Biomarkers for survival prospects of AML patients

Abstract

This invention relates to a method for diagnosing the survival prospects of patients with acute myeloid leukemia (AML) in vitro or in vitro, comprising the step of detecting the expression of at least newly identified AML biomarker genes.

Description

Technical Field

[0001] This invention belongs to the field of diagnosis and treatment of acute myeloid leukemia (AML) and relates to a novel method for in vitro or ex vivo diagnosis of the survival prospects of AML patients. The invention also relates to the use of kits and biomarkers for such in vitro or ex vivo diagnosis. Background Technology

[0002] Acute myeloid leukemia (AML – also known as acute myeloblastic leukemia) is a group of malignant blood cancers that affect hematopoietic cells in the bone marrow. They are characterized by an overproduction of primitive cells (also called “immature blood cells”), which are characterized by their inability to differentiate into mature cells and their uncontrolled proliferation. As a result, this dysfunction of the bone marrow prevents the normal production of blood cells and leads to a variety of clinical syndromes, which can sometimes be very severe and potentially fatal. AML typically occurs in adults over 40 years of age, especially older adults (over 60). However, it also affects younger patients and has even been reported in pediatric cancers (known as childhood AML).

[0003] The diagnosis of AML is primarily based on a percentage of blast cells greater than 20% in the bone marrow, or on the presence of certain identified genetic abnormalities such as t(8;21), inv(16), or t(15;17) (Pelcovits and Niroula 2020 PMID32236160). The choice of treatment for AML patients depends heavily on the molecular and cytogenetic characteristics of the leukemia (Dinardo and Lachowies 2019 PMID 31350639), as well as the patient's clinical characteristics (age, comorbidities). Treatment typically involves induction cytotoxic chemotherapy or hypomethylating agent-based therapies, sometimes supplemented by targeted therapy. Additionally, bone marrow transplantation (also known as "hematopoietic stem cell allogeneic transplantation") has been proposed as consolidation therapy to prevent relapse following chemotherapy. Allogeneic transplantation significantly increases the patient's chances of a cure. However, it is an invasive procedure that can be difficult for patients to tolerate and carries a significant risk of graft-versus-receptor reactions. In fact, on average, only 50% of AML patients survive more than one year after initial diagnosis. Five years after initial diagnosis, the percentage of surviving patients drops to an average of approximately 27%. Furthermore, the survival prospect declines sharply with increasing patient age. Therefore, in order to develop the best treatment plan for each patient, healthcare teams need reliable indicators that allow them to estimate the patient's survival prognosis at the time of diagnosis.

[0004] The World Health Organization (WHO) classifies AML into several subtypes based on its hematological, morphological, cytogenetic (karyotype), and gene mutation characteristics (Arber et al., 2016 PMID 27069254). Based on this classification, the European Leukemia Network (ELN) expert consortium proposed a prognostic classification system with three risk levels: good, intermediate, and poor (Döhner et al., 2022 PMID 35797463). While this classification is widely used in clinical practice, it is insufficient to define treatment strategies applicable to all patients, for example, when deciding on allogeneic transplantation for patients in the intermediate-risk category. Therefore, it is necessary to find new indicators to improve the survival prognosis of AML patients, thereby improving the existing classification and enabling the development of the most appropriate treatment plan for each patient.

[0005] In this regard, although diagnostic methods related to AML have improved considerably in recent decades, they have not taken into account transcriptomic data on gene expression. However, these data provide new and supplementary information to existing classifications and therefore represent a promising source of indicators for predicting the survival prospects of cancer patients.

[0006] In this context, building upon the work of researchers at the Grenoble Institute for Advanced Biosciences (IAB)—who demonstrated that cells undergo severe dysregulation at both the genomic and epigenomic levels during tumorigenesis, affecting gene expression profiles—the inventors unexpectedly and surprisingly identified five novel biomarkers by focusing on the diagnosis of survival prospects in AML patients. More specifically, the inventors have developed an innovative method and associated diagnostic tools that enable the identification of novel biomarkers capable of predicting the survival prospects of AML patients, alone or in combination. The inventors have tested their method in numerous AML patient cohorts, validating it for each individual. Furthermore, the inventors have demonstrated that the survival predictions obtained through this novel method represent a reliable and robust indicator in themselves and can also be used in conjunction with known indicators in the prior art to improve existing classifications. Summary of the Invention

[0007] Therefore, the present invention relates to a method for diagnosing the survival prospects of a patient with acute myeloid leukemia (AML) in vitro or in vitro, comprising the step of detecting the expression of at least one gene selected from ADSS1, FAM171B, MLF1, SLITRK5 and TNNT1 in a biological sample of the patient.

[0008] Therefore, by detecting the ectopic expression of at least one of the aforementioned genes in a biological sample, the method of the present invention itself provides healthcare teams with a robust indicator of the survival prospects of patients with AML awaiting treatment. Furthermore, this additional indicator can be combined with existing indicators (ELN indicators, age-related indicators) to enhance the robustness of the established predictions. Another advantage of this indicator is that it is rapidly and easily obtained using techniques already employed in numerous medical centers (e.g., RT-qPCR testing). Therefore, obtaining this additional indicator using the method according to the invention is inexpensive and can be standardized in laboratories at said medical centers in a short period of time.

[0009] This invention makes a significant contribution to the range of diagnostic tools available to healthcare teams for acute myeloid leukemia (AML) in order to develop the most appropriate treatment plan for each AML patient.

[0010] In the context of this invention, the terms "in vitro diagnostics" and "ex vivo diagnostics" refer to tests performed outside the patient after a biological sample has been collected from the patient. Tests performed on biological samples collected from the patient and stored in a laboratory (typically under sterile conditions and unchanged) for a short period (usually less than 24 hours) fall within the scope of ex vivo diagnostics according to the invention. Tests performed on such samples over longer periods generally fall within the scope of in vitro diagnostics according to the invention because they require a cell culture step to maintain the cell viability of the biological sample.

[0011] In the context of this invention, the term "biological sample" refers to tissues, body fluids, and components of said tissues and body fluids (e.g., cells) that may contain cancerous blast cells. According to the field of application of this invention, and as a non-exhaustive example, the biological sample may be a bone marrow sample or a blood sample, particularly a peripheral blood sample.

[0012] In the context of this invention, the terms "survival prospects," "survival prognosis," "life outcome," "survival probability," or "survival prediction" are synonyms, all referring to an estimate of the patient's survival based on specific indicators for AML patients.

[0013] In the context of this invention, the expression "detection of expression of at least one gene" means quantifying at least one expression product of that gene. In the sense of this invention, a gene expression product is any biomolecule produced by the expression of said gene. For example, a gene expression product can be an RNA transcript or a protein obtained after ribosomal translation of a transcript. "Transcript" refers to RNA produced by gene transcription, particularly messenger RNA (mRNA). More precisely, a transcript is RNA produced by post-transcriptional modification of the pre-RNA form following gene transcription. In the context of this invention, the expression levels of one or more RNA transcripts of the same gene and / or the expression levels of one or more isoforms of proteins encoded by the same gene can be quantified. In some cases, the quantitative expression of the detected gene can be compared to a reference value. In the sense of this invention, below this value, the gene is considered not expressed. For example, this could be random and / or isolated expression of the gene, representing background noise, which should not be considered within the scope of this invention. Conversely, above the reference value, gene expression is considered detected. This is ectopic expression, also known as out-of-context expression, because the five genes identified by the inventors are not expressed in the original cells of healthy individuals. Gene expression can be detected by any method known to those skilled in the art. In the case of mRNA transcripts, detection can be performed by a direct method, i.e., by any method known to those skilled in the art capable of determining the presence of the transcript in a sample; or by an indirect method, i.e., after the transcript has been converted into DNA, or after the transcript has been amplified, or after amplifying the DNA obtained after the transcript has been converted into DNA. Many methods exist for detecting nucleic acids (see, for example, Kricka et al., Clinical Chemistry, 1999, no. 45(4), pp. 453-458; Relier GH et al., DNA Probes, 2nd edition, Stockton Press, 1993, sections 5 and 6, pp. 173-249). Gene expression can be detected, in particular, by reverse transcription-polymerase chain reaction (RT-PCR), preferably by quantitative RT-PCR (RT-qPCR) (e.g., using FilmArray). ® Measurements are performed using either the technology or Fluidigm's Biomark™ platform; by sequencing (preferably high-throughput sequencing) or by hybridization techniques (e.g., using hybridization microarrays or via NanoString). ® nCounter ® The measurement is performed using the technology.

[0014] In the context of this invention, the term "reference value" refers to the expression level of a given gene in one or more healthy individuals (i.e., individuals who do not have AML and / or do not exhibit any specific medical condition). This reference value can be determined directly from the analysis of biological samples from healthy individuals, or it can be the result of multiple values ​​obtained from the same healthy individual and / or from several biological samples from several healthy individuals.

[0015] In the context of this invention, the ADSS1 gene refers to the gene known as "adenosylsuccinate synthase 1" (Ensembl identifier: ENSG00000185100) located on chromosome 14 (according to GRCh38 / hg38, the chromosomal location of this gene is: chr 14: 104,724,174-104,747,325). Its nucleotide sequence is SEQ ID NO: 1 or a sequence having at least 65%, or even 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with sequence SEQ ID NO: 1.

[0016] In the context of this invention, the FAM171B gene refers to the gene known as “Sequence Similarity Family 171 Member B” (Ensembl identifier: ENSG00000144369) located on chromosome 2 (according to GRCh38 / hg38, the chromosomal location of this gene is: chr 2: 186,694,060-186,765,959). Its nucleotide sequence is SEQ ID NO: 2 or a sequence having at least 65%, or even 70%, 75%, 80%, 85%, 90%, 95%, or even 99% identity with sequence SEQ ID NO: 2.

[0017] In the context of this invention, the MLF1 gene refers to the gene known as "myeloid leukemia factor 1" (Ensembl identifier: ENSG00000178053) located on chromosome 3 (according to GRCh38 / hg38, the chromosomal location of this gene is: chr3: 158,571,163-158,607,252). Its nucleotide sequence is SEQ ID NO: 3 or a sequence having at least 65%, or even 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity with sequence SEQ ID NO: 3.

[0018] In the context of this invention, the SLITRK5 gene refers to the gene known as "SLIT and NTRK-like family member 5" (Ensembl identifier: ENSG00000165300) located on chromosome 13 (according to GRCh38 / hg38, the chromosomal location of this gene is: chr 13: 87,671,371-87,696,272). Its nucleotide sequence is SEQ ID NO: 4 or a sequence having at least 65% or even 70%, 75%, 80%, 85%, 90%, 95%, or even 99% identity with sequence SEQ ID NO: 4.

[0019] In the context of this invention, the TNNT1 gene refers to the gene known as "troponin T1, slow skeletal muscle type" (Ensembl identifier: ENSG00000105048) located on chromosome 19 (according to GRCh38 / hg38, the chromosomal location of this gene is: chr19: 55,132,698-55,149,206). Its nucleotide sequence is SEQ ID NO: 5 or a sequence having at least 65% or even 70%, 75%, 80%, 85%, 90%, 95%, or even 99% identity with sequence SEQ ID NO: 5.

[0020] Preferably, the present invention relates to the method described above, and has the following technical features, individually or in combination:

[0021] The step of detecting the expression of at least one gene selected from ADSS1, FAM171B, MLF1, SLITRK5, and TNNT1 includes measuring the expression of said gene and comparing said measurement with a reference value for the expression of said gene in a healthy individual.

[0022] Gene expression was detected using RT-qPCR or high-throughput RNA-seq sequencing.

[0023] The detection step includes detecting the expression of at least two of the genes, preferably at least three of the genes, more preferably at least four of the genes, and even more preferably the expression of all five genes.

[0024] - The detection steps include detecting the expression of two genes selected from FAM171B, MLF1, and SLITRK5.

[0025] - The detection steps include detecting the expression of the following three genes: FAM171B, MLF1, and SLITRK5.

[0026] - The detection steps include detecting the expression of the following four genes: FAM171B, MLF1, SLITRK5, and TNNT1.

[0027] - The detection steps include detecting the expression of five genes.

[0028] -The biological sample from the patient is a bone marrow sample from the patient, and

[0029] -The patients are between 50 and 80 years old, preferably between 60 and 70 years old.

[0030] The present invention also relates to a kit for in vitro or ex vivo diagnosis of the survival prospects of patients with acute myeloid leukemia (AML), comprising a tool for detecting the expression of at least one gene selected from ADSS1, FAM171B, MLF1, SLITRK5 and TNNT1 in a biological sample from said patient.

[0031] Preferably, the detection tool of the kit according to the invention is capable of detecting the expression of at least one gene selected from ADSS1, FAM171B, MLF1, SLITRK5 and TNNT1 in bone marrow samples from the patient.

[0032] The present invention also relates to the use of at least one of ADSS1, FAM171B, MLF1, SLITRK5 and TNNT1 as a biomarker for diagnosing the survival prospects of a patient in vitro or in vitro in biological samples from a patient with acute myeloid leukemia (AML).

[0033] Preferably, the present invention relates to the uses described above, and has the following technical features, alone or in combination:

[0034] - At least two of the genes, preferably at least three of the genes, more preferably at least four of the genes, and even more preferably all five genes, are used as biomarkers for the in vitro or ex vivo diagnosis of the survival prospects of patients with acute myeloid leukemia (AML) in biological samples.

[0035] -The biological sample from the patient is a bone marrow sample from the patient. Attached Figure Description

[0036] [ Figure 1 ]: Represents the average survival over time for patients with AML belonging to different cohorts, which is a function of MLF1 gene expression detection;

[0037] [ Figure 2]: represents the average survival over time for patients with AML belonging to different cohorts, which is a function of MLF1 and FAM171B gene expression detection;

[0038] [ Figure 3 ]: Represents the average survival over time for patients with AML belonging to different cohorts, which is a function of MLF1 and SLITRK5 gene expression detection;

[0039] [ Figure 4 ]: represents the average survival over time for patients with AML belonging to different cohorts, which is a function of MLF1, FAM171B and SLITRK5 gene expression detection;

[0040] [ Figure 5 ]: represents the average survival over time for patients with AML belonging to different cohorts, which is a function for detecting the expression of MLF1, FAM171B, SLITRK5, and TNNT1 genes;

[0041] [ Figure 6 ]: represents the average survival over time for patients with AML belonging to different cohorts, which is a function of MLF1, FAM171B, SLITRK5, TNNT1 and ADSS1 gene expression detection;

[0042] [ Figure 7 ]: represents the average survival over time for patients with AML belonging to different cohorts, which is a function of MLF1, FAM171B and ADSS1 gene expression detection;

[0043] [ Figure 8 ]: represents the average survival over time for patients with AML belonging to different cohorts, which is a function of ADSS1, FAM171B and SLITRK5 gene expression detection;

[0044] [ Figure 9 ]: represents the average survival over time for patients with AML belonging to different cohorts, which is a function of ADSS1, FAM171B and TNNT1 gene expression detection;

[0045] [ Figure 10 ]: Represents the average survival over time for patients with AML belonging to different cohorts, which is a function of ADSS1, MLF1, and SLITRK5 gene expression detection;

[0046] [ Figure 11 ]: Represents the average survival over time for patients with AML belonging to different cohorts, which is a function for detecting ADSS1, MLF1, and TNNT1 gene expression;

[0047] [ Figure 12]: represents the average survival over time for patients with AML belonging to different cohorts, which is a function of ADSS1, SLITRK5 and TNNT1 gene expression detection;

[0048] [ Figure 13 ]: represents the average survival over time for patients with AML belonging to different cohorts, which is a function of FAM171B, MLF1 and TNNT1 gene expression detection;

[0049] [ Figure 14 ]: represents the average survival over time for patients with AML belonging to different cohorts, which is a function of FAM171B, SLITRK5 and TNNT1 gene expression detection;

[0050] [ Figure 15 ]: represents the average survival over time for patients with AML belonging to different cohorts, which is a function of MLF1, SLITRK5 and TNNT1 gene expression detection;

[0051] [ Figure 16 ]: represents the average survival over time for patients with AML belonging to different cohorts, which is a function of ADSS1, FAM171B, MLF1 and SLITRK5 gene expression assays;

[0052] [ Figure 17 ]: represents the average survival over time for patients with AML belonging to different cohorts, which is a function for detecting the expression of ADSS1, FAM171B, MLF1, and TNNT1 genes;

[0053] [ Figure 18 ]: represents the mean survival over time for patients with AML belonging to different cohorts, which is a function of ADSS1, FAM171B, SLITRK5, and TNNT1 gene expression assays; and

[0054] [ Figure 19 ]: represents the average survival over time for patients with AML belonging to different cohorts, and is a function for detecting the expression of ADSS1, MLF1, SLITRK5, and TNNT1 genes.

[0055] The invention is illustrated in a non-limiting manner by way of the following examples. Detailed Implementation

[0056] Example 1: Identification and Validation of Five Biomarkers

[0057] The inventors established a list of tissue-specific genes that are not expressed or are weakly expressed in the bone marrow of healthy patients. This step involved selecting more than 3,000 genes.

[0058] The inventors further selected genes from the chosen genes, specifically those showing ectopic activation exceeding 10% in leukemia cases. This step selected 314 genes.

[0059] In the next step, the inventors identified candidate genes capable of individually predicting the survival prognosis of AML patients. To this end, they defined an activation threshold for each of the 314 genes in the training dataset (corresponding to a hypothetical cohort of AML patients) and assessed their relationship to survival by defining their activation state (ON: expressed, or OFF: inactive) in consideration of gene expression levels. The log-rank test (a recognized and commonly used test in survival analysis) and the Cox proportional hazards model (also a well-established method in this field, described in the publication "Analysis of survival data. Cox & Oakes. 1984. ISBN 978-0-412-24490-2") were used for the survival analysis that enabled the identification of these candidate genes.

[0060] At the end of this step, five candidate genes—ADSS1, FAM171B, MLF1, SLITRK5, and TNNT1—were selected based on predictive values ​​for AML patient survival, such as p-values, hazard ratios, and robustness of their ON / OFF activation states. These genes were subsequently validated on two independent AML datasets.

[0061] Example 2: Results

[0062] To demonstrate the robustness of the diagnostic methods and related prognostic tools according to the invention, five biomarkers selected after the validation phase were used alone or in various combinations in a cohort of AML patients. These biomarkers were able to classify patients based on the number of biomarker genes with ectopic expression (and therefore ON activation). Among the five ultimately selected biomarker genes, the greater the number of biomarker genes with an activated state (ON state), the worse the patient's prognosis, as shown in the following results.

[0063] For each test dataset (cohort), patients were divided into two groups based on the activation status of the selected genes. The difference in survival probability between the two groups was calculated using a log-rank test, and this was found to be statistically significant in all considered datasets (log-rank p-value < 0.05). The effect of the number of activated genes among the five biomarker genes on survival was also calculated using a Cox model. The inventors have demonstrated that the number of activated genes is significantly correlated with the probability of patient survival in all considered datasets (Cox p-value < 0.05), thus demonstrating robust prediction of the survival prospects of patients with acute myeloid leukemia using the method and related tools according to the invention.

[0064] The five identified biomarker genes were examined based on different distributions and on the following dataset (AML patient cohort):

[0065] - Public datasets GSE10691, GSE146173, GSE37642, BEATAML, and TCGA-LAML (RNA-seq transcriptome data and DNA microarrays).

[0066] - The public dataset TARGET-AML, in which patients are minors, and

[0067] - A dataset created by the inventors, TRANSCRI-LAM. This cohort consists of 100 AML patients diagnosed with and followed up at Grenoble Alpes University Hospital (CHUGA) and who consented to participate in the AC-2014-2094 CRB09 clinical hematology collection. In this cohort, high-throughput RNA-seq sequencing was performed. Figures 1 to 6 The TRANSCRI-LAM-RNA-seq version of this cohort (or via standardized RT-qPCR assay) Figures 1 to 6 The TRANSCRI-LAM RT-qPCR version of this cohort was used to quantify biomarker expression levels. In clinical practice, RT-qPCR testing offers several advantages over high-throughput RNA-seq sequencing. It is significantly cheaper, standardized, and widely used in hospital laboratories. Therefore, the inventors verified that their method and related tools can be advantageously standardized to this technology. Thus, the expression levels of five biomarker genes were measured in all patient samples from the TRANSCRI-LAM cohort using RT-qPCR. The results were normalized relative to four housekeeping genes, ABL, ACTB, U6, and GAPDH, and expressed relative to their respective reference tissues (muscle for ADSS1 and TNNT1 genes, brain for FAM171B and SLITRK5 genes, and testis for MLF1 gene). The activation threshold (defined as ON / OFF detection state) for each gene was established relative to reference values ​​obtained using six healthy bone marrow samples and was equal to the mean of the gene expression in the six samples plus two standard deviations. The results show that the method (and related tools) according to the present invention are well-suited for RT-qPCR technology. Therefore, a standardized and reproducible RT-qPCR test can be proposed for use in hospital practice.

[0068] 2.1: Using the MLF1 gene as a biomarker ( Figure 1 )

[0069] In this test, all the above cohorts were analyzed, and patients in each cohort were distinguished according to the following criteria:

[0070] In their respective bone marrow samples (it is known that the MLF1 gene is expressed ectopically only in bone marrow cells).

[0071] - Expression of the MLF1 gene was detected. Figure 1 Group 1) and

[0072] - No expression of this gene was detected. Figure 1 Group 0 in the middle.

[0073] For each cohort, patients in group 0, where ectopic expression was not detected, were observed to have better survival prospects than patients in group 1, where the gene expression was detected.

[0074] 2.2: Using the combination of MLF1 and FAM171B genes as a biomarker ( Figure 2 )

[0075] In this test, all the above cohorts were analyzed, and patients in each cohort were distinguished according to the following criteria:

[0076] In their respective bone marrow samples (it is known that the MLF1 or FAM171B gene is expressed ectopically only in bone marrow cells).

[0077] - Expression of at least one of the MLF1 or FAM171B genes was detected. Figure 2 Groups 1-2 and

[0078] - No expression of these genes was detected. Figure 2 Group 0 in the middle.

[0079] For each cohort, patients in group 0, where no ectopic expression was detected, were observed to have better survival prospects than patients in groups 1-2, where expression of at least one of the two genes was detected.

[0080] 2.3: Using the combination of MLF1 and SLITRK5 genes as a biomarker ( Figure 3 )

[0081] In this test, all the above cohorts were analyzed, and patients in each cohort were distinguished according to the following criteria:

[0082] In their respective bone marrow samples (it is known that the MLF1 or SLITRK5 genes are expressed ectopically only in bone marrow cells).

[0083] - Expression of at least one of the two genes, MLF1 or SLITRK5, was detected. Figure 3 Groups 1-2 and

[0084] - No expression of these genes was detected. Figure 3 Group 0 in the middle.

[0085] For each cohort, patients in group 0, where no ectopic expression was detected, were observed to have better survival prospects than patients in groups 1-2, where expression of at least one of the two genes was detected.

[0086] 2.4: Using a combination of MLF1, FAM171B, and SLITRK5 genes as biomarkers ( Figure 4 )

[0087] In this test, all the above cohorts were analyzed, and patients in each cohort were distinguished according to the following criteria:

[0088] In their respective bone marrow samples (it is known that the MLF1, FAM171B, or SLITRK5 genes are expressed ectopically only in bone marrow cells).

[0089] - Expression of at least two of the genes MLF1, FAM171B, or SLITRK5 was detected. Figure 4 Groups 2-3 and

[0090] - No expression of these genes was detected, or only expression of one of these three genes was detected. Figure 4 Groups 0-1 in the middle.

[0091] For each cohort, patients in group 0-1, where at most one of the three genes was expressed, were observed to have better survival prospects than patients in group 2-3, where at least two of the three genes were expressed.

[0092] 2.5: Using a combination of MLF1, FAM171B, SLITRK5, and TNNT1 genes as biomarkers ( Figure 5 )

[0093] In this test, all the above cohorts were analyzed, and patients in each cohort were distinguished according to the following criteria:

[0094] In their respective bone marrow samples (it is known that the MLF1, FAM171B, SLITRK5, and TNNT1 genes are expressed ectopically only in bone marrow cells).

[0095] - Expression of at least two of the genes MLF1, FAM171B, SLITRK5, and TNNT1 was detected. Figure 5 Groups 2-4 in the middle) and

[0096] - No expression of these genes was detected, or only expression of one of these four genes was detected. Figure 5 Groups 0-1 in the middle.

[0097] For each cohort, patients in groups 0-1, where at most one of the four genes was expressed, were observed to have better survival prospects than patients in groups 2-4, where at least two of the four genes were expressed.

[0098] 2.6: Using a combination of MLF1, FAM171B, SLITRK5, TNNT1, and ADSS1 genes as biomarkers (Figure)6）

[0099] In this test, all the above cohorts were analyzed, and patients in each cohort were distinguished according to the following criteria:

[0100] In their respective bone marrow samples (it is known that the MLF1, FAM171B, SLITRK5, TNNT1, and ADSS1 genes are expressed ectopically only in bone marrow cells),

[0101] - Expression of at least two of the genes MLF1, FAM171B, SLITRK5, TNNT1, and ADSS1 was detected. Figure 6 Groups 2-5 in the middle) and

[0102] - No expression of these genes was detected, or only expression of one of these five genes was detected. Figure 6 Groups 0-1 in the middle.

[0103] For each cohort, patients in groups 0-1, where at most one of the five genes was expressed, were observed to have better survival prospects than patients in groups 2-5, where at least two of the five genes were expressed.

[0104] 2.7: Using a combination of MLF1, FAM171B, and ADSS1 genes as a biomarker ( Figure 7 )

[0105] In this test, all the above cohorts were analyzed, and patients in each cohort were distinguished according to the following criteria:

[0106] In their respective bone marrow samples (it is known that the MLF1, FAM171B, or ADSS1 genes are expressed ectopically only in bone marrow cells).

[0107] - Expression of at least two of the genes MLF1, FAM171B, or ADSS1 was detected. Figure 7 Groups 2-3 and

[0108] - No expression of these genes was detected, or only expression of one of these three genes was detected. Figure 7 Groups 0-1 in the middle.

[0109] For each cohort, patients in group 0-1, where at most one of the three genes was expressed, were observed to have better survival prospects than patients in group 2-3, where at least two of the three genes were expressed.

[0110] 2.8: Using a combination of ADSS1, FAM171B, and SLITRK5 genes as biomarkers ( Figure 8 )

[0111] In this test, all the above cohorts were analyzed, and patients in each cohort were distinguished according to the following criteria:

[0112] In their respective bone marrow samples (the ADSS1, FAM171B, or SLITRK5 genes are known to be ectopically expressed only in bone marrow cells),

[0113] - Expression of at least two of the genes ADSS1, FAM171B, or SLITRK5 was detected. Figure 8 Groups 2-3 and

[0114] - No expression of these genes was detected, or only expression of one of these three genes was detected. Figure 8 Groups 0-1 in the middle.

[0115] For each cohort, patients in group 0-1, where at most one of the three genes was expressed, were observed to have better survival prospects than patients in group 2-3, where at least two of the three genes were expressed.

[0116] 2.9: Using a combination of ADSS1, FAM171B, and TNNT1 genes as biomarkers ( Figure 9 )

[0117] In this test, all the above cohorts were analyzed, and patients in each cohort were distinguished according to the following criteria:

[0118] In their respective bone marrow samples (the ADSS1, FAM171B, or TNNT1 genes are known to be ectopically expressed only in bone marrow cells),

[0119] - Expression of at least two of the genes ADSS1, FAM171B, or TNNT1 was detected. Figure 9 Groups 2-3 and

[0120] - No expression of these genes was detected, or only expression of one of these three genes was detected. Figure 9 Groups 0-1 in the middle.

[0121] For each cohort, patients in group 0-1, where at most one of the three genes was expressed, were observed to have better survival prospects than patients in group 2-3, where at least two of the three genes were expressed.

[0122] 2.10: Using a combination of ADSS1, MLF1, and SLITRK5 genes as biomarkers ( Figure 10 )

[0123] In this test, all the above cohorts were analyzed, and patients in each cohort were distinguished according to the following criteria:

[0124] In their respective bone marrow samples (the ADSS1, MLF1, or SLITRK5 genes are known to be ectopically expressed only in bone marrow cells),

[0125] - Expression of at least two of the genes ADSS1, MLF1, or SLITRK5 was detected. Figure 10 Groups 2-3 and

[0126] - No expression of these genes was detected, or only expression of one of these three genes was detected. Figure 10 Groups 0-1 in the middle.

[0127] For each cohort, patients in group 0-1, where at most one of the three genes was expressed, were observed to have better survival prospects than patients in group 2-3, where at least two of the three genes were expressed.

[0128] 2.11: Using a combination of ADSS1, MLF1, and TNNT1 genes as a biomarker ( Figure 11 )

[0129] In this test, all the above cohorts were analyzed, and patients in each cohort were distinguished according to the following criteria:

[0130] In their respective bone marrow samples (the ADSS1, MLF1, or TNNT1 genes are known to be ectopically expressed only in bone marrow cells),

[0131] - Expression of at least two of the genes ADSS1, MLF1, or TNNT1 was detected. Figure 11 Groups 2-3 and

[0132] - No expression of these genes was detected, or only expression of one of these three genes was detected. Figure 11 Groups 0-1 in the middle.

[0133] For each cohort, patients in group 0-1, where at most one of the three genes was expressed, were observed to have better survival prospects than patients in group 2-3, where at least two of the three genes were expressed.

[0134] 2.12: Using a combination of ADSS1, SLITRK5, and TNNT1 genes as biomarkers ( Figure 12 )

[0135] In this test, all the above cohorts were analyzed, and patients in each cohort were distinguished according to the following criteria:

[0136] In their respective bone marrow samples (the ADSS1, SLITRK5, or TNNT1 genes are known to be ectopically expressed only in bone marrow cells),

[0137] - Expression of at least two of the genes ADSS1, SLITRK5, or TNNT1 was detected. Figure 12 Groups 2-3 and

[0138] - No expression of these genes was detected, or only expression of one of these three genes was detected. Figure 12 Groups 0-1 in the middle.

[0139] For each cohort, patients in group 0-1, where at most one of the three genes was expressed, were observed to have better survival prospects than patients in group 2-3, where at least two of the three genes were expressed.

[0140] 2.13: Using a combination of FAM171B, MLF1, and TNNT1 genes as a biomarker ( Figure 13 )

[0141] In this test, all the above cohorts were analyzed, and patients in each cohort were distinguished according to the following criteria:

[0142] In their respective bone marrow samples (it is known that the FAM171B, MLF1, or TNNT1 genes are expressed ectopically only in bone marrow cells).

[0143] - Expression of at least two of the genes FAM171B, MLF1, or TNNT1 was detected. Figure 13 Groups 2-3 and

[0144] - No expression of these genes was detected, or only expression of one of these three genes was detected. Figure 13 Groups 0-1 in the middle.

[0145] For each cohort, patients in group 0-1, where at most one of the three genes was expressed, were observed to have better survival prospects than patients in group 2-3, where at least two of the three genes were expressed.

[0146] 2.14: Using a combination of FAM171B, SLITRK5, and TNNT1 genes as biomarkers ( Figure 14 )

[0147] In this test, all the above cohorts were analyzed, and patients in each cohort were distinguished according to the following criteria:

[0148] In their respective bone marrow samples (it is known that the FAM171B, SLITRK5, or TNNT1 genes are expressed ectopically only in bone marrow cells).

[0149] - Expression of at least two of the genes FAM171B, SLITRK5, or TNNT1 was detected. Figure 14 Groups 2-3 and

[0150] - No expression of these genes was detected, or only expression of one of these three genes was detected. Figure 14 Groups 0-1 in the middle.

[0151] For each cohort, patients in group 0-1, where at most one of the three genes was expressed, were observed to have better survival prospects than patients in group 2-3, where at least two of the three genes were expressed.

[0152] 2.15: Using a combination of MLF1, SLITRK5, and TNNT1 genes as a biomarker ( Figure 15 )

[0153] In this test, all the above cohorts were analyzed, and patients in each cohort were distinguished according to the following criteria:

[0154] In their respective bone marrow samples (it is known that the MLF1, SLITRK5, or TNNT1 genes are expressed ectopically only in bone marrow cells).

[0155] - Expression of at least two of the genes MLF1, SLITRK5, or TNNT1 was detected. Figure 15 Groups 2-3 and

[0156] - No expression of these genes was detected, or only expression of one of these three genes was detected. Figure 15 Groups 0-1 in the middle.

[0157] For each cohort, patients in group 0-1, where at most one of the three genes was expressed, were observed to have better survival prospects than patients in group 2-3, where at least two of the three genes were expressed.

[0158] 2.16: Using a combination of ADSS1, FAM171B, MLF1, and SLITRK5 genes as biomarkers ( Figure 16 )

[0159] In this test, all the above cohorts were analyzed, and patients in each cohort were distinguished according to the following criteria:

[0160] In their respective bone marrow samples (the ADSS1, FAM171B, MLF1, and SLITRK5 genes are known to be expressed ectopically only in bone marrow cells),

[0161] - Expression of at least two of the genes ADSS1, FAM171B, MLF1, and SLITRK5 was detected. Figure 16 Groups 2-4 in the middle) and

[0162] - No expression of these genes was detected, or only expression of one of these four genes was detected. Figure 16 Groups 0-1 in the middle.

[0163] For each cohort, patients in groups 0-1, where at most one of the four genes was expressed, were observed to have better survival prospects than patients in groups 2-4, where at least two of the four genes were expressed.

[0164] 2.17: Using a combination of ADSS1, FAM171B, MLF1, and TNNT1 genes as biomarkers ( Figure 17 )

[0165] In this test, all the above cohorts were analyzed, and patients in each cohort were distinguished according to the following criteria:

[0166] In their respective bone marrow samples (the ADSS1, FAM171B, MLF1, and TNNT1 genes are known to be expressed ectopically only in bone marrow cells),

[0167] - Expression of at least two of the genes ADSS1, FAM171B, MLF1, and TNNT1 was detected. Figure 17 Groups 2-4 in the middle) and

[0168] - No expression of these genes was detected, or only expression of one of these four genes was detected. Figure 17 Groups 0-1 in the middle.

[0169] For each cohort, patients in groups 0-1, where at most one of the four genes was expressed, were observed to have better survival prospects than patients in groups 2-4, where at least two of the four genes were expressed.

[0170] 2.18: Using a combination of ADSS1, FAM171B, SLITRK5, and TNNT1 genes as biomarkers ( Figure 18 )

[0171] In this test, all the above cohorts were analyzed, and patients in each cohort were distinguished according to the following criteria:

[0172] In their respective bone marrow samples (the ADSS1, FAM171B, SLITRK5, and TNNT1 genes are known to be ectopically expressed only in bone marrow cells),

[0173] - Expression of at least two of the genes ADSS1, FAM171B, SLITRK5, and TNNT1 was detected. Figure 18 Groups 2-4 in the middle) and

[0174] - No expression of these genes was detected, or only expression of one of these four genes was detected. Figure 18 Groups 0-1 in the middle.

[0175] For each cohort, patients in groups 0-1, where at most one of the four genes was expressed, were observed to have better survival prospects than patients in groups 2-4, where at least two of the four genes were expressed.

[0176] 2.19: Using a combination of ADSS1, MLF1, SLITRK5, and TNNT1 genes as a biomarker ( Figure 19 )

[0177] In this test, all the above cohorts were analyzed, and patients in each cohort were distinguished according to the following criteria:

[0178] In their respective bone marrow samples (the ADSS1, MLF1, SLITRK5, and TNNT1 genes are known to be ectopically expressed only in bone marrow cells),

[0179] - Expression of at least two of the genes ADSS1, MLF1, SLITRK5, and TNNT1 was detected. Figure 19 Groups 2-4 in the middle) and

[0180] - No expression of these genes was detected, or only expression of one of these four genes was detected. Figure 19 Groups 0-1 in the middle.

[0181] For each cohort, patients in groups 0-1, where at most one of the four genes was expressed, were observed to have better survival prospects than patients in groups 2-4, where at least two of the four genes were expressed.

[0182] 2.20: Conclusion

[0183] These experimental data clearly demonstrate that the methods, kits, and uses according to the invention can provide indicators related to the survival prospects of AML patients, which 1 / complement and improve upon those already available to healthcare teams, 2 / are robust and reliable in themselves, and 3 / can be rapidly obtained using technologies already employed in numerous medical centers (e.g., via RT-qPCR testing). These experimental data, particularly those related to the TARGET-AML cohort, also indicate that the methods, kits, and uses according to the invention are suitable for pediatric AML, as they can provide such indicators for underage patients.

[0184] Furthermore, the data provided also indicate that the robustness of this indicator increases with the number of the five biomarker genes used (whose expression was detected).

Claims

1. A method for diagnosing the survival prospects of a patient with acute myeloid leukemia (AML) in vitro or in vitro, comprising the step of detecting the expression of at least one gene selected from ADSS1, FAM171B, MLF1, SLITRK5 and TNNT1 in a biological sample of the patient.

2. The method according to the preceding claim, wherein the step of detecting the expression of at least one gene selected from ADSS1, FAM171B, MLF1, SLITRK5 and TNNT1 includes measuring the expression of the gene and comparing the measured value with a reference expression value of the gene in a healthy individual.

3. The method according to any one of the preceding claims, wherein the expression of the gene is detected using RT-qPCR technology or by high-throughput RNA-seq sequencing.

4. The method according to any one of the preceding claims, wherein the detection step includes detecting the expression of at least two of the genes, preferably detecting the expression of at least three of the genes, more preferably detecting the expression of at least four of the genes, and even more preferably detecting the expression of all five genes.

5. The method according to the preceding claim, wherein the detection step comprises detecting the expression of two genes selected from FAM171B, MLF1, and SLITRK5.

6. The method of claim 4, wherein the detection step comprises detecting the expression of the following three genes: FAM171B, MLF1, and SLITRK5.

7. The method of claim 4, wherein the detection step comprises detecting the expression of the following four genes: FAM171B, MLF1, SLITRK5, and TNNT1.

8. The method of claim 4, wherein the detection step includes detecting the expression of the five genes.

9. The method according to any one of the preceding claims, wherein the biological sample of the patient is a bone marrow sample from the patient.

10. A kit for in vitro or ex vivo diagnosis of the survival prospects of a patient with acute myeloid leukemia (AML), comprising a tool for detecting the expression of at least one gene selected from ADSS1, FAM171B, MLF1, SLITRK5, and TNNT1 in a biological sample from said patient.

11. The kit according to the preceding claim, wherein the biological sample of the patient is a bone marrow sample from the patient.

12. Use of at least one gene selected from ADSS1, FAM171B, MLF1, SLITRK5 and TNNT1 as a biomarker for in vitro or ex vivo diagnosis of the survival prospects of a patient in a biological sample from a patient with acute myeloid leukemia (AML).

13. The use according to the preceding claim, wherein at least two of the genes, preferably at least three of the genes, more preferably at least four of the genes, and even more preferably all five genes, are used as biomarkers for in vitro or ex vivo diagnosis of the survival prospects of a patient in a biological sample from a patient with acute myeloid leukemia (AML).

14. The use according to claim 12 or 13, wherein the biological sample of the patient is a bone marrow sample from the patient.

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