Bone architecture, bone material properties, and bone turnover in non-osteoporotic post-menopausal women with fragility fracture.

Macro- and microarchitectural, bone material property, dynamic histomorphometric, and bone turnover marker data were studied in normal bone mineral density (BMD) post-menopausal women with fragility fracture. Women with fracture had thinner iliac cortices and more homogeneous bone material properties in cortical bone than age/BMD-matched non-fracture women. Low cortical thickness and bone tissue heterogeneity in normal BMD women are associated with prevalent fragility fracture. INTRODUCTION: Bone mass (bone mineral density, (BMD)) of the spine and hip is today's best single measurement for evaluating future fragility fracture risk. However, the majority of fragility fractures occur in women with BMD T-score above the WHO osteoporotic BMD threshold of - 2.5, indicating that non-BMD endpoints may play a role in their fragility fractures. We hypothesize that in non-osteoporotic women, bone micoarchitecture, bone material properties, dynamic histomorphometric endpoints, and bone turnover markers are related to fragility fracture. METHODS: Two groups (N = 60 each) of post-menopausal women with total hip BMD T-score ranging from + 0.3 to -2.49 were recruited: fragility fracture and age/BMD-matched, non-fragility fracture women. Normal (T-score > - 0.99) and osteopenic (T-score ≤ - 1.0) BMD cohorts were designated within both the fracture and non-fracture groups. Transiliac biopsy specimens were obtained to evaluate dynamic histomorphometric and microarchitectural endpoints and bone material properties by static and dynamic nanoindentation testing. All variables for fracture and non-fracture women within each BMD cohort were compared by the Wilcoxon signed-rank test (P < 0.01). RESULTS: Compared to non-fracture/normal BMD women, fracture/normal BMD women display lower iliac cortical thickness (- 12%, P = 0.0041) and lower heterogeneity of hardness (- 27%, P = 0.0068), elastic modulus (- 35%, P = 0.0009), and storage modulus (- 23%, P = 0.0054) in the cortical bone tissue, and lower heterogeneity of hardness (- 13%, P = 0.0088) in the trabecular bone tissue. Osteopenic women had no abnormalities related to fracture status. CONCLUSION: Post-menopausal women with normal BMD and fragility fracture have low cortical thickness and heterogeneity of several bone material properties in cortical and trabecular mineralized bone tissue. These differences may explain a portion of the excess bone fragility in women with normal BMD and fragility fracture.

Effect of Macroanatomic Bone Type and Estrogen Loss on Osteocyte Lacunar Properties in Healthy Adult Women.

This is the first study to examine clinical human bone specimens by three-dimensional imaging to characterize osteocyte lacunar properties as a function of macroanatomic bone type and estrogen loss. We applied laboratory-based instrumentation [3D X-ray microscope (3DXRM), MicroXCT-200; Carl Zeiss/Xradia, Inc.] that reaches the same resolution as synchrotron microscopy. We used serial transiliac bone biopsy specimens to examine the effect of macroanatomic bone type and estrogen status on osteocyte lacunar properties. These properties include lacunar size (volume, axes lengths of the ellipsoidal lacunar voids), distribution (density, average near-neighbor lacunar distance), and shape factors (sphericity ratio, average eigenvalues, degree of equancy, elongation, and flatness) in both cortical and trabecular bone tissue. The lacunar properties (volume, surface area, density, near-neighbor distance, etc.) and the shape factors (E1, L1, L2, degree of equancy, degree of elongation) were different between cortical and trabecular bone regardless of estrogen status. In cortical bone and trabecular nodes, the lacunar void volume and surface area were either smaller or tended to be smaller in postmenopausal as compared to premenopausal women. The void volume-to-bone volume ratio of cortical bone showed declining trends with estrogen loss. While there were differences between trabecular and cortical bone tissue, the lacunar void sphericity ratio for trabecular struts shows decreasing trends in postmenopausal women. These data suggest that using 3DXRM can provide new insight into osteocyte lacunar properties in transiliac bone biopsies from patients with various skeletal disease/conditions and pharmaceutical treatments.

Bone loss in the oestrogen-depleted rat is not exacerbated by sitagliptin, either alone or in combination with a thiazolidinedione.

Antihyperglycaemic therapy on bone was evaluated in the ovariectomized (OVX), non-diabetic adult rat. Animals were treated daily for 12 weeks with various doses of sitagliptin, pioglitazone, rosiglitazone, combinations of sitagliptin with pioglitazone or vehicle alone. Sitagliptin target engagement was confirmed by assessing inhibition of plasma dipeptidyl peptidase-4 (DPP-4) and oral glucose tolerance. Parameters related to bone health were evaluated in femur and vertebrae by dual-energy X-ray absorptiometry and histomorphometry. Bone mineral density (BMD) generally did not differ significantly between OVX-sitagliptin-treated animals and OVX-vehicle controls. In lumbar vertebrae, however, there was significantly less BMD loss with increasing sitagliptin dose. Thiazolidinedione (TZD) treatment generally resulted in lower BMD; OVX-TZD-treated (but not OVX-sitagliptin-treated) animals also had lessened cortical thickness in central femur and profoundly greater bone marrow adiposity in lumbar vertebrae. These findings support prior findings with TZDs and suggest a neutral or beneficial impact of DPP-4 inhibition on bone health.

Intermittent parathyroid hormone enhances the healing of medication-related osteonecrosis of the jaw lesions in rice rats.

Medication-related osteonecrosis of the jaw (MRONJ) is a potentially severe adverse event in patients treated with antiresorptives. Management of MRONJ is challenging, and no non-antibiotic, established medical treatment exists. Intermittent parathyroid hormone (iPTH) has been used off-label to treat MRONJ with favorable results. However, its medical efficacy has rarely been substantiated in clinical or preclinical experiments. Using a validated rice rat, infection-based model of MRONJ, we evaluated the effects of iPTH on established MRONJ. We hypothesize that iPTH contributes to MRONJ resolution by enhancing alveolar bone turnover and healing oral soft tissues. Eighty-four rice rats began a standard rodent chow diet at age 4 weeks to induce localized periodontitis. Rats were simultaneously randomized to receive saline (vehicle, VEH) or zoledronic acid (ZOL, 80 µg/kg IV) every 4 weeks. Oral exams were conducted bi-weekly to assign a gross quadrant grade (GQG, 0-4) to evaluate any lesion at the lingual aspect of the interdental space between maxillary molar (M2) and M3. 14 of 20 VEH-treated rice rats (70%) developed maxillary localized periodontitis with GQG 2-3 after 30 ± 10 weeks of saline. Additionally, 40 of 64 ZOL-treated rice rats with periodontitis developed MRONJ-like lesions after 30 ± 10 weeks of ZOL treatment. Rice rats with localized periodontitis or MRONJ-like lesions were treated with saline or iPTH (40 µg/kg) subcutaneously (SC) 3 times/week For 6 weeks until euthanasia. We found that iPTH -treated ZOL rats had a lower prevalence of MRONJ (p < 0.001), with lower severity extent of oral lesions (p = 0.003) and percentage of empty osteocyte lacunae (p < 0.001). ZOL rats treated with iPTH displayed a higher osteoblast surface (p < 0.001), more osteoblasts (p < 0.001), higher osteoclast surface (p < 0.001) and more osteoclasts (p = 0.002) at alveolar bone surfaces than ZOL/VEH rats. Greater gingival epithelial thickness and epithelial cell proliferation rate was found in the oral mucosa and gingiva of ZOL/PTH rats than in ZOL/VEH rats (p < 0.001). Our data suggest that iPTH is an efficacious non-operative medicinal therapy that accelerates oral healing and enhances the resolution of MRONJ lesions in ZOL-treated rice rats.

Enhanced alveolar bone loss in a model of non-invasive periodontitis in rice rats.

OBJECTIVE: The rice rat (Oryzomys palustris) develops periodontitis-like lesions when fed a diet rich in sucrose and casein (H-SC). We aimed to establish whether this model can accurately mimic the development of human periodontitis. MATERIALS AND METHODS: For this purpose, 28-day-old rice rats (15/group) were assigned to standard (STD) or H-SC diets and sacrificed after 6, 12, and 18 weeks. Jaws were processed for morphometric, histometric, histologic, histomorphometric, and micro-CT analyses. RESULTS: We found a progressive increase in horizontal alveolar bone loss (ABL) with age in maxillae of rats fed the STD diet as determined by morphometry. The H-SC diet exacerbated horizontal ABL at the palatal surface at 12 and 18 weeks. Furthermore, increased vertical ABL was detected in mandibles and maxillae of rats fed the H-SC diet for 12 and/or 18 weeks by histometry and micro-CT. Remarkably, the H-SC diet significantly increased bone remodeling at the interproximal alveolar bone of mandibles from rats fed for 6 weeks, but not in those fed for longer periods. CONCLUSIONS: These findings indicate that the H-SC diet induced a transient increase in alveolar bone remodeling, which is followed by ABL characteristic of moderate periodontitis.

Biologic and pathologic aspects of osteocytes in the setting of medication-related osteonecrosis of the jaw (MRONJ).

Medication-related osteonecrosis of the jaw (MRONJ) is a potentially severe, debilitating condition affecting patients with cancer and patients with osteoporosis who have been treated with powerful antiresorptives (pARs) or angiogenesis inhibitors (AgIs). Oral risk factors associated with the development of MRONJ include tooth extraction and inflammatory dental disease (e.g., periodontitis, periapical infection). In bone tissues, osteocytes play a bidirectional role in which they not only act as the "receiver" of systemic signals from blood vessels, such as hormones and drugs, or local signals from the mineralized matrix as it is deformed, but they also play a critical role as "transmitter" of signals to the cells that execute bone modeling and remodeling (osteoclasts, osteoblasts and lining cells). When the survival capacity of osteocytes is overwhelmed, they can die. Osteocyte death has been associated with several pathological conditions. Whereas the causes and mechanisms of osteocyte death have been studied in conditions like osteonecrosis of the femoral head (ONFH), few studies of the causes and mechanisms of osteocyte death have been done in MRONJ. The three forms of cell death that affect most of the different cells in the body (apoptosis, autophagy, and necrosis) have been recognized in osteocytes. Notably, necroptosis, a form of regulated cell death with "a necrotic cell death phenotype," has also been identified as a form of cell death in osteocytes under certain pathologic conditions. Improving the understanding of osteocyte death in MRONJ may be critical for preventing disease and developing treatment approaches. In this review, we intend to provide insight into the biology of osteocytes, cell death, in general, and osteocyte death, in particular, and discuss hypothetical mechanisms involved in osteocyte death associated with MRONJ.

Preclinical models of medication-related osteonecrosis of the jaw (MRONJ).

Medication-related osteonecrosis of the jaw (MRONJ) is a potentially severe adverse event affecting patients with cancer and patients with osteoporosis who have been treated with powerful antiresorptives (pARs) or angiogenesis inhibitors (AgIs). pARs, including nitrogen-containing bisphosphonates (N-BPs; e.g., zoledronic acid, alendronate) and anti-RANKL antibodies (e.g., denosumab), are used to manage bone metastases in patients with cancer or to prevent fragility fractures in patients with osteoporosis. Though significant advances have been made in understanding MRONJ, its pathophysiology is still not fully elucidated. Multiple species have been used in preclinical MRONJ research, including the rat, mouse, rice rat, rabbit, dog, sheep, and pig. Animal research has contributed immensely to advancing the MRONJ field, particularly, but not limited to, in developing models and investigating risk factors that were first observed in humans. MRONJ models have been developed using clinically relevant doses of systemic risk factors, like N-BPs, anti-RANKL antibodies, or AgIs. Specific local oral risk factors first noted in humans, including tooth extraction and inflammatory dental disease (e.g., periodontitis, periapical infection, etc.), were then added. Research in rodents, particularly the rat, and, to some extent, the mouse, across multiple laboratories, has contributed to establishing multiple relevant and complementary preclinical models. Models in larger species produced accurate clinical and histopathologic outcomes suggesting a potential role for confirming specific crucial findings from rodent research. We view the current state of animal models for MRONJ as good. The rodent models are now reliable enough to produce large numbers of MRONJ cases that could be applied in experiments testing treatment modalities. The course of MRONJ, including stage 0 MRONJ, is characterized well enough that basic studies of the molecular or enzyme-level findings in different MRONJ stages are possible. This review provides a current overview of the existing models of MRONJ, their more significant features and findings, and important instances of their application in preclinical research.

Preventing or controlling periodontitis reduces the occurrence of osteonecrosis of the jaw (ONJ) in rice rats (Oryzomys palustris).

INTRODUCTION: Osteonecrosis of the jaw (ONJ) is an adverse event that requires association of both systemic risk factors, such as powerful anti-resorptives (pARs; e.g. zoledronic acid [ZOL]), and local oral risk factors (e.g. tooth extraction, periodontitis). Whereas optimal oral health prior to initiate pARs is recognized as critically important for minimizing ONJ risk, the efficacy of preventive/maintenance measures in patients who are taking pARs is understudied. Rice rats fed a standard diet (STD), rich in insoluble fiber, develop localized periodontitis. STD-rats with localized periodontitis treated with ZOL for 18-24 wk develop ONJ. Hence, we hypothesized that controlling/preventing localized periodontitis in the ZOL-treated rats, reduces ONJ occurrence. METHODS: We used two approaches to attempt reducing periodontitis prevalence: 1) periodontal cleaning (PC); and 2) replacing the STD-diet with a nutritionally-equivalent diet high in soluble fiber (SF). 75 four-week-old male rats were weight-randomized into five groups (n = 15) in a 24-week experiment. Three groups ate the STD-diet and two the high SF-diet. STD-diet groups received intravenous (IV) vehicle (VEH) q4wks (STD + VEH), 80 µg/kg ZOL q4wks IV (STD + ZOL), or ZOL plus PC q2wks (STD + ZOL + PC). The SF-diet groups received VEH (SF + VEH) or ZOL (SF + ZOL). Jaws were processed for histopathology and evaluated for ONJ prevalence and tissue-level periodontitis. RESULTS: 1) 40% of STD + VEH rats developed maxillary localized periodontitis with no ONJ; 2) 50% of STD + ZOL rats developed ONJ; 3) 7% of STD + ZOL + PC rats developed ONJ (p < 0.01 vs. STD + ZOL); and 4) one SF + ZOL rat developed localized periodontitis, and no SF + VEH or SF + ZOL rats developed ONJ (p < 0.001 vs. STD + ZOL). CONCLUSIONS: 1) Periodontal cleaning in ZOL-treated rats decreases localized periodontitis severity and reduces ONJ prevalence; and 2) feeding a SF-diet to ZOL-treated rats reduces both incidence of localized periodontitis and ONJ. Our data indicates strong oral microbial community shifts according to oral health condition and trends in the shifts associated with diet.

Anti-vascular endothelial growth factor antibody monotherapy causes destructive advanced periodontitis in rice rats (Oryzomys palustris).

OBJECTIVE: Angiogenesis inhibitors (AgI) are commonly used in combination chemotherapy protocols to treat cancer, and have been linked to osteonecrosis of the jaw (ONJ). However, it is unknown if AgI therapy alone is sufficient to induce ONJ. We have previously established an ONJ model in rice rats with localized periodontitis that receive zoledronic acid (ZOL). The purpose of this study was to use this model to determine the role of anti-vascular endothelial growth factor A (anti-VEGF) antibody treatment of rice rats with localized maxillary periodontitis. We hypothesized that rice rats with localized maxillary periodontitis given anti-VEGF monotherapy will develop oral lesions that resemble ONJ, defined by exposed, necrotic alveolar bone. METHODS: At age 4 weeks, 45 male rice rats were randomized into three groups (n = 15): 1) VEH (saline), 2) ZOL (80 µg/kg body weight, intravenously once monthly), and 3) anti-VEGF (5 mg B20-4.1.1/kg body weight, subcutaneously twice weekly). After 24 weeks, rats were euthanized, jaws were excised and a high-resolution photograph of each quadrant was taken to assign a severity grade based on gross appearance. Jaws were then fixed, scanned by MicroCT, decalcified and sectioned for histopathologic and immunohistochemical analyses. RESULTS: 40-80% of the rats in the three groups developed gross oral lesions. 50% of ZOL rats developed ONJ. In contrast, 80% of the anti-VEGF rats developed destructive advanced periodontitis that was characterized by extreme alveolar bone loss and fibrosis. Anti-VEGF rats never developed exposed, necrotic bone. Furthermore, only anti-VEGF rats developed mild to severe mandibular periodontitis. Compared to VEH rats, more T-cells were found in periodontal lesions of anti-VEGF rats and more cells of the monocyte lineage were found in ONJ lesions of ZOL rats. CONCLUSIONS: Anti-VEGF monotherapy administered to a validated rodent model of ONJ caused a destructive advanced form of periodontitis that differed significantly from ONJ.

Inhibition of vascular endothelial growth factor in young adult mice causes low bone blood flow and bone strength with no effect on bone mass in trabecular regions.

OBJECTIVE: To determine the effect of an antibody to vascular endothelial growth factor (VEGF) on bone blood flow, bone strength, and bone mass in the young adult mouse. METHODS: Ten-week-old male BALB/cJ mice were body weight-randomized into either a rodent anti-VEGF monoclonal antibody (anti-VEGF, B20-4.1.1; 5 mg/kg 2×/wk.; n = 12) group or a vehicle (VEH; n = 12) group. After 42 days, mice were evaluated for bone blood flow at the distal femur by 18F-NaF-PET/CT and then necropsied. Samples from trabecular and cortical bone regions were evaluated for bone strength by mechanical testing, bone mass by peripheral quantitative computed tomography (pQCT), and micoarchitecture (MicroCT). Hydration of the whole femur was studied by proton nuclear magnetic resonance relaxometry (1H NMR). RESULTS: Distal femur blood flow was 43% lower in anti-VEGF mice than in VEH mice (p = 0.009). Ultimate load in the lumbar vertebral body was 25% lower in anti-VEGF than in VEH mice (p = 0.013). Bone mineral density (BMD) in the trabecular region of the proximal humeral metaphysis by pQCT, and bone volume fraction and volumetric BMD by MicroCT were the same in the two groups. Volume fraction of bound water (BW) of the whole femur was 14% lower in anti-VEGF than in VEH mice (p = 0.003). Finally, BW, but not cortical tissue mineral density, helped section modulus explain the variance in the ultimate moment experienced by the femur in three-point bending. CONCLUSION: Anti-VEGF caused low bone blood flow and bone strength in trabecular bone regions without influencing BMD and microarchitecture. Low bone strength was also associated with low bone hydration. These data suggest that bone blood flow is a novel bone property that affects bone quality.

Zoledronic acid increases the prevalence of medication-related osteonecrosis of the jaw in a dose dependent manner in rice rats (Oryzomys palustris) with localized periodontitis.

OBJECTIVE: Investigate role of dose/duration of zoledronic acid (ZOL), a powerful anti-resorptive (pAR), on prevalence of medication-related osteonecrosis of the jaw (MRONJ) in rice rats (Oryzomys palustris), a species with natural susceptibility to food impaction-induced localized periodontitis (FILP). We hypothesize that ZOL induces MRONJ lesions in rice rats with FILP, and that the prevalence of MRONJ rises with increasing dose and duration of ZOL treatment. METHODS: We performed a toxicology experiment with clinically-relevant doses of ZOL in female rats (N=230) fed standard (STD) rodent chow. At age 4weeks (baseline), 12 rats were necropsied. The rest were randomized into five groups that began to receive 0, 8, 20, 50 or 125µg/kg ZOL IV/q 4weeks. After 12, 18, 24 and 30weeks, subgroups (N=9-16) from each of the dose groups were necropsied. High-resolution macroscopic photos of all jaw quadrants were given a gross quadrant grade (GQG) (0-4 or MRONJ) that classified FILP lesion severity and determined presence of gross MRONJ. Quadrants with GQG≥1 were examined histopathologically. Logistic regression analysis (ZOL dose/duration) of MRONJ prevalence was completed. RESULTS: We found: 1) 75% of 0µg/kg ZOL rats developed FILP lesions; 2) baseline rats and rats treated with 0µg/kg ZOL had no MRONJ; 3) 29 gross MRONJ cases were identified; 4) all gross MRONJ cases were confirmed histopathologically by the observation of exposed necrotic bone, and 53 new cases were discovered (total=82); 5) ZOL dose (P<0.001), but not duration (P=0.326), was a significant predictor of MRONJ prevalence; 6) 13% prevalence of gross MRONJ among all rats, with 22% prevalence among rats exposed to ZOL oncologic doses (20-125µg/kg); 7) 38% prevalence of histopathologic MRONJ among all rats, with 73% prevalence among rats exposed to ZOL oncologic doses. CONCLUSIONS: This is the first experiment to show a dose response relationship between clinically relevant doses of ZOL and MRONJ prevalence.

Mechanism of action, pharmacokinetic and pharmacodynamic profile, and clinical applications of nitrogen-containing bisphosphonates.

Nitrogen-containing bisphosphonates (nBPs) are bone-specific agents that inhibit farnesyl diphosphate synthase. nBPs' strong affinity for bone, and not for other tissues, makes them potent inhibitors of bone resorption and bone remodeling activity, with limited potential for side-effects in non-skeletal tissues. Five nBPs are currently approved in the United States. The primary indications are for treatment of osteoporosis (alendronate, ibandronate, and risedronate) and treatment/prevention of skeletal-related events (SREs) in multiple myeloma and breast and prostate cancer patients (ibandronate, pamidronate, and zoledronic acid). nBPs are the most efficacious drugs available for these diseases, reducing osteoporotic fracture risk by 50-60% in persons with low bone mass or prior osteoporotic fracture, and SREs by one-third in cancer patients. The absorbed nBP dose for cancer patients is from seven to ten times that in osteoporosis patients. nBPs are unique in that they first exert profound pharmacodynamic effects long after their blood levels reach zero. Current pharmacokinetic studies indicate that approximately half of any nBP dose reaches the skeleton, with an early half-life of ten days, and a terminal half-life of about ten years. Practical study design limitations and theoretical considerations suggest that both the half-life and the amount of nBP retained in the skeletons of patients on long-term nBP therapy are substantially overestimated by extrapolation directly from current pharmacokinetic data. In fact, the amount of nBP being released from skeletal tissues of long-term-treated patients, particularly in osteoporosis patients, becomes insufficient to maintain full pharmacodynamic efficacy relatively soon after dosing is interrupted.

Age-related periodontitis and alveolar bone loss in rice rats.

OBJECTIVE: To characterize in rice rats: (a) periodontitis (PD) progress with feeding of standard laboratory rat chow (STD) during ages 4-80 weeks; and (b) PD progress with feeding of a high sucrose-casein (H-SC) diet during young adulthood. METHODS: One group (N=12) was euthanized at age 4 weeks (Baseline). Four groups (N=8-16) consumed a STD diet from baseline and were necropsied at ages 22, 30, 52, and 80 weeks. Three groups (N=10-16) consumed an H-SC diet from baseline. Two were necropsied at ages 22 and 30 weeks, respectively. The third switched to the STD diet at age 22 weeks and was necropsied at age 30 weeks. All mandibles/maxillae were assessed by histometry for degree of periodontal inflammation (PD Score), alveolar crest height (ACH, mm), and horizontal alveolar bone height (hABH, mm2). RESULTS: In STD diet rats aged ≥30 weeks, all endpoints were worse (P<0.05) than at Baseline. In H-SC diet rats aged ≥22 weeks, all endpoints were worse than at Baseline (P<0.05). At age 22 weeks, all endpoints were worse in the H-SC group than in the STD group (P<0.05). By age 30 weeks, the STD and H-SC groups did not differ. CONCLUSIONS: 1) STD diet fed rice rats develop moderate/severe PD by age 30 weeks; 2) an H-SC diet accelerates moderate/severe PD development; and 3) switching to a STD diet does not halt/reverse PD that was accelerated by an H-SC diet. These data further clarify use of the rice rat as a PD model.

Germ-line deletion involving the INK4 locus in familial proneness to melanoma and nervous system tumors.

Joint predisposition to malignant melanoma and nervous system tumors (NSTs) is a puzzle. Several melanoma susceptibility genes have been identified, including p16, a clustered tumor suppressor. However, the molecular bases of inherited proclivity to NSTs in the absence of a recognizable genetic syndrome are unknown. We analyzed two families with joint proneness to melanoma and NSTs in view of genetic linkage and identification of the causal molecular lesions. Highly informative linkage markers were used for segregation analyses of the predisposition alleles in the two pedigrees. Characterization of the molecular lesions required hemizygosity mapping based on microsatellite markers physically mapped to contigs of the 9p21 region and a Southern blot approach using several PCR-generated probes. Both families were found to be allelic and linked to p16 markers. In the family segregating the melanoma/NST syndrome, a large germ-line deletion ablated the whole p16, p19, and p15 gene cluster (or INK4 locus), whereas a more circumscribed molecular lesion disrupting p16 and p19 but leaving p15 unaltered segregated with the melanoma-astrocytoma syndrome (MIM 155755). Our results suggest that multiple cancer susceptibility in these two families ensues from contiguous tumor suppressor gene deletion. Indeed, known phenotypes associated with germ-line p16 mutations and an apparent correlation between the deletion span and tumor spectrum in the two families suggest a new model of cancer pathogenesis based on the inactivation of contiguous tumor suppressor genes, an alternative to the established pleiotropic effects of single-gene disruption.

Localization of common deletion regions on 1p and 19q in human gliomas and their association with histological subtype.

Allelic alterations of chromosomes 1 and 19 are frequent events in human diffuse gliomas and have recently proven to be strong predictors of chemotherapeutic response and prolonged survival in oligodendrogliomas (Cairncross et al., 1998; Smith et al., submitted). Using 115 human diffuse gliomas, we localized regions of common allelic loss on chromosomes 1 and 19 and assessed the association of these deletion intervals with glioma histological subtypes. Further, we evaluated the capacity of multiple modalities to detect these alterations, including loss of heterozygosity (LOH), fluorescence in situ hybridization (FISH), and comparative genomic hybridization (CGH). The correlation coefficients for detection of 1p and 19q alterations, respectively, between modalities were: 0.98 and 0.87 for LOH and FISH, 0.79 and 0.60 for LOH and CGH, and 0.79 and 0.53 for FISH and CGH. Minimal deletion regions were defined on 19q13.3 (D19S412-D19S596) and 1p (D1S468-D1S1612). Loss of the 1p36 region was found in 18% of astrocytomas (10/55) and in 73% (24/33) of oligodendrogliomas (P < 0.0001), and loss of the 19q13.3 region was found in 38% (21/55) of astrocytomas and 73% (24/33) of oligodendrogliomas (P = 0.0017). Loss of both regions was found in 11% (6/55) of astrocytomas and in 64% (21/33) of oligodendrogliomas (P < 0.0001). All gliomas with LOH on either 1p or 19q demonstrated loss of the corresponding FISH probe, 1p36 or 19q13.3, suggesting not only locations of putative tumor suppressor genes, but also a simple assay for assessment of 1p and 19q alterations as diagnostic and prognostic markers.

Alterations of chromosome arms 1p and 19q as predictors of survival in oligodendrogliomas, astrocytomas, and mixed oligoastrocytomas.

PURPOSE: A recent report suggests that alterations of chromosome arms 1p and 19q are associated with chemotherapeutic response and overall survival in anaplastic oligodendroglioma patients treated with procarbazine, lomustine, and vincristine chemotherapy. We set out to further clarify the diagnostic and prognostic implications of these alterations in a broader set of diffuse gliomas, including astrocytic neoplasms and low-grade oligodendrogliomas. PATIENTS AND METHODS: Fluorescence in situ hybridization (FISH) signals from DNA probes mapping to 1p and 19q common deletion regions were enumerated in 162 diffuse gliomas (79 astrocytomas, 52 oligodendrogliomas, and 31 mixed oligoastrocytomas), collected as part of an ongoing prospective investigation of CNS tumors. RESULTS: The oligodendroglial phenotype was highly associated with loss of 1p (P =.0002), loss of 19q (P <.0001), and combined loss of 1p and 19q (P <.0001). Combined loss of 1p and 19q was identified as a univariate predictor of prolonged overall survival among patients with pure oligodendroglioma (log-rank, P =.03) and remained a significant predictor after adjusting for the effects of patient age and tumor grade (P <.01). This favorable association was not evident in patients with astrocytoma or mixed oligoastrocytoma. CONCLUSION: Combined loss of 1p and 19q is a statistically significant predictor of prolonged survival in patients with pure oligodendroglioma, independent of tumor grade. Given the lack of this association in patients with astrocytic neoplasms and the previously demonstrated chemosensitivity of oligodendrogliomas, a combined approach of histologic and genotypic assessment could potentially improve existing strategies for patient stratification and management.

Phase II trial of primary chemotherapy followed by reduced-dose radiation for CNS germ cell tumors.

PURPOSE: A prospective phase II study was initiated to assess the response rate, survival, and late effects of treatment in patients with newly diagnosed CNS germ cell tumors (GCT), using etoposide plus cisplatin followed by radiation therapy prescribed by extent of disease, histology, and response to chemotherapy. PATIENTS AND METHODS: Seventeen patients aged 8 to 24 years with histologically proven CNS GCT received etoposide (100 mg/m2/d) plus cisplatin (20 mg/m2/d) daily for 5 days every 3 weeks for four cycles, followed by radiation therapy. Nine patients had germinomas; eight had mixed GCT. Four patients (three with germinomas and one with mixed GCT) presented with leptomeningeal dissemination. RESULTS: Radiographically, 14 of 17 patients were assessable for response; 11 patients experienced complete regression, and three had major partial regression before radiation. Six of seven assessable patients with elevated CSF levels of alpha-fetoprotein or betahuman chorionic gonadotropin had normalization with chemotherapy alone; all normalized with combined chemotherapy and radiation therapy. All 17 patients are alive without evidence of disease (median follow-up, 51 months). One patient developed a relapse in the spinal leptomeninges and was rendered free of disease with spinal radiation more than 5 years ago. One patient developed carotid stenosis requiring surgery. Thus far, only minimal long-term deterioration in neurocognitive function has been detected as a consequence of protocol treatment. CONCLUSION: Conventional-dose intravenous chemotherapy with etoposide and cisplatin can effect tumor regression in a high proportion of patients with CNS GCT, including those with leptomeningeal metastases. Acute and long-term toxicities are acceptable. Progression-free survival and overall survival are excellent.

Trabecular bone microarchitecture after alendronate treatment of osteoporotic women.

OBJECTIVE: To compare the microarchitecture of iliac crest trabecular bone from women treated for two to three years with alendronate versus that of women treated with placebo. RESEARCH DESIGN AND METHODS: Three-dimensional micro-computed tomography (micro-CT; resolution 20 microm) and two-dimensional histomorphometry (resolution 5-7 microm) were used to examine trabecular bone from single transilial biopsies obtained at the completion of clinical trials. MAIN OUTCOME MEASURES: Microarchitectural variables, including bone volume, trabecular number, trabecular thickness, and trabecular spacing in specimens from alendronate- and placebo-treated women were examined. Three-dimensional images of trabecular bone from both groups were constructed from CT images. Correlations among variables and between techniques were also calculated. RESULTS: Eighty-eight specimens were suitable for evaluation by both techniques. As measured by two-dimensional histomorphometry, bone volume fraction (as a proportion of total volume) and trabecular thickness were significantly greater in alendronate specimens, 17.1 +/- 5.5% vs. 13.4 +/- 5.5% (p = 0.0043) and 127 +/- 29 microm vs. 109 +/- 28 microm (p = 0.0090), respectively, and trabecular spacing was significantly smaller, 729 +/- 227 microm vs. 862 +/- 338 microm (p = 0.005). Micro-CT yielded similar findings: bone volume and trabecular number were significantly greater in alendronate specimens: 19.4 +/- 6.2% vs. 16.2 +/- 6.3% (p = 0.0412) and 1.46(+/-) 0.32 vs. 1.31(+/-) 0.33 per mm (p = 0.0346). Two-dimensional and micro-CT measured characteristics correlated strongly with one another, with Pearson product moment correlation coefficients ranging from 0.60 (for trabecular thickness) to 0.83 (for bone volume). CONCLUSIONS: Trabecular microarchitecture of the ilium, whether studied by two- or three-dimensional methods, is better (greater bone volume, greater trabecular thickness, decreased trabecular spacing) after alendronate treatment than after two to three years of treatment with placebo. Bone volume in a trabecular region is strongly correlated to its microarchitecture, suggesting that bone quantity predicts values for these microarchitectural endpoints.

Lack of germ-line mutations of CDK4, p16(INK4A), and p15(INK4B) in families with glioma.

Gliomas are tumors of the central nervous system that may be inherited in some patients. The gene(s) responsible for the clustering of gliomas in families have not yet been identified. Molecular studies of sporadic high-grade gliomas have revealed mutations or deletions of the genes encoding the protein kinase inhibitors p16(INK4A) and p15(INK4B) in a large proportion of tumors. Moreover, those tumors without deletions frequently display gene amplification and/or over-expression of mRNA encoding the protein kinase cdk4. We hypothesized that germ-line mutations in the p16(INK4A), p15(INK4B), or CDK4 genes might contribute to some cases of familial gliomas. To address this issue, we analyzed 36 kindreds with a predisposition to glial tumors. Genomic DNA from index members of these families was screened by PCR-single-strand conformational polymorphism analysis. We did not detect any functional mutations in the p16(INK4A), p15(INK4B), or CDK4 genes, although two individuals did have a previously described A140T polymorphism in p16(INK4A). Thus, despite the association between the sporadic forms of high-grade glioma and abnormalities of p16(INK4A), p15(INK4B), or CDK4, we found no evidence that germ-line mutations in the coding region of these three genes predispose to inherited glial tumors.

The health care status of the diabetic population as reflected by physician claims to a major insurer.

BACKGROUND: Conventional epidemiologic data suggest that diabetic patients use more health care resources than nondiabetic patients, yet overall health care use by diabetic individuals has never been fully quantitated. We took a new approach to this issue based on the actual economics of the provision of health care to diabetic insured individuals. METHODS: The claims records in the Mutual of Omaha Current Trends database, which contains information on more than 400,000 individuals, were surveyed to identify patients with diabetes and create the contrast population of nondiabetic patients by exclusion. International Classification of Diseases, Ninth Revision, Clinical Modification, codes and Physicians' Current Procedural Terminology, Fourth Edition, codes were used to determine all diagnoses recorded and all physician services rendered to the contrast populations. Age- and sex-adjusted comparisons were performed using Mantel-Haenszel procedures to determine an adjusted odds ratio (AOR). RESULTS: A total of 13,304 diabetic individuals and 388,053 nondiabetic individuals who received health care services from January 1, 1988, to January 1, 1989, were identified. Diabetic insured individuals constituted 3.1% of the overall insured population yet accounted for 8.3% of the charges (P < .01). Inpatient charges accounted for 81% of total diabetic charges but only 61.5% of total nondiabetic charges (P < .001). Diabetic insured individuals had twice as many physician office visits (AOR = 1.87; 95% confidence interval [CI], 1.79 to 1.96), with 2.5 times more physician hospital visits [AOR = 2.50; 95% CI, 2.27 to 2.75). However, the increases in physician care were not uniformly distributed across the diagnostic spectrum. The frequencies of well-established complications of diabetes, such as ischemic heart disease (AOR = 3.32; 95% CI, 3.12 to 3.53), peripheral vascular disease (AOR = 3.14; 95% CI, 2.79 to 3.53), and eye disease (AOR = 3.10; 95% CI, 2.94 to 3.27), were threefold higher in the diabetic group, with parallel increases in related medical services, such as cardiac catheterization (AOR = 3.02; 95% CI, 2.27 to 4.0), vascular surgery (AOR = 2.94; 95% CI, 2.64 to 3.27), and ophthalmologic procedures (AOR = 2.94; 95% CI, 2.72 to 3.18). In contrast, most diagnostic categories showed little or no increase. For example, the frequency of neoplasms (AOR = 1.11; 95% CI, 1.03 to 1.19) was minimally increased, and the associated procedural concomitants of therapeutic radiology (AOR = 0.81; 95% CI, 0.47 to 1.39) and chemotherapy (AOR = 0.98; 95% CI, 0.60 to 1.60) were not increased in the diabetic group. CONCLUSIONS: Our most important new finding is that diabetic patients have neither an elevated risk for a wide spectrum of diseases nor an increase in the receipt of physician services for diagnostic categories without increased risk, despite more frequent physician encounters. We provide real-world risk estimates that help in calculating the effect of offering specific insurance to diabetic individuals or including them in group health plans. The techniques we have developed to analyze computerized claims databases in this way may serve to better quantify the true impact of chronic diseases on the health care system.