Detection of Minimal Residual Disease in Leukemic Patients with the T(10;14)(Q24;Qll) Chromosomal Translocation1

[CANCER RESEARCH 50, 5240-5244. September 1, I990| Detection of Minimal Residual Disease in Leukemic Patients with the t(10;14)(q24;qll) Chromosomal Translocation1

Jacob Kagan, Lawrence R. Finger, Emanuel Besa, and Carlo M. Croce The Fels Institute for Cancer Research and Molecular Biology, School of Medicine, Temple University, Philadelphia, Pennsylvania 19140 fJ. K., L. R. F., C. M. C.], and Department of Medicine, Medical College of Pennsylvania, Philadelphia, Pennsylvania 19129 (E. B.Â¡

ABSTRACT clustered within a 263 base pair region of the TCL-3 locus on chromosome 10q24. (17) Early relapse and minimal residual disease during clinical remission In the present study we have applied the polymerase chain was examined in two patients having acute T-cell leukemia/lymphoma reaction (18) to amplify the t(10;14)(q24;ql 1) chromosomal with the t(10;l4Xq24;qll) chromosomal translocation. Molecular probes juncture for the detection of minimal residual disease during which can detect T-cell receptor a/6 cianai rearrangements and a TCL-3 remission. probe which can detect the clonal rearrangement due to the chromosomal translocation failed to detect the leukemic clones during clinical remission by Southern filter hybridization. MATERIALS AND METHODS However, application of the polymerase chain reaction technology in amplification of the t(10;14Xq24;ql 1) chromosomal juncture during clin Patient Material ical remission permitted us to increase the detection level of neoplastic Peripheral blood lymphocyte samples were collected during the cells up to 1 leukemic cell/125,000 normal cells using l ng of DNA. course of the disease from two patients, DW and JM, with T-cell Amplified junction fragments were detected in both patients. In one case, lymphoblastic leukemia/lymphoma and acute T-cell leukemia, respec during the period of clinical remission no amplified fragments were tively. Both patient's leukemic cells carry the t(10;14)(q24;ql 1) chro detected. mosomal translocation (16, 17). Samples. DW and JM are blood samples collected before treatment, during the course of disease. DW-R,, DW-R2, DW-R,, and JM-R blood INTRODUCTION samples were collected monthly during clinical remission. Lymphocytes were isolated from heparinized blood by Ficoll-Hy- Similarly to other hematopoietic malignancies the majority paque separation (Pharmacia). Other cell lines included in the present of lymphoid neoplasms are of monoclonal origin (1). Previ study were: 639 AD4, a somatic cell hybrid derived from the leukemic ously, the determination of monoclonality within lymphoid T-cells of DW which has retained the chromosome 10q+ (16); 648 neoplasms has been largely restricted to B-cell neoplasms which BD4, a somatic cell hybrid derived from the leukemic T-cells of JM display either one of the immunoglobulin light chain isotypes which has retained all the relevant chromosomes (10, 10q+, 14, 14q-) (K or A), a specific cytogenetic abnormality, or two different (17); and 6488E 10 (JM), a somatic cell hybrid derived from the polymorphic forms of glucose-6-phosphate dehydrogenase iso- leukemic T-cells of JM which has retained the chromosome 10q+. (17). zyme in the patient (2-4). Southern Blot Analysis Recently the development of a genetic approach, based upon molecular analysis of immunoglobulin and T-cell receptor genes Ten n% of high molecular weight DNA from patients or human rearrangement, has been used to determine monoclonality and placenta! DNA and 20 n<Â¿ofhigh molecular weight 648 li1)4 (JM) lineage specificity in tumors without lineage-specific surface somatic cell hybrid DNA were digested to completion by various determinants (5-13). In addition, molecular probes have been restriction enzymes, fractionated on a 0.6% agarose gel (Sigma), and transferred to either a nitrocellulose or a PVDF Immobiline membrane used in the detection of residual or recurrent disease not de (Millipore) as previously described by Southern (19). Hybridization tected by routine diagnostic methods (14). The sensitivity of conditions of the nick-translated DNA probes to a specific activity of 5 Southern filter analysis is useful in the detection of a clonal cell x 10* cpm/fig with the DNA-bound filters were previously described population only if it makes up 1-5% of the total cell population. (16). Attempts to amplify the immunoglobulin or T-cell receptor rearrangements are limited due to the large variety of alternative DNA Probes Variable (V), Diversity (D), and Joining (J) segments which (a) PSKW0.6H is a 0.6H fragment located 27 kilobases 5' to TCR2 might be also present in the normal cells in addition to the CÂ«segment3; (ft) PJK J02 2.4 E/B (17); (c) PJK J5, 3.0 Sad (17); (d) neoplastic cells. PlOUa, 5.OE (17); (e) PJK 1.7 E/B, a TCL-3 probe (17). On the other hand, cytogenetic abnormalities, such as translocations, inversions, and deletions, are nonrandomly associ Amplification of the t(10;14Xq24;ql 1) Chromosome Juncture ated with specific types of human leukemias and lymphomas Amplification with Taq DNA polymerase was performed according (3, 15). In these cases detection of patient genomic DNA to the manufacturer's instructions (Perkin Elmer Cetus, Norwalk, CT). rearrangements due to the chromosomal abnormality is a Target sequences were amplified from 1 /jg of genomic DNA in a 100- unique tumor-specific marker. Recently we have defined a novel Â¿il1x PCR buffer ( 10 miviTris HC1, pH 8.3-50 mM KC1-1.5 mM MgCl2- gene rearrangement in acute T-cell leukemia/lymphoma with a 0.01% (w/v) gelatin-2 mM dNTP-1 JIM of each of the 2 amplimers) t(10;14)(q24;ql 1) chromosomal translocation (16, 17). These with 2.5 n\ of Taq DNA polymerase. The reaction mixtures were overlaid with 100 ^' of paraffin oil and the reaction-capped tubes were translocation breakpoints occur within the T-cell receptor 5- incubated for 10 min at 94"C followed by serial cycles of denaturing (1 chain locus at the D52 segment on chromosome 14ql 1 and are min at 94Â°C),annealing (1 min at 65Â°C),and primer extensions (2 min at 72Â°C).After the final cycle the tubes were incubated for 10 min at Received 3/20/90; revised 5/21/90. the extension temperature (72Â°C).The PCR products were extracted 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 with 18 U.S.C. Section 1734 solely to indicate this fact. 2The abbreviations used are: TCR, T-cell receptor; PCR, polymerase chain 1Supported by CA 25875, CA 39860, American Cancer Society CH 378A. reaction. and W. W. Smith Charitable Trust. ' L. Finger, unpublished data. 5240

Fig. 1. Clonal rearrangements at the TCR D<5,1DÃ”2 J<5i1 CÃ¡II a/o locus. A, genomic organization of the TCR â€¢HEBE Â«HHE â€¢ IIH a/o locus. Top line: boxes, structural genes; closed and hatched boxes, coding exons and EII E HHH EHBHEBi/HB EHEHHEH 8â€¢^\ltf1/ifTm\EEHHBEHBEH 1 W 8HIBHBH the 3'-untranslated region, respectively. Re IIp0III I101 1 v Yiw//^lYiW_â€”_ II I\lI0 I striction sites: B, BamH\, E, EcoRÃŒ,H, JÃ“FM)mmm1100J<52J<534_â€” pJKJijf/SS PSKW0.6H1 Hindlll. PSKW0.6H, PJKJ62 E/B 2.4, PJKJ5 PJKJÃ•3OSdd1 3.0 Sad, and P101JSES.O, the locations of the probes used to detect rearrangements. />'.clonal 190 rf 120 " 30JaHHBEHHB rearrangement of the TCR Ja locus in the DW 10Coâ€¢ kbBUSM DNA samples. Southern blot hybridization of /yÂ«;Â«IIldigested DNAs probed with the PSKW 0.6H probe. C, clonal rearrangement at TCR Jo: locus in JM's DNA samples. Southern blot hybridization of EcoRI digested DNAs probed with the PJKJ52 E/B 2.4 probe. Å’ er (D) Clonal rearrangement at the TCRJÃ•locus 555 associated with a t(10;14)(q24;ql 1) chromo OOO kb somal translocation. Southern blot hybridiza tion of Ã„omHI-digested DNAs probed with -23 the PJKJ5 3.0 Sad probe. PL, human placental DNA; DW and JM, patient DNA samples -9.4 carrying the t(10;14)(q24;qll) chromosomal translocation; DW-R1, DW-R2, JM-R, DNA -6.6 GL- samples collected during clinical remission R- from both patients; 648 BD4 (JM), a somatic -4.4 cell hybrid derived from JM's leukemic T-cells GL â€” which has retained all of the relevant chro R â€” mosomes (10, 10q+, 14,14q-). Germline DNA fragments (GL) and rearranged DNA frag ments (R) are indicated, kb, molecular sizes in â€”2.3 kilobases. -2.0

SamHI EcoRI BamHl twice with Chloroform and 15 n\ of each sample was subjected to size complementary primer sequence located 120 base pairs 3' to the T-cell fractionation on a 4% agarose gel (1% SeaKem GTG agarose, 3% receptor DS2segment on chromosome 14ql 1 (20). Nusieve GTG agarose, FMC). DNA fragments were resolved on an agarose gel, transferred to an Immobiline membrane (Millipore), and hybridized with T4 polynucle- RESULTS otide kinase (Pharmacia) end-labeled oligonucleotides which are com plementary to an internal segment of the amplified sequence. Detection of Clonal Rearrangement. During our search for the t(10;14)(q24;qll) chromosomal translocations in acute T-cell Oligonucleotides leukemias and lymphomas we have applied a battery of TCR a/6 probes which spans the entire TCR a/6 locus to detect Oligonucleotides were synthesized and high performance liquid chro- DNA rearrangements by Southern blotting analysis. matography purified by Research Genetics (Huntsville, AL). Using a probe PSKW0.6H (Fig. I, A and B) located on Probe A. 17-mer5'-TCGCGCTGTCATTCACC-3' is a chromosome chromosome 14 ~27 kilobases 5' to Ca, we have detected a 10 sequence which is complementary to the segment adjacent to the 4.7-kilobase fiawzHI-rearranged fragment and a 3.6-kilobase l( 10;14) chromosomal breakpoints (17). BamHl germline fragment. The rearranged fragment was de Probe B. 19-mer 5'-ATCCGTCAAGGAAGCGGCT-3' is a chro tected only in DWs DNA and not in two remission samples of mosome 10 sequence complementary to the internal sequence in the DW (DW-R, and DW-R2), obtained for evaluation while the 263-base pair additional segment of DWs chromosomal juncture se patient was in remission. Another probe PJKJ52 2.4 E/B which quence (17). spans the chromosome 14 J62 segment detects a 5-kilobase Amplimers were designed to include both a primer sequence that is EcoRI germline fragment and a 3-kiIobase Â£coRI-rearranged complementary or reverse complementary to a flanking sequence of the fragment in the JM somatic cell hybrid 648BD4 (Fig. I, A and chromosomal breakpoint and an anchor sequence having several infre quent endonucleases restriction sites (S//I, Noti, Eagl, Smal in ampli- C) which has retained all of the relevant chromosomes (10, 10q+, 14, 14q). This clonal rearrangement is specific for JM's mers C and F or Sail in amplimers D, E, and G) to facilitate subcloning of the amplified fragments into PBluescript SK (Stratagene) for further leukemic T-cells and was not detected in other leukemic sam studies. ples or in the JM-R DNA sample obtained for evaluation while the patient was in clinical remission (Fig. 1C). Amplimer C. S'-GCGCGGCCGCGGAGGCCCGGGTCTGTCTC- A third clonal rearrangement was detected using the chro GGCTTCTGGCCTT- ' 3. mosome 14 TCRÃ´-probe PJKJ6 3.0 Sad. A 13.9-kilobase Amplimer D. 5'-TTGTCGACAAGAGACGTTCGCGGTTTCG-'3. /to/wHI-rearranged fragment compared to a 16.5-kilobase Amplimer E. 5'-TTGTCGACGTGTTTGTCTCCTGAGGCAT-'3. Amplimer F. 5'-GCGCGGCCGCGGAGGCCCGGGTAAGCTGG- BamHl germline fragment was detected in both patients with GATTAAG-'3. the t(10;14)(q24;qll) chromosomal translocation (Fig. ID). Amplimer G. 5'-TTGTCGACGGCCCAGCTTAAGGTTGGCAG- The same size fia/wHI-rearranged fragment was detected using A-'3. the TCL-3 probe PJK 1.7E/B derived from the chromosome Amplimers C, D, F, and G primer sequences are either complemen 10q24 region adjacent to the chromosomal breakpoint (Fig. 2). tary or reverse complementary to the t(10;14) chromosomal transloca These results confirm that both probes are located on the same tion breakpoint-flanking sequence (17). Amplimer E has a reverse rearranged fragment which spans the chromosomal transloca- 5241

D A Probe A -a CH 10 I' c" 'Q J20] C -aE r- CM JM 10q* h eh. 10 oo ÃœC~- OC DC o. co -3 o Q Ã» kb DWIOq1

D- Probe B â€”23 F R â€” â€¢ â€¢Â«â€”Primer l l AN sequences â€”9.4

M1234567M 1234567 1234567 GL â€” â€”6.6

â€”4.4

â€”2.3 â€”2.0 Fig. 3. The amplification of the t( 10:14)(q24;q 11) chromosomal translocation breakpoint junction. A, the amplification strategy. Amplimer C is located 5' to the breakpoint region on chromosome 10q24 and has a complementary primer to the flanking sequence: amplimer E is located 3' to the breakpoint region on chromosome 14qll and has a reverse complementary primer to the flanking sequence. Amplimer D has a primer reverse complementary to chromosome 10 sequence located 3' to the t(10:14) breakpoint. Amplimer F has a complementary 8amHI primer to the 5' end of the extra 263-base pair sequence on chromosome 10 in Fig. 2. Clonal rearrangements at the TCL-3 locus. Southern blot hybridization DWs breakpoint junction and amplimer G has a reverse complementary primer of Ã„amHI-digested DNAs probed with the PJK 1.7 E/B probe. PL, human to the 3' end of the extra 263-base pair sequence on chromosome 10 in DWs placenta! DNA; 6488E10 (JM); JM-R. JM patient DNA collected during clinical remission: DW, DW patient DNA; DH'-RI and DH'-R2, DWs remission samples. breakpoint junction. An internal oligonucleotide hybridization probe. Probe A, identifies amplified fragments between amplimer C and E or C and D, while GL, Germline DNA fragments: R, rearranged DNA fragments, kb, molecular Probe B identifies only amplified fragments which contain the 263-base pair sizes in kilobases. additional sequence adjacent to the breakpoint in DWs DNA. All the amplimers include an anchor (AN) sequence with endonucleases restriction sites. B, ethidum bromide-stained PCR-amplified DNA fragments separated on a 4% agarose gel. tion breakpoint. PJK 17 E/B probe also detects a 5.7-kilobase Lane I, negative control, human placenta! DNA amplified between amplimers C and E: Lane 2: positive control, amplification of the chromosome 10 sequence germline BamHl fragment from the normal chromosome 10 from human placental DNA between amplimers C and D: Lane 3, JM's DNA alÃelein the patient samples and in the human placenta! DNA sample amplified between amplimers C and E; Lane 4, JM-R DNA sample control (Fig. 2). As expected this probe detected only the 13.9- amplified between amplimers C and E; Lane 5, DWs DNA sample amplified kilobase Ã„awHI-rearranged fragment in the hybrid between amplimers C and E; Lane 6, DW-R, DNA sample amplified between amplimers C and E; Lane 7, DW-R2 DNA sample amplified between amplimers 6488E10(JM) since this hybrid retained the chromosome 10q+ C and E; M. markers. OX 174 Hae\\\ DNA fragments. C, Southern blot analysis and not a normal chromosome 10 alÃele(17). The rearranged of the ethydium bromide-stained gel (B) probed with Probe A. D, Southern blot 13.9-kilobase fiamHI-rearranged fragment was not detected in analysis of the ethydium bromide-stained gel (B) probed with Probe B. the samples JM-R, DW-R,, and DW-R; obtained during clini cal remission (Fig. 2). flanking sequence on chromosome 14qll (amplimer E, Fig. Although the clonal rearrangement at the TCR a/5 locus 3A). detected in DWs and JM's DNA may serve as individual clonal Amplification across the t(10;14)(q24;ql 1) chromosomal markers for each patient in monitoring the progression of the junction was carried out using amplimers C and E as a means disease, only probes which detect a rearranged fragment due to to monitor the level of the leukemic cells carrying the translo the chromosomal translocation will serve as a tumor-specific cation in the patient's peripheral blood lymphocytes. Amplified marker for patients with t( 10;14)(q24;q 11) chromosomal trans- fragments were detected in both patients prior to clinical re location. mission. As expected, DWs 486-base pair amplified fragment Amplification of the t(10;14) Chromosomal Juncture. The is larger than JM's 233-base pair amplified fragment because molecular cloning of the breakpoint sequences revealed that the of the additional 263 nucleotides adjacent to DWs breakpoint translocation occurred into the D<52segment of TCR<5on chro on chromosome 10 (17) (Fig. 3, A and B, Lanes 3-5). Ampli mosome 14qll and within a 263-base pair segment of chro fication of the chromosome 10 sequence from human placental mosome 10 (17). In order to amplify the translocation break DNA between amplimers C and D served as a positive control point junctions we have designed one set of amplimers with a and gave rise to a 149-base pair fragment (Fig. 3, A and B, Lane complementary primer to the 5' end of the translocation break 2). point-flanking sequence on chromosome 10q24 (amplimer C, By Southern blot analysis using "P-end-labeled Probe A we Fig. Â¡A)and another set of amplimers with a reverse comple were able to detect all of the amplified fragments which span mentary primer to the 3' end of the translocation breakpoint- the translocation junctions amplified between amplimers C and 5242

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1990 American Association for Cancer Research. MINIMAL RESIDUAL DISEASE IN LEUKEMIC PATIENTS 1 234567 As for the second patient DW, PCR amplification across the chromosomal juncture of the samples confirmed the initial Southern hybridization results (Fig. IÃŸand Fig. 2). A 486-base pair amplified fragment was detected only in the sample DW probed with Probe A (Fig. 3C, Lanes 5-7, and Fig. 4, Lane 1) or with Probe B (Fig. 3D, Lanes 5-7) and not in samples DW- RI and DW-R2 obtained while the patient reached clinical remission (Fig. 3, Lanes 6 and 7, and Fig. 4, Lanes 2 and 3). Shortly after those examinations patient DW relapsed. A 485-base pair fragment resulting from amplification between amplimers C and E (Fig. 4, Lanes I and 4) was detected in peripheral blood sample DW-R,. Controls in the experiment included a JM 233-base pair fragment amplified between am plimers C and E (Fig. 4, Lane 5), a normal chromosome 10 149-base pair fragment amplified between amplimers C and E (Fig. 4, Lane 7), and a human placenta! DNA negative control, without a t(10;14) chromosomal translocation (Fig. 4, Lane 6). Subsequently, patient DW relapsed and died due to T-cell lymphoblastic leukemia/lymphoma. Amplification Sensitivity. The amplification sensitivity of our amplification assay was estimated in a mixing experiment. Patient DW's DNA containing the t(10;14)(q24;ql 1) chromo somal translocation was serially diluted with human placenta! DNA and 1 ^g of the mixed DNA was amplified for 30 cycles Fig. 4. Identification of early relapse by PCR amplification of the t(10;14) (q24;ql 1) chromosomal juncture. Lanes 1-4: DW. DW-R,. DW-R2. and DW-R3 between amplimers C and E. As demonstrated in Fig. 5, we can patient DNA samples amplified between amplimers C and E: Lane 5: JM's DNA easily detect an amplified fragment in the 1 ng DNA mixture amplified between amplimers C and E; Lane 6: human placenta! DNA amplified which contained only 10 pg of the patient's DNA carrying the between amplimers C and E; Lane 7: human placenta! DNA amplification of the chromosome 10 sequence between amplimers C and D. Amplified products were t( 10;14) chromosomal translocation. These results indicate that separated on a 4% agarose gel, transferred to an Immunobiline membrane, and our resolution allows the detection of 1 leukemic cell out of detected by hybridization with Probe A. 125,000 normal cells. This resolution can be further improved 10-fold by using 10 ng DNA in the amplification process (data not shown).

DISCUSSION Clonal rearrangements of immunoglobulin or T-cell receptor genes have been detected in the vast majority of lymphoprolif- erative disorders (14). We have detected and cloned4 the phys iological rearrangements of the TCR a/5 locus in both patients (Fig. 1). Each of these clonal rearrangements might be used as an individual specific marker in detecting the neoplastic clone. However, recent observations have described a secondary re Fig. 5. Sensitivity and specificity of the PCR-amplified t(10;14)(q24;qll) chromosomal juncture. DW patient's DNA (1 fig) was serially diluted in human arrangement within the TCR a/o locus to a preexisting and functional Va-Ja segment of the TCRÂ«gene in a T-cell line placenta! DNA [lacking a id0:14) chromosomal translocationj and amplified between amplimers C and E. Amplified DNA (I >

By Southern blot analysis we are limited by the detection antibody repertoire. Science (Wash. DC), 238: 1079-1087, 1987. 7. Yanagi, Y., Yoshikai, Y., LeggeÂ«,K., Clark, S. P., Alexander, I., and Mak, level of a clonal population of cells which have to account for T. W. A human T cell specific cDNA clone encodes a protein having extensive 1-5% of the total cell population. In order to improve the homology to immunoglobulin chain. Nature (Lond.), 308: 145-149, 1984. detection level of leukemic T-cells with a t(10;14)(q24;qll) 8. Yanagi, Y., Chan, A., Chin, B., Minden, M., and Mak, T. W. Analysis of cDNA clones specific for human T cells and a and 0 chains of the T cell chromosomal translocation within the lymphocyte cell popu receptor hetrodimer from human T cell line. Proc. Nati. Acad. Sci. USA, 82: lation of the patient we have applied the polymerase chain 3430-3434. 1985. 9. Lefranc. M. P., Foster, A., Baer, T., Stinson. M. A., and Rabbits, T. H. reaction to amplify the chromosomal juncture. Diversity and rearrangement of human T cell rearranging y genes: nine germ- The specificity of our amplification was demonstrated by (a) line variable genes belonging in two subgroups. Cell, 45: 237-246, 1986. 10. Flag, F.. Pelicci, P. G., Bonetti, F., Knowles, D. M., II, and Dalla Pavera, R. the inability to amplify t(10;14) chromosomal junction frag T cell receptor gene rearrangements as markers of lineage and clonality in T ments in normal placenta! DNA serving as the negative control, cell neoplasms. Proc. Nati. Acad. Sci. USA, 82: 3460-3464, 1985. (6) detection of the expected size fragments which spans the 11. HÃ¢ta,S., Brenner, M. B., and Krangel, M. S. Identification of putative human T cell receptor Ã¤complementary DNA clones. Science (Wash. DC), 238: chromosomal translocation (233 base pairs in .IM and 486 base 678-682, 1987. pairs in DW), (c) specific detection of the amplified fragments 12. Chien, Y., Iwashima, M., Wettstein, D. A., Kaplan, K. B., Elliot, J. F.. Born, by a chromosome 10 internal probe which flanks the translo W., and Davis, M. M. T cell receptor Ã¨rearrangements in early thymocytes. cation breakpoint, (

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Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1990 American Association for Cancer Research. Detection of Minimal Residual Disease in Leukemic Patients with the t(10;14)(q24;q11) Chromosomal Translocation

Jacob Kagan, Lawrence R. Finger, Emanuel Besa, et al.

Cancer Res 1990;50:5240-5244.

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