长模板PCR反应检测BCR-ABL融合基因 1999

Long-Template DNA Polymerase Chain Reaction for the Detection of the bcr/abl Translocation in Patients with Chronic Myelogenous Leukemia1

Cornelius F.Waller,Gereon Dennebaum, Christoph Feldmann,and Winand Lange2 Department of Internal Medicine I,Hematology/Oncology,Albert Ludwigs University Freiburg Medical Center,D-79106Freiburg, Germany

ABSTRACT

In most patients with chronic myelogenous leukemia (CML)primitive hematopoietic progenitors carry the ac-quired reciprocal bcr/abl gene rearrangement t(9;22)(q34.1; q11.21).However,not all of the progenitor cells express the bcr/abl hybrid mRNA or the p210fusion protein.These cells, therefore,might escape detection by techniques that are based on expression of the fusion gene.To circumvent this problem,we established a new detection method for the rearrangement at the DNA level.Because breakpoints might occur in a very large genomic region(>200kb),we devel-oped a long-template DNA-PCR(LT-DNA-PCR).In22of59 CML patients,fragments of up to19kb could be amplified. Furthermore,6of7leukapheresis products of three bcr/abl-positive patients which were collected after mobilization chemotherapy and had been shown to be negative for the bcr/abl rearrangement by FISH and by RT-PCR were clearly positive by ing a specific pair of primers,it is possible to detect the presence of,and to characterize,the individual gene rearrangement.This ap-proach could serve for diagnostic purposes as well as detec-tion of minimal residual disease under cytotoxic therapy or after purging regimens,being independent of expression of the bcr/abl hybrid mRNA or the fusion protein. INTRODUCTION

CML3is a clonal hematological stem cell malignancy. Usually,a typical peripheral blood count leads to the diagnosis.In95%of patients the Philadelphia chromosome(Ph)is present (1,2).This is the result of an acquired reciprocal translocation t(9;22)(q34.1;q11.21)of the c-abl oncogene from chromosome 9to the bcr on chromosome22(3).Due to this positioning effect a patient-specific chimeric fusion gene ually, fusion gene products of two slightly different sized mRNAs of approximately8.5kb are expressed,b2a2and b3a2.Both of them encode for a M

r

210,000fusion protein(p210bcr/abl),a protein with increased tyrosine kinase activity(4,5).

In the bcr,the translocation occurs somewhere in an intron within a5.8-kb genomic region(6,7).In abl,the chromosomal breakpoints are dispersed over an intron region as large as about 200kb(8).Complete intron sequence is available only for the bcr introns,whereas in abl,approximately95kb of the200kb are yet unknown(Fig.1A).

Two research groups independently reported that primitive CML progenitors carry the rearrangement but do not necessarily express the bcr/abl hybrid mRNA or the p210fusion protein(9, 10).Because of this,these cells might escape detection by techniques that are based on expression of the fusion gene. Therefore,we intended to establish a method for detection of patient-specific rearrangements at the DNA level.Unfortu-nately,the breakpoints in CML are dispersed over such a large genomic region that at present,it is not possible to cover them by conventional PCR methods.In1994,Cheng et al.(11) showed effective amplification of long targets from human genomic DNA using a recombinant Thermus thermophilus DNA polymerase.In accordance with their approach,we de-signed a set of LT-PCRs with one constant bcr primer and10abl primers at a distance of approximately15kb each.Depending on the translocation site the first abl primer3Јof the transloca-tion together with the constant5Јbcr primer should be able to amplify the fusion region(Fig.1B).

MATERIALS AND METHODS

Patient Samples.Peripheral blood from59patients with CML,4healthy volunteers,3control patients and three Phϩcell-lines(BV173,K-562,and LAMA84)were analyzed (Table1).Furthermore,samples from leukapheresis products of three patients with CP-CML collected after mobilization chem-otherapy consisting of idarubicin,cytosine arabinoside,and etoposide were analyzed(Table3).Samples were drawn after informed consent was obtained according to institutional guide-lines.

RNA Isolation.Total cellular RNA from1ϫ105to1ϫ106cells from peripheral blood or thawed leukapheresis samples was extracted using a RNeasy Total Kit(Quiagen,Hilden, Germany)according to the manufacturer’s instructions.

RT-PCR.RT-PCR amplification using nested primers was performed according to standard protocols as described previously(12).

Received5/6/99;revised8/24/99;accepted9/10/99.

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 with18U.S.C.Section1734solely to

indicate this fact.

1Supported in part by the W.Sander-Stiftung(C.F.W.,W.L.),the

Deutsche Krebshilfe(W61/93/La1),and the Zentrum Klinische For-

schung I,Albert Ludwigs University Freiburg(C.F.W.,W.L.).

2To whom requests for reprints should be addressed,at Department of

Hematology/Oncology,University of Freiburg,Hugstetter Strasse55,

D-79106Freiburg,Germany.Phone:49-761-270-3852;Fax:49-761-

270-3418;E-mail:Lange@l.uni-freiburg.de.

3The abbreviations used are:CML,chronic myelogenous leukemia;CP,

chronic phase;Ph,Philadelphia chromosome;bcr,breakpoint cluster

region;LT-PCR,long template-PCR;RT-PCR,reverse transcription-

PCR;FISH,fluorescence in situ hybridization.

4146Vol.5,4146–4151,December1999Clinical Cancer Research