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Journal of Clinical Oncology, Vol 25, No 6 (February 20), 2007: pp. 682-689 © 2007 American Society of Clinical Oncology. DOI: 10.1200/JCO.2006.06.8213 Clinical and Molecular Characteristics of Malignant Transformation of Low-Grade Glioma in Children
From the Departments of Oncology, Developmental Neurobiology, Biochemistry, Biostatistics, Radiological Sciences, and Pathology, St Jude Children's Research Hospital; Department of Interdisciplinary Sciences, University of Tennessee Health Sciences Center, Memphis, TN; and the Northern Institute for Cancer Research, University of Newcastle, Newcastle-upon-Tyne, United Kingdom Address reprint requests to Alberto Broniscer, MD, Department of Oncology, St Jude Children's Research Hospital, Mail Stop 260, 332 North Lauderdale St, Memphis, TN 38105; e-mail: alberto.broniscer{at}stjude.org
PURPOSE: To analyze the clinical and molecular characteristics of malignant transformation (MT) of low-grade glioma (LGG) in children. PATIENTS AND METHODS: The clinical, radiologic, and histologic characteristics of children treated at our institution who experienced MT of LGG were reviewed. Molecular alterations in these tumors were analyzed by fluorescent in situ hybridization, immunohistochemistry, and TP53 sequencing. Cumulative incidence estimate and risk factors for MT were determined for 65 patients with grade 2 astrocytoma treated at our institution during the study interval. RESULTS: Eleven patients who experienced MT were identified (median age at diagnosis of LGG, 13.3 years). Initial diagnoses were grade 2 astrocytoma (n = 6) and other grade 1/2 gliomas (n = 5). The median latency of MT was 5.1 years. Histologic diagnoses after MT were glioblastoma (n = 7) and other high-grade gliomas (n = 4). The 15-year cumulative incidence estimate of MT among 65 patients with grade 2 astrocytoma was 6.7% ± 3.9%; no risk factor analyzed, including radiotherapy, was associated with MT. Tissue was available for molecular analysis in all patients, including nine with samples obtained before and after MT. TP53 overexpression was more common after MT. Deletions of RB1 and/or CDKN2A were observed in 71% of LGGs and in 90% of tumors after MT. PTEN pathway abnormalities occurred in 76% of patients. One of five oncogenes analyzed (PDGFRA) was amplified in one patient. CONCLUSION: The molecular abnormalities that occur during MT of LGG in children are similar to those observed in primary and secondary glioblastoma in adults.
Gliomas are the most common group of CNS neoplasms in children and adults.1 Whereas low-grade gliomas (LGGs) comprise the majority of these neoplasms in children, high-grade gliomas (HGGs), particularly glioblastoma (WHO grade 4), predominate in adults.1 Glioblastoma in adults is classified as secondary or primary on the basis of progression from pre-existing LGGs and on distinct patterns of molecular abnormalities.2 Unlike adults, little is known about the mechanisms of tumorigenesis of HGG in children.3 Although some molecular abnormalities encountered in HGG in children are reminiscent of secondary glioblastoma,4 these neoplasms rarely originate from pre-existing LGGs.5-10 Although one study suggested an association between radiotherapy (RT) and malignant transformation (MT) of LGG in children,8 no information is available about cumulative incidence, risk factors, and molecular abnormalities associated with this event. We conducted extensive clinical and molecular analysis in the largest cohort to date of children who experienced MT of LGG to improve the understanding of this important phenomenon in pediatric gliomas.
Once institutional review board approval was obtained, we selected patients younger than age 22 years with HGG treated at our institution between January 1, 1985 and December 31, 2003 who experienced MT of LGG. Our criteria for MT were based on: consecutive histologic diagnosis of LGG and HGG in at least two distinct tumor samples obtained from the same patient at different time points; exclusion of second neoplasms by radiologic review; and compatible clinical history. Our radiologic criterion for MT was the presence of contiguous or overlapping sites of involvement of LGG and subsequent HGG. HGGs that arose in areas distinct from LGG were considered second neoplasms.11 Central histologic review (C.E.F.) of all tumor samples at diagnosis of LGG and HGG confirmed MT. An independent histologic review was conducted by a second neuropathologist (D.W.E.) who was blinded in regards to sample identity and timing of collection. Clinical information and treatment data were ascertained from chart review. All available tumor samples were collected for molecular studies.
Immunohistochemistry and Fluorescent in Situ Hybridization Studies Immunohistochemical studies for the epidermal growth factor receptor (EGFR), other EGFR-family members (ERBB2, ERBB3, and ERBB4), TP53, phospho-AKT (pAKT) and phospho-S6 (pS6) were performed using standard techniques as previously reported.4,12,13 Controls included a panel of healthy human tissues and tumor cells with previously characterized overexpression of each EGFR-family protein. For pAKT and pS6, brain tissue from Pten conditional knockout and healthy mice were used as positive and negative controls, respectively, for activation of the PTEN pathway. Dual-color fluorescent in situ hybridization (FISH) analyses were conducted as previously described.14 Table 1 lists the bacterial artificial chromosome and P1 bacteriophage clones used to produce FISH probes. Probes were paired for dual-target hybridization as follows: EGFR/7q, 17p/ERBB2, PTEN/DMBT1, RB1/13q34, CDKN2A/9q, CDK4/12p, 4p/PDGFRA, 5p/PDGFRB, 1p/1q, and 19p/19q. Sections showing sufficient hybridization efficiency (signals in the majority of nuclei) were considered informative, and 100 to 200 intact, non-overlapping nuclei were scored for fluorescent signals by two independent reviewers. Thresholds indicating abnormalities/deletions were determined for each probe on the basis of counts from non-neoplastic control specimens (healthy brain from autopsy patients).
Deletion was defined as more than 40% of tumor nuclei containing a test:control ratio  0.5. Homozygous CDKN2A deletion was defined by the simultaneous lack of both CDKN2A signals and presence of 9q signals in more than 20% of tumor cells. CDKN2A and 9q signals had to be seen within vascular endothelial cells (ie, internal non-neoplastic control) to rule out the possibility of partial hybridization failure. Amplification was defined as more than 5% nuclei containing a test:control probe ratio higher than 2.
TP53 Sequencing
Control LGGs After institutional review board approval, tumor samples from 33 patients with LGG (juvenile pilocytic astrocytoma [JPA], n = 20; WHO grade 2 infiltrative astrocytoma, n = 7; pleomorphic xanthoastrocytoma, n = 3; ganglioglioma, n = 2; and oligodendroglioma, n = 1) who did not experience MT underwent PTEN analysis by FISH and immunohistochemical evaluation of pAKT and pS6. Their median age at diagnosis of LGG was 8.9 years (range, 0.2 to 16.5 years). The predominant tumor locations were the diencephalon (n = 13; 39%), cerebral hemispheres (n = 10; 30%), and posterior fossa (n = 8; 24%).
Statistical Analysis The exact log-rank test was used to compare the incidence of MT in patients with WHO grade 2 infiltrative astrocytoma (n = 65) on the basis of sex, race, tumor location, extent of resection, and use of RT or chemotherapy.16 Fisher's exact test was used to evaluate the association of molecular abnormalities. Clinical characteristics, treatment, and outcome of 65 patients with WHO grade 2 infiltrative astrocytoma are provided in Appendix Table A1 (online only).
Of 165 patients with HGG treated at our institution during the study interval, 11 patients met our criteria for MT of LGG (Table 3). Whereas both reviewers agreed on the diagnosis of MT in nine of 11 patients, a minor disagreement occurred in one patient where MT was confirmed by a third neuropathologist.17 No independent review was performed in one patient. The median age at diagnosis of LGG was 13.3 years.
Only three patients received no additional therapy after surgery for LGG (Table 3). Six children received external-beam RT for LGG (median dose, 54 Gy; range, 50.4 to 55.8 Gy; dose unknown for one patient). Additional local RT consisted of brachytherapy (50 Gy) for one patient and a stereotactic radiosurgical boost (12.5 Gy) for another patient. Five children received chemotherapy for LGG (Table 3). Three patients (patients 6, 7, and 8 in Table 3) experienced treatment failure of LGG before the diagnosis of MT. Two patients (patients 6 and 7) received chemotherapy after surgery and local RT at the time of treatment failure. Another patient (patient 8) received RT after first surgery for LGG; he received chemotherapy and gene therapy with vector producer cells containing herpes simplex thymidine kinase and ganciclovir at subsequent treatment failures of LGG (Table 3).18
Characteristics of MT, Treatment, and Outcome
Table 3 provides details about treatment after MT. Only one patient remained alive without evidence of disease 6.5 years after the diagnosis of MT. All deaths resulted from tumor progression. The median survival after MT was 0.6 years.
Incidence and Risk Factors None of the potential risk factors analyzed in 65 patients with WHO grade 2 infiltrative astrocytoma, including use of RT (P = .54), tumor location (P = .37), extent of resection (P = .12), and use of chemotherapy (P = .11) were found to be statistically associated with increased incidence of MT.
Molecular Studies
In 11 tumor samples analyzed from eight patients who experienced MT, we observed TP53 mutation in one LGG and in two HGGs. Two of these three tumor samples harbored a homozygous single base-pair mutation at codon 273 (C  T), a common site of missense mutations in secondary glioblastoma in adults.19 One patient demonstrated a heterozygous single base-pair mutation at codon 275 (GT). One patient (patient 11) whose paired samples were analyzed had a TP53 mutation only after MT. Abnormalities of the retinoblastoma tumor-suppressor pathway (CDKN2A and/or RB1 deletion) were observed in five (71%) of seven LGGs, and in nine (90%) of 10 tumors after MT. RB1 and CDKN2A loss were mutually exclusive in 77% of patients, and RB1 loss was observed in combination with both hemizygous and homozygous deletions of CDKN2A. All but one tumor showing loss of 1p had concomitant loss of 19q. There was no obvious relation of loss of 1p and 19q and histologic diagnosis. Thirteen (76%) of 17 tumor samples analyzed showed evidence of PTEN pathway alteration (Table 4). Concomitant overexpression of pAKT and pS6 consistent with inactivation of PTEN was common (P = .015), although three tumors (two LGG and one HGG) showed combined overexpression of pAKT and pS6 without concomitant PTEN deletion. Therefore, a borderline statistical association was found between PTEN deletion and pAKT (P = .14) and pS6 (P = .085) overexpression suggesting that PTEN deletion alone does not identify all tumors in which PTEN pathway signaling is dysregulated. Representative examples of FISH evaluation of PTEN status and corresponding pAKT and pS6 immunohistochemical studies are shown in Figure 2.
Of five oncogenes tested, PDGFRA amplification was detected in only one patient after MT. Although EGFR and ERBB4 overexpression were seen in both LGGs and HGGs, they occurred more commonly in the latter group. Neither ERBB2 nor ERBB3 expression was documented in any of the tumors analyzed.
Control LGGs
Whereas half to more than 90% of adults with WHO grade 2 infiltrative astrocytoma experience MT,20,21 we showed that the long-term risk of MT in histologically identical neoplasms in children is less than 10%. Although the cumulative incidence of MT among older children seemed to be higher than that for all patients, the small number of assessable patients and the large SEs of our estimates preclude any conclusions about differences in the occurrence of this phenomenon based on age. Of 65 patients with WHO grade 2 infiltrative astrocytoma in this study, 46 (71%) remained alive without tumor progression after a median follow-up of almost 11 years. Only two of 46 patients had not had follow-up within the past 2 years. Whereas two of 17 patients with ganglioglioma experienced MT in this study, this phenomenon was observed in only three of 184 predominantly adult patients in a recent study.22 Another report described the occurrence of MT in one of 24 patients younger than age 21 years with ganglioglioma.23 We did not draw any conclusions about MT in children with ganglioglioma because of the small number of assessable patients. Unlike two previous studies that found an approximately 10% cumulative incidence of MT in children with JPA,7,10 in our experience this event was exceedingly rare. Three of 36 patients (28 patients < 18 years of age) with JPA experienced MT in one of these previous studies7; however, no details were provided about the characteristics of MT. In a second study, Krieger et al10 reported four patients with JPA (age range at diagnosis of LGG, 15 months to 14 years) who experienced MT. Treatment after MT consisted of chemotherapy only (n = 2) and the combination of chemotherapy and RT (n = 1); one patient received no therapy after MT. All four patients remained alive at the time of publication (follow-up, 1 to 5 years). Their favorable outcome raises the question whether these tumors had truly undergone MT. We acknowledge that the strict selection criteria for MT in this study and the lack of histologic confirmation at autopsy in patients who died of progressive LGG may have caused underestimation of the cumulative incidence of this phenomenon. In contrast, it is possible that the cumulative incidence of MT may have been overestimated by the inadequate representation of areas with more malignant characteristics at the time of diagnosis of LGG. However, the thorough and prolonged follow-up of our patients, combined with the radiologic and central histologic review of all instances of MT strengthened the reliability of our results. None of the risk factors analyzed in this study demonstrated a statistically significant association with MT. The small number of assessable patients limited the power of our analysis to confirm or refute the previously proposed association between RT and MT of LGG in children.8 Whereas the molecular abnormalities associated with MT in adults have been extensively characterized,2 limited molecular analyses have been previously described in only four patients younger than 22 years who experienced MT.24-26 We were able to perform molecular studies in all tumor samples obtained after MT, and in nine tumor samples at the diagnosis of LGG and after MT. Our choice of molecular studies was based on abnormalities commonly seen in glioblastoma in adults. TP53 overexpression was observed more frequently after than before MT in this study; however, this association did not reach statistical significance. As described previously,4 we found that TP53 overexpression did not strictly overlap with the presence of TP53 mutations. PTEN deletion was observed after MT in more than half of our patients, a rate similar to that described in both primary and secondary glioblastoma in adults and primary glioblastoma in children.15,19,27 Unlike LGGs in adults,28,29 we observed an unexpectedly high frequency of PTEN deletion in LGGs from patients who experienced MT as well as patients with LGG in the control group. Although deletions of chromosome 10q that encompass PTEN could target an alternative tumor-suppressor locus, the common combined overexpression of pAKT and pS6 observed in LGGs support dysregulation of the PTEN pathway. Future studies should analyze other molecular determinants of PTEN pathway regulation in LGG in children, particularly inactivating mutations of PTEN and activating mutations of PIK3CA. The latter have been reported in high-grade astrocytoma and would be predicted to cause coordinate increases in pAKT and pS6.30,31 Combined loss of 1p and 19q was uncommon among our patients, particularly in those samples obtained after MT, and similar to the results of a recent pediatric study.32 Although HGGs in children rarely originate from pre-existing LGGs, the molecular abnormalities found in pediatric de novo HGGs seem to be most reminiscent of secondary glioblastoma in adults.4,33 Conversely, some of the genetic alterations observed in our patients after MT, including deletion of key genes in the retinoblastoma tumor-suppressor and PTEN pathways, overlap with those of both primary and secondary glioblastoma in adults. Although this study revealed several key aspects of MT of LGG in children, our results were limited by the small number of affected patients. Therefore, larger series of children with MT of LGG are warranted to validate our findings.
The authors indicated no potential conflicts of interest.
Conception and design: Alberto Broniscer, Suzanne J. Baker, Larry E. Kun, Amar Gajjar, Richard J. Gilbertson, Christine E. Fuller Financial support: Amar Gajjar Administrative support: Amar Gajjar Provision of study materials or patients: Alberto Broniscer, Larry E. Kun, Amar Gajjar Collection and assembly of data: Alberto Broniscer, Suzanne J. Baker, Alina N. West, Melissa M. Fraser, Erika Proko, James Dalton, Gerard P. Zambetti, Amar Gajjar, Richard J. Gilbertson, Christine E. Fuller Data analysis and interpretation: Alberto Broniscer, Suzanne J. Baker, Alina N. West, Melissa M. Fraser, Erika Proko, Mehmet Kocak, James Dalton, Gerard P. Zambetti, David W. Ellison, Amar Gajjar, Richard J. Gilbertson, Christine E. Fuller Manuscript writing: Alberto Broniscer, Suzanne J. Baker, Alina N. West, Gerard P. Zambetti, Larry E. Kun, Amar Gajjar, Richard J. Gilbertson, Christine E. Fuller Final approval of manuscript: Alberto Broniscer, Suzanne J. Baker, David W. Ellison, Larry E. Kun, Amar Gajjar, Richard J. Gilbertson, Christine E. Fuller
We thank Pam Hays for her assistance with patient follow-up and Sharon Naron for her assistance with scientific editing.
Supported in part by National Institutes of Health grants (Cancer Center Support [CORE] Grant No. P30 CA21765 and Grant No. P01 CA096832 to S.J.B); Musicians Against Childhood Cancer; the Noyes Foundation; the Ryan McGhee Foundation; and by the American Lebanese Syrian Associated Charities (ALSAC). Presented in part at the 12th International Symposium on Pediatric Neuro-Oncology, Nara, Japan, July 6-9, 2006. Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article.
1. Central Brain Tumor Registry of the United States: 2004-2005 Statistical Report: Primary Brain Tumors in the United States Statistical Report, 1997-2001. http://www.cbtrus.org/reports//2004-2005/2005report.pdf 2. Kleihues P, Burger PC, Collins VP, et al: Glioblastoma, in Kleihues P, Cavenee WK (eds): Pathology and Genetics: Tumours of the Nervous System. Lyon, France, IARC Press, 2000, pp 29-39 3. Broniscer A, Gajjar A: Supratentorial high-grade astrocytoma and diffuse brainstem glioma: Two challenges for the pediatric oncologist. Oncologist 9:197-206, 2004 4. Pollack IF, Finkelstein SD, Woods J, et al: Expression of p53 and prognosis in children with malignant gliomas. N Engl J Med 346:420-427, 2002 5. Phuphanich S, Edwards MS, Levin VA, et al: Supratentorial malignant gliomas of childhood: Results of treatment with radiation therapy and chemotherapy. J Neurosurg 60:495-499, 1984[Medline] 6. Dropcho EJ, Wisoff JH, Walker RW, et al: Supratentorial malignant gliomas in childhood: A review of fifty cases. Ann Neurol 22:355-364, 1987[CrossRef][Medline] 7. Wallner KE, Gonzales MF, Edwards MS, et al: Treatment results of juvenile pilocytic astrocytoma. J Neurosurg 69:171-176, 1988[Medline] 8. Dirks PB, Jay V, Becker LE, et al: Development of anaplastic changes in low-grade astrocytomas of childhood. Neurosurgery 34:68-78, 1994[Medline] 9. Pollack IF, Claassen D, al-Shboul Q, et al: Low-grade gliomas of the cerebral hemispheres in children: An analysis of 71 cases. J Neurosurg 82:536-547, 1995[Medline] 10. Krieger MD, Gonzalez-Gomez I, Levy ML, et al: Recurrence patterns and anaplastic change in a long-term study of pilocytic astrocytomas. Pediatr Neurosurg 27:1-11, 1997[Medline] 11. Broniscer A, Ke W, Fuller CE, et al: Second neoplasms in pediatric patients with primary central nervous system tumors: The St Jude Children's Research Hospital experience. Cancer 100:2246-2252, 2004[CrossRef][Medline] 12. Gilbertson RJ, Perry RH, Kelly PJ, et al: Prognostic significance of HER2 and HER4 coexpression in childhood medulloblastoma. Cancer Res 57:3272-3280, 1997 13. Kwon CH, Zhu X, Zhang J, et al: MTor is required for hypertrophy of Pten-deficient neuronal soma in vivo. Proc Natl Acad Sci U S A 100:12923-12928, 2003 14. Fuller CE, Wang H, Zhang W, et al: High-throughput molecular profiling of high-grade astrocytomas: The utility of fluorescence in situ hybridization on tissue microarrays (TMA-FISH). J Neuropathol Exp Neurol 61:1078-1084, 2002[Medline] 15. Kalbfleisch JD, Prentice RL: Competing risks and multistate models, in Kalbfleisch JD, Prentice RL (eds): The Statistical Analysis of Failure Time Data (ed 2). New York, NY, John Wiley & Sons, 2002, pp 252-255 16. Mantel N, Haenszel W: Statistical aspects of the analysis of data from retrospective studies of disease. J Natl Cancer Inst 22:719-748, 1958 17. Fouladi M, Jenkins J, Burger P, et al: Pleomorphic xanthoastrocytoma: Favorable outcome after complete surgical resection. Neuro-Oncol 3:184-192, 2001[Abstract] 18. Kun LE, Gajjar A, Muhlbauer M, et al: Stereotactic injection of herpes simplex thymidine kinase vector producer cells (PA317-G1Tk1SvNa. 7) and intravenous ganciclovir for the treatment of progressive or recurrent primary supratentorial pediatric malignant brain tumors. Hum Gene Ther 6:1231-1255, 1995[Medline] 19. Ohgaki H, Dessen P, Jourde B, et al: Genetic pathways to glioblastoma: A population-based study. Cancer Res 64:6892-6899, 2004 20. Vertosick FT Jr, Selker RG, Arena VC: Survival of patients with well-differentiated astrocytomas diagnosed in the era of computed tomography. Neurosurgery 28:496-501, 1991[CrossRef][Medline] 21. McCormack BM, Miller DC, Budzilovich GN, et al: Treatment and survival of low-grade astrocytoma in adults–1977-1988. Neurosurgery 31:636-642, 1992[Medline] 22. Luyken C, Blumcke I, Fimmers R, et al: Supratentorial gangliogliomas: Histopathologic grading and tumor recurrence in 184 patients with a median follow-up of 8 years. Cancer 101:146-155, 2004[CrossRef][Medline] 23. Im SH, Chung CK, Cho BK, et al: Intracranial ganglioglioma: Preoperative characteristics and oncologic outcome after surgery. J Neurooncol 59:173-183, 2002[CrossRef][Medline] 24. Hayashi Y, Iwato M, Hasegawa M, et al: Malignant transformation of a gangliocytoma/ganglioglioma into a glioblastoma multiforme: A molecular genetic analysis: Case report. J Neurosurg 95:138-142, 2001[Medline] 25. Kim NR, Wang KC, Bang JS, et al: Glioblastomatous transformation of ganglioglioma: Case report with reference to molecular genetic and flow cytometric analysis. Pathol Int 53:874-882, 2003[CrossRef][Medline] 26. Hulsebos TJ, Troost D, Leenstra S: Molecular-genetic characterisation of gliomas that recur as same grade or higher grade tumours. J Neurol Neurosurg Psychiatry 75:723-726, 2004 27. Korshunov A, Sycheva R, Gorelyshev S, et al: Clinical utility of fluorescence in situ hybridization (FISH) in nonbrainstem glioblastoma of childhood. Mod Pathol 18:1258-1263, 2005[CrossRef][Medline] 28. Rasheed A, Herndon JE, Stenzel TT, et al: Molecular markers of prognosis in astrocytic tumors. Cancer 94:2688-2697, 2002[CrossRef][Medline] 29. Fan X, Munoz J, Sanko SG, et al: PTEN, DMBT1, and p16 alterations in diffusely infiltrating astrocytomas. Int J Oncol 21:667-674, 2002[Medline] 30. Samuels Y, Wang Z, Bardelli A, et al: High frequency of mutations of the PIK3CA gene in human cancers. Science 304:554, 2004 31. Broderick DK, Di C, Parrett TJ, et al: Mutations of the PIK3CA in anaplastic oligodendrogliomas, high-grade astrocytomas, and medulloblastomas. Cancer Res 64:5048-5050, 2004 32. Pollack IF, Finkelstein SD, Burnham J, et al: Association between chromosome 1p and 19q loss and outcome in pediatric malignant gliomas: Results from the CCG-945 cohort. Pediatr Neurosurg 39:114-121, 2003[CrossRef][Medline] 33. Alonso M, Hamelin R, Kim M, et al: Microsatellite instability occurs in distinct subtypes of pediatric but not adult central nervous system tumors. Cancer Res 61:2124-2128, 2001 Submitted March 29, 2006; accepted November 28, 2006. This article has been cited by other articles:
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Copyright © 2007 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
