Humoral hypercalcemia of malignancy caused by parathyroid hormone-related peptide-secreting neuroendocrine tumors: Report of six cases

We report the clinical characteristics and management of six patients with metastatic gastroentero-pancreatic neuroendocrine tumor (NET) presenting with severe hypercalcemia due to elevation of parathyroid hormone-related protein (PTHrP). All patients had histological confirmation of NET, five well-differentiated and one poorly differentiated. In 5 patients, hypercalcemia developed after years after the initial diagnosis of NET. One patient presented with concomitant elevation of PTHrP and intact parathyroid hormone (PTH) in the setting of multiple endocrine neoplasia 1 (MEN1). In all the other cases, PTH levels were low or undetectable. Management of malignant hypercalcemia due to PTHrP-producing NET is challenging, and optimal therapy depends on the extent of metastatic disease and the grade of malignancy. Aggressive tumor cytoreduction in addition to the systemic treatment modalities is frequently used to control disease progression and endocrine symptoms. To our knowledge, this is the largest series to date of hypercalcemia mediated by PTHrP-secreting NET.     

Lung cancer associated with hypercalcemia induced by concurrently elevated parathyroid hormone and parathyroid hormone-related protein levels

In general, many cases of malignancy-associated hypercalcemia are due to HHM. In patients with humoral hypercalcemia of malignancy (HHM), it has been reported that plasma parathyroid hormone-related protein (PTHrP) and cyclic adenosine monophosphate (cAMP) levels were elevated, while plasma PTH and active vitamin D(3) levels were suppressed. Our patient showed hypercalcemia with a concurrent increase in plasma and tumor tissue PTHrP and PTH concentrations and also high cAMP and low 1-25(OH)(2)VD(3) levels in the plasma. These data suggest that the hypercalcemia exhibited by our patient was consistent with HHM due to lung cancer and its liver metastasis. Moreover, diagnostic imaging and autopsy findings showed no appreciable lesions of the parathyroid gland. In addition, histopathologic examination of the primary and metastatic tumors revealed the existence of PTH immunohistochemically stained with anti-PTH antibodies, suggesting an ectopic-PTH-producing lung tumor. From these data, our patient was diagnosed with a rare case of lung cancer, which produced both ectopic PTH and PTHrP.     

Circulating PTH and PTHrP levels before and after treatment of tumor induced hypercalcemia with Pamidronate Disodium (APD).

The effect of lowering ionized calcium on circulating parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP) was assessed in twenty patients with hypercalcemia of malignancy following treatment with Pamidronate Disodium. Ionized calcium levels fell rapidly in all treated patients. PTH concentrations were initially suppressed below normal in 18 patients, but rose from 0.48 +/- 0.42 pmol/L to 3.63 +/- 3.13 pmol/L (p less than 0.01) after treatment, reaching higher than normal values in some patients even in the presence of persistent hypercalcemia. PTHrP concentrations did not change significantly after treatment. These findings are consistent with an increased sensitivity of parathyroid tissue to changes in ionized calcium following prolonged exposure to hypercalcemia. Regulation of tumor secretion of PTHrP by calcium was not apparent within the range of calcium concentrations in this study.     

Production of parathyroid hormone-related protein by a rat parathyroid cell line

Parathyroid hormone-related protein (PTHrP), the peptide associated with humoral hypercalcemia of malignancy, has been identified in fetal and adult parathyroid glands. We here report a sub-clone of a rat parathyroid cell line which secretes a single peptide species corresponding in size to PTHrP(1-84). Biological activity of the secretion product was blocked by a specific antiserum against PTHrP, but not by parathyroid hormone (PTH) antiserum. Secretion of PTHrP by these cells was regulated by extracellular calcium in the physiological range. A single messenger RNA species for PTHrP was identified, though PTH mRNA could not be shown in these cells. Hybrid CAT genes containing 700-1000 bp of 5'-flanking DNA from the human PTH or PTHrP genes were transfected into these cells, and the PTHrP gene was expressed at 10-fold higher levels than the PTH gene. These cells thus provide a valuable model system for investigation expression of PTHrP in a non-transformed cell line.     

Historical Perspective and Evolutionary Origins of Parathyroid Hormone-Related Protein

After the discovery of parathyroid hormone-related protein (PTHrP) as the cause of the hypercalcemia of malignancy, it was found to be distributed widely in tissues, with its actions driving many physiologic and pathologic conditions. Its involvement in cancer extended to a contribution to the ability of cancer cells to promote bone resorption and establish as metastases. It was found to have multiple activities within the sequence, including a nuclear localizing sequence and a specific nuclear transport system. PTHrP and parathyroid hormone (PTH) appear to have arisen from a common ancestral gene and the comparative endocrinology and genomics studies focused on finding where the two genes appeared. PTHrP has been identified in bony and cartilaginous fish in the same tissues as in humans, indicating that PTHrP has fundamental and basic physiological roles in all vertebrates. PTHrP has been localized in fish neoplasms suggesting that PTHrP’s role in tumor formation is a conserved role from at least fish to humans. Interestingly, PTH has been identified in both bony and cartilaginous fish even though they lack a parathyroid gland and indicate that PTH’s evolutionary history is much longer too. So the point where PTHrP and PTH were duplicated is still unknown. A comparison among the analogous human, mouse, chicken, xenopus, and fugu sequences of the PTHrP gene demonstrates elements of conservation. When coupled with human Encode data and knowledge of interspecies gene structure, it offers rudimentary insights into function and underwrites hypotheses on physiology.     

Gastric cancer associated with overexpression of parathyroid hormone-related peptide (PTHrP) and PTH/PTHrP receptor in relation to tumor progression

Parathyroid hormone-related peptide (PTHrP) is involved in cell proliferation in both neoplastic and non-neoplastic tissues. We describe an autopsy case of gastric cancer in a patient who showed serum hypercalcemia and overexpression of PTHrP and PTH/PTHrP receptor in the metastatic tumor cells. The primary gastric tumor was poorly differentiated adenocarcinoma, and multiple metastases were present in the bone, multiple visceral organs, peritoneum, and lymph nodes. PTHrP and its mRNA were detected only in the metastatic tumor cells, but not in primary gastric tumor. PTH/PTHrP receptor was also demonstrated immunohistologically in metastatic tumor cells. This case suggests that the expression of PTHrP is related to tumor progression and the poor prognosis in tumors associated with humoral hypercalcemia.     

The Role of PTHrP in Skeletal Metastases and Hypercalcemia of Malignancy

Since its discovery as the principal mediator of humoral hypercalcemia of malignancy, parathyroid hormone-related protein (PTHrP) has emerged as a key player in skeletal complications associated with solid tumor metastasis to bone. In addition to functioning as an endocrine factor, this pleiotropic peptide mediates its actions locally on tumor and stromal cells in paracrine, autocrine, and intracrine fashion when cancer metastasizes to the bone compartment. Multiple splice variants and newly described PTHrP fragments confer diverse functions to PTHrP that extend beyond binding to its common receptor with parathyroid hormone. Here, we summarize the causal role of PTHrP in humoral hypercalcemia of malignancy and its local involvement in the progression of osteolytic and osteoblastic cancer bone metastases. Clinical and preclinical findings describing how PTHrP regulates tumor and stromal cell interactions are summarized, with emphasis on emerging evidence of PTHrP’s role in tumorigenesis and cancer cell proliferation. Finally, we examine therapeutic opportunities and limitations to targeting PTHrP directly and indirectly in cancer patients.     

A case of myeloma with hypercalcemia caused by high serum concentrations of both parathyroid hormone-related peptide (PTHrP) and macrophage inflammatory protein-1α (MIP-1α)

A 62-year-old woman was admitted with dry mouth, general fatigue, and severe back pain. Biochemistry examination showed extreme hypercalcemia (21.2 mg/dL). Bone marrow examination was negative, but needle biopsy of a metastatic lung tumor revealed abnormal plasma cells; thus, multiple myeloma stage III-A was finally diagnosed. Serum concentrations of both parathyroid hormone-related peptide (PTHrP) and macrophage inflammatory protein-1α (MIP-1α) were markedly elevated (PTHrP 7.2 pmol/L, normal <1.1 pmol/L; MIP-1α 84.9 pg/mL, normal <46.9 pg/mL). Her myeloma appeared to have simultaneously caused two mechanisms producing hypercalcemia: humoral hypercalcemia of malignancy (HHM) by PTHrP and local osteolytic hypercalcemia (LOH) by MIP-1α. Therefore, the combination of two calcium-modulating abnormalities likely aggravated her hypercalcemia.     

Primary hyperparathyroidism

Primary hyperparathyroidism is the third most frequent endocrine disorder. The condition required for diagnosis is inappropriately elevated secretion of parathyroid hormone (PTH) with respect to calcemia. Most often, the disease is due to a parathyroid adenoma, i.e. a monoclonal benign parathyroid tumor, less often to a parathyroid hyperplasia. The main tumorogenic mechanisms currently proposed are a DNA rearrangement in the PTH locus (transposition of the PTH promoter upstream to Cyclin D1/PRAD 1 gene) and a mutation of the gene responsible for multiple endocrine neoplasia type I. The clinical presentation has strikingly evolved towards a milder, asymptomatic form, frequently diagnosed on systematic screenings. Though the mechanism of hypercalcemia is better understood, several hypothesis are still being considered about the regulation of tumoral PTH secretion: the role of the expression of calcium-receptor in parathyroid gland cells, vitamin D receptor and estrogen receptor polymorphisms, etc. Surgery is still advised for symptomatic forms of the disease, either because of a bone involvement, or because of an evolutive nephrolithiasis. In the near future, the new calcium-receptor agonists could be a relevant therapeutic approach.     

Expression of parathyroid hormone-related protein in abnormal human parathyroids

The expression of parathyroid hormone-related protein (PTHrP) in abnormal human parathyroids was investigated. Northern blot analysis of RNA extracted from human benign parathyroid adenomata (n = 4) revealed multiple PTHrP mRNA species ranging in size from 1.8 to 4 kb. The relative abundance of PTHrP mRNA expressed in two of the adenomata was similar to that of a tumour (DAF) associated with humoral hypercalcaemia of malignancy, whereas PTHrP mRNA was of low abundance in a third and was undetectable in the fourth. PTHrP-like immunoreactivity was detected in extracts of abnormal parathyroid tissue (benign adenoma (n = 7), hyperplasia (n = 5) and parathyroid carcinoma (n = 2] using a sensitive specific two-site immunoradiometric assay for human (h) PTHrP(1-86) and a radioimmunoassay for hPTHrP(1-34). Ratios of hPTHrP(1-86)- and hPTHrP(1-34)-like immunoreactivities relative to hPTH(1-84)-like immunoreactivity in the parathyroid tissue extracts were, on average, less than 1%. PTHrP bioactivity in the extracts could not be distinguished from that of PTH, by an osteosarcoma cell bioassay. We conclude that, despite reports of over-expression of PTHrP mRNA in parathyroid adenomata, the potential contribution of PTHrP to the total PTH-like activity of adenomata and other abnormal parathyroid tissue may be insignificant relative to PTH.     

Severe growth retardation and early lethality in mice lacking the nuclear localization sequence and C-terminus of PTH-related protein

Parathyroid hormone (PTH) plays a central role in the regulation of serum calcium and phosphorus homeostasis, while parathyroid hormone-related protein (PTHrP) has important developmental roles. Both peptides signal through the same G protein-coupled receptor, the PTH/PTHrP or PTH type 1 receptor (PTH1R). PTHrP, normally a secreted protein, also contains a nuclear localization signal (NLS) that in vitro imparts functionality to the protein at the level of the nucleus. We investigated this functionality in vivo by introducing a premature termination codon in Pthrp in ES cells and generating mice that express PTHrP (1–84), a truncated form of the protein that is missing the NLS and the C-terminal region of the protein but can still signal through its cell surface receptor. Mice homozygous for the knock-in mutation (Pthrp KI) displayed retarded growth, early senescence, and malnutrition leading postnatally to their rapid demise. Decreased cellular proliferative capacity and increased apoptosis in multiple tissues including bone and bone marrow cells were associated with altered expression and subcellular distribution of the senescence-associated tumor suppressor proteins p16^INK4a and p21 and the oncogenes Cyclin D, pRb, and Bmi-1. These findings provide in vivo experimental proof that substantiates the biologic relevance of the NLS and C-terminal portion of PTHrP, a polypeptide ligand that signals mainly via a cell surface G protein-coupled receptor.     

Parathyroid Hormone-Related Protein Expression in Human Gastric Adenocarcinomas Not Associated with Hypercalcemia

Objective: Our evaluation of a patient with a parathyroid hormone-related protein (PTHrP)-producing gastric adenocarcinoma and hypercalcemia prompted us to study the expression of PTHrP in 13 additional patients with gastric cancer and in 10 control cases. Our objective was to investigate by immunohistology the expression of PTHrP in gastric cancer.
Methods: Immunohistology studies were conducted with two murine monoclonal antibodies to synthetic peptides of human PTHrP, 9H7, and 8BI2. The 9H7 antibody was raised to the carboxyterminal amino acid fragment (109-141) of PTHrP, and the 8B12 antibody was raised to the ammo-terminal amino acid fragment (1-34) of PTHrP.
Results: Paraffin-embedded tumor specimens from 13 of 14 cases of gastric adenocarcinoma stained positively with the antibody to the carboxy terminus of the PTHrP peptide, but none stained positively with the antibody directed against the amino terminus. None of 10 control cases stained positively with either antibody. The staining was predominately evident in the cytoplasm of the tumor cells. Except for the sentinel case, none of the other patients with gastric adenocarcinoma had hypercalcemia. Thus, gastric adenocarcinoma seems to rarely result in systemic hypercalcemia.
Conclusions: Our studies demonstrated that abnormal PTHrP production can occur in malignant cells without producing hypercalcemia. PTHrP may play a role in the pathogenesis of gastric adenocarcinoma that is independent of its hypercalcemic effects. PTHrP measurements may be clinically valuable in patients with cancer who are not hypercalcemic.     

Parathyroid hormone-related protein in adult T-cell leukemia-lymphoma

OBJECTIVE: To determine whether parathyroid hormone-related protein is synthesized and secreted by the tumor cells of patients with adult T-cell leukemia-lymphoma. DESIGN AND PATIENTS: Convenience sample of three patients with adult T-cell leukemia-lymphoma. Two patients developed hypercalcemia, and one patient was normocalcemic. SETTING: Inpatient facilities at two university-affiliated medical centers. INTERVENTION: All patients had a lymph node biopsy. In addition, samples of ascitic or pleural fluid, or both, were obtained from these patients. MEASUREMENTS AND RESULTS: Using RNA blot analysis, we showed that parathyroid hormone-related protein (PTHrP) messenger RNAs (mRNAs) were constitutively expressed in the tumor cells from all patients. Cyclic adenosine monophosphate (cAMP) production-stimulating activity, assessed using osteoblast-like UMR106 cells, was demonstrated in the pleural and ascitic fluids from the two patients who developed hypercalcemia. The elution profiles of the cAMP production-stimulating activity in the ascitic fluid extracts were very similar to those of the tumor extracts from hypercalcemic nude rats that had been implanted with a human cancer tumor. CONCLUSIONS: Parathyroid hormone-related protein is produced by tumor cells in adult T-cell leukemia-lymphoma, which may be an important factor in the development of hypercalcemia in the patients with this disease. However, the development of hypercalcemia may depend on other factors such as the number of tumor cells, access of the protein into systemic circulation, and the presence of some additional substances.     

The Role of PTHrP in Regulating Mineral Metabolism During Pregnancy,Lactation, and Fetal/Neonatal Development

Parathyroid hormone-related protein (PTHrP) was originally identified as the cause of humoral hypercalcemia of malignancy (HHM), a condition that resembles primary hyperparathyroidism and the effects of excess parathyroid hormone (PTH). But HHM is an unusual situation because PTHrP is normally undetectable in the circulation of the child or adult. Instead, most of PTHrP’s actions are now understood to be paracrine or autocrine, and not humoral. However, PTHrP is present in the circulation at measurable levels during fetal development, pregnancy, and lactation. During these time periods, PTHrP has humoral actions that regulate mineral and bone homeostasis independently of PTH. In fact, the existence of PTHrP was also predicted by the characteristic pattern of serum chemistries and PTH in cord blood of normal newborns, and by the normalization of calcium metabolism that temporarily occurs in hypoparathyroid women who breast-feed. This article reviews our present understanding about PTHrP’s role to control mineral and bone metabolism during pregnancy, lactation, and fetal development. Excess PTHrP can also be produced by the placenta or the breasts during pregnancy, or by the breasts during lactation, and in both situations it can lead to hypercalcemia and other clinical features that are indistinguishable from HHM. The highest concentrations of PTHrP are found in milk, and recent evidence indicates that milk-based PTHrP may reduce mineral accretion by the newborn skeleton, but whether it does this through local actions in the neonatal gut or after absorption into the neonatal circulation is unknown.     

Parathyroid hormone-related protein a peptide of diverse physiologic functions.

Parathyroid hormone-related protein (PTHrP) is the factor responsible for the syndrome of humoral hypercalcemia of malignancy. PTHrP is produced by a multitude o f normal as well as malignant cells, and exerts both classic parathyroid hormone (PTH)-like and PTH-unlike effects. The molecular cloning of the PTHrP gene, and the subsequent recognition of its widespread expression in normal tissues under normal physiologic conditions, has prompted intense inquiry into its biologic function. PTHrP appears to act in an autocrine or paracrine fashion in (a) normal embryogenesis and neonatal development, (b) cellular growth and differentiation, (c) reproduction and lactation, (d) epithelial calcium transport, and (e) smooth muscle relaxation. These five key emerging physiologic roles of PTHrP are the focus of this review.     

Hypercalcemia secondary to leprosy

Despite the high prevalence of leprosy in undeveloped countries, hypercalcemia secondary to leprosy is rare. One of most important mechanisms responsible for this disorder seems to be high serum concentrations of 1,25-dihydroxyvitamin D produced extrarenally by the granulomatous tissue. Serum levels of parathyroid hormone-related protein (PTHrP) have never been analyzed in this disorder. We report here a case of hypercalcemia in a patient with leprosy. Serum levels of 1,25-dihydroxyvitamin D were normal in spite of low levels of 25-dihydroxyvitamin D and acute renal failure. Suppressed serum levels of parathyroid hormone and PTHrP were also remarkable. In this case, PTHrP seems not to play an important role in the pathogenesis of hypercalcemia. Our data indicate that this disorder may be due, at least in part, to abnormal calcitriol overproduction by granulomatous tissue. Further investigations of the prevalence and pathogenesis of this type of hypercalcemia are needed.     

Hypercalcemia due to sun exposure in a patient with multiple myeloma and elevated parathyroid hormone-related protein

A patient with multiple myeloma who developed hypercalcemia during three different stages of his disease, with a different hypercalcemic agent elevated in his serum on each occasion, is described. The initial episode of hypercalcemia was associated with high serum interleukin-6 (IL-6). After treatment for myeloma normocalcemia was achieved. Subsequently, a relapse of hypercalcemia occurred, this time characterized by frankly elevated plasma parathyroid hormone-related protein (PTHrP) but normal IL-6. Monotherapy with pamidronate infusions resulted in remission of the hypercalcemia and a significant fall in PTHrP levels. A third spell of hypercalcemia characterized by an acute rise in serum 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D to abnormally high levels occurred during the summer season after prolonged and intense exposure to the sun.     

Non-Hodgkin's lymphoma presenting as multiple bone lesions and hypercalcemia

We report a rare case of a patient with non-Hodgkin's lymphoma who developed multiple bone lesions and hypercalcemia. A 50-year-old woman complained of drowsiness and multiple bone pain on admission. Radiographic examination revealed multiple bone fractures and osteolytic lesions. She was diagnosed with diffuse large B cell lymphoma by biopsy of an inguinal lymph node. Elevation of parathyroid hormone-related protein (PTHrP) and hypercalcemia were confirmed pretreatment, and those serum levels decreased during chemotherapy for lymphoma. However, the disease was resistant to chemotherapy combined with rituximab. These findings suggest that hypercalcemia is associated with PTHrP and the prognosis of patients with bone lymphoma in advanced stage is poor, although it is thought to be a relatively favorable prognosis in localized primary lymphoma of bone.     

Expressions of parathyroid hormone-related protein (PTHrP) and PTH/PTHrP-receptor (PTH/PTHrP-R) in gastrointestinal stromal tumours (GISTs), leiomyomas and schwannomas

BACKGROUND: Gastrointestinal stromal tumours (GISTs) are rare, c-kit and CD34 positive, and different from other mesenchymal tumours of the gastrointestinal tract (e.g. leiomyomas and schwannomas). The purpose of this study was to investigate the roles of parathyroid hormone-related protein (PTHrP) and parathyroid hormone/parathyroid hormone-related protein-receptor (PTH/PTHrP-R) in the growth and differentiation of GISTs. METHODS: Nineteen GISTs, six leiomyomas and five schwannomas were examined in this study. RESULTS: All of the GISTs and leiomyomas, and four of the schwannomas (80.0%) were positive for PTHrP. Immunohistochemical staining revealed that all of the leiomyomas, 90% of the GISTs and 80% of the schwannomas expressed PTH/PTHrP-R. Furthermore, both PTHrP and PTH/ PTHrP-R were expressed in the cytoplasm of identical cells in all of these tumours. CONCLUSION: Our results suggest that both PTHrP and PTH/PTHrP-R play an important role in the growth and differentiation of GISTs, leiomyomas and schwannomas.     

PTHrP secretion is stimulated by CT and inhibited by CgA peptides

We studied the regulation of the secretion in vitro of the parathyroid hormone-related protein (PTHrP) associated with the hypercalcemia of malignancy by Chromogranin A (CgA)-derived peptides and by human calcitonin (CT) in the BEN human lung tumor cell line. The amino terminal peptide of CgA, CgA1-40, inhibited the secretion of PTHrP, whereas other peptides had no such effect. Human CT stimulated the secretion of PTHrP, whereas other hormones had no such effect. Both effects occurred in a dose-dependent manner. These studies reveal novel regulatory pathways among peptides and proteins that are commonly associated with each other and can have paracrine interactions. CgA may be processed at its multiple dibasic sites to peptides that regulate the secretion of its co-resident hormones, in this case PTHrP. In addition to a paracrine effect, CT may be clinically useful as a provocative agent for PTHrP secretion. Complex interactions are present among the calcium-regulating hormones and their associated proteins.