Software-based risk stratification of pulmonary adenocarcinomas manifesting as pure ground glass nodules on computed tomography.

OBJECTIVES: To assess the performance of the "Computer-Aided Nodule Assessment and Risk Yield" (CANARY) software in the differentiation and risk assessment of histological subtypes of lung adenocarcinomas manifesting as pure ground glass nodules on computed tomography (CT). METHODS: 64 surgically resected and histologically proven adenocarcinomas manifesting as pure ground-glass nodules on CT were assessed using CANARY software, which classifies voxel-densities into three risk components (low, intermediate, and high risk). Differences in risk components between histological adenocarcinoma subtypes were analysed. To determine the optimal threshold reflecting the presence of an invasive focus, sensitivity, specificity, negative predictive value, and positive predictive value were calculated. RESULTS: 28/64 (44%) were adenocarcinomas in situ (AIS); 26/64 (41%) were minimally invasive adenocarcinomas (MIA); and 10/64 (16%) were invasive ACs (IAC). The software showed significant differences in risk components between histological subtypes (P<0.001-0.003). A relative volume of 45% or less of low-risk components was associated with histological invasiveness (specificity 100%, positive predictive value 100%). CONCLUSIONS: CANARY-based risk assessment of ACs manifesting as pure ground glass nodules on CT allows the differentiation of their histological subtypes. A threshold of 45% of low-risk components reflects invasiveness in these groups. KEY POINTS: • CANARY-based risk assessment allows the differentiation of their histological subtypes. • 45% or less of low-risk component reflects histological invasiveness. • CANARY has potential role in suspected adenocarcinomas manifesting as pure ground-glass nodules.

Selective regulation of arterial branching morphogenesis by synectin.

Branching morphogenesis is a key process in the formation of vascular networks. To date, little is known regarding the molecular events regulating this process. We investigated the involvement of synectin in this process. In zebrafish embryos, synectin knockdown resulted in a hypoplastic dorsal aorta and hypobranched, stunted, and thin intersomitic vessels due to impaired migration and proliferation of angioblasts and arterial endothelial cells while not affecting venous development. Synectin(-/-) mice demonstrated decreased body and organ size, reduced numbers of arteries, and an altered pattern of arterial branching in multiple vascular beds while the venous system remained normal. Murine synectin(-/-) primary arterial, but not venous, endothelial cells showed decreased in vitro tube formation, migration, and proliferation and impaired polarization due to abnormal localization of activated Rac1. We conclude that synectin is involved in selective regulation of arterial, but not venous, growth and branching morphogenesis and that Rac1 plays an important role in this process.

High-resolution quantitative computed tomography demonstrating selective enhancement of medium-size collaterals by placental growth factor-1 in the mouse ischemic hindlimb.

BACKGROUND: The process of arteriogenesis after occlusion of a major artery is poorly understood. We have used high-resolution microcomputed tomography (mu-CT) imaging to define the arteriogenic response in the mouse model of hindlimb ischemia and to examine the effect of placental growth factor-1 (PlGF-1) on this process. METHODS AND RESULTS: After common femoral artery ligation, mu-CT imaging demonstrated formation of collateral vessels originating near the ligation site in the upper limb and connecting to the ischemic calf muscle region. Three-dimensional mu-CT and quantitative image analysis revealed changes in the number of segments and the segmental volume of vessels, ranging from 8 to 160 microm in diameter. The medium-size vessels (48 to 160 microm) comprising 85% of the vascular volume were the major contributor (188%) to the change in vascular volume in response to ischemia. Intramuscular injections of Ad-PlGF-1 significantly increased Sca1+ cells in the circulation, alpha-actin-stained vessels, and perfusion of the ischemic hindlimb. These effects were predominantly associated with an increase in vascular volume contributed by the medium-size (96 to 144 microm) vessels as determined by mu-CT. CONCLUSIONS: High-resolution mu-CT delineated the formation of medium-size collaterals representing a major vascular change that contributed to the restoration of vascular volume after ischemia. This effect is selectively potentiated by PlGF-1. Such selective enhancement of arteriogenesis by therapeutically administered PlGF-1 demonstrates a desirable biological activity for promoting the growth of functionally relevant vasculature.

Low bone mineral density is associated with bone microdamage accumulation in postmenopausal women with osteoporosis.

Marked suppression of bone turnover by bisphosphonates is associated with increased bone microdamage accumulation in animal models. The purpose of this study was to test the hypothesis that long-term treatment with alendronate (ALN) results in accumulation of microdamage in bone in women after menopause. Sixty-six postmenopausal women with osteoporosis (mean age of 68.0 years and mean BMD T-score of -1.7 at total hip and -2.8 at lumbar spine; 62% with prevalent fractures) were evaluated in this cross-sectional analysis. Thirty-eight had been treated previously with ALN (10 mg/day or 70 mg/week for a mean duration of 63.6 months) while twenty-eight were treatment naive (TN). Without adjustments, crack surface density (Cr.S.Dn) and crack density (Cr.Dn) were not different between ALN and TN patients. After adjustment for potential confounders (age, prevalent fractures, femoral neck BMD, activation frequency and center), Cr.Dn was elevated in ALN patients (P=0.028 and P=0.069 for Cr.S.Dn). In ALN patients only, lower femoral neck BMD (Cr.S.Dn, r=-0.58, P=0.003; Cr.Dn, r=-0.54, P=0.005) and increased age (Cr.S.Dn, r=0.43, P=0.03; Cr.Dn, r=0.43, P=0.03) were associated with microdamage accumulation. Among potential confounders, femoral neck BMD was the only independent predictor for these correlations (P=0.04 for Cr.Dn and P=0.03 for Cr.S.Dn). We conclude that increased microdamage accumulation may occur in low BMD patients treated with alendronate.

Properties of bisphosphonates in the 13762 rat mammary carcinoma model of tumor-induced bone resorption.

Bone metastasis from primary tumors is a clinically important complication of neoplastic progression. The role of parathyroid hormone-related protein (PTHrP) and transforming growth factor (TGF)-beta1 in this process has been clearly established. The current study describes an in vivo model of 13762 rat mammary carcinoma tumor cell-induced osteolysis in which PTHrP and TGF-beta1 expression is observed. Exposure of in vitro-cultured 13762 cells to doxorubicin, cis-platinum, carboplatin, methotrexate, 5-fluorouracil, paclitaxel, alendronate, risedronate, or pamidronate for 72 h resulted in varying effects on cell proliferation (IC(50) values of 0.005, 0.4, 1.9, >40, 17.9, 0.003, >40, >40, and 33.6 micro M, respectively). Tumor cells were implanted into the intramedullary space of the proximal tibia of rats, and the time course of tumor progression was evaluated using radiographic and microcomputed tomography scanning techniques. Trabecular bone mineral density, cortical bone mineral density, and whole bone mineral density were measured (in mg/cm(3)). In untreated animals, radiographic evidence of osteolysis was evident 7 days after implantation. Trabecular bone mineral density and whole bone mineral density were significantly decreased by 21 days after implantation (48% and 26%, respectively). Bisphosphonates showed broad protective activity against tumor-driven osteolysis, Immunohistochemical evaluation of s.c. and intratibially implanted cells demonstrated the expression of PTHrP and TGF-beta1. The results of this study demonstrate the ability of 13762 rat mammary carcinoma cells to elicit a measurable osteolysis and that bisphosphonates inhibit the tumor-induced bone resorption in this model.

Enhancement of experimental fracture-healing by systemic administration of recombinant human parathyroid hormone (PTH 1-34).

BACKGROUND: Recombinant human parathyroid hormone (PTH [1-34]; teriparatide) is a new treatment for postmenopausal osteoporosis that can be systemically administered for the primary purpose of increasing bone formation. Because several studies have described the enhancement of fracture-healing and osteointegration in animals after use of PTH, we sought to critically analyze this skeletal effect. METHODS: Two hundred and seventy male Sprague-Dawley rats underwent standard, closed femoral fractures and were divided into three groups that were administered daily subcutaneous injections of 5 or 30 mug/kg of PTH (1-34) or vehicle (control). The dosing was administered for up to thirty-five days. Groups were further divided into three subgroups and were killed on day 21, 35, or 84 after the fracture. The bones were subjected to mechanical torsion testing, histomorphometric analysis, or microquantitative computed tomography. RESULTS: By day 21, calluses from the group treated with 30 mug of PTH showed significant increases over the controls with respect to torsional strength, stiffness, bone mineral content, bone mineral density, and cartilage volume. By day 35, both groups treated with PTH showed significant increases in bone mineral content and density and total osseous tissue volume, and they demonstrated significant decreases in void space and cartilage volume (p < 0.05). Torsional strength was significantly increased at this time-point in the group treated with 30 mug of PTH (p < 0.05). While dosing was discontinued on day 35, analyses performed after eighty-four days in the group treated with 30 mug of PTH showed sustained increases over the controls with respect to torsional strength and bone mineral density. No change was noted in osteoclast density at the time-points measured, suggesting that treatment with PTH enhanced bone formation but did not induce bone resorption. CONCLUSIONS: These data show that daily systemic administration of PTH (1-34) enhances fracture-healing by increasing bone mineral content and density and strength, and it produces a sustained anabolic effect throughout the remodeling phase of fracture-healing.

Teriparatide [PTH(1-34)] strengthens the proximal femur of ovariectomized nonhuman primates despite increasing porosity.

UNLABELLED: OVX monkeys treated for 18 months with 1 or 5 microg/kg/d teriparatide [PTH (1-34)] had significantly stronger proximal femora relative to ovariectomized controls. Teriparatide enhancement of cortical area, cortical width, and trabecular bone volume seemed to more than compensate for the dose-dependent increase in cortical porosity. Beneficial effects of teriparatide treatment on the proximal femur persisted beyond the treatment period and may extend to the marrow. INTRODUCTION: We conducted a detailed quantitative analysis of the effects of teriparatide on the proximal femur of ovariectomized monkeys. Teriparatide increased bone mass, enhanced structural architecture, and strengthened the hip, despite increasing cortical porosity. MATERIALS AND METHODS: Monkeys were treated with vehicle (sham or OVX controls), 1 microg/kg/day teriparatide [parathyroid hormone (1-34); PTH1], or 5 microg/kg/day teriparatide (PTH5) for 18 months or for 12 months followed by 6 months of treatment withdrawal (PTH1W and PTH5W, respectively). Excised proximal femora were analyzed by microCT, conventional histomorphometry, and biomechanics. RESULTS AND CONCLUSIONS: The femoral neck showed significant reduction in trabecular bone volume (BV/TV) for OVX compared with sham, whereas PTH1 BV/TV was restored to sham levels and PTH5 BV/TV was greater than sham and OVX. The withdrawal groups had BV/TVs intermediate between sham and OVX. PTH1 had trabecular number (Tb.N) greater than OVX, and PTH5 Tb.N was greater than sham and OVX. The withdrawal groups had Tb.Ns intermediate between sham and OVX. No differences between groups were observed for trabecular orientation or trabecular thickness. Teriparatide dose-dependently increased bone formation rate and activation frequency in the femoral neck. Cellular composition analyses suggested a tendency of ovariectomy to increase adiposity of marrow by 100%, whereas PTH tended to reduce adipocyte number and increase osteoblast number compared with OVX. Analyses of the cortex showed dose-dependent elevation of cortical porosity, which was consistent with enhanced bone turnover with treatment. Cortical porosity was reduced after withdrawal of teriparatide, because PTH1W cortical porosity was lower than OVX, whereas PTH5W cortical porosity was intermediate between sham and OVX. Increased cortical porosity did not weaken the proximal femora. Biomechanics showed that ovariectomy weakened proximal femora compared with sham, but PTH1, PTH5, and PTH1W were stronger than OVX and not different from sham. PTH5W strength was intermediate between sham and OVX. Therefore, teriparatide had beneficial effects on the proximal femur, despite increasing cortical porosity. Cortical porosity did not adversely affect the mechanical integrity of the proximal femora, because enhanced cortical area and trabecular bone volume more than compensated for the porosity. Much of the beneficial effects of teriparatide were retained after 6 months withdrawal from treatment. PTH effects on the femoral neck were not limited to bone but may include inhibition of OVX-stimulated adiposity of the marrow.

Raloxifene, estrogen, and alendronate affect the processes of fracture repair differently in ovariectomized rats.

We investigated the effects of inhibitors of bone resorption (estrogen, raloxifene, and alendronate) on the processes of fracture repair in ovariectomized (OVX) rats. One hundred forty female Sprague-Dawley rats at 3 months of age were either OVX or sham-operated and divided into five groups: sham control, OVX control, estrogen (17alpha-ethynyl estradiol [EE2], 0.1 mg/kg), raloxifene (Rlx, 1.0 mg/kg), and alendronate (Aln, 0.01 mg/kg) groups. Treatment began immediately after the surgery. Four weeks postovariectomy, prefracture controls were killed and bilateral osteotomies were performed on the femoral midshafts and fixed with intramedullary wires. Treatment was continued and fracture calluses were excised at 6 weeks and 16 weeks postfracture for evaluation by X-ray radiography, quantitative computed tomography (QCT,) biomechanical testing, and histomorphometry. At 6 weeks postfracture, Aln and OVX had larger calluses than other groups. Sham and OVX had higher ultimate load than EE2 and Rlx, with Aln not different from either control. Aln calluses also contained more mineral (bone mineral content [BMC]) than all other groups. By 16 weeks postfracture, OVX calluses were smaller than at 6 weeks and the dimensions for Aln had not changed. Aln had higher BMC and ultimate load than OVX, EE2, and Rlx. EE2 and Rlx had similar biomechanical properties, which were similar to sham. Interestingly, OVX and Aln animals were heavier than other groups at all time points; therefore, ultimate load was normalized by body weight to show no significant differences in strength of the whole callus between groups at either 6 weeks or 16 weeks postfracture. However, Aln strongly suppressed remodeling of the callus, resulting in the highest content of woven bone, persistent visibility of the original fracture line, and lowest content of lamellar bone, compared with other groups. Therefore, the larger Aln callus appeared to be a remarkable, morphological adaptation to secure the fracture with inferior material. In conclusion, OVX-stimulated bone turnover resulted in the fastest progression of fracture repair that was most delayed with Aln treatment, consistent with marked suppression of bone resorption and formation activity. Estrogen and Rlx had similar effects that were generally similar to sham, indicating that mild suppression of bone turnover with these agents has insignificant effects on the progression of fracture repair.

Longitudinal in vivo analysis of the region-specific efficacy of parathyroid hormone in a rat cortical defect model.

PTH has been shown to enhance fracture repair; however, exactly when and where PTH acts in this process remains to be elucidated. Therefore, we conducted a longitudinal, region-specific analysis of bone regeneration in mature, osteopenic rats using a cortical defect model. Six-month-old rats were ovariectomized, and allowed to lose bone for 2 months, before being subjected to bilateral 2-mm circular defects in their femoral diaphyses. They were then treated for 5 wk with hPTH1-38 at doses of 0, 3, 10, or 30 microg/kg . d and scanned weekly by in vivo quantitative computed tomography. Quantitative computed tomography analyses showed temporal, dose-dependent increases in mineralization in the defects, intramedullary (IM) spaces, and whole diaphyses at the defect sites. Histomorphometry confirmed PTH stimulation of primarily woven bone in the defects and IM spaces, but not the periosteum. After necropsy, biomechanical testing identified an increase in strength at the highest PTH dose. Serum procollagen type I N-terminal propeptide concentration showed a transient increase due to drilling, but procollagen type I N-terminal propeptide also increased with PTH treatment, whereas tartrate-resistant acid phosphatase unexpectedly decreased. Analyses of lumber vertebra confirmed systemic efficacy of PTH at a nonfracture site. In summary, PTH dose dependently induced new bone formation within defects, at endocortical surfaces, and in IM spaces, resulting in faster and greater bone healing, as well as efficacy at other skeletal sites. The effects of PTH were kinetic, region specific, and most apparent at high doses that may not be entirely clinically relevant; therefore, clinical studies are necessary to clarify the therapeutic utility of PTH in bone healing.

Lack of bone neoplasms and persistence of bone efficacy in cynomolgus macaques after long-term treatment with teriparatide [rhPTH(1-34)].

In rats, teriparatide [rhPTH(1-34)] causes marked increases in bone mass and osteosarcoma. In primates, teriparatide causes lesser increases in bone mass, and osteosarcomas have not been reported. Previous studies in primates were not designed to detect bone tumors and did not include a prolonged post-treatment observation period to determine whether tumors would arise after cessation of treatment. Ovariectomized (OVX), skeletally mature, cynomolgus monkeys (n = 30 per group) were given teriparatide for 18 mo at either 0 or 5 microg/kg/d subcutaneously. After 18 mo of treatment, subgroups of six monkeys from both groups were killed and evaluated, whereas all remaining monkeys entered a 3-yr observation period in which they did not receive teriparatide. Surveillance for bone tumors was accomplished with plain film radiographs, visual examination of the skeleton at necropsy, and histologic evaluation of multiple skeletal sites. Quantitative assessments of bone mass, architecture, and strength were also performed. After the 18-mo treatment period, vertebral BMD, BMC, and strength (ultimate load) were increased by 29%, 36%, and 52%, respectively, compared with OVX controls. Proximal femur BMD, BMC, and strength were also increased by 15%, 28% and 33%, respectively. After 3 yr without treatment, no differences in bone mass or strength at the vertebra were observed relative to OVX controls; however, the femoral neck showed significant persistence in stiffness (20%), BMC (14%), and trabecular BV/TV (53%), indicating a retention of teriparatide efficacy at the hip. Radiographs and histology did not identify any bone proliferative lesions or microscopic lesions of osteosarcoma at the end of the treatment or observation period. These data indicate that teriparatide did not induce bone proliferative lesions over a 4.5-yr interval of observation, including 18 mo of treatment and 3 yr of follow-up observation. Bone analyses confirmed that teriparatide caused increases in bone mass and strength, consistent with previous studies. During the withdrawal phase, beneficial effects of teriparatide treatment on the vertebra were lost; however, some of the beneficial effects on the proximal femur persisted for 3 yr after cessation of treatment. Although the lack of bone tumors in this study provides some additional reassurance regarding the safety of teriparatide for the primate skeleton, the small group size and other limitations of this, or any other animal study, limit the ability to draw definitive conclusions regarding the risk of bone tumor developments in patients.

Bone neoplasms in F344 rats given teriparatide [rhPTH(1-34)] are dependent on duration of treatment and dose.

A long-term study was conducted in female F344 rats to determine the relative importance of dose, treatment duration, and age at initiation of treatment on the incidence of teriparatide [rhPTH[1-34)]-induced bone proliferative lesions. Treatment groups consisted of different combinations of dose (0, 5, or 30 microg/kg/d), treatment duration (6, 20, or 24 months) and age at initiation of treatment (2 or 6 months of age). The primary endpoints were the incidence of bone neoplasms and effects on bone mass and structure as evaluated by quantitative computed tomography and histomorphometery. Significant increases in the incidence of bone tumors (osteoma, osteoblastoma, and osteosarcoma) occurred in rats treated with 30 microg/kg for 20 or 24 months. No neoplasms were found when the 5 microg/kg treatment was initiated at 6 months of age and continued for either 6 or 20 months (up to 70% of life span). This treatment regimen defined a "no-effect" dose for neoplasm formation that nevertheless resulted in substantial increases in bone mass. These results demonstrate that treatment duration and administered dose are the most important factors in the teriparatide-induced bone tumors in rats.

Skeletal changes in rats given daily subcutaneous injections of recombinant human parathyroid hormone (1-34) for 2 years and relevance to human safety.

Fischer 344 rats (60/sex/group) were given daily subcutaneous injections of recombinant human parathyroid hormone (PTH)(1-34) for 2 years at doses of 0, 5, 30, or 75 microg/kg. Treatment caused substantial increases in bone mass consistent with the known pharmacologic effects of once-daily administration. As determined by quantitative computed tomography (QCT) and histomorphometry, bone mass was markedly increased. Substantial new bone formation resulted in a large decrease in marrow space accompanied by altered bone architecture. Bone proliferative lesions were observed in all PTH( 1-34)-treated groups. Osteosarcoma occurred in 3, 21, and 31 male rats and in 4, 12, and 23 female rats in the 5-, 30-, and 75-microg/kg treatment groups, respectively. Focal osteoblast hyperplasia, osteoma, and osteoblastoma were much less frequent. Although the specific cellular or molecular mechanisms responsible for the rat bone tumors have not been fully elucidated, the data suggest that these lesions resulted from the long duration of treatment and the exaggerated pharmacologic response of the rat skeleton to daily treatment with PTH(1-34). Important differences between the rat study and clinical use in adult humans suggest that the increased incidence of bone neoplasia in rats treated for 2 years is likely not predictive of an increased risk of bone cancer in skeletally mature adult humans being given PTH(1-34) for a limited period of time in the treatment of osteoporosis.