A Case of Neuroendocrine Oncogenic OsteomalaciaAssociated With a PHEX and Fibroblast Growth Factor-23Expressing Sinusidal Malignant SchwannomaM. R. JOHN,1* H. WICKERT,1* K. ZAAR,2 K. B. JONSSON,3 A. GRAUER,1 P. RUPPERSBERGER,4
H. SCHMIDT-GAYK,4 H. MURER,5 R. ZIEGLER,1 and E. BLIND6
1Department of Internal Medicine I, Endocrinology and Metabolism, University of Heidelberg, Heidelberg, Germany2Department of Anatomy and Cell Biology, University of Heidelberg, Heidelberg, Germany3Endocrine Unit, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA4Laboratory Group, Heidelberg, Germany5Department of Anatomy, Physiology, University of Zurich, Zurich, Switzerland6Department of Medicine, Endocrinology, University of Wurzburg, Wurzburg, Germany
Oncogenic osteomalacia is a rare paraneoplastic syndrome char-acterized by hypophosphatemia due to elevated renal phosphateclearance and inappropriately low serum 1,25-dihydroxyvitaminD3. Since its first description in 1947 by McCance26 approxi-mately 120 cases have been reported.
Briefly, the clinical presentation of the syndrome typicallyincludes bone pain, fractures, fatigue, and proximal muscleweakness, and is often mistaken for a variety of other disordersresulting in late diagnosis and debilitating progress of the dis-ease. The underlying tumors are usually benign but may occa-sionally be malignant. Most are of mesenchymal origin and about50% are pathologically classified as highly vascularized heman-giopericytomas.39 They are often difficult to localize due to theirsmall size. Upon localization and complete removal of the tumorthe patient is cured from the metabolic disorder with subsequentrapid healing of the bone phenotype. Experimental findings haveindicated that these tumors secrete or metabolize a humoralfactor responsible for the biochemical abnormalities in thesepatients and which seems to be a unknown component of phos-phorus and vitamin D metabolism.5–7,20,25,28,29,42 This putativefactor has already been termed “phosphatonin,” even though itstill awaits actual discovery.19
In patients with X-linked hypophosphatemia (XLH), a dom-inant genetic disorder with overlapping clinical and biochemicalabnormalities, a mutated gene has recently been identified.10
Surprisingly, a membrane-bound metalloendopeptidase was iso-lated and named PHEX (formerly PEX), an abbreviation forphosphate-regulating gene with homologies to endopeptidaseslocated on the X-chromosome. Numerous heterozygous inacti-vating mutations in the PHEX gene have been described thus far,and larger genomic deletions are known to be the cause of thedisease in the equivalent genetic disorder in mice (hyp and Gy).38
Another related hereditary disorder of phosphate homeostasis,autosomal-dominant hypophosphatemic rickets (ADHR), hasbeen associated very recently with mutations in a new member of
Address for correspondence and reprints: Dr. Markus R. John, Massa-chusetts General Hospital, Endocrine Unit, 50 Blossom St., WEL 501,Boston, MA 02114. E-mail: [email protected]. Presented inpart at the second joint meeting of the American Society for Bone andMineral Research and the International Bone and Mineral Society, SanFrancisco, CA, 1998 (Abstract F208).
*M.R.J. and H.W. contributed equally to this work.
the fibroblast growth factor (FGF) family, FGF-23.40 AlthoughFGF-23 appears to be expressed in only a few tissues,40,43 arecent report has demonstrated it to be a secreted protein that isoverexpressed in primary tissue of tumors, causing phosphatewasting.41
In this study we present clinical data from a patient withoncogenic osteomalacia followed over several years; we eval-uated the characteristics of the underlying tumor tissue andinvestigated whether this tumor might express PHEX orFGF-23 genes or mimic any of the genetic disorders men-tioned earlier.
Materials and Methods
Case Data
A 54-year-old woman was referred to our hospital for evaluationof low serum phosphate (1.39 mg/dL; normal range 2.6–4.5).She also had elevated alkaline phosphatase (313 U/L; normalrange �170), low 1,25-dihydroxyvitamin D3 level (13 ng/mL;normal range 30–90), and elevated renal phosphate clearance(23.9 mL/min; normal range 5.4–16.2) in the context of normalintact parathyroid hormone (iPTH; 39.3 pg/mL; normal range
Figure 1. Biochemical markers of bone and mineral homeostasis before and after tumor resection. Areas between horizontal lines within the graphsrepresent normal ranges. Black dots represent single measurements, whereas small horizontal bars represent mean values of these measurements.Absolute bone mineral content (BMC) and bone mineral density (by DXA) at the lumbar spine before and after tumor treatment. BMC is expressedas grams per square centimeter; DXA values are age-matched Z values. Note the different time scales. The graph represents a 7 year preoperative anda 3 year postoperative time-frame. Shaded regions represent a 3 week immediate postoperative time course. The arrows indicate tumor treatment. Thehatched boxes at the bottom of the graph indicate periods of pharmacotherapy with phosphate and 1,25-dihydroxyvitamin D3.
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10–65) and normal serum calcium (9.1 mg/dL; normal range8.8–10.6). The time course for her biochemical parameters isshown in Figure 1.
Clinically, she presented with fatigue, weakness, and severebone pain in her skeletal thorax and spine, pain in both knees,and pain upon palpation of the metatarsophalangeal joints. Bonescintigraphy revealed hot spots in the ribs bilaterally and in theleft dorsal os ischii. Like most prominent sequelae of osteoma-lacious bone disease, conventional X-rays showed a Looser zonein the right trochanter major, a spontaneous fracture in theleft-sided third metatarsal, and codfish vertebrae with coarsetrabeculae in the lumbar spine. After differential diagnosis,which included exclusion of a Fanconi syndrome, a diagnosis ofoncogenic osteomalacia was made. However, no X-rays, com-puted tomography (CT), or magnetic resonance imaging (MRI)scans of the head were performed.
The patient was treated symptomatically with pain medica-tion and with oral administration of 3.5 g inorganic phosphateand 0.5 �g 1,25-dihydroxyvitamin D3 (calcitriol) per day, whichresulted in reduced bone pain and significantly improved bonemineral density (BMD) (Figure 1). No causative tumor wasdiscovered until 6 years later, when she presented with severefatigue, headache radiating into her right eye, double vision, anda protusio of her right ocular bulbus. MRI (Figure 2) and bonescan (Figure 3) showed a large destructive tumor reaching fromthe right frontal sinus via the nasal ethmoidal cells to the rightmaxillary sinus with infiltration of the head of the orbita, repre-senting a malignant schwannoma. A complete resection of thetumor was attempted, and adjuvant radiotherapy was alsoapplied.
Two years postoperatively, serum phosphate levels remainedsubnormal, indicating that the surgical and radiotherapeutic in-tervention had been only palliative. However, serum phosphatelevels, renal phosphate clearance, and alkaline phosphatase be-came normal about 2.5 years postoperatively. Three years post-operatively, MRI studies revealed no signs of recurrent disease.However, complete healing of the oncogenic osteomalacia due tofinal necrosis of any tumor remnants remains unproven, because
the patient still has decreased 1,25-dihydroxyvitamin D, despitenormal levels of 25(OH)D.
Laboratory Data
Blood and urine chemistry, as well as markers of vitamin D,calcium, and bone metabolism, were analyzed by routine meth-ods. Osteodensitometry was determined by dual-energy X-rayabsorptiometry (DXA).
Cell Culture
Fresh tumor tissue obtained during surgery was mechanicallyminced under sterile conditions and transferred to poly-D-lysine-coated Petri dishes containing Dulbecco’s modified eagle me-dium (DMEM), 10% fetal calf serum (FCS), and 1% penicillin/streptomycin (all from Gibco, Gaithersburg, MD), and kept in ahumidified atmosphere at 37°C, 5% CO2. After four passagesand mechanical isolations, a fast-growing fibroblast-free tumorcell line was established. Passaging was performed every 5 daysthereafter. The cell line was subsequently given the nameHMS-97.
Immunohistochemistry
For the evaluation of Schwann-cell-specific epitopes, immuno-histochemistry was performed. Cultured tumor cells were platedonto chamber slides (Falcon, Becton Dickinson, Franklin Lakes,NJ) coated with poly-D-lysine and grown until subconfluency.The cells were then fixed with 4% paraformaldehyde, washedwith phosphate-buffered saline (PBS), and blocked with 2%
Figure 2. Preoperative magnetic resonance imaging (MRI) of the skull.Circumscribed gadolinium contrasted tumor tissue reaches from the rightfrontal sinus via the nasal ethmoidal cells to the right maxillary sinus.The right orbita is invaded, leading to a protusio bulbi. Periorbital boneis also invaded and partially destroyed.
Figure 3. Preoperative bone scintigraphy with 99mtechnecium showingincreased multifocal tracer uptake of the ribs, skull, spine, pelvis, and hipjoints. The inset provides a close-up view of the head region withincreased uptake in the area of the right-sided ethmoidal cells andsinuses.
395Bone Vol. 29, No. 4 M. R. John et al.October 2001:393-402 Neuroendocrine oncogenic osteomalacia
bovine serum albumin (BSA), 2% FCS, and 0.2% fish gelatin (allfrom Sigma, St. Louis, MO). They were then incubated withprimary epitope-specific antibodies (diluted 1:80–1:100), includ-ing monoclonal mouse antivimentin (Sigma), monoclonal anti-CNPase (Sigma), and monoclonal mouse anti-human neuron-specific enolase (NSE; Dako, Carpinteria, CA), andneurofilaments NF200a and NF200b (Dako). The latter threewere briefly counterstained with 4�,6�-diamidine-2-phenylindoledihydrochloride (DAPI; Molecular Probes, Inc., Eugene, OR) toallow clearer identification. After washing, cells were incubatedwith species-specific FITC-labeled secondary antibodies (anti-mouse immunoglobulin G [IgG] FITC, 1:80–1:100; Sigma).After another wash, cells were mounted in a solution consistingof 40 mL PBS, 10 g Moviol, and 20 mL glycerol (Calbiochem,Inc., San Diego, CA), allowed to harden, and examined on afluorescent microscope (Zeiss Axiophot, Zeiss, Thornwood, NJ).
Histology and Electron Microscopy
Hematoxylin–eosin stains of both primary tumor tissue andcultured tumor cells were performed by standard techniques.
For electron microscopy, cultured tumor cells were grown in3.5 cm Petri dishes until confluency and initially fixed with 2%glutaraldehyde in PIPES buffer (pH 7.4). After cutting, sectionswere washed in PIPES buffer, postfixed by the Karnovskyprocedure for osmium reduction, dehydrated, and embedded inEpon 812 (Fluka, Ulm, Germany). Ultrathin sections, contrastedwith uranylacetate and alkaline lead citrate, were examined usinga transmission electron microscope (Philips 301; Philips NV,Eindhoven, The Netherlands).
Karyotyping and Cell-cycle Evaluation
Metaphase arrest of the cell line and chromosome banding wasperformed using the standard trypsin–Giemsa technique.11 Chro-mosome numbers were counted from 12 spreads. Cell-cycleevaluation was done by staining cell DNA with propidium iodideand subsequent automated fluorescence-activated cell sorting(FACS) analysis (FACScalibur, Becton Dickinson). Both studieswere performed at the Department of Cytogenetics, LaboratoryGroup, Heidelberg.
Cell Culture Supernatant and Bioassays
Tumor cells were plated in multiple 15 cm Petri dishes andgrown in 35 mL of standard culture medium, as described earlier,until 90% confluent. Aspirated culture medium containing FCSwas collected and stored at �20°C. Cells were then washed threetimes with 50 mL of PBS and incubated for 2 days with 35 mLof DMEM containing 1% penicillin/streptomycin and either 1 �serum replacement solution 3 (Sigma) or FCS. The resultingsupernatant was aspirated and transferred to 15 mL Centriprepconcentrators (Millipore, Bedford, MA) with a cutoff of 3000 or10,000 MW and centrifuged at 4°C. The retentate was furtherpooled and concentrated with the same concentrators until anapproximately 50� concentration was achieved. The resultingretentates and filtrates were stored at �80°C until in vitro and invivo assays were performed.
Human embryonic kidney cells (293-HEK EBNA, Invitro-gen, Carlsbad, CA), stably transfected with either the humanPTH1 or PTH2 receptor, were used to measure the accumulationof the intracellular second messengers upon exposure with su-pernatant to test for receptor-activating bioactivity, as describedelsewhere.18
Phosphate uptake was measured in opossum kidney (OK)cells (clone 3B/2) as described elsewhere.33
Isolation of Tumor Total and Messenger RNA and TumorGenomic DNA
Total tumor (HMS-97) RNA was extracted from cells using atotal RNA extraction kit (Qiagen, Inc., Valencia, CA) andmRNA was extracted with the Pharmacia QuickPrep mRNApurification kit (Pharmacia, Piscataway, NJ). Human kidney totalRNA was a kind gift from Dr. Ernestina Schipani (Boston, MA),and human lymphoblastoid total RNA was a kind gift from Dr.Murat Bastepe (Boston). Genomic DNA from HMS-97 cells wasisolated using the QiaAmp Blood and Tissue Kit (Qiagen).
PHEX-specific Polymerase Chain Reaction and Sequencing
Reverse transcription-polymerse chain reaction (RT-PCR). Onemicrogram of mRNA was reverse-transcribed using oligo(dT)primer and SuperScript II (Gibco) reverse transcriptase for 1 h at42°C in a reaction volume of 30 �L. Based on the publishedcDNA sequence,14 four sets of overlapping PCR primer pairswere designed.
Two overlapping PCR products (P1, N-terminal PHEX se-quence; P2, C-terminal PHEX sequence) were obtained from thefollowing primer pairs: primer 1 forward, 5�-GATGGAAGCA-GAAACAGGGAG-3� and primer 1 reverse, 5�-CCATTGAG-GCAGCAAAGTTG-3�; and primer 2 forward, 5�-GACCATT-GCCAACTATTTGG-3�, and primer 2 reverse, 5�-CATGCCTCTGTTCATCGTGG-3�. The expected product sizeswere P1 1214 bp, and P2 1266 bp. The optimized PCR condi-tions for both primer sets were: 20 �L cDNA (200 ng reverse-transcribed mRNA); 50 pmol forward and reverse primer each; 1�L nucleotide mix (10 mmol/L; Roche, Indianapolis, IN); 5 �LPfu buffer; 1 �L cloned Pfu-polymerase (Stratagene, La Jolla,CA); 21�L H2O. Cycling conditions were: (i) 5 min denaturationat 95°C; (ii) 1 min denaturation at 95°C, 1 min annealing at 69°C,and 2.5 min extension at 72° (35 cycles); and (iii) 10 min finalextension at 72°; on a GeneE thermocycler (Techne, FisherScientific, Pittsburgh, PA). The two PCR reactions were frac-tionated on a 1% agarose gel and purified using the QIAquick(Qiagen) gel extraction kit.
To obtain the complete PHEX gene sequence of the tumor,exons 1 and 22 were amplified from genomic tumor DNA. Exon1 � 22-specific PCRs were performed according to publishedrecommendations,10 utilizing Pfu-polymerase (Stratagene). Asexpected, two PCR products were obtained: exon 1 (249 bp) andexon 22 (237 bp). PCR bands were fractionated on a 2% agarosegel and purified as noted earlier.
Purified RT-PCR products were custom sequenced on bothstrands (Lion Bioscience, Heidelberg, Germany). Genomic tu-mor cell PCR products were sequenced on both strands at thecore sequencing facility, Massachusetts General Hospital.
FGF-23/Autosomal-dominant Hypophosphatemic Rickets(AHDR) Locus Amplification From Genomic Tumor DNAand Sequencing
We amplified exon 3 of FGF-23, which contains the three mutationsimplicated as the cause of ADHR,40 from HMS-97 genomic tumorDNA: forward primer A (5�-GGTTCGCTCTTGTCCTTCC-3�) andreverse primer B (5�-CCGATTTCCTCTTCCCTAC-3�), for theinitial PCR. A nested PCR was performed with primer A andreverse primer C (5�-TCCATACATGCCCCTGTCACC-3�).
The optimized PCR conditions for the initial PCR were: 200ng HMS-97 tumor DNA; 100 pmol forward and reverse primereach; 1 �L nucleotide mix (10 mmol/L; Roche); 5 �L PCRbuffer; 10 �L Q-solution; 0.5 �L Hotstar Taq-polymerase (Qia-gen); 30.5�L H2O. Cycling conditions were: (i) 15 min denatur-
396 M. R. John et al. Bone Vol. 29, No. 4Neuroendocrine oncogenic osteomalacia October 2001:393-402
ation at 95°C; (ii) 1 min denaturation at 95°C; 45 sec annealingat 60°C, 1.5 min extension at 72°C (35 cycles); and (iii) 10 minfinal extension at 72°C; on a Mastercycler Gradient (Eppendorf,Hamburg, Germany). The nested PCR reaction was carried outusing the same reaction profile and conditions, except templatewas 5 �L of a 1:100 dilution of the initial PCR reaction. ThePCR reactions were fractionated on a 1.5% agarose gel. Thenested PCR product was purified as noted earlier and sequencedon both strands at the Massachusetts General Hospital coresequencing facility using internal primer D (5�-TACGACGTC-TACCACTCTC-3�) and primer C.
Northern Blot Analysis
Twenty micrograms of HMS-97 total RNA and human kidney orlymphoblastoid total RNA was size-fractionated on 1% agarose-formaldehyde gels. RNAs were transferred and immobilized ona nylon membrane (Ambion, Austin, TX). Random (�32P)-dCTPlabeled PHEX and FGF-23 probes were prehybridized for 1.5 hand then hybridized for 2 h at 68°C in ExpressHyb hybridizationsolution (Clontech, Palo Alto, CA). Washing was performedunder stringent conditions (final wash: 0.1 � SSC, 0.1% SDS,50°C 40 min). The blots were exposed for 48 or 72 h at �70°Con a Biomax MS film (Kodak, Rochester, NY). The PHEX probewas an almost full-length cDNA, derived from the aforemen-tioned RT-PCR reactions. The FGF-23 probe comprised exon 3and was the same as that obtained from the aforementionednested FGF-23 ADHR locus amplification.
Results
Case Report
The present case report is that of an adult patient with oncogenicosteomalacia, most likely due to a malignant schwannoma.Treatment of this patient during the years preceding the discov-ery of the tumor consisted of 3.5 g of oral phosphate and 0.5 �gof calcitriol per day, which stabilized phosphate levels at about40% below the lower limit of normal. The condition was due toan inhibition of tubular reabsorption of phosphate (Figure 1).
Alkaline phosphatase, as a marker of osteoblast activity,decreased from values of �400 U/L to an almost normal range of200 U/L postoperatively (Figure 1), whereas urinary deoxypyri-dinium cross-links, as a marker of osteoclast activity, was con-sistently within the upper normal range. Spinal BMD and bonemineral content (BMC) increased significantly over the years andno additional fractures took place; DXA values rose from 0.89 to1.15 g/cm2 (BMC), with an age-matched Z score (BMD) risingfrom �0.5 to �2.4 (Figure 1). Generally, iPTH and calciumlevels were normal but increased over time within the normalrange, except for transient episodes of secondary hyperparathy-roidism (HPT).
The patient’s tumor was detectable on a bone scan (Figure 3),which was strikingly similar to a previously published case,23 butarose rather unexpectedly. The resection of the neoplastic tissueresulted in almost complete suppression of detectable serumphosphate on the first postoperative day, possibly due to tissuemanipulation during surgery with the subsequent release ofexcess bioactive phosphaturic factors into the circulation. Duringthe 3 weeks following resection of a sinusidal malignant schwan-noma, serum phosphate levels and 1,25-dihydroxyvitamin D3
transiently reached the low-normal range. Such a normalizationof serum phosphate was previously achieved on only one occa-sion (at �4 years), and only temporarily while the patient wassubjected to a “Coca Cola diet,” a beverage rich in phosphoricacid and possibly enhanced phosphate resorption (Figure 1).
Postoperatively, serum phosphate levels again began to de-crease, suggesting that the disease was not completely cured atthis time. Persistent mild 2° HPT might have contributed to thesedecreasing phosphate levels. Two months postoperatively, adju-vant localized radiotherapy was applied. However, serum phos-phate again only transiently increased to subnormal values 2weeks after radiation (Figure 1). It was not until 2 years afteradjuvant radiotherapy that consistent, normalized serum phos-phate and alkaline phosphatase levels were seen.
Six months postoperatively, the patient was again started ondaily oral doses of 3.5 g phosphate and 0.5 �g 1,25-dihydroxyvi-tamin D3. During 1998, the patient remained hypophosphatemicwith elevated alkaline phosphatase and mild 2° HPT. Two and ahalf years postoperatively, serum phosphate levels normalizedand a further gain in BMC and BMD was observed (Figure 1).These measurements were repeated 6 months later andconfirmed.
Experimental Findings
A hematoxylin-eosin-stained section of the primary tissueshowed characteristic Schwann cell nuclear palisading (Figure4A), although not to the same extent as expected in benign tissuesuch as an acoustic neuroma. The cells also showed positiveimmunohistochemistry staining for S-100.
Tumor tissue obtained during surgery was used to establish anontransformed stable primary malignant schwannoma cell line,HMS-97 (human malignant schwannoma cell line, 1997).HMS-97 cells divided rapidly, making it necessary to split thecells every 5 days; these have remained stable in culture for �12months. A subconfluent spread of these cells showed occasionalmitotic and megakaryotic cells and cells with multiple nuclei(Figure 4B). Electron microscopy revealed organelle-rich cells(Figure 4C) with a rather prominent Golgi apparatus and plentyof presumably neurosecretory granules (Figure 4D), a featuresuggesting an active endocrine cell. Cell-cycle determination ofapproximately 13,000 cultured cells using FACS analysisshowed 24.4% diploid cells (of these were 4.1% in S phase) and75.6% aneuploid cells (of which were 9.4% in S phase) (data notshown). Metaphase spreads and basic chromosome banding bystandard techniques showed a variety of different karyotypes. Onthe 11 spreads obtained, chromosomal numbers varied between53 and 65, indicating a hyperdiploid or hypotriploid karyotype(Figure 4E,F).
On immunohistochemistry, cultured cells stained positive forvimentin, a ubiquitous intermediate filament, predominant innerve sheath neoplasms,13 as well as for the neuronal markersneurofilament 200a, neurofilament 200b, CNPase, and neuron-specific enolase (NSE) (Figure 5).
To address the heterogeneous findings from material thatcaused oncogenic osteomalacia (reported previously), we col-lected several liters of conditioned tumor medium over a shorttime period from early passages (fewer than eight) of the cell lineHMS-97.
We first sought to detect PTH1- or PTH2-receptor-activatingactivity in the conditioned medium in three different cell lines.Stimulation of human embryonic kidney 293-HEK EBNA cells,stably expressing either the human PTH1 or PTH2 receptor, orstimulation of opossum kidney cells with 50� concentratedconditioned tumor medium retentate, did not increase intracellu-lar cAMP or inositol-phosphate accumulation after incubationfor 4 h (data not shown). This was the case with both thehigh-molecular-weight fraction (MW �10,000 and MW�30005,35) and filtrate corresponding to a low-molecular-weightfraction (MW �3000 and MW �10,00030).36
Cai et al.5 reported parathyroid hormone (PTH)-like immu-
397Bone Vol. 29, No. 4 M. R. John et al.October 2001:393-402 Neuroendocrine oncogenic osteomalacia
noreactivity in tumor cell-conditioned media.16 However, wecould not demonstrate a positive reaction by performing dot blotson previously lyophilized and reconstituted conditioned mediausing antibodies against N-terminal, midregional, or C-terminalPTH or PTH-related protein (antibody source).4,12
In addition, we could not detect any significant inhibition ofphosphate transport in a standard phosphate-transport uptakeassay, by preincubating opossum kidney cells with tumor-de-rived retentates or filtrates for 4 h and 20 h.5,21,22,29,42 This wasalso the case with equally fractionated standard conditionedculture medium that contained FCS. In comparison, mediumspiked with human parathyroid hormone [hPTH(1-34)] as apositive control elicited an expected response in the assaysystems.
In one animal experiment, conditioned medium was injectedintraperitoneally into three adult female Fisher rats. Five milli-liters of either 50� concentrated retentate (MW �3000), or
unconcentrated filtrate (MW �3000) from serum-free condi-tioned tumor medium, was used and serum phosphate levels weremeasured after 3 and 7 h, but no significant change was detected(data not shown).
To detect PHEX gene mutations in the tumor, mRNA fromcultured tumor cells (passaged four times) was isolated andreverse-transcribed. Ninety-eight percent of the coding region ofthe PHEX gene could be amplified by two overlapping 1214 bpand 1266 bp RT-PCR products. The remaining 5� and 3� codingsequence was obtained by isolation of genomic DNA from tumortissue and amplification of PHEX exons 1 and 22 by PCR.Alignment of the 249 bp and 237 bp products obtained with thetwo larger overlapping cDNA products thus covered 100% of thecoding exonic region (Figure 6A,B). Sequence analysis revealedthat the tumor-derived PHEX gene sequence was identical tothose published previously,10,14 with the exception of a silentnucleotide substitution at position 1758 from cytosine to thymi-
Figure 4. (A) Light micrograph of the patient’s stained resected malignant schwannoma, showing compact bundles of fusiform cells with elongatednuclei, which are sometimes regimented in a palisading pattern. Several blood vessels are visible. The appearance is rather solid. (B) Culturedtumor-derived HMS-97 cells. Note occasional multinucleic cells and mitosis. (C) Electron microscopy of a representative cultured malignant HMS-97schwannoma cell. (D) Ultrastructural findings show an organelle-rich cytoplasm with a prominent Golgi apparatus and abundant granules (presumablyneurosecretory). (E,F) Two selected metaphase spreads of the cultured malignant schwannoma cells, showing massive chromosomal rearrangementswith translocations and hyperdiploidy (53 and 63 chromosomes) to different degrees.
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dine (nucleotide sequence data not shown). Although there havealready been some reports on PHEX gene expression usingsensitive RT-PCR expression analysis, this is, to our knowledge,the first time that two faint PHEX transcripts have been visible inan oncogenic osteomalacia tumor-derived cell line upon northernblot analysis (Figure 6C).
We also analyzed in this tumor cell line the FGF-23 genemutations reported just recently in ADHR patients by amplifyingexon 3 of FGF-23 from genomic tumor DNA. Sequence analysisrevealed that the tumor-derived FGF-23 gene sequence did notshow any of the three previously reported mutations in two
Figure 5. Positive immunohistochemical epitope characteristics of cul-tured HMS-97 tumor cells from the resected malignant schwannoma. (A)CNPase. (B) Neurofilament 200a. (C) Neurofilament 200b. (D) neuron-specific enolase (NSE). (E) Vimentin. For (B)–(D), nuclei counterstainedwith DAPI.
Figure 6. (A) Schematic representation of the selected PCR strategy toamplify the tumor-derived PHEX gene. RT-PCR on cultured malignantschwannoma cells could be performed between exon 1 and exon 22.However, anchoring primers had to be laid into these exons. Therefore,exons 1 and 22 had to be sequenced from genomic tumor DNA to coverthe regions hidden in the annealing region of the initial primers. (B)Ethidium bromide-stained agarose gel showing the obtained PHEX PCRproducts. (A) RT-PCR products: product I (1214 bp) and product II(1266 bp) (size marker: 1 kb ladder [Gibco], 1% agarose gel). (B)Genomic tumor DNA PCR: exon 1 (249 bp) and exon 22 (237 bp) (sizemarker: 100 bp ladder [Gibco], 2% agarose gel). (C) Northern blotanalysis of the PHEX gene in total RNA from human kidney (controltissue, left lane) and cultured HMS-97 cells (right lane). Two distincttranscripts are visible in HMS-97 RNA.
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relatively adjacent arginines (Figure 7A). However, using north-ern blot analysis, for the first time we were able to detect twodistinct FGF-23 transcripts from an oncogenic osteomalacia-derived tumor cell line in total RNA (Figure 7B).
Discussion
Schwannomas as a Cause of Oncogenic Osteomalacia
In this study, the underlying tumor in our patient with oncogenicosteomalacia, a malignant schwannoma, was examined exten-sively and her progress monitored over several years. Malignantschwannomas as a cause of oncogenic osteomalacia are rare, buthave been described in the literature.3,15 Commonly, underlyingneurofibromatosis (von Recklinghausen disease) accounts formost cases of malignant schwannomas,8 and there have beenseveral reports on the development of oncogenic osteomalacia inneurofibromas of these patients.7 However, sporadic malignantschwannomas, as in our case, have a better prognosis in terms ofsurvival (16% vs. 53% 5 year survival8).
The HMS-97 cell line derived from this tumor, is, to ourknowledge, the first permanent human cell line of this type ofmalignancy that is not derived from a patient with neurofibro-matosis.2 The staining with various markers in cultured schwan-noma cells strongly suggests that this cell line has neuroendo-crine characteristics.37
Attempts to Reproduce Previous Findings
We sought to address a number of previously reported experi-mental findings, which have been reported after analysis ofoncogenic osteomalacia tumors, by analyzing select morpholog-ical and functional properties of our HMS-97 cells. We did notdetect any PTH1- or PTH2-receptor-activating bioactivity inthese cells using two different assay systems. Attempts to detectphosphate-transport-regulating bioactivity using OK cells in tu-mor-conditioned media and related animal experiments were alsounsuccessful. This may have been due to a loss of specificphosphatonin gene expression of the cell line during early pas-sages, as has been reported elsewhere for some other cell linesunder similar circumstances, or due to an indirect mechanism ofaction. Alternatively, our concentrated supernatant might stillhave been too dilute to induce bioactivity.
PHEX Expression
In patients with the related disorder XLH, mutations in the PHEXgene have recently been identified.10 In our patient, PHEX wasexpressed in the tumor cell line by RT-PCR and as two tran-scripts on northern blot. We could exclude a mutation of thecoding sequence, resulting in a change of the amino acid se-quence and thus the possibility that the tumor mimicked XLH.
At this point, it is unclear whether the expression of PHEX insome of these tumors24,30,32 is an in vitro artifact leading toimmediate degradation of any released phosphatonin (which is apresumed substrate of PHEX) and, in our case, sabotaged ourefforts to purify the hormone. An alternative explanation couldbe that these usually highly vascularized tumors overexpressPHEX, which in term inactivates an unknown phosphate-con-serving hormone in a dose-dependent manner, leading to a netloss of phosphate.
FGF-23 Expression
Another related genetic disorder, autosomal-dominant hypophos-phatemic rickets (ADHR), has recently been mapped to chromo-some 12p13,9 and mutations of a novel gene, FGF-23, have beenidentified as the cause of this rare disease.40 How FGF-23 causesthis disturbance in phosphate metabolism is unknown at present.We analyzed the tumor cell line for the known mutations inFGF-23, but no mutations in that region could be detected. Wedid not expand our search to the entire gene, because thepreviously reported mutations are either in the same or adjacentarginines where computer analysis predicts a cleavage site forproteases. In this sense, XLH and ADHR may be sibling dis-eases, where, in the former syndrome, there may be no degrada-tion of a phosphaturic factor, and in the latter there may be adegradation-resistant phosphaturic hormone. So far, the literaturesuggests only very limited FGF-23 expression in a few tissues.The FGF-23 expression observed by our group may be the resultof a translocated alternative or constitutive active promoter; forexample, chromosomal rearrangements have been observed withsome parathyroid adenomas.1 Because the HMS-97 cells dem-onstrated a very complex karyotype, we consider the latter to bethe likely explanation and, in addition, the observed transcriptswere larger than those previously reported.40,41 However, acomplete cytogenetic evaluation of these complex cytogenetic
Figure 7. (A) Partial sequence chromatogram of exon 3 of the tumor cell-derived FGF-23 gene, covering the region where mutations have been foundin ADHR patients. The sites of the ADHR mutations are indicated with arrows, but they are not mutated in HMS-97 cells. (B) Northern blot analysisof the FGF-23 gene in total RNA from human lymphoblastoid (control tissue, right lane) and cultured HMS-97 cells (left lane). Two distinct transcriptsare visible in HMS-97 RNA.
400 M. R. John et al. Bone Vol. 29, No. 4Neuroendocrine oncogenic osteomalacia October 2001:393-402
aberrations would have to include comparative genome hybrid-ization with, for example, fibroblast metaphases from the samepatient. Because of the different chromosome numbers obtained,we did not consider this to be a feasible option. Genomicimbalances with, for example, overrepresentation of segmentshave been found in at least four other malignant peripheral nervesheath tumors.27,34 However, it was not reported whether any ofthese tumors also led to oncogenic osteomalacia.
It is also possible that neither PHEX nor FGF-23 is directlyinvolved in the phosphaturic paraneoplastic syndrome of onco-genic osteomalacia tumors. It is conceivable that some of thesetumors secrete a hormone precursor that requires activation indistant tissues. Because direct phosphaturic effects of FGF-23have not been demonstrated so far, it might be possible thatFGF-23, or another tumor-derived hormone, leads to the releaseof phosphatonin from other tissues such as bone.31 Gene expres-sion profiles of tumors associated with oncogenic osteomalaciamay provide more objective and less selective data in the fu-ture,17 and may ultimately provide evidence that there is nohomogeneity with respect to the secreted factors.
However, at this point, we prefer to believe that the observedFGF-23 expression, which in two previous publications40,43 wasonly detectable in organ RNA of normal tissues using verysensitive methods, might have led to a state of “hyper-FGF-23ism” in our patient, subsequently leading, directly or indi-rectly, to hypophosphatemia and decreased 1,25-dihydroxyvita-min D3. A very recent report, demonstrating FGF-23 expressionin primary tumor tissue of four patients with oncogenic osteo-malacia, supports this hypothesis.41
Conclusions
Surveillance of patients with suspected oncogenic osteomalaciashould include repeated imaging of at least the head, because thisis one of the most common locations of oncogenic osteomalaciatumors. Presurgical palliative long-term treatment of our patientwith phosphate and 1,25-dihydroxyvitamin D3 resulted in: (i)stabilized or improved biochemical parameters; (ii) a favorableeffect on bone abnormalities and BMD, especially prevention ofadditional fractures; (iii) significantly improved pain manage-ment; and (iv) no adverse side-effects of the combination ther-apy, such as nephrolithiasis/nephrocalcinosis or hypercalcemia.
The malignant peripheral nerve sheath tumor is a rare exam-ple of an ectodermal tumor causing oncogenic osteomalacia. Theestablishment of a primary, untransformed, permanent cell lineof the underlying malignancy might prove useful in the future forthe genetic characterization of this mysterious disease. In addi-tion, it could be a valuable tool for cytotoxicity studies, investi-gation of neuroendocrine Schwann cell function, and in theassessment of autoimmune peripheral nerve disease. HMS-97cells show significant PHEX and FGF-23 gene expression,which, when mutated, play a major role in phosphate homeosta-sis. Whether FGF-23 is indeed involved in the pathogenesis ofoncogenic osteomalacia and comparable to phosphatonin re-mains to be determined once recombinant hormone and specificantibodies are available to allow further experimentation.
Acknowledgments: This work was supported in part by grants of theDeutsche Forschungsgemeinschaft to E.B. (Bl 291/4-1) and to M.R.J.(JO 315/1-1 and JO 315/1-2). The authors thank Gabi Kraemer andJuliane Schroth for expert technical assistance as well as Dr. Markus J.Seibel, Dr. Johannes Pfeilschifter, and Dr. Urs Benck for help with thebiochemical workup. We also thank Dr. Harald Juppner, Boston, forhelpful suggestions.
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Date Received: December 20, 2000Date Revised: April 3, 2001Date Accepted: April 27, 2001
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