Ubiquitous Aberrant RASSF1A Promoter Methylation in Childhood Neoplasia1

994 Vol. 10, 994–1002, February 1, 2004 Clinical Cancer Research

Ivy H. N. Wong,1 Jacqueline Chan,1 Joyce Wong,2 during, and after treatment, correspondingly, from pediat- and Paul K. H. Tam2 ric patients with neuroblastoma, thyroid carcinoma, hepa- Departments of 1Biochemistry and 2Surgery, Faculty of Medicine, tocellular carcinoma, rhabdomyosarcoma, Burkitt’s lym- The University of Hong Kong, Hong Kong Special Administrative phoma, T-cell lymphoma, or acute lymphoblastic leukemia. Region Concordantly, RASSF1A methylation was found during treatment in plasma of the same patients, suggesting cell death and good response to chemotherapy. ABSTRACT Conclusions: RASSF1A methylation in tumor or buffy Purpose and Experimental Design: The role of coat did not correlate strongly with age, tumor size, recur- RASSF1A has been elucidated recently in regulating apo- rence/metastasis, or overall survival in this cohort of pedi- ptosis and cell cycle progression by inhibiting cyclin D1 atric cancer patients. Of importance, epigenetic inactivation accumulation. Aberrant RASSF1A promoter methylation of RASSF1A may potentially be crucial in pediatric tumor has been found frequently in multiple adult cancer types. initiation. Using methylation-specific PCR and reverse transcription- PCR, we investigated epigenetic deregulation of RASSF1A in primary tumors, adjacent nontumor tissues, secondary INTRODUCTION metastases, peripheral blood cells, and plasma samples from Childhood neoplasms are biologically different from adult children with 18 different cancer types, in association with tumors, in that childhood neoplasms resemble the embryonic their clinicopathologic features. precursors of the cell types they arise, similar to undifferentiated Results: Regardless of the tumor size, ubiquitous cells appearing during normal embryonic development (1, 2). RASSF1A promoter methylation was found in 67% (16 of Genetic or epigenetic alterations in pediatric tumors may poten- 24) of pediatric tumors, including neuroblastoma, thyroid tially lead to arrested differentiation or dedifferentiation. The carcinoma, hepatocellular carcinoma, pancreatoblastoma, biological characteristics of pediatric malignancies, such as the adrenocortical carcinoma, Wilms’ tumor, Burkitt’s lym- ability to proliferate, invade, migrate, and exhibit differential phoma, and T-cell lymphoma. A majority (75%) of pediatric sensitivity to cytotoxic agents, may provide insights into normal cancer patients with tumoral RASSF1A methylation was cellular and developmental processes. Pediatric tumors are very male. Methylated RASSF1A alleles were also detected in 4 of often highly invasive, metastasizing early in the course of the 13 adjacent nontumor tissues, suggesting that this epigenetic development, but they are very responsive to current therapies change is potentially an early and critical event in childhood (1, 2). neoplasia. RASSF1A promoter methylation found in 92% In contrast to inconsistent chromosomal aberrations found (11 of 12) of cell lines largely derived from pediatric cancer in various adult cancer types, recurring cytogenetic abnormali- patients was significantly associated with transcriptional ties are observed in pediatric cancers (1, 2). As opposed to adult silencing/repression. After demethylation treatment with epithelial tumors, pediatric solid tumors possessing only a few 5-aza-2-deoxycytidine, transcriptional reactivation was genetic mutations can develop after short latent periods (2, 3). shown in KELLY, RD, and Namalwa cell lines as analyzed The methylation patterns in adult tumors have been studied by reverse transcription-PCR. For the first time, RASSF1A extensively, and each tumor type appears to have a distinct methylation was detected in 54% (7 of 13), 40% (4 of 10), methylation profile (4). However, little has been known about and 9% (1 of 11) of buffy coat samples collected before, the methylation profiles in childhood malignancies. RAS plays an important role in the signal transduction from cell surface receptors to an array of intracellular signaling pathways. Mutations leading to constitutive activation of RAS Received 3/12/03; revised 9/19/03; accepted 10/23/03. are commonly found in human cancers (4, 5). RAS binds and Grant support: Research Grants Council Grant No. HKU7484/03M activates a diverse array of effectors and mediates tumor sup- from the Hong Kong Research Grants Council and Research Grants pressive effects in addition to oncogenic effects (6). Activated Council Direct Allocation No. 10204245 from the University of Hong RAS mediates the induction of DNA synthesis (7), tumorigenic Kong. transformation (8), metastasis/invasion (9), reduction of growth The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked factor dependence (10), loss of contact inhibition (11), inhibi- advertisement in accordance with 18 U.S.C. Section 1734 solely to tion of terminal differentiation (12), and resistance to apoptosis indicate this fact. (13). On the other hand, RAS can induce growth inhibitory Requests for reprints: Ivy H. N. Wong, at the Department of Biochem- effects, such as senescence (14), necrosis (15), apoptosis (16), istry, 3/F, Laboratory Block, Faculty of Medicine Building, 21 Sassoon Road, The University of Hong Kong, Hong Kong Special Administra- and terminal differentiation. tive Region. Phone: (852) 2819 9472; Fax: (852) 2712 2719; E-mail: Loss of heterozygosity of chromosome 3p21.3 is one of the [email protected]. most frequent alterations in solid tumors (17, 18). Located

within this 3p21.3 locus, RASSF1 encodes a novel RAS effector, chronic myeloid leukemia (n ϭ 5). With curative intent, these which has been identified recently as a tumor suppressor of patients underwent surgical resection, chemotherapy, radiother- many different cancer types (19–21). The RASSF1 gene has two apy, peripheral blood stem cell or bone marrow transplantation. CpG islands within two known promoters controlling gene Pediatric Tumors, Adjacent Nontumor Tissues, and expression (19). RASSF1 encodes two major transcripts, 1A and Secondary Metastases. A total of 39 surgically resected spec- 1C, by alternative promoter usage and alternative RNA splicing. imens, including 24 primary tumors, 13 matched adjacent non- RASSF1A and 1C transcripts have four common exons, which tumor tissues, and two secondary metastases, were collected encode a COOH-terminal RAS association domain (19, 22). from 24 pediatric patients with neuroblastoma (n ϭ 8), thyroid RASSF1A and RASSF1C have PEST sequences with a serine carcinoma (n ϭ 2), hepatocellular carcinoma (n ϭ 2), Langer- residue as a putative phosphorylation target for ataxia-telangi- hans cell histiocytosis (n ϭ 2), desmoplastic small round cell ectasia-mutation (23). As differed from RASSF1C, RASSF1A tumor (n ϭ 1), pancreatoblastoma (n ϭ 1), adrenocortical carcinoma (n ϭ 1), Wilms’ tumor/nephroma (n ϭ 2), rhabdomy- has an NH2-terminal SH3 domain and a putative cysteine-rich diacylglycerol/phorbolester-binding domain (24). RASSF1A in- osarcoma (n ϭ 1), Burkitt’s lymphoma (n ϭ 3), or T-cell activation can be a tumorigenic mechanism distinct from the lymphoma (n ϭ 1). oncogenic activation of RAS signaling. Loss of RASSF1A ex- Peripheral Blood Cells and Plasma Samples from Pe- pression may shift the balance of RAS activities toward a diatric Cancer Patients. Forty-nine buffy coat samples (n ϭ growth-promoting effect (25). 34) and plasma samples (n ϭ 15) were collected before, during, Frequent RASSF1A promoter methylation has been ob- and after treatment from 23 pediatric cancer patients, who served recently in tumor types with uncommon RAS mutations suffered from neuroblastoma (n ϭ 5), medulloblastoma (n ϭ 1), and associated with transcriptional silencing of RASSF1A (18, primitive neuroectodermal tumor (n ϭ 1), thyroid carcinoma 19, 26, 27). In fact, RASSF1A blocks cell cycle progression (n ϭ 1), hepatocellular carcinoma (n ϭ 2), adrenocortical carcinoma (n ϭ 1), ovarian dysgerminoma (n ϭ 1), rhabdomyo- from G1 phase to S phase by controlling the entry at the retinoblastoma restriction point and inhibiting cyclin D1 protein sarcoma (n ϭ 3), Burkitt’s lymphoma (n ϭ 2), T-cell lymphoma accumulation at the post-transcriptional level (28). RASSF1A (n ϭ 1), or acute myeloid leukemia/acute lymphoblastic leuke- has been implicated in suppressing tumorigenesis in vitro and in mia/chronic myeloid leukemia (n ϭ 5). As a control, 20 buffy vivo (19). Reactivation of RASSF1A transcription in lung carci- coat samples and 20 plasma samples were collected from 20 noma cells reduced colony formation, suppressed cell growth pediatric patients with no cancer. dependent or independent of anchorage, and inhibited tumor Cell Lines Derived from Pediatric Cancer Patients. formation in nude mice (19). Oncogenic RAS does not alter Neuroblastoma (SK-N-AS, SK-N-DZ, SK-N-SH, SK-N-MC, RASSF1A-induced growth inhibitory effects in an immortalized and KELLY), hepatocellular carcinoma (Hep3B), hepatoblas- cell line, but the effects of RASSF1A are dominant to oncogenic toma (HepG2), rhabdomyosarcoma, Burkitt’s lymphoma RAS in human mammary epithelial cells (28). Thus, loss of (JIYOYE, DAUDI, Namalwa), and papillary thyroid carcinoma RASSF1A can be a determining step for oncogenic transforma- (K1) cell lines were purchased from the American Type Culture tion without RAS-activating mutations. Collection (Manassas, VA). All of the cell lines, except K1, In the present study, we analyzed RASSF1A promoter were derived from pediatric cancer patients. SK-N-AS was methylation in a wide range of pediatric tumors and cell lines cultured in DMEM supplemented with 4.5 grams/liter glucose derived from pediatric cancer patients. The association between and 0.1 mM nonessential amino acids. SK-N-DZ was cultured in promoter methylation and transcriptional repression was studied DMEM supplemented with 4.5 grams/liter glucose and 1 mM in the cell lines before and after demethylation treatment. In sodium pyruvate (Life Technologies, Inc., Grand Island, NY). relation to cancer progression, we studied whether this epige- SK-N-SH and SK-N-MC were cultured in Eagle’s Minimal netic alteration could be detected in blood cells or plasma Essential Medium supplemented with 0.1 mM nonessential samples collected from pediatric cancer patients before, during, amino acids and 1 mM sodium pyruvate (Life Technologies, and after treatment. Moreover, the clinical relevance of aberrant Inc.). K1 was cultured in DMEM:Ham’s F12:MCDB 105 in RASSF1A methylation was investigated among the pediatric 2:1:1 proportions (Life Technologies, Inc., Sigma Chemical Co., cancer patients studied. St. Louis, MO). HepG2 and Hep3B were cultured in DMEM medium. RD was cultured in DMEM supplemented with 2% nonessential amino acids and 2% vitamins (Life Technologies, MATERIALS AND METHODS Inc.). The remaining cell lines were cultured in RPMI 1640 Profile of Pediatric Cancer Patients. With written con- (Life Technologies, Inc.). All media were also supplemented sent and ethics approval, we recruited a total of 35 pediatric with 2 mM glutamine, 100 units/ml penicillin, 100 ␮g/ml strep- cancer patients, who suffered from neuroblastoma (n ϭ 8), tomycin, and 10% fetal bovine serum (Life Technologies, Inc.). medulloblastoma (n ϭ 1), primitive neuroectodermal tumor Demethylation Treatment. KELLY, RD, Namalwa, and (n ϭ 1), thyroid carcinoma (n ϭ 2), hepatocellular carcinoma DAUDI cell lines were treated with 3 ␮M 5-aza-2Ј-deoxycyti- (n ϭ 3), Langerhans cell histiocytosis (n ϭ 2), desmoplastic dine (Sigma Chemical Co.) for 3–10 days in the corresponding small round cell tumor (n ϭ 1), pancreatoblastoma (n ϭ 1), growth media (29). adrenocortical carcinoma (n ϭ 1), Wilms’ tumor/nephroma DNA Extraction. Total genomic DNA was extracted (n ϭ 2), ovarian dysgerminoma (n ϭ 1), rhabdomyosarcoma from cancer cell lines, primary tumors, nontumor tissues, and (n ϭ 3), Burkitt’s lymphoma (n ϭ 3), T-cell lymphoma (n ϭ 1), secondary metastases from pediatric cancer patients using the or acute myeloid leukemia/acute lymphoblastic leukemia/ QIAamp Tissue Kit (Qiagen, Hilden, Germany). Total genomic

DNA was extracted from blood cells and plasma samples using the QIAamp Blood Kit (Qiagen). Bisulfite Modification and Methylation-Specific PCR for Analyzing the Promoter Region. Bisulfite modification of genomic DNA would convert unmethylated cytosine residues into uracil residues (30, 31). Conversely, methylated cytosine residues would remain unmodified. Thus, methylated and unmethylated DNA sequences would be distinguishable by using sequence-specific PCR primers (30, 31). Bisulfite modification was conducted using the CpGenome DNA Modification Kit (Intergen Co., Purchase, NY). Bisulfite-modified DNA was amplified using primers specific for the methylated sequence, 5Ј-GTGTTAACGCGTTGCGTATC-3Ј and 5Ј-AACCCCGC- GAACTAAAAACGA-3Ј. All bisulfite-modified DNA samples were also amplified using primers specific for the unmethylated sequence, 5Ј-TTTGGTTGGAGTGTGTTAATGTG-3Ј and 5Ј- CAAACCCCACAAACTAAAAACAA-3Ј. PCR was conducted for 35 or 55 cycles using the GeneAmp DNA Amplifi- cation Kit and AmpliTaq Gold polymerase (Applied Biosystems, Perkin-Elmer, Foster City, CA). The optimized thermal profile included initial denaturation at 95°C for 12 min, followed by 35 or 55 cycles of 95°C for 45 s, 60°C for 45 s, 72°C for 1 min, and a final extension at 72°C for 10 min. Each sample was analyzed in triplicate. PCR products were analyzed by agarose gel electrophoresis and ethidium bromide staining. RNA Extraction. After washing in PBS and centrifuga- tion, the cell pellet was resuspended in 0.5 ml of guanidinium Fig. 1 Ubiquitous RASSF1A promoter methylation in pediatric tumors thiocyanate solution. Total RNA was extracted using a single- and adjacent nontumor tissues. A, Lanes 1–19, tumors; Lane M, molec- step method (32). ular weight markers. B, Lanes 1–10, adjacent nontumor tissues; Lane M, Reverse Transcription-PCR. Total RNA (2 ␮g) was molecular weight markers. Arrow, the expected size of PCR products. denatured at 65°C for 2 min and annealed with 1 ␮g of random primers (Invitrogen, Carlsbad, CA) at 37°C for 10 min (33). cDNA was synthesized at 37°C for 1 h using 200 units of Moloney murine leukemia virus reverse transcriptase (Invitro- of one), Wilms’ tumor (one of one), Burkitt’s lymphoma (one of ␤ three), and T-cell lymphoma (one of one). In addition, aberrant gen). 2-microglobulin cDNA was amplified as a control to ensure that a similar amount of high-integrity RNA was reverse RASSF1A promoter methylation was found in both lung metas- transcribed in each reaction (33). PCR was conducted using tases from the two hepatocellular carcinoma patients studied. primers specific for RASSF1A, 5Ј-CAGATTGCAAGTTCAC- Moreover, methylated RASSF1A alleles were detected in 4 of 13 CTGCCACTA-3Ј and 5Ј-GATGAAGCCTGTGTAAGAAC- nontumor tissues, which were resected from 2 hepatocellular CGTCCT-3Ј. The optimized thermal profile included initial carcinoma patients, 1 Wilms’ tumor patient, and 1 adrenocorti- denaturation at 95°C for 12 min, followed by 40 cycles of 95°C cal carcinoma patient (Fig. 1B). However, this epigenetic for 1 min, 65°C for 1 min, 72°C for 1 min, and a final extension change was not detected in blood cells from 20 pediatric patients at 72°C for 10 min. Each sample was analyzed in triplicate. PCR with no cancer. Unmethylated RASSF1A alleles were detected in products were analyzed by agarose gel electrophoresis and all of the nontumor tissues, tumors, and secondary metastases ethidium bromide staining. from pediatric cancer patients and blood cells from 20 pediatric Statistical Analyses. Association of RASSF1A promoter patients with no cancer. methylation in tumor/buffy coat with gender, age, tumor size, RASSF1A Methylation Patterns in Peripheral Blood recurrence/metastasis, and overall survival was analyzed using Cells and Plasma Samples from Pediatric Cancer Patients Fisher’s exact test and Kaplan-Meier Log-rank test. Before, During, and After Treatment. Among 34 buffy coat samples collected before, during, and after treatment, RASSF1A promoter methylation was detected in 12 samples from pediatric RESULTS patients with neuroblastoma (n ϭ 4), thyroid carcinoma (n ϭ 2), Ubiquitous RASSF1A Promoter Methylation in Pediat- hepatocellular carcinoma (n ϭ 2), rhabdomyosarcoma (n ϭ 1), ric Tumors, Adjacent Nontumor Tissues, and Secondary Burkitt’s lymphoma (n ϭ 1), T-cell lymphoma (n ϭ 1), or acute Metastases. RASSF1A promoter methylation was found in lymphoblastic leukemia (n ϭ 1; Fig. 2). Before treatment, 54% 67% (16 of 24) of primary pediatric tumors of eight cancer types (7 of 13) of buffy coat samples from pediatric cancer patients (Fig. 1A), including neuroblastoma (seven of eight), thyroid showed RASSF1A promoter methylation. During treatment, carcinoma (two of two), hepatocellular carcinoma (two of two), 40% (4 of 10) of the buffy coat samples analyzed showed this pancreatoblastoma (one of one), adrenocortical carcinoma (one epigenetic alteration. Concordant with the positive results on the

RASSF1A Promoter Methylation in Nearly All of the Cell Lines Derived from Pediatric Cancer Patients. RASSF1A promoter methylation was found in 92% (11 of 12) of cell lines largely derived from pediatric cancer patients, including neuroblastoma (SK-N-AS, SK-N-DZ, SK-N-SH, SK-N- MC, and KELLY), hepatocellular carcinoma (Hep3B), hepatoblastoma (HepG2), rhabdomyosarcoma, Burkitt’s lymphoma (DAUDI, Namalwa), and papillary thyroid carcinoma (K1) cell lines (Fig. 4A). Only JIYOYE cell line did not possess RASSF1A promoter methylation. SK-N-AS, SK-N-DZ, SK-N-SH, SK-N- MC, KELLY, Hep3B, HepG2, and RD cell lines showed com- plete RASSF1A methylation. Unmethylated RASSF1A alleles

Fig. 2 RASSF1A methylation patterns in peripheral blood cells from pediatric cancer patients before, during, and after treatment. A and B, Lanes 1–34, buffy coat samples collected from pediatric cancer patients; Lane M, molecular weight markers. Arrow, the expected size of PCR products. RASSF1A methylation was detected before (Lanes 2, 8, 9, 12, 22, 28, and 31), during (Lanes 7, 18, 21, and 23), and after treatment (Lane 19).

Fig. 3 RASSF1A methylation patterns in plasma samples from pediatric cancer patients before, during, and after treatment. Lanes 1–2, control plasma samples collected from pediatric patients with no cancer; Lanes 3–17, plasma samples collected from pediatric cancer patients; Lane M, molecular weight markers. Arrow, the expected size of PCR products. RASSF1A methylation was detected during treatment (Lanes 3 and 17).

buffy coat from 2 of these 4 patients, RASSF1A promoter methylation was also found during treatment in plasma of a neuroblastoma patient and a hepatocellular carcinoma patient (Fig. 3). This epigenetic change was not detectable in the remaining 13 plasma samples collected before (n ϭ 6), during (n ϭ 3), or after (n ϭ 4) treatment from the pediatric cancer patients studied. Only one of six buffy coat samples collected 1 month after treatment possessed the same alteration, whereas Fig. 4 RASSF1A promoter methylation in nearly all of the cell lines none of five buffy coat samples collected 2 months after treat- derived from pediatric cancer patients. A, detection of methylated RASSF1A alleles by methylation-specific PCR; Lane N, normal blood ment showed RASSF1A promoter methylation. As a control, cell DNA; Lanes 1–12, Hep3B, HepG2, K1, DAUDI, JIYOYE, Nama- methylation-specific PCR was also performed on buffy coat and lwa, RD, SK-N-AS, SK-N-DZ, SK-N-SH, SK-N-MC, and KELLY cell plasma samples from 20 pediatric patients with no cancer, and lines; Lane M, molecular weight markers. B, detection of unmethylated RASSF1A promoter methylation was not detected. However, RASSF1A alleles by methylation-specific PCR; Lanes 1–12, SK-N-AS, SK-N-DZ, SK-N-SH, SK-N-MC, KELLY, RD, K1, Namalwa (barely unmethylated RASSF1A alleles were detected in all of the buffy detectable unmethylated RASSF1A alleles), DAUDI, JIYOYE, Hep3B, coat and plasma samples from the 20 noncancer pediatric pa- and HepG2 cell lines; Lane M, molecular weight markers. Arrow, the tients and all of the 23 pediatric cancer patients studied. expected size of PCR products.

they both had tumoral RASSF1A methylation. On the other hand, all of the 7 pediatric cancer patients with unmethylated RASSF1A alleles were alive and well. Using Kaplan-Meier Log-rank test, RASSF1A methylation in tumors was not significantly associated with overall survival in this series of pediatric cancer patients (P ϭ 0.302, n ϭ 22).

DISCUSSION RASSF1A promoter methylation and transcriptional repression have been found in many different cancer types, including lung and breast cancers (19, 20, 26, 27, 34–40). In the present study, we demonstrated frequent and ubiquitous RASSF1A pro- Fig. 5 Reactivation of RASSF1A transcription in KELLY, RD, Nama- moter methylation in childhood neuroblastoma, thyroid carci- lwa, and DAUDI cell lines by demethylation treatment as analyzed by noma, hepatocellular carcinoma, pancreatoblastoma, adrenocor- reverse transcription-PCR. Lane B, before demethylation treatment with 5-aza-2Ј-deoxycytidine; Lane D, after demethylation treatment with tical carcinoma, Wilms’ tumor, Burkitt’s lymphoma, and T-cell 5-aza-2Ј-deoxycytidine; Lane H, HepG2 cell line; Lane M, molecular lymphoma. Furthermore, aberrant RASSF1A methylation was weight markers. Arrow, the expected size of PCR products. found in secondary lung metastases from hepatocellular carcinoma patients. Thus far, RASSF1A methylation is the first molecular abnormality that is common to a large and diverse were only detected in JIYOYE, DAUDI, and K1 and were group of pediatric tumors. Our findings implicate that aberrant barely detectable in Namalwa (Fig. 4B). RASSF1A promoter methylation may contribute to the develop- Reactivation of RASSF1A Transcription by Demethy- ment of a wide range of pediatric tumors. lation Treatment. RASSF1A promoter methylation correlated RAS promotes both cell transformation and death, leading with transcriptional silencing or repression in KELLY, RD, to a hypothesis that signal transduction pathways driving pro- HepG2, and Namalwa cell lines (Fig. 5). JIYOYE cell line liferation and death are tightly linked to protect against onco- containing solely unmethylated RASSF1A alleles showed genic transformation. Of interest, an inverse correlation has RASSF1A mRNA. KELLY, RD, Namalwa, and DAUDI cell been found between oncogenic RAS mutations and RASSF1A lines were treated with 5-aza-2Ј-deoxycytidine to examine the promoter methylation. The frequency of RASSF1A methylation relationship between demethylation and transcriptional reacti- (20%) in colorectal cancer with common RAS mutations was vation. RASSF1A transcription was examined by reverse tran- lower than the frequencies observed in other tumor types (62– scription-PCR before and after treatment with 5-aza-2Ј-deoxy- 91%) with uncommon RAS mutations (5, 20, 26, 27, 34–37, cytidine. RASSF1A transcripts were not detected in KELLY and 41–44). This is consistent with the fact that concomitant genetic RD but were minimally detectable in Namalwa before 5-aza- and epigenetic alterations in the same signaling pathway are 2Ј-deoxycytidine treatment (Fig. 5). After demethylation treat- rarely observed in a single tumor type. Cancer cell lines har- ment, reactivation of RASSF1A transcription was seen in all of boring oncogenic RAS mutations also showed epigenetic silenc- the three cell lines. These results confirmed that transcriptional ing of RASSF1A, suggesting that RAS activation and RASSF1 silencing or repression was directly associated with RASSF1A inactivation are not necessarily mutually exclusive (45). How- promoter methylation. Conversely, DAUDI cell line possessing ever, the majority of colorectal cancers with RAS mutations both methylated and unmethylated RASSF1A alleles showed lacked RASSF1A promoter methylation; thus, the coexistence of RASSF1A mRNA before and after demethylation treatment. RASSF1A and RAS alterations might be stochastic events (41). Correlation of RASSF1A Methylation with Clinicopath- Transcriptional repression of RASSF1A was identified in ologic Profile of Pediatric Cancer Patients. Correlations 17% of paired bladder carcinomas and nontumor bladder tissues were analyzed between RASSF1A methylation status and (43). In this study, we also found aberrant RASSF1A promoter clinicopathologic features, including gender, age, tumor size, methylation in nontumor tissues adjacent to hepatocellular car- recurrence/metastasis, and overall survival (Table 1). Of cinoma, adrenocortical carcinoma, or Wilms’ tumor, suggesting note, 12 of 16 pediatric cancer patients with RASSF1A pro- that epigenetic alteration of RASSF1A is an early and critical moter methylation in tumors were male (Fisher’s exact test, event during childhood neoplasia. Methylation of CDH1, P ϭ 0.058, n ϭ 23). However, RASSF1A promoter methyl- CDH13, and RASSF1A has been demonstrated previously in ation in tumors was not significantly associated with age nonmalignant prostatic tissues (46), and p16 promoter methyl- (Fisher’s exact test, P ϭ 0.685, n ϭ 23) or tumor size ation was detected in preneoplastic lung tissues (47). Further- (Fisher’s exact test, P ϭ 0.361, n ϭ 21). In addition, more, minimal RASSF1A promoter methylation has been de- RASSF1A methylation status in tumor or buffy coat was not tected in normal lung, breast, colon and kidney tissues, and associated with metastasis development or tumor recurrence normal prostate epithelial cells from cancer patients (20, 21, 34, in this cohort of pediatric cancer patients (Fisher’s exact test, 36, 41). This might be explained by the presence of tumor cells P ϭ 0.667, n ϭ 24; P ϭ 1.000, n ϭ 23). During a median in some “nontumor” tissues, or RASSF1A methylation is indeed follow-up of 19 months for 22 pediatric cancer patients with an early and premalignant alteration. informative RASSF1A methylation status in tumor, only 2 RASSF1A promoter methylation was detected in 36–100% pediatric cancer patients died of cancer or metastasis, and of cell lines derived from lung cancer, breast cancer, or ovarian

Table 1 Clinicopathologic features of pediatric cancer patientsa Tumor Survival RASSF1A RASSF1A Age size Metastasis/recurrence (follow-up methylation methylation Children Gender (years) (cm) (months after diagnosis) months) in tumor in blood Neuroblastoma M 1 14 Intraabdominal lymph node (6) 18D b ● Neuroblastoma F 3 10 Nil 22 b Neuroblastoma M 12 days 16.4 Yes Yes (ND) b NA Neuroblastoma M 5 1.5 Bone (Ͻ1) 21 b NA Neuroblastoma M 5 months 9 Pleural effusion/pericardium (7) 18 b NA Neuroblastoma M 4 months 16.4 Nil 22 b Neuroblastoma* M 3 4 Bone marrow, left suprarenal lymph 19 ● node (1) Neuroblastoma M 2 7 Nil 11 b ● Medulloblastoma M 6 5 Spine (2) 14 NA Primitive neuroectodermal tumor F 17 8.5 Lung and pleura (24), recurrence 41 NA Papillary thyroid carcinoma ND ND 2.6 Right cevial lymph node (5) 40 b NA Thyroid carcinoma F 16 2.6 Cevial lymph node (1) 17 b ● Hepatocellular carcinoma M 13 8 Lung (8), recurrence 8D b NA Hepatocellular carcinoma F 4 1.5 Yes 22D NA ● Hepatocellular carcinoma M 11 2 Nil 16 b ● Langerhan’s cell histiocytosis F 4 3 Nil 22 NA Langerhan’s cell histiocytosis F 11 months 0.3 Nil 19 NA Desmoplastic small round F 9 ND Retroperitoneal lymph node/peritoneal 18 NA cell tumor seedling (Ͻ1), recurrence (15) Pancreatoblastoma M 49 days 10 Nil 33 b NA Adrenocorticol carcinoma M 6 4 Liver and para-aortic lymph node (12), 26 b recurrence (12) Wilms’ tumor F 2.5 12 Nil 22 b NA Mesoblastic nephroma F 14 days 8 Nil Yes (ND) NA Ovarian dysgerminoma F 12 ND Peritoneal seedling (Ͻ1) 15 NA Rhabdomyosarcoma F 3 3 Axillary lymph node (1), 26 recurrence (18) Rhabdomyosarcoma M 8 ND Nil 16 NA Rhabdomyosarcoma M 2 ND Nil 10 NA ● Burkitt’s lymphoma M 3 4 Nil 19 b NA Burkitt’s lymphoma* M 9 ND Nil 41 ● Burkitt’s lymphoma M 9 6 Nil 13 T-cell lymphoma M 11 ND Lung and peritoneum (Ͻ1) 11 b ● AML M 14 ND Nil 2D NA AML M 2 ND Relapse (17) 23D NA ALL F 13 ND Optic nerve infiltration (28), 43 NA relapse (28) ALL F 3 ND Nil 12 NA ● CML M 16 ND Relapse (1) 54 NA a ND, not documented; D, deceased; NA, Not available; b, Tumoral RASSF1A methylation; ●, RASSF1A methylation in blood cells; , RASSF1A methylation in plasma; , unmethylated RASSF1A;*,RASSF1A methylation in blood cells but not tumors; CML, chronic myeloid leukemia; AML, acute myeloid leukemia; ALL, acute lymphoblastic leukemia.

cancer (26, 27). Consistently, 92% (11 of 12) of cell lines Methylation and loss of heterozygosity are the major loss- largely derived from pediatric cancer patients, including neuro- of-function mechanisms for RASSF1A inactivation (19). blastoma, hepatoblastoma, hepatocellular carcinoma, rhab- RASSF1A mutations appear to be rare in human cancers (18, 20, domyosarcoma, and Burkitt’s lymphoma cell lines, showed 26). Consistent with Knudson’s two-hit hypothesis, 77% of RASSF1A promoter methylation in correlation with transcription small cell lung cancers with 3p21.3 allelic loss also had silencing or repression. The absence of unmethylated RASSF1A RASSF1A methylation (25, 26). Similarly, clear cell renal cell alleles and RASSF1A mRNA in KELLY, RD, and HepG2 cell carcinomas with RASSF1A methylation also demonstrated 3p21 lines suggest homozygous inactivation by biallelic methylation. allelic loss, but some tumors with 3p21 allelic loss did not show After demethylation treatment with 5-aza-2Ј-deoxycytidine, RASSF1A methylation (37). These results suggest that RASSF1A RASSF1A expression was reactivated in KELLY, RD, and Na- inactivation by two hits (methylation and 3p loss) is a critical malwa. In fact, RASSF1A blocked cell cycle progression from step in tumorigenesis. However, concurrent loss of heterozygos-

G1 phase to S phase and post-transcriptionally inhibited cyclin ity and methylation may be stochastic events during tumor D1 accumulation in cancer cell lines (28), implicating the bio- initiation. According to Knudson’s two-hit model, the first “hit” logical and physiological relevance of RASSF1A in cell cycle is often a point mutation, small deletion, or epigenetic event, regulation by inducing G1 arrest at the retinoblastoma restriction which is followed by the second chromosomal loss or loss of point and inhibiting cell growth or suppressing tumorigenicity. heterozygosity (25). Most likely, the first hit in RASSF1A during

carcinogenesis is epigenetic silencing, which may be followed icopathologic features. RASSF1A inactivation is common in by the second 3p loss. grade I tumors; RASSF1A methylation may thus be an early RASSF1A methylation was rarely found in non-small cell event during breast cancer progression (20). No statistically lung, ovarian, and cervical cancer with 3p21.3 loss (26). Fre- significant association has been found between RASSF1A meth- quent epigenetic inactivation of RASSF1A occurred in papillary ylation and gender, age, Tumor-Node-Metastasis pathological renal cell carcinomas, despite rare 3p21.3 allelic loss (37). In stage, or tumor histology in patients with non-small cell lung or line with this reciprocal relationship, there is increasing evi- bladder cancer (27, 43). RASSF1A methylation was not associ- dence supporting that RASSF1A haploinsufficiency by either ated with gender or Dukes’ stage in colorectal cancer patients promoter methylation or 3p21 allelic loss might promote tumor- (41). In our cohort of pediatric cancer patients, those with igenesis without the need for a “second hit” (18). In addition, tumoral RASSF1A promoter methylation were predominantly growth suppression experiments indicate that even partial male. Consistent with the lack of correlation between RASSF1A RASSF1A repression may provide selective growth advantage methylation and tumor stage or survival in neuroblastoma pa- (36). tients (18), there was no relationship between RASSF1A pro- For the first time, we demonstrated frequent RASSF1A moter methylation in tumor or buffy coat and other clinicopath- promoter methylation in buffy coat from pediatric patients with ologic parameters, including age, tumor size, recurrence/ neuroblastoma, thyroid carcinoma, hepatocellular carcinoma, metastasis, or overall survival in this series of pediatric cancer rhabdomyosarcoma, Burkitt’s lymphoma, T-cell lymphoma, or patients. The lack of association between RASSF1A methylation acute lymphoblastic leukemia. RASSF1A promoter methylation and clinicopathologic characteristics may support the notion that was shown in 54%, 40%, and 9% of buffy coat samples col- this frequent and ubiquitous epigenetic alteration may poten- lected from pediatric cancer patients before, during, and after tially be a very early and critical event deregulating apoptosis treatment, correspondingly. Of note, most of the pediatric cancer and cell cycle progression in childhood neoplasia (28). How- patients studied underwent chemotherapy with or without sur- ever, further investigation of a larger cohort of pediatric cancer gical resection. Concordantly, RASSF1A promoter methylation patients is required to confirm the clinical relevance of was found during chemotherapy in plasma of 2 patients with RASSF1A methylation. neuroblastoma or hepatocellular carcinoma, possessing the iden- Conversely, grade III renal cell carcinomas showed a tical epigenetic alteration in buffy coat, suggestive of cell death higher rate of RASSF1A promoter methylation than grades I and and good response to chemotherapy. RASSF1A methylation was II tumors (35). Down-regulation of RASSF1A correlated with only found in 1 of 11 buffy coat samples after treatment but not advanced tumor stage and grade in patients with bladder or in plasma of the same patient, suggesting poor response to gastric cancer (38, 43). Moreover, RASSF1A inactivation was treatment. Indeed, this patient had developed metastasis. Con- more frequent in advanced and poorly differentiated tumors than versely, RASSF1A methylation was not detected in the remain- in early stage well-differentiated or moderately differentiated ing three plasma samples collected after treatment or the corre- tumors (38). In prostate cancer, RASSF1A promoter methylation sponding buffy coat samples, and these patients did not have was also associated with high GS grade (46). In addition, recurrence or metastasis. RASSF1A hypermethylation was associated with poor prognosis Of particular interest, RASSF1A promoter methylation was in patients with bladder or lung cancer (27, 44). Apparently, detected in blood cells from neuroblastoma and Burkitt’s lym- epigenetic inactivation of RASSF1A may play different roles phoma patients who did not possess the identical methylation during the progression of different childhood and adult cancer abnormality in tumors. These circulating tumor cells may rep- types. In particular, RASSF1A promoter methylation may pos- resent more malignant clones in blood. These circulating tumor sibly be associated with pediatric tumor initiation in an early cells with RASSF1A promoter methylation and reduced expres- stage. The tumor suppressor function of RASSF1A is implicated sion might possess the antiapoptotic mechanism, in that based on the fact that chromosome transfer of 3p fragment RASSF1A can normally interact with RAS-binding NORE1 suppressed tumorigenicity (52). Frequent RASSF1A inactivation protein, proapoptotic protein kinase MST1, and activated RAS in multiple pediatric cancer types may provide new opportuni- to induce apoptosis (48). This may also explain why RASSF1A ties to develop anticancer drugs to reactivate RASSF1A expres- methylation was not detected in plasma of all of the pediatric sion and, hence, downstream tumor suppressive effects of cancer patients before treatment, implicating no signs of cellular RASSF1A. apoptosis. 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Ivy H. N. Wong, Jacqueline Chan, Joyce Wong, et al.

Clin Cancer Res 2004;10:994-1002.

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