Luminal Phenotype Research Articles

Functional and molecular characterisation of EO771.LMB tumours, a new C57BL/6-mouse-derived model of spontaneously metastatic mammary cancer.

The translation of basic research into improved therapies for breast cancer patients requires relevant preclinical models that incorporate spontaneous metastasis. We have completed a functional and molecular characterisation of a new isogenic C57BL/6 mouse model of breast cancer metastasis, comparing and contrasting it with the established BALB/c 4T1 model. Metastatic EO771.LMB tumours were derived from poorly metastatic parental EO771 mammary tumours. Functional differences were evaluated using both in vitro assays and spontaneous metastasis assays in mice. Results were compared to non-metastatic 67NR and metastatic 4T1.2 tumours of the 4T1 model. Protein and transcript levels of markers of human breast cancer molecular subtypes were measured in the four tumour lines, as well as p53 (Tp53) tumour-suppressor gene status and responses to tamoxifen in vivo and in vitro. Array-based expression profiling of whole tumours identified genes and pathways that were deregulated in metastatic tumours. EO771.LMB cells metastasised spontaneously to lung in C57BL/6 mice and displayed increased invasive capacity compared with parental EO771. By immunohistochemical assessment, EO771 and EO771.LMB were basal-like, as was the 4T1.2 tumour, whereas 67NR had a luminal phenotype. Primary tumours from all lines were negative for progesterone receptor, Erb-b2/Neu and cytokeratin 5/6, but positive for epidermal growth factor receptor (EGFR). Only 67NR displayed nuclear estrogen receptor alpha (ERα) positivity. EO771 and EO771.LMB expressed mutant p53, whereas 67NR and 4T1.2 were p53-null. Integrated molecular analysis of both the EO771/EO771.LMB and 67NR/4T1.2 pairs indicated that upregulation of matrix metalloproteinase-3 (MMP-3), parathyroid hormone-like hormone (Pthlh) and S100 calcium binding protein A8 (S100a8) and downregulation of the thrombospondin receptor (Cd36) might be causally involved in metastatic dissemination of breast cancer.

Molecular Markers as Prognostic Factors in DCIS and Small Invasive Breast Cancers.

Ductal carcinoma in situ (DCIS) accounts for up to half of screen-detected breast cancers and thus constitutes a major public health problem. Despite effective current treatment many patients with DCIS are either over- or undertreated because of the paucity of precise models to predict recurrence or progression. The combination of clinical and molecular factors as already applied for invasive disease may help to build such models also for DCIS. We compared 53 DCIS (36.6 %) and 92 (63.4 %) invasive breast cancer cases and found no significant differences in age, receptor status of ER, PR, and HER2, and the use of radiotherapy. Interestingly, the proportion of disseminated tumor cells (DTC) did also not significantly differ between DCIS and invasive cases (p = 0.57). A negative PR status was associated with the detection of DTCs (p = 0.026). We then compared relationships of clinical parameters and biomarkers with patients' prognosis in 43 DCIS and 40 small invasive tumors ≤ 5 mm (T1a). ER negativity was associated with shorter relapse free survival in the complete cohort (p = 0.004) and showed a trend in both subgroups (p = 0.053 for DCIS and p = 0.046 for T1a, respectively). In conclusion, we found markedly similar properties of both DCIS and small invasive breast cancers with respect to the distribution of several parameters as well as to the prognostic value of biomarkers. DCIS with a luminal phenotype seem to be characterized by a favourable prognosis.

Abstract 2099: Cellular and molecular determinants of retinoic acid sensitivity in breast cancer

Abstract All-trans-retinoic acid (ATRA) is the primary metabolite of vitamin A and a promising agent in the treatment/prevention of solid tumors, including breast cancer. Breast cancer is a very heterogeneous disease that can be classified according to the global gene-expression profiles into various subgroups. A rational use of ATRA in the clinics requires definition of the breast cancer subtypes characterized by sensitivity/natural-resistance to this compound and derived retinoids. In addition, it is fundamental to define the cellular and molecular mechanisms underlying the sensitivity/resistance to ATRA. As a first step in this direction, 44 breast cancer cell lines, belonging to the Cancer Cell Line Encyclopedia (CCLE) of the Broad Institute and recapitulating the heterogeneity of this tumor, were evaluated for their sensitivity to the anti-proliferative activity of ATRA. Individual cell lines were challenged with increasing concentrations of the retinoid for 3, 6 and 9 days. The concentrations of ATRA causing 50% of the maximal growth inhibition (IC50) and the maximal effect observed were calculated. The two parameters, along with the duplication time of the cell lines, were used to define a novel algorithm (ATRA SCORE). The ATRA score is a continuous parameter which defines the sensitivity of the cell lines to ATRA in a quantitative fashion. The lower the ATRA score the higher the sensitivity of the cell to the anti-proliferative action of ATRA. Using this score, an enrichment in cell lines characterized by a luminal phenotype and/or estrogen receptor (ER)-positivity was observed in the group showing sensitivity to ATRA. In contrast an enrichment in cell lines with a basal phenotype and/or ER-negativity was evident in the group characterized by natural resistance to the retinoid. Measurement of the mRNAs corresponding to various isoforms of the retinoid receptors belonging to the RAR and RXR family in the 44 cell lines allowed us to establish correlations between the expression of these transcripts and ATRA sensitivity. With respect to this, we found a significant and direct correlation with the expression levels of RARalpha1. This indicates that the receptor represents a determinant and a predictor of ATRA- sensitivity in breast cancer cells. The correlation was confirmed also at the protein level using specific RARalpha antibodies. Most of these findings were validated with the use of short-term cultures of breast cancer tissue slices obtained from surgical specimens. The availability of the global gene-expression profiles of each cell line permitted the construction of a gene fingerprint consisting of approximately 130 elements that has the potential to predict sensitivity to ATRA not only in breast cancer cell lines, but also in tumor samples. In addition, the identified gene fingerprint allowed the identification of various and unexpected molecular determinants of sensitivity which do not belong to the classic retinoid pathway. Citation Format: Enrico Garattini, Floriana Centritto, Silvio K. Garattini, Gabriela Paroni, Marco Bolis, Maddalena Fratelli, Mineko Terao. Cellular and molecular determinants of retinoic acid sensitivity in breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2099. doi:10.1158/1538-7445.AM2014-2099

Abstract 3914: Tcfap2c regulates growth and oncogenesis in neu-activated breast cancer

Abstract TFAP2C and mouse homolog tcfap2c are transcription factors that play a critical role in mammary gland development and in maintaining the luminal breast cancer phenotype. Interestingly, several human HER2-positive breast cancer cell lines display genetic dependency on TFAP2C. However, the role of TFAP2C in carcinogenesis and progression in neu-activated breast cancer remains unknown. We examined the role of tcfap2c in mammary gland carcinogenesis by examining tumor formation in mice expressing an activated rat neu gene using MMTV-neu with and without mammary gland conditional knockout (KO) of tcfap2c using MMTV-Cre. The time until spontaneous palpable tumor formation was measured in mice harboring the neu oncogene with and without tcfap2c. Mice with homozygous conditional deletion of tcfap2c demonstrated a significant delay in tumor formation compared to littermate controls that retained tcfap2c expression (42 vs. 25 weeks, p<0.004). Immunohistochemistry of the tumors demonstrated a luminal phenotype; cytokeratin 8 (CK8), a luminal marker, was more strongly expressed compared to cytokeratin 5 (CK5), a basal marker, in tumors from both control (883 vs. 0.1 cells/HPF, p<0.0005) and KO animals (845 vs. 0, p<0.0005). Cleaved caspase-3 (CC3) showed that apoptosis was increased in tumors from KO compared to control animals (5 vs. 0.3 CC3-positive cells/HPF, p<0.0008). Ki-67 showed that proliferation was decreased in KO compared to control (348 vs. 142 cells/HPF, p<0.009). To validate the effects of tcfap2c, we established a cell line from a tumor obtained from an animal expressing activated neu with homozygous floxed tcfap2c lacking Cre expression. Paired cell lines with differential expression of tcfap2c were established by infecting the cells with Adenovirus-Cre (Ad-Cre) or Adenovirus-Empty (Ad-Empty). Proliferation was assessed by MTT and demonstrated that loss of tcfap2c expression resulted in a 30% decrease in proliferation rate (p<0.006). The difference in proliferation was confirmed in vivo through xenograft experiments of these paired cell lines inoculated into athymic (nude) mice. Mice xenografted with the paired cell lines demonstrated that KO of tcfap2c resulted in a significant delay in tumors reaching a 2 cm threshold requiring euthanasia (27.8 vs. 19.8 days post-inoculation, p<0.003). In order to identify a potential mechanism accounting for the differences in proliferation and delay in carcinogenesis, we identified tcfap2c target genes by comparing the binding sites of tcfap2c by chromatin-immunoprecipitation-sequencing (ChIP-seq) and differential expression by microarray/RT-PCR. We identified erbb3 and egfr as two tcfap2c target genes by ChIP-seq. KO of tcfap2c resulted in a 3-fold and 12-fold reduction in erbb3 and egfr expression, respectively (p<0.05). Taken together, these findings indicate that tcfap2c plays critical roles in oncogenesis and progression of HER2-positive mammary cancer. Citation Format: Jung M. Park, Tong Wu, George Woodfield, Anthony Cyr, James De Andrade, Weizhou Zhang, Frederick Domann, Ronald J. Weigel. Tcfap2c regulates growth and oncogenesis in neu-activated breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3914. doi:10.1158/1538-7445.AM2014-3914

Abstract LB-222: Differentiation state heterogeneity in breast cancer and its role in therapeutic response

Abstract Despite the expansion of the therapeutic arsenal available to oncologists, resistance to chemo and targeted therapies remains a critical issue. Recent studies interrogating the intratumor heterogeneity of solid tumors suggest that phenotypic heterogeneity between the cells of a single tumor can provide both de novo and acquired resistance. Genetic, epigenetic and allelic differences provide unique phenotypes to tumor subpopulations, creating a landscape of differential sensitivity to therapeutics throughout the tumor. Through examining the expression of markers of the luminal, basal, and mesenchymal differentiation phenotypes of normal breast tissue, we discovered that breast cancer tumors can display dramatic intratumor cellular heterogeneity of these differentiation state markers. We hypothesized that this phenotypic heterogeneity of differentiation state was also present within breast cancer cell lines; Interrogation of the RNAseq data of 50+ breast cancer cell lines did in fact suggest that some cell lines exhibit simultaneous expression luminal, basal, and mesenchymal differentiation state markers. Further analysis of the differentiation markers through quantified immunofluorescence confirmed the presence of heterogeneity of marker expression amongst subpopulations of many of the cell lines. Our results reveal that this differentiation state heterogeneity is prominent in basal-like tumors and cell lines, and that the heterogeneity persists in 3D culture, 3D-bioprinted tumors, and in vivo. Comparison of our findings with results of previous large-scale drug studies revealed that heterogeneous lines display increased insensitivity to targeted therapeutics compared to less heterogeneous lines, suggesting that the differentiation state heterogeneity affords these lines some resistance. To determine the therapeutic consequences of this observed heterogeneity within the breast cancer cell lines, top ranked heterogeneous lines were run through high-throughput screens to assess the effects of >120 drugs on cell phenotypes including proliferation and differentiation-marker expression. We observed that certain therapeutics had significant effects on population heterogeneity. Importantly, we have found that small-molecule kinase inhibitors, in a target dependent manner, can shift the population to homogeneity in marker expression. It is an intriguing possibility that by reducing the intratumoral heterogeneity, we could increase the sensitivity of these cells to a second therapeutic. Therefore, our ongoing efforts are focused on identifying therapies that effectively eliminate the homogenous population we can now drive, as well as on understanding the mechanisms by which these therapies alter the differentiation phenotypes. Citation Format: Tyler Risom, Juha Rantala, Ellen Langer, Nicholas Wang, Laura Heiser, Megan Troxell, Joe Gray, Rosalie Sears. Differentiation state heterogeneity in breast cancer and its role in therapeutic response. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-222. doi:10.1158/1538-7445.AM2014-LB-222

Abstract 3589: Adipose stem cell-derived interferon gamma alters breast epithelial stem cell hierarchy

Abstract Autologous adipose stem cell transplantation is increasingly being used for soft tissue reconstruction, particularly after mastectomy. However, a recent study demonstrated recurrence of invasive ductal carcinoma in the breast within four months of autologous fat grafting. Previous studies have documented how cancer cells commandeer adipose stem cells (ASC) to support their survival by altering extracellular matrix composition and stiffness. This study focused on delineating the effects of ASCs on the self-renewal capacity of breast epithelial cells. The immortalized breast epithelial cell line MCF-10A, MCF-10A overexpressing Slug, and MCF-10A overexpressing SRC were examined using a mammosphere assay and flow cytometry for the effects of ASCs on their self-renewal and stem-luminal progenitor-differentiated cell surface marker profile. MCF-10A-Slug cells provided a unique model system to study breast epithelial cell dedifferentiation as Slug functions downstream of the breast cancer susceptibility gene BRCA1 and is a major suppressor of progenitor cell lineage commitment and differentiation. Interestingly, results with these established cell lines were reproduced in primary breast epithelial cells isolated from a high-risk patient. We show that secreted factors from ASCs promote self-renewal of all cell types examined. ASC co-culture or treatment with ASC conditioned media (CM) altered the number of CD49fhigh/EpCAMlow stem-like and CD49fmedium/EpCAMmedium luminal progenitor cells in 2D culture and mammosphere assays. ASCs secrete Interferon gamma (IFNγ), CD40 ligand, IL-23 and SERPINE1. Among these factors, only IFNγ treatment altered stem cell hierarchy; while MCF-10A-Slug cells displayed only luminal progenitor phenotype, cells treated with IFNγ contained a distinct stem-like cell population. These results suggest that paracrine activity of adipose stem cell-derived factors can either enhance dedifferentiation and promote proliferation of dormant tumor cells or predispose normal cells to transformation by blocking differentiation. Financial support: Susan G. Komen For the Cure SAC110025 and Zeta Tau Sorority (HN) Citation Format: Manjushree Anjanappa, Riesa M. Burnett, Stephanie Merfeld-Clauss, William A. Wooden, Keith L. March, Sunil Tholpady, Harikrishna Nakshatri. Adipose stem cell-derived interferon gamma alters breast epithelial stem cell hierarchy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3589. doi:10.1158/1538-7445.AM2014-3589

Abstract SY13-03: Signal pathways in prostate cancer stem cells

Abstract New therapeutic strategies are needed to treat prostate cancer as it progresses to the castration resistant stage following treatment with drugs to reduce androgen production or interfere with androgen receptor function. Genetic analyses of DNA alterations and RNA expression show that multiple pathways can be involved in this cancer progression. An understanding of the cellular components needed to initiate prostate cancer and sustain it during the process of metastasis may help to elucidate new pathways for therapy. We have developed techniques to transform mouse and human benign prostate epithelial basal stem cells and define them as one cell of origin which can respond to multiple stimuli to produce cancers which are capable of maturing to more differentiated cell types including luminal, squamous and neuroendocrine phenotypes. Different subtypes of cells with the properties of cancer stem cells capable of transplantation of the cancer phenotype have been defined. Current work is focusing on the activation of specific kinase driven pathways to provide new targets for therapy in metastatic prostate cancer. We previously demonstrated that castration resistant prostate cancer (CRPC) tissues exhibit increased tyrosine phosphorylation, suggesting that enhanced tyrosine kinase activity may contribute to advanced disease. To identify specific kinase activation patterns in metastatic CRPC, we utilized phospho-tyrosine peptide enrichment and quantitative mass spectrometry using tissue from CRPC patients obtained at rapid autopsy. Evaluation of these rare metastatic CRPC samples for tyrosine phosphorylation and kinases revealed activated SRC, EGFR, RET, ALK, and other targets. Importantly, these kinase activation patterns exhibited intrapatient similarity and interpatient heterogeneity implying clonal origins of these lesions. To evaluate the function of these kinases in metastatic CRPC, we are analyzing their role during specific steps of the metastatic cascade. We hope our approach will improve an understanding of the mechanistic role of kinase activation in prostate cancer and identify promising therapeutic strategies targeting non-mutated kinases for metastatic CRPC. Citation Format: Owen N. Witte. Signal pathways in prostate cancer stem cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr SY13-03. doi:10.1158/1538-7445.AM2014-SY13-03

Abstract 3053: Epigenetic control of cell phenotypes in the stem cell compartments of human prostate and prostate cancer: Implications for enhancement of prostate cancer therapies

Abstract There is increasing evidence that the phenotype of heterogeneous prostate tumors can be determined to some extent by reversible (non-mutagenic/epigenetic) genetic mechanisms. In the complex microenvironment of a developing cancer, this is an excellent survival strategy. The target cell (and molecular targets) for primary therapeutic intervention in prostate cancer is frequently determined by hormone responsiveness. We have shown that, in cells fractionated from human cancers (relative to cell lines or multiply passaged xenografts), the human prostate cancer-inducing cell (TIC) is a primitive, more basal-like cell with stem cell characteristics and limited hormone sensitivity. The TICs, and indeed normal tissue stem cells, have proven remarkably resistant to both radiotherapy and chemotherapy, as a result of their unique stem cell-like chromatin configuration, which can be ‘unlocked’ using common epigenetic control disruptors (e.g. HDAC inhibition), resulting in differentiation and the enhancement of therapeutic efficacy. This result prompted us to explore the nature of epigenetic control in multiple cellular fractions of both basal and luminal cells purified from established prostate cell lines, primary tumor cultures, primary xenografts of prostate cancer and small directed biopsies of freshly biopsied prostate tumor tissues. The data indicate a remarkable difference between multiple ‘standard’ cell line models of prostate cancer and cell types from near-patient models. Chromatin status in terms of histone modification, genomic methylation patterns, microRNA expression are all cell-type specific, in contrast to the variable data reported in extracts of unfractionated human tumor biopsies, reflecting tissue heterogeneity. The TIC fraction has retained (or acquired) the bivalent chromatin of embryonic stem cells in a fully undifferentiated state. Dominant epigenetic signals are associated with cellular differentiation and maintenance of the proliferating luminal phenotype, which can mask underlying changes responsible for tumor induction, stem cell maintenance and metastatic capability. Therapeutic targeting of the former simply shrinks tumors without affecting metastatic induction and patient survival. Unlinked clusters of coregulated genes define the differentiation process, and contain novel molecular targets such as stem cell maintenance genes, which could render all cells in a cancer susceptible to current therapies as part of a combination treatment strategy. The lower concentrations of stem cell modifying agents (e.g. retinoic acid and HDAC inhibitors), when used as phenotype modifiers rather than toxins, in combination with more established (cytotoxic) therapies, are more likely to be well tolerated in patients, compared to the toxic clinical doses of current ‘differentiating’ agents. Citation Format: Norman J. Maitland, Davide Pellacani, Jayant K. Rane, Fiona M. Frame, Anne T. Collins. Epigenetic control of cell phenotypes in the stem cell compartments of human prostate and prostate cancer: Implications for enhancement of prostate cancer therapies. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3053. doi:10.1158/1538-7445.AM2014-3053

Differential expression of genes involved in the epigenetic regulation of cell identity in normal human mammary cell commitment and differentiation.

The establishment and maintenance of mammary epithelial cell identity depends on the activity of a group of proteins, collectively called maintenance proteins, that act as epigenetic regulators of gene transcription through DNA methylation, histone modification, and chromatin remodeling. Increasing evidence indicates that dysregulation of these crucial proteins may disrupt epithelial cell integrity and trigger breast tumor initiation. Therefore, we explored in silico the expression pattern of a panel of 369 genes known to be involved in the establishment and maintenance of epithelial cell identity and mammary gland remodeling in cell subpopulations isolated from normal human mammary tissue and selectively enriched in their content of bipotent progenitors, committed luminal progenitors, and differentiated myoepithelial or differentiated luminal cells. The results indicated that, compared to bipotent cells, differentiated myoepithelial and luminal subpopulations were both characterized by the differential expression of 4 genes involved in cell identity maintenance: CBX6 and PCGF2, encoding proteins belonging to the Polycomb group, and SMARCD3 and SMARCE1, encoding proteins belonging to the Trithorax group. In addition to these common genes, the myoepithelial phenotype was associated with the differential expression of HDAC1, which encodes histone deacetylase 1, whereas the luminal phenotype was associated with the differential expression of SMARCA4 and HAT1, which encode a Trithorax protein and histone acetylase 1, respectively. The luminal compartment was further characterized by the overexpression of ALDH1A3 and GATA3, and the down-regulation of NOTCH4 and CCNB1, with the latter suggesting a block in cell cycle progression at the G2 phase. In contrast, myoepithelial differentiation was associated with the overexpression of MYC and the down-regulation of CCNE1, with the latter suggesting a block in cell cycle progression at the G1 phase.

Pathological features and prognosis of lobular carcinoma in Egyptian breast cancer patients.

To illustrate the differences between invasive lobular and ductal carcinomas (ILCs and IDCs) in terms of baseline demographics, pathologic features and recurrence in Egyptian breast cancer patients. Retrospective analysis of breast cancer patients diagnosed and treated between 2000 and 2008 was performed. 176 (8.5%) and 1758 (85%) cases were diagnosed with ILC and IDC, respectively. Compared with IDC, ILC was less observed in patients under 35 years of age (3.4 vs 9.3%; p = 0.009), and was associated with more bilaterality (p = 0.001), advanced tumor stage (p = 0.027) and nodal involvement (p = 0.004). On the other hand, IDC was significantly associated with more luminal B-like phenotype (16.9 vs 8.1%; p < 0.001) and more HER2-enriched disease (11.5 vs 2.7%; p < 0.001). At a median follow-up time of 64 months, ILC histology was independently associated with better disease-free survival (hazard ratio: 0.58; 95% CI: 0.36-0.93; p = 0.023). Bone and peritoneal relapses were more common in ILC, while lung relapses were more common in IDC. ILC has distinct biologic and prognostic features that may warrant different therapeutic approaches.

Anorectal stricture in 102 patients with Crohn's disease: natural history in the era of biologics.

The natural history of nonfistulising perianal Crohn's disease (PCD) remains unknown. To assess the long-term outcome of anorectal strictures. All outcomes of PCD patients with anorectal stricture followed in a single unit from 2005 to 2013 were reviewed, as well as subsequent therapeutic management. Cumulative incidence of stricture healing (disappearance of the anal stricture) was estimated using a Kaplan-Meier method and factor associated with an unfavourable course (persistent stricture S2, persistent stoma or proctectomy) with nonparametric test. A total of 102 patients (M/F: 37/65) were included. The duration of CD at diagnosis was 8.9 years. After a median follow-up period of 2.8 years, 52 of the 88 followed patients (59%) achieved anorectal stricture healing. Two patients (2%) developed anal adenocarcinoma. Female gender [HR 2.05 (1.1-4.03), P = 0.0221], disease duration of CD of less than 10 years [HR 1.94 (1.01-3.63), P = 0.0271], and anal fistula at stricture diagnosis [HR 2.36 (1.21-5.05), P = 0.0106) were significantly associated with anorectal stricture healing in a multivariate analysis model. Twenty-eight patients (32%) had an unfavourable course at the end of follow-up. Gender and introduction or optimisation of TNFα antagonist treatment decreased the risk of unfavourable course in multivariate analysis. Conversely, the Luminal B2 phenotype at CD diagnosis was the only factor associated with unfavourable course. Anorectal stricture does not imply a nonreversible and complicated condition related to severe perianal Crohn's disease. However, both the diagnosis of cancer and sepsis drainage remain challenging in this situation.

PIK3CA mutations in androgen receptor-positive triple negative breast cancer confer sensitivity to the combination of PI3K and androgen receptor inhibitors.

IntroductionTriple negative breast cancer (TNBC) is a heterogeneous collection of biologically diverse cancers, which contributes to variable clinical outcomes. Previously, we identified a TNBC subtype that has a luminal phenotype and expresses the androgen receptor (AR+). TNBC cells derived from these luminal AR + tumors have high frequency phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) mutations. The purpose of this study was to determine if targeting phosphoinositide 3-kinase (PI3K) alone or in combination with an AR antagonist is effective in AR + TNBC.MethodsWe determined the frequency of activating PIK3CA mutations in AR + and AR- TNBC clinical cases. Using AR + TNBC cell line and xenograft models we evaluated the effectiveness of PI3K inhibitors, used alone or in combination with an AR antagonist, on tumor cell growth and viability.ResultsPIK3CA kinase mutations were highly clonal, more frequent in AR + vs. AR- TNBC (40% vs. 4%), and often associated with concurrent amplification of the PIK3CA locus. PI3K/mTOR inhibitors had an additive growth inhibitory effect when combined with genetic or pharmacological AR targeting in AR + TNBC cells. We also analyzed the combination of bicalutamide +/- the pan-PI3K inhibitor GDC-0941 or the dual PI3K/mTOR inhibitor GDC-0980 in xenograft tumor studies and observed additive effects.ConclusionsWhile approximately one third of TNBC patients respond to neoadjuvant/adjuvant chemotherapy, recent studies have shown that patients with AR + TNBC are far less likely to benefit from the current standard of care chemotherapy regimens and novel targeted approaches need to be investigated. In this study, we show that activating PIK3CA mutations are enriched in AR + TNBC; and, we show that the growth and viability of AR + TNBC cell line models is significantly reduced after treatment with PI3K inhibitors used in combination with an AR antagonist. These results provide rationale for pre-selection of TNBC patients with a biomarker (AR expression) to investigate the use of AR antagonists in combination with PI3K/mTOR inhibitors.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-014-0406-x) contains supplementary material, which is available to authorized users.

Acid ceramidase is associated with an improved prognosis in both DCIS and invasive breast cancer

Acid ceramidase (ASAH1) a key enzyme of sphingolipid metabolism converting pro-apoptotic ceramide to sphingosine has been shown to be overexpressed in various cancers. We previously demonstrated higher expression of ASAH1 in ER positive compared to ER negative breast cancer. In the current study we performed subtype specific analyses of ASAH1 gene expression in invasive and non invasive breast cancer. We show that expression of ASAH1 is mainly associated with luminal A – like cancers which are known to have the best prognosis of all breast cancer subtypes. Moreover tumors with high ASAH1 expression among the other subtypes are also characterized by an improved prognosis. The good prognosis of tumors with high ASAH1 is independent of the type of adjuvant treatment in breast cancer and is also detected in non small cell lung cancer patients. Moreover, even in pre-invasive DCIS of the breast ASAH1 is associated with a luminal phenotype and a reduced frequency of recurrences. Thus, high ASAH1 expression is generally associated with an improved prognosis in invasive breast cancer independent of adjuvant treatment and could also be valuable as prognostic factor for pre-invasive DCIS.

Metaplastic carcinoma of the breast: an immunohistochemical study.

BackgroundMetaplastic breast carcinoma is a rare entity of breast cancer expressing epithelial and/or mesenchymal tissue within the same tumor. The aim of this study is to evaluate the clinicopathological features of metaplastic breast carcinoma and to confirm the triple negative, basal-like and/or luminal phenotype of this type of tumor by using immunohistochemical staining.MethodsSeven cases of MBC were evaluated for clinico-pathological features including follow up data. Cases were studied immunohistochemically by CK-Pan, Vimentin, ER, PR, HER2, basal markers (CK5/6, p63, EGFR, SMA and S-100), luminal cytokeratins (CK8, CK18 and CK19), markers for syncytial cells (β-HCG and PLAP), as well as prognostic markers (p53, ki-67 and calretinin).ResultsThe mean age of the patients was 36 years. Three cases showed choriocarcinomatous features. All of our cases were negative for ER, PR and HER2. Six out of the 7 cases showed basal-like differentiation by demonstrating positivity with at least one of the basal/myoepithelial markers. Also 6 out of the 7 cases expressed luminal type cytokeratins (CK8, CK18 and/or CK19). P53 was positive in 3 cases, ki-67 was strongly expressed in only one case, while calretinin was expressed in 6 cases.ConclusionMetaplastic breast carcinoma presents in our population at a younger age group than other international studies. All cases are categorized immunohistochemically under the triple negative group of breast cancer and 86% of them exhibited basal-like and luminal phenotype. Majority of cases developed local recurrence and distant metastasis in a relatively short period of time.Virtual SlidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1101289295115804

C-FLIP(L) contributes to TRAIL resistance in HER2-positive breast cancer

Breast cancers with HER2 amplification have a poorer prognosis than the luminal phenotypes. TRAIL activates apoptosis upon binding its receptors in some but not all breast cancer cell lines. Herein, we investigated the expression pattern of c-FLIP(L) in a cohort of 251 invasive breast cancer tissues and explored its potential role in TRAIL resistance. C-FLIP(L) was relatively high-expressed in HER2-positive breast cancer in comparison with other molecular subtypes, co-expressed with TRAIL death receptors, and inversely correlated with the apoptosis index. Downregulation of c-FLIP(L) sensitized SKBR3 cells to TRAIL-induced apoptosis in a concentration- and time-dependent manner and enhanced the activities and cleavages of caspase-8 and caspase-3, without altering the surface expression of death receptors. Together, our results indicate that c-FLIP(L) promotes TRAIL resistance and inhibits caspase-3 and caspase-8 activation in HER2-positive breast cancer.

VGLL1 expression is associated with a triple-negative basal-like phenotype in breast cancer.

Vestigial-like 1 (VGLL1) is a poorly characterized gene encoding a transcriptional co-activator structurally homologous to TAZ and YAP that modulates the Hippo pathway in Drosophila. In this study, we examined the expression of VGLL1 and its intronic miRNA, miR-934, in breast cancer. VGLL1 and miR-934 expression miRNA profiling was carried out on frozen samples of grade 3 invasive ductal carcinomas. VGLL1 protein was also examined in 433 sporadic and BRCA1-associated breast carcinomas on tissue microarrays. RNA-seq data from The Cancer Genome Atlas (TCGA) was used to confirm differences in VGLL1 and miR-934 expression in different breast cancer subtypes, and to correlate their expression with that of other genes and miRNAs. Of 28 miRNAs differentially expressed in estrogen receptor (ER)-positive and ER-negative grade 3 breast carcinomas, miR-934 was most strongly upregulated in ER-negative carcinomas, and its expression was correlated with that of VGLL1. Nuclear VGLL1 expression was observed in 13% of sporadic breast carcinomas, and while VGLL1 was only occasionally found in luminal A (0.70%) and B (5.60%) carcinomas, it was often expressed in HER2-positive (17%), triple-negative (TN) breast carcinomas (>40%) and BRCA1-associated TN carcinomas (>50%). These findings were confirmed in the TCGA dataset, which revealed positive associations with luminal progenitor genes (GABRP, SLC6A14, FOXC1, PROM1, and BBOX1) and strong negative correlations with ER-associated genes (ESR1, C6ORF211, GATA3, and FOXA1). Moreover, VGLL1 expression was associated with reduced overall survival. In conclusion, VGLL1 and miR-934 are mainly expressed in sporadic and BRCA1-associated TN basal-like breast carcinomas, and their coordinated expression, at least partially mediated by the direct modulation of ESR1, might be involved in the maintenance of a luminal progenitor phenotype.

45P - Methylation of DNA Free in Blood Sample as Prognostic Factor in Breast Cancer Subtype

Background: The impact of methylation as a factor on tumor progression and its potential predictive value remain relatively unknown. The aim of this study was to correlate methylation levels of promoter ESR1 with association to known prognostic factors in breast cancer and their correlation with expression of estrogen receptor (ER) in tumor and luminal phenotype.

Invasive breast carcinomas of no special type with osteoclast-like giant cells frequently have a luminal phenotype.

Invasive carcinoma of no special type (NST) with osteoclast-like giant cells (OGCs) represents a unique type of breast neoplasm, characterized by the presence of multinucleated OGCs and a vascularized, hemorrhagic stroma. Because of its rarity, the literature regarding this tumor remains limited and a detailed immunophenotype of this tumor has not been established as yet. We report a clinicopathological and immunohistochemical study of 42 patients with invasive carcinoma NST with OGCs. Macroscopically, these tumors presented as a well-delimited red-brown mass. A remarkable feature of the tumor was the presence of OGCs in the fibroblastic or hemorrhagic vascular stroma, as well as in the adjacent tumor nests or glandular lumina. The number of OGCs varied from 8 to 105 per 10 high-power fields with an average of 48. The tumors were well to moderately differentiated. Cribriform architecture was observed in 27 tumors (63%). All of the 36 available tumors were of luminal phenotype, according to the Ki67 labeling index 89% luminal A and 11% luminal B. With a mean follow-up time of 46.4months, lung metastasis was found in 2 patients (5%) at 7 and 11years after the operation, respectively. None of the other cases had presented with evidence of recurrence or metastasis. To the best of our knowledge, this is the largest reported series of invasive carcinoma NST with OGCs as yet. Our study revealed that invasive carcinoma NST with OGCs exhibit a luminal phenotype with luminal A subtype as the major group.

Distinct Pathways Regulated by RET and Estrogen Receptor in Luminal Breast Cancer Demonstrate the Biological Basis for Combination Therapy

We investigated directed therapy based on TFAP2C-regulated pathways to inform new therapeutic approaches for treatment of luminal breast cancer. TFAP2C regulates the expression of genes characterizing the luminal phenotype including ESR1 and RET, but pathway cross talk and potential for distinct elements have not been characterized. Activation of extracellular signal-regulated kinases (ERK) and AKT was assessed using phosphorylation-specific Western blot. Cell proliferation was measured with MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] after siRNA (small interfering RNA) gene knockdown or drug treatment. Cell cycle, Ki-67, and cleaved caspase 3 were measured by fluorescence-activated cell sorting. Tumorigenesis was assessed in mice xenografts. Knockdown of TFAP2C or RET inhibited GDNF (glial cell line-derived neurotrophic factor)-mediated activation of ERK and AKT in MCF-7 cells. Similarly, sunitinib, a small-molecule inhibitor of RET, blocked GDNF-mediated activation of ERK and AKT. Inhibition of RET either by gene knockdown or by treatment with sunitinib or vandetanib reduced RET-dependent growth of luminal breast cancer cells. Interestingly, knockdown of TFAP2C, which controls both ER (estrogen receptor) and RET, demonstrated a greater effect on cell growth than either RET or ER alone. Parallel experiments using treatment with tamoxifen and sunitinib confirmed the increased effectiveness of dual inhibition of the ER and RET pathways in regulating cell growth. Whereas targeting the ER pathway altered cell proliferation, as measured by Ki-67 and S-phase, anti-RET primarily increased apoptosis, as demonstrated by cleaved caspase 3 and increased TUNEL (terminal deoxyneucleotidyl transferase dUTP nick end labeling) expression in xenografts. ER and RET primarily function through distinct pathways regulating proliferation and cell survival, respectively. The findings inform a therapeutic approach based on combination therapy with antiestrogen and anti-RET in luminal breast cancer.

ESR1gene promoter region methylation in free circulating DNA and its correlation with estrogen receptor protein expression in tumor tissue in breast cancer patients

BackgroundTumor expression of estrogen receptor (ER) is an important marker of prognosis, and is predictive of response to endocrine therapy in breast cancer. Several studies have observed that epigenetic events, such methylation of cytosines and deacetylation of histones, are involved in the complex mechanisms that regulate promoter transcription. However, the exact interplay of these factors in transcription activity is not well understood. In this study, we explored the relationship between ER expression status in tumor tissue samples and the methylation of the 5′ CpG promoter region of the estrogen receptor gene (ESR1) isolated from free circulating DNA (fcDNA) in plasma samples from breast cancer patients.MethodsPatients (n = 110) with non-metastatic breast cancer had analyses performed of ER expression (luminal phenotype in tumor tissue, by immunohistochemistry method), and the ESR1-DNA methylation status (fcDNA in plasma, by quantitative methylation specific PCR technique).ResultsOur results showed a significant association between presence of methylated ESR1 in patients with breast cancer and ER negative status in the tumor tissue (p = 0.0179). There was a trend towards a higher probability of ESR1-methylation in those phenotypes with poor prognosis i.e. 80% of triple negative patients, 60% of HER2 patients, compared to 28% and 5.9% of patients with better prognosis such as luminal A and luminal B, respectively.ConclusionSilencing, by methylation, of the promoter region of the ESR1 affects the expression of the estrogen receptor protein in tumors of breast cancer patients; high methylation of ESR1-DNA is associated with estrogen receptor negative status which, in turn, may be implicated in the patient’s resistance to hormonal treatment in breast cancer. As such, epigenetic markers in plasma may be of interest as new targets for anticancer therapy, especially with respect to endocrine treatment.