Serum leucine-rich alpha-2-glycoprotein-1 with fucosylated triantennary N-glycan: a novel colorectal cancer marker.

BACKGROUND: Carcinoembryonic antigen (CEA) and carbohydrate antigen (CA)19-9 are used in clinical practice as tumor markers to diagnose or monitor colorectal cancer (CRC) patients, However, their specificities and sensitivities are not ideal, and novel alternatives are needed. In this study, mass spectrometry was used to search for screening markers, focusing on glycan alterations of glycoproteins in the sera of CRC patients. METHODS: Glycopeptides were prepared from serum glycoproteins separated from blood samples of 80 CRC patients and 50 healthy volunteers, and their levels were measured by liquid chromatography time-of flight mass spectrometry (LC-TOF-MS). RESULTS: Leucine-rich alpha-2-glycoprotein-1 with fucosylated triantennary N-glycan (LRG-FTG) was identified as CRC marker after evaluating 30,000 candidate glycopeptide peaks. The average LRG-FTG level in CRC patients (1.25 ± 0.973 U/mL) was much higher than that in healthy volunteers (0.496 ± 0.433 U/mL, P < 10- 10), and its sensitivity and specificity exceeded those of CA19-9. The combination of CEA and LRG-FTG showed a complementary effect and had better sensitivity (84%), specificity (90%), and AUC (0.91 by ROC analysis) than each marker alone or any other previously reported marker. LRG-FTG alone or combined with CEA also corresponded well with patient response to treatment. CONCLUSIONS: We identified LRG-FTG as a new CRC marker, with a sensitivity and specificity exceeding CA19-9. The combination of LRG-FTG and CEA showed much higher sensitivity and specificity than each marker alone. Further validation beyond this initial exploratory cohort is warranted.

Impact of M-protein detection on the response evaluations of patients undergoing treatment with the IgG-κ monoclonal antibodies daratumumab or isatuximab, and discrepancies between immunofixation electrophoresis (IFE) systems and reagents.

BACKGROUND: Immunofixation electrophoresis (IFE) is the standard method for confirming the presence of a monoclonal protein (M-protein) at multiple myeloma (MM) diagnosis. IFE is also essential at assessment of complete response (CR) and stringent CR during treatment. As the CR assessment is influenced by daratumumab and isatuximab, HYDRASHIFT assays were developed. METHODS: Samples from patients under treatment that included daratumumab or isatuximab were tested and monitored by IFE on the HYDRASYS system using HYDRASHIFT assays (HYDRASYS/HYDRASHIFT) and by IFE on the Epalyzer2 system (Epalyzer). RESULTS: The IFE using HYDRASYS/HYDRASHIFT avoided a false positive caused by drug-related IgG-κ and contributed to accurate assessment of CR. Furthermore, HYDRASYS/HYDRASHIFT detected small M-proteins at early relapse and detected free light chains (FLCs) in patients with renal impairment exhibiting high serum FLCs despite being often missed on Epalyzer. CONCLUSION: Sensitivity and specificity of M-protein detection vary greatly depending on the IFE system and reagents used.

A unique mutation of ALK2, G356D, found in a patient with fibrodysplasia ossificans progressiva is a moderately activated BMP type I receptor.

Fibrodysplasia ossificans progressiva (FOP) is a rare autosomal dominant congenital disorder characterized by progressive heterotopic bone formation in muscle tissues. A common mutation among FOP patients has been identified in ALK2, ALK2(R206H), which encodes a constitutively active bone morphogenetic protein (BMP) receptor. Recently, a unique mutation of ALK2, ALK2(G356D), was identified to be a novel mutation in a Japanese FOP patient who had unique clinical features. Over-expression of ALK2(G356D) induced phosphorylation of Smad1/5/8 and activated Id1-luc and alkaline phosphatase activity in myoblasts. However, the over-expression failed to activate phosphorylation of p38, ERK1/2, and CAGA-luc activity. These ALK2(G356D) activities were weaker than those of ALK2(R206H), and they were suppressed by a specific inhibitor of the BMP-regulated Smad pathway. These findings suggest that ALK2(G356D) induces heterotopic bone formation via activation of a BMP-regulated Smad pathway. The quantitative difference between ALK2(G356D) and ALK2(R206H) activities may have caused the phenotypic differences in these patients.

[ASBMR 2006 Report. Pathophysiology and diagnosis of osteoporosis].

This is a brief report on the papers related to pathophysiology and diagnosis of osteoporosis, which were presented in the 28th annual meeting of American Society for Bone and Mineral Research (ASBMR) held in Pennsylvania Convention Center, Philadelphia. In this meeting, there were numerous studies on risk factors for osteoporosis, since they are indispensable to estimate bone quality which is still impossible to quantify. In addition, the osteocytes are being dug out from the deep and dark bone lacunae for scientific examination. Very remarkable advancement in the mechanisms of the age-related inhibition of bone formation are also reported.

[Osteoporosis and intake of carbohydrates].

Adequate energy intake including carbohydrates is essential to maintain bone mass. Emaciation along with deficiency in nutrients, such as calcium, vitamin D, and protein is a significant risk factor for bone loss, and should be avoided. However, there is no clinical evidence that shows the direct effects of carbohydrate on bone mass. On the other hand, excessive intake of carbohydrates results in obesity, which causes other metabolic diseases such as diabetes mellitus (DM). Therefore, dietary regimen must be balanced in general, and complications and conditions of individual patients should be taken well into account. In addition, energy intake is a basis for adequate exercise in order to maintain physical activity and ideal body weight, which will further decrease the risk of bone fracture. Some indigestible carbohydrates, such as inulin and oligofructose, are shown to increase the availability of minerals from foods, and thus can be beneficial to bone mass.

Parathyroid hormone-related protein induced coupled increases in bone formation and resorption markers for 7 years in a patient with malignant islet cell tumors.

Parathyroid hormone-related protein (PTHrP) and PTH share the common PTH/PTHrP receptor. Although an elevated level of circulating PTHrP in patients with malignancies causes hypercalcemia as does PTH, chronic and systemic effects of PTHrP on bone metabolism in humans are not well understood because tumor-burden patients showing hypercalcemia usually have a poor prognosis. We investigated bone and calcium metabolism in a patient with malignant islet cell tumors showing hypercalcemia due to the elevated plasma PTHrP level for 7 years. Hypercalcemia and hypercalciuria continued throughout the clinical course in spite of frequent infusions of bisphosphonates. Bone resorption markers and a bone formation marker were consistently elevated as seen in primary hyperparathyroidism, a disease caused by an autonomous hypersecretion of PTH. Based on biochemical measurements including bone markers and serum 1,25-dihydroxyvitamin D, the clinical features of this case essentially are the same as those of primary hyperparathyroidism except for the elevated level of plasma PTHrP with suppressed intact PTH level. Therefore, it is suggested that chronic and systemic effects of PTHrP on bone as well as calcium metabolism are indistinguishable from those of PTH in human.

Receptor tyrosine kinases inhibit bone morphogenetic protein-Smad responsive promoter activity and differentiation of murine MC3T3-E1 osteoblast-like cells.

Growth factors such as fibroblast growth factor-2 (FGF-2) and epidermal growth factor (EGF) that activate extracellular signal-regulated kinases (ERKs) through receptor tyrosine kinases (RTKs) stimulate proliferation but suppress differentiation of osteoblasts. To study the mechanism of this inhibitory action of these growth factors on osteoblastic differentiation, we evaluated Smad1 transactivity in MC3T3-E1 osteoblast-like cells by reporters of promoter activity of mouse Smad6, an early response gene to bone morphogenetic proteins (BMPs). FGF-2 and EGF inhibited alkaline phosphatase activity and Smad6 promoter activity stimulated by BMP-2. Overexpression of constitutively active MEK by adenovirus mimicked, but that of dominant negative Ras or treatment with a MEK1 inhibitor, PD098059, reversed, the inhibitory effects of these growth factors on both activities. These effects are mediated by BMP-responsive elements (BMPREs) on Smad6 promoter, because an artificial reporter driven by three tandem BMPREs gave similar results, and these effects were all abolished when the BMPREs were mutated. RTK-ERK activation inhibited the promoter activity even when BMP signal was mediated by a mutant Smad1, which lacks phosphorylation sites by ERKs, or by a Smad1 fused to Gal4 DNA binding domain, which constitutively localizes in the nucleus. These results show that the RTK-Ras-ERK pathway suppresses BMP signal by interfering with Smad1 transactivity. Because direct phosphorylation of Smad1 by ERKs is not required for the inhibition, other transcriptional factors that are phosphorylated by ERKs might be involved in the regulation of osteoblastic differentiation by ERKs.

Constitutively activated ALK2 and increased SMAD1/5 cooperatively induce bone morphogenetic protein signaling in fibrodysplasia ossificans progressiva.

Fibrodysplasia ossificans progressiva (FOP) is a rare autosomal dominant disorder characterized by congenital malformation of the great toes and by progressive heterotopic bone formation in muscle tissue. Recently, a mutation involving a single amino acid substitution in a bone morphogenetic protein (BMP) type I receptor, ALK2, was identified in patients with FOP. We report here that the identical mutation, R206H, was observed in 19 Japanese patients with sporadic FOP. This mutant receptor, ALK2(R206H), activates BMP signaling without ligand binding. Moreover, expression of Smad1 and Smad5 was up-regulated in response to muscular injury. ALK2(R206H) with Smad1 or Smad5 induced osteoblastic differentiation that could be inhibited by Smad7 or dorsomorphin. Taken together, these findings suggest that the heterotopic bone formation in FOP may be induced by a constitutively activated BMP receptor signaling through Smad1 or Smad5. Gene transfer of Smad7 or inhibition of type I receptors with dorsomorphin may represent strategies for blocking the activity induced by ALK2(R206H) in FOP.

Interleukin-11 as a stimulatory factor for bone formation prevents bone loss with advancing age in mice.

Cytokines in interleukin (IL)-11 subfamily participate in the regulation of bone cell proliferation and differentiation. We report here positive effects of IL-11 on osteoblasts and bone formation. Overexpression of human IL-11 gene in transgenic mice resulted in the stimulation of bone formation to increase cortical thickness and strength of long bones, and in the prevention of cortical bone loss with advancing age. Bone resorption and osteoclastogenesis were not affected in IL-11 transgenic mice. In experiments in vitro, IL-11 stimulated transcription of the target gene for bone morphogenetic protein (BMP) via STAT3, leading to osteoblastic differentiation in the presence of BMP-2, but inhibited adipogenesis in bone marrow stromal cells. These results indicate that IL-11 is a stimulatory factor for osteoblastogenesis and bone formation to conserve cortical bone, possibly by enhancing BMP actions in bone. IL-11 may be a new therapeutic target for senile osteoporosis.