Proc. Natl. Acad. Sci. USA Vol. 88, pp. 5413-5417, June 1991 Medical Sciences p53 mutations in human lymphoid malignancies: Association with Burkitt lymphoma and chronic lymphocytic leukemia (tumor suppressor genes) GIANLUCA GAIDANO*, PAOLA BALLERINI*, JERRY Z. GONG*, GIORGIO INGHIRAMI*, ANTONINO NERI*t, ELIZABETH W. NEWCOMB*, IAN T. MAGRATH§, DANIEL M. KNOWLES*, AND RICCARDO DALLA-FAVERA* *Department of Pathology and Cancer Center, College of Physicians and Surgeons, Columbia University, New York, NY 10032; tDepartment of Pathology and Cancer Center, New York University School of Medicine, New York, NY 10032; tCentro Malattie del Sangue "G. Marcora," Ospedale Maggiore, 20122 Milan, Italy; and §Pediatric Branch, National Cancer Institute, Bethesda, MD 20892 Communicated by Michael Potter, February 25, 1991 (receivedfor review December 13, 1990)

ABSTRACT We have investigated the frequency of p53 alteration may contribute to the deregulated growth charac- mutations in B- and T-cell human lymphoid malancies, teristics ofcancer cells. First, several types ofhuman tumors including acute lymphoblastic leukemia, the major subtypes of display the monoallelic loss of variable portions of the short non-Hodgkin lymphoma, and chronic lymphocytic leukemia. arm of chromosome 17 (5). In the case of colon cancer, it has p53 exons 5-9 were studied by using genomic DNA from 197 been shown that the region 17p13.1, the site of the p53 locus primary tumors and 27 cell lines by single-strand conformation (8), is consistently lost (4). Second, consistent with Knud- polymorphism analysis and by direct sequencing of PCR- son's model of tumor suppressor genes (9), it was found that amplified fragments. Mutations were found associated with (i) the 17p13.1 loss was consistently associated with mutations Burkitt lymphoma (9/27 biopsies; 17/27 cell lines) and its of the residual p53 allele, which are thought to inactivate the leukemic counterpart L3-type B-cell acute lymphoblastic leu- remaining p53 function (4-6). Finally, functional evidence for kemia (5/9), both of which also carry activated c-myc onco- the tumor suppressor role of p53 was achieved by showing genes, and (ii) B-cell chronic lymphocytic leukemia (6/40) and, that normal p53 can inhibit the transformation of rodent cells in particular, its stage of progression known as Richter's and can revert the malignant human transformation (3/7). Mutations were not found at any signif- phenotype of carcinoma icant frequency in other types of non-Hodgkin lymphoma or cells in vitro (10-12). However, p53 may differ in some acute lymphoblastic leukemia. In many cases, only the mutated respects from the prototype of tumor suppressor genes, in allele was detectable, implying loss of the normal allele. These that at least some p53 mutant alleles can behave as dominant results suggest that (i) signicant differences in the frequency oncogenes by transforming target cells in vitro and causing of p53 mutations are present among subtypes of neoplasms tumorigenesis in transgenic mice even in the presence of the derived from the same tissue; (ii) p53 may play a role in tumor normal allele (13, 14). Regardless of the exact mechanism progression in B-cell chronic lymphocytic leukemia; (ii) the involved, p53 mutations and allelic losses can now be con- presence of both p53 loss/inactivation and c-myc oncogene sidered as a relatively frequent pathogenetic feature of sev- activation may be important in the pathogenesis of Burkitt eral types of human malignancies, including those of the lymphoma and its leukemic form L3-type B-cell acute lympho- colon, breast, lung, brain, and soft tissues (refs. 4-6; for blastic leukemia. review, see ref. 15). Several lines ofindirect evidence suggest that alterations of Neoplasia involving the lymphoid system is characterized by the p53 gene might be relevant to the development of lym- marked biological and clinical heterogeneity including malig- phoid neoplasia in humans, including the development of nancies as distinct as acute lymphoblastic leukemia (ALL), lymphomas in transgenic mice carrying mutant p53 alleles non-Hodgkin lymphoma (NHL), chronic lymphocytic leuke- (14), the presence of high levels of p53 expression, typical of mia (CLL), and multiple myeloma. The pathogenesis ofthese tumors harboring p53 mutations, in lymphoproliferative dis- malignancies is also heterogeneous, since distinct molecular orders (16), and the presence of p53 mutations in a few lesions have been found associated with specific subtypes of T-ALL cell lines tested (17). These results have prompted our lymphoid tumors (for review, see ref. 1). These lesions involve comprehensive analysis of the frequency of p53 mutations in "dominantly acting" oncogenes activated either by chromo- a large panel of human lymphoid malignancies, including the somal translocations, affecting c-myc in high-grade NHL and most common types of B- and T-cell tumors. The results affecting bcl-2 in low- and intermediate-grade NHL, or by suggest that p53 mutations are associated with Burkitt lym- point mutations, affecting N-ras in ALL and multiple myeloma phoma (BL), its leukemic counterpart L3-type B-ALL, and (1). However, recent evidence has implied a role for "tumor the late stages of B-CLL. suppressor genes" in tumorigenesis (reviewed in ref. 2), the disruption or loss of which is thought to relieve the cell from negative regulatory signals. While various studies (3-6) have MATERIALS AND METHODS shown that tumor suppressor genes-namely, Rb and p53- Pathologic Samples. Peripheral blood, bone marrow, or are the site of frequent lesions in multiple types of human lymph node samples from 197 patients were collected during tumors, their involvement in human lymphoid malignancies standard diagnostic procedures. For most cases, the fraction has not been comprehensively investigated. of malignant cells in the pathologic specimen was at least The p53 gene (7) encodes a 53-kDa nuclear phosphoprotein 60%, and for all cases it was at least 30%, as determined by that may be involved in the negative regulation ofcell growth. cytofluorometric analysis of cell-surface markers, antigen- Several lines of evidence support the notion that its loss or receptor gene rearrangement analysis, or both (18). DNA was

The publication costs of this article were defrayed in part by page charge Abbreviations: ALL, acute lymphoblastic leukemia; NHL, non- payment. This article must therefore be hereby marked "advertisement" Hodgkin lymphoma; CLL, chronic lymphocytic leukemia; BL, Bur- in accordance with 18 U.S.C. §1734 solely to indicate this fact. kitt lymphoma; SSCP, single strand conformation polymorphism. 5413 Downloaded by guest on September 25, 2021 5414 Medical Sciences: Gaidano et al. Proc. Natl. Acad. Sci. USA 88 (1991) purified by digestion with proteinase K, extraction with Table 1. Frequency of p53 mutations in lymphoid malignancies phenol/chloroform, and precipitation by ethanol (19). Diagnosis Positive/tested Cell Lines. The following BL cell lines were used in the present study: CW678, MC116, KK125, JD38, As283A, B-cell tumors EW36, Ramos, ST486, PA682, PP984, CA46, NAB9B, EB3, ALL Daudi, Namalwa, Ag876, P3HR1, Mwika, BL2, BL37, BL47, Precursor B 2/25 BL104, BL84, BL99, BL49, BL60, and BL113 (20, 21). For B-ALL (L3) 5/9 four BL cases (GS 111, BL37, BL60, and BL113), DNA from NHL nontumor cells from the same patients was obtained from Small lymphocytic 0/9 three lymphoblastoid cell lines (IARC176A, IARC277, and Follicular* 0/20 IARC632, normal counterparts of BL37, BL60, and BL113, Diffuset 0/14 respectively) and a T-cell clone (normal counterpart of GS Burkitt 111). Biopsiest 9/27 Oligonucleotide Primers. All of the oligonucleotides used Cell lines§ 17/27 for PCR amplification in this study were synthesized by the B-CLL 6/40 solid-phase triester method (22). Names and sequences ofp53 Richter's transformation 3/7 primers derived from published sequences (23) are as follows: HCL 1/8 P5-5, 5'-TTCCTCTTCCTGCAGTACTC-3' (nucleotides PLL 0/2 911-930); P6-3, 5'-AGTTGCAAACCAGACCTCAG-3' (nu- T-cell tumors cleotides 1249-1268); P7-5, 5'-GTGTTGTCTCCTAGGT- T-ALL 0/7 TGGC-3' (nucleotides 1269-1288); P8-5, 5'-TATCCTGAG- T-CLL 0/9 TAGTGGTAATC-3' (nucleotides 1411-1430); P9-3, 5'- CTCL 0/8 AAGACTTAGTACCTGAAGGGT-3' (nucleotides 1654- PTCL 1/12 1674). In addition, the following primers were used: P5-3, HCL, hairy cell leukemia; PLL, prolymphocytic leukemia; CTCL, 5'-ACCCTGGGCAACCAGCCCTGT-3', derived from in- cutaneous T-cell lymphoma; PTCL, peripheral T-cell lymphoma. tron 5 sequence (spanning nucleotides 26-46 3' to the exon *Follicular small cleaved (13 cases) and follicular mixed (7 cases). 5/intron 5 boundary); P6-5, 5'-ACAGGGCTGGTTGC- tDiffuse mixed (5 cases) and diffuse large cell (9 cases). CCAGGGT-3', derived from intron 5 sequence (spanning tSporadic Burkitt lymphoma (13 cases) and endemic Burkitt lym- 26-46 to exon 5 phoma (14 cases). nucleotides 3' the 5/intron boundary); P7-3, §Sporadic Burkitt lymphoma (19 cell lines) and endemic Burkitt 5'-GTCAGAGGCAAGCAGAGGCT-3', derived from intron lymphoma (8 cell lines). 7 sequence (G.G., unpublished observation; spanning nucle- otides 46-65 3' to exon 7/intron 7 boundary); P8-3, 5'- equivocal diagnosis and a high percentage of malignant cells. AAGTGAATCTGAGGCATAAC-3', derived from intron 8 In addition, 27 BL cell lines and 4 germ-line control tissues, sequence (spanning nucleotides 45-64 3' to exon 8/intron 8 obtained from the corresponding tumor patients, were in- boundary); P9-5, 5'-GCAGTTATGCCTCAGATTCAC-3', cluded in this study. derived from intron 8 sequence (spanning nucleotides 42-62 A two-step experimental approach was devised. By SSCP 3' to exon 8/intron 8 boundary). analysis (24), all samples were analyzed for mutations within Single-Strand Conformation Polymorphism (SSCP) Analy- exons 5-9 of the p53 gene, which are the regions most sis. SSCP analysis was accomplished according to an adapted version of a previously reported method (24). Briefly, PCRs frequently affected by mutations in other types of human were performed with 100 ng of genomic DNA, 10 pmol of tumors (4-6). Under our experimental conditions, the SSCP each primer, 2.5 ,uM dNTPs, 1 ,Ci of [a-32P]dCTP (NEN; method is sensitive at the level ofat least 1% and it is specific, specific activity, 3000 Ci/mmol; 1 Ci = 37 GBq), 10 mM as evaluated by the 100% concordance between the results Tris HCI (pH 8.8), 50 mM KCI, 1 mM MgCl2, 0.01% gelatin, obtained by direct sequencing versus SSCP analysis of 69 0.5 unit of Taq polymerase (Cetus), in a final vol of 10 ,ud. distinct PCR-amplified fragments (50 negative/19 positive for Thirty cycles of denaturation (94°C), annealing (63°C for mutations) representative ofp53 exons 5-9 from BL cell lines reactions amplifying exons 5, 6, 7, and 9; 58°C for exon 8), (data not shown). Fragments displaying an altered electro- and extension (72°C) were done on an automated heat-block phoretic mobility by SSCP analysis were subsequently ream- (DNA thermal cycler; Perkin-Elmer/Cetus). The reaction plified in a separate reaction and analyzed by direct sequenc- mixture (2 ul) was diluted 1:25 in 0.1% NaDodSO4/10 mM ing to confirm and characterize the nature of the mutations. EDTA and further mixed 1:1 with a sequencing stop solution Frequency of p53 Mutations in Lymphoid Neoplasia. The (19) containing 20 mM NaOH. Samples were heated at 95°C results ofthe SSCP analysis are summarized in Table 1, while for S min, chilled on ice, and immediately loaded (3 ,ul) onto representative results are shown in Fig. 1. The overall a 6% acrylamide/TBE gel containing 10% (vol/vol) glycerol. frequency of p53 mutated samples among the primary lym- Gels were run at 8 W for 12-15 h at room temperature. phoid malignancies studied is 13.7% (27/197); however, Autoradiography was performed with an intensifying screen mutations do not follow a random distribution among the for 6-72 h. different subtypes of lymphoid tumors, but instead cluster Direct Sequencing of PCR Products. PCR was performed into two main groups: (i) BL, in which 9/27 (33%) biopsies with 500 ng ofgenomic DNA, 20 pmol ofeach primer, 200,uM and 17/27 (63%) cell lines are mutated, and its leukemic dNTPs, 10 mM Tris-HCl (pH 8.8), 50 mM KCl, 1 mM MgCl2, equivalent L3-type B-ALL, in which 5/9 (55%) cases are 0.01% gelatin, and 2.5 units of Taq polymerase. The number mutated; and (ii) B-CLL, in which 6/40 (15%) cases are and conditions of amplification cycles were as described mutated; interestingly, a higher percentage of mutations is above. Direct sequencing of the amplified product was per- observed in the B-CLL stage of progression known as formed as described (25). Richter's transformation (3/7; 42%). Occasional positive cases were found in hairy cell leukemia, precursor B-ALL, and peripheral T-cell lymphoma, while other types of malig- RESULTS nancies-e.g., low- and intermediate-grade NHL-were Experimental Plan. We selected 197 cases, representative found completely negative for p53 mutations. of the spectrum of human lymphoid malignancies (Table 1), Type and Position of p53 Mutations. The characteristics of from our collection of pathologic specimens based on un- the p53 mutations detected by SSCP analysis were further Downloaded by guest on September 25, 2021 Medical Sciences: Gaidano et al. Proc. Natl. Acad. Sci. USA 88 (1991) 5415

BDLBLITcells BL

~ a

N~~~~~~~~~~~~~~~~

..; B-CLL~~~~~~

0 41M*40k.'w -

EXON 6 -A .~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

BL

FIG. 1. SSCP analysis of p53 mutations in +' lymphoid malignancies. PCR-amplified frag- _owa" ments corresponding to individual exons 5-9 were amplified from genomic DNA in the pres- woo ence of [a-32P]dCTP, denatured by heat and al- a. kaline treatment, and run on a 6% acrylamide gel containing 10%1 glycerol. Representative samples mmM- iww are shown for exons 5-8. Although three main bands (corresponding to the residual undenatured B-CLL BL BL/LCL BL/LCCL BL fragment and the two . .a II single-stranded fragments) are expected in this assay for wild-type samples, N Jr @ NN-+-:c,>$ an additional band, probably representing a dif- ferent conformer of the fragment, was consis- .__ _ _~~~~~~~~~'r _ommom tently observed (see ref. 24). Samples were scored positive for mutations when bands differ- __._ _i ent from the normal control (N) were detectable.

- Om 4 - GS 111/GS 115 (exon 5) and BL60/IARC277 and EXON 8 101 0-4m ll ~6::as.000*-A m _i ,; PW 0 e' '* BL113/IARC632 (exon 8) represent pairs of neo- _. _mm plastic and normal tissue DNAs obtained from the same patient. LCL, lymphoblastoid cell line.

examined by direct sequencing of the PCR-amplified exons the mutation site was either not detectable or was present at (see Table 2 for summary and Fig. 2 for representative low intensity, presumably reflecting the loss of the normal results). In the vast majority of samples (41/44), p53 muta- allele (Fig. 2; see below). In two BL primary tumors (HO4024 tions were represented by single nucleotide changes, most and H03876) and two BL cell lines (P3HR1 and BL49), two frequently missense mutations (39 events), and occasionally...... mutations...... _ were observed at different codons, all in the nonsense mutations (4 events), point deletions (2 events), or presence of the wild-type band (Fig. 2); in two other cases point insertions (2 events). Mutations occurred more fre- (Ramos and Namalwa), one codon displayed two mutations. quently (48.8% of cases) at CpG dinucleotides, in the form of Whether these mutations reside on the same allele or on C to T or G to A transitions, although 12 transitions (mainly different chromosomes has not been assessed. G to A or A to G) not occurring at CpGs and 10 transversions Finally, it is noteworthy that almost one-third (4/13) of were also detected. The high frequency of mutations at CpGs cases displaying an abnormal SSCP migration pattern for is in accordance with previous data from various human exon 6 (i.e., 1.78% of all cases analyzed) contained a silent tumors (5), with the exception of lung carcinomas, where G mutation at codon 213 when assayed by direct sequencing, to T transversions are most frequently detected (15). Three suggesting the presence of a polymorphism. samples (RDF 378, L3-4, DK 1361) displayed gross rear- rangements, which could also be detected as a shift in the size DISCUSSION of the band following electrophoresis in 1.8% agarose gels (data not shown; see Table 2). Our data indicate that point mutations in the p53 gene do not The location of mutations affecting the p53 gene has been represent a general lesion in lymphoid neoplasia but, rather, reported to correspond to regions of the gene displaying high are specifically associated with certain subtypes of malig- homology among different species (26). Thirty-five point nancies such as BL and B-CLL. Previously available data mutations fell into these regions or their immediate bound- were limited to the analysis of a few T-ALL cell lines (17) in aries, while 12 were located at other sites, mainly between which mutations were detectable at a relatively high fre- codons 205 and 216. quency. This observation, however, was not confirmed by To confirm the assumption that p53 mutations in BL our analysis of primary T-ALL samples. There may be represent a somatic event, we analyzed the DNA from three multiple reasons for this discrepancy, but a higher frequency lymphoblastoid cell lines and one T-cell clone obtained from of mutations in cell lines versus primary samples is also four corresponding BL patients that had been shown to observed in this study for BL (63% vs. 33%). While it remains harbor p53 mutations. In each case, the mutation was de- possible that p53 mutations and deletions may frequently tected only in the tumor tissue, thus suggesting that it had occur during in vitro establishment or culture (10, 27), it is arisen during the neoplastic process (Fig. 1). also conceivable that those cases that carry p53 mutations/ In 24 cases (marked with asterisk in Table 2; 13/27 mutated deletions may be more suitable for in vitro establishment as biopsies; 11/17 mutated cell lines), the wild-type sequence at permanent cell lines. In general, these results suggest caution Downloaded by guest on September 25, 2021 5416 Medical Sciences: Gaidano et al. Proc. Natl. Acad Sci. USA 88 (1991) Table 2. p53 mutations in lymphoid malignancies has been restricted to the p53 sites most often implicated in Amino acidI human tumors. Evidence from studies on solid tumors suggest Sample Diagnosis ('-odon Mutation substitutiona that other regions could be mutated, albeit at lower frequency (28). Second, the SSCP method may not detect every possible DK 428 Precursor 273 TGT* B-ALL CGT Arg Cys mutation in a given DNA fragment, although in trial experi- L3-1 B-ALL L3 282 TGG* CGG Arg Trp ments performed on restricted panels of BL cases during this L3-2 B-ALL L3 175 CGC CAC His Arg study, there has been a perfect concordance between screen- L3-5 B-ALL L3 205 TAT TGT Tyr CGys L3-8 B-ALL L3 175 CGC CAC Arg His ing conducted independently by SSCP and PCR sequencing analysis. Overall, we believe that although the possibility ofan H03931 sBL 213 CGA TGA* EndI Arg underestimate of p53 mutations must be recognized, it is H04024 sBL 236 TAC TGC Tyr Cys 239 AAC AGC Asn Ser probably not of significant proportions. Several of indirect a GS 602 sBL 175 CGC CAC* Arg His lines evidence suggest pathogenetic GS 111 sBL 173 GTG -GGG ValW Gly role for the observed mutations. First, most mutations found IM 6 eBL 144 CAG CCG Gln Pro in this study (32/43 missense and nonsense mutations) in- H02218 eBL 133 ATG AAG* Met Lys volve codons that are found mutated in other tumors (4-6, H02209 eBL 175 CGC CAC* Arg His 15); among the remaining 11 mutations, 8 involve highly H03876 eBL 175 CGC CAC Arg His conserved domains of the p53 protein (26). Second, the vast 216 GTG GAG Val Glu majority of predicted p53 amino acid substitutions in lym- H02217 eBL 258 GAA AAA* Glu Lys phoid malignancies involve regions of the protein that are KK125 sBL cell line 248 CGG CAG* Arg Gln highly conserved through evolution (26) and/or correspond MC116 sBL cell line 238 TGT TAT* Cys Tyr to regions of the mouse p53 gene whose mutations result in JD38 sBL cell line 234 TAC TGC* Tyr Cys its oncogenic activation (10). Finally, the recent evidence (29) AS283A sBL cell line 206 TTG TAG* Frameshift of germ-line p53 mutations may raise the issue of the germ- Ramos sBL cell line 254 ATC GAC* Ile -* Asp line versus somatic nature of the observed mutations. For CW678 sBL cell line 175 CGC CAC* Arg His four cases, the absence of mutations in non-tumor DNA ST486 sBL cell line 158 CGC CAC Arg His could be demonstrated. In addition, no evidence of familiar PP984 sBL cell line 273 CGT TGT Arg Cys predisposition has been reported for most lymphoid malig- BL37 sBL cell line 237 ATG ATA* Met Ile nancies (30), suggesting that the observed p53 mutations BL99 sBL cell line 213 CGA TGA* Arg EndI represent somatic events occurring during tumorigenesis. BL49 sBL cell line 176 TGC TAC Cys Tyr The results presented here have implications for the role of 248 CGG TGG Arg Trp p53 as either a recessive or a dominant-negative oncogene. In P3HR1 eBL cell line 163 TAC CAC Tyr His a sizable proportion of cases only the mutated p53 allele was 287 GAG TAG Glu EndI found, while the normal allele has presumably been lost. EB3 eBL cell line 206 TTG TAG* Frameshift Although deletions of the short arm of chromosome 17 have Daudi eBL cell line 213 CGA TGA Arg EndI been described in lymphomas (31), BL and B-CLL in par- Namalwa eBL cell line 248 CGG CAG Arg GlnO ticular are not frequently associated with cytogenetic alter- 248 CGG TGGt Arg Trp ations of region 17p (32, 33); we conclude that, in these two BL60 eBL cell line 282 CGG TGG* Arg Trp types of lymphoid malignancies, the loss of the normal p53 BL113 eBL cell line 273 CGT TGT* Arg Cys allele may reflect cytogenetically undetectable events such as DK 1640 B-CLL 194 CTT CGGT Leu Arg small deletions within the 17p13 region or the loss of one DK 1631 B-CLL 248 CGG CGAG Arg Gln chromosome and the duplication of the remaining mutated DK 1617 B-CLL 205 TAT TGT Tyr Cys chromosome. In all these cases, however, it appears that DK 1198 B-CLL 152 CCG CCCG* Frameshift mutated p53 alleles act as typical recessive oncogenes. Con- (inserted a G) versely, the interpretation of those cases in which both a DK 885 B-CLL 232 ATC AGC* Ile -- Ser normal and a mutated p53 allele are detectable is more DK 981 B-CLL 275 TGT TTGT* Frameshift difficult. In these cases we could not distinguish whether this (inserted a T) finding reflects: (i) hemizygosity for the mutated allele, the DK 1317 Richter's 282 CGG TGG* Arg TrpI normal one being present only in contaminating normal cells; DK 1732 Richter's 237 ATG ATA* Met Ile or (ii) true p53 heterozygosity of the tumor cells, with the DK 2042 Richter's 281 GAC GAG Asp Glu mutated allele acting as a dominant-negative oncogene; or DK 490 PTLC 177 CCC CGC Pro Arg (iii) "first step" p53 heterozygosity (possibly with no phe- RDF 378 Precursor B-ALL A976-990* Frameshift notypic effect), which may be followed by "second step" loss L3-4 B-ALL L3 IExon S -15-bp deletiont ofthe normal allele, allowing for the recessively acting action DK 1361 HCL IExon 8 -25-bp of the mutated allele. Further studies combining the analysis insertiont of p53 mutation and loss of heterozygosity along the 17p PTLC, peripheral T-cell lymphoma; HCL, hairy cell leukemia; region in sequential tumor samples may help in addressing sBL, sporadic BL; eBL, endemic BL; bp, base pair. these issues. *Wild-type sequence at the mutation site could not be detected or The association between B-CLL and p53 mutation occurs was faintly visible. at low frequency, yet it is noteworthy in view of the paucity tThe observed mutations would lead to different amino acid substi- of information tutions if both mutations resided on the same allele. regarding the involvement of either dominant tThe PCR amplified fragment displayed an abnormal size on agarose or recessive oncogenes in this malignancy. On the other gel and the mutation was confirmed by PCR-SSCP technique; hand, a number of cytogenetic abnormalities, presumably further analysis by PCR direct sequencing could not be informative reflecting unknown oncogene activation events, have been because of the superimposition of the wild-type sequence. identified and shown to have prognostic significance in this disease (33). It may be interesting to note that, within the in extrapolating the frequency ofp53 mutations in vivo based general B-CLL category, p53 mutations appear to be more on data derived from cell lines. frequent in Richter's syndrome, which represents the evo- The results reported here may slightly underestimate the lution of B-CLL into a highly aggressive malignancy. Further frequency ofp53 mutations for two reasons. First, our analysis studies based on a larger number ofcases will clarify whether Downloaded by guest on September 25, 2021 Medical Sciences: Gaidano et al. Proc. Natl. Acad. Sci. USA 88 (1991) 5417

G A T C G A T C iC G A T C G A T C wA G A T C G A T C Cs "M , ~~T _ _ o~~~AG CODON N 4 T CODON 213 t j i G 239 1 CODON - T 248 __ = T C

CODON - - _ "I CODON asA _- CG 216 _ C \ G A N N KK 125 BL 47 G H03931 H03876 C HO2217 H04024 C

GJ A TC G A T C SC G A T C G A T C , T G A TC G A T C A

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N DK 1640 DK 1617 C 1104024 1102217 C DK 1198 RDF 407 C(

FIG. 2. p53 mutations detected by PCR-amplified fragments. Each mutation shown is matched to a control DNA. The codon at which the mutation occurs is indicated. Each sequence is shown 5' (bottom) to 3' (top). Coding strands are shown for cases H02217/H04024 (codons 236 and 239), DK 1198/RDF 407; noncoding strands are shown for cases KK125/BL47, H03931/H03876, DK 1640/DK 1617, H04024/H02217 (codon 258). Arrows point to bands corresponding to mutated base pairs. p53 mutations significantly correlate with B-CLL neoplastic 8. McBride, 0. W., Merry, D. & Givol, D. (1986) Proc. Natl. Acad. Sci. USA 83, 130-134. progression. 9. Knudson, A. G. (1985) Cancer Res. 45, 1437-1443. Among all types of lymphoid neoplasia, the most frequent 10. Finlay, C. A., Hinds, P. W. & Levine, A. J. (1989) Cell 57, 1083- targets for p53 mutations are BL and L3-type B-ALL. BL 1093. includes two pathogenetically distinct forms-namely, the 11. Eliyahu, D., Michalovitz, D., Eliyahu, S., Pinhasi-Kimhi, 0. & Oren, M. (1989) Proc. Natl. Acad. Sci. USA 86, 8763-8767. endemic African type characterized in virtually all cases by 12. Baker, S. J., Markowitz, S., Fearon, E. R., Willson, J. K. V. & Epstein-Barr virus infection and the sporadic American type Vogelstein, B. (1990) Science 249, 912-915. characterized by Epstein-Barr virus infection in only 309o 13. Halevy, O., Michalovitz, D. & Oren, M. (1990) Science 250, 113-116. of cases. The L3-type B-ALL leukemic cells are morpholog- 14. Lavigueur, A., Maltby, V., Mock, D., Rossant, J., Pawson, T. & ically similar to BL, suggesting that this tumor is a leukemic Bernstein, A. (1989) Mol. Cell. Biol. 9, 3982-3991. manifestation ofBL. A common denominator among all these 15. Iman, D. S. & Harris, C. C. (1991) Crit. Rev. Oncogen. 2, 161. diseases is the consistent presence of chromosomal translo- 16. Prokocimer, M., Shaklai, M., Ben Bassat, H., Wolf, D., Goldfinger, N. & Rotter, V. (1986) Blood 68, 113-118. cations leading to c-myc oncogene activation (reviewed in 17. Cheng, J. & Haas, M. (1990) Mol. Cell. Biol. 10, 5502-5509. ref. 1). Interestingly, p53 mutations have also been found at 18. Knowles, D. M., Pelicci, P. G. & Dalla-Favera, R. (1986) Hum. high frequency in those cases ofAIDS-associated lymphoma Pathol. 17, 546-551. that an activated c-myc oncogene 19. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989) Molecular carry (P.B., G.G., G.I., Cloning: A Laboratory Manual (Cold Spring Harbor Lab., Cold J.Z.G., G. Saglio, D.M.K., and R.D.-F., unpublished data). Spring Harbor, NY). Whether p53 mutation/loss and c-myc activation by chro- 20. Lenoir, G. M., Preud'homme, J. L., Bernheim, A. & Berger, R. mosomal translocation act synergistically in the pathogenesis (1982) Nature (London) 298, 474-476. of the Burkitt type phenotype remains to be determined. 21. Benjamin, D., Magrath, I. T., Maguire, R., Janus, C., Heather, D. T. & Parsons, R. G. (1982) J. Immunol. 129, 1336-1342. We are grateful to Michael Dean for advice for the SSCP protocol, 22. Seliger, J., Ballas, K., Merold, A., Kotschi, U., Lyons, J., Eisen- to Lionel Crawford and Peter Lamb for communicating unpublished beiss, F., Sinha, N. D. & Talwar, G. P. (1986) Chem. Scr. 26, 569-577. p53 sequences, to Giuseppe Saglio for contributing some DNA 23. Buchman, V. L., Chumakov, P. M., Ninkina, N. N., Samarina, samples, and to John Krolewski for critical reading of the manu- 0. P. & Georgiev, G. P. (1988) Gene 70, 245-252. script. This work was supported by National Institutes of Health 24. Orita, M., Suzuki, Y., Sekiya, T. & Hayashi, K. (1989) Genomics Grants CA44029 (to R.D.-F.) and EY06337 and CA40236 (to 5, 874-879. D.M.K.). G.G. is partially supported by the Gigi Ghirotti Founda- 25. Neri, A., Knowles, D. M., Greco, A., McCormick, F. & Dalla- tion. P.B. is supported by afellowship from the Associazione Italiana Favera, R. (1988) Proc. Natl. Acad. Sci. USA 85, 9268-9272. 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