Genetic and Epigenetic Alterations in Tumor Progression in a Dedifferentiated Chondrosarcoma

PATHOLOGY

Original Paper

RESEARCH AND PRACTICE © Urban & Fischer Verlag http://www.urbanfischer.de/journals/prp

Genetic and Epigenetic Alterations in Tumor Progression in a Dedifferentiated Chondrosarcoma M. Röpke1, C. Boltze2, H.W. Neumann1, A. Roessner2, R. Schneider-Stock2 1

2

Department of Orthopedics, Department of Pathology, Otto-von-Guericke University, Magdeburg, Germany

Summary

Introduction

In this case of a dedifferentiated chondrosarcoma, we searched for genetic or epigenetic alterations in both components of the tumor, the low grade chondroblastic component, and the high grade osteosacomatous component. To date, only little is known about aberrant patterns of DNA methylation in chondrosarcomas. Microdissection was used as a valuable method for clearly separating the tissues. We examined CpG island methylation of 8 tumor suppressor genes and candidate tumor suppressor genes, which are involved in different pathways: cell cycle (p21WAF1, p16INK4, p14ARF), apoptosis (DAPK, FHIT), DNA repair (hMLH1), and cell adherence (E-Cadherin). We found p16INK4 and E-cadherin promotor methylation in the low grade chondroid compartment of the dedifferentiated chondrosarcoma. P16INK4, FHIT, and E-cadherin were methylated in the highly malignant osteosarcomatous compartment of the tumor. Earlier investigations of this chondrosarcoma showed p53 mutation and p53-LOH in the anaplastic component. As shown in this case, it was accompanied by Rb-LOH. Early methylation of p16INK4 and E-cadherin in the chondroid compartment could point to the monoclonal origin of demonstrated dedifferentiated chondrosarcoma. Further alterations, as shown in p53, Rb and FHIT, are responsible for the “switch” to a high grade anaplastic sarcoma.

The term “dedifferentiated chondrosarcoma” refers to a primary bone tumor that consists of two different compartments. One component morphologically resembles enchondroma or conventionally low grade chondrosarcoma, the other contains a variant of high grade mesenchymal neoplasm, such as malignant fibrous histiocytoma, fibrosarcoma, osteosarcoma, or angiosarcoma. The poor prognosis of the so-called dedifferentiated chondrosarcoma is determined by the nonchondroid high grade component caused by invasive growth and formation of metastases [2, 15]. Dedifferentiated chondrosarcomas were found in 15% of all chondrosarcomas [8]. Nevertheless, the origin of the nonchondroid high grade areas is still unclear. Some authors presume that two separate cell clones develop from multipotent mesenchymal stem cells into different forms of differentiated neoplastic cells components (collision tumor) [1, 6, 29]. However, other investigators believe that genomic instability causes a common mesenchymal stem cell precursor to develop into a differentiated and a dedifferentiated cell population (monoclonal origin) [6, 13]. Only little is known about genetic or epigenetic alterations that play a role in tumor

Key words: Dedifferentiated chondrosarcoma – Microdissection – DNA methylation – Rb-LOH – p53-LOH – p53 mutation Pathol. Res. Pract. 199: 437–444 (2003)

Address for correspondence: Martin Röpke, Department of Orthopedics, Otto-von-Guericke-University Magdeburg, Leipziger Straße 44, D-39120 Magdeburg, Germany. Phone: +49-39167-14032; Fax: +49-39167-14006. E-mail: [email protected] 0344-0338/03/199/06-437 $15.00/0

438 · M. Röpke et al.

progression from low-malignant chondroblastic to highly malignant anaplastic chondrosarcoma. There are some reports suggesting a role of p53 mutations and p53 loss of heterozygosity (LOH) in causing this transition [15, 35, 36, 38]. Rb-LOH and p16INK4 gene alterations were reported to occur frequently in chondrosarcomas, but they were not separately investigated in the two parts of dedifferentiated chondrosarcomas [11, 38]. Regarding the real causal relationship between tumor progression and genetic alterations, it is necessary to clearly separate both tumor components. In this study, we investigated a case of a dedifferentiated chondrosarcoma, in which both tumor components, i.e., the low malignant chondroid and the highly-malignant anaplastic part, were separated by laser microdissection, followed by an investigation of a panel of genetic and epigenetic markers that possibly predict tumor progression (Fig. 1, Table 1). In this case, a functionally relevant p53 mutation/p53-LOH was detected only in the dedifferentiated component [15], and this alteration was suggested to participate in the ‘switch’ from low-malignant to highly malignant dedifferentiation.

physis of the left femur, for which the patient had to undergo surgery. After resection of the proximal left femur, the defect was reconstructed by a modular arthroprosthesis. Histology showed structures of a low grade conventional condrosarcoma in association with an anaplastic sarcoma, with production of tumor osteoid. Therefore, a dedifferentiated chondrosarcoma with a high grade osteosarcomatous component was diagnosed. The surgical therapy was followed by radiation therapy of the upper leg. Adjuvant chemotherapy was planned. However, the patient died of a pulmonary embolism under radiation therapy.

Materials and Methods Clinical history

After a history of 6 weeks with pain in the left knee and upper leg, a 53-year-old woman suffered a pathological fracture of the proximal femur. Radiological examinations (x-ray and MRI) showed an agressive osteolytic tumor with a soft tissue component in the proximal dia-

Fig. 1. Pathways of Rb and p53 in cell cycle and apoptosis (products of the genes examined are indicated by firmer frames).

Table 1. Locus of the genes and funktion of the gene products Gene

Locus

FHIT

3p14.2

P16INK4 (CDKN2A) P14ARF

9p21 9p21

P73 (TP73)

1p36

P21WAF1 (CDKN1A) 6p21.2 E-cadherin (CDH1) 16q22.1 hMLH1 3p21.3 DAPK Rb

9q34.1 13q14.2

Function

References

Involved in the regulation of apoptosis and in cell cycle control, molecular mechanism or functional pathway is still unknown, Inhibitor of cdk4 and cdk6 Interaction with the mdm2 oncoprotein blocks p53 degradation and, enhancing p53-dependent transactivation, induces G1 phase arrest (p21) or apoptosis Member of the p53 family, has high homology with p53, that can mimic its ability to bind DNA and transactivate the gene(s) related to cell cycle regulation and/or apoptosis. Universal cdk inhibitor Cell adherence DNA mismatch repair gene. Tumor-specific promotor hypermethylation of hMLH1 may be an early event in carcinogenesis of the stomach Death-associated protein kinase is a positive mediator of interferon-gamma -induced programmed cell death and belongs to the serine/threonine protein kinase family. Retinoblastoma protein control gene expression mediated by E2F, which can transactivate genes whose products are important for S phase entry

10, 19, 26, 30 33 5, 27 18 37 23 25 9, 20 33

Genetic and Epigenetic Alterations in Tumor Progression in a Dedifferentiated Chondrosarcoma · 439

Fig. 2. Overview of laser microdissection process: A: H&E staining of the well-differentiated cartilaginous part (1) and the dedifferentiated focus (2) of the chondrosarcoma (x200), selected areas (A–C, LMP), a single laser shot ejected the microdissected samples from the object slide (D, E) and catapulted the tissue directly into the cap of a PCR tube (F).

440 · M. Röpke et al. Molecular investigations

DNA-isolation Chondroblastic and dedifferentiated regions as well as non-neoplastic reference tissue were selected from formalin-fixed, paraffin-embedded sections using laser microdissection (P.A.L.M.), (Fig. 2). DNAs were prepared according to the manufacturer’s instructions (Machery and Nagel, Germany). MSP (methylation-specific PCR) Extracted DNA was bisulfite-modified by using the CpGenome DNA Modification kit (Q-Biogene, Heidelberg, Germany). The modified DNA was subject to MSP using specific primers. Primer sequences, annealing temperature, and expected product sizes are listed in Table 2. Two µl of bisulfate-modified DNA were ampli-

fied in a total volume of 25 µl containing 1 × PCR buffer, 0.4 mM dNTPs each, 10 pmol each primer, 2 mM MgCl2 (p14ARF meth and unmeth, Rb-in20) or 3 mM MgCl2 (all other genes), 0.5 units Hot GoldStar DNA polymerase (Eurogentech, Seraing, Belgium). PCR conditions for p16-MSP were given in detail recently [31]. PCR was done in an automated thermocycler (PTC200, MA. USA) using hot start programs and 35 cycles of 95 °C for 1 min, annealing (Table 2) for 1.5 min, and extension at 72 °C for 2 min, followed by a final 72 °C step for 10 min. We used the restriction enzyme-related polymerasechain reaction (RE-PCR) for detecting methylation status in the p73 gene promotor, as described by Kawano et al. [22]. Genomic DNA (0.2 µg) was digested with 20 u of either the methyl-sensitive (HpaII) or its methy-

Table 2. Primer sequences and annealing temperature Gene p14 meth. p14 unmeth p16 meth p16 unmeth p21 meth p21 unmeth E-cad meth E-cad unmeth MLH1 meth MLH1 unmeth DAPK meth DAPK unmeth FHIT meth FHIT unmeth p73 Rb-in20

sense antisense sense antisense sense* antisense sense* antisense sense antisense sense antisense sense antisense sense antisense sense* antisense sense* antisense sense antisense sense antisense sense antisense sense antisense sense antisense sense antisense

* sense primer was Cy5 labeled

Sequences

Length of PCRproduct

Tm

5′-TAggTTTTTggTgATTTTTC-3′ 5′-ACCTAATCTTCTAAAAAACg-3′ 5′-TAggTTTTTggTgATTTTTT-3′ 5′-ACCTAATCTTCTAAAAAACA-3′ 5′-FAM-TTA TTA gAg ggT ggg gCg gAT CgC-3′ 5′-gAC CCCgAA CCg CgA CCg TAA-3′ 5′-FAM-TTA TTA gAg ggT ggg gTg gAT TgT-3′ 5′-CAA CCC CAA ACC ACA ACC ATA A-3′ 5′-TTg gAA TTC ggT TAg gTT TAg T-3′ 5′-gCg CTC gAC CCA CCg Cg-3′ 5′-gTT ggA ATT Tgg TTA ggT TTA gT-3′ 5′-ACA CTA ACC CAC CAC Agg A-3′ 5′-TTA ggT TAg Agg gTT ATC gCg T-3′ 5′-TAA CTA AAA ATT CAC CTA CCg AC-3′ 5′- TAA TTT TAg gTT AgA ggg TTA TTg T-3′ 5′-CAC AAC CAA TCA ACA ACA CA-3′ 5′-AgC ggA TAg CgA TTT TTA ACg C-3′ 5′-FAM-TCT TCg TCC CTC CCT AAA ACg-3′ 5′-gAA gAg Tgg ATA gTg ATT TTT AaT gT-3′ 5′-FAM-ATC TCT TCA TCC CTC CCT AAA ACA-3′ 5′-ggA TAg TCg gAT CgA gTT AAC gTC-3′ 5′-CCC TCC CAA ACg CCg A-3′ 5′-ggA ggA TAg TTg gAT TgA gTT AAT gTT-3′ 5′-CAA ATC CCT CCC AAA CAC CAA-3′ 5′-TTg ggg CgC ggg TTT ggg TTT TTA CgC-3′ 5′-CgT AAA CgA CgC CgA CCC CAC TA-3′ 5′-TTg ggg TgT ggg TTT ggg TTT TTA Tg-3′ 5′-CAT AAA CAA CAC CAA CCC CAC TA-3′ 5′-ggg gAC gCA gCg AAA CCg-3′ 5′-CTg CAg CCg TCg CAg CC-3′ 5′-TgA gCA CCC AgA ATT AgA AC-3′ 5′-TTA ACA Agg TgT ggT ggT AC-3′

187-bp

50 °C

187-bp

50 °C

150-bp

60 °C

151-bp

58 °C

89-bp

56 °C

89-bp

56 °C

116-bp

61 °C

97-bp

59 °C

78-bp

60 °C

85-bp

60 °C

114-bp

60 °C

119-bp

60 °C

74-bp

68 °C

74-bp

63 °C– 56 °C

76-bp

65 °C

393-bp

57 °C

Genetic and Epigenetic Alterations in Tumor Progression in a Dedifferentiated Chondrosarcoma · 441

lation-resistant isoschizomer (MspI) restriction enzyme overnight at 37 °C in a 10 µl reaction mix. An aliquot of 2 µl restriction mixture was directly used for a subsequent PCR amplification of the potential CpG island of the p73 exon 1. Standardized methylated DNA (Q-Biogene, Heidelberg, Germany) was used as a positive control for methylation, and normal blood DNA was used as a negative control. Aliquots of PCR products were loaded onto polyacrylamide gels cross-linked with piperazindiacrylamid [7]. The gels were stained with silver according to Goldman and Merril [16].

Fig. 3. Results of methylation-specific PCR for methylation status of the E-cadherin (A), FHIT (B), and p16 (D) promotor region. m and un indicate reactions using primers specific for methylated and unmethylated sequences, respectively. 1: osteosarcomatous component, 2: chondroblastic component of the dedifferentiated chondrosarcoma; NT: patient’s blood. C: PCR for Rb-intron 20 marker showing allelic loss in the osteosarcomatous component of the dedifferentiated chondrosarcoma (← ); PCR products were detected on silver-stained polyacrylamide gels (A–C) or using the capillary sequencer ABI 310 with dye-labeled forward primers (D).

Results Pathological findings

Gross features We investigated a part of the proximal femur diaphysis (17 cm in length), which was fractured into two parts. A well-circumscribed, ovoid, lobulated, and nodular mass (3.5 × 3.8 × 3.2 cm) with a distinct fibrous capsule was seen in the fracture zone. On section, it had a gelatinous, gray to tan-brown surface. Histological features The paraffin-embedded sections, all stained with hematoxylin and eosin (H&E), showed a multinodular pattern. The tumor was composed of two basic cellular elements, undifferentiated round or spindle-shaped mesenchymal cells, and small well to poorly circumscribed islets of fairly well-differentiated cartilaginous tissue, frequently with central calcification and with production of tumor osteoid (Fig. 2A). The primitive mesenchymal cells possessed ovoid and elongated hyperchromatic nuclei and scanty, poorly outlined cytoplasm. In contrast, the cartilaginous foci consisted of rounded or slightly elongated cells of uniform shape and size, separated by variable amounts of mucoid material. The cells possesed small nuclei and, in the narrow rim of deeply eosinophilic cytoplasm, features characteristic of chondroblasts. In the undifferentiated part, the proliferation index (Ki-67 antigene) increased by < 30%, but only by 5% in the well-differentiated part. These findings led to the diagnosis of osteosarcomatoid dedifferentiated chondrosarcoma. Genetic findings

Both tumor components showed aberrantly methylated p16INK4 and E-cadherin promotors (Table 3, Fig. 3A, D). Methylation of the FHIT promotor was exclusively detectable in the highly malignant dedifferentiated tumor component (Fig. 3B). We did not find any aberrant hypermethylation for p21WAF1, hMLH1, p73, and p14ARF gene promotors. Loss of heterozygosity at the Rb (Fig. 3C) and p53-locus [15] was shown only in the dedifferentiated component of the chondrosarcoma.

Table 3. Results (m, CpG island methylation) Tumor component p16

p21

FHIT

hMLH1

DAPK

p73

p14

E-cad

Rb-LOH

p53-Mut* p53-LOH*

Chondroid Anaplastic

– –

– m

– –

– –

– –

– –

m m

– +

– +

m m

* data are published in Grothe et al. [15]

– +

442 · M. Röpke et al.

Discussion Both tumor components of a dedifferentiated chondrosarcoma were separated by laser microdissection and investigated for aberrant methylated promotor regions of different genes as well as for Rb-LOH. In the last few years, it has been shown that CpG island promotor hypermethylation is an effective mechanism that inactivates genes by disturbing their transcription. Tumor cells have aberrant patterns of DNA methylation, and many cellular pathways are inactivated in this way [11, 12]. Reports on methylation of CpG islands in the genome of chondrosarcomas are rare. Asp et al. [3, 4], examining the methylation status of p16INK4 gene and p14ARF gene, reported aberrant methylation of p16INK4 gene, but not of p14ARF gene. Analysis of 22 chondrosarcomas revealed methylated p16INK4 gene in one central grade II chondrosarcoma, in three grade III chondrosarcomas, and in one dedifferentiated chondrosarcoma. That investigation includes three dedifferentiated chondrosarcomas, but only tissue samples taken from the more differentiated parts were analyzed, and a comparative examination of both parts was not carried out [3, 4]. In this case of a dedifferentiated chondrosarcoma, we searched for gene alterations in both components of the tumor, the chondroblastic component, and the high grade osteosarcomatous component. Microscopically, the junction between the different tissues is sharp and without transitional zone. Therefore, microdissection was a valuable method for the separation of the two areas. We examined CpG island methylation of 8 tumor suppressor genes and candidate tumor suppressor genes, which are involved in different signaling pathways (cell cycle, apoptosis, DNA-repair). Why did we decide in favor of these genes? The INK4a-ARF locus, encoding the growth suppressor genes p14ARF and p16INK4, represents a convergence of the two major pathways of tumorigenesis: the Rb and p53 pathways. Alterations of the INK4aARF locus in chondrosarcomas have been described [3, 4]. In this case of a dedifferentiated chondrosarcoma, we proved methylation of p16INK4 in both the chondroid and the nonchondroid component of the tumor. P14ARF gene failed to give evidence of methylation in both tumor components. Nevertheless, the p53 pathway was defect, because alterations of the p53 gene were previously detected exclusively in the anaplastic component of this tumor [15]. The p53 gene showed a point mutation in codon 193 of exon 6 (CAT to CTT). This mutation was found only in the high grade osteosacomatous component of the tumor and was functionally relevant because of simultaneous loss of heterozygosity [15]. P73 is a member of the p53 family and has a structure similar to that of p53 [18]. Also, the activities of p73 are remarkably similar to those of p53 in many respects [28]. Jost et al. [21] reported that p73 can activate the

p53-responsive elements in p21WAF1 and can also induce apoptosis when overexpressed. We did not find methylation of this gene either in the cartilaginous component or in the anaplastic component of the dedifferentiated chondrosarcoma. FHIT is regarded as the most commonly altered gene in human cancer [14, 26], and it has been reported that loss of FHIT function is a very early and highly frequent event in carcinogenesis [24, 26]. FHIT protein overexpression alters the cell cycle profile of malignant cells and triggers apoptosis [32]. It was shown that transfection of FHIT in H460 lung cells correlated with a high rate of apoptosis-induced DNA strand breaks, as well as with a significant G0-G1 cell cycle arrest, which could be mediated by p21WAF1 induction [30]. Most examinations have concentrated on epithelial tumors, but Hinohara et al. [17] reported that there were also abnormalities of the FHIT transcripts in osteosarcomas and Ewing sarcomas. In our case, FHIT promotor methylation was found in the anaplastic component of the dedifferentiated chondrosarcoma; there was no methylation in the cartilaginous tumor component. Furthermore, no methylation was detectable in the promotor of p21WAF1 gene. P21WAF1is a universal inhibitor of cyclindependent kinases and is a down-stream effector of different genes as described above. Moreover, we did not find any methylation in the promotor of the DAPK gene. Cancer is a disease of multiple pathways and genetic or epigenetic lesions, and all of them are necessary to develop a fully established tumor [11]. Having investigated the dedifferentiated chondrosarcoma, we were able to prove several alterations of different signaling pathways. Interestingly, inactivation of cell cycle genes was predominantly observed (p53, Rb, p16INK4), playing a major role at G1/S arrest. However, genes associated with common genetic instability (FHIT) or adhesion molecules (E-cadherin) also seem to be involved in tumor progression of dedifferentiated chondrosarcomas. In the low malignant cartilaginous part of the tumor, promotors of p16INK4 and E-cadherin were methylated. Regarding the development of dedifferentiated chondrosarcomas, we prefer the hypothesis according to which both components are derived from a single abnormal clone or cell, which shows inactivation of p16INK4 gene and E-cadherin gene by CpG-methylation in the case described. In this genetic instability, further alterations are responsible for the “switch” to a high grade sarcoma. Our molecular genetic data of both components of dedifferentiated chondrosarcomas strongly support this hypothesis.

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