Functional folate receptor alpha is elevated in the blood of ovarian cancer patients.

BACKGROUND: Despite low incidence, ovarian cancer is the fifth leading cause of cancer deaths and it has the highest mortality rate of all gynecologic malignancies among US women. The mortality rate would be reduced with an early detection marker. The folate receptor alpha (FRalpha) is one logical choice for a biomarker because of its prevalent overexpression in ovarian cancer and its exclusive expression in only a few normal tissues. In prior work, it was observed that patients with ovarian cancer had elevated serum levels of a protein that bound to a FRalpha-specific monoclonal antibody relative to healthy individuals. However, it was not shown that the protein detected was intact functional FRalpha. In the current study, the goal was to determine whether ovarian cancer patients (n = 30) had elevated serum levels of a fully functional intact FRalpha compared to matched healthy controls (n = 30). METHODOLOGY/PRINCIPAL FINDINGS: FRalpha levels in serum were analyzed by two methods, immunoblotting analysis and a radiolabeled folic acid-based microfiltration binding assay. Using the immunoassay, we observed that levels of FRalpha were higher in serum of ovarian cancer patients as compared to controls. Similar results were also observed using the microfiltration binding assay, which showed that the circulating FRalpha is functional. Importantly, we also found that the levels of FRalpha were comparable between early and advanced stage patients. CONCLUSIONS: Our results demonstrate that ovarian cancer patients have elevated levels of functional intact FRalpha. These findings support the potential use of circulating FRalpha as a biomarker of early ovarian cancer.

Characterization of circulating osteoblast lineage cells in humans.

We recently identified circulating osteoblastic cells using antibodies to osteocalcin (OCN) or alkaline phosphatase (AP). We now provide a more detailed characterization of these cells. Specifically, we demonstrate that 46% of OCN positive (OCN(pos)) cells express AP, and 37% also express the hematopoietic/endothelial marker CD34. Using two different anti-OCN antibodies and forward/side light scatter characteristics by flow cytometry, we find that OCN(pos) cells consist of two distinct populations: one population exhibits low forward/side scatter, consistent with a small cell phenotype with low granularity, and a second population has higher forward/side scatter (larger and more granular cell). The smaller, low granularity population also co-expresses CD34, whereas the larger, more granular cells are CD34 negative. Using samples from 26 male subjects aged 28 to 68 years, we demonstrate that the concentration of circulating OCN(pos) cells increases as a function of age (R=0.59, P=0.002). By contrast, CD34(pos) cells tend to decrease with age (R=-0.31, P=0.18); as a consequence, the ratio of OCN(pos):CD34(pos) cells also increase significantly with age (R=0.54, P=0.022). These findings suggest significant overlap between circulating cells expressing OCN and those expressing the hematopoietic/endothelial marker CD34. Further studies are needed to define the precise role of circulating OCN(pos) cells not only in bone remodeling but rather also potentially in the response to vascular injury.

Remodeling and vascular spaces in bone.

In recent years, we have come to appreciate that the close association between bone and vasculature plays a pivotal role in the regulation of bone remodeling and fracture repair. In 2001, Hauge et al. characterized a specialized vascular structure, the bone remodeling compartment (BRC), and showed that the outer lining of this compartment was made up of flattened cells, displaying all the characteristics of lining cells in bone. A decrease in bone turnover leads to a decrease in surfaces covered with remodeling compartments, whereas increased turnover causes an increase. Immunoreactivity for all major osteotropic growth factors and cytokines including osteoprotegerin (OPG) and RANKL has been shown in the cells lining the BRC, which makes the BRC the structure of choice for coupling between resorption and formation. The secretion of these factors inside a confined space separated from the bone marrow would facilitate local regulation of the remodeling process without interference from growth factors secreted by blood cells in the marrow space. The BRC creates an environment where cells inside the structure are exposed to denuded bone, which may enable direct cellular interactions with integrins and other matrix factors known to regulate osteoclast/osteoblast activity. However, the denuded bone surface inside the BRC also constitutes an ideal environment for the seeding of bone metastases, known to have high affinity for bone matrix. Reduction in BRC space brought about by antiresorptive therapies such as bisphosphonates reduce the number of skeletal events in advanced cancer, whereas an increase in BRC space induced by remodeling activators like PTH may increase the bone metastatic burden. The BRC has only been characterized in detail in trabecular bone; there is, however, evidence that a similar structure may exist in cortical bone, but further characterization is needed.

Circulating cells with osteogenic potential.

While osteoclast lineage cells are clearly present in the peripheral circulation, whether there is a comparable pool of circulating osteoblast lineage cells has remained controversial. Using assays requiring adherence to plastic (as originally described by Friedenstein and colleagues for bone marrow stromal cells over four decades ago), several studies have shown that plastic adherent cells with osteogenic potential are, indeed, present in the circulation of a number of species, but at extremely low concentrations. Work from a number of independent groups over the past decade has also identified a population of nonadherent bone marrow cells with osteogenic potential. Since these nonadherent cells may be much more likely to access the peripheral circulation than plastic adherent cells, we tested for the presence of circulating osteoblast lineage cells in humans using flow cytometry to identify cells in the peripheral blood expressing bone-related proteins. Our findings indicate that these cells are present in the circulation in significant numbers, are markedly increased in the peripheral blood of adolescent boys going through the growth spurt, and may also increase following fractures. These circulating osteogenic cells express bone-related proteins, can mineralize in vitro, and form bone in vivo. The identification of these osteogenic cells in peripheral blood opens up new questions regarding the possible role of these cells in bone remodeling, in fracture repair, and possibly in vascular calcification.

Circulating osteoblast-lineage cells in humans.

BACKGROUND: Although current evidence suggests that only a minuscule number of osteoblast-lineage cells are present in peripheral blood, we hypothesized that such cells circulate but that their concentration has been vastly underestimated owing to the use of assays that required adherence to plastic. We further reasoned that the concentration of these cells is elevated during times of increased bone formation, such as during pubertal growth. METHODS: We used flow cytometry with antibodies to bone-specific proteins to identify circulating osteoblast-lineage cells in 11 adolescent males and 11 adult males (mean [+/-SD] age, 14.5+/-0.7 vs. 37.7+/-7.6 years). Gene expression and in vitro and in vivo bone-forming assays were used to establish the osteoblastic lineage of sorted cells. RESULTS: Cells positive for osteocalcin and cells positive for bone-specific alkaline phosphatase were detected in the peripheral blood of adult subjects (1 to 2 percent of mononuclear cells). There were more than five times as many cells positive for osteocalcin in the circulation of adolescent boys (whose markers of bone formation were clearly increased as a result of pubertal growth) as compared with adult subjects (P<0.001). The percentage of cells positive for osteocalcin correlated with markers of bone formation. Sorted osteocalcin-positive cells expressed osteoblastic genes, formed mineralized nodules in vitro, and formed bone in an in vivo transplantation assay. Increased values were also found in three adults with recent fractures. CONCLUSIONS: Osteoblast-lineage cells circulate in physiologically significant numbers, correlate with markers of bone formation, and are markedly higher during pubertal growth; therefore, they may represent a previously unrecognized circulatory component to the process of bone formation.

Graves' dermopathy and acropachy are markers of severe Graves' ophthalmopathy.

It is generally considered that thyroid dermopathy and acropachy almost always occur with Graves' ophthalmopathy and that these two extrathyroidal manifestations are indicators of severe autoimmune disease and hence of more severe ophthalmopathy. However, documentation of these anecdotal impressions is needed. We assessed the presence of optic neuropathy and frequency of orbital decompression in 2 referral cohorts: 40 patients with acropachy and dermopathy (acropachy group) and 138 patients with Graves' dermopathy and no acropachy (dermopathy group). We compared those cohorts with a cohort of 114 patients who had ophthalmopathy without dermopathy and acropachy (control group). We considered optic neuropathy and the need for orbital decompression to be indicators of severe Graves' ophthalmopathy. The frequency of orbital decompression was significantly higher in the dermopathy group than in the control group (odds ratio, 3.55) and even higher in the acropachy group (odds ratios: 20.68 for acropachy group compared with control group; 5.83 for acropachy group compared with dermopathy group). The same trend occurred with optic neuropathy but was not statistically significant (alpha = 0.05; p = 0.07). Five patients were exceptions: they had definite Graves' dermopathy without clinically obvious ophthalmopathy. In conclusion, dermopathy and acropachy appear to be markers of severe ophthalmopathy. Occasionally, however, Graves' dermopathy occurs without clinical ophthalmopathy.