Minimal residual disease in acute leukaemia: preclinical studies in a relevant rat model (BNML)

3 Minimal residual disease in acute leukaemia: preclinical studies in a relevant rat model (BNML) ANTON HAGENBEEK ANTON C. M. MARTENS 'Minimal residual disease' (MRD) in leukaemia is defined as the relatively few leukaemic cells which have survived successful remission-induction chemotherapy hiding below the conventional cytological detection level. As is illustrated in Figure 1, in man their number may vary between 1 and as rem ission · Induction chemo ther op y

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much as 1010 cells. Obviously, the presence of MRD heralds a leukaemic relapse at a later stage and therefore today 's leukaemia research and treatment are focused on (1) developing methods to decrease the detection level of residual leukaemic cells; and (2) defining strategies to eradicate MRD. As much of the questions to be answered are difficult to approach in human leukaemia, there is a great need for realistic animal models to translate clinical problems into laboratory experiments and to extrapolate new diagnostic and therapeutic tools from model to man. ANIMAL MODELS A particular animal model should be chosen based upon the specific question to be answered. In bone marrow transplantation research various animal species are employed, such as rodents, dogs and subhuman primates (Figure 2). However, if leukaemia is the main subject of investigation only rodents remain as there are no reproducibly growing (transplantable) leukaemias available in the larger mammals. Apart from investigations in 'spontaneously' arising rodent leukaemias, the majority of experimental studies are performed in transplantable leukaemias. Two important prerequisites for evaluating the efficacy of a given treatment are (a) a reproducible growth pattern upon injection of

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leukaemic cells; and (b) a linear relationship between the number of inoculated leukaemic cells and the survival time. Two major groups of transplantable rodent leukaemias are recognized, i.e. the fast-growing leukaemias (e.g. L1210) which are suitable to study the cycle- or phasespecificity of (new) cytostatic drugs and the slow-growing leukaernias, which from a kinetic point of view come close to the human counterpart. BN ACUTE MYELOCYTIC LEUKAEMIA (BNML) In 1974 we developed an acute myelocytic leukaemia (AML) transplantable in the inbred Brown Norway rat strain (BNML). The major characteristics of this model have been described (Hagenbeek and Van Bekkum, 1977; Van Bekkum and Hagenbeek, 1977; Martens et al, 1990a,b) and are summarized below: Induced by DMBA Analogy with human acute (pro )myelocytic leukaemia: 1. Cytology-cytochemistry 2. Slow growth rate (107 cells i.v.: death at day 23) 3. Severe suppression of normal haemopoiesis due to an absolute numerical decrease in normal haemopoietic stem cells 4. Diffuse intravascular coagulation (DIC) 5. Response to chemotherapy as in human AML 6. Leukaemic clonogenic cells present 7. Low antigenicity 8. No virus In particular as regards its biology of growth and its sensitivity to chemotherapy, the BNML has now been widely accepted as a most relevant model for human AML. So far, 20 leukaemia research centres in Europe, the USA and Canada are employing the BNML in their programmes. The leukaemia was classified as an acute promyelocytic leukaemia (Figure 3B), with signs of diffuse intravascular coagulation as the leukaemia progresses. The leukaemia can be transferred by intraperitoneal (i.p.), subcutaneous (s.c.), intrathecal (i.t.) or intravenous (i.v.) injection of single-cell suspensions. The i.p. injection of leukaemic cells results in the formation of greenish colonies of leukaemic cells in the mesenterium (chloromas), followed eventually by disseminated leukaemia growth. No ascites fluid is formed. The s.c. transfer of leukaemic cells results in the slow development of a subcutaneous tumour which eventually results in disseminated leukaemia growth. The i.p. and s.c. transfer of leukaemia cells were not investigated in depth, in contrast to the i. t. transfer of leukaemic cells (Hoogerbrugge and Hagenbeek, 1985) which was studied extensively with the purpose of the development of a model for central nervous system (CNS) leukaemia. Most studies, however, were done after intravenous transfer of leukaemia, The survival time after i, v. injection of leukaemic cells is linearly correlated with the injected cell number (Figure 3A). After injection leukaemic cells

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spread to all organs, but they predominantly home to the bone marrow, liver and spleen where they start to proliferate and increase in number (Hagenbeek and Martens, 1980). The EDso, which represents the number of cells that is required to induce leukaemia in 50% of the animals, was found to be 25 cells. The leukaemic cells are transferred in a syngeneic system, i.e. the inbred BN rat strain and were found not to be immunogenic. The growth pattern of the leukaemia appeared to be highly reproducible throughout a long period of exploitation. The BNML could be successfully transferred to certain F1 hybrid combinations with varying degrees of hybrid resistance (Vaughan et aI, 1978; Singer et al, 1980; Williams et al, 1980; Burke et al, 1986; Tutschka et al, 1987). The liver and spleen increase in size and weight during the progression of

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the disease (Figure 3C) . During the development of leukaemia the bone marrow is rapidly replaced by leukaemic blast cells. At this stage leukaemic cells are appearing in the peripheral blood leading to increased numbers of peripheral white blood cells, predominantly leukaemic blast cells. The fate of the pluripotent haemopoietic stem cells during this process has been studied . Haemopoietic stem celis are detected by a spleen colony assay (SCA) (Figure 3D) . Concurrently with the replacement of the normal bone marrow, the number of haemopoietic stem cells decreases and reaches very low levels during the terminal stage of the disease. Simultaneously, the number of stem cells in the spleen and in the peripheral blood increases (Figure 3E). Another important characteristic of the BNML is the clonogenic property of the leukaemic cells. The injection of low numbers of cells « lOS) results in the development of leukaemic colonies in the spleen, defined as LCFU-S (leukaemic colony forming unit spleen) clearly visible on the surface 19-20 days after injection (Figure 3D). Every viable leukaemic cell has clonogenic potential (Van Bekkum et ai, 1978). However, after the i.v. injection , a certain proportion of the cells home to places which are unfavourable for leukaemic cell growth . This explains the discrepancy between an EDso value of 25 cells and a clonogenic potential of close to one . Finally, the BNML has the advantage that the leukaemic cells can be discriminated from the normal cells by the use of a mouse anti-BNML monoclonal antibody (MCA) . The MCA reacts with an antigen, present in low quantitie s on normal granulocytes and in high qu antities on leukaemic blast cells, but which is absent on normal lymphocytes and normal blast cells. Flow cytometry enables the detection of leukaemic cells (Figure 3F). METHODS TO DETECT AND QUANTIFY MINIMAL RESIDUAL DISEASE

Various methods are being evaluated for their potency in decreasing the detection level of MRD . The different appro aches aimed at selective discrimination between normal and leukaemic cells are shown in Figure 4. Several of these have been developed in the BNML model and will be discussed here. Based on the previously discussed growth characteristics, five independent methods are available by which the leukaemic cell content in animals, organs or cell suspensions can be determined. Cytology/cytochemistry

Simple total nucleated cell counting in combination with May-GriinwaldGiemsa staining for differential cell counting is sufficient to determine leukaemic cell numbers when they are present in frequencies between 1 % and 100%. Cytochemical characterization revealed that the leukaemic cells were promyelocytes (peroxidase reaction + + ; esterase + ; sudan black + + +; acid phosphatase + +, alkaline phosphatase -).

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Obviously it makes a difference which type of organ is studied. In spleen cell suspensions the detection level will be low (1 % ) because promyelocytes are rarely observed in the spleen under normal conditions. In the bone marrow, however, detection of leukaemic promyelocytes will be difficult when they are present in a frequency below 5%, because this is the background value of normal promyelocytes.

The organ weight parameter The growth of leukaemic cells in liver and spleen results, during the later stages of leukaemia development, in an increase in the weight of both or~ans. It was found that 1g increase in weight corresponds to an increase of 10 cells (Figure 5). Once the organ weight is significantly increased, the leukaemic cell number can be deduced from it. The spleen increases in weight from about 500 mg to 3-4 g towards the terminal stages of leukaemia. The liver increases in weight from 10 to 20-25 g. For the spleen the increase has to be at least 200 mg (corresponding to 2 x 108 cells) to become significantly different from the normal background organ weight. For the liver the increase has to be at least 1 g to become significant, which corresponds with 1 x 109 cells. By weighing the spleen and the liver the effectiveness of a treatment can be quantified. The method is valuable for studing advanced disease. Changes in the tumour load in the range 5 x 10 to 1-2 X 1010 can be studied corresponding to one and a half decade (expressed as 1.5 log).

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The survival time assay A more sensitive method is based on the relation between survival time and number of injected cells (Figure 6). A single cell suspension can be prepared from the total content of a femoral bone by flushing the marrow cavity with a physiological fluid (e.g. phosphate-buffered saline). The total volume of this suspension is measured as well as its cell content. A fraction of the total volume (e.g. 1/10) is injected i.v. into normal recipient rats. If leukaemic cells are present in the cell suspension, the animals will eventually die from leukaemia. The survival time is recorded. Based on the known relation between the number of leukaemic cells injected and the survival time, the originally injected leukaemic cell content can be deduced. To determine the leukaemic cell content of any organ, the survival time assay can be used which implies that the total leukaemic cell content for each organ of choice can be determined. For very low numbers of leukaemic cells (i.e. below 100

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Figure 6. The dose-survival assay for measuring small numbers of leukaemic cells.

cells) the survival times deviate from the straight line, i.e. the survival times increase disproportionately (Van Bekkum and Hagenbeek, 1977). However, by using the results from the dose-survival curve, the leukaemic cell number can be deduced from the percentage of animals that develop leukaemia. The limits of detection of this dose-survival bioassay span 8-9 logs.

Leukaemic colony forming unit-spleen assay (LCFU-S) After the i.v. injection of low numbers of leukaemic cells into normal recipient rats they distribute to all tissues of the body. Cells seeding into the spleen form colonies. When monitored at the appropriate time, i.e. 19-20 days after the injection, these colonies (composed of leukaemic cells) are clearly visible on the surface of the rat spleen. In analogy to spleen colony formation of normal stem cells it is assumed that each colony is derived from one clonogenic leukaemic cell, termed leukaemic colony forming unit spleen (LCFU-S). The fraction of leukaemic cells which enters the spleen could be determined. This so-called f-factor was found to be 0.002 (Van Bekkum, 1977). By counting the number of spleen colonies and correcting this value for the f-factor, the number of clonogenic cells in a cell suspension can be calculated. The limits of detection of this method span 5-6 logs. The LCFU-S assay shows a strong resemblance with the spleen colony assay by which normal haemopoietic stem cells can be measured (Till and McCulloch, 1961). Normal stem cells also form colonies on the surface of the spleen and are therefore named colony forming unit spleen (CFU-S). However, there are two major differences between normal and leukaemic spleen colony

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formation: (1) to determine the normal CFU-S the recipients have to be conditioned with lethal whole body irradiation while the LCFU-S assay is performed in non-irradiated recipients; and (2) the normal CFU-S colonies can be counted between days 7 and 15 after injection of the cells while in the LCFU-S assay the colonies are counted at days 19-20. This indicates that normal haemopoietic stem cells proliferate approximately twice as fast as clonogenic leukaemic cells. These discriminative assays are shown in Figure 7. In summary, one of the great advantages of the BNML is the fact that it is possible to enumerate normal CFU-S as well as LCFU-S in the same cell suspension by choosing the appropriate spleen colony assay. These two methods allow the determination of the therapeutic index of all treatments which depend on the differences in response between leukaemic cells and normal haemopoietic cells. Chemotherapy and total body irradiation have extensively been studied with these methods while current research with recombinant haemopoietic growth factors will indicate whether stimulation of haemopoietic cells also results in stimulation of the growth of leukaemic cells. Monoclonal antibody labelling and flow cytometry A monoclonal antibody (MCA) was raised against BNML cells, i.e. MeARm124 (Dr R. J. Johnson, Johns Hopkins University, Baltimore, MD,

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USA), which detects an antigen that is present in a high density on leukaemic cells, in a low density on normal granulocytes and absent on lymphocytes and normal blast cells (Martens et al, 1984; Martens and Hagenbeek, 1985). After labelling of cells with the FITC conjugated MCA under standard conditions it is possible to discriminate between normal and leukaemic cells on the basis of fluorescence intensity using a flow cytometer. The principle of the method is shown in Figure 8. A typical fluorescence distribution histogram -of a bone marrow cell suspension containing low numbers of leukaemic cells is shown in Figure 9. The fraction of leukaemic cells can easily be determined. This type of analysis allows exact quantitation of the leukaemic cell content in cell suspensions prepared from any organ. If, however, the frequency of leukaemic cells is low, e.g. in minimal residual disease (MRD), accurate analysis is hampered due to the presence of dead cells which aspecifically bind the MeA. The addition of propidium iodide (PI) to counter-stain the dead cells with the use of a special setting on the fluorescence-activated cell sorter (FACS II) (using a 570nm dichroic mirror and the FITC filter combination), enabled selective measurement of the fluorescence intensity of the viable cells while retaining the possibility to measure two other parameters, i.e. forward light scatter (FLS) and perpendicular light scatter (PLS). The detection limit of leukaemic cells using flow

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cytometry is in the order of one leukaemic cell per 10000 to 100000 normal cells and thus spans four to five decades (Martens and Hagenbeek, 1985). All five methods, independently allow the enumeration of leukaemic cell numbers in various organs. The sensitivity is, however, different for each of them. The lower limits and the range of detection ofthe various methods are shown in Figure 10. In addition, several new methodologies are being developed, two of which are briefly discussed next. Flow karyotyping

BNML cells appeared to be characterized by a typical pattern of chromosomal aberrations, i.e. 1p+, 2p+, 8q+, -9, 12q-, 20q+, XX (A. Hagemeijer, Erasmus University, Department of Cell Biology and Genetics, Rotterdam, The Netherlands). The method of flow karyotyping is being explored. With a specially designed dual laser beam flow cytometer it is possible to quantitate objectively leukaemia-associated specific chromosomal anomalies. Chromosomes in suspension are stained with fluorescent dyes, e.g. chromomycin A3 binding to C-G base pairs in the DNA and Hoechst 33258, binding to A-T base pairs, and subsequently run through the flow cytometer, the lasers exciting fluorescence, specific to the configuration of the particular single chromosome passing the laser beams. An example is given in Figure 11. The BNML-associated markers 1p+ and 2p+ are clearly visible. In contrast to conventional methods of karyotyping, thousands of

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chromosomes can be processed per second and in this way 'minimal residual disease' can be detected quantitatively (Arkesteijn et ai, 1987, 1988, 1990).

Genetically marked leukaemic cells Currently, gene transfer techniques to introduce an indicator gene into mammalian cells or embryos as a single cell marker for the study of cell lineages or embryonic development are widely employed . Owing to its high efficiency, retroviral vector-mediated gene transfer is quite often the method of choice to genetically modify mammalian cells. The advantages of retroviral vectors include their unequalled high transfer efficiency, expression in most cell types, accurate and stable integration of a single vector copy into chromosomal DNA, a wide choice of different vectors with different host ranges, and the ease of manipulation of the retroviral genome. As a

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reporter gene, Escherichia coli f3-galactosidase lacZ has proven to be effective in genetic tagging of cells and subsequent tracing of their progeny using cytochemical staining procedures (Goring et al, 1987; Liu et al, 1989; Lin et al, 1990). To study and trace minimal residual leukaemia, we have used a retroviral shuttle vector (BAG) encoding E. coli lacZ as well as the neomycin

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resistance gene (neo'') (Cepko et al, 1984) to infect LT12 cells, an in vitro growing sub line of the BN rat acute myelocytic leukaemia (Lacaze et ai, 1983). The lucZ and neo'' genes were successfully integrated into the chromosomal DNA of LT12 cells and the leukaemic cell lines with lucZ and neo'' (LT12nl) were cloned and expanded from a selective agar culture system with the neomycin analogue G418. For the study of MRD in the BNML model using genetically marked leukaemic cells, the establishment THE PRINCIPLE

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of LT12nl cell lines with a stable and high expression of the marker genes was found to be feasible. The principle of retrovirus-mediated gene transfer as well as the various methods to detect genetically marked leukaemic cells are shown in Figure 12. Studies to apply this new approach in vivo for the detection of MRD are ongoing. In conclusion, a variety of methods has been described here each with its

own sensitivity in terms of detecting MRD. Obviously, extrapolation of these techniques to human acute leukaemia-as far as feasible-has significant implications for the clinical management of this disease as is summarized below:

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Quantification of the efficacy of a given treatment (log leukaemic cell kill) (Dis)continuation of treatment Early detection of arising drug-resistant leukaemic cell clones Establishment of new prognostic factors: which patients will/will not benefit from high-dose chemoradiotherapy and BMT? Quantification of the number of leukaemic cells in autologous marrow grafts Study of the kinetics of regrowth of residual leukaemia

THE DISTRIBUTION OF MRD DURING REMISSION AND AT RELAPSE

Employing the monoclonal antibody Rm124 and flow cytometry, a survey of extensive marrow sampling showed that regrowing leukaemia cells are inhomogeneously disturbed (Martens et al, 1987). Analysis of marrow samples obtained from various bones, e.g. femora, tibiae, ribs, vertebrae, scapulae and sternum, yielded vast differences in leukaemic cell frequencies up to a factor of 28 ODD-fold. This variation was most pronounced in the group of so-called smaller bones, e.g. ribs, vertebrae and scapulae. It became clear that the measured leukaemic cell frequency in one specific marrow sample does not reflect the concentration in the other marrow compartments. Subsequently, a detailed analysis was done in individual animals before as well as after the induction of remission. The study revealed that recurrent leukaemia in the BNML was characterized by an initial phase of 'clonal relapse' followed after a certain delay by generalized spreading of leukaemic cells (Figure 13). The clonal regrowth foci originating from the cells which survived treatment were called 'primary relapse' CPR) sites in contrast to 'secondary relapse' (SR) sites, which developed from cells that migrated out from PR sites. Typical of PR sites was that they consisted of relatively large foci of leukaemic cells which were randomly distributed in the bone marrow compartment. The number of the PR sites and hence the number of clonal regrowth foci, was related to the fraction of leukaemic cells that survived treatment. Typically, once SR sites were identified, they were found to be present in all investigated marrow samples. For example, a femur could

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contain few foci with large numbers of leukaemic cells (representing PR sites), while in the remaining marrow cavity space, much lower concentrations (lOO-fold or more) were found. However, they were all in the same range. Similar low concentrations were in those cases found in all the ribs of the animals, suggesting that these marrow compartments were infiltrated practically at the same moment and by the same route, most likely the blood circulation. Time intervals of up to 20 days between the end of treatment and the spreading of leukaemic cells from the PR sites were observed in the BNML model. The cell population doubling time after treatment was found to be 0.97 days (Schultz et ai, 1986a,b). Assuming the survival of one single leukaemic cell, it can be deduced that at the PR site originating from this single cell a total offspring of 3 X 105 to 4 X 105 are produced before the leukaemia started to disseminate. The results discussed here have important implications for the strategy of marrow sampling in patients. When leukaemia regrowth during the initial phase is also characterized by clonal development in patients, the chance to puncture PR sites is extremely small. In cases where leukaemic cells are detected, they will most likely be leukaemic cells from SR sites. Improvement of detection methods willincrease the likelihood of earlier detection of leukaemic cells at SR sites. Once SR leukaemia has been identified, an unknown number of PR sites is also present. However, estimation of the total tumour load will remain difficult. The clonal regrowth pattern of leukaemia has similarities in this respect with micro metastases growth from solid tumours, e.g. lymphoma, lung or mammary carcinomas after removal of the primary tumour. For these types of tumours, the methods employed for detection of the metastases and hence 'MRD' detection 'in situ', e.g. by nuclear magnetic resonance techniques or by radiolabelling the tumours with specific markers and subsequent 'total body scanning' are reported to become more sensitive. The application of these methods in the follow-up of leukaemia remission patients is recommended to improve the detection, localization and possibly quantification of the 'primary' leukaemia relapse sites. In any case, it is advisable to reconsider the strategy for detection of residual disease in the light of the findings in the BNML model. No matter what modem sampling and analysis technique may be proposed for the detection of residual cells, they all face the difficulty of inhomogeneity of distribution. Provided that the methods for the detection of residual disease in leukaemia patients will indeed be improved in the future and that residual leukaemic cells might be detected at frequencies in the order of one per 1 X 105 or 1 X 106 normal cells, the relevance of the measured values to quantify the residual tumour load has to be questioned. TREATMENT OF MINIMAL RESIDUAL DISEASE

One of the major problems in leukaemia treatment employing either allogeneic or autologous bone marrow transplantation (BMT) is leukaemia relapse. Apparently, leukaemic cells have survived high-dose conditioning

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further intensification? purging? T-cell depletion? low dose chemotherapy - growth factors - BRM?

Figure 14. How to prevent a relapse after autologous or allogeneic bone marrow transplantation (BMT) in acute leukaemia? (BRM, biological response modifiers).

treatment before BMT. In case of autologous BMT, leukaemic cells reinfused with the graft might in addition contribute to leukaemia relapse after BMT. Figure 14 summarizes the crucial points in time during the treatment of acute leukaemia at which the leukaemic cell load can be reduced step by step. Studies in the BNML have contributed to the development of more effective remission-induction and maintenance regimens, including anthracyclines (Sonneveld and Van den Engh, 1981; Sonneveld and Van Bekkum, 1981; Nooter et al, 1983, 1984, 1986), AMSA (Hagenbeek and Martens, 1986) and high-dose cytosine arabinoside timed sequential treatment (Aglietta and Sonneveld, 1978; Aglietta and Colly, 1979; Burke et al, 1980; Colly et al, 1984a,b). These data will not be discussed here. In addition, in the BNML a variety of high-dose marrow ablative conditioning regimens has been studied prior to BMT (Hagenbeek and Martens, 1981; Hagenbeek and Martens, 1982a,b; Martens and Hagenbeek, 1982; Hagenbeek and Martens, 1987a). With methods described before their efficacy could be evaluated in terms of 'log leukaemic cell kill'. A summary of the data obtained so far is given in Table 1. It is of interest to see that the efficacy of the various regimens in the BNML shows an inverse relationship with relapse rates in human acute myelocytic leukaemia treated with similar conditioning regimens. Obviously, this points at the relevance of the BNML in predicting the outcome of treatment in man. Furthermore, the BNML has served as the preclinical model to develop the concept of eliminating leukaemic cells from autologous marrow grafts with in vitro chemotherapy (Sharkis et al, 1980; Hagenbeek and Martens, 1983a; 1984). In the subsequent studies to be presented, two questions will be addressed: (1) what is the contribution of leukaemic cells in the autologous marrow graft to a leukaemia relapse after autologous BMT; and (2) what is

627

BN ACUTE MYELOCYTIC LEUKAEMIA

Table 1. Efficacy of ablative conditioning regimens prior to bone marrow transplantation in acute myelocytic leukaemia. A Log leukaemia cell kill (BNML) Bu+TBI TBI+Bu 0+TID TID+Cy Cy+Bu HDAC+Cy+TBI Bu+Cy

B % Relapse after allo-BMT

in first remission (AML)

5* 6 ~

9-10** 10 >10*** >10

~

10

5-10 8

A, rat BNML; B, human AML. * 70%; ** 20%; *** 25% treatment-related mortality. Cy, cyclophosphamide; TBI, total body irradiation; Bu, busulphan; HDAC, high-dose cytosine arabinoside.

the role of the so-called graft-versus-leukaemia reaction in preventing a leukaemia relapse after allogeneic BMT? AUTOLOGOUS BONE MARROW TRANSPLANTATION So far, clinical results show that 50-60% of patients with AML receiving an autologous marrow graft in first remission will relapse (Lowenberg et al, 1990). Obviously, this may be either due to residual leukaemia in the host or to leukaemic cells reinfused with the graft or to a combination of both. Arguments in favour of residual disease in the host being the major determinant as regards the occurrence of a relapse (first remission AML) are summarized below: 1.

2. 3. 4.

Similar relapse rates after autologous and isologous BMT Leukaemia relapses occur at (about) the same time after autologous, isologous and allogeneic BMT (median: 9 months) ED so for human AML: 103_104 cells (extrapolated from preclinical data in the BNML; Leukemia Research 9: 1389-1395 (1985) So far: no beneficial effect from in vitro 'purging'

In particular the ED so concept will now be further analysed. In the BNML model it is known that 25 leukaemic cells are needed to induce leukaemia in 50% of normal recipient rats after i. v. injection (ED so) . If a total inoculum of, for example, 1000 leukaemic cells is regarded as 40 ED so units, an average of 20 BNML cells would grow out in vivo. Indeed after i. v. injection of 107 BNML cells, 2 x lOs leukaemic cells were recovered at the start of the leukaemia growth process in the major target organs for leukaemia growth in the rat, i.e. bone marrow, spleen and liver. If an inoculum contains x ED so units, with each unit having a chance of 0.5 to grow out and cause overt leukaemia, the chance that leukaemia will not or will develop is 0.5x or 1-05", respectively. If these mathematics are applied to data derived from

628

A. HAGENBEEK AND A. C. M. MARTENS

III

'a
10 10

---- ------------------@.._-------

..!!! 4i u

u

e OJ

'S

j

.::t. ....

.::t. ~

....0

10 8

al

_

.. e OJ ...

10 6

.t:J

+'

.Q

..

j

...

OJ +'

Co

E E

j

'" iO s

~ 01

'0 .,

eOJ

. .~ e

"0

~

OJ ..

C

OJ

j

"0 1Il

..

.o

1_ _] time after start treatment

* graft: 2 x 10 8 cells/kg (SW: 75 kg) CR

: "complete remission"

Figure 15. Relationship between the log leukaemic cell kill induced by remission-induction chemotherapy and the leukaemic cell content of the human autologous marrow graft,

experiments where the incidence of leukaemia was related to the i.v. injection of graded low numbers of leukaemic cells, there is a perfect agreement. With the ED so model, computer simulations can be performed yielding the chance that leukaemia will develop from injected leukaemic cells as a fraction of (1) the number of cells injected; and (2) the ED so value. The ED so for human AML is not known. It seems reasonable to assume that remission-induction treatment on average induces a 410g leukaemic cell kill (Figure 15). Thus an autologous marrow graft will contain one leukaemic cell per 104 normal cells, or 1.5 x 106 in a graft containing a total of 1.5 x 1010 cells (2 X 108 cells/kg; 75 kg patient). In the BNML it was found that 1% of in vivo clonogenic leukaemic cells survive cryopreservation. Furthermore, from in vitro studies with human AML it is concluded that only 0.1-1 % of all leukaemic cells can be considered clonogenic. Taking these two factors together, only 15-150 clonogenic leukaemic cells out of 1.5 x 106 are reinfused with the marrow graft. Assuming now an ED so value for human AML to be 1000 or 10000 clonogenic cells, which seems realistic based on previous BNML studies (Hagenbeek and Martens, 1985, 1987b), it can be calculated employing the computer simulation model that the chance that reinfused leukaemic cells indeed cause leukaemia is 1-10% or 0.1-1 %, respectively. This can be derived from the data presented in Table 2.

629

BN ACUTE MYELOCYTIC LEUKAEMIA

Table 2. Theoretical chance (%) of leukaemia development as function of assumed ED so value and the number of leukaemic cells in the human autologous bone marrow graft. Number of leukaemic cells in graft

ED50 value 10 100 1000 5000 10000

10

15

100

150

500

1000

1500

5000

10000

50.0 6.7 0.7 0.1 <0.1

64.6 9.9 1.0 0.2 0.1

99.9 50.0 6.7 1.4 0.7

100.0 64.6 9.9 2.1 1.0

100.0 96.9 29.3 6.7 3.4

100.0 99.9 50.0 12.9 6.7

100.0 100.0 64.6 18.8 9.9

100.0 100.0 96.9 50.0 29.3

100.0 100.0 99.9 75.0 50.0

The theoretical probability of leukaemia development, i.e. the chance that at least one unit of ED50 cells yields a cell to grow out, is given by (1-0.Y) x 100%, where x denotes the number of ED,o units injected.

Thus, to prevent a leukaemia relapse after autologous BMT, major emphasis should be given to more effective pretreatment of the patient. From the previous studies in the BNML it is known that high-dose cytosine arabinoside and total body irradiation or high-dose busulphan followed by cyclophosphamide are the most effective conditioning regimens, both inducing a more than 10log leukaemic cell kill, the first regimen, however, causing a 25% treatment-related mortality (Table 1). Based on the findings reported above, the current Dutch autologous BMT study in first remission AML employs non-purged marrow after conditioning with busulphancyclophosphamide. ALLOGENEIC BONE MARROW TRANSPLANTATION The mechanism by which MRD is eradicated with allogeneic BMT is not completely clear (Figure 16). In principle, both cytotoxic chernoradiotherapy (conditioning before BMT) and a graft-versus-leukaemic (GvL) reaction playa role. Arguments in favour of the role of these two entities are summarized below: 1.

Cytotoxic treatment -rapidity of the response to remission-induction chemotherapy -40-50% cures after autologous/isologous BMT -less relapses after flash TBI versus fractionated TBI -low-dose chemotherapy after allo BMT -a one log leukaemic cell kill by GvL only 2. Graft-versus-leukaemia -less relapses after aUo BMT versus auto/iso BMT -GvRD ~ ti } relapse rate i - T ce11 d ep1e lOn -less or no immunosuppression after BMT ~relapse rate t Since the introduction of T cell depletion a number of centres have reported an increase in leukaemia relapse rate after allogeneic BMT. The

630

A. HAGENBEEK AND A. C. M. MARTENS

.-- -_._-------~:._--- --------------.-, -_.__.

103

time

Figure 16. Cure after allogeneic bone marrow transplantation: chemoradiotherapy and/or graft-versus-leukaemia? (GvL, graft-versus-leukaemia; BMT, bone marrow transplantation; CR, complete remission; MRD, minimal residual disease).

other way around: lower relapse rates have been observed if patients develop graft-versus-host disease (GvHD). Furthermore, evidence for a so-called graft-versus-leukaemia reaction (GvLR) is derived from the observation in AML that the leukaemia relapse rate after allogeneic BMT in first remission is lower than after autologous or isologous BMT. The probabilities of leukaemia cure, Pr{cure} determined directly from Kaplan-Meier plots obtained by long-term follow-up of large numbers of patients, are approximately 0.9 and 0.4 in patients with severe GvHD and without GvHD respectively (Weiden et al, 1981). In other words, GvHD apparently is related to a decrease in the number of surviving clonogenic leukaemia cells. It is not known whether this antileukaemia effect is merely alloreactivity or that an immunological reaction occurs specifically against leukaemic cells. Experimental data indicate that the graft-versus-host reaction and the GvLR can be separated, although the evidence is not convincing (Meredith and O'Kunewick, 1983). While having a beneficial effect as far as leukaemia cure probability is concerned, unfortunately, GvHD in itself causes serious morbidity and mortality, thus reducing the net success rate again. In this context, it might be useful to evaluate what extra decrease, quantitatively, in survival of clonogenic leukaemic cells is correlated with GvHD. A theoretical probability of leukaemia cure can be derived using either Poisson statistics or the binomial probability distribution. Let the tumour burden of a patient be M clonogenic leukaemic cells. Owing to treatment, let

631

BN ACUTE MYELOCYTIC LEUKAEMIA

each leukaemic celI have a chance, SF, to survive, and let X denote the number of surviving clonogenic leukaemic cells after treatment. If the conditions for Poisson statistics are satisfied, i.e. if M is large (greater than 100) and SF is small (less than 0.1) and SFx M is constant, then the probability of cure can be written as: Pr{cure} = Pr{X= O} = e- S F X M

(1)

Several initial tumour loads, M, and surviving fractions, SF, as listed in Table 3, were chosen such that Poisson statistics apply. The theoretical probability of cure was computed using equation (1). It decreases with either increasing original tumour load or increasing surviving fraction. Although not tabulated here, the binomial distribution too yields a decrease in theoretical probability of cure with increasing M and/or SF. Table 3. Theoretical probability of cure according to Poisson statistics, as function of initial leukaemic cell load and 'log cell kill' effect. Initial cell load (M) Surviving fraction (SF) 10- 1 10- 2 10- 3 10- 4 10- 5 10- 6 10- 7

4E 10- 9 1 -

1012

1010

~

0

0 0

0 0 0 0 0

0 0 0 0 0 0 0 0

4x 10- 44

()

0 0 0 0

4 X 10- 44 5 X 10- 5 0.37

4 X 10- 44 5 X 10- 5

~ 0.90 0.99

106

104

0

0

0

4 X 10- 44 5 X 10- 5

0

4x 10- 44 5 X 10- 5 0.37 0.90 0.99

1 1

0.37 0.90 0.99 1

1 1 1

From Table 3, it is seen that a rise from about 40 to 90% cures corresponds with a difference of 1log cell kill for several combinations of M and two subsequent SF values. In particular, this is the case when assuming-as suggested by (pre)clinical findings-that the initial leukaemic population just after remission-induction therapy has a size of M = 108 (Figure 15) cells and that the log cell kill induced by the conditioning regimen has the value eight Or nine, respectively. Thus, the GvLreaction at the most induces a one log leukaemic cell kill. This theoretically derived conclusion was recently supported by studies on allogeneic BMT in the BNML model (Hagenbeek et ai, 1990). As the graft-versus-leukaemia reaction contributes a one log leukaemic cell kill only, it is strongly advocated to seek other ways of achieving this extra kill instead of reintroducing unmanipulated allogeneic marrow grafting (i.e. going back to the 1970s) with the major drawback of graft-versushost related mortality. Conclusions from studies in autologous and allogeneicBMT are givenbelow:

Autologous BMT 1. Residual leukaemia in the host is the major determinant for leukaemia relapse after BMT

632 2.

A. HAGENBEEK AND A. C. M. MARTENS

The chance that leukaemic cells reinfused with the marrow graft contribute to a leukaemia relapse varies between 0.1 and 10%.

Allogeneic BMT 1. The graft-versus-leukaemia reaction at the most induces a one log leukaemic cell kill 2. Therefore, avoiding T cell depleted BMT and thus re-introducing graftversus-host related mortality is strongly discouraged. Instead: In general Major emphasis should be given to more effective means of in vivo leukaemic cell load reduction prior to or after BMT In summary, the development of more effective means to reduce the leukaemic cell load prior to BMT seems to be most logical. Achievement of an additional one log cell kill in conditioning therapy is presently being explored in our BMT centre in Rotterdam by increasing the dose of total body irradiation from 2 x 5.0 Gy to 2 x 6.0 Gy in conjunction with high-dose chemotherapy before T cell depleted allogeneic BMT. The addition of 2 X 1.0 Gy to the conditioning regimen is thought to induce an extra 1-2 log leukaemic cell kill. An alternative may be to apply biological response modifiers/interleukin-Z or low-dose chemotherapy after BMT (Figure 14). Optimal timing employing appropriate doses were shown in the BNML model to eradicate the few logs of leukaemic cells which remain after BMT without jeopardizing the graft (Hagenbeek and Martens, 1982, 1983b). In this way a significant increase in cure rates could be achieved.

SUMMARY

The AML model in the BN rat has contributed considerably to improved understanding of the various aspects of leukaemia growth, responses to chemotherapy, application of BMT as treatment modality and the possibilities and limitations for the detection of residual disease during the remission phase. Obviously, there are restrictions with regard to the extrapolation of the rat data to the human situation. Leukaemia growth in inbred rats is highly reproducible, while in humans it presents a high degree of individual variation. However, several characteristics are shared and the aim should be to identify the similarities as well as the dissimilarities between human and rat leukaemia. In that way progress may be envisaged with respect to reaching the final goal of curing human leukaemia. Acknowledgements These studies were partly supported by the Dutch Cancer Foundation/Queen Wilhelmina Fund. Our sincere thanks are due to the other members of the Department of HaematoOncology for providing us with their data: D. W. Van Bekkum, O. J. A. Arkesteijn, C. J. de Groot, F. W. Schultz, P. J. Hendrikx, Y. Yan, T. C. Kloosterman, P. M. Hoogerbrugge, C. Ophorst, J. Gaiser, M. Tielemans and T. Brazier.

BN ACUTE MYELOCYTIC LEUKAEMIA

633

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