Rapid Escalation of High-Volume Exercise during Caloric Restriction; Change in Visceral Adipose Tissue and Adipocytokines in Obese Sedentary Breast Cancer Survivors.

Aerobic exercise reduces risk for breast cancer and recurrence and promotes visceral adipose tissue (VAT) loss in obesity. However, few breast cancer survivors achieve recommended levels of moderate to vigorous physical activity (MVPA) without supervision. In a two-cohort study, feasibility of 12 weeks of partially supervised exercise was started concomitantly with caloric restriction and effects on body composition and systemic risk biomarkers were explored. In total, 22 obese postmenopausal sedentary women (including 18 breast cancer survivors) with median age of 60 and BMI of 37 kg/m2 were enrolled. Using personal trainers twice weekly at area YMCAs, MVPA was escalated to ≥200 min/week over 9 weeks. For cohort 2, maintenance of effect was assessed when study provided trainer services were stopped but monitoring, group counseling sessions, and access to the exercise facility were continued. Median post-escalation MVPA was 219 min/week with median 12-week mass and VAT loss of 8 and 19%. MVPA was associated with VAT loss which was associated with improved adiponectin:leptin ratio. In total, 9/11 of cohort-2 women continued the behavioral intervention for another 12 weeks without trainers. High MVPA continued with median 24-week mass and VAT loss of 12 and 29%. This intervention should be further studied in obese sedentary women.

Change in Blood and Benign Breast Biomarkers in Women Undergoing a Weight-Loss Intervention Randomized to High-Dose ω-3 Fatty Acids versus Placebo.

The inflammation-resolving and insulin-sensitizing properties of eicosapentaenoic (EPA) and docosahexaenoic (DHA) fatty acids have potential to augment effects of weight loss on breast cancer risk. In a feasibility study, 46 peri/postmenopausal women at increased risk for breast cancer with a body mass index (BMI) of 28 kg/m2 or greater were randomized to 3.25 g/day combined EPA and DHA (ω-3-FA) or placebo concomitantly with initiation of a weight-loss intervention. Forty-five women started the intervention. Study discontinuation for women randomized to ω-3-FA and initiating the weight-loss intervention was 9% at 6 months and thus satisfied our main endpoint, which was feasibility. Between baseline and 6 months significant change (P < 0.05) was observed in 12 of 25 serum metabolic markers associated with breast cancer risk for women randomized to ω-3-FA, but only four for those randomized to placebo. Weight loss (median of 10% for trial initiators and 12% for the 42 completing 6 months) had a significant impact on biomarker modulation. Median loss was similar for placebo (-11%) and ω-3-FA (-13%). No significant change between ω-3-FA and placebo was observed for individual biomarkers, likely due to sample size and effect of weight loss. Women randomized to ω-3-FA exhibiting more than 10% weight loss at 6 months showed greatest biomarker improvement including 6- and 12-month serum adiponectin, insulin, omentin, and C-reactive protein (CRP), and 12-month tissue adiponectin. Given the importance of a favorable adipokine profile in countering the prooncogenic effects of obesity, further evaluation of high-dose ω-3-FA during a weight-loss intervention in obese high-risk women should be considered. PREVENTION RELEVANCE: This study examines biomarkers of response that may be modulated by omega-3 fatty acids when combined with a weight-loss intervention. While focused on obese, postmenopausal women at high risk for development of breast cancer, the findings are applicable to other cancers studied in clinical prevention trials.

Randomized Phase IIB Trial of the Lignan Secoisolariciresinol Diglucoside in Premenopausal Women at Increased Risk for Development of Breast Cancer.

We conducted a multiinstitutional, placebo-controlled phase IIB trial of the lignan secoisolariciresinol diglucoside (SDG) found in flaxseed. Benign breast tissue was acquired by random periareolar fine needle aspiration (RPFNA) from premenopausal women at increased risk for breast cancer. Those with hyperplasia and ≥2% Ki-67 positive cells were eligible for randomization 2:1 to 50 mg SDG/day (Brevail) versus placebo for 12 months with repeat bio-specimen acquisition. The primary endpoint was difference in change in Ki-67 between randomization groups. A total of 180 women were randomized, with 152 ultimately evaluable for the primary endpoint. Median baseline Ki-67 was 4.1% with no difference between arms. Median Ki-67 change was -1.8% in the SDG arm (P = 0.001) and -1.2% for placebo (P = 0.034); with no significant difference between arms. As menstrual cycle phase affects proliferation, secondary analysis was performed for 117 women who by progesterone levels were in the same phase of the menstrual cycle at baseline and off-study tissue sampling. The significant Ki-67 decrease persisted for SDG (median = -2.2%; P = 0.002) but not placebo (median = -1.0%). qRT-PCR was performed on 77 pairs of tissue specimens. Twenty-two had significant ERα gene expression changes (<0.5 or >2.0) with 7 of 10 increases in placebo and 10 of 12 decreases for SDG (P = 0.028), and a difference between arms (P = 0.017). Adverse event incidence was similar in both groups, with no evidence that 50 mg/day SDG is harmful. Although the proliferation biomarker analysis showed no difference between the treatment group and the placebo, the trial demonstrated use of SDG is tolerable and safe.

Effects of Weight Loss and Weight Regain on Circulating Biomarkers in Overweight/Obese Breast Cancer Survivors Enrolled in a Weight Loss Trial in the Rural Midwest.

BACKGROUND: Obesity is associated with worse breast cancer prognosis, however little is known about the level of weight loss required to improve pathway biomarkers. The effects of weight regain on biomarkers are also largely unknown. METHODS: Overweight/obese breast cancer survivors enrolled in an 18-month behavioral weight loss trial provided weight and serum biomarkers [leptin, adiponectin, insulin, plasminogen activator inhibitor-1 (PAI-1), IL-6, TNFα, and hepatocyte growth factor HGF] at baseline, 6, and 18 months (n = 138). Change in biomarkers over time and by weight loss thresholds were examined. RESULTS: Mean weight loss at 6 months was 13.3 ± 5.0 kg; from 6 to 18 months, mean regain was 4.0 ± 5.2 kg. Favorable biomarker modulations were observed at 6 months for leptin, adiponectin, insulin, PAI-1, IL-6, and HGF (P < 0.006 to P < 0.0001). These changes remained significant overall at 18 months despite attenuation in some. Women who lost <10% of baseline weight showed significantly smaller modulation effects for leptin (P < 0.0001), adiponectin:leptin (A/L) ratio (P < 0.0001), PAI-1 (P < 0.001), and insulin (P = 0.003) compared with women who lost >10%. Women who lost >10% observed a significant increase in adiponectin (P < 0.0001), and these women continued to show improved adiponectin from 6 to 18 months despite weight regain. Physical activity contributed additional effects on biomarker change for leptin, A/L ratio, and PAI-1. CONCLUSIONS: These findings are consistent with a clinical target of 10% weight. IMPACT: Sustained increases in adiponectin likely confer benefits for breast cancer prognosis even with weight regain.

Effect of Bazedoxifene and Conjugated Estrogen (Duavee) on Breast Cancer Risk Biomarkers in High-Risk Women: A Pilot Study.

Interventions that relieve vasomotor symptoms while reducing risk for breast cancer would likely improve uptake of chemoprevention for perimenopausal and postmenopausal women. We conducted a pilot study with 6 months of the tissue selective estrogen complex bazedoxifene (20 mg) and conjugated estrogen (0.45 mg; Duavee) to assess feasibility and effects on risk biomarkers for postmenopausal breast cancer. Risk biomarkers included fully automated mammographic volumetric density (Volpara), benign breast tissue Ki-67 (MIB-1 immunochemistry), and serum levels of progesterone, IGF-1, and IGFBP3, bioavailable estradiol and testosterone. Twenty-eight perimenopausal and postmenopausal women at increased risk for breast cancer were enrolled: 13 in cohort A with baseline Ki-67 < 1% and 15 in cohort B with baseline Ki-67 of 1% to 4%. All completed the study with > 85% drug adherence. Significant changes in biomarkers, uncorrected for multiple comparisons, were a decrease in mammographic fibroglandular volume (P = 0.043); decreases in serum progesterone, bioavailable testosterone, and IGF-1 (P < 0.01), an increase in serum bioavailable estradiol (P < 0.001), and for women from cohort B a reduction in Ki-67 (P = 0.017). An improvement in median hot flash score from 15 at baseline to 0 at 6 months, and menopause-specific quality-of-life total, vasomotor, and sexual domain scores were also observed (P < 0.001). Given the favorable effects on risk biomarkers and patient reported outcomes, a placebo-controlled phase IIB trial is warranted.

Circulating adipose stromal cells as a response biomarker in phase II energy balance trials of obese breast cancer survivors and high-risk women.

PURPOSE: Circulating adipose stromal cells (CASC) are thought to be increased in obesity and facilitate angiogenesis, and tumor metastases. METHODS: CASC were identified from buffy coat peripheral blood mononuclear cells (PBMCs) by flow cytometry as CD34brightCD31- CD45- and CASC frequency was compared to adiposity measures in 33 women at increased risk for breast cancer. Feasibility of CASC as a response biomarker for a diet and exercise intervention in ten breast cancer survivors was then explored. RESULTS: For 33 high-risk women, median CASC frequency was 9.7 per million PBMCs and trended positively with body mass index, fat mass index (FMI), and percent android fat. Correlation was significant when BMI was dichotomized at > versus < 35 kg/m2 (p = 0.02). For ten breast cancer survivors with a median BMI of 37 kg/m2, median CASC frequency was 16.4 per million PBMCs. In univariate analyses, change in BMI, total fat and visceral fat were significantly correlated with change in CASC frequency. On multivariate analysis, change in visceral adipose had the strongest association with change in CASC frequency (p < 0.00078). CONCLUSIONS: The association between the reduction in visceral adipose tissue and the decrease in frequency of circulating adipose stromal cells suggests that the latter might be a useful biomarker in clinical trials of obese breast cancer survivors undergoing a weight loss intervention.

Modulation of Breast Cancer Risk Biomarkers by High-Dose Omega-3 Fatty Acids: Phase II Pilot Study in Premenopausal Women.

Higher intakes of the omega-3 eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) relative to the omega-6 arachidonic acid (AA) have been variably associated with reduced risk of premenopausal breast cancer. The purpose of this pilot trial was to assess feasibility and explore the effects of high-dose EPA and DHA on blood and benign breast tissue risk biomarkers before design of a placebo-controlled phase IIB trial. Premenopausal women with evidence of hyperplasia ± atypia by baseline random periareolar fine needle aspiration were given 1860 mg of EPA + 1500 mg of DHA ethyl esters daily for 6 months. Blood and benign breast tissue were sampled during the same menstrual cycle phase prestudy and a median of 3 weeks after last dose. Additional blood was obtained within 24 hours of last dose. Feasibility, which was predefined as 50% uptake, 85% retention, and 70% compliance, was demonstrated with 46% uptake, 94% completion, and 85% compliance. Cytologic atypia decreased from 77% to 38% (P = 0.002), and Ki-67 from a median of 2.1% to 1.0% (P = 0.021) with an increase in the ratio of EPA + DHA to AA in erythrocyte phospholipids but no change in blood hormones, adipokines, or cytokines. Exploratory breast proteomics assessment showed decreases in several proteins involved in hormone and cytokine signaling with mixed effects on those in the AKT/mTOR pathways. Further investigation of EPA plus DHA for breast cancer prevention in a placebo-controlled trial in premenopausal women is warranted.

Clinical Trial of Acolbifene in Premenopausal Women at High Risk for Breast Cancer.

The purpose of this study was to assess the feasibility of using the selective estrogen receptor modulator (SERM) acolbifene as a breast cancer prevention agent in premenopausal women. To do so, we assessed change in proliferation in benign breast tissue sampled by random periareolar fine-needle aspiration (RPFNA) as a primary endpoint, along with changes in other risk biomarkers and objective and subjective side effects as secondary endpoints. Twenty-five women with cytologic hyperplasia ± atypia and ≥2% of breast epithelial cells staining positive for Ki-67, received 20 mg acolbifene daily for 6-8 months, and then had benign breast tissue and blood risk biomarkers reassessed. Ki-67 decreased from a median of 4.6% [interquartile range (IQR), 3.1%-8.5%] at baseline to 1.4% (IQR, 0.6%-3.5%) after acolbifene (P < 0.001; Wilcoxon signed-rank test), despite increases in bioavailable estradiol. There were also significant decreases in expression (RT-qPCR) of estrogen-inducible genes that code for pS2, ERα, and progesterone receptor (P ≤ 0.026). There was no significant change in serum IGF1, IGFBP3, IGF1:IGFBP3 ratio, or mammographic breast density. Subjective side effects were minimal with no significant increase in hot flashes, muscle cramps, arthralgias, or fatigue. Objective measures showed a clinically insignificant decrease in lumbar spine bone density (DEXA) and an increase in ovarian cysts but no change in endometrial thickness (sonography). In summary, acolbifene was associated with favorable changes in benign breast epithelial cell proliferation and estrogen-inducible gene expression but minimal side effects, suggesting a phase IIB placebo-controlled trial evaluating it further for breast cancer prevention.

Modulation of Breast Cancer Risk Biomarkers by High-Dose Omega-3 Fatty Acids: Phase II Pilot Study in Postmenopausal Women.

Associational studies suggest higher intakes/blood levels of the omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) relative to the omega-6 arachidonic acid (AA) are associated with reduced breast cancer risk. We performed a pilot study of high-dose EPA + DHA in postmenopausal women to assess feasibility before initiating a phase IIB prevention trial. Postmenopausal women with cytologic evidence of hyperplasia in their baseline random periareolar fine needle aspiration (RPFNA) took 1,860 mg EPA +1500 mg DHA ethyl esters daily for 6 months. Blood and breast tissue were sampled at baseline and study conclusion for exploratory biomarker assessment, with P values uncorrected for multiple comparisons. Feasibility was predefined as 50% uptake, 80% completion, and 70% compliance. Trial uptake by 35 study entrants from 54 eligible women was 65%, with 97% completion and 97% compliance. Favorable modulation was suggested for serum adiponectin (P = 0.0027), TNFα (P = 0.016), HOMA 2B measure of pancreatic ß cell function (P = 0.0048), and bioavailable estradiol (P = 0.039). Benign breast tissue Ki-67 (P = 0.036), macrophage chemoattractant protein-1 (P = 0.033), cytomorphology index score (P = 0.014), and percent mammographic density (P = 0.036) were decreased with favorable effects in a proteomics array for several proteins associated with mitogen signaling and cell-cycle arrest; but no obvious overall effect on proteins downstream of mTOR. Although favorable risk biomarker modulation will need to be confirmed in a placebo-controlled trial, we have demonstrated feasibility for development of high-dose EPA and DHA ethyl esters for primary prevention of breast cancer.

The prognostic value of BRCA1 promoter methylation in early stage triple negative breast cancer.

INTRODUCTION: Methylation of the BRCA1 promoter is frequent in triple negative breast cancers (TNBC) and results in a tumor phenotype similar to BRCA1-mutated tumors. BRCA1 mutation-associated cancers are more sensitive to DNA damaging agents as compared to conventional chemotherapy agents. It is not known if there is an interaction between the presence of BRCA1 promoter methylation (PM) and response to chemotherapy agents in sporadic TNBC. We sought to investigate the prognostic significance of BRCA1 PM in TNBC patients receiving standard chemotherapy. METHODS: Subjects with stage I-III TNBC treated with chemotherapy were identified and their formalin-fixed paraffin-embedded (FFPE) tumor specimens retrieved. Genomic DNA was isolated and subjected to methylation-specific PCR (MSPCR). RESULTS: DNA was isolated from primary tumor of 39 subjects. BRCA1 PM was detected in 30% of patients. Presence of BRCA1 PM was associated with lower BRCA1 transcript levels, suggesting epigenetic BRCA1 silencing. All patients received chemotherapy (anthracycline:90%, taxane:69%). At a median follow-up of 64 months, 46% of patients have recurred and 36% have died. On univariate analysis, African-American race, node positivity, stage, and BRCA1 PM were associated with worse RFS and OS. Five year OS was 36% for patients with BRCA1 PM vs. 77% for patients without BRCA1 PM (p=0.004). On multivariable analysis, BRCA1 PM was associated with significantly worse RFS and OS. CONCLUSIONS: We show that BRCA1 PM is common in TNBC and has the potential to identify a significant fraction of TNBC patients who have suboptimal outcomes with standard chemotherapy.

Favorable modulation of benign breast tissue and serum risk biomarkers is associated with > 10 % weight loss in postmenopausal women.

We conducted a phase II feasibility study of a 6-month behavioral weight loss intervention in postmenopausal overweight and obese women at increased risk for breast cancer and the effects of weight loss on anthropomorphic, blood, and benign breast tissue biomarkers. 67 women were screened by random peri-areolar fine-needle aspiration, 27 were registered and 24 participated in the interventional phase. The 24 biomarker evaluable women had a median baseline BMI of 34.2 kg/m(2) and lost a median of 11 % of their initial weight. Significant tissue biomarker modulation after the 6-month intervention was noted for Ki-67 (if restricted to the 15 women with any Ki-67 at baseline, p = 0.041), adiponectin to leptin ratio (p = 0.003); and cyclin B1 (p = 0.001), phosphorylated retinoblastoma (p = 0.005), and ribosomal S6 (p = 0.004) proteins. Favorable modulation for serum markers was observed for sex hormone-binding globulin (p < 0.001), bioavailable estradiol (p < 0.001), bioavailable testosterone (p = 0.033), insulin (p = 0.018), adiponectin (p = 0.001), leptin (p < 0.001), the adiponectin to leptin ratio (p < 0.001), C-reactive protein (p = 0.002), and hepatocyte growth factor (p = 0.011). When subdivided by <10 or >10 % weight loss, change in percent total body and android (visceral) fat, physical activity, and the majority of the serum and tissue biomarkers were significantly modulated only for women with >10 % weight loss from baseline. Some factors such as serum PAI-1 and breast tissue pS2 (estrogen-inducible gene) mRNA were not significantly modulated overall but were when considering only those with >10 % weight loss. In conclusion, a median weight loss of 11 % over 6 months resulted in favorable modulation of a number of anthropomorphic, breast tissue and serum risk and mechanistic markers. Weight loss of 10 % or more should likely be the goal for breast cancer risk reduction studies in obese women.

Assessment of RNA in human breast tissue sampled by random periareolar fine needle aspiration and ductal lavage and processed as fixed or frozen specimens.

Ductal lavage (DL) and random periareolar fine needle aspiration (RPFNA) have both been proposed as minimally invasive techniques to sample breast tissue during breast cancer prevention trials. Laser capture microdissection (LCM), linear RNA amplification and quantitative real-time polymerase chain reaction (qPCR) theoretically overcome the limitations of small specimen size obtained with DL and RPFNA. In order to test the yield, relative stability and amplifiability of RNA from fixed and archived RPFNA and DL specimens, breast tissue was sampled from individual high risk women (n=9) by both DL and RPFNA. RPFNA samples showed good RNA/cDNA yield and amplification while only 2 of 9 of the paired DL specimens had cDNA of adequate quality for subsequent PCR. One and two rounds of linear amplification provided approximately a 200- and 20,000-fold enrichment of RNA, respectively. PCR analysis consistently detected ER and COX-1 mRNA in the majority of RPFNA samples examined while pS2, PCNA, VEGF and survivin expression varied with subject. RNA yield and/or stability was greater for fixed and archived RPFNA than DL specimens of breast tissue. In a subsequent study examining an expanded biomarker gene panel in fixed vs. frozen RPFNA samples, mRNA profiles and ranked relative mRNA abundance were similar (r=0.89) for frozen and fixed RPFNA specimens. In summary, frozen RPFNA samples may be optimal for RNA endpoints in human breast cancer prevention trials but fixed RPFNA specimens allow similar analyses with greater convenience.

Reduction in Ki-67 in benign breast tissue of high-risk women with the lignan secoisolariciresinol diglycoside.

Preclinical and correlative studies suggest reduced breast cancer with higher lignan intake or blood levels. We conducted a pilot study of modulation of risk biomarkers for breast cancer in premenopausal women after administration of the plant lignan secoisolariciresinol given as the diglycoside (SDG). Eligibility criteria included regular menstrual cycles, no oral contraceptives, a >3-fold increase in 5-year risk, and baseline Ki-67 of ≥2% in areas of hyperplasia in breast tissue sampled by random periareolar fine-needle aspiration (RPFNA) during the follicular phase of the menstrual cycle. SDG (50 mg/d) was given for 12 months, followed by repeat RPFNA. The primary end point was change in Ki-67. Secondary end points included change in cytomorphology, mammographic breast density, serum bioavailable estradiol and testosterone insulin-like growth factor-I and IGF-binding protein-3, and plasma lignan levels. Forty-five of 49 eligible women completed the study with excellent compliance (median = 96%) and few serious side effects (4% grade 3). Median plasma enterolactone increased ∼9-fold, and total lignans increased 16-fold. Thirty-six (80%) of the 45 evaluable subjects showed a decrease in Ki-67, from a median of 4% (range, 2-16.8%) to 2% (range, 0-15.2%; P < 0.001, Wilcoxon signed rank test). A decrease from baseline in the proportion of women with atypical cytology (P = 0.035) was also observed. Based on favorable risk biomarker modulation and lack of adverse events, we are initiating a randomized trial of SDG versus placebo in premenopausal women.

CCN5/WISP-2 expression in breast adenocarcinoma is associated with less frequent progression of the disease and suppresses the invasive phenotypes of tumor cells.

Although previous in vitro studies predicted that CCN5/WISP-2 may act as an anti-invasive gene in breast cancer, the distribution pattern of CCN5 in breast cancer samples is conflicting. Thus, we systematically investigated the CCN5 expression profile in noninvasive and invasive breast tumor samples and its functional relevance in breast cancer progression. The studies showed that CCN5 expression is biphasic, such that in normal samples CCN5 expression is undetectable, whereas its expression is markedly increased in noninvasive breast lesions, including atypical ductal hyperplasia and ductal carcinoma in situ. Further, CCN5 mRNA and protein levels are significantly reduced as the cancer progresses from a noninvasive to invasive type. Additionally, we showed that CCN5 mRNA and protein level was almost undetectable in poorly differentiated cancers compared with the moderately or well-differentiated samples and its expression inversely correlated with lymph node positivity. The result was further supported by evaluating the RNA expression profile in microdissected sections using real-time PCR analysis. Therefore, our data suggest a protective function of CCN5 in noninvasive breast tumor cells. This hypothesis was further supported by our in vitro studies illuminating that CCN5 is a negative regulator of migration and invasion of breast cancer cells, and these events could be regulated by CCN5 through the modulation of the expression of genes essential for an invasive front. These include Snail-E-cadherin signaling and matrix metalloproteinase (MMP)-9 and MMP-2. Collectively, these studies suggest that the protective effect of CCN5 in breast cancer progression may have important therapeutic implications.

Reduction in proliferation with six months of letrozole in women on hormone replacement therapy.

The objective of this study was to determine if 6 months of the aromatase inhibitor letrozole, administered to postmenopausal women taking a stable dose of hormone replacement remedy, would be safe and would modulate biomarkers of breast cancer risk. The intent was to reduce the proliferation marker Ki-67 while maintaining adequate systemic levels of estradiol so as to avoid perimenopausal symptoms. Postmenopausal women at high risk for development of breast cancer and taking a stable dose of estrogen or estrogen plus progestin were screened by random periareolar fine needle aspiration (RPFNA). To be eligible, the acquired breast epithelial cells had to be characterized as cytologic atypia or borderline atypia with > or =1,000 epithelial cells on the cytomorphology slide; plus > or =500 epithelial cells on a slide processed for Ki-67 immunocytochemistry. Forty-two women were enrolled in the one arm study and received 2.5 mg letrozole per day for 6 months, followed by repeat assessment of biomarkers. Ki-67 was reduced by a median relative value of 66%. There was no significant change in breast cell cytomorphology; ER weighted index score; serum estradiol, testosterone, or IGF-1:IGFBP-3 ratio; mammographic breast density, or frequency or severity of perimenopausal symptoms. Given the dramatic reduction in proliferation, the effect of letrozole on risk and response biomarkers should be explored further in a randomized, placebo-controlled Phase IIB breast cancer chemoprevention trial.

Detection of biomarker gene expression by real-time polymerase chain reaction using amplified ribonucleic acids from formalin-fixed random periareolar fine needle aspirates of human breast tissue.

OBJECTIVE: To address the detection of breast cancer biomarker gene expression in formalin-fixed random periareolar fine needle aspiration (RPFNA) samples of benign breast tissue collected during breast cancer prevention trials by quantitative real-time polymerase chain reaction (qPCR). STUDY DESIGN: Formalin-fixed breast epithelial cells collected by RPFNA and processed as thin layer preparations were isolated by laser capture microdissection (LCM). Ribonucleic acid (RNA) was extracted and amplified using a single round of T7-based linear amplification followed by quality assessment and biomarker assay using TaqMan chemistry. RESULTS: More than 80% of RPFNA samples yielded RNA of sufficient quantity and quality for measurement of a panel of biomarker genes following a single round of linear amplification. RNA and protein expression for estrogen receptor alpha, as assessed by LCM/qPCR and immunohistochemistry, were correlated. Amplification plots were similar for cDNA standards and cDNA derived from RPFNA samples. CONCLUSION: Assessment of gene expression using amplified RNA from microdissected formalin-fixed RPFNAs can increase the number of biomarkers used during breast cancer chemoprevention trials.

In vitro models for studying human uterine and placental macrophages.

Human monocytes and macrophages, which are also called mononuclear phagocytes, represent a major arm of the innate immune system. These cells not only protect against infection but are also central to tissue remodeling and production of chemokines, cytokines, and growth factors. Tissue macrophages reside in the human placenta and uterine decidua throughout pregnancy, where they comprise part of the host defense network and facilitate placental and extraembryonic development. The purpose of this chapter is to describe methods for establishing useful models of human uteroplacental macrophages: (1) differentiated U937 myelomonocytic cells, (2) peripheral blood monocytes, (3) peripheral blood monocyte-derived macrophages, (4) decidual macrophages, and (5) placental macrophages.

Analysis of the soluble isoforms of HLA-G mRNAs and proteins.

The human major histocompatibility complex (MHC) contains genes encoding the Human Leukocyte Antigens (HLA). Of these antigens, placental immunologists need study only the HLA class I molecules, because HLA class II expression is repressed in the fetal placental cells that are in direct contact with maternal blood and tissues containing maternal immune cells. The class I antigens are subdivided into two general categories. The class Ia antigens are highly polymorphic and are typified by HLA-A, -B, and -C; these are expressed by nearly all somatic cells and stimulate graft rejection when foreign to the host. By contrast, the HLA class Ib antigens, HLA-E, -F, and -G, have restricted expression, few variants, and appear rarely to be immunostimulatory. One class Ia antigen, HLA-C, and the three class Ib antigens are differentially expressed by trophoblast cell subpopulations. In order to understand immune privilege in the pregnant uterus and placenta, it is essential to study the unique structural and functional features of these four genes and their glycoprotein products. In this chapter, we focus on the first class Ib gene identified in human placentas, HLA-G, with emphasis on its two soluble isoforms, HLA-G5 and HLA-G6. We describe methods developed in our laboratory to distinguish mRNAs encoding HLA-G5 and HLA-G6, and antibody-based protocols for identification of the soluble isoforms.

Isolation and culture of term human trophoblast cells.

Experimentation with most human cell types is restricted to the use of cell lines, and this limits our ability to extrapolate interpretations to the in vivo condition. However, in studying human trophoblast cells, we have a unique opportunity to obtain large quantities of readily available human tissue. In this chapter, we outline the methodology for purification of human trophoblast cells from term placentas. The procedures are based on enzymatic dissociation of villous placental tissue, followed by gradient centrifugation and immunomagnetic bead purification. Purity may be assessed by immunocytochemistry or flow cytometry using a number of markers to identify both cytotrophoblast cells and cellular contaminants. The resulting cytotrophoblast cell populations have excellent viability and purity, and may be subjected to long-term culture.

Placental cell expression of HLA-G2 isoforms is limited to the invasive trophoblast phenotype.

The HLA-G message is alternatively spliced into multiple transcripts, two of which encode soluble isoforms. To initiate studies on the specific functions of the soluble isoforms, we produced soluble rHLA-G1 (rsG1) and rsG2 in human embryonic kidney 293 cells and characterized the proteins. Both isoforms were glycosylated and formed disulfide-bonded oligomers. Recombinant sG1 associated with beta(2)-microglobulin, whereas rsG2 did not. Mouse mAb generated to rsG1 (1-2C3), which identified exclusively sG1, and mAb generated to rsG2 (26-2H11), which identified both soluble and membrane G2 (m/sG2), were used for immunohistochemical isoform mapping studies on placental tissue sections. Soluble G1 protein was abundant in many subpopulations of trophoblast cells, whereas m/sG2 protein was present exclusively in extravillous cytotrophoblast cells. Although both isolated placental villous cytotrophoblast cells and chorion membrane extravillous cytotrophoblast cells contained mRNAs encoding sG1 and sG2, protein expression was as predicted from the immunostains with m/sG2 present only in the invasive trophoblast subpopulation. Analysis of function by Northern and Western blotting demonstrated that both rsG1 and rsG2 inhibit CD8alpha expression on PBMC without changing CD3delta expression or causing apoptotic cell death. Collectively, the studies indicate that: 1) both sG1 and m/sG2 are produced in placentas; 2) transcription and translation are linked for sG1, but not G2; 3) expression of G2 is exclusively associated with the invasive phenotype; and 4) the two isoforms of sG may promote semiallogeneic pregnancy by reducing expression of CD8, a molecule required for functional activation of CTL.