incidences of level 4 neutropenia, thrombocytopenia and anemia were 46.6%, 28.6% and 5.3%, respectively. The most common non‐hematologic AE was fever with neutropenia (36.5% of total cycles), while others were mild and manageable. Conclusions: GLIDE is an effective regiment for newly diagnosed stage IV and relapsed ENKTL. Up‐front ASCT after achieving CR can reduce relapse and prolong survival. Treatment related adverse reactions and support care need concerns.

Abstract P6-01-05: Potential biomarkers of response to primary antiangiogenic and hormonal therapy in post-menopausal women with hormone-positive, HER2-negative primary breast cancer

Introduction The role of antiangiogenic therapy in primary hormonal therapy in HER2-negative ER-positive early breast cancer is unknown. Potential biomarkers of response to antiangiogenic therapy are lacking. A phase I clinical trial was conducted with sunitinib and exemestane given at conventional dose (25 mg/d) during 6 months, before surgery. 18 patients were enrolled, 15 in dose level 0 of sunitinib (25 mg/d) and 3 in dose level 1 (37.5 mg/d). Results were presented in SABCS 2011. Main toxicities were: asthenia (50%), leucopenia (28%, all grade 2), diarrhea (28%), mucositis (22%), and hypertension (22%). 10 patients achieved radiological partial response (56%) and 8 patients stable disease (44%). 7 patients (38.89%) obtained a pathological downstaging. Potential biomarkers of response to antiangiogenic therapy are presented. Materials and methods Tissue samples were obtained by fine-needle aspiration or core needle biopsy before starting treatment, one month after and at surgery. All samples were formalin-fixed paraffin-embedded. Ki67, phospho-ERK and mean vessel density (by CD34), were analyzed by immunostaining. At the same time points, plasma levels of angiopoietin 2 (ANG2), soluble VEGFR2 and VEGF were analyzed by ELISA. Basal levels and its changes over time were evaluated and correlated with clinical outcomes. Results Changes in Ki67 were observed, with a median value of 16.44%pre surgery and 12.78% post surgery (p=0.062). A significant decrease in mean vessel density was not observed. Basal levels of plasmatic biomarkers are shown in the next table: Values of biomarkers are average in ng/ml ± Standard Deviation. Differences analyzed by student9s t-test. Abreviations: SD= stable disease. PR= partial response. p = p value. Path. Downst.= Pathological downstaging. Furthermore, there was a significant decrease in VEGFR2 mean levels after one month of treatment (p=0.0046): 12284±3449 ng/ml at baseline; 8148±3216 ng/ml at one month and 7732±3052 ng/ml at 6 months. Differences between basal and one month determination were significant (p Conclusions Baseline ANG2 levels have a promising predictive value of response in this phase I trial of neoadjuvant combination of sunitinib plus exemestane. There is a significant early decrease in VEGFR2 with treatment with sunitinib. These results should be validated in further studies to improve the selection of patients for antiangiogenic+hormonal therapy. Citation Format: Helena Verdaguer, Serafin Morales, Valenti Navarro, Alba Martinez Lopez, Anna Petit, Fina Climent, Oriol Casanovas, Sonia Pernas. Potential biomarkers of response to primary antiangiogenic and hormonal therapy in post-menopausal women with hormone-positive, HER2-negative primary breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-01-05.

Clinical Characteristics and Risk of Relapse for Patients with Stage I-II Diffuse Large B-cell Lymphoma Treated in First Line with Immunochemotherapy.

Diffuse large b-cell lymphoma (DLBCL) is an aggressive and potentially curable lymphoma that presents itself as stage I-II in 30% of all cases. It is known that in these localized stages, 15-20% of patients treated without rituximab eventually relapse, but less data exist regarding rituximab era. We have analyzed clinico-pathological features and risk of relapse in 98 patients with I-II stage DLBCL in complete response (CR) or unconfirmed CR (CRu) after first-line treatment consisting of immunochemotherapy. Twelve patients (12.2%) eventually relapsed. Late relapse, more than two years after diagnosis, occurred in three patients, and early relapse, less than two years after diagnosis, was documented in nine patients. Median time from diagnosis to relapse was 0.61 years for patients with early relapse and 3.66 years for patients with late relapse. The second CR rate obtained was similar in the late and in early relapsing patients, being 33% versus 44% (p = 0.072), respectively. Three-year overall survival (OS) was 22% for early relapsing patients and 33% for late relapsing patients (p = 0.65). In conclusion, patients who are diagnosed with stage I-II DLBCL and achieve a CR/CRu with first line immunochemotherapy have a good prognosis. However, a proportion of patients relapse, and this is less frequent in patients treated with first line with immunochemotherapy. These patients have a poor prognosis.

Breast Cancer OncoGuia: surgical pathology report guidelines.

Location† Laterality: ❐ Right breast ❐ Left breast† Quadrant: ❐ Upper outer ❐ Upper inner ❐ Lower outer ❐ Lower inner ❐ Nipple ❐ Others Type of sample† Needle biopsy: ❐ CNB (core needle biopsy) ❐ VAB (vacuum-assisted biopsy) No. of cores: † Surgical biopsy: ❐ With wire-guided localisation ❐ Without wire-guided localisation† Surgical treatment: ❐ Lumpectomy ❐ Quadrantectomy❐ Simple mastectomy ❐ Modifi ed radical❐ Others (specify) mastectomy † Sample size ........ mm† Sample weight: ........ (gr)† Skin involvement: ❐ Yes ❐ No† Size of the lesion/tumour ......... mm† Preoperative examination† Resection margin (distance and specifi ed margins): ...† Lymph node sampling:❐ No lymph nodes present❐ Sentinel lymph node(s) present❐ Axillary dissection (partial or complete)❐ Lymph nodes present within the breast specimen❐ Other lymph nodes, specify location: † Macro photo: ❐ Yes ❐ No† Tumour bank: ❐ Yes ❐ No† Fixation time: ........ (h)† Key to sections: ........