Leukemia (1999) 13, 453–459  1999 Stockton Press All rights reserved 0887-6924/99 $12.00 http://www.stockton-press.co.uk/leu Aberrations of the pathway components p53, MDM2 and CDKN2A appear independent in diffuse large B cell lymphoma MB Møller1, Y Ino2, A-M Gerdes3, K Skjødt4, DN Louis2 and NT Pedersen1

Departments of 1Pathology, 3Clinical Genetics, and 4Medical Microbiology, Odense University, Denmark; and 2Molecular Neuro-Oncology Laboratory, Department of Pathology (Neuropathology) and Neurosurgical Service, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA

The two products of the CDKN2A gene, and p19ARF, ways together through the 9p21 .5,6 In this locus resides have recently been linked to each of two major tumour sup- two -dependent kinase inhibitor , CDKN2A and pressor pathways in human carcinogenesis, the RB1 pathway CDKN2B. The CDKN2A gene has the rare ability to encode and the p53 pathway. p16 inhibits the phosphorylation of the ARF retinoblastoma gene product by -dependent kinases, two different , p16 and p19 . This is due to the whereas p19ARF targets MDM2, a p53 inhibitory , for unusual organisation of the gene with two alternative first degradation. A deletion of CDKN2A would therefore disturb , 1␣ and exon 1␤, and common exons 2 and 3.7,8 both pathways. To explore the p53 pathway genes as a func- Because they are translated in different reading frames p16 tional unit in diffuse large B cell non-Hodgkin’s lymphomas and p19ARF are structurally unrelated. Despite this difference (DLCL), we wanted to see whether there exists mutually exclus- iveness of aberrations of CDKN2A, MDM2 and p53, since this both can arrest the in G1, albeit through different has not been analysed previously. We investigated 37 DLCL for mechanisms. p16 inhibits CDK4/6 in the RB1 pathway, aberrations of p15, p16, p19ARF, MDM2, and p53 at the epigen- whereas p19ARF interacts with the p53 pathway by binding to etic, genetic and/or protein levels. Homozygous deletion of MDM2.5,6 The binding of p19ARF to MDM2 promotes MDM2 CDKN2A was detected in seven (19%) of 37 tumours, and degradation, thereby annulling MDM2’s inhibition of p53.5,6 ′ another three cases were hypermethylated at the 5 CpG island Hence, a mutation in CDKN2A potentially disrupts both the of p16. No point mutations were found in CDKN2B or CDKN2A. Immunohistochemical staining of formalin-fixed, paraffin- RB1 and p53 pathways. In the RB1 pathway mutations of embedded tissue for p16 confirmed these results, as all CDKN2A and RB1 are inversely correlated in various human tumours with alterations of CDKN2A were p16 immunoneg- tumours1 as are p53 mutations and MDM2 amplifications in ative. We found p53 mutations in eight (22%) cases and MDM2 soft tissue sarcomas9 and gliomas10 supporting that the genes overexpression in 16 (43%) tumours. Twenty-three (62%) are part of a functional unit.1,9,10 The same could be expected tumours had alterations of one or more p53 pathway compo- for p53 and p19ARF.5 Diffuse large B cell lymphomas (DLCL), nents (p53, p19ARF and MDM2). Furthermore, 7/9 (78%) p16- immunonegative tumours showed co-aberration of p53 and/or the non-Hodgkin’s lymphoma subtype with the highest mor- MDM2. The lack of correlation between these aberrations sug- tality and the largest increase in annual incidence gests that DLCL acquire additional growth advantage by inacti- increase,11,12 carry CDKN2A mutations (almost exclusively vating both of these critical regulatory pathways. deletions) in 15–20% of cases,13 mutated p53 15–20%,14 and Keywords: diffuse large B cell non-Hodgkin’s lymphoma; p53; p16; overexpression of MDM2 in up to 50%.15,16 To determine the ARF p19 ; p15; MDM2 status and correlations of the RB1 and p53 pathways in DLCL, we undertook a mutational analysis of p15, p16, p19ARF and p53, as well as immunohistochemical detection of p16 and Introduction MDM2 in 34 patients with DLCL from a single institution. The present study is the first that reports the interrelations of aber- The RB1 pathway and the p53 pathway are two major growth rations of these cell cycle regualtory components in DLCL. regulatory pathways involved in human tumorigenesis. Both pathways consist of a number of tumour suppressor genes and proto-oncogenes, which – if deregulated – promote the forma- Materials and methods tion of cancer. The two most commonly altered genes in human cancer are CDKN2A (or p16INK4a) and p53 (or TP53) Tissue material from the RB1 and the p53 pathway, respectively. The RB1 pathway exerts its tumour suppressor function primarily in the Tumour specimens were obtained at the time of diagnosis of the cell cycle by inhibiting transcription of genes (n = 34) and relapse (n = 3) from 34 patients at Odense Uni- necessary for transition from G1 to .1 The p53 pathway versity Hospital. Tumours were snap-frozen in liquid nitrogen regulates two end points: and cell cycle arrest in and stored at −80°C in OCT compound (Miles, Elkhart, IN, G1,2 depending on cell type and cell state. p53 acts as a tran- USA). Adjacent non-frozen blocks were fixed in 10% buffered scription factor and interacts with other cellular proteins ther- formalin and embedded in paraffin. The tumours were classi- eby exerting its tumour suppressor functions.2 The protein fied as DLCL according to the REAL classification.17 The cases product of the proto-oncogene MDM2 targets p53 for degra- were subclassified as diffuse centroblastic (CB) or immuno- dation through ubiquitin-mediated proteolysis.3 Furthermore, blastic (IB) lymphomas according to the updated Kiel classi- MDM2 also binds p53 thereby transcriptionally silencing it, fication.18 Two cases, 12.03 and 12.05, were IB, the remain- resulting in block of p53’s apoptosis- and growth arrest- ing 35 tumours were CB. DNA was extracted from frozen inducing functions.4 Recent research has linked the two path- tumour tissues using the QIAamp Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. Prior to DNA extraction, the tumour tissues were examined to ensure Correspondence: MB Møller, Department of Pathology, Odense Uni- versity, Winsløwparken 15, DK-5000 Odense C, Denmark; Fax that they consisted of viable tumour tissue with at least 50% +45 65 91 29 43 tumour cells. Received 7 August 1998; accepted 9 October 1998 For determination of immunohistochemical reaction pat- p53 pathway aberrations in DLCL MB Møller et al 454 terns of normal lymphoid tissues, two lymph nodes, one phoresis on 3% agarose gels. All samples were run with both spleen and one tonsil without malignancy were randomly assays for confirmation. selected from the tissue files at Odense University Hospital. Single-strand confirmation polymorphism (SSCP) analysis of The tissues had been fixed in 10% buffered formalin and CDKN2A exons 1, 2 and 3, and CDKN2B exon 2 was perfor- embedded in paraffin. med as published previously22 except for modifications of the PCR amplification protocols: all five fragments (CDKN2A exon 1, 5′-half of exon 2, 3′-half of exon 2, exon 3, CDKN2B Immunohistochemistry for p16 and MDM2 exon 2) were amplified using the touch-down protocol described. The non-commercial mouse monoclonal anti-p16 antibody SSCP of the alternative spliced exon 1␤ of CDKN2A was JC8 was evaluated along with the two commercial antibodies performed using previously published primers amplifying the against p16, the monoclonal G175-405 and the polyclonal exon 1␤ including intron–exon borders.23 Amplification was ␮ ␮ rabbit anti-human p16INK4 (PharMingen, San Diego, CA, done with 200 M dNTPs, 1.5 mM MgCl2, 1.0 M CDKN2A USA). JC8 proved to be the superior antibody and was used in exon 1␤ primers, 0.5 U Taq polymerase, 5–50 ng genomic the present study. Formalin-fixed paraffin-embedded sections DNA, and 5% dimethylsulfoxide in 10-␮l reaction volumes. were stained using the assay described in detail elsewhere.19 A touch-down PCR protocol was used with a first annealing Two monoclonal antibodies against MDM2, Ab-1 clone IF2 temperature of 67°C and a last annealing temperature of 60°C. (Oncogene Research, Cambridge, MA, USA) and NCL-MDM2 To increase the sensitivity of the SSCP assay the resulting 412- (Novocastra Laboratories, Newcastle upon Tyne, UK) were bp fragment was digested with FokI to generate fragments of evaluated. IF2 was chosen for this study because of its greater 185 and 227 bp, respectively. reproducibility and reduced cytoplasmic staining (data not The PCR products were separated on 6% non-denaturing shown). Briefly, 4 ␮m sections were deparaffinized, micro- polyacrylamide gels with 10% glycerol overnight at 4–8 W. wave oven antigen retrieved in T-EG buffer (10 mM Tris Cases with mobility shifts were reamplified and the fragments (Sigma, St Louis, MO, USA), 0.5 mM EGTA (Sigma), pH 9.0) were gel purified from 2% agarose gels using the Compass Kit and incubated overnight at 4°C with IF2 primary antibody in (American Bioanalytical, Natick, MA, USA). Purified frag- 1:100 dilution. TechMate 1000 (DAKO, Glostrup, Denmark) ments were sequenced with the T7 Sequenase kit (Amersham, was used for automated staining using the LSAB+ kit (DAKO) Cleveland, OH, USA) using the SSCP primers. with diaminobenzidine as chromogen and brief counterstain- For identification of hypermethylation of CpG islands of the ing in Mayer’s hematoxylin. promoter region of p16 we used a modification of the MSP Staining of normal germinal centres from tonsils and lymph assay24 described previously.19 nodes show weak staining of variable numbers of stromal cells PCR-RFLP analysis of CDKN2A exon 3 for identification of such as follicular dendritic cells and weak staining of a common C to G polymorphism in the 3′ UTR was performed occasional lymphoid cells (present study and Nielsen, as described.22 Stemmer-Rachamimov, Shaw, Koh, Louis, unpublished results). Based on this, a case was scored as p16 immunoneg- ative if all malignant cells were unstained, while stromal cells Mutation analysis of p53 or reactive non-malignant cells in the same section showed nuclear staining. If any malignant cells had nuclear staining Denaturing gradient gel electrophoresis (DGGE)-based the tumour was regarded as p16 positive. Tumours with strong screening for mutations in the entire p53 coding sequence, ie p16 expression, ie Ͼ50% immunoreactive nuclei, were exons 2–11, and splice junctions followed by cycle sequen- scored as strongly p16 immunopositive. cing of fragments with deviant DGGE patterns was done as For evaluation of MDM2 immunoreactivity, only nuclear described25 with the following exceptions: during PCR cresol reaction was considered. An image analysis system consisting red and sucrose were omitted, and DGGE was performed on of an Olympus BX60 light microscope, a Sony CCD camera, gels containing a 30–80% gradient of urea and formamide. and the image analysis software package ImagePro Plus The gels were run at 150 V for 5 h. (Media Cybernetics, Silver Spring, MD, USA) was used. A total of 22 high power fields (×400) of normal lymphoid tissue rep- resenting germinal centres, mantle zones and interfollicular Statistical analysis areas were counted. On average 1.2% (0–4.4%) of the cells were MDM2 immunopositive. The germinal centres had the Fisher’s exact test was used to test for correlations of CDKN2A highest average percentage, 2.7% (0.2–4.4%), while the aver- and p53/MDM2. age percentage of MDM2-positive non-germinal centre fields was 0.3% (0–0.7%). Based on these controls, we defined MDM2 overexpression (MDM2+) as nuclear MDM2 immuno- Results positivity of у10% of tumour cells. CDKN2A and CDKN2B mutations

Genetic analysis of CDKN2A and CDKN2B HD of CDKN2A was detected in seven (19%) of 37 DLCL biopsies from 34 patients (Table 1; Figure 1(a) ). In one of Two previously decribed comparative multiplex PCR assays three patients with sequential biopsies available HD was were used with minor modifications to detect homozygous present at relapse (11.08R) but not at the time of diagnosis deletions (HDs) of CDKN2A.20,21 The PCR programming was (11.08). SSCP analysis of CDKN2A exons 1␣,1␤, 2 and 3 as changed to a touch-down procedure with gradually decreas- well as CDKN2B exon 2 revealed no tumour-specific ing annealing temperature from 62°Cto55°C. The products migration shifts (Table 1). DNA sequencing showed that a shift were visualised by ethidium bromide staining after electro- in the 3′-half exon 2 amplicon of case 11.09 was a common p53 pathway aberrations in DLCL MB Møller et al 455 Table 1 Molecular genetic and immunohistochemical findings in the 37 DLCL analysed

Case No.a p15 p16 p19ARF p53 MDM2 SSCPb SSCPd DGGE IHC (%) HD SSCPc MSP IHC

11.03 ns n ns nd + ns w Ͻ10 11.05 ns n ns nd + ns M 44 11.06 ns n ns nd + ns w Ͻ10 11.08 ns n pm3 nd + ns w 47 11.08R ns DEL pm3 nd − ns w 20 11.09 ns DEL pm2/pm3 nd − ns w 29 11.12 ns n ns nd ++ ns w Ͻ10 11.13 ns n ns nd + ns w Ͻ10 11.14 ns n ns nd ++ ns M Ͻ10 11.15 ns n pm3 nd + ns w 16 11.17 ns n ns U + ns w Ͻ10 11.18 ns n pm3 nd + ns w 12 11.20 ns n ns nd ++ ns w 39 11.21 ns n pm3 M na ns M Ͻ10 11.21R ns n pm3 M − ns M Ͻ10 11.22 ns n ns nd + ns w Ͻ10 11.22R ns n ns nd + ns w Ͻ10 11.23 ns n ns nd ++ ns M Ͻ10 11.24 ns n ns nd + ns w Ͻ10 11.25 ns n ns nd ++ ns w 23 11.26 ns n ns nd + ns w 14 11.27 ns n ns nd + ns w 47 11.28 ns n ns U + ns w Ͻ10 11.29 ns n ns nd + ns w 46 11.31 ns n ns nd + ns w Ͻ10 11.33 ns DEL ns U − ns M 15 11.34 ns n ns nd ++ ns w 13 11.35 ns n ns nd ++ ns w Ͻ10 11.36 ns n ns nd + ns w Ͻ10 11.37 ns n ns nd + ns w 24 11.38 ns n ns U + ns w Ͻ10 11.39 ns DEL ns nd − ns w Ͻ10 11.40 ns n ns M − ns M Ͻ10 11.41 ns DEL ns nd − ns w 45 11.42 ns DEL ns nd − ns M 33 12.03 ns n ns nd na ns w Ͻ10 12.05 ns DEL ns nd − ns w Ͻ10 ns, no shifts; n, normal; nd, not done; w, wild type; M, mutated; pm3, polymorphism in exon 3; DEL, deletion; pm2, polymorphism in exon 2; U, unhypermethylated at 5′ CpG island; M, hypermethylated at 5′ CpG island; na, tissue not available for analysis. a11.08R, 11.21R and 11.22R are biopsies from lymphoma relapses. 12.03 and 12.05 are immunoblastic, the remaining cases are centroblas- tic lymphomas. bSSCP mutational analysis of p15 exon 2. cSSCP mutational analysis of p16 exons 1␣, 2 and 3. dSSCP mutational analysis of p16 exon 1␤.

GCG to ACG polymorphism, changing alanine to threonine. Hypermethylation of the CDKN2A promoter region Seven identical shifts were identified in the exon 3 fragments, one homozygous and six heterozygous, suggesting a frequent polymorphism. PCR-RFLP analysis of two of these cases con- Seven DLCL cases that lacked CDKN2A HD, two p16 immu- firmed the presence of the C/G polymorphism in base 494 of nonegative, four weak p16 positive, and one not investigated the 3′ UTR (data not shown). immunohistochemically, were subjected to the MSP assay (Figure 1(b) ). Both p16-negative tumours as well as the tumour not investigated immunohistochemically amplified p16 and MDM2 immunohistochemistry with the methylation-specific primers (Table 1), as did the two colon carcinoma-positive control cell lines with known pro- moter region methylation, HT-29 and CaCo2. Conversely, All seven tumours with HD of CDKN2A were p16 immu- none of the p16 weak-positive cases nor DNA from normal nonegative (Table 1, Figure 2(a) and (b) ). Of the remaining 28 tumours evaluated for p16, two were p16 negative and four lymphoid tissue amplified with these primers, but showed were weakly p16 positive. In six cases, the p16 expression amplification with the unmethylated base-specific primers. was strong. MDM2 was overexpressed in 16 of 37 (43%) lymphomas (Table 1; Figure 2(c) and (d) ). p53 pathway aberrations in DLCL MB Møller et al 456 of the p53 and RB1 pathways. Previous studies of CDKN2A in DLCL have shown that HD are moderately common, present in 18 (11%) of 165 cases,26–31 similar to our 18% in primary DLCL (and 19% overall). Point mutations of CDKN2A are rare in DLCL, seen in only one of 125 cases reported,27–29,32 and not detected in our material. A third major means of CDKN2A inactivation is promoter silencing by hypermethylation of the 5′ CpG island. Although some reports have shown hypermethylation in 11 (39%) of 28 cases,29,33,34 we found hypermethylation in only 6% of primary DLCL (and 8% overall). The finding of MDM2 overexpression in 43% of DLCL in this study is comparable to previous studies.15,16,35 The mech- anism behind this overexpression is obscure in NHL as ampli- fication of MDM2 is rarely seen in lymphoid neoplasms over- expressing MDM215,16,35 indicating that other post- transcriptional mechanisms are responsible.36–40 Splicing vari- ation is a mechanism identified in some tumour types. Tumour-associated splice variants can retain their trans- Figure 1 Genetic and epigenetic analysis of CDKN2A. (a) Com- parative multiplex PCR analysis showing deletion of CDKN2A in lane forming ability even if they harbour partial deletions of the 39 3 (case 11.39) as preferential amplification of the control band (top region encoding the p53 binding domain. These findings band) and reduced amplification of the CDKN2A fragment (bottom suggest a tumorigenic function independent of p53. Our find- band) is seen. Lanes 1 and 2 shows tumours (11.37 and 11.38) without ing of MDM2 overexpression in 43% of p53-mutated DLCL HD. MSP analysis (b) of CDKN2A using bisulfite-treated DNA from could also indicate that there is an additive effect on lympho- three tumours using primer sets selectively amplifying unmethylated magenesis of simultaneous p53 mutation and MDM2 over- (U) or methylated (M) DNA, shows successful amplification with the M primer set in tumours 11.21 and 11.21R as evidence of hyperme- expression. thylated 5′ CpG island. MW, pUC18/HaeIII digest size marker. MDM2 levels are also subject to regulation by proteolytic degradation mediated by p19ARF.6 Therefore one would p53 mutations expect that tumours with HD of CDKN2A (thereby inactivat- ing p19ARF) would be defective in MDM2 degradation and Seven mutations were found in 37 tumours (Table 1). All express elevated levels of MDM2. Consistent with this, the seven mutations were base substituting missense mutations present study shows overexpression of MDM2 in 71% (five of affecting the conserved hot-spot region of exons 5–8 (Table 2). seven) of DLCL with CDKN2A deletions. Although the In patient 11.21 the primary tumour and the recurrent tumour remaining two tumours have very low to undetectable levels showed an identical p53 mutation. (Ӷ1% of nuclei immunopositive) of MDM2, it cannot be excluded that these tumours harbour non-sense or frameshift mutations of MDM2 that could preclude detection of the ARF Interrelations of alterations of p16, p19 , p53 and protein.41 MDM2 The relative importance of p16 and p19ARF in lymphoma/ leukemogenesis is at present unresolved. Deletions of all of A summary of the alterations in the 37 DLCL is shown in the coding sequences of p16 and p19ARF will of course inacti- Table 3. One or more genes were altered in 23 (62%) of 37 vate both genes, whereas deletions or point mutations only lymphomas, and in nine (24%) tumours two or three genes affecting exons 2 and 3 could selectively inactivate p16. These were aberrant. Twenty-three (62%) tumours had alterations of exons are important for p16 tumour suppressive function one or more p53 pathway components (p53, p19ARF and while intact exon 1␤ has proved sufficient for p19ARF effect MDM2), with two (5%) patients (11.33 and 11.42) having in murine and human cell lines.42,43 Moreover, functionally alterations of all three components. MDM2 overexpression analysed cancer-associated point mutations in exon 2 may and p53 mutation were independent events (P = 1.00) with inactivate p16 and not p19ARF.5,42,43 That exon 1␤ encodes three tumours having both aberrations, five with p53 mutation p19ARF’s MDM2 binding region6 could explain its functional only and 13 with MDM2 overexpression only. CDKN2A- deleted cases showed a trend towards correlation with MDM2 importance. The fact that mutations in lymphoid neoplasms rarely are point mutations but most often co-deletions of p15 overexpression as five (71%) of seven DLCL with CDKN2A ␤ HD had MDM2 overexpression (P = 0.21). Four (44%) of nine and p16 (including exon 1 ) or deletions of p16 alone (most including exon 1␤44), combined with identification of p16-immunonegative tumours harboured p53 mutations, ␤ showing a trend towards a positive correlation (P = 0.14). Of inclusion of exon 1 in all of the rarer ‘p15-only’ deletions in 45 ARF seven tumours with CDKN2A deletions two had concomitant acute lymphoblastic leukaemia, suggests a role for p19 p53 mutations (P = 0.80). Interestingly, the majority, seven in lymphoma/leukemogenesis. To date, no point mutations ␤ 6 (78%) of nine, of p16-immunonegative tumours showed alter- specific to exon 1 have been identified, suggesting that co- ation of p53 and/or MDM2 (P = 0.29). inactivation of p16, and perhaps p15 as well, confers a selec- tive growth advantage to a tumour cell. Genetic alteration may not even be necessary for inactivation of p19ARF. Neo- Discussion plastic haematopoietic cell lines expressing abundant ␤ tran- script without apparent mutations can be negative for the pro- We analysed a group of 34 primary DLCL and three recurrent tein suggesting there could exist an uncoupling of DLCL for the status of several genes functioning at the nexus transcription and translation.46 This mechanism could be p53 pathway aberrations in DLCL MB Møller et al 457

Figure 2 Immunostaining patterns for p16 (a) and (b) and MDM2 (c) and (d) in DLCL. (a) Anti-p16 study of case 11.15 showing heterogeneous nuclear staining of tumour cells. Case 12.05 shows p16-immunonegative tumour cells (b). Note the distinct nuclear staining of admixed small lymphocyte (arrowhead). (c) Anti-MDM2 study of normal splenic germinal centre shows occasional weak MDM2-positive lymphoid cells (arrowheads), whereas case 11.29R shows intense nuclear MDM2-immunopositivity of the lymphoma cells (d). (a)–(d), ×200.

Table 2 Description of the eight p53 mutations in 37 DLCL found by DGGE analysis of exons 2–11

Case No. Exon Codon Nucleotide Mutation Amino acid Table 3 Summary of molecular genetic and immunohistochemical 11.05 5 175 524 G→A Arg→His findings in 37 DLCL 11.14 6 194 581 T→C Leu→Pro 11.21 8 281 841 G→A Asp→Asn Aberration Co-aberration No. (%) 11.21R 8 281 841 G→A Asp→Asn → → 11.23 7 248 743 G A Arg Gln a 11.33 6 193 578 A→G His→Arg CDKN2A 10 (27) 11.40 5 151 452 C→A Pro→His None 2 (5) 11.42 7 259 775 G→A Asp→Asn p53 3 (8) MDM2 3 (8) Both 2 (5) p53b 8 (22) ′ None 2 (5) active in cases with selective p16 inactivation, eg 5 CpG CDKN2A 3 (8) island hypermethylation. MDM2 1 (3) The hypothesis that p19ARF is a tumour suppressor protein Both 2 (5) that functions in the p53 pathway predicts that tumours with MDM2c 16 (43) inactivation of p19ARF are less likely to harbour p53 alter- None 10 (27) 5 CDKN2A 3 (8) ations. A re-examination of the literature of published p53 1 (3) mutations of CDKN2A and p53 could not definitely confirm Both 2 (5) a reciprocal relationship.5 Reviewing the 263 cases (including the 37 reported in this study) of lymphoid neoplasia analysed aHomozygous deletion or promoter hypermethylation. for both p53 mutations and CDKN2A deletions47–50 also did bMutation. not show a sufficiently low rate of co-alterations to substan- cOverexpression. p53 pathway aberrations in DLCL MB Møller et al 458 tiate the hypothesis. On the contrary, our results imply that a The Non-Hodgkin’s Lymphomas. Edward Arnold: London, 1990, second alteration of the p53 pathway components p53, pp 1–14. p19ARF and MDM2, results in a substantial additional growth 13 Siebert R, Willers CP, Opalka B. Role of the cyclin-dependent kin- ase 4 and 6 inhibitor gene family p15, p16, p18 and p19 in leuke- advantage of the tumour, as compared to alteration of only mia and lymphoma. Leuk Lymphoma 1996; 23: 505–520. one of these components. 14 Preudhomme C, Fenaux P. The clinial significance of mutations In conclusion, p16, p19ARF, p53 and MDM2 are frequently of the p53 in haematological malignancies. aberrant in DLCL and can be involved in disease progression. Br J Haematol 1997; 98: 502–511. The results support an interaction between p19ARF and MDM2 15 Maestro R, Gloghini A, Doglioni C, Gasparotto D, Vukosavljevic but alterations of the p53 pathway genes are not mutually T, De Re V. MDM2 overexpression does not account for stabiliz- ation of wild-type p53 protein in non-Hodgkin’s lymphomas. exclusive. Future identification of new components and inter- Blood 1995; 85: 3239–3246. actions will undoubtedly further elucidate the biology of this 16 Kawamata N, Miller C, Levy V, Shintaku IP, Koeffler HP, Said JW. important aspect of lymphomagenesis. Relations to other mdm-2 oncogene expression in non-Hodgkin’s lymphomas. genes involved in the RB1 pathway as well as clinical data Diagn Mol Pathol 1996; 5: 33–38. are currently being investigated, and may be translated into 17 Harris NL, Jaffe ES, Stein H, Banks PM, Chan JK, Cleary ML, Delsol new therapeutic initiatives. G, De Wolf-Peeters C, Falini B, Gatter KC, Grogan TM, Isaacson PG, Knowles DM, Mason DY, Mu¨ller-Hermelink HK, Pileri SA, Piris MA, Ralfki{r E, Warnke RA. A revised European–American classification of lymphoid neoplasms: a proposal from the Inter- Acknowledgements national Lymphoma Study Group. Blood 1994; 84: 1361–1392. 18 Stansfeld AG, Diebold J, Noel H, Kapanci Y, Rilke F, Kelenyi G, We are grateful to Jim Koh, University of Vermont Cancer Sundstrom C, Lennert K, van Unnik JA, Mioduszewska O, Wright Center, Burlington, VT, for the provision of the JC8 antibody, DH. Updated Kiel classification for lymphomas. 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