Rearrangements of the tal 1 locus as clonal markers for T cell acute lymphoblastic leukemia

Rearrangements of the tal-1 locus as clonal

markers for T cell acute lymphoblastic

leukemia.

O G Jonsson, … , G R Buchanan, R G Smith

J Clin Invest.

1991;

87(6)

:2029-2035.

https://doi.org/10.1172/JCI115232

.

Normal and aberrant immune receptor gene assembly each produce site-specific DNA

rearrangements in leukemic lymphoblasts. In either case, these rearrangements provide

useful clonal markers for the leukemias in question. In the t(1;14)(p34;q11) translocation

associated with T cell acute lymphoblastic leukemia (T-ALL), the breakpoints on

chromosome 1 interrupt the tal-1 gene. A site-specific deletion interrupts the same gene in

an additional 26% of T-ALL. Thus, nearly one-third of these leukemias contain clustered

rearrangements of the tal-1 locus. To test whether these rearrangements can serve as

markers for residual disease, we monitored four patients with T-ALL; three of the leukemias

contained a deleted (tald) and one a translocated (talt) tal-1 allele. These alleles were

recognized by a sensitive amplification/hybridization assay. tald alleles were found in the

blood of one patient during the 4th mo of treatment but not thereafter. Using a quantitative

assay to measure the fraction of tald alleles in DNA extracts, we estimated that this month 4

sample contained 150 tald copies per 10(6) genome copies. The patient with t(1;14)

(p34;q11) (talt) leukemia developed a positive assay during the 20th mo of treatment. By

standard criteria, all four patients remain in complete remission 11-20 mo into treatment. We

conclude that tal-1 rearrangements provide useful clonal markers for approximately 30% of

T-ALLs.

Research Article

Rearrangements of the

tal-1 Locus

as

Clonal

Markers

for T Cell Acute Lymphoblastic Leukemia

OlafurG.Jonsson,*Richard L.

Kitchens,*

Richard J.

Baer,'

GeorgeR.Buchanan,*andR. Graham

Smith*

Departments of*Pediatrics, tInternalMedicineandMicrobiology, University ofTexas SouthwesternMedical Center, Dallas, Texas 75235

Abstract

Normal and aberrantimmunereceptor geneassembly each producesite-specificDNA rearrangements in leukemiclympho-blasts. In either case, these rearrangements provide useful clonal markers for the leukemias in question. In the

t(l;14Xp34;q1l)translocationassociated with T cellacute lym-phoblastic leukemia(T-ALL),thebreakpointsonchromosome 1interruptthe tal-i gene. Asite-specificdeletioninterruptsthe

samegene inanadditional 26% of T-ALL.Thus, nearly

one-third oftheseleukemias contain clustered rearrangements of thetal-)locus.Totestwhetherthese rearrangementscan serve as markers for residual disease, we monitored four patients

withT-ALL; threeoftheleukemias containedadeleted(tald)

and one atranslocated (tal) tal-i allele. These alleles were

recognized by a sensitiveamplification/hybridization assay. tald alleleswerefoundin the bloodofonepatient duringthe 4th mooftreatmentbutnotthereafter.Usingaquantitativeassay

to measure the fraction oftald alleles in DNA extracts, we

estimatedthat this month 4samplecontained 150

tall

copies

per106genome

copies.

Thepatientwith

t(1;14Xp34;q11)

(tat)

leukemiadeveloped apositiveassayduringthe20thmo of

treatment.By standardcriteria,allfourpatients remainin

com-plete remission 11-20 mo into treatment. We conclude that

tal-irearrangementsprovide usefulclonal markersfor - 30%

ofT-ALLs.(J. Clin.Invest.1991.87:2029-2035.)Key words: minimal residual disease * helix-loop-helix proteins

Introduction

T cell acute lymphoblastic leukemia (T-ALL)' comprises

- 15%ofall casesofALL. Featuresthatdistinguish this

dis-ease from B-lineage ALLinclude higher incidence in males,

oldermeanageatdiagnosis, highmeanbloodleukocytecount,

andthe

frequent

presence ofa mediastinalmass(1-3). Al-though theprognosis for both children (4, 5)andadults (6, 7)

hasimproved with increased therapeutic intensity, - 40% of

childrenand at least60% ofadultsstillrelapse and dieof

drug-resistant disease.

Addresscorrespondence andreprint requests to R. Graham Smith,

Department of InternalMedicine,University ofTexas Southwestern

Medical Center,Dallas, TX 75235-8852.

Dr. Jonsson'scurrentaddressisDepartmentofPediatrics, St. Jo-seph'sHospital,Landakot, 101 Reykjavik, Iceland.

Receivedforpublication 2 October 1990and in revisedform 9 Jan-uary1991.

1.Abbreviationsused in this paper: T-ALL,T cell acute lymphoblastic leukemia;taldt,deleted,translocatedtal.

Because induction of complete remission is achieved in the vastmajority of patients, current effortstopreventtreatment

failure focus upon modifications of postremission consolida-tion and/or maintenance chemotherapy. Unfortunately, the disease isnotdetectable by routine analysis during the remis-sion period; thus, the effect of therapy on tumor burden is difficultto assess.A sensitiveclonal assay for the residual leu-kemicpopulation during remission would help guide therapeu-ticdecisions beyond the induction period.

A number ofchromosomal translocations are associated with T-ALL (8-10). Theoretically, the underlying molecular rearrangementscould form the basis for sensitive clonal assays for minimal residual disease, justasBCR-ABL gene fusions have servedasmarkers in chronicmyelogenous leukemia (1

1-15). However, nosingletranslocation is found inmorethan 5-10% of cases, and 20-30% of these leukemias display no

karyotypicabnormalitiesatall(8-10).Thiscytogenetic

hetero-geneitysuggests thatanumber ofloci will needtobe

character-izedindetail beforespecificmolecularprobessuitable for de-tection of occult leukemia in themajorityofcases canbe

pre-pared.

We have recently isolated and sequenced thebreakpoint regions derived from one recurrent cytogenetic defect in T-ALL, namely the t(l ;l4Xp34;qI1)translocation (16, 17).In six

cases analyzed in detail, the breakpoints on chromosome 1 clustered within a 1-kbregion. This translocation cleaves the

tal-igeneonchromosome1,separatingits 5' end from therest

of the gene, which is transposed into the Tcell receptora/6 locusonchromosome 14.Thetal-Igenepotentiallyencodes a

protein containingahelix-loop-helix domain, which isfound

in agrowing number of highlyconserved DNAbinding pro-teins involved in the regulationofgrowthand development (18).Severalgenes inthis familyareknown to bedisruptedin subsets of ALL(19-23). Althoughofpotential pathogenic

sig-nificance, the t(1;14)(p34;ql1)translocation is foundin only

3% of T-ALLs (24).

To investigatethe possibility ofa widerrole forthe tal-i

gene inT-ALL,wesearchedforrearrangementsof thisgene in theblastcellsfromagroupof 50 patients withT-ALL.These

leukemias didnot harborthet(l;l4Xp34;ql 1) translocation. Surprisingly, 13(26%) of these leukemiascontained

rearrange-ments atthis locus, all of whichwereidenticalatthe levelof

Southern hybridization analysis (25). Detailed analysis re-vealeda

site-specific

- _{90-kb deletion}onchromosome

1,

one end of whichlies -1 kb from theclustered translocation breakpointsinthe

t(1;l4)(p34;ql

1) cases. As demonstrated by

nucleotidesequenceanalysis, thedeletions are all remarkably

site-specific, differingattheir ends by onlyafewbasesfromone

leukemiatoanother.Thesedeletionswere notfoundin remis-sionperipheral blood leukocytes and therefore appear to be

leukemia specific.Thus,site-specificrearrangements atthe tal-I locus characterize

nearly

30% of T-ALLs: 3% aredueto

translocation

(talt

alleles), while 26% result fromaninterstitial

J.Clin. Invest.

K TheAmerican Society forClinicalInvestigation, Inc. 0021-9738/91/06/2029/07 $2.00

deletion that istoo smalltobe detectedcytogenetically (tald alleles). Theserearrangementsprovide the opportunityto de-velopsensitive clonalassaysfor the relevant leukemias. In this

paper, wedescribeassaysthatcandetect 10rearranged tal-1 cells inabackground of 106 normal cells. Moreover,a modifica-tion of theassay is presented which quantitates tald alleles. Using theseassays, wehave monitored fourpatients with T-ALLwhoarecurrently in remission.

Methods

Patientsand cells. Four patients(L14, L23, L54, and L8 1) with T-ALL were studied. Ages at diagnosis were 10, 7, 9, and 28 yr, respectively. Theimmunophenotype of these leukemias wasCD5 + CD7 + CD19

-TdT+(terminal deoxynucleotidyl transferase). Patients were treated

accordingtoPediatric Oncology Group protocol 8704 (26) (patients L14, L23, and L54) or a minor modification of the Linker regimen for adult ALL (27) (patient L8 1). Normal peripheral blood mononuclear cellswereobtained from consenting adults. Small samples of thymus wereobtained from children (ages 1-20 mo) undergoing cardiac sur-gery. Verterbral bone marrow was obtained at autopsy from young adultaccident victims within 12 h postmortem. T-ALL cell lines RPMI 8402 (28) and MOLT 16 (29) were grown in RPMI 1640 medium containing 10% fetal bovine serum. Blood and bone marrow leuko-cytes werelysed with ammonium chloride and high molecular weight DNApurified by standard methods (30).

DNAamplification. PCR werecarried out as recommended by the manufacturer (Perkin-Elmer Cetus Corp., Norwalk, CT), except thatI

Uof Perfect Match (Stratagene Inc., San Diego, CA) was included in eachreaction.Inearlyexperiments,apairofoligonucleotideprimers, C and D,wereusedtoamplifyatald fragmentof - 225 bp (25). To

increasespecificityofamplification,two newamplimers, P1 and P2, wereconstructedfrom the nucleotide sequences on either side of the

commondeletion(Fig. I A).Relativetotheorientationof the tal-i gene,oligonucleotide P1 begins76 bases 3'tooligonucleotideC, and

oligonucleotide P2 is displaced 12 bases 3' to oligonucleotide D. Oligo-nucleotides P1 and P2, amplifying tatd fragments of - 165 bp, were

routinely used for the detection and quantitation assays reported in this study. Anothersetofoligonucleotides, Xl and X2,wasprepared for the detection of thetaP translocation in leukemia L23 (Fig. 2). These oligomers amplify a 251-bpfragment from the L23 leukemia.High molecularweight DNA (0.1-10 Mg) was added to PCR mixtures and the corresponding number of genome copieswascalculated assuming that 1.5X I05diploid cells containI /gDNA.Amplificationwas

ac-complished inaPerkin-Elmer Cetus Corp. thermalcyclerin 60 cycles. Thefirst cycle consisted of 3 min at 940,1minat610 and 2minat72°; in subsequent cycles the melting step lasted I min. Control PCR

mix-tures were runwith everyexperiment and'included normal liveror

thymus DNA and- _{Ing tatd DNA.Oligonucleotides}P1and F(Fig.I

A) were used toconfirm the integrity of theDNAsamples; all samples containatleastonenormalgermline chromosome Ip, which yieldsa

250-bpfragmentuponPCRcontainingthese primers.Standard pre-cautionsweretakentoavoid contamination of PCR reaction mixtures with PCR products (31).

Hybridization. Aliquotsofthe PCR mixtureswereseparatedon3% NuSeive/1% SeaKemGTG agarose gels (FMCBioproducts,Rockland, ME), denatured, transferredtoHybond-N filters (Amersham Corp., Arlington Heights, IL) and fixedtothe filterby baking. Two kinds of

oligonucleotideswereused forhybridization.Thefirst,oligonucleotide H,was an18-merwhichdetected all tat" alleles (Fig.IA). The second were 4 oligonucleotides which detected specific rearrangements in thetat"leukemias L14, L54 and L81 (Fig. IB)orthetal' leukemia L23(oligonucleotide H2;Fig. 2). End labeling of oligonucleotides, hy-bridization andstringent washingwascarried out aspreviously de-scribed (32).

QuantitativePCR assayfor taldalleles. PCRwascarriedoutas

describedabove, except that internal standard DNAswereaddedto

each reaction(33, 34).The standardswerechosentoyieldanamplified

productthatdiffered insizefrom the targetproduct by -10bp.Thus,

RPMI8402 DNAwasusedasastandard in assays for L14alleles,while MOLT-16 DNAwasused in assays for L54 alleles(Fig. 1 B).DNA standardswereseriallydiluted into normal liverorthymusDNA(12

-_centromere germlineip

TTTGCAGTCGATAACGTGCCATTTAAGTTGTTTTTACGGTGGGAATTTCTTGAGGACTGAAACCTTGAATGCTCGCTCTTGCATTCCTCACATTTCTGGCTCACACTCTGCTACGTAGT...ACGGTGTCTGGGAGAAGGTTTCACAGAGGAAGTGA

oligonucleotide F d

taL junction rearranged

TTTGCAGTCGATAACGTGCCATTTAAAGTTGTTTTTACGGTGGGAATTTCTTGAGGACTGAAACCTTGAATGCTCGCTCTTGCATTCCTCACAATTTCcgratcaaaTCATTTCTTCTTCGTGGTTGTGTGCATGCGGTGGGATTGTGAGAGTGCGTTC

No- site <

oLigonucLeotideP1 oLigonucleotide N oligonucLeotideP2

germline1p telomere0

germline centromeric lp

Cell line RPMI 8402 MOLT 16

Patient L14 L54 L81

CATTCCTCACAATTTCTGGCTCACACTCTGCTACGTAGTAAGGG CATTCCTCACAATTTC

CATTCCTCACAATTTCTGGCTCTA CATTCCTCACAATTTCTGG CATTCCTCACAATTTCTGGC CATTCCTCACAATTTCTGG

cggatcaaa ttaggggttc gaaacgactt

g

TCATTTCTTC

GGTTTTCATTTCTTC

TTGGTTTTCATTTCTTC

GTTGGTTTTCATTTCTTC GGTTTTCATTTCTTC

germline telomeric lp

Figure1. (A) Sequence of the deletedtal-i (tald)allelederived from the culturedT-ALL celllineRPMI8402.Thegermlinesequencesof the

centromeric(top line)and telomeric(bottom line) sides of therearrangementarealigned with thetald allele (middle line). About 90 kb of the

normalsequenceisdeleted duetotherearrangement(reference 25). The distal end of this deletion liesinthe first intron ofthetal-igene. Junc-tionalnucleotidesnotfound in thegermlinesequencesareinlowercaseletters. OligonucleotidesP1andP2areusedtoamplifytaldalleles. Oli-gonucleotidesP1 and Famplifya250-bp fragmentonthe 5'(centromeric)side of normaltal-Ialleles andserve ascontrolprimersinPCRassays.

OligonucleotideHis usedtodetectamplified talt alleles inhybridizationassays.Dots(... ) in thetoplinesignifynucleotides omitted from thefigure. (B)taldalleles from fiveT-ALLs, includingthreepatients currentlyinremission(L 14, L54,L81)andtwocell lines(RPMI8402 and MOLT16).Asin A, thetaldsequences arealigned with germline centromeric(topline)andtelomeric (bottom line)sequences,and nongermline nucleotidesareshowninlowercaseletters.The sizes of fragments amplified with oligonucleotidesP1and P2areshownattheright.

159 bp

172 bp

170 bp 162 bp

159 bp

TGCCGCCGCCCCTCZ.GCCTCGGAGACTCTCTTCCTTCCTTCCCCCTTTTCCTTACGCAATATACAGAAATGCGCGAGGCTGTGGTTGGTTTTCATTTCTTCTTCGTGGTTGTGTGCATGCGGTGGGATTGTGAGAGTGCGTTC

- centromere germLine 1p

AAACACCTGCAGTTGGGGGCGGAAAGCTGTTTG.. .AAACTCGCT1TTGGGCGCGGCAGATCGCCCAGGACCACACCGCAGCGTMCTGCAG

der ip AAACACCTGCAGTTGGGGGCGGAAAGCTGTTTG....AA

oligonucLeotideXl

I D62 JS1

RACTCGCTTTGGGCGCGGCAGATCGCCCAGGmawangagcgagatggACTGGGGG&ATtttaagaATACTCATCTTTGGAAAGGAACCCGTGTGACTGTGGAACCAAGTAA

oligonucLeotide H

germl ine14q

2

...

2

D82

otigonucleotideX2

... ....teLomere

-kCCTGTACAAAAACTGCAGGGGCAAAAGT

Figure 2. Sequence of the translocated tal-i(talP)allele in T-ALL frompatientL-23. Chromosome I p(top line)and 14q (bottomline) sequences

arealignedwith thetaltallele(middle line).ThebreakpointonchromosomeIpis in the first known intron of thetal-Igene, 2.5 kb 3' of the distal side of thetalt rearrangements(references 16 and 17). Thetranslocationcreates aderIp chromosome,whichjoinsthe firstexonof the tal-I geneto arearranged D62-J51 geneonchromosome 14q11.Nongermlinejunctional nucleotidesareshown in lower case letters.D62and

J61 segmentsareoverlined. Heptamer-nonamer recombination sequencessurroundingthegermlineD62 segmentarehighlightedwithoverlying

dots.OligonucleotidesXl and X2areusedtoamplifythe L23talt allele; oligonucleotideH2 is usedtodetect thisamplified productina

hy-bridization assay. Dots(... )in the top and middle linessignifynucleotides omitted from thefigure.

yg/ml). The dilutions of standardDNA wereaddedtoPCRmixtures containingtargetDNAswhosetaldallelesweretobequantitated.The productswereseparated byelectrophoresisthrough 10%

polyacryl-amide gels and visualizedby stainingwith ethidiumbromide. The

origi-nal number of targettaldalleleswasestimatedbydensitometric

com-parison ofstainingintensities of the standard and targetproducts.

Equal numbers of standard and target alleles presentatthe startof

amplificationwereshowntogenerateequalyieldsofproducts.Refined estimates of target allele numberwereobtainedbyrepeatingassays in the presence ofanarrowrange of twofolddilutionsof standard DNAs.

Results

Basisfor detection oftal/- gene rearrangementsinT-ALL.The rearrangements ofthe tal-I gene exploited in this study are shown inFigs. 1 and2. The

tald

alleles are the consequence of

an 90 kbdeletion which amputatesatleastoneexonof the

tal-igeneonchromosome lp

(25).

The5'and3'ends of these

deletionsareremarkablyclustered withinafew basepairsinall

tald

allelessequencedsofar (25).Forthefive T-ALLsstudied herein, thesequences near these ends areshown inFigure 1B.

Each

tald

allele differs by several nucleotidesattheproximal anddistalendsof the deletionand in the sequence of the extra

nongermline nucleotides that replacethedeletion. These

fea-tures enablethe

specific

detection ofthesealleles by PCRand

hybridization

assays, asdescribedinMethods.Asimilar strat-egy was used to detect thet(l

14Xp32;q

11)

(tal')

allele in

mate-rialfrom patientL23(Fig. 2).

Distribution of tald alleles. Using Southern hybridization

analysis,

wepreviously reported that thetald rearrangement wasfoundin13of 50T-ALLdiagnostic bloodorbonemarrow

samples (25). Inthis study,wefounda

perfect

correlation be-tweenresultsoftheSouthernandPCRassaysofthese50

leuke-mias. A sample ofthe data is shown in Fig. 3. In Southern

hybridization to atal-i probe, two

tald-positive

leukemias

(lanes 4 and8)yieldeda novel4.5-kbEcoRI fragment. PCR assayscontaining oligonucleotidesC and D generated products

- 225bpinlength from the same twoleukemias(see

Mieth-ods) (25). The other seven leukemias shown in Fig. 3 were

negative byboth Southern and PCRanalysis.

In orderforthe

tald

PCR assay to be ausefultool for the

detection of minimalresidual disease,

talt-bearing

cellsshould

notbefound innormalhematopoietic tissues. UsingthePCR

assay, wefoundnoevidence of suchcellsinnormalperipheral

blood mononuclear cells (n = 40), thymus(n = 5), or bone

marrow(n =5) (datanotshown). Thesensitivityof this assay was 5-10 genomecopies(cells) per 106. Therefore, within the limitsofdetection ofthisassay,taldrearrangements arespecific for leukemic cells.

Detection

ofresidual

diseasein T-ALL.Four

patients

with

T-ALLwerestudied. Threeoftheseleukemias (L14,L54,and L8 1) contained the

tald

deletion, whilethefourth(L23)hadthe talt translocation. The results of surveillance for taldalleles are

showninFig.4andsummarizedinTableI. All fourpatients

were in complete remission at the time of collection ofthe

samples forassay.

Characteristicbandsrepresenting tald fragmentswere

am-plified in PCRs containing the diagnostic bonemarrow sam-plesfromeachofthese threepatients (Fig.4, lanes 1, 6, and10). These bands differ slightly in size, as predicted from the se-quencesofthe corresponding taldalleles(Fig.1B). Oligonucle-otideprobesHand P1, butnotP2,hybridizedto faster-migrat-ing bandsaswell(Fig.4 C, lanes 1, 3, 4, and 7;and data not

shown). Therefore,these faster-migratingbands areprobably single-stranded

tald

DNA produced during the PCR reaction (35). Dilution experiments demonstratedasensitivity of

detec-tion of10

copies

per 106(Fig.4 AandC, lanes 2-4).

tald

alleles

were not detected in blood or bone marrow samples drawn

from patients L14and L81 between the 4th and 20th mo of

1 2 3 4 5 6 7 8 9 Figure3.Assayfortalt

A alleles in bonemarrow samplesobtainedatthe

225-

time

of diagnosis from

patientswith T-ALL. (A) Products of PCR

re-B actions containing

oli-250- gonucleotides C andD,

which amplifyatald C _fragmentof 225 bp

(reference 25). Size

4.4

- _{markers(bp)areshown} byhorizontallinesat

theleft side of the fig-2.0 - _{ure. (B)PCRproducts}

amplifiedwith oligonu-cleotides PI andF toconfirmDNAintegrity. (C) Southern analysis ofgenomicDNAdigestedwithEcoRIandhybridized withtal-i probeB2EE-2.0(reference25). Size markers (kb) are shown by hori-zontallinesattheleft side of thefigure.

..-I ... ---

L14

L54 L81 appearance of a minimal tumor burden rather late in the

courseoftreatmentof patient L23. Furtherobservation is

nec-essary to understand the significance of this finding.

LO~t

LO LO LO

co>LOt LO)

co

)

Quantitativeassay

for

tald alleles. The

availability

of

sev-0 0 0 0 0 0 0 0 eraltaldalleles,eachofwhich yieldedaPCR productofdistinct x x x x x x x x size, allowed us to establish a quantitative assay for genomes O of) C

_containing

these rearrangements. In this assay, known amountsofa

tald-containing genomic

DNAservedasinternal

C] C)D

CDL

standards for themeasurementof

target

alleles

(33,

34).

Stan-dard andtargetPCR productsweredistinguishedbytheir

dif-12 3 4 5 6 7 8 9 10 11 _{ferent lengths on polyacrylamide gels. The ratio ofthe yields of}

thetwoproductswasestimatedbyvisual inspectionand scan-ning densitometry. As shown in Figure6 A, thisassay accu-rately measured 50 and 250 copies of Ll4D

tald

alleles diluted

into 1.2 x

106

normalthymus genomecopies.In this case, the RPMI 8402 taldallele served as theinternal standard for the

Ll4D target allele, the PCR products differing by 11 bp in

length (Fig. 1 B). To measure the number of

tald

alleles in

bloodsample

L54F1,

MOLT 16 DNA waschosenas a

stan-dard. Approximately 30 copies of theL54alleleper1.9x

l01

U

genomes

(150/106)

were

present

atthis time

_point

_(Fig.

6

_B).

As

noted, samples obtained from patient L54 after this timewere

negative in the

amplification/hybridization

assayandtherefore contained <10 tald alleles persample analyzed (Fig. 4, Ta-ble

I).

118

-Figure 4.Detection of tald alleles by amplification/hybridization as-say.PCR mixtures contained: highmolecularweightDNAderived

frombloodorbonemarrowcells ofpatients L14, L54,orL81;and

either(A and C) oligonucleotidesP1 and P2(Fig. A)fordetection oftaldalleles;or(B)oligonucleotidesP1 andF(Fig. A)tocontrol for DNAintegrity.D indicatesdiagnostic sample;Fsamplesare

fol-low-upperipheralbloodleukocytescollectedduringclinical remission

(Table I).Thenumber ofgenomecopiesaddedtoPCRmixtures,

shown above thelanes,wascalculatedassuming I ggDNA= 1.5 x l0 genomes.Sizemarkers(bp)areshownbyhorizontal lineson

the left side of thefigure. (AandB)Ethidiumbromide-stained 10%

polyacrylamide gelsof PCRproducts. (C)Southernhybridizationof

PCRproducts amplifiedwitholigonucleotidesPI andP2,probed with oligonucleotideH(Fig. IA).

treatment(Table I). Examples of these negative assays are showninFig. 4, lanes5 and 11.Bycontrast, tald alleleswere detectedinabloodsample drawnfrompatientL54duringthe 4thmo oftherapy(Fig. 4, lane 7). However, specimens col-lected frompatient L54 from the 5th through the 11thmoof

treatmentwerenegative (Table I).

Detectionoftal'alleles in material frompatient L23 is shownin Fig. 5 and TableI. The characteristic 25l-bp band

was seenupon amplification of DNA isolated from the diag-nosticmarrow sample (Fig. 5 A, lane 1). This bandwasnot obtainedwith normalthymusDNA(lane 5) andwasdistinct fromthecontrol 250-bp fragment from the5'region ofthe tal-1 locusamplified with oligonucleotides P1 and F (B). The sensi-tivity of detection, 5-10 copies per 106, was similartothat obtainedinthe

tald

assay(lanes 2-4).Abloodsampledrawn in

the 19thmooftherapywasnegative (lane 6). However, mo

latertheassay wasclearlypositive (lane 7). Because this patient showednoclinicalorhematological signs of relapseatthetime

the latter sample was collected, this assayresult implies the

TableI.Rearranged

tal-1

AllelesinBlood andBone Marrow

Samplesfrom T-ALLPatients

Monthof

Patient Sample treatment Detection

L14* Do 1 +

F1 4

-F2 17

-F3 18

-F4 20

-L54* D 1 +

F1 4 +(150/106)

F2 5

-F3 6

-F4 8

-F5 9

F6 9

-F7§ 10

-F8 11

-L81* D 1 +

F3 13

F4 14

F6 15

L23t D 1 +

F2 19

F3 20 +

DNAwasextracted fromdiagnostic(D)orfollow-up(F)peripheral

bloodorbone marrows leukocytesandassayed by the

amplification/hybridization procedurefortald (*)ortap($)alleles. +, allelesdetected; -, allelesnotdetected. Both bloodand bonemarrow

frompatient L54wereassayedatmonth 10.taldalleles insample

L54F1 werequantitatedasdescribed in Methods.

A

194

-

118-B

250

-C

-A 271

234

-B

I

250- 1

C

271 -|

234

-L23 NT L23 Figure5. Detection of

taltalleles by

amplifica-Cs Ln tion/hybridization as-X x' x say.PCR

mixturescon-C1 _{CD LO} 0~ OC, -Cl

CO O ° 6c 6 tained:highmolecular

anaLL

weight

DNA

derived

1 2 3 4 5 6 7 from blood or bone

marrowcells ofpatient L23; andeither (A and C)oligonucleotidesXl and X2(Fig. 2) for

de-5 tection oftaltalleles;or

(B)oligonucleotides PI

andF(Fig. IA)to

con-trolforDNAintegrity. Dindicatesdiagnostic sample;Fsamples arefollow-upperipheral blood leukocytes collected

duringclinical remission(TableI).NTindicates normalthymus

DNA.The numberofgenomecopiesadded to PCRmixtures,shown above thelanes, was calculatedassuming 1_jugDNA= 1.5X l0

genomes.(A andB) Ethidium bromide-stained 10%polyacrylamide

gel of PCR products. (C) Southernhybridizationof PCRproducts

amplified witholigonucleotidesXlandX2,probed with

oligonucle-otide H2(Fig. 2).

Discussion

Although significant improvements in longterm disease-free survival have resulted fromcontemporary

therapeutic

pro-grams, - 40%of childrenand60%of adults withT-ALLstill

relapseanddieofdrug-resistant leukemia.Atthe same

time,

an

increasing variety of therapeutic modalitieshasappeared, in-cluding novel doses and schedules of existing drugs, newer agents such aspentostatin

(deoxycoformycin), biologic

re-sponsemodifiers suchas

a-interferon, immunotoxins,

and

allo-geneic or autologous bone marrow

transplantation.

Because thetotalpoolofpatients is relatively smallandprognostic strati-fication isnotwelldeveloped, progress inselection of

optimal

therapy for individualpatientshasbeen limited.Accurate

de-tection andquantitation ofdisease

during

the intensive

postin-duction phase of therapy could facilitate selection ofthe best

consolidation/maintenance approach foreach individual

pa-tient.

Site-specificDNA rearrangementsinALLcellsprovide

clonal markersforthedetection of

residual

disease.These rear-rangements areoftwo sorts:physiologic immunereceptor gene rearrangementsandpathologic recombinationaleventssuchas

chromosomaltranslocations,

deletions,

andinsertions.

Exam-ples oftheformerprocess areTCRbVDJ segment rearrange-mentswhich havebeenexploitedasclonal markers forT-ALL

populations

(36, 37). Potential

disadvantages

of this approach include the requirement for specific probes for each clone, the dominance ofnewrearrangements

during

clonal

progression,

and doubtsregarding true tumor

specificity

of the particular rearrangement

being

monitored. Use of the

latter,

pathological

kinds ofrearrangements asclonal markersmay overcomethese limitations, especially ifasite-specific abnormalitywerefound inalarge

fraction

ofT-ALLs.

The

t(1;14)(p34;ql

1) translocation is foundinonly 3% of

T-ALL(24). Thefinding ofagene

tal-i,

encodingacandidate

helix-loop-helix

DNAbinding proteinatthe site of the

break-points

onchromosome 1 p,raisedthequestionwhether other,

nontranslocational rearrangements of thisgene mightbe in-volved in leukemias of this type. Remarkably, 13 of 50 such

leukemias were found to contain nearly identical deletions of

- 90 kb from the upstream region of this gene (25). The

dele-tions were not found in remission peripheral blood leukocytes, indicating that tald alleles are not germline genetic polymor-phisms in these patients (25). The site of this common rear-rangementwithinthefirstknownintron ofthetal/- locus is -1 kb 5' of a cluster of breakpoints found in the

t(l;l4Xp34;ql 1) translocations inT-ALL(25).Inadditionto adding strong circumstantial evidence for a role of thesetal-i

rearrangementsin the pathogenesisofT-ALL, the remarkably focused natureof therearrangementsprovidesanopportunity

tomonitortheleukemiccloneswith straightforwardgenomic PCR assays. A single pair of amplimers sufficed for all tald

alleles,whileafewadditional pairs should be adequate to

de-tectthetaltcases.Atotalof 30%of T-ALLs should thus be amenable todisease monitoring based upon thesetal-i rear-rangements.

The successof thisapproach depends upon the absenceof

tal-i rearrangementsin normalhematopoieticcells. The site-specificnatureofthese rearrangements raisesthequestion whetherthey could play a roleinnormal T cell development or

function. Moreover, aberranttrans-rearrangements involving immune receptor genes have been demonstrated by PCR of

normalthymocyte DNA (38). Sincebothkinds of tal-i rear-rangementsstudied hereinmayoriginate from misdirected

ac-A

L14D

K

RPMI8402 CD

194-U

50 250

10 0 LO C) a

cm_c0 LO 10 c 0Ln 10

CY) cam c C\ cm u \rL

C)

C)

-L14D

-RPMI8402

L14D U1 h,- I c

RPMI8402 60

B

L54F1

F

MOLT 16

co

14 10 0 C) 0 ) C\4

6 C) _C)

C.o

1.9x10

5

.

1

COCo

co CD

-

MOLT 16

-

L54Fl

L54F1

MOLT1 6

CY) r'- v- (.0

c:i IC)

Figure 6. Quantitationoftaldallelesusinganinternal standard in PCR assay. (A)Quantitationof L14Dtaldalleles.Varyingamounts

ofRPMI 8402 DNA, shownasgenomecopiesabove thelanes,were

addedtoPCR mixturescontainingeither 50or250 genomecopiesof LI4D DNA. Theproductswereanalyzedon a10%polyacrylamide gelandstained withethidium bromide. The L14D and RPMI 8402 productsaremarkedontherightsideof thefigure;asize marker

(194bp) is shownontheleft. The band intensitieswerequantitated

tion of the immune receptor recombinase, we examined cell

populations containingmature and developing T lymphocytes

for evidence oftalt alleles. Usingthe

amplification/hybridiza-tionassay,whichdetects 10 rearranged alleles among 106 cells, wedidnot detect any

talt

alleles in normal mature or

differen-tiating hematopoietictissues.Thus,it is unlikelythat rearrange-ments atthetal-I locus play any important developmental or

functional roleinnormalTcells.Of practicalsignificance, the low backgroundoftheserearrangementsin normal

hematopoi-etic cells shouldnotinterfere with sensitiveand specific

detec-tionof the leukemic clones.

Inthisstudy, we have tested in four patients the suitability

oftal-Igene rearrangements as clonal markers for T-ALL. Of

the threesubjects withtalt leukemia, evidence ofresidual dis-ease wasfoundearlyinremissionin one (L54). However, the

leukemic

talt

marker was not detected during an ensuing fol-low-up periodof7mo. On the other hand, the one patient with

t(l;14)(p32;ql 1)

(talt)

disease hadapositive PCR assay in his

20thmooftreatmentwhichwas preceeded by a negative assay.

Onlyfurther observation of these and other patients willtest

thepredictivepowerof theseassaysfor diseaserecurrence.

Previous experiencesuggeststhat theabilitytoquantitate thesealleles inserial samples should increase the

reliability

of relapseprediction.Forexample, inpatients with chronic

my-elogenous leukemia followed aftermarrowablation and alloge-neicbone marrowtransplantation, the finding of isolated Ph' translocations either by

cytogenetic

orPCRassayhasnot

pre-dicted subsequent relapse. On the otherhand, serial increases in the Ph'markerorits molecular

equivalent

havecorrelated withhematologic relapse (1 1-15, 39).Therefore,wesoughtto

developa

quantitative

assayfortalt allelesbasedonthe PCR method. The modified assay relies on an internal standard which contains a second tald allele

distinguishable from

the

first

basedonthesizeof the

amplified product.

The

reliability

of thisassaydependsonthe

assumption

that

amplification

of thesetwoclosely relatedsequencesis

equally

efficient

through-outthe PCR.Under these

conditions,

therelative

yield

of the

twoproducts

depends solely

uponthe ratio of

copies

presentat

the startof the reaction

(33, 34).

This

assumption

shouldbe trueifno

cis-acting

factors favor the

amplification

ofoneallele

overthe other. To ensure such

equality,

we used

only

rigor-ously

purified

DNA

samples

inthisassay.Under these

condi-tions,

the

quantitative

assay

accurately

measured 50 to 250

copies

of

talt

allelespresentina

large

excess

of

normalDNA.

30

copies (150/106)

of the L54

talt

allele were found in the L54F1 sample

during

the 4th mo of treatment. We are

currently

adapting

this

approach

tothe

dsetection

oftattaswell

as

talt

alleles.

Nowthat

talt-

rearrangements havebeen showntobe

feasi-bleclonalmarkersfora

large

subsetof

T-ALLs,

expanded

clini-caltrialsare

appropriate

to testthe accuracyof

relapse

predic-tion. One

ancillary question

tobeaddressedinsuchtrials is the

stability

of theserearrangementsasclonal markers

during

dis-ease

progression.

Asmall subset ofT-ALL

patients relapse

with

ordevelopde novo

myeloid

leukemia

(40).

Whether

tal-I

rear-rangementspersist during such shifts inphenotype isan

intrigu-ing issue

touching

on both the

biology

and management of these

lineage-switching leukemias.

Acknowledgments

Wethank Jayne Cash for bloodsamplesfrompatientsL14,L23,and L54, BarryCooperfor bloodsamplesandclinical datafrompatient

L8 1, andEugeneFrenkelforacriticalreadingof themanuscript.

This workwassupportedbytheNational Cancer Institute(grants CA-42891, CA-44016, CA-46593, CA-47975, CA-33625,and

CA-21675[Core]),theChildren's CancerFund ofDallas,the Weekendto WipeOutCancer,and the NasherFamilyCancerResearch Fund.

References

1.Sen,L.,and L.Borella. 1975. Clinicalimportanceoflymphoblastswith T-markers inchildhood leukemia.N.Engl.J.Med.292:828-832.

2.Tsukimoto, I.,K. Y.Wong,and B. C.Lampkin.1976.Surfacemarkers and

prognosticfactorsinacutelymphoblasticleukemia.N.Engl.J.Med. 294:245-248.

3.Roper, M.,W. M.Crist,R.Metzgar,A.H.Ragab,S.Smith,K._Starling,J.

Pullen,B.Leventhal,A. A.Bartolucci,and M. D.Cooper. 1983.Monoclonal

antibodycharacterization of surfaceantigensinchildhood T-celllymphoid

ma-lignancies.Blood. 61:830-837.

4.Shuster,J.J.,J. M.Falletta,D. J.Pullen,W. M.Crist,G. B.Humphrey,

B. L.Dowell,M. D.Wharam,and M.Borowitz. 1990.Prognosticfactorsin

childhoodT-cellacutelymphoblasticleukemia:apediatric oncologygroupstudy.

Blood.75:166-173.

5.Pui,C.H.,F.G.Behm,B.Singh,M. J.Schell,D.L.Williams,G. K.Rivera,

D.K.Kalwinsky,J. T.Sandlund,W. M.Crist,and S. C.Raimondi.1990.

Hetero-geneityofpresentingfeaturesandtheirrelationto treatment outcomein 120

children withT-cellacutelymphoblasticleukemia.Blood.75:174-179. 6.Hoelzer, D.,E.Thiel,H._Lofller,T.Bitchner,A.Ganser,G.Heil,P.Koch,

M.Freund,H.Diedrich,H.RMhl,etal. 1988.Prognosticfactorsinamulticenter

studyfortreatmentofacutelymphoblasticleukemiainadults. Blood. 71:123-131.

7.Gaynor, J.,D.Chapman,C.Little,S.McKenzie,W.Miller,M.Andreeff,

Z.Arlin,E.Berman,S.Kempin,T.Gee,and B. Clarkson. 1988.Acause-specific

hazardrateanalysisofprognosticfactors among 199 adults withacute

lympho-blasticleukemia: the memorialhospitalexperiencesince 1969. J. Clin.Oncol. 6:1014-1030.

8.Raimondi,S.C.,F.G.Behm,P. K.Roberson,C. H.Pui, G.K.Rivera,S. B.

Murphy,and D.L. Williams. 1988.Cytogeneticsof childhoodT-cellleukemia. Blood. 72:1560-1566.

9.Kaneko, Y.,G.Frizzera,T._Shikano,H.Kobayashi,N._Maseki,and M.

Sakurai. 1989.Chromosomal andimmunophenotypicpatternsinTcellacute

lymphoblasticleukemia(T-ALL)andlymphoblastic lymphoma (LBL).

Leuke-mia.3:886-892.

10.Ucken,F.M.,K.J.Gajl-Peczalska,A.J.Provisor,and N.A.Heerema.

1989.Immunophenotype-karyotypeassociationsinhumanacutelymphoblastic leukemia.Blood.73:271-280.

11.Gabert, J.,I.Thuret,M.Lafage,Y.Carcassonne,D.Maraninchi,and P.

Mannoni.1989. Detectionof residualbcr/abltranslocation_bypolymerasechain

reactioninchronicmyeloidleukaemiapatientsafterbone-marrow

transplanta-tion. Lancet. ii: 1125-1128.

12.Lange, W.,D.S.Snyder,R.Castro,J. J.Rossi,and K. G. Blume. 1989. Detectionbyenzymaticamplificationof bcr-abl mRNA inperipheralblood and bonemarrowcellsofpatientswith chronicmyelogenousleukemia. Blood. 73:1735-1741.

13.Delfau,M.H.,J.P.Kerckaert,M._{Collyn d'Hooghe,}P.Fenaux,J.L. La,

J. P.Jouet,and B.Grandchamp. 1990.Detection of minimal residual diseasein chronicmyeloidleukemiapatientsafter bonemarrowtransplantation by

poly-merasechainreaction. Leukemia.4:1-5.

14.Kohler, S.,N.Galili,J.L.Sklar,T.A.Donlon,K. G.Blume,and M. L.

Cleary. 1990.Expressionof bcr-ablfusiontranscripts followingbonemarrow transplantationforPhiladelphiachromosome-positiveleukemia. Leukemia.

4:541-547.

15.Sawyers,C.L.,L.Timson,E.S.Kawasaki,S. S.Clark,0.N.Witte,andR. Champlin.1990. Molecularrelapseinchronicmyelogenousleukemiapatients

after bonemarrowtransplantationdetectedbypolymerasechainreaction. Proc.

Natl.Acad.Sci. USA87:563-567.

16.Chen, Q.,J.-T.Cheng,L.-H.Tsai,N.Schneider,G.Buchanan,A.Carroll,

W.Crist,B.Ozanne,M.Siciliano,and R. Baer. 1990. The tal geneundergoes

chromosometranslocationinTcellleukemia andpotentiallyencodesa

helix-loop-helixprotein.EMBO(Eur.Mol. Biol.Organ.)J.9:415-424.

17.Chen, Q.,C. Y.-C.Yang,J. T.Tsan,Y.Xia,A.H.Ragab,S. C.Peiper,A.

Carroll,and R.Baer. 1990.Codingsequences of thetal-Igeneare_{disrupted by}

chromosome translocation in human T cellleukemia.J.Exp.Med.

172:1403-1408.

18.Murre, C.,P.S.McCaw,and D.Baltimore. 1989.AnewDNAbinding

anddimerization motifinimmunoglobulinenhancerbinding, daughterless,

MyoD,and mycproteins.Cell. 56:777-783.

19.Leder, P.,J.Battey,G.Lenoir,C.Moulding,W._Murphy,H.Potter,T.

Stewart,and R. Taub. 1983.Translocationsamongantibodygenesin human

cancer.Science(Wash.DC).222:765-771.

20. Mellentin, J. D., S. D. Smith, and M. L. Cleary. 1989.Iyl-l,anovel gene alteredbychromosomaltranslocationin T cellleukemia,codes fora_proteinwith

21. Mellentin, J. D., C. Murre, T. A. Donlon, P. S. McCaw, S. D. Smith, A. J. Carroll, M. E. McDonald, D. Baltimore, and M. L. Cleary. 1989. The gene for enhancer binding proteins E l 2/E47 liesatthet( 1; 19) breakpoint in acute

leuke-mias.Science (Wash. DC). 246:379-382.

22.Nourse, J., J.D.Mellentin, N. Galili, J. Wilkinson, E. Stanbridge, S. D. Smith, andM.L.Cleary. 1990. Chromosomal translocation t(I;19)results in

synthesis ofahomeobox fusionmRNAthat codes forapotentialchimeric

tran-scription factor. Cell.60:535-545.

23. Kamps, M. P., C. Murre,X. H.Sun, and D. Baltimore. 1990.Anew

homeobox gene contributes theDNAbinding domain ofthet(1; 19) translocation protein in pre-BALL.Cell.60:547-555.

24.Carroll, A., W. Crist, M. P. Link, M. D. Amylon, D. J. Pullen, A. H. Ragab, G. R. Buchanan, R. S. Wimmer, and T. J. Vietti. 1990. The t( 1; l4Xp34;q11) translocationis non-random and restricted to T-cell acute

lym-phoblasticleukemia.Blood.76:1220-1224.

25. Brown, L., J.-T. Cheng, Q. Chen,M.Siciliano,W.Crist, G. Buchanan, and R.Baer. 1990.Site-specificrecombination of the tal-l gene isacommon occurrence in human T cellleukemia.EMBO (Eur.Mol.Biol. Organ.) J. 9:3343-3351.

26. Amylon, M., S. Murphy, and J. Pullen. 1988. Treatment oflymphoid

malignanciesaccordingto immune phenotype:preliminaryresults in T-cell dis-ease.Proc. Am.Soc. Clin.Oncol.7:225a.(Abstr.)

27.Linker, C. A., L. J. Levitt, M. O'Donnell, C. A. Ries, M. P. Link, S. J. Forman, andM.J. Farbstein. 1987. Improved resultsoftreatment of adult acute lymphoblastic leukemia. Blood. 69:1242-1248.

28. Minowada, J., and G. E. Moore. 1975. T lymphocyte cell lines derived from patients with acute lymphoblastic leukemia. In Comparative Leukemia Research 1973:Leukemogenesis.Y. Itoand R. Dutcher, editors. University of Tokyo Press, Tokyo. 251-261.

29. Kohno, K, T. Otani, and J. Minowada. 1986. Human T-cell leukemia cell lines (MOLT 16 and MOLT 17): production of IL2-like factor. Proc. Annu. Meet. Jpn. Cancer Assoc. 45:203a. (Abstr.)

30.Herrmann, B.G.,and A.-M.Frischauf.1987. Isolationof genomicDNA. MethodsEnzymol. 152:180-183.

31.Kwok, S., and R. Higuchi. 1989. Avoiding false positives with PCR. Na-ture(Lond.).339:237-238.

32.Jonsson, 0.G., R. L. Kitchens, F. C. Scott, and R. G. Smith. 1990. Detection of minimal residual disease in acutelymphoblasticleukemia using immunoglobulin hypervariable region specific oligonucleotide probes.Blood.

76:2072-2079.

33.Gilliland, G., S. Perrin,K.Blanchard, andH. F.Bunn.1990.Analysisof

cytokinemRNAand DNA:detectionandquantitationbycompetitive polymer-ase chain reaction. Proc.Natl. Acad.Sci. USA. 87:2725-2729.

34. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular Cloning:A

Laboratory Manual. Cold Spring Harbor Laboratory (CSH), Cold Spring Harbor, NY. 14.30-14.33.

35.Gyllensten,U.B., and H.A.Erlich. 1988. Generationofsingle-stranded

DNAby the polymerase chain reaction and itsapplicationtodirectsequencing of

the HLA-DQA locus. Proc.Nati.Acad.Sci. USA. 85:7652-7656.

36. Hansen-Hagge,T.E., S. Yokota, and C.R. Bartram. 1989.Detectionof minimalresidual disease in acutelymphoblasticleukemia by in vitro

amplifica-tionofrearranged T-cell receptor delta chain sequences.Blood. 74:1762-1767.

37. Campana, D., S. Yokota, E.Coustan-Smith,T. E. Hansen-Hagge, G. Janossy, and C. R. Bartram. 1990.Thedetection of residualacutelymphoblastic

leukemia cells with immunologic methods and polymerase chainreaction:a

com-parative study. Leukemia. 4:609-614.

38.Tycko,B., J. D. Palmer, andJ.Sklar. 1989.Tcell receptor gene

trans-rearrangements: chimeric gamma-delta genes in normal lymphoid tissues.

Science (Wash. DC). 245:1242-1246.

39. Arthur, C. K.,J. F.Apperley,A. P.Guo, F. Rassool,L.M.Gao, and J. M. Goldman.1988.Cytogeneticeventsafter bone marrow transplantation for chronicmyeloidleukemia in chronic phase.Blood.71:1179-1186.

40.Pui, C. H., F. G. Behm, S. C.Raimondi,R.K.Dodge, S. L. George, G. K. Rivera, J. Mirro, Jr., D. K. Kalwinsky, G.V.Dahl, and S. B. Murphy. 1989. Secondaryacutemyeloidleukemia in children treated foracutelymphoid