From www.bloodjournal.org by guest on February 6, 2015. For personal use only. p53 Gene Mutation in B-Cell Chronic Lymphocytic Leukemia Is Associated With Drug Resistance and Is Independent of M D R l / M D R 3 Gene Expression By Soumaya El Rouby, Anju Thomas, Dan Costin, Carl R . Rosenberg, Milan Potmesil, Robert Silber, and Elizabeth W. Newcomb W e studied 5 3 patients with B-cell chronic lymphocytic leukemia (B-CLL) and found mutations of the p53 gene in 15%. Patients with p53 gene mutations were found to have an aggressive form of B-CLL disease characterized by advanced Rai stage, rapid lymphocytedoublingtime (LDT), and resistance to chemotherapy. While 27 of 29 treated patients (93%) without p53 mutations achieved a partial remission, only one of seven treated patients (14%)with p53 mutations achieved a partial remission (P = .00009). Adjusting for prognostic factors (age, sex, race, and Rai stage), patients with p53 gene mutations had a 13-fold greater risk of death than patients without p53 mutations (P = .013). In addition to examining the clinical relevance of p53 gene mutations in B-CLL, w e investigated the possible role of p53 gene regulation in the expression of the multidrug resistance genes M D R I and MDR3. W e quantitated MDRI and MDR3 mRNA expression by reverse transcription-polymerase chain reaction (RT-PCR). Expression of both the MDRI and MDR3 genes was independent of p53 gene mutation or prior drug treatment, and did not predict for clinical response. Our findings indicate that p53 gene mutations in B-CLL are associated with a poor clinical outcome and may be a prognostic indicator for drug resistance. 0 1993 by The American Society of Hematology. B mia (ANLL),l6-l9acute lymphoblastic leukemia (ALL),”,” chronic myelogenous leukemia (CML),22 multiple mye l ~ m a , ’and ~ non-Hodgkin’s lymphoma.24While the product of the MDR3 gene has been c h a r a c t e r i ~ e dthe , ~ ~function of its product in drug resistance is less well understood than that of the MDR 1 gene. The expression of both these genes has been analyzed in B-CLL patient^.^^.^' Although no link between MDRI expression and aggressive disease has been observed, a possible association between MDR3 overexpression and advanced Rai stages has been suggested.” In the present study, we investigated the role of p53 gene mutations in the regulation of MDR gene expression and its possible clinical relevance. We correlate the occurrence of p53 gene mutations with Rai stages, responsiveness to chemotherapy, and overall prognosis. In addition, we show that MDR gene overexpression in these patients is independent of p53 gene mutation and clinical response to chemotherapy. -CELL CHRONIC lymphocytic leukemia (B-CLL) is a disease characterized by the clonal expansion of CD5’ B cells. While chemotherapy can effectively control lymphocyte proliferation in most patients, there are few complete remissions. In addition, a small percentage of patients do not respond to any therapy. Prognostic factors that indicate shorter survival duration have been described. These include advanced clinical stage,’-3a diffuse pattern of bone marrow in~olvement,~ a lymphocyte doubling time (LDT) of less than 12 month^,^ and the presence of chromosomal abn~rmalities.~,~ Attention has recently focused on tumor-suppressor genes as possible prognostic indicators in human cancer.’-’’ Structural alterations and point mutations of the p53 tumor-suppressor gene have been demonstrated in 10% to 15% of B-CLL patients.”-’3 Wild-type p53 protein has been shown to repress the activity of the human MDRl gene promoter in vitro, while the mutant p53 protein hasa stimulatory effect on this a ~ t i v i t y .Increased ’~ expression of the MDR 1 gene is considered a common mechanism for drug resistan~e.’~ Overexpression of the MDR 1 gene product, P- 170, has been associated with drug resistance in hematologic malignancies, including acute nonlymphocytic leuke- From the Departments of Pathology, Medicine, Radiology, and Environmental Medicine, New York University School of Medicine and Kaplan Comprehensive Cancer Center, New York, NY. Submitted April 26, 1993; accepted August 12, 1993. Supported in part by National Cancer Institute Grants No. CA535 72, CA-50529, and CA-54484, and by the Marcia Slater Society for Research in Leukemia and the Harry and Gussie Wallerstein Foundation. D.C. is a Fellow of the American Cancer Society. Address reprint requests to Elizabeth W. Newcomb, PhD, Department of Pathology MSB531, New York University Medical Center and Kaplan Comprehensive Cancer Center, 550 First Ave, New York, NY 10016. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C.section 1734 solely to indicate this fact. 0 I993 by The American Society of Hematology. 0006-4971/93/82l0-0028$3.00/0 3452 MATERIALS AND METHODS Patient population. Fifty-three patients with B-CLL, seen at New York University Medical Center, were studied. The diagnosis of B-CLL required the demonstration of at least 10 X 109/Lmonoclonal B lymphocytes positive for CD5, CD19, and CD20. The disease was staged according to Rai et al.’ Some of the clinical characteristics of the patient population, including LDT, are listed in Table 1. Indications for treatment included symptomatic adenopathy, hepatomegaly or splenomegaly, or disease progression to Rai stage 111 or IV. Most patients were treated either with chlorambucil or with chlorambucil and prednisone. Nine patients received cyclophosphamide, five patients fludarabine, two patients 2‘-deoxycoformycin, and one patient an Adriamycin-containing regimen (Adria Labs, Columbus, OH). Several patients were treated with multiple drug regimens. A complete response to treatment was defined by a lymphocyte count of less than 4 X 109/L, a granulocyte count greater than 1.5 X 109/L, a platelet count greater than 100 X IO9, and a normal bone marrow examination.” A partial response was defined by a decrease of at least 50%in the diameter of the enlarged lymph nodes and a decrease of the lymphocyte count by 75%.30,31 Enrichment of B lymphocytes and DNA extraction. After obtaining informed consent, samples of heparinized blood were obtained from B-CLL patients or normal healthy volunteers. MononuBlood, Vol82, No 11 (December 1). 1993: pp 3452-3459 From www.bloodjournal.org by guest on February 6, 2015. For personal use only. p53 MUTATION AND DRUG RESISTANCE IN B-CLL 3453 Table 1. Summarv of Clinical Parameters of Age at Diagnosis (vr) LDT (mo) Patients with p53 mutations 1 2 3 4 5 6 7 8 75 49 76 55 76 75 67 67 36 3 18 6 7 2 <1 12 Patients without p53 mutations 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 36 64 59 68 65 69 56 80 56 65 79 58 57 53 60 60 55 36 65 60 55 70 62 47 65 60 60 58 41 49 61 74 73 65 74 70 37 54 76 42 76 68 77 48 54 6 8 20 Patient No 20 34 NA 18 20 30 14 12 NA NA 24 24 NA NA 36 36 NA 37 6 NA 15 12 9 NA 24 NA 18 >38 >42 >92 >18 41 >38 >80 >26 >23 48 >28 >85 >79 NA >92 53 B-CLL Patients Rai Stage Therapy Response II II 111 I1 111 II IV II NT 247 112 103 124 28 NT 109 CP, c FLU, CHOP, CAE, SPX CP, C E CP, FLU CP CP CP, CVP, FLU, M, E, dCF None No No No No No Yes No II 34 8 NT 19 NT NT NT NT 15 NT 66 NT NT 13 NT NT NT 41 11 NT NT NT NT 23 NT 38 NT NT NT 23 36 24 NT 66 33 110 46 13 23 34 123 NT NT NT 36 CLB, C CP, CLB CP CP CP, SPX CP, CLB, FLU CP CLB CLB CLB, CP CLB CLB, C CLB CP CLB CLB CP, c P, c CLB CLB CP CP FLU CP CLB, CP CP, CLB, C, C E, C CLB CL8 dCF P CLB c, CVP None None None None None None None None None None None None None None None None Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No 111 I I II Ill I1 II I I I II I I II Ill II I II II I I I I IV II I 1 I II II 0 I I 0 I 0 I 0 I II 0 I 0 I I + + + + + +P - Abbreviations: NA. not assessable; IC50(CLB),concentration of CLB in pmol/L that inhibits viability of 50% of the lymphocytes in vitro; C, cytoxan (Bristol-Myers. Evansville. IN); CAE, Cytoxan Adriamycin etoposide-16; CLB, chlorambucil; CP, chlorambucil + prednisone; CVP, cytoxan vincristine + prednisone; CHOP, Cytoxan + Adriamycin vincristine + prednisone; FLU, fludarabine; dCF, 2’-deoxycoformycin; M, mitoxantrone; E, etoposide; C P, Cytoxan + prednisone; P, prednisone; SPX, splenectomy; NT, not tested before initial chemotherapy. + + + + + From www.bloodjournal.org by guest on February 6, 2015. For personal use only. EL ROUBY ET AL 3454 clear cells were isolated by centrifugation on a Ficoll-Hypaque gradient. B lymphocytes were further enriched by rosetting T cells with sheep red blood cells (SRBC rosetting) (Crane Labs, NY) as described.” The final preparation from B-CLL patients contained greater than 90% B lymphocytes. B lymphocytes or monocytes from normal healthy donors were further purified by sorting using fluorescein isothiocyanate (F1TC)-labeled monoclonal antibody to CD19 (clone 89B, IgGlK; Coulter, FL) and phycoerythrin-labeled mAb to CD14 (clone Cris-6, IgGI; Olympus Corp, NY), respectively. Cells were incubated for 30 minutes with the appropriate dilution of the mAbs in ice-cold phosphate-buffered saline (PBS) containing 1% bovine serum and 1% human AB serum (GIBCO, BRL, NY). Cells were washed, resuspended in PBS, and then applied to an Ortho Cytofluorograph equipped with an argon ion laser (Ortho Instruments, MA). Sorted cells were collected into ice cold RPMI with 20% fetal calf serum (FCS). High-molecular weight DNA was isolated by digestion with proteinase K (Boehringer, Indianapolis, IN), extraction with phenol/chloroform, and ethanol pre~ipitation.~’ In vitro chemosensitivity assay. B lymphocytes obtained from patients before initial therapy were cultured on microtiter plates (2.5 X lo6cells/well) in 180 pL of RPMI 1640 plus 10%FCS in the presence ofchlorambucil or without the drug for 72 hours followed by addition of 3-(43 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) as previously de~cribed.’~.’~ The IC,, is defined as that concentration ofthe drug (pmol/L) that inhibits viability by 50%.” Oligonucleotide primers. Primers used to amplify exons 4 through 9 of the p53 gene have been previously de~cribed.’~ Amplimers used for amplification of MDRl and 0-actin-specific sequences have also been rep~rted.”.’~MDR3-specific sequences were amplified using the following primers: sense strand, GCTTCAGGAATTGTTGA (residues 2602 to 262 I), and antisense strand, TCGAAAACAACCGGCATAGG (residues 285 1 to 2870). These primers generate a 268-kb PCR product. All oligonucleotide primers were either synthesized with an Applied Biosystems Synthesizer (model 380A) or purchased from OPERON (Technologies Inc, CA). Detection ofp53 mutations. Mutations of p53 in B-CLL patients were detected by single-strand conformation polymorphism (SSCP) analysis of PCR products. Primer pairs for exons 4 through 9 were used to amplify p53 coding sequence, using 100 ng of genomic DNA isolated from the lymphocytes of each patient. The PCR mixture, the amplification conditions, and direct sequencing of PCR products performed with the sequencing kit (United States Biochemical) were described p r e v i o ~ s l yWhen . ~ ~ screening for mutations in the SSCP assay and DNA sequencing, genomic DNA from samples containing known wild-type and mutant p53 alleles were amplified by PCR and run in parallel as controls. Detection of gene expression by reverse transcription and PCR. Total cellular RNA was prepared by guanidine isothiocyanate e~traction.’~ Poly A mRNA was purified by affinity adsorbtion to oligo-(dT)-cellulose using the Fast Track mRNA isolation kit (In Vitrogen, CA). Complementary DNA (cDNA) was prepared by reverse transcription (RT) of mRNA (0.5 pg) from purified B lymphocytes or cell lines using 50 ng of random hexadeoxynucleotide primer and 200 U of MO MULV reverse transcriptase (Superscript RNase H-Reverse transcriptase; BRL) under conditions recommended by the supplier. cDNA aliquots equivalent to 0.05 pg @-actin) and 0.25 pg (MDRI and MDR3) RNA were used for enzymatic amplification by PCR using 2 U of Amplitaq polymerase (Perkin-Elmer/Cetus, Nonvalk, CT). PCR was performed in 50 pL containing I pmol/L of specific primers. Twenty-five cycles were + performed: each cycle included I minute ofdenaturation at 94”C, I minute of primer annealing (55”C, MDRI; 57”C, MDR3; 60°C, 0-actin) and 2 minutes of extension at 72°C. PCR products (20 pL/reaction) were analyzed by electrophoresis on 1.2% agarose gel and visualized after ethidium bromide staining. Gels were processed for Southern blotting. The membranes containing the DNA fragments were hybridized with specific oligonucleotides end-labeled with [y3’P]-ATP,’’ washed, and exposed to x-ray film at -70°C for 2 hours: MDR I , ACTAGAAGGTGCTGGGAAG; MDR3, GGACAGTTGTGTCTTTGACC; 0-actin, GGAGTCCTGTGGCATCCAC. The hybridization signals were quantified by densitometric scanning of the autoradiographs (LKB Ultrascan XL Laser Densitometer). The human leukemia cell line CCRFCEM and its vinblastine-resistant derivative CEM/VLB 100 served as negative and positive controls, respectively, for MDRl gene expression.40The human liver cell line HepC2 served as a positive control for MDR3 gene expression4’ and the CEM/VBL100 cell line was used as the negative control. Statistical analysis. Comparison between patients by p53 status was assessed using the Mann-Whitney test (continuous variables) and Fisher’s exact test (nominal variable^).^^ Survival distributions for the two groups of patients were compared using the Kaplan-Meier method. The difference between the two survival curves was assessed using Gehan’s generalized Wilcoxon test. To control for the effects of confounding variables, ie, prognostic factors that differed by p53 status, the data were subjected to the Cox proportional hazards model. Using this procedure, a time-weighted relative risk for death was estimated and was adjusted for pertinent confound er^.^' P values in all cases were two-tailed. All statistical calculations were performed using SAS procedure^.^^ RESULTS Identgfication ofp53 mutations. Mutations of p53 were detected in B lymphocytes from eight of 53 patients ( 1 5%) by SSCP analysis (Fig I). At the time of p53 gene analysis, six of these eight patients had been untreated. Mutations were confirmed by DNA sequencing (Fig 2) and the data are listed in Table 2. Six of the mutations were missense mutations, resulting in an amino acid substitution, and two ofthe mutations were nonsense mutations, resulting in premature termination of protein translation. The mutations were located in the highly conserved domains of the p53 gene. Three mutations occurred in exon 5 (37%), two in exon 6 (25%), and one each in exons 4, 7, and 8. Patient characteristics and clinical course. The 45 patients without p53 mutations had a median age at diagnosis of 62 years (range, 36 to 80) and the eight patients with mutations had a median age of 67 (range, 49 to 81) ( P = NS). There was no difference in gender between the two groups. As shown in Table I , no patient with a p53 gene mutation was in Rai stage 0 or I, compared with 57% of patients without p53 mutations ( P = .002). A LDT of less than 8 months was observed in five of eight patients (63%) with p53 mutations, compared with only 9% of the assessable patients without mutations ( P = .003). Of the 45 patients without p53 mutations, 29 (64%) required therapy, and 27 of these patients (93%) had a partial remission. In contrast, 7 of 8 patients (87%)with p53 mutations required therapy, but only one patient (14%) achieved a partial remission (P= .00009). A shorter survival duration From www.bloodjournal.org by guest on February 6, 2015. For personal use only. p53 MUTATION AND DRUG RESISTANCE IN B-CLL 3455 WILD-TYPE MUTATED 1 2 3 4 5 6 A C G T - m- G cC-l;i - EXON 4 A C G T G C C -M ~- c- EXON 5: Codon 152 1 ( 1 2 3 4 5 6 7 8 1 EXON 6:Codon 209 Fig 2. Representative DNA sequence analyses show p53 gene mutations detected by SSCP in 6-CLL patients. Mutation in codon 152 results from insertion of a C (GCC to GCCC); mutation in codon 209 (AGA to ACA) results from a single base change. EXON 5 I 1 2 3 4 5 6 7 8 EXON 8 was also observed in patients with p53 mutations (Fig 3). Six of eight patients (75%) with p53 mutations have died, compared with eight of45 patients ( 18%) without p53 mutations (relative risk, 4.66: P = .I5 by Gehan's generalized Wil- apy, the B lymphocytes from 28 patients (six with p53 mutations) were tested for drug resistance with the MTT chemosensitivity assay (Table I). Because IC,, values are significantly increased following chlorambucil treatment:' Patient No.' Fig 1. Detection of p53 gene mutations in 6-CLL patients by PCR-SSCP analysis. DNA from each patient's lymphocytes was amplified by PCR in the presence of w3*PdCTP for each of the corresponding exons 4 through 9 of the p53 gene. The radiolabeled DNA fragments are separated on a nondenaturinggel. Representative SSCP migration patterns of some samples are shown for exons 4, 5, and 8. DNA samples with a p53 mutation show a shift in electrophoretic mobility as compared with other DNA samples on the same gel: exon 4 (lane 6, patient no. 8). exon 5 (lane 7, patient no. 5). and exon 8 (lane 5, patient no. 1). 8 4 5 6 2 3 7 1 Exon Codon Mutation Amino Acid 4 5 5 5 6 6 7 8 47 168 152 177 209 209 241 286 CCG-ICG CAC-CSC Insert of C Pro to Ser His to Pro ccc-CIC Pro to Leu Arg to Lys Arg to Thr Ser to Phe Glu to stop AGA-AAA AGA-ACA TCC-EC GAA-IAA Six of eight patients did not receive chemotherapy before DNA analysis for p53 gene mutations. From www.bloodjournal.org by guest on February 6, 2015. For personal use only. EL ROUBY ET AL 3456 loo L I 0 I 40 I 80 I 120 I I I 160 200 240 Months After Diagnosis Fig 3. Kaplan-Meier survival curves after diagnosis of 6-CLL based on the presence or absence of p53 gene mutations. expression (Fig 4 and Table 3). There was no association between MDR 1 expression and the following: p53 gene mutations, prior drug treatment, clinical course of the disease, and Rai stages. MDR3 gene expression was sevenfold greater in the human liver cell line HepG2 relative to the negative cell line CEM/VBLIOO. A twofold to threefold increase in MDR3 expression was observed in sorted CDI 9' cells and no expression was observed in CD14' cells from normal healthy donors (Table 3). Of 16 patients analyzed for MDR3 gene expression (Fig 4 and Table 3), 12 expressed the MDR3 gene. Eight patients showed intermediate to high levels, four showed low expression (absorbance value less than sorted CD 19' cells), and four showed no expression of the MDR3 gene. Intermediate to high expression of the MDR3 gene was detected in 43% of patients with p53 mutations and 56% of patients without p53 mutations (Table 3). There was no association between prior drug treatment, Rai stage, clinical response to treatment, and MDR3 gene expression. In addition, there was no association between the levels of expression of MDR3 and MDRl in the same patient. DISCUSSION ICs0 data as a predictive indicator are presented only for those patients who were tested before therapy. The mean ICs0of chlorambucil was 3.4-fold greater when p5 3-positive lymphocytes were tested compared with p53-negative lymphocytes (P= .006). MDRl and MDRS gene expression. RNA samples obtained from B lymphocytes of 16 B-CLL patients were analyzed for coexpression of the MDRI, MDR3, and 0-actin genes using the RT-PCR method. The results of Southern blot analysis of gene expression for MDR I , MDR3, and 0-actin are shown in Fig 4. The oligonucleotides used to probe the Southern blots for either the MDRl or the MDR3 gene were specific and hybridized only with their respective cDNA products. Seven of the patients had p53 mutations (lanes a through g). Of the nine patients without p53 mutations (lanes h through p), six had received drug treatment (lanes h through m), while three other patients were without drug treatment (lanes n through p). mRNA from human cell lines specific for MDRl (CEM/VBLlOO) and MDR3 (HepG2) gene expression served as positive controls (lanes 1). mRNA from human cell lines with basal levels of MDR 1 (CCRF-CEM) and MDR3 (CEM/VBL100) expression served as negative controls (lanes 2). Measurement of P-actin gene expression was used as an internal control for RNA recovery from each patient. Densitometric quantitation of MDRl and MDR3 gene expression of B lymphocytes from B-CLL patients and sorted CD 19' and CD 14' cells from normal healthy donors are summarized in Table 3. The drug-resistant cell line CEM/VLB 100 expressed a 12-fold greater level ofthe MDR 1 gene relative to the parental drug-sensitive CCRF-CEM cell line. A twofold increase in MDRl expression was observed in sorted CD19+ cells and no expression was observed in sorted CD14+ cells from normal healthy donors (Table 3). All 16 patients analyzed for MDRl gene expression showed an intermediate to high Mutations in the p53 tumor suppressor gene were identified in eight of 53 B-CLL patients. It is less likely that chemotherapy selected out p53 mutant clones of B lymphocytes, since six of eight patients with p53 mutations were untreated at the time of DNA analysis. In the other two patients, DNA was not available before treatment. Patients with p53 gene mutations differed from those without mutations in several ways: they were more likely to be in Rai stage I1 or higher, to have a decreased LDT, to require chemotherapy, to show resistance to chlorambucil in vitro, to have a poor clinical response to therapy, and to have a shorter survival duration. In view ofthe report that p53 mutations may be involved in regulation ofthe MDRl gene,14 we examined if p53 mutations were associated with modulation of the genes involved in multidrug resistance in B-CLL patients. The B lymphocytes from all patients expressed high levels of the MDR I gene. MDR 1 gene overexpression was not dependent on the presence of p53 mutations, suggesting that MDRI overexpression in B-CLL lymphocytes is not regulated by the p53 gene product. Similarly, it has been recently suggested that MDR 1 in AML expression is not affected by the presence of p53 mutations.46 Overexpression of MDR 1 has been associated with resistance to chemotherapy in many m a l i g n a n ~ i e sDrugs . ~ ~ used for ANLL therapy, such as the anthracyclines, are susceptible to the action of the P-170 efflux pump. In untreated ANLL, 67% of patients with high MDRI levels fail to go into complete remission after one cycle of anthracyclinebased treatment, compared with 33% of ANLL patients with low MDR 1 expres~ion.'~ In a prospective study of 122 patients with untreated ANLL, 68% of P- 170-positive patients did not achieve complete remission compared with 19% of the P-170-negative patients.'* In our study, the absence of any association between MDRl expression and From www.bloodjournal.org by guest on February 6, 2015. For personal use only. . 3457 p53 MUTATION AND DRUG RESISTANCE IN B-CLL 1 2 a b c d e MDR1 m - f g h i j k l m n o p 0 - Fig 4. Southem blot analysisof gene expression by RT-PCR of RNA from E-CLL patients. The coexpression of MDRl ,MDRB. and &actin genes was analyzed in 1 6 B-CLL patients (lanes a-g, patients no. 1-7; lanes h-m, patients no. 9-1 4; lanes n-p, patients no. 38-40) whose clinical parameters are detailed in Table 1. mRNA from human cell lines CEM/VLBlOO and HepG2 sewed as positive controls (lane 1)for the expression of MDRl and MDR3 genes, respectively. mRNA from human cell lines with basal levels of M D R l (CCRF-CEM) and MDR3 (CEM/VBL100) sewed as negative controls (lane 2) for MDRl and MDR3 gene expression, respectively. PCR-amplified products were separated on agarose gels, Southern blotted and hybridized to 32P-labeledoligonucleotideprobes specific for each gene product. Films were exposed for 2 hours at 70°C. The signals on the autoradiographs were scanned by a laser densitometerto quantitate the relative levels of gene expression for each patient (see Table 3). The expression of @-actinsewed as an internal control for the recovery of RNA from each patient. - clinical response to therapy may be due to the fact that most of the drugs used to treat B-CLL are not affected by the MDR 1 gene product, P- 170. Our results show intermediate to high levels of MDRI gene expression in B lymphocytes from all 16 B-CLL patients tested. High expression of the MDRl gene in B lymphocytes from 90% to 100%of B-CLL patients has been shown in two other In contrast, we and ~ t h e r sfound ~ ~ . low ~ ~expression of MDR I in sorted C D 19' B lymphocytes from normal healthy donors. Similar results of low MDRl expression have been reported for enriched populations of normal B lymphocytes.26All ofthe studies to date show that MDRl expression is usually low in B lymphocytes obtained from normal donors compared with B lymphocytes obtained from B-CLL patients. Further studies on MDR I expression in CD5' B lymphocytes from normal donors and B-CLL patients are needed to understand this difference in the pattern of MDR I gene expression and its role in the disease. MDR3 is the second human multidrug resistance gene to be identified by its homology with the MDRl gene. A physiological role of MDR3 in the phenomenon of multidrug resistance is not yet established. In this study, we observed a low expression of MDR3 in normal sorted C D 19' cells and highly variable MDR3 gene expression in B-CLL patients. Previous studies have showed high levels of MDR3 expression in untreated patients with prolymphocytic leukemia," or in patients in advanced Rai stages 111 and IV?' Our study showed no association between the level of MDR3 expression and Rai stage, previous drug treatment or the presence of p53 gene mutations. Additional studies of MDR3 expression in a large group of B-CLL patients will be required to determine whether there is an association with any of the clinical parameters. This report demonstrates an association between p53 gene mutations and an aggressive form of B-CLL characterized by advanced Rai stage, rapid LDT, increased drug resistance as measured by a poor response to chemotherapy, and shortened survival duration. Mutations of the p53 gene have been associated with disease progression and decreased survival in many other human cancers, including colon adenocarcinoma,' breast cancer,' CML,'' multiple m y e l ~ m a , ~ ' transitional cell bladder cancer,52gastric cancer,53and lung ~ a n c e r . No ~ . ~other ~ studies to date have linked the presence of p53 mutations in B-CLL with decreased response to chemotherapy, in vitro drug resistance, and a shortened survival duration. Our results suggest that p53 mutations may become a marker for drug resistance in B-CLL. Since the From www.bloodjournal.org by guest on February 6, 2015. For personal use only. EL ROUBY ET AL 3458 Table 3. Summary of MDR Gene Expression MDR Gene Expression' Rai Stage Sample Controls CEM/VBL100 CEM HepG2 CD19+cells Donor 1 Donor 2 Donor 3 CD14+cells Donor 1 Donor 2 Patients with p53 mutations 1 2 3 4 5 6 7 Patients without p53 mutations 9 10 11 12 13 14 38 39 40 II II 111 II Ill II IV II Ill I I II Ill II 0 I Clinical Response MDRl MDR3 2.4 0.2 ND 0.2 0.4 0.5 0.1 0.5 0.1 0.1 0.0 0.0 No No No No No Yes No 1.4 1.4 2.0 1.7 1.9 1.8 2.0 0.4 0.0 0.3 0.1 1.4 1.o 1.o Yes Yes Yes Yes Yes Yes NT NT NT 1.7 1.2 1.3 1.6 0.0 0.1 0.5 1.5 1.5 1.3 0.6 2.1 0.8 2.0 2.0 2.0 2.2 2.4 0.5 1.5 0.6 0.0 Abbreviations: NT, not treated; ND, not determined. The values given are the difference between the minimum and maximum absorbance of each sample. overall frequency of p53 mutations is relatively low in BCLL, further studies in a larger population of patients will be needed to establish the reliability of a p53 mutation as a prognostic indicator. ACKNOWLEDGMENT The authors thank Laura Morse and John Drygas for their assistance in separation of B lymphocytes used in this study, Henry Cohen for graphical and statistical assistance, and Drs Joel Buxbaum and Riccardo Dalla-Favera for their critical reading of the manuscript. The human cell lines CCRF-CEM and CEM/VBL 100 were generously provided by Dr William T. Beck. We are grateful to Dr John Hint of the Cell Sorting Unit of the Kaplan Comprehensive Cancer Center for help with cell sorting. REFERENCES 1. Rai KR, Sawitsky A, Cronkite EP, Chanana AD, Levy RN, Pasternack BS: Clinical staging of chronic lymphocytic leukemia. Blood 46919, 1975 2. 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For personal use only. 1993 82: 3452-3459 p53 gene mutation in B-cell chronic lymphocytic leukemia is associated with drug resistance and is independent of MDR1/MDR3 gene expression S el Rouby, A Thomas, D Costin, CR Rosenberg, M Potmesil, R Silber and EW Newcomb Updated information and services can be found at: http://www.bloodjournal.org/content/82/11/3452.full.html Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. 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