P53 mutations in hairy cell leukemia - Nature

Leukemia (2000) 14, 706–711  2000 Macmillan Publishers Ltd All rights reserved 0887-6924/00 $15.00 www.nature.com/leu

P53 mutations in hairy cell leukemia EA Ko¨nig1,2, WC Kusser2, C Day2, F Porzsolt4, BW Glickman2, G Messer4, M Schmid4, R de Chaˆtel1,3, ZL Marcsek1,3 and J Demeter3,4 1

Department of Molecular Genetics, Semmelweis University, School of Medicine, Budapest, Hungary; 2Centre for Environmental Health, University of Victoria, Victoria BC, Canada; 3First Department of Medicine, Semmelweis University Medical School, Hungary; and 4 Department of Internal Medicine III, University of Ulm, Germany

We have studied the frequency of p53 mutations in genomic DNA extracted from peripheral blood or the spleen of 61 patients with hairy cell leukemia using PCR-SSCP and automated cycle sequencing. We identified exon 5–8 mutations in 17 cases, corresponding to a frequency of 28%. In four cases, mutations were localized in exon 5; one patient with atypical HCL had a mutation in exon 6 at the 3⬘ boundary; five cases showed mutations in exon 7, while exon 8 was found to be mutated in seven cases. The mutations found could be divided into three major categories: structural (n = 9), inactivating (n = 6), and neutral (n = 2) mutations. None of the three transitions found occurred at CpG dinucleotides. The rate of p53 mutations found in this large cohort of HCL patients is unexpectedly high as in other non-Hodgkin lymphomas p53 mutations predict for poor treatment outcome. The character of the mutations we have found is entirely different from that described in other hematologic malignancies. Leukemia (2000) 14, 706–711. Keywords: p53; hairy cell leukemia; mutation

Introduction Hairy cell leukemia (HCL) is a chronic B cell lymphoproliferative disorder characterized by pancytopenia, splenomegaly and the absence of lymphadenopathy. Leukemic blood picture is rare, occurring in about 8 to 10% of all cases. HCL is unique among the low-grade lymphomas not only because of its peculiar biological properties, but also because treatment responses differ basically from all the other non-Hodgkins lymphomas. In the last two decades, two different classes of drugs have been shown to be effective in its treatment: interferon-␣ (IFN-␣) results in partial remission of the disease in the majority of cases while pentostatin (2⬘-deoxycoformycin, 2⬘-DCF) or 2-chlorodeoxyadenosine (2-CdA) treatment often result in lasting complete remission.1–3 The presence of minimal residual disease (MRD) in patients treated with either DCF or 2-CdA is associated with an increased risk of relapse.4 Furthermore, it has been claimed that IFN-␣ might play a role in the development of second malignancies following HCL.5 p53, the product of the gene TP53 plays a key role in cell cycle arrest in the G1 phase, and thus behaves as a tumor suppressor. The TP53 gene has been found to be one of the most frequently mutated genes in human cancers. Mutations within this gene occur mainly between exons 5 and 8.6 Overall, the prevalence of p53 mutations in hematological malignancies appears to be moderate compared to that seen in solid tumors.7 Despite the large number of investigations on p53 in malignant diseases, in HCL only p53 allele deletion has been stud-

Correspondence: J Demeter, First Department of Medicine, Semmelweis University Medical School, 1083 Budapest Kora´nyi S u 2/a, Hungary; Fax: 36 1 2100279 Received 28 June 1999; accepted 7 December 1999

ied, while the frequency of p53 mutations in this disease is unknown. HCL has been poorly characterized at the molecular level. The expression of the c-src proto-oncogene has been shown to be increased in spleen samples from five patients with HCL.8 The PRAD-1/CCND1 gene was found to be overexpressed at mRNA and protein levels in a high number of hairy cell leukemias, however the levels of expression were much lower than in mantle cell lymphomas.9 It is well known, however, that both light and heavy chain areas of the immunoglobulin loci are rearranged, with evidence of partial, but atypical, class-switching, though without any evidence for enhanced somatic mutations. In terms of malignant cell ontogeny the leukemic cells in HCL have differentiated beyond the level seen in CLL and prolymphocytic leukemia (PLL), in which class switching has not occurred. However, the lack of somatic mutations and lack of formation of immunoglobulin for secretion place the HC at an earlier stage of B cell development than the myeloma cell.1 We have studied the frequency of p53 mutations after extraction of genomic DNA from the peripheral blood or the spleen of 61 patients with HCL. We have identified mutations in exons 5–8 in 17 cases, corresponding to a frequency of 28%. In other hematologic malignancies such as CLL, mantle cell lymphoma, PLL and diffuse large cell lymphoma, the presence of p53 mutations predicts for poor treatment outcome,10–14 thus the high rate of p53 mutations found in HCL, an indolent NHL with a good response to several therapeutic options is surprising. Materials and methods

Patients Sixty out of 67 patients with HCL and one patient with HCL-variant (total n = 61) from the Department of Hematology/Oncology, University of Ulm, for whom detailed clinical data were available, were selected for this study. Criteria for the diagnosis of HCL included the characteristic clinical features, presence of hairy cells in the peripheral blood smear, bone marrow histology, tartrate resistant acid phosphatase positivity, as well as characteristic histological picture of the spleen in the case where a splenectomy has been performed. Hairy cell leukemia variant was characterized with a very high number of tartrate resistant acid phophatase negative ‘hairy’ cells in the peripheral blood. The majority of genomic DNA was extracted from freshly collected peripheral blood, though several samples were extracted from PBMNC or spleen cells stored in liquid nitrogen or at −80°C. DNA was extracted from the peripheral blood as well from the removed spleen of one patient. In three further cases DNA was extracted from the removed spleen only. The number of circulating hairy cells was less than 5% in 41 patients, between 5

p53 and hairy cell leukemia EA Ko¨nig et al

In addition to the above-mentioned DNA samples we have also used control DNA from the peripheral blood randomly collected from seven healthy blood donors. The following cell lines were also used as mutant and wild-type controls: retinoblastoma cell line Y79, ATCC HTP 18 with exons 5 to 9 wild type, and a RFLP at codon 72, exon 4,16 colo 320 DM, ATCC CCL220, with a point mutation in exon 7, codon 24816 and a tumor sample with a Taq I polymorphism in exon 6 of p53.17,18 DNA from homogenized tissue or washed cell suspensions was extracted by standard proteinase K digestion and phenol extraction followed by agarose gel electrophoresis and OD 260/280 measurements for quality control. The mutant and wild-type controls were included in the SSCP analysis performed in the course of this study to ascertain optimal electrophoresis running conditions for the separation of mutant and wild-type bands.

Polymerase chain reaction (PCR) was performed in a Perkin-Elmer Cetus 9600 series thermocycler (Perkin-Elmer, Norwalk, CT, USA). dNTPs were used at 1 mM final concentration (Pharmacia, Bromma, Sweden), oligonucleotide primers of Dalton Chemicals (North York, Ontario, Canada) as described previously.15 Oligonucleotide primer sequences used in the performance of the polymerase chain reaction (PCR) were as follows: 5F(13009) CACTT GTGCC CTGAC TTTCA, 5R(13246) CTGCT CACCA TCGCT ATCTG; 6F (13276) GCCCA GGGTC CCCAG GCCTC, 6R (13476) CCGGA GGGCC ACTGA CAACC; 7F (13941) CTTGC CACAG GTCTC CCCAA, 7R(14158) AGGGG TCAGC GGCAA GCAGA; 8F (14346) AAAGG ACAAG GGTGG TTGGG, 8R (14614) CTGCA CCCTT GGTCT CCTCC. One Upper sample of Taq DNA polymerase and PCR buffer supplied by the manufacturer (Perkin-Elmer AmpliTaq) supplemented with 3 mm MgCl2 and 150–250 ng of genomic DNA were used in 50 ␮l reaction volumes. Amplification program: denaturation at 95°C for 3 min, 35 cycles of 92°C 35 s, 61°C 45 s, 72°C 45 s, finished by 72°C for 5 min and hold at 4°C. The PCR fragment size for p53 exon 5 was 237 bp, for exon 6 was 200 bp, for exon 7 was 217 bp and for exon 8 was 268 bp.

Table 1

Case/Sex

707

Control samples and cell lines

and 25% in seven cases and above 25% in nine patients. At the time of DNA sampling several patients had already been treated, but among the samples with p53 point mutations, only six patients had received specific anti-neoplastic treatment: four had been treated with recombinant IFN-␣ and two with 2-chlorodeoxyadenosine. One of the patients (No. 14) was found to have renal cell carcinoma 6 months following treatment with 2-CdA for HCL in September 1993. None of the other patients were found to develop a second malignancy. Clinical information on the 16 HCL patients and the HCL-variant case with p53 mutations is given in Table 1.

Single-strand conformational polymorphism (SSCP) analysis SSCP was performed according to Hongyo et al19 and Sugano et al20 with the following modifications: 10 ␮l PCR samples have been denaturated by adding 10 ␮l formamide containing buffer (0.05% saturated xylene cyanol–bromphenol blue, 20 mm EDTA, 0.5% SDS in formamide) at 96°C for 5 min and placed on ice until loaded to 10% acrylamide–bisacrylamide gels (29:1, BioRad Laboratories, Hercules, CA, USA) in TBE. All 20 ␮l of samples were loaded to the gel, run at 200 V at 4°C for 2 h. The gels were silver stained according to Switzer et al.21 Shortly after, the gels were removed and fixed in 50% ethanol:10% acetic acid for 15 min and subsequently washed three times in dH20 for 10 min each, cross-linked in 10% glutardialdehyde for 25 min, washed under continuous agitation and silver stained (in 140 ml dH20, 3.1 ml 1 m NaOH, 2.1 ml 25% NH4OH and 6 ml 1.14 m AgNO3). Gels were

Clinical and laboratory features of the 16 HCL patients and the patient with HCL-variant (case No. 5) with p53 mutations

Age at diagnosis

WBC (×109/l)

1/M

60

9.5

2/F 3/M 4/M 5/M 6/M 7/F 8/F 9/M 10/M 11/M 12/M 13/M 14/M 15/M 16/F

68 73 38 57 50 53 50 36 68 63 41 39 53 56 39

23.8 1.9 1.9 100.0 2.5 12.9 3.1 2.1 3.1 3.6 2.9 2.8 1.4 5.6 3.1

17/M

71

2.1

% hairy cells in the peripheral blood

Time of sampling related to treatment status

Survival (months) from diagnosis

35.0

Pretreatment (purified T cells) Pretreatment Pretreatment After 2CDA Pretreatment During IFN-␣ Pretreatment Pretreatment Pretreatment Pretreatment During IFN-␣ After IFN-␣ After IFN-␣ After 2CDA Pretreatment After splenectomy Pretreatment

83

58.0 2.0 0 99.0 13.0 52.0 1.0 14.0 8.0 2.0 1.0 No data 4.0 6.0 1.0 0

23+ 22 88+ No data 30 2+ 128+ 115+ 170+ 204+ 69+ 166+ 59+ 45+ 85 102+

Leukemia


708

‘developed’ in 0.24 m citric acid and 0.06% formaldehyde for approximately 5 min and stopped in 1% acetic acid. The gels were plastic wrapped in plastic foil and stored at room temperature. Mutants found in the first round of analysis were confirmed by repeating the PCR-SSCP analysis followed by DNA sequencing. ssDNA purification of mutant candidate bands showing differential migration through SSCP analysis were purified using the Wizard PCR Preps DNA Purification System (Promega, Madison, WI, USA) according to the kit instructions and the supernatants were used as templates to reamplify the mutant strands by PCR.

Automated cycle sequencing (ALF cycle sequencing) The assay included 1 ␮l of 10× sequencing buffer (10 mm TrisHCl (pH 8.5), 20 mm KCI, 3 mm MgCl2), 4 ␮l appropriate termination mix (Pharmacia-LKB Biotechnology AB, Bromma, Sweden), 0.25 ␮l fluorescent labeled forward or reverse oligonucleotide primer (at OD 0.5), 0.25 ␮l Taq polymerase (AmpliTaq Perkin-Elmer), 1 ␮l template DNA and H2O to a final volume of 9 ␮l. ALF cycle sequencing program: denaturation: 94°C for 2 min; 25 cycles of 94°C/10 s, 46°C/20 s, 72°C/30 s, soak cycle: 72°C for 15 min and hold at 4°C. Fluorescently labeled forward and reverse oligonucleotide primers were used in the performance of ALF Cycle Sequencing as follows: 5F(13024) TTTCA ACTCT GTCTC CTTCC, 6R (13476) CCGGA GGGCC ACTGA CAACC, 7R(14158) AGGGG TCAGC GGCAA GCAGA, 8R (14614) CTGCA CCCTT GGTCT CCTCC: (forward primer was used for exon 5 and reverse primers were used for exons 6, 7 and 8; all primers were synthesized by Dalton Chemicals. Stop dye solution was added after completion of the ALF cycle sequencing reaction (Pharmacia LKB). The DNA sequence was read on a Pharmacia-LKB ALF DNA sequencer using a ReadyMixGel (ALF grade from Pharmacia Bio Tech). The sequence reading was performed on Lasergene (by DNASTAR). Multiple sequence alignments were performed to determine the final mutation results.

Results Mutations were detected in 17 out of 61 HCL patients (28%, with a total number of 29 mutations) but one of these patients had a variant form of HCL (Table 2). In four cases the mutations were localized in exon 5, the patient with HCLvariant had a mutation in exon 6 at the 3⬘ boundary, in five cases in exon 7 while in seven cases mutations were found in exon 8. The following types of mutations could be detected: five cases of missense mutations (including one case of deletion and four cases of insertions), a deletion leading to a frameshift. Among the base substitution mutations there were three transitions (two of them silent), three cases of simple transversion (one of which remained silent), and seven double transversions. As a result of these double transversions a polar amino acid (Cys) is exchanged by an apolar one (Phe) in codon 275, while, conversely, in codon 276 an apolar amino acid (Ala) is being exchanged to a polar one (Ser). Though the mutations found in codon 275 and 276 do not necessarily lead to an extended change in protein structure itself but because they are located in the DNA binding domain, in the absence of 275Cys, the DNA binding activity of p53 may Leukemia

decrease. Therefore, the transversion in codons 275 and 276 of the p53 gene might play a role in protein function. When determining the number of mutations found in the individual patients, six were found to have one mutation, 10 had two mutations and one patient (patient No. 1) had three different mutations. These mutations could be divided into three major categories: structural (n = 9), inactivating (n = 6) and neutral (n = 2) mutations. Out of the 41 patients with less than 5% circulating hairy cells, mutations were identified in 13 cases. Of interest, all the seven double tranversions occurred in this group of patients. In the group of patients with 5 to 25% hairy cells in their peripheral blood, one out of seven patients was found to carry a mutation. Two out of eight patients with more than 25% hairy cells were found to carry a mutation. Twelve out of 16 patients with p53 mutations are alive and well 2 to 204 (median, 95) months from diagnosis of typical HCL (Table 1). Among these 16 cases with p53 mutations patients Nos 1, 3, 4, 6, 8, 9–12, 16 and 17 (Table 1) had been treated with IFN-alpha. Three of these patients (Nos 3, 7 and 16) were resistant to the drug. Treatment of patient No. 6 was continued with pentostatin, but hemolysis precluded further treatment with this drug. A splenectomy was performed but the disease remained progressive and the patient died 30 months following diagnosis of HCL as a consequence of severe infection. In HCL patient No. 16, the disease was resistant to IFN-␣, pentostatine treatment resulted in a partial remission only; 2-CdA was not available at that time. The patient died 85 months following the diagnosis of HCL because of fungal infection. Patient No. 3 also died of fungal sepsis in 1988. Thus the cause of death in the three patients with p53 mutations and resistance to IFN-␣ was sepsis. On the other hand patient No. 1 (with three different p53 mutations in his pretreatment purified T cell sample) responded well to IFN-␣ treatment which he received for 3 years, his disease became non-symptomatic and stable in the course of the treatment. He died as a consequence of gastrointestinal bleeding. Discussion HCL is unique among the low-grade non-Hodgkin’s lymphomas also because of its relatively indolent clinical course. The disease responds well to splenectomy, treatment with IFN-alpha and purine-nucleoside analogues in that order with potential cure in patients treated with the latter class of drugs. Remission duration with 2-CdA extends beyond 8 years for patients responding to this agent.22,23 Although a blastic variant of HCL has been described, the progression of the disease to a more malignant form is extremely rare.24,25 In patients with progressive disease, death is usually the consequence of infection and not of disease transformation. Accordingly, we have expected the frequency of p53 mutations in HCL to be low. Contrary to our expectations we have found a relatively high frequency (28%) of p53 mutations in a large group of HCL patients. The likelihood of the introduction of this frequency of mutations by the Amplitaq is extremely low.26 The high frequency of p53 mutations found compares to the 10–15% frequency found in patients with chronic lymphocytic leukemia.27–29 B-PLL is a malignancy of B cells frozen at an even more mature stage of maturation, and in this leukemia the frequency of p53 mutations is the highest among all hematological malignancies, 53% (10 per 19 cases).30 This is


Table 2

709

Mutations in the p53 exons 5–8 in the 16 HCL patients and the patient with HCL-variant (case No. 5)

Patient No.

% hairy cells

Exon

Codon

Nucleotide

1 (separated T cells)

⬍5%

5

151

CCC-CCA

Pro-Pro

(Tranv)

156 185 177 186 184 187 241 238 240 234 252 235 240 241 275 276 275 276 275 276 275 276 275 276 275 276 275 276

CGC-ACG AGC-TGC CCC-CTC GAT-TGA GAT-GTA GGT-GTG TCC-CC TGT-TGC AGT-AGA TAC-TAT CTC-CT AAC-AacC AGT-AGA TCC-ACC TGT-TTT GCC-TCC TGT-TTT GCC-TCC TGT-TTT GCC-TCC TGT-TTT GCC-TCC TGT-TTT GCC-TCC TGT-TTT GCC-TCC TGT-TTT GCC-TCC

Arg-Thr (stop) Ser-Cys Pro-Leu Asp-Stop Asp-Val (stop) Gly-Val Ser-Pro (stop) Cys-Cys Ser-Arg Tyr-Tyr Leu-Leu Asn-Asn (stop) Ser-Arg Ser-Thr Cys-Phe Ala-Ser Cys-Phe Ala-Ser Cys-Phe Ala-Ser Cys-Phe Ala-Phe Cys-Ser Ala-Ser Cys-Phe Ala-Ser Cys-Phe Ala-Ser

(Ins) (Tranv) (Ts) (Ins) (Ins) (Ins) (Del) (Ts) (Transv) (Ts) (Del) (Ins) (Transv) (Transv) (Transv) (Transv) (Transv) (Transv) (Transv) (Transv) (Transv) (Transv) (Transv) (Transv) (Transv) (Transv) (Transv) (Transv)

2 3 4 5 6 7

58% 70% ⬍5% ⬎95% 13% 52%

8

⬍5%

9 10

⬍5% ⬍5%

11

⬍5%

12

⬍5%

13

⬍5%

14

⬍5%

15

⬍5%

16

⬍5%

17

⬍5%

6 7

8

in concordance with the very aggressive clinical course and general resistance to chemotherapy in PLL. In multiple myeloma, the frequency of p53 mutations is low (2 to 10%) and their presence is associated with advanced forms of the malignancy.31,32 Mutations of the p53 gene were studied using SSCP and direct sequencing of any suspicious PCR products. The sensitivity of SSCP at the DNA level is only about 70%, so that mutations that involve fewer than 20% leukemic cells may remain undetected by this method. As the majority of HCL samples in which mutations were found derived from patients with the aleukemic form of the disease, with less than 5% of hairy cells in the peripheral blood, we conclude that an inherited mutation may facilitate the loss of the second allele. Further studies are necessary to determine whether the mutations found destroy the structure of the p53 protein. The overwhelming majority (25 out of 28) of the mutations we have found in HCL occurred in the hot spots but not in codons 175, 248 or 273, which are characteristic of other hematological malignancies. Three out of 28 mutations (found in the same patient) were outside the mutational hot spots. These mutations were found in the DNA extracted from the purified T cells of a patient with the aleukemic form of the disease. Furthermore, in seven aleukemic cases double transversions were found in exon 8. This seems to be a new and very consistent finding, and represents an event that does not seem to occur frequently in any of the leukemias. As the majority of the p53 mutations (including all the double transversions in exon 8) occurred in the aleukemic cases as shown in Table 1, it seems to be necessary to look at the T cell interactions found in HCL. These findings may support our pre-

Amino-acid (type of mutation)

viously published hypothesis that a paracrine pathway plays a major role in the growth regulation of hairy cells.33 The existence of a T cell clone (beside the hairy cell clone) responsive to treatment can not be excluded. Of interest, T cells in blood and spleen of HCL patients showed clonal excess by the TCR-PCR and a very restricted and skewed T cell repertoire.34,35 The mechanism of the mutations in HCL differed basically from that found in other hematological malignancies: in our HCL samples none of the three transition mutations occurred at CpG dinucleotides. In contrast, in the majority of hematological malignancies the transitions usually occur at the CpG dinucleotides suggesting the involvement of DNA methylation induced cytosine deamination.7 HCL patients seem to have certain chromosome changes not found in the other B cell leukemias. Haglund and coworkers36 found chromosome 5 to be involved in clonal aberrations in 12 out of 30 (40%) of HCL patients, most commonly as trisomy, or pericentric inversions and interstitial deletions involving band 5q13. The same group suggested that 5q13.3 is likely to harbor a gene involved in the transformational event of HCL.37 Catovsky’s group showed clonal chromosome abnormalities in 12 out of 15 HCL patients, of which five had a 14q+. In that study neither chromosome 5, nor 17 were found to be involved.38 Recently they demonstrated p53 allele loss through monosomy and/or monoallelic deletion in 15/20 cases of typical HCL using FISH. Three of these patients had aggressive disease (with features of transformation or lymphadenopathy or resistant disease) but the proportion of p53 deletion was lower in these three cases.39 The loss of the p53 allele was found to have greater significance in the clinical Leukemia


710

outcome in patients with HCL-variant. Moreover, in two out of six HCL-variant cases abnormalities of chromosome 17 have been identified.40 In hematological malignancies point mutations of p53 are usually associated with deletion of the other p53 allele, through 17p deletion. We did not directly assess the loss of heterozygosity, but the clear sequencing results suggest that the other allele may well have been deleted. Though the rate of p53 mutations in our patients with HCL is unexpectedly high, p53 is certainly only one of the prognostically important factors in malignant lymphomas. In conclusion, the high rate of p53 mutations (28%) we have found in HCL is surprising as in other non-Hodgkin lymphomas p53 mutations predict for poor treatment outcome. Moreover, the character of these mutations is entirely different from that described in other hematologic malignancies. Prospective evaluation of uniformly treated patients will help to further assess the clinical significance of p53 mutations in HCL.

12

13

14

15

16

17

Acknowledgements Dr J Demeter is a scientific fellow of the Alexander von Humboldt Foundation, Bonn, Germany.

18

19

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