ICANCER RESEARCH57, 837-841. March 1, 9971

Advances in Brief

Distinct Patterns of Inactivation of @15lN@@4Band @J6IN@@4ACharacterize the Major

Types of Hematological Malignancies1

James G. Herman,2 Curt I. Civin, Jean-Pierre J. Issa, Michael I. Collector, Saul J. Sharkis, and Stephen B. BaylinThe Oncology Center Ii. G. H., C. I. C., f-P. f. I., M. 1. C., S. f. S., and S. B. B.] and Departments of Medicine (S. B. B.) and Pediatrics [C. 1. C.), The fohns Hopkins UniversitySchool of Medicine. Baltimore, Maryland 2/205

In hematological malignancies, homozygous deletion of @/6â@NK4Ais primarily limited to ALL3 and non-Hodgkin's lymphoma (10—13).Although ALL has among the highest reported rates of homozygousdeletion at 9p2l in primary neoplasia (6, 9—11, 14), assigning a tumorsuppressor role to p16@―@4―alone in these malignancies is difficult,because most cases include both p15@―4'@and p16@―4―in the deletedregion (15). A subset of malignancies, including ALL, have selectivehomozygous deletion of@ (16, 17), but these malignanciesgenerally represent only a minority of those with homozygous deletion.

Our recent findings demonstrate that inactivation of p15―@―4'@andp/6@―4@is a much more frequent event in neoplasia than indicatedsolely by the presence of homozygous deletions. We and others haveobserved that aberrant methylation of 5' region CpG islands in thesegenes represents an alternative mechanism to homozygous deletionand coding region mutations for inactivation (18—22).For each gene,this hypermethylation is found in neoplastic cells and is associatedwith transcriptional loss that was reversed by treatment with theDNA-demethylating agent 5-aza-2'-deoxycytidine (18—20,22). Unlike homozygous deletions, inactivation of these genes through promoter region hypermethylation is selective, typically limited to only

@j5INK4B or @J6INK4A (20), and usually limited to only p/61t@4@' in

solid tumors (20). However, in a small series of acute leukemias,selective hypermethylation of @j5@'@4B,without hypermethylation ofp16@@K4A, was observed (20), suggesting that inactivation@

might be an important event in the development of hematologicalmalignancies. To further define the importance of both pj5hl@@@(4Band

@ in the development of hematological malignancies, we ex

amined the inactivation of these genes in a large number of primaryleukemias and lymphomas. Our study demonstrates that by examiningboth homozygous deletion and hypermethylation as inactivatingevents, important selective roles for @J5@@@K4Band @j6@'K4@@can bedefined for subtypes of hematological malignancy, and that one, andin some tumor types, both, genes are virtually always involved.

Materials and Methods

Samples and DNA Preparation. High molecular weight DNA was isolated from primary leukemia and lymphoma as described previously (19).Classification of leukemia or lymphoma phenotype was based on the clinicaldiagnosis. B precursor versus I precursor ALL was determined by surface

markers (B with positive staining for CD19, CDIO, and HLA-DR and negativefor CD4, CD7, and CD34). NHL was divided into low grade (follicular inseven cases, follicular and diffuse in four cases) or high grade (diffuse largecell lymphoma in four cases, 10-1+ in one case, and immunoblastic in onecase). Burkitt's lymphoma samples were sporadic (3) and endemic (5) tumors.Fifteen of the 175 samples included in Table 1 were reported previously (20).

Southern Hybridization. Five @gof genomic DNA from cancer cell lines,normal tissues, and primary neoplasia were isolated and digested overnight

3 The abbreviations used are: ALL, acute lymphocytic leukemia; NHL. non-Hodgkin's

lymphoma; MSP, methylation-specific PCR; AML, acute myelogenous leukemia; CML,chronic myelogenous leukemia; TGF, transforming growth factor.

Abstract

Inactivation of the cyclin-dependent kinase inhibitors pJ6I@W@4d@andpJ5INK4B are frequent alterations in neoplasia, often resulting from ho

mozygous deletion or promoter region hypermethylation. We have ana

lyzed both modes of inactivation of p15@'48and pJ6INK4A in the majortypes of adult and pediatric hematological malignancies. Hypermethylation 0fp151NK48,without alteration 0fpJ6INK4A,was an almost universalfinding in adult acute myelogenous leukemia, and occurred very frequently in adult acute lymphocytic leukemia and pediatric acute myelogenous leukemia and acute lymphocytic leukemia. In contrast, neitherpJ5INK4B nor pJ6INK4A were inactivated in any stage of chronic myelogenous leukemia. Hypermethylation ofp16―@―4@,often without alterations ofp1S―@'48,was found in non-Hodgkin's lymphoma and was much morefrequent in cases with high-grade than low-grade histology. Enrichednormal bone marrow stem cells had no detectable promoter region methylation of these genes, as analyzed by a newly developed PCR method.Remarkably distinct patterns of inactivation of@ and p16@4―characterize different types of hematological malignancy, and alterationsin these tumor suppressor genes are one of the most common alterationsin hematological malignancies.

Introduction

Loss of cell cycle control is a frequent alteration in neoplasia, andchanges in the cyclin D/retinoblastoma gene (Rb) pathway are ofteninvolved. Rb is fundamentally important for the G1-S transition of thecell cycle and is inactivated in many forms of human cancer. However, within the hematological malignancies, inactivation of Rb isinfrequent (1), suggesting that other genes involved in cell cyclecontrol might be altered. The cyclin-dependent kinase inhibitor

@j6@lK4@@ normally inhibits the phosphorylation of Rb by cyclin D and

cyclin-dependent kinases 4 and 6 (2). The@ gene, located atchromosome 9p2l, is lost through homozygous deletion in manymalignancies (3, 4), including hematological malignancies (4). Homozygous deletions usually include the adjacent gene plS―@4'@',which encodes another cyclin-dependent kinase inhibitor (5). Mostevidence has suggested that pi6@―4'@is the target of inactivation at9p2l, because inactivating point mutations of p16'@―4―,althoughinfrequent, are found in some tumors (3, 6) and in the germ line ofpatients with familial melanoma (4, 7), whereas point mutations havenot been found in p15 (8, 9). Similarly, pl6@@@(4Ahas been reported tobe deleted without deletion@ whereas the opposite patternis seldom observed.

Received 11/4/96; accepted 1/18/97.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

I Supported by Grant CA433 1 8 and NIH Core Grant 2P30-CA06973—34. J. G. H. and

S. B. B. receive research funding and are entitled to sales royalty from ONCOR, which isdeveloping products related to research described in this paper. The terms of thisarrangement have been reviewed and approved by The Johns Hopkins University inaccordance with its conflict of interest policies.

2 To whom requests for reprints should be addressed, at The Johns Hopkins Oncology

Center, 424 North Bond Street, Baltimore, MD 21231.

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Table I inactivation 0f@/5!@V@l@4Band @j6INK4Ain a@75 cases ofpriman hematologicalmalignancy, aceording to clinicaldiagnosis@j5INK4B

@j6INK4AInactivation

p/S or ploInactivation p/S andp16Diagnosis HD― Meth―HD―Meth―Adult

AML (60) 0 53 (88%) 0Pediatric AML (27) 0 18 (67%) 0AdultALL(7) 0 5(71%) 0PedBprecursorALL(23) 4(17%) 11(48%) 3―(13%)Ped T precursor ALL (12) 4 (33%) 6 (50%) 9 (75%)AduItCML(2l) 0 0 0NHL-lowgrade(ll) 0 1(9%) 0NHL-high grade (6) 0 1 0Burkitt's lymphoma (8) 1 ( I3%) 4 (50%) 00

1 (4%)00001(9%)5 (83%)6 (75%)53

(88%)18 (67%)5(71%)

15(65%)12 (100%)01(9%)5 (83%)7 (88%)0

1 (4%)02@'(9%)7(58%)01(9%)1 (17%)4(50%)a

HD, homozygous deletion; Meth, hypermethylated in the 5' promoter region.

h Two additional B-precursor ALL had homozygous deletion of @15Lv.@'4@@, which extended to

PATTERNSOF INACTtVATIONOF p/51'°48AND @/6INK4A

with excess restriction endonuclease (HindIll and EagI) and hybridized as

described (18, 20). All blots were first hybridized to the@ exon 1probeand then stripped and reprobed with the pJ6INK4Aexon 1 probe.

MSP. Bisulfite treatment of DNA and MSP were performed using primerpairs for pJ5INK4B (pl5U and pl5M) and pJ6INK4A (pl6U and pl6M) as

described (23). DNA isolated from 50,000 bone marrow cells, fractionated asdescribed below, was used. Controls without DNA and positive controls for U

and M reactions were performed for each set of PCR. Ten @dof each PCRreaction was directly loaded onto nondenaturing 6% polyacrylamide gels,

stained with ethidium bromide, and visualized under UV illumination.

Stem Cell Isolation. Humanbonemarrowcells wereobtainedfromnormalvolunteers, and the small-sized cells containing enriched populations of hematopoietic stem/progenitor cells were collected at 25 ml/min (FR25) as

described (24). These cells were subjected to staining with an antibody to theCD34 molecule to further enrich for stem/progenitor cells as described (24).Positive cells were those brighter than 99% of the control antibody staining.

Results

Selective Hypermethylation of p151―48 Characterizes AMLbut not CML. Restrictionof genomicDNAfromprimarymalignancies with the flanking enzyme Hindu!, plus the methylation-sensitiveenzymeEagI,uponSouthernhybridizationwitheitheraexon1or @16INK4Aexon1probe,monitorsthemethylationstatusofCpG islands in the 5' regions of these genes, as well as detectinghomozygous deletion (20). As shown previously by this technique,

methylation of these sites does not occur in normal tissue and isassociated with transcriptional loss of the associated gene in neoplasms (I 8 —20).We observed no homozygous deletions of@or p16@―4―in any of 87 cases of AML examined, confirming theresults of others that homozygous deletion of these genes is aninfrequent event in AML (10—13).However, we found that hypermethylation 0fpJ5INK4B occurred in 88% of adults and 67% of childrenwith AML (Fig. 1; summarized in Table I ). In one of these cases, apediatric patient, the@ gene was also methylated, but theremaining 70 AML samples with@ methylation were notmethylated at the 5' CpG island of@

In contrast to the results in acute myelogenous leukemias, we foundno alterations of either p15@―4'@or @J6INK4Ain any patient at anystage of CML. There were no homozygous deletions or cases withhypermethylation in 2 1 samples from 20 patients with CML ( I I inchronic phase, 2 in accelerated phase, and 8 in blast crisis; 1 patienthad samples from both accelerated phase and blast crisis). We diddetect, in many cases of CML, altered size of a cross-hybridizingfragmentto thep15INK4Bprobewhichdoesnot representp15 (20).The significance of this finding is presently unknown.

Both @J5INK4Band pJ6h1@hI@4AAre FrequentlyInactivatedinALL. In adultALL,hypermethylationof p/5―4―@was evidentinfive of seven cases, but there were no homozygous deletions of either

Fig. I . Southern Analysis of @J5@w48(a) andpiót @‘4/@(b) in primary hematological malignancies. The numbered lanes in a and b correspond toDNA from the same sample. The clinical diagnosisof malignancy is shown above. Among the lymphoma samples. Lane 27 is a Burkitt's lymphoma.Lane I is DNA digested with HindIII alone.whereas all other lanes are digested with both HindIll and EagI. In a, the unmethylated pattern of

@j5INK48 is observed in Lanes 2, 6, /2. and /9—26

(presence of 2.2- and 0.5-kb bands), as seen previously for normal tissue (20). Hypermethylation ofp15 NK4B @sobserved in Lanes 3—5,7, 8, 11, /4, 15,/8. and 27 (retention of the 2.8-kb band). Homozygous deletion of @j5INK4I1is seen in Lanes /3, 16,and 17. *, a cross-hybridizing fragment recognizedby the@ exon I probe (20). b, the normalunmethylated pattern of @j6!NK4/@is observed inLanes 2—12.14. /6. 19—22,23, and 24 (presence of3.9-, 2. 1-. and 0.55-kb bands). Hypermethylation of

@ (retention of 6.0-kb band) is observed in

Lanes 23, 26. and 27. Homozygous deletion of@ is seen in i.i.,nes s'S. 17, and 18. Lane 13

reveals a rearrangement Øf@J6@v@5@4Aexon I , whichoccurs with loss of @j5INK4IJin this tumor, supgesting that the homozy@ous deletion of @15@wBapproaches the @j6!NA4gene promoter region.

a

b

Pediatric Adult PediatricAML AML B-ALL T-ALL Lymphoma

@:@iipi@!!;I @sfÃw̧* __ . ‘a@ @i@ *@ •o@@ _ .@@ , .. 0@ ‘- —@

0.5kb@ - 0@.@ @.@

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

fr@ @. .@a*W0S'@

‘. *1@@.@

*

11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26276 7 8 9 10

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:@ , :-@@@@@@ * III .@ 1@@ , I@ •@

@@@ .. @.•. . .•@ . . .@ .@ @.@..@ @•...@. @..@ .. .@@@

PATI'ERNS OF INACTIVATION OF plS―°48AND @j6INK4A

1 2 3 4KGIA Lymph 1 2 3 4

p16―@4―or piS. Previous reports have documented homozygousdeletion in piS@―4'@and pi61@4― and have, in general, suggestedmore frequent homozygous deletion in T-precursor ALL than inB-precursor ALL (6, 9, 14, 16, 17). Therefore, we examined a largernumber of pediatric ALL samples to allow us to determine whetherthe pattern of inactivation 0fp/5INK4B and@ differed betweenthe B-precursor and T-precursor phenotypes of ALL. In B-precursorALL, homozygous deletion ofpiSh1@@K4B occurred in 17% of patients,

whereas hypermethylation of piS@'@(4Bwas found in another 48%.Thus, @J5INK4Bis inactivated in 65% of the B-precursor ALL samples, an incidence very similar to that found in pediatric AML. In the11 pediatric B-precursor ALL samples with hypermethylation of

@/51NK4B the @J6INK4A gene was not affected by either homozygous

deletion or hypermethylation in any case.The pattern of inactivation for piSh1W@4@@and@ in T-precur

sor ALL was quite differentfromthat in B-precursorALL.Inactivation of either @J5i@NK4Bor pi6@―4―,and most frequently both, wasfound in all 12 of the pediatric T-precursor samples examined. Theanalysis of T-precursor ALL confirmed the higher rate of homozygous deletion ofp/6―t―4―in this subtype (75%) observed by others (6,14, 16, 17). Somewhat surprisingly, these homozygous deletions ofp161―4―included the p/5JNK4B gene in only two of nine T-precursorALL. However, of the seven leukemias with homozygous deletionlimited to@ five were hypermethylated at the piS1―4'@gene(Fig. 1; Table 1). This unusual pattern of dual inactivation 0fpJ5m@@4@@and pi6@―4'@occurred in two of two patients with the clinical diagnosis of lymphoblastic lymphoma. The only time we have previouslyobserved homozygous deletion of pi6@―4―and hypermethylation ofpi51―4'@was in leukemic cell lines also ofT-precursor origin (A3.0l,Molt3, and ML-!; Ref. 20), suggesting that this unusual pattern ofinactivation of both p/5INK4B and pi6@―4@'may be unique to TprecursorALL.

p16@―4―@Is Frequently Hypermethylated in NIIL. We exammed inactivation of pi5―@―4'@and@ in another group ofhematological malignancies, the NHLs. Although inactivation ofthese cyclin-dependent kinase inhibitors was found, the pattern wasstrikingly different from that in acute leukemia. In 25 cases of NHL,we found inactivation of piSI@V@@4Bby homozygous deletion in onlyone sample (4%), but frequentinactivationof pi6―@―@4―by hypermethylation (12 samples, 48%) and less frequent hypermethylation ofpJ5INK4B (28%). Most importanfly, in analyzing different types of

non-Burkitt's NHL, we found that low-grade NHL cases had infrequent hypermethylationof pi6@―4'@(9%) whereashigh-gradeNHLcases had frequent hypermethylation (83%) of this gene (Fig. 1; Table

1, different at P < 0.005 by Fisher's exact test). The association ofhypermethylation ofp/6@―4―with more aggressive phenotype is alsoevident if these lymphomas are classified according to the workingformulation [low grade, 0 of 7; intermediate grade, 4 of 8 (50%); highgrade, 2 of 2 (100%)].

Another distinct pattern 0fpJ5AtNK4Band@ inactivation wasfound in lymphomas of the Burkitt's type. A high frequency ofinactivation of both genes, including one homozygous deletion of

@/5INK4B alone, two samples with selective hypermethylation of

pi6@@@4A,and four examples of hypermethylation of both@and pi6―@―4'@.We have previously found hypermethylation of both

@/5INK4B and@ to be very unusual in a primary malignancy.

Interestingly, one of the previous examples of this pattern was theBurkitt's lymphoma cell line Raji (20).

Hypermethylation 0fpJ5I@W@4@@andp167―4'@ Is a de Novo Eventin Hematological Malignancies. One important consideration, forboth the biology of neoplasms and considerations for clinical markerstudies, is whether the promoter region hypermethylation of pi6m!c4@@and@ is an absolutely de novo event in tumor progression.Although homozygous deletions or point mutations of a gene shouldnot be found in any normal cell, the changes in methylation couldtheoretically occur. If present in a small normal cell population, suchas a stem/progenitor cell, from which a malignancy may arise, thiswould not be detected by the Southern analysis usually employed forassay of methylation status.

To address this issue directly, we used a newly developed method,MSP (23), to examine stem cells from normal bone marrow. MSP iswell suited for this type of study because it can detect one methylatedallele in 1000 unmethylated alleles (23). In a number of primaryleukemia and lymphoma samples, the pauem of hypermethylationseen by Southern analysis was reproduced by MSP (Ref. 23 and datanot shown), confirming that MSP can detect aberrant methylation inprimary malignancies. We examined four samples of normal bonemarrow fractionated on the basis of CD34 expression, which characterizes early hematopoietic progenitor cells (25). Neither CD34— norCD34+ cells had any evidence of methylation of the pl51―@4'@gene(data not shown). To provide an even more sensitive method fordetection of aberrantly methylated piS@'K4@@alleles, we examinedthree additional marrows from normal donors that were not onlyCD34 selected but also were fractionated by cell elutriation to obtainmore highly enriched stem cell populations (24). Even for thesesamples, we found no evidence of aberrant methylation 0fp/5INK4B inany fraction (Fig. 2). These samples were similarly negative forpi6@―4'@hypermethylation (data not shown).

839

Normalmarrow-i Normalmarrow-2

UMUMUMUMUMUMUMUMUMUM

Fig. 2. MSP of @J5INK4Bin normal marrow stem/progenitor cells. PBR322 Msp digest is shown at left as a molecular weight marker. with fraction number shown for normalmarrows. Fraction 1, total bone marrow (Ficoll isolated); Fraction 2, rotor off; Fraction 3, FR25, CD34—; Fraction 4, FR25, CD34+. Amplification of the appropriately sized PCRproduct (154 bp) occurred only with unmethylated (U)1rimers in all fractionated samples from the normal marrow and from normal peripheral lymphocytes. KG1A, a leukemia cellline determined previously to be methylated at @15@w4exon I by Southern analysis, produced amplification (l48-bp product) only with the methylated (hf) set of primers and servesas a positive control.

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———I@———@——-@

-1@

PATFERNSOF INACtiVATION OF p15@@@8AND @j61@@t44

of @J6INK4nwas low (26). In that study, however,@ was notexamined. The importance in hematological transformation of loss of

@J5INK4B function may be linked to the role for this gene in control of

Normal proliferation in normal hematopoiesis. Expression of @j5@'K48 is a

key event in a nonconstitutive cyclin D-Rb pathway induced byNH Lymphoma, TGF-f3 (5). Normal hematopoietic progenitor cells appear to be sen

sohdtumors sitive to growth inhibition by TGF-@ (27). We have noted previously

AdultAMLALL that inactivation of @j5@@@c'4B@ leukemic cell lines correlated with

Pediat,IcAML loss of sensitivity to growth inhibition by TGF-f3 (20).

PediatricB-ALL In contrast to acute leukemias, selective inactivation of@

Pediatric1-ALL most frequently associated with hypermethylation, plays a prominent

role in high-grade but not low-grade NHL. Previous studies have alsofound that homozygous deletions were limited to high-grade lympho-.mas, mainly the diffuse large cell type (10, 15, 28). Homozygousdeletions in these studies were not as frequent as the hypermethylationwe observed. Homozygous deletions typically include both@and @j6INK4Awhereas promoter region hypermethylation was mostoften selective for p16@―4―in NHL, suggesting that @J6a@NK4@@is theprimary target of inactivation for this region in NHL. Point mutationswithin @J6lNK4Ain 7% of B-lineage NHL (28) are consistent with ourresults, suggesting that@ is the target of inactivation at 9p2lin this type of malignancy. However, p151―@4'@was not studied forpoint mutations in this report (28). Further supporting a central role of

@J6INK4A inactivation in the development of lymphoma is the frequent

occurrence of lymphomas in mice homozygous for loss of @J6a@NK4@@gene function (29).

Loss of function of both @j5@'i@@K48and @J6lNK4Acould have profound consequences, and our results are consistent with this. Suchdual loss is frequent in two subtypes of hematological malignancies,pediatric T-precursor ALL and Burkitt's lymphoma (Table 1; Fig. 3).Both of these malignancies have extremely high proliferative indices.Dual hypermethylation of the p15―@4'@and @j6@K4@@genes in Burkitt's, unusual for any cancer type, may emphasize the consequencefor loss of both genes. Furthermore, the dual inactivation@otherwise frequent in lymphomas, and @j5@FK48,more frequent inacute leukemias, is interesting, because Burkitt's lymphoma overlapsin clinical characteristics with leukemia and lymphoma. Lymphoblastic lymphomafT-precursor ALL may represent a similar situation.

Given the virtually obligate changes in the @j5lNK41@and/or@J6@@K4Agenes in the hematological disorders studied, the absence of

inactivation events for either gene would also be revealing for pathogenesis. This is exemplified by our findings for CML where@or @j6@K4@@inactivation was not observed, even for patients in blastcrisis. This finding emphasizes the different cellular events operativein CML versus AML throughout initiation and tumor progression.Previous studies have suggested that a subset of lymphoid blast crisisof CML may demonstrate homozygous deletion of the 9p2l region(30). The blast crisis samples in our present study are predominantlyof the myeloid type, and further studies of cases of CML withlymphoid properties are now warranted. However, failure to findmethylation of the @J5@@K4Bgene in patients presenting with apparently de novo acute leukemia may be useful in identifying those whohave progressed through CML. In support of this, one of the adultALL samples without@ hypermethylation was Philadelphiachromosome positive.

Our present findings have clinical as well as biological significance.The frequent occurrence of @J5INK4t@1and @J6!NK4nchanges in hematological neoplasms makes these alterations attractive as diagnosticand prognostic markers. This is especially so for the hypermethylationchanges, because the sensitive PCR-based technique used did notdetect this alteration in any normal hematopoietic cell, suggesting itcould be used to monitor residual disease or the reappearance ofdisease after therapy. Our observations are potentially important for

p16@5'-.@ p15―5'@

-@- -.@- BurNS'slyniphoma

Fig. 3. Schematic representation of predominant patterns of inactivation of @j5IN@4Band @16/NK4@@malignancy. Homozygous deletions are depicted as the absence of allexons. Hypermethylation of exon I is shown by a large M intersecting the arrow markingtranscription. Not shown is homozygous deletions of both @j5INK4Band@ alsoobserved in a subset of T-precursor and B-precursor ALL.

Discussion

In this study, we have developed a detailed map of the pattern ofinactivation of two related genes within an array of neoplasms arisingwithin a single cell system (i.e., hematological malignancies). Byexamining the two most common modes for inactivation of the

@J5!NK4B and p16―4@' genes in malignancy, homozygous deletions

and 5' region CpG island hypermethylation, distinct and selectiveinvolvement of these genes in specific subtypes of hematologicalmalignancies have been appreciated, suggesting a contribution of eachgene, and in some instances both genes, to development and/orprogression of specific types of leukemia and lymphoma.

In the hematological disorders studied, 5' region hypermethylationof both the @J5INK4Band @J6!NK4Agenes is especially important.Although homozygous deletions of one or both genes is not uncommon, aberrant methylation is either the most frequent, or exclusive,form of inactivation in most types of leukemia and lymphoma invesligated. However, to invoke a pathogenetic contribution to a molecular event observed in neoplasia, the absence of this change from anynormal stage in the parent cell population is important. Inactivation ofthe@ or @J6INK4Agenes by homozygous deletion wouldcertainly not be expected for any normal cell population. Likewise innormal cells, many genes contain promoter region CpG islands thatare not methylated, allowing a transcription-ready state. Consistentwith this, we now show that the CpG islands of the p151―@48andpI6@―4―genes are not methylated in normal hematopoietic cells,including highly enriched populations of the stem/progenitor cells thatare the likely cells of origin for transformation in the development ofhematological malignancy. Thus, the methylation events that havebeen shown previously to correlate with transcriptional inactivationfor these genes (18—20, 22) arise de novo during hematopoietictransformation or progression.

The selective or dual inactivation of @J5lNK4Band/or @J6lNK4Aobserved provide insight into the importance of these genes to different types of hematological malignancy. Distinct patterns and types ofinactivation of the @J5Th@K4Band @J6INK4Agenes occur in specificsubtypes of hematological malignancies (summarized in Fig. 3). Selective loss of the @J5INK4Bgene, virtually always due to hypermethylation rather than homozygous deletion, appears to have a veryspecial role in hematological neoplasia.@ loss is characteristicof most types of acute leukemias, including adult and pediatric AMLand ALL. Recently, another group reported absence of hypermethylation 0fpJ6INK4A in AML, even in those leukemias where expression

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PA1TERNS OF INACTIVATION OF @J5l5K48AND @j61NK4A

future therapeutic approaches in these diseases. Transcriptional mactivation associated with aberrant promoter region hypermethylation isat least partially reversible with demethylating agents such as 5-azacytidine and 5-aza-2'-deoxycytidine (18—20, 22). Although theseagents have been used in clinical trials for acute leukemias andmyelodysplasia, the specific mechanisms underlying beneficial resultshave not been explored. The extremely frequent methylation of

@J5INK4B and/or @J6JNK4A in leukemias and lymphomas make reacti

vation of expression of these cell cycle-controlling genes an invitingtherapeutic opportunity. Future studies of 5-azacytidine and newstrategies for demethylation as a treatment for hematological malignancies should include careful monitoring of the pI5@―@4'@and

@j6hiW@4@@genes as a guide to therapeutic efficacy.

Acknowledgments

We thank Drs. Connie Griffin and Angela Manns for tumor samples andDrs. Jeremy Graff and Richard Ambinder for review of the manuscript.

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INK4Ap16 and INK4Bp15Distinct Patterns of Inactivation of

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