Molecular studies in patients with chronic ... - Wiley Online Library

British Journal of Haematology, 2001, 115, 569±574

Molecular studies in patients with chronic myeloid leukaemia in remission 5 years after allogeneic stem cell transplant define the risk of subsequent relapse Tariq I. Mughal, Agnes Yong, Richard M. Szydlo, Francesco Dazzi, Eduardo Olavarria, Frits van Rhee, Jaspal Kaeda, Nick C. P. Cross, Charles Craddock, Ed Kanfer, Jane Apperley and John M. Goldman Department of Haematology, Hammersmith Hospital, Imperial College School of Medicine, London, UK Received 8 May 2001; accepted for publication 17 July 2001

Summary. We identified 103 consecutive patients who, 5 years after allogeneic transplantation for chronic myeloid leukaemia (CML), were in molecular remission (MR). The 103 patients were divided into three groups on the basis of reverse transcription±polymerase chain reaction (RT±PCR) studies for BCR-ABL transcripts in the first 5 years post transplant: Group A comprised 63 patients who had been continuously PCR negative; Group B comprised 20 patients with one or more positive PCR result but only at a low level; and Group C comprised 20 patients who had fulfilled the criteria for molecular relapse, been treated with donor lymphocyte infusions (DLI) and had thereafter regained complete MR within the 5-year post-transplant period. The median follow-up for all 103 patients was 8´4 years from

transplant (range 5±17´6 years). In group A only one patient relapsed at 9´2 years. In group B eight patients (40%) relapsed: six at molecular, one at cytogenetic and one haematological levels. The actuarial probabilities of survival at 10 years for patients in Groups A, B and C were 97´4%, 92´9% and 100% respectively; the probabilities of relapse were 3%, 54% and 0% respectively. We conclude that molecular studies during the first 5 years post transplant can help to predict long-term leukaemia-free survival and, possibly, cure of CML.

Allogeneic haemopoietic stem cell transplantation (alloSCT) from a human leucocyte antigen (HLA)-identical sibling or a suitable unrelated donor using bone marrow or peripheral blood as source of stem cells offers the possibility of long-term molecular remission, and probably of cure, for patients with chronic myeloid leukaemia (CML) who are eligible for this treatment (Goldman et al, 1986; Thomas et al, 1986). However, leukaemia sometimes recurs after allo-SCT; the risk factors for relapse are well defined and include disease-specific factors, notably phase of CML at time of transplant, intensity type of conditioning, use of Tcell depletion of donor cells, and absence of graft-versus-host disease post SCT. The majority of relapses occur within 3 years but relapses at much later intervals have been identified and the kinetics of `early' and `late' relapses may be different. Until recently the management of relapse included hydroxyurea, interferon-alpha or a second transplant procedure using the original donor; donor lymphocyte

infusions (DLI) have also proved remarkably effective in restoring remission for patients in relapse, although they are complicated in some cases by graft-versus-host disease or graft failure (Kolb et al, 1995; Collins et al, 1997). The recent introduction of the Abl tyrosine kinase inhibitor STI571 (imatinib mesylate) offers another therapeutic possibility (Druker et al, 2001). In practice the precise definition of relapse and consequently the optimal timing of further therapeutic manoeuvres are controversial (Faderl et al, 1999; Goldman et al, 1999; Giralt et al, 2001). The reverse transcription± polymerase chain reaction (RT±PCR) is widely used to monitor patients and the first suspicion of relapse may be the detection of BCR-ABL transcripts in a patient in whom they could not previously be detected (Cross et al, 1993a; Lion et al, 1993; Radich et al, 1995; Lin et al, 1996). In some cases the numbers of BCR-ABL transcripts rises steadily thereafter and, once the Ph chromosome is identified in the patient's marrow, the diagnosis of relapse is universally agreed; in other cases transcript numbers remain low or disappear. The problem is how to recognize the patient with a low level of BCR-ABL transcripts whose

Correspondence: Professor John Goldman, Department of Haematology, Hammersmith Hospital/ICSM, Du Cane Road, London W12 0NN, UK. E-mail: jgoldman@ic.ac.uk q 2001 Blackwell Science Ltd

Keywords: chronic myeloid leukaemia, reverse transcription±polymerase chain reaction, stem cell transplant.

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disease is likely to progress. In one study the authors used a two-step RT±PCR to monitor 64 patients post allo-SCT for CML and concluded that they could define the subgroups at risk for disease progression (Roth et al, 1992), while in another similar study the authors could detect no association between PCR positivity and subsequent `relapse' (Miyamura et al, 1993). In our experience PCR positivity within 6 months after allo-SCT did not predict a worse outcome, whereas PCR positivity later than 6 months after allo-SCT did (Lin et al, 1996; Olavarria et al, 2001). We and others believe that rising numbers of BCR-ABL transcripts should be taken as an indication for therapeutic intervention, but even this can be disputed. The sequence of molecular events is poorly defined in patients who survive $ 5 years after allo-SCT. We therefore studied subsequent BCR-ABL transcript levels in a cohort of patients who were in molecular remission at 5 years post allo-SCT for CML, some of whom had received treatment with DLI. Our principal aim was to define any specific pattern of BCR-ABL transcript levels within 5 years of alloSCT that might predict later relapse. A secondary aim was to assess the durability of molecular remissions after DLI administered before the 5-year landmark. PATIENTS AND METHODS Patients. Of all patients with Philadelphia (Ph) chromosome-positive CML subjected to allo-SCT at the Hammersmith Hospital in London between 1 May 1981 and 1 October 1994, 120 were alive and in molecular remission (defined below) 5 years after allografting. Seventeen of these patients had no molecular follow-up data and were excluded from further study; 103 patients were included in this analysis. The phase of CML was classified at the time of transplant: 93 patients were in chronic phase, 5 were in accelerated phase and 5 were in second chronic phase after treatment for blast crisis. All patients were transplanted using bone marrowderived stem cells from an HLA-identical sibling donor (n 71) or from a phenotypically HLA-matched unrelated donor (n 32). All unrelated donor±recipient pairs were serologically matched at class I and II loci but molecular typing for DRB1 was not generally available. No patient received peripheral blood-derived stem cells. The transplant conditioning regimen included cyclophosphamide and fractionated total body irradiation (TBI) (n 93), busulphan and cyclophosphamide (n 5) and TBI, cyclophosphamide and busulphan (n 5) (Spencer et al, 1995). Bone marrow aspirates or peripheral blood samples or both were obtained at regular intervals post transplant for cytogenetic and molecular studies. The study cohort was defined on the basis of survival without any BCR-ABL transcripts detectable by semiquantitative RT±PCR performed on peripheral blood during the interval extending from 3 months before to 3 months after the 5-year landmark. The patients were divided into three groups: group A comprised 63 patients who had been continuously RT±PCR negative post transplant; group B comprised 20 patients who had one or more positive PCR studies but did not meet the criteria for relapse; and group C comprised 20 patients who had had a

molecular relapse and had then regained complete molecular remission within the 5-year post-transplant period following treatment with DLI. The median follow-up for all 103 patients was 8´4 years (range 5±17´6 years). All patients had a minimum of three RT±PCR results, including one in the final year of follow-up; the median number of results per patient was four (range 3±21). Quantitative RT±PCR for BCR-ABL transcript. White cell pellets isolated from peripheral blood (10±20 ml) and/or bone marrow aspirate (1±5 ml) were transferred to 1±2 ml of guanidinium thiocyanate (GTC) solution containing bmercaptoethanol and homogenized using a needle and syringe. Total RNA was extracted using the RNA easy kit (Qiagen, Crawley, UK) in accordance with the manufacturer's instructions or using the ultracentrifuge-caesium chloride method (Cross et al, 1993b). The RNA was reverse transcribed using Moloney murine leukaemia virus (MMLV) reverse transcriptase as described previously and the cDNA tested for BCR-ABL by multiplex PCR (Cross et al, 1994). Only samples found to be BCR-ABL negative by multiplex PCR were subjected to testing by nested PCR. In samples found to be positive, BCR-ABL transcripts were quantified by competitive PCR relative to the number of ABL transcripts as an endogenous control (Hughes & Goldman, 1990). The result was then expressed as a percentage BCR/ABL:ABL ratio. If on occasion in a given sample BCR-ABL transcripts were not detected but the ABL transcripts were measured as , 1 104, the sensitivity of the assay was deemed to have been suboptimal and the result was ignored. Cytogenetic analysis. Bone marrow aspirates were obtained at various intervals and the presence or absence of the Ph chromosome was sought in G-banding metaphase preparations. A minimum of 20 metaphases was examined. Definition of relapse. The finding of a positive RT±PCR result within the first 6 months post allo-SCT or the finding thereafter of one or more very weakly positive RT±PCR tests was not by itself regarded as indicating relapse. Thus molecular relapse could only be recognized more than 6 months post SCT and was somewhat arbitrarily defined as three RT±PCR results over a minimum of 4 weeks with either a BCR-ABL/ABL ratio in excess of 0´02% or showing clearly rising levels with the last two higher than 0´02% or two results over a minimum period of 4 weeks higher than 0´05%. Cytogenetic relapse was defined as one or more Phpositive metaphases detected without evidence of haematological relapse. Haematological relapse was defined as peripheral blood leucocytosis with predominance of myelocytes and neutrophils in the differential count accompanied by a hypercellular bone marrow and Ph positivity on cytogenetic analysis. For patients treated by DLI, molecular remission was defined by finding two RT±PCR negative results at least 12 weeks apart. Statistics. Survival was calculated using the method of Kaplan & Meier (1958) and relapse using the cumulative incidence method (Gooley et al, 1999). RESULTS The patient characteristics are summarized in Table I. The

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molecular remission after DLI and none has subsequently relapsed during the median follow-up period of 42 months (range 5±101 months). Thus, the actuarial survival at 10 years was 100% and the probability of relapse was 0%.

median duration of disease prior to allo-SCT was shorter for group C patients than for patients in groups A and B. A higher proportion of group C patients were in chronic phase at the time of the SCT. In group A only one patient fulfilled criteria for molecular relapse at 9´2 years; the actuarial survival at 10 years was 97´4% and the probability of relapse was 2´6%. In group B eight patients (40%) satisfied criteria for relapse, six at the molecular level, one at the cytogenetic level and one at the haematological level. The actuarial survival for Group B patients was 92´9% and the probability of relapse at 10 years was 54´3% (Fig 1). A schematic representation of serial RT±PCR ratios for two of the group B patients is shown in Fig 2. Both patients demonstrated fluctuating positivity and negativity but were negative at the time of the 5-year landmark analysis. Patient Y fulfilled criteria for molecular relapse at 6´5 years post allo-SCT, was treated with DLI and achieved molecular remission which has remained durable since then. Patient Z fulfilled criteria for molecular relapse at 7´5 years post allo-SCT and also achieved sustainable molecular remission following treatment with DLI. The 20 patients in Group C all achieved

DISCUSSION In this study of 103 patients transplanted for CML, 20 patients satisfied our criteria for molecular relapse within 5 years of the SCT and were treated with DLI. The remaining 83 patients received no further treatment for leukaemia during the first 5 years post SCT. Of these, 63 had been persistently PCR-negative and among these there was one molecular relapse during the period of subsequent follow-up. In contrast, 20 patients had one or more positive RT±PCR tests within the first 5 years post allo-SCT but did not satisfy our criteria for relapse and thus did not receive DLI or other treatment for leukaemia. Of the these 20 patients, eight eventually satisfied our criteria for relapse, six at the molecular level, and one each at the cytogenetic and haematological levels. Thus, the actuarial probability of relapse for these 20 patients was 54%. These eight patients

Table I. Characteristics of patients. Group A (n 63)

B (n 20)

C (n 20)

Disease status: CP Advanced

59 4

15 5

19 1

Donor: HLA-identical sibling MUD

46 17

13 7

12 8

Patient sex: Male Female

36 27

9 11

12 8

Patient age (years): median (range)

31´6 (10±50)

33´6 (4±59)

35´6 (17±55)

Duration of disease pre-SCT (years): median (range) 8

Cell dose 10 /kg: median (range)

1´5 (0´5±7´80)

1´4 (0´4±2´7)

1´1 (0´5±3´9)

3´0 (1´3±5´1)

2´9 (1´9±5´0)

2´9 (1´6±4´8)

GVHD prophylaxis: CSA 1 MTX TCD

41 22

11 9

7 13

Acute GVHD grade: 0 1 2 3±4

11 18 21 13

4 10 3 3

2 10 6 2

Chronic GVHD: Nil Limited Extensive

28 9 26

9 5 6

9 9 2

SCT, stem cell transplant; CSA, cyclosporin A; MTX, methotrexate; TCD, T-cell depletion of donor marrow; CP, chronic phase; GVHD, graftversus-host disease; MUD, matched unrelated donor; y, years. q 2001 Blackwell Science Ltd, British Journal of Haematology 115: 569±574

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Fig 1. Cumulative incidence estimation of the probability of relapse for patients surviving in remission more than 5 years post SCT.

were treated with DLI and all achieved molecular remission. They were alive and in molecular remission at the time of analysis. The 20 patients in Group C all achieved molecular remission after DLI administered within 5 years of transplant and none has subsequently relapsed during the median follow-up period of 42 months. We believe it possible that the majority of patients in Group A will eventually prove to have been cured of CML, although, in practice, it is extremely difficult to define `cure' after allo-SCT. The International Bone Marrow Transplant Registry (IBMTR) reported that patients in the early stages of CML subjected to allo-SCT using blood- or marrowderived stem cells had a probability of 5-year leukaemia-free survival of 55±60% (Horowitz et al, 1996). The probability of relapse was 15% but the definition of relapse was based generally on cytogenetic or clinical criteria; the probability would almost certainly have been higher if molecular criteria had been used. The majority of the relapses occurred within the first 3 years post SCT, but patients were still at risk for relapse thereafter and a relapse as late as 14 years after SCT has been reported (Yong & Goldman, 1993; van Rhee et al, 1994a). Thus, any method such as serial

monitoring by RT±PCR for a protracted period after alloSCT that allowed the clinician to predict with reasonable certainty `permanent' freedom from relapse would be valuable. Patients in Group B had one or more positive RT±PCR test in the interval from 6 months to 5 years post allo-SCT but never satisfied our criteria for molecular relapse and were RT±PCR negative at 5 years. On subsequent follow-up they proved, however, to have a significantly higher incidence of relapse than patients in Group A and some subsequently received treatment with DLI. We have previously shown that patients treated in early relapse, i.e. in molecular or cytogenetic relapse, have a higher response rate to DLI than those treated in haematological relapse (van Rhee et al, 1994b). As the patient cohorts in that study were not strictly comparable, we cannot conclude that early treatment of relapse is therefore mandatory and cannot say whether patients in Group B in this study should or should not have received earlier treatment with DLI. Moreover, we cannot therefore say whether or not our current definition of molecular relapse should be revised. The availability of other methods for treating relapse, such as interferon-alpha

Fig 2. Schematic representation of serial RT±PCR ratios for two of the group B patients demonstrating fluctuating positivity and negativity before and after the 5year landmark. q 2001 Blackwell Science Ltd, British Journal of Haematology 115: 569±574

Molecular Studies Can Predict Long-term Survival in CML or STI571, further complicates the decision as to whether patients with low level transcripts after allografting should receive further treatment. The issue could be addressed by a prospective study but its design would be complex. In this study, all patients who had a molecular relapse within 5 years and who were then treated with DLI achieved molecular remission, which was sustained at 10 years. This is in accordance with our previous experience in a larger group of patients, in whom we saw very few (3/44) relapses with a median and maximal follow-up of 29 and 89 months respectively (Dazzi et al, 2000a). This supports the notion that, until we have further experience with STI571, the use of DLI in compliance with the escalating dose regimen pioneered by the Memorial Sloan±Kettering group (Mackinnon et al, 1995; Dazzi et al, 2000b) may be the best way of treating CML patients who relapse after allo-SCT, defining relapse as one will. We conclude that molecular studies during the first 5 years post transplant help to predict long-term leukaemiafree survival and possibly cure of CML. Patients who show transient evidence of relapse at the molecular level within 5 years of SCT are at relatively high risk of subsequent relapse, but those who do sustain a molecular relapse can be restored to durable molecular remission by DLI. It is noteworthy that with a median follow-up of 9´9 years for group A patients, one subsequent relapse was observed. This emphasizes the need to indefinitely monitor all patients allografted for CML. We suggest therefore that patients should be monitored by RT±PCR for BCR-ABL transcripts at intervals no greater than 3 monthly during the first year post transplant and thereafter at 6-monthly intervals for at least 5 years. After the 5-year point, patients who had been persistently RT±PCR negative could be checked at yearly intervals, while those who had been transiently positive could be monitored more frequently, perhaps at 6-month intervals. If any patient were found to be positive by RT± PCR, then the frequency of molecular monitoring should be increased and the possibility of further treatment with DLI or with STI571 would need to be considered. ACKNOWLEDGMENTS We thank the staff of the Stem Cell Laboratory, the Minimal Disease Group and the Cytogenetics Group for their valuable contributions to this paper. We thank especially the nursing staff responsible in large part for the clinical care of the patients included in this study. This work was supported in part by the Leukaemia Research Fund (UK) and the Kay Kendall Research Fund. REFERENCES Collins, R.H., Shpilberg, O., Drobyski, W.R., Porter, D.L., Giralt, S., Champlin, R., Goodman, S.A., Wolff, S.N., Hu, W., Verfaillie, C., List, A., Dalton, W., Ognoskie, N., Chetrit, A., Antin, J.H. & Nemunaitis, J. (1997) Donor leukocyte infusions in 140 patients with relapse malignancy after allogeneic bone marrow transplantation. Journal of Clinical Oncology, 15, 433±444. Cross, N.C.P., Hughes, T.P., Lin, F., O'Shea, P., Bungey, J., Marks, D.I.,

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