ES2873049T3 - Method, sequences, compositions and kit for the detection of mutations in the promoter of the Htert gene - Google Patents

Abstract

Set of primers and probes for the detection of mutations c.-124 C> T and c.-146 C> T in the promoter of the hTERT gene characterized in that it comprises the primers with SEQ ID NO: 1 and SEQ ID NO: 2 and the probes with SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6.

Description

DESCRIPTION

Method, sequences, compositions and kit for the detection of mutations in the promoter of the Htert gene

The present invention relates to a method for the detection of mutations c.-124 C> T and c.-146 C> T in the promoter of the Htert gene. The method referred to uses a reaction composition comprising primers for amplification and probes for genotyping.

Another aspect of this invention refers to the primers and probes used in the performance of the aforementioned method with sequences, identified as SEQ ID NO: 1 to SEQ ID NO: 6, that show high specificity for these mutations, as well as compositions that contain them.

The present invention also relates to a kit comprising the aforementioned compositions for detecting the mutations c.-124 C> T and c.-146 C> T in the promoter of the Htert gene carrying out the method of the present invention.

The method, gene sequences, compositions and kit of the present invention can be used advantageously to detect trace amounts of the c.-124 C> T and c.-146 C> T mutations, present in biological samples due to their high sensitivity and specificity for such mutations.

The present invention, therefore, can be applied in early detection, identification, detection of recurrence or prediction and monitoring of diseases associated with these mutations, such as bladder carcinomas, thyroid carcinomas, squamous cell carcinoma, basal cell carcinomas, cancer. of skin, cancers of the central nervous system and hepatocellular carcinoma, among others, and finally provide the basis for an appropriate treatment environment.

Therefore, the present invention is within the technical field of medicine, pharmaceuticals, molecular biology, biochemistry and related human genetics.

DESCRIPTION

Method, sequences, compositions and kit for the detection of changes in the promoter of the Htert gene

Technical field of the invention

The present invention relates to a method for the ultrasensitive detection of mutations c.-124 C> T and c.-146 C> T in the promoter of the Htert gene, the reaction compositions comprising primers for amplification and probes for genotyping, denominating the sequences SEQ ID NO: 1 to SEQ ID NO: 6, which were designed to present a high specificity for these mutations, as well as the kit comprising such compositions to carry out the method referred to.

The present invention can be used to advantage to detect trace amounts of such c.-124C> T and c.-146C> T mutations present in biological and in vitro samples due to the high sensitivity of the method and the specificity of the gene sequences created, and thereby allowing early detection, identification of recurrence or prediction and monitoring of diseases associated with these mutations, such as bladder carcinomas, thyroid carcinomas, squamous cell carcinoma, basal cell carcinomas, skin cancer, nervous system cancers central and hepatocellular carcinoma, among others and finally provide the basis for the appropriate treatment environment.

Therefore, the present invention is within the technical field of medicine, pharmaceuticals, molecular biology, biochemistry and related human genetics.

State of the art

Somatic mutations that occur during DNA replication that precede mitotic division are at the origin of certain human disorders, particularly certain types of cancers. The presence of telomerase enzyme was observed in a high percentage, approximately 75-80%, of tumor cell lines. Recently, applicants have identified mutations in the promoter of the telomerase gene that encodes this enzyme, the TERT gene, in particular, mutations at positions c.-124C> T and c.-146C> T (from ATG initial). These mutations have been associated with various malignant tumors, such as central nervous system cancers (43-51%), bladder carcinomas (59-66%), hepatocellular carcinomas (59%), thyroid carcinomas (10%), cancers skin (melanoma 29% -73%, basal cell carcinoma (BCC) 73% and squamous cell carcinomas (SCC) 45%) and gastrointestinal stromal tumors (GIST) (4%) [1-7].

The fact that mutations in the telomerase promoter are present with a relatively high frequency in certain cancers makes them possible biomarkers for early detection, diagnosis, prognosis, detection of recurrence, or prediction / monitoring of therapeutic response of tumors, such as central nervous system tumors, bladder carcinomas, hepatocellular carcinomas, thyroid carcinomas and skin cancers, among others. For example, in bladder cancer, somatic mutations at positions c.-124C> T and c.-146C> T (from ATG) of the telomerase gene have a prevalence that reaches 85% and, together with the R248C and S249C mutations in the fibroblast growth receptor 3 (FGFR3) gene [9] are the most reliable biomarkers of bladder cancer.

However, for the in vitro use of biological fluid samples (for example, urine, plasma, cerebrospinal fluid, aspiration cytology, among others), the method of detecting mutations at positions c.-124 C> T and c .-146C> T (from ATG) of the telomerase gene must have a high analytical sensitivity in order to detect trace amounts of the mutated alleles, even when they are “diluted” in a sample with a higher abundance of “wild-type” alleles. ”.

Document WO2015153808 discloses methods and compositions useful in the detection of some diseases, in particular cancer, more specifically thyroid cancer, related to mutations in the positions c.-124C> T and c.-146C> T of the TERT gene . However, the nucleotide sequences disclosed in that document are used for the detection of such mutations by direct, sequence-dependent amplification methods, and have limited sensitivity when the presence of normal sequences is relatively high compared to those of mutant sequences. On the other hand, they are susceptible to the appearance of nonspecific amplification, so they do not show high specificity for this type of mutation, which causes, in their general method, that they are not sufficiently sensitive and specific.

Document WO2014160834 discloses methods and compositions useful in the detection and treatment of different types of thyroid cancer, related to mutations in the c.-124 C> T and c.-146 C> T positions of the TERT gene. However, in this document, the nucleotide sequences disclosed did not show high sensitivity since the presence of normal sequences is very abundant compared to mutant sequences, and the proposed method is based on DNA sequencing using the kit. “BigDye terminator v3.1” ready sequencing tool and on an Applied Biosystems ABI PRISM 3730 kit, which has limited sensitivity to detect rare alleles in the reaction. In addition, the presence of the mutation must be confirmed by sequencing in both the sense and antisense directions, making the method impractical and time consuming and applicable only to thyroid cancers.

Yves Allory et al: "Telomerase Reverse Transcriptase Promoter Mutations in Bladder Cancer: High Frequency Across States, Detection in Urine, and Lack of Association with Outcome", EUROPEAN UROLOGY, vol. 65, No. 2, February 1, 2014 (02-01-2014), pages 360-366, describe a SNaPshot assay to detect mutants referred to in the present invention. The fragment comprising the mutations is amplified with a pair of primers and the mutations are each detected with a probe, which is used in a single nucleotide extension reaction. In the present invention, other primers are used and the probes are allele specific with no single nucleotide extension.

Christian Koelsche et al: "Tert promoter hotspot mutations are recurrent in myxoid liposarcomas but rare in other soft tissue sarcoma entities", JOURNAL OF EXPERIMENTAL & CLINICAL CANCER RESEARCH BIOMED CENTRAL LTD, LONDON, UK, vol. 33, No. 1, April 11, 2014 (04-11-2014), page 33, describe a method that uses PCR and direct sequencing to detect mutations. The primers and probes used in the method described in this document are different from those defined in the present invention.

Kun Wang et al: "Tert promoter mutations are associated with distant metastases in upper urothelial tract carcinomas and serve as urinary biomarkers detected by a sensitive castPCR", Oncotarget, vol. 5, No. 23, December 9, 2014 (12-09-2014), pages 12428-12439, describe Sanger sequencing to detect the mutations described in the present invention. In particular, this reference describes a highly specific and sensitive castPCR method for detecting C228T, using allele-specific primers and blockers and a detection probe.

Document WO2015 / 049063, in the name of Deutsches Krebsforschung, (2015-04-09), describes a widely used method for the detection of the two mutations, which comprises an amplification step followed by a sequencing step. However, the primer / probe combinations used in D4 are different from the present invention.

The methods described in the art to discriminate mutations at the c.-124C> T and c.-146C> T positions of the TERT gene that comprise sequencing or single-base extension have the disadvantage that, after amplification, it is necessary to perform a second reaction (sequencing or SNaPshot or single base extension), thus posing the problem of a high risk of contamination derived from the manipulation of amplification products when discrimination is not performed in a single reaction.

Furthermore, in single-base sequencing or extension, equimolar amounts of dideoxynucleotides are used and there is no bias towards the detection of the mutant allele, with the disadvantage that reaction resources are consumed by extensions of wild-type alleles and raising the problem of the lack of a reaction bias towards the detection of mutant alleles.

To achieve biased amplification of mutant alleles, methods in the art rely on either the use of a wild-type sequence blocker or sequence-specific primers for amplification of the mutant allele with specific PCR primers from allele.

Methods that use PCR blockers to suppress wild-type allele amplification, known as Cast PCR, favor the use of reaction resources toward amplification of mutant alleles. However, because the amplification of the wild-type allele is suppressed in the reaction, there is no internal control in the reaction for the amplification of the specific target sequence and, in this condition, the lack of amplification of the mutant alleles cannot distinguished from inhibition of target sequence amplification due to, for example, contaminants or impurities. Typically, when performing Cast PCR, a reaction with probes to detect the wild-type allele must be performed in parallel, in a separate reaction using the same sample, to certify that the sample is suitable for amplification. However, this does not allow a true endogenous control to be present in the mutant detection reaction and endogenous control and target sequences cannot be detected within a single reaction.

Allele-specific PCR-based methods use sequence-specific primers such that the 3 'nucleotide of the primer is complementary to the mutant nucleotide in the target sequence, thus inhibiting the possibility of DNA polymerases extending the primers with 3' projections. . However, it is known that, even in the absence of 3 'complementarity, DNA polymerases can extend mismatched primers and, since the primer is integrated into a sequence, which is then targeted in a new round of amplification, this can generate exponential nonspecific amplification.

Other methods, such as real-time PCR for allelic discrimination, comprise amplification of a target sequence spanning a specific mutant site followed by hybridization of the produced amplimers with wild-type and mutant sequence-specific labeled probes, which are they degrade in the extension phase by the exonuclease activity of the 3'-5 'DNA polymerase to produce fluorescence. Equimolar concentrations of probes are used for wild-type and mutant alleles and this has the disadvantage that there is no bias for the use of reaction resources in favor of detection of the mutant allele.

Furthermore, no method for discriminating mutations at the c.-124C> T and c.-146C> T positions of the TERT gene known in the art can be calibrated to a desired limit of detection / analytical sensitivity and this cannot be differentially established for one u another allele based on the reaction composition. This is a disadvantage of current methods, as it is known that analytical sensitivity does not necessarily follow clinical sensitivity and detection of very low levels of a biomarker may occur in a subclinical setting and not necessarily translate into clinical expression. A method in which analytical detection limits could be calibrated for clinical expression would have a higher positive predictive value and greater clinical specificity, as few false positives would be generated.

Therefore, it is necessary to develop a method with sufficient sensitivity and specificity that allows the detection of such mutations in biological samples even under conditions where the mutated alleles are present in very low amounts.

The present invention proposes an ultrasensitive method that allows the detection of mutations c.-124 C> T and c.-146 C> T in the promoter of the Htert gene, by designing primers and probes whose sequences are specific for this purpose. .

Summary of the invention

The present invention refers to the set of primers, probe sequences, compositions, kits and methods as defined in claims 1 to 14. These DNA primers and probes, because they have a high specificity in the detection of such mutations , allow its use in a method for the detection of high sensitivity of these mutations.

The compositions of the present invention are applicable to the detection of mutations c.-124 C> T and c.-146 C> T in the promoter of the Htert gene, in combinations that allow the detection of both the wild-type allele of endogenous control such as mutant alleles to be detected in the same reaction, at differential analytical sensitivities, biased towards the detection of mutant alleles and with detection limits that can be calibrated by manipulating the reaction composition that can be prepared previously or can be prepared in the moment of the performing the detection method of the invention.

In these compositions, the amount of DNA in the test sample can be quite low, given the high specificity of the primers and probes of the present invention designed for that purpose.

The kit of the present invention has the advantage of developing an ultrasensitive method for detecting in vitro mutations c.-124 C> T and c.-146 C> T in the promoter of the Htert gene, in a fast and reliable way, adding a sample human DNA test for the detection of said mutation.

The method of the present invention can be used advantageously to detect trace amounts of such mutations c.-124 C> T and c.-146 C> T present in biological samples due to its high sensitivity conferred by the genetic sequence designed for this purpose and which has a high specificity for such mutations.

Therefore, the present invention can be applied in early detection, identification, detection of recurrence or prediction and monitoring of diseases associated with such mutations, such as bladder carcinomas, thyroid carcinomas, skin cancers, central nervous system cancers and hepatocellular carcinoma. among others and finally provide the basis for the appropriate treatment environment.

Description of the invention

The present invention relates to a method for the in vitro detection of mutations c.-124 C> T and c.-146 C> T M in the promoter of the Htert gene. The reaction method referred to uses compositions comprising primers for amplification and probes for genotyping, the sequences of which (SEQ ID NO: 1 to SEQ ID NO: 6) are specific for these mutations.

Thus, primer sequences for amplification and probes for genotyping are created in order to build the basis for said amplification reaction, for example, a real-time PCR (RT-PCR) or digital-time PCR assay. 6 sequences were designed for the primers and probes, as shown in the sequence listing. This includes sequences for the following purposes:

to. Detection of the mutation located at -124 bases downstream of the ATG of the hTERT gene

• SEQ ID NO: 1: Initiator F

• SEQ ID NO: 2: Initiator R

• SEQ ID NO: 3: Probe -124C

• SEQ ID NO: 4: Probe -124T

b. Detection of the mutation located at -146 bases downstream of the ATG of the hTERT gene

• SEQ ID NO: 1: Initiator F

• SEQ ID NO: 2: Initiator R

• SEQ ID NO: 5: Probe -146C

• SEQ ID NO: 6: Probe -146T

The above sequences can be prepared by the nucleotide synthesis method using solid phase phosphoramidite nucleosides as described in the publication: Beaucage, SL; Iyer, RP (1992). "Advances in the Synthesis of Oligonucleotides by the Phosphoramidite Approach". Tetrahedron 48 (12): 2223. The synthesis can be carried out in columns with solid support (controlled pore glass or polystyrene) functionalized with the first base of the 3 'end of each oligonucleotide. The preparation of each oligonucleotide follows after several synthesis cycles, each consisting of chemical reactions, usually four chemical reactions: i) release (detritylation), ii) coupling, iii) protection, and iv) oxidation. At each cycle, nucleotide residues corresponding to the desired sequence are added to the 5 'terminal end of the growing strand.

These can be further modified in the 5 'and 3' sequences of the probes by adding corresponding fluorophores such as FAM, Yakima Yellow, quencher (TAMRA), etc. known to experts, for the purpose of multiplex PCR amplification and hybridization reactions. Therefore, suitable fluorophores in the present invention are, for example, compounds known from Alexa Fluor FAM, TET, JOE, VIC, HEX, Cy3, ATTO 550, TAMRA, ROX, Cy5, Cy5.5.

Nucleotides refer to nucleotides of natural or synthetic origin, capable of base-pairing hybridization with complementary nucleotides, and may include, without limitation, DNA, RNA, and nucleotide analogs (for example, nucleic acids with closed conformation, known as "Blocked nucleic acids" - LNA) or nucleotides without phosphodiester-type junctions between nucleotides (eg peptide nucleic acid - PNA) and nucleic acids with thiodiesel bonds, or the like for the same purpose.

Compositions for DNA amplification and hybridization (PCR compositions) were prepared by adding commercially available PCR reactant solutions with different nucleotide sequences with SEQ ID NO: 1 to SEQ ID NO: 6 in different concentrations, and bidistilled water and bidesionized water.

The PCR reaction solutions used vary with the desired type of amplification reaction. Examples of reactant solutions suitable for PCR for carrying out the present invention are, for example, the "TaqMan® Universal PCR Master Mix" solution from Thermofischer Scientific or similar solutions for the purpose of DNA amplification and hybridization.

Probe concentrations refer to the molar concentration of the solution, corresponding to the number of moles of each probe per volume of solution, where nanoMolar (nM) corresponds to 1x10-9 moles per liter.

Compositions for PCR were prepared comprising various combinations of different concentrations of the probes with sequences SEQ ID NO: 3 to SEQ ID NO: 6. As mentioned above, probes were designed whose sequences are defined by SEQ ID NO: 3 and SEQ ID NO: 4 for the specific detection of the c.-124 C> T mutation in the promoter of the Htert gene while the probes SEQ ID NO: 5 and SEQ ID NO: 6 were designed for the specific detection of the c. -146 C> T in the promoter of the Htert gene.

Therefore, the compositions of the invention for PCR can comprise two sets of probes mentioned above, that is, the compositions of the PCR of the invention comprise all probes with SEQ ID NO: 3 to SEQ ID NO: 6 (complete set c .-124 c.-146).

Both sets of probes are present in the PCR compositions, so these compositions are specific for the detection of both c.-124 C> T and c.-146 C> T mutations.

The concentration of nucleotide probes of the present invention in the solutions is adjusted by PCR, in order to promote the sensitivity of the method of the invention in the detection of such mutations. Preferably, the final concentration of each probe is adjusted to values between 400 and 1600 nM, more preferably with final concentration values of 800 to 1600 nM and in a further preferred embodiment of the invention the final concentration values of each probe are of about 1600 nM.

Therefore, it is possible to obtain PCR compositions comprising different combinations of final concentration values of each of the probes defined by SEQ ID NO: 3 and SEQ ID NO: 6.

Therefore, in a preferred form, the PCR compositions comprising the following concentrations of the probes with SEQ ID NO: 3 to SEQ ID NO ^: 6:

i. from 250 nM to 500 nM of SEQ ID NO: 3, from 400 nM to 1600 nM of SEQ ID NO: 4, from 250 nM to 500 nM of SEQ ID NO: 5 and from 400 nM to 1600 nM of SEQ ID NO: 6,

or

ii. from 250 nM to 500 nM of SEQ ID NO: 3, from 800 nM to 1600 nM of SEQ ID NO: 4, from 250 nM to 500 nM of SEQ ID NO: 5 and from 800 nM to 1600 nM of SEQ ID NO: 6.

The compositions of the reaction mixtures for the detection of the mutations c.-124 C> T and c.-146 C> T in the promoter of the Htert gene were prepared by adding DNA from the biological sample to be tested to the compositions mentioned above for PCR.

Test DNA can be obtained from tissue, urine, circulating tumor DNA, germ line, or other biological sources using standard methods known in the art such as those available from Qiagen QIAamp® or similar methods. The amount of DNA to be tested may be present in the composition to detect mutations c.-124 C> T and c.-146 C> T in the promoter of the Htert gene, at concentrations below 500 ng, less than 100 ng, 50 ng and greater than 1 ng.

The adjustment referred to in the concentration of the probes used is intended to enhance the analytical sensitivity of the method of the present invention, demonstrated by a minimum number of mutant alleles (MUT DNA) detected. relative to the number of "wild-type" alleles (TN DNA). It is assumed that the increase in analytical sensitivity results from the increase in the concentrations of the probes used in the reaction favoring the amplification of the mutant allele.

Simultaneously, the efficiency of the hybridization and amplification of the "wild-type" allele was reduced in order to maintain as much as possible the equilibrium of the reaction in favor of the amplification of the mutant allele.

Therefore, it was possible to develop a method in which various combinations of different concentrations of the probes used give rise to conditions that enhance analytical sensitivity and can detect very low levels of mutant alleles.

Up to certain levels, increasing the concentration of mutated probe does not result in nonspecific detection of the "wild-type" allele as evidenced by the absence of signal from the -124T and -146T probes when using only type sample. TN DNA (Figure 5 - panels C and D). Therefore, based on manipulation of the probe concentrations used, it is possible to calibrate the sensitivity of the analytical method while preserving its specificity.

Reflecting this increased efficiency in the detection of the mutated alleles -124T and -146T, is the significant increase in the analytical sensitivity of the method, which passes to minimum detection values of 1.56% for the allele -124T and the 0.78% for the -146T allele (Figures 6A and B, respectively).

Therefore, the method of the present invention comprises the steps of: i) preparing a composition to detect mutations c.-124 C> T and c.-146 C> TM in the promoter of the Htert gene, as described above, ii) promotion of DNA extracted from a biological sample to be tested for mutation, a composition for PCR amplification and hybridization, also as described above, iii) DNA amplification and hybridization of the composition of sample to detect mutations c.-124 C> T and c.-146 C> T in the promoter of the Htert gene by PCR reaction, real-time PCR, RT-PCR or digital PCR or even multiplex PCR reaction and iv) analysis of the amplification curves thus obtained.

The presence of mutated c.-124 C> T and c.-146 C> T in the promoter of the Htert gene of the test sample is analyzed to determine the existence of an exponential growth of a fluorescence signal, which corresponds to the presence of at least one of the mutations referred to.

The method of the present invention is useful in the detection of trace amounts of the c.-124 C> T and c.-146 C> T mutations in various biological fluids, eg, urine, plasma, cerebrospinal fluid, aspiration cytology , among others, having as input DNA obtained from these different sources. In these examples, the advantage is being able to make a determination from a less invasive kind of sample without having to rely on a biopsy or surgery.

It can be applied in the surveillance of patients with bladder cancer by means of a DNA-based analysis result of a urine sample. This application is based on the fact that the mutation c.-124C> T and c.-146C> T are highly frequent in bladder cancer and can be detected in DNA derived from urine (Figure 7). For patients who are under clinical surveillance because they have a 50-70% probability of recurrence, the possibility of detecting recurrence by a urinalysis has advantages over the conventional method based on cystoscopy as it is completely non-invasive and more convenient. for the patient.

The method of the present invention can also be applied to the detection of other types of cancer in which Htert gene mutations are present and for other clinical purposes. An example is the analysis of fine needle aspirates of thyroid nodules in order to provide useful prognostic information in deciding the need for ablative treatment or extension of lymphadenectomy.

Another example of application of the present invention relates to early detection, diagnosis, prognosis, detection of recurrence or prediction / monitoring of the response to the therapy of tumors such as cancers of the central nervous system, bladder carcinomas, hepatocellular carcinomas, carcinomas of thyroid, skin cancers, among others.

The present invention may also be useful for the analysis of tumor tissue or circulating DNA in the context of "liquid biopsy" for the possible selection and treatment of candidate patients, response and prediction to treatment and monitoring, for example, with telomerase inhibitors for the promoter of the Htert gene.

Brief description of the figures

Figure 1: Demonstration of the assay's ability to detect mutations in the promoter of the Htert gene (TERTp) c.-124 C> T.

In this figure, it is presented that the assay is specific for the detection of alleles at position -124. Assay performance is confirmed by testing for the detection of the c.-124 C> T mutation in DNA samples heterozygous for the c.-124 C> T mutation (HET124 DNA) and "wild-type" cases (TN DNA ). The results demonstrate that the specific probe for the mutant allele (probe -124 T) generates a signal in the HET-124 DNA sample and not in the TN DNA sample and the probe for the "wild-type" allele (probe -124 C) generates an amplification of the signal in both samples.

1 - Amplification curve obtained with the -124C probe ("wild type" allele) in TN DNA.

2 - Amplification curve obtained with the -124C probe ("wild type" allele) in HET-124 DNA.

3 - Amplification curve obtained with the probe -124T (mutant allele) in HET-124 DNA.

4 - Amplification curve obtained with the probe -124T (mutant allele) in TN DNA.

X axis - Amplification cycle

Y axis - Fluorescence change

Figure 2: Demonstration of the assay's ability to detect mutations in TERTp c.-146 C> T

In this figure, it is presented that the assay is specific for the detection of alleles at position -146. The performance of the test in detecting the mutation c. -146 C> T is confirmed by analysis of heterozygous DNA samples for c.-146 mutated C> T (HET146 DNA) and wild-type (TN DNA) cases. The results show that the probe specific for the mutant allele (probe -146 T) generates a signal in the HET-146 DNA sample and not in the TN DNA sample and that the probe for the "wild-type" allele ( probe -146 C) generates an amplification of the signal in both samples.

1 - Amplification curve obtained with the -146C probe ("wild type" allele) in TN DNA.

2 - Amplification curve obtained with the -146T probe (mutant allele) in HET-146 DNA.

3 - Amplification curve obtained with the -146C probe ("wild type" allele) in HET-146 DNA.

4 - Amplification curve obtained with the probe -146T (mutant allele) in TN DNA.

X axis - Amplification cycle

Y axis - Fluorescence change

Figure 3: Assessment of the analytical specificity of the tests:

(A) Specific detection of the TERTp c.-124 C> T mutation

(B) Specific detection of the TERTp c.-146 C> T mutation

In this figure, the assay was shown to have no cross-reactivity between the mutations tested. Figure 3-A: The heterozygous DNA sample for the TERTp -146C> T mutation is not amplified by the -124T probe and the only probe that fluoresces is in fact the -124C probe (corresponding to the "wild-type" allele in position -124).

Figure 3-B: On the other hand, a heterozygous DNA sample for the TERTp -124C> T mutation is not amplified by the -146T probe and the only probe that emits fluorescence is in fact the -146C probe (corresponding to the allele "of wild type ”at position -146).

1 - Amplification curve obtained with the -124C probe ("wild type" allele) in HET-146 DNA.

2 - Amplification curve obtained with the probe -124T (mutant allele) in HET-146 DNA.

3 - Amplification curve obtained with the -146C probe ("wild type" allele) in HET-124 DNA.

4 - Amplification curve obtained with the -146T probe (mutant allele) in HET-124 DNA.

X axis - Amplification cycle

Y axis - Fluorescence change

Figure 4: Assessment of analytical sensitivity for the detection of the -124T (A) and -146T (B) allele under conditions that mimic DNA samples derived from biological fluids, in which there are small numbers of mutated alleles in a global context in where the predominant allele is "wild type". DNA samples with the c.-124C> T mutation or samples with the c.-146C> T mutation serially diluted in "wild-type" DNA samples were used.

This figure reports the efficiency and sensitivity of the test in detecting the mutated alleles -124T and -146T.

1 - Amplification curve obtained with the -124T probe at 50% MUT124 DNA / TN DNA.

2 - Amplification curve obtained with the -124T probe at 25% MUT 124 DNA / TN DNA.

3 - Amplification curve obtained with the 12.5% MUT124 DNA / TN DNA probe -124T.

4 - Amplification curve obtained with the -124T probe at 6.25% MUT124 DNA / TN DNA.

5 - Amplification curve obtained with the -124T probe at 3.125% MUT124 DNA / TN DNA.

6 - Amplification curve obtained with the -146T probe at 50% MUT 146 / TN DNA.

7 - Amplification curve obtained with the -146T probe at 25% MUT 146 DNA / TN DNA.

8 - Amplification curve obtained with the -146T probe at 12.5% MUT146 DNA / TN DNA.

9 - Amplification curve obtained with the -146T probe at 6.25% MUT 146 / TN DNA.

X axis - Amplification cycle

Y axis - Fluorescence change

Figure 5: Enhancement of analytical sensitivity in the detection of the allele -124T (A) and -146T (B).

DNA samples with the c.-124C> T mutation or samples with the c.-146C> T mutation serially diluted in “wild-type” DNA samples were used in a ratio of 25% MUT DNA / TN-DNA .

It can be seen that, under a condition of low probe concentration for the "wild-type" allele (eg 250 nM), the use of a higher probe concentration for the mutated alleles -124T and -146T (400-800 -1600 nM) results in a significant increase in the detection signal of the mutated alleles (panels A and B of Figure 5, respectively).

In panels C and D of this figure, it can be seen that, up to certain levels, the increase in the concentration of mutated probe does not result in nonspecific detection of the "wild-type" allele as evidenced by the absence of signal from the -124T and -146T probes when using only TN-DNA as sample.

1 - Amplification curve, probe -124T (mutant) at a concentration of 400 nM.

2 - Amplification curve, probe -124C (wild type) at a concentration of 250 nM.

3 - Amplification curve, probe -124T (mutant) at a concentration of 800 nM.

4 - Amplification curve, probe -124C (wild type) at a concentration of 250 nM.

5 - Amplification curve, probe -124T (mutant) at a concentration of 1600 nM.

6 - Amplification curve, probe -124C (wild type) at a concentration of 250 nM.

7 - Amplification curve, probe -146T (mutant) at a concentration of 400 nM.

8 - Amplification curve, probe -146C (wild type) at a concentration of 250 nM.

9 - Amplification curve, probe -146T (mutant) at a concentration of 800 nM.

10 - Amplification curve, probe -146C (wild type) at a concentration of 250 nM.

11 - Amplification curve, probe -146T (mutant) at a concentration of 1600 nM.

12 - Amplification curve, probe -146C ("wild type") at a concentration of 250 nM.

13 - Amplification curve, probe -124T (mutant) at a concentration of 1600 nM.

14 - Amplification curve, probe -124C ("wild type") at a concentration of 250 nM.

15 - Amplification curve, probe -1246T (mutant) at a concentration of 400 nM.

16 - Amplification curve, probe -146C ("wild type") at a concentration of 250 nM.

X axis - Amplification cycle

Y axis - Fluorescence change

Figure 6: Evaluation of the analytical sensitivity for the detection of the allele -124T (A) and -146T (B) when the concentration of the probe for the mutant allele is increased and the concentration of the probe used for the allele "of wild type ”. This change in the relative composition of the probes results in an increase in analytical sensitivity.

This figure shows the increased efficiency and sensitivity in detecting the mutated alleles -124T and -146T: the minimum detection values were 1.56% for the -124T allele and 0.78% for the allele. -146T.

1 - Amplification curve obtained with the -124T probe at 25% MUT124 DNA / TN DNA.

2 - Amplification curve obtained with the 12.5% MUT124 DNA / TN DNA probe -124T.

3 - Amplification curve obtained with the -124T probe at 6.25% MUT124 DNA / TN DNA.

4 - Amplification curve obtained with the -124T probe at 3.125% MUT124 DNA / TN DNA.

5 - Amplification curve obtained with the -124T probe at 1.56% MUT124 DNA / TN DNA.

6 - Amplification curve obtained with the -146T probe at 50% MUT 146 / TN DNA.

7 - Amplification curve obtained with the -146T probe at 25% MUT 146 DNA / TN DNA.

8 - Amplification curve obtained with the -146T probe at 12.5% MUT146 DNA / TN DNA.

9 - Amplification curve obtained with the -146T probe at 6.25% MUT 146 / TN DNA.

sonda -146T al 3,125% de ADN MUT146 / ADN TN. 11 - Curva de amplificación obtenida con

sonda -146T al 1,56% de ADN MUT 124 / ADN TN. 12 - Curva de amplificación obtenida con

sonda -146T al 0,78% de ADN MUT146 / ADN TN. Eje X - Ciclo de amplificación10 - Amplification curve obtained with

-146T probe at 3.125% MUT146 DNA / TN DNA. 11 - Amplification curve obtained with

-146T probe 1.56% MUT 124 DNA / TN DNA. 12 - Amplification curve obtained with

-146T probe at 0.78% MUT146 DNA / TN DNA. X axis - Amplification cycle

Y axis - Fluorescence change

Figure 7: Demonstration of the assay's ability to detect TERTp c.-124 C> T and c.-146 C> T mutations in urine samples from bladder cancer patients. In this figure, the detection of the c.-124 C> T mutation or the c.-146 C> T mutation in urine samples can be seen. The results show that, in this type of samples obtained from patients with bladder cancer, mutated alleles for the -124 (A) or -146 (B) mutation are detected, as well as signals for the corresponding "wild-type" alleles. .

1 - Amplification curve obtained with the -124C probe ("wild type" allele) in DNA obtained from a urine sample from a patient with bladder cancer.

2 - Amplification curve obtained with the probe -124T (mutant allele) in DNA obtained from a urine sample from a patient with bladder cancer.

3 - Amplification curve obtained with the -146C probe ("wild type" allele) in DNA obtained from a urine sample from a patient with bladder cancer.

4 - Amplification curve of -146T obtained with the probe (mutant allele) in DNA obtained from a urine sample from a patient with bladder cancer.

X axis - Amplification cycle

Y axis - Fluorescence change

EXAMPLES

Example 1 - Preparation of primer and probe sequences

Two primer sequences, SEQ ID NO: 1 and SEQ ID NO: 2, and 4 other sequences of the probes, SeQ iD NO: 3 SEQ ID NO: 6 were prepared as follows:

The sequences indicated above were prepared by the nucleotide synthesis method using solid phase phosphoramidite nucleoside. The synthesis was carried out in columns packed with the solid support functionalized with the first base of the 3 'end of each oligonucleotide. The preparation of each oligonucleotide followed after several cycles of synthesis, each consisting of four chemical reactions:

Unblocking (detritylation); coupling; protection and oxidation. In each cycle, nucleotide residues corresponding to the desired sequence were added step by step to the 5 'terminal end of the growing strand. The concentration of the probes was adjusted to values of 400 to 1600 nM by diluting a lyophilized preparation in double distilled water and double deionized water.

Example 2 - Preparation of PCR reaction compositions

Various PCR reaction compositions were prepared comprising the nucleotide according to sequences with SEQ ID NO: 1 to SEQ ID NO: 6 in different concentrations (eg 400nm, 800nm, 1600nM) and as described below. For comparison purposes, solutions were also prepared with an alternative concentration of probe for the "wild-type" allele (eg, 250 nM, 500 nM).

I - Compositions for the detection of mutations located at bases -124 in the 5 'sense of the ATG initiation codon of the hTERT gene

Composition of the invention:

• Solution reagent for real-time PCR “TaqMan ® Universal PCR Master Mix” at 1X concentration;

• oligonucleotide with the sequence: SEQ ID NO: 1, at a concentration of 900 nM;

• oligonucleotide with the sequence: SEQ ID NO: 2, at a concentration of 900 nM;

• oligonucleotide probe with the sequence: SEQ ID NO: 3, at a concentration of 250 nM, which contains modifications such as the incorporation of the compound known as YAKIMA YELLOW ® and tetra-methylrhodamine (TAMRA) at the 5 'and 3' terminal ends , respectively;

• oligonucleotide probe with the sequence: SEQ ID NO: 4, at a concentration of 1600 nM, which contains modifications such as the incorporation of the compound known as 6-carboxy-fluorescein (6-FAM) and tetramethyl-rhodamine (TAMRA) in the terminal ends 5 'and 3', respectively;

• bidistilled and bidesionized water.

Composition of the invention Ib:

This composition is prepared in a similar way to composition la but replacing the oligonucleotide probe concentration with that identified as: SEQ ID NO: 4, at a concentration of 800 nM.

Composition of the invention Ic:

This composition is prepared in a similar way to composition Ia but replacing the oligonucleotide probe concentration with that identified as: SEQ ID NO: 4, at a concentration of 400 nM.

Comparative composition Id:

This composition is prepared in a similar way to composition Ia but replacing the oligonucleotide probe with the sequence: SEQ ID NO: 3, at a concentration of 500 nM.

II - Compositions for the detection of mutations located at -146 bases in the 5 'sense of the ATG initiation codon of the hTERTgene gene

Composition of the invention IIa:

• Solution reagent for real-time PCR “TaqMan ® Universal PCR Master Mix” at 1X concentration;

• oligonucleotide with the sequence: SEQ ID NO: 1, at a concentration of 900 nM;

• oligonucleotide with the sequence: SEQ ID NO: 2, at a concentration of 900 nM;

• oligonucleotide probe with the sequence: SEQ ID NO: 5, at a concentration of 250 nM, containing modifications at the 5 'and 3' terminal ends, respectively;

• oligonucleotide probe with the sequence: SEQ ID NO: 6, at a concentration of 1600 nM, containing modifications at the 5 'and 3' terminal ends, respectively;

• bidistilled and bidesionized water.

The modifications mentioned in the sequences of the 5 'and 3' terminal ends of the probes correspond to the addition of fluorophores (FAM, Yakima yellow) and quencher (TAMRA), etc. known to the expert.

Composition of the invention IIb:

This composition is prepared in a similar manner to composition IIa, however, the oligonucleotide probe with the sequence: SEQ ID NO. 6 is present at a concentration of 800 nM.

Composition of the invention I Ic:

This composition is prepared in a similar manner to composition IIa, however, the oligonucleotide probe with the sequence: SEQ ID NO: 6 is present at a concentration of 400 nM.

Comparative composition IId:

This composition is prepared in a similar way to composition Ia, however, the oligonucleotide probe with the sequence: SEQ ID NO: 5 is present at a concentration of 500 nM.

III Compositions for the simultaneous detection of mutations located at bases -124 and -146 in the 5 'sense of the ATG initiation codon of the hTERT gene:

Composition of the invention IIIa:

• Solution reagent for real-time PCR “TaqMan ® Universal PCR Master Mix” at 1X concentration;

• oligonucleotide with the sequence: SEQ ID NO: 1, at a final concentration of 900 nM;

• oligonucleotide with the sequence: SEQ ID NO: 2, at a final concentration of 900 nM;

• oligonucleotide with the sequence: SEQ ID NO: 3, at a final concentration of 900 nM;

• oligonucleotide probe with the sequence: SEQ ID NO: 4, at a concentration of 1600 nM, containing modifications at the 5 'and 3' terminal ends, respectively;

• oligonucleotide probe with the sequence: SEQ ID NO: 5, at a concentration of 250 nM, containing modifications at the 5 'and 3' terminal ends, respectively;

• oligonucleotide probe with the sequence: SEQ ID NO: 6, at a concentration of 1600 nM, containing modifications at the 5 'and 3' terminal ends, respectively

• bidistilled and bidesionized water.

Composition of the invention IlIb:

This composition is prepared in a similar manner to composition IIIa, however, the oligonucleotide probe with the sequence: SEQ ID NO: 4 is present at a concentration of 800 nM.

Composition of the invention IIIc:

This composition is prepared in a similar way to composition IIIa, however, the oligonucleotide probe with the sequence: SEQ ID NO: 4 is present at a concentration of 400 nM.

Composition of the invention II Id:

This composition is prepared in a similar way to composition IIIa, however, the oligonucleotide probe with the sequence: SEQ ID NO. 6 is present at a concentration of 800 nM.

Composition of the invention IIIe:

This composition is prepared in a similar manner to composition IIIa, however, the oligonucleotide probe with the sequence: SEQ ID NO: 6 is present at a concentration of 400 nM.

Comparative composition IIIf:

This composition is prepared in a similar way to composition Ia, however, the oligonucleotide probe with the sequence: SEQ ID NO: 3 is present at a concentration of 500 nM.

Comparative composition IIIg:

This composition is prepared in a similar way to the composition but replacing the oligonucleotide probe with the sequence: SEQ ID NO: 5, present at a concentration of 500 nM.

Example 3 - Preparation of compositions to detect mutations

The compositions of the reaction mixtures for the detection of mutations c.-124 C> T and c.-146 C> T in the promoter of the hTERT gene were prepared with the addition of DNA from a biological test sample, approximately 100 ng of DNA (a smaller amount can be used, ranging from 1 ng to 500 ng) to the PCR compositions mentioned above, with the concentration of the probes with SEQ ID NO 3 and SEQ ID NO: 6, adjusted to values ranging between 400 and 1600 nM, as described in the previous example. Compositions for detection were therefore prepared with DNA samples containing the c.124 C> T mutation or samples containing the c.146 C> T mutation "serially diluted" in "wild-type" DNA samples.

Likewise, compositions for detection were prepared for comparative purposes, through the addition of DNA from the biological test sample to the comparative solutions of the previous example.

Example 4 - Method for the detection of mutations c.-124 C> T and c.-146 C> T located in the prompter of the hTERT gene

To carry out the method of the present invention, different compositions were prepared to detect mutations as described in the previous example.

In this example, DNA from cell lines or DNA from tumor tissue was used, either fresh or formalin-fixed and embedded in paraffin.

The compositions for the detection of the previous example were subjected to amplification and hybridization processes in real time PCR and digital PCR. The compositions were also subjected to simultaneous (multiplex) assays using probes for both assays mutations in the same reaction, the probes being labeled with different fluorophores. Among the most common fluorophores are compounds known as Alexa Fluor FAM, TET, JOE, VIC, HEX, Cy3, ATTO 550, TAMRA, ROX, Cy5, Cy5.5.

To carry out the method in real time PCR, the ABI PRISM ® 7500 Fast machine marketed by Scientific ThermoFischer was used.

Amplification and hybridization conditions:

1st phase: 1 cycle of 10 minutes at 95 ° C

2nd phase: 45 cycles, comprising 30 seconds at 92 ° C, 1 minute at 60 ° C, with a temperature drop of 0.2 ° C per cycle starting at cycle 25

3rd phase: 1 cycle of 1 minute at 57 ° C, with signal acquisition

Example 5 - Method for the detection of mutations c.-124 C> T and c.-146 C> T located in the prompter of the hTERT gene in a urine sample

Patient urine samples were obtained from bladder cancer patients in clinical or monitoring (cystoscopy) programs for the prevention of tumor recurrence. The urine was centrifuged at a force exceeding 1000 times the gravitational force (1000 G) for a period of time exceeding 5 minutes to pellet the bladder epithelial cells present in the urine. DNA was extracted from the pelleted cells obtained using common methods known in the art such as QlAamp® available from Qiagen or similar methods. Between 1 and 500 ng of the DNA obtained were added to the compositions for amplification and detection of mutations prepared as described in examples 1,2 and 3.

The compositions for the detection of Example 3 were subjected to amplification and hybridization processes in real time PCR and digital PCR. The compositions were also subjected to simultaneous (multiplex) assays using in the same reaction probes for both mutations in analysis, which are the probes labeled with different fluorophores. Among the most common fluorophores, there are compounds known as Alexa Fluor FAM, TET, JOE, VIC, HEX, Cy3, ATTO 550, TAMRA, ROX, Cy5, Cy5.5.

To carry out the method in real time PCR, the ABI PRISM ® 7500 Fast machine marketed by Scientific ThermoFischer was used.

Amplification and hybridization conditions:

1st phase: 1 cycle of 10 minutes at 95 ° C

2nd phase: 45 cycles, comprising 30 seconds at 92 ° C, 1 minute at 60 ° C, with a temperature drop of 0.2 ° C per cycle starting at cycle 25

3rd phase: 1 cycle of 1 minute at 57 ° C, with signal acquisition

Using this method, the changes c.-124 C> T and c.-146 C> T in the promoter of the hTERT gene were detected in patients with both low and high-grade bladder cancer, as well as in patients with cancer of the recurrent bladder (figure 7).

In conclusion:

The specificity of the probes was verified, that is, it was evaluated whether the probes for the c.-124 C> T mutation (probes with SEQ ID NO: 3 and SEQ ID NO: 4) did not produce amplification of a heterozygous DNA sample. for the alteration c.-146 C> T (at Dn MUT 146) and vice versa (with the probes with SEQ ID NO: 5 and SEQ ID NO: 6) (Figures 3A and 3B). Then it was verified that there are no cross-detection or false detection phenomena between the probes referred to and the respective mutations.

In DNA samples derived from biological fluids, in which there are small numbers of mutated alleles in an environment dominated by "wild-type" alleles, the analytical sensitivity of the -124T and -146T probes at a final concentration of 400 nM in their The respective mutation detection capacity is maintained up to a maximum of 3.125% in the case of the -124T probe and 6.25% for the -146T probe (Figures 4a and b, respectively), which clearly shows the high efficiency of the probes for the detection of mutations under conditions of high dilution of "wild type" alleles and, therefore, the ultrasensitivity of the method of the present invention.

Under conditions that used 25% MUT DNA / TN DNA, it can be seen that, at low concentration of the probe for the "wild-type" allele (eg 250 nM), the use of a higher concentration of the probe to the alleles mutated -124T and -146T (400-800-1600 nM) results in a significant increase in the detection signal of the mutated alleles (panels A and B of Figure 5, respectively), clearly indicating that, by manipulating the concentration of the probes, it is possible to adjust the sensitivity of the method of the present invention, to detect the mutations in question.

The increase in the detection efficiency of the mutated alleles -124T and -146T, corresponded a significant increase in the sensitivity of the analytical method of the invention, going to minimum detection values of 1.56% in the case of the allele -124T and of 0.78% in the case of the -146T allele (Figures 6A and B, respectively), which clearly indicates that, by manipulating the concentration of the probes, it is possible to enhance the analytical sensitivity of the method of the invention to detect mutations in question, while maintaining its specificity.

SEQUENCE LIST

I - Primers

SEQ ID NO: 1:

5'-CCACGTGCGCAGCAGGAC-3 '

SEQ ID NO: 2:

5'-CCGTCCTGCCCCTTCACCTT-3 '

II - Specific probes for c.-124 C> T mutations in the prom otor of the hTERT gene

SEQ ID NO: 3:

Yakima Yellow-5'-AGGGCCCGGAGGGGGCT-3'-TAMRA Dye

SEQ ID NO: 4:

FAM-5'-AGGGCCCGGAAGGGGCT-3'-TAMRA

III - Specific probes for c.-146 C> T mutations in the prompter of the hTERT gene

SEQ ID NO: 5:

Yakima Yellow-5'-CGGGGACCCGGGAGGGGT-3'-TAMRA Dye

SEQ ID NO: 6:

FAM-5'-CGGGGACCCGGAAGGGGT-3'-TAMRA

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1. Hayflick, L. and P.S. Moorhead, The serial cultivation of human diploid cell strains. Exp Cell Res, 1961. 25: pp.

585-621.

2. Blackburn, E.H., Structure and function of telomeres. Nature, 1991. 350 (6319): pp. 569-73.

3. Greider, C.W., Telomerase is processive. Mol Cell Biol, 1991. 11 (9): pp. 4572-80.

4. Szostak, J.W. and E.H. Blackburn, Cloning yeast telomeres on linear plasmid vectors. Cell, 1982. 29 (1): pp. 245-55.

5. Gunes, C. and K.L. Rudolph, The role of telomeres in stem cells and cancer. Cell, 2013. 152 (3): pp. 390-3 6. Murnane, J.P., Telomere dysfunction and chromosome instability. Mutat Res, 2012. 730 (1-2): pp. 28-36.

7. Cesare, A.J. and R.R. Reddel, Alternative lengthening of telomeres: models, mechanisms and implications. Nat Rev Genet, 2010. 11 (5): pp. 319-30.

8. Kim, N.W., et al., Specific association of human telomerase activity with immortal cells and cancer. Science, 1994.

266 (5193): pp. 2011-5.

Claims (11)

1. Set of primers and probes for the detection of mutations c.-124 C> T and c.-146 C> T in the promoter of the hTERT gene characterized in that it comprises the primers with SEQ ID NO: 1 and SEQ ID NO: 2 and the probes with SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6. 2. Composition for DNA amplification and hybridization by PCR in an in vitro method for detecting mutations c.-124 C> T and c.-146 C> T in the promoter of the hTERT gene in a DNA sample characterized by It comprises the set of primers and probes according to claim 1 in addition to the PCR reagent solutions. Composition according to claim 2, characterized in that at least one probe with SEQ ID NO: 3 to SEQ ID NO: 6 is present in the composition in a final concentration of 400 nM to 1600 nM. Composition according to any one of claims 2 to 3, characterized in that at least one probe with SEQ ID NO: 3 to SEQ ID NO: 6 is present in the composition in a final concentration of 800 nM to 1600 nM. Composition according to any of claims 2 to 4, characterized in that the probes with SEQ ID NO: 3 and SEQ ID NO: 5 are present in the composition in the final concentration of 250 nM to 500 nM and that the probes with SEQ ID NO: 4 and SEQ ID NO: 6 are present in the composition in a final concentration of 800 nM to 1600 nM. Composition according to any of claims 2 to 5, characterized in that the probes with SEQ ID NO: 3 to SEQ ID NO: 6 are present in the composition in the following concentrations: i) from 250 nM to 500 nM of SEQ ID NO: 3, from 400 nM to 1600 nM of SEQ ID NO: 4, from 250 nM to 500 nM of SEQ ID NO: 5 and from 400 nM to 1600 nM of SEQ ID NO 6, or ii) from 250 nM to 500 nM of SEQ ID NO: 3, from 800 nM to 1600 nM of SEQ ID NO: 4, from 250 nM to 500 nM of SEQ ID NO: 5 and from 800 nM to 1600 nM of SEQ ID NO 6. 7. Composition for use in an in vitro method for detecting mutations c.-124 C> T and c.-146 C> T in the promoter of the hTERT gene in a subject, characterized in that it comprises at least one composition according to any of claims 2 to 6 in addition to a human DNA sample isolated from a human biological sample. Composition for use according to claim 7, characterized in that the DNA is present in the composition at a final concentration of less than 500 ng, less than 100 ng or less than 50 ng and more than 1 ng. 9. Kit for use in an in vitro method for detecting mutations c.-124 C> T and c.-146 C> T in the promoter of the hTERT gene in a subject, characterized in that it comprises at least one composition according to the Claims 2 to 6. Kit for use according to claim 9, characterized in that it further comprises at least one DNA sample containing the mutation c.-124 C> T and c.-146 C> T in the promoter of the hTERT gene. 11. In vitro method for the detection of mutations c.-124 C> T and c.-146 C> T in the promoter of the hTERT gene in a DNA sample characterized in that it comprises the following steps: a) Preparation of a composition for detection as described in claims 7 to 8, by promoting a contact of the DNA sample extracted from a biological sample, to be tested for the mutation to which reference is made, with a composition for amplification and hybridization by PCR as described in claims 2 to 6, b) amplification and hybridization of the DNA present in the sample by PCR reaction using the compositions according to claims 2 to 6, c) analysis of the amplification curves obtained in (b) and verification of the presence of exponential amplification of a fluorescence signal, which corresponds to the positivity for the presence of the c.-124 C> T mutation and the c mutation. -146 C> T in the promoter of the hTERT gene in the sample under analysis. A method according to claim 11, wherein the DNA is isolated from a selected biological sample of urine, plasma, cerebrospinal fluid or aspiration cytology of a human. Method according to any of claims 11 and 12, wherein the amount of DNA in the DNA sample from step (a) varies at a final concentration of less than 500 ng, less than 100 ng, or less than 50 ng and more. of 1 ng. Method according to any one of claims 11 to 13, wherein in step b) the amplification conditions are as follows: • 1st phase: 1 cycle of 10 minutes at 95 ° C • 2nd phase: 45 cycles, comprising 30 seconds at 92 ° C, 1 minute at 60SC, with a drop in temperature of 0.2 ° C per cycle starting at cycle 25 • 3rd phase: 1 cycle of 1 minute at 57 ° C, with signal acquisition.