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Document Type and Number:
WIPO Patent Application WO/1985/000812
Kind Code:
Conjugates of transferrin or ceruloplasmin with anti-tumour agents. Such conjugates are useful in the treatment of tumours. Suitable anti-tumour agents include adriamycin, daunomycin, methotrexate, vincristin, 6-mercaptopurine, cytosine arabinoside and cyclophosphamide. Transferrrin or ceruloplasmin in preferably coupled to the anti-tumour agent by means of glutaraldehyde.

Application Number:
Publication Date:
February 28, 1985
Filing Date:
August 10, 1983
Export Citation:
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International Classes:
A01N25/04; A01N53/00; A61K47/48; C07K14/79; C08F2/00; C08F2/22; C08F2/44; C12N9/02; G01N33/50; A61K38/00; (IPC1-7): C07G7/00; C07G7/04; A61K37/02; A61K37/14; A61K47/00; C12Q1/18
Foreign References:
Other References:
The Lancet, Vol. 2, No. 8191, issued 23 August 1980 (London, GB) W.P. FAULK et al.: "Transferrin and Transferrin Receptors in Carcinoma of the Breast", pages 350-392, see page 392, left-hand column, line 60 - right-hand column, line 2, lines 10-14
Die Makromolekulare Chemie, Supplement Vol. 2, issued 3 September 1979 M. WILCHEK: "Affinity Therapy and Polymer Bound Drugs", pages 207-214 see page 208, lines 1-5 and 15-21; page 213, lines 18-25; page 209, lines 7-9 and table 1
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1. A conjugate of a protein with a cytotoxic agent characterised in that the conjugate is a conjugate of a from traasf.err.i_n..,and. ceruloplasmin with ent.
2. A conjugate according to claim 1, characterised in that the protein is linked to the antitumour agent by means of glutaraldehyde.
3. A conjugate according to claim 1 or claim 2, characterised in that the antitumour agent is selected from adriamycin, daunomycin, methotrexate, vincristin, 6mercaptopurine, cystosine arabinoside, and cyclophos phamide.
4. A conjugate according to claim 3, characterised in that the antitumour agent is adriamycin, and the conjugated protein is apotransferrin.
5. A conjugate according to any one of claims 1 to 4 for use in the treatment of tumours.
6. A process for forming a conjugate of a protein with a cytotoxic agent characterised in that a protein selected from transferrin and ceruloplasmin is conjugated with an antitumour agent, and the resulting conjugate of native transferrin or ceruloplasmin is then separated from conjugates comprising aggregated protein molecules or fragments of protein molecules.
7. A process according to claim 6, characterised in that said conjugates are separated by gel filtration chromatography.
8. A process according to claim 6 or claim 7, characterised in that the protein is conjugated with the antitumour agent in the presence of glutaraldehyde.
9. A process according to any one of claims 6 to 8, characterised in that the antitumour agent is selected from adriamycin, daunomycin, methotrexate, vincristin, 6mercaptoρurine, cytosine arabinoside, and cyclophosphamide.
10. A kit of reagents for the treatment of tumours characterised in that it comprises (a) ironbearing transferrin, and (b) a conjugate according to any of claims 1 to 4.
11. A process for determining the susceptibility of tumour cells to antitumour agents characterised in that it comprises administering separately to respective portions of said tumour cells a plurality of conjugates of transferrin or ceruloplasmin each with a different antitumour agent.
12. A kit of reagents for performing the process of claim 11, characterised in that it comprises a plurality of conjugates of transferrin or ceruloplasmin each with a different antitumour agent.
13. A method of treating a subject suffering from cancer characterised in that it comprises administering to the subject a pharmacologically effective amount of a conjugate according to claim 5. S3 ?"_..«« τ., _„.„; .
CONJUGATES OF PROTEINS WITH ANTI-TUMOUR AGENTS This invention relates to conjugates of proteins wit anti-tumour agents, and more particularly to conjugate of transferrin or ceruloplasmin with anti-tumour agents. The invention also relates to methods for preparing suc conjugates.

Transferrin is a protein which occurs in bloo plasma, including human blood plasma, and which has a important function in the transport of iron. The role o transferrin in he e synthesis in developing red blood cells has been widely studied, and the presence of transferrin receptors on reticulocytes has been noted.

Ceruloplasmin is a copper-binding glycoprotein which also occurs in blood plasma. It has been suggested that modified ceruloplasmin might fit into transferrin receptors.

It has been noted recently that transferrin receptors are to be found on the surfaces of tumour cells. It has been suggested that research into breast cancer therapy might be approached through the use of transferrin labelled with cytotoxic agents (Faulk et al, The Lancet, August 23, 1980, page 392).

A favoured approach to producing tumour-specific drugs has involved the linking of cytostatic drugs such as daunomycin, adriamycin and methotrexate, to antibodies against tumour-associated surface antigens. The ability of such complexes to kill malignant cells selectively has, so far, been limited. An alternative, and more recent approach has been 'to link a toxin, instead of a cytostatic drug-, to the antibody.

Following the identification of transferrin . receptors on the surfaces of tumour cells by Faulk and Galbraith

(Proc. Roy. Soc. (B) 204: 83-97, 1979), Trowbridge and

Domingo (Nature, Vol. 294, page 171, 1981) have obtained monoclonal antibodies against the transferrin

receptor of human cells, and have coupled such antibodies to ricin or diphtheria toxin sub-units. They found that the growth of human tumour cells was specifically inhib¬ ited in vitro by such conjugates. However, in experi- 5 ments designed to test the effectiveness of the antibody- toxin conjugates ir^ ______} they found that anti- transferrin receptor antibody alone inhibits the growth of human melanoma cells in nude mice. Indeed, they found no evidence that the conjugate is more effective

10 than unmodified antibody in inhibiting growth of M21 melanoma cells in nude mice. That is to say, the cyto- toxic properties of the ricin A moiety of the antibody- toxin conjugate were not manifest in ^ vivo.

In surprising contrast to the results of Trowbridge

15. and Domingo, it has now been found that when an anti- tumour agent is conjugated with transferrin or cerulo¬ plasmin, not only does the protein moiety retain its affinity for transferrin receptors, but, moreover, the anti-tumour agent retains its anti-tumour properties.

20 According to the present invention, there is provided a conjugate of transferrin or ceruloplasmin with an anti-tumour agent- Preferably, the anti-tumour agent is adriamycin, methotrexate, vincristin, daunomycin, 6-mercaptopurine, cytosine arabinoside, or cyclo

25 phospha ide. More preferably, the anti-tumour agent is adriamycin, and the protein with which it is conjugated is preferably apotransferrin.

It is particularly preferred that the transferrin or ceruloplasmin be linked to the anti-tumour agent 0 by means of glutaraldehydel

The transferrin or ceruloplasmin is preferably of human origin, though any transferrin or ceruloplasmin may be used which can bind to transferrin receptors on the surface of the tumour cell which it is desired to 5 treat.

Preferably, the transferrin or ceruloplasmin conjugate is separated from conjugates which comprise aggregated transferrin or ceruloplasmin molecules and/or fragments of transferrin or ceruloplasmin molecules cross-linked by glutaraldehyde. Such aggregates and/or fragments may be separated from the desired conjugates by, for example, gel filtration chromatography.

Also provided by the present invention are con¬ jugates of transferrin or ceruloplasmin as described above, for use in the treatment of tumours.

According to a further aspect of the present invention, there is provided a reagent kit for the treatment of tumours, comprising iron-bearing transferrin and a conjugate of transferrin or ceruloplasmin with an anti-tumour agent. Such of the patient's normal cells which have transferrin receptors may be protected against the effects of the conjugate by saturating these receptors with the iron-bearing transferrin before administration of the conjugate. The present invention also provides a process for determining the susceptibility of tumour cells to anti-tumour agents, comprising administering separately to portions of said tumour cells conjugates of transferrin with a number of different anti-tumour agents. A reagent kit comprising a number of such different conjugates may be provided for this purpose.

It has been found that the conjugates of the present invention are taken up extremely rapidly by tumour cells. This means that within a matter of hours of removal from the patient, tumour cells may be tested against a range of conjugates of transferrin or cerulo¬ plasmin with different anti-tumour agents. Such a process would enable the chemotherapy which is most effective for a given patient to be determined as soon as possible after isolation of the

tu our cells.

There are now described, by way of example, con¬ jugates of transferrin and ceruloplasmin according to the present invention, and a method for preparing the same, with reference to the accompanying drawings, in which:-

Figure la shows the results of gel filtration chromatography of native transferrin.

Figure lb shows the results of similar gel filtration chromatography of a transferrin control, Figure lc shows the results of similar gel filtration chromatography of a transferrin-adriamycin conjugate according to the invention.

Figure 2 is a graph showing the effects of different quantities of conjugate on tumour cells, and Figure 3 is a graph showing the time-course of -- H-thymidine uptake inhibition by conjugate.

EXAMPLE Cells HL-60 (acute promyelocytic leukemia) and Daudi (Burkitt's lymphoma) cells, were grown in suspension at 37°C in RPMI-1640 medium with L-glutamine, 25mM HEPES (Gibco European, Glasgow, Scotland), 50 mcg/ml gentamicin (Flow Laboratories, Ayrshire, Scotland) and supplemented with 10% fetal calf serum (Sera-Lab Ltd., Crawley Down, Sussex, England), this combination being hereafter referred to as complete medium. Human venous blood was collected into acid citrate dextrose solution, and peripheral blood mononuclear cells (PBM) were separated from venous blood samples with the use of Isolymph (Gallard-Schlesinger Chemical Mfg. Corp., Carle Place, New York, USA). Viability was determined by trypan blue exclusion.

Preparation of Conjugates To a solution containing 10 mg of either human transferrin (Trf) (99% iron-free, from Behringwerke AG, Marburg, W. Germany) or human cerulo- plasmin (Cer) (prepared as described by Pegandier, Z. e_t al, Clin. Che . Acta 30, 387-394 (1970)), and 3 mg of

adria ycin (Adr) hydrochloride (Far italia Co., Milan,

Italy) in 1 ml of 0.1M phosphate buffered saline (PBS), pH 7.0 was added dropwise to 0.5 ml of an aqueous solution of 0.25% glutaraldehyde (BDH Chemicals Ltd., Poole, England) at room temperature (RT) with gentle mixing. After 2 hours incubation at RT in the dark, 0.5 ml of 1M ethanolamine (Sigma London Chemical Co. Ltd.,

Poole), pH 7.4., was added and incubated at 4°C overnight.

The mixture was then centrifuged at l,000g for 15 minutes and the supernatant was collected and chromatographed through a column (2 cm x 20 cm) of Sepharose CL-6B

(Pharmacia Fine Chemicals, Uppsala, Sweden) equilibrated in 0.15M PBS, pH 7.2. Protein and Adr were identified as they emerged from the column by spectrophotometric readings taken at OD28O -*-- or Tr - { - r or Ce and at

OD495 for Adr, respectively. Spectrophoto- metrically defined 1.2 ml fractions were pooled and sterilized by irradiation in a Gamma Cell 1000 irradiator

(Atomic Energy of Canada) and stored at 4°C in the dark until used.

Immunocytology and Fluorescence Localization of Trf and Adr. Cells (1 x 10 6 ) were reacted with 100 ul of Adr, Trf or Trf-Adr conjugate for 20 minutes at 4°C or for 3 hours at 37°C, washed twice in Hank's balanced salt solution (HBSS, Gibco Europe) at 4°C and reacted with 100 ul of a 1:20 dilution of a fluorescein isothiocyanate (FITC) conjugate of goat anti-human Trf (Atlantic Anti¬ bodies, Maine, USA). This antibody did not react with Daudi or HL-60 cells prior to their exposure to human Trf. Following incubation with anti-Trf, the cells were washed three times in HBSS at 4°C, suspended in 50% glycerol/PBS and mounted on glass slides to be studied by epi- illu ination with a Zeiss Universal microscope fitted with an HBO 50 mercury arc lamp. This microscope was equipped with three different optical systems for the analysis of Trf and Adr: (a) the FITC labelled anti-Trf could be identified by


using a blue interference filter (BP455-490) with a FT510 chromatic beam splitter and a BP520-560 barrier filter; (b) Adr was identified by using a green inter¬ ference filter (BP546/7) with a FT 580 chromatic beam splitter and a LP590 barrier filter; and (c) anti-Trf and Adr could be simultaneously identified by using a (BP455-490) interference filter, a FT510 chromatic beam splitter and a LP 520 barrier filter.

Measurements of Cellular Proliferation and Via- bility For a direct inhibition assay, cells (0.5 x10 ) were incubated with 100 pl of sterile Trf-Adr or with Cer-Adr conjugates containing 1 μq of Adr, or with complete medium at 37°C in 5% C0„/air for 3 hours, following which they were washed twice in complete medium for 7 minutes per wash at 4°C, and centrifuged at 400g. For a competitive inhibition assay, cells (0.5 x 10 ) were incubated with or without 100 JJI of Trf (1mg/ml) at 4°C for 30 minutes, followed by incu¬ bation with Trf-Adr, Cer-Adr or PBS at 4°C for 30 minutes. They were then washed twice at 4°C and further incubated at 37°C for 2 hours. For the quantification of cellular proliferation, 300 jjl of complete medium was added to the washed cell pellet, and the cells were resuspended and distributed in triplicate as 100 l aliquots in flat-bottom microtiter plates (Flow

Laboratories), each well of which was pre-loaded with 100 μl of complete medium and 25 μl of 2 /iCi of H- thymidine (specific activity, 2 Ci/mmole, Radiochemical Center, Amersham, Bucks, England). Plates were then incubated at 37°C in 555 CO ' /air for 16 hours, and the cells were harvested on glass fiber filters in a MASH II multiple harvester (Microbiological Associates, Walkersville, Md. , USA). The amount of 3 H-thymidine incorporated into DNA was measured in a LKB 1216 Rackbeta II liquid scintillation counter and expressed as counts

per minute (cpm). Quantitative measure ' s of the via¬ bility and number of cells were also done in all experi¬ ments.

Chromatographic" Properties of Trf, Trf-Adr . .Conjugates and 'Adr. In preparing the conjugates, it was found that native Trf (1 ml of 5mg/ml in PBS) consis¬ tently emerged from a column of Sepharose CL-6B between fractions 22 and 30, peaking at fraction 26 (Fig. 1a). However, when Trf, which had been carried through the conjugation procedure in the absence of Adr, was chromatographed on the same column, a more complex pattern was obtained (Fig. 1b). The acromolecular peak (A in Fig. 1b) was precipitated by anti-Trf anti¬ body in Ouchterlony and immunoelectrophoresis gels, as did the samples which emerged at the same place as native Trf, i.e., between fraction numbers 20-26 (B in Fig. 1b), but the late appearing peak (C in Fig. 1b) was not precipitated by anti-Trf in gels. It was therefore considered that "A" was Trf aggregates, "B" was chromato- graphically and antigenically native Trf and "C" was Trf fragments. When Trf-Adr conjugates were passed through the column, the 0D„ Rn tracing was similar to the controls, but reading at 0D, - revealed Adr in both peaks A and B (Fig. 1c), whereas nothing was detected in 0D, g , in the absence of Adr. Accordingly, peak B was used as Trf-Adr conjugate in the following experiments. It should be noted that Adr has a molecular weight of 579, and that it does not chromatograph within the mole¬ cular weight range of Sepharose CL-6B.

> Immunological Identification of Trf on Plasma

Membranes and Fluorescence Localization of Adr in Nuclei. Trf, Adr and Trf-Adr conjugates were investigated for their ability to be bound by plasma membrane receptors and to intercalate with nuclear DNA. This was done by using two different manipulations; the first being the

incubation of Trf-receptor-positive cells with the above reagents for 20 minutes at 4°C, and the second being incubation of cells with the same reagents for 3 hours at 37°C. For these experiments, Daudi and HL-60 cells were used, since it has previously been reported that their plasma membranes have receptors for Trf, and PBM were used at Trf receptor-negative cells. When HL-60 or Daudi cells were incubated with Trf which had been carried through the conjugation and chromatography in the absence of Adr, or with Trf-Adr conjugates for 20 minutes at 4°C, a uniformly speckled pattern of fluorescence could be identified on their plasma membranes following incubation with FITC labelled antibodies to human Trf. In contrast, only 8% of PBM were found to find Trf or Trf-Adr conjugates, and none of the PBM, HL-60 or Daudi cells reacted with FITC anti-Trf after being incubated with Adr alone (Table 1). However, incubation of PBM, HL-60 or Daudi cells with free Adr for 20 minutes at 4°C resulted in a red fluorescence of their nuclei, a result which was not obtained by incubation of any of the target cells with Trf or Trf-Adr conjugates for 20 minutes at 4°C.

When these experiments were repeated with the use of the same cells and the same reagents, but with the incubation time changed to 3 hours and the temperature changed to 37°C, the results were different. In the first instance, the pattern of im unofluorescence follow¬ ing incubation of HL-60 or Daudi cells with Trf or Trf- Adr conjugates was no longer homogeneous, but appeared j-o be clustered into islands of fluorescent patches on plasma membrane ' s. The most striking difference found with these changed conditions of time and temperature for incubation was that obtained with the Trf-Adr conjugates, for Adr could now be identified in the nucleus.

Effects of Trf-Adr Conjugates on H-Thymidine

Uptake and Viability -of Cells. . It is thought that Adr acts by inserting itself between base pairs of DNA, resulting in an inhibition of DNA synthesis and eventual cell death, so the inhibition of H-thymidine uptake as 5 well as trypan blue exclusion were chosen as measures of cellular proliferation and viability, respectively. Incubation of Trf-Adr conjugates for 3 hours at 37°C with Daudi and HL-60 cells followed by two washes and a 16 hour culture period in tritiated thymidine had. the 10 striking effect of diminishing both proliferation and viability, whereas minimal effects were observed for both thymidine incorporation and viability when PBM were tested under the same conditions (Table 2). The Trf control manifested moderate suppresion of Daudi and HL-60 15 cells and augmentation of PBM proliferation, while Trf- Adr conjugates consistently recorded more than 90ΪΌ suppression of both cell lines in thirty experiments. Regarding the death of PBM, it should be pointed out that, unlike Daudi and HL-60, very few PBM were found to con- 20 tain Adr in their nuclei, indicating that dead cells resulted as a consequence of manipulation iji vitro.

In order to provide additional evidence for the specific inhibition of cellular proliferation by Trf-Adr conjugates, Cer-Adr conjugates were used as a protein 25 control for Trf. The choice of Cer was predicated by the observation that other proteins such as human al¬ bumin, Gc and alpha fetal protein are immunologically cross reactive with unfolded Trf but not with Cer. Although Cer-Adr conjugates were found to kill target


30 cells i vitro, when Trf-Adr or Cer-Adr conjugates were allowed to displace Trf, Trf-Adr conjugates were twice as efficient as Cer-Adr conjugates (Table 3). These findings confirm the suggestion that modified Cer might fit into Trf receptors, but they additionally show that

35. Trf-Adr conjugates have a higher affinity than Cer-Adr conjugates for Trf receptors. Native Trf alone in the

growth media could not have been solely responsible fo " r these results, since test and control experiments contained the same amount of Trf (Table 3). Dose and Time Variables in Cell Killing by Trf-Adr Con ugates. rf-Adr conjugates were serially diluted to test the end-point of their ability to inhibit the uptake of ^H-thymidine. The results of these experiments showed that the proliferation of HL-60 cells was directly associated with the amount of Trf-Adr conjugate used in the initial 3 hour preincubation step of the assay (Figure 3). Conjugate binding was rapid and achieved a plateau at 30 minutes preincubation (Figure 4), although the irt vitro assay for inhibition of cellular proliferation employed a preincubation step that was studied by using a fixed amount of conjugate with varied periods of time.

Effects of ^H-Thymidine Uptake of Leuke ic Cells Using techniques similar to those described in relation to Table 2 the effect of Trf-Adr on *-*H-thymidine uptake by leukemic cells taken from six different patients suffering variously from acute myelocytic leukemia (AML) or from acute lymphatic leukemia (ALL) were studied. The results are set out in Table 4. Significant inhibition was noted in each case. Trf Binding by PBM from Normals and Patients

Experiments were carried out to test the ability to bind transferrin of cells taken from a number of healthy people as well as from a number of patients with a lymphoma or a myeloma or suffering from AML, ALL, chronic myelocytic leukemia (CML), or chronic lymphatic leukemia (CML). Some of these patients were in remission (R) whilst others were not in remission (NR) . In these experiments the technique used was generally as described above in relation to Table 1. The results are summarised in Table 5.

In vivo Tests of the Effects of Trf-Adr Treatment in

Peripheral Blood of AML Patients.

Table 6 summarises the effects on a number of patients of treatment with 1 mg. Trf-Adr administered by the intravenou route in the form of conjugate in physiological saline. Patients Nos. 1, 4 and 5 were the same patients as are referred to in Table 4. The effects of this dosage on the counts of both blasts and polymorphonucleated neutrophiles (PMN) are listed.

Table 1. Plasma Membrane and Nuclear Reactivity of Trf, Adr and Trf-Adr Conjugates with HL-60 Cells at Different Temperatures and Times of Incubation

Reagents Temperature Time Membrane Nuclear fluorescence fluorescence r

• for Trf for Adr I

Trf 4°C 20 minutes 70-80/3+ 1 0

Adr II •1 0 100 2

Trf-Adr II II 70-80/3+ 0

Trf 37°C 3 hours 70-80/3+ 0

Adr II II 0 100

Trf-Adr II II 70-80/3+ 70-80

* •* Percentage of cells fluorescing/intensity of fluorescence 2 Percentage of positive cells.

§ ι-rtl

Table 2

Effects of Trf-Adr on 3 H-Thymidine Uptake and Viability of Cells

-H-thymidine uptake (cpm) Viability (%)

Cells Control Trf Control Trf-Adr Control Trf Trf-Ad Control

Daudi 155,511.33+6,793.22* 72,339.66+3,159.67 14,850.00+812.43 89 73 63

HL-60 199,918.66+7,489.50 32,058.33+1,899.52 3,515.00+124.65 92 88 56

PBM 316.33+87.61 672.67+215.42 98.67+25.01 94 87 86

*Mean +_ SEM from data of triplicate cultures obtained from 30 experiments. Cells (0.5 x 10-5) we re incubated with growth medium (control), Trf control (B in Figure lb) or Trf-Adr (B in. Figure lc) for 3 hours at 37°C, washed and incubated with •^H-thymidine for 16 hours at 37°C, after which the uptake of 3H-thymidine and viability were determined.

Discordance between the effects of Trf on thymidine incorporation and cell viability i

Table 3

Competitive Inhibition of Trf-Adr and Cer-Adr with Trf on ***H-thymidine Uptake of Daudi Cells


No preincubation Preincubation with Trf


Trf-A r 87000.67 + 5857.78 48624.33 + 3724.19 Cer-Adr 80687.33 + 2025.06 85563.33 + 5677.34 Control 145987.00 + 6266.32 122467.33 + 7625.16

Cells were preincubated with or without 100 ul of Trf for 30 minutes at 4°C, followed by incubation with Trf-Adr, Cer-Adr or PBS for 30 minutes at 4°C, washed twice at 4°C, then further incubated for 2 hours at 37°C. The measurement of incorporation of 3 H-thymidine was as detailed in materials and methods.

Table 4

Effects of Trf-Adr on ---H-thymidine Uptake of Leukemic Cells

- H-thymidine uptake (cpm)

Inhibition (%)

Cells Control Trf-Adr

Patient No. 1 (AML)

PBM 6,259+357 75+22 98.8 .

Marrow 4,747+821 1,334+57 71.9 Patient No. 2 (AML) . | PBM 1,582+59 238+15 85.0

- Marrow 5,733+219 657+286 88.5 • Patient No. 3 (ALL) 1

PBM 412+72 186+34 ιi 54.9

Marrow 1,659+56 923+79 44.4 Patient No. 4 (AML)

PBM 5,426+242 1,995+173 63.3

Marrow 23,972+2,183 3,845+256 84.0 Patient No. 5 (AML)

PBM 3,451+88 597+38 82.8

Marrow 14,836+957 1,894+91 87.2 Patient No. 6 (Anemia/AML)

PBM 1,475+100 449+108 69.5 o. Marrow 46,974+4,836 17,208+819 63.4 a • t

Table 5

Trf Binding by PBM from Normals and Patients

Diagnosis No, of cases Anti-Trf (%) Trf+Anti-Trf (%) _ ;

Normal 23 2.83 + 3.02 8.30 + 3.55

Lymphoma 18 14.72 +14,00 30.44 +14.96 ; Myeloma 7 15.28 + 9.21 23.71 + 7.65 I

AML (R) 6 9.33 + 3.33 18.17 + 5.56 ; J AML (NR) 1 65.00 + 0.00 81.00 + 0.00 ' ' CML 6 10.83 + 9.66 20.50 + 7.76

CLL 10 6.10 + 4.48 14.80 + 7.24