Metastatic Retroperitoneal Paraganglioma in a 16-year-old Girl. Case report, Molecularpathological and Cytogenetic Findings

PATHOLOGY RESEARCH AND PRAOlCE

Teaching Case

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Metastatic Retroperitoneal Paraganglioma in a 16-year-old Girl. Case report, Molecularpathological and Cytogenetic Findings S. Blasius, C. Brinksehmidt, C. Poremba, H. J. Terpe, H. Halm 1, J. Sehleef2 , J. Ritter 3 , K. Wortler4 , W. Boeker and B. Doekhorn-Dworniezak Institute of Pathology, 'Dept. of Orthopedics, 2Dept. of Pediatric Surgery, 3Dept. of Pediatric Oncology, 4Dept. of Radiology, University of MOnster, Germany

Summary

Introduction

Retroperitoneal paraganglioma is a rare tumor, especially occurring in childhood and adolescence, with a marked tendency to become biologically malignant. It has not been possible to predict the clinical outcome of paraganglioma patients by conventional histology, hence malignancy can only be demonstrated by the occurrence of metastatic lesions. Currently, only limited information on the genetics of this tumor is available. We report on a 16-year-old girl with a large retroperitoneal paraganglioma and an osseous metastasis to the first lumbar vertebra. In addition to morphological and immunohistochemical examinations, a molecular cytogenetic analysis was performed. Comparative genomic hybridization (CGH) revealed imbalanced chromosomal aberrations with a loss of chromosome Ip and a gain of Iq, indicating isochromosome lq. A loss of chromosome 3 as well as low-level gains of chromosomes 4, 5, 6q, llq and 13q were detected. A PCRbased microsatellite analysis of 1p confirmed the loss of heterozygosity, including NBI and NB2 putative tumor-suppressor gene regions. Telomerase activity, which is found in the majority of malignant tumors, could not be detected. The case presented here is the first more comprehensive molecular genetic analysis of a sporadic malignant paraganglioma.

Paraganglioma is a rare tumor mainly presenting between the third and fifth decades of life, and yet no age is spared. Ten percent of the tumors occur in children [15]. Sporadic and familiar tumors (as part of MEN 2) are well-known. Historically, adrenal (phaeochromocytoma) and extra-adrenal paragangliomas were discussed separately. Extra-adrenal paragangliomas were divided on the basis of their anatomical distribution into cervical, thoracic and intra-abdominal tumors [8J. Fifteen to twenty percent of childhood paragangliomas are extraadrenal [16J. The intra-abdominal extra-adrenal paraganglioma is the most aggressive paraganglioma with malignant behavior in up to 50% of the cases [21]. Vascular invasion and extensive local invasion are fundamental characteristics of malignant neoplasms. These features are, however, of limited value in assessing neuroendocrine tumors such as paraganglioma [6]. So far, there has been no generally accepted cytopathological, immunohistochemical, ultrastructural or gross pathological feature suitable for predicting the biological behavior of these tumors. A few cytogenetic findings have been reported on familiar or sporadic paragangliomas [9, 13, 25]. In the case presented here, molecular cytogenetic analysis by applying comparative genomic hybridization (CGH) was complemented by molecular

Key words: Malignant paraganglioma - CGH - Cytogenetics - Telomerase activity Pathol. Res. Pract. 194: 439-444 (1998)

Address for correspondence: Dr. med. S. Blasius, Institute of Pathology, Domagkstf. 17,48149 MUnster, Germany. Tel.: ++49 -(0)251/835-5443, Fax: ++49-(0)251/835-5460 0344-0338/98/0194-0439$5.00/0

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genetic examinations of specific gene regions on chromosome 1 and the determination of telomerase activity.

Material and Methods Tissue Samples

Tumor material obtained directly at surgery was immediately snap-frozen and stored at -80°C until use. To obtain constitutive DNA, a peripheral blood sample was acquired additionally. For histopathological examination, tumor samples were fixed in 4% buffered formalin, paraffin-embedded and routinely processed. Immunohistochemical examinations were performed according to standard procedures by using a slightly modified APAAP-method and by employing the following primary antibodies: chromogranin A (LK2H 10, 1: 10), vimentin (1:2), cytokeratin (KL-l, 1:100) (all Immunotech, Hamburg, FRO), synaptophysin (1 :20, Boehringer, Mannheim, FRO) and S100 (1:6000, Dako Diagnostica Hamburg, FRO).

extreme varicosis of the internal genital tract was seen with otherwise normal findings. Postoperative phlebography performed in order to exclude thrombosis with consecutive collateral circulation showed a massive dislocation of the vena cava inferior. A retroperitoneal tumor was suspected. MRI tomography revealed a tumor measuring 14xlOx14 cm in the right abdomen with a cranio-dorsal dislocation of the kidney (Fig. 1). Laboratory parameters were within the normal range, especially tumor markers, such as catecholamines and metabolites, dopamine, AFP, NSE and ~-human chorionic gonadotropin (~-HCG). Explorative laparotomy was performed and a huge retroperitoneal tumor was resected which was adhesive at the distal thoracic and lumbar spine. The postoperative course was uneventful. Tc-99-MDP- and 1-123MIBG-scintigraphy, performed 10 days and three weeks later, revealed an increased uptake in the first lumbar vertebra. MRI tomography showed a lesion in

CGH and Microsatellite Analysis

Tissue sections (10-20 /lm) for DNA extraction were prepared with a cryomicrotome. Representative frozen sections were examined to ensure that tumor cells were present. DNA was extracted, based on a proteinase-K-digestion and followed by phenol-chloroform extraction according to a standard protocol. With slight modifications, COH analysis [2J was performed as described by Du Manoir et al. [5]. Tumor and constitutional DNA were used as a template for PCR amplification with specific sets of CY5 fluorescently labeled oligonucleotides at the microsatellite regions D1S243, DlS214, O1S234, O1S255, O1S200 and DlS203 [IOJ according to standard procedures [I9]. The PCR-products were separated electrophoretically with on-line detection, subsequent fragment analysis and data collection on an automated DNA sequencer (ALFexpress; Pharmacia, Freiburg, FRO). Telomerase Activity-Assay

Fig. 1. Contrast-enhanced Tl-weighted MR-image showing a huge tumor in the right abdomen, adjacent to the spine, with central cystic region.

For in vitro detection of telomerase activity, a modified version of the TRAP (telomeric repeat amplification) assay was used [14]. The analysis of TRAP amplification products was performed on denaturing 8% polyacrylamid gels on an automated laser-fluorescence sequencer (ALFexpress, Pharmacia, Freiburg, FRO). In order to exclude a false-negative result, the viability of the tumor material was confirmed in control RT-PCR for ~­ actin as an indirect marker of tissue suitability for RNA-based assays.

Case Report Clinical History

A 16-year-old girl without any previous complaints presented with a sudden onset of a cramp-like, lancinating pain in the right lower abdomen. At laparoscopy, an

Fig. 2. Osteolytic lesion in the left dorsal third of the vertebra with a destruction of the dorsal cortical lamellae on CT-scan.

Metastatic Retroperitoneal Paraganglioma . 441 the dorsal third of the vertebral corps and the pedicle (Fig. 2). A metastatic tumor was suspected and demonstrated by CT-guided needle biopsy. Chemotherapy was applied using a neuroblastomaprotocol (NB90 of the German Society of Pediatric Oncology and Hematology). Five months later, the tumorbearing corps of the first lumbar vertebra was removed and the defect was filled up with a spacer. There were no postoperative complications. The patient is doing well without evidence of disease at ten months followup. Pathology

Macroscopy: The resection specimen of the retroperitoneal tumor (817 g) measured 15xl1.5x9.5 cm and was thinly en-

Fig. 3. Cut surface of the resection specimen showing a centrally cystic, homogeneous tumor with hemorrhages and prominent vascularization.

capsulated. The cut surface showed a brownish, glossy tumor with central liquid areas, a yellowish necrosis and hemorrhages (Fig. 3). The vertebral corporectomy specimen showed a reddish tumor in the left-hand sided dorso-lateral position with a focally complete destruction of the posterior cortical lamellae, engaging about a third of the volume (Fig. 4). Especially on the right-hand side, lateral aspects of the cortex were preserved. Microscopy: The primarily resected retroperitoneal tumor was composed of moderately polymorphous tumor cells arranged in a "Zellballen" pattern. The tumor cells showed hyperchromatic round to oval nuclei and clear or slightly eosinophilic granular cytoplasm (Fig. 5). Rarely, mitotic figures could be seen. The "Zellballen" were surrounded by sustentacular cells with elongated cytoplasmic processes. The diagnosis of paraganglioma was confirmed by immunohistochemical examination showing S-lOO-protein positivity in sustentacular cells and chromogranin A expression in chief cells (Fig. 6). Vascular invasion as well as focal infiltration of the fibrous capsule could be shown, and a marginal resection was supposed. Histologically, similar morphologic features were found in the vertebral specimen resected in the second operation. The tumor spread through the dorsal cortical lamellae and reached the medullary cavity. The tumor cells did not show any response to preoperative chemotherapy. Cytogenetic and Molecular Pathological Findings

A representative pattern of CGH analysis is shown in Fig. 7. CGH analysis revealed a loss of chromosome Ip and a gain of lq, indicating isochromosome 1. Furthermore, a loss of the entire chromosome 3 and, with the 50% threshold, low-level gains of chromosomes 4, 5, 6q, 9p, llq and 13q could be detected. The deletion of chromosome 1p, detected by CGH, was confirmed by a PCR-based microsatellite analysis, applying six different microsatellite markers. A reduction to homozygosity was found in both the NbRI and NbR2 region, spanning chromosomes 1p35 and 1p32-35, respectively [19]. Five areas of the primarytumor material were analyzed for telomerase activity by the TRAP-assay. No detectable activity was found when several areas of the tumor were repeatedly analyzed.

Discussion Fig. 4. Vertebrectomy specimen showing the hemorrhagic osteolytic lesion in the left dorsal third of the specimen.

Morphological examination of paraganglioma is of limited value with respect to the prediction of the clinical outcome. In our case, the tumor was found in the

442 . S. Blasius et al.

Fig. 5. Typical paraganglioma with "Zellballen" arrangement of tumor cells surrounded by fibrous connective tissue. HE 360x.

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Fig. 7. CGH ratio profile with loss of chromosome lp and gain of lq, as well as loss of the entire chromosome 3 and low level gains of chromosomes 4, 5, 6q, 9p, llq and 13q. The middle line on the ratio profiles reflects a balanced state, whereas the first lines to the right and left side represent the 50% thresholds.

Metastatic Retroperitoneal Paraganglioma . 443 lumbar spine after the resection of the main intraabdominal tumor. The question arises of whether the vertebral tumor had grown in continuity from the main tumor or had rather represented a metastatic lesion. The intraosseous location in the left dorsal third of the vertebral corps and the pedicle is typical of a hematogenic metastasis [1, 18]. Since no destruction of the righthand side cortical lamellae could be seen, it seems very unlikely that the vertebral lesion developed by continuous tumor growth. These findings led us to the diagnosis of metastatic (malignant) paraganglioma. Few reports have been published on the cytogenetic findings in paraganglioma. In the malignant paraganglioma presented here, we found copy-number abnormalities indicative of i(1q) and also a loss of chromosome 3 and low level gains of chromosomes 4,5, 6q, 9p, llq and 13q by means ofCGH. The loss of Ip was confirmed by microsatellite analysis. Performing short-term cell cultures, Jin et al. (1996) [13J observed i(1q) in one of two sporadic paragangliomas studied. Two other studies performed by Granger and Houn (1990) [1 OJ and Zaslav et al. (1995) [26J revealed either no or nonrandom abnormalities on chromosomes 5 and 7, respectively. Isochromosome 1 is the third most frequent genomic alteration in malignant neoplasms and very rarely seen in benign tumors [17]. As the follow-up of the case reported by Jin et al. was short (three months), malignancy cannot be excluded. Genomic instability in 1p can be found in a variety of human malignancies, and the existence of cancer-related genes in Ip has been suggested [20]. 1p deletion is a common event in neuroblastoma, found in about 30% of the cases [3]. The existence of a composite sympatho-adrenal paraganglioma (phaeochromocytoma), which is also known to be observed in rare extra-adrenal, intra-abdominal locations [21], suggests an evolution of paraganglioma and neuroblastoma from a common progenitor cell with divergent differentiation [7]. Compared to the area of genomic alterations in 1p in neuroblastoma, which includes the distal and proximal putative tumor-suppressor gene [3, 4J, CGH analysis showed a larger region of deletion. Characteristic findings of aberrations on chromosome 2 (N-myc locus) and on chromosome 17 in neuroblastoma [2J were not shown in this case. Therefore, our molecular genetic analysis of an intra-abdominal paraganglioma suggests a different genetic background of these tumor entities. Telomerase activity has not yet been studied in paraganglioma. Telomerase is expressed in gerrnline cells, immortalized cells and in 60-94% of malignant tumors [11, 12,23, 24J, whereas telomerase activity is usually negative in normal somatic cells [22J. In our case, telomerase activity could not be detected in repeatedly performed telomerase assays. However, since no relevant data on paraganglioma exist, it is difficult to evaluate the telomerase negativity in our case.

At present, no characteristic cytogenetic or molecular genetic data on paraganglioma are available. Our case revealed divergent results without telomerase activity, but certain genomic alterations, of which i(1q) might be indicative of malignancy. The recognition of malignancy by the demonstration of metastatic lesions will remain a clinical effort, unless the examination of more tumors will support our finding of i(1q) inmalignant paraganglioma.

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Received: January 28, 1998 Accepted in revised form: April 20, 1998