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Background: Inflammatory breast cancer (IBC) is the most lethal and aggressive form of breast cancer, yet no targeted therapy has been approved specifically for this disease. There is a critical need for innovative treatment approaches for patients with IBC. There is a paucity of data on immune checkpoint inhibitors (ICIs) in IBC, yet a strong biological rationale exists to lay the groundwork for testing the efficacy of ICIs in IBC. We have previously shown that an anti–epidermal growth factor receptor (EGFR) antibody, panitumumab (sponsored by Amgen), combined with Abraxane (sponsored by Bristol Myers Squibb), and carboplatin followed by 5-fluorouracil, epirubicin, and cyclophosphamide achieves a very high pathologic complete response (42%) in patients with triple-negative receptor status (TN-IBC) (NCT01036087). We hypothesize that inducing a broad shift in the IBC tumor microenvironment (TME), a critical driver of the IBC clinical phenotype and metastasis, from an immunosuppressive to an immunoreactive phenotype can enhance the efficacy of ICIs. Here we report the impact of targeting EGFR on ICI effectiveness by modulating the immunosuppressive TME in TN-IBC. Methods: We examined the effects of panitumumab on components of the immune TME in IBC patient tissues collected before and after panitumumab treatment. Furthermore, we analyzed panitumumab effects in an IBC SUM149 humanized mouse model (SUM149-hu-NSG-SGM3) by using multiplex immunofluorescence staining (mIF) of single-cell RNA-sequencing (scRNA-seq) and multicolor flow cytometry. We examined the changes in chemokines in humanized mouse tissue after panitumumab treatment using a cytokine antibody array and enzyme-linked immunosorbent assay (ELISA). We also studied the mechanism of EGFR regulation of immunosuppressive chemokine expression via transcription factor EGR1 by using reverse transcription-polymerase chain reaction (RT-PCR), Western blotting, and chromatin immunoprecipitation (ChIP) assays. In addition, we tested the efficacy of panitumumab combined with anti-PD-L1 antibody (Bio X Cell, clone 29E.2A3) in reducing IBC tumor growth in a humanized IBC SUM149 mouse model. Results: IBC patients who achieved pathologic complete response after receiving panitumumab/neoadjuvant chemotherapy had increased levels of CD8+ T-cells and decreased levels of M2 macrophages and T-reg cells. In the humanized mouse model, panitumumab treatment reduced IBC tumor growth and increased immune response that favors an antitumor effect, as shown by scRNA-seq, flow cytometry, and mIF analyses. In addition, we observed that panitumumab treatment increased the expression of chemokines that function as a chemoattractant for CD8+ T-cells, including CXCL10, CCL4, and CXCL9. We also observed that panitumumab treatment reduced EGR1 expression, leading to the downregulation of chemokines involved in the recruitment of immunosuppressive M2 macrophages and T-reg cells, including CCL2, CCL20 CXCL5, and IL-8. Finally, panitumumab enhanced the reduction in tumor growth by anti-PD-L1 antibody treatment; furthermore, the combination of panitumumab and anti-PD-L1 antibody increased the presence of CD8+ T-cells and reduced the presence of T-reg cells and M2 macrophages more effectively than either treatment alone. Conclusion: Panitumumab treatment converts the immunosuppressive IBC TME to an immunoreactive status by modulating the global expression of chemokines by downregulating the EGR1 transcription factor. This modulation of the TME by panitumumab improves the inhibition of IBC tumor growth by an ICI. Our study is the first to demonstrate that targeting the EGFR/EGR1 axis remodels the IBC TME by regulating chemokine expression, thus boosting the antitumor immune response in IBC and response to ICI. Citation Format: Xiaoping Wang, Takashi Semba, Ganiraju C. Manyam, Jing Wang, Shan Shao, Francois Bertucci, Pascal Finetti, Savitri Krishnamurthy, Lan ThiHanh Phi, Troy Pearson, Jared K. Burks, Evan N. Cohen, James M. Reuben, Fei Yang, Hu Min, Nicholas Navin, Toshiaki Iwase, Yichao Shen, Xiang Zhang, Debu Tripathy, Naoto T. Ueno. Remodeling the inflammatory breast cancer tumor microenvironment to enhance immunotherapy: Novel therapeutic development [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 PD10-08.

Background: Inflammatory breast cancer (IBC) and triple negative breast cancer (TNBC) are more aggressive than other breast cancer subtypes, and up until recently lacked therapy options that maintain acceptable quality of life. While chemotherapy is a treatment option for all subtypes of breast cancer, it is used long-term as maintenance therapy for metastatic TNBC (mTNBC) and often produces cumulative toxicity resulting in discontinuation of treatment. Moreover, responses are rarely durable after discontinuation of chemotherapy. Immune checkpoint blockade has the potential to maintain therapy. To determine factors associated with response to anti-PD-1 therapy (pembrolizumab), we performed minimally invasive blood-based analyses of T-cell repertoire and phenotype to evaluate their association with progression-free survival (PFS) in 15 patients with metastatic IBC (mIBC) or mTNBC. Materials and Methods: Fifteen patients with mIBC (n=6) or mTNBC (n=9) were enrolled on an ongoing phase II study to receive pembrolizumab as maintenance therapy after achieving a clinical response or stable disease to systemic chemotherapy for metastatic disease. We performed analyses of T-cell repertoire and phenotype on peripheral blood mononuclear cells (PBMC) from samples obtained post induction therapy but before initiation of pembrolizumab (baseline) therapy. Expression of T-cell exhaustion markers 2B4, CTLA4, BTLA, Lag3, PD-1, and Tim3 was evaluated on PBMC by flow cytometry. T-cell receptor (TCR) beta chain CDR3 DNA sequencing by ImmunoSEQ and richness (diversity) and clonality (reactivity) were evaluated. Patients were followed through 5 months of treatment with pembrolizumab to evaluate the association between T-cell repertoire and phenotype with PFS. Results: Seven patients had stable disease (SD) and 8 had progressive disease (PD) by 5 months. The median follow-up was 14.2 months (range: 4.5 to 27.7 months). Among the patients who progressed within 5 months, the earliest and the latest time to progression was at 1.38 months and 4.82 months, respectively. CTLA4 expression in CD4+ T cells at baseline was significantly higher in patients with PD than in patients with SD (p = 0.040). Patients with a low percentage of CD4+ T cells expressing exhaustion markers (CTLA4, Tim3, and 2B4) at baseline were more likely to have SD (chi-square p = 0.041) and significantly longer median PFS than patients with PD (median time to progression was not reached in SD vs. 4.1 months in PD, p = 0.018). Additionally, patients with high clonality and low CD4+ T exhaustion markers were more likely to have SD (chi-square p = 0.041) and a longer median time to progression (median time to progression was not reached in SD vs. 4.1 months in PD, p = 0.015). Conclusions: Baseline T-cell clonality and T-cell exhaustion markers have significant translational relevance and can help to explain variable responses to immune checkpoint blockade. Our data suggest that T-cell reactivity at baseline, and a lower percentage of CD4+ T cells expressing CTLA4/Tim3/2B4 were favorable prognostic factors for pembrolizumab maintenance therapy. This proof-of-concept provides compelling data that T-cell clonality and phenotyping of T-cell exhaustion markers can be useful and should be applied to future trials with immune checkpoint inhibitors. Citation Format: Hui Gao, Kumiko Kida, Evan N Cohen, Angela Alexander, Bora Lim, Charla Parker, Sanda Tin, Vicente Valero, Debu Tripathy, Alexandre Reuben, Naoto T Ueno, James M Reuben. Peripheral T cell clonality and exhaustion as novel biomarkers for anti-PD-1 (pembrolizumab) maintenance therapy in patients with metastatic inflammatory breast cancer (mIBC) and non-IBC triple negative breast cancer (mTNBC) [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 P3-09-12.

Background: Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer characterized by a high rate of metastatic recurrence and poor prognosis. Molecular mechanism underlying the metastatic behavior of TNBC has not been well elucidated, and newer approaches addressing drivers of metastasis are crucial to improving patient outcomes. PEA15 (Phosphoprotein enriched in astrocytes-15) regulates cell proliferation, apoptosis, and autophagy. In breast cancer, PEA15 expression inhibits invasion by binding to ERK and preventing its nuclear translocation. The biological function of PEA15 is tightly regulated by its phosphorylation at Ser104 and Ser116. However, the effect of PEA15 phosphorylation status on TNBC remains unknown. In this study, we tested the hypothesis that unphosphorylated PEA15 will prevent metastasis in TNBC through inhibition of the epithelial-to-mesenchymal transition (EMT). Method: We established stable cells overexpressing unphosphorylatable (PEA15-AA) and phospho-mimetic (PEA15-DD) PEA15 mutants in MDA-MB-468 cells. To dissect specific Cellular Mechanisms regulated by PEA15 phosphorylation, we performed RT-PCR immune and metastasis arrays. In vivo mouse models were used to see effects of PEA15 phosphorylation on tumor growth. Results: The clonogenic growth of PEA15-AA–expressing cells was significantly reduced by 80% compared with empty vector-transfected cells (PEA15-V). Anchorage-independent growth, an indicator of in vivo tumorigenicity, was inhibited in cells expressing PEA15-AA by 60% compared with PEA15-V. PEA15-AA upregulated the expression of E-cadherin and decreased the expression of mesenchymal markers, suggesting that PEA15-AA reverses EMT. Compared with PEA15-V, migration and invasion of cells expressing PEA15-AA were reduced by 65% and 72%, respectively. In contrast, PEA15-DD promoted migration, invasion, and expression of mesenchymal markers. To determine the in vivo effect of PEA15-AA, we injected stable PEA15 transfectants of MDA-MB-468 cells into the mammary fat pad of NOD/SCID mice. The PEA15-DD–injected group showed greater tumor volumes than PEA15-V and PEA15-AA groups, suggesting that PEA15-AA has antitumor effects both in vitro and in vivo. From the immune and metastasis arrays, we found that expression level of IL-8, which is known to induce EMT, was greatly decreased by PEA15-AA, while IL-8 was highly expressed in PEA15-DD cells. Addition of recombinant IL-8 to the cells expressing PEA15-AA partially rescued mesenchymal characteristics, increasing migration and expression of mesenchymal markers. By contrast, IL-8 knockdown in PEA15-DD–expressing cells decreased the mesenchymal phenotype. These findings indicate that IL-8 may play an important role as a mediator of phosphorylation of PEA-15 in breast cancer cell migration and invasion and suggest that PEA15-AA inhibits the expression of IL-8, thereby reversing EMT. Conclusion: Taken together, our results show that PEA15 phosphorylation serves as an important regulator, having a dual role as an oncogene or tumor suppressor. Further studies are warranted to evaluate the impact of PEA15 phosphorylation status on metastasis in vivo. These findings support the development of PEA15-AA as a potential therapeutic strategy for TNBC. Citation Format: Park J, Chauhan G, Cohen EN, Ueno NT, Battula VL, Tripathy D, Reuben JM, Bartholomeusz C. PEA15-AA, an unphosphorylatable mutant of PEA15, as a novel therapeutic gene for triple-negative breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-06-22.

Background: Most cancer cells depend more on glycolysis for their energy need compared with their normal counterparts incubated under identical condition. In addition to glycolysis, tumor cells also engage mitochondrial ox-phos to support growth. Here, we show that PCa cells surviving under anti-androgen enzalutamide (ENZA) treatment switch from glycolysis to ox-phos for their energy need and are more vulnerable to mitochondria-targeted metabolic inhibitors. We have also standardized a high-resolution quantitative fluorescence microscopic method to detect this metabolic switch in patient circulating tumor cells (CTCs). Methods: Seahorse assay has been used to compare mitochondrial metabolism in ENZA treated anti-androgen-sensitive LNCaP and -resistant C4-2 and PCa2b cells. The Seahorse data were supported by mass spectroscopic analysis of corresponding metabolites and metabolic fluxes. An ex vivo fluorescence staining for the CTC mitochondria with high ox-phos followed by high-resolution quantitative microscopic image analysis method has been standardized to follow such changes in cultured cells and patient CTCs. Results: Seahorse, mass spectroscopy and high-resolution microscopy of mitochondrial ox-phos showed that ENZA significantly decreases glycolysis and increases ox-phos in all surviving PCa cells within 24h of treatment. These cells are more vulnerable to treatment with mitochondrial ox-phos inhibitor IACS-010759 and a glutaminase inhibitor CB-839. High-resolution microscopic analysis of CTCs has thus far been performed in 18 patient blood samples. Six out of the 18 patients developed resistance to anti-androgen therapy within 0-6 months of sample collection. CTCs from all six patients showed a relatively higher average fluorescence due to high mitochondrial ox-phos as compared with the rest of the patients. Discussion: The data presented here may lead to informed combination therapy for selected PCa patients developing resistance to anti-androgen therapy for better clinical outcome. Citation Format: Hirak S. Basu, Nathaniel Wilganowski, Samantha Robertson, Sumankalai Ramachandran, Amado Zurita-Saavedra, Mark Titus, Evan Cohen, James Reuben, George Wilding. Metabolic switch from glycolysis to oxidative phosphorylation (ox-phos) provides survival advantage to anti-androgen-treated prostate cancer cells and make them vulnerable to mitochondrial metabolism inhibitors IACS-010759 and CB-839 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4791.

Immune checkpoint inhibitors (ICIs) is now a standard of care for metastatic TNBC, the most aggressive subtype of breast cancer. However, rates of tumor response to ICIs are low. Evidence suggests that an immunosuppressive TME weakens the efficacy of ICIs. We previously showed that JNKs can regulate migration, invasion, and stemness of TNBC, and JNKs are known to mediate inflammatory signaling. However, the role of JNK signaling in the immunosuppressive TME of TNBC is unclear. We studied the role of JNK signaling in modulating the immunosuppressive TME of TNBC by treating syngeneic TNBC mice with a JNK inhibitor and determining the chemokine profiles related to the aggressive nature of TNBC. Method: The effect of JNK inhibitor, JNK-IN-8, on TNBC tumor growth and lung metastasis was measured in 4T1.2 syngeneic mouse model. Cytokine expression in tumor tissues from vehicle- and JNK-IN-8-treated mice was measured by antibody array and qPCR. Tumor-infiltrating immune cells were measured by flow cytometry. For a rescue experiment, 4T1.2 mice were treated with JNK-IN-8 and administered either PBS or recombinant mouse CCL2 via an osmotic pump. In addition, CCL2 levels were measured in sera of 46 TNBC patients by Luminex assay. Results: JNK-IN-8 treatment significantly reduced tumor growth (p = 0.0065), lung metastasis (p = 0.0364), and tumor-infiltrating leukocytes, including regulatory T cells (Tregs) (p = 0.0469) and myeloid-derived suppressor cells (MDSCs) (p = 0.0143), while increasing infiltration of CD8+ T cells (p = 0.0152). JNK-IN-8 also reduced levels of chemokines, including CCL2, CCL11, CCL19, CCL21, CCL22, and CXCL1, and increased levels of cytokines involved in T cell activation and effector function, including IL-2 and IFN-γ. CCL2 is involved in attracting Tregs, MDSCs, and other cells. Indeed, systemic CCL2 administration mitigated the inhibitory effect of JNK-IN-8 on tumor-infiltrating Tregs and MDSCs, TNBC tumor growth, and lung metastasis in the 4T1.2 mouse model. Indeed, in patient serum, the median CCL2 level was significantly higher in patients with metastatic TNBC than in patients with non-metastatic TNBC (988.72 pg/mL vs 414.86 pg/mL, p = 0.0053), and a higher CCL2 level was associated with poor progression-free and overall survival (univariate Cox, p = 0.004 and p = 0.008). Conclusion: We have determined that the CCL2 is one of the important critical cytokines contributing to the immunosuppressive TME of TNBC. Our data suggest that JNK inhibition can improve an immunosuppressive TME through CCL2 reduction and may have the potential to boost the efficacy of ICIs. Studies of the therapeutic efficacy of JNK inhibitor-ICI combination treatment in syngeneic mouse models are in progress. Citation Format: Takashi Semba, Xuemei Xie, Evan N. Cohen, James M. Reuben, Kevin N. Dalby, Xiaoping Wang, Naoto T. Ueno. Suppression of tumorigenicity and metastasis of triple-negative breast cancer (TNBC) by c-Jun N-Terminal Kinase (JNK) inhibitor via reversing immunosuppressive tumor microenvironment (TME) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5067.

Background:Circulating tumor cells (CTCs) are an independent predictor of survival in patients with breast cancer. In addition, mesenchymal (EMT-CTC) and stem-like (Stem-CTC) CTCs contribute to disease progression. The objective of the overall study is to determine whether a comprehensive lifestyle intervention program started prior to radiotherapy can modulate changes in CTC subsets that are correlated with disease recurrence and progression. For these analyses we examined the association between medical and treatment-related factors and CTCs. Patients and Methods: Seventy-eight patients with stage II/III breast cancer were recruited and randomized to either the intervention group or a standard care group. The intervention group (n=42) had in-person lifestyle counseling across the 4-6 weeks of radiotherapy (XRT) followed by video counseling for the subsequent 12 months. The standard care group (n=36) was provided patient-education materials for cancer prevention including information on diet, exercise, and stress management, without counseling. Blood samples were collected prior to initiation of XRT, end of XRT, and at 3-month intervals thereafter for up to 5 years. CTC subsets were detected by AdnaTest EMT2 kit (Qiagen, Venlo, Netherlands). Samples were considered positive for CTCs if any one of breast (EPCAM, MUC1, and HER2), EMT (TWIST1), or stem cell-related (ALDH1, AKT2, and PI3Kalpha) genes were detected by PCR above the manufacturer's suggested threshold. Results: The median age of patients was 49 years (range 26-82 years). Thirty-four patients were overweight (BMI 24.4-30) and 44 patients were obese (BMI >30). Forty-five patients were HR+Her2-, 12 patients were HR+Her2+, 5 patients were HR-Her2+, and 16 patients were TNBC. Sixteen patients were stage IIA or IIB, 34 patients were stage IIIA or IIIB, 27 patients were stage IIIC, and 1 was stage IV. Sixty-seven of 78 patients received neoadjuvant chemotherapy (NACT); 13 patients achieved a complete pathological response (pCR). The median follow-up was 21.6 months. CTC data of both intervention and standard groups were similar at baseline. Presence of CTCs at baseline or follow-up time points was not correlated to HR/Her2 status, stage, obesity, or pCR, but was significantly correlated with receiving NACT. Patients without NACT had significantly higher CTCs than patients who underwent NACT (Fisher Exact Test p=0.010). Furthermore, CTCs by the detection of any gene 3 months after completing XRT was associated with shorter PFS (log-rank p=0.016) and OS (p=0.03). Conclusions:This is an interim analysis of the prognostic potential of CTCs detected by AdnaTest EMT2 kit in non-metastatic breast cancer. We observed a lower proportion of patients with CTCs following neoadjuvant chemotherapy. However, the relative small sample size and short follow-up time preclude drawing conclusions to the efficacy of using CTCs as surrogate measures for lifestyle interventions, although the presence of CTCs in peripheral blood of patients 3 months after radiation therapy can be a promising indicator of disease relapse and overall survival. Citation Format: Gao H, Cohen EN, Yang P, Austin TA, Haddad R, Wu Q, Basen-Engquist KM, Ochoa JM, Arun BK, Perkins GH, Tin S, Vallone VS, Mallaiah SG, West CB, Thompson AS, Chaoul A, Cohen L, Reuben JM. Circulating tumor cell subset analysis to assess lifestyle interventions for breast cancer patients after neoadjuvant chemotherapy [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-01-15.

Background: Capecitabine is an established radiosensitizer in rectal and other cancers. We conducted a prospective single arm phase II study to examine the response rate of gross chemo-refractory breast cancer treated with concurrent capecitabine and radiotherapy. Methods: Patients who had inoperable or marginally operable gross disease in the breast and/or lymph node(s) after chemotherapy or gross disease on the chest wall or in the regional lymphatics after mastectomy were eligible. Patients 1-9 received capecitabine 825 mg/m2 BID daily beginning on the first day of radiotherapy. Excess grade 3 toxicity (%) was observed; the protocol was amended and subsequent patients received drug only on radiation treatment days. Radiation dose was at the discretion of the treating physician (50Gy-72 Gy, with no more than 2.5 Gy/fraction). Response was assessed by a single physician using paired radiation planning CTs (pretreatment and on-treatment after 45 Gy). Clinical correlation to all other available imaging was also made. Kaplan-Meier curves were used to estimate overall survival (OS) and local recurrence-free survival (LRFS). Circulating tumor cells (CTCs) in blood were examined in consenting patients. Results: The trial was stopped early after an unplanned interim analysis prompted by slow accrual suggested futility independent of response. From 2009-2012, 32 patients were accrued; 26 completed protocol specific treatment (17 post-mastectomy radiation with gross nodes, 4 pre-op, 5 aggressive palliation) and are included in this analysis. Median follow up was 7.3 months (interquartile range 6.7 – 17.4). Nineteen patients (73%) had a partial or complete response. Fourteen patients (53.9%) experienced at least one grade 3 non-dermatitis toxicity including 7/9 treated with continuous dosing. Four inoperable patients were treated with pre-op radiation therapy and 3 converted to operable. None achieved a pCR or near pCR. One-year actuarial OS was 52%. There was no difference in OS comparing among PMRT vs. preoperative or palliative RT (P = 0.90). One-year actuarial local recurrence free survival among PMRT patients was 38%. Ten patients had triple negative (TN) receptor status. There was no difference in radiation response by receptor status (P = 0.56); however, treatment was deemed subjectively futile (i.e., converted to operable but death secondary to new widespread M1 disease immediately post-op) in 9 of the 10 patients with TN disease versus 6 of the 16 patients with non-TN disease (P = 0.014). Median OS and 1-yr actuarial OS, among non-TN vs. TN patients were not reached vs. 6.1 months and 77% vs. 10% (P < 0.001), respectively. Eight/fifteen patients tested were positive for CTCs. CTCs did not correlate to receptor status, futility of RT or OS. Conclusions: Capecitabine can be safely administered as a daily concurrent chemoradiation regimen with weekend holidays. However, in this small, prospective and selected cohort, concurrent chemoradiation with capecitabine was futile among patients with TN breast cancer. Alternative strategies are urgently needed in TN patients. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-14-08.

BACKGROUND AND RATIONALE Inflammatory breast cancer (IBC) is the most aggressive form of breast cancer and patients frequently present with metastases at the time of their diagnosis. Although a robust IBC-specific molecular signature remains elusive, the disease is frequently characterized by persistent expression of the adhesion molecule, E-cadherin. This is highly counterintuitive as epithelial to mesenchymal transition (EMT), frequently associated with metastasis, results in decreased E-cadherin expression and highly aggressive cancers frequently express low levels of E-cadherin. We hypothesized that persistent inflammation, mediated by immune activation, increases the plasticity of IBC cells, inducing EMT and allowing the re-acquisition of epithelia characteristics once removed from the inflammatory foci. In support of this hypothesis, previous in vitro work showed that soluble factors from activated immune cells induce EMT-related transcripts in both IBC and non-IBC cell lines. However, uniquely in 3 of 4 IBC cell lines but none of the non-IBC cell lines, this program included an increase of E-cadherin expression. RESULTS We used real-time cell analysis (RTCA) from Acea Biosciences (San Diego, CA) to probe the effect of immune conditioned media, produced by stimulating healthy donor peripheral blood mononuclear cells through the T-cell receptor or through toll-like receptor-4, on SUM149 inflammatory breast cancer cells. Consistent with the increased expression of E-cadherin, we observed rapid and strong increases in cellular adhesion as measured by the RCTA cell-index following culture with immune inflammatory factors. However, using the CIM chip, the same cells also showed strong increases in invasion and migration. To determine the inflammatory factors involved in this process, we screened the immune conditioned media using a Luminex array (Millipore, Billerica, MA). TGF-b, TNF-α, and IL-6, previously shown to induce EMT, were all found at elevated levels. In 5 culture supernatants of healthy donor PBMC activated for 48h with anti-CD3 antibody, TGF-β had a modest 1.6-fold increase; TNF-α had an average 101-fold increase; while IL-6 had an average 347-fold increase. When added to cultures of SUM149 cells, these factors recapitulated the EMT gene expression signature in SUM149 including the increase in E-cadherin expression. Furthermore, the addition of neutralizing antibodies against TNF-α, TGF-β, and IL-6 to immune conditioned media prior to exposure to SUM149 cells resulted in less EMT. CONCLUSIONS Inflammatory factors may induce both the migratory ability and the characteristic persistent E-cadherin expression of IBC cells. This is mediated in part by TNF-α, TGF-β, and IL-6. However, the molecular basis for this unique IBC response requires further study hindering the development of optimal therapies. Ongoing studies at MD Anderson are exploring both the tumor and stromal components of inflammatory breast cancer. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-06-07.

Background: Breast cancer metastasis which ultimately results in breast cancer death, is an event believed to be initiated by the migration of tumor initiating cells (TIC) from the primary tumor to niches for micrometastatic disease. Recent data suggests the tumor microenvironment promotes TIC. The clinical relevance of secreted factors from the microenvironment on TIC surrogate, mammosphere (MS) formation and MS sensitivity to drug therapy was investigated using breast cancer patient fluids inherently conditioned by the tumor microenvironment: post-operative seromas and malignant pleural effusions. Methods: Fluids from 48 patients with breast cancer (15 seromas and 33 pleural effusions) and mesenchymal stem cells (MSC) from healthy donors were collected on IRB approved protocols. Cellular components were eliminated from patient-derived fluids using density-gradient centrifugation. MSC conditioned media (MSC-CM) was collected from 3D cultures of primary MSC. Luminex multiplex array platform was used to characterize 79 cytokine and growth factor components of all fluids. In addition, MSC-CM and patient-derived fluids were added to cultures of breast cancer cell lines: MCF-7, an estrogen receptor (ER)-positive cell line; SUM149, a triple-negative inflammatory breast cancer cell line; and SUM159, a triple-negative metaplastic breast cancer cell line and MS forming efficiency was examined. Results: Our results show that pleural effusions and seromas are enriched for factors also secreted by MSC such as MCP-1, GRO, IL-6, and VEGF-A. We found remarkable similarities regarding the cytokines and growth factors profile in pleural effusions and seromas. Both patient-derived fluids have comparable amount of Angiopoetin-2, Leptin, TNF-beta, VEGF, IL-2, IL-3, IL-4 and IL-10. EGF, TNF-alpha, IL-1, IL-6, IL-8 and IL-16 were significantly different between pleural effusions and seromas. Seroma fluid from bilateral drains in a patient with an invasive cancer and a contralateral benign mastectomy had very similar cytokine concentrations. Moreover, MSC-CM and pleural fluids from ER-positive and ER-negative patients increased the MS formation efficiency of both triple-negative cell lines while seroma fluids from ER-positive and ER-negative patients increased the MS formation efficiency of ER-positive cell line MCF-7. Finally, we evaluated the impact of a panel of drugs (simvastatin, pravastatin and erlotinib) on cell cultures grown with MSC-CM and patient-derived fluids. We found that the effect of chemotherapies on MS formation can be attenuated by patient-derived fluids. Conclusions: Seroma and pleural effusion fluids from breast cancer patients have similar cytokine profiles, change MS formation efficiency of standard breast cancer cell models, and mediate sensitivity to therapy. Here we demonstrate that host and microenvironmental factors are critical for determining resistance to therapy and may be independent of obvious tumor related factors. Future studies will investigate the prognostic implications of factors that promote TIC survival in the fluid tumor microenvironment. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-06-05.

Background: Inflammatory breast cancer (IBC) is a rare but highly aggressive form of breast cancer, responsible for 8%–10% of breast cancer–related deaths. To date, a unique molecular signature for IBC able to explain the dramatic differences in survival and tumor aggressiveness compared with non-IBC has not been identified. Triple-negative (TN) subtype is associated with worse clinical outcome in IBC patients. Tumor-associated dendritic cells (DCs) are the central regulators of immune responses in the tumor microenvironment, where the crosstalk with IBC cells may induce proinflammatory immune responses responsible of the development of highly aggressive tumor cells: cancer stem cells with epithelial mesenchymal transition (CSCs/EMT) phenotype. The two miR-19a target genes PTEN and SOCS1 regulate DC activation and maturation by inhibiting toll-like receptor (TLR) and CD40 signaling. As we found that the TN-IBC SUM149 cells expressed and secreted higher levels of miR-19a compared with the TN non-IBC SUM159 cells, we hypothesized that miR-19a secreted by SUM149 cells can be taken up by DCs and downregulate PTEN and SOCS1, leading to increased maturation of DC and production of proinflammatory cytokines upon TLR-mediated activation. Methods: DCs were generated from healthy donor-derived monocytes cultured for 5 days in RPMI 10% FBS supplemented with IL-4 and GM-CSF 1000 U/ml. To track miRNA transfer to DCs, SUM149 cells were transfected with either Dy547-labeled non-targeting miRNA or miR-19a-mimic, then co-cultured with DCs in 1.0 mm pore size transwell chambers for 24 h. MiRNA uptake by DCs was assessed by confocal fluorescence microscopy and qRT-PCR. The effect of miR-19a uptake by DCs were assessed by measuring: the levels of PTEN and SOCS1 mRNA after 24 h of DC co-culture with miR-19a-transfected SUM149; the expression levels of the costimulatory-activation markers CD80, CD86, CD40, CD83, HLA-DR (by FACS); and the production of IL-6, TNF-α (by ELISA) after DC activation by TLR4/LPS (100 ng for 18 h). As IL-6 induces STAT3, a transcription factor for miR-19a, we also evaluated the effect of IL-6 on miR-19a expression in SUM149 cells. Results: Fluorescent Dy547-labeled miRNA and miR-19a were transferred from SUM149 to DCs. The uptake of miR-19a induced the downregulation of both PTEN and SOCS1 mRNA in DCs, leading to increased expression of costimulatory-activation markers and production of IL-6 and TNF-α after DC activation by TLR4/LPS vs control. Stimulation of SUM149 cells with IL-6 upregulated miR-19a expression that was associated with an increased expression of CSC markers (CD44+CD24−Aldefluor+) and upregulation of EMT-regulating transcription factors. Conclusion: DCs can uptake miR-19a secreted by SUM149 cells, leading to increased production of IL-6 and TNF-α upon DC activation by TLR stimulation. IL-6 upregulated miR-19a expression in SUM149 cells. Therefore, miR-19a/IL-6 self-sustaining loop may represent a novel way by which TN-IBC cells maintain a proinflammatory tumor microenvironment able to induce the development of tumor cells with CSCs/EMT phenotype responsible of the poor prognosis of patients with TN-IBC. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-12-12.

Background: Cytokines and chemokines are known to be involved in tumor growth and progression of disease. Sialyl LewisX (sLeX), a ligand for adhesion molecule E-selectin, is known to affect inflammatory processes and an elevated level is associated with tumor metastasis. Therefore, we assessed serum levels of sLeX and cytokines/chemokines in patients with non-invasive ductal carcinoma in situ (DCIS), early invasive breast cancer (EBC), or metastatic breast cancer (MBC). Patients and Methods: Sera from 250 patients (26 DCIS, 157 EBC, 67 MBC) and 43 healthy donors (HD) were assayed for sLeX using an immunoassay kit (CSLEX; Nittobo Medical Co. Ltd., Japan) and a panel of cytokines and chemokines using a multiplex assay kit. Differences in serum markers between patients and HD, and among patient groups were determined using the Kruskal-Wallis and Mann-Whitney tests. Spearman's correlation determined the non-parametric correlation between the serum levels of sLeX and the inflammatory mediators. The receiver operating characteristic (ROC) curves and the corresponding area under the curve (AUC) analyses were used to determine the sensitivity and specificity of a given cut-off value for a particular serum marker. Results: The median sLeX level tended to increase with the stage of disease: MBC > EBC > DCIS albeit without significant differences among the disease stages. Among MBC patients, patients with sLeX below 1.75 U/mL had significantly improved overall survival (OS, mean survival 11.1 vs. 33.7 months, P = 0.002) and progression-free survival (PFS, mean survival 9.7 vs. 20.9 months, p = 0.042). The Hazard Ratio of high sLeX for OS was 5.5 (95% CI 1.6 to 18.9, p = 0.007) and 2.3 for PFS (95% CI 1.0 to 5.2, P = 0.048). EBC and MBC patients have significantly higher serum levels of IL-1, IL-1RA, IL-6, IL-8, MCP-1, MCP-3, and MIP-1βthan those of HD. In addition, there were positive correlations between the serum levels of sLeX and cytokines IL-1β, IL-1RA, IL-2, IL-8, MIP-1β, and MCP-3. The AUC for sLeX was 0.598 (P = 0.016), and a cut-off of 3.13 pg/mL distinguished hormone receptor (HR)-positive from HR-negative patients (χ2 = 4.0, P = 0.045). Likewise, the AUC for TNF-α was 0.620 (P = 0.003), and a cut-off 7.18 pg/mL distinguished HR-positive from HR-negative patients (χ2 = 12.6, P < 0.001). Using a cut-off value established by ROC curves, few MBC patients (9 of 66, 13.6%) had a serum IL-2 level > 7.1 pg/mL compared to 57 of 185 (30.8%) non-MBC patients (χ2 = 7.4, P = 0.007), suggesting that metastatic disease may be associated with immune suppression related to low serum IL-2. Conversely, 31of 66 (47%) MBC patients had a serum MCP-1 level > 750 pg/mL vs. 37 of 185 non-MBC patients (20%) (χ2 = 23.8, P < 0.0001), suggesting that a high level of MCP-1 may play an important role in metastasis. Conclusion: Serum levels of sLeX were able to distinguish HR-positive from HR-negative patients and predict overall survival in metastatic patients. Serum sLeX and some inflammatory mediators tended to increase with the severity of disease, and together may facilitate local invasion of tumor cells. Furthermore, serum levels of MCP-1 and IL-2 may have prognostic value in breast cancer patients. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-10-32.

Background: Increased expression of miR-19a plays an important role in tumor progression by regulating angiogenesis and apoptosis. As miRs can be a valuable diagnostic and prognostic serum biomarker, we assessed if high serum levels of miR-19a were associated with the shorter overall survival (OS) of patients with metastatic inflammatory breast cancer (MIBC) and metastatic non-inflammatory breast cancer (MNIBC). Methods: We analyzed 27 MNIBC (10 HER2 normal [HER2−] and 17 HER2 amplified [HER2+]), 37 MIBC (18 HER2− and 19 HER2+) patients, and 30 healthy donors (HDs). Patients' sera were collected before starting a new line of treatment. MiR-19a levels were measured in patients' sera and cell lines (MCF-7, SKBR3, MA-231, KPL-4, SUM-149) by qRT-PCR to assess differences in expression levels between IBC and non-IBC. MiR-192 and U6 snRNA were used to normalize miR-19a expression levels in serum and cell lines, respectively. Fold-changes in expression of miR-19a were calculated using the 2−DCt method. Mann-Whitney U test, ROC curve analysis, Kaplan-Meier, and Student's t-test were used for statistical analysis. Results: Median levels of miR-19a were higher in sera of both MNIBC and MIBC patients than in HDs (p = .001, p < 001, respectively). Median serum level of miR-19a was higher in MIBC than in MNIBC (p = .013). ROC curve analysis revealed that miR-19a could differentiate MNIBC from MIBC (AUC = 0.683; p = 0.013). With a cutoff value of 1.62 set by ROC, the sensitivity, specificity and positive predictive value (PPV) were 56.7%, 85.2%, and 84.0%, respectively. The triple receptor negative (TN) IBC cell line SUM-149 had the highest level of miR-19a and it was higher than the non-IBC: MDA-231 (TN; p = .031); SKBR3 (HER2+; p = .021); and MCF-7 (ER/PR+; p = .003). KPL-4 (IBC, HER2+) had the second highest miR-19a levels after SUM-149. MIBC patients had a shorter median OS than MNIBC patients (18.8 vs 27.8 months; p = .041; range, 0.7–40.3 months; median follow-up 19.5 months). MIBC patients with HER2− tumor (n = 11) and high serum levels of miR-19a (>1.62) had a shorter median OS than MNIBC patients with HER2− tumors (n = 9) and low serum level of miR-19a ( 1.62) and HER2+ MNIBC patients with low miR-19a ( Discussion: In this pilot study, miR-19a may be a potential biomarker for inflammatory breast cancer, as significantly higher miR-19a levels were present in the sera of MIBC compared with MNIBC as well as in IBC compared with non-IBC cell lines. Furthermore, miR-19a may be predictive of OS, as HER2− MIBC patients with high serum levels of miR-19a had shorter OS than HER2− MNIBC patients with low serum levels of miR-19a. Our data suggest that high expression of miR-19a: is a feature of IBC tumor cells; can be detected in the serum; may favor tumor progression and predict poor outcome of HER2− MNIBC patient. A larger cohort of patients is required to confirm these observations and examine predictive potential. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-10-02.

Background: Expression of cancer related genes and proteins in clinical specimens are the mainstay of personalized targeted therapy. In this study we investigated a blood-based non-invasive and sensitive technique to map biomarkers in breast cancer patients at the protein level instead of expression of cancer related genes. Other multi-platform assays require a large amount of clinical material, multi-step sample processing and complicated data analysis. Proximity Extension Assay performs a harmonious blending of immunoassay and PCR to amplify protein expression signal, thereby enabling multiplexing with small sample input (1 µl). Material and methods: Plasma samples (n=25) from patients with inflammatory breast cancer (IBC) or non-IBC, metastatic and non-metastatic, collected prior to starting a new therapy (treatment naive) or a new line of therapy were analyzed using the Proseek Multiplex Oncology I v2 panel (Olink Proteomics, Uppsala, Sweden) for simultaneous detection of 92 human protein biomarkers. In the assay, each protein biomarker is detected by a matched pair of antibodies coupled to unique DNA-tags. Upon binding to the proteins, the correctly hybridized DNA-tags form an amplicon that is measured by a digital PCR. The data was subjected to a dynamic Principal Component Analysis (PCA) with a T-test filter using Multid GenEx software allowing the identification of a specific protein signature with the greatest inter-group differences. Plasma from seven healthy normal donors (HD) was also included in the analysis as comparison. Results: A plasma protein signature consisting of MK, elf-4B, VIM biomarkers delineated all breast cancers from normal healthy donors. IL-8, CD40L, GDF-15, MCP-1, PARK7, CXCL11, FADD, CAIX, CD69, MIC-A, VEGF-D, EGFR, elf-4B, VIM, and PRSS8 distinguished patients with metastatic breast cancer from normal healthy donors. PCA also identified differences between IBC patients, non-IBC patients, and healthy donors. Clustered association of several protein biomarkers (MCP-1, VIM, HGF, PRSS8 and HER2) distinguished IBC from healthy donors. Plasma from IBC and non-IBC patients differed in their distribution of TNFSF14, CAIX, KN1A, CDHE4 and EGFR. All protein comparisons have p values < 0.05. Conclusion: These preliminary data suggest that it is possible to distinguish between cancer patients and healthy normal donors, and IBC and non-IBC patients based on a plasma protein profile using the Proseek Multiplex Oncology I v2 panel from OLINK with just 1 µl of plasma. This pilot study will lead to the establishment of a training set for a protein signature to be applied in a subsequent validation in a larger cohort to assess treatment induced biomarker profile changes. Citation Format: Jayachandran G, Cohen EN, Gao H, Alvarez RH, Valero V, Lim B, Woodward WA, Ueno NT, Reuben JM. Delineation of breast cancer based on circulating protein biomarker profile using the OLINK proteomics multiplexed proximity extension assay [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-02-07.

Background: Impaired immunosurveillance and immune dysregulation contribute to the pathogenesis and progression of breast cancer (BC). Upon activation, T cells synthesize inflammatory cytokines such as TNF-α that can promote or inhibit tumor growth. We therefore investigated T-cell cytokine syntheses as a predictor of disease progression. Methods: We recruited 115 BC patients [25 with locally advanced breast cancer (LABC), 21 with metastatic breast cancer (MBC), 25 with non-metastatic inflammatory breast cancer (IBC), and 44 with metastatic IBC (mIBC)] and 31 healthy donors (HD) for this ongoing study. The tumor phenotype consisted of 69 hormone receptor (HR) positive (including 26 patients with HER2 positive disease), 16 HR negative but HER2+, 30 triple negative BC (TNBC). To evaluate T cell function, peripheral blood mononuclear cells from patients and HD were stimulated overnight with immobilized anti-CD3 and soluble anti-CD28 antibodies and assessed for the percentage of T cells that synthesized cytokines by multi-parameter flow cytometry. The associations of T cell cytokine production profile with patient progression free survival (PFS) were analyzed by Kaplan Meier Test. Results: The median follow-up (FU) of 113 evaluable patients was 14.1 months with a median time to relapse of 10.5 months; 54 patients had stable disease (SD) and 59 patients had progression of disease (PD). In the entire cohort, on univariate analysis, metastasis, IBC, stage, and previous treatment predicted for worse PFS (p< 0.05). In non-metastatic patients (LABC+IBC), absolute count of anti-CD3 activated CD8+ T cells producing IL-17 was significantly higher in the SD patients compared with patient with PD (p=0.038), but it did not predict PFS (p=0.073). Similarly in metastatic patients, anti-CD3 activated CD4+ T cells producing TNF-α were significantly higher in patients with SD (p=0.025) and was predictive of longer PFS (p=0.033). Considering all patients with IBC (IBC + mIBC), although patients with PD had significantly fewer (percent and absolute number) anti-CD3 activated T cells capable of producing cytokines, this immune impairment was mostly related to metastasis and previous treatment. However, the percentage of anti-CD3 activated CD8+ T cells producing TNF-α was an independent positive prognostic indicator of PFS (p=0.002). Conclusion: Higher than average cytokine syntheses by anti-CD3 activated T cells are significantly associated with longer PFS. These data are consistent with the hypothesis that an adaptive immune response can control disease progression. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-20-03.

Background: Deficiencies in innate and adaptive immune responses by plasmacytoid dendritic cells (pDC) and myeloid DC (mDC) have been linked to poor clinical outcome in breast cancer (BC) (Treilleux, Clin Cancer Res, 2004, PMID 15569976). pDC produce IFN-a and pro-inflammatory cytokines that regulate innate and adaptive immunity in breast cancer. mDC present in blood and secondary lymphoid organs secrete IL-12 and induce inflammatory cytokine production by T cells. Therefore, we studied DC activity in the peripheral blood and assessed their function with clinical outcome in breast cancer patients. Methods: We recruited 115 BC patients [25 with locally advanced non-IBC (LABC), 25 with IBC, 21 with metastatic breast cancer (MBC), and 44 with metastatic IBC (mIBC)] and 31 healthy donors (HD) for this study. Peripheral blood pDC and mDC were activated through toll-like receptor (TLR)-7 to assess IFN-α and IL-10 production whereas mDC were activated through TLR-8 to assess production of IL-12 and TNF-α by multi-parameter flow cytometry. Associations between cytokine production by TLR-activated pDC and mDC with progression free survival (PFS) and overall survival (OS) of patients were analyzed by Kaplan Meier Test. Results: The median follow-up (FU) of 113 evaluable patients was 14.1 months with a median time to progression of 10.5 months; 54 patients had stable disease (SD) and 59 had progression of disease (PD). Metastasis, previous treatments, and IBC contributed to shorter PFS and OS. Compared to HD, BC patients had significantly fewer total DC (p=0.008), mDC (p=0.008), and pDC (p=0.003) per μL. In general, the number of TLR-7-activated pDC per μL that produced IFN-a(p=0.023) or IL-10 (p=0.027) and the number of TLR-8-activated mDC per μL that produced IL-12 (p Conclusion: BC patients had significantly fewer pDC and mDC in peripheral blood than HD. IBC patients with above average numbers of TLR-activated DC capable of producing proinflammatory cytokines had a significantly shorter PFS or OS. Disease progression in IBC is related to an increased number of activated dendritic cells producing inflammatory cytokines. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-20-04.

Background MicroRNAs (miRNAs) play an important role in the regulation of tumorigenesis and metastatic process. MiR-21 is an oncomiR that is overexpressed by breast cancer tumor tissue and is associated with high tumor grade and negative hormone receptor status. MiR-21 can control angiogenesis by regulating vascular endothelial growth factor (VEGF) as well as the expression and secretion of basic fibroblast growth factor (bFGF). MiR-19a is a member of the miR-17-92 family that was shown to suppress breast cancer cell proliferation. As angiogenesis factors act as attractants of circulating tumor cells (CTCs), a strong predictor of overall survival in metastatic breast cancer (MBC), we investigated the relationship between CTC count, levels of VEGF and bFGF, and the expression of miR-21 and miR-19a in sera of MBC patients. Methods RNA was extracted from sera collected from 33 MBC patients and 10 healthy donors (HD) using Total RNA purification Kit (Norgene Biotek Corporation, ON, Canada). The expression levels of miR-21, miR-19a and miR-192 were evaluated in triplicate by qRT-PCR using the TaqMan MicroRNA Assay (Applied Biosystems, Foster City, CA). Mir-192 was used to normalize the expression levels of miR-21 and miR-19a. The fold-change values were calculated using 2-DCt, where DCt= mean CTtarget-miRNA - mean CTmiR-192. CTCs were detected by CellSearch System (Veridex LLC, Warren, NJ). Serum VEGF and bFGF were determined using a multiplex bead assay (Millipore, Billerica, MA). Mann-Whitney U test was used to compare the miRNAs expression level between MBC patients and HD. Spearman's correlation was used to determine the association between the expression of miRNAs and growth factors. Results There was a significantly higher median level of miR-21 in MBC patients compared with HD (22.8 versus 7.2, P=0.003), while the expression level of miR-19a was similar for both groups. On average, the relative abundance of miR-21 in sera (ratio of MBC versus HD) is 3.1-fold overexpression. Furthermore, levels of miR-21 was positively correlated with the serum levels of VEGF (Spearman Rho= 0.364; P= 0.038) and bFGF (Spearman Rho= 0.367; P = 0.036). CTCs were detected in 23 patients, ranged from 1 to 134 per 7.5 mL of peripheral blood. Patients with no CTCs had a significantly higher median level of miR-19a than patients with no CTC (6.5 vs 2.8, P =0.047). Conclusion Our results support the notion that miR-21 overexpression may play a role in angiogenesis and metastasis by upregulating VEGF and bFGF in MBC. Undetectable CTC count may be related to overexpression of miR-19a, an epigenetic suppressor of breast tumor proliferation. Whereas overexpression of miR-19a may limit the tumor cells from entering the peripheral circulation, increasing miR-21 in the serum could accelerate the disease progression by promoting angiogenesis in MBC patients. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-09-01.

Background: Expression of cancer related genes and proteins in clinical specimens are the mainstay of personalized targeted therapy; however, a diagnostic signature for inflammatory breast cancer (IBC) remains elusive. In this study, we employed a blood-based, non-invasive, sensitive technology to map biomarkers in patients with IBC at the protein level. Proximity Extension Assay consists of a harmonious blending of immunoassay and PCR to amplify protein expression signal, thereby enabling multiplexing with small sample input (1 μl). Other multi-platform assays require a large amount of clinical material, multi-step sample processing and complicated data analysis. Materials and methods: Serum samples (n= 159) from patients with primary IBC (IBC, n= 30), metastatic IBC (MIBC, n= 54), locally advanced breast cancer (LABC, n= 24) and metastatic breast cancer (MBC, n= 27) were prospectively collected from subjects prior to starting a new therapy (treatment naive) or a new line of therapy between 2009 and 2012. Sera from 24 healthy normal donors (HD) were included in the analysis for comparison. The samples were analyzed using two panels: Proseek Multiplex Oncology II and Proseek Multiplex Inflammation I (Olink Proteomics, Uppsala, Sweden) for simultaneous detection of 92 human protein biomarkers in each panel. In the assay, each protein biomarker is detected by a matched pair of antibodies coupled to unique DNA-tags. Upon binding to the proteins, the correctly hybridized DNA-tags form an amplicon that can be measured by PCR.For initial analysis, sample populations were compared using the Mann-Whitney-U test. Results: In comparison with HD sera, sera of breast cancer patients had 41 proteins from the oncology panel and 28 from the inflammation panel that were significantly higher, whereas 5 from the inflammation panel were significantly lower. From the inflammation panel, 11 proteins (PD-L1, IL-2, IL-7, IL-18, uPA, CCL4, CCL23, CXCL9, CXCL10, CXCL11 and TNF-alpha) showed significant differential expression between IBC and non-IBC derived samples (irrespective of metastatic status); for each marker, levels were higher in IBC than in non-IBC. In contrast, 9 proteins from the oncology panel (CRNN, CTSV, ERBB4, FR-gamma, ITGAV, MIA, PODXL, SCF and SEZ6L) were differentially expressed; however, each of these proteins was higher in non-IBC than in IBC. Among the aforementioned proteins, CCL4, IL-2, IL-7, PD-L1, TNF-alpha, uPA, CRNN, CTSV, FR-gamma, ITGAV, MIA, SCF and SEZ6L did not differentiate cancer and HD, but were uniquely characteristic of the IBC vs non-IBC comparison. Conclusion: These preliminary data suggest that it is possible to distinguish between cancer patients and healthy normal donors, and also to delineate between IBC and non-IBC patients based on expression of serum proteins. Validation of this serum protein signature is planned in a larger patient cohort. Citation Format: Cohen EN, Jayachandran G, Gao H, Tin S, Alvarez RH, Valero V, Lim B, Woodward WA, Ueno NT, Reuben JM. Circulating protein biomarker profile for inflammatory breast cancer using a multiplexed proximity extension assay [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-02-04.

Triple-negative breast cancer (TNBC) is an aggressive subtype with no proven active targeted therapies available. Patients with TNBC have a very poor prognosis because the disease often metastasizes. Newer approaches to preventing metastasis and inhibiting tumor growth, are crucial to improving prognosis for these patients. Previously it has been shown that PEA-15 binds to ERK, preventing ERK from being translocated to the nucleus and hence blocking its activity. Our previous studies showed that overexpression of wild type PEA-15 inhibited ERK activity and reduced tumor volume in a TNBC xenograft model. We also showed that PEA-15 being unphosphorylated at both Ser104 and Ser116 inhibited ovarian cancer cell tumorigenicity. However, the function and impact of PEA-15 phosphorylation on TNBC is not well understood. From these observations, we hypothesized that unphosphorylated PEA-15 will prevent metastasis in TNBC through the inhibition of EMT. To study the effect of the phosphorylation status of PEA-15 on metastasis in TNBC, we established stable cell lines overexpressing nonphosphorylatable (PEA-15-AA) and phosphomimetic (PEA-15-DD) mutants in MDA-MB-468 cells. The clonogenic growth of PEA-15-AA was significantly reduced by 80% compared with vector control, PEA-15-V. Anchorage-independent growth of PEA-15-AA was inhibited by 60% compared with PEA-15-V (P Citation Format: Jihyun Park, Evan N. Cohen, Gaurav Chauhan, Jangsoon Lee, Naoto T. Ueno, Debu Tripathy, James M. Reuben, Chandra Bartholomeusz. PEA-15 regulates epithelial-mesenchymal transition and invasive behavior through its phosphorylation in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 897. doi:10.1158/1538-7445.AM2017-897

Background: CTCs play a crucial role in tumor dissemination and are prognostic factor in primary and metastatic breast cancer patients. Immune cells in peripheral blood (PB) contribute to an unfavorable microenvironment for the CTCs survival. As such, effective host innate and adaptive immune surveillance systems could adversely influence tumor dissemination whereas dysfunctional immune systems could provide a favorable microenvironment for the dissemination of CTCs and cancer progression. This study aimed to correlate CTCs with the functions of innate [natural killer (NK) cells] and adaptive (T-cells) immune effector cells in PB of IBC patients. Methods: This prospective study included 65 IBC (21 non-metastatic, 14 de novo metastatic and 30 recurrent metastatic) patients treated between October 2008 and April 2012 at the MD Anderson Cancer Center. CTCs were enumerated before patients started a new line of chemotherapy using the CellSearch® system, 33 (50.8%) of patients were treatment naïve at the time of blood collection. The phenotype of T cells, their ability to secrete cytokines following activation through the T-cell receptor (TCR) and the NK cell subsets were analyzed by multiparameter flow cytometry and the results were correlated with CTCs and clinical outcome. For survival analysis immune cell counts were dichotomized to low or high category using the median count. Results: At least 1 CTC (≥ 1) or ≥ 5 CTCs per 7.5 mL of PB was detected in 40 (61.5%) or 21 (32.3%) of patients, respectively. Patients with at least 1 CTC or ≥ 5 CTCs had a significantly inferior overall survival (OS) [HR=2.48, p=0.003 and HR=1.85, p=0.045] than patients with no CTCs or with Conclusions: IBC patients with CTCs in PB had abnormalities in both innate and adaptive immunity as evidenced by low percentages of NK cell subsets, and low percentage of TCR-activated CD8+ T cells producing TNF-α, respectively. These data illustrate an inverse relationship between CTCs and both innate and adaptive immune cells in the PB microenvironment that could potentially impact tumor cell dissemination and initiation of the metastatic cascade. Moreover, immune cell profiling could add further prognostic value to CTCs in IBC patients. Citation Format: Michal Mego, Hui Gao, Evan N Cohen, Simone Anfossi, Antonio Giordano, Sanda Tin, Tamer M Fouad, Wendy A Woodward, Ricardo H Alvarez, Vicente Valero, Naoto T Ueno, Gabriel N Hortobagyi, Massimo Cristofanilli, James M Reuben. Circulating tumor cells (CTC) are associated with defects in innate and adaptive immunity in inflammatory breast cancer (IBC) patients [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-01-04.

Background: Cancer stem cells (CSCs) in bone marrow (BM) are epithelial tumor cells that express CD44+CD24−/lo. CSCs can be further divided into those that have ALDH activity (Aldefluor+) and those that do not. We hypothesized that if CSCs are responsible for tumor dissemination, their presence in BM would be prognostic in early stages of breast cancer (EBC) patients. Therefore, using multiparameter flow cytometry (FACS), we assessed epithelial cells for expression of CD44+CD24−/low and Aldefluor in BM of EBC patients and correlated these findings with circulating tumor cells (CTC), disseminated tumor cells (DTC) and clinical outcome. Methods: This is a prospective laboratory study (Protocol 04–0657) conducted in the Departments of Surgical Oncology and Hematopathology, at The University of Texas MD Anderson Cancer Center in Houston, TX, USA, and was approved by the institutional review board. BM aspirates were collected at the time of surgery from 108 patients with EBC. BM was analyzed for CSCs and ALDH activity by FACS analysis. Ten cytospin slides of the bone marrow mononuclear cells were prepared from each BM aspirate and reacted with a cocktail of antibodies, including AE1/AE3, CAM5.2, MNF116, CK8 and CK18, to identify epithelial cells (DTC) by immunohistochemistry (IHC). In addition, the CellSearch system (Veridex, Raritan, NJ) was used to enumerate CTCs in each of 3 tubes of 7.5 mL of peripheral blood per patient. Overall survival (OS) and disease-free survival (DFS) were calculated from the date of diagnosis and analyzed with Kaplan-Meier survival plots. Cox multivariate proportional hazards model was also performed. Results: Of the 103 evaluable patients, 45 patients received neoadjuvant chemotherapy, 35 adjuvant chemotherapy and 23 no chemotherapy. The median follow-up was 40 months (range 7–66 months). Patients with CSCs in BM had a hazard ratio (HR) of 8.8 for DFS (p = 0.002); patients with Aldefluor+ CSCs had a HR of 5.9 (p = 0.052) for DFS. Seven patients have expired and all of them had CSCs in BM. Moreover, we evaluated the presence of DTCs by FACS (CD326+CD45−) in 104 patients. Sixty-one (75%) of patients had positive BM samples for DTC (CD326+CD45- BM cells ≥ 0.53%). No association was shown between DTC in BM as determined by FACS and CTCs in PB. In a multivariate model, after adjusting for age (< 45 years old), clinical T stage, N stage, ER, PR, HER2 status, and nuclear grading, the presence of CSCs was found to be an independent predictor of DFS (HR = 15.8, P = 0.017) as was the presence of CTCs (HR = 13.9, P = 0.007). In this subset of patients, the presence of Aldefluor+ CSCs and DTCs was not found to be predictive of DFS in the multivariate model including the same factors as listed above. Conclusions: Our data indicate that the presence of BM metastasis is correlated to CSCs, and CSCs irrespective of ALDH activity are independent adverse prognostic factors in EBC patients. Moreover, the presence of CTCs was a strong independent predictor of DFS. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-03-01.

Background: Inflammation contributes to the increased invasiveness and poor prognosis in breast cancer (BC) patients. Specifically, the expression of the proinflammatory cytokines interleukin-6 (IL-6), tumor necrosis factor alpha (TNFα) and interleukin-1 (IL-1) have all been linked to increased invasiveness and poor prognosis. Interestingly, the increased invasiveness was associated with an increase in the acquisition of markers of epithelial-mesenchymal transition (EMT). Therefore, we determined whether the levels of circulating proinflammatory cytokines (IL-1, IL-6, TNFα) and antiinflammatory cytokines (IL10) were correlated with the induction of EMT transcription factors (TFs), Snail1, Zeb1, Twist1, in breast cancer patients. Materials and Methods: From two laboratory-based ongoing studies at the MD Anderson Cancer Center, 41 BC patients were assessed for EMT-TFs in circulating CD45-ve cells (EMT-CTCs) and for serum proinflammatory cytokines before starting any treatment. 32 of 41 patients assessed for EMT had metastatic BC. EMT-CTCs were detected by qRT-PCR for the EMT-TFs Snail1, Zeb1 and Twist1 (Mego 2011; PMID 21387303) and serum cytokines were measured by Luminex bead array assay (MILLIPLEX™ MAP Human Cytokine/Chemokine Panel). Cytokine serum concentrations were compared with the median cytokine levels of healthy donors (HD). We examined the association of serum cytokines above the median HD levels and the presence of EMT-CTCs by non-parametric Mann-Whitney test with a statistical significance for p Results: Of the 41 patients assessed for both serum cytokines and EMT-CTCs, 14 (34%) were positive for at least one EMT-TF, including 3 of 9 (33%) patients with no-metastatic BC and 11 of 32 (34%) patients with metastatic BC. We found that serum levels of IL1a, IL2, TGFα, and TNFβ in patients that were above the median levels of HD sera were higher in patients with EMT-CTCs in the blood (higher IL1a concentration in patients with over expression of Snail1, Zeb1, and Twist1; IL2 with Zeb1; TGFα with Snail1; TNFβ with Zeb1, and Twist1). Further, the higher ratio of proinflammatory/anti-inflammatory cytokines, was associated with the presence of at least one EMT-TF, e.g., IL8/IL10 (p=.005) and TNFα/IL10 (p=.037). Discussion: Patients with proinflammatory cytokine (IL1a, IL2, TGFα, and TNFβ) levels above the median levels of HD or who had a predominantly proinflammatory cytokine profile were more likely to have at least one EMT-TF in their blood. These data are consistent with the hypothesis that proinflammatory cytokines promote EMT, which may be involved in tumor aggressiveness and disease progression. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-02-07.

Rationale: Inflammatory breast cancer (IBC) is the most insidious form of locally advanced disease. Emerging evidence suggests that host factors in the microenviromement may interact with underlying IBC genetics to promote the aggressive nature of the tumor. An integral part of the metastatic process involves epithelial to mesenchymal transition (EMT) where primary breast cancer cells gain motility and stem cell features that allow distant seeding. Interestingly, the IBC consortium microarray data found no clear evidence for EMT in IBC tumor tissues. However, it is unknown if soluble factors secreted by activated immune cells mediate EMT in the IBC microenvironment that may account for the absence of EMT in studies of the tumor cells themselves. Therefore, we tested whether the conditioned media of activated immune cells were capable of inducing EMT in IBC cells. Methods: Conditioned media (CM) were generated using healthy donor peripheral blood mononuclear cells that were activated with anti-CD3 antibody immobilized to plastic and soluble anti-CD28 antibody to activate T cells through the T-cell receptor (TCR) or left unstimulated for 48 hours. Thereafter, CM from each of the cultures was harvested and filtered. Next, 48-hour pre-seeded SUM149 IBC cells were grown in culture medium consisting of 25% CM and 75% IBC culture medium for an additional 2 days. Unconditioned media and TGF-β were used as negative and positive controls, respectively for EMT. Following treatment with CM, RNA was extracted from the target cells and analyzed for the presence of EMT-related transcription factors (EMT-TF) and markers of epithelial and mesenchymal states by TaqMan® qRT-PCR. Subsequently, a panel of 24 genes was tested on 4 IBC cell lines (SUM149, SUM190, KPL4 and IBC-3) and 5 non-IBC cell lines (MCF-10a, MCF-7, MDA-231, and MDA-453) treated with immune-activated CM using the Fluidigm® Dynamic Array integrated fluidic circuit (“chip”) gene expression platform which allows for the simultaneous quantification of 2,304 data points using TaqMan® assays. Formalin-fixed, paraffin embedded blocks were prepared from trypsinized cells for immunohistochemical (IHC) staining to detect E-cadherin and vimentin expression. Results: SUM149 cells cultured in the presence of TCR-activated CM for two days showed upregulation in EMT-TFs (SNAIL1, ZEB1, and TG2), vimentin and fibronectin by qRT-PCR. IHC staining showed increases in both vimentin and E-cadherin expression after 48-hour exposure to anti-TCR CM. Fluidigm® gene expression analysis of multiple cell lines exposed to anti-TCR CM showed that E-cadherin expression was unchanged or slightly decreased in non-IBC cell lines, whereas 3 of 4 IBC cell lines showed an increase in E-cadherin. Discussion: These data suggest that soluble factors secreted by activated immune cells are capable of inducing EMT in IBC cells and may mediate the persistent E-cadherin expression observed in IBC. Such processes may contribute to the highly aggressive nature of the disease; however, an immune competent in vivo model is warranted to fully understand the implications of these findings. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-04-06.

Breast cancer metastasis which ultimately results in breast cancer death, is an event believed to be initiated by the migration of tumor initiating cells (TIC) from the primary tumor to niches for micrometastatic disease. Recent data suggests the tumor microenvironment promotes TIC. The clinical relevance of secreted factors from the microenvironment on TIC surrogate, mammosphere (MS) formation and MS sensitivity to drug therapy was investigated using breast cancer patient fluids inherently conditioned by the tumor microenvironment: post-operative seromas and malignant pleural effusions. Fluids from 20 patients with breast cancer (8 seromas and 13 pleural effusions) and mesenchymal stem cells (MSC) from healthy donors were collected on IRB approved protocols. Cellular components were eliminated from patient-derived fluids using density-gradient centrifugation. MSC conditioned media (MSC-CM) was collected from 3D cultures of primary MSC. Luminex multiplex array platform was used to characterize 79 cytokine and growth factor components of all fluids. In addition, MSC-CM and patient-derived fluids were added to cultures of breast cancer cell lines: MCF-7, an estrogen receptor (ER)-positive cell line; SUM149, a triple-negative inflammatory breast cancer cell line; and SUM159, a triple-negative metaplastic breast cancer cell line and MS forming efficiency was examined. Our results show that pleural effusions and seromas are enriched for factors also secreted by MSC such as MCP-1, GRO, IL-6 and IL-8. We found remarkable similarities regarding the cytokines and growth factors profile in pleural effusions and seromas. Both patient-derived fluids have comparable amount of IL-2, IL-3, IL-4 and IL-10, as well as, VEGF, EGF and FGF-b. IL-1β and IL-16 were significantly different between pleural effusions and seromas. Seroma fluid from bilateral drains in a patient with an invasive cancer and a contralateral benign mastectomy had very similar cytokine concentrations. Moreover, MSC-CM and pleural fluids from ER+ and ER- patients increased the MS formation efficiency of both triple-negative cell lines while seroma fluids from ER+ and ER- patients increased the MS formation efficiency of ER-positive cell line MCF-7. Finally, we evaluated the impact of a panel of drugs on cell cultures grown with MSC-CM and patient-derived fluids. We found that the effect of chemotherapies on MS formation can be attenuated by patient-derived fluids. Seroma and pleural effusion fluids from breast cancer patients have similar cytokine profiles, change MS formation efficiency of standard breast cancer cell models, and mediate sensitivity to therapy. Here we demonstrate that host and microenvironmental factors are critical for determining resistance to therapy. Future studies will investigate the prognostic implications of factors that promote TIC survival in the fluid tumor microenvironment. 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 5210. doi:1538-7445.AM2012-5210

Background: MicroRNAs (miRs) have oncogenic and tumor-suppressor functions. MiR-21, miR-19a and miR-10b are overexpressed in breast cancer and regulate tumor cell migration, invasion and angiogenesis. We assessed the levels of miR-21, -19a, and -10b in sera of breast cancer patients and their association with the stage, histological type, hormonal receptor (HR) status and HER2 amplification in the primary tumor. Since circulating tumor cells (CTC) detected by CellSearch® are an independent and strong predictor of overall survival in metastatic breast cancer (MBC), we assessed the relationship between circulating miRs and CTCs. Methods: The study consisted of 30 healthy donors (HD) and 95 breast cancer (BC) patients. Patients’ sera were collected before starting a new line of treatment. Total RNA was isolated, reverse transcribed to cDNA and then subjected to qRT-PCR for the detection of miR-21, -19a, -10b and -192 using the TaqMan MicroRNA Assay (Applied Biosystems, Foster City, CA). Mir-192 was used to normalize the expression levels of the other miRs. Fold-changes in expression of miRs were calculated using the 2−DCt method, where DCt= mean CTtarget-miRNA -mean CTmiR-192. CTCs were enumerated using CellSearch™ (Veridex LLC, Warren, NJ). Mann-Whitney U test was used to determine differences in serum miR expression levels between patients and HD. Results: Of the 95 BC patients, 39 were non-MBC and 56 MBC. Patients grouped according to the receptor expression by immunohistochemical staining consisted of 27 HR+HER2−, 30 HR+HER2+, 20 HRHER2+, and 18 HRHER2− triple negative BC (TNBC). MiR-21 and miR-19a were higher in non-MBC patients than in HD (p=.001, p Discussion: High expression levels of miR-21, miR-19a and miR-10b in sera are observed in breast cancer patients, especially with advanced disease. HER2+ BC patients had higher serum levels of miR-21 and miR-10b than HER2−. IBC patients had a higher serum level of miR-19a than non-IBC patients. Moreover, patients with Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-03-02.

Background: The detection of more than 5 CTCs/7.5 ml of blood in MBC pts predicts worse progression-free survival (PFS) and overall survival (OS). We investigated time-related clinical trend of CTCs according to types of 1st line therapy (hormonal therapy, HTx; chemotherapy CTx, anti-HER2 therapy; CTx with Bevacizumab) and immunohistochemical (IHC) subtypes of disease. Methods: Enumeration of CTCs was performed by the CellSearch™ technology as standard of care for 235 MBC pts receiving 1st line treatment for MBC. The evaluation of CTCs was performed as follow: baseline CTC value defined as assay taken within 30 days before starting therapy, post-treatment CTC value defined as the lowest CTC value after starting therapy; progression CTC value defined as assay taken within 30 days before documented radiological progression of disease (PD). We analyzed the effect of treatments on CTC count by Wilcoxon matched pair test and the progression CTC value within all subtypes by Kruskal-Wallis one-way analysis of variance. Results: A total of 1,052 CTC assays were performed from September 2004 to June 2010 in 235 MBC pts. One hundred thirty pts (55%) had HR+/HER2- MBC, 20 (9%) HR+/HER2+, 23 (10%) HR-/HER2-, and 62 (26%) triple-negative MBC (TNBC). Forty-seven pts received HTx (25 with available post-treatment CTC, median PFS for all group: 10.6 months), 41 pts were treated with anti-HER2 treatment (30 post-treatment CTC, median PFS: 12 months), 39 received Bevacizumab (31 post-treatment CTC, median PFS: 7.3 months), 87 CTx combination with taxanes/anthracyclines or other (epothilone or platinum salts) (57 post-treatment CTC, median PFS: 9.4 months) and 21 mono-chemotherapy (16 post-treatment CTC, median PFS: 3.6 months). Table 1 describes treatment distribution among subtypes. All administrated 1st line treatments decreased the CTC number with exception of HTx (66% patients decreased post-treatment CTCs value after HTx, p=.31). According to IHC subtypes, post-treatment CTC value decreased in all subtypes, while progression CTC at radiological PD increased in pts with HR+/HER2- and TNBC (45% and 51% pts had < 5 progressing CTCs, respectively, p.05). Conclusion: This large retrospective study showed that monitoring of CTCs is an appropriate tool for predicting response to different types of treatments, but is least useful for pts receiving HTx. Clinical trend of CTC value consistently reflects disease course in pts with HR+/HER2- and TNBC, while pts with HER2+ MBC treated with trastuzumab or lapatinib presented a low number of CTCs at PD. Our data may suggest phenotypical heterogeneity of CTCs in pts with HER-2 amplified and HR+ disease. Table 1 Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr PD04-02.

Background: Circulating tumor cells (CTCs) are an independent predictor of survival in metastatic breast cancer patients and patients with ≥5 CTC per 7.5 mL of whole blood (WB) have a poor prognosis and overall survival than patients with fewer than 5 CTC. Dendritic cells (DCs), the most efficient antigen presenting cells (APC), are essential for a robust host immune response. Therefore, we investigated the relationship between CTCs and the immunophenotype and function of DCs in breast cancer (BC) patients. Methods: This ongoing prospective study consists of 47 patients, 26 with metastatic breast cancer, 19 with inflammatory breast cancer; and 2 with locally advanced breast cancer, with a median age of 52 years (range: 34-71 years). Primary tumor characteristics of patients included 28 hormone receptor positive, 18 HER2 amplified, and 12 triple receptor negative. Fluorescence-activated cell sorting analysis was used to enumerate circulating DC subsets, myeloid (mDC) and plasmacytoid (pDC), that express costimulatory molecules (CD40, CD80, and CD86). Additionally, we assessed the ability of DCs to synthesize interleukin (IL)-10, IL-12, interferon-alpha (IFN-a) and tumor necrosis factor (TNF)-a constitutively and following activation with toll-like receptor (TLR) agonists. CTCs were enumerated in 7.5 mL of WB using CellSearch (Raritan, NJ). Results: CTCs were detected in 26 patients (range from 1 to 1009) including 12 patients with ≥5 CTCs. Compared with patients with < 5 CTCs, those with ≥5 CTCs have a significantly higher median number of mDC (P = 0.007) and higher median percentages (%) of TLR-activated mDC that expressed CD80 (P = 0.047) and CD86 (P = 0.063). Additionally, compared with patients who had < 5 CTCs, patients with ≥5 CTCs had a lower median % of TLR-activated mDC that synthesized IL-12 (P=0.028), TNF-α (P=0.006), or IL-10 (P = 0.031); and there was a higher median % of TLR-activated pDC that synthesized IL-10 (P = 0.000). Conclusions: Even though BC patients with ≥5 CTCs have more mDC, activated mDCs are less capable of synthesizing inflammatory factors (TNF-a and IL-12) responsible for adaptive immunity. Moreover, there was increased % of activated pDC that synthesized IL-10, a cytokine responsible for downregulating adaptive immune responses. These preliminary data suggest that activated DCs from BC patients with ≥5 CTCs are likely to have a suboptimal ability to sustain an efficient adaptive immune response compared with activated DCs of BC patients with < 5 CTCs. This is the first report linking CTCs, a proven poor prognostic indicator, with potential dysfunctional APC in BC patients. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-02-04.

Background Transforming growth factor (TGF)-b1 has pleiotropic effects in cancer. In the early stages of breast cancer, TGF-b may be responsible for immune tolerance through the activation of T-Regulatory cells (TR). On the other hand, in the late stages of disease, it may induce angiogenic factors [vascular endothelial growth factor (VEGF), interleukin-8 (IL-8) (in Bonnomet J Mammary Gland Biol Neoplasia 2010); and IL-17 (Pickens J Immunol 2010)], and epithelial to mesenchymal transition (EMT) which may lead to an increase in the number of circulating tumor cells (CTC) (Kim, Cell 2009). Therefore, we investigated the possible correlation between TGF-b1, CTC count, angiogenic factors and T-cell function of patients with locally advanced or metastatic breast cancer. Methods As an interim analysis of an on-going prospective study, sera and peripheral blood mononuclear cells (PBMC) were collected from breast cancer patients starting a new line of therapy. At analysis, study enrollment included 78 patients with breast cancer [19 with locally advance disease (LABC), 23 with non-metastatic inflammatory breast cancer (IBC), and 32 with metastatic disease (MBC) including 19 with IBC features], and 28 healthy donors (HD). Serum TGF-b1, VEGF, and IL-8 levels were measured by Milliplex Luminex kits (Millipore, Billerica, MA). CD4+CD25+CD127dim TR cells were enumerated in whole blood by FACS (BD Biosciences, San Jose, CA). PBMC were used to study the ability of T cells to synthesize cytokines following activation of the T-cell receptor by immobilized anti-CD3 antibodies. CTC were enumerated by CellSearch (Veridex, Raritan, NJ). The Spearman Rho was calculated for nonparametric correlations and the Mann-Whitney U test was used to determine significant differences between median values. Results LABC patients and HD had a median serum TGF-b1 that was significantly higher than that of MBC patients (P = 0.042). There were statistically significantly positive correlations between serum TGF-b1 and the number of CD4+ T cells (rho = .226, P = .029) and IL-10 produced by %CD4+ (rho = .388, P = .002) and %CD8+ (rho = .459, P < .001) T cells. Furthermore, there was a positive correlation between serum TGF-b1 and the % of CD8+, but not CD4+, T cells that produced IL-17 (rho = .250, P = .022). Serum TGF-b1 levels did not correlate with either % or number of TR cells. Although serum TGF-b1 level of MBC patients was independent of CTCs (24.4 ng/mL vs. 24.0 ng/mL, P = .317), MBC patients with CTC had significantly higher serum levels of angiogenic factors such as VEGF (530 ng/mL vs. 240 ng/mL, P=0.037) and IL-8 (45.6 pg/ml vs. 20.0 pg/ml P= .006) than those of patients with no CTC. Even so, MBC patients with or without CTC have similar % of CD4 and CD8 T-cell subsets that could be activated to produce IFN-g, IL-2, TNF-a, IL-17, or IL-10. Conclusion: The concomitant presence of elevated serum TGF-ß1 levels and IL-10 producing T cells suggest immune suppression could facilitate disease progression of breast cancer. T-cell function is independent of CTC in MBC patients; however, serum VEGF and IL-8 levels were significantly elevated in MBC patients with CTC suggesting that vascular changes can facilitate tumor dissemination. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-02-07.

Background: High dose chemotherapy (HDCT) in patients with metastatic breast cancer (MBC) followed by autologous hematopoietic stem cell transplantation (ASCT) offers high complete response rates compared to standard therapy. However, the bone marrow (BM) is a reservoir for disseminated tumor cells (DTC) and the inadvertent recruitment from BM of DTC, cancer stem cells (CSC), and non-leukocytes undergoing epithelial mesenchymal transition (EMT) during the CD34+ hematopoietic progenitor cell (HPC) mobilization could adversely impact clinical outcome. As epithelial cells in EMT possess stem-like properties, we evaluated the apheresis product for CSC and expression of EMT to ascertain if they had an impact on clinical outcome in the transplant setting. Methods: Twenty-one MBC patients treated with HDCT underwent apheresis to harvest HPC and subsequent ASCT. A peripheral blood (PB) sample was collected before apheresis (baseline) and again after ASCT (post-treatment) for enumeration of CTC by CellSearch (Veridex, LLC, Raritan, NJ). Furthermore, mononuclear cells (MNC) were isolated from 19 HPC-depleted apheresis products. An aliquot of MNC was further depleted of CD45+ leukocytes and then interrogated for expression of the EMT-inducing transcription factor TWIST1 by quantitative RT-PCR. Another aliquot of each MNC sample was analyzed by 6-color flow cytometry for the presence of CSC (CD326+CD44hiCD24lo) and with ALDH-1 activity (Aldefluor+ CSC), measured by Aldefluor (StemCell Technologies, Vancouver, BC). The logrank test was used to determine if progression-free survival (PFS) was associated with the number of CTCs at baseline and post-ASCT; baseline percentages of CSC and Aldefluor+ CSC; and expression of TWIST1 by non-leukocytes. Results: The median follow-up was 17 months with a median time to disease progression of 9.4 months. The median overall survival was 12.9 months. At follow-up, 7 patients had non-progressive disease (NPD) and 12 had progressive disease (PD). CTCs were detected in 6 pts (range, 1-115) at baseline and in 9 patients post-ASCT (range, 1-131). PD patients had statistically significantly higher % of CD326+ epithelial cells but not CSC within the HPC-depleted apheresis products when compared with identical apheresis products of NPD patients (0.20% vs. 0.11%; P=0.033). Aldefluor test was performed in 12 of 19 samples. Four samples with TWIST1 transcripts contained a significantly higher % of CD326+ Aldefluor+ CSC than the samples without TWIST (9.58% vs. 1.58%; P =0.007). The presence of CTC at baseline was associated with shorter progression-free survival (PFS) (P =0.027). However, PFS was not associated with the TWIST1 expression or with % of Aldefluor+ CSC detected in apheresis products. Conclusion: Baseline CTC number was predictive of PFS. HDCT followed by ASCT did not reduce the number of CTC in patients with MBC. Finally, leukocyte-depleted apheresis products that expressed EMT-inducing TWIST1 transcripts contained higher % CSCs suggesting a strong linkage between circulating epithelial cells undergoing EMT and the production of cancer stem cells. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-02-08.

Background: Impaired immunosurveillance and immune dysregulation contribute to the pathogenesis and progression of breast cancer. We recently reported that breast cancer (BC) patients have compromised immunity with significantly fewer T cells, B cells, and dendritic cells compared to normal healthy donors. To further investigate immune function, T cells were assessed for their ability to synthesize cytokines prior to initiation of therapy and 3 months later. Methods: We recruited 46 BC patients [3 with locally advanced breast cancer (LABC), 13 with metastatic breast cancer (MBC), 15 have newly diagnosed inflammatory breast cancer (IBC), and 15 with metastatic IBC (MIBC)] and 13 healthy donors (HD) for this ongoing study. The tumor phenotype consisted of 28 hormone receptor (HR) positive (including 10 patients with HER2 positive disease), 7 HR negative but HER2+, 11 triple negative (TN). Thirty-nine patients are alive and have both baseline and follow up data at 3 months; 7 deceased patients had baseline data only. Peripheral blood mononuclear cells (PBMC) from patients and HD were stimulated overnight with immobilized anti-CD3 and soluble anti-CD28 antibodies and cytokine (IL-2, IL-4, IL-10, and IL-17, TNF-α, and IFN-γ) syntheses by activated (CD69+) CD4 and CD8 T cells were measured by multi-color flow cytometry. Statistical differences between the immunoresponses of patients and HD were analyzed by Mann-Whitney U test. Results: Compared with HD, IBC and MIBC patients had a statistically significantly lower percentage (%) of TNF-α+ CD8+ T cells (p=0.041 and p=0.037 respectively) at baseline. MIBC patients also had significantly lower % IFN-γ+ CD8+ T cells (p=0.046) than HD at baseline visit. But there were no significant differences among LABC, IBC, MBC, and MIBC patients. TN patients had statistically significantly lower % of IL-2+ CD4+ T cells (p=0.047) and TNF-α+ CD8+ T cells (p=0.041) at baseline than HD. The 7 deceased patients (3 IBC, 3 MIBC, and 1 MBC) had significantly lower %'s of CD4+ and CD8+ T cells that synthesized IL-2, TNF-α, IFN-γ, IL-4, and IL-10 than the 39 living patients (all p < 0.05) at baseline. At 3 months, all patient groups except IBC had T-cell responses that were similar to those of HD. Specifically, IBC patients had persistently significantly lower % of TNF-α+ CD8+ T cells than HD (p=0.048). Conclusion: In summary, T cell function is essential for maintaining immunosurveillance against tumor progression. Our data suggest that BC patient, especially IBC patients, have impaired T-cell responses and that therapeutic intervention may restore immune function only in a subset of those patients. Immunomodulatory therapy can play a role in IBC and contribute to improve patient outcome. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5589.

Background: Inflammatory breast cancer (IBC) is a rare but highly aggressive form of the disease but the immune profile associate with pathology and disease progression is poorly characterized. The cells of the innate immune system including natural killer (NK) and dendritic cells (DC) can establish the conditions for an adaptive response to the tumor and can actively kill tumor cells. Alternatively, the inflammatory conditions created by these cells can promote processes like metastasis and invasion. Here we report that immune activation in IBC is associated with a poor prognosis. Methods: A total of 76 breast cancer (BC) patients starting a new line of therapy (22 IBC, 23 metastatic IBC, 8 locally advanced breast cancer, and 23 metastatic) were evaluated for immune phenotypes and cytokine synthesis by DC activated through toll-like receptors (TLR) 7 and 8. Results: Among evaluable patients, 39 (58%) had hormone receptor positive (HR+) primary tumors, 20 (31%) were Her2 positive, and 18 (26%) were triple negative (TN). Among IBC patients, 23 (49%) were HR+, 15 (33%) were Her2+, and 14 (31%) were TN. Among non-IBC patients, 16 (73%) were HR+, 5 (22%) were Her2+, and 4 (17%) were TN. With a mean follow-up time of 7.8 months, 11 (16%) of the patients (10 IBC) died of disease. Most strikingly, BC survivors had a higher median CD4 count than deceased patients (p = .023) and was a significant predictor of outcome (p = 0.01). CD3 and CD19 lymphocytes of BC survivors had higher expression of CXCR4 than deceased patients (p = .038 and p = .029). In contrast, survivors also had lower lymphocyte percentages of all NK sub-types including ADCC NK (p = .008), non-ADCC NK (p = .001), exhausted (p = .014) and non-exhausted NK (p = .005) than deceased patients. Although DC counts were not significantly different, the percentage of TLR-induced pDC producing IFN-α was lower in BC survivors than deceased patients (p = 0.025). Conversely, the percentage of pDC constitutively producing the anti-inflammatory cytokine IL-10 was higher in survivors compared to non-survivors (p = .007). Compared with IBC non-survivors, IBC survivors had a higher % of pDC producing IL-10 (p = 0.005) and fewer mDC producing TNF-α (p = .030) following TLR activation. With respect to antigen presentation, mDC of BC survivors had lower expressions of the co-stimulatory molecules CD40 (p= .036), CD80 (p = .013) and CD86 (p = .006) than mDC of deceased patients. Conclusion: We observed low CD4 counts in IBC that were associated with a poor prognosis. Additionally, immune activation as evidenced by DC pro-inflammatory cytokine production, expression of costimulatory molecules, and increased NK counts was associated with poor prognosis. Together, these data suggest that the innate immune system is highly active in the late stages of disease but the adaptive response is severely compromised by factors still unknown that can contribute to the peculiar clinical presentation and aggressive features of the disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5583.

Background: Inflammatory breast cancer (IBC) is an insidious form of breast cancer. Cytokines and chemokines direct the metastatic potential of disease and serve as biomarkers for disease progression. Circulating tumor cells (CTC) are an independent prognostic factor in metastatic disease. Further, TGF-β is involved in the induction of the epithelial-mesenchymal transition (EMT) which regulates the metastatic ability of IBC. As prognostic factors of aggressive disease, we measured serum cytokines and leukocyte phenotypes, and correlated the values with known CTC results of IBC and non-IBC patients.Methods: Peripheral blood (PB) from 35 IBC patients (18 non-metastatic and 17 metastatic) and 19 non-IBC patients (7 LABC, 12 MBC) was collected prior to starting a new therapy to measure a panel of 33 cytokines, chemokines, and growth factors in serum by Luminex; CTC by CellSearch™; and PB leukocyte immunophenotype by FACS. The Kruskal-Wallis and Mann-Whitney U tests determined the differences in cytokine levels between IBC, non-IBC and healthy donors (HD) and presence of CTCs.Results: There were no differences in serum cytokine, chemokine or growth factor levels between IBC and non-IBC patients. EGF, IP-10, MIG, Eotaxin, MCP-1 and TNF-RI were significantly elevated in breast cancer patients compared to HD. TNF-RI, EGF, HGF, IP-10, MIG, Eotaxin, MCP-1 and interleukin (IL)-10 were higher in IBC patients than in HD. Compared to IBC patients, non-IBC patients had fewer dysregulated cytokines relative to HD including higher EGF, Eotaxin, MCP-1 and IL-8.Perhaps counter-intuitively, non-metastatic IBC patients had higher plasma levels of IL-2, -2R, -4, -5, -10, -12p70, -15, -17, FGF-b, IFN-γ, GM-CSF, and MIP1-α than metastatic IBC patients. Moreover, these differences were not observed between LABC and MBC patients. Furthermore, compared to HD, metastatic IBC had lower IL-4, -7, -17, -12p70, IFN-γ, RANTES, but higher levels of IP-10, Eotaxin, MCP-1, and TNF-RI. These data suggest that non-metastatic IBC patients are more immune competent than metastatic IBC patients. Finally, there were differences in the immunophenotype as well as cytokine levels between IBC patients with and without CTCs. IBC patients with CTC had a lower %T-cells (p=0.003) and higher %B-cells (p=0.008) and TNF-RI (p=0.01) than IBC patients without CTCs which may lead to a decrease in cellular immunity. Cell-mediated immunity may be further compromised by the elevated levels of serum TGF-β (p= 0.064) that can also promote EMT and metastatic progression.Conclusion: We report a comprehensive analysis of the serum cytokine and chemokine profiles in IBC patients. More importantly, this is the first report of potential interactions between soluble factors, CTC, and immune parameters in IBC patients. Non-metastatic IBC patients are more immune competent than metastatic IBC patients as evidenced by the high levels of pro- and anti-inflammatory factors; however, the presence of CTC in IBC tends to shift the immune response to a TH2 polarization with a decrease in T-cells, and a concomitant increase in B-cells and serum TGF-β and TNF-RI levels. Additional studies are needed to determine the role of soluble factors in the pathogenesis and progression of IBC and the impact on clinical outcome. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2135.

Background: Inflammatory breast cancer (IBC) is characterized by an acute inflammation of the skin of the affected breast due to blockage of the dermal lymphatics by tumor emboli. Cytokines and chemokines affect the migration of tumor cells and immune cell function that regulates the pathogenesis of IBC. Preliminary data suggest that sera of IBC patients contain several inflammatory cytokines and chemokines capable of regulating innate and adaptive cellular immune responses. Since there is a paucity of data on the characteristics and function of immune cells of IBC patients, we determined the immunophenotype and cytokine production by T cells and dendritic cells.Methods: From October 2008 through May 2009, peripheral blood (PB) from 34 IBC patients (18 non-metastatic and 16 metastatic), 18 non-IBC patients and 24 healthy donors (HD) were analyzed to determine the immunophenotype of T-cell subsets, activated and regulatory T-cells, B-cells, natural killer cell subsets (NKC), and dendritic cell (DC) subsets. Additionally, we assessed the ability of T-cells and DCs to synthesize cytokines following activation through the T-cell receptor (TCR) and toll-like receptors (TLR), respectively. The Kruskal-Wallis and Mann-Whitney U tests determined the differences between IBC patients, non-IBC patients, and HD.Results: IBC and non-IBC patients were well-matched in terms of ER, PR, Her2, high-grade tumor, tumor size, and menopause status. Both IBC and non-IBC patients had significantly fewer lymphocytes, total T-cells (CD3+), T-helper (CD4+), T-cytotoxic/suppressor (CD8+), and B (CD19+) than HD (all p < 0.01). Non-IBC patients had a significantly lower CD4/CD8 ratio than HD (p = 0.016) while IBC patients had significantly fewer T-regulatory (CD4+CD25hiCD127-) cells than HD (p = 0.02) and non-IBC patients (p < 0.05). With respect to innate immunity markers, IBC patients also had significantly lower percentages of ADCC/NK (CD16+CD56+, p = 0.034) and non-exhaustive NK (CD56+CD57+, p = 0.02) than HD and a significantly higher percentage of non-ADCC/NK cells than non-IBC patients (p = 0.03). Non-IBC patients and HD had similar innate immunity markers. Even though all groups had similar percentages of myeloid (mDC) and plasmacytoid (pDC) dendritic cells, mDC of IBC patients displayed a higher level of constitutive activation than non-IBC with increased expression of CD40 and CD80, and decreased CCR5 expression. IBC patients had more mDC that constitutively produced TNF-a (p 0.041) and IL-10 (p = 0.029) than HD, and more mDC than non-IBC patients that produced IL-10 (p = 0.028).Conclusion: The immune profile of patients with breast cancer suggests multiple abnormalities involving T-, B-, NK- and dendritic cells. Furthermore, IBC patients exhibited constitutive immune activation as measured by increased expression of co-stimulatory receptors (CD80, CD40) and constitutive production of TNF-a and IL-10 by mDC as well as fewer T-regulatory cells. Taken together, these preliminary data suggest that IBC patients are more likely than non-IBC patients to have a dysregulated immune function. Additional studies on T-cell activation and function are warranted to better understand factors associated with host defense mechanisms of IBC patients and possible therapeutic strategies to restore immune function. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4129.

Abstract #4031 Background The presence of epithelial cells in bone marrow (BM), disseminated tumor cells (DTC), that are CD326+CD45- is a negative independent prognostic factor in primary breast cancer (PBC). Cancer-initiating stem cells (CSC) are purported to be CD44+CD24-, express aldehyde dehydrogenase 1 (ALDH), and possess self-renewal potential. We examined the expression of CD44, CD24, and ALDH on BM aspirates of patients with PBC. Methods As part of an ongoing prospective study, from September 2006 to May 2008, BM aspirates were collected from 47 PBC patients, median age of 52 yrs (range: 30-76 yrs). At time of BM collection, 9 patients had received neoadjuvant therapy. BM mononuclear cells were analyzed for DTC and CSC phenotype and ALDH function (Aldefluor® assay kit) by 6-color FACS analysis and data correlated with primary lesion tumor size, tumor markers (ER and PR; over-expressed HER-2 by FISH), nodal status, and neoadjuvant chemotherapy. Results The mean (±SEM) percentages of DTC and ALDHbr cells were 1.07% ± 0.09% and 2.17% ± 0.21%, respectively. Within the DTC population, the mean proportion of CSC and ALDHbr cells were 79.6% ±1.69% and 43.8% ± 3.4%, respectively. There was a statistically significant positive correlation between size of the primary tumor and the percentages of DTC (rho =0.364, p = 0.016, n = 43), DTC/ALDHbr (rho = 0.533, p = 0.28, n = 17), and CSC within the DTC/ALDHbr population (rho = 0.790, p = 0.004, n = 11). Specifically, the BM from patients whose primary tumors were 2cm (T2). Addition of Aldeflour® to the immunophenotype analysis further enriched the proportion of CSC phenotype from 79.1% in the DTC population to 92.3% in the DTC/ALDHbr population (p = 0.047). Of particular clinical relevance, patients who received neoadjuvant therapy had a near statistically significantly higher percentage of DTC than untreated patients (adjuvant 0.99% vs. neoadjuvant 1.41 %, p = 0.066) without a significant decrease in the proportion of CSC within the DTC population (adjuvant 78.6% vs. neoadjuvant 84.6%, p = 0.312), suggesting CSC were not eliminated by neoadjuvant therapy. Conclusion Epithelial cells with cancer-initiating stem cell-like phenotypes can be identified in BM aspirates of patients with PBC. The DTC population contained a larger subset of CD44+CD24- cells that overlapped with a smaller subset of ALDHbr cells. The increased percentage of CSC within the ALDHbr population and persistence following therapy further suggests that the relevance of both phenotypes to the mechanism responsible for recurrent disease in breast cancer warrants further investigation and validation. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4031.

Abstract #5044 Background: Malignant pleural effusion is a debilitating complication of metastatic breast cancer (MBC), including inflammatory breast cancer (IBC). The cellular components in pleural effusion are important indicators of pathologic process. Although pleural fluids from patients with IBC contain tumor cells and immune cells, little is known about the phenotype and function of the immune cell infiltrate. The objective of the study was to determine the immunophenotype and function of the cellular immune cells in pleural effusion of patients with pleural metastases. Methods and Patients: Pleural effusions from 9 women with MBC, 6 with IBC, median age 56 years (range 44-75 yrs), were submitted for pathological review. Mononuclear cells (MNC) were reacted with a cocktail of monoclonal antibodies to detect surface antigens by 8-parameter flow cytometry. In addition, MNC were activated through the T-cell receptor (TCR) with immobilized anti-CD3 and soluble anti-CD28 to assess intracytoplasmic syntheses of pro-inflammatory [interleukin (IL)-2, tumor necrosis factor (TNF)-a and interferon-gamma (IFN-γ)] and anti-inflammatory (IL-10) cytokines by TCR-activated CD4+ and CD8+ T cells. Data are presented as the mean (±SEM) of TCR-activated CD4+ and CD8+ T-cells that synthesized inflammatory and anti-inflammatory cytokines. Results: Upon pathological review, the mean (±SEM) number of tumor cells, MNC, and neutrophils were 20.2% ± 7.4% (range, 2%-72%), 62.4% ± 7.7% (range, 25%-96%), and 17.3% ± 5.9% (range, 0%-50.0%), respectively. The MNC consisted of 83.1% ± 8.2% CD3+ pan-T, 58.2% ± 7.0% CD4+ T, 24.2% ± 5.2% CD8+ T, 2.8% ± 0.9% CD19+ B, 4.0% ± 0.7% natural killer (NK), 3.4% ± 1.8% NKT, and 0.9% ± 0.2% dendritic cells, respectively. The CD4/CD8 T-cell ratio was 3.62 ± 0.78. The percentages of CD4+ and CD8+ T cells that synthesized inflammatory and anti-inflammatory cytokines following activation through the TCR-activated are listed in Table 1. Conclusion: There were no statistically significant differences between pleural fluids of IBC and non-IBC subjects with respect to tumor cell involvement, MNC phenotype, or T-cell function. Moreover, the function of pleural fluid T cells of MBC patients were similar to that of normal peripheral blood T cells (historical data, not shown). These data suggest that T-cells in the pleural fluids of patients with MBC are immune competent and can be activated through the TCR to synthesize pro- and anti-inflammatory cytokines. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5044.

Abstract #3084 BACKGROUND: Inflammatory breast cancer (IBC) is the most aggressive manifestation of primary breast cancer and its clinical presentation and the peculiar geographic distribution have suggested a possible viral etiology. Mouse Mammary Tumor, Human Herpes Virus 8 or Epstein-Barr virus (EBV) may play significant roles in the pathogenesis of the disease and account for the peculiar lymphangiogenic mechanism of growth and disease progression. In particular, EBV late membrane protein (LMP)-1 plays a critical role in tumor invasion lymphangiogenesis. In this study, EBV DNA was measured in each FNA sample from patients with IBC and locally advanced breast cancer (LABC) using real time PCR directed against 5 different targets within the EBV genome. PATIENTS AND METHODS: Sixty-eight fine needle aspirates (FNA) of breast cancer from patients with newly diagnosed IBC (n=18) and LABC (n=50) were examined. The FNA specimens were collected for the pharmacogenomic research program before any systemic therapy started. Median age of subjects was 53 yrs (range: 26-83 yrs). Total DNA was extracted from the FNA using the QIAmp Kit. EBV DNA were detected by quantitative polymerase chain reaction (QPCR) directed at five different targets within the EBV genome (BamH1W, LMP1, EBNA1, LMP2, and BZLF1) that encode products that are highly conserved across all EBV strains and important for viral pathogenesis. DNA extracted from Namalwa cells were used to construct the standard (R2 = 0.993); HL60 cells were used as EBV-negative controls. Data were collected and analyzed by Sequence Detection System software (Applied Biosystems Incorporated, ABI, Foster City, CA). RESULTS: EBV DNA was detected in freshly obtained breast cancer FNA samples using Q-PCR. The Q-PCR assays were sensitive to as low as 1 copy of EBV DNA per reaction. Of 68 breast tumor FNA specimens, EBV genes were detected in FNA from 4/18 (22%) IBC patients (range, 1-4 genes) and 5/50 (10%) LABC patients (range, 1-3 genes). Among the 9 positive specimens, BamH1W was detected in 6; EBNA1 in 5; LMP1 in 4; BZLF1 in 2; and LMP2 in 1, respectively. DISCUSSION AND CONCLUSIONS: Q-PCR is an effective method for detecting EBV DNA in FNA of breast cancer patients. The two most frequently detected EBV DNA sequences were BamH1W (6 of 9) and EBNA1 (5 of 9). BamH1W is a sequence that is frequently replicated throughout the EBV genome and EBNA1 is a reliable latent gene responsible for maintaining infection for life of the host. LMP-1 is a potential viral oncogene and was detected in 4/9 patients. The detection of these EBV sequences suggest latent EBV infections in IBC and LABC patients. The clinical significance of these findings warrants further investigation. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3084.

Abstract #4030 Background: The presence of epithelial cells (CD326+CD45dim) in the bone marrow (BM), i.e. disseminated tumor cells (DTC), are related to poor prognosis of primary breast cancer (PBC). Notch signaling pathway is involved in the development of the breast and aberrant activation of Notch signaling is an early event in breast cancer. Others have shown that breast cancer stem cells (BCSC) with the putative phenotype of CD44+CD24- can be identified within cell lines and primary tumor and have upregulated genes including Notch-1. However, it is unclear if Notch-1 is highly expressed by DTC and BCSC in BM of patients with PBC. We hypothesized that the Notch-1 expression is correlated with the level of BCSC within DTC. In this study, we enumerated BCSC in DTC-enriched populations in patients with PBC and assessed the expression of Notch-1 by RT-PCR. Methods and Patients: As part of an ongoing prospective study, from September 2006 to May 2008, 121 BM aspirates were collected from as many patients with PBC. Thirty-seven patients, median age of 52 years (range, 30-76 years), provided BM aspirates under anesthesia at time of surgery. At the time of BM collection, 9 patients had received neoadjuvant therapy. BM mononuclear cells (BMC) were enriched for DTC using anti-CD326 antibody-coated magnetic beads, and isolated using the AutoMACS-Pro cell separator system. DTC and BCSC were identified by multi-color FACS analysis. Thereafter, DTC-enriched cell preparations were analyzed for expression of ER, PR, HER-2, Notch-1, and Numb by RT-PCR; level of each transcript was normalized to a ratio to the house keeping gene GAPDH. Results: The mean ± SEM DTC in BMC was 1.05 % ± 0.09% and the majority was BCSC (79.7% ± 1.87%). The proportion of DTC-enriched fractions that expressed Notch-1, Numb, ER, PR, and HER-2 transcripts was 27/37 (73.0%), 33/37 (89.2%), 24/37 (64.9%), 15/37 (40.5%), and 20/37 (54.1%), respectively. The expression of Notch-1, Numb, ER, PR, and HER-2 transcripts did not correlate with the percentage of DTC. Contrary to our hypothesis, Notch-1 and Numb mRNA expression in DTC-enriched cells negatively correlated with the percentage of BCSC (Spearman's rho = -0.384, p= 0.027; and rho= -0.347, p= 0.048, respectively). Conclusions: Our results show no correlation between the level of Notch-1 expression and the percentage of BCSC in DTC-enriched populations in BM of patients with PBC. Since these studies were conducted in CD326-enriched populations, it underscores the necessity to further purify the CD44+CD24- cells and conduct studies in isolated single cells to definitively delineate the gene expression of BCSC from patients with PBC. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4030.