The mechanical environment of a cell, which profoundly influences cell behaviors, includes loading parameters such as magnitude, rate cycle number and duration. Time-varying, or dynamic, levels of load are generally associated with anabolic responses, while time invariant, or static, forces often increase catabolic pathophysiology. How cells differentiate between static and dynamic loading parameters is unclear. In mesenchymal stem cells (MSC), we found that static strain (SS) increased nuclear YAP at 3 h, whereas 200 cycles of dynamic strain (DS) did not. Differences in nuclear YAP were directed by phosphorylation of Hippo enzymes: SS promoted association of PP2Ac dephosphorylase with nuclear MST1/2. In contrast, DS activated Hippo signaling with phosphorylation of MST1/2 and YAP. Actin remodeling was differentially affected by DS and SS: DS generated thicker fibers with greater cross-sectional area and enhanced parallel alignment. In contrast, static strain reinforced the existing isotropic actin network by increasing fiber density and focal adhesion count. DS induced actin remodeling was associated with phosphorylation of both LATS1/2 and AMOT, which binds both actin and nuclear MST1/2. When LATS1/2 was inhibited, or AMOT was knocked down, nuclear PP2Ac increased and YAP was dephosphorylated. This suggests that DS induced actin remodeling provokes release of phosphorylated AMOT from actin binding sites, while SS induced actin reinforcement sequesters AMOT. In sum, our data shows that MSC are equipped to differentiate between load components, resulting in nuanced mechanical effects on the Hippo pathway.

Inflammasome initiates inflammation via the maturation of interleukin-1 beta (IL-1β). Periodontitis is a prevalent, male-biased disease characterized by inflammation-driven bone loss, yet the mechanism(s) of this sex bias is unknown. This study explored whether enhanced inflammasome represents a causal mechanism for this bias. Analyses of three separate human studies (>6,200 samples) show that males have significantly higher IL-1β in the gingival crevicular fluid than females during health and periodontitis. This pattern is experimentally reproduced with different versions of the ligature-induced periodontitis mouse model where males show greater IL-1β secretion than females. The inflammasome drives bone resorption in males but not females as revealed by analyses of inflammasome gene-deletion mice. Pharmacologic treatment with a caspase-1/4 inhibitor reduces inflammatory cell infiltration, dampens osteoclastogenesis signaling (via the receptor activator of nuclear factor-kappa B pathway), and prevents bone resorption in males but not females during experimental periodontitis. While ovariectomized females show no change in their nonresponsiveness to caspase-1/4 inhibition, orchiectomized males no longer respond to the inhibition, suggesting the importance of an intact male reproductive system in the mediation of this inhibition. Thus, our study identifies inflammasome activation as causal for male-biased experimental periodontitis and supports sex-stratified studies to foster future advancement of inflammasome therapeutics in periodontics.

AbstractPolymerized β-actin may provide a structural basis for chromatin accessibility and actin transport into the nucleus can guide mesenchymal stem cell (MSC) differentiation. Using MSC, we show that using CK666 to inhibit Arp2/3 directed secondary actin branching results in decreased nuclear actin structure, and significantly alters chromatin access measured with ATACseq at 24 h. The ATAC-seq results due to CK666 are distinct from those caused by cytochalasin D (CytoD), which enhances nuclear actin structure. In addition, nuclear visualization shows Arp2/3 inhibition decreases pericentric H3K9me3 marks. CytoD, alternatively, induces redistribution of H3K27me3 marks centrally. Such alterations in chromatin landscape are consistent with differential gene expression associated with distinctive differentiation patterns. Further, knockdown of the non-enzymatic monomeric actin binding protein, Arp4, leads to extensive chromatin unpacking, but only a modest increase in transcription, indicating an active role for actin-Arp4 in transcription. These data indicate that dynamic actin remodeling can regulate chromatin interactions.

Introduction: Longitudinal effect of diet-induced obesity on bone is uncertain. Prior work showed both no effect and a decrement in bone density or quality when obesity begins prior to skeletal maturity. We aimed to quantify long-term effects of obesity on bone and bone marrow adipose tissue (BMAT) in adulthood. Methods: Skeletally mature, female C57BL/6 mice (n = 70) aged 12 weeks were randomly allocated to low-fat diet (LFD; 10% kcal fat; n = 30) or high-fat diet (HFD; 60% kcal fat; n = 30), with analyses at 12, 15, 18, and 24 weeks (n = 10/group). Tibial microarchitecture was analyzed by µCT, and volumetric BMAT was quantified via 9.4T MRI/advanced image analysis. Histomorphometry of adipocytes and osteoclasts, and qPCR were performed. Results: Body weight and visceral white adipose tissue accumulated in response to HFD started in adulthood. Trabecular bone parameters declined with advancing experimental age. BV/TV declined 22% in LFD (p = 0.0001) and 17% in HFD (p = 0.0022) by 24 weeks. HFD failed to appreciably alter BV/TV and had negligible impact on other microarchitecture parameters. Both dietary intervention and age accounted for variance in BMAT, with regional differences: distal femoral BMAT was more responsive to diet, while proximal femoral BMAT was more attenuated by age. BMAT increased 60% in the distal metaphysis in HFD at 18 and 24 weeks (p = 0.0011). BMAT in the proximal femoral diaphysis, unchanged by diet, decreased 45% due to age (p = 0.0002). Marrow adipocyte size via histomorphometry supported MRI quantification. Osteoclast number did not differ between groups. Tibial qPCR showed attenuation of some adipose, metabolism, and bone genes. A regulator of fatty acid β-oxidation, cytochrome C (CYCS), was 500% more abundant in HFD bone (p < 0.0001; diet effect). CYCS also increased due to age, but to a lesser extent. HFD mildly increased OCN, TRAP, and SOST. Conclusions: Long-term high fat feeding after skeletal maturity, despite upregulation of visceral adiposity, body weight, and BMAT, failed to attenuate bone microarchitecture. In adulthood, we found aging to be a more potent regulator of microarchitecture than diet-induced obesity.

Abstract Disclosure: T.L. Cater: None. M. Styner: None. Atypical femur fractures (AFF), so-named due to an unusual diaphyseal location, have been linked to antiresorptives (AR), though this remains unclear. AFF case reports are vital due to a lack of certainty about pathogenic mediators and management. Case: A 52-yr-old-female presented with symptomatic T6/T7/T8 compression sustained while raking acorns. Ten years prior, bilateral knee/thigh pain developed; X-ray showed mild arthritis; bone scan noted slight activity in lateral femoral cortices with later X-ray read as negative for fracture; DEXA indicated lowest T of −1.5 in the spine. She was managed with PT and bracing. Medical history included hip dysplasia s/p casting, endometriosis, infertility, hypothyroidism, and 2 full-term NSVD in her late 30s. She worked as an administrator before becoming a stay-at-home mom and denied alcohol or tobacco use. Family history was notable for hip fracture (mother) and hypermobility termed as Ehlers-Danlos (son). After thoracic healing, at 53, she was referred to endocrinology; exam showed Ht 69 in (175 cm), Wt 185 lb (83 kg), normal dentition, mild thoracic kyphosis without tenderness. Labs indicated normal Ca, Mg, PO4, Creat, 25(OH)D, Alk Phos, and SPEP. She was current on cancer screening including mammograms. She declined osteoporosis therapy, wishing to work on lifestyle, yet, when she returned to our clinic at 55, after new compression (T11/T12/L1), she was amenable to begin abaloparatide. Despite the anabolic, new L2/L3 fracture arose and thigh pain advanced. Genetic Invitae OI/bone fragility panel was negative for causal variants in 46 genes. DEXA showed 20% improved spine BMD: 5-15% in other sites. After 2 years of abaloparatide and 4 months after one-time dose of denosumab, a new L4 deformity arose, and thigh pain remained. She reported legs were giving way under her, prompting X-rays, which were concerning for bilateral AFF (confirmed with bone scan, MRI). In lieu of a 2nd dose of denosumab, romosozumab was started and she was promptly scheduled for prophylactic, orthopedic femoral nailing. Conclusions: This bisphosphonate-naïve-female began experiencing symptoms of AFF >5 years prior to presentation, concomitant with spine fragility. Skeletal deterioration occurred in absence of, as well as contemporaneous with, osteoporosis therapies, anabolic as well as one dose of AR despite improved BMD via DEXA. Her personal history of hip dysplasia might supply clues about geometry that favors AFF as her family history. Notably, long-standing discomfort in the thighs persisted and progressed during anabolic therapy. More research is needed to understand AFF pathogenesis, and this might also shed light on other severe forms of early bone fragility, as demonstrated herein. Our case also highlights a multi-year, sub-clinical period in which fragility progresses until a clinical diagnosis is reached, as well as the clinical conundrum of AFF management. Presentation: Thursday, June 15, 2023

Exercise, typically beneficial for skeletal health, has not yet been studied in lipodystrophy, a condition characterized by paucity of white adipose tissue, with eventual diabetes, and steatosis. We applied a mouse model of global deficiency of Bscl2 (SEIPIN), required for lipid droplet formation. Male twelve-week-old B6 knockouts (KO) and wild type (WT) littermates were assigned six-weeks of voluntary, running exercise (E) versus non-exercise (N=5-8). KO weighed 14% less than WT (p=0.01) and exhibited an absence of epididymal adipose tissue; KO liver Plin1 via qPCR was 9-fold that of WT (p=0.04), consistent with steatosis. Bone marrow adipose tissue (BMAT), unlike white adipose, was measurable, although 40.5% lower in KO vs WT (p=0.0003) via 9.4T MRI/advanced image analysis. SEIPIN ablation’s most notable effect marrow adiposity was in the proximal femoral diaphysis (-56% KO vs WT, p=0.005), with relative preservation in KO-distal-femur. Bone via μCT was preserved in SEIPIN KO, though some quality parameters were attenuated. Running distance, speed, and time were comparable in KO and WT. Exercise reduced weight (-24% WT-E vs WT p<0.001) but not in KO. Notably, exercise increased trabecular BV/TV in both (+31%, KO-E vs KO, p=0.004; +14%, WT-E vs WT, p=0.006). The presence and distribution of BMAT in SEIPIN KO, though lower than WT, is unexpected and points to a uniqueness of this depot. That trabecular bone increases were achievable in both KO and WT, despite a difference in BMAT quantity/distribution, points to potential metabolic flexibility during exercise-induced skeletal anabolism.

All organisms exist within a physical space and respond to physical forces as part of daily life. In higher organisms, the skeleton is critical for locomotion in the physical environment, providing a carapace upon which the animal can move to accomplish functions necessary for living. As such, the skeleton has responded evolutionarily, and does in real-time, to physical stresses placed on it to ensure that its structure supports its function in the sea, in the air, and on dry land. In this article, we consider how those cells responsible for remodeling skeletal structure respond to mechanical force including load magnitude, frequency, and cyclicity, and how force rearranges cellular structure in turn. The effects of these forces to balance the mesenchymal stem cell supply of bone-forming osteoblasts and energy storing adipocytes are addressed. That this phenotypic switching is achieved at the level of both gene transactivation and alteration of structural epigenetic controls of gene expression is considered. Finally, as clinicians, we consider this information as it applies to a prescriptive for intelligent exercise.

Aging induces alterations in bone structure and strength through a multitude of processes, exacerbating common aging- related diseases like osteoporosis and osteoarthritis. Cellular hallmarks of aging are examined, as related to bone and the marrow microenvironment, and ways in which these might contribute to a variety of age-related perturbations in osteoblasts, osteocytes, marrow adipocytes, chondrocytes, osteoclasts, and their respective progenitors. Cellular senescence, stem cell exhaustion, mitochondrial dysfunction, epigenetic and intracellular communication changes are central pathways and recognized as associated and potentially causal in aging. We focus on these in musculoskeletal system and highlight knowledge gaps in the literature regarding cellular and tissue crosstalk in bone, cartilage, and the bone marrow niche. While senolytics have been utilized to target aging pathways, here we propose non-pharmacologic, exercise-based interventions as prospective “senolytics” against aging effects on the skeleton. Increased bone mass and delayed onset or progression of osteoporosis and osteoarthritis are some of the recognized benefits of regular exercise across the lifespan. Further investigation is needed to delineate how cellular indicators of aging manifest in bone and the marrow niche and how altered cellular and tissue crosstalk impact disease progression, as well as consideration of exercise as a therapeutic modality, as a means to enhance discovery of bone-targeted therapies.

Mesenchymal stem cells (MSC) respond to environmental forces with both cytoskeletal re-structuring and activation of protein chaperones of mechanical information, β-catenin and Yes-Associated Protein 1 (YAP1). To function, MSCs must differentiate between dynamic forces such as cyclic strains of extracellular matrix due to physical activity and static strains due to ECM stiffening. To delineate how MSCs recognize and respond differently to both force types, we compared effects of dynamic (200 cycles x 2%) and static (1 × 2% hold) strain on nuclear translocation of β-catenin and YAP1 at 3h after force application. Dynamic strain induced nuclear accumulation of β-catenin, and increased cytoskeletal actin structure and cell stiffness, but had no effect on nuclear YAP1 levels. Critically, both nuclear actin and nuclear stiffness increased along with dynamic strain-induced β-catenin transport. Augmentation of cytoskeletal structure using either static strain or lysophosphatidic acid (LPA) did not increase nuclear content of β–catenin or actin, but induced robust nuclear increase in YAP1. As actin binds β-catenin, we considered whether β-catenin, which lacks a nuclear localization signal, was dependent on actin to gain entry to the nucleus. Knockdown of cofilin-1 (Cfl1) or importin-9 (Ipo9), which co-mediate nuclear transfer of G-actin, prevented dynamic strain-mediated nuclear transfer of both β-catenin and actin. In sum, dynamic strain induction of actin re-structuring promotes nuclear transport of G-actin, concurrently supporting nuclear access of β-catenin via mechanisms utilized for actin transport. Thus, dynamic and static strain activate alternative mechanoresponses reflected by differences in the cellular distributions of actin, β-catenin and YAP1. Significance statement Cells integrate both static and dynamic mechanical signals through the actin cytoskeleton which is attached to the nuclear envelope, affecting nuclear transport of β-catenin and YAP1. Dynamic strain induces nuclear translocation of β-catenin, but not YAP1, while static strain causes nuclear translocation of YAP1, but not β-catenin. Importantly, nuclear transport of actin is induced by dynamic but not static force. Furthermore, nuclear import of β-catenin depends on cofilin/importin-9 dependent actin transport mechanisms. Thus the presence of β-catenin and YAP1 in the nucleus represent specific responses to regulatory mechanical signals.

Capable of generating excess catecholamines, untreated extra-adrenal paragangliomas (PGLs) result in severe cardiovascular morbidity and mortality. Increasingly, a hereditary basis can be identified to underlie PGLs, though such data are largely absent in populations of non-European descent. We present two patients with PGL, both exhibiting similar age, sex, and geographic ancestry. Our patients are unrelated, Kinyarwanda-speaking females from the Democratic Republic of the Congo. The first patient presented with lower extremity edema and poorly controlled hypertension and was found to have multifocal PGL in the abdomen and bladder, proven by biopsy and treated with surgical excision. Our second patient presented with palpitations, shortness of breath, headache, and hypertension, was found to have mediastinal PGL, and underwent surgical excision. Genetic testing was negative in both cases. The first patient has not shown recurrence based on active surveillance with imaging and biochemical testing. There is a concern for recurrence in the second patient, eight years after diagnosis, which is currently being investigated. Our second patient lived at a high altitude for most of her life, pointing toward a possible role of hypoxia in the pathogenesis of her tumor development. Our cases raise questions that require active inquiry regarding additional environmental and/or genetic factors that might predispose to PGLs in uncommon anatomic sites and in understudied, vulnerable populations.

Abstract Context The contribution of lumbar scoliosis to osteoporosis is unknown. Objective This work aimed to determine the prevalence and relationship of lumbar scoliosis to osteoporosis in aging women. Methods A cross-sectional analysis used dual-energy x-ray absorptiometry (DXA) scans of randomly selected groups of postmenopausal women (64-68, 74-78, and 84-88 years; N = 300 each) in a university teaching hospital from 2014 to 2019. Lumbar Cobb angle was tested for an association to femoral neck (FN), total hip (TH), and spine T score, age, weight, and ethnicity. Logistic regression tested an association between scoliosis (Cobb angle > 10°) and osteoporosis (T score ≤ –2.5). Available sequential DXA scans (N = 51) were analyzed for changes in Cobb angle using a linear mixed model of these longitudinal data. Results Osteoporosis and Cobb angle both increased with age: from 22% and 4.4 (SD = 7.8) respectively in 64- to 68-year-olds to 32.9% and to 9.7 (SD = 9.2) in women age 84 to 88 years. The prevalence of clinically significant scoliosis rose from 11.5% in the youngest group, to 27.3% and 39.4% in the age 74 to 78 and 84 to 88 cohorts, respectively. Cobb angle increased 0.7° per year of follow-up. After adjusting for covariates, there was no significant association between T scores at any site (TH, FN, or spine) and Cobb angle. Conclusion Based on screening DXAs, the incidence and degree of lumbar scoliosis increases significantly in women between age 65 and 85 years. There was no association between the incidence of lumbar scoliosis and FN bone density.

ABSTRACT Tumor‐induced osteomalacia (TIO) is a rare cause of impaired bone mineralization mediated by the osteocyte‐derived, phosphaturic hormone: fibroblast growth factor 23 (FGF23). The case is presented of a previously healthy 45‐year‐old man who developed fragility fractures at multiple sites (initially metatarsals, eventually ribs, hips, spine, scapula, and sacrum) resulting in rapid functional deterioration, weakness, and the inability to bear weight and ambulate without a walker. Workup for secondary causes of bone loss was negative except for mild hypogonadotropic hypogonadism with normal pituitary MRI and hypophosphatemia that persisted despite aggressive supplementation. Testosterone was initiated but discontinued 6 months later because of deep vein thrombosis and pulmonary embolism, likely provoked by his new sedentary state, in addition to smoking history and possibly testosterone usage. Serum FGF23 was nonelevated at 138 mRU/mL (44–215). A genetic panel for OI variants was negative for a causal mutation. At the age of 48, 3 years after his initial fracture, he was referred to our academic endocrine clinic. We ruled out additional mutations that lead to hypophosphatemic rickets, including phosphate‐regulating endopeptidase homolog, X‐linked. PET/CT looking for a potential TIO locus revealed uptake in the left suprapatellar recess. Biopsy was consistent with a phosphaturic mesenchymal tumor. FGF23 was repeated for a preoperative baseline and now found to be elevated at 289 mRU/mL. In retrospect, it is likely that the initial level was inappropriately elevated for the degree of hypophosphatemia. After resection, he experienced marked improvement in physical function, decreased pain, and resolution of renal phosphate wasting. The principals of establishing a robust clinical diagnosis of TIO should be emphasized, excluding other entities and avoiding pitfalls in the interpretation of laboratory testing. © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.

ABSTRACTUse of the selective estrogen receptor modulator Tamoxifen (TAM) is a mainstay to induce conditional expression of Cre recombinase in transgenic laboratory mice. To excise β‐cateninfl/fl in 28‐day‐old male and female Prrx1‐CreER/β‐cateninfl/fl mice (C57BL/6), we utilized TAM at 150 mg/kg; despite β‐catenin knockout in MSC, we found a significant increase in trabecular and cortical bone volume in all genders. Because TAM was similarly anabolic in KO and control mice, we investigated a dose effect on bone formation by treating wild‐type mice (WT C57BL/6, 4 weeks) with TAM (total dose 0, 20, 40, 200 mg/kg via four injections). TAM increased bone in a dose‐dependent manner analyzed by micro–computed tomography (μCT), which showed that, compared to control, 20 mg/kg TAM increased femoral bone volume fraction (bone volume/total volume [BV/TV]) (21.6% ± 1.5% to 33% ± 2.5%; 153%, p < 0.005). With TAM 40 mg/kg and 200 mg/kg, BV/TV increased to 48.1% ± 4.4% (223%, p < 0.0005) and 58% ± 3.8% (269%, p < 0.0001) respectively, compared to control. Osteoblast markers increased with 200 mg/kg TAM: Dlx5 (224%, p < 0.0001), Alp (166%, p < 0.0001), Bglap (223%, p < 0.0001), and Sp7 (228%, p < 0.0001). Osteoclasts per bone surface (Oc#/BS) nearly doubled at the lowest TAM dose (20 mg/kg), but decreased to <20% control with 200 mg/kg TAM. Our data establish that use of TAM at even very low doses to excise a floxed target in postnatal mice has profound effects on trabecular and cortical bone formation. As such, TAM treatment is a major confounder in the interpretation of bone phenotypes in conditional gene knockout mouse models. © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.

Abstract BACKGROUND: The phosphaturic, bone-derived hormone FGF23, mediates bone loss in TIO. Tumor resection typically results in skeletal healing and reversal of biochemical defects. Case: A 45-year-old Caucasian man with no past medical history presented with non-traumatic fractures of bilateral metatarsals. Over the subsequent 2 years, he sustained fragility stress fractures in bilateral femurs, eventually rendering him non-ambulatory; though he continued to work as a property manager in an office setting. History included 30 pack-years of smoking as well as osteochondrosarcoma in his mother. Labs showed a low phosphorus at 2.1 mg/dL [2.7–4.5], along with calcium 8.8 mg/dL [8.4–10.4], iPTH 48 pg/mL [12–88], and ALP of 155 [IU]/L [34–104]. 25-OH vitamin D and 1,25-(OH)2 vitamin D were 27 ng/mL [30–80] and 12 pg/mL [20–80], respectively. Free testosterone was 4.4 ng/mL [5–21] and LH 2.2 mIU/mL [3.0–10.0]. Other pituitary hormones and brain MRI were unremarkable. Vitamin D3, calcitriol, phosphate and testosterone were prescribed. Testosterone was discontinued 6 months later, after diagnosis with DVT/PE. Incidental rib fractures on CXR prompted a 3-phase 99Tc-MDP bone scan, revealing multiple sites of uptake: ribs, scapulae, sternum, thoracolumbar spine, sacrum, bilateral ankles and feet. DXA revealed T-scores of -2.8 in the spine and -1.9 in the femoral neck. Labs pointed to ongoing phosphate wasting, despite compliance with calcitriol 0.25 mcg, cholecalciferol 5000 IU and phosphorus 2250 mg in divided doses. Calculated TRP was 64% [>80%] and TmP/GFR 1.74 mg/dL [2.5–4.5], consistent with low phosphate reabsorption. Urine 24-hour calcium was 244.8 mg. Testing for causal mutations of hypophosphatemic rickets and osteogenesis imperfecta was negative. FGF23 was within the reference range at 138 RU/mL [LabCorp ELISA 44–215], interpreted as inappropriately normal given ongoing phosphate loss. This prompted a search for a suspected TIO locus. Two years after presentation, 18F-FDG-PET exhibited a hypermetabolic focus at the left suprapatellar recess. Biopsy was consistent with a mesenchymal tumor and chromogenic in situ hybridization (Mayo Clinic) was positive for FGF23 mRNA. After surgical resection of the 1.7 cm tumor, serum FGF23 declined to <50 RU/mL and was 90 RU/mL 3 weeks later [Mayo <180 RU/mL]. Calcitriol and phosphorus supplementation were discontinued. One year post-operatively phosphorus was 3.6 mg/dL [2.5–5.0], with a normal calcium, iPTH and 25-OH vitamin D. Lumbar spine T score improved to -1.0 (+46.43%). Conclusion: A high clinical index of suspicion is required for TIO in the setting of phosphaturic osteomalacia, particularly with a normal serum FGF23. Recently, similar microRNA profiles were noted in osteosarcomas and TIO1. This, along with our patient’s family history raises the question of a possible predisposition to skeletal neoplasms. 1. Green et al. Bone Reports, 2017.

Abstract Introduction: Capable of generating excess catecholamines, untreated extra-adrenal paragangliomas (PGL) result in severe cardiovascular morbidity and mortality. Increasingly, a hereditary basis can be identified to underlie PGLs, though such data is largely absent in non-Caucasian populations. Case 1: A 43 yr. old Kinyarwanda-speaking woman from DR Congo presented with left lower extremity edema and hypertension, with blood pressure of 154/86 while on spironolactone, HCTZ and furosemide. Ultrasound was negative for a DVT; abdominal CT revealed a 3 cm necrotic mass, inferior to the duodenum and abutting the IVC and aorta, as well as 2 bladder wall lesions. EUS-guided FNA revealed a keratin-negative neuroendocrine tumor. Urinary 24hr norepinephrine (NE) was high at 185 mcg [15–80]. Urinary 24hr normetanephrine (NM) was high at 1404 mcg [119-451; hypertensive <900]. MIBG scan confirmed avidity in the aortocaval mass. Despite lack of bladder uptake on MIBG, pathology similarly pointed to PGL. Surgery included excision of bladder, pelvic nodes, uterus, and aortocaval tumor. Post-op, urinary 24hr NE was 18 mcg and NM was 297 mcg, both normal. One year later, MIBG/SPECT and CT of the abdomen were negative for recurrence. A GeneDx panel of 12 PGL/PCC mutations was negative. Case 2: An unrelated 41 yr. old Kinyarwanda-speaking woman from Rwanda, with prior history of preeclampsia and multiple miscarriages, presented with palpitations, headaches and hypertension. Echo showed a 4 cm mass posterior to the left atrium; the mass was18F-FDG PET-avid. Video-assisted thoracoscopy was performed yet the tumor’s vascularity precluded a biopsy. Biopsy of mediastinal mass after performing thoracotomy was consistent with PGL. Plasma NM was high at 7.1 nmol/l (<0.90), consistent with PGL and she underwent complete removal of the tumor. Testing for SDHB mutation was negative. Symptoms resolved and antihypertensives were discontinued. Follow-up plasma NM was 0.55 nmol/l 1-year post op and remained normal for six years of follow-up. Discussion: Less than 10% of PGLs are known to involve the mediastinum or bladder (1). In familial PGL, the most commonly identified non-syndromic mutations involve SDHD, SDHAF2, SDHB, SDHD, SDHC, VHL, and MAX. Tumorigenesis in a sizable fraction of PGLs is not well understood. Conclusion: We present two cases of extra-adrenal PGL, both exhibiting similar age, sex and geographic ancestry. Our cases raise questions that require active investigation regarding additional environmental and/ or genetic factors which might predispose to PGLs in uncommon anatomic sites. References: (1) Erickson D et al. J Clin Endocrinol Metab, 2001 (2) Martins et al. Int J of Endocrinol, 2014