Bone Marrow Transplantation, (1998) 22, 1137–1143  1998 Stockton Press All rights reserved 0268–3369/98 $12.00 http://www.stockton-press.co.uk/bmt FLAG-idarubicin and allogeneic stem cell transplantation for Ph-positive ALL beyond first remission

M Deane1,2, M Koh1, L Foroni1, G Galactowicz1, AV Hoffbrand1, M Lawler3, L Secker-Walker1 and HG Prentice1

2Department of Haematology, Norfolk and Norwich Hospital, Norwich, UK; 1Department of Haematology, Royal Free Hospital, London, UK; and 3Department of Haematology, Sir Patrick Dun Research Laboratory, St James’s Hospital, Dublin, Republic of Ireland

Summary: median CR duration of 7 months and 13% 3-year disease- free survival (DFS) probability in adults.1,2,4 BMT from a We describe a single centre experience of eight consecu- sibling donor is a more effective treatment with 2-year DFS tive patients with relapsed or refractory Ph+ ALL probabilities of 38 and 46% for patients in 1st CR and 41 treated with the FLAG/idarubicin regimen followed by and 28% for patients beyond 1st CR in the IBMTR series5 BMT or PBSCT. Following FLAG/idarubicin, one achi- and the series reported by Chao et al.6 Radich et al7 eved a partial response and seven CR. All patients sub- reported 30 patients who received BMT in CR or relapse, sequently received allogeneic transplants: one sibling of whom nine were disease-free beyond 2 years. Casper et BMT, three matched unrelated (MUD) BMT and four al8 reported 68% DFS at 13 months in 14 paediatric sibling PBSCT. Two patients received second trans- patients who received BMT in CR. plants with PBSC from their original BM donors follow- The results of chemotherapy in patients with relapsed ing FLA/Ida with no further conditioning. Three acute leukaemia after BMT are poor especially in those patients are alive in CR 9, 24 and 32 months after trans- who relapse early.9 Second BMT is associated with a high plant. Seven of eight patients had a cytogenetic response mortality and morbidity especially when performed within following FLAG/Ida induction and one of seven became 1 year of the initial transplant with only 10% long-term bcr-abl negative. All eight patients had a complete cyto- survivors in the IBMTR series.10–12 The role of DLI as genetic response following transplant. Four of five adoptive immunotherapy for relapsed CML after BMT has assessable patients became p190 bcr-abl negative after been established in recent years.13–19 This approach has also transplant; three of these subsequently relapsed. Both been used in relapsed AML after BMT but has been less patients with the p210 bcr-abl transcript remained bcr- successful in ALL.19–23 However, there are reports of suc- abl positive in CR after transplant. FLAG/Ida was well cess in ALL when DLI has been combined with cytokines tolerated and appears to be effective in inducing or preceded by chemotherapy.24,25 Although the feasibility remission in relapsed Ph+ ALL. The use of FDR-con- of allogeneic PBSCT as a primary transplant is estab- taining chemotherapy without further conditioning lished,26–28 its use as second transplant has so far been prior to PBSCT deserves further study in heavily pre- described only in individual cases.29–34 treated patients and, in patients with relapsed ALL fol- There is relatively little data available on the pattern of lowing BMT, may be a safer option than DLI (donor bcr-abl detection by PCR in Ph+ ALL following treatment. lymphocyte infusion) by avoiding the associated risk In the largest series reported,7 the risk of relapse was of aplasia. significantly associated with p190 but not p210 bcr-abl Keywords: fludarabine; FLAG; acute lymphoblastic leu- positivity after BMT. kaemia; Philadelphia chromosome; bcr-abl; peripheral The FLAG or FLAG-idarubicin regimens, which com- blood stem cell transplantation bine fludarabine (FDR), high-dose ara-C and G-CSF with or without idarubicin (Ida), have recently been used with good responses in poor risk AML and myelodysplasia and refractory or relapsed ALL.35–38 The rationale of these regi- The Ph chromosome, t(9;22)(q34;q11), is detected in up to mens is that the agents appear to be synergistic.39,40 FDR 25% of adults and in 2–6% of children with ALL and the triphosphate, the active metabolite of FDR, inhibits ribonu- bcr-abl transcript is detectable by PCR in up to 32% of cleotide reductase with a subsequent accumulation of intra- adults with ALL.1,2 Most childhood and more than 50% of + cellular ara-CTP. A positive correlation has been found adult Ph ALL express the p190 rather than the p210 bcr- between intracellular ara-CTP and remission rates.41 G-CSF 3 abl fusion transcript. The prognosis with chemotherapy prior to FDR increases the fraction of cells in cycle when alone is very poor with almost no long-term survivors, they are most vulnerable to ara-C.40 Ida may not be as sus- ceptible to multidrug resistance as other anthracyclines as Correspondence: Dr M Deane, Department of Haematology, Norfolk and it does not induce P-glycoprotein expression in leukaemic 42 Norwich Hospital, Brunswick Road, Norwich, NR1 3SR, UK cell lines. + Received 22 April 1998; accepted 30 July 1998 We report eight patients with relapsed or refractory Ph FLAG-idarubicin and allogeneic transplantation for Ph+ ALL M Deane et al 1138 ALL, reinduced with FLAG/Ida followed by allogeneic plex clones thought to have evolved from the original transplantation, and describe the results of cytogenetic and Ph+ clone. molecular monitoring. Reinduction chemotherapy Patients, donors and methods Seven patients were reinduced with FLAG Ida (fludarabine 30 mg/m2, ara-C 2 g/m2 days 1–5, G-CSF 300 ␮g/m2 day Patients Ϫ1 to neutrophil recovery, Ida 8 mg/m2 days 1–3) and Eight consecutive patients treated for relapsed or refractory patient 2 with FLAG. Four patients had a second course of Ph+ ALL between August 1995 and July 1997 at a single FDR ara-C (patients 3, 6 and 8) or FDR ara-C Ida (patient centre are described. Patient details are shown in Table 1. 7) according to the same schedule. Patients 3 and 7 had Patient 2 was treated with FLAG/Ida at the time of cyto- CNS disease and received additional local chemotherapy. genetic relapse 5 months after remission induction with the MRC UKALL 12 schedule. Patient 7 had a BMT in 2nd Donors CR, a testicular relapse 2 years later and was treated with FLAG/Ida at the time of CNS relapse a year later. The Patients 1, 2 and 8 received bone marrow from matched remaining six patients had morphological BM relapse at the unrelated donors: the donor for patient 1 was fully HLA- time of treatment with FLAG/Ida. Patients 1 and 5 relapsed matched; patient 2 received BMT from a DRB2-mis- on maintenance treatment at 11 and 23 months following matched donor with a CTLp of one in 10 000; patient 8 remission induction. Patient 5, a 20-year-old male, refused received BMT from a C-mismatched donor. The donors for a matched sibling BMT in 1st CR. Patient 6 relapsed 10 the other patients were fully HLA-matched siblings. Patient weeks following BMT; he received DLI (1 ϫ 107 CD3 3 received BM and patients 4, 5, 6 and 7, PBSC allografts. cells/kg) in cytogenetic relapse but progressed to develop Patients 1, 3 and 8 received sex-mismatched BMTs and frank morphological relapse when he was treated with the remaining patients, sex-matched transplants. Informed FLAG/Ida. Patient 3 had been refractory to standard induc- consent was obtained using institutional guidelines. tion and subsequent combination chemotherapy over a per- iod of 5 months following diagnosis. Patient 4 was treated Mobilisation, collection and transplantation of PBSC with four courses of ara-C and daunorubicin because of an initial diagnosis of AML and was treated with FLAG/Ida PBSC donors were given 10 ␮g/kg G-CSF as a single daily at the time of relapse 3 months later. Patient 8 presented subcutaneous injection for 4 or 5 days, with day 5 timed with a biphenotypic leukaemia and was treated with to coincide with day 0 of the allograft. Leukapheresis was FLAG/Ida following a partial response to ADE10 with commenced once the number of peripheral blood CD34 weekly VCR. cells exceeded 20/␮l.43 Donor venous access was obtained In addition to the Ph chromosome, seven of eight patients by bilateral antecubital venepuncture. Leukapheresis was (patients 1–7) had additional cytogenetic abnormalities performed using a continuous flow blood cell separator associated with adverse prognostic significance. Cyto- (Cobe Laboratories, Lakewood, CO, USA), processing 15 genetic results on two patients were first available at the l of blood on each occasion. The donors tolerated G-CSF time of refractory disease or relapse: patient 3 had complex and leukapheresis without complications. The results of the cytogenetic abnormalities including monosomy 7 and leukaphereses are shown in Table 2. The leukapheresis pro- patient 4 had del(11q23). Five patients developed additional ducts were administered fresh in all cases. In the cases of cytogenetic abnormalities at relapse: patient 1, an additional patients 6 and 7, the leukapheresis products were aliquoted Ph chromosome and monosomy 7; patient 2, 6q−; patient and an aliquot containing 2 ϫ 106/kg CD34 cells was 5, 7q−; patient 6, additional clonal abnormalities and administered. The leukapheresis product for patient 4 was additional Ph chromosomes; patient 7, independent com- partially T cell-depleted by treatment of half of the product

Table 1 Clinical characteristics and response to FLAG/Ida

Patient Age/Sex Disease Relapse Previous Haem Cyto PCR Prev No. phase on treatment CR (months) response response response BMT

1 39/F Rel 1 Yes 11 PR No No No 2 16/M Rel 1 Yes (cyto) 5 CR Yes No No 3 7/M Ref NA NA CR Yes No No 4 38/M Rel 1 No 7 CR Yes No No 5 20/M Rel 1 Yes 23 CR Yes No No 6 30/M Rel 1 No 5 CR Yes Yes Yes 7 17/M Rel 3 No 12 CRa Yesa NA Yes 8 31/M Ref NA NA CR Yes No No

In the disease phase column, Rel and Ref refer to patients in first or subsequent relapse or with refractory disease at the time of treatment. NA = not applicable; PR = partial response. aPatient 7, remission of CNS disease; reduction in proportion of BM abnormal cytogenetic clones. FLAG-idarubicin and allogeneic transplantation for Ph+ ALL M Deane et al 1139 Table 2 Characteristics of G-CSF-mobilised PBSC

Donor No. of Total MNC × 108/kg CD34-infused × 106/kg CD3-infused × 108/kg leukaphereses

4 1 4.14 3.6 0.75a 5 1 10.1 3.8 1.4 6 2 7.87 2 1.5 7 1 13.8 2 0.6 aHalf of the harvest was incubated with Campath Ig 0.1 ␮g/106 MNC. with Campath IG 0.1 ␮g/106 MNC (P Jacobs, personal the eight patients were p190 bcr-abl positive and two communication). patients (patients 4 and 8), p210 bcr-abl-positive. Chimeric studies were performed in the cases of patients 6 and 7 using pre-transplant DNA extracted from donor and Bone marrow transplantation recipient. The DNA profile was compared using a panel of Patient 1 received a MUD BMT at another institution; short tandem repeat markers as previously described.45 Two details were unavailable. The unrelated bone marrow for informative markers were used in each case. patients 2 and 8 was fully T cell-depleted using Campath IgM (0.24 and 0.01 ϫ 106 CD3 cells/kg infused, respectively). Patient 3 received unmanipulated bone Results marrow. Response to reinduction chemotherapy Conditioning and prophylaxis Five (patients 3, 4, 5, 6 and 8) of the six patients with The details for patient 1 were not available. Patients 2, 3, morphological bone marrow disease achieved morphologi- 4 and 5 received TBI 750 cGy as a single fraction and cal CR following a single course of FLAG/Ida. Patient 1 cyclophosphamide 120 mg/kg. Patients 2 and 8, who were had a partial response with a reduction in bone marrow MUD BMT recipients, in addition received TLI 900 cGy blasts from 90 to 50%. in six fractions and a 5-day course of Campath 1G. Patients Patient 7 had CNS disease associated with complex 3, 6 and 7 were transplanted at the time of nadir counts, abnormal cytogenetic clones in the BM, thought to have day 10, following a second course of FDR ara-C (patient evolved from the original Ph+ clone, and responded to local 6) or FDR ara-C Ida (patient 7). Patients 6 and 7 received and systemic treatment. The patient had previously had a no additional conditioning. All patients were given intra- BMT: chimaeric studies before and 6 weeks after FLAG- venous CY from day Ϫ1 and patient 8 short MTX (see Ida reinduction showed a reduction in the proportion of BM Table 3). recipient haemopoiesis from 30 to Ͻ10% with a parallel reduction in the proportion of the abnormal cytogenetic clones (data not shown). Documentation of engraftment and remission The results of the cytogenetic and PCR responses to rein- Haemopoietic recovery was defined by recovery of the neu- duction chemotherapy are illustrated in Table 1 and Figure trophil count to Ͼ0.5x109/l and an unsupported platelet 1. Seven of eight patients achieved a cytogenetic response count of Ͼ20x109/l. and one of seven patients tested, patient 6, became bcr- Reverse transcriptase PCR was used to detect bcr-abl abl-negative. transcripts as previously described.44 At diagnosis, six of FLAG/Ida was generally well tolerated in all patients,

Table 3 Details of transplant procedure and outcome

Patient Donor/ Conditioning ANC Ͼ0.5 Plt Ͼ20 GVHD GVHD Event-free No. Graft (days) (days) prophylaxis acute/chronic survival (months)

1 MUD/BM NA NA NA NA III 7 (CR) 2 MUD/BM CY/TBI/TLI/Camp 19 19 CYA/TCD I/nil 7 (rel) 3 Sib/BM CY/TBI/FDR/Ara-C 16 35 CYA II/nil 8 (rel) 4 Sib/PBSC CY/TBI 300 Ͼ300 CYA/PTCD I/nil 12 (CR) 5 Sib/PBSC CY/TBI 11 12 CYA II/ext 24+ CR 6 Sib/PBSC FDR/ara-C 17 17 CYA II/ext 13 (rel) 7 Sib/PBSC FDR/ara-C/Ida 17 42 CYA II/lim 32+ CR 8 MUD/BM CY/TBI/TLI/Camp 22 22 CYA/MTX/TCD No 9+ CR

NA = details not available; CY/TBI = cyclophosphamide 120 mg /kg, single fraction TBI 750 cGy; Camp = Campath Ig 20 mg daily × 5 days pre- BMT; TLI = total lymphoid irradiation 900 cGy in six fractions; TCD = T cell depletion; PTCD = partial T cell depletion; ext = extensive; lim = limited; rel = relapse. FLAG-idarubicin and allogeneic transplantation for Ph+ ALL M Deane et al 1140 1 D 2 R 3 R 4 D 5 6 R Patient Number 8 Months 1234 56 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 FLAG PCR pos PCR neg BM/PBSCT Ph pos Ph neg

Figure 1 Time course of seven patients from induction showing induction chemotherapy as a solid arrow, BMT as an open arrow. Positive PCR results for bcr-abl are indicated by a solid circle and negative results by an open circle; positive cytogenetic results for Philadelphia chromosome are indicated by a solid box and negative results by an open box. R indicates relapse; D indicates died. The line indicates length of survival in months.

the main side-effects being a self-limiting skin rash and negative bcr-abl PCR assays 11 weeks and 10 months prior abnormal liver function tests. There were no toxic deaths. to bone marrow relapse. Chimaeric studies on patient 6 at the time of the negative bcr-abl assay 11 weeks post-trans- plant and 10 months prior to relapse showed complete Results of PBSCT and BMT donor chimaerism (data not shown). Patient 3 had a nega- The outcome and details of haemopoietic recovery are sum- tive bcr-abl assay 4 weeks prior to a local orbital relapse marised in Table 3. Six patients had grade I or II GVHD and had a bone marrow relapse 4 months later. Patient 8 of the skin and/or gut which responded to treatment. Patient remained p210 bcr-abl-positive at 3 months and is in CR 1 had severe GvHD of skin and gut. Patients 5, 6 and 7 5 months after BMT. Patient 4 remained p210 bcr-abl-posi- developed chronic GVHD. Patients 6 and 7 both received tive, Ph-negative at 7 months and died of infection 12 PBSCT as second transplants from their original BM months post-transplant. donors and developed GVHD after the second but not the Patient 6 had a second CR of 13 months after PBSCT first transplant. having relapsed 10 weeks following initial BMT in 1st CR. Patients 8, 5 and 7 remain in CR 9, 24 and 32 months Patient 7 who had a CNS relapse has been in CR for 28 after transplant. Patients 1 and 4 died of transplant compli- months after a second transplant with a preceding CR of 12 cations in CR at 7 and 12 months. Patients 2, 3 and 6 months; cytogenetic studies which had previously shown relapsed 7, 8 and 13 months after transplant. The event- complex abnormal clones have been persistently normal free and disease-free survivals beyond 2 years were 30 and from 3 weeks after PBSCT. 47.6% (see Figure 2). The results of cytogenetic and bcr-abl analysis of seven patients are shown in Figure 1. Patient 7 had a CNS relapse Discussion and was not assessable by bcr-abl monitoring. All patients had complete cytogenetic responses following transplant. We chose to use the FLAG/Ida regimen in this series of Four patients (patients 6, 2, 3 and 5) were p190 bcr-abl- high risk and heavily pre-treated patients because of its negative at 3, 5, 7 and 16 months following transplantation. reported efficacy and our own experience in its use in treat- Patient 5 remains in CR at 20 months. Patients 2 and 6 had ment of refractory leukaemia, as well as an acceptable tox- icity profile. The main complication of FDR therapy is pro- 1.0 longed immunosuppression which becomes irrelevant after allogeneic stem cell transplantation. Seven of eight patients 0.9 EFS 30% achieved CR, including two with previously refractory dis- 0.8 ease, and one, a partial response, following a single course DFS 47.6% of FLAG/Ida. Seven patients had a cytogenetic response 0.7 following FLAG/Ida reinduction and one became bcr-abl negative. Gehly et al46 reported Ph negativity in three of 0.6 eight patients in CR following chemotherapy all of whom 47 0.5 remained bcr-abl-positive. Mitterkauer et al reported transient bcr-abl negativity in two patients following 0.4 chemotherapy or BMT. 0.3 Although the number of patients in this series is small, Cumalative proportion surviving the 2-year DFS of 47.6% compares well with the reported 0.2 2-year DFS of 41 and 28% in patients transplanted beyond 0 5 10 15 20 25 30 35 1st CR,5,6 particularly as ours is a high risk group of Months patients on the basis of cytogenetic abnormalities. Figure 2 Event-free and disease-free survival of eight patients in In our series, bcr-abl negativity was not predictive of months. relapse risk. Of four patients p190 bcr-abl negative at 3– FLAG-idarubicin and allogeneic transplantation for Ph+ ALL M Deane et al 1141 16 months post-transplant, three relapsed. Miyamura et al48 our patients were receiving second transplants, we used reported PCR negativity in nine of 15 patients who received PBSC products containing 2 ϫ 106 CD34 cells/kg and auto- or allo-BMT, seven of whom remained PCR-negative Ͻ2 ϫ 108 CD3 cells/kg, in the absence of definitive data and disease-free at follow-up of 15–64 months. Radich et on whether the risk of GVHD correlates with the dose of al7 reported PCR negativity in 14 of 31 patients after BMT. T cells in PBSCT. In this study, the risk of relapse correlated with p190 but Although the follow-up of some of the surviving patients not p210 bcr-abl positivity. in this series is relatively limited, the results illustrate that Two of the patients in this series relapsed following FLAG/Ida regimens can be useful in providing rapid dis- BMT and had second transplants with PBSC from the same ease control in patients with relapsed or refractory Ph+ ALL donors, one 4 months and the second more than 3 years prior to allogeneic transplantation. The use of these regi- after BMT. One relapsed 13 months following the second mens in heavily pre-treated patients without further con- transplant; the second patient remains in CR at 28 months. ditioning prior to transplantation avoids the toxicity of con- Both patients engrafted in the expected time-scale, with res- ventional conditioning regimens. Further studies are needed toration of complete donor chimaerism demonstrated in to define the optimum cellular composition of second trans- both. Neither patient suffered from significant procedure plants using PBSC which provide engraftment with mini- related toxicity, although both patients have developed mal risk of GVHD. As in other studies of Ph+ ALL, treat- chronic GVHD, neither having had GVHD after their initial ment of this disorder remains inadequate. However, we BMTs. The results of reinduction chemotherapy for would suggest that the use of PBSCT following FDR/ara- relapsed acute leukaemia after BMT are very poor with 7% C induction and conditioning deserves further study in ALL CR in patients given chemotherapy for ALL relapsing relapsed after BMT in the context of a multi-centre study within 100 days of transplant compared to 65% CR for and may be a safer and more effective option than DLI. relapse beyond 1 year in the series reported by Mortimer et al.9 Similarly the results of second BMT are very poor in patients with early relapse of acute leukaemia. Sanders Acknowledgements et al11 reported one survivor among 15 patients transplanted within 2 years of the initial transplant and six survivors We would like to thank Neil Watson for his help in collating among 11 patients transplanted more than 2 years after the data. initial BMT. Given the poor prognosis in patients with early relapse after BMT, the use of DLI has seemed an attractive approach. However, the EBMT data on use of DLI for relapsed ALL following BMT shows no CR in 12 patients References who either failed to respond to chemotherapy or were treated with DLI alone, whereas three of nine patients 1 Lestingi TM, Hooberman AL. Philadelphia chromosome-posi- treated with chemotherapy prior to DLI remained in CR at tive acute lymphoblastic leukemia. Hematol/Oncol Clin N Am 70–298 days.19 In the case of the patient in this series 1993; 7: 161–175. treated with DLI, DLI alone was not effective when given 2 Secker-Walker LM, Prentice HG, Durrant J et al. Cytogenetics adds independent prognostic information in adults with acute in early relapse, whereas PBSCT following chemotherapy lymphoblastic leukemia on MRC trial UKALL XA. Br J induced complete donor chimaerism and a CR which lasted Haematol 1997; 96: 601–610. for 13 months. Marrow aplasia can occur in more than 50% 3 Kantarjian H, Talpaz M, Dhingra K et al. Significance of the of patients given DLI for relapsed disease without prior P210 vs P190 molecular abnormalities in adults with Philadel- chemotherapy.49 In the context of the rapid disease kinetics phia chromosome positive acute leukemia. Blood 1991; 78: of relapsed ALL, there are clear advantages to the use of 2411–2418. chemotherapy followed by PBSCT rather than DLI alone, 4 Kantarjian HM, Walters RS, Keating MJ et al. Results of the in that this approach avoids the risks of aplasia and provides vincristine, doxorubicin and dexamethasone regimen in adults rapid haemopoietic reconstitution and restoration of with standard- and high-risk acute lymphoblastic leukemia. J chimaerism. Clin Oncol 1990; 8: 994–1004. 5 Barrett AJ, Horowitz MM, Ash RC et al. Bone marrow Three patients were transplanted at the time of nadir ransplantation for Philadelphia chromosome-positive acute counts following FDR ara-C/Ida. Two of these patients had lymphoblastic leukemia. 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