Edward R. Sauter

Circulating microRNAs in breast cancer and healthy subjects

BackgroundIt has been demonstrated that extracellular mRNA can be detected in the circulation. Our hypothesis was that circulating miRNAs are also present and differentially expressed in the serum of breast cancer patients compared to controls.FindingsWe measured miRNA in the serum of samples with and without the addition of miRNA prior to analysis. To test our RNA extraction efficiency, we spiked-in serial dilutions of single-strand C elegens miR-39 (cel-miR-39) and human miR-145 (has-miR-145) into goat serum and a 10 year old human serum specimen. We next analyzed miR-16, -145, and -155 in archived serum specimens from 21 participants, 13 of whom did and 8 of whom did not have breast cancer. We were able to detect the miRNAs from all the serum samples to which the miRNAs had been added. We were also able to detect endogenous miR-16, -145, and -155 in all serum samples. While the expression of all three miRNAs was similar in samples from healthy women compared to those with breast cancer, women with progesterone receptor (PR, p = 0.016) positive tumors had higher miR-155 expression than tumors that were negative for these receptors.Conclusion1) RNA species can be detected in archived serum; 2) miR-155 may be differentially expressed in the serum of women with hormone sensitive compared to women with hormone insensitive breast cancer. Screening serum for miRNAs that predict the presence of breast cancer is feasible, and may be useful for breast cancer detection.

Vitamin D and Skin Physiology: A D-Lightful Story

Throughout evolution, exposure to sunlight and the photosynthesis of vitamin D3 in the skin has been critically important for the evolution of land vertebrates. During exposure to sunlight, the solar UVB photons with energies 290–315 nm are absorbed by 7‐dehydrocholesterol in the skin and converted to previtamin D3. Previtamin D3 undergoes a rapid transformation within the plasma membrane to vitamin D3. Excessive exposure to sunlight will not result in vitamin D intoxication because both previtamin D3 and vitamin D3 are photolyzed to several noncalcemic photoproducts. During the winter at latitudes above ∼35°, there is minimal, if any, previtamin D3 production in the skin. Altitude also has a significant effect on vitamin D3 production. At 27° N in November, very little (∼0.5%) previtamin D3 synthesis was detected in Agra (169 m) and Katmandu (1400 m). There was an ∼2‐ and 4‐fold increase in previtamin D3 production at ∼3400 m and at Everest base camp (5300 m), respectively. Increased skin pigmentation, application of a sunscreen, aging, and clothing have a dramatic effect on previtamin D3 production in the skin. It is estimated that exposure in a bathing suit to 1 minimal erythemal dose (MED) is equivalent to ingesting between 10,000 and 25,000 IU of vitamin D2. The importance of sunlight for providing most humans with their vitamin D requirement is well documented by the seasonal variation in circulating levels of 25‐hydroxyvitamin D [25(OH)D]. Vitamin D deficiency [i.e., 25(OH)D < 20 ng/ml] is common in both children and adults worldwide. Exposure to lamps that produce UVB radiation is an excellent source for producing vitamin D3 in the skin and is especially efficacious in patients with fat malabsorption syndromes. The major cause of vitamin D deficiency globally is an underappreciation of sunlights role in providing humans with their vitamin D3 requirement. Very few foods naturally contain vitamin D, and those that do have a very variable vitamin D content. Recently it was observed that wild caught salmon had between 75% and 90% more vitamin D3 compared with farmed salmon. The associations regarding increased risk of common deadly cancers, autoimmune diseases, infectious diseases, and cardiovascular disease with living at higher latitudes and being prone to vitamin D deficiency should alert all health care professionals about the importance of vitamin D for overall health and well being.

Nipple aspirate fluid: a promising non-invasive method to identify cellular markers of breast cancer risk

To evaluate the feasibility of nipple aspiration and to identify intermediate markers of breast cancer risk, nipple aspirate fluid (NAF) was collected from 177 subjects using a modified breast pump. The first 33 subjects demonstrated that we could obtain NAF quickly, reliably and repeatedly. Specimens from the remaining 144 subjects were collected to evaluate promising cellular biomarkers. NAF was obtained in 167 out of 177 (94%) subjects overall and in 99% of the 144 most recent subjects. Sufficient NAF was obtained to evaluate cytology in 160 out of 167 (96%) cases and specimens were sufficiently cellular to analyse DNA markers in 53% of cases. Among the last 144 subjects, menopausal status did not influence the ability to obtain NAF. NAF cytology correlated with increased breast cancer risk (P = 0.002). Using computerized image analysis of NAF epithelial cells, DNA index (P = 0.0002), percentage of cells in G2M (P = 0.05) and percentage of cells with hypertetraploidy (P = 0.002) increased as cytology became more abnormal. Our data indicate that NAF can be obtained in essentially all eligible subjects; that breast epithelial cells are evaluable in > 95% of NAF samples for cytology and in over half of NAF samples for DNA index (ploidy) and cell cycle analysis; and that abnormal NAF cytology correlates with increased breast cancer risk. This suggests that biomarkers identified in nipple aspirate fluid may prove useful either as an adjunct to currently accepted breast cancer screening methods, or to evaluate response to a chemopreventive agent.

Proteomic Analysis to Identify Breast Cancer Biomarkers in Nipple Aspirate Fluid

Purpose: Proteomic analysis of breast nipple aspirate fluid (NAF) holds promise as a noninvasive method to identify markers of breast cancer. The objectives of the study were to: (a) describe the NAF proteome, (b) identify candidate markers of breast cancer in NAF by using proteomic analysis, and (c) validate the markers identified by using a quantitative, high-throughput ELISA analysis. Experimental Design: For proteome analysis, NAF proteins from a single subject without breast cancer were separated by two-dimensional PAGE and were subjected to matrix-assisted laser desorption ionization time-of-flight mass spectometry identification. A total of 41 different proteins were identified, 25 of which were known to be secreted. To identify breast cancer markers, we separated 20 NAF samples (10 normal, 10 cancer) by two-dimensional PAGE. Three protein spots were detected that were up-regulated in three or more cancer samples. These spots were identified to be gross cystic disease fluid protein (GCDFP)-15, apolipoprotein D (apoD), and α1-acid glycoprotein (AAG). To validate these three potential biomarkers, 105 samples (53 from benign breasts and 52 from breasts with cancer) were analyzed using ELISA. Results: Among all of the subjects, GCDFP-15 levels were lower (P < 0.001) and AAG levels were higher (P = 0.001) in breasts with cancer. This was also true in premenopausal (GCDFP-15, P = 0.011; AAG, P = 0.002) but not in postmenopausal women. GCDFP-15 levels were lowest (P = 0.003) and AAG levels highest (P < 0.001) in women with ductal carcinoma in situ (DCIS). Menopausal status influenced GCDFP-15 and AAG more in women without breast cancer than in women with breast cancer. apoD levels did not correlate significantly with breast cancer. Conclusions: Our study revealed that the NAF proteome, as defined by two-dimensional PAGE, consists of a limited number of proteins, and that the expression of AAG and GCDFP-15 correlates with disease presence and stage.

Detection of Breast Cancer in Nipple Aspirate Fluid by CpG Island Hypermethylation

Purpose: New approaches to the early detection of breast cancer are urgently needed as there is more benefit to be realized from screening. Nipple aspiration is a noninvasive technique that yields fluid known to contain breast epithelial cells. Silencing of tumor suppressor genes such as p16INk4a, BRCA1, and hMLH1 have established hypermethylation as a common mechanism for tumor suppressor inactivation in human cancer and as a promising target for molecular detection. Experimental Design: Using sensitive methylation-specific PCR, we searched for aberrant promoter hypermethylation in a panel of six normally unmethylated genes: glutathione S-transferase π 1 (GSTP1); retinoic acid receptor-β2 (RARβ2); p16INk4a; p14ARF; RAS association domain family protein 1A (RASSF1A); and death-associated protein kinase (DAP-kinase) in 22 matched specimens of tumor, normal tissue, and nipple aspirate fluid collected from breast cancer patients. Results: Hypermethylation of one or more genes was found in all 22 tumor DNAs (100% diagnostic coverage) and identical gene hypermethylation detected in 18 of 22 (82%) matched aspirate fluid DNAs. In contrast, hypermethylation was absent in benign and normal breast tissue and nipple aspirate DNA from healthy women. Conclusions: Promoter hypermethylation of important cancer genes is common in breast cancer and could be detected in matched aspirate DNAs from patients with ductal carcinoma in situ or stage I cancer. Promoter hypermethylation represents a promising marker, and larger studies may lead to its useful application in breast cancer diagnosis and management.

Proteomic analysis of nipple aspirate fluid to detect biologic markers of breast cancer.

The early detection of breast cancer is the best means to minimise disease-related mortality. Current screening techniques have limited sensitivity and specificity. Breast nipple aspirate fluid can be obtained noninvasively and contains proteins secreted from ductal and lobular epithelia. Nipple aspirate fluid proteins are breast specific and generally more concentrated than corresponding blood levels. Proteomic analysis of 1 μl of diluted nipple aspirate fluid over a 5–40 kDa range from 20 subjects with breast cancer and 13 with nondiseased breasts identified five differentially expressed proteins. The most sensitive and specific proteins were 6500 and 15 940 Da, found in 75–84% of samples from women with cancer but in only 0–9% of samples from normal women. These findings suggest that (1) differential expression of nipple aspirate fluid proteins exists between women with normal and diseased breasts, and (2) analysis of these proteins may predict the presence of breast cancer.

Effects of high-protein diets on fat-free mass and muscle protein synthesis following weight loss: a randomized controlled trial

The purpose of this work was to determine the effects of varying levels of dietary protein on body composition and muscle protein synthesis during energy deficit (ED). A randomized controlled trial of 39 adults assigned the subjects diets providing protein at 0.8 (recommended dietary allowance; RDA), 1.6 (2X‐RDA), and 2.4 (3X‐RDA) g kg–1 d–1 for 31 d. A 10‐d weight‐maintenance (WM) period was followed by a 21 d, 40% ED. Body composition and postabsorptive and postprandial muscle protein synthesis were assessed during WM (d 9‐10) and ED (d 30‐31). Volunteers lost (P<0.05) 3.2 ± 0.2 kg body weight during ED regardless of dietary protein. The proportion of weight loss due to reductions in fat‐free mass was lower (P<0.05) and the loss of fat mass was higher (P<0.05) in those receiving 2X‐RDA and 3X‐RDA compared to RDA. The anabolic muscle response to a protein‐rich meal during ED was not different (P>0.05) from WM for 2X‐RDA and 3X‐RDA, but was lower during ED than WM for those consuming RDA levels of protein (energy × protein interaction, P<0.05). To assess muscle protein metabolic responses to varied protein intakes during ED, RDA served as the study control. In summary, we determined that consuming dietary protein at levels exceeding the RDA may protect fat‐free mass during short‐term weight loss.—Pasiakos, S. M., Cao, J. J., Margolis, L. M., Sauter, E. R., Whigham, L. D., McClung, J. P., Rood, J. C., Carbone, J. W., Combs, G. F., Jr., Young, A. J. Effects of high‐protein diets on fat‐free mass and muscle protein synthesis following weight loss: a randomized controlled trial. FASEB J. 27, 3837–3847 (2013). www.fasebj.org

Proteomic analysis of nipple aspirate fluid using SELDI‐TOF‐MS

Proteomic analysis of body fluids, including breast nipple aspirate fluid (NAF), holds promise to aid in early cancer detection. We conducted a prospective trial that collected NAF from women scheduled for diagnostic breast surgery to determine 1) the consistency of proteomic results, 2) protein masses associated with breast cancer, 3) subsets of women with a unique proteomic profile and 4) a breast cancer predictive model. NAF was collected preoperatively in 114 women and analyzed by SELDI‐TOF mass spectrometry over a 3–50 kDa range using H4, NP and SAX ProteinChips. For all 3 chips, the same protein peaks were detected over 90% of the time in duplicate samples. The overall coefficient of variation was ≤ 0.17% for each chip for the internal standard and ≤ 0.29% for the unknown proteins. Seven candidate protein ion masses frequently expressed in NAF were identified. Three (5,200‐H4, p=.04, 11,880‐H4, p=.07 and 13,880 Da‐SAX, p=.03) were differentially expressed in women with/without breast cancer. Protein expression differed between women with/without pathologic nipple discharge (PND), but the 5,200, 11,880 and 13,880 proteins remained associated with breast cancer even if PND samples were excluded. Subset analysis identified differences in expression between benign disease and DCIS and between DCIS and invasive cancer for the 5,200 and 33,400 Da proteins. The best cancer detection model included age, parity and the 11,880 Da protein, and excluded women with PND. 1) NAF proteomic analysis using SELDI‐TOF is reproducible with the same sample set across different platforms, 2) differential proteomic expression exists between women/without breast cancer and 3) combining proteomic and clinical information that are available before surgery optimizes the prediction of which women have breast cancer.

Soy Isoflavones Have an Antiestrogenic Effect and Alter Mammary Promoter Hypermethylation in Healthy Premenopausal Women

We determined if soy isoflavones have dose-related estrogenic and methylation effects. Thirty-four healthy premenopausal women were randomized to 40 mg or 140 mg isoflavones daily through one menstrual cycle. Breast specific and systemic estrogenic effects were assessed measuring the estrogenic marker complement (C)3 and changes in cytology, whereas methylation assessment of 5 cancer related genes (p16, RASSF1A, RAR β 2, ER, and CCND2) was performed on intraductal specimens. Serum genistein significantly increased after consuming both isoflavone doses. Cytology did not significantly change at either isoflavone dose. Serum C3 levels posttreatment were inversely related to change in serum genistein ( r =–0.76, P = 0.0045) in women consuming low but not high dose isoflavones. The RAR β 2 hypermethylation increase posttreatment correlated with the posttreatment genistein level considering the entire group ( r = 0.67, P = 0.0017) and those receiving high-dose isoflavones ( r = 0.68, P = 0.021). At the low but not the high isoflavone dose, CCND2 hypermethylation increase correlated with posttreatment genistein levels ( r = 0.79, P = 0.011). In summary, the inverse correlation between C3 and genistein suggests an antiestrogenic effect. Isoflavones induced dose-specific changes in RAR β 2 and CCND2 gene methylation, which correlated with genistein levels. This work provides novel insights into estrogenic and methylation effects of dietary isoflavones.

Quantitative evaluation of DNA hypermethylation in malignant and benign breast tissue and fluids

The assessment of DNA had demonstrated altered methylation in malignant compared to benign breast tissue. The purpose of our study was to (i) confirm the predictive ability of methylation assessment in breast tissue, and (ii) use the genes found to be cancer predictive in tissue to evaluate the diagnostic potential of hypermethylation assessment in nipple aspirate fluid (NAF) and mammary ductoscopic (MD) samples. Quantitative methylation specific (qMS)‐PCR was conducted on three specimen sets: 44 malignant (CA) and 34 normal (NL) tissue specimens, 18 matched CA, adjacent normal (ANL) tissue and NAF specimens, and 119 MD specimens. Training and validation tissue sets were analyzed to determine the optimal group of cancer predictive genes for NAF and MD analysis. NAF and MD cytologic review were also performed. Methylation of CCND‐2, p16, RAR‐β and RASSF‐1a was significantly more prevalent in tumor than in normal tissue specimens. Receiver operating characteristic curve analysis demonstrated an area under the curve of 0.96. For the 18 matched CA, ANL and NAF specimens, the four predictive genes identified in cancer tissue contained increased methylation in CA vs. ANL tissue; NAF samples had higher methylation than ANL specimens. Methylation frequency was higher in MD specimens from breasts with cancer than benign samples for p16 and RASSF‐1a. In summary, i) routine quantitative DNA methylation assessment in NAF and MD samples is possible, and ii) genes hypermethylated in malignant breast tissue are also altered in matched NAF and in MD samples, and may be useful to assist in early breast cancer detection.