Expanding the role of blood progenitor cells

Annals ofOncology 6: 759-767, 1995. © 1995 Kluwer Academic Publishers. Printed in the Netherlands.

Review Expanding the role of blood progenitor cells R. Pettengell Department of Developmental Hematopoiesis Memorial Sloan-Kettering Cancer Center, New York, NY, U.S.A.

Key words: blood progenitor cells, mobilisation, transplantation

haemopoiesis following myeloablative therapy. In contrast, 'committed', lineage-specific progenitor cells, can In the adult, haemopoietic progenitor cells principally differentiate only in a predetermined pathway. These reside in the bone marrow. Bone marrow was therefore cells are identified as 'colony-forming cells' (CFC) in used as a source of haemopoietic progenitors for trans- clonogenic assays. In animal models, in vivo repopulaplantation when myeloablative therapies were intro- tion assays can detect stem cells capable of sustaining duced for the treatment of leukaemia and lymphoma. haemopoiesis. The stem cells constitute a subset of Haemopoietic progenitors were first demonstrated in cells that resemble small transitional lymphocytes. human peripheral blood in 1971 [1]. In normal indivi- Flow cytometric cell sorting, using monoclonal antiduals, blood progenitor cells (BPC) circulate in low bodies directed against cell surface glycoproteins, has numbers, and many apheresis procedures are needed been used in conjunction with functional assays, to to collect enough for transplantation. Such cells how- show that both committed progenitors and stem cells ever can engraft just as well as bone marrow cells, with are among the population that express the cell surface similar patient recovery times. Richman et al. [2] were antigen CD34. Subfractionation of the CD34+ cells the first to report that BPC could be mobilised from the has further characterised the stem cell population by marrow into the circulation, after observing a 20-fold the absence of a range of antigens found on maturing increase in blood GM-CFC following treatment with and committed haemopoietic cells (HLA-DR, CD33, cyclophosphamide and doxorubicin. More recently, a CD38, or CD45-RA antigen) as 'lineage negative' variety of different regimens, using cytotoxics and cyto- (Lin-), to express Thy-1 antigen and not to stain with kines alone and in combination, have been shown to the supra-vital dye Rhodamine 123 (Rhl23dull). The stimulate BPC mobilisation. BPC are increasingly used population with these characteristics constitutes only in place of bone marrow for transplantation after 0.01%-0.1% of normal human marrow mononuclear myeloablative therapy, because BPC transplantation is cells. The most primitive haemopoietic cells that can be associated with more rapid recovery of both platelet assessed in humans are the long term culture initiating and neutrophil numbers and with less morbidity. cells (LTCIC), which are capable of sustained producRecent studies have shown that different subsets of tion of the different CFC in long term culture and are BPC can be obtained from different BPC sources, such the closest approximation to human stem cells [3]. Such as bone marrow, umbilical cord blood and leukaphere- cells circulate in the peripheral blood at approximately sis product, or by using different BPC mobilising regi- 3 per ml, and constitute about 0.25%-l% of the mens, or by cell sorting and ex vivo expansion. These CD34+ population and have now been shown to have diverse BPC subpopulations will find specific applica- the same cell surface phenotype and proliferative capation in autologous and allogeneic transplantation, or to bilities as bone marrow LTCIC [4]. support dose intensive chemotherapy regimens, or for Haematological recovery following myeloablative use in gene therapy. Here, we will review the expanding therapy and haemopoietic progenitor cell transplantarole of BPC in oncology. tion depends on the presence in the graft of committed progenitors, to effect early engraftment, and primitive progenitors, to effect long term reconstitution. TransCharacterisation of BPC — primitive and committed plantation of CD34+ cells from bone marrow or peripheral blood has demonstrated that both primitive progenitors and committed progenitor populations reside in the In the bone marrow, the different cell lineages derive CD34+ fraction [5]. Karyotyping of recipients after from the putative pluripotent stem cells. In theory, a sex-mismatched allogeneic bone marrow transplantasingle such cell would be capable of re-establishing tion in humans has proved that donor cells are responIntroduction

760 sible for long term engraftment. Until recently, it remained uncertain whether long term engraftment following transplantation of autologous BPC was achieved by infused stem cells or endogenous cells which had survived the conditioning regimen. A few studies had shown that early progenitors were present in the leukapheresis product and able to produce sustained haemopoiesis in an in vitro transplantation model [6]. Retroviral gene marking techniques have now been used in children undergoing autologous BPC transplantation to show that cells of both myeloid and lymphoid lineages were derived from a common precursor and that such cells are capable of sustained haemopoietic reconstitution over 18 months [7]. Sexmismatched allogeneic transplantation using mobilised BPC is expected to confirm that functional blood stem cells are collected from the peripheral blood at apheresis. Because different proportions of CFC and more primitive cells are mobilised by different stimuli, and with different time-courses, it is likely that primitive and committed progenitor cell mobilisation occur by distinct mechanisms. In patients receiving doxorubicin/ etoposide and G-CSF, both LTCIC and CFC appear to be released at the same time [6]. The quality of these cells in long term cultures is equivalent whether sampled at the GM-CFC peak or 5 days earlier. In contrast, Sutherland et al. [8] reported that earlier mobilisation of LTCIC than GM-CFC can occur after cyclophosphamide alone. BPC kinetics have not been studied with other mobilising regimens but these data demonstrate that primitive and committed progenitors cannot be assumed to share the same kinetics. A high level of CD34+ cells does not necessarily correlate with a high level of cells with stem cell phenotype [9]. This indicates that the measurement of a single progenitor subpopulation, such as GM-CFC or CD34+ cells, should be used with caution to assess the capacity of a drug for mobilisation of the various populations of primitive haemopoietic progenitor cells. In heavily pretreated patients, previous therapy may have compromised marrow reserve, leading to poor yields even for an established mobilising regimen [10-12].

BPC in normal donors for allogeneic transplantation. The populations of BPC obtained with different mobilising regimens will however determine their suitability for the task. Cytokines The use of cytokines alone to mobilise BPC is particularly attractive for cancer patients in remission, and normal donors for gene therapy or allogeneic transplantation. Cytokines differ in their ability to mobilise blood progenitor cells, the timing of mobilisation and the cell populations released. For example, erythropoietin and G-CSF are both lineage-specific growth factors, but striking differences have been reported in their abilities to mobilise BPC. Erythropoietin is a weak stimulus [13], but G-CSF is a very potent stimulus to BPC mobilisation. GM-CSF alone increased CFC 3-18 fold [14-18]. In contrast, growth factors such as IL-3 and the c-kit ligand (stem cell factor, SCF) [19], which act on early haemopoietic cells, are weak BPC mobilisers when administered alone, but they are potent when combined with later acting factors such as sequential IL-3 and GM-CSF [20-22] or SCF with G-CSF which resulted in a further 2-5 fold rise than was seen with G-CSF alone. As receptors for G-CSF are found only on cells of the committed granulocyte lineage, the finding that it can mobilise primitive blood progenitor cells, including multipotent precursors and cells capable of long term myeloid and lymphoid reconstitution, is unexplained. G-CSF treatment increases the numbers of circulating clonogenic cells 6- to 58-fold, and CD34+ cells 4- to 62-fold [23]. G-CSF in doses from 2-15 jig/kg given to normal volunteers increased circulating CFU-GM 4-46 fold [24,25]. Bone pain is associated with the higher doses. A peak in progenitor release is observed after 4 to 6 days of administration. Some groups have used G-CSF alone to mobilise BPC in donors for allogeneic transplantation [26,27]. The optimal combination of cytokines for mobilisation has not yet been established. Although considerable work has been done to determine the best schedules of G-CSF to use to abrogate chemotherapy-induced neutropenia, those giving optimal BPC yields have not been determined.

BPC mobilisation Chemotherapy A wide variety of cytokines and pharmacological agents, alone and in combination, can mobilise BPC from the marrow to the peripheral blood. Although many different mobilisation protocols are now in clinical use, there are few data comparing the quality and the types of haemopoietic progenitor cells mobilised. Because apheresis is such an efficient method of collecting mononuclear cells however, sub-optimal mobilising regimens can be compensated for by performing repeated leukaphereses. The optimum BPC mobilising regimen will depend on their proposed application. For example, cytotoxics will not be acceptable to mobilise

The majority of patients for whom autologous BPC are currently required are already receiving cytotoxic chemotherapy. A number of drug combinations have been used to mobilise BPC [23], based on popular therapeutic regimens. Comparisons between them are difficult because the degree of BPC mobilisation varies widely with pretreatment, disease extent and pathology, and the mobilising chemotherapy used. Even within a single treatment centre there can be considerable variability. This is not surprising in the light of laboratory and assay variables and the high interpatient variability

761 (up to 100-fold) in numbers of progenitors mobilised. Furthermore, most publications record apheresis yields, rather than circulating BPC numbers. As for cytokines, the mechanisms by which cytotoxic drugs stimulate BPC release remain unknown. No relationship has yet emerged with the mode of antirumour action, class of drug or degree of myelosuppression induced. The alkylating agent cyclophosphamide is the best documented and most widely used drug for CFC mobilisation. It has been shown to effectively mobilise early and committed progenitors into the circulation while sparing the most primitive progenitor cells. Other alkylating agents however, such as melphalan and busulphan, are toxic to haemopoietic stem cells. Etoposide is a topoisomerase-II inhibitor and an effective CFC mobilising drug, even in pretreated patients. Like cyclophosphamide, etoposide appears to spare haemopoietic stem cells [6, 28, 29]. This contrasts with the anthracycline doxorubicin, which appears to mobilise BPC well after the first dose and less well thereafter, suggesting that it is toxic to marrow stem cells [N.G. Testa, unpublished data]. In general, it seems that drugs such as melphalan, busulphan or platinum, that induce severe thxombocytopenia, are most likely to be toxic to haemopoietic stem cells. High doses of cytotoxics have previously been thought necessary for BPC mobilisation. Although these have the advantage of antitumour activity, they carry significant risks of infectious complications, and even death. There is some evidence that higher doses of cyclophosphamide, that are more myelosuppressive, stimulate greater progenitor release than smaller doses. In our study using chemotherapy and G-CSF, we found that a low nadir ANC count was not a prerequisite for the release of primitive cells [6]. Increasing the dose of cytotoxics used does not result in increases in progenitor yields comparable to those obtained by the addition of G-CSF. From the limited data available, we cannot determine whether drug combinations are superior to single agents for BPC mobilisation or merely risk additional toxicity. Combinations of chemotherapy and cytokines The majority of studies suggest that the combination of chemotherapy and cytokines stimulates more BPC into the circulation than either alone [6, 30, 31]. An additive or synergistic effect on CFC mobilisation has been seen in all studies using G- or GM-CSF following combination chemotherapy [23]. Such combinations have the advantage (for cancer patients) of antitumour effects and improved safety. It is worth noting however that the addition of GM-CSF to cyclophosphamide increased the yield of CFC and CD34+ cells in blood during recovery from the chemotherapy-induced nadir, but did not further augment the number of LTCIC that could be collected [8]. Following the licensing of filgrastim for BPC mobilisation in several countries,

G-CSF alone and cyclophosphamide/G-CSF have become the most popular regimens. Novel sources of BPC Bone marrow has conventionally been used for haemopoietic rescue following myeloablative treatments. Early studies used freshly collected allogeneic bone marrow, but the development of cryopreservation made autologous bone marrow transplantation possible. Modern controlled-rate cryopreservation with DMSO in liquid nitrogen leads to loss of only 20% of CFC during the freeze-thaw process. Bone marrow transplantation has the disadvantage that harvesting requires a general anaesthetic. In addition, patients with hypoplastic marrow (following previous chemotherapy or radiotherapy) or bone marrow involvement with tumour, are unsuitable for autologous bone marrow transplantation. Recently interest has focused on the use of 5-fluorouracil [32] or cytokine priming before bone marrow harvest or ex vivo [33,34] to improve the yield of specific haemopoietic subpopulations [35]. In some studies there has been accelerated engraftment after transplantation of primed bone marrow, but this has not been shown in randomised studies or comparisons with leukapheresis products. The use of G-CSF or GM-CSF after bone marrow transplantation can shorten the period of neutropenia to that obtained following BPC transplantation, and it seems likely that further improvements in thrombocytopenia will be obtained when recombinant thrombopoietins become available for clinical use [36]. Bone marrow remains the standard against which novel sources of BPC must be assessed. Mobilised BPC collected by apheresis The collection of BPC from the peripheral blood by apheresis is an attractive alternative to bone marrow harvesting. Apheresis is an outpatient procedure, but it requires a co-operative subject, good venous access and a skilled operator. It is not without risks. Citrate toxicity can cause symptomatic hypocalcaemia and, rarely, cardiac arrhythmias. BPC reinfusion can also be hazardous. The reinfusion of high volume products (after multiple apheresis procedures) can lead to DMSO toxicity and haemolysis [37]. Autologous BPC transplantation has been clearly shown to lead to earlier recovery of neutrophil and platelet counts than bone marrow transplantation, reducing hospital costs and stays [38]. Two randomised studies have shown that G-CSF given following mobilised BPC transplantation also accelerate neutrophil engraftment [39,40]. By using effective BPC mobilising regimens, and careful timing of apheresis to coincide with maximal circulating levels of BPC, enough cells for transplantation can be obtained with a single apheresis [6, 41, 42]. Although single apheresis transplantation appears feasible even in heavily pretreated patients with lym-

762 phomas, it may not be possible in patients with primary disorders of haemopoietic system such as myeloma [10]. The optimum timing and frequency of collection in patients may vary depending on whether prior chemotherapy is used for priming, which growth factor is used, and the state of the patient's bone marrow. The minimum number of BPC required to effect haemopoietic reconstitution after myeloablation has not been determined. As discussed above, this is partly due to the incomplete information available on the composition of the haemopoietic product obtained after various BPC mobilising regimens. In addition, factors such as the disease and age of the patient, and the degree of marrow stromal damage incurred by prior chemotherapy and radiotherapy will influence engraftment. In most clinical studies 3-20 x 104 CFUGM or 2 x 106 CD34+ cells/kg has been used as a minimum inoculum size and rapid engraftment has consistently occurred [43,44]. Although leukapheresis product contains primitive and committed BPC, it does not contain the stromal elements present in bone marrow. There is considerable concern about possible tumour contamination of BPC collected by apheresis in cancer patients. A recent study of particular interest describes the release of cancer cells into the circulation with mobilised blood progenitors in patients with small cell lung and breast cancer [45]. The likelihood of marrow contamination varies for different tumours. For example, bone marrow metastases are common in NHL and small-cell lung cancer but uncommon in ovarian cancer [46]. It is likely that these differences will be reflected in the apheresis product, but the risks may also depend upon the choice of BPC mobilising regimen [47,48]. The presence of receptors for haemopoietic growth factors on some tumour cells suggests that direct stimulation could occur. In assessing tumour contamination of the apheresis product, data from functional assays of clonogenicity or tumorigenicity are more persuasive than histology, cytogenetics, immunologic and gene rearrangement studies because with these techniques it is difficult to evaluate the significance of a positive result [46,49]. Genetic marking of tumour cells will help elucidate the role of minimal malignant contamination of apheresis product [50]. Franklin et al. [51] have shown that positive selection of CD34+ BPC from breast cancer patients resulted in a marked reduction in the number of tumour cells reinfused after high dose chemotherapy. Future studies will determine the extent to which CD34+ BPC contain residual tumour cells and their clinical significance [49]. Umbilical cord blood Volume for volume, human umbilical cord blood is as rich a source of haemopoietic progenitor cells as bone marrow [52-54]. The proliferative potential of LTCIC from umbilical cord blood exceeds that of adult bone marrow, compensating in part for the lower number of

cells that can be obtained from a single donor compared with a conventional bone marrow harvest. Indeed, sustained haemopoietic engraftment after myeloablation has been obtained with as few as 2 x 104 LTCIC from umbilical cord blood [55]. Umbilical cord blood can be harvested without hazard or inconvenience to the donor, and with a low risk of important infectious diseases, eg CMV, to the recipient. Among 50 reported umbilical cord blood transplants, the median number CFC-GM infused was 1.9 x 104/kg (range <0.1-25.6 x 104/kg). The recipients ranged in age from 1.3 to 47.8 years and had a median weight of 20 kg (7.5-98.8 kg). Engraftment of donor cells was seen in 39 of 44 evaluable transplants, with neutrophil recovery to 0.5 x 109/l at a median of 22 days and platelet recovery to 50 x 109/l at a median of 49 days. There was no correlation between nucleated cell count or CFC content of the graft and time to neutrophil recovery or probability of engraftment [55]. These data indicate that umbilical cord blood provides sufficient transplantable haemopoietic stem cells for children with HLA-identical or 1-HLA antigen-disparate sibling donors but whether this will prove adequate for 2- or 3-FfLA antigen-disparate sibling donors and adults remains to be determined. Controversy continues over whether umbilical cord blood BPC should be maintained or expanded in bioreactors. In one report, umbilical cord blood mononuclear cells and LTCIC expanded in a bioreactor in the presence of irradiated bone marrow stroma, IL-3, IL-6 and kitligand yielded sufficient cells for transplantation from an innoculum of 3-4 x 108 mononuclear cells (10-15 mis) [56]. In reported umbilical cord blood transplants, the incidence of Graft Versus Host Disease was low. One of the unanswered questions about umbilical cord blood transplantation is whether the reduced immunogenicity of the graft (compared with bone marrow from an unrelated donor) will also result in less Graft Versus Leukaemia effect [57]. A recent report suggested that in umbilical cord blood, a Graft Versus Leukaemia effect was mediated by natural killer and LAK cells, independent of T cell mediated Graft Versus Host Disease [58]. Ex-vivo expansion of BPC Ex vivo expansion is a technique using nutrients and cytokines (added or supplied by a nutrient stromal layer) to favour the growth of haemopoietic progenitors in vitro that can then be reinfused to patients. Ongoing studies have four general goals. The first is to expand lineage-committed progenitor cells with the purpose of totally preventing pancytopenia following myeloablative chemotherapy. This is based on the belief that relatively mature cells are responsible for early engraftment and that if they can be expanded to sufficiently high numbers, the period of pancytopenia can be progressively reduced. For the expansion of committed

763 progenitors, the optimum combination of cytokines includes foMigand, IL-3, IL-6 and G- or GM-CSF [5961]. hi one study, allogeneic transplantation of marrow cultured with IL-3 and GM-CSF led to a reduction in cytopenia by four days. Clinical trials are in progress to determine whether it is possible to improve on BPC transplantation recovery times and whether sufficient numbers of appropriate cells can be produced to provide clinical benefit [60]. The second goal is to reduce the number and length of BPC collection procedures, so that one apheresis could suffice for repeated transplantations, or so that enough cells for transplantation could be obtained from a small sample of umbilical cord blood. Recent studies have shown that significant expansion of CFC are possible in ex vivo cultures of mononuclear or purified CD34+cells [62]. The third goal is to expand selected subsets of cells. These might be 'stem cells' required for gene therapy, or specific committed progenitors, such as megakaryocytes, or lymphocytes for adoptive cancer immunotherapy [63-65]. Whilst it has been demonstrated that CD34+ cells can be expanded in culture [4,62], controversy continues over whether LTCIC can be expanded and whether LTCIC represent true 'stem cells' [3, 62,66]. The fourth goal is to use ex vivo BPC expansion as a method of purging malignant cells whilst maintaining transplantation potential by favouring the growth of BPC over tumour cells. This remains speculative, and has been challenged by the finding that cytokine regimens that mobilise BPC can also stimulate the release of tumour cells, which suggests that tumour growth may also be favoured by the conditions of ex vivo expansion [45]. Thus, the infusion of ex vivo manipulated and expanded primitive and committed BPC appears to be feasible and safe but clinical benefit is still unproven. Mobilised BPC in whole blood The use of non-cryopreserved bone marrow for autologous transplantation is well documented [67]. We have recently demonstrated that committed progenitors in whole blood and leukapheresis product can be stored for up to 48 hours at 4 °C with similar losses (25%39%) to those occurring through freeze-thawing [68]. Primitive progenitors survived even longer. BPC mobilised by chemotherapy and G-CSF circulate in sufficient numbers that 500-750 ml of blood contains enough to support subablative chemotherapy. We have used sequential collections and reinfusions of haemopoietic progenitors in whole blood to support doseintensive, multicyclic chemotherapy for solid tumours [69]. The collection of mobilised BPC by venesection is a simple procedure, that does not require the expensive techniques of leukapheresis and cryopreservation. Our data suggest that chemotherapy regimens of short duration, utilising drugs of short half-life, which are effective

in mobilising blood progenitors and have low toxicity for blood stem cells, can be dramatically dose intensified using this outpatient procedure. This attractive approach however severely restricts the choice of chemotherapy regimen used. Another potential advantage over the use of cryopreserved aliquots of leukapheresis product obtained at the start of treatment is that malignant contamination of BPC obtained in whole blood at each treatment cycle will be sequentially reduced by in vivo purging. New applications for BPC

Substantial laboratory and animal data indicate that many anticancer agents have steep dose-response curves. Undoubtedly, low cytotoxic dose intensity is associated with poor results. Reviews of clinical data support the hypothesis that increased cytotoxic dose intensity can improve response rates and survival in some haematological malignancies, but this is unproven for the common solid tumours. Prospective randomised trials are now in progress to test this hypothesis in different tumours. The finding that cytotoxic drugs and haemopoietic growth factors can stimulate the release of progenitor cells into the circulation has permitted exploration of high dose intensity schedules using autologous BPC support Myeloablative therapy and BPC transplantation Myeloablative therapy was first routinely used with bone marrow transplantation to eliminate minimal residual disease in patients with chemosensitive leukaemias. Its use has now been extended to other chemosensitive tumours, but in most of these (with the exception of relapsed lymphomas) [70] it remains experimental. In recent years however, autologous transplantation has been more widely used. It is in this setting that the advantages of BPC over bone marrow transplantation have been demonstrated. Rapid and sustained haemopoietic recovery occurs after autologous BPC transplantation, with a shorter period of thrombocytopenia, thus improving the risk/benefit ratio of the procedure [38, 71, 72]. Other advantages include the avoidance of a general anaesthetic for bone marrow harvesting, and the ability to offer high dose therapy to patients with poor marrow reserve after previous chemo-radiotherapy, or with bone marrow involvement by tumour. For these reasons, BPC transplantation has replaced autologous bone marrow transplantation in many centres and as a result, high dose treatments are now being offered to a wider range of patients. There are however no prospective randomised trials showing improved disease outcome in patients treated with BPC transplantation over bone marrow transplantation. One study in non Hodgkin's lymphoma showed a significantly better survival for a poor prognosis group of patients with bone marrow involve-

764 ment receiving BPC transplantation compared with a better prognosis group receiving bone marrow transplantation [73]. Whether this is due to a lower likelihood of occult tumour cells, a greater number of cytotoxic effector cells or a different and advantageous pattern of immunologic recovery remains unknown. The issue of whether dose intensive chemotherapy with BPC support is more effective as induction or consolidation therapy is not resolved. The rationale for early treatment is to minimize tumour resistance and ensure adequate BPC yields. However, the increased risk of tumour contamination must be weighed against this. An increased rate of lymphoid reconstitution has been reported following BPC transplantation in humans and may be important, given the probability that part of the anti-tumour effect of allogeneic bone marrow transplantation results from an immunological graft versus tumour effect [74,75]. The ratio of T-cells to progenitors, and the distribution of T-cell subsets differs between apheresis and bone marrow harvests [64]. Autologous BPC rescue results in earlier immunologic reconstitution than bone marrow transplantation, with apparently complete recovery within six months. Whether this will result in a graft versus tumour effect and enhanced disease-free survival, as suggested by the Nebraska group [73], is yet to be determined. The potential to manipulate the graft, either in vitro or in vivo, with immunomodulatory agents may also prove beneficial. Before these important questions can be answered, it will be essential to know more about the composition of the haemopoietic product collected. We are only beginning to understand the different immunologic effects of various forms of transplantation [75]. Allogeneic BPC transplantation Early concerns that infusion of BPC would increase the incidence and severity of GVHD because of the large number of T lymphocytes in the product have not been confirmed. Allogeneic BPC are well tolerated and may accelerate engraftment [76,77]. Accessory cells in the product are more accessible to ex vivo manipulation. However, the cellular composition of the graft is extremely important to haemopoietic recovery and for graft versus tumour effect. Therefore immunologically tailored grafts will need to be extensively evaluated both in vitro and in vivo. The use of human umbilical cord blood for allogeneic transplantation is an exciting research area (see above). The use of donor BPC avoids concerns about the quantity and quality of prior therapy and contaminating tumour cells when autologous bone marrow or BPC are used.

successful in other chemosensitive cancers (myeloma and relapsed germ cell tumours) or the common solid tumours (breast cancer, ovarian cancer and small cell lung cancer) [79-81]. Indeed, multicyclic chemotherapy has been the foundation of the successful treatment of these cancers. The role of increasing cytotoxic dose intensity is as yet unproven, but new methods of maximising cytotoxic dose intensity for these tumours are being explored. Multiple cycles of myeloablative treatment, each supported by autologous BPC transplantation offers the opportunity to maximise cytotoxic dose intensity for solid tumours in which a single high dose treatment is unsatisfactory. Phase I and II studies of dose escalation by high dose sequential therapy with repeated transplantation show that this approach is possible, with manageable haematological toxicity, but with significant non-myeloid toxicity [82]. Such an approach carries substantial morbidity and mortality, in addition to requiring prolonged hospitalisation. Most centres using this approach are planning to collect enough BPC for all the transplantation procedures before embarking upon the first myeloablative treatment. This has the advantage of ensuring that the haemopoietic cells are not damaged by the treatment, but damage to the marrow stroma may still delay re-engraftment in later cycles. Furthermore, careful screening and purging will be needed to minimise the risk of tumour contamination in the BPC product. It remains to be determined whether multiple high dose cycles will prove better than a single cycle of myeloablative treatment. Multiple subablative treatments with BPC support Multicyclic chemotherapy has conventionally been used in the common solid tumours. Haemopoietic growth factors have been used to improve the delivery of the planned dose intensity, but their capacity to increase cytotoxic dose intensity is limited [83]. There is therefore interest in using BPC to support increased dose intensity of sub-myeloablative chemotherapy [8486]. In most studies published to date, the same strategy has been adopted as that used for multiple transplants — that of using aliquots of BPC collected before starting the dose intensive treatment. An alternative strategy is that of sequential collection and reinfusion of BPC at each treatment cycle [69]. This has the advantage of reducing the risks of malignant cell contamination, because the tumour cell kill will in theory purge the blood of malignant cells at each cycle of chemotherapy. Although the risks of BPC contamination in patients with solid tumours are thought to be lower than those of bone marrow contamination, tumour contamination still occurs.

Multiple high dose treatments with transplantation Gene therapy Although a single high dose treatment may be enough to eradicate residual leukaemia, and possibly lymphoma, [48,78] it is unlikely that such a strategy will be

Haemopoietic progenitor cells are among the most accessible stem cells in the body. Well-characterised

765 methods are available for their ex vivo cultivation. They are therefore an attractive target for the transfer of therapeutic genes in patients with single-gene disorders, such as thalassaemias or mucopolysaccharidoses. In addition, the transfection of haemopoietic stem cells with drug resistance genes is being investigated as a means to increasing the risk-benefit ratio of high dose therapy or to give a selection advantage to the transfected population with another gene of interest [87]. The ability to isolate and expand autologous BPC facilitates gene transfer strategies. Summary

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