Annals of Clinical & Laboratory Science 39:295-302 (2009)
© 2009 Association of Clinical Scientists
Molecular Genetic Analysis of a Primitive Neuroectodermal Tumor Arising after Intracranial Radiation and Chemotherapy for Leukemia
Mahlon D. Johnson1,5,
Paul L. Moots3,
Zhengping Zhuang4 and
Robert J. Weil2,6
1 Departments of Pathology, 2 Neurosurgery, and 3 Neurology, Vanderbilt University Medical Center, Nashville, Tennessee; 4 Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland; 5 Department of Pathology, University of Rochester Medical Center, Rochester, New York; 6 Brain Tumor & Neuro-Oncology Center, Cleveland Clinic Foundation, Cleveland, Ohio
Address correspondence to Robert J. Weil, M.D., Brain Tumor & Neuro-Oncology Center, Cleveland Clinic Foundation, ND4-40 Lerner Research Institute, 9500 Euclid Ave, Cleveland, OH 44195, USA; tel 216 444 2007; fax 216 444 2683; e-mail weilr{at}ccf.org; or Mahlon D, Johnson, M.D., Ph.D., Dept. of Pathology & Laboratory Medicine, Univ. of Rochester Medical Center, 601 Elmwood Ave, Box 626, Rochester, NY 14623, USA; tel 585 276 3087; fax 585 273 1027; e-mail mahlon_johnson{at}urmc.rochester.edu.
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Abstract
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Primitive neuroectodermal tumors are aggressive tumors of the central nervous system (CNS), yet their etiology remains unclear. We report a case of a primitive neuroectodermal tumor (PNET) arising in the cerebellum and pons 7 yr after intracranial radiation and chemotherapy for leukemia involving the CNS. This case suggests a possible link between radiation, chemotherapy, and the formation of these tumors, with a potential new pathogenetic role for somatic inactivation of the protooncogene RET.
Keywords: chemotherapy, primitive neuroectodermal tumor, leukemia, loss of heterozygosity, allelic imbalance, radiation-induced tumors, secondary neoplasms, microdissection, RET protooncogene
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Introduction
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Secondary tumors of the central nervous system are a well-recognized phenomenon, especially after treatment for leukemia [1–7]. The principal etiologic factor for secondary tumor formation is thought to be radiation-induced injury to normal cells during treatment of the primary tumor. Intrathecal chemotherapy may also play a role in secondary tumor formation [4,5]. The most common radiation-induced secondary neoplasms of the central nervous system are sarcomas, meningiomas, and gliomas [4,7]. We present a case of a multifocal primitive neuroectodermal tumor arising in the posterior fossa of a patient who had previously received cranial radiation and intrathecal methotrexate therapy, as well as stem cell transplantation after supplemental total body irradiation to treat an acute lymphocytic leukemia (ALL) with central nervous system involvement
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Materials and Methods
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Microscopy.
H&E-stained paraffin sections of tumor and normal cerebellum were available for analysis by light microscopy and immunohistochemistry. Additional fresh tumor tissue was fixed in glutaraldehyde for analysis by transmission electron microscopy (data not shown). The tissue samples and clinical and radiographic information were obtained as part of an Institutional Review Board-approved study of brain tumors at Vanderbilt University School of Medicine. Tissue from the anonymous (male) donor of bone marrow was not available.
Loss of heterozygosity analysis using microsdissection.
Selective tissue microdissection was performed, as previously described, to obtain pure populations of normal cerebellar granule cells and tumor cells [8]. We used the polymorphic marker D10S215, which maps to the PTEN locus on chromosome 10q23.3, and the markers D9S287 and D9S303, both of which map to the PTCH gene locus at 9q23 (Research Genetics, Huntsville, AL), in quantitative PCR analysis with genomic DNA extracted from microdissected, pure populations of tumor and normal cerebellar tissue. We performed PCR amplifications in the presence of [
-32P] dCTP (0.1 µCi/µl) (Amersham Biosciences, Piscataway, NJ), using Ampli-Taq Gold DNA polymerase (Perkin Elmer Roche, Foster City, CA). PCR conditions for marker 10S215 were as follows: initial denaturation at 95°C for 10 min, then 30 cycles, each with 1 min of denaturation at 95°C, 1 min of annealing at 55°C, and 1 min of extension at 72°C; PCR was completed with a final extension at 72°C for 10 min. For the markers D9S303 and D9S287, the annealing temperature was 58°C. The amplicons were resolved on a 6% polyacrylamide gel. Gels were dried and exposed to Kodak (Rochester, NY) XAR film. All PCR reactions were performed in triplicate and were repeated twice.
Microsatellite analysis for loss of heterozygosity on chromosome 17.
For nonradioactive microsatellite analysis, primers D17S520 (17p12) and the intragenic marker, TP53 (17p13.1) (Research Genetics), were used under the conditions described above for D10S677. PCR products were separated on 8% denaturing gels and analyzed after silver staining, as described.
Quantitative PCR amplification of microsatellites at chromosome 10q11.
We used two polymorphic markers, D10S677 and RET (Research Genetics), in quantitative PCR analysis, with genomic DNA extracted from microdissected normal cerebellum and tumor, as previously described [17,18]. In brief, PCR amplifications were performed in the presence of [-32P] dCTP (0.1 Ci/l) as described above. PCR conditions were as follows: initial denaturation at 95°C for 10 min, then 30 cycles, each with 1 min of denaturation at 95°C, 1 min of annealing at 55°C, and 1 min of extension at 72°C; PCR was completed with a final extension at 72°C for 10 min. The amplicons were resolved on a 6% polyacrylamide gel. Gels were dried and exposed to Kodak XAR film. Quantitative analysis of allelic intensities/imbalance was performed using phosphorimage analysis (Molecular Dynamics, Amersham Biosciences). A ratio of 1.5: 1 or greater of the tumor DNA peak: germline DNA peak was defined as allelic imbalance. All PCR reactions were performed in triplicate and were repeated twice. Each densitometric measurement was performed 3 times.
Microsatellite analysis at other loci.
Genotypes for multiple loci were determined by PCR amplification using primers for markers on 1p (D1S80 on 1p36-p35), 3p26-p25 (D3S1110), and 19q13 (D19S601), all from Research Genetics. Nucleotide sequences and mapping information were retrieved from the Human Genome Database (GDB). All amplifications were performed in 15 µl reaction volumes and included an initial denaturation at 94°C of 6 min and a final extension at 72°C for 10 min. PCR conditions for marker D19S601 included 30 cycles with denaturation at 94°C for 45 sec, annealing at 57°C for 45 sec, and 1 min extension at 72°C. PCR conditions for marker D1S80 were 35 cycles of 94°C for 1 min, 65°C for 1 min, and 72°C for 1.5 min. PCR conditions for D3S1110 were the same as for D1S80 except for annealing at 64°C Amplicons were subjected to 6% polyacrylamide denaturing gel electrophoresis at 60 watts for 1.5 hr and analyzed as described above.
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Case Report
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Clinical Summary
The patient was a 29-yr-old man with a history of acute lymphocytic leukemia, which was diagnosed 7 yr prior to this presentation. Initial treatment at an outside facility began with systemic chemotherapy. Malignant leukemic cells were identified in his cerebrospinal fluid and he underwent whole-brain radiation with 2400 cGy, followed by intrathecal methotrexate. His leukemia relapsed 3 yr later. At that time, he underwent a second round of chemotherapy with high-dose ara-C, mitoxantrone, cyclophosphamide, VP-16, and total body irradiation to 1000 cGy. This was followed by an allogeneic bone marrow transplant, at an outside institution, from an anonymous, matched male donor. The patient experienced a mild case of graft-versus-host disease, but otherwise did well.
Six mo prior to this presentation, the patient developed personality changes including swings in temperament and mood. Over the previous 3 wk, the patient reported episodic light-headedness with activity and bilateral facial numbness. No abnormalities were found on neurological examination. An MRI of the brain revealed a 3.0 x 3.0 x 2.5 cm lesion involving the pons and left cerebellar peduncle and a second lesion measuring 2.5 x 2.5 x 2.5 cm in the right cerebellar hemisphere (Fig. 1
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Fig. 1. Magnetic resonance imaging. A, axial; B, coronal; and C, sagittal T1-weighted, gadolinium-enhanced views, which show both the right cerebellar hemispheric tumor and the pontine and left cerebellar peduncle lesions.
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A bone marrow biopsy was normal. CSF cytology was negative for tumor or microorganisms and polymerase chain reaction on the CSF was negative for Epstein-Barr virus, JC virus, or other infectious agents. A suboccipital craniectomy with open biopsy of the right cerebellar lesion was performed using frameless stereotactic guidance; normal-appearing cerebellum, approximately 4 cm away from the tumor, was also biopsied. The patient was neurologically stable postoperatively and was discharged home without neurological deficit. He refused additional treatment. Three mo later, the patient died of respiratory failure related to tumor infiltration and brainstem compression. An autopsy was not performed.
Pathological findings.
The microscopic sections (Fig. 2) revealed cerebellar folia with white matter replaced by a cellular, primitive neuroectodermal tumor. Cells with round or oval nuclei, minimal eosinophilic cytoplasm, and granular or coarse nucleoplasm populated the tumor. Numerous mitoses and apoptotic nuclei were seen and focal necrosis was present. There were no rhabdomyoblasts or sarcomatous elements and vascular proliferation was absent. Scattered monocytes and rare microglia were present; a paucity of reactive astrocytes flanked the tumor. There was no desmoplastic component. Tumor cells exhibited extensive circumnuclear and cytoplasmic synaptophysin immunoreactivity as well as PAS staining. Moderate numbers of cells exhibited cytoplasmic chromogranin, CD99, and glial fibrillary acidic protein staining. No distinct neurofilament, desmin, epithelial membrane antigen, CD20, CD3, CD45RO, CD45, CD68, 1gG, IgM, IgA, kappa chain, or lambda chain immunostaining was seen; these findings are consistent with a primitive neuroectodermal tumor. CD34 immunostaining was negative. Ultra-structurally, the cells exhibited a high nuclear/cytoplasmic ratio with clumped and dispersed chromatin. No cell junctions were seen. The cytoplasm contained polysomes and secondary lysosomes. Normal hematopoietic cells were seen. The ultrastructural findings were consistent with a CNS origin of the tumor cells.
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Fig. 2. Histological and immunohistological views of the tumor. A, PNET with anaplasia, hyperchromatic nuclei, and mitoses are numerous (400x). B, Tumor with extensive synaptophysin staining (400x). C, MIB-1 labeling (brown nuclei) of tumor is approximately 20% (200x).
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Genetic findings.
Molecular analysis of tumor tissue showed loss of heterozygosity on chromosome 17p13 at the p53 locus and on the long arm of chromosome 10, involving the RET locus, as well as at the PTEN locus (Fig. 3 and data not shown). There was no loss of heterozygosity at the PTCH1 locus using 2 separate polymorphic markers. There was no evidence of genetic heterogeneity between tumor and normal cerebellum at a variety of micro-satellite markers on chromosomes 1, 3, and 19, as well as at the PTCH1 locus on chromosome 9, which suggests that the tumor did not arise from donor tissue.
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Fig. 3. Loss of heterozygosity analysis of microdissected tissues. A, LOH shown at the RET locus, using the flanking polymorphic marker D10S667. Lane 1 is a normal control; lanes 2 and 3, different portions of the microdissected tumor; lane 4 contained no Taq polymerase; lanes 5–7 are normal cerebellum from the patient; and lane 8 is a known control with loss of the WT allele. B, LOH shown at the RET locus, using the intragenic polymorphic marker RET. T, tumor tissue; N, normal cerebellum. Arrowheads denote the proper products. As shown by A and B, the tumor demonstrates allelic imbalance/loss of heterozygosity at chromosome 10q11, including the RET gene locus, when compared with the normal cerebellum (N). Microsatellite, polymorphic genomic DNA was amplified from normal and tumor cells. The two alleles represent the two polymorphic genomic sequences, which are separated by gel electrophoresis. The differing densities in the tumor, T, in panel B represent allelic loss (loss of heterozygosity) at the intragenic locus, RET; this is seen in panel A as loss of the lower bands in tumor in lanes 2 and 3.
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Discussion
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Primitive neuroectodermal tumors are a category of World Health Organization (WHO) grade IV CNS tumors named because of their primitive cellular appearance. The term primitive neuroecto-dermal tumor (PNET) is used to characterize an undifferentiated, "small cell," embryonal neoplasm with minimal cytoplasm, indistinct architectural features, and an immunophenotype indicative of extensive neural, glial and, on occasion, mesenchymal differentiation. Some refer to such neoplasms as "embryonal" tumors [11]. The developmental biology of PNETs has expanded in recent years with the growth of molecular genetics [12]. The most common abnormality (30–50% of tumors) is loss or mutation on the short arm of chromosome 17 (17p), near but frequently distinct from the p53 gene [12].
In a recent study of 12 pediatric patients with sporadic supratentorial PNETs, mutations, or alterations of p53 and PTEN were found in only 1 (8%) patient each. Alterations of CDKN2A, EGFR, CDK4, and MDM2 genes, commonly implicated in gliomagenesis, were not identified in any tumor [9]. Recent studies by Schuerlen et al [14] and Mollenhauer et al [15] suggest, however, that while LOH on the distal arm of 10q is a common event in PNETs, it tends to involve the DMBT1 locus at 10q25.3-qter rather than PTEN, or in rare situations, to involve both.
In patients who have undergone cranial irradiation, there is an association between radiation and the formation of cranial tumors such as sarcomas, meningiomas, and gliomas [1–7]. It has been proposed that faulty repair of DNA damage caused by ionizing radiation underlies neoplastic degeneration [7]. Secondary tumor formation following cranial irradiation, however, seems to be a multifactorial process and comprises variables such as radiation type and quantity; the nature of the primary disease; sex; age at irradiation; type and duration of chemotherapy; and the concept that patients with one tumor may have a genetic disposition to develop a second neoplasm that is stimulated by radiation [5,7].
One subgroup of patients with primary neoplasms who are prone to develop secondary tumors is comprised of those treated with cranial radiation for leukemia. The increased risk for nervous system tumors in these patients has been estimated to be 5 to almost 30 times normal [1–3,5,16]. Intrathecal chemotherapy, specifically methotrexate, may also potentiate neoplastic formation [4]. Malignant glial tumors, sarcomas, and meningiomas are the most common secondary tumors in patients with leukemia [4]. Finally, it has been suggested that the risk of developing second tumors at 10 yr after intensive chemo-irradiation and bone marrow transplantation may be as high as 20% [16].
The association between cranial radiation, chemotherapy, and neoplasia has been less commonly implicated in the pathogenesis of PNETs. Only 16 cases have been identified in the literature describing intracranial PNETs after radiation [2,3,5,16–19] (Table 1). Barasch et al [2] reported the first case, a patient who underwent cranial radiation and intrathecal methotrexate for leukemia. Relling et al [16] described a patient who developed a left frontal PNET 6 yr after radiotherapy [16]. Additional patients have been described after irradiation for lymphoma, retinoblastoma, and gliomas, with doses ranging from 1800 to 5500 cGy, alone or with intrathecal methotrexate. The range of latency between treatment and discovery of a PNET was 5–18 yr [2,3,5,16–19].
Brüstle et al [3] reported 3 patients who had tumors resembling PNETs after CNS treatment for ALL (n = 2) and malignant T-cell lymphoma. Activating mutations of another proto-oncogene, RET, which encodes a receptor tyrosine kinase that activates both the Ras/MAPK and PI3-kinase pathways, have been identified in familial and sporadic medullary thyroid carcinoma (MTC) and other tumors of neural origin that comprise multiple endocrine neoplasia, type 2 [10,20]. Although the presence of multiple, activating mutations of RET has previously been thought to be the source of tumorigenesis, recent work has shown that RET allelic imbalance is similarly tumorigenic [10,20]. In this setting, the wild type allele, which has been offsetting the mutant, activated (oncogenic) allele, is now lost, and thus the unrestrained mutant allele promotes tumorigenesis [10,20]. While for some tumors, somatic RET mutations may represent a primary event (as with familial MTC), for sporadic tumors, it may be involved in tumor progression. And, as suggested elsewhere, allelic imbalance of RET, with or without inactivating mutations of PTEN, may be even more tumorigenic in the setting of other genetic abnormalities [20]. Interestingly, RET is more sensitive to fragmentation/rearrangements after ionizing radiation than several proto-oncogenes [21].
One final possible explanation is that radiation induces activation of either leukemic cells or of hematopoietic stem cell precursors originating from the previous allogeneic stem cell transplant [22,23]. However, the absence of CD34 staining–a marker of hematopoietic stem cells–as well as the lack of microglial cell proliferation, which has been shown in animal models to be the form that hematological precursors take in the CNS, diminishes the likelihood that these PNETs are the result of transformation of either the original leukemic cells or the transplanted bone marrow cells [23]. Ultrastructural analysis supported a neural origin for the tumor cells. Furthermore, hematopoietically-derived cells do not express synaptophysin. There was no evidence of micro-satellite instability at several loci analyzed, when tumor and normal cerebellar cells were analyzed simultaneously, which confirms that the tumor cells did not derive from another individual.
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Table I. Characteristics of Patients Developing PNETs after Cranial IrradiationIn summary, we present a patient who developed a multifocal, CNS primitive neuroectodermal tumor 7 yr after being diagnosed and treated for ALL with cranial radiation and intrathecal methotrexate, and then by total body irradiation and allogeneic bone marrow transplantation. Genetic analysis suggests that multiple genetic abnormalities may be necessary to initiate radiation-induced PNET formation. Tumorigenesis in this setting may share some similarities with the genesis of sporadic medulloblastomas or other primary brain tumors (for example, alterations of p53 and PTEN) as well as novel features (allelic imbalance of the proto-oncogene, RET). Although it is difficult to define the exact etiology and mechanism behind the formation of secondary, post-radiation tumors of the CNS, there is evidence that these tumors may be induced after normal neuronal development has been completed. This work intimates that a multifocal genetic origin for secondary primitive neuroectodermal tumors is likely and suggests some additional mechanisms that may underlie the genesis of sporadic PNETs.
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Acknowledgements
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We thank the Melvin Burkhardt chair in neurosurgical oncology and the Karen Colina Wilson research endowment within the Brain Tumor and Neuro-oncology Center at the Cleveland Clinic Foundation for partial support and funding.
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Abstract
Introduction
