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WO2017168031A1 - Utilisation de tcfl5/cha comme nouveau marqueur poue le pronostic et/ou le diagnosic différentiel des leucémies lymphoblastiques aiguës - Google Patents

Utilisation de tcfl5/cha comme nouveau marqueur poue le pronostic et/ou le diagnosic différentiel des leucémies lymphoblastiques aiguës Download PDF

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WO2017168031A1
WO2017168031A1 PCT/ES2017/070201 ES2017070201W WO2017168031A1 WO 2017168031 A1 WO2017168031 A1 WO 2017168031A1 ES 2017070201 W ES2017070201 W ES 2017070201W WO 2017168031 A1 WO2017168031 A1 WO 2017168031A1
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seq
tcfl5
cha
isoforms
patients
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Manuel Fresno Escudero
Núria GIRONÈS PUJOL
Manuel RAMÍREZ ORELLANA
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Fundacion Para La Investigacion Biomedica Del Hospital Infantil Universitario Nino Jesus
Universidad Autonoma de Madrid
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Fundacion Para La Investigacion Biomedica Del Hospital Infantil Universitario Nino Jesus
Universidad Autonoma de Madrid
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    • C—CHEMISTRY; METALLURGY
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids

Definitions

  • the present invention belongs to the technical field of biotechnology and medicine.
  • it provides a prognostic method of acute lymphoblastic leukemia (ALL) based on the determination of the expression levels of each and every isoform of the TCFL5 gene, including CHA.
  • ALL acute lymphoblastic leukemia
  • kit for the implementation of said method It also refers to a method for differential diagnosis, classification or stratification of patients based on said prognosis; a method for treatment; and a method for monitoring disease progression and / or treatment efficacy in patients with ALL.
  • T-LLA and B-LLA The acute lymphoblastic leukemias T and B (T-LLA and B-LLA) are a group of malignant neoplasms characterized by bone marrow invasion of hematopoietic precursors (Inaba, H., M. Greaves, and CG Mullighan, Acute lymphoblastic leukaemia Lancet, 2013. 381 (9881): p. 1943-55). Immature leukemic cells usually invade the blood quite quickly. These cells can spread to other parts of the body, such as lymph nodes, liver, spleen, central nervous system (the brain and spinal cord) and testicles (in men).
  • ALL represents 12% of leukemias diagnosed in the US and Europe and 60% of all cases occur in people under 20 years. ALL is the most frequent leukemia in children (3 out of 4) and the cost of treatment is approximately 100,0006 per patient. The LLA exceeds 6,000 new cases a year in the US and a similar percentage in Europe. About 20% of patients have treatment failure, including patients who were initially considered low risk. Relapsing ALL can be considered the fifth most common cancer in children and represents a significant percentage of annual deaths of children associated with cancer (Bhojwani, D. and CH Pul, Relapsed childhood acute lymphoblastic leukaemia. Lancet Oncol, 2013. 14 (6 ): p. e205-17).
  • T-ALL represents 10% -15% of pediatric ALL and 25% of adult ALL cases and remains an aggressive disease with 5-year disease-free survival between 20 and 50%. The prognosis is still poor in children and adults. Early relapses are common in T-ALL and are associated with a poor prognosis.
  • the basis of the current treatment is the hemopathological diagnosis and can be grouped into subtypes, based on chromosomal translocations and gene expression profiles (Inaba, H., et al., Acute lymphoblastic leukaemia. Lancet, 2013. 381 (9881): p .1943-55).
  • TCFL5 is a transcription factor of the basic helix-loop-helix family and one of the 14 genes that characterize TEL / AML1-positive leukemias (Gandemer V, Rio AG, from Tayrac M, et al. Five distinct biological! Processes and 14 differentially expressed genes characterize TEUAML1-positive leukemia BMC Genomics. 2007; 8: 385). It has also been identified as one of the 5 genetic factors associated with vincristine resistance in ALL (Silveira VS, et al. Gene expression pattern contributing to prognostic factors in childhood acute lymphoblastic leukemia. Leuk Lymphoma. 2013; 54: 310-314 , Table 1). Silveira VS, et al. They also describe a significant association between higher levels of TCFL5 gene expression and bone marrow status and with a negative B-LLA ETV6 / RUNX1 phenotype.
  • TCFL5 has also been described as a NOTCH-1 target gene, an important mediator in the development of T-ALL leukemia (Weerkamp, F., et al., Identification of Notch target genes in uncommitted T-cell progenitors: No direct induction of a T-cell specific gene program Leukemia, 2006. 20 (11): p. 1967-77).
  • the CHA transcription factor was first cloned by the inventors, being described as an isoform of the TCFL5 gene (Rodr ⁇ guez, CI, N. Girones, and M. Fresno, Cha, a basic helix-loop-helix transcription factor involved in the regulation of upstream stimulatory factor activity J Biol Chem, 2003. 278 (44): p. 43135-45).
  • the stratification of ALL patients allows the intensity of the therapy to be adapted to the risk of relapse of the patient, thus contributing to improving the survival rate of the patients and / or preventing relapses.
  • Gene expression profiles can provide a good tool for subclassification and therapeutic stratification of patients (Zhou, Y., et al., Advances in the molecular pathobiology of B-lymphoblastic leukemia. Hum Pathol 2012. 43: 1347-1362 ; Mullighan, CG, Molecular genetics of B-precursor acute lymphoblastic leukemia. J Clin Invest 2012. 122: 3407-3415; Mullighan, CG, Genomic profiling of B- progenitor acute lymphoblastic leukemia.
  • genetic markers associated with high risk were identified in B-ALL patients and their use for diagnosis, prognosis and / or in determining the efficacy of treatment. More specifically the methods described comprise the determination of the expression levels of genes selected from the following group: CENTG2 (ArfGAP with GTPase domain, ankyrin repeat and PH domain 1); PTPRM (protein tyrosine phosphatase, receptor type, M); STAPI (signal transducing adapter family member 1); CCNJ (cyclin J); PCDH17 (procadherin 17); MCAM (melanoma cell adhesion molecule); CAPN3 (calpain 3); CABLESI (Cdk5 and Abl enzyme substrate 1); GPR155 (G protein-coupled receptor 155); MUC4 (mucin 4); GPRI 10 (G protein-coupled receptor 1 10); IGJ (immunoglobulin J polypeptide); NRXN3 (neurexin 3); CD99 (CD99 molecule);
  • WO2015 / 092755 reports a method for the diagnosis or prognosis of acute high hyperdiploidy lymphoblastic leukemia (HeH-ALL) by simultaneous determination of the presence / absence of chromosomes 6, 18 and 21 using iFISH technology.
  • WO2008 / 019872 refers to a method for the diagnosis of acute lymphoblastic leukemia in children by determining the expression levels of at least 14 genes of those described in Tables 1-4 or 5-16, especially those shown in Figure 3 of said publication: DNTT, SBGR101, DEFA3, CAMP, FCER2, DEFA4, BPI, PGLYRP1, LTF, SBGR2, NM_003526, CEACAM8, RNASE3, and ELA2.
  • the inventors performed a bioinformatic analysis based on data from TCFL5 mRNA sequences published in the RNAseq RNA sequencing databases (NCBI, Ensemble, Vega and EC gene) in which the exon junctions were analyzed, and established consensus messengers expressed in human (see Figure 1 and Table I of Example 1).
  • the inventors established that the TCFL5 gene has 6 different major isoforms at the mRNA level: (1) TCFL5R, (2) TCFL5R4b, (3) TCFL5R7, (4) TCFL5R4b6 (5) TCFL5R6 and (6) CHA (Tcfl5R2b or R2b), increasing the number of isoforms with respect to the 2 isoforms previously described: TCFL5 and CHA.
  • TCFL5 when referring to the TCFL5 isoform, it refers to that which has E1, E2a, E3, E4 (E4a or E4b), E5 and E8; that is, it encompasses TCFL5R and TCFL5R4b.
  • CHA isoform
  • CHA uses an alternative promoter located between exon 1 and 2, presenting as initial exon E2b.
  • E2b E3, E4, E5 and E8.
  • TCFL5 the absence of E1 and by presenting E2b instead of E2a (the portion of the E2a sequence other than E2b has been indicated in italics in Figure 3).
  • exon 2b contains a 5'UTR and a coding region coincident with E2a so that at the protein level the sequence corresponding to E2 is identical in TCFL5 and CHA, and therefore CHA differs only by the absence of the sequence amino acid corresponding to E1.
  • TCFL5R2b TCFL5R2b
  • TCFL5R2b TCFL5R2b
  • Fig. 2 and 7B mRNA level
  • Fig. 7A protein
  • TCFL5 as a prognostic marker and / or differential diagnosis in ALL will depend on whether the expression levels of the TCFL5 isoform are determined, on the CHA isoform (TCFL5R2b ) or both.
  • Fig. 6C and Table VI of Example 5 The inventors established an association between low levels of TCFL5 / CHA expression and a high-risk ALL prognosis (Fig. 6C and Table VI of Example 5). In particular, they observed that the expression of mRNA of TCFL5 / CHA at the time of diagnosis is much lower in high-risk ALL than in non-high-risk (medium or low) ALL (Table VII of Example 5). In Fig. 6A (Table IV of Example 5) it is shown that at the time of diagnosis the expression of TCFL5 / CHA in patients with ALL-T is less than in patients with ALL-B, although the difference is not statistically significant.
  • the inventors determined that there is a correlation between the levels of expression of TCFL5 / CHA in patients with ALL and the progression of the disease and / or efficacy of the treatment. More specifically, they observed that the expression of TCFL5 / CHA mRNA in ALL is significantly lower in relapse than in diagnosis (Fig. 6B and Table V of Example 5), said association is especially observed in B-ALL samples (Table VIII of Example 5).
  • the present invention relates in a first aspect to a method for the prognosis and / or differential diagnosis of patients with acute lymphoblastic leukemia (ALL), which comprises the determination of the expression levels of the TCFL5 gene where said determination includes the quantification of the CHA isoform.
  • ALL acute lymphoblastic leukemia
  • it refers to a method for the prognosis and / or differential diagnosis of patients with acute lymphoblastic leukemia (ALL) comprising the following stages:
  • TCFL5 / CHA expression levels refers to the expression levels of TCFL5 and CHA together.
  • the present invention relates to the in vitro use of the expression levels of the TCFL5 and CHA isoforms of the TCFL5 gene determined, preferably simultaneously, in an isolated biological sample of a patient with acute lymphoblastic leukemia (ALL) for the prognosis and / or differential diagnosis of patients with ALL, where a reduction in the values of TCFL5 / CHA in the patient sample with respect to reference values is indicative of high-risk ALL.
  • ALL acute lymphoblastic leukemia
  • the present invention also relates to a method for monitoring disease progression and / or efficacy of treatment in patients with ALL comprising the following stages:
  • the present invention relates to the in vitro use of the expression levels of the TCFL5 and CHA isoforms of the TCFL5 gene determined, preferably simultaneously, in an isolated biological sample of a patient with acute lymphoblastic leukemia (ALL) for the monitoring of disease progression and / or efficacy of treatment in patients with ALL, where a reduction in the values of the patient sample with respect to reference values is indicative of relapse.
  • ALL acute lymphoblastic leukemia
  • the invention also refers to a method for determining the most suitable treatment for an ALL patient comprising the classification of said patient according to the method for the prognosis and / or differential diagnosis of the first aspect of the invention.
  • the present invention relates to a method for the treatment of ALL patients where said treatment is determined based on the classification of said patient according to the method for the prognosis and / or differential diagnosis of the first aspect of the invention.
  • the invention refers to a kit for the prognosis, differential diagnosis and / or monitoring of patients with acute lymphoblastic leukemia (ALL) comprising:
  • a primer and / or probe comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 5 to SEQ ID NO: 76, and sequences identical to any of them at least 75%; Y
  • the invention also relates to the use of a kit for the prognosis, differential diagnosis and / or monitoring of patients with acute lymphoblastic leukemia (ALL) in a method according to the first aspect of the invention, wherein said kit comprises: a. a reagent to determine, preferably simultaneously, the expression levels of the TCFL5 and CHA isoforms of the TCFL5 gene; Y
  • the invention is related to a polynucleotide consisting of a nucleotide sequence selected from the group consisting of SEQ ID NO: 5 to SEQ ID NO: 76, and sequences identical to any of them at least 75%.
  • Fig. 1 Transcribed mRNA consensus majority of the TCFL5 gene. They were determined by analysis of exon junctions on already published databases of RNAseq sequences of the TCFL5 gene.
  • Fig. 2 TCFL5 mRNA transcripts corresponding to exon E1 vs E2 encoders present in different cells / tissues. They were determined by analysis of the already published RNAseq databases referring to the TCFL5 gene and are represented by color codes (darker higher frequency), with respect to the 2 exons. A high expression of coding E2 is observed in cells and tissues where the expression of E1 is low, indicating a differential expression of CHA with respect to the other isoforms of the TCFL5 gene (they present E1).
  • Fig. 3 Sequence of TCFL5 RNAm (SEQ ID NO: 1). The 5'UTR and 3'UTR regions are indicated. Likewise, the sequence corresponding to the coding region in which the different exons E1, E2, E3, E4, E5 and E8 are indicated with alternating colors (gray and black) is specified. In this sequence, exon 2 is E2a, the part of exon 2a used by TCFL5 but not by CHA is represented in italics and the CHA initiator ATG within exon 2 has been highlighted in bold (the untranslated part of this exon for CHA is different and represents exon 2b, not shown). The common region for both isoforms is shown shaded in gray. Likewise, some of the sequences used for the design of primers where the forward primer joins E5 and the reverse to E8 have been framed.
  • Fig. 4 Alignment of the coding sequences of TCFL5 and CHA (mRNA).
  • Fig. 5 Expression of TCFL5 and CHA in Jurkat cells: effects of DAPT (Notchl inhibitor) on the levels of TCFL5 / CHA expression at the protein level (Western Blot).
  • DAPT Notchl inhibitor
  • Fig. 6 Expression of TCFL5 / CHA in ALL samples obtained from patients: The expression levels of TCFL5 / CHA at the mRNA level are shown individually for each of the study groups. The graphs compare the values obtained in samples (A) of B-ALL vs T-ALL, (B) collected at the time of diagnosis vs. relapses and (C) classified according to the degree of low, medium or high risk of according to the criteria specified in materials and methods.
  • Fig. 7 Expression of TCFL5 / CHA in ALL leukemia samples obtained from patients at the time of diagnosis (LLA012, LL014) and relapse (LLA751, LLA783).
  • A Expression of CHA and TCFL5 proteins by Western Blot;
  • B Quantification of the mRNA of TCFL5 / CHA by qRT-PCR.
  • monitoring or monitoring of disease progression refers to determining the evolution of the disease, for example determining whether there is a relapse.
  • efficacy of a treatment refers to the degree to which a treatment achieves the desired or intended result, for example, the ability of a drug to achieve the desired effect.
  • treatment encompasses both a prophylactic and therapeutic treatment.
  • therapeutic treatment refers to one whose objective is to move from a disease or pathological state to a state of health.
  • prophylactic treatment refers to the prevention of a pathological state.
  • terapéuticaally effective amount refers to an amount that is effective, after the administration of a single or multiple dose to a subject (eg, a human patient) in prophylactic treatment or therapeutic of a disease, disorder or pathological condition.
  • probe refers to synthetically or biologically produced nucleic acids, between 10 and 285 nucleotides in length that contain specific nucleotide sequences that allow specific and preferred hybridization under predetermined conditions to the sequences. of target nucleic acid, and optionally have been modified for detection or to enhance assay performance.
  • a minimum of ten nucleotides is necessary to obtain statistically specificity and to form stable hybridization products, and a maximum of 285 nucleotides generally represents an upper limit for the length at which the reaction parameters can be easily adjusted to determine erroneously paired sequences and preferential hybridization.
  • the probes may contain certain constituent elements that contribute to their correct or optimal operation under certain test conditions.
  • the probes can be modified to improve their resistance to degradation by nucleases, to carry out the detection of ligands (for example, fluorescein marking) or to facilitate their capture on a solid support (for example, polyA tail ).
  • ligands for example, fluorescein marking
  • a solid support for example, polyA tail
  • the term "primers” as used herein refers to oligonucleotides or probes that can be used in an amplification procedure, such as a polymerase chain reaction (“PCR”), to amplify a nucleotide sequence. Primers are designed based on the polynucleotide sequence of a particular target sequence, for example, a specific mRNA sequence. The design and validation of primers and probes is well known in the art.
  • hybridization refers to a process whereby, under predetermined reaction conditions, two partially, substantially or completely complementary strands of nucleic acid are allowed to come into contact antiparallel to form a double-stranded nucleic acid with specific and stable hydrogen bonds, following explicit rules that make the nucleic acid bases can match each other.
  • partially complementary refers to a nucleotide sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary to a reference nucleotide sequence.
  • substantially complementary refers to a nucleotide sequence that is at least about 95%, 96%, 97%, 98%, or 99% complementary to a reference nucleotide sequence.
  • substantially hybridization means that the amount of hybridization observed will be such that someone who observes the results considers the positive result with respect to hybridization data in positive and negative controls. Data that are considered “background noise” are not substantial hybridization.
  • restrictive hybridization conditions means from about 35 ° C to 65 ° C in an approximately 0.9 molar NaCl saline solution.
  • the restriction can also be governed by such reaction parameters as the concentration and type of ionic species present in the hybridization solution, the types and concentrations of agents Denaturants present, and hybridization temperature. In general, as hybridization conditions become more restrictive, longer probes are preferred if stable hybrids are to be formed. As a rule, the restriction of the conditions under which hybridization will take place will determine certain characteristics of the preferred probes to be used.
  • antibody includes monoclonal and polyclonal antibodies, as well as recombinant antibodies.
  • recombinant antibody refers to an antibody produced or expressed using a recombinant expression vector, wherein the expression vector comprises a nucleic acid encoding the recombinant antibody, such that the introduction of the expression vector in an appropriate host cell results in the production or expression of the recombinant antibody.
  • the recombinant antibodies can be chimeric or humanized antibodies, mono- or multi-specific antibodies.
  • antibody also refers to fragments and derivatives of all of the foregoing, and may further comprise any variant thereof that retain the ability to specifically bind an epitope.
  • Antibodies may include, but are not limited to monoclonal antibodies (mAbs), single domain antibodies (sdAb), single chain antibodies (scFv), Fab fragments, F (ab ') 2, Fv fragments (sdFv) disulfides, anti - idiotype (anti-ld antibodies), intra-bodies, synthetic antibodies, and epitope binding fragments of any of the foregoing.
  • mAbs monoclonal antibodies
  • sdAb single domain antibodies
  • scFv single chain antibodies
  • Fab fragments fragments
  • F (ab ') 2 Fv fragments (sdFv) disulfides
  • anti-ld antibodies anti-idiotype antibodies
  • kit indicates a set of reagents and adjuvants required for an analysis. Although a kit consists of most cases of several units, the various elements of analysis presented in a single unit, which should be considered as kits, may also be available.
  • ALL acute lymphoblastic leukemia
  • the invention relates to a method for the prognosis and / or differential diagnosis of patients with acute lymphoblastic leukemia (ALL) comprising the determination of the levels of expression of the TCFL5 gene where said determination includes the quantification of the isoform CHA.
  • ALL acute lymphoblastic leukemia
  • ALL acute lymphoblastic leukemia
  • the method according to the first aspect of the invention allows differential diagnosis, classification or stratification of ALL patients based on prognosis e.g., high-risk ALL.
  • the invention relates to a method for the differential diagnosis of patients with acute lymphoblastic leukemia (ALL) which comprises the determination of the levels of expression of the TCFL5 gene where said determination includes the quantification of the CHA isoform.
  • ALL acute lymphoblastic leukemia
  • ALL acute lymphoblastic leukemia
  • simultaneous determination refers to the joint determination and by a single reaction, for example by the use of primers that amplify the common region to both isoforms (mRNA) and / or the use of affinity reagents that specifically bind to the polypeptides encoded by them.
  • the TCFL5 gene (NCBI Gene ID: 10732) has been described in humans and encodes a basic helix-loop-helix transcription factor. It is located on chromosome 20 (20q13.33) and has 8 exons (E1-E8) that undergo alternative splicing giving rise to 6 majority forms according to bionformatic analyzes carried out by the inventors of RNA sequencing databases "RNAseq" (NCBI, Ensemble, Vega and EC gene).
  • Such isoforms of the TCFL5 gene are: (1) TCFL5R, (2) TCFL5R4b, (3) TCFL5R7, (4) TCFL5R4b6 (5) TCFL5R6 and (6) CHA (Tcfl5R2b or R2b), increasing the number of isoforms with respect to the 2 isoforms previously described: Tcfl5R and CHA (Tcfl5R2b).
  • Tcfl5R2b TCFL5R
  • CHA Tcfl5R2b
  • sequence of the transcription variant or isoform CHA is identified in the present invention as SEQ ID NO: 2 (NCBI: AJ271337.1).
  • polypeptides encoded by the TCFL5 and CHA (mRNA) isoforms correspond to the amino acid sequences SEQ ID NO: 3 and SEQ ID NO: 4, respectively.
  • the determination of the TCFL5 and CHA isoforms according to the method of the invention is preferably carried out with reagents that detect both isoforms of the TCFL5 gene (at the mRNA or protein level) simultaneously, more preferably the detection and / or quantification of said Isoforms are specific for TCFL5 and CHA, not detecting and / or quantifying other isoforms of the TCFL5 gene.
  • the expression levels of the TCFL5 and CHA isoforms are determined at the level of messenger RNA (mRNA).
  • Molecular biology methods for quantifying target nucleic acid sequences are well known in the art. These procedures include, but are not limited to endpoint PCR, competitive PCR, reverse transcriptase associated with PCR (RT-PCR), quantitative PCR (qPCR), reverse transcriptase associated with qPCR (RT-qPCR), PCR-pyrosequencing, PCR -ELISA, DNA microarrays, mass spectrometry, in situ hybridization assays such as dot blotting or fluorescence in situ hybridization assay (FISH), branched DNA (bDNA; Nolte, Adv. Clin.
  • Such methods may also include pre-conversion of mRNA into cDNA through reaction with a reverse transcriptase (RT), for example the PCR reaction is usually preceded by the conversion of mRNA into cDNA and is referred to as RT-PCR.
  • RT reverse transcriptase
  • Primers and / or probes in general, react by offering a direct and linear response to increasing amounts of target nucleic acid sequences. Thanks to the comparison with appropriate standards, the amount of a nucleic acid sequence given in a sample can be easily quantified.
  • said molecular procedure for gene quantification is selected from the group consisting of quantitative polymerase chain reaction (qPCR), PCR-pyrosequencing, fluorescence in situ hybridization (FISH), DNA microarrays and PCR-ELISA.
  • a preferred quantification procedure is FISH, which combines the hybridization of probes with fluorescent optical microscopy, confocal laser microscopy or flow cytometry for the direct quantification of individual target sequences.
  • qPCR quantitative PCR
  • the qPCR or real-time PCR is well known by an expert in the field.
  • Different instruments for carrying out said reaction are marketed, such as ABI Prism 7700 SDS, GeneAmp 5700 SDS, ABI Prism 7900 HT SDS of Applied Biosystems; iCycler iQ from Bio-Rad; Cepheid Smart Cycler; Rotor-Gene from Corbett Research; LightCycler by Roche Molecular Biochemicals and Mx4000 Multiplex by Stratagene.
  • the qPCR procedure allows the exact quantification of the PCR product in real time by measuring the accumulation of the PCR product very early in the exponential phase of the reaction, thus reducing the bias in the quantification linked to the efficiency of the PCR amplification that is produces in endpoint PCR.
  • Real-time PCR is well known in the art and therefore, is not described in detail herein.
  • An overview of the technology and protocols for qPCR are available, for example, from the providers mentioned above, for example, http://www.sigmaaldrich.com/technical- documents / protocols / biology / sybr-green-qpcr.
  • biochemical detection refers to a procedure for reporting the amplification of the specific PCR product in real-time PCR. These biochemical detection are classified into two main groups.
  • the first group comprises double stranded DNA interleaving molecules: such as SYBR Green I and EvaGreen; and the second group includes oligonucleotides typically labeled with a fluorophore.
  • the latter has been divided into three subgroups: (i) primers-probes (Scorpions, Amplifluor®, LUX TM, Cyclicons, Angler®); (ii) hydrolysis (TaqMan, MGB-TaqMan, Snake assay) and hybridization (Hybprobe or FRET, Molecular Beacons, HyBeacon TM, MGB-Pleiades, MGB-Eclipse, ResonSense®, Yin-Yang or displacing); and (iii) nucleic acid analogs (PNA, LNA®, ZNA TM, unnatural bases: Plexor TM primer, Tiny-Molecular Beacon), see E. Navarro eta I.. Cl ⁇ nica Chimica Acta, Volume 439,
  • said probes are double-labeled oligonucleotides, such as hydrolysis probes or molecular beacons.
  • the 5 'end of the oligonucleotide is typically labeled with a fluorescent indicator (reporter) molecule such as FAM, TET or JOE while the 3' end is labeled with a quencher (quencher) molecule, such as TAM or BHQ1.
  • the probe sequence is specific for a region of interest in the amplified target molecule.
  • said probe is a hydrolysis probe that is designed so that the length of the sequence places the fluorophore 5 'and the deactivating molecule 3' in close proximity sufficiently narrow to suppress fluorescence.
  • indicator and extinguishing molecules for use in qPCR probes are well known in the art. These are available, for example, from https://www.eurofinsgenomics.eu/en/dna-rna-oligonucleotides/optimised-application-oligos/qpcr- probes.aspx.
  • probes and / or primers are used to quantify nucleotide sequences.
  • the term "a primer and / or probe” specifically includes “primers and / or probes", encompassing for example a primer, a probe, a primer and a probe, a pair of primers, and a pair of primers and a probe. Both terms are used interchangeably in the present invention.
  • the probes and / or primers used in the method of the invention specifically hybridize with SEQ ID NO: 1 and / or SEQ ID NO: 2.
  • Such probes and / or primers are partially complementary, preferably substantially or completely complementary to SEQ ID NO: 1 and / or SEQ ID NO: 2 or fragments thereof as described herein.
  • a probe and / or primer is a polynucleotide sequence of between 10 and 30 nucleotides, more preferably between 15 and 26 nucleotides, even more preferably between 18 and 22 nucleotides, and still much more preferably of about 20 nucleotides.
  • said primers and / or probes have been modified for detection or to enhance assay performance.
  • said primers and / or probes comprise or consist of a sequence selected from the group consisting of SEQ ID NO: 5 to SEQ ID NO: 76 (see Table III), and sequences identical to any of them in at least 75%.
  • said primers and / or probes comprise or consist of a sequence selected from the group consisting of SEQ ID NO: 5 to SEQ ID NO: 76 (see Table III).
  • said primers and / or probes are specific for the quantification / amplification of a region between exons 3 and 8 of TCFL5R (SEQ ID NO: 1) allowing to quantify specifically and simultaneously the expression of TCFL5 (independently that exon 4 presents the variant E4a or E4b) and CHA (TCFL5R2b).
  • the expression levels of the TCFL5 / CHA isoforms at the mRNA level are determined by quantifying a sequence between the first nucleotide of E3 and the last nucleotide of E8.
  • said primers and / or probes specifically hybridize with sequences comprised in any of exons E3, E4, E5 and / or E8, including when both primers hybridize to the same exon and any combination thereof.
  • said primers and / or probes comprise or consist of a sequence selected from the group consisting of SEQ ID NO: 5 to SEQ ID NO: 42; SEQ ID NO: 45 to SEQ ID NO: 62 and SEQ ID NO: 71 to SEQ ID NO: 74 (see Table III), and sequences identical to any of them at least 75%. More preferably, said primers and / or probes comprise or consist of a pair of primers selected from the group consisting of SEQ ID NO: 5 to SEQ ID NO: 42; SEQ ID NO: 45 to SEQ ID NO: 62 and SEQ ID NO: 71 to SEQ ID NO: 74 (see Table III).
  • said primers and / or probes specifically hybridize with sequences comprised in any of exons E3, E4 and / or E5 (amplifying a region common to all isoforms), including when both primers hybridize to the same exon and any combination thereof.
  • said primers and / or probes comprise or consist of a sequence selected from the group consisting of SEQ ID NO: 5 to SEQ ID NO: 42 and SEQ ID NO: 71 to SEQ ID NO: 72 (see Table III), and sequences identical to any of them in at least 75%.
  • said primers and / or probes comprise or consist of a pair of primers selected from the group consisting of SEQ ID NO: 5 to SEQ ID NO: 42 and SEQ ID NO: 71 to SEQ ID NO: 72 (see Table III ).
  • said primers and / or probes amplify the common and specific region of the TCFL5R and CHA isoforms, more specifically a primer and / or hybrid probe at E3 or E5 and the other at E8, or both at E8.
  • said primers and / or probes comprise or consist of a sequence selected from the group consisting of SEQ ID NO: 45 to SEQ ID NO: 62 and SEQ ID NO: 73 to SEQ ID NO: 74 (see Table III), and sequences identical to any of them in at least 75% More preferably, said primers and / or probes comprise or consist of a pair of primers selected from the group consisting of SEQ ID NO: 45 to SEQ ID NO: 62 and SEQ ID NO: 73 to SEQ ID NO: 74 (see Table III ).
  • one of the primers and / or probes hybrid specifically with a sequence comprised in E5 and the other in E8.
  • said primers and / or probes comprise or consist of a sequence selected from the group consisting of SEQ ID NO: 45 to SEQ ID NO: 54 and SEQ ID NO: 73 to SEQ ID NO: 74 (see Table III), and sequences identical to any of them in at least 75%.
  • said primers and / or probes comprise or consist of a pair of primers selected from the group consisting of SEQ ID NO: 45 to SEQ ID NO: 54 and SEQ ID NO: 73 to SEQ ID NO: 74 (see Table III ).
  • one of the primers and / or probes hybrid specifically with a sequence comprised in E3 and the other in E8.
  • said primers and / or probes comprise or consist of a sequence selected from the group consisting of SEQ ID NO: 55 to SEQ ID NO: 56 (see Table III), and sequences identical to any of them at least 75% .
  • said primers and / or probes comprise or consist of a pair of primers selected from the group consisting of SEQ ID NO: 55 to SEQ ID NO: 56 (see Table III).
  • both primers and / or probes hybridize to E8.
  • said primers and / or probes comprise or consist of a sequence selected from the group consisting of SEQ ID NO: 57 to SEQ ID NO: 62 (see Table III), and sequences identical to any of them at least 75% .
  • said primers and / or probes comprise or consist of a pair of primers selected from the group consisting of SEQ ID NO: 57 to SEQ ID NO: 62 (see Table III).
  • said primers and / or probes comprise or consist of a sequence selected from the group consisting of SEQ ID NO: 45 (First TCFL5 / CHA 01 Forward): GAGACTGACAAGGCCACAACT, SEQ ID NO: 46 (First TCFL5 / CHA 01 Reverse) : CCGCAAAATACGCTCTCAA, and sequences identical to any of them in at least 75%.
  • the oligonucleotide sequences with an identity of at least 75% mentioned in the present invention preferably have an identity of at least 80%, at least one 85%, at least 90%, at least 95%, more preferably, 96%, 97%, 98%, 99% or 100% with the respective reference sequences.
  • these sequences with an identity of at least 75% may have the same number of nucleotides, or have more or less nucleotides than the reference sequence.
  • identity refers to an exact correspondence of nucleotide with nucleotide or amino acid with amino acid of two polypeptide or polynucleotide sequences or, respectively. Two or more sequences (of polynucleotides or amino acids) can be compared by determining their "percent identity.”
  • the "percent identity” of two sequences, whether nucleic acid or amino acid sequences, is the number of exact matches between two aligned sequences divided by the length of the shortest sequence and multiplied by 100.
  • Suitable programs to calculate identity in percentage or similarity between sequences they are well known in the art, such as the NCBI BLAST program, used for example with the default parameters (http://www.ncbi.nlm.gov/cgi-bin/BLAST).
  • Quantification levels can be absolute or relative. In general, it is preferred that the expression levels be normalized. Normalization can be performed with respect to different measurements in the sample, such as by sample weight, quantification of human cells, quantification of total DNA, and / or quantification of the expression levels of a constitutive expression gene. These procedures are well known to one skilled in the art.
  • normalization is carried out with respect to the quantification of the expression levels of a constitutive expression gene.
  • genes that are constitutively expressed or “genes of constitutive expression” are understood to be those genes that have been described that are constantly transcribed. Examples of constitutively expressed genes are 2-myoglobulin, ubiquitin, 18S ribosomal protein, cyclophilin A, GAPDH, tyrosine activation protein 3- monooxygenase / tryptophan 5-monooxygenase (YWHAZ), beta-actin, ⁇ -2 - microglobulin or hypoxanthine-guanine phosphoribosyltransferase (HPRT).
  • the quantification of the TCFL5 / CHA expression levels is performed by RT-qPCR and comprises the normalization of the expression levels with respect to a constitutive expression gene.
  • the detection and / or quantification of the TCFL5 / CHA isoforms can also be carried out at the protein level.
  • Polypeptides encoded by the TCFL5 and CHA (mRNA) isoforms correspond to the amino acid sequences referred to as SEQ ID NO: 3 and SEQ ID NO: 4 in the present invention.
  • immunoassays There are various methods for quantifying peptides and proteins well known to a person skilled in the art, such as immunoassays. Various types of immunoassays are known to a person skilled in the art for the specific quantification of proteins of interest, either in solution or using a solid phase assay.
  • affinity reagents which may be specific receptors or ligands, for example antibodies, preferably labeled.
  • Western blotting or immunoblotting allows comparing abundances of proteins separated by an electrophoretic gel, eg. SDS-PAGE.
  • the proteins separated by gel electrophoresis are transferred onto a sheet of polymeric material (generally nitrocellulose, nylon, or polyvinylidene difluoride), where they are immobilized.
  • Target proteins are revealed by using a solution that contains a specific antibody.
  • the antibody can be conjugated directly with a radioactive, fluorescent or enzymatic marker (direct detection method) or a secondary antibody can be used that recognizes the primary antibody and therefore amplifies the signal (indirect detection method or sandwich type assay) .
  • a specific capture antibody for the target protein is immobilized on a polymeric or plastic surface and a solution containing the protein of interest (eg, serum or cell lysate) is added to the support. Finally, the sample is incubated on the support for a time to allow antigen-antibody complexes to form. Next, one or more washes are usually performed to remove the solution and the target protein is detected with a second antibody that recognizes a different protein epitope than the one recognized by the capture antibody. As in the case of Western Blot, this detection antibody can be directly labeled or can be recognized with a secondary antibody.
  • the protein of interest eg, serum or cell lysate
  • An immunoassay commonly used for protein quantification is the enzyme-linked immunosorbent assay (ELISA) in which the detection antibody carries an enzyme that converts a commonly colorless substrate into a colored compound or a non-fluorescent substrate into a fluorescent compound. Also, in other solid phase immunoassays, the antibody may be labeled with a radioactive or fluorescent isotope.
  • ELISA enzyme-linked immunosorbent assay
  • MS mass spectrometry
  • LC / MS liquid chromatography coupled to mass spectrometry
  • LC / MS tandem LC-MS / MS
  • the quantification of TCFL5 / CHA is performed by an immunoassay comprising the use of any antibody that detects both isoforms, such as HPA055223 ( Sigma) or SAB4500152 (Sigma), or antibodies specific to each of them.
  • the degree of variation (increase or decrease) in the expression values of TCFL5 / CHA in the patient sample with respect to the reference values can be, for example, at least 5%, 10%, 15%, 20% , 25%, 30%, 35%, 40%, 45%, 50%, 75%, 90%, 100%, 110%, 120%, 130%, 140%, 150% or more.
  • said variation is statistically significant.
  • statically significant refers to a p-value of less than 0.05, for example, a p-value of less than 0.025, a p-value of less than 0.01 or a value of p of less than 0.005, using an appropriate statistical test. A person skilled in the art will know how to define the most appropriate statistical tests.
  • a parametric model such as the student's t test or the ANOVA test is used; and when at least one of these two requirements is not achieved, then, in general, a non-parametric model such as the Mann-Whitney U test or the Kruskal-Wallis test is used.
  • Biological samples for use in the methods of the invention can be obtained from a variety of biological tissues or fluids, in particular blood, but bone marrow, lymph, cerebrospinal fluid, synovial fluid, and the like can also be used. . Such samples can be separated by centrifugation, decantation, density gradient separation, apheresis, affinity selection, FACS, etc.
  • the biological samples used in the method of the present invention are preferably samples of bone marrow, peripheral blood or cerebrospinal fluid. Said types of sample are routinely used in clinical practice and a person skilled in the art will know how to identify the appropriate means for obtaining and preserving them (Coustan-Smith E et al., Blood. 2002 Oct 1; 100 (7): 2399 -402; Martinez-Laperche C et al., American journal of hematology 88: 359-64).
  • the biological sample Once the biological sample is obtained, it can be used directly, frozen, or kept in an appropriate culture medium. Several culture media can be used to keep cells in culture. Samples can be obtained by any suitable procedure, such as blood collection, venous puncture, biopsy, or the like.
  • Mononuclear cells are generally isolated from said biological sample, by methods known in the state of the art. Mononuclear cells are typically isolated by density gradient centrifugation, for example with Ficoll® or Percoll®.
  • the determination of the expression levels of the TCFL5 and CHA isoforms is performed in a cell population that has been isolated from a biological sample of the patient by a method comprising a density gradient centrifugation.
  • the purification of a cell population of interest in a biological sample comprises a positive and / or negative selection based on the expression of characteristic cell surface markers.
  • the determination of the expression levels of the TCFL5 and CHA isoforms is performed in a population of ALL cells that has been isolated from a biological sample of the patient by a method that further comprises positive selection by the use of cell surface markers associated with leukemia.
  • LLA-B cells can be selected by removal of other MCs such as T lymphocytes (CD2 +, or CD3 + cells), and / or by the presence of specific surface markers, such as CD19 (B lymphocyte specific ), and / or CD10 (antigen associated with lymphoblastic leukemia).
  • the LLA-B cells are isolated by a selection comprising the expression of CD19, and / or CD10.
  • the population of ALL cells can be selected based on the expression of at least one cell surface marker.
  • Selection is usually carried out using proteins that specifically bind to one of said cell surface proteins, typically antibodies, and that can be linked to solid supports (e.g., particles or plastic surfaces) or can be conjugated with tidal molecules (for example, fluorochrome) that can be detected, for example, by flow cytometry.
  • proteins that specifically bind to one of said cell surface proteins typically antibodies, and that can be linked to solid supports (e.g., particles or plastic surfaces) or can be conjugated with tidal molecules (for example, fluorochrome) that can be detected, for example, by flow cytometry.
  • the patient's biological sample and the reference biological sample are of the same type, that is, they have the same biological origin and have been isolated using the same procedures.
  • Said biological samples can be taken around the time of diagnosis, before, during or after treatment, preferably they are taken around the time of diagnosis.
  • the reference values may be the expression levels determined for the expression product in one or more reference samples (for example, mean values +/- sme) or predetermined values.
  • said reference value is referred to as cut-off or threshold value.
  • a variety of statistical and mathematical methods to establish the threshold or cut-off value are known in the state of the art.
  • a threshold or cut-off expression value for a particular biomarker can be selected, for example, using a Receiver Operating Characteristic (ROC) analysis.
  • ROC Receiver Operating Characteristic
  • cut-off value can be varied, for example, by moving it along the ROC chart, to obtain different sensitivity or specificity values and therefore affecting the overall performance of the assay.
  • the best cut-off point refers to the value obtained from the ROC chart for a biomarker that produces the best sensitivity and specificity. Sensitivity and specificity values are calculated over the range of thresholds (cut-offs).
  • the threshold or cut-off values can be selected so that the sensitivity and / or specificity values are at least about 70%, and can be, for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 100% in at least 60% of the study population, or in at least 65%, 70%, 75% or 80% of the study population.
  • the reference or control samples for obtaining the reference values correspond to one or more samples of lymphocytes isolated from healthy donors (eg, peripheral blood or bone marrow) or to established lymphocyte lines B (Raji, ATCC® CCL-86 TM) or human T (Jurkat, Clone E6-1 (ATCC® TIB-152 TM)
  • the reference samples for obtaining the reference or control values correspond to one or more cell samples of patients with ALL or to established cell lines of ALL
  • the cell lines and / or cell samples of donors (healthy or patient) used have been previously characterized, for example, according to their phenotype (eg, LLA-B or LLA-T), immunophenotype (a standard panel includes: CD10, CD19, CD20, CD34, CD38 and CD45), genetic markers of LLA-B (eg PAX5, IKZF1, or EBF1) and cytogenetics (eg, presence of translocations MLL-AF4, ETV6-RUNX1 or BCR
  • LLA-T eg HOX11 L2, LYL1 plus LM02, TAL1 plus LM01 or LM02, HOX1 1 , and MLL-EN, NOTCH1, c-MYC, etc.
  • Characteristic cell lines of LLA-B include but are not limited to CCL-120 (ATCC).
  • characteristic cell lines of LLA-T include but are not limited to CCL-1 19, CCL-120.1, CRL-1552, CRL-2264, CRL-2265, PTS-CCL-119, CRM-CCL-1 19, CRM -CCL- 1 19D, CRL-1 1386, TI B-195 (all of ATCC).
  • said patient samples have been associated with patients at medium and / or low risk both LLA-B and LLA-T.
  • the method of the invention is used for the prognosis and / or differential diagnosis of patients with ALL-B or ALL-T.
  • patients with ALL-B Preferably, in patients with ALL-B.
  • said patients are children.
  • the infant population in ALL is typically defined as those patients who are under 20 years old.
  • said patients are between 0 and 15 years old, and even between 0-12 months.
  • said patients are between 1 and 19 years old.
  • the classification of the disease can be, for example, a preference classification based on the risk of relapse (remission vs. therapeutic failure); but that can also be based on the clinical characteristics of the patients, on the cytogenetic data; in the subtype of leukemia; the response to treatment and / or the etiology of the disease. Typically, those with the highest chance of relapse are defined as high-risk patients.
  • These criteria for the classification and / or stratification of patients are well known by a person skilled in the art and are described for example in Ceppi F, et al., (2015) Risk factors for relapse in childhood acute lymphoblastic leukemia: prediction and prevention.
  • ALL patients are usually classified into one of the following risk groups:
  • STANDARD RISK or LOW RISK The patient must meet each and every one of the following criteria:
  • MEDIUM RISK Those patients who do not meet the criteria of Standard Risk or High Risk.
  • said method further comprises the detection or quantification of one or more Genetic markers described with prognostic value for ALL, preferably comprises determining the presence of one or more of the translocations selected from the group consisting of t (12; 21), t (1; 19), t (9; 22) and t (4; 11), Hypodiploidy ⁇ 44 chromosomes or DNA index ⁇ 0.81.
  • Said method may further comprise the determination of one or more of the biochemical and / or clinical parameters described above commonly used for stratification of ALL patients according to risk.
  • the method of the invention may further comprise the storage of the results obtained in a data storage device.
  • said Data storage device is a sheet of paper.
  • said data storage device is a computer readable medium.
  • a computer-readable medium may be any apparatus that may include, store, communicate, propagate or transport the results of the determination of the process of the invention.
  • the medium may be an electronic, magnetic, optical, electromagnetic, infrared or semiconductor system (or apparatus or device) or a propagation medium.
  • a further aspect of the invention relates to a computer-implemented method, in which the method is any of the methods described herein or any combination thereof.
  • any computer program capable of implementing any of the methods of the present invention or used to implement any of these methods or any combination thereof is also part of the present invention.
  • any device or apparatus comprising or carrying a computer program capable of carrying out, or for the application of any of the methods of the present invention or any combination thereof is also included as part of the present specification.
  • the present invention relates to the in vitro use of the expression levels of the TCFL5 and CHA isoforms of the TCFL5 gene determined, preferably simultaneously, in an isolated biological sample of a patient with acute lymphoblastic leukemia (ALL) for the prognosis and / or differential diagnosis of patients with ALL, where a reduction in the expression values of TCFL5 / CHA in the patient sample with respect to reference values is indicative of high-risk ALL.
  • ALL acute lymphoblastic leukemia
  • the invention also refers to a method for the personalization of the treatment or for the determination of the most appropriate treatment based on the patient's risk profile, wherein said method comprises the prognosis and / or differential diagnosis according to the risk according to the method of the first aspect of the invention.
  • the invention refers to a method for the treatment of ALL patients that comprises the administration of a therapeutically effective amount of a drug or combination of drugs where said treatment is determined based on the classification or stratification of said patient. depending on the risk according to the method of First aspect of the invention.
  • Such therapy is typically formed by the association of several drugs, including, among others, vincristine, prednisone and anthracyclines (daunorubicin or idarubicin or doxorubicin), L-asparaginase, and methotrexate, etc ((lnaba, H., M. Greaves, and CG Mullighan , Acute lymphoblastic leukaemia, Lancet, 2013. 381 (9881): p. 1943-55).
  • drugs including, among others, vincristine, prednisone and anthracyclines (daunorubicin or idarubicin or doxorubicin), L-asparaginase, and methotrexate, etc ((lnaba, H., M. Greaves, and CG Mullighan , Acute lymphoblastic leukaemia, Lancet, 2013. 381 (9881): p. 1943-55).
  • a greater intensity of treatment is characterized by a greater dosage and / or frequency of administrations with respect to the administration of the same drug or combination of drugs in a patient classified as medium or low risk.
  • a treatment of greater intensity may also consist of the administration of other drugs whose use is considered more aggressive, for example, being associated with a higher toxicity.
  • the present invention also relates to a method for monitoring disease progression and / or efficacy of treatment in patients with ALL comprising the determination of levels of expression of the TCFL5 gene where said determination includes quantification of the CHA isoform.
  • it refers to a method for monitoring disease progression and / or efficacy of treatment in patients with ALL comprising the following stages:
  • the sampling is done at the beginning of the treatment, throughout the treatment and / or once the treatment is finished, in a cyclic or timely manner.
  • the invention refers to the use in vitro of the expression levels of the TCFL5 and CHA isoforms of the TCFL5 gene determined, preferably simultaneously, in an isolated biological sample of a patient with acute lymphoblastic leukemia (ALL) for the monitoring of patients with ALL, where a reduction in values TCFL5 / CHA expression in the patient sample with reference values is indicative of relapse.
  • ALL acute lymphoblastic leukemia
  • the invention relates to a method of obtaining useful data for the prognosis, differential diagnosis and / or monitoring of patients with acute lymphoblastic leukemia (ALL) comprising: a. determine, preferably simultaneously, the expression levels of the TCFL5 and CHA isoforms of the TCFL5 gene in a biological sample isolated from said patient.
  • ALL acute lymphoblastic leukemia
  • the invention refers to a kit for the prognosis, differential diagnosis and / or monitoring of patients with ALL in a method according to the previous aspects of the invention, wherein said kit comprises: a. a reagent to determine the expression levels of the TCFL5 gene where said determination includes the quantification of the CHA isoform; Y
  • said kit comprises: a. a reagent to determine, preferably simultaneously, the expression levels of the TCFL5 and CHA isoforms of the TCFL5 gene; Y
  • kits for the prognosis, differential diagnosis and / or monitoring of patients with acute lymphoblastic leukemia comprising:
  • a primer and / or probe comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 5 to SEQ ID NO: 76, and sequences identical to any of them at least 75%; Y b. optionally, instructions for the use of said primer and / or probe for the determination of the expression levels of said isoforms in a biological sample isolated from said patient. More preferably, said primers and / or probes comprise or consist of a sequence selected from the group consisting of SEQ ID NO: 5 to SEQ ID NO: 42; SEQ ID NO: 45 to SEQ ID NO: 62 and SEQ ID NO: 71 to SEQ ID NO: 74, and sequences identical to any of them at least 75%.
  • said primers and / or probes comprise or consist of a sequence selected from the group consisting of SEQ ID NO: 45 to SEQ ID NO: 62 and SEQ ID NO: 73 to SEQ ID NO: 74, and sequences identical to any of them at least 75%.
  • kit for the prognosis, differential diagnosis and / or monitoring of patients with ALL comprising:
  • a pair of primers selected from the group consisting of the SEQ sequences
  • said primer and / or probe comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 45, SEQ ID NO: 46 and sequences identical to any of them at least 75%.
  • SEQ ID NO: 45 amino acid sequence
  • SEQ ID NO: 46 amino acid sequence sequences identical to any of them at least 75%.
  • Other preferred sequences have been described under the first aspect of the invention.
  • said kit further comprises reagents suitable for quantitative PCR quantification.
  • reagents suitable for quantitative PCR quantification includes a DNA polymerase, such as Taq DNA polymerase (for example, the hot-start Taq DNA polymerase), a suitable buffer (for example Tris-HCI, pH 8-9), magnesium (for example, MgCI 2 ) , deoxynucleotides (dNTPs) and optionally other reagents, such as gelatin and / or bovine albumin.
  • a DNA polymerase such as Taq DNA polymerase (for example, the hot-start Taq DNA polymerase)
  • a suitable buffer for example Tris-HCI, pH 8-9
  • magnesium for example, MgCI 2
  • dNTPs deoxynucleotides
  • optionally other reagents such as gelatin and / or bovine albumin.
  • said kit In addition to reagents for carrying out the PCR amplification reaction, said kit also usually carries reagents for the detection and quantification of the amplified products as described herein, for example double DNA intercalating fluorescent molecules. labeled chain or oligonucleotides (for example, fluorescent hydrolysis probes).
  • said kit further comprises reagents suitable for carrying out the mRNA transotranscription.
  • a reverse transcnptase such as the murine leukemia virus (MuLV RT) reverse transcnptase
  • a suitable buffer for example Tris-HCI, pH 8-9
  • magnesium eg, MgCI 2
  • dNTPs eg, RNase inhibitors
  • RNase inhibitors e.g, RNase inhibitors
  • said reagent for the determination, preferably simultaneously, of the expression levels of the TCFL5 and CHA isoforms of the TCFL5 gene is a polypeptide, preferably an antibody, capable of specifically binding to the TCFL5 and CHA isoforms to protein level
  • Said kit may comprise in a particular embodiment reagents for performing an immunohistochemical (ICH) assay, which typically comprises a secondary antibody conjugated with enzyme (for example conjugated to horseradish peroxidase) or alkaline phosphatase), an enzyme substrate and a dye, for example, hematoxylin.
  • ICH immunohistochemical
  • the invention also relates to the use of a kit for the prognosis, differential diagnosis and / or monitoring of patients with ALL in a method according to the above aspects of the invention, wherein said kit comprises: a. a reagent to determine the expression levels of the TCFL5 gene where said determination includes the quantification of the CHA isoform; Y
  • kit for the prognosis, differential diagnosis and / or monitoring of patients with ALL in a method according to the above aspects of the invention, wherein said kit comprises: to a reagent to determine, preferably simultaneously, the expression levels of the TCFL5 and CHA isoforms of the TCFL5 gene; Y
  • step a) for the determination of the expression levels of said isoforms in a biological sample isolated from said patient has been described herein.
  • Particular embodiments and preferred features of this aspect of the invention have been defined in previous aspects, in particular in the first aspect of the invention.
  • the invention relates to a nucleotide or polynucleotide molecule, preferably a probe and / or primer, which specifically hybridizes to SEQ ID NO: 1 and / or SEQ ID NO: 2.
  • Said probes and / or primers are partially complementary, preferably substantially or completely complementary to SEQ ID NO: 1 and / or SEQ ID NO: 2 or fragments thereof as described in other aspects of the invention.
  • the present invention refers to a composition comprising said polynucleotide and one or more excipients.
  • said sequence is selected from the group consisting of SEQ ID NO: 5 to SEQ ID NO: 76, and sequences identical to any of them at least 75%.
  • said primers and / or probes comprise or consist of a sequence selected from the group consisting of SEQ ID NO: 5 to SEQ ID NO: 42; SEQ ID NO: 45 to SEQ ID NO: 62 and SEQ ID NO: 71 to SEQ ID NO: 74, and sequences identical to any of them at least 75%.
  • said primers and / or probes comprise or consist of a sequence selected from the group consisting of SEQ ID NO: 45 to SEQ ID NO: 62 and SEQ ID NO: 73 to SEQ ID NO: 74, and sequences identical to any of them at least 75%.
  • said sequence is selected from the group consisting of SEQ ID NO: 45, SEQ ID NO: 46, and sequences identical to any of them at least 75%.
  • Other preferred sequences have been described under the first aspect of the invention.
  • the invention relates to the use of a nucleotide sequence that specifically hybridizes to SEQ ID NO: 1 and / or SEQ ID NO: 2 as a primer and / or probe in an in vitro method for prognosis, diagnosis and / or monitoring of patients with ALL according to the previous aspects of the invention.
  • said sequence is selected from the group consisting of SEQ ID NO: 5 to SEQ ID NO: 76, and sequences identical to any of them in at least 75%.
  • said primers and / or probes comprise or consist of a sequence selected from the group consisting of SEQ ID NO: 5 to SEQ ID NO: 42; SEQ ID NO: 45 to SEQ ID NO: 62 and SEQ ID NO: 71 to SEQ ID NO: 74, and sequences identical to any of them at least 75%.
  • said primers and / or probes comprise or consist of a sequence selected from the group consisting of SEQ ID NO: 45 to SEQ ID NO: 62 and SEQ ID NO: 73 to SEQ ID NO: 74, and sequences identical to any of them at least 75%.
  • said sequence is selected from the group consisting of SEQ ID NO: 45, SEQ ID NO: 46 and sequences identical to any of them at least 75%.
  • Other preferred sequences have been described under the first aspect of the invention.
  • the invention relates to a method for the determination in vitro, preferably simultaneously, of the expression levels of the TCFL5 and CHA isoforms of the TCFL5 gene in an isolated biological sample of a patient with acute lymphoblastic leukemia (ALL) ); where the expression levels of the TCFL5 and CHA isoforms are determined at the mRNA level by the use of a primer and / or probe selected from the group consisting of the sequences SEQ ID NO: 5 to SEQ ID NO: 76, and identical sequences to any of them at least 75%.
  • ALL acute lymphoblastic leukemia
  • said primers and / or probes comprise or consist of a sequence selected from the group consisting of SEQ ID NO: 5 to SEQ ID NO: 42; SEQ ID NO: 45 to SEQ ID NO: 62 and SEQ ID NO: 71 to SEQ ID NO: 74, and sequences identical to any of them at least 75%. Even more preferably, said primers and / or probes comprise or consist of a sequence selected from the group consisting of SEQ ID NO: 45 to SEQ ID NO: 62 and SEQ ID NO: 73 to SEQ ID NO: 74, and sequences identical to any of them at least 75%.
  • said primer and / or probe consists of a nucleotide sequence selected from the group consisting of SEQ ID NO: 45, SEQ ID NO: 46 and a sequence identical to any of them at least 75%.
  • SEQ ID NO: 45 SEQ ID NO: 45
  • SEQ ID NO: 46 SEQ ID NO: 46
  • sequence identical to any of them at least 75% Other preferred sequences have been described under the first aspect of the invention.
  • any embodiment analyzed in this specification can be implemented with respect to any method, kit, a polynucleotide, reagent or use of the invention, and vice versa.
  • those detailed features and particular embodiments relating to the first aspect of the invention may also be implemented with respect to other aspects of the invention.
  • the particular embodiments described herein are shown by way of illustration and not as limitations of the invention. The main characteristic features of the present invention can be used in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize, or will be able to determine using no more than routine experimentation, numerous equivalents to the specific procedures described herein. These equivalents are considered to be within the scope of the present invention and are contemplated by the claims.
  • the words “understand” (and any way of understanding, such as “understand” and “understand”), “have” (and any form of having, such as “have” and “ has “),” include “(and any way of including, such as” include “and” include ”) or” contain “(and any way of containing, such as” contains “and” contain ”) are inclusive or open and do not exclude elements or stages of the procedure not mentioned, additional.
  • the term “comprises” encompasses and specifically describes “consists essentially of” and “consists of”.
  • the term “consisting essentially of” limits the scope of a claim to the specified materials or stages and those that do not materially affect the basic (s) and novel (s) feature (s). s) of the claimed invention.
  • the term “consisting of” excludes any element, step or ingredient not specified in the claim except, for example, impurities usually associated with the element or limitation.
  • A, B, C or combinations thereof refers to all permutations and combinations of the enumerated points preceding the term.
  • A, B, C or combinations thereof is intended to include the minus one of: A, B, C, AB, AC, BC or ABC, and if the order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC or CAB.
  • combinations containing repetitions of one or more points or terms such as BBB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, and so on, are expressly included.
  • the person skilled in the art will understand that there is typically no limit on the number of points or terms in any combination, unless it is evident otherwise from the context.
  • approximation words such as, without limitation, "about”, “about”, “approximately” refer to a condition that, when modified as such, is understood to be not necessarily absolute or perfect It would be considered close enough for those skilled in the art to ensure the designation of the condition as present.
  • the extent to which the description can vary will depend on how large a change can be instituted and a person skilled in the art still recognizes that the modified characteristic trait still has the required characteristics and capabilities of the unmodified characteristic trait.
  • a numerical value in this document that is modified by an approximation word such as “approximately” may vary from the value set at ⁇ 1, 2, 3, 4, 5, 6, 7 , 10, 12 or 15%, or less, preferably represents the set value ( ⁇ 0%).
  • Bone marrow aspirate samples were obtained at the time of diagnosis or relapse of 60 children with ALL (ages 0-15 years) from the Ni ⁇ o Jes ⁇ s University Hospital in Madrid. 18 LLA-T and 42 LLA-B
  • Three risk groups were distinguished based on an evaluation of the data related to the patient's age and the results obtained in one or more of the following genetic and / or biochemical tests:
  • STANDARD RISK or LOW RISK The patient must meet each and every one of the following criteria:
  • MEDIUM RISK Those patients who do not meet the criteria of Standard Risk or High Risk. Western blot
  • the membrane was developed using the SuperSignal kit (Thermo Scientific) or the Immun-Star HRP kit (BIO_RAD) following the manufacturer's recommendations.
  • the extraction of mononucleated cells from patients was performed by density gradient centrifugation. Bone marrow samples from patients are diluted in half in PBS. In a separate tube, 5 ml of Ficoll (Ficoll® Paque Plus, Sigma-Aldrich) are prepared. The diluted bone marrow sample is placed on the ficoll slowly supporting the tip on the tube wall to generate two phases. It is centrifuged at 1500 rpm for 25 minutes with low deceleration at room temperature. We are left with the white phase that is where the mononucleated cells are found. Wash with PBS 1400 rpm for 7 minutes to remove traces of ficoll. After washing, the pellet is resuspended in fetal bovine serum (FBS) and cryopreserved in vials of a maximum of 12.5x10 6 cells.
  • FBS fetal bovine serum
  • RNA extraction was carried out with RNeasy Plus Mini Kit (Qiagen) following the manufacturer's instructions.
  • RNeasy Plus Mini Kit Qiagen
  • For the retrotranscription the TaqMan Reverse Transcription Reagents Kit (Applied Biosystem, Life Technologies) was used following the program: one cycle at 42 ° C for 45 minutes and one last cycle at 99 ° C for 3 minutes.
  • SYBR GREEN PCR Master Mix (Applied Biosystem, Life Technologies) was used following the program: one cycle 50 ° C for 2 min, 95 ° C for 10 minutes, 40 cycles at 95 ° C for 15 seconds and 58 ° C for 1 minutes, continuous melting curve.
  • TCFL5 / CHA_F (SEQ ID NO: 45): 5 'GAGACTGACAAGGCCACAACT 3'
  • TCFL5 / CHA_R (SEQ ID NO: 46): 5 'CCGCAAAATACGCTCTCAA 3'
  • RNA extraction and RT-qPCR (Protocol used in example 6)
  • RNA samples were stored in TRIzol (Life Technologies) at 70 ° C until use.
  • Total RNA extraction was performed with RNeasy Plus Mini Kit (Qiagen) following the manufacturer's recommendations.
  • Quantitative RT-qPCR The mRNA was retrotranscribed to complementary DNA (cDNA) and the genes were quantified with specific oligonucleotides (SEQ ID NO: 45 (TCFL5 / CHA 01 Forward) and SEQ ID NO: 46 (TCFL5 / CHA 01 reverse) of Table III) with the GoTaq 2-Step RT-PCR System kit (Promega) following the manufacturer's recommendations.
  • the values obtained for each gene were normalized with the House keeping HPRT (Hypoxanthine-guanine phosphoribosyltransferase) gene to obtain AC T , after which they were normalized with the value of the control sample to obtain ⁇ ⁇ .
  • the primers used to quantify HPRT expression were HPRT-Forward (SEQ ID NO: 79): CTGGAAAGAATGTCTTGATTGTGG; and HPRT-Reverse (SEQ ID NO: 80): CATCTTGGATTATACTGCCTGAC.
  • Example 1 Determination of consensus messengers and identification of new isoforms of TCFL5
  • Table I shows the consensus mRNAs or isoforms identified from the mRNA sequences published in the different databases.
  • Table I The access codes for each of the TCFL5 mRNAs described in databases and the association according to the bioinformatic analysis performed by the inventors are indicated.
  • Table II provides additional data regarding the variants or isoforms and the corresponding mRNA sequences.
  • H20C8515.3 features a longer 3'UTR.
  • R AB012124.1 begins 39 bases after skipping the 1st ATG, so it could be a partial messenger. In addition, it has a shorter 3'UTR.
  • Q9UL49-1 is the translation of this messenger.
  • AF070992.1 starts 170 bases after skipping the 1st ATG, so it could be a messenger
  • exon 4 has 3 bases less at its 5 'end (exon 4b).
  • exon 7 It presents an alternative final exon (called exon 7).
  • R7 XM_005260184.1, ENST00000217162, BC046933.1 and H20C8515.8 show differences in the length of their UTRs.
  • exon 6 It presents an alternative final exon (called exon 6) and uses alternative exon 4 (3 bases less in
  • BC065520.1 has some sequencing errors.
  • CHA uses an alternative initial exon (exon 2b) that contains a 5'UTR and a coding region
  • H20C8515.2 and H20C8515.5 have 3'UTRs longer.
  • the TCFL5 gene is composed of 8 exons (see Figure 1) with exons 3 (E3) and 5 (E5) common to all isoforms.
  • Exon 1 (E1) is present in 5 of the 6 isoforms, its expression missing only in the isoform called CHA (R2b).
  • the last exon may be represented by exon 8, in the case of the consensus isoform TCFL5 (TCFL5R) and the CHA isoform, by exon 7 or by exon 6.
  • the consensus isoform TCFL5 (TCFL5R) and the isoform that ends with exon 6, can present an exon 4 that lacks the first codons (R4b6).
  • the CHA isoform has an exon 2 different from the rest (E2b).
  • Example 2 Design of oligonucleotides for the detection of TCFL5 / CHA
  • primers were designed that quantify TCFL5, CHA independently and TCFL5 / CHA together.
  • TCFL5 / CHA primers were designed that amplify a region common to both isoforms, that is, the region between exons 3 and 8 (see Figs. 1 and 3).
  • E1 and E2 in the databases cited above was analyzed and a preferential expression of E1 was observed in brain, heart, liver, lung, kidney and testis, while E2 was preferentially expressed in the Jurkat T cell line , peripheral blood mononucleated cells (PBMNCs), monocytes and T and B cells. The results are shown in Fig. 2.
  • PBMNCs peripheral blood mononucleated cells
  • monocytes monocytes
  • T and B cells T and B cells.
  • Fig. 2 Example 4.- Determination of the expression levels of TCFL5 and CHA in cell lines treated with DAPT
  • TCFL5 The expression levels of the TCFL5, CHA and HSP90 isoforms (as a control) in Jurkat cells (Clone E6-1, ATCC® TIB-152 TM) were determined by Western Blot. The results in Fig. 5 show that a difference in expression is observed between the CHA and TCFL5 isoforms for different concentrations of DAPT.
  • Example 5 Comparison of TCFL5 / CHA mRNA expression levels in samples of ALL patients (ALL-B vs. ALL-T, diagnosis vs. relapse, high risk vs. medium vs. low, and non-high risk vs. high risk)
  • TCFL5 / CHA were determined by Real Time qPCR.
  • Example 6 Determination of TCFL5 / CHA mRNA in ALL patients (diagnosis vs. relapse)

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Abstract

La présente invention concerne une méthode de pronostic et/ou de diagnostic différentiel de patients présentant une leucémie lymphoblastique aiguë (LLA), comprenant les étapes suivantes: a) détermination simultanée des niveaux d'expression des isoformes TCFL5 et CHA du gène TCFL5 dans un échantillon biologique prélevé sur ledit patient ; et b) comparaison des niveaux d'expression de TCFL5/CHA dans l'échantillon du patient à des valeurs de référence, une réduction des valeurs de l'échantillon du patient par rapport aux valeurs de référence étant une indication d'une LLA à risque élevé, lesdits échantillons de référence correspondant de préférence à un ou plusieurs échantillons de patients atteints de LLA ou à des lignées établies de LLA. L'invention concerne également une trousse pour la mise en oeuvre de cette méthode. L'invention concerne de plus une méthode de diagnostic différentiel, de classification ou de stratification des patients sur la base dudit pronostic ; une méthode de traitement ; et une méthode de surveillance de la progression de la maladie et/ou de l'efficacité du traitement de patients atteints de LLA:
PCT/ES2017/070201 2016-04-01 2017-04-03 Utilisation de tcfl5/cha comme nouveau marqueur poue le pronostic et/ou le diagnosic différentiel des leucémies lymphoblastiques aiguës Ceased WO2017168031A1 (fr)

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Publication number Priority date Publication date Assignee Title
WO2003083140A2 (fr) * 2002-03-22 2003-10-09 St.Jude Children's Research Hospital, Inc. Classification et prevision du pronostic d'une leucemie aigue lymphoblastique par profilage de l'expression genique
EP2213738A2 (fr) * 2002-11-14 2010-08-04 Dharmacon, Inc. Molécules siRNA anti Bcl-2

Patent Citations (2)

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Publication number Priority date Publication date Assignee Title
WO2003083140A2 (fr) * 2002-03-22 2003-10-09 St.Jude Children's Research Hospital, Inc. Classification et prevision du pronostic d'une leucemie aigue lymphoblastique par profilage de l'expression genique
EP2213738A2 (fr) * 2002-11-14 2010-08-04 Dharmacon, Inc. Molécules siRNA anti Bcl-2

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Title
GANDEMER VIRGINIE ET AL.: "Five distinct biological processes and 14 differentially expressed genes characterize TEL/AML1-positive leukemia", BMC GENOMICS, vol. 8, no. 1, 23 October 2007 (2007-10-23), London, UK, pages 385, XP055427430, ISSN: 1471-2164 *
SILVEIRA VANESSA S ET AL.: "Gene expression pattern contributing to prognostic factors in childhood acute lymphoblastic leukemia", LEUKEMIA & LYMPHOMA ENGLAND, vol. 54, no. 2, 31 January 2013 (2013-01-31), pages 310 - 314, ISSN: 1029-2403 *

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