Bone tissue as the site of action of oncological drugs

Bone tissue is of special importance in the pathophysiology and the clinical course of neoplastic diseases. Metastases to bones do not pose a direct threat to the patient’s life, yet they markedly lower the quality of life and may be associated with complications leading to severe disability and, in consequence, to shortening of the patient’s life. In the clinical picture of certain neoplasms, such as multiple myeloma, prostate cancer, thyroid cancer or sometimes breast cancer, the dominating symptoms are those related to bone infiltration. On the other hand, there are also effects of the oncological treatment on the bone – both those concerning the physiological bone cycle (of increasing importance with the observed prolongation of patient life) and the interactions between the neoplastic cells and bone cells. Below, we present a literature review on this subject. The issue of steroid therapy’s effects on bones is not addressed here as the multitude of usages of this drug class resulted in the subject being already extensively discussed in other publications.

Chemotherapy

Adverse effects of chemotherapy concerning bones may in a longer-term perspective lead to health complaints markedly lowering the quality of life, particularly in patients with good prognosis, even more so because this approach is often combined with different hormone and radiation therapies. As early as in 1965, a report was published presenting the results of a study on the effects of methotrexate (MTX) treatment on calcium metabolism, demonstrating elevated levels of this element in urine and stool, its lowered levels in blood serum, and indirectly its increased bone resorption [1]. The above data have been reflected in a clinical setting, in patients treated with MTX for acute myeloid leukaemia, where there have been observed pain and difficult uniting of fractures during the course of treatment and remission upon its completion [2-8]. Although the high doses of steroids have been of significance, the cytostatic agents have been deemed to play an important role here. Reduction in the bone mineral density (BMD) has been observed as well as qualitative features of osteopenia, seen in radiograms as shrinking of the cortical bone, lower Singh index, more distinct (due to thinning) trabeculae or – in contrast – lack of the trabecular structure in imaging. Similar reports have been published based on observations in patients treated with MTX for collagenoses [9-17]. Apart from clinical symptoms, there was also described in this group of patients inhibition of osteoblast activity [13], as well as a significant reduction in osteocalcin synthesis by osteoblasts [14] – seen in both of the above studies after a weekly administration of MTX at low doses. Those data are in agreement with the observed in vitro effects of MTX on osteoblasts, where this cytostatic agent has proven to be a potent inhibitor of osteoblast proliferation, affecting bone metabolism and regeneration [15, 16]. On the other hand, no effects of the drug on osteoblast differentiation have been found [16]. The MTX potential for directly affecting bones is indicated by its high level in the cortical and trabecular bone one day after its intramuscular administration [17]. Katz and colleagues [18] have reported a significant reduction in the parathyroid hormone and total calcium levels in the blood in response to low MTX doses, suggesting a direct influence on secretion of this hormone. However, no significant changes in the blood levels of free calcium and osteocalcin or in the BMD as measured by double-photon absorption (DPA) have been detected. Meanwhile, osteopathy was found in children with brain tumours treated with MTX at a cumulative dose of 20 g/m² to 135 g/m², suggesting that the intracellular accumulation of this cytostatic agent and its polyglutamate derivative might have caused this [19]. The radiological and scintigraphic evaluation of bones of 87 patients with osteosarcoma [20] revealed symptoms, present in 8 of them, analogous to those accompanying leukaemia treatment. Those were as follows: osteopenia, pseudocalcification zones and multifocal nature of the process. The bones most commonly affected by the above complications were the humeral, calcaneal, pubic and tibial ones. A significant difference in age was observed between the patients with bone lesions (mean age: 9.2) and those without them (mean age: 14.9): p < 0.001. No direct effects of the dose size on osteopathy were seen. Yet another reported observation was the difference in BMD measurement results at 6 and 9 months after the completion of chemotherapy for osteosarcoma with doxorubicin and cisplatin in combination with a low (750 mg/m²) or high (7.5 g/m²) MTX dose, in comparison with the control group. A significant reduction in BMD in the high-dose group indicates a correlation between osteopenia and the dose. The absence of detectable effects of low drug doses on bones may stem from their intensive growth in patients with osteosarcoma, a neoplasm occurring more often in tall individuals. On the other hand, the presence of distinct osteopenia at high MTX doses in sites where the trabecular structure dominates is in agreement with the fact that this bone type is particularly sensitive to the effects of other osteopenia-inducing factors as well, such as oestrogen deficiency or steroid therapy [21]. Of note are the adverse effects on the bones in paediatric patients of ifosfamide (IFO) and the chemotherapy regimens including it. The renal tubule damage occurring during this drug use leads to impaired phosphate resorption, and in consequence to metabolic acidosis and to phosphate and calcium loss with urine [22]. The result of this may be the BMD reduction [23] and osteomalacia [24, 25] observed in children treated with IFO. Chemotherapy with IFO may also be associated with lower osteocalcin levels in blood serum [26]. An important factor causing osteopenia in females who underwent chemotherapy is the inhibition of ovarian activity. This is confirmed by studies conducted on patients treated for Hodgkin’s lymphoma – there has been observed a statistically significant reduction in the density of trabecular and cortical bone in cases of ovarian failure, as compared with patients retaining normal ovarian function [27, 28]. The effects of chemotherapy on gonads have also been implicated in the reduction of BMD in males treated for the same condition, as suggested by the correlation between BMD and testosterone levels [29]. Impaired gonad function occurred more often in patients treated for Hodgkin's lymphoma as compared with the group with non-Hodgkin’s lymphoma, due to the difference in the frequency of radiation therapy and procarbazine use [30] – however, reduced BMD is also found in females treated for non-Hodgkin’s lymphoma. This is believed to be caused not only by high steroid doses but also by cytostatic agents [31]. Reduced BMD seen in patients undergoing chemotherapy for breast cancer at pre-menopausal age should also be correlated with ovarian failure. In the study by Bruning [32], chemotherapy was associated with higher incidence of premature menopause and significantly reduced BMD as compared with the control group of female patients at pre-menopausal age after mastectomy and not undergoing chemotherapy. In the ZEBRA study, an improvement in bone mineralization was not observed even after three years of completion of adjuvant chemotherapy according to the CMF (cyclophosphamide, methotrexate, 5-fluorouracil) regimen [33]. Its effects in the bones of females at pre-menopausal age treated for breast cancer have also been found in radiological structure examination [34]. The reduction in trabeculae thickness and the relative field occupied by trabeculae correlates with the clinical and experimental observations of the inhibitory effect of MTX on osteoblast proliferation and activity. The mineralization impairment is reflected in the reduction in the mean radiological bone density. The comparison of the results for a group of patients after chemotherapy with the control group of similar age and with patients with clinically manifesting senile or pre-menopausal osteoporosis indicates that this group is similar in terms of radiological structure to females at an advanced age. Thus, it is not surprising that similar symptoms are caused by high-dose chemotherapy with bone marrow or circulating progenitor cell transplantation [35-38]. Following treatment with chemotherapy only, 30% of female patients retain their ovary function [35], while when used in combination with radiation therapy (total body irradiation – TBI) only 10% of patients do so [36]. Of significance here are high doses of busulfan [37]. Castaneda and colleagues have observed that osteopenia occurs in 33% and osteoporosis in 18% of patients treated with bone marrow transplantation [38].

Oncological hormone therapy

As mentioned above, one of the crucial mechanisms causing bone metabolism disorders in the course of oncological treatment is the inhibition of gonad function. This occurs not only during cytotoxic treatment, but it mainly accompanies the hormone therapy used in treating hormonedependent neoplasms – breast cancer and prostate cancer. In patients of both genders, sex hormones play a crucial role in the maintenance of optimal bone mass [39, 40]. Its volume depends on the balance between bone formation and bone resorption. In recent years, the effects of oestrogen on those processes have been studied in great detail [41]. The regulator of bone remodelling is a signalling pathway, the main components of which are osteoprotegerin (OPG), the receptor activator of nuclear factor B (RANK) and its ligand (RANKL) – a protein from the tumour necrosis factor (TNF) family of which OPG is also a member [42]. The latter is produced by osteoblasts and its progenitors, and it is the main activator of osteoclast differentiation and maturation, influencing their activity, function and life span [43-45]. The binding of RANK with its ligand initiates a signalling cascade in osteoclasts and leads to the formation of a mature and fully functional resorption cell. Osteoprotegerin, produced, among others, in osteoblasts, is a soluble form of RANK and is capable of binding the ligand, by which it inhibits the RANK-RANKL binding and the process of osteoclast maturation. The osteoclast maturation and activity, and, thus, also the intensity of the bone resorption process, depend on the balance between RANKL and OPG. Oestrogens stimulate the OPG synthesis in osteoblasts, which leads to lowering of the RANKL level. The result is inhibition of bone remodelling and resorption, and stimulation of bone formation. In cases of oestrogen deficiency, the RANKL level goes up and the sensitivity of RANK to its ligand increases, which leads to the stimulation and hyperactivity of osteoclasts, accompanied by an increase in bone resorption and calcium release from bones. Apart from influencing the OPGRANKL-RANK pathway, oestrogens also stimulate the synthesis of transforming growth factor  (TGF-) and insulin-like growth factor 1 (IGF-1) [46, 47]. The effects of androgens on bone metabolism are also dependent on TGF- and IGF-1, but they are associated to a greater extent with testosterone conversion into oestradiol and the activity of the latter. The testosterone-oestradiol conversion is effected through aromatisation, and the aromatase level as well as the oestrogen receptor function are crucial for bone metabolism in males. There have been cases reported of pathological bone mass loss in males with oestrogen receptor mutation causing its inactivation and in those with genetically determined aromatase deficiency [48, 49]. Bone remodelling caused by oncological hormone treatment is similar to that seen in the course of natural menopause, with a rapid loss of bone mass (by approx. 2-3% each year). The loss affects mainly the trabecular bone (2030% in 10 years), while to a lesser extent the cortical bone (5-10% over the same period).

Hormone therapy in breast cancer

The main points of hormone therapy in breast cancer are the inhibition of ovary function, lowering the levels of circulating oestrogens, and the inhibition of cellular activity. To this end, analogues of luteinizing hormone-releasing hormone (LHRH), surgical oophorectomy, oestrogen receptor modulators and inhibitors of aromatase (the enzyme catalysing the conversion of androgens into oestrogens) are used. LHRH analogues are widely recognised drugs used in pre-menopausal patients. These derivatives of the natural peptide (characterised by increased activity and longer half-life as compared to the natural hormone) block the LHRH receptors on pituitary cells and thus lead to decreased gonadotropin secretion and pharmacological castration. It has been shown that after 6 months of such treatment there occurs a marked reduction in spinal BMD [50]. Tamoxifen belongs to the class of selective oestrogen receptor modulators (SERMs). In breast cancer cells, it acts as an antagonist of oestrogens, while in bones and endometrium it acts as a weak oestrogen (agonist activity). Still, it exerts a dual clinical effect, depending on the patient’s hormone status. In premenopausal females, it leads to BMD loss [51], while in post-menopausal patients the use of tamoxifen results in a significant increase in the bone density, as measured in the lumbar spine region [52, 53]. On the other hand, it has not been found that the use of tamoxifen in breast cancer prophylaxis significantly lowers the risk of fractures. The NSABP P-1 study has demonstrated a 21% reduction in the risk of fractures in a group of patients aged above 50 and receiving tamoxifen as a preventive measure, in comparison with the group receiving placebo; yet this difference did not prove significant (HR 0.79, 95% CI: 0.60-1.05) [54]. Meanwhile, the IBIS-1 study, evaluating tamoxifen in breast cancer prophylaxis in groups of pre- and post-menopausal females, did not show any differences in the fracture incidence [55]. Although certain aspects of treatment with aromatase inhibitors remain to be elucidated, their use has been increasing steadily in the last decade. These preparations are divided into non-steroid (letrozole and anastrozole) and steroid ones (exemestane). Their mode of action consists in reversible (non-steroid ones) or irreversible (steroid ones) inhibition of aromatase, the enzyme catalysing the testosterone-oestradiol conversion. This leads to a lowering of oestrogen levels in circulating blood in post-menopausal females by approx. 98% [56, 57]. This mechanism is responsible for adverse effects different to those seen in the treatment with tamoxifen. Numerous clinical studies have shown that tamoxifen results in lowered levels of bone metabolism markers and in higher BMD, while aromatase inhibitors have a reverse effect here. A direct comparison of three third-generation aromatase inhibitors has revealed that they have a similar effect on the levels of bone metabolism markers [58], although some researchers suggest that the use of exemestane results in a smaller loss of bone mass as compared with the use of letrozole or anastrozole [59]. This may stem from the steroid structure of exemestane – its main metabolite, 17-hydroxyexemestane, possesses internal androgen activity, thus providing a protective effect. Additional data may be derived from the MA.27 – bone protocol study. In the studies concerning adjuvant treatment (ATAC, BIG 1-98, IES), the use of all of the above-mentioned drugs resulted in an increased risk of fractures as compared with the use of tamoxifen [60-62]. The absolute differences have not been large (0.8-4%), yet proved significant. In the ATAC study, it was found that the use of anastrozole, as compared with tamoxifen, resulted in a significantly higher incidence of osteoporosis with complications in the form of fractures (11% vs. 7.7%, p < 0.0001), and that the incidence of adverse effects increased with time [61]. Also, the other non-steroid aromatase inhibitor – letrozole – caused fractures more often than tamoxifen (5.8% vs. 4%, p = 0.0006) [60]. In studies with sequential (following prior application of tamoxifen) use of aromatase inhibitors (IES, MA.17), the differences were smaller (in MA.17 the difference was not significant), which might be related to the protective action of tamoxifen on the bone density [62, 63]. The use of letrozole following 5-year treatment with tamoxifen (MA.17) resulted in a higher incidence of osteoporosis and fractures as compared with placebo [respectively: 8.1% and 6.0% for osteoporosis (p = 0.003), and 5.3% and 4.6% for fractures] [64, 65]. In the IES study, it was found that the use of exemestane, as compared with tamoxifen, caused a higher incidence of osteoporosis (7.4% and 5.7%, respectively, p = 0.05) and factures (3.1% and 2.3%, respectively) [62]. Also the ABCSG 8 and ARNO 95 studies demonstrated a higher percentage of fractures in the group of patients treated sequentially with an aromatase inhibitor (anastrozole), as compared with the treatment with tamoxifen only (2.4% vs. 2.1%), but the difference was not significant [66]. Summing up: the data from the abovementioned randomised trials indicate unambiguously that the use of aromatase inhibitors in adjuvant treatment is associated with a higher risk of osteoporosis and fractures. When analysing the risk of such complications in patients treated with aromatase inhibitors, the coexistence of other risk factors should be taken into account. These include: baseline T-score < –1.5, age > 65, body mass index (BMI) < 20, positive family history for hip fractures, oral steroid therapy for over 6 months, and tobacco smoking (at present or in the past). The importance of all the above factors has been confirmed in large clinical trials conducted on a population of healthy post-menopausal females [67-72].

Hormone therapy in prostate cancer

Antiandrogen treatment has found use in all stages of prostate cancer. Its basic idea is to limit the effects of androgens on neoplastic cells, achieved by surgical orchiectomy, the use of LHRH analogues, as well as androgen receptor antagonists and drugs directly inhibiting the synthesis of adrenal gland androgens (ketoconazole and aminoglutethimide). The antiandrogen treatment usually causes a marked BMD loss, most pronounced in the first year of therapy (2.4-10%) and growing by 1-2% each year, as compared with the loss associated with age [42, 73]. The retrospective Medicare 1992-2001 analysis (4494 patients) has shown that the use of antiandrogen hormone therapy increases the risk of BMD reduction to the osteopenia/osteoporosis level and the risk of fractures to 42%, as compared with 16% in the group not subject to antiandrogen treatment [74]. The bone mass loss in the course of antiandrogen treatment is also dependent on lifestyle: bad diet, alcohol abuse, tobacco smoking or a significant reduction in exercise. The oldest drug class in use is oestrogens (mainly diethylstilbestrol, DES – a half-synthetic oestrogen derivative), yet nowadays they are losing significance due to the high risk of thromboembolic complications [75, 76]. As regards bone, oestrogens do not exhibit toxicity. This is because their use, as opposed to surgical orchiectomy or LHRH analogue use, does not have a significant lowering effect on bone density [77], and may even exhibit a protective action on bones [78]. LHRH analogues currently constitute an alternative – one widely used and preferred by patients – to surgical orchiectomy [79]. In the prospective studies evaluating the effects of castration on the bone status, usually both methods (pharmacological and surgical) have been assessed together, and it has been shown that the treatment accelerates bone remodelling and results in a BMD reduction by 4-10% in the first year of therapy [73, 77, 80-83]. The above processes progress over the course of treatment and result in a higher risk of bone complications. Several studies have demonstrated an increase in the risk of fractures in patients treated with LHRH analogues [74]. In the population of patients with prostate cancer, fractures occur often, in several or a dozen or so patients [80, 81]. There is observed a significant correlation between fractures and shorter survival [86]. However, their causes are complex – apart from osteoporosis, there coexist bone metastases and injuries, often all in one patient [84, 85]. Surgical or pharmacological castration results in the lowering of circulating testosterone levels by approx. 90%, since peripheral conversion of adrenal gland androgens into testosterone accounts for the remaining 10% [87], hence the concept of therapy consisting in blocking of the androgen receptors on neoplastic cells by the so-called antiandrogens. There are distinguished two classes of these preparations: steroid (blocking the androgen receptor and inhibiting the testosterone production – cyproterone acetate) and non-steroid ones (blocking the receptor only – flutamide, nilutamide, bicalutamide). It has been shown that flutamide acts on osteoblast colonies as an androgen receptor agonist, which in these cells leads to increased synthesis of interleukin-6 and inhibition of synthesis of cytokines involved in bone resorption [86]. This mechanism may result in a reduction of bone mass in the course of testosterone deficiency. In this context, the combined use of flutamide and LHRH (maximum androgen blockage – MAB) would have a protective effect on bones. Unfortunately, this is not always the case in practice, as it has been shown that the use of MAB results in a bone density reduction by approx. 6% after 6 months of treatment [88]. Bicalutamide used in monotherapy may result in an increased bone density, as shown by prospective studies [89]. This is likely to be caused by elevated levels of circulating oestradiol during the treatment with bicalutamide.

Targeted therapy

The recently distinguished class of drugs defined as targeted therapy encompasses mainly monoclonal antibodies (usually directed against the extracellular receptor domains) and inhibitors of tyrosine kinase forming the intracellular portion of the membrane receptor. Adverse effects on bones in the course of treatment with the above preparations are not addressed, although some of those drugs are targeted at bones precisely. One of them is denosumab – a monoclonal antibody against RANKL, inhibiting the RANK/RANKL signalling pathway, which results in inhibited bone resorption in vitro and in vivo [90, 91]. Apart from its other uses, the drug produced promising results in studies on patients with advanced neoplastic disease affecting bones [92, 93].

Yet another class of substances belonging to targeted therapy are the inhibitors of Src tyrosine kinase which in an in vivo model have proven essential in forming the ruffled borders of osteoclast membrane, thus affecting their resorption potential [94].

The above preparations exhibit activity in pre-clinical studies and in early clinical trials, providing in a longer-term perspective the possibility of using them in cases of neoplasm-induced osteolysis [95, 96].

Of interest are observations on the bone-associated effects of targeted therapy (monoclonal antibodies and tyrosine kinase inhibitors) directed against insulin-like growth factor 1 (IGF-1), related to its activity stimulating RANKL synthesis and thus bone resorption, to its significance for the integrity of cortical bone (circulating IGF-1) and trabecular bone (skeletal IGF-1), and to the maintenance of bone structure in adults. It is also known that the IGF-1 level correlates with bone density in post-menopausal females [97-102].

A special role in the pathogenesis of neoplastic diseases affecting bones is played by a protein resembling the parathyroid hormone (PTHrP), responsible for rapid calcium release from bones, hypercalcaemia and weakening [103]. Hence, understandably, attempts are made at neutralising this factor. In in vitro studies, humanised monoclonal antibodies against PTHrP have proven effective in counteracting hypercalcaemia caused by this factor derived from osteosarcoma, lung cancer and breast cancer cells [104-106].

Bisphosphonates

Bisphosphonates are a class of drugs of established standing in oncology, also used in the treatment of osteoporosis and – in its labelled form – in bone scintigraphy. As pyrophosphate derivatives, bisphosphonates are classified in two main categories, depending on the presence or absence of a nitrogen atom in the molecule. Of crucial importance for their activity are radicals – particularly the presence of nitrogen in the R2 radical. Bisphosphonates are a standard of care in neoplastic diseases affecting bones [107]. The above substances are capable of inhibiting the bone metabolism by reducing the intensity of resorption. This stems from the effects they have on bone cells, particularly on osteoclasts. Bisphosphonates not only inhibit the activity of osteoclasts but also delay their maturation and accelerate apoptosis [108]. They are also capable of affecting the cell ultrastructure, mainly through disturbing the formation of ruffled membrane borders mentioned above [109, 110].

Bisphosphonates also act on osteoblasts, slowing down their proliferation, facilitating their maturation and stimulating their bone-forming activity, while by also changing the secretory potential of osteoblasts they indirectly decrease the osteoclast activity [111-113]. It is also worth mentioning that bisphosphonates induce apoptosis in the breast cancer cell line in vitro [114].

All the above makes this class of substances not only recommended as effective in preventing complications of neoplastic bone infiltration and the occurrence of new disease foci in bones, but also capable of reducing bone loss in patients after treatment of early breast cancer, and in addition it may prove of importance in the prevention of early breast cancer recurrence [107, 115-122].

References

  1. Nevinny HB, Krant MJ, Moore EW. Metabolic studies of the effects of methotrexate. Metabolism 1965; 14: 135-9.   

2. Ragab AH, French RS, Vietti TJ. Osteoporotic fractures secondary to methotrexate therapy of acute leukemia in remission. Cancer 1970; 25: 580-5.   

3. O’Regan S, Melhorn DK, Newman AJ. Methotrexate-induced bone pain in childhood leukemia. Am J Dis Child 1973; 126: 489-90.   

4. Stanisavljevic S, Babcock AL. Fractures in children treated with methotrexate for leukemia. Clin Ortho 1977; 125: 139-44.   

5. Hui L, Wiernik PH. Avascular necrosis of bone after adult acute lymphocytic leukemia treatment with methotrexate, vincristine, L-asparaginase, and dexamethasone (MOAD). Am J Hematol 1996; 52: 184-8.   

6. Nesbit M, Krivit W, Heyn R, Sharp H. Acute and chronic effects of methotrexate on hepatic, pulmonary, and skeletal systems. Cancer 1976; 37: 1048-54.   

7. Schwartz A M, Leonidas JC. Methotrexate osteopathy. Skeletal Radiol 1984; 11: 13-6.   

8. Halton JM, Atkinson SA, Fraher L, Webber C, Gill GJ, Dawson S, Barr RD. Altered mineral metabolism and bone mass in children during treatment for acute lymphoblastic leukemia. J Bone Miner Res 1996; 11: 1774-83.   

9. Zonneveld IM, Bakker WK, Dijkstra PF, Bos JD, van Soesbergen RM, Dinant HJ. Methotrexate osteopathy in long-term, low-dose methotrexate treatment for psoriasis and rheumatoid arthritis. Arch Dermat 1996; 132: 184-7.  

10. Maenaut K, Westhovens R, Dequeker J. Methotrexate osteopathy, does it exist? J Rheumatol 1996; 23: 2156-9.  

11. Dequeker J, Maenaut K, Verwilghen J, Westhovens R. Osteoporosis in rheumatoid arthritis. Clin Exp Rheumatol 1995; 13 (Suppl 12): S21-S26.  

12. Shapira D, Scharf Y. Insufficiency fracture of the distal tibia mimicking arthritis in a rheumatoid arthritis patient. The possible role of methotrexate treatment. Clin Exp Rheumatol 1995; 13: 130-1.  

13. Preston SJ, Diamond T, Scott A, Laurent MR. Methotrexate osteopathy in rheumatic disease. Ann Rheum Dis 1993; 52: 582-5.  

14. Ward SB, Smith JB, Maccario D, Abruzzo JL. Serum osteocalcin (OC) in assessment of methotrexate (MTX) induced osteoporosis. Arthritis Rheum 1992; 35 (suppl 5): R5.  

15. Scheven BA, van der Veen MJ, Damen CA, Labefer FP, van Rijn HJ, Bijlsma JW, Duursma SA. Effects of methotrexate on human osteoblasts in vitro: modulation by 1,25-dihydroxyvitamin D3. J Bone Miner Res 1995; 10: 874-80.  

16. van der Veen MJ, Scheven BA, van Roy JL, Damen CA, Lafeber FP, Bijlsma JW. In vitro effects of methotrexate on human articular cartilage and bone-derived osteoblasts. Br J Rheumatol 1996; 35: 342-9.  

17. Bologna C, Edno L, Anaya J-M, et al. Methotrexate Concentration in Synovial Membrane and Trabecular and Cortical Bone in Rheumatoid Arthritis Patients. Arthritis Rheum 1994; 12: 1770-3.  

18. Katz JN, Le Boff MS, Wade JP, Brown EM, Liang MH. Effect of methotrexate on bone density and calcium homeostasis in rheumatoid arthritis. Clin Res 1989; 37: 509A.  

19. Meister B, Gassner I, Streif W, Dengg K, Fink FM. Med Pediatr Onkol 1994; 23: 493-6.  

20. Ecklund K, Laor T, Goorin AM, Connoly LP, Jaramillo D Methotrexate osteopathy in patients with osteosarcoma. Radiology 1997; 202: 543-7.  

21. Gnudi S, Butturini L, Ripamonti C, Avella M, Bacci G. The effects of Methotrexate (MTX) on bone. Ital J Orthop Traumatol 1988; 14: 227-31.  

22. Smeitink J, Verreussel M, Schroder C, Lippens R. Nephrotoxicity associated with ifosfamide. Eur J Pediatr 1988; 148: 164-6.  

23. De Schepper J, Hachimi-Idrissi S, Louis O, Maurus R, Otten J. Bone metabolism and mineralisation after cytotoxic chemotherapy including ifosfamide. Arch Dis Child 1994; 71: 346-8.  

24. Burk CD, Restaino I, Kaplan BS, Meadows AT. Ifosfamide-induced renal tubular dysfunction and rickets in children with Wilms tumour. J Pediatr 1990; 117: 331-5.  

25. Skinner R, Pearson AD, Price L, Coulthard MG, Craft AW. Nephrotoxicity after ifosfamide. Archives of Disease in Childhood, 1990; 65: 732-8.  

26. Kother M, Schindler J, Oette K, Berthold F. Abnormalities in serum osteocalcin values in children receiving chemotherapy including ifosfamide. In Vivo 1992; 6: 219-21.  

27. Kreuser ED, Felsenberg D, Behles C, Seibt-Jung H, Mielcarek M, Diehl V, Dahmen E, Thiel E. Long-term gonadal dysfunction and its impact on bone mineralization in patients following COPP/ABVD chemotherapy for Hodgkin's disease. Ann Oncol 1992; 3 suppl 4: 105-10.  

28. Redman JR, Bajorunas DR, Wong G, McDermott K, Gnecco C, Schneider R, Lacher MJ, Lane JM. Bone mineralization in women following successful treatment of Hodgkin's disease Bone mineralization in women following successful treatment of Hodgkin's disease. Am J Med 1988; 85: 65-72.  

29. Holmes SJ, Whitehouse RW, Clark ST, Crowther DC, Adams JE, Shalet SM. Reduced bone mineral density in men following chemotherapy for Hodgkin's disease. Br J Cancer 1994; 70: 371-5.  

30. Bokemeyer C, Schmoll HJ, van Rhee J, Kuczyk M, Schuppert F, Poliwoda H. Long-term gonadal toxicity after therapy for Hodgkin's and non-Hodgkin's lymphoma. Ann Hematol 1994; 68: 105-10.  

31. Ratcliffe MA, Lanham SA, Reid DM, Dawson AA. Bone mineral density (BMD) in patients with lymphoma: the effects of chemotherapy, intermittent corticosteroids and premature menopause. Hematol Oncol 1992; 10: 181-7.  

32. Bruning PF, Pit MJ, de Jong-Bakker M, van den Ende A, Hart A, van Enk A. Bone mineral density after adjuvant chemotherapy for premenopausal breast cancer. Br J Cancer 1990; 61: 308-10.  

33. Fogelman I, Blake GM, Blamey R, et al. Bone mineral density in premenopausal women treated for node-positive early breast cancer with 2 years of goserelin or 6 months of cyclophosphamide, methotrexate and 5-fluorouracil (CMF). Osteoporos Int 2003; 14: 1001-6.  

34. Leśniewski-Kmak K, Zieliński KW, Szczylik C. Quantitative assessment of the clavicle radiostructure as a tool for estimation of the osteopathic effect of breast cancer chemotherapy. Breast Cancer Research and Treatment 2002; 73: 189-97.  

35. Chatterjee R, Mills W, Katz M, McGarrigle HH, Goldstone AH. Prospective study of pituitary-gonadal function to evaluate short-term effects of ablative chemotherapy or total body irradiation with autologous or allogenic marrow transplantation in post-menarcheal female patients. Transplant 1994; 13: 511-7.  

36. Schimmer AD, Quatermain M, Imrie K, et al. Ovarian function after autologous bone marrow transplantation. J Clin Oncol 1998; 16: 2359-63.  

37. Teinturier C, Hartmann O, Valteau-Couanet D, Benhamou E, Bougneres PF. Ovarian function after autologous bone marrow transplantation in childhood: high-dose busulfan is a major cause of ovarian failure. Bone Marrow Transplant 1998; 22: 989-94.  

38. Castaneda S, Carmona L, Carvajal I, Arranz R, Diaz A, Garcia-Vadillo A. Reduction of bone mass in women after bone marrow transplantation. Calcif Tissue Int 1997; 60: 343-7.  

39. Turner RT, Riggs BL, Spelsberg TC. Skeletal effects of estrogen. Endocr Rev 1994; 15: 275-300.  

40. Hofbauer LC, Khosla S. Androgen effects on bone metabolism: Recent progress and controversies: Eur J Endocrinol 1999; 140: 271-86.  

41. Riggs BL, Khosla S, Melton LJ 3rd. Sex steroids and the construction and conservation of the adult skeleton. Endocr Rev 2002; 23: 279-302.  

42. Hofbauer LC, Schoppet M. Clinical implications of the osteoprotegerin/RANKL/RANK system for bone and vascular disease. JAMA 2004; 292: 490-5.  

43. Matsuzaki K, Udagawa N, Takahashi N, et al. Osteoclast differentiation factor (ODF) induces osteoclast-like cell formation in human peripheral blood mononuclear cell cultures. Biochem Biophys Res Commun 1998; 246: 199-204.  

44. Nakagawa N, Kinosaki M, Yamaguchi K, Shima N, Yasuda H, Yano K, Morinaga T, Higashio K. RANK is the essential signaling receptor for osteoclast differentiation factor in osteoclastogenesis. Biochem Biophys Res Commun 1998; 253: 395-400.  

45. Yasuda H, Shima N, Nakagawa N, et al. Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis inhibitory factor and is identical to TRANCE/RANKL. Proc Natl Acad Sci USA 1998; 95: 3597-602.  

46. Ernst M, Heath JK, Rodan GA. Estradiol effects on proliferation, messenger ribonucleic acid for collagen and insulin-like growth factor-I, and parathyroid hormone-stimulated adenylate cyclase activity in osteoblastic cells from calvariae and long bone. Endocrinology 1989; 125: 825-33.  

47. Oursler MJ, Cortese C, Keeting PE, et al. Modulation of transforming growth factor-beta production in normal human osteoblast-like cells by 17beta-estradiol and parathyroid hormone. Endocrinology 1991; 129: 3313-20.  

48. Smith EP, Boyd J, Frank GR, et al. Estrogen resistance caused a mutation in the estrogen receptor gene in a man. N Engl J Med 1994; 331: 1056-61.  

49. Carani C, Qin K, Simoni M, Faustini-Fustini M, Serpente S, Boyd J, Korach KS, Simpson ER. Effect of testosterone and estradiol in a man with aromatase deficiency. N Engl J Med 1997; 337: 91-5.  

50. Johansen JS, Riis BJ, Hassager C, Moen M, Jacobson J, Christiansen C. The effect of gonadotropin-releasing hormone agonist analog (nafarelin) on bone metabolisme. J Clin Endocrinol Metab 1988; 67: 701-6.

51. PowlesTJ, HickishT, Kanis JA, Tidy A, Ashley S. Effect of tamoxifen on bone mineral density measured by dual-energy X-ray absorptiometry in healthy premenopausal and postmenopausal women. J Clin Oncol 1996; 14: 78-84.  

52. Love RR, Mazess RB, Barden HS, Epstein S, Newcomb PA, Jordan VC, Carbone PP, DeMets DL. Effects of tamoxifen on bone mineral density in postmenopausal women with breast cancer. N Engl J Med 1992; 326: 852-6.  

53. Kristensen B, Ejlertsen B, Dalgaard P, Larsen L, Holmegaard SN, Transbol I, Mouridsen HT. Tamoxifen and bone metabolism in postmenopausal low-risk breast cancer patients. Oncol 1994; 12: 992-7.  

54. Fisher B, Costantino JP, Wickerham DL, et al. Tamoxifen for prevention of breast cancer: report of the National Surgical Adjuvant Breast and Bowel Project P-1 Study. J Natl Cancer Inst 1998; 90: 1371-88.  

55. Cuzick J, Forbes J, Edwards R, et al. First results from the International Breast Cancer Intervention Study (IBIS-I): a randomised prevention trial. Lancet 2002; 360: 817-24.  

56. Joensuu H, Ejlertsen B, Lonning PE, Rutqvist LE. Aromatase inhibotors in the treatment of early and advanced breast cancer. Acta Oncol 2005; 44: 201-2.

 57. Strasser-Weippl K, Goss PE. Advances in adjuvant hormonal therapy for postmenopausal women. J Clin Oncol 2005; 23: 1751-9.

 58. McCloskey E, Hannon R, Lakner G et al. Effects of third generation aromatase inhibitors on bone health and other safety parameters: results of an open, randomised, multi-centre study of letrozole, exemestane and anastrozole in healthy postmenopausal women. Eur J Cancer 2007; 43: 2523-31.

 59. Chien AJ, Goss PE. Aromatase inhibitors and bone health in women with breast cancer. J Clin Oncol 2006; 24: 5305-12.

 60. Coates AS, Keshaviah A, Thurlimann B, et al. Five years of letrozole compared with tamoxifen as initial adjuvant therapy for postmenopausal women with endocrine-responsive early breast cancer: update of study BIG 1-98. J Clin Oncol 2007; 25: 486-92.

 61. Baum M, Buzdar A, Cuzick J, et al. Anastrozole alone or in combination with tamoxifen versus tamoxifen alone for adjuvant treatment of postmenopausal women with early-stage breast cancer: results of the ATAC (Arimidex, Tamoxifen Alone or in Combination) trial efficacy and safety update analyses. Cancer 2003; 98: 1802-10.

 62. Coombes RC, Hall E, Gibson LJ, et al. A randomized trial of exemestane after two to three years of tamoxifen therapy in postmenopausal women with primary breast cancer. N Engl J Med 2004; 350: 1081-92.  

63. Perez EA, Josse RG, Pritchard KI, et al. Effect of letrozole versus placebo on bone mineral density in women with primary breast cancer completing 5 or more years of adjuvant tamoxifen: a companion study to NCIC CTG MA.17. J Clin Oncol 2006; 24: 3629-35.  

64. Goos PE, Ingle JN Martino S, et al. A randomized trial of letrozol in postmenopausal women after five years of tamoxifen therapy for early stage breast cancer. N Eng J Med 2003; 349: 1793-802.  

65. Goos PE, Ingle Jn Martino S, et al. Randomized trial of letrozol following tamoxifen as extended adjuvant therapy in receptor-positive breast cancer: updated findings fram NCIC CTG MA. 17 J Natl Cancer Inst 2005; 97: 1262-71.  

66. Jakesz R, Jonat W, Gnant M, et al. Switching of postmenopausal women with endocrine responsive early breast cancer to anastrozole after two years’adjuvant tamoxifen: combined results of ABCSG trial 8 and ARNO 95 trial. Lancet 2005; 366: 445-62.  

67. Siris ES, Chen YT, Abbott TA, Barrett-Connor E, Miller PD, Wehren LE, Berger ML. Bone mineral density thresholds for pharmacological intervention to prevent fractures. Arch Intern Med 2004; 164: 1108-12.  

68. Albrand G, Munoz F, Sornay-Rendu E, DuBoeuf F, Delmas PD. Independent predictors of all osteoporosis-related fractures in healthy postmenopausal women: the OFELY study. Bone 2003; 32: 78-85.  

69. De Laet C, Kanis JA, Oden A, et al. Body mass index as a predictor of fracture risk: a metaanalysis. Osteoporos Int 2005; 16: 1330-8.

 70. Kanis JA, Johansson H, Oden A, et al. A family history of fracture and fracture risk: a meta-analysis. Bone 2004; 35: 1029-37.  

71. van Staa TP, Leufkens HGM, Cooper C. The epidemiology of corticosteroid-induced osteoporosis: a meta-analysis. Osteoporos Int 2002; 13: 777-87.  

72. Tattersfield AE, Harrison TW, Hubbard RB, Mortimer K. Safety of inhaled corticosteroids. Proc Am Thorac Soc 2004; 1: 171-5.  

73. Daniell HW, Dunn SR, Ferguson DW, Lomas G, Niazi Z, Stratte PT. Progressive osteoporosis during androgen deprivation therapy for prostate cancer. J Urol 2000; 163: 181-6.

 74. Smith MR, Lee WC, Brandman J, Wang Q, Botteman M, Pashos CL. Gonadotropinreleasing hormone agonists and fracture risk: a claims-based cohort study of men with nonmetastatic prostate cancer. J Clin Oncol 2005; 23: 7897-903.  

75. Byar DP, Corle DK. Hormone therapy for prostate cancer: results of the Veterans Administration Cooperative Urological Research Group studies. NCI Monogr 1988; 7: 165-70.  

76. Bishop MC, Lemberger RJ, Selby C, Lawrence WT. Oestrogen dosage in prostatic cancer: the threshold effect? Br J Urol 1989; 64: 290-6.

 77. Eriksson S, Eriksson A, Stege R, Carlström K. Bone mineral density in patients with prostatic cancer treated with orchidectomy and with estrogens. Calcif Tissue Int 1995; 57: 97-9.  

78. Ockrim JL, Lalani EN, Banks LM, et al. Transdermal estradiol improves bone density when used as single agent therapy for prostate cancer. J Urol 2004; 172: 2203-7.  

79. Hellerstedt BA, Pienta KJ. The current state of hormonal therapy for prostate cancer. CA Cancer J Clin 2002; 52: 154-79.  

80. Maillefert JF, Sibilia J, Michel F, Saussine C, Javier RM, Tavernier C. Bone mineral density in men treated with synthetic gonadotropin-releasing hormone agonists for prostatic carcinoma. J Urol 1999; 161: 1219-22.

 81. Smith MR, McGovern FJ, Zietman AL, Fallon MA, Hayden DL, Schoenfeld DA, Kantoff PW, Finkelstein JS. Pamidronate to prevent bone loss during androgen-deprivation therapy for prostate cancer. N Engl J Med 2001; 345: 948-55.

 82. Berruti A, Dogliotti L, Terrone C, et al. Changes in bone mineral density, lean body mass and fat content as measured by dual energy x-ray absorptiometry in patients with prostate cancer without apparent bone metastases given androgen deprivation therapy. J Urol 2002; 167: 2361-7.

 83. Smith MR, Eastham J, Gleason DM, Shasha D, Tchekmedyian S, Zinner N, et al. Randomized controlled trial of zoledronic acid to prevent bone loss in men receiving androgen deprivation therapy for nonmetastatic prostate cancer. J Urol 2003; 169: 2008-12.  

84. Townsend MF, Sanders WH, Northway RO, Graham SD Jr. Bone fractures associated with luteinizing hormone-releasing hormone agonists used in the treatment of prostate carcinoma. Cancer 1997; 79: 545-50.  

85. Oefelein MG, Ricchiuti V Conrad W, Resnick MI. Skeletal fractures negatively correlate with overall survival in men with prostate cancer. J Urol 2002; 168: 1005-7.  

86. Hofbauer LC, Khosla S. Androgen effects on bone metabolism: Recent progress and controversies. Eur J Endocrinol 1999; 140: 271-86.  

87. Hellerstedt BA, Pienta KJ. The current state of hormonal therapy for prostate cancer. CA Cancer J Clin 2002; 52: 154-79.

 88. Diamond T, Campbell J, Bryant C, Lynch W. The effect of combined androgen blockade on bone turnover and bone mineral densities in men treated for prostate carcinoma: Longitudinal evaluation and response to intermittent cyclic etidronate therapy. Cancer 1998; 83: 1561-6.

 89. Sieber PR, Keiller DR, Kahnoski RJ, Gallo J, McFadden S. Bicalutamide 150 mg maintains bone mineral density during monotherapy for localised or locally advanced prostate cancer. J Urol 2004; 171: 2272-6.

 90. Gerstenfeld LC, Sacks DJ, Pelis M, et al. Comparison of Effects of the Bisphosphonate Alendronate Versus the RANKL Inhibitor Denosumab on Murine Fracture Healing. J Bone Miner Res 2009; 24: 196-208.  

91. Kostenuik P, Nguyen H, McCabe J, et al. Denosumab, a fully human monoclonal antibody to RANKL, inhibits bone resorption and increases bone density in knock-in mice that express chimeric (murine/human) RANKL. J Bone Miner Res 2009; 24: 182-95.

 92. Lipton A, Steger GG, Figueroa J, et al. Randomized active-controlled phase II study of denosumab efficacy and safety in patients with breast cancer-related bone metastases. J Clin Oncol 2007; 25: 4431-7.  

93. Fizazi K, Lipton A, Mariette X, Body JJ, Rahim Y, Gralow JR, et al. Randomized phase II trial of denosumab in patients with bone metastases from prostate cancer, breast cancer, or other neoplasms after intravenous bisphosphonates. J Clin Oncol 2009; 27: 1564-71.  

94. Boyce BF, YonedaT, Lowe C, Soriano P, Mundy GR. Requirement of pp60c-src expression for osteoclasts to form ruffled borders and resorb bone in mice. J Clin Invest 1992; 90: 1622-7.  

95. Boyce BF, Xing L, Shakespeare W, Wang Y, Dalgarno D, Iuliucci J, Sawyer T. Regulation of bone remodeling and emerging breakthrough drugs for osteoporosis and osteolytic bone metastases. Kidney Int Suppl 2003; S2-5.  

96. Hannon RA, Clack G, Rimmer M, Swaisland A, Lockton JA, Finkelman RD, Eastell R. Effects of the Src kinase inhibitor saracatinib (AZD0530) on bone turnover in healthy men: a randomized, double-blind, placebo-controlled, multiple-ascending-dose phase I trial. J Bone Miner Res 2010; 25: 463-71.  

97. Wang Y, Nishida S, Elalieh HZ, Long RK, Halloran BP, Bikle DD. Role of IGF-I signaling in regulating osteoclastogenesis. J Bone Miner Res 2006; 21: 1350-8.  

98. Yakar S, Canalis E, Sun H, et al. Serum IGF-1 determines skeletal strength by regulating sub-periosteal expansion and trait interactions. J Bone Miner Res 2009; 24: 1481-92.  

99. Zhang M, Xuan S, Bouxsein ML, et al. Osteoblast-specific knockout of the insulin-like growth factor (IGF) receptor gene reveals an essential role of IGF signaling in bone matrix mineralization. J Biol Chem 2002; 277: 44005-12.

100. Zhao G, Monier-Faugere MC, Langub MC, et al. Targeted overexpression of insulin-like growth factor I to osteoblasts of transgenic mice: increased trabecular bone volume without increased osteoblast proliferation. Endocrinology 2000; 141: 2674-82.

101. Giustina A, Mazziotti G, Canalis E. Growth hormone, insulin-like growth factors, and the skeleton. Endocr Rev 2008; 29: 535-59.

102. Langlois JA, Rosen CJ, Visser M, Hannan MT, Harris T, Wilson PW, Kiel DP. Association between insulin-like growth factor I and bone mineral density in older women and men: the Framingham Heart Study. J Clin Endocrinol Metab 1998; 83: 4257-62.

103. Martin TJ, Grill V. Hypercalcemia in cancer. J Steroid Biochem Mol Biol 1992; 43: 123-9.

104. Onuma E, Sato K, Saito H, et al. Generation of a humanized monoclonal antibody against human parathyroid hormone-related protein and its efficacy against humoral hypercalcemia of malignancy. Anticancer Res 2004; 24: 2665-73.

105. Sato K, Onuma E, Yocum RC, Ogata E. Treatment of malignancy-associated hypercalcemia and cachexia with humanized anti-parathyroid hormone-related protein antibody. Semin Oncol 2003; 30, supl. 16: 167-73.

106. Saito H, Tsunenari T, Onuma E, Sato K, Ogata E, Yamada-Okabe H. Humanized monoclonal antibody against parathyroid hormone-related protein suppresses osteolytic bone metastasis of human breast cancer cells derived from MDA-MB-231. Anticancer Res 2005; 25: 3817-23.

107. Aapro M, Abrahamsson PA, Body JJ, et al. Guidance on the use of bisphosphonates in solid tumours: recommendations of an international expert panel. Ann Oncol 2008; 19: 420-32.

108. Ito M, Amizuka N, Nakajima T, Ozawa H. Ultrastructural and cytochemical studies on cell death of osteoclasts induced by bisphosphonate treatment. Bone 1999; 25: 447-52.

109. Murakami H, Takahashi N, Sasaki T, et al. A possible mechanism of the specific action of bisphosphonates on osteoclasts: tulidronate preferentially affects polarized osteoclasts having ruffled border. Bone 1995; 17: 137-44.

110. Sato M, Grasser W, Endo N, Akins R, Simmons H, Thompson DD, Golub E, Rodan GA. Bisphosphonate action. Alendronate localization in rat bone and effects on osteoclasts structure. J Clin Invest 1991; 88: 2095-105.

111. Reinholz GG, Getz B, Pederson L, Sanders ES, Subramaniam M, Ingle JN, Spelsberg TC. Bisphosphonates directly regulate cell proliferation, differentiation, and gene expression in human osteoblasts. Cancer Res 2000; 60: 6001-7.

112. Giuliani N, Pedrazzoni M, Passeri G, Girasole G. Bisphosphonates inhibit IL-6 production by human osteoblast-like cells. Scand J Reumatol 1998; 27: 38-41.

113. Vitté C, Fleisch H, Guenther HL. Bisphosphonates induce osteoblasts to secrete an inhibitor of osteoclasts-mediated resorption. Endocrinology 1996; 137: 2324-33.

114. Fromigue O, Lagneaux L, Body JJ. Bisphosphonates induce breast cancer cell death in vitro. J Bone Miner Res 2000; 15: 2211-21.

115. Diel IJ, Solomayer EF, Costa SD, Gollan C, Goerner R, Wallwiener D, Kaufmann M, Bastert G. Reduction in new metastases in breast cancer with adjuvant clodronate treatment. N Engl J Med 1998; 339: 357-63.

116. Powles TJ, McCloskey E, Paterson AH, Ashley S, Tidy VA, Nevan-taus A, Rosenqvist K, Kanis J. Oral clodronate and reduction in loss of bone mineral density in women with operable primary breast cancer. J Natl Cancer Inst 1998; 90: 704-8.

117. Gnant MF, Mlineritsch B, Luschin-Ebengreuth G, et al Adjuvant endocrine therapy plus zoledronic acid in premenopausal women with early-stage breast cancer: 5-year follow-up of the ABCSG-12 bone-mineral density substudy. Lancet Oncol 2008; 9: 840-9.

118. Brufsky AM, Bosserman LD, Caradonna RR, et al. Zoledronic acid effectively prevents aromatase inhibitor-associated bone loss in postmenopausal women with early breast cancer receiving adjuvant letrozole: Z-FAST study 36-month follow-up results. Clin Breast Cancer 2009; 9: 77-85.

119. Eidtmann H, de Boer R, Bundred N, et al. Efficacy of zoledronic acid in postmenopausal women with early breast cancer receiving adjuvant letrozole: 36-month results of the ZO-FAST Study. Ann Oncol 2010; 21: 2188-94.

120. Hershman DL, McMahon DJ, Crew KD, Cremers S, Irani D, Cucchiara G, Brafman L, Shane E. Zoledronic acid prevents bone loss in premenopausal women undergoing adjuvant chemotherapy for early-stage breast cancer. J Clin Oncol 2008; 26: 4739-45.

121. Powles T, Paterson A, McCloskey E, et al. Reduction in bone relapse and improved survival with oral clodronate for adjuvant treatment of operable breast cancer [ISRCTN83688026]. Breast Cancer Res 2006; 8: R13.

122. Kokufu I, Kohno N, Takao S, et al. Adjuvant pamidronate (PMT) therapy for the prevention of bone metastasis in breast cancer (BC) patients (pts) with four or more positive nodes. Proc Am Soc Clin Oncol 2004; 23: 9.





Address for correspondence

Krzysztof Leśniewski-Kmak MD

Medical University of Gdańsk

Department of Oncology Propaedeutics

Powstania Styczniowego 9b

81-519 Gdynia, Poland

e-mail: klkmak@gumed.edu.pl