Seminars in Hematology
Volume 40, Issue 1 , Pages 72-78, January 2003

Autografting in chronic myeloid leukemia☆☆☆

Department of Hematology/Oncology, IRCCS, Ospedale “Casa Sollievo della Sofferenza,” San Giovanni Rotondo (FG), Italy

Article Outline

Abstract 

Autografting (or autologous stem cell transplant [ASCT]) followed by “rescue” with Philadelphia chromosome (Ph)-negative hematopoietic progenitor cells (HPC) remains a good procedure to guarantee prolonged survival for patients mobilized and autografted soon after diagnosis. Among 50 autografted patients who were treated with interferon alpha (IFN-α) and imatinib (for cytogenetic relapse after IFN-α), 41 are alive at a median of 51 months (range, 8 to 106 months). Twenty-eight (56%) patients maintain major cytogenetic remission after ASCT + IFN-α ± imatinib. Such results are probably better than those achieved by IFN-α alone and are similar to the best results obtained in younger patients after allografting with human leukocyte antigen (HLA)-identical sibling donors. The integration of imatinib, during the coming years, into an autografting procedure could represent important progress towards developing a cure for chronic myeloid leukemia (CML) patients who cannot undergo conventional allografting. Semin Hematol 40:72-78. Copyright 2003, Elsevier Science (USA). All rights reserved.

 

It is conventional to assume that the acquisition of the Philadelphia chromosome (Ph) by target stem cells confers on their progeny a proliferative advantage whereby the leukemic clone expands inexorably while the proliferation of normal hematopoietic progenitor cells (HPC) is suppressed indefinitely. More recently, both laboratory10, 22, 39 and clinical17, 26, 33 evidence supported the persistence of Ph-negative HPC in many patients. Various markers of clonality have been used to show that at least some of these cells do not belong to the leukemic clone and are presumably normal.14, 21, 35 Residual normal HPC represent a quantitatively useful reservoir from a therapeutic standpoint, particularly early in the course of disease. Clinical evidence for the persistence of HPC in chronic myeloid leukemia (CML) has been provided by the fact that Ph-negative cells are mobilized into the blood of patients treated with chemotherapy and granulocyte colony-stimulating factor (G-CSF), and by Ph-negative recovery of patients treated with interferon alpha (IFN-α). The rationale to apply autografting in CML derives from the assumption that benign HPC coexist with their malignant counterparts and can have a proliferative advantage in some conditions. These Ph-negative or predominantly negative HPC can be collected after debulking of the leukemic clone with myelosuppressive chemotherapy plus G-CSF and can be used as a rescue treatment after myeloablative chemoradiotherapy protocols that reduce the leukemic burden and, most importantly, the clonogenic cells responsible for blastic transformation. In this way it is possible to “set back the clock” so that the cells that have already mutated are reduced and replaced by new nontransformed cells after autografting. Over the last 13 years our group has demonstrated the feasibility of this strategy.3, 6

The advent of imatinib mesylate has made it more difficult to determine which patients should be advised to undergo autografting, which type of autograft should be used, and when it should be performed.

In this report, we will update our experience and that of others with autografting using Ph-negative HPC (“in vivo” purging); subsequently, we will discuss the role of autografting in the imatinib era.

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What is the rationale for autografting in CML? 

The main objective of autografting is to prolong survival, since there is no clear indication that this procedure cures patients with CML. A number of lines of evidence indicate that “diploid” (Ph-negative) stem cells coexist in many newly diagnosed CML patients. Long-term culture-initiating cells (LTC-IC) from CML patients have a very poor self-maintenance capacity10, 22, 39; therefore, steps that temporarily reduce to low levels the numbers of both diploid and leukemic stem cells can offer a proliferative advantage for regeneration of diploid as compared to leukemic cells. Since it appears that the normal hematopoietic reservoir declines with time, mobilization and collection of peripheral hematopoietic progenitor cells to store Ph-negative progenitors should be performed as soon after diagnosis as possible.

A variety of mobilization and pretransplant cytoreductive regimens have been employed. Our team pioneered this procedure and demonstrated that in many patients it was possible to collect enough stem cells for use as “rescue” after high-dose chemotherapy.

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Choice of progenitor cells 

Peripheral HPC have the advantage that they can be harvested by leukapheresis, thus bypassing the need for general anesthesia. In addition, peripheral HPC result in faster hematologic recovery as compared to bone marrow–derived stem cells. In recent years several groups have analyzed different mobilization protocols for stem cell content. Sutherland et al reported the kinetics of committed and primitive HPC mobilized after chemotherapy and cytokines and demonstrated the presence of LTC-IC in these harvests.32 Breems et al analyzed the leukapheresis products with the cobblestone area–forming cell (CAFC) assay and standard clonogenic assays. They demonstrated a good correlation between the number of colony-forming units (CFUs) and different subsets of CAFCs; the quality of the progenitor cell harvests, in terms of engraftment, correlated well with the numbers of mobilized HPC.2 Prosper et al evaluated the phenotypic and functional aspects of LTC-IC and demonstrated a 60-fold increase of LTC-IC at week 5 and a 12-fold increase of CFUs after G-CSF therapy30; the combination of stem cell factor with chemotherapy and G-CSF was able to mobilize an increasing number of LTC-IC.40 Finally, Ho et al demonstrated that pluripotent and lineage-committed CD34+ subsets in leukapheresis products were mobilized by G-CSF, granulocyte-macrophage colony-stimulating factor (GM-CSF), or a combination of both.19 Taken together, these results indicate that primitive as well as more mature hematopoietic progenitors can easily be mobilized depending on the mobilization protocol used.

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Clinical results with the “in vivo” technique employed in genoa 

One hundred ninety-four patients with Ph-positive CML in different phases of the disease entered our protocol (Table 1).

Table 1. Mobilization of progenitor cells in CML: 13 years of experience (1989-2002)
No. of patients194
Median age (yr)47 (range, 21-62)
Phase of disease
Blastic phase (BP)38 patients
Accelerated phase (AP)28 patients
Chronic phase (CP)128 patients
Early chronic phase60 patients
Procedure-related deaths8 (4%) (BP, 5; AP, 2; CP, 1)
Thirty-eight patients were mobilized during blastic phase, 28 patients in accelerated phase, and 128 patients in chronic phase. Sixty patients in chronic phase were in the first year of disease and had not been pretreated with IFN-α; 68 other patients had previously received IFN-α therapy and had proved to be cytogenetically refractory and/or intolerant to it. The treatment regimens for mobilization consisted of idarubicin, cytarabine, and etoposide for 5 days (ICE) or mini-ICE (ICE for 3 days) protocols (Table 2).6
Table 2. Patients mobilized within 12 months and not pretreated with IFN-α
No. of patients60
Median age (yr)46 (range, 21-62)
Sokal index
Low23 patients (38%)
Intermediate22 patients (37%)
High15 patients (25%)
Median interval from diagnosis to mobilization (mo)2 (range, 1-12)
Mobilization regimen
ICE17
Mini-ICE43

We have updated (to June 2002) the results achieved in patients in early phase of disease (ECP) and not previously treated with IFN-α.6 All patients completed the mobilization protocol and none died of the procedure. Cytogenetic analysis of collected peripheral HPC showed only diploid cells in 36 patients (61%) and ≤ 34% Ph-positive cells in 14 patients (22%). Fifty (83%) of the 60 patients were Ph-negative and had a major cytogenetic remission. Comparison of these data with those achieved in patients in late chronic phase (LCP), cytogenetically refractory to IFN-α, revealed a higher rate of complete cytogenetic remission among patients mobilized early at diagnosis (ECP: 36/60 [60%] v LCP: 23/68 [33%]). These results were supported by the significantly greater numbers of CD34+ cells, granulocyte-macrophage colony-forming units (CFU-GM), and LTC-IC.29

The high rate of Ph-negative collections obtained led us to employ quantitative competitive reverse-transcriptase polymerase chain reaction (RT-PCR) to determine their leukemic content.9, 11, 20 We analyzed 50 consecutive patients, 35 of them in ECP. Our main goal was to evaluate the importance of minimal residual disease in the graft as a prognostic factor for outcome after autografting. A BCR-ABL/ABL ratio less than 0.01 in the graft was strongly correlated with a longer duration of cytogenetic response in the marrow after transplant.9 Twenty-three patients who received grafts with low-level contamination had a sustained cytogenetic response, none experienced prolonged neutropenia and/or thrombocytopenia, and nine had long-lasting complete or major cytogenetic remissions after transplant. The actuarial plots relative to the duration of the cytogenetic response in 50 patients are shown in Fig 1.

To date, 50 patients in ECP, who achieved complete or major cytogenetic remission of peripheral HPC, underwent autografting (Table 3).
Table 3. Autografting with mobilized peripheral HPC: Outcome
Follow-up (mo)
No. of PatientsMedianRange
Autografted50
Alive41 (82%)518-106
Major cytogenetic remission28 (56%)
Ph-positive > 34%13 (26%)
Died9 (18%)4,17,18,21,28,29,38,63,99
Blastic phase7
Fulminant hepatitis1
Cardiac stroke1
High-dose therapy consisted of busulfan (4 mg/kg/d × 4 days) in 44 patients and a total-body irradiation (TBI)-containing regimen (idarubicin or etoposide and single-dose TBI) in six patients. All engrafted and no patient died of the procedure. After hematopoietic recovery, all patients were initially treated with low-dose IFN-α (3 MU three times weekly); subsequently, when possible, patients received full-dose IFN-α (5 MU/d). The median follow-up after autografting is 51 months (range, 8 to 106 months). Nine patients died at a median of 28 months (range, 4 to 99 months) after autologous stem cell transplant (ASCT). Seven patients died of blastic transformation, one of fulminant hepatitis, and one of cardiac arrest. Subsequently, 19 patients relapsed cytogenetically while on IFN-α and seven of them also experienced hematologic relapse; they were treated with imatinib at a dosage of 400 mg/kg/d. Fourteen of 19 patients obtained another cytogenetic remission, which was complete in 10, but there were no molecular remissions. At present, 18 of 19 patients are alive in hematologic remission. Figure 2 shows the overall survival of 50 patients autografted during ECP.
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  • Fig. 2. 

    Autografting in 50 patients with ECP-CML: overall survival. Arrows indicate patients who relapsed after IFN-α and received imatinib. The other patients (+) are now alive receiving IFN-α therapy.

The arrows show the patients who relapsed after IFN-α and received imatinib. All patients are alive with the exception of one death due to cardiac arrest 1 month after starting imatinib. In conclusion, treatment with ASCT/IFN-α ± imatinib was able to control the disease in many patients: 41 patients are alive in hematologic remission and 56% are also in major cytogenetic remission.

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International results achieved with in vivo purging 

Several groups have attempted to reproduce these results and improve on mobilization protocols. More than 500 patients have undergone this procedure. First, Kantarjian et al treated 55 patients with advanced or late chronic phase disease who were cytogenetically refractory to IFN-α and other therapies, using two protocols more intensive than mini-ICE.25 Despite the irrefractory state, 44% of leukaphereses were completely Ph-negative (27%) or with less than 34% Ph-positive cells (17%); moreover, 29% of patients in accelerated phase achieved complete disappearance or less than 35% Ph-positive cells on peripheral blood collections. Van den Berg et al treated 14 patients with the ICE protocol; patients in advanced phase obtained 100% Ph-positive harvests, whereas chronic-phase patients achieved 100% Ph-negative leukaphereses.36 Chalmers et al summarized results of 40 patients treated at different UK institutions with an idarubicin-cytarabine (IDAC) regimen. Forty-three percent of the aphereses were 100% Ph-negative using standard cytogenetics. Subsequently, 20 patients were autografted and 15 are alive at a median follow-up of 24 months.8 Fisher et al investigated the feasibility and the toxicity of this procedure in patients with LCP or advanced-phase disease.15 Different protocols were used to mobilize HPC (ICE, mini-ICE, daunorubicin-containing regimens 3+7, 2+5). Complete and major responses were achieved in nine of 13 (69%) patients treated with mini-ICE and three of 23 (13%) treated with 3+7/2+5 protocols. Of 21 patients autografted, seven achieved complete/major cytogenetic remission. Transplantation-related mortality was absent. Heinzinger et al selected Ph-negative progenitor cells, which do not express HLA-DR molecules. Eight patients in ECP were mobilized with ICE protocol followed by G-CSF and interleukin-3. The content of Ph-positive cells in the leukapheresis product as well as after CD34+ enrichment and after in vitro culture was analyzed with fluorescence in situ hybridization (FISH) and RT-PCR. A substantial number of peripheral HPC samples were negative for BCR-ABL transcripts.18 More recently, the same group treated 17 patients.38 High numbers of CD34+ HLA-DR− cells were obtained. Autografting using predominantly Ph-negative apheresis products was performed in 16 of 17 patients after high-dose therapy. Ten patients achieved complete or major cytogenetic remission. The median follow-up after transplantation was 18 months. Lennard et al collected peripheral HPC in 30 patients after intensive chemotherapy and G-CSF.27 Chemotherapy was well tolerated and progenitor cells were collected early during recovery of the white blood cell count. In 10 patients the collections showed greater than 75% Ph-negativity (range, 79% to 100%), but the apheresis products were positive for BCR-ABL transcripts. Adequate numbers of progenitor cells were collected for engraftment in 21 of 30 patients. Recently, Sureda et al treated 20 consecutive patients with the mini-ICE regimen shortly after diagnosis.31 Complete cytogenetic remission on peripheral blood was observed in 25% of patients and 50% achieved a major remission for a total response rate of 75%. After autografting, many patients did not develop febrile neutropenia and four were treated on an outpatient basis. No patient died as a result of the transplant. Morton et al treated 21 patients with advanced-phase disease with the ICE protocol and G-CSF. Despite their advanced phase and heavy pretreatment, 52% of patients (11/21) achieved a complete/major cytogenetic response assessed in peripheral blood.28 Johnson et al treated 10 patients (nine in stable chronic phase and one in accelerated phase) with high-dose hydroxyurea (3 to 5 g/m2 daily for 7 days) followed by 300 μg of G-CSF daily until the last day of harvesting.23 Five patients required admission to the hospital with neutropenic fever; four patients developed rash and mucositis. All aphereses were PCR-positive but half of the patients had greater than 98% normal metaphases.

A recently reported European multicenter study of advanced-phase patients sought to evaluate the quantity and quality of mobilized progenitor cells in patients ineligible for allografting and cytogenetically refractory to IFN.7 Thirty-two patients received intensive chemotherapy and G-CSF. A total of 119 leukaphereses were performed. Six (21%) of 29 evaluable patients achieved complete cytogenetic remission and four (14%) a major response, for a total major response rate of 35%. Sixteen patients were autografted and all were alive at a median follow-up of 9 months (range, 3 to 12 months). Despite the high-risk characteristics of this patient population, Ph-negative and partially negative HPC were successfully mobilized in more than one third. The possibility has recently been addressed that an additional cycle of intensive chemotherapy would have increased the number of Ph-negative progenitors in the harvest: HPC were collected from 19 patients during ECP following each of two consecutive cycles with an ICE-modified protocol (first cycle) and high-dose cytarabine (Ara-C) and amsacrine (second cycle). After the first cycle, all patients showed a cytogenetic response in peripheral blood collections (complete in three patients and major in five); after the second cycle, seven patients obtained complete (n = 1) and major (n = 6) cytogenetic remissions. Seven patients were autografted and maintained with IFN. Currently, 16 patients are alive with 50% of them having a major cytogenetic remission. In conclusion, a second cycle did not appear to be effective in improving Ph-negativity of the HPC graft (R. Janssen, personal comunication).

We conclude that premobilization chemotherapy provide preferential in vivo reduction of the Ph-positive stem cell population and synergized the effects of G-CSF to stimulate the release of primitive Ph-negative HPC into the blood. The entire procedure is well tolerated. Notwithstanding the different phases of disease, 13 (2%) of 529 patients, many of whom were heavily pretreated and in an advanced phase of disease, died of mobilization therapy and nine (3%) of 237 died of autografting. During ECP, the HPC collections yielded significantly more CD34+ cells than from steady-state or mobilized bone marrow.37 Ph-negative mobilized cells proved to be polyclonal (by X-chromosome inactivation) in the majority of patients in ECP.1 Following these results, an international randomized trial was started under the aegis of the European Group for Blood and Marrow Transplantation, in order to investigate the role of autografting followed by IFN-α versus IFN-α/Ara-C. A few months from the initial inclusion of patients, the first preliminary data on imatinib were published, making it difficult to recruit patients to the study. Thus we lost the opportunity to evaluate the role of autografting/IFN-α as a possible new arm to be compared with imatinib.

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Is there a role for autografting in the imatinib era? 

In 1999 the first preliminary results with imatinib were presented and it was soon clear that this drug would revolutionize the treatment of CML.12, 13 The data continue to be impressive and the first results of a prospective randomized study comparing imatinib with IFN-α + Ara-C (Novartis CSTI 106) showed more favorable results in terms of complete hematologic remissions and major cytogenetic responses after imatinib (F. Guilhot et al: EHA 2002 Meeting). The situation is different in advanced phases of disease, where the drug is not effective in all patients and many develop resistance.24, 34 In the next few years, if results with imatinib maintain the promise, large-scale studies of autografting in CML will not be easily viable. However, since imatinib will not produce cytogenetic remissions in all patients and only rare patients so far have achieved complete molecular remissions, the role of autografting could be revisited and possibly included in new therapeutic strategies. One of the most convincing arguments in favor of imatinib is, besides its efficacy, its easy administration and the apparent absence of major side effects. This concept was possibly exaggerated by the media which led patients to nearly universally prefer treatment with imatinib rather than other transplant or nontransplant procedures. Thus the recent randomized trial (Novartis CSTI 106) shows that very few patients have abandoned imatinib, whereas more than 70% of patients assigned to the IFN-α/Ara-C arm have done so. At the same time new studies are evaluating the combination of imatinib with other drugs. We cannot ascertain whether the addition of other drugs such as pegylated-IFN and high-dose Ara-C, characterized by different toxicity profiles with respect to imatinib, offers a real benefit to patients, but they will increase toxicity. Moreover, only 16 months have passed from the start of the Novartis CSTI 106 trial and more time is required before we can determine whether imatinib will perform better than conventional therapy to increase survival. An alternative approach (at least for intermediate- to high-risk patients) could be autografting as soon as possible after imatinib induction therapy, in an attempt to kill the residual leukemic clonogenic cells. Although autografting may induce toxicity and increase the need for hospitalization, it could offer the advantage of killing leukemic progenitors responsible for blastic transformation.

The following procedure, proposed originally by our team and accepted by seven other Italian groups, is presented as an alternative and more feasible approach for all risk categories. This protocol should be appropriate in patients for whom an allograft is deemed inappropriate by virtue of age, medical fitness, or lack of donor. All patients will receive imatinib; after 3 months, if the patient achieves complete hematologic remission with or without a major cytogenetic response, imatinib is stopped for 1 week and then G-CSF at 10 μg/kg/d is started for 3 to 4 days to collect ≥2 × 106/kg HPC. Subsequently, these cells will be studied for cytogenetics and BCR-ABL/ABL ratio and will be cryopreserved; in the meantime, the patient resumes imatinib until disease progression. In this phase, high-dose chemotherapy (like busulfan) followed by previously collected Ph-negative or predominantly negative HPC, will be given. After engraftment, the patient will be “maintained” with IFN-α (since progression occurred on imatinib). If, on the contrary, the patient achieves complete hematologic remission but not a major cytogenetic response, imatinib will be stopped, and in vivo mobilization chemotherapy with mini-ICE + G-CSF begun; HPC will be collected for use as “rescue” after high-dose busulfan. After engraftment, IFN-α will be offered to the patient (Fig 3).

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  • Fig. 3. 

    Flow chart of the protocol proposed by our team. HR, hematological remission; MCyR, major cytogenetic remission; NoCyR, no cytogenetic remission; HD, high-dose; Glivec (Novartis Pharmaceuticals, Hanover, NJ), imatinib mesylate.

Our experience with five patients mobilized in the above conditions showed in three of them an excellent collection in terms of CD34+ cells (>2 × 106/kg) and a content of leukemic cells lower than those found in the bone marrow, using RT-PCR for BCR-ABL transcripts. Two of these patients who had high-risk characteristics were able to auto-engraft after marrow ablation with the high-dose therapy regimen.

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Conclusion and overall management strategy 

Residual normal hematopoiesis is present at diagnosis in many patients. During chronic phase, the leukemic cells can be destroyed quite easily by therapy. Various markers exist for leukemia cells, which can readily be recognized and quantitated and thus exploited for therapeutic purposes. We and others have confirmed that it is possible to mobilize nonclonal HPC in CML and in other hematologic diseases with cytogenetic markers such as Ph-positive acute lymphoblastic leukemia and myelodysplastic syndromes.4, 5 Thus it is likely that attention in the next years could focus on this aspect, giving new impetus to the role of autografting.

For overall management, there seems to be general agreement that younger patients (<50 years) with high-risk CML who have an HLA-identical sibling should still receive an allografts after remission induced by imatinib or IFN-α.16 This procedure should also be offered to younger patients (≤40 years) for whom a matched unrelated donor is available. Since it has been clearly demonstrated that the reservoir of normal hematopoiesis declines with time, it may be desirable to mobilize, collect, and store Ph-negative or prevalently Ph-negative progenitors as soon after diagnosis as possible, after the disease is controlled by imatinib and while the search for a matched unrelated donor proceeds. After 3 to 6 months, if no donor is found, the patient continues imatinib. In case of relapse autografting with the previously stored Ph-negative progenitors followed by IFN-α may be undertaken.

Unfortunately, the unsolved questions related to imatinib are so numerous that we still need many further studies. Meanwhile, autografting followed by “rescue” with Ph-negative hematopoietic progenitor cells collections remains a good procedure to guarantee a prolongation of survival to patients mobilized and autografted early after diagnosis. Forty-one of 50 patients autografted, who were treated with IFN-α and imatinib (for cytogenetic relapse after IFN-α) are alive at a median of 51 months (range, 8 to 106 months). Twenty-eight (56%) patients maintain a major cytogenetic response after ASCT + IFN-α ± imatinib. Such results are probably better than those achieved by IFN-α alone and are similar to the best results obtained in younger patients after allografting with HLA-identical sibling donors. The integration of imatinib into an autografting procedure could represent important progress toward curing CML patients who cannot undergo conventional allografting. At present, autografting can be used as salvage therapy in patients who progress despite imatinib.

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☆ Supported by “Un Taglio per la vita.”

☆☆ Address reprint requests to Angelo M. Carella, MD, IRCCS, Ospedale “Casa Sollievo della Sofferenza,” Department of Hematology/Oncology-Viale Cappuccini, 71013 San Giovanni Rotondo (FG), Italy.

PII: S0037-1963(03)70044-4

Seminars in Hematology
Volume 40, Issue 1 , Pages 72-78, January 2003

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