PTEN Mutations and Activation of the PI3K/Akt/mTOR Signaling Pathway in Papillary Tumors of the Pineal Region

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1 J Neuropathol Exp Neurol Copyright Ó 2014 by the American Association of Neuropathologists, Inc. Vol. 73, No. 8 August 2014 pp. 747Y751 ORIGINAL ARTICLE PTEN Mutations and Activation of the PI3K/Akt/mTOR Signaling Pathway in Papillary Tumors of the Pineal Region Tobias Goschzik, PhD, Marco Gessi, MD, Dorota Denkhaus, and Torsten Pietsch, MD Abstract Papillary tumors of the pineal region (PTPR) are recognized as a distinct entity in the World Health Organization classification of CNS tumors. Papillary tumors of the pineal region frequently show loss of chromosome 10, but no studies have investigated possible target genes on this chromosome. Chromosome 10 harbors the PTEN (phosphatase and tensin homolog) gene, the inactivation of which, by mutation or epigenetic silencing, has been observed in different brain tumors, including high-grade gliomas. In this study, we investigated copy number changes by molecular inversion probe (MIP) analysis and the mutational status of PTEN in 13 PTPR by direct sequencing. MIP analysis of 5 PTPR showed chromosome 10 loss in all cases. In addition, there were losses of chromosomes 3, 14, 22, and X, and gains of whole chromosomes 8, 9, and 12 in more than 1 case. One case had a homozygous PTEN deletion; and 2 point mutations in exon 7 of PTEN (G251D and Q261stop) were found. Immunohistochemistry revealed decrease or loss of the PTEN protein and increased expression of p-akt and p-s6. These results indicated that PTEN mutations and activation of the PI3K/Akt/mTOR signaling pathway may play a role in the biology of PTPR. This evidence may lead to the possible use of PI3K/Akt/mTOR inhibitors in therapy for patients with PTPR. Key Words: Homozygous deletion, Molecular inversion probe analysis, Mutations, Papillary tumors of the pineal region, PI3K/Akt/ mtor signaling pathway, PTEN. INTRODUCTION Papillary tumors of the pineal region (PTPR) are uncommon neuroepithelial tumors of the pineal region, mostly arising in adults and characterized by papillary architecture and epithelial cytology (1, 2), and have been included as a distinct pathologic entity in the 2007 World Health Organization (WHO) classification of CNS tumors. Their immunohistochemical profile and ultrastructural features have been From the Institute of Neuropathology, University of Bonn Medical Center, Bonn, Germany. Send correspondence and reprint requests to: Torsten Pietsch, MD, Institute of Neuropathology, University of Bonn Medical Center, Sigmund-Freud-Str 25, Bonn 53127, Germany; t.pietsch@uni-bonn.de The authors declare no conflicts of interest. Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal s Web site ( J Neuropathol Exp Neurol Volume 73, Number 8, August 2014 widely investigated in recent, indicating a possible origin from the ependymal cells of the subcommissural organ (SCO). By contrast, the molecular features of PTPR have been only partially evaluated (3), and few studies have highlighted their cytogenetic alterations (4, 5). Notably, more than 75% of PTPR showed loss of chromosome 10 (4, 5), but no studies to date have identified possible target genes on this chromosome. Chromosome 10 harbors (among others) the PTEN gene (10q23), the deletion and/or functional loss of which is frequently observed in several brain tumors (6). The PTEN gene encodes a ubiquitously expressed tumor suppressor phosphatase that antagonizes the PI3K/Akt/mTOR (phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin) signaling pathway, thereby modulating apoptosis, cell cycle progression, proliferation, and angiogenesis (6). PTEN is frequently altered by somatic mutations and/or deletions in various types of neoplasms. Among brain tumors, PTEN mutations are frequently observed in high-grade gliomas (6). In addition, germline mutations in the PTEN gene were found in Cowden disease, in several other rare cancerhamartoma syndromes, and in Lhermitte-Duclos disease (6). Data regarding the possible presence of PTEN mutations in PTPR are not available. In this study, after elucidating copy number changes with molecular inversion probe (MIP) analysis, we investigated the mutational status of PTEN in 13 PTPR. MIP technology was used because this probe hybridization-based method is applicable for the analysis of formalin-fixed paraffin-embedded (FFPE) tissue-derived DNA, which is highly fragmented. This is in contrast to whole-genome/whole-exome sequencing or single nucleotide polymorphism arrays that have, in principal, a higher coverage but do not give sufficient results with fragmented DNA. MATERIALS AND METHODS Tissue Samples and Immunohistochemistry Thirteen FFPE tissue specimens from PTPR of 11 patients were studied. For 1 patient, only tissue from the relapsed tumor was available; for another patient, only tissue from the metastatic PTPR and the corresponding relapse of this metastasis was available. Histologic material was retrieved from the archives of the University of Bonn Institute of Neuropathology and of the German Brain Tumor Reference Center (Bonn, Germany). All tumors were classified according to the WHO classification of CNS tumors (1). 747

2 Goschzik et al J Neuropathol Exp Neurol Volume 73, Number 8, August 2014 Confirmatory immunohistochemical analyses were performed on a semiautomated immunohistochemical stainer (Tecan, Crailsheim, Germany) or a Benchmark XT Ventana immunostainer (Roche-Ventana, Darmstadt, Germany) with antibodies against microtubule-associated protein-2 (clone HM- 2; Sigma, St Louis, MO), pan-cytokeratin (clone Lu-5; Bachem, Weil am Rhein, Germany), S-100 protein (polyclonal), epithelial membrane antigen (clone E29), glial fibrillary acidic protein (clone 6F2), neurofilament protein (clone 2F11), synaptophysin (clone SY38), vimentin (clone Vim 3B4), and NSE (clone BBS/ NC/VI-H14) (all from Dako, Glostrup, Denmark). Proliferation indices were evaluated with antibody to the Ki67 antigen (clone MIB1; Dako). For immunohistochemistry, antibodies against p-akt (clone 736E11; Cell Signaling, Danvers, MA), p-s6 (Clone 91B2; Cell Signaling), and PTEN protein (clone PN37; Invitrogen, Darmstadt, Germany) were used. The antiyp-akt and antiyp-s6 antibodies were directed to Ser473 and Ser235/236 phosphorylation sites, respectively. After microwave treatment (30 minutes in 0.01 mol/l sodium citrate, ph 6.0), slides for PTEN, p-akt, and p-s6 immunohistochemistry were incubated in 3% H 2 O 2 for 5 minutes to block endogenous peroxidase activity and washed with distilled water and then Tris-buffered saline solution with 0.1% Tween 20. The slides were incubated in a serum-free blocking solution (Catalyzed Signal Amplification II kit; Dako) for 30 minutes at room temperature. After removal of the blocking solution, the samples were incubated with antiyp- Akt, antiyp-s6, or anti-pten antibody (at 1:200, 1:200, or 1:1000 dilution, respectively) overnight at 4-C. After rinses with Tris-buffered saline solution with 0.1% Tween 20, the bound antibody was detected using the Catalyzed Signal Amplification II biotin-free tyramide signal amplification system (Dako) and visualized using diaminobenzidine. The slides were lightly counterstained with hematoxylin. Immunohistochemical results were scored semiquantitatively by 2 neuropathologists. The intensity of staining and the percentages of PTEN-, p-akty, and p-s6ystained cells were evaluated. An arbitrary scale was chosen for scoring staining intensity as follows: 0, negative; 1+, faintly positive; 2+, moderately positive; 3+, strongly positive. The cumulative percentage of positive cells was assessed on the entire tumor slide. MIP Analysis To identify copy number gains and losses, we used a MIP array including approximately 330,000 inversion probes (v2.0; Affymetrix, Santa Clara, CA). Molecular inversion probe assay was performed on 5 cases as previously described (7). This method could not be applied to the remaining cases because of the low amount and poor quality of available total FFPE-derived DNA. The raw MIP data files were analyzed using the Nexus Copy Number 7.0 Discovery Edition software (BioDiscovery, El Segundo, CA). BioDiscovery s SNP- FASST2-Segmentation algorithm was used to make copy number and loss-of-heterozygosity calls. GISTIC (Genomic Identification of Significant Targets in Cancer) analysis was also used to distinguish significant chromosomal aberrations from random background. Gains were defined as more than 2.3 copies (high copy gains 93.2), and losses were defined as less than 1.7 copies. Amplification was defined as more than 10 copies of a gene locus. PTEN Mutational Analysis DNA from FFPE tumor tissue was extracted using the QIAamp DNA Mini Tissue Kit (Qiagen, Düsseldorf, Germany), FIGURE 1. A virtual karyotype of 5 PTPR analyzed with MIP array. Molecular inversion probe analysis in individual cases shows losses of chromosomes 3, 14, 22, and X, and gains of chromosomes 8, 9, and 12. Notably, all cases investigated show a loss of chromosome 10, including a case with evidence of a homozygous deletion of PTEN (data not shown). Gains are indicated by blue bars on the right side of each chromosome; losses are shown in red on the left side. Thicknesses of the bars indicate the frequency of alterations. Chr, chromosome. 748 Ó 2014 American Association of Neuropathologists, Inc.

3 J Neuropathol Exp Neurol Volume 73, Number 8, August 2014 according to the manufacturer s instructions. PTEN exons 1 to 9 were amplified as previously described (8). Evaluation of the size of amplified products was performed by separation using 2% agarose gel electrophoresis. Polymerase chain reaction products were visualized and documented on a Geldoc 1000 system (BioRad, Munich, Germany). Polymerase chain reaction products were purified using a polymerase chain reaction purification kit (Qiagen). Direct sequencing reactions were performed in duplicate (forward and reverse) as custom service by Eurofins MWG Operon (Ebersberg, Germany) using 30 ng of the polymerase chain reaction product. RESULTS The 11 patients (male, 4; female, 7) with PTPR who were included in this study had a mean age of 30.2 years (range, 17Y49 years) at diagnosis. Three patients developed relapsing or metastatic disease (1 case with local relapse and 2 cases with distant metastases). The metastases affected the cerebral hemisphere (frontal right) and the spinal cord. Histologically, PTPR consisted of alternating solid or papillary areas. The papillary areas showed 1 or more layers of cuboidal to columnar epithelial cells lining fibrovascular cores. The tumor cells showed variable cytologic features, mostly with uniform nuclei and moderate polymorphism. Only 3 cases had at least focal pleomorphic cell nuclei. Mitotic activity varied between 1 and 8 mitoses in 10 high-power fields. Necrosis was observed in 6 cases. Patient clinical data and immunohistochemical profiles are summarized in Table, Supplemental Digital Content 1, Molecular inversion probe analysis of 5 cases showed losses of chromosomes 3, 14, 22, and X, and gains of whole chromosome 8, 9, and 12 in more than 1 case (Fig. 1). Notably, all cases investigated showed a loss of chromosome 10. One case showed a homozygous deletion of PTEN. No evidence of high copy gains of PDGFR, KIT, EGFR, ormet or of other PI3K/Akt/mTOR pathway members (e.g. AKT1 and AKT2) or MYC genes (e.g. MYCC and MYCN) was found. Furthermore, no alteration of CDK4, CDK6, orcdkn2a was evident on MIP analysis. The mutational analysis of PTEN revealed the presence of point mutations in exon 7 in 2 tumors (Figs. 2A, B) (ggtygat [G251D] and cagytag [Q261stop] mutation, respectively) (Figs. 2C, D). The mutations were uncovered in tumors affecting patients aged 17 and 49 years. Both mutations are already listed in the COSMIC (Catalogue of Somatic Mutations in Cancer) database (cancer.sanger.ac.uk). Immunohistochemistry showed a reduced expression of PTEN protein (Fig. 2E) and positive staining for p-akt (Fig. 2F) and p-s6 in most PTPR cases, including PTEN mutated cases. This suggests activation of the PI3K/Akt/mTOR signaling pathway (Table). DISCUSSION The PTEN tumor suppressor gene, located on chromosome 10q23, is one of the most frequently mutated genes in human cancer (9). Its germline mutations cause Cowden syndrome (6, 8, 9), which is characterized by an increased risk for the development of benign and malignant tumors. Sporadic PTEN Mutations in PTPR PTEN mutations have been observed frequently in a variety of human neoplasms, including endometrial carcinoma, glioblastoma, and skin and prostate cancers (6, 9, 10). Mutations may occur along the entire gene, are predominantly monoallelic, and result frequently in truncation of the protein (9). Among brain tumors, PTEN mutations are most frequently identified in gliomas (6). Whereas mutations are observed in approximately 10% to 40% of primary glioblastomas and in a lower number of anaplastic astrocytomas and oligodendrogliomas, they are rare or absent in WHO grade I or II gliomas, glioneuronal tumors, and ependymal tumors (6), which conversely may show PTEN promoter methylation (11). PTEN mutations are very uncommon among brain tumors other than gliomas and have only been rarely identified in meningiomas (12) and medulloblastomas (8). Besides mutations, homozygous PTEN deletions are present in about 5% to 10% of glioblastomas. Although loss of chromosome 10 represents the most common documented cytogenetic alterations in PTPR (4, 5), no studies have been performed to identify the target gene on this chromosome. Here, we identified for the first time PTEN mutations in PTPR, including a homozygous deletion and 2 somatic point mutations. Both point mutations were located in exon 7 (G251D and Q261stop, respectively). They affected the sequence of PTEN that encodes the C2 domain, which is important for PTEN-membrane interaction, bringing the active site to the membrane-bound PIP3 to dephosphorylate it. The frequent loss of the PTEN locus may play an important role in the pathogenesis of PTPR. However, at this time, functional studies of cell lines or genetic animal models designed to prove the functional consequences of PTEN inactivation are not possible. Loss of function of the PTEN tumor suppressor can lead to constitutive activation of the PI3K/Akt/mTOR pathway (13). In our series, immunohistochemistry with antibodies against p-akt and p-s6 yielded positive results in PTPR, confirming the activation of the downstream signaling pathway in these tumors. In addition to PTEN mutations, other possible mechanisms leading to PI3K/Akt/mTOR pathway activation can therefore be hypothesized (i.e. PTEN promoter methylation). Such epigenetic inactivation has been found in medulloblastomas (8). The PI3K/Akt/mTOR pathway has been shown to play an important role in cancer by regulating apoptosis, neoangiogenesis, and proliferation, and has become an important target for anticancer drug development (6, 9, 10, 14). Papillary tumors of the pineal region are characterized by a high risk of local recurrence and are usually treated with a combination of surgery and radiotherapy. The role of chemotherapy in the treatment of these patients is poorly documented (3, 15). Only a few cases of chemotherapy-treated PTPR have been reported. In a large study, chemotherapy did not seem to influence overall survival or progression-free survival (3, 15). The possible use of alkylating agents has also been hypothesized (15). Given pathway activation, the use of PI3K/Akt/mTOR inhibitors could also be hypothesized for the treatment of PTPR even though these agents have demonstrated different efficacies (i.e. in subependymal giant cell astrocytomas and high-grade gliomas), probably as a consequence Ó 2014 American Association of Neuropathologists, Inc. 749

4 Goschzik et al J Neuropathol Exp Neurol Volume 73, Number 8, August 2014 FIGURE 2. Histopathologic, immunohistochemical, and molecular features of 2 PTPR harboring PTEN mutations. (AYD) PTEN mutated tumors. Case 1 (A) and Case 6 (B) show a typical PTPR appearance with papillary architecture and epithelial cytology. Case 1 (C) and Case 6 (D) show point mutations in exon 7: Case 1, ggtygat (G251D); Case 6, cagytag (Q261stop). (E) Tumors show loss of PTEN protein (Case 6). (F) Papillary tumors of the pineal region are p-aktyimmunopositive (Case 3), indicating activation of the PI3K/Akt/mTOR pathway. 750 Ó 2014 American Association of Neuropathologists, Inc.

5 J Neuropathol Exp Neurol Volume 73, Number 8, August 2014 PTEN Mutations in PTPR TABLE. PTEN Protein, p-akt, and p-s6 Immunohistochemical Expression and PTEN Status in PTPR Case No. PTEN p-akt p-s6 PTEN Mutation PTEN Homozygous Deletion 1 1+ (980%) 3+ (70%) 3+ (980%) G251D No 2 1+ (20%) 0 2+ (20%) WT No (50%) 2+ (40%) Yes 4 1+ (20%) 3+ (980%) 3+ (40%) WT No 5 1+ (70%) 2+ (10%) 3+ (10%) WT NA (60%) 3+ (10%) Q261stop No 7 2+ (30%) 3+ (80%) 3+ (80%) WT NA 8 NI 3+ (5%) 3+ (70%) WT NA 9 3+ (80%) 2+ (50%) 3+ (20%) WT NA (70%) 1+ (30%) NA WT NA (80%) 3+ (40%) 3+ (70%) WT NA (60%) 2+ (50%) 3+ (980%) WT NA 13 NA 3+ (980%) 3+ (50%) WT NA Immunohistochemical staining scores in the indicated percentages of cells: 0, negative; 1+, faintly positive; 2+, moderately positive; 3+, strongly positive. NA, not analyzed; NI, not informative; WT, wild type. of the presence of feedback loops and cross-talk with other signaling pathways (14, 16). In conclusion, PTEN mutations and PI3K/Akt/mTOR signaling pathway activation may play a role in the biology of PTPR. This evidence may lead to the clinical use of PI3K/ Akt/mTOR inhibitors as a possible option for therapy for such tumors. REFERENCES 1. Jouvet A, Nakazato Y, Scheithauer BW, et al. Papillary tumors of the pineal region. In: Louis DN, Ohgaki H, Wiestler OD, et al, eds. WHO Classification of Tumours of the Central Nervous System. 4th ed. Lyon, France: IARC Press, 2007:128Y29 2. Dahiya S, Perry A. Pineal tumors. Adv Anat Pathol 2010;17:419Y27 3. Fèvre-Montange M, Hasselblatt M, Figarella-Branger D, et al. Prognosis and histopathologic features in papillary tumors of the pineal region: A retrospective multicenter study of 31 cases. J Neuropathol Exp Neurol 2006;10:1004Y11 4. Hasselblatt M, Blümcke I, Jeibmann A, et al. Immunohistochemical profile and chromosomal imbalances in papillary tumours of the pineal region. Neuropathol Appl Neurobiol 2006;32:278Y83 5. Gutenberg A, Brandis A, Hong B, et al. Common molecular cytogenetic pathway in papillary tumors of the pineal region (PTPR). Brain Pathol 2011;21:672Y77 6. Knobbe CB, Merlo A, Reifenberger G. Pten signaling in gliomas. Neuro-oncology 2002;4:196Y Wang Y, Cottman M, Schiffman JD. Molecular inversion probes: A novel microarray technology and its application in cancer research. Cancer Genet 2012;205:341Y55 8. Hartmann W, Digon-Söntgerath B, Koch A, et al. Phosphatidylinositol 3 -kinase/akt signaling is activated in medulloblastoma cell proliferation and is associated with reduced expression of PTEN. Clin Cancer Res 2006;12:3019Y27 9. Ortega-Molina A, Serrano M. PTEN in cancer, metabolism, and aging. Trends Endocrinol Metab 2013;24:184Y Shi Y, Paluch BE, Wang X, et al. PTEN at a glance. J Cell Sci 2012;125: 4687Y Wiencke JK, Zheng S, Jelluma N, et al. Methylation of the PTEN promoter defines low-grade gliomas and secondary glioblastoma. Neuro-oncology 2007;9:271Y Peters N, Wellenreuther R, Rollbrocker B, et al. Analysis of the PTEN gene in human meningiomas. Neuropathol Appl Neurobiol 1998;24:3Y8 13. Song MS1, Salmena L, Pandolfi PP. The functions and regulation of the PTEN tumour suppressor. Nat Rev Mol Cell Biol 2012;13:283Y Massacesi C, di Tomaso E, Fretault N, et al. Challenges in the clinical development of PI3K inhibitors. Ann N Y Acad Sci 2013;1280:19Y Fauchon F, Hasselblatt M, Jouvet A, et al. Role of surgery, radiotherapy and chemotherapy in papillary tumors of the pineal region: A multicenter study. J Neurooncol 2013;112:223Y Markman B, Tao JJ, Scaltriti M. PI3K pathway inhibitors: Better not left alone. Curr Pharm Des 2013;19:895Y906 Ó 2014 American Association of Neuropathologists, Inc. 751