Clinical Reviews in Bone and Mineral Metabolism, vol. 1, no. 1, 51–63, Spring 2002 © Copyright 2002 by Humana Press Inc. All rights of any nature whatsoever reserved. 1534-8644/02/1:51–63/$13.25

Hypercalcemia of Malignancy Epidemiology and Pathogenesis

T. John Martin, MD St. Vincent’s Institute of Medical Research, Melbourne, Victoria 3065, Australia

Epidemiology

Table 1 Diseases Associated with Hypercalcemia

Hypercalcemia is a very common metabolic complication of malignant disease. Indeed, malignancy is the most frequent cause of hypercalcemia in a general hospital patient population, whereas primary hyperparathyroidism is a more common cause of elevated blood calcium in the community at large. Figures obtained in the early 1980s indicated that the annual incidence rate for hypercalcemia in the entire population is 250 per million population per year, whereas the annual incidence rate of the hypercalcemia of malignancy is approximately 150 new patients per million population per year (1,2) (Table 1). It follows that in any study of hypercalcemia in hospitalized patients, cancer is likely to be the most prominent cause. To this needs to be added the fact that the pattern of patients in an individual hospital will determine the association of tumor types with hypercalcemia. Thus, in a hospital that is a specialized cancer center with radiation therapy facilities, cancers that are susceptible to radiation will feature most prominently. In a hospital that gathers many patients with chest diseases, lung cancer will predominate, as will breast cancer in a hospital focused upon the diseases of women. These are examples of the confounding factors that need to be considered in any studies of the types of hypercalcemia of malignancy.

Primary hyperparathyroidism Malignant disease Unknown Others (sarcoid, thyrotoxicosis, vitamin D intoxication, renal failure, immobilization)

No. of patients

Percent of total

111 72 12 12

54 34 6 6

Note: A study of 207 patients in an urban population of 1 million people, presenting with hypercalcemia over a 5-mo period (1). Source: ref. 2.

Hypercalcemia is not distributed evenly throughout the cancer population, in that certain types of malignant disease are more commonly associated with hypercalcemia than would be expected from their relative frequency. Primary tumors of the lung, breast, head and neck, kidney, and ovary have a higher incidence of hypercalcemia than other cancers. Hypercalcemia occurs most typically with squamous cell carcinoma of the lung despite the fact that adenocarcinoma and small-cell carcinoma frequently metastasize to the bone. Hypercalcemia occurs in 30–40% of patients with malignant tumors of the breast, but rarely in patients with colorectal cancers and prostate cancer. Hematological malignancies can be complicated by hypercalcemia, which can occur in up to one-third of patients with multiple myeloma, but the increasing use of bisphosphonates in therapy in

* Address correspondence to T. John Martin, M.D., St. Vincent’s Institute of Medical Research, Melbourne, Victoria 3065, Australia.

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52 the last few years has reduced this incidence. Primary bone tumors such as osteogenic sarcoma virtually never produce hypercalcemia. The association of specific tumors with hypercalcemia is related to their ability to secrete factors that act systemically and/or to their ability to metastasize to bone. This will be the subject of further discussion in the following sections.

Clinical Features When hypercalcemia develops in patients with cancer, it can present with symptoms referable to almost any organ system (Table 2), with the severity of the symptoms depending on the level of the plasma calcium, on how rapidly it rose, and on the general medical condition of the patient. The hypercalcemia of malignancy is usually steadily and rapidly progressive, with progression enhanced by dehydration, for example, which, in turn, can result from the effects of the underlying illness itself (reviewed in ref. 3). Symptoms may be experienced at a lower plasma concentration than might otherwise be expected, because patients are often symptomatic from the disease process itself. Patients with malignant disease and hypercalcemia usually have a significant tumor burden, sometimes with widespread metastases. It is important to recognize those symptoms and signs that may be caused by hypercalcemia because it is reversible with appropriate treatment and its progression can often be prevented if it is recognized early. Gastrointestinal symptoms such as anorexia, nausea, and vomiting are early and frequent features in the hypercalcemia associated with malignancy and can be mistakenly attributed to the underlying disease or to cytotoxic or radiation therapy. Neurological manifestations occur in over 50% of patients, consisting of cognitive and behavioral changes, alteration in the level of consciousness, and neuromuscular disturbances. The milder symptoms are difficulty in concentrating, fatigue, and depression. Psychiatric symptoms may resemble mania, schizophrenia, acute confusion, and even catatonic stupor. A familiar sequence of events is that of a patient with moderate hypercalcemia whose fluid intake decreases because of some mental confusion, perhaps compounded by anorexia and nausea. The further fluid depletion results in more

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Martin Table 2 Symptoms and Signs of Hypercalcemia Gastrointestinal Nausea and anorexia Vomiting Constipation Abdominal pain Neurological Headache Confusion Psychosis Drowsiness Coma Renal Polyuria and thirst Dehydration

severe hypercalcemia, with progression to drowsiness and coma. Renal manifestations are polyuria and polydipsia, which occur because hypercalcemia interferes with antidiuretic hormone action at the distal nephron, causing a syndrome like diabetes insipidus. The resulting dehydration further exacerbates the hypercalcemia. In elderly subjects, nocturia is common, and the clinician needs to be alert to the significance of a requirement for fluid at the time of nocturnal voiding. The prognosis of patients with hypercalcemia of malignancy is generally poor, in that it so often occurs in patients with advanced malignancy. Treatment of the hypercalcemia has a marked effect on symptoms without improvement of survival, and it is a very useful palliative measure in symptomatic patients.

Mechanisms of Hypercalcemia Associated with Malignancy The plasma calcium level is maintained within a very narrow range in normal individuals through a homeostatic system under the hormonal control of parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D [1,25(OH)2D]. PTH acts directly on bone to promote bone resorption and on the kidney to decrease calcium excretion, increase phosphorus excretion, and promote renal generation and excretion of cyclic AMP. Since the early 1920s, the biochemical features of primary hyperparathyroidism

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Hypercalcemia of Malignancy were recognized as high plasma calcium and low plasma phosphorus. In malignancy, the disturbance of normal regulation of calcium homeostasis leads to increased bone resorption in all cases and sometimes decreased renal excretion of calcium. The single most important pathogenetic factor contributing to hypercalcemia in patients with malignant disease is increased bone resorption, either generalized throughout the skeleton or confined to the bone surrounding advancing lytic metastases. The alternative possibilities are that hypercalcemia could primarily be the result of reduced renal calcium clearance or increased absorption of calcium from the intestine. These latter two possibilities are unlikely because renal calcium clearance is actually increased in patients with hypercalcemia of malignancy, even more than in patients with primary hyperparathyroidism (4). Increased calcium absorption from the intestine is unlikely because direct measurements show reduced calcium absorption in cancer (5) and glucocorticoids, which directly inhibit calcium absorption (6), are usually ineffective in the hypercalcemia of cancer. Although renal tubular reabsorption of calcium is not likely to be the proximal mechanism in cancer, it is, nevertheless, an important part of the overall process. Despite the very markedly increased bone resorption in many patients with cancer, normal homeostatic mechanisms, in which the kidney plays a central role by increasing calcium clearance, are able to regulate the serum calcium. Any alteration in renal function could lead to hypercalcemia in a patient with increased bone resorption by causing a decrease in filtered calcium load or an increase in proximal tubular reabsorption of calcium. The latter is frequently responsible for acute precipitation of hypercalcemia and is likely to occur whenever the glomerular filtration rate declines, because a decline in the glomerular filtration rate leads to sodium reabsorption in the proximal convoluted tubules (7). Sodium and calcium reabsorption in the proximal tubules are closely linked, with increased sodium reabsorption leading to increased calcium reabsorption, favoring hypercalcemia. The changes in renal function in patients with cancer favoring this occur because of intercurrent infections and fever, dehydration, or vomiting, which may be the result of the disease itself or precipitated by cytotoxic therapy.

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Historical Perspective Among patients with cancer-associated hypercalcemia, those with solid tumors without bone metastases have received most attention in the literature, but they constitute the smallest group. Hypercalcemia in these patients has been long recognized to be the result of the secretion by tumor cells of factor(s) that act generally upon the skeleton to promote bone resorption to the extent that the capacity of normal homeostatic mechanisms is overwhelmed. Despite the fact that this comprises the smallest group of patients, it is the pursuit of its cause, together with improved understanding of many aspects of bone cell biology, that has led to much greater appreciation of the pathogenesis of hypercalcemia in all forms of malignancy. Hypercalcemia occurring as a metabolic complication of malignancy was first described in 1924 (8). In 1936, Gutman et al. (9) reported a case of a 57-yr-old male with bronchogenic carcinoma and marked hypercalcemia with no metastases evident radiologically or at postmortem and normal parathyroids. The hypercalcemia was attributed to “complicating factors as yet wholly obscure.” In 1941, Albright proposed that the hypercalcemia in a patient with renal carcinoma that had resolved after irradiation of a single bone metastasis might be the result of the production by the cancer of PTH (10). In succeeding years, this idea gained acceptance, and the term “ectopic PTH syndrome” became widely used to apply to patients with cancer who had a high plasma calcium, low phosphorus, and minimal or no bony metastases. Support for this appeared to come in 1966 when Berson and Yalow (11) published results with the first radioimmunoassay for PTH, in which they found significant elevations of the PTH level in a number of unselected patients with lung cancer. Another study reported a primary hepatoma with an arterio-venous gradient in PTH levels across the tumor bed (12) and PTH release from renal carcinoma cells in culture was also reported (13), although the immunoreactivity of ‘PTH’ detected in the latter study was of greater molecular weight than that of PTH itself. Throughout this time, it was evident, however, that the radioimmunoassay of PTH presented technical problems, and in none of the above instances were circulating levels of PTH convincingly very high—

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54 certainly not at the levels frequently found with corresponding degrees of elevation of plasma calcium in patients with primary hyperparathyroidism. With the improvement of radioimmunoassays for PTH in the early 1970s, some doubt arose regarding the nature of the hormone(s) producing the biochemical features of this cancer syndrome. In 1971, Riggs et al. (14) studied hypercalcemic patients with nonparathyroid cancer or primary hyperparathyroidism and noted that, although the hypercalcemia was greater in the tumor group, the latter had lower serum immunoreactive concentrations of an apparently different immunologic identity than the PTH measured in primary hyperparathyroidism. In studying a breast cancer from a hypercalcemia patient, Melick et al. (15) extracted PTH-like immunoreactivity that was nonparallel to standard purified PTH in the assay. Roof et al. (16) used several radioimmunoassays (RIAs) to demonstrate heterogeneity and a lack of immunological identity of PTH-like peptides in sera from both normocalcemic and hypercalcemic cancer patients. They concluded that “some tumors elaborate peptides with an amino acid sequence different from that of normal parathyroid hormone.” A crucial study by Powell et al. (17) used multiple RIAs that enabled detection of PTH, proPTH, or PTH fragments in the study of tumor extracts and sera from patients with hypercalcemia and malignancy. PTH immunoreactivity was not detectable despite significant PTH-like bioactivity in tumor extracts and it was concluded that a humoral substance other than PTH must be responsible for the hypercalcemia. The use of the term “humoral hypercalcemia of malignancy” (HHM) was proposed in 1979 by Martin and Atkins (18) to describe the syndrome, on the basis that the responsible factor behaved in some respects like PTH but that it appeared to be chemically different. Comprehensive clinical and biochemical investigations followed (19–21), indicating that patients with HHM also exhibited increased renal production of cAMP despite low or undetectable plasma levels of PTH, making it abundantly clear that the tumors were producing a factor with biological effects strikingly similar to those of PTH, but that the factor was not PTH itself. Newly available bioassays for PTH led to further developments in understanding the syndrome.

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Martin The bioassays revealed that these tumor extracts could stimulate adenylate cyclase in PTH- responsive renal cortical membranes (22). A sensitive cytochemical assay for PTH in kidney cells could detect PTH-like bioactivity in the serum of patients in whom immunoreactive PTH was undetectable (23). Studies in PTH-responsive osteogenic sarcoma cells showed that tumor extracts of rat and human origin could also stimulate adenylate cyclase in this system (24). Peptide antagonists of PTH blocked biological activity, but preincubation with PTH antisera was ineffective in blocking biological activity, indicating that the active material acted on PTH receptors but was immunologically distinct from PTH. Finally, when nucleotide probes for PTH became available, it was not possible to demonstrate mRNA for PTH in tumors associated with the HHM syndrome (25). In attempts to identify and purify the “PTH-like factor,” animal models of HHM were developed and cell lines were established from human tumors associated with HHM, in anticipation that these would provide a plentiful supply of material for purification. Sequence was finally obtained (26) and cloning was achieved in 1987 (27) from a cultured human squamous cell line established from a tumor taken from a patient with hypercalcemia. A confirmatory partial amino acid sequence was obtained also from human renal carcinoma cells (28) and a breast tumor (29). Much is now known about the structure–activity relationships of parathyroid hormone-related protein (PTHrP) and the complex nature of its gene. Its precise functional role in cancer patients and normal physiology is under continuing investigation, but it is clear that its excessive production is the major cause of hypercalcemia in patients with the humoral hypercalcemia of malignancy.

PTHrP Protein and Gene Structure The N-terminal amino acid sequence revealed that this protein shared substantial sequence homology with PTH, with 8 of the first 13 amino acids identical. Beyond this, the sequence is unique, showing no conserved stretches of amino acid homology with PTH. Molecular cloning revealed this protein to be larger than PTH, the mature protein 141 amino acids in length, with a prepro sequence of 36 amino acids. Additional isoforms of 139 and 173 amino acids in

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Hypercalcemia of Malignancy length, identical in sequence up to residue 139, were also predicted. The gene for human PTHrP is complex, with nine exons and three promoters, and, together with the 3′ alternate splicing, is able to generate multiple mRNA transcripts (30). The presence of several promoters suggests the potential use of different promoter regions, which may result in tissue-specific expression of PTHrP. Apart from specifying different isoforms of PTHrP, the alternative transcripts confer different properties as mRNA species. However, all three isoforms contain multiple copies of an AUUUA instability motif, which is associated with rapid turnover mRNA (31). The instability of the mRNA and the fact that its expression is subject to regulation by so many cytokines and growth factors are consistent with PTHrP functioning as a cytokine. PTHrP can, indeed, act locally in several tissues, although initially discovered as a hormone produced in excess by certain malignancies.

Actions of PTHrP and Roles in Hypercalcemia of Malignancy Actions of PTHrP Studies with synthetic and recombinant PTHrP peptides indicate that its effect in bone and kidney closely parallel those of PTH itself and it is now accepted that these events occur via authentic PTH receptors. The PTH-like bioactivity of PTHrP, like that of PTH, resides in the first 34 amino acids. Because non-PTH-like actions of PTHrP have also been recognized, it must be assumed that PTHrP activates pathways, distinct from those influenced by PTH, but mediated by specific PTHrP receptors that recognize nonhomologous regions of the molecule. The mechanisms by which PTHrP promotes bone resorption have been clarified greatly over the last 3 yr, with the discovery of crucial molecules involved in control of osteoclast formation and activity. Osteoprotegerin (OPG) is a secreted member of the tumor necrosis factor (TNF) receptor family, which is produced by osteoblastic stromal cells and profoundly inhibits osteoclast formation at a late stage of their development (32,33). The ligand for OPG is a cell-surface molecule of the stromal cell that is a member of the TNF superfamily. Given the name

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55 RANK ligand (RANKL), this molecule is a powerful inducer of osteoclast formation by acting directly on hematopoietic cells in the presence of macrophage colony-stimulating factor (M-CSF) and without accompanying stromal cells (34–36). RANKL was discovered independently as a T-cell molecule (37), and interestingly, activated T-cells when cocultured with spleen cells generate authentic, functional osteoclasts (38). The production of RANKL by osteoblastic stromal cells is enhanced by treatment with PTHrP, PTH, and other stimulators of osteoclast formation, and the production of OPG is inhibited by these same agents (39). It seems that the progression of hemopoietic precursors to the formation of osteoclasts depends on the balance between these two critical molecules, with the amount of RANKL available to act upon its receptor, RANK, being the ultimate determinant of osteoclast formation. In the case of the humoral hypercalcemia of malignancy, the generalized great increase in osteoclast formation most commonly results from the stimulus provided by PTHrP to this process. In the case of breast cancer metastasis to bone, it has also been shown that PTHrP production by cancer cells can promote RANKL production by host bone cells, thus favoring osteoclast formation (40), but it should be emphasized that there are several other cancerderived cytokines that are also capable of promoting production of RANKL in bone to favor osteoclast formation, for example, interleukin (IL)-6, IL-11, and prostaglandin E2. They have not been investigated to the same extent as PTHrP. The implication of these pathogenetic mechanisms for the development of new approaches to treatment will be discussed later.

Localization of PTHrP in Malignant Tissues Subsequent to the original identification of PTHrP in tumor extracts using sensitive PTH bioassays, the development of antibodies raised to synthetic PTHrP peptides and the preparation of cDNA probes have enabled PTHrP to be detected in tumors using immunocytochemistry, Northern blot analysis, and in situ hybridization (41). Immunohistochemical studies using highly specific polyclonal antibodies have enabled identification of PTHrP in a variety of tumors. Although PTHrP is most commonly produced by squamous

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56 cell tumors, other tumors, which may or may not generally be associated with hypercalcemia, are also sources of this hormone. Other tumors in which PTHrP has been detected include breast cancer, renal cell carcinoma, melanoma, skin tumors, neuroendocrine tumors, and medullary thyroid carcinoma. The universal presence of PTHrP in squamous cell carcinoma is reflected by its presence in the spinous keratinocytes of normal skin (41), the cell considered to be the differentiating feature of squamous cell tumors. No immunoreactive PTHrP could be detected in basal cells of skin or in basal skin tumors.

Cancer Hypercalcemia Syndromes and Involvement of PTHrP Hypercalcemia associated with malignancy generally falls into three syndromes: the humoral hypercalcemia of malignancy, that associated with skeletal metastases, and the hypercalcemia of hematological malignancy. Humoral Hypercalcemia of Malignancy The term “humoral hypercalcemia of malignancy” (HHM) describes those patients with certain cancers in whom the blood calcium is elevated in the absence of skeletal metastases. There is increased bone resorption, decreased renal calcium clearance, increased renal excretion of phosphate and cAMP, and often a mild hypokalemic hypochloremic alkalosis. Animal models of HHM comprising human or animal tumors associated with HHM transplanted into nude mice exhibit all of the features of the syndrome. It has been clearly demonstrated that PTHrP antibodies raised to synthetic PTHrP(1–34) or PTHrP(1–16), which are highly specific for PTHrP and which block adenylate cyclase stimulation by PTHrP in PTH-responsive osteoblasts, can also reverse the hypercalcemia in such models (42). In nude mice bearing transplanted squamous cell carcinomata of the lung or of the larynx, infusion of antibodies produced significant reduction of serum calcium and urinary cyclic AMP within 6 h and persisted for 24–48 h. No changes were seen in serum calcium or cyclic AMP excretion in animals given nonimmune serum or antibody-free serum derived from affinity extraction of the antibody. In addition to providing evidence of the PTHrP contribution to HHM, these studies also demonstrate the possible use of antibodies for therapeutic purposes. Using an

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Martin N-terminal RIA (43), we have detected circulating PTHrP in 100% of hypercalcemic patients with solid tumors and no bone metastases, and other immunoassays have detected elevated levels in the vast majority of patients with this syndrome. There is little doubt that PTHrP is the major mediator if not the sole mediator, of hypercalcemia in patients with HHM. It is still possible that in some cases, other bone-resorbing factors could contribute to the development of hypercalcemia on a humoral basis. Colony-stimulating factors, epidermal growth factor, IL-1α, IL-1β, and IL-6, transforming growth factor (TGF)-α and TGFβ, and TNF have all been shown to promote bone resorption in vitro and/or be associated with hypercalcemia. The significance of these factors and their possible interplay with PTHrP should become clear as our knowledge of the role of cytokines in bone metabolism is increased.

Hypercalcemia and Skeletal Metastases One of the unforeseen results of the discovery of PTHrP as the cause of the HHM syndrome was the light it shed, on the pathogenesis of lytic bone metastases in breast cancer. For a long time, it was considered that in patients with radiological evidence of skeletal metastases, local osteolysis produced by tumor cells was responsible for the hypercalcemia. There is now evidence for a humoral contribution in these patients also. Particularly in breast cancer, the extent of metastatic bone disease correlates poorly with both the occurrence and the degree of hypercalcemia (44). In 80–90% of hypercalcemic patients with unselected solid tumors, irrespective of whether bone metastases are present, there is evidence of an underlying humoral mechanism (44). Measurement of plasma PTHrP concentrations by direct RIA confirmed this finding, with 65% of a series of hypercalcemic breast cancer patients having detectable levels (43) (Fig. 1). PTHrP levels above those of normal subjects were also found in 64% of patients with hypercalcemia and metastatic malignancy to bone, from primary sources other than breast (Fig. 1). This latter group included several patients in whom the mechanism of hypercalcemia was likely to be humoral. Consistent with this is the finding that in the metastatic group, all squamous cell cancer patients had elevated PTHrP levels. The presence or absence of bone metastases, a feature that was used Volume 1, 2002

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Fig. 1. Plasma PTHrP concentrations in patients with diagnoses as indicated. (Based on data from ref. 43.)

to distinguish between humoral and osteolytic mechanisms of hypercalcemia, can no longer be used to define the syndrome of HHM. It is now well established that a large proportion of patients with hypercalcemia and skeletal metastases have high circulating levels of PTHrP. In the case of breast cancer, another possible role arises for PTHrP. The avidity of breast cancer metastases for bone has long been recognized. Approximately 70% of patients dying from breast cancer have bone metastases and, of these, 10–20% are hypercalcemic (45). Currently, there is no single accurate predictor to identify which patients will develop bone metastases. PTHrP has been demonstrated by immunohistochemistry in approx 60% of breast tumors (46). PTHrP has been detected by immunohistochemical staining in 92% of breast cancer metastases to bone compared with only 17% of metastases to other sites (47). PTHrP mRNA has been detected by in situ hybridization in 73% of bone metastases from breast cancer and 20% of metastases to other sites (48). One possibility that is of particular interest is that PTHrP production might contribute to the ability of breast cancers to erode bone and establish there as metastases (47). These clinical observations have been extended by using a Clinical Reviews in Bone and Mineral Metabolism

mouse model of bone metastases in which inoculation of a human cancer cell line into the left ventricle of the mouse reliably produces osteolytic metastases. An antibody against PTHrP(1–34) blocked the formation of osteolytic bone lesions and the growth of metastatic deposits (49) in nude mice injected with a PTHrP-producing breast cancer cell line. These data strongly suggest that PTHrP expression by breast cancer cells enhances their metastatic potential to bone. The mechanisms of osteolytic metastasis formation in cancer have been reviewed recently (50). Prostate cancers also have a predilection for metastasis to bone and may produce osteolytic and/or osteoblastic lesions. Unlike breast cancer, hypercalcemia is a rare complication of prostate cancer (less than 2% of cases) and is usually associated with tumors of unusual histology (51). Despite this, PTHrP was detected immunohistochemically in 100% of prostate cancers (52). PTHrP protein and mRNA have been detected in both the neuroendocrine and epithelial cells of the prostate (53,54) and PTHrP is a prostate-derived component of seminal plasma (55). Some preliminary evidence has been obtained to suggest that PTHrP might be an autocrine growth regulator in prostate cancer cells (56) and a role for PTHrP in

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58 the progression of prostatic intraepithelial neoplasia has been suggested (57). Hypercalcemia in Hematological Malignancy Hematological malignancies may be associated with osteolytic bone destruction and with hypercalcemia. Hypercalcemia is relatively uncommon in both non-Hodgkin’s and Hodgkin’s lymphoma. In a series of 165 consecutive patients admitted to a hematology unit, we have documented hypercalcemia in 18. It was the result of primary hyperparathyroidism in three cases. In the remainder, it was associated with multiple myeloma in nine, high grade B-cell non-Hodgkin’s lymphoma in five and with myeloid neoplasia in one (58). A number of case reports have documented hypercalcemia without lytic bone lesions in both Hodgkin’s and nonHodgkin’s lymphoma that was associated with elevated 1,25(OH)2D levels and low PTH levels in plasma (59,60). We have detected circulating levels of PTHrP of the order of those associated with HHM in cases of non-Hodgkin’s lymphoma of B-cell lineage (55). Immunohistochemical staining demonstrated intracellular PTHrP in some of the neoplastic cells from a lymph node section in one of the cases. PTHrP gene expression has been extensively studied in adult T-cell leukemia/lymphoma, a malignancy associated with human T-cell leukemia virus type 1 (HTLV-1) infection. This malignancy is frequently associated with the HHM syndrome (61). Expression of PTHrP mRNA within HTLV-1infected T-cells in culture has been demonstrated, and immunohistochemical staining detected PTHrP in neoplastic lymph nodes. Hypercalcemia occurs in approximately one-third of all patients with multiple myeloma (62). Histological examination of the lytic lesions in multiple myeloma has shown increased bone resorption by normal osteoclasts in the areas near myeloma cells (63). This observation suggests that myeloma cells secrete a factor or factors that stimulate the osteoclast either directly or via the osteoblast to resorb bone. Because the initial demonstration of the secretion of an “osteoclast activating factor” by myeloma cells, several cytokines have been postulated to be responsible for increased bone resorption, among them TNF-β, IL-1, and IL-6. In our series of consecutive hypercalcemic patients admitted to the

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Martin hospital with hematological malignancies, elevated plasma levels of PTHrP were measured in one-third of patients with hypercalcemic multiple myeloma (58). We have also demonstrated PTHrP gene transcripts and protein in the bone marrow plasma cells from a hypercalcemic patient with elevated plasma PTHrP levels, indicating PTHrP production by the myeloma cells (64). It seems likely that PTHrP can be added to the list of several cytokines able to contribute to the hypercalcemia and the bone loss resulting in osteoporosis, which is a common complication of this disease. Interesting light has been shed on this mechanism, with recent data showing that myeloma cells from unselected patients express mRNA and protein for both RANKL and OPG (65), raising the possibility that the myeloma cells themselves may be able to promote osteoclast formation, without necessarily making use of osteoblastic stromal cells as intermediaries. It would be interesting indeed if myeloma cells were to prove capable also of processing extracellular RANKL to reach the circulation. Such a possibility needs to be investigated when appropriate assays become available. Thus, the mechanism of skeletal invovement in multiple myeloma, as well as the mechanism of hypercalcemia in the disease, has several possible contributing agents, including PTHrP, but a unifying factor could be RANKL, if preliminary data are confirmed with further study.

Implications for Diagnosis and Treatment Diagnosis A large number of immunoassays for PTHrP have been developed using antibodies directed against amino-terminal, mid-molecule, and carboxy-terminal sequences, as well as two-site assays spanning amino-terminal and mid-molecule sequences. The first assays showed the potential diagnostic application of amino-terminal PTHrP RIAs, finding elevated levels in a significant proportion of patients with tumor-induced hypercalcemia (66–68). Using an N-terminal RIA (43), we have detected circulating PTHrP in 100% of hypercalcemic patients with solid tumors and no bone metastases, and in 65% of

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Hypercalcemia of Malignancy patients with hypercalcemia and breast cancer metastatic to bone. Patients with solid tumors other than breast who had hypercalcemia and bone metastases also had elevated PTHrP levels, particularly those with squamous cell carcinoma where 100% had detectable PTHrP immunoreactivity (Fig. 1). In attempts to increase analytical sensitivity and specificity, immunoradiometric assays for PTHrP were developed (69–71). Immunoradiometric assays use two antibodies and only those species containing both target epitopes are detected, thus increasing the assay specificity. Because the hypercalcemic action of PTHrP is contained within the amino terminus, a PTHrP immunoassay must provide a sensitive and reliable measure of all circulating forms containing this region of the PTHrP molecule in order to reflect circulating bioactivity in tumor-induced hypercalcemia. A large number of potential posttranslational cleavage sites exist within the mature protein and very little is known about the PTHrP forms released into plasma and their subsequent catabolism. In vivo tumor-specific processing of PTHrP may limit the diagnostic utility of IRMAs. PTHrP assays have a role to play in the investigation of hypercalcemia, but the assay used must be able to provide a true indication of the concentration of circulating PTHrP fragments able to produce hypercalcemia by their actions upon kidney and bone, actions contained within the amino-terminal portion of the molecule. The use of currently available assays is often to confirm a diagnosis that is suspected or to monitor cancer treatment.

Treatment Despite PTHrP having a dual action on bone and kidney, the agents available for the treatment of hypercalcemia in cancer are primarily aimed at reducing bone resorption and no agent has a substantial effect on calcium excretion. A poor response to pamidronate occurs in patients with hypercalcemia of malignancy who have evidence of renal tubular stimulation as indicated by a low tubular threshold for phosphate or high threshold for calcium. It is therefore not surprising that in studies of patients with tumor-induced hypercalcemia, the PTHrP level was the best determinant for the calcemic response to pamidronate, with high levels correlating with poor response and vice versa (72) (Fig. 2).

Clinical Reviews in Bone and Mineral Metabolism

59 The failure to inhibit the renal mechanism of hypercalcemia is the major cause of a poor response in patients with high circulating PTHrP levels. Currently, the prediction of a poor response will not change clinical practice because agents are not available that conveniently increase calcium excretion. The development of drugs that inhibit the tubular reabsorption of calcium, specific inhibitors of PTH or PTHrP action, humanized monoclonal antibodies to PTHrP, or inhibitors of PTHrP production may allow better control of hypercalcemia in these patients when used in combination with the available inhibitors of osteolysis. Of particular interest in this category may be the new analogs of vitamin D that have low calcemic activity yet retain the property of inhibition of PTH gene expression. The first reported analog of this type was 22-oxa-1,25(OH)2D, or 22-oxacalcitriol (OCT) (73), which has been shown to inhibit expression of the PTH gene in vitro and in vivo (74) and is currently under investigation in the treatment of secondary hyperparathyroidism associated with chronic renal failure. 1,25(OH)2D is known to inhibit PTHrP gene expression (75), noncalcemic 1,25(OH)2D analogs have the same effect on PTHrP in vitro (76) and, hence, may offer a valuable option in addition to bisphosphonates in the treatment of PTHrP-mediated hypercalcemia. Now that so much more is known of the control of osteoclast formation and activity, with the discovery of OPG, RANKL, and RANK, and the pathogenetic significance of these in the skeletal complications of cancer, totally new targets for drug development are available. Thus, in animal models of humoral hypercalcemia of malignancy, OPG is effective in treating and preventing the hypercalcemia (77,78), as also, unsurprisingly, is treatment with a RANK–immunoglobulin fusion protein (79). Although these proteins do not of themselves provide readily developed drugs, they do provide the proof of concept to lead to searches for drugs based on other points in the RANKL/RANK axis. These would include modulating local production of OPG or RANKL, modifying the interaction of RANKL with RANK, or approaches through the RANK signaling pathways (80).

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Martin

Fig. 2. Pretreatment PTHrP levels in patients with hypercalcemia of malignancy in relation to response to treatment and the number of infusions of pamidronate given. (Based on data from ref. 72.)

Summary and Perspective The discovery of PTHrP has provided an explanation for much of the pathogenesis of the hypercalcemia of cancer, contributing not only to the humoral syndrome certainly but also to the hypercalcemia accompanying bony metastases and hematological malignancies. Immunoassays that measure PTHrP levels in the circulation indicated that PTHrP-mediated hypercalcemia not only occurs with solid tumors without skeletal metastases but also in association with hematological malignancies and with cancers that have metastasized to bone. In the case of breast cancer, another possible role arises for PTHrP. The high incidence of PTHrP production by primary breast cancers, and higher incidence of PTHrP expression in skeletal than nonskeletal metastases, have led to the hypothesis that PTHrP might contribute to the development of breast cancer metastases in bone. Clinical Reviews in Bone and Mineral Metabolism

Experimental evidence is now available to support this. It is useful to keep in mind, however, that even in HHM, there may in many instances be contributions from other bone-resorbing factors. That is certainly so in metastatic bone disease and hematological cancers, where other bone-resorbing cytokines (e.g., IL-11, IL6) could be significant contributors. Whatever the contributing cytokines and hormones, it is clear that increased osteoclast formation is a prime pathogenetic mechanism in the hypercalcemia of cancer, whether this be a generalized increase, as in the case of HHM, or localized at sites of tumor invasion of bone, where enhanced osteoclast formation facilitates the growth of tumor in bone. Recent discoveries of mechanisms involved in osteoclast formation will provide further insights into these processes, as well as providing exciting new targets for drug development. Volume 1, 2002

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62 34. Yasuda H, Shima N, Nakagawa N, Yamaguchi K, Kinosaki M, Mochizuki S-I, et al. 1998 Osteoclast differentiation factor is a ligand for osteoprotogerin/osteoclastogenesis inhibitory factor and identical to TRANCE/RANKL. Proc Natl Acad Sci USA 95:3597–3602. 35. Lacey DM, Timms E, Tan HL, Kelley MJ, Dunstan CR, Burgess T, et al. 1998 Osteoprotogerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell 93:165–176. 36. Quinn J MW, Elliott J, Gillespie MT, Martin TJ. 1998 A combination of osteoclast differentiation factor and macrophage-colony stimulating factor is sufficient for both human and mouse osteoclast formation in vitro. Endocrinology 139:4424–4427. 37. Wong BR, Rho J, Arron J, Robinson E, Orlinick J, Chao M, et al. 1997 TRANCE is a novel ligand of the tumor mecrosis factor receptor family that activates c-Jun N-terminal kinase in T-cells. J Biol Chem 272:25,190–25,194. 38. Horwood NJ, Kartsogiannis V, Quinn JMW, Romas E, Martin TJ, Gillespie MT. et al. 1999 Activated T lymphocytes support osteoclast formation in vitro. Biochem Biophys Res Commun 265:144–150. 39. Horwood NJ, Elliott J, Martin TJ, and Gillespie MT. 1998 Osteotropic agents regulate the expression of osteoclast differentiation factor and osteoprotegerin in osteoblastic stromal cells. Endocrinology 139:4743–4746. 40. Thomas RJ, Guise TA, Yin JJ, Elliot J, Horwood NJ, Martin TJ, et al. 1999 Breast cancer cells interact with osteoblasts to support osteoclast formation. Endocrinology 140:4451–4458. 41. Danks JA, Ebeling PR, Hayman JA, Chou ST, Moseley JM, Dunlop J, et al. 1989 Parathyroid hormone-related protein: immunohistochemical localization in cancers and in normal skin. J Bone Miner Res 4:273–278. 42. Kukreja SC, Schavin DH, Wimbuscus S, et al. 1998 Antibodies to parathyroid hormone-related protein lower serum calcium in athymic mouse models of malignancy associated hypercalcemia due to human tumors. J Clin Invest 82:1798–1802. 43. Grill V, Ho P, Body JJ, Lee SC, Kukreja SC, Moseley JM, et al. 1991 Parathyroid hormone-related protein: elevated levels in both humoral hypercalcemia of malignancy and hypercalcemia complicating metastatic breast cancer. J Clin Endocrinol Metab 73:1309–1315. 44. Ralston SH, Fogelman I, Gardner MD, Boyle IT. 1984 Relative contribution of humoral and metastatic factors to the pathogenesis of hypercalcemia of malignancy. Br Med J 288:1405–1408. 45. Coleman RE, Rubens RD. 1987 The clinical course of bone metastases from breast cancer. Br J Cancer 55:61–66. 46. Southby J, Kissin MW, Danks JA, Hayman JA, Moseley JM, Henderson MA, et al. 1990 Immunohistochemical localization of parathyroid hormone-related protein in human breast cancer. Cancer Res 50:7710–7716. 47. Powell GJ, Southby J, Danks JA, Stillwell RG, Hayman JA, Henderson MA, et al. 1991 Localization of parathyroid hormone-related protein in breast cancer metastases; increased incidence in bone compared with other sites. Cancer Res 51:3059–3061.

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