WO1996015263A1

WO1996015263A1 - Monitoring malignant disease

Info

Publication number: WO1996015263A1
Application number: PCT/GB1995/002647
Authority: WO
Inventors: James Stephen Wainscoat; Jacqueline Boultwood; Carrie Fidler; Paul Austin Henry Moss
Original assignee: James Stephen Wainscoat; Jacqueline Boultwood; Carrie Fidler; Paul Austin Henry Moss
Priority date: 1994-11-11
Filing date: 1995-11-10
Publication date: 1996-05-23
Also published as: GB9422836D0

Abstract

The state of a malignant disease, such as a leukaemia, of an individual may be determined by comparing the level of mitochondrial DNA (mtDNA) in cells from the individual with normal cells of the same type, for example using nucleic acid probes specific for mtDNA and probes specific for genomic DNA. The ratio of mtDNA: genomic DNA is increased in malignant cells in acute myeloid leukaemia (AML). AML patients in remission have normal amounts of mtDNA in their cells. In chronic granulocytic leukaemia (CGL) the ratio increases on progression of the disease to AML.

Description

MONITORING MALIGNANT DISEASE

The present invention relates to monitoring malignant diseases, diagnostically and/or for assessment of timing and/or nature of treatment.

The present invention is founded upon the surprising discovery that mitochondrial (mt) DNA is amplified (ie there is more of it) in malignant cells. Also, the level of mtDNA in cells is shown to increase concomitantly with transformation of cells from a pre- malignant dysplastic state to a malignant state. Indeed, mtDNA levels are shown to rise and fall reproducibly with progression and remission of disease. These results indicate a number of practical utilities for determining and monitoring the level of mtDNA in cells of individuals who may have a malignant disease, such as a leukaemia. These are discussed infra .

Previously, electron microscopy techniques have shown the presence of morphological abnormalities of mitochondria in acute myeloid leukaemia (AML) . Grossly enlarged mitochondria, termed giant mitochondria in association with AML were first described by Bessis and Breton-Gorius¹ in 1969. Although giant mitochondria are infrequently reported other structural abnormalities including dilation, deformation, aberrant cristae and contact with nucleus are among morphological changes of mitochondria most often cited in AML^2-6. mtDNA has also been shown to be abnormal in AML. This phenomenon was first reported by Clayton et al in 1967⁷ who demonstrated the presence of circular dimers • and complex catenated forms of mtDNA using the ethidium bromide centrifugation method in three patients with chronic or acute leukaemia. Clayton et al reported similar observations in a group of patients with chronic granulocytic leukaemia (CGL)^8- The presence of the circular dimers and catenated dimers in the mtDNA from the leukaemic patient studied and the absence of this form in the mtDNA from normal mature human leukocytes, Hela cells, and a variety of normal mammalian tissues prompted this group to suggest a correlation between the occurrence of the abnormal mtDNA and human leukaemia. Electron microscopic studies on mtDNA by these^7,8 and other workers⁹ have confirmed the presence of circular dimers and catenated forms in acute leukaemias.

It has been suggested on steric grounds that these aberrant forms of mtDNA are non-functional and unsuitable for RNA synthesis^{9, 10}. Normal haematopoiesis is dependent upon the transcription/translation of mtDNA into functional protein and it has been speculated that the presence of these complex forms of mtDNA in AML may be relevant to pathogenesis.

It should be noted that the existence of abnormal and/or dimerised or catenated mtDNA in cells provides no suggestion of amplification of the DNA, as is confirmed by experiments reported herein.

Differences in amounts of mtDNA in different cell types have been reported, with minor, apparently insignificant variations in the level in a leukaemic cell line as compared with white blood cells¹⁶. The ratio between mtDNA and nuclear DNA has been reported to be the same in tumorigenic derivatives and the parent cell lines of mouse origin¹⁷.

In the work described herein, "an investigation was carried out designed to quantitate the amount of mtDNA in cells of individuals with malignant disease relative to that observed in the same cell type from normal individuals. As examples of easily studied malignant diseases, Acute Myeloid Leukaemia (AML) and Chronic Granulocytic Leukaemia (CGL) have been investigated.

Thus, the amount of mtDNA in AML relative to that observed in peripheral blood leucocytes from normal individuals has been determined. This study has demonstrated consistent amplification of mtDNA in AML. Amplification of mtDNA in AML is therefore an intrinsic feature of the malignant myeloid phenotype and this is a novel observation. The level of mtDNA amplification varied between AML samples from 2 to 50 fold.

A number of marrow samples taken from AML patients at disease presentation and remission were investigated. Amplification of mtDNA was observed in the bone marrow samples from these cases at presentation but mtDNA levels essentially equivalent to that observed in peripheral blood leucocytes from normal healthy controls were observed at remission.

The finding that a range of infections or inflammatory disorders giving rise to abnormal white cell counts showed no corresponding increase in mtDNA levels supports the specificity of amplifications of mtDNA with malignant disease.

Three cases of AML with a preceding myelodysplasia (MDS) were also included in these study. Interestingly, mtDNA amplification was shown to occur with transformation from MDS to AML. This observation suggests a role for amplification of mtDNA in malignant transformation, eg of myeloid cells. Further studies will establish whether the observed increase in mtDNA is a cause or a consequence of transformation, though knowledge of this is not required for purposes of the present invention.

A number of CGL samples in the chronic phase of the disease were also included in this study. All showed mtDNA dosage levels equivalent to those obtained in the peripheral blood samples from normal healthy controls, ie amplification of mtDNA does not occur in this myeloid malignancy in the chronic phase. This observation is significant because the presence of circular dimers and catenated forms of mtDNA have been previously reported in 14 patients with CGL using non-quantitative methods. Thus whilst abnormal forms of mtDNA occur in CGL they do not result in an increase in the quantity of total mtDNA as occurs in AML.

However, CGL in transformation to AML (accelerated phase and/or blasts crisis) was also studied and showed amplification of mtDNA, indicating that amplification of mtDNA as a marker for progression in CGL. Furthermore, in 2/5 cases of CGL studied, where the chronic phase although not formally classified as being in acceleration showed very early signs of progression into accelerated phase, mtDNA amplification was observed in the chronic phase as well as in the accelerated phase and blast crisis. This implicates mtDNA amplification as a marker for identification of patients likely to transform in the near future. Accordingly, the present invention provides a number of aspects, generally concerned with determination of the level of mtDNA in cells of individuals who have, or might have, eg are suspected of having, a malignant disease, as compared with the level of mtDNA in cells of the same type in normal individuals. Generally, the methods are preferred in vitro on a sample of cells removed from the individual.

According to a first aspect of the present invention there is provided a method of determining the state of malignant disease of an individual, the method comprising determining the level of mtDNA in cells of the individual in comparison with the normal level of mtDNA in cells of the same type. Thus, the amount of mtDNA in cells of the individual may be determined and compared with the amount of mtDNA determined in cells of the same type known to be normal. A comparative ratio which signifies amplification has the indications discussed herein. Examples of malignant diseases which may be investigated include AML and CGL, in which case the level of mtDNA in haematopoietic cells will be analysed (eg comparing bone marrow eelIs/peripheral blood leucocytes) , cervical cancer, colonic tumours or other endothelial dysplasias, or solid tumours, e.g. in an organ or gland such as salivary gland. Point mutation or gross abnormalities of mtDNA have been shown to be associated with a number of human diseases and there is evidence to suggest that alterations in mtDNA may contribute to the tumorigenic phenotype of human malignancy¹⁹. The mitochondria of tumour cells have been shown to differ functionally and morphologically from those of non-malignant cells. The mitochondria of colonic tumours for example, are aberrant in both structure and function²⁰. The presence of abnormal circular dimers of mtDNA have been reported in some solid tumours e.g. salivary gland carcinoma¹⁹.

The method may provide information which can be used to diagnose malignant disease in an individual not known to have the disease, even though it is perhaps suspected. For example, the presence of mtDNA amplification in 100% of cases of Acute Myeloid Leukaemia (AML) studied, indicates use in the diagnosis of AML. Alternatively, the method may be used to monitor progression of disease in a patient, for example to follow change from a pre-malignant condition to malignancy (eg myelodysplasia (MDS) to AML) , or to monitor periods of remission and relapse (eg in AML where mtDNA levels in patients in remission are normal) . Also, changes in the nature of disease may be followed, as in the example of progression of CGL to AML wherein mtDNA levels increase.

Currently, only blood counts are used in a clinical context to monitor progression in AML. Molecular methods are used in research centres where the interest and resources exist. However, even then remission/progression cannot be monitored in 50% of patients because only about 50% of AML patients have chromosomal abnormalities, the rest having no mutations. The present invention provides an alternative, and indeed more widely useful, approach.

CGL patients are currently monitored by cytogenetics and blood count. However, using these procedures it is not always obvious when patients are going to change from the chronic to the acute phase of the disease. Amplification of mtDNA provides a marker which may be of assistance to the clinician, enabling therapeutic intervention at a stage earlier than would otherwise be, and currently is, possible. Such treatment may involve use of chemotherapy, either single or combination, such as interferon and bulsulphan¹⁸, and/or radiotherapy, which may, upon indication of progression, be increased in intensity.

Conveniently, the level of mtDNA is determined as a ratio of mtDNA:genomic DNA (i.e. the relative amounts of mtDNA and genomic DNA are compared) . Probing with suitable nucleic acid probes specific for mtDNA and genomic DNA respectively is the preferred mode of determination. Such probes are well-known and available in the art, and include the probes used in the experiments reported herein, and fragments thereof which retain specificity. New probes may easily be obtained and or designed based on specificity for mitochondrial DNA or genomic DNA respectively. Essentially any sequence found in either the mitochondrial or the nucleus but not both can be used, e.g. any known gene or fragment thereof retaining specificity as between the mitochondrial and chromosomal genomes. Such specificity can be routinely determined by those skilled in the art. Standard techniques for probing and other procedures of use in the present invention are exemplified in Sambrook et al¹⁵.

Generally, probes are labelled (e.g. radioactively) to allow quantitation of hybridisation between probe and target DNA (either mitochondrial or genomic) , eg by autoradiography or other techniques well-known to those skilled in the art.

Another aspect of the invention provides the use of a mtDNA-specific nucleic acid probe in diagnosis and/or monitoring of a malignant disease. This may be in accordance with any embodiment or variation of the present invention as disclosed.

Aspects and embodiments of the present invention will now be discussed in more detail, by way of example and not limitation. Further aspects and embodiments of the present invention will be apparent to those skilled in the art.

All documents mentioned herein are hereby incorporated by reference. EXAMPLE 1 - GENE DOSAGE ANALYSIS

Gene dosage analysis is a well established method for the determination of gene copy number^13,14. Gene dosage experiments designed to allow for a quantitative assessment of total mtDNA relative to human genomic DNA were carried out.

High molecular weight DNA was obtained by phenol/chloroform extraction using standard methods¹⁵ from various cells of various individuals. The DNA was digested with the restriction enzyme EcoRI (EcoRI cleaves mtDNA at three different sites) and size fractionated by electrophoresis through 1% agarose gels. The DNA was transformed to Hybon-N (Amersham) according to standard procedures for Southern blotting¹⁵. DNA probes were labelled to high specific activity by random hexanucleotide priming (Boehringer Mannheim) and hybridization was carried out at 65°C for 16 hours.

Filters containing patient and control DNA were simultaneously hybridised to two probes; a 16.5kb total mtDNA genome probe (purified total mtDNA isolated from human placenta and the 1.9kb genomic Eco Rl-Sst I fragment from the renin gene (pHRn ESI.9, ATCC) . The renin gene is localised to chromosome lq32 or lq42 (a region karyotypically normal in the AML, MDS and CGL patients included in the study) , and acts as an internal hybridisation standard for human genomic DNA filters were washed for 20 min in 0.1X standard saline citrate, 0.1% SDS at 65°C and autoradiographed between intensifying screens at -70°C. The total mtDNA genomic probe gave 3 hybridisation fragments (8kb.7.4 kb and 1.1 kb) and the renin probe a single 5.5 kb fragment.

After autoradiography, the film was scanned with an enhanced laser densitometer (LKB Ultrascan XL) to quantitate the relative intensities of the mtDNA and renin gene hybridisation signals, ie the signal from the renin probe and the signal from the mtDNA fragment (a single 7.4kb hybridisation fragment of the mtDNA was chosen) .

A comparative densitometric ratio of one was derived from the two hybridization signals in 20 peripheral blood samples from normal healthy individuals. An approximately two fold (100%) increase in this ratio indicates a two fold increase in the dosage of mtDNA and is consistent with two fold amplification. A greater than 100% increase in this ratio is consistent with a corresponding increase in mtDNA amplification.

EXAMPLE 2 - DETERMINATION OF MTDNA LEVELS IN INDIVIDUALS High molecular weight DNA was obtained from peripheral blood leucocytes (blast samples) from AML patients, bone marrow samples (blast samples) from AML patients, bone marrow samples from AML patients in remission, peripheral blood leucocytes from patients with myelodysplasia (MDS) , peripheral blood leucocytes from patients with CGL and peripheral blood leucocytes from 20 normal healthy individuals and peripheral blood leucocytes from 20 individuals suffering from a range of infections or inflammatory disorders giving rise to abnormal white cell counts. Gene dosage analysis was carried out in accordance with procedures described in Example 1.

Results

A comparative densitometric ratio of one was derived was obtained in 20 peripheral blood samples from normal health individuals. There were no exceptions. Comparative densitometric ratios in excess of one were obtained in all the AML samples included in this study. Therefore there was no overlap between the normal and leukaemic samples and no false positive or false negative rates have been observed.

All 25 AML samples (blast samples) showed amplification of mtDNA relative to mtDNA quantities in peripheral blood leucocytes from normal healthy individuals. The level of mtDNA amplification varied from 2 to 50 fold in the AML samples and over 50% showed mtDNA amplification of 8 fold or greater (see Table I) . An additional 4 cases of AML were investigated at presentation and remission and showed 3-10 fold amplification of mtDNA at presentation and mtDNA dosage levels equivalent to normal peripheral blood controls at remission (see Table 1) .

Three cases of AML with a preceding myelodysplastic phase were investigated. In each case amplification of mtDNA (3-10 fold) followed transformation to AML (see Table 1) . Comparative densitometric ratios essentially the same as those observed in the peripheral blood samples from healthy controls, ie. one, were observed in all 11 CGL samples in the chronic phase of the disease.

Transformation of CGL to AML was also studied. In one case 19 fold amplification of mtDNA was observed. Out of 5 other cases, 3/5 in the chronic phase of the disease showed mtDNA dosage levels equivalent to those obtained with the peripheral blood samples from normal healthy controls. However, samples taken from the same patients in accelerated phase and/or blasts crisis all showed amplification of mtDNA (5-10 fold) . These data indicate that amplification of mtDNA may be a marker for progression in CGL. These observations suggest a role for amplification of mtDNA in malignant transformation of myeloid cells. In the other 2 cases the chronic phase of the disease (although not formally classified as being in acceleration) showed very early signs of progression into accelerated phase, i.e. either karyotypic changes associated with progression or the increase of Ber-abl transcripts by RT-PCR analysis. Significantly, mtDNA amplification was observed in both these cases in the chronic phase as well as in the accelerated phase and blast crisis, indicating value of the observation of mtDNA amplification not only as a marker for progression in CGL but also for the identification of patients likely to transform in the near future (see Table 2 for results) . Clearly this has important implications for patient treatment and therapy.

Peripheral blood leucocytes from 20 individuals suffering from a range of infections or inflammatory disorders giving rise to abnormal white cell count were also investigated and showed mtDNA levels essentially the same as those obtained for the 20 peripheral blood leucocytes from normal healthy controls, ie one (see Table 1) . TABLE 1 (overleaf)

MtDNA dosage analysis in various haematological samples and controls .

Optical densitometric readings were obtained from both the 7.4 mtDNA fragment and the 5.5 kb renin gene fragment. A comparative densitometric ratio of approximately one was derived from the two hybridization signals in 20 peripheral blood samples from normal healthy individuals. An approximately two fold (100%) increase in this ratio indicates a two fold increase in the dosage of mtDNA and is consistent with two fold amplification. A greater than 100% increase in this ratio is consistent with a corresponding increase in mtDNA amplification.

Ratio

Peripheral Blood

Case Normal Misc Infection CGL CGL→AML AML→remission MDS→AML Number Control

1 0.91 0.95 0.89 18.81 14.62 1.20→9.98

2 1.23 1.0 1.17 50.0 1.32→4.6

3 1.25 0.99 0.89 2.75 0.89→3.0

4 1.28 1.07 0.91 2.9

5 1.44 0.91 0.71 5.8

6 1.22 1.34 0.71 9.1

7 1.56 1.07 0.90 9.5

8 0.72 1.57 1.01 11.13

9 1.00 1.38 0.93 6.1

10 1.31 1.21 0.87 3.75

11 0.85 1.31 1.09 7.4

12 0.99 1.09 8.44

13 1.07 1.32 8.25

14 1.1 1.58 2.8

15 1.0 1.17 10.5

16 1.1 1.25 8.68

17 0.81 1.04 12.47

18 1.0 1.01 7.56

19 0.96 1.08 3.55

20 1.01 1.12 2.625

21 2.0

22 8.1

23 4.5

24 8.25

25 8.44

26 7.4-M .21

27 10.09→1.7

28 3.15→1.3

29 2.9-M .55 Table 2

Representative mtDNA dosage analysis of CGL patients in the chronic phase of the disease and transformation.

CP - chronic phase;

AC - accelerated phase;

BC - blast crisis.

MtDNA levels in chronic phase were essentially same as normal controls (Cl & C2) . Amplification of mtDNA 5-10 fold was observed following transformation. CP4 also showed amplification of mtDNA in chronic phase, this is consistent with other early signs of progression.

Case Ratio

CP, 0.9

AC, 6.6

CP₂ 1.5

AC₂ 4.7

CP₃ 1.2

AC₃ 7.8

BC₃ 10.0

Ci 1.1 c₂ 1.1

CP₄ 4.7

AC₄ 8.1 REFERENCES

I. Bessis M, and Breton-Gorius J.R. (1969) Nouv. Rev.Fr.Hematol.9, 245-278. 2. Schumacher et al (1972) Blut 25,169-178.

3. Schumacher et al(1974) Am J. Pathol 74, 71-82.

4. Szekely et al (1972) Blut 25,376-384.

5. Ghadially F.N., Skinnider L.F. (1974) J Pathol 114,11. 6. Herrera-Goepfert et al (1986) Human Pathology 17, 748-753.

7. Clayton, D.A. Vinograd J. (1967) Nature 216,652- 657.

8. Clayton D.A. , Vinograd J. (1969) PNAS 62,1077-1084. 9. Firkin F.C., Clarke-Walker G.D. (1979) Br.J. Hae at .

43,201-206. 10. Hatfill et al (1993) Leukemia Research 17,907-913.

II. Attardi et al (1982) In mitochondrial genes

(Slonimski P and Borst P., Eds.) Cold Spring Harbor, New York.

12. Manyan et al (1972) J. Lab. Clini. Med. 89, 137.

13. Kere et al (1987) N.Engl.J. Med. 316,499-503.

14. Boultwood et al (1993) Genomics 19, 425-432.

15. Sambrook et al (1989) "Molecular cloning: a laboratory manual" . Cold Spring Harbor Laboroatory Press, Cold Spring Harbor. New York. 16. Van den Bogert et al . (1993) Biochimica et Biophysica Acta, 1144, pp 177-183.

17. Shay et al. (1990) J. Biol. Chem. 265, pp 14802- 14807. 18. Goldman J.M., (1994) Blood Reviews 8, 21-29.

19. Baggetto L.G., (1993) Eur. J. Cancer 29, 156-159.

20. Modica-Napolitano et al (1989) Cancer Res. 49, 3369-3373.

Claims

1. A method of determining the state of malignant disease of an individual, the method comprising determining the level of mitochondrial DNA in cells of the individual in comparison with the normal level of mitochondrial DNA in cells of the same type.

2. A method according to claim 1 used to monitor the progression of malignant disease in the individual.

3. A method according to claim 2 wherein the individual is monitored for remission and relapse.

4. A method according to claim 2 wherein the individual is monitored for progression from a pre- malignant to a malignant state.

5. A method according to claim 2 wherein the nature of the disease is monitored.

6. A method according to claim 1 used in diagnosis of malignant disease in the individual.

7. A method according to claim 1 wherein the malignant disease is a leukaemia.

8. A method according to claim 7 wherein the leukaemia is acute myeloid leukaemia.

9. A method according to claim 7 wherein the leukaemia is chronic granulocytic leukaemia.

10. A method according to any preceding claim wherein the level of mitochondrial DNA in a sample of cells is determined as a ratio of mitochondrial DNA:genomic DNA in the sample.

11. A method according to claim 10 wherein DNA is extracted from the sample and probed with a nucleic acid probe specific for mitochondrial DNA and a nucleic acid probe specific for genomic DNA.

12. A method according to claim 11 wherein the probes are labelled.

13. Use of a nucleic acid probe specific for mitochondrial DNA in determining the state of a malignant disease of an individual.

14. Use according to claim 13 wherein progression of the malignant disease is monitored.

15. Use according to claim 13 wherein the malignant disease is diagnosed.