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From www.bloodjournal.org by guest on June 17, 2017. For personal use only. REVIEWARTICLE p53 in Hematologic Malignancies By Jun Imamura, lsao Miyoshi, and H. Phillip Koeffler T HE p53 HAS BEEN CHOSEN as molecule of the year for 1993 by the journal Science.' The protein's illustrious history began quietly in 1979, when a 53-kD eukaryotic protein was shown to bind to SV40 large T The protein was named p53 becauseofitssize.Initial studies suggested that p53 was an oncogene because it could transform rodent ~ e l l s . ~The - ' ~p53 genes used in these studies hadbeenisolated from cancer cell lines;these p53 genes were subsequently discovered to containmissense mutations andthe resultant mutant proteins had propertiesdifferent from thoseofwild-type (wt) ~ 5 3 . ' . ' ~Investigators "~ also noted that several virally transformed murine cell lines as well as a human myeloid leukemic cell line (HL-60) had major deletions of the p53 gene.",'" We found that the p53 gene was mutated in human cancer; four of five osteosarcomas frompatients had major disruptionsof their p53 genes." Other studies foundthat loss of heterozygosity (LOH) of the short arm of human chromosome 17 in the region coding for p53 occurred in a number of human cancers.22-24 Additional careful analysis of the p53 gene has shownthat it is frequently mutated in more than 50 varieties of human cancers including lung, breast, thyroid, gastrointestinal, and ovarian cancers, lymphomas/leukemias,and brain tumors.2',2s-44 Furtherfunctional studies determinedthat wt-p53 suppressed transformation of cells's"sand overexpression of wt-p53 blocked cells in the G , phase of the cell cycle.J',46 Taken together, p53 fulfills the criteria of a tumor-suppressor gene, including the finding of LOH in the region of p53 in tumors, the presence of p53 mutations in human and murine tumors and transformed cell lines, and the ability of wt-p53 to suppress transformation of cells having p53 mutations. In one decade, a protein discovered through an effort to understand how SV40 transforms cells was initially characterized as an oncogene, only to find that it is a pivotal tumor-suppressor that is the guardian of DNA-damaged cells by halting their proliferation, pushing badly damaged cells into an apoptotic cell death and preventing unwanted DNA amplification. This reviewwill characterize the p53 abnormalities in hematopoietic malignancies and discuss the clinical significance of these alterations. In addition, potential therapeutic approaches will be briefly mentioned. DETECTING p53 ABNORMALITIES IN HUMAN CANCERS Southern blottinganalysis was the first methodused to detect p53 mutations in various cancers." However, this is a cumbersome technique that can onlydetect gross alterations. An indirect detection method is the analysis of LOH, taking advantage of DNA p~lymorphisms.'~ The paradigm for LOH is that one p53 allele develops a point mutation; then, through one of several genetic mechanisms, including recombination or duplication, the normal p53 allele is lost. Therefore, LOH in the region of a tumor-suppressor gene is analogous toa tombstone marking thelethally injured tumorsuppressor gene. Several very informative polymorphic sites are present in the region of the p53 gene.40,4x This indirect approach of identifying tumors with potential p53 mutations is flawed because LOH in the region of p53 gene can occur in the absence of a detectable p53 mutation, perhaps because another tumor-suppressor gene exists in the same chromosomal region." Also,thisanalysisrequires normal tissue from the same individual. Analysis of single-strandconformationalpolymorphism (SSCP) using polymerase chainreaction (PCR) allowsa relativelysimpledetection system for point mutations.4y~54 The PCR-SSCP technique can detect an abnormality in less than 10% DNA containing mutant p53 in a background of 90% DNA containing ~ t - p . 5 3 . ~ ~The ~ ' ' specificity of PCRSSCP ismore than 95% for 100- to 300-bp PCR fragments.5" Thus, this protocol is very useful to screen for mutations in a short regionof a gene. Those sampleswith abnormal SSCP require nucleotide sequencing toassert if the abnormal SSCP representsamutationthat eitheralters an amino acid or merely represents a polymorphism or silent mutation. A potential false-negative result can occur if the p53 mutation is located outside the area examined by SSCP. More than 90% of mutations of p53 probably occur in exons 5 through 8 andmostanalysishas focused onthis r e g i ~ n . ' ~ . ' " ~An ~-~' enormous problem is that the SSCP and sequencingtechniques are very time-consuming and require refined expertise. Less frequently used techniques, such as either denaturing gradient gel electrophoresis, RNase protection assay, and detection of basepair mismatches with hydroxylamine and osmium tetroxide, have their own inherit problems and usually are even more labor-intensive than PCR-SSCP.sx-62 Immunohistochemistry for p53 is the simplest analysis for p53 integrity, but this approach cannot directly detect p53 mutations. Because ofashorthalf-life of about 6 to 20 From the Cedars-Sinai Medical Center/UCLASchool of Medicine, minutes, wt-p53 usually does not accumulate in most normal Division of Hematology/Oncology, Los Angeles, CA; and the Dein amounts detectable by immunohistochemical partment of Internal Medicine, Kochi Medical School, Kochi, Japan. tissues Submitted March 16, 1994; accepted June 17, 1994. methods. However, most missense mutations of p53 prolong Supported by National Institutes of Health Grants No. CA42710, the half-life of the protein, permitting it to be immunohistoCA33936, and DK42792 as well as bythe Parker Hughes Leukemia chemically detectable in those tumors containing a p53 misFund and Concern Foundation. sense m ~ t a t i o n . ~ ~The . ~ ' technique ~~" is rapid and easily perAddress reprint requests to H. Phillip Koefler, MD, Chiej Hemaformed by many pathology laboratories. Nevertheless, tology/Oncology Division, Cedars-Sinai Medical CenterIUCLA quantitation is difficult and false-positives and -negatives School of Medicine, 8700 BeverlyBlvd, 8210, Los Angeles,CA can occur dependingon the tissue. False-negatives are partic90048. ularly the case if the p53 mutation results in either a prema0 1994 by The American Socier): of Hematology. ture stopcodon, frame-shift, or alteration of a splice site 0006-4971/94/8408-03$3.00/0 2412 Blood, Vol 84, No 8 (October 15), 1994: pp 2412-2421 From www.bloodjournal.org by guest on June 17, 2017. For personal use only. 2413 p53 IN HEMATOLOGICMALIGNANCIES Table 1. p53 Alterations in Hematopoietic Malignancies Disease Frequency of Mutation (%) MDS AML C-ALL ALL L3 T-ALL T-ALL relapsed CLL Richter's syndrome B-low-grade lymphoma B-high-grade lymphoma Burkitt's lymphoma HD ATL CML Chronic phase Blast crisis CTCL HCL T-CLL Multiple myeloma 5 15 3 50 Rare 30 15 40 Rare 30 40 70* 40 Rare 20-30 Rare 10 Rare 5 ~ ~~ Data are from primary malignancies and do not include data from cell lines. Abbreviations: MDS, myelodysplastic syndrome; AML, acute myelogenous leukemia; ALL, acute lymphocytic leukemia; C-ALL, common ALL; ALL LS,Burkitt's type ALL; CLL, chronic lymphocytic leukemia; ATL, adult T-cell leukemia; CML, chronic myelogenous leukemia; CTCL, cutaneous T-cell leukemia; HCL, hairy cell leukemia. * Sixty percent to 80% of cases of mixed cellularity and nodular sclerosing type HD have RS cells that are p53 positive by immunohistochemistry. or enhancer/promoter region. These types of p53 mutations represent about 10% of the total p53 alterations. False-positives also can occur. For example, in one series of lymphomas, we found 50% of tumors with immunohistochemically detectable p53, but no mutations were detected in the p53 gene.7' This finding could be explained in part because we did not analyze for alterations in every region of the gene. Also, some rapidly dividing normal tissue express p53, such as activated T lymphocytes. In addition, p53 might be detected if wt-p53 was bound to another protein that inactivated but prolonged its half-life (ie, SV40 lzrge T antigen). Ideally, a p53 antibody that can detect only mutant p53 is required for immunohistochemistry, but this reagent is not yet available. The monoclonal antibody known as pAb 240 has specificity for many mutant p53s by immunoprecipitation of nondenatured p53, but the antibody cannot differentiate mutant from wt-p53 on either Western blot or immunohistochemistry because both denature the p53 pr~tein.~' p53 ALTERATIONS IN HEMATOLOGIC MALIGNANCY A summary of the incidence of p53 mutations in hematologic malignancies is provided in Table 1. Several of the notable features of p53 alterations in these diseases are summarized in Table 2 and these features are discussed within the context of the individual disease. Chronicmyelogenousleukemia (CML). The structure and expression of the p53 gene is altered in about 20% to 30%of samples from patients in myeloid blast crisis of CML, whereas chronic-phase CML cells only rarely have detectable p53 a l t e r a t i ~ n s . ~ ~ ~Several ' , ~ * ~ features ' * ~ ~ of p53 and CML are the following: (1) CML is analogous to osteosarcoma in so far as the p53 gene can be altered by either point mutations or major DNA rearrangements. Why these two diseases frequently have major rearrangements of p53 is unclear. Another malignancy that usually has a major p53 rearrangement is murine erythroleukemia, which is associated with a viral infection. (2) The p53 alterations almost always occur in myeloid, not lymphoid, blast crisis. (3) The p53 mutations are associated most frequently with samples in which one of the short arms of chromosome 17 (1 7p) has been loss, usually through formation of either an isochromosome 17q [i(17q)] or unbalanced tran~location.~~ The i(17q) chromosome occurs in about 30% of cases of myeloid blast crisis of CML and about 40% of these have p53 mutations on the remaining p53 allele.74The loss of a 17p (containing p53) may precede the p53 mutation of the remaining allele in CML.74 In contrast, the p53 mutations in gliomas and colorectal and breast tumors occur on one p53 allele and then the remaining normal p53 allele is lost. In either case, these observations emphasize the strong selection for complete loss of p53 function in the process of carcinogenesis. (4) Circumstantial evidence strongly suggests that a p53 mutation in the CML clone can result in disease transformation to myeloid blast crisis?' Pari passu, when wt-p53 is transfected and stably expressed in the p53 null CML erythroblastic cell line K562, growth of the cells slows and they undergo partial differentiation suggesting an involvement of wt-p53 in the differentiation processes.75Although rare, the finding of a p53 mutation in myeloidcells during the chronic phase probably is a grave prognostic sign. Acute myelogenous leukemia (AML) and meylodysplastic syndrome (MDS). In 1986, we found that 8 of 33 patient samples from a variety of hematopoietic malignancies showed increased accumulation of p53 using immunoprecipitation analy~is.'~ Seven of the eight samples occurred in cells of patients with either preleukemia or AML. These and similar results suggested the notion that p53 may contribute Table 2. Notable Features of p53 Alterations in Hematopoietic Malignancies 1. Development of p53 mutations is often correlated with worsening or relapsing of the hematopoietic malignancy. 2. Loss of short arm of chromosome 17 is associated with a p53 mutation on the remaining allele in several hematopoietic malignancies. 3. B-cell lymphomas with p53 mutations often have c-myc activation, but EBV infection does not appear to correlate with the presence of a p53 mutation. 4. HD has p53-positive RS cells but accompanied lymphocytes, eosinophils, and macrophages do not overexpress p53 consist with these cells being a reaction to the malignant process. Lymphocyte-predominant HD does not have p53-positive RS cells. 5. Individuals with LFS have a p53 mutation in their germline and have an increase incidence of leukemias and lymphomas. From www.bloodjournal.org by guest on June 17, 2017. For personal use only. 2414 to the phenotype of certain leukemias Frequent in p53 abnormalities have been reported in cell lines derived from acute myeloid leukemia samples, suggesting that p53 gene inactivationmayhavea role in theestablishmentof these This cell was first observed in HL-60 cells thathaveamajorrearrangement of p53 resulting in absence of p53 expression." Although frequent in AML cell lines, the mutational frequency of p53 in AML cells from individualsabout is The mutational frequency increases to about 50% in AML samples having 17p monosomy, similar to what is observed in myeloid blast crisis of IMAMURA, MIYOSHI, AND KOEFFLER and overexpression of the protein. These tumors appear to have normal p53 alleles, supportingtherole of mdm-2 in the inactivation of wt-p53 resulting in tumor progression." Conceivably overexpressed mdm-2 or another as yet unidentified protein is binding and inactivating p53, which contributes to the process of leukemogenesis. However, evidence for this hypothesis is presently lacking. Of note, the introduction of wt-p53 into a murine p53 null AML line (MI) had no effect on differentiation of the cells, but induced their apoptosis; this was inhibitable by a growth factor (interleukin-6 [IL-6])."x Furthermore, myeloid progenCML,X(J.XI itor cells and thymocytes from pS3-deficient mice are more The p53 gene is also infrequently altered in MDS, with a resistant than their normal counterparts to development of frequency of about 5% to 10%; most of these are missense apoptosis."".""' Together, theseresultssuggestthatnormal point mutations.JZ~X2~8" Characteristics of p53 in theMDS myeloid progenitor cells might continuously undergo apoclone include the following. (1) p53 mutations occur in the ptotic death in the absence of appropriate differentiation and proliferation signals. Loss of p53 in these cells could provide subtypes of MDS with a prognostically poor French-American-British (FAB) classification (eg, refractory anemia with them with a growth advantage by decreasing theirrate of excess blasts [RAEB], RAEB in transition, chronic myelodeath. These cellswould continue to cycleuntil further oncomonocytic leukemia). Mutations of p53 have not been regenic events occurred. ported in patients with either refractory anemia (RA) or RA Acute lymphoblasticleukemia (ALL). We examined 330 with ring sideroblasts. (2) Many of the patients with a p53 samples of common-ALL, pre-pre B-ALL, and pre B-ALL mutation havemonosomy of chromosome 17, similar to and found a 2% to 3% incidence of p53 mutations in these leukemias." Several smaller series found either a similar or CML and AML.(3) Samples with p53 mutations have promslightly higher frequency of alterations of p53.47.1"1.1"2 These inent p53 accumulation in their blast cells but not in their p53 mutations appear mostoften in patientswithrelapse mature and presumably MDS-derived myeloidcells,sugphase of In contrast,type (Burkitt's) Lz Bgesting that transcription of even mutated p53 is under norALL have a 50% incidence of p53 mutation^^^.^^' and often mal control. have activation of the c-myc gene. Although T-cell leukemia Cases of AML and MDS have been reported that have cell lines have about a 50% frequency of p53 mutations,"" high levels of p53 protein, as determined by either immunoprecipitation or immunochemistry, buthaveno detectable p53 mutations are rarely found in patients with newly diagp53 m~tations.~"."' One study found that the protein in AML nosed T-ALL."" Interestingly, about 30% of samples from patients with relapse T-ALL have p53mutations, suggesting cells, although not mutated, often adopted the conformation the importance of this alteration with progressive disease. of mutant pS3 as identified by antibody pAb240 in AML.Xs Patients with Li-Fraumeni syndrome (see below)have a 5 % Additionally, p53 has been found in mutant conformation in to 1 0 % frequency of childhood ALL, with the leukemic cells normal human activated T lymphocytes and CD34' hematohaving a homozygous p53 mutations. poietic stem cells.*' The wt-p53 is an allosteric protein that Most of the p53 mutations in these hematopoietic maligcan exist potentially in two conformations that may be denanciesare G:Cto A:Ttransitionalnucleotidealterations pendent on the cell cycle. This alteration of p53 conformaand most of these occur at CpG dinucleotides. This pattern tion in normal dividing myeloid and lymphoid cells may be of mutations probably results from spontaneousdeamination associated with a temporary inability of the protein to halt suggestingthat,in ALL and hemato~ cells at the G, phase allowing these cells to p r o l i f e r a t i ~ n . ~ ~at~ ~5-methylcytosine, logic malignancies in general, the p53 mutations may not be In AML, this "permanent" alteration in conformation could induced by exogenous carcinogens. The codon location of be a mechanism causing the preferential proliferation over these mutations in any of the hematopoietic malignancies do differentiation of AML cells. Related or independent is the not appear toinfluence either the phenotype or clinical course observation that about half of all AML sampleshave elevated of these leukemias and lymphomas. expression of mdm-2 (murine double minute).x' The human Chronic lymphocytic leukerniu (CLL). The leukemic homologue of mdm-2 gene may act in a negative feedback cells have p53 mutations in about 15% of individuals with loop with pS3. The mdm-2 gene was originally identified on CLL." A minority of patients with CLL transform to a rapdouble-minutechromosomes thatwereamplifiedapproxiidly aggressive lymphoproliferative disorder that is known mately %-fold in a spontaneously arisingtumorigenic Balb/c as Richter's syndrome. At least 40% of these patients have 3T3 cellline") and overexpressionof this gene can transform a p53 mutation in their more malignant cells." Therefore, normal murine fibroblasts." The product of the mdm-2 gene alterations of p53 are closely associated with transformation binds towt-p53and negativelyregulatestranscriptional of CLL into a very aggressive lymphoma. activation of ~ t - p 5 3 . ' ~ .It' ~can overcome wt-p53-mediated Adult T-cell leukemia (ATL). About 30% to 50% of indisuppression of transformed cellular growth.'J In addition, viduals with ATLhave p53 mutations in theirleukemic wt-p53 can induce mdm-2 expression," suggesting an cel]s.<l,.34.?5 In our series, leukemiccells of 4 of 10 acute autoregulatory model for functionalactivity of p53."'."' Five ATL cases had homozygous p53 mutations. In addition. we percent to 30% of sarcomas have mdm-2 gene amplification From www.bloodjournal.org by guest on June 17, 2017. For personal use only. p53 IN HEMATOLOGIC MALIGNANCIES studied an informative patient whose ATL cells had no detectable p53 mutation in the chronic phase of the disease. A novel clone emerged that quickly expanded to acute ATL and rapidly led to the death of the patient; these cells had a homozygous missense mutation of ~ 5 3 The . ~ data ~ suggest that alterations of the p53 gene are frequently acquired abnormalities in acute ATL and may occur in the transition to the more aggressive leukemic phenotype. The natural history of development of acute ATL suggests that HTLV-1 infection alone is not sufficient to cause leukemia. Many individuals infected with the virus do not develop ATL; those who do often have a 20- to 30-year latency period. HTLV-1 does not contain an oncogene, probably does not activate proto-oncogenes by insertional activation, nor does it inactivate tumor-suppressor genes by disrupting them.'" A possible hypothesis concerning the development of ATL can be stated as follows. HTLV-1 infection results in acute expression of the tax product encoded from the X region of HTLV-1, perhaps resulting in autocrine growth stimulation through production of IL-2 and other lymphokines. 105.106 This stimulation may cause a polyclonal increase of the HTLV-l-infected T lymphocytes. These cells may periodically expand by various stimuli such as infections that might enhance tax production. This polyclonally and then oligoclonal expanded population of cells may have a slight growth advantage over normal cells. Evolution from chronic to acute ATL may result from additional mutations such as a homozygous mutational alteration of p53. This model is analogous to the progression of CML to blast crisis. Leukemic lymphocytes staining positive for p53 are frequently found in patients with ATL even when the leukemic cells have no p53 mutation. These cells are also Ki-67 positive, suggesting that the proliferating cells are staining positively for p53 protein. We examined normal, uninfected phytohemagglutinin (PHA)-stimulated T cells and HTLV-Itransformed T cells from the same normal individual and found prominent p53 expression in both.Io7Further studies are required to determine if the prominent expression of p53 in ATL cells represents either an aberrant p53 or a normally expressed p53 in a rapidly proliferating population of T cells. Similarities exist between HTLV and Epstein-Barr virus (EBV). Both can easily immortalize lymphocytes in vitro. Neither contain oncogenes nor transform by insertional mutagenesis. Most people infected with either of these viruses are asymptomatic and only rarely develop a malignancy. Infection withboth viruses can at least initially stimulate polyclonal cellular proliferation. In both ATL and Burkitt's lymphoma, p53 mutations are intimately associated with progression to malignancy. Burkitts lymphoma also is almost always associated with activation of c-myc. Activation of an oncogene is also likely to occur in ATL, butit has not been identified as yet. Therefore, both viruses may provide a growth advantage to a large cohort of cells. This active proliferation over a long period of time may enhance the opportunities of developing a p53 mutation. Hodgkin ' S disease (HD). Immunohistochemical staining for p53-protein on frozen- and paraffin-embedded lymph node samples shows that a significant proportion of p53positive neoplastic cells are detected in about 60% to 80% 2415 of cases with mixed cellularity and nodular sclerosing type ~ ~ . 6 7 , 6 8 , 1 0 8I-O1 Immunoreactivity is localized to the nuclei of Reed-Sternberg (RS) cells or its mononuclear variants. The number of positive cells vary between 10%to 60%of recognizable RS cells. Mutations of p53 have been detected in enriched RS cell preparations." Accumulation of mutant p53 only in RS cells suggests an important role of p53 in the tumorigenic process of HD disease. No correlation has been found between EBV infection and p53 reactivity in RS cells.67The background of small lymphocytes, plasma cells, eosinophils, and histiocytes in the HD samples are unstained for p53. This finding is consistent with the view that RS cells (and variants) are the neoplastic components of HD. In addition, no p53 staining is noted in lymphocyte-predominant HD. This finding is of interest in view of the possibility that this disease may really be a form of low-grade B-cell lymphoma rather than a subtype of HD. Non-Hodgkin lymphoma (NHL). Aggressive, high-grade B-cell NHL has about a 30% incidence of p53 mutations, whereas indolent B-cell NHLrarely have alterations of p53~68.70.71.113-115 About10% of T-cell NHL also have p53 mutations. We found that patients with acquired immunodeficiency syndrome (AIDS) have an increased incidence of development of B-cell immunoblastic lymphomas (BIBL). However, their incidence or type of p53 mutations does not differ when compared with BIBL in individuals without human immunodeficiency virus (HIV) infe~tion.~' In another study of AIDS-related lymphoma, p53 was mutated only in those samples histologically classified as small, noncleaved cell lymphoma (SNCCL) and 63% of SNCCL samples had p53 mutations."' Almost all of these samples hadc-myc activation and were usually not infected with EBV.'16 The c-myc is also dysregulated in Burkitt's lymphoma and L3type B-ALL, which also frequently have p53 alterations, suggesting that those tissues with an abnormally expressed c-myc protein may gain an additional growth advantage by mutating the p53 gene. Whereas 50% of aggressive B-cell NHL express high levels of p53, only 20% to 50% of these had demonstratable p53 mutation^.^' Similarly, postthymic T-cell lymphomas and CD30 (Ki-l)-positive, anaplastic large-cell lymphomas frequently overexpress p53 in the absence of detectable p53 In contrast, cells of nonmalignant hyperplastic lymph nodes have negligible p53 expre~sion.~' Therefore, immunohistochemical analysis of p53 may help discriminate between normal andneoplastic lymph nodes, but cannot distinguish wild-type from mutant p53 protein in this disease. The nonmutated, but overexpressed, p53 may be stabilized in these lymphoma cells by alternate mechanisms such as binding to an additional protein, for example, mdm-2 or a viral product. Low-grade NHL rarely have p53 alterations, but their progression to high-grade lymphoma canbe associated with development of p53 mutations. For example, serial biopsies of patients with follicular NHL who underwent histologic transformation showedthat one-third of the transformed samples acquired a p53 mutation that was not detected in the follicular stage of the disease."' Another study found From www.bloodjournal.org by guest on June 17, 2017. For personal use only. 2416 IMAMURA, MIYOSHI. AND KOEFFLER Table 3. Hematopoietic Malignancies in Which p53 Mutations Are that 4 of 5 cases of transformation of follicular to diffuse Associated With Disease Progression large-cell NHL were associated with p53mutations.”’ Interestingly, in this study, one sample offollicular NHL had 1. Evolution from chronic phase to myeloid blast crisis ofCML. regions of transformation to high-grade NHL; cells of this 2. Evolution from myelodysplastic syndrome to acute myelogenous region,butnot those of the follicularareas, contained a leukemia. mutant ~ 5 3 . ’ ”Particularly intriguing, p53-positive staining 3. Evolution from follicular to high-grade lymphoma. 4. Evolution from CLL to high-grade Richter’s-type lymphoma. cellscanbedetectedbefore transformation tohigh-grade 5. Progression t o a refractory phase of multiple myeloma. lymphoma, but they represent a minority of the lymphoma6. Development of relapsed B- or T-ALL. tous cells.”’ The percentage of p53 positive cells increased either just before or after histologic progres~ion.’’~ Therefore, individuals with low-grade follicular NHL thathave p53 staining cells may be at increased risk for transformation tions. p53 mutations have also been associated with progresto an aggressive NHL. These patients may be appropriate sion of solid malignancies suchas the transition from benign candidates to receive intensive therapy, but further studies adenoma to malignantcolon carcinoma,”* evolution of glioare required to confirm this impression. The routine staining m a ~ , ’ ~ ’ . and ’ ’ ~ development of metastatic prostate cancers. of lymph nodes for p53 may become standard practice both However, mutationsof p53havealso beenfoundin the for prognosis and therapy of lymphomas. precancerous phases of adenocarcinomas, including adenoBurkitt’s lymphoma. We reported that Burkitt’s a matous polyps ofpatientswith familialpolyposis coli,”’ lymphoma cell line (Raji) showed increased amount of ~ 5 3 . ~suggesting ~ that p53 mutations may occur as an early event Afterwards, several groups found that p53is usually mutated in carcinogenesis as well. Burkitt’s in lymphoma More cell importantly, Li-Fraumeni syndrome (LFS) and cancer families. LFS the frequency of p53 mutations isabout35%to45% in isa rare autosomal dominantly inherited syndrome conBurkitt’s lymphomas from patient^.*^*^*' More than 50% of sisting of the following clinicalcharacteristics:aproband these mutations are clustered in a small stretch of 33 amino with either acute lymphocytic leukemia, sarcoma, breast canacids (codons 213 to 248), with codons 213 and 248 being cer, brain tumor, andor adrenocortical carcinoma before the the most frequently mutated spots; codon273, which is often age of 45; a first-degree relative with a cancer in this group; mutated in solid tumors, is rarely altered.”’ The p53 mutant and a first- or second-degree relative with sarcoma at any genesclonedfromthesecellshave losttheirabilities to age or any cancer before age 45.’’‘ These individuals have inhibit DNA synthesis, a characteristic of wt-p53.’” a germline mutation of p53,which is consistent with the Almost all cases of Burkitt’s have a chromosomal translofirst hit in Knudson’s two-hit mutational modelof hereditary cancer.i37.13s The developing cancers have loss of the wtcation resulting in c-myc activation. Taken together with the above presented SNCCL data, activation of myc and mutap53 allele (second hit) and retain the mutant p53 allele.’’9 tion of p53 may play a critical function in the development Paradoxically, even though all the cells of these individuals of a subgroup of B-cell lymphomas. The EBNA5 can bind have a p53 mutation, they have a propensity to develop only both RB and p53 and is required for B-cell transformation; several types of cancer. Soft-tissue sarcomasusually develop the role thatthisviralproteinplays in pathogenesis of in the first 5 years of life. Acute leukemias andbrain tumors lymphomas requires further s t ~ d y . ~Clearly, ~ ~ , ~ more ~ ’ than occur throughout childhood and young adulthood; the rare one mechanism canlead to lymphomas, because, unlike Afadrenalcortical carcinomas occur primarilyininfancy. In rican Burkitt’s, American Burkitt’s can often arise without young adults, breast cancer is by far the most common neoEBV infection. The EBV-associated nasopharynegeal carciplasm. The incidence of ALL inindividuals with LFS is nomas appear to have a normal ~ 5 3 . ~ * ’ . ’ * ~ about 6%. Multiple myeloma. The p53 is mutatedin about 5% to LFS is a rare genetic disorder. The prevalence of a germ10% of cases of multiple m y e l ~ m a . ’ ~ ~ Approximately ”*~ linep53 mutation is approximately 0.01% in the general 80% of human multiple myeloma cell lines have p53 mispopulation, 0.1% to 1% among various cancer patients, and sense mutations.’” Little is known of p53 expression in the 5% to10% among young patientswith multiple cancer^.'^".^^' plasma cells of patients with multiple mye10rna.l~’ Although Roughly 50% of family members with LFS develop neoplanumbers are small, data suggest that development of a p53 sia by the ageof 30, in contrastto a 1% incidence of developmutation is a late event in the disease and is associated with ment of cancer by the same cohortin the general population. an aggressive c o ~ r s e . ~ ~ * , ~ * ~ Astriking feature of the affected members of families p53 mutations and progression of hematopoietic maligwith LFS is their high frequency of second malignancies, nancies. As shown on Table 3, p53 mutation is often assowith almost 50% of affected members developing morethan ciatedwithprogressionof the hematopoieticmalignancy. one neoplasia. This observation has prompted several investigations of the incidence of germline p53 mutations in indiFor example, evolution of CML and MDS to myeloid blast vidualswithmultiple cancers. In one study examining 59 crisis and AML, respectively, has been associated with loss children and young adults who would not be considered as of the short arm of chromosome 17 and mutationof the having LFS, but who had developed two malignancies, the remaining p53 allele. Also, several of the lymphoproliferaoverallfrequency of p53 mutationsintheir germline was tive disorders, such as CLL and follicular lymphoma, can approximately 7%.14’ One of the four tumors in this cohort acquire a p53 alteration as they progress toa more malignant was an NHL. In another study, 10 families whose relatives phenotype. Relapsed T- and B-ALLoften develop p53muta- From www.bloodjournal.org by guest on June 17, 2017. For personal use only. 2417 p53 IN HEMATOLOGIC MALIGNANCIES had a high incidence of leukemias and lymphomas were examined for p53 germline mutations and none was found.14' In contrast, analysis for germline p53 mutations in individuals with childhood ALL containing p53 mutations in their leukemic cells found that one (pre-B-ALL) of the four individuals had a germline alteration of p53. Several observations of LFS can be made. (1) Germline p53 mutations have been observed mostly in patients with an unusual history of cancer, ie, either multiple malignancies or family histones of cancers. (2) Most of the germline p53 mutations have been inherited; only 2 of 18 germline p53 mutations have been shown to occur de novo.'" In contrast, germline retinoblastoma mutations most often (85%) occur de novo in cases of retinoblastomas. (3) Although germline p53 mutations can be widely distributed within the gene, an increase predominance is found between codons 240 and 285. Almost all are missense point mutations. (4)Because of the very low prevalence of germline p53 mutations in the general population and the intensive analysis required to detect germline p53 mutations, general screening of individuals is not indicated. An individual with cancer and a strong family history of cancer or an individual with multiple neoplasias of the type occurring in LFS should be screened for p53 mutations and, if positive, relatives at risk should then be ~creened.'~'In addition, in the next few years, clinicians must determine what type of surveillance should be given to presymptomatic, germline carriers of p53 mutations. Gene therapy and othertherapeuticapproaches. Because of the pivotal role played by p53 in the regulation of cell replication, the therapeutic restoration of expression of wt-p53 in tumor cells having a p53 mutation might eventuallyplay a role in cancer gene therapy. In several model systems, features of the tumor phenotype can be suppressed in vitro through the restoration of expression of the mutant tumor suppressor gene such as p53 and Rb. However, before this method can serve as the basis for gene therapy of cancer, many conceptual and technical problems must be solved. Because such genetically modified cells will continue to contain and express other mutations, the mechanisms and frequency of reversion to the tumor phenotype must be examined. Experimentally, the transduced wt-p53 frequently becomes mutated in transformed cells already expressing a mutant ~ 5 3 . l Nevertheless, ~ ~ " ~ ~ colon adenocarcinoma cells with other genetic defects in addition to the p53 gene became nontumorigenic after being transfected with plasmids expressing a normal p53 allele.'46In the future, highly efficient and targeted gene delivery vectors mustbe developed to be clinically useful.'49 Experiments have shown that p53 retroviral vectors are capable of penetrating multiple cell layers of three-dimensional tumor masses and mediating potential therapeutic effects.'" The regional administration of viral supernatant containing p53 could be conceivably useful therapeutically in established tumors and premalignant lesions of the upper aerodigestive tract and gastrointestinal mucosa that had p53 mutations. At this time, none of the delivery systems are powerful enough to be useful for in vivo therapy. Furthermore, normal cells that are transduced with the p53 expression vector may become dysfunctional because of either inappropriate levels or timing of expression of p53. Immunotherapeutic approaches may also be possible. If the mutant p53 epitopes were displayed on the malignant cells and could be presented to T lymphocytes by class 1 major histocompatibility complex molecules on the plasma membrane, a cytotoxic immune response might be selective against these cancer cells. Indirect evidence suggests that this may OCCUI-.'~' A murine model has shown that a cytotoxic immune response can occur against cells expressing mutant ~53.'~ In' addition, autoantibodies to p53 have been identified in about 9% to 16% of cancer patients, including those with breast cancer and lymphomas; no evidence exists that these antibodies are directed against mutant p53 or that these patients have an improved clinical response to their cancer,1s3-lss Although untested, the use of endogenously expressed mutant p53 as a target antigen remains a potential approach for vaccine immunotherapy of selected cancers. Identification of the genes that are regulated by p53 permits another future avenue of therapy. For example, p53 induces expression of a cyclin-dependent kinase inhibitor known variously as either WAF-l, Picl, Cipl, SDI-l, or p21, which helps regulate the cell cycle; cells with mutant p53 lose this control. Perhaps therapeutically induced expression of WAF-l in p53 mutant cells will correct alterations in the cell cycle. For example, cells with p53 mutations do not stop at the GI phase of the cell cycle after DNA damage. This contrasts to cells with wt-p53, which enforces a pause at the G , phase of the cell cycle for DNA repair, before beginning DNA synthesis. Finally, this difference in cell cycle control after DNA damage between malignant cells having mutant p53 and normal cells expressing wt-p53 can be used to design novel chemotherapy andor irradiation treatment schedules. In summary, p53 mutations occur moderately often in hematopoietic malignancies. They are particularly associated with progression of disease in both lymphoid and myeloid leukemias as well as lymphomas. In addition, p53 mutations occur very frequently in Burkitt's and other high-grade Bcell lymphomas. 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For personal use only. 1994 84: 2412-2421 p53 in hematologic malignancies J Imamura, I Miyoshi and HP Koeffler Updated information and services can be found at: http://www.bloodjournal.org/content/84/8/2412.citation.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. Copyright 2011 by The American Society of Hematology; all rights reserved.