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Letters to the Editor |
1 Endocrine Unit and 3
Pediatric Nephrology Unit, Massachusetts General Hospital and Harvard Medical School, Boston, MA
2 Department of Oral and Maxillofacial Surgery, Massachusetts General Hospital and Dental Department, Harvard Vanguard Medical Associates, Boston, MA
aAddress correspondence to this author at: Massachusetts General Hospital, Endocrine Unit, Thier 1051, 55 Fruit Street, Boston, MA 02114, Fax (617) 726-7543, E-mail hjueppner{at}partners.org
To the Editor:
Oncogenic osteomalacia (OOM)1 is a rare tumor-induced disease characterized by hypophosphatemia due to a decreased renal threshold of phosphate reabsorption, low 1,25-dihydroxyvitamin D concentrations, and osteomalacia (1). Determining the location of OOM tumors, which often produce excess amounts of the phosphaturic hormone fibroblast growth factor-23 (FGF-23), can be difficult, and confirmation of successful tumor removal may require prolonged postoperative observation until the return of serum indicators to reference-interval concentrations (2). Here, we report the modification of a commercially available intact FGF-23 assay (3), which enabled us to rapidly document high FGF-23 content in OOM tumor extracts. The assay takes <30 min to complete, and visual inspection of the test plate is sufficient to distinguish positive from negative samples, therefore allowing fast intraoperative assessment of FGF-23 content in OOM tumor extracts.
Tumor tissue from 6 patients with OOM was used in this study. Tumor 1 tissue was from a 63-year-old woman with biochemical abnormalities characteristic of OOM. Ten years before the current study, this patient underwent resection of a small mesenchymal tumor in the maxilla, and thereafter her blood and urine chemistry indicators returned to reference intervals. The patient again developed hypophosphatemia, however, and underwent a second operation. During this procedure, tissue from the regrown tumor was obtained for this study. We also obtained tissue from 5 tumors that have previously been described (4): tumor 2 from the mandible (mixed connective tissue tumor), tumor 3 from the thigh (angiodysplastic tumor), tumor 4 from the nose (hemangiopericytoma), tumor 5 from the thigh (hemangiopericytoma), and tumor 6 from the foot (hemangiopericytoma). All tumors had been immediately frozen after surgical removal and stored at –80° C. In healthy individuals, FGF-23 is predominantly produced by bone, and therefore bone was chosen as a control tissue; in addition, tissue was obtained from an ovarian cancer tumor. The Partners Human Research Committee approved the use of discarded human tissue.
Small amounts of tissue (32–160 mg per tumor; Table 1
) were removed from the frozen tumors and placed into 800 µL of ice-cold 0.9% NaCl containing protease inhibitors (Sigma protease inhibitor cocktail P2714, 1 µL/10 mg tissue). Tissue was crushed by hand using a pestle. In a tabletop centrifuge (Eppendorf centrifuge 5415C), samples were spun at room temperature for 1 min at 16 000g in microfuge tubes, and FGF-23 was measured in the supernatants. Extracts from the 2 control tissues and from each of the 6 tumors were measured using 2 variations of an assay for intact human FGF-23 (Immutopics) (3). The capture antibody was coated onto a 96-well plate, and the detection antibody was conjugated with horseradish peroxidase. The regular assay with an upper calibrator of 400 ng/L takes about 4 h to complete, and dilutions of tumor extract with 0 ng/L calibrator were required to yield results within the calibration curve. For the rapid assay, 3 calibrators (0, 120, and 400 ng/L) or undiluted tumor extracts were incubated with the 2 FGF-23–specific antibodies for 10 min (instead of the 3-h period used in the regular assay) on a rocking platform at room temperature. After rinsing, incubation with the horseradish peroxidase substrate was performed for 10 min (instead of 30 min), the plate was visually inspected, and photographs were taken. The absorbance at 450 nm was measured by a spectrophotometer after the addition of 50 µL of 1 mol/L sulfuric acid. When measured by regular protocol, OOM tumor extracts had intact FGF-23 concentrations of 3609 to 11 187 ng/L, whereas concentrations in extracts from bone and ovarian tumor were much lower (Table 1
). The modified rapid FGF-23 assay revealed in all undiluted tumor extracts an intense signal visible to the naked eye. The highest concentration calibrator (400 ng/L) and extract from bone tissue showed only small visible signals, whereas extracts from the ovarian cancer tumor and from the 0 and 120 ng/L calibrators were indistinguishable. When measured by spectrophotometer, readings for all samples were higher than those for the 0 ng/L calibrator; OOM extracts provided signals that were well above those of the 400 ng/L calibrator.
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In summary, simple aqueous extracts from small portions of 6 different OOM-associated tumors revealed very high FGF-23 concentrations as assessed by the calibrator assay; FGF-23 concentrations were also readily detectable by a modified rapid test that takes <30 min to complete. Like intraoperative parathyroid hormone assays (5), this rapid assay could be performed in or near the operating room, especially because visual inspection of the test plate was sufficient to detect FGF-23 in all 6 tumors tested. The assay may furthermore help define, intraoperatively, the disease-free margins of tumors located in areas that are difficult to access surgically.
Acknowledgments
Grant/Funding Support: This work was supported by grants from the National Institute of Diabetes and Digestive and Kidney Disease (RO1-46718-10 to H. Jüppner).
Financial Disclosures: None declared.
Acknowledgment: The authors would like to thank Makoto Okazaki for assistance with photographs.
Footnotes
1 Nonstandard abbreviations: OOM, oncogenic osteomalacia; FGF-23, fibroblast growth factor-23. 
References
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