PTHrP Levels Research Articles

SAT-491 Severe Hypercalcemia Secondary to Primary Skeletal Sarcoidosis

Introduction: Sarcoidosis is an autoimmune disorder that is characterized by non-caseating granulomas found throughout affected tissues. It has an incidence of 10-12 per 100,000 patients per year (2). Common clinical manifestations of sarcoidosis include hilar adenopathy, abnormally high angiotensin-converting enzyme (ACE), abnormally high vitamin D, and respiratory symptoms such as cough. While the lungs are commonly affected by sarcoidosis, extrapulmonary involvement occurs in over 30% of cases of sarcoidosis (1). The incidence of musculoskeletal involvement, specifically, is approximately 14%, however, most cases are asymptomatic (2). Case Presentation: A 79-year-old female with a past medical history of asthma, dementia, CAD, CKD, and HTN presents with asymptomatic hypercalcemia. She was directly admitted for hypercalcemia, AKI, and UTI and was started on Keflex and IV fluid therapy. Endocrinology, rheumatology, oncology, and nephrology were consulted. Over the course of her evaluation, further labs revealed: continued hypercalcemia: with a nadir of 13.5, elevated 1,25 vitamin D with a nadir of 100.0, and elevated ACE levels with a nadir of 125. Phosphorus, magnesium, PTH, and PTHrP levels were all within normal limits. The patient’s only complaint was dry mouth. Repeat physical examinations were stable and within normal limits other than increasing blood pressure, as high 172/70. She was started on dexamethasone to decrease conversion of Vitamin D to 1,25 hydroxyvitamin D. Throughout her stay, the patient had a negative NM parathyroid scan and a negative full body PET-CT. A CT chest w/o contrast revealed nonsp ecific ground-glass opacification with interval resolution of a small pulmonary nodule. Her diagnosis of sarcoidosis was only confirmed with a bone marrow biopsy of her right posterior iliac crest that revealed multiple non-necrotizing granulomas. Discussion: Atypical presentations of illnesses warrant exhaustive diagnostic workup. Although this patient’s laboratory values classically pointed towards a granulomatous process - elevated vitamin D and ACE levels - her diagnostic imaging studies, including full body PET-CT, were negative, steering away from a diagnosis of skeletal sarcoidosis. If it were not for a bone marrow biopsy, this patient would have potentially suffered greatly with the development of cystic bony degeneration and consequences such as fracture needing invasive orthopedic surgical intervention.

SAT-498 Use of Cinacalcet for PTHrP-Mediated Hypercalcemia of Malignancy in Penile Squamous Cell Carcinoma

Background: PTHrP-mediated hypercalcemia accounts for 80% of hypercalcemia of malignancy (HCM), and is traditionally treated with bisphosphonates or hemodialysis. We report a patient with penile squamous cell carcinoma (SCC) and bisphosphonate resistant PTHrP-mediated HCM that responded to calcimimetic therapy. Clinical Case: A 75-year-old man with metastatic penile SCC was admitted with altered sensorium. He had no prior history of hypercalcemia or fractures. His exam was significant for disorientation and hypovolemia. Labs revealed an elevated ionized calcium [iCa] of 1.65 mmol/L [0.95-1.32 mmol/L], phosphorus 2.6 mg/dl [2.5-4.6 mg/dL], magnesium 1.8 mg/dl [1.6-2.7 mg/dL], appropriately low intact PTH 8.1 pg/ml [8.0-85.0 pg/mL], 25-hydroxy vitamin D of 24 ng/ml [30-100 ng/mL], TSH 3.3 uIU/mL [0.350-4.940 uIU/mL], elevated PTHrP level 47 pg/ml [14-27 pg/mL], 1, 25-dihydroxy vitamin D 25 pg/ml [18 - 64 pg/mL], and serum immunofixation negative for paraproteins. Due to concomitant SIADH, intravenous (IV) fluids were used cautiously. He received IV zoledronic acid (ZA) 4mg on Day 2, following which iCa normalized in two days. One month later, the patient was readmitted with pneumonia. On Day 7, he was noted to have an elevated iCa of 1.4 mmol/L. On Day 8 of admission, he was given a second dose of IV ZA 4mg and iCa normalized 48 hours later. However, his hospital course was complicated by recurrences of hypercalcemia on day 14 (iCa 1.41 mmol/L) and day 24 (iCa 1.41 mmol/L) requiring 2 additional doses of IV ZA 4mg (Days 14 and 24) resulting in brief normalization of iCa. On day 26, iCa increased to 1.36 with further ZA unable to be administered due to worsening creatinine clearance. Use of IV fluids was limited due to worsening hyponatremia. Denosumab or calcitonin were not available in the inpatient formulary. Prior to considering hemodialysis, the patient was started on a trial of oral cinacalcet 30mg daily on Day 28. Within 24 hrs of initiation of cinacalcet, iCa normalized. On Day 40 hemodialysis was initiated. iCa increased to 1.36 on Day 43 requiring 60 mg of cinacalcet daily. Throughout the remainder of the patient’s hospitalization, further titration of cinacalcet was required with 90 mg twice daily prescribed on Day 54 resulting in normalization of iCa. On day 60, iCal was again found to be abnormal with a repeat PTHrP level of 1318 pg/ml. iCal levels continued to trend up until day 64, when the patient had a cardiac arrest and expired. Conclusion: Cinacalcet is an allosteric activator of the calcium sensing receptor. The mechanism of effect on PTHrP-mediated HCM is still under investigation, but in murine models, increased calcitonin secretion appears to play a role. The use of cinacalcet for HCM has been described in only four prior case reports. Further studies are needed to further elucidate the role of cinacalcet as a therapeutic option in HCM.

SAT-503 A Rare Case of Hypercalcemia Due to PTH Secreting Lung Adenocarcinoma

Introduction: Hypercalcemia can be present in up to 20% of malignancies. The majority are caused by humoral hypercalcemia of malignancy, mediated by secretion of PTHrP, typically by squamous cell carcinomas. Other etiologies of hypercalcemia in malignancy include local osteolysis, and increased production of 1,25(OH)2D. In rare cases, hypercalcemia can be caused by secretion of PTH by tumors. Clinical Case: A 60-year-old man with metastatic lung adenocarcinoma presented with altered mental status and was noted to have hypercalcemia to 19.2 mg/dL (reference range, 8.5-10.1). Imaging revealed a 12.0 cm right mediastinal soft tissue mass. He was initially treated with isotonic saline, calcitonin, and zolendronic acid. PTH was elevated to 594 pg/mL (reference range, 14-72), 25OHD was low at 13.2 ng/mL (reference range, 30-100), and PTHrP was normal at 22 pg/mL (reference range, 14-27). Serum calcium reached a nadir of 9.3 mg/dL (reference range, 8.5-10.1). During the course of hospitalization, serum calcium began to rise despite repeated infusions of zolendronic acid. Given inappropriately elevated PTH, hypercalcemia, and normal PTHrP, it was assessed that primary hyperparathyroidism may be the cause of hypercalcemia, and potentially treatable with parathyroid surgery. Endocrine surgery was consulted, and recommended a sestamibi scan, which failed to reveal parathyroid adenomas; however, there was significant uptake over the right upper lung region, left axillary region and bony structures, spatially concordant with the known malignancy. Calcium continued to rise, at which time the health care proxy decided against any further interventions. The patient expired on day 30. Conclusion: PTHrP causes hypercalcemia via similar mechanisms to PTH. In this patient, primary hyperparathyroidism was considered unlikely as the sestamibi scan did not reveal any evidence of parathyroid adenomas; instead, there was increased uptake over the patient’s lung mass, axillary lymph nodes, and metastatic bone lesions. There are known case reports of PTH secreting tumors, which were demonstrated on both sestamibi and immunohistochemistry for intact PTH on tumor tissue. PTH secreting tumors, whilst rare, should be considered in patients with elevated PTH, normal PTHrP levels, and no evidence of primary hyperparathyroidism. Sestamibi scan should be considered as a modality to help diagnose PTH secreting tumors if suspected.

PTHrP May Contribute to Cachexia Accompanying Multiple Myeloma Bone Disease

Background: Cachexia, an energy-wasting disorder of adipose and skeletal muscles, occurs commonly in advanced cancers and is a serious problem that interferes with response to treatment and affects quality of life. Treatment or cachexia is yet available. Chemotherapy and radiation may further worsen cachexia. Cachexia in cancer bone metastasis was described in breast and prostate cancers where cancer cells induce osteolysis and systemic muscle wasting, with PTHrP secreted by tumor cells as a driver of the process. PTHrP increases RANKL-induced osteolysis and stimulates browning of white fat, which in turn, induces skeletal muscle wasting via unknown secreted factor(s). Since MM cells express PTHrP, we asked whether PTHrP drives white to brown fat conversion in MM.Methods: First, we established an ex vivo organ co-culture assay (EVOCA) of neonatal mouse bone and cancer cells to study this conversion, using solid tumor osteolytic cell lines; PC3 prostate cancer cells and MDA231 breast cancer cells. GFP-expressing tumor cells engrafted onto the mouse calvariae causing lytic lesions within 7-14 days of culture. Bone (mouse) and tumor (human) gene expression was analyzed by RT-PCR using species-specific PCR primers, compared to RPL32 loading control. Tumor burden was measured by human RPL32. Mean gene changes between conditions were compared using paired t-tests. Tumor PTHrP and bone TRAP levels increased in tumor/bone co-culture compared to tumor alone and bone alone. Bone thermogenic genes UCP1 and DiO2 were increased in PC3 and MDA231 co-culture compared to bone alone (5.3 and 4.1 folds), respectively (p=0.05, and 0.04), supporting white to brown fat conversion. We then applied the EVOCA for MM studies.Results: EVOCA of MM cell lines 8226 and JJN3 caused similar histologic lytic lesions and TRAP induction of bones, similar to the PC3 and MDA231 findings. Both cell lines significantly induced bone UCP1 and DiO2, compared to bone alone. UCP1 and DiO2 induction by 8226 was 5.4 and 3.2 folds, respectively (p=0.02, and 0.04), while induction of bone UCP1 and DiO2 by JJN3 was 3.8 and 2.8 folds, respectively (p=0.05, and 0.03). Bone treated with recombinant PTHrP (positive control) strongly induced UCP1 and DiO2 (7.2 and 4.6 folds). EVOCA of MM cell lines were treated with available anti-myeloma agents and bisphosphonate. Bortezomib and lenalidomide dramatically decreased tumor growth and TRAP induction. Zoledronic strongly inhibited TRAP induction and marginally inhibited tumor growth. However, these agents had marginal effects on tumor-induced UCP1 and DiO2 upregulation. PTHrP neutralizing antibody partially blocked UCP1 and DiO2 induction by MM cells and had no effects on MM cell growth.To further evaluate for a direct contribution of PTHrP on fat browning, preadipocyte cell lines 3T3-L1 were differentiated into fat-laden adipocytes and treated with recombinant PTHrP, conditioned media (CM) from 8226 culture, CM from EVOCA of bone alone and CM from bone+8226 cells for 24 hours. UCP1 and DiO2 were upregulated by both PTHrP and CM of bone+8226 cells, compared to untreated 3T3-L1 cells, and 3T3-L1 treated with CM from bone alone. The UCP1 and DiO2 upregulation by CM of bone+8226 cells was modestly blocked by PTHrP neutralizing antibody.Conclusion: MM cells colonized to bones activate osteolysis, changes of fat genes compatible with fat browning; characterics of cachexia. Cachexia in MM may be attributed to other factors in addition to PTHrP. The backbone anti-myeloma drugs, Lenalidomide and Bortezomib, and the antiresorptive Zoledronic acid have limited effect on tumor-induced fat browning. Bone+MM coculture allows concurrent assessment of tumor, bone and fat changes, and can be applied to studies of novel agents to combat cachexia in myeloma. DisclosuresSuvannasankha:Glaxo Smith Klein: Research Funding; Janssen: Research Funding; Celgene: Honoraria; Amgen: Research Funding.

Association of PTHrP levels in CSF with Alzheimer’s disease biomarkers

BackgroundParathyroid hormone-related protein (PTHrP) is involved in intracellular calcium regulation, neural cell proliferation and synaptic transmission. To date, no studies have been performed to evaluate the potential of PTHrP concentrations in cerebrospinal fluid (CSF) as a biomarker of brain pathophysiology. In this study we evaluated the association between CSF concentrations of PTHrP with the core CSF biomarkers of Alzheimer’s disease (AD). MethodsPTHrP and calcium were analysed using validated mass spectrometry based methods in a set of CSF samples that tested positive (n = 45) and negative (n = 45) for the AD biomarkers, including total tau protein (T-tau), phosphorylated tau protein (P-tau) and amyloid-β 42 (Aβ42). The measured CSF concentrations of PTHrP and calcium (Ca) were evaluated for association with AD CSF biomarkers. ResultsPTHrP and Ca concentrations in CSF samples ranged between 25 and 137 pmol/L and 0.92–1.53 mmol/L, respectively. Higher concentrations of PTHrP were observed in association with increased concentrations of T-tau and P-tau in the AD and the control group; while a stronger correlation was observed in the control group (ρ = 0.6, p < 0.0001; and ρ = 0.72, p < 0.0001, for T-tau and P-tau, respectively). Negative correlation was observed between concentrations of PTHrP and Aβ42 in the AD group (ρ = 0.27, p = 0.015). A statistically significantly lower ratio Aβ42/PTHrP was observed in the AD group (p < 0.0001). ConclusionIn the current study, we observed an association of PTHrP concentrations with concentrations of clinically used CSF biomarkers of AD. Concentrations of PTHrP were positively correlated with concentrations of T-tau and P-tau, suggesting an association with neuronal secretion and function, which is reduced upon progression to AD pathology. Our data suggest potential utility of the Aβ42/PTHrP ratio in assessment of AD progression.

Abstract P3-05-05: PTHrP affects breast cancer initiation and progression through Stat5 signaling pathway

Abstract PTHrP affects mammary development and breast cancer. PTHrP is expressed in basal myoepithelial cells until late pregnancy, when it also becomes expressed in alveolar epithelial cells. During lactation, PTHrP is secreted into milk and into the circulation, and regulates systemic calcium and bone metabolism. Secretion of PTHrP by breast cancers stimulates bone resorption, promoting the skeletal metastases and/or development of hypercalcemia. Less is known about the contribution of PTHrP to development and/or progression of primary breast cancers. Recent GWAS reports have identified the PTHrP gene as a breast cancer susceptibility locus. Using data from the Cancer Genome Atlas (TCGA) Project and a Yale tissue microarray (YTMA49) we have found that increased breast tumor PTHrP expression predicts a more aggressive phenotype and increased mortality, signifying the clinical relevance of high tumor PTHrP levels. In an attempt to define how PTHrP affects breast cancer, we assessed the effects of PTHrP overexpression on the development of mammary tumors in mice. We developed a tetracyline-regulated, MMTV-driven transgenic model of PTHrP overexpression targeted to mammary epithelial cells (MMTV-rtTA;tetO-hPTHrP). Surprisingly, we found that PTHrP overexpression in luminal epithelial cells causes alveolar hyperplasia, secretory differentiation and milk production in virgin mice, associated with a lower number of luminal progenitor cells and basal stem cells upon FACS analysis. Additionally, Signal Transducer and Activator of Transcription 5 (Stat5) is highly activated in the setting of PTHrP-induced alveolar hyperplasisa. This activation is functionally relevant as evidenced by higher levels of β-Casein mRNA and protein, the transcription of which is regulated by activated Stat5 (pStat5). This effect of PTHrP was reversible upon withdrawal of doxycycline (Dox). Addition of Dox to primary mammary epithelial cell cultures from these mice recapitulated the findings. Knocking out PTHrP receptor (PTH1R) in these mice did not affect this phenotype, suggesting that PTHrP acts in a receptor-independent manner. Overexpression of PTHrP in MMTV-PyMT mice dramatically accelerated formation of mammary tumors reducing both latency of tumor formation and survival of the mice. It caused a higher rate of proliferation and a lower rate of apoptosis in vivo. All MMTV-rtTA;tetO-hPTHrP;MMTV-PyMT mice developed palpable tumors in all mammary glands within 3 weeks of age, became hypercalcemic and died before 4.5 weeks of age. These mice had higher levels of pStat5 and βCasein. Tumor cells cultured ex vivo and cell lines established from these tumors overexpressed PTHrP when stimulated with Dox. PTHrP overexpression, and not exogenous PTHrP, caused higher levels of proliferation, pStat5, and β-Casein. This PTHrP-induced activity of Stat5 was blocked by treatment of these cells with a pharmacological inhibitor of Jak2, a tyrosine kinase upstream of Stat5. Additionally, T47D cells transfected with PTHrP showed higher levels of pStat5, suggesting that PTHrP could activate Stat5 signaling regardless of the cell type. We show that PTHrP overexpression results in Stat5 activation and increased proliferation in breast cancer cells in a cell-autonomous fashion and independent of its receptor. Citation Format: Takyar FM, Boras-Granic K, Kim W, Dann P, Wysolmerski JJ. PTHrP affects breast cancer initiation and progression through Stat5 signaling pathway [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-05-05.

Calcium-Sensing Receptor Promotes Breast Cancer by Stimulating Intracrine Actions of Parathyroid Hormone-Related Protein.

Parathyroid hormone-related protein (PTHrP) contributes to the development and metastatic progression of breast cancer by promoting hypercalcemia, tumor growth, and osteolytic bone metastases, but it is not known how PTHrP is upregulated in breast tumors. Here we report a central role in this process for the calcium-sensing receptor, CaSR, which enables cellular responses to changes in extracellular calcium, through studies of CaSR-PTHrP interactions in the MMTV-PymT transgenic mouse model of breast cancer and in human breast cancer cells. CaSR activation stimulated PTHrP production by breast cancer cells in vitro and in vivo Tissue-specific disruption of the casr gene in mammary epithelial cells in MMTV-PymT mice reduced tumor PTHrP expression and inhibited tumor cell proliferation and tumor outgrowth. CaSR signaling promoted the proliferation of human breast cancer cell lines and tumor cells cultured from MMTV-PyMT mice. Further, CaSR activation inhibited cell death triggered by high extracellular concentrations of calcium. The actions of the CaSR appeared to be mediated by nuclear actions of PTHrP that decreased p27(kip1) levels and prevented nuclear accumulation of the proapoptotic factor apoptosis inducing factor. Taken together, our findings suggest that CaSR-PTHrP interactions might be a promising target for the development of therapeutic agents to limit tumor cell growth in bone metastases and in other microenvironments in which elevated calcium and/or PTHrP levels contribute to breast cancer progression. Cancer Res; 76(18); 5348-60. ©2016 AACR.

Serum PTHrP Predicts Weight Loss in Cancer Patients Independent of Hypercalcemia, Inflammation, and Tumor Burden.

Recent animal studies showed that tumor-derived PTHrP induced cancer cachexia by fat browning with increased energy expenditure; however, clinical evidence from human data is insufficient. We investigated whether serum PTHrP levels independently predicts weight loss (WL) in cancer patients. From a longitudinal observational cohort, body mass index (BMI) of patients with measured serum PTHrP levels (n = 624) was assessed (median follow-up of 327 d). Cox hazard models were used to examine the predictive value of PTHrP for WL defined by consensus definition (WL [consensus], percentage WL < -5% or percentage WL < -2% plus BMI < 20 kg/m(2)) and by BMI-adjusted grades (WL [BMI adjusted]). The overall risk of WL (consensus) was 34.4%. Compared with PTHrP-negative subjects, patients with higher PTHrP levels (PTHrP ≥ median 5.7 pmol/L) had more WL (percentage WL, -6.9% vs -1.1%, P = .010) at follow-up. A higher PTHrP level was associated with an increased loss of body weight (β = -2.73), muscle (β = -1.85), and fat (β = -2.52) after controlling for age, sex, and BMI. Kaplan-Meier analysis demonstrated that subjects with higher PTHrP had increased WL risk compared with lower PTHrP or PTHrP-negative groups (52.0% vs 38.9% vs 29.7%, P < .001). Serum PTHrP was independently associated with an increased WL risk (hazard ratio [HR]1.23, P = .005) adjusted for potent predictors including serum levels of calcium, C-reactive protein, albumin, cancer stage, and performance status of patients. Consistent results were observed when BMI-adjusted WL was applied. Serum PTHrP levels predicted cancer-associated WL independent of the presence of hypercalcemia, inflammation, tumor burden, and other comorbidities.

Co-expression of parathyroid hormone related protein and TGF-beta in breast cancer predicts poor survival outcome

BackgroundBetter methods to predict prognosis can play a supplementary role in administering individualized treatment for breast cancer patients. Altered expressions of PTHrP and TGF-β have been observed in various types of human cancers. The objective of the current study was to evaluate the association of PTHrP and TGF-β level with the clinicopathological features of the breast cancer patients.MethodsImmunohistochemistry was used to examine PTHrP and TGF-β protein expression in 497 cases of early breast cancer, and Kaplan-Meier method and COX’s Proportional Hazard Model were applied to the prognostic value of PTHrP and TGF-β expression.ResultsBoth over-expressed TGF-β and PTHrP were correlated with the tumor in larger size, higher proportion of axillary lymph node metastasis and later clinical stage. Additionally, the tumors with a high TGF-β level developed poor differentiation, and only TGF-β expression was associated with disease-free survival (DFS) of the breast cancer patients. Followed up for a median of 48 months, it was found that only the patients with negative TGF-β expression had longer DFS (P < 0.05, log-rank test). Nevertheless, those with higher PTHrP expression tended to show a higher rate of bone metastasis (67.6 % vs. 45.8 %, P = 0.019). In ER negative subgroup, those who developed PTHrP positive expression presented poor prognosis (P < 0.05, log-rank test). The patients with both positive TGF-β and PTHrP expression were significantly associated with the high risk of metastases. As indicated by Cox’s regression analysis, TGF-β expression and the high proportion of axillary lymph node metastasis served as significant independent predictors for breast cancer recurrence.ConclusionsTGF-β and PTHrP were confirmed to be involved in regulating the malignant progression in breast cancer, and PTHrP expression, to be associated with bone metastasis as a potential prognostic marker in ER negative breast cancer.

Abstract 1041: PTHrP(12-48) localization is intracellular and it inhibits cell proliferation and osteoclastogenesis

Abstract Background: PTHrP is produced by various osteotropic malignancies and promotes progression of bone metastasis. PTHrP(12-48) is a unique, proteolytically-derived fragment of full length PTHrP and is the first PTHrP fragment, identified in the serum of breast cancer patients. Serum PTHrP(12-48) levels in breast cancer patients, correlate with a high sensitivity and specificity with the presence of bone metastasis. The purpose of this study was to define the role of PTHrP(12-48) in the metastatic breast tumor microenvironment. PTHrP(12-48) activity at the cellular level, was examined by PTHrP(12-48) agonist/antagonist activity at the PTH1 receptor (PTH1R), examination of subcellular localization and the effects of PTHrP(12-48) on cell proliferation, survival and osteoclast differentiation. Methods: To test the agonist/antagonist activity of PTHrP(12-48), c-AMP and IP3 assays were performed in cells expressing the PTH1R, in the presence of potent PTH1R agonists (PTH 1-34) and antagonists (PTH7-34). Immunolocalization of PTHrP(12-48) was examined by immunohistochemistry (IHC) using a specific anti-PTHrP(12-48) antibody and by immunofluorescence in PTH1R expressing human cells and human peripheral blood mononuclear cell (PBMC)-derived osteoclasts, which lack PTH1R. Cellular proliferation was measured in PTH1R expressing and PTH1R lacking cells. In addition, the effect of PTHrP(12-48) on human osteoclast precursor survival and osteoclast differentiation was assessed in receptor activator of nuclear factor kappa beta ligand (RANKL) treated PBMC cultures. Results: The data demonstrate that PTHrP(12-48) is not an agonist or an antagonist of the PTH1R receptor. IHC demonstrated positive immunostaining in primary breast tumors and bone metastases, whereas normal breast tissue was negative. Immunofluorescence successfully identified the cytoplasmic localization of PTHrP(12-48), 90 minutes after addition of ligand in PTH1R expressing cells. In contrast, no cytoplasmic staining was observed in PTH1R lacking cells. In addition PTHrP(12-48) significantly inhibited serum-induced proliferation of PTH1R expressing cells, but not PTH1R lacking cells. Interestingly, although human osteoclast precursors lack PTH1R expression, PTHrP(12-48) treatment strongly inhibited the survival and proliferation of PBMCs as well as their differentiation into osteoclasts. Conclusions: PTHrP(12-48) is a circulating bone metastasis biomarker that is expressed by primary breast tumors as well as bone metastasis. PTHrP(12-48) does not interact productively with the PTH1R in terms of second messenger activation. However, treatment of cells with PTHrP(12-48) results in cellular uptake by PTH1R expressing cells, with a resultant inhibition of serum-induced cell proliferation and osteoclastogenesis. Citation Format: Varinder Kaur, Archana Kamalakar, Nisreen Akel, Kim Leitzel, Allan Lipton, Larry J. Suva. PTHrP(12-48) localization is intracellular and it inhibits cell proliferation and osteoclastogenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1041. doi:10.1158/1538-7445.AM2015-1041

PDE3A mutations cause autosomal dominant hypertension with brachydactyly.

Cardiovascular disease is the most common cause of death worldwide, and hypertension is the major risk factor. Mendelian hypertension elucidates mechanisms of blood pressure regulation. Here we report six missense mutations in PDE3A (encoding phosphodiesterase 3A) in six unrelated families with mendelian hypertension and brachydactyly type E (HTNB). The syndrome features brachydactyly type E (BDE), severe salt-independent but age-dependent hypertension, an increased fibroblast growth rate, neurovascular contact at the rostral-ventrolateral medulla, altered baroreflex blood pressure regulation and death from stroke before age 50 years when untreated. In vitro analyses of mesenchymal stem cell-derived vascular smooth muscle cells (VSMCs) and chondrocytes provided insights into molecular pathogenesis. The mutations increased protein kinase A-mediated PDE3A phosphorylation and resulted in gain of function, with increased cAMP-hydrolytic activity and enhanced cell proliferation. Levels of phosphorylated VASP were diminished, and PTHrP levels were dysregulated. We suggest that the identified PDE3A mutations cause the syndrome. VSMC-expressed PDE3A deserves scrutiny as a therapeutic target for the treatment of hypertension.

Anti-TNFα treatment decreases the previously increased serum Indian Hedgehog levels in patients with ankylosing spondylitis and affects the expression of functional Hedgehog pathway target genes

ObjectiveIndian Hedgehog (Ihh) is the ligand that activates the Hedgehog pathway (HH) in the skeleton—the main controller of endochondral ossification. We aimed at assessing serum levels of Ihh in patients with ankylosing spondylitis (AS) and the effect of serum from patients with AS on HH pathway activation. MethodsSerum Ihh levels were measured in 59 patients with AS, 70 patients with rheumatoid arthritis (RA), and 53 healthy subjects. The effect of serum from patients with AS on HH pathway activation was evaluated using an osteoblast-like cell line model. ResultsPatients with AS not on anti-TNFα treatment had significantly higher Ihh levels compared to patients with RA not on anti-TNFα treatment (mean ± SEM of OD: 0.370 ± 0.025 vs. 0.279 ± 0.026 for patients with AS and RA, respectively, p = 0.027) and healthy subjects (p = 0.031). Patients with AS on anti-TNFα treatment had significantly lower Ihh levels compared to patients with AS not on such treatment (p = 0.028). Patients with RA on anti-TNF treatment had higher levels of Ihh compared to patients not on such treatment (p = 0.013). PTHrP levels were similar in patients with RA, AS, and healthy subjects and were not affected by anti-TNFα treatment. We next assessed HH pathway activation in Saos2 cells following incubation with serum from AS patients prior to and following anti-TNF treatment. The HH pathway was downregulated following treatment. ConclusionsIhh levels are increased in patients with AS and decrease following anti-TNFα treatment; this finding may have pathogenic and clinical implications.

Rare Case of a Patient Presenting with Parathyroid Hormone-Related Peptide Mediated Hypercalcemia and IgG Heavy Chain Disease: Case Report and Review of Literature

IgG Heavy Chain Disease (γHCD) is a rare plasma cell disorder. Hypercalcemia related to plasma cell dyscrasias is related to non-PTHrP related mechanisms. Here we describe the first case of a patient with γHCD and PTHrP related hypercalcemia. Methods: Patient case derived from chart review from 2011 to 2015. Literature review performed searching PubMed 1968-current. Results: The patient was diagnosed with hypercalcemia with elevated PTHrP and exclusion of other etiologies of hypercalcemia. She was diagnosed with (γHCD) by M-spike 0.64 g/dL, IFE showing a broad band of IgG heavy chain, without associated light chains and severe depression of the non-mono-clonal IgG. Serum immunoglobulins demonstrated elevated IgG (2110 mg/dL), normal IgA (46 mg/dL) and decreased IgM (<21 mg/dL). Bone marrow biopsy showed 5% PCs, non-clonal by kappa/lambda, but exclusive for IgG by IHC, without any staining for IgA or IgM. The patient was started on therapy with improved hypercalcemia and PTHrP levels. Conclusions: This is the first reported case of γHCD presenting with PTHrP related hypercalcemia. Given that skeletal involvement is uncommon in γHCD, hypercalcemia secondary to γHCD may at times be a PTHrP driven phenomenon and we recommend that this test be ordered in such cases.

Patient With IgG Heavy Chain Disease Presents With Parathyroid Hormone-Related Peptide Mediated Hypercalcemia

BackgroundMultiple myeloma is associated with excessive tumor-induced, osteoclast-mediated bone destruction. Hypercalcemia remains the most frequent metabolic complication of myeloma in patients, and excessive osteolysis plays a major contributory role in its pathogenesis. Hypercalcemia caused by increased blood levels of PTHrP have been found in patients with solid tumors and are uncommon in patients with hematologic malignancies including multiple myeloma. Here we present a unique case of a rare variant of multiple myeloma, IgG heavy chain disease, presenting with PTHrP associated hypercalcemia. MethodsA retrospective chart review of one patient from 2011-2013 including clinical history, laboratory data, imaging, and pathology was performed. ResultsA 60 year old female with past medical history of stage IIA Hodgkin's lymphoma diagnosed in 1992, treated with total nodal radiation with recurrence in 1997 treated with 6 cycles of ABVD achieving complete remission. The patient was found to have hypercalcemia (11.0 mg/dL) and renal failure (1.29 mg/dL) in March 2011. Hypercalcemia workup revealed suppressed PTH (<3 pg/ml), elevated ionized calcium (6.1 mg/dL) elevated parathyroid related peptide (38pg/ml), normal vitamin D and ACE levels. Serum protein electrophoresis (SPEP) showed M-spike of 0.9 g/dL. Immunofixation electrophoresis (IFE) demonstrated an IgG monoclonal immunoglobulin without a corresponding light chain (figure 1). Free serum kappa and lamda light chains were within normal limits. Serum IgG was elevated (4678 mg/dL), normal IgA (127 mg/dL) and low IgM (39mg/dL). Also, Beta-2 microglobulin was elevated (14.3 mg/L). The patient was seen by hematology for monoclonal gammopathy and hypercalcemia and a solid tumor work up was recommended given that the most common the mechanism of hypercalcemia for multiple myeloma is osteoclast-driven and not PTHrP related. CT chest/abdomen/pelvis, bone scan, bone survey, mammography, pelvis ultrasound were negative. Bone marrow biopsy was performed November 2011 showing 10% plasma cells (PC) by CD138, non clonal by kappa/lambda. Patient was placed on zolendonic acid for hypercalcemia and her renal function and hypercalcemia continued to worsen over a period of months. A kidney biopsy was performed in April, 2012 and revealed acute and chronic tubulointerstitial nephritis with secondary glomerulosclerosis and mild interstitial fibrosis and tubular atrophy suggestive of sarcoidosis and the patient was placed on a course of prednisone with transient improvement in calcium and renal function. The patient presented in emergency room in April 2013 with altered mental status. She was found to have hypercalcemia (12.0 mg/dL), renal failure (1.55 mg/dL),). SPEP revealed M-spike 0.64 g/dl. IFE displayed a broad band of IgG heavy chain, without associated light chains and severe depression of the non-monoclonal IgG. Serum immunoglobulins demonstrated elevated IgG (2110 mg/dL), normal IgA (46 mg/dL) and decreased IgM (<21 mg/dL). Bone marrow biopsy showed 5% PCs (figure 2), non clonal by kappa/lambda- but exclusive for IgG by IHC, without any staining for IgA or IgM (figure 3). Cytogenetics were normal. Based on the constellation of findings and similarity to prior workup the patient was diagnosed with IgG heavy chain disease and therapy with cytoxan, dexamethasone, bortezomib was initiated. Calcium levels improved and she has recovered clinically; she is currently completing her second cycle of therapy. We plan to follow PTHrP levels along with routine paraprotein assessments. [Display omitted] [Display omitted] [Display omitted] ConclusionsPTHrP mediated hypercalcemia is rare in patients with multiple myeloma and the main mechanism of hypercalcemia in multiple myeloma is osteoclast-mediated bone destruction. This is the first reported case of IgG heavy chain disease presenting with PTHrP related hypercalcemia. Another critical point related to this case, is that kappa/lambda staining, the primary mechanism of determining plasma cell clonality, is not useful in exclusively heavy chain disease- and can, in such cases, obscure the diagnosis of this plasma cell malignancy. Disclosures:No relevant conflicts of interest to declare.

Hu/elav RNA-binding protein HuR regulates parathyroid hormone related peptide expression in human lung adenocarcinoma cells.

In 54 stage I and II human lung adenocarcinomas, HuR and PTHrP levels were positively correlated and the PTHrP-HuR status of the tumor was an independent prognostic marker of the clinical outcomes of patients. The possibility that HuR could upregulate PTHrP expression in lung adenocarcinoma was investigated by immunohistochemical, Western blot and RT-PCR analyses in HCC44 and DV90 human lung adenocarcinoma cell lines. In both cell lines, knockdown of HuR by specific siRNAs reduced PTHrP mRNAs and both cellular and secreted protein. Moreover, it inhibited cell growth and induced cell apoptosis, as revealed by the increase of caspase-3 activity. These effects were partially rescued by the addition of exogenous PTHrP (1-34). Analysis by actinomycin D assay revealed that in both cell lines HuR silencing produced a decrease of PTHrP mRNA half-life by about 70%. These findings add PTHrP to the list of lung cancer-associated genes, whose mRNA is stabilized by HuR.

The Abnormal Phenotypes of Cartilage and Bone in Calcium-Sensing Receptor Deficient Mice Are Dependent on the Actions of Calcium, Phosphorus, and PTH

Patients with neonatal severe hyperparathyroidism (NSHPT) are homozygous for the calcium-sensing receptor (CaR) mutation and have very high circulating PTH, abundant parathyroid hyperplasia, and severe life-threatening hypercalcemia. Mice with homozygous deletion of CaR mimic the syndrome of NSHPT. To determine effects of CaR deficiency on skeletal development and interactions between CaR and 1,25(OH)2D3 or PTH on calcium and skeletal homeostasis, we compared the skeletal phenotypes of homozygous CaR–deficient (CaR−/−) mice to those of double homozygous CaR– and 1α(OH)ase–deficient [CaR−/−1α(OH)ase−/−] mice or those of double homozygous CaR– and PTH–deficient [CaR−/−PTH−/−] mice at 2 weeks of age. Compared to wild-type littermates, CaR−/− mice had hypercalcemia, hypophosphatemia, hyperparathyroidism, and severe skeletal growth retardation. Chondrocyte proliferation and PTHrP expression in growth plates were reduced significantly, whereas trabecular volume, osteoblast number, osteocalcin-positive areas, expression of the ALP, type I collagen, osteocalcin genes, and serum ALP levels were increased significantly. Deletion of 1α(OH)ase in CaR−/− mice resulted in a longer lifespan, normocalcemia, lower serum phosphorus, greater elevation in PTH, slight improvement in skeletal growth with increased chondrocyte proliferation and PTHrP expression, and further increases in indices of osteoblastic bone formation. Deletion of PTH in CaR−/− mice resulted in rescue of early lethality, normocalcemia, increased serum phosphorus, undetectable serum PTH, normalization in skeletal growth with normal chondrocyte proliferation and enhanced PTHrP expression, and dramatic decreases in indices of osteoblastic bone formation. Our results indicate that reductions in hypercalcemia play a critical role in preventing the early lethality of CaR−/− mice and that defects in endochondral bone formation in CaR−/− mice result from effects of the marked elevation in serum calcium concentration and the decreases in serum phosphorus concentration and skeletal PTHrP levels, whereas the increased osteoblastic bone formation results from direct effects of PTH.

A Male Patient with Humoral Hypercalcemia of Malignancy (HHM) with Leukocytosis Caused by Cutaneous Squamous Cell Carcinoma Resulting from Recessive Dystrophic Epidermolysis Bullosa

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe skin disorder. Although the patients are at risk for cutaneous squamous cell carcinoma (SCC), no case of cutaneous SCC derived from RDEB with humoral hypercalcemia of malignancy (HHM) has been reported. We present the first case report of a male patient with HHM with leukocytosis caused by cutaneous SCC resulting from RDEB. A 20-yr-old Japanese male patient with RDEB; the diagnosis was confirmed by electron microscopic examination, suffered an intractable skin ulcer and hypercalcemia and leukocytosis. PTH-rP, SCC antigen and Granulocyte colony-stimulating factor (G-CSF) levels were elevated. The histological diagnosis of the skin lesion was made well-differentiated SCC. Immunohistochemical staining showed the expression of PTH-rP in atypical tumor cells. For the control of hypercalcemia before an amputation, we used zoledronate safely and could control the serum Ca concentration in the normal range. After the amputation of his right leg including SCC, leukocytosis improved immediately and PTH-rP in blood decreased to the normal range. One month after the amputation, local recurrence of cutaneous SCC and multiple lung metastases were observed. PTH-rP increased gradually associated with hypercalcemia. Although the patient reached an unfortunate turning point about 4 mo after the amputation, we propose that zoledronate is an effective and safe treatment for HHM with cardiorenal complications.

Increased PTHrP and Decreased Estrogens Alter Bone Turnover but Do Not Reproduce the Full Effects of Lactation on the Skeleton

During lactation, calcium is mobilized from the maternal skeleton to supply the breast for milk production. This results in rapid but fully reversible bone loss. Prior studies have suggested that PTHrP, secreted from the breast, and estrogen deficiency, due to suckling-induced central hypogonadism, combine to trigger bone resorption. To determine whether this combination was sufficient to explain bone loss during lactation, we raised PTHrP levels and decreased levels of estrogens in nulliparous mice. PTHrP was infused via osmotic minipumps and estrogens were decreased either by using leuprolide, a long-acting GnRH agonist, or by surgical ovariectomy (OVX). Bone mineral density declined by 23.2 ± 1.3% in the spine and 16.8 ± 1.9% in the femur over 10 d of lactation. This was accompanied by changes in trabecular architecture and an increase in both osteoblast and osteoclast numbers. OVX and PTHrP infusion both induced a modest decline in bone mineral density over 10 d, but leuprolide treatment did not. The combination of OVX and PTHrP was more effective than either treatment alone, but there was no interaction between PTHrP and leuprolide. None of the treatments reproduced the same degree of bone loss caused by lactation. However, both forms of estrogen deficiency led to an increase in osteoclasts, whereas infusion of PTHrP increased both osteoblasts and osteoclasts. Therefore, although the combination of PTHrP and estrogen deficiency contributes to bone loss, it is insufficient to reproduce the full response of the skeleton to lactation, suggesting that other factors also regulate bone metabolism during this period.

A cup half-full or half-empty? when PTHrP levels matter

A cup half-full or half-empty? when PTHrP levels matter

Association between calcium intake, parathormone levels and blood pressure during pregnancy.

Purpose: To evaluate the association between calcium intake from diet, calciotropic hormones (PTH, PTH-rp), vasoactive regulators (endothelin, nitric oxide) and blood pressure levels during pregnancy, birth and puerperium. Method: In a prospective study 149 healthy normotensive primigravidas were followed-up from 15 weeks of gestation to puerperium. Daily calcium intake, calciuria, PTH, PTH-rp, endothelin, nitrite-nitrate, and Holter Test were assessed. Linear regression models were performed to evaluate the association between calcium intake, blood pressure levels and the laboratory tests. Multivariate regression models were performed to control potential confounders. Results: A significant increase of calcium intake during pregnancy was observed (931±301 mg/day to 1,195±467 mg/day, p&lt; 0.001). Plasma PTH-rp, endothelin, and nitrite-nitrate levels did not change during pregnancy. Among the women 38 (25.4%) had low calcium intake (&lt; 800 mg/day) with a larger increase of systolic and diastolic blood pressure during pregnancy (p=0.04) birth (p=0.006) and puerperium (p=0.01). After adjusting for other factors the multivariate analyses showed statistical association between low calcium intake, high parathormone levels and high systolic blood pressure levels during pregnancy (p=0.002). Conclusion: Low calcium intake during pregnancy is associated with a larger increase of systolic blood pressure and high parathormone levels.