Autografting in Philadelphia (Ph)+ chronic myeloid leukaemia using ...

Bone Marrow Transplantation, (1997) 19, 969–976  1997 Stockton Press All rights reserved 0268–3369/97 $12.00

Autografting in Philadelphia (Ph)+ chronic myeloid leukaemia using cultured marrow: an update of a pilot study LH Coutinho1, J Chang1, ML Brereton2 , GR Morgenstern3, JH Scarffe4, CJ Harrison5 , JA Liu Yin2, PJ Darbyshire6, S Burdach7, TM Dexter1 and NG Testa1 1

CRC Department of Experimental Haematology, 5 CRC Department of Cancer Genetics, Paterson Institute for Cancer Research; Department of Haematology, 4 Department of Medical Oncology, Christie Hospital, Manchester; 2Department of Clinical Haematology, Manchester Royal Infirmary; 6Birmingham Children’s Hospital, UK; and 7Kinderklinik, Universitat Dusseldorf, Germany

3

Summary: Incubation of CML marrow in long-term culture (LTC) conditions may result in selection of normal (Ph−) LTCinitiating cells (LTC-IC) as early as 10 days, and in production of Ph− clonogenic cells and mature end cells within 5 weeks. This was the rationale for using marrow cells from 10-day-old LTC to autograft nine chronic phase CML patients, ineligible for HLA-matched sibling donor transplant, and who were selected on the basis of a pre-transplant screening LTC test. Of the transplanted patients three died; two of graft failure and one of therapy-related toxicity with 97% Ph− cells 16 months following the autograft. The reconstituting haemopoietic cells in the seven engrafted patients were 100% Ph− in four, > 90% Ph− in two and 71% Ph− in the seventh, with a duration of complete cytogenetic response of 6–12 months. Three patients reverted to chronic phase and 100% Ph+ haemopoiesis 27–36 months post-autograft. The other three patients remain in continuous haematological remission with 22% Ph− cells in one and complete cytogenetic remission in the other two 3–4 years post-autograft. IFN therapy was generally introduced on the first evidence of recurrence of Ph+ cells or of cytogenetic deterioration. Further strategies to modulate immune surveillance in vivo may improve the outcome of cultured marrow autografts which give an initial and rather prolonged bias towards Ph− haemopoiesis. Keywords: autologous marrow transplantation; purging; LTBMC; CML

Chronic myeloid leukaemia (CML) is a clonal myeloproliferative disorder that arises from a somatic mutation of a pluripotent haemopoietic stem cell characterised as the bcrabl fusion gene.1 This underlying genetic rearrangement results, in most cases, in the formation of the Philadelphia chromosome or the 9;22 translocation that is present in all Correspondence: Dr NG Testa, CRC Department of Experimental Haematology, Paterson Institute for Cancer Research, Christie Hospital, Wilmslow Road, Manchester, M20 4BX, UK Received 1 November 1996; accepted 26 January 1997

dividing haemopoietic cells of non-lymphoid lineages and, in some patients, also in B lymphocytes.2,3 The haematological manifestation of the disease can be easily controlled with cytotoxic drugs, such as busulphan and hydroxyurea, but there is little evidence that they delay further progression to accelerated or blastic phase.4 However, interferon-a has been reported to delay disease progression and improve survival in CML,5,6 and in a small proportion of patients to induce partial or complete cytogenetic remission.7 Allogeneic bone marrow transplantation, by inducing complete eradication or at least long-term suppression of the Ph+ clone, constitutes the most effective option and the only known curative treatment in CML.8,9 However, it can only be offered to a small proportion of patients because of lack of a suitable donor or advanced age. For patients aged less than 50 years, only 15% have HLA-matched sibling donors.4 The role of matched unrelated donor transplants remains uncertain, but the procedure may be useful for young patients.10 A recent multicentre analysis of autologous transplants in CML suggested that autografting might be a possible alternative for many of these patients.11 The preliminary results for patients autografted in first chronic phase indicate a survival plateau of 58% up to 7 years. Although not curative, the survival may be better than that achieved by treatment with conventional drugs or IFN-a. Long-lasting molecular remissions have been documented in syngeneic transplants despite the lack of graftversus-leukaemia effect.12 In addition, a recent report on CML patients autografted with retrovirally marked stem cells showed that leukaemic cells in the infused marrow contributed to their relapse.13 Thus, it is at least theoretically beneficial to autograft Ph− marrow. In view of this, various centres have conducted purging procedures in order to decrease the ratio of Ph+ to Ph− cells in the autografts, namely: in vivo chemotherapy with peripheral blood progenitor cell (PBPC) collection in the early phase of haematopoietic recovery,14 marrow exposed to IFN-g15 or mafosfamide,16 or marrow cultured for 10 days.17 Encouraged by previous reports 18,19 and our own results of a selective enrichment of Ph− cells in long-term culture of marrow from Ph+ CML patients,20,21 we have used a similar procedure to that of Barnett et al17 to autograft nine

Pilot study of autografting in CML using cultured marrow LH Coutinho et al

970

Ph+ CML patients in chronic phase. We now report the results of this pilot study after a follow-up of 3–7 years. Patients and methods

Culture assays

Eligibility The following criteria were applied: (1) Ph+ CML in chronic phase; (2) ,50 years old; (3) lack of an HLAmatched sibling donor; and (4) previous evidence in longterm bone marrow cultures (LTBMC) of the presence of residual Ph− progenitors and depletion of Ph+ progenitors (see Results). All patients were required to have adequate renal, hepatic, pulmonary and cardiac function. Patients Of the CML patients who had their marrow assessed in LTC, 20 (40% of the total) met the culture criteria (see Results) used for entry in this study.21 A clinical summary of the nine CML patients who subsequently received their cultured marrow following myeloablative chemotherapy between February 1989 and January 1993 is found in Table 1. There were six males and three females with an age range of 9–48 years (median 38). The median interval from diagnosis to autograft was 15 months (7–42). Before the transplant all patients, except one initially managed with busulphan, were treated with hydroxyurea (HU) alone at intermittent high doses, or at conventional doses in combination with IFN-a or with thioguanine (one case). None of the four patients who received IFN-a therapy for 6–24 months had had a complete or major cytogenetic response. IFN was stopped at least 1 month before collection of marrow for culture, and if necessary, patients were managed in the meantime with conventional doses of HU. Four patients referred to the centre as newly diagnosed were treated with pulse high-dose HU as the recommended regimen with a view to performing autografting with cultured cells. This was based on data21,22 showing that pulse HU has a selective effect on Ph− haemopoiesis in CML which results in improved recovery of Ph− cells from LTC (see Results). Details of dose and schedule have been previously reported. 23 None of the patients selected for culture autografting had undergone splenectomy. The protocol was Table 1

Patient clinical details

UPN

Sex/Age

Months from diagnosis to autograft

Previous therapy

891 892 901 902 911 912 921 922 931

M/48 M/47 M/12 M/38 F/31 F/48 M/47 F/9 M/12

7 13 42 15 26 18 11 15 12

pulse HU pulse HU HU + IFN-a BU + IFn-a HU + IFN-a pulse HU pulse HU HU + IFN-a HU + thioguanine

UPN = unique patient number.

approved by the local ethical committee and patients or their legal guardians gave informed consent before entry into the study.

Standard LTBMC were established in 25 cm 2 tissue flasks with a preliminary sample of marrow from each patient to evaluate the genotype of clonogenic cells and mature end cells. Cells were inoculated in a 10 ml volume of culture medium at a concentration of 2 × 106 nucleated cells/ml and the cultures were maintained for 4–5 weeks. At weekly feeding the content of clonogenic cells, including those originating multilineage (CFU-GEMM), erythroid (BFU-E) and granulocyte–macrophage colonies (GM-CFC), was determined. The methodology of the LTBMC system and of clonogenic assay has been described in detail elsewhere.24 Cytogenetic analyses We used conventional cytogenetic methods to obtain metaphase spreads of marrow aspirates and of non-adherent cells from LTBMC. In addition, metaphase preparations were made of colonies, predominantly of the GM-CFC lineage, as described before.25 Colonies were plucked out individually or were pooled from the entire culture dish. Preparations were Giemsa banded before analysis. Pre-transplant schedule and conditioning regimen On day −10 marrow was harvested under general anaesthesia and allopurinol and anti-fungal prophylaxis started. On days −9 and −5, respectively, prophylactic treatment was started with co-trimoxazole against Pneumocystis carinii and with acyclovir against viral infections. Patients were conditioned with a regimen of busulphan administered orally at 1 mg/kg every 6 h on days −9 to −6, followed by cyclophosphamide given intravenously on days −5 and −2 at 50 mg/kg per day with mesna and an adequate hydration regimen to prevent uroepithelial toxicity. Processing and culture of marrow for autografting A volume of 1.0–1.5 l of marrow was aspirated from the posterior iliac crests under general anaesthesia to aim for a collection of about 4 × 108/kg body weight. The aspirated marrow was collected in Fenwall blood bags (Baxter Healthcare, Norfolk, UK) containing 10 000 U of preservative-free heparin as the anticoagulant. A buffy coat fraction was then prepared on a COBE 2991 blood cell processor (COBE Labs, Centrewood, CO, USA) and after counting the cells in a Coulter counter (S890) an aliquot of 2 × 108/kg body weight was used for LTBMC. The remainder, a minimum of 1.3 × 108/kg, was frozen in liquid nitrogen in autologous plasma and 20% DMSO (v/v) (Scintillation grade; Koch Light Ltd, Aldrich, UK) on a controlled rate cell freezer (PTC 200; Planar Biomed Products Ltd, Middlesex, UK) for use as a backup if necessary. At least one LTBMC flask was established per kilogram of body weight for each patient to ensure adequate numbers


of cultured cells for re-infusion. An aliquot of 2 × 108 buffy coat cells was placed into 175 cm2 flask (Falcon, Becton Dickinson, NJ, USA) containing 100 ml of Iscove’s modified Dulbecco’s medium (IMDM) (Gibco, Grand Island, NY, USA) supplemented with 200 mm l-glutamine, 300 mg/l penicillin, 50 mg/l of streptomycin sulphate, 7.5% sodium bicarbonate, 40 U/ml of preservative-free heparin, 10% (v/v) fetal calf serum (pre-tested batch) (Flow Labs, Irvine, CA, USA), 10% (v/v) horse serum (pre-tested batch) (Flow Labs), and 5 × 10−7 m hydrocortisone sodium succinate (Sigma, Dorset, UK). Thus, the final cell concentration was similar to that of the standard 25 cm 2 LTBMC flask (2 × 106 nucleated cells/ml). The flasks were gassed with 5% CO2 in air and incubated at 33°C for 10 days. An aliquot of LTBMC medium before cell inoculation and aliquots from a random sample of flasks after 5 days of culture were screened bacteriologically. No contamination of cultured material was ever established in this group of patients. All culture procedures were carried out in a designated room with positive pressure filtered air ventilation in level II microbiological safety cabinets. After 10 days of culture the adherent layer of each flask was detached by mechanical scraping with a ‘rubber policeman’ and collected together with the non-adherent cell suspension. The contents of 20 flasks were pooled at a time and transferred to a 2 l Fenwal transfer bag (R2041 Travenol, Norfolk, UK). The cells were washed in IMDM on a COBE 2991 blood cell processor and the final volume of the total proof reduced to 200–300 ml. The final pool was loaded into a 600 ml transfer pack (R2021 Travenol) containing 2% (v/v) autologous serum and infused into the patient via the intravenous long line without a filter. Minor reactions of fever and chills occurred occasionally and were managed with anti-histamine and hydrocortisone. An aliquot of each cultured autograft was taken for assessment of incidence of GM-CFC, and in the last cases, for evaluation of Mk-CFC numbers in the relevant clonogenic assays.24 The incubation period of 10 days was chosen based on our previous experience of cultured autografts in AML patients who successfully reconstituted with karyotypically normal cells following myeloablative chemotherapy.26,27 Later, a similar culture time was used in CML by the Vancouver group and it was shown to contain maximal numbers of selectively enriched normal (Ph−) stem cells.17,28

intervals of 3–4 months during the 1st year, and every 6– 12 months during the subsequent years. Haematologic remission was defined as the absence of morphologic evidence of CML in blood and marrow. Complete and major cytogenetic remission were defined respectively as the presence of 100% Ph− cells and of at least >65% to ,100% Ph− cells in the marrow. With one exception, maintenance therapy with IFN-a was introduced at 6–12 months postautograft when Ph- cells were still in the majority or at least detectable. The dose of IFN ranged from 2–5 mega units subcutaneously once daily 3–7 days/week.

Patients care and follow-up after autografting

UPN

The management of these patients with regard to antibiotic usage, irradiated blood products and intravenous nutrition was similar to that of conventional autologous BMT. Prophylactic use of acyclovir and co-trimoxazole was maintained for 6 weeks and 6 months, respectively. Patients seronegative for cytomegalovirus (CMV) received CMV blood products. Patients were allowed home when their absolute neutrophil counts exceeded 0.5 × 109/l and there was no clinical sign of infection. Platelet support continued on an out-patient basis and it was stopped when the patient’s own platelet count exceeded 20 × 109/l. BM aspirates for morphologic and cytogenetic assessment were obtained at the time of engraftment and thereafter at

Results Pre-transplant screening LTBMC test A preliminary sample of marrow for each patient was tested in LTC to assess the presence of residual Ph− cells and the degree of cytogenetic conversion within 4–5 weeks. The populations assessed were developing non-clonogenic cells and also colony-forming cells (mainly from the supernatant of the cultures). We made the assumption that an enrichment of Ph− cells in such mature populations after that time in culture would mirror the selection of a primitive haemopoietic cell population of similar genotype, present in the initial inoculum and preserved for at least 10 days. Patients were considered eligible if the percentage of Ph− cells was >75% in colony preparations by 3 weeks in culture (Table 2). However, for patients in whom culture cytogenetic preparations were only obtained from supernatant mononuclear cells, a conversion of .50% Ph− metaphases at 3 weeks was considered adequate. This was based on previous studies of the kinetics of Ph− restoration in the different LTBMC cell compartments (with the content of normal genotype in the dividing supernatant mononuclear cell population being on average a third of that of the clonogenic cells).21 Four of these patients (UPNs 891, 892, 912 and 921) initially failed the culture criteria to enter the study since their marrow showed marginal levels (2%) or no detectable Ph− cells in LTBMC at diagnosis. These patients were put on a regimen of pulse high-dose hydroxyurea and their marTable 2

Cytogenetic studies in LTBMC prior to autografting

Months from diagnosis

% Ph− metaphases or colonies (No. analysed) Initial marrow inoculation

891 892 901 902 911 912 921 922 931 a

4 11 37 11 21 14 10 14 6

3 (30)b 10 (50)b 0 (30)b 0 (30)b 17 (30) 30 (10) 25 (60)a 0 (30)b 6 (31)

LTBMC 81 57 100 90 93 100 88 94 94

(29)b (56)b (30)b (30)b (30) (13) (60) a (17)b (17)

Weeks 3 3 3 3 4 4 5 2 2

Pooled colonies. Cytogenetic preparations of mononuclear cells from the initial aspirate or the culture supernatant.

b

971


972

Ph– metaphases %

100 80 60 40 S3 20

S2

0

S1

UPN 891

UPN 892

UPN 912

UPN 921

Figure 1 Series of LTC tests for each patient showing % Ph− metaphases (of a total of 23–50 analysed) in preparations of mononuclear cells or pooled colonies from LTC established with a diagnostic sample (S1 = at 2–4 weeks) and from LTC established 5–7 days after a pulse of high-dose HU (S2 = at 2–4 weeks and S3 = at 5 weeks). For comparison the same cell population (either supernatant mononuclear cells, pooled colonies from the supernatant or pooled colonies from the adherent layer) was analysed after similar time in culture of flasks established from the same individual before and after HU therapy, as previously reported.21

row re-assessed in LTBMC after 2–5 months of treatment. This time they all showed a high degree of cytogenetic conversion to Ph− haemopoiesis following LTBMC (Figure 1). In two cases, 57% and 90% Ph− metaphases were obtained from preparations of supernatant mononuclear cells (UPNs 891 and 921), and in the other two colony cytogenetic preparations showed 79% and 93% Ph− metaphases (UPNs 892 and 912). Repeated samples were obtained later and continued to show similar levels of cytogenetic response in LTBMC even after 4–14 months of pulse HU therapy (Table 2). Cell dose and time of engraftment With an average reduction in culture of 50% of the original cell inoculum, the autograft after 10 days in culture contained a median of 2.4 × 108 nucleated cells/kg body weight (range 1–4). The median GM-CFC dose was 6.5 × 104 cells/kg body weight (range 0.9–36) representing only 0.02% of the total nucleated cells infused (Table 3). Table 3 UPN

891 892 901 902 911 912 921 922 931 Median (range)

In addition aliquots of autografts from three patients which were also assayed for megakaryocyte progenitors produced no colonies, despite the incidence of Mk-CFC in the original marrow harvest being within the normal range (4–25 per 105 mononuclear cells) (data not shown). One patient died of sepsis 1 month after the autograft before any signs of engraftment occurred. Another patient failed to engraft despite the infusion of the back-up marrow and died 3 months after the autograft. Evidence of myeloid engraftment was observed in seven patients, with normal (or near normal) blood counts being subsequently achieved in all. One case had prolonged thrombocytopenia of .1 year duration, which proved to be fatal. The median time for the recovery of neutrophils to 0.5 3 109/l in seven patients was 26 days (range 15–39), and for the recovery of platelets in six patients was 95 days (54–252) with a median duration of platelet support of 8 weeks (range 4– 21) (Table 3). Time-course cytogenetic analyses Table 4 shows the cytogenetic results of the original bone marrow harvest, the reinfused cultured cell suspension and of follow-up marrow samples obtained after the autograft. The infused cells were in most patients still predominantly Ph+. However, these are mainly mature end cells and as such are distant in the differentiation pathway from the long-term culture-initiating cells (LTC-IC), which are thought to overlap or be closer to the cells responsible for long-term engraftment. Although the number of LTC-IC infused was not measured,29 the cytogenetic profile of the progeny of these cells (ie clonogenic cells from 4- to 5week-old LTC) was shown to be predominantly Ph− (data not shown). This was reflected in the composition of the marrow at the time of engraftment, with four patients showing exclusively (100%) Ph− haemopoiesis and three having predominantly (71–92%) Ph− cells. The duration of complete Ph− haemopoiesis ranged from 6 to 12 months. IFN therapy was started at 6–12 months after autografting, except for one case (UPN 891) who received IFN at the return to clinically apparent chronic phase. For those who were started earlier on IFN, the Ph+ clone continued to increase in two (UPNs 901 and 911), while in three who actually had not reconstituted with

Cell dose and time of engraftment Cell dose /kg b.w. (×108 /kg)

GM-CFC /kg b.w. (×104 /kg)

Days to ANC >0.5 × 109/l

Days to platelets >50 × 109/l

Platelet support (weeks)

1.0 1.0 2.8 4.0 2.4 2.2 1.4 2.5 3.6 2.4 (1–4)

0.9 7.8 11.2 2.8 2.9 5.3 36.0 15.0 1.1 6.5 (0.9–36)

25 NA 39 NA 15 32 24 26 28 26 (15–39)

252 NA 54 NA 70 95 126 NA 180 95 (54–252)

21 12 7 4 4 8 8 10 6 8 (4–21)

ANC = absolute neutrophil count; NA = not attained.


Table 4

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Time-course cytogenetic analyses

UPN

% Ph− marrow metaphases (No. analysed) Harvest

Reinfusion

Months following autograft 1–2

891 901 911 912 921 922 931

19 0 0 17 20 0 0

(43) (30) (30) (30) (50) (30) (30)

100 (5) 3 (30) 14 (7) — 50 (30) 26 (23) —

100 100 100 92 71 100 90

(46) (30) (30) (50) (14) (50) (50)

4–6

8–9

100 (50) 95 (30) 100 (30) 100 (80) 83 (30) 100 (22) 86 (50)a

97 86 100 85 57 96 92

(60) (30)a (30) (60) (30) (50) a (30)

12–18 87.5 (32) 78 (27) 50 (30)a 57 (30)a 16 (30)a 97 (63) 99 (100)

24–28 0 67 50 97 40

(30)a (30) (30) (30) (25)

100 (30)

30–36 23 0 0 100 59

(30) (30) (30) (30) (22)

48 0 (30) 100 (30) 22 (30)

100 (28)

Patients started on IFN-a maintenance. Two of the original patients not included due to graft failure and early death (1–3 months).

a

100% Ph− cells the Ph+ clone was further suppressed, in two of them to undetectable levels (UPNs 912 and 931). The remaining patient (UPN 922) is not evaluable since she died 7 months later as a consequence of profound thrombocytopenia. Outcome and survival

Discussion

The survival and present status of each patient at the last follow-up (May 1996) is described in Table 5. There were three transplant-related deaths: two patients died of graft failure at 1 and 3 months post-autograft, and one died of cerebral vascular accident as a consequence of profound thrombocytopenia at 16 months. This patient then had 97% Ph− haemopoiesis. Three patients reverted to chronic phase and 100% Ph+ haemopoiesis at 27 (UPN 891) and 36 (UPNs 901 and 911) months post-autograft. Unrelated HLA-matched donors were found for two (UPNs 901 and 911) and in a second attempt to cure their disease, MUD transplants were carried out at 43 and 46 months respectively after the autograft. Unfortunately both succumbed to infection and died 7 and 12 months later. Four patients are alive 40–87 months post-autograft: one Table 5

in chronic phase controlled by HU (UPN 891), one remaining in haematological remission but with evidence of Ph+ clone evolution despite being on IFN-alpha (UPN 921), and two in complete cytogenetic remission 40 and 53 months post-autograft (UPNs 912 and 931).

Our study confirms that autografts of cultured marrow in CML result in a high rate of marrow reconstitution with entirely or predominantly Ph− cells and a prolonged period of cytogenetic remission in some patients.17 Although we have not quantitatively evaluated the numbers of Ph− LTC-IC and their leukaemic counterparts after 10 days in culture for the infused autograft or as part of the pre-harvest evaluation, our indirect assessment of these progenitor populations proved to be adequate to predict engraftment with total or predominantly Ph− cells. The culture criteria for eligibility were a minimum content of >75% Ph− metaphases from colony (GM-CFC) preparations or .50% Ph− metaphases from supernatant mononuclear cells at 3 weeks. This would entail, by the kinetics of Ph− conversion in LTBMC,18 a greater percentage still

Outcome and survival after autografting

UPN

% Ph− cells at engraftment

Duration of marked Ph suppressiona (months)

892 902 922 891 901 911 921 912 931

— — 100 100 100 100 71 92.5 90

— — >16 27 36 36 18 >48 >36

Outcome of autograft

early death early death late death Rel→CP Rel→CP Rel→CP HR CCR CCR

Survival postautograft (months)

Comments and present status

3 1 16 87 55 53 50 53 40

graft failure graft failure prolonged thrombocytopenia (CVA) IFN-a 30 months post-autograft for 6 months, no response, put on HU MUD transplant at 43 months after autograft, died of ARDS (post-viral) 12 months later MUD transplant 46 months after autograft, died 7 months later of septicaemia on IFN-a maintenance (22% Ph− cells) on IFN-a maintenance (100% Ph− cells) on IFN-a maintenance (100% Ph− cells)

CVA = cerebral vascular accident; Rel = relapse; CP = chronic phase; MUD = matched unrelated donor; ARSD = acute respiratory distress syndrome; CCR = complete cytogenetic remission; HR = haematologic remission. a Defined as the time up to the first cytogenetic evaluation with Ph− cells being ,50%.


974

of Ph negativity by 5 weeks. Since at 5 weeks, the GMCFC cells are not part of a pre-existing GM-CFC population, but are derived from a primitive progenitor cell population (ie the LTC-IC),30 the conversion to maximal or total Ph− haemopoiesis among the former cells would suggest a large differential in the relative numbers of Ph− and Ph+ LTC-IC in the initial inoculum. This differential becomes even greater after 10 days in culture, because the maintenance of leukaemic LTC-IC is very compromised in such conditions compared to normal (Ph−) LTC-IC cells.19 By using a quantitative assessment of the content of primitive progenitors after 10 days in LTBMC with a minimum requirement of 20 Ph− LTC-IC per 2.5 × 107 initial marrow cells and the numbers of Ph+ stem cells being less than the limit of the sensitivity of the assay, it is possible that Barnett et al17 have selected a better group of patients, ie with a greater reserve of normal marrow than the cohort of patients selected in this study. The greater percentage of evaluable cases reported by Barnett et al17 who reconstituted with entirely Ph− haemopoiesis (13 out of 16, 81%) compared to the one in this study (four out of seven, 57%) is consistent with this postulate. Recently, the degree of Ph− haemopoiesis in the reconstituting marrow after transplantation has been shown to correlate with the percentage of Ph− cells in the infused autograft.31 However, the other three evaluable patients in this study still attained high levels of Ph negativity (71–92%) as reported for the remaining three patients (75–94% Ph− cells) in the Vancouver study. Therefore, all patients in our study who successfully engrafted showed a regenerating marrow with only or mainly Ph− cells. Data presented here and in a previous report show that treatment with pulse high-dose HU prior to harvest helped four patients to achieve the degree of Ph negativity in LTBMC considered necessary for the autograft programme.21 Others utilising a similar regimen of high-dose HU have also observed marked suppression of the Ph+ clone in three out of five patients which allowed collection of predominantly normal cells by leukapheresis.32 In addition, we have previously shown that IFN-a therapy in early stage disease is associated with a significantly higher number of patients who show a major degree of Ph− conversion (>50% Ph− cells) in LTBMC compared to untreated patients, even in cytogenetic non-responders.21 It is possible then to increase the proportion of eligible CML patients for this procedure by using these additional therapeutic regimens to reduce the leukaemic burden in vivo prior to LTBMC purging. Three patients died due to procedure-related toxicity (33% mortality), two of these as a consequence of graft failure. The incidence of graft failure (22%) was similar to that reported by Barnett et al17 (23%), and no different from the rates of graft failure in recipients of autografts or peripheral blood mononuclear cells (19–21%),33,34 or in autologous transplantation of PBPC collected in the recovery phase of myelosuppressive treatment (19% to 22%).14,31 The time of haematological recovery in the present study was very similar to that reported previously for patients who received cultured autografts17 and for recipients of unmanipulated marrow autografts.33,35 On the other hand, the speed of haematological reconstitution was slow if com-

parison is made with autografts of PBPC with a median time for neutrophil recovery being 8–11 days more prolonged than the latter.14,31 In addition, recovery of platelets was rather slow. The absence in culture from three patients of megakaryocyte progenitors after 10 days raises the possibility that the conditions of the system might select against megakaryocytes. However, the identification of thrombopoietin may allow improvements in the survival and development of megakaryocyte progenitors in vitro, and thus reduce the period of thrombocytopenia following cultured autografts. Laboratory and clinical studies along these lines and to further improve purging36 are now being carried out. Importantly, in this study, reconstituted haemopoiesis was sustained with no occurrence of late graft failure, suggesting that engraftment with cultured autografts was mediated by haemopoietic stem cells. Durable Ph− haemopoiesis has been reported to occur after unmanipulated autografts of marrow or peripheral blood.33–35 In those cases, however, restoration to Ph− haemopoiesis is reported to occur sporadically and only 4 months to 2 years after the autograft. This suggests that the origin of Ph− haemopoiesis is endogenous rather than autograft-derived. In addition, haemopoietic reconstitution in conventional autografts is mainly with Ph+ cells. Thus, the speed and the high rate of Ph− reconstitution in our series of patients, together with an absence of late appearance of Ph− cells, strongly suggest, as previously reported 17 that Ph− haemopoiesis in recipients of cultured autografts is mainly, if not all, culture-derived. Another procedure generating wide interest as an effective treatment to induce durable complete cytogenetic remission in CML is the autografting of PBPC collected in the recovery phase of chemotherapy-induced aplasia.14 This group reported that five out of the seven evaluable patients reconstituted with Ph− haemopoiesis and remained in complete cytogenetic remission during the 8–12 months of the autograft follow-up.37 Nevertheless, others have not been able to reproduce these results due to considerable technical and logistic difficulties such as complications of myelosuppression requiring frequent hospitalizations, unpredictable time of mobilization necessitating repeated out-patient visits and resulting in the Ph negative ‘window’ being missed or low number of progenitor cells being collected.38 Relapse is the major cause of treatment failure in autologous transplantation due to graft contamination and lack of graft-versus-leukaemia reactions. The former issue may at least in part be addressed by ‘purging’ techniques. However, the lack of immunological effector cells still remains, posing a greater risk for relapse than in syngeneic transplants which are associated with 50% relapse rate.39 The duration of complete cytogenetic responses in this study ranged from 6 to 12 months. This is comparable with the period of complete cytogenetic remission reported by others using purged autografts.15–17,31 In three patients of the present series, cytogenetic relapse was associated with the return to chronic phase, but none progressed to blast phase disease within the 4–7 years following the autograft and 6.5–8 years since diagnosis. This exceeds the median duration of chronic phase in CML suggesting that these patients might have benefited from the treatment. In another three patients, IFN-a introduced 6–12 months post-auto-


graft was effective in further suppressing the Ph+ clone. In two of the latter patients a status of complete Ph negativity was attained after interferon administration and the patients remain in cytogenetic remission 40 and 53 months after autografting and more than 46 and 62 since diagnosis. Despite rather prolonged Ph− haemopoiesis being maintained in some patients with IFN-a following autografting with cultured cells, further improvements in immunomodulation of GVL effectors are required to reduce the probability of relapse and disease evolution.40,41 Only then will it be possible to assess if autografting procedures such as the one reported here can be considered as a curative strategy for the majority of CML patients who are not eligible for allogeneic transplantation.

13

14

15

16

Acknowledgements LHC was supported by the Leukaemia Research Fund and the North West Regional Health Authority.

17 18

References 1 Shtivelman E, Lifshitz B, Gale RP, Canaani E. Fused transcript of abl and bcr genes in chronic myelogenous leukaemia. Nature 1985; 315: 550–554. 2 Fialkow PJ, Jacobson RJ, Papayannopoulou T. Chronic myelocytic leukemia: clonal origin in a stem cell common to the granulocytic, erythrocytic, platelet and monocyte/ macrophage. Am J Med 1977; 63: 125–130. 3 Martin PJ, Najfeld V, Hansen JA et al. Involvement of the Blymphoid system in chronic myelogenous leukaemia. Nature 1980; 287: 49–50. 4 Savage DG, Goldman JM. Approaches to the treatment of chronic myeloid leukemia. Int J Hematol 1994; 60: 1–21. 5 The Italian Cooperative Study Group on Chronic Myeloid Leukemia. Interferon alfa-2a as compared with conventional chemotherapy for the treatment of chronic myeloid leukemia. New Engl J Med 1994; 330: 820–825. 6 Allan NC, Richards SM, Shepherd PCA. UK Medical Research Council randomised, multicentre trial of interferonan1 for chronic myeloid leukaemia: improved survival irrespective of cytogenetic response. Lancet 1995; 345: 1392– 1397. 7 Giralt S, Kantarjian H, Talpaz M. Treatment of chronic myelogenous leukemia. Semin Oncol 1995; 22: 396–404. 8 Clift RA, Buckner CD, Thomas ED et al. Marrow transplantation for chronic myeloid leukemia: a randomized study comparing cyclophosphamide and total body irradiation with busulfan and cyclophosphamide. Blood 1994; 84: 2036–2043. 9 Van Rhee F, Lin F, Cross NCP et al. Detection of residual leukaemia more than 10 years after allogeneic bone marrow transplantation for chronic myelogenous leukaemia. Bone Marrow Transplant 1994; 14: 609–612. 10 Spencer A, Szydlo RM, Brookes PA et al. Bone marrow transplantation for chronic myeloid leukaemia with volunteer unrelated donors using ex vivo or in vivo T-cell depletion: major prognostic impact of HLA class I identity between donor and recipient. Blood 1995; 86: 3590–3597. 11 McGlave PB, De Fabritiis P, Deisseroth A et al. Autologous transplants for chronic myelogenous leukaemia: results from eight transplant groups. Lancet 1994; 343: 1486–1488. 12 Fefer A, Radich J, Pavletic Z et al. Syngeneic bone marrow

19

20

21

22

23

24

25

26

27

transplantation (BMT) for chronic myelogenous leukemia in chronic phase: update of the original 14 Seattle patients including PCR results. Blood 1994; 84 (Suppl. 1): 252a. Deisseroth AB, Zu Z, Claxton D et al. Genetic marking shows that Ph+ cells present in autologous transplants of chronic myelogenous leukemia (CML) contribute to relapse after autologous bone marrow in CML. Blood 1994; 83: 3068–3076. Carella AM, Podesta M, Frassoni F et al. Collection of ‘normal’ blood repopulating cells during early hemopoietic recovery after intensive conventional chemotherapy in chronic myelogenous leukemia. Bone Marrow Transplant 1993; 12: 267–271. McGlave PB, Arthur D, Miller WJ et al. Autologous transplantation for CML using marrow treated ex vivo with recombinant human interferon gamma. Bone Marrow Transplant 1990; 6: 115–120. Carlo-Stella C, Mangoni L, Almici C et al. Autologous transplant for chronic myelogenous leukemia using marrow treated ex vivo with mafosfamide. Bone Marrow Transplant 1994; 14: 425–432. Barnett MJ, Eaves CJ, Phillips GL et al. Autografting with cultured marrow in chronic myeloid leukemia: results of a pilot study. Blood 1994; 84: 724–732. Coulombel L, Kalousek DK, Eaves CJ et al. Long-term marrow culture reveals chromosomally normal hematopoietic progenitor cells in patients with Philadelphia chromosomepositive chronic myelogenous leukemia. New Engl J Med 1983; 308: 1493–1498. Udomsakdi C, Eaves CJ, Swolin B et al. Rapid decline of chronic myeloid leukemic cells in long-term culture due to a defect at the leukemic stem cell level. Proc Natl Acad Sci USA 1992; 89: 6192–6196. Coutinho LH, Testa NG, Chang J et al. The use of cultured bone marrow cells in autologous transplantation. In: Gross A, Gee AP, Worthington-White DA (eds). Bone Marrow Purging and Processing. Alan R Liss: New York, 1990, pp 415–433. Coutinho LH, Brereton ML, Santos AMW et al. Evaluation of cytogenetic conversion to Ph− haemopoiesis in long-term bone marrow culture for patients with chronic myeloid leukaemia on conventional hydroxyurea therapy, on pulse highdose hydroxyurea and on interferon-alpha. Br J Haematol 1996; 93: 869–877. Chang J, Coutinho LH, Harrison CJ et al. High-dose pulsed hydroxyurea treatment conserves Philadelphia chromosome negative (Ph(−)) haemopoiesis in chronic myeloid leukaemia. Br J Haematol 1989; 71 (Suppl. 1): 6. Coutinho LH, Chang J, Testa NG, Dexter TM. Autologous bone marrow transplantation using cultured cells in patients with leukaemia. in: Testa NG, Molineux G (eds). Haemopoiesis. A Practical Approach. Oxford University Press: Oxford, 1993, pp 219–231. Coutinho LH, Gilleece MH, De Wynter EA et al. Clonal and long-term cultures using human bone marrow. In: Testa NG, Molineux G (eds). Haemopoiesis. A Practical Approach. Oxford University Press: Oxford, 1993, pp 75–105. Dube ID, Eaves CJ, Kalousek DK, Eaves AC. A method for obtaining high quality chromosome preparations from single hemopoietic colonies on a routine basis. Cancer Genet Cytogenet 1981; 4: 157–168. Chang J, Coutinho LH, Morgenstern G et al. Reconstitution of haematopoietic system with autologous marrow taken during relapse of acute myeloblastic leukaemia and grown in longterm culture. Lancet 1986; i: 294–295. Chang J, Morgenstern GR, Coutinho LH et al. The use of bone marrow cells grown in long-term culture for autologous bone marrow transplantation in acute myeloid leukaemia: an update. Bone Marrow Transplant 1989; 4: 5–9.

975


976

28 Barnett MJ, Eaves CJ, Phillips GL et al. Successful autografting in chronic myeloid leukaemia after maintenance of marrow in culture. Bone Marrow Transplant 1989; 4: 345–351. 29 Sutherland HJ, Lansdorp PM, Henkelman DH et al Functional characterization of individual human hematopoietic stem cells cultured at limiting dilution on supportive marrow stromal layers. Proc Natl Acad Sci USA 1990; 87: 3584–3588. 30 Andrews NC, Singer JW, Berstein ID. Precursors of colonyforming cells in humans can be distinguished from colonyforming cells by expression of CD33 and CD34 antigens and light scatter properties. J Exp Med 1989; 169: 1721–1731. 31 Talpaz M, Kantarjian H, Liang J et al. Percentage of Philadelphia chromosome (Ph)-negative and Ph-positive cells found after autologous transplantation for chronic myelogenous leukemia depends on percentage of diploid cells induced by conventional dose chemotherapy before collection of autologous cells. Blood 1995; 85: 3257–3263. 32 Kuss BJ, Sage RE, Shepherd KM et al. High dose hydroxyurea in collection of Philadelphia chromosome-negative stem cells in chronic myeloid leukaemia. Leuk Lymphoma 1993; 10: 73–78. 33 Brito-Babapulle F, Bowcock SJ, Marcus RE et al. Autografting for patients with chronic myeloid leukaemia in chronic phase: peripheral blood stem cells may have a finite capacity for maintaining haemopoiesis. Br J Haematol 1989; 73: 76– 81. 34 Hoyle C, Gray R, Goldman J. Autografting for patients with CML in chronic phase: an update. Br J Haematol 1994; 86: 76–81.

35 Kantarjian HM, Talpaz M, Andersson B et al. High doses of cyclophosphamide, etoposide and total body irradiation followed by autologous stem cell transplantation in the management of patients with chronic myelogenous leukemia. Bone Marrow Transplant 1994; 14: 57–61. 36 Hunter MG, Bawden L, Brotherton D et al. BB-10010: an active variant of human macrophage inflammatory protein-1a with improved pharmaceutical properties. Blood 1995; 86: 4400–4408. 37 Bergamaschi G, Podesta M, Frassoni F et al. Restoration of normal polyclonal haemopoiesis in patients with chronic myeloid leukaemia autografted with Ph-negative peripheral stem cells. Br J Haematol 1994; 87: 867–870. 38 Mehta J, Mijovic A, Powles R et al. Myelosuppressive chemotherapy to mobilize normal stem cells in chronic myeloid leukemia. Bone Marrow Transplant 1996; 17: 25–29. 39 Horowitz MM, Gale RP, Sondel PM et al. Graft-versus-leukemia reactions after bone marrow transplantation. Blood 1990; 75: 555–562. 40 Scheffold C, Brandt K, Johnston V et al. Potential of autologous immunologic effector cells for bone marrow purging in patients with chronic myeloid leukemia. Bone Marrow Transplant 1995; 15: 33–39. 41 Massumoto C, Benyunes MC, Sale G et al. Close simulation of acute graft-versus-host disease by interleukin-2 administered after autologous bone marrow transplantation for hematologic malignancy. Bone Marrow Transplant 1996; 17: 351–356.