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Background: Inflammatory breast cancer (IBC) is characterized by a young age at diagnosis, high metastatic potential, and poor overall survival. To better understand the etiology of this aggressive disease, we performed a retrospective study to evaluate genomic alterations and tumor microenvironment in IBC patients. Methods: Targeted next generation sequencing (NGS) was performed using blood cell-free DNA (cfDNA) (n=32), paired DNA from tumor tissues (n=29) and DNA from whole blood or normal tissue (n=20). We also characterized the tumor infiltrating lymphocytes (TILs) in tumor tissue biopsies by studying several markers (CD4, CD8, FoxP3, CD20, PD-1 and PD-L1) through immunohistochemistry (IHC) staining. Results: Our results shown a high incidence of germline variants in patients with IBC that could be associated with an increased risk of developing the disease. Germline variants were found more frequently in patients with TN (38.9%) than non-TN IBC (28.6%). IHC staining revealed that in 7 of 24 (29%) patients, tumor biopsies were positive for PD-L1 and PD-1 expression on TILs. Patients with PD-L1&PD-1 positive biopsies had a lower OS (31 months) than patients with PD-L1&PD-1 negative (38 months) tumors, though the difference was not statistically significant. PD-L1&PD-1 positive tumors had significantly more CD8+ (cytotoxic T-cells), CD20+ (B-cells) and FoxP3+ (Tregs) cells than PD-L1&PD-1 negative ones. Furthermore, most IBC patients showed to have deficiencies in the homologous recombination repair (HRR) pathway (BRCA1, BRCA2, PALB2, RAD51C, ATM, BARD1) making them sensitive to poly (ADP-ribose) polymerase (PARP) inhibitors. All tumor biopsies showed positive staining for p-FAK1(Tyr 397). Conclusion: A subset of IBC patients (29%) expressed both PD-L1 and PD-1 on tumor immune cells infiltrates, providing rationale for testing PD-1/PD-L1 blocking therapies. IBC patients could have benefits from PD-1/PD-L1 blocking therapies alone or in combination with PARP or FAK (Tyr397) kinase inhibitors, both having the capacity of altering the tumor microenvironment. Citation Format: Maria F. Arisi, Yulan Gong, Rajeswari Nagarathinam, Lorenzo Gerratana, Jianming Pei, Kathy Q. Cai, Jennifer Winn, Zachary Hasse, Elias Obeid, Julio Noriega, Christopher Sebastiano, Eric Ross, R. Katherine Alpaugh, Massimo Cristofanilli, Sandra V Fernandez. Genetic variants and tumor microenvironment in inflammatory breast cancer: Clues for targeted therapies [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-24-02.

Supplementary Figure 1 from Epithelial to Mesenchymal Transition in Human Breast Epithelial Cells Transformed by 17β-Estradiol

The estrogen dependence of breast cancer has long been recognized; however, the role of 17β-estradiol (E2) in cancer initiation was not known until we showed that it induces complete neoplastic transformation of the human breast epithelial cells MCF-10F. E2 treatment of MCF-10F cells progressively induced high colony efficiency and loss of ductulogenesis in early transformed (trMCF) cells and invasiveness in Matrigel invasion chambers. The cells that crossed the chamber membrane were collected and identified as bsMCF; their subclones were designated bcMCF; and the cells harvested from carcinoma formation in severe combined immunodeficient mice were designated caMCF. These phenotypes correlated with gene dysregulation during the progression of the transformation. The highest number of dysregulated genes was observed in caMCF, being slightly lower in bcMCF, and lowest in trMCF. This order was consistent with the extent of chromosome aberrations (caMCF > bcMCF >>> trMCF). Chromosomal amplifications were found in 1p36.12-pter, 5q21.1-qter, and 13q21.31-qter. Losses of the complete chromosome 4 and 8p11.21-23.1 were found only in tumorigenic cells. In tumor-derived cell lines, additional losses were found in 3p12.1-14.1, 9p22.1-pter, and 18q11.21-qter. Functional profiling of dysregulated genes revealed progressive changes in the integrin signaling pathway, inhibition of apoptosis, acquisition of tumorigenic cell surface markers, and epithelial-mesenchymal transition. In tumorigenic cells, the levels of E-cadherin, epithelial membrane antigen, and various keratins were low and CD44E/CD24 were negative, whereas SNAI2, vimentin, S100A4, FN1, HRAS, transforming growth factor β1, and CD44H were high. The phenotypic and genomic changes triggered by estrogen exposure that lead normal cells to tumorigenesis confirm the role of this steroid hormone in cancer initiation. [Cancer Res 2007;67(23):11147–57]

Supplementary Figure 2 from Epithelial to Mesenchymal Transition in Human Breast Epithelial Cells Transformed by 17β-Estradiol

Supplementary Figure 3 from Epithelial to Mesenchymal Transition in Human Breast Epithelial Cells Transformed by 17β-Estradiol

Background Inflammatory breast cancer (IBC) is a rare but aggressive carcinoma characterized by severe erythema and edema of the breast, with many patients presenting in advanced metastatic disease. The “inflammatory” nature is not due to classic immune-mediated inflammation, but instead results from tumor-mediated blockage of dermal lymphatic ducts. Previous work has shown that expression of PD-L1 on tumor cells can suppress T cell activation in triple-negative (TN) non-IBC breast cancer. In the present work, we investigated immune parameters in peripheral blood of metastatic IBC patients to determine whether cellular components of the immune system are altered, thereby contributing to pathogenesis of the disease. These immune parameters were also compared to PD-1 and PD-L1 expression in IBC tumor biopsies. Methods Flow cytometry-based immune phenotyping was performed using fresh peripheral blood from 14 stage IV IBC patients and compared to 11 healthy age-similar control women. Immunohistochemistry for CD20, CD3, PD-1, and PD-L1 was performed on tumor biopsies of these metastatic IBC patients. Results IBC patients with Stage IV disease had lymphopenia with significant reductions in circulating T, B, and NK cells. Reductions were observed in all subsets of CD4+ T cells, whereas reductions in CD8+ T cells were more concentrated in memory subsets. Immature cytokine-producing CD56bright NK cells expressed higher levels of FcγRIIIa and cytolytic granule components, suggesting accelerated maturation to cytolytic CD56dim cells. Immunohistochemical analysis of tumor biopsies demonstrated moderate to high expression of PD-1 in 18.2% of patients and of PD-L1 in 36.4% of patients. Interestingly, a positive correlation was observed between co-expression levels of PD-L1 and PD-1 in tumor biopsies, and higher expression of PD-L1 in tumor biopsies correlated with higher expression of cytolytic granule components in blood CD4+ T cells and CD56dim NK cells, and higher numbers of CD8+ effector memory T cells in peripheral blood. PD-1 expression in tumor also correlated with increased infiltration of CD20+ B cells in the tumor. Conclusions Our results suggest that while lymphocyte populations are severely compromised in stage IV IBC patients, an immune response toward the tumor had occurred in some patients, providing biological rationale to evaluate PD-1/PD-L1 immunotherapies for IBC.

To better understand the etiology of inflammatory breast cancer (IBC) and identify potential therapies, we studied genomic alterations in IBC patients. Targeted, next-generation sequencing (NGS) was performed on cell-free DNA (cfDNA) (n = 33) and paired DNA from tumor tissues (n = 29) from 32 IBC patients. We confirmed complementarity between cfDNA and tumor tissue genetic profiles. We found a high incidence of germline variants in IBC patients that could be associated with an increased risk of developing the disease. Furthermore, 31% of IBC patients showed deficiencies in the homologous recombination repair (HRR) pathway (BRCA1, BRCA2, PALB2, RAD51C, ATM, BARD1) making them sensitive to poly (ADP-ribose) polymerase (PARP) inhibitors. We also characterized the tumor-infiltrating lymphocytes (TILs) in tumor tissue biopsies by studying several markers (CD4, CD8, FoxP3, CD20, PD-1, and PD-L1) through immunohistochemistry (IHC) staining. In 7 of 24 (29%) patients, tumor biopsies were positive for PD-L1 and PD-1 expression on TILs, making them sensitive to PD-1/PD-L1 blocking therapies. Our results provide a rationale for considering PARP inhibitors and PD-1/PDL1 blocking immunotherapy in qualifying IBC patients.

Circulating tumor DNA (ctDNA) is a component of cell-free DNA (cfDNA) that is shed by malignant tumors into the bloodstream and other bodily fluids. ctDNA can comprise up to 10% of a patient’s cfDNA depending on their tumor type and burden. The short half-life of ctDNA ensures that its detection captures tumor burden in real-time and offers a non-invasive method of repeatedly evaluating the genomic profile of a patient’s tumor. A challenge in ctDNA detection includes clonal hematopoiesis of indeterminate potential (CHIP), which can be distinguished from tumor variants using a paired whole-blood control. Most assays for ctDNA quantification rely on measurements of somatic variant allele frequency (VAF), which is a mutation-dependent method. Patients with certain types of solid tumors, including colorectal cancer (CRC), can have levels of cfDNA 50 times higher than healthy patients. ctDNA undergoes a precipitous drop shortly after tumor resection and therapy, and rising levels can foreshadow radiologic recurrence on the order of months. The amount of tumor bulk required for ctDNA detection is lower than that for computed tomography (CT) scan detection, with ctDNA detection preceding radiologic recurrence in many cases. cfDNA/ctDNA can be used for tumor molecular profiling to identify resistance mutations when tumor biopsy is not available, to detect minimal residual disease (MRD), to monitor therapy response, and for the detection of tumor relapse. Although ctDNA is not yet implemented in clinical practice, studies are ongoing to define the appropriate way to use it as a tool in the clinic. In this review article, we examine the general aspects of ctDNA, its status as a biomarker, and its role in the management of early (II–III) and late (IV; mCRC) stage colorectal cancer (CRC).

In order to better understand the etiology of inflammatory breast cancer (IBC), we performed a retrospective study to evaluate genomic alterations in 32 IBC patients using circulating DNA from blood. In 20 patients, mutation analyses were also conducted on tumor tissue or tumor cells from pleural effusions; in 15 of these patients, blood and tissue or pleural effusions were collected at the same time. Next generation sequencing (NGS) and a panel of 93 breast cancer related genes (Qiagen) were used for these studies. We found that of the 32 total patients, 11 (34.4%) carried at least one putative pathogenic germline variant, and another 8 patients (25%) carried at least one likely pathogenic germline variant. Ten patients (31.3%) carried only germline variants of uncertain significance and in 3 patients (9.4%) germline variants were not detected. Putative germline pathogenic variants were observed in the BRCA2 (2/32), TP53 (2/32), BRCA1 (1/32), PALB2 (1/32), CHEK2 (1/32), ATM (1/32), PMS1 (1/32), MUTYH (1/32), and MSH2 (1/32) genes. In addition, putative germline likely pathogenic variants were observed in the BARD1 (10/32), RB1 (2/32), APC (1/32), FANCC (1/32), AR (1/32), RAD51C (1/32), RAD51D (1/32) and CASP8 (1/32) genes. Most of the patients in our study had a family history of cancer and seventeen of the patients (53.1%) had family history of breast cancer in particular. Of those patients, 7 had a family history of breast cancer at a young age ( Citation Format: Jennifer S. Winn, Zachary Hasse, Jianming Pei, Michael Slifker, Sebastian M. Arisi-Fernandez, Jacqueline N. Talarchek, Elias Obeid, Karthik Devarajan, Hong Wu, Yulan Gong, Massimo Cristofanilli, R.Katherine Alpaugh, Sandra Viviana Fernandez. Germline and somatic variants in patients with inflammatory breast cancer (IBC) detected using cell-free DNA [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-03-11.

We studied genomic alterations in 19 inflammatory breast cancer (IBC) patients with advanced disease using samples of tissue and paired blood serum or plasma (cell-free DNA, cfDNA) by targeted next generation sequencing (NGS). At diagnosis, the disease was triple negative (TN) in eleven patients (57.8%), ER+ Her2- IBC in six patients (31.6%), ER+ Her2+ IBC in one patient (5.3%), and ER- Her2+ IBC in one other patient (5.3%). Pathogenic or likely pathogenic variants were frequently detected in TP53 (47.3%), PMS2 (26.3%), MRE11 (26.3%), RB1 (10.5%), BRCA1 (10.5%), PTEN (10.5%) and AR (10.5%), other affected genes included PMS1, KMT2C, BRCA2, PALB2, MUTYH, MEN1, MSH2, CHEK2, NCOR1, PIK3CA, ESR1 and MAP2K4. In 15 of the 19 patients in which tissue and paired blood were collected at the same time point, 80% of the variants detected in tissue were also detected in the paired cfDNA. Higher concordance between tissue and cfDNA was found for variants with higher allele fraction in tissue (AFtissue &ge, 5%). Furthermore, 86% of the variants detected in cfDNA were also detected in paired tissue. Our study suggests that the genetic profile measured in blood cfDNA is complementary to that of tumor tissue in IBC patients.

Inflammatory breast cancer (IBC) is a distinct clinicopathologic entity that carries a worse prognosis relative to non-IBC breast cancer even when matched for standard biomarkers (ER/PR/HER2). The objective of this study was to identify opportunities for benefit from targeted therapy, which are not currently identifiable in the standard workup for advanced breast cancer. Comprehensive genomic profiling on 53 IBC formalin-fixed paraffin-embedded specimens (mean, 800× + coverage) using the hybrid capture-based FoundationOne assay. Academic and community oncology clinics. From a series of 2208 clinical cases of advanced/refractory invasive breast cancers, 53 cases with IBC were identified. The presence of clinically relevant genomic alterations (CRGA) in IBC and responses to targeted therapies. CRGA were defined as genomic alterations (GA) associated with on label targeted therapies and targeted therapies in mechanism-driven clinical trials. For the 44 IBCs with available biomarker data, 19 (39 %) were ER-/PR-/HER2- (triple-negative breast cancer, TNBC). For patients in which the clinical HER2 status was known, 11 (25 %) were HER2+ with complete (100 %) concordance with ERBB2 (HER2) amplification detected by the CGP assay. The 53 sequenced IBC cases harbored a total of 266 GA with an average of 5.0 GA/tumor (range 1-15). At least one alteration associated with an FDA approved therapy or clinical trial was identified in 51/53 (96 %) of cases with an average of 2.6 CRGA/case. The most frequently altered genes were TP53 (62 %), MYC (32 %), PIK3CA (28 %), ERBB2 (26 %), FGFR1 (17 %), BRCA2 (15 %), and PTEN (15 %). In the TNBC subset of IBC, 8/19 (42 %) showed MYC amplification (median copy number 8X, range 7-20) as compared to 9/32 (28 %) in non-TNBC IBC (median copy number 7X, range 6-21). Comprehensive genomic profiling uncovered a high frequency of GA in IBC with 96 % of cases harboring at least 1 CRGA. The clinical benefit of selected targeted therapies in individual IBC cases suggests that a further study of CGP in IBC is warranted.

Retinoids have been used as potential chemotherapeutic or chemopreventive agents because of their differentiative, anti-proliferative, pro-apoptotic and antioxidant properties. We investigated the effect of all trans-retinoic acid (ATRA) at different stages of the neoplastic transformation using an in vitro model of breast cancer progression. This model was previously developed by treating the MCF-10F human normal breast epithelial cells with high dose of estradiol and consists of four cell lines which show a progressive neoplastic transformation: MCF-10F, normal stage; trMCF, transformed MCF-10F; bsMCF, invasive stage; and caMCF, tumorigenic stage. In 3D cultures, MCF-10F cells form tubules resembling the structures in the normal mammary gland. After treatment with estradiol, these cells formed tubules and spherical masses which are indicative of transformation. Cells that only formed spherical masses in collagen were isolated (trMCF clone 11) and treated with ATRA. After treatment with 10 or 1 μM ATRA, the trMCF clone 11 cells showed tubules in collagen; 10 and 43% of the structures were tubules in cells treated with 10 and 1 μM ATRA, respectively. Gene expression studies showed that 207 genes upregulated in transformed trMCF clone 11 cells were downregulated after 1 μM ATRA treatment to levels comparable to those found in the normal breast epithelial cells MCF-10F. Furthermore, 236 genes that were downregulated in trMCF clone 11 were upregulated after 1 μM ATRA treatment to similar levels shown in normal epithelial cells. These 443 genes defined a signature of the ATRA re-programming effect. Our results showed that 1 μM ATRA was able to re-differentiate transformed cells at early stages of the neoplastic process and antagonistically regulate breast cancer associated genes. The invasive and tumorigenic cells did not show any changes in morphology after ATRA treatment. These results suggest that ATRA could be used as a chemopreventive agent to inhibit the progression of premalignant lesions of the breast.

Introduction: Inflammatory breast cancer (IBC) is a very aggressive variant of breast cancer with a poor prognosis.. Mouse Mammary Tumor-associated Virus (MMTV) and other infectious agents have been considered as possible etiological agents of IBC particularly related to the initial description of higher incidence in women living in rural areas in North Africa. The etiological role of bacteria in this disease has never been explored in spite of the evidence that chronic infections with certain bacteria can facilitate tumors development. We retrospectively evaluated tissue samples from patients with recurrent IBC to identify potential bacterial agents that could play a role in the development and progression of the disease. Methods: DNA was isolated from formalin-fixed paraffin embedded samples of 24 Inflammatory Breast Cancer (IBC) patients whose specimen had been submitted for genomic analysis using next-genomic sequencing (Foundation One™). An additional 3 non-IBC patients (lymph node, lung metastatic lesions) were included in the study. Unselected DNA libraries from these samples were pooled and sequenced on a HiSeq-2000 sequencer. Comparing the sequence data to a reference of 5,569 bacterial and viral genomic sequences identified the presence of Propionibacterium acnes, Ralstonia pickettii and Methylobacterium consequently, we enriched the DNA libraries for the presence of these species as well as bacterial rRNA genes using hybrid capture with synthesized oligonucleotide baits and sequenced the enriched libraries. Results: Twenty three IBC patients and 3 non-IBC breast cancer patients were included in the study. Tissue specimens included, 14 chest wall and skin;6 breast, 2 lymph nodes; 1 liver, 1 lung, 1 pleural fluid, 1 brain. In 16 out of the 23 IBC (70%) of specimens we detected bacteria DNA. Propionibacterium acnes were detected in 12 cases. Bartonella henselae was detected in 1 out of the 23 IBC specimens. Furthermore, additional detected species included Ralstonia pickettii (3 cases) and Pseudomonas aeruginosa (2). No bacteria were detected in samples from non-IBC breast cancer patients. Conclusions: In this study, we identified Propionibacterium acnes and Bartonella henselae in samples from IBC patients. The anaerobic Gram-positive bacterium P. acnes is ubiquitously found in sebaceous follicles of the human skin. Recent reports showed that P. acnes was present in a high number of cancerous prostate tissue samples. It has been suggested that P. acnes may be a contributing factor to the initiation or progression of prostate cancer. Bartonella is a Gram-negative bacteria usually associated with cat-scratch disease, urban trench fever, bacillary angiomatosis-peliosis and endocarditis. Some reports that have showed some similarities between cat scratch disease and inflammatory breast cancer. Our results suggested that P. acnes or Bartonella henselae infections might contribute to the clinical and pathological characteristics of IBC, associated with rapid spread of the breast tumor cells through the lymphatic system. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-12-07.

Analogues structurally related to anaplastic lymphoma kinase (ALK) inhibitor 1 were optimized for metabolic stability. The results from this endeavor not only led to improved metabolic stability, pharmacokinetic parameters, and in vitro activity against clinically derived resistance mutations but also led to the incorporation of activity for focal adhesion kinase (FAK). FAK activation, via amplification and/or overexpression, is characteristic of multiple invasive solid tumors and metastasis. The discovery of the clinical stage, dual FAK/ALK inhibitor 27b, including details surrounding SAR, in vitro/in vivo pharmacology, and pharmacokinetics, is reported herein.

Inflammatory breast cancer (IBC) is a very aggressive type of advanced breast cancer with a poor prognosis. Clinical symptoms involve a rapid onset of changes in the skin overlying the breast, including edema, redness and swelling including a wrinkled and orange peel appearance in the skin. This particular presentation is due to the invasion of the skin dermal lymphatics by breast cancer cells that obstructed the lymph channels producing the characteristic skin changes that mimic an inflammatory process. Mouse Mammary Tumor-associated Virus (MMTV) and other infectious agents have been consider as possible etiological agents of IBC particularly related to the initial description of higher incidence in women living in rural areas in North Africa. Although, the etiopathological role of bacteria in this disease has never been explored in spite of the evidence that chronic infections with certain bacteria can facilitate tumors development. Several bacterial infections promote cell proliferation and could increase the rate of cell transformation. Bartonella spp. is an emerging pathogen that can cause conditions which are characterized by the development of proliferative lesions. Bartonella might cause vasculoproliferative disorders by triggering the proliferation of endothelial cells and inducing the secretion of proliferative cytokines from infected host cells. It cause persistent infection of erythrocytes and endothelial cells in their mammalian hosts and their transmission occur mainly by blood-sucking arthropods. Bartonella are Gram-negative bacteria usually associated with cat-scratch disease, urban trench fever, bacillary angiomatosis-peliosis and endocarditis. Some reports have showed some similarities between cat scratch disease and inflammatory breast cancer (Povoski et al., Breast J 9: 497–500, 2003). Methods and Results: In the present work, we report the identification of Bartonella henselae in the pleural effusions of two patients with IBC. These patients had both estrogen-receptor negative (ER−) and progesterone-receptor negative (PR−) disease and have failed a number of previous chemotherapy regimens. Furthermore, they had metastatic disease to the pleura, skin, lymph nodes and lung. Cells from the pleural fluids of these patients stained positive for Bartonella sp. using the Warthin-Starry staining kit. Moreover, we used PCR and sequencing of the 16S–23S intergenic spacer (ITS) region to identify the etiological agent as Bartonella henselae. By immunofluorescence using an antibody that specifically recognized Bartonella henselae, the bacteria were found in the nucleus of IBC tumor cells confirming the infection and their intracellular localization. Conclusions: These results suggested that bacteria infections such as the one produced by Bartonella henselae might contribute to the clinical and pathological characteristics of IBC, associated with rapid spread of the breast tumor cells through the lymphatic system. The infection and activation of endothelial lymphatics by Bartonella might contribute to the highly metastatic process observed in IBC patients. An acute inflammatory reaction triggered by the Bartonella infected endothelium may be crucial for initiating the chronic inflammation in IBC patients and the rapid spread of tumor cells. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P3-10-03.

Background: Inflammatory breast cancer (IBC) is the most metastatic variant of locally advanced breast cancer. Although IBC is diagnosed less commonly than other types of breast cancer, it is extremely aggressive, and accounts for a disproportionate number of breast cancer related deaths annually. IBC exhibits very specific patterns of lymphangiogenesis and vasculogenic mimicry, however detailed studies of the genes and proteins involved in these angiogenic processes are lacking. This study performed whole unbiased gene transcription studies with validation by protein arrays using all available pre-clinical cell lines and in vivo xenograft models of IBC, including a new model of IBC, FC-IBC01, which exhibits lymphovascular invasion, to identify the specific pathways involved in the distinctive angiogenesis observed in IBC. Materials and Methods: Real-time quantitative RT-PCR, cDNA microarray gene profiling, immunofluorescence with confocal imaging and protein arrays were used to examine differential expression of specific angiogenic gene families including VEGFA,B,C,D, VEGF Receptor genes, and ANG/TIE genes linked to angiogenesis and lymphangiogenesis. Results: Activity of the matrix metalloproteinase, MMP-2, is required for IBC tumor cells to undergo vasculogenic mimicry (VM), which is associated with a loss of TIMP-2, a well known inhibitor of angiogenesis. Therapeutics that target MMP activity can successfully inhibit this VM. Furthermore, pre-clinical models of IBC that form IBC tumor emboli exhibit lymphovascular invasion that is associated with distinct patterns of expression of genes that encode for distinct receptor tyrosine kinases that may represent important therapeutic targets for IBC. Discussion: Identification of the distinct angiogenic pathways that are activated in IBC provides insight into the therapeutic targets that may abrogate the distinct lymphovascular invasion and vasculogenic mimicry that are linked to the aggressive metastasis of IBC. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-05-04.

Inflammatory breast cancer (IBC) currently accounts for 2% to 6% of all breast cancer cases in the United States and up to 20% of all breast cancer cases globally. IBC exhibits distinctively aggressive clinical features compared to all breast cancers, and accounts for a disproportionally high mortality rate—15% of breast cancer-related deaths in U.S. In addition, the survival rates between stage-matched IBC and non-IBC differ drastically. Therefore, we are in need of better understanding the molecular abnormalities driving IBC aggressive phenotype. We performed a study to evaluate the genomic alterations in cell free DNA (cfDNA) from plasma from 13 IBC patients, including 9 with triple-negative disease. In 6 patients, mutation analyses were also studied in tumor samples (tumor tissue or tumor cells from pleural effusions). Mutation analysis was performed by next-generation sequencing (NGS) using a unique molecular identifiers (UMI) assay and a panel of 93 breast cancer-related genes (Qiagen). The data were analyzed using the Qiagen's GeneGlobe portal and Biomedical Genomics Workbench and interpretation was performed with Qiagen's QCI. Somatic mutations detected in cfDNA samples were seen in: TP53 (7/13), RB1 (2/13), GEN1 (2/13) and EP300 (2/13); additional somatic mutations were found in PIK3CA (1/13), ERBB2 (1/13), PALB2 (1/13) and MUC16 (1/13). In 4 patients with plasma and tumor samples taken at the same time of the disease progression, complete concordance was not found in the somatic mutations detected in cfDNA and tumor cells DNA. Interestingly, in 12 of 13 IBC patients some variants were observed with high variant allele frequencies (VAF), ~50% or ~100% at a total coverage depth ≥230 reads, in the following genes: BRCA2 (1/13), RAD51D (2/13), PALB2 (1/13), RAD51C (1/13), AR (1/13) and MUTYH (1/13), which were classified as pathogenic or likely pathogenic, and BARD1 (3/13), SYNE1 (2/13), KMT2C (2/13), BRIP1 (1/13), XRCC3 (1/13), RET (1/13), APC (1/13), RAD50 (1/13) and MUC16 (1/13), classified as variants of uncertain significance. Moreover, 11 of these patients had a family history of different cancers including breast, colon, prostate, stomach, bladder, cervical cancers, and melanoma and myeloma. In the 6 patients where cfDNA and tumor samples were available, mutations with high VAF were found in both samples (100% concordance), suggesting that variants with high VAF are germline variants. These results suggest that the described germline variants could increase the risk of IBC and somatic mutation information obtained from cfDNA is complementary to that obtained from tissue samples. Studies on more samples are in progress. Citation Format: Jianming Pei, Jacqueline Talarchek, Jennifer S. Winn, Katherine Alpaugh, Massimo Cristofanilli, Sandra V. Fernandez. Genomic profiling of cell free DNA (cfDNA) from patients with inflammatory breast cancer (IBC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4593.

There is growing concern that estrogenic environmental compounds that act as endocrine disrupting chemicals might potentially have adverse effects on hormone-sensitive organs such as the breast. This concern is further fueled by evidence indicating that natural estrogens, specifically 17 β-estradiol (E2), are important factors in the initiation and progression of breast cancer. We have developed an in vitro- in vivo model in which we have demonstrated the carcinogenicity of E2 in the human breast epithelial cells MCF-10F. Hypermethylation of NRG1, STXBP6, BMP6, CSS3, SPRY1 and SNIP were found at different progression stages in this model. The utilization of this powerful and unique model has provided a tool for exploring whether bisphenol A (BPA) and butyl benzyl phthalate (BBP) have relevance in the initiation of breast cancer. These studies provide first hand evidence that the natural estrogen 17 β-estradiol and xenoestrogenic substances like BPA are able to induce neoplastic transformation in human breast epithelial cells.

The estrogen dependence of breast cancer has long been recognized; however, the role of 17β-estradiol (E2) in cancer initiation was not known until we showed that it induces complete neoplastic transformation of the human breast epithelial cells MCF-10F. E2 treatment of MCF-10F cells progressively induced high colony efficiency and loss of ductulogenesis in early transformed (trMCF) cells and invasiveness in Matrigel invasion chambers. The cells that crossed the chamber membrane were collected and identified as bsMCF; their subclones were designated bcMCF; and the cells harvested from carcinoma formation in severe combined immunodeficient mice were designated caMCF. These phenotypes correlated with gene dysregulation during the progression of the transformation. The highest number of dysregulated genes was observed in caMCF, being slightly lower in bcMCF, and lowest in trMCF. This order was consistent with the extent of chromosome aberrations (caMCF > bcMCF >>> trMCF). Chromosomal amplifications were found in 1p36.12-pter, 5q21.1-qter, and 13q21.31-qter. Losses of the complete chromosome 4 and 8p11.21-23.1 were found only in tumorigenic cells. In tumor-derived cell lines, additional losses were found in 3p12.1-14.1, 9p22.1-pter, and 18q11.21-qter. Functional profiling of dysregulated genes revealed progressive changes in the integrin signaling pathway, inhibition of apoptosis, acquisition of tumorigenic cell surface markers, and epithelial-mesenchymal transition. In tumorigenic cells, the levels of E-cadherin, epithelial membrane antigen, and various keratins were low and CD44E/CD24 were negative, whereas SNAI2, vimentin, S100A4, FN1, HRAS, transforming growth factor β1, and CD44H were high. The phenotypic and genomic changes triggered by estrogen exposure that lead normal cells to tumorigenesis confirm the role of this steroid hormone in cancer initiation. [Cancer Res 2007;67(23):11147–57]

Metabolic conversion of endogenous estrogens, estradiol (E2) and estrone (E1), to the catechol estrogens 4-hydroxyE1(E2) [4-OHE1(E2)] has been implicated in the initiation of cancer in rodents and humans. Evidence collected in our laboratories has shown that 4-OHE1(E2) are enzymatically oxidized to E1(E2)-3,4-quinones [E1(E2)-3,4-Q], which have the potential to damage DNA by forming predominantly depurinating adducts, 4-OHE1(E2)-1-N3Ade and 4-OHE1(E2)-1-N7Gua, leading to the accumulation of mutations and probably cell transformation. The human breast epithelial cell line MCF-10F has been transformed by treatment with E2 or 4-OHE2. We have used MCF-10F cells to study the presence of adducts and conjugates after treatment with 4-OHE2. To mimic the intermittent exposure of breast cells to endogenous estrogens, MCF-10F cells were treated with 1 μM 4-OHE2 for a 24-h period at 72, 120, 192 and 240 h postplating. Culture media were collected at each point, extracted by solid-phase extraction and analyzed by HPLC connected with a multichannel electrochemical detector and/or ultraperformance liquid chromatography/tandem mass spectrometry. Media from successive treatments with 4-OHE2 showed the formation of methoxy and cysteine conjugates, and the depurinating adducts 4-OHE1(E2)-1-N3Ade. The amount of 4-OHE1(E2)-1-N3Ade adducts was higher after the third treatment; smaller amounts of the 4-OHE1(E2)-1-N7Gua adducts were detected after the second and third treatments. These results demonstrate that MCF-10F cells oxidize 4-OHE2 to E1(E2)-3,4-Q, which react with DNA to form the depurinating N3Ade and N7Gua adducts. This DNA damage can play an important role in the 4-OHE2-induced mutations and transformation of MCF-10F cells to malignant cells. © 2007 Wiley-Liss, Inc.

An elevated incidence of breast cancer in women has been associated with prolonged exposure to high levels of estrogens. Our laboratory demonstrated that treatment of the immortalized human breast epithelial cells MCF-10F with 17β-estradiol (E2), 4-hydroxyestradiol (4-OHE2) or 2-hydroxyestradiol (2-OHE2) induces phenotypical changes indicative of neoplastic transformation. MCF-10F cells treated with E2, 4-OHE2 or 2-OHE2 formed colonies in agar methocel and lost their ductulogenic capacity in collagen, expressing phenotypes similar to those induced by the carcinogen benzo[a]pyrene. To investigate whether the transformation phenotypes were associated with genomic changes, cells treated with E2, 4-OHE2 or 2-OHE2 at different doses were analyzed using microsatellite markers. Since microsatellite instability (MSI) and loss of heterozygosity (LOH) in chromosomes 13 and 17 have been reported in human breast carcinomas, we tested these parameters in MCF-10F cells treated with E2, 2-OHE2, or 4-OHE2 alone or in combination with the antiestrogen ICI182780. MCF-10F cells treated with E2 or 4-OHE2, either alone or in combination with ICI182780, exhibited LOH in the region 13q12.3 with the marker D13S893 located at ∼0.8 cM telomeric to BRCA2. Cells treated with E2 or 4-OHE2 at doses of 0.007 and 70 nM and 2-OHE2 only at a higher dose (3.6 μM) showed a complete loss of 1 allele with D13S893. For chromosome 17, differences were found using the marker TP53-Dint located in exon 4 of p53. Cells treated with E2 or 4-OHE2 at doses of 0.007 nM and 70 nM and 2-OHE2 only at a higher dose (3.6 μM) exhibited a 5 bp deletion in p53 exon 4. Our results show that E2 and its catechol estrogen metabolites are mutagenic in human breast epithelial cells. ICI182780 did not prevent these mutations, indicating that the carcinogenic effect of E2 is mainly through its reactive metabolites 4-OHE2 and 2-OHE2, with 4-OHE2 and E2 being mutagenic at lower doses than 2-OHE2. © 2005 Wiley-Liss, Inc.

Introduction Circulating tumor cells (CTCs) are tumor cells shed from either primary tumors or its metastases that circulate in the peripheral blood of patients with metastatic cancers. The molecular characterization of the CTCs is critical to identifying the key drivers of cancer metastasis and devising therapeutic approaches. However, the molecular characterization of CTCs is difficult to achieve because their isolation is a major technological challenge. Methods CTCs from two triple negative breast cancer patients were enriched using CellSearch and single cells selected by DEPArray™. A TP53 R110 fs*13 mutation identified by next generation sequencing in the breast and chest skin biopsies of both patients was studied in single CTCs. Results From 6 single CTC isolated from one patient, 1 CTC had TP53 R110 delC, 1 CTC showed the TP53 R110 delG mutation, and the remaining 4 single CTCs showed the wild type p53 sequence; a pool of 14 CTCs isolated from the same patient also showed TP53 R110 delC mutation. In the tumor breast tissue of this patient, only the TP53 R110 delG mutation was detected. In the second patient a TP53 R110 delC mutation was detected in the chest wall skin biopsy; from the peripheral blood of this patient, 5 single CTC and 6 clusters of 2 to 6 CTCs were isolated; 3 of the 5 single CTCs showed the TP53 R110 delC mutation and 2 CTCs showed the wild type TP53 allele; from the clusters, 5 showed the TP53 R110 delC mutation, and 1 cluster the wild type TP53 allele. Single white blood cells isolated as controls from both patients only showed the wild type TP53 allele. Conclusions We are able to isolate uncontaminated CTCs and achieve single cell molecular analysis. Our studies showed the presence of different CTC sub-clones in patients with metastatic breast cancer. Some CTCs had the same TP53 mutation as their matching tumor samples although others showed either a different TP53 mutation or the wild type allele. Our results indicate that CTCs could represent a non-invasive source of cancer cells from which to determine genetic markers of the disease progression and potential therapeutic targets. Electronic supplementary material The online version of this article (doi:10.1186/s13058-014-0445-3) contains supplementary material, which is available to authorized users.

Introduction Inflammatory breast cancer (IBC) is the most metastatic variant of breast cancer with the poorest survival in all types of breast cancer patients and presently therapeutic targets for IBC are very limited. Enhancer of zeste homolog 2 (EZH2) is frequently expressed in human IBC and its expression positively correlates with worse clinical outcome. However, the molecular basis for EZH2 promoting IBC has not been explored. Here, we investigated the functional role of EZH2 in IBC cells by examining the effects of its knockdown on the formation of tumor spheroids and invasion of these cells in vitro and in vivo in an orthotopic xenograft model. Methods SUM149 and a new IBC cell line-FC-IBC-02 derived from pleural effusion fluid of an IBC patient were used in this study. Specific knockdown of EZH2 was performed using short hairpin RNA (shRNA) specific to the human EZH2 gene. Cell growth and the formation of tumor spheroids were examined in vitro. The effects of EZH2 knockdown on IBC cell migration and invasion were examined by a Boyden chamber assay. For the in vivo tumor growth studies, IBC cells were orthotopically transplanted into the mammary fat pads of immunodeficient mice. Results The results showed that EZH2 is expressed at higher levels in human IBC cell lines compared with normal human mammary epithelial cells, and the knockdown of EZH2 expression significantly suppressed cell growth and tumor spheroid formation of human IBC cells in vitro. In addition, EZH2 knockdown inhibited the migration and invasion of IBC cells. Significantly, EZH2 knockdown suppressed the angiogenesis and tumor growth of IBC cells in vivo. Conclusions Our results provide direct evidence that EZH2 is critical for the formation of tumor spheroids and invasion of human IBC cells and could be a potential target for developing novel therapeutic strategies for human IBC.

Inflammatory breast cancer (IBC) is the most lethal variant of locally advanced breast cancer and carries with it a very low survival rate of 40 % at 5 years. IBC does not present as a lump but rather mimics characteristics of an inflammation that first appears as swelling of the breast, with edema, redness, and common lymph node involvement. The physical changes in the breast are associated with the presence of nests of aggregated tumor cells, defined as tumor emboli that are encircled by lymphatic vessels, effectively blocking lymphatic drainage. Little is understood about IBC, in part due to the lack of preclinical models that recapitulate its distinct characteristics. This chapter provides an overview of our studies that have profiled all available preclinical models of IBC, including two new models recently developed, to elucidate the molecular underpinnings of this lethal variant of breast cancer. Our studies demonstrate that IBC is enriched for cells that express CD44+ and CD133+ and have aldehyde dehydrogenase-1 (ALDH1) activity, supporting a cancer stem cell/tumor initiating phenotype, associated with a very high metastatic potential to multiple distant organ sites. IBC has a distinct gene signature including E-cadherin expression with associated loss of expression of ZEB1, a transcriptional repressor of E-cadherin. IBC is also characterized by loss of expression of genes within the transforming growth factor-beta (TGFβ) signaling pathway, which is permissive for cohesive invasion by IBC tumor emboli. Taken together, these studies suggest that IBC is a very distinct variant of breast cancer characterized by epithelial plasticity, enrichment of a stem cell phenotype, and cohesive invasion as an adaptive survival mechanism, consistent with the definition of IBC as the most metastatic variant of breast cancer.

It has been suggested that xenoestrogens, a group of agents termed endocrine disruptors, may contribute to the development of hormone-dependent cancers, such as breast and endometrial cancers. We previously demonstrated that the xenoestrogen, bisphenol A (BPA), was able to induce the transformation in vitro of human breast epithelial cells. The normal-like human breast epithelial cell line, MCF-10F, formed tubules in collagen (3-D cultures), although after treatment with BPA (10-5 M and 10-6 M BPA) the cells produced less tubules (73% and 80%, respectively) and some spherical masses (27% and 20%, respectively). In the present study, expression and DNA methylation analyses were performed in these cells after exposure to BPA. These cells showed an increased expression of BRCA1, BRCA2, BARD1, CtIP, RAD51 and BRCC3, all of which are genes involved in DNA repair, as well as the downregulation of PDCD5 and BCL2L11 (BIM), both of which are involved in apoptosis. Furthermore, DNA methylation analysis showed that the BPA exposure induced the hypermethylation of BCL2L11, PARD6G, FOXP1 and SFRS11, as well as the hypomethylation of NUP98 and CtIP (RBBP8). Our results indicate that normal human breast epithelial cells exposed to BPA have increased expressions of genes involved in DNA repair in order to overcome the DNA damage induced by this chemical. These results suggest that the breast tissue of women with BRCA1 or BRCA2 mutations could be more susceptible to the effects of BPA.

Study background: Inflammatory Breast Cancer (IBC) is the most metastatic variant of breast cancer. Although IBC is recognized as a distinct variant of breast cancer, the molecular basis for the rapid progression of IBC remains largely undefined, in part due to the lack of preclinical models that recapitulate the human disease as well as a lack of comprehensive analysis of the preclinical models of IBC that are available. Methods: All available 7 pre-clinical models of IBC, including 2 new models, FC-IBC01 and FC-IBC02 developed from pleural effusion, were used to identify genes and molecular pathways that are selectively altered compared to non IBC breast tumor models. Laser capture micro dissection of biopsy tissue from core biopsy and skin punch biopsies were also analyzed by whole transcriptome analysis. Results: Whole transcriptome analysis defined 7 pre-clinical models of IBC as being within either the triple negative or ErbB2/Her-2 expressing subtypes, similar to the prevalence of these subtypes of breast cancer observed in IBC patients. Comparative analysis of the FC-IBC01, FC-IBC02 and Mary-X models of IBC demonstrated that each of these recapitulate the formation of tumor emboli with encircling lymphovasculogenesis. The majority (6/7) of the pre-clinical models of IBC express CDH1, which encodes for E-cadherin, which was associated with a loss of ZEB1, a transcriptional repressor of E-cadherin. The lack of ZEB1 expression was validated in a limited set of 4 skin punch biopsy samples from IBC patients that were isolated by laser capture micro-dissection, demonstrating concordance with loss of ZEB1 in pre-clinical models of IBC. Expression of other transcription factors involved in acquisition of a cancer stem cell phenotype, including Snai1, which encodes for Snail, SNAI2, which encodes for Slug and TWIST1, was retained in pre-clinical models of IBC. Maintenance of E-cadherin in pre-clinical models of IBC was associated with a loss of genes within the transforming growth factor beta (TGFβ signaling pathway, with expression of SMAD6, a known repressor of TGFβ. This is similar to a recent study reporting the persistence of E-cadherin and loss of TGFβ signaling in IBC patient tumors based on gene profiling of 3 independent data sets. Conclusion: The present studies provide first time comparison of gene signatures of 7 pre-clinical models of IBC, including our 2 newly developed pre-clinical models, FC-IBC01 and FC-IBC02, that recapitulate formation of tumor emboli with encircling lymphatic vessels, similar to that observed in biopsy tissues of IBC patients. We demonstrate that E-cadherin expression was associated with both loss of ZEB1 and diminished expression of multiple genes within the TGFβ signaling pathway, with retention of expression of transcription factors and surface markers consistent with maintenance of a cancer stem cell phenotype, as has been reported to be a characteristic of IBC tumors. Collectively, these observations provide first time characterization of the molecular signatures of all available pre-clinical models of IBC, and suggest that IBC has a signature of epithelial plasticity, with characteristics of their ability to undergo the mesenchymal to epithelial reverting transition. The loss of genes within the TGFβ signaling is also consistent with the tight cell: cell aggregation of IBC tumor cells within tumor emboli that exhibit “cohesive invasion”. The new pre-clinical models of IBC that recapitulate the human disease will serve as useful tools to accelerate our understanding of the molecular underpinnings and therapeutic targets of IBC as the most lethal form of breast cancer.

Most breast carcinomas that are estrogen receptor (ER) and progesterone receptor (PR) positive respond initially to an endocrine therapy, but over time, they develop resistance (acquired hormone resistance). Others, however, fail to respond from the beginning (constitutive resistance). Overcoming hormone resistance is one of the major desirable aims in breast cancer treatment. Using the medroxyprogesterone acetate (MPA)-induced breast cancer mouse model, we have previously demonstrated that antiprogestin-responsive tumors show a higher expression level of PR isoform A (PRA) than PR isoform B (PRB), while tumors with constitutive or acquired resistance show a higher expression level of PRB. The aim of this study was to investigate whether PRA silencing in resistant tumors was due to PRA methylation. The CpG islands located in the PRA promoter and the first exon were studied by methylation-specific PCR (MSP) in six different tumors: two antiprogestin-responsive, two constitutive-resistant, and two with acquired resistance. Only in constitutive-resistant tumors, PRA expression was silenced by DNA methylation. Next, we evaluated the effect of a demethylating agent, 5-aza-2´-deoxycytidine, on PRA expression and antiprogestin responsiveness. In constitutive-resistant tumors, 5-aza-2´-deoxycytidine treatment in vitro and in vivo restored PRA expression and antiprogestin RU-486 responsiveness. Furthermore, high levels of DNA methyltransferase (Dnmts) 1 and 3b were detected in these tumors. In conclusion, our results suggest that methyltransferase inhibitors in combination with antiprogestins may be effective in the treatment of constitutive-resistant carcinomas with a high DNA methyltransferase level. Fil: Wargon, Victoria. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina; Argentina Fil: Fernandez, Sandra V.. Fox Chase Cancer Center. Breast Cancer Research Laboratory; Estados Unidos Fil: Goin, Mercedes. Laboratorios Beta S.A.; Argentina Fil: Giulianelli, Sebastian Jesus. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina; Argentina Fil: Russo, José. Fox Chase Cancer Center. Breast Cancer Research Laboratory; Estados Unidos Fil: Lanari, Claudia Lee Malvina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina; Argentina

Epigenetic inactivation of genes by DNA hypermethylation plays an important role in carcinogenesis. An in vitro model of human breast epithelial cell transformation was used to study epigenetic changes induced by estradiol during the neoplastic process. Different stages of tumor initiation and progression are represented in this model being MCF-10F the normal stage; trMCF cells, the transformed stage; bsMCF cells, the invasive stage and, caMCF cells, the tumor stage. Global methylation studies by restriction landmark genomic scanning (RLGS) showed an increased DNA methylation during the in the invasive and tumor stages. Expression studies showed that NRG1 (neuregulin 1), CSS3 (chondroitin sulfate synthase 3) and SNIP (SNAP-25-interacting protein) were downregulated in the invasive and tumor cells. The transformed cells showed low expression of STXBP6 (amysin) compared to the parental cells MCF-10F. The treatment of these cells with the demethylating agent 5-aza-dC alone or in combination with the histone deacetylase inhibitor trichostatin increased the expression of NRG1, STXBP6, CSS3 and SNIP confirming that DNA methylation plays an important role in the regulation of the expression of these genes. The NRG1 exon 1 has a region located between −136 and +79 (considering +1, the translational initiation site) rich in CpG sites that was analyzed by methylation specific PCR (MSP). NRG1 exon 1 showed progressive changes in the methylation pattern associated with the progression of the neoplastic process in this model; NRG1 exon 1 was unmethylated in MCF-10F and trMCF cells, becoming hypermethylated in the invasive (bsMCF) and tumor (caMCF) stages. Studies of human breast tissue samples showed that NRG1 exon 1 was partially methylated in 14 out of 17 (82.4%) invasive carcinomas although it was unmethylated in normal tissues (8 out of 10 normal breast tissue samples). Furthermore, NRG1 exon 1 was partially methylated in 9 out of 14 (64.3%) morphologically normal tissue samples adjacent to invasive carcinomas.

Human chorionic gonadotropin (hCG), a hormone produced during pregnancy, can elicit life-long refractoriness to carcinogenesis by differentiation of the breast epithelium. Human breast epithelial cells MCF-10F form tubules in collagen, mimicking the normal ductules. We have shown that 17 beta-estradiol (E2) alter the ductulogenic pattern of these cells. The effect of the recombinant hCG (rhCG) in vitro was evaluated on the transformation of MCF-10F induced by E2. MCF-10F cells were treated with 70 nM E2 alone or in combination with 50 IU/ml rhCG during 2 weeks, while the controls were treated with DMSO (the solvent in which E2 was dissolved) or rhCG alone. At the end of treatment, the cells were plated in type I collagen matrix (3D-cultures) for detecting 2 main phenotypes of cell transformation, namely the loss of ductulogenic capacity and the formation of solid masses. Although E2 significantly increased solid mass formation, this effect was prevented when MCF-10F cells were treated with E2 in combination with rhCG. Furthermore, E2 increased the main duct width (p < 0.001), and caused a disruption of the luminal architecture, whereas rhCG increased the length of the tubules (p < 0.001) and produced tertiary branching. In conclusion, rhCG was able to abrogate the transforming abilities of estradiol, and had the differentiating property by increasing the branching of the tubules formed by breast epithelial cells in collagen. These results further support our hypothesis, known as the terminal differentiation hypothesis of breast cancer prevention, that predicts that hCG treatment results in protection from tumorigenic changes by the loss of susceptible stem cells 1 through a differentiation to refractory stem cells 2 and increase differentiation of the mammary gland. (C) 2009 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved.

Epithelial-mesenchymal transition (EMT) in epithelial cells has been indicated as an important component of neoplastic transformation although, the genetic mechanism involved in this process has not been defined. The aim of this study was to evaluate the expression of different genes related to EMT such as E-cadherin, TGFbeta1, TGFbeta2, h-RAS, TWIST1, SNAIL2, SMAD5, FN1, CEACAM1 and JAG1 using the in vitro-in vivo model of the estrogen induced cell transformation developed in our laboratory. The E2-transformed MCF-10F (E2 70) cells and the tumorigenic cell line C5-A8-T8 (C5-T8) exhibit progressive loss of ductulogenesis as demonstrated by growth in collagen matrix. MCF-10F cells form ductal structures while E2 70 cells form solid spherical masses that in histological sections exhibit a pattern of growth resembling ductal hyperplasia or carcinoma in situ. The tumorigenic cells C5-T8 did not form structures on collagen acquiring an invasive pattern with spindle like features. We have observed a reduction in E-cadherin expression in E2 70 cells and a complete loss in C5-T8 cells. TGFbeta1, TGFbeta2, CEACAM1 and JAG1 were down-regulated in E2 70 and C5-T8 cells. SMAD5 and h-RAS were up-regulated in the tumorigenic C5-T8 cells whereas FN1, Twist1 and Snail2 were up-regulated in C5-T8 and down-regulated in E2 70. We conclude that the loss of expression of TGFbeta1, TGFbeta2, CEACAM1 and JAG1 are related to ductulogenesis and branching and the overexpression of h-RAS with loss of E-cadherin expression and up-modulation of TWIST1, SNAIL2 and SMAD5 expressions are involved in the EMT modulation.

Cancer cells in peripheral blood, known as circulating tumor cells (CTCs), play an important role in tumor dissemination. CTCs are shed from either the primary tumor or its metastases and circulate in the peripheral blood of patients; thus they can be regarded as “liquid biopsies” of metastasizing cells. Although the exact origin and physiology of CTCs is unknown, a fraction of these cells are thought to be viable metastatic precursors capable of initiating a clonal metastatic lesion. The molecular characterization of CTCs is important because it may provide insights into the molecular biology of metastasis, the association of their molecular profiles with treatment outcomes, and reveal the presence of potential therapeutic targets. The process of CTC enrichment represents a significant step toward the isolation of CTCs from whole blood. Given that there are approximately 5×109 erythrocytes and 107 leukocytes per ml of whole blood, purification must represent a reduction of many orders of magnitude. In this study, we show a method to isolate CTCs which combines a microfluidic cell separation device (Parsortix system) as an enrichment method followed by the DEPArray selection system. The Parsortix system is versatile in its ability to separate, capture and harvest CTCs in an enriched form compatible with the needs of downstream processes such as DEPArray. Here we demonstrate enrichment of CTCs via the Parsortix system using both breast cancer cell spiked healthy blood donor samples and blood from metastatic breast cancer patients. For these experiments, two milliliters of blood were processed. The cells were harvested and stained to identify CTCs in the enriched harvest product. Cells were stained with fluorescently labeled monoclonal antibodies specific for pan cytokeratin (CK-8/18/19-PE), leukocyte common antigen (CD45-APC), and nuclear stained with [4′,6-diamidino-2-phenylindole (DAPI)]. With spiked samples, tumor cells were isolated by DEPArray selection, which yielded a pure population of tumor cells for molecular characterization. Tumor cells were defined by presence of a clear DAPI-stained nucleus, CK-PE-positive cytoplasm and CD-45-APC negative. We demonstrated that by using a combination of enrichment and isolation/selection methods, we are able to isolate single, uncontaminated tumor cells to achieve single cell molecular analysis. The use of single cells is emerging as a powerful approach to molecular analysis in oncology, and this study demonstrates its potential application with circulating tumor cells. Citation Format: Sandra V. Fernandez, Christopher Wagner, Zahida Parveen, Lucy Aburto, Carmela Paolillo, George Hvichia, Zhaomei Mu, Laura Austin, Massimo Cristofanilli. Enrichment and isolation of uncontaminated breast cancer cells from human blood samples. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 224. doi:10.1158/1538-7445.AM2015-224

Inflammatory breast cancer (IBC) is a very aggressive type of advanced breast cancer with a poor prognosis. Due to its propensity to rapidly metastasize, IBC is responsible for a disproportionate number of breast cancer-related deaths; 15% of all breast cancer deaths result from this disease. Approximately 30% of IBC patients have distant metastases at time of diagnosis, in contrast to 5% of patients with non-IBC breast cancers. Currently, there is no adequate adjuvant therapy to reduce the risk of recurrence and mortality in IBC patients. We recently found that FAK1, also known as PTK2, is amplified, up-regulated and phosphorylated (active) in some inflammatory breast cancer cells [1]. The focal adhesion kinase FAK1 is a cytoplasmic tyrosine kinase that localizes to focal adhesions, and controls a number of cell pathways including proliferation, viability and survival. Activated FAK1 is absent or low in normal tissue and benign neoplasms and up-regulated in invasive and metastatic tumors. In this study, we investigated a new dual FAK1/ALK inhibitor, CEP-37440, in vitro and in vivo using preclinical models of IBC. We found that 300 nM CEP-37440 was able to decrease the proliferation of the triple negative FC-IBC02 cell line and, 1000 nM CEP-37440 was able to complete inhibit the proliferation of these cells. SUM190 showed a 50% decreased in proliferation when these cells were treated with 1000 nM CEP-37440. Genes involved in functions such as cellular growth and proliferation, cell cycle, cell death and survival and cellular movement and cell-to cell signaling and interaction were differentially regulated by CEP-37440 in sensitive IBC cell lines. Among them, TGFβ1 and MMP3 were down-regulated and DKK3, CAV1 and TFPI2 were up-regulated by CEP-37440. In FC-IBC02 orthotopic breast cancer xenograft models, CEP-37440 was able to reduce the growth of the primary tumor and inhibit the development of spontaneous metastases in brain. In conclusion, CEP-37440 was highly effective in reducing the proliferation in vitro and in vivo of IBC cells that expressed high levels of phospho-FAK (Tyr397). References 1. Fernandez S, Robertson F, Pei J, Aburto-Chumpitaz L, Mu Z, Chu K, Alpaugh K, Huang Y, Cao Y, Ye Z et al: Inflammatory breast cancer (IBC): clues for targeted therapies. Breast Cancer Res Treat 2013, 140:23-33. Citation Format: Israa Salem, Lucy Aburto, Sankar Addya, Inna Chervoneva, Zhaomei Mu, Bruce Ruggeri, Massimo Cristofanilli, Sandra V. Fernandez. CEP-37440, a highly selective and potent dual inhibitor of ALK and FAK1 inhibits the proliferation of inflammatory breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3232. doi:10.1158/1538-7445.AM2015-3232

Breast cancer is a malignancy whose dependence on estrogen exposure has long been recognized even though the mechanisms whereby estrogens cause cancer are not clearly understood. This work was performed to determine whether 17beta-estradiol (E2), the predominant circulating ovarian steroid, is carcinogenic in human breast epithelial cells and whether nonreceptor mechanisms are involved in the initiation of breast cancer. For this purpose, the effect of four 24 h alternate periods of 70 nM E2 treatment of the estrogen receptor alpha (ER-alpha) negative MCF-10F cell line on the in vitro expression of neoplastic transformation was evaluated. E2 treatment induced the expression of anchorage-independent growth, loss of ductulogenesis in collagen, invasiveness in Matrigel, and loss of 9p11-13. Only invasive cells that exhibited a 4p15.3-16 deletion were tumorigenic. Tumors were poorly differentiated ER-alpha and progesterone receptor-negative adenocarcinomas that expressed keratins, EMA, and E-cadherin. Tumors and tumor-derived cell lines exhibited loss of chromosome 4, deletions in chromosomes 3p12.3-13, 8p11.1-21, 9p21-qter, and 18q, and gains in 1p, and 5q15-qter. The induction of complete transformation of MCF-10F cells in vitro confirms the carcinogenicity of E2, supporting the concept that this hormone could act as an initiator of breast cancer in women. This model provides a unique system for understanding the genomic changes that intervene for leading normal cells to tumorigenesis and for testing the functional role of specific genomic events taking place during neoplastic transformation.

An elevated incidence of breast cancer in women has been associated with prolonged exposure to high levels of estrogens. Our laboratory demonstrated that treatment of the immortalized human breast epithelial cells MCF-10F with 17beta-estradiol (E2), 4-hydroxyestradiol (4-OHE2) or 2-hydroxyestradiol (2-OHE2) induces phenotypical changes indicative of neoplastic transformation. MCF-10F cells treated with E2, 4-OHE2 or 2-OHE2 formed colonies in agar methocel and lost their ductulogenic capacity in collagen, expressing phenotypes similar to those induced by the carcinogen benzo[a]pyrene. To investigate whether the transformation phenotypes were associated with genomic changes, cells treated with E2, 4-OHE2 or 2-OHE2 at different doses were analyzed using microsatellite markers. Since microsatellite instability (MSI) and loss of heterozygosity (LOH) in chromosomes 13 and 17 have been reported in human breast carcinomas, we tested these parameters in MCF-10F cells treated with E2, 2-OHE2, or 4-OHE2 alone or in combination with the antiestrogen ICI182780. MCF-10F cells treated with E2 or 4-OHE2, either alone or in combination with ICI182780, exhibited LOH in the region 13q12.3 with the marker D13S893 located at approximately 0.8 cM telomeric to BRCA2. Cells treated with E2 or 4-OHE2 at doses of 0.007 and 70 nM and 2-OHE2 only at a higher dose (3.6 microM) showed a complete loss of 1 allele with D13S893. For chromosome 17, differences were found using the marker TP53-Dint located in exon 4 of p53. Cells treated with E2 or 4-OHE2 at doses of 0.007 nM and 70 nM and 2-OHE2 only at a higher dose (3.6 microM) exhibited a 5 bp deletion in p53 exon 4. Our results show that E2 and its catechol estrogen metabolites are mutagenic in human breast epithelial cells. ICI182780 did not prevent these mutations, indicating that the carcinogenic effect of E2 is mainly through its reactive metabolites 4-OHE2 and 2-OHE2, with 4-OHE2 and E2 being mutagenic at lower doses than 2-OHE2.

The breast attains its maximum development during pregnancy and lactation. After menopause the breast regresses in both nulliparous and parous women containing lobular structures that have been designated lobules type 1. Despite the similarity in the lobular composition of the breast at menopause, the fact that nulliparous women are at higher risk of developing breast cancer than parous women, indicates that Lobules type 1 in these two groups of women might be biologically different, or exhibit different susceptibility to carcinogenesis. Based on these observations it was postulated that the Lobule type 1 found in the breast of nulliparous women and of parous women with breast cancer never went through the process of differentiation, retaining a high concentration of epithelial cells that are targets for carcinogens and therefore susceptible to undergo neoplastic transformation, these cell are called Stem cells 1, whereas Lobules type 1 structures found in the breast of early parous postmenopausal women free of mammary pathology, on the other hand, are composed of an epithelial cell population that is refractory to transformation called Stem cells 2. It was further postulated that the degree of differentiation acquired through early pregnancy has changed the "genomic signature" that differentiates the Lobule type 1 from the early parous women from that of the nulliparous women by shifting the Stem cell 1 to a Stem cell 2 that is refractory to carcinogenesis, making this the postulated mechanism of protection conferred by early full term pregnancy. The identification of a putative breast stem cell (Stem cell 1) has reached in the last decade a significant impulse and several markers also reported for other tissues have been found in the mammary epithelial cells of both rodents and humans. Although still more work needs to be done in order to better understand the role of the Stem cell 2 and its interaction with the genes that confer it a specific signature, collectively, the data presently available provides evidence that pregnancy, through the process of cell differentiation, shifts the Stem cell 1 to Stem cell 2, cells that exhibit a specific genomic signature that could be responsible for the refractoriness of the mammary gland to carcinogenesis.

Prolonged exposure to 17beta-estradiol (E2) is a key etiological factor for human breast cancer. The biological effects and carcinogenic effects of E2 are mediated via estrogen receptors (ERs), ERalpha and ERbeta. Anti-estrogens, e.g. tamoxifen, and aromatase inhibitors have been used to treat ER-positive breast cancer. While anti-estrogen therapy is initially successful, a major problem is that most tumors develop resistance and the disease ultimately progresses, pointing to the need of developing alternative drugs targeting to other critical targets in breast cancer cells. We have identified that Na+, K+-ATPase, a plasma membrane ion pump, has unique/valuable properties that could be used as a potentially important target for breast cancer treatment: (a) it is a key player of cell adhesion and is involved in cancer progression; (b) it serves as a versatile signal transducer and is a target for a number of hormones including estrogens and (d) its aberrant expression and activity are implicated in the development and progression of breast cancer. There are several lines of evidence indicating that ouabain and related digitalis (the potent inhibitors of Na+, K+-ATPase) possess potent anti-breast cancer activity. While it is not clear how the suggested anti-cancer activity of these drugs work, several observations point to ouabain and digitalis as being potential ER antagonists. We critically reviewed many lines of evidence and postulated a novel concept that Na+, K+-ATPase in combination with ERs could be important targets of anti-breast cancer drugs. Modulators, e.g. ouabain and related digitalis could be useful to develop valuable anti-breast cancer drugs as both Na+, K+-ATPase inhibitors and ER antagonists.

The elevated incidence of breast cancer in women has been associated with prolonged exposure to high levels of estrogens. Our laboratory has demonstrated that treatment of the immortalized human breast epithelial cells MCF-10F with 17β-estradiol (E 2 ) or its metabolites 4-hydroxy-estradiol (4-OH-E 2 ) and 2-hydroxy-estradiol (2-OH-E 2 ) induces phenotypical changes indicative of neoplastic transformation. The MCF-10F cells treated with E 2 , 2-OH-E 2 and 4-OH-E 2 form colonies in agar methocel and lost their ductulogenic capacity in collagen matrix, expressing phenotypes similar to those induced by the carcinogen benz(a)pyrene (BP). To investigate whether these phenotypic changes were associated with genomic changes, MCF-10F cells treated with either E 2 , 2-OH-E 2 , or 4-OH-E 2 at different doses (0.007 nM, 70 nM and 3.6 μM) were analyzed using a combination of standard G-banding and comparative genomic hybridization (CGH). Whereas no aneuploidy was observed in any of the transformed cells, the CGH revealed instead that only cells treated with 4-OH-E 2 at the highest concentration (3.6 μM) exhibited DNA gains at 8q24, 9q34 and 20q 13 and losses at 13q21.

Retinoids have been used as potential chemotherapeutic or chemopreventive agents because of their differentiative, anti-proliferative, pro-apoptotic, and anti-oxidant properties. The effects of retinoids are mediated by retinoic acid receptors (RARβ;,RARα,RARγ) and the retinoid X receptors (RXR) that bind the DNA as heterodimers. The objective of this project was to study the potential of retinoic acid to revert the neoplastic process using an in vitro model of breast cancer progression. This model was previously developed by treating the human normal breast epithelial MCF-10F cells with high dose of estradiol and consists of four cell lines which show a progressive neoplastic transformation: (a) the spontaneously immortalized cell line MCF-10F, which does not show any characteristic of invasiveness or tumor formation and therefore is considered to be a normal-like breast epithelial cell line; (b) the transformed trMCF cells; (c) the invasive bsMCF cells and; (d) the caMCF tumor cells. The bsMCF cells induced tumors when they were injected in the mammary fat pad of SCID mice. We previously showed that RARβ was unmethylated in MCF-10F and trMCF cells, but became hypermethylated in bsMCF and caMCF. We studied the effect of retinoic acid at the different stages in this model by cultivating the cells in 3D-cultures (collagen matrix). In collagen, the MCF-10F cells form tubules resembling the normal mammary gland. After treatment with estradiol, cells formed tubules and spherical masses which are indicative of transformation. Cells that only formed spherical masses in collagen were isolated (trMCF clone 11) and treated with different concentrations of ATRA (10-5M to 10-8M). We found that trMCF clone 11 cells showed a reduction of solid masses and increased number of tubules in collagen after being treated with 10-6M ATRA. A total of 43% of the structures were tubules in trMCF clone 11 cells treated with 10-6M ATRA. Gene expression studies showed that 207 genes up-regulated in transformed trMCF clone 11 cells were down-regulated after ATRA treatment to levels comparable to those found in the normal breast epithelial cells MCF-10F. Furthermore, 236 genes that were down-regulated in trMCF clone 11 were up-regulated after ATRA treatment to similar levels shown in normal epithelial cells. These 443 genes defined a signature of the ATRA re-programming effect. Our results showed that 10-6M ATRA was able to re-differentiate transformed cells at early stages of the neoplastic process to a normal stage and antagonistically regulate breast cancer associated genes. The invasive and tumorigenic cells did not show any changes in morphology after treatment with ATRA alone or in combination with 5-aza-dC. These results suggest that ATRA could be used as chemopreventive agent to inhibit the progression of premalignant lesions of the breast. This work was supported by The Pennsylvania Breast Cancer Coalition and Friends for an Earlier Breast Cancer Test. Citation Format: Maria F. Arisi, Rebecca A. Starker, Sankar Addya, Yong Huang, Sandra V. Fernandez. All trans-retinoic acid (ATRA) induces redifferentiation of early transformed breast epithelial 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 2109. doi:10.1158/1538-7445.AM2014-2109

Inflammatory breast cancer (IBC) is the most aggressive type of advanced breast cancer characterized by rapid proliferation, early metastatic development, and poor prognosis. The peculiar clinical presentation of IBC characterized by breast erythema and swelling is caused by the invasion of aggregates of tumor cells (tumor emboli) into the dermal lymphatics, causing an obstruction of the lymph channels. Although, tumor emboli are also occasionally demonstrated in non-IBC tumors, they are more frequently detected and more numerous in IBC. Tumor emboli expressed cell-cell adhesion molecules that maintain the tumor cells together. The high risk of disease recurrence in soft tissue and lymph nodes shortly after completion of multidisciplinary treatment particularly in patients with residual disease supports the hypothesis that these tumor emboli are critically related to the development of micro metastatic disease. We developed a new model of IBC derived from the pleural effusion of a woman with metastatic secondary IBC. FC-IBC02 cells are triple negative and form clusters in suspension that were strongly positive for E-cadherin, β-catenin, and TSPAN24, all adhesion molecules that play an important role in cell migration and invasion. The maintenance of cell-cell adhesions allow the migration of tumor cells through the lymphatic and blood vessels as clusters. FC-IBC02 cells shown a partial or incomplete epithelial-mesenchymal transition (EMT); these cells express some epithelial markers (EpCAM, E-cadherin) and also mesenchymal markers (VIM, FN1, Snai2, Twist1). Furthermore, circulating tumor cells (CTC) from IBC patients are present in the blood as single cells and clusters supporting the collective migration of tumor cells in IBC. FC-IBC02 cells were highly metastatic when injected in the fat mammary pad of SCID mice and these cells were able to produce brain metastasis in mice by intracardiac or intra-peritoneal injections. Genomic studies of FC-IBC02 and other IBC cell lines showed that IBC cells had important amplification of 8q24 where MYC, ATAD2 and the focal adhesion kinase FAK1 are located. MYC and ATAD2 showed between 2.5-7 copies in IBC cells. FAK1, which plays important roles in anoikis resistance and tumor metastasis, showed 6-4 copies in IBC cells. FAK1 is amplified, upregulated and phosphorylated (active) in inflammatory breast cancer. Additionally, FC-IBC02 showed amplification of ALK and NOTCH3. Our results indicate that MYC, ATAD2, CD44, NOTCH3, ALK and/or FAK1 may be used as potential targeted therapies against IBC. We are currently evaluating an ALK/FAK inhibitor using the novel established IBC model. Citation Format: Sandra V. Fernandez, Fredika M. Robertson, Sankar Addya, Zhaomei Mu, Massimo Cristofanilli. Clues for targeted therapies in inflammatory breast cancer (IBC). [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 4988. doi:10.1158/1538-7445.AM2014-4988

BACKGROUND: Inflammatory breast cancer (IBC) is an aggressive type of advanced breast cancer with a poor prognosis and lower survival rate. IBC is characterized by peculiar clinical and biological features and resistance to standard treatments. IBC has shown the capacity to spread early through lymphatic channels and blood vessels causing the specific inflammatory signs. Currently, there are no specific therapeutic options for IBC patients because of very few molecular alterations associated with IBC. Thus, there is an unmet need to identify distinguished features of IBC that differentiate the disease from non-inflammatory breast cancer (nIBC). Metabolomic analysis of cancer cells holds the promise to identify unique chemical fingerprints of important cellular processes. In this study we investigated the global biochemical profiles of IBC and nIBC cells. METHODS: Three IBC cell lines, SUM190, SUM149, FC-IBC-02, and three nIBC cell lines, MCF-7, MDA-MB-231, MDA-MB-468, with five replicates for each cell line, were analyzed in this study. Metabolomic profiling analysis was performed by Metabolon, Inc (Durham, NC) on the gas chromatography/mass spectroscopy and ultrahigh performance liquid chromatography/mass spectroscopy platforms as previously described (Proc. Natl. Acad. Sci. U. S. A. 108, 3270-3275, 2011). A total of 347 metabolites were detected. Welch's two-sample t-test was used to identify biochemicals that differed significantly between experimental groups (p≤0.001), as well as those approaching significance (0.001 RESULTS: Comparison of global biochemical profiles for IBC and nIBC cell lines revealed numerous statistically changed metabolites at a p-value threshold of p CONCLUSIONS: Our study is the first to demonstrate difference of metabolic profiles between IBC and nIBC cells. Future studies comparing global biochemical profiles in tissue biopsy and blood from IBC and nIBC patients may help to confirm the relevance of these findings in cell lines. Metabolic targets may provide more effective and specific therapeutic strategy for the treatment of IBC patients. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-01-05.

Inflammatory breast cancer (IBC) is the most aggressive type of advanced breast can[[Unsupported Character - Codename ­]]cer and it is associated with a poor prognosis in spite of appropriate multidisciplinary treatments. The disease is characterized by peculiar molecular and clinical features and usually affects younger patients, mostly under the age of 50 years at diagnosis. IBC has shown the capacity to spread early, primarily through lymphatic channels and secondarily through blood vessels causing the inflammatory signs and development of early metastasis. The high incidence of metastatic disease at presentation, the persistence of residual disease after induction chemotherapy associated with significant risk of disease recurrence immediately surgery strongly support our hypothesis that these patients develop micro metastatic disease early in the disease course (Cristofanilli et al., Cancer 2007;110:1436-44). Moreover, the lower survival rate of IBC patients may be due to the early activation of metastatic process associated with a peculiar phenotype of cancer stem cells (CSCs) (Cristofanilli et al., Oncologist 2003;8:141-8). Although IBC research has been ongoing for over 15 years, very few molecular alterations have been associated specifically with IBC and few preclinical models are currently available to evaluate the peculiar biology of IBC. The majority of IBC studies have been performed using the cell lines SUM149 and SUM 190 which were developed from the primary tumor and, KPL-4 isolated from the pleural effusion of IBC patients. Although both the SUM149 and KPL-4 injected into immuno- compromised mice form primary tumors, there are currently only two in vivo xenograft models of IBC, the Mary-X and the WIBC-9 models that recapitulate the tumor emboli that are the signature of IBC in humans. We have developed another IBC model, FC-IBC-02, derivate from the pleural effusion of a 49 years old IBC triple negative patient. Tumor cells from the pleural effusion were ER(-) Pgr(-) and Her2(-) and strongly positive for tetraspanin CD151 and E-cadherin. Cells from the pleural effusion of this patient were growth under non-adherent conditions in serum-free mammary epithelial growth medium (MEGM). These tumor spheroids have Aldhehyde Dehydrogenase 1 (ALDH-1) activity and characteristics of cancer stem cells (CSC) and they rapidly developed tumors when they were injected in the mammary fat pad of SCID mice. These tumors were poorly differentiated carcinomas ER-negative PgR-negative Her2-negative and the mice developed micro metastasis in the lungs. In contrast, cells isolated under adherent conditions were unable to produce tumor in SCID mice. In summary, our studies demonstrated that IBC is a disease enriched for cells with a stem cell phenotype and these cells are highly tumorigenic. IBC may represent an ideal model to evaluate stem cell targeting therapeutic modalities. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5342. doi:1538-7445.AM2012-5342

Background: Inflammatory breast cancer (IBC) is the most metastatic variant of breast cancer. It is associated with a poor survival rate (40% 5-year survival) despite appropriate multidisciplinary care. For such an aggressive type of cancer, IBC has been understudied, in part due to the lack of adequate numbers of cell lines and mouse models that recapitulate the human disease. To expand our understanding of IBC, we have obtained all of the previously developed and characterized IBC cell lines and models including Mary-X, SUM149, SUM190, KPL-4, MDA-IBC-3 and have developed two new IBC models, designated as FC-IBC01 and FC-IBC02, using tumor cells derived from pleural effusion of IBC patients. Materials and Methods: Each of these IBC cell lines has been luciferase (LUC)-tagged, allowing the growth of orthotopic injection or subcutaneous implantation to be evaluated by bioluminescent imaging (BLI). Alternatively, the LUC-tagged IBC cells can by injected via either intra-cardiac or intravenous route of delivery, which promotes rapid tumor colonization, resulting in both visceral and skeletal metastasis. Growth of IBC tumors can then be monitored immediately using BLI, thus eliminating the lag time needed for the physical detection of palpable tumors. BLI imaging also allows for monitoring of the kinetics and location of development of metastatic lesions. Whole transcriptome analysis was performed on IBC cell lines and xenograft tissues to define the heterogeneity of IBC as a distinct variant of breast cancer Results: These models have allowed us to identify micro-metastatic foci in multiple sites distant from the IBC primary tumor in each of these models of IBC and allow the quantitation of anti-tumor and anti-metastatic effects of targeted therapeutics as single agents as well as the potential synergy of combinations of agents. As an example, injection of LUC-tagged IBC cell lines such as SUM149-Luc, into the left ventricle of NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice allows the metastatic tumor burden to be monitored longitudinally by whole animal BLI, which can be validated at necropsy and by immunohistochemical analysis. Whole transcriptome analysis of pre-clinical models of IBC reflect the molecular subtypes observed in IBC patients, with the majority of IBC models being of the basal like, luminal B and Her2 amplified. Discussion: First time analysis of known and newly developed pre-clinical models of IBC allows a more complete analysis of IBC as a distinct variant of breast cancer. Furthermore, these approaches allow rapid evaluation of the promising targeted therapeutics identified based on whole transcriptome analysis of both IBC patient tumors and pre-clinical models developed from IBC patients. We believe that this extensive collection of LUC-tagged IBC cell lines is an invaluable tool for IBC research since the cell lines encompass the broad spectrum of IBC heterogeneity. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-03-06.

We have developed an in vitro model of breast cancer progression that consists of four derived ER-negative PR-negative cell lines: (a) the normal-like breast epithelial cell line MCF-10F; (b) the transformed trMCF cells; (c) the invasive bsMCF cells and; (d) the tumorigenic caMCFs cells which shown all characteristics of a fully malignant breast cancer cells [1,2]. Briefly, the normal human breast epithelial MCF-10F cells were treated with high concentration of 17β-estradiol (70 nM) giving origin to trMCF cells; trMCF cells were seeded in Boyden chambers and the cells that crossed the membrane were selected and expanded giving origin to bsMCF. The bsMCF cells were injected in the mammary fat pad of SCID mice and they induced tumors that were poorly differentiated adenocarcinomas that were ESR, progesterone receptor (PR) and ERBB2 negatives. Cells were isolated from 4 different tumors growth in four animals and they gave origin to the tumorigenic caMCFs cells (caMCF1, caMCF2, caMCF3 and caMCF4) [1,2]. This in vitro model of breast cancer progression was used to study DNA-methylation changes at the different stages using sensitive methylation specific PCR (MSP) and, we found that NRG1 and RARβ showed progressive changes in their methylation pattern. NRG1 and RARβ were unmethylated in MCF-10F and trMCF cells, becoming hypermethylated in the invasive (bsMCF) and tumor (caMCF) stages. The clinical relevance of these findings were pursued by studying NRG1 and RARβ methylation in 17 cases of invasive ductal carcinomas (IDC) and 11 normal breast tissue samples (controls). NRG1 was hypermethylated in 14 out of 17 IDC (82.4%) vs. 2 out of 10 (20%) controls. All the ER-negative PR-negative tumors (10/10) showed NRG1 hypermethylated. From the seven ER-positive IDC, only 4 (57%) showed NRG1 hypermethylated. RARβ was methylated in 9 out of 16 IDC (56.3%) vs. 2 out of 11 (18.2%) controls. Most of the ER-negative PR-negative invasive ductal tumors showed RARβ hypermethylated (9/10). None of the ER-positive IDC shown RARβ hypermethylated. Altogether our data shown that the majority (90%) of the ER-negative PR-negative invasive ductal carcinomas shown hypermethylation of both NRG1 and RARβ. None of the ER-positive invasive ductal carcinomas showed RARβ hypermethylation and, only 57% showed hypermethylation of NRG1. (This work was supported by The Pennsylvania Breast Cancer Coalition). REFERENCES [1] S.V. Fernandez, K.E. Snider, Y.Z. Wu, I.H. Russo, C. Plass and J. Russo DNA methylation changes in a human cell model of breast cancer progression, Mutat Res 688 (2010) 28-35. [2] Y. Huang, S.V. Fernandez, S. Goodwin, P.A. Russo, I.H. Russo, T.R. Sutter and J. Russo Epithelial to mesenchymal transition in human breast epithelial cells transformed by 17beta-estradiol, Cancer Res 67 (2007) 11147-11157. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 75. doi:10.1158/1538-7445.AM2011-75

Retinoids, usually formed from dietary vitamin A, play a key role during embryonic development, cellular proliferation and differentiation, and are potent inhibitors of cell growth. Retinoids have been recognized as promising anticancer compounds. The effects of retinoids are mediated by retinoic acid receptors (RARβ, RARα, RARγ) and the retinoid X receptors (RXR) that binds the DNA as heterodimers. The objective of this project is to study the potential of retinoic acid to revert the neoplastic process using an in vitro model of breast cancer progression developed in our laboratory. The MCF-10F progression model consists of four derived cell lines: (a) the spontaneously immortalized cell line MCF-10F, which does not show any characteristic of invasiveness or tumor formation and therefore is considered to be a normal-like breast epithelial cell line; (b) the transformed trMCF cells; (c) the invasive bsMCF cell line and; (d) the tumor cell lines, caMCFs, which shown all characteristics of a fully malignant breast cancer cell types. The bsMCF cells induced tumors when they were injected in the mammary fat pad of SCID mice; these tumors were poorly differentiated adenocarcinomas that were estrogen receptor (ER), progesterone receptor (PR) and ERBB2 negatives [1]. Using this model, DNA-methylation studies were performed and we have found that RARβ (retinoic acid receptor β) was unmethylated in MCF-10F and trMCF cells becoming hypermethylated at the invasive (bsMCF) and tumorigenic (caMCF) stages. We studied the effect of retinoic acid at the different stages of the in vitro model of breast cancer progression using 3D-cultures (collagen matrix). Cultivation of normal breast epithelial cells within collagen enables the cells to polarize and to spontaneously differentiate tubular structures. In collagen, the MCF-10F cells form tubules resembling the normal mammary gland. The transformed cells trMCF form tubules and spherical masses; spherical masses are an indication of cell transformation. One solid mass was isolated and the cells expanded, giving origin to trMCF clone 11, which only form solid masses in collagen. The trMCF clone 11 cells were treated with different concentrations of retinoic acid during 15 days and, after the treatments the cells were plated in collagen. The trMCF clone 11 treated with 10-6M and 10-7M retinoic acid shown a reduction of solid masses and increased of tubules in collagen. Although bsMCF and caMCF were treated with 5-aza-dC to reactivate the expression of RARβ in combination with retinoic acid, a change in the phenotype of these cells in collagen was not observed. Our results indicated that retinoic acid was able to re-differentiate transformed cells at early stages of the neoplastic process. (This work was supported by The Pennsylvania Breast Cancer Coalition). REFERENCES. [1] S.V. Fernandez et al. DNA methylation changes in a human cell model of breast cancer progression, Mutat Res 688 (2010) 28-35. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4551. doi:10.1158/1538-7445.AM2011-4551