[CANCER RESEARCH 51. 3059-3061. June 1. 1991] Advances in Brief Localization of Parathyroid Hormone-related Protein in Breast Cancer Métastases: Increased Incidence in Bone Compared with Other Sites' Gerard J. Powell,2 Justine Southby, Janine A. Danks,1 Ross G. Stillwell, John A. Hayman, Michael A. Henderson,4 Richard C. Bennett, and T. John Martin5 I'nirersily of Melbourne Department of Surgery ¡(j.J. P., M. A. //., R. C. B.¡,St. lïncenl'sInstitute of Medical Research and L'niversity of Melbourne Department of Medicine [J. S.. J. A. D., T. J. M.], and Department oj Anatomical Pathology /R. G. S.J, St. I 'incenl's Hospital, Melbourne, I'¡doria3065, Australia, anil L'niversity of Melbourne Department of Pathology, Repatriation Cenerai Hospital, Heidelberg, Victoria 3081, Australia [J. A. H.J Abstract Parathyroid hormone-related protein (FI'HrP) has recently been iden tified in 60% of a series of primary breast cancers. The detection of a bone-resorbing factor in tumors with a propensity to metastasize to bone prompted study of PTHrP in breast cancer metastasis. PTHrP »as localized by immunohistology in 12 of 13 (92%) breast cancer métastases in bone and in 3 of 18 (17%) métastases in non-bone sites. The statistical difference »ashighly significant (P < 0.0001). Production of PTHrP as a bone-resorbing agent may contribute to the ability of breast cancers to grow as bone métastases. Introduction PTHrP6 is recognized as the principal agent responsible for the increased bone résorptionand reduced calcium excretion accompanying the humoral hypercalcemia of malignancy (1). In addition, it has been identified in normal human skin (2), as well as in a series of squamous cell cancers in normocalcemic subjects (2). We have demonstrated PTHrP immunohistochem- ically in 60% of primary tumors from 101 unselected patients with breast cancer (3). Seven of these patients have developed bone métastasesin the early period of follow-up and all have had primary tumors that have stained positively for PTHrP. These observations and the frequency with which breast cancer metastasizes to bone (4) prompted the present investigation, in which immunohistochemistry has been used to determine the incidence of PTHrP staining in a retrospective series of breast cancer métastasesin bone and non-bone sites. The metastatic process is a complex, nonstochastic sequence of essential steps, in which a metastatic cancer cell must interact with the microen- vironment of the target organ in a manner that allows tumor growth if this process is to be completed. PTHrP is a powerful stimulator of bone résorption(5). This has led us to consider whether PTHrP produced by breast cancers might contribute to their ability to establish and grow in bone. Materials and Methods Paraffin blocks of breast cancer métastaseswere obtained from pathology archives for all biopsy-proved métastases. Those arising from Received 2/26/91; accepted 4/11/91. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 L'.S.C. Section 1734 solely to indicate this fact. 1Supported by the Anti-Cancer Council of Victoria and the National Health and Medical Research Council of Australia. 2 Recipient of a Cockburn Research Scholarship of the Royal Australasian College of Surgeons Foundation and the R. J. Fletcher Research Scholarship of the University of Melbourne. ' Recipient of an Australian Research Council Individual Fellowship. 4 Recipient of a Florance Cancer Research Fellowship of the Royal Australa sian College of Surgeons Foundation. * To whom requests for reprints should be addressed. 6 The abbreviation used is: PTHrP. parathyroid hormone-related protein. breast primaries were positively identified and métastasesto regional lymph nodes and local recurrences were excluded from study. Post mortem specimens were also excluded inasmuch as these have previ ously been found to be unsuitable for immunohistochemistry due to the effects of autolysis. The patient's records were used to confirm the findings and to provide relevant clinical data. A total of 42 biopsy- proved, distant breast cancer métastaseswere identified from 40 pa tients over the period 1986 to 1990 for which there were complete records. Of these, sufficient material was available for immunohisto- chemical study in 31 cases. Tumor Samples. In the case of soft tissue métastases,the tumor tissue had been formalin fixed, routinely processed, and embedded in paraffin. Tissue samples taken from bone had been decalcified in citric (2.5%)/formic acid (8.75%) solution (6) following fixation before proc essing and paraffin embedding. Sections (5 urn thick) at either end of those cut serially were stained in hematoxylin and eosin and reviewed by a pathologist to ensure that representative tumor was present in the sections. Immunohistochemistry. The primary antibody was a polyclonal anti- serum raised against the amino-terminal portion of the PTHrP mole cule by injecting synthetic PTHrP(l-34) into New Zealand White rabbits. The antiserum (255.5) was characterized as described previ ously (2. 3) and demonstrated no detectable cross-reactivity with para thyroid hormone. This antiserum compares favorably with other PTHrP antisera used previously in ¡mmunohistochemistry (2, 7). The peroxidase-antiperoxidase method of tissue antigen localization used was modified from that described by Danks et al. (2), based on the technique of Sternberger el al. (8). Controls. To ensure specificity of staining for PTHrP, antibody and method controls were performed (2) including (a) deletion of alternate layers of the antibody sandwich (primary antiserum, secondary anti- serum, and PAP complex) and (b) preabsorption of anti-PTHrP(l-34) antiserum overnight with 0.5 mg/ml synthetic PTHrP(l-34) and hu man PTH(l-34), respectively, prior to immunohistochemistry on skin and breast cancer specimens previously found to stain positively for PTHrP. To ascertain that the décalcificationtechnique did not alter PTHrP staining, a fresh parathyroid adenoma was placed in décalcifi cation solution, processed, and used as a positive control for immuno- staining (7). All experiments included samples of human skin as a positive control (2) and the substitution of primary antibody with preimmune rabbit serum as a negative control. Evaluation of Staining. The stained sections were reviewed by three independent observers and compared with controls and hematoxylin and eosin-stained slides. Tumor métastaseswere called positive if any of the tumor cells stained brown. Sections involving bone marrow were reviewed by a pathologist to ensure that marrow stem cells were not confused with tumor. There was concordance of opinion in all cases except one specimen from bone marrow which was equivocal and therefore judged to be negative. Results Of the 31 specimens obtained for study. 13 were from bone and 18 were from various non-bone sites (Table 1). The 13 bone métastasesincluded 6 diagnostic trephines, reamings from 4 3059 Research. on August 31, 2017. © 1991 American Association for Cancer cancerres.aacrjournals.org Downloaded from