METHOD FOR DIAGNOSING HEMATOLOGICAL DISORDERS
Disclosed is a method for the diagnosis, and/or the classification, of a hematological disorder, including the steps of: a). measuring, the expression level of at least the genes of a sub-group of 6 genes, b). comparing the expression level of each genes measured in step a)., with the expression level of the same genes in healthy control sample, and c). determining the status of the biological sample.
The present invention relates to a method for diagnosing hematological disorders in a patient.
The great quantity of hematopoietic cells and the many stages of differentiation through which they pass further complicate the classification of the neoplasis originating from this type of cells. Despite the efforts to establish a classification based on “real” entities, some of the categories are ambiguous and in many cases contain very heterogeneous groups as regards a response to therapy of clinical course. This heterogeneity is that responsible for, on the one hand, the incessant search for markers capable of differentiating some behaviours from others and, on the other hand, that the disputed classification of this type of neoplasia is subjected to continuous revisions.
An ideal classification system should be precise, reproducible, easy to use and should especially have biological and clinical significance (Chan W C et al., Croat Med J. 2005; 46:349-59). The current diagnosis systems and the classification of the hematological neoplasias are based on the recognition of histological and morphological, immunophenotypical and cytogenetic characteristics and study of a molecular marker with prognostic value. However, in some of the diagnostic categories defined in this way, the following is observed:
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- A marked heterogeneous therapy response: within the same disease, patients either reach full remission, or partial remission, or do not respond, or relapse after a certain therapy. The capacity to predict a response is especially important in this type of neoplasias since the transplant of stem cells is an effective but toxic alternative response. The capacity to determine which patients would respond to a conventional therapy before giving it may be beneficial to be able to apply the most effective treatment to each patient.
- A variable clinical behaviour: within this category for some patients the disease is going to remain stable for long periods of time and that are not going to need therapy and whereas for others the disease is going to progress rapidly requiring aggressive therapy.
These variations point to the existence of molecular heterogeneity within the diagnostic categories, differences which the conventional methods of diagnosis are not capable of determining and hence, the search for new forms of analysis which provide a greater resolution in the characterization of this type of neoplasias.
In this line, the use of expression arrays have demonstrated being effective not only in deciphering the biological and clinical diversity which is found in many tumours, but in understanding the biological and pathological processes which affect many symptoms and, in particular, the hematopoietic system. The expression arrays are ordered arrays of sequences associated to a solid support, complementary to mRNA or to its corresponding cDNA or cRNA, which allow the analysis of the differential expression of hundreds or thousands of genes simultaneously. One of the supports to which they are frequently bound is to rectangular fragments of glass similar to slides, a format which is frequently alluded to by the terms microarray, biochip or, simply, chip. Their use is becoming increasingly frequent for the diagnosis of various diseases or for the evolution of the evaluation of the susceptibility of suffering from them.
In 1999, the Golub group published one of the first articles referring to the role of arrays in the classification of hematological neoplasias (Golub T R et al, Science. 1999; 286:531-7). An array with 6817 genes represented was used for the study of expression profiles in acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL). A group of 50 genes was selected with the capacity of predicting the type of leukemia (class predictor) and they were used to classify a group of unknown samples in the correct categories. The study of the expression of these 50 genes is sufficient for the classification of a sample of AML or ALL. Despite the fact that the distinction between AML and ALL is well established with the current diagnostic methods, the study revealed the existence of specific expression patterns associated with each type of acute leukemia and proved the use.
The European patent application EP1947194 has proposed the use of specific oligonucleotides for diagnosing chronic lymphoid leukemia (CLL) diseases.
The international application WO 2005/080601 discloses methods of genetic analysis for the classification, diagnosis and prognosis of acute myeloid leukemia (AML). This application provides a method for differentiating AML subtypes, but never provides methods for the discrimination between preleukemic and leukemic states.
The international application WO 2006/125195 discloses a group of 24 genes whose expression allows to classify a sample as myelodysplastic syndrome (MDS), AML, or not diseased. However, this document does not provide a method for classifying different grade of MDS.
So there is a need to provide a new method for diagnosing, classifying the malignant and premalignant states of cancer.
Oxidative stress is generally defined as an imbalance between the generation of reactive oxygen species (ROS) and impaired antioxidant defense systems. It has long been known to be involved in the pathophysiology of cancer.
High level of ROS produced either endogenously or exogenously can attack lipids, proteins, and nucleic acids simultaneously in living cells. This has led to cells developing various antioxidant defense mechanisms to both prevent the excessive formation of ROS and limit their harmful effects. The appropriate redox balance is maintained via the combined action of antioxidant enzymes.
Myelodysplastic syndroma and acute leukemia are characterized by a pathological hematopoieisis. ROS certainly plays an important role in human hematopoiesis. For example, it is well established in murine models that ROS-induced p38 MAPK activation is crucial in hematopoiesis and increased ROS levels are required to trigger hematopoietic stem cells (HSC) exit from quiescence and to drive maturation and differentiation. Moreover, a high ROS level induces a perturbation in the self-renewal activity of HSC. It is now well established that the progression of normal cells to neoplastic transformation results from the accumulation of mutations in genes that control cellular proliferation, survival, and differentiation. Approximately, 30% myelodysplastic syndroma cases progress to acute leukemia. Indirect evidences suggest a role for oxidant DNA damage in the pathogenesis of myelodysplasia. Moreover, the flow cytometric quantification of ROS in bone marrow cells from myelodysplastic and leukemic patients reveals an increase in ROS level in all cases.
So, studying the antioxidant response in premalignant and malignant state seems to be a good start for providing a new useful method.
The cancer stem cell (CSC) hypothesis suggests that a subset of cells within a tumor has the ability to recapitulate the generation of a continuously growing tumor (Clarke M F et al, Cancer Research. 2006; 66:9339-44). CSCs are best described in human in which the rare so-called leukemia stem cells (L-HSCs) can be prospectively isolated and shown to transmit the disease when introduced into immuno-compromised mice (Lap/dot T et al, Nature. 1994; 645-8). Cells which do not share this phenotype often represent the bulk of the leukemic clone, but fail to transmit the disease upon transplantation. The early work on L-HSC has now been extended to a list of tumors which is rapidly expanding (Bomken S, Br. J. Cancer. 2010; 103.439-45). Because they appear to be resistant to drugs that are commonly used to treat leukemia in humans, L-HSCs may be responsible for relapse in some patients (Ishikawa F, Nat. Biotechnol 2007; 25:1315-21). Genes which are functionally significant for L-HSC expansion may therefore represent the ultimate therapeutic targets. This raises the possibility that other ROS scavenging systems are of regulatory importance in other cancer stem cells. Indeed a low ROS level in breast CSCs has recently been reported, where it was associated with increased expression of the glutathione biosynthesis genes (Diehn M, Nature. 2009; 458:780-3).
Therefore, one aim of the invention is to provide a new method for diagnosing cancer.
Another aim of the invention is to provide a rapid efficient method for classifying pathologic sample, which cannot be classified by other method.
Another aim of the invention is to provide a kit for the implementation of the above methods.
Another aim of the invention is to provide composition allowing the implementation of the above method.
The invention relates to a method for the diagnosis, and/or the classification, preferably in vitro, of an hematological disorder, in particular myeloid and/or lymphoid hematological disorder, preferably myeloid hematological disorder,
said method comprising the steps of:
a). measuring, from cells contained in a biological sample of a subject, preferably from blood cells or bone marrow cells containing sample, the expression level of at least the genes of a sub-group of 6 genes belonging to a set of genes chosen among a group of 24 genes, said group of 24 genes comprising or being constituted by the nucleic acid sequences SEQ ID NO:1 to 24,
wherein said subject is suspected to be afflicted by an hematological disorder, in particular myeloid and/or lymphoid hematological disorder, preferably myeloid hematological disorder,
said 6 genes belonging to said sub-group comprising or being constituted by the nucleic acid sequences SEQ ID NO: 1 to 6,
b). comparing the expression level of each genes measured in step a)., with the expression level of the same respective genes from cells contained in a control sample preferably from blood cells or bone marrow cells containing sample, said control sample being of the same nature than said biological sample, to establish a gene expression level ratio for each genes of said sub-group, and
c). determining the status of said biological sample such that if the ratio established in step b). for each genes of any combination of at least 3 genes from said sub-group is either ≧2 or ≦0.5, said biological sample is representative of an hematological disorder cells.
In other words, the step b). according to the invention consists of:
comparing the expression level of each genes measured in step a)., with the expression level of the same respective genes from cells contained in a control sample, preferably from a control sample containing blood cells or bone marrow cells, said control sample being of the same nature than said biological sample, to establish a gene expression level ratio Ri between the expression level of each genes i measured in step a) and the expression level of the same respective genes i from cells contained in a control sample, for each genes of said sub-group.
The step c). according to the invention consists of determining the status of said biological sample such that if the ratio Ri for each genes of any combination of 3 genes from said sub-group is
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- either ≧2,
- or ≦0.5,
- said biological sample is representative of an haematological disorder cells.
The invention is based on the unexpected observation made by the Inventors that at least 6 specific genes, i.e. the genes comprising or being constituted by the nucleic acid sequences SEQ ID NO 1-6, belonging to a group of 24 specific genes comprising or being constituted by the nucleic acid sequences SEQ ID NO 1-24 are sufficient to determine the status a of an hematological disorder.
The invention is preferably carried out with sample from patients who have not been previously treated for an hematological disorder.
These genes are specifically genes coding for enzymes involved in the detoxification of cells, in which ROS accumulate.
The natural process involved in the elimination of ROS is represented in
According to the invention the group (C) of 24 genes comprises a set (B) of genes, said set comprising the subgroup (A) of 6 specific genes as defined above. The imbrications of the group/set/subgroup according to the invention are represented in
The method according to the invention is thus carried out as follows:
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- from a sample of a patient, the nucleic acid molecules contained in said sample are extracted, preferably the RNA molecules, according to extraction methods known in the art,
- the amount of specific nucleic acid molecules, corresponding to the nucleic acid molecules comprising or being constituted by at least SEQ ID NO: 1-6, is quantified, by well known techniques as illustrated hereafter,
- the amount quantified in the above step is compared with the amount of the same nucleic acid molecules contained in a control sample, and a ratio is established.
Accordingly, the control sample, which is used as reference, is a sample of an healthy individual, said healthy sample being of the same nature than the sample of the patient. Advantageously, the control sample corresponds to a pool of numerous samples of different healthy individuals, i.e. the control sample represents the mean of numerous healthy individual.
As mentioned above, the sample of the patient and the control sample are of the same origin. This means that if the sample of the patient is originated from blood of the patient, the control sample is originated from blood of one or many healthy individuals. Blood, in the invention, means total blood, plasma, serum, peripheral blood mononuclear cells (PBMC) . . . .
In the same way, if the sample of the patient is originated from bone marrow, the control sample is originated from bone marrow of one or many healthy individuals.
In the invention, the biological of the patient, from whom the biological sample is used, is suspected to be afflicted by an hematological disorder, in particular myeloid and/or lymphoid hematological disorder, preferably myeloid hematological disorder.
This means that the pathological status of the patient is:
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- either undetermined (the pathologist does not know if said patient is afflicted by an hematological disorder),
- or determined (the pathologist knows that the patient is afflicted by an hematological disorder).
This also means that the pathological status of the patient is:
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- first determined (the pathologist identify if the patient is afflicted by an hematological disorder, or not),
- and if the patient is afflicted by an hematological disorder, said hematological disorder is classified.
If the pathological status is undetermined, then the pathologist measure the expression levels of the genes consisting of SEQ ID NO: 1-6 in a biological sample from said patient and compare said expression levels with the expression levels of the same genes (i.e. genes SEQ ID NO: 1-6) in a control sample of the same nature, as defined above, (for instance a pool of cells from healthy donors that is used as control sample or reference), in order to establish the ratios Ri, and to determine if the patient is afflicted by hematological disorder or not.
If the pathological status is determined (for instance by cytological studies), the pathologist measures the expression levels of the genes consisting of SEQ ID NO: 1-6 in a biological sample from said patient and compares said expression levels with the expression levels of the same genes (i.e. genes SEQ ID NO: 1-6) in a control sample of the same nature, as defined above, (for instance a pool of cells from healthy donors that is used as control sample or reference), in order to establish the ratios Ri, and can classify the hematological disorder according to the method of the invention.
The method according to the invention provides an easy to use, rapid and efficient process to evaluate the status of a sample identified as, or suspected to be, a sample corresponding to an hematological disorder, in particular a myeloid disorder.
If the ratio between the expression level of at least 3 genes, of the above 6 genes belonging to the above defined subgroup, of a patient sample and the expression level of the same at least 3 genes of a control sample is either ≧2 or ≦0.5, then the sample of the patient would be considered as presenting the features of a sample corresponding to an hematological disorder.
Above and hereafter, the ratio is defined as follows:
Ri=[Amount(expression level) of a gene i of SEQ ID NO:i in the patient sample]/[Amount(expression level) of a gene i of SEQ ID NO:i in the control sample]
i varying from 1 to 24.
Therefore R3 represents the ratio as defined above, relative to the gene SEQ ID NO: 3, and thus Ri represents the ratio as defined above, relative to the gene SEQ ID NO: i, i varying from 1 to 24.
In the invention, “the ratio Ri of each gene of any combination of at least 3 genes of the 6 genes of said subgroup is ≧2 or ≦0.5” means that the ratio Ri of each gene of any combination of 3, or 4 or 5 or 6 genes is ≧2 or ≦0.5.
All the 20 combinations of 3 genes chosen among the 6 genes are listed hereafter:
SEQ ID NO: 1+SEQ ID NO: 2+SEQ ID NO: 3, SEQ ID NO: 1+SEQ ID NO: 2+SEQ ID NO: 4, SEQ ID NO: 1+SEQ ID NO: 2+SEQ ID NO: 5, SEQ ID NO: 1+SEQ ID NO: 2+SEQ ID NO: 6, SEQ ID NO: 1+SEQ ID NO: 3+SEQ ID NO: 4, SEQ ID NO: 1+SEQ ID NO: 3+SEQ ID NO: 5, SEQ ID NO: 1+SEQ ID NO: 3+SEQ ID NO: 6, SEQ ID NO: 1+SEQ ID NO: 4+SEQ ID NO: 5, SEQ ID NO: 1+SEQ ID NO: 4+SEQ ID NO: 6, SEQ ID NO: 1+SEQ ID NO: 5+SEQ ID NO: 6, SEQ ID NO: 2+SEQ ID NO: 3+SEQ ID NO: 4, SEQ ID NO: 2+SEQ ID NO: 3+SEQ ID NO: 5, SEQ ID NO: 2+SEQ ID NO: 3+SEQ ID NO: 6, SEQ ID NO: 2+SEQ ID NO: 4+SEQ ID NO: 5, SEQ ID NO: 2+SEQ ID NO: 4+SEQ ID NO: 5, SEQ ID NO: 2+SEQ ID NO: 5+SEQ ID NO: 6, SEQ ID NO: 3+SEQ ID NO: 4+SEQ ID NO: 5, SEQ ID NO: 3+SEQ ID NO: 4+SEQ ID NO: 6, SEQ ID NO: 3+SEQ ID NO: 5+SEQ ID NO: 6 and SEQ ID NO: 4+SEQ ID NO: 5+SEQ ID NO: 6.All the 15 combinations of 4 genes among the 6 genes are the following ones:
SEQ ID NO: 1+SEQ ID NO: 2+SEQ ID NO: 3+SEQ ID NO: 4, SEQ ID NO: 1+SEQ ID NO: 2+SEQ ID NO: 3+SEQ ID NO: 5, SEQ ID NO: 1+SEQ ID NO: 2+SEQ ID NO: 3+SEQ ID NO: 6, SEQ ID NO: 1+SEQ ID NO: 3+SEQ ID NO: 4+SEQ ID NO: 5, SEQ ID NO: 1+SEQ ID NO: 3+SEQ ID NO: 4+SEQ ID NO: 6, SEQ ID NO: 1+SEQ ID NO: 2+SEQ ID NO: 4+SEQ ID NO: 5, SEQ ID NO: 1+SEQ ID NO: 2+SEQ ID NO: 4+SEQ ID NO: 6, SEQ ID NO: 1+SEQ ID NO: 2+SEQ ID NO: 5+SEQ ID NO: 6, SEQ ID NO: 1+SEQ ID NO: 2+SEQ ID NO: 3+SEQ ID NO: 5, SEQ ID NO: 1+SEQ ID NO: 2+SEQ ID NO: 3+SEQ ID NO: 6, SEQ ID NO: 2+SEQ ID NO: 3+SEQ ID NO: 4+SEQ ID NO: 5, SEQ ID NO: 2+SEQ ID NO: 3+SEQ ID NO: 4+SEQ ID NO: 6, SEQ ID NO: 2+SEQ ID NO: 3+SEQ ID NO: 5+SEQ ID NO: 6, SEQ ID NO: 2+SEQ ID NO: 4+SEQ ID NO: 5+SEQ ID NO: 6 and SEQ ID NO: 3+SEQ ID NO: 4+SEQ ID NO: 5+SEQ ID NO: 6.All the 6 combinations of 5 genes among 6 genes are the following ones:
SEQ ID NO: 1+SEQ ID NO: 2+SEQ ID NO: 3+SEQ ID NO: 4+SEQ ID NO: 5, SEQ ID NO: 1+SEQ ID NO: 2+SEQ ID NO: 3+SEQ ID NO: 4+SEQ ID NO: 6, SEQ ID NO: 1+SEQ ID NO: 2+SEQ ID NO: 3+SEQ ID NO: 5+SEQ ID NO: 6, SEQ ID NO: 1+SEQ ID NO: 2+SEQ ID NO: 4+SEQ ID NO: 5+SEQ ID NO: 6, SEQ ID NO: 1+SEQ ID NO: 3+SEQ ID NO: 4+SEQ ID NO: 5+SEQ ID NO: 6 and SEQ ID NO: 2+SEQ ID NO: 3+SEQ ID NO: 4+SEQ ID NO: 5+SEQ ID NO: 6.Finally, the combination of the six genes is SEQ ID NO: 1+SEQ ID NO: 2+SEQ ID NO: 3+SEQ ID NO: 4+SEQ ID NO: 5+SEQ ID NO: 6.
According to the invention, a sample of a patient wherein the ratio Ri of each gene belonging to any combination of least 3 genes of the subgroup of 6 genes is ≧2 or ≦0.5 will be considered as a sample corresponding to an hematological disorder.
For instance, if the combination SEQ ID NO: 1+SEQ ID NO: 2+SEQ ID NO: 3 is studied, if the respective ratios R1, R2 and R3 as described above are as follows:
R1≧2 and R2≧2 and R3≧2, or R1≦0.5 and R2≧2 and R3≧2, or R1≧2 and R2≦0.5 and R3≧2, or R1≧2 and R2≧2 and R3≦0.5, or R1≦0.5 and R2≦0.5 and R3≧2, or R1≦0.5 and R2≧2 and R3≦0.5, or R1≧2 and R2≦0.5 and R3≦0.5, or R1≦0.5 and R2≦0.5 and R3≦0.5,then the sample of the patient in which the ratios are calculated will be considered as a sample corresponding to an hematological disorder.
The above example applies mutatis mutandis to the combinations of at least 3 genes mentioned above.
Consequently, if the combination SEQ ID NO: 1+SEQ ID NO: 2+SEQ ID NO: 3+SEQ ID NO: 4 is studied, 4 combinations of 3 genes exist:
SEQ ID NO: 1+SEQ ID NO: 2+SEQ ID NO: 3, SEQ ID NO: 1+SEQ ID NO: 2+SEQ ID NO:4, SEQ ID NO: 1+SEQ ID NO: 3+SEQ ID NO: 4, and SEQ ID NO: 2+SEQ ID NO: 3+SEQ ID NO: 4.Therefore, if the respective ratios R1, R2, R3 and R4 are as follows:
-
- for combination 1:
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- for combination 2
-
- for combination 3,
-
- for combination 4
then the sample of the patient in which the ratios are calculated will be considered as a sample corresponding to an hematological disorder.
In the invention, the genes for which the expression level is measured are represented by the RNA molecules obtained by the transcription of said genes. The transcription process is well known in the art.
Therefore, the invention relates to a process as defined above, in which the expression level of the above genes is measured by determining the amount of RNA molecules that are the products of the transcription of said genes, i.e. which are the products of the expression of said genes.
Some genes in the invention are able to express many variants, i.e. many RNA molecules that differ in their sequences. Theses variants generally differ in there sequence after alternative splicing, said alternative splicing having as consequence to add, to delete and/or to modify one or more parts of the nucleic acid sequence contained in the gene in the resulting RNA molecule. The skilled person knows the mechanisms of alternative splicing.
Therefore some genes according to the invention can express more than one RNA molecule, and provide variants.
The PRDX gene is able to express 3 different variants: the first variant comprising or consisting of the nucleic acid sequence SEQ ID NO: 5, a second variant comprising or consisting of the nucleic acid sequence SEQ ID NO: 73 and a third variant comprising or consisting of the nucleic acid sequence SEQ ID NO: 74.
The SOD2 gene is able to express 3 different variants: the first variant comprising or consisting of the nucleic acid sequence SEQ ID NO: 7, a second variant comprising or consisting of the nucleic acid sequence SEQ ID NO: 75 and a third variant comprising or consisting of the nucleic acid sequence SEQ ID NO: 76.
The GSR gene is able to express 4 different variants: the first variant comprising or consisting of the nucleic acid sequence SEQ ID NO: 8, a second variant comprising or consisting of the nucleic acid sequence SEQ ID NO: 77, a third variant comprising or consisting of the nucleic acid sequence SEQ ID NO: 78 and a fourth variant comprising or consisting of the nucleic acid sequence SEQ ID NO: 79.
The GLRX gene is able to express 2 different variants: the first variant comprising or consisting of the nucleic acid sequence SEQ ID NO: 9 and a second variant comprising or consisting of the nucleic acid sequence SEQ ID NO: 80.
The PDRX5 gene is able to express 3 different variants: the first variant comprising or consisting of the nucleic acid sequence SEQ ID NO: 11, a second variant comprising or consisting of the nucleic acid sequence SEQ ID NO: 81 and a third variant comprising or consisting of the nucleic acid sequence SEQ ID NO: 82.
The GPX4 gene is able to express 3 different variants: the first variant comprising or consisting of the nucleic acid sequence SEQ ID NO: 14, and a second variant comprising or consisting of the nucleic acid sequence SEQ ID NO: 83.
The PDRX5 gene is able to express 3 different variants: the first variant comprising or consisting of the nucleic acid sequence SEQ ID NO: 16, a second variant comprising or consisting of the nucleic acid sequence SEQ ID NO: 84 and a third variant comprising or consisting of the nucleic acid sequence SEQ ID NO: 85.
Thus, according to the invention, the measure of the expression level of the gene comprising or being constituted by SEQ ID NO: 5, can be evaluated by the measure of the expression level of the gene comprising or being constituted by SEQ ID NO: 73 or SEQ ID NO: 74.
In the same manner, the measure of the expression level of the gene comprising or being constituted by SEQ ID NO: 7, can be evaluated by the measure of the expression level of the gene comprising or being constituted by SEQ ID NO: 75 or SEQ ID NO: 76.
Moreover, the measure of the expression level of the gene comprising or being constituted by SEQ ID NO: 8, can be evaluated by the measure of the expression level of the gene comprising or being constituted by SEQ ID NO: 77, SEQ ID NO: 78 or SEQ ID NO: 79.
Moreover, the measure of the expression level of the gene comprising or being constituted by SEQ ID NO: 9, can be evaluated by the measure of the expression level of the gene comprising or being constituted by SEQ ID NO: 80.
Moreover, the measure of the expression level of the gene comprising or being constituted by SEQ ID NO: 11, can be evaluated by the measure of the expression level of the gene comprising or being constituted by SEQ ID NO: 81 or SEQ ID NO: 82.
Moreover, the measure of the expression level of the gene comprising or being constituted by SEQ ID NO: 14, can be evaluated by the measure of the expression level of the gene comprising or being constituted by SEQ ID NO: 83.
Moreover, the measure of the expression level of the gene comprising or being constituted by SEQ ID NO: 16, can be evaluated by the measure of the expression level of the gene comprising or being constituted by SEQ ID NO: 84 or SEQ ID NO: 85.
Therefore, as disclosed before and hereafter in the invention, the genes comprising or being constituted by the following sequences: SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 14 and SEQ ID NO: 16 can be replaced by their respective variants, as defined above.
The genes used according to the invention are represented in the following table 1:
Table 1 represents SEQ ID of the genes, and the variant when they exist, used in the invention.
According to the invention, hematological disorders correspond to disorders which primarily affect the blood. In particular, hematological disorders according to the invention encompass all cytopenias (anemia: decrease in red blood cell count or hemoglobin, thrombopenias: decrease in blood platelet count, and leukopenias: decrease in leukocyte count) whatever the mechanism such as hemoglobinopathies and myelodysplastic syndrome, myeloproliferative disorders (increased numbers of myeloid cells or myelofibrosis, including chronic myeloid leukemia, polycythemia vera, essential thrombocythemia, idiopathic myelofibrosis), lymphoproliferative disorders (increased numbers of lymphoid cells, including chronic lymphocytic leukemia, lymphomas, myeloma, plasmacytoma), acute leukemias and coagulopathies (disorders of bleeding and coagulation).
In one advantageous embodiment, the invention relates to a method as defined above, wherein if the ratio established in step b). is
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- ≦0.3, for the gene comprising or being constituted by the nucleic acid sequence SEQ ID NO: 1, and
- ≧3.0, for the genes comprising or being constituted by the nucleic acid sequences SEQ ID NO: 2 and 3,
then said biological sample is representative of an acute myeloid leukemia.
In other words, an embodiment of the invention relates to a method as defined above, wherein if
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- the ratio R1 is ≦0.3, for the gene comprising or being constituted by the nucleic acid sequence SEQ ID NO: 1, and
- the ratios R2 and R3 are ≧3.0, for the genes comprising or being constituted by the nucleic acid sequences SEQ ID NO: 2 and 3, then said biological sample is representative of an acute myeloid leukemia.
In an embodiment of the invention, when the ratio R1 is ≦0.3 and the ratios R2 and R3 are both ≧3, the sample originating from the patient is representative of an acute myeloid leukemia (AML). Said 3 criterions are cumulative.
AML are clonal proliferation of immature cells of the myeloid origin. They may appear de novo or secondary in patients with myelodysplastic syndrome (MDS). The classification prepared by the French-American-British group (FAB) considers eight varieties (M0-M7) based on morphological criteria and on the immunophenotype of the neoplastic cells (Bennett J M, et al, 1976).
The World Health Organisation (WHO) classifies AML by incorporating morphological, immunophenotypical, genetic and clinical data to be able to define biological homogeneous entities and with clinical relevance. Thus, AML is classified into four large categories:
1.—AML with recurrent genetic anomalies,
2.—AML with multilineage dysplasia,
3.—AML related to treatment and
4.—non-classifiable AML.
Before the invention, the cytogenetic analysis represented the most powerful prognosis factor. It is used to identify subgroups of AML with different prognosis: low risk with favourable response to treatment (t(8;21), t(15;17) or inv(16)), intermediate risk (normal karyotype or t(9;11) or high risk (inv(3), del(5q) or del(7q), or more than three alterations). There is molecular heterogeneity within the risk group. In some cases of patients with normal karyotype, the presence of mutations has been found in some genes.
Advantageously, the invention relates to the method as defined above, wherein if the ratio established in step b). is
-
- ≦0.3, for the gene comprising or being constituted by the nucleic acid sequence SEQ ID NO: 1, and
- ≧3.0, for the genes comprising or being constituted by the nucleic acid sequences SEQ ID NO: 2 and 3,
and further wherein the ratio R10 between the expression level of the gene consisting of SEQ ID NO: 10 measured in said sample and in said control sample is lower than 0.5, (R10≦0.5), preferably is lower than 0.3, (R10≦0.3)
then said biological sample is representative of an acute myeloid leukemia.
In another embodiment, the invention relates to a method as defined above, wherein step c.) is such that
if the ratio established in step b). for each genes of any combination of at least 3 genes from said sub-group is either ≧2 or ≦0.5, provided that
-
- the ratio between the expression level of the gene comprising or being constituted by the nucleic acid sequence SEQ ID NO: 1 measured in said biological sample and measured in said control sample, is not ≦0.3, or
- the ratios between the expression level of each of genes comprising or being constituted by the nucleic acid sequences SEQ ID NO: 2 or 3 measured in said biological sample and measured in said control sample, is not ≧3,
then said biological sample is representative of a myelodysplasic disorder, in particular myelodysplasia chosen among refractory anemia (RA), refractory anemia with ringed sideroblasts (RARS), refractory cytopenia with multilineage dysplasia (RCMD), refractory anemia with excess of blasts (RAEB), 5q-syndrome and myelodysplasia unclassifiable.
In other words, in another advantageous embodiment, the invention relates to a method as defined above, wherein
if the ratio established in step b). for each genes of any combination of at least 3 genes from said sub-group is either ≧2 or ≦0.5,
provided that if the combination of at least 3 genes corresponds to the genes comprising or being constituted by the nucleic acid sequence SEQ ID NO: 1, 2 and 3, wherein
-
- the ratio RI is ≦0.3, for the gene comprising or being constituted by the nucleic acid sequence SEQ ID NO: 1, and
- the ratios RI are ≧3.0, for the genes comprising or being constituted by the nucleic acid sequences SEQ ID NO: 2 and 3, this combination is excluded
then said biological sample is representative of a myelodysplasic disorder, in particular myelodysplasia chosen among refractory anemia with ringed sideroblasts (RARS), refractory cytopenia with multilineage dysplasia (RCMD), refractory anemia with excess of blasts (RAEB) or 5q-syndrome and unclassifiable myelodysplasia.
According to the invention, if the ratio of at least 3 genes belonging to the sub-group constituted by the genes comprising or being constituted by the nucleic acid SEQ ID NO: 1-6 is either ≧2 or ≦0.5, excluding the particular combination of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 2 wherein the ratio of SEQ ID NO: 1 is ≦0.3 and the ratios of SEQ ID NO: 2 and 3 are ≧3, then biological sample is representative of a myelodysplasic disorder, in particular myelodysplasia chosen among refractory anemia with ringed sideroblasts (RARS), refractory cytopenia with multilineage dysplasia (RCMD), refractory anemia with excess of blasts (RAEB) or 5q-syndrome and unclassifiable myelodysplasia.
According to the invention, myelodysplasic disorders are defined as preleukemia, and correspond to diverse collection of hematological conditions united by ineffective production (or dysplasia) of myeloid blood cells and risk of transformation to acute myelogenous leukemia (AML).
The French-American-British (FAB) classification has subdivided the myelodysplasic disorders as follows:
Refractory anemia (RA), characterized by less than 5% primitive blood cells (myeloblasts) in the bone marrow and pathological abnormalities primarily seen in red cell precursors,
Refractory anemia with ringed sideroblasts (RARS), also characterized by less than 5% myeloblasts in the bone marrow, but distinguished by the presence of 15% or greater red cell precursors in the marrow being abnormal iron-stuffed cells called “ringed sideroblasts”,
Refractory anemia with excess blasts (RAEB), characterized by 5-20% myeloblasts in the marrow,
Refractory anemia with excess blasts in transformation (RAEB-T), characterized by 21-30% myeloblasts in the marrow (>30% blasts is defined as acute myeloid leukemia), and
Chronic myelomonocytic leukemia (CMML), not to be confused with chronic myelogenous leukemia or CML, characterized by less than 20% myeloblasts in the bone marrow and greater than 1000×109/μL monocytes (a type of white blood cell) circulating in the peripheral blood.
More recently, the World Health Organization (WHO) has classified dysplastic syndromes as follows:
Chromosome 5q deletion syndrome (chromosome 5q monosomy, 5q-syndrome) is a rare disorder caused by loss of part of the long arm (q arm) of human chromosome 5.
The 5q-syndrome is characterized by macrocytic anemia and often thrombocytosis, erythroblastopenia, megakaryocyte hyperplasia with nuclear hypolobation and an isolated interstitial deletion of chromosome 5. The 5q-syndrome is found predominantly in females of advanced age.
In still another embodiment, the invention relates to a method above defined, wherein
said set comprises 10 genes, said 10 genes comprising or being constituted by the nucleic acid sequences SEQ ID NO: 1 to 10, and the step c). is such that
-
- if the ratio established in step b). for each genes of any combination of 3 genes from said sub-group is either ≧2 or ≦0.5,
provided that - the ratio between the expression level of the gene comprising or being constituted by the nucleic acid sequence SEQ ID NO: 1 measured in said biological sample and measured in said control sample, is not ≦0.3, or
- the ratios between the expression level of each of genes comprising or being constituted by the nucleic acid sequences SEQ ID NO: 2 or 3 measured in said biological sample and measured in said control sample, is not ≧3
and further - if the ratio established in step b) for at least one gene of said set that does not belong to said subgroup is ≧2 and the ratio established in step b) of at least one other gene of said set that does not belong to said subgroup is ≦0.5,
then said biological sample is representative of a refractory anemia with excess of blast or of a 5q-syndrome.
- if the ratio established in step b). for each genes of any combination of 3 genes from said sub-group is either ≧2 or ≦0.5,
In other words, in still another advantageous embodiment, the invention relates to a method above defined, wherein
said set comprises 10 genes, said 10 genes comprising or being constituted by the nucleic acid sequences SEQ ID NO: 1 to 10, and the step c). is such that
-
- if the ratio established in step b). for each genes of any combination of 3 genes from said sub-group is either ≧2 or ≦0.5,
provided that if the combination of at least 3 genes corresponds to the genes comprising or being constituted by the nucleic acid sequence SEQ ID NO: 1, 2 and 3, wherein - the ratio RI is ≦0.3, for the gene comprising or being constituted by the nucleic acid sequence SEQ ID NO: 1, and
- the ratios RI are ≧3.0, for the genes comprising or being constituted by the nucleic acid sequences SEQ ID NO: 2 and 3,
this combination is excluded,
and further - if the ratio established in step b) for at least one gene of said set that does not belong to said subgroup is ≧2 and the ratio established in step b) of at least one other gene of said set that does not belong to said subgroup is ≦0.5,
then said biological sample is representative of a refractory anaemia with excess of blast or of a 5q-syndrome.
- if the ratio established in step b). for each genes of any combination of 3 genes from said sub-group is either ≧2 or ≦0.5,
In this advantageous embodiment of the invention, the genes belonging to the set (i.e. genes comprising or being constituted by SEQ ID NO: 1 to 10) are helpful for discriminating the myelodysplastic disorders.
More precisely, the genes belonging to the set but that do not belong to the sub group (i.e. genes comprising or being constituted by SEQ ID NO: 7 to 10-D in
Then, if, by measuring the expression level of the genes SEQ ID NO: 1-6, the biological sample is considered to be representative of myelodyplastic disorder, it is possible according to the invention to separate refractory anemia with excess of blast and of a 5q-syndrome from the other pathologies, when the ratio of the expression of at least one gene of the group consisting of SEQ ID NO: 7 to 10 is ≧2 and when the ratio of the expression of at least one gene of the group consisting of SEQ ID NO: 7 to 10 is ≦0.5.
Another advantageous embodiment of the invention relates to a method previously defined, wherein
said set comprises 10 genes, said 10 genes comprising or being constituted by the nucleic acid sequences SEQ ID NO: 1 to 10, and the step c). is such that
-
- if the ratio established in step b). for each genes of any combination of 3 genes from said sub-group is either ≧2 or ≦0.5, provided that
- the ratio between the expression level of the gene comprising or being constituted by the nucleic acid sequence SEQ ID NO: 1 measured in said biological sample and measured in said control sample, is not ≦0.3, or
- the ratios between the expression level of each of genes comprising or being constituted by the nucleic acid sequences SEQ ID NO: 2 or 3 measured in said biological sample and measured in said control sample, is not ≧3,
and - if the ratio established in step b) for at least one gene of said set that does not belong to said subgroup is ≧2 and the ratio established in step b) of at least one other gene of said set that does not belong to said subgroup is ≦0.5,
and further
if the ratio established in step b) for at least 4 genes of the group of 24 genes that does not belong to said set is ≧3,
then said biological sample is representative of a refractory anemia with excess of blast.
In other words, another advantageous embodiment of the invention relates to a method previously defined, wherein
said set comprises 10 genes, said 10 genes comprising or being constituted by the nucleic acid sequences SEQ ID NO: 1 to 10, and the step c). is such that
-
- if the ratio established in step b). for each genes of any combination of 3 genes from said sub-group is either ≧2 or ≦0.5,
provided that if the combination of at least 3 genes corresponds to the genes comprising or being constituted by the nucleic acid sequence SEQ ID NO: 1, 2 and 3, wherein - the ratio Ri is ≦0.3, for the gene comprising or being constituted by the nucleic acid sequence SEQ ID NO: 1, and
- the ratios Ri are ≧3.0, for the genes comprising or being constituted by the nucleic acid sequences SEQ ID NO: 2 and 3,
this combination is excluded,
and - if the ratio established in step b) for at least one gene of said set that does not belong to said subgroup is ≧2 and the ratio established in step b) of at least one other gene of said set that does not belong to said subgroup is ≦0.5,
and further - if the ratio established in step b) for at least 4 genes of the group of 24 genes that does not belong to said set is ≧3,
then said biological sample is representative of a refractory anaemia with excess of blast.
- if the ratio established in step b). for each genes of any combination of 3 genes from said sub-group is either ≧2 or ≦0.5,
In this advantageous embodiment of the invention, the genes belonging to the group (i.e. genes comprising or being constituted by SEQ ID NO: 1 to 24) are helpful for discriminating between refractory anaemia with excess of blast and of a 5q-syndrome.
More precisely, the genes belonging to the group but that do not belong to the set (i.e. genes comprising or being constituted by SEQ ID NO: 11 to 24-E in
The invention also relates to a method for the diagnosis, and/or the classification, preferably in vitro, of an hematological disorder, in particular myeloid and/or lymphoid hematological disorder, preferably myeloid hematological disorder,
said method comprising the steps of:
a). measuring, from cells contained in a biological sample of a subject, preferably from blood cells or bone marrow cells containing sample, the expression level of at least the genes of a sub-group of 6 genes belonging to a set of genes chosen among a group of 24 genes, said group of 24 genes comprising or being constituted by the nucleic acid sequences SEQ ID NO:1 to 24,
said 6 genes belonging to said sub-group comprising or being constituted by the nucleic acid sequences SEQ ID NO: 1 to 6,
b). comparing the expression level of each genes measured in step a)., with the expression level of the same respective genes from cells contained in a control sample, preferably from a control sample containing blood cells or bone marrow cells, said control sample being of the same nature than said biological sample, to establish a gene expression level ratio Ri between the expression level of each genes measured in step a) and the expression level of the same respective genes from cells contained in a control sample, for each genes of said sub-group, and c). determining the status of said biological sample such that if the ratio Ri for each genes of any combination of 3 genes from said sub-group is
-
- either ≧2,
- or ≦0.5,
- said biological sample is representative of an hematological disorder cells.
An advantageous embodiment of the invention relates to a method as defined above, wherein if
-
- the ratio Ri is ≦0.3, for the gene comprising or being constituted by the nucleic acid sequence SEQ ID NO: 1, and
- the ratios Ri are ≧3.0, for the genes comprising or being constituted by the nucleic acid sequences SEQ ID NO: 2 and 3,
then said biological sample is representative of an acute myeloid leukemia.
In this advantageous embodiment of the method according to the invention, in step c). when the ratio Ri of the expression level of the genes GPX3 (SEQ ID NO: 1) ≦0.3, the ratio of the expression level of the genes GPX1(1) (SEQ ID NO: 2) is ≧3.0 and the ratio of the expression level of the genes GLRX2(2) (SEQ ID NO: 3) is ≧3.0, then the biological sample is representative of an acute myeloid leukemia (AML).
In another advantageous embodiment, the invention relates to a method as defined above, wherein step c.) is such that
if the ratio Ri established in step b). for each genes of any combination of at least 3 genes from said sub-group is either ≧2 or ≦0.5,
provided that if the combination of 3 genes corresponds to the genes comprising or being constituted by the nucleic acid sequence SEQ ID NO: 1, 2 and 3, wherein
-
- the ratio Ri is ≦0.3, for the gene comprising or being constituted by the nucleic acid sequence SEQ ID NO: 1, and
- the ratios Ri are ≧3.0, for the genes comprising or being constituted by the nucleic acid sequences SEQ ID NO: 2 and 3,
said combination is excluded
then said biological sample is representative of a myelodysplasic disorder, in particular myelodysplasia chosen among refractory anemia with ringed sideroblasts (RARS), refractory cytopenia with multilineage dysplasia (RCMD), refractory anemia with excess of blasts (RAEB) or 5q-syndrome and unclassifiable myelodysplasia.
In other words, when the ratio Ri, established in step c)., of the expression level of each gene of any combination of at least 3 genes chosen among the genes comprising or being constituted by SEQ ID NO: 1-6 is either ≧2 or ≦0.5, and said combination does not corresponds to the combination that defines a biological sample as representative of an AML, then said sample is representative of a myelodysplastic disorder or syndrome.
Another advantageous embodiment of the invention relates to a method previously defines, wherein
said set comprises 10 genes, said 10 genes comprising or being constituted by the nucleic acid sequences SEQ ID NO: 1 to 10, and further, in step c.)
if the ratio Ri of all the genes of said set that do not belong to said subgroup is comprised between 0.3 to 2, the extremity of the interval being excluded
then said biological sample is representative of a refractory anemia with ringed sideroblasts or a refractory cytopenia with multilineage dysplasia.
In the invention “the genes of said set that do not belong to said subgroup” corresponds to the genes belonging to the group D as defined in
Since the subgroup consists of the genes comprising or being constituted by the nucleic acid sequences SEQ ID NO: 1-6 and the set consists of the genes comprising or being constituted by the nucleic acid sequences SEQ ID NO: 1-10, consequently, the genes of said set that do not belong to said subgroup correspond to the genes comprising or being constituted by the nucleic acid sequences SEQ ID NO: 7-10.
In the above embodiment, if the ratio Ri for each gene represented by the nucleic acid sequences SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, i.e. all the ratios of said genes, is comprised between 0.3 and 2, 0.3 and 2 being excluded from the interval, said biological sample is representative of a refractory anemia with ringed sideroblasts or a refractory cytopenia with multilineage dysplasia.
It is possible to write that if 0.3<R7<2, and 0.3<R8<2, and 0.3<R9<2, and 0.3<R10<2, R7, R8, R9 and R10 representing the respective ratio for the genes represented by SEQ ID NO: 7, 8, 9 and 10), further to the evaluation of the ratio of the genes SEQ ID NO: 1-6, then said biological sample is representative of a refractory anemia with ringed sideroblasts or a refractory cytopenia with multilineage dysplasia.
The terms “comprised between 0.3 to 2, the extremity of the interval being excluded” refer to the interval represented by the mathematical symbol: ]0.3;2.0[. This interval includes all the values comprised between 0.3 and 2.0, but excludes the specific values 0.3 and 2.
If Ri=0.3, or Ri=2.0, Ri does not belong to the interval ]0.3;2.0[.
The evaluation of the ratio of the expression level of the genes represented by SEQ ID NO: 7-10, in a specific interval, allows to discriminate some myelodysplastic syndrome.
To summarise,
if the ratio for each gene of any combination of 3 genes chosen among the 6 genes represented by SEQ ID NO: 1, 2, 3, 4, 5 and 6, is either ≧2.0 or ≦0.5, excluding the combination that defines leukemia, and the ratio of each gene represented by SEQ ID NO: 7, 8, 9 and 10 is comprised in the interval ]0.3; 2.0[, then the biological sample is representative of a refractory anemia with ringed sideroblasts or a refractory cytopenia with multilineage dysplasia.
In one another advantageous embodiment, the invention relates to a method according the definition mentioned above, wherein further, in step c.)
-
- if the ratio Ri of at least one gene belonging to said group of genes that does not belong to said set is ≦0.3, then said biological sample is representative of a refractory anemia with ringed sideroblasts, and
- if the ratio RI of at least one gene belonging to said group of genes that do not belong to said set is ≧3.0,
- then said biological sample is representative of a refractory cytopenia with multilineage dysplasia.
In the invention “gene belonging to said group of genes that does not belong to said set” corresponds to the genes belong ing to the group E as defined in
Since the subgroup consists of the genes comprising or being constituted by the nucleic acid sequences SEQ ID NO: 1-6, the set consists of the genes comprising or being constituted by the nucleic acid sequences SEQ ID NO: 1-10, and the group consists of the genes comprising or being constituted by the nucleic acid sequences SEQ ID NO: 1-24, consequently, the genes of said group that do not belong to said set correspond to the genes comprising or being constituted by the nucleic acid sequences SEQ ID NO: 11-24.
In the above embodiment, the genes represented by SEQ ID NO: 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 and 24 provide supplemental information regarding the nature of the tested biological sample.
To summarise,
1) a) if the ratio for each gene of any combination of 3 genes chosen among the 6 genes represented by SEQ ID NO: 1, 2, 3, 4, 5 and 6, is either ≧2.0 or ≦0.5, excluding the combination that defines leukemia, and
b) the ratio of each gene represented by SEQ ID NO: 7, 8, 9 and 10 is comprised in the interval ]0.3; 2.0[, and
c) the ratio of at least 1 gene, i.e. 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14 genes, represented by SEQ ID NO: 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 and 24 is ≧3.0, then said biological sample is representative of a refractory cytopenia with multilineage dysplasia, and
2)) a) if the ratio for each gene of any combination of 3 genes chosen among the 6 genes represented by SEQ ID NO: 1, 2, 3, 4, 5 and 6, is either ≧2.0 or ≦0.5, excluding the combination that defines leukemia, and
b) the ratio of each gene represented by SEQ ID NO: 7, 8, 9 and 10 is comprised in the interval ]0.3; 2.0[, and
c) the ratio of at least 1 gene, i.e. 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14 genes, represented by SEQ ID NO: 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 and 24 is ≦0.3, then said biological sample is representative of a refractory anemia with ringed sideroblasts.
In one other embodiment, the invention relates to a method as defined above, wherein
said set comprises 10 genes, said 10 genes comprising or being constituted by the nucleic acid sequences SEQ ID NO: 1 to 10, and further
if
-
- the ratio Ri of at least one gene of said set that does not belong to said subgroup is ≧2.0, or
- the ratio Ri of at least one gene of said set that does not belong to said subgroup is ≦0.3, or
- the ratio Ri of at least one gene of said set that does not belong to said subgroup is ≧2.0 and the ratio Ri of another gene that does not belong to said subgroup is ≦0.3,
- then said biological sample is representative of chosen among refractory cytopenia with multilineage dysplasia, refractory anemia with excess of blasts, 5 q-syndroma and unclassified myelodysplasia.
In this advantageous embodiment, it is taking account of the case wherein at least one gene represented by SEQ ID NO: 7, 8 9 or 10 does not belong to the interval ]0.3; 2.0[ as defined above.
In a first case, at least one gene has a ratio ≧2.0, whatsoever the ratio of the other genes. In a second case at least one gene has a ratio ≦0.3, whatsoever the ratio of the other genes. In a third case, both at least one gene has a ratio ≧2.0 and another gene has a ratio ≦0.3, whatsoever the ratio of the other genes.
In one other advantageous embodiment, the invention relates to a method previously defined, wherein
if the ratio Ri of at least one gene of said set that does not belong to said subgroup is ≧2.0 and the ratio Ri of at least another gene of said set that does not belong to said subgroup is ≦0.5,
then said biological sample is a representative refractory anemia with excess of blasts or of 5q-syndrome.
This embodiment concerns the case in which at least one gene has a ratio ≧2.0, whatsoever the ratio of the other genes, and in particular the case in which at least one gene has a ratio ≧2.0 and another gene has a ratio ≦0.5, and therefore possibly ≦0.3.
This particular situation allows to detect, or to identify, myelodysplastic syndromes that are representative refractory anemia with excess of blasts or of 5q-syndrome.
In one other advantageous embodiment, the invention relates to a method previously defined, wherein
if the ratio Ri of at least one gene of said set that does not belong to said subgroup is ≧2.0 and the ratio Ri of at least another gene of said set that does not belong to said subgroup is ≦0.5,
then said biological sample is a representative refractory anemia with excess of blast or 5q-syndrome,
and further
-
- if the ratios Ri of at least four genes belonging to said group of genes that do not belong to said set are ≧3.0, then said biological sample is representative of a refractory anemia with excess of blasts, and
- if the ratios Ri of at most three genes belonging to said group of genes that do not belong to said set are ≧3.0, then said biological sample is representative of a 5q-syndrome.
To summarise,
1) a) if the ratio for each gene of any combination of 3 genes chosen among the 6 genes represented by SEQ ID NO: 1, 2, 3, 4, 5 and 6, is either ≧2.0 or ≦0.5, excluding the combination that defines leukemia, and
b) the ratio of at least one gene represented by SEQ ID NO: 7, 8, 9 and 10 is ≧2.0, and at least one other gene represented by SEQ ID NO: 7, 8, 9 and 10 is ≦0.5, and
c) the ratios of at least 4 genes, i.e. 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14 genes, represented by SEQ ID NO: 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 and 24 are ≧3.0, then said biological sample is representative of refractory anemia with excess of blasts, and
2)) a) if the ratio for each gene of any combination of 3 genes chosen among the 6 genes represented by SEQ ID NO: 1, 2, 3, 4, 5 and 6, is either ≧2.0 or ≦0.5, excluding the combination that defines leukemia, and
b) the ratio of at least one gene represented by SEQ ID NO: 7, 8, 9 and 10 is ≧2.0, and at least one other gene represented by SEQ ID NO: 7, 8, 9 and 10 is ≦0.5, and
c) the ratio of at most 3 genes, i.e. 0, or 1, or 2, or 3, genes, represented by SEQ ID NO: 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 and 24 are ≧3.0, then said biological sample is representative of a 5q-syndrome.
In one other advantageous embodiment, the invention relates to a method according the above definition,
wherein if
-
- the ratio Ri of at least one gene of said set that does not belong to said subgroup is ≧2.0 and
- the ratio Ri of no gene of said set that does not belong to said subgroup is ≦0.5,
- then said biological sample is representative of a refractory cytopenia with multilineage dysplasia,
In this specific embodiment, if the ratio of at least one gene represented by SEQ ID NO: 7, 8 9 or 10 is ≧2.0 and the ratio of the other genes is included in the interval [ 0.5; +∞[, then said biological sample is representative of a refractory cytopenia with multi lineage dysplasia.
To summarize:
a) if the ratio for each gene of any combination of 3 genes chosen among the 6 genes represented by SEQ ID NO: 1, 2, 3, 4, 5 and 6, is either ≧2.0 or ≦0.5, excluding the combination that defines leukemia, and
b) the ratio of at least one gene represented by SEQ ID NO: 7, 8, 9 and 10 is ≧2.0, and the ratio of no gene represented by SEQ ID NO: 7, 8, 9 and 10 is ≦0.5, then said biological sample is representative of a refractory cytopenia with multilineage dysplasia.
In one other advantageous embodiment, the invention relates to a method according the above definition,
wherein further if
-
- the ratio Ri of at least one gene of said set that does not belong to said subgroup is ≦0.5 and
- the ratio RI of no gene of said set that does not belong to said subgroup is ≧2.0,
- then said biological sample is representative of refractory anemia with ringed sideroblasts, refractory cytopenia with multilineage dysplasia, refractory anemia with excess of blasts, 5q-syndrome or unclassified myelodysplasia,
In this specific embodiment, if the ratio of at least one gene represented by SEQ ID NO: 7, 8 9 or 10 is ≦0.5 and the ratio of the other genes is included in the interval ]−∞; 2.0], then said biological sample is representative of refractory anemia with ringed sideroblasts, refractory cytopenia with multilineage dysplasia, refractory anemia with excess of blasts, 5q-syndrome or unclassified myelodysplasia.
To summarize:
a) if the ratio for each gene of any combination of 3 genes chosen among the 6 genes represented by SEQ ID NO: 1, 2, 3, 4, 5 and 6, is either ≧2.0 or ≦0.5, excluding the combination that defines leukemia, and
b) the ratio of at least one gene represented by SEQ ID NO: 7, 8, 9 and 10 is ≦0.5, and the ratio of no gene represented by SEQ ID NO: 7, 8, 9 and 10 is ≧2.0, then said biological sample is representative of refractory anemia with ringed sideroblasts, refractory cytopenia with multilineage dysplasia, refractory anemia with excess of blasts, 5q-syndrome or unclassified myelodysplasia.
In one other advantageous embodiment, the invention relates to a method as defined above, wherein, in step c.)
if
-
- the ratio Ri of at least one gene of said set that does not belong to said subgroup is ≦0.5 and
- the ratio Ri of no gene of said set that does not belong to said subgroup is ≧2.0,
- and further
- if
- the ratios Ri of all the genes belonging to said group of genes that do not belong to said set are comprised between 0.5 to 2, the extremity of the interval being excluded,
- then said biological sample is representative of a unclassified myelodysplasia,
In this advantageous embodiment the genes represented by SEQ ID NO: 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 are useful for discriminate unclassified myelodysplasia from refractory anemia with ringed sideroblasts, refractory cytopenia with multilineage dysplasia and 5q-syndrome.
In one other advantageous embodiment, the invention relates to a method as defined above, wherein, in step c.)
if
-
- the ratio Ri of at least one gene of said set that does not belong to said subgroup is ≦0.3 and
- the ratio Ri of no gene of said set that does not belong to said subgroup is ≧2.0,
- and further
- if
- the ratios Ri of at least one gene belonging to said group of genes that do not belong to said set is ≧2.0, then said biological sample is representative of a 5q-syndrome or refractory cytopenia with multilineage dysplasia.
To summarize:
a) if the ratio for each gene of any combination of 3 genes chosen among the 6 genes represented by SEQ ID NO: 1, 2, 3, 4, 5 and 6, is either ≧2.0 or ≦0.5, excluding the combination that defines leukemia, and
b) the ratio of at least one gene represented by SEQ ID NO: 7, 8, 9 and 10 is ≦0.3, and the ratio of no gene represented by SEQ ID NO: 7, 8, 9 and 10 is ≧2.0, and
c) the ratios RI of at least one gene represented by SEQ ID NO: 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 is ≧2.0,
then said biological sample is representative of a 5q-syndrome or refractory cytopenia with multilineage dysplasia.
Another advantageous embodiment of the invention relates to a method as defined above, wherein
if the ratio Ri of at least one gene of said set that does not belong to said subgroup is ≦0.3 and the ratio Ri of no gene that does not belong to said subgroup is ≧2.0,
then said biological sample is representative of a unclassified myelodysplasia, a 5q-syndrome, or of a refractory cytopenia with multilineage dysplasia.
To summarize:
a) if the ratio for each gene of any combination of 3 genes chosen among the 6 genes represented by SEQ ID NO: 1, 2, 3, 4, 5 and 6, is either ≧2.0 or ≦0.5, excluding the combination that defines leukemia, and
b) the ratio of at least one gene represented by SEQ ID NO: 7, 8, 9 and 10 is ≦0.3, and the ratio of no gene represented by SEQ ID NO: 7, 8, 9 and 10 is ≧2.0, said biological sample is representative of a unclassified myelodysplasia, a 5q-syndrome, or of a refractory cytopenia with multilineage dysplasia.
Another advantageous embodiment of the invention relates to a method as defined above, wherein
if the ratio Ri of at least one gene of said set that does not belong to said subgroup is ≦0.3 and the ratio Ri of no gene that does not belong to said subgroup is ≧2.0,
and further
if the ratios Ri of at least two genes of said set that do not belong to said subgroup are ≦0.3 and the ratio Ri of no gene of said set that does not belong to said subgroup is ≧2.0,
then said biological sample is representative of a 5q-syndrome, or of refractory cytopenia with multilineage dysplasia.
To summarize:
a) if the ratio for each gene of any combination of 3 genes chosen among the 6 genes represented by SEQ ID NO: 1, 2, 3, 4, 5 and 6, is either ≧2.0 or ≦0.5, excluding the combination that defines leukemia, and
b) the ratio of at least one gene represented by SEQ ID NO: 7, 8, 9 and 10 is ≦0.3, the ratio of at least one other gene represented by SEQ ID NO: 7, 8, 9 and 10 is ≦0.3 and the ratio of no gene represented by SEQ ID NO: 7, 8, 9 and 10 is ≧2.0, said biological sample is representative of a 5q-syndrome, or of a refractory cytopenia with multilineage dysplasia.
Another advantageous embodiment of the invention relates to a method as defined above, wherein
if the ratio Ri of at least one gene of said set that does not belong to said subgroup is ≦0.3 and the ratio Ri of no gene that does not belong to said subgroup is ≧2.0,
and
if the ratios Ri of at least two genes of said set that do not belong to said subgroup are ≦0.3 and the ratio Ri of no gene of said set that does not belong to said subgroup is ≧2.0,
then said biological sample is representative of a 5q-syndrome, or of refractory cytopenia with multilineage dysplasia.
and
-
- if further the ratios Ri of at least two genes belonging to said group of genes that do not belong to said set are ≧2.0, then said biological sample is representative of a 5q-syndrome.
To summarize:
a) if the ratio for each gene of any combination of 3 genes chosen among the 6 genes represented by SEQ ID NO: 1, 2, 3, 4, 5 and 6, is either ≧2.0 or ≦0.5, excluding the combination that defines leukemia, and
b) the ratio of at least one gene represented by SEQ ID NO: 7, 8, 9 and 10 is ≦0.3, the ratio of at least one other gene represented by SEQ ID NO: 7, 8, 9 and 10 is ≦0.3 and the ratio of no gene represented by SEQ ID NO: 7, 8, 9 and 10 is ≧2.0, and
c) the ratios of at least two genes represented by SEQ ID NO: 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 are ≧2.0,
said biological sample is representative of a 5q-syndrome.
The invention also relates to a method for the diagnosis, and/or the classification, preferably in vitro, of an hematological disorder, in particular myeloid and/or lymphoid hematological disorder, preferably myeloid hematological disorder,
said method comprising the steps of:
a). measuring, from cells contained in a biological sample of a subject, preferably from blood cells or bone marrow cells containing sample, the expression level 24 genes, said 24 genes comprising or being constituted by the nucleic acid sequences SEQ ID NO:1 to 24,
b). comparing the expression level of each genes measured in step a)., with the expression level of the same respective genes from cells contained in a control sample preferably from blood cells or bone marrow cells containing sample, said control sample being of the same nature than said biological sample, to establish a gene expression level ratio for each genes of said sub-group, and
c). determining the status of said biological sample such that if the ratio established in step b).
More advantageously, the invention relates to a method previously defined, wherein the expression level of the genes is measured by a method allowing the determination of the amount of the mRNA or of the cDNA corresponding to said genes. Preferably said method is a quantitative method.
Levels of mRNA can be quantitatively measured by northern blotting which gives size and sequence information about the mRNA molecules. A sample of RNA is separated on an agarose gel and hybridized to a radio-labeled RNA probe that is complementary to the target sequence. The radio-labeled RNA is then detected by an autoradiograph. Northern blotting is widely used as the additional mRNA size information allows the discrimination of alternately spliced transcripts.
Another approach for measuring mRNA abundance is reverse transcription quantitative polymerase chain reaction (RT-PCR followed with qPCR). RT-PCR first generates a DNA template from the mRNA by reverse transcription, which is called cDNA. This cDNA template is then used for qPCR where the change in fluorescence of a probe changes as the DNA amplification process progresses. With a carefully constructed standard curve qPCR can produce an absolute measurement such as number of copies of mRNA, typically in units of copies per nanolitre of homogenized tissue or copies per cell. qPCR is very sensitive (detection of a single mRNA molecule is possible), but can be expensive due to the fluorescent probes required.
Northern blots and RT-qPCR are good for detecting whether a single gene or few genes are expressed.
Other methods known for one skilled in the art include DNA microarrays or technologies like Serial Analysis of Gene Expression (SAGE).
SAGE can provide a relative measure of the cellular concentration of different messenger RNAs. The great advantage of tag-based methods is the “open architecture”, allowing for the exact measurement of any transcript are present in cells, the sequence of said transcripts could be known or unknown.
The preferred method used according to the invention is RT-qPCR.
In still another advantageous embodiment, the invention relates to a method above defined, wherein the measure of the expression level of the genes is carried out by using the at least 6 pairs of oligonucleotides belonging to a group of 24 pairs of oligonucleotides comprising or being constituted by the nucleic acid sequences SEQ ID NO: 25 to 72, said at least 6 pairs of oligonucleotides comprising or being constituted by the nucleic acid sequences SEQ ID NO: 25 to 36.
In the invention:
-
- the pair of oligonucleotides comprising of being constituted by the nucleic acid sequences SEQ ID NO: 25 and SEQ ID NO: 26, are used for measuring the expression level of the gene comprising or being constituted by the nucleic acid sequence SEQ ID NO: 1,
- the pair of oligonucleotides comprising of being constituted by the nucleic acid sequences SEQ ID NO: 27 and SEQ ID NO: 28, are used for measuring the expression level of the gene comprising or being constituted by the nucleic acid sequence SEQ ID NO: 2,
- the pair of oligonucleotides comprising of being constituted by the nucleic acid sequences SEQ ID NO: 29 and SEQ ID NO: 30, are used for measuring the expression level of the gene comprising or being constituted by the nucleic acid sequence SEQ ID NO: 3,
- the pair of oligonucleotides comprising of being constituted by the nucleic acid sequences SEQ ID NO: 31 and SEQ ID NO: 32, are used for measuring the expression level of the gene comprising or being constituted by the nucleic acid sequence SEQ ID NO: 4,
- the pair of oligonucleotides comprising of being constituted by the nucleic acid sequences SEQ ID NO: 33 and SEQ ID NO: 34, are used for measuring the expression level of the gene comprising or being constituted by the nucleic acid sequence SEQ ID NO: 5,
- the pair of oligonucleotides comprising of being constituted by the nucleic acid sequences SEQ ID NO: 35 and SEQ ID NO: 36, are used for measuring the expression level of the gene comprising or being constituted by the nucleic acid sequence SEQ ID NO: 6.
In one advantageous embodiment, the inventions relates to the method as defined above,
using the at least 10 pairs of oligonucleotides belonging to a group of 24 pairs of oligonucleotides comprising or being constituted by the nucleic acid sequences SEQ ID NO: 25 to 72, said at least 6 pairs of oligonucleotides comprising or being constituted by the nucleic acid sequences SEQ ID NO: 25 to 44.
In the invention:
-
- the pair of oligonucleotides comprising of being constituted by the nucleic acid sequences SEQ ID NO: 36 and SEQ ID NO: 37, are used for measuring the expression level of the gene comprising or being constituted by the nucleic acid sequence SEQ ID NO: 7,
- the pair of oligonucleotides comprising of being constituted by the nucleic acid sequences SEQ ID NO: 38 and SEQ ID NO: 39, are used for measuring the expression level of the gene comprising or being constituted by the nucleic acid sequence SEQ ID NO: 8,
- the pair of oligonucleotides comprising of being constituted by the nucleic acid sequences SEQ ID NO: 41 and SEQ ID NO: 42, are used for measuring the expression level of the gene comprising or being constituted by the nucleic acid sequence SEQ ID NO: 9,
- the pair of oligonucleotides comprising of being constituted by the nucleic acid sequences SEQ ID NO: 43 and SEQ ID NO: 44, are used for measuring the expression level of the gene comprising or being constituted by the nucleic acid sequence SEQ ID NO: 10.
In one advantageous embodiment, the inventions relates to the method as defined above,
using 24 pairs of oligonucleotides comprising or being constituted by the nucleic acid sequences SEQ ID NO: 25 to 72.
In the invention:
-
- the pair of oligonucleotides comprising of being constituted by the nucleic acid sequences SEQ ID NO: 45 and SEQ ID NO: 46, are used for measuring the expression level of the gene comprising or being constituted by the nucleic acid sequence SEQ ID NO: 11,
- the pair of oligonucleotides comprising of being constituted by the nucleic acid sequences SEQ ID NO: 47 and SEQ ID NO: 48, are used for measuring the expression level of the gene comprising or being constituted by the nucleic acid sequence SEQ ID NO: 12,
- the pair of oligonucleotides comprising of being constituted by the nucleic acid sequences SEQ ID NO: 49 and SEQ ID NO: 50, are used for measuring the expression level of the gene comprising or being constituted by the nucleic acid sequence SEQ ID NO: 13,
- the pair of oligonucleotides comprising of being constituted by the nucleic acid sequences SEQ ID NO: 51 and SEQ ID NO: 52, are used for measuring the expression level of the gene comprising or being constituted by the nucleic acid sequence SEQ ID NO: 14,
- the pair of oligonucleotides comprising of being constituted by the nucleic acid sequences SEQ ID NO: 53 and SEQ ID NO: 54, are used for measuring the expression level of the gene comprising or being constituted by the nucleic acid sequence SEQ ID NO: 15,
- the pair of oligonucleotides comprising of being constituted by the nucleic acid sequences SEQ ID NO: 55 and SEQ ID NO: 56, are used for measuring the expression level of the gene comprising or being constituted by the nucleic acid sequence SEQ ID NO: 16,
- the pair of oligonucleotides comprising of being constituted by the nucleic acid sequences SEQ ID NO: 57 and SEQ ID NO: 58, are used for measuring the expression level of the gene comprising or being constituted by the nucleic acid sequence SEQ ID NO: 17,
- the pair of oligonucleotides comprising of being constituted by the nucleic acid sequences SEQ ID NO: 59 and SEQ ID NO: 60, are used for measuring the expression level of the gene comprising or being constituted by the nucleic acid sequence SEQ ID NO: 18,
- the pair of oligonucleotides comprising of being constituted by the nucleic acid sequences SEQ ID NO: 61 and SEQ ID NO: 62, are used for measuring the expression level of the gene comprising or being constituted by the nucleic acid sequence SEQ ID NO: 19,
- the pair of oligonucleotides comprising of being constituted by the nucleic acid sequences SEQ ID NO: 63 and SEQ ID NO: 64, are used for measuring the expression level of the gene comprising or being constituted by the nucleic acid sequence SEQ ID NO: 20,
- the pair of oligonucleotides comprising of being constituted by the nucleic acid sequences SEQ ID NO: 65 and SEQ ID NO: 66, are used for measuring the expression level of the gene comprising or being constituted by the nucleic acid sequence SEQ ID NO: 21,
- the pair of oligonucleotides comprising of being constituted by the nucleic acid sequences SEQ ID NO: 67 and SEQ ID NO: 68, are used for measuring the expression level of the gene comprising or being constituted by the nucleic acid sequence SEQ ID NO: 22,
- the pair of oligonucleotides comprising of being constituted by the nucleic acid sequences SEQ ID NO: 69 and SEQ ID NO: 70, are used for measuring the expression level of the gene comprising or being constituted by the nucleic acid sequence SEQ ID NO: 23,
- the pair of oligonucleotides comprising of being constituted by the nucleic acid sequences SEQ ID NO: 71 and SEQ ID NO: 72, are used for measuring the expression level of the gene comprising or being constituted by the nucleic acid sequence SEQ ID NO: 24.
The following table 3 represents genes/variants/oligonucleotides used according to the invention.
The oligonucleotides defined above are preferably used for carrying out a qPCR reaction.
qPCR is well known in the art, and can be carried out by using, in association with oligonucleotides allowing a specific amplification of the target gene, either with dyes or with reporter probe.
Both techniques are briefly summarized hereafter.
Real-Time PCR with Double-Stranded DNA-Binding Dyes as Reporters:
A DNA-binding dye binds to all double-stranded (ds) DNA in PCR, causing fluorescence of the dye. An increase in DNA product during PCR therefore leads to an increase in fluorescence intensity and is measured at each cycle, thus allowing DNA concentrations to be quantified.
However, dsDNA dyes such as SYBR Green will bind to all dsDNA PCR products, including nonspecific PCR products (such as Primer dimer). This can potentially interfere with or prevent accurate quantification of the intended target sequence.
The reaction is prepared as usual, with the addition of fluorescent dsDNA dye.
The reaction is run in a Real-time PCR instrument, and after each cycle, the levels of fluorescence are measured with a detector; the dye only fluoresces when bound to the dsDNA (i.e., the PCR product). With reference to a standard dilution, the dsDNA concentration in the PCR can be determined.
Like other real-time PCR methods, the values obtained do not have absolute units associated with them (i.e., mRNA copies/cell). As described above, a comparison of a measured DNA/RNA sample to a standard dilution will only give a fraction or ratio of the sample relative to the standard, allowing only relative comparisons between different tissues or experimental conditions. To ensure accuracy in the quantification, it is usually necessary to normalize expression of a target gene to a stably expressed gene (see below). This can correct possible differences in RNA quantity or quality across experimental samples.
Fluorescent Reporter Probe Method
Fluorescent reporter probes detect only the DNA containing the probe sequence; therefore, use of the reporter probe significantly increases specificity, and enables quantification even in the presence of non-specific DNA amplification. Fluorescent probes can be used in multiplex assays—for detection of several genes in the same reaction—based on specific probes with different-coloured labels, provided that all targeted genes are amplified with similar efficiency. The specificity of fluorescent reporter probes also prevents interference of measurements caused by primer dimers, which are undesirable potential by-products in PCR. However, fluorescent reporter probes do not prevent the inhibitory effect of the primer dimers, which may depress accumulation of the desired products in the reaction.
The method relies on a DNA-based probe with a fluorescent reporter at one end and a quencher of fluorescence at the opposite end of the probe. The close proximity of the reporter to the quencher prevents detection of its fluorescence; breakdown of the probe by the 5′ to 3′ exonuclease activity of the Taq polymerase breaks the reporter-quencher proximity and thus allows unquenched emission of fluorescence, which can be detected after excitation with a laser. An increase in the product targeted by the reporter probe at each PCR cycle therefore causes a proportional increase in fluorescence due to the breakdown of the probe and release of the reporter.
The PCR is prepared as usual, and the reporter probe is added.
During the annealing stage of the PCR both probe and primers anneal to the DNA target.
Polymerisation of a new DNA strand is initiated from the primers, and once the polymerase reaches the probe, its 5′-3′-exonuclease degrades the probe, physically separating the fluorescent reporter from the quencher, resulting in an increase in fluorescence.
Fluorescence is detected and measured in the real-time PCR thermocycler, and its geometric increase corresponding to exponential increase of the product is used to determine the threshold cycle (CT) in each reaction.
In one particular embodiment, the measure of the expression level of the genes as defined above is achieved, in addition to the above defined specific oligonucleotides, by using a probe commercially available. Each gene for which the expression level is expected is associated with a specific probe, a probe recognizing one gene is not able to recognize another gene. Moreover a probe specific of one gene can also detect, when they exist, variants of said genes.
The advantageous probes used in the invention are listed in the table A.
The association between gene/variant/oligonucleotides and probes are represented in the following table 4.
Table 4 can be read as follows: The expression level of GPX3 (SEQ ID NO: 1) can be quantitatively measured by using the oligonucleotides SEQ ID NO: 24 and 25, and using as probe, coupled with a fluorescent dye and a quencher, having the following sequence CCAGCCGC.
Also, another example: The expression level of PDRX (SEQ ID NO: 5), or one of its variants (SEQ ID NO 73 or 74) can be quantitatively measured by using the oligonucleotides SEQ ID NO: 33 and 34, and using as probe, coupled with a fluorescent dye and a quencher, having the following sequence CTGGCTGG.
The above definitions apply mutatis mutandis for the other genes.
Quencher and dye mentioned above can be chosen by the skilled person, depending of the assay.
In another advantageous embodiment, the invention relates to the method as defined above, using at least one of the oligonucleotide allowing the measurement of the expression level of each of at least 6 genes comprising or being constituted by the nucleic acid sequences SEQ ID NO: 1-6, preferably using at least one of the oligonucleotide allowing the measurement of the expression level of each of at least 10 genes comprising or being constituted by the nucleic acid sequences SEQ ID NO: 1-10, in particular using at least one of the oligonucleotide allowing the measurement of the expression level of each of the 24 genes comprising or being constituted by the nucleic acid sequences SEQ ID NO: 1-24.
In this embodiment, the method is adapted for northern blot assay.
The invention also relates to a composition comprising oligonucleotides allowing the measure of the expression of at least the genes of a sub-group of 6 genes belonging to a set of genes chosen among a group of 24 genes, said group of 24 genes comprising or being constituted by the nucleic acid sequences SEQ ID NO:1 to 24, said 6 genes belonging to said sub-group comprising or being constituted by the nucleic acid sequences SEQ ID NO: 1 to 6.
In one advantageous embodiment, the invention relates to a composition as defined above, comprising oligonucleotides allowing the measure of the expression of at least the genes of a set of 10 genes chosen among a group of 24 genes, said group of 24 genes comprising or being constituted by the nucleic acid sequences SEQ ID NO:1 to 24,
said 10 genes belonging to said set comprising or being constituted by the nucleic acid sequences SEQ ID NO: 1 to 10.
In one other advantageous embodiment, the invention relates to a composition as defined above, comprising oligonucleotides allowing the measure of the expression of the 24 of said group of 24 genes.
In one other advantageous embodiment, the invention relates to a composition as defined above, said composition comprising at least 12 oligonucleotides chosen among a library of 48 oligonucleotides comprising or consisting of the nucleic acid sequences SEQ ID NO: 25-72, allowing the measure of the expression of at least the genes of a sub-group of 6 genes belonging to a set of genes chosen among a group of 24 genes comprising or being constituted by the nucleic acid sequences SEQ ID NO:1 to 24,
said at least 12 oligonucleotides comprising or consisting of the nucleic acid sequences SEQ ID NO: 25-36.
In one other advantageous embodiment, the invention relates to a composition as defined above, said composition comprising at least 20 oligonucleotides chosen among a library of 48 oligonucleotides comprising or consisting of the nucleic acid sequences SEQ ID NO: 25-72, allowing the measure of the expression of at least the genes a set of 10 genes chosen among a group of 24 genes comprising or being constituted by the nucleic acid sequences SEQ ID NO:1 to 24,
said at least 20 oligonucleotides comprising or consisting of the nucleic acid sequences SEQ ID NO: 25-44.
In one other advantageous embodiment, the invention relates to a composition as defined above, said composition comprising 48 oligonucleotides comprising or consisting of the nucleic acid sequences SEQ ID NO: 25-72, allowing the measure of the expression of a group of 24 genes comprising or being constituted by the nucleic acid sequences SEQ ID NO:1 to 24.
The above composition may further comprise probes as defined above in Table 4.
In another aspect, the invention relates to the composition as mentioned above, for its use for the diagnosis, and/or the classification, preferably in vitro, of an hematological disorder, in particular myeloid and/or lymphoid hematological disorder, preferably myeloid hematological disorder.
Therefore, the invention relates to the above composition per se, and relates to said composition for its use as mentioned above.
The invention relates to a kit comprising at least 12 oligonucleotides chosen among a group of 48 oligonucleotides comprising or consisting of the nucleic acid sequences SEQ ID NO: 25-72, said at least 12 oligonucleotides comprising or consisting of the nucleic acid sequences SEQ ID NO: 25-36.
In one advantageous embodiment, the invention relates to the kit as defined above, comprising at least 20 oligonucleotides, said at least 20 oligonucleotides comprising or consisting of the nucleic acid sequences SEQ ID NO: 25-44.
In one advantageous embodiment, the invention relates to a kit as defined above comprising 48 oligonucleotides comprising or consisting of the nucleic acid sequences SEQ ID NO: 25-72.
The invention also relates to, in one advantageous embodiment, a kit as defined above, further comprising at least 6 specific probes that respectively interact the nucleic acid molecules comprising or consisting of SEQ ID NO: 1-6, preferably further comprising at least 10 specific probes that respectively interact the nucleic acid molecules comprising or consisting of SEQ ID NO: 1-10, in particular further comprising at least 24 specific probes that respectively interact the nucleic acid molecules comprising or consisting of SEQ ID NO: 1-24.
In one another advantageous embodiment, the invention relates to a kit as defined above, further comprising nucleic acid molecules corresponding to the genes SEQ ID NO: 1-24, in an amount representative at least one pathology chosen among: acute myeloid leukemia (AML), refractory anemia with ringed sideroblasts (RARS), refractory cytopenia with multilineage dysplasia (RCMD), refractory anemia with excess of blasts (RAEB) or 5q-syndrome and unclassifiable myelodysplasia.
In another advantageous embodiment, the invention also relates to the kit as defined above, further comprising nucleic acid molecules of a control sample as defined above.
The invention also relates to a positive control sample comprising or being constituted by at least the nucleic acid molecules corresponding to the genes represented by SEQ ID NO: 1-6, chosen among the group of 24 genes represented by SEQ ID NO: 1-24, said nucleic acid molecules being present in said sample in an amount as represented in the 6 first lines of table 5 or table 6, compared to an healthy sample in which each of the respective nucleic acid molecules are present in an amount of 1.
Table 5 represents, for each gene of SEQ ID NO: 1-24, the specific interval corresponding to the mentioned pathology.
Table 6 represents, for each gene of SEQ ID NO: 1-24, the mean±the standard deviation corresponding to the mentioned pathology.
The invention also relates to a method for determining the efficacy of a treatment of an hematological disorder, said treatment being liable to be administered to a patient,
said method comprising
a). a step of contacting, preferably in vitro, a biological sample of a subject afflicted by an hematological disorder, preferably a myeloid and/or a lymphoid hematological disorder, more preferably myeloid hematological disorder, with a drug liable to be used to treat, or liable to treat, said hematological disorder,
b). a step of measuring, in the biological sample contacted with a drug in step a)., the expression level of at least the genes of a sub-group of 6 genes belonging to a set of genes chosen among a group of 24 genes, said group of 24 genes comprising or being constituted by the nucleic acid sequences SEQ ID NO:1 to 24,
said 6 genes belonging to said sub-group comprising or being constituted by the nucleic acid sequences SEQ ID NO: 1 to 6,
and
c). a step of comparing the expression level of said at least the genes of a sub-group of 6 genes belonging to a set of genes chosen among a group of 24 genes obtained in step b). with the expression level of said at least the genes of a sub-group of 6 genes belonging to a set of genes chosen among a group of 24 genes obtained measured in said biological sample which has not been contacted with said drug liable to be used to treat, or liable to treat, said hematological disorder.
The above method is easy to carry out, and allows to evaluate the AML sample susceptibility to a drug. This is very important to reduce the cost of treatments, that can be ineffective in patient, because the tumor is resistant to the drug.
The above method is advantageously used to screen, in vitro, drugs having an effect on AML progression, and that could be used in vivo for the treatment of the patient.
This is more advantageous important for screening drugs, or compounds that are able to modulate the epigenetic modification, in particular demethylating agents such as azacytidine (5-azacytidine) or decitabine (5-azadeoxycytidine).
Azacytidine and decitabine are powerful chemotherapeutic agents used for treating AML and high grade MDS, but only about 30% of AML and high grade MDS are sensitive to their effects. Thus, in order to reduce the costs, and the side effects of ineffective treatment, it is advantageous to verify in vitro, if the AML and high grade MDS that has to be treated is responsive to these compounds.
The example 9 shows that the treatment of AML sample with azacytidine can modulate the expression ratio of the genes SEQ ID NO: 1-24, (and consequently at the genes SEQ ID NO: 1-6) demonstrating that azacytidine, in this particular patient from which the AML sample derives, would be effective if it is used in vivo.
Advantageously, the invention relates to a method as defined above, wherein said set consists of 10 genes consisting of the nucleic acid sequences SEQ ID NO: 1-10.
Advantageously, the invention relates to a method as defined above, the expression level of the 24 genes consisting of SEQ ID NO: 1-24 is measured.
Y-axis represents the ratio ΔCTgene (ΔCTgene=CTgene−CTGAPDH).
All the samples used in the following examples have been first tested according to the invention, in a blind test, and compared with control samples.
All the SMD and leukemic samples satisfy the provisions of the method according to the invention, i.e. in each of the samples at least 3 genes of the genes SEQ ID NO: 1-6 are expressed such that their ratio compared to the expression of the same corresponding genes in control samples is either lower than 0.5 or higher to 2.
Example 1In the following examples, the patient's samples and the analysis genes expressions are analysed as follows.
Material and Methods Sample HarvestNormal bone marrow (BM) samples (used as a reference) were obtained from patients undergoing orthopedic surgery. Bone marrow samples from MDS and AML patients were obtained at the time of diagnosis and during the follow up. AML and MDS cells were classified according to morphological, cytochemical and cytogenetical findings. Patients were informed and consenting following a procedure approved by the ethical committee. BM samples were aspirated into heparinized syringes and transferred to EDTA tubes.
Erythrocyte LysisLysis was performed in 47 mL lysis buffer containing EDTA (0.12 mM), potassium bicarbonate (KHCO3) (10 mM), and ammonium chloride (NH4Cl) (150 mM) for 3 mL of BM. Following incubation at room temperature for 15 min, cells were centrifuged at 700 g for 10 min, and washed, twice, in 20 mL of Phosphate Buffered Saline (PBS) (Invitrogen). The pellet was resuspended in Trizol® (Invitrogen) (1 mL/8.106 cells), mixed vigorously for 15 minutes. The lysate was stored at −80° C. until the RNA extraction step.
RNA ExtractionTotal RNA extraction was performed according to the chloroform/isopropanol/ethanol method. The phase separation was obtained by adding chloroform (0.2 mL per 1 mL of TRIzol® purchased from Invitrogen) to the lysate. After mixing for 50 sec, the solution was separated into three phases by centrifugation at 12,000 g for 15 min at 4° C. RNA was precipitated, from the aqueous phase, by adding isopropanol (0.5 mL per 1 mL of TRIzol®). Following incubation at room temperature for 10 min and centrifugation at 12,000 g for 10 min at 4° C., RNA was washed in 75% ethanol (1 mL per 1 mL of TRIzol®) and centrifugated at 7500 g for 5 min at 4° C. This step was performed twice. After removing ethanol supernatant, the RNA pellet was air-dried for 20 min. Then, RNA was dissolved in 50 μL of UltraPure™ DEPC-treated water (Invitrogen) and stored at −80° C.
RNA Quantification and QualificationTotal cellular RNA was quantified using a Nano-Drop 1000 spectrophotometer (Nano-Drop Technologies) and RNA purity was analyzed using an Agilent 2100 Bioanalyzer (Agilent Technologies).
Reverse Transcription and Quantitative Real-Time PCR (qRT-PCR) Analysis
Three micrograms of total RNA from each sample were reverse transcribed using the SuperScript® VILO™ cDNA Synthesis kit (Invitrogen) according to the protocol of the supplier. The relative quantification of gene expression was done by real-time PCR on the LightCycler® 480 microwell plate-based cycler platform (Roche Applied Science) using Universal ProbeLibrary assays designed with the ProbeFinder software (Roche Applied Science, www.roche-applied-science.com/sis/rtpcr/upl/ezhome.html). Primers were purchased from Invitrogen and Universal ProbeLibrary probes from Roche Applied Science. The nucleotide sequences of the primers and probes of each target are shown in Table A. All targets were concomitantly analyzed. qRT-PCR reactions were carried out in a total volume of 10 μL on 20 ng of cDNA using LightCycler® 480 Probes Master (Roche Applied Science). The LightCycler® 480 was programmed to an initial denaturation (95° C., 10 min) following by 45 cycles of 10 sec at 95° C., 30 sec at 60° C., 1 sec at 72° C. and a final cooling step at 40° C. for 10 sec. All reactions were run in triplicate, and average values were used for quantification. Results were analyzed by the relative quantification method (ΔΔCT=ΔCTpatient−ΔCTreference) using the Cycle threshold (CT) values determined with the LightCycler® 480 software (release 1.5.0) from Roche Applied Science. The human glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) was used as the endogenous control to normalize the expression of the target (ΔCT=CTtarget−CTendogenous control). Change of relative mRNA expression between a patient and the reference was determined for each target using the 2exp(−ΔΔCT) method (Livak K J and Schmittgen T D. Analysis of relative gene expression data using real time quantitative PCR and the 2exp(−ΔΔCT) method. Methods 2001; 25:402-408).
Example 2 Use of 6 Genes for Diagnosing Myelodysplasia and LeukemiaIn order to test the validity of the method, patient samples identified as representative of leukemic samples where used, and the expression level of each of the genes represented by SEQ ID NO: 1 to SEQ ID NO: 6 was evaluated by RT-qPCR as disclosed in Example 1, by using oligonucleotides and probes of the Table 4.
The results are represented in the following table 7:
Table 7 represents the ratio of the expression level of each indicated genes (SEQ ID NO: 1-6) for each AML sample #1-#5
The criterions defined in the invention are satisfied:
A sample is considered as representative of an hematological disorder when the ratio of the expression level of each genes of any combination of 3 genes among the genes represented by SEQ ID NO: 1-6 is either ≧2.0 or ≦0.5.
Whatever the combination of 3 genes taken in consideration, the above criterions are satisfied.
The other criterion is that: if ratio of SEQ ID NO: 1 is ≦0.3 and the ratio of both SEQ ID NO: 2 and 3 are ≧3.0, the sample is representative of an AML.
Again, for all the 5 above AML samples tested (Table 7), the criterions are satisfied.
All the tested samples satisfy the criterions regarding AML. Therefore, the method according to the invention allows the discrimination between AML and myelodysplastic disorders.
Example 3 Validation of the Method with Leukemic Cell LinesIn order to validate the method, the expression level of the genes represented by SEQ ID NO: 1-6 was evaluated in 11 cell lines corresponding to almost all the AML subtypes defined according to the FAB classification.
The following cell lines have been tested:
KG1a and KG1 (FAB M0/M1), HL60 (FAB M2), KASUMI-1 (FAB M2), ML-2 (FAB M4), MV4-11 (FAB M5), THP-1 (FAB M5), U937 (FAB M5), K562 (FAB M6), TF-1 (FAB M6) and UT7 (FAB M7).The expression levels are indicated in the following table 8.
Table 8 represents the ratios Ri between the expression levels of the indicated gene in the corresponding cell line, compared to the expression levels of the corresponding gene in control samples (healthy bone marrow samples).
The criterions defined in the invention as satisfied:
A sample is considered as representative of an hematological disorder when the ratio of the expression level of each genes of any combination of 3 genes among the genes represented by SEQ ID NO: 1-6 is either ≧2.0 or ≦0.5.
Whatever the combination of 3 genes taken in consideration, the above criterions are satisfied.
The other criterion is that: if ratio of SEQ ID NO: 1 is ≦0.3 and the ratio of both SEQ ID NO: 2 and 3 are ≧3.0, the sample is representative of an AML.
Again, for all the 11 above cell lines tested (Table 3), the criterions are satisfied, and the method confirm that these results obtained with this cell lines correspond to those obtained with primary leukemic cells.
Example 4 Classification of Myelodyplastic SampleAs defined above, it has been proposed that the measure of the expression level of the genes represented by SEQ ID NO: 1-24 allows to identify subtype of myelodysplastic syndrome.
A panel of patient sample, classified by other techniques, has been tested to validate the method according to the invention.
3 RARS, 6 RCMD and 3 RAEB have been used, and the expression level of the genes represented by SEQ ID NO: 1-24 have been evaluated.
Results are presented hereafter:
All the tested samples satisfy the criterions defined in the method according to the invention.
Example 5 Schematic Representation of RARS, RCMD, RAEB and AML Specific ProfilesThe results corresponding to the above classification can be illustrated by an “antioxidogram”, corresponding to areas representatives of a determined sample.
The expression of each gene of SEQ ID NO: 1-24 is measured by qRT-PCR as defined above, and compared to the expression level of an housekeeping gene GAPDH.
The antioxidogram for RARS sample (
Indeed, by measuring the ratio for each genes represented by SEQ ID NO: 1-24, a curve can be drawn. This curve can then be compared to the antioxidograms, and thus, it is easy to determine into what type of pathology belongs the studied sample.
Example 6 Classification of Unclassifiable Myelodysplastic SyndromesThe above antioxidogram can be used to classify myelodysplastic syndromes for which other classification techniques fails.
The expression level of each of the genes of SEQ ID NO: 1-24 has been measured according to the invention, and an curve has been established.
This curve (hashed line) has been then compared to the “specific” antioxidogram of RARS, RCDM, RAEB and AML.
Chronic myelomonocytic leukemia (CMML) is a form of leukemia featuring monocytosis. The categorization of this disease has been controversial.
Patients with CMML can present with various clinical features, mimicking either myelodysplastic syndroms or myeloproliferative neoplasms depending upon a patient's specific presentation.
Due to this controversy it was classified by the World Health Organization in a “myelodysplastic/myeloproliferative” category of medical conditions in the early 2000s.
The oxydogram according to the invention can be helpful to determine the status of a CMML sample, which is close to RAEB by histological analysis.
As shown in
These data demonstrate that CMML are distinct from SDM.
Example 8 Use of Oxydogram for the Follow-Up of Evolutive MDSAs mentioned previously, MDS evolve progressively toward AML. It is thus important to know if this progression is slow or rapid.
The oxydogram can be used to determine if the molecular expression of the genes SEQ ID NO: 1-24 has evolved from diagnosis to a determined date.
An example is shown in
Twelve months from the diagnosis, the expression level of the genes SEQ ID NO: 1-24 have also been measured.
The
New therapeutic agents are actively searched in order to treat AML. These compounds are expensive, due to extensive searches, and unfortunately are not universally effective in all AML samples.
An average of 30% of AML subtypes are responsive to the new therapeutic drugs, and could effectively be used in the patient, in order to slow down the leukemic progression.
However, positive or negative in vivo response to a drug cannot be obtained before 6 to 9 months after the beginning of the treatment.
The oxydogram according to the invention can be used to evaluate, in vitro, if the drug will be effective on the leukemic sample, by studying the modulation of the expression level of the genes SEQ ID NO: 1-24.
Claims
1. A composition comprising at least 12 oligonucleotides chosen among a library of 48 oligonucleotides comprising or consisting of the nucleic acid sequences SEQ ID NO: 25-72.
2. The composition according to claim 1, wherein said composition comprises at least the 12 oligonucleotides that comprise or consist of the nucleic acid sequences SEQ ID NO: 25-36.
3. A kit comprising at least 12 oligonucleotides chosen among a group of 48 oligonucleotides comprising or consisting of the nucleic acid sequences SEQ ID NO: 25-72, said at least 12 oligonucleotides comprising or consisting of the nucleic acid sequences SEQ ID NO: 25-36.
4. A method for diagnosis, and/or the classification, preferably in vitro, of an hematological disorder, in particular myeloid and/or lymphoid hematological disorder, preferably myeloid hematological disorder comprising, comprising measuring an expression level of a gene using the composition according to claim 1.
Type: Application
Filed: Sep 28, 2016
Publication Date: Jan 12, 2017
Inventors: Olivier HERAULT (Veigne), Christine VIGNON (Chambray-Les-Tours)
Application Number: 15/278,353
