Heat shock protein 70 (Hsp70) is frequently overexpressed in many different tumor types. However, Hsp70 has also been shown to be selectively presented on the plasma membrane of tumor cells, but not normal cells, and this membrane form of Hsp70 (mHsp70) could be considered a universal tumor biomarker. Since viable, mHsp70-positive tumor cells actively release Hsp70 in lipid micro-vesicles, we investigated the utility of Hsp70 in circulation as a universal tumor biomarker and its potential as an early predictive marker of therapeutic failure. We have also evaluated mHsp70 as a target for the isolation and enumeration of circulating tumor cells (CTCs) in patients with different tumor entities. Circulating vesicular Hsp70 levels were measured in the peripheral blood of tumor patients with the compHsp70 ELISA. CTCs were isolated using cmHsp70.1 and EpCAM monoclonal antibody (mAb)-based bead approaches and characterized by immunohistochemistry using cytokeratin and CD45-specific antibodies. In two out of 35 patients exhibiting therapeutic failure two years after initial diagnosis of non-metastatic breast cancer, progressively increasing levels of circulating Hsp70 had already been observed during therapy, whereas levels in patients without subsequent recurrence remained unaltered. With regards to CTC isolation from patients with different tumors, an Hsp70 mAb-based selection system appears superior to an EpCAM mAb-based approach. Extracellular and mHsp70 can therefore serve as a predictive biomarker for therapeutic failure in early-stage tumors and as a target for the isolation of CTCs in various tumor diseases.

Background: During the menopausal transition, around 25% of women experience a particularly accelerated loss of bone mineral density. These so-called “fast bone losers” represent a group of patients with an increased risk of osteoporosis. The precise mechanisms underlying this extraordinary level of bone mass reduction have not yet been conclusively elucidated. The PeKnO study (Perimenopausale Knochendichte und Ovulation; Perimenopausal Bone Density and Ovulation) was a 2-year prospective study investigating menstrual cycle changes, hormonal levels, markers of bone metabolism, and changes in bone mineral density (BMD) in perimenopausal women. The PeKnO study specifically focused on the questions of when the maximum of bone loss occurs, whether the decreasing number of ovulatory cycles correlates with increased bone density loss, and which hormones play a role during these processes. Methods: Healthy women aged ≥45 years with menstrual cycles of ≤42 days and without any exogenous hormonal intake continually self-assessed the lengths of their menstrual cycles and the occurrence of LH peaks with the help of a commercially available electronic fertility monitoring device. At baseline and at 6, 12, 18, and 24 months, hormones (LH, FSH, 17β-estradiol, progesterone, cortisol) and markers of bone metabolism (bone-specific alkaline phosphatase (BAP), osteocalcin (OC), and CTX (C-terminal telopeptide) were assessed during the luteal phase. Trabecular bone density was measured in the lumbar spine (vertebrae L1 through L3) by means of quantitative computed tomography (QCT) at the beginning and at the end of the 2-year study period. Patients were divided into 3 groups according to the changes in bone mineral density (BMD) that occurred within the period of 2 years: group I with an increase in BMD, group II with a decrease in BMD of ≤7%, and group III with a decline in BMD of >7%. Women in the latter group were defined as fast bone losers. Results: From a total of 72 recruited patients with an average age of 48.1 (±2.4) at baseline, complete 2-year data were available from 49 participants. Over the course of 24 months, mean bone mineral density decreased by −4.26 (±4.65). In the same time period, the proportion of ovulatory cycles declined from 67% to 33%. The decrease in the ovulatory rate significantly correlated with an enhanced BMD loss (r = 0.68; p < 0.05). Twelve of the forty-nine participants (24.3%) showed a BMD loss of >7% and were identified as fast bone losers. Levels of the luteal phase hormones LH, FSH, 17β-estradiol, and progesterone were significantly different between the three groups. Conclusion: The PeKnO study confirms a marked decline of the ovulatory rate during perimenopause, which is associated with an increased bone density loss while estrogen levels are still adequate.

Although a fragility fracture family history (FFFH+) has repeatedly been shown to be associated with lower bone mineral density (BMD), its relationship to human BMD change is unclear. Animal research, however, documented that different purebred strains within rodent species have wide ranges in rates of bone acquisition during growth as well as in change post-ovariectomy. Our objective was to compare the rate of premenopausal spinal trabecular BMD change between women with and without a general family history of fragility fracture.Healthy premenopausal community women participated in prospective observational studies at two academic medical research centres: Vancouver, Canada (n = 66) and Munich, Germany (n = 20). The primary outcome was annual spinal BMD change, measured by quantitative computed tomography (QCT). The two studies employed similar methodologies for assessing QCT and FFFH.Volunteer community participants had a mean age of 36.0 (SD, 6.9) years, body mass index 22.5 (2.4) and baseline QCT of 150.2 (22.5) mg/cm(3) trabecular bone. The rates of BMD change were similar in both cities: - 3.5 (5.1)/year Vancouver, - 2.0 (3.4)/year Munich (95 % CI of difference: - 3.9, 0.9). Over a third of the women (31 of the 86, 36 %) reported FFFH+. Those with and without a FFFH were similar in demographics, nutrition, exercise, menstrual cycle and luteal phase lengths and physiological measures (serum calcium, osteocalcin and estradiol). However, women with FFFH+ lost trabecular BMD more rapidly: FFFH+, - 4.9 (5.0), FFFH-, - 2.2 (4.4) mg/cm(3)/year (95 % CI diff - 0.7 to - 4.8, F1.83 = 7.88, p = 0.006). FFFH+ explained 7.7 % of the variance in QCT volumetric trabecular spinal bone change/year in these healthy premenopausal women.This study shows for the first time that having a history of a fragility fracture in a family member is associated with a greater rate of premenopausal spinal trabecular bone loss.

Stress-inducible heat shock protein 70 (Hsp70), which functions as a molecular chaperone and is frequently overexpressed in different cancer cell types, is present on the cell surface of tumor cells and is actively released into the circulation in free and extracellular lipid vesicle-associated forms. Since the exact pathomechanism of endometriosis has not yet been elucidated (although it has been associated with the development of endometrial and ovarian cancer), we asked whether extracellular Hsp70 and circulating endometriotic cells (CECs) reflect the presence and development of endometriosis. Therefore, circulating levels of free and lipid microvesicle-associated Hsp70 were measured using the Hsp70-exo ELISA, and the presence of circulating CECs in the peripheral blood of patients with endometriosis was determined using membrane Hsp70 (mHsp70) and EpCAM monoclonal antibody (mAb)-based bead isolation approaches. Isolated CECs were further characterized by immunofluorescence using reagents directed against cytokeratin (epithelial marker), CD45 (leukocyte marker), CD105/CD44 (mesenchymal stemness markers) and by comparative RNA analysis. Similar to the situation in patients with cancer, the levels of circulating Hsp70 were elevated in the blood of patients with histologically proven endometriosis compared to a healthy control cohort, with significantly elevated Hsp70 levels in endometriosis patients with lesions outside the uterine cavity. Moreover, CECs could be isolated using the cmHsp70.1 mAb-based, and to a lesser extent EpCAM mAb-based, bead approach in all patients with endometriosis, with the highest counts obtained using the mHsp70-targeting procedure in patients with extra-uterine involvement. The longevity in cell culture and the expression of the cytokeratins CD105 and CD44, together with differentially expressed genes related to epithelial-to-mesenchymal transition (EMT), revealed similarities between mHsp70-expressing CECs and circulating tumor cells (CTCs) and suggest a mesenchymal stem cell origin. These findings support the involvement of mHsp70-positive stem cell-like cells in the development of endometriotic lesions. In summary, elevated levels of Hsp70 and CECs in the circulation could serve as liquid biopsy markers for endometriosis with extra-uterine involvement and help to elucidate the underlying pathomechanism of the disease.

Background Some studies have already proposed an inverse association between vitamin D levels and breast density. As breast density is already considered an established risk factor for breast cancer, such a connection could offer a new starting point for the prevention of breast cancer. Material and Methods To investigate this suggested connection, a total of 412 pre- and 572 post-menopausal women for whom mammography was indicated were recruited into this cross-sectional study. In addition to a questionnaire-based interview on the patientʼs general and gynecological medical history, her eating habits and lifestyle, serum levels of 25-hydroxyvitamin D [25(OH)D], calcium, phosphate and creatinine were determined. Breast density was determined by mammography and categorized as 1 to 4 according to the ACR classification. In addition to performing descriptive analysis to get a better overview of the data, a number of multivariate regression models were developed to determine the impact of confounders and the connection between vitamin D and mammographic density. Results More than half of all participants had low levels of 25(OH)D (< 20 ng/ml) and only a small minority of women (5.7 %) had what are currently considered to be optimal serum levels of 25(OH)D of at least 30 ng/ml. The significant majority of the cohort had a medium mammographic density (n = 463 had ACR 2; n = 343 had ACR 3). Logistic regression analysis showed that lower 25(OH)D serum levels were associated significantly more often with high rather than medium breast density. This association remained, even after adjusting for other factors which influence breast density such as age, BMI and menopausal status (p = 0.032 for ACR 4 vs. ACR 2; p = 0.028 for ACR 4 vs. ACR 3). When the same analysis was done separately for pre-menopausal and post-menopausal women, BMI in both groups was found to be inversely correlated with breast density and this inverse correlation was highly significant. In post-menopausal women, age was found to be similarly correlated while 25(OH)D did not appear to be associated with ACR. In pre-menopausal women the opposite was the case: although there was no correlation between age and breast density, higher vitamin D levels tended to be associated with lower breast density (p = 0.06 for ACR 2 vs. ACR 4) in this smaller sample (n = 412). When vitamin D-rich food and food supplements were also taken into account, regular intake of vitamin D preparations was associated with lower breast density; this association achieved borderline statistical significance (p = 0.05 for ACR 3 vs. ACR 4). When the analysis also took menopausal status into account, the breast density of pre-menopausal women was lower following regular vitamin D intake and this lower breast density of pre-menopausal women was statistically highly significant (p < 0.001 for ACR 1 and ACR 2 vs. ACR 4, respectively). This effect was not found in post-menopausal women. Frequent intake of vitamin D-containing nutrition had no significant impact on ACR in either of the groups. Conclusion These results reinforce the assumption previously proposed by several authors that higher levels of 25(OH)D pre-menopause and vitamin D substitution are associated with lower breast density and could reduce the risk of breast cancer. The findings did not confirm any post-menopausal association between vitamin D and mammographic breast density.

Introduction Known characteristics of patients with PCOS include infertility, menstrual disorders, hirsutism and also often insulin resistance. These symptoms increase with increasing body weight. In the LIPCOS study (Lifestyle Intervention for Patients with Polycystic Ovary Syndrome [PCOS]) long-term changes of the PCOS in dependence on pregnancy and parenthood were systematically assessed. In the framework of the LIPCOS study, PCOS patients were given a standardised carbohydrate-rich test meal in order to examine glucose homeostasis and insulin secretion. The results were compared with those of a eumenorrhoeic control group who all had corresponding BMI values and corresponding ages. Methods and Patients 41 PCOS patients (without diabetes) and 68 controls received a standardised carbohydrate-rich test meal (260 kcal, 62 % carbohydrates, 32 % fat, 6 % proteins) in order to generate a submaximal insulin and glucose stimulation. The values were determined at baseline and postprandial after 60, 120 and 180 minutes. In addition, the corresponding C-peptide levels were recorded. Results In the PCOS patients (n = 41), the insulin secretion test after a standardised test meal showed almost identical baseline and postprandial insulin levels when compared with those of the age- and BMI-matched eumenorrhoeic controls (n = 68). In the PCOS patients, the baseline and postprandial glucose levels were significantly elevated (92.88 ± 10.28 [PCOS] vs. 85.07 ± 9.42 mg/dL [controls]; p < 0.001) so was C-peptide (p < 0.025). Conclusions In the present study we have shown for the first time that, after consumption of a standardised test meal, PCOS patients formally exhibit a higher fasting insulin resistance than controls. In spite of the higher stimulated C-peptide levels, the insulin levels did not increase more strongly with increasing glucose levels than in controls which may be indicative of a higher insulin clearance in PCOS patients.