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BACKGROUND: Excess body fatness and hyperinsulinemia are both associated with an increased risk of postmenopausal breast cancer. However, whether women with high body fatness but normal insulin levels or those with normal body fatness and high levels of insulin are at elevated risk of breast cancer is not known. We investigated the associations of metabolically defined body size and shape phenotypes with the risk of postmenopausal breast cancer in a nested case-control study within the European Prospective Investigation into Cancer and Nutrition.METHODS: Concentrations of C-peptide-a marker for insulin secretion-were measured at inclusion prior to cancer diagnosis in serum from 610 incident postmenopausal breast cancer cases and 1130 matched controls. C-peptide concentrations among the control participants were used to define metabolically healthy (MH; in first tertile) and metabolically unhealthy (MU; >1st tertile) status. We created four metabolic health/body size phenotype categories by combining the metabolic health definitions with normal weight (NW; BMI RESULTS: Women classified as MUOW/OB were at higher risk of postmenopausal breast cancer compared to MHNW women considering BMI (OR = 1.58, 95% CI = 1.14-2.19) and WC (OR = 1.51, 95% CI = 1.09-2.08) cut points and there was also a suggestive increased risk for the WHR (OR = 1.29, 95% CI = 0.94-1.77) definition. Conversely, women with the MHOW/OB and MUNW were not at statistically significant elevated risk of postmenopausal breast cancer risk compared to MHNW women.CONCLUSION: These findings suggest that being overweight or obese and metabolically unhealthy raises risk of postmenopausal breast cancer while overweight or obese women with normal insulin levels are not at higher risk. Additional research should consider the combined utility of anthropometric measures with metabolic parameters in predicting breast cancer risk.

Many sources indicate that smallholder tree-crop commodity farmers are poor, but there is a paucity of data on how many of them are poor and the depth of poverty. The living income concept establishes the net annual income required for a household in a place to afford a decent standard of living. Based on datasets on smallholder cocoa and tea farmers in Ghana, Ivory Coast and Kenya and literature, we conclude that a large proportion of such farmers do not have the potential to earn a living income based on their current situation. Because these farmers typically cultivate small farm sizes and have low capacity to invest and to diversify, there are no silver bullets to move them out of poverty. We present an assessment approach that results in insights into which interventions can be effective in improving the livelihoods of different types of farmers. While it is morally imperative that all households living in poverty are supported to earn a living income, the assessment approach and literature indicate that focussing on short- to medium-term interventions for households with a low likelihood of generating a living income could be: improving food security and health, finding off-farm and alternative employment, and social assistance programmes. In the long term, land governance policies could address land fragmentation and secure rights. Achieving living incomes based on smallholder commodity production requires more discussion and engagement with farmers and their household members and within their communities, coordination between all involved stakeholders, sharing lessons learnt and data.

Background: Banana by-products are undervalued and their potential remains untapped. They are often composted after the fruits are harvested, reducing the cause of environmental pollutions due to open burning. Objectives: The study aims to identify the bioactive compounds in banana [Musa acuminate x balbisiana Colla cv. Saba (Musaceae)] inflorescence buds that are responsible for the antibacterial activity on selected foodborne pathogens. Methods: Dried inflorescence buds were extracted using methanol and subsequently partitioned into chloroform, ethyl acetate, butanol and deionized water. Further isolation of bioactive components was based on a bioassay-guided fractionation and the inhibitory activity at various concentrations against selected foodborne pathogens was determined. The compounds were identified using highperformance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC-MS/MS). Results: The SPE-fraction 3 (BWF-3) purified from the methanolic water partition of banana inflorescence showed the most prominent inhibition against Staphylococcus aureus with minimum inhibitory concentration at 12.0 μg/ml. The BWF-3 was later identified as proanthocyanidins with epigallocatechin as the main extension unit. Additionally, the survival of Listeria monocytogenes increased with the fortification of ferum (II) and (III) at a concentration as low as 1 mM but not for the calcium, magnesium, manganese and glucose. Conclusion: The methanolic partition of banana inflorescence buds could be a potential source of natural antibacterial for food and pharmaceutical applications.

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.

PURPOSE: The carbohydrate Sialyl Lewis(X) (sLe(X)) mediates cell adhesion, is critical in the normal function of immune cells, and is frequently over-expressed on cancer cells. We assessed the association, differential levels, and prognostic value of sLe(X) and inflammatory cytokines/chemokines in breast cancer sera. METHODS: We retrospectively measured sLe(X) and a panel of cytokines/chemokines in the sera of 26 non-invasive ductal carcinoma in situ (DCIS), 154 invasive non-metastatic breast cancer (non-MBC), 63 metastatic breast cancer (MBC) patients, and 43 healthy controls. Differences in sLe(X) and inflammatory cytokines among and between patient groups and healthy controls were assessed with nonparametric tests and we performed survival analysis for the prognostic potential of sLe(X) using a cut-off of 8 U/ml as previously defined. RESULTS: Median serum sLe(X) was significantly higher than controls for invasive breast cancer patients (MBC and non-MBC) but not DCIS. In univariate analysis, we confirmed patients with serum sLe(X) >8 U/ml have a significantly shorter progression free survival (PFS) (P=0.0074) and overall survival (OS (P=0.0003). Similarly, patients with high serum MCP-1 and IP-10 had shorter OS (P=0.001 and P

Background Anthropometric and lifestyle factors may influence cancer risks through hormonal changes but the evidence to date is not conclusive. We therefore investigated associations of body size and composition, physical activity and sedentary time with serum hormone concentrations in pre- and post-menopausal women. Methods Design: Cross-sectional. Setting: UK Biobank, a large prospective cohort study involving about 500,000 adults aged between 40–69 years when recruited in 22 assessment centres between 2006 and 2010. Participants: 20,758 pre-menopausal and 71,101 post-menopausal women, of whom 4,803 (23%) and 15,469 (22%) respectively had accelerometer data. Exposures: body mass index (BMI), height, waist to height ratio, waist to hip ratio, body fat mass, trunk fat mass, self-reported and accelerometer-measured physical activity and self-reported sedentary time. Outcomes: serum concentrations of total and calculated free oestradiol, total and calculated free testosterone, sex hormone binding globulin (SHBG) and insulin-like growth factor-1 (IGF-1). Statistical analysis: Multivariable linear regression analysis. Results The exposure-outcome associations are reported only if there was at least a 5% difference in hormone concentrations between the highest and lowest exposure groups. In pre-menopausal women, higher BMI was associated with a lower concentration of total oestradiol (15% difference in the highest vs. lowest BMI group, i.e., 35+ kg/m2 vs Self-reported physical activity was associated with somewhat lower concentrations of total and calculated free testosterone (pre-menopausal 10% (free testosterone), post-menopausal 5% and 11% respectively in the most vs. least active quartile) and a higher concentration of SHBG (premenopausal 11%, postmenopausal 10%), and the opposite was true for self-reported sedentary time. The associations were slightly stronger with accelerometer-measured physical activity, but were attenuated after adjustment for BMI. Ptrend for all reported associations was Conclusion This study confirms strong associations between adult anthropometric factors and hormone and SHBG concentrations in both pre- and post-menopausal women; this may partly explain the effects of these factors on cancer risks. The associations with physical activity and sedentary time were at most modest.

SUMMARY Deep space exploration will require real-time, minimally invasive monitoring of astronaut health to mitigate the potential health impairments caused by space radiation and microgravity. Genotoxic stress in humans can be monitored by quantifying the amount of DNA double-strand breaks (DSBs) in immune cells from a simple finger prick. In a cohort of 674 healthy donors, we show that the endogenous level of DSBs increases with age and with latent cytomegalovirus infection. To map the range of human responses to space radiation, we then study DSB induction and repair in immune cells from 319 healthy donors after the cells are exposed to galactic cosmic ray components and lymphocytes from 30 cancer patients after radiotherapy. Individuals with low baseline DSB have fewer clinical complications, enhanced DNA damage repair responses, and a functional dose-dependent cytokine response in healthy donor cells. This supports the use of DSB monitoring for health resilience in space., Graphical Abstract, In Brief Pariset et al. find that individuals with a lower baseline level of DNA damage are resilient to clinical complications after radiotherapy and present higher radiation-induced levels of DNA repair foci and inflammatory cytokines. These findings support the use of baseline DNA damage as a biomarker for radiation sensitivity.

Magnetic resonance imaging is considered low risk, yet recent studies have raised a concern of potential damage to DNA in peripheral blood leukocytes. This prospective Institutional Review Board-approved study examined potential double-strand DNA damage by analyzing changes in the DNA damage and repair markers γH2AX and 53BP1 in patients who underwent a 1.5 T gadolinium-enhanced cardiac magnetic resonance (MR) exam. Sixty patients were enrolled (median age 55 years, 39 males). Patients with history of malignancy or who were receiving chemotherapy, radiation therapy, or steroids were excluded. MR sequence data were recorded and blood samples obtained immediately before and after MR exposure. An automated immunofluorescence assay quantified γH2AX or 53BP1 foci number in isolated peripheral blood mononuclear cells. Changes in foci number were analyzed using the Wilcoxon signed-rank test. Clinical and MR procedural characteristics were compared between patients who had a >10% increase in γH2AX or 53BP1 foci numbers and patients who did not. The number of γH2AX foci did not significantly change following cardiac MR (median foci per cell pre-MR = 0.11, post-MR = 0.11, p = .90), but the number of 53BP1 foci significantly increased following MR (median foci per cell pre-MR = 0.46, post-MR = 0.54, p = .0140). Clinical and MR characteristics did not differ significantly between patients who had at least a 10% increase in foci per cell and those who did not. We conclude that MR exposure leads to a small (median 25%) increase in 53BP1 foci, however the clinical relevance of this increase is unknown and may be attributable to normal variation instead of MR exposure.

Purpose To determine the role of macrophage polarization on the response of inflammatory breast cancer (IBC) cells to radiation and whether modulation of macrophage plasticity can alter radiation response. Methods and Materials The human THP-1 monocyte cell line and primary human monocytes isolated from peripheral blood mononuclear cells were differentiated into macrophages and polarized to either an “antitumor” (M1) or a “protumor” (M2) phenotype. These polarized macrophages were co-cultured with IBC cells (SUM149, KPL4, MDA-IBC3, or SUM190) without direct contact for 24 hours, then subjected to irradiation (0, 2, 4, or 6 Gy). Interleukin (IL)4/IL13-induced activation of STAT6 signaling was measured by Western blotting of phospho-STAT6 (Tyr641), and expression of M2 polarization gene markers (CD206, fibronectin, and CCL22) was measured by quantitative polymerase chain reaction. Results Expression of M2 polarization markers was higher in M2-polarized macrophages after IL4/IL13 treatment than in control (M0) or M1-polarized macrophages. Co-culture of IBC cell lines with M1-polarized THP-1 macrophages mediated radiosensitivity of IBC cells, whereas co-culture with M2-polarized macrophages mediated radioresistance. Phosphopeptide mimetic PM37, targeting the SH2 domain of STAT6, prevented and reversed IL4/IL13-mediated STAT6 phosphorylation (Tyr641) and decreased the expression of M2 polarization markers. Pretreatment of M2-THP1 macrophages with PM37 reduced the radioresistance they induced in IBC cells after co-culture. Targeted proteomics analysis of IBC KPL4 cells using a kinase antibody array revealed induction of protein kinase C zeta (PRKCZ) in these cells only after co-culture with M2-THP1 macrophages, which was prevented by PM37 pretreatment. KPL4 cells with stable short hairpin RNA knockdown of PRKCZ exhibited lower radioresistance after M2-THP1 co-culture. Conclusions These data suggest that inhibition of M2 polarization of macrophages by PM37 can prevent radioresistance of IBC by down-regulating PRKCZ.

Relatively little is known about the antiproliferative effects of artemisinin, a naturally occurring antimalarial compound from Artemisia annua, or sweet wormwood, in human endometrial cancer cells. Artemisinin induced a G1 cell cycle arrest in cultured human Ishikawa endometrial cancer cells and downregulated cyclin-dependent kinase-2 (CDK2) and CDK4 transcript and protein levels. Analysis of CDK4 promoter-luciferase reporter constructs showed that the artemisinin ablation of CDK4 gene expression was accounted for by the loss of CDK4 promoter activity. Chromatin immunoprecipitation demonstrated that artemisinin inhibited nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) subunit p65 and p50 interactions with the endogenous Ishikawa cell CDK4 promoter. Coimmunoprecipitation revealed that artemisinin disrupts endogenous p65 and p50 nuclear translocation through increased protein-protein interactions with IκB-α, an NF-κB inhibitor, and disrupts its interaction with the CDK4 promoter, leading to a loss of CDK4 gene expression. Artemisinin treatment stimulated the cellular levels of IκB-α protein without altering the level of IκB-α transcripts. Finally, expression of exogenous p65 resulted in the accumulation of this NF-κB subunit in the nucleus of artemisinin-treated and artemisinin-untreated cells, reversed the artemisinin downregulation of CDK4 protein expression and promoter activity, and prevented the artemisinin-induced G1 cell cycle arrest. Taken together, our results demonstrate that a key event in the artemisinin antiproliferative effects in endometrial cancer cells is the transcriptional downregulation of CDK4 expression by disruption of NF-κB interactions with the CDK4 promoter.

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 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: 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.

CTCs are involved in tumor dissemination and are an independent prognostic factor in primary and metastatic breast cancer patients. Dendritic cells (DCs) are the most efficient antigen presenting cells and are comprised of plasmacytoid-(pDC) and myeloid-(mDC) derived DC subsets. This study aimed to correlate CTC counts with the peripheral blood DC immunophenotypes and functions of inflammatory breast cancer (IBC) patients. This study included 65 IBC patients. Peripheral blood (PB) was obtained from patients prior to starting a new line of chemotherapy for CTCs enumeration by CellSearch® and DC phenotype and function by flow cytometry; the characteristics of DCs were then correlated with CTC counts and clinical outcome. Twenty-one (32.3%) patients with CTCs ≥5 had a significantly inferior overall survival (OS) compared to patients with

Objectives: To determine the effects of thalidomide and placebo on anorexia-cachexia and its related symptoms, body composition, resting metabolic rate, and serum cytokines and their receptors in patients with advanced cancer. Methods: Included in the study were patients with advanced cancer with weight loss greater than 5% in 6 months and who reported anorexia, fatigue, and one of the following: anxiety, depression, or sleep disturbances. Patients on chemotherapy within 2 weeks prior or during the study were excluded from the study. Patients were randomly assigned to either 100 mg thalidomide or placebo once a day for 14 days. The Edmonton Symptom Assessment Scale (ESAS), Functional Assessment of Anorexia/Cachexia Therapy (FAACT), Functional Assessment of Cancer Illness Therapy (FACIT-F), Hospital Anxiety Depression Scale (HADS) Pittsburgh Sleep Quality Index (PSQI) were utilized, and in addition body composition, Resting Energy Expenditure (REE), and serum cytokine levels were assessed. Results...

Background: Circulating tumor cells (CTCs) are an independent prognostic factor in metastatic breast cancer (MBC) patients treated by conventional dose chemotherapy. The aim of this study was to determine the role of CTCs and CTCs undergoing epithelial-mesenchymal transition (EMT) in metastatic breast cancer. We used the platform of high-dose chemotherapy (HDCT) and autologous hematopoietic stem cell transplantation (AHSCT) to study the CTCs and CTCs with EMT. Patients and methods: CTCs were enumerated in 21 MBC patients before apheresis and 1 month after AHSCT. CD34-depleted apheresis products were analyzed for CD326+ epithelial and Aldefluor+ cancer stem cells (CSC) by flow cytometry and were depleted of CD45+ cells and assessed for EMT-inducing transcription factors (EMT-TF) by quantitative RT-PCR. Results: Patients with ≥ 5 CTCs/7.5 mL of peripheral blood 1 month after AHSCT had shorter progression-free survival (PFS) (P=0.02) and overall survival (OS) (P=0.02). Patients with apheresis products containing high percentages of CD326+ epithelial cells or overexpressing EMT-TF had shorter PFS. In multivariate analysis, low percentage of CD326+ epithelial cells and response to HDCT with AHSCT were associated with longer PFS, whereas lower CTCs after AHSCT was associated with longer OS. High CTCs, 1 month after AHSCT correlated with shorter PFS and OS in MBC patients undergoing HDCT and AHSCT, while CTCs with EMT and CSCs phenotype in apheresis products are associated with relapse. Conclusion: Our data suggest that CTC and CTCs with EMT are prognostic in MBC patients undergoing HDCT followed by AHSCT.

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.

Indole-3-carbinol (I3C), a naturally occurring hydrolysis product of glucobrassicin from cruciferous vegetables such as broccoli, cabbage and Brussels sprouts, is an anticancer phytochemical that triggers complementary sets of antiproliferative pathways to induce a cell cycle arrest of estrogen-responsive MCF7 breast cancer cells. I3C strongly downregulated transcript expression of the catalytic subunit of the human telomerase (hTERT) gene, which correlated with the dose-dependent indole-mediated G(1) cell cycle arrest without altering the transcript levels of the RNA template (hTR) for telomerase elongation. Exogenous expression of hTERT driven by a constitutive promoter prevented the I3C-induced cell cycle arrest and rescued the I3C inhibition of telomerase enzymatic activity and activation of cellular senescence. Time course studies showed that I3C downregulated expression of estrogen receptor-alpha (ERα) and cyclin-dependent kinase-6 transcripts levels (which is regulated through the Sp1 transcription factor) prior to the downregulation of hTERT suggesting a mechanistic link. Chromatin immunoprecipitation assays demonstrated that I3C disrupted endogenous interactions of both ERα and Sp1 with an estrogen response element-Sp1 composite element within the hTERT promoter. I3C inhibited 17β-estradiol stimulated hTERT expression and stimulated the production of threonine-phosphorylated Sp1, which inhibits Sp1-DNA interactions. Exogenous expression of both ERα and Sp1, but not either alone, in MCF7 cells blocked the I3C-mediated downregulation of hTERT expression. These results demonstrate that I3C disrupts the combined ERα- and Sp1-driven transcription of hTERT gene expression, which plays a significant role in the I3C-induced cell cycle arrest of human breast cancer cells.

Background: Given the benefits in using epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI), clinical practice guidelines recommend EGFR mutation testing of nonsquamous non–small cell lung cancer (NSCLC). However, not all patients get tested, which may have an impact on the prevalence of EGFR mutation previously estimated. Aim: To determine the trends of EGFR mutation testing in patients with nonsquamous NSCLC in New Zealand, and to explore the possible associations between the proportions tested, selectivity and the prevalence of EGFR mutation. Methods: This population-based study involves all patients who were diagnosed with nonsquamous NSCLC in the four health regions of New Zealand between January 2010 and July 2016. We identified eligible patients from the New Zealand Cancer Registry and obtained information on EGFR testing from TestSafe, a clinical information sharing service. We then calculated the proportions of patients tested for EGFR mutation and computed selectivity indices for eleven periods. We used a log-linear model to assess the associations between the proportions tested, selectivity and the prevalence of EGFR mutation. Results: Of the 2986 patients involved in this analysis, 1280 (42.9%) were tested for EGFR mutation. The proportion tested increased from 3.7% in 2010 to 74.0% in 2016. Testing was more prevalent in younger age group, female, Asian and patients with adenocarcinoma, and when specimens for testing was available. Such selectivity, however, decreased from 2010 to 2016. The prevalence of EGFR mutation varied widely across the periods, ranging from 16.8% in January-June 2014 to 43.8% in 2010. It was negatively associated with the proportion tested ( P = 0.02), and positively associated with the selectivity of testing ( P = 0.03). The log linear models estimated that the prevalence of EGFR mutation would be at most 16.1% (95% CI: 9.5%–27.1%) if 100% of patients were tested. Conclusion: In New Zealand, the uptake of EGFR mutation testing has improved over time but there is still room for improvement. Incomplete and selective testing may result in an overestimation of the prevalence of EGFR mutation in patients with nonsquamous NSCLC.

Background: Women diagnosed with breast cancer, their doctors, and their families, would find a valid estimate of her prognosis helpful in planning treatment and support. Assessing prognosis is complex as many factors influence it. Several predictive models have been produced, but none has been developed or tested on patients in New Zealand (NZ). Aim: We aimed to develop and validate a NZ predictive model (NZPM) for breast cancer, and compare its performance to a widely used UK-developed model, the Nottingham Prognostic Index (NPI). Methods: We developed a model to predict 10-year breast cancer-specific survival, using data collected prospectively in the largest population-based breast cancer registry in NZ (Auckland, 9182 patients), and assessed its performance in this data set (internal validation) and in an independent NZ population-based series of 2625 patients in Waikato (external validation). The data included all women with primary invasive breast cancer diagnosed from 1 June 2000 to 30 June 2014, with follow-up to death or to 31 December 2014. We used multivariate Cox proportional hazards regression to assess predictors and to estimate the probability of breast cancer mortality within 10 years, and therefore 10-year survival, for each patient. We assessed observed survival by the Kaplan-Meier method. We assessed discrimination by the C-statistic, and calibration by comparing predicted and observed survival rates for patients in 10 groups ordered by predicted 10-year survival. We compared this NZPM with the NPI in the validation data set. Results: The final NZPM used continuous variables of age, tumor size, and number of positive lymph nodes, and categorical variables of ethnicity, tumor stage, tumor grade, ER and PR receptors, HER2 status, and histologic type of tumor. Discrimination was good: C-statistics were 0.84 for internal validity and 0.83 for independent external validity. For calibration, for both internal and external validity, the predicted 10-year survival probabilities in 10 groups of patients, ordered by predicted survival, were all within the 95% confidence intervals (CI) of the observed Kaplan-Meier survival probabilities. The NZPM showed good discrimination even within the prognostic groups defined by the NPI. Conclusion: These results for the NZPM show good internal and external validity, transportability, potential clinical value, and its clear superiority over the NPI. Further research will assess other potential predictors, other outcomes, performance in specific subgroups of patients, and compare the NZPM to other models, which have been developed in other countries and have not yet been tested in NZ.

Cells within the tumor microenvironment, including but not limited to macrophages and mesenchymal stem cells (MSCs), can promote the phenotype and aggressiveness of inflammatory breast cancer (IBC). For example, co-injection of MSCs with SUM149 IBC cells significantly increased the clinical features of IBC such as skin invasion and metastasis. Our preliminary work showed that MSCs can be educated by co-culture with M1 polarized (anti-tumor) or M2 polarized (pro-tumor) mouse Raw macrophages. Such education of MSCs by M2 Raw macrophages leads to increased IL6 secretion by MSCs, relative to M1-educated or uneducated MSCs. M2-educated MSCs also have increased migration toward IBC cell lines SUM149 and IBC3, effects that can be blocked by an anti-IL6 antibody. Co-culture with M2-educated MSCs also enhances migration and mammosphere formation of IBC cells. Radiation response of IBC cells upon interactions with cells from the tumor microenvironment was also analyzed. Preliminary work shows that co-culture of IBC cells (SUM149 and KPL4) with M2-polarized human THP1 macrophages, prior to ionizing radiation, mediates radiation resistance of IBC cells, and this effect can be decreased by either adding HDL lipoproteins during co-culture period or by STAT6 inhibitors that block IL4/IL13-mediated phosphorylation of STAT6 and M2-polarization in THP1 macrophages. MSCs can also be polarized into either a MSC1 phenotype or a MSC2 phenotype by exposure to toll-like receptor (TLR) ligands TLR4 or TLR3, respectively. Indoleamine-pyrrole 2,3-dioxygenase (IDO) expression in MSCs is a marker of MSC2 polarization that is induced after exposure with TLR3 ligand (PolyIC) relative to MSC1 (TLR4 stimulated; LPS-treated) or parental MSCs. Similar to macrophage polarization, while MSC1 mediates anti-tumor effects, MSC2 are immunosuppressive and thus contribute to tumor growth. Preliminary work also shows that co-culture of IBC cells with MSC2 mediates radioresistance and this can be decreased as well by exposure to HDL during co-culture period prior to radiation. HDL protective effects, in part, can be explained by decreased expression of TLR3-induced IDO mRNA levels in MSC2. In the present work, we extended the above mentioned observations regarding the crosstalk between mouse Raw macrophages and MSCs by analyzing the effect of co-culture of human THP1 macrophages (parental designated as M0, M1- or M2-polarized) with MSCs on the IDO mRNA expression in MSCs, a marker of MSC2 polarization. Surprisingly, co-culture of M1-polarized THP1 with MSCs resulted in a robust increased expression of IDO mRNA in MSC relative to parental MSC (uneducated) or MSCs co-cultured with M2-THP1. Further studies are needed to determine the effects of increased IDO expression in MSC, upon M1-THP1 co-culture, on the aggressive behavior of IBC cells and whether this could be altered with IDO inhibitors. Our results suggest that there could be inter-species differences between mouse and human macrophages on the education of human MSCs. Based on our findings we propose testing a combination of STAT6 inhibitors that reverse M2-polarization of macrophages and IDO inhibitors that can decrease MSC2 phenotype mediated by TLR3 exposure and/or M1-THP1 education. Citation Format: Rahal OM, Wolfe AR, Mandal PK, Larson R, Tin S, Reuben JM, McMurray JS, Woodward WA. Understanding the complexity of macrophage and mesenchymal stem cell interactions to improve treatment outcome for IBC patients [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 PD6-06.

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.

Inflammatory breast cancer (IBC) is the most insidious form of locally advanced breast cancer; about a third of patients have distant metastasis at initial staging. Emerging evidence suggests that host factors in the tumor microenvironment may interact with underlying IBC cells to make them aggressive. It is unknown whether immune cells associated to the IBC microenvironment play a role in this scenario to transiently promote epithelial to mesenchymal transition (EMT) in these cells. We hypothesized that soluble factors secreted by activated immune cells can induce an EMT in IBC and thus promote metastasis. In a pilot study of 16 breast cancer patients, TNF-α production by peripheral blood T cells was correlated with the detection of circulating tumor cells expressing EMT markers. In a variety of IBC model cell lines, soluble factors from activated T cells induced expression of EMT-related genes, including FN1, VIM, TGM2, ZEB1. Interestingly, although IBC cells exhibited increased invasion and migration following exposure to immune factors, the expression of E-cadherin (CDH1), a cell adhesion molecule, increased uniquely in IBC cell lines but not in non-IBC cell lines. A combination of TNF-α, IL-6, and TGF-β was able to recapitulate EMT induction in IBC, and conditioned media preloaded with neutralizing antibodies against these factors exhibited decreased EMT. These data suggest that release of cytokines by activated immune cells may contribute to the aggressiveness of IBC and highlight these factors as potential target mediators of immune-IBC interaction.

Dear Editor,We would like to discuss on the publication on ‘Tuberculin skin test versus interferon-gamma release assays.’1 De Keyser et al. concluded that ‘IGRAs, especially T-SPOT.TB, are more eff...

Background Nucleostemin is a GTPase residing in the nucleolus that is considered to be an important cancer stem/progenitor cell marker protein due to its high expression levels in breast cancer stem cells and its role in tumor initiation of human mammary tumor cells. It has been proposed that nucleostemin may represent a valuable therapeutic target for breast cancer; however, to date evidence supporting the cellular mechanism has not been elucidated. Results Expression of exogenous HER2, a member of the EGF receptor gene family, in the human MCF-10AT preneoplastic mammary epithelial cell line, formed a new breast cancer cell line, 10AT-Her2, which is highly enriched in cells with stem/progenitor cell-like character. 10AT-Her2 cells display a CD44+/CD24-/low phenotype with high levels of the cancer stem/progenitor cell marker proteins nucleostemin, and active aldehyde dehydrogenase-1 (ALDH-1). The overall expression pattern of HER2 protein and the stem/progenitor cell marker proteins in the 10AT-Her2 cell population is similar to that of the luminal HER2+ SKBR3 human breast cancer cell line, whereas both MCF-7 and MDA-MB-231 cells display reduced levels of nucleostemin and no detectable expression of ALDH-1. Importantly, in contrast to the other well-established human breast cancer cell lines, 10AT-Her2 cells efficiently form tumorspheres in suspension cultures and initiate tumor xenograft formation in athymic mice at low cell numbers. Furthermore, 10AT-Her2 cells are highly sensitive to the anti-proliferative apoptotic effects of indole-3-carbinol (I3C), a natural anti-cancer indole carbinol from cruciferous vegetables of the Brassica genus such as broccoli and cabbage. I3C promotes the interaction of nucleostemin with MDM2 (murine double mutant 2), an inhibitor of the p53 tumor suppressor, and disrupts the MDM2 interaction with p53. I3C also induced nucleostemin to sequester MDM2 in a nucleolus compartment, thereby freeing p53 to mediate its apoptotic activity. Small interfering RNA knockdown of nucleostemin functionally documented that nucleostemin is required for I3C to trigger its cellular anti-proliferative responses, inhibit tumorsphere formation, and disrupt MDM2–p53 protein–protein interactions. Furthermore, expression of an I3C-resistant form of elastase, the only known target protein for I3C, prevented I3C anti-proliferative responses in cells and in tumor xenografts in vivo, as well as disrupting the I3C-stimulated nucleostemin–MDM2 interactions. Conclusions Our results provide the first evidence that a natural anti-cancer compound mediates its cellular and in vivo tumor anti-proliferative responses by selectively stimulating cellular interactions of the stem/progenitor cell marker nucleostemin with MDM2, which frees p53 to trigger its apoptotic response. Furthermore, our study provides a new mechanistic template that can potentially be exploited for the development of therapeutic strategies targeted at cancer stem/progenitor cells.

Introduction Altered serum microRNA (miRNA) levels may be correlated with a dysregulated expression pattern in parental tumor tissue and reflect the clinical evolution of disease. The overexpression of miR-21, miR-10b, and miR-19a is associated with the acquisition of malignant characteristics (increased tumor cell proliferation, migration, invasion, dissemination, and metastasis); thus, we determined their utility as serum biomarkers for aggressive breast cancer (HER2-overexpressed or -amplified [HER2(+)] and inflammatory breast cancer [IBC]). Experimental design In this prospective study, we measured miR-21, miR-10b, and miR-19a levels using quantitative reverse transcriptase-polymerase chain reaction in the serum of 113 breast cancer patients and determined their association with clinicopathologic factors and clinical outcome. Thirty healthy donors with no history of cancer were enrolled as controls. Results Patients with non-metastatic HER2(+) breast cancer had higher serum miR-21 median levels than patients with non-metastatic HER2(-) disease (p = 0.044); whereas patients with metastatic HER2(+) breast cancer had higher serum miR-10b median levels than patients with metastatic HER2(-) disease (p = 0.0004). There were no significant differences in serum miR-19a median levels between HER2(+) and HER2(-) groups, regardless of the presence of metastases. High serum miR-19a levels were associated with IBC (p = 0.039). Patients with metastatic IBC had significantly higher serum miR-19a median levels than patients with metastatic non-IBC (p = 0.019). Finally, high serum miR-19a levels were associated with longer progression-free survival time (10.3 vs. 3.2 months; p = 0.022) and longer overall survival time (median not reached vs. 11.2 months; p = 0.003) in patients with metastatic HER2(+) IBC. Conclusion High levels of miR-21 and miR-10b were present in the serum of patients with non-metastatic and metastatic HER2(+) breast cancer, respectively. High levels of serum miR-19a may represent a biomarker for IBC that is predictive for favorable clinical outcome in patients with metastatic HER2(+) IBC.

Objective: This study investigated the prevalence of posttraumatic stress among survivors of serious injury-producing car crashes. Methods: This population-based prospective cohort study, conducted in New Zealand, recruited hospitalized car occupants (passengers and drivers) as well as nonhospitalized drivers after a crash in which at least one occupant was hospitalized. Fifty-nine hospitalized passengers (62%) and 209 drivers (72%) completed five- and 18-month interviews. The Impact of Event Scale assessed symptoms of posttraumatic stress. Results: At five months 28% of hospitalized passengers, 24% of hospitalized drivers, and 24% of nonhospitalized drivers reported symptoms consistent with posttraumatic stress disorder. At 18 months, 23% of hospitalized passengers, 11% of hospitalized drivers, and 7% of nonhospitalized drivers reported significant levels of stress. Conclusions: Strategies to prevent disabling sequelae of crashes must address the needs of hospitalized and nonhospitalized survivors. (Psychiatric Services 60:402–404, 2009)

Background: Cancer stem cells (CSCs) are epithelial tumor cells that express CD44 + CD24 −/lo . CSCs can be further divided into those that have aldehyde dehydrogenase (ALDH) activity (Aldefluor + ) and those that do not. We hypothesized that if CSCs are responsible for tumor dissemination, their presence in bone marrow (BM) would be prognostic in early stages of breast cancer (EBC) patients. Patients and methods: BM aspirates were collected at the time of surgery from 108 patients with EBC. BM was analyzed for CSCs and ALDH activity by flow cytometry. Overall survival 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 carried out. Results: 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. All deceased patients (n = 7) had CSCs in BM. In multivariate analysis, the presence of CSCs in BM was a prognostic factor of DFS (HR = 15.8, P= 0.017). Conclusions: The presence of BM metastasis is correlated with CSCs and these CSCs irrespective of ALDH activity are an independent adverse prognostic factor in EBC patients.

Currently, there is extensive information about circulating tumor cells (CTC) and their prognostic value; however, little is known about other characteristics of these cells. In this prospective study, we assessed the gene transcripts of epithelial-to-mesenchymal transition—inducing transcription factors (EMT-TF) and cancer stem cell (CSC) features in patients with HER2+ metastatic breast cancer (MBC). Epithelial cells were enriched from peripheral blood mononuclear cells (PBMC) using antibody-coated anti-CD326 antibody (CD326+) magnetic beads, and the residual CD326− PBMCs were further depleted of leukocytes using anti-CD45 antibody-coated magnetic beads (CD326−CD45−). RNA was extracted from all cell fractions, reverse transcribed to cDNA, and subjected to quantitative reverse transcription PCR to detect EMT-TFs (TWIST1, SNAIL1, ZEB1, and TG2) as a measure of CTCs undergoing EMT (EMT-CTCs). In addition, PBMCs were analyzed using multiparameter flow cytometry for ALDH activity and CSCs that express CD24, CD44, and CD133. Twenty-eight patients were included in this study. At least one EMT-TF mRNA was elevated in the CTCs of 88.2% of patients and in the CD326−CD45− cell fraction of 60.7% of patients. The CD326−CD45− fraction of patients with elevated SNAIL1 and ZEB1 transcripts also had a higher percentage of ALDH+/CD133+ cells in their blood than did patients with normal SNAIL1 and ZEB1 expression (P = 0.038). Our data indicate that patients with HER2+ MBCs have EMT-CTCs. Moreover, an enrichment of CSCs was found in CD326−CD45− cells. Additional studies are needed to determine whether EMT-CTCs and CSCs have prognostic value in patients with HER2+ MBCs treated with trastuzumab-based therapy. Mol Cancer Ther; 11(11); 2526–34. ©2012 AACR.

Artemisinin, a naturally occurring component of Artemisia annua, or sweet wormwood, is a potent anti-malaria compound that has recently been shown to have anti-proliferative effects on a number of human cancer cell types, although little is know about the molecular mechanisms of this response. We have observed that artemisinin treatment triggers a stringent G1 cell cycle arrest of LNCaP (lymph node carcinoma of the prostate) human prostate cancer cells that is accompanied by a rapid down-regulation of CDK2 and CDK4 protein and transcript levels. Transient transfection with promoter-linked luciferase reporter plasmids revealed that artemisinin strongly inhibits CDK2 and CDK4 promoter activity. Deletion analysis of the CDK4 promoter revealed a 231-bp artemisinin-responsive region between -1737 and -1506. Site-specific mutations revealed that the Sp1 site at -1531 was necessary for artemisinin responsiveness in the context of the CDK4 promoter. DNA binding assays as well as chromatin immunoprecipitation assays demonstrated that this Sp1-binding site in the CDK4 promoter forms a specific artemisinin-responsive DNA-protein complex that contains the Sp1 transcription factor. Artemisinin reduced phosphorylation of Sp1, and when dephosphorylation of Sp1 was inhibited by treatment of cells with the phosphatase inhibitor okadaic acid, the ability of artemisinin to down-regulate Sp1 interactions with the CDK4 promoter was ablated, rendering the CDK4 promoter unresponsive to artemisinin. Finally, overexpression of Sp1 mostly reversed the artemisinin down-regulation of CDK4 promoter activity and partially reversed the cell cycle arrest. Taken together, our results demonstrate that a key event in the artemisinin anti-proliferative effects in prostate cancer cells is the transcriptional down-regulation of CDK4 expression by disruption of Sp1 interactions with the CDK4 promoter.

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.

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.