P ATIENTS WITH ACUTE myeloid leukemia (AML)

Competitive CBF[i/MYHll Reverse-Transcriptase Polymerase Chain Reaction for Quantitative Assessment of Minimal Residual Disease During Postremission T...
Author: Griffin Freeman
2 downloads 0 Views 783KB Size
Competitive CBF[i/MYHll Reverse-Transcriptase Polymerase Chain Reaction for Quantitative Assessment of Minimal Residual Disease During Postremission Therapy in Acute Myeloid Leukemia With Inversion(16): A Pilot Study By Klaus Laczika, Michael Novak, Bernadette Hilgarth, Margit Mitterbauer, Gerlinde Mitterbauer, Alexander Scheidel-Petrovic, Christine Scholten, Renate Thalhammer-Scherrer, Stefan Brugger, Felix Keil, Ilse Schwarzinger, Oskar A. Haas, Klaus Lechner, and Ulrich Jaeger Purpose: (1) Quantification of minimal residual disease (MRD) by competitive CBFp/MYH 11 reverse-transcriptase polymerase chain reaction (RT-PCR) in patients with acute myeloid leukemia (AML) and inversion(16) [inv(16)] during postremission therapy, (2) comparison of this method with conventional two-step RT-PCR, and (3) evaluation of a potential prognostic value. Patients and Methods: MRD of six consecutive adult patients with AML and inv(16)(p1 3;q22) or t( 16;1 6)(p 13; q22) who entered complete remission (CR) was monitored by competitive CBFP/MYHI I RT-PCR in their bone marrow (BM) during postremission therapy with highdose cytarabine (HiDAC) or after BM transplantation with a matched unrelated-donor marrow (MUD-BMT) during an observation period of 4.5 to 27 months after initiation of treatment. Results: Competitive PCR showed a gradual decline by at least 4 orders of magnitude after 7 to 9 months in patients in continuous CR (CCR), while one patient who

P

ATIENTS WITH ACUTE myeloid leukemia (AML) with a pericentric inversion of chromosome 16 [inv(16)(p13;q22)] have a favorable prognosis when treated with chemotherapy.'1-6 Almost all patients achieve complete remission (CR) with standard induction therapy and remissions can be maintained in more than 50% of patients who receive postremission therapy with high-dose cytarabine (HiDAC). 7-10 Because of the high efficacy of conventional chemotherapy, patients with this AML subtype may not be regarded as candidates for bone marrow transplantation (BMT) in first CR, even when a suitable donor is available.11, 12 Nevertheless, almost half of the patients relapse despite optimal postremission therapy. Early identification of these patients could allow the risk-adapted management of prognostic subgroups.12,13 Inv(16)(p13;q22) and t(16;16)(p13;q22) can be detected on a molecular level by reverse-transcriptase polymerase chain reaction (RT-PCR) amplification of CBF,/MYHII fusion transcripts.14,15 The diagnostic value of CBFJ3/ MYHll RT-PCR is well established.16-22 Because of the high sensitivity of RT-PCR, it appears attractive to monitor patients during and after chemotherapy and to use this test as a predictor of outcome.23 However, conflicting information has been obtained for conventional two-step CBFl3/MYH11

relapsed after 13.5 months only achieved a reduction by 2 orders of magnitude at the end of consolidation therapy. A rapid decrease below the detection limit was observed within 1 month in two patients after MUDBMT. A temporary reappearance of molecular MRD was observed in these patients during immunosuppression for graft-versus-host disease (GvHD). After reduction of immunosuppression, the level of MRD dropped again below the PCR detection limit. Molecular monitoring by conventional two-step RT-PCR yielded comparable results only when multiple assays per time point were performed, while single-assay RT-PCR gave misleading results. Conclusion: Competitive RT-PCR is a valuable tool for molecular monitoring during postremission chemotherapy, as well as after BMT. J Clin Oncol 16:1519-1525. © 1998 by American Society of Clinical Oncology.

RT-PCR monitoring. A number of patients have been studied in CR after various treatments and with sensitivities of one in 104-5 for type A transcripts. 16,18,19, 2 1 ,23 Thirteen of 18

patients in CR investigated between 21 days and 9 years

From the Department of Medicine I, Division of Hematology, Clinical Institute of Medical and Chemical Laboratory Diagnosis, Divisions of Hematology and MolecularBiology,Department ofMedicine I, Bone Marrow Transplantation Unit, University of Vienna; and Children's CancerResearch Institute,St Anna Children'sHospital, Vienna, Austria. SubmittedJune 20, 1997; acceptedDecember 10, 1997. K.L. and M.N. contributed equally to this work and should both be consideredfirst authors. Supported by grantsfrom the Kommission Onkologie of the Medical Faculty of the University of Vienna ("Max Kellner Stipendium, "A.S.P and U.J.), the Anton Dreher-Gedaechtnisschenkung(#258/95; U.J.), the OesterreichischeKinderkrebshilfe, and the European Community, Vienna, Austria (grant no. CA-CT94-1703; O.A.H.). M.M. was the recipient of a young investigator'sresearch grantfrom the University of Vienna, Vienna, Austria. Presented in part at the Thirty-Eighth Meeting of the American Society ofHematology, Orlando, FL, December 6-10, 1996. Address reprint requests to Ulrich Jaeger MD, Klinik fuer Innere Medizin I, Abt. Haematologie und Haemostaseologie, Waehringer Guertel 18-20, A-1090 Vienna, Austria; Email [email protected]. © 1998 by American Society of Clinical Oncology. 0732-183X/98/1604-0017$3.00/0

Journalof ClinicalOncology,Vol 16, No 4 (April), 1998: pp 1519-1525

Downloaded from ascopubs.org by 37.44.207.160 on January 28, 2017 from 037.044.207.160 Copyright © 2017 American Society of Clinical Oncology. All rights reserved.

1519

1520

LACZIKA ET AL respectively. Three patients (no. 2, 4, and 5) had a continuous CR (CCR) for 30, 28, and 19 months, respectively; patient no. 1 died in CR from an unrelated cause; and patient no. 3 relapsed after 13.5 months and underwent MUD-BMT in second CR. Patient no. 6 was refractory to two cycles of standard induction therapy and achieved a partial remission after salvage treatment with cytarabine, mitoxantrone, and HiDAC (HAM) (Table 1).26 He underwent MUD-BMT and achieved CR, which now lasts for 2 years. RNA isolation. Mononuclear cells from BM and PB samples were isolated by Ficoll-Hypaque centrifugation. RNA was extracted from 1 X 107 cells according to the acid guanidinium thiocyanate-phenol27 chloroform method reported by Chomczynski and Sacchi. RNA pellets were resuspended in 25 tcL diethylpyrocarbonate (DEPC)treated water and used directly for reverse transcription or stored at -80 0 C for later analysis. RT-PCR amplification. RT-PCR amplification was performed according to a standard protocol28: for reverse transcription, 20-pL reactions were set up, which contained 2 pg RNA, 50 mmol/L Tris hydrochloride, pH 8.3, 75 mmol/L potassium chloride, 10 mmol/L dithiothreitol (DTT) 3 mmol/L magnesium chloride (MgCl 2), 20 U RNase inhibitor (RNA guard; Pharmacia, Uppsala, Sweden), 2 pg RNase-/DNase-free bovine serum albumin (Pharmacia), 1 mmol/L of each dATP, dCTP, dGTP, and dTTP, 100 pmol random hexamer primers (Pharmacia), and 200 U of Moloney murine leukemia virus (M-MLV) 0 RT (Gibco BRL, Gaithersburg, MD). After 60 minutes at 37 C, this cDNA solution (2 pL from mononuclear cell [MNC]-derived cDNA) was used for PCR. Nested or seminested two-step PCR was performed with the follow5 ing primers (Fig lA): KL 20-CBFP' : GCA GGC AAG GTA TAT 5 TTG AAG G; KL 23-CBFP' : GAA TTT GAA GAT AGA GAC AG; 1 5 KL 24-CBFSt : GTC TCA TCG GGA GGA AAT GG; KL 3029 MYH11 : TCG TGA TCG TGT CTC TGC AGT (reverse); KL 21 -MYH1115,29: CTC TTC TCC TCA TTC TGC TC (reverse); and KL 21 31--CBFP/MYHll : TTC TCC AGC TCA TGG ACC TCC (reverse). Combinations of first-step (KL20/30, 23/30, and 20/21) and secondstep primers (KL20/21, 23/21, 24/21, and 23/31) were tested under various conditions in order to achieve optimal sensitivity. A GeneAmp PCR Reagent Kit with AmpliTaqR DNA Polymerase (Perkin Elmer Cetus, Norwalk, CT) was used under conditions recommended by the supplier, except that 2.5 mmol/L MgC12 was used in a total reaction volume of 50 pL. The reaction was performed in a Perkin Elmer GeneAmp PCR System 2400 thermocycler. The conditions for the

after chemotherapy alone were still PCR-positive in their BM. PCR-negative results were only obtained in five patients after more than 7 months in CR. Moreover, two of the BM-positive patients were PCR-negative in their peripheral blood (PB).1 9 These data suggest that PCR positivity gradually decreases over time, but that most patients will remain positive until the first year in CR, at least in their BM. PCR positivity may even be compatible with long-term remission or cure. Therefore, conventional PCR results seem to have limited predictive value. This prompted us to quantify CBF/3/MYHII transcripts by a competitive PCR assay 24,25 in the BM of patients in CR after standard induction therapy followed by consolidation with a widely used HiDAC regimen. 9 The major goal of this monitoring study was to establish the first-year kinetics of minimal residual disease (MRD) to define reasonable checkpoints for further decisions based on PCR results. Moreover, we investigated the time course of CBFI3/MYH11 in two patients who underwent BMT with a matched unrelateddonor marrow (MUD-BMT). PATIENTS AND METHODS Patientsand Therapeutic Regimens Six adult patients were diagnosed to have AML with an inv(16)(p13; q22) or t(16;16)(p13;q22) and various French-American-British (FAB) subtypes: M4Eo (patients no. 1, 3, and 4), MO (no. 5), MI (no. 6), and M2 (no. 2). Patient characteristics, cytogenetic findings, and therapies are listed in Table 1. All six patients displayed a type A PCR 1 product, 4,16 were first-step CBF3/MYHII RT-PCR-positive at diagnosis, and were prospectively PCR-monitored during an observation period of 4.5 to 27 months. Five of six patients achieved a CR after one cycle of induction therapy (daunorubicin, cytarabine, and etoposide 26 [DAV]). Patients no. 1 to 4 received consolidation treatment with 9 intermittent HiDAC as described. Patient no. 5 received only one third of the dose per cycle due to his age. The scheduled four cycles of consolidation therapy could only be completed in patients no. 3 and 5, while therapy was terminated due to toxicity (infection and thrombocytopenia) after three and two cycles in patients no. 1, 4, and 2,

Table 1. Patient Characteristics Poatient No.

Age (years)

FAB

Induction

Cytogenetics

1 2 3

29 47 28

M4Eo M2 M4Eo

inv(1l6) inv(16), del(3)q(21) inv(16), + 8, + 12

4 5 6

18 64 36

M4Eo MO M1

inv(16), + 22 inv(16) t(16;16)

DAV 3 DAV3 DAV 3 DAV3 DAV3 DAV3 DAV 3 DAV 2

+ + + + + + + +

5 5 5 5 5 5 5 5

+ + + + + + + +

7 7 7 Reinduction 7 7 7 7, 5, HAM

Consolidation

Outcome

3x HiDAC 2x HiDAC 4x HiDAC DAV2 + 5 + 5 3x HiDAC 4x 1/3 HiDAC

Death in CR, 8 months CCR 30+ months CR 12.5 months, relapse MUD-BMT in 2nd CR, CR 13+ months CCR 28+ months CCR 19+ months Primary refractory MUD-BMT, CCR 24+ months

2 Abbreviations: DAV 3 + 5 + 7, daunorubicin 45 mg/m days 1 to 3, cytarabine 2 x 100 mg/m2 days 1 to 7, and etoposide 100 mg/m2 days 1 to 526; DAV 2 + 2 5 + 5, daunorubicin 45 mg/m2 days 1 to 2, cytarabine 2 x 100 mg/m2 days 1 to 5, and etoposide 100 mg/m2 days 1 to 5; HAM, cytarabine 2 x 1 g/m days 1to 2 2 bone unrelated-donor matched MUD-BMT, 1/3/5; days x 1 g/m 2 1/3 HiDAC, 1/3/59; days 2 x 3 g/m HiDAC, 2 to 5; days 10 mg/m2 4 and mitoxantrone marrow transplantation.

Downloaded from ascopubs.org by 37.44.207.160 on January 28, 2017 from 037.044.207.160 Copyright © 2017 American Society of Clinical Oncology. All rights reserved.

1521

COMPETITIVE RT-PCR FOR CBFp/MYH I1

amount of competitor that produced an equivalent PCR signal between competitor and patient cDNA from a diagnostic BM-MNC sample that contained greater than 90% blast cells was determined in repeated experiments. A total of 101 to 102 ng of plasmid DNA added to 2 pL of first-step reaction was needed. Therefore, serial log-fold dilutions of the competitor from 102 down to 10-4 ng, which corresponds to 3.3 x 1010 to 3.3 x 104 plasmids (calculated for a size of 2,900 nt) were mixed with 2 pL of the first-step reaction and amplified with KL24/21. We chose a DNA competitor, because RNA competitors are subject to degradation. 24,25 An RNA competitor could also be transcribed from our plasmid. 33 In contrast to monitoring of BCR/ABL in chronic myeloid leukemia (CML), 34 serial log-fold competitor dilutions were used for CBFP/MYHII, because of the more rapid and steeper kinetics. The competitor was only added to the second-step reaction, because the calculated number of competitor molecules that would have to be added to a first-step reaction in the lowest dilution step is less than 10. Competitor dilutions in this range are unreliable. The products were visualized on ethidium bromide-stained agarose gels and scored using the equivalence point between the 66- and 216-nt fragments (Fig 2A). Competitive assays were performed in duplicate. Correlationbetween competitive PCR and number of leukemic cells. Log-fold dilutions of leukemic cells in normal MNC down to a ratio of 1 in 105 were investigated by competitive PCR. A representative experiFig 1. (A)Localization of PCR primers. (B)Dilution experiment. A 216-nt band corresponding to a type A CBFI/MYHI I transcript is generated by a 2-step PCR with primers KL23/30 (first step, not shown) and KL24/21 (second step).

first-step PCR with KL23/30 consisted of 40 cycles with 30 seconds at 94°C, 1 minute at 58 0C, and 1.5 minutes at 72oC. Two microliters of the first-step reaction was then used for the second step, with KL24/21 under the same conditions. Ten microliters of the PCR product was visualized on ethidium bromide-stained agarose gels. PCR controls. Precautions against cross contamination were taken following the recommendations of Kwok and Higuchi. 30 Negative controls included the cDNA-and PCR-reagent mixes with water instead of cDNA in each experiment. To avoid false-negatives, the presence of intact RNA and adequate cDNA generation was evaluated for each sample by a control PCR using p-actin sequence-specific primers, 2 which allow discrimination between genomic DNA and cDNA. 8,31 Sensitivity of RT-PCR. The sensitivity of a single PCR assay was determined by serial dilution of a leukemic BM sample (> 90% blast cells in MNC) from a patient with a type A transcript with MNC from a normal individual down to a ratio of 1:107 as described.31 RNA from these mixtures was RT-PCR-amplified (Fig IB) with various nested primer combinations (Fig 1A). The primer pairs KL 23/30 and 24/21 were selected because they allowed the constant detection of one in 102-3 in the first and one in 105 cells in the second step in repeated experiments (Fig lB). The use of a junctional oligonucleotide specific for type A transcripts (KL31)21 did not further improve the sensitivity in our assays. Specificity of the assay was assured by hybridization with an internal oligonucleotide (KL31) (data not shown). Competitive PCR. Competitor DNA consisted of a CBFP/MYHII cDNA fragment cloned into bluescript (Strategene, La Jolla, CA) from a patient with an aberrant transcript. 32 This fragment can be amplified with the same second-step primers (KL24/21) as regular type A transcripts, but gives a PCR product of 66 nt instead of 216 nt. Since PCR assays during consolidation treatment were only second-steppositive, a regular first-step PCR with 2 pL of patient cDNA (corresponding to 0.2 pg of sample RNA) was performed with KL23/30. The

Fig 2. (A) Competitive PCR. (Top) Diagnostic BM (patient no. 4): 101 ng of competitor-generated equivalent signals (A) between the patient's cDNA (216 nt) and competitor (66 nt). (Bottom) BM at 2 months in CR: the equivalence point has shifted to 10-1 ng of competitor. (B) Correlation between dilution experiment of conventional 2-step PCR and competitive PCR.

Downloaded from ascopubs.org by 37.44.207.160 on January 28, 2017 from 037.044.207.160 Copyright © 2017 American Society of Clinical Oncology. All rights reserved.

1522

LACZIKA ET AL

ment that shows good correlation between the amount of MRD and decreasing competitor concentrations is presented in Fig 2B. Cytogenetic analysis. Unstimulated isolated BM cells were cultured for 24 to 48 hours. Chromosomes were prepared and G-banding with trypsin was performed according to standard techniques as described. 35 If possible, 20 metaphases were analyzed. Karyotyping was performed on a Genevision 121 chromosome analysis system (Applied Images, Santa Clara, CA) and the results described according to the International System for Human Cytogenetic Nomenclature 3 (ISCN). 6

RESULTS Kinetics of MRD DuringHiDAC Consolidation Quantificationby competitive PCR. BM samples of five patients who entered hematologic remission after induction therapy with DAV were monitored over a period of 4.5 to 27 months after treatment was started. Patients less than 60 years of age received two (patient no. 2), three (no. 1 and 4), or four (no. 3) cycles of HiDAC, while patient no. 5 received four cycles of a reduced HiDAC (one third of the dose). At diagnosis, all five patients showed an equivalence point between patient and competitor PCR product when 101 to 102 ng of plasmid was added to the second-step reaction (Figs 2A, 3, and 4). During consolidation, the amount of the transcripts decreased in all patients, with considerable variability in the kinetics of the decline (Fig 3). After 3 to 4.5 months two patients (no. 1 and 5) already had transcript levels below the detection limit of the competitive assay, which corresponds to a reduction by greater than 5 logs. The other three patients (no. 2, 3, and 4) only had a reduction by 2 to 3 logs. The further course in these three patients was quite different. In patients no. 2 and 4, the amount of MRD declined slowly and fell below the detection limit after 9 and

Fig 4. Comparison of MRD in BM and PB in patient no. 3. The lowest level of MRD in the PB was 10-3. Hematologic relapse (REL) was preceded by an increase in transcript numbers 1.5 months earlier.

16 months, respectively. In contrast, the amount of CBFf3/ MYH11 transcripts in patient no. 3 did not further decrease and the patient finally relapsed. Thus, the point of discrimination was reached at 7 to 9 months (after the end of HiDAC therapy), when good responders had a more than four log-fold decrease, while patient no. 3 remained at a level of approximately -2 logs despite CCR. All patients in stable CR finally had a reduction of the transcript by greater than 5 logs. In patient no. 3, MRD in BM and PB was compared by competitive PCR (Fig 4). Although the reduction in transcript levels was more pronounced in the PB, it never crossed the limit of -5 logs. The clinical relapse was also preceded by an increase in transcript levels in the PB (Fig 4). Multiple PCRs. To compare the results of the competitive PCR with a conventional PCR, PB and BM samples of patients no. 1 to 4 were monitored by multiple two-step PCR assays per time point (n = 4 to 14) over a period of 7 to 10 months. As shown in Table 2, all patients were positive in four of four assays in their BM and PB at the start of treatment. A gradual decrease in positivity was only observed after 2 to 3 months, with the PB preceding the BM. While the PB became negative in two patients, at least one positive result was obtained in every BM tested during the Table 2. Results of Multiple PCRs at Various Time Points

Fig 3. Kinetics of MRD in the BM of 5 patients during and after HiDAC or intermediate-dose cytorabine. All patients had equivalence points at 101 to 101-5 ng of competitor at diagnosis. At 7 to 9 months, patients in CCR had already a reduction of transcript numbers by - 4 logs (< 10-3), while patient no. 3 showed a reduction by only 2 logs, despite being in morphologic remission. REL, relapse.

Patient

Diagnosis

1 BM PB 2 BM PB 3 BM PB 4 BM PB

4/4 4/4 4/4 4/4 4/4 4/4 4/4 4/4

1 Month

2 Months

4/4

4/4

4/4 3/4 4/4 4/4 4/4

4/4 3/4

3 Months

4 Months

5/6 Months

2/4 2/4 4/4 1/4 3/4 1/4 3/4 3/4

3/4 0/4

Downloaded from ascopubs.org by 37.44.207.160 on January 28, 2017 from 037.044.207.160 Copyright © 2017 American Society of Clinical Oncology. All rights reserved.

7/8 Months

9/10 Months

1/4

1/4 3/4 0/4 1/4

10/14 1/4 13/14 1/14 1/14 0/14

1/4 1/4 1/14 1/14

1523

COMPETITIVE RT-PCR FOR CBFP3/MYHI 1 first 10 months, which indicates incomplete eradication of the leukemic clone. Kinetics of MRD After MUD-BMT Measured by Competitive PCR The course of MRD was studied in two patients who underwent MUD-BMT. Patient no. 3 was transplanted in second CR. Patient no. 6 was transplanted in partial remission after several induction therapies. He achieved hematologic CR after MUD-BMT. A rapid reduction of MRD was seen in both patients after 1 month (Fig 5A and B). However, both patients showed a recurrence of molecular MRD after 4 and 14 months while still in CCR. In patient no. 3, cyclosporine had to be terminated due to seizures; she received prolonged immunosuppression with prednisone for skin graft-versus-host disease (GvHD) (Fig 5A). However, following amelioration of GvHD and reduction of prednisone CBF /MYH11, RT-PCR converted to negativity again. In patient no. 6, the increase of detectable MRD coincided with reinstitution of immunosuppression due to liver GvHD. However, 4 months later, after control of GvHD, termination

Fig 5. MRD in BM of patients no. 3 and 6 after MUD-BMT. GvHD, Graft-v-host disease; CSA, cyclosporine; Pred, Prednisone.

of prednisolone, and reduction of cyclosporine, no PCRdetectable MRD was observed (Fig 5B). DISCUSSION We have established a competitive RT-PCR assay for the semiquantitative assessment of CBFf3/MYHII transcripts in patients with AML with an inv(16). Competitive PCR was first used for monitoring of CML. 24 ,33 The method is particularly helpful when transcripts are constantly detected by conventional PCR, as is the case with BCR/ABL in CML treated with interferon. 34 In acute leukemia, competitive PCR has been used in BCR/ABL-positive acute lymphoblastic leukemia (ALL), 37 as well as in AML with a t(8;21), 25 where the transcript is found in long-term remission or even 28 38 39 after allogeneic BMT. , , While the rapid kinetics of MRD in promyelocytic leukemia with a PMIJRARa fusion transcript treated with all-trans retinoic acid (ATRA) and chemotherapy do not warrant quantification beyond a regular two-step PCR,40 the published data on CBF3/MYH11 in inv(16) have already suggested that most patients remain PCR-positive in their BM during the first few months in CR. 16 -18,21 In a recent study, Costello et al 23 found molecular conversion of the BM in two patients 7 and 16 months after start of chemotherapy. Using a competitive CBF3/MYH11 RT-PCR assay, Evans et al41 recently showed a gradual decline of MRD down to PCR negativity between 24 and 80 weeks in patients treated with various protocols. The results of the multiple PCRs in our patients confirmed that BM samples will remain strongly positive during the first 3 months. After that, a gradual decrease in PCR positivity was observed. As described by Tobal et al, 19 negative results occurred preferentially in the PB. The fact that this partial negativity in our homogeneously treated group appeared as early as 4 to 8 months after diagnosis may be attributable to the aggressive HiDAC postremission protocol.9 As in other studies, none of our patients became completely PCR-negative in the BM during the initial observation period. Therefore, BM testing seems to be preferable for monitoring. The multiple assays also demonstrate that single two-step PCR assays will yield unreliable random results that do not reflect the molecular status during follow-up evaluation. Multiple testing of one sample gave a more reliable impression of the amount of MRD. Nevertheless, the best quantification was achieved by the competitive method. Using this method, we were able to detect pronounced differences in CBF)3/MYHll transcript levels, especially during the first few months, when conventional PCR gave uniformly positive results. The slope of decline was quite variable among the five patients. While the number of transcripts dropped below the detection limit of

Downloaded from ascopubs.org by 37.44.207.160 on January 28, 2017 from 037.044.207.160 Copyright © 2017 American Society of Clinical Oncology. All rights reserved.

1524

LACZIKA Er AL

competitive PCR as early as 3 to 4.5 months in patients no. 1 and 5, relatively high levels were still detectable in the other three patients, who had all received two cycles of HiDAC at this time. Interestingly, two patients showed a further decline, while the patient who relapsed still had considerable MRD. The checkpoint for discrimination between good and poor responders was reached after 7 to 9 months, when all patients except no. 3 had a reduction by more than 4 logs in the BM. At this time, the transcript level in patient no. 3 had only decreased by 2 orders of magnitude in the BM and by 4 orders of magnitude in the PB. This patient, who had additional chromosomal abnormalities, clinically relapsed 6 months later, which indicates a considerable predictive value for competitive PCR in inv(16). Early prediction of clinical outcome by quantifying MRD in acute leukemia has been proven useful in children with ALL.42 Interestingly, the study reported by Brisco et a142 showed a comparable reduction in the amount of MRD in good-risk patients. Adult ALL also shows similar kinetics of MRD.43 We suggest that in the case of AML with an inv(16), an inadequate molecular response of CBFJ3/MYH11 in hematologic CR at 7 to 9 months should prompt a search for matched unrelated donors in patients without human leukocyte antigen (HLA)-compatible siblings. On the other hand, good responders may represent the particular subgroup of patients likely to be cured by chemotherapy alone.7,8 Mayer et a19 reported that the aggressive HiDAC postremission regimen could only be completed in 56% of patients. Only one of our patients received four cycles of the

scheduled dose, while the treatment was stopped in three patients because of infection or thrombocytopenia. Patient no. 5 received four cycles of a reduced HiDAC therapy. There was no significant correlation between number of cycles, dose of cytarabine received, and kinetics of CBFJ3/ MYHll transcripts. Patient no. 5 had a reduction of CBFJ3/ MYH11 by greater than 5 logs after the first consolidation, while patient no. 4 still had considerable MRD after three cycles of HiDAC (at 5.5 months). MUD-BMT was able to eradicate MRD below the limit of sensitivity of the competitive assay within 1 month, even in a patient who had been refractory to various induction therapies. The transient reappearance of MRD in patients no. 3 and 6 vanished again after reduction of immunosuppression, which thus suggests a potential graft-versus-leukemia effect responsible for eradication of MRD. Due to the limited number of patients and the short observation period, it is not possible to draw definitive conclusions as to the usefulness of competitive PCR for treatment decisions. However, our data clearly demonstrate that it allows accurate monitoring of MRD during the early phase of CR. The introduction of automated technologies such as real-time PCR44,45 will allow study of a large number of samples and patients in the near future and will hopefully contribute to risk-adapted management of patients. ACKNOWLEDGMENT The expert technical assistance by Gertrude Ferstl and Uli Fischer is

greatly appreciated.

REFERENCES 1. Rowley JD: Recurring chromosomal abnormalities in leukemia and lymphoma. Semin Hematol 27:122-136, 1990 2. Bitter MA, LeBeau MM, Rowley JD, et al: Association between morphology, karyotype, and clinical features in myeloid leukemias. Hum Pathol 18:211-215, 1987 3. Berger R, Flandrin G, Bernheim A, et al: Cytogenetic studies on 519 consecutive de novo acute nonlymphocytic leukemias. Cancer Genet Cytogenet 29:9-21, 1987 4. Tashiro S, Kyo T, Tanaka K, et al: The prognostic value of cytogenetic analyses in patients with acute nonlymphocytic leukemia treated with the same intensive chemotherapy. Cancer 70:2809-2815, 1992 5. Haferlach T, Gassmann W, L6ffler H, et al: Clinical aspects of acute myeloid leukemias of the FAB types M3 and M4Eo. Ann Hematol 66:165-170, 1993 6. Marosi C, K611er U, Koller-Weber E, et al: Prognostic impact of karyotype and immunologic phenotype in 125 adult patients with de novo AML. Cancer Genet Cytogenet 61:14-25, 1992 7. Bloomfield CD, Lawrence D, Arthur DC, et al: Curative impact of intensification with high-dose cytarabine (HiDAC) in acute myeloid leukemia (AML) varies by cytogenetic group. Blood 84:111a, 1994 (suppl 1, abstr 431) 8. Bloomfield CD, Baer MR, Herzig GP: Acute myeloid leukemia in

adults: An update. Education Programme of the Second Meeting of the European Haematology Association, Paris, France, 1996, p 91 9. Mayer RJ, Davis RB, Schiffer CA, et al: Intensive postremission chemotherapy in adults with acute myeloid leukemia. N Engl J Med 331:896-903, 1994 10. Ghaddar HM, Plunkett W, Kantarjian HM, et al: Long-term results following treatment of newly-diagnosed acute myelogenous leukemia with continuous-infusion high-dose cytosine arabinoside. Leukemia 8:1269-1274, 1994 11. L6wenberg B: Post remission treatment of acute myelogenous leukemia. N Engl J Med 332:260-262, 1995 (editorial) 12. Bartram CR: Detection of minimal residual leukemia by the polymerase chain reaction: Potential implications for therapy. Clin Chim Acta 217:75-83, 1993 13. Mayer RJ: Allogeneic transplantation versus intensive chemotherapy in first remission acute leukemia: Is there a "best choice"? J Clin Oncol 6:1532-153 6 , 1988 (editorial) 14. Liu PP, Hajra A, Wijmenga C, et al: Molecular pathogenesis of the chromosome 16 inversion in the M4Eo subtype of acute myeloid leukemia. Blood 85:2289-2302, 1995 15. Liu P, Tarl6 SA, Hajra A, et al: Fusion between transcription factor CBF3/PEBP2p and myosin heavy chain in acute myeloid leukemia. Science 261:1041-1044, 1993

Downloaded from ascopubs.org by 37.44.207.160 on January 28, 2017 from 037.044.207.160 Copyright © 2017 American Society of Clinical Oncology. All rights reserved.

1525

COMPETITIVE RT-PCR FOR CBFp/MYHI 1 16. Claxton DF, Liu P, Hsu HP, et al: Detection of fusion transcripts generated by the inversion 16 chromosome in acute myelogenous leukemia. Blood 83:1750-1756, 1994 17. van der Reijden BA, Lombardo M, Dauwerse HG, et al: RT-PCR diagnosis of patients with acute nonlymphocytic leukemia and inv(16)(p13q22) and identification of new alternative splicing in CBF3-MYH11 transcripts. Blood 86:277-282, 1995 18. Poirel H, Radford-Weiss I, Rack K, et al: Detection of chromosome 16 CBF3-MYH11 fusion transcript in myelomonocytic leukemias. Blood 85:1313-1322, 1995 19. Tobal K, Johnson PRE, Saunders MJ, et al: Detection of CBFJ3/MYHII transcripts in patients with inversion and other abnormalities of chromosome 16 at presentation and remission. Br J Haematol 91:104-108, 1995 20. Shurtleff SA, Meyers S, Hiebert SW, et al: Heterogeneity in CBF13/MYHII fusion messages encoded by the inv(16)(p13;q22) and the t(16;16)(p13;q22) in acute myelogenous leukemia. Blood 85:36953703, 1995 21. H1bert J, Cayuela JM, Daniel MT, et al: Detection of minimal residual disease in acute myelomonocytic leukemia with abnormal marrow eosinophils by nested polymerase chain reaction with allele specific amplification. Blood 84:2291-2296, 1994 22. Campana D, Pui C-H: Detection of minimal residual disease in acute leukemia: Methodological advances and clinical significance. Blood 85:1416-1434, 1995 23. Costello R, Sainty D, Blaise D, et al: Prognosis value of residual disease monitoring by polymerase chain reaction in patients with CBFR/MYH11-positive acute myeloblastic leukemia. Blood 89:22222223, 1997 (letter) 24. Cross NCP, Feng L, Chase A, et al: Competitive polymerase chain reaction to estimate the number of BCR-ABL transcripts in chronic myeloid leukemia patients after bone marrow transplantation. Blood 82:1929-1936, 1993 25. Muto A, Mori S, Matsushita H, et al: Serial quantification of minimal residual disease of t(8;21) acute myelogenous leukemia with competitive PCR-assay. Br J Haematol 95:85-94, 1996 26. Thalhammer F, Geissler K, Jager U, et al: Duration of second complete remission in patients with acute myeloid leukemia treated with chemotherapy: A retrospective single center study. Ann Hematol 72:216-222, 1996 27. Chomczynski P, Sacchi N: Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156-159, 1987 28. Kusec R, Laczika K, Knobl P, et al: AMLl/ETO fusion mRNA can be detected in remission blood samples of all patients with t(8;21) acute myeloid leukemia after chemotherapy or autologous bone marrow transplantation. Leukemia 8:735-739, 1994 29. Matsuoka R, Yoshida MC, Furutani Y, et al: Human smooth muscle myosin heavy chain gene mapped to chromosomal region 16q12. Am J Med Genet 46:61-67, 1993 30. Kwok S, Higuchi R: Avoiding false positives with the PCR. Nature 339:237-238, 1989

31. Ng S-Y, Gunning P, Eddy R, et al: Evolution of the functional human p-actin gene and its multi-pseudogene family: Conservation of noncoding regions and chromosomal dispersion of pseudogenes. Mol Cell Biol 5:2720-2732, 1985 32. Novak M, Laczika K, Mitterbauer M, et al: Novel CBFPMYHlI fusion transcripts and alternative splicing in acute myeloid leukemia with inversion of chromosome 16. Blood 86:2449-2450, 1995 (letter) 33. Lion T, Henn T, Gaiger A, et al: Early detection of relapse after bone marrow transplantation in patients with chronic myelogenous leukemia. Lancet 341:275-276, 1993 34. Gaiger A, Henn T, H6rth E, et al: Increase of BCR-ABL chimeric mRNA expression in tumor cells of patients with chronic myeloid leukemia preceding disease progression. Blood 86:2371-2178, 1995 35. Haas OA, Schwarzmeier JD, Nacheva E, et al: Investigations on karyotype evolution in patients with chronic myeloid leukemia (CML). Blut 48:33-43, 1984 36. Mitelman F, Rowley JD, Sakurai M: Long term survival in acute myelogenous leukemia: A second follow-up of the Fourth International Workshop on chromosomes in leukemia. Cancer Genet Cytogenet 73:1-7, 1994 37. van Rhee F, Marks DI, Lin F, et al: Quantification of residual disease in Philadelphia-positive acute lymphoblastic leukemia: Comparison of blood and bone marrow. Leukemia 9:329-335, 1995 38. Nucifora G, Rowley JD: AML1 and the 8;21 and 3;21 translocations in acute and chronic leukemia. Blood 86:1-14, 1995 39. Jurlander J, Caligiuri MA, Ruutu T, et al: Persistence of the AML1/ETO fusion transcript in patients treated with allogeneic bone marrow transplantation for t(8;21) leukemia. Blood 88:2183-2191, 1996 40. Laczika K, Mitterbauer G, Korninger L, et al: Rapid achievement of PML/RARac polymerase chain reaction (PCR)-negativity by combined treatment with all-trans-retinoic acid and chemotherapy in acute promyelocytic leukemia: A pilot study. Leukemia 8:1-5, 1994 41. Evans PAS, Short MA, Jack AS, et al: Detection and quantification of the CBFO/MYH I1 transcripts associated with the inv(16) in the presentation and follow-up samples from patients with AML. Leukemia 11:364-369, 1997 42. Brisco MJ, Condon J, Hughes E, et al: Outcome prediction in childhood acute lymphoblastic leukemia by molecular quantification of residual disease at the end of induction. Lancet 343:196-200, 1994 43. Scholten C, Fidinger M, Mitterbauer M, et al: Kinetics of minimal residual disease during induction/consolidation therapy in standard-risk adult B-lineage acute lymphoblastic leukemia. Ann Hematol 71:155-160, 1995 44. Heid CA, Stevens J, Livak KJ, et al: Real time quantitative PCR. Genome Res 6:986-994, 1996 45. Marcucci G, Livak KJ, Bi WL, et al: Detection of AML1/ETO transcript in patients with t(8;21)-associated AML using a novel "real time" quantitative RT-PCR assay. Blood 90:390a, 1997 (suppl 1, abstr 1735)

Downloaded from ascopubs.org by 37.44.207.160 on January 28, 2017 from 037.044.207.160 Copyright © 2017 American Society of Clinical Oncology. All rights reserved.

Suggest Documents