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Keywords:

  • desmoid;
  • ossification;
  • BMP and activin membrane-bound inhibitor;
  • hypermethylation

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. CASE REPORT
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

A rare case of desmoid-type fibromatosis with focal metaplastic bone in the chest wall suggested that enhanced responsiveness to BMP signaling by decreasing BAMBI expression through promoter hypermethylation plays a crucial role in the formation of metaplastic bone.

Introduction: Desmoid-type fibromatosis, originating from mesenchymal cells with myofibroblastic features, is a locally aggressive and frequently recurring infiltrative lesion. One such sporadic case with metaplastic ossification in the chest wall is presented.

Materials and Methods: A 43-year-old man was referred to the hospital with a gradually enlarging hard mass in the left anterolateral chest wall. A thoracotomy was carried out, and histopathological specimens were used for immunohistochemical, genetic, and methylation studies.

Results: Accumulation of altered β-catenin associated with a somatic heterozygous activating mutation in codon 41 was detected in the typical desmoid-type fibromatosis and at the ossifying focus. Among factors related to bone formation and the classical wnt-β-catenin signaling pathway, BMP and activin membrane-bound inhibitor (BAMBI) expression was specifically downregulated at the ossifying focus. Hypermethylation of the BAMBI promoter was observed in microdissected tissue from the ossifying focus but not in that from the typical desmoid-type fibromatosis.

Conclusions: Because both BMP and classical Wnt/β-catenin/LEF1 signaling cooperatively and mutually induce differentiation of mesenchymal cells into osteoblastic cells and promote bone formation, the epigenetic event leading to the enhanced responsiveness to BMP signaling may play a crucial role in the formation of metaplastic bone.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. CASE REPORT
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

DESMOID-TYPE FIBROMATOSIS (desmoid tumor or aggressive fibromatosis) is a locally aggressive and infiltrative lesion characterized by lack of capacity to metastasize distantly but by frequent local recurrence.(1–4) It is composed of spindle-shaped florid cells originating from mesenchymal cells of myofibroblastic features, occurring most frequently in the abdominal wall during or after pregnancy.(5) Here, we describe a sporadic case with metaplastic ossification in the left anterolateral chest wall. BMP and activin membrane-bound inhibitor (BAMBI) gene inactivation by promoter hypermethylation was speculated to contribute to the formation of metaplastic bone.

CASE REPORT

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. CASE REPORT
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

A 43-year-old man was referred to our hospital with a gradually enlarging hard mass in the left anterolateral chest wall with progressive pain of insidious onset, continual over a 7-year period. The medical history and the review of the patient's systems were unremarkable. A plain chest CT scan revealed a tumor with a relatively clear margin in the left anterolateral chest wall, with its CT number consistent with that of homogenous muscle content (Fig. 1A, arrows). A T1-weighted MRI scan also showed a well-circumscribed tumorous mass involving the fifth and sixth ribs. Percutaneous aspiration cytology indicated a nonsarcomatous fibrous increase with spindle-polygonal cells, although no definitive diagnosis could be established because of the small quantity of cells collected. Based on these findings, under a preoperative diagnosis of primary, locally aggressive fibrous tumor, presumably desmoid-type fibromatosis of the chest wall, a thoracotomy was carried out through an anterolateral skin incision. At surgery, the tumor was found to originate from the nonbony portion of the left chest wall and to have a well-defined capsule, with some areas of adhesion. Neither dissemination nor pleural invasion in the thoracic cavity were evident, but because the tumor was firmly attached to the fifth and sixth costal ribs, it was removed in conjunction with the resection of the chest wall to which it adhered. The resected tumor measured 9.5 × 5.5 × 4.5 cm, and a cross-section revealed it to be yellowish-white with relatively well-defined outlines (Fig. 1B, arrows). Histopathologically, the tumor was a typical desmoid-type fibromatosis composed of florid spindle-shaped fibroblast-like cells with abundant collagen fibers (Fig. 2A). Interestingly, a small ossifying focus was found inside the fibromatosis (Fig. 2B). By immunohistochemistry using the anti-β-catenin antibody (DakoCytomation, Kyoto, Japan) that recognizes the C-terminal portion, β-catenin was found accumulated within the nuclei of both the spindle-shaped tumor cells in the fibromatosis (Fig. 2C) and the osteoblastic cells in the ossifying focus (Fig. 2D). From formalin-fixed paraffin-embedded tumor tissue samples of both the fibromatosis and the ossifying focus, exon 3 of the β-catenin gene was amplified using the following set of primers as previously described(6): CAT-3 sense, 5′-ATTTGATGGAGTTGGACATGGC-3′; CAT-3 antisense, 5′-CCAGCTACTTGTTCTTGAGTGAAGG-3′. Of the 10 independent sequences of the PCR product from both the fibromatosis and the ossifying foci, four and five products, respectively, contained an A to G point mutation resulting in threonine to alanine transition at codon 41, one of the phosphorylation sites by axin (Fig. 2E, right). The altered β-catenin must, therefore, be resistant to ubiquitination signaling by the adenomatous polyposis coli (APC)/axin/glycogen synthase kinase-3β (GSK) complex.(7) On the other hand, sequences of the PCR product from surrounding nontumorous tissue contained no such mutation (Fig. 2E, left). To further investigate the factors related to bone formation and the classical wnt-β-catenin signaling pathway, the expression of BMP-2, BMP receptor type 1A and 1B, RANKL, osteoprotegerin (OPG), Runx2, Msx2, and Dlx5 was analyzed immunohistochemically using goat polyclonal anti-BMP-2, -BMPR-I, -RANKL, -OPG, -Runx2, -Msx2, and -Dlx5 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and rabbit polyclonal anti-human BAMBI(8) (raised against C-terminal peptide, a kind gift from Dr T Akiyama, Laboratory of Molecular and Genetic Information, Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo, Japan). The result of the immunohistochemical analysis is summarized in Table 1. Among these factors, BMP-2 (Figs. 3A and 3B), BMP receptors, RANKL, and OPG were positive in the tumor cells of the fibromatosis and slightly enhanced in the ossifying focus. Runx2, Msx2, and Dlx5 were almost negative in both areas, except for a weak staining of Msx2 in the ossifying focus. On the other hand, BAMBI expression was positive in the tumor cells of the fibromatosis, but was diminished in the spindle-shaped osteoblastic cells around the ossifying focus and was negative in the osteocytes embedded in the mineralizing bone matrix (Figs. 3C and 3D). Negative controls prepared by nonimmunized animal serum matching the primary antibody did not show any significant staining in the immunohistochemical studies (Figs. 2 and 3, insets). Because in the 5′-flanking region of the BAMBI gene,(8–10) numerous CpG loci are clustered and form a typical CpG island (as schematically shown in Fig. 4A), where methylation generally accounts for the epigenetic mechanism of gene silencing, we extended our study to determine the methylation status of the BAMBI promoter region at both the ossifying focus and the surrounding fibromatosis. Sections (7 μm thick) were cut and stained with HE, and the laser capture microdissection system (LM200; Olympus, Tokyo, Japan) was used to isolate the ossifying focus from typical desmoid-type fibromatosis (Fig. 4B). Dissected samples were lysed overnight in 25 μl of lysis solution containing 0.2 mg/ml Proteinase K, 10 mM Tris-HCL (pH 8.0), 10 mM EDTA, and 1% Tween 20. Low-melting agarose beads (10 μl) containing 7.5 μl of cell lysate were formed (final 1.6%), and methylated CpG was analyzed by the sodium bisulfite method as described.(11) Bead fragments were analyzed by methylation-specific nested PCR using the sets of primers listed in Table 2. The first PCR conditions were as follows: denaturation at 95°C for 1 minute, 30 cycles of 95°C for 30 s, 60°C for 30 s, 70°C for 1 minute, and a final elongation step of 5 minutes at 70°C. The nested PCR conditions were as follows: denaturation at 95°C for 1 minute, 25 cycles of 95°C for 30 s, 60°C for 30 s, 70°C for 1 minute, and a final elongation step of 5 minutes at 70°C. Consistent with the immunohistochemical analysis, the three independent methylation-specific PCR assays showed that DNA amplification was observed in the conventional desmoid tumor with strong BAMBI expression in case of using primers matching the unmethylated sequence pattern and in the ossifying focus with weak BAMBI expression in case of using primers matching the totally CpG-methylated sequence pattern. One of the representative results is shown in Fig. 4C.

Table Table 1.. Results of Immunohistochemical Analysis
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Table Table 2.. Primer Sequences Used in the Methylation-Specific PCR Studies
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Figure FIG. 1.. (A) The tumor, with relatively clear margins, in the left anterolateral chest wall (white arrows). (B) Measuring 9.5 × 5.5 × 4.5 cm, the tumor was removed with the costal ribs (right, black arrows).

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Figure FIG. 2.. Histologic features of the typical desmoid-type fibromatosis and ossifying focus. (A) HE staining showing typical desmoid-type fibromatosis consisting of spindle-shaped cells and collagen fibers (×200). (B) Fibromatosis cells merging into spindle-shaped osteoblastic cells around the mineralized bone (top portion; HE, ×200). β-catenin is accumulated within the nuclei of (C) tumor cells in the typical fibromatosis (×200) and (D) osteoblastic cells (arrows, ×200). Negative controls prepared by nonimmunized mouse serum show no significant staining (insets in C and D). (E) Sequences for exon 3 of the β-catenin gene by PCR. A to G point mutation (marked *) at codon 41 was observed in about one-half of the sequences from tumor tissues (right), indicating that the alteration of the β-catenin gene is an acquired somatic heterozygous activating point mutation, whereas no mutation was found in sequences from surrounding nontumorous tissue (left).

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Figure FIG. 3.. Immunohistochemical evaluation of BMP-2 and BAMBI expression in both the fibromatosis (Desmoid) and the bone-forming areas (Bone-forming). (A and B) BMP-2 expression is positive in both areas (×200). (C) BAMBI expression is positive in the fibromatosis (×200) but diminished in the spindle-shaped osteoblastic cells around the ossifying focus, and (D) negative in the osteocytes in the mineralizing bone matrix (×200). Negative controls show no significant staining (insets in A-D).

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Figure FIG. 4.. Schema of the human BAMBI gene promoter and its methylation status by methylation-specific PCR on microdissected tissue samples. (A) Numerous CpG loci (small vertical lines) are clustered in the 5′-flanking region of the BAMBI gene. A TATA-box, three tandem repeats of Smad binding elements (3×SBE), and a transcription start site (arrow) are mapped on the promoter. Four pairs of primers (horizontal bars at the bottom) were used for methylation-specific PCR (Table 2). Methylation-specific PCR was done, first by PCR amplification by either the combination of ms1 and ma1 or us1 and ua1 and second by nested PCR using either the combination of ms2 and ma2 or us1 and ua2, respectively. (B) The portion of the bone-forming area separated by microdissection. Left and middle photos show HE staining before and after microdissection, and the right pictures shows a portion of the bone-forming area specifically captured by microdissection. (C) The BAMBI promoter is not methylated in the fibromatosis (Desmoid) but is methylated specifically in the microdissected sample from the bone-forming area (Bone-forming). Negative and positive controls were prepared using bisulfite nonconverted and converted placental DNA, respectively, as PCR templates. The smaller band that appears in the U lane is derived from a primer dimer. U and M indicate unmethylated and methylated patterns.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. CASE REPORT
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Because the autosomal-recessive osteoporosis-pseudoglioma syndrome attributed to inactivating mutations in the Wnt receptor, low-density lipoprotein receptor-related protein 5 (LRP5), shows reduced bone by decreasing osteoblast proliferation,(12, 13) and conversely, because autosomal-dominant high bone mass traits attributed to activating mutations in LRP5 show increased bone mass by increasing osteoblast proliferation,(14) the classical Wnt/β-catenin/lymphocyte-enhancing factor 1 (LEF1) signaling pathway must play important roles in regulating BMD by modulating postnatal osteoblast proliferation. On the other hand, somatic mutations of either the APC or the β-catenin gene that result in the stabilization and accumulation of β-catenin by escaping from the phosphorylation by axin,(15, 16) consequently preventing the ubiquitination of β-catenin,(6) have been shown as causative genetic alterations in desmoid-type fibromatosis.(17–20) The increased β-catenin translocates in the nucleus, displaces co-repressors from LEF1, binds LEF1, and recruits co-activators to induce numerous genes related to the cell cycle including LEF1 itself, c-myc, and cyclin D1.(21, 22) Desmoid-type fibromatosis, therefore, provides an in vivo constitutive active model for the classical Wnt/β-catenin/LEF1 signaling pathway in mesenchymal cells. Indeed, a somatic monoallelic mutation (A41G) in the β-catenin gene was shown in the fibromatosis of our patient; this mutation occurs frequently in female breast fibromatoses.(23) The resulting altered and stabilized β-catenin accumulated in the nucleus. Moreover, the same somatic mutation was found in samples from both the typical desmoid-type fibromatosis and the ossifying focus, indicating that ectopic ossification is probably caused by the direct transdifferentiation of tumor cells into the osteoblastic phenotype.

To explain the mechanism of metaplastic bone formation, we hypothesized that, in the presence of active wnt signaling, mesenchymal cells transdifferentiate into an osteoblastic phenotype when additional events leading to the activation of BMP signaling occur either through the increased expression of BMP, their receptors and intercellular signal transducers, or the decreased expression of the inhibitors of BMP signaling. Immunohistochemically, the expression of BAMBI was selectively diminished at the site of the ossifying focus (Fig. 3D), whereas that of BMP-2 and its receptor remained unchanged. Hypermethylation of CpG-islands in the promoter region, an important epigenetic event, leads to the silencing of the gene during embryogenesis, the differentiation of cells and tissues, tumorigenesis, and the progression of the tumor by changing the chromosomal structure or affinity of the transcriptional factors.(24) We therefore examined the methylation status of the BAMBI promoter by methylation-specific PCR, and observed CpG island hypermethylation in the promoter specifically in the microdissected samples taken from the ossifying focus, indicating that selective reduction of BAMBI expression is probably caused by the selective hypermethylation of the BAMBI promoter region in the ossifying focus.

BAMBI, originally isolated from melanoma cells with low metastatic potential by a differential display cloning technique as a novel gene (nma), correlates inversely with the metastatic potential of human melanoma cell lines and xenografts.(25) It is a transmembrane glycoprotein structurally similar to the TGF-β family serine/threonine protein kinase type I receptor, except that it lacks an intracellular kinase domain.(10, 25–27) Consequently, as a pseudoreceptor, BAMBI is known to interact with many of the type I and II TGF-β receptors, including ALK 1, 3, 4, 5, and 6, but not with ALK 2 in vitro, and to function as a negative regulator of TGF-β signaling by decreasing type I and II TGF-β receptor complexes.(26) The BAMBI gene, shown to be the direct target of both TGF-β(8, 10) and classical Wnt/β-catenin/LEF1 signaling pathways,(9) is therefore thought to play an important role as part of the negative feedback loop system of TGF-β signaling.(8) Although the role of BAMBI in calcified tissues is not clearly understood, reflecting that BAMBI is a direct target gene of both TGF-β and classical Wnt/β-catenin/LEF1 signaling pathways, its expression is observed among ameloblasts, odontoblasts, and osteoblasts.(28, 29) Because both BMP and classical Wnt/β-catenin/LEF1 signaling cooperatively and mutually induce mesenchymal cells into osteoblastic differentiation and promote bone formation,(30–33) such epigenetic events that lead to the increased responsiveness to BMP signaling in mesenchymal cells with constitutively active classical Wnt/β-catenin/LEF1 signaling may play an important role in metaplastic bone formation. It is noteworthy that only one case, other than this one, with metaplastic bone formation has been reported to date.(34) Although the assays performed in this study do not allow of going beyond correlations, and the incidence of bone formation in desmoid-type fibromatosis itself is very rare, we believe that morphology-oriented epigenetic studies on the genes related to BMP and Wnt/β-catenin/LEF1 signaling pathways, especially to the BAMBI gene, would provide useful information for elucidating not only the molecular mechanism of metaplastic bone formation in tumors but also osteoblastic differentiation under physiologic conditions.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. CASE REPORT
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

The authors thank Dr T Akiyama (Laboratory of Molecular and Genetic Information, Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo, Japan) for kindly providing the antibody against human BAMBI; Dr Takeshi Kondo, Dr Kenta Kishimoto, Shuichi Matsuda, and Noriko Sakamoto in our laboratory for excellent technical assistance; and Drs Takashi Marui, Toshihiro Akisue, Kotaro Nishida, Tetsuya Nakatani, and Hanako Murakami (Department of Orthopedic Surgery), Masahiro Yoshimura, Yoshimasa Maniwa (Department of Cardiovascular Respiratory Surgery), Naoki Kanomata, and Naoko Noda (Division of Diagnostic Pathology, Kobe University Hospital) for kind and helpful cooperation. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Health and Welfare, Japan (SK) and by a Grant-in-Aid for Scientific Research (SK, 16590278; RK, 16590313), and for 21st Century COE Program, “Center of Excellence for Signal Transduction Disease: Diabetes Mellitus as Model” from the Ministry of Education, Culture, Sports, Science and Technology of Japan (SK).

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  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. CASE REPORT
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES
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