Molecular features of primary mediastinal B-cell lymphoma: involvement of p16INK4A, p53 and c-myc

Molecular features of primary mediastinal B-cell lymphoma:

involvement of p16INK4A, p53 and c-myc

ALDO SCA RPA,1 PATRIC K S. MO OR E,1 GILDA S RI GAUD,1 GIORGIO INGHIRAMI,4 MA RI NA MONTRE SOR,1

MA RTA ME NEGAZZI,2 GI US EPPE TODESCHINI3

AN D FABIO MENE STRINA1 1Dipartimento di Patologia,

Sezione Anatomia Patologica, 2Dipartimento di Scienze Neurologiche e della Visione, Sezione Chimica Biologica,

and 3Dipartimento di Medicina Clinica e Sperimentale, Sezione di Ematologia, UniversitaÁ di Verona, Verona,

Italy, and 4Department of Pathology and Kaplan Comprehensive Cancer Center,

New York University School of Medicine Medical Center, New York, U.S.A.

Received 10 May 1999; accepted for publication 8 July 1999

Summary. Primary mediastinal B-cell lymphoma (PMBL)shows chromosome 9p anomalies in 50% of cases. Based onreports that p16INK4A gene, located on this chromosomalarm, is frequently altered in aggressive lymphomas, weanalysed for alterations of this gene in 27 cases of PMBL,which were part of a series of 32 PMBL cases that have beencharacterized for alterations in c-myc, p53, N-ras, bcl-1, bcl-2,bcl-6 and for Epstein-Barr virus (EBV) infection. Four casesshowed p16INK4A gene anomalies, including three withpromoter methylation and one homozygous deletion. EightPMBLs showed c-myc rearrangements. Three additionalcases showed sequence variations in the c-myc P2 promoter,two of which consisted of the same germline variation

involving a novel polymorphic XhoI site. Four tumourscontained p53 gene mutations and three had clonal EBVinfection. One case had a bcl-6 rearrangement. In conclusion,our study shows that p16INK4, c-myc and p53 alterationsoccur in 15%, 25% and 13% of PMBLs, respectively. EBVmonoclonality was found in 9% of cases, whereas noabnormality was detected in bcl-1, bcl-2 and N-ras. Thus,none of the common genetic aberrations seen in other typesof non-Hodgkin's lymphomas appears to be stringentlyinvolved in the pathogenesis of this unique lymphoma type.

Keywords: mediastinal lymphoma, thymic lymphoma,p16INK4A, p53, c-myc.

Primary mediastinal B-cell lymphoma (PMBL) is an aggres-sive non-Hodgkin's lymphoma (NHL) affecting mainly youngadult patients (Addis & Isaacson, 1986; Cazals-Hatem et al,1996; Menestrina et al, 1986; MoÈller et al, 1986b; Yousemet al, 1985). It expresses B-lineage surface molecules such asCD19 and CD20, has its immunoglobulin genes rearranged,and displays severe defects in expression of immunoglobulinconstituents and HLA class I and II molecules (Menestrinaet al, 1986; MoÈller et al, 1986a, 1987a, b; Momburg et al,1987; Scarpa et al, 1987).

To date, little is known about the molecular alterations inPMBL (Scarpa et al, 1991; Tsang et al, 1996). The onlyinformation about chromosomal alterations in this diseasehas been obtained by comparative genomic hybridizationanalysis, which showed that the most consistent anomaly

was gains on chromosome 9 in 50% of cases (Joos et al,1996; Scarpa et al, 1999). However, regions of chromosomalampli®cation or duplication may be also accompanied bylosses (Achille et al, 1996). In fact, we recently demonstratedthat among a series of nonrandom chromosomal imbalancesdetected by DNA ®ngerprinting of paired normal/tumoursamples of PMBL, both gains and losses on chromosome 9were observed (Scarpa et al, 1999).

It has been recently reported that the p16INK4A gene,located on chromosome 9p, is frequently altered inaggressive large cell variants of NHL and particularlyamong those representing the transformation of marginalzone and mucosa-associated low-grade lymphomas (Pinyolet al, 1998; Villuendas et al, 1998). Interestingly, PMBL isconsidered to arise from a noncirculating population ofintramedullary thymic B lymphocytes (Addis & Isaacson,1986; Davis et al, 1990; MoÈller et al, 1987b; Paulli et al,1997) as it shows an immunophenotypic pro®le similar tothe family of `extrafollicular B cells', including monocytoid Bcells, and parafollicular cells of the mucosa-associated

British Journal of Haematology, 1999, 107, 106±113

106 q 1999 Blackwell Science Ltd

Correspondence: Dr Aldo Scarpa, Dipartimento di Patologia-Anatomia Patologica, UniversitaÁ di Verona, Strada Le Grazie,

I-37134 Verona, Italy. e-mail: [email protected].

107Thymic B Lymphoma

q 1999 Blackwell Science Ltd, British Journal of Haematology 107: 106±113

lymphoid tissue. A possible low-grade counterpart has alsobeen described (Isaacson et al, 1990).

These considerations prompted us to analyse a series of 27PMBL cases for alterations in the p16INK4 gene, includingmutation, deletion, and de novo methylation of the CpGisland within the promoter. Potentially inactivating altera-tions were found in four cases (15%), suggesting a role for thep16INK4 tumour suppressor gene not described previously inPMBL.

The 27 cases studied were part of a larger series of 32PMBL that have been collected during the last 10 years andprogressively characterized for genetic alterations commonlyinvolved in the pathogenesis of various types of NHL,including c-myc, p53, N-ras, bcl-1, bcl-2, bcl-6, and for thepresence of Epstein-Barr virus (EBV).

Based on the suggestion that c-myc is overexpressed in aproportion of PMBLs not showing translocation (Menegazziet al, 1999), we also analysed the c-myc P2 promoter, since

mutations in this region may lead to disruption of thenegative c-myc autoregulation mechanism (Facchini et al,1997; Taub et al, 1984). We describe novel sequencevariants in the c-myc P2 promoter region, including anewly discovered polymorphism.

MATERIALS AND METHODS

Tumour samples, immunohistochemistry and DNA extraction.Frozen tumour samples were retrieved from the ®les of thePathology Department of Verona University and includedcases observed at our University Hospital or received forconsultation. All samples were obtained from untreatedpatients suffering from a mediastinal mass. Diagnosis ofPMBL was established in each case by standard clinical andhistopathologic criteria and by cell marker analysis (Addis &Isaacson, 1986; Menestrina et al, 1986; MoÈller et al, 1986a;Paulli et al, 1997). In all cases immunohistochemical

Table I. Molecular genetic lesions in 32 primary mediastinal B-cell lymphomas.

Ig gene

Patient Age (yr) Sex JH K EBV p53 p16 c-myc

1 30 F R R ÿ ÿ Methyl ÿ

2 30 F G R ÿ ÿ ÿ ÿ

3 25 F R G ÿ ÿ ÿ ÿ

4 25 F R R ÿ I255F ÿ Exon 1

5 41 F R R ÿ ÿ ÿ Exon 1

6 27 M R R ÿ ÿ ÿ Exon 1

7 52 F R R ÿ ÿ ÿ Promoter8 30 F R R � R248Q ÿ ÿ

9 23 M R G ÿ ÿ Methyl Promoter

10 30 F R R ÿ ÿ ÿ ÿ

11 67 M R R ÿ Y220L Methyl ÿ

12 26 F R R ÿ ÿ ÿ ÿ

13 26 F R R ÿ ÿ nd ÿ

14 32 M G R ÿ ÿ HD ÿ

15 37 F R R ÿ R273L ÿ Exon 1

16 30 M R R ÿ ÿ ÿ Exons 2±3

17 28 M R G ÿ ÿ ÿ ÿ

18 29 F R G ÿ ÿ ÿ Promoter19 26 F R R � ÿ ÿ ÿ

20 27 M R R ÿ ÿ ÿ ÿ

21 36 F R R � ÿ ÿ ÿ

22 29 M R R ÿ ÿ ÿ ÿ

23 20 M R R ÿ ÿ ÿ ÿ

24 32 F R G ÿ ÿ Intronic Exon 1

25 29 M R R ÿ ÿ ÿ ÿ

26 35 F R R ÿ ÿ A148T ÿ

27 28 F R R ÿ ÿ nd ÿ

28 30 F R G ÿ ÿ ÿ Exons 2±3

29 58 M R R ÿ ÿ nd ÿ

30 58 M R R ÿ ÿ nd ÿ

31 30 M R R ÿ ÿ ÿ ÿ

32 33 F R R ÿ nd nd Exon 1

R, rearranged; G, germline; �, positive; ÿ, negative; nd, not done; HD, homozygous deletion; methyl, methylated

50 CpG island.

The putative mutational effects or location are indicated.

analysis con®rmed the lymphoid nature of the neoplasticcells, which expressed panleucocyte antigen CD45 andseveral B-cell-related markers, including CD20 and CD22.In no case could surface immunoglobulins be detected. Thepresence of p53 protein nuclear accumulation was examinedusing the anti-p53 monoclonal antibodies as previouslydescribed (Chilosi et al, 1996). The DNA was extracted asdescribed (Scarpa et al, 1991).

Southern blot analysis and DNA probes. Genomic DNA wasdigested with the appropriate restriction enzymes andelectrophoresed on 0´8% agarose gels, denatured, neutra-lized, and transferred to nitrocellulose ®lters according tostandard procedures. The organization of the c-myc genewas analysed by hybridization of EcoRI, HindIII andBamHI digested genomic DNA with the MC413RC probe(Scarpa et al, 1999), which contains the third exon of thec-myc locus. Point mutations or small internal rearrange-ments of the c-myc gene are detected in about 60% ofcases by a polymorphic PvuII site located in the 30 end ofexon 1 by hybridization of the 50-Pv probe to PvuIIdigested genomic DNA (Scarpa et al, 1991). Bcl-1rearrangements were analysed by hybridization of the2´3 kb SstI fragment from the bcl-1 locus to HindIIIdigested DNA (Tsang et al, 1996). Bcl-2 rearrangements

were analysed by hybridization of the pFL-1 and pFL-2probes to BamHI, HindIII, EcoRI and PstI digested DNA(Scarpa et al, 1991). Bcl-6 rearrangements were visualizedby hybridization of the 0.96E-E probe with BamHI and XbaIdigested DNA (Tsang et al, 1996). The presence of EBVsequences in genomic DNA was shown by the use of the longinternal direct repeat probe (BamHI-W/V) with BamHIdigested DNA (Dambaugh et al, 1980) and its monoclonalnature using a DNA probe speci®c for EBV genomic termini(Weiss et al, 1987).

PCR-SSCP and sequencing analysis. Primers and conditionsfor ampli®cation of the p53 (exons 4±9), c-myc and N-ras(exons 1 and 2) genes were as described (Chilosi et al, 1996;Inghirami et al, 1994; Scarpa et al, 1994). For the p16INK4

gene, exon 1 was ampli®ed in its entirety, whereas exon 2was divided into two fragments as described (Zhang et al,1994). PCR reactions contained 0´1 mg DNA, 0´5 pmol eachof the appropriate primers, 200 mM dNTP, 1 ´ type II reactionbuffer (Perkin Elmer), 1´5 mM MgCl2, 0´1 ml [a-32P]dCTP (IIITBq/mmol) and 0´5 U Taq polymerase in a ®nal volume of10 ml. For SSCP analysis, samples were run on 5%polyacrylamide gels containing 0´125% bis-acrylamide for2±6 h at 30 W. Bands exhibiting aberrant migration werecut from the gel, re-ampli®ed and sequenced. Direct DNA


108 Aldo Scarpa et al

Fig 1. Analysis of p16INK4 gene alterations in

PMBL. (A) Duplex PCR showing deletion of

exon 1 of the p16INK4 gene in patient 14. Themolecular weight marker is on the left side of

the gel. Arrows indicate the position of the

bands ampli®ed from the p16INK4 and p53

genes. (B) SSCP analysis of exon 1 of thep16INK4 gene showing an aberrantly migrating

band in patient 24. (C) SSCP analysis of exon 2

of the p16INK4 gene showing an aberrantlymigrating band in case 26. (D) Representative

methylation-speci®c PCR analysis of the 50 CpG

island. U, PCR with primers speci®c for

unmethylated DNA. M, PCR with primersspeci®c for methylated DNA. The molecular

weight marker is on the left of the gel. Cases 1,

9 and 11 are positive for methylation. For all

panels, the relative case numbers areindicated.



sequencing was performed as described (Chilosi et al, 1996),or alternatively, using an ABI Prism 377 instrument.

Methylation-speci®c PCR for p16INK4 was carried outessentially as described (Herman et al, 1996). Brie¯y, 1 mg ofDNA was incubated for 30 min at 378C with 0´2 M NaOH in a®nal volume of 50 ml. To this mixture, 30 ml 10 mM

hydroquinone and 520 ml 3 M Na bisulphite were added.After incubation overnight at 508C, the reactions weredesalted, NaOH was added to 0´3 M , and then brie¯yincubated at room temperature. DNA was then precipitatedand resuspended in H2O. PCR with primers speci®c formethylated and unmethylated DNA was performed asdescribed (Herman et al, 1996).

RESULTS

The results of the molecular genetic characterization aresummarized in Table I. Clonal immunoglobulin heavy and/or light chain gene rearrangement products were found inall cases.

p16INK4 geneTwenty-seven cases were analysed for molecular alterationsin the p16INK4 gene. Potentially inactivating alterations ofthe p16INK4 gene were found in four cases (15%). Case 14had a homozygous deletion and three cases had methylationof the 50 CpG island (Fig 1). Additionally, PCR-SSCP analysisrevealed an abnormally migrating band in cases 24 and 26,due to a mutation in intron 1 (�27, C to A) (Fig 1) and a G-A

polymorphism at codon 148 (ala to thr) (Aitken et al, 1999),respectively.

p53 geneFour of the 31 cases analysed had a mutated p53 gene (13%)as detected by PCR-SSCP analysis of exons 4±9 (Fig 2).Subsequent re-ampli®cation and DNA sequence analysisshowed that missense mutations were present in all fourcases (Table II). Immunostaining for p53 protein revealedpositively staining nuclei in >10% of neoplastic cells in thefour cases bearing p53 gene mutations, whereas theremaining 28 cases scored negative.

c-myc geneEight of the 32 cases had alterations in the c-myc gene(25%), as exempli®ed in Fig 3. Southern blot analysis of the

Fig 2. Analysis of p53 gene alterations in PMBL. (A) H&E stain of case 11. (B) Immunohistochemistry with anti-p53 antibody showing nuclear

accumulation. (C) Representative SSCP analysis of exon 6 of p53 gene showing an aberrantly migrating band in case 11. (D) Sequence analysis ofthe normal and aberrantly migrating bands from the SSCP bands shown in C. The mutation is indicated by the arrow.

Table II. Mutation of p53 gene in four PMBL cases.

Patient IHC* Exon Codon Mutation Predicted effect

4 80% 7 255 ATC to TTC Ile to Phe

8 10% 7 248 CGG to CAG Arg to Gln

11 90% 6 220 TAT to TGT Tyr to Leu

15 60% 8 273 CGT to CTT Arg to Leu

* IHC, p53 immunohistochemistry; %, ratio of positive cells to the

neoplastic population.

c-myc gene detected rearrangements of the second/thirdexon in two cases, characteristic of the translocations foundin sporadic Burkitt's lymphomas. Small mutations near the30 end of non-coding exon 1 were detected in six additionalcases by either Southern blot or PCR-SSCP analysis. We alsodecided to enlarge our analysis of molecular alterations ofthe c-myc gene to include the P2 promoter region. Threecases were found to harbour deviations from the publishedsequence (Fig 4) (Battey et al, 1983). Case 9 had a C-Gtransversion at �4. Cases 7 and 18 both showed a G-Atransition at ÿ106 (Fig 4B). DNA sequence analysis ofnormal DNA from both these patients con®rmed thatthis sequence deviation was heterozygotic in nature andgermline in origin (Fig 4B). This sequence difference lies inthe recognition site for XhoI at ÿ107 (CTCGAG-CTCAAG).To determine the frequency of this polymorphism amongnormal individuals, PCR ampli®ed DNA from 100 subjectswas analysed by either XhoI digestion or direct sequenceanalysis. Among these, one heterozygote was found andwas thus observed in 0´5% of normal chromosomes(n�200).

Epstein-Barr virusEBV DNA sequences were found in three cases (9%) and theirclonal origin was demonstrated by Southern blot analysisusing the terminal repeat probe (data not shown).

Bcl-1, bcl-2, bcl-6 and N-rasNo case showed bcl-1, bcl-2 or N-ras alterations. A bcl-6 generearrangement was found in one case (patient 2) (data notshown).



Fig 3. Southern blot and SSCP analysis of the

c-myc gene in PMBL. (A) Southern blot analysis

with the MC413RC probe to EcoRI digested

DNA (germline band of 12´8 kb) showing amajor rearrangement in cases 16 and 28.

(B) Southern blot analysis with the 50-Pv probe

probe to PvuII digested DNA (germline bandof 0´86 kb) showing rearrangements in cases

32, 6 and 24. (C) and (D) SSCP analysis of the

®rst intron of the c-myc gene showing

mutations in cases 15 and 4. Case numbers areas indicated.

Fig 4. SSCP analysis of the promoter region of the c-myc gene.

(A) SSCP analysis showing aberrantly migrating bands in cases 7, 9

and 18. Case numbers are as indicated. (B) Representative sequence

analysis. Panels 1 and 2 show the sequence of the normal andaberrantly migrating bands in case 18. Panel 3 shows the sequence

analysis of PCR ampli®ed DNA from the same tumour, demonstrating

the heterozygosity of the sequence polymorphism.



DISCUSSION

The novel ®ndings of the present study included thedemonstration of p16INK4 gene alterations in 15% andthe occurrence of monoclonal EBV infection in 9% of PMBL.In addition, we described sequence variants in the c-mycP2 promoter region, including a newly discovered poly-morphism. Our data also con®rmed previous observationson a larger series that alterations of the c-myc and p53 genesoccur in 25% and 13% of cases, respectively, and that bcl-6gene rearrangements are rarely encountered, whereas noalterations were found in the N-ras, bcl-1 or bcl-2 genes(Scarpa et al, 1991; Tsang et al, 1996).

Alterations of the p16INK4gene have been found in varyingpercentages among different primary NHLs (5±45%) andhave been associated with aggressive variants and/ortumour progression (Klangby et al, 1998; Pinyol et al,1998; Villuendas et al, 1998). In our series of PMBL, p16INK4

gene alterations were found in a proportion not dissimilarto that seen in the aggressive variants of NHL. Methylationof the promoter was the most frequent alteration andwas found in 11% (3/27) of cases, whereas a homozygousdeletion was found in one case. An additional case (tumour24) showed an intronic mutation whose effect cannotbe predicted with certainty given its distance from theexon±intron junction.

For the ®rst time we report the detection of clonal EBVsequences in PMBL in 3/32 cases. Cazals-Hatem et al (1996)reported EBV infection in a similar proportion of cases (2/41)by in situ hybridization, but its clonal nature was notdetermined. EBV is only rarely associated with B-cell NHLsother than Burkitt's and AIDS-associated lymphomas and ithas been hypothesized that it is most likely to be present inonly a proportion of tumour cells, suggesting that infectionoccurred late in the oncogenic process (Hamilton-Dutoit et al,1993; Hummel et al, 1995; Pallesen et al, 1993; Young et al,1989). However, the clonality of EBV termini in our threePMBL strongly suggests that EBV infection had preceded andthus most probably contributed to clonal expansion in thesemalignancies (Neri et al, 1991). Furthermore, although therole of EBV on gene expression is at present unclear, it isnoteworthy that increased levels of c-myc mRNA were foundin those PMBL with EBV infection (Menegazzi et al, 1999).

C-myc gene anomalies were detected in a somewhatgreater percentage of cases (8/32, 25%) than observed byTsang et al (1996). Six cases showed mutations or smallrearrangements at the 30 end of the ®rst exon, reminiscent ofthose observed in the translocation 8;14 of the endemicBurkitt's lymphomas or in its variants t(2;8) and t(8;22).Two additional cases showed a major rearrangement ofc-myc gene, with truncation within its ®rst intron, similar tothose observed in sporadic Burkitt's and acquired immuno-de®ciency-associated lymphomas. In 9% of cases (3/32) wealso detected deviations from the published sequence of thec-myc P2 promoter (Battey et al, 1983). In particular, twocases showed a heterozygotic germline G to A transition atÿ106, abolishing the XhoI restriction site. This XhoIpolymorphism lies a few bases upstream from a poorlycharacterized purine-rich element shown to be necessary for

optimal c-myc transcriptional initiation (Moberg et al, 1991,1992) and was found to have a frequency of 0´5% in normalsubjects in the present study. No speculation can be madeabout the signi®cance of this polymorphism as familyhistories were not available for these cases. One additionalcase showed a C to G transversion at �4, lying directlywithin the protein binding element for the transcriptionfactor YY-1 (TFII-I, USF) (Li et al, 1994) which interactsdirectly with the c-myc protein to inhibit transcriptionalinitiation from the P2 promoter (Roy et al, 1993).Unfortunately, normal DNA was not available from thispatient.

Consistent with the ®ndings of Tsang et al (1996), weobserved no alterations in the bcl-1, bcl-2 or N-ras genes.Similarly, rearrangement of the bcl-6 gene was found in onlyone case, af®rming that it is not a signi®cant pathogeneticevent in these tumours. Missense point mutations in the p53tumour suppressor gene were detected in a small proportionof cases (13%; 4/31) and showed excellent correlation withp53 nuclear accumulation as determined by immunohisto-chemistry. Taken together, our data give further support tothe concept that PMBL represents a distinct clinicopatholo-gical entity, whose molecular genetic pathway is apparentlyunrelated to that of diffuse large B-cell lymphoma or otherB cell malignancies which are characterized by bcl-6 andbcl-2 gene rearrangements. Likewise, the absence of N-rasmutations would seem to exclude the possibility that PMBLis a tumour corresponding to terminal steps of B-celldifferentiation (MoÈller et al, 1987b), as plasma cell disordersshow N-ras mutations in up to 30% of cases (Corradini et al,1993; Portier et al, 1992).

Despite the analysis of a signi®cant number of genes formolecular alterations commonly involved in other haemo-poietic malignancies, the identi®cation of crucial genesinvolved in the pathogenesis of PBML still awaits discovery.At present the most frequent molecular anomalies foundremain the alterations of the c-myc, p16INK4 and p53 genesin a reasonably modest 25%, 15% and 13% of cases,respectively. Thus, although these genes are involved in aproportion of cases of this disease, none by itself wouldappear to be stringently associated with PMBL pathogenesis.

ACKNOWLEDGMENTS

This study was supported by grants from the AssociazioneItaliana Ricerca sul Cancro (AIRC), Italy, the University ofVerona and the Ministero UniversitaÁ e Ricerca Scienti®cae Tecnologica (COFIN), Italy, and the NIH CA66229,CA14462 and CA76584 grants to G.I., U.S.A.

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Molecular features of primary mediastinal B-cell lymphoma: involvement of p16INK4A, p53 and c-myc