WO2006111586A2

WO2006111586A2 - Method for the in vitro determination of the degree of methylation of the line-1 promoter

Info

Publication number: WO2006111586A2
Application number: PCT/ES2005/000205
Authority: WO
Inventors: Xabier Aguirre Ena; GÓMEZ José ROMÁN; Antonio JIMÉNEZ VELASCO; Felipe Prosper Cardoso
Original assignee: Proyecto De Biomedicina Cima, S.L.; Asociación Medica E Investigación (A.M.I.); Fundación Imabis
Priority date: 2005-04-20
Filing date: 2005-04-20
Publication date: 2006-10-26
Also published as: WO2006111586B1; WO2006111586A3

Abstract

The invention relates to a method for the in vitro determination of the degree of methylation of the LINE-1 promoter, which is characterised in that it comprises genomic DNA amplification with specific amplification primers.

Description

PROCEDURE FOR THE IN VITRO DETERMINATION OF THE DEGREE OF METHODATION OF THE 'PROMOTER OF LINE-I.

TECHNICAL FIELD OF THE INVENTION

The present invention falls within the scope of the methods of detecting the degree of methylation of the LINE-I promoter.

STATE OF THE TECHNIQUE

Chronic Myeloid Leukemia (CML) is a malignant disease of hematopoietic stem cells, which has an incidence of 1.2: 100,000 inhabitants and years. It has three distinct phases characterized both at the phenotypic level and from the point of view of the treatment and the prognosis of the disease (chronic phase -3 to 4 years- accelerated phase -1 to 2 years- and finally a blastic phase -6 to 12 months - in which the disease is refractory to treatment). The transition from a Chronic Phase to a Blastic Crisis is characterized by genomic instability in which an accumulation of molecular and chromosomal alterations is observed, but the mechanism by which this genomic instability occurs is still unknown. The possibility of determining mechanisms involved in genomic instability would favor the development of new therapeutic and diagnostic strategies.

From the point of view of treatment, a drug directed specifically against the molecular target of the disease called Imatinib has recently been discovered, capable of inhibiting the BCR-ABL oncogene responsible for the disease. The Philadelphia chromosome is the specific marker of the disease and appears as a consequence of a reciprocal translocation between chromosomes 9 and 22 resulting in an abnormal protein with tyrosine activity

- '/ O "> REPLACEMENT JA (Rule 23) BCR-ABL kinase and on which the alterations of the leukemic cells depend. The only treatment that has demonstrated the possibility of curing patients with this disease is the allogeneic transplantation of hematopoietic progenitors, treatment on the other hand associated with significant morbidity and mortality. The transplant has proven to be more effective as soon as possible in the course of the disease.

As for the treatment with Imatinib, although 70% of the patients respond to the treatment obtaining a complete cytogenetic response when administered at the time of diagnosis of the disease, there are 30% of patients who develop resistance to the treatment and these percentages increase very important in advanced stages of the disease (patients in accelerated phase, less than 20% get a long-term response).

There is an important interest in defining biological markers that help predict: 1) which patients will respond to Imatinib treatment; 2) which patients will progress to advanced stages of the disease early. Being able to define these two aspects would condition the possibility of choosing new treatments for patients earlier and therefore with greater guarantees of success.

In the case of CML, there are predictive or resistance factors to Imatinib treatment, the presence of ABL1 mutations being the most frequently described, and predictive factors of disease progression, mainly related to the clinical presentation of the disease.

Several studies show that DNA methylation plays a very important role in maintaining genomic stability and that in particular the hypomethylation of DNA in tumor cells is associated with instability. DNA methylation changes can

REPLACEMENT SHEET (Rule 23) occur both in regions close to promoters and first exons of different genes (as a mechanism to control their transcription) and in repetitive regions of the genome rich in CpG dinucleotides. Abnormal (increased) methylation of said promoters results in the silencing of their expression.

In addition to the specific regulation of genes through the methylation of their promoters, in the human genome up to 20% of the sequences are formed by a series of repetitive elements called retrotransposons and that under normal conditions are hypermethylated so that they are not expressed. These retrotransposons are characterized by their ability to transcribe to RNA, retrotranscribe to DNA and that that DNA is inserted into a new region of genomic DNA contributing to the regulation of some genes, and in turn, facilitating alterations at the gene level. One of the most frequent retrotransposons is LINE-I. When retrotrasposons jump from one region of the genome and are inserted into another, they can contribute to inducing genome alterations, especially when they are not silenced by hypermethylation of their promoter regions. It is unknown, however, which genes are affected as a result of LINE-I hypomethylation.

DNA methylation changes can occur both in regions close to promoters and first exons of different genes and in repetitive regions of the genome rich in CpG dinucleotides. In regions close to the promoters and first exons of different genes, the alteration of the methylation that usually occurs is a hypermethylation that normally leads to a decrease or loss of expression of those genes. Different studies have shown that hypermethylation of different genes plays a very important role in the progression and response of different types of tumors (Román-Gómez et

REPLACEMENT SHEET (Rule 2S) to the. Promoter hypermethylation of cancer related genes is a strong independent prognostic factor in Acute Lymphoblastic Leukemia; Blood 2004; 104: 2492-2498).

Within repetitive regions, the LINE-I ₁ elements are one of the most important elements. These elements are usually methylated, in this case their hypomethylation is the mechanism of alteration. Different studies have described that these elements can be inserted within the regions of the genes, being able to control and modify the normal transcription of these genes (Druker et al. Complex patterns of transcription at the insertion site of a retrotransposon in the mouse. Nucleic Acids Research ; 2004; 32: 5800-5808; Han et al. Transcriptional disruption by the Ll retrotransposon and implications for mammalian transcriptomes. Nature, - 2004; 429: 268-274). In different types of tumors, it has been observed that LINE-I is hypomethylated and in the case of prostate carcinoma it has been observed that this hypomethylation may be involved in the progression of the disease (Chalitchagorn et al. Distinctive pattern of LINE-I methylation level in normal tissues and the association with carcinogenesis; Oncogene; 2004; 23: 8841-8846; Florl et al. Coordinate hypermethylation at specific genes in prostate carcinoma precedes LINE-I hypomethylation; British Journal of Cancer; 2004; 91: 985- 994). But so far it was not known what role hypomethylation of LINE-I plays in predicting the prognosis of the disease.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1: Methylation status of the LINE-I promoter in patients and CML cell lines.

A) Analysis of the CpG island of LINE-I by MSP-PCR in healthy individuals and patients with CML. CP: chronic phase of the CML; BC: blastic crisis of the CML; UM:

REPLACEMENT SHEET (Rule 26) unmethylated sequence; M: methylated sequence. In healthy individuals, hypermethylation of the LINE-I promoter is observed (lack of amplification of the non-methylated sequence) while in the samples of patients with CML, different degrees of hypotnetylation of the LINE-I promoter are observed (amplification of both the methylated sequence as of the unmethylated sequence).

B) Analysis of the CpG island of the LINE-I promoter by means of MSP-PCR in six Ph positive cell lines. UM: unmethylated sequence; M: methylated sequence. In lines K562 and KU81, hypomethylation of the LINE-I promoter is observed.

Figure 2: Distribution of the disease-free survival curve (PFS) by the Kaplan and Meier method in patients with CML according to the methylation status of the LINE-I promoter.

Figure 3: Effect of hypomethylation of LINE-I on the expression of ORF-I and c-MET. A) Treatment of the TOM-I line (hypermethylated LINE-I) with the 5-aza-2'-deoxycytidine demethylating agent. The cells were treated for 4 days with 2 μM and 4 μM of 5-aza-2'-deoxycytidine. This treatment resulted in the hypomethylation of the LINE-I promoter and the sense expression of ORF1 and c-MET antisense. UM: unmethylated sequence; M: methylated sequence ^' . B) Expression of the c-MET protein on the surface of bone marrow cells from healthy individuals and individuals with CML.

DESCRIPTION OF THE INVENTION

The present invention relates to a method for in vitro determination of the degree of methylation of the LINE-I promoter, characterized in that it comprises amplifying genomic DNA by means of a pair of primers.

REPLACEMENT SHEET (Rule 26) specific amplification of unmethylated sequences and a pair of amplification primers specific for methylated sequences, where both pairs:

- they have a size between 10 and 30 nucleotides,

-selectively inhibited with a sequence between nucleotides 49 and 420 of the consensus sequence of the LINE-I promoter, -at least one selectively hybridized with a sequence between nucleotides 100 and 150, or between nucleotides 240 and 290 of said promoter consensus sequence.

In this procedure for in vitro determination of the degree of methylation of the LINE-I promoter, said primers preferably have a size of between 15 and 25 nucleotides.

In a specific embodiment of the present invention, at least one of the amplification primers is selected from: SEQ. ID. NO: 1, SEQ. ID. NO: 2, SEQ.

ID. NO: 3, and SEQ. ID. NO: 4. In another specific embodiment of the invention, primers specific for methylated sequences possess the sequences corresponding to SEQ. ID. NO: 1 and 2. Furthermore, in another specific embodiment of the invention, primers specific for non-methylated sequences have sequences corresponding to SEQ. ID. NO: 3 and 4.

In a preferred embodiment of the present invention, the method for in vitro determination of the degree of methylation of the LINE-I promoter is a methylation sensitive PCR. And, in a more preferred embodiment, said method is a methylation sensitive PCR by real time quantification.

In another preferred embodiment of the present invention, the process for in vitro determination of the degree of methylation of the LINE-I promoter is

SHEET LEAVE (Rule 26) characterized in that a degree of methylation less than 80% is related to the presence of leukemia. In a preferred embodiment, the leukemia is related to a degree of methylation of the LINE-I promoter of less than 70%. In a more preferred embodiment said degree of methylation related to the presence of leukemia is less than 50%.

In a preferred embodiment of the invention, said leukemia is acute leukemia or chronic leukemia. In addition, said leukemia is selected from: a chronic myeloid leukemia, an acute myeloid leukemia, an acute lymphocytic leukemia or a chronic lymphocytic leukemia.

In a specific embodiment of the invention, the in vitro determination of the degree of methylation of the LINE-I promoter makes it possible to judge the evolution of said leukemia. In a preferred embodiment, said determination may also allow to evaluate the evolution of said leukemia in response to various treatments. In addition, the present invention relates to an oligonucleotide selected from SEQ. ID. NO: 1, SEQ. ID. NO: 2, SEQ. ID. NO: 3 and SEQ. ID. NO: 4.

On the other hand, the present invention relates to a kit for determining the degree of methylation of the LINE-I ₇ promoter characterized in that it comprises a pair of amplification primers specific for unmethylated sequences and a pair of amplification primers specific for methylated sequences .

In a preferred embodiment, said kit for determining the degree of methylation of the LINE-I promoter is characterized in that both pairs of primers:

- they have a size between 10 and 30 nucleotides,

- selectively inhibited with a sequence between nucleotides 49 and 420 of the consensus sequence of the LINE-I promoter,

REPLACEMENT SHEET (Rule 28) - at least one of them selectively hybridizes with a sequence comprised between nucleotides 100 and 150, or between nucleotides 240 and 290 of said promoter consensus sequence.

In a specific embodiment of said kit, said pair of amplification primers specific to methylated sequences are characterized in that at least one of the primers is selected from: SEQ. ID. NO: 1, SEQ. ID. NO: 2, SEQ. ID. NO: 3 and SEQ. ID. NO: 4. In a preferred embodiment of said kit, the pair of amplification primers specific for methylated sequences are

I KNOW THAT. ID. NO: 1 and 2, and the pair of amplification primers specific for unmethylated sequences are SEQ. ID. NO: 3 and 4.

In a specific embodiment of said kit for determining the degree of methylation of the LINE-I promoter, it comprises appropriate reagents for performing a methylation sensitive PCR method. On the other hand, the present invention relates to a method of diagnosis of leukemia, by determining the presence of hypomethylation of the LINE-I promoter, so that a hypomethylation is considered indicative of leukemia, when its levels are below 80 %. In a preferred embodiment said hypomethylation is less than 70%, and in a more preferred embodiment, said hypomethylation is less than 50%. In a specific embodiment, said method allows to predict the evolution of said leukemia, and / or determine the response of said leukemia to various treatments.

EMBODIMENT OF THE INVENTION

The following examples are intended to illustrate, in no way limitative, the embodiment of the invention object of the present patent application.

REPLACEMENT SHEET (Rule 26) Example 1: Quantification of quantitative MSP methylation

a.- Obtaining the biological sample and extraction of genomic DNA

In this study, the analysis of the methylation status of the LINE-I promoter was carried out in 140 patients affected by chronic myeloid leukemia (CML) diagnosed between 1982 and 2003. For the specific analysis, bone marrow was obtained from each patient in the time of diagnosis (before receiving any treatment) and healthy bone marrow donors. In all cases, the bone marrow sample was obtained after informed consent. From the bone marrow sample, the population of mononucleated cells was obtained after sedimentation in a Ficoll-Hypaque gradient. The bone marrow sample at diagnosis was obtained in all patients, while in 47 both the sample was obtained at diagnosis and the sample at the time of the blast crisis (34 in myeloid blast crisis and 13 in lymphoid blast crisis). Blastic crisis was defined as the presence of 30% blasts in peripheral blood or bone marrow. The DNA for subsequent analysis of the methylation status of the LINE-I promoter was obtained from each bone marrow sample by using the QIAmp DNA Mini Kit (Qiagen, Hilden, Germany).

b.- Pretreatment with sodium bisulfite

The quantitative MSP technique was used to analyze the methylation status of the LINE-I promoter. The MSP (Methylation Specific PCR) technique is based on a first treatment of the DNA sample with sodium bisulfite, which modifies unmethylated cytosines to uracil, while cytosines

REPLACEMENT SHEET (Rule 26) methylated do not undergo any change after this treatment. In our study, 1 μg of DNA from each bone marrow sample of patients with CML was treated and modified using the CpGenomic ™ DNA modification Kit (Intergen Company, Purchase, NY). The protocol of this modification kit consists briefly of the following:

- Mix in a 1.5 ml microtube, 1 μg of DNA, 7 μl of 3M NaOH, 2 μl of Reagent IV (provided by the kit) and water to complete 100 μl of the reaction.

- Incubate the DNA 10 minutes at 37 ⁰ C.

- Add 550 μl of DNA Modification Reagent I (provided by the kit) prepared at the time and mix using vortex. Preparation of DNA Modification Reagent

I: Add 571 μl of water to 227 mg of DNA Modification Reagent for each sample

I and adjust the pH to 5.0 with NaOH.

- Incubate from 16 to 20 hours at 50 ⁰ C. - Add 5 μl of the DNA Modification Reagent III (provided by the kit). Add 750 μl of DNA Modification Reagent

II (provided by the kit) and mix. Preparation of DNA Modification Reagent II: First add 1 μl of β-mercaptoethanol to 20 ml of deionized water. For each sample to be modified, add 750 μl of the β-mercaptoethanol solution to 1.35 g of the DNA Modification Reagent II. - Incubate for 10 minutes at room temperature.

- Centrifuge 10 seconds 5000 X g to precipitate the DNA III Modification Reagent and remove the supernatant.

REPLACEMENT SHEET (Rule 26) - Add 1 ml of 70% ethanol, vortex, centrifuge 10 seconds 5000 xg and remove the supernatant. Repeat this process 3 times.

- After removing the supernatant from the third wash with 70% ethanol, centrifuge 3 minutes at 13000 rpm. and remove the supernatant using a micropipette.

- Add to each sample 50 μl of 20 mM NaOH / 90% ethanol. Preparation of 20 mM NaOH / 90% ethanol: For the preparation of 1 ml of this solution, add 900 μl of 100% ethanol, 93.4 μl of water and 6.6 μl of 3M NaOH.

- Vortex and incubate the mixture for 5 minutes at room temperature.

- Centrifuge 10 seconds 5000 x g to precipitate the sample. Add ImI of 90% ethanol and vortex

'to wash the sample. Centrifuge again 10 seconds 5000 x g and remove the supernatant. Repeat this process twice.

- After removing the supernatant from the second wash with 90% ethanol, centrifuge the sample at 13000 rpm. for 5 minutes.

- Remove all the supernatant and resuspend the precipitate in 30 μl of TE (10 mM Tris / 0.1 mM EDTA, pH 7.5). Incubate the sample at 55 ⁰ C for 15 minutes.

- Centrifuge at 13000 r.p.m for 3 minutes and transfer the supernatant to a new 1.5 ml microtube.

- Proceed to the MSP technique or save the sample at -20 ⁰ C.

PCR amplification

REPLACEMENT SHEET (Rule 26) After modification of the DNA by means of sodium bisulfite, the MSP was performed for the analysis of the methylation status of the LINE-I promoter. This MSP consists of two PCRs, one performed with specific primers for amplification of the LINE-I promoter in the methylated state and a second PCR performed using specific primers for amplification of the LINE-I promoter in the non-methylated state. Quantitative MSP was performed using a LightCycler (Roche Molecular Biochemicals) with the ability to discriminate three fluorescent colors using 1 µl of DNA modified with sodium bisulfite in a final reaction volume of 10 µl with 0.4 µmol / 1 of each primer, 3 , 5 mmol / 1 Mg ²⁺ and 1 μl of the 1Ox LightCycler FastSatr DNA Master SYBR Green I (Roche Molecular Biochemicals).

The amplification of the LINE-I promoter in the methylated state was carried out by using the primers: sense, 5 '-GTCGAATAGGAATAGTTTCGG-3'; antisense, 5 '-ACTCCCTAACCCCTTACGCT-3'; and amplification of the LINE-I promoter in a non-methylated state by using the primers: sense, 5 '-GTTGAATAGGAATAGTTTTGGTTT-3 •; antisense, 5'- ACTCCCTAACCCCTTACACTT-3 '. The amplification conditions consisted of: denaturation program consisting of a cycle _ 95 ° C 10 minutes; amplification program, consisting of 45 cycles at 95 ° C 1Os, 65 ° C 1Os and 72 ° C 1Os, ^• melting program, consisting of a cycle at 95 ° C Os, 40 ⁰ C 6Os and 90 ⁰ C for 6Os, ^• and a cycle cooling program at 40 ⁰ C 6Os. The temperature transition was 20 ° C / s, except in the melting program that was 0.4 ° C / s between 40 ⁰ C and 90 ⁰ C.

d.- Quantification of methylation

REPLACEMENT SHEET (Rule 26) For the quantification analysis of LINE-I methylation, the procedure of the relative quantification of the problem or target gene was used

(methylated sequence of the LINE-I promoter) versus its relationship with the control gene (non-methylated sequence of the LINE-I promoter). These calculations were directly performed by the LightCycler software

(RealQuant, version 1.0, Roche). The normal methylation ratio of the LINE-I promoter was obtained by the following equation and expressed as a percentage of the control gene:

Normalised ratio (N _L1 ) = (E _target ) ^{ΔCp target (contro1 "} sample) ΔCp ref (control - sample)

normalised ratio: normalized ratio, target: target, control: control, sample: sample.

To obtain the cut-off point that specified the hypomethylation of the LINE-I promoter, the bone marrow of 50 healthy individuals was analyzed. As expected, the LINE-I promoter was methylated in healthy subjects, but in some cases amplification of both the methylated and non-methylated sequence was obtained. Due to these data, the sample of healthy individual that presented the smallest difference in amplification between the methylated and nonmethylated sequences was taken as a control, considering that in this case the LINE-I promoter was 100% methylated. Based on this prerequisite, in the analysis of the 50 samples of healthy individuals, values between 100% and 231% were obtained, the average being 160% ± 45%. Thanks to this data, the value of 70% (determined as the mean minus 2 SD) was chosen as the cut-off point, defining

REPLACEMENT SHEET (Rule 26) ratios equal to or less than this value as hypomethylation of the LINE-I promoter.

& .- Statistical Methods All statistical calculations were performed using the SPPS statistical program (SPPS, Chicago, IL). The overall survival (OS) was calculated from the diagnosis of CML until the death of the patient. Progression-free survival (PFS) was calculated from the diagnosis of CML until the onset of a blastic crisis or death without disease progression. The distribution of the OS and PFS curves was done using the Kaplan and Meier method, with a 95% confidence interval calculated by the Greenwoods formula average. The comparison of the OS and PFS between the different groups was based on the Log-Rank test. The comparison of the significance of the prognostic factors was based on the Cox and hazard regression models.

Example 2: Design of amplification primers

The design of the primers for the analysis of the methylation of the LINE-I promoter using the MSP-PCR technique was carried out using the computer program designed by the author Long-Cheng Li and located at the Internet address: http : // www. dahivauroloqy. com / cgi-bin / methprimer / methprimer. CGI

This program allows to design primers for the analysis of methylation by means of MSP-PCR, analysis of methylation by restriction enzymes sensitive to methylation and sequencing analysis after treatment with sodium bisulfite. For the analysis of the methylation status of LINE-I through MSP-PCR, the parameters that were taken into account were the following:

REPLACEMENT SHEET ÍReαla 26) - First, the region of the LINE-I promoter was defined that had a CpG island with a content greater than 60% of C + G and a frequency of CpG dinucleotides observed compared to the expected greater than 0.6.

- Once the region within the promoter was defined, primers were designed on this region. These primers had to comply with the following:

• Primers that amplify a fragment of between 100 and 300 base pairs.

• Primers with a Tm between 55 ° C and 65 ° C.

^• Primers with a length of between 20 and 30 base pairs.

• Primers with a minimum of two CpG dinucleotides in their sequence.

• Primers in which one of the CpG dinucleotides is located in the last three bases of its sequence.

Example 3: Chronic myeloid leukemia. Degree of methylation of LINE-I.

LINE-I methylation was analyzed in 50 samples of normal bone marrow. As expected, the LINE-I promoter sequences were strongly methylated, which is manifested by the amplification of the methylated sequences, according to the method described in the previous examples, with the absence of amplification of non-amplified sequences. in most samples (72%). The analysis of the methylation of the LINE-I promoter in samples of patients with chronic myeloid leukemia, resulted in the amplification of unmethylated promoter sequences, said hypomethylation being more pronounced in samples from patients in blast crisis than in patients in phase chronic (Figure IA).

REPLACEMENT SHEET (Rule 26) On the other hand, it was observed that the LINE-I promoter in LMC derived cell lines positive for the Philadelphia chromosome (K562, KU812, TCC-s and KYO-I) was strongly hypomethylated, while B cell precursor cell lines positive for the Philadelphia chromosome, they showed normal levels of LINE-I methylation (MY, TOM-I, BV173 and NALM-20) (Figure IB).

Example 4: Chronic myeloid leukemia. Determination of cut-off or reference values

To obtain the cut-off point that specified the hypomethylation of the LINE-I ₁ promoter, the bone marrow of 50 healthy individuals was analyzed. As expected, the LINE-I promoter was methylated in healthy subjects, but in some cases amplification of both the methylated and non-methylated sequence was obtained. Due to these data, the sample of healthy individual that presented the smallest difference in amplification between the methylated and nonmethylated sequences was taken as a control, considering that in this case the LINE-I promoter was 100% methylated. Based on this prerequisite, in the analysis of the 50 samples of healthy individuals, values between 100% and 231% were obtained, the average being 160% + 45%. The value of 70% (determined as the mean minus 2 SD) was chosen as the cut-off point, defining ratios equal to or less than this value as hypomethylation of the LINE-I promoter.

Example 5: Chronic myeloid leukemia. Analysis of patients diagnosed with CML.

By means of the MSP-PCR analysis performed for the study of the methylation status of the LINE-I promoter in the samples of patients with CML, the hypomethylation of the LINE-I promoter was observed with a higher

REPLACEMENT SHEET (Rule 26) frequency in CML samples in blast crisis

(74.5%) than in chronic phase CML samples (38%).

The difference between the groups of patients with CML in blast crisis and chronic phase was statistically very significant (P <0.0001). In addition, by the technique of

Quantitative MSP showed that among the hypomethylated CML samples, the samples of CML patients in blast crisis had a higher hypomethylation compared to chronic CML samples (37.5% versus 46% P = O, 07).

In this same study, it was observed that the samples of patients with CML who presented hypomethylation of the LINE-I promoter presented a worse response to the usual treatments used in the clinic (interferon and imatinib). In the case of interferon treatment, of the 76 patients treated, a complete cytogenetic remission was observed in 32% of patients with hypermethylated LINE-I, but this complete cytogenetic remission was only observed in 3% of patients with LINE -I hypomethylated (P = O, 004). Multivariate statistical analysis showed that the methylation status of the LINE-I promoter was the only independent factor of the cytogenetic response to interferon in this group of patients. In the case of patients with chronic phase CML treated with Imatinib (n = 25), a greater cytogenetic response was observed in 100% of patients in the group with hypermethylated LINE-I, while in the group presenting the LINE-I promoter hypomethylation showed a greater cytogenetic response in only 47% of cases (P = O, 034). In addition, the molecular response was significantly higher in patients presenting with the hypermethylated LINE-I promoter (80%) compared to those presenting with the hypomethylation of the LINE-I promoter (18%, P = O, 036).

REPLACEMENT SHEET (Rule 26) In the study conducted on disease-free survival (PFS) it was shown that in the case of CML patients who presented hypomethylation of the LINE-I promoter, the mean PFS was 58.3 months, while patients with CML with the hypermethylated LINE-I promoter had a mean PFS of 96.8 months, significantly higher than those of the hypomethylated group (P = O, 005) (Figure 2).

Example 6: Chronic myeloid leukemia. LINE-I hypomethylation is associated with the activation of c-MET antisense transcription.

LINE-I has two transcription regulatory regions located in the 5 'untranslated region: ^• an internal or sense promoter that directs the complete LINE-I transcription (ORF-I), and an antisense promoter

(ASP) that directs transcription in the opposite direction to adjacent cell sequences. To determine the relationship between LINE-I hypomethylation and ORF-I expression, TOM-I cells (hypermethylated) were treated with the 5'-aza-2 'deoxycytidine demethylating agent. This treatment induced the hypomethylation of LINE-I and the expression of ORF-I transcripts (Figure 3A). On the other hand, it was found that the complete LINE-I sequence is inserted in intron -2 of the c-MET gene between nucleotides 46113 and 52058, so that the c-MET gene has the same transcription direction that the antisense promoter (ASP) of LINE-1. Thus, it was studied whether the hypomethylation of the promoter of

LINE-I could affect the expression of c-MET. It was observed that c-MET expression was significantly higher in those patients with chronic myeloid leukemia who had the hypomethylated LINE-I promoter. Similarly, c-MET overexpression was detected in 61% of patients with chronic myeloid leukemia with

REPLACEMENT SHEET (Rule 26) LINE-I hypomethylation but in no patient who possessed the methylated LINE-I promoter (Figure 3, A and B).

From the previous examples we can conclude that: - a patient with a chronic phase CML who has hypomethylation of LINE-I has a high risk of developing early to an advanced stage of the disease (blast crisis);

- in addition, said chronic phase patient with LINE-I hypomethylation has an increased risk of resistance to current reference treatments (interferon or Imatinib); whereby

- Transplantation of hematopoietic progenitors could be a more appropriate therapeutic indication for such patients.

REPLACEMENT SHEET (Rule 26)

Claims

1. Procedure for in vitro determination of the degree of methylation of the LINE-I promoter, characterized in that it comprises amplifying genomic DNA by means of a pair of amplification primers specific for unmethylated sequences and a pair of amplification primers specific for methylated sequences, where both pairs of primers: - have a size between 10 and 30 nucleotides,

2. Procedure for in vitro determination of the degree of methylation of the LINE-I promoter according to claim 1, characterized in that said primers have a size between 15 and 25 nucleotides.

3. Procedure for the in vi tro the degree of methylation of the promoter determination LINE-I _'according to claim 1 or 2, wherein at least one of the amplification primers is selected from: SEQ. ID. NO: 1, SEQ. ID. NO: 2, SEQ. ID. NO: 3, and SEQ. ID. NO: 4.

4. Procedure for in vitro determination of the degree of methylation of the LINE-I promoter according to claim 1, characterized in that said primers specific for methylated sequences possess the sequences corresponding to SEQ. ID. NO: 1 and 2.

REPLACEMENT SHEET (Rule 26)

5. Method for in vitro determination of the degree of methylation of the LINE-I promoter according to claim 1, characterized in that said primers specific for non-methylated sequences have sequences corresponding to SEQ. ID. NO: 3 and 4.

6. Procedure for in vitro determination of the degree of methylation of the LINE-I promoter according to one of claims 1 to 5, characterized in that said method is a methylation sensitive PCR.

7. Method for in vitro determination of the degree of methylation of the LINE-I promoter according to one of claims 1 to 6, characterized in that said methylation-sensitive PCR is real-time quantification.

8. Method for in vitro determination of the degree of methylation of the LINE-I promoter according to claim 1, characterized in that a degree of methylation of less than 80% is related to the presence of leukemia.

9. Method for in vitro determination of the degree of methylation of the LINE-I promoter according to claim 8, characterized in that said degree of methylation is less than 70%.

10. Method for in vitro determination of the degree of methylation of the LINE-I promoter according to claim 8, characterized in that said degree of methylation is less than 50%.

11. Procedure for in vitro determination of the degree of methylation of the LINE-I promoter according to one of claims 8 to 10, characterized in that said leukemia is an acute leukemia or a chronic leukemia.

12. Procedure for in vitro determination of the degree of methylation of the LINE-I promoter according to the

REPLACEMENT SHEET (Rule 26) claim 11, characterized in that said leukemia is selected from: a chronic myeloid leukemia, an acute myeloid leukemia, an acute lymphocytic leukemia or a chronic lymphocytic leukemia.

13. Procedure for the in vitro determination of the degree of methylation of the LINE-I promoter according to one of claims 11 or 12, characterized in that said determination allows to determine the evolution of said leukemia.

14. Method for in vitro determination of the degree of methylation of the LINE-I promoter according to one of claims 11 or 12, characterized in that it allows determining the evolution of said leukemia in response to treatments.

15. An oligonucleotide selected from SEQ. ID. NO: 1, SEQ. ID. NO: 2, SEQ. ID. NO: 3 and SEQ. ID. NO: 4.

16. Kit for determining the degree of methylation of the LINE-I promoter, characterized in that it comprises a pair of amplification primers specific for unmethylated sequences and a pair of amplification primers specific for methylated sequences.

17. Kit for determining the degree of methylation of the LINE-I promoter according to claim 16, characterized in that both pairs of primers:

- they have a size between 10 and 30 nucleotides,

REPLACEMENT SHEET (Rule 26)

18. Kit for determining the degree of methylation of the LINE-I promoter according to one of claims 16 or 17, characterized in that at least one of the primers is selected from: SEQ. ID. NO: 1, SEQ. ID. N0: 2, SEQ. ID. NO: 3 and SEQ. ID. NO: 4.

19. Kit for determining the degree of methylation of the LINE-I promoter according to one of claims 16 to 18, characterized in that the pair of specific amplification primers of methylated sequences are SEQ. ID. NO: 1 and 2, and the pair of amplification primers specific for unmethylated sequences are SEQ. ID. NO: 3 and 4.

20. Kit for determining the degree of methylation of the LINE-I promoter according to one of claims -16 to 19, characterized in that it comprises appropriate reagents for performing a methylation sensitive PCR method.

REPLACEMENT SHEET (Rule 26)