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Frequent Alterations of the p14^ARF and p16^INK4a Genes in Primary Central Nervous System Lymphomas

Second Department of Pathology [M. N., E. I., K. S., N. K.] and Department of Neurosurgery [T. S., H. H., H. N.], Nara Medical University, 634-8521 Nara, Japan

	ABSTRACT

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To elucidate the role of p53/p16^INK4a/RB1 pathways in the tumorigenesis of primary central nervous system lymphomas (PCNSLs), we have analyzed p14^ARF, p16^INK4a, RB1, p21^Waf1, and p27^Kip1 status in a series of their 18 sporadic cases of diffuse large B-cell lymphoma, using methylation-specific PCR, differential PCR, and immunohistochemistry. Homozygous deletion or methylation of p14^ARF was detected in 10 (56%) PCNSLs, and they were almost entirely deletions (except 1 case). A total of 11 (61%) PCNSLs demonstrated homozygous deletion (6 cases) or methylation (5 cases) of p16^INK4a. Six tumors showed both p14^ARF and p16^INK4a homozygous deletions. Hypermethylation of the RB1 and the p27^Kip1 promoter region was detected in 2 (11%) cases, whereas p21^Waf1 methylation was not detected in any. Immunohistochemistry revealed loss of p14^ARF and p16^INK4a expression in 10 (56%) samples, correlating with the gene status. Four cases showed independent negative immunoreactivity for pRB and p27^Kip1, and nearly one-half of cases (8 of 18; 44%) were characterized by lack of p21^Waf1 expression. These results indicate that inactivation of p14^ARF and p16^INK4a by either homozygous deletion or promoter hypermethylation represents an important molecular pathogenesis in PCNSLs. Hypermethylation of RB1, p21^Waf1, and p27^Kip1 appears to be of minor significance, these genes being independently methylated in PCNSLs.

	Introduction

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The incidence of PCNSLs² in nonimmunodeficient patients has been markedly increasing over the past decades, and currently they are estimated to account for >6% of all primary brain tumors (1) . Morphologically, the vast majority of PCNSLs are high-grade non-Hodgkin’s lymphomas of diffuse large B-cell type, according to the revised European American Lymphoma classification (2) . There are no lymph nodes or lymphatics within the nervous system, and therefore the pathogenesis and histogenetic origin of PCNSLs in immunocompetent patients are still poorly understood.

Molecular genetic studies have been conducted that revealed the p16^INK4a gene to be frequently inactivated by either homozygous deletion (40–50%) or 5'-CpG hypermethylation (15–30%; Refs. 3 , 4 ). Mutations in the p53 gene were observed in a small fraction of PCNSLs, whereas genetic alterations such as MDM2, CDK4, CCND1, MYC, and REL were not detected (3) .

p14^ARF interacts physically with MDM2 and stabilizes p53 protein in the nucleus by blocking its cytoplasmic transport and MDM2-mediated degradation (5 , 6) so that it may act as an upstream regulator of p53 function. Homozygous deletion of p14^ARF has been reported in 25–60% of glioblastomas (7 , 8) and 8% of systemic non-Hodgkin’s lymphomas (9) . The human p14^ARF promoter has also been shown to be aberrantly methylated in gliomas, colorectal adenomas and carcinomas (7 , 10) , and esophageal carcinomas (11) but was not detected in one series of systemic non-Hodgkin’s lymphomas (12) .

Inactivation of the retinoblastoma gene (RB1) product by mutation, deletion, and/or promoter hypermethylation has been reported as an alternative molecular mechanism leading to p16^INK4a inactivation, CDK4 amplification, and CCND1 amplification/rearrangement in human tumors, including gliomas (8) . Several previous studies have implicated alterations of RB1 in systemic non-Hodgkin’s lymphomas (13) . Thus, genetic alterations of the INK4a/ARF locus may cause impairment in both p14^ARF/p53 and p16^INK4a/RB1 pathways in the development and progression of human non-Hodgkin’s lymphomas.

To cast light on the presence of p14^ARF alterations in human PCNSLs and their possible alternative or coordinate inactivation with p53/p16^INK4a/RB1 pathways, we have studied a series of 18 PCNSLs for genetic aberrations and expression of several genes shown previously to be altered and/or aberrantly expressed to some extent in systemic non-Hodgkin’s lymphomas.

	Materials and Methods

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Tumor Samples and DNA Extraction.
Eighteen primary malignant non-Hodgkin’s lymphomas of the central nervous system were obtained from immunocompetent patients treated between 1984 and 2000 in the Department of Neurosurgery, Nara Medical University. Tumor samples were fixed in buffered formalin and embedded in paraffin. Pathological diagnosis was made according to the revised European American Lymphoma classification of lymphoid neoplasms (2) . DNA was extracted from paraffin sections as described previously (7 , 14) . In one tumor (case 11), p14^ARF and p16^INK4a immunopositive and negative tumor areas could be clearly recognized. These areas were carefully microdissected and analyzed separately.

MSP.
DNA methylation patterns in the CpG islands of the p14^ARF, p16^INK4a, RB1, p21^Waf1, and p27^Kip1 genes were determined by MSP (15) . Sodium bisulfite modification was performed using a CpGenome DNA Modification kit (Intergen, Oxford, United Kingdom) according to the manufacturer’s protocol with minor modifications (7 , 14) . The primer sequences for p14^ARF, p16^INK4a, and RB1 with methylated and unmethylated PCR have been reported previously (10 , 15 , 16) . Other primer sequences were as follows: 5'TTG GGC GCG GAT TCG TC-3' (sense) and 5'-CTA AAC CGC CGA CCC GA-3' (antisense) for the p21^Waf1 methylated reaction; 5'TTA GTT TTT TGT GGA GTT G-3' (sense) and 5'-CTC AAC TCT AAA CCA CCA A-3' (antisense) for the p21^Waf1 unmethylated reaction, and 5'-AAG AGG CGA GTT AGC GT-3' (sense) and 5'-AAA ACG CCG CCG AAC GA-3' (antisense) for the p27^Kip1 methylated reaction; 5'-ATG GAA GAG GTG AGT TAG T-3' (sense) and 5'-AAA ACC CCA ATT AAA AAC A-3' (antisense) for the p27^Kip1 unmethylated reaction. MSP conditions for p14^ARF, p16^INK4a, and RB1 were described in detail previously (7 , 14) . The annealing temperature for p21^Waf1 methylated and unmethylated reactions was 62°C, and for p27^Kip1 methylated and unmethylated reactions was 66°C. Amplified products were electrophoresed on 2% agarose gels and visualized by ethidium bromide staining.

Differential PCR for p14^ARF and p16^INK4a Deletions.
To assess homozygous deletions, we carried out differential PCR with primers covering exon 1ß of the p14^ARF gene, using the GAPDH gene as a reference. Differential PCR for homozygous deletion of p16^INK4a (exon 1) was carried out using the ß-actin gene as a reference. The primer sequences and PCR conditions were as described previously (7) , and PCR products were analyzed in 8% acrylamide gels, photographed using a DC290 Zoom Digital Camera (Eastman Kodak, Rochester, NY). Densitometry of the PCR fragments was performed using Kodak Digital Science ID Image Analysis Software (Ver. 3.5.2; Eastman Kodak). Samples presenting <20% of the control signal were considered homozygously deleted (7) .

PCR-Single Strand Conformation Polymorphism Analyses for p53 Mutations.
PCR amplification of exons 5, 6, 7, 8, and 9 of the p53 gene, single-strand conformation polymorphism analysis, and DNA sequencing was conducted as described previously (17) .

Immunohistochemistry.
Expression was assessed immunohistochemically, using a polyclonal antihuman p14^ARF antibody (FL-132: SC1661; Santa Cruz Biotechnology, Santa Cruz, CA) and monoclonal antibodies to p16^INK4a (F-12: SC1661; Santa Cruz Biotechnology), pRB (clone G3–245; PharMingen, San Diego, CA), p21^Waf1 (F-5: SC-6246; Santa Cruz Biotechnology), p27^Kip1 (clone 57; Transduction Laboratories, Lexington, KY), and MDM2 (clone IF2; Oncogene, Boston, MA). After deparaffinization, sections were heated to boiling for 5 min in 10 mM sodium citrate (pH 6.0) buffer in a pressure cooker. They were then incubated for overnight at 4°C with antibodies for p14^ARF, pRB, and p27^Kip1 at a dilution of 1:500, p16^INK4a at a dilution of 1:1000, p21^Waf1, and MDM2 at a dilution of 1:100. Binding reactions were visualized using a Histofine SAB-PO kit and diaminobenzidine (Nichirei, Tokyo, Japan), and sections were counterstained with hematoxylin.

Statistical Analysis.
The Fisher’s exact test was used to examine possible associations between p14^ARF and other genetic alterations.

	Results

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p14^ARF and p16^INK4a Alterations.
Homozygous deletion of the p14^ARF gene was detected by differential PCR in 9 of 18 (50%) PCNSLs examined (Table 1 and Fig. 1 ) and hypermethylation of the p14^ARF promoter in 1 of 18 (6%). Methylated and unmethylated control DNAs showed the expected fragment sizes of 122 and 132 bp, respectively (Fig. 2) . Homozygous p16^INK4a deletion was detected in 6 (33%) PCNSLs (Table 1 and Fig. 1 ) and promoter hypermethylation of the p16^INK4a gene in 5 (28%) cases. Methylated and unmethylated control DNAs showed the expected fragment sizes of 150 and 151 bp, respectively (Fig. 2) .

View larger version (52K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Differential PCR to assess p14ARF and p16INK4a homozygous deletions in PCNSLs. Case 2 has a normal gene status. Case 4 shows p14ARF and p16INK4a deletion. Cases 10, 13, and 14 have p14ARF homozygous deletions without p16INK4a deletion.

View larger version (55K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. Methylation specific PCR of CpG islands of the p14ARF and p16INK4a promoter in PCNSL. In case 3, there was only unmethylated DNA (U). In case 11, p14ARF and p14INK4a methylation (M) was restricted to areas (shown as *) lacked p14ARF and p16INK4a immunoreactivity. In patient 17, the biopsy sample showed only unmethylated status for p14ARF, whereas the tumor revealed methylated DNA for p16INK4a.

RB1, p21^Waf1, and p27^Kip1 Alterations.
RB1 and p27^Kip1 promoter hypermethylation was detected in 2 of 18 (11%) PCNSLs (Table 1 and Fig. 2 ). Methylated and unmethylated control DNAs showed the expected fragment sizes of 163 bp for RB1 and 195 and 212 bp for p27^Kip1, respectively (Fig. 2) . p21^Waf1 methylation was not detected in any PCNSL (Table 1 and data not shown). Methylation patterns of these genes appeared independent of each other.

Immunohistochemistry and Correlation with Genetic Analyses.
Nuclear immunoreactivity to p14^ARF was observed in neurons and glial cells in peritumoral brain tissues. Ten cases (56%) of PCNSLs showed loss of p14^ARF expression, and of the remaining cases, 4 showed immunoreactivity in >20% of the neoplastic cells (Table 1) . Loss of p16^INK4a expression was observed in 10 of 18 cases (56%; Table 1 ). There was a close correlation between loss of p14^ARF/p16^INK4a expression, as detected by immunohistochemistry and homozygous deletion/promoter methylation. All 8 cases with p14^ARF expression showed a normal p14^ARF gene status, whereas one case (case 6) with p16^INK4a promoter methylation showed p16^INK4a expression (Table 1) . In one case (case 11), some tumor areas showed p14^ARF/p16^INK4a expression, but there were also focal areas with neoplastic cells that lacked p14^ARF/p16^INK4a expression. In this case, only microdissected areas with loss of p14^ARF/p16^INK4a expression showed promoter hypermethylation of both genes (Table 1 ; Figs. 2 and 3 ).

View larger version (147K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. A, p14ARF immunohistochemistry showing nuclear immunoreactivity in the majority of tumor cells (case 11). B, the same case with focal loss of p14ARF expression. C and D, case 11 exhibits extensive areas (C) with marked immunoreactivity for p16INK4a but focal tumor cell clusters (D) with loss of p16INK4a, counterstained with hematoxylin. x300.

p53 Gene Mutations and Correlation between p14^ARF and p53 Status.
We failed to find any p53 mutations in any of the 18 cases examined in this study. There was no significant correlation between p14^ARF alterations, p53 mutations, and MDM2 overexpression.

	Discussion

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The present study showed the most frequent abnormality in our series of PCNSLs to be homozygous deletion or promoter hypermethylation of the p14^ARF (10 of 18; 56%) and p16^INK4a (11 of 18; 61%) genes. We detected frequent p14^ARF homozygous deletion (9 of 18; 50%), whereas the p14^ARF promoter was unmethylated, even when the p16^INK4a gene was methylated, except in 1 case (case 11). In this context, it is of interest that Baur et al. (12) found methylation silencing of p15^INK4b and p16^INK4a in human B-cell and T-cell lymphomas to be frequent, whereas methylation silencing of p14^ARF was extremely rare. Meléndez et al. (18) also pointed out that p19^ARF (the murine homologue of p14^ARF) expression was lost or reduced in a significant percentage of murine primary lymphomas, whereas the p19^ARF CpG island was infrequently methylated. Thus, p14^ARF homozygous deletion, rather than promoter hypermethylation, is the most likely to be the essential event for p14^ARF inactivation in PCNSLs.

The p16^INK4a and p14^ARF genes are frequently co-deleted in human neoplasms, and this was also the case for 6 PCNSLs in our series. However, cases with p14^ARF deletion alone were also encountered, and a higher frequency of p14^ARF than p16^INK4a deletions has been reported for other human neoplasms (7 , 11) , suggesting that p14^ARF is the primary target with 9p21 deletions. This conclusion is supported by studies of mice, lacking p19^ARF (the murine homologue of p14^ARF) expression alone through selective disruption of exon 1ß, which develop tumors at several sites, including lymphomas, sarcomas, and gliomas (19) .

p14^ARF plays a major role in the p53 pathway by binding specifically to MDM2, resulting in stabilization of both p53 and MDM2 (5 , 6) . With regard to MDM2, PCNSLs appear similar to systemic lymphomas, which only exceptionally show amplification (3 , 20) . Growth arrest induced by p14^ARF is therefore p53 dependent. Recent studies of the INK4a/ARF locus as a regulatory region for both p16^INK4a/RB1 and p14^ARF/p53 pathways indicated that p53 mutations may be more rare in tumors with inactivation of this locus than in those with wild-type INK4a/ARF genes (5) . In the present series, p14^ARF alterations and p53 mutations appeared to be unrelated, and an inverse correlation between p14^ARF and p53 is not always detected, e.g., in leukemia-lymphoma cell lines and large B-cell lymphomas (21) . Although inactivation of the p53 gene is a relatively common phenomenon (20–40%) in lymphomas outside central nervous system (22) , p53 mutations appear extremely rare in PCNSLs (3 , 4) . It has been suggested that the pattern of these alterations in tumors may depend on the order of events (6) . When p14^ARF alterations occur early in the development of the PCNSLs, the tumors may retain wild-type p53 genes.

For the majority of human neoplasms, a clear correlation has been reported between deletion/promoter methylation and loss of gene expression detected by immunohistochemistry (7 , 14 , 16) . The present study also revealed a close link between gene inactivation and expression. All 8 PCNSLs with p14^ARF expression showed a normal p14^ARF gene status, whereas all 10 PCNSLs with p14^ARF deletion or promoter methylation showed loss. There was also 1 case (case 6) with p16^INK4a promoter methylation that expressed p16^INK4a, but this may be explained by incomplete gene silencing because of insufficient density and extent of methylation (23) . The relation was also exemplified by the finding that in 1 case, promoter hypermethylation was detected only in areas lacking p14^ARF and p16^INK4a immunoreactivity but not in the areas with expression. Regarding alternative explanations, one common feature of p16^INK4a regulation is that tumors with increased levels have RB1 alterations. Although there was no inverse correlation between p16^INK4a and RB1 in our series, the p16^INK4a immunopositive case (case 6) with p16^INK4a promoter hypermethylation showed RB1 alteration.

More than 50% of high-grade systemic non-Hodgkin’s lymphomas lack pRB expression (13) , whereas Cobbers et al. (3) reported that PCNSLs showed strong nuclear immunoreactivity in all of their 20 samples. We showed that loss of pRB expression was found in 4 (22%) cases examined. This is agreement with the former report on systemic lymphomas but at variance with the latter one on PCNSLs, although our cases are central nervous lymphomas. In glioblastomas (14) and pituitary adenomas (16) , a clear correlation has been reported between RB1 homozygous deletion and/or promoter hypermethylation and loss of pRB expression detected by immunohistochemistry. The present study suggests that this might also be the case for PCNSLs as the underlying cause of promoter methylation with loss of RB1 expression.

Regarding lymphomas, p21^Waf1 alterations have been analyzed in only a few studies. A role for deletions and loss of expression of p21^Waf1 in aggressive lymphomas has been proposed (24) , but others failed to identify mutations of this gene in a large series of lymphoid neoplasms (25) . Our findings for PCNSLs are concordant with Cobbers’ immunohistochemical results (3) . p21^Waf1 expression was relatively low in many tumors (44%). However, promoter hypermethylation of p21^Waf1 was not detected, even in those without p21^Waf1 expression, and p21^Waf1 point mutations seem to be very rare in human tumors (26) . p21^Waf1 expression might be regulated at the transcriptional level, although any significance of p21^Waf1 in the development of lymphomas is still unclear.

p27^Kip1 is also important as a cyclin-dependent kinase inhibitor impacting on passage through the G₁ as well as G₂ restriction points. Specific alterations of the p27^Kip1 gene, including mutations and homozygous deletions, are exceedingly rare in human cancers, including systemic non-Hodgkin’s lymphomas (27) , and Worm et al. (28) indicated that p27^Kip1 methylation may be a cause of monoallelic p27^Kip1 silencing in malignant melanomas. We found 4 cases (22%) to be negative for p27^Kip1 expression by immunohistochemistry, and p27^Kip1 hypermethylation was detected in 2 cases. However, regulation of p27^Kip1 expression may occur at various stages. Some osteosarcomas and breast and colon cancers may express p27^Kip1 mRNA, but p27^Kip1 protein is absent because of rapid proteasome-mediated degradation in the tumor cells (29) . These findings suggest that aberrant p27^Kip1 methylation is not the only mechanism causing reduced p27^Kip1 levels, and epigenetic changes could also play a role in PCNSL pathogenesis.

In summary, the present study showed that alterations of p14^ARF and p16^INK4a genes are frequent in PCNSLs. Other related genes (RB1 and p27^Kip1) were also found to be independently methylated, although infrequently, suggesting that de novo methylation during PCNSL tumorigenesis results from independent and sequencespecific mechanisms.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom requests for reprints should addressed, at Second Department of Pathology, Nara Medical University, 634-8521, 840 Shijo-cho, Kashihara, Nara, Japan. Phone: 81-744-22-3051, extension 2237; Fax: 81-744-23-5687; E-mail: nkonishi{at}nmu-gw.naramed-u.ac.jp

² The abbreviations used are: PCNSL, primary central nervous system lymphoma; MSP, methylation-specific PCR.

Received 5/10/01. Accepted 7/18/01.

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